pmid stringlengths 8 8 | pmcid stringlengths 8 11 ⌀ | source stringclasses 2
values | rank int64 1 9.78k | sections unknown | tokens int64 3 46.7k |
|---|---|---|---|---|---|
36967572 | PMC10214270 | pmc | 6,309 | {
"abstract": "Abstract The rapid rise of triboelectric nanogenerators (TENGs), which are emerging energy conversion devices in advanced electronics and wearable sensing systems, has elevated the interest in high‐performance and multifunctional triboelectric materials. Among them, cellulosic materials, affording high efficiency, biodegradability, and customizability, are becoming a new front‐runner. The inherently low dielectric constant limits the increase in the surface charge density. However, owing to its unique structure and excellent processability, cellulose shows great potential for dielectric modulation, providing a strong impetus for its advanced applications in the era of Internet of Things and artificial intelligence. This review aims to provide comprehensive insights into the fabrication of dielectric‐enhanced cellulosic triboelectric materials via dielectric modulation. The exceptional advantages and research progress in cellulosic materials are highlighted. The effects of the dielectric constant, polarization, and percolation threshold on the charge density are systematically investigated, providing a theoretical basis for cellulose dielectric modulation. Typical dielectric characterization methods are introduced, and their technical characteristics are analyzed. Furthermore, the performance enhancements of cellulosic triboelectric materials endowed by dielectric modulation, including more efficient energy harvesting, high‐performance wearable electronics, and impedance matching via material strategies, are introduced. Finally, the challenges and future opportunities for cellulose dielectric modulation are summarized.",
"introduction": "1 Introduction Cellulose is one of the oldest natural polymers on Earth; it has a unique structure and special properties. [ \n \n 1 \n \n ] Cellulose has been used for centuries as a key material in various practical applications and has contributed to human evolution and technological developments. [ \n \n 2 \n \n ] With the rapid development of the Fourth Industrial Revolution, several requirements have been put forward for modern advanced materials in regard to convenience and intelligence. [ \n \n 3 \n \n ] Cellulose is a renewable, low‐cost, and biodegradable natural biomass material, with promising properties: suitable mechanical properties, easy processability, dielectric property, piezoelectricity, and convertibility. [ \n \n 4 \n \n ] Additionally, nanocellulose has offered great advantages in the synthesis of advanced functional materials owing to its excellent properties and unique shape. Hence, cellulose shows much adaptability and is highly competitive among many polymer materials that have been widely used in cutting‐edge technology fields, such as renewable energy, environmental protection, biomedicine, aerospace, advanced electronic equipment, and green chemistry. [ \n \n 5 \n \n ] \n Among prospective cellulose‐based materials, cellulosic triboelectric materials have received extensive attention owing to their percolation energy harvesting and conversion capabilities. [ \n \n 6 \n \n ] In 2012, Wang et al. [ \n \n 7 \n \n ] developed a triboelectric nanogenerator (TENG) capable of converting mechanical energy into electrical energy. TENG has now proven to be a reliable technology for green energy harvesting and conversion because of its low cost, multiple structures, high fabrication efficiency, excellent environmental adaptability, and stable output. [ \n \n 8 \n \n ] The fundamental of TENGs is the coupling effect of contact electrification (CE) and electrostatic induction caused by the periodic contact and separation of triboelectric materials, respectively. [ \n \n 9 \n \n ] As the main component for CE, triboelectric materials directly affect the energy conversion efficiency and application prospects of TENGs; hence, they have become a trending research topic in recent years. [ \n \n 10 \n \n ] A large number of lone electron pairs of oxygen atoms endows cellulose with extremely high electron‐donating ability, i.e., the tendency to lose electrons, making cellulose a promising triboelectric material. [ \n \n 11 \n \n ] As the most abundant biomass resource on Earth, the sustainability of cellulose enables it to meet the needs of the Internet of Things (IoT) for large‐scale self‐powered sensing arrays. [ \n \n 12 \n \n ] Additionally, the inherent biodegradability and biocompatibility of cellulose render it environmentally friendly and harmless to human health, unlike most petroleum‐based polymer triboelectric materials. [ \n \n 13 \n \n ] In addition, the excellent processability enables the customization of cellulose into triboelectric materials with different properties, shapes, and sizes to suit specific environmental and functional requirements. [ \n \n 14 \n \n ] This is highly instrumental in expanding the application prospects of TENGs. To date, research on cellulosic triboelectric materials has made great progress, including simple paper‐based, wood‐based, and gel‐structured triboelectric materials, as well as chemical functionalization of the surface of cellulosic materials. Numerous efforts have been made to improve the triboelectric properties of cellulosic triboelectric materials. [ \n \n 15 \n \n ] However, much research is needed to achieve advanced cellulosic triboelectric materials toward completely harnessing the great potential of cellulose. A large number of research teams have proposed many methods to enhance and improve the output performance of TENGs, such as charge excitation of TENG, [ \n \n 16 \n \n ] structure optimization and innovation, [ \n \n 17 \n \n ] circuit design and management, [ \n \n 18 \n \n ] etc. These methods also play a positive role in expanding the application prospects of TENGs. In the choice of materials, the surface charge density of triboelectric materials determines the output performance of TENGs. [ \n \n 19 \n \n ] Based on dielectric theory, the dielectric constant ( k ) is the ability of a material to polarize in an alternating electric field. [ \n \n 20 \n \n ] It also reflects the ability of triboelectric materials to generate and retain triboelectric charges during the working process. [ \n \n 21 \n \n ] According to the frequency range, the polarization types can be divided into electronic polarization, ionic polarization, dipole polarization, and interface polarization. Most polymeric materials are composed of covalent bonds such as C—H that do not induce polarization, so their inherently low dielectric constants largely limit the surface charge density. [ \n \n 20 \n \n ] Therefore, dielectric modulation is typically performed to improve the surface charge density of triboelectric materials by enhancing their polarization response, thereby fundamentally increasing their effective dielectric constant. [ \n \n 22 \n \n ] Cellulose has a high capability of dielectric modulation because of its triboelectric properties and wide availability. In addition, owing to its unique structure and processability, cellulose has great potential for dielectric modulation via advanced material design strategies and processing technologies. [ \n \n 23 \n \n ] In recent years, numerous effective modulation methods have been proposed, such as chain‐structure modification, [ \n \n 24 \n \n ] polymer blending, [ \n \n 25 \n \n ] and conductive‐ or high‐k‐filler doping. [ \n \n 26 \n \n ] These direct approaches increase the dielectric constant and surface charge density, boosting the development of cellulosic triboelectric materials. Owing to dielectric modulation, the application of cellulose‐based TENGs has been significantly broadened from self‐powered multifunctional sensing to wearable health care systems, [ \n \n 27 \n \n ] microwave devices to electromagnetic interference (EMI) shielding, [ \n \n 28 \n \n ] and human‐machine interfaces to intelligent robots. [ \n \n 29 \n \n ] High‐k cellulosic triboelectric materials exhibit promising application potential in numerous emerging smart technologies and advanced manufacturing fields. This review focuses on fabricating dielectric‐enhanced cellulosic triboelectric materials via dielectric modulation. The dielectric properties of cellulosic triboelectric materials are improved by doping with conductive or high‐k fillers, thereby obtaining better triboelectric properties. This modulation method applies to cellulosic triboelectric materials of different dimensions as well as different types of fillers ( Figure \n \n 1 \n ). The excellent properties and unique advantages of high‐k cellulosic triboelectric materials are demonstrated by analyzing the preparation methods of cellulosic triboelectric materials. Furthermore, the principles and strategies of dielectric modulation are elaborated from both theoretical and strategic perspectives. Several commonly used dielectric characterization methods are introduced in detail, and their technical characteristics are analyzed. Subsequently, the performance enhancements endowed by dielectric modulation to cellulosic triboelectric materials are reviewed, including more efficient energy harvesting, high‐performance wearable electronics, and impedance matching via material strategies. Finally, the challenges and prospects are discussed in view of the large‐scale commercialization of advanced high‐k cellulosic triboelectric materials. Figure 1 Construction of dielectric‐enhanced cellulosic triboelectric materials with performance enhancement via dielectric modulation."
} | 2,351 |
31787952 | PMC6853845 | pmc | 6,310 | {
"abstract": "Microbes in various aquatic ecosystems play a key role in global energy fluxes and biogeochemical processes. However, the detailed patterns on the functional structure and the metabolic potential of microbial communities in freshwater lakes with different trophic status remain to be understood. We employed a metagenomics workflow to analyze the correlations between trophic status and planktonic microbiota in freshwater lakes on Yun-Gui Plateau, China. Our results revealed that microbial communities in the eutrophic and mesotrophic-oligotrophic lake ecosystems harbor distinct community structure and metabolic potential. Cyanobacteria were dominant in the eutrophic ecosystems, mainly driving the processes of aerobic respiration, fermentation, nitrogen assimilation, nitrogen mineralization, assimilatory sulfate reduction and sulfur mineralization in this ecosystem group. Actinobacteria, Proteobacteria (Alpha-, Beta-, and Gammaproteobacteria), Verrucomicrobia and Planctomycetes, occurred more often in the mesotrophic-oligotrophic ecosystems than those in the eutrophic ecosystems, and these taxa potentially mediate the above metabolic processes. In these two groups of ecosystems, a difference in the abundance of functional genes involved in carbohydrate metabolism, energy metabolism, glycan biosynthesis and metabolism, and metabolism of cofactors and vitamins significantly contribute to the distinct functional structure of microbiota from surface water. Furthermore, the microbe-mediated metabolic potentials for carbon, nitrogen and sulfur transformation showed differences in the two ecosystem groups. Compared with the mesotrophic-oligotrophic ecosystems, planktonic microbial communities in the eutrophic ecosystems showed higher potential for aerobic carbon fixation, fermentation, methanogenesis, anammox, denitrification, and sulfur mineralization, but they showed lower potential for aerobic respiration, CO oxidation, nitrogen fixation, and assimilatory sulfate reduction. This study offers insights into the relationships of trophic status to planktonic microbial community structure and its metabolic potential, and identifies the main taxa responsible for the biogeochemical cycles of carbon, nitrogen and sulfur in freshwater lake environments.",
"conclusion": "Conclusion Our research reports on the planktonic microbial communities of five plateau freshwater lakes with different trophic status, located in Yunnan, China. The trophic alterations caused by anthropogenic activities are not only related to microbial community composition, but also to the genetic potential for important carbon, nitrogen and sulfur biogeochemical cycling reactions mediated by microbes in the surface waters. The overall differences in metabolic functions and the genetic potential for elemental cycling were strongly related to divergence in the taxonomic structure and diversity of the planktonic microbial communities. Energy metabolism and cofactors and vitamin metabolism had strong representation in the eutrophic ecosystems; carbohydrate metabolism and glycan biosynthesis and metabolism had strong representation in the mesotrophic-oligotrophic ecosystems. Moreover, the phylum Cyanobacteria, dominant in the eutrophic ecosystems, mainly mediated the processes of aerobic respiration, fermentation, nitrogen assimilation, nitrogen mineralization, assimilatory sulfate reduction and sulfur mineralization in this system. The phyla Actinobacteria and Proteobacteria (Alpha-, Beta-, and Gammaproteobacteria), Verrucomicrobia and Planctomycetes showed higher relative abundance in the mesotrophic-oligotrophic ecosystems than those in the eutrophic ecosystems. In the mesotrophic-oligotrophic ecosystems, aerobic respiration, nitrogen assimilation, nitrogen mineralization and assimilatory sulfate reduction were mainly mediated by the phylum Actinobacteria, sulfur mineralization was mainly driven by Alphaproteobacteria, and fermentation was mainly driven by Planctomycetes. Planktonic microbial communities in the eutrophic ecosystems had higher potential for aerobic carbon fixation, fermentation, methanogenesis, anammox, denitrification and sulfur mineralization than those in the mesotrophic-oligotrophic ecosystems. Besides, planktonic microbial communities in the mesotrophic-oligotrophic ecosystems had higher metabolic potentials for aerobic respiration, CO oxidation, nitrogen fixation and assimilatory sulfate reduction than those in the eutrophic ecosystems. Overall, trophic preference of some key taxonomic groups leads to communities with distinct taxonomy and functions, corresponding to ecosystem-specific carbon, nitrogen and sulfur cycles in Yun-Gui Plateau freshwater lakes characterized by different trophic status.",
"introduction": "Introduction The microbiota in aquatic ecosystems plays an important role in elemental cycling and global energy fluxes ( Falkowski et al., 2008 ; Clark et al., 2018 ; Cronan, 2018 ). The relations between the taxonomic structure of microbial communities in aquatic environments and complex environmental factors such as trophic status ( Llirós et al., 2014 ; Wan et al., 2017 ), seasons ( Zhu et al., 2019 ), elevation gradient ( Li H. et al., 2017 ), and salinity ( Eiler et al., 2014 ) have been well studied. However, little is known about the correlations of these factors with community functions. Therefore, improving our knowledge about the link between taxonomy and function of microbial communities can contribute to a better understanding of the response mechanisms of microbiota to key environmental changes and gradients ( Logue et al., 2015 ; Arora-Williams et al., 2018 ). The Yun-Gui Plateau Lake Zone is the smallest of the five lake-zones in China ( Ma et al., 2011 ). About half of the lakes in this zone, accounting for 90% of the lake area, are located in Yunnan Province which is a biodiversity hotspot ( Zhou et al., 2019 ), and these lakes are sensitive areas for recording regional ecology and global climate change ( Li et al., 2015 ). The plateau lake ecosystems are vulnerable and not easily restored once damaged because of the relatively low rate of water exchange and resilience, and the steep and little-developed lakeshores ( Wang and Dong, 1998 ; Liao et al., 2016 ). In the past few decades, some of these lakes have been seriously damaged by intensification of human activities, leading to deterioration of water quality and degradation of ecosystem function ( Li W. et al., 2017 ; Liu et al., 2017 ; Gao et al., 2018 ; Wu et al., 2019 ). Eutrophication is one of the biggest of such challenges; it changes the diversity and composition of lake organisms and poses a serious threat to ecosystem service function ( Liu et al., 2012 ; Shi et al., 2016 ; Dong et al., 2018 ). To date, most studies have concentrated on microbial communities in sediment from Yun-Gui Plateau lakes with different trophic levels ( Bai et al., 2012 ; Dai et al., 2016 ; Yang et al., 2017a , b ). Only a few studies have focused on microbiota in lake surface waters, in which the microorganisms are more sensitive to lake eutrophication than those in sediment ( Zeng et al., 2019 ). Bacterioplankton compositions in eutrophic Lake Dianchi ( Wen et al., 2012 ; Dai et al., 2016 ; Han et al., 2016 ), mesotrophic Lake Erhai ( Hu et al., 2013 ) and oligotrophic Lake Haixihai ( Dai et al., 2016 ) were investigated by analyzing 16S rRNA gene sequences. Dai et al. (2016) and Han et al. (2016) demonstrated that trophic status may play important roles in shaping the taxonomic structure of bacterioplankton communities in the Yun-Gui Plateau freshwater lakes. Nevertheless, the relations of lake trophic status to the functional structure of the microbial communities and the ecological processes within freshwater systems have seldom been examined. Because of decreased cost and increased throughput of sequencing technology ( Neufeld, 2017 ; Quince et al., 2017 ), the powerful approach of metagenomics is now widely applied in studies of microbial communities from many diverse environments, including soil ( Diamond et al., 2019 ), sediment ( Vavourakis et al., 2018 ), hosts ( Rothschild et al., 2018 ), seawater ( Sunagawa et al., 2015 ), and freshwater ( Arora-Williams et al., 2018 ). A curated set of metabolic marker genes was used to quantify the genetic potential for microbe-mediated biogeochemical cycles in a meromictic lake by Lauro et al. (2011) . This method has since been widely used in different types of ecosystem, including salt marsh ( Dini-Andreote et al., 2016 ), sediments ( Hamilton et al., 2016 ), an estuary ( Kieft et al., 2018 ), and a stratified euxinic lake ( Llorens-Marès et al., 2015 ). Therefore, besides the characterization of community structure and reconstruction of genomes in individual samples, comparative analysis of the samples at multiple time points or of parallel samples across different environmental gradients using metagenomics facilitates the elucidation of complex microbial processes in the community, which are difficult to simulate in the laboratory. In this study, we applied shotgun metagenomics to examine the taxonomic and functional structure of surface-water microbial communities from five freshwater lakes on the Yun-Gui Plateau. These lakes had four trophic levels: eutrophic, meso-eutrophic, oligo-mesotrophic and oligotrophic. The relative abundance of metabolic marker genes was used to assess the genetic potential for each conversion step of the carbon, nitrogen, and sulfur cycles in the freshwater lake ecosystems. We explored the links between microbial composition and metabolic potential, and inferred the response mechanisms of microbe-mediated carbon, nitrogen, and sulfur cycles to lake trophic-level changes. We addressed the following two questions: (a) How does trophic status relate to distinct taxonomic and functional structures of planktonic microbial communities? (b) To what extent is it related to trophic status that each conversion step of microbe-mediated biogeochemical cycling pathways?",
"discussion": "Discussion Distinct Taxonomic Structure and Diversity of Communities in Each Ecosystem In this study, the percentage of reads that could be taxonomically classified was relatively low. This is a reasonable outcome, explained by the incomplete information contained in reference databases and eukaryotic contamination in environmental metagenomes ( Gori et al., 2011 ; Miller et al., 2019 ). Nevertheless, the annotation results reflect the composition of the microbial communities in the samples based on high-quality assignments. Only a few studies have shown that there are remarkable differences in planktonic microbial community structure in freshwater lakes with different trophic status ( Dai et al., 2016 ; Han et al., 2016 ; Hanson et al., 2017 ; Ji et al., 2018 ). In this study, we found that there were large differences in the taxonomic structures of the microbial communities from eutrophic (Group I) and mesotrophic-oligotrophic (Group II, the trophic level from mesotrophy to oligotrophy) freshwater ecosystems in the Yun-Gui Plateau. Moreover, our results suggested that the abundance of the phylum Cyanobacteria (order Chroococcales), which was dominant in eutrophic conditions, was significantly higher in eutrophic environments than that in mesotrophic-oligotrophic environments. In the mesotrophic-oligotrophic ecosystems, the phyla of Actinobacteria and Proteobacteria (Alpha-, Beta-, and Gammaproteobacteria) became dominant, indicating that they have a distinct preference for less eutrophic conditions. Thus, we focused on the correlation between these key taxonomic groups and trophic status. The results of RDA revealed that the occurrence of the phylum Cyanobacteria correlated with trophic status ( McMahon and Read, 2013 ), and the occurrence of Actinobacteria and Proteobacteria (Alpha-, Beta-, and Gammaproteobacteria) with less eutrophic states ( Haukka et al., 2006 ; Ji et al., 2018 ). The co-occurrence of the key taxa and the particular trophic level indicates that each taxonomic group has unique characteristics in freshwater lake ecosystems. For example, Alphaproteobacteria are competitive in conditions of low nutrient/substrate utilization rate ( Newton et al., 2011 ), and Cyanobacteria outcompete other planktonic microbes for nutrients in eutrophic systems ( McMahon and Read, 2013 ). Liu et al. (2012) reported that deeper lakes usually have better water quality than shallow lakes, and lake depth plays an important role in explaining the spatial dynamic of water quality in Yunnan Plateau. In our study, we observed the same findings that eutrophic ecosystems were shallow lakes and mesotrophic-oligotrophic ecosystems were deep lakes. It may be due to the deep lakes are associated with higher nutrient dilution ability than shallow lakes ( Liu et al., 2012 ). The correlation analysis between environmental factors indicated that lake depth has significant relationships with TN and TP concentrations. Thus, we propose the average water depth of a lake can be used as a predictor of eutrophication. Additionally, previous studies have reported that the diversity pattern of planktonic bacterial communities in freshwater systems could be significantly correlated with TN and TP concentrations when subjected to eutrophication ( Dai et al., 2016 ; Zeng et al., 2019 ), and this is consistent with the results of our RDA and variation partitioning. Although there were some differences in sampling time, location and size fraction of samples from the same lake in our study, the clustering of all samples still showed a significant pattern. Samples could be divided into two groups according to the trophic status of the lake. In addition, we observed that there were important differences in taxonomic alpha- and beta-diversity patterns across trophic gradients. Consequently, we conclude that the taxonomic diversity of planktonic microbial communities in freshwater lakes may be related to trophic status. Horner-Devine et al. (2003) observed that the diversity of planktonic bacteria exhibits a downward arched (parabolic) pattern along a gradient of primary productivity. Zeng et al. (2019) also found that the planktonic bacterial community has a positive quadratic relationship with the trophic level. Our results reflected a similar trend, that the alpha-diversity of planktonic microbiota in the eutrophic systems was significantly lower than that in mesotrophic-oligotrophic conditions, and within the mesotrophic-oligotrophic ecosystems, the alpha-diversity in the mesotrophic lake was higher than that in the oligotrophic lake. Distinct Functional Structure of Communities in Each Ecosystem Previous studies suggested that the functional structure of the microbial community is strongly associated with the taxonomic structure across the soil, estuary water and lake ecosystems ( Dini-Andreote et al., 2016 ; Ren et al., 2017 ; Kieft et al., 2018 ). The profile of microbial community functions during a Cyanobacterial bloom in a eutrophic freshwater lake has been reported ( Steffen et al., 2012 ; Chen et al., 2018 ). However, no comparative metagenomics study has been performed revealing the differences in microbial communities in lakes with different trophic status. Using metagenomic analysis, we observed a large difference in the functional structure of the planktonic microbial community between eutrophic and mesotrophic-oligotrophic freshwater ecosystems in the Yun-Gui Plateau lakes, which was strongly correlated with the differences in the taxonomic structures of the communities. By correlation analysis between environmental factors and functional categories, we found that the functional profiles of lakes with different trophic status were mainly correlated to TN and TP concentrations. Our results showed that genes encoding carbohydrate metabolism and glycan biosynthesis and metabolism were abundant in mesotrophic-oligotrophic freshwater ecosystems, suggesting that microbial communities in surface water of mesotrophic-oligotrophic freshwater ecosystems may have higher utilization rates of organic carbon and higher carbon flux than those of eutrophic systems ( Biddanda et al., 2001 ). Furthermore, genes involved in energy metabolism and cofactors and vitamin metabolism were abundant in the eutrophic ecosystems, which probably related to the high abundance of Cyanobacteria driving rapid energy conversion in this ecosystem and the need for heterotrophic bacteria to produce a large number of cofactors and vitamins ( Tang et al., 2010 ; Li et al., 2018 ). Accordingly, we inferred that trophic status may contribute to changes in ecosystem function by driving the taxonomic and functional divergence of the microbial community. Metabolic Potential of Communities in Each Ecosystem Owing to variance in the overall functional potential distributions of microbial communities, it can be hypothesized that microbe-mediated biogeochemical cycles are ecosystem-specific, resulting in differences in genetic potential for carbon, nitrogen and sulfur cycling processes in the overlying water of freshwater lakes with different trophic states. In our study, two high abundance metabolic processes, nitrogen assimilation and nitrogen mineralization, had equal potential across all lakes, indicating that differences in taxonomic composition do not influence the potential of the community to drive these processes. However, the relative abundance of markers of some processes was not constant between ecosystems. For instance, the potential for aerobic respiration and assimilatory sulfate reduction was relatively more abundant in the mesotrophic-oligotrophic freshwater ecosystems, while aerobic carbon fixation, fermentation and sulfur mineralization genes were relatively more abundant in the eutrophic freshwater ecosystems. Although lakes only account for a small fraction of the surface of the Earth ( Chen et al., 2015 ), changes in these processes caused by trophic alteration in freshwater lakes may affect global biogeochemical cycles. The phylum Cyanobacteria plays a crucial role as a primary producer in freshwater ecosystems, and it provides organic matter through photosynthesis to support the growth of various heterotrophic planktonic bacteria ( Fujii et al., 2016 ). Therefore, it is reasonable that eutrophic ecosystems with a high abundance of Cyanobacteria have a stronger potential for aerobic carbon fixation. Furthermore, in shallow eutrophic lakes, the occurrence of algal blooms in summer not only provides abundant organic matter, but also forms a local dark and anaerobic environment in the overlying water. Stal and Moezelaar (1997) reported that in dark, anoxic conditions, Cyanobacteria use fermentation instead of aerobic respiration as an alternative means of energy generation. Hence, Cyanobacteria in eutrophic ecosystems drive fermentation processes to produce energy to compensate for the relatively low potential of aerobic respiration. There have been few studies on CO oxidation in lake surface waters. CO in water mainly comes from photochemical degradation of Chromophoric/Colored dissolved organic matter ( Stubbins, 2001 ), which is accelerated by nutrient accumulation ( Zhang et al., 2010 ; Zhou et al., 2018a ). Therefore, a eutrophic ecosystem should have more CO flux. However, the abundance of marker genes related to CO oxidation in the mesotrophic-oligotrophic freshwater ecosystems was higher than that in the eutrophic lakes, indicating that the CO oxidation potential in the mesotrophic-oligotrophic lakes was higher. This may be because of microorganisms need more efficient energy harvesting in conditions of low nutrition, and higher primary productivity can reduce the dependence of planktonic microorganisms on exogenous carbon in eutrophic waters. Furthermore, we found that methanogenesis was driven by Euryarchaeota in the eutrophic surface water of Dianchi Lake. Recent works have revealed that a large fraction of CH 4 oversaturation in aquatic environments is produced in oxygenated surface waters ( Townsend-Small et al., 2016 ; Zhou et al., 2018b ). Thus, we suspect that a local anaerobic environment caused by Cyanobacterial blooms in eutrophic lakes may promote the production of CH 4 in aerobic overlying water to some extent ( Xing et al., 2012 ). Evans et al. (2017) reported that eutrophication causes lakes to transition from sinks to sources of carbon. Our data suggest carbon accumulation in the eutrophic lake because of increased carbon fixation potential relative to respiratory potential. Wu et al. (2019) found that algal blooms could accelerate the nitrogen cycling rate. Our results showed that there was no dramatic divergence in the potential for N-cycle processes between the eutrophic and the mesotrophic-oligotrophic freshwater ecosystems, but there were some noteworthy differences in anammox, denitrification and nitrogen fixation. Rich organic matter produced by algal blooms can be converted into ammonia and nitrate for anammox and denitrification ( Wu et al., 2019 ). Hence, we infer that there are high potentials for these two processes in eutrophic ecosystems, which may be the result of an accelerated N-cycle within this ecosystem. In addition, the lower potential for nitrogen fixation in eutrophic ecosystems is the result of the presence of rich organic matter, while the higher potential for nitrogen fixation in the mesotrophic-oligotrophic ecosystems is most likely related to a lack of organic matter. Although sulfur cycling in freshwater sediments and vertical water columns has been well studied ( Cai et al., 2019 ; Ren et al., 2019 ), the genetic potential for sulfur transformation in surface waters of lakes with different trophic status has not been studied. With the death of a large number of Cyanobacteria in eutrophic lakes, the high content of sulfur-containing amino acids in their cells might be released ( Lu et al., 2013 ), resulting in a water column enriched with organic sulfur. Our results showed that planktonic microbial communities in the eutrophic ecosystems exhibited less abundance of assimilatory sulfur reduction-related genes to produce organic sulfur; on the contrary, planktonic microbial communities in the eutrophic ecosystems exhibited higher potential for sulfur mineralization than those in the mesotrophic-oligotrophic environments, which may lead to the tendency of the eutrophic ecosystems to release H 2 S gas."
} | 5,629 |
21060738 | PMC2975365 | pmc | 6,311 | {
"abstract": "A plasmid-based expression system wherein mekB was fused to a constitutive Methanosarcina acetivorans promoter was used to express MekB, a broad-specificity esterase from Pseudomonas veronii , in M. acetivorans . The engineered strain had 80-fold greater esterase activity than wild-type M. acetivorans . Methyl acetate and methyl propionate esters served as the sole carbon and energy sources, resulting in robust growth and methane formation, with consumption of >97% of the substrates. Methanol was undetectable at the end of growth with methyl acetate, whereas acetate accumulated, a result consistent with methanol as the more favorable substrate. Acetate was consumed, and growth continued after a period of adaptation. Similar results were obtained with methyl propionate, except propionate was not metabolized.",
"introduction": "Introduction Methane-producing microbes, methanogens, constitute the largest described group within the domain Archaea and are among the most ancient of extant life forms. The phylogenetic diversity of methanogens is immense, consistent with the extremes of anaerobic habitats in which they proliferate. Methanogens grow at temperatures ranging from 0 to 110°C ( 1 , 2 ), salinities ranging from those in freshwater to those in hypersaline ( 3 , 4 ), pHs ranging from 4.5 to 9.5 ( 5 , 6 ), and anaerobic environments ranging from deep sea hydrothermal vents to the intestinal tracts of humans ( 7 , 8 ). This remarkable diversity is in stark contrast to the few simple substrates, primarily one-carbon substrates, utilized for growth and methanogenesis. Most described species reduce CO 2 to methane with H 2 , formate, or carbon monoxide (CO 2 reduction pathway). A few species convert the methyl groups of acetate (aceticlastic pathway) or the one-carbon substrate methanol, methylamines, or methyl sulfide (methylotrophic pathway) to methane ( 4 ). In nature, methanogens are terminal organisms of anaerobic microbial food chains where other microbes, primarily from the domain Bacteria , break down complex biomass to supply simple substrates for methanogens. In true symbiotic fashion, microbes acting on the complex biomass are also dependent on methanogens to remove products of their thermodynamically unfavorable reactions ( 9 ). The process occurs in diverse anaerobic environments and is a vital link in the global carbon cycle, producing nearly 1 billion metric tons of methane each year ( 10 ). Here, anaerobic microbial food chains have evolved to efficiently coordinate their metabolism in natural environments where biomass is most often limiting. Conversion of organic wastes and renewable biomass to methane is a viable alternative to the use of fossil fuels ( 11 ); however, in large-scale reactors receiving high loading rates of biomass, the fragile community interactions are easily disrupted, hindering efficient implementation of this technology. A potential route to overcoming this impediment is the engineering of methanogens with hybrid pathways incorporating enzymes from the domain Bacteria , thereby simplifying microbial food chains. Although genetic exchange systems have been available for nearly a decade ( 12 ), this technology has not been applied to successful engineering of novel methanogenic pathways. We set out to broaden the substrate range of Methanosarcina acetivorans C2A through the rational design of a pathway that allows growth and production of methane with nonnative substrates. M. acetivorans C2A ( 13 ) is a particularly good candidate, as it employs all three methanogenic pathways ( 14 – 18 ) and a robust genetic exchange system is available ( 19 ). We chose the methyl esters of acetate (methyl acetate [MeAc]) and propionate (methyl propionate [MePr]), both of which are widely used industrial solvents and also produced biologically ( 20 , 21 ). Furthermore, both compounds are relatively hydrophobic and likely to be passively transported across the cytoplasmic membrane. Importantly, methanogens are not documented to metabolize either compound. Recently, the aerobic isolate Pseudomonas veronii MEK700 was shown to utilize an esterase (MekB) with broad substrate specificity in the aerobic metabolism of 2-butanone ( 22 ). MekB was shown to hydrolyze a variety of esters, including MeAc and MePr. Thus, the metabolic pathway shown in Fig. 1 , whereby the expression of mekB within M. acetivorans C2A would confer growth with methyl esters when combined with the methylotrophic pathway of methanogenesis, was designed. FIG 1 Design of a methylotrophic pathway for metabolism of methyl esters by Methanosarcina acetivorans C2A. Reactions in red are from the domain Bacteria and those in blue from the domain Archaea . (A) Oxidation of one methyl group of methanol to CO 2 by a reversal of the CO 2 reduction pathway of methanogens. (B) Reduction of three methyl groups to methane by the electrons generated from section A. R represents CH 3 or CH 3 CH 2 (methyl acetate or methyl propionate). A plasmid-based expression system was used to express MekB within wild-type M. acetivorans C2A. The mekB gene was PCR amplified from pJOE5358.1 ( 22 ) and fused to the promoter (P tbp ) for the gene encoding the TATA binding protein of M. acetivorans ( 23 ). The DNA fragment containing P tbp - mekB was then cloned into the XhoI/BamHI sites of the Escherichia coli - M. acetivorans shuttle vector pWM321 to form pDL203. Plasmids were transformed into M. acetivorans C2A using a liposome-mediated protocol ( 19 ). Transformants were selected by plating cells on solid high-salt (HS) medium ( 19 ) containing 125 mM methanol and 2 µg/ml puromycin. The resultant strains, C2A(pWM321) and C2A(pDL203), were grown with HS medium containing the indicated growth substrate. Cell lysates from methanol- or acetate-grown C2A(pWM321) had low but detectable esterase activity (~25 units/mg protein), whereas the activities for lysates from methanol- or acetate-grown C2A(pDL203) were approximately 80-fold higher (~2,000 units/mg protein). Activity was measured as previously described ( 22 ), with units defined as nmol of 4-nitrophenyl acetate hydrolyzed/min. Protein was determined as previously described ( 24 ), with bovine serum albumin as the standard. These data reveal that wild-type M. acetivorans lacks a highly active esterase and that MekB is expressed in an active form in C2A(pDL203). Expression of MekB did not adversely affect growth of C2A(pDL203) with methanol or acetate (data not shown). The ability of C2A(pWM321) and C2A(pDL203) to utilize MeAc or MePr as the sole carbon and energy sources was examined ( Fig. 2 ). MeAc, MePr, methanol, and methane were measured using a Shimadzu gas chromatograph (GC-14A) equipped with a flame ionization detector (FID) and a thermal conductivity detector. A silico steel 100/120 ShinCarbon-ST column (Restek) with He as the carrier gas was used at a constant temperature of 100°C for determination of methane. A glass 80/100 Porapak QS column (Alltech) with He as the carrier gas was used at 150°C to measure methanol, MeAc, and MePr. Acetate and propionate were detected by high-performance liquid chromatography using an Aminex HPX-87H column (Bio-Rad). Strains C2A(pWM321) and C2A(pDL203) were grown in HS medium supplemented with 150 mM morpholinepropanesulfonic acid (MOPS; pH 7.5) to increase the buffering capacity. Medium containing either substrate was left uninoculated (abiotic control) or inoculated with C2A(pWM321) or C2A(pDL203). Approximately 10% of the MeAc and 20% of the MePr were abiotically hydrolyzed to methanol and acetate or propionate in the uninoculated controls during the time course of the experiment. The decrease of MeAc or MePr in C2A(pWM321) cultures was nearly identical to the level for the uninoculated abiotic controls, indicating no hydrolysis of the esters by C2A(pWM321), a result consistent with the intrinsically low esterase activity in the native strain. However, a low level of growth, which could have been due to abiotically produced methanol, was observed for the C2A(pWM321) culture. FIG 2 Comparison of rates of methyl ester metabolism by Methanosarcina acetivorans C2A containing pWM321 or pDL203. Growth, substrate concentration, and product formation were monitored during metabolism of 100 mM MeAc (A1 to A3) or 50 mM MePr (B1 to B3) by strain C2A(pWM321) (■) and strain C2A(pDL203) (▲), compared to the levels for uninoculated abiotic controls (♦). (A3 and B3) Methanol (solid line), acetate or propionate (dashed line), and methane (dotted line). The reported data represent the means of results from triplicate experiments. In contrast to C2A(pWM321), the C2A(pDL203) culture consumed >97% of the MeAc or MePr initially added, resulting in robust growth and methane production ( Fig. 2 ). Approximately 50% of the MeAc was hydrolyzed within 60 h and 95% within 74 h, at which time growth was only half maximal (optical density at 600 nm [OD 600 ] = 0.41), consistent with the idea that MekB has high specific activity with MeAc. Rapid hydrolysis of MeAc by C2A(pDL203) resulted in accumulation of methanol and acetate to similar levels in the medium (230 µmol and 270 µmol, respectively) before any significant growth occurred (50 h; OD 600 = 0.09). Methanol was subsequently consumed during exponential growth, concomitant with accumulation of methane ( Fig. 2 ) and CO 2 (data not shown). At the end of growth, methanol levels were below the detection limit, whereas acetate was not significantly metabolized, consistent with methanol as a more energetically favorable substrate. The generation time for growth with MeAc was 10.7 ± 0.2 h ( Fig. 2 ), compared to 10.2 ± 0.2 h for growth with methanol (data not shown). The total amount of products at the end of growth (acetate, methane, and CO 2 ) accounted for 89% of the carbon in the MeAc consumed, with the balance assumed to be cell carbon. Accumulated acetate was consumed after a lag period (data not shown), characteristic of the diauxic growth parameters for this species ( 23 ). Unlike the metabolism of MeAc, during metabolism of MePr, hydrolysis and propionate accumulation paralleled growth and methane production ( Fig. 2 ). Further, methanol never accumulated in the medium, suggesting that the methanol liberated from hydrolysis of MePr by MekB in the cytosol was immediately consumed. It took approximately 80 h to hydrolyze 50% of the MePr added and approximately 115 h to hydrolyze 97%, consistent with the idea that MekB has lower esterase activity with MePr. The generation time for growth with MePr was 18.5 ± 0.2 h ( Fig. 2 ), compared to 10.2 ± 0.2 h for growth with methanol, consistent with the idea that hydrolysis is the limiting factor. The total amount of products at the end of growth (propionate, methane, and CO 2 ) accounted for 88% of the carbon in the MePr consumed, with the balance assumed to be cell carbon. Propionate was not significantly metabolized, even after prolonged incubation (data not shown). Cell lysates from MeAc- and MePr-grown C2A(pDL203) had esterase activity (~1,500 to 3,000 units/mg protein) comparable to the activity determined with methanol- and acetate-grown cells, suggesting that MeAc and MePr do not affect the expression or activity of MekB. These data are consistent with the pathway illustrated in Fig. 1 , whereby expression of active MekB in C2A(pDL203) confers the ability to hydrolyze the methyl esters with the liberated methanol metabolized through the methylotrophic pathway. Metabolism of MeAc and MePr by C2A(pDL203) through the methylotrophic pathway was further supported by molar growth yields when C2A(pDL203) was cultured with either MeAc (4.93 ± 0.05 g [dry weight]/mol methane) or MePr (5.33 ± 0.30 g [dry weight]/mol methane), that were similar to those observed for methanol-grown cultures (5.23 ± 0.05 g [dry weight]/mol methane) but not acetate-grown cultures (2.68 ± 0.06 g [dry weight]/mol methane). Metabolism of MeAc and MePr by C2A (pDL203) was further examined with resting cell suspensions ( Table 1 ). M. acetivorans strains were grown in HS medium containing the indicated substrates to mid-exponential phase (OD 550 = 0.4 to 0.6). Cells were harvested by centrifugation at 5,000 × g for 10 min in an anaerobic chamber (Coy Manufacturing, Inc.), washed with substrate-free HS medium, and resuspended in HS medium containing 150 mM MOPS (pH 7.5) to give a final OD 550 of 3 to 4. Cell suspensions (1 ml) were anaerobically aliquoted into 10-ml serum bottles containing 1 atm N 2 and sealed with butyl rubber stoppers. Substrates were added from anoxic stock solutions to give a final concentration of 50 mM and the bottles incubated at 37°C. Gas samples were withdrawn every 30 min for 6 h for measurement of methane. Cells of C2A(pDL203) grown with either methanol, MeAc, or MePr supported substantial rates of methane production when supplied with methanol but did not produce significant amounts when supplied with acetate ( Table 1 ). However, acetate-grown C2A (pDL203) produced methane at comparable initial rates with both acetate and MeAc but did not produce significant amounts when supplied with methanol or MePr, confirming that acetate liberated from MeAc was metabolized through the aceticlastic pathway. TABLE 1 Rates of methane production by M. acetivorans cell suspensions Strain Growth substrate Specific activity with substrate added (nmol methane/min/mg [dry wt] cells) a Methanol Acetate MeAc MePr C2A(pWM321) Methanol 135 ± 7 BDL BDL BDL C2A(pWM321) Acetate BDL 48 ± 4 BDL BDL C2A(pDL203) Methanol 166 ± 2 BDL 155 ± 2 86 ± 2 C2A(pDL203) Acetate BDL 92 ± 3 103 ± 3 BDL C2A(pDL203) MeAc 210 ± 18 BDL 183 ± 25 60 ± 7 C2A(pDL203) MePr 158 ± 3 BDL 130 ± 17 123 ± 17 a MeAc, methyl acetate; MePr, methyl propionate; BDL, below the detection limit of 1 nmol methane/min/mg (dry weight) of cells. Implications. The results demonstrate the heterologous expression of a catabolic enzyme from an aerobic species of the domain Bacteria in a strictly anaerobic methanogen from the domain Archaea that confers robust metabolism of substrates more complex than previously reported for any methanogen. This work inaugurates the engineering of metabolic pathways expanding the narrow range of simple substrates for methanogens, leading to the simplification of anaerobic microbial food chains converting complex biomass to methane."
} | 3,610 |
38143871 | PMC10748501 | pmc | 6,313 | {
"abstract": "Arbuscular mycorrhizal fungi (AMF) have demonstrated the potential to enhance the saline-alkali tolerance in plants. Nevertheless, the extent to which AMF can ameliorate the tolerance of salt-sensitive plants to alkaline conditions necessitates further investigation. The current study is primarily centered on elucidating the impact of AMF on the growth of the Huayu22 (H22) when cultivated in saline-alkaline soil. We leveraged DNA of rhizosphere microorganisms extracted from saline-alkali soil subjected to AMF treatment and conducted high-throughput sequencing encompassing 16S rRNA gene and ITS sequencing. Our findings from high-throughput sequencing unveiled Proteobacteria and Bacillus as the prevailing phylum and genus within the bacterial population, respectively. Likewise, the predominant fungal phylum and genus were identified as Ascomycota and Haematonectria . It is noteworthy that the relative abundance of Proteobacteria, Actinobacteria, Chloroflexi, Bacteroidetes, and Ascomycota exhibited significant increments subsequent to AMF inoculation. Our investigation into soil enzyme activity revealed a remarkable surge post-AMF inoculation. Notably, the amounts of pathogen growth inhibitory enzymes and organic carbon degrading enzymes rise, as predicted by the putative roles of microbial communities. In saline-alkali soil, inoculation of AMF did boost the yield of H22. Notable improvements were observed in the weight of both 100 fruits and 100 grains, which increased by 20.02% and 22.30%, respectively. Conclusively, this study not only provides a theoretical framework but also furnishes empirical evidence supporting the utilization of AMF as a viable strategy for augmenting the yield of salt-sensitive plants grown in alkaline conditions.",
"conclusion": "5 Conclusion This study highlights how AMF can enhance the yield of salt-sensitive plants in saline-alkali conditions. After AMF application, there was a positive shift in the soil microbial community, favoring aerobic and facultative anaerobic bacteria over anaerobic species. Additionally, AMF significantly increased soil enzyme activity, particularly those inhibiting pathogens and aiding organic carbon degradation. It also improved metabolic pathways related to amino acids and carbohydrates, mineralization, and reduced enzymes involved in fatty acid biosynthesis, while boosting pathogen-inhibiting enzymes in the rhizosphere. In saline-alkaline soil, AMF led to a remarkable 20.02% increase in 100-fruit weight and a 22.30% rise in 100-grain weight compared to the control. This improvement is attributed to the creation of a more diverse microenvironment and heightened enzyme activity facilitated by AMF. Importantly, AMF played a vital role in shaping the microbial population structure around peanut roots, creating an environment conducive to plant growth. These findings offer valuable insights into how AMF enhance salt resistance and overall health of peanut plants in saline-alkaline soil.",
"introduction": "1 Introduction In the semi-arid tropics (SAT), which include parts of Africa, Asia, North America, and South America, where extremes of drought and soil salinity are common, approximately 60% of the world’s peanut crop is produced ( Mace et al., 2006 ; Cuc et al., 2008 ). Unfavorable environmental factors significantly impact peanut output and growth. High salt concentrations also lead to physiological dryness and ion toxicity, inhibit peanut growth, biomass, yield, photosynthesis, and water use efficiency ( Chao et al., 2022 ). Consequently, peanut roots, stems, and leaves are unable to develop properly. To address the shortage of cultivable land and promote regional agriculture, attempts have been made to cultivate peanuts in saline-alkaline soil in northern China, especially in the coastal regions of the Yellow River Delta ( Ci et al., 2021 ). However, since peanuts are particularly sensitive to salinity, salinity stress interferes with their growth at various stages, including seed germination, chlorophyll production, pod development, and fodder production, ultimately reducing crop yield ( Avishek et al., 2018 ). Being a saline-alkali-sensitive plant, H22 production and quality suffer due to the impact of saline-alkali soil. Consequently, one of the main breeding objectives in the peanut industry and saline-alkali land usage projects is to further improve peanut salt tolerance. Worldwide, soil salinity is becoming a significant problem as it is encountered in all climates, seriously restricting crop yields and plant growth ( Evelin et al., 2019 ). Approximately 9 × 10 8 m 2 of land, or 25% of the world’s surface area, is affected by salinization. According to research, 30% of cultivated land will convert into saline soil within 20 years, and this figure may rise to 50% within 30 years. This will have an increasingly negative impact on plant growth and the ecological environment worldwide ( Porcel et al., 2012 ). Recent years have seen an increase in soil salinity due to over-cultivation and growing industrialization, leading to secondary soil salinization. One of the most significant environmental problems affecting humanity is salinization. Biodiversity is significantly impacted by salinization, and saline habitats require more time to restore than other soil ecosystems ( Bless et al., 2018 ). High salt concentrations cause physiological dryness and ion toxicity, in addition to limiting plant growth, biomass, yield, photosynthesis, and water usage efficiency. High salinity forces plants to absorb more harmful ions like Na + and Cl − while dramatically reducing their ability to absorb phosphorus and potassium ( Khan et al., 2019 ). Plant roots, stems, and leaves grow and develop at a much slower rate. As a result, improving saline soil and increasing plant salt tolerance are currently key priorities. Important plant activities, such as nitrogen fixation and phosphorus solubilization depend heavily on the rhizosphere microbiome ( Ceja-Navarro et al., 2021 ). On the other hand, root-associated microbiota carried out specific functions, including element metabolism and transformation (for instance, nitrogen and phosphate cycling), which are beneficial for the growth of their host and affect plant fitness ( Huang et al., 2022 ). The bacterial community structure associated with peanuts may be unique, while changes in composition and their relationships with salt stress and peanut cultivars remain unknown. Soil pollution contributes uncertain effects and may cause different shifts in the root-associated microbiome of various niches (e.g., bulk soil, rhizosphere, rhizoplane, and endosphere), which need to be clarified. The application of arbuscular mycorrhizal fungi (AMF) is one of the most significant, environment friendly, and economically advantageous bioremediation technique for managing abiotic stress in soil and plants ( Dhalaria et al., 2020 ). The various metabolic abilities of rhizosphere-associated microorganisms enable them to actively influence plant growth and tolerance to biotic and abiotic stresses ( Dennis et al., 2010 ). Most plants can establish a healthy symbiotic connection with AMF, one of the significant soil microbes, which enhances plant water metabolism and salt tolerance ( Wang et al., 2019 ). Most of the research on arbuscular mycorrhizal fungi (AMF) conducted thus far has focused on how AMF inoculation affects plant performance or the general composition and functioning of microbial communities in pots or agricultural soil ( Elliott et al., 2021 ; Basiru and Hijri, 2022 ). However, the effects of AMF application on salt-sensitive plants and the microbial population in saline-alkaline soils are less well understood. Given the wide-ranging impact of the soil microbiome on plant performance, it is crucial to investigate how AMF affects the microorganisms associated with peanut roots and how this correlates with crop growth in order to increase peanut yields in saline-alkaline soil. Our research encompassed an examination of root-associated microorganisms, soil enzyme activity, peanut growth, and pod yield, with the aim of shedding light on the intricate interplay between AMF and these critical components in saline-alkaline soil conditions.",
"discussion": "4 Discussion The primary threat to global agricultural productivity is soil salinity, which significantly hinders the growth and yield of groundnut crops. Therefore, it is crucial to explore effective strategies for mitigating the adverse effects of saline soil on global peanut production. Studies have shown that rhizosphere-associated bacteria play a vital role in influencing a plant’s ability to tolerate salt stress ( Ullah et al., 2017 , 2019 ). To address the detrimental consequences of saline soil on groundnut cultivation, the utilization of AMF emerges as a potentially valuable and efficient approach. In this study, we employed functional prediction and high-throughput sequencing techniques to investigate the characteristics of the microbial community structure in the rhizosphere soil of peanut plants following mycorrhizal inoculation. AMF inoculation significantly increased the relative abundance of Proteobacteria, Actinobacteriota, Chloroflexi, and Bacteroidetes, which are among the top 10 phyla. Protozoa are abundant, widely distributed, and play a crucial role in agricultural soils ( Fierer, 2017 ). Additionally, Actinobacteria are highly beneficial in decomposing organic matter, making molecules more readily absorbable by plants ( Servin et al., 2008 ). Bacteroidetes are saprophytic organisms responsible for breaking down complex organic compounds ( Thomas et al., 2011 ). In our research, we observed higher densities of Firmicutes in RSA and RIA. Maheshwari et al. (2011) found that various members of the Firmicutes phylum inhabit different agricultural niches, enhancing crop yield through processes such as phytohormone production, antibiotic release, phosphate solubilization, atmospheric N 2 fixation, NH 3 release, and other mechanisms. Actinomycetes and Bacteroides have been shown to have high salinity tolerance in previous studies ( Zhao et al., 2020 ; Chi et al., 2021 ). These changes in the microbial community structure have the potential to enhance the saline-alkali tolerance of the H22 variety and subsequently increase its productivity in saline-alkali soil. The soil–plant compartment had an impact on the composition of the fungal community. Our research revealed that the AMF groups exhibited lower fungal diversity. Ascomycota and Basidiomycota, which accounted for between 62.00% and 89.00% of the relative abundance in the soil–plant compartments, constituted the majority of the major fungal phyla in our study. In contrast to the root endosphere, Mucoromycota fungi were more commonly found in the rhizosphere and bulk soil. Among these, the Mortierellaceae family, particularly the genus Mortierella , was dominant. According to our findings, the Mortierella accounted for 6.14% of the relative abundance of all genera, making it one of the most prevalent genera. Notably, Mortierella plays a significant role in phosphorus cycling in the rhizosphere ( Osorio and Habte, 2001 ). One significant AMF genus that influences plants is known as Glomus ( Landwehr et al., 2002 ). Previous research has demonstrated that inoculation with Glomus promotes plant growth and biomass ( Campos-Soriano et al., 2010 ; Naseer et al., 2022 ). In our study, Glomus was the most prevalent AMF genus in both the CK and AMF groups ( Figure 5 ). AMF offers a wide range of advantages and potential value in harsh environments ( Guo et al., 2022 ). According to a recent study by Salvioli et al. (2016) , bacteria associated with AMF enhance AMF fitness and soil nutrient uptake. Estrada et al. (2013) demonstrated how mycorrhizal inoculation improved potassium accumulation and reduced sodium ion levels in plants experiencing salt stress. These findings collectively suggest that AMF inoculation may enhance the nitrogen and phosphorus cycles and promote the development of H22 in saline-alkaline soil. Soil enzymes, primarily produced by microorganisms, play vital roles in organic material decomposition and ecosystem processes. In saline-alkaline soil areas, key enzymes like catalase, urease, phosphatase, sucrase, and cellulase are used as indicators to predict soil ecosystem function and environmental quality ( Qu et al., 2020 ). Catalase, for example, breaks down hydrogen peroxide and enhances soil oxidation ( Guo et al., 2015 ). Urease activity indicates potential changes in soil nitrogen content ( Jia et al., 2017 ). Phosphatase generates phosphate ions, contributing to soil nutrient availability. Sucrase hydrolyzes sucrose, improving soil nutrients availability. AMF enhances plant root nitrogen uptake by boosting soil enzyme activity in the rhizosphere ( Liu and Chen, 2021 ). Soil dehydrogenase activity, representing soil microbial metabolism, can reflect microbial redox capacity ( Xu et al., 2016 ). Soil phosphatase, which catalyzes the mineralization of soil organic phosphorus, marginally increased following AMF inoculation ( Hu et al., 2014 ). AMF, by chelating trace elements, can alter the macro-to-trace element ratio in the soil. This promotes the production of sugars, minerals, lipids, and various vitamins through biosynthesis. AMF also enhances microbial mineralization in the soil, particularly the breakdown of polysaccharides into soluble monomers. Consequently, cultivating H22 and applying AMF inoculation on saline-alkaline lands have significant potential to improve soil properties, a critical task for increasing crop production capacity and preserving soil fertility. These empirical findings have been unequivocally confirmed through yield assessments, demonstrating that the implementation of AMF inoculation can substantially increase H22 yields on saline-alkaline terrain. The majority of soil metabolites come from microbes and plant roots. Depending on the plant species, genotype, and environmental factors, different root exudates have different compositions. Our functional prediction suggests that AMF encourage various microbial enzyme creations, which could quicken soil metabolism. Furthermore, based on a functional prediction ( Supplementary Figure S7B ), the activities of the dioxygenases flavanone 3-dioxygenase and hydroxyquinol 1, 2-dioxygenase increased in the presence of AMF. These enzymes efficiently degrade aromatic compounds by initiating the breakage of aromatic rings and hydroxylating the benzene rings of aromatic compounds with the help of molecular oxygen. AMF can thereby expedite the decomposition of aromatic chemicals. According to prior research ( Vacheron et al., 2013 ), these aromatic chemicals may induce chemotactic responses in soil microorganisms, particularly plant growth-promoting rhizobacteria (PGPR). AMF therapy thereby affects the microbial community’s structure and function, as well as the host’s ability to attract PGPRs, consequently promoting H22 expansion."
} | 3,763 |
27387069 | null | s2 | 6,314 | {
"abstract": "Assimilatory and dissimilatory utilisation of autotroph biomass by heterotrophs is a fundamental mechanism for the transfer of nutrients and energy across trophic levels. Metagenome data from a tractable, thermoacidophilic microbial community in Yellowstone National Park was used to build an in silico model to study heterotrophic utilisation of autotroph biomass using elementary flux mode analysis and flux balance analysis. Assimilatory and dissimilatory biomass utilisation was investigated using 29 forms of biomass-derived dissolved organic carbon (DOC) including individual monomer pools, individual macromolecular pools and aggregate biomass. The simulations identified ecologically competitive strategies for utilizing DOC under conditions of varying electron donor, electron acceptor or enzyme limitation. The simulated growth environment affected which form of DOC was the most competitive use of nutrients; for instance, oxygen limitation favoured utilisation of less reduced and fermentable DOC while carbon-limited environments favoured more reduced DOC. Additionally, metabolism was studied considering two encompassing metabolic strategies: simultaneous versus sequential use of DOC. Results of this study bound the transfer of nutrients and energy through microbial food webs, providing a quantitative foundation relevant to most microbial ecosystems."
} | 342 |
24278322 | PMC3835567 | pmc | 6,315 | {
"abstract": "Methanogenic archaea produce methane as a metabolic product under anoxic conditions and they play a crucial role in the global methane cycle. In this study molecular diversity of methanogenic archaea in the hyporheic sediment of the lowland stream Sitka (Olomouc, Czech Republic) was analyzed by PCR amplification, cloning and sequencing analysis of the methyl coenzyme M reductase alpha subunit ( mcrA ) gene. Sequencing analysis of 60 clones revealed 24 different mcrA phylotypes from hyporheic sedimentary layers to a depth of 50 cm. Phylotypes were affiliated with Methanomicrobiales, Methanosarcinales and Methanobacteriales orders. Only one phylotype remains unclassified. The majority of the phylotypes showed higher affiliation with uncultured methanogens than with known methanogenic species. The presence of relatively rich assemblage of methanogenic archaea confirmed that methanogens may be an important component of hyporheic microbial communities and may affect CH 4 cycling in rivers.",
"conclusion": "Conclusion To the best of our knowledge, this study is the first analysis of the methanogenic community composition in river hyporheic sediments with respect to the process of methanogenesis. The presence of methanogenic archaea was detected using mcrA gene marker and FISH (MPB-1 oligonucletide probe) to a 50 cm river sediment depth. The data from the mcrA gene sequencing, retrieved a relatively large number of methanogenic phylotypes. These results support our previous measurements and suggest that methanogens contributes significantly to the hyporheic microbial community and may affect CH 4 cycling in the Sitka stream sediments. The results also indicate the presence of both hydrogenotrophic and acetoclastic metabolic pathways in the Sitka river sediment. We hope these findings will be helpful for further research on the ecological function of methanogens in the carbon cycle in river hyporheic sediments.",
"introduction": "Introduction The decomposition of organic matter in aquatic sediments is an important process in global and local carbon budgets, as it ultimately recycles complex organic compounds from terrestrial and aquatic environments into carbon dioxide and methane. The latter is a major component in the carbon cycle of anaerobic aquatic systems. Since a relatively large amount of methane production has been observed in river sediments [ 1 - 4 ], we hypothesised that river sediments may act as a considerable source of methane gas emission into the environment [ 5 ]. Methane (CH 4 ) is produced mostly by methanogenic archaea [ 6 , 7 ] as a final byproduct of anaerobic respiration and fermentation but there is also aerobic formation of methane by the aerobic degradation of methyl phosphonates [ 8 ] or by oxidation of ascorbic acid using iron compounds and hydrogen peroxide [ 9 ]. Methanogenic archaea belonging to the Euryarchaeota phylum are divided into seven orders: Methanopyrales, Methanococcales, Methanobacteriales, Methanomicrobiales , Methanosarcinales and the recently recognized groups Methanocellales and Methanoplasmatales [ 10 , 11 ]. Methanogenic archaea are ubiquitous in anoxic environments and require a redox potential of less than -300 mV for their growth [ 12 ]. They can be found in moderate habitats such as rice paddies [ 13 ], soils [ 14 ], lake sediments [ 15 ], in extreme conditions such as hydrothermal vents [ 16 ], permafrost soils [ 17 , 18 ] and also in the gastrointestinal tract of animals [ 19 ]. Freshwater sediments, including wetlands, rice paddies and lakes, are thought to contribute 40 to 50% of the annual atmospheric methane flux [ 20 ]. Rates of methane production and consumption in sediments are controlled by the relative availability of substrates for methanogenesis. The most important immediate precursors of methanogenesis are acetate and H 2 /CO 2 . The acetate is converted into CH 4 and CO 2 by acetoclastic methanogens while hydrogenotrophic methanogens convert CO 2 and H 2 or formate to CH 4 [ 21 ]. Acetate is consumed by a limited number of strains such as Methanosarcina spp. and Methanosaeta spp., the latter are incapable of using hydrogen. A large quantity of the acetate is produced in natural ecosystems and acetoclastic methanogens are responsible for 30–70% of methane production from freshwater sediments [ 22 ]. Hydrogenotrophic methanogens of the genus Methanobacterium , are important in maintaining low levels of atmospheric H 2 [ 23 ]. According to Conrad et al. [ 24 ], the degradation pathway of polysaccharides in methanogenic sediments (lakes, bogs, paddy fields, marine etc) is such that about two-thirds of the produced CH 4 is theoretically derived from acetate and one third from H 2 /CO 2, if steady state conditions exist. The universal distribution of the hydrogenotrophic pathway suggests that hydrogenotrophic methanogenesis may be the ancestral form of biological methane production and that hydrogenotrophic methanogenesis appears only once in evolution [ 25 ]. Some studies showed that temperature conditions can be helpful for defining the structure and function of the methanogenic microbial community. Noll et al. [ 26 ] observed functional changes in rice fields soil from a mixture of acetoclastic and hydrogenotrophic methanogenesis to exclusively hydrogenotrophic methanogenesis over a temperature range of 42–46°C. Another study indicated that at 30°C, the methanogenic community in soil consists mainly of Methanosarcinaceae , whereas at 15°C, the diversity of methanogenic archaea is greater and includes for example members of the Methanosaetaceae family [ 27 ]. However, despite the fact that anaerobic metabolism is described in many lakes, estuaries and wetland sediments, there is a paucity of information on the methanogen diversity in river ecosystems. Methanogenic archaea express the enzyme methyl-coenzyme M reductase which catalyzes the terminal step in biogenic methane production [ 28 , 29 ]. This enzyme complex is present in methanogens and methane oxidizers, making it a suitable tool for specific detection of methanogens. Methyl-coenzyme M reductase ( mcr ) constitutes about 5-12% of methanogen cellular protein and has been resolved into three components - A, C, and a small cofactor B. Component C is thought to be the site for methyl reduction and it is composed of three subunits; α, β and γ which are coded for by mcrA, mcrB , and mcrG genes respectively [ 30 ]. The genomes of all methanogenic archaea encode at least one copy of the mcrA operon [ 28 ]. The gene coding for mcr has been the target for many molecular ecological studies of methanogens [ 30 - 32 ]. The mcr operon exists in two forms, mcrA and mrtA gene coding. The mcrA gene is thought to be present in all methanogens, while the mrtA gene has only been demonstrated in members of the orders Methanobacteriales and Methanococcales [ 33 ]. The aim of this study was to identify and investigate the distribution of methanogens in two sediment depths (0-25 cm and 25-50 cm sediment layer) using the functional gene marker (the α-subunit of the methyl-coenzyme M reductase – mcrA gene ). We used PCR, cloning and sequencing analysis for the determination of the methanogenic phylogenetic composition. In addition, analysis of dissolved methane, total cell numbers, abundance of methanogens and potential methane production were also measured at the chosen locality simultaneously. The results are part of a long-term study of organic carbon dynamics and associated microbial communities in hyporheic sediments of the small lowland Sitka stream in Olomouc, a city in the Czech Republic. Earlier measurements of relatively high methane production confirmed the suitability of the field site for the study of methane cycling [ 1 , 34 ].",
"discussion": "Results and Discussion Environmental and microbial parameters of the river hyporheic sediment Generally, interstitial water revealed relatively high dissolved oxygen saturation with the exceptions of localities V and IV where the concentration of dissolved oxygen sharply decreased with depth. However, it never dropped below ∼ 10% of the oxygen saturation (data not shown). Vice versa, these two localities were also characterized by much higher concentrations of dissolved ferrous iron and dissolved methane than sites located upstream ( Table 1 ). Concentration of ferrous iron reflected the anaerobic conditions of the interstitial environment and showed the highest concentration in the deepest sediment layer (25-50cm) (p=0.02; n=5) at locality IV ( Table 2 ). In aquatic environments, iron can be found in two forms. In oxic environments iron occurs in the form of ferric iron (Fe 3+ ) and in anoxic enviroment ferrous iron (Fe 2+ ) dominates. Ferric iron is reduced to ferrous iron in anoxic conditions by bacteria which utilize this reduction process for their growth. This reduction can be carried out using one of several reductans such as hydrogen, pyruvate, lactate, acetate etc. [ 48 ]. Localities IV and V are situated in the lowland part of the stream which naturally meanders through an intensively managed agricultural landscape with increasing trophic level in the environment. The river bed sediment in lowland parts is characterised by fine sediment with organic matter accumulation. These budgets of organic matter allow local anoxic conditions. Locality IV, in particular, showed high concentration of organic carbon, ferrous iron and acetate in the sediment. These parameters facilitate the presence of methanogens in sediment and this is supported by the high concentration of dissolved methane in intestitial water and the methanogenic potential measurements ( Table 1 ). The average annual temperature of interstitial water at localities in the downstream parts of the Sitka stream was about 2.5°C higher than in localities upstream and this may result in higher methane production in the region. The precursor of methanogenesis, acetate was found in the interstitial water at all study sites and measured regularly at higher concentration with maximum concentration usually during the summer period ( Table 2 ). However, the concentration of other precursors such as propionate, valerate and butyrate were also measured but the values were under detection limits (data not shown). At locality no. IV the mean methane concentration in the interstitial water ranged between 2262.65 - 3856.01 µg L -1 at 0-25 and 25-50 cm depths, respectively ( Table 2 ). However the differences were not significant (p=0.11; n=5). Generally, the methanogenic potential (MP) varied around 180 pmol CH 4 g -1 DW h -1 (0,21 nmol CH 4 .g.WW.h -1 , respectively) at the study site. The methanogenic potential was found to be similar in both sediment layers (p=0.82; n=5) ( Table 2 ). These results show decreased readings compared to our previous study [ 34 ], in which a considerable amount of methane production was found in the upper sediment layer. To date, there is no standardised approach to measuring potential methane production and hence different results could be due to different methodologies. The MP range is quite broad and may differ by up to three orders of magnitude (10 – 2 to 10 1 µmol m -3 s -1 ,, 10 3 -10 6 nmol m -3 h -1 respectively); however, depending on temperature or availability of electrons, it can reach up to 10 8 nmol m -3 h -1 . Increasing temperature apparently raises the ability to produce methane [ 41 ]. Study of anaerobic incubation of sediment acceptors for methanogens of chalk streams revealed maximum MP at a depth of 6 cm (16.5 CH 4 nmol g -1 wet sed. h -1 ) with MP decrease with increasing sediment depth. However these authors only investigated a depth 0-8 cm under the river bed [ 2 ]. Although there was found a lot of variability in interstitial methane concentration and methanogenic potential, the values of both the parametres suggest that the studied locality (no. IV) produces a lot of methane and hence would be suitable for analysis of methanogen diversity in the region. Fluctuation in the data even during measurements conducted at the same day should not be suprising when considering the very dynamic system of hyporheic zone. Concentrations of methane may differ by up to several orders of magnitude both in horizontal and vertical profile of the sediments [ 49 ]. Total prokaryote cell numbers showed a significantly higher value in the deeper sediment layer (6.18×10 6 cells g -1 DW) than in the upper sediment layer (4.28×10 6 cells g -1 DW) (p=0.02; n=15). The abundance of methanogenic archaea identified with probe MPB1 was higher in deeper sediment layer, however the values were not significantly different (p=0.06; n=15) ( Table 2 ). The values of the total cell numbers obtained from river sediment were relatively low compared to that of other sediments which varied from 10 6 - 10 10 per g -1 DW [ 50 , 51 ]. This could be explained by the use of density centrifugation and sonication which can potentially damage prokaryote communities and influence total cell numbers and diversity. However, additional purification of cells by a combination of sonication and detergent treatment, followed by density gradient centrifugation is often recommended for soil, sediment or biofilm samples [ 52 ]. Even if the total cell numbers are slightly underestimated, sonication and density centrifugation techniques are still powerful enough to enable comparison between equally treated samples [ 53 ]. Further, checking of direct microscopic cell counts after sonication showed that the efficiency of the sonication was 85–90% (as shown in our previous experiments). Direct counting of bacteria in sediment is limited due to masking of bacteria by sediment particles. Density centrifugation results in the separation of bacteria from sediment particles and improves the purity of cell suspensions [ 54 ]. Identification of methanogens based on mcrA genes The methanogenic community in the hyporheic sediment of the Sitka stream was also analyzed by PCR amplification, cloning, and sequencing of the methyl coenzyme M reductase ( mcrA ) gene. A total of 60 mcrA gene sequences revealed 24 different phylotypes. These phylotypes were clustered into four groups and they confirmed affiliation to Methanosarcinales (10 phylotypes), Methanomicrobiales (10 phylotypes, one phylotype was found in both sediment layer, respectively) and Methanobacteriales orders (3 phylotypes) and uncultutured group (1 phylotype) of methanogens. Six members of the Methanosarcinales order were affiliated to the Methanosarcinaceae family and the nearest identical uncultured sequences were obtained from fen soil in Germany [ 55 ], Tibetian Zoige wetland sediment [ 56 ] and rice roots grown in Holland [ 57 ]. Four phylotypes were related to the acetoclastic methanogen Methanosaeta concilii and showed the highest similarity with sequences retrieved in a meromictic lake sediment in France [ 58 ] and flooding soil in Holland [ 59 ]. Within the Methanomicrobiales order, ten different phylotypes were detected and clustered along with the uncultured sequences, which were obtained from humic bog lake [ 60 ], acidic peatland [ 61 ], peat soil from Finland [ 62 ], Tibetian wetland soil [ 56 ] and rice roots in Holland [ 57 ]. Three phylotypes were related to the Methanobacteriales order and of these three phylotypes, one was closely affiliated to Methanobacterium sp. which was isolated from a Western Siberian peat bog [ 63 ] and the second one was clustered with clone originating in flooding soil [ 59 ] and rice root samples in Holland [ 57 ]. One single clone remains unclassified and was clustered with a sequence, which came from a biogas plant reactor in India [ 64 ]. It however showed 76% identity with Methanobacterium sp. Most of the clones assigned in this study showed low affiliation with known methanogenic species and were closely related to uncultured methanogens obtained from other similar environments ( Figure 1 ). Some environmental studies also confirmed that some clones may constitute an unclassified methanogenic cluster [ 56 – 58 ]. 10.1371/journal.pone.0080804.g001 Figure 1 Phylogenetic tree of mcrA gene clone (phylotype) sequences retrieved from hyporheic river sediment. The clones come from upper sediment layer (0-25 cm depth) are described as „P“, clones come from deeper sediment layer (25-50 cm depth) are described as „H“. The numbers in parenthesis indicate the number of clones. The phylogenetic tree is rooted with Methanopyrus kandleri . Members of all three orders were detected in a whole bottom sediment irrespective of depth. The number of clones affiliated with Methanomicrobiales predominated in the deeper layer while numbers of Methanosarcinales clones in general were higher in the upper sediment layer. However, higher number of Methanosarcinaceae -like clones were found in the upper layer and Methanosaetaceae -like clones prevalenced in the deeper layer of sediment ( Table 3 ). 10.1371/journal.pone.0080804.t003 Table 3 Number of clones and phylotypes and their phylogenetic affiliation to each library. \n Variable/ Depth \n Phylogenetic affiliation Methanomicrobiales Methanosarcinales (Methanosarcinaceae/ Methanosaetaceae) Methanobacteriales unclassified No. of clones [depth 0-25 cm] 11 21 (20/1) 1 1 [depth 25-50 cm] 14 7 (1/6) 5 0 No. of phylotypes [depth 0-25 cm] 5 6 (5/1) 1 1 [depth 25-50 cm] 6 4 (1/3) 2 0 Clones occurrence [%] [depth 0-25 cm] 32 59/3 3 3 [depth 25-50 cm] 54 4/23 19 0 The coverage (C) of each clone library, a measure of captured diversity, was calculated as: C=1-(n/N), where n is the number of different phylotypes from a clone library that were encountered only once and N is the total number of sequenced clones in the library [ 65 ]. The coverage of each library was 76,5% for upper sediment layer (0-25 cm) and 76,9% for deeper sediment layer (25-50 cm). Of the total number of 24 methanogenic phylotypes identified from hyporheic sediment, 13 phylotypes (34 clones, 57% from total number of clones) were found in the upper sediment layer (0-25 cm). One phylotype obtained from the upper layer (1 clone, 3%) was affiliated to the Methanobacteriales . Phylotypes related to the Methanosarcinales order including 5 phylotypes (20 clones, 59%) Methanosarcinaceae -like and one phylotype (1 clone, 3%) Methanosaetaceae -like member. Larger number of Methanosarcinales -like archaea in the upper sediment layer was also confirmed by FISH analyses (unpublished data). Five phylotypes (11 clones, 32%) retrieved from the upper layer were related to the Methanomicrobiales . A single unclassified methanogen clone (3%) was retrieved from the upper sediment layer. Twelve phylotypes (26 clones, 43% from total number of clones) were detected in the deeper sediment layer (25-50 cm). Six phylotypes (14 clones, 54%) were related to the Methanomicrobiales . Four phylotypes (7 clones, 27%) were affiliated to the Methanosarcinales order, Methanosarcinaceae -like member including one phylotype (1 clone, 4%) and Methanosaetaceae -like member consists of three phylotypes (6 clones, 23%). Two phylotypes (5 clones, 19%) of these were associated with the genus Methanobacterium , Our results indicate the presence of both hydrogenotrophic and acetoclastic methanogens in river sediment. These observations are supported by the stable carbon isotope signature of methane (δ 13 CH 4 ) which shows that both acetoclastic and hydrogenotrophic pathways take part in methanogenesis along the vertical profile of the Sitka stream [ 66 ]. The latest results show that the acetoclastic pathway predominates over the hydrogenotrophic pathway in a whole bottom sediment irrespective of depth and contributes to the methanogenesis in the Sitka stream with approximately 70 - 80% (unpublished data). \n Methanomicrobiales group of methanogens only grow in the presence of hydrogen, formate and alcohols with the exception of methanol. The Methanosarcinaceae can utilize all methanogenic substrates except for formate but the Methanosaetaceae grow exclusively using acetate as an energy source [ 67 ] whereas Methanobacteriales grow by CO 2 reduction. In the Sitka stream sediment, the number of phylotypes related to Methanomicrobiales and Methanosarcinales was equivalent. However the number of Methanosarcinales clones (n=28) was higher over Methanomicrobiales clones (n=25). As suggested in an earlier study [ 68 ], members of these two orders may be efficient syntrophic partners in the complete degradation of organic biomass in freshwater sediments. Only one study mentions methanogens in river sediment. This research investigated microbial populations in the extremely metal-contaminated Coeur d'Alene River sediments but the authors found just three methanogen phylotypes related to the Methanosarcinales order [ 69 ]. Most of the earlier reports on methanogens diversity were came from ruminants [ 70 – 72 ]. Moreover, DGGE analyses based on 16S rDNA of the methanogen community of the Sitka stream hyporheic sediments also retrieved a resembling number of taxonomic units at locality no. IV and this supports the results of this study (unpublished data). The application of PCR-based technologies for the investigation of naturally occurring methanogen populations has several advantages [ 72 ]. These methods are effective for detecting novel sequences, indicating unculturable new species and providing more complete description of the methanogen community structure. However, molecular methods introduce their own bias, such as the favoured lysis of one cell type over another, leading to the recovery of unrepresentative DNA fractions or skewed PCR amplification, where certain bands are favoured over others [ 33 ]. Another form of PCR bias is template reannealing which may occur during PCR when a high concentration of a product has accumulated and similar products and templates reanneal to each other, inhibiting primer binding and further amplification of a product [ 73 ]. It has also been suggested that the variability in copy numbers and intraspecies and interspecies heterogeneity of functional genes may represent a source of biases in microbial ecological studies [ 74 ]."
} | 5,578 |
36791061 | PMC9931117 | pmc | 6,317 | {
"abstract": "Critical phenomena are wildly observed in living systems. If the system is at criticality, it can quickly transfer information and achieve optimal response to external stimuli. Especially, animal collective behavior has numerous critical properties, which are related to other research regions, such as the brain system. Although the critical phenomena influencing collective behavior have been extensively studied, two important aspects require clarification. First, these critical phenomena never occur on a single scale but are instead nested from the micro- to macro-levels (e.g., from a Lévy walk to scale-free correlation). Second, the functional role of group criticality is unclear. To elucidate these aspects, the ambiguous interaction model is constructed in this study; this model has a common framework and is a natural extension of previous representative models (such as the Boids and Vicsek models). We demonstrate that our model can explain the nested criticality of collective behavior across several scales (considering scale-free correlation, super diffusion, Lévy walks, and 1/ f fluctuation for relative velocities). Our model can also explain the relationship between scale-free correlation and group turns. To examine this relation, we propose a new method, applying partial information decomposition (PID) to two scale-free induced subgroups. Using PID, we construct information flows between two scale-free induced subgroups and find that coupling of the group morphology (i.e., the velocity distributions) and its fluctuation power (i.e., the fluctuation distributions) likely enable rapid group turning. Thus, the flock morphology may help its internal fluctuation convert to dynamic behavior. Our result sheds new light on the role of group morphology, which is relatively unheeded, retaining the importance of fluctuation dynamics in group criticality.",
"introduction": "Introduction The critical phenomena of collective animal behavior, which are widely observed [ 1 – 4 ], elucidate the criticality of living systems [ 5 – 8 ]. However, the study of these phenomena is hindered by two core, interrelated problems: (1) the critical phenomena are nested across several scales, and (2) the functional roles of group criticalities are not clearly understood from an adaptive perspective. The first problem reveals the statistical properties required for systems analyzed in research studies. The second problem indicates that if the role of criticality is not well understood, it is possible that it is not treated with sufficient importance. Criticality in collective behavior occurs on at least two levels: macro and micro. The macro-scale criticality is the criticality of the system as a whole, as represented by the scale-free correlation of the system [ 8 – 12 ]. Cavagna et al. [ 9 ] found that a flock (of starlings, for example) has size-independent correlation domains for its direction and speed, and that high correlation raises the system susceptibility [ 13 – 15 ]. Thus, with scale-free correlation, the members of the flock can quickly share information and achieve optimal responses to external stimuli as a group. Overall, macro-scale criticality maximizes the benefits of group membership in various ways [ 6 – 8 ]. In contrast, micro-scale criticality is the one that occurs for an element or individual as represented by a Lévy walk [ 16 – 23 ], which is an optimal search strategy (i.e., the optimal balance between exploration and exploitation) for a given space. Some researchers have also suggested that Lévy walks contribute to smooth communication between individuals in flocks [ 24 , 25 ]. Notably, each fish in a group tries to search for and communicate with the new neighbors within the group. The micro-scale criticality may also therefore be related to group benefits. Although these critical properties have been separately reported, several systems seem to coexist (e.g., in bacteria [ 10 , 18 ], fish schools[ 12 , 24 ], and proteins[ 11 , 22 ]). At first glance, group and individual benefits appear to be in conflict each other, because there is generally a trade-off between the two [ 26 , 27 ]; however, the nested critical system seems to address these disagreements. The concept of “nested criticality” considered in this paper is derived from these observations. Next, we consider the second problem noted above, i.e., the lack of clarity regarding the relationship between criticalities and the observed collective behavior. Group turning is one of the most debated topics of collective behavior. Although some researchers have suggested that criticalities and group turning are related [ 9 , 28 ], their suggestions seem to be more suitable to options other than criticality. For instance, Attanasi et al.[ 29 , 30 ] believe that the quantum effect causes rapid group turning and have also suggested that individual fluctuations may trigger this behavior [ 31 ]. These solutions are attractive in themselves; however, the relationship between collective behavior and critical phenomena remains obscure. Unfortunately, the currently existing technologies do not provide sufficient data (only time intervals of a few seconds for analysis [ 31 , 32 ]) to describe the relationship between group turning and critical phenomena. In a previous study, we developed a collective behavioral model based on ambiguous interactions as one possible solution [ 33 ]. We applied this model to several flocking models and showed that the interaction between the alignment and attraction can be regarded as a single interaction with time-scale differences. Alignment pertains to the infinitely long predicted neighbor positions of a given agent, whereas attraction relates to the interactions of that agent with the current neighbor positions. We showed that the medium region of this time scale could play a vital role in group criticality. However, our result was restricted to a two-dimensional system, and we did not investigate the functional role of criticality. Additionally, as positional relations in three dimensions are more complex than those in two dimensions, it was unclear whether our model could yield meaningful results for the three-dimensional case. Especially, the noise definition of our model must be reconsidered for further extension to general situations. In the present study, we propose a model that can simultaneously explain nested criticality in collective behavior and its functional abilities (i.e., group turning); our model is a natural extension of our previous model to three dimensions. The self-tuned noise through ambiguous interactions make it possible to bridge the gap between micro- and macro-scale criticality. Moreover, our newly developed model elucidates the two features discussed above (i.e., the nested criticality and its functional roles) via an information theoretic analysis. The remainder of this paper is structured as follows. First, we present our model algorithm and explain why our model is a natural extension of the previous representative models. Second, we examine the criticality of group behavior. For various kinds of criticality (super diffusion, scale-free correlation, Lévy walks, and 1/ f fluctuation), we confirm that nested criticality holds without strict parameter tunings. This suggests that the critical phenomena are universal and can be observed in the broader range of parameter regions rather than only in a restricted region as conventionally thought. Third, we divide a flock into two scale-free induced subgroups and examine the information transfer between them, evaluating the influence of this coarse-grained information (the average vectors of the fluctuation and velocity vectors) on the future behavior of the group. Our analysis reveals that these coarse-gained average vectors significantly impact the rapid group turning behavior. Finally, we present a new method of applying partial information decomposition (PID) [ 34 , 35 ] to the group behavior and find that combining group morphology (i.e., the velocity distribution) with internal fluctuations can likely enable rapid group turning. Our findings imply that flock morphology may aid in the conversion of internal fluctuations into dynamic behavior.",
"discussion": "Discussion In this study, we demonstrated the nested criticality that emerged from a single algorithm employed our proposed ambiguous interaction model, which is a natural extension of the representative flocking model. We re-interpreted the attraction and alignment as short- and long-term predictions, respectively, and as a key concept of our model, we considered these predictions as regions (i.e. C ) rather than points. This vagueness offered the agents with several options in specific contexts. As our model does not contradict the concepts of the Boids model or other models, appropriate statistical properties could be confirmed (e.g., scale-free correlation and super diffusion). We also confirmed several micro-criticalities (i.e., Lévy walks and 1/ f fluctuation) caused by self-tuning noise. In our model, the next direction of the focal agent is provided as a region C ; thus, the behavior of that agent strongly depends on the behavior of its neighbors. If C decreases or increases (e.g., with local high/low polarity), the next direction accordingly becomes deterministic or indeterministic. This continuous oscillation between deterministic and indeterministic behavior yields the criticality of each individual. Furthermore, the two observed criticalities are in stark contrast. While the Lévy walk relates to the criticality in space, the 1/ f fluctuation is related to the criticality in time. Some researchers suggest that the self-similar structure of a time series for jittering behavior is related to the Lévy-walk result [ 71 – 73 ]. Although it is uncertain whether our result can be applied to actual data, we will confirm this relationship in future research. Before discussing the group turning behavior, we consider the meaning of the micro-scale criticality, which pertains to system robustness. Generally, robustness is quite a different concept to stability [ 74 ]. System stability can be described as its “persistence” in the original state, whereas system robustness relates to the “interplay between organization and dynamics” [ 74 ]. There is no original state to which the flock can return. In this sense, the flock is not a stable system; however, it is robust, as its shape and behavior change dynamically depending on the situation. In fact, some studies have suggested that robust systems should include micro-level criticality. For example, Ros et al. showed that, even for simple models such as the self-propelled particle model, various collective behaviors can emerge through the application of the logistic map update rule at Feigenbaum critical points [ 75 ]. Another example is the inverse Bayesian inference [ 76 ]. Unlike Bayesian inference, inverse Bayesian inference involves permanently changing a hypothesis, and Gunji et al. successfully reproduced Lévy walks and various collective phenomena by applying Bayesian and inverse Bayesian inference [ 76 – 78 ]. All these studies have shown that micro-level criticality constitutes the robustness of group behavior. The nested criticality confirmed in this study is also consistent with these findings. Next, we considered the functional role of criticality. Although the scale-free correlation may relate to group turning behavior, the lack of experimental data has prevented further investigations. However, our scale-free induced coarse-gain group method elucidated the relationship between criticality and group turning behavior. Our method assumes a macro-criticality that is stable for a certain interval. Using this approach, we showed that the interactions between correlated domains contribute to rapid group turning involving both velocity and fluctuation distributions. The PID analysis conducted in this study provided us with a more detailed description of this mechanism. The PID uniquely decomposed the mutual information I ( X , Y ; Z ) into four types of information: redundancy, two unique information flows, and synergy (where X and Y are inputs and Z is the output). Recall that redundancy means that the system contains a compensatory input for an output. In other words, if one input (i.e., X ) is missing, the same output can be expected from the other source as well (i.e., Y ). In the case of continuous inputs, redundancy refers to correlational inputs. As inputs are symmetrical in such cases, one side of the information can be recovered from the other. The unique information flow is the remainder of the mutual information; that is, I ( X ; Z ) (or I ( Y ; Z )) minus the redundancy. This quantity resembles the transfer entropy, but we must not confuse the two as the transfer entropy sometimes over- or under-estimates the net information [ 67 ]. The unique information flow complements this disadvantage of transfer entropy. Finally, synergy refers to the remainder of the mutual information, i.e., I ( X , Y ; Z ) minus the two unique information flows and redundancy. In contrast to redundancy, the pair of synergy inputs contribute to the output. The effect of the inputs on the output is non-linear. In this case, the loss of one source of information cannot reproduce the same output. The above-mentioned discussion explains why high redundancy emerges in the fluctuation inputs, R ({ F lead ( t ), F follow ( t )}; K ( t )). The strong correlation between the two groups (i.e., F lead and F follow ) comes from the scale-free correlation. This input symmetry of F lead and F follow also resonates with the critical-system susceptibility. The flock certainly contributes to the group turning by increasing its susceptibility. In contrast, the synergy for the velocity inputs, S ({ V lead ( t ), V follow ( t )}; K ( t ))), is more substantial than its redundancy (paired t -test; t (99) = −2.76, p < 0.001). This tendency means the asymmetrical input relation of V lead and V follow is the key to the group turning behavior for the velocity distribution. Our analysis suggests that the velocity and fluctuation distributions contribute to the group turning according to their different roles. The strong correlation between S ({ V lead ( t ), V follow ( t )}; K ( t )) and R ({ F lead ( t ), F follow ( t )}; K ( t )) provides a more detailed picture. No other combination showed such a strong relationship, which suggests a structural coupling. Although the influence of R ({ F lead ( t ), F follow ( t )}; K ( t )) means that the correlated fluctuation inputs contribute to the group turning, the meaning of S ({ V lead ( t ), V follow ( t )}; K ( t )) remains unclear. Intuitively, the two subgroups contribute independently (i.e. non-correlated) to the group turning. Considering this asymmetric input relation, the velocity vector distribution should be considered here. We call this type of velocity distribution “torsion,” as the two velocity inputs exhibit high torque during the rapid group turning (see S1 Table ; alternatively, the distorted formation during group turning is visible in S1 Video ). Considering the torque as object rotation, the high torque velocity distribution can relate to the group turn. Therefore, the correlation between S ({ V lead ( t ), V follow ( t )}; K ( t )) and R ({ F lead ( t ), F follow ( t )}; K ( t )) should be interpreted as follows: A high fluctuation correlation and an appropriate flock morphological structure (i.e., velocity torsion) are needed to generate a rapid group turn. Thus, a suitable morphology coupled with correlated fluctuations to transform the fluctuation power are required for group turning behavior. This proposition also indicates that the inverse relation (i.e., between S ({ F lead ( t ), F follow ( t )}; K ( t )) and R ({ V lead ( t ), V follow ( t )}; K ( t ))) does not hold. High R ({ V lead ( t ), V follow ( t )}; K ( t ))) yields a symmetric distribution of the velocity vectors only, whereas high S ({ F lead ( t ), F follow ( t )}; K ( t )) indicates low R ({ F lead ( t ), F follow ( t )}; K ( t )) (Pearson’s correlation test: n = 100, r = −0.92, p < 10 −40 ). Therefore, the inverse relation states that the heterogeneous fluctuation distribution has no relation with the homogeneous velocity distribution in terms of generating a group turn. The coupling with the fluctuation power may be disconnected when the sub-groups work as correlational units. In this study, we considered only one parameter, v max / R . It remains to be seen whether the same results can be obtained by adding more realistic constraints (e.g., a maximum turning rate, visual field, and gravity effect) to our model. However, the behavior of our model is statistically close to those of real flocks. We also noted that the realistic conditions implemented using the Boids model cannot replicate a more accurate description of group criticality [ 42 ]. Furthermore, few models focus on criticality across several levels. The effect of further adding more realistic assumptions to our model remains to be investigated in a future study. Our flock model suggests that the nested criticality within the flock has dual effects on the group turning behavior, via the fluctuation power in the form of symmetric fluctuation inputs and via the group morphology in the form of asymmetric velocity inputs. Both the aspects have functional roles when coupled. The flock morphology may help its internal fluctuation convert to dynamic behavior. However, several studies have paid a significant amount of attention to the former but not to the latter [ 9 , 28 – 32 , 40 , 79 , 80 ]. This study therefore suggested the importance of group morphology and that group criticality may support such couplings. The functional role of group criticality described in this study is a verifiable hypothesis if sufficient data become available."
} | 4,514 |
30641952 | PMC6356539 | pmc | 6,320 | {
"abstract": "In this study, a novel superhydrophobic nano-aluminum/iron (III) oxide composite has been prepared by a facile one-step process of electrophoretic deposition, with wide potential applications. The optimal suspension included ethanol, acetyl-acetone, and the additives of fluorotriphenylsilane and perfluorodecyltriethoxysilane. The microstructure, wettability, and exothermic performance were analyzed by field emission scanning electron microcopy (FESEM), X-ray diffraction (XRD), water contact angle measurements, and the differential scanning calorimetry (DSC) technique. The water contact angle and the heat-release of the target composites could reach to ~170° and 2.67 kJ/g, and could still keep stable, after exposure for six months, showing a great stability. These results provided an exquisite synthesis of ideas, for designing other superhydrophobic energetic materials with self-cleaning properties, for real industrial application.",
"conclusion": "4. Conclusions In brief, SAFFs, with wide applications, have been fabricated by a facile one-step-process-controllable EPD technique. The resulting energetic product exhibited an outstanding superhydrophobicity, with a contact angle up to ca. 170°, and a great heat-release performance with Q up to 2.67 kJ/g, respectively. Moreover, the hydrophobic stability and exothermic stability of the SAFFs could be retained for at least six months, in changeable circumstances. Thus, this work provided a new perspective for designing novel energetic material with a high real-environment tolerance, for real industrial applications.",
"introduction": "1. Introduction Nano energetic materials or metastable interstitial composites (MICs), including Al/CuO, Al/Fe 2 O 3 , Al/Bi 2 O 3 , Al/MO 3 , and unconventional substances of Al/AgIO 3 , Al/I 2 O 5 , and Al/KMnO 4 etc., have attracted steadily growing attentions, due to their higher energy density, faster energy release rates, higher explosion intensity, and more efficient reaction process, resulting from fuller interface contact between the reactants [ 1 , 2 , 3 , 4 , 5 ]. Up to now, based on a great number of advantages, they have been the subject of intense research work for fields of blasting, welding, automotive air-bag propellants, hardware destruction, gas sensor, etc. [ 6 , 7 , 8 , 9 , 10 ]. Notably, the heat-release (Q) of the theoretical stoichiometric Al/Fe 2 O 3 , as a classical thermite system, is more than 900 cal/g, and its adiabatic temperature is up to 3135 K, with wide potential applications [ 11 ]. Recently, abundant efforts have been devoted to fabricate Al/Fe 2 O 3 energetic materials, by using various techniques, including simple physical mixing [ 12 , 13 ], magnetron sputtering [ 14 ], arrested reactive milling (ARM) [ 15 ], electrospinning [ 16 ], vapor deposition [ 17 ], sol-gel technique [ 18 ], etc. Most relevant research works are mainly concerned with simplifying the synthesis technique, optimizing their morphology, or designing new structures to improve the exothermic capacity or develop the utilization rate of energy. For example, Dadbakhsh and Hao designed an Al/Fe 2 O 3 powder mixture distributed uniformly, by using selective laser melting [ 19 ]. The self-assembly and solvent-based mixing techniques have been used to prepare an Al/Fe 2 O 3 nanothermite with the Fe 2 O 3 as nanotubes [ 20 ]. In addition, the AP/Al/Fe 2 O 3 ternary energetic materials have been successfully fabricated by sol-gel, wet impregnation, and solvent anti-solvent processes, by Gao et al. [ 21 ]. However, the key components of nano-Al and Fe 2 O 3 powders in energetic materials are hydrophilia or surperhydrophilia, leading to performance attenuation. Thus, it is rather interesting to develop the exothermic stability and nature environment resistance to develop in energetic materials. The one commonly used method for preserving target energetic materials is by virtue of a nitrogen or argon gas seal bag or equipment. Moreover, Zhou et al. have proposed the glancing angle deposition technique and magnetron sputtering deposition process, to obtain the highly superhydrophobic Mg/Fluorocarbon core/shell nano-energetic arrays, with a static contact angle up to 162° [ 22 ]. The Al/CuO, with excellent superhydrophobicity, has been fabricated by chemical vapor deposition with an atomic layer deposition technology, by Collins et al. [ 23 ]. However, the most recent reported technologies, generally, are high in cost and complicated. Thus, it is still an impassable bottle-neck, to design novel Al/Fe 2 O 3 energetic materials with self-protection and high-exothermic capacity. The electrophoretic deposition (EPD) method have been reported in the literature, to be a low-cost and highly efficient technique for fabricating promising films or coatings [ 24 , 25 , 26 ]. As for the Al/Bi 2 O 3 thermite system, a facile two-step method of EPD and surface modification was introduced, in our previous research work, to construct a superhydrophobic Al/Bi 2 O 3 ; their exothermic stability could be maintained for two years, which is of great benefit for practical applications [ 27 ]. Moreover, the mentioned method has also been applied to the Al/CuO system [ 28 ]. The focus of this work was to attempt to prepare self-protected or superhydrophobic Al/Fe 2 O 3 materials, by using an improved one-step process of EPD, based on the two-steps technique, to enhance their adaptive capacity in the real, natural environment. The corresponding mechanism diagram is displayed in Figure 1 . Additionally, the superhydrophobicity and exothermic stability and water-proof or self-cleaning property of the product composite films have been systematically studied in detail.",
"discussion": "3. Results and Discussion 3.1. Characterization of the Product—SAFFs Figure 2 shows the XRD result of the fabricated SAFFs. Clearly, all mainly diffraction lines for the Al (04-0787, the Fm-3m (225)), and the Fe 2 O 3 (33-0664, R-3c (167)) were identified, demonstrating the presence of Al and Fe 2 O 3 , in the product, which is characteristic of nano-composite films deposited by the EPD technique. In addition, no peak for Al 2 O 3 or Fe indicated no reaction between the Fe 2 O 3 and the Al, during a typical EPD process. Figure 3 displays the top-view optical and macroscopic SEM images of the target SAFFs. As shown in Figure 3 a, the product surface (in the black part) was relatively uniformly-distributed, with no locally macroscopic agglomerate areas, indicating that a suspension including ethanol, acetyl-acetone, and fluorotriphenylsilane and perfluorodecyltriethoxysilane as additives, was the suitable dispersant for this electrophoresis assembly. The higher resolution FESEM image in Figure 3 b shows the special mesh-like microstructures in the SAFFs, which provided the structural foundation for improving the superhydrophobicity or weather-proof property, and contributed to the heat-release during the exothermic reaction (Equation (1)).\n (1) 2 Al + Fe 2 O 3 → Fe 2 O 3 + 2 Fe + Δ Q Moreover, the component composition (Al and Fe 2 O 3 particles) of the product were still nano-scale, as can be clearly seen in Figure 3 c,d, which were conducive to the largely increasing contact areas among the reactants, and the decreasing mass-transfer length, during the exothermic chemical process [ 27 , 29 ]. 3.2. Wettability The wettability of the product was systematically analyzed for investigating its hydrophobic performance. A water droplet with a volume of 5 μL on the product surface (it was difficult to do this due to the rather small rolling angle, <1°, as shown in Table 1 ) was close to a sphere in the typical Cassie state [ 28 , 30 , 31 ], as seen in the photo embedded in Figure 3 b. The corresponding water contact angle was measured at ca. 170°, which meant that the SAFFs were outstandingly superhydrophobic [ 32 , 33 , 34 , 35 ]. It is worth mentioning that the samples from the different parallel experiments showed similar results, as seen in Table 1 . In addition, the water droplet dynamic impact test was used here, to examine the water-proof property of the samples. The whole impact process of a dyed water droplet on the target surface process included the five steps of the initial state (I), the falling process (II), the contact process (III), the seceding state (IV), and the rebounding process (V). Due to the abundant air bubbles captured by the porous structures in the SAFFs [ 36 ], the droplet could secede quickly after a rather short contact time, with the superhydrophobic surface, and bounce off, which is demonstrated in Video S1 in the Supplementary Materials . Moreover, when the SAFFs were placed at a small angle, the impact process of the water droplet was also realized at a fast speed, as clearly seen in Video S2 in the Supplementary Materials . 3.3. Thermal Analysis In order to analyze the heat-release performance of the SAFFs, all samples were characterized by the DSC technique. Generally speaking, the output of the heat is essential to the energetic materials or other kinds of explosive materials. In this special energetic system, energy release from the SAFFs was due to the process shown in Equation (1), and the corresponding specific exothermic process is recorded in Figure 4 . Clearly, there is a sharp exothermic peak at ca. 600 °C, due to the strong chemical reaction between the nano-Al and the Fe 2 O 3 , in the composite films. There was a small endothermic peak at ca. 600 °C, resulting from the melting process of the nano-Al [ 37 ]. The total heat-release was up to 2.67 kJ/g, fitted by the DSC assistant software, which provided the thermal source theoretical foundation for the various potential applications of the SAFFs. 3.4. Stability Analysis For practical purposes, the effect of the variable environment on the water-proof property of product were analyzed, in detail, by adjusting the exposure time and the humidity. By comparing with Figure 5 a,b, it can be seen that after going through a long exposure period of half a year, the SAFFs were almost unchanged, with an even distribution in the nano-scale and vast fascinating porous structures. The relationship of the contact angle and the exposure time is displayed in Figure 6 a, where the contact angle of the target SAFFs was nearly 170° and barely got smaller with an increasing exposure time. Figure 6 b displays the contact angle as a function of humidity, which was used to simulate a realistic environment. Clearly, there were few fluctuations on the contact angle of the samples, after six months of exposure, and the corresponding contact angle remained at a high level of 170°. Moreover, as the pH increased from 1 to 11, the contact angle also remained almost stable ( Figure 6 c), showing only a marginal effect of the pH, on the water-proof property of the product. What needed to be specially mentioned was that different droplets, with different surface tensions, including water, diiodomethane, ethylene glycol, peanut oil, olive oil, and hexadecane, were used to examine the practicability of the SAFFs. As shown in Figure 6 d, the contact angle of the product decreased with the surface tension in the droplet. However, the contact angle of the SAFFs was still more than 150° (the “superhydrophobic” materials) even when the surface tension of the hexadecane was as low as 27.5 mN/m [ 30 ]. Thus, all results indicated the outstanding superhydrophobicity and stability of the product. Figure 7 shows the transformation law for the heat-release (Q) of the product, for various exposure times in the natural environment, and different humidity levels. As seen from Figure 7 a, the internal chemical energy of SAFFs had a very small fluctuation, even after six months of exposure, and the fluctuation rate ( F r , calculated by the Equation (2)) was as low as 0.75%, showing a great heat stability.\n (2) F r = Q h − Q l Q i × 100 % \nwhere the Q h , Q l , and Q i represent the highest, lowest, and the initial heat-release value. In addition, the effect of the changeable humidity on the exothermic performance of the product was almost negligible, as shown in Figure 7 b, and the corresponding F r was only 1.01%, which also indicated that the fabricated novel energetic materials, with ultra-long lifespan would have great potential applications in lots of domains."
} | 3,087 |
24904294 | PMC4033042 | pmc | 6,322 | {
"abstract": "We present a preliminary study of a thalamo-cortico-thalamic (TCT) implementation on SpiNNaker (Spiking Neural Network architecture), a brain inspired hardware platform designed to incorporate the inherent biological properties of parallelism, fault tolerance and energy efficiency. These attributes make SpiNNaker an ideal platform for simulating biologically plausible computational models. Our focus in this work is to design a TCT framework that can be simulated on SpiNNaker to mimic dynamical behavior similar to Electroencephalogram (EEG) time and power-spectra signatures in sleep-wake transition. The scale of the model is minimized for simplicity in this proof-of-concept study; thus the total number of spiking neurons is ≈1000 and represents a “mini-column” of the thalamocortical tissue. All data on model structure, synaptic layout and parameters is inspired from previous studies and abstracted at a level that is appropriate to the aims of the current study as well as computationally suitable for model simulation on a small 4-chip SpiNNaker system. The initial results from selective deletion of synaptic connectivity parameters in the model show similarity with EEG power spectra characteristics of sleep and wakefulness. These observations provide a positive perspective and a basis for future implementation of a very large scale biologically plausible model of thalamo-cortico-thalamic interactivity—the essential brain circuit that regulates the biological sleep-wake cycle and associated EEG rhythms.",
"introduction": "1. Introduction Computational models are being adopted at an increasing rate as a tool to investigate the cellular mechanisms of brain rhythms in both normal and pathological conditions (Aradi and Érdi, 2006 ; Breakspear et al., 2010 ; Terry et al., 2011 ). While computational resource is an obvious constraint in such endeavors, two further significant obstacles in mimicking the biology are parallelizing neuronal activity, and “de-syncing” the population activity from the master-clock of the computer. Our longer-term interest is in mimicking electroencephalogram (EEG) signatures of the sleep-wake cycle, by simulating biologically plausible computational models using biologically plausible computational techniques. In recent years the University of Manchester has been developing SpiNNaker (Spiking Neural Network architecture), a bespoke massively parallel machine to mimic the inherent parallelism of neuronal activity in real time (Furber et al., 2013 ). The brain-inspired parallel and asynchronous architecture of SpiNNaker permits biologically plausible computation of brain models—a feature that would otherwise rely on heavyweight software and its compilation on conventional Von-Neumann architectures, and yet achieve minimal parallelism. The study presented here is an initial attempt to design and implement a thalamo-cortico-thalamic (TCT) circuitry on the intrinsically parallel SpiNNaker, which can then be scaled up to mimic biologically plausible EEG signatures of the sleep-wake cycle. The purpose of this work is to demonstrate, as a proof of concept, that such a model can be implemented on SpiNNaker, and to investigate the benefits and drawbacks of this approach. It is not our intention here to produce a model which fully and correctly replicates all brain rhythms measured by EEG in regard to the TCT circuitry; capturing the complex dynamics involved in that system is beyond the scope of the current work. Neuronal dynamics recorded in EEG, often termed brain rhythms (Buzsáki, 2006 ), are an inexpensive and popular means of correlating brain activity with its various functional states (Wright and Liley, 1996 ; Nunez, 2000 ). The feed-forward and feed-back circuitry between the thalamus and the cortex has long since been known to play a key role in modulating brain rhythms associated with the various sleep stages as well as the sleep-wake transition (Steriade et al., 1993 ; Steriade, 2003 , 2005 ; Crunelli et al., 2011 ). Computational models of the TCT brain circuit have therefore been the basis for studying neuronal mechanisms in sleep (Lumer et al., 1997a ; Hill and Tononi, 2005 ; Traub et al., 2005 ; Bojak et al., 2011 ; Olbrich et al., 2011 ; Robinson et al., 2011 ) as well as in conditions where the EEG is qualitatively similar to certain sleep stages such as epilepsy (Breakspear et al., 2006 ) and under anaesthesia (Hutt and Longtin, 2010 ). While all such models refer to a similar holistic structure of the thalamocortical circuit, the models' internal structure, simulation platforms and parameterizations are significantly diverse. Thus, a fundamental aspect in computational modeling of the brain is the level of abstraction; the level of biological detail incorporated in a model needs to be appropriate to the problem at hand. For example, Olbrich et al. ( 2011 ) has attempted a multi-scale (time) model architecture in sleep, while (Bojak et al., 2011 ) has stressed on multi-modal models. On the other hand, (Hill and Tononi, 2005 ) have based their model on that of Lumer et al. ( 1997a , b ) and have looked into a multi-columnar model of the thalamocortical circuit to mimic brain rhythms of sleep and wakefulness as well as to understand memory consolidation during sleep (Nere et al., 2013 ). Another key aspect is the source of experimental data for both model structure and parameterizations. Comprehensive data on synaptic connectivity in the mammalian visual cortex is available in the works of Binzegger et al. ( 2004 ); Douglas and Martin ( 2004 ) and Neymotin et al. ( 2011 ) with some estimation for parameters which were not available from physiological studies. Further, extensive physiological data on rodent and other mammalian lateral geniculate nucleus (LGN: the thalamic nucleus in the visual pathway) is available in Horn et al. ( 2000 ); Sherman and Guillery ( 2001 ); and Jones ( 2007 ). Based on these thalamic and cortical physiological datasets as well as DTI (Diffusion Tensor Imaging) data obtained from two human samples, Izhikevich and Edelmann ( 2008a ) have presented a comprehensive TCT circuit using minimal parameter spiking neural models (Izhikevich, 2003 ) to mimic spiking population behavior. The SpiNNaker-based TCT model presented here is at the level of abstraction of the model in Izhikevich ( 2003 ), and has two modules viz. a thalamic module and a cortical module. The design and layout of the thalamic module is as in Bhattacharya et al. ( 2011 ) and is based on physiological data obtained from Sherman ( 2006 ). The cortical module layout and parameterizations are based on a previous implementation on SpiNNaker (Sharp et al., 2012 ) that was designed to test fast, stable and power-efficient performance on SpiNNaker when compared with other available platforms. The detailed modeling approach and parameterizations is covered in section 2. To the best of our knowledge, we are not aware of any prior instance of mimicking EEG signals using the SpiNNaker machine; similarly, this is the first instance of implementation of a TCT model within the SpiNNaker framework. In section 3, we present the preliminary results from this study based on our observation of the membrane potential time-series and power spectra of the cell populations. Specifically, the output of the excitatory cells of the thalamus and the cortical layer 4 are studied as a part of the first set of results from the TCT model simulation on SpiNNaker. An average of three trial runs of the model with all parameters at their initial values showed the membrane potential of both cell populations as noisy time series outputs with the dominant frequency of oscillation within the alpha band (8–12 Hz), a characteristic of quiet wakefulness. Next, we performed preliminary engineering of the model parameters to induce a sleep-wake transitional behavior in the model. The particular case we examined, which is outlined in more detail in section 3, was that of disconnecting the thalamic reticular nucleus (TRN) cell population in the model. This was designed to alter the thalamo-cortico-thalamic loop, which is responsible for the maintenance of the quiet wakefulness alpha rhythm, and simulate the situation during sleep in which cortical areas become functionally disconnected (Massimini et al., 2005 ). It thus provides a good test of the neuronal dynamics of the model in a situation in which the real dynamics are reasonably well understood. In previous (Bhattacharya, 2013 ; Bhattacharya et al., 2013 ) as well as ongoing (unpublished) work, lumped parameter models of neuronal population of the thalamocortical circuits [also known as neural mass models (Marreiros et al., 2009 )] have shown dependence on the TRN connectivity for mimicking qualitative dynamics as seen in EEG patterns of sleep and quiet wakefulness. Our results showed some important similarities with real sleep EEG time series data (also shown) when the TRN population is disconnected. However, significant differences with sleep power spectral data have also been observed; this suggests the model requires further tuning before it can fully capture sleep/wake thalamocortical dynamics. It is important to note that the purpose of the work presented here is to design a working model structure of the TCT circuit on SpiNNaker such that the model dynamics show some similarity to known dynamics of sleep and wake EEG in terms of characteristic spectral power; the intention is not to present a fully tuned model or a detailed exploration of those dynamics. A discussion on the motivation of the current work, the drawbacks, the implications of the initial results presented and future work plans is provided in section 4.",
"discussion": "4. Discussion Sleep and its biological relevance and mechanisms have been of interest in research (Rasch and Born, 2013 ) and beyond; a “healthy” sleep pattern have tremendous impact on daily activities (Mednick and Ehrman, 2006 ). Thus it is not surprising that sleep disturbances are a common accompaniment of several neurological and psychiatric disorders (Brown et al., 2012 ). Additionally, the time and frequency signatures of sleep electroencephalography (EEG) in neurological disorders often provide a better understanding of the disease conditions [for example in schizophrenia (Gardner et al., 2014 ); Alzheimer disease (Jonkman, 1997 )]. Furthermore, rapid-eye-movement (REM) sleep is thought to play a role in memory consolidation involving the non-hippocampal brain parts (Born et al., 2006 ). The thalamo-cortico-thalamic circuitry plays a key role in generating brain rhythms (Steriade et al., 1993 ; McCormick and Bal, 1997 ). Several studies on thalamocortical dynamics have used mesoscopic scale lumped parameter models to mimic EEG in healthy conditions (Robinson et al., 2002 ; Zavaglia et al., 2006 ; Deco et al., 2008 ; Modolo et al., 2013 ; Moran et al., 2013 ), as well as to investigate anomalous EEG in neurological disorders (Suffczyński et al., 2004 ; Roberts and Robinson, 2008 ; Pons et al., 2010 ; de Haan et al., 2012 ). In recent research (Bhattacharya, 2013 ), which is along similar lines as in Lytton ( 1996 ); Erdi et al. ( 2006 ), the need for detailed synaptic mechanisms in thalamocortical lumped parameter models to facilitate biologically realistic mapping of model features is emphasized. While extended work on the lumped parameter model implementing synaptic dynamics remains ongoing, we believe it is necessary to have a parallel line of investigation using a population model comprising of network(s) of single neuron models (i.e., single-neuron-level population model as opposed to lumped parameter population models) that is similar in structure to the former. This gives a “two-scale” architecture to the thalamo-cortico-thalamic framework. The endeavor will be to use the framework for realistic simulation of EEG dynamics in sleep-wake transition. Here, we have presented a preliminary study on inducing a transition from quiet wakefulness to a “slow wave” (higher amplitude, lower frequency) pattern in the model output, and have shown the similarity and dissimilarity of the model output with real EEG data of sleep and wakefulness; these are discussed further below. The primary issue in building a single-neuronal-level population model is the deficiency in available computational resources in terms of implementing biologically plausible parallel and asynchronous information transmission and exchange within the model framework. Another key aspect is energy-efficiency whereby maximal information processing is carried out using minimal resources, a mechanism that allows biology to deal with massive amounts of data in a fast and power efficient manner. This necessitates specialized computational tools to provide a low-power, parallel asynchronous framework for building very-large-scale-biologically-plausible models (VLSBm). The SpiNNaker (Spiking Neural Network architecture) chip is a platform designed to occupy this space; it meets all of the above criteria for building VLSBm and has been tested to outperform current available software and hardware platforms when building a cortical model of spiking neural networks (Sharp et al., 2012 ). In this work we have built a thalamo-cortico-thalamic spiking neural network for implementation on SpiNNaker. The mini-framework consists of 1090 neurons to mimic approximately 0.15 mm 2 of thalamocortical tissue. We have focussed on the thalamocortical relay (TCR) cells and the cortical Layer 4 pyramidal (PY4) cells; the layer 4 cells are known to be dominated by the sensory pathway input from the thalamus compared to inputs from other cortical areas (Gil et al., 1999 ). With all model parameters at their base values, the TCR time series output and its power spectra resembles the EEG characteristics of quiet wakefulness. Observation of the corresponding PY4 cell outputs indicate that the behavior of these cells are largely driven by the TCR cells. Next, we endeavored to vary specific model parameters to simulate non-rapid eye movement (non-REM) sleep stages. The thalamic reticular nucleus (TRN) neurons are implicated in playing a vital role in effecting slow wave oscillation in the EEG such as observed during slow wave sleep (SWS). To test this feature in the model, we disconnect all efferents from and afferents to the TRN cell population. We observe a distinct transition in the time series behavior of both the TCR and PY4 cells that resemble the EEG time series in SWS, albeit at a slightly higher frequency of oscillation (observed by visual inspection). This observation is reflected in the power spectra where the dominant frequency of oscillation for both population outputs are within the theta band, unlike the dominant delta band frequency seen in all stages of sleep EEG data. We speculate that the current disagreement in the power spectra of the SWS simulation on the TCT model may be addressed by dynamically changing the spiking behavior of the model cell populations (see below for further discussion on this). Furthermore, it will be interesting to observe how the intracortical afferents affect the PY4 cells in comparison to the TCR afferents (Destexhe, 2008 ; Lee and Sherman, 2008 ) and whether the model behavior conforms to experimental observations. Nonetheless, we note that the framework presented herein is a pilot study only, designed primarily to test the ability of the hardware to capture thalamocortical dynamics. We believe that the outcome from this study will provide a “basis” for simulating EEG signals on SpiNNaker-based computational models. Thus, at this stage, we do not attempt to simulate a true replication of the sleep-wake dynamics on the model. The larger goal of the work is to lay the foundations for building a VLSBm of thalamocortical interactivity to simulate biologically realistic sleep rhythms as observed in EEG. However, further testing and simulation on SpiNNaker will be required before scaling up the model for realistic simulation of EEG rhythms; we will take this up as an extension of the current work. Altogether, we believe this is a promising first demonstration of SpiNNaker as a platform for investigating thalamocortical circuits in humans. A widespread current concern in the computational neuroscience community is the non-trivial task of populating the parameter space of computational models; the task gets harder with increasing model size as experimental data with definitive values for specific parameters are difficult to acquire. We have sourced appropriate model parameter values from Binzegger et al. ( 2004 ); Izhikevich and Edelmann ( 2008b ); Bhattacharya et al. ( 2011 ); Galbraith ( 2011 ); and Sharp et al. ( 2012 ). Model layout and neuronal dynamics are from Sherman ( 2006 ) and Bhattacharya et al. ( 2011 ) and Izhikevich ( 2003 , 2004 ), respectively. The absolute values of the model parameters often require appropriate scaling for the simulation platform, and a common approach to deal with this aspect has been to normalize all model parameters to a “simulator-friendly” scale. Along these lines, several assumptions and simplifications have been made in this study: First, burst spiking dynamics of the thalamic cells that are crucial for generating slow wave oscillations (Jeanmonod et al., 1996 ; Magnin et al., 2005 ) are explored minimally. The thalamo-cortical relay (TCR) cells are tested for tonic spiking behavior in this work, which best align with the awake state of the brain. We speculate that the results reflect this behavioral mode of the TCR cells, clearly showing a resemblance with both time-series and power spectra of EEG in quiet awake state. However, the TCR displays burst spiking dynamics during the stages of sleep. Similarly, the TRN cells are known to show rich spiking dynamics (e.g., rebound bursting, low threshold spiking) that underlie sleep-wake oscillatory activity. These variant dynamics of the TCR and TRN cells will be further investigated in our ongoing work. Thalamic interneurons are more problematic; there are to our knowledge no references in the modeling literature relating specifically to the spiking dynamics of the thalamic interneurons (Destexhe et al., 1998 ). However the cortical basket cells, which are also categorized as local interneurons depending on their function and dendritic arborization, are described in Izhikevich and Edelmann ( 2008a ) using Fast Spiking (FS) dynamics. We have arbitrarily adopted this spiking behavior for the IN cells. Overall, much more detailed exploration and simulation of the individual thalamic cell spiking dynamics needs to be performed to preview the parameter space that would allow full replication of EEG in different sleep stages and the sleep-wake transition. It needs to be mentioned here that a high number of synaptic efferents from the thalamic interneurons are dendro-dendritic (Cox and Sherman, 2000 ). However, this aspect does not affect the synaptic transmission in the TCT framework as it comprises of spiking neuron models, and does not take into account the detailed axonal and dendritic attributes related to spike transmission and reception. Second, the Izhikevich model uses common excitatory and inhibitory synaptic parameters for all cell populations of excitatory and inhibitory types. This is a significant limitation and requires modification in future versions of the model to enable a direct comparison with the current lumped parameter models that include neurotransmitter and receptor dynamics. Third, the neuronal population in the thalamus represents a loose estimate as no definitive data on the number of thalamic cells within a cortical column is available from literature. We preserve the (intra-thalamic) proportion of thalamic cells in the (Izhikevich and Edelmann, 2008a ) thalamocortical model (only “specific nucleus” parameters are considered; the “non-specific nucleus” parameters are ignored), but scale this up by a factor of 10 2 . This may be contrasted with a factor of 10 scaling of the number of cortical cells. Thus the model is designed to place increased emphasis on the thalamic behavior and its effects on cortical oscillations for our test purposes. Fourth, our objective is to simulate EEG in sleep and quiet wakefulness. Thus, the simulated retinal input to the model needs to conform to discharge rates of the retinal spiking neurons during the resting state. In an early work on the cat retina (Kuffler, 1953 ), it is observed that the resting state discharge rate of a single retinal neuron is approximately 25 Hz. This is in agreement with the spike source rate provided as input to the TCT model in this work. However, in a relatively recent work (Robinson et al., 2004 ), it is estimated that the resting state firing rate of retinal input is 11 Hz, while in an alert awake state this is in the range 12–20 Hz. Thus, it would need further work to test these variations in experimental data and the effects on the model output in context to mimicking sleep-wake EEG. Fifth, the probability of connection between the intra-thalamic cells as well as for the feedforward and feedback connections between the thalamus and the cortex is arbitrarily set at 0.25 by empirical study on SpiNNaker. This will need further attention and more detailed tuning in future work. Finally, the conduction delay for thalamocortical and corticothalamic communication is implemented using a uniformly distributed function to generate a random delay. However, data acquired from physiology and tested on computational models is available in literature (Roberts and Robinson, 2008 ). This will be explored for implementation in future work. In conclusion, we have presented a pilot study which involved building biologically plausible networks on a biologically plausible computational platform—SpiNNaker. The study examines the feasibility of simulating EEG rhythms of sleep and wakefulness by implementing a thalamo-cortico-thalamic framework. The longer-term aim is to build a VLSBm of thalamo-cortico-thalamic synaptic interactivity on SpiNNaker, which will then be validated with real EEG data collected during sleep (Durrant et al., 2013 ). The work presented here gives a preliminary study of this approach. Ongoing work to build a similar framework with the lumped parameter approach will provide a “multi-scale” architecture to the model in both space and time. Together these models should provide new insights into the mechanisms which give rise to the rich thalamocortical dynamics seen in the human brain. Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."
} | 5,712 |
39046963 | PMC11268599 | pmc | 6,327 | {
"abstract": "The purple nonsulfur bacteria, Rhodospirillum rubrum , is recognized as a potential strain for PHAs bioindustrial processes since they can assimilate a broad range of carbon sources, such as syngas, to allow reduction of the production costs. In this study, we comparatively analyzed the biomass and PHA formation behaviors of R . rubrum under 100% CO and 50% CO gas atmosphere and found that pure CO promoted the PHA synthesis (PHA content up to 23.3% of the CDW). Hydrogen addition facilitated the uptake and utilization rates of CO and elevated 3-HV monomers content (molar proportion of 3-HV up to 9.2% in the presence of 50% H 2 ). To elucidate the genetic events culminating in the CO assimilation process, we performed whole transcriptome analysis of R . rubrum grown under 100% CO or 50% CO using RNA sequencing. Transcriptomic analysis indicated different CO 2 assimilation strategy was triggered by the presence of H 2 , where the CBB played a minor role. An increase in BCAA biosynthesis related gene abundance was observed under 50% CO condition. Furthermore, we detected the α-ketoglutarate (αKG) synthase, converting fumarate to αKG linked to the αKG-derived amino acids synthesis, and series of threonine-dependent isoleucine synthesis enzymes were significantly induced. Collectively, our results suggested that those amino acid synthesis pathways represented a key way for carbon assimilation and redox potential maintenance by R . rubrum growth under syngas condition, which could partly replace the PHA production and affect its monomer composition in copolymers. Finally, a fed-batch fermentation of the R . rubrum in a 3-l bioreactor was carried out and proved H 2 addition indeed increased the PHA accumulation rate, yielding 20% ww -1 PHA production within six days.",
"conclusion": "Conclusions Syngas fermentation presents an economical and effective mode for PHA production in R . rubrum . Deeply exploring the metabolic mechanisms of carbon assimilation under different gas environments offers an opportunity to improve growth and PHA production of R . rubrum . Our data support previous results indicating that R . rubrum could use the BCAAs biosynthesis pathway to help maintain redox homeostasis during photoheterotrophic metabolism [ 27 , 33 ]. In addition to some well-described electron sinks, such as calvin cycle, H 2 production and PHA biosynthesis, BCAA biosynthesis has been recently highlighted as one of alternatives to those pathways. We have demonstrated here that the presence of H 2 is profit for the photoheterotrophic assimilation of CO and make a significant change for the cellular carbon assimilation pathway. Moreover, accompanied by the occurrence of water-gas reaction, the accumulation of co-producing H 2 was also optimized and opens a possibility of exploring co-generation of bio-H 2 during syngas fermentation. Based on transcriptome comparative analysis, we revealed that the addition of H 2 triggered a series of BCAA related metabolism instead of CBB cycle to regulate the redox balance. To date, PHA industrial production from syngas has been limited by the variability of syngas and the insufficient productivities of PHA. This finding has important implication for the biotechnological use of R . Rubrum for PHA production on syngas. H 2 /CO ratio in syngas might act as a key adjuster knob to balance the cellular various metabolisms to regulate the PHA productivity. In the actual application process, with only a few adaptations (H 2 /CO ratio regulation), various sources of syngas can be applied to biosynthesize a large variety of chemicals.",
"introduction": "Introduction Valorization and reuse of wastes through bioconversion into value-added products plays an extremely important role in releasing the current energy crisis and environmental pollution [ 1 , 2 ]. Syngas, which is a blend of CO and H 2 , is widely formed during thermochemical conversion of various wastes and is considered an energy vector for a sustainable energy future. Syngas could be transformed into value-added products including ethanol, butanol, acetic acid or butyric acid by carbon-fixing microorganisms, in a process known as syngas fermentation [ 3 , 4 ]. So, syngas fermentation presents a highly attractive potential for biofuel, fine chemicals and biopolymer production nowadays [ 5 , 6 ]. Poly(3-hydroxyalkanoates) (PHAs) considered “green plastics”, are one of the potential products that can be synthesized by syngas fermenting bacteria [ 7 , 8 ]. Rhodospirillum rubrum ( R . rubrum ), a purple non-sulfur bacteria (PNSB) alpha-proteobacterium, can use syngas as carbon and energy source for growth and for PHAs accumulation under anaerobic [ 9 , 10 ]. R . rubrum is equipped with CO monoxide dehydrogenase (CODH) and hydrogenase (H 2 ase) activity to fix syngas [ 11 , 12 ]. Applying the water-gas shift reaction, it oxidizes CO with H 2 O into CO 2 and H 2 . Then, the produced H is later used to assimilate carbon molecules from CO and CO 2 into the biomass. CO 2 can then be fixed by various carboxylases into the ethylmalonyl-CoA pathway, the Calvin-Benson-Bassham cycle or the reductive tricarboxylic acid cycle to produce PHA [ 10 , 13 , 14 ]. During syngas fermentation, R . rubrum mainly synthesizes the short-chain-length PHAs (PHA SCL ; 3 to 5 carbon atoms), where 3-hydroxybutyrate (3HB) is a dominant monomer of the product. The product in RRNCO medium with syngas was reported to contain 86% of 3HB and 14% 3HV (3-hydroxyvalerate) of total PHA [ 10 ]. The presence of 3HV occurrence in the polymer enhanced physicochemical properties (e.g., higher elasticity, flexibility, etc.) [ 15 ]. Furthermore, the components of carbon in the precursor substrate greatly influences the amount of carbon in the monomers in the PHA chain. Currently, the main challenges of the industrial-scale syngas to PHAs conversion were the low syngas conversion efficiency and the slower PHA synthesis rate of the associated microbes [ 16 ]. It was reported that CO conversion efficiency was around 50% when artificial syngas was used as the carbon source [ 10 , 17 ]. To overcome this limitation, more information about the assimilation metabolism of syngas is needed. Previous research showed that the ribulose 1,5 biphosphate carboxylase/oxygenase (Rubisco) was mainly involved in CO 2 fixation during photoautotrophic growth [ 18 , 19 ]. In addition to the Calvin–Benson–Bassham (CBB) cycle, ethylmalonyl-CoA pathway (EMC) is now well accepted as an alternative pathway for the CO 2 -fixing to achieve electron balance during phototrophic growth on acetate [ 20 , 21 ]. Moreover, acetate has been shown to be a good co-substrate for R . rubrum growth in the process of syngas fermentation. It is mainly assimilated through the EMC pathway to provide the carbon skeleton for PHAs and biomass synthesis [ 21 , 22 ]. In addition to microbial metabolism, the composition of syngas (commonly referred to in terms of H 2 /CO ratio), which varies depending on its origin or substrate, is another non-ignored factor in the actual fermentation process. Moreover, previous studies have demonstrated that different syngas concentrations have a significant impact on the growth of R . rubrum [ 14 ]. The experiments with different initial CO concentrations have revealed that a p CO of 0.60 atm was optimal condition for R . rubrum cultivation in darkness and its maximum polyhydroxybutyrate (PHB)production reached 26% CDW [ 23 ]. Additionally, research on R . rubrum grown in different partial pressures of CO has also shown that the maximal biomass and PHA yield (8.3% CDW) were obtained at ppCO 0.6 bar under light condition [ 24 ]. The main objective of this research was to study the assimilation of syngas in R . rubrum under different atmosphere conditions directed towards CO utilization rates, PHAs production, as well as, to demonstrate CO 2 assimilation strategy. In order to reveal the assimilation of CO into cellular central metabolites biomass, a comparative transcriptomic analysis of 100% CO and 50% CO fermentation was carried out. We highlighted two different assimilation pathways for CO photo-assimilation. Under pure CO condition, Rubisco was actively incorporating CO 2 from gas substrate into biomass and the bioproduction of PHA acted as an electron sink in the balance of reduction equivalents during photo-heterotrophic growth. Instead, the branched-chain amino acids (BCAA) biosynthesis as an unexpected new CO 2 assimilation pathway was significantly induced under 50% CO (50% H 2 ) condition and partly substituted for PHA synthesis contributing to electron balance. Finally, a fed-batch fermentation revealed a decrease in cellular PHA content but a prominent increase in PHA synthesis rate with 25% H 2 added in initial gas. And its whole fermentation period could be cut down by half to six days, yielding the final PHA yield around 20% w w −1 .",
"discussion": "Result and discussion PHA production of R . rubrum for using CO as carbon substrate R . rubrum is able to utilize CO as carbon and energy sources by the water-gas shift reaction and various carbon-fixing metabolisms to produce PHA. The biomass and PHA formation behaviors of R . rubrum under different carbon substrate were investigated ( Fig 1 ). The bacteria were firstly cultured in growth medium 3 days to achieve a high cell density, then transferred to the induced medium containing fructose or CO as the sole source of carbon and energy. After 132 h of incubation, the cultures with 10 g l −1 fructose had grown to the highest concentration (2.2 g l −1 ) ( Fig 1A ). And the highest PHA content of 24.4% (w/w) was detected at 108 h. Moreover, its species of PHA was PHBV which was composed of approximately 53.5% 3HB and 46.5% 3HV monomers. Interestingly, although the biomass was maintained at the initial value of 1.2 g l −1 in the CO cultivation, a similar maximum PHBV production (23.3%) was obtained after 12 d ( Fig 1B ). However, the component analysis shown that the content of 3HV in this PHBV was only 2.2%, which was much lower than that on fructose medium. Furthermore, we monitored the CO consumption during gas fermentation and found the CO conversion was around 25%, which is similar to the report of Karmann et al [ 14 ]. 10.1371/journal.pone.0306222.g001 Fig 1 PHA fermentation analysis of R . rubrum cultivated under different growth conditions. (A) Growth of R . rubrum , PHA production and fructose uptake rate during 10g/l fructose fermentation process; (B) CO uptake rate, PHA content and 3-HV monomers proportion during pure CO fermentation process; CO uptake rate (C), PHA content and 3-HV monomers proportion (D) under different hydrogen addition ratio. Data are mean ±SD, n = 3. Syngas is rich in CO and H 2 and its CO/H 2 ratio varies depending on its origin. Therefore, different syngas mixtures were applied to investigate whether variation in CO/H 2 ratio offers an opportunity to optimize the PHA production and CO assimilation efficiency of R . rubrum . We added diversity proportions of H 2 to achieve different proportional syngas ranging from 100% CO to 20% CO and used it as the gas phase for R . rubrum cultivation. Then, the CO consumption ratio was monitored. As shown in Fig 1C , the addition of H 2 could facilitate the uptake of CO. The CO absorption speed was largely accelerated by adding 20% hydrogen and the CO conversion was enhanced to 42%. Moreover, when the H 2 /CO ratio was increased to 50%, CO was completely assimilated after 8 d of incubation. We speculated that the initial addition of H 2 was oxidized to provide energy for cells by hydrogenase, and further improved energy conversion efficiency in R . rubrum cells. Despite improved CO utilization, the maximum PHA concentration was gradually decreased with the increase of H 2 addition after 8 d of fermentation ( Fig 1D ). Interestingly, a significantly higher molar proportion of 3HV was observed with H 2 addition, which was reached 11.8% in the presence of 80% H 2 , suggesting that cells were growing with different metabolic strategy. Consequently, the regulation of a syngas with the desired CO/H 2 ratio will be a key factor to optimize PHA production in R . rubrum . What’s more, the effects of H 2 also need to be in-depth assessed. Global changes in gene expression during fructose, CO and CO/H 2 fermentation processes To get a comprehensive view of CO assimilation into biomass, we monitored gene expression changes in R . rubrum cultivated with an initial gas atmosphere of 100% CO or 50% CO (50% H 2 ) using RNA sequencing. 12 cell samples under three fermentation conditions (including 10g L −1 fructose, 100% CO, 50% CO induced fermentation and pre-culture cultivation samples, named R2YG, R2YC, R2YH and R2K respectively) were collected and subjected to Illumina high-throughput sequencer. After cleaning and filtering out low quality and ambiguous reads, 91 million clean reads containing 13.6 Gb of valid data were acquired ( Table 1 ). The sequencing data were deposited in the National Center of Biotechnology Information (NCBI) database (accession number: PRJNA943070). 10.1371/journal.pone.0306222.t001 Table 1 Summary of transcription data obtained by Illumina sequencing. Sample name Raw reads Clean reads clean bases Error rate (%) Q20 (%) Q30 (%) GC content (%) \n R2K1 \n 7647650 7556418 1.1G 0.02 98.06 94.5 64.58 \n R2K2 \n 7825864 7678970 1.2G 0.03 97.92 94.2 64.66 \n R2K3 \n 7956798 7862684 1.2G 0.02 98.04 94.45 64.73 \n R2YC1 \n 7560608 7443368 1.1G 0.03 97.91 94.26 66.6 \n R2YC2 \n 7940112 7867056 1.2G 0.03 96.89 91.48 65.58 \n R2YC3 \n 9028798 8878318 1.3G 0.03 97.88 94.15 65.64 \n R2YG1 \n 7640584 7545428 1.1G 0.02 98.03 94.44 65.31 \n R2YG2 \n 7605020 7518366 1.1G 0.03 97.18 92.29 66.28 \n R2YG3 \n 8597972 8471042 1.3G 0.03 97.79 93.91 64.8 \n R2YH1 \n 7717202 7560448 1.1G 0.03 97.85 94.07 63.48 \n R2YH2 \n 7609948 7507340 1.1G 0.02 98.04 94.5 64.44 \n R2YH3 \n 5372448 5219664 0.8G 0.03 97.77 93.99 64.56 Comparisons of the expression profiles under different fermentation conditions revealed substantial differences. Compared with the cell growth stage, fructose induced medium led to up-regulation of 1384 genes, whereas 1222 genes were found to be down-regulate ( Fig 2A ). The highly gene modification suggest that microbial cells trigger different metabolisms to response nutrient stress. Similarly, the numbers of differential expression genes (DEGs) were found to be up to 2731and 2554 after induction culture with pure CO and 50% CO, respectively. Furthermore, pairwise comparisons were conducted among the three induction phases to identify DEGs involving in different carbon source assimilation process. Compared with 50% CO, 1744 transcripts were showed significant differential expression under 100% CO condition, of which ~50% were up regulated. By contrast, only 660 transcripts showed significant differential expression in fructose fermentation versus pure CO fermentation. This seemed to imply that the more similar metabolic pathway might be used to assimilate those two different carbon sources during PHA accumulation phase. While the presence of H 2 could make a different for some metabolisms of bacteria at this phase. Cluster analysis of the DEGs in a heatmap revealed that R . rubrum cell exhibited highly similar expression patterns under fructose and pure CO fermentation conditions ( Fig 2C ). However, bacterial cellular metabolism patterns under 50% CO condition were similar with control group, to a certain extent, which suggesting addition of H 2 might partially alleviate the stress response and restore the growth mode of R . rubrum cell. 10.1371/journal.pone.0306222.g002 Fig 2 Global transcriptome during fructose, CO and CO/H 2 fermentation processes. (A) The number of differentially expressed genes in each condition; (B) The Venn diagram shows the overlap of up-regulated and down-regulated genes in various induced condition and growth stage samples. The numbers in the circles indicate the number of genes changed in each condition; (C) Dissimilar effects of fructose, pure CO and 50% CO upon global gene expression. Metabolic and functional shifts in response to changing gas atmosphere To determine the functional implication during the different fermentation stages, GO analysis was carried out on those DEGs of cell growth stage and PHA production phase. The biological functions of those DEGs were classified into three categories: molecular function, cellular component and biological process. The results showed that similar transcriptional induction under three PHA fermentation conditions were observed for the genes related to transmembrane transport (membrane, transmembrane transport complex, plasma membrane protein complex, etc.), cellular macromolecule biosynthesis, ATP biosynthesis (ATPase activity, nucleoside-triphosphatase activity, hydrolase activity, etc.) ( S1 Fig ), suggesting a high degree of metabolism changes and substance exchange in response to nitrogen deficiency in the medium. Instead, a number of genes linked to peptide biosynthetic process, ribonucleoprotein complex, oxidoreductase activity, NADH dehydrogenase subunits were well expressed in the growth phase, which are consistent with their importance of cell assembly and energy conservation for cell growth. In addition, several flagellum-dependent cell motility related genes were also strongly induced, that corresponds to Raberg et al [ 25 ]. Reported that flagellation was strongly occurred during growth and stagnated during PHA synthesis. To gain insight into the specific mechanism of R . rubrum under different gas atmosphere, we comparatively analyzed the cellular metabolic adaptations to pure CO and 50% CO fermentation ( Fig 3A ). At a first glance, it seemed to imply that the pure CO atmosphere triggered intense cellular stress response. Large amounts of DEPs annotated into cellular response to stimulus, signal transduction, ATPase activity was significantly expressed, as well as several transcription factors and regulators. Such as the genes coding for aldehyde dehydrogenase ( Rru_A0656 , Rru_A1542 , Rru_A0914 ), iron-containing alcohol dehydrogenases ( Rru_A0904 ) and several NADH dehydrogenases (e.g., Rru_A0320 , Rru_A0314 , Rru_A0321 , etc.), which are belonging to the reactive oxygen species (ROS) detox system, were significantly induced ( S1 Appendix ). Aldehyde dehydrogenases are known as a critical component in response to various environmental stresses, particularly the ROS stress [ 26 ]. Taken together, these stress related genes reflected an intracellular oxidative stress reaction induced in pure CO atmosphere, which was consistent with the high PHA yield under CO fermentation. There is an agreement that the responses of R . rubrum to stress pressure are firstly triggered by the activation of ROS signal transduction pathways and PHA biosynthesis plays a key role in balancing the redox state. In addition, comparing with cell growth phase, genes enriched in glycolysis/gluconeogenesis, citrate cycle (TCA cycle), carbon metabolism pathways were significantly downregulated in pure CO fermentation stage, which was corresponds with the stunted bacterial growth ( S1 Appendix ). Interestingly, hydrogen addition significantly induced protein biosynthesis process and the expression of genes linked to translation, peptide biosynthetic process, tRNA aminoacylation for protein translation and ribonucleoprotein complex. All these changes suggested a strong induction in protein synthesis might lead to a gradual recovery of the growth restarted. Combined with KEGG pathway enrichment analysis, we picked 11 pathways enriched by DEPs (padj<0.05) ( Fig 3B ). Most of the H 2 up-regulated genes were enriched in the pathways of ribosome, carbon metabolism and biosynthesis of secondary metabolism (59%, total 224 genes) ( S2 Appendix ). Apart from these three pathways, the remaining pathways (e.g., citrate cycle (TCA cycle), glycolysis/gluconeogenesis, glyoxylate and dicarboxylate metabolism, C5-Branched dibasic acid metabolism, etc.) were almost related to carbon metabolism and carbon storage. These results imply that carbon metabolism can act as the central response to the change of fermentation gas atmosphere. 10.1371/journal.pone.0306222.g003 Fig 3 Categories analysis of DEG in pure CO and 50% CO fermentation. (A) GO biological processes in genes upregulated under pure CO fermentation; (B) Top 11 pathways enriched by KEGG analysis for upregulated genes in 50% CO fermentation. Metabolic pathways of CO assimilation and PHA production in pure CO and 50% CO fermentation (i) CO assimilation and H 2 production as a first step of the R . rubrum gas fermentation During gas fermentation, H 2 addition accelerated the CO absorption and elevated molar proportion of 3HV in PHAs, although PHA concentration was decreased. To gain insight into the specific mechanism of CO assimilation into biomass under different gas atmosphere, an expression analysis of genes potentially involved in the assimilation/fixation of CO was carried out. R . rubrum can use CO under anaerobic conditions as carbon and energy source. When exposed to CO, CODH is induced to oxidate CO into CO 2 . Evaluation of expression profiles of the genes coding for CODH ( Rru_A1427 ) and its key regulator CooA (CO-sensing transcription activator, Rru_A1431 ) revealed that transcripts of CODH and CooA were more abundant at gas fermentation phase compared to cellular growth phase ( Table 2 ). During the CO oxidation to CO 2 , two protons are reduced to H 2 catalyzed by a CO-tolerant membranous energy-conserving [NiFe]-hydrogenase (ECH). Then, H 2 is released as a co-production of the water gas-shift reaction. Consistently, the genes coding for CO-linked ECH ( Rru_A1425 ), a formate-linked hydrogenase ( Rru_0326 ) and a H 2 uptake hydrogenase ( Rru_A1161 ) were found up-regulated in different degree upon CO exposure. To get a comprehensive view of CO assimilation, the expression levels of CODH and CooA were monitored by RT-PCR at different time points under pure CO and 50% CO conditions. As shown in Fig 4 , the expression levels of CODH and CooA were upregulated in gas fermentation process about 100-fold and sevenfold respectively, indicating a strong upregulation of CODH and CooA by CO. In agreement with the phenotypic observations, the expression of CODH and CooA was significantly higher in the presence of 50% H 2 than that in pure CO at the initial stage of fermentation. Moreover, the expression of CODH and CooA was exhibited a gradual decrease with the fast CO consumption after 8 days of fermentation. This observation suggests that 50% CO fermentation may have more advantages than pure CO fermentation, at least from the perspective of gas utilization efficiency. 10.1371/journal.pone.0306222.g004 Fig 4 Changes of CODH and CooA expression during different gas fermentation process. 10.1371/journal.pone.0306222.t002 Table 2 Genes involved in CO assimilation, CO 2 fixation and redox homeostasis highlighted by transcriptome analysis. Gene name Description Role in R . rubrum Pure CO induction vs. growth phase 50% CO induction vs. growth phase p-value log2 Fold change p-value log2 Fold change \n CO assimilation \n \n Rru_A1427 \n Carbon-monoxide dehydrogenase 5.91E-86 6.089 1.39E-83 4.606 \n Rru_A1431 \n Crp/Fnr family transcriptional regulator 6.66E-21 2.681 6.35E-07 1.324 \n Rru_A1425 \n ECH hydrogenase subunit E 1.76E-93 5.544 4.74E-87 4.161 \n Rru_A0326 \n transcriptional regulator 1.64E-09 1.091 ND a ND \n Rru_A1161 \n Ni-Fe hydrogenase 1.53E-25 3.968 1.11E-11 2.518 \n Calvin-Benson-Bassham cycle \n \n Rru_A2400 \n Ribulose bisphosphate carboxylase 0.015 1.137 ND ND \n methylmalonyl-CoA (MMC) pathway \n \n Rru_A0052 \n Biotin carboxylase ND ND 1.18E-90 2.350 \n Rru_A0053 \n Carboxyl transferase ND ND 2.41E-83 2.305 \n Rru_A2318 \n 2-methylcitrate dehydratase 1.72E-49 5.708 4.62E-28 3.536 \n Rru_A2320 \n Phosphoenolpyruvate phosphomutase 6.41E-28 4.305 6.84E-11 1.857 \n Rru_A2319 \n 2-methylcitrate synthase/Citrate synthase 4.49E-15 5.863 8.30E-11 2.855 \n ethylmalony-CoA (EMC) pathway \n \n Rru_A1201 \n Mesaconyl-CoA hydratase ND ND 0.00013 0.8 \n Rru_A3062 \n Methylmalonyl-CoA mutase 1.63E-08 1.338 1.21E-16 1.947 \n Rru_A3063 \n Crotonyl-CoA carboxylase/reductase 2.38E-08 1.800 3.65E-54 3.723 \n Rru_A3064 \n Isovaleryl-CoA dehydrogenase: Acyl-CoA dehydrogenase ND ND 4.74E-11 1.429 \n pyruvate ferredoxin oxidoreductase (PFOR) \n \n Rru_A2398 \n Pyruvate- flavodoxin oxidoreductase ND ND 0.002392 0.561 \n Threonine dependent branched-chain amino acid (BCAA) biosynthesis pathways \n \n Rru_A1135 \n Aminotransferase ND ND 0.003836 0.325 \n Rru_A0743 \n Aspartate kinase ND ND 0.013116 0.284 \n Rru_A1196 \n Aspartate semialdehyde dehydrogenase ND ND 2.20E-34 2.005 \n Rru_A3053 \n Homoserine kinase 1.21E-06 0.938103 ND ND \n Rru_A0467 \n Acetolactate synthase large subunit ND ND 5.35E-20 1.689 \n Rru_A0468 \n Acetolactate synthase small subunit ND ND 0.005039 0.561 \n branched-chain amino acid (BCAA) degradation pathways \n \n Rru_A1977 \n Pyruvate ferredoxin/flavodoxin oxidoreductase 1.95E-24 4.686015 6.32E-17 3.731 \n Rru_A1978 \n Indolepyruvate ferredoxin oxidoreductase 4.37E-56 3.829587 1.13E-45 3.322 \n TCA \n \n Rru_A2721 \n 2-oxoglutarate synthase ND ND 0.011457 0.342 \n Rru_A2129 \n fumarase ND ND 3.09E-13 1.052 \n PHA \n \n Rru_A0278 \n Transcriptional regulator ND ND 7.46E-11 1.442 \n Rru_A1816 \n Poly(R)-hydroxyalkanoic acid synthase (PhaC3) 1.55E-07 1.052 ND ND \n Rru_A2111 \n Hypothetical phasin protein ND ND 1.32E-05 0.746 \n Rru_A2817 \n Phasin protein 9.57E-12 1.795 1.66E-18 1.322 \n Rru_A1585 \n Polyhydroxyalkanoate depolymerase PhaZ1 2.62E-07 0.921 3.14E-17 1.861 \n Rru_A3356 \n Polyhydroxyalkanoate depolymerase 2.40E-06 1.590 5.57E-06 1.267 a Not detected (ii) Identification of potential routes involved in CO 2 fixation into organic compounds during different gas fermentation Subsequently, those CO 2 generated by oxidation is fixated into organic compounds via various carboxylases. Our study indicated that R . rubrum adopted different carboxylation strategies under different gas fermentation conditions, thus affecting the distribution of intracellular components. Related carboxylases were compiled in Table 2 . The ribulose bisphosphate carboxylase (RuBisCO), the main enzyme of the Calvin-Benson-Bassham (CBB) cycle, showed a significantly higher abundance in pure CO induced fermentation ( Rru_A2400 , p = 0.015, fold change = 1.137), which implicated the necessity of CBB cycle for CO 2 incorporation to central metabolites upon CO exposure. Moreover, the CBB pathway was also used as a redox balancing pathway to dissipate the reduced equivalents generated during 100% CO induced fermentation [ 27 , 28 ]. Besides Rubisco other carboxylases belonging to enthylmalony-CoA (EMC) pathway ( Rru_A3062 , p = 1.63E-08, fold change = 1.338; Rru_A3063 , p = 2.38E-08, fold change = 1.800) also participates in the assimilation of catalytic CO 2 . Those are consistent with the previous reports that the EMC pathway acts as a CO 2 assimilation and redox balancing route for acetate growing cells [ 10 , 21 ]. However, in contrast to what was observed in the pure CO fermentation, the addition of H 2 make a great change to the carbon assimilation and metabolism pathway. The transcriptomic data and RT-PCR expression assay both revealed that the expression level of RuBisCO showed no variation between growth phase and 50% CO fermentation phase, which implied the CBB pathway was not essential for CO 2 fixation under 50% CO gas atmosphere ( Fig 5 ). Significantly, a higher abundance of genes belonging to branched-chain amino acid (BCAA; Ile, Leu, Val) biosynthesis and degradation pathways were found to by transcriptome analysis, which represent another alternative route to managing the redox balance ( Rru_A0467 , p = 5.35E-20, fold change = 1.689; Rru_A0468 , p = 0.005, fold change = 0.561; Rru_A1977 , p = 6.32E-17, fold change = 3.731; Rru_A1978 , p = 1.13E-45, fold change = 3.322) ( Fig 6 ). These results implied the potential involvement of ILV biosynthesis in the metabolism of syngas (CO/H 2 ) in R . rubrum . According to this clue, we discovered that the reversal tricarboxylic acid (TCA) cycle was operated, specifically, α-ketoglutarate (αKG) synthase ( Rru_A2712 , p = 0.011, fold change = 0.342) which oxidizes succinyl-CoA to form αKG and meanwhile fixes one molecule of CO 2 . This pathway and the subsequent synthesis of αKG-derived amino acids were previously proposed as an important mechanism for electron balance in a R . rubrum Calvin- cycle mutant [ 29 , 30 ]. Moreover, the fumarase which plays a key role in the TCA cycle to catalyze the reversible dehydration of fumaric acid to malate also showed a massively higher relative abundance in syngas fermentation condition ( Rru_A2129 , p = 3.09E-13, fold change = 1.052). Its enzymatic products fumarate/malate are important precursors for the threonine-dependent isoleucine synthetized [ 27 , 30 ]. In addition, our RT-PCR analysis revealed that two other carboxylases related to TCA cycle were also highly upregulated in gas fermentation stage. Propionyl-CoA carboxylase (Rru_A1943) catalyzed propionyl-CoA yielding methylmalonyl-CoA linking to succinyl-CoA synthesis and pyruvate carboxylase (Rru_A2317) catalyzed pyruvate yielding oxaloacetate ( Fig 5 ). 10.1371/journal.pone.0306222.g005 Fig 5 Expression analysis of CO 2 fixation related genes under different gas growth condition by RT-PCR. 10.1371/journal.pone.0306222.g006 Fig 6 Schematic representation of the central carbon metabolism and threonine-dependent isoleucine biosynthesis pathways in 50% CO fermentation condition, as highlighted by transcriptome data. The colored circles indicate the fold changes between 50% CO fermentation and growth condition, ranging from red (genes are more abundant in the 50% condition) to green (genes are less abundant in the 50% condition). Nonsignificant genes are represented by grey circle. Currently, isoleucine pathway has always been considered as a reduced equivalent consuming pathway. Very interestingly, our data also revealed that a series of enzymes in reductive threonine-dependent pathway were found to be upregulated in the syngas condition ( Rru_A1135 , p = 0.003, fold change = 0.325; Rru_A0743 , p = 0.013, fold change = 0.284; Rru_A1196 , p = 2.20E-34, fold change = 2.005). One oxaloacetate and one pyruvate were used to initiate the threonine-dependent pathway and finally product threonine which was subsequently catalyzed by deaminase to form oxobutanoate. Then the acetolactate synthase (Rru_A0467; Rru_A0468) are responsible for the catalysis of oxobutanoate to start the BCAA biosynthesis pathway. Those results highlighted the cellular preferred the reductive threonine-dependent pathway for isoleucine synthesis instead of the citramalate-dependent pathway. Additionally, pyruvate is another key metabolite of the BCAAs biosynthesis [ 31 ]. The pyruvate ferredoxin oxidoreductase (PFOR) catalyzes a reversible reaction and can combine CO 2 with acetyl-CoA to form pyruvate. Coherently, PFOR coded by the Rru_A2398 , were observed to be more abundant in 50% CO fermentation condition, while no differences were found under pure CO atmosphere. And as in previous study, starting from acetyl-CoA, de novo synthesis of BCAA consumes reduced ferredoxin and NADPH contributing to cellular electron balance [ 30 , 31 ]. These all reflected that the BCAA biosynthesis pathway and its related metabolic processes TCA cycle represent a key way for CO 2 fixation and redox potential maintenance in R . rubrum during syngas fermentation process. To further validate this assertion, we analyzed the accumulation of intracellular amino acids in R . rubrum growing in pure CO or syngas conditions. For the concentrations of BCAAs analysis, results showed that only leucine was detected and significantly different in these two fermentation conditions ( S3 Appendix ). As previous study showed that the synthesis of BCCAs was impacted by various environmental changes and the accumulation of isoleucine-leucine-valine (ILV) was transient [ 32 ]. Further detailed experimentation will be required to reveal the accumulation regular of BCAAs and in-depth understand their role in redox state balance. Besides, other amino acids also exhibited obvious changes in concentration. In accordance with previous studies, the synthesis of these amino acids via specific carboxylation reactions (e.g., Glu derives from αKG, Ser from 3-phosphoglycerate, Ala from pyruvate, etc.) could also contributed to electron balance [ 30 ]. (iii) The production of the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Short-chain-length (SCL) PHAs (monomer units of C3 to C5) are synthesized from acetyl-CoA via three enzymatic reactions by three different enzymes. The first reaction is the condensation of two acetyl-CoA molecules into acetoacetyl-CoA by β ketothiolase (PhaA). In the second reaction, acetoacetyl-CoA reductase (PhaB) reduces the acetoacetyl-CoA to form 3-hydroxyalkanoate-CoA. Finally, PHA synthase (phaC and phaE) catalyzes the polymerization of 3-hydroxyalkanoate-CoA monomers to form a polyhydroxyalkanoate. The gas substrate used to fermentation and its assimilation pathway directly impact the production and composition of the PHA polymers. Our PHA quantitation analysis revealed a higher production of PHAs in the pure CO fermentation than that of syngas. However, transcriptomic data revealed that there were no significant differences in the expression of the three key enzymes of PHA synthesis pathway (phaA, phaB and phaC; separately coded by Rru_A0274 , Rru_A0273 , and Rru_A0275 ) under both fermentation conditions. Interestingly, one of the homologous PHA polymerase gene phaC3 ( Rru_A1816 ) and a phasin ( Rru_2817 ) associated with PHA granules were upregulated in pure CO fermentation. In case of syngas cultivation, the regulator of the phaCAB cluster ( Rru_A0278 ) and two phasins ( Rru_2111 , Rru_2817 ) were shown more abundant. Meanwhile, two depolymerases ( Rru_A1585 and Rru_A3356 ) were highly upregulated under both conditions. The enzyme of polymerization and depolymerization of PHA work together to determine the final production of PHA. Those above gene expression changes were influenced by the specific time points of sampling. Samples of 8 th day gas fermentation were selected for RNA sequencing. And at this time point the cellular PHA formation might reach to platform stage, especially in syngas fermentation. Moreover, in the late fermentation, PHA would undergo a rapid mobilization with the shortage of carbon sources. Since transcriptome assay only reflected the expression of PHA synthesis-related gene at a specific time, we further analyzed the expression of the serval key enzymes of PHA synthesis pathway at different fermentation times. RT-PCR analysis showed that the Rru_A0275 , Rru_A1816 and Rru_A0274 transcript levels were elevated after 4 days in pure CO culture, whereas Rru_A0273 gene expression was slightly decreased ( Fig 7 ). Moreover, the expression of Rru_A1816 and Rru_A0274 were evident at least 8 days, which is consistent with the gradually increased intracellular PHA accumulation during pure CO fermentation. Unlike 100% CO fermentation, only the PHA polymerase coding gene Rru_A0275 and Rru_A1816 were induced and reached the highest spot on the fourth day after transferred into syngas medium and then shown a gradually decreasing trend, which might finally lead to a decrease in PHA production. We speculated that this might be due to the rapid consumption of CO during 50% CO fermentation process. With the shortage of carbon sources PHA would undergo a rapid mobilization. 10.1371/journal.pone.0306222.g007 Fig 7 Expression analysis of PHA biosynthesis related genes during 100% CO and 50% fermentation process by RT-PCR. PHA production has been considered as a mechanism to maintain the redox homeostasis of the cell and its biosynthesis is induced by the redox stress [ 27 , 33 ]. Indeed, there are other alternative pathways in R . rubrum competing with PHA synthesis to help balance the redox pool state. Moreover, studies show that PHA production may be blocked while another electron sink metabolism is available. We proposed that under syngas fermentation condition, the BCAA biosynthesis pathway might act as a key electron-sinking pathway to bypass PHA production, and then eventually lead to the decrease of intracellular PHA accumulation. Even though, besides the PHA polymer yield improvement, the physicochemical properties and biodegradability optimizing, which are dependent on their monomeric constituents and relative order in the polymer, are other key points for the PHA research [ 34 , 35 ]. As our phenotypic characteristics result shown, H 2 addition induced a significantly higher molar proportion of 3-HV, which triggers our more consideration about gas fermentation for PHA production. Previous research showed that the 3-HV monomers of the copolymer (3-hydroxybutyrate-co-3-hydroxyvalerate) might originate from the condensation of acetyl-CoA and propionyl-CoA into 3-ketovaleryl-CoA and the odd number of carbon sources, such as propionate, could induce the production of the 3-HV content [ 36 , 37 ]. Moreover, the pyruvate synthesized by PFOR could also be converted to 3-HV precursor molecule propionyl-Co-A. Meanwhile the biosynthesis and degradation pathways of L-isoleucine (ILV) could convert acetyl-CoA into propionyl-CoA, which constituted a new photoheterotrophic carbon assimilation pathway. These results suggested that the production of PHA might be modified by the suitable addition of H 2 for the desired copolymer synthesis. Fed-batch cultivation with R . rubrum on syngas The results above revealed that H 2 addition could accelerate the CO absorption and improve gas utilization efficiency in R . rubrum . However, due to the activation of the ILV pathway which partly replaced PHA biosynthesis, the final production of PHA was decreased. In the actual fermentation process, we should comprehensively consider fermentation rate and PHA yield. With the above factors, we regulated the CO:H 2 ratio at 3:1 (simulated syngas) as initial gas feed and compared it with the pure CO fermentation. A fed-batch bioprocess in a 3-l bioreactor was performed and the final broth composition, regarding biomass production, PHA content and acetate concentration were monitored for each culture ( Fig 8 ). Similarly, R . rubrum was cultivated by the two-step method as described in Methods. To achieve rapid growth, the cells were first cultured in broth medium to the stationary phase, and then transferred to the 3-l bioreactor with RRNCO mineral medium (containing 1.38 g l -1 sodium acetate as co-substrate) for gas fermentation experiments. 10.1371/journal.pone.0306222.g008 Fig 8 Fed-batch fermentation of R . rubrum in a 3-l bioreactor. (A) Schematic layout of the bioreactor for gas fermentation; Biomass concentrations and PHA yields under different gas conditions: (B) pure CO fermentation, (C) 75% CO fermentation; (D) Acetate concentrations during different fermentation conditions; After10mM acetate added in the mid-stage, biomass concentrations, PHA yields and acetate content in pure CO condition (E), or 75% CO condition (F). Data are mean ±SD, n = 3. acetate-feeding strategies. The most remarkable change occurred with 25% H 2 added was a significant increase in PHA accumulation rate, yielding the maximum PHA content (16.7% ww -1 ) after 6 days of fermentation ( Fig 8C ). During pure CO growth, although its final PHA yield up to 24.3% ww -1 , its fermentation time was extended to 12 days ( Fig 8B ). In addition to the gas substrate, acetate in liquid culture also has a positive effect on PHA accumulation. Revelles et al. reported acetate providing the carbon skeleton for PHAs and promoting PHA accumulation, and therefore, the effect of acetate during gas fermentation was assessed [ 10 ]. As shown in Fig 8D , the initial acetate was gradually consumed along with the extension of fermentation time. Differently, the maximum values of acetate consumption rates (0.3 g l -1 d -1 ) were observed 3 days after inoculation in CO/H 2 atmosphere, and acetate content was decreased to 31% on the 4 th day of cultivation ( Table 3 ). At this point, only 24% of CO was consumed from the initial syngas which implied that the accumulation of PHA was tightly related to acetate concentration. While in pure CO condition, acetate content was decreased to 43% on the 6 th day of cultivation and 20% of CO was conversed at this moment. In this sense, the initial H 2 addition indeed excited carbon sources utilization (including CO assimilation and acetate conversion) and in part accelerated PHA accumulation. Although the cellular PHA was gradually accumulated during gas fermentation process, the biomass yield had not significant increase. This might be due to the period of bacterial cell growth. The stationary phase cells were used for gas fermentation in our experiment. In this condition, as Godoy et al. reported polymer accumulation prevailed over residual biomass formation [ 38 ]. Therefore, accompanied by PHA accumulation biomass formation had a slight increase at earlier part of fermentation, and at the end of the fermentation with the mobilization of PHA biomass yields shown a downward trend. 10.1371/journal.pone.0306222.t003 Table 3 General profiling of R . rubrum grown in a 3-l bioreactor under different gas compositions. Fed-batch Cultivation days CDW (g l -1 ) PHBV (% ww -1 ) HV monomer composition (% a ) CO consumption (% b ) Acetate consumption (% c ) \n 100% CO \n 6 2.32±0.18 7.90±1.24 2.2±0.29 20.38±0.03 56.52±1.40 12 2.95±0.19 28.64±1.74 2.4±0.17 62.86±1.62 \n 75% CO \n 4 2.45±0.18 8.8±1.17 6.3±0.23 24.06±1.57 68.84±0.62 6 2.65±0.17 20.09±1.17 6.2±0.27 50.20±5.57 Values represent the mean ± standard deviation of three independent biological replicates. The cell dry weight (CDW), PHA (% cell dry weight), CO and acetate consumptions during fermentation process (100% CO and 75% CO gas atmosphere) were determined. a Percentage of HV in polymer (mol%) b Percentage of gas conversion (%) from the initial concentration on syngas. c Percentage of acetate absorption (%) from the initial concentration on RRNCO media. Considering the high consumption rate of acetate in R . rubrum gas fermentation process, acetate-feeding strategies were established to optimize the PHA production process. And 10 mM acetate was respectively added on the 4 th day of CO/H 2 cultivation when the initial residual acetate amount was only 31% or the 7 th day of pure CO condition with 29% residual acetate ( Fig 8E and 8F ). With a controlled acetate feeding, cells re-started PHA formation at a faster rate, eventually reaching platform stage. The most remarkable results were the highly efficient accumulation of PHA in case of CO/H 2 cultivation, yielding the maximum PHA production (PHBV around 20% ww -1 , with 6%mol 3HV) in only six days ( Table 3 ). Compared with pure CO condition, its fermentation period was decreased by half, although its PHA production was not as high as that of the pure CO cultivation (PHBV around 28% ww -1 , with 2% mol 3HV). These results suggested that H 2 plays an important role during the start-up stage of gas fermentation, and it may serve as an energy supply to activate cellular carbon assimilation and accelerate PHA synthesis. Table 4 shows that there is a large variation in the PHA content of R . rubrum , ranging from 8% to 81%, mainly depending on the type of carbon sources and growth conditions. Using aerobic-anaerobic two-phase growth with fructose (C6) as carbon sources, co-polymer PHBV production finally reached 81% CDW (with 86% mol 3HV) in 12-days fermentation [ 38 ]. This was the highest PHBV yield and proportion of HV monomer by utilizing C6 substrate. However, the cost of the raw materials (especially carbon source) required in the mass PHA production is a main limiting factor for industrial applications [ 41 ]. Syngas fermentation offered an attractive economic prospect for PHA production. The cost of producing the PHA with syngas (C1 substrates) is not only less expensive than that by sugar but also environmentally friendly. According to Table 4 , our procedures indeed increased PHA production when compared to previous works operating in the same culture medium. Our pure CO fermentation results were very close to those achieved with R . rubrum on syngas in continuous light or dark cultures [ 10 , 23 ]. Furthermore, our PHBV productivity in syngas atmosphere is better than others achieved with syngas or CO fermentation and achieve its maximum PHBV yield in 6 days. Therefore, it is interesting for further developing new strategies for initial syngas selection and gas composition regulation to improve PHA production of R . rubrum . 10.1371/journal.pone.0306222.t004 Table 4 Comparative PHA production in Rhodospirillum rubrum under different fermentation conditions. Growth conditions Cultivation modes Carbon sources PHA yields (% ww -1 ) and detection time-point References 550 R8AH, light, anaerobic, N limitation Photo-heterotrophic Yeast extract (1g l -1 ) + β-Hydroxybutyrate (30 mM) PHB: 46.8 10 days [ 39 ] Yeast extract (1g l -1 ) + Acetate (30 mM) PHB: 19.2 10 days Yeast extract (1g l -1 ) + Propionate (30 mM) PHBV: 2.2 (HV: 58.5%) a 10 days RRNCO (with 10 g l -1 fructose), dark, not strictly anaerobic, N limitation Chemo-heterotrophic Fructose (10 g l -1 ) PHBV: NM b (HV: 46.5%) NM b [ 40 ] RRNCO (with fructose, 1 g l -1 yeast extract), dark, aerobic-anaerobic transition, N limitation Chemo-heterotrophic Yeast extract (1 g l -1 ) + Aerobic condition: fructose (13.3 mM) Anaerobic condition: fructose (40 mM) PHBV: 29 (HV: 75%) 4 days [ 38 ] Yeast extract (1 g l -1 ) + Aerobic condition: fructose (13.3 mM) Anaerobic condition: fructose (40 mM) + bicarbonate (12 mM) PHBV: 81 (HV: 86%) 12 days RRNCO (with yeast extract), light, anaerobic, N limitation Photo-heterotrophic Yeast extract + Artificial gas (56.0% N 2 , 17.2% CO, 16.3% CO 2 , 8.8% H 2 ) PHA: 34.8 6 days [ 42 ] RRNCO, dark, anaerobic, N limitation Chemo-heterotrophic Syngas (50% CO and 50% N 2 ) + Acetate (15 mM) PHB:26 9 days [ 23 ] RRNCO, light, anaerobic, N limitation Photo-heterotrophic Syngas (40% CO, 40% H 2 , 10% CO 2 and 10% N 2 ) + Acetate (10 mM) PHB: 28 NM b [ 10 ] RRNCO (with 15mM fructose), light, anaerobic, P limitation Photo-heterotrophic syngas (25% CO, 25% H 2 , 5% CO 2 , and 45% N 2 ) + Acetate (10 mM) + fructose (15 mM) PHB: 30 NM b [ 43 ] RRNCO, light, anaerobic, N limitation Photo-heterotrophic CO (initial ppCO 0.6 bar) + Acetate (10 mM) PHB: 8.3±3.4 12 days [ 24 ] RRNCO, light, anaerobic, N limitation Photo-heterotrophic CO + Acetate (16.8 mM) PHBV: 28.6 (HV:2.4) 12 days This work syngas (75% CO and 25% H 2 ) + Acetate (16.8 mM) PHBV:20 (HV:6.2) 6 days a Monomers found in polymer (mol%) b Not mentioned"
} | 11,833 |
26678754 | PMC4683463 | pmc | 6,329 | {
"abstract": "A fluorinated silyl functionalized zirconia was synthesized by the sol-gel method to fabricate an extremely durable superhydrophobic coating on cotton fabrics by simple immersion technique. The fabric surfaces firmly attached with the coating material through covalent bonding, possessed superhydrophobicity with high water contact angle ≈163 ± 1°, low hysteresis ≈3.5° and superoleophilicity. The coated fabrics were effective to separate oil/water mixture with a considerably high separation efficiency of 98.8 wt% through ordinary filtering. Presence of highly stable (chemically and mechanically) superhydrophobic zirconia bonded with cellulose makes such excellent water repelling ability of the fabrics durable under harsh environment conditions like high temperature, strong acidic or alkaline solutions, different organic solvents and mechanical forces including extensive washings. Moreover, these coated fabrics retained self-cleanable superhydrophobic property as well as high water separation efficiency even after several cycles, launderings and abrasions. Therefore, such robust superhydrophobic ZrO 2 coated fabrics have strong potential for various industrial productions and uses.",
"discussion": "Discussion A fluorinated silyl functionalized zirconia (fsZr) based superhydrophobic and superoleophilic coating was fabricated on cotton fabric. The FTIR spectral analysis ( Fig. 2 ) of the coating material reveals the existence of signature peaks of zirconia along with the characteristic peaks originated from –CF 2 groups present in fluorinated silyl functions. The molar ratio of Zr:Si in fsZr coating obtained from semi-quantitative analysis using TEM-EDX ( Fig. 3d ) is consistent with the nominal composition used for sol preparation. The coated cotton fabric shows excellent stain-resistant and self-cleaning abilities ( Fig. 4 ) along with superoleophilic property ( Supplementary Fig. S5d ) due to the combined effect of C–F groups possessing very low surface energy in addition with the micro level rough fibrous exterior surface. The superhydrophobic character of this fsZr coated fabric surface can be explained by the Cassie-Baxter model where the water droplets form spheres and reside on the top of such durable dense rough surface (as observed in Fig. 3a,b ) but do not fill up the nanogrooves 44 . The important criteria for the practical uses of the superhydrophobic textiles are their durability and reusability. However, it is observed that the various superhydrophobic materials (silica and other) lose their hydrophobicity in presence of strong acid, alkali and different organic solvents within certain hours. This drop of water repellency could happen due to the instability of the components present in those superhydrophobic materials in strong acidic and alkaline medium, and the non-polar–non-polar interactions in between the materials and organic solvents 12 24 . Whereas this fsZr coating showed outstanding chemical stability after immersion in strong acidic and alkaline solutions ( Fig. 5 ), and different organic solvents ( Supplementary Table S1 ) for a longer period of time. The persistence of superhydtophobicity of the coated cotton fabric even after immersion into strong alkaline medium up to one month was not shown by any research group. In this case, such excellent chemical stability and reusability of the coatings were achieved mainly due to the use of zirconia based (fsZr) coating which is chemically inert as well as strong alkali-acid resistant 28 29 . More elaborately, due to the highest bond dissociation energy and strong covalent nature of Zr–O–Zr compared to Si–O–Si, Ti–O–Ti, Al–O–Al etc, the hydrolysis and dissolution of that network is prohibited at very low and high pH, respectively 26 which facilitate the chemical stability of fsZr coating. Further, the less reactive fluorinated silyl functional groups having lower surface energy than other non-polar components successfully restrict the contact of acidic/alkaline aqueous solutions to the Zr–O–Si linkage side in coating material 32 33 . As a result, the whole coating remained firmly bonded with the cellulose units even under a very harsh chemical environment. The difference between water and oil on this type of surface with special wettability results in one intrinsic application in oil/water separation. Moreover, the textiles could be a better candidate due to their soft and flexible nature. Since the fluorinated silyl functionalized zirconia on fabrics showed excellent water repelling as well as superoleophilic property with enormous chemical stability, we performed the water separation experiments on coated “filter cloth” ( Fig. 6 ). This coated fabric exhibits considerably high water separation efficiency without deteriorating its original WCA and CAH even after several cycles of separations. To the best of our knowledge, few successful superhydrophobic fabrics for oil/water separation have been reported due to the instability of superhydrophobic material in non-polar solvents 12 24 . The excellent stability and reusability of the fsZr coated fabrics under different conditions could provide more opportunities for numerous practical applications. Similarly, this coated fabric retained its superhydrophobicity and high oil/water separation efficiency also after several cycles of sand paper abrasion test ( Fig. 7 ) and standard launderings ( Fig. 8 ). It is noteworthy here that such excellent abrasion and laundering durability as well as reusability of the coating can also be described as the combination of mechanical robustness and chemical inertness of the fabricated zirconia based superhydrophobic coating covalently bonded with cellulose of the fabric. Besides repeated oil/water separations, such washing durability also recommends the utility of these coated fabrics in superhydrophobic garment manufacturing for military, different labs and daily uses. In conclusion, we have demonstrated a simple, new and innovative approach to fabricate an exceptionally stable zirconia based superhydrophobic as well as superoleophilic coating on cotton fabric. The existence of chemically inert and mechanically durable fluorinated silyl functionalized zirconia bonded with cellulose makes the superhydrophobicity of coating material sustainable under severe environment conditions such as high temperature, corrosive solutions, various organic solvents, and mechanical forces for longer period of time. The coated fabric possesses high water separation efficiency even after several cycles of treatment in different conditions. Undoubtly, zirconia present in this fsZr coating plays the key role for generation of such extraordinary stability for day-to-day practical uses which cannot be achievable by any other superhydrophobic composite. Due to these outstanding mechanical and chemical robustness as well as self-cleanable features, these fsZr coated superhydrophobic fabrics could be employed to manufacture oil/water separation apparatus, military suits, lab coats, medical clothing and daily garments. Thus this newly designed fsZr coating has immense potential to being revolution in the field of technical textiles with various functionalities for the benefit of humanity."
} | 1,807 |
33522615 | PMC8048686 | pmc | 6,331 | {
"abstract": "Abstract The phenomenon that organisms can distinguish genetically related individuals from strangers (i.e., kin recognition) and exhibit more cooperative behaviours towards their relatives (i.e., positive kin discrimination) has been documented in a wide variety of organisms. However, its occurrence in plants has been considered only recently. Despite the concerns about some methodologies used to document kin recognition, there is sufficient evidence to state that it exists in plants. Effects of kin recognition go well beyond reducing resource competition between related plants and involve interactions with symbionts (e.g., mycorrhizal networks). Kin recognition thus likely has important implications for evolution of plant traits, diversity of plant populations, ecological networks and community structures. Moreover, as kin selection may result in less competitive traits and thus greater population performance, it holds potential promise for crop breeding. Exploration of these evo‐ecological and agricultural implications requires adequate control and measurements of relatedness, sufficient replication at genotypic level and comprehensive measurements of performance/fitness effects of kin discrimination. The primary questions that need to be answered are: when, where and by how much positive kin discrimination improves population performance.",
"conclusion": "8 CONCLUSIONS AND OUTLOOK Since the pioneering work of Dudley and File ( 2007 ), kin discrimination has been documented to affect many traits in a wide variety of plant species among different taxonomic clades, life forms and origins (see citations in the preceding sections). Many cases have been documented of plants exhibiting more cooperative behaviour towards kin than towards strangers. These behaviours can reduce competition for a common resource pool or expand the availability of resources. While concerns still remain about some of the methods used, it is probably safe to say that belowground kin recognition in plants exists. However, our understanding of the mechanisms has so far been limited, except for the idea that, in general, root exudates play a role in kin recognition. More biochemical and molecular research is needed to identify the chemical cues that associate with or signify genetic relatedness, and how and where this is sensed in plants. Most of the work so far has been conducted with rather limited sets of genetic material and are being limited to (half‐)sibs versus strangers without quantifying genetic relatedness (but see, e.g., Karban et al., 2013 ). Expanding on this will not only help overcome some of the experimental biases that have been associated with research on kin discrimination (see examples of elegant designs from Bhatt et al., 2011 ; Ehlers & Bilde, 2019 ; Semchenko et al., 2014 ) but would also give a much better indication of the level at which plants can detect genetic relatedness. Kin recognition may also have far‐reaching implications for the structure and composition at the community level, as it can modify multi‐trophic interactions and potentially select populations with lower genetic diversity. Many more field experiments are needed to explore these effects. Finally, the existence of kin discrimination has been linked to breeding more cooperative crops that could form more productive stands with higher resource‐use efficiency. More research is needed to determine the extent to which the positive effects indeed exist and whether it would not be more effective to breed crops that express these traits constitutively rather than in a relatedness‐dependent way.",
"introduction": "1 INTRODUCTION Plants often grow in dense vegetation stands, such as grasslands, woodlands, forests or agricultural fields, where there are usually intensive interactions with neighbours. These interactions can occur in the form of resource competition, facilitative habitat modification or communication. The ubiquity and diversity of plant–plant interactions entail that the effects of a given set of traits for an individual cannot be viewed independently of the characteristics of its neighbours (e.g., Cabal, Martínez‐García, de Castro Aguilar, Valladares, & Pacala, 2020 ; Riechert & Hammerstein, 1983 ). Plant–plant interactions in turn also play a key role in driving ecosystem processes such as carbon uptake, water and nutrient balances, interactions with other non‐plant organisms as well as crop yields (see review Anten & During, 2011 ). Much plant ecological research is built on the concept of individual selection, that is, plant traits of a given individual are (at least implicitly) viewed from the perspective of how these traits directly benefit the fitness of that individual. Cooperative behaviour (i.e., actions that benefit the group rather than the individual) and even altruistic behaviour (i.e., actions that benefit other individuals at a cost to the actor) are, however, quite common in nature. To explain the evolution of such ‘non‐selfish’ behaviour, Hamilton ( 1964 ) introduced the idea of ‘kin selection’. The basic concept of kin selection is that if one individual helps another with whom it shares a certain number of alleles, those alleles will be passed to the next generation not only through its own fitness but also through the enhanced fitness of that recipient. An important prerequisite for relatedness‐dependent behaviour (i.e., kin discrimination) to occur, in turn, is kin recognition (i.e., the ability to detect the difference in level of relatedness between oneself and another individual), though, alternatively, kin selection also can be favoured by population viscosity (Waldman, 1988 ). Kin recognition has been demonstrated in a wide variety of organisms including animals (both vertebrates and invertebrates, Waldman, 1988 ), fungi (Malik & Vilgalys, 1999 ) and even bacteria (Smith & Dworkin, 1994 ). However, the idea that it may also occur in plants was long considered outlandish. This scepticism occurred despite the well‐known facts that plants can sense and respond to the presence of other plants, for example, through light signals (Pierik & de Wit, 2014 ) and physical contact (de Wit et al., 2012 ). Plants can even detect the status of neighbours, for example, whether a neighbour is attacked by a herbivore being conveyed through volatiles (Karban, Yang, & Edwards, 2014 ) or whether the neighbour is stressed by drought being conveyed through sounds (Jeong et al., 2014 ). The fact that plants can distinguish between self‐ and non‐self, and are thus capable of some level of identity recognition, has been evident from the observation that many species prevent self‐pollination (Fujii, Kubo, & Takayama, 2016 ). Research indicates that plants may also be able to distinguish between their own roots and those of a non‐self neighbour (see Chen, During, & Anten, 2012 for a review) and between their own herbivore‐induced volatiles and those produced by another plant (Karban & Shiojiri, 2009 ). The first evidence of belowground kin recognition in plants came from a study of the annual Cakile edentula (Dudley & File, 2007 ), finding that plants reduced root allocation (i.e., produced less roots for a given aboveground mass) when interacting with half‐sibs from the same mother rather than with strangers from a different mother. A series of subsequent findings have strengthened the idea that kin recognition and associated kin discrimination occur in plants (Figure 1 ), though there is a heavy debate about methodology used (e.g., Ehlers & Bilde, 2019 ; Klemens, 2008 ; Simonsen, Chow, & Stinchcombe, 2014 , and see details in Section 5 ). Kin discrimination has by now been studied for different traits and functions in a wide variety of wild as well as domesticated species. FIGURE 1 Summary of the physiological mechanisms for kin recognition and the associated kin discrimination effects in plants. Signals and pathways that are proven to mediate belowground kin recognition include root exudates and probably also common mycorrhizal networks. In addition, signals like volatiles and probably also profiles of reflected light can mediate aboveground kin recognition, but they are not the focus of this review. Effects of kin discrimination include not only a reduction of resource competition intensity but also cooperation to increase resource availability, such as more investments in common mycorrhizal network, and probably also in aboveground pollinator attraction (which is not the focus here) [Colour figure can be viewed at wileyonlinelibrary.com ] The existence of kin recognition in plants has important consequences for plant ecology and evolutionary biology, which go far beyond the interesting fact that plants can recognize their kin. First, kin selection may affect the genetic structure and diversity of a population, as it tends to favour lower genetic diversity at the group level (Platt & Bever, 2009 ). Second, cooperative traits tend to lead to greater performance at the population level (Anten & During, 2011 ) and thus may have important implications for ecosystem functioning. This, in turn, may also have implications for agriculture (Murphy, Swanton, Van Acker, & Dudley, 2017 ), since farmers aim for group performance (high yields or resource‐use efficiency at the crop‐stand level) rather than individual performance. It further raises the question, to what extent kin discrimination could contribute to better crop performance and could thus be a breeding target (Anten & Vermeulen, 2016 ; Denison, 2011 ). The objectives of this paper are therefore to (a) critically appraise the evidence for kin discrimination and its underlying mechanisms in plants, (b) discuss potential ecological implications of kin discrimination and (c) discuss the extent to which kin discrimination could be a favourable trait in agricultural crops. While the degree of relatedness/kinship can range from the same genetically identical clone to different phylogenetic clades, we mainly focus on kin recognition at the intraspecific level. We first give a brief introduction to kin selection and related concepts and describe under what conditions it is most likely to occur. We then explore the evidence for kin recognition and discrimination in plants and critically appraise the methods that were used. In so doing, we explore different implications of kin recognition for resource acquisition. The first, and the one having received most attention in the literature, is plants exhibiting less competitive traits by producing smaller, shorter‐lived or less efficient resource harvesting structures when interacting with kin than when interacting with non‐kin. Secondly, we move beyond the direct plant–plant resource competition and explore how kin discrimination in plants may result in increasing the availability of resources to a group of related plants, and show how this can involve interactions with other organisms such as mycorrhizal symbionts. Thirdly, we briefly touch upon the environmental dependency and wider ecological implications of kin discrimination. We also raise concerns about limitations and drawbacks of the methodologies and interpretations in the current research field. Finally, we explore potential applications of kin selection in crops, discussing to what extent kin recognition would be a desirable crop trait."
} | 2,841 |
37741957 | PMC10579274 | pmc | 6,333 | {
"abstract": "High-temperature geothermal springs host simplified microbial communities; however, the activities of individual microorganisms and their roles in the carbon cycle in nature are not well understood. Here, quantitative stable isotope probing (qSIP) was used to track the assimilation of 13 C-acetate and 13 C-aspartate into DNA in 74 °C sediments in Gongxiaoshe Hot Spring, Tengchong, China. This revealed a community-wide preference for aspartate and a tight coupling between aspartate incorporation into DNA and the proliferation of aspartate utilizers during labeling. Both 13 C incorporation into DNA and changes in the abundance of taxa during incubations indicated strong resource partitioning and a significant phylogenetic signal for aspartate incorporation. Of the active amplicon sequence variants (ASVs) identified by qSIP, most could be matched with genomes from Gongxiaoshe Hot Spring or nearby springs with an average nucleotide similarity of 99.4%. Genomes corresponding to aspartate primary utilizers were smaller, near-universally encoded polar amino acid ABC transporters, and had codon preferences indicative of faster growth rates. The most active ASVs assimilating both substrates were not abundant, suggesting an important role for the rare biosphere in the community response to organic carbon addition. The broad incorporation of aspartate into DNA over acetate by the hot spring community may reflect dynamic cycling of cell lysis products in situ or substrates delivered during monsoon rains and may reflect N limitation.",
"conclusion": "Conclusions Carbon cycling in hydrothermal systems has rarely been explored in situ. In this study, we applied qSIP to investigate the utilization of distinct pools of labile DOC, acetate and aspartate, by thermophiles in Gongxiaoshe Hot Spring. The microbial community responded to both substrates, although only aspartate was coupled to net increases in abundance, and most taxa preferentially incorporated aspartate over acetate into DNA, possibly reflecting the dynamic cycling of cell lysis products within the spring ecosystem (e.g., due to viral lysis) or the favorable C:N stoichiometry of aspartate over acetate. We also leveraged rich metagenomic datasets from these springs, which allowed us to go beyond typical qSIP studies that focus on 16S rRNA gene variants to look into phylogenetic and genomic determinants for the utilization of the different labile DOC pools, revealing a correlation between high aspartate assimilation and fast growth rate. The broad utilization of aspartate was congruent with the near ubiquity of the polar amino acid ABC transport system. Fast growth rate and membrane transporters for organic compounds may endow some members of thermophilic communities, including rare taxa, to respond quickly to influxes of organic matter during monsoons or other ecosystem perturbations. Further studies could probe the fates of a more diverse suite of organic compounds to determine whether observed resource partitioning extends to other substrates and whether specialist/generalist paradigms reported in other ecosystems extend to simplified microbial communities that inhabit extreme environments.",
"introduction": "Introduction Dissolved organic carbon (DOC) is an important component of Earth’s carbon cycle. Labile DOC is the most dynamic organic matter pool in any aquatic system and plays an important role in all biogeochemical cycles [ 1 , 2 ]. In the ocean, heterotrophic microorganisms can quickly assimilate and respire labile DOC [ 3 ], and in turn, the growth and activity of planktonic heterotrophs is impacted by the bioavailability and characteristics of labile DOC [ 4 – 6 ]. One study tracked the assimilation of six 13 C-labeled labile substrates into DNA, including acetate and amino acids, by microbial communities in coastal seawater, showing that microorganisms that assimilate specific organic substrates are phylogenetically related [ 7 ]. Such phylogenetic conservation of resource utilization reflects similar distribution patterns among related microorganisms and/or similar lifestyles [ 8 ]. Similar results were obtained using 11 different organic substrates, also in coastal seawater [ 9 ], and the authors proposed two different resource utilization strategies: generalists and specialists. While carbon source assimilation into DNA was either high or low in specialists, generalists incorporated intermediate levels of most or all substrates. Despite numerous studies investigating the roles of specific microorganisms in labile DOC dynamics in marine and other aquatic systems, very little is known about the utilization of labile DOC in hydrothermal systems that may resemble environments in which life first arose [ 10 – 12 ]. In terrestrial hydrothermal systems, DOC can originate from biological sources including nitrogen-depleted allochthonous organic matter from plants and soils, from autochthonous organic matter from thermophilic autotrophs, or from subsurface abiotic processes such as Fischer-Tropsch synthesis and Sabatier-type reactions that require high temperature and pressure [ 13 – 15 ]. Regardless of the source, both fresh and ancient organic materials are susceptible to thermal alteration and decomposition, which also impact labile DOC composition [ 16 , 17 ]. The relative contributions of DOC with allochthonous and autochthonous origins differ among hot springs. Nye and colleagues characterized DOC in 222 terrestrial springs, 30 from the Tengchong hydrothermal region in China and 192 from Yellowstone National Park in the USA [ 18 ]. DOC concentrations ranged from 16 µM to 3 mM, with allochthonous organic matter (i.e., humic-like component) dominating in weakly acidic and circumneutral springs, and low-molecular-weight organic matter (i.e., protein-like component) interpreted to be of hydrothermal origin in alkaline springs with lower DOC concentrations. Humic-like and protein-like components were the main organic carbon signatures in these springs, in addition to an abundant acid-soluble lignin derivative that was exclusively in acidic springs [ 18 ]. Moreover, the amount of allochthonous organic carbon in hot springs can be influenced by seasonal precipitation [ 19 , 20 ], which in turn affects both the quantity and character of labile DOC pools available for heterotrophic thermophiles. Although few studies have assessed labile DOC utilization in terrestrial geothermal springs, whole-community studies suggest heterotrophic activity of thermophiles in situ may be underappreciated. One study of acidic and circumneutral springs in Yellowstone National Park described high rates of formate and acetate mineralization and formate-induced suppression of autotrophy, suggesting that facultative autotrophs and mixotrophs favor organic carbon assimilation [ 21 ]. A separate study found up to 49-fold increases in the instantaneous rate of oxygen consumption by microbial communities in ~80 °C sediments and spring water amended with organic acids or yeast extract and peptone, providing indirect evidence for the use of different organic carbon pools by native communities [ 22 ]. Another study demonstrated mineralization of 13 C-labeled organic compounds, including glucose, citrate, succinate, pyruvate, acetate, and amino acids, in 65 to 95 °C sediment microcosms [ 23 ]. It is worth noting that these studies focused on the bulk community. In contrast, the activities of individual thermophiles in situ have rarely been explored. One method to identify microorganisms that assimilate specific components of labile DOC is stable-isotope probing (SIP). SIP was developed as a tool in microbial ecology over twenty years ago to identify populations actively assimilating labeled compounds of interest into DNA [ 24 , 25 ]. However, traditional nucleic acid SIP is not quantitative because the DNA or RNA is only separated into two fractions – “heavy” (active) and “light” (inactive) – and guanine and cytosine content is not accounted for, such that high-GC organisms can erroneously be identified as active and low-GC organisms can be misidentified as inactive. Recently, quantitative stable isotope probing (qSIP) was developed to address these deficiencies [ 26 ]. qSIP has subsequently been applied to demonstrate that most taxa are active in wet soils [ 27 ], better understand organic matter priming in soils [ 26 , 28 ], illuminate the interactions between soil minerals and bacteria [ 29 ], identify highly active bacterial predators and symbionts [ 30 , 31 ], and probe autotrophy and DOC assimilation in benthic lacustrine sediments [ 32 ]. Yet, to date, qSIP has not yet been applied to probe the functions of microorganisms in extreme environments. In this study, qSIP was applied to assess microbial activity in ~74 °C, pH 7.3 carbonate sediments in Gongxiaoshe Hot Spring during the winter dry season. The geochemistry of Gongxiaoshe Hot Spring has been reported on several different dates [ 19 , 20 , 33 ]. It is relatively stable, with source pool pH and temperatures ranging from pH 7.29-7.7 and 73.8-75 °C. The dissolved oxygen concentration is low (1.5 mg/L), as are dissolved organic carbon (DOC ≤ 1.5 mg/L) and nitrogen (NH 4 + /NH 3 ≤ 0.1 mg/L; NO 2 - /NO 3 - ≤ 0.1 mg/L; total N ≤ 0.4 mg/L). The sediments also have low total organic carbon (TOC ≤ 11.6 mg/g) and very little total organic nitrogen (TON ≤ 0.1 mg/g). Gongxiaoshe Hot Spring is one of many geothermal springs in the Indo-Burma Range of southwest China that are situated in a subtropical climate and are thought to be driven by latent heat from volcanic activity during the Pliocene and Miocene [ 34 ]. Springs in this region are exposed to large influxes of terrestrial organic carbon during the summer monsoon season [ 19 ]. This increase in DOC during the monsoon season is accompanied by large increases in soil mesophiles in the springs, suggesting that monsoon rains deliver both terrestrial soil organic matter and microorganisms to the springs through surface runoff and/or shallow recharge [ 19 , 20 ]. In our experiments, two components of the labile DOC pool – the organic acid acetate and the amino acid aspartate – were used in qSIP experiments to assess the responses of specific thermophiles to pulses of labile DOC. While DOC in geothermal systems is complex and poorly understood, acetate was chosen because it is a key intermediate in the carbon cycle as a product of both primary and secondary fermentations and aerobic processing of complex organic carbon such as plant biomass [ 35 – 37 ] and it is commonly detected in hydrothermal systems [ 21 , 38 , 39 ]. Aspartate was chosen as an intermediate in the degradation of proteinaceous biomass – including microbial necromass made available by cell lysis – and because it is a hub for both catabolic and anabolic pathways, as the key intermediate in the aspartate pathway [ 40 , 41 ]. Aspartate also contains both C and N atoms and could potentially relieve nitrogen limitation. In addition to qSIP, metagenomics was applied to probe the genetic determinants of carbon source utilization. Our results showed strong resource partitioning, a community-wide preference for aspartate incorporation into DNA over acetate, a community-wide increase in biomass only in aspartate-amended microcosms, and near-universal presence of the polar amino acid ABC transporter in the genomes of species that incorporate 13 C atoms from aspartate into DNA, hereafter termed “utilizers”.",
"discussion": "Discussion Sources of acetate and aspartate in geothermal springs The sources and compositions of DOC in geothermal systems are complex [ 18 ]. Organic acids have been measured in high-temperature geothermal water in both acidic and circumneutral springs [ 21 , 38 ] and in interstitial fluids of phototrophic mats, along with polar metabolites such as amino acids, sugars, and nucleotide bases [ 39 ]. Here, acetate and aspartate were chosen to represent two different components of the labile DOC pool to assess the organotrophic potential of a thermophilic microbial community. Acetate is a key metabolite for both intra- and intercellular metabolism and can be found universally in both oxic and anoxic environments, including geothermal springs [ 35 – 37 ]. Acetate could be produced biologically by microbes through fermentation or overflow metabolism during heterotrophic growth or by leakage of photosynthate by photoautotrophs [ 85 ]. Indeed, acetate is a major metabolite in hot spring chlorophototrophic mats [ 39 , 86 ] that could supply DOC to microbes in hotter parts of spring systems, and acetogenesis from HCO 3 - was detected in hot springs over a wide pH (3.5–8.5) and temperature range (60–80 °C) [ 87 ]. Thus, acetate could be both allochthonous or autochthonous in origin. Aspartate, a negatively charged amino acid, could be generated through cell lysis caused by viruses [ 88 ] or other mechanisms leading to cell lysis, such as parasitism or predation. But, unlike acetate, aspartate provides both carbon and nitrogen. The importance of aspartate for thermophiles Our results showed higher bulk incorporation of aspartate over acetate into community DNA. Aspartate was also used more widely by the microbial community and was coupled to increases in the abundance of microorganisms in aspartate-amended microcosms and the specific taxa that incorporated it. Together, these data may suggest a broader importance of amino acid metabolism over organic acid metabolism in high-temperature systems. This result was somewhat surprising because acetate is a well-known carbon and energy source for prokaryotes [ 89 ] that can be readily converted to the fundamental metabolite acetyl-CoA via ACK-PTA or ACS, and because organic acids have been shown to be metabolized by cultivated thermophiles [ 90 – 93 ] and in situ in terrestrial geothermal springs [ 21 , 22 ]. One possible explanation for the poor incorporation of acetate into DNA is that the acetate switch may have occurred because the initial concentration of acetate in our samples (~3.2 mM) was similar to the minimum acetate concentration known to induce the acetate switch (~1 mM) [ 94 , 95 ]. However, this acetate concentration is lower than the concentrations that support the growth of many hyperthermophiles (6.1 to 12.2 mM) [ 90 – 93 ]. Another possible explanation is that acetate may have been assimilated broadly, but incorporated into other macromolecules, such as bacterial lipids. This caveat extends to all SIP approaches (e.g., DNA, RNA, lipids). While it is probable that different results would be obtained by examining other macromolecules, those would also suffer from biases according to the metabolism of each community member. Ultimately, the increase in 16S rRNA gene copy number and the strong positive correlation between aspartate incorporation into DNA and change in ASV abundance argue that there is indeed a community preference for aspartate in these experiments. Finally, it is possible that acetate amendment may have led to N starvation given that dissolved inorganic N concentrations are low in Gongxiaoshe Hot Spring [ 19 , 20 ], so that acetate addition could result in the uncoupling of acetate assimilation from growth. On the contrary, aspartate has a C:N stoichiometry of 4:1, which is close to the Redfield ratio [ 96 ] and may be more favorable for growth of heterotrophic thermophiles. This interpretation is consistent with the heavy labeling of DNA from ammonia-oxidizing archaea (i.e., Candidatus Nitrosocaldus [ 97 ]) and nitrite-oxidizing bacteria (i.e., Candidatus Nitrotheca and Candidatus Nitrocaldera [ 98 ]) only in aspartate-amended incubations, which could reflect deamination of aspartate followed by oxidation of the resulting ammonia. Aspartate is an important substrate for thermophiles. First, aspartate is the central metabolite in the aspartate metabolic pathway and is therefore a central metabolite for protein synthesis, nucleotide metabolism, tricarboxylic acid cycle, glycolysis, and other biosynthetic pathways [ 40 , 41 ]. It can be converted into pyruvate and other intermediates in the TCA cycle, generating free energy for microbial growth. Additionally, aspartate is an important compatible solute to manage heat stress [ 99 ], and part of a large cytoplasmic pool of aspartate could flux into DNA synthesis [ 100 ]. However, it should be noted that our experiments do not rule out an important role for acetate, because acetate could be preferentially incorporated into other macromolecules such as bacterial lipids [ 101 , 102 ]. 13 C-acetate assimilation into lipids has previously been exploited to identify acetate utilizers in alkaline hot springs [ 38 ]. Heterotrophy among thermophiles Although high-temperature ecosystems are often discussed as ‘chemoautotrophic systems’ [ 103 ], our experiments show that many thermophiles readily assimilated aspartate and/or acetate. Some of the highly labeled taxa are well-known for their broad heterotrophic activities, such as Thermus [ 104 ], and members of the Desulfurococcaceae , including Ignisphaera [ 105 , 106 ]. In contrast, although some members of the Aquificae were highly active assimilating both substrates, they are often thought of in the context of primary production. Yet, the assimilation of labile DOC, observed here in situ, is consistent with broad heterotrophic activity of some members of the Aquificaceae [ 107 – 109 ] and a member of the Aquificaceae isolated from nearby springs that uses acetate as an electron donor [ 93 ]. Other taxa that were highly labeled belong to uncultivated groups lacking known autotrophic pathways, such as Candidatus Fervidibacter [ 110 ] and Candidatus Kryptobacter [ 111 ]. The assimilation of aspartate by Candidatus Micrarchaeota is consistent with previous reports that some members of this phylum have the genomic capacity to utilize amino acids [ 112 ]. Role of low-abundance taxa in geothermal systems and caution interpreting SIP data The rare biosphere is often thought to be a transient or persistent “seed bank” of cells that is not fit to grow in a particular environment under the conditions in which it was sampled [ 113 ]. Yet, in our study, some low-abundance ASVs had the highest amounts of isotope incorporation, while the most abundant species had low isotope incorporation rates. This uncoupling of activity from cell abundance has been noted in other environments, such as the coastal marine pelagos, where low cell respiration rates for abundant Pelagibacter species were noted [ 114 ], despite other studies reporting high in situ rates of Pelagibacter growth, protein synthesis, and substrate assimilation [ 115 ]. The authors [ 114 ] suggested that the low respiration rate by Pelagibacter may not reflect low activity in general but instead the preferential use of rhodopsins for ATP synthesis over respiration. By extension, caution is warranted not to equate substrate incorporation into DNA in any stable isotope probing study to activity in general. For example, in our study, we measured high rates of incorporation of C atoms from aspartate into DNA of a low-abundance Thermocrinis ASV, but that same ASV did not incorporate C atoms from acetate into DNA. Some species of Thermocrinis use acetate as a carbon source [ 109 ], and 13 C atoms from acetate have previously been traced into Thermocrinis lipids in situ in streamer communities [ 38 ]. Acetate incorporation into lipids is expected given the key role of acetate as a building block for acyl chains of bacterial lipids, but not archaeal lipids. Thus, we urge caution not to overinterpret low isotope incorporation because the choice of substrates may influence which taxa assimilate the substrate and how the substrate is processed in each species into macromolecules. These caveats regarding low activity withstanding, the high activity of some members of the rare biosphere in our study is still remarkable. In our study, all of the microorganisms with high substrate assimilation rates (AFE > 0.2) were rare in the community at the time of sampling (<0.5%), and while some became more abundant during the corresponding incubation ( Micrarchaeales (J92), Ignisphaera (J21), and Acetothermiia (J56)), others did not. Other studies have also noted increases in the abundance of some rare taxa in response to environmental perturbations [ 116 ], which can be interpreted in the context of relieving growth limitations. On the other hand, other studies have noted poor correlations between activity and abundance [ 117 ], which may be due to viral lysis, parasitism, or predation of the most active microorganisms. Indeed, many of the active taxa in our study belonged to the Patescibacteria and Micrarchaeota , which are typically inferred to be epibiontic symbionts of other prokaryotes, possibly acting as parasites or predators [ 118 , 119 ], although some studies have challenged this broad interpretation [ 120 ]. Ultimately, our study reveals a complex relationship between activity and abundance that requires further study, particularly in geothermal systems."
} | 5,288 |
22624091 | PMC3354671 | pmc | 6,337 | {
"abstract": "Human impact on biodiversity usually is measured by reduction in species abundance or richness. Just as important, but much more difficult to discern, is the anthropogenic elimination of ecological interactions. Here we report on the persistence of a long ecological interaction chain linking diverse food webs and habitats in the near-pristine portions of a remote Pacific atoll. Using biogeochemical assays, animal tracking, and field surveys we show that seabirds roosting on native trees fertilize soils, increasing coastal nutrients and the abundance of plankton, thus attracting manta rays to native forest coastlines. Partnered observations conducted in regions of this atoll where native trees have been replaced by human propagated palms reveal that this complex interaction chain linking trees to mantas readily breaks down. Taken together these findings provide a compelling example of how anthropogenic disturbance may be contributing to widespread reductions in ecological interaction chain length, thereby isolating and simplifying ecosystems.",
"discussion": "Discussion Ecological interaction chains in the native forests of Palmyra connect processes in these forests to the ecology of manta rays through a diverse series of trophic, non-trophic (behavioral), and physical mechanisms. Native trees provide needed nesting/roosting habitat for seabirds and thus help to maintain high local abundances of seabirds. These seabirds vector large quantities of marine-derived materials into the nutrient impoverished atoll terrestrial communities defining biogeochemical patterns of both plants and soils in native forest areas. Many of the nutrients concentrated in these native forests are returned to the adjacent oligotrophic ocean waters via rain and tidal vectoring. Sampling of the plankton communities directly along these native forest coastlines revealed that phytoplankton are more productive, zooplankton are more abundant, and key zooplankton taxa achieve larger sizes. The most parsimonious explanation for these observed patterns in the plankton is that they are responding to the forest-facilitated, seabird-vectored nutrient additions. The last key observation that we made in these native forest associated habitats was that manta rays, which feed exclusively on plankton, were more abundant and active along these plankton rich native forest coasts. While manta rays are wide ranging animals, this attraction to and persistence along these native forest coasts represents an important and unexpected link between their foraging ecology and forest dynamics. The detectable presence of seabird-derived δ 15 N materials in terrestrial, intertidal, subtidal, and pelagic organisms situated along this interaction pathway provides compelling support that this is indeed a unified long chain of dependant interactions. Sampling of other potential N sources in this system has revealed no evidence of alternative allochthonous or autochthonous origin materials which could have otherwise created these δ 15 N patterns 14 . This series of connections defines one of the longest ecological interaction chains yet observed in nature 20 21 22 23 . Other work has demonstrated that the majority of species involved in ecological interactions are only two links apart 24 . The interaction chain linking trees to manta rays in Palmyra's native forests is at least five linkages long. The circuitous architecture of this particular interaction chain is as noteworthy as its length. This interaction presents an interesting route through which oceans affect change on land, and changes on land can feed back to influence ecological processes in the oceans ( Figure 1 ). Reports of unidirectional transboundary ecological connections have garnered much attention 25 26 – but this example of a complex bi-directional interaction adds to our understanding of the degree to which ecosystems can be interconnected. This interaction includes an interesting mix of both top-down (i.e. loss of birds affects plant and soil ecology) and bottom-up ecological effects (i.e. increases in bird-derived nutrients appears to increase plankton abundance). Instances of complex top-down and bottom-up interactions may be quite common in nature, but good empirical examples of these dynamics are as yet still emerging 27 . We posit that this long interaction chain present in and near the native forests of Palmyra is maintained by the relative lack of human disturbance in the better protected parts of this unusually remote site. Data collected from our altered palm sites support this conclusion by demonstrating that forest alteration severely degrades the efficacy of this series of interactions ( Figure 1 ). The corruption of these interactions very likely has a major negative affect upon the strength of the cross-taxonomic and cross-system connections that they supported. Observations from other systems suggest that many complex ecological interaction chains and associated sources of connectivity may be similarly vulnerable to anthropogenic perturbation. The introduction of non-native predators to Aleutian Islands caused the disintegration of ecologically important sea to land nutrient connections 20 . Intense increases in nutrient inputs associated with human sewage and agriculture contributed to the collapse of the biologically, structurally, and interactively complex coral reef communities in Kaneohe Bay, Hawaii 28 . Overfishing of salmon in the Pacific Northwest USA compromised the transfer of nutrients from marine to freshwater ecosystems affecting terrestrial plant and animal communities in a variety of ways 26 29 . While numerous other such examples exist, anthropogenic disturbances should not universally be expected to cause contractions in ecological interaction chain length or reductions in system connectivity. The character of the disturbance in question as well as the properties of the recipient system will both determine the final effects that human change has on networks of ecological interactions. However, because many sources of anthropogenic change have the effect of rapidly altering the overall and relative abundance of particular species, directly removing species, introducing species foreign to established ecological interaction networks, eliminating habitat, and changing the physical and chemical properties of local environments – we argue that human-induced constrictions or eliminations of ecological interaction series are likely to have occurred and be occurring much more commonly than is presently appreciated. Recognizing the effects on anthropogenic activities on ecological interaction chains is more difficult than documenting more tangible disturbance effects (e.g. species extinctions or introductions) because interactions between species and ecosystems do not fossilize and leave little material evidence behind to chronicle their disappearance. However, observations made in more-intact ecosystems, such as those reported herein, help bring these losses in interaction chain length to light and highlight the implications that this type of environmental change may be having upon ecosystem connectedness. Sustained investigation of our remaining uniquely pristine environments will help to extend our understanding of the ubiquity and importance of this intangible, but potentially important type of shifting baseline."
} | 1,842 |
25505459 | PMC4243572 | pmc | 6,338 | {
"abstract": "In many ecosystems, global changes are likely to profoundly affect microorganisms. In Southern California, changes in precipitation and nitrogen deposition may influence the composition and functional potential of microbial communities and their resulting ability to degrade plant material. To test whether such environmental changes impact the distribution of functional groups involved in leaf litter degradation, we determined how the genomic diversity of microbial communities in a semi-arid grassland ecosystem changed under reduced precipitation or increased N deposition. We monitored communities seasonally over a period of 2 years to place environmental change responses into the context of natural variation. Fungal and bacterial communities displayed strong seasonal patterns, Fungi being mostly detected during the dry season whereas Bacteria were common during wet periods. Most putative cellulose degraders were associated with 33 bacterial genera and predicted to constitute 18% of the microbial community. Precipitation reduction reduced bacterial abundance and cellulolytic potential whereas nitrogen addition did not affect the cellulolytic potential of the microbial community. Finally, we detected a strong correlation between the frequencies of genera of putative cellulose degraders and cellulase genes. Thus, microbial taxonomic composition was predictive of cellulolytic potential. This work provides a framework for how environmental changes affect microorganisms responsible for plant litter deconstruction.",
"introduction": "INTRODUCTION Establishing the connection between community structure and function has been a longstanding yet elusive goal in microbial ecology. Making such a connection is especially critical for predicting how communities and functions will respond to global environmental change ( Allison and Martiny, 2008 ; Trivedi et al., 2013 ). Meeting this challenge depends on linking traits that control responses of microbial taxa to the environment (“response” traits) with those that determine ecosystem function (“effect” traits; Lavorel et al., 1997 ; Gross et al., 2009 ; Webb et al., 2010 ). This linkage has been elusive due to the difficulty of isolating microorganisms and characterizing their traits in complex communities. The advent of high-throughput – omics approaches offers a means of linking response and effect traits in microbial communities. When coupled with experimental manipulations of environmental conditions, (meta)genomic approaches can provide a window into both microbial community responses and concurrent changes in functional potential. Local communities are increasingly being confronted by global-scale environmental changes. For instance, drought and nitrogen deposition are predicted to affect many ecosystems ( Cook et al., 2004 ; Hole and Engardt, 2008 ; Fenn et al., 2010 ; Seager and Vecchi, 2010 ). Nitrogen deposition is known to change plant diversity and to increase primary production ( Lamarque et al., 2005 ; Cleland and Harpole, 2010 ), whereas drought can lead to reduced primary production and plant diversity ( Mueller et al., 2005 ). Previous studies have shown that microorganisms also respond to changes in water availability ( Pesaro et al., 2004 ; Sheik et al., 2011 ; Cregger et al., 2012 ; Barnard et al., 2013 ; Bouskill et al., 2013 ). Similarly, N-addition can promote the growth of copiotrophic microbes, whereas other lineages may be negatively affected ( Fierer et al., 2012 ; Philippot et al., 2013 ). However, we currently have a limited understanding of how environmental changes directly impact the genomic diversity of microbial communities and their associated functional potential. Plant polymer degradation is a key microbial function that channels plant litter into microbial biomass, where it can be mineralized to CO 2 or stabilized as soil carbon ( Cebrian, 1999 ). Cellulose is one of the most abundant polymers in plant litter, and therefore the breakdown of this compound is a key step in the decomposition of plant material. Cellulose-degrading microbes produce cellulase enzymes that catalyze the first step of cellulose hydrolysis and release oligosaccharides that are accessible for many other lineages ( Lynd et al., 2002 ; Goldfarb et al., 2011 ; Berlemont and Martiny, 2013 ). Cellulases belong to glycoside hydrolases (GH) families 5, 6, 7, 8, 9, 12, 44, 45, and 48 ( Berlemont and Martiny, 2013 ). These enzymes are frequently associated with carbohydrate binding modules (CBMs) assumed to increase cellulose hydrolysis. In both isolated microorganisms and complex microbial communities, the redundancy of seemingly similar proteins is assumed to promote synergistic interactions among enzymes with varying regulatory mechanisms and/or biochemical properties (substrate specificity, pH, etc.; Wilson, 2011 ). Bacteria that carry cellulase genes are commonly associated with specific genera within the phyla Acidobacteria , Actinobacteria , Proteobacteria , Bacteroidetes , and Firmicutes ( Haichar et al., 2007 ; Ulrich et al., 2008 ; Schellenberger et al., 2010 ; Goldfarb et al., 2011 ; Barnard et al., 2013 ; Berlemont and Martiny, 2013 ). The initial enzymatic breakdown of cellulose typically results in the release of oligosaccharides like cellobiose. To use oligosaccharides, microorganisms need to express β-glucosidase, which is associated with GH families 1 and 3. A recent genomics analysis suggests that more than 80% of sequenced bacterial lineages carry β-glucosidase ( Berlemont and Martiny, 2013 ) and therefore most lineages may opportunistically benefit from the enzyme production of cellulose degraders. In arid and semi-arid ecosystems, cellulose degradation and litter decomposition rates may depend on the responses of cellulose-degrading lineages to water and nutrient availability. Previously at our study site in a semi-arid California grassland ecosystem, experimentally induced drought significantly reduced litter decomposition rates and bacterial biomass ( Allison et al., 2013 ). Likewise, decomposition rates and bacterial biomass were markedly lower during the summer dry season. Nitrogen addition had weaker effects on litter decomposition, but there was some evidence for adaptation of microbial communities to nitrogen availability. These results raise the question of whether changes in the abundance of cellulose-degrading lineages contributed to changes in overall litter decomposition rates. To understand how cellulose degradation might respond to environmental changes, we identified the microbial metagenomic content in leaf litter across seasons and under experimentally manipulated water and nitrogen availability. Specifically, we aimed to characterize the genetic diversity of the leaf litter microbial community, the organisms carrying cellulases, and cellulase genes. We hypothesized that most of the cellulolytic potential (i.e., the collection of detected genes coding for cellulases) would be associated with fungal lineages, consistent with past studies of leaf litter ( Schneider et al., 2012 ). Next, we investigated how cellulolytic potential responded to seasonal and experimental precipitation reduction in this environment. We hypothesized that cellulolytic traits would be correlated with responses to precipitation and nitrogen availability owing to physiological tradeoffs. Enzyme expression requires cellular resources, particularly nitrogen, so cellulolytic traits should be more prevalent in nitrophilic, copiotrophic taxa ( Treseder et al., 2011 ). In contrast, cellulolytic traits should correlate negatively with drought tolerance due to the high resource cost associated with cell walls, osmolytes, and other tolerance traits. Finally, we evaluated if the environmental responses of microbial lineages were correlated with changes in cellulolytic potential. We hypothesized that the frequency of cellulolytic traits in the microbial community is predictable based on the taxa present in the community (e.g., at the genus level). Such a relationship is expected if cellulolytic traits are phylogenetically conserved and would be useful for linking cellulose degradation with other traits conserved among microbial taxa ( Allison and Martiny, 2008 ; Berlemont and Martiny, 2013 ; Martiny et al., 2013 ; Zimmerman et al., 2013 ).",
"discussion": "DISCUSSION PATTERNS IN DIVERSITY AND CELLULOLYTIC POTENTIAL Generally, Fungi are assumed to be the most active microorganisms in litter deconstruction ( Boer et al., 2005 ; Deacon et al., 2006 ; van der Wal et al., 2013 ). For example, a recent proteomic analysis suggests that Fungi express many hydrolytic enzymes ( Schneider et al., 2012 ), and another study shows the incorporation of labeled substrates into mainly fungal biomarkers ( van der Wal et al., 2013 ). Here, we observe that the majority of sequences for GHs in grassland leaf litter metagenomes are associated with bacterial lineages. This result is consistent with relatively high bacterial biomass in the leaf litter from Loma Ridge ( Alster et al., 2013 ) and the representation of bacterial GHs in fosmid libraries derived from the same litter and shown to be active on plant polymers ( Nyyssönen et al., 2013 ). As described for forest litter ( Stursová et al., 2012 ), the microbial communities involved in cellulose deconstruction in grassland leaf litter may also be enriched in bacteria due to differences in leaf litter chemistry and climatic conditions. Thus, cellulolytic bacteria may be important for litter decomposition. We detected sequences in leaf litter communities affiliated with every known cellulolytic GH family. In contrast, cellulases are less diverse in microbial communities involved in plant cell wall deconstruction in the cow rumen ( Hess et al., 2011 ), a fungus garden ( Suen et al., 2010 ), or the termite hindgut ( Warnecke et al., 2007 ). Leaf litter potentially contains more complex and heterogeneous substrates and displays more fluctuations in environmental parameters (e.g., substrate chemistry and availability, temperature, and water/oxygen availability). This variation may lead to genetically and functionally diverse communities. Many of the bacterial potential cellulose degraders we identified are commonly involved in cellulose deconstruction including Streptomyces ( Semêdo et al., 2000 ), Sorangium ( Hou et al., 2006 ), Myxococcus ( Bensmail et al., 1998 ), Acidothermus ( Barabote et al., 2009 ), and Thermobispora ( Anderson et al., 2012 ). In most cases, these lineages are associated with cellulolytic activity resulting from the expression of multiple and diverse genes for cellulases ( Wilson, 2011 ). In agreement with the CAZY-genome database ( Lombard et al., 2014 ), some less frequent potential cellulose degraders harbor a higher frequency of reads for putative cellulases including Herpetosiphon aurantiacus , Saccharophagus degradans , and Sorangium cellulosum . This suggests that potential cellulolytic lineages that are less frequent may have an impact on litter deconstruction as shown for some specific fungal taxa ( Deacon et al., 2006 ). On the other hand, the most abundant potential cellulose degraders (e.g., Streptomyces ) display a lower frequency of cellulase sequences. This result is in good agreement with the hypothesis that reducing the number of cellulase genes in bacteria may reduce the cost of enzyme production and allow a higher growth rate ( Allison, 2012 ). We speculate that such a high variability of the cellulolytic potential, together with other adaptations [i.e., filamentous growth of Streptomyces ( Boer et al., 2005 ) and cellulolosome production of Saccharophagus and Clostridium ( Sabathe et al., 2002 ; Taylor et al., 2006 )] increases the decomposition of plant material by providing synergistic biochemical activities that target different fractions of the substrate ( Hättenschwiler et al., 2011 ; van der Wal et al., 2013 ). Sequences for putative β-glucosidases in bacterial potential cellulose degraders account for 52% of the detected sequences from GH families 1 and 3. As previously suggested, it is likely that these enzymes are broadly distributed in bacteria including many non-cellulolytic lineages (i.e., opportunists or cheaters; Berlemont and Martiny, 2013 ). Indeed, many bacterial lineages, including some non-degraders, are stimulated when subjected to labile oligosaccharides ( Goldfarb et al., 2011 ). As a consequence, β-glucosidase activity in environmental samples is likely a poor proxy for the degradation of complex cellulose polymers. However, opportunists may contribute indirectly to plant litter degradation by processing cellulose deconstruction byproducts. MICROBIAL RESPONSE TO ENVIRONMENTAL MANIPULATIONS Our data are consistent with prior findings suggesting that Fungi are less negatively affected by low water availability than bacterial populations and are thus more frequent under seasonally occurring or experimental drought ( Allison et al., 2013 ; Barnard et al., 2013 ; van der Wal et al., 2013 ). Indeed, experimental precipitation reduction marginally, but significantly, increases fungal relative abundance. At our site, this increased frequency of reads from fungi under dry conditions is likely to result from a reduction in bacterial biomass ( Allison et al., 2013 ). Although most bacterial relative abundances declined under low water availability, there was some variation in response at the genus level within the Actinobacteria and Firmicutes . In some genera within these phyla, tolerance to desiccation is likely achieved through multiple strategies to survive reduced water potential [e.g., production of a surfactant in Bacillus ( Straight et al., 2006 ), exopolysaccharides in Pseudomonas ( Roberson and Firestone, 1992 ), osmolytes in Cyanobacteria ( Rajeev et al., 2013 )]. LINKS BETWEEN TAXONOMY AND FUNCTION Across seasons and treatments, bacterial responses to reduced precipitation and nitrogen addition were not directly correlated with cellulolytic potential. However, the ratio of Bacteria to Fungi was reduced under seasonal drought and experimental precipitation reduction. Fungi have relatively few cellulases, and litter decomposition experiments show that litter decay rates are lower during seasonal drought periods and in the reduced precipitation treatment ( Allison et al., 2013 ). This pattern could be interpreted as a reduced role of Fungi in comparison to Bacteria in litter decomposition in this ecosystem, but further studies are needed to quantify the contribution from the two groups. Also contrary to our physiological tradeoffs hypothesis, copiotrophic Bacteria that were favored in nitrogen-enriched plots did not show higher genetic cellulolytic potential. Consistent with our initial hypothesis, the genetic potential for cellulose deconstruction was highly predictable based on the taxonomic composition of the microbial community. This finding aligns with the previously described conservatism at the genus-species level of genes for cellulases in sequenced bacterial genomes ( Berlemont and Martiny, 2013 ). Our current study generalizes this pattern to diverse communities containing poorly sequenced (e.g., Acidobacteria and Chloroflexi ) or hyper-variable taxa (e.g., Bacillus ; Berlemont and Martiny, 2013 ), thereby reducing the dependence of trait prediction on sequenced genomes. Furthermore, a phylogeny-function relationship allows for the prediction of the cellulolytic potential in taxonomically resolved communities. The sequencing of metagenomes involves some important potential limitations. The approach may have a bias due to variations in extraction efficiency among different lineages. Also, poorly characterized bacterial phyla (e.g., Acidobacteria ) and complex genomes from fungi are unevenly detected in metagenomic studies due the reliance on a small number of previously annotated genes and genomes. Thus, the frequencies and metabolic potentials of phyla with few genome sequences are likely underestimated. In addition, we specifically recognize that some genes from GHs identified as potential cellulases may possibly have other enzymatic functions ( Berlemont and Martiny, 2013 ; Nyyssönen et al., 2013 ). In addition, some enzymes identified as cellulases may be involved in cellulose biosynthesis or in the interaction between microorganisms and plants ( Medie et al., 2012 ; Berlemont and Martiny, 2013 ). For example, genera described as plant pathogens [e.g., Clavibacter ( Jahr et al., 2000 )] or plant growth promoting rhizo-bacteria [e.g., Rhizobium ( Robledo et al., 2008 )] may not contribute to cellulose degradation. As is the case for most metagenomic analyses, these biases may influence our results in unknown ways as most traits have not been fully characterized genetically and/or biochemically. Despite these caveats, our metagenomic approach provides a powerful tool for linking microbial community composition and potential function under environmental change. When combined with experimental confirmation of biochemical function ( Nyyssönen et al., 2013 ), highly robust linkages between microbial composition and ecosystem processes may be achieved. Our data show that fungi are drought resistant, but they are likely not the primary contributors to the cellulolytic potential in this grassland litter community. Rather, changes in cellulolytic potential due to seasonality and experimental precipitation reduction are driven by the dynamics of bacterial taxa that are highly sensitive to drought. Together with previous study ( Allison et al., 2013 ), these results suggest that drought-associated reductions in litter decay and cellulose deconstruction may be related to shifts in microbial community composition and not simply direct moisture limitation. Under nitrogen addition, litter decay is not likely to increase through effects on cellulolytic potential. Describing the effect of precipitation reduction and nitrogen deposition, across seasons, on microbial communities involved in plant material deconstruction is a prerequisite for future investigation of combined effects of these perturbations. More broadly, the phylogenetic conservatism of functional traits and the response of microbial taxa to simulated environmental changes provide a robust conceptual framework to predict how microbial communities will respond to global changes and impact ecosystem functioning."
} | 4,620 |
26527720 | PMC4702813 | pmc | 6,341 | {
"abstract": "MetaNetX is a repository of genome-scale metabolic networks (GSMNs) and biochemical pathways from a number of major resources imported into a common namespace of chemical compounds, reactions, cellular compartments—namely MNXref—and proteins. The MetaNetX.org website ( http://www.metanetx.org/ ) provides access to these integrated data as well as a variety of tools that allow users to import their own GSMNs, map them to the MNXref reconciliation, and manipulate, compare, analyze, simulate (using flux balance analysis) and export the resulting GSMNs. MNXref and MetaNetX are regularly updated and freely available.",
"conclusion": "CONCLUSION The www.metanetx.org resource provides a comprehensive suite of tools for the analysis of genome-scale metabolic models, based on a single integrated namespace of metabolites and metabolic reactions that integrates the most widely used biochemical databases and model repositories – MNXref. The reconciliation process used in MNXref greatly simplifies the development and analysis of genome-scale metabolic models, allowing users to concentrate on model analysis rather than the time-consuming problem of identifier mapping. Future developments will include the provision of tools and the integration of new resources such as the SwissLipids knowledgebase ( 17 ), which provides lipid structures and curated data on enzymatic reactions.",
"introduction": "INTRODUCTION A genome-scale metabolic network (GSMN), or stoichiometric model, describes the set of biochemical reactions which may occur in a given organism, as well as the requisite enzymes, and may also include information on sub-cellular compartments, transport reactions and transporters. By design GSMNs are focused on the metabolism of small molecular weight compounds when energy and mass conservation law can be applied, and are not suited to represent gene regulation or signaling pathways. In practice, a GSMN has a double purpose, as it is both a repository of knowledge about an organism's metabolism, and a model that can be simulated, using flux balance analysis (FBA). Such simulations can address different questions: (i) establish the essentiality of genes in specific growth conditions; (ii) reveal opportunities for metabolic engineering and optimization; (iii) suggest new drug targets ( 1 ). To permit simulations, a GSMN usually includes artificial reactions that describe the growth medium, a growth equation (which implies the composition of the biomass) and possibly hypothetical reactions not (yet) supported by experimental biology but required to make a model functional. A relatively small number of high quality GSMNs have been published to date, essentially for model organisms, and are made available by a few dedicated databases ( 2 – 6 ). The development of such models requires significant human effort and curation, and the fully automated reconstruction of a GSMN from an annotated genome sequence remains a challenge ( 7,8 ). Such methods require the integration of high quality curated data covering the known biochemistry of a vast range of organisms, as well as methods that address the specific requirements of a functional GSMN, including the elemental balancing of individual reactions. These considerations form the major motivation for the development of the resource presented here."
} | 824 |
37583477 | PMC10423689 | pmc | 6,343 | {
"abstract": "Highlights • Report of a high throughput screening (HTP) method to identify efficient lignin degrading microbes from large number of biomasses. • HTP screening process of large number of natural biomasses based on liquid culture and specific enzyme activities. • Inclusion of a quantitative 96-well plate assay in the primary screening process resulted in isolating efficient lignin degrading consortia. • Promising microbial consortium gained through HTP screening included bacterial strains : Serratia, Enterobacter, Raoultella and Bacillus and fungal counterparts included: Mucor, Trametes, Conifera and Aspergillus . • Application of the developed HTP process in practical isolation of new ligninolytic strains for bioprocessing and bioremediation initiatives.",
"conclusion": "4 Conclusion Innovative screening techniques allow the isolation of microbes and enzymes that have previously eluded detection and extend the capability to discover novel enzymes. Essential to this endeavor, an HTP screening technique using a modified culture enrichment method for screening and selection of microbial strains from a large number of natural biomasses based on laccase activities was proposed and demonstrated in the study. This two-step strategy quickly identified promising consortia and isolated individual strains for efficient lignin and lignocellulose valorization, resulting in the identification of ten new bacterial and five new fungal strains with high lignin-degrading potential, including Aspergillus sydowii and Raoutella ornitholytica with the highest laccase activities. Moreover, the study evaluated the capability of the selected strains to produce other hydrolyzing enzymes, showing promising results for reducing enzyme costs in lignocellulosic biorefinery processes and discovering new enzymes with novel activities for the valorization of not only woody biomass but also biological waste, crops, and grains.",
"introduction": "1 Introduction Novel and innovative screening approaches are uncovering the existence of a wide range of previously unknown enzyme activities from active microbial communities distributed in nature. Biomass conversion utilizes microorganisms and their enzymes, such as cellulases, xylanases and ligninases, to transform lignocellulose into platform chemicals which can then be converted to various products, including biofuels, chemicals, and materials. Microorganisms that can effectively break down biomass in high yields are vital for achieving sustainable and economically viable production of value-added products from lignocellulosic feedstock [ [1] , [2] ]. Lignin is an abundant natural phenolic polymer with immense potential to be valorized into high-value bioproducts after degradation into short oligomers or monomeric subunits. More so, biological and enzymatic degradation of lignin has the potential to advance sustainable green biotechnology as a highly selective and environmentally benign substitute for conventional fossil-based fuel production [ [3] , [4] , [5] ]. Indeed, adequate removal of lignin from green waste, a vital byproduct in the paper industry and biofuel manufacture, occupies the center stage in lignin biodegradation and bioremediation initiatives [ 6 ]. In plant cell walls, cellulose, hemicellulose, and lignin are united in a tight embrace, and denuding them of lignin provides microbial consortia with larger accessibility for cellulose bioprocessing. The lignin separated from woody biomass is converted into target compounds like vanillin, polyhydroxyalkanoates, lipids, and cis-muconic acid, rendering it a promising feedstock. However, the structural complexity of lignin and expensive enzymes are the two main obstacles that reduce the process’ efficiency and make it unprofitable [ 7 ]. Microbes that break down lignin can produce a range of enzyme combinations comprising several types of lignin-degrading enzymes, such as laccases, manganese peroxidases, and lignin peroxidases [ 8 ]. Proficient lignin degradation can be achieved by the concerted action of multiple enzymes that work in cascade reactions which enhance the saccharification of polysaccharides of lignocellulosic biomass. Screening of microorganisms with significant lignin degradation ability allows the opportunity to discover and characterize novel enzymes that potentially allow low-cost biological processes. However, discovering lignin-degrading microorganisms with a high performance largely depends on the screening strategy. A variety of microbial screening approaches and strategies were employed and adapted to identify novel enzymes for specific functionalities and targets [ [9] , [10] , [11] , [12] ]. Culture-dependent screening strategies mainly utilize a culture enrichment method which involves growing microorganisms in a cultivating medium with lignin as the carbon source, and the screening is solely activity based without relying on the prior knowledge of the enzyme. After the culture enrichment process, the most common practice is to perform a qualitative screening using an agar plate containing dyes resembling lignin fragments, followed by a quantitative screening of selected microbes based on activity [ [13] , [14] , [15] , [16] ,]. The agar plate screening method is laborious, time-consuming, has low throughput, and lacks precision as it may not accurately detect the target enzyme. Submerged screening methods, on the other hand, offer better control over the culture conditions, which can lead to higher enzyme production. They also allow the possibility for high throughput (HTP) screening of a vast number of samples simultaneously. Additionally, through the use of specific assay conditions or detection methods, the target enzymes can be identified with better specificity and sensitivity compared to agar plate screening methods. Moreover, screening in liquid media also mimics industrial submerged culture conditions, thus allowing the identification of potential microbes and enzymes for industrial applications [ 17 ]. There are no studies reported thus far on the HTP screening process of a large number of natural biomasses based on liquid culture and specific enzyme activities. This study aimed to 1) develop an HTP process that speeds up the identification of efficient lignin-degrading consortia, requiring fewer iterations and labor and facilitating the quick isolation of a large number of efficient microbial strains; 2) application of the developed process in practical isolation of new ligninolytic strains from natural biomasses. In the preliminary screening step, decomposed wood samples were obtained from the Ottawa River banks. Each sample was separately incubated using an enriched culture approach, and the total laccase activities of each culture were quantitatively screened on 96-well plates. This initial step allows quick HTP screening of a vast number of samples. For secondary screening, the majority of samples were rejected due to low or negative laccase activities, and only a small number of samples with high laccase activities were selected for the identification of individual strains with high enzyme activities. This technique significantly reduced the time and labor to go through a large number of samples, meanwhile increasing the success rate of identifying new bacterial and fungal strains with superior lignin-degrading ability. More interestingly, some lignin-consuming strains also exhibited significant xylanase and β -glucanase activities, suggesting that this technique may enhance the opportunity to identify super strains that express multiple enzymes simultaneously. In addition, the proposed strategy can be easily modified and adapted to isolate new strains based on different enzyme activities.",
"discussion": "3 Results and discussion 3.1 Establishing a high throughput screening process Traditionally, lignin-degrading microorganisms have been selected based on qualitative methods such as direct screening using culture plates from environmental samples or by first enriching the environmental samples and then performing a plate-based screening [ 27 ]. The conventional screening process followed in previous studies is illustrated in the steps (left) shown in Fig. 1 . To improve the reliability and efficiency of selected strains that highly express laccase enzymes using an HTP process, the methodology was changed by including a quantitative detection of laccase activity in the primary screening step as illustrated in the steps (right) shown in Fig. 1 . The process applied in this study allows for the screening of large numbers of natural biomass samples (50 samples, 100 cultures), allowing quick identification of promising consortia. The HTP process was further applied in secondary screening by growing multiple colonies in a small volume that allowed quick isolation of high-activity strains. The identity of the selected strains was confirmed by sequencing, and the reliability of the screening process was further confirmed by growing up some of the selected strains in larger volumes and testing expressed enzyme activities. Fig. 1 Schematic of the screening workflow for ligninolytic microorganisms using the conventional method [ 6 ] and the HTP technique in this study. Fig 1 3.2 Isolation and preliminary screening of laccase-producing bacterial and fungal strains This study aimed to apply the HTP process to isolate potential ligninolytic microorganisms from the decayed wood samples collected near the Ottawa River in Ontario, Canada. A total of fifty wood samples were incubated by submerged fermentation and checked for total laccase activity quantitatively. Antimicrobials were added in the culturing medium separately for bacterial and fungal cultures, thus separating mixed microbial communities into each individual monoculture. Out of 50 decomposed wood samples, eight bacterial cultures (A10, A12, A14, A16, A28, A32, A41, A50) and six fungal cultures (F10, F12, F14, F32, F41, F50) demonstrated high laccase activities ( ≥ 50 U/L), with six samples overlapping in both groups and the other two only showed high activities in bacterial cultures and not in fungal cultures. Additionally, nine bacterial cultures (A2, A6, A18, A24, A30, A34, A36, A46, A48) and nine fungal cultures (F2, F6, F16, F28, F30, F34, F36, F46, F48) showed moderate laccase activity ( ≥ 20 U/L), with seven overlapping samples ( Fig. 2 A, B). The remaining 19 bacterial cultures and 21 fungal cultures showed low activity (< 20 U/L), and 14 wood samples showed no laccase activity under either culturing condition ( Table 1 ). As a result, only a small percentage of the biomass samples - 16% for bacterial cultures and 12% for fungal cultures - underwent secondary screening, with the majority of samples (84% for bacterial cultures and 88% for fungal cultures) being excluded. Yet, a good number of both bacterial and fungal strainswith high laccase activities were isolated in secondary screening as presented in Section 3.3 . Therefore, this methodology led to a significant reduction in labor and a considerable increase in the probability of identifying highly positive isolates. Fig. 2 Bar graph representing laccase activity of decomposed wood samples under bacterial (A) and fungal (B) culture conditions with high and moderate laccase activities. Wood samples with laccase activity ≥ 50 U/L were selected for secondary screening. Fig 2 Table 1 Distribution of wood samples based on laccase activity in primary screening. Table 1 Laccase activity of wood samples in primary screening Samples under bacterial growth conditions Samples under fungal growth conditions Low active wood samples (<20 U/L) 19 21 Moderately active wood sample (≥ 20 U/L) 9 9 High active wood samples (≥ 50 U/L) 8 6 Wood samples with no activity 14 14 Total wood samples 50 50 3.3 Secondary screening for high laccase-producing strains In the secondary screening, the wood samples from primary screening that exhibited high levels of total laccase activities were selected for isolating individual bacterial and fungal strains that express at least 20 U/L laccase activity. Subsequently, ten out of 82 bacterial isolates were chosen after isolating axenic cultures from primary screened wood samples. The potential bacterial strains selected were designated as AORB9, AORB10, AORB12, AORB28, AORB37, AORB46, AORB55, AORB25, AORB19, and AORB48 with laccase activity ranging from 22.31 to 65.77 U/L ( Fig. 3 A). Similarly, in the case of fungal strains, five out of 46 strains were selected that included AORF12, AORF21, AORF39, AORF43, and AORF39, as they revealed laccase activities ranging from 33.19 to 41.51 U/L ( Fig. 3 B). Fig. 3 (A) Scatter plot representing the total protein content and laccase activity on secondary screening of bacterial isolates. Bacterial strains which showed laccase activity ≥ 20 U/L were selected for molecular identification (B) Scatter plot representing the total protein content and laccase activity on secondary screening of fungal isolates. Fungal strains which showed laccase activity ≥ 30 U/L were selected for molecular identification. Black dots represent selected strains and grey dots represent non-selected strains. Blue dots showed representative strains with different enzyme activities and protein concentrations (color should be used). Fig 3 Total protein concentration in the microbial cultures was also examined in addition to the enzyme activity, and the majority of the strains ( P < 0.001) with low enzyme activity exhibited low protein concentrations. It has been reported that, generally, high protein concentrations might be associated with higher enzyme activity [ 28 ]. However, it should be noted that protein concentration is not specific to a particular enzyme and can be affected by other factors, such as the growth conditions and the presence of other enzymes and proteins. For example, in the case of a few strains, high enzyme activity was noticed despite low protein concentration (AORB55, AORF21), and high protein concentrations with low enzyme activities were also observed (AORB73, AORF2) as depicted in blue dots in Fig. 3 A, B. Assuming the majority of the expressed protein is composed of enzymes, then the laccase-specific activity of AORB55 would be much higher than AORB46, which showed similar activity but a much higher protein content. Future detailed identification and characterization of the specific enzymes would address this question. 3.4 Time course profile of laccase production by selected microbes The selected microbial isolates were assayed to determine the time course of laccase production. To this end, time course studies were carried out for ten bacterial strains over a 120-h period with 24-h time intervals and five fungal strains over a 240-h period with 48-h time intervals. The results of the laccase activity of selected fungal and bacterial strains are shown in the heat map plot, Fig. 4 A and B. The onset of laccase production occurred at different times and was not the same for all bacterial strains. Most tested bacterial strains showed significant laccase activities at 24 h and increased significantly at later hours, with AORB46 showing the highest activity at 96 h (69.42 U/L), followed by AORB55 (66.03 U/L). Further extended cultures after 96 h led to decreased activities in most strains except AORB10, AORB19, AORB28, and AORB37. In the case of fungal isolates, laccase production started at 96 h of incubation for AORF39 and AORF43, the other three stains started at 144 h, and enzyme activities kept increasing during the culturing period up to 240 h. Strain AORF21 (133.44 U/L) showed the highest activity, followed by AORF3 (121.10 U/L) and AORF12(119.35 U/L), respectively. AORF21 from the fungal isolates and AORB46 from the bacterial isolates demonstrated the highest laccase activity among each group. Fig. 4 Heatmap of the time course of laccase activity profiles of (A) fungal strains (B) bacterial strains. The purple-yellow color scheme was used to represent the laccase activity in the culture media of individual strains . Fig 4 3.5 Molecular identification of selected laccase-producing fungal strains After the secondary screening, five potential fungal isolates were selected for genotypic identification based on internal transcribed spacer (ITS) gene sequencing analyses (Supplementary Figure. 2). BLAST search results of five ligninolytic fungal strains were distributed among four genera in 3 different taxa. This includes an ascomycete Aspergillus sydowii 49G11 (AORF21), two basidiomycetes: Trametes versicolor ANT213 QFB286 (AORF39) and Coniferaporia weirii JV0309 (AORF12) and two zygomycetes: Mucor circenelloides Pub005 (AORF39) and Mucor sp. CMRP 3219 (AORF43). Phylogenetic tree of fungal strains was constructed using the neighbor joining criteria of MEGA-X software (Supplementary Figure. 4 (a) to (e)). Many species of the genus Trametes sp. including T. versicolor are known for their laccase production and its applications in denim bleaching [ 29 ]. Among the identified fungal strains (see Supplementary Table 2), the two Basidiomycetes - AORF39 and AORF12 are typical white rot fungi that have been reported as the most efficient lignin degraders in nature by the production of extracellular lignin-degrading enzymes [ 30 ]. Moreover, the white rot fungus Coniferaporia weirri (AORF12), formerly Phellinus weirii , is a destructive root pathogen of conifers, and its laccase production has been studied previously [31] . Ascomycetes also play an integral role in the recycling of lignin in nature. The strain Aspergillus sydowii NYKA 510 has been isolated from agricultural soil and has been reported as a powerful laccase producer, and has been successfully employed in a microbial fuel cell [ 32 ]. There are few studies on the involvement of lower fungi in laccase production, as Zygomycota do not utilize lignin and cellulose in general. However, few members of this phylum are confined to the outer layers of decomposing plant tissue and can break down lignin [ [33] , [34] ]. Moreover, there are very few studies on the role of lower fungi, zygomycetes, in lignin degradation. Among them, an indigenous litter-dwelling fungus Mucor circinelloides GL1 is reported as a promising laccase producer [ 35 ]. 3.6 Molecular identification of selected laccase-producing bacterial strains The BLAST search results of 16S rRNA gene sequence of ten chosen bacterial strains revealed gammaproteobacterial strains under three genera and one strain under phylum Firmicutes, class Bacilli as follows: Serratia ( Serratia sp. CT197(AORB9), Serratia proteamaculans 336X(AORB19), Serratia marcescens AS09 (AORB28), Enterobacter ( Enterobacter sp. XBGRY7(AORB10), Enterobacter ludwigii JUQ409 (AORB25), Enterobacter hormaechei EN 336X (AORB55), and Raoutella ( Raoultella ornitholytica 4625 (AORB12), Raoultella ornitholytica Sch7(AORB37), Raoutella ornitholytica RT 1902(AORB9) and Bacillus sp. Ti1 (AORB48) (Supplementary Figure. 3). Phylogenetic tree of bacterial strains was constructed using the neighbor joining criteria of MEGA-X software (Supplementary Figure. 5 (a) to (j)). All the ten potential laccase-producing bacterial species identified belonged to the phylum Gammaproteobacteria and class Bacilli (Supplementary Table 1). Gammaproteobacteria is a large and diverse category of bacteria with a wide range of phenotypes and metabolic capabilities. Their degradative traits are displayed in various nutrient-restrictive and xenobiotic habitats [ 36 ]. They have been isolated as one of the predominant bacterial phyla, which have been associated with lignin degradation from tropical forest soil [ [37] , [38] ]. Among the genus Serratia, S. marcescens isolated from a glacial site in India has been shown to produce laccase, which can tolerate wide pH and temperature range [ 39 ]. The ability of Serratia proteamaculans sp . to secrete the laccase enzyme has been studied, and its cultural conditions were optimized [ 25 ]. In addition, Enterobacter ludwigii , isolated from decayed wood samples, has been shown to produce laccase and dye-decolorizing properties [40] . The bacterial species Raoultella ornithinolytica OKOH-1, isolated from sediment samples using culture enrichment, showed ligninolytic properties and held significant promise for use in various industrial applications, including the treatment of dye-contaminated wastewater management processes [ 41 ]. More so, there are several reports of the ability of bacterial strains under the genus Bacillus on lignin degradation [ [42] , [43] ]. Together, among the identified microbial strains, bacterial candidates AORB10, AORB12, AORB28 and fungal isolates AORF3 showed a sequence identity of 98%. Furthermore, fungal strains AORF43 and AORF39 along with bacterial strain AORB48 displayed sequence identities of 97%, 96% and 95%, respectively. Additionally, bacterial strains AORB55 and AORB37 showed sequence identities of 94% and 91%. Microbial isolates with sequence identity less than 99% compared to reference sequences may potentially represent novel species, and further studies are warranted for their classification. Moreover, a sequence identity below 95% identity is an indication of the possibility of a novel genus [ 44 ]. Thus, this methodology translates into an efficient and successful HTP screening process and isolation of unique lignin-degrading microorganisms. Although high throughput screening may expeditiously isolate an ensemble of microbes, they essentially might not unlock all entourage of microbes in an environmental sample due to the need for the preparation of varied culture media types based on nutritional requirements that promote growth to enhance their detection and isolation. Thus, the preparation of specific culture media takes precedence allowing for more efficient HTP screening of microbes, including unique isolates from the target samples. Furthermore, while biological lignin degradation is achieved by the combined action of multiple enzymes through diverse degradation pathways, this study was constrained by using laccase enzyme as the exclusive marker for evaluating the lignin-degrading traits of the isolates, consequently, may miss strains producing other enzymes instead of the detected one given the high throughput nature of the study. 3.7 Xylanase and β-glucanase activity profiles of selected microbial strains The selected microbial strains were also assessed for their ability to produce other biomass hydrolyzing enzymes, such as β -glucanase and xylanase, on different culture media. The β-glucanase activity was determined in separate media using β-glucan and lignin as carbon sources. In the presence of β-glucan as the carbon source, Aspergillus sydowii 49G11 (AORF21) and Trametes versicolor ANT213QFB286 (AORF3) displayed β-glucanase activity of 3.13 U/ml and 2.92 U/ml, respectively. On the other hand, Mucor circinellodes Pub005 (AORF39) exhibited β-glucanase activity of 3.5 U/ml when lignin was used as the carbon source, while no enzyme activity was detected when β-glucan was the carbon source ( Fig. 5 ). Fig. 5 Bar graph representing β -glucanase activity by screened microbial strains using β -glucan as carbon source (Black) and lignin (Grey) as carbon source. Error bars indicate the standard error of the mean of three replicates. Fig 5 Following this approach, Xylanase activity was measured in separate media using xylan and lignin as carbon sources. When xylan was the carbon source, Aspergillus sydowii 49G11 (AORF21) and Trametes versicolor ANT213 QFB286 (AORF3) exhibited xylanase activity of 4.09 U/ml and 3.22 U/ml, respectively. In contrast, Mucor sp. CMRP 3219 (AORF43) showed xylanase activity of 1.16 U/ml when lignin was the carbon source, with no enzyme activity detected when xylan was used. Furthermore, the bacterial strain Enterobacter hormaechei EN 314T (AORB55) demonstrated xylanase activity of 0.66 U/ml and 0.69 U/ml when xylan and lignin were used as the carbon sources, respectively ( Fig. 6 ). Fig. 6 Bar graph representing xylanase activity by screened microbial strains using xylan as carbon source (Black) and lignin (Grey) as carbon source. Error bars indicate the standard error of the mean of three replicates. Fig 6 Aspergillus sydowii proved to be one of the best xylanase-producing strains isolated from shrimp shells [ 45 ]. Trametes versicolor has been extensively studied and is considered a highly promising strain due to its ability to produce numerous biodegrading enzyme [ 46 ]. Mucor Circinellodes Pub005 and Mucor sp. CMRP 3219 exhibited β -glucanase and xylanase while using lignin as a carbon source. Mucor circinelloides is a fungus species that are dimorphic and extensively mentioned as a model producer of different enzymes that are useful for various industrial applications [ 47 ]. Additionally, the bacterial strain Enterobacter sp. exhibited xylanase activity while using both xylan and lignin separately. A heat and alkali-tolerant Enterobacter sp. was obtained from a sediment sample gathered from the Mandovi estuary located on the west coast of India and was determined to be an efficient xylanase-producing strain [ 48 ]. Biological lignin valorization is a complex multi-enzymatic process [ 49 ], including laccase, which is known to oxidize lignin employing molecular oxygen as the exclusive cofactor which sets laccase apart from other lignin-degrading enzymes - lignin peroxidases, manganese peroxidases and versatile peroxidase; that depend on H 2 O 2 [ 50 ]. Laccase can cause the oxidative breakdown of phenolic units within lignin, resulting in the oxidation of the Cα position, cleavage of the Cα-Cβ bond, and cleavage of aryl-alkyl bonds. It can also attack the non-phenolic subunits of lignin through synthetic and natural mediators [ [51] , [52] ]. Several earlier studies have implicated laccase in the biodegradation of lignin, such as in Sporotrichum pulverulentum , Coriolus versicolor and Trametes versicolor , where laccase constitutes part of medley of enzymes causing lignin degradation in model compounds and treated lignin [ [53] , [54] , [55] ]. Additionally, Pycnoporus cinnabarinus , a fungal mutant strain devoid of laccase failed to secrete manganese peroxidase and lignin peroxidase, thus deteriorating its ability to metabolize synthetic lignin [ [56] , [57] ]. Furthermore, it was observed that a mutant strain of fungus, Pleurotus , lacking laccase enzyme activity exhibited inefficient degradation of lignin [ 50 ]. In line with these observations and findings, laccase was selected as a marker where microbial isolates are screened to identify the targeted lignin degrading microorganisms using a high throughput screening process. In the primary screening, lignin was used as the sole carbon source that acts as a selective pressure to limit the growth of microorganisms that cannot use lignin as a single carbon source. This step focused the screening process towards strains that secreted high level ligninolytic enzymes needed to break down lignin polymer into smaller, more easily metabolizable compounds. Unexpectedly, a few, including both bacterial and fungal strains, also showed high-level cellulase and hemicellulase activities. Due to the fact that all enzymes are highly expressed under a single condition, these few strains may have significant valuable applications to be used for their combined or selective delignification ability in lignocellulosic biomass degradation. Zhang et al. [ 58 ] obtained four lignin-degrading microbial consortia from 40 antique wooden samples in a tedious way. Each sample was grown up in a separate flask, and selections were based on subjective, visual color change. Even though the main fungal and bacterial compositions of the four consortia were identified by genomic sequencing, no specific strain was isolated. Similarly, from twelve samples of decayed tree trunks, stumps, and surrounding soil samples, Fang et al. [ 6 ] isolated a microbial consortium that demonstrated the ability to selectively break down lignin from tree trimmings where a chromogenic substrate was incorporated in the culture media that produced a color change when laccase is produced. The reported process is laborious and time-consuming as it takes months of culture and many generations of subcultures. Xiong et al. [ 59 ] identified four bacterial strains from the soil, silage, and straw samples by first flask extraction of microorganism mixtures, followed by growth on ligninolytic selection agar media. Colonies that demonstrated aniline blue decolorization were chosen individually, and the strains were identified through 16S rDNA sequencing. There is no selection and screening of the starting biomass before single colony selection, and the total numbers of biomass samples tested were not reported. Recently, Elframawy et al. [ 60 ] screened 23 samples (14 soil samples and 9 old black liquor samples) to identify lignin degrading Actinobacteria ( Streptomyces strains). Soil samples were first pretreated for 10 days, followed by the soil dilution plate technique (AGA plates with nystatin to reduce fungal growth), while black liquor samples were directly spread onto agar plates after dilution. Colonies were selected after 21 days of incubation, transferred to new AGA plates, and further cultured for 14 days to purify the isolates. The reported HTP approach not only allowed the quick identification of multiple fungal and bacterial strains but also some particularly interesting strains that can produce multiple, synergistic, and cohesive enzymes for efficient lignocellulose hydrolysis."
} | 7,438 |
22983037 | null | s2 | 6,345 | {
"abstract": "Insects, the most diverse group of macroorganisms with 900,000 known species, have been a rich playground for the evolution of symbiotic associations. Symbionts of this enormous animal group include a range of microbial partners. Insects are prone to establishing relationships with intracellular bacteria, which include the most intimate, highly integrated mutualisms known in the biological world. In recent years, an explosion of genomic studies has offered new insights into the molecular, functional, and evolutionary consequences of these insect-bacterial partnerships. In this review, I highlight some insights from genome sequences of bacterial endosymbionts and select insect hosts. Notably, comparisons between facultative and obligate bacterial mutualists have revealed distinct genome features representing different stages along a shared trajectory of genome reduction. Bacteria associated with the cedar aphid offer a snapshot of a transition from facultative to obligate mutualism, illustrating the genomic basis of this key step along the symbiotic spectrum. In addition, genomes of stable, dual bacterial symbionts reflect independent instances of astonishing metabolic integration. In these systems, synthesis of key nutrients, and perhaps basic cellular processes, require collaboration among co-residing bacteria and their insect host. These findings provide a launching point for a new era of genomic explorations of bacterial-animal symbioses. Future studies promise to reveal symbiotic strategies across a broad ecological and phylogenetic range, to clarify key transitions along a spectrum of interaction types, and to fuel new experimental approaches to dissect the mechanistic basis of intimate host-symbiont associations."
} | 437 |
22983037 | null | s2 | 6,346 | {
"abstract": "Insects, the most diverse group of macroorganisms with 900,000 known species, have been a rich playground for the evolution of symbiotic associations. Symbionts of this enormous animal group include a range of microbial partners. Insects are prone to establishing relationships with intracellular bacteria, which include the most intimate, highly integrated mutualisms known in the biological world. In recent years, an explosion of genomic studies has offered new insights into the molecular, functional, and evolutionary consequences of these insect-bacterial partnerships. In this review, I highlight some insights from genome sequences of bacterial endosymbionts and select insect hosts. Notably, comparisons between facultative and obligate bacterial mutualists have revealed distinct genome features representing different stages along a shared trajectory of genome reduction. Bacteria associated with the cedar aphid offer a snapshot of a transition from facultative to obligate mutualism, illustrating the genomic basis of this key step along the symbiotic spectrum. In addition, genomes of stable, dual bacterial symbionts reflect independent instances of astonishing metabolic integration. In these systems, synthesis of key nutrients, and perhaps basic cellular processes, require collaboration among co-residing bacteria and their insect host. These findings provide a launching point for a new era of genomic explorations of bacterial-animal symbioses. Future studies promise to reveal symbiotic strategies across a broad ecological and phylogenetic range, to clarify key transitions along a spectrum of interaction types, and to fuel new experimental approaches to dissect the mechanistic basis of intimate host-symbiont associations."
} | 437 |
38399867 | PMC10892470 | pmc | 6,347 | {
"abstract": "The accumulation of microorganisms, plants, algae, or small animals on wet surfaces that have a mechanical function causes biofouling, which can result in structural or other functional deficiencies. The maritime shipping industry must constantly manage biofouling to optimize operational performance, which is a common and long-lasting problem. It can occur on any metal structure in contact with or submerged in ocean water, which represents additional costs in terms of repairs and maintenance. This study is focused on the production of antifouling coatings, made with nanoparticles of copper selenide (CuSe NPs) modified with gum arabic, within a water-base acrylic polymeric matrix. During the curing of the acrylic resin, the CuSe NPs remain embedded in the resin, but this does not prevent the release of ions. The coatings released copper and selenium ions for up to 80 days, and selenium was the element that was released the most. The adhesion of film coatings to metallic substrates showed good adhesion, scale 5B (ASTM D3359 standard). Antimicrobial activity tests show that the coatings have an inhibitory effect on Escherichia coli and Candida albicans . The effect is more noticeable when the coating is detached from the substrate and placed on a growing medium, compared to the coating on a substrate. Scanning electron microscopy (SEM) observations show that nanostructured CuSe coatings are made up of rod-shaped and spherical particles with an average particle size of 101.6 nm and 50 nm, respectively. The energy dispersive X-ray spectroscopy (EDS) studies showed that the ratio of selenium nanoparticles is greater than that of copper and that their distribution is homogeneous.",
"conclusion": "4. Conclusions CuSe nanostructured coatings were successfully obtained employing previously synthesized CuSe nanoparticles modified with gum arabic, embedded in a commercial resin. Despite the surface inhomogeneity of the coatings, the scratch adhesion tests showed great adhesion, in part due to the interaction present among the CuSe nanoparticles, the gum arabic, and the commercial resin determined by FTIR. Elemental analysis using EDS shows that the nanostructured coating is composed mainly of oxygen and carbon, elements that belong to the resin and gum arabic. Besides these elements, selenium and copper appear. Selenium is in a higher proportion than copper. On the other hand, elemental mapping shows that both selenium and copper are well dispersed in the coating, with selenium found in a higher proportion. The micrographs obtained by electron microscopy show that the CuSe nanostructured coatings are composed of some agglomerates and mainly of spherical particles with an average particle diameter in the range of 32.1 to 49.8 nm. The nanostructured CuSe coating was shown to have excellent adhesion (ASTM D3359 standard), with a rating of 5B, which is the highest (having only detachment in the scratched area). This result indicates that the mechanical properties of the coating are outstanding, so it would be expected to be very resistant and not easily detached under hostile environmental conditions. The release of selenium and copper ions in these coatings was verified, where selenium ions were released in greater proportion. During the drying and curing of the coating, the CuSe NPs remain embedded in the resin matrix; this process leads to the good mechanical resistance of the coating and favors the slow release of the ions, which are responsible for the inhibition of the microbial growth of the microorganisms studied, Escherichia coli and Candida albicans .",
"introduction": "1. Introduction Biofouling can be defined as the adhesion of micro- and macroorganisms to a metal structure submerged in ocean waters, and more than 4000 species have been identified in this process [ 1 ]. Its main consequence is the deterioration of the affected metals, derived from the corrosion induced by microorganisms (CIM), a fact that causes damage and weakening of the structures, increasing maintenance costs and their periodicity [ 2 ]. It is estimated that the damage associated with corrosion per year ranges from 30 to 50 billion US dollars [ 3 ]. Biofouling is also associated with the increase in the use of fuel from ships and its consequent increase in environmental pollution; due to the increase in friction during their displacement, economic studies have established that biofouling can increase fuel consumption between 40 and 77% [ 3 ]. An analysis of the economic impact of biofouling in the US Navy fleet indicates an additional cost of between USD 180 and 260 million per year [ 4 ]. The advantages of using antifouling coatings on yachts have been evaluated, taking into account the reduction in fuel consumption and the corresponding reduction in CO 2 emissions, and it was found that the fuel reduction in one year can be approximately 13.7 × 10 3 kg and CO 2 emissions can be reduced by 43.3 tons [ 5 ]. Other damages caused by biofouling include the clogging of sewage pipes and biofouling overgrowth on offshore oil or power generation platforms, which become heavier and less resistant to marine wear [ 6 ]. Another important consequence of the formation of the biofouling would be the ecological alteration caused by the artificial introduction of organisms (through dragging) from one ecosystem to another [ 4 , 7 ]. The prevention of biofouling phenomena is a widespread issue dealing with a plethora of research fields focused on the design of highly performing nanocomposite materials. Research on antifouling coatings involves water purification systems, marine equipment, biomedical devices, food packaging, fabrics, and heritage materials [ 1 , 8 , 9 ]. The increasing interest in the development of antifouling coatings has led to the development of different types of coatings that are used today. Decades ago, coatings made with paints embedded in cytotoxic agents were freely employed, whose function was the gradual release of these agents into the environment, but their use was restricted/limited due to the environmental deterioration they caused [ 10 ]. Coatings that facilitate removal (foul-release) have a hydrophobic surface where aquatic organisms have low adherence, which facilitates their removal. Some of these coatings can be gradually hydrolyzed, releasing their surface layer, thus limiting biofouling [ 11 , 12 ]. Finally, another type of antifouling coatings are those that usually employ a polymer matrix endowed with nanoparticles, which in combination have an effect that limits or inhibits microbial growth [ 13 ]. In addition, the surface roughness characteristics of these coatings are also a limitation for biofouling [ 14 ]. The type of nanoparticles and their interaction with the polymer matrix are the main factors to consider in the development of antifouling coatings. The use of copper as an antimicrobial agent has been known since antiquity and its use as nanoparticles is well documented [ 15 , 16 ]. In the last decade, the interest in synthesizing copper nanoparticles [ 17 , 18 ] and copper nanocomposites has increased significantly [ 19 , 20 ], since their excellent properties allow them to have applications in different fields of science. Selenium is an element with biological importance, since it forms part of most living beings, it has low toxicity and excellent antimicrobial properties, and it is used in various products related to the safety of human health, for example, in the generation and handling of food [ 8 ], as an antioxidant [ 21 , 22 ], for its anti-carcinogenic properties [ 22 ], and therapeutical applications [ 23 ], among others. Copper selenide (CuSe), on the other hand, is a chalcogenide commonly used in applications such as photovoltaics, thermoelectrics, and electronics [ 24 , 25 , 26 ]. It is a compound capable of being presented in different compositions and very complicated crystalline structures [ 25 , 26 , 27 , 28 ]. The Cu/Se ratio plays an important role in the properties of the material, and it was reported that hexagonal α-CuSe formed with 60% selenium is converted to hexagonal γ-CuSe at ~130 °C [ 28 ]. In the case of selenium NPs, the transformation can occur at lower temperatures [ 25 ]. Coatings with metal nanoparticles such as Ag and Cu have been proven to have excellent antibacterial properties; however, this property can be inefficient over time because bacteria can develop resistance to the nanoparticles. In the last decade, superhydrophobic (self-cleaning) coatings have emerged as a durable solution to control the spread of bacteria, because they are purely structural and do not develop bacterial resistance. Recent research combines the antimicrobial effects of superhydrophobic coatings and coatings with biocides such as Cu, Ag, Zn, or TiO 2 [ 29 , 30 ]. Superhydrophobic coatings have a physical antibacterial action, they prevent the adhesion of bacteria because they have contact angles greater than 150°, and they are considered environmentally friendly and long-lasting; this last property has recently been controversial [ 30 , 31 ]. Little has been said about its antimicrobial properties, but considering that copper and selenium present excellent antimicrobial properties separately, CuSe may be a promising candidate in this field. In the present work, we developed nanostructured antifouling coatings that consist of copper selenide (CuSe) nanoparticles and a commercial acrylic resin named Rhoplex Ac-261 and their surface, mechanical, and antimicrobial properties were evaluated.",
"discussion": "3. Results and Discussion 3.1. CuSe Nanostructured Coatings’ Characterization Figure 1 presents the optical image of CuSe antifouling coating at 4×. It can be appreciated that this coating presents a not-homogeneous distribution due to the formation of bubbles during the drying and curing of the coating. Imperfections on the surface can be observed even when different layers of the coating are applied. Figure 2 presents the FTIR spectra of the CuSe nanostructured coating and the Rhoplex Ac-261 commercial resin as a blank. The Rhoplex Ac-261 acrylic resin spectrum presents bands between 3000 and 2800 cm −1 , 1730 cm −1 , and 1030 and 1250 cm −1 , which can be assigned to bands of C-H bonds, carbonyl groups, and C-O, respectively. The detailed analysis of the spectrum shows characteristic signals at 2958 cm −1 , 2882 cm −1 , 1730 cm −1 , 1448 cm −1 , and 1152 cm −1 , where the first two are attributed to the stretching of the -CH group, the third one belongs to the stretching of the C=O group, the fourth signal is due to the stretching of the -CH 3 group, and the last one is due to the stretching and vibrations of O-CH 3 corresponding to the ester group. The presence of the band at 3446 cm −1 suggests the presence of hydroxyl groups (-OH) or water molecules trapped in the test material [ 35 ]. The bands described are characteristic of polymethylmethacrylate (PMMA); however, the absorption band at 961 cm −1 is typical of polybutylacrylate (PBA), and this band is due to the oscillation of the carbon of the -COO group of the PBA, as found in the literature [ 36 ]. PMMA and PBA had similar molecular backbones; only slight differences were found in their FTIR spectra, and the presence of these segments suggests the presence of the copolymer P(MMA-co-BA) [ 37 ]. The spectrum of the nanostructured coating with CuSe nanoparticles turned out to be very similar; the main difference found is the peak localized at 611 cm −1 , which is attributed to the bending vibrations of CuSe [ 26 ]. Modifications and shifts in the nanostructured CuSe coating spectrum compared to the Rhoplex Ac-261 spectrum are attributed to an interaction with CuSe-GA (see Section 2.1 ) nanoparticles. As reported, predominant groups of gum arabic can be shown in the spectrum of the modified nanoparticles [ 38 ]. Figure 3 shows the SEM micrograph of the CuSe nanostructured coating for elemental analysis. In this analysis, five zones were selected to carry out the elemental analysis, and zones 1, 2, and 3 were the ones that presented a greater number of particles compared to zones 4 and 5. According to the analysis, the presence of copper and selenium was observed in all areas. This demonstrates that the copper and selenium particles are embedded in the resin and that the dispersion and depth of the particles are varied. Figure 4 shows a comparison of the spectra of the Rhoplex Ac-261 commercial resin and the CuSe coating obtained from the elemental analysis (EDS), where the signals that are not labeled correspond to the elements gold and palladium used in the preparation of the sample. Table 1 shows the concentration in weight percentage of the elements carbon, oxygen, selenium, and copper. The average of the first three zones showed that the amount of selenium in the coating was greater than the amount of copper (Se/Cu = 1.35); on the other hand, the carbon and oxygen elements are derived from the chemical structure and functional groups of resin and gum arabic. On the other hand, elemental mapping was carried out to determine the presence of copper and selenium nanoparticles in the coating. Figure 5 a presents yellow (selenium) and red (copper) dots that indicate the homogeneous distribution of the elements present in the coating. The elemental mapping of each element presented in Figure 5 b,c confirms the perfect distribution of selenium and copper particles. In the right area of the image, the presence of these points was not clearly observed because the substrate has a hole in this area. Both analyses were carried out in the same area of the substrate and it was observed that the number of copper particles present in the coating is lower than the number of selenium particles; this is in agreement with the percentages by weight presented in Table 1 . Scanning electron microscopy micrographs at 50,000× and 100,000× of the CuSe nanostructured coating at 3.0% wt. of CuSe nanoparticles are shown in Figure 6 a and Figure 6 b, respectively. In both magnifications, they show two types of morphologies: rod-shaped nanoparticles and spherical nanoparticles. The nanoparticles with spherical morphology have an average diameter in the range of 32.1 nm and 49.8 nm, while rod-shaped nanoparticles mainly presented longitudes of around 101.6 nm. Although both types of nanoparticles showed a tendency to form agglomerates, it was not possible to observe micron-sized agglomerates; the original spherical morphology was preserved even when the CuSe coating was subjected to heat treatment during deposition. It has been reported that selenium and CuSe nanoparticles are structurally unstable and can change their size and morphology depending on the synthesis conditions, temperature, and composition, causing the formation of micrometric agglomerates [ 21 , 25 ], and the transformation can occur at low temperatures such as 25 °C [ 28 ]. This phenomenon was not observed in the coatings with CuSe nanoparticles even though they were subjected to temperatures of 130 °C during curing. The results of the elemental analysis of the ion release test are shown in Table 2 . These show that the nanoparticles were able to release ions through the polymer matrix and when the exposure time increases, the ion release also increases. A clear tendency can be seen in the release of ions, with selenium being released in a higher concentration in all cases, with respect to copper. The phenomenon of ion release from nanoparticles embedded in a polymer matrix is achieved in several stages, but mainly three are involved in soluble matrices, which are the following: The release by “burst”: This occurs when the ions that are closest to the surface are released; this could be due to a release due to the swelling of the material [ 39 ], to the bad interaction between the matrix and the active principle, or to the porosity of the matrix when it comes into contact with an environment that dissolves it [ 40 ]. The diffusional release: It is the stage where the ions begin to diffuse through the polyacrylate matrix [ 39 , 41 ]. Erosional release: This occurs when the material degrades due to environmental causes, which releases ions [ 39 , 41 ]. For systems with nanoparticles homogeneously distributed in the medium, only the last two stages are considered, while for those that are heterogeneously distributed, all three occur. For this case, it is considered that the nanoparticles are homogeneously distributed and settled on the coating surface. Between days 40 and 80, the release of selenium ions decreased; this can be due to many factors as follows. First, the substrate could present limitations of ion release, where there could be erosion or diffusion through the membrane. Another explanation could be that at a time point between 20 and 80 days of exposure, there are impediments to the release of selenium ions because they are bonded as a compound with copper in addition to the fact that, as previously reported, the use of biopolymers as stabilizers in nanoparticles could slow down the release of these [ 42 ]. The results obtained of the release of ions from the coating show that the release occurs very slowly. This release rate suggests that there is a good interaction between the resin and the CuSe-GA nanoparticles, since if there were weak interactions between them, we would observe a sudden initial release [ 40 , 43 ], which is not optimal for applications such as coatings or paintings. These results are in agreement with the findings found in FTIR. Figure 7 presents the surface roughness of CuSe nanostructured coatings. The arithmetic mean height (Sa) starts with a value of 17.0 µm and increases to 117.3 µm after 10 days. From here, a decrease in the arithmetic mean is recorded, reaching 85.2 µm at 80 days. On the other hand, the maximum height obtained (Sz) exhibits a behavior similar to the previous one, which confirms having the maximum height at 10 days and after that, there is a decrease in both coatings. This observed behavior may be due to the interaction of water with the coating. In the first days of immersion, the coating tends to swell, which under the roughness analysis, indicates an increase in it. After 10 days, the maximum peak of the roughness is observed, which would correspond to the maximum capacity of the coating to swell due to water, and from there, the coating begins to return to its original shape. The fact that the coating does not recover its original roughness indicates that the interaction with water generates an irreversible increase in roughness. Figure 8 presents the contact angle measurements of CuSe nanostructured coatings. The measurement of the contact angle of the CuSe nanostructured coating without water immersion (0 days) was 39.6 degrees. At 5, 10, and 20 days, different values of 75, 74, and 78 degrees were observed. These results indicate that the changes obtained in the surface of the nanostructured CuSe coating by immersion tend to exhibit a decrease in their hydrophilic behavior. However, at 40 and 80 days, there is a decrease in the contact angle of 49 and 51 degrees, respectively, indicating that the hydrophilic character increases after 20 days of immersion. These coatings show a minor hydrophilic character between 5 and 20 days, indicating that immersion during the first days significantly alters their nature. The fact that this character is lost after 20 days suggests that the coating returns to a state similar to the initial one, and therefore that it recovers its hydrophilic character [ 44 ]. This experimental evidence explains the strong release of selenium and copper ions observed after 20 days. 3.2. Mechanical Tests Analysis Figure 9 presents the results of the CuSe nanostructured coatings measured with an adhesion probe by a tape test. It can be appreciated that after the scratching and adhesion of the tape, the coating is maintained (having only detachment in the scratched area), which places it at a 5B rating, this being the highest. This indicates that the adhesion of these coatings is high, so it is expected that they do not come off easily under uncontrolled conditions. We want to strongly emphasize that CuSe-coated steel substrates can be mechanically cut without peeling off the coating. Although acrylic resin produces coatings with excellent mechanical resistance and allows the release of CuSe ions, the low values of the contact angle suggest that the adhesion of bacteria may be favored. Chemical surfaces that eliminate bacteria on contact require humidity from the medium, but this condition degrades the coating and its average lifetime decreases [ 29 ]. To extend the antimicrobial efficiency of CuSe coatings, it is necessary to apply a second layer of a superhydrophobic coating (θ ≥ 150°). This prevents the adhesion of bacteria by a physical mechanism and at the same time prevents the diffusion of moisture or contaminants in the CuSe coating layer. Once the superhydrophobic coating loses its hydrophobic property, the CuSe coating will be activated by releasing copper and selenium ions. Strategies that combine coatings with different mechanisms of action are attractive and present reduced environmental risks [ 29 , 30 , 31 ]. 3.3. Microbiological Tests Results The results of microbiological analyses of CuSe nanostructured coatings are shown as follows. First, Figure 10 shows images of CuSe nanostructured coatings at different weight concentrations of CuSe nanoparticles covered with the culture medium (without inoculation). These coatings will serve as a blank control to compare with the inoculated coatings, for comparing color, texture, and possible microbial growth. The culture medium has a transparent, slightly yellow appearance. However, as the concentration of CuSe nanoparticles increases, the slightly yellow appearance becomes slightly darker. Figure 11 and Figure 12 exhibit images of the microbial growth of both E. coli and C. albicans strains in contact with nanostructured coatings at different CuSe concentrations. In Figure 11 , we can observe images that show the CuSe nanostructured coatings inoculated with Escherichia coli . Comparing Figure 11 a and Figure 10 a, it can be seen that Figure 11 a shows a yellow coloration and a milky white halo around the inoculation zone; this suggests that microbial growth spread throughout the culture medium due to the moist conditions present on the surface. In Figure 11 b–f, the substrates did not present a yellow coloration on the entire surface and only maintained the white halo in the incubation zone. This again suggests microbial growth only in the incubation zone (possibly due to the removal of the CuSe coating), and the proliferation of Escherichia coli bacteria through the culture medium was not favorable due to the release of copper and selenium ions. These results suggest that CuSe nanostructured coatings in concentrations of 0.5 to 3.0% by weight of CuSe nanoparticles have an inhibitory effect on the growth of E. coli on nutrient agar. Figure 12 presents nanostructured CuSe coatings inoculated with Candida albicans studied under the same conditions as the bacteria Escherichia coli. The substrate without CuSe NPs, illustrated in Figure 12 a, presented characteristics similar to those observed in Figure 11 a, that is, a yellow coloration and a white halo at the inoculation site. The substrates with CuSe NPs ( Figure 12 b–f) also presented a white halo with greater intensity that indicates microbial growth exclusively in the inoculation zone. The CuSe nanostructured coatings were cleaned with 70% ethanol and washed with water to determine the number of colony-forming units (CFUs), following a previous report [ 34 ]. In the plates seeded from the dilutions, a decrease in the logarithmic number was evident with the presence of the growth of bacteria placed in contact with coatings compared to the control. The antimicrobial activity studies illustrated in Figure 11 and Figure 12 suggest that CuSe coatings have hydrophilic characteristics (contact angles less than 90°) that favor the dispersion of moisture on the surface and the release of copper and selenium ions preventing the proliferation of Escherichia coli and Candida albicans . After biological contamination, Figure 11 and Figure 12 , the substrates were cleaned with ethanol and water. The CuSe nanostructured coatings were then removed in the form of a film from the metal substrates by scraping and placed directly on Petri dishes with nutrient agar inoculated with Escherichia coli and Candida albicans ( Figure 13 and Figure 14 , respectively). The concentrations of CuSe nanostructured coatings employed were the same as in the previous experiment, 0, 0.5, 1.0, 1.5, 2.0, and 3.0% wt. of CuSe nanoparticles. Figure 13 and Figure 14 show the presence of two zones separated by a central line, the zone located on the left corresponds to microbial growth under optimal conditions and the zone on the right corresponds to microbial growth in the presence of the nanostructured CuSe coating. Figure 13 shows the images of CuSe nanostructured coatings at different concentrations at wt.% in direct contact with the E. coli strain. Figure 13 a represents a blank control where no CuSe nanostructured coating was used. On the side exposed to the CuSe nanostructured coatings, microbial growth is lower than on the opposite side. This inhibition increases as CuSe nanoparticles’ concentration increases, as seen in the image of CuSe 2.0 wt. % and higher concentrations. These results match with the previous experiment, where the microbial growth inhibition caused by CuSe nanostructured coatings for Escherichia coli was demonstrated. Figure 14 presents images of the inhibition evaluation of the direct contact of a CuSe nanostructured coating with the Candida albicans strain. Figure 14 a shows the image of the blank where no CuSe nanostructured coating was used. As in the counterpart with E. coli , the inhibition produced by the CuSe nanostructured coatings can be appreciated. This inhibition is observed clearly in all the treatments and becomes clearer as the CuSe concentration increases. These results confirm that the CuSe nanostructured coatings have an inhibitory effect, in direct contact, on microbial growth in Candida albicans . The results of antimicrobial activity against Escherichia coli and Candida albicans indicated that the CuSe NPs are distributed over the entire surface of the coating (confirmed by SEM studies) and it is likely that a good number of NPs settle at the bottom of the coating during the application and curing process of the coating [ 39 , 40 , 41 ]. The use of CuSe biocides in antifouling coatings could be an excellent alternative due to the environmental regulations imposed on copper-based coatings, but it will be necessary to carry out toxicity studies to know the potential of these materials [ 45 ]."
} | 6,760 |
20226723 | null | s2 | 6,348 | {
"abstract": "Engineering industrial microbes has been hampered by incomplete knowledge of cell biology. Thus an iterative engineering cycle of modeling, implementation, and analysis has been used to increase knowledge of the underlying biology while achieving engineering goals. Recent advances in Systems Biology technologies have drastically improved the amount of information that can be collected in each iteration. As well, Synthetic Biology tools are melding modeling and molecular implementation. These advances promise to move microbial engineering from the iterative approach to a design-oriented paradigm, similar to electrical circuits and architectural design. Genome-scale metabolic models, new tools for controlling expression, and integrated -omics analysis are described as key contributors in moving the field toward Design-based Engineering."
} | 211 |
26290073 | PMC4632617 | pmc | 6,349 | {
"abstract": "Cold-water corals, such as Lophelia pertusa , are key habitat-forming organisms found throughout the world's oceans to 3000 m deep. The complex three-dimensional framework made by these vulnerable marine ecosystems support high biodiversity and commercially important species. Given their importance, a key question is how both the living and the dead framework will fare under projected climate change. Here, we demonstrate that over 12 months L. pertusa can physiologically acclimate to increased CO 2 , showing sustained net calcification. However, their new skeletal structure changes and exhibits decreased crystallographic and molecular-scale bonding organization. Although physiological acclimatization was evident, we also demonstrate that there is a negative correlation between increasing CO 2 levels and breaking strength of exposed framework (approx. 20–30% weaker after 12 months), meaning the exposed bases of reefs will be less effective ‘load-bearers’, and will become more susceptible to bioerosion and mechanical damage by 2100.",
"introduction": "1. Introduction Cold-water corals (CWCs), such as Lophelia pertusa, form complex three-dimensional framework that support high biodiversity [ 1 , 2 ] and commercially important species [ 3 ]. These vulnerable marine ecosystems [ 4 ] are found throughout the world's oceans to 3000 m deep [ 1 , 2 ]. Projections of ocean acidification suggest that ocean pH will decrease by another approximately 0.3–0.4 pH units by the end of the century [ 5 ] and decrease the saturation state of aragonite (calcium carbonate polymorph). The cold-water Scleractinia of the deep-sea live at lower temperature (4–12°C) [ 2 ] and aragonite saturation states ( Ω Aragonite ) than tropical species. It is estimated that up to 70% of CWC reefs, which currently live at low saturation states relative to tropical corals ( Ω Aragonite < 2) will be in aragonite-undersaturated water by the end of the century, and as such are at greater risk than tropical Scleractinia from the projected shallowing of the aragonite saturation horizon [ 2 , 5 , 6 ]. Given their importance, [ 4 ] a key question is how both the living and the dead framework will fare under projected climate change [ 7 ]. Studies have examined the response of the abundant CWC L. pertusa to single stressors, ocean acidification or warming, using coral respiration or calcification rates as response variables [ 6 , 8 – 14 ]. The general consensus was that considerable variability exists between individuals [ 10 ], but L. pertusa has the ability to tolerate single stressors ( figure 1 ). However, there are major knowledge gaps to be explored before any inferences can be made as to the long-term survival and ecological role of CWC reefs [ 15 ]. Since the most likely future climate scenario involves changes in both temperature and CO 2 [ 16 ], it is vital to understand whether CWCs can acclimatize to multiple stressors simultaneously, and whether this is at a cost to other processes [ 17 ]. While adaptation to changing conditions can occur over subsequent generations, the slow growth of CWCs coupled with the projected rapid change in ocean acidification and warming [ 18 ], means that reef survival will depend heavily on the acclimatization capacity of currently living CWCs. Their acclimatization capacity is their ability to respond plastically to their environment [ 19 ] and is genetically constrained [ 20 ]. To assess acclimatization ability, it is vital to conduct relatively long-term experiments, as short-term experiments may produce results (e.g. detrimental impacts of ocean acidification upon key processes), which may not appear in long-term studies, as organisms have undergone alterations in key regulatory processes to acclimatize [ 19 ]. While L. pertusa carbon budgets are not fully understood due to limitations of obtaining and experimenting on live samples, energetic inputs (food) and reserves will be used for calcification, respiration, reproduction, maintenance, and particulate and dissolved organic matter release (electronic supplementary material, figure S1).\n Figure 1. Chart showing locations and summarizing physiological results from research on projected future impacts of temperature and ocean acidification on Lophelia pertusa . ‘R’, Respiration; ‘C’, calcification; ‘↑’, an increase; ‘↓’, a decrease; ‘/’, no statistically significant change. Symbols represent experiment endpoint results, pH is recorded in the total scale. Even if corals can acclimatize sufficiently, net reef accretion in aragonite-undersaturated conditions ( Ω Aragonite < 1) will only occur if coral calcification exceeds dissolution and bioerosion of exposed dead skeleton, and if skeletons can continue to physically support the living reefs sitting above them. This is critical to understand, since the coral's skeletal framework provides an important ecosystem function, and may persist for millennia. To explore these issues, we conducted a 1-year study on L. pertusa to address three main questions in a long-term experimental context:\n (1) Can L. pertusa acclimatize to elevated temperature and CO 2 with continued calcification and respiration at present-day rates? (2) Do elevated temperature and CO 2 conditions impact the biomineralization of newly produced skeleton? (3) Does ocean acidification have a detrimental impact on the strength of exposed L. pertusa skeletons?",
"discussion": "4. Discussion (a) Long-term acclimatization and changes in biomineralization Under ocean acidification scenarios (single or combined stressors), there was no significant difference in calcification or respiration rates from controls at three, six or 12 months. Both of these processes usually positively correlate [ 11 ], and this was observed here at three and six months. However, this relationship breaks down with the inclusion of 12-month data. Considering that these energetically costly processes are usually coupled, a deviation from this relationship may indicate that ‘normal’ energetic strategies are no longer applying, possibly due to other processes using energetic reserves. The significant reduction in respiration at 12 months in corals in the elevated temperature treatment may represent such a change, and highlights that many processes may be occurring of which we have poor understanding and/or cannot easily measure. In the most likely future climate scenario, where both temperature and CO 2 levels increase, calcification and metabolic rates of L. pertusa do not differ from controls. In all climate change studies to date on L. pertusa , significant changes in respiration and calcification are only seen in the short term ( figure 1 ), from 24-h experiments to four weeks. Beyond four weeks, changes are not observed ( figure 1 ) [ 6 , 11 , 13 , 14 ]. While this study did not observe any decrease in calcification or respiration at the earliest time point (three months), comparable shorter-term studies performed on L. pertusa collected on the same expedition did observe significant impacts of ocean acidification on respiration [ 9 ]. Given that L. pertusa has been demonstrated to be significantly impacted by ocean acidification in short-term exposures [ 6 , 9 , 10 , 12 ], we can infer from non-significant differences here between controls and ocean acidification treatments that corals have acclimatized to their new conditions. It is likely that in the days to months following a perturbation, energetic pathways in this species may prioritize protective or acclimation pathways (e.g. induction of heat shock proteins [ 17 ]). It is currently unknown whether energetic costs are involved in changing corallite shape and crystal molecular bonding, or whether this is a consequence of reducing resource allocation to cytoskeletal organization (which may vary by species [ 37 ]), but this question remains a priority for future investigations. Here, it was demonstrated that crystal organization changes under elevated CO 2 levels. Under control conditions and under elevated temperatures, typical diffraction and organization of skeletal aragonite was observed. As CO 2 increased, diffraction decreased and only scattered and disjointed aragonite crystals were imaged. The COC typically acts as the biomineralization ‘scaffold’ [ 34 ] and is surrounded by fibrous aragonitic bundles. The decrease in diffraction observed here indicates that a change has occurred to biomineralization processes under high CO 2 leading to less organization in the fibrous aragonite crystals. Interestingly, corals that were exposed to elevated temperature and CO 2 did not show the same crystal disorganization as corals exposed to high CO 2 alone. Evidence is now emerging in tropical corals that organic matrix protein incorporation in coral skeletons, which may promote calcification in less favourable calcifying fluid conditions [ 38 ], significantly changes under elevated CO 2 conditions. Along with results here, it highlights the need for multi-parameter analysis of calcification and biomineralization in an ocean acidification and warming ocean context. Multi-stressor experiments are also very important, as increased temperature in conjunction with ocean acidification may impact biomineralization differently than ocean acidification alone, such as demonstrated in crystal organization here. It is noteworthy that L. pertusa is potentially a relatively plastic coral species as demonstrated by its large bathymetric range spanning a variety of aragonite saturation states (30–3000 m [ 1 , 2 ]), its occurrence in different temperature habitats [ 6 , 26 , 28 ], and its ability to thrive in areas such as the Mingulay Reef Complex, where tidally driven downwellings cause daily variations in carbonate chemistry equivalent to ca 25 year jump in atmospheric CO 2 [ 39 ]. The growth of longer and thinner corallites in high CO 2 treatments might represent one plastic ability of L. pertusa to alter morphology. Longer and thinner corallites could enhance prey capture opportunities for individual polyps [ 40 ], which would be key to meeting potentially increased acclimation costs under high CO 2 conditions. However, such corallites may be more easily damaged or snapped, thereby decreasing future framework stability. (b) The future of deep-sea coral reefs It is encouraging that the CWC L. pertusa appears to be able to physiologically acclimate to future projected environmental changes. However, this by no means guarantees their long-term survival. Acclimatization comes at a cost, and this cost either has to be met by increased energetic inputs [ 41 ], or by re-allocation of energy from other processes. Some of these costs may be met by changes to biomineralization processes and needs to be investigated further, but the change observed in the linear relationship between respiration and calcification indicates that changes could be happening in energetic allocation. The reduction in respiration shown by corals exposed to high-temperature treatment after 1 year also indicates that processes may be changing, and highlights that many processes may be occurring of which we currently have poor understanding. Of great concern is that all studies to date on L. pertusa do not account for any impact on coral reproductive fitness. Indeed, Albright & Mason [ 42 ] demonstrated how the fertilization success of a tropical coral was negatively impacted by increased CO 2 . Acclimatization may therefore not ensure the long-term survival of corals if reproductive fitness also decreases. Another great concern for the future of deep-water coral reefs is the dissolution of exposed skeleton if the surrounding water becomes undersaturated with respect to aragonite, with a corresponding reduction in load-bearing capacity. While extracellular pH upregulation of the calcifying fluid allows corals to actively grow in undersaturated water [ 43 ], this does not protect exposed skeleton [ 44 ], which frequently forms the largest proportion of any deep-water reef framework [ 1 ]. Although the imperforate skeletons of L. pertusa will likely have slower dissolution rates than many perforate corals [ 44 ], and continuous framework may be colonized and potentially ‘protected’, bio-eroding sponges may become more efficient under future conditions thereby weakening framework colonized by other epifauna further [ 45 ]. While the ecologically significant ability of adult L. pertusa to skeletally fuse [ 28 ] helps strengthen the framework as a whole, the fact that over 95% of CWC reefs are found above the saturation horizon depth [ 5 ] infers that, in the long-term, net reef growth cannot normally be maintained in undersaturated water. The importance of skeletal dissolution with regard to ocean acidification has largely been overlooked in the discussion on how coral ecosystems will fare under future climate change [ 46 ]. Importantly, given that no adaptation can happen with regard to dissolution as it is a biogeochemical response [ 46 ], it is potentially increased dissolution of exposed aragonite, rather than a reduction in calcification rates of the live coral that could lead to future net CWC reef loss. A loss of positive reef accretion has been observed in tropical coral ecosystems along natural CO 2 gradients [ 47 ], and it is feasible to assume that a similar reduction would happen in CWC reefs as the aragonite saturation horizon shoals. Overall, we demonstrate here for the first time that L. pertusa can acclimatize to multiple stressors of temperature and CO 2 , but that significant changes happen to its skeletal biomineralization, molecular-scale bonding and structure. We also demonstrate that exposed coral framework, which forms the structural base of CWC reefs becomes structurally weaker even after 12 months of high CO 2 conditions. The question remains as to whether L. pertusa can continue to calcify at a rate which supports net reef growth, or whether potential energetic reallocation from key processes will result in decreased fitness and a long-term loss in ecosystem function when combined with weakening and dissolution of their foundation framework. Given our new evidence that CWC biomineralization changes under projected conditions, and that exposed framework becomes weakened, it is premature to assume that the impacts of ocean acidification on CWCs will be minimal based solely on the ability of the live coral to physiologically acclimatize in the short term. Strategies to reduce CO 2 emissions are still needed to minimize impacts of ocean acidification on CWCs as well as other marine biodiversity [ 18 ]."
} | 3,661 |
38898047 | PMC11186829 | pmc | 6,350 | {
"abstract": "One of the focal points in the field of intelligent transportation is the intelligent control of traffic signals (TS), aimed at enhancing the efficiency of urban road networks through specific algorithms. Deep Reinforcement Learning (DRL) algorithms have become mainstream, yet they suffer from inefficient training sample selection, leading to slow convergence. Additionally, enhancing model robustness is crucial for adapting to diverse traffic conditions. Hence, this paper proposes an enhanced method for traffic signal control (TSC) based on DRL. This approach utilizes dueling network and double q-learning to alleviate the overestimation issue of DRL. Additionally, it introduces a priority sampling mechanism to enhance the utilization efficiency of samples in memory. Moreover, noise parameters are integrated into the neural network model during training to bolster its robustness. By representing high-dimensional real-time traffic information as matrices, and employing a phase-cycled action space to guide the decision-making of intelligent agents. Additionally, utilizing a reward function that closely mirrors real-world scenarios to guide model training. Experimental results demonstrate faster convergence and optimal performance in metrics such as queue length and waiting time. Testing experiments further validate the method's robustness across different traffic flow scenarios.",
"conclusion": "Conclusion This study proposes an innovative TSC method that integrates dueling network, noise network, PER, and double q-learning mechanisms. This method effectively extracts key features of intersection traffic flows by analyzing discretized state space and utilizes vehicle queue length, which is easier to obtain in practical applications, as the reward function to guide the model training process. Action selection relies on a cyclic phase action space to provide decision support for phase transitions. In this method, PER accelerates the convergence speed of the model through non-uniform sampling; meanwhile, the introduction of noise network enhances the robustness of the model; the application of dueling network and double q-learning effectively alleviates the problem of overestimation that may occur in the DQN algorithm. Through training and ablation experiments in scenario 1, it is verified that this method can quickly learn better decision strategies π and exhibit accelerated convergence characteristics. In the testing experiments of scenarios 2–5, it is further demonstrated that this method can signirove traffic flow efficiency and reduce vehicle waiting time and lane queue length in different scenarios. However, our study still has some limitations and should be further improved in future research. One major drawback of the DRL-based TSC framework is the cold-start problem during early training, where random exploration leads to poor performance, worse than even basic FTC methods. This poses significant challenges for real-world applications. Future solutions may involve using offline DRL or safe DRL. In future research, more field data should be introduced to further validate the model proposed in our study. Additionally, future studies should extend single-agent intersection signal control to multi-agent intersection collaborative signal control.",
"introduction": "Introduction With the flourishing development of the global automotive industry, the prevalence of private cars continues to rise, thus resulting in frequent traffic congestion 1 . According to statistics, in 2019, the total greenhouse gas emissions resulting from congestion reached 36 million tons 2 , with over half generated by light vehicles, causing adverse effects on the economic development of the United States during peak traffic congestion periods 3 . Traffic congestion can be addressed through improvements in public transportation systems, road expansions, and the implementation of traffic management policies 4 . However, factors such as geographical conditions, government policies, financial constraints, technological deficiencies, and societal acceptance limit the effectiveness of these methods in alleviating traffic congestion, thus necessitating active exploration of more efficient solutions. Existing infrastructure cannot be changed, urban TSC is an economical and efficient way to address traffic congestion 5 . Today, urban TS are typically optimized using fixed-time control (FTC), induction control, and Adaptive Traffic Signal Control (ATSC) methods. FTC is a repetitive pattern that does not change with real-time traffic conditions. Its cycle continues regardless of dynamic traffic changes in the area 6 . Induction control methods operate TS based on data from loop detectors. However, during the process of collecting and analyzing traffic flow data, the actual conditions at intersections may have changed, induction control methods unable to fully meet dynamic traffic demands 7 . In contrast, ATSC dynamically adjusts signal timing strategies based on real-time traffic information to reduce potential congestion in saturated road networks 8 . Early ATSC methods addressed optimization issues by seeking effective coordinated control strategies, such as SCOOT 9 and TUC 10 . Reinforcement Learning (RL) is a method that holds promise for adaptively adjusting TSC strategies based on real-time traffic conditions 11 . Deep Learning is a machine learning method that learns data features through hierarchical abstraction 12 . The combination of deep learning and RL is termed DRL 13 . The development of DRL provides additional technical support for ATSC, further driving the success of intelligent transportation 2 , 14 . Despite some successes in the field of TSC, DRL also faces notable drawbacks and challenges. For instance, DRL suffers from inefficient training sample selection because uniform sampling is unscientific due to the varying importance of each sample 15 , leading to slow convergence. Additionally, enhancing model robustness is crucial for adapting to diverse traffic conditions, as real-world traffic flows are dynamically changing 16 . Therefore, it is necessary to further enhance training speed and improve model robustness. This paper makes the following contributions: A TSC model is proposed in this paper, which integrates a comprehensive strategy involving dueling network, noisy network, prioritized experience replay, and double q-learning, and is referred to as PN_D3QN. A safer phase-cycle action space is designed for intelligent agent action decision-making, with a more realistic reward function guiding model training. This approach considers vehicle positions, velocities, and the current phase state space to extract traffic environment features. Multiple traffic flow scenarios are used to validate the effectiveness and robustness of the proposed model.\n\nIntroduction noise To enhance the adaptability of the model to different traffic flow scenarios, consider introducing noise in the fully connected network. Specifically, replace the original neural network parameters w with µ + ε ⊙ ξ , where µ , ε , and ξ have the same shape as w . The symbols µ and ε represent the mean and standard deviation, respectively, which are parameters of the neural network learned from the samples. ξ represents random noise, with each of its elements independently sampled from the standard normal distribution N(0,1). The symbol “⊙” denotes element-wise multiplication. At this point, the function Q ( s , a , w ) is updated to: 15 \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$Q\\left( {s,a;w} \\right) = Q\\left( {s,a,\\xi ;\\mu ,\\varepsilon } \\right)$$\\end{document} Q s , a ; w = Q s , a , ξ ; μ , ε Accordingly, the loss function is updated to: 16 \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$J(\\mu ,\\varepsilon ) = \\frac{1}{m}\\sum\\limits_{i}^{m} {\\left[ {Q_{t\\arg et} (s,a) - Q(s,a,\\xi ;\\mu ,\\varepsilon )} \\right]^{2} }$$\\end{document} J ( μ , ε ) = 1 m ∑ i m Q t arg e t ( s , a ) - Q ( s , a , ξ ; μ , ε ) 2 The target network function is updated to: 17 \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$Q_{t\\arg et} (s,a) = r + \\gamma Q(s^{\\prime},\\arg \\max Q(s^{\\prime},a^{\\prime};\\mu ,\\varepsilon );\\mu^{ - } ,\\varepsilon^{ - } ))$$\\end{document} Q t arg e t ( s , a ) = r + γ Q ( s ′ , arg max Q ( s ′ , a ′ ; μ , ε ) ; μ - , ε - ) ) The gradient descent updates parameters µ and ε of the main network: 18 \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\mu \\leftarrow \\mu - \\alpha \\cdot \\nabla_{\\mu } J(\\mu ,\\varepsilon )\\;\\;\\;\\varepsilon \\leftarrow \\varepsilon - \\alpha \\cdot \\nabla_{\\varepsilon } J(\\mu ,\\varepsilon )$$\\end{document} μ ← μ - α · ∇ μ J ( μ , ε ) ε ← ε - α · ∇ ε J ( μ , ε ) The parameters µ - and ε - of the target network are updated according to the following equation: 19 \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\mu_{new}^{ - } = \\tau \\mu_{new} + (1 - \\tau )\\mu_{now}^{ - } \\;\\;\\;\\varepsilon_{new}^{ - } = \\tau \\varepsilon_{new} + (1 - \\tau )\\varepsilon_{now}^{ - }$$\\end{document} μ new - = τ μ new + ( 1 - τ ) μ now - ε new - = τ ε new + ( 1 - τ ) ε now - By using µ + ε ⊙ ξ to replace the original parameter w , the robustness of the model can be significantly improved. During training, if ε and ξ are not introduced, the obtained parameters will be µ . When the parameters strictly equal µ , the model can make a more accurate estimate of the optimal action-value function. However, when µ is perturbed, the model output may exhibit significant bias. By forcing the neural network to minimize the loss function J ( µ , ε ) during training with parameters containing noise, the model's resistance to interference is enhanced. As long as the parameters are within the neighborhood of µ , the model can make a reasonably accurate estimate of the optimal action-value function."
} | 2,730 |
33655883 | PMC8032392 | pmc | 6,352 | {
"abstract": "Horizontal gene transfer is a major force in bacterial evolution. Mobile genetic elements are responsible for much of horizontal gene transfer and also carry beneficial cargo genes. Uncovering strategies used by mobile genetic elements to benefit host cells is crucial for understanding their stability and spread in populations. We describe a benefit that ICE Bs1 , an integrative and conjugative element of Bacillus subtilis , provides to its host cells. Activation of ICE Bs1 conferred a frequency-dependent selective advantage to host cells during two different developmental processes: biofilm formation and sporulation. These benefits were due to inhibition of biofilm-associated gene expression and delayed sporulation by ICE Bs1 -containing cells, enabling them to exploit their neighbors and grow more prior to development. A single ICE Bs1 gene, devI (formerly ydcO ), was both necessary and sufficient for inhibition of development. Manipulation of host developmental programs allows ICE Bs1 to increase host fitness, thereby increasing propagation of the element.",
"introduction": "Introduction Conjugative elements and phages are abundant mobile genetic elements in bacteria, capable of transferring DNA between cells ( Frost et al., 2005 ). Integrative and conjugative elements (ICEs) appear to be the most widespread type of conjugative element ( Guglielmini et al., 2011 ). ICEs are found integrated in a host genome. When activated, they excise and produce conjugation machinery that transfers the element DNA from the host cell to recipients ( Carraro and Burrus, 2015 ; Johnson and Grossman, 2015 ; Wozniak and Waldor, 2010 ). ICEs often carry ‘cargo’ genes that are not necessary for transfer but confer a phenotype to host cells. In fact, ICEs (conjugative transposons) were first identified because of the phenotypes conferred by cargo genes ( Franke and Clewell, 1981 ). Cargo genes include those encoding antibiotic resistances, metabolic pathways, and determinants of pathogenesis and symbiosis ( Johnson and Grossman, 2015 ). Transfer of mobile elements between cells contributes to rapid evolution and spread of associated cargo genes and phenotypes ( Frost et al., 2005 ; Treangen and Rocha, 2011 ). Despite the benefits cargo genes can provide, the maintenance and transfer of mobile genetic elements requires host cellular resources and in some cases is lethal ( Baltrus, 2013 ). Maintenance of a mobile genetic element in host cells requires balancing the costs and benefits to the host or a sufficiently high transfer frequency. Many mobile elements, especially ICEs, have been identified bioinformatically ( Bi et al., 2012 ; Guglielmini et al., 2011 ). Many of these ICEs contain putative cargo genes. However, the phenotypes conferred by these genes cannot be inferred from sequence nor are they easily detected experimentally ( Cury et al., 2017 ). ICE Bs1 , a relatively small (~20 kb) ICE found in most strains of Bacillus subtilis , was identified bioinformatically ( Burrus et al., 2002 ) and experimentally based on its regulation by cell-cell signaling ( Auchtung et al., 2005 ). Most of the ICE Bs1 genes needed for conjugation are grouped together in an operon that is repressed until activating signals are sensed ( Figure 1 ). Two pathways activate ICE Bs1 , both of which lead to cleavage of the repressor ImmR by the protease and anti-repressor ImmA ( Auchtung et al., 2007 ; Bose et al., 2008 ; Bose and Grossman, 2011 ). ICE Bs1 contains the cell-cell signaling genes, rapI and phrI , which regulate ICE Bs1 activation by sensing population density and the relative abundance of ICE Bs1 -containing host cells ( Auchtung et al., 2005 ). RapI is produced at high cell density and during the transition to stationary phase and stimulates the proteolytic cleavage of the repressor ImmR by the protease ImmA ( Bose and Grossman, 2011 ). Overproduction of RapI stimulates activation of ICE Bs1 in >90% of cells ( Auchtung et al., 2005 ). RapI activity (and therefore ICE Bs1 activation) is inhibited by PhrI, a peptide that is secreted by cells that contain ICE Bs1 . PhrI levels indicate the relative abundance of ICE Bs1 -containing cells in the population, preventing the activation and possible redundant transfer of ICE Bs1 if most nearby cells already contain the element. ICE Bs1 is also activated during the RecA-dependent DNA damage response ( Auchtung et al., 2005 ). Figure 1. Genetic map and regulatory pathways of ICE Bs1. Genes are represented by horizontal block arrows indicating the direction of transcription. Vertical right-angle arrows mark the positions of promoters, and the arrowhead indicates the direction of transcription. Genes known to be involved in the conjugative life cycle of ICE Bs1 are shaded in gray. The 60 bp direct repeats that mark the ends of ICE Bs1 are shown as black rectangles. (Inset) A partial genetic map that highlights factors involved in the regulation of ICE Bs1 . The major promoter Pxis drives expression of most genes in ICE Bs1 . Pxis is repressed by the ICE-encoded repressor ImmR. Repression is relieved when ImmR is cleaved by the protease ImmA, and proteolytic cleavage is stimulated by activated RecA (RecA*) in response to DNA damage, or, independently by the cell signaling regulator RapI. RapI is made when cells are crowded by potential recipients, but repressed by the ICE-encoded secreted peptide PhrI if the neighboring cells already contain a copy of ICE Bs1. devI (formerly ydcO ) is the third open-reading frame downstream of Pxis. DevI inhibits sporulation and expression of biofilm matrix genes, likely by inhibiting Spo0A (directly or indirectly). In the genetic pathways, black arrows indicate activation and red T-bars indicate inhibition. Biofilms appear to be hotspots of horizontal gene transfer for bacteria growing in natural settings ( Madsen et al., 2012 ; Molin and Tolker-Nielsen, 2003 ). Undomesticated strains of B. subtilis form complex biofilms on agar plates and at the air-liquid interface in standing cultures ( Vlamakis et al., 2013 ). There is also extensive spore formation in B. subtilis biofilms ( Branda et al., 2001 ; Vlamakis et al., 2008 ). In addition, during growth in a biofilm, ICE Bs1 is naturally activated and transfers efficiently, generating on the order of 10 new ICE Bs1 -containing host cells (transconjugants) per donor cell under appropriate conditions ( Lécuyer et al., 2018 ). B. subtilis biofilms are held together by a matrix composed of secreted exopolysaccharides, protein fibers, and DNA ( Vlamakis et al., 2013 ). This matrix reinforces cell-cell contacts, likely promoting rapid spread of ICE Bs1 by conjugation. Additionally, the conditions that promote biofilm formation (high cell density) also promote activation and transfer of ICE Bs1 and sporulation ( Auchtung et al., 2005 ; Grossman and Losick, 1988 ). Although biofilm growth is clearly beneficial to conjugation, it is unknown how ICE Bs1 impacts its host cells under these conditions. In this study, we describe a selective advantage provided by ICE Bs1 to its host cells during growth in biofilms. This fitness benefit was due to inhibition of host biofilm and spore development. We identified the ICE Bs1 gene devI (formerly ydcO ) as necessary and sufficient to inhibit host development and provide a selective advantage to ICE Bs1- containing cells. We also provide evidence indicating that devI likely inhibits the key developmental transcription factor Spo0A, reducing its ability to stimulate biofilm and sporulation gene expression. devI ( ydcO ) is conserved in other ICE Bs1 -like elements, indicating that manipulation of host development may be a conserved strategy among this family of mobile genetic elements. We postulate that manipulation of host pathways may be a common function of many of the as yet uncharacterized cargo genes in ICEs.",
"discussion": "Discussion Our work demonstrates that ICE Bs1 confers a selective advantage on its host cells by delaying biofilm and spore development, enabling the host to grow more than cells without ICE Bs1 . When ICE Bs1- containing cells are the minority in a mixed population, ICE Bs1 genes are induced. One of these genes, devI , is necessary and sufficient to inhibit biofilm- and sporulation-associated gene expression, likely by inhibiting the key developmental regulator Spo0A, either directly or indirectly. Together with previous findings we conclude that ICE Bs1 encodes at least three distinct strategies to benefit its host cells. (1) Inhibition of development (described here) provides a growth advantage in biofilms and during sporulation. (2) Exclusion, mediated by yddJ , blocks the conjugation machinery and protects the host cell from lethal excessive transfer ( Avello et al., 2019 ). (3) An abortive infection mechanism, mediated by spbK ( yddK ) protects populations of ICE Bs1 host cells from predation by the lysogenic phage SPβ ( Johnson et al., 2020 ). We propose that all three strategies provide a competitive advantage for ICE Bs1 and its host cells in different conditions. Expression of devI reduces biofilm matrix expression and delays the initiation of sporulation. Production of the biofilm matrix is a public good, benefiting the whole community ( Dragoš et al., 2018 ). Avoidance of matrix production can therefore be considered an exploitative behavior. Exploitation can be detrimental to the population as a whole ( Smith and Schuster, 2019 ), but we did not observe any negative effects on populations under conditions where ICE Bs1 host cells had an advantage. This is in agreement with the facultative nature of ICE Bs1 cheating ( Even-Tov et al., 2016 ; Pollak et al., 2016 ). Quorum-sensing by rapI-phrI ensures that ICE Bs1 cheats only as a minority, where its impact on total public goods levels is negligible. Interestingly, the pBS32 plasmid utilizes direct regulation of biofilm formation by a Rap receptor to its benefit ( Omer Bendori et al., 2015 ; Pollak et al., 2015 ), while in ICE Bs1 this regulation was moved from the Rap receptor to one of its regulated genes. The fitness consequences of sporulation inhibition are complicated ( Mutlu et al., 2018 ). Delaying sporulation too long would result in a loss of viability of the starved cells. Inhibition of sporulation by ICE Bs1 appears to be transient; ICE Bs1 host cells eventually sporulate and do not lose significant viability as a consequence of delaying sporulation. Regulation of devI expression by the cell-cell signaling genes rapI-phrI is likely critical for transient developmental inhibition. Because commitment to sporulation is irreversible, sporulating too early is detrimental if nutrient deprivation is short-lived. B. subtilis cells with activated Spo0A that have not yet committed to sporulate also delay commitment to sporulation by killing sibling cells to liberate nutrients (‘cannibalism’) ( González-Pastor et al., 2003 ). Cannibalism is regulated by Spo0A and the subpopulation of cannibal cells (those doing the killing) overlaps with those producing the biofilm matrix ( López et al., 2009 ). Because of this overlap, it seems unlikely that devI delays sporulation by stimulating cannibalism. ICE Bs1 stability during sporulation We hypothesize that in addition to providing a fitness advantage to its host cell, delaying sporulation may also improve stability of ICE Bs1 in the host during development. Sporulation involves the formation of an asymmetric division septum generating the larger mother cell and the smaller forespore ( Errington, 2001 ; Higgins and Dworkin, 2012 ). Sporulation is induced when cells are at a high population density and running out of nutrients, conditions that also activate ICE Bs1 ( Auchtung et al., 2005 ; Grossman and Losick, 1988 ). The plasmid form of ICE Bs1 that is generated after excision from the chromosome is not known to have a mechanism for active partitioning and is more likely to remain in the larger mother cell if the cells do enter the sporulation pathway and divide asymmetrically. Therefore, the ability of ICE Bs1 to delay the initiation of sporulation under conditions where the element is activated could help prevent loss of the element and maintain ICE Bs1 in host cells. Mobile genetic elements employ various strategies to promote their maintenance during sporulation. Rates of curing during sporulation for various plasmids in Bacillus species vary widely and do not necessarily correlate with their stability during normal cell division ( Tokuda et al., 1993 ; Turgeon et al., 2008 ). Mechanisms encoded by plasmids to promote their stability during growth and sporulation include the production of dynamic cytoskeletal filaments ( Becker et al., 2006 ) and post-segregational killing of plasmid-cured pre-spores with toxin-antitoxin systems ( Short et al., 2015 ). Interestingly, even lytic phage genomes can be incorporated into spores (first described in the 1960 s) by co-opting the host’s chromosomal partitioning system ( Meijer et al., 2005 ). Diversity of cargo genes and associated phenotypes Mobile genetic elements, especially ICEs, are widespread in bacteria ( Frost et al., 2005 ; Guglielmini et al., 2011 ). Many known mobile genetic elements encode cargo genes that confer easily recognizable phenotypes, notably antibiotic resistance. Other cargo genes provide less obvious phenotypes but still fundamentally alter the physiology of the host cell. A large (500 kb) ICE was discovered in Mesorhizobium loti because its horizontal transfer conferred the ability to form nitrogen-fixing symbiotic rood nodules on Lotus plant species ( Sullivan and Ronson, 1998 ). In many pathogens, cargo genes in mobile elements are largely responsible for virulence. For example, Vibrio cholerae is capable of a pathogenic lifestyle in human hosts due to the toxin-encoding phage CTXΦ ( Waldor and Mekalanos, 1996 ). In the sporulating pathogen Bacillus anthracis , mobile genetic elements regulate both virulence and host development. Two plasmids, pXO1 and pXO2, provide the genes for toxin synthesis and production of a protective capsule, respectively ( Green et al., 1985 ; Mikesell et al., 1983 ). pXO1 also contains a regulatory gene, atxA , that regulates virulence factor production and inhibits host cell sporulation ( Dale et al., 2018 ). Co-regulation of virulence factors and sporulation is likely important during infection, as B. anthracis spores are thought to be more susceptible than vegetative cells to eradication by the immune system ( Mock and Fouet, 2001 ). Mobile elements are also known to alter the host’s interaction with other horizontally acquired DNA, which has implications for the fitness and evolvability of the host. For example, the plasmid pBS32 in B. subtilis encodes an inhibitor of the host’s DNA uptake machinery, blocking natural transformation ( Konkol et al., 2013 ). Interestingly, genes with roles in defense against foreign DNA, CRISPR-Cas systems, are also identified within mobile elements ( Faure et al., 2019 ; McDonald et al., 2019 ; Millen et al., 2012 ). Competition between mobile elements not only shapes the repertoire of cargo genes in a given cell, but it may also protect the host from harmful elements. Many mobile genetic elements have been identified bioinformatically from genome sequences or discovered by means other than the phenotypes they provide ( Bi et al., 2012 ; Guglielmini et al., 2011 ; Johnson and Grossman, 2015 ). Many elements lack obvious cargo genes, or at least lack cargo genes that have recognizable functions ( Cury et al., 2017 ). We suspect that many elements with uncharacterized cargo genes provide important traits to their hosts beyond the scope of the phenotypes currently attributed to mobile elements. Although mobile genetic elements can have remarkably broad host ranges, such as the Tn 916 -Tn 1545 group of ICEs ( Clewell et al., 1995 ; Roberts and Mullany, 2009 ) and the IncP-1 group of plasmids ( Popowska and Krawczyk-Balska, 2013 ), cargo genes and their associated functions could be highly specific to certain hosts. Characterization of unknown cargo genes is likely to expand the diversity of traits currently attributed to mobile genetic elements. We speculate that many of these genes modulate normal host functions rather than provide entirely new phenotypes. Understanding cargo gene function is critical for understanding interactions between and co-evolution of mobile elements and their hosts."
} | 4,141 |
35559312 | PMC9091625 | pmc | 6,357 | {
"abstract": "In this study, an anaerobic baffled reactor (ABR) coupled with a microbial electrolysis cell (MEC) was set up to treat carbohydrate-containing wastewater at 55 ± 1 °C. The MEC was employed to accelerate the degradation of volatile fatty acids (VFAs). The removal of chemical oxygen demand (COD) and production of methane and the corresponding kinetics were determined for different organic load rates (OLRs). The highest COD removal rate was 95.8% at an OLR of 7.0 kg COD m −3 d −1 , but it declined to 90.4% when the OLR was 19.4 kg COD m −3 d −1 and finally stabilized at 65.3% when the OLR was increased to 34.3 kg COD m −3 d −1 . The volumetric production of methane was 1.5 L (L −1 d −1 ) when the OLR was 7.0 kg COD m −3 d −1 and increased to 4.1 L (L −1 d −1 ) at an OLR of 34.3 kg COD m −3 d −1 , when the methane yield stabilized at 0.20–0.25 L g −1 COD removed . The kinetics and predictions according to the Stover–Kincannon and Van der Meer–Heertjes models closely agreed with the experimental data for the removal of COD and volumetric production of methane, respectively. An analysis of the microbial community suggested that hydrolytic bacteria, syntrophic fatty acid-oxidizing bacteria (SFOB), exoelectrogens and hydrogenotrophic methanogens achieved a significant synergistic effect and enhanced the degradation of VFAs, which made the thermophilic anaerobic system stable and efficient at high OLRs.",
"conclusion": "4. Conclusion An investigation of the performance of an ABR-MECs system in the thermophilic digestion of carbohydrate-containing wastewater was conducted in this study in qualitative and quantitative terms. The highest COD removal efficiency was 95.8% at an OLR of 7.0 kg COD m −3 d −1 . It decreased to 65.3% when the OLR was 34.3 kg COD m −3 d −1 , and the methane yield stabilized at 0.20–0.25 L g −1 COD removed . The kinetics and predictions according to the Stover–Kincannon and Van der Meer–Heertjes models closely agreed with the removal of COD and volumetric production of methane, respectively, with high correlation coefficients ( R 2 ) of 0.979 and 0.968. The proposed models enabled precise control of the system. A microbial analysis revealed that the dominant microbes found in C1 were hydrolytic bacteria ( e.g. , Thermohydrogenium and Chloroflexi), whereas SFOB ( e.g. , Syntrophobacter ), exoelectrogens ( e.g. , Clostridium and Methanobacterium ) and hydrogenotrophic methanogens ( e.g. , Methanobacterium ) were found to be dominant in the MEC compartments. The microbial community suggested that the rapid degradation of VFAs and the production of considerable quantities of methane were achieved by the synergistic effect of different functional microbes in the thermophilic ABR-MECs system.",
"introduction": "1. Introduction The accumulation of VFAs is a key issue that affects the stability of an anaerobic system when used to treat high-concentration organic wastewater. 1 Specifically, the accumulation of acids is more likely to occur in a thermophilic system because hydrolysis is more rapid. 2,3 In the past, researchers have tried to enhance the capacity and stability of anaerobic digestion (AD) by regulating the microbial community and engineering. 4 As for regulating the microbial community, bioaugmentation technology has been widely employed to increase the abundance of functional microbes. For instance, in order to alleviate the accumulation of VFAs that results from an imbalance in metabolic rates between hydrolytic bacteria, acidogens and methanogens, SFOB and acid-resistant methanogens were acclimated to improve the degradation of VFAs. 5 Nevertheless, the basic biological characteristics ( i.e. , long generations, slow metabolic rate and narrow niche) of methanogens are difficult to change via acclimation. 6 Besides, how to ensure the sustainability and enrichment of target microorganisms in an AD system is a major challenge for conventional bioaugmentation. A microbial electrochemical system could provide a new way to solve these problems. 7 In a typical microbial electrolysis cell (MEC), exoelectrogens transfer electrons to the anode by oxidizing organic matter, and then the electrons pass through the circuit to the cathode to produce H 2 . MECs have also been employed in AD systems. Whereas previous studies of AD-MECs mainly focused on improvements in the production of hydrogen, 8 the microbial community in the cathode for the generation of methane, 9 the removal efficiency for specific pollutants, 10 and the electron transfer mechanism of the exoelectrogens, 11 little work has been done to investigate the synergistic degradation of VFAs by functional bacteria. However, acetic acid is a favorable substrate for exoelectrogens. 12 It is a fact that exoelectrogens efficiently enhance the degradation of acetic acid and relieve suppression by acetic acid for the decomposition of propionic and butyric acid by SFOB, which provides the theoretical possibility of the rapid dissociation of VFAs. Furthermore, early studies declared that exoelectrogens have many advantages over methanogens, such as longer generations, a faster metabolic rate and a wider niche. 13 Hence, it would be a novel idea to intensify the degradation of VFAs via an AD system coupled with MECs (AD-MECs). This not only overcomes the shortcomings of methanogens but also ensures the robustness of target microorganisms in the system by providing continuous electrical stimulation. Although a single-phase reactor such as an up-flow anaerobic sludge bed (UASB) has been adapted to perform an AD-MEC process by the insertion of electrodes, the different niches of acidogenic bacteria, methanogens and exoelectrogens make it difficult to optimize the different microbes in an integral reactor and further limit its efficiency. 14 It is feasible to set up a coupled AD-MEC process by connecting a continuous stirred-tank reactor (CSTR) with an MEC in series. 15 Nevertheless, the construction of the MEC reactor will increase the cost. Owing to its baffled structure, an ABR is an ideal reactor for achieving the phase separation and distribution of dominant microorganisms in different regions. 16 It provides a structural support for better coupling with an MEC in an integral reactor. Therefore, an ABR was used to construct an ABR-MECs system. On the other hand, although AD technology is widely applied, its optimum operation is seldom achieved because of the empiricism that is prevalent in the design and daily operation of an anaerobic system. With the increasing demand for efficient operation and model-based design, kinetic modeling of the AD process has received extensive attention and has been addressed via substrate utilization models, microbial growth models and product formation models. 17,18 The determination of the conversion of methane is the most common on-line measurement and is easily performed as it is directly proportional to the degradation of the substrate. 19 In many studies, the production of methane and removal of COD were used to estimate model parameters. 20 Many types of mathematical models have been developed. Among these, the anaerobic digestion model 1 (ADM1) is the most advanced owing to its precise predictability and strong generalizability. 21 The ADM1 model reflects the major processes in the conversion of complex organic substrates into methane. 22 However, the model requires a large number of constants and coefficients, which should be calibrated according to the characteristics of the substrate. Such calibration requires the use of special assays and computing skills, which is difficult for scientists and engineers in engineering applications. 23 Therefore, simplified models that consist of only a few variables have been widely studied. The first-order rate equation, Stover–Kincannon model, and Van der Meer–Heertjes model have been used to satisfactorily predict the production of methane and removal of the substrate in an AD process. 24,25 In this study, an ABR-MECs system was set up to treat synthetic carbohydrate-containing wastewater at 55 ± 1 °C. The efficiencies of the removal of COD and production of methane at different OLRs were analyzed qualitatively and quantitatively. The first purpose was to reveal the intrinsic relationship between the microbial composition and degradation of VFAs so as to establish a theoretical foundation for improving the stability of an anaerobic system when used to treat an easily acidified substrate. The second aim was to optimize the operation of the system and improve the efficiency of regulation via the establishment of dynamic models.",
"discussion": "3. Results and discussion 3.1. Effect of OLR on substrate removal In Phase I ( Fig. 2a and b ), the system was operated for different HRTs of 24 h, 21 h and 18 h with an influent COD concentration of 6 g L −1 . Correspondingly, the COD removal rate was 95.4%, 94.6% and 80.5%, respectively. The COD removal efficiency declined sharply in Stage 3 when the HRT was changed from 21 h to 18 h. The COD removal efficiency in the five compartments at an HRT of 18 h was about 50.6%, 13.9%, 7.6%, 7.4% and 1.0%, respectively. The COD reduction rate in the last four compartments was low because the VFAs produced in the first compartment could not be effectively used in the subsequent compartments. This was consistent with the findings of Wu et al. , who declared that exoelectrogens had longer generations and the efficiency of the degradation of VFAs decreased when the HRT was reduced. 27 After Phase I, the operational scheme that was adopted is shown in Table 1 . In Phase II, a longer HRT of 48 h was introduced for the acclimation of microbes, and the COD removal rate stabilized at about 95.8% at an OLR of 7.0 kg COD m −3 d −1 . Thereafter, the HRT was reduced to 24 h and 21 h with OLRs of 14.0 and 16.0 kg COD m −3 d −1 , respectively. Correspondingly, the COD removal efficiency declined instantly to about 91.8% and 90.3%, respectively, and then efficiencies of 95.5% and 94.7% were observed after the system attained a stable condition. Notably, although the OLR increased from 7.0 to 16.0 kg COD m −3 d −1 , the COD removal rate declined slightly. Therefore, the influent COD concentration was further increased to 17 g L −1 in Phase III. Similar performance in terms of removal efficiency was observed with new OLRs in the range of 8.5–19.4 kg COD m −3 d −1 , and the COD removal rate stabilized at 92.4%, 91.6% and 90.0% at HRTs of 48 h, 24 h and 21 h, respectively. Clearly, the COD removal efficiency decreased from 95.8% to 90.0% when the OLR increased from 7.0 to 19.4 kg COD m −3 d −1 . In order to investigate the performance of the system at high OLRs, a rapid increase in the OLR to 22.9, 28.6 and 34.3 kg COD m −3 d −1 was carried out in Phase IV, and the COD removal rate finally reached 84.9%, 77.2% and 65.3%, respectively. Consequently, the highest removal efficiency was 95.8% when the OLR was 7.0 kg COD m −3 d −1 , and the lowest COD reduction rate was 65.3% at an OLR of 34.3 kg COD m −3 d −1 . When the OLR was less than 19.4 kg COD m −3 d −1 , the COD removal rate exceeded 90.0%. Fig. 2 Performance of the ABR-MECs system at different COD concentrations and HRTs: COD removal rate and COD concentration (a), pH and ALK (b), methane content and methane yield (c), and volumetric yield of biogas and methane (d). C1, C2, C3, C4 and C5 denote the first to the fifth compartments of the reactor, respectively, in the direction of flow. In terms of favorable OLRs, Fang et al. (2011) 32 and Jing et al. (2013) 33 reported that the best OLR ranges for anaerobic digestion were 2.5–3.2 kg COD m −3 d −1 and 1.4–16.0 kg COD m −3 d −1 , respectively. Obviously, the OLRs of 7.0–19.4 kg COD m −3 d −1 that were found to be favorable in this experiment were higher than the values in these reports. Higher OLRs resulted from the effective degradation of VFAs ( Fig. 3a–c ). Acetic acid and butyric acid were dominant when the OLR was less than 19.4 kg COD m −3 d −1 , and VFAs were rapidly degraded so that less than 500 mg L −1 remained in the effluent. In contrast, the concentration of propionic acid increased rapidly with a further increase in the OLR, and it eventually became dominant in all the compartments when the OLR increased to 34.3 kg COD m −3 d −1 ( Fig. 3d–f ). The degradation of VFAs at high OLRs could result from the synergistic action of microorganisms. Hydrolytic bacteria in the first compartment degraded organics to VFAs, which were further consumed by exoelectrogens and SFOB in the subsequent compartments. This was the reason why the concentration of propionic acid and acetic acid always remained low in the effluent of the system. This was consistent with the findings of a previous study, namely, that propionic acid could be rapidly oxidized to acetic acid by propionate oxidation bacteria in an AD process. 34 One direct evidence was the fact that Syntrophobacter (5.0%), which is a propionic acid-degrading bacterium, 35 was well enriched in the MEC compartments. Moreover, acetic acid can be substantially degraded in MECs. 36 During the utilization of acetate for biogas production, a significant increase in the removal of COD, which was 1.7 times higher than that in the control group, was observed in an AD-MECs system. 37 Another reason could be that their shorter generations and faster metabolic rate made exoelectrogens adapt to variations in the HRT more effectively than methanogens. Thus, the growth of exoelectrogens was boosted in the AD-MECs, and the bacterial population increased to a greater extent in the AD-MECs than in an AD system. 38 This confirmed that SFOB and exoelectrogens were well enriched and performed well in the ABR-MECs system. Hence, an effective way to improve the stability of a thermophilic AD system comprises efficiently degrading VFAs by the introduction of the MECs. The results also suggested that 19.4 kg COD m −3 d −1 was the most favorable OLR and 34.3 kg COD m −3 d −1 was the maximum value (critical point for further acidification) for the ABR-MECs system used to treat carbohydrate-containing wastewater in thermophilic conditions. Fig. 3 Contents of VFAs in the five compartments (C1–C5) at different OLRs. 3.2. Effect of OLR on methane production The content and production rate of methane are illustrated in Fig. 2c and d . As the HRT was reduced from 48 h to 21 h with the variation in the COD concentration in the feed, the OLR increased from 7.0 to 34.3 kg COD m −3 d −1 . Correspondingly, the methane production rate rose from 1.2 to 4.1 L (L −1 d −1 ) and the biogas production rate rose from 1.6 to 6.1 L (L −1 d −1 ). The results indicated that the increase in the volumetric production of methane was related to the increase in the OLR, whereas the methane yield was stable at 0.20–0.25 L g −1 COD removed . Besides, the methane content in each compartment ranged from 60.4% to 85.3% in Phase II and Phase III and from 53.2% to 80.8% in Phase IV. The maximum methane content decreased from 85.3% to 80.8% when the OLR increased from 19.4 to 34.3 kg COD m −3 d −1 . The results suggested that the performance of the ABR-MECs system was impaired when the OLR exceeded 19.4 kg COD m −3 d −1 . The decrease was due to the reduction in pH with the increase in the OLR. The pH was stable at 6.50–7.70 when the OLR was lower than 19.4 kg COD m −3 d −1 , which was favorable for acidogenic bacteria, methanogens and exoelectrogens, 39 and the phenomenon of an imbalance between acid producers and consumers did not exist. The methane content was higher than the value of 73.0–75.4% recorded in a pilot-scale UASB. 40 Owing to the efficient utilization of VFAs via extracellular electron transfer by exoelectrogens, more carbon was converted into methane in situ . 41 Therefore, purer methane was detected in the MEC compartments. This could have been due to the significant enrichment of hydrogenotrophic methanogens ( Methanobacterium , 58.7%) in the ABR-MECs system. The proportion of Methanobacterium in the system was twice that in the inoculum (25.8%). The percentage of hydrogenotrophic methanogens was also much higher than that in a traditional AD system in which aceticlastic methanogens were always dominant among archaea. The increase in hydrogenotrophic methanogens was due to the enhanced production of H 2 by exoelectrogens. Nevertheless, if this was the only way in which hydrogen was consumed by hydrogenotrophic methanogens, hydrogen should have been detected in the system, as hydrogenotrophic methanogens cannot completely remove hydrogen if the production of methane is only caused by the conversion of hydrogen. 42 In fact, hydrogen was not detected in the ABR-MECs system. There could be other ways in which methane was generated. Electrochemical systems provide possibilities for new methanogenic pathways. Exoelectrogens are defined as kinds of microorganism that perform extracellular respiration. In particular, Methanobacterium could be a kind of methanogen with electrochemical activity. 41 Methane could be produced by Methanobacterium via direct electron transfer via the reaction CO 2 + 8H + + 8e − → CH 4 + 2H 2 O. This provides the theoretical possibility of the purification of carbon in situ , and more carbon can be converted stably into methane. Thus, the methane yield stabilized in the range of 0.20–0.25 L g −1 COD removed when the OLR was in the wide range of 7.0–34.3 kg COD m −3 d −1 . Moreover, the stable yield of methane provided a guarantee for simulating the dynamic process and predicting the volumetric production of methane. 3.3. Microbial community analysis Sequence clustering analysis was conducted in accordance with similarity and the representation of OTUs. 43 To confirm the diversity and richness of the microbial community, the Shannon, ACE and Chao indices were estimated. The Chao and ACE indices are related to the richness of microbes, whereas the Shannon index is negatively related to the diversity of species. 44 The average values of the Chao, ACE and Shannon indices in C2–C4 were 298, 301 and 3.55, respectively, which were lower than those in C1 (352, 358, 3.59) and C5 (382, 395, 4.01). This also suggested that although the richness of microbes was lower in C2–C4 than in C1 and C5, the diversity increased in C2–C4. A visual expression is presented in Fig. 4 . PCA analysis visually indicates that the bacteria in C2–C4 are far away from those in C1 and C5. The microbes in C2–C4 were greatly different from those in C1 and C5 owing to the introduction of the MECs. This was further elaborated as follows. Fig. 4 PCA analysis of eubacteria in the five compartments (C1–C5). At the phylum level, the bacterial components in C1 were Firmicutes (31.8%), unclassified bacteria (28.2%), Chloroflexi (12.5%), Spirochaetes (10.1%), Verrucomicrobia (4.3%) and Proteobacteria (3.2%). However, in C2 (which represents the MEC compartments), they were unclassified bacteria (52.6%), Firmicutes (14.7%), Proteobacteria (9.7%), Acetothermia (4.6%), Chloroflexi (3.9%), Planctomycetes (3.5%), Verrucomicrobia (2.6%), Spirochaetes (1.9%), Thermotogae (1.8%) and Actinobacteria (1.3%). From these results, it can be observed that Firmicutes were the dominant bacteria in both the inoculum and the MEC compartments of the ABR-MECs system, but the abundance of Firmicutes in the MECs was less than that in the inoculum, and the proportion of unclassified bacteria in the MECs increased significantly. Moreover, the dominant archaea changed from Methanosaeta in the inoculum to Methanobacterium in the MECs. These phenomena confirmed the influence of the MECs on the microbial community in the AD system. Besides, more unclassified bacteria and Proteobacteria and fewer Chloroflexi were detected in C2 in comparison with C1. Recently, similar phyla, including Proteobacteria, Firmicutes, Chloroflexi and Bacteroidetes, were reported to be the dominant phyla in anaerobic sludge. 44 Firmicutes and Proteobacteria were reported to be the dominant phyla in high-temperature electrochemical systems and anaerobic systems. 45 Therefore, the presence of Chloroflexi in C1 was attributed to hydrolytic fermentative functionalities. 46 The synergistic coexistence of Chloroflexi and Firmicutes in C1 was suggested to have contributed to the hydrolysis and acidogenesis processes, and Proteobacteria and unclassified bacteria exercised heterotrophic and electrochemical functionalities in the MEC compartments. Thus, the high abundance of dominant bacteria in different regions supplied the biological basis for the enhancement in the performance of the ABR-MECs system. At the genus level, the relative abundances are shown in Fig. 5 . The dominant bacteria in C1 were hydrolytic acidification bacteria such as Thermohydrogenium (17.1%), unclassified Chloroflexi (8.7%), and Treponema (6.7%), whereas the main bacteria in C2 were Clostridium (6.0%), Syntrophobacter (5.0%) and Acetothermia (4.6%). The content of unclassified bacteria increased from 28.2% in C1 to 52.9% in C2 and then decreased to 48.3%, 40.7% and 35.7% in C3, C4 and C5, respectively. The obvious downward trend could be due to the concentration of the substrate in the different compartments. Notably, the microbial communities were significantly different in the MEC compartments in comparison with those in C1 and C5, and unclassified bacteria grew in large quantities. The thermophilic members of the exoelectrogen group need further investigation in future work. Fig. 5b shows the content of archaea. Eight species of archaea were identified in the thermophilic ABR-MECs system. The dominant archaea, taking C4 as an example, were Methanobacterium (58.7%), Methanosaeta (18.2%), Methanomassiliicoccus (14.9%) and Methanolinea (3.2%), which belong to the phylum Euryarchaeota. Specifically, Methanobacterium in the ABR-MECs was suggested to exercise hydrogenotrophic and extracellular electron transfer functionalities. 47 Methane could be produced by Methanobacterium by direct electron transfer in the biocathode via the reaction CO 2 + 8H + + 8e − → CH 4 + 2H 2 O. 48 In all, high contents of hydrolytic bacteria ( e.g. , Thermohydrogenium and Chloroflexi) were found in C1, whereas more SFOB ( e.g. , Syntrophobacter ), exoelectrogens ( e.g. , Clostridium and Methanobacterium ) and hydrogenotrophic methanogens ( e.g. , Methanobacterium ) were detected in C2–C4. Fig. 5 Distribution of functional bacterial and archaeal populations (sequence reads ≥ 1%) in the anaerobic sludge obtained from the five compartments (C1–C5): genus of eubacteria (a) and genus of archaea (b). The reciprocity of hydrolytic bacteria, SFOB, exoelectrogens and hydrogenotrophic methanogens in the ABR-MECs system smoothly degraded organic matter via the following metabolic pathways: (1) hydrolytic bacteria in the first compartment degraded the substrate into micromolecular organic matter. (2) Organic substances that are difficult to utilize ( e.g. , propionic acid and butyric acid) were rapidly degraded to acetic acid by SFOB and in turn exploited by exoelectrogens in the anode. (3) Electrons were transferred to the cathode, directly obtained by Methanobacterium or transferred to H 2 and then used by hydrogenotrophic methanogens to generate methane. Thus, the consumption of acetic acid by exoelectrogens relieved the inhibition of SFOB by acetic acid and further promoted the enrichment of hydrogenotrophic methanogens. The MECs in the AD system provided continuous electrical stimulation for the enrichment of target microorganisms. The microbial communities revealed that hydrolytic acidification bacteria, SFOB, exoelectrogens and hydrogenotrophic methanogens could form dominant communities in different regions of the system and synergistically degrade VFAs at high OLRs. The distribution of microorganisms theoretically explained the phenomena that the efficient removal of the substrate and production of methane could be achieved in the ABR-MECs system, but the dynamic relations need to be further revealed to optimize the system. 3.4. Modeling of substrate removal and methane production For a quantitative analysis of the relationship between the OLR, the removal of the substrate and the generation of methane, the kinetic coefficients of the first-order and Stover–Kincannon models were used for determining the removal of COD. Then, the coefficients and models that were obtained were evaluated and the removals of COD were predicted. 3.4.1. First-order and Stover–Kincannon models for substrate removal As shown in Fig. 6a , data for the steady state of each stage were used to determine the kinetic coefficients of the first-order model. A linear equation was achieved with a kinetic constant K 1 of 3.08789, and the correlation coefficient ( R 2 ) was equal to 0.597. It is clear that the linear fitting was poor and the degradation of the substrate was not consistent with the first-order model. Fig. 6 Determination of kinetic constant in first-order model for removal of the substrate (a). Determination of kinetic constant in Stover–Kincannon model for removal of the substrate (b). Kinetic constants for the production of biogas and methane determined by the modified Stover–Kincannon model (c). Kinetic constant for the production of methane determined by the Van der Meer–Heertjes model (d). Observed and predicted COD concentrations in effluent determined by the Stover–Kincannon kinetic model (e). Observed and predicted production of methane determined by the modified Stover–Kincannon and Van der Meer–Heertjes models (f). The results of a kinetics study using the Stover–Kincannon model 30 are presented in Fig. 6b . A positive linear correlation is observed between the reduction of COD and the OLR. From the intercept and slope of the plotted line, the maximum utilization rate ( U max ) and saturation constant ( K B ) for the removal of COD were 107.5 and 104.3 g COD L −1 d −1 , respectively, and the correlation was strong ( R 2 = 0.997). The maximum utilization rate ( U max ) in each compartment was also estimated to determine the rate of degradation of the substrate. The U max values were 95.7 and 45.4 g COD L −1 d −1 in C1 and C2, respectively, which indicated that the maximum rate of consumption of COD in C1 was twice that in C2. Therefore, a large percentage of the COD in the reactor was consumed in the first compartment. Besides, the U max values in C3, C4 and C5 were 26.3, 11.4 and 9.8 g COD L −1 d −1 , respectively. This reveals that the maximum rate of consumption of COD gradually decreased from C1 to C5. According to the Stover–Kincannon model, the relationship between S e , S i and the HRT is described by eqn (9) . To further confirm the accuracy of the Stover–Kincannon model, eqn (9) was used to predict the concentration of COD ( Fig. 6e ). Notably, the predicted values agree with the experimental values with a strong correlation ( R 2 = 0.979). This suggests that eqn (9) based on the Stover–Kincannon model is able to make reliable predictions of the removal of COD: 9 where S i and S e are the COD concentrations of the influent and effluent (g L −1 ) and HRT is the hydraulic retention time ( d ). 3.4.2. Modified Stover–Kincannon model and Van der Meer–Heertjes model for the production of biogas The results of a kinetics study using the modified Stover–Kincannon model are presented in Fig. 6c . The coefficients ( R 2 ) of the modified Stover–Kincannon model for the production of biogas and methane were 0.987 and 0.984, respectively. The maximum specific gas production rate ( G max ) and the constant of proportionality ( G B ) were 26.8 L (L −1 d −1 ) and 95.492 (dimensionless). The highest methane gas production rate ( M max ) and the constant of proportionality ( M B ) were 17.5 L (L −1 d −1 ) and 84.298 (dimensionless), respectively. On the other hand, the results of kinetics studies using the Van der Meer–Heertjes model are presented in Fig. 6d . The R 2 coefficient was 0.972. It is difficult to judge these two models because their R 2 coefficients were similar. Predicted values of methane yields obtained from the two models are plotted against experimental values for the production of methane ( Fig. 6f ). The R 2 value of the modified Stover–Kincannon model was 0.939, whereas it was 0.968 for the Van der Meer–Heertjes model. The output of the Van der Meer–Heertjes model agrees better with the experimental results. Hence, the Van der Meer–Heertjes model was employed to estimate the production of methane. According to the Van der Meer–Heertjes model, the relationship between V m , S i , S e and HRT can be described by eqn (10) to make reliable predictions of the production of methane: 10 where V m is the methane production rate (mL d −1 ), S i and S e are the COD concentrations of the influent and effluent (g L −1 ), respectively, and HRT is the hydraulic retention time ( d ). By the establishment of these two models, the relationship between the removal of COD, the production of methane and the OLR was clearly determined for the treatment of such carbohydrate-containing wastewater. It further revealed the quantitative relationships between the concentrations of organics (in and outside the substrate, measured in terms of COD) and the HRT. Plotting the variables is helpful for determining the optimal control and providing precise predictions for further improving the performance of a thermophilic ABR-MECs system. In addition, it is beneficial for predicting trends in the removal of organics and the production of methane at a high OLR and avoiding decreases in performance with an increase in the OLR when dealing with easily acidified substrates."
} | 7,473 |
20569124 | PMC3014662 | pmc | 6,358 | {
"abstract": "We investigated whether the physical castes of the dimorphic ant Pheidole pallidula (Nylander) (Hymenoptera: Formicidae), are involved in determining within-nest activities and how their social investment in everyday tasks is influenced by large changes in the colony's caste ratio. Although the large-headed majors are morphologically distinct from minors, they are similar in size, exhibit similar behavioral repertoires and carry out nearly the same tasks as minors. Changes, even large ones, in the colony's caste ratio have no significant effect on the repertoire size of either caste. Majors do not compensate for the depletion of minors by expanding their repertoire or increasing their activity level. Instead of being an idle stand-by caste as suggested for other Pheidole specie s, P. pallidula majors are nearly as totipotent as minors. Moreover, their performance rate of social behaviors is remarkably high and constant regardless of the colony caste ratio. Such high investment of the major caste helps the colony to keep social behaviors at a baseline even in colonies undergoing large demographic changes. Alternative schemes of social regulation in polymorphic ant species are discussed. A possible methodological bias accounting for between-species differences in the level of majors' specialization is described.",
"introduction": "Introduction A common way of organizing work in insect societies is through the division of labor in which individuals consistently perform a subset of tasks for periods ranging from a few days to their whole lives. At the colony level, the simultaneous performance of different tasks by different groups of specialized individuals is assumed more efficient than if tasks were performed sequentially by unspecialized πindividuals ( Oster and Wilson 1978 ; Jeanne 1986 ; Gordon 1989 ; Robinson 1992 ; Bourke and Franks 1995 ). Task specialization is known to be influenced by the age of workers, their morphology, their genetic background and their individual experience in bees and in ants (for bees, see Robinson and Page 1988 ; Seeley 1995; Dreller and Page 1999 ; Huang and Robinson 1999 ; Fewell and Bertram 2002 ; Hurd et al. 2007 ; and for ants, see Gordon 1989 , 1996 ; Hölldobler and Wilson 1990 ; Theraulaz et al. 1998 ; Beshers et al. 1999 ; Tripet and Nonacs 2004 ; Heredia and Detrain 2005 ; Seid and Traniello 2006 ; Ravary et al. 2007 ). Several studies have shown that division of labor between workers is a dynamic phenomenon: the species-specific behavioral profile of each caste can be altered by day-to-day or seasonal changes in the colony composition due to predation, competitive pressure or changes in environmental resources ( Davidson 1978 ; Sorensen et al. 1984 ; Johnston and Wilson 1985 ; Wheeler 1991 ; Aarab and Jaisson 1992 ; Schmid-Hempel 1992 ; Tschinkel 1993 ; Gordon 1996 ; Passera et al. 1996 ). In order to maintain colony organization at a high efficiency level even after large perturbations in its caste ratio, one colony should ideally be able to fill-in for missing individuals. Over a long time-scale, a colony can increase the production of new adults belonging to the depleted caste through an adaptive demography response ( Calabi and Traniello 1989 ; Passera 1977 ; Passera et al. 1996 ). Over a shorter time-scale, a more flexible response can be achieved by the behavioral plasticity of individuals that perform tasks considered atypical for their caste ( Wilson 1980 , 1983 , 1984 , 1986 ; Detrain and Pasteels 1991 ; Cassill and Tschinkel 1999 ) and/or change their activity rate ( Wilson 1984 ; Brown and Traniello 1998 ). Regarding dimorphic ant species, physical castes are expected to differ in their behavioral capabilities. From an ergonomic perspective ( Oster and Wilson 1978 ) it is assumed that the numerically dominant minor caste consists in “generalists,” workers that take care of all tasks necessary to colony development. Contrastingly, majors are “specialists” that perform only a subset of the minors' behavioral repertoire ( Wilson 1976a b , 1984 ; Calabi and Traniello 1989 ; Brown and Traniello 1998 ). Species of the ant genus Pheidole typically possess such dimorphic workers: minor workers perform most tasks within the nest and forage while majors, with their disproportionately large head, are specialized for colony defense, seed milling and food storage ( Wilson 2003 ). With its worldwide distribution and with its diversity (more than 900 described species, see Bolton 1995 ; Wilson 2003 ), Pheidole has become a key genus for investigating the adaptive nature of caste morphology among species living under different ecological conditions. Furthermore, the diversity of Pheidole has provided a framework to investigate questions concerning the evolutionary ecology of morphological variation as well as the interplay between morphological and behavioral specialization ( Pie and Traniello 2006 ; Moreau 2008 ). Several authors have suggested that the breadth of the behavioral repertoire of minors has been a stabilizing factor, buffering the need for morphological specialization in specific tasks such as brood care and nest construction ( Bolton 1995 ; Pie and Traniello 2006 ; Moreau 2008 ). Conversely, the distinct head morphology of majors appears related to their behavioral specialization for a limited number of tasks. In conditions of highly disturbed caste ratio, majors may enlarge their repertoire size and/or increase their performance rate of some behaviors to compensate for a depletion of the minor caste ( Wilson 1984 , 1986 ; Calabi and Traniello 1989 ; Brown and Traniello 1998 ; Burkhardt 1998 ). However, this behavioral flexibility of majors needs to be examined by separating possible sampling size effects ( Sempo and Detrain 2004 ) from actual changes in their behavioral capacities. The close relationship between caste morphology and behavioral specialization in the Pheidole genus has been highlighted on several species of the New World, which is assumed to be the cradle of this “hyperdiverse” genus ( Moreau 2008 ). The Old World species, Pheidole pallidula (Nylander) (Hymenoptera: Formicidae), offers a contrasting picture of division of labor. With a degree of morphological specialization similar to New World species, P. pallidula majors show an unexpectedly large behavioral repertoire: they are involved in defense ( Detrain and Pasteels 1992 ) as well as foraging ( Detrain and Pasteels 1991 ; Detrain and Deneubourg 1997 ), food storage ( Lachaud et al. 1992 ) and brood care ( Sempo and Detrain 2004 ). With such an extended behavioral repertoire of both minor and major castes, whether and how does social regulation occur in P. pallidula colonies? Would majors further enlarge their behavioral repertoire following deep changes in the colony's caste ratio? If their behavioral repertoire remains unchanged, would majors change their activity rate or their engagement in social behaviors within the nest?",
"discussion": "Discussion It is commonly admitted that, in the dimorphic Pheidole ant genus, minors carry out nearly all colony tasks while majors are specialized and display only a small part (13% to 19%) of the minors' behavioral repertoire ( Wilson 1984 ; Brown and Traniello 1998 ). Figure4:: Total number of social behaviors performed by minors (black histogram) and majors (white histogram) as a function of the fraction of majors in the colony. Bars = mean ± standard deviation, n = 20 scanning sessions for each bar. High quality figures are available Besides, it was reported that majors can act as an emergency stand-by caste by enlarging their repertoire in order to compensate for minors depletion due to demographic changes or predation ( Wilson 1984 ). However, the role of majors in social regulation should be reconsidered by paying attention to possible bias due to sampling size. For instance, the largest majors' repertoire ( Wilson 1984 ) was reported for colonies composed of only majors which also implies the largest number of observations (more than 500 majors observed). As this sampling size was up to 20 times higher than on colonies with “natural” caste ratio, the extended repertoire of majors can simply result from the observation of less frequent behaviors. Here it is shown that, in P. pallidula , changes in the size of majors' repertoire with colony caste ratio can be explained simply by sampling size effects without invoking qualitative changes in majors behavioral profile (see also Jaisson et al. 1988 ; Sempo and Detrain 2004 ). Whatever the caste ratio, the majors' repertoire is strikingly similar to that of minors. Even though they are morphologically specialized with their hypertrophied head and powerful mandibles, P. pallidula majors perform all tasks excepting some brood care behaviors and are nearly as totipotent as the minors. Besides changes in the behavioral repertoire size, social regulation can also take place through an increase in the number of active individuals and/or through an enhancement of individual activity rate. Indeed, inactive individuals constitute a reserve workforce that can assume new tasks depending on colony needs. Those inactive individuals that have no fixed role belong mainly to a transition group intermediate in behavior and age between nurses and foragers (e.g. Lasius niger ( Lenoir and Ataya 1983 ) and Cataglyphis cursor ( Retana and Cerda 1991 )). In polymorphic ant species, differences in the activity level between workers are usually related to body size, with minors being more active than majors as shown in Pheidole guilelmimuelleri and P. pubiventris ( Wilson 1984 ), P. morrisi ( Patel 1990 ), Atta sexdens ( Wilson 1980 ), Erebomyrma nevermanni ( Wilson 1986 ), Megaponera foetens ( Villet 1990 ) or Solenopsis invicta ( Mirenda and Vinson 1981 ; Sorensen et al. 1984 ). Likewise, in the dimorphic P. pallidula ant species, inactive individuals are nearly twice more frequent among majors than among minors. Nevertheless, whatever the colony caste ratio, the activity level of P. pallidula majors is rather high and quite stable in comparison with other Pheidole species (see Wilson 1984 ; Brown and Traniello 1998 ). For instance, the ratio of the majors to minors activity rate remains stable at around 0.65 in P. pallidula , while it varies between 0.05 and 0.52, depending on the caste ratio in P. guilelmimuelleri ( Wilson 1984 ). The relatively high activity level of P. pallidula majors cannot be related to a lower degree of morphological specialization as predicted by the ergonomic optimization theory ( Oster and Wilson 1978 ). Indeed, the relative head widths of the minor to the major caste are remarkably similar being 0.39 for P. guilelmimuelleri ( Patel 1990 ) and 0.42 for P. pallidula ( Detrain 1989 ). One may question the adaptive value of maintaining a higher proportion of inactive ants among majors since, in P. pallidula , resting majors do not participate in social regulation even after large perturbations of the colony caste ratio. Part of the answer lies in the existence of replete majors with distended gaster that account for up to 32% of the total P. pallidula majors population ( Lachaud et al. 1992 ) and for nearly 50% of inner-nest majors (personal observation). This sub-caste of majors is mainly located in quiet nest areas far from the nest entrance (personal observation) or near the brood area ( Sempo et al. 2006a ), does not forage outside the nest or defend it, and is characterized by a very low activity level ( Lachaud et al. 1992 ). Replete majors were also found in other polymorphic ants ( Camponotus spp. ( Wilson 1974 ; Espadaler et al. 1990 ; Hasegawa 1993 ), Myrmecocystus mexicanus \n( Conway 1990 ), P. hortensis ( Calabi et al. 1983 ), P. morrisi ( Yang 2006 ) and Solenopsis invicta ( Mirenda and Vinson 1981 )). In polymorphic ants, a distinction can thus be made between two types of inactive majors: (1) idle unspecialized majors that become active due to colony need for an additional workforce as in many Pheidole species ( Wilson 1984 ; Brown and Traniello 1998 ) and (2) replete majors that stay inactive even after large demographic perturbations (as for P. pallidula majors), acting as living reservoirs that deaden food shortages. As shown above, P. pallidula societies do not compensate for the depletion of minor caste either by extending the behavioral repertoire of majors or by increasing their activity level. However, among the active individuals, some social regulation may also occur by switching toward activities that are essential for social cohesion (i.e. allogrooming) or for colony survival (i.e. food exchanges and brood care). For instance, following the removal of one age-class or a traumatic change in the age structure, workers are first and foremost reallocated to social tasks ( Lenoir 1979 ; Meudec and Lenoir 1982 ; McDonald and Topoff 1985 ; Calabi and Traniello 1989 ). Similarly, in polymorphic ants, major workers can show a high sensitivity to a shift in the colony's caste-ratio (i.e. from less than 10% to 90%) majors) as they increased by 15 to 30 times their rate of social behaviors (as shown for three Pheidole species in Wilson 1984 ). Unexpectedly, in P. pallidula , the rate of social behaviors performed by majors stayed stable regardless of the colony caste ratio. However, due to their high social investment (≈ 60% of the total number of acts), majors maintain brood care and other essential tasks at a satisfactory baseline level, even in colonies almost deprived of its minor caste. The ergonomic resiliency of an ant colony relies on its ability to cope with changes occurring over short and long time-scales ( Oster and Wilson 1978 ). In polymorphic ant species, short-term regulation can rely upon the ability of specialized majors to express new, atypical behaviors and/or to increase their performance rate of social activities ( Wilson 1984 ). This short-term behavioral flexibility of the major worker, increasing the rate of activity as well as the behavioral repertoire in case of minor depletion is well described for different species such as Pheidole guilelmimuelleri and P. pubiventris ( Wilson 1984 ), P. morrisi ( Patel 1990 ) and P. dentata ( Burkhardt 1998 ; Seid and Traniello 2006 ). However, this stand-by caste status of majors is not the common rule for all Pheidole species. Indeed, P. pallidula majors perform an almost full minors repertoire and exhibit a relatively high level of social activities under all circumstances (not only in severe crisis of minors depletion). Secondly, an adaptive demography process may occur to face long-term changes in colony needs and/or environmental constraints (season, predation, etc.). Ideally, the production of majors is expected to be finely tuned to fill-in tasks for which they are specialized, as shown by the increased number of P. pallidula majors in the presence of competitors ( Passera et al. 1996 ). One, however, may notice that, even in similar environmental conditions, there is still a high variability in the caste ratio between colonies of the same species ( P. dentata ( Oster and Wilson 1978 ), P. morrisi (Bhatkar and Whitcomb in Patel 1990 ; Yang 2006 ), P. pallidula ( Passera 1974 )). Hence, it could be more advantageous for an ant society, as observed in P. pallidula , to dispatch each caste into almost all colony tasks except those which cannot be physiologically or morphologically achieved (for example, in P. pallidula : majors do not lay the recruitment trail ( Ali et al. 1988 ; Detrain et al. 1991 )). Without denying the existence of behavioral flexibility among physical castes in ants, this paper has stressed alternative schemes of social regulation. Independent of any qualitative or quantitative changes in the behavioral profile of one caste, other factors, such as a spatial reorganization of ants within the nest, could participate in social regulation. In this respect, differences between castes in aggregative patterns ( Sempo et al. 2006a , b ) would deserve further investigations in order to be coupled to the efficiency and flexibility of task performance by each worker caste."
} | 4,078 |
27668873 | PMC5036821 | pmc | 6,361 | {
"abstract": "The immune response by T cells usually discriminates self and non-self antigens, even though the negative selection of self-reactive T cells is imperfect and a certain fraction of T cells can respond to self-antigens. In this study, we construct a simple mathematical model of T cell populations to analyze how such self/non-self discrimination is possible. The results demonstrate that the control of the immune response by regulatory T cells enables a robust and accurate discrimination of self and non-self antigens, even when there is a significant overlap between the affinity distribution of T cells to self and non-self antigens. Here, the number of regulatory T cells in the system acts as a global variable controlling the T cell population dynamics. The present study provides a basis for the development of a quantitative theory for self and non-self discrimination in the immune system and a possible strategy for its experimental verification.",
"introduction": "Introduction The problem of self/non-self discrimination is a key issue in immunology. Interactions among a variety of immune cells enable them to recognize and to attack non-self antigens such as bacteria and viruses, whereas they normally remain tolerant to self antigens such as tissues. Self and non-self antigens are recognized by T cells via antigen presentation. Antigen presenting cells (APCs) capture antigens, break them into small peptides, and present them on MHC molecules [ 1 ]. T cells interact with the presented antigenic peptides via T cell receptors (TCRs) on their surface, which have structural diversity generated by gene rearrangement [ 2 ]. The affinity between antigen and TCR depends on their structures, and controls whether a T cell is activated (i.e., antigen-specific proliferation of T cells) or not [ 3 ]. As the number of potential antigens is huge, the number of possible interactions among antigens and TCRs is likewise enormous. An essential question here is how the immune system recognizes unpredictable non-self antigens to which it responds and self antigens to which it is tolerant. The classical idea of the self/non-self discrimination is that self-reactive T cells, i.e., T cells having TCRs with high affinity to self antigens, are eliminated in their developmental process(here, the term “affinity” is used to describe the relative responsiveness of a TCR to an antigen rather than biophysical properties). The result is that only T cells tolerant to self tissues are allowed to circulate. This assumption is partially true, as T cells that recognize self antigens undergo clonal deletion in the thymus, which is the so-called negative selection process [ 4 ]. However, it has been understood that the negative selection is not always complete, i.e., the negative selection only partially deletes self-reactive T cells. Self-reactive T cells exist in healthy individuals, and they are non-activated even in the presence of their cognate self antigens [ 5 ]. This fact indicates that the immune response cannot be captured by the reactivity of a single T cell to self/non-self antigens. Rather, the mechanism of self/non-self discrimination should be described by behavior at the cell population level, including various antigens and T cells, activation and suppression of cellular proliferation, and complex cell-cell interactions. Regulatory T (Treg) cells play an essential role in suppressing aberrant immune reactions against self antigens [ 6 ]. Treg cells constitute approximately 10% of peripheral T cells, and depletion of the Treg-fraction from a normal immune system can induce autoimmune diseases [ 7 , 8 ]. Genetic defects in Treg development also cause fatal autoimmune diseases [ 9 ]. These facts show that a substantial number of self-reactive T cells are retained among conventional T (Tconv) cells even after the negative selection in the thymus and further indicate that Treg cells inhibit the proliferation of these self-reactive Tconv cells. Treg cells have as much variety of TCRs as Tconv and are suggested to be selected from self-reactive T cells in the thymus [ 9 ]. According to the tracking of T cells with a particular TCR and deep sequencing of TCR genes of T cell subpopulations, TCR repertoires of Treg and Tconv are partially overlapped but not identical, and TCRs of Treg cells tend to have higher affinity to self-antigens than those of Tconv cells [ 10 ]. Also, stimulated Treg cells, which are exposed to their specific antigen, are able to suppress proliferation of Tconv with irrelevant antigens [ 11 ]. Therefore, despite the necessity of suppression by Treg to avoid autoimmunity, too much intensification of Treg can disturb a beneficial immune response to non-self antigens. Adequate control of Treg population is important to maintain immune response only for non-self antigens. Given this background, how does the immune system maintain both tolerance to self tissues and responses to any non-self antigens? The important condition here is that the selection of T cells in the thymus is imperfect. Suppose that there is a self antigen and a non-self antigen presented on MHC, and that we can measure affinities of these antigens to all Tconv cells in a body, i.e., obtain affinity distributions. Due to the negative selection in the thymus, the peak of the affinity distribution might be larger for the non-self antigen than for the self antigen, while incomplete negative selection means these two affinity distributions can have a significant overlap. Intuitively, the overlap makes it difficult to set a single threshold affinity level beyond which the Tconv cells are activated only for any non-self antigens. Here, the problem is how robust discrimination of self and non-self antigen is possible by such imperfect selections, which generate only biases in the affinity distributions. Although several mathematical models based on interactions of Tconv, Treg, and APC have been studied [ 12 – 15 ], the variety of antigens and TCRs were not considered in those studies. Thus, the mechanism of immune response which responds only to non-self antigens remains unclear. In this study, by using a simple stochastic population model of immune cells, we demonstrate that robust discrimination of self and non-self antigens is possible based on slight differences in the affinity distributions to self and non-self antigens, with the aid of suppression by Treg cells. The results provide a novel mechanism of self/non-self discrimination based on a global control of T cell immune activity by Treg cells.",
"discussion": "Results and Discussion To investigate the mechanism of self/non-self discrimination, we consider the case that one randomly chosen self or non-self target antigen is presented on a certain fraction of APCs, while the other APCs present various self antigens selected randomly. Fig 2a shows the average number of Tconv cells in the system as a function of the fraction of the target antigen denoted by r t . The number of Tconv cells was obtained after the system falls into a steady state of the cell number. In the case of non-self antigen presentation, the number of Tconv cells sharply increases by increasing r t . In contrast, the number of cells is almost unchanged when self target antigen is presented. In the former case, the actively dividing Tconv cells have significantly lower k off (higher affinity) for the presented target antigen than Tconv cells supplied from outside the system. Fig 2b shows how the distribution of k off of Tconv for the non-self target antigens changes by increasing r t . As shown, k off of Tconv cells significantly decreases when r t exceed a threshold level (∼0.1). In this region, Tconv cells which have higher affinities to the target antigen are selectively amplified. The resulting population is dominated by Tconv cells which are the offspring of a few such Tconv cells. The threshold level is determined by a balance between the basal reproduction activity α and the suppression of reproduction by Treg cells. We confirmed that such amplification of reactive T cells to the non-self target and tolerance to the self target are independent of the choice of self and non-self target antigens. 10.1371/journal.pone.0163134.g002 Fig 2 Response of T cell populations with respect to antigen presentation. (a) The average number of Tconv cells as a function of the fraction of the target antigen r t . Each dot represents the result obtained by presentation of different randomly chosen target antigen. The average number of Tconv cells is obtained by averaging the number over 1000 time units after the system settled down to a steady state. (b) The distribution of k off of Tconv cells to the non-self target antigens. The distribution is obtained after the system falls into a steady state. When r t is small (≲ 0.1), the distribution of k off is almost identical to that of supplied Tconv cells to the environment (the distribution is shown in Fig 1b ). In contrast, in the region of r t ≳ 0.1, the dissociation rate to the target antigen significantly decreases, indicating that T cells having high affinity to the target are selectively amplified. (c) The average number of Treg cells as a function of the fraction of the target antigen r t . Each dot represents the result obtained by presentation of a different target antigen. The parameters used in these calculations are N = 1000, M self = M non−self = 5000, K Tconv = K Treg = 5000, k on = 0.1, α = 30, and β = 10. Tconv and Treg cells with randomly chosen TCRs are continuously supplied to the environment at a ratio of 9:1. The flow rate of T cell supply to the environment is 0.5 cell per unit time, while cells which are not attached to an APC are randomly discarded from the environment with a probability of 0.05 per unit time. The mechanism for the self and non-self discrimination in Fig 2 is as follows: when the fraction of the non-self target antigen increases, the affinity between Tregs and antigens on APCs decreases “on average”, due to the affinity bias Δ Treg . Then, the number of Tregs on APCs decreases as shown in Fig 2c , resulting in increasing the division probability D . As a result, a competition between T cell populations arises on the APCs, and eventually Tconv cells which have a relatively higher affinity to the target non-self antigen occupy the APCs. In contrast, when a self-antigen is presented, the number of Treg cells increases by increasing r t as shown in Fig 2c . In this case, Treg cells having relatively higher affinities to the presented antigen are amplified. However, the increase of cell number is limited due to auto-suppression by the division probability. Here, the number of Tregs on APCs can be regarded as a macroscopic variable that controls the immune response, by enabling the threshold response of T cell proliferation with robust self/non-self discrimination, even though the affinity distributions to self and non-self antigens have a significant overlap. It should be stressed that, without the regulation of cell proliferation by Treg cells, the clear self/non-self discrimination such as in Fig 2 is difficult when the negative selection is imperfect. To demonstrate this, we simulated the response to self or non-self antigen presentations when there are no Treg cells and where the affinity distribution of Tconv is identical to that in Fig 1b (Δ Tconv = 0.75 and σ = 1). To evaluate the accuracy of self/non-self discrimination, we define the discrimination score as follows:\n S ≡ ∫ 0 1 2 { ⟨ N n s ( r t ) ⟩ - ⟨ N s ( r t ) ⟩ } d r t . (2) \nHere, 〈 N s ( r t )〉 and 〈 N ns ( r t )〉 represent the average numbers of Tconv cells when a self or a non-self target antigen is presented in the ratio r t , respectively, where the average is taken over various target antigens. This discrimination score corresponds to the area between the curves of self and non-self antigen presentations in Fig 2a , which takes a larger value when the system can discriminate self and non-self antigens accurately. Fig 3a shows the discrimination score S as a function of the parameter α representing the basal reproduction activity. As shown in the figure, in the case without Treg cells, the discrimination score S has a peak around α ∼ 0.2. However, the maximum value of S is significantly smaller than in the case with Treg cells, indicating lower discrimination accuracy in the case without Treg cells. Fig 3b shows the number of Tconv cells in the case without Treg as a function of r t , where the parameter α is set to 0.1 which is close to that which results in the maximum value of S . As shown, the increase in Tconv cell number occurs in both cases of self and non-self antigen presentations, and thus the clear self/non-self discrimination as in Fig 2a is difficult. We have performed numerical experiments using various different parameter sets and confirmed that as long as the affinity distributions of TCRs to self and non-self antigens have a significant overlap as in Fig 1b , the maximum discrimination score is generally smaller in the case without Treg cells than in that with Treg cells, as shown in S1 Fig . The results suggested that when the affinity bias is small, clear self/non-self discrimination as in Fig 2 is possible only with the aid of Treg regulation. 10.1371/journal.pone.0163134.g003 Fig 3 Self/non-self discrimination accuracy. (a) The discrimination score S as a function of basal reproduction activity α . (b) The average number of Tconv cells in the case without Treg regulation. The reproduction activity α is set to 0.1 at which the discrimination score becomes maximum. The parameters are set to those used in Fig 2 , except for α . \n Fig 4 shows the maximum value of the discrimination score S as a function of Δ Tconv and Δ Treg obtained with Treg regulation. The maximization of S is taken over the basal reproduction activity α . As shown, the maximum discrimination score takes larger values in a relatively narrow range of Δ Treg (e.g., 0.25 < Δ Treg < 0.75). The maximum discrimination score becomes small when Δ Treg is large, because in this region the active proliferation of Tconv cells is suppressed by Treg cells for both self and non-self antigen presentation. In contrast, the maximum discrimination score monotonically increases with increasing Δ Tconv . The dependency of discrimination performance on the affinity biases Δ Tconv and Δ Treg is robust on changing model rules and parameters, suggesting it is a general feature of this Treg-driven self/non-self discrimination. 10.1371/journal.pone.0163134.g004 Fig 4 Discrimination score as a function of Δ Tconv and Δ Treg . The color represents the maximum value of the discrimination score S . The maximization of S is taken over the basal reproduction activity α . μ Tconv,self and μ Treg,non−self are set to −3. \n Fig 5 presents the dynamic change of T cell number over time in response to antigen presentation. Fig 5(a) shows the response to a non-self antigen presentation at time = 0 (denoted by arrow), in which time series data obtained by different initial conditions are overlaid. As shown, the response time to reach the new steady state fluctuates over samples. In contrast, when the non-self antigen presentation stops and all APCs start to present randomly chosen self antigens, the number of T cells quickly falls into the original steady state as shown in Fig 5(b) . These results indicated that, in the former case, some time is necessary to find and to amplify Tconv cells which have high affinity to the presented non-self antigen. 10.1371/journal.pone.0163134.g005 Fig 5 The dynamic change of T cell number in response to antigen presentation. (a) Response to a non-self antigen presentation. At time = 0 (denoted by arrow), an non-self antigen is presented in APCs with the ratio r t = 0.3. In the figures, time series data obtained by 10 different initial conditions are overlaid. (b) Response to stopping a non-self antigen presentation. At time = 0, the presented non-self antigen on APCs is replaced by randomly chosen self antigens. The parameters are set to those used in Fig 2 . To sum up, in this study we demonstrated that self/non-self discrimination is possible based on regulation by Treg cells, even when the affinity distributions of TCRs to self and non-self antigens have a significant overlap. The number of Treg cells on APCs plays the role of a macroscopic variable controlling the activation of T cells responding to presented non-self antigens. We emphasize that the results presented herein are independent of details of the modeling and valid over a broad class of models, as long as the model includes stochastic dynamics of T cell populations, the broad affinity distributions, and the suppression of T cell proliferation on APCs by Treg cells. For example, in the simulations presented in this paper, we assumed a fixed association rate constant k on which is independent of presented antigens and TCRs. We confirmed that this assumption can be relaxed, i.e., that similar behavior of self/non-self discrimination emerges when k on depends on the combination of antigens and TCRs, as experimental studies demonstrated [ 17 ]. Furthermore, we assumed that the affinity distribution depends only on the cell type (i.e., Tconv or Treg) and antigen type (i.e., self or non-self), and is independent of each presented antigen. Again, we confirmed that this assumption can be relaxed, i.e., even when the affinity distributions are different among presented antigens, discrimination is possible within a certain range of affinity biases, as shown in S2 Fig for example. For another example of the generality of the results, we analyzed a different model in which each APC presents multiple (e.g., ∼100) antigens and same number of Tconv or Treg cells can attach on the APC simultaneously. As shown in S3 Fig , we confirmed that robust self/non-self discrimination is also possible by this model when Treg cells suppress the proliferation of T cells which attach on the same APC, i.e., the division probability D of T cells on an APC is a function of the number of Treg cells on the corresponding APC. In such local suppression models, self/non-self discrimination was observed as long as the migration of T cells among APCs is active enough. Also, we relaxed the assumption that after cell division, one daughter cell keeps binding to the same APC. We performed the simulation in which both daughter cells are released into the environment after cell division event, and found that the Treg facilitated self/non-self discrimination is possible as shown in S4 Fig . However, we also found that the ranges of parameter values in which the self/non-self discrimination occurs generally shrink in this model, which might suggest the importance of sequestration of APC by T cells for the self/non-self discrimination. There are several previous studies for theoretical analysis of the self/non-self discrimination [ 18 ]. For example, McKeithan [ 19 ] discussed that by assuming multiple reaction steps in TCR and antigen interaction, a small affinity difference between self and non-self antigens to a TCR can be amplified in a way analogous to the error reduction in kinetic proofreading [ 20 ], which can bring about an accurate self/non-self discrimination. However, this approach and many other studies generally rely on the assumption that TCRs have a greater affinity for non-self antigens than for self-antigens. This is not always the case for each combination of TCR and antigens, instead, such affinity differences should rely on the statistical difference of a huge number of combinations of TCR and antigens, as in the affinity bias shown in Fig 1b . The present study is the first to demonstrate that a small bias in the affinity distribution can maintain robust and accurate self/non-self discrimination with the suppression of T cell activation by Treg cells. Also, Freitas and colleagues discussed homeostasis of T cells by quorum-sensing mechanism [ 21 ]. Although our model neglected such complicated interactions among Tconv and Treg cells and included the simplified growth suppression by Treg cells, the inclusion of the quorum-sensing mechanism into our model might facilitate robustness of the Tconv/Treg population dynamics. Of course, there is limited experimental support for this mechanism of discrimination. To verify the Treg-regulation based self/non-self discrimination we proposed, the most important experimental data is the affinity distribution between TCRs and self/non-self antigens. Although the affinities between TCRs and antigens have been quantified for some specific combinations, for better modeling to provide quantitative predictions, a larger number of TCR-antigen combinations should be quantified to clarify the nature of the affinity distribution and how the affinity affects the proliferation of T cells. Furthermore, the suppression of T cell proliferation by Treg cells should also be quantified to evaluate the contribution of Treg regulation to accurate self/non-self discrimination. In our model, the suppression of T cell proliferation by Treg cells was assumed to be global, i.e., the division probability of T cells depends on the total number of Treg cells attaching to APCs in the system. This is based on the assumption that secretion factors such as interleukin-10 produced by Treg cells contribute to suppression. However, in addition to such secretion factors, direct cell-cell interactions might also play a role in Treg regulation. Quantitative evaluation of the cell-cell interaction is necessary to develop more precise models describing the immune response. To obtain these quantitative data, the dynamics of T cell and APC populations should be analyzed at single-cell resolution. Recent advances in time-lapse single-cell imaging might enable us to obtain a large amount quantitative data to analyze dynamics of T cell proliferations and interactions in near future."
} | 5,530 |
36229605 | PMC9579051 | pmc | 6,362 | {
"abstract": "Photosystem I (PSI) enables photo-electron transfer and regulates photosynthesis in the bioenergetic membranes of cyanobacteria and chloroplasts. Being a multi-subunit complex, its macromolecular organization affects the dynamics of photosynthetic membranes. Here we reveal a chloroplast PSI from the green alga Chlamydomonas reinhardtii that is organized as a homodimer, comprising 40 protein subunits with 118 transmembrane helices that provide scaffold for 568 pigments. Cryogenic electron microscopy identified that the absence of PsaH and Lhca2 gives rise to a head-to-head relative orientation of the PSI–light-harvesting complex I monomers in a way that is essentially different from the oligomer formation in cyanobacteria. The light-harvesting protein Lhca9 is the key element for mediating this dimerization. The interface between the monomers is lacking PsaH and thus partially overlaps with the surface area that would bind one of the light-harvesting complex II complexes in state transitions. We also define the most accurate available PSI–light-harvesting complex I model at 2.3 Å resolution, including a flexibly bound electron donor plastocyanin, and assign correct identities and orientations to all the pigments, as well as 621 water molecules that affect energy transfer pathways."
} | 325 |
37483217 | PMC10357540 | pmc | 6,364 | {
"abstract": "Anisotropic photonic hydrogels with alternatively stacked\npoly(dodecyl\nglyceryl itaconate) (PDGI) bilayers and polyacrylamide (PAAm) gel\nlayers are unique soft materials with various functions. It is known\nthat to form the lamellar phase of bilayers, a small amount of co-surfactant\nsodium dodecyl sulfate (SDS) should be present in the precursor monomer\nsolutions of the gels. However, little is known about the influence\nof the co-surfactant on the structure of bilayers and on the mechanical\nproperties of such photonic hydrogels. Herein, we chose several co-surfactants\nand studied the effect of the co-surfactants on the self-assembly\nbehavior of the bilayers and on the mechanical properties of the resulting\nphotonic hydrogels. A macroscopically aligned lamellar phase could\nbe induced for all the co-surfactants. Interestingly, the mechanical\nresponse of the photonic hydrogels sensitively depends on the chemical\nstructure of the co-surfactant, especially at large deformation. We\nhypothesize that doping by small amounts of co-surfactants dramatically\nchanges the anchoring strength and density of PAAm strands onto the\nbilayer surface, thereby influencing the load transfer efficiency\nfrom the bilayer to the PAAm gel layer at large deformation and the\nrupture of the bilayer. This work provides new understanding in the\nmolecular mechanisms of deformation and strengthening in this soft\nand anisotropic nanocomposite, helping to design more robust photonic\nhydrogels.",
"conclusion": "Conclusions Self-assembled PDGI/PAAm hydrogels exhibiting\nstructural colors\nwere successfully synthesized in the presence of small amounts of\ndifferent co-surfactants. Even at such small molar ratios to DGI,\nthe concentration and chemical structure of co-surfactants were found\nto influence significantly the structure and mechanical behavior of\nthe photonic hydrogels. At small strain, the concentration of co-surfactants\nwas found to impact DGI packing within bilayers, that is, bilayer\nstiffness but the co-surfactant structure had limited influence. At\nlarge strain, the change in bilayer density did not account for large\ndifferences in tensile stress between samples at the as-prepared state\nand equilibrium swelling state. Thus, swelling mismatch between the\nPAAm gel layer and the bilayers dramatically weakens the adsorption\nof PAAm strands onto bilayers. PDGI/PAAm hydrogels with neutral or\ncationic co-surfactants exhibited a pronounced strain-hardening at\nlarge deformation, not present with anionic co-surfactants. The strain-hardening\ncorrelated with the rupture of bilayers into smaller rods as analyzed\nby USAXS, which enhances energy dissipation. We hypothesize that,\nin contrast with anionic co-surfactants, neutral or cationic co-surfactants\nwould promote strong adsorption of PAAm strands onto PDGI bilayers,\nthus improving load transfer after rupture of the bilayers at large\ndeformation. This study highlights the importance of interfacial interactions\nin this lamellar bilayer/hydrogel composite system, thus providing\nclues to the design of tougher photonic hydrogels only by small changes\nin their composition.",
"introduction": "Introduction Under certain conditions, surfactants\nare known to form different\nstructures in aqueous solution through self-assembly, 1 e.g., micellar and lamellar. Such organizations rely on\na critical packing parameter 2 defined by\nthe ratio between the volume occupied by the hydrophobic tail on the\none hand and the product between the surface area of the hydrophilic\nhead and the length of the hydrophobic tail on the other hand. These\ngeometrical parameters depend on the chemical structure of the surfactant\nincluding ionic and non-ionic but can also be tuned to some extent\nby the physico-chemical environment, e.g., pH, temperature, or doping\nby co-surfactants in small quantities. 3 , 4 Very small\namounts of sodium dodecyl sulfate (SDS), a widely used anionic surfactant,\nwere found to enable the non-ionic surfactants to form lamellar phases. 5 , 6 By doping in non-ionic amphiphiles, SDS prevents phase segregation\nin aqueous solution by lowering the energy of the system. Moreover,\nSDS plays a role as a stabilizer in the case of defects in lamellar\norganizations (such defects are inherent to liquid crystalline organizations 7 ), thus increasing the lifetime of the assemblies.\nIn this work, we chose monomeric dodecylglyceryl itaconate (DGI) as\na non-ionic surfactant. DGI molecules alone phase-separate into micelles\nin water. In the presence of small amounts of the co-surfactant SDS,\nDGI molecules form lamellar liquid crystal phases above the Krafft\npoint of DGI (43 °C), exhibiting iridescent color. The color\nof such solutions depends on the SDS concentration relative to DGI. 8 Specifically, the solution is red at low SDS\ncontent (0.025 mol % of DGI) and undergoes a blue-shift at higher\nSDS concentrations (2.5 mol % of DGI). Indeed, SDS enables to reduce\nthe distance between neighboring lamellae and compensates the appearance\nof subsequent defects as mentioned above. Tsujii and co-workers\nimmobilized the lamellar liquid crystal phases\nof monomeric DGI in the chemically crosslinked polyacrylamide (PAAm)\nnetwork through one-pot polymerization of the DGI monomer and acrylamide\nmonomer. They found that DGI and AAm are independently polymerized\nand the polymeric DGI (PDGI) bilayers are embedded in the PAAm gel\nmatrix physically without covalent bonding between the two networks. 9 By applying shear to the precursor monomer solution,\nHaque and co-workers succeeded in synthesizing hydrogels with uniaxially\naligned bilayers. 10 − 13 These PDGI/PAAm hydrogels showed unique properties, such as one-dimensional\nswelling, tunable structural colors, and anisotropic mechanical properties.\nThe adsorption of the PAAm gel phase onto PDGI bilayers through strong\nhydrogen bonding 14 endows the photonic\nhydrogels with high strength and toughness. Although Tsujii and co-workers\ninitially reported the possible use of different co-surfactants to\nobtain iridescent DGI solutions, 6 solely\nSDS at fixed amounts relative to DGI (0.025 mol % of DGI) has been\nused so far in the studies of PDGI/PAAm hydrogels. Therefore, the\ninfluence of co-surfactants on the self-assembly of DGI and on the\nproperties of PDGI/PAAm hydrogels remains poorly understood. Herein, we report the effect of different co-surfactants on the\nformation of lamellar liquid crystal phases of DGI and on the mechanical\nproperties of PDGI/PAAm hydrogels as composite materials. We first\ninvestigated in which concentration range these co-surfactants allow\nto form uniaxially aligned bilayers in the PDGI/PAAm hydrogels. Then,\nwe studied the mechanical behavior of the PDGI/PAAm hydrogels by tensile\ntests at the equilibrium swelling state. USAXS analysis enabled to\nunravel the structure change at large deformation. By comparing with\nthe behavior at the as-prepared state, we hypothesize on the possible\nmechanisms induced by different co-surfactants.",
"discussion": "Results and Discussion Structure of PDGI/PAAm Hydrogels with Different Co-surfactants The chemical structure of co-surfactants studied in this work is\nshown in Figure 1 a.\nGeometrical parameters of the co-surfactants, that is, headgroup area a 0 and length of their hydrophobic tail in terms\nof carbon atom number l t , are displayed\nin Figure 1 b, along\nwith the data of DGI. SDS and SST are anionic, DTAC is cationic, and\nthey are fully dissociated in water. The p K a of NBA is around ∼10, meaning that its headgroup is slightly\nprotonated in MilliQ water. Compared to DGI, SDS has the same tail\nlength but its headgroup area is larger, SST has the same headgroup\narea but a longer tail, and DTAC has a smaller headgroup but the same\ntail length, while NBA has both smaller headgroup area and shorter\ntail. In order to investigate the concentration range of each co-surfactant\nfor the formation of the lamellar phase of DGI bilayers, the co-surfactant\nto DGI molar ratio R was varied in the range R = 1:8000 to 1:64 and other formulations for hydrogel synthesis\nwere fixed ( Figure 1 a). When uniaxially aligned bilayers are formed in the PDGI/PAAm\nhydrogels ( Figure 1 c), the hydrogels should show swelling only in the thickness direction,\nperpendicular to the bilayers. This is because the alternatively stacked\nPDGI bilayers are impermeable to water, and swelling is only allowed\nin the thickness direction of the PAAm gel layers between the bilayers. 10 Thus, characterizing the swelling ratios of\nthe hydrogels in the three directions (length, width, and thickness)\nspecifies the structure of the bilayers. Figure 1 (a) Chemical structures\nof molecules used in this work. (b) Summary\nof the geometrical characteristics of monomeric DGI and the four co-surfactants\nused in this study. Headgroup areas in water were calculated or taken\nfrom the literature. 16 − 18 (c) Illustration of the 1D photonic lamellar structure\nof PDGI/PAAm hydrogels synthesized by one-pot polymerization of self-assembled\nmonomeric DGI bilayers in PAAm hydrogel precursor solutions. Small\namounts of co-surfactants were added to the solutions. As plotted in Figure 2 a, the hydrogels exhibited one-dimensional\nswelling behavior at low\nco-surfactant concentration but lateral swelling occurred at increased\nco-surfactant concentration, except for NBA. The concentration up\nto which the 1D swelling was maintained depends on the chemical structure\nof co-surfactants: for SDS up to R = 1:250, for SST\nup to R = 1:500, for DTAC up to R = 1:4000, while NBA exhibited one-dimensional swelling even at the\nhighest concentration investigated ( R = 1:64). For\nPDGI/PAAm hydrogels exhibiting 1D swelling, the bilayers are uniaxially\naligned in a continuous phase to a macroscopic scale of several centimeters\n(sample size). However, it is worth mentioning that at the highest\nNBA concentration ( R = 1:64), both uniaxially aligned\nand curved bilayers were observed in the sample by cryo-SEM ( Figure S1 ). Therefore, according to the structure\nof the co-surfactant, there is a concentration threshold above which\nthe lamellar bilayer structure is disrupted, which allows 3D swelling.\nThis is consistent with macroscopic observations of iridescence in Figure 2 c. When the co-surfactant\nconcentration increases, self-assembled DGI structures would tend\nto deviate from lamellar to curved ones (hexagonal, micellar) due\nto increased head-to-head repulsion, especially for charged co-surfactants.\nThus, more defects will be induced at increased co-surfactant concentration.\nSpecifically, it was recently shown that swelling PDGI/PAAm hydrogels\nin SDS solution tend to transform the lamellar bilayers into micelles\n(above the critical micellar concentration of SDS), which is accompanied\nby 3D swelling. 19 In our case, since DGI\nmonomers are initially not polymerized when self-assembled in the\npresence of SDS, a much lower SDS concentration is likely to disturb\nthe lamellae. Figure 2 Structure characterization of the PDGI/PAAm hydrogels\nprepared\nwith different co-surfactant to DGI molar ratio R for the four co-surfactants.\n(a) Swelling ratios in thickness, width, and length direction relative\nto the as-prepared state. (b) Photographs of the hydrogels placed\non a black substrate (each square of the photos is 5 mm × 5 mm),\nwith increasing R from left to right, together with\nreflectance intensity spectra of the hydrogels. (c) Table summarizing\nthe structural properties of the hydrogels. Upper row: 1D swelling\nand iridescence properties; lower row: bilayer modulus in MPa estimated\nfrom Equation (1) (see Figure S5 ). The\ndata of SDS at R = 1:4000 is from the literature. 10 We then characterized the structural color to clarify\nthe lamellar\nstructure of the hydrogels. Figure 2 b shows photographs of the hydrogels prepared at various R for the four co-surfactants, together with reflectance\nintensity spectra. In general, hydrogels with SDS, SST, and NBA were\ntransparent, showing bright structural color especially above R = 1:1000, while those with DTAC were slightly opaque,\nshowing dim structural color. Above R = 1:1000, hydrogels\nwith SDS and NBA exhibited reflectance peaks around 400∼500\nnm, while the reflectance peaks of hydrogels with SST and DTAC were\nmuch weaker. Specifically, hydrogels with DTAC showed peaks in the\nnear-infrared region (around 700∼800 nm), indicating large\ninter-bilayer spacing d , consistent with their low\nco-surfactant content. The peaks of hydrogels with SDS, NBA, and SST\nexhibited a slight blue-shift with increasing amounts of co-surfactants\n( Figure S2 ), corresponding to a decrease\nin the inter-bilayer distance d ( Figure S3 ), in accordance with previous observations for DGI\nin solution with SDS. 8 In contrast, hydrogels\nwith DTAC showed a slight red-shift with DTAC concentration increase.\nInterestingly, the hydrogel with NBA at R = 1:64\nexhibited a broader peak, confirming its structural inhomogeneity\nat large scale. The effect of co-surfactant concentration on anisotropic\nswelling and structural color is summarized in Figure 2 c. For all co-surfactants considered, small R induces both 1D swelling and iridescence, that is, formation\nof continuous bilayers. However, differences appear at higher R , which depend on the chemical structure of the co-surfactant.\nFor example, the hydrogel with SDS at R = 1:64 was\nvery soft and almost colorless, while some hydrogels exhibited structural\ncolor at high R even with an imperfect 1D swelling.\nSpecifically, for SST at R = 1:250, the hydrogel\nshowed lateral swelling and was much softer but exhibited a slightly\nblueish structural color, meaning that the hydrogel has discontinuous\nbilayers. 13 , 19 This observation indicates that a longer\nhydrophobic tail destabilizes the bilayers, which is also noticed\nin other liquid crystals. 20 Our work further\nsuggests that in the case of a long hydrophobic tail, a larger headgroup\narea of the co-surfactant would counter-balance the destabilizing\neffect. For example, 1D swelling is obtained with DTAC or SDS at R = 1:4000 but not with DTAC at higher R . When the hydrophobic tail is short enough as in the case of NBA,\nthe co-surfactant stabilizes the bilayers over a wider range of concentrations\neven though the headgroup area is small. Effect of Co-surfactant Concentration and Swelling on the Mechanical\nBehavior of the Photonic Hydrogels To characterize the mechanical\nproperties of the hydrogels, we chose to compare hydrogels with SDS,\nNBA, and SST above R = 1:1000 and DTAC at R = 1:8000 and 1:4000 since these hydrogels used DGI synthesized\nfrom the same batch. In fact, previous work noticed slight variation\nin tensile behavior of the hydrogels at large deformation for different\nbatches of DGI ( Figure 2 a; 21 Figure 1 b inset 22 ). Figure 3 a,b show the co-surfactant\nconcentration effects on the tensile stress–strain curves.\nAll samples showed overlapped tensile behaviors at small strain (strain\n<2.5) but the tensile behaviors at large strain (strain >2.5)\nwhere\nstrain-hardening starts to occur was highly dependent on the co-surfactant\nchemical structure and concentration. Before the yielding strain (ca.\n0.25), bilayers deform without rupture, while at a large strain, bilayer\nrupture occurs. 22 Therefore, the effect\nof the co-surfactant on the self-assembly of bilayers and on their\nrupture can be elucidated by analyzing the stress–strain curves\nat small and large strain, respectively. Figure 3 Tensile behavior of PDGI/PAAm\nhydrogels with uniaxially aligned\nlayered structure prepared with various co-surfactants. (a and b)\nRepresentative tensile behavior of the hydrogels at equilibrium swelling.\n(c) Tensile behavior of hydrogels with SDS or NBA at the as-prepared\nstate, as typical examples. (d) Comparison between the tensile behavior\nat the as-prepared state and that at the equilibrium swelling state,\nwhere the nominal stress for the swollen samples was corrected by\ntheir thickness swelling ratio. The tensile response was obtained\nalong the bilayer direction. The numbers in the figures indicate the\nco-surfactant to DGI molar ratio ( R ). The insets\nin each figure show the enlarged plot at small strain. The small strain behavior is shown in Figure 3 a,b as insets. Concentration\ndependence is\nobserved for both anionic co-surfactants SDS and SST but not for DTAC\nand NBA as emphasized in semi-log plots in Figure S4 . If the elasticity of the hydrogels at small strain is dominated\nby the inter-lamellar distance, we would expect that the modulus E and interlayer spacing d follow the relation E ∼ 1/ d . However, such correlation\nis not observed ( Figure S5 ). This result\nsuggests that DGI molecular packing slightly depends on R . To verify this speculation, we calculated the Young modulus of\nthe bilayer from the Young modulus of the PDGI/PAAm hydrogels ( Figure S6 ) and that of the pure PAAm hydrogel.\nThe results of bilayer modulus are shown in Figure 2 c. As expected, hydrogels with SST ( R = 1:250) that showed lateral swelling have relatively\nsoft PDGI bilayers (1 MPa), while bilayers with DTAC are significantly\nstiffer (4.8–6.1 MPa). Hydrogels with SDS and NBA exhibit a\nsimilar range of bilayer stiffness (2.8–4.7 MPa). Interestingly,\nwhen compared at the same hydrophobic tail length ( l t = 12 for SDS and DTAC), it seems that the smaller headgroup\narea of the co-surfactant induces higher stiffness of the bilayers\n(DTAC) than the larger headgroup area (SDS). Noticeably, increasing\nNBA concentration up to R = 1:64 does not further\nstabilize the bilayer. At large strain, samples with NBA or\nDTAC ( Figure 3 b) showed\nstronger strain-hardening than\nsamples with SDS or SST ( Figure 3 a). SDS concentration at R = 1:1000\nseemed to enhance strain-hardening, while hydrogels with SST at 1:250\nand 1:1000 molar ratio to DGI appeared distinctly softer (see the\nsemi-log plot in Figure S7 ). Noticeably,\nthe hydrogel with SST at R = 1:1000 exhibited a much\nhigher Young’s modulus than that of the hydrogel at R = 1:250 (almost 3 fold), stressing that the behavior at\nsmall and large strains originates from different mechanisms. Surprisingly,\nhydrogels with NBA or DTAC exhibited a concentration-dependent strain-hardening\nbehavior (except for NBA at R = 1:64): strain-hardening\nwas enhanced at higher NBA concentration ( R = 1:250)\nand at lower DTAC concentrations ( R = 1:8000) ( Figure 3 b and Figure S8 ). Therefore, small changes in co-surfactant\nconcentration dramatically affected the tensile response of PDGI/PAAm\nhydrogels at small and large strains. It is worth mentioning\nthat during swelling, PAAm gel layers exert\na biaxial tension onto the bilayers because the gel layers intend\nto swell in an isotropic manner. Consequently, bilayers might become\nless stable in a hydrogel at the equilibrium swelling state and more\nsusceptible to rupture under tensile deformation. In order to skirt\nswelling-induced effects, the tensile behavior of the hydrogels was\nfurther investigated in their as-prepared state. For comparison purposes,\nhydrogels with SDS or NBA were chosen as models for each type of mechanical\nbehavior (with R = 1:1000, 1:500, and 1:250). It\nshould be noted that the mechanical behavior of pure PAAm hydrogels\nwithout DGI is independent of the presence of SDS or NBA ( Figure S9 ). As plotted in Figure 3 c, the tensile response of hydrogels with\nNBA at the as-prepared state showed weak dependence on its concentration.\nIn contrast, a lower SDS content correlated with enhanced strain-hardening\nat large strains. Small strain behavior and Young’s moduli\nof the hydrogels increased with decreasing SDS concentration but showed\nslight inverse dependence on NBA concentration ( Figure 3 c, inset and Figure S10 ). To investigate how swelling affects the mechanical behavior\nof\nthe hydrogels, we compared samples at the as-prepared and equilibrium\nswelling states. Since small strain behavior mostly arises from physical\ninteractions within PDGI bilayers, we plotted tensile curves of hydrogels\nwith NBA or SDS at R = 1:500 at the swollen state\nwith correction of the nominal stress by their thickness swelling\nratio in Figure 3 d\n(see Figure S11 for a comparison of non-corrected\ncurves). At small deformation ( Figure 3 d, inset), the tensile behavior at the as-prepared\nstate and corrected swollen state collapsed within experimental error.\nThis indicates that swelling of the PAAm gel layer actually has little\neffect on the PDGI bilayers. However, at large deformation, there\nis a significant difference even after correction. This means that\nthe interactions between PAAm gel layers and PDGI bilayers are reduced\nto a greater extent than what would be expected from the softening\ninduced only by swelling. This result suggests that the hydrogen bonds\nbetween PAAm strands and PDGI bilayers are weakened by swelling mismatch. From the swelling-induced softening at large deformation, we speculate\nthat the chemical structure of the co-surfactant also plays a role\nin the physical interactions between PAAm gel layers and PDGI bilayers. Co-surfactant-Dependent Toughening Mechanism at Large Strain\nin Photonic Hydrogels Figure 4 a compares the large strain tensile behavior of the\nselected hydrogels at R = 1:500 for SDS, NBA, and\nSST and R = 1:8000 for DTAC. This concentration was\nchosen because of similar bilayer stiffness (or highest one in the\ncase of SST). A previous study on PDGI/PAAm hydrogels with the SDS\nco-surfactant at R = 1:4000 showed that stretching\nalong the direction of the lamellae induces structure transformation\nto fibrous structures aligned along the deformation axis. The rupture\nof PDGI bilayers occurred with the formation of rod-like structures\nat small elongation (strain ∼1–4). 22 In order to understand the origin of strain-hardening or\nsoftening at large elongation, we performed USAXS to compare the structural\nchanges in the hydrogels with different co-surfactants. To reveal\nhow elongation affects the rupture of the bilayers, we studied the\nin-plane structure by imposing the X-ray beam perpendicular to the\nplane of the lamellae ( Figure 4 a, schematics). The 2D USAXS patterns of PDGI/PAAm gels are\nshown in Figure 4 b\nfor strain ∼0 and strain ∼7.3, where large differences\nin strain-hardening behavior were observed for different co-surfactants.\nAt strain ∼0, the 2D patterns showed a very weak and diffuse\nisotropic scattering, confirming the in-plane isotropic structure\nof the bilayers independent of the co-surfactant ( Figure S12 ). Such isotropic patterns strongly contrast with\nthe ones observed perpendicular to the bilayers ( Figure S13 ). At strain ∼7.3, the 2D patterns were strongly\nanisotropic, and two scattering streaks in the equatorial direction\nwere clearly observed perpendicular to the deformation direction.\nSuch streaks have been observed before in tough hydrogels where the\nstructure, initially isotropic, becomes anisotropic during uniaxial\nstretching (alignment along the stretching direction). 23 In PDGI/PAAm hydrogels, streaks are a characteristic\nsignature of rod-like structures, indicating the rupture of bilayers\nalong the elongation direction. 22 Therefore,\nrupture of the bilayers and alignment of the PAAm chains along the\ntensile direction occur simultaneously at large strains. Figure 4 Mechanism of\ndeformation and energy dissipation during tensile\nloading of PDGI/PAAm hydrogels. (a) Uniaxial tensile behavior of the\nhydrogels with different co-surfactants compared at fixed R (at similar bilayer stiffness). (b) USAXS patterns correlate\nwith the strain-hardening or softening at large deformation. (c) Ruland’s\nstreak method was applied at 730% strain for the hydrogels at fixed R , (d) from which the length of rod-like structures in the\ndifferent hydrogels was determined. (e) Cyclic tensile behavior at\nroom temperature or 50 °C of PDGI/PAAm hydrogels with either\nSDS or NBA co-surfactants at different R (swollen\nat equilibrium). (f) Photos of the photonic hydrogels at different\ntemperatures. Interestingly, USAXS patterns at strain ∼7.3\ncorrelate well\nwith the co-surfactant-dependent large-strain tensile behavior. The\nstreaks of hydrogels with NBA or DTAC that showed distinct strain-hardening\nare much longer than those of SDS and SST, indicating that rod-like\nstructures are relatively shorter for the gels showing strain-hardening.\nTo quantitatively estimate the rod length, we adopted the Ruland streak\nmethod. 24 An example of Lorentzian fit\nof experimental data from the SDS 1:500 sample (integrated profile\nof the 2D azimuthal plot at q = 2.7 m Å –1 ) is shown in Figure 4 c. Figure 4 d shows the Ruland plot, that is, the azimuthal widths, Δψ 1/2 along the equatorial axis (ψ = 90) against the inverse\nof scattering vector, 1/ q . The rod length L estimated\nfrom the slope of the Ruland plot is shown in the table below the\nplot. It is worth mentioning that USAXS intensity is weaker than that\nof SAXS, explaining lower r 2 during line\nfitting. From these results, we can state that hydrogels with NBA\nand DTAC have shorter rod-like structures (below 80 nm), while those\nwith SDS and SST have longer ones (112 and 393 nm, respectively).\nTherefore, we may hypothesize that the breaking of the bilayers into\nrods shorter than 80 nm efficiently contributes to the strain-hardening\nbehavior of the hydrogel. Indeed, breaking more physical bonds between\nDGI monomers induces more energy dissipation. Such behavior reminds\nthat of spider-silk under uniaxial tension, where similarly, strain-hardening\nat large deformations was associated to enhanced H-bond breakage within\nsilk protein β-sheets. 25 To\ntest this hypothesis, we compared the cyclic tensile behavior\nof PDGI/PAAm hydrogels with SDS or NBA at large deformation (up to\nstrain ∼8.3). As plotted in Figure 4 e (first cycle only), PDGI/PAAm hydrogels\nwith SDS did not exhibit significantly different cyclic behavior nor\npeak stress with respect to their SDS content. In contrast, hydrogels\nwith NBA reached higher peak stress and larger hysteresis loop with\nincreased NBA content. All PDGI/PAAm hydrogels exhibited larger hysteresis\nloop during the first cyclic loading and unloading, followed by smaller\nloops that are almost overlapped in the later cycles ( Figure S14 ). Such behavior is typical of pure\nphysical hydrogels, 26 confirming that energy\ndissipation processes mostly originate from breaking of non-covalent\ninteractions, like sacrificial bonds. In order to probe the strength\nof physical interactions, the same set of experiments was conducted\nat a high temperature (∼50 °C), above the Krafft point\nof DGI (43 °C), where bilayers are expected to behave like a\nliquid crystal. First, the Young modulus of all hydrogels dropped\nby a ∼9 fold factor and did not seem to be influenced by the\nconcentration of co-surfactants ( Figure S15 ). It became close to that of a single PAAm hydrogel, about 10 kPa,\nconfirming that bilayers lost their cohesion at the macroscopic scale.\nSecond, Figure 4 f shows\nthat all hydrogels still exhibited bright structural color after heating,\nindicating that the lamellar organization is preserved at ∼50\n°C. Finally, the cyclic tensile behavior (first loop) in Figure 4 e shows that hydrogels\nwith increasing NBA content still exhibited higher peak stress at\nlarge deformation, while hydrogels with increasing SDS concentration\nshowed opposite trend and lower peak stresses. The hysteresis of the\nfirst loop appeared reduced independent of the co-surfactant, suggesting\na decrease in the strength of physical interactions. However, the\namount of dissipated strain energy at 50 °C during the first\ncycle is still somewhat preserved at the highest NBA content ( Figure S16 ). At the solution state, it is known\nthat higher temperatures tend to decrease the solubility of amphiphiles\ndue to reduced hydration of the hydrophilic headgroups. 27 As a consequence, headgroup/headgroup repulsion\nbecomes weaker, which decreases their occupied surface area and may\nchange their optimal configuration, that is, from lamellae to inverted\nmicelles in the case of DGI. This phenomenon could reduce the strength\nof hydrophobic interactions within PDGI bilayers, thus reducing both\npeak stress and hysteresis at 50 °C. However, the somewhat preserved\nphysical interactions at high temperature and highest NBA concentration\nreveal the strength of interfacial interactions in this system. Knowing that PAAm strands were shown to adsorb strongly via hydrogen\nbonds onto PDGI bilayers, 14 we propose\nthe following mechanism. At the as-prepared state, both the co-surfactant\nconcentration and the chemical structure determine the number of anchorage\npoints between PAAm strands and PDGI bilayers. More specifically,\ncationic co-surfactants (DTAC and NBA) would tend to promote such\nphysical bonds more than anionic co-surfactants (SDS, SST) due to\nthe affinity of PAAm chains for positively charged surfaces. 28 Since the number of adsorption sites of PAAm\nstrands onto PDGI bilayers was shown to increase with decreasing amounts\nof water molecules, 15 it is also possible\nthat co-surfactants with a smaller headgroup area than DGI (NBA and\nDTAC) tend to hydrate DGI headgroups less than SDS or SST, thus promoting\nPAAm adsorption. After swelling, some of the hydrogen bonds between\nPAAm strands and PDGI bilayers are irreversibly broken due to swelling\nmismatch between the bilayer and the PAAm gel layer, independent of\nthe co-surfactant. During uniaxial tensile deformation, the rigid\nPDGI bilayers carry the load first and then break into rod-like objects\nof about 800 nm length until strain ∼4. 22 Then, the load is transferred to the soft PAAm gel layers\nthrough hydrogen bonding of PAAm strands onto a hard bilayer surface,\nas reported in another nanocomposite system. 29 PAAm strands with more anchorage points (NBA and DTAC) tend to reach\ntheir limit of extensibility at lower strains, thus inducing strain-hardening.\nDue to strain-hardening of PAAm strands, PDGI bilayers further rupture\ninto smaller rods as strain increases, which dissipates energy. When\ncompared at strain ∼7.3, more adsorption induces stronger strain-hardening.\nOverall, this mechanism reminds that of double network hydrogels, 30 where physical bonds between the two networks\nprovide synergistic reinforcement mechanisms. 31 , 32 In the case of hydrogels with SDS or SST, less anchorage points\nimply that PAAm strands carry load mostly in the bulk of the PAAm\ngel layer and PDGI bilayers break into longer rods (less efficient\nload transfer and lower energy dissipation)."
} | 7,662 |
29084554 | PMC5663086 | pmc | 6,365 | {
"abstract": "Background 3-Hydroxypropionic acid (3-HP) is an important platform chemical, serving as a precursor for a wide range of industrial applications such as the production of acrylic acid and 1,3-propanediol. Although Escherichia coli or Saccharomyces cerevisiae are the primary industrial microbes for the production of 3-HP, alternative engineered hosts have the potential to generate 3-HP from other carbon feedstocks. Methylobacterium extorquens AM1, a facultative methylotrophic α-proteobacterium, is a model system for assessing the possibility of generating 3-HP from one-carbon feedstock methanol. Results Here we constructed a malonyl-CoA pathway by heterologously overexpressing the mcr gene to convert methanol into 3-HP in M. extorquens AM1. The engineered strains demonstrated 3-HP production with initial titer of 6.8 mg/l in shake flask cultivation, which was further improved to 69.8 mg/l by increasing the strength of promoter and mcr gene copy number. In vivo metabolic analysis showed a significant decrease of the acetyl-CoA pool size in the strain with the highest 3-HP titer, suggesting the supply of acetyl-CoA is a potential bottleneck for further improvement. Notably, 3-HP was rapidly degraded after the transition from exponential phase to stationary phase. Metabolomics analysis showed the accumulation of intracellular 3-hydroxypropionyl-CoA at stationary phase with the addition of 3-HP into the cultured medium, indicating 3-HP was first converted to its CoA derivatives. In vitro enzymatic assay and β-alanine pathway dependent 13 C-labeling further demonstrated that a reductive route sequentially converted 3-HP-CoA to acrylyl-CoA and propionyl-CoA, with the latter being reassimilated into the ethylmalonyl-CoA pathway. The deletion of the gene META1_4251 encoding a putative acrylyl-CoA reductase led to reduced degradation rate of 3-HP in late stationary phase. Conclusions We demonstrated the feasibility of constructing the malonyl-CoA pathway in M. extorquens AM1 to generate 3-HP. Furthermore, we showed that a reductive route coupled with the ethylmalonyl-CoA pathway was the major channel responsible for degradation of the 3-HP during the growth transition. Engineered M. extorquens AM1 represents a good platform for 3-HP production from methanol. Electronic supplementary material The online version of this article (10.1186/s12934-017-0798-2) contains supplementary material, which is available to authorized users.",
"conclusion": "Conclusions The engineered M. extorquens AM1 demonstrated the production of 3-HP on methanol with a titer of 6.8 mg/l in shake flask cultivation, which was further improved over tenfold by increasing the promoter strength and copy number of mcr \n 550–1219 . Although further strain optimization is required to make this system industrially relevant, metabolic engineering precedents exist that have resulted in similar magnitudes of increase [ 7 ]. It has been shown in engineered P. denitrificans that 3-HP was degraded through an oxidative route, in which 3-HP was first oxidized to malonate semialdehyde and then metabolized to acetyl-CoA [ 17 , 18 ]. However, our metabolomics, 13 C-labeling analysis, in vitro enzymatic assays and knockout experiment demonstrated that 3-HP was mainly reduced to 3-HP-CoA and then sequentially converted to acrylyl-CoA and propionyl-CoA during the growth transition in engineered M. extorquens AM1. This novel work makes a good start for bioconversion of methanol into economically important product of 3-HP.",
"discussion": "Discussion \n Methylobacterium extorquens AM1 has been considered as a potential platform strain for industrial production of valuable chemicals such as mevalonate, 1-butanol and 2-hydroxyisobutyrate [ 30 , 32 , 33 , 41 ]. In this work, we first optimized a 3-HP synthetic pathway in M. extorquens AM1, and then focused on the demonstration of the mechanism of 3-HP reassimilation. It has been reported that tuning of gene expression levels was critical for proper functioning of a heterologous synthetic pathway in M. extorquens AM1. For instance, Hu et al. found that the strain expressing the adhE2 and ter from a promoter of intermediate strength produced the highest 1-butanol [ 32 ]. In our case, four different promoter strengths were tested and the strongest promoter mxaF was shown to generate the highest 3-HP, comparable with the preliminary titer of other engineered microorganisms [ 5 ]. The pool size of precursor acetyl-CoA was similar between the YHP5 strains and the other three recombinant strains, implying that upstream metabolic fluxes were not significantly affected by the introduction of the malonyl-CoA pathway. Moreover, overexpressing mcr \n 550–1219 resulted in further improvement of 3-HP production and decrease of the acetyl-CoA pool size, suggesting that this synthetic pathway drew more acetyl-CoA flux into 3-HP synthesis and the supply of acetyl-CoA may become insufficient. In the YHP6 strain carrying dual strongest promoters of mxaF – mxaF , the titer of 3-HP and the pool of acetyl-CoA were both lower than that in the YHP5 strain. This was presumably because high expression of Mcr could cause metabolic imbalance and in turn interrupt the entire flux distribution. In order to pull more flux to acetyl-CoA, we overexpressed the gene pyk encoding pyruvate kinase which was predicted to lead to higher relative fluxes into acetyl-CoA in M. extorquens AM1 [ 42 ]. However, the improvement of 3-HP production was not observed (Additional file 2 : Figure S6). In addition, the constructed malonyl-CoA pathway led to the net consumption of 2 molecules of NADPH per 1 molecule of 3-HP produced. In M. extorquens AM1 grown with methanol, cell growth is limited by reducing power [ 36 ], suggesting that an insufficient supply of NADPH is another bottleneck for 3-HP production. Further multiple genes manipulation for improving acetyl-CoA and reducing power supply is necessary to enhance the 3-HP production in M. extorquens AM1. In addition, a partial β-alanine pathway has been constructed to be able to produce the 3-HP (Additional file 2 : Figure S2), it would be interesting to combine full β-alanine pathway with malonyl-CoA pathway to figure out whether the combined pathways would improve the titer of 3-HP further. Recent reports have demonstrated that 3-HP was steadily produced by utilizing either the malonyl-CoA pathway or the β-alanine pathway [ 5 – 10 , 43 ]. However, in M. extorquens 3-HP was rapidly degraded upon the transition from exponential to stationary growth phase. The addition of 3-HP into the medium of the cell culture further demonstrated that 3-HP degradation was lower than the release during exponential phase but higher during stationary phase. This degradation phenomenon was also observed in the recombinant P. denitrificans , Meyerozyma guilliermondii and Rhodococcus erythropolis [ 15 , 44 , 45 ]. Lee et al. demonstrated that the genes hpdh and hbdh encoding 3-HP dehydrogenase and 3-hydroxyisobutyrate dehydrogenases in P. denitrificans were mainly responsible for 3-HP degradation through the oxidative route, in which 3-HP was first oxidized to malonate semialdehyde and then metabolized to acetyl-CoA [ 17 ]. However, neither untargeted nor targeted metabolome analysis detected the accumulation of malonate semialdehyde, which might be due to its instability in the extraction or low pool abundance in cell. But 3-HP-CoA, propionyl-CoA and its downstream intermediates were observed to build up in the cell, suggesting 3-HP was reduced to 3-HP-CoA via the reductive route and then reassimilated by the EMC pathway. Previously we have demonstrated a highly efficient pyrophosphate-mediated glycolytic pathway for methane assimilation in Methylomicrobium alcaliphilum strain 20Z by tracing the pyruvate 13 C-labeling pattern [ 46 ]. Here, if 3-HP reassimilation flux through the reductive pathway produced triply labeled 3-HP-CoA and propionyl-CoA, and the latter enters the EMC pathway, malate would maintain the same labeled pattern and have faster 13 C-incorporation than its downstream metabolite, acetyl-CoA. If 3-HP was reassimilated by the oxidative route, it would be expected to generate more amounts of doubly labeled acetyl-CoA and fewer amounts of doubly and triply labeled propinyl-CoA in a time course. Our results demonstrated that both routes operated but the reductive route functioned as the major one for reassimilating the 3-HP based on two possible explanations. Firstly 3-HP-CoA and propionyl-CoA were more significantly labeled compared to acetyl-CoA, proposing that reductive route flux was higher than oxidative route. Secondly by 5 and 12 h, the labeled pool of acetyl-CoA was much higher than its precursor of malate, suggesting part of labeled acetyl-CoA might come from the oxidative route. Moreover, the enzymatic assay not only confirmed that the reductive route for 3-HP assimilation in M. extorquens AM1 was through acrylyl-CoA as intermediates, but also suggested that the genes encoding enzymes for the reductive route were unlikely able to be induced by 3-HP. The latter finding differed with the previous observation that the degradation of 3-HP by the oxidative route in P. denitrificans was increased greatly when cells were exposed to 3-HP [ 47 ]. In that study, the transcription of hpdh and hbdh involved in the oxidative route was found to be induced by a LysR-type transcriptional regulator binding with 3-HP molecule. \n Methylobacterium extorquens AM1 harbors a predicted DctA dicarboxylic acid transporter, which has been proposed to uptake C4 or C5 dicarboxylic acid and even C3 pyruvate [ 35 , 38 , 48 ]. Therefore, a knock out mutant of the dctA homologue (META1_3271) is expected to have an impact on the production of 3-HP. Unfortunately, dctA mutant was still able to degrade the 3-HP, which may either be due to an incomplete disruption of acid transport or to the existence of at least one additional system that can transport 3-HP into the cell [ 48 , 49 ]. In addition, the transport function of some DctA examples has been shown to be sodium ion dependent, and reduced sodium ion has been demonstrated to block the reuptake of mesaconic acid and methylsuccinic acid in engineered M. extorquens AM1 [ 48 ]. However, in our study 3-HP degradation was not decreased when the sodium ion was reduced to 60-fold lower, suggesting either DctA is not playing a role in 3-HP degradation or it is independent of sodium ion. An acrylyl-CoA reductase (AcuI) catalyzing the reduction of acrylyl-CoA to propionyl-CoA has been identified in Ruegeria pomeroyi and R. sphaeroides (Table 2 ) [ 50 ]. The acuI mutant was unable to grow on 3-HP as sole carbon source in R. sphaeroides [ 14 ]. The deletion of gene META1_4251 encoding a protein with 59% amino acid sequence identity to AcuI of R. sphaeroides resulted in slower degradation of 3-HP in late stationary phase. One possible explanation is that low accumulation of acrylyl-CoA in the beginning of stationary phase would not be capable of disrupting the reaction equilibrium between 3-HP-CoA and acrylyl-CoA, but high accumulation of acrylyl-CoA would generate a strong feedback to shift the equilibrium against acrylyl-CoA production. Hence, we propose that META1_4251 is involved in the third reaction of 3-HP assimilation. In addition, acyl-CoA transferase or acetyl-CoA/propionyl-CoA synthetase was reported to be responsible for converting 3-HP to 3-HP-CoA in engineered E. coli for the production of acrylic acid (Table 2 ) [ 51 ]. An attempt was made to delete the gene META1_2054 encoding a putative acyl-CoA transferase with 51% amino acid sequence identity to YdiF of Cupriavidus necator . However, we failed to obtain the null mutant, suggesting this gene might have an essential function for M. extorquens AM1 growth. The purified protein was shown to exhibit poor catalysis with 3-HP as a substrate, suggesting that META1_2054 was unlikely a key gene for the first reaction in the reductive route. Therefore, once other genes are identified and biochemically characterized, it may become clearer how M. extorquens AM1 reassimilates 3-HP and will prevent the degradation and enhance product formation rate."
} | 3,074 |
39450158 | null | s2 | 6,366 | {
"abstract": "Finding the shortest path in a graph has applications in a wide range of optimization problems. However, algorithmic methods scale with the size of the graph in terms of time and energy. We propose a method to solve the shortest-path problem using circuits of nanodevices called memristors and validate it on graphs of different sizes and topologies. It is both valid for an experimentally derived memristor model and robust to device variability. The time and energy of the computation scale with the length of the shortest path rather than with the size of the graph, making this method particularly attractive for solving large graphs with small path lengths."
} | 165 |
36321143 | PMC9552755 | pmc | 6,369 | {
"abstract": "Triboelectric nanogenerators (TENGs) have garnered considerable attention as an emerging energy harvesting technology. To improve the electrical properties of the triboelectric materials in TENGs, various micro- and nanomaterials with strong charge-trapping capabilities are introduced as filler materials. However, the fillers generally perform a single function and lack long-term operational durability. Hence, further research is required to achieve stable and efficient TENGs. In this study, NH 2 metal–organic frameworks (NH 2 -MOFs) were combined with a cellulose nanofiber (CNF) to prepare a composite film. NH 2 -MOFs have an aminated bimetallic organic backbone with strong charge-induction and charge-trapping capabilities. Thus, their addition significantly improved the stability, positive triboelectric properties and charge-trapping performance of the composite film. The optimized composite film and a fluorinated ethylene propylene film were used as triboelectric pairs to assemble a TENG. The electrical performance of the TENG was approximately 230% greater than that of a TENG with a pure CNF film and remained very stable for at least 90 days. These results demonstrate that NH 2 -MOFs are promising fillers for improving the performance of TENGs and expanding the range of materials used in TENG construction.",
"conclusion": "Conclusions In this study, to enhance the electrical properties of TENGs, aminated bimetallic MOFs (NH 2 -MIL-101(Fe,Cu)) were introduced as fillers. Unlike conventional fillers, the NH 2 -MOFs had bifunctionality owing to the –NH 2 groups, which improved the charge-induction capability, and bimetals, which increased the number of charge-trapping sites. The mass ratio of NH 2 -MOFs in the CNF film was optimized. The output voltage and current reached 194 V and 14 μA, respectively, at a M 3 mass fraction of 0.5 wt%, with a maximum output power density of 20.3 mW cm −2 at a load resistance of 10 7 Ω. In addition, the C M 3 -TENG exhibited superior stability and durability, could light 84 LEDs simultaneously, and could charge six different capacitors according to its self-powering characteristics. In summary, given the structural versatility and designability of MOFs, embedding NH 2 -MOF fillers into CNFs is a direct and effective way to improve the electrical performance of TENGs.",
"introduction": "Introduction As the popularity of electronic devices grows, so does the demand for electricity. Electricity is typically generated via the combustion of fossil fuels. However, conventional fossil fuels are limited in supply and non-renewable. Therefore, in order to replace conventional fossil fuels, it is imperative that renewable energy is harvested. 1–5 In 2012, Fan et al. 6 proposed the concept of a triboelectric nanogenerator (TENG) that converts mechanical energy from the environment into electrical energy through the coupling of contact and electrostatic induction. 7 TENGs are low-cost, small, portable, and applicable to a variety of scenarios. 8,9 Thus, they have been used to power wearable electronics, 10,11 sensors, 12,13 and other small electronic devices. 14 However, the relatively low output power density of TENGs limits their wider application. Researchers have made numerous attempts to enhance the electrical properties of TENGs by selecting suitable triboelectric materials, 15–17 introducing functional groups, 18,19 doping with materials with high dielectric constants, 20,21 and designing micropatterned surfaces. 22,23 Among these approaches, the selection of suitable electrode materials to increase the surface charge density is the most effective way of improving the fundamental performance of TENGs. Many synthetic polymers have advantageous properties applicable to enhancing TENG electrical performance, but their long-term use causes plastic pollution. In contrast, cellulose, which is the most abundant natural polymer on Earth, is widely available, biodegradable, and renewable. 24,25 However, cellulose becomes positively charged due to triboelectric electrification. Hence, the direct use of cellulose as an electrode material does not fulfill practical requirements, necessitating its modification. Embedding nano- and microscale materials (“fillers”) into a cellulose matrix not only leads to desirable properties, but also improves the surface properties and enhances the output performance of TENGs. 26–28 Existing fillers typically perform only a single function in capturing triboelectric charges, and there are few systematic studies on their charge-induction capabilities. Although amino groups are suitable electron-releasing groups that can improve the triboelectric properties of cellulose, 29 it is still a challenge to perfectly introduce amino groups into the filler to prepare a composite with strong charge-induction and charge-trapping capabilities. 30 Metal–organic frameworks (MOFs) are an emerging class of porous materials with multiple crystallinities formed by ligating metal ions or clusters with multifunctional organic ligands. 31 Owing to the structural and functional versatility of MOFs, their utilization as functional fillers is promising for the development of high-performance TENG composites. 32 Considering that iron is an environmentally friendly, abundant, inexpensive, and non-toxic metal, we explored the use of an iron-based MOF (MIL-101(Fe)) with a topological network structure of an MTN molecular sieve composed of Fe 3+ and 1,4-phenylene terephthalic acid. However, the poor stability of MIL-101(Fe) (due to the inherent lack of unsaturated sites) hinders its efficient application and the stability of its long-term operation. Therefore, in order to develop high-performance stable TENG composites, we doped Cu 2+ ions into MIL-101(Fe) to further improve its stability and increase the number of the unsaturated metal sites. In this study, we prepared aminated bimetallic MOFs (NH 2 -MIL-101(Fe,Cu)) as a bifunctional filler. In addition to playing a charge-trapping role similar to that of conventional fillers, it enhanced charge induction by introducing –NH 2 groups and increasing the surface roughness. In order to develop a high-performance and stable TENG, NH 2 -MIL-101(Fe, Cu) was combined with a cellulose nanofiber (CNF) to prepare a composite film for use as the triboelectric layer. The composite film was characterized by scanning electron microscopy (SEM), Fourier-transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), thermogravimetric (TG) analysis, and atomic force microscopy (AFM). The resultant TENG could charge and power external devices and exhibited potential for use in self-powered sensing systems. These results provide a factual basis for further expanding the application scope of triboelectric materials used in TENGs.",
"discussion": "Results and discussion Characterization of MOFs and composite films \n Fig. 2 shows the surface morphology and energy-dispersive X-ray spectroscopy (EDS) energy spectra of the MOFs and composite films. Fig. 2(a) shows that all the M 1 particles had a uniform three-dimensional octahedral structure and smooth surface. The relative percentages of C, O, and Fe were 50.56%, 35.10%, and 14.33%, respectively ( Fig. 2(b) ). As shown in Fig. 2(d) , the synthesized M 2 particles had a hexagonal spindle-shaped body with a uniform particle distribution. The relative percentages of C, N, O, and Fe were 49.26%, 6.75%, 36.61%, and 7.38%, respectively ( Fig. 2(e) ). The presence of N proves that the –NH 2 group was successfully grafted onto the MOFs in M 2 . As shown in Fig. 2(g) , the doping of Cu also affected the morphology of the material, as M 3 had a slightly rougher surface than M 2 . The relative percentages of C, N, O, Fe, and Cu in M 3 were 43.47%, 9.41%, 30.76%, 15.84%, and 0.52%, respectively ( Fig. 3(h) ). The presence of N and Cu indicates that Cu 2+ and –NH 2 were successfully introduced into the MOFs in M 3 . As shown in Fig. 2(c), (f) and (i) , the MOF nanoparticles were clearly visible in the C M 1 , C M 2 , and C M 3 composite films, with uniform distributions in the CNF matrix. This should provide mechanical stability to the composite films. Fig. 2 Surface morphology of the MOFs and composite films. (a) SEM image of M 1 , the (b) EDS energy spectrum of M 1 , the (c) SEM image of the C M 1 composite film, the (d) SEM image of M 2 , the (e) EDS energy spectrum of M 2 , the (f) SEM image of the C M 2 composite film, the (g) SEM image of M 3 , the (h) EDS energy spectrum of M 3 , and the (i) SEM image of C M 3 composite film. Fig. 3 FTIR spectra from (a) 4000 to 400 cm-1 and (b) 2000 to 400 cm −1 ; (c) TG analysis from 30 to 600 °C; XRD spectra from (d) 5° to 30° and (e) 8° to 11°; and (f) AFM images of the CNF membrane and C M 3 composite membrane. \n Fig. 3(a) and (b) show the FTIR spectra of the samples. The spectra of M 2 and M 3 contained two low-intensity absorption peaks at 3350 and 3467 cm −1 , which originate from asymmetric and symmetric N–H stretching vibrations, respectively. In addition, they contained an absorption peak at 1255 cm −1 , which originates from C–N stretching vibrations. 35 These peaks indicate that –NH 2 was successfully grafted onto the MOFs. The peaks corresponding to N–H and C–N stretching vibrations were also observed in the C M 3 composite film, indicating that M 3 was successfully compounded with the CNF. No new absorption bands or peak shifts were found in the C M 3 spectrum, indicating that no chemical interactions occurred between the CNF and MOFs. The FTIR spectra of MOFs contained peaks at 1598 and 1578 cm −1 , which originate from C \n \n\n<svg xmlns=\"http://www.w3.org/2000/svg\" version=\"1.0\" width=\"13.200000pt\" height=\"16.000000pt\" viewBox=\"0 0 13.200000 16.000000\" preserveAspectRatio=\"xMidYMid meet\"><metadata>\nCreated by potrace 1.16, written by Peter Selinger 2001-2019\n</metadata><g transform=\"translate(1.000000,15.000000) scale(0.017500,-0.017500)\" fill=\"currentColor\" stroke=\"none\"><path d=\"M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z\"/></g></svg>\n\n O stretching vibrations; 36 at 1388 and 1380 cm −1 from C–O stretching vibrations and at 552 and 524 cm −1 from Fe–O stretching vibrations. 37 All the M 2 peaks had lower intensities compared to those of M 3 , which was attributed to electrostatic interactions between the copper ions and organic ligands in the benzene ring. The peaks of both M 2 and M 3 were shifted compared to those of M 1 , which was primarily due to the interaction between –NH 2 and the MOFs. 38 The stability of the MOFs was analyzed via TG analysis, as shown in Fig. 3(c) . All the samples had two main stages of weight loss. The first stage occurred from 30 to 305 °C, with a mass loss of 10.1% to 24.2%. This is mainly due to the removal of guest water molecules. The second stage occurred from 305 to 600 °C, with a mass loss of 31.8% to 40.1%. This is mainly caused by the removal of hydroxyl groups from the skeleton and by structural collapse. The order of thermal stability was M 3 > M 2 > M 1 (as determined by TG analysis), indicating that the introduction of –NH 2 and Cu 2+ improved the stability of the MOFs. The crystal structures of the samples were determined via XRD. As shown in Fig. 3(d) and (e) , the characteristic diffraction peaks of M 2 and M 3 were in agreement with those reported previously, 33 indicating the successful synthesis of NH 2 -MOFs. The XRD peak intensities of the C M 3 composite film differed somewhat from those of the CNF film. These changes were attributed to the interaction between the NH 2 -MOFs and CNF. However, the XRD spectrum of the C M 3 composite film confirms that the crystallinity of the composite film remained unchanged. As shown in Fig. 3(f) , the root-mean-square (RMS) roughness of the CNF film was increased from 47.52 to 82.13 nm after the addition of M 3 nanoparticles, indicating an increase in the roughness of the C M 3 composite film after the addition of MOF nanofillers. Structure and the working mechanism of TENGs As shown in Fig. 4(a) , the C M 3 -TENG consisted of a C M 3 composite and FEP membranes as triboelectric pairs. NH 2 -MOFs have high porosities, high specific surface areas, and rigid MTN-type crystal structures. They also consist of two spherical cages: one with a large cage diameter of 3.4 nm and one with a small cage diameter of 2.9 nm. 39 The coupling between contact and electrostatic induction in TENG devices is based on the C M 3 composite films, and the working mechanism is shown in Fig. 4(b) . No charge is generated in the initial stage. When an external force is applied, the upper and lower layers come into contact, and the surface charge is transferred due to electrostatic induction. It can be concluded from the electronegativity sequence that C M 3 is more likely to lose electrons, and FEP is more likely to gain electrons; when electrons are transferred from the C M 3 membrane to the FEP membrane, negative charges accumulate on the surface of the FEP membrane. When the two layers are separated as a result of the external force, a distance-dependent potential difference is generated, which reaches its maximum at the maximum distance. The point imbalance between the two layers causes electrons to flow in the external circuit until an equilibrium state is reached. When the direction of the external force changes, the two layers are pressed further together, and the potential difference begins to decrease. As a result, the electrons move in opposite directions during the pressurization process, and the electron flow is driven back and forth during the periodic contact and separation process, resulting in an output of AC voltage and current. Fig. 4 (a) Structure and the (b) working mechanism of TENGs. NH 2 -MOF enhancement of TENG electrical performance To investigate the effect of the MOFs on the contact initiation performance of the CNF film, the open-circuit voltage, short-circuit current, and charge density of C-TENG, C M 1 -TENG, C M 2 -TENG, and C M 3 -TENG were probed. A linear motor was used to oscillate two TENG electrodes into periodic contact and separation at an acceleration of 0.5 m s −2 with a friction area of 4 × 4 cm. As shown in Fig. 5(a)–(c) , the open-circuit voltage of the C-TENG was 63 V, its short-circuit current was 3.5 μA, and its charge density was 2 nC cm −2 . In comparison, the open-circuit voltage of the C M 1 -TENG increased from 63 to 142 V (a 125% improvement), the short-circuit current increased from 3.5 to 11 μA (a 214% improvement), and the charge density increased from 2 to 4.2 nC cm −2 (a 110% improvement). These improvements resulted from the MIL-101(Fe) nanoparticles, which have strong dielectric-trapping and dielectric-induction capabilities; thus, they enhanced the triboelectric properties of the composite and the electrical performance of the TENG. The open-circuit voltage, short-circuit current, and charge density of the C M 2 -TENG were 182 V, 14 μA, and 5 nC cm −2 , respectively, corresponding to 189%, 300%, and 150% improvements over those of the C-TENG, respectively. These improvements resulted from the electron-giving function of the –NH 2 groups, which further improved the triboelectric positive polarity of the CNF film and thus the electrical performance of the TENG. Finally, the C M 3 -TENG had an open-circuit voltage, short-circuit current, and charge density of 194 V (266% increase compared to that of the C-TENG), 14 μA (300% improvement), and 5 nC cm −2 (150% improvement), respectively. Fig. 5 TENG electrical performance. (a) Open-circuit voltage, (b) short-circuit current, and (c) charge density for different composite films. (d) Open-circuit voltage, (e) short-circuit current, and (f) charge density for different addition ratios. (g) Open-circuit voltage, (h) short-circuit current, and (i) charge density for different accelerations. We also investigated the relationship between the mass ratio of M 3 nanoparticles and the TENG electrical performance to optimize the mass loading of the active filler. As shown in Fig. 5(d)–(f) , the output voltage and current initially increased as the M 3 mass ratio increased. When the mass ratio was 0.5 wt%, the open-circuit voltage, short-circuit current, and charge density reached their maximum values of 194 V, 14 μA, and 5 nC cm −2 , respectively. The positive correlation between the mass loading of C M 3 and the electrical performance was due to the charge transfer caused by the –NH 2 groups. However, as the mass ratio increased above 0.5 wt%, the electrical output decreased. At a mass ratio of 5 wt%, the open-circuit voltage, short-circuit current, and charge density of the TENG were 150 V, 11 μA, and 4.3 nC cm −2 , respectively. This was attributed to the large-scale agglomeration of C M 3 particles in the polymer matrix when the mass loading exceeded 0.5 wt%, which also led to the formation of voids between the C M 3 particles and CNF matrix. Therefore, the optimal mass ratio of the C M 3 nanoparticles in the CNF matrix was 0.5 wt%. As shown in Fig. 5(g)–(i) , when the acceleration increased from 0.5 to 1.4 m s −2 , the open-circuit voltage increased from 194 to 220 V (a 13.4% increase), the short-circuit current increased from 14 to 21 μA (a 50% increase), and the charge density increased from 5 to 5.9 nC cm −2 (an 18% increase). This increase of electrical performance with acceleration was due to the increased charge transfer rate. However, when the acceleration was greater than 1.4 m s −2 , the electrical performance decreased, which was primarily due to the insufficient contact area between the positive and negative materials. The latter was caused by the rapid motion of the TENG electrodes as they were driven by a linear motor, which decreased the rate at which the surface charge could be transferred. Applications of the NH 2 -MOF-incorporated TENG As shown in Fig. 6(a) , we also used the C M 3 -TENG as a power source for LED lamps and capacitors to explore the potential for extending its range of applications. The C M 3 -TENG was connected to a resistor box, and the changes in the output voltage and current as well as the output power in the 10 3 –10 8 Ω range were measured. The results show that the output voltage increased as the external resistance increased, while the current exhibited the opposite trend. As shown in Fig. 6(b) , the output power density initially increased and then decreased; the maximum output power density was 20.3 μW cm −2 when the external resistance was 10 7 Ω. For practical applications, stability is one of the most important parameters of TENGs. Therefore, we conducted a stability test on the C M 3 -TENG, as shown in Fig. 6(d) . The current remained essentially unchanged after 15 000 contact–separation cycles at an acceleration of 0.5 m s −2 , which proved that the C M 3 -TENG was durable. As shown in the SEM images in Fig. 6(c) , the morphology of the C M 3 -TENG did not change significantly after the 90 day cycling test. Additionally, as shown in Fig. 6(e) , we tested the stability of the C-TENG and C M 3 -TENG separately. The output current of the C M 3 -TENG remained essentially unchanged after 90 days, while that of the C-TENG decreased slightly, indicating that the C M 3 -TENG was more stable. More importantly, the C M 3 -TENG directly powered 84 LEDs simultaneously, as shown in Fig. 6(f) . To simulate other practical applications, the C M 3 -TENG was connected to capacitors of six different sizes (1, 2.2, 3.3, 4.7, 10, and 33 μF) for charging, as shown in Fig. 6(g) . This test demonstrated the real-time variation in the voltage of the charging process for the six capacitors over the course of 120 s, and as shown in Fig. 6(h) , the voltage increased during each cycle. These practical results demonstrate that the design of the C M 3 -TENG has a promising future in the field of self-powered devices. Fig. 6 Applications of the NH 2 -MOF-incorporated TENG. (a) Dependence of the TENG output current and voltage on the external load resistance. (b) Instantaneous TENG power output as a function of the external load resistance. (c) SEM before and after comparison of the C M 3 -based TENG over 15 000 cycles. (d) Stability of the C M 3 -based TENG over 15 000 cycles. (e) Electrical transport performance over 90 days. (f) Eighty-four commercial LEDs powered by the NH 2 -MOF-incorporated TENG. (g) Rectifier circuit. (h) Output voltage on different capacitors (1, 2.2, 3.3, 4.7, 10, and 33 μF). (i) Enlarged view of the voltage changes."
} | 5,177 |
25028427 | PMC4161262 | pmc | 6,371 | {
"abstract": "ABSTRACT Microbial communities typically contain many rare taxa that make up the majority of the observed membership, yet the contribution of this microbial “rare biosphere” to community dynamics is unclear. Using 16S rRNA amplicon sequencing of 3,237 samples from 42 time series of microbial communities from nine different ecosystems (air; marine; lake; stream; adult human skin, tongue, and gut; infant gut; and brewery wastewater treatment), we introduce a new method to detect typically rare microbial taxa that occasionally become very abundant (conditionally rare taxa [CRT]) and then quantify their contributions to temporal shifts in community structure. We discovered that CRT made up 1.5 to 28% of the community membership, represented a broad diversity of bacterial and archaeal lineages, and explained large amounts of temporal community dissimilarity (i.e., up to 97% of Bray-Curtis dissimilarity). Most of the CRT were detected at multiple time points, though we also identified “one-hit wonder” CRT that were observed at only one time point. Using a case study from a temperate lake, we gained additional insights into the ecology of CRT by comparing routine community time series to large disturbance events. Our results reveal that many rare taxa contribute a greater amount to microbial community dynamics than is apparent from their low proportional abundances. This observation was true across a wide range of ecosystems, indicating that these rare taxa are essential for understanding community changes over time.",
"introduction": "INTRODUCTION Microbial communities predominate Earth’s diverse ecosystems, contributing immense biomass and underpinning integral biogeochemical processes. They sustain the bases of food webs, provide key natural products that support human health and energy needs, and recycle carbon and nutrients that would otherwise stagnate. Despite the central role of microbial communities in biological systems, we are just beginning to understand the intricate interactions between their members and how these interactions contribute to ecosystem functions. Of particular interest is the role of rare microorganisms within a community, which make up the majority of the observed membership at any given time ( 1 – 5 ) (see Fig. S1 in the supplemental material). Determining whether these taxa remain rare or periodically bloom to abundance will change our understanding of each organism’s role in microbially mediated ecosystem functions and, importantly, in the stability of ecosystems in general. Rare microbial community members encompass an immense diversity (the “rare biosphere”) ( 6 – 9 ). Still, the ecological roles of the vast majority of rare microorganisms remain unclear. Some rare microorganisms are likely on their way to local extinction ( 8 ) or are transient taxa that are “passing through” an environment ( 10 – 13 ). Some rare taxa may even be active, providing important functions that are disproportionate to their abundance or growth rate ( 14 – 16 ), and others may be dormant or inactive, awaiting favorable environmental conditions to grow ( 17 , 18 ). An increase in the abundance of rare microorganisms that “wait” for favorable environmental conditions could be attributed to growth from low abundance, to awakening from dormancy, or to differential survival (i.e., escape from predation). Though there are a variety of ecological explanations for rare-to-prevalent dynamics, we still lack general empirical documentation of the phenomenon among microbial communities, and so their general incidence remains uncertain. Because rare microbial taxa are difficult to observe, even less is known about their dynamics than is known about their ecological roles. A key unknown is how often rare taxa become abundant and hence play a potentially greater role in the ecology of a given system. However, there are a small but growing number of studies that have documented the dynamics of rare microbial taxa and provide some insights. For example, in the Arctic Ocean, rare microorganisms exhibited biogeography, indicating that some rare taxa, like more abundant taxa, have distributions based on their ecological requirements ( 19 ). In a sulfide-rich artesian spring, rare taxa exhibited patchiness over 1 mm ( 20 ), which also suggests that rare taxa can have clear distributions at fine spatial scales. Additionally, some coastal sand communities have rare members that do not often become abundant, suggesting that these members have a minimal influence on biogeochemical processes ( 21 ). Conversely, in other coastal sand communities, rare microbial taxa were shown to be as sensitive as prevalent taxa to environmental changes caused by an off-shore oil spill ( 22 ). The discrepancy between the latter two studies highlights our modest knowledge of the potential contributions of rare taxa and especially calls into question whether such conclusions are transferable to other ecosystems. Therefore, to understand the general importance of rare microbial taxa, their contributions to the larger community and their dynamics, we must systematically interrogate microbial communities from a variety of ecosystems by using consistent methods. The availability of inexpensive, high-throughput sequencing technologies has led to an increased number of temporal studies of microbial communities ( 23 ). One of these studies identified a microbial taxon that bloomed to abundance from an apparently persistently rare state ( 24 , 25 ). The dynamic of rarity to prevalence has also been observed in two other studies of marine bacterioplankton ( 14 , 26 ). Here, we asked how the pattern of microbial rarity to prevalence is manifested in communities inhabiting very different ecosystems. We refer to microbial taxa that are typically in low abundance in one locality but occasionally become prevalent over time as “conditionally rare.” Our objective was to understand the incidence of conditionally rare taxa (CRT) and their contribution to changes in microbial communities through time. We introduce a simple method for identifying CRT from temporal studies of diverse microbial communities and apply this method to a suite of time series data sets generated by using 454 pyrosequencing or Illumina sequencing of 16S rRNA gene fragments. Each data set contained a large percentage of very rare taxa, as typical for microbial communities (see Fig. S1 in the supplemental material). These data sets were previously analyzed by using a closed-reference operational taxonomic unit (OTU)-picking protocol ( 27 ) for direct comparison of their temporal patterns (see Table S1 in the supplemental material) ( 28 ). Because this OTU-picking protocol discards reads that do not match reference sequences at a minimum of 97% identity, it minimizes the rare OTUs arising from sequencing or PCR errors. The closed-reference protocol also avoids the “OTU splitting” that may occur when OTUs are defined by using a de novo protocol. We show that within many ecosystems, CRT contributed to temporal patterns of microbial diversity disproportionately to their relative abundances, suggesting an important role for CRT in structuring microbial communities over time. We also explicitly examine the influences of sampling frequency, study duration, and sequencing depth on the detection of CRT.",
"discussion": "DISCUSSION Our results show that CRT can influence changes in microbial community structure. CRT contributed from 0 to 97% of the variability in the observed temporal community dissimilarity. Though it may seem obvious that CRT would contribute the most to temporal community dissimilarity during their transitions, it was unexpected that they would contribute so disproportionately (i.e., up to 97%) compared with their relative abundance during a “bloom” (mean relative abundance during a bloom, 2.7%; median, 1%). Our previous analysis suggested that the longer a community is observed, the more the perceived magnitude of the changes in community structure is reduced, suggesting very low rates of community change over long-term observations ( 28 ). Together, these results indicate that many baseline temporal changes in bacterial and archaeal diversity may be attributed to changes in the relative contributions of taxa that already exist within the community, including CRT transitions. We provide a simple tool for identifying CRT and suggest that, on the whole, CRT comprise taxa that are always present and that it is less common for these taxa to be introduced by a dispersal event. However, while our strategy identifies taxa that can be targeted for further analysis, it does not explicitly reveal the ecological mechanisms of CRT within a community. These mechanisms are diverse and numerous, and determining the ecological properties of individual taxa is difficult and costly ( 33 – 35 ). However, we provide one example in which we capitalized on a temporal lake study to deduce CRT ecology by contrasting routine dynamics with a disturbance. In doing so, we were able to distinguish CRT that responded to both natural and forced mixing events from those that responded only to a forced event. These methods provide a springboard for hypothesis generation and are useful for understanding the contributions of CRT to different types of ecological dynamics. For example, in the context of human microbial consortia, similar analyses may be done in instances of pathogen invasion or pathobiont formation to understand when, how, and under what environmental conditions a typically rare or invasive member of the human microbiome is able to thrive following such a disturbance. Though we cannot prove that one-hit-wonder CRT are not artifacts due to PCR ( 36 ) or sampling anomalies ( 37 ), the fact that the majority of CRT were observed multiple times within a series suggests that this scenario is not common and asserts that CRT would remain important contributors to community dynamics despite occasional misidentification due to artifacts. In reality, one-hit-wonder CRT likely comprise a combination of newly dispersed taxa that fail to thrive long term, rare but persistent taxa that fall below the level of detection when not blooming, CRT that were not observed long enough to detect subsequent blooms (insufficient time series), and artifacts. There have been two distinct approaches to considering the rare biosphere in microbial ecology: (i) deep sequencing to detect as many rare members as possible ( 6 ) and (ii) omission of the entire rare “tail” to clarify overarching community patterns, whether arbitrary (e.g., 50 or fewer sequences) or methodological (e.g., after determining the abundance cutoff at which rare taxa do not contribute substantially to community dissimilarity) ( 38 ). Although the ecological roles of many rare taxa are unknown, it has been suggested that rare taxa are not necessarily important for the comparison and interpretation of microbial community patterns ( 10 , 38 ). As more data from temporal studies of microbial communities are collected, it is likely that the dynamics of CRT will play an increasingly important role in our understanding of both the subtle temporal variability ( 39 ) and the disturbance responses of microbial communities. Furthermore, we know that some rare taxa play critical ecological roles in ecosystems, for example, diazotrophs in seawater ( 40 ), bacterial and archaeal ammonia oxidizers in soils ( 41 , 42 ), and methanogens in guts ( 43 ). Thus, detection of CRTs will provide clues as to the identities of rare taxa that play previously unknown but equally critical ecological roles. Finally, studies that use unsaturated sequencing efforts to infer community assembly rules may attribute the appearance of new taxa to dispersal, when these taxa may instead already persist in the community in low abundance or in a dormant state ( 24 ). Therefore, close inspection of CRT dynamics in sufficiently sequenced communities will provide insights into the different roles of dispersal and blooms in community dynamics. Given the ubiquity of CRT detected across an array of ecosystems and the large contribution of CRT to community dissimilarity, our results show that rare-to-prevalent dynamics are generally important and that these dynamics are especially critical for the community at the time points of CRT transitions. These data provide evidence that not all of the members of the microbial rare biosphere are always rare but that many contribute to the larger community at key time points. Furthermore, our analysis revealed synchronous dynamics of many CRT within a community and suggests that some CRT may be indicators of environmental changes that are unmeasured, providing clues about the identities of more subtle physical, chemical, or biological drivers of microbial dynamics. Finally, as transient members of the rare biosphere, CRT likely contribute to the high alpha diversity observed in many microbial communities."
} | 3,240 |
33881981 | PMC8289221 | pmc | 6,372 | {
"abstract": "Micro-organisms contribute to Earth’s mineral deposits through a process known as bacteria-induced mineral precipitation (BIMP). It is a complex phenomenon that can occur as a result of a variety of physiological activities that influence the supersaturation state and nucleation catalysis of mineral precipitation in the environment. There is a good understanding of BIMP induced by bacterial metabolism through the control of metal redox states and enzyme-mediated reactions such as ureolysis. However, other forms of BIMP often cannot be attributed to a single pathway but rather appear to be a passive result of bacterial activity, where minerals form as a result of metabolic by-products and surface interactions within the surrounding environment. BIMP from such processes has formed the basis of many new innovative biotechnologies, such as soil consolidation, heavy metal remediation, restoration of historic buildings and even self-healing concrete. However, these applications to date have primarily incorporated BIMP-capable bacteria sampled from the environment, while detailed investigations of the underpinning mechanisms have been lagging behind. This review covers our current mechanistic understanding of bacterial activities that indirectly influence BIMP and highlights the complexity and connectivity between the different cellular and metabolic processes involved. Ultimately, detailed insights will facilitate the rational design of application-specific BIMP technologies and deepen our understanding of how bacteria are shaping our world.",
"introduction": "Introduction Bacterial activity is evident in our landscapes and throughout the geological record, where it has helped shape Earth’s mineral deposits [ 1 ]. This has occurred, to some degree, via a process known as bacteria-induced mineral precipitation (BIMP). The variety of mineral deposits that are formed through bacterial activity can take on the form of stalactites and stalagmites [ 2 ], microbialites, stromatolites and thrombolites [ 3, 4 ] as well as large-scale sedimentation [ 4 ]. More recently, the ability of bacteria to induce mineral formation has gained attention for biotechnological application. In particular, the precipitation of calcium carbonate in the form of calcite, the mineral that forms limestone, has been exploited in innovative technologies in civil engineering. The first patented application is considered to have been by Adolphe and colleagues in 1990 for biological treatment of degrading stone surfaces [ 5 ]. Since then, more technologies have been developed, with a lot of attention surrounding the concept of self-healing concrete [ 6–8 ]. Other applications of BIMP include soil consolidation or heavy metal bioremediation, and excellent recent reviews exist that cover the spectrum of such technologies in detail [ 9–14 ]. For the purposes of this review, BIMP is defined as a process by which bacterial activity indirectly induces mineral formation via the release of metabolic by-products and surface interactions with ions in the open environment [ 15–17 ]. This is in contrast to bacteria-controlled biomineralization, e.g. the formation of magnetite by magnetotactic bacteria, which is metabolically and genetically controlled by the bacteria and occurs in defined locations, e.g. magnetosomes [ 18–20 ]. The latter has been reviewed in detail elsewhere [ 15, 21 ] and will not be covered here. The minerals formed by BIMP generally have no specific function (aside from some potential ecological benefits) and can be considered an unintended and uncontrolled consequence of bacterial activity [ 22, 23 ]. Depending on the author, indirect biomineralization is sometimes subdivided further into more nuanced ‘bacteria-induced’ versus ‘bacteria-influenced’ mineral precipitation [ 13, 20, 24 ]. The boundaries between the two are, however, not clear cut and in this review no such division is made. Bacteria-induced mineral precipitation Precipitation of mineral species in an aqueous system occurs when the ion concentration exceeds solubility and reaches a degree of super-saturation. Once the activation energy barrier is overcome, initial crystal nucleation occurs, in which metastable critical nuclei form that may dissolve back into the bulk phase. Subsequent aggregation of individual nuclei describes the process of crystal growth and precipitation [ 25–27 ]. Nucleation can take place either homogeneously, whereby nucleation occurs when critical nuclei form in the absence of foreign particles (via random collisions of ions or atoms in solution), or heterogeneously, whereby nucleation takes place when critical nuclei form on surfaces of foreign particles [ 25–27 ]. Such particles lower the activation energy by providing templates with spacing that enhances nucleation and thus, precipitation [ 25–27 ]. Furthermore, during the nucleation process foreign particles may aggregate, leading to the formation of mixed precipitates [ 28 ]. In BIMP, bacteria can induce biomineralization by modulating precipitation-relevant parameters like local ion concentrations or pH in the environment and/or by bacterial cells themselves providing nucleation sites for crystal formation. In general, this bacterial process involves the attraction of cations to negative charges on the cell surfaces, while metabolic activity provides the appropriate microenvironment and counter-anions so that these cations may precipitate as minerals [ 29 ]. The BIMP trait is common amongst bacteria across environments [ 9, 30–33 ], and, depending on bacterial species and environment, it can lead to a range of precipitated minerals ( Table 1 ). The bacteria-induced formation of some of these minerals can further lead to co-precipitation of additional divalent metal cations and anions [ 34–36 ]. Indirect bacterial influence on precipitation parameters of saturation state and nucleation catalysis can be broadly separated into two contributing areas: cell surface and metabolic activity, and our current understanding of the mechanisms of these will be reviewed here. Table 1. Minerals precipitated in association with bacterial activity * \n \n Mineral \n \n Chemical formula \n \n Reference \n \n \n Carbonates \n \n \n \n \n Calcite \n \n CaCO 3 \n \n \n [ 30 ] \n \n Dolomite \n \n CaMg(CO 3 ) 2 \n \n \n [ 111, 112 ] \n \n Kutnahorite \n \n CaMn(CO 3 ) 2 \n \n \n [ 113 ] \n \n Siderite \n \n FeCO 3 \n \n \n [ 114 ] \n \n Magnesite \n \n MgCO 3 \n \n \n [ 54, 115 ] \n \n Otavite \n \n CdCO 3 \n \n \n [ 116 ] \n \n Strontianite \n \n SrCO 3 \n \n \n [ 72 ] \n \n Rhodochrosite \n \n MnCO 3 \n \n \n [ 117 ] \n \n Cerussite \n \n PbCO 3 \n \n \n [ 118 ] \n \n Hydrozincite \n \n Zn 5 (CO 3 ) 2 (OH) 6 \n \n \n [ 36, 119 ] \n \n Dypingite \n \n Mg 5 (CO 3 )(OH) 2 ·5H 2 O \n \n [ 120 ] \n \n Witherite \n \n BaCO 3 \n \n \n [ 121 ] \n \n \n Phosphates \n \n \n \n \n Tricalcium phosphate \n \n Ca 3 (PO 4 ) 2 \n \n \n [ 78 ] \n \n Struvite \n \n NH 4 MgPO 4 ∙6H 2 O \n \n [ 74, 113 ] \n \n Bobierrite \n \n Mg 3 (PO 4 ) 2 ·8H 2 O \n \n [ 74, 122 ] \n \n Baricite \n \n (MgFe) 3 (PO 4 ) 2 ·8H 2 O \n \n [ 74 ] \n \n Vivianite \n \n Fe 3 (PO 4 )·2H 2 O \n \n [ 114 ] \n \n Autunite \n \n Ca(UO 2 ) 2 (PO 4 ) 2 ∙10-12H 2 O \n \n [ 44 ] \n \n Uramphite \n \n NH 4 UO 2 PO 4 \n \n \n [ 101, 123 ] \n \n Apatite \n \n Ca 10 (PO 4 ) 6 (OH) 2 \n \n \n [ 124 ] \n \n Pb-hydroxyapatite \n \n Ca 2.5 Pb 7.5 (OH) 2 (PO 4 ) 6 \n \n \n [ 125 ] \n \n Strengite \n \n FePO 4 ·2H 2 O \n \n [ 126, 127 ] \n \n Variscite \n \n AlPO 4 ·2H 2 O \n \n [ 97 ] \n \n \n Silicates \n \n \n \n \n Gehlenite \n \n Ca 2 Al(AlSiO 7 ) \n \n [ 128 ] \n \n Silica \n \n SiO 2 \n \n \n [ 129 ] \n \n Nontronite \n \n Na 0.3 Fe 3+ \n 2 (Si,Al) 4 O 10 (OH) 2 ·nH 2 O \n \n [ 130 ] \n \n Chamosite \n \n (Fe 5 Al)(Si 3 Al) 10 (OH) 8 \n \n \n [ 126 ] \n \n Kaolinite \n \n Al 4 (Si 4 O 10 )(OH) 4 \n \n \n [ 126 ] \n \n \n Sulphides \n \n \n \n \n Mackinawite \n \n FeS \n \n [ 76 ] \n \n Greigite \n \n Fe 3 S 4 \n \n \n [ 76, 131 ] \n \n Pyrite \n \n FeS 2 \n \n \n [ 132 ] \n \n Covellite \n \n CuS \n \n [ 133, 134 ] \n \n Sphalerite \n \n ZnS \n \n [ 135 ] \n \n Galena \n \n PbS \n \n [ 134 ] \n \n Digenite \n \n Cu 9 S 5 \n \n \n [ 136 ] \n \n \n Sulphates \n \n \n \n \n Gypsum \n \n CaSO 4 ·2H 2 O \n \n [ 41, 54 ] \n \n Celestite \n \n SrSO 4 \n \n \n [ 72 ] \n \n Barite \n \n BaSO 4 \n \n \n [ 121, 137 ] \n \n \n Oxides \n \n \n \n \n Magnetite \n \n Fe 3 O 4 \n \n \n [ 114 ] \n \n Hematite \n \n Fe 2 O 3 \n \n \n [ 23, 138 ] \n \n Ferrihydrite \n \n Fe 2 O 3 ·0.5H 2 O \n \n [ 138 ] \n \n Geothite \n \n α-FeO(OH) \n \n [ 138 ] \n \n Manganite \n \n MnOOH \n \n [ 139 ] \n \n Vernadite \n \n MnO 2 \n \n \n [ 57, 140, 141 ] \n \n Hausmannite \n \n Mn 3 O 4 \n \n \n [ 142 ] \n \n Todorokite \n \n (Ca,Na,K) x (Mn 4+ ,Mn 3+ ) 6 O 10 ·3.5H 2 O \n \n [ 138 ] \n \n Birnessite \n \n (Na,Ca,K) x (Mn 4+ ,Mn 3+ ) 2 O 4 ·1.5H 2 O \n \n [ 138 ] \n \n Uraninite \n \n UO 2 \n \n \n [ 143–145 ] \n \n Calcium Arsenate \n \n CaHAsO 3 \n \n \n [ 146 ] \n *Note that while these minerals have all been reported to be formed in association with bacterial activity, the mechanisms for their formation are not always known, and some minerals can be formed by multiple different mechanisms. The minerals listed and accompanying sources are non-exhaustive of the examples available in the literature. Cell surface: nucleation catalysis, saturation state and nucleation template The large surface area to volume ratio of bacteria make them ideal crystal nucleation sites. Covered by functional groups with a net negative charge, their surface acts as a metal cation scavenger concentrating dilute cations attracted from the environment [ 29, 37, 38 ]. Net negative surface charge is imparted by carboxyl (R-CO 2 H) and phosphate groups (R-PO 4 H 2 ) of teichoic acids in Gram-positive bacteria, and phospholipids and lipopolysaccharides (LPS) in Gram-negative bacteria [ 39 ]. Bacterial S-layers further influence net surface charge depending on the presence or absence of S-layer glycol proteins with glycosylated long carbohydrate chains, and depending on the structural groups exposed within their lattice pores [ 40–42 ]. These bacterial surface structures are illustrated in Fig. 1 . Extracellular polymeric substances (EPS), capsules, sheaths, slimes and biofilm matrices may further surround Gram-positive or Gram-negative bacteria. These are also usually associated with a net negative charge imparted by carboxyl and phosphate groups, which are free to interact with soluble cations [ 43 ]. Fig. 1. Schematic of the major supramolecular structures on the surface architecture of (a) Gram-positive and (b) Gram-negative bacteria, which provide sites for metal cation interaction. The red circles represent sites of negative charge, the grey circle represent sites of neutral charge, the blue circles represent positively charged cations, and dotted lines illustrate the attraction between negative and positive charges. Adapted from [ 42, 147 ]. The extent of the surface negative charge is governed by the deprotonation of functional groups with an increase in pH: carboxyl, phosphate, hydroxyl (R-OH) and sulphate (R-SO 4 ) groups increase their negative charge, while amine (R-NH 2 ) groups decrease their positive charge. For bacteria living in environments with neutral pH ranges, this means that surfaces tend to be negatively charged and have a high affinity for cationic species [ 44, 45 ]. Carboxyl groups in particular have been found to contribute strongly to the metal-binding capability. Studies on \n Bacillus subtilis \n used chemical modification of phosphate and carboxyl functional groups to demonstrate their importance in and relative contribution to metal ion binding [ 46, 47 ]. More recent studies of Gram-positive cell walls support this role, with half the binding of calcium and magnesium coming from polyphosphate groups of teichoic acids and half from carboxyl groups of peptidoglycan [ 48 ]. Teichoic and teichuronic acids, as well as LPS are natively stabilized by the presence of divalent cations, providing starting nucleation sites for mineral formation [ 43 ]. Surface cation binding sites are assumed to form the centre of crystal growth. Mineral precipitation occurs from nucleation of cations to previously adsorbed surface cations. The formation of these critical nuclei is stabilized by the surface functional groups through a reduction of tension between the bulk water phase and mineral nucleus [ 43 ]. Once bound, supersaturation is achieved by lowering the free energy necessary for precipitation, often with the help of metabolism-induced changes in pH. Consequently precipitation can then occur faster than in systems without bacteria [ 49 ]. For example, in the precipitation of the calcium-magnesium mineral dolomite, the dehydration of the magnesium ion and subsequent carbonation are the rate-limiting step of nucleation [ 50 ]. In the presence of carboxyl groups, [Mg(H 2 O) 6 ] 2+ binds and dehydrates to [Mg(H 2 O) 5 (R-COO)] + . This lowers the activation energy for subsequent carbonation and attachment of Ca 2+ to form dolomite [CaMg(CO 3 ) 2 ] [ 50–52 ]. Thus, bacteria provide a mechanism of heterogeneous precipitation, with their surfaces acting as a nucleation catalyst and template, as well as increasing the saturation state through local attraction of cations. Beyond the direct influence of bacterial surfaces, the microenvironment they create also plays a very important role in influencing ion saturation state. All submerged surfaces, such as those of micro-organisms, are surrounded by a thin-filmed water envelope called the hydrodynamic boundary layer [ 53 ]. Bacteria live at an extremely low Reynolds number, that is, the viscous forces of the environment dominate over their ability to move. As a consequence, these bacteria experience greater viscous drag and so struggle to escape their thin water envelope [ 53 ]. Within this surrounding water envelope, concentration gradients of ions can form where local concentrations are higher than in the bulk aqueous environment. Supersaturation will vary with ion concentration and so precipitation will be favoured within the cell-surface vicinity where the concentration is highest. The concentration gradient is the combined result of cell surfaces lowering thermodynamic activation energies, sequestering cations, as well as metabolic activity providing anions such as HCO 3 \n - , all of which occurs within the surrounding water layer [ 29, 54 ]. This principle can be extended further to other layers surrounding microbial surfaces such as biofilm matrices, slimes, sheaths, filaments, capsules and EPS secretions. These layers can create a microenvironment that favours supersaturation and thus precipitation via local changes in ion mobility, viscosity and nucleation kinetics ( Fig. 2 ) [ 55 ]. For example, mineralization has been seen on bacterial sheaths and filaments [ 56, 57 ], slimes [ 58, 59 ], biofilms and EPS [ 60 ]. Some findings even showed that purified EPS alone could contribute to mineral precipitation, while other studies found that EPS production was not always associated with mineral precipitation [ 61–63 ]. This emphasizes the complexity of the process dependent on the bacterium, environment, mineral formed and underlying mechanism. In cyanobacterial systems, EPS has been shown to inhibit the precipitation in the bulk phase of the environment by trapping large amounts of divalent cations in its sugars, acidic residues and negatively charged functional groups. Only upon degradation of EPS and liberation of the cations does the saturation index increase, allowing for the precipitation of minerals [ 24, 64, 65 ]. Fig. 2. Mineral encasement of a bacterial cell. (a) Bacterial cell (green) surrounded by a microenvironment (light blue) created by an extremely low Reynolds number and/or sheaths, capsules, slimes, biofilm matrices or extracellular polymeric substances. (b) Accumulation, stabilization and slow diffusion of ions within the microenvironment close to the cell occurs from metabolism and cell-surface interactions creating a high local ion concentration (dark blue). (c) Within the cell-surface vicinity, at a high ion concentration, the equilibrium is shifted in favour of supersaturation and thus precipitation (grey shapes). (d) Onset of precipitation can lead to the breakdown of the microenvironment and, along with the degradation of some extracellular organic components, leaves behind a mineral-organic phase encasing the cell (grey shapes). Further to creating favourable conditions, the microenvironment is not subject to the same kinetics as the bulk environment and therefore also protects against inhibiting factors such as ion complexing and cation hydration [ 66 ]. Thus, bacterial surfaces and their microenvironments allow precipitation to occur even in unfavourable conditions such as acidic environments [ 67 ]. Over the course of precipitation and with the eventual degradation of some extracellular organic components, the microenvironment is broken down and leaves behind a mineral organic phase encasing the cell, illustrated in Fig. 2 . Active mechanisms by which the bacteria can avoid or escape such encasement are discussed later in this article. Cell surface: polymorph ratio, crystal morphology, mineral type, and crystal size In addition to providing nucleation sites and concentrating ions, surface structures can influence mineral polymorph ratio, crystal morphology and the type of minerals precipitated. Polymorphs have the same chemical structure but differ in their crystal structure [ 68 ]. Calcium carbonate mainly encompasses the polymorphs' calcite, vaterite and aragonite, and their ratios can be affected by cell-surface chemistry. For example, the presence of carboxylic groups, phosphonates, sulfonates and amino acids has been found to promote formation of vaterite [ 69 ]. The morphology of calcium carbonate crystals has been reported to be influenced through the presence of organic matter, e.g. by an increase in acidity of l -amino acids and xanthan content, where calcite crystals transitioned from rhombohedra to fibro-radial spherulites, and the monocrystals that make up the typical vaterite crystal spheres evolved from clustered short needles to clustered large hexagons [ 58 ]. The type of mineral precipitated is in part determined by the selective adsorption of metals to certain functional groups. Different metals were found to bind cell-surface components with different affinities. For example, it was reported that Mg 2+ bound with a higher affinity than Ca 2+ to cell walls of the Gram-positive \n B. subtilis \n [ 46, 47, 70 ] as well as to cell envelopes of the Gram-negative \n Escherichia coli \n [ 71 ]. The selective adsorption of calcium and strontium cations versus that of magnesium to pores within S-layers of \n Synechococcus \n sp. governed the preferred precipitation of the sulphate minerals gypsum and celestite [ 72 ]. Cell surface or metabolism: are precipitating bacteria dead or alive? Different observations have been reported regarding whether BIMP is strictly dependent on bacterial activity, specifically whether dead cells may be able to facilitate biomineralization. This leads to different interpretations of how important cell-surface structures are for the process of mineral precipitation. While materials science studies showed that precipitation can occur on functional group monolayers [ 73 ], absence of precipitation on dead cells suggests that the organic material is not simply a nucleation seed, but that metabolic activity also plays a key role [ 74, 75 ]. In contrast, other work found that minerals do form on dead cells and their debris [ 61, 76, 77 ]. This discrepancy may simply be a result of the differences between bacterial species, environmental conditions or methodologies used to prepare the dead cells, as this may affect structural properties [ 2 ]. Systematic studies would be required to determine how much of this variability in mineral precipitation on live versus dead cells is genuinely due to specific properties of the particular species investigated, or if other factors of experimental design or conditions are the main drivers of the outcome. While an unequivocal answer to the question is currently lacking, considerations of the implications of BIMP in bacterial communities may shed some light. As described above, mineral precipitation on the cell surface leads to encasement of the cell ( Fig. 2 ). Therefore, if only living cells precipitated minerals, the whole population could run the risk of entombment and death. To allow for the continued growth of a population, precipitation might therefore be assumed to occur only on dead cells and/or a restricted number of live cells [ 78 ]. On the other hand, mechanisms exist for active evasion of entombment by shedding encrusted S-layers [ 41 ], forming mineral sheaths/capsules [ 56 ], forming nanoglobules to act as decoy precipitation targets [ 79, 80 ], or even controlling surface functional group distribution to control precipitation occurrence [ 81 ]. The role of metabolism in evading entombment is also unclear. One observation has been that induction of a proton motive force by metabolic activity of live cells reduced the cell-wall metal-binding ability [ 82 ]. Metabolism as an active mechanism against entombment also has been proposed in cyanobacteria and suggested that dead cells could potentially be better at mineral precipitation because they retained more of their negative surface charge [ 83 ]. Zeta potential analysis was used to approximate the net surface charge of the bacteria by measuring the potential differences between the cell and fluid interface [ 84 ]. In these studies, metabolic activity was found to contribute to a more positive surface charge, likely regulated to attract anions for metabolism. On the other hand, dead cells retained a constant negative charge on their surface structures [ 84 ]. At a community level, i.e. a mixture of live and dead bacteria, one explanation for the ability of these bacteria to precipitate minerals on their surface may be as a result of cations binding to negatively charged surfaces of dead or inactive cells. Alternatively, a somewhat counterintuitive explanation might be the attraction of carbonate anions to metabolically active cells and letting these act as the seed for nucleation rather than the typical cations [ 84 ]. Evidence of changes in cell-surface charge between dead and live cells is still limited, and there is likely to be variability among bacterial species depending on their surface structures. Taking into account these observations, the more likely explanation is that most often both surface structure and bacterial metabolism are required as catalysts to modulate precipitation parameters by influencing saturation state and nucleation ability. Precipitation should occur under conditions of supersaturation, when cations attracted to the bacterial surface react with counter anions in the environment. Anion concentration is in turn environment dependent or may be supplemented by metabolism, suggesting both live and dead bacteria may be needed. Bacterial metabolism: pH and anions, including dissolved inorganic carbon (DIC) Apart from the availability of nucleation sites, mineral precipitation also depends on (i) availability of anions, (ii) availability of cations and (iii) pH [ 85 ]. Bacterial metabolism plays an integral role in BIMP whereby it chemically alters the environment through the production of metabolites and by-products that influence the local pH and ion concentrations (e.g. carbonate, phosphate or metal cations). Modulation of these parameters ultimately affects supersaturation conditions and thus precipitation. A key parameter to consider in mineral precipitation is the ion activity product (IAP), which for low-solubility minerals can be approximated as the product of the concentrations of the anion and cation composing the mineral, as exemplified for calcium carbonate in Equation 1. Supersaturation is achieved when the IAP of the mineral exceeds its solubility product constant ( K \n sp ), as defined in the saturation index (SI) (Equation 2). A system is considered supersaturated when SI>0 [ 13, 86, 87 ]. \n (Equation 1) I A P ( C a C O 3 ) = [ C a 2 + ] × [ C O 3 2 − ] \n \n (Equation 2) S I = l o g ( I A P K s p ) \n While K \n sp is a constant for a given system, IAP depends on effective concentrations and can be influenced by environmental factors such as bacterial metabolism. The precise value of SI at which precipitation occurs spontaneously for a given system can vary, depending, for example, on the presence of organics that can promote precipitation or even inhibit it despite high saturation states [ 69, 86, 88–90 ]. In that regard, SI only predicts the point at which precipitation is thermodynamically favoured but not when it actually begins. In BIMP, the point at which precipitation is observed can in part depend on cell density and nucleation points [ 13, 60, 91–93 ], but it also critically depends on the effects of bacterial metabolism on IAP. Metabolic activity is furthermore accompanied by changes in pH due to the production of various metabolic by-products. This in turn affects precipitation potential, with a higher pH directly contributing to the availability of anions through deprotonation and supersaturation. For example, in the case of mineral carbonates, the precipitation potential is dependent on both the pH and the carbonate anion concentration, known as the total dissolved inorganic carbon (DIC), which is the sum of the dissolved forms of CO 2 , HCO 3 \n - and CO 3 \n 2- . Moreover, the concentration of anions is directly related to the pH through the dissociation constants as seen in the carbonate equilibrium (Equation 3) [ 94 ]. At higher pH, the carbonate equilibrium is shifted to the right and carbonate species are deprotonated. As a result, more bicarbonate (HCO 3 \n - ) and carbonate (CO 3 \n 2- ) ions are available for precipitation. Similarly, phosphate groups will be subject to changes in protonation state, depending on environmental pH (Equation 4). Sulphate groups will typically be present in their deprotonated state due to their low pKa values (usually below 2.5) (Equation 5), which will generally be exceeded by environmental pH [ 60 ]. Precipitation at low pH is possible in theory, but mostly applies to phosphate and sulphate-containing minerals where the anion component has a lower pKa. However, in practice, low pH often leads to dissolution of minerals. \n (Equation 3) C O 2 + H 2 O ↔ p K a 6.35 H C O 3 − + H + ↔ p K a 10.3 C O 3 2 − + 2 H + \n \n (Equation 4) H 3 P O 4 ↔ p K a 2.16 H 2 P O 4 − + H + ↔ p K a 7.21 H 2 P O 4 2 − + H + ↔ p K a 12.32 H 2 P O 4 3 − + H + \n \n (Equation 5) H 2 S O 4 ↔ p K a − 3.0 H S O 4 − + H + ↔ p K a 1.99 S O 4 2 − + H + \n Bacterial metabolism, through a modulation in pH and the production of anions such as phosphates, sulphates and carbonates, therefore has a direct influence on IAP and can increase the likelihood of anions and cations precipitating together as minerals [ 87 ]. Which anions are produced ultimately also depends on the availability of nutrients and metabolic capabilities of the specific bacteria present. For example, bacteria capable of reducing sulphate can produce sulphide ions that can directly precipitate as minerals, while bacteria that break down urea or amino acids increase the local pH, which in turn favours formation of carbonates for mineral precipitation ( Fig. 3 ). For reasons of brevity, however, only the key contributing factors in terms of net ion production and pH effects created by different metabolic pathways contributing to mineral precipitation are discussed here. The specific physicochemical details of the various individual metabolic pathways that can induce mineral precipitation have been reviewed elsewhere [ 33, 95 ]. Fig. 3. Metabolic pathways associated with bacteria-induced mineral precipitation. Various products of metabolism result in a net effect, shown on the right, that primes the environment for mineral precipitation. AA refers to anion availability, typically bicarbonate and carbonate. Products in green are increased and those in red are decreased as a result of metabolic activity. Adapted from [ 98 ]. Autotrophic metabolic pathways Autotrophic metabolic pathways such as non-methylotrophic methanogenesis or oxygenic and anoxygenic photosynthesis utilize CO 2 to produce organic matter. This causes a depletion in CO 2 that alters the bicarbonate equilibrium through a shift to the left (Equation 3), leading to removal of H + as bicarbonate concentration increases, as well as dissociation of bicarbonate ions to CO 2 and OH - . The resulting increase in pH favours precipitation under conditions of low DIC but high concentrations of suitable cations ( Fig. 3 ) [ 24, 85 ]. Aerobic heterotrophic metabolism Aerobic heterotrophic metabolism can cause local increases in anion concentration and pH. As mentioned above, aerobic heterotrophs break down organic carbon to produce CO 2 that partially converts to carbonate and bicarbonate and increases DIC and pH in the bulk phase [ 64, 96 ]. Nitrogen cycle Dissimilatory reduction of nitrate under anoxic conditions and deamination of amino acids for their catabolic use both lead to production of ammonium and hydroxide ions and consumption of H + ions. This causes an increase in pH and thus shifts dissociation equilibria of anions that are relevant for mineral formation ( Fig. 3 ) [ 96 ]. The role of ureolysis in mineral precipitation is explained below. Sulphur cycle Dissimilatory reduction of sulphate, carried out in anoxic conditions by sulphate-reducing bacteria, results in the production of carbonate, bicarbonate and hydrogen sulphide (H 2 S) ( Fig. 3 ). Whether this leads to biomineralization depends on the fate of the H 2 S produced. Excreted sulphide can lead to authigenic precipitation in the bulk phase by directly reacting with metal cations in the environment to precipitate sulphide minerals [ 97 ]. Alternatively, loss of H 2 S can occur through degassing or consumption by anoxygenic sulphide phototrophic bacteria that oxidize H 2 S to elemental sulphur and form intra- or -extracellular deposits. The removal of H 2 S increases the pH and thus favours precipitation ( Fig. 3 ) [ 98 ]. On the other hand, autotrophic sulphide-oxidizing aerobic bacteria use H 2 S (and other reduced sulphur compounds, S 0 and S 2 O 3 \n 2- ) to produce sulphate ions that form sulphuric acid, decreasing the pH and dissolving precipitates [ 64, 98 ]. The balance between precipitation and dissolution therefore will be dependent on environmental conditions such as oxygen availability, light and pH, which serve to decouple the different metabolic processes in time and space and establish local conditions where net precipitation can occur [ 99 ]. Single enzyme-mediated reactions Aside from broader metabolic pathways, specific enzymes can also contribute to precipitation. Acid phosphatases liberate phosphoryl groups, thus accelerating formation of phosphate mineral species, and strains overproducing this enzyme were shown to precipitate uranium phosphate species [ 34, 100–102 ]. However, not all bacteria with phosphatase activity can precipitate minerals, lending weight to the idea that specific cell-surface structures are likely required to provide nucleation sites for precipitation [ 103 ]. Carbonic anhydrase, catalysing the interconversion of CO 2 to HCO 3 \n - and H + , has been suggested as a key enzyme in precipitation due to its effect on local HCO 3 \n - concentration. The presence of extracellular carbonic anhydrase was found to govern the location of crystal precipitates in biofilms of \n Alcanivorax borkumensis \n [ 96 ]. Indeed, carbonate precipitation was restricted to areas with high extracellular concentration and activity of carbonic anhydrase. Ureolysis as part of the nitrogen cycle is also an enzymatically driven process. This enzymatic activity may potentially be strong enough to increase supersaturation to such high levels that precipitation can occur without the need for nucleation sites provided by bacterial cell surfaces [ 104 ]. Indeed, it was observed that some strongly ureolytic bacteria could induce calcite precipitation at a considerable distance to the bacterial colony [ 32 ]. While the processes described in this review, i.e. the complex interplay between physical properties of bacterial cells and their metabolic activity, explain why in BIMP one is more likely to encounter heterogenous precipitation, strongly ureolytic bacteria may, in fact, be an exception and capable of driving homogenous nucleation. Cell metabolism: provision of cations Apart from the generation of anion species needed for precipitation, cation availability can also be influenced through metabolic activity. As defined within the IAP, the concentration of the metal cation is also important for the precipitation of a mineral species (Equation 1). Bacteria, often via enzymatic activities, may reduce a mineral compound to produce divalent cations that can then react with anions to precipitate as a different mineral [ 105, 106 ]. Some bacteria utilize metal ions as terminal electron acceptors in microaerobic or anaerobic conditions to produce cations, for example Fe 2+ through reduction of oxidized iron (Fe 3+ ), usually from dissolution of other iron oxides, as reviewed in detail elsewhere [ 106–108 ]. The resulting Fe 2+ can subsequently interact with various anions to form a variety of iron minerals ( Table 1 ). Many iron-reducing bacteria are also capable of reduction of manganese (Mn 4+ to Mn 2+ ), providing Mn 2+ cations for mineral formation [ 59 ]. Metal oxidation can also occur under anoxic conditions through the activity of some phototrophic bacteria and some nitrate-respiring bacteria [ 59 ]. Local cation concentration can also fluctuate due to active bacterial processes such as intracellular metal ion homeostasis via ionic pumps and channels. In high-calcium environments, such as calcareous caves and limestone soils, the need to maintain a low intracellular calcium concentration is essential to ensure bacterial survival and growth [ 109 ]. Microbes can achieve this through active efflux of intracellular calcium by ATP-dependent antiporters, increasing the local calcium availability and pH near the cell surface and thus contributing to precipitation [ 85, 110 ]. Thus, active calcium efflux could be seen to influence biomineralization in two ways: it ensures bacterial growth to provide nucleation sites while simultaneously increasing the local cation concentration. Indeed, active processes of ion excretion may precede the passive precipitation discussed previously and allow microbes to act as nuclei for subsequent crystal growth [ 98 ]. Prospects In exploring the underlying processes enabling BIMP, a lot of benefit has been gained from research across multiple disciplines investigating the different aspects of organic-mineral interphases. While there are mechanistic differences in the way bacteria induce mineralization dependent on their surface architecture and metabolism, understanding the contributing components is important for biotechnological application. An additional layer of complexity is introduced when considering that bacteria do not occur in isolation, and that metabolic processes of one group of organisms are often interdependent with the activities of other groups. Indeed, in nature precipitation results from the activities of mixed populations, which often grow as biofilms rather than planktonic cells [ 24, 98 ]. This could possibly be exploited in utilizing communities and biofilm growth of micro-organisms to maximize precipitation potential. BIMP has seen increased applications in civil engineering and biotechnology over recent years, as extensively reviewed elsewhere [ 10, 12–14, 87 ]. In brief, mineral precipitation mainly has two different roles in these technologies. For applications that include soil consolidation, heritage conservation and self-healing concrete, precipitated minerals and embedded cells and organic components become the ‘glue’ that binds and/or seals the surrounding matrix. For applications of bioremediation such as of toxic heavy metals or radionuclides or in carbon dioxide sequestration, the elements in question are directly precipitated or co-precipitated, rendering them bio-unavailable [ 13, 14 ]. Fundamental mechanistic insight will therefore be important in making more informed decisions in choosing the appropriate bacteria for a specific application in terms of strain characteristics and minerals precipitated. This could allow for selective mineral precipitation, dependent on preferential surface binding and metabolic anion production of the chosen bacterium. Additionally, one could even modulate the speed of precipitation through the choice of metabolic capability, depending on application need. In the future, detailed mechanistic insights may inform rational directed evolution or genetic engineering approaches for application-driven strain development. The complexity of BIMP and its dependency on precise bacterial properties may therefore even be viewed as a benefit. Nature may reveal a useful and versatile toolbox of different bacteria, supplemented by systematic strain engineering to meet future needs for sustainable BIMP technologies."
} | 9,326 |
33241850 | null | s2 | 6,373 | {
"abstract": "Lignocellulosic biofuels and chemicals have great potential to reduce our dependence on fossil fuels and mitigate air pollution by cutting down on greenhouse gas emissions. Chemical, thermal, and enzymatic processes are used to release the sugars from the lignocellulosic biomass for conversion to biofuels. These processes often operate at extreme pH conditions, high salt concentrations, and/or high temperature. These harsh treatments add to the cost of the biofuels, as most known biocatalysts do not operate under these conditions. To increase the economic feasibility of biofuel production, microorganisms that thrive in extreme conditions are considered as ideal resources to generate biofuels and value-added products. Halophilic archaea (haloarchaea) are isolated from hypersaline ecosystems with high salt concentrations approaching saturation (1.5-5 M salt concentration) including environments with extremes in pH and/or temperature. The unique traits of haloarchaea and their enzymes that enable them to sustain catalytic activity in these environments make them attractive resources for use in bioconversion processes that must occur across a wide range of industrial conditions. Biocatalysts (enzymes) derived from haloarchaea occupy a unique niche in organic solvent, salt-based, and detergent industries. This review focuses on the use of haloarchaea and their enzymes to develop and improve biofuel production. The review also highlights how haloarchaea produce value-added products, such as antibiotics, carotenoids, and bioplastic precursors, and can do so using feedstocks considered \"too salty\" for most microbial processes including wastes from the olive-mill, shell fish, and biodiesel industries."
} | 430 |
33241850 | null | s2 | 6,374 | {
"abstract": "Lignocellulosic biofuels and chemicals have great potential to reduce our dependence on fossil fuels and mitigate air pollution by cutting down on greenhouse gas emissions. Chemical, thermal, and enzymatic processes are used to release the sugars from the lignocellulosic biomass for conversion to biofuels. These processes often operate at extreme pH conditions, high salt concentrations, and/or high temperature. These harsh treatments add to the cost of the biofuels, as most known biocatalysts do not operate under these conditions. To increase the economic feasibility of biofuel production, microorganisms that thrive in extreme conditions are considered as ideal resources to generate biofuels and value-added products. Halophilic archaea (haloarchaea) are isolated from hypersaline ecosystems with high salt concentrations approaching saturation (1.5-5 M salt concentration) including environments with extremes in pH and/or temperature. The unique traits of haloarchaea and their enzymes that enable them to sustain catalytic activity in these environments make them attractive resources for use in bioconversion processes that must occur across a wide range of industrial conditions. Biocatalysts (enzymes) derived from haloarchaea occupy a unique niche in organic solvent, salt-based, and detergent industries. This review focuses on the use of haloarchaea and their enzymes to develop and improve biofuel production. The review also highlights how haloarchaea produce value-added products, such as antibiotics, carotenoids, and bioplastic precursors, and can do so using feedstocks considered \"too salty\" for most microbial processes including wastes from the olive-mill, shell fish, and biodiesel industries."
} | 430 |
31969660 | PMC6976594 | pmc | 6,375 | {
"abstract": "Coral reefs are vital for the marine ecosystem and their potential disappearance can have unequivocal consequences on our environment. Aside from pollution-related threats (changes in water temperature, plastics, and acidity), corals can be injured by diseases, predators, humans and other invasive species. Diseases play an important role in this decline, but so far very few mitigation strategies have been proposed and developed to control this threat. In this work, we demonstrate that recently developed bi-layer human skin wound treatment patches containing antiseptics and natural antioxidants with controlled-release capacity can be adapted to treat scleractinian coral wounds effectively. A hydrophilic bilayer film based on polyvinylpyrrolidone (PVP) and hyaluronic acid was used to cover the open wounds while delivering the antiseptics for rapid action. Afterwards, the hydrophilic bi-layer covered wound was sealed with an antioxidant and hydrophobic ε-caprolactone-p-coumaric acid copolymer by melt injection at low temperatures. Treated coral injuries were monitored both in aquaria system and in natural environment in Maldives for over 4 months to reduce the number of entry points for organisms that could lead to diseases. The corals well-tolerated both biomaterials as well as the antiseptics incorporated in these materials. The treatments displayed self-adhering properties, tuneable dissolution time, and biocompatibility and stimulated regeneration properties within the coral wound. As such, this work demonstrates that certain human skin wound treatment materials can be successfully adapted to the curing of coral wounds and delivery of specific drugs to slow down, reduce or even stop the spread of diseases in scleractinian corals as well as in all other benthic organisms affected by uncontrolled pathologies.",
"introduction": "Introduction Coral reefs are declining worldwide with coral diseases emerging as one of the most distressing threats 1 . In the last 50 years, this ecosystem is subjected to constant and extensive degradation, and a reduction of 25% of the global functional coral reefs has been recorded 2 . Among the coral reefs dwellers, reef-building corals are one of the most fragile components affected by changes in the marine realm 3 . Environmental stressors, including rising of the seawater temperatures, nutrient input of runoff, and sedimentation exacerbate the declining health of corals. Diseases play an important role in this apocalyptic scenario with the increased sea temperature that expedites the frequent diffusion of infections caused by microorganisms 3 , 4 . Currently, the number of known and identified coral diseases varies from 24 to 40, but it might be even more due to limited analytical characterization methods, deprived knowledge of the putative pathogens and the consequent deficiency of epizootiological data 5 , 6 . Therefore, the lack of well-defined knowledge makes the control of coral diseases a real challenge, especially when they are caused by bacteria 6 . One of the most dramatic and recent examples of such diseases is the Stony Coral Tissue Loss Disease (SCTLD), which has been destroying an entire ecosystem on the Florida Reef Tract since 2014. So far, its effect is unprecedented, and all the efforts carried out by researches have not been successful in discovering the relevant agents and taking the appropriate countermeasures 7 . For instance, mitigation techniques have already been proposed to reduce or slow down the progression of many coral diseases, however, most of them have not yielded and effective solution on a global scale 8 yet. In particular, coral wounds can be considered as the starting point of some coral diseases. Since the wound practically exposes the coral skeleton, pathogens settle on the skeleton and transform it into a proliferation substrate, increasing the likelihood of infections 9 . The first method to treat and manage a coral disease was developed in the late 1980s to control an outbreak of black-band disease (BBD) in Looe Key National Marine Sanctuary 10 (lower Florida Keys, USA). Later on, Hudson et al . 10 developed an aspirator device to remove the filament mats produced by the BBD bacterium, and then they covered the wound with a clay-based sealant. However, as they reported, in some cases, this passive covering did not ensure efficient disinfection of the underlying area leading to post-treatment infections of the corals. In the meantime, several other treatments and strategies have been proposed to manage and mitigate the damage inflicted by BBD and similar syndromes 11 , 12 . One of the most common strategy suggested was the removal of the lesion, considered as a common form of medical intervention to cure some diseases affecting both vertebrates and invertebrates 11 , 12 . Other than this, researchers used biological control techniques such as probiotics and phage therapy to manage bacterial pathogens 13 , the mechanical removal of disease vectors, such as the corallivore Coralliophila abbreviata 14 , and the excision of healthy coral branch tips from diseased Acropora cervicornis colonies 15 . In addition, researchers also used epoxy adhesives to mechanically block the progression of a tissue-loss disease on A. cervicornis 15 . Aeby et al. reported the application of antibacterial chlorine powder embedded within epoxy to treat and mechanically block the progression of BBD in Montipora 12 , but considering the polymicrobial origin of several coral diseases, the employment of more specific type of drugs can be a better strategy to complete eradicate the infection. A recent work of Sweet et al . 16 showed as some antibiotics can be employed for the treatment of the White band disease (WBD), a polymicrobial infection of the corals. Here, the authors simply added the antibiotics at a suitable concentration in the water of the aquarium. Although the results were positive in mitigating the WBD diffusion, the same authors highlighted how this type of treatment could not be applied at a large-scale level. Indeed, to reach an effective concentration of antibiotic in the sea, we would risk compromising the marine ecosystem, limiting this approach to emergency cases in the aquarium environment. Therefore, the ever-increasing spread of coral diseases and the lack of adequate healing strategies bring up the urgent necessity to design new treatments for the in situ delivery of drugs such as antiseptics, antibacterial agents or anti-protozoans, and, at the same time, avoiding the spread of potential pollutions in the sea. One of the approaches could be the fabrication of biocompatible, biodegradable, and bioactive polymeric drug delivery systems suitable for the coral application. In particular, the modern strategies and treatments applied for human wounds can be a point of inspiration and constitute some potential guidelines also for the coral disease prevention 17 – 20 . In humans, the main role of the skin tissue is to protect the body from external pathogenic agents, acting as a shield 21 . After a skin injury, the loss of this barrier can cause rapid infections and bacterial colonization. The presence of bacteria in the wound bed is one of the reasons why the healing process is delayed 21 . Therefore, in order to avoid bacterial infection and promote the healing, an ideal wound dressing, firstly, should ensure a covering of the wound. At the same time, the wound dressing should be able to kill the bacteria that have already infected the wound 22 . Similarly, for coral injuries or wounds, the potential wound dressings must be made from materials that are not bio-persistent and toxic for marine environment in general. Intelligent wound dressings can be made from several polymers and active compounds that can be infused with medicinal compounds to accelerate and manage the healing process. Among them, the biocompatible synthetic polymer polyvinylpyrrolidone (PVP) has gained interest in the topical application not only because it can modulate the crystallinity of several drugs favourably but also for its capacity to form transparent films and ensure good self-adhesion to the moist skin 23 – 25 . In recent years, the natural polymer hyaluronic acid (HA) has also played an important role in the pharmaceutical field due to its anti-inflammatory, regenerative and anti-age properties. By the same token, polycaprolactone (PCL) is a biodegradable synthetic polyester 26 that has been broadly applied in biomedical fields for the preparation of controlled drug delivery systems such as implants and scaffolds 27 , 28 . It has been recently combined or co-polymerized with effective natural antioxidants such as vanillic, ferulic, p-coumaric and syringic acids 29 . In this work, we attempted to use polymeric smart wound dressings in tandem, inspired by human skin wound care and management to cure coral wounds. In particular, we used a dual-drug carrying PVP-HA-based bilayer construct, loaded with eosin-based antiseptic and the antibiotic Ciprofloxacin, as well as a ε-caprolactone-p-coumaric acid copolymer (PCL-PCA) developed for skin regeneration 25 , 30 . Dual drug-carrying bilayer construct was applied directly onto the wound and acted as material for the in situ delivery of two antibacterial drugs 25 (see Fig. 1 ). Afterwards, the PCL-PCA copolymer with antioxidant capacity was melted to seal and shield the wound. The two materials were applied and tested in combination (see Fig. 1 ) and separately as control samples, and monitored during two distinct phases: ex-situ and in-situ . The first part of the experiments consisted of 10-days of observation ex-situ , and we focused on understanding the ability of the two materials to adhere to the coral surface, to resist underwater dissolution and to evaluate potential toxic effects on the coral, if any. Afterwards, the survival and the health of the corals were followed for four months continuously in-situ . Figure 1 Schematic representation of a coral wound and its potential treatment. Illustration of a coral wound in the sea. In the first close-up, all the potential risks when a coral loses its protection are presented. In the other two close-ups, the two potential steps of the treatment suggested in this work are shown. The bilayer should act as a bioactive dressing for the delivery of drugs in order to disinfect and treat the wound (Step 1). Instead, the PCL-PCA paste should act as a shield against all the external agents and the fast dissolution of the bilayer dressing (Step 2).",
"discussion": "Results and Discussion Coral wounds may increase susceptibility to some diseases 31 , hence, the materials developed and used in this study are expected to prevent disease outbreaks in the short term relevant to the early stages of the coral wound. During the monitoring period, the applied materials were well-tolerated by the coral system and exhibited proper and healthy restoration properties. In total, 52 coral wounds were created and they were monitored within 4 experimental groups: untreated, treated with only bilayer, treated with only PCL-PCA copolymer, and treated with both bilayer and PCL-PCA copolymer (Figs. 2 and S1 ). Figure 2 Coral wounds. Photographs in tank immediately after inflicted injuries of the control ( a ), bilayer-treated sample ( b ), PCL-PCA-treated sample ( c ) and bilayer + PCL-PCA-treated sample. ( d ) The arrows indicate the position of the injuries. The first treatment tested consisted of the application of the dual drug-carrying PVP-HA-based bilayer film directly on the open coral wounds. In total, 8 lesions have been treated with this material, where each of them has been carefully covered by a strip of bilayer film wrapped over the wound (red-colored foils in Fig. 3a ). As expected, due to the hydrophilic nature of its components PVP and HA, the bilayer dissolved and disintegrated in few hours after the total immersion in the water. The dissolution process started about 10–15 minutes after the immersion (Fig. 3b ) in all coral-induced wounds where the material has been located. Eventually, the bilayer film disappeared, revealing the bare skeleton underneath after about 3 hours (Fig. 3b–g ). The treatment has been monitored for 10 days to observe any further changes on the coral fragments before termination. Figure 3 Application of bilayer materials on coral wounds. Photographs of bilayer applied on different coral wound monitored for the first 3 hours. For the other 3 experimental groups, during this ex-situ phase, we took into account different parameters, reported in Table S1 , to investigate the state of health of the corals. In particular, we evaluated the injury condition, the colony health, and the biopaste condition. For each condition, three variables were ranked with values from 0 to 4 (see Table S1 ). In Fig. 4 , we reported the main results of the monitoring parameters at different time points. For the injury condition, we reported results about the percentage of covered injuries, the general condition of the injuries, and the progression of the healing. As colony health, we evaluated the formation of mucus, the bleaching, and the necrosis. Instead, regarding the biopaste condition, we monitored the hardening, the adhesion, and the dissolution parameters. Figure 4 Monitoring parameters. Graphs of the observed parameters during the ex-situ phase for the PCL-PCA, PCL-PCA + Bilayer and for the Control expressed as “Injury Condition” ( a–c ), “Colony Health” ( d–f ), and “Biopaste Condition” ( g–i ). The PCL-PCA copolymer treatment consisted of the application of a melted material directly on the 16 induced coral wounds. The PCL-PCA copolymer is a solid film at room temperature, thus to obtain a conformal coverage of the coral wound, it was heated to 60 °C and immediately applied in the molten state. After the immersion in water, the material solidified, leaving a cup-shaped self-adherent structure on all coral wounds (Fig. 5b ). Immediately after the application and during the whole initial phase of monitoring period (10 days), the PCL-PCA copolymer remained adherent to the wounds and no signs of dissolution and detachment were noticed for all the coral wounds under analysis except for a single wound that lost few small pieces from the top portion of the cup without exposing the wound. Furthermore, even if no detectable anomalies have been observed regarding the mucus production and presence of necrosis tissue, a slight variation in color in three of 16 fragments was observed between day 8 and day 10. In particular, those fragments showed a slightly pale tissue diffuse spread throughout their surface (Fig. 4d–f ). Figure 5 Coral wounds status after 10 days. Photographs in tank after 10 days from the injuries of the control ( a ), PCL-PCA sample ( b ) and bilayer + PCL-PCA sample. ( c ) The arrows indicate the position of the injuries. Based on these observations, it appears that the application of the melt copolymer does not injure the coral. Note that the copolymer is not constantly kept at its melting point during its application on the coral. Hence, it already cools down during application. However, we are aware that the melted copolymer can prematurely close the wound or kill some bacteria. At this stage, it is difficult to quantify this. However, during the application, the wound does not experience a constant high temperature. In relation to this, some works 32 , 33 demonstrated that a similar temperature maintained at a longer time can have a weak antibacterial activity. We cannot exclude that the molten biopolymer injection process can affect the microorganisms present in the wound. Hence, further in-depth studies will be planned and conducted to this effect. For the third set of experiments, a dual treatment of bilayer and PCL-PCA was applied on 12 coral-induced wounds (Fig. 5c ). This treatment combines the possibility of a short time in situ drug delivery ensured by the bilayer film and the sealing and shielding activity of the PCL-PCA cup. This tandem treatment demonstrated superior self-adhesion to the wound surface with respect to the PCL-PCA treatment. In support of this, none of the applied PCL-PCA structures came off the induced coral wounds and, moreover, no variations in the consistency or dissolution of the copolymer have been detected (Fig. 4g,h ). In line with this, the condition of the lesion zones after 10 days of monitoring remained at an optimal state, with no changes in the coverage status and including the backside of the lesion zone. A small alteration observed was related to three fragments that appeared slightly paled between day 9 and day 10. No relevant signs of mucus production and the presence of tissue necrosis were noticed; see Figs. 4d,f and 5b,c . On the untreated coral wounds, no significant changes occurred in the inflicted wounds during 10 days of monitoring (Fig. 5a ). However, light pale conditions and patches over the wound surfaces formed almost on all the fragments monitored (Fig. 4e ). During these 10 days of indoor aquaria system monitoring, all fragments survived, and no clear differences were observed among the untreated control, the PCL-PCA and bilayer/PCL-PCA-treated samples in terms of healing or progression of the injuries, see Fig. 4 . This may be attributed to the particular coral species and its resistance, the number of wounds produced, absence of infection, use of aquaria controlled systems and more likely to a combination of these scenarios. Moreover, to the best of our knowledge, several causes can trigger the host’s immune responses involved in wound healing such as the toll-like pathway, the melanin synthesis for tissue regeneration, the complementary system resulting in apoptosis of damaged cells, and cell activation to move amoebocytes to the wound 34 . Furthermore, tissue regeneration and its associated immune pathways require extensive cellular resources (e.g., carbon products and amoebocytes) to be translocated to the wound 35 . Therefore, to further explore the effectiveness of the treatments, in the second part of the study, all the coral fragments were attached on a rope and dipped 5–20 meters down the ocean waters (December 2018, Maldives) and monitored till April 2019 (see Figure S2 ). The in-situ monitoring revealed that all treated fragments survived and that most of the injuries showed complete tissue recovery after about 4 months. By contrast, after 2 months of observations on untreated wounds (the control samples) demonstrated some lesions with algal overgrowth (see white arrow in Fig. 6a ), suggesting a failure in the natural healing process compared to the treated samples where no algae growth and invasion were noticed (Fig. 6b,c ). Figure 6 Coral wounds status after 2 months. Photographs underwater after 2 months from the injuries of the control ( a ), PCL-PCA-treated sample ( b ) and bilayer + PCL-PCA-treated sample. ( c ) The arrows indicate the position of the injuries. After 4 months underwater, we used the generation of new tips “branchiness” as measure of a positive healing effect on the coral fragments treated with PCL-PCA and bilayer/PCL-PCA materials and compared the results with untreated coral fragments. All fragments showed formation of new tips (including the control samples), confirming the suitability of Maldivian lagoons for the restoration of branching Acropora corals, see Fig. 7 . Figure 7 Coral wounds status after 4 months. Photographs underwater after 4 months from the injuries of the control ( a ), PCL-PCA sample ( b ) and bilayer + PCL-PCA sample ( c ). The arrows indicate the position of the new tips. An average of 16.4 new tips per fragment for the untreated corals was observed. Instead, in the case of corals treated with PCL-PCA and PCL-PCA/bilayer an average of 19.4 and 21.4 new tips per coral fragment were recorded, respectively (Fig. 8 ). Figure 8 Branchiness. The number of new tips for fragment formed after 4 months from the wound injury. Although the branchiness per fragment was not statistically significant between the treatments (H- Kruskal Wallis p = 0.323), this trend was also observed in the total number of new tips formed. Indeed, the untreated coral produced in total 82 new tips, while the corals treated with PCL-PCA and PCL-PCA/bilayer, generated 97 and 107 new tips, respectively. Moreover, the highest number of new tips (n = 34) has been observed on a fragment treated with PCL-PCA/bilayer. As such, although the tandem treatment appears to have a positive impact on the coral fragment restoration, the next steps will be focused on verifying the efficacy of the treatment in an infected coral system. However, the slight difference observed may be indicative of possible future development of similar tools for the large-scale treatment of coral diseases. In the real ocean habitat, the number of new tips may be influenced by many factors such as the number of starting branches, the total size of the fragment, the presence of predators, nonetheless, present observations indicate that the fragments treated with PCL-PCA/bilayer grow and heal faster than the untreated and PCL-PCA-treated samples. It might be possible that the fragments without treatments needed more energy to cover the injuries with the new tissue. In fact, corals use the same finite cellular resources for both tissue regeneration and reproduction 35 , creating a tradeoff between these metabolic processes. Immediately after the damage, polyps neighboring lesions experience a limited supply of cellular resources, reducing local reproductive output within the colony as resources are shunted toward wound healing 36 . On the other hand, we cannot exclude that the competition between algae invasion or other organisms that colonize the open wound and healing may slow the recovery process in the control coral fragments compared with the other two treatments. In line with this, the use of injectable PCL-PCA copolymer as a seal for injuries not only closes the injury but may also hinder the settlement of other organisms, including the algae. Although more observations and data would be required, this may be attributed to the inherent antibacterial, antifungal and antioxidant properties of p-coumaric acid 37 . More habitat observations will be needed to confirm these preliminary but encouraging observations toward controlling coral reef damage. Similarly, finding the proper coral wound dressing treatments may not only reduce the corallivores activity but also may function to enhance coral growth. Indeed, protection from corallivores with various foraging strategies can increase coral growth 38 and coral reproductive potential 39 . Moreover, beyond affecting corals through mechanical damage, corallivores provide entry points for opportunistic bacteria (e.g., coral diseases) and can aid in parasite transmission on the reef 40 . Further understanding of how the use of antiseptic bilayer and the injectable PCL-PCA copolymer might shape the effects of corallivores on coral health will also be important and will need to be elucidated in the next future. It is widely acknowledged that the systems of large-scale coral reefs are declining 41 . Strategies for ecosystem-scale reefs conservation is now an indispensable component for the recovery of this ecosystem. Given the inability of these species 2 to adapt to rapid environmental changes 42 , vigorous efforts have been initiated in various areas of the world to enhance the recovery of reefs or increase their resilience 43 . One example is the coral reef restoration practice that consists of the active recovery of an area that has been degraded, damaged, or destroyed by direct transplantation, gardening and seeding approach 44 , 45 . This requires open wound coverage to avoid settlement of pathogens or reducing the spread of infectious disease as well as the algal overgrowth. Thus, the use of new mitigation tools aimed to cure open wounds will be at the forefront of these efforts. In conclusion, our preliminary tests reveal that treating scleractinian coral injuries/wounds by combining a rapidly dissolving antiseptic bilayer film with an injectable antioxidant copolymer can be a promising method to reduce the number of wound entry points for organisms that could lead to diseases. Scleractinian coral well-tolerated both biomaterials tested as well as the antiseptics incorporated in these materials. In addition, the treatments had very good self-adhering properties, tuneable dissolution time, and biocompatibility and promoted regeneration of coral tissues. PCL-PCA copolymer not only acts as a hydrophobic seal for the wound but also keeps the treated zone free from invasive proliferation. This work could be a first step toward applying active biomaterials developed for human wound treatment to the coral restoration field. However, it is stressed that future work related to this tandem treatment should be conducted in more detail on already infected coral wounds in their real habitats in order to assess its efficiency. Nonetheless, these preliminary observations are encouraging in the sense that certain human skin wound treatment soft materials can be adapted to the sealing and subsequent curing cure of coral wounds while delivering specific drugs to the potential infection zones. This would pave the way to slow down, reduce or even stop the spread of diseases in scleractinian corals as well as in all other benthic organisms affected by uncontrolled pathologies."
} | 6,400 |
29574330 | null | s2 | 6,376 | {
"abstract": "One promise of synthetic biology is to provide solutions for biomedical and industrial problems by rational design of added functionality in living systems. Microbes are at the forefront of this biological engineering endeavor due to their general ease of handling and their relevance in many potential applications from fermentation to therapeutics. In recent years, the field has witnessed an explosion of novel regulatory tools, from synthetic orthogonal transcription factors to posttranslational mechanisms for increased control over the behavior of synthetic circuits. Tool development has been paralleled by the discovery of principles that enable increased modularity and the management of host-circuit interactions. Engineered cell-to-cell communication bridges the scales from intracellular to population-level coordination. These developments facilitate the translation of more than a decade of circuit design into applications."
} | 234 |
25991679 | PMC4442139 | pmc | 6,379 | {
"abstract": "ABSTRACT Wetland restoration on peat islands previously drained for agriculture has potential to reverse land subsidence and sequester atmospheric carbon dioxide as peat accretes. However, the emission of methane could potentially offset the greenhouse gas benefits of captured carbon. As microbial communities play a key role in governing wetland greenhouse gas fluxes, we are interested in how microbial community composition and functions are associated with wetland hydrology, biogeochemistry, and methane emission, which is critical to modeling the microbial component in wetland methane fluxes and to managing restoration projects for maximal carbon sequestration. Here, we couple sequence-based methods with biogeochemical and greenhouse gas measurements to interrogate microbial communities from a pilot-scale restored wetland in the Sacramento-San Joaquin Delta of California, revealing considerable spatial heterogeneity even within this relatively small site. A number of microbial populations and functions showed strong correlations with electron acceptor availability and methane production; some also showed a preference for association with plant roots. Marker gene phylogenies revealed a diversity of major methane-producing and -consuming populations and suggested novel diversity within methanotrophs. Methanogenic archaea were observed in all samples, as were nitrate-, sulfate-, and metal-reducing bacteria, indicating that no single terminal electron acceptor was preferred despite differences in energetic favorability and suggesting spatial microheterogeneity and microniches. Notably, methanogens were negatively correlated with nitrate-, sulfate-, and metal-reducing bacteria and were most abundant at sampling sites with high peat accretion and low electron acceptor availability, where methane production was highest.",
"introduction": "INTRODUCTION Wetlands cover about 5 to 8% of the earth’s land surface ( 1 ) and provide important ecosystem services such as wildlife habitat, water purification, and flood control. As a major terrestrial carbon reservoir, estimated at 20 to 30% of the global soil carbon pool ( 2 ), wetlands play an important role in global carbon cycling, yet around the world wetlands are shrinking due to agricultural and industrial development and urbanization ( 3 ), releasing stored carbon into the atmosphere and accelerating climate change. In the Sacramento-San Joaquin (SSJ) Delta area, California, historic freshwater tidal marshes were drained and converted to agriculture for their fertile organic-rich soils between the late 19th and early 20th centuries ( 4 ). Substantial land surface subsidence has since occurred, largely due to accelerated microbial oxidation of peat as drainage increased soil aeration ( 5 ), causing significant carbon loss to the atmosphere and imposing a risk of levee failures in the SSJ Delta ( 6 ). One potential means to mitigate these risks is to restore these historical wetlands, as waterlogged anoxic conditions are expected to slow microbial decomposition and favor peat accumulation from wetland plant detritus. To evaluate the long-term carbon storage rates and land subsidence reversal potential of reestablished wetlands, in 1997 the U.S. Geological Survey (USGS) and the California Department of Water Resources (DWR) started a pilot-scale restoration project on Twitchell Island in the SSJ Delta with managed hydrology. Data collected from 1997 to 2006 demonstrated that rapid peat accretion and land surface elevation were achievable, with an average rate of ~4 cm/year ( 7 ). In addition to reversing land subsidence, the high primary production and low decomposition rates in restored wetlands may result in a net atmospheric carbon dioxide (CO 2 ) sequestration, allowing them to act as “carbon farms.” However, one major concern is the emission of methane (CH 4 ), a common decomposition end product in anoxic environments when terminal electron acceptors are depleted. CH 4 is a potent greenhouse gas (GHG) with a 100-year global warming potential 25 times higher than that of CO 2 , and natural wetlands contribute ~20 to 39% of global CH 4 emissions ( 8 ), making them the largest nonanthropogenic source of atmospheric CH 4 . When CH 4 emission is large enough to counterbalance the CO 2 captured by primary production, a wetland may effectively change from a GHG sink to a GHG source ( 9 ). CH 4 and CO 2 flux data collected during the first 6 years (1997 to 2003) from the pilot-scale restoration wetlands on Twitchell Island indicated that these wetlands could mitigate carbon loss and even become a net GHG sink ( 10 ). However, their long-term carbon storage potential and GHG budget are the subject of ongoing investigation. Net CH 4 emission is governed by production, oxidation, and transportation and varies widely among wetlands due to differences in vegetation, soil type, pH, organic carbon composition, water chemistry, hydrology, and climate, as discussed in reviews ( 11 – 14 ). In wetland ecosystems, microbial communities play an important role in governing carbon flux, as dead plant biomass is either stored as peat or decomposed through microbial activities. Under conditions depleted of oxygen and other electron acceptors, methanogenic archaea use CO 2 or small organic compounds (e.g., acetate and methylamines) as the terminal electron acceptor to produce CH 4 . The amount of carbon diverted to methanogenesis can be influenced by anaerobic respiration processes dependent on nitrate, manganese(IV), iron(III), and sulfate, which commonly occur in wetland environments. The produced CH 4 can be consumed by methanotrophs, which generate energy through oxidation of CH 4 with oxygen at the water-sediment interface or rhizosphere, where both CH 4 and oxygen are available. All these microbial processes can affect the net production and release of CH 4 . Variations in peat accretion within the pilot-scale restored wetland on Twitchell Island have been attributed to the hydraulic design (a gradient from inlet to outlet), and we expected to also observe biogeochemical gradients associated with the hydrology. Therefore, we hypothesized that peat microbial community composition and functions, as well as CH 4 emission, would exhibit patterns associated with the biogeochemical gradients due to distance from the inlet and proximity to plant roots. To test this, we collected biogeochemical data, evaluated CH 4 emission from different sites and sample types within the wetland, and applied a high-throughput sequencing approach to characterize peat microbial community composition and functional profiles, focusing on the microbial populations and processes influencing CH 4 flux. We aimed to identify community patterns, indicator species, genes, and pathways that are associated with biogeochemical variables and CH 4 emission along the gradient.",
"discussion": "RESULTS AND DISCUSSION Site biogeochemistry. We selected sites with a decreasing proximity to the inlet and differing rates of peat accretion: an “inlet” site with low accretion (A), a “transitional” site with intermediate accretion (B), and two “interior” sites with high accretion (C and L) ( 7 ) (see Fig. S1 in the supplemental material). From the inlet to interior sites, pH, sulfate, nitrate, and dissolved oxygen (DO) at the standing water-peat interface decreased, and soluble iron (Fe) and manganese (Mn) increased in February ( Fig. 1a and b ). Increasing solubilization of Fe and Mn results from solid-phase Fe(III) and Mn(IV) reduction ( 12 ). These physicochemical patterns across sites were similar for August ( Fig. 1d and e ), although weaker than those in February. The pH of river water inputs was ~7.7 and decreased from the inlet to interior sites (from 7.0 to 6.5 in February and from 6.5 to 6.2 in August), likely due to anoxic decomposition of plant detritus, which releases carbonic, fulvic, humic, and other organic acids ( 15 ). River water had a DO concentration of ~8 mg/liter and was the primary source of nitrate, sulfate, and oxidized Fe ( 10 ). The observed decreases in electron acceptors in conjunction with increases in reduced Fe and Mn suggest that a variety of electron acceptors were being consumed along the water passage, although at these concentrations nitrate is predicted to be the most energetically favorable electron acceptor. Additionally, interior sites (“backwater” areas) experienced less hydraulic mixing and chemical exchange with river water than did the inlet site, which likely further decreased the influx of electron acceptors to the interior. FIG 1 Physicochemical gradients across sites A (inlet), B (transitional), and L/C (interior) at the standing water-peat interface in February (a and b) and August (d and e) and peat pore water chemical profiles along the depth at site A (inlet) in February (c) and in August (f). Iron (Fe) and manganese (Mn) were measured as the soluble fraction, which is mostly as Fe(II) and Mn(II), respectively, under in situ pH. At the inlet site, with increasing peat depth, we observed decreases of nitrate and sulfate and increases of soluble Fe(II) and Mn(II) for both seasons ( Fig. 1c and f ), indicating that the environment becomes more reduced with depth as is common for wetland pore waters ( 16 ). Other sampling sites displayed similar depth-dependent redox gradients (see Fig. S1 in the supplemental material). CH 4 flux. From laboratory incubations of February samples, statistically significant differences in CH 4 production were observed among the three sites ( Fig. 2a ). As the incubations were conducted in airtight jars, CH 4 oxidation was expected to be minimal. The lower net production of CH 4 from samples collected at the inlet than from the transitional or the interior site suggests lower methanogenic potential at the inlet site FIG 2 Box plot of net CH 4 production from the laboratory incubation of samples collected in February (a) and box plot of noon CH 4 flux measured on-site during August sampling (b) for the different sites. Letters within the plots (“a,” “b,” and “c”) indicate groups by Duncan’s new multiple range test. Sites within the same group are not significantly different. In August, CH 4 flux was monitored in situ using static chambers. Significantly lower CH 4 emissions were observed at the inlet and transitional sites than at the interior site ( Fig. 2b ). Although CH 4 emission at the inlet site averaged lower than that at the transitional site, this difference was not significant. Overall, both February laboratory incubation and August on-site measurement suggest similar variation in CH 4 emission among sites, which was lower at the inlet and higher at the interior. Microbial community composition. Wetland microbial communities as assessed by 16S rRNA gene sequencing harbored members of numerous phyla and averaged a Shannon diversity index of 5.8, higher than the average Shannon index of 5.0 in the adjacent cornfield soil. Wetland microbial community composition was also different from that of the adjacent cornfield soil. Specifically, wetlands displayed higher abundances of Proteobacteria , Chloroflexi , Bacteroidetes , and Euryarchaeota , particularly Methanomicrobia (see Fig. S2 in the supplemental material), and this is consistent with the expectation that after inundation, soil environments become more anoxic and therefore enriched in these phyla commonly found in anoxic environments ( 17 ). At the operational taxonomic unit (OTU) level, community composition was more influenced by sample site and sample type (bulk peat versus plant rhizome) and less influenced by depth or season ( Fig. 3 ). Among February samples, the largest site-associated community difference was between the inlet and the interior site, with communities from the transitional site in between on the nonmetric multidimensional scaling (NMDS) plot ( Fig. 3a ). A similar site-associated community pattern was observed for August samples, but communities from the transitional and the interior sites were not significantly different ( Fig. 3c ), which may be attributed to the weaker site chemical gradients in August than in February ( Fig. 1 ). Site-associated community differences were also revealed on the correspondence analysis biplot (see Fig. S3 in the supplemental material), where the environmental variables, particularly the electron acceptor availabilities (such as DO, sulfate, and nitrate), were strongly correlated with the community patterns. FIG 3 Nonmetric multidimensional scaling (NMDS) analysis of microbial communities based on relative abundance of OTUs for February (a and b) and August (c and d) wetland samples. (a and c) Data points colored by sample site; (b and d) data points colored by sample type. The indicated ANOSIM R statistics and PerMANOVA P values for panels a and c and for panels b and d were based on the comparisons among sample sites and among sample types, respectively. Sample type also influenced microbial communities. Tule and cattail rhizomes contain microbial communities distinct from bulk peat communities ( Fig. 3b and d ), likely reflecting the microenvironmental and/or chemical differences between rhizomes and bulk peat. Bulk peat is anoxic and mainly comprised of decomposed plant material. However, wetland vascular plants transport oxygen to roots to support root aerobic respiration and oxidation of Fe(II) and Mn(II) ( 18 ); oxygen leakage from roots and rhizomes can cause elevated oxygen levels immediately surrounding roots and rhizomes ( 19 ). In addition, labile carbon is released from plant roots as exudates ( 20 ), leading to elevated levels of exudate-derived metabolites, such as acetate, in the rhizosphere ( 21 ). Further, plant-microbial interactions, including symbioses, can select for unique microbial communities within the rhizosphere ( 22 ). Community differences between low- and high-CH 4 -flux sites. Over 100 OTUs are differentially represented between the low-CH 4 -flux communities (from the inlet site) and the high-CH 4 -flux communities (from the transitional and the interior sites) for February samples (see Table S1A in the supplemental material). Of particular interest are methanogens, which are anaerobic archaea belonging to the Euryarchaeota . Three microbial pathways have been found for methanogenesis depending on substrates used: hydrogenotrophic (from H 2 and CO 2 ), methylotrophic (from methylated compounds), and acetoclastic pathways (from acetate) ( 23 ). Consistent with the higher CH 4 flux observed in transitional and interior sites, we observed higher relative abundances of methanogenic OTUs at these two sites (see Table S1A and Fig. S4a ), including OTUs belonging to the hydrogenotrophic Methanoregula and Methanobacterium genera ( 23 – 25 ), the metabolically versatile Methanosarcina genus that can use all three methanogenic pathways ( 23 ), and the acetoclastic specialist Methanosaeta , whose cultivated representatives use only acetate and have a high affinity for acetate compared to that of Methanosarcina ( 26 ). In contrast, many proteobacterial OTUs were more abundant at the inlet site (see Table S1A and Fig. S4b in the supplemental material). These include OTUs classified as Dechloromonas , many members of which are able to use nitrate as an electron acceptor; Desulfobacteraceae , a sulfate-reducing family belonging to the Deltaproteobacteria ( 27 ); Thiobacillus (16S rRNA V8 region 100% identical to Thiobacillus denitrificans ), an obligate autotroph that can couple thiosulfate oxidation to denitrification ( 28 , 29 ); and Geobacter , a genus encompassing species capable of reducing insoluble Fe and Mn oxides in soils and sediments ( 30 ). Higher representation of these OTUs at the inlet site corresponds well with the higher availability of sulfate, nitrate, Fe(III), and Mn(IV) at the inlet than at transitional or interior sites and likely contributes to lower methanogenesis and higher carbon mineralization via alternative terminal electron-accepting pathways. The coexistence of all these OTUs also suggests that a variety of electron acceptors are being used at this site, possibly within spatial and temporal microniches where electron acceptor availability is not accurately represented by bulk biogeochemistry. Community differences between bulk peat and rhizomes. A number of alphaproteobacterial OTUs had higher relative abundance in rhizome samples (see Table S1B in the supplemental material), particularly in the order Rhizobiales , which includes nitrogen-fixing symbionts of plant roots and methanotrophs in the family of Methylocystaceae , such as Methylosinus (see Fig. S4c ), a type II aerobic methanotroph ( 31 , 32 ), which may find a niche at the rhizome surface, where low levels of oxygen are present. A number of methanogenic OTUs classified as Methanobacterium , Methanosarcina , Methanosaeta , and the family Methanospirillaceae were also more abundant in rhizomes, despite the overall lower number of methanogens (see Table S1B and Fig. S4c ). Similarly, Cadillo-Quiroz and colleagues found higher relative abundances of Methanosarcina and Methanosaeta , the only two genera known to include members capable of using acetate for methanogenesis, in rhizosphere than in bulk peats ( 33 ), a pattern likely linked to root exudation of acetate ( 21 ). Though methanogens are often regarded as strict anaerobes, oxygen is present near the rhizome ( 19 ), and so methanogens found on rhizomes must be able to tolerate oxygen. Indeed, Kiener and Leisinger ( 34 ) determined that some members of Methanosarcina and Methanobacterium are able to survive after an exposure to oxygen. In addition, Methanosarcina was found to be able to produce CH 4 even under oxic conditions, and its catalase genes were actively transcribed for oxygen detoxification ( 35 ). OTUs more abundant in bulk peats include those classified as Methanoregula , Crenarchaeota C2 group, Bacteroidales , and Thermodesulfovibrionaceae (see Fig. S4d in the supplemental material). Previously, Methanoregula was also found to be more abundant in bulk peats than in rhizosphere and was the dominant methanogen in bulk peats ( 33 ). Members of Methanoregula are hydrogenotrophic methanogens ( 24 , 25 ) and sensitive to even trace amounts of oxygen ( 25 ) and therefore are more likely to occur in bulk peats than under the microaerobic conditions surrounding rhizomes and roots. The Crenarchaeota C2 group, also known as rice cluster IV ( 36 ), is a deeply branched lineage initially identified from lake and marsh sediments ( 37 ) and has since been found in many anoxic environments, including sediment, soil, rice paddies, and anaerobic digesters ( 38 ). No cultured representative is available for this group, and their physiology is yet to be investigated. Interspecies interactions. Interspecies interactions have the potential to affect wetland belowground processes through cometabolic or syntrophic interactions. We explored potential interspecific interactions using correlation and network analyses (see Table S2A and B and Fig. S5 in the supplemental material). Methanogen OTUs were positively correlated with OTUs belonging to Planctomycetes and Firmicutes (particularly Clostridia ), presumably due to methanogens’ consumption of the carbohydrate fermentation products (e.g., acetate and hydrogen) generated by these populations. A number of methylotrophic alphaproteobacterial OTUs and a few Syntrophaceae OTUs in the Deltaproteobacteria cooccurred with methanogen OTUs. Methanogens were negatively correlated with many OTUs in Proteobacteria and Nitrospirae (especially Thermodesulfovibrionaceae ), which contain anaerobic respiring populations. In total, there are 1,019 positive and 286 negative pairs of significant correlations among the analyzed OTUs. Interestingly, about one-third of the positive correlations were between OTUs in the same phylum, whereas only 3% of negative correlations occurred within the same phylum. For example, 26 pairs of methanogen OTUs were positively correlated, yet no negative correlations were found among methanogens. This indicates that species with the same functional guild tend to cooccur and may suggest that habitat filtering is a strong factor shaping microbial communities in this carbon- and nutrient-rich but electron-acceptor-poor environment. Functional gene abundance profiles by metagenomics. Metagenome sequencing was conducted on 11 selected samples collected from the surface 0- to 12-cm peats in February, including different combinations of sample site and type, and two additional samples from a replicate core at site B, because a large variation of community composition was observed between the duplicate cores at this site. At least 52 Gb of sequence data was generated from each site. However, fewer than 10% of reads assembled into contigs of >200 bases in length for most samples (see Text S1A in the supplemental material), which reflects the high complexity of wetland peat microbial communities. Indeed, under comparable sequencing efforts, the percentage of reads assembled was inversely correlated to the Shannon diversity index ( H ) of microbial communities estimated by 16S rRNA gene analysis (see Text S1A ). Therefore, to best analyze the data, the assembled contigs and unassembled reads were both used for gene prediction and functional annotation, with the abundance of each contig adjusted by its read depth (fold coverage) for quantitative analyses. Genes and pathways involved in microbial processes in wetland peats, such as lignocellulose degradation, fermentation, anaerobic respiration, and production and oxidation of CH 4 , were present in each metagenome as expected. We used gene-centric analysis to reveal major differences in community functional profiles. The equal representation of housekeeping clusters of orthologous groups (COGs) among metagenomes indicates that bias associated with the average genome size in different samples was minimal and thus verifies the comparability of these metagenomes (see Text S1B in the supplemental material). Gene families involved in CH 4 metabolism, denitrification, dissimilatory sulfate reduction, dissimilatory metal reduction, nitrogen fixation, and hydrogen production and consumption were among the differentially represented gene families among sample sites and types. These are important functions generally found in wetland peats and are relevant to the biogeochemistry measurements that we collected. Therefore, we focused on gene families in these pathways, and a heat map was generated for these gene families to show their distribution patterns ( Fig. 4 ). FIG 4 Heat map of gene families in important pathways generated with R and its “gplots” package, using the odds ratios between an individual metagenome and the combined average. Note that there are no COGs and Pfams specific for nitrite reductase genes nirK and nirS . As pfam02239 and pfam00034 are domains constituting nirS and COG2132, pfam00394, and pfam07732 are domains constituting nirK , we included them in this heat map, but note that they can also be components of some other redox genes. Metagenomes from the interior site had the highest relative abundance of genes in methanogenesis pathways, consistent with higher CH 4 emission and higher abundance of methanogen OTUs at the interior site than at the inlet site. Metagenomes from the inlet site, on the other hand, had the highest representation of genes involved in dissimilatory sulfate reduction, denitrification, and metal reduction. Unlike the [NiFe] hydrogenase genes (pfam00374), the iron-only hydrogenase genes (pfam02256 and pfam02906) increased from the inlet to the interior site, suggesting that the environment became more fermentative and that more hydrogen was likely produced from fermentation through the iron-only hydrogenases in the interior site. As observed in the community composition data, the replicate cores from the transitional site (B1 and B2) showed large differences in functional gene abundance profiles ( Fig. 4 ). Nevertheless, the abundance patterns of genes in these anaerobic respiration pathways are consistent with the chemical gradients and the differentially represented OTUs among these sites. Such functional gene distributions have not, to our knowledge, been previously demonstrated in wetland environments. Nitrogenase genes required for nitrogen fixation were more abundant in rhizomes than in bulk peats, consistent with the overrepresentation of nitrogen-fixing Rhizobiales OTUs in rhizome samples from the 16S rRNA gene data. Similar to the nitrogenase gene pattern, genes involved in aerobic oxidation of CH 4 were more abundant in rhizomes than in bulk peats, reflecting the availability of oxygen near rhizomes and consistent with the overrepresentation of aerobic methanotrophs in rhizome samples from 16S rRNA gene analysis. The pattern of genes in CH 4 oxidation supports the idea that the oxygen leakage from roots and rhizomes creates a niche for aerobic methanotrophs to oxidize CH 4 to CO 2 , thereby mitigating CH 4 emissions ( 39 , 40 ). Because diffusion and gas-bubble ebullition through the water column typically contribute a minor proportion to CH 4 emission from wetlands compared to plant aerenchyma transport ( 10 , 41 ), methanotrophs associated with plant roots and rhizomes can be a mitigating barrier that significantly reduces wetland CH 4 emission. Diversity and distribution of methanogens. The genes encoding methyl coenzyme M reductase (MCR), which catalyzes the terminal step in methanogenesis, are functional markers of methanogenesis, and the gene for its subunit A, mcrA , is often used as a methanogen phylogenetic marker. We performed phylogenetic analysis using metagenome mcrA sequences from the assembled part, presumably derived from abundant methanogen populations in this wetland ( Fig. 5a ). All analyzed metagenome mcrA sequences were affiliated with Methanomicrobiales , Methanobacteriales , and Methanosarcinales , spanning three out of the six major orders of known methanogens. The Methanoregulaceae family within Methanomicrobiales harbors more than half of metagenome mcrA sequences, some of which have high coverage (e.g., >50×). Particularly, an mcrA gene closely affiliated with Methanoregula boonei 6A8 has a very high coverage (i.e., 186×), which allowed the recovery of a near-complete genome of the methanogen containing it (data not shown). This genome has only the hydrogenotrophic methanogenesis pathway and lacks the methylotrophic pathway and the hdrDE genes needed for growing on acetate, confirming it as a hydrogenotrophic specialist. As members of Methanomicrobiales and Methanobacteriales are mostly hydrogenotrophic ( 23 – 25 ), their abundant presence suggests the importance of hydrogenotrophic methanogenesis in this wetland. FIG 5 Diversity of mcrA genes and abundance of methanogens. (a) Phylogenetic tree of mcrA , including all sequences recovered from metagenome assemblies with lengths of >150 amino acids. Wetland sequences are labeled with colors indicative of different sample sites, and their fold coverage in metagenomes is indicated in the parentheses. The accession numbers of reference sequences are in brackets. (b) Relative abundance of methanogens in the total community estimated by mcr genes. Among Methanosarcinales , the acetoclastic specialist genus, Methanosaeta , mainly contains sequences from site A, and the metabolically versatile genus, Methanosarcina , exclusively contains sequences from cattail rhizomes on sites B and L. The Methanobacteriaceae branch within the Methanobacteriales is represented only by rhizome samples from site L. These site- and type-associated methanogen distribution patterns remain even when we included additional shorter sequences from wetland metagenomes (data not shown). The type-associated patterns likely reflect acetate and oxygen availabilities as discussed in the above 16S rRNA gene analysis session. We estimated the relative abundance of methanogens and several other functional guilds in the community by comparing their specific functional marker gene abundance to general single-copy housekeeping genes, using their length-normalized abundances (see Text S1C in the supplemental material for details on the normalization method). We expect this method to be much less biased than traditional PCR-based quantification, which is subject to PCR bias and depends on the design of functional gene and housekeeping gene primers inclusive enough for different taxa. To validate this method, we compared methanogen estimates by relative abundance of mcr genes against the estimates by 16S rRNA gene amplicon sequencing and by the classification of 16S rRNA gene reads in the metagenomes ( 42 ) (see Text S1D for details). We demonstrate that the estimate by metagenome mcr was very comparable to the estimate by metagenome 16S rRNA genes and was more quantitatively accurate than 16S rRNA gene amplicon sequencing. The estimated methanogens ranged from 2% to 10% of the total community and were highest at the interior and lowest at the inlet ( Fig. 5b ). Diversity and distribution of methanotrophs. Methanotrophs use methane as a sole carbon and energy source and can be classified as type I (belonging to Gammaproteobacteria ) and type II (belonging to Alphaproteobacteria ) methanotrophs ( 43 ). For example, these types differ in carbon assimilation pathways, cell morphology, and ultrastructure. In addition, a verrucomicrobial methanotroph ( 44 ) and a nitrite-dependent anaerobic methane oxidizer, “ Candidatus Methylomirabilis oxyfera” in candidate division NC10 ( 45 ), were also reported. The first step in methane oxidation is catalyzed by methane monooxygenase (MMO), which has two forms: a particulate membrane-bound form (pMMO) and a soluble cytoplasmic form (sMMO). Nearly all methanotrophs (with some exceptions such as Methylocella ) possess pMMO, whereas sMMO is present in only a few methanotroph genera ( 32 , 46 ). The alpha subunits of pMMO ( pmoA ) and sMMO ( mmoX ) genes are often used to study methanotroph phylogeny ( 32 ). To reveal the diversity of methanotrophs, we first constructed a pmoA phylogenetic tree ( Fig. 6a ). The majority of wetland pmoA genes were affiliated with Methylocystis and Methylosinus within Methylocystaceae (type II), and some were affiliated with Methylococcaceae (type I). No sequence was affiliated with Verrucomicrobia or “ Ca . Methylomirabilis oxyfera” (tree not shown), and this was confirmed by blasting Verrucomicrobia and “ Ca . Methylomirabilis oxyfera” pmoA genes against our metagenomes. Notably, a branch of pmoA consists only of metagenome rhizome sequences, and these sequences are less than 65% identical to any sequence in the nr database. It is not clear whether they are true pmoA genes or pmoA -homologous amoA (ammonia monooxygenase) genes, as they match to Nitrosococcus and methanotrophs almost equally. However, their adjacent pmoB components are more similar to methanotrophs than to Nitrosococcus , suggesting that these novel sequences are likely from an unidentified methanotroph group. FIG 6 Diversity of pmoA genes and abundance of methanotrophs. (a) Phylogenetic tree of pmoA , including sequences recovered from metagenome assemblies with lengths of >80 amino acids. Wetland sequences were labeled with colors indicative of different sample types, and their fold coverage in metagenomes was indicated in the parentheses. The accession numbers of reference sequences are in brackets. (b) Relative abundance of methanotrophs in the total community estimated by pmoA genes. It was previously reported that all pMMO operons were organized as pmoCAB , whereas some gammaproteobacterial methanotrophs possess a novel pmoA gene in a pmoABC operon, in addition to the traditional pmoA gene in the pmoCAB operon ( 47 ). This pmoA was proposed to have an unknown function different from the traditional pmoA genes and was referred to as “ pxmA .” A number of our wetland sequences were within the newly identified Methylococcaceae \n pxmA cluster, and they also have a pmoABC operon structure (if the operon is recovered), confirming their affiliation with pxmA . In addition, by searching reference methanotroph genomes, we also identified Methylocystis strain SB2 and Methylocystis rosea SV97T possessing such operon structures, and together with Methylococcaceae , they form a pxmA cluster, different from the traditional pmoA in either type I or II methanotrophs. A number of our wetland sequences were affiliated with these two Methylocystis \n pxmA sequences, and this finding suggests that this novel pxmA is present not only in type I as previously thought ( 47 ) but also in type II methanotrophs, although its function is still unclear. The distribution of pmoA exhibited some patterns associated with sample types ( Fig. 6a ). Clearly, the number of pmoA sequences recovered from bulk peats is much lower than that from rhizomes. In addition, pmoA sequences affiliated with Methylosinus are mainly from tule rhizomes, whereas the Methylocystaceae \n pmoA2 (an isozyme of pmoA in some Methylocystis genomes [ 48 ]) was found only in cattail rhizomes, suggesting some differential recruitments by the two plants. As most methanotrophs possess pMMO ( 32 ), the abundance of methanotrophs was estimated by pmoA genes (see method details in Text S1E in the supplemental material) and ranged from 0.7 to 3.4% of the total community ( Fig. 6b ). In general, methanotrophs were present at higher abundances in the rhizomes than in the bulk peats within the same site. Overall, there is no correlation between methanotroph abundance and methanogen or methane production, suggesting that oxygen availability in the microenvironment is probably the limiting factor controlling methanotroph populations in this wetland. As we consider the bulk sediment to be anoxic, the presence of aerobic methanogens, although at low levels, may be due to the many fine roots in the sediment, allowing oxygen to penetrate peats surrounding these roots, therefore increasing the microheterogeneity of the sediment. We also constructed an mmoX phylogenetic tree (see Fig. S6 in the supplemental material). As expected, fewer mmoX sequences were recovered from the wetland, and in particular, no mmoX sequences were obtained from bulk peats. About two-thirds of the wetland mmoX sequences are in the Methylocystaceae and Methylococcaceae families, and about one-third belong to Beijerinckiaceae , including members such as Methylocella and Methyloferula , which lack the pmoA genes in their genomes. Therefore, the above methanotroph abundance estimation by pmoA overlooked the contribution of Beijerinckiaceae methanotrophs, although they are minor based on the number and sequence coverage of their mmoX genes. Abundance of anaerobic respiring populations. We estimated the abundances of sulfate reducers using the dissimilatory sulfite reductase genes ( dsrAB ) and of denitrifiers using nitrous oxide reductase genes ( nosZ ), and they ranged from 7% to 17% and from 3% to 8% of the total community, respectively ( Fig. 7a and b ; see also Text S1E in the supplemental material for method details). We used the mtrB/pioB gene family to estimate metal reducers that possess mtrB , encoding an outer membrane protein associated with multiheme c -type cytochromes involved in extracellular electron transfer in dissimilatory metal reduction ( 49 , 50 ), acknowledging that this might have led to overestimation of mtrB -containing metal reducers, because this gene family is also found in a few iron-oxidizing bacteria ( 51 ). In addition, metal reducers using other mechanisms were not evaluated in our study, partly because their metal reduction genetic machinery is less well understood. Overall, the inlet site had the highest abundances of denitrifiers, sulfate reducers, and metal reducers, and the interior site had the lowest abundances of these guilds (see Fig. S7 ). Correlations were observed between nitrate concentration and denitrifier relative abundance ( r = 0.78), between the reduced Fe and Mn and metal reducers ( r = −0.93), and between sulfate and sulfate reducers ( r = 0.96) (see Fig. S7 ), indicating a strong relationship between site chemistry and corresponding microbial populations. Interestingly, populations of anaerobic respiring populations were positively correlated with each other (average r = 0.67), suggesting that these guilds may not be in direct competition. FIG 7 Relative abundances of sulfate-reducing prokaryotes (SRP) estimated by dissimilatory sulfite reductase genes ( dsrAB ) (a), denitrifiers by nitrous oxide reductase genes ( nosZ ) (b), and metal reducers by the multiheme c -type cytochrome-associated mtrB/pioB gene family (c) show negative correlations with methanogen abundance estimated by methyl coenzyme M reductase genes ( mcr ). In contrast, methanogen abundance was negatively correlated with the abundances of sulfate reducers ( r = −0.67 [ Fig. 7a] ), denitrifiers ( r = −0.64 [ Fig. 7b] ), and metal reducers ( r = −0.78 [ Fig. 7c] ), respectively, suggesting that these anaerobic respiration processes may serve as electron sinks diverting electrons from methanogenesis in this wetland. These respiration processes are thermodynamically more favorable than methanogenesis and are expected to outcompete methanogenesis ( 52 ). Furthermore, sulfate reducers have higher affinities for H 2 and acetate than do methanogens ( 53 , 54 ), and iron reducers also have a higher affinity for acetate and compete effectively with methanogens in wetland surface sediments ( 55 ). In addition, denitrification intermediates such as nitrite and nitric oxide may be toxic to methanogens ( 56 , 57 ). Therefore, these electron acceptors, if present at sufficient concentrations, can both inhibit methanogenesis and suppress methanogen abundance, thus mitigating the global warming potential from wetlands. Although wetland restoration recovers valuable ecosystem services, restoration projects need to be carefully designed and managed to minimize GHG emissions. Our results indicate that methanogens were present throughout this wetland, ranging from 2% to 10% of the total community, and redox conditions favoring peat accretion also encouraged methanogens and methane emission. Therefore, carbon sequestration and storage as peat need to be carefully balanced with GHG emission throughout the management of the restoration. In general, net GHG emission may depend on the interplay of a number of factors, including climate, soil type, plant species, hydrology, and water chemistry. From the perspective of the microbial contribution to GHG flux, increasing the water inflow rate to increase electron acceptor influxes or draining the wetland intermittently to reoxidize reduced electron acceptors may be promising in mitigating CH 4 emissions. In addition, as methanogenesis was more significant at the backwater sites, which experienced less water flow and less influx of electron acceptors, changing the wetland hydraulic design to minimize backwater areas might be effective to reduce CH 4 emission but must be evaluated against benefits of plant productivity and habitat quality. In summary, the differences in microbial community composition and functional profiles reveal complex interactions among functional guilds and among wetland plants, microorganisms, and their environment. Analyses of 16S rRNA and functional genes showed distribution patterns associated with biogeochemistry and indicated microspatial heterogeneity of wetland sediments. Phylogeny of marker genes revealed a diversity of major methane-producing and -consuming populations and discovered novel diversity within methanotrophs. Quantitative comparative analyses of shotgun sequence data reveal the competition with and inhibition of methanogens by anaerobic respiring microorganisms without biases introduced by cultivation or PCR amplification and provided molecular evidence explaining the spatial variations in biogeochemistry and methane production. This information is useful in planning and operating wetland restoration projects in order to reduce CH 4 emission to the atmosphere and maintain the carbon storage potential of restored wetlands. Based on these findings, more specific studies can be carried out to evaluate the impact of wetland water chemistry and hydrology on CH 4 emission and carbon sequestration before large-scale restoration projects are implemented."
} | 10,236 |
37463961 | PMC10353996 | pmc | 6,380 | {
"abstract": "With climate projections questioning the future survival of stony corals and their dominance as tropical reef builders, it is critical to understand the adaptive capacity of corals to ongoing climate change. Biological mediation of the carbonate chemistry of the coral calcifying fluid is a fundamental component for assessing the response of corals to global threats. The Tara Pacific expedition (2016–2018) provided an opportunity to investigate calcification patterns in extant corals throughout the Pacific Ocean. Cores from colonies of the massive Porites and Diploastrea genera were collected from different environments to assess calcification parameters of long-lived reef-building corals. At the basin scale of the Pacific Ocean, we show that both genera systematically up-regulate their calcifying fluid pH and dissolved inorganic carbon to achieve efficient skeletal precipitation. However, while Porites corals increase the aragonite saturation state of the calcifying fluid (Ω cf ) at higher temperatures to enhance their calcification capacity, Diploastrea show a steady homeostatic Ω cf across the Pacific temperature gradient. Thus, the extent to which Diploastrea responds to ocean warming and/or acidification is unclear, and it deserves further attention whether this is beneficial or detrimental to future survival of this coral genus.",
"introduction": "Introduction Ocean warming and acidification threaten the health and survival of tropical coral reefs 1 – 3 . Projections based on possible future climate scenarios range from a significant decline to the complete disappearance of coral reefs by 2100 (IPCC Special Report, 2018—“Global Warming of 1.5 °C; IPCC Special Report, 2019—Ocean and Cryosphere in a Changing Climate”). For more than a century, increasing emissions of anthropogenic CO 2 4 and other greenhouse gases have caused the temperature of the shallow ocean to rise by 0.3–0.6 °C and the pH to fall by ~ 0.1 units (i.e., ocean acidification, OA) 5 . At the same time, the carbonate ion concentration (CO 3 2− ) and the aragonite saturation state (Ω) in the surface ocean have decreased by ~ 16% 6 , 7 . Depending on the specific CO 2 emission scenario 8 , models predict a rise in temperature of several degrees and a further decline in seawater pH (pH sw ) of 0.14–0.43 by 2100 6 , 9 . All this could have severe implications for the formation of aragonite in stony corals, including a decline in the calcification rate and skeletal density 9 – 12 . Several studies have shown that scleractinian (aragonite) corals have an adaptive capacity to maintain calcification under unfavorable environmental conditions 13 – 15 . They precipitate their calcium carbonate in a biologically controlled manner within a semi-isolated space, known as the extracellular calcifying fluid (cf), located between the skeleton and the calicoblastic epithelium 16 . Corals have developed biological mechanisms to actively concentrate dissolved inorganic carbon (DIC) into the cf and remove protons (i.e., increase the pH cf relative to the ambient seawater). This shifts the DIC equilibrium in favor of [CO 3 2− ], thus enabling the coral to achieve higher Ω cf values. In particular, by upregulating their cf carbonate chemistry, corals achieve aragonite saturation state levels 4 to 6 times higher than that of seawater 15 , 17 , 18 , which promotes the precipitation of CaCO 3 . Moreover, recent intra-colony studies of the genus Porites suggested that cf carbonate chemistry varies seasonally, with such variations being regulated by a combination of environmental drivers (e.g., light, temperature, nutrients) and metabolic processes (e.g., metabolic carbon from symbiotic photosynthesis) 19 – 23 . We investigated the carbonate chemistry of the calcifying fluid of two massive and long-lived coral genera ( Porites and Diploastrea ) to identify differences and similarities between taxa under identical climatic and hydrological conditions. These coral genera, prevalent reef builders of the Pacific Ocean 24 , have been targeted because of their wide latitudinal distribution, longevity (on the order of centuries), and great potential as palaeoceanographic archives. While Porites is known to be among the most resilient corals 25 – 27 , less is known about the genus Diploastrea regarding its stress tolerance. In this study, we compared the calcification and carbonate chemistry up-regulation of Diploastrea heliopora and Porites corals from across a range of environments. To this, we analyzed the skeletal geochemistry and growth parameters of 39 colonies of Porites (n = 33) and Diploastrea (n = 6) collected across the tropical Pacific Ocean during the Tara Pacific expedition (2016–2018). The collected corals represent a suite of cores exposed to various hydrological conditions of seawater temperature (SST: 22.4–29.8 °C), salinity (SSS: 31.5–36.1), and carbonate chemistry (total scale pH sw : 8.01–8.09) (Fig. 1 , Table S1 , S2 ). The average chemical composition of the calcifying fluid (pH cf , [CO 3 2− ] cf , DIC cf , Ω cf ) was derived from paired boron isotope (δ 11 B) and B/Ca analyses of core-top samples corresponding to the last 6 years of growth (2010–2016; Methods). Based on these data, we assessed the impact of the ambient seawater properties (SST, salinity, carbonate chemistry) on the cf composition of these slow-growing reef-building genera at the Pacific basin scale. Figure 1 Map of the Pacific Ocean showing the sampling locations of the 39 coral colonies of Porites (n = 33) and Diploastrea (n = 6), cored during the Tara Pacific expedition (2016–2018) used to investigate the chemical properties of the calcification fluid. The 27 white dots correspond to sites where only Porites were collected and the 6 black dots correspond to sites where both Porites and Diploastrea were collected. Numbering, geographical locations, and coral cores are detailed in Table S1 . ( A ) Mean sea surface temperature (SST) along the Pacific Ocean for the period 2010–2016. Global annual SSTs were extracted from the MODIS-Aqua satellite and global mapped climatologies established for the period from 2002 to 2018 (NASA Goddard Space Flight Center) ( B ) Mean seawater pH for the period 2010–2016. We used monthly global reconstructed surface ocean p CO 2 , air-sea fluxes of CO 2 and pH to calculate seawater pH and associated uncertainties on a 1° × 1° regular grid 28 . These maps were obtained from an ensemble-based forward feed neural network approach mapping in situ data for surface ocean fugacity (SOCAT data base 29 , https://www.socat.info/ ) and sea surface salinity, temperature, sea surface height, chlorophyll a, mixed layer depth, and atmospheric CO 2 mole fraction.",
"discussion": "Results and discussion Coral samples were collected from 33 sites in the Pacific Ocean characterized by different environmental conditions. The mean SST values (integrated over the period 2010–2016) varied between 22.44 °C in Easter Island and 29.76 °C in Papua New Guinea (> 7 °C difference). Mean pH exhibited a relatively small difference between 8.01 in Kiribati and 8.09 in Heron Island (ΔpH = 0.08). Thus, the calculated seawater saturation states (Ω SW ) varied from 3.21 in Coiba to 3.95 in Moorea (“integrated seawater properties” in Table S2 , Fig. S1 ). Boron-derived values of the cf carbonate chemistry revealed significant differences in [CO 3 2− ] cf and Ω cf ( P < 0.05) between Porites and Diploastrea, with the latter showing lower values (Table S1 ). Cores of the two genera also showed significantly different linear extension and calcification rates ( P < 0.05). The comparison between environmental data, growth parameters, and boron-derived cf estimates for Porites (Figs. 2 , S2 ) indicates that average pH cf was not controlled by spatial differences in seawater pH or aragonite saturation state ( P > 0.05). Instead, our data suggest that spatially average pH cf is linked to SST (R = − 0.63, P < 0.001) and DIC sw (R = 0.41, P = 0.017). While DIC sw showed a significant correlation with salinity (R = 0.98, P < 0.001), pH cf was also related to salinity but to a lesser degree (R = 0.35, P = 0.046). Similarly, DIC cf was related to SST (R = 0.71, P < 0.001). Thus, on spatial scales a strong negative correlation exists between pH cf and DIC cf (R = − 0.81, P < 0.001), consistent with other studies at a seasonal scale 20 , 30 , 31 . Our results suggest that seawater temperature explains most of the variance in pH cf and DIC cf in Porites colonies at a basin-scale (Fig. 2 ). Similarly, overall observations apply to Diploastrea samples, since B/Ca, δ 11 B, DIC cf , and pH cf were significantly correlated with seawater temperature (Fig. 3 A–D). However, this contrasts with other studies that have shown that seawater pH is the main driver of pH cf on annual and longer time scales, while temperature only plays a secondary role 32 , 33 . This suggests that the magnitude of SST variations (seasonal vs. annual and temporal vs. spatial) is what effectively controls the relationship between temperature and cf carbonate chemistry. At large, as expected and previously observed in various Indo-Pacific regions 20 , 30 – 34 , Porites calcification was positively correlated with SST (R = 0.37, P = 0.034) and displayed a positive correlation with DIC cf (R = 0.35, P = 0.044). Figure 2 Chord diagram showing the relevant relationships between seawater, calcifying fluid (cf) chemistry, and coral growth parameters for Porites across the Pacific Ocean. Each variable is displayed as a node, with the size of the arc corresponding to the strength of the correlation (correlation coefficients are also reported close to each node and in Fig. S2 ). Links between two nodes displaying a correlation coefficient < 0.5 (for positive correlation) and <|− 0.5| (for negative correlation) are not shown to keep the graph readable and not overwhelming. SST, Sea Surface Temperature; SSS, Sea Surface Salinity; pH sw , seawater pH (total scale); [CO 3 2− ] sw , seawater carbonate ion concentration; DIC sw , seawater dissolved inorganic carbon; Ω sw , aragonite saturation state in seawater; pH cf , calcifying fluid pH (total scale); [CO 3 2− ] cf , calcifying fluid carbonate ion concentration; DIC cf , calcifying fluid dissolved inorganic carbon; Ω cf , aragonite saturation state in the calcifying fluid. Figure 3 Coral skeletal isotopic composition. ( A ) B/Ca and ( B ) δ 11 B values of Porites (n = 33, blue) and Diploastrea (n = 6, red) corals across the Pacific Ocean plotted against SST. ( C – F ) Carbonate chemistry variables of calcifying fluid calculated for each colony studied here (DIC cf , pH cf , [CO 3 2− ] cf , Ω cf , respectively) plotted against SST. The filled blue and red dots represent the 6 sites where both Porites and Diploastrea were sampled. Accordingly, the solid blue and red lines correspond to the regression lines for these 6 sites, while the dashed blue lines correspond to the regression lines for all sites where Porites were sampled (n = 33). SST values were obtained from the AVHRR-OISSTv2 (0.25 × 0.25°) dataset and correspond to mean values calculated for the period 2010–2016. X and Y errors correspond to 2σ standard deviations of mean SST and 2σ standard errors of measurements or calculations, respectively. Statistical parameters are reported in Table S4 . In agreement with recent studies focused on Porites at a seasonal scale (Fig. S3 ; 20 , 22 ), our bulk 6 yr-integrated results show a strong negative relationship between pH cf and SST as well as a positive correlation between DIC cf and SST for both coral genera (Fig. 3 ). These opposing relationships suggest that corals up-regulate their internal pH in response to temperature-related changes in metabolic DIC, as already posited in previous studies (e.g., by means of higher metabolic DIC availability from algal symbiont photosynthesis at warmer temperatures and/or light) 23 , 32 . In this study, for Porites we determined a DIC cf increase of 128 ± 23 μmol kg −1 per °C, while pH cf decreased by 0.015 ± 0.004 per °C (Fig. 3 , Table S4 ), resulting in an increase in [CO 3 2 ] cf of 29 ± 5 μmol kg −1 per °C and higher Ω cf values (~ 21 vs. ~ 16, Fig. 3 ). The decrease in pH cf with temperature observed at a spatial scale is around three times lower than previous estimates observed at a seasonal scale 32 , and therefore steadier (homeostatic). Besides the notion that temperature influences pH cf up-regulation, our study demonstrates the pH cf up-regulation capacity of Porites across stable and warm regions as well as in regions with a large seasonal temperature amplitude and low mean annual temperatures (or mean annual light) (i.e., sub-equatorial vs. equatorial regions). Thus, pH cf up-regulation overcomes the decrease in DIC cf due to colder SSTs (Fig. 3 ) in sub-equatorial areas to enable coral calcification. Seasonally-resolved Porites records of δ 11 B and B/Ca have shown that DIC cf is lower during winter months (i.e., colder temperatures) due to lower metabolic supply of DIC within the calcifying fluid 20 . The supply of this metabolically derived carbon is driven by light and temperature through the respiration of algal symbiont photosynthates 35 , as colder temperatures reduce zooxanthellae activity and reduce the concentration of metabolic DIC in the calcifying fluid. However, higher nutrient availability at higher latitudes may contribute to partially offsetting the detrimental effects caused by the lower metabolic supply of DIC cf . The negative correlation between pH cf and DIC cf at a spatial scale in our study is consistent with intracolonial seasonal variations reported in previous studies 20 , 33 . The up-regulation of pH cf is one way for corals to compensate for the reduced metabolic carbon input from the algal symbiont and to maintain supersaturated conditions in a biologically controlled compartment with respect to aragonite (Ω cf ~ 5 × Ω SW ) 20 . This explains why Porites corals living in equatorial and sub-equatorial regions display similar Ω cf values, despite their different internal pH cf , driven by temperature-dependent DIC cf regulation. Since the photosynthetic activity of the coral associated algal symbiont is presumably reduced at higher latitudes (~ 27° N/S in this study) due to lower light availability compared to equatorial latitudes 36 , corals may use their energy to regulate their cf chemistry, in particular their pH cf , to maintain active growth and skeletal accretion. The results of our study, based on a multi-year sampling strategy of coral core-tops across the Pacific Ocean, are consistent with the calcification model proposed by Ross et al. 30 , 31 (Fig. S4 ), based on a seasonal timescale. The primary mechanism for the up-regulation of pH cf involves the Ca 2+ -ATPase pump, which exchanges one calcium ion for two protons across the cell membrane 37 – 40 . The removal of H + from the cf increases the diffusion of metabolic CO 2 38 , which is either protonated to bicarbonate (HCO 3 − ) by carbonic anhydrase (CA) and/or transported in the form of HCO 3 − by bicarbonate anion transporters (BATs, i.e., through active transport) 41 . Up-regulation of pH cf shifts the DIC equilibrium in favor of CO 3 2− , thereby increasing the internal aragonite saturation state to promote skeletal formation 20 , 38 , 42 , 43 . Our study provides evidence that to maintain growth Porites corals up-regulate their pH cf and increase their DIC cf concentration in response to changes in SST across the Pacific Ocean. This physiological mechanism has already been observed for Porites on a seasonal timescale in the Great Barrier Reef 20 (Fig. S3 ) and Galapagos 22 , as well as during the 1998 bleaching event and associated thermal stress 19 . Here, for the first time we demonstrate that this mechanism applies across a wide range of latitudes and longitudes. The ability of corals to modulate their calcifying fluid chemistry explains their sustained calcification rates, which are primarily driven by temperature and DIC cf . Porites corals in warmer environments display lower pH cf but higher DIC cf and [CO 3 2− ] cf (Fig. 3 ), leading to significantly higher Ω cf compared to the surrounding seawater (Fig. 3 ) and increasing calcification rates. In contrast to Porites , branching corals 31 , 32 exhibit higher calcification rates at lower temperatures and higher pH cf and [CO 3 2− ] cf . It is now recognized that the internal modulation of coral calcifying fluid is genus-specific (if not species-specific) 21 , 31 , 44 . Our study demonstrates that Porites colonies living across a wide range of environments across the Pacific Ocean can modulate their cf chemistry in response to prevalent regional temperature regimes to maintain calcification rates, as previously suggested 20 , 22 . Conversely to Porites , the capacity of the long-lived massive coral Diploastrea to regulate its internal pH cf had not been studied yet. While Diploastrea and Porites showed similar decreases in B/Ca ratios with increasing temperature, Diploastrea consistently exhibited lower δ 11 B values (and therefore pH cf ) at the same temperature (Fig. 3 ), indicating taxa-specific differences with regard to internal pH cf regulation (Fig. 4 ). In both coral genera, pH cf and DIC cf were positively correlated with the Pacific Ocean temperature. However, at higher temperatures, Diploastrea showed a reduced pH cf up-regulation due to a pH cf decrease of − 0.036 ± 0.006 per °C (n = 6), resulting in lower [CO 3 2− ] cf and Ω cf values (Table S1 ). This newly discovered finding suggests different mechanisms of calcification control in Porites and Diploastrea . Our interpretation is that either the Diploastrea Ca 2+ -ATPase pump is less effective than that of Porites in removing H + from the calcifying cell, or that Diploastrea has a mechanism for conserving energy by maintaining stable levels of [CO 3 2− ] cf and Ω cf (~ 16–18), particularly in regions of higher temperatures (Fig. 3 , Table S1 ). Figure 4 Correlations between SST and calcifying fluid composition in co-occurring Porites (n = 6, blue dots) and Diploastrea (n = 6, red dots) specimens across the Pacific Ocean. Solid blue and red lines in the left panels indicate significant correlations ( P < 0.05) for Porites and Diploastrea , respectively. The dashed lines are not significant at the 95% level. ( A ) pH cf , ( C ) DIC cf , ( E ) [CO 3 2− ] cf , and ( G ) Ω cf . Differences between the two genera are reported in the right panels as Δ (i.e. Δ = Porites values – Diploastrea values). Black solid lines indicate significant correlations (R 2 = 0.69–0.82; P < 0.05). ( B ) ΔpH cf , ( D ) ΔDIC cf , ( F ), Δ[CO 3 2− ] cf , and ( H ) ΔΩ cf . SST values are from AVHRR-OISSTv2 (0.25 × 0.25°) and correspond to annual integrated 6-yr values during the period 2010–2016. X and Y errors correspond to 2σ standard deviations of mean SST and 2σ standard errors of measurements or calculations, respectively. Statistical parameters are reported in Table S4 . Response differences of corals to fluctuating temperature (e.g., based on a regional gradient, seasonality, thermal stress) in relation to their calcifying mechanisms have already been documented. It is worth noting that the observed pH cf decrease with SST for Diploastrea (− 0.036 per °C, Table S4 ) is comparable to the mean drop (− 0.03 per °C) recorded for seven symbiotic coral species (4 genera) studied on a seasonal scale in Western Australia over a wide range of latitudes (~ 11°) 31 . However, although the magnitude of change is equivalent, the underlying mechanisms are different. In particular, since DIC cf up-regulation was lower in the Australian corals, the resulting Ω cf values were lower (~ 10–12), and the Ω cf change with temperature varied among species. A notable difference has also been observed between aquaria-reared colonies of Pocillopora damicornis and Stylophora pistillata grown under various temperature and pCO 2 conditions 44 that indicate that only Pocillopora damicornis lose its compensatory ability under thermal stress (31 °C vs. 28 °C) with Ω cf values clearly below 10 for different pH sw conditions. Further, during a local thermal stress and bleaching event 45 , the branching coral Acropora aspera continued to up-regulate pH cf at high temperatures, while DIC cf up-regulation was significantly impaired, which is in contrast to the response of massive corals examined here. A species-specific response of pH cf and DIC cf up-regulation relative to seawater carbonate chemistry variation and ocean acidification has already been described at a seasonal timescale, showing marked differences in calcification control between massive corals such as Porites , Acropora , Psammocora , and Pocillopora 46 , 47 . A summary of the taxon-specific responses of cf carbonate chemistry to temperature for the massive corals here studied as Δ (i.e., Porites values— Diploastrea values) is provided in Fig. 4 B,D,F,H. It is apparent that an increase in temperature leads to a substantial increase in Δ, especially for the key parameters [CO 3 2− ] cf and Ω cf that are directly linked to coral calcification. Thus, despite the elevation of DIC cf at high temperatures, the capacity of Diploastrea to increase Ω cf under warmer conditions is clearly different from Porites . However, based on our data, Diploastrea maintain their capacity to regulate Ω cf and exhibit homeostatic control of the aragonite saturation state independently of geographic location or temperature. This indicates that the pronounced drop of pH cf up-regulation with temperature (− 0.036 ± 0.006 per °C, n = 6) is sufficiently compensated by the buffering capacity and DIC cf increase (129 ± 30 per °C, n = 6). Therefore, the calcification ability of Diploastrea is less sensitive to ocean temperature changes compared to Porites when we consider the key parameters [CO 3 2− ] cf and Ω cf . This may suggest that calcification rate for Diploastrea is potentially less variable in space and time (seasonal amplitudes) compared to Porites . The mechanism observed in Diploastrea appears to resemble the one described by Georgiou et al. (2015) 46 , which demonstrated the capacity to maintain calcification irrespective of environmental differences (i.e., homeostasis), suggesting a similar mechanism in Diploastrea . By stabilizing its chemical composition, even at high temperatures, Diploastrea can achieve optimal calcification. Further studies are required to evaluate such a hypothesis but also better understand the potential impact of the lower Ca 2+ -ATPase pump efficiency and pH cf up-regulation posited for Diploastrea at higher temperatures on calcification, especially in the context of climate change. It should also be noted that Ω cf values (~ 16–18) were calculated assuming Ca 2+ concentrations in the calcifying fluid similar to that of the seawater. However, this assumption requires further investigation for Diploastrea and Porites , as recent studies have shown substantial variations in Ca 2+ concentration of the calcifying fluid 15 . Our study across the Pacific Ocean confirms the ability of the massive reef-building Porites genus to modulate the composition of its calcifying fluid in response to seawater temperature and carbonate chemistry, as observed for other scleractinian corals at different locations or exposed to disparate environmental conditions. For Porites , an upward shift in pH cf and DIC cf (relative to seawater) driven by temperature changes is the presumed mechanism for Porites to compensate for the impact of future thermal stress events or ocean warming on calcification in the Pacific Ocean. Further, our study demonstrates that SST rather than pH sw or Ω sw , is the key parameter controlling Porites calcifying fluid properties, through the activity of the zooxanthellae. Thus, Porites is able to adapt its metabolism to increases in seawater temperature, heralding the adaptive potential of Porites to maintain or reinforce a high aragonite saturation state Ω cf and calcification capacity in the face of climate change. Importantly, however, our results do not rule out that ocean acidification 12 , 33 , 46 – 50 or other environmental factors, including changes in light conditions 23 , may affect coral calcification locally in the near future. Our study also demonstrates biological control of the calcification process is taxon-specific. We show that Diploastrea displays a different strategy than Porites at high temperatures (28–30 °C), in that it maintains consistent calcification rates irrespective of the prevailing environment. Species-specific differences need to be thus considered when forecasting coral future survival 51 . Further investigations of the response of Diploastrea corals at annual and seasonal timescales will reduce uncertainties and better constrain the range of their homeostatic ability to calcify at warming water temperatures. A part of these future research works will be conducted in the new program COR-Resilience (2023–2028) recently funded by the French National Research Agency."
} | 6,365 |
38252290 | PMC10803391 | pmc | 6,381 | {
"abstract": "Abstract The microbial production of polyhydroxyalkanoate (PHA) block copolymers has attracted research interests because they can be expected to exhibit excellent physical properties. Although post-polymerization conjugation and/or extension have been used for PHA block copolymer synthesis, the discovery of the first sequence-regulating PHA synthase, PhaC AR , enabled the direct synthesis of PHA–PHA type block copolymers in microbial cells. PhaC AR spontaneously synthesizes block copolymers from a mixture of substrates. To date, Escherichia coli and Ralstonia eutropha have been used as host strains, and therefore, sequence regulation is not a host-specific phenomenon. The monomer sequence greatly influences the physical properties of the polymer. For example, a random copolymer of 3-hydroxybutyrate and 2-hydroxybutyrate deforms plastically, while a block copolymer of approximately the same composition exhibits elastic deformation. The structure of the PHA block copolymer can be expanded by in vitro evolution of the sequence-regulating PHA synthase. An engineered variant of PhaC AR can synthesize poly( d -lactate) as a block copolymer component, which allows for greater flexibility in the molecular design of block copolymers. Therefore, creating sequence-regulating PHA synthases with a further broadened substrate range will expand the variety of properties of PHA materials. This review summarizes and discusses the sequence-regulating PHA synthase, analytical methods for verifying block sequence, properties of block copolymers, and mechanisms of sequence regulation. Key points • Spontaneous monomer sequence regulation generates block copolymers • Poly(D-lactate) segment can be synthesized using a block copolymerization system • Block copolymers exhibit characteristic properties Graphical abstract",
"introduction": "Introduction Polyhydroxyalkanoates (PHA) are bacterial storage polyester accumulated as intracellular insoluble inclusions (Zhang et al. 2022 ). PHAs are synthesized from various hydroxyacyl-coenzyme A (CoA)s via successive ester exchange reactions catalyzed by PHA synthases. PHA synthases are classified into four major classes based on their subunit structure and the range of substrates. The range of substrate of PHA synthase is a key factor in determining the structure of PHA (Neoh et al. 2022 ). Research interest in PHAs has increased in recent years due to their biobased and biodegradable thermoplastics applications. The most abundant PHAs are poly(3-hydroxybutyrate) [P(3HB)] and its copolymer with 3-hydroxyvalerate (3HV) (PHBV). Although the brittleness of these polymers has long been recognized as a weakness, recent advances in processing technology have allowed them to be used as a hard material for containers and other applications (Pandey et al. 2022 ). However, the characterizations of many commercial PHAs are yet to be published. The random copolymer P[3HB- co -3-hydroxyhexanoate (3HHx)] (PHBH) is a material whose brittleness is improved by reducing its crystallinity (Tang et al. 2022 ). PHBH is used to manufacture products such as bags, straws, utensils, and brushes. 4-hydroxybutyrate (4HB)-based polymers are also attracting significant research interest due to their high flexibility and bioabsorbability (Utsunomia et al. 2020 ). As these studies demonstrate, random copolymerization and composite are the current major strategies to regulate the physical properties of PHAs. Block copolymers have attracted research attention in polymer chemistry for their ability to form microphase separation by self-assembly. Due to immiscibility and chain connectivity, the phase separation between two or more segments occurs in the tens of nanometers range (Kim et al. 2010 ). The structure of the microdomains, such as lamellae, cylinders, and spheres, changes depending on the monomer composition and degree of segregation. Block copolymers exert characteristic properties due to microphase separation (Hillmyer and Tolman 2014 ). Since the usefulness of block copolymers has been well known in the field of synthetic polymers, it makes sense that interest and attempts to synthesize block copolymers were made in the biosynthesis of PHAs. However, the PHA block copolymer biosynthesis was more complex than initially thought. This review discusses attempts to synthesize block copolymers, the discovery of sequence-regulating PHA synthase, analytical methods for verifying block sequence, properties of block copolymers, and mechanisms of sequence regulation."
} | 1,132 |
34863247 | PMC8645107 | pmc | 6,382 | {
"abstract": "Background As methane is 84 times more potent than carbon dioxide in exacerbating the greenhouse effect, there is an increasing interest in the utilization of methanotrophic bacteria that can convert harmful methane into various value-added compounds. A recently isolated methanotroph, Methylomonas sp. DH-1, is a promising biofactory platform because of its relatively fast growth. However, the lack of genetic engineering tools hampers its wide use in the bioindustry. Results Through three different approaches, we constructed a tunable promoter library comprising 33 promoters that can be used for the metabolic engineering of Methylomonas sp. DH-1. The library had an expression level of 0.24–410% when compared with the strength of the lac promoter. For practical application of the promoter library, we fine-tuned the expressions of cadA and cadB genes, required for cadaverine synthesis and export, respectively. The strain with P rpmB - cadA and P DnaA - cadB produced the highest cadaverine titre (18.12 ± 1.06 mg/L) in Methylomonas sp. DH-1, which was up to 2.8-fold higher than that obtained from a non-optimized strain. In addition, cell growth and lysine (a precursor of cadaverine) production assays suggested that gene expression optimization through transcription tuning can afford a balance between the growth and precursor supply. Conclusions The tunable promoter library provides standard and tunable components for gene expression, thereby facilitating the use of methanotrophs, specifically Methylomonas sp. DH-1, as a sustainable cell factory. Graphical Abstract \n \n Supplementary Information The online version contains supplementary material available at 10.1186/s13068-021-02077-8.",
"conclusion": "Conclusions For the first time, a tunable library consisting of 33 promoters of different strengths was constructed for the gene expression optimization of Methylomonas sp. DH-1. The results showed that the fine-tuning of transcription rather than overexpression allows efficient production of recombinant proteins and regulation of metabolic pathways in cells. In future studies, an inducible system based on the tunable promoter library should be designed for more dynamic control of transcription. This system will help make methanotrophs a major sustainable platform for producing value-added products via C1 assimilation. Overall, the promoter library discussed here will facilitate the genetic manipulation of Methylomonas sp. DH-1 for successful implementation of methanotroph biotechnology.",
"discussion": "Results and discussion Construction of the promoter library for Methylomonas sp. DH-1 Among the various parameters that determine the expression level of a gene, transcription is the first step and the main target for gene regulation. As a result, promoters have been widely examined and used to control gene expression [ 20 – 22 ]. To identify potential promoters that can be used for gene expression optimization, we first utilized computational models to predict promoter sequence regions from the genomic sequence of Methylomonas sp. DH-1 [ 23 , 24 ]. The computational prediction could not identify all the promoters of Methylomonas sp. DH-1 because the tools used have not been developed for Methylomonas sp. DH-1. To complement that, we also evaluated the promoters of M. trichosporium OB3b, a model organism of type II methanotroph [ 25 ], because Methylomonas sp. DH-1 also contains genes related to type II methanotroph [ 11 ] and they may share similar consensus promoter sequences. Therefore, the prediction was also performed on the genomic sequence of M. trichosporium OB3b to find more promoter candidates that could be applied to Methylomonas sp. DH-1. A total of 110 promoter candidates were predicted: 93 sequences from Methylomonas sp. DH-1 and 17 from M. trichosporium OB3b. When the predicted promoters were functionally categorized based on their downstream coding sequences, most of them can be grouped into genetic regulators, metabolism, and gene expression (Fig. 1 A). Detailed information on the promoters is available in Additional files 2 and 3 : Tables S1 and S2. In these tables, a promoter sequence has been defined as an upstream 100-bp sequence from the transcription start site, including -35 and -10 elements. Fig. 1 Construction of a library of promoter candidates in Methylomonas sp. DH-1. A In silico approach to predict promoters from the genomic sequences of Methylomonas sp. DH-1 and M. trichosporium OB3b and the functional classification of the coding sequences under the control of the predicted promoters. B 2D-PAGE approach to identify strong native promoters that allow for the overexpression of Methylomonas sp. DH-1. C Addition of four well-known promoters of M. trichosporium OB3b and Escherichia coli to the library To find promoters with enough strength for efficient overexpression, the total cellular proteome of Methylomonas sp. DH-1 was quantitatively analysed by 2D gel electrophoresis (2D-PAGE) [ 26 ] (Fig. 1 B; Additional file 1 : Fig. S1). We chose five highly dense protein spots (indicated by arrows in Additional file 1 : Fig. S1), which were then excised and digested with trypsin. The resulting peptides were analysed by matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) mass spectrometry, and the corresponding proteins were identified by peptide mass fingerprinting. The five highly expressed proteins were pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (encoded by the mxaF gene), glutamine synthetase (encoded by the glnA gene), elongation factor Tu (encoded by the tuf gene), transaldolase (encoded by the tal gene), and 3-hexulose-6-phosphate synthase (encoded by the hps gene). We extracted the 5′ upstream region of the protein-coding genes to obtain their promoter sequences, including − 35 and − 10 elements. The identified proteins and their promoter sequences are listed in Additional file 4 : Table S3. Interestingly, none of the promoters identified by 2D-PAGE analysis overlapped with the promoters predicted by the computational tool. In addition to the predicted and identified promoters, we included known promoters in our library: two M. trichosporium OB3b promoters (methanol dehydrogenase ( mxaF ) and methane monooxygenase ( pmoC )) and two E. coli promoters ( lac and tac ) (Fig. 1 C). Finally, we compiled a library of 119 promoter candidates to construct a tunable promoter library (Fig. 1 ) . Evaluation of promoter candidates in Methylomonas sp. DH-1 To evaluate the promoter candidates, in addition to the promoters identified from 2D-PAGE analysis and the known promoters, we randomly selected several promoters from each category. The transcriptional activities of 38 out of 119 candidate promoters in Methylomonas sp. DH-1 were quantitatively measured. For the strength measurement, a plasmid harbouring the green fluorescent protein ( gfp ) gene under the control of a promoter candidate was constructed. Protein production level is determined not only by promoter strength but also by translational efficiency. To exclude the effect of translation on protein production, we used the same 50-nt long UTR, SD, and GFP coding sequences, because translation initiation region (around 30-nt before and after the start codon) is a determinant of translational efficiency [ 27 , 28 ]. The promoter- gfp gene was integrated into the non-coding region of the genome of Methylomonas sp. DH-1 for fluorescence measurement (Fig. 2 A). Fig. 2 Strength of promoter candidates measured by a GFP reporter. A Construction of a GFP expression plasmid under the control of a predicted promoter for integration into a Methylomonas sp. DH-1 chromosome. B Promoter strength measurement in Methylomonas sp. DH-1 by flow cytometry. Promoters showed up to 410-fold differences in GFP expression. The strength was normalized by that of the lac promoter (100%). As the y-axis is drawn at log scale, five promoter candidates that showed no detectable fluorescence were excluded from the graph. Data indicate mean ± SEM ( n = 3) The strength of the promoters varied from 0.24% to 410% in Methylomonas sp. DH-1 compared with that of the lac promoter, which shows that the library can cover approximately 1708-fold range of expression levels in Methylomonas sp. DH-1 and can be used to fine-tune the gene expression. The transcriptional activities of the promoters are shown in Fig. 2 B and Table 1 . Five of the 38 evaluated promoters displayed no detectable fluorescence at all. We also found that the five promoters identified from 2D-PAGE analysis exhibited high transcriptional activity. In particular, the promoter of the mxaF gene from Methylomonas sp. DH-1 showed the highest expression level, indicating that this promoter can be used for the overexpression of a target gene. Because the mxaF gene encodes the α-subunit of methanol dehydrogenase, a key enzyme in the methanol-to-formaldehyde conversion for methane utilization, the promoter could be highly active. In contrast, the promoter strength of the mxaF gene from M. trichosporium OB3b—a strong promoter [ 29 ]—was < 1% in Methylomonas sp. DH-1. The GFP intensity under the control of P tal showed the second-highest intensity. Typically, transaldolases function in the non-oxidative phase of the PP pathway in carbohydrate metabolism to generate nicotinamide adenine dinucleotide phosphate (NADPH) and ribose, which are essential for the biosynthesis of secondary metabolites and amino acids. Table 1 Tunable promoter library for Methylomonas sp. DH-1 Promoter identification method Gene name Promoter strength (%) In silico prediction ( Methylomonas sp . DH-1) 50S ribosomal protein L28 ( rpmB ) 22.09 Fur family transcriptional regulator 14.99 50S ribosomal protein L21 ( rplU ) 7.16 Shikimate kinase ( aroK ) 6.49 SsrA-binding protein ( smpB ) 3.79 Phosphate ABC transporter permease 2.94 TetR family transcriptional regulator 2.7 ABC transporter 2.21 LysR family transcriptional regulator 1.68 16S ribosomal RNA 0.54 Glycosyl transferase 0.39 Cytochrome c oxidase subunit 2 0.0 Elongation factor P ( efp ) 0.0 50S ribosomal protein L31 ( rpmE ) 0.0 10 kDa chaperonin ( groS ) 0.0 In silico prediction ( M. trichosporium OB3b) ATP-binding protein 191.08 (2Fe–2S)-binding protein 41.54 Ribosomal RNA small methyltransferase I ( rsmI ) 28.42 Peptidase S8 12.92 Iron–sulphur cluster assembly scaffold protein 7.45 Integrase 3.45 tRNA-dihydrouridine(20/20a) synthase ( dusA ) 2.91 Chromosomal replication initiator protein DnaA 2.81 Cytochrome c peroxidase 1.84 Thioredoxin peroxiredoxin 1.71 dTDP-glucose 4,6-dehydratase ( rfbB ) 0.56 Restriction endonuclease 0.43 Carbonic anhydrase 0.24 GGDEF domain-containing protein 0.0 2D-PAGE ( Methylomonas sp. DH-1) Methanol dehydrogenase ( mxaF ) 411.18 Transaldolase ( tal ) 262.9 Glutamine synthetase ( glnA ) 68.74 3-hexulose-6-phosphate synthase ( hps ) 67.37 Elongation factor Tu ( tuf ) 16.12 M. trichosporium OB3b promoters Methane monooxygenase ( pmoC ) promoter C 1.58 Methanol dehydrogenase (mxaF) promoter 0.51 E. coli promoters lac promoter (without operator) 100 tac promoter (without operator) 20.54 In general, there is an overall correlation between transcript level and protein level, but there are also many reports on the inconsistency of the correlation [ 30 ]. For example, highly transcribed mRNAs often failed to be highly translated [ 31 ]. To investigate if the high expression of the proteins was due to the high strength of their promoters, not high efficiency in translation, we confirmed their transcript levels by using the previous transcript data of Methylomonas sp. DH-1 [ 11 ]. According to the previous gene expression profile, of the five highly expressed proteins identified by the 2D-PAGE, only the transcription levels of three proteins were available (methanol dehydrogenase, transaldolase, and 3-hexulose-6-phosphate synthase). The three proteins were involved in the methanol oxidation, the PP pathway, and the RuMP cycle, respectively, and were grouped into a high transcription category (Fig. 2 B). Interestingly, the GFP intensities expressed from the P ATP-binding protein of M. trichosporium OB3b and the lac promoter of E. coli took third and fourth places, respectively. Because of the wide range of expression levels, the developed library of promoters can be utilized for fine-tuning gene expression in Methylomonas sp. DH-1. It should be noted that promoters may display different activity under different conditions due to regulatory effects. Thus, for the consistent measurement of promoter activity, we chose a defined expression context, such as exponential growth phase and methane as a carbon source [ 32 ], and the developed library was ensured to operate under the defined context. Influence of the expression level of cadA on cell fitness and cadaverine production Because Methylomonas sp. DH-1 has various metabolic pathways, such as the RuMP pathway, the PP pathway, the EMP pathway, the TCA cycle, and the serine cycle, it has been engineered as a promising host platform for C1 microbial cell factories [ 12 – 16 ]. As a practical application of our tunable promoter library, we diversified the expression level of the cadA gene to maximize the cadaverine titre from Methylomonas sp. DH-1. Generally, the microbial production of cadaverine, a natural nylon monomer used for polyamide production, is achieved through the overexpression of the E. coli lysine decarboxylase (CadA), which bioconverts lysine to cadaverine. Hence, it can be used in the chemical industry [ 33 , 34 ]. The biosynthetic pathway towards lysine/cadaverine synthesis in Methylomonas sp. DH-1 is illustrated in Fig. 3 . In native Methylomonas sp. DH-1, lysine synthesis involves oxaloacetate (OAA) derived from the serine and TCA cycles. However, because of the lack of the cadA gene, the bacterium cannot synthesize cadaverine. Thus, we introduced the cadA gene from E. coli into the genome of Methylomonas sp. DH-1 for cadaverine production. Fig. 3 Metabolic pathways of Methylomonas sp. DH-1 for the production of cadaverine from methane. Reactions mediated by heterologous enzymes are shown in red. CadA, l -lysine decarboxylase; CadB, lysine/cadaverine antiporter protein Fine-tuning the CadA expression is important for efficient cadaverine production and cell fitness, because overexpressed CadA might deplete lysine, which is also used as a precursor for cell wall synthesis [ 35 ]. In addition, the highly accumulated cadaverine could inhibit the activity of CadA [ 36 ] or induce cytotoxicity. Thus, the level of CadA expression should be balanced for optimum cell growth/toxicity and cadaverine production. We employed five promoters of different strengths from our library to diversify the expression level of CadA (Fig. 4 A). The promoters and their relative strengths were P DnaA (2.81%), P Integrase (3.45%), P rpmB (22.09%), P (2Fe–2S)-binding protein (41.54%), and P mxaF (411%). We then introduced the five constructed genes into the genome of Methylomonas sp. DH-1 and tested the resulting strains for the desired bioconversion in shake-flask cultures with a supplementation of 30% methane (v/v) as the sole carbon source. Therefore, we measured cadaverine and its precursor lysine titres along with cell growth. As shown in Fig. 4 B, C, the CadA overexpression by two strong promoters (P mxaF and P (2Fe–2S)-binding protein ) failed to produce cadaverine and greatly retarded the cell growth (Fig. 4 B, C). On the contrary, the strains expressing CadA under the control of three weeks or moderate promoters (P DnaA , P Integrase , and P rpmB ) displayed normal growth and higher cadaverine titres of 2.85 ± 0.13, 11.55 ± 2.70, and 8.32 ± 0.81 mg/L, respectively, after 72 h of cultivation. In a previous study, the same tendency of cadaverine titre change with respect to the level of CadA had been observed in E. coli [ 37 ]: when CadA was highly expressed, the cadaverine titre was decreased. One plausible hypothesis for the decreased cadaverine titre is lysine depletion. As shown in Fig. 4 D, when the two strong promoters were employed, the lysine titre inside Methylomonas sp. DH-1 was remarkably decreased. Since lysine is an important amino acid for growth, the depletion also induced cell growth defect [ 38 ], which in turn resulted in a decrease in overall cadaverine titre (Fig. 4 B). Interestingly, the lysine concentrations in the strains of the three weak or moderate promoters were not statistically different from that of wild type when tested by t-test ( p -value > 0.1), even though the lysine-consuming cadA gene was introduced. It is thus likely that CadA induced a metabolic flux in the lysine biosynthetic pathway by consuming lysine at an appropriate rate. Collectively, these results suggest that fine-tuning the expression of a target gene, rather than its overexpression, is a key to engineer metabolic pathways and that our tunable library can provide a genetic tool for optimization. Fig. 4 Effects of the expression level of the cadA gene on cell growth and lysine/cadaverine biosynthesis in Methylomonas sp. DH-1. A Genetic structure of cadA under the control of five promoters. Cadaverine production ( B ), growth ( C ), and lysine synthesis ( D ) of wild-type and five engineered Methylomonas sp. DH-1 strains in an NMS medium with a supplementation of 30% methane (v/v) as the sole carbon source. Data indicate mean ± SEM ( n = 3) Improved cadaverine production by optimizing cadB expression The optimization of cadA gene expression in Methylomonas sp. DH-1 using a promoter library helped enhance cadaverine production without redesigning the lysine synthetic pathway. It has been reported that the co-expression of CadA and CadB (a lysine/cadaverine antiporter) could simultaneously enhance lysine and cadaverine production because CadB exports intracellular cadaverine, a feedback inhibitor of CadA [ 33 , 35 , 36 ]. To further enhance cadaverine production, we introduced the cadB gene into three cadaverine-producing strains, P DnaA - cadA , P Integrase - cadA , and P rpmB - cadA . To investigate whether the fine-tuning of CadB gene expression could also affect cell growth and lysine/cadaverine production, we co-expressed the cadB gene under three different promoters (P DnaA , P (2Fe–2S)-binding protein , and P mxaF ) and constructed nine strains in total (Fig. 5 A). Fig. 5 Effect of the expression level of the cadB gene on lysine/cadaverine biosynthesis in three selected cadA knock-in strains. A Genetic structure for cadA and cadB expression. Three different promoters (P DnaA , P (2Fe–2S)-binding protein , and P mxaF ) were employed to diversify the expression level of cadB . Cadaverine ( B ) and lysine titres ( C ) in the engineered Methylomonas sp. DH-1 strains. Data indicate mean ± SEM ( n = 3) As shown in Fig. 5 B, the highest cadaverine titres, 11.18 ± 0.07 and 14.46 ± 1.01 mg/L, were achieved from the P DnaA - cadA /P DnaA - cadB and P rpmB - cadA /P DnaA - cadB strains, respectively, after 72 h of cultivation. The cadaverine titres were improved by 3.92- and 1.73-fold compared with those of their parental strains, P DnaA - cadA and P rpmB - cadA , respectively. Interestingly, although the introduction of cadB increased the lysine titre in all cadA / cadB knock-in strains, its introduction into the P Integrase - cadA strain reduced cadaverine production in all promoters. To investigate the reason for the inefficient production of cadaverine in these strains, the cell growth and cadaverine production were monitored for 120 h (Fig. 6 A, B). Interestingly, when cadB was overexpressed, the cells showed a major growth defect and decreased the cadaverine titre. Unlike the cadA gene, the overexpression of the cadB gene did not deplete the intracellular lysine concentration (Fig. 5 C). One plausible explanation is that the additional overproduction of CadB consumed intracellular resources and posed a burden to the cell, thereby retarding the cell growth; however, this should be elucidated further. After 96 h of cultivation, the engineered P rpmB - cadA /P DnaA - cadB strain afforded the maximum cadaverine titre (18.12 ± 1.06 mg/L), which was 2.18-fold higher than that (8.32 ± 0.81 mg/L) of its parental strain (P rpmB - cadA strain) and 2.78-fold higher than that (6.50 ± 0.02 mg/L) of the non-optimized cadA/cadB strain (P rpmB - cadA /P (2Fe–2S)-binding protein - cadB ). Fig. 6 Cell growth ( A ) and cadaverine titre ( B ) over time with respect to the employed promoter for the cadB gene. Data indicate mean ± SEM ( n = 3)"
} | 5,211 |
27148218 | PMC4838734 | pmc | 6,383 | {
"abstract": "Keeping in view, the challenges concerning agro-ecosystem and environment, the recent developments in biotechnology offers a more reliable approach to address the food security for future generations and also resolve the complex environmental problems. Several unique features of cyanobacteria such as oxygenic photosynthesis, high biomass yield, growth on non-arable lands and a wide variety of water sources (contaminated and polluted waters), generation of useful by-products and bio-fuels, enhancing the soil fertility and reducing green house gas emissions, have collectively offered these bio-agents as the precious bio-resource for sustainable development. Cyanobacterial biomass is the effective bio-fertilizer source to improve soil physico-chemical characteristics such as water-holding capacity and mineral nutrient status of the degraded lands. The unique characteristics of cyanobacteria include their ubiquity presence, short generation time and capability to fix the atmospheric N 2 . Similar to other prokaryotic bacteria, the cyanobacteria are increasingly applied as bio-inoculants for improving soil fertility and environmental quality. Genetically engineered cyanobacteria have been devised with the novel genes for the production of a number of bio-fuels such as bio-diesel, bio-hydrogen, bio-methane, synga, and therefore, open new avenues for the generation of bio-fuels in the economically sustainable manner. This review is an effort to enlist the valuable information about the qualities of cyanobacteria and their potential role in solving the agricultural and environmental problems for the future welfare of the planet.",
"conclusion": "Conclusion and Future Recommendations It is imperative for the healthy agro-ecosystem to gain sustainability in the true sense in order that it conserves the nature and natural resources, and also maintains the complexity and diversity of the ecosystems. It supports and sustains sufficient food production for the increasing world population, ensures economic viability, and safer living for both humans as well as other livestock. Above all, it addresses the present day environmental concerns. For poor farmers (especially in developing countries), it is not quite easy to afford the costly chemical fertilizers and pesticides and also feel concerned for the environmental issues. Cyanobacteria in this context can be very effective for enriching soil organic carbon and nitrogen and enhancing phosphorus bioavailability to the plants. Cyanobacteria are excellent accumulators or degraders of various environmental contaminants such as heavy metals, pesticides, and oil containing compounds. Such ubiquitous bio-agents can also be used for capturing and storage of CO 2 that may also lead to climate change mitigations through photosynthesis and biological calcification. They are also the ideal source of variety of bioactive compounds with marked antagonistic properties. There is enormous scope for the development of bio-agents including cyanobacteria for sustainable agriculture which also takes care of the improvement in the nutrient status of soil and biological control of pest and diseases that may ultimately lead to reductions in the agricultural costs ( Singh, 2013b ; Singh and Singh, 2013b ). However, it is necessary to carry out further investigations for exploitation of cyanobacteria with the futuristic goal to achieve the target of sustainable agriculture and environment. In view of the declining soil health and productivity due to increased human activities, the maintenance of environmental sustainability is the challenging task ahead. The cyanobacteria are multi-functional bio-agents for safe and eco-friendly agriculture and environmental sustainability, along with several other uses. To improve their utility in agriculture and associated sectors needs serious attention. Thus there is an urgent need to address certain key issues of exploiting cyanobacteria, the better way. Further, the application of molecular biology has improved our understanding of the effectiveness for betterment of healthy and sustainable agro-ecosystems. Since the use of cyanobacteria to produce valuable chemicals including food supplements is still little explored, there seems a long way to go. In addition to product developments, future research must address the strain improvement of useful cyanobacteria to achieve high quality food and fuel products, maintain high growth rates and survival under harsh environmental conditions. These will be the key factors to leap from laboratory studies to large-scale and profitable bio-fuel production for sustainable agriculture, ecosystem and environmental development. The utility of cyanobacteria in sustainable agriculture and environment can be enhanced by genetic manipulations ( Golden et al., 1987 ; Koksharova and Wolk, 2002 ; Huang et al., 2010 ; Heidorn et al., 2011 ). However, the application of genetic engineering to improve bio-fuel production in cyanobacteria is still in its infancy. In future, genetic and metabolic engineering of cyanobacteria are likely to play important roles in improving the economics of cyanobacteria-mediated bio-fuel production. Cyanobacteria can be genetically modified to potentially increase their growth and photosynthetic efficiency, biomass yield, lipid and carbohydrate productivity, improve temperature tolerance, and reduce photo-inhibition and photo-oxidation ( Volkmann and Gorbushina, 2006 ; Volkmann et al., 2006 ). However, from lab to field condition shift will not be as easy as it has to addressed several issues such as social relevance, political lobbying and fulfillments with the regulatory norms. Besides these, problems related to cross-contamination through use of closed-photo-bioreactors as a substitute of open ponds, it is recommended to be thoroughly examined prior to execution.",
"introduction": "Introduction The present world population of about 7.2 billion is expected to cross 9.6 billion by the end of year 2050. In order to provide food to all by that times, the annual production of cereals needs a jump of about 50%, i.e., from 2.1 billion tons per year to ∼3 billion tons per year. This onerous target puts enormous pressure on agriculture sector to achieve the food security. But such a quantum leap in food production can be achieved either by bringing more and more land under cultivation or by enhancing the productivity of cultivable land available. The first option remains a distant dream in the light of limited land and growing population. The option of increasing soil fertility and agricultural productivity with the help of better eco-friendly management tools, promises a successful food security. The current agricultural practices are heavily dependent on the application of synthetic fertilizers and pesticides, intensive tillage, and over irrigation, which have undoubtedly helped many developing countries to meet the food requirement of their people; nevertheless raised environmental and health problems, which include deterioration of soil fertility, overuse of land and water resources, polluted environment, and increased cost of agricultural production. A big question before the present day agriculture is to enhance the agricultural production to meet the present and future food requirements of the population within the available limited resources, without deteriorating the environmental quality ( Singh and Strong, 2016 ). The sustainable agriculture practices can fulfill the growing need of food as well as environmental quality ( Mason, 2003 ). The present philosophy of sustainable agriculture includes eco-friendly, low-cost farming with the help of native microorganisms. It also emphasizes that the farmers should work with natural processes to conserve resource such as soil and water, whilst minimizing the cost of agricultural production and waste generation that adversely affects the environment quality. Such sustainable agricultural management practices will make the agro-ecosystem more resilient, self-regulating and also maintain the productivity and profitability. Since long, the microbes have been known to contribute to the soil fertility and sustainable green energy production ( Koller et al., 2012 ). During the last decades, the microbial processes of green energy production have gained interest as the sustainable tool for the generation of bio-fuels, namely methane (CH 4 ), ethanol, H 2 , butanol, syngas, etc. Current investigations witnessed noteworthy surge growth in the production of cyanobacterial biomass for bio-fuels, food supplements (super foods), and bio-fertilizers for safe agriculture ( Yamaguchi, 1997 ; Benson et al., 2014 ). They have been classified as beneficial as well as harmless bio-agents based on their role in regulating plant productivity. In reality, these two diverse groups of microorganisms coexist in nature, and predominance of one at any point of time, depends mainly on the environmental conditions. For many years, soil microbiologists and microbial ecologists have been studying the effect of beneficial or efficient soil microorganisms for sustainable agriculture which not only contribute to soil fertility, crop growth and yield, but also improve the environment quality. Nowadays, sustainable agriculture practices have envisaged an important role of these tiny microorganisms in achieving the food security without creating environmental problems. The recent trends of using the bio-inoculants containing beneficial soil microbes over synthetic fertilizers, insecticides, and pesticides for enhancing crop productivity is a welcome step. As a beneficial microbe, cyanobacteria could play a potential role in the enhancement of agriculture productivity and mitigation of GHG emissions ( Singh, 2011 ; Singh et al., 2011a ). Very recently, it has been proposed that cyanobacteria could be the vital bio-agents in ecological restoration of degraded lands ( Singh, 2014 ). Cyanobacteria are the group of photosynthetic organisms which can easily survive on bare minimum requirement of light, carbon dioxide (CO 2 ) and water ( Woese, 1987 ; Castenholz, 2001 ). They are phototrophic, and naturally occur in several agro-ecosystems like paddy fields and from Antarctica to Arctic poles ( Pandey et al., 2004 ). They fulfill their own nitrogen requirement by nitrogen (N 2 )-fixation, and produce some bioactive compounds, which promote the crop growth/protect them from pathogens and improve the soil nutrient status. Cyanobacteria are also useful for wastewater treatment, and have the ability to degrade the various toxic compounds even the pesticides ( Cohen, 2006 ). A conceptual model about the role of cyanobacteria in sustainable agriculture and environmental management has been proposed ( Figure 1 ). This review highlights the role of cyanobacteria in bio-energy production, ecological restoration, agriculture and environmental sustainability. FIGURE 1 A hypothetical model exhibiting the potential roles of cyanobacteria in sustainable agriculture and environmental management ."
} | 2,766 |
25345760 | PMC5381374 | pmc | 6,384 | {
"abstract": "The ability of coral reefs to engineer complex three-dimensional habitats is central to their success and the rich biodiversity they support. In tropical reefs, encrusting coralline algae bind together substrates and dead coral framework to make continuous reef structures, but beyond the photic zone, the cold-water coral Lophelia pertusa also forms large biogenic reefs, facilitated by skeletal fusion. Skeletal fusion in tropical corals can occur in closely related or juvenile individuals as a result of non-aggressive skeletal overgrowth or allogeneic tissue fusion, but contact reactions in many species result in mortality if there is no ‘self-recognition’ on a broad species level. This study reveals areas of ‘flawless’ skeletal fusion in Lophelia pertusa , potentially facilitated by allogeneic tissue fusion, are identified as having small aragonitic crystals or low levels of crystal organisation, and strong molecular bonding. Regardless of the mechanism, the recognition of ‘self’ between adjacent L. pertusa colonies leads to no observable mortality, facilitates ecosystem engineering and reduces aggression-related energetic expenditure in an environment where energy conservation is crucial. The potential for self-recognition at a species level, and subsequent skeletal fusion in framework-forming cold-water corals is an important first step in understanding their significance as ecological engineers in deep-seas worldwide.",
"discussion": "Discussion The ability of L. pertusa to skeletally fuse 12 between individuals has facilitated their roles as deep-sea ecosystem engineers. Here we demonstrate that this can occur between genetically distinct adult individuals and not just between closely related individuals. It is likely that this ability has been driven by two main factors: 1) evolutionary pressure for cold-water corals to stabilise their own framework, much like the role of calcifying encrusting algae on tropical reefs, and 2) the benefit of reducing energetic investment into aggressive competition interactions, which can lead to mortality or reductions in growth and gonad development in reef forming corals 11 . Creation of continuous reef frameworks would ensure suitable substrate is formed for the settlement of subsequent generations providing a selective advantage. Skeletal fusion would also act to prevent unnecessary coral death if the underlying framework is broken e.g. from strong currents and/or bioerosion, as live coral branches falling into other colonies would be more likely to survive and grow. Since deep-water corals rely purely on heterotrophic feeding (i.e. feeding on passing prey items, which varies spatially and temporally) avoiding unnecessary energetic expenditure, which could be reallocated to growth and reproduction, would be of significant benefit to the success and continuation of L. pertusa reefs. Further work is needed to identify whether this skeletal fusion is driven by: 1) allogeneic fusion between individuals, or 2) an efficient, minimally aggressive ‘overgrowth’ strategy across individuals that does not result in mortality of any polyps. Both of these mechanisms are represented here, and both rely on a high degree of self-recognition at a species level. Efficient overgrowth with no observable mortality is apparent in Fig. 2B and C , and potential allogeneic tissue fusion is identifiable in Fig. 3A , leading to seamless skeletal fusion ( Fig. 3.D ). The skeletal fusion reported here between genetically distinct individuals with unique multi-locus genotypes at 15 microsatellite loci, underpins the fact that while L. pertusa can discriminate ‘self’ on a species level, they do not reject between individuals. The occurrence of both overgrowth and potential allogeneic fusion in data presented here complements observations that allogeneic fusion is complex, and can happen in a series of cascading events 6 . For the rare instances that adult allogeneic fusion has been noted in tropical corals 16 , they are typically explained by a close genetic similarity of the colonies involved 4 5 . The difference between overgrowth and tissue allogeneic fusion may be represented in: 1) the strength or molecular organisation of the aragonite bonds 14 15 , and 2) the organisation or size of aragonite crystals. For 1) the decreased FWHM at the suture-less fusion zone indicates decreased molecular organisation but stronger bonding within the aragonite. FWHM in areas with small micro-sutures and larger micro-sutures increased respectively. It seems that full skeletal fusion where no suture is formed, results in strong molecular bonding, and potentially small, or disorganised crystal bundles compared to areas where sutures were apparent. Allogeneically fused tissue could thus result in different biomineralisation properties of the coral skeleton. In areas that may be the result of overgrowth, decreased aragonite bond strength of one or both of the polyps, or increased crystallographic organisation is observed. It is unknown how these factors are controlled when contact does occur between corals, and whether the cavities that are observed near the fusion zones are a result of fusion-skeletal effectors, or the result of overgrowth of organic debris while the coral was growing. Aside from the specific mechanisms that facilitate this skeletal fusion, it is also unknown whether this ability is limited to the deep-water coral L. pertusa or whether other deep-water reef framework-forming species (see Fig. 1 ) also exhibit skeletal, and potential allogeneic fusion, between non-kin adult individuals. Regardless of whether the large, multi-colony frameworks which are often characteristic of L. pertusa reefs are mostly a result of allogeneic tissue fusion or efficient low aggression overgrowth, the ability of L. pertusa to self-recognise at a species level to routinely undergo skeletal fusion has made them one of the most significant ecosystem engineers of the deep seas."
} | 1,494 |
36443458 | PMC9712116 | pmc | 6,385 | {
"abstract": "Despite advances in sequencing, lack of standardization makes comparisons across studies challenging and hampers insights into the structure and function of microbial communities across multiple habitats on a planetary scale. Here we present a multi-omics analysis of a diverse set of 880 microbial community samples collected for the Earth Microbiome Project. We include amplicon (16S, 18S, ITS) and shotgun metagenomic sequence data, and untargeted metabolomics data (liquid chromatography-tandem mass spectrometry and gas chromatography mass spectrometry). We used standardized protocols and analytical methods to characterize microbial communities, focusing on relationships and co-occurrences of microbially related metabolites and microbial taxa across environments, thus allowing us to explore diversity at extraordinary scale. In addition to a reference database for metagenomic and metabolomic data, we provide a framework for incorporating additional studies, enabling the expansion of existing knowledge in the form of an evolving community resource. We demonstrate the utility of this database by testing the hypothesis that every microbe and metabolite is everywhere but the environment selects. Our results show that metabolite diversity exhibits turnover and nestedness related to both microbial communities and the environment, whereas the relative abundances of microbially related metabolites vary and co-occur with specific microbial consortia in a habitat-specific manner. We additionally show the power of certain chemistry, in particular terpenoids, in distinguishing Earth’s environments (for example, terrestrial plant surfaces and soils, freshwater and marine animal stool), as well as that of certain microbes including Conexibacter woesei (terrestrial soils), Haloquadratum walsbyi (marine deposits) and Pantoea dispersa (terrestrial plant detritus). This Resource provides insight into the taxa and metabolites within microbial communities from diverse habitats across Earth, informing both microbial and chemical ecology, and provides a foundation and methods for multi-omics microbiome studies of hosts and the environment.",
"discussion": "Discussion Here we discuss some of the caveats and limitations of our study, and further highlight how our approach advances understanding of microbial community dynamics and functional diversity. Due to their extensive nature, we provide additional important points of discussion as Supplementary Information . We begin by recognizing that certain environments included in EMPO are represented here by only a handful of samples (Fig. 1 ) and/or a single sample set (Supplementary Table 1 ), and note that we had to exclude them from some of our analyses due to low representation (for example, machine learning and co-occurrence analyses). We recommend that future efforts focus on additional sampling of these environments to further generalize our findings to those habitats. Similarly, we hope to expand sampling geographically to broaden our scope of inference, as many important environments and locations could not be included here (or, indeed, in the EMP’s 27,000-sample dataset 1 ). We also note that the inherent design of the EMP (that is, crowd-sourced samples from experts in respective fields) prevented us from explicitly exploring causation with respect to the environment in our analysis, and thus our findings are based largely on observations and correlations among feature sets and associated metadata. In our example analysis, we explored whether every metabolite is everywhere but the environment selects (that is, the Baas Becking hypothesis 42 , 43 , but for microbially related metabolites). Whereas we interpret our findings as strong evidence that every metabolite is everywhere but the environment selects, our study was not designed to address this hypothesis explicitly, and further evidence is needed to support this hypothesis. For example, features at abundances below the detection limit of our approach could not be considered here, but may alter our view of these patterns. Similarly, although input sample volumes were normalized as best as possible, they may influence estimates of alpha-diversity, and the values reported here probably exhibit some error in part due to this influence. We also identified metabolite–microbe co-occurrences, and note that our approach for characterizing co-occurrences, ‘mmvec’ 56 , does not currently allow for controlling for covariates and this may influence results. However, in our analysis we were able to include EMPO as a variable, which we designed to account for variation among environments that may not be captured by available metadata. Here we described patterns of turnover, nestedness and co-occurrence of metabolites and microbes across a diverse set of environments while addressing ecological questions surrounding the distribution of metabolites and their relationships with microbial taxonomic and functional diversity. One outstanding question in microbial ecology asks how microbial taxon profiles can be integrated with functional ones 57 . Here, in addition to describing microbial taxa, their functions and their metabolites, we explicitly tested for metabolite–microbe co-occurrences and explored how they relate to the environment, for which we have outlined our approach (Extended Data Fig. 1 ). Our analysis provides insight into biological processes including microbial community assembly and links microbial taxonomic profiles with metabolism and functional diversity (that is, enzymes) at planetary scale. Our work provides an initial view of how microbially related metabolites are structured with respect to factors including host association, salinity and the presence of certain microbes (Figs. 3 and 5 ). Importantly, we identified the most abundant and highly ranked pathway representing the metabolites best able to distinguish environments to be terpenoids 58 , highlighting the importance of this group of metabolites in distinguishing Earth’s environments (Fig. 4a and Supplementary Table 7 ). We acknowledge that previous studies describing microbial taxa and function using globally distributed sample sets, such as for the human gut, soils and the ocean, have shown that both can vary across locations 59 – 62 . Similarly, studies examining metabolite profiles across changes in microbial community composition, or environmental stress such as from heat, have shown variation associated with either 20 , 21 or both 23 . Furthermore, among previous multi-omics studies combining metagenomics with metatranscriptomics, metaproteomics and/or metabolomics, some of which have shown the correlation between data layers to vary across sites, the majority are focused on a single environment 63 – 73 . Here we performed multi-omics integration of a dataset encompassing a diversity of environmental sample types representing several habitats, generated using standardized methods allowing for robust meta-analysis with data from other studies using the same approach. Our approach illustrates that recent advances in computational annotation tools offer a powerful toolbox to interpret untargeted metabolomics data 41 . We anticipate that parallel advances in metagenomic sequencing, genome assembly and genome mining will improve the discovery and classification of functional products from among microbes and provide additional insight into these findings. By following standardized methods available on GitHub and making this dataset publicly available in Qiita and GNPS, this study will serve as an important resource for continued collaborative investigations. In the same manner, the development of optimized instrumentation and computational methods for metabolomics will expand the depth of metabolites surveyed in microbiome studies."
} | 1,954 |
26104283 | PMC4522039 | pmc | 6,386 | {
"abstract": "Alien species can severely disrupt the structure, function and stability of native communities. We evaluated the structure of pollination networks in the three main habitats and in the two seasons on the two most disturbed Galápagos Islands, and investigated how such structure is influenced by invasive plants. Alien plants integrate easily into the communities, but show low impact on overall network structure, except for an increase in network selectiveness. The highly invaded and low diversity humid zone showed the highest nestedness and the lowest modularity. Both pollinators and plants were more generalized during the hot season, when most plants were flowering and networks became more nested.",
"conclusion": "Conclusions The structure of pollination networks is highly consistent on the two most disturbed islands of the Galápagos archipelago. Differences in network structure exist across the main habitats. The most widespread arid habitat consistently bears the largest pollination networks and differs strongly from the humid habitat in descriptors such as interaction evenness, nestedness and modularity. The transition habitat between the arid and the humid zone shows pollination networks more similar in structure to those in the arid than in the humid areas. The humid habitat is also the most invaded by alien species and this could partly explain some of the differences in its network structure, such as its more nested pattern and its lower modularity level compared with the arid and the transition zones. Pollinators appear to interact with more plants in the humid habitat than in the arid one. The incidence of alien flowers might actually increase the level of pollinator generalization, although results are inconclusive as this was observed in only one of the two study years. Overall, the level of invasion has a weak influence on pollination network structure and seems to be associated with only one metric, H 2 ′ which measures the level of selectiveness; thus, as invasion progresses, species in the network appear to become more selective in their choice of partners, interacting with less abundant species more than would be expected by chance. Pollination networks are larger during the hot/rainy season, when most flowers are in bloom and more insects are flying, than in the cold/dry season. They are also more nested in the hot season, and thus probably more robust to disturbances. Pollinators visit more plant species, and plants are visited by more pollinator species, during the hot season. In the cold season, the number of insects is especially low in the humid zones and thus the number of pollinators visiting plants is also lower in that season and habitat. In contrast, both pollinator and plant selectiveness ( d ′) and strength ( st , importance to the plant and pollinator community, respectively) were spatially and temporally consistent and not influenced by alien plants. Alien pollinators interacted with fewer plants, were less selective in their choice (i.e. tended to visit the most abundant species) and were less important to the plant community (i.e. showed lower species strength) than endemic and native pollinators. They, however, infiltrated the native communities of all habitats and in both seasons and currently represent over 40 % of all recorded pollination interactions. Alien plants, on the other hand, were visited by approximately the same number of pollinators as natives—but less than endemic plants—implying that they are also well integrated into the native communities. In this study, we found a rather feeble effect of alien plants on the structure of pollination networks. As previously mentioned in the methods, our study intentionally considered sites that are not completely disturbed by highly invasive species (e.g. Psidium guava , Rubus nivaeus , Syzygium jambos ) which have displaced many native species in the invaded areas, mainly in the humid zones ( Guézou et al . 2010 ). Hence, the overall weak effect we found does not imply a weak influence of plant invasions on the reproductive success of native species. The fact that alien plant species are present in all habitats and in both seasons and that they are involved in ∼25 % of all pollination interactions, actually leads us to think that their effect on the functioning of native communities is far from negligible.",
"introduction": "Introduction Sexual reproduction is an essential step for the life cycle of most plant species, and is chiefly limited by the quantity and quality of pollen grains arriving to their stigmas ( Ashman et al . 2004 ). Pollination is thus a critical step in plant reproduction, and many animals, mostly insects, have a vital role in facilitating this step in ∼90 % of the worlds' plant species ( Ollerton et al . 2011 ). Islands harbour a disproportionate part of the worlds' biological diversity and are particularly rich in endemic and threatened species ( Sax and Gaines 2008 ). Oceanic islands, in particular, are generally characterized by low insect diversity ( Gillespie and Roderick 2002 ) and simplified pollination networks when compared with mainland systems ( Olesen and Jordano 2002 ; Traveset et al . 2015 b ). This low abundance and diversity of pollinators on islands is likely to translate into reduced pollinator redundancy, potentially leading to highly vulnerable communities when faced with disturbances, e.g. El Niño Southern Oscillations ( Traveset and Richardson 2006 ). Ecological networks offer a most valuable solution to evaluate the overall changes in community structure and function as a response to disturbances affecting species composition ( Bascompte 2010 ; Heleno et al . 2014 ). For example, in order to survive in such low diversity ecosystems, some animal species that successfully colonize isolated islands tend to broaden their trophic niches, thus interacting with more species (mutualistic partners or prey) than their continental counterparts ( Carlquist 1974 ; Olesen et al . 2002 ). This expansion of the feeding niche can characterize entire island communities, a phenomena coined ‘interaction release’ and that tends to have a stabilizing effect on insular interaction networks ( Traveset et al . 2015 a ). Apart from their low diversity and high generalization when compared with continental communities, oceanic island interaction networks tend to be characterized by an increased nestedness, i.e. an ordered interaction distribution pattern where specialist species interact with specific sub-sets of the partners of most generalist species ( Olesen and Jordano 2002 ; Padrón et al . 2009 ; Kaiser-Bunbury et al . 2010 ; Traveset et al . 2013 ). While increased generalization and nestedness may increase network stability ( Sebastián-González et al . 2015 ), overall low biodiversity and the existence of small endemic populations suggest high species vulnerability at least to some specific sources of disturbance, such as invasive species ( Berglund et al . 2009 ; Traveset and Richardson 2014 ). In this study we focus on the impacts of alien plants on pollination networks. Biological invasions are a growing threat to the worlds' biodiversity ( Lambertini et al . 2011 ) and particularly worrying on oceanic islands, where the arrival of alien species frequently triggers serious disruptive effects on the intricate network of interactions established between native species throughout their shared evolutionary history ( Kaiser-Bunbury et al . 2011 ; Traveset et al . 2014 ). Specifically, applying a network approach to frame biological invasions at the community level is particularly suitable for clarifying how invasive species can integrate into the existing interaction networks, the likely consequences for community structure, and the consequences for the most vulnerable species ( Memmott et al . 2007 ; Bascompte 2009 ; Traveset and Richardson 2014 ). In fact, if pollination networks vary naturally in space and time, it is likely that opportunities for alien pollinators and plants to ‘infiltrate’ those networks will also vary in space and time. Thus, aliens may find particularly favourable biotic and abiotic conditions under which their integration into the native communities (and potential invasion) is more likely. Recent studies have begun to evaluate the temporal and spatial variability of pollination network structure (e.g. Olesen et al . 2008 ; Petanidou et al . 2008 ; Dupont et al . 2009 ); however, we are only starting to understand such patterns, how they are related to each other and, particularly, how spatio-temporal dynamics might affect the capacity of alien species to infiltrate into and impact pollination networks. For example, an invasion might be more likely during a particular season, year (e.g. during particularly wet years), or in certain habitats. As in most archipelagos throughout the World, the number of alien species in the Galapagos began to accumulate even before the first permanent human settlement of the islands, increased exponentially over the last 50 years in step with increasing human pressure ( Tye 2006 ) and currently forms over 60 % of the vascular flora ( Jaramillo et al . 2014 ). Indeed, alien species, both plants and animals, are generally considered the main threat to the conservation of the unique Galápagos biodiversity ( Bensted-Smith 2002 ), and predicting the effects of alien plants and pollinators on the reproduction of native vegetation is a major conservation and scientific goal ( Tapia et al . 2009 ; Traveset et al . 2013 ). A recent compilation of plant–animal pollination interactions retrieved data from 38 studies published in the last 100 years in highly scattered literature ( Chamorro et al . 2012 ). This study concluded that most interactions were documented by observations highly limited in space and time, and thus identified strong biases in the sampling effort dedicated to different islands, times of day, focal plants and functional groups of visitors, reducing our ability to derive solid generalizations from these incomplete datasets ( Chamorro et al . 2012 ). While alien invasive plants may have a direct negative effect on native plants due to direct competition for space ( Magee et al . 2001 ), repercussions may also cascade throughout the entire network of biological interactions of an island or archipelago without necessarily leading to local extinction of native species ( Jäger et al . 2009 ). Such a disturbance scenario can be better understood with a network approach ( Traveset et al . 2013 ), such as the one we apply here. Human pressure is not evenly distributed across the islands but is heavily concentrated on the two large central islands: Santa Cruz, the most populous island, and San Cristóbal, which holds the administrative capital of the archipelago. Human developments are restricted to a small proportion of each island's area; however, an extensive use of the transition and highland zones for agriculture boosted the number of alien plant species. Thus, these two islands offer suitable models to improve our understanding of the disruptive effect of alien species on the native interaction networks of the Galápagos and to forecast short- and mid-term impacts on the islands with low human presence (Isabela and Floreana), and long-term impacts on the most pristine uninhabited islands. The main objective of this study was thus to assess the spatio-temporal variation of pollination interactions in the two most disturbed Galápagos islands and to determine whether and how alien plants may modify such interaction patterns. In addition, we investigated if alien species (both animals and plants) differ from endemic and non-endemic natives in their integration into the pollination networks.",
"discussion": "Discussion Spatio-temporal network patterns and influence of plant invasion Despite network size being larger in Santa Cruz than in San Cristóbal, especially in 2011 when it was twice as large, the overall pollination network structure was similar between the two islands. Strong spatial variation in network structure was detected, however, across habitats. The arid zone, which includes the vast majority of the land area and bears the highest species richness, supported the largest pollination networks. In contrast, the transition and the humid zone were more similar in size, though this was not consistent between islands or years. Flower and insect abundance are known to be influenced by abiotic conditions such as temperature or rainfall which can vary much spatially and temporally ( Ziegler 1995 ; Trueman and d'Ozouville 2010 ). Alien plant species represented up to 40 % of the plants in some networks, particularly in the humid zone of Santa Cruz and in the transition zone in San Cristóbal. However, overall network size was not affected by the level of invasion—measured as the proportion of alien flowers—suggesting both that alien plants do not differ from natives with respect to the diversity of their pollinators and that aliens do not displace native plant species in the pollination networks. Habitats also differed in interaction evenness, nestedness and modularity. The uniformity in the distribution of interaction frequencies was higher in the humid habitat than in the two other habitats. In a previous study in the Galápagos ( Traveset et al . 2013 ), a decrease in interaction evenness was observed along a gradient of invasion intensity at the island scale, being attributed to shifts in the proportion of strong and weak interactions in the network. However, the present work showed no effect of invasion level on this network parameter and, actually, the humid habitat is that bearing the highest fraction of alien species. Interaction evenness has been reported to increase after an invasion in one study on seed dispersal networks ( Heleno et al . 2013 b ) but not in another ( Heleno et al . 2013 a ). Hence, further data are needed to generalize about how this network parameter, known to be inversely related to network stability ( Rooney and McCann 2012 ), is influenced by alien invasions. The humid habitat showed the strongest nested pattern (in which specialist species link to a subset of species with which generalists also interact), which could also be attributed, at least partly, to its high level of invasion. The degree of nestedness has been found to increase with the integration of alien species ( Padrón et al . 2009 ; Santos et al . 2012 ); this is because aliens tend to be generalist species and/or are linked to generalist species ( Aizen et al . 2008 ; Traveset et al . 2013 ). Thus, although the level of invasion overall was a poor predictor of nestedness, we cannot discard the possibility that a higher incidence of alien flowers enhances a nested pattern in a habitat. Modularity—another common parameter that informs us on how cohesive the network is and how vulnerable it can be to different types of disturbances ( Olesen et al . 2007 )—was lower in the humid zone, i.e. this zone had a weaker segregation of species into cores of strong interactions, than the arid and transition zones. The lower modularity in the humid zone might be associated with its lower plant and animal diversity compared with the transition and arid zones, and also with its relatively higher linkage levels (see below). The level of invasion has been documented to decrease modularity, and thus to enhance network cohesiveness in some studies ( Santos et al . 2012 ; Albrecht et al . 2014 ). It is thus possible that the lower modularity in the humid habitat is partly due to its higher incidence of aliens. A low modularity has potential effects on network functioning, reciprocal selection regimes and the cascade of perturbations throughout the network ( Albrecht et al . 2014 ). Other network descriptors, such as connectance, interaction strength asymmetry and network complementary specialization ( H 2 ′ ) , did not vary much either in space or time. The level of network connectance, which is inversely related to network size, was both spatially and temporally consistent, despite species richness in each network varying across islands, habitats and seasons. This parameter is a measure, albeit crude, of network generalization level and, as expected from other island studies ( Olesen and Jordano 2002 ; Traveset et al . 2013 ), we found relatively high values (∼18 % on average, ranging from 12 to 40 %, across the 36 matrices analysed). No effect of invasion level on connectance was observed, which is consistent with previous studies ( Forup and Memmott 2005 ; Heleno et al . 2012 ), although network rewiring can actually occur and, as a result, the number of interactions between native species can decrease ( Aizen et al . 2008 ; Padrón et al . 2009 ; Kaiser-Bunbury et al . 2011 ). Besides being consistent in space and time, most values of interaction strength asymmetry were positive which indicates that animals are more dependent upon plants than vice versa ( Blüthgen et al . 2007 ), a result commonly found in other oceanic archipelagos ( Kaiser-Bunbury et al . 2010 ; Traveset et al . 2015 b ), and a pattern not found to be influenced by invasion level in this study. Finally, an interesting finding from our study was that H 2 ′ increased with the level of plant invasion, implying that species become more selective in their choice of mutualists by being compelled to interact with less abundant partners as invasion progresses. This finding contrasts with results from other studies which have reported a decrease in H 2 ′ after an invasion ( Heleno et al . 2013 b ). Regarding the species-level parameters, pollinators tended to visit more plant species in Santa Cruz than in San Cristóbal, what can be attributed to the higher plant species richness in the former. Pollinators were also more generalist in the humid zone even though here the number of plants is lower than in the other two zones. It is possible, thus, that the lower amount of floral resources in the humid zone promotes insects visiting more plant species, as has been found in a number of island studies ( Olesen et al . 2002 ; Kaiser-Bunbury et al . 2009 ; Padrón et al . 2009 ; Traveset et al . 2013 ). Interestingly, one of the years (2011), pollinators visited more plant species at sites with a greater fraction of alien flowers, suggesting that pollinators might be attracted to the new species which in turn would enhance their visitation to the other native plants in the community. Such ‘facilitative’ effects of alien plant species on native ones have been often reported in different systems (e.g. Moeller 2005 ; Jakobsson et al . 2009 ). Plant species, on the other hand, showed higher generalization levels in their pollination interactions in Santa Cruz than in San Cristóbal, at least in 2011 when more insect species were found on the former island. Plants were visited by less pollinator species in the humid zone, as the total number of pollinators is also lower in this zone compared with the other two zones. The other two parameters, species specialization d ′ and strength st , were highly consistent in space. Both pollinators and plants had a similar level of selectiveness in their flower or pollinator use, respectively, and were also equally important to the plant or pollinator communities, respectively, in the two islands and in the three habitats. A fairly constant value of d ′ for both plants and pollinators has been previously reported across five of the Galápagos Islands ( Traveset et al . 2013 ). Moreover, those two parameters were not influenced by plant invasion level. In contrast, at least one study (of seed-dispersal networks) has reported the level of invasion to decrease species specialization d ′ of native species ( Heleno et al . 2013 a ). Except for a few differences between pollination networks of different habitats, our results were highly consistent between the 2 years of the study, which were both considered ‘normal’ years in terms of precipitation and sea surface patterns (FCD Weather report, data not shown), despite the usual fluctuations in flower production and flower-visitors' presence/abundance. Thus, we focus on the temporal differences observed between seasons. All pollination networks were larger during the hot rainy season, when more plant species are blooming and more insects are flying, than in the cold dry season. Both pollinators and plants actually showed higher linkage levels in the hot than in the cold season, given the greater availability of partner species in the former. Moreover, networks were more nested in the cold than the hot season after controlling for network size, which influences this parameter. Such temporal difference in the degree of nestedness suggests that the interactions in the hot season tend to be more specific, with specialist species interacting more than expected with each other and less so with generalists. Integration of alien species on pollination networks Alien pollinators were consistently found to visit fewer plant species than endemic pollinators and, at least one year, also visited fewer plant species than native pollinators, which suggests that these newly-arrived species are focusing flower visitation on species with particular traits. However, the fact that alien pollinators also showed lower levels of selectiveness than endemic pollinators implies that they tend to visit more abundant flower resources compared with endemic pollinators, which visit even rare flowers. Likewise, species strength was consistently lower for alien than endemic pollinators, indicating that the former are less important to plants. In a previous study focusing on the pollination networks of the arid zone in five Galápagos islands, we found that alien insects had more links than either endemics or non-endemic natives ( Traveset et al . 2013 ), which suggests that the inclusion of the two other habitats, transition and humid, in the present study masks that pattern and/or that Santa Cruz and San Cristóbal are somewhat outliers in archipelago wide patterns, possibly due to the high level of disturbance. Alien plants were also consistently more specialized than endemic plants, although they were similar to native species. In contrast to pollinators, plants showed similar selectiveness regardless of their origin, but again, endemic plant species were more important to the pollinator community than alien plants. These findings were consistent with our previous study ( Traveset et al . 2013 ). It might be possible that aliens do not rely as much on pollinators as native species do. However, no data are currently available on the breeding system for the large majority of plants and, thus, future studies are needed to test this hypothesis."
} | 5,714 |
36145739 | PMC9501341 | pmc | 6,387 | {
"abstract": "The nature of plant–fungi interaction at early stages of arbuscular mycorrhiza (AM) development is still a puzzling problem. To investigate the processes behind this interaction, we used the Medicago lupulina MlS-1 line that forms high-efficient AM symbiosis with Rhizophagus irregularis . AM fungus actively colonizes the root system of the host plant and contributes to the formation of effective AM as characterized by a high mycorrhizal growth response (MGR) in the host plant. The present study is aimed at distinguishing the alterations in the M. lupulina root metabolic profile as an indicative marker of effective symbiosis. We examined the root metabolome at the 14th and 24th day after sowing and inoculation (DAS) with low substrate phosphorus levels. A GS-MS analysis detected 316 metabolites. Results indicated that profiles of M. lupulina root metabolites differed from those in leaves previously detected. The roots contained fewer sugars and organic acids. Hence, compounds supporting the growth of mycorrhizal fungus (especially amino acids, specific lipids, and carbohydrates) accumulated, and their presence coincided with intensive development of AM structures. Mycorrhization determined the root metabolite profile to a greater extent than host plant development. The obtained data highlight the importance of active plant–fungi metabolic interaction at early stages of host plant development for the determination of symbiotic efficiency.",
"conclusion": "5. Conclusions To sum up the data, we note that, at early stages of AM establishment, the metabolite profiles of M. lupulina roots significantly differ in their spectrum from those in the leaves. The roots contain fewer sugars and organic acids. Hence, compounds supporting the growth of mycorrhizal fungus (especially amino acids, the number of lipids, and specific carbohydrates) accumulated and coincided with intensive development in AM structures. This result clearly confirms the intensive development of AM fungus in the roots of young host plants. Such an observation is crucial for determining the further development of M. lupulina plants under phosphate deprivation and, in turn, reveals one of the mechanisms of plant adaptation based on symbiosis.",
"introduction": "1. Introduction The efficient symbiosis between higher plants and arbuscular mycorrhiza (AM) fungi is one of the more intriguing questions in modern biology. AM is a widespread symbiosis formed by more than 80% of land plants and Glomeromycetes fungi [ 1 ]. AM plays an important role in terrestrial ecosystems. Such plant–fungal association enhances plant growth and its adaptive capabilities [ 1 , 2 , 3 ]. This phenomenon has both biological and agricultural importance, as environmental stresses can reduce crop production by up to 70% [ 4 ]. Nevertheless, the lack of mycorrhizal growth response (MGR) was shown for Pisum sativum , Plantago lanceolata, P. major, M. truncatula, Veronica chamaedrys, Poa annua, and Vitis vinifera inoculated with R. irregularis [ 5 , 6 , 7 ]. Thus, the importance of determining new parameters that reflect the intensity of interactions between AM partners is self-evident. The intensification of AM biology research is based on the employment of new so-called “omic” technologies. AM fungi effect the transcriptome of M. truncatula, Nicotiana attenuata, Sorghum bicolor [ 8 , 9 , 10 ], and the proteome of M. truncatula and Sorghum bicolor [ 11 , 12 , 13 , 14 ]. Special attention was paid to AM manifesting metabolic rearrangements [ 5 , 15 , 16 , 17 ]. Its significant species specificity has been revealed for plants [ 6 , 18 , 19 , 20 , 21 , 22 ]. This was demonstrated for a number of model plants: Pisum sativum L. [ 23 , 24 , 25 ], Medicago truncatula Gaertn. [ 26 , 27 ], M. lupulina L. [ 17 , 28 ], Vicia faba L. [ 23 ], Lotus japonicus L. [ 29 ], Phaseolus vulgaris L. [ 30 ], Zea mays L. [ 31 ], Solanum lycopersicum L. [ 32 ], Petunia×hybrida hort. ex Vilm. [ 33 ], etc. At the same time, the mycosymbiont, being an obligate symbiotroph, receives photosynthates from the host plant [ 34 ] and is known to synthesize a number of specific metabolites [ 35 , 36 , 37 ]. Nevertheless, AM fungi species specificity has not yet been truly confirmed. In more than half of studies, Rhizophagus irregularis strains were used to elucidate the effect of AM fungi on the diversity of primary and secondary metabolites of the host plant [ 16 ]. AM fungi induce a wide range of interactions from mutualistic to parasitic [ 38 ]. The highest symbiotic efficiency is manifested under low phosphorus (P) level in the substrate. The contribution of inorganic P absorption due to AM fungi through hyphae and arbuscules can reach 90–99% of the total uptake of the host plant [ 39 , 40 ]. Under conditions of P deficiency, the plant–fungal interaction triggers a shift in the profile of primary metabolites: a decrease in leucine, isoleucine, phenylalanine, aspartic acid, tryptophan, and tyrosine in the roots of Solanum lycopersicum and an increase in glutamic acid [ 41 ], as well as an increase in the total content of proteins and carbohydrates in the leaves of Anadenanthera colubrina [ 42 ]. Analysis of the mycorrhization effect of R. irregularis (previously ascribed erroneously to Glomus intradices ; [ 43 ]) on M. truncatula ’s metabolism under conditions of P deficiency revealed higher levels of trehalose, asparagines [ 27 ], aspartic acid, glutamic acid [ 6 , 27 ], oleic acid, palmitic acid, palmitvaccenic acid, vaccenic acid [ 27 ], homoserine, isoleucine, ornithine, phenylalanine, serine, and threonine [ 6 ]. On the contrary, P excess coupled with R. irregularis inoculation prompted lower levels of asparagine, fatty acids, and their esters, glutamine, phenylalanine, and alanine but a higher level of xylitol in Triticum durum plants [ 44 ]. Besides AM-induced alterations in the primary metabolism of host plants, a number of studies indicate an increase in the content of a number of secondary metabolites: apocarotenoids, especially blumenols [ 16 , 38 , 45 , 46 , 47 ], mycorradicins [ 45 ], cyclohexenone derivatives [ 27 ], and abscisic acid [ 48 ]; isoflavonoids such as daidzein, malonylononin and ononin [ 27 ]; phenylpropanoids, tomatine [ 15 ], and other secondary metabolites [ 16 ] and species-specific metabolites such as glabridin, withaferin-A, alpha terthienyl [ 49 ], and steviol glycosides [ 50 ]. Revealed metabolic alterations commonly reflect the sum of ongoing metabolic changes specific to a plant’s organs and tissue. These are also prompted by both host plant development and establishment of AM symbiosis. Thus, the detection of time points for the metabolic profile analysis is crucial. Usually, this is calculated as the number of days after inoculation (dai). Most studies related to the plant metabolome were provided once at late stages of AM symbiosis: analysis of plant metabolites at 180 dai in Glycyrrhiza glabra [ 49 ]; 127 dai in Leymus chinensis [ 51 ]; 90 dai in Withania somnifera and Tagetes erecta [ 49 ]; 85 dai in Elymus nutans and E. sibiricus [ 52 ]; 84, 77, and 70 dai in Lotus japonicus [ 53 ]; 70 dai in Senecio jacobaea [ 47 ]; 62 dai in Plantago lanceolata , P. major , Veronica chamaedrys , M. truncatula , and Poa annua [ 6 ]; 60 dai in Vitis vinifera [ 7 ]; 50 dai in Solanum lycopersicum [ 15 ]; ~50 dai in Lactuca sativa [ 54 ]; 49 dai in Eclipta prostrate [ 55 ]; 42 dai in Medicago truncatula [ 56 ]. Only limited investigations were focused on plant metabolomes throughout development. As an example, the analysis of Stevia rebaudiana leaves at 69, 89, and 123 dai [ 50 ]; P. sativum leaves and roots from 7 to 110 dai at six stages of the pea plant development [ 5 , 25 ]; M. lupulina leaves from 14 to 52 dai at seven stages of black medic growth [ 17 ]. Moreover, a literature review indicates that authors do not commonly associate the selected dai with a specific transition of the host plant to a new developmental stage and/or the intensification of AM symbiotic invasion. This makes it difficult to compare the results obtained over different studies. Special attention is merited by the rare data obtained at early stages of AM symbiosis development because this is the period when AM fungus is actively colonizing the host plant’s root system [ 27 , 48 ]. These results can contribute to discovering the mechanisms that appear at a biochemical level over the intense interaction of symbiotic partners. Among such investigations are: Anchusa officinalis stems at 9 dai [ 57 ], M. truncatula leaves at 28 dai [ 40 ], Sorghum caudatum , and S. bicolor roots at 30 dai [ 38 ]. This study aims to uncover the metabolic response of M. lupulina roots in mycorrhization with R. irregularis. For research purposes, a highly responsive M. lupulina line, MlS-1 (up to MGR > 350% depending on growth conditions), was selected see [ 17 , 28 , 58 ]. This line expresses the dwarf phenotype under conditions of low P level in the substrate, a deficiency which is significantly reduced owing to mycorrhization. The AM effect on the metabolic profile of M. lupulina roots was detected with GS-MS at early stages of plant growth (14 and 24 dai) under conditions of P deficiency. This period was characterized by active root mycorrhization. The results obtained clarified the intensity of metabolic response triggered by both partners: the host plant’s early development (stages of the first and second leaf) and AM symbiosis. The assumption is that the root metabolic profile alterations caused by mycorrhization are prioritized over those that result from the host plant growing.",
"discussion": "3. Discussion Phosphorus (P) deprivation is one of the most widespread environmental stressors that plants are faced with over the course of their growth. To elucidate the mechanisms as to how plants adapt to P starvation is of a great importance. It was clearly shown that such mechanisms could be detected at transcriptomic and proteomics levels and could easily be phenotypically distinguished by an increase in root architecture and root/shoot ratio elevation [ 59 , 60 ]. In our study, we used a special M. lupulina line, MlS-1, which demonstrated a dwarf phenotype under a condition of low P level in the substrate. It was of an interest as to how this black medic would behave at early stages of development, namely at first and second leaf. This period is characterized by the appearance of new leaves. This stage, of course, facilitates photosynthesis, but it has not yet been determined how it affects root development under conditions of limited nutrients. Our results indicated that P deprivation, even at early stages of development, facilitated the accumulation of root fresh weight and almost double the increase of root/shoot ratio ( Figure 1 ). These data are in agreement with the literature. Such a reaction, but at later stages, was distinguished for some other plants such as Arabidopsis, maize, Lupinus albus , Stylosanthes , and others [ 61 , 62 , 63 , 64 ]. Further GS-MS analysis revealed that the root metabolic profile of young M. lupulina plants is a changeable parameter. The profile had just over 300 compounds. The largest group is represented by sugars. The diversity of this group of root metabolites was slightly smaller than in the leaves, whose profile we distinguished earlier [ 17 ]. A similar correlation was previously shown for pea plants [ 25 ]. The number of investigations devoted to the metabolic profiles of plant roots at early development stages, including that at P deprivation, is very limited. One of the well-documented reactions is the intensification of organic acid synthesis. Accumulation of organic acids is supported with the increase of expression of genes encoding enzymes of TCA [ 65 ]. The elevation of the synthesis and further secretion of organic acids from roots lead to an activation of insoluble soil phosphate. According to our data, black medic roots increase the concentration of malic acid at the second week, which was a switch to accumulation of lactic acid with plant aging. We detected a slight increase of glyceric acid. This is known as an important precursor for several phosphorylated compounds such as 2-phosphoglyceric acid, 3-phosphoglyceric acid, and 1,3-bisphosphoglyceric acid. P limitation might result in the accumulation of this acid, as well as AMP. At 14 DAS, roots synthesize threonine, glycine, and proline. All three are known as metabolites that have a significant role in defense against such abiotic stressors as osmoprotectants. Another distinguished reaction under P limitation is the accumulation of sugars. It had earlier been shown that P deficiency can lead to a serious violation of carbohydrate metabolism [ 42 , 66 ]. In black medic roots, we detected the elevation of both fructose and glucose in the roots of the youngest plants. At 24 DAS, a decrement in monosaccharides in roots of non-mycorrhizal black medic plants might be the result of intensive exudation of primary metabolites, including sugars, into the rhizosphere. It possibly includes different mechanisms, such as passive losses and active exudation. Moreover, besides its nutritive role, glucose has important signaling functions [ 67 ]. Previously, it has been shown that sugars in young plants perform a regulatory function, intensifying root elongation [ 68 ]. Taken together, performed analysis clearly indicates metabolic adaptation of M. lapulina roots to P starvation. This adjustment includes different groups of metabolites: carbonic and amino acids, as well as sugars. This reprogramming of plant metabolism coincides with a limitation in the pool of phosphorylated compounds. The most important is that all these effects were detected at the second week of black medic development and varied at a later period with prolongation of P deprivation. Another well-known mechanism for plants to tolerate P deficiency is to develop arbuscular mycorrhiza: effective symbioses between land plants and fungi. The model object of this study is the highly responsive M. lupulina MlS-1 line in which P starvation triggers dwarfism. The AM symbiotic interaction prevents this phenotype from appearing [ 28 , 69 ]. The metabolic background of such reprogramming is not truly understood. It is well documented that AM fungi might consume up to 20% of host plant photosynthates but simultaneously enhance plant growth [ 1 ]. Thus, AM implies an intensive exchange of metabolites between symbionts. Of particular interest is discovering such a swap at the earliest stages of host plant development, when metabolites and especially carbohydrates are so necessary for the development of the plant itself. Thereby, we provided a GS-MS analysis of the changes in M. lupulina root metabolome after inoculation with the Rhizophagus irregularis at 14 and 24 DAS. We determined that, already at the earliest investigated period in the roots of young plants, active formation of all symbiotic structures is observed: the formation of appressories on the surface of the roots, as well as arbuscules, vesicles, and intraradical mycelium ( Figure S1 ). At the next stage (24 DAS), the development of these structures was only intensified. In conditions of low P content in the substrate, the functioning of AM led to an acceleration of the development of the aboveground parts of the plant: an increase in the fresh and dry mass of shoots, as well as the number of leaves. This latter is well combined with the previously revealed increase in the level of cytokinins in the shoots of mycorrhized plants, which indicates the stimulating effect of AM on shoot development, mainly through the development of leaves, an organ that provides an increase in photosynthate levels [ 69 ]. The appearance of new leaves and an increase in photosynthesis will prompt the enrichment of the metabolic profile of black medic roots. Moreover, it should also be taken into account that the metabolome of mycorrhized roots reflects the metabolic rearrangements of both symbiotic partners. This distinguishes the roots of mycorrhized plants from the leaves, where the age and the development stage have a greater effect on the metabolome than mycorrhization [ 17 ]. It should be noted that the accumulation of sugars differs significantly in the control and mycorrhized plants. A mycorrhiza-dependent shift in the spectrum of sugars most likely depends also on the intensification of its transport from the leaf and because of its further metabolization by both symbionts in the roots. The intensive alteration at two tested dates (14th and 24th DAS) is illustrated in Figure 3 and Figure 4 . This supposition coincides with AM-induced accumulation of “complex sugars”, which are metabolites with sugar residues but distinct from monosaccharides. Such an accumulation of different forms of sugars is a quite common case in many host plants over mycorrhization ([ 16 ] and citations therein). The importance of glucose was shown for arbuscule and intraradical fungal development [ 70 ] and was supported by the intensification of fungal hexose transporter activity. In addition, among others, we detected trehalose, which is known as a typical fungal metabolite. Accumulation of this disaccharide in AM roots indicated vigorous carbohydrate metabolism of the mycosymbiont [ 71 ]. The result supported the idea that sugars from plant shoots are a source of sugar synthesis in AM fungus. One very interesting fact caught our attention: a decrease in the level of intermediates of the Krebs cycle (citrate, succinate, malate, and fumarate) at the early stages of mycorrhization. A similar decrease in the content of organic acids was demonstrated earlier for dicotyledonous plants but in leaves [ 6 , 53 ]. Organic acids of the TCA cycle such as aconitic acid and fumaric acid reduction in mycorrhizal roots of M. truncatula were suggested to be implied with mycorrhiza-induced stimulation of the mitochondrial and plastidial metabolisms [ 27 ]. Along with this, the reason for such changes may be related to the inhibition of the early stages of the Krebs cycle, as well as increased transamination reactions, leading to rapid depletion of the ketoacid pool. Unlike organic acids, the level of a number of amino acids increased during mycorrhization. These include leucine, glycine, histidine, tyrosine, tryptophan, and lysine ( Figure 3 ). Accumulation of a number of amino acids was noted earlier [ 27 , 72 ]. The role of amino acids in the development of the host plant root system is indisputable, especially at such an early stage. At the same time, one more interesting aspect can be considered: the active synthesis of proteins by the mycosymbiont. These secreted effector proteins are supposed to regulate processes in the host plant and are hypothesized to be factors that control symbiotic efficiency and/or host range [ 73 , 74 , 75 ]. Another important group of primary metabolisms is lipophilic compounds. AM fungi is apparently unable to synthesize a sufficient amount of 16:0 FAs (fatty acids) but has enzymes for further elongation of 16:0 FA to a higher chain length and for FA desaturation [ 35 , 76 ]. Moreover, the application of a visualization approach demonstrated that lipid-producing plastids increase in number and, together with the endoplasmic reticulum, change their position and gather in the neighborhood of arbuscules [ 36 ]. M. truncatula mutants in AM-specific paralogs of two lipid biosynthesis genes were used to demonstrate the importance of plant lipid biosynthesis for arbuscule development [ 77 ]. Unfortunately, our analysis did not reveal an increase in the level of 16:0 and 16:1 FAs in the roots of 14 DAS mycorrhizal plants, which are marker lipids of arbuscular mycosymbionts. However, there was a significant increase in the content of seven different sterols. However, our study did not detect an accumulation of ergosterol, which is suggested as a key factor in arbuscular mycorrhizal fungi growth [ 37 ]. Our data coincide with some of the literature which indicates that representatives of the phylum Glomeromycota that form AM contain sterols other than ergosterol [ 78 ]. Further development of M. lupulina plants led to a significant change in the spectrum of metabolites that reflected the increased role of mycorrhization. The accumulation of amino acids in M. lupulina roots increases at 24 DAS, which indicates an upregulation in nitrogen metabolism. Note that accumulation of monosaccharides and the general alteration in the spectrum of carbohydrates was caused by the mycorrhization. It is no wonder that formation of AM stimulated the accumulation of phosphates in roots both at 14 and 24 DAS. Such an effect was described earlier for leaves of M. lupulina [ 17 ] and other species, for example, Sorghum caudatum , S. bicolor [ 38 ], Kennedia coccinea , K. stirlingii , K. carinata , K. prostrata [ 79 ], and others. The analysis of the data obtained at different stages of seedling development using the SUS plot ( Figure 6 ) showed the same signs of loadings for most of the metabolites, which confirms the role of mycorrhization in determining root metabolism. At the final step, we compared the changes, initiated by mycorrhization, in roots and leaves of the M. lupulina MlS-1 line at the early stages of its development (a detailed analysis of the metabolic profiles of the leaves was carried out earlier by [ 17 ]). Organ specificity was the strongest factor compared to the development stage and even mycorrhization ( Figure 7 A). We also note a greater variance in the metabolic profiles of leaves in comparison to roots. The reasons of such a difference could be both internal and external. The transformation of energy during photosynthesis determines a wide variety of synthetic processes, which defines a wider range of metabolites and more complicated regulatory responses. In addition, less diversity in the root may be the result of a more constant environment in the soil compared to the air environment of the leaf. Similar conclusions were drawn during an analysis of the metabolomics profiles of P. sativum roots and leaves [ 5 , 25 ]. It is shown that the most striking distinguishing feature was the accumulation of amino acids and a number of nitrogen-containing compounds in M. lupulina roots ( Figure 7 B). This was less pronounced in the leaves but intensified with mycorrhization development. While the effect of mycorrhization on the profiles of black medic MIS-1 leaves and roots was quite similar ( Figure 7 C), AM effects are not always integral and so may represent a high degree of responsiveness on the part of the host plant."
} | 5,702 |
36630500 | PMC9833677 | pmc | 6,389 | {
"abstract": "Evolution prediction is a long-standing goal in evolutionary biology, with potential impacts on strategic pathogen control, genome engineering, and synthetic biology. While laboratory evolution studies have shown the predictability of short-term and sequence-level evolution, that of long-term and system-level evolution has not been systematically examined. Here, we show that the gene content evolution of metabolic systems is generally predictable by applying ancestral gene content reconstruction and machine learning techniques to ~3000 bacterial genomes. Our framework, Evodictor, successfully predicted gene gain and loss evolution at the branches of the reference phylogenetic tree, suggesting that evolutionary pressures and constraints on metabolic systems are universally shared. Investigation of pathway architectures and meta-analysis of metagenomic datasets confirmed that these evolutionary patterns have physiological and ecological bases as functional dependencies among metabolic reactions and bacterial habitat changes. Last, pan-genomic analysis of intraspecies gene content variations proved that even “ongoing” evolution in extant bacterial species is predictable in our framework.",
"introduction": "INTRODUCTION Uncovering rules and predicting the future of life are foundational objectives in evolutionary biology and many related fields. Naturally, genomic changes, such as substitutions, insertions/deletions, and horizontal gene transfers (HGTs), occur stochastically. While these changes can randomly dominate populations by genetic drift ( 1 ), evolutionary pressures can also preferentially select specific changes constrained or biased by developmental ( 2 ), physiological ( 3 ), ecological ( 4 ), and phylogenetic effects ( 5 ). If these evolutionary pressures and constraints are universal and shared across different lineages, then similar evolutionary patterns will be repetitively observed and can be used to predict future evolution. It was also envisioned that uncovering these patterns and predicting evolution would empower us to design effective strategies in bioengineering and synthetic biology ( 6 ). The predictability of evolution has been extensively studied for short-term and sequence-level evolution, that is, at the microevolution scale. Laboratory evolution of Escherichia coli and Saccharomyces cerevisiae traced by DNA sequencing and targeted genetic modification has shown that many sequence-level mutations can independently and repetitively accumulate ( 7 , 8 ) and are contingent upon previous genetic changes ( 9 – 11 ). These evolutionary patterns reflect the fitness landscapes shaped by epistatic interactions, thus enabling the prediction and control of short-term evolution ( 9 , 12 , 13 ). Predictive modeling of antibiotic resistance evolution has been applied in selecting drugs to suppress the emergence of resistance in pathogens ( 14 , 15 ). In addition, cancer cell evolution has predictable, sequential patterns of mutations, which will help the diagnosis of cancer stages and the selection of effective treatments ( 16 ). Furthermore, a recent study showed the predictability of viral sequence mutations to evade the immune system, providing insights into the design of antibody drugs that avoid tolerance evolution ( 17 ). On the other hand, few studies have investigated the predictability of more marked, long-term, and system-level evolution at the macroevolutionary scale, such as metabolic system evolution by tens to hundreds of gene gains and losses. While limited exceptions include findings of specific HGT order patterns, such as those in the Rubisco subunit genes ( 18 ) and prediction of losable genes on streamlining genomes ( 19 ), it is still impossible to know which species will likely gain and lose a given gene in the future. Such a lack of studies is principally because system-level evolution is seldom directly not observable in laboratories. However, abundant genome data, large-scale phylogenetic trees, ancestral gene content reconstruction methods, and machine learning techniques are now becoming available to systematically reconstruct the gene content (i.e., gene sets on genomes) of ancestral species, enumerate gene gain/loss events across diverse phylogenetic clades, and reveal universal patterns of gene gain/loss events ( 20 – 23 ). Despite growing datasets and technologies, no study has comprehensively examined the predictability of biological system evolution. This study developed a versatile analysis framework that enables us to learn the patterns and rules of gene content evolution using machine learning approaches. Analysis of the reconstructed gene content evolution based on 2894 extant bacterial genomes in diverse phyla demonstrated that the gene gain/loss events of various metabolic genes were substantially predictable. These evolutionary patterns are confirmed to have solid physiological and ecological bases and can be used to predict ongoing evolution using pan-genomic datasets. We envision that our approach will enable forecasting of the future evolution of genomes and provide insights into the design principles of biological systems.",
"discussion": "DISCUSSION This study developed a computational framework, Evodictor, for learning the general patterns behind gene gain/loss events at the macroevolutionary scale and enabling gene-by-gene prediction of future gene gain/loss events based on the genomes of extant species. Cross-validation and pan-genome analysis showed that gene gain/loss events are universally predictable for various metabolic OGs using our framework. Our framework enables the prediction of gene gains and losses, discussed separately in previous studies ( 18 , 19 ). Because of this characteristic, gene losses were revealed to be less predictable than gene gains ( Figs. 2A , 3B , and 6C and figs. S2A, S3, B to D, and S4), consistent with previous reports on gene losses under relaxed evolutionary pressures associated with environmental changes ( 42 ). Further investigation of metabolic pathway architectures and meta-analysis of metagenomic datasets demonstrated that our method detects evolutionary patterns whose universality has solid physiological and ecological bases. From a physiological aspect, the downstream-to-upstream evolutionary order in metabolic pathway evolution is consistent with the retrograde model originally proposed to explain pathway evolution by gene duplication, where the fitness effect of gene gain is determined by functional dependencies ( 43 ). From an ecological aspect, habitat transition patterns from soil to plant-associated environments were found to correlate with gene gain orders. This also suggests that Evodictor performance may be improved by inferring past environments based on abundant metagenomic datasets and explicitly using that information for the evolutionary prediction. There are several rooms to improve and extend the Evodictor framework. It is envisioned to train predictive models by considering uncertainties in reconstructing phylogenetic trees and ancestral gene content. Consideration of time (e.g., branch length) information would also be promising, although dating prokaryotic phylogenetic trees is intrinsically difficult because of few fossil records ( 44 ). Furthermore, while the present method uses a gene content vector only of the parental node for prediction (i.e., Markov property assumption), those of grand parental nodes would provide information about how the environment is changing at the parent node and what genes will be likely gained/lost next. In addition, instead of the presence/absence data of OGs, we may embed protein sequence data into a vector by protein language models to consider inter-/intraortholog sequence diversities ( 45 – 47 ). Last, the iterative application of Evodictor may let us simulate long-term genome evolution and conduct orbit analysis in a genomic state space. Whether the dynamics of bacterial genome evolution will be caught in some attractors and oscillate, converge to equilibrium, or continue to diverge would be of special interest in biophysics of genome evolution. Evodictor is a versatile analysis framework that requires only a lineage tree (e.g., phylogenetic tree) and a trait table (e.g., ortholog table) as inputs. Thus, in addition to metabolic system evolution, it is envisioned that Evodictor will predict pathogenic species that will likely gain antibiotic resistance genes via HGTs to mitigate the potential risks of resistance disseminations ( 3 , 48 , 49 ). Predicting the gain/loss potential of various genes would be beneficial for estimating the feasibility of knockin/knockout experiments and stability of edited genomes in bioengineering and synthetic biology ( 6 , 50 ). Furthermore, Evodictor may also be useful in estimating the future influence of synthetic genomes in the context of ethical, legal, and social issues. In addition to genome evolution, Evodictor can be applied to evolution of transcriptomes by organs ( 51 ) and of phenotypic traits such as butterfly wing patterns ( 52 ) and morphology of plants and Aves ( 53 , 54 ). From a broader perspective, single-cell transcriptomics will also be empowered if a cell lineage tree and a cell-gene matrix of expression levels (as a trait table) are given to Evodictor to decipher the universal rules behind cell differentiation by taking advantage of the rapidly progressing cell lineage tracing technologies ( 55 , 56 ). Evodictor, as a framework for modeling and predicting diversification of complex biological systems, would become more beneficial as we obtain larger lineage trees and trait databases, which are growing with various omics technologies."
} | 2,425 |
27903820 | PMC5264505 | pmc | 6,391 | {
"abstract": "Biological systems exhibit complex behaviours that emerge at many different levels of organization. These span the regulation of gene expression within single cells to the use of quorum sensing to co-ordinate the action of entire bacterial colonies. Synthetic biology aims to make the engineering of biology easier, offering an opportunity to control natural systems and develop new synthetic systems with useful prescribed behaviours. However, in many cases, it is not understood how individual cells should be programmed to ensure the emergence of a required collective behaviour. Agent-based modelling aims to tackle this problem, offering a framework in which to simulate such systems and explore cellular design rules. In this article, I review the use of agent-based models in synthetic biology, outline the available computational tools, and provide details on recently engineered biological systems that are amenable to this approach. I further highlight the challenges facing this methodology and some of the potential future directions.",
"introduction": "Introduction Synthetic biology aims to apply engineering principles to biological systems to enable the more rational design of novel functionalities. This has resulted in the engineering of cells able to perform complex computations [ 1 , 2 ], act as biosensors of disease [ 3 ] and, building on the success of metabolic engineering, sustainably produce valuable drugs and chemicals [ 4 ]. In most cases, it is impractical to construct and test every possible design of a synthetic biological system. To address this issue, mathematical modelling and computational simulations form an essential part of the design process. They enable large-scale in silico investigations into the robustness of specific designs, help to identify key parameters, and can filter out designs that are likely to be non-functional [ 5 ]. This reduces the costly and time-consuming laboratory work required to develop a functional system. Owing to our ability to observe and measure many diverse aspects of individual cells, much of the modelling in synthetic biology to date has focused on intracellular dynamics (i.e. capturing changes in the rates of transcription and translation, and variations in the concentrations of chemicals, mRNAs and proteins over time). However, there is growing realization that the robustness of natural biological systems is often derived from collective population-level features that extend beyond individual cells. Colonies of bacteria are known to communicate and co-ordinate their growth during infection [ 6 , 7 ], and exploit collective behaviours to enable the emergence of antibiotic resistance [ 8 ]. To unravel these mechanisms and make use of them in our own synthetic systems, models must extend beyond intracellular dynamics and encompass the interactions between cells and their shared environment. Agent-based modelling (also referred to as individual-based modelling) attempts to bridge this gap by considering large numbers of autonomous ‘agents’ that can interact within a virtual environment [ 9 ] ( Figure 1 A). Agents can represent any entity of interest, such as a molecule, cell or multicellular organism, and each independently follows a prescribed set of rules. In a biological setting, these rules are often encoded as genetic circuits that drive cellular responses to particular stimuli. By simulating the behaviour of these virtual populations in realistic environments, it is possible to gain an understanding of how low-level cellular rules lead to the emergence of collective population-level behaviours [ 9 ] ( Figure 1 B). Figure 1 Principles of agent-based modelling ( A ) An agent-based simulation consists of a virtual environment where large numbers of autonomous agents can interact. A model of a bacterial colony is shown with agents representing cells. Each cell contains a synthetic genetic circuit that controls its behaviour. In this case, the genetic circuit takes two chemicals as inputs (Q1 and aTc) and produces a single chemical output (Q2) if both inputs are absent (a NOR logic operation). A range of common cellular inputs and outputs are shown. To ensure that simulations faithfully reproduce the biological system, key physical processes encountered or utilized by the agents must be implemented within the virtual environment. Those relevant to bacteria are shown. ( B ) Interactions between agents implementing specific rules and the shared environment can lead to the emergence of collective behaviours. These include dynamic co-ordination (e.g. synchronization of gene expression; see Figure 2 A) and population-level encodings of continuous inputs (e.g. cells are either in an ‘ON’ or ‘OFF’ state and the fraction of the population in an ‘ON’ state corresponds to the continuous concentration of the input, similar to the bimodality of the lactose utilization network in E. coli [ 10 ]). A major benefit of using agents to model the discrete elements of a system is the ability to capture minor differences that exist or can arise between them. For example, intracellular noise causes the expression of the same protein to vary across a population, and for cells that are motile, differences in the history of their movement can lead to subtle changes in the way they respond to new stimuli. Other modelling approaches often average out these differences, assuming cells behave in a uniform way across the entire system. Although such simplifications are sometimes appropriate, many processes in biology actively make use of cellular differences to achieve novel functions. One of the most famous is the bimodality of the lactose utilization network in Escherichia coli . In this system, mixed populations can emerge with a cell being either fully active or inactive, with the fraction of both controlled by the lactose concentration [ 10 ] ( Figure 1 B). This is useful because a diverse population ensures that at least some cells are poised to exploit potential changes in environmental conditions, improving the fitness of the population as a whole. Averaging the state of cells across the population would miss this vital feature. Another aspect of agent-based modelling that is difficult to reproduce using other methodologies is the multiple ways that interactions between agents can occur. The most basic type of interaction is physical, where two agents meet. However, because not all interactions may lead to a behavioural response, the rules controlling how an agent reacts are often probabilistic. This is akin to the chance that you might fall ill after meeting a colleague that is feeling unwell. In addition to direct encounters, the environment itself can also act as a means for indirect interactions [ 11 ]. In Nature, pheromones are often deposited into the environment to be sensed later by other individuals. This allows the environment itself to become an indirect channel for communication. In both cases, modelling the range of interactions that take place is a challenge for many methods, but is easily handled by agent-based models because these events are explicitly captured. In this review, I discuss the general principles of agent-based modelling and show how it can support the rational engineering of collective behaviours in synthetic biology. Although agent-based modelling has begun to see applications in diverse areas of this field (e.g. in the design of co-operating nanoparticles for medicine [ 12 ]), in this review, I focus exclusively on cellular systems with agents representing individual cells. Recent synthetic biology examples are used to illustrate how population-level features can arise from simple cellular programs, and a full list of currently available computational tools will be provided. This review aims to give a general introduction to the field of agent-based modelling, some of its applications to synthetic biology and outline the challenges and future directions of this methodology."
} | 1,992 |
32093403 | PMC7076665 | pmc | 6,392 | {
"abstract": "Nitrogen is one of the essential plant nutrients and a major factor limiting crop productivity. To meet the requirements of sustainable agriculture, there is a need to maximize biological nitrogen fixation in different crop species. Legumes are able to establish root nodule symbiosis (RNS) with nitrogen-fixing soil bacteria which are collectively called rhizobia. This mutualistic association is highly specific, and each rhizobia species/strain interacts with only a specific group of legumes, and vice versa. Nodulation involves multiple phases of interactions ranging from initial bacterial attachment and infection establishment to late nodule development, characterized by a complex molecular signalling between plants and rhizobia. Characteristically, legumes like groundnut display a bacterial invasion strategy popularly known as “crack-entry’’ mechanism, which is reported approximately in 25% of all legumes. This article accommodates critical discussions on the bacterial infection mode, dynamics of nodulation, components of symbiotic signalling pathway, and also the effects of abiotic stresses and phytohormone homeostasis related to the root nodule symbiosis of groundnut and Bradyrhizobium . These parameters can help to understand how groundnut RNS is programmed to recognize and establish symbiotic relationships with rhizobia, adjusting gene expression in response to various regulations. This review further attempts to emphasize the current understanding of advancements regarding RNS research in the groundnut and speculates on prospective improvement possibilities in addition to ways for expanding it to other crops towards achieving sustainable agriculture and overcoming environmental challenges.",
"introduction": "1. Introduction Leguminosae, after Asteraceae and Orchidaceae, the third-largest family of angiosperms and includes many agronomically and economically important crops [ 1 ]. The legume family consists of 770 genera and 19,500 species [ 2 ]. Papilionoideae, the largest subfamily within the Fabaceae, is divided into distinct phylogenetic clades, namely genistoids, dalbergioids, indigoferoids, milletioids, robinioids, and inverted repeat-lacking clade (IRLC) [ 3 ]. Popularly studied model legumes within these clades include Aeschynomene and Arachis (groundnut) within the dalbergioids, Glycine (soybean) and Phaseolus (common bean) in milletioids, Lotus amongst the robinioids and Medicago (alfa-alfa), and Pisum (pea) in the IRLC clade. Legumes are distinct from non-legume species in terms of the nitrogen acquisition by developing root nodules that fix atmospheric nitrogen through symbiotic N 2 -fixing rhizobia. This trait is both ecologically and agriculturally important. Legume rhizobia symbiosis initiated about 58 million years ago [ 4 ]. Interestingly, humans would require an extra 288 billion kilograms of fuel to produce the same amount of nitrogen that is fixed by legumes each year through the process of biological nitrogen fixation (BNF) [ 5 ]. BNF assimilates atmospheric nitrogen in the form of organic compounds as a sustainable source of nitrogen in an agricultural context. Organically fixed nitrogen can be utilized directly by the plant and, advantageously, it is less susceptible to denitrification, volatilization, and leaching [ 6 , 7 ]. Root-nodule symbiosis (RNS) allows legume plants to house diazotrophic bacteria in an intracellular manner [ 8 ]. RNS establishment involves rhizobial invasion through root epidermis, and nodule organogenesis across the root cortical cells. The most common strategy of invasion is through root hair curling and infection thread (a cellulosic tube that allows rhizobial cells to migrate and infect root cells) formation, where the nodule primordia are induced from a distance [ 9 ]. Infection Thread (IT) formation is common among temperate legumes such as Vicia sp., Trifolium sp. and Pisum sp. Model legumes Medicago truncatula and Lotus japonicus also display IT-mediated rhizobial invasion [ 10 , 11 ]. However, there is an alternative mode of rhizobial invasion, crack-entry, where rhizobia enter via an intercellular route at the lateral root base. Approximately 25% of legumes are adapted to ‘crack-entry’, which is a characteristic feature for some subtropical legumes belonging to dalbergoid/genistoid clades such as Arachis sp., Stylosanthes sp. and Aeschynomene sp. [ 12 ]. In these crop species, rhizobia directly access the cortical cells to develop nodule primordia, and the infected cells divide repeatedly to form a mature aeschynomenoid nodule in which the core of the infected zone remains separated from uninfected cells [ 13 ]. Groundnut, an important crop species belonging to the Fabaceae (Leguminosae) family, subfamily Papilionoideae, is globally one of the major oilseed crops. Importantly, the N 2 fixation efficiency of groundnut is relatively low as compared to other legume species, thus leaving scope for improvement [ 14 ]. Contrary to the model legume plants, the molecular mechanism of root nodule symbiosis is less studied in groundnut. The latest evidences came from recent studies which involved molecular-omics approaches to identify key factors controlling the inception and progress of symbiosis through ‘crack-entry’ in selected legumes including groundnut [ 15 , 16 ]. These studies provided evidence of the interactions of the Nod factor (specific signal molecules secreted by rhizobia) with several signalling and hormonal biosynthesis-related genes during rhizobial infection. However, information is still limited on the ‘crack-entry’ mechanism for legumes belonging to the dalbergioid/genistoid clade, which are basal in their divergence within the Papilionoideae. Therefore, understanding the evolution of nitrogen-fixation by root nodules and the identification of key genes involved in this phenomenon in different legume species is important for sustainable crop production. Within legumes, groundnut can be used as a model crop plant to understand the crack entry mechanism. Further, combining the available knowledge and the molecular explanations from legumes species such as groundnut, M. truncatula and L. japonicus will help in developing an in-depth understanding of the molecular networks involved in RNS. In this article, we reviewed the advances in research pertaining to symbiotic interaction in a complex ecological system and the progress made in the molecular aspects of RNS, with special reference to groundnut."
} | 1,626 |
34180604 | PMC8236291 | pmc | 6,393 | {
"abstract": "Abstract Natural isolates of the soil‐dwelling bacterium Bacillus \n subtilis form robust biofilms under laboratory conditions and colonize plant roots. B. subtilis biofilm gene expression displays phenotypic heterogeneity that is influenced by a family of Rap‐Phr regulatory systems. Most Rap‐Phr systems in B. subtilis have been studied independently, in different genetic backgrounds and under distinct conditions, hampering true comparison of the Rap‐Phr systems’ impact on bacterial cell differentiation. Here, we investigated each of the 12 Rap‐Phr systems of B.subtilis NCIB 3610 for their effect on biofilm formation. By studying single ∆ rap ‐ phr mutants, we show that despite redundancy between the cell–cell communication systems, deletion of each of the 12 Rap‐Phr systems influences matrix gene expression. These Rap‐Phr systems therefore enable fine‐tuning of the timing and level of matrix production in response to specific conditions. Furthermore, some of the ∆ rap ‐ phr mutants demonstrated altered biofilm formation in vitro and colonization of Arabidopsis \n thaliana roots, but not necessarily similarly in both processes, indicating that the pathways regulating matrix gene expression and other factors important for biofilm formation may be differently regulated under these distinct conditions.",
"introduction": "1 INTRODUCTION In nature, biofilms are the predominant lifestyle of bacteria and are known as surface‐associated microbial communities embedded in a self‐produced matrix (Flemming & Wingender, 2010 ; Hall‐Stoodley et al., 2004 ; López et al., 2010 ). Biofilms have been widely studied since they represent a fascinating example of microbial development in response to environmental cues (O'Toole et al., 2000 ). Furthermore, studying biofilms is of special interest due to their detrimental impact in clinical and industrial settings (Di Pippo et al., 2018 ; Stewart, 2002 ) as well as their promising potential within the biotechnology industry (Blake et al., 2021 ; Singh et al., 2006 ). Regarding the latter, the gram‐positive bacterium Bacillus \n subtilis has in the last two decades gained interest due to its promising potential as a biocontrol agent within agriculture (Kiesewalter et al., 2021 ; Ongena & Jacques, 2008 ). In its natural habitat, the soil‐dwelling bacterium colonizes plants by forming a biofilm on the root (Bais et al., 2004 ; Beauregard et al., 2013 ; Chen et al., 2012 ). After successfully colonizing the root, B.subtilis exerts its plant‐beneficial properties, including directly promoting plant growth and protecting the plant against diseases (Blake et al., 2021 ). Furthermore, B. subtilis forms spores that are highly resistant to extreme environments (Piggot & Hilbert, 2004 ) facilitating easy formulation (Ongena & Jacques, 2008 ). \n Bacillus \n subtilis can easily be isolated from the rhizosphere of plants (Fall et al., 2004 ), and a study performed by Chen et al. ( 2013 ) showed that the majority of natural strains isolated from the rhizosphere formed architecturally complex biofilms under laboratory conditions, indicating that biofilm formation is an important trait for B. subtilis to thrive in its natural habitat. In the laboratory, B. subtilis has long been studied using different kinds of biofilm models including colonies at the air‐agar interface and floating biofilms formed at the air–liquid interface, termed pellicles (Arnaouteli et al., 2021 ). A prevalent feature of B. subtilis biofilms is that they display complex phenotypic heterogeneity, where genetically identical cells differentiate into distinct cell types in response to external cues (López & Kolter, 2010 ; Lopez et al., 2009 ). The variation in environmental conditions throughout the biofilm (Costerton et al., 1994 ) thereby leads to a heterogeneous population with different cell types performing distinct tasks and occupying different micro‐niches. The extracellular signals triggering cell differentiation include quorum‐sensing molecules, natural products, and nutrient availability that activate a set of sensor kinases (Arnaouteli et al., 2021 ; Mhatre et al., 2014 ). Once activated, the sensor kinases phosphorylate their respective master transcriptional regulators, Spo0A, DegU, and ComA, each of which activates different sets of genes (López & Kolter, 2010 ). The Spo0A pathway governs differentiation into matrix‐producing cells and sporulating cells. In response to external cues, one or more of five histidine kinases, KinA‐E, are activated which results in phosphorylation of Spo0F. Spo0F~P then transfers its phosphoryl group to Spo0B, which in turn transfers the phosphoryl group to and thereby activates Spo0A (Fujita et al., 2005 ; Jiang, Shao, et al., 2000 ). At low Spo0A~P levels, the genes involved in the synthesis of matrix components, exopolysaccharide (EPS) and TasA protein fiber, are expressed (Cairns et al., 2014 ; Fujita et al., 2005 ). These two matrix components are well known to be required for biofilm formation in vitro and on the plant root (Beauregard et al., 2013 ; Branda et al., 2006 ). When high levels of Spo0A~P are reached, genes involves in sporulation are expressed (Fujita et al., 2005 ). The DegU response regulator is phosphorylated by its cognate histidine kinase DegS. Studies have indicated that inhibition of flagellar rotation, as may take place upon contact with a surface, acts as a mechanical trigger to activate the DegS‐DegU two‐component signaling pathway (Cairns et al., 2013 ). At very low levels of DegU~P, genes related to swarming motility are expressed, while elevated levels of DegU~P induce exoprotease production and at the same time represses motility genes (Belas, 2013 ; Verhamme et al., 2007 ). Finally, the pheromone ComX activates the histidine kinase ComP, which phosphorylates ComA, resulting in the expression of genes involved in competence development and surfactin production (Comella & Grossman, 2005 ). This regulatory network governing cell differentiation in B. subtilis is further regulated by a family of response regulator aspartyl‐phosphate (Rap) phosphatases and their associated phosphatase regulator (Phr) peptides (Perego, 2013 ). In the B. subtilis group, 80 distinct putative rap ‐ phr alleles have been identified with a strain having on average 11 rap genes (Even‐Tov et al., 2016 ). The abundance of Rap and Phr peptides is transcriptionally controlled in response to different cellular signals (Auchtung et al., 2005 ; Jarmer et al., 2001 ; Jiang, Grau, et al., 2000 ; Lazazzera et al., 1999 ; McQuade et al., 2001 ; Mueller et al., 1992 ; Ogura et al., 2001 ; Perego et al., 1994 ). The genes encoding the Rap‐Phr pairs are found as gene cassettes with the phr gene immediately downstream of the rap gene and the expression of these being transcriptionally coupled, with some phr genes also being transcribed independently of their cognate rap genes from promoters controlled by σ H (McQuade et al., 2001 ; Pottahil & Lazazzera, 2003 ; Reizer et al., 1997 ). Some exceptions to this exist, for example, the rapB gene is not followed by an active peptide encoding gene (Perego et al., 1996 ). Moreover, some Rap proteins are regulated by Phr peptides encoded in other cassettes, for example, RapB and J are both controlled by PhrC (Parashar, Jeffrey, et al., 2013 ). When expressed, the Rap phosphatases exert their effect within the cell by either dephosphorylating Spo0F~P (thus hindering Spo0A phosphorylation) or inhibiting the DNA‐binding activity of ComA or DegU (Perego, 2013 ). In contrast, the product of the phr gene is secreted out of the cell through the Sec‐dependent export pathway and processed into mature five to six amino acid signaling peptides. At high cell density, the Phr peptides reach threshold concentrations at which they are transported back into the cell by the oligopeptide permease (Opp) (Perego, 2013 ; Pottahil & Lazazzera, 2003 ). Once within the cell, the Phr peptides will inhibit their cognate Rap proteins, thereby relieving the inhibition of the master regulators resulting in altered gene expression (Perego, 2013 ; Pottahil & Lazazzera, 2003 ). The Rap‐Phr systems thereby act as cell–cell signaling systems in B.subtilis , allowing the bacteria to respond to environmental changes only at sufficient cell densities. As expected by the diversity and abundance of multiple Rap‐Phr systems regulating the activity of these three master regulators, the Rap phosphatases show high redundancy in their regulatory function: RapA, B, E, H, I, J, and P have been shown to dephosphorylate Spo0F~P, RapC, D, F, H, K, and P regulate ComA, while RapG has been shown to regulate the activity of DegU (Auchtung et al., 2006 ; Ogura & Fujita, 2007 ; Perego, 2013 ; Omer Bendori et al., 2015 ) (Table A1 ). Furthermore, RapI is involved in the regulation of mobile genetic elements, as it activates the propagation of the mobile genetic element that encodes it (Auchtung et al., 2005 ). The regulation of the master regulators by multiple Rap phosphatases allows the integration of diverse signals to control cell differentiation in response to different conditions. However, this overall overview of the Rap‐Phr signaling network in B. subtilis is based on studies where most Rap‐Phr systems have been tested in different genetic backgrounds and under distinct cultivation conditions (Perego, 2013 ). Additionally, previous investigations in B. subtilis have directed their study toward certain targets of Rap‐Phr regulation, with RapA and B being mostly studied for their impact on sporulation (Perego & Hoch, 1996 ), while RapC and F are involved in competence development (Bongiorni et al., 2005 ; Core & Perego, 2003 ). So far, only RapP has been demonstrated to impact biofilm formation (Parashar, Konkol, et al., 2013 ; Omer Bendori et al., 2015 ). We previously studied all 12 Rap‐Phr systems of the undomesticated strain B. subtilis NCIB 3610 in the same genetic background by following the relative abundance of all possible single and double ∆ rap ‐ phr mutants as well as the wild‐type WT (79 strains) in populations subjected to different selective conditions. This study highlighted that the variability in Rap‐Phr systems affected the ability to compete in diverse environments (Gallegos‐Monterrosa et al., 2021 ). In this study, we systematically investigated the contribution of each of the 12 Rap‐Phr systems in B. subtilis 3610 to biofilm formation. We assessed wild type (WT) and the 12 single ∆ rap ‐ phr mutants for matrix gene expression and biofilm formation under different conditions. We found that all 12 mutants showed altered matrix gene expression compared with the WT. Furthermore, we observed that the Rap‐Phr modules affect not only in vitro biofilm formation but also the colonization of plant roots that represents an ecologically relevant environment.",
"discussion": "4 DISCUSSION For decades, the Rap‐Phr regulatory systems which control the activity of the three master regulators governing cell differentiation in B. subtilis have been extensively studied (Perego, 2013 ; Pottahil & Lazazzera, 2003 ). However, since most studies have investigated the Rap‐Phr networks of B. subtilis independently from each other, in different genetic backgrounds, and under distinct conditions, it is difficult to compare the results from these studies. Here, we investigated all 12 Rap‐Phr systems found in B. subtilis 3610 for their effect on matrix gene expression and biofilm development in vitro . In addition, we examined the impact of Rap‐Phr systems in the colonization of plant roots for the first time. Several of the Rap phosphatases have been reported to dephosphorylate Spo0F~P (Perego, 2013 ; Omer Bendori et al., 2015 ) which is expected to influence matrix production, but only RapP has previously been demonstrated to affect matrix gene expression (Parashar, Konkol, et al., 2013 ; Omer Bendori et al., 2015 ). We were, therefore, interested in testing the effect of each of the 12 Rap‐Phr systems on matrix gene expression. Inspired by a previously established method (Kearns et al., 2005 ), expression from the promoter of the tapA ‐ sipW ‐ tasA operon was measured for WT and the 12 ∆ rap ‐ phr mutants in MSgg under shaking conditions. Only ∆ rapA showed a significantly reduced relative ON population compared with the WT. Such reduced relative proportion of ∆ rapA cells in the ON state may be due to more cells committing to activation of the sporulation pathway, and therefore, attenuating induction of matrix genes (Bischofs et al., 2009 ). Although only some of the Rap phosphatases have been reported to dephosphorylate Spo0F~P (Perego, 2013 ; Omer Bendori et al., 2015 ), we observed that all mutants except ∆ rapA showed increased matrix gene expression. This indicates that despite the diversity of targets among Rap‐Phr systems, and the seeming redundancy of several Rap phosphatases regulating the same master regulator (Perego, 2013 ), each of the 12 Rap‐Phr systems has a regulatory role that affects matrix gene expression under the tested conditions. The involvement of 12 Rap‐Phr systems in influencing matrix gene expression suggests that the production of these costly public goods is under complex control, and allows the integration of multiple signals to fine‐tune the timing of matrix production in response to different conditions (Auchtung et al., 2006 ; Dragoš et al., 2018 ). Matrix production is well known to be required for the formation of architecturally complex biofilms under laboratory conditions (Arnaouteli et al., 2021 ; Branda et al., 2006 ). Moreover, matrix production and localized cell death are responsible for the formation of wrinkles during biofilm development (Asally et al., 2012 ; Branda et al., 2006 ; Gallegos‐Monterrosa et al., 2017 ). We, therefore, speculated that the increased matrix gene expression observed for all mutants, except ∆ rapA , would manifest in these 11 mutants forming more wrinkled colonies and more complex, robust pellicles compared with the WT. However, only some of the mutants were affected in biofilm formation. In accordance with increased matrix gene expression, the ∆ rapB , J , and K mutants formed more wrinkled colonies, and ∆ rapI and P formed complex, though very small colonies compared with the WT. Moreover, ∆ rapC formed a larger, smoother, and more transparent colony. Furthermore, ∆ rapA , with the similar mean expression of the tapA ‐ sipW ‐ tasA operon and a smaller relative ON population compared with WT, also formed a more wrinkled colony. Surprisingly, none of the mutants displayed increased pellicle robustness or complexity compared with the WT. In contrast, ∆ rapA , C , I , and P formed thinner and/or non‐homogenous pellicles. Next, we were interested in studying how Rap‐Phr systems affect biofilm formation of B. subtilis in a more ecologically relevant environment, that is, the plant root. Similar to biofilm formation in vitro , biofilm formation on the plant root by B. subtilis depends on matrix gene expression regulated by Spo0A (Beauregard et al., 2013 ; Chen et al., 2013 ). We, therefore, hypothesized that the increased matrix gene expression observed for most mutants would allow more bacterial cells to attach to and colonize the root. However, only ∆ rapD , J , and P showed increased root colonization, while ∆ rapI was reduced in root colonization. The biofilm and root colonization experiments of the ∆ rap ‐ phr mutants thus show that the magnitude of matrix gene expression under shaking conditions does not directly correlate with the ability to develop complex biofilms in vitro (on agar and at the air–liquid interface) or to colonize the plant root (Table A1 ). A lack of positive correlation between matrix gene expression and biofilm formation was observed in a previous study which showed that the magnitude of expression of epsA ‐ O and tasA ‐ sipW ‐ tasA in B. subtilis 168 variants did not directly correlate with the formation of wrinkled biofilms (Gallegos‐Monterrosa et al., 2016 ). These experiments could thus support that biofilm formation in i n vitro and on plant roots is influenced by additional factors than just matrix gene expression. For example, surfactin production, which is regulated by ComA—a target of several Rap phosphatases (Perego, 2013 )—was shown to influence the colony structure of B. subtilis NCIB 3610 on MSgg, though this secondary metabolite was not essential for pellicle formation and root colonization (Thérien et al., 2020 ). However, it has to be noted that matrix gene expression was measured under heavily shaking conditions (220 rpm), while colonies and pellicles were developed under static conditions, and root colonization was assayed under mildly shaking conditions (90 rpm). An alternative explanation for the lack of correlation between matrix gene expression under shaking conditions and biofilm formation in vitro and on the root could be that the effect of the rap ‐ phr deletions on tapA operon expression may vary between these different conditions. Further work is needed to fully explain the discrepancies observed in this study between tapA operon expression and biofilm formation. Interestingly, several studies have reported a correlation between the ability of strains to form robust biofilms in vitro and to colonize the root—both within and among strains (Chen et al., 2013 ; Gallegos‐Monterrosa et al., 2016 ). However, the ability of the ∆ rap ‐ phr mutants to form biofilm in vitro did not necessarily reflect the ability to colonize the root (compare Figures 2 and 3 and the summary in Table A1 ). For example, ∆ rapA and C formed thin and non‐homogenous pellicles but were able to colonize the root to similar levels as the WT. ∆ rapD displayed comparable biofilm in vitro to the WT but was significantly better in root colonization. In addition, ∆ rapJ formed a highly wrinkled colony, but a pellicle similar to the WT, and was increased in root colonization. Finally, both ∆ rapI and P showed reduced colony size and thin and/or non‐homogenous pellicle formation, but while ∆ rapI showed reduced root colonization, ∆ rapP was increased in root colonization compared with the WT. These results indicate that the effect of the rap ‐ phr deletions on biofilm formation varies between in vitro and root conditions. This was similarly shown for a ∆ tagE mutant (deficient in glycosylating wall teichoic acid), which was affected in root colonization but displayed similar biofilm formation on agar and at the air–liquid interface as the WT (Tzipilevich & Benfey, 2021 ). In the study by Gallegos‐Monterrosa et al. ( 2016 ), showing that strains forming complex colonies and robust pellicles also efficiently colonize the root, the B. subtilis 168 stocks displayed genetic variation in distinct loci (e.g., epsC that encodes an enzyme that is directly involved in matrix production), resulting in large differences among the strains in their ability to form biofilm and colonize the root. In contrast, the ∆ rap ‐ phr mutations studied here might only slightly modulate the regulatory pathways of B. subtilis ; therefore, the ability of the mutants to form biofilm and colonize the root is less altered compared with WT. Nonetheless, the same study also demonstrated that biofilm development is influenced by medium composition (Gallegos‐Monterrosa et al., 2016 ). Besides static vs mild agitation and a temperature difference (30 vs. 24°C), the media used for testing colony and pellicle formation, and for testing root colonization also slightly differ. First, the MSgg medium used for colony and pellicle formation contains a 10 times higher concentration of glycerol (0.5 %) compared with the MSNg medium used for plant root colonization (0.05%). During plant root colonization, the bacteria thus depend on plant polysaccharides and root exudates as carbon sources. In addition, in vitro biofilm development depends on the availability of iron and manganese (Kolodkin‐Gal et al., 2013 ; Mhatre et al., 2016 ; Shemesh & Chai, 2013 ), while during plant root colonization in MSNg, biofilm formation is induced by plant polysaccharides and root exudates (Beauregard et al., 2013 ; Chen et al., 2013 ). The presence of plant polysaccharides may, therefore, allow ∆ rap ‐ phr mutants that form weak pellicle biofilms in vitro to efficiently colonize the root ( ∆rapA , C and P , Figures 2 and 3 ). Thereby, the pathways regulating matrix gene expression and other factors important for biofilm formation may be differently regulated under distinct conditions. Finally, the disparate results obtained in this study may be understood in light of the full set of 12 Rap‐Phr systems with redundant functions: the influence of a single rap ‐ phr deletion might be masked by the function of another redundant Rap‐Phr system. Furthermore, if such potential redundancy varies between the different conditions employed in this study (e.g., if the rap ‐ phr genes are differentially expressed under the distinct conditions tested), this could (partly) explain the observed discrepancy, for example, between biofilm formation in vitro and on the root. To conclude, we here show that all 12 Rap‐Phr systems have an impact on matrix gene expression in liquid culture. Thereby, the diversity in Rap‐Phr systems in B. subtilis 3610 could function to integrate multiple signals to fine‐tune the timing and level of matrix gene expression in response to new ecological niches, such as those it will encounter in soil. Furthermore, we show that the ability to form biofilm in vitro not necessarily reflects the ability to colonize the root under the tested conditions. These findings thus support that the pathways involved in matrix gene expression and other components important for biofilm establishment could be differently influenced under distinct conditions."
} | 5,528 |
33396244 | PMC7824734 | pmc | 6,394 | {
"abstract": "Growing evidence showed that efficient acquisition and use of nutrients by crops is controlled by root-associated microbiomes. Efficient management of this system is essential to improving crop yield, while reducing the environmental footprint of crop production. Both endophytic and rhizospheric microorganisms can directly promote crop growth, increasing crop yield per unit of soil nutrients. A variety of plant symbionts, most notably the arbuscular mycorrhizal fungi (AMF), nitrogen-fixing bacteria, and phosphate-potassium-solubilizing microorganisms entered the era of large-scale applications in agriculture, horticulture, and forestry. The purpose of this study is to compile data to give a complete and comprehensive assessment and an update of mycorrhizal-based inoculant uses in agriculture in the past, present, and future. Based on available data, 68 mycorrhizal products from 28 manufacturers across Europe, America, and Asia were examined on varying properties such as physical forms, arbuscular mycorrhizal fungal composition, number of active ingredients, claims of purpose served, mode of application, and recommendation. Results show that 90% of the products studied are in solid formula—powder (65%) and granular (25%), while only 10% occur in liquid formula. We found that 100% of the products are based on the Glomeraceae of which three species dominate among all the products in the order of Rhizophagus irregularis (39%), Funneliformis mosseae (21%), Claroideoglomus etunicatum (16%). Rhizophagus clarus is the least common among all the benchmark products. One third of the products is single species AMF and only 19% include other beneficial microbes. Of the sampled products, 44% contain AMF only while the rest are combined with varying active ingredients. Most of the products (84%) claimed to provide plant nutrient benefits. Soil application dominates agricultural practices of the products and represents 47%. A substantial amount of the inoculants were applied in cereal production. Recommended application doses varied extensively per plant, seed and hectare. AMF inoculant seed coating accounted for 26% of the products’ application and has great potential for increased inoculation efficiency over large-scale production due to minimum inoculum use. More applied research should also be conducted on the possible combination of AMF with other beneficial microbes.",
"conclusion": "5. Conclusions This study is one of the foremost to examine commercial mycorrhiza inoculants extensively. Overall, the study shows that commercial AMF inoculants vary in terms of physical forms, species compositions, claims of functions, methods of application and recommendations. All the examined commercial products are based on Glomeraceae and are in three physical formulae, powder, granular, and liquid. Liquid inoculants are mainly based on single AMF species and have the least proportion among the inoculants while solid inoculants are more diverse in species composition and account for most of the AMF inoculants available. Many of the inoculants consist of other beneficial microbes, and this is believed to increase benefits and purposes served by these inoculants. AMF inoculant market is lopsided toward Asia, Europe, and the Americas. Production and application are still very low in developing countries especially Africa. Much still needs to be done to create awareness and investment to bring Africa among relevant players in the AMF inoculant industry. In addition, the claims of AMF inoculant products provide at least three main benefits to plants; nutrient uptake, plant growth induction, and climate stress alleviation, which are extensively supported by scientific data. Most of these inoculants are applied to plants through the soil and very few coated on seeds. Most of these products are mainly applied to cereals; however, AMF inoculation faces the challenge of having short product shelf life, which often discourages long-term storage and transportation. Introduction of liquid inoculants is believed to address some drawbacks related to inoculant production and application, but the cost of liquid formulation is higher. Therefore, new technologies offering a longer shelf life such as seed coating, free drying and nano-encapsulation need to be examined. Seed coating is an emerging technology that has the potential for increased efficiency and is relatively cost effective due to minimal inoculum needed. Future biofertilizer research prospects should also focus on practical and cheap inoculum production based on a combination of in vitro AMF co-culture and co-inoculation with other plant beneficial microbes, such as plant growth-promoting bacteria (PGPB).",
"introduction": "1. Introduction There has been a remarkable surge in development of the mycorrhizal-based inoculants market in the last two decades, essentially in horticulture and field crop production. The biofertilizer market in agriculture is estimated to reach USD 2.3 billion by 2022, at a Compound Annual Growth Rate (CAGR) of 14.08% during this period [ 1 ]. Arbuscular mycorrhizal fungi (AMF) are especially used in most bioinoculant production as they have been known for establishing symbiotic relationships with more than 85% of plant species of agricultural interest [ 2 ]. They have been linked to several benefits including macro- and micro-nutrient uptake, water absorption, soil aggregate stability [ 3 , 4 ], salinity and drought stress suppression, trace metal detoxification, and protection against pathogens and herbivores [ 5 ]. AMF provides these numerous benefits to plants in exchange for carbohydrates and other photosynthetic derivatives [ 6 ]. Several reasons are attributed to the unprecedented booming of the mycorrhizal inoculant industry. There is a growing scientific evidence proving various benefits provided to crops by the mycorrhizal inoculants in terms of growth and yield, which has attracted much interest from end users. Rising global population with corresponding food demand and a growing concern for the environment has also increased the need for bio-fertilization. The United Nations predicted that the global population would increase from the current 7.7 billion to 8.5 billion in 2030 and to 9.7 billion in 2050 [ 7 ] with food demand forecasted to rise by 70% in 2050 [ 8 ]. Agricultural intensification is the main solution to overcome impending food crisis, but also constitutes corresponding threat to the environment. Therefore, there is need for paradigm shift to a sustainable agricultural production system that advocates for environmentally friendly practices [ 9 ]. Biofertilizers, especially arbuscular mycorrhizal fungi, are becoming an integral part of sustainable agriculture. They have been recognized as ecologically and economically important, performing the roles of fertilizers and pesticides [ 5 ]. AMF have become a key component of organic farming and have contributed to the success of the farming system by maintaining long-term soil health and fertility [ 10 , 11 ]. AMF are increasingly dominating the biofertilizer market space and have proven to be practicable options to improve crop productivity [ 12 ]. According to Berruti et al. (2015) [ 6 ], AMF inoculation can provide immense benefits by cutting down expenses for growers, and land recovery projects. It has been observed that mycorrhizal-based products are more cost-effective than conventional fertilizers especially in regions where phosphorus depletion in soils is a serious plant nutrition problem, thus driving the demand for large scale production. For instance, in India, commercial mycorrhizal-based inoculants are being widely used in rice production to thwart the effects of low phosphorus levels in the soil combined with the rising cost of synthetic P fertilizers. Despite these benefits and justifiable reasons for the market explosion, only a few AMF strains are marketed globally as biofertilizers because of significant limitations hindering mass production of AMF inoculants given their obligate biotrophic lifecycle, meaning that they require a host plant to grow and reproduce. In addition, there is great concern about the quality and quantity of the mycorrhizal inoculants [ 13 , 14 ]. Efficacy of inoculants is also affected by the different field conditions such as compatibility to various soil characteristics, different crop species, indigenous microbial communities, and environmental factors as well as the soil fertility management practices of the native soils [ 15 , 16 ]. These constraints influence soil microbial dynamics and functional processes impacting the performance of commercial bioinoculants. Consequently, there has been a conflicting stance on their efficacy in field conditions [ 17 ]. Owen et al. (2015) [ 18 ] mentioned that there was a distinct lack of robust field-based testing of commercial bioinoculants as most studies have focused on greenhouse pot trials. Therefore, for AMF inoculant industry to thrive, rigorous research must be conducted to provide best practices to the inoculant companies regarding composition, quality, quantity, and application methods of the products [ 19 ]. Although lack of efficacy or negative impacts of AMF inoculation has been reported [ 20 , 21 , 22 ], recent studies conducted under field conditions have shown promising results [ 12 , 23 , 24 , 25 , 26 ]. A growing number of market players are investing in mycorrhizal inoculant production, but very limited information is publicly available regarding commercial inoculants in the market. In this study, we collected and analyzed important data available on benchmark mycorrhizal products to synthesize and compare their characteristics such as composition, formulation of products, propagule contents, claimed crop benefits, active ingredients, and mode and types of applications. We also reviewed recent trials of commercial AMF inoculants under greenhouse and field conditions. Lastly, we revealed the status quo in the AMF inoculant markets and industry and share our perspectives on potential market-based research opportunities.",
"discussion": "4. Discussion 4.1. Product Breakdown by Country of Production Success in mycorrhiza inoculant production has reached a new phase and the industry has been growing rapidly. This survey is the first to assess the variability in commercial mycorrhizal inoculants. Our study focused on 68 products from 28 manufacturers mainly in Europe, North America, South America and Asia. Our sampling is not exhaustive and was based on information available during the study. However, there is no valid data on the actual number of firms producing inoculants, the number of propagules produced, or the number of areas treated with mycorrhizal inoculants. Our study is in line with previously published work [ 27 , 28 ]. For example, in 2016, Pal et al. (2016) [ 27 ] listed more than sixty manufacturers of mycorrhizal inoculants across Asia, Europe, North America, South America and South Africa. In 2018, Chen et al. [ 28 ] also identified more than seventy-five firms producing and marketing mycorrhizal-based inoculants in Europe alone, an increment over the small number of (ten to forty) firms involved in the business in 2000 and 2010, respectively ( Supplementary Materials Figure S4 ). Major players in the mycorrhizal industry were found in the United States, Canada, Germany, Italy, Czech Republic, United Kingdom, and Spain. Reasons for this could be attributed to available data as well as to increased awareness about the environmental benefits of mycorrhizal fungi, increased demand for organic food products and the availability of modern technology for inoculant production. Reports indicated Asia-Pacific as the third largest player (after North America and Europe) in the global biofertilizer market with increasing demand in India, China and Taiwan [ 1 ]; however, local data on AMF inoculants was unavailable for inclusion in this study. Despite a long history of AMF research in Australia, AMF is rarely considered by farmers in management decisions due to a lack of agronomic-relevant recommendations, which resulted from a lack of dialogue between AMF researchers and agronomists [ 29 ]. Thus, the status of commercial mycorrhiza production in Australia is still unclear although much literature has been published on the role of mycorrhizal fungi in crop production and forestry [ 29 , 30 , 31 , 32 , 33 ]. Mycorrhiza production in Africa is still at a medium or small scale due to technological limitations but Pal et al. (2016) [ 27 ] indicated South Africa and Kenya as being among the major players in Africa. 4.2. AMF-Based Inoculant Formulation Our survey found that more than 60% of the products are in powder formulation while only 29% are in liquid. This could be explained by the methods of mass production in vivo versus in vitro as well as the conservation and stability of mycorrhizal propagules. Most of the solid inoculants contain more than one species of AMF most likely produced in vivo using conventional co-culture with a host plant in a substrate usually under greenhouse; however, all of the liquid inoculants are only available in one single strain Rhizophagus irregularis isolate DAOM197198 as the only active ingredient ( Supplementary Materials Table S1 ). So far, R. irregularis is the most successful strain that is produced in vitro using a large-scale mycoreactor with transformed roots [ 34 ], although many other strains have also been cultured successfully in vitro but at a laboratory scale [ 35 ]. Large-scale AMF inoculants are produced in vivo by co-cultivating AMF and host plants in inert substrates to allow for propagation, and substrates rich in propagules are harvested at the end of cycle [ 36 ]. The conventional culture technique is cheap and often leads to solid products, but the main drawback is high-risk contamination and a low concentration of AMF propagules. In vitro production techniques offer contamination-free propagules due to the sterile culture conditions. Products can be handled to contain more propagules with recent data indicating that propagule numbers have been increasing significantly from hundreds of spores produced initially to up to several thousand per mL [ 36 ]. Solid substrate inoculants have gained wider application possibly due to the ease of handling (mixing), conservation and richness of AMF species and inclusion of other beneficial microbes such as plant growth promoting bacteria, but application in large field is faced with some drawbacks. First major challenge with a solid substrate inoculant is the labor-intensive application especially in large-scale operation. It requires special machinery that suits different varieties of plant, soil and fertilization program to have a homogeneous inoculation process [ 37 ]. Miguel et al. (2007) [ 38 ] also reported a number of limitations with solid inoculants: spore germination is affected by long dormancy periods due to the packaging conditions; propagule concentration in a solid inoculant is also natural and cannot be increased to the desired size; and glomalin (glomalin-related soil proteins) that accelerates formation of stable soil aggregates is not excreted on solid substratum. Introducing a liquid substrate helps to overcome some of these limitations. It reduces the dormancy period of spores; it enables the propagule concentration to be increased to the desired amount; and improves the formation of soil aggregates by stimulating glomalin secretion at high concentrations. Glomalin secretion at high amounts in liquid media earns additional marketing claims for manufacturers. Therefore, it is not surprising that most inoculants that claimed to contribute to the improving soil structure are in liquid forms ( Supplementary Materials Table S1 ). Liquid inoculants also allow easy handling and low transportation costs as they can be designed to contain more propagules than solid products. The liquid inoculant is better suited for fertigation and irrigation than solid inoculants [ 37 , 38 ]. It may be essential to highlight that the liquid inoculant is not a complete substitute of the solid-based inoculant as it does not solve all the problems. It has a short shelf life due to its liquid form and this may limit its commercial application. It is a sterile product lacking beneficial microbes compared to inoculants in solid form. The negative impact of the sterile condition and artificial way of production as well as the lack of beneficial microbes on inoculants have been reported, although there is little published data available. Calvet et al. (2013) [ 39 ] reported that in vitro -based inoculants produced less spores and recorded lower mycorrhization than in vivo inoculants in leek plants. Moreover, only R. irregularis is available in liquid form as confirmed by this study. It may be perceived that future demand will favor liquid inoculants due to its various advantages under field condition, but solid inoculants are well-established products, especially in forestry, gardening, and horticulture. 4.3. Inoculant Composition Arbuscular mycorrhizal fungi belong to the phylum Glomeromycota which consists of four orders, 12 families, 41 genera and approximately 338 species [ 40 ]. Gigasporaceae, Glomeraceae, and Acaulosporaceae represent the most diverse genera within the phylum, containing 82% of the entire species [ 41 ]. Glomeraceae family includes the abundant genera such as Glomus, Rhizophagus, Funneliformis and Septoglomus, which have been reported in all continents [ 41 , 42 , 43 ]. Most of the commercial inoculants evaluated in the present study were found to include species belonging to Glomus, Funneliformis, Rhizophagus and Septoglomus genera. Among all the AMF species present in the products, R. irregularis and F. mosseae were the dominant species. R. irregularis was the most widespread, occurring in more than one-third of the products ( Supplementary materials Figure S2A ). Members of the Glomeraceae have displayed differential efficacy in terms of host root colonization and performance under varying field conditions. For example, species of Glomus and Rhizophagus have been reported to outperform other genera such as Gigaspora and Scutellospora under different management practices [ 44 , 45 ]. R. irregularis and F. mosseae are very productive members of the Glomeraceae family in terms of root colonization, nutrient foraging and association with other microbes. R. irregularis was dubbed an aggressive and quick colonizer of plants at low soil phosphorus (P) compared to many other species such as Claroideoglomus claroideum ( G. claroideum ) and R. eutenicum [ 46 ]. Since R. irregularis and F. mosseae are generalist mycobionts, they currently seem to be the best candidates to provide farmers and manufacturers maximum return on investment because they can be applied to varied hosts and survive long-term storage. Espinosa et al. (2005) [ 47 ] reported the effectiveness of seven species of AMF ( R. irregularis, R. fasciculatus, F. mosseae, R. clarus, Paraglomus occultum, Acaulospora scrobiculata and Diversispora spurcum ) evaluated using different tropical crops (potato, cassava, sweet potato, malanga, pepper, cucumber, tomato and banana), and found that R. irregularis performed consistently well for all crops evaluated. F. mosseae and R. fasciculatus also showed adequate performance in the same experiment. In another study, R. irregularis was found to be the most efficient in P foraging in patchy environments compared to Gigaspora magarita and F. mosseae [ 48 ]. Resistance of F. mosseae and R. irregularis to soil disturbance [ 46 ] makes them most suitable in both till and no-till fields. Rouphael et al. (2015) [ 34 ] mentioned that for effective root colonization AMF inoculants should [ 49 ] (1) contain a mixture of AMF species, (2) a high number of propagules, (3) be free of pathogens, (4) include beneficial bacteria, and (5) must have a long shelf life. Although many of the examined products contain a mixture of AMF, not all of them contain a consortium of AMF, high propagule concentrations and beneficial bacteria. The commercial inoculants consist of two or more strains of AMF species, but a larger percentage is based on single strains only. Inoculants with a single species of AMF should not be considered inferior to those with multiple fungi species because some AMF single species ( F. mosseae , R. irregularis ) are generalists (capable of colonizing a large variety of host plants), have a longer shelf life, and are geographically distributed all over the world [ 43 ], thus these species may serve multiple functions and colonize multiple crops. Wagg et al. (2015) [ 50 ] also suggested that the composition of species within a consortium could be more important to improving productivity. Apart from root colonization of host plants, AMF develop associations with vast communities of mycorrhyzospheric microbes that promote plant growth either by solubilizing P or secreting growth-promoting substances such as siderophore or indoleacetic acid (IAA) [ 51 ]. Our study shows that 19% of the inoculants comprise beneficial microbes while the rest do not. AMF inoculants consisting of consortium of P-solubilizing and N-fixing bacteria will be an added value for the industry. Moreover, long-term in vitro propagation of AMF has the potential to domesticate AMF species and alter their genetic functionalities, but co-culture with other microbes can help to mitigate putative genetic variation and function by activating AMF genes that would otherwise be silent or deleted [ 52 ]. Although inoculants containing beneficial microbes may confer many benefits to crops, more work needs to be done to characterize and isolate complementary growth-promoting bacteria for AMF inoculant production. 4.4. Propagule Concentration The presence of viable propagules is important to the colonization of plant roots by AMF inoculants. After the AMF species composition, propagule concentration is the most interesting characteristic of commercial inoculants. Species composition may be more important to scientists but propagule number is a vital technical tool for the end users. Equivalent to nutrient composition of chemical fertilizers, the number of viable propagules may be used to determine the quantity of inoculants applied per hectare. After considering effectiveness of AMF species, it will be reasonable to expect inoculants with a higher propagule number to be more cost effective in terms of transportation costs and doses, leading to a better return on investment for manufacturers and growers. Our study showed that most products contain more than 100 propagules per gram, with 1 to 8 different AMF species. However, propagule concentration is independent of the number of AMF species. 4.5. Industrial Claims on the Effects of AMF-Based Inoculants Nutrient uptake, stress alleviation, growth and crop quality enhancements are the dominant benefits attributed to most commercial inoculants ( Figure 1 ). This is not surprising as many results from greenhouse and field trials have also proven the positive effects of AMF inoculation. AMF inoculation primarily boost nutrient mobilization and uptake [ 26 , 53 , 54 , 55 , 56 , 57 ]. AMF inoculation enhances uptake and transportation of nutrients such as P, N, Mg, K, Fe, Cu, Zn and Mo. [ 58 , 59 , 60 ]. Up to 75-90% P and 5-80% N uptake by mycorrhizal plants were attributed to AMF in the soil, but this amount may vary depending on crop species and field conditions [ 61 , 62 ]. Plant nutrition benefits are mainly attributed to the extraradical hyphae of AMF, which can spread to farther distances (where normal roots cannot reach), thereby increasing the surface area for nutrient uptake. For example, a study conducted by Karaca et al. (2013) [ 24 ] showed tremendous increase in soybean root and leaf growth when it was treated with a combination of AMF inoculant, phosphorus and sulfur as compared to the control. Uptake of macro- and micro-elements by AMF host plants is expected to result in improved growth, yield and crop quality. Stoffel et al. (2020) reported that inoculating maize crops ( Zea mays ) with AMF significantly increased P uptake, biomass and grain yield in low or medium soil P levels. Hijri (2016) [ 12 ] also demonstrated that inoculating potato crops with AMF inoculants increased average yield by 3.9 tons/ha, representing 9.5 % increment of total crop yield. Mycorrhiza inoculation especially in the field does not always produce positive effects on biomass and grain yield. For example, Faye et al. (2020) [ 22 ] showed that inoculating soybean ( Glycine max (L.) Merr .) enhanced root nodulation but had no significant effect on grain yield. Similarly, Rosa et al. (2020) [ 21 ] showed that inoculation of grapevine rootstocks resulted in increased biomass production only in greenhouse but not in the field. N nutrition via AMF is still under debate. Bücking and Kafle (2015) [ 63 ] hypothesized that AMF are able to transfer N to their host plants through the mycorrhizal interface, but it has been suggested that higher N contents in inoculated plants is a consequence of the synergistic effect resulting from improved P uptake [ 64 ]. Additionally, Wang et al, (2018) [ 65 ] reported that AMF reduced the acquisition of N by plants in N limiting soils thereby affecting the plant health. AMF-mediated nitrogen acquisition was reported in some grass species but no effect was observed on total N uptake in the plant community [ 66 ]. Depression in growth of AMF inoculated crops in some cases may be linked to mutualism-parasitism-continuum whereby carbon cost of plant exceeds nutritional benefit obtained from AMF symbiosis, leading to increased cost to benefit ratio [ 45 , 62 , 64 , 67 ]. Climate stress alleviation in crops was also widely claimed by inoculant manufacturers. Smith et al. [ 68 ] mentioned that apart from the direct nutrition function, AMF also play a significant role in regulating the biochemical processes of a plant in the presence of abiotic and biotic stresses. Since abiotic stresses, such as drought, salinity, extreme temperature and trace elements, among others, have tremendous negative impacts on crop performance, AMF inoculants are becoming a very important tool to rely on considering the projected future consequences of climate change and environmental degradation. Liu et al. (2016) [ 25 ] reported that AMF inoculation of crops grown under lower temperature conditions had significant positive impact on the physiological features of the crops. Under drought stress, arbuscular mycorrhizal symbiosis promotes tolerance through stomatal control, direct water and nutrient uptake by the hyphae [ 69 , 70 ], adjustment of osmotic and antioxidant protection systems [ 71 , 72 ] and by increasing the regulatory function of the stress-responsive hormones [ 73 ]. In saline soils, mycorrhiza inoculation was reported to enhance plant survival through maintenance of cell homeostasis. Conversely, AMF effect under saline condition is not always positive; reduced viability and symbiotic function of AMF in extreme salt conditions have been reported. Romero-Munar et al. (2019) [ 74 ] reported reduced leaf growth and root colonization of A. donax (giant reed) inoculated with a commercial inoculant under moderate to high salt conditions. Despite numerous claims by AMF inoculant dealers, economic gains from independent studies based on AMF inoculation remains unclear. Inoculation of crop with AMF often leads to increased root colonization but effect on yield is not always predictable despite improvement in plant nutrient composition and crop quality. For this reason, yield increase may not be the only criteria to justify the efficiency of bioinoculants. Moreover, AMF inoculant performance has not been consistent and depends on factors such as soil type, nutrient concentration, AMF species, crop genotype as well as biotic and abiotic stress conditions. As such, growers may need to identify crop type and operating environments including soil disturbance level and fertilization plan before selecting commercial inoculants. There may be need to choose single species inoculants which were suggested to be the best in controlled environment [ 75 ] or consortium of AMF species which is less host specific in the field. We present in Supplementary materials Table S2 the results obtained in studies that evaluated some commercial AMF inoculants including some that were mentioned in our study. 4.6. Recommended Inoculant Application Methods and Doses Soil application is a traditional method that has been practiced for decades and is more common than other bioinoculation pathways. According to Rocha et al. (2019) [ 76 ], direct soil application decreases physical damage to fragile seeds and cotyledons, minimizes the effects of pesticides and fungicides on the seeds, and gives smaller seeds the opportunity to be inoculated. Soil inoculation via liquid or powdered inoculants depends on the type of crops. It has been reported that powdered inoculants work best with grassy seeds such as wheat, barley, and oats, among others, because they are hairy textured, which means that the powder easily sticks to them [ 76 , 77 ]. On the other hand, liquid inoculants are often ideal for smooth-surfaced seeds such as corn, beans, and alfalfa because they form suitable adhesion. As per this survey, the application rates of inoculant products varied but application per hectare was commonly used. A range of 0.12 kg to 30 kg per hectare was considered as ideal depending on the strain composition and the crops they were applied to. This rate tallies with the quantity used in some studies conducted on coffee and horticultural crops, which showed an increase in dry matter yield at an application rate of 4–5 kg per hectare [ 2 , 78 ]. Seed treatment is a novel pathway for inoculant application and accounted for 26% of all the products surveyed. The main advantage of this technique is its ability to achieve immense precision in delivering the agents or active ingredients but the technique is faced with some challenges that may hinder its application and scaling up to commercial levels. Treated or coated seeds often lack uniformity in the quantity of inoculant received and may be contaminated by other microbes. Thick coatings may also hinder seed germination [ 79 ]. Selection and maintenance of viable inoculants in coated seeds are important areas that need to be addressed [ 80 ], especially for long-term storage. Moreover, due to lack of awareness in rural areas, the adoption rate among farmers remains low [ 17 ]. The doses for treating the seeds were a lot smaller, at maximum 2 g per 1 kg of seeds compared to soil treatment because only minimum inoculants are required for several seeds. The least common application method reported in this study is the co-application of inoculants and chemical fertilizers, which only accounted for 11% of the total applications. Availability of P in the plant tissues resulting from application of chemical fertilizers may inhibit the colonization of roots by AMF [ 81 , 82 ]. In addition, excess P levels have been reported to cause toxicity to AMF. These reasons may limit large scale adoption of this method. Cereal production clearly dominated the inoculation market space due to relatively greater quantities of cereal crops cultivated on large arable lands globally. Furthermore, during the early growth stages of production, P becomes a limiting nutrient for cereals, especially for root development, therefore, mycorrhizal-based inoculants would be vital in P acquisition [ 55 ]. Horticulture is the other major target market for several inoculant products. Inoculation of horticultural crops with AMF is increasing rapidly owing to improvement of the quality of the natural contents of inoculated crops. Rouphael et al. (2010) [ 83 ] reported that arbuscular mycorrhizal symbiosis can induce changes leading to the enhanced biosynthesis of phytochemicals, which are known to provide health benefits. Healthy dieting has led to increased demand for natural crops and this is likely to lead to a surge in inoculant production and usage."
} | 8,092 |
28986532 | PMC5630616 | pmc | 6,397 | {
"abstract": "A pressing challenge for ecologists is predicting how human-driven environmental changes will affect the complex pattern of interactions among species in a community. Weighted networks are an important tool for studying changes in interspecific interactions because they record interaction frequencies in addition to presence or absence at a field site. Here we show that changes in weighted network structure following habitat modification are, in principle, predictable. Our approach combines field data with mathematical models: the models separate changes in relative species abundance from changes in interaction preferences (which describe how interaction frequencies deviate from random encounters). The models with the best predictive ability compared to data requirement are those that capture systematic changes in interaction preferences between different habitat types. Our results suggest a viable approach for predicting the consequences of rapid environmental change for the structure of complex ecological networks, even in the absence of detailed, system-specific empirical data.",
"introduction": "Introduction Anthropogenic land-use intensification reduces habitat complexity, with profound consequences for plant and animal species 1 . The most immediate effects of habitat simplification are shifts in the frequency and specificity of interactions between consumer and resource species 2 . These shifts result in changes to weighted network structure 3 , 4 and can have significant practical consequences, as species interactions underpin crucial ecosystem services such as biological control, pollination and seed dispersal 5 – 8 . Field studies have begun to quantify how interaction frequencies differ among habitat types 9 – 13 , but exhaustive collection of these data can be laborious and a bottleneck to understanding community responses to environmental changes, especially for species-rich communities containing rare and undocumented species 11 . Models that could predict interaction frequencies in modified habitats would help alleviate this problem, but several hurdles need to be overcome 14 – 16 . In particular, some changes to interaction patterns will result simply from changes in random encounter rates when species’ abundances change, whereas others will result from altered foraging behaviour. It would be useful to describe how changes in relative species abundance 17 vs. changes in species behaviour 18 contribute to changing network structure. Furthermore, it is important to describe these changes at the level of individual field sites, and not just for aggregated networks built from interaction data collected across multiple field sites. Separating relative species abundance and species behaviour is important because large differences in recorded interaction frequencies can be attributed to random encounter among species even when there are large differences in relative species abundance 19 , without the need to appeal to more complex ecological processes or mechanisms 20 , 21 . In other cases, assuming only random encounters may be insufficient to fully explain changes in weighted network structure, so by separating out the contribution of relative species abundance it will be easier to identify and investigate the effects of habitat modification on species behaviour. Such clarifications are especially relevant for understanding major structural alterations of a habitat, such as deforestation: in addition to changes in relative species abundance, predator foraging efficiency and strategy are affected by decreases in habitat complexity 22 and prey switching, in turn, depends on resource availability and accessibility 23 . In this study, we test whether we can accurately predict the effects of habitat modification on the structure of weighted host-parasitoid networks 10 – 13 (parasitoids are insects that live in or on the body of their host, eventually killing it). Our approach involves networks sampled at field sites in both modified and relatively unmodified habitat types (hereafter ‘unmodified habitat types’), and uses mathematical models that both estimate differences in relative species abundance between field sites and separate random-encounter effects from differences in species behaviour. We represent the combined effect of host and parasitoid species behaviour by interaction preferences. Interaction preferences were originally designed to improve measurements of nestedness in weighted networks 24 ; here, we use them to describe differences in species behaviour between field sites in similar and different habitat types, and to make predictions of weighted network structure. We hypothesise that species behaviour does not change significantly between field sites in similar habitat types but does change significantly between field sites in different habitat types. This hypothesis would correspond to small differences in individual interaction preferences between field sites in similar habitat types, with larger differences between field sites in different habitat types. It also suggests that predicting weighted network structure at new field sites in a similar habitat type to existing data should be more straightforward than if new field sites are in a different habitat type. Because interaction preferences may change as habitats are modified, we focus on predicting weighted network structure in modified habitat types using models primarily calibrated with data collected from unmodified habitat types. We consider a total of seven models with different complexities and data requirements, and show that neglecting to separate changes in relative species abundance from changes in species behaviour results in poor predictions of weighted network structure. We then assess the performance of five models based on ecological mechanisms that do make this separation and show that including increasingly more information from modified habitat types results in progressively better predictions. We find that models that capture systematic, community-wide changes in interaction preferences offer the best combination of model complexity and performance. These changes could, for example, relate to altered resource selectivity by consumers in habitat types with minimal forest coverage. Our new modelling approach represents a simple yet powerful way of scaling up existing data to predict weighted network structure across multiple field sites of a given habitat type, predict the effects of habitat modification, and inform the amount and type of additional data that should be collected in novel environments to improve predictions.",
"discussion": "Discussion A wealth of information about behaviour and species’ responses to the environment is contained in weighted interaction networks 2 . However, predictions cannot be made based on empirical networks alone. Ecologists and conservation practitioners need models that combine information from existing networks with other data and theory to make accurate predictions in novel environments. In this study, we compared the performance of seven models and found that simpler models were sufficient to predict network structure at field sites in similar habitat types to existing data, but more complex models were required when field sites were in different habitat types. This result is consistent with our hypothesis that host and parasitoid species behaviour does not change significantly between field sites in similar habitat types but does change significantly between field sites in different habitat types. Our findings suggest that if network data representative of new field sites are readily available then predicting weighted network structure is straightforward: interaction preferences are likely to be similar and the alternative preferences model can be used with empirical estimates of species abundance, such as those collected during biodiversity monitoring programmes. For example, the interaction preferences inferred here for rice and pasture habitat types could be used to make predictions at new but similar field sites in Ecuador. Of course, it must be recognised that interaction preferences can only be determined if pairs of species have been observed co-occurring already, which may be a limiting factor for predicting weighted network structure in systems with frequent spatial and temporal turnover of community composition. Prediction is more difficult if new field sites are in modified habitat types with limited existing data to inform models, as is the case with most urban habitat types like parks and community gardens. Interaction preferences are likely to be different, and accurate prediction requires understanding which ecological processes and mechanisms are driving these differences. But, as our results for the correlated preferences model show, consistent changes in species behaviour can be mapped to systematic changes in interaction preferences, with measurable benefits for prediction. In addition, the specified preferences model highlights how targeted data collection of particular species and interactions can make predicting the effects of habitat modification more efficient. And given that our models span a range of data requirements, it is possible to customise the trade-off between prediction accuracy and sampling effort depending on the practical question of interest. In this study, independent measurements of relative species abundance were not available and so predictions were based on effective abundances estimated from network data. By design, our method for estimating relative species abundances will tend to favour an explanation of interaction frequencies in terms of mass action, potentially at the expense of under-estimating genuinely strong or weak interaction preferences. In this regard, it is a conservative method that could under-attribute changes in network structure to species behaviour. It is not currently known how effective abundances correspond to more direct measurements or estimates of species abundance in the field. Although our general approach to prediction is valid either way, determining how effective abundances relate to more direct measurements will be necessary to ensure accurate predictions of weighted network structure. Finding clear relationships between inferred and measured species abundances would also bring about time and cost savings, as only interaction data or abundance data would need to be collected, as appropriate. Identifying such relationships will help with the practical side of prediction, but other kinds of data are needed to clarify the role of species behaviour in determining network structure. This is because our current definition of interaction preferences does not separate ‘inherent’ preferences from complicating factors due to the local environment. By ‘inherent’ preferences, we mean some kind of baseline expectation for how often, for example, a parasitoid would select a particular host given a choice of alternative hosts from different species, but described at the population level rather than the more usual individual level. These ‘inherent’ preferences are best measured in the controlled setting of laboratory experiments, and doing so would also help untangle the issue of potential and realised niche (Supplementary Note 2 ). Once measurements have been made, it will then be possible to test more nuanced hypotheses, such as whether ‘inherent’ preferences are masked in forested habitat types but revealed in open habitat types. We used a likelihood function based on the multinomial distribution to calculate model performance. This probability distribution is useful because it directly compares model predictions for multiple species to a recorded set of interaction counts. It does so by representing the probability that a parasitoid picks a given host, conditioned on information about other hosts in the community. This conditioning is necessary if, for example, the abundances of particular host species lead to parasitoids forming search images 28 that affect their per capita probabilities of attacking other hosts in the community. As such, the multinomial distribution relies on species richness and community composition being relatively stable over the time period of data collection. Alternatively, one could use a likelihood function based on the binomial distribution, which represents the probability of recording a successful parasitism event given a host-parasitoid encounter in the field, independent of community composition (we discuss other possible probability distributions for the likelihood function in Supplementary Note 2 ). The binomial distribution assumes that network structure is primarily a pairwise phenomenon, whereas the multinomial distribution assumes that it is primarily a community phenomenon, and likely it is a mixture of the two. In future work, it will be useful to compile general patterns of shifting interaction preferences between different habitat types, and, indeed, patterns that arise from other forms of environmental change. For example, interaction data could be collected along an altitudinal gradient as a proxy for temperature change, using differences between sets of inferred interaction preferences as the basis of predictive models for climate warming. Identifying which interactions need to be characterised and hardcoded in models is also important for prediction; and the fact that some interactions deviate so strongly from mass action suggests that they may be worth deeper investigation in their own right. Promisingly, we found that only a small fraction of interactions may need to be sampled in modified habitats to significantly improve predictions of network structure, and these interactions likely involve common species with many interaction partners. It will be interesting to apply our models, based on host-parasitoid networks, to other classes of weighted interaction network, such as plant-pollinator networks (in which weights represent the number of recorded visits between species). Although many biological details will of course vary between network classes, separating relative species abundance from other factors affecting network structure will still be useful because our general approach, at its core, represents a fundamental modelling step that is now taken for granted in population dynamical models 29 . With our new methods and models, we can now begin to predict how human-driven change could impact species’ interactions in novel environments and unfamiliar conditions. By separating abundance and behaviour, we are better able to compare the functional roles of rare and specialist species to the roles of more abundant and generalist species in a community, both in terms of ecosystem service output and also their relative contributions to network persistence and stability 30 – 33 . Our approach is also relevant as the final step in a more ambitious sequence of predictions. Species distribution and demographic models use environmental variables and species’ vital rates (e.g., survival, growth, and reproduction) to predict the geographical distribution and abundance of species 34 , 35 . The models we have presented can convert these abundances into weighted interaction networks. In this way, we can begin to predict the composition and structure of communities, and, therefore, start assessing and predicting the effects of environmental changes on the global provision of ecosystem services."
} | 3,874 |
35736294 | PMC9228305 | pmc | 6,399 | {
"abstract": "Developing an effective phycoremediation system, especially by utilizing microalgae, could provide a valuable approach in wastewater treatment for simultaneous nutrient removal and biomass generation, which would help control environmental pollution. This research aims to study the impact of low-voltage direct current (DC) application on Chlorella vulgaris properties and the removal efficiency of nutrients (N and P) in a novel electrokinetic-assisted membrane photobioreactor (EK-MPBR) in treating synthetic municipal wastewater. Two membrane photobioreactors ran in parallel for 49 days with and without an applied electric field (current density: 0.261 A/m 2 ). Mixed liquid suspended soils (MLSS) concentration, chemical oxygen demand (COD), floc morphology, total phosphorus (TP), and total nitrogen (TN) removals were measured during the experiments. The results showed that EK-MPBR achieved biomass production comparable to the control MPBR. In EK-MPBR, an over 97% reduction in phosphate concentration was achieved compared to 41% removal in the control MPBR. The control MPBR outperformed the nitrogen removal of EK-MPBR (68% compared to 43% removal). Induced DC electric field led to lower pH, lower zeta potential, and smaller particle sizes in the EK-MPBR as compared with MPBR. The results of this novel study investigating the incorporation of Chlorella vulgar is in an electrokinetic-assisted membrane photobioreactor indicate that this is a promising technology for wastewater treatment.",
"conclusion": "4. Conclusions A modified membrane photobioreactor that incorporated a low-voltage electric field and the algae C. vulgaris was developed. This novel study compared the biomass production and the nitrogen and phosphorus removal efficiency of C. vulgaris in the electrokinetic-assisted membrane bioreactor with that of the control. The biomass production in the EK-MPBR was comparable to that in the MPBR. Nutrient removal was lower and significantly higher in EK-MPBR for total nitrogen and total phosphorus, respectively. This can be explained by electrochemical reactions around the electrodes. The results also showed that increased cell charge and formation of smaller particles under the applied electric field was observed, which may affect biomass production. The work presented here has implications for future studies of the electric field in MPBRs and may help modify the design of membrane photobioreactors for membrane fouling control. Further research identifying the mortality rate of the algae under different applied currents molecular changes and electrokinetically affected assimilation efficiency of the cells will be beneficial for improving electro-phycoremediation techniques in wastewater treatments.",
"introduction": "1. Introduction Wastewater treatment is a growing concern because wastewater contains pollutants such as nitrogen and phosphorus, which in excess can threaten wildlife and marine life [ 1 , 2 ]. Algae can assimilate these nutrients from wastewater and prevent eutrophication [ 3 , 4 , 5 , 6 ]. Phycoremediation, or biological treatment that utilizes algae for nutrient removal from wastewater, is one of the recent technologies gaining attention due to its low cost and environmental footprint [ 5 , 7 , 8 ]. Membrane photobioreactor technology (MPBR), as one of the biological wastewater treatment systems, is widely used for simultaneous wastewater treatment and microalgae production [ 9 ]. This technology has gained momentum due to promising nutrient removal and the high quality of effluent, together with the production of concentrated microalgae [ 10 , 11 ]. The biomass production of MPBRs has industrial applications, including biofuel, foods, and feeds [ 12 , 13 , 14 ]. Several studies on MPBR have been conducted by various research groups with different microalgae species and MPBR configurations [ 15 , 16 , 17 , 18 ]. These studies have demonstrated the advantages of MPBR systems for nutrient removal and microalgae biomass production compared to the conventional microalgae system [ 19 ]. Chlorella vulgaris ( C. vulgaris ) is an extensively used microalgae in MPBRs for wastewater treatment [ 8 , 20 , 21 ]. It is also produced for human nutrition and biodiesel feedstock applications [ 12 , 14 , 22 ]. C. vulgaris can grow in diverse environments [ 23 , 24 , 25 ] such as high temperature, e.g., up to 40 °C [ 14 ], acidic and alkaline (pH from 3 to 11.5) [ 20 , 21 , 22 ], light intensity [ 22 ], and high salinity. The most studied condition affecting their growth is light, but few studies focus on the effect of a low-voltage continuous electric field on photosynthesis and growth efficiency [ 14 ]. There are several parameters that affect the phycoremediation process [ 8 ]. Wastewater characteristics are one of the factors that have been studied. One recent study investigated the implications of urban wastewater concentration and induced stress on the growth of Chlorella fusca [ 26 ]. Using real wastewater instead of synthetic wastewater is another recent research focus on the growth study of C. vulgaris and its bioremediation of primary (PE) and secondary (SE) urban effluents [ 27 ]. Some reported researchers have focused on operating conditions such as hydraulic retention time (HRT), solid retention time (SRT), and turbulent pulsation [ 28 , 29 , 30 ]. Some studies investigated the effect of a low-voltage electric field on the growth and nitrogen and phosphorus removal efficiency of C. vulgaris [ 31 , 32 ]. Other research groups have studied the application of moderate and short-term electric fields in stimulating the growth and metabolism of C. vulgaris [ 13 , 33 ]. They found that in batch culture, a short-term applied electric field could improve the biomass growth of microalgae [ 13 , 33 ]. A study using an applied pulse electric field recently revealed the potential for increased lipid content from C. vulgaris [ 34 ]. Studies that incorporate electric fields in membrane bioreactors (MBR) have found improved chemical oxygen demand removal (COD) as well as nitrogen and phosphorus removal were observed by applying a short-term electric field in membrane bioreactors (MBR), where activated sludge was used [ 35 , 36 , 37 , 38 ]. To date, only one study has integrated an electric field into an MBR with algae and activated sludge [ 2 ]. However, the combination of an electric field with a membrane photobioreactor utilizing algae as biomass has not yet been investigated. The current study examines the effect of a low-voltage continuous electric field on the microalgae growth rate, biomass quality, and overall nutrient removal (N and P) of an MPBR with C. vulgaris in treating synthetic municipal wastewater. An MPBR and an electrokinetic-assisted MPBR (EK-MPBR) with C. vulgaris were operated in parallel for 49 days to investigate the electric field effects on biomass production, biomass productivity, COD removal, and nutrient (N and P) removals. This is the first study on EK-MPBR, and the results demonstrate that it is a promising technology that simultaneously removes nutrients and reproduces microalgae.",
"discussion": "3. Results and Discussion In this study, the performance of the electrokinetic-assisted photobioreactor on phycoremediation is investigated and compared with a control photobioreactor. The effect of the electric field is classified in terms of biomass production, nutrient removal, zeta potential, pH, and floc morphology. 3.1. Effect of EF Treatment on Biomass Production We investigated the effect of the applied electric field on algae growth in photobioreactors. The MLSS concentration was measured and used as an indicator of algal growth throughout the study. The time course measuring the MLSS concentration of the reactor is shown in Figure 2 . Both MPBRs operated in parallel with an initial concentration of 1.16 ± 0.4 g/L of MLSS. The biomass value and productivity of the control reactor (MPBR) varied from 0.97 g/L to 2.12 g/L and 32.33–70.66 g/Ld, respectively. For the EK-MPBR, the corresponding values were lower than the control, with biomass ranging from 0.62 g/L to 1.59 g/L of MLSS and productivity ranging from 18–53 mg/Ld. However, these values were not significantly different ( p > 0.05). In the EK-MPBR, MLSS fluctuation over the first 39 days of the experiment was smoother and relatively higher compared to the control MPBR. This improved productivity suggests that the applied electric field stimulated the growth of microalgae. The present study is consistent with the findings of others that the electric field increased the productivity of Chlorella vulgaris by enhancing the transport of substances across the algae cell membrane [ 45 , 46 ]. The hormetic response of low-dose stimulation and high-dose inhibition seen in Figure 2 is an adaptive cell response that is stimulatory in the short term and inhibitory in long-term exposure [ 47 ] was also observed in the pre-treatment of Chlorella vulgaris with the application of a short-term moderate electric field [ 13 ]. Corpuz et al. studied the effect of a long-term applied electric field bioreactor, where they observed a similar trend in the Algae-Activated sludge bioreactor [ 2 ]. In the study of Corpuz et al., it was mentioned that more prolonged exposure to the electric field inhibited microalgae growth starting on day 28. This retardation of microalgae, which is caused mainly by electrochemical reactions around the cathode, was delayed in this study until day 39. Indirect oxidation due to the modified design of the cathode in our study could play a role in growth inhibition caused by hydroxyl radicals. By placing the cathode behind the membrane, the released ions in the permeate could be removed by the permeate pump, and therefore, their accumulation over time is controlled. The indirect oxidation effect on the molecular level in MPBRs could be the focus of future studies. 3.2. Nutrient Removal and Wastewater Treatment Potential The wastewater treatment performance of EK-MPBR was compared to the control MPBR to determine electric field efficiency in terms of nutrient removal from the wastewater. The efficiency of a phycoremediation system is defined by how well algae can remove nitrogen, phosphorus, and COD from wastewater. Figure 3 shows the percentage of nutrient removal of EK-MPBR and MPBR over time. The concentrations of N, P, and COD in the influent were maintained constant at the levels of 25n ± 2 mg/L, 3.5 ± 0.3 mg/L, and 20 ± 2.5 mg/L, respectively. EK-MPBR showed a statistically significant higher phosphorous removal with an overall removal of 97.98n ± 0.02% ( p < 0.05). This demonstrates the advantage of EK-MPBR for phosphorus removal compared to the overall removal of 41.81 ± 0.05% of the control reactor. The main two phosphorus removal mechanisms in algal systems are biomass adsorption and precipitation of phosphorus [ 2 , 25 ]. In an electric field-assisted system, electrochemical oxidation on the surface of the electrodes and electrochemical reactions in the suspension can also contribute to phosphorus removal. Given a pH range of 7.5 to 8.5 for EF-MPBR, phosphorus adsorption on the surface of the anode (graphite) is not the dominant mechanism [ 48 ]. The improved phosphorus removal in EK-MPBR can be attributed to the occurrence of electrochemical reactions in the suspension and the overall ion strength in the mixed liquor solution [ 33 , 49 ]. A recent study showed that in biomass combined with activated sludge, the applied electric field improved phosphorus removal by 65% compared to its control reactor and was mainly due to electrochemical reactions [ 2 ]. The released aluminum ions from the aluminum anode and the generation of phosphate aluminum complex contribute to the removal of phosphorus [ 50 , 51 ]. Although a number of studies have evaluated the effect of electric field on MBRs [ 2 , 33 , 52 , 53 ], its effect on molecular adsorption in MPBRs needs to be verified in future studies. Limited phosphate concentration in the suspension in EK-MPBR can lower the biomass productivity and removal efficiency caused by a low biomass concentration [ 54 ]. However, this is in contrast to the MLSS concentration ( Figure 2 ). As such, it is likely that the stimulating effect of the electric field outweighed the inhibitory effect of P depletion. Furthermore, the limited phosphate concentration in the suspension in EK-MPBR is beneficial for reducing membrane fouling. The correlation between P depletion and biofilm growth has been reported by other studies [ 55 , 56 ]. The low concentration of P, ranging from 0.05 mg/L to 1.09 mg/L in EK-MPBR, is below the concentration needed for biofilm growth compared to the control MPBR, ranging from 1.79 to 2.39 mg/L [ 49 ]. This suggests the existing potential of EK-MPBR for enhanced membrane performance. Despite the significant phosphorus removal of EK-MPBR, the nitrogen removal efficiency of EK-MPBR was depressed to some extent. The better phosphorous removal efficiency compared to the nitrogen removal efficiency in EK-MPBR might be because of their different removal mechanisms. At the water-oxide interface, phosphate removal utilizes an inter-sphere adsorption mechanism that is less affected by ionic strength as compared to nitrogen removal, which has an outer-sphere adsorption mechanism [ 48 ]. As shown in Figure 3 b, EK-MPBR TN removal ranged from 17.82% to 85%, whereas in control MPBR, TN was removed by 58% to 85% ( p < 0.05). The concentration of TN in the influent was kept at a constant value of 25 ± 2 mg/L for both reactors. The lower nitrogen removal efficiency agrees with other studies that showed that the electric field might interfere with the nitrogen removal process [ 33 , 57 , 58 ]. The fluctuation over the 49 days in TN removal in EK-MPBR could be attributed to the change of MLSS concentration ( Figure 3 a), zeta potential ( Figure 4 ), and ionic strength of the suspension, which may have interfered with nitrification. The lower nitrogen removal in EK-MPBR due to the presence of an electric field agrees with the study by Zhang et al. [ 33 ]. The potential changes in ionic properties of the microalgae could be due to the electrochemical reactions and the incorporation of the electric field, which further have an inhibitory impact on the removal of total nitrogen [ 33 ]. However, both EK-MPBR and MPBR showed nitrogen removal comparable to other studies ( Table 3 ). Figure 3 c represents the COD concentration over the experimental period. The influent COD concentration for both reactors was kept constant at 20 ± 1.8 mg/L. The COD reduction can be attributed to the thickness of the biofilm, electrochemical oxidation of organic substances, and oxidation of the organic compounds by electrochemically generated oxidants such as hydrogen peroxide [ 2 ]. In this study, due to the induced multiple factors, further investigation is needed before highlighting any underlying reasons as the main mechanism of COD removal in EK-MPBR when comparing it with MPBR. 3.3. Effect of EF on the Physiology of Microalgae Zeta potential, pH, and the morphology of biomass are measured as indicators of changes in physiology under the electric field and their effect on the phycoremedation efficiency [ 60 , 61 ]. In both MPBRs, the zeta potential remained negative over the experimental period. Zeta potential is dependent on factors such as pH and ion type and strength [ 62 ]. In alkaline conditions, zeta potential increases (i.e., becomes more negative) with pH increase as particles are surrounded by more negative charge in the suspension. The zeta potential values, represented in Figure 3 , along with the pH data depicted in Figure 4 , agree with the above statement. The zeta potential in the control MPBR was higher than that of EK-MPBR. This can be attributed to the higher pH in the reactor compared to EK-MPBR [ 62 ]. Furthermore, zeta potential is a function of other factors, such as the composition and concentration of metabolites in the suspension [ 62 ]. This could explain the fluctuating behavior, especially over the first days of the operation, when the microalgae have unstable conditions due to adaptation to the new environment. Surface charge, as represented by the zeta potential here, can contribute to the nutrient uptake efficiency of the system [ 63 ]. In alkaline solution, the predominant phosphate ions are HPO 4 2 − and PO 4 3 − [ 64 ]. The higher the surface charge, the stronger the electrostatic repulsion would interfere with the adsorption of these ions on the algal cell surface [ 64 , 65 ]. Reportedly, the lower surface charge positively affects the adsorption of the orthophosphate by C. vulgaris [ 65 ]. Sedimentation is another mechanism of phosphorus removal from wastewater that is also affected by the surface charge and electrostatic repulsion [ 66 , 67 ]. The phosphate ions form complex salts in wastewater, such as calcium phosphate in the form of sediments [ 67 ]. The lower surface charge would enhance sedimentation and phosphorus removal. Therefore, as shown in Figure 4 , the decreased zeta potential in EK-MPBR (−27 compared to −20 mv in MPBR) can be attributed to the better phosphorus removal efficiency in EK-MPBR (97.98 ± 0.02% in EK-MPBR compared to 41.81 ± 0.05% in MPBR). The nitrogen removal, however, was affected differently by the surface charge. Due to the increased surface charge, the adsorption of negatively charged hydroxyl ions (OH) that are part of the denitrification process could be decreased [ 68 ]. The effect of the electric field on this removal pathway can be further verified through electrochemical analysis of the cells in future studies. The variation of pH with time is shown in Figure 5 . While both reactors started with the same pH, the applied electric field lowered pH over time. The electrochemical reaction around the cathode is a determining factor [ 69 ]. In alkaline conditions, the following reaction at the cathode can be expected: (2) 2 H 2 O + 2 e − → H 2 ( g ) + 2 OH − ( aq ) As a result of this electrolysis reaction, the pH near the cathode is expected to increase under the applied electric field. However, the results show that other factors may be involved in pH changes. One of the underlying reasons for pH changes could be the materials used and impurities that arise during the strengthening of the carbon in the manufacturing process, which could be released from the electrode to the suspension when placed under the electric field [ 70 ]. pH is also sensitive to the mechanism of microalgae growth. Considering glucose in the influent, the suspension provides a mixotrophic and/or heterotrophic culture for C. vulgaris growth. In both cases, mixotrophic and heterotrophic, pH depends on the microalgae’s preferred growth kinetics. While the pH remained unchanged in the mixotrophic condition, heterotrophic culture showed a gradual decrease in the suspension [ 71 ]. As shown in Figure 6 , the lighter suspension over time under the electric field demonstrates heterotrophic dominancy, and therefore decreased pH. Figure 7 shows the morphology of the suspensions in MPBR and EK-MPBR. One of the main objectives of this investigation is to study possible morphology changes due to the applied electric field. As shown in Figure 6 , the floc size in the EK-MPBR is smaller than that of the MPBR. This agrees with the higher fraction of smaller particles seen in the particle size distribution (PSD) analysis ( Figure 8 ). The fraction of the smaller particles in EK-MPBR ( Figure 8 ) can be attributed to floc breakage and disintegration due to the applied electric field and the electrophoresis phenomenon. Electrophoresis and movement of the charged particles could result in more breakage of the flocs [ 49 ]. In electrophoresis, the charged particles tend to move toward the electrode with the opposite charge, which can cause collisions and smaller particle formation. This potentially explains the formation of smaller flocs in EK-MPBR. The conceptual image of the phenomena is presented in Figure 9 ."
} | 5,033 |
30554875 | null | s2 | 6,400 | {
"abstract": "Vibrio cholerae uses a quorum-sensing (QS) system composed of the autoinducer 3,5-dimethylpyrazin-2-ol (DPO) and receptor VqmA (VqmA"
} | 33 |
37796897 | PMC10591310 | pmc | 6,401 | {
"abstract": "Abstract Sulfate/sulfite-reducing microorganisms (SRM) are ubiquitous in nature, driving the global sulfur cycle. A hallmark of SRM is the dissimilatory sulfite reductase encoded by the genes dsrAB . Based on analysis of 950 mainly metagenome-derived dsrAB -carrying genomes, we redefine the global diversity of microorganisms with the potential for dissimilatory sulfate/sulfite reduction and uncover genetic repertoires that challenge earlier generalizations regarding their mode of energy metabolism. We show: (i) 19 out of 23 bacterial and 2 out of 4 archaeal phyla harbor uncharacterized SRM, (ii) four phyla including the Desulfobacterota harbor microorganisms with the genetic potential to switch between sulfate/sulfite reduction and sulfur oxidation, and (iii) the combination as well as presence/absence of different dsrAB- types , dsrL -types and dsrD provides guidance on the inferred direction of dissimilatory sulfur metabolism. We further provide an updated dsrAB database including > 60% taxonomically resolved, uncultured family-level lineages and recommendations on existing dsrAB -targeted primers for environmental surveys. Our work summarizes insights into the inferred ecophysiology of newly discovered SRM, puts SRM diversity into context of the major recent changes in bacterial and archaeal taxonomy, and provides an up-to-date framework to study SRM in a global context.",
"conclusion": "Conclusion Metagenome-driven discoveries have opened a new window into the hidden diversity of SRM. We can now start to appreciate that besides the four bacterial and two archaeal phyla harboring cultured SRM, the potential to perform dissimilatory sulfate/sulfite reduction extends to a total of 23 bacterial and 4 archaeal phyla. Many of the phyla now recognized to play a role in sulfur cycling were represented by DsrAB-encoding MAGs recovered from low-sulfate environments, supporting the notion that hidden or cryptic sulfur cycling in low-sulfate environments is an understudied area. For a few of these potential SRM, such as members of the Acidobacteriota , mesophilic Nitrospirota , and Bacteriodata family UBA2268 (Kapabacteria), meta-omics based studies under constrained environmental conditions could provide strong evidence of a sulfate-reducing lifestyle. However, the large majority of novel, putative SRM still await experimental confirmation of their physiology. Furthermore, we could show that the primers used in dsrAB gene-based approaches cover a large fraction of the novel diversity of SRM, with many of the previously taxonomically unresolved DsrAB lineages now anchored by DsrAB-encoding MAGs. As such, dsrAB gene-based surveys can be used with confidence in the future to explore the enigmatic world of a functional microbial guild that has shaped biogeochemical cycling on Earth since the Archaean (Shen et al. 2001 , Wacey et al. 2011 ).",
"introduction": "Introduction The sulfur cycle is one of the most important biogeochemical cycles on Earth (Canfield and Farquhar 2012 ) tightly interacting with carbon, nitrogen, and metal cycling (Jørgensen 2021 ). It is mainly regulated by activities of sulfate/sulfite-reducing microorganisms (SRM) and sulfur-oxidizing microorganisms (SOM) as their counterparts (Dahl et al. 2008 , Rabus et al. 2013 , Rabus et al. 2015 , Wasmund et al. 2017 , Jørgensen 2021 ), which cycle sulfur between its most oxidized (sulfate, +VI) and its most reduced state (sulfide, -II). On a global scale, sulfate reduction is one of the dominant processes driving the mineralization of organic matter in anoxic environments. Of the estimated 260 Tmol C org reaching the global seabed each year, one third is mineralized through sulfate reduction in marine sediments (Jørgensen 2021 ). About 90% of the end product, sulfide, is re-oxidized to sulfate either directly or indirectly at the expense of oxygen (Jørgensen 2021 ). This represents 25% of global oxygen consumption in sediments and has a direct impact on the redox state of Earth's surface. The relevance of sulfur cycling increases further in coastal sediments, where sulfate reduction accounts for 50% of C org mineralization and re-oxidation of sulfide consumes 50% of the oxygen entering the sediment (Jørgensen 2021 ). In addition to marine sediments, marine oxygen minimum zones represent environments of active sulfur cycling. In these oxygen-depleted waters, sulfide produced by sulfate reduction is rapidly re-oxidized to sulfate by sulfide oxidation coupled to nitrate reduction (Canfield et al. 2010 , Johnston et al 2014 , Callbeck et al. 2018 , van Vliet et al. 2021 ). Here, the term “cryptic sulfur cycle” was coined for the first time—“cryptic” because it was not evident from the spatial concentration profiles of inorganic sulfur compounds, in particular sulfide (Canfield et al. 2010 ). While the importance of sulfate reduction in marine environments is well explained by the high availability of sulfate (ca. 28 mM), its role in biogeochemical cycling of anoxic freshwater environments such as lake sediments, groundwater, peatlands, or rice paddy fields is less obvious because of the low prevailing sulfate concentrations (typically 10–300 µM) (Pester et al. 2012 ). Nevertheless, the rates at which sulfate reduction proceeds can be equally high in marine and freshwater settings, resulting in rapid cycling of sulfur species in anoxic freshwater environments. Because of its less obvious relevance and high variability in space and time, the sulfur cycle in freshwater systems is often referred to as a cryptic or hidden sulfur cycle as well (Pester et al. 2012 ). The contribution of sulfate reduction to C org mineralization in anoxic freshwater environments has not been evaluated so systematically as in marine environments, but single studies report values of 17–35% in lake sediments (Urban et al. 1994 , Thomsen et al. 2004 ) and 36–50% in peatlands (reviewed in Pester et al. 2012 ). Yet another low-sulfate environment with cryptic sulfur cycling are deep marine subsurface sediments below the sulfate-methane transition zone. Here, sulfur cycling operates at very slow sulfate reduction rates. These slow rates are maintained by the re-supply of sulfate mediated by Fe(III)-driven sulfide oxidation (Holmkvist et al. 2011a , b , Pellerin et al. 2018 , Jørgensen et al. 2019 , Findlay et al. 2020 ). Besides their relevance for biogeochemical cycling, SRM represent an important symbiotic guild in the mammalian intestinal tract (Barton et al. 2017 ) and are also beneficial in bioremediation, such as degrading hydrocarbons and removing heavy metals from sulfate-containing groundwater and wastewater (Muyzer and Stams 2008 , Qian et al. 2019 ). However, they can also be an economic burden by causing steel corrosion or oil souring (Muyzer and Stams 2008 , Rey et al. 2013 , Rabus et al. 2015 , Singh and Lin 2015 , Wolf et al. 2022 ). In the context of climate change and human-induced eutrophication, it is noteworthy that oxygen concentrations in pelagic zones of the global ocean, coastal waters, and lakes have been declining for decades (Jenny et al. 2016 , Breitburg et al. 2018 ). The resulting oxygen-deficient zones can turn euxinic (anoxic conditions with > 0.1 μM sulfide) upon release of toxic sulfide by SRM, which further aggravates the negative effects of oxygen shortage causing death to fauna including economically relevant fish, shrimp and crabs (Diaz and Rosenberg 2008 , Jenny et al. 2016 , Bush et al. 2017 , Diao et al. 2018 , van Vliet et al. 2021 ). On the other hand, SRM can also exert positive climate change effects. Especially in low-sulfate habitats with active cryptic sulfur cycling, such as rice paddy fields, peatlands and lake sediments, SRM compete for substrates with microorganisms involved in the methanogenic degradation network (Pester et al. 2012 , Wörner et al. 2016 , Wörner and Pester 2019 ). This leads to a partial diversion of the carbon flux from CH 4 to CO 2 , which is the less potent greenhouse gas on a per molecule basis (Pester et al. 2012 ). Stimulation of cryptic sulfur cycling, e.g. by the addition or intrinsic activity of sulfide-oxidizing cable bacteria can thus exert positive effects on mitigation of methane emissions (Sandfeld et al. 2020 , Scholz et al. 2020 ) or delay the development of euxinia (Seitaj et al. 2015 ). Most SRM share a canonical core enzyme repertoire for carrying out dissimilatory sulfate reduction (Fig. 1 ). This intracellular pathway includes the enzymes sulfate adenylyltransferase (Sat), adenylyl phosphosulfate reductase (AprAB), dissimilatory (bi)sulfite reductase (DsrAB), and the sulfide releasing DsrC. The complexes QmoAB(C) and DsrMK(JOP) complement the pathway by transferring reducing equivalents towards AprAB and DsrC, respectively (Pereira et al. 2011 , Ramos et al. 2012 , Santos et al. 2015 ). Hereafter, we refer to this pathway as the Dsr-pathway. Most SRM (with the exception of early diverging archaea) and microorganisms relying on a partial sulfate reduction pathway such as sulfite-, thiosulfate-, and organosulfonate reducers as well as sulfur disproportionating microorganisms utilize in addition DsrD, which is an allosteric activator of DsrAB (Ferreira et al. 2022 ). Among these enzymes, DsrAB can be used not only as a functional but, with some limitations, also as a phylogenetic marker for SRM. Phylogenetically, this enzyme comprises three major lineages that largely differentiate between (i) reductively-operating DsrAB of archaeal origin, (ii) reductively-operating DsrAB of bacterial origin, and (iii) oxidatively or reverse-operating DsrAB (rDsrAB), which occur in a variety of phototrophic and chemotrophic SOM (Loy et al. 2009 , Müller et al. 2015 ). SOM that rely on rDsrAB for sulfur oxidation also share a number of additional enzymes with SRM, including Sat, AprAB, QmoABC, DsrC, and DsrMKJOP (Dahl 2017 , Tanabe and Dahl 2022 ). Figure 1. The pathway of dissimilatory sulfate reduction. The Dsr-pathway includes the enzymes sulfate adenylyltransferase (Sat), adenylyl phosphosulfate reductase (AprAB), dissimilatory sulfite reductase (DsrAB), and the sulfide releasing DsrC protein. The complexes QmoAB (C) and DsrMK(JOP) complement the pathway by transferring reducing equivalents towards AprAB and DsrC, respectively (Pereira et al. 2011 , Ramos et al. 2012, Santos et al. 2015 ). Reducing equivalents required by DsrAB can be delivered by a yet unknown oxidoreductase or DsrL (Löffler et al. 2020 ). DsrD acts as an allosteric activator of DsrAB in sulfate/sulfite-, thiosulfate-, and organosulfonate reducers as well as sulfur disproportionating microorganisms (Ferreira et al. 2022 ). The phylogenetic distinction of reductively and oxidatively operating DsrAB was initially also supported by the presence of additional, presumably SOM-specific enzymes. These include DsrEFH as a sulfur donor protein for DsrC in SOM (Stockdreher et al. 2012 ) and DsrL as an essential oxidoreductase in sulfur oxidation (Lübbe et al. 2006 ) that transfers reducing equivalents from rDsrAB to NAD + (Löffler et al. 2020 ). However, metagenome-assembled genomes (MAGs) from a variety of habitats questioned this clear distinction, with DsrEFH, DsrL, or both being co-encoded together with reductive DsrAB (Anantharaman et al. 2018 , Hausmann et al. 2018 , Tan et al. 2019 , Thiel et al. 2019 , Ye et al. 2022 ). The recent identification of two discrete DsrL types, with DsrL-1 occurring only in SOM, while DsrL-2 occurring in organisms with either a reductive/disproportionating or oxidative sulfur metabolism (Löffler et al. 2020 ), highlights the difficulties in delineating the energy metabolism solely from genomic data. Functional gene prediction is further complicated by the examples of Desulfurivibrio alkaliphilus (Thorup et al. 2017 ) and the so-called cable bacteria affiliated to the Desulfobulbaceae (Pfeffer et al. 2012 , Risgaard-Petersen et al. 2015 ). Both can oxidize sulfide by operating the canonical pathway of sulfate reduction in reverse, including a reductive-type DsrAB, and couple this either with intracellular nitrate reduction in the case of D. alkaliphilus (Thorup et al. 2017 ) or to electrogenic oxygen or nitrate reduction in spatially separated cells along filaments in the case of cable bacteria (Kjeldsen et al. 2019 ). Despite these constraints, dsrAB gene-based molecular approaches have become an important tool for studying the diversity and ecology of SRM in the environment. First introduced by Wagner et al. 1998 , cumulative evidence from a large variety of marine, terrestrial, and man-made environments revealed that the diversity of SRM extends massively beyond cultured representatives in the four bacterial phyla Desulfobacterota (formerly known as Deltaproteobacteria and Thermodesulfobacteria , Waite et al. 2020 ), Bacillota (formerly known as Firmicutes, Oren and Garrity 2021 ), Thermodesulfobiota (Frolov et al. 2023 ), and Nitrospirota (Oren and Garrity 2021 ) as well as the two archaeal phyla Thermoproteota (formerly known as Crenarchaeota, Oren and Garrity 2021 ) and Halobacterota (formerly part of the Euryarchaeota, Rinke et al. 2021 ). A systematic review of environmental dsrAB genes encoding the reductive bacterial-type DsrAB revealed at least 13 lineages at the approximate family level that could not be related to any cultured SRM or higher-rank taxa (Pester et al. 2012 , Müller et al. 2015 ). At the species level, a broad census based on dsrB gene amplicon sequencing identified 167 397 species-level operational taxonomic units (OTUs) across 14 different environments (Vigneron et al. 2018 ). If compared to the approximately 460 described SRM listed in the LPSN database (lpsn.dsmz.de), this means that > 99% of SRM diversity is represented by uncultured microorganisms without taxonomic assignment. Members of well characterized Desulfobacterota ( Desulfobacteraceae, Syntrophobacteraceae, Desulfovibrionaceae, Desulfobulbaceae ) often dominate the SRM community in marine and freshwater surface sediments (Vigneron et al. 2018 , Wörner and Pester 2019 , Jørgensen 2021 ) and the uncharted dsrAB gene sequence space largely represents low-abundance taxa. However, in certain environments representatives of uncultured dsrAB lineages can constitute numerically relevant members of the SRM community (Vigneron et al. 2018 ), including coastal sediments in the Arctic (Flieder et al. 2021 ), wetlands (Pester et al. 2012 ), and deep subsurface marine sediments with active but cryptic sulfur cycling (Leloup et al. 2009 ), to name a few. Therefore, there is a need to identify these yet unknown SRM and to understand their ecophysiology and evolution. In recent years, an increasing number of new DsrAB-encoding taxa have been discovered by metagenomic surveys of environmental samples and the delineation of MAGs. Here, we provide a systematic review of these novel findings, give insights into the increased diversity of (putative) SRM, and place this in the context of the recently proposed overarching changes to bacterial and archaeal taxonomy (Parks et al. 2018 , Parks et al. 2020 , Oren and Garrity 2021 , Rinke et al. 2021 ). Detailed overviews of well-studied phyla harboring SRM, including cultured and environmental representatives, have been provided in excellent reviews elsewhere (Rabus et al. 2013 , Rabus et al. 2015 , Langwig et al. 2022 )."
} | 3,883 |
38651372 | PMC11036299 | pmc | 6,402 | {
"abstract": "Microscopic, photosynthetic prokaryotes and eukaryotes, collectively referred to as microalgae, are widely studied to improve our understanding of key metabolic pathways (e.g., photosynthesis) and for the development of biotechnological applications. Omics technologies, which are now common tools in biological research, have been shown to be critical in microalgal research. In the past decade, significant technological advancements have allowed omics technologies to become more affordable and efficient, with huge datasets being generated. In particular, where studies focused on a single or few proteins decades ago, it is now possible to study the whole proteome of a microalgae. The development of mass spectrometry-based methods has provided this leap forward with the high-throughput identification and quantification of proteins. This review specifically provides an overview of the use of proteomics in fundamental (e.g., photosynthesis) and applied (e.g., lipid production for biofuel) microalgal research, and presents future research directions in this field.",
"conclusion": "8. Conclusions In summarizing the recent advances in microalgal proteomics, this review underscores the crucial role of these technologies in understanding both fundamental biological processes and their biotechnological applications. Yet the importance of rigorously maintaining key steps from sample preparation to data analysis for successful proteomics analyses cannot be overstated. Recent years have seen substantial efforts to develop and streamline proteomics frameworks, making this field increasingly accessible to non-experts. This evolution has been complemented by the emergence of companies specializing in affordable data analysis, curation and visualization. With the ongoing integration of AI, these processes are set to become even more efficient and reliable. The insights gained from functional enrichment analyses and protein–protein interactions have been instrumental in addressing complex biological questions and will certainly guide future research directions (e.g., strain improvement). As we transition into the post-omics era, the increasing availability of advanced software and databases promises a broad spectrum of discoveries in both fundamental and applied microalgal research. This journey into the future of microalgal proteomics is not just about enhancing our understanding but also about harnessing the potential of microalgae in innovative and sustainable ways. Notably, microalgae can produce a wide array of chemicals (e.g., nutraceuticals, pharmaceuticals); therefore, the field would benefit from more proteomics studies focusing on conditions triggering the production of these chemicals.",
"introduction": "1. Introduction Photosynthetic microorganisms can be found in all aquatic ecosystems, in which, as primary producers, they have important ecological functions, either positive (e.g., biogeochemical cycle [ 1 , 2 , 3 ]), or negative (e.g., when they bloom at the wrong time and place [ 4 , 5 ]). Although they encompass both prokaryotes (cyanobacteria) and eukaryotes (“true” microalgae), they will be collectively referred to as microalgae in the present review [ 6 ]. Microalgae such as the model Chlamydomonas reinhardtii are widely studied to improve our understanding of key plant functions such as chloroplast-based photosynthesis and nutrient assimilation, and the structure and function of the eukaryotic flagella [ 7 , 8 , 9 , 10 ]. In addition, due to their physiology and biochemistry, significant research is focusing on the development of microalgae-based environmental biotechnologies for food and high-value molecules production, biofuels generation, wastewater treatment and nutrient recovery [ 11 , 12 , 13 , 14 ]. In recent years, the so-called “omics” technologies have been shown to be valuable tools in microalgal research [ 15 , 16 , 17 , 18 ]. Omics, which includes genomics, transcriptomics, proteomics and metabolomics, broadly refers to the comprehensive analyses of classes of biological molecules, i.e., DNA, RNA, proteins and metabolites, and their interactions (interactomics, Figure 1 ). Omics allow us to determine and study the whole makeup of a cell/biological system at a given time and, therefore, to correlate molecular signatures with phenotypes [ 17 , 19 , 20 , 21 , 22 , 23 ]. With a wide range of applications, these technologies have considerably accelerated the rate of discoveries and provided a leap forward in fundamental and applied research. For example, the sequencing and study of the C. reinhardtii genome in 2007 unraveled the evolution of the eukaryotic flagellum and plastid [ 7 ]. In the past decade, technological developments in hardware and software have allowed omics technologies to become more affordable and more accessible [ 22 , 24 , 25 ]. Consequently, it is now possible to study the whole genome, transcriptome, proteome and metabolome of an organism in a matter of weeks (assuming the proper steps are considered and followed). As clear evidence, whereas until 2008 the genome of only three microalgae had been sequenced [ 16 ], many omics datasets have now been deposited in public repositories. While genomics and transcriptomics deliver significant information about genes and their expressions, these approaches do not provide an indication of protein levels, protein turnover and post-translational modifications. Consequently, proteomics, i.e., the study of proteins (from their structure to their interactions with other molecules in a cell), corroborates and builds on genomics and transcriptomics and it is, therefore, a comprehensive approach to characterize a biological system. As proteins are effectors of biological functions, the levels (and forms) of proteins in a cell indeed represent comprehensive information about cellular function, defining the phenotype of a cell in response to genetic or environmental changes [ 13 , 19 , 25 ]. This review aims to give an overview of proteomics technologies and their applications for microalgal research and focuses on the recent improvements in the use of proteomics in microalgal research. It also presents the future of proteomics in the post-omics era, i.e., the development and use of artificial intelligence (AI)-based technologies. Recent advancements in machine learning, particularly deep learning, have enabled researchers to accurately predict protein structures and functions from their amino acid sequences, improving the quality and reliability of analytical workflows in mass spectrometry-based proteomics [ 26 ]. This approach is becoming central to biomarker discovery from proteomics data, and is beginning to outperform existing assays [ 27 ]. Furthermore, AI algorithms have been instrumental in analyzing large-scale proteomic datasets [ 27 , 28 , 29 , 30 ], ultimately enabling the identification of novel protein interactions and pathways that are critical for microalgal adaptation to environmental stresses [ 16 , 31 ]. It is noteworthy that the potential of microalgal biotechnology and that of proteomics technologies, along with their respective advantages and disadvantages, have been reviewed elsewhere [ 11 , 16 , 19 , 21 , 32 , 33 ]. Therefore, these topics are not the primary focus of this review."
} | 1,816 |
39888896 | PMC11785343 | pmc | 6,404 | {
"abstract": "Herbivorous fishes play a crucial role in the conservation of coral reefs threatened by thermal stress ( e . g ., marine heatwaves and long-term ocean warming) by helping to maintain reefs in a coral-dominated state via the removal of algae. However, as thermally sensitive ectotherms, rising thermal stress may also pose a serious threat to these fishes and the critical ecosystem functions they deliver. Here we evaluate the consequences of thermal stress on the capacity of a common herbivorous coral reef fish ( Acanthurus triostegus ) to control finely filamentous matrices of Caulerpa sertularioides and C . verticillata algae in Hawai ʻ i, by characterizing in-vivo changes in metabolic demands, diurnal foraging rates, activity patterns and individual condition in a laboratory setting during winter (24.0±0.1°C), summer (27.5±0.1°C), and at the peak of a representative marine heatwave, (31.0±0.1°C). Rising temperatures caused significant increases in standard metabolic rate (from ~135 O 2 kg -1 h -1 in winter to 224 O 2 kg -1 h -1 at the peak of a marine heatwave), but not in the proportion of time spent active (~83–96%) or foraging (~2.4 bites min -1 ). Consequently, A . triostegus gained body mass during summer and winter, but lost ~0.8% body mass per day during the marine heatwave. Given marine heatwaves can last for weeks to months, these results indicate that while herbivorous coral reef fishes may continue to remove algae during periods of thermal stress, their ability to control many macroalga may be limited due to precipitous reductions in individual performance. Therefore, in addition to algal types, the thermal sensitivity in herbivorous reef fishes will need to be considered for the successful implementation of coral-algal management strategies in a warmer world.",
"introduction": "Introduction Coral reefs, often referred to as the “rainforest of the sea,” occupy less than 1% of the ocean floor yet are home to over 25% of all marine life, making them among the most biodiverse and productive ecosystems on Earth [ 1 ]. Given their ecological significance, it is crucial to understand the impact of forceful agents of disturbance such as marine heatwaves (MHWs) [ 2 ]. MHWs, defined as “discrete prolonged anomalously warm water events with sea-surface temperatures exceeding the 90 th percentile threshold calculated across 30 years and persisting for at least 5 days” [ 3 ], are known to have increasingly devastating and long-lasting impacts on ecosystem resilience and services [ 4 – 7 ]. The extreme warming associated with MHWs has caused widespread coral devastation globally [ 8 – 10 ] including recurring mass coral bleaching events [ 10 – 13 ], with notable impacts in the Pacific [ 8 , 14 , 15 ]. Alarmingly, these disturbances have been increasing in frequency, intensity, severity, and duration over the past century and are projected to continue to do so throughout the next century [ 5 , 6 , 16 ]. It is anticipated that the escalation of MHWs may eventually lead to the irreversible loss of many coral reef ecosystems worldwide [ 5 , 8 , 16 , 17 ]. In light of these projections, understanding the ecological impacts of MHWs on coral reef ecosystems becomes of utmost importance. The severe or prolonged disturbances caused by MHWs can lead to a collapse of habitat structure and a transition from coral to algal dominance [ 18 , 19 ]. Since the shift from coral to algal dominance fundamentally alters the ecosystem, the role of herbivorous reef fishes is regarded to be essential for the conservations of coral reefs in a warmer future [ 19 – 21 ]. By removing algae that may otherwise hinder the settlement, growth, and survival of corals and coral recruits [ 18 , 19 , 21 – 23 ], herbivorous reef fishes help prevent and mitigate transitions from coral to algal-dominated states [ 18 , 24 , 25 ]. Therefore, reductions in their abundance may directly undermine reef resilience. In fact, herbivorous coral reef fishes, including ’browsers’ ( i . e ., species that consume fleshy, rapidly growing macroalgae encroaching on established coral), ’grazers’ ( i . e ., species that feed on algal turf covering coral settlement surfaces), and ’scrapers’ ( i . e ., species that actively scrape the reef matrix to reveal fresh coral settlement areas), are often thought to serve as the final line of defense against uncontrolled algal proliferation [ 18 ]. However, most reef fishes are also highly sensitive to rising temperatures [ 26 – 28 ], leading to uncertainty about species health and resilience, including the retention and delivery of herbivorous reef fish functions, as these ecosystems are increasingly exposed to temperatures beyond those for which they have evolved [ 26 , 29 , 30 ]. As ectotherms, the metabolic energy demands and condition of herbivorous reef fishes is regulated by ambient ocean temperatures [ 31 – 34 ]. Specifically, the minimum oxygen uptake required by an ectotherm to maintain homeostasis (denoted as standard metabolic rate, SMR) [ 35 ], typically increases at a rate of 2x for every 10°C rise (defined as a Q 10 ) [ 36 , 37 ]. In thermally sensitive species, including tropical coral reef fishes, SMR often increases at a Q 10 rate of 2-3x [ 38 ], suggesting ocean warming of 3–4°C—such as might occur during a marine heatwave—could increase basal energetic demands for survival by up to 55% [ 38 , 39 ]. Current projections indicate a global average temperature increase of approximately 2.7°C above preindustrial levels (or about 1.7°C above present day) [ 16 ], underscoring the importance of understanding the potential impacts of both chronic warming and acute thermal stress events on these species. Studies have already demonstrated that temperatures just 2–3°C above annual summer maxima can have detrimental effects on multiple traits of tropical coral reef fishes, including swimming, growth, activity, reproduction and foraging patterns [ 28 , 31 , 40 – 44 ]. Accordingly, rising temperatures pose a potential threat to the retention and function of herbivorous reef fishes as increased energetic demands will necessitate increased energy acquisition ( e . g ., by increasing algal foraging rates), reduced energy expenditure ( e . g ., by reducing activity) [ 40 , 45 , 46 ] or shift in diets to more energetic items [ 47 ]. However, herbivorous reef fishes already spend over 80% of their time feeding during peak summer conditions [ 48 , 49 ] and are not expected to have the ability to process food more quickly through their digestive system [ 44 , 48 , 50 – 53 ] suggesting substantial increases in foraging rates may be infeasible. Given these constraints, there is great uncertainty of how herbivorous reef fishes may cope with rising energetic demand and the resultant impacts on herbivore functions [ 54 , 55 ]. Therefore, as we look to these organisms to help safeguard reef ecosystems into the future [ 24 , 44 , 56 ], this study aims to investigate the consequences of rising ocean temperatures on the retention and delivery of herbivorous reef fish functions. Using an ecologically important herbivorous reef fish, the convict tang ( Acanthurus triostegus ) and a turf matrix of filamentous green algae ( Caulerpa sertularioides and C . verticillata ) as model representatives, we hypothesize that as ocean temperatures increase ( i . e ., from winter, to summer, to the peak of a MHW), herbivores will show two responses to cope with elevated energy requirements: 1) algal foraging rates will increase, and 2) energy expenditure will reduce ( i . e ., minimizing activity time). By investigating these thermal responses, we aim to gain insight into whether an important herbivorous reef fish can maintain its body condition while continuing to remove algae in a warmer future.",
"discussion": "Discussion This study investigated the impacts of rising temperature on rates of energetic demand ( i . e ., SMR), acquisition ( i . e ., foraging rates) and expenditure ( i . e ., activity levels) of an ecologically important herbivorous coral reef fish foraging on a finely filamentous matrix of Caulerpa sertularioides and C . verticillata . While herbivorous coral reef fishes are expected to increase foraging rates or reduce activity to meet rising metabolic demands brought on by ocean warming, this premise has to our knowledge not been explicitly tested. Contrary to our expectations, we demonstrate that despite rising metabolic demands, A . triostegus does not increase foraging rates, and instead, increases levels of activity as temperatures increase. Consequently, the temperature driven increase in metabolic demands, alongside steady feeding rates and increased activity levels, resulted in a significant reduction in body mass when exposed to a simulated MHW. Since feeding rates remained constant, algae consumption rates would not decrease directly due to temperature. However, the thermally induced reductions in the condition of A . triostegus combined with expectations of faster growth rates of many algae in warmer waters [ 91 ] could indirectly affect their long-term ability to control algae. This could have important implications for our reliance on herbivorous functions in a warming world, with potential downstream consequences for reef resilience and the trophic structure [ 55 ]. Energetic demands of A . triostegus , here evaluated as oxygen consumption, increased by ~28% from winter to summer (Q 10 of 2.0) and an additional ~29% from summer to the peak of a simulated MHW (Q 10 of 2.1). These thermally induced increases in energetic demand were similar to that observed in a range of other coral reef fish species ( e . g ., Q 10 of 1.1–5.7 (29–33°C) [ 31 , 92 , 93 ] and matched the global mean projections for most marine organisms of Q 10 = 2–3 [ 39 , 86 , 94 ]. To overcome these thermally driven energetic demands many species are expected to increase energy acquisition. However, contrary to the anticipated increase, the foraging rates of A . triostegus remained consistent across temperatures. Previous studies of herbivore responses to temperature have shown comparable results. Some herbivorous fish, especially parrotfishes, exhibit increased foraging rates with changing latitudinal or seasonal temperatures [ 44 , 50 , 51 , 53 ]. However, at temperatures outside their optimal range, these rates can plateau or even decrease [ 50 , 95 ]. For instance, feeding rates of Acanthurus bahianus have been shown to decrease more rapidly than metabolic rates when temperatures drop, suggesting an inability to maintain feeding efficiency at suboptimal temperatures. This diminished feeding rate in cooler waters highlight a temperature-dependent physiological constraint [ 96 ]. Here, the examined MHW conditions are beyond the temperatures under which most reef fishes have evolved and a plateau in feeding rates suggests temperature-dependent physiological constraints may be mirrored at temperatures above optimum. Emerging studies suggest that the diet of herbivores may influence their thermal tolerance due to changes in e.g. fatty acid composition needed for cardiac function [ 97 ]. Accordingly, the nutritional quality of C . sertularioides and C . verticillata , and their specific metabolic requirements for digestions, might not only affect the energy availability but also the physiological capacity of A . triostegus to withstand elevated temperatures. In this context, it is important to consider that fish experiencing higher energetic demands during MHWs may be compounded by the concurrent inability to increase their foraging rates to boost intake. The resulting nutritional and energetic deficiencies, combined with higher SMR, could easily limit energy allocation to processes like growth and reproduction as seen in this study. Indeed, the fact that fish in summer conditions maintained body weight, while MHW fish could not, suggest that it was no longer possible to balanced increased metabolic demands with digestive and assimilative efficiencies. Future research should explore these potential trade-offs, as digestive constraints may further complicate the relationship between foraging, temperature, and energy balance. The reason for unaltered foraging rates in herbivorous fishes is uncertain but may be caused by foraging mode and/or the algal type examined. Unlike piscivores, which may have the flexibility to opt for larger and more frequent meals, herbivores adopt a fermentation-based digestion process resembling an assembly line [ 52 , 58 , 98 ]. Many species are thought to operate at near maximum foraging capacity under current day temperatures, typically spending >80% of their time in this activity [ 48 ], potentially making further increases in foraging rates infeasible [ 52 ]. Interestingly, herbivorous fish in Hawai ʻ i consist of multiple families, each with different digestive strategies. Conducting similar studies on other functional groups of herbivorous reef fish could reveal varying responses to thermal stress due to differences in their digestive physiology and their roles in coral-algal interactions. For instance, unicornfish ( Naso spp .) and parrotfish ( Scarus spp .) are abundant herbivores in Hawai ʻ i that primarily rely on foregut fermentation. In contrast, surgeonfish ( Acanthurus spp .) and chubs ( Kyphosus spp .) exhibit hindgut fermentation [ 99 ]. The foregut fermentation process, involving microbial breakdown of plant material early in digestion, may allow these herbivores to better extract nutrients from algae [ 99 ]. This could theoretically leave some herbivores less impacted by ocean warming. Additionally, Specific Dynamic Action (SDA), which represents the energy used for meal digestion and assimilation likely escalates under increased temperatures, further complicating the energy budget during MHWs [ 83 ]. As SDA varies with diet composition, understanding its impact under different thermal conditions could provide insights into how A . triostegus and similar species manage their energy needs when foraging cannot be increased. This point underscores the need for comparative studies across different herbivore guilds to better understand their ecological roles and resilience to environmental changes. In addition to foraging rates, the algae matrix examined here, Caulerpa sertularioides and C . verticillata contain terpenoid metabolites thought to diminish attractiveness to herbivores [ 78 , 100 ]. It is possible, that these chemicals may diminish palatability or impede rapid digestion in herbivores [ 78 , 100 ], potentially inhibiting increases in herbivore foraging rates even in scenarios where increased foraging is necessary for condition and survival. Indeed, this scenario seems to be supported by our data, as the recorded foraging rates of ~2.4 bites min -1 are substantially below typical foraging rates observed for A . triostegus on other algae species in the wild (typical ~10–50 bites min -1 [ 101 – 103 ]. Here, A . triostegus maintained body mass by foraging on these algae during winter and summer alike, but experienced significant body mass loss during a MHW. As a result, our findings indicate that less attractive algae species may be the first to escape top-down herbivore grazing due to ocean warming, raising concerns about steadily diminished control of problematic algae as oceans warm. It is important to acknowledge that the experiment was conducted in an aquarium setting, which inherently isolates the fish from natural competitors and predators. This controlled environment could potentially result in altered activity rates compared to those observed in natural settings. Additionally, the preference for certain algae may change as temperatures increase, as different diet items can influence the thermal tolerance due to changes in e.g. fatty acid composition needed for cardiac function [ 97 ]. Furthermore, as food was only available between 08:30 and 16:30 HST, this schedule may not fully encapsulate the natural feeding periods of some coral reef fish, which may feed more actively from sunrise to sunset [ 104 ]. In nature, individuals will also have to spend time searching for food and recurrently hide from predator, none of which was issues in our predator-free system with ad-libitum food. However, individuals might still have faced an added challenge of feeding during a shorter time period than possible in the wild, with potentially lower total food intake than theoretically possible. Regardless, the reduced conditions of fishes during the MHW compared to winter and summer conditions reveal that MHWs can drastically change the status quo for feeding patterns of herbivorous fishes, likely demanding either faster or longer feeding periods each day to maintain condition. While the broader implications for different algal species remain uncertain, our data suggest less attractive algae, such as C . verticillata , could face unique challenges as the effectiveness of top-down algal control may diminish as oceans warm. Dietary flexibility in response to changing thermal conditions may also play a key role in the ability of these fishes to maintain fitness and condition during such events. The activity data for A . triostegus revealed that individuals did not attempt to conserve energy during marine heatwaves (MHWs), the only condition where they experienced a substantial change in temperature from their accustomed ambient conditions. Instead, the fish spent 83 to 96% of their time outside the shelter, actively swimming and foraging, with significant increases in activity observed during MHW conditions. Given their constant need to feed, reducing activity levels may not be a practical strategy. The increased activity during elevated MHW temperatures may suggest avoidance behavior, where A . triostegus attempted to escape the warmer conditions. This situation places A . triostegus and other herbivores in a challenging position, where they must constantly forage without any opportunity to conserve energy, while their current foraging strategy cannot match the thermally induced elevations in energy demand. Given the inability to increase food intake or reduce activity, a viable third strategy for herbivorous coral reef fishes to cope with periods of ocean warming may involve relocating to more thermally suitable habitats. Specifically, poleward migration or movement to cooler deeper regions of the reef located below the thermocline could allow a species to remain within optimal thermal conditions [ 34 , 105 ]. However, while scientific studies have demonstrated that some coral reef fish species can successfully migrate to more thermally optimal habitats during extreme temperatures [ 34 , 105 – 108 ], most adult coral reef fishes do not possess the ability to partake in poleward migration as this phenomenon predominantly occur during the dispersal of larval juvenile fishes. Similarly, refuge seeking in deeper sections of the reef may be equally implausible, as MHWs commonly span the entire water column of coral reef environments (including those in our study sites in Hawai ʻ i [ 13 ]. The mismatch between energy demands and energy acquisition caused a precipitous loss of body mass with potential downstream consequences for condition. A 0.8% decrease in body mass on a daily basis is undoubtedly unsustainable, and our findings therefore suggest that these fishes may only be able to continue their trait of top-down algal control for a limited period of time during severe MHWs. If this pattern holds true in the wild, a decrease in the condition and local abundance of herbivorous reef fishes during prolonged warming periods might lead to reduced rates of algal removal, particularly those with herbivore defenses. This reduction could in turn increase the likelihood of ecosystems transitioning from coral dominance to algal dominance [ 19 , 20 ]. This is particularly worrying, as MHWs are projected to increase significantly in frequency, intensity, duration, and expansiveness in the years to come [ 5 , 6 , 16 ]. Overall, our findings indicate that herbivorous coral reef fishes may be more adversely affected by rising ocean temperatures than previously expected, as elevated energetic demands coupled with limitations in energy acquisition severely compromised the condition of a common herbivorous reef fish and/or its ability to help control algal proliferation, at least for some species of algae. Considering the association between rising ocean temperatures and the increased likelihood of transitions from coral dominance to algal dominance, foraging adaptations that underpin the ability of herbivores to mitigate these risks may be in jeopardy. Our results reveal an important relationship between energy demands, foraging patterns, and the condition of herbivorous reef fish in the context of global change ecology. They also highlight that effective herbivore conservation and management strategies, aimed at supporting the resilience of tropical marine ecosystems, would benefit from further research in this field. In particular, more study is needed to determine whether the patterns observed in the present study are consistent across different algal types and the various functional traits of other herbivorous fishes."
} | 5,344 |
34611168 | PMC8492667 | pmc | 6,406 | {
"abstract": "Artificial microbial consortia seek to leverage division-of-labour to optimize function and possess immense potential for bioproduction. Co-culturing approaches, the preferred mode of generating a consortium, remain limited in their ability to give rise to stable consortia having finely tuned compositions. Here, we present an artificial differentiation system in budding yeast capable of generating stable microbial consortia with custom functionalities from a single strain at user-defined composition in space and in time based on optogenetically-driven genetic rewiring. Owing to fast, reproducible, and light-tunable dynamics, our system enables dynamic control of consortia composition in continuous cultures for extended periods. We further demonstrate that our system can be extended in a straightforward manner to give rise to consortia with multiple subpopulations. Our artificial differentiation strategy establishes a novel paradigm for the creation of complex microbial consortia that are simple to implement, precisely controllable, and versatile to use.",
"introduction": "Introduction The evolutionary transition from single cell to multicellular organisms marked a critical turning point in biology 1 . Such shift relied on optimizing fitness and productivity through division of labour and specialization 2 , 3 . The same principle can be extended to microorganisms living together to form microbial communities or consortia. Engineered microbial consortia hold enormous potential and have been hailed as the next frontier in synthetic biology 4 , 5 . Proof of concept studies have concretely established applications in bioproduction 6 , 7 , bioremediation 8 , 9 , and soil microbiome engineering 10 , paving the way for therapeutic applications using human microbiome engineering 11 , 12 . In the context of bioproduction, microbial consortia possess several advantages over traditional monocultures as functional specialization allows metabolic burden to be shared across different species. Diversification thus allows yields to be optimized simply through tuning consortia composition, rather than re-engineering the strain itself 13 . Moreover, by including multiple species, toxic by-products produced by one species can be sequestered and/or metabolized by another, thereby improving the efficiency of the overall process 14 . Microbial consortia are typically generated by culturing two or more species together. Such co-culturing approaches rely on various inter-species interactions to ensure the co-existence of different species like mutualism 15 , emergent cooperation 16 , competitive amensalism 17 , and predation 18 . Despite considerable advances in our ability to engineer microbial consortia 6 , 8 , 9 , 19 – 21 and in our understanding of community interactions 15 , 16 , 18 , 21 , 22 , dynamic control of consortium composition remains a key challenge in the field 19 . Typically, stable consortia are based on syntrophic or quorum sensing interactions that, albeit being autonomous, remain critically dependent on cell density, thus limiting the applicability for dynamic control. Additionally, scaling the consortium to include more than two species requires non-trivial considerations that may not lead to stable co-existence 20 . In light of these limitations, an externally controllable differentiation system could be well suited to address this challenge. In recent years, advances in biological control have come from coupling computers with growing cells carrying the engineered system, made possible by special platforms that integrate biological systems with the computer via a feedback loop 23 – 27 . The development of optogenetics, i.e. the use of light to trigger cellular processes, has contributed significantly to control applications by increasing the spatiotemporal resolution of the control signal 23 , 24 , 28 – 38 . Control of protein expression using light has been demonstrated both at the population level 23 , 28 , 31 and in single cells 30 , 33 , 34 , 37 . Optogenetics has been used to control cellular processes in other contexts, for instance, signalling dynamics 24 , morphogenesis 36 , neuroscience 38 , bioproduction, and metabolic engineering 29 , 35 . However, control of population dynamics using optogenetics in a multispecies environment has not been demonstrated yet. Here, we present an artificial differentiation system in S. cerevisiae capable of generating a microbial consortium composed of functionally different subpopulations emerging from a single population akin to differentiation in multicellular organisms. Concretely, we achieve differentiation into genetically distinct subpopulations—henceforth referred to as species to highlight the analogy to natural microbial consortia—via recombination-based genetic rewiring that can be externally controlled via light. We demonstrate that our system shows desirable features including low background activity, high efficiency for optogenetic recombinases in budding yeast, graded response to varying light signals, absence of hysteresis, and dynamics that are fast, predictable, and tunable. The system reaches >99% differentiation after 4 h of light stimulation and can be stably maintained at any given intermediate level of differentiation for long periods of time (>48 h). Owing to its fast and predictable dynamics, our differentiation system enables rapid and robust bidirectional control of a microbial consortium arising from a single strain at user-defined compositions in continuous cultures for extended periods in dynamic setups. Coupling the system to a growth arrest module allows us to control population growth rates in continuous culture in different physiological contexts. We show that our system can be extended to give rise to complex multispecies microbial consortia. We engineer two differentiation programmes that can be used to control the total number of species. Finally, we show that our system allows for spatial structuring of microbial consortia by imprinting patterns in 2D cultures with high resolution. To the best of our knowledge, this is the first report of light-driven system for control of a microbial consortium.",
"discussion": "Discussion Microbial consortia are expected to be of great utility for biotechnology and hold immense potential for diverse applications 4 – 11 . However, dynamic control of consortium composition remains relatively unexplored despite being a key challenge in the field 19 . In the present study, we address this challenge with the help of an artificial differentiation system in S. cerevisiae capable of generating microbial consortia with custom composition. The system is based on blue light inducible expression of Cre recombinase driven by EL222 from a non-leaky promoter 40 . We characterized the system in small-scale liquid culture (cells growing in a microfluidic chamber), larger-scale liquid cultures (batch and continuous), and short-term solid cultures (monolayer in µIbidi slide) and found it to be functional despite changes in the context. Moreover, we established that it possesses several desirable characteristics: fast, reproducible and tunable dynamics, high efficiency for light inducible recombinases in budding yeast (Supplementary Table 2 ) 41 – 44 , low leakage, and graded response of the population to light (Figs. 1 and 2 ). The efficiency of our system allowed us to achieve high levels of differentiation with short transient pulses that eliminate the risk of phototoxicity. Graded population responses to light were critical for achieving control of consortia composition (Fig. 3 ). Moreover, the high degree of reproducibility in response to light stimuli allowed us to develop a predictive model that could be used as a basis to precisely control microbial community dynamics. Using the developed model in an MPC framework allowed us to achieve bidirectional control of consortia composition in a dynamic setup (Fig. 3 ). We note that Klavins and colleagues 51 have developed another differentiation system in yeast, that, in principle at least, could have been used to generate consortia. This system uses a toggle switch to implement memory, and chemical inducers to toggle the switch. We believe that using light as inducer and a DNA implementation of memory allowed us to precisely characterize and select systems with appropriate properties, and drive them with the needed precision to obtain subpopulations in desired organization and proportions in space and in time, respectively. Several solutions for the stable maintenance of microbial consortia have been proposed recently. In particular, Hasty and colleagues 52 , 53 , Lu and colleagues 21 , 54 , and Barnes and colleagues 17 achieved this by using synthetic biology approaches. These authors demonstrate the capacity to maintain co-cultures of several bacterial subpopulations over extended durations. However, none of these approaches succeeded at precisely controlling consortia composition. Moreover, the functioning of these systems relies on the release of signalling molecules in the environment (quorum-sensing molecules or bacteriocin) that trigger cell death. The fact that signalling molecules are released by cells creates de facto a strong dependency of the functioning on growing conditions, and notably on the density of cell cultures, an important aspect for bioproduction applications. Lastly, previous designs use elaborate genetic engineering solutions for the molecular implementation of control mechanisms, thus making extension and scaling up of these designs potentially challenging. Moreover, external control by light is inexpensive and compatible with most media composition. In summary, in comparison to previously existing solutions, our system is simple to implement, quantitatively predictable and actionable, and versatile to use. The efficiency of the optogenetically inducible recombinase developed in this work exceeds any reported in the literature for optogenetic recombinases in yeast (Supplementary Table 2 ). Previous optogenetic recombination systems are based on engineering a photoactivable Cre that is typically split into two subdomains tagged with the respective photosensitive heterodimers that can be brought together upon light illumination to form a functional Cre 41 – 43 . However, such approaches result in activity loss as functional Cre is a tetramer and the probability of four dimerized split-Cre molecules to assemble together hinges on the relative concentrations of the two subunits 44 . A recent study reported a monogenic photoactivable Cre that is based on fusion of a LOV domain with a destabilized Cre variant 44 . The authors reported higher efficiency and stronger activation when compared to previous systems. This monogenic photoactivable Cre matched the efficiency reported for our system for up to 40 min of induction. After 40 min of induction, the activity seemed to plateau. An optogenetically inducible recombinase has been recently reported in bacteria which uses split-Cre tagged to vivid homodimers 55 . The authors demonstrate the high efficiency and low leakage of their system at the population level. However, these properties are not quantified at the single-cell level, so a precise comparison of efficiency and leakage is not possible. Chemically inducible recombinase based systems have been employed more prominently in bacteria 56 – 58 and show high efficiency (>90%) but graded response or bimodal behaviour has not been reported. Based on the principle of division of labour, microbial consortia have been employed to increase bioproduction by distributing the metabolic burden. Such approaches necessitate functional specialization in the constituent species of consortia. We provided evidence that our design can be implemented in different physiological contexts by coupling it to a growth arrest module (GAuDi system) to allow optogenetic control of growth rate and consortium composition in self-contained continuous cultures (Fig. 4 ). A GAuDi-like system has the potential to facilitate the switch to continuous bioproduction, touted to be the future of bioproduction 59 , by separating growth and production across different subpopulations. In the context of metabolic engineering, our system could serve as a pathway switch, with the potential of compartmentalizing metabolic flux in the population. This could be achieved, for instance, by replacing fluorescent proteins by orthogonal TFs that drive entire pathways 60 , leading to division of labour paradigms in consortia engineering and opening up possibilities for population level metabolic engineering. To show that complex multispecies consortia can be created using our system, we engineered asynchronous or sequential differentiation programmes based on multiple recombination cassettes that extended the core system to generate and stably maintain multispecies consortia from a single strain in continuous liquid cultures (Fig. 5 ). These programmes could be scaled exponentially for applications requiring dynamic control of complex multispecies consortia and do not require intricate genetic circuits spread over multiple populations to ensure stability. Finally, the capacity to optogenetically control cell fate decisions with spatiotemporal precision has the potential to become a critical tool for dissecting signalling pathways 24 or understanding developmental programmes 36 . Here, we showed pattern generation in 2D cultures in a microfluidic plate (Fig. 1 and Supplementary Fig. 20 ). Since we are not restricted to patterns attainable in nature 61 , our system can provide a unique tool to study how equilibria are reached in multispecies ecosystems and elucidate microbial interactions in complex spatially structured communities. In conclusion, we show that the system has highly desirable characteristics making it a practical tool for robustly generating and maintaining functionally distinct subpopulations both in space and in time."
} | 3,499 |
39936903 | PMC11915797 | pmc | 6,407 | {
"abstract": "ABSTRACT Gelatinous zooplankton (GZ) represents an important component of marine food webs, capable of generating massive blooms with severe environmental impact. When these blooms collapse, considerable amounts of organic matter (GZ-OM) either sink to the seafloor or can be introduced into the ocean’s interior, promoting bacterial growth and providing a colonizable surface for microbial interactions. We hypothesized that GZ-OM is an overlooked marine hotspot for transmitting antimicrobial resistance genes (ARGs). To test this, we first re-analyzed metagenomes from two previous studies that experimentally evolved marine microbial communities in the presence and absence of OM from Aurelia aurita and Mnemiopsis leidyi recovered from bloom events and thereafter performed additional time-resolved GZ-OM degradation experiments to improve sample size and statistical power of our analysis. We analyzed these communities for composition, ARG, and mobile genetic element (MGE) content. Communities exposed to GZ-OM displayed up to fourfold increased relative ARG and up to 10-fold increased MGE abundance per 16S rRNA gene copy compared to the controls. This pattern was consistent across ARG and MGE classes and independent of the GZ species, indicating that nutrient influx and colonizable surfaces drive these changes. Potential ARG carriers included genera containing potential pathogens raising concerns of ARG transfer to pathogenic strains. Vibrio was pinpointed as a key player associated with elevated ARGs and MGEs. Whole-genome sequencing of a Vibrio isolate revealed the genetic capability for ARG mobilization and transfer. This study establishes the first link between two emerging issues of marine coastal zones, jellyfish blooms and ARG spread, both likely increasing with future ocean change. Hence, jellyfish blooms are a quintessential “One Health” issue where decreasing environmental health directly impacts human health. IMPORTANCE Jellyfish blooms are, in the context of human health, often seen as mainly problematic for oceanic bathing. Here we demonstrate that they may also play a critical role as marine environmental hotspots for the transmission of antimicrobial resistance (AMR). This study employed (re-)analyses of microcosm experiments to investigate how particulate organic matter introduced to the ocean from collapsed jellyfish blooms, specifically Aurelia aurita and Mnemiopsis leidyi , can significantly increase the presence of antimicrobial resistance genes and mobile genetic elements in marine microbial communities by up to one order of magnitude. By providing abundant nutrients and surfaces for bacterial colonization, organic matter from these blooms enhances ARG proliferation, including transfer to and mobility in potentially pathogenic bacteria like Vibrio . Understanding this connection highlights the importance of monitoring jellyfish blooms as part of marine health assessments and developing strategies to mitigate the spread of AMR in coastal ecosystems.",
"introduction": "INTRODUCTION Gelatinous zooplankton represents an important component of marine food webs inhabiting tropical to polar marine ecosystems. They represent ~30% of the total biovolume, corresponding to 8%–9% of the globally stored carbon in planktonic communities ( 1 ). The most common groups among marine gelatinous zooplankton include medusae (jellyfish), ctenophores (comb jellies), salps, and chaetognaths. Within the context of climate change, given future ocean projections and considering the adaptability of gelatinous zooplankton to a wide range of environmental conditions these organisms will likely increasingly dominate planktonic marine ecosystems, leading to significant changes in the ocean’s carbon cycle ( 1 – 3 ). An increase in gelatinous zooplankton abundance has already been recorded worldwide, particularly in anthropogenically impacted coastal areas, which is threatening marine ecosystem health and services ( 2 , 4 ). Due to a combination of life-history traits and low metabolic requirements certain gelatinous zooplankton species (e.g., Mnemiopsis leidyi, Aurelia aurita ) ( 5 , 6 ) are capable of generating massive gelatinous zooplankton blooms (hereinafter GZ-blooms), representing an important perturbation to marine ecosystems ( 7 – 9 ). These GZ-blooms are often followed by a sudden collapse of the entire population resulting in a large influx of gelatinous zooplankton detrital organic matter (hereinafter GZ-OM) that transforms the ambient seawater organic matter pool by releasing considerable amounts of bloom-specific particulate and dissolved organic and inorganic matter compounds ( 10 – 12 ). GZ-OM can then be degraded and consumed at different rates in a cascade by specific microbial assemblages, dominated by copiotrophic bacterial lineages, with consistent metabolic fingerprints ( 7 , 9 , 13 ). In this study, we hypothesize that the GZ-OM introduced into ocean ecosystems upon the decay of GZ-bloom events serves as a yet overlooked hotspot for transmitting antimicrobial resistance genes (ARGs) in marine environments. Antimicrobial resistance (AMR) and the spread of ARGs is one of the major global health challenges ( 14 ) with globally already 4.71 million deaths associated and 1.14 million deaths directly attributable to bacterial AMR in 2021 ( 15 ). To mitigate the predicted future rise in these numbers, it is important to understand AMR evolution, selection, and transmission within and across all interconnected “One Health” compartments (humans, animals, and the environment) ( 16 , 17 ). Especially, understanding the biotic and abiotic drivers underlying this spread is crucial to creating targeted intervention measures ( 18 , 19 ). With abundance of ARGs increasing in many ecosystems due to anthropogenic activities ( 20 , 21 ), marine ecosystems and their microbiomes are no exception ( 16 , 22 ). In particular, coastal zones as a likely entry point of ARG-carrying microbes from, for example, wastewater effluents to the marine environments are in the spotlight as they provide exposure points to humans who are using them recreationally ( 23 ). For example, increased colonization of marine surfers with AMR bacteria has previously been proven ( 24 ). Moreover, various pollutants of marine ecosystems ranging from chemicals to microplastics can contribute to the spread of AMR in non-coastal marine ecosystems which can accumulate in marine animal microbiomes and subsequently through the food chain be conveyed back to terrestrial animals and humans ( 25 ). Yet, potential links between these two emerging issues of anthropogenically impacted marine zones, bloom-forming gelatinous zooplankton species, and ARG have not been explored. One of the most important ecological mechanisms underlying this spread of ARGs is the conjugative transfer of ARG-encoding mobile genetic elements (MGEs) such as plasmids ( 26 , 27 ). These conjugative plasmids can spread between closely related bacteria but also be transferred to phylogenetically distant bacterial groups ( 28 – 31 ). Plasmid transfer rates in aquatic environments are particularly elevated when bacterial abundances and activity are high, ensuring high bacterial encounter rates such as in biofilm formed on the surfaces of particles ( 32 – 34 ). Both these factors are given during the decay of GZ-bloom events as the released GZ-OM represents an abundant substrate to promote bacterial growth and copious colonizable surfaces for interactions. Furthermore, microbial degraders of GZ-OM, potentially enriched in AMR, could hitchhike on these organic particle surfaces by drifting with ocean currents over long distances into the oceanic interior ( 35 , 36 ) and/or connect the microbiomes of upper and bottom ocean layers upon sinking to the ocean floor ( 37 , 38 ). Thus, GZ-bloom events could provide a yet overlooked hot spot for the spread of AMR and a potential vector of AMR/ARG transmission in marine environments. Consequently, to address the hypothesis that GZ-OM degrading microbial communities provide a hot spot for the spread of AMR, we re-analyzed existing metagenomic data sets ( 7 , 13 ) from microcosm experiments previously conducted and performed and analyzed new, time-resolved, and replicated GZ-OM degradation experiments to increase the statistical power of the analysis. This provided insights into how the microbial degradation of biomass from different bloom-forming gelatinous zooplankton species affects the abundance, diversity, and dynamics of AMR and MGEs.",
"discussion": "DISCUSSION With this study, we established the first link between two emerging marine issues, jellyfish blooms and AMR spread, both likely increasing in projected future ocean scenarios ( 5 , 80 ). Metagenomic analysis of marine microbial communities exposed to GZ-OM confirmed our hypothesis that decaying GZ-blooms represent a yet overlooked hot spot of AMR proliferation in marine environments. Already after 2–4 days of exposure to GZ-OM, we recorded an up to fourfold increase in relative ARG abundance per 16S rRNA gene copy in the degrader communities compared to ambient marine microbiomes. This increase becomes particularly relevant when considering that bacterial production rates due to the nutrient influx through degradable GZ-OM in the otherwise nutrient-poor marine environment can be up to one order of magnitude elevated ( 5 ) with absolute bacterial biomass increasing 10- to 100-fold in the microcosms ( 7 , 12 , 13 ), with equal numbers being reported for natural ecosystems exposed to GZ-OM after bloom events ( 81 ). Combining this increase in absolute bacterial and relative ARG abundances, GZ-blooms are predicted to result in an absolute increase of ARG abundance by several orders of magnitude compared to the surrounding marine microbiomes. The observed trait was consistent, independent of the gelatinous zooplankton species and the year of the experiment, suggesting that the underlying mechanism of this increase in AMR is based on the general influx of nutrients and colonizable surfaces through GZ-OM. Still future work should aim at disentangling the individual contributions of these two general mechanisms, which are further supported by the phylogenetic diversity of the colonizing bacterial communities being highly similar across GZ-OM from different species both in our analysis as well as in studies performed in similar regions of the Mediterranean Sea ( 82 – 85 ). It furthermore proved to be a significant explanatory variable for the observed ARG diversity and increased ARG abundance. Potential carriers of these increasing ARGs were consistent in all our data sets and included Pseudoalteromonas , Vibrio , and Alteromonas , known key GZ-OM degraders ( 7 , 8 , 13 , 84 , 85 ), as well as Thalassotalea , Colwellia associated with psychrophilic lifestyle ( 69 ), Algicola, regular colonizers of algal surfaces ( 70 ), and other potential degraders of hydrocarbons in marine environment ( Oleiphilus , Anaerosinus ). More importantly, when considering the risks associated with the observed enrichment of ARGs, several genera that contain known potential human or animal pathogens ( Enterobacter , Escherichia-Shigella , Acinetobacter , Vibrio , Pajaroellobacter , Francisella , and Arsenophonus ) ( 67 ) were identified to not only be significantly increased in relative abundance in the degrading communities but also correlated with specific ARGs as their potential carriers. This is consistent with previous reports that jellyfish-colonizing microbiomes regularly include elevated proportions of potential human pathogenic strains ( 84 , 85 ). The simultaneous increased abundances of potential pathogens and ARGs in the degrading communities do not, on their own, immediately translate into an elevated risk if ARGs are not transferred. Our data provides a strong indication that such horizontal acquisition of ARGs by these potential pathogenic strains is indeed taking place. First, similar to ARGs, the relative abundance of MGEs in the GZ-OM degrading communities was significantly elevated. These ARG-encoding MGEs have the potential to be transferred even to phylogenetically distant bacterial groups ( 28 – 31 ) and horizontal gene transfer rates are particularly elevated when bacterial abundances and activity are high and bacteria have high encounter rates ( 32 , 33 , 86 , 87 ) such as in biofilms formed on GZ-OM particles. The observed high connectivity between marine environmental and potentially pathogenic species in the ARG-genera co-occurrence network as co-hosts of specific identical ARGs suggests that this scenario indeed occurs. The feasibility of increased ARG transfer is moreover supported by Vibrio and Alteromonas, identified as main players during GZ-OM degradation, being well known for their ability to engage in marine horizontal gene transfer through diverse pathways including conjugation ( 88 , 89 ), transduction ( 90 ), or transformation ( 91 , 92 ). Many Vibrio strains are naturally competent and can take up and integrate free DNA through transformation ( 91 ). Their ability to obtain such DNA for incorporation through competitive mechanisms (e.g., type VI secretion systems) from other bacteria ( 91 , 93 ) might play a major role in the high bacterial density scenarios that are found in biofilms formed on GZ-OM particles. Second, genomic analysis of the A. aurita -associated V. splendidus strain revealed a high number of insertion sequences, and numerous multidrug-efflux pumps but also the ARGs tet ( 33 ) and qnrS carried in the two identified chromosomes. The quinolone resistance encoding qnr genes are generally plasmid-associated ( 94 ), while tet ( 33 ) has been observed in a broad range of environmental hosts ( 95 ), suggesting its general mobility. In addition, a yet unknown plasmidic structure was identified that hosts several insertion sequences, a copy of the hip A gene able to induce a dormant state that favors the persistence of ARGs ( 77 , 78 ), and a bacteriocin efflux pump as part of a compound transposon. Together, this provides a strong indication that the GZ-OM degrading communities have members that indeed possess the necessary genomic plasticity that provides a high potential of acting as donors and recipients of horizontally transferable ARGs. This could constitute a significant risk, as these GZ-OM colonizing communities enriched in AMR and potential pathogens that could acquire novel ARGs can hitchhike on these particle surfaces by drifting with ocean currents over long distances in the ocean interior and coastal environments where exposure to marine organisms of higher complexity such as fish, crustaceans or mollusks (e.g., also commercially important groups) and/or humans is likely ( 35 , 36 ). When considering gelatinous zooplankton detritus as a hotspot for the marine spread of AMR, it is also likely that living gelatinous zooplankton colonized by bacteria could equally play a role. Here it is additionally relevant to consider their life stage-specific features. During the polyp stage, meroplanktonic gelatinous zooplankton species are mostly found in coastal areas that frequently are highly anthropogenically impacted (e.g., pillars of industrial ports), where they can accumulate different types of pollutants ( 96 – 98 ). These could provide a (co-)selective potential for ARGs of their microbial colonizers ( 99 , 100 ) while also being in more direct contact with potential colonizers enriched in ARGs (e.g., from wastewater discharged into the ocean ( 24 )). After polyp strobillation, syphozoan medusae transition into the planktonic stage of their life cycle, with ephyrae developing into the adult medusa stage. These planktonic stages can drift and/or swim with ocean currents over long distances, and in this way represent an overlooked route of AMR (and ARG) to otherwise not impacted environments. Similarly, ctenophores, which spend their entire life cycle in the planktonic stage, can contribute to this dispersal. For instance, invasive species like Mnemiopsis leidyi , which invaded many coastal marine ecosystems globally, might present a special threat. During their planktonic life, they are efficient grazers of a significant part of the ocean’s planktonic production ( 2 , 101 ) and can further accumulate nanoparticles, microplastic debris ( 102 – 105 ), heavy metals, and pollutants ( 106 ) which have the potential to (co-)select for ARGs and increase horizontal gene transfer rates of the colonizing microbes ( 29 , 30 , 32 , 99 ). These unexplored aspects regarding the spread of AMR in connection with GZ need to be studied and taken into account, especially when considering harvesting GZ for food, fertilizers, medicine, and cosmetics, or considering their use in wastewater treatment applications ( 107 , 108 ). Still, it needs to be taken into consideration that the here employed short-term microcosm experiments may not fully replicate natural scenarios, but they can serve as an important first step toward understanding the complex interactions that occur in marine ecosystems exposed to GZ-blooms. These controlled experiments provide valuable insights and form a foundation for future studies under more natural conditions. Moreover, such future studies could employ deeper sequencing and long-read-based techniques as well as novel, molecular, PCR-based techniques ( 109 , 110 ) to gain further insights into ARG-host and ARG-MGE associations during GZ-OM degradation. In conclusion, we here provide evidence that jellyfish blooms are a quintessential “One Health” issue where decreasing environmental health is immediately connected to benign effects on human health by amplifying the spread of antimicrobial resistance genes and their potential transfer to human pathogens. This is of particular relevance as both issues are likely to increase in importance with current climate change projections."
} | 4,502 |
33565580 | PMC8136488 | pmc | 6,408 | {
"abstract": "Abstract Horizontal gene transfer (HGT) is central to prokaryotic evolution. However, little is known about the “scale” of individual HGT events. In this work, we introduce the first computational framework to help answer the following fundamental question: How often does more than one gene get horizontally transferred in a single HGT event? Our method, called HoMer , uses phylogenetic reconciliation to infer single-gene HGT events across a given set of species/strains, employs several techniques to account for inference error and uncertainty, combines that information with gene order information from extant genomes, and uses statistical analysis to identify candidate horizontal multigene transfers (HMGTs) in both extant and ancestral species/strains. HoMer is highly scalable and can be easily used to infer HMGTs across hundreds of genomes. We apply HoMer to a genome-scale data set of over 22,000 gene families from 103 Aeromonas genomes and identify a large number of plausible HMGTs of various scales at both small and large phylogenetic distances. Analysis of these HMGTs reveals interesting relationships between gene function, phylogenetic distance, and frequency of multigene transfer. Among other insights, we find that 1) the observed relative frequency of HMGT increases as divergence between genomes increases, 2) HMGTs often have conserved gene functions, and 3) rare genes are frequently acquired through HMGT. We also analyze in detail HMGTs involving the zonula occludens toxin and type III secretion systems. By enabling the systematic inference of HMGTs on a large scale, HoMer will facilitate a more accurate and more complete understanding of HGT and microbial evolution.",
"introduction": "Introduction The transfer of genetic information between organisms that are not in a direct ancestor–descendant relationship, called horizontal gene transfer (HGT), is a crucial process in microbial evolution. For instance, HGT of pathogenicity and other genomic islands facilitate adaptation to new ecological niches ( Hacker et al. 1997 ; Gogarten et al. 2002 ; Dobrindt et al. 2004 ; Papke and Gogarten 2012 ); HGT helps maintain cohesion within groups or phylotypes of organisms ( Papke et al. 2004 ; Polz et al. 2013 ); gene transfer, not autochtonous gene duplication, is the most important process for gene family expansion in bacteria and archaea ( Treangen and Rocha 2011 ); and gene transfer together with vertical inheritance shaped the microbial tree of life ( Hilario and Gogarten 1993 ; Doolittle 1999 ; Andam and Gogarten 2011 ; Pace et al. 2012 ). In fact, HGT is so common that the number of distinct genes present in a species far exceeds the number of genes present in any individual genome ( Lapierre and Gogarten 2009 ; Puigbo et al. 2014 ; Fullmer et al. 2015 ; Soucy et al. 2015 ); for example, less than 10% of the nonoverlapping gene set from 61 Escherichia coli is present in all the genomes that were included in the analysis ( Lukjancenko et al. 2010 ). Despite the importance of HGT to microbial evolution, surprisingly little is known about the scale of individual HGT events. Specifically, an HGT event may involve the transfer of a gene fragment, a single complete gene, or multiple complete genes, and very little is currently known about the units of HGT events. Chan, Beiko, et al. (2009) were among the first to conduct a systematic study of the scale of HGT events. The study considered gene families from 144 prokaryotic species and distinguished between HGTs that transferred a complete gene and those that transferred only a part of gene based on finding recombination breakpoints in gene family alignments. The study found that both gene-level and subgene-level HGTs were common and that pathogens were more likely to engage in gene-level HGT than nonpathogens. However, this study only considered single-copy gene families and did not study transfers involving multiple genes. A related study by Chan, Darling, et al. (2009) , using the same methods as Chan, Beiko, et al. (2009) , rejected the hypothesis that protein domains acted as units of HGT. Szöllősi et al. (2015) studied single-gene HGT among fungi and cyanobacteria and, based on gene order information for terminal taxa, they observed that many HGTs between terminal branches appeared clustered together on genomes, suggesting the presence of multigene transfers. Phylogenetic analysis coupled with either sequence similarity analysis or phylogenetic reconciliation techniques have also been used to identify some instances of plasmid-borne horizontal transfer of gene clusters ( Petersen and Wagner-Dobler 2017 ; Brinkmann et al. 2018 ). More recently, Dunning et al. (2019) used multiple grass genomes and phylogenetic comparative analysis to find 59 single-gene HGTs into Alloteropsis semialata that were organized into 23 acquired genome fragments, suggesting horizontal transfer of genomic fragments containing multiple genes. Although these previous studies have helped establish the presence of multigene horizontal transfers, there do not currently exist any rigorous computational frameworks for systematically detecting and quantifying plausible multigene horizontal transfers. Researchers have also previously explored “highways of gene sharing” in microbes ( Beiko et al. 2005 ; Zhaxybayeva et al. 2009 ; Bansal, Banay, et al. 2013 ). These highways represent pairs of species or species groups that are connected to each other by a multitude of HGT events. Highways result when divergent organisms share an ecological niche and engage in gene transfer for extended periods of time. Highways capture the magnitude of HGT that has occurred between a pair of species or species groups but do not shed light on the units of transfer for individual HGT events. In this work, we focus on the problem of systematic, automated discovery of high-confidence instances where multiple complete genes were transferred in a single horizontal transfer event; we refer to such horizontal transfers as horizontal multigene transfers (HMGTs). We develop a novel computational framework, called HoMer (for horizontal multigene transfer), that builds upon recent computational advances in the detection of single-gene HGTs and leverages large-scale availability of microbial genomic data sets to infer plausible HMGTs. HoMer infers single-gene HGT events across the given set of species or strains using phylogenetic reconciliation, uses several techniques to account for (single-gene) HGT inference uncertainty, combines that information with gene order information, and uses statistical analysis to identify candidate (multigene) HMGTs. HoMer can infer HMGTs not only between terminal taxa but also between ancestral species (internal edges) on the species tree, allows for easy adjustment of the stringency of detected HMGTs, and can be used to estimate statistical support for the inferred HMGTs. It is also highly scalable and can be applied to hundreds of taxa in a matter of hours. We apply HoMer to a genome-scale data set of over 22,000 gene families (or consolidated homologous groups) from 103 Aeromonas strains representing 28 different species ( Rangel et al. 2019 ), and infer a large number of plausible HMGTs of various scales at both small and large phylogenetic distances. Aeromonas are a genus of Gram-negative bacteria that are known to cause disease in humans and fish. They are found in water and sediments and live in beneficial associations with fish and leeches ( Janda and Abbott 2010 ; Milligan-Myhre et al. 2011 ; Marden et al. 2016 ; Fernandez-Bravo and Figueras 2020 ). The Aeromonas genus serves as an excellent test case for this study because of the availability of genomes from 28 distinct species with multiple strain genomes available for several of these species, resulting in a broad data set with sufficient breadth and depth to assess both inter- and intraspecies HGTs and HMGTs. Moreover, the presence of frequent HGT within the Aeromonads has been previously established ( Morandi et al. 2005 ; Silver et al. 2011 ; Colston et al. 2014 ). Analysis of HMGTs inferred on the Aeromonas data set reveals several fundamental insights and interesting relationships between gene function, phylogenetic distance, and frequency of multigene transfer. For instance, we find that 1) the observed relative frequency of HMGT increases as divergence between genomes increases, 2) genes transferred together in an HMGT often belong to the same COG functional category, and 3) rare genes are frequently acquired through HMGT. We also analyze in detail some specific HMGTs involving type III secretion systems (T3SS) and the zonula occludens toxin (ZOT). This work makes it feasible, for the first time, to systematically infer HMGTs on a large scale, and demonstrates the prevalence and significance of HMGTs in microbial evolution. The systematic discovery of HMGTs, enabled by HoMer, will help advance our understanding of horizontal gene transfer and microbial evolution. HoMer is freely available open-source from https://compbio.engr.uconn.edu/software/homer/ . The Aeromonas data set used in this work and a complete list of putative HMGTs discovered for this data set are also freely available from the same URL.",
"discussion": "Discussion In this work, we introduced a new computational framework, HoMer, for the systematic discovery of HMGTs at a large scale. Its application to the Aeromonads demonstrates the prevalence of HMGTs as well as their significance to microbial evolution. For instance, we found that HMGTs are ubiquitous and a large fraction of transferred genes are transferred as part of HMGTs, at both short and large phylogenetic distances. We also found that the observed relative frequency of HMGT increases as divergence between genomes increases, that HMGTs often have conserved gene functions, that genes from all functional categories appear to be roughly equally likely to be transferred as part of HMGTs, and that rare genes acquired from outside a particular clade of interest are frequently acquired through HMGT. Our analysis of HMGTs involving the ZOT and T3SS shows that within-genus HMGTs play an important role in diversifying host–symbiont interactions, and that in the case of the ZOT, phages appear to play a major role in shuffling the ZOT gene neighborhood via repeated recombination and invasion events. These analyses also have some limitations, as we discuss below. Selection, Drift, and the Bacterial Pan-Genome It is important to conceptually distinguish genes that, following a gene transfer event, are found in a genome but that do not provide a selective advantage to the organism or to themselves, from genes that are either selfish genetic elements and/or contribute to the fitness of organism or population harboring them. The situation is comparable to distinguishing mutations (or single nucleotide polymorphisms) observed in a population from substitution events (i.e., mutations fixed in a lineage). This distinction becomes especially important if one considers rates of gene acquisition over time. In our analyses, when studying recent transfers into branches leading to leaves, we cannot distinguish between genes that will be only transient residents in the recipient lineage from genes that will be fixed in the lineage due to genetic drift, due to selection at the gene level (selfish genetic elements), and/or selection due to increased fitness of the recipient organism. The first quantitative assessments of HGT ( Lawrence and Ochman 1998 ) already observed that a large fraction of genes acquired in a lineage reside in the recipient lineage only temporarily. Lawrence and Ochman (1998) estimates that the E. coli lineage since divergence from Salmonella acquired about 1,600 kb of DNA through HGT, of which only 548 kb persist in the lineage today ( Lawrence and Ochman 1998 ; Lawrence 1999 ). Williams et al. (2012) made the surprising observation that genes that are part of operons frequently integrate into the recipient genome through homologous recombination, resulting in homologous replacement even between species belonging to different genera. This illustrates that coevolution between genes that are part of an operon does not necessarily result in a strong selective force against gene transfers that break up coevolution, suggesting that at least some of the acquired genes, including those that are fixed in the recipient lineage, may be selectively neutral with respect to the gene they replace and may be fixed due to genetic drift. The discussion of fixed and only transiently acquired HGTs is further complicated by the fact that many bacterial and archaeal species possess pan-genomes much larger than the genome of an individual ( Tettelin et al. 2005 ). If one considers the pan-genome as a shared genomic resource ( Soucy et al. 2015 ), or if an ecotype has a selective advantage only under temporary but recurring environmental conditions ( Viver et al. 2020 ) a gene or variant genome may persist in a population for a long time, without ever being fixed in the population. Many genes that adapt organisms to a particular ecological niche are present on genomic islands, their mobility often facilitated by flanking selfish genetic elements. Although these genes may be fixed in organisms occupying a particular niche, they are not necessarily fixed in the species ( Papke and Gogarten 2012 )—obviously, this discussion is complicated by the lack of a generally accepted prokaryotic species concept. In case organisms are engaging in frequent gene transfer followed by homologous recombination, the biological species concept can be extended to prokaryotes ( Dykhuizen and Green 1991 ); however, the boundaries of exchange communities are less strict in bacteria and archaea than in eukaryotes, making the delineation less precise ( Gogarten et al. 2002 ; Retchless and Lawrence 2007 ). Selfish genetic elements provide a particular challenge. They might persist for some time in a population due to their selfishness but they also facilitate the transfer of genomic islands, and it is often not clear if an element persists due to its selfishness or due to its contribution to within species variation. The bacterial defense systems that we observe as transferred illustrate this point. Restriction modification systems are addiction cassettes and thus may be considered selfish; however, their presence in only part of a population prevents the whole population from being wiped out by a virus ( Seshasayee et al. 2012 ; Kong et al. 2013 ; Fullmer et al. 2019 ). Given the high frequency of genes in our study that did not have a clearly identified function, we were not able to analyze HMGTs that did not involve selfish or MGEs. Genomic islands include ecological and pathogenicity islands, and islands exclusively consisting of a MGEs ( Langille et al. 2010 ). We find less than 15% of across-species HMGTs and less than 5% of within-species HMGTs associated with MGEs; nevertheless, we expect that most HMGTs between species, especially between divergent ones, represent genomic islands. A more detailed analysis of genome sequences surrounding the integration sites that also pays attention to nonprotein encoding features such as direct repeats and tRNA coding genes will be needed to verify this hypothesis. Similarly, a comprehensive identification of HMGTs that aid in ecological adaptation remains difficult at present because most of the genes that are part of HMGTs have no identified function. Methodological Limitations and Biases HoMer is easy to use, scalable, and effective, and makes it feasible to systematically infer HMGTs on a large scale. We expect that the systematic discovery of HMGTs, enabled by this work, will lead to enhanced understanding of horizontal gene transfer and microbial evolution. Nonetheless, the current HMGT inference framework implemented in HoMer has some limitations and potential biases worth understanding. A key limitation is that our ability to infer HMGTs depends on there being sufficient phylogenetic resolution in the gene trees to reasonably detect (single-gene) HGT events. This limitation makes it harder to infer HGTs and HMGTs between closely related pairs of strains or species, and can thus bias HMGT inference results by resulting in a greater false-negative rate for such pairs. Another important limitation is that our approach is focused on finding HMGTs that are “large enough” to be unlikely to occur by chance. In other words, to control for the false-positive rate, the 〈 x , y , z 〉 parameter values have to be set conservatively. However, as our statistical analysis ( table 2 ) suggests, the vast majority true HMGTs may be smaller than are detectable using our default 〈 x , y , z 〉 parameter setting of 〈 3 , 4 , 1 〉 . Future Directions Although our experimental analysis with the Aeromonads sheds light on the prevalence of HMGT and provides several fundamental insights, many important questions remain unanswered. For instance, in addition to the hypothesis related to genomic islands posed above, it would be interesting to investigate if HMGTs tend to correspond to operon boundaries or to functional pathways. It would also be useful to extend our computational framework to make it more suitable for detecting HMGTs between more distantly related species with little gene order conservation. This may be achieved by combining HoMer with methods that model genome rearrangement and/or infer ancestral genome orderings. Finally, it would be useful to develop and apply appropriate statistical tests to determine the statistical significance of inferred individual HMGTs or groups of HMGTs of interest."
} | 4,437 |
37425797 | PMC10327127 | pmc | 6,409 | {
"abstract": "Most biomolecular systems are dependent on a complex interplay of forces. Modern force spectroscopy techniques provide means of interrogating these forces. These techniques, however, are not optimized for studies in constrained or crowded environments as they typically require micron-scale beads in the case of magnetic or optical tweezers, or direct attachment to a cantilever in the case of atomic force microscopy. We implement a nanoscale force-sensing device using a DNA origami which is highly customizable in geometry, functionalization, and mechanical properties. The device, referred to as the NanoDyn, functions as a binary (open or closed) force sensor that undergoes a structural transition under an external force. The transition force is tuned with minor alterations of 1 to 3 DNA oligonucleotides and spans tens of picoNewtons (pN). This actuation of the NanoDyn is reversible and the design parameters strongly influence the efficiency of resetting the initial state, with higher stability devices (≳10 pN) resetting more reliably during repeated force-loading cycles. Finally, we show that the opening force can be adjusted in real time by the addition of a single DNA oligonucleotide. These results establish the NanoDyn as a versatile force sensor and provide fundamental insights into how design parameters modulate mechanical and dynamic properties.",
"introduction": "INTRODUCTION Biomolecular functions are often driven by inter- and intramolecular forces. Thus, elucidating the forces within and between biomolecular systems provides critical insight into the mechanisms of their functions 1 – 3 . Molecular force spectroscopy has been a powerful approach for probing the interactions that are responsible for these forces and providing mechanistic insight into function 4 – 7 . However, current force spectroscopy techniques have limitations such as challenges with force measurements in constrained or crowded environments. For instance, both magnetic and optical tweezers necessitate the use of large (>1 µM) beads, which act as handles for applying forces on nanoscale samples 6 – 9 . Atomic force microscopy requires the sample be attached to a cantilever tip 4 , 6 , 10 – 13 . These methodologies are limited to systems where space is available for the handles, which makes it challenging to implement these approaches within cells 14 , 15 and nanofluidic devices 16 , 17 . Here we present the development and investigation of a DNA Origami (DO) nanodevice that has the potential to address these limitations. DO nanotechnology has significant promise in developing nanodevices for complex functions including drug delivery 18 – 20 , molecular sensing 21 , 22 , and probing single molecule dynamics and interactions 23 – 28 . More specifically, DO has been established as a useful approach for single molecule force sensing, with demonstration of DO devices applying and detecting both tensile and compressive forces 29 – 32 . Complex and dynamic 3-dimensional DO nanodevices can perform prescribed functions through controlled actuation, making their use precise and reproducible 30 , 33 – 35 . DO devices are biocompatible, functionalizable, and on the nanometer (nm) size scale, which are key characteristics that position them to function within complex nanoscale environments. In addition, the intrinsic modularity of DO devices allows them to be modified and tuned without the need for redesign of the primary structure 36 . In this study we focus on a DO force sensor, the NanoDyn (ND), which has been previously shown to be sensitive to compressive depletion forces 30 . Hudoba et al . introduced the ND as a sensitive reporter of compressive depletion forces due to local molecular crowding on the order of 100 femtoNewtons (fN) and with a lower limit of force detection of 40 fN. Furthermore, this work demonstrated the viability of this device operating in molecularly crowded environments. Here, we build on that research and demonstrate the utility of the ND not only as a highly sensitive reporter of compressive depletion forces, but also as a robust, dynamic device capable of detecting tensile forces from picoNewtons to tens of picoNewtons (pN) where device design parameters allow tunable control of the force response. Taking advantage of the modular nature of the ND, we show that an individual single stranded DNA molecule, which we refer to as a zipper strand, can be modified to set the force-sensing capabilities and be incorporated after folding and purifying the ND. This allows for rapid and efficient tuning of the device and eschews the need to fold and purify a separate structure for different force applications. We investigated its response to tensile forces and determined that it can be tuned to be sensitive to a range of forces through the adjustment of 1 to 3 zipper strands. We show that the ND detection force can be adjusted between 5–13 pN by changing a single zipper strand within the device. We then demonstrate that by incorporating multiple zippers in parallel, forces of about 30 pN with the potential of even higher forces can be detected. We find that more stable interactions (opening forces ≳ 10 pN) lead to a higher reclosure probability. Finally, we show that the force-sensing range of the ND can be adjusted in real time by iteratively incorporating DNA zippers in situ . This study lays the groundwork for a modular and versatile force probe that has the potential to be used in complex biological systems where traditional force spectroscopy techniques are challenging or impractical to implement.",
"discussion": "DISCUSSION In this work, we demonstrated that the DNA origami ND can function as a modular nanodevice that can be tuned to detect a range of tensile forces. The modular design allowed the base-structure to be folded, purified, and stored without force-sensing loops. Then, immediately before use, the force detection range was customized by incorporating one or more zipper stands. We demonstrated that varying the zipper region length within a single force-sensing loop modulated the opening force over a range of 3-fold up to a maximum unzipping force of about 15 pN 39 , 40 . We then showed that including multiple force-sensing loops in parallel enabled a wider range of opening forces up to 26 pN with only 3 relatively weak zippers, which exceeds the inherent unzipping force of 15 pN. We found that the interaction stability affected the reversibility of ND, with median opening forces below 10 pN not reclosing reliably, indicating that they will not function well for the sensing of repeated force cycling. However, devices with a median opening force above 10 pN repeatedly reclosed, which confirms their utility in detecting repeated force applications. Finally, we showed that single DNA zipper strands can be iteratively incorporated into the ND during a force measurement. This opens the possibility for tuning the force-sensing range of single NDs in real time during a measurement. This work expands the utility of using nanoscale force sensors as a complementary approach to existing force spectroscopy techniques. The ND has the potential to probe a wide range of forces in constrained environments where it can be difficult to implement other force spectroscopy techniques 4 , 6 – 12 . We previously showed that the ND can operate in crowded environments and detect compressive depletion force in the range of 0.05 to 1 pN 30 . Here in this work, we demonstrated the same ND base structure can also be used to detect tensile forces from 6 to at least 26 pN. The ability for the ND to operate in different modes for detecting both compressive and tensile forces with order of magnitude different force ranges indicates its high versatility for a DNA origami device 23 , 25 , 26 , 29 – 31 , 46 , 47 . In comparison to the previous study presented in Dutta et al. 29 , our results indicate the ND provides a wider dynamic range of force sensing. This is consistent with the idea that integrating the force-sensing loops between the two-barrel structures allows for a more balanced distribution of the force on these force-sensing loops. There is the potential for further versatility of the ND since up to six force-sensing loops could be included in the ND, each with independent nucleotide sequences to which DNA zippers can be incorporated independently and reproducibly. Assuming an opening force of 10 pN of force per force-sensing loop, using six force-sensing loops should result in an opening force of more than 60pN of force. These large forces do occur in biological systems including the forces on phage genomes during viral packaging 48 , 49 and the forces on mitotic chromosomes during mitosis 50 . However, measurements of these high forces will require covalent attachments or multiple non-covalent attachments to prevent failure of the attachment before device opening and force detection 13 , 51 . The overall length of the ND at 100 nm in length is advantageous for constrained environments. However, for experiments requiring smaller devices, the overall length of the ND could be reduced by designing shorter barrels, while retaining the loop regions. The shortened length could be accomplished with the same DNA scaffold by increasing the width, or with a shorter DNA scaffold. In addition to our current method of monitoring relative length change with magnetic tweezers, a fluorophore pair that undergoes Förster Resonance Energy Transfer (FRET) can be incorporated into the ND with 2 fluorophore labeled oligos, as reported in Hudoba et al . 30 , where high FRET reports a closed state and low FRET reports the open state. This will allow detection of a force range in environments where attaching a force handle is not possible. In the broader context of applications for the ND, there is significant potential for investigating cell-cell and cell-surface interactions based on previous studies. ssDNA hairpins 52 , 53 and DNA origami platforms 29 have been successfully implemented to investigate intercellular forces. The ND could be used similarly where the modularity of the ND could complement these previously published elegant studies by enabling a wider range of cell adhesion force-sensing. Furthermore, iterative zipper incorporation would allow the force sensor to be tuned in conjunction with changes in the extracellular environment that cause the cells to adapt by changing their cell-surface interactions. Future studies will be needed to investigate these potential applications of this versatile nanoscale device."
} | 2,635 |
24074355 | PMC3850637 | pmc | 6,410 | {
"abstract": "Background While most resources in biofuels were directed towards implementing bioethanol programs, 1-propanol has recently received attention as a promising alternative biofuel. Nevertheless, no microorganism has been identified as a natural 1-propanol producer. In this study, we manipulated a novel metabolic pathway for the synthesis of 1-propanol in the genetically tractable bacterium Escherichia coli . Results E. coli strains capable of producing heterologous 1-propanol were engineered by extending the dissimilation of succinate via propionyl-CoA. This was accomplished by expressing a selection of key genes, i.e. (1) three native genes in the sleeping beauty mutase (Sbm) operon, i.e. sbm - ygfD - ygfG from E. coli , (2) the genes encoding bifunctional aldehyde/alcohol dehydrogenases (ADHs) from several microbial sources, and (3) the sucCD gene encoding succinyl-CoA synthetase from E. coli . Using the developed whole-cell biocatalyst under anaerobic conditions, production titers up to 150 mg/L of 1-propanol were obtained. In addition, several genetic and chemical effects on the production of 1-propanol were investigated, indicating that certain host-gene deletions could abolish 1-propanol production as well as that the expression of a putative protein kinase (encoded by ygfD/argK ) was crucial for 1-propanol biosynthesis. Conclusions The study has provided a novel route for 1-propanol production in E. coli , which is subjected to further improvement by identifying limiting conversion steps, shifting major carbon flux to the productive pathway, and optimizing gene expression and culture conditions.",
"conclusion": "Conclusions In this study, we demonstrated the manipulation of the homologous Sbm operon for extended dissimilation of succinate in E. coli , leading to 1-propanol production. Using the engineered E. coli strains for anaerobic cultivation in a shaker, 1-propanol titers up to 150 mg/L could be obtained. However, ethanol, acetate, and lactate represented the major metabolites, potentially limiting the productivity of 1-propanol. To improve the efficiency and applicability of this biocatalytic system, further studies have to be conducted to derive superior production strains by eliminating key conversion bottlenecks, metabolic imbalances, and undesirable byproducts as well as to optimize gene expression and culture conditions.",
"discussion": "Discussion To date, metabolic engineering of E. coli for 1-propanol biosynthesis has been conducted through two major pathways, i.e. (1) the keto-acid biosynthetic pathway [ 6 - 8 ] and (2) the extended 1,2-propanediol pathway [ 5 ]. Unlike these approaches, our strategy focused on activation of the endogenous but often silent Sbm operon for extended conversion of succinate into 1-propanol. The 1-propanol-producing capacity was implemented by transforming a wild-type E. coli strain, BW25141, with three plasmids respectively harboring the Sbm operon genes (with the exception of ygfG ), sucCD , and adhE2 for expression of these key genes. Using the metabolically engineered strains for anaerobic fermentation, we obtained 1-propanol titers up to 150 mg/L which is comparable to those of other studies [ 5 , 9 ]. In addition, we identified several potential factors limiting 1-propanol production, in particular the abundance of precursors and the conversion step catalyzed by a bi-functional alcohol/aldehyde dehydrogenase. While it is possible to perform this biotransformation aerobically, anaerobic cultivation was chosen for two reasons. Firstly, the two TCA intermediates of succinate and succinyl-CoA are the precursors for 1-propanol biosynthesis and their abundance can potentially limit 1-propanol production. Under anaerobic, but not aerobic, conditions, E. coli generates both succinate and succinyl-CoA as fermentation end products via a reductive reverse TCA pathway (Figure 1 ). Secondly, potential oxygen-sensitivity of AdhE2 and other ADHs is another limitation for oxygenic production of 1-propanol. While the expression of enzymes encoded by the Sbm operon is potentially detectable, their levels are far too low to form a functional pathway [ 13 , 14 , 23 ]. Moreover, due to E. coli ’s inability to produce coenzyme B 12 , the expressed Sbm remains as an inactive apo-enzyme, but nano-molar supplementation of cyanocobalamin can result in the formation of active Sbm [ 24 , 25 ]. Our observations of no detectable titers of propionate and 1-propanol for wild-type BW25141 as well as the production of 1-propanol upon heterologous expression of the Sbm operon genes with proper supplementation of cyanocobalamin was associated with the activation of the Sbm-pathway. While the activated Sbm-pathway can result in 1-propanol production, the expression of SucCD was deemed crucial to increase the succinyl-CoA pool and consequently the 1-propanol titer. In addition, 1-propanol production was enhanced by exogenous supplementation of succinate. These results suggest that 1-propanol production can be limited by the availability of various precursors and key enzymes along this 1-propanol-producing pathway. While the metabolic context for the three enzymes encoded by the four-gene Sbm operon, i.e. Sbm, YgfG, and YgfH, has been unraveled, the biological role of the other member, i.e. YgfD/ArgK, remains ambiguous. Earlier studies determined that YgfD/ArgK is a putative arginine kinase interacting with Sbm in vivo and in vitro [ 14 ] and involved in the phosphorylation of periplasmic binding proteins for amino acid translocation [ 11 ]. The activity of YgfD/ArgK was shown to be potentially essential for 1-propanol biosynthesis since the 1-propanol titer was significantly reduced by the ygfD/argK deletion. Interestingly, propionate production was hardly affected by the ygfD/argK deletion, and this result is consistent with a previous report [ 26 ], where propionate was derived from fatty acids by expressing the Sbm-operon genes excluding ygfD/argK in an engineered E. coli strain. A selection of native and non-native ADHs were heterologously expressed for evaluation of their effects on 1-propanol-producing capacity of various metabolically engineered E. coli strains, with AdhE2 and BdhB being identified as the most prominent ones for 1-propanol production. Nevertheless, our consistent observation that ethanol titers were significantly higher than 1-propanol implies that propionyl-CoA or propionaldehyde might have less affinity towards ADHs than acetyl-CoA or acetaldehyde. Several native E. coli ADHs (e.g. YqhD, AdhP, and AdhE MUT ) were also active in driving 1-propanol production, but in a much lower titer. In particular, the generation of the aerotolerent AdhE mutant (AdhE MUT ) opens an avenue for aerobic production of 1-propanol. Under anaerobic conditions, the maximum theoretical yield (on the molar basis) of 1-propanol from glucose is less than one due to limited NADH availability. Thus, developing an oxygenic production system would be beneficial as it increases the carbon throughout whilst improving cell growth and physiology. Under anoxic conditions for anaerobic fermentation in E. coli , the carbon flux at the PEP node favors reduction into pyruvate rather than carboxylation into oxaloacetate (OAA), with lactate, acetate, and ethanol as major metabolites (Figure 1 ). Note that there are four NADH-consuming steps along the 1-propanol-producing pathway downstream of phosphoenolpyruvate (PEP), whereas only one or two NADH-consuming steps for the other pathways associated with the major metabolites. The anaplerotic reactions within the metabolic network are optimized in order to balance the cell’s energy budget and electrons. Consequently, only ~10% of glucose consumed is channeled towards succinate and cell mass [ 27 ]. Our results suggest that the production of 1-propanol was potentially hampered by the inherent limitation in succinate production and a metabolic deficiency in NADH generation. Interestingly, propionate was also concomitantly produced with 1-propanol in our metabolically engineered strains (Tables 1 and 2 ). Additional studies are needed to elucidate the dichotomy between 1-propanol and propionate accumulation. There is an apparent need to reduce the amounts of major metabolites, i.e. ethanol, acetate, and lactate. This could be achieved by knocking out relevant native genes in the hope to redirect the carbon flux into the 1-propanol-producing pathway. While deletions of both adhE and pta were previously found to improve succinate titers [ 28 ], these mutations abolished 1-propanol production in our study (data not shown). Deletion of pta resulted in the channeling of the carbon flux towards lactate accumulation. In addition, heterologous expression of E. coli AdhE or other ADH homologs failed to complement the adhE genomic knockout in terms of restoring 1-propanol production, potentially due to unknown perturbations in the metabolite pool or gene regulation. While the lactate level was significantly reduced for the ldhA null mutants, they produced considerable levels of both acetate and ethanol, thus reducing the carbon flux towards 1-propanol production (Table 3 ). Nonetheless, the ldhA mutation was deemed beneficial since it offers an additional NADH source and greatly reduces the acidification of the medium, thus improving cell growth. Another critical factor limiting the production of 1-propanol (and other desired metabolites, such as succinate [ 28 ] and malate [ 29 ]) is the energetically favored diversion of carbon flux at the node of PEP towards pyruvate, resulting in the production of the major metabolites ethanol, lactate, and acetate. Blocking the production of one of these major metabolites (i.e. lactate, acetate, or ethanol) causes the accumulation of the others without improving the overall production of 1-propanol since these major metabolites all share the same precursor of pyruvate. Therefore, the implementation of a “driving force” diverting the carbon flux from pyruvate to OAA appears to be inevitable. Several metabolic engineering strategies to improve this are currently under our investigation Since a considerable amount of succinate accumulated in the extracellular medium potentially due to the poor affinity of succinate to SucCD (K m of ~0.25 mM with succinyl-CoA as the substrate in comparison to K m of ~4 mM with succinate as the substrate [ 30 ]), we are also identifying novel succinyl-CoA synthethases with a higher affinity for succinate to alleviate this limitation in 1-propanol production."
} | 2,646 |
36770451 | PMC9920594 | pmc | 6,412 | {
"abstract": "Thermal contact resistance between the microprocessor chip and the heat sink has long been a focus of thermal management research in electronics. Thermally conductive gel, as a thermal interface material for efficient heat transfer between high-power components and heat sinks, can effectively reduce heat accumulation in electronic components. To reduce the interface thermal resistance of thermally conductive gel, hexagonal boron nitride and graphene oxide were hybridized with a low-melting-point alloy in the presence of a surface modifier, humic acid, to obtain a hybrid filler. The results showed that at the nanoscale, the low-melting-point alloy was homogeneously composited and encapsulated in hexagonal boron nitride and graphene oxide, which reduced its melting range. When the temperature reached the melting point of the low-melting-point alloy, the hybrid powder exhibited surface wettability. The thermal conductivity of the thermally conductive gel prepared with the hybrid filler increased to 2.18 W/(m·K), while the corresponding thermal contact resistance could be as low as 0.024 °C/W. Furthermore, the thermal interface material maintained its excellent electric insulation performance, which is necessary for electronic device applications.",
"conclusion": "4. Conclusions TIM increased the heat transfer path and improved the heat dissipation efficiency by serving as a “bridge” between the heating device and the heat sink. LMPA was introduced in the BNG-based TCG for improved thermal management. LMPA and BNG were uniformly compounded on a nanometer scale based on a self-designed preparation. Using the phase change characteristics of BNG-loaded LMPA, an effective heat transfer path was fabricated, resulting in significantly improved thermal conductivity and TCR of the TIM. More specifically, the thermal conductivity of BNG 1 LMPA 2 /SG increased to 2.18 W/(m·K), which was 12.8 times that of the matrix, while the TCR decreased to 0.024 °C/W, which was 63% lower than that of BN 1 LMPA 2 /SG with the same filling amount. Hybridization of a composite of LMPA–BN–OG in the presence of HA can provide a strategy for ensuring TCG with high thermal conductivity and low thermal resistance while maintaining volume resistivity and breakdown voltage within a specific range. BNG-LMPA, a ternary hybrid material, provided a significant contribution to thermal conductivity, which is conducive to subsequent research of multi-hybrid materials. Additionally, this work provides guidance for the comprehensive improvement of TIM thermal management performance from the aspects of building an efficient thermal conduction path and improving the interface wetting effect.",
"introduction": "1. Introduction Thermal management is crucial in electronic cooling due to constant increases in the power density of electronic devices. Impeded heat transfer capacity due to a bump or corrugated interface between electronic components and radiators severely reduces the reliability of electronic devices. Eliminating the air gap between the interfaces and providing more heat conduction points will reduce thermal contact resistance (TCR) and improve heat transfer efficiency [ 1 , 2 ]. Thermal interface materials (TIM), with properties such as thermal conductivity, interface wettability, durability, etc., are used to fill the interface gap and are required to have excellent heat dissipation effects, thereby improving reliability [ 3 ]. Thermally conductive gel (TCG) is a new type of TIM with these characteristics, particularly in terms of durability for weak oil pump-out, compared with thermally conductive grease. Conventionally, TCG is prepared using reactive crosslinking silicone oil and heat-conductive particles [ 4 ]. Some key issues must be addressed to obtain excellent thermal conductivity for TIM. First, the thermally conductive properties of TCG mainly depend on the filling materials. When fillers are incorporated into the polymer matrix, they form a thermally conductive network, thus improving the composite’s thermal conductivity. Graphene has a significantly higher inherent thermal conductivity than carbon nanotubes, making it a suitable thermally conductive filler [ 5 , 6 ]. Adding graphene nanosheets (10 vol%) to pure gel sheets increased the thermal conductivity by 90% [ 7 ]. However, graphene’s electrical conductivity limits its applications, especially in electronic packaging where electronic insulation is required. Graphene oxide (GO) prepared following the Hummer method has electronic insulation properties due to severe functionalization of the conjugate network [ 8 ]. Hexagonal boron nitride (h-BN), which has a similar crystal structure to graphene, is attractive as an alternative filler due to its excellent thermal conductivity, electrical insulation, and high thermal stability. Compared to an epoxy resin substrate, its thermal conductivity coefficient can be significantly improved through hybridization with h-BN and GO fillers [ 9 ]. That is, the hybridization of different fillers is one of the possible ways to improve the performance of fillers and TIM. Second, filler surface modification is performed to improve filler dispersion in the matrix and achieve better hybrid filler interface connection to improve the composite’s thermal conductivity. As previously reported, the hybrid structure of surface-modified BN and GO was synthesized by surface modification of the filler, which has a stable interface [ 10 ]. The silver nanoparticle served as a bridging agent for the phonon transport of the hybrid structure. BN–Ag–GO is an effective thermally conductive filler in epoxy matrices [ 11 ]. Referring to this scheme, phase change materials with tunable melting temperatures provide a new design for thermal energy storage applications. Low-melting-point alloy (LMPA) has recently emerged as one of the directions of thermal conductivity reinforcement [ 12 , 13 ]. It has medium-high thermal conductivity and many applications, particularly excellent thermal physical properties, including wettability and adhesion, which are essential for surface conformability with little or no thermal resistance [ 14 ]. LMPA can be used as a new electronic packaging material by introducing it into a polyvinylidene fluoride matrix [ 15 ]. To date, LMPA is used for thermal management in various applications, such as desktop computers, light-emitting diodes, electronic cooling, and biological heat transfer [ 16 , 17 ]. By combining LMPA and BN, Ge Xin et al. found that the thermal conductivity of thermal grease reached 1.8 W/(m·K) and effectively reduced its TCR from 13.8 to 0.547 °C/W [ 18 ]. Due to the poor surface energy of BN, its microspheres need to be modified by dopamine to form a compact inorganic network structure with LMPA. Adding LMPA can improve the coherence of the inorganic filler in the polymer matrix, moderate agglomeration in the polymer matrix, and improve the thermal conductivity of the polymer material. Electronic equipment must have efficient heat dissipation capacity and maintain a certain insulation performance in various scenarios to ensure long-term safe operation [ 19 ]. The published literature suggests that LMPA-based composites may have higher electrical conductivity than those based on BN or carbon nanotubes [ 12 , 20 ]. To improve the thermal conductivity of TCG and ensure electrical insulating properties, we mixed LMPA with an inorganic electrically insulating material (BN) to prepare thermally conductive fillers. During filler modification [ 15 , 21 ], LMPA melts after the temperature reaches the melting point. As a bridging agent, LMPA, combined with other nonmetallic fillers, forms a micro continuous thermally conductive network, effectively reducing the TCR of TCG [ 22 ]. If LMPA is applied to interfaces, inappropriate surface wettability may lead to unacceptable performance deterioration [ 14 ]. In this article, we focus on the recent results of the research group.",
"discussion": "3. Results and Discussion 3.1. Microstructure and Element Distribution of the Hybrid Filler As depicted in Figure 2 a, BN–LMPA and BNG–LMPA appeared white and gray, respectively. h-BN, BNG, LMPA, and BNG–LMPA were characterized by XRD. The diffraction peaks (002), (100), (101), (102), (004), and (112) shown in Figure 2 b were the XRD characteristic diffraction peaks of h-BN and BNG. The XRD characteristic peaks of LMPA were Sn (29.8°, 32.1°), Bi (36.3°, 47.4°), and In (58.7°, 66.4°) [ 24 ]. The presence of Ga in LMPA was not directly detected by XRD, thus indicating that the Ga signal was too weak or Ga was absent in LMPA. Due to the Ga-driven degradation of many metal microelectronic components, the corrosiveness and toxicity of Ga-based LMPA affect its use. An LMPA without Ga, an inorganic insulating filler, and polymer matrix three-component composites are more likely to provide a cheaper, noncorrosive solution [ 25 ]. The XRD spectra of BN–LMPA and BNG–LMPA showed that the strength of (100), (101), (004), and (102) crystal planes gradually decreased, and weak characteristic diffraction peaks of LMPA appeared, indicating that h-BN and BNG combined well with LMPA. HA contains several active functional groups, such as carboxyl and phenolic hydroxyl groups, which are excellent ceramic dispersants. Its hydrophobic properties reduced the surface free energy of the powder. Thus, the BN and GO particles modified by the HA surface repelled each other, and the dispersed particles were hindered by space, ensuring that the stability of the dispersion system was maintained. After modification, GO combined with BN by winding and surface action to form an elliptical sphere (BNG) ( Figure S1 ). As shown in Figure 2 c–e, BNG was compounded with LMPA through an independently designed preparation method to maintain the shape of the elliptical particulate matter of BNG. It has been proven that LMPA does not alter the structure of BNG but can form a unique structure, with BNG as the skeleton, by adhering to or even penetrating BNG. Element distribution analysis of BNG 1 LMPA 2 revealed that Figure 2 f–k elements (N, C, O, Bi, In, and Sn) were uniformly distributed in the whole space, indicating that LMPA and BNG were uniformly recombined in the microstructure. 3.2. Phase Change of LMPA in Hybrids The latent heat of phase change and phase change temperature are two of the most vital thermal physical energy parameters of phase change materials that influence their applicability. TIM is made from a phase change material, which can rapidly absorb heat and cool down electronic devices when heated. The DSC test on BNG–LMPA was used to investigate the influence of the different fillers and their ratios on the melting point. Figure 3 a shows that the phase transition temperature range of LMPA was 47–72 °C, which was within the heating temperature range of general electronic devices, and the maximum absorption peak was reached at 67 °C. When BNG was combined with LMPA, the maximum absorption peak temperature was reduced, and the LMPA melting range decreased [ 18 ]. The energy absorbed by melting was proportional to the LMPA content in the composites, and the latent heat of phase change of the two composites increased with increasing LMPA content. Since BNG has a higher heat transfer capacity than LMPA [K Sn = 67.0 W/(m·K), K Bi = 7.9 W/(m·K), and K In = 82.0 W/(m·K)] when it is uniformly mixed with LMPA at the microscopic level, heat can be transferred quickly to reach the initial melting temperature of LMPA. The results showed that BNG–LMPA/SG could rapidly absorb heat, thereby protecting electronic devices before the temperature reached the limit. The spreading (interfacial bonding) process of BNG 1 LMPA 2 under a certain pressure and heating program (5 °C/s) was observed using the transmission light of an optical microscope. As shown in Figure 3 b, BNG 1 LMPA 2 blocked the light, so the sample part appeared in shadow while other parts without the sample appeared white. During heating, the TCG temperature reached the melting point of LMPA at approximately 150 s, and the image changed to varying degrees. BNG 1 LMPA 2 also underwent a phase transformation at 150 s. Under external pressure, LMPA spread easily and had a large diffusion range. At 450 s (80 °C), the black area gradually expanded, at which point LMPA infiltrated from the inside of BNG and covered the entire slide surface. This indicated that BNG 1 LMPA 2 had good wettability in TIM, enabling it to rapidly transmit heat flow and effectively protect heating devices. 3.3. Thermal Conductivity of BN–LMPA/SG and BNG–LMPA/SG The thermal conductivity of composites is mainly determined by the heat transfer capacity of the filler, thermal network density, and TCR. BN–LMPA and BNG–LMPA of different proportions were prepared to compare the effect of the LMPA content on the thermal conductivity of TCG with or without GO. On the basis of h-BN and BNG, LMPA was added to prepare different proportions of BN-LMPA and BNG-LMPA. From Table 1 , it can be seen that h-BN with GO initially improved the thermal conductivity of TCG, while LMPA further improved the thermal conductivity of TCG. Figure 4 a,b indicates the thermal conductivity and TCR of BN–LMPA/SG, respectively. When the LMPA content was low, the thermal conductivity increased slowly with increasing BN 4 LMPA 1 filling amount. In this case, BN 4 LMPA 1 , and the surrounding matrix had high TCR and interfacial phonon scattering, which was not conducive to heat transfer. When the LMPA content was further increased, the contribution of the composite filler to thermal conductivity was significantly improved. When the filling amount of BN 1 LMPA 1 was 60 wt.%, the thermal conductivity reached 1.47 W/(m·K), which was approximately 8.7 times that of the pure gel matrix (0.17 W/m·K). An appropriate amount of LMPA promoted the formation of a heat conduction path; thus, it synergistically enhanced the thermal conductivity of TCG with h-BN. When the filling amount of BN 1 LMPA 2 /SG was 60 wt.%, the thermal conductivity reached 1.62 W/(m·K) and the TCR decreased to 0.039 °C/W, which was 97% higher and 128% lower than that of BN 4 LMPA 1 /SG, respectively. This indicated that BN 1 LMPA 2 -filled composites had a certain TC enhancement, particularly TCR reduction. Figure 4 c,d indicates the thermal conductivity and TCR curves of BNG–LMPA/SG, respectively. When the filling amount of BNG 2 LMPA 1 /SG was 60 wt.%, the thermal conductivity was 1.59 W/(m·K) and the TCR decreased to 0.042 °C/W, which was similar to that of BN 1 LMPA 2 . The preliminary results showed that the presence of GO resulted in a more significant contribution of BNG–LMPA to the composite’s thermal conductivity than BN–LMPA. When used as TIM, LMPA exchanged heat flow between the thermal filler and matrix, significantly improving the composite’s thermal conductivity. Meanwhile, BNG–LMPA/SG hot pressing formed a coherent network structure, which reduced the interface thermal resistance. The maximum thermal conductivity of BNG 1 LMPA 2 /SG filled with 60 wt.% was approximately 2.18 W/(m·K) and the TCR was as low as 0.024 °C/W, which increased by 35% and decreased by 63%, respectively, when compared with BN 1 LMPA 2 /SG filled with the same amount. To visualize the heat transfer effects of BN 1 LMPA 2 /SG and BNG 1 LMPA 2 /SG, the two composite materials with 60 wt.% filling volume were uniformly coated on the slide, and the temperature response difference was recorded by infrared thermography. Figure 4 e depicts the heat transfer efficiency of BN 1 LMPA 2 /SG and BNG 1 LMPA 2 /SG. At 5 s, the surface temperature of BN 1 LMPA 2 /SG (left) was 38.3 °C, and it reached equilibrium after approximately 45 s, similar to BNG/SG, but at a lower equilibrium temperature. Alternatively, the surface temperature of BNG 1 LMPA 2 /SG rose rapidly to 42.3 °C in 5 s, and it only took 35 s to reach equilibrium, with a lower equilibrium temperature than that of BN 1 LMPA 2 /SG. This was attributed to the fact that BNG 1 LMPA 2 could accelerate heat emission and had a higher heat dissipation effect. Lattice vibration (phonon) is the primary mechanism of heat conduction of nonmetallic materials, but it contributes less to the heat conduction of metallic materials. Here, LMPA can provide a pathway for electron heat conduction. As the temperature rises, lattice vibration and free-electron movement combine to form a new equilibrium state, increasing heat transfer via phonons and electrons [ 26 ]. Based on the above analysis, BNG 1 LMPA 2 /SG can play a better heat transfer role in the system due to the actions of LMPA. The formation of an effective three-dimensional seepage network of heat flow via the synergistic effect is the key criterion for determining the thermal conductivity of composite materials [ 27 ]. As shown in Figure 5 a, BN 1 LMPA 2 was less evenly distributed in TCG, where the blend filler of LMPA and h-BN had made a relatively limited contribution to the thermal conductivity of TCG. When used as TIM, the heat flow could not be effectively transmitted between the thermally conductive filler due to heat flow network defects, resulting in poor thermal conductivity. Meanwhile, as shown in Figure 5 b, LMPA adhered to the surface of h-BN and GO in the presence of HA, and the hybridization interaction between LMPA and h-BN was significantly improved. Figure 5 c,d demonstrates the microscopic morphology of BNG 1 LMPA 2 /SG. The continuity of BNG/SG and BN 1 LMPA 2 /SG was stronger, and the thermally conductive network was more complete. This indicated that the melting flow of LMPA inside BNG 1 LMPA 2 /SG, which ran through the BNG interior, connected adjacent fillers in the matrix to form a new heat conduction path, which could still be maintained after the temperature was cooled. The comparison between h-BN and BNG–LMPA/SG showed that the discontinuity defect of h-BN in TCG was compensated by LMPA, with BNG–LMPA/SG contributing more significantly to reducing the interface thermal resistance. 3.4. Electric Insulation Performance of TCG In addition to heat accumulation, a bad electromagnetic wave caused by electronic equipment is also a potential hazard [ 28 ]. Therefore, TIM with mutually multifunctional properties, such as a high dielectric constant, high breakdown strength, and high TC, are preferred. The structure of h-BN is similar to that of electrically conductive graphene or graphite, except no free electron exists. GO prepared following the Hummers method is functionalized due to the conjugate network, showing insulation characteristics [ 29 , 30 ]. The conductivity of materials is usually expressed by resistivity or conductivity independent of size. In this experiment, two methods were selected to test the conductivity of TCG, namely, breakdown voltage ( Figure 6 ) and volume resistivity ( Table 2 ). GO and h-BN take advantage of the excellent inplane thermal conductivity of two-dimensional fillers to reduce electrical conduction along the thickness direction, which effectively translates into high thermal conductivity and breakdown strength of composites. Compared with BN/SG, the volume resistivity and breakdown voltage of BNG/SG were enhanced. The addition of LMPA inhibited the insulation performance of BN 1 LMPA 2 /SG, in which the volume resistivity decreased by an order of magnitude and the breakdown voltage decreased by approximately 50%. The BNG 1 LMPA 2 composite filler prepared by combining BNG with LMPA still had high volume resistivity and the breakdown voltage was higher than that of BN 1 LMPA 2 /SG, which met the requirements of insulating occasions. Compared with the pristine polymer, BNG 1 LMPA 2 /SG exhibited multifunctional properties under various synergistic mechanisms, including high thermal conductivity, low thermal resistance, and medium insulation performance."
} | 4,977 |
32654271 | null | s2 | 6,413 | {
"abstract": "Efficient symbiotic colonization of the squid Euprymna scolopes by the bacterium Vibrio fischeri depends on bacterial biofilm formation on the surface of the squid's light organ. Subsequently, the bacteria disperse from the biofilm via an unknown mechanism and enter through pores to reach the interior colonization sites. Here, we identify a homolog of Pseudomonas fluorescens LapG as a dispersal factor that promotes cleavage of a biofilm-promoting adhesin, LapV. Overproduction of LapG inhibited biofilm formation and, unlike the wild-type parent, a ΔlapG mutant formed biofilms in vitro. Although V. fischeri encodes two putative large adhesins, LapI (near lapG on chromosome II) and LapV (on chromosome I), only the latter contributed to biofilm formation. Consistent with the Pseudomonas Lap system model, our data support a role for the predicted c-di-GMP-binding protein LapD in inhibiting LapG-dependent dispersal. Furthermore, we identified a phosphodiesterase, PdeV, whose loss promotes biofilm formation similar to that of the ΔlapG mutant and dependent on both LapD and LapV. Finally, we found a minor defect for a ΔlapD mutant in initiating squid colonization, indicating a role for the Lap system in a relevant environmental niche. Together, these data reveal new factors and provide important insights into biofilm dispersal by V. fischeri."
} | 339 |
31211205 | PMC6562174 | pmc | 6,414 | {
"abstract": "Photosynthetic microorganisms offer promising perspectives for the sustainable production of value-added compounds. Nevertheless, the cultivation of phototrophic organisms to high cell densities (HCDs) is hampered by limited reactor concepts. Co-cultivation of the photoautotrophic Synechocystis sp. PCC 6803 and the chemoheterotrophic P. taiwanensis VLB 120 enabled HCDs up to 51.8 g CDW L −1 . Respective biofilms have been grown as a biofilm in capillary flow-reactors, and oxygen evolution, total biomass, as well as the ratio of the two strains, have been followed under various cultivation conditions. Furthermore, biofilm formation on a microscopic level was analyzed via confocal laser scanning microscopy using a custom made flow-cell setup. The concept of mixed trophies co-cultivation was coupled to biotransformation, namely the oxyfunctionalization of cyclohexane to cyclohexanol. For benchmarking, the performance of the phototrophic reaction was compared to the chemical process, and to a biotechnological approach using a heterotrophic organism only. The data presented refer to our research paper “Mixed-species biofilms for high-cell-density application of Synechocystis sp. PCC 6803 in capillary reactors for continuous cyclohexane oxidation to cyclohexanol” Hoschek et al., 2019."
} | 326 |
24698742 | null | s2 | 6,416 | {
"abstract": "Lignocellulosic biomass has been recognized as a promising feedstock for the fermentative production of biofuel. However, the pretreatment of lignocellulose generates a number of by-products, such as furfural, 5-hydroxylmethyl furfural (5-HMF), vanillin, vanillic acids and trans-p-coumaric acid (TPCA), which are known to inhibit microbial growth. This research explores the ability of Rhodococcus opacus PD630 to use lignocellulosic biomass for production of triacylglycerols (TAGs), a common lipid raw material for biodiesel production. This study reports that R. opacus PD630 can grow well in R2A broth in the presence of these model inhibitory compounds while accumulating TAGs. Furthermore, strain PD630 can use TPCA, vanillic acid, and vanillin as carbon sources, but can only use TPCA and vanillic acid for TAG accumulation. Strain PD630 can also grow rapidly on the hydrolysates of corn stover, sorghum, and grass to accumulate TAGs, suggesting that strain PD630 is well-suited for bacterial lipid production from lignocellulosic biomass."
} | 261 |
35808095 | PMC9268176 | pmc | 6,417 | {
"abstract": "The nanostructure composed of nanomaterials and subwavelength units offers flexible design freedom and outstanding advantages over conventional devices. In this paper, a multifunctional nanostructure with phase-change material (PCM) is proposed to achieve tunable infrared detection, radiation cooling and infrared (IR)-laser compatible camouflage. The structure is very simple and is modified from the classic metal–dielectric–metal (MIM) multilayer film structure. We innovatively composed the top layer of metals with slits, and introduced a non-volatile PCM Ge 2 Sb 2 Te 5 (GST) for selective absorption/radiation regulation. According to the simulation results, wide-angle and polarization-insensitive dual-band infrared detection is realized in the four-layer structure. The transformation from infrared detection to infrared stealth is realized in the five-layer structure, and laser stealth is realized in the atmospheric window by electromagnetic absorption. Moreover, better radiation cooling is realized in the non-atmospheric window. The proposed device can achieve more than a 50% laser absorption rate at 10.6 μm while ensuring an average infrared emissivity below 20%. Compared with previous works, our proposed multifunctional nanostructures can realize multiple applications with a compact structure only by changing the temperature. Such ultra-thin, integratable and multifunctional nanostructures have great application prospects extending to various fields such as electromagnetic shielding, optical communication and sensing.",
"conclusion": "4. Conclusions In summary, this paper proposes a multifunctional nanostructure with phase-change materials for tunable infrared detection, radiative cooling and infrared-laser compatible camouflage. The structure uses a combination of gold arrays with slits in the top-layer, and innovatively introduces the nonvolatile phase-change material GST. The study shows that by combining the plasmon resonance and nanogap resonance excited from the classical MIM structure, the device can selectively tune the absorptivity/emissivity at specific wavelengths. Due to the symmetry of the structure, the proposed device has the remarkable features of wide-angle absorption and polarization insensitivity, which is beneficial for practical applications. At the same time, according to the analysis of the energy distribution of the electromagnetic field and the study of various resonance theories, we can expand the strong interaction between light and matter to other wavelengths and achieve more applications. It should be emphasized that, different from the single function of other traditional structures, the proposed multifunctional structure can realize complex multiple functions without changing the structure by only changing the temperature. In addition, our device is extremely simple and easy to fabricate, which will greatly benefit the needs of integration and miniaturization. It has potential applications in various fields such as electromagnetic absorbers, electromagnetic shielding, infrared stealth, optical communication and sensors.",
"introduction": "1. Introduction As we all know, thermal radiation is a natural phenomenon everywhere in our life. It propagates in the form of electromagnetic (EM) waves and undertakes the important function of heat transfer at the same time, among which the infrared (IR) band has the most significant thermal effect. Since most of the infrared radiation of objects in nature is incoherent light, their radiation is unpolarized in the whole wide spectrum, and does not change with angle and random direction. The control of thermal radiation can only be achieved by changing the temperature of the object, which has great limitations in practical applications. In fact, the ability to control EM absorption and thermal radiation is important in many applications, including infrared detection [ 1 , 2 ], radiative cooling [ 3 , 4 , 5 ], solar steam engines [ 6 , 7 ], spectral sensors [ 8 ], thermal management [ 9 , 10 ] and thermal camouflage [ 11 , 12 , 13 , 14 , 15 , 16 , 17 ]. In recent decades, with the rapid development of nanotechnology, many interesting phenomena of manipulating EM waves have been generated. Through the interaction between subwavelength size structures and light by using metasurfaces (MSs), the free regulation of the infrared thermal radiation of objects has become a reality [ 18 , 19 , 20 , 21 ]. More and more attention has been paid to the law of light–matter interaction at the micro/nano scale and its application in light modulation and detection [ 22 , 23 , 24 ]. Therefore, by combining the modulation effect of photonic devices with the infrared photothermal effect, the design of effective micro/nano structures is conducive to manipulating the optical form of the thermal radiation of objects in the fields of infrared stealth [ 25 ], nano manufacturing [ 26 ] and solar energy utilization [ 27 ]. For example, based on the principles of metamaterials (MMs) [ 28 , 29 , 30 ], PCMs and plasmonics [ 31 ], thermal radiation is enhanced or suppressed at specific wavelengths. These photon structures show strong absorption dependence based on the resonant wavelength of electromagnetic waves. By reasonably designing the shape and size of micro-nano structures, we can independently choose the intensity, wavelength and bandwidth of radiation according to Kirchhoff’s law of thermal radiation, so as to obtain the adjusted thermal radiation spectrum. At present, with these characteristics, the response wavelength of micro-nano structures can be extended to various bands of visible light [ 32 , 33 ], infrared [ 34 , 35 ], terahertz [ 36 , 37 ], millimeter wave [ 38 , 39 , 40 ] and microwave [ 41 , 42 , 43 ]. Based on this, micro-nano devices are widely used in optical computing [ 44 ], night infrared imaging [ 45 ], infrared camouflage [ 46 , 47 ], optical communication [ 48 ] and other applications. According to Wien’s displacement law of thermodynamics,\n (1) λ m a x ⋅ T = 2898 μ m ⋅ K , For the black body, the peak wavelength of thermal radiation is in the infrared band (wavelength range: 700 nm–1 mm) under normal conditions, especially in the mid-infrared band (wavelength range: 1.4–14 μm). Therefore, the reconnaissance, detection and imaging technology in the mid-infrared band began to develop rapidly, and has been widely applied in various fields, such as infrared photoelectric sensors, infrared detectors and infrared thermal imagers [ 49 ]. In addition, the applications for military reconnaissance and stealth related to the middle infrared have also made this band become the focus of competing research. Due to the physical effects, such as surface plasmon resonance (SPR) and Fabry Perot (FP) cavity resonance, the intrinsic emissivity of the object can be changed by changing the material, structure and size of the micro-nano photonic device. Thus, adjustable infrared absorption and thermal radiation can be realized, and even compatible camouflage of two different bands can be integrated in the same device. For example, Du Kaikai et al. have proposed a selective microbolometer based on metamaterial absorbers (MAs); MAs provide a new method for heat distribution adjustment and monitoring of microbolometers, and show great promise in photothermal imaging systems. Therefore, micro-nano structures make great contributions to infrared detection in the specific wavelength region of mid-far infrared [ 50 ]. Zhu Huanzheng et al. realized multispectral camouflage, wavelength selective emission and microwave absorption by using micro-nano structures such as multilayer films (including ZnS/Ge) and metasurfaces (including Cu-ITO-Cu), providing an idea for multifunctional compatible stealth [ 51 ]. It is important to note, however, that the selectivity of specific wavelengths in these studies all depends on the shape, size and period of the corresponding micro-nano structure. That is to say, once these micro-nano structures are manufactured, their corresponding resonant wavelength and absorption spectrum are determined at the same time and cannot be changed. Therefore, how to realize an adjustable multifunctional application on the basis of a fixed structure is very important for dynamic compatibility stealth. PCMs have a good prospect in active dynamic tuning, such as Ge 2 Sb 2 Te 5 (GST), VO 2 , Ge 2 Se 2 Sb 4 Te 1 (GSST) and Sb 2 S 3 , which have two states (crystalline and amorphous) with physical properties that are obviously different. In particular, the dielectric constant of GST varies greatly before and after phase transformation, and GST has been widely used in a variety of photoelectric devices, such as absorbers, chiral metamaterials, optical switches [ 52 , 53 ], tunable optical metasurfaces [ 54 ] and multifunctional devices [ 55 ]. Based on the above requirements, we consider combining PCMs and MSs to achieve complex multifunctional functions on a very simple micro/nano structure, while controlling the absorption/radiation intensity only by temperature changes without changing the structure and size, so as to achieve different practical applications. In this work, we design and study a multifunctional metal–dielectric–metal (MIM) sandwich structure. Firstly, the conventional MIM multilayer film structure is composed of Au/Al 2 O 3 /Au on the bottom silicon substrate, and the top layer of the structure is innovatively composed of metals with periodic slits. It can be used to stimulate nanogap plasmon resonance and surface plasmon resonance in combination with a dielectric spacer to realize an infrared detection function. Subsequently, the PCM GST is added to the multilayer structure to form the Au/Al 2 O 3 /GST/Au/Si structure. By controlling the temperature, the GST changes dynamically and continuously between amorphous, intermediate and crystalline states, and changes the absorptivity/emissivity of the device to the IR-laser band, so as to achieve different functions. Compared with previous work, our design overcomes the disadvantages of being untunable, single function and difficult to integrate. It can realize many functions such as tunable infrared detection, radiation cooling and infrared-laser compatible camouflage under a simple structure with active control. This will greatly contribute to the development of optical communication, radiation cooling, adjustable electromagnetic wave control and various thermal camouflage technologies for military purposes.",
"discussion": "3. Results and Discussion Since the photon energy in the infrared band is not much different from the chemical bond energy of various substances in the air, the atmosphere has a strong absorption effect on the light in the infrared band. Figure 2 a shows the atmospheric transmission spectrum in the infrared band. Due to the high transmittance of the atmosphere in the mid-wave infrared (MWIR) and long-wave infrared (LWIR), light in the 3–5 μm and 8–14 μm bands can pass through atmosphere; these are called atmospheric windows. In the atmospheric window, the energy of an object can be directly radiated into the distance. The higher the radiation intensity, the easier it is for the infrared detector to absorb the energy of the corresponding band, and the easier it is to find the target. This is the principle of infrared detection. According to Kirchhoff’s law, under the condition of thermal equilibrium, we have\n (3) α = ε , \nwhere α is the infrared absorption rate of the surface of the object and ε is the infrared radiation rate of the object. If one wants to avoid being detected by infrared detectors, the structure must have a low absorption rate/radiance rate (high reflectivity) in the atmospheric window to achieve the effect of infrared camouflage. Different from the principle of infrared camouflage, the physical mechanism of laser stealth is to achieve high absorption on the target surface. The working band of the laser is generally 10.6 μm. The light detection and ranging (LiDAR) detector emit laser light to the surface of the target, and then receives the reflected signals to detect the target. For LiDAR detection, we need to increase the absorptivity (reduce the reflectivity) of the target surface to achieve LiDAR stealth. As can be seen in Figure 2 a, 10.6 μm is just in the atmospheric window band to achieve infrared camouflage. To achieve infrared-LiDAR compatible camouflage, it is necessary to ensure that the target has a high absorption rate at 10.6 μm and low absorption at 8–14 μm (except 10.6 μm). Figure 2 b exhibits the dielectric constants of aGST and cGST in the infrared band. From the curves in the figure, we can observe that since the imaginary part of the dielectric constant (short blue line) is close to zero, aGST is a transparent medium in the infrared band without electromagnetic losses. When the GST phase changes to the crystalline state, the real part of the dielectric constant (solid blue and red lines) increases significantly, and the imaginary part also changes from zero. This phenomenon causes GST to generate electromagnetic losses in the infrared band and transform into an infrared absorbing material. In our study, selective infrared absorption of the device was first achieved through metasurface and nanogap resonance. Then, the nonvolatile PCM GST is used to perform dynamic infrared thermal radiation regulation and realize multifunctional applications. 3.1. “Wide-Angle, Polarization-Insensitive Dual-Band Infrared Detection” under the Four-Layer Structure The first application of the designed multifunctional device is wide-angle, polarization-insensitive dual-band infrared detection. Infrared detection refers to finding the target by absorbing the radiation power of the target object in the mid-infrared band. Current infrared detectors can be roughly divided into two categories according to physical effects: the photothermal category and photonic category. They work in different ways, but the core principle is to determine the intensity of the target’s infrared radiation/absorption through changes in physical signals (such as geometry, conductivity) or electrical signals. Here, we only consider the radiation intensity of the target itself, while ignoring the radiation intensity of the surrounding environment and the solar radiation energy reflected by the target. The spectral radiation intensity of the black body and the target object can be expressed by Planck’s law of black body (BB) radiation, as follows [ 66 ]: (4) I 0 θ , λ , T = ε θ , λ , T I B B λ , T , \nwhere ε θ , λ , T is the intrinsic emissivity of the object and I B B λ , T represents the BB radiation spectrum. Simultaneously,\n (5) I B B ( λ , T ) = 2 π h c 2 λ 5 ⋅ 1 e h c λ κ T − 1 , This shows that I B B is only related to the temperature T , and all objects with a temperature higher than absolute zero can generate thermal radiation, such as the sun (6000 K), incandescent lamps (3000 K) or the human body (310 K) spontaneously radiating heat outward. Using this feature, the reconnaissance of target objects can be achieved by designing a device with a high absorption rate of thermal radiation at the atmospheric window. As shown in Figure 3 a, the designed four-layer structure has obvious absorption peaks in the two atmospheric windows: the absorption rate reaches 73% at λ = 3.6 μm and the absorption rate is 83% at λ = 8.5 μm, respectively. It shows that this structure has a strong detection ability for infrared radiation of the target, and can be regarded as a mid-infrared detector. This is due to the introduction of narrow slits and nano-gap Al 2 O 3 layers on the MIM structure. We found that they provide an important channel for controlling the absorption behavior; the former is to help the local energy leak out of the slit to be used away from the resonant unit, thus balancing the absorption of the material and the leakage rate of radiation and maintaining the perfect absorption of the structure, while the latter acts as a capacitor that can red-shift the absorption peak by adjusting the thickness and period [ 67 ]. In each constituent unit of Figure 1 b, the dielectric spacing between the top gold array and the bottom metal can be viewed as a MIM waveguide. The round-trip between the ends of the metal array forms a waveguide-type FP cavity, where the lowest-order resonant mode results in high absorption at the corresponding wavelength. The absorption of this structure can be understood through time-domain coupled mode theory, which treats resonances as discrete modes that are weakly coupled to the environment. This device can be regarded as a typical gapped surface plasmon ( GSP ) resonator, and its resonance position can be described by a simple FP resonator equation [ 68 , 69 ]: (6) w 2 π λ 0 n G S P = m π − ϕ , \nwhere λ 0 is the wavelength in free space, w is the band width, n G S P is the real part of the effective refractive index of the GSP , m is the mode order of the GSP and ϕ is an additional phase shift. It is worth pointing out that the GSP resonator is primarily an absorbing element when the gap is at subwavelength dimensions. Once the gap size becomes larger, the GSP resonance will gradually become weak enough to achieve high absorption. In order to explore the generation mechanism of the two absorption peaks located in the atmospheric window, we analyzed the electric and magnetic field distributions of the designed structure at λ = 3.6 μm and λ = 8.5 μm. Figure 3 b,d show the electromagnetic field distribution at λ = 3.6 μm; we can find that the device excites high-order resonances at short wavelengths, and most of the electric field energy is trapped in the dielectric Al 2 O 3 layer, indicating that nanogap resonances are generated and GSPs lead to absorption peaks. From Figure 3 c, it can be seen that at λ = 8.5 μm, there is a clear coupling between the top gold array and the bottom metal, and opposite charges are observed at the corresponding positions, indicating the generation of localized surface plasmon resonance (LSPR). Combined with the magnetic field distribution in Figure 3 e, it is found that the magnetic field energy is mainly concentrated in the gold array and the Au layer, indicating that there is a strong magnetic resonance (MR) in the dielectric region along the magnetic field direction. We believe that the emergence of MR can enhance the localized field at the resonance wavelength, thereby achieving high absorption at the corresponding wavelength through the enhanced LSPR. Therefore, we can use this phenomenon to realize dual-band infrared detection in the atmospheric window. Subsequently, we varied the thickness of the dielectric layer used to excite the nanogap resonance and found a significant change in absorptivity. This part of the work is detailed in Supplementary Materials Information S1 . At the same time, we also verified that when the slit width is unchanged and the period becomes larger, the absorption peak has a red shift. It can extend this local resonance phenomenon to other wavelength bands, and it is fascinating for some applications that require subwavelength metasurface absorbing structures or strong interactions between light and matter. Figure 4 a shows the absorptivity of the device at different incident angles, and the absorptivity does not decrease significantly with the increase of the incident angle. Up to the large incident angle of about 60 degrees, the device can still maintain the absorption rate of more than 50% in the dual-band, indicating that it has good wide-angle absorption. Furthermore, since the designed structures are highly symmetric gold arrays and multilayer thin film structures, they are theoretically insensitive to polarization angles. Through the simulation calculation, we can know the influence of the polarization angle on the structure from Figure 4 b, which verifies the polarization insensitivity of the device. Consequently, the designed four-layer structure exhibits wide-angle absorption and polarization insensitivity, which is beneficial for practical applications in infrared detection. 3.2. “Multifunctional Infrared Stealth, Radiation Cooling and Laser Stealth” under the Five-Layer Structure The means of opposing infrared detection is infrared stealth technology, which is a matchup of “spear and shield”. Infrared stealth is to reduce the probability of the target being detected. The purpose is to make the radiation intensity of the target in the mid-infrared band consistent with the radiation intensity of the background, so that the detection imaging technology cannot distinguish between the two. In the mid-infrared band, because the radiation intensity of most of the background is very low, it is a key factor for infrared stealth to reduce the external radiation rate of the target through technical means. At the same time, detection systems on the battlefield no longer work only in the infrared band: multi-band detection is gradually becoming the mainstream thinking. With the continuous development of LiDAR technology, weapon systems using laser detection and guidance technology have high precision. Once the target is tracked by relevant weapons, the probability of survival will be extremely small. Therefore, the development of laser camouflage technology has great strategic significance. Laser camouflage technology is mainly aimed at 10.6 μm and other laser working bands. The purpose is to reduce the reflectivity of the target in these bands, so that the laser detection and guidance system cannot receive the reflected echo or identify the target. According to Kirchhoff’s law, the emissivity is equal to the absorptivity. At the same time, in traditional devices, it can be known from Equation (2) that for opaque materials, the sum of absorptivity and reflectivity is 1. Thus, to achieve compatible stealth in the IR band (3–5 and 8–14 μm) and the LiDAR band (10.6 μm), there is a natural conflict: the target needs to achieve both low absorbance/emissivity at 8–14 μm to achieve IR stealth and high absorbance (low reflectance) at 10.6 μm (in the 8–14 μm) to achieve LiDAR stealth. Therefore, it is still a very challenging task to achieve compatible camouflage in the IR-laser band. In response to the above practical requirements, we have researched and designed a multifunctional device, as shown in Figure 1 d, which has a simple structure that can integrate multiple functions in a fixed structure. On the basis of the four-layer structure, we introduce the nonvolatile PCM GST, which can dynamically adjust the absorptivity of the device by using the change of its dielectric constant before and after the phase change, thereby realizing multifunctional applications. It can be observed from Figure 5 that after adding the GST film, the position and number of the main absorption peaks of the device have changed. This is because the resonance frequency and loss have changed, and the position and intensity of the resonance wavelength have also changed. Figure 5 a shows the absorbance and reflectance of the designed five-layer structure in the IR-laser band. There is a clear absorption peak in each of the two atmospheric windows (3–5 and 8–14 μm) and in the non-atmospheric window (5–8 μm). Among them, the two absorption peaks located in the atmospheric window can reach the absorption rate of 95% and 55%, which still provides the basis for infrared detection. It is worth noting that the absorption/radiation intensity of the device in the non-atmospheric window can reach about 66%. At the same time, according to Boltzmann’s law [ 70 ],\n (7) E b = ∫ 0 ∞ E b λ d λ = ∫ 0 ∞ c 1 λ − 5 e c 2 λ T − 1 d λ = σ T 4 , \nwhere the proportionality coefficient σ is the Stefan constant, which is 2 π 5 k 4 15 c 2 h 3 ≈ 5.67 × 10 − 8 W ⋅ m 12 ⋅ K − 4 , and it can be clearly found that the thermal radiation intensity of a black body is proportional to the fourth power of the temperature. The higher the temperature of the object, the greater the total energy radiated. Thus, we can control the radiation spectrum of objects by adjusting their intrinsic emissivity and their temperature. As shown in Figure 5 , if we adjust the absorption/radiation wavelength to the non-atmospheric window by selective control, we can achieve effective thermal management by a higher absorption/radiation rate, so that heat can be released from the non-atmospheric window and heat buildup can be avoided. In this way, the device can achieve the effect of radiation cooling without being detected by the infrared detector. Based on the above phenomena, the first function of the five-layer structure we designed is infrared detection and radiation cooling. Then, simply by changing the temperature, after annealing the GST film at 160 °C (433.15 K), we obtained the five-layer structure containing the cGST film (thumbnail in Figure 5 b). It can be observed from Figure 5 b that the absorption rate of the device in the atmospheric window is significantly reduced, especially at 3–5 μm, where the maximum absorption rate is significantly reduced from 95% to 22%, and the average absorption rate is also reduced to about 15%. This makes it possible to transform functions of the device from infrared detection to infrared stealth. In order to explore the reason for this phenomenon, we simulated and analyzed the electromagnetic field distribution of the device in two phase states. In the comparison of Figure 6 a,b and Figure 7 a,b, it can be found that the electromagnetic field intensity of the device at the first absorption peak decreases with the phase transition of GST. When the GST film is in an amorphous state, plasmon resonance occurs at the interface between the top surface of the underlying metal and GST, and the same resonance phenomenon is also excited at the interface between the top gold array and the nano-spaced Al 2 O 3 layer. In addition, since the thickness of the dielectric layer is changed from 20 nm in the four-layer structure to a narrower 10 nm, a stronger nanogap resonance is excited, and the resonance wavelength is slightly red-shifted and the absorption effect becomes more intense. When the GST film phase changes to a crystalline state, various resonance effects are weakened, which leads to the transition of the device from high infrared absorption to low absorption. Similarly, by comparing Figure 6 c,d with Figure 7 c,d, we can observe that the electric field energy in the GST layer is significantly weakened, so that the absorption rate in the non-atmospheric window appears decreased: the absorption peak at λ = 6.6 μm decreased from 66% to 34%. However, since the magnetic field distribution is mainly concentrated at the slit and the connection area between the inner surface of the gold array and the thick gold layer, the change of the magnetic field strength is not large, and the effect of MR on the absorption spectrum is small. Although the average emissivity of the device in the non-atmospheric window has decreased at this time, it still maintains a certain radiative cooling effect. Interestingly, although the first two absorption peaks changed significantly, the absorption peak located at LWIR is exactly at λ = 10.6 μm, which is in a higher absorption state both before and after the phase transition. The position of the resonance wavelength is just the detection wavelength of LiDAR, so that better laser stealth function can be achieved. Combining Figure 6 and Figure 7 as a whole, it seems that both in the aGST and cGST states, higher order resonances are excited at short wavelengths, and the resonance order gradually decreases as the wavelength increases, which is also consistent with the properties of nano-gap resonance. Because gap plasmon resonance creates electric dipoles between parallel metal plates, smaller gap sizes result in lower energy states [ 71 , 72 ]. To sum up, it can be seen from Figure 5 b that the average absorptivity/emissivity of the device in both atmospheric windows is around 20%. Especially under the premise of maintaining the high absorption in the 10.6 μm band, the average emissivity in the 8–14 μm band remains low. It shows that the five-layer structure can allow for the better infrared-laser stealth function, and at the same time has a certain radiation cooling effect, realizing multifunctional applications. Furthermore, the stability of the device under different incident and polarization angles is investigated. As shown in Figure 8 , the reflectance spectra of the device under various conditions were obtained through simulation calculations. Regardless of whether the GST film is in the crystalline or amorphous state, the designed five-layer structure has good wide-angle absorption, and can still maintain high absorption at a large incident angle of about 60°. Meanwhile, the device also has good polarization insensitivity, and can maintain multifunctional applications well at a polarization angle of about 50°. This provides robust conditions for practical applications. 3.3. “Tunble Infrared-LiDAR Compatible Camouflage” by Different Crystalline Fractions of GST It can be found in previous studies [ 63 ] that the main reason why the device can achieve multifunctional applications under the five-layer structure is the addition of GST [ 73 ]. In fact, the phase transition process of GST is continuous and reversible. The aGST and cGST can achieve mutual conversion under the stimulation of external temperature changes, electric field changes and direct laser irradiation. Since GST is a nucleation-oriented material, the amorphous GST is first obtained by magnetron sputtering. Then, the aGST will gradually form many small nuclei inside under the stimulation of external factors. Finally, these small nuclei will join together to form a regular crystal structure and become the cGST [ 74 , 75 ]. Therefore, it is theoretically possible to estimate the proportion of crystalline molecules in the GST films by the corresponding spectral simulations to derive the degree of GST phase transition. We assume that the GST films in the mesophase state consist of different proportions of amorphous and crystalline molecules with dielectric constants ε a G S T λ and ε c G S T λ at wavelength λ , respectively [ 76 ]. At the same time, we can apply the relevant effective dielectric theory to estimate the effective dielectric constant ε G S T λ , C of this GST film in the mesophase state, where C is 0% to 100% of this GST film (from aGST to cGST) crystalline fraction ratio. In this paper, we use Equation (8) to define the Lorentz–Lorentz relation to approximate the effective permittivity of the GST intermediate state [ 73 ]: (8) ε G S T λ , C − 1 ε G S T λ , C + 2 = C × ε c G S T λ − 1 ε c G S T λ + 2 + 1 − C × ε a G S T λ − 1 ε a G S T λ + 2 , Thus, we can use the simple means of changing the temperature to stimulate the GST to be in different intermediate states. Further, the absorptivity of the device containing GST films with different crystalline fractions can be approximately calculated according to Equation (8). After simulation, we obtained a three-dimensional schematic diagram of the absorption rate of the designed five-layer structure in the infrared band, as shown in Figure 9 . Figure 9 clearly shows the continuous changes of the three absorption peaks under each crystalline fraction of GST. It can be directly observed from Table 1 that there is a significant decrease in the absorptivity of the first absorption peak from aGST to cGST, and a weakening of the second absorption peak, while the third absorption peak changes very little. Consequently, according to the dynamic change of the absorption rate of the device in different states of GST, we can use the significantly decreased absorbance of the first absorption peak to realize the functional transformation from IR detection to IR stealth in atmospheric windows. Good radiative cooling is maintained with good absorption/emissivity of the second absorption peak. The stable laser stealth function is realized by utilizing the almost invariant property of the third absorption peak. In addition, according to the continuous change of the GST phase transition process, we can achieve the corresponding IR-LiDAR compatible camouflage according to the actual emissivity of the background. The infrared emissivity of the device is made to be dynamically consistent with the background, so as to achieve dynamic and continuous infrared stealth and stable laser stealth. At the same time, the entire dynamic adjustment process can be achieved only by a simple temperature change, and the device can also maintain a certain radiative cooling when the temperature changes, so that the internal heat will not accumulate all the time, which is more in line with the requirements of practical applications. In practice, our proposed optical design can first be used for passive detectors in the infrared band. The metasurface detector can absorb the infrared radiation energy from the atmospheric window, and when the target enters the detection range, it causes a change in the absorbed energy. By comparing with the ambient energy, passive infrared detection can be achieved. Furthermore, the proposed optical design can be mounted on the outer surface of the protected device to achieve active laser stealth and passive IR stealth. By the higher absorption at 10.6 μm, this design can absorb most of the active LiDAR detection signals and make the signals impossible to return. At the same time, the device can avoid detection by IR detectors due to its low emissivity in the atmospheric window when the design contains the cGST. Finally, the proposed optical design can also wrap the device inside and provide energy release and passive radiative cooling for the device using the emissivity in the non-atmospheric window. In conclusion, our device realizes complex multi-band, multi-scenario and multifunctional applications on a fixed and compact structure, providing important ideas and references for electromagnetic shielding, perfect absorption, thermal management and infrared stealth."
} | 8,561 |
23110428 | PMC3533583 | pmc | 6,418 | {
"abstract": "Background Microalgae hold promise for yielding a biofuel feedstock that is sustainable, carbon-neutral, distributed, and only minimally disruptive for the production of food and feed by traditional agriculture. Amongst oleaginous eukaryotic algae, the B race of Botryococcus braunii is unique in that it produces large amounts of liquid hydrocarbons of terpenoid origin. These are comparable to fossil crude oil, and are sequestered outside the cells in a communal extracellular polymeric matrix material. Biosynthetic engineering of terpenoid bio-crude production requires identification of genes and reconstruction of metabolic pathways responsible for production of both hydrocarbons and other metabolites of the alga that compete for photosynthetic carbon and energy. Results A de novo assembly of 1,334,609 next-generation pyrosequencing reads form the Showa strain of the B race of B. braunii yielded a transcriptomic database of 46,422 contigs with an average length of 756 bp. Contigs were annotated with pathway, ontology, and protein domain identifiers. Manual curation allowed the reconstruction of pathways that produce terpenoid liquid hydrocarbons from primary metabolites, and pathways that divert photosynthetic carbon into tetraterpenoid carotenoids, diterpenoids, and the prenyl chains of meroterpenoid quinones and chlorophyll. Inventories of machine-assembled contigs are also presented for reconstructed pathways for the biosynthesis of competing storage compounds including triacylglycerol and starch. Regeneration of S -adenosylmethionine, and the extracellular localization of the hydrocarbon oils by active transport and possibly autophagy are also investigated. Conclusions The construction of an annotated transcriptomic database, publicly available in a web-based data depository and annotation tool, provides a foundation for metabolic pathway and network reconstruction, and facilitates further omics studies in the absence of a genome sequence for the Showa strain of B. braunii , race B. Further, the transcriptome database empowers future biosynthetic engineering approaches for strain improvement and the transfer of desirable traits to heterologous hosts.",
"conclusion": "Conclusions and outlook Functional annotation of the transcriptome of the B. braunii race B, strain Showa uncovered diverse biological processes operational in this hydrocarbon-producing green alga. Global comparisons with other algal transcriptomes reveal similar degrees of annotation coverage, with the number and the range of annotations supporting a strong degree of conservation between algal genomes and transcriptomes. The level of annotation coverage in B. braunii is supported by the identification of the majority of predicted enzymes within diverse biological pathways, for example within those related to the production of terpenes and lipids. This study describes reconstituted metabolic pathways related to the biosynthesis of terpenoid hydrocarbons in the non-model green microalga B. braunii Showa, following the fate of photosynthetic carbon from 3-phosphoglycerate to the general terpenoid precursors IPP and DMAPP, the production of linear polyprenyl backbones, the biosynthesis of triterpenoids, the decoration/tailoring of botryococcene and squalene to yield liquid hydrocarbon compounds and matrix structural materials, and possible routes for the extracellular localization of these compounds. A recurrent theme is the expansion of particular gene families. This allows the adaptation of the paralogs to structurally orthogonal substrates (botryococcene methyltransferases), and permits neofunctionalization to support novel biochemical reactions (botryococcene synthase). Paralogs may yield increased metabolic flux, or may provide additional flexibility in terms of regulation, compertmentalization, and biochemical properties (deoxyxylulose phosphate synthase, and possibly ABC transporters for hydrocarbon export). Metabolic pathways leading to other terpenoids have also been reconstructed, and anabolic pathways for competing storage compounds (TAG and polysaccharides) were similarly mapped. The reconstructed metabolic networks, their participating enzymes and the corresponding cDNA sequences provide a genetic and metabolic framework that should empower biosynthetic engineering approaches targeting the increased production of hydrocarbons in B. braunii , or the mobilization of these pathways into genetically tractable photosynthetic (algal or land plant) hosts or heterotrophic microbial strains [ 50 , 120 - 122 ]. In particular, increasing the flux of photosynthetic carbon towards terpene precursors in the chloroplast or the cytosol of B. braunii by transplanting the cytoplasmic mevalonate pathway, or by fine tuning the expression of DXR (deoxyxylulose phosphate reductase) and Idi (isopentenyl-diphosphate delta-isomerase) may be interesting approaches [ 60 , 120 , 123 ]. Further experiments should investigate the importance of the pentose phosphate cycle as an anaplerotic pathway feeding into the MEP/DOXP pathway, and clarify the actual metabolite(s) and enzyme(s) connecting these two routes [ 62 ]. Conversely, tuning down, or blocking the biosynthesis of competing storage carbons (like that of starch in Chlamydomonas [ 20 , 124 , 125 ]) may also increase the accumulation of liquid hydrocarbons. Varying the level and timing of the expression for the two-component botryococcene synthase [ 34 , 38 ] and various storage hydrocarbon decorating enzymes (including the recently described methyltransferases for C 32 triterpenoids [ 33 ], the still unknown methyltransferases for C 34 triterpenoids, and the various oxidases that yield oxidized (methyl)squalenes, ether polymers and matrix materials) should help to tailor liquid hydrocarbon biosynthesis for particular biofuel applications. Investigations into the export of hydrocarbons into the extracellular matrix of B. braunii should uncover a particularly valuable trait that may have a widespread application to increase yield, reduce end product toxicity and simplify product recovery during biofuel manufacture by fermentation. Interesting questions are presented by the interconnection of the biosyntheses of terpenoid hydrocarbons and very long chain fatty acids with the formation of the extracellular matrix materials in B. braunii that provide the building blocks for an extracellular carbon storage organ, but also the physical basis for colony organization in this organism. Further transcriptomic, proteomic, metabolomic, and metabolic flux analyses that compare varied growth conditions influencing hydrocarbon accumulation would shed light on the regulatory networks and pathway interactions channeling carbon and energy flow in algal cells. In addition to biofuel biosynthetic pathway discovery, the integrated data-mining environment offered by the publicly available web annotation tool described here allows researchers to query the B. braunii transcriptome for any specific transcript sets to rapidly and efficiently extract biologically relevant information related to different contexts. As the transcriptome dataset is revised and supplemented with addtional manually curated annotations, the most current functional data will be made available to the community via the public portal and annotation tool.",
"discussion": "Results and discussion Biomass sample collection and RNA isolation from the Showa strain of B. braunii The advent of massively parallel high throughput cDNA sequencing techniques now allows the cost-effective de novo assembly and analysis of the transcriptomes of organisms with still unsequenced genomes [ 23 , 24 ]. The genome size of B. braunii Showa has recently been measured at 166 Mbp, significantly larger than that of the largest sequenced Chlorophyta alga, V. carteri at 138 Mbp [ 36 ]. This large genome, and the long and frequent repeat regions present in the B. braunii genome (A. Koppisch, personal communication) make the sequencing and analysis of this genome challenging. Although a transcriptome sequence database cannot capture untranscribed genomic regions (for example promoters) and does not reflect post-transcriptional regulation, it can still serve as a useful tool for gene discovery and metabolic pathway and network reconstruction, and will inform further proteomic and genomic analyses. As a first step to construct such a database, we isolated total RNA from seven time points (days 0, 3, 5, 8, 14, 18, and 22) during the four week culture cycle of the Showa strain of B. braunii , race B. These time points are centered on the early portion of the culture cycle (days 0–8) because previous studies have shown that the biosynthesis and accumulation rate of botryococcenes is maximal in this period [ 35 , 37 , 38 ]. Additionally, both enzyme activity and gene expression associated with botryococcene biosynthesis have been shown to be maximal during these early time points [ 35 , 37 , 38 ]. Because of this, we hoped that the resulting transcriptomic database would be enriched for transcripts related to liquid hydrocarbon biosynthesis. Total RNA from each time point was purified using TRIzol and LiCl precipitation to eliminate co-purifying polysaccharides. Each sample was analyzed for protein and polysaccharide contaminations ( Additional file 1 : Table S1) and 5-μg aliquots from the samples with the most RNA (days 0, 3, and 5) were analyzed on a denaturing agarose gel ( Additional file 1 : Figure S1A). To further confirm successful isolation of good quality RNA, RT-PCR was carried out to amplify cDNA fragments for several B. braunii Showa genes (Additional file 1 : Figure S2), including squalene synthase (SS) [ 37 ] and squalene synthase-like-1 (SSL-1) [ 34 ]. All of the RNA samples were then pooled into a single sample, treated with DNAse (Additional file 1 : Figure S1B), and analyzed for any remaining RNase contamination (Additional file 1 : Figure S1C). These analyses indicated that the isolated RNA was of high quality and did not contain active nucleases. The pooled RNA sample was submitted to the Department of Energy Joint Genome Institute (JGI) for transcriptome sequencing. De novo assembly of the B. braunii Showa transcriptome 454 pyrosequencing yielded 1,334,609 reads (620 Mb of data) representing the ESTs from mRNA isolated from whole cells of a near-axenic B. braunii Showa culture [ 36 ] as described above. The reads have been deposited by the JGI into two publicly available Sequence Read Archive accessions, SRX028986 and SRX028987. We assembled these reads into 46,422 contigs with an average length of 756 bp (Additional file 1 : Figure S3) using a multi-step, recursive sequence assembly protocol [ 39 , 40 ] as described in the Methods. This new transcriptome assembly provides significantly improved coverage over that of Watanabe et al. for a different B race strain of B. braunii (27,427 contigs with an average length of 267 bp for strain BOT-22 [ 32 ]). Contig coverage in the Showa transcriptome assembly spans three orders of magnitude, from 1X to 8,231X. The most highly represented transcripts either encode proteins related to photosynthesis, or do not show significant similarity to sequences in the GenBank non-redundant database (E-value < 1e-5, Additional file 1 : Table S2). To benchmark the quality of our assembly, we compared the B. braunii Showa transcriptome to the core set of 458 conserved proteins (CEGs) that occur in a wide variety of eukaryotes [ 41 ]. Using the Core Eukaryotic Genes Mapping Approach (CEGMA) algorithm [ 41 , 42 ], we recovered 451 out of the 458 core proteins (98.4%, E value cutoff ≤ 1e-5), with 325 out of the 458 CEGs (71.0%) yielding alignments whose lengths exceed 60% of either the CEG or the contig sequence. Functional annotation, web-based annotation tool and data depository Functional annotations were assigned to all unique transcript sequences using a previously described annotation pipeline [ 28 ]. The Kyoto Encyclopedia of Genes and Genomes (KEGG) [ 25 ], MetaCyc [ 43 ], Reactome [ 44 ], Panther [ 45 ], and Pfam [ 46 ] annotation databases were chosen to provide biological pathway, ontology, and protein domain annotations. Orthologous proteins from C. reinhardtii (a model green alga) and Arabidopsis thaliana (thale cress) were also used to infer ontology identifiers from their respective Gene Ontology and MapMan Ontology sets [ 47 ] (Additional file 1 : Table S3). Functional annotations were assigned to 20,906 transcripts (45%), while 8,575 sequences yielded significant BlastX hits (E value <1e-5) against the non-redundant protein database of GenBank. Species in the green plant lineage were the most frequent sources from which functional annotations were derived. Top-hit analysis of KEGG protein alignments producing pathway annotations confirmed the algal character of the transcripts (Additional file 1 : Table S4). Eleven of the top 15 organisms are algae or land plants, with the two top organisms, V. carteri and C. reinhardtii , together contributing 25% of all KEGG pathway assignments. An analysis of the contigs by the Metagenomics RAST server [ 48 ] found top Blast hits to proteins from fungi (Ascomycota and Basidiomycota, 19.8% of the contigs), animals (Chordata, Arthropoda, and Nematoda, 10.7% of the contigs), and bacteria (Firmicutes, Proteobacteria, and Actinobacteria, 10.1% of the contigs). However, this analysis likely provides a highly inflated estimate of non-Botryococcal transcripts in our database. While some of these transcripts may indeed originate from contaminating organisms reflecting the non-axenic nature of the algal culture and/or sample handling mistakes introduced during the sequencing process, others still likely represent genuine B. braunii transcripts. These transcripts may highlight gaps and biases in the GenBank database, reflect localized high similarities to phylogenetically distant homologues, originate from sequencing/assembly mistakes, or represent genes from recent horizontal gene transfer. Four-way comparisons amongst the genome sequences of selected green algae and the Showa transcriptome revealed 1,297 KEGG objects that are shared by B. braunii , Micromonas sp. RC299, Chlorella variabilis NC64A, and C. reinhardtii (Figure 2A ), while 260 KEGG objects were found only in B. braunii Showa ( Additional file 2 : Table S5). This is likely an overestimate of the true number of KEGG-annotated genes unique to B. braunii . First, several plastid-encoded transcripts (e.g. psbA, psbB) are present in our transcriptome assembly that are not found in the nuclear genome annotations of the other three species. Second, contigs representing transcripts of non- Botryococcus origin are also present in our database due to the non-axenic nature of the culture from which the mRNA was isolated (see examples in the later sections). Nevertheless, KEGG ontology terms assigned to contigs in our transcriptome show a distribution similar to those in the sequenced genomes of other Chlorophyta algae (Figure 2B ).\n Figure 2 Comparisons of KEGG annotations for Chlorophyta algae. A . Overlap amongst KEGG annotation objects assigned to one or more genes/contigs in the transcriptome of B. braunii Showa, and the genomes of C. reinhardtii , Ch. variabilis NC64A, and Micromonas RC299. Contigs with KEGG annotations found only in the B. braunii Showa transcriptome are listed in Additional file 1 : Table S5. B. Distribution of genes/contigs annotated with KEGG ontology categories. Circles on left: first-level ontologies; circles on right: second-level metabolic process ontologies. B , B. braunii Showa; Ch, Ch. variabilis NC64A; C , C. reinhardtii ; M, Micromonas RC299. Inspection of the completeness of pathway assignments shows that most reactions in the majority of KEGG pathways are catalyzed by at least one predicted enzyme in the B. braunii Showa transcriptome. The distribution of molecular function GO (Gene Ontology) identifiers reveals the diversity of annotation categories assigned to the contigs (Figure 3 ). Thus, the annotation shows that we have assembled a comprehensive database of the B. braunii Showa transcriptome that may serve as a valuable reference for investigating the metabolic capabilities of this organism.\n Figure 3 B. braunii Showa contigs annotated with Gene Ontology terms for molecular function. In order to facilitate the exploration of the data and to expedite future B. braunii omics and functional genetic studies, the sequences and their annotations have been deposited into the Algal Functional Annotation Tool [ 28 ] and are publicly accessible from a web-based portal at http://pathways-pellegrini.mcdb.ucla.edu/botryo1 . A combined view of annotations from all primary databases for any particular transcript can be accessed by the transcript ID, and provides pathway, ontology, and protein domain data alongside primary sequence data. Functional enrichment testing and dynamic pathway visualization may be performed for lists of transcripts from within the annotation tool. Transcripts may also be looked up by biological function using a keyword search tool that returns lists of transcripts with annotations matching a keyword or a phrase. Lastly, a pathway browser tool allows visualization of transcripts for any KEGG pathway of interest. The public repository of the B. braunii Showa transcriptome assembly and the utilities provided to query the data will furnish a platform to update annotations as these are made available by future studies and characterization of additional strains. Manual curation and pathway reconstruction of the terpenome Biosynthesis of terpenes yields a large variety of essential primary metabolites and specialized secondary metabolites in plants. Terpene biosynthesis provides membrane sterols, phytohormones, carrier molecules for N-glycan biosynthesis, pigments and antioxidants, volatile oils, aroma compounds and resins, and various toxins. Terpene biosynthesis also provides the prenyl side chains of photosynthetic pigments and meroterpenoid carrier molecules in oxidative phosphorylation, as well as polyprenyl compounds for the prenylation of proteins [ 49 , 50 ]. Crucially, it also underpins the production of extracellular liquid hydrocarbons and contributes to the polymeric extracellular matrix materials in race B strains of B. braunii [ 11 , 51 ]. C 30 -C 37 triterpenes, in the form of methylated, oxidized, and cyclized botryococcenes, as well as methylated squalene, are initially synthesized inside the cells and at least botryococcenes can be found in intracellular oil bodies [ 11 ]. However, the majority (95%) of the botryococcenes are deposited into the colony extracellular matrix [ 10 ]. These oils that may be described as “bio-crude” are of prime interest for the biofuel industry as petroleum replacements. To derive a comprehensive picture of the biosynthesis of the terpenome of B. braunii Showa, contigs in our databases that encode proteins for metabolic pathways yielding terpenes and their precursors (Figure 1 ) were collected and supplemented by further contigs identified by targeted Blast searches and Pfam keyword inquiries, as described in the Methods section. Contigs were extended and assembled into isotigs, and sequence discrepancies and potential frameshifts in the derived protein models were resolved using manual alignments and scrutiny of the assemblies. Manually curated transcript sequences (contigs and isotigs) are referred heretofore as “curated contigs”, while transcripts assembled without human supervision are simply called “contigs”. Contigs and curated contigs were considered as potentially originating from a different organism of the non-axenic culture if their deduced protein products show the highest similarities to fungal, animal or bacterial proteins with no or very low similarities to plant proteins, and if at the same time their codon usage significantly differs from that of B. braunii ( http://www.kazusa.or.jp/codon/index.html ). Subcellular localization of the deduced proteins was also predicted using TargetP ( http://www.cbs.dtu.dk/services/TargetP/ ). Based on databank similarities and the prediction of facile translational start and stop codons, full-size protein models were derived for approximately 30% of the manually curated transcripts. A distinguishing feature of B. braunii Showa transcripts is their surprisingly long (>1-2 kb) 3’ untranslated regions (UTRs), as already noted by Okada et al. [ 35 ]. Machine-assembled contigs with moderate to high sequence coverage, apparently derived from these long 3’ UTRs, are abundant in the transcriptome database. These may be partially responsible for the relatively large number of contigs in the dataset, and are also in apparent congruence with the large genome size of B. braunii [ 36 , 52 ]. Transcript abundances were approximated from sequence coverage, i.e. the number of primary sequence reads for a particular contig divided by the length of that contig. True estimation of expression levels would require the utilization of other techniques like qRT-PCR, DNA microarray analysis, or proteomics [ 18 , 21 , 22 ]. Biosynthesis of terpene precursors Terpenes are biosynthesized from the universal C 5 building blocks isopentenyl diphosphate (IPP 13 , Figure 4 ) and dimethylallyl diphosphate (DMAPP, 14 ). These precursors originate from the mevalonate (MVA) pathway in the cytosol of animal, fungal, archaeal, Gram-positive coccus, and higher plant cells, while the methylerythritol 4-phosphate/deoxyxylulose phosphate (MEP/DOXP) pathway is operational in plant plastids and many Gram-positive and Gram-negative Eubacteria. Genomic and biochemical evidence indicates that green algae (Chlorophyta) may have lost the MVA pathway, while other photosynthetic eukaryotes including land plants (Streptophyta) generally retain a functional contingent of the MVA pathway enzymes [ 49 ]. Experimental evidence from B. braunii also argues for the exclusive utilization of the MEP/DOXP pathway for terpene precursor biosynthesis in this alga [ 53 ]. Fittingly, exhaustive searches of the assembled B. braunii Showa transcriptome (and the unassembled singletons) identified ESTs only for the first two of the six enzymes of the MVA pathway (Additional file 1 : Table S6) [ 54 ]. These two enzymes, a predicted acetyl-CoA carboxylase (AtoB, E.C. 2.3.1.9) and a deduced hydroxymethylglutaryl-CoA synthase (HMGS, E.C. 2.3.3.10), were found to be encoded by a single curated contig each with low sequence coverage (<25 reads/kb), hinting at low abundance of the corresponding mRNA species in B. braunii Showa. Further curated contigs in our assembly for presumed AtoB and HMGS, and for a third predicted MVA pathway enzyme, hydroxymethylglutaryl-CoA reductase (HMGR, E.C. 1.1.1.34) display very high identities to fungal sequences and show a highly divergent codon usage compared to that of B. braunii . These transcripts may have originated from cohabiting fungi of the non-axenic culture used for RNA isolation, or may result from very recent horizontal gene transfers into the alga. Both AtoB and HMGS are involved in various catabolic processes, including the degradation of ketone bodies and branched-chain amino acids. Thus, their deduced presence in the B. braunii Showa transcriptome is not an indication for the presence of a functional MVA pathway.\n Figure 4 Reconstruction of the biosynthesis of terpenoid precursors in B. braunii Showa. 1, 3-phospho-D-glycerate; 2, 3-phospho-D-glyceroyl phosphate; 3, D-glyceraldehyde 3-phosphate; 4, 2-phospho-D-glycerate; 5, phosphoenolpyruvate; 6, pyruvate; 7, 1-deoxy-D-xylulose 5-phosphate; 8, 2- C -methyl-D-erythritol 4-phosphate; 9, 4-diphosphocytidyl-2- C -methyl-D-erythritol; 10, 4-diphosphocytidyl-2- C -methyl-D-erythritol 2-phosphate; 11, 2- C -methyl-D-erythritol 2,4-cyclodiphosphate; 12, ( E )-4-hydroxy-3-methylbut-2-en-1-yl diphosphate; 13, isopentenyl diphosphate; 14, dimethylallyl diphosphate. PGK, phosphoglycerate kinase; GAPDH, glyceraldehyde phosphate dehydrogenase; GAPN, NADP + -dependent glyceraldehyde 3-phosphate dehydrogenase; PGAM, phosphoglycerate mutase; ENO, phosphopyruvate hydratase; PK, pyruvate kinase; DXS, 1-deoxy-D-xylulose 5-phosphate synthase; DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase; IspD, 2- C -methyl-D-erythritol 4-phosphate cytidylyltransferase; IspE, 4-(cytidine 5’-diphospho)-2- C -methyl-D-erythritol kinase; IspF, 2- C -methyl-D-erythritol 2,4-cyclodiphosphate synthase; IspG, ( E )-4-hydroxy-3-methylbut-2-enyl diphosphate synthase; IspH, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; Idi, isopentenyl-diphosphate Δ-isomerase. Fd, ferredoxin. In contrast, a complete contingent of deduced enzymes for the MEP/DOXP pathway [ 49 , 55 , 56 ] is well represented in the B. braunii Showa transcriptome. The MEP/DOXP pathway uses D-glyceraldehyde 3-phosphate and pyruvate as its metabolic input. Photosynthetic 3-phospho-D-glycerate ( 1 ) may be converted to D-glyceraldehyde 3-phosphate ( 3 ) via 3-phospho-D-glyceroyl phosphate ( 2 ) by the collective action of phosphoglycerate kinase (PGK, E.C. 2.7.2.3) and glyceraldehyde phosphate dehydrogenase (GAPDH, Figure 4 , Additional file 1 : Table S6). PGK is represented in our transcriptome by three nonredundant curated contigs for three presumed isoenzymes, one of which may have originated from a fungal cohabitant. Curated contigs for one predicted isoform of the NADP + -dependent GAPDH (E.C. 1.2.1.13, Benson-Calvin cycle) and five inferred isoforms (three of them probably of fungal origin) of the NAD + -dependent GAPDH (E.C. 1.2.1.12, glycolysis and gluconeogenesis) were also identified. During glycolysis, D-glyceraldehyde 3-phosphate ( 3 ) may also be converted to ( 1 ) by the non-phosphorylating NADP + -dependent glyceraldehyde 3-phosphate dehydrogenase (GAPN, E.C. 1.2.1.9), encoded by a curated contig with moderate coverage (25–99 reads/kb) in the dataset (Additional file 1 : Table S6). TargetP predictions for the subcellular localization of these enzymes provided support for the mitochondrial targeting of the putative NAD + -dependent GAPDH (E.C. 1.2.1.12) isoforms, and cytosolic localization for the PGK isozymes. Some transcripts for inferred PGK and NAD + -dependent GAPDH (E.C. 1.2.1.12) are present at very high abundance (>250 reads/kb) in the transcriptome, suggesting a high flux involving D-glyceraldehyde 3-phosphate in the glycolysis/gluconeogenesis pathways in B. braunii Showa. Contigs encoding short fragments of PGK and GAPDH orthologs have recently been described from the race B B. braunii strain BOT-22 [ 32 ]. These show 89-99% identity at the amino acid level with the corresponding enzymes from B. braunii Showa predicted in this study. Pyruvate ( 6 ) is formed from ( 1 ) in three steps by phosphoglycerate mutase (PGAM, E.C. 5.4.2.1), phosphopyruvate hydratase (ENO, E.C. 4.2.1.11) and pyruvate kinase (PK, E.C. 2.7.1.40) during glycolysis/gluconeogenesis (Figure 4 ). All these enzymes are encoded in the transcriptome as multiple deduced isozymes (Additional file 1 : Table S6). Curated contigs 10955, 16949, 23205, and 41366 show the highest similarities to individual domains of PKs with four similar catalytic domains each, thus probably representing a single multifunctional enzyme. All these curated contigs have moderate coverage. Curated contig 43373, encoding a predicted cytoplasmic ENO, is the only exception and is represented by very abundant ESTs. Two contigs from B. braunii BOT-22 (FX085139 and FX085140) encoding short regions of PK [ 32 ] show 94% amino acid identity to two inferred PK isozymes encoded in our dataset (curated contig 10955 and 41736, respectively). Four curated contigs code for three predicted isozymes of the first enzyme of the MEP/DOXP pathway, the thiamine diphosphate-dependent 1-deoxy-D-xylulose 5-phosphate synthase (DXS, E.C. 2.2.1.7) that produces 1-deoxy-D-xylulose 5-phosphate ( 7 ) from ( 3 ) and ( 6 ) (Figure 4 , Additional file 1 : Table S6). Multiple isoenzymes of DXS are routinely found in land plants [ 57 ], and clade into three phylogenetically distinct families [ 58 ]. Constitutively expressed DXS isozymes of these plants produce precursors for essential terpenoids, while certain inducible DXS isozymes specialize in stress response and ecological interactions with symbionts or pathogens [ 57 ]. In contrast, genomic evidence shows that strains of green algae harbour only a single DXS each. These proteins form a sister clade to the three DXS clades of land plants [ 35 , 49 ]. The Okada group has recently reported the cloning and biochemical characterization of three isozymes of DXS from B. braunii Showa [ 35 ]. Interestingly, these three isoenzymes all fall into the basal clade for Chlorophyta DXSs, thus representing paralogous sequences resulting from gene duplications within the green algal lineage [ 35 ]. All three isozymes were found to be expressed simultaneously, and show similar kinetic parameters except for a higher temperature tolerance for DXS-III [ 35 ]. Our data also indicate similar, moderate transcript abundances for DXS-I (curated contig 10163, 66.6 reads/kb) and DXS-II (curated contig 42027, 93.7 reads/kb), with a somewhat lower abundance for DXS-III-related ESTs (curated contigs 07667 and 11032, 4.0 reads/kb and 29.4 reads/kb, respectively). Short curated contigs representing orthologs of DXS-II (FX085276 and FX085277) and DXS-III (FX085274 and FX085275) were also identified in the preliminary transcriptomic analysis of the B. braunii race B strain BOT-22 [ 32 ], with amino acid identities to the Showa enzymes in the 72-84% range ( Additional file 1 : Table S6). All three DXS isozymes of the Showa strain have been found to contain chloroplast targeting sequences at their N-termini [ 35 ], in agreement with our TargetP predictions ( Additional file 1 : Table S6). DXS has been described as one of the rate-limiting steps of the MEP/DOXP pathway in plants [ 49 , 57 ], thus the expression of three isoforms of this enzyme in B. braunii Showa might provide an increased metabolic flux for the production of terpenoid precursors. Alternatively, each DXS isoform might be associated with the production of a specific class of terpenoids. Considering the absence of the cytoplasmic mevalonate pathway as an alternative to furnish isoprene precursors, evolutionary optimization of the DXS step by repeated gene duplications providing parallel capacity for the production of ( 7 ) might have been beneficial for B. braunii race B strains. 1-deoxy-D-xylulose 5-phosphate ( 7 ) is converted to 2- C -methyl-D-erythritol 4-phosphate ( 8 , Figure 4 ) by the NADPH-dependent enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR, E.C. 1.1.1.267). A single deduced isozyme of DXR is encoded in our dataset by a curated contig with moderate coverage (Additional file 1 : Table S6). The DXR-catalyzed reaction is the first committed step towards the production of isoprene precursors since ( 7 ) is also utilized for the biosynthesis of thiamine diphosphate and pyridoxal phosphate [ 57 ]. The rest of the pathway involves the CTP-dependent conversion of ( 8 ) to 4-diphosphocytidyl-2- C -methyl-D-erythritol ( 9 ) by IspD (2- C -methyl-D-erythritol 4-phosphate cytidylyltransferase, E.C. 2.7.7.60), and the ATP-dependent phosphorylation of ( 9 ) to 4-diphosphocytidyl-2- C -methyl-D-erythritol 2-phosphate ( 10 ) by IspE (4-(cytidine 5’-diphospho)-2- C -methyl-D-erythritol kinase, E.C. 2.7.1.148). Curated contigs for these two inferred enzymes are present in the Showa transcriptome at moderate coverage. Formation of 2- C -methyl-D-erythritol 2,4-cyclodiphosphate ( 11 ) from ( 10 ) by IspF (2- C -methyl-D-erythritol 2,4-cyclodiphosphate synthase, E.C. 4.6.1.12) releases CMP, followed by the two-electron reduction of ( 11 ) to ( E )-4-hydroxy-3-methylbut-2-en-1-yl diphosphate ( 12 ) by the [4Fe-4S] enzyme IspG (4-hydroxy-3-methylbut-2-enyl diphosphate synthase, E.C. 1.17.7.1). The last step of the MEP/DOXP pathway is the formation of both isopentenyl diphosphate (IPP, 13 ) and dimethylallyl diphosphate (DMAPP, 14 ) from ( 12 ) by another [4Fe-4S] enzyme, IspH (also known as LytB, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, E.C. 1.17.1.2)[ 59 ]. Both IspG and IspH accept electrons from a ferredoxin. These electrons may originate directly from the photo-oxidation of water during photosynthetic conditions in the chloroplast without the involvement of reducing cofactors, while a ferredoxin reductase is required in the dark to channel electrons from cellular pools of NADPH [ 57 ]. The branching reaction catalysed by IspH is in stark contrast to the MVA pathway that yields IPP ( 13 ) exclusively [ 59 ], which has to be later isomerised to DMAPP ( 14 ) by Idi (isopentenyl-diphosphate delta-isomerase, E.C. 5.3.3.2). Each of the predicted enzymes for the downstream half of the MEP/DOXP pathway (IspF and onwards) are encoded by single nonredundant curated contigs with high to very high sequence coverage in the B. braunii Showa transcriptome (200.7, 242.9, and 426.6 for IspF, IspG and IspH, respectively), indicating vigorous transcription and perhaps robust metabolic flow through these enzymes. This is in contrast to some plant systems where the same enzymes were found to be rate-limiting [ 49 , 57 ]. Two presumed isozymes of Idi are encoded by two nonredundant curated contigs with only low coverage in the Showa transcriptome. Another curated contig with moderate coverage for a putative fungal IPP isomerase was also identified (34876, Additional file 1 : Table S6). Although Idi is generally present in organisms utilizing the MEP/DOXP pathway for terpenoid precursor biosynthesis, it has not been found strictly essential and plays only a supplementary role in optimizing IPP and DMAPP ratios [ 57 ]. Type II IPP isomerases, detected in Streptomyces spp. and in Synechocystis spp. [ 60 , 61 ], were not found in the Showa transcriptome. TargetP predictions for all MEP/DOXP enzymes suggest chloroplast targeting, with the exception of IspH where both chloroplast and mitochondrial targeting seems equally plausible (Additional file 1 : Table S6). The cyanobacterium Synechocystis sp. PCC6803 contains no MVA pathway but features a full complement of the MEP/DOXP pathway. Nevertheless, neither pyruvate ( 6 ) nor 1-deoxy-D-xylulose 5-phosphate ( 7 ) has been observed to stimulate IPP biosynthesis in cell extracts. Similarly, fosmidomycin has not been seen to inhibit DXR by reducing IPP biosynthesis in vitro or by reducing cellular growth in vivo [ 62 ]. On the other hand, metabolites of the reductive pentose phosphate cycle, especially D-xylulose 5-phosphate, increased IPP formation even in the presence of the DXR inhibitor fosmidomycin [ 62 ]. While the immediate entry point of the pentose phosphate cycle metabolites into the MEP/DOXP pathway is currently not known, it is assumed to be downstream of DXR (Figure 4 ). A survey of the Showa transcriptome identified curated contigs encoding putative chloroplast-targeted RPE (ribulose phosphate 3-epimerase, E.C. 5.1.3.1) and thiamine diphosphate-dependent TKTL (transketolase, E.C. 2.2.1.1) enzymes that yield D-xylulose 5-phosphate in the pentose phosphate cycle (Additional file 1 : Table S6). Short contigs that encode fragments of the TKTL enzyme have recently been identified from B. braunii BOT-22 (FX085315 and FX085314) [ 32 ], and show 87-90% amino acid identities with the enzyme encoded by curated contig 32329 of the Showa transcriptome. Transcripts of putative fungal origin were also detected for both RPE and TKTL. Thiamine diphosphate-dependent phosphoketolase (XFP, E.C. 4.1.2.9) was represented only by a single curated contig of potential fungal origin. This enzyme generates ( 3 ) and acetyl phosphate from D-xylulose 5-phosphate and inorganic phosphate. Curated contig 30447 for RPE has moderate sequence coverage at 55.8 reads/kb, while reads for curated contig 32329 for TKTL are highly abundant at 436.6 reads/kb. While the importance, or even the dominance, of the pentose phosphate cycle for isoprene precursor biosynthesis has been speculated upon for the BOT-22 and BOT-70 strains of B. braunii race B [ 31 , 32 ], these recent studies employed limited transcriptomic and EST datasets. Our data support the presence of a fully functional MEP/DOXP pathway in B. braunii Showa, with multiple paralogous DXSs with moderate EST coverage for each, providing a reasonable entry for isoprene precursor biosynthesis. The sequence coverage of the inferred downstream half of the MEP/DOXP pathway enzymes (IspF and onwards) is higher than that of the upstream half of the pathway, which would be consistent with an anaplerotic feed of metabolites from the pentose phosphate cycle. Future studies of the dynamics of transcription of the MEP/DOXP pathway and the pentose phosphate cycle enzymes by e.g. qRT-PCR, in relation to the age of the culture and hydrocarbon production levels would be necessary to shed more light on this issue. Investigating the inhibition by fosmidomycin of B. braunii cultures in vivo and/or the isolated Showa DXR enzyme in vitro may also provide further clues to map the flux of metabolites through these alternative biosynthetic routes. Terpene backbone biosynthesis Terpenoids are derived from linear polyprenyl diphosphate chains that are generated by the stepwise recursive head-to-tail (1’-4) condensation of IPP ( 13 ) first with DMAPP ( 14 ) and further with allylic polyprenyl diphosphates with the concomitant release of pyrophosphate in every condensation cycle (Figure 5 ). These alkylations are catalysed by a family of polyprenyl diphosphate synthases (prenyl transferases) that are present in widely varying numbers in plants [ 63 ]. Prenyl transferases may have relatively relaxed substrate and product specificities, accepting DMAPP or longer allylic prenyl diphosphates and producing polyprenyls of different chain lengths [ 63 - 65 ].\n Figure 5 Reconstruction of the biosynthesis of linear terpene backbones in B. braunii Showa. 15, Geranyl diphosphate; 16, 2- trans , 6- trans -farnesyl diphosphate; 17, all-trans -geranylgeranyl diphosphate; 18, all-trans -nonaprenyl diphosphate; 19, all-trans -decaprenyl diphosphate; 20, di-trans , poly-cis -polyprenyl diphosphate (n=10-55). GDPS, geranyl diphosphate synthase; FDPS, farnesyl diphosphate synthase; GGDPS, geranlylgeranyl diphosphate synthase; SDPS, solanesyl diphosphate synthase; PDSS1, decaprenyl diphosphate synthase; DHDDS, dehydrodolichyl diphosphate synthase. C 10 geranyl diphosphate ( 15 , Figure 5 ) is synthesized by geranyl diphosphate synthase (GDPS, E.C. 2.5.1.1) from ( 13 ) and ( 14 ). 15 yields monoterpenes in plants, and serves as a precursor for solanesyl diphosphate synthase (SDPS, E.C. 2.5.1.84) that affords all-trans -nonaprenyl diphosphate ( 18 ) for the polyprenyl chains of terpenoid quinones in the mitochondria (ubiquinones) and in the chloroplast (plastoquinones). Both GDPS and SDPS are predicted to be present in the Showa transcriptome as a single nonredundant curated contig each with low sequence coverage (Additional file 1 : Table S7). Farnesyl diphosphate synthase (FDPS, E.C. 2.5.1.10) generates C 15 farnesyl diphosphate ( 16 ) in two reaction steps via 15 . 16 is the precursor for the sesquiterpenes and the triterpenoids (including squalene and phytosterols), and provides the farnesyl side chains for post-translational modification of proteins. 16 is also the substrate for decaprenyl diphosphate synthase (PDSS1, E.C. 2.5.1.91). This enzyme produces all-trans -decaprenyl diphosphate ( 19 ) for the side chain of the mitochondrial electron carrier ubiquinone-10 (coenzyme Q 10 ). The cis- prenyltransferase dehydrodolichyl diphosphate synthase (DHDDS, E.C. 2.5.1.-) also uses 16 as its substrate to generate dehydrodolichyl diphosphates ( di-trans , poly-cis -polyprenyl diphosphate, 20 ) that serve as precursors to the glycosyl carrier lipid dolichol for N -glycan biosynthesis. Crucially, 16 is also the precursor for the liquid hydrocarbon triterpenoid botryococcenes and methylated squalenes, and the cell wall ether lipids and some of the matrix polymers of B. braunii Showa [ 9 , 11 ]. Two nonredundant curated contigs for two putative isozymes of FDPS with 72% amino acid identity were identified in the Showa transcriptome, both with moderate sequence coverage (Additional file 1 : Table S7). The FDPS isozyme encoded by curated contig 15137 is predicted to be localized outside the chloroplast, the mitochondrion, or the secretory apparatus of the cell. A third curated contig for a presumed FDPS with extremely low sequence coverage may stem from a fungal source (Additional file 1 : Table S7). Deduced DHDDS isozymes are encoded by four curated contigs of low sequence coverage, one of which is potentially of fungal origin. A single curated contig with low sequence coverage encodes a putative PDSS1 that may also have been derived from a fungal cohabitant (Additional file 1 : Table S7). Geranylgeranyl diphosphate ( 17 ) is produced by geranylgeranyl diphosphate synthase (GGDPS, E.C. 2.5.1.29), encoded in the Showa transcriptome by only a single nonredundant curated contig with moderate sequence coverage (Additional file 1 : Table S7). In higher plants, this enzyme may be present as three isoenzymes - one cytosolic, one plastidic and one mitochondrial [ 49 ]. TargetP prediction supports mitochondrial localization for the single GGDPS identified in the Showa transcriptome. The expected plastidic GGDPS might be encoded by the chloroplast genome and thus likely missed by our transcriptome database. Alternatively, the other isoforms may arise from multiple targeting, or simply represent gaps in the current transcriptome database due to lower levels of expression or sequencing/assembly artifacts. Plant GGDPS enzymes may initiate synthesis of 17 from 14 (Figure 5 ), but the most effective substrate of GGDPSs from animals and fungi is 16 [ 49 , 65 ]. 17 serves as the precursor for the C 20 diterpenes including gibberellins, the phytyl side chains of chlorophyll, phylloquinone and tocopherol (vitamin E), and the geranylgeranyl chains of prenylated proteins. 17 is also the precursor for tetraterpenoid carotenoids, including lycopene, lutein, canthaxanthin and others isolated from race L and race B B. braunii strains [ 11 , 66 ]. Liquid triterpenoid hydrocarbon biosynthesis Farnesyl diphosphate ( 16 ) serves as the precursor for the biosynthesis of the C 30 triterpenoid structural isomers squalene and botryococcene, catalyzed by squalene synthase (SQS, E.C. 2.5.1.21) and squalene synthase-like enzyemes, respectively, in a two-step reaction. First, head-to-head condensation of two molecules of 16 yields presqualene diphosphate ( 21 , Figure 6 ) with a C1’-2-3 cyclopropyl moiety. Next, 21 undergoes a reductive rearrangement in the presence of NADPH to afford either squalene ( 22 ) with a C1’-1 linkage, or botryococcene ( 23 ) with a C1’-3 bond connecting the two farnesyl moieties [ 67 ]. The B. braunii Showa SQS for the biosynthesis of squalene (termed BSS) has been cloned and expressed in E. coli [ 37 ]. Recombinant BSS catalysed both half-reactions in the presence of NADPH and afforded 22 , but not 23 . BSS is encoded in the Showa transcriptome by a curated contig with high sequence coverage (Additional file 1 : Table S8). While botryococcene synthase activity has been demonstrated in B. braunii Showa cell extracts [ 38 ], the corresponding enzyme(s) and gene(s) remained elusive until the recent identification of three squalene synthase-like enzymes (SSL-1 to SSL-3) from this strain [ 34 ]. Unlike any other known natural SQS, SSL-1 catalyses only the first half reaction to produce 21 . Reactions combining SSL-1, SSL-2 and NADPH afforded squalene 22 , while those with SSL-1, SSL-3 and NADPH provided botryococcene 23 as the main product with minor amounts of 22 [ 34 ]. Thus, the solution for the problem of the biosynthesis of both squalene and botryococcene in B. braunii Showa involved the evolution of a novel enzyme system in addition to a conserved 22 -producing SQS by repeated gene duplications and neofunctionalization of the paralogs. In this ancillary system, the two half reactions of the canonical SQS reaction are dissociated into separate enzymes that presumably form subunits for a catalytic complex. Production of 22 by two separate enzyme systems (BSS and the SSL-1 + SSL-2 complex) might provide separate pools of this compound for primary triterpenoids (membrane sterols) and secondary triterpenoids (liquid hydrocarbons and matrix polymers) [ 34 ]. All three SSL enzymes are represented by curated contigs of moderate sequence coverage in the Showa transcriptome (Additional file 1 : Table S8), with no additional SQS isozymes detectable. While BSS, SSL-1 and SSL-3 do not appear to be targeted to the chloroplast, mitochondrion or the lumen of the endoplasmic reticulum based on predictions by TargetP, botryococcene synthase activity has been shown to be associated with the membrane fraction [ 38 ]. Additionally, BSS (but not SSL-1 or SSL-3) has been described to associate with the ER using a C-terminal membrane anchoring sequence [ 34 ].\n Figure 6 Reconstruction of the biosynthesis of linear tri- and tetraterpenoids in B. braunii Showa. 21, presqualene diphosphate; 22, squalene; 23, botryococcene; 24, (1 R , 2 R , 3 R )-prephytoene diphosphate; 25, 15 -cis -phytoene. SQS, squalene synthase; SSL-1, -2, -3, squalene synthase-like enzymes; CrtB, phytoene synthase. In addition to SQSs and phytoene synthases (CrtB, see next section), genome sequence surveys of algae often identify a gene encoding a putative, uncharacterized protein with the head-to-head trans-isoprenyl diphosphate synthase fold. These predicted proteins (Class 1 isoprenoid biosynthesis-related proteins [ISR]), form a clade that is distinct from SQSs and CrtB enzymes. ISRs had been hypothesized earlier to constitute the then-unknown botryococcene synthase [ 68 ], but the recent in vitro studies from the Chappell laboratory, as described above, conclusively showed that these proteins are not necessary for botryococcene biosynthesis [ 34 ]. A single nonredundant curated contig with low sequence coverage represents this cryptic ISR enzyme in the Showa transcriptome ( Additional file 1 : Table S8). The extracellular liquid hydrocarbons of B. braunii , race B are dominated by variously methylated botryococcenes, with C 32 and C 34 botryococcenes as the most abundant liquid hydrocarbon oil (Figure 7 ) [ 69 - 71 ]. Methylated squalene (C 34 tetramethylsqualene) represents approximately 4.5% of the liquid hydrocarbons in race B [ 72 ], with a higher level of tetramethylsqualene (10%) found covalently linked to polyacetals in the polymers of the colony extracellular matrix [ 51 ]. These methylated botryococcenes (C 31 – C 37 ) and methylsqualenes (C 31 – C 34 ), are biosynthesized by S-adenosylmethione (SAM)-dependent methyltransferases acting upon botryococcene 23 and squalene 22 , respectively. The biosynthesis of C 32 botryococcene ( 26 ) and C 32 methylsqualene ( 29 ) has recently been clarified by the Chappell group [ 33 ] while this manuscript was in preparation. This paper described the cloning, heterologous expression and functional characterization of six enzymes showing similarity to sterol 24- C -methyltransferases (SMT, E.C. 2.1.1.41) that catalyse single methyl additions onto the linear prenyl side chains of sterols using SAM as the methyl donor [ 73 , 74 ]. Three of these putative SMTs that harboured variant sterol binding domains were found to conduct two successive methyl transfers onto linear triterpenoids. Triterpenoid methyltransferase-1 (TMT-1) and TMT-2 both yielded terminal mono- and dimethylated squalene (e.g. 29 ), while TMT-3 afforded terminal mono- and dimethylated botryococcene (e.g. 26 ). TMT-1 and TMT-2 displayed very little activity towards C 30 botryococcene 23 , while squalene 22 was a similarly weak substrate for TMT-3. The remaining three SMT-like enzymes (SMT-1 to SMT-3) did not accept 22 or 23 as their substrates, nor did they methylate common plant sterols in spite of harbouring apparently canonical sterol binding domains. None of the six identified enzymes, nor any of their pairwise combinations accepted the C 32 linear triterpenoids 26 or 29 , thus the biosynthesis of the C 34 linear triterpenoids 27 and 30 , or that of the higher botryococcene homologs like 28 remains to be elucidated. All six SMTs identified by Niehaus et al. [ 33 ] were well represented in the Showa transcriptome analysed here (Additional file 1 : Table S8). Sequence coverage ranged from 71.7 reads/kb for TMT-2 to 1196.7/kb for SMT-1, with the botryococcene methyltransferase TMT-3 also exhibiting an extremely high sequence coverage at 768.4 reads/kb. Thus, the genes for these SMTs are apparently very actively transcribed, perhaps reflecting the fact that >70% of the liquid hydrocarbon oils of B. braunii Showa are composed of C 32 – C 34 botryococcenes [ 33 ]. All three TMTs are predicted by TargetP here to localize into the secretory system (endoplasmic reticulum, Golgi apparatus), and were found by Niehaus et al . to associate with yeast microsomes (originating from the ER) upon heterologous expression in that host [ 33 ]. Indeed, C 33 – C 34 botryococcenes are overwhelmingly localized in the extracellular matrix, while 23 and lesser methylated botryococcenes predominate in intracellular oil bodies [ 13 , 75 ]. In contrast, SMT-1, SMT-2 and SMT-3 are predicted here to be targeted to a compartment outside the chloroplast, mitochondrion or the secretory system (Additional file 1 : Table S8). Our analysis has also uncovered four additional nonredundant curated contigs encoding deduced SMTs, one of them (32241) of putative fungal origin, all with low (5.7 - 15.0 reads/kb) sequence coverage. These predicted enzymes might be candidates for the missing SMTs for the biosynthesis of 27 and 30 . The biosynthetic basis for the double bond isomerization and terminal cyclization reactions that lead to the various C 34 botryococcene isomers in the B race of B. braunii ( 27 , Figure 7 ) remain unknown [ 9 , 11 ]. A curated contig with low sequence coverage (31093, 11.2 reads/kb) encodes an enzyme with similarity to 24-methylenesterol C -methyltransferases (MSMT, E.C. 2.1.1.143). These enzymes afford C24-ethyl sterols by methylating C24-methylsterols like 24-methylenelophenol [ 76 ]. It remains to be determined whether the corresponding enzyme takes part in phytosterol biosynthesis or in the production of higher botryococcenes like the C 37 botryococcene 28 (Figure 7 ).\n Figure 7 Biosynthesis of liquid triterpenoid compounds and matrix polymer materials in B. braunii Showa. 26, C 32 botryococcene; 27, representative C 34 isomeric botryococcenes; 28, a C 37 botryococcene; 29, C 32 dimethylsqualene; 30, C 34 tetramethylsqualene; 31, dihydroxy-tetramethylsqualene; 32, diepoxy-tetramethylsqualene; 33, botryolin; 34, braunixanthin 1; 35, complex polymeric matrix lipid from B. braunii race B . TMT, terpenoid methyltransferase, SMT, squalene methyltransferase. Hydroxylation, epoxidation, and formation of O-containing heterocycles (for example compounds 31 , 32 , and 33 , Figure 7 ) increase the complexity of race B liquid and polymer hydrocarbons. These oxidized methylsqualenes, together with carotenoids and very long chain fatty acids (and to a smaller degree oxidized botryococcenes) also support the biosynthesis of ether lipids like braunixanthin 1 ( 34 ) and matrix polymers ( 35 ) as precursors [ 9 - 11 , 51 , 77 ]. Candidates for the introduction of the oxygen functionality into linear triterpene structures might be enzymes similar to the flavoprotein squalene monooxygenase (SQLE, E.C. 1.14.13.132) that produces (3 S )-2,3-epoxy-2,3-dihydrosqualene from 22 by incorporation of molecular oxygen. Enzymes similar to SQLE might also accept tetramethylsqualene 30 as their substrate and catalyse epoxide formation towards the centre of the long terpene chain, yielding compounds like diepoxy-tetramethylsqualene ( 32 ). Reducing equivalents are channelled to these enzymes by NADPH-dependent hemoprotein reductases. The Showa transcriptome contains seven nonredundant curated contigs with very low to low sequence coverage (3.4 to 20.2 reads/kb, Additional file 1 : Table S8) encoding SQLE-like enzymes: these putative enzymes are candidates to channel squalenes and maybe botryococcenes towards the production of extracellular matrix materials. Biosynthesis of other terpenoids The terpenome of B. braunii Showa includes meroterpenoid quinones, the side chain of chlorophyll, diterpenoid gibberellins, triterpenoid phytosterols, tetraterpenoid carotenoids, polyprenyl carrier molecules (dolichol, see above), and the prenyl chains of proteins. All these primary and secondary metabolites draw on the common isoprene (IPP and DMAPP) pool generated by the chloroplast-based MEP/DOXP pathway, and divert these precursors away from the production of liquid and matrix hydrocarbons. We have curated and catalogued contigs representing putative enzymes involved in these competing pathways. Squalene 22 is the precursor for the triterpenoid phytosterols, chloroplast membrane cholesterol and its esters [ 73 , 78 ], and vitamin D 3 . Following oxidation of 22 to ( S )-squalene 2,3-epoxide ( 36 , Figure 8 ) by SQLE (see previous section), 2,3-oxidosqualene cyclases catalyse a cationic cyclization cascade converting linear triterpenes to fused ring compounds. A single curated contig with moderate sequence coverage (Additional file 1 : Table S9) encodes a putative cycloartenol synthase (CAS, E.C. 5.4.99.8) which may produce cycloartenol ((3 S )-2,3-epoxy-2,3-dihydrosqualene, 37 ), an intermediate in the biosynthesis of phytosterols. The Showa transcriptome contains curated contigs for a full contingent of enzymes (Additional file 1 : Table S9) to afford 24-methylenecholesterol ( 38 ) and isofucosterol ( 39 ), the main sterols in B. braunii strains [ 79 ]. Stigmasterol and β-sitosterol, close structural analogues of 38 and 39 , have also been identified in this alga by pyrolysis-GC/MS [ 80 ], while conventional GC/MS has identified campesterol and β-sitosterol [ 79 ]. In C. reinhardtii , precursors of the predominant membrane sterol ergosterol were shown to be synthesized by an identical pathway [ 81 ]. Similar enzymes, with the addition of sterol-4α-carboxylate 3-dehydrogenase (ERG26, E.C. 1.1.1.170) and vitamin D 25-hydroxylase (CYP2R1, E.C. 1.14.13.15), may also be involved in the biosynthesis of cholesterol ( 40 ) [ 79 , 82 ] and calcidiol (25-hydroxyvitamin D3, 41 ) (Additional file 1 : Table S9). A curated contig for a putative sterol esterase (LIPA, E.C. 3.1.1.13) may take part in the esterification of sterols with long-chain fatty acids – this enzyme may alternatively be involved in the production of ester polymers or matrix materials ( 34 and 35 , see previous section).\n Figure 8 Reconstruction of the biosynthesis of triterpenoid sterols and vitamin D 3 in B. braunii Showa. 36, ( S )-2,3-epoxisqualene; 37, cycloartenol; 38, 24-methylenecholesterol; 39, isofucosterol; 40, cholesterol; 41, 25-hydroxyvitamin D 3 (calcidiol). SQLE, squalene monooxygenase; CAS, cycloartenol synthase; SMT, sterol 24- C -methyltransferase; ERG25, methylsterol monooxygenase; CPI1, cycloeucalenol cycloisomerase; CYP51, sterol 14-demethylase; ERG24, Δ-14-sterol reductase; EBP, cholestenol Δ-isomerase; MSMT, 24-methylenesterol C -methyltransferase; SC5DL, lathosterol oxidase; DHCR7, 7-dehydrocholesterol reductase. Geranylgeranyl diphosphate 17 serves as the precursor for the biosynthesis of the tetraterpenoid carotenoids that are important photoprotectants, antioxidants and membrane protein function modulators for the photosynthetic complexes [ 83 - 85 ]. Tetraterpenoid biosynthesis initiates with a trans-isoprenyl diphosphate synthase, phytoene synthase (CrtB, E.C. 2.5.1.32), catalysing the head-to-head condensation of two molecules of 17 in a two-step reaction with the concomitant release of pyrophosphate, paralleling the reactions of squalene synthase in triterpenoid biosynthesis discussed earlier. In the first step, (1 R , 2 R , 3 R )-prephytoene diphosphate ( 24 ) with a C1’-2-3 cyclopropyl moiety is produced (Figure 6 ). Next, 24 undergoes a rearrangement, this time with no reduction, to afford 15- cis- phytoene ( 25 ) with a C1’-1 linkage. CrtB is encoded in the Showa transcriptome by a curated contig with low sequence coverage (Additional file 1 : Table S10). 25 may be converted to a variety of carotenoids in B. braunii Showa by a complex network of enzymes, with curated contigs encoding enzymes presumed to be involved in the production of lycopene ( 42 ), zeaxanthin ( 43 ), violaxanthin ( 44 ), and lutein ( 45 ) (Figure 9 , Additional file 1 : Table S10). The majority of the deduced carotenoid tailoring enzymes listed in Additional file 1 : Table S10 are represented by multiple nonredundant isozymes encoded in the Showa transcriptome. Some of these deduced enzymes are predicted by TargetP to be localized outside of the plastid, the mitochondrion or the secretory systems. All these contigs have low sequence coverage (less than 25 reads/kb), with the sole exception 09778, which codes for a β-carotene 3-hydroxylase (CrtR, E.C. 1.14.13.129) that has moderate sequence coverage (94.1 reads/kb). Considering the variety of predicted isozymes, and the fact that many of the carotenoid biosynthetic enzymes have somewhat broad substrate specificities and may catalyse multiple and overlapping reaction steps [ 85 ], this alga may biosynthesize a large variety of these pigments. Indeed, 43 45 , as well as various other β-carotenes, echinenone, canthaxanthin, loroxanthin, and neoxanthin have been described from race B strains [ 66 , 77 , 86 - 89 ]. Some of these carotenoids may also be incorporated into the ether polymers and insoluble extracellular matrix materials, thus may have a structural role [ 9 - 11 , 51 ].\n Figure 9 Reconstruction of tetraterpenoid biosynthesis in B. braunii Showa. 42 , lycopene; 43 , zeaxanthin; 44 , violaxanthin; 45 , lutein; 46 , trans,trans -lycopadiene. CrtI, phytoene desaturase; CrtP, 15- cis- phytoene dehydrogenase; CrtQ, ζ-carotene desaturase; CrtH, prolycopene isomerase; CrtY, lycopene β-cyclase; CrtR, β-carotene 3-hydroxylase; ZEP, zeaxanthin epoxidase; VDE, violaxanthin de-epoxidase; CrtL2, lycopene ε-cyclase; LUT1, carotene ε-monooxygenase, ?, unidentified enzyme(s). The prominent liquid hydrocarbon trans,trans- lycopadiene ( 46 , Figure 9 ) of race L strains of B. braunii is probably biosynthesized by reduction of an acyclic carotenoid such as 15- cis- phytoene ( 25 ) or lycopene ( 42 ) [ 10 ]. Lycopadiene ( 46 ) is considered a biomarker for race L strains of B. braunii as there are no reports for the occurrence of this compound in race B strains [ 90 ]. Indeed, we could not identify plausible tetraterpene reductases in the Showa transcriptome for the biosynthesis of 46 . Meroterpenoid quinones play indispensable roles as electron carriers in photosynthesis in the chloroplast and oxidative phosphorylation in the mitochondria. The terpene chains of these molecules, as well as that of the photosynthetic pigment chlorophyll, are derived from polyprenyl diphosphates. The C 20 prenyl diphosphate geranylgeranyl diphosphate ( 17 ) is reduced to phytyl diphosphate ( 47 , Figure 10 ) by geranylgeranyl reductase (ChlP, E.C. 1.3.1.83) represented by three nonredundant curated contigs in the Showa transcriptome. One of these contigs, 30757, features high sequence coverage and the encoded protein is predicted to be directed into the chloroplast, as expected (Additional file 1 : Table S11). 47 is the precursor for the phytyl side chain of prenylated proteins and that of chlorophylls ( 48 ). A single curated contig with moderate sequence coverage encodes the putative prenyl transferase chlorophyll synthase (ChlG, E.C. 2.5.1.62) that attaches the phytyl side chain onto chlorophyllide-a to yield 48 . 47 is also the precursor for the terpenoid side chains of α-tocopherol (vitamin E, 49 ) and phylloquinone (vitamin K1, 50 ). The vitamin E family of antioxidants including α-tocopherol ( 49 ) contributes to the integrity of photosynthetic membranes and may influence plant responses to many physiological stressors [ 91 ]. Phylloquinone (vitamin K1) is present in all photosynthetic plants as a cofactor for photosystem-I-mediated electron transport [ 92 ]. All the enzymes necessary for the biosynthesis of vitamin E from 47 and homogentisate, and vitamin K1 from 47 and 1,4-dihydroxy-2-naphthoate are predicted to be represented in the Showa transcriptome by curated contigs of low to moderate sequence coverage, some of them (VTE1, GTMT, and UbiE, Additional file 1 : Table S11) encoded as multiple isozymes. The polyprenyl diphosphates 18 and 19 , derived from geranyl diphosphate ( 15 ) and geranylgeranyl diphosphate ( 16 ) provide the terpene side chains of the electron carriers plastoquinone-9 ( 51 ) in the chloroplast, and ubiquinone ( 52 ) and menaquinone in the mitochondria ( 53 , Figure 10 ) [ 93 ]. Proteins similar to the enzymes necessary for the biosynthesis of plastoquinone from 18 and homogentisate, and for the production of coenzyme Q (ubiquinone 52 ) and vitamin K2 (menaquinone 53 ) from 4-hydroxybenzoate and 1,4-dihydroxy-2-naphthoate, respectively, and from 18 and 19 are encoded in the Showa transcriptome by curated contigs of low to moderate sequence coverage, many as multiple isozymes (Additional file 1 : Table S11). However, we could not identify contigs for methylsolanyl-benzoquinone methyltransferase (VTE3) for 51 biosynthesis, and a presumed ubiquinone biosynthesis methyltransferase (Coq5, E.C. 2.1.1.201) is encoded only by a single curated contig of predicted fungal origin (Additional file 1 : Table S11).\n Figure 10 Reconstruction of meroterpenoid quinone biosynthesis in B. braunii Showa. 47, phytyl diphosphate; 48, chlorophyll a (R=CH 3 ) and b (R=CH 2 O); 49, α-tocopherol (vitamin E); 50, phylloquinone (vitamin K1); 51, plastoquinone-9; 52, ubiquinone (Coenzyme Q); 53, menaquinone (vitamin K2). ChlP, geranylgeranyl reductase; ChlG, chlorophyll synthase; HPT, homogentisate phytyltransferase; VTE1, tocopherol cyclase; GTMT, tocopherol methyltransferase; MenA, dihydroxynaphthoate octaprenyltransferase; UbiE, phylloquinone / menaquinone methyltransferase; HST, homogentisate solanesyltransferase; VTE3, methylsolanyl-benzoquinone methyltransferase (not found); Coq2, 4-hydroxybenzoate hexaprenyltransferase; Coq3, hexaprenyldihydroxybenzoate methyltransferase (two methylation reactions); Coq6, ubiquinone biosynthesis monoonxygenase; Coq5, ubiquinone biosynthesis methyltransferase; Coq7, ubiquinone biosynthesis monooxygenase. Terpenoid side chains are highlighted in gray. The diterpenoid growth hormones gibberellic acids [ 94 ] are derived from geranylgeranyl diphosphate ( 17 ) by cyclization followed by multiple oxidations in land plants. There are no unequivocal reports on the production of gibberellins in green algae [ 95 ], nor have genes with high similarity to key gibberellin production pathways been located in genome sequences [ 15 ]. Fittingly, the first enzyme of the gibberellic acid pathway that generally harbours both ent- copalyl diphosphate synthase (E.C. 5.5.1.13) and ent- kaurene synthase (E.C. 4.2.3.19) activities could not be identified in the B. braunii Showa transcriptome. On the other hand, curated contigs that may encode enzymes for the oxidative processing of the tetracyclic intermediate ent -kaurene to gibberellin A4 are present in the transcriptome at low sequence coverage (Additional file 1 : Table S12). S -adenosylmethione regeneration In addition to the biosynthesis of many primary metabolites of terpenoid or other origin, the production of large amounts of higher botryococcenes and methylated squalenes (Figure 7 ) in B. braunii Showa requires a robust supply of S -adenosylmethionine (SAM) that is used as a donor for methylation reactions. Transfer of the methyl group of SAM to the substrate by these methyltransferases yields S -adenosylhomocysteine that is hydrolysed by S -adenosyl-L-homocysteine hydrolase (AhcY, E.C. 3.3.1.1) to homocysteine and adenosine (Additional file 1 : Table S13). Homocysteine is methylated by 5-homocysteine S -methyltransferases, including the S -methylmethionine-dependent MmuM (E.C. 2.1.1.10), the 5-methyltetrahydrofolate- and cobalamin-dependent MetH (E.C. 2.1.1.13), and the 5-methyltetrahydropteroyl-triglutamate-utilizing but cobalamin-independent MetE (E.C. 2.1.1.14), yielding L-methionine. Finally, S -adenosylmethionine synthase (MetK, E.C. 2.5.1.6) transfers the adenosyl moiety of ATP to methionine, yielding SAM and releasing phosphate and pyrophosphate. Machine-assembled contigs for all these deduced enzymes have been identified in the Showa transcriptome, with low (MetK, MmuM), low to moderate (MetH) and high to extremely high sequence coverage (AhcY, MetE, Additional file 1 : Table S13). Competing storage compounds: biosynthesis of triacylglycerols Photosynthetic carbon and energy intended for storage is partitioned in B. braunii Showa amongst terpenoid hydrocarbons, triacylglycerols (TAGs), and carbohydrates. We have generated inventories for machine-assembled contigs predicted to encode crucial enzymes in hydrocarbon-competing storage compound biosynthetic pathways. Fatty acid biosynthesis for polar membrane lipids (various glycosyl-glycerolipids and phosphoglycerolipids) and neutral TAGs in microalgae like B. braunii primarily occur in the chloroplast (with limited synthesis also occurring in the mitochondria, [ 96 ]) by a type II (multiprotein complex) fatty acid synthase (FAS) enzyme system. Malonyl-CoA, the substrate for FAS, is derived from the primary metabolite acetyl-CoA by the biotin-containing multienzyme complex acetyl-CoA carboxylase (Acc, E.C. 6.4.1.2) in the chloroplast (Figure 11 ) [ 19 , 97 ]. Using ATP, the biotin carboxylase subunit AccC carboxylates biotin on the biotin carboxyl carrier protein AccB, followed by the transfer of the carboxyl moiety to acetyl-CoA by the carboxyl transferase subunit AccA and AccD. Contigs of moderate to high sequence coverage encode the presumed AccA, B and C (Additional file 1 : Table S14), while contigs for AccD (carboxyl transferase β-subunit) was missing from the Showa transcriptome as expected for a plastid-encoded gene [ 98 ]. A multifunctional, most likely cytosolic Acc isozyme (ACAC, E.C. 6.3.4.14) [ 19 , 98 ] is encoded by multiple contigs with various sequence coverage. Malonate from malonyl-CoA is transferred to the acyl carrier protein (ACP1 and ACP2) of the type II FAS by ACP S -malonyltransferase (FabD, E.C. 2.3.1.39) [ 23 , 97 ]. Malonyl-ACP is the acyl donor for the subsequent recursive, decarboxylative Claisen condensations catalysed by the β-ketoacyl:ACP synthase components of the type II FAS, generating the growing fatty acyl chain intermediates that remain bound to the ACP as thioesters. KAS III (β-ketoacyl:ACP synthase III, FabH, E.C. 2.3.1.180) conducts the first condensation using acetyl-CoA and malonyl-ACP. Acyl-ACP (C 4 -C 14 ) serves as the acceptor for further condensations with malonyl-ACP as the donor to produce long chain fatty acids (most frequently palmitic acid, C16:0) by KAS I (β-ketoacyl:ACP synthase I, FabB, E.C. 2.3.1.41). The final condensation to yield the long chain fatty acid stearic acid (C18:0) is catalysed by KAS III (β-ketoacyl:ACP synthase II, FabF, E.C. 2.3.1.179) [ 23 ]. Each condensation cycle also involves three consecutive reductions to yield the fully saturated fatty acyl-ACP from the nascent β-ketoacyl-ACP. These steps are catalysed by the NADPH-dependent β-ketoacyl-ACP reductase (FabG, E.C. 1.1.1.100), β-hydroxyacyl-ACP dehydratase (FabZ, E.C. 4.2.1.-), and the NADH or NADPH-dependent enoyl-ACP reductase (E.C. 1.3.1.-). Desaturation to generate long chain fatty acids with n-9 cis double bonds is carried out at the acyl-ACP thioester stage by stearoyl-ACP Δ 9 desaturase (DesA, E.C. 1.14.19.2) [ 23 ], yielding palmitoleic or more frequently oleic acid (C16:1 n-9 and C18:1 n-9, respectively). Long chain acyl-ACPs may be utilized for a direct transfer of the acyl group to afford phosphatidic acid towards the synthesis of various polar membrane glycerolipids in the plastid. Alternatively, long chain acyl-ACPs are hydrolysed by thioesterases (including oleoyl-ACP hydrolase [FatA, E.C. 3.1.2.14]) to release free fatty acids that are exported from the plastid. These fatty acids are reactivated as CoA thioesters by long chain fatty acid:CoA ligases (FadD, E.C. 6.2.1.3) in the cytosolic face of the ER for the biosynthesis of various lipids [ 19 ] (Figure 11 ). Multiple contigs for two deduced isoforms of ACP, and a full contingent of predicted enzymes for a type II FAS, a Δ 9 desaturase, a thioesterase and fatty acyl CoA ligases have been identified in the Showa transcriptome ( Additional file 1 : Table S14), some with very high sequence coverage (contig 35624 with 725.5 reads/kb for an ACP, and contig 43176 with 378.2 reads/kb for a Δ 9 desaturase). A short contig (FX085405) for FabB from the race B strain B. braunii BOT-22 shows 98% identity to the FabB encoded by contig 14404 of the Showa strain [ 32 ]. Interestingly, contigs with low sequence coverage, coding for assumed animal and fungal Type I (multifunctional enzyme) FASs have also been identified in the Showa transcriptome. Based on the high similarities of the encoded proteins to animal or fungal enzymes, and the divergent codon usage of the contigs, the corresponding transcripts may have originated from other organisms present in the non-axenic culture.\n Figure 11 Reconstruction of triacylglycerol biosynthesis in B. braunii. Deduced enzymes are shown in white boxes, enzymes not represented in the transcriptome of the Showa strain of B. braunii are in grey boxes. AccABC, acetyl-CoA carboxylase, subunits A, B, and C; AccD, acetyl-CoA carboxylase, subunit D; ACAC, multifunctional protein acetyl-CoA carboxylase; FabD, ACP: S -malonyltransferase; ACP, acyl carrier protein; FabH, β-ketoacyl:ACP synthase III; FabB, β-ketoacyl:ACP synthase I; FabF, β-ketoacyl:ACP synthase II; FabG, β-ketoacyl:ACP reductase; FabZ, β-hydroxyacyl:ACP dehydratase; FabI, enoyl-ACP reductase; DesA, stearoyl-ACP δ-9 desaturase; FatA, oleoyl-ACP hydrolase; FadD, long chain fatty acid:CoA ligase; GlpK, glycerol kinase; GPAT, glycerol-3-phosphate O -acyltransferase; LPAAT, lysophosphatidic acid acyltransferase; PAP, phosphatidic acid phosphatase; DGAT, diacylglycerol acyltransferase; PDAT, phospholipid:diacylglycerol acyltransferase; FAD6, Δ-12 fatty acid desaturase; FAD8, Δ-15 fatty acid desaturase; FADS2, Δ-6 fatty acid desaturase; ELOVL5, very long chain fatty acid elongase; KAR, β-ketoacyl-CoA reductase; PHS1, 3-hydroxyacyl-CoA dehydratase; TER, enoyl-CoA reductase, VLC-TE, very long chain fatty acyl-CoA hydrolase. CoA, coenzyme A; LCSFA, long chain saturated fatty acid (C16-18:0); LCUFA, long chain unsaturated fatty acid (C16-18:1 n-9); LC-PUFA, long chain polyunsaturated fatty acid (C16-18:2–4); VLC-PUFA, very long chain polyunsaturated fatty acid (C20-24:2–4), TAG, triacylglycerol. Long chain fatty acids may undergo further desaturations and chain elongations to afford long chain and very long chain polyunsaturated fatty acids (LC-PUFA and VLC-PUFA, respectively, Figure 11 ). Oxygen-dependent Ω 6 (Δ 12 ) fatty acid desaturases (FAD6, E.C. 1.14.19.-) and Ω 3 (Δ 15 ) fatty acid desaturases (FAD8, E.C. 1.14.19.-) in the chloroplast and the endoplasmic reticulum generate linoleic acid (C18:2 n-6) and α-linolenic acid (C18:3 n-3) and their longer chain equivalents, respectively, using acyl-glycerolipid substrates [ 23 , 99 , 100 ]. Both of these predicted enzymes are encoded in the Showa transcriptome at low to moderate sequence coverage. An apparent front-end desaturase, FADS2 (Δ 6 fatty acid desaturase, E.C. 1.14.19.-) that may yield stearidonic acid (C18:4 n-3) or its longer chain equivalents, perhaps by using acyl-CoA esters [ 19 ], is also present in the transcriptome with low sequence coverage. For VLCFA and VLC-PUFA, recursive cycles of 2-carbon additions from malonyl-CoA and the following three desaturation steps outside of the chloroplast (probably in the microsomes) parallel that of de novo long-chain fatty acid biosynthesis in the chloroplast, but without the involvement of acyl carrier proteins [ 19 ]. Thus, multiple contigs with moderate to high sequence coverage that encode putative very long chain fatty acid elongase (ELOVL5, E.C. 2.3.1.-), β-ketoacyl-CoA reductase (KAR, E.C. 1.1.1.-), 3-hydroxyacyl-CoA dehydratase (PHS1, E.C. 4.2.1.-), and enoyl-CoA reductase (TER, E.C. 1.3.1.-) enzymes have been found in the Showa transcriptome. However, a very long chain fatty acyl-CoA hydrolase (E.C. 3.1.2-) that may act as a thioesterase has not been identified. VLCFA, VLC-PUFA and hydrocarbon pyrolysis products of these have been observed in B. braunii , albeit primarily in the A race strains [ 80 , 101 - 103 ]. Triacylglycerol storage lipids are assembled in the endoplasmic reticulum by two sequential acylations of sn -glycerol-3-phosphate, followed by dephosphorylation and a final acyl transfer (Figure 11 ). Exchange of acyl chains amongst TAG, glycerolipids and the acyl-CoA pool (acyl editing) provides flexibility to channel carbons for storage or for functional lipid biosynthesis [ 23 ]. Following the ATP-dependent phosphorylation of glycerol by glycerol kinase (GlpK, E.C. 2.7.1.30), glycerol-3-phosphate O -acyltransferase (GPAT, E.C. 2.3.1.15) and lysophosphatidic acid acyltransferase (LPAAT, E.C. 2.3.1.51) generate phosphatidic acid using the acyl-CoA pool (Additional file 1 : Table S14). Phosphatidic acid phosphatase (PAP, E.C. 3.1.3.4) affords sn- 1,2-diacylglycerol, to be acylated by diacylglycerol acyltransferase (DGAT, E.C. 2.3.1.20) to produce TAG. Diacylglycerols are also the precursors for the various polar lipids (glycosyl-glycerolipids and phosphoglycerolipids), while phospholipid:diacylglycerol acyltransferase (PDAT, E.C. 2.3.1.158) shuttles acyl groups amongst phosphoglycerolipids, betaine lipids, and TAG in an acyl-CoA independent process [ 19 , 97 , 103 ]. All these key TAG biosynthetic enzymes are predicted to be present in the Showa transcriptome with low sequence coverage ( Additional file 1 : Table S14) which may be reflective the low level of TAGs predicted to be present in the B race of B. braunii [ 79 ]. In land plants, TAGs accumulate in oil bodies whose lipid/water interface features the structural proteins oleosins. Hydrophobic proteins (MLDP, major lipid droplet protein) that may be functionally equivalent to, but not structurally similar to plant oleosins have recently been identified by proteomic approaches in the oil bodies of Chlorophyta algae [ 20 , 104 ]. Multiple contigs encoding presumed MLDPs are also featured in the Showa transcriptome, some with very high sequence coverage (contigs 0772, 35177 and 42893, with 237–295 reads/kb) that may indicate active transcription of the corresponding genes. Competing storage compounds: biosynthesis of starch and other carbohydrates A major sink for photosynthetic carbon intended for storage in algae are polysaccharides including starch. The biosynthesis of these compounds competes with those of hydrocarbon oils and TAG lipids in B. braunii , thereby reducing biofuel yield. On the other hand, starch and cellulosic biomass, after hydrolysis, may be utilized as a feedstock for the fermentative production of biofuel using non-photosynthetic microorganisms [ 105 , 106 ]. Starch biosynthesis in the chloroplast is initiated by the phosphorylation of α-D-glucose at the C6 position by hexokinase (HK, E.C. 2.7.1.1) and glucokinase (Glk, E.C. 2.7.1.2) in ATP-dependent reactions. D-glucose 6-phosphate is then converted by phosphoglucomutase (PGM, E.C. 5.4.2.2) to α-D-glucose 1-phosphate that serves as a substrate for the ATP-dependent glucose-1-phosphate adenylyltransferase (GlgC, E.C. 2.7.7.27), a major rate-controlling enzyme of the pathway in plants and bacteria [ 107 ]. The resulting ADP-glucose is then polymerized by starch synthase (GlgA, E.C. 2.4.1.21) to generate amylose with linear α-1,4-glycosidic linkages. Branched α-1,6 glycosidic linkages between α-1,4-glucan chains is generated by the 1,4-α-glucan branching enzyme (GlgB, E.C. 2.4.1.18) to yield water-insoluble amylopectin. Conversely, amylo-α-1,6-glucosidase (AGL, E.C. 3.2.1.33) hydrolyses α-1,6 glycosidic linkages to limit branching and to mobilise glucose from starch. Multiple contigs with low to moderate sequence coverage encoding these predicted enzymes are present in the Showa transcriptome, with the deduced glycogen debranching enzyme AGL encoded by a single contig that might have originated from a fungal cohabitant (Additional file 1 : Table S15). Considering that cellulose is a major constituent of the cell wall in green algae (up to 80% in Chlorella sp., [ 108 ]), the biosynthesis of this 1,4-β-D-glucan presents a major demand from the available photosynthetic carbon. Since Chlorophyta cell walls do not contain lignin [ 109 , 110 ], cellulose from the biomass of these algae may provide a relatively more easily accessible source of sugar for the production of biofuels by fermentation. For the biosynthesis of cellulose, α-D-glucose 1-phosphate is activated by uridylation by UTP:α-D-glucose-1-phosphate uridylyltransferase (UGP, E.C. 2.7.7.9). The resulting UDP-glucose is the substrate for cellulose synthase (BcsA, E.C. 2.4.1.12). Orthologous genes are present in single copies in green algae [ 111 ]. Contigs with low to moderate sequence coverage have been identified for these two inferred enzymes in the B. braunii Showa transcriptome (Additional file 1 : Table S15). Localization of liquid terpenoid compounds into the extracellular matrix Apart from the biosynthesis of hydrocarbons with fuel characteristics and utility resembling that of fossil crude oil, B. braunii is also remarkable in depositing these compounds into a communal extracellular matrix that holds the colony together. Forming a fibrous network, this matrix consists of highly complex cross-linked polymers, originating from methylated squalenes and/or very long chain fatty acids [ 9 ]. The hydrocarbons initially accumulate within the cells as oil bodies, but the large majority (95%) of the extractable liquid oils are found in the matrix [ 9 ]. Traffic of these hydrocarbons seems to coincide with maturation of botryococcenes to C 34 and higher homologues in B. braunii , race B [ 13 , 75 ]. While the exact mechanism of the excretion of hydrocarbons into the extracellular space is unknown, the characterization and subsequent engineering of this trait into other (micro)organisms holds great promise for relieving product toxicity and simplifying extraction of advanced biofuels from biomass. Efflux pumps [ 112 ] are one of the obvious candidates for the cellular export system of hydrocarbons. The Showa transcriptome contains numerous contigs encoding potential ABC (ATP-binding cassette) transporters (also known as multidrug-resistance-related proteins, MRPs) that mediate ATP-dependent transport of a bewildering array of molecules across organellar and cellular membranes (Additional file 1 : Table S16). Plant ABC transporters fall into several subfamilies with varied (and often uncharacterized) functions but with a substantial functional redundancy of the family members within a single organism [ 113 ]. Subfamily A members take part in the transport of various lipids including sterols and lipoproteins across membranes in animals. Although members of this subfamily have been identified in plants, their functions have not been characterized. Subfamily B members are multidrug resistance factors that take part in auxin, secondary metabolite, and xenobiotic traffic in plants, with mitochondrion-located members involved in iron-sulphur cluster trafficking. Subfamily C members play a role in detoxification and in vacuolar transport of glucuronides, chlorophyll degradation products and anthocyanins. Subfamily D members are essential for the import of VLCFA into the peroxisomes for β-oxidation. Subfamily G transporters are involved in the export of alkanes and other lipids that form the waxy cuticle and in the secretion of volatile compounds in flowers and roots of plants. Other group members convey resistance to various substances including terpenoids and herbicides [ 113 , 114 ]. Subfamily G is generally expanded in plants compared to animals [ 114 ], with a large number of transcripts encoding presumed members of this subfamily also present in the Showa transcriptome. The proteins represented by these contigs, and to a lesser extent those of putative Subfamily D and A members (Additional file 1 : Table S16), are prime candidates for the role of a liquid hydrocarbon exporter in B. braunii race B strains including Showa. Programmed cell death may be an alternative or auxillary mechanism to release liquid hydrocarbon products from intracellular vesicles into the extracellular matrix, or to generate the outer matrix-derived “cell cap” material that covers the outer edge of the B. braunii cells in the colony [ 115 ] . However, given the large amount of liquid hydrocarbon in the extracellular matrix and that we infrequently see dead cells of B. braunii Showa within a colony that is rapidly accumulating extracellular hydrocarbons, the contribution of cell death to the extracellular localization of these compounds may only be limited. While caspase-mediated apoptotic Type 1 cell death pathway transcripts are only sporadically represented in the Showa transcriptome, a significant number of contigs encode putative proteins related to the Type 2 (autophagic) cell death pathway (Additional file 1 : Table S17). Autophagy recycles cellular constituents (from cytosolic macromolecules to whole organelles) by proteolytic degradation in response to cell aging or various stress conditions including nutrient deprivation and oxidative stress. Autophagy plays a housekeeping role and is one of the coping mechanisms of the cell, and its deficiencies were linked to several diseases in mammals [ 116 ]. Over-activation of autophagy on the other hand promotes cell death in a caspase-independent pathway or by a complex interplay with apoptosis [ 117 ]. Autophagy pathways are evolutionarily conserved, and have also been characterized in green algae including C. reinhardtii [ 118 , 119 ]. Stress signals are transmitted by 5'-AMP-activated protein kinase (AMPK, E.C. 2.7.11.11) towards the FKBP12-rapamycin complex-associated protein (mTOR) and the mTOR associated protein (GβL), causing de-suppression of autophagy (Figure 12 ). Activation of autophagy-related protein 1 (unc51-like kinase ATG1, E.C. 2.7.11.1) and Beclin 1 (VPS30 or ATG6) activates the ATP-dependent phosphatidylinositol 3-kinase (VPS34, E.C. 2.7.1.137) that phosphorylates 1-phosphatidyl-1D-myo-inositol. The resulting 1-phosphatidyl-1D-myo-inositol 3-phosphate recruits further ATG proteins to the phagophore membrane. These include ATG8-phosphatidylethanolamine (itself generated by ATG4, ATG3 and ATG7), and the complex of ATG5 and ATG12 whose conjugation is catalysed by ATG7 and ATG10. The binding of these complexes facilitates selection of cargo to be degraded, and leads to the expansion of the autophagosome and its fusion with the vacuolar membrane (Figure 12 ) [ 116 , 119 ]. Contigs with low to moderate sequence coverage encoding predicted autophagy pathway proteins are present in the Showa transcriptome (Additional file 1 : Table S17). Further experiments are necessary to determine whether this reconstructed autophagy pathway contributes to hydrocarbon excretion in B. braunii Showa, or whether its presence in the transcriptome simply reflects the abundance of cells that are in stationary phase approaching senescence and may be stressed by nutrient limitation in the long term culture.\n Figure 12 Reconstruction of autophagy regulation in B. braunii. Deduced enzymes are shown in white boxes, enzymes not represented in the transcriptome of the Showa strain of B. braunii are in grey boxes. AMPK, 5'-AMP-activated protein kinase, catalytic alpha subunit; GβL, mTOR associated protein; mTOR, FKBP12-rapamycin complex-associated protein; ATG1, unc51-like kinase; ATG6, Beclin1; VPS34, phosphatidylinositol 3-kinase; ATG3, -4, -5, -7, -10, -12, autophagy-related proteins; ATG8-PE, phosphatidylethanolamine-modified autophagy-related protein 8. PI, 1-phosphatidyl-D-myo-inositol; PI3P, 1-phosphatidyl-D-myo-inositol 3-phosphate."
} | 21,402 |
39732882 | PMC11682423 | pmc | 6,420 | {
"abstract": "The management and creation of Marine Protected Areas (MPAs) is currently under great focus, with international organisations aiming to protect 30% of our oceans by 2030. The success of MPAs depends on a nuanced understanding of local ecological dynamics and threats, which can significantly influence ecosystem balance. Herbivory can be a stressor for foundation species, namely kelp forests, contributing to their decline in several regions of the globe. However, the dynamics inherent to herbivory and MPA’s implementation are still poorly understood. Here, the impact of protection status, depth, kelp species, and grazer type on herbivory (occurrence, rate, and grazer frequency) was assessed through a comprehensive experimental approach involving tethering experiments and faunal characterisation of macro-herbivores. The research was conducted in habitats off the central coast of Portugal: Peniche (PEN) and the MPA Berlengas Archipelago (MPA-BER). Our findings revealed that herbivory occurrence and rate are higher within the MPA, especially at greater depths. Instead of urchins, fish are the significant contributors to kelp consumption, showing a preference for the kelp S. polyschides . Results provide the first experimental evidence in the Atlantic region identifying fish as the dominant herbivores driving increased kelp biomass loss, a relationship potentially magnified by MPA implementation. Hence, protection status may not benefit all ecosystem components, enhancing the need for robust MPA management to balance trophic interactions and support biodiversity and ecosystem resilience. Supplementary Information The online version contains supplementary material available at 10.1038/s41598-024-82557-7.",
"introduction": "Introduction The dynamic balance of marine ecosystems is maintained through intricate interactions among their biological and physical components. Within this context, Marine Protected Areas (MPAs) have emerged as a cornerstone of marine conservation efforts. The effectiveness of MPAs in conserving marine biodiversity and safeguarding critical habitats has been increasingly recognised 1 – 3 . This prompted a global effort to expand MPA coverage as part of broader strategies to protect marine ecosystems and ensure the sustainability of ocean resources 4 , and international organisations have set the goal of protecting 30% of the ocean area by 2030 5 . In Europe, a recently approved Restoration law also targets the protection of 20% of land/ocean by 2030 and all ecosystems in need of restoration by 2050 6 . MPAs’ most general purpose is to act as an effective management tool to reverse and mitigate the impacts of overfishing and increase resilience to other stressors, such as climate change 3 , 7 – 9 . They not only facilitate the recovery of species directly affected by harvesting but can also indirectly promote broader ecosystem recovery and improved ecosystem health by re-establishing lost trophic interactions, such as trophic cascades 9 , 10 . However, a broad understanding of all ecosystem dynamics is required to achieve its full potential and benefits. Beyond fish protection, in several regions of the globe the conservation of marine forests, particularly kelp forests, has increasingly become a crucial point of MPA design and management 11 . Kelp forests are among the ocean’s most productive and biodiverse ecosystems, providing essential services such as habitat provision for commercially important species and nutrient cycling. However, they are currently degraded and jeopardised by several stressors 12 – 14 . Climate change, herbivory, and harvesting are the most common drivers of change affecting kelp forests negatively 15 , and the efficacy of MPAs is contingent upon the nuanced understanding of these threats 7 , 16 , 17 . Recent advances in marine conservation have highlighted the role of MPAs in mitigating some of the critical threats to kelp forests, including overfishing, herbivory and habitat degradation, and shown that well-designed MPAs, with effective enforcement and management plans, can lead to significant increases in biomass and biodiversity within kelp forests 9 , 11 . Nonetheless, climate change (heatwaves) and disturbance caused directly by abiotic factors are more difficult to mitigate with MPAs 11 , which is why it is rarely a central goal considered when planning and designing them. Like in terrestrial forests 18 , herbivory is a key aspect to consider while perceiving the dynamics of kelp forests 19 – 21 . The grazing activities of macro-herbivores, such as sea urchins and certain fish species, can significantly influence the health and sustainability of kelp forests. In the absence of natural predators, herbivore populations can explode, namely sea urchins, leading to overgrazing and the potential devastation of kelps—a phenomenon known as “urchin barrens” 13 , 20 , 22 . Conversely, controlled levels of herbivory are necessary for the natural cycling and renewal of kelp forests, illustrating the complex nature of these interactions. Herbivory can also be potentiated by other biotic and abiotic factors, including species composition of herbivores, the availability of kelp species, and environmental conditions such as depth, temperature, and light availability 23 – 25 . These interactions have significant implications for the health and stability of kelp habitats, affecting their capacity to support diverse marine communities and provide other ecosystem services 12 , 14 , 26 . With the crescent need for MPA implementation, understanding how MPAs can affect herbivory is pivotal. Equally crucial is understanding how fish-oriented marine protected areas will affect or are currently affecting pre-existing foundation species. Removing fishing pressures may lead to changes in herbivore populations and impact the dynamics of these ecosystems, as seen in some Mediterranean seagrass and other brown algae forests where MPAs increased herbivorous fish populations, leading to a decrease in primary producers 27 , 28 . Based on recent research and field observations, we know that this may be the case in kelp forests in the Northeast Atlantic. Kelp forests are degraded inside MPAs while healthy in the adjacent areas, which is paradoxical 29 . Here, the protection from fishing pressures within MPAs is hypothesised to increase the abundance of herbivorous fish 29 , which, in turn, can lead to changes in herbivory rates and potentially affect the structure and resilience of kelp forest ecosystems. Given the crucial importance of understanding these intricate dynamics, our study focuses on assessing the effects of protection status, depth, kelp species, and grazer type on herbivory rates and macro-herbivore abundance. This contributes to a growing body of evidence that emphasises the need for integrated management approaches that consider both the protection of kelp forests and the regulation of herbivore populations within MPAs. In light of these considerations, this work contributes to the ongoing debate on the ecological effects of MPAs, providing insights into the complex interactions between marine herbivores and kelp forest ecosystems. Through a comprehensive analysis of herbivory dynamics inside and outside an MPA, this research aims to enhance our understanding of marine ecosystem functioning and inform the development of effective conservation strategies that support marine biodiversity and ecosystem resilience.",
"discussion": "Discussion Our study provides significant insights into the dynamics of herbivory in marine ecosystems, particularly within and outside Marine Protected Areas (MPAs). By examining the influence of protection status, depth, kelp species, and grazer type on herbivory occurrence, herbivory rates, and macro-herbivore abundance in the surroundings, we have uncovered patterns that could have broad implications for the management and conservation of these crucial habitats. Importantly, this research offers the first empirical evidence from the Atlantic region identifying fish as the predominant grazers in kelp forests and that this grazing can be intensified by MPA implementation. As initially hypothesised, most herbivory occurred inside the Marine Protected Area (MPA) and was performed by fish. In addition, it was found that a higher herbivory rate happened in depth and especially in the kelp species S. polyschides . Importantly, protection status also significantly affected grazer marks’ frequency. Fish bites were significantly more abundant inside the MPA-BER, and urchin ones were more abundant in PEN (Fig. 7 ). The enhanced abundance of fish in deeper areas within the MPA can be explained by the increased protection from any fishing and recreational activities that happen at the surface, allowing for a safer habitat for these species 30 . Alternatively, the higher urchin abundance in shallower areas outside the MPA highlights potential differences in habitat quality or predation pressures. Fish preference for the kelp species S. polyschides can be due to the higher abundance of this species in the surroundings 12 , possibly creating a consumption habit. Our analysis of grazer marks frequency and herbivory rates is further supported by the higher abundance of fish found through UVC and DOV surveys inside the MPA-BER, particularly at greater depths. In contrast, urchin abundance was more pronounced in PEN, especially in shallower waters. \n Fig. 7 Graphical representation of the higher herbivory rates and fish grazers found inside the MPA – BER (Berlengas), especially at higher depths (image partially created with BioRender.com). \n Results showed that MPAs with fisheries restrictions alongside depth gradients significantly influence the distribution and abundance of different herbivore groups. This underscores the importance of protection status and grazers’ identity in shaping the dynamics of kelp forests. It suggests that herbivorous fish might play a more dominant role in these ecosystems than previously thought, despite potential limitations in the conclusions due to using tethered kelp to assess herbivory rates. While this method provides a standardised measure of grazing pressure across different sites and depths, it may introduce an element of artificial food availability. However, it is important to note that within the MPA, there is already an abundance of various algal species, including smaller kelps like Phyllariopsis spp. Despite this natural diversity, herbivorous fish actively consumed the tethered Laminaria ochroleuca and Saccorhiza polyschides , indicating a selective feeding choice rather than a forced consumption due to the absence of alternatives. In addition, this method is widely used in various studies 19 , 31 – 33 . Outside the MPA, the algal community also includes a mix of kelp species and other algae 34 , providing herbivores with the same natural food options. This reduces the likelihood that the observed herbivory is solely an artifact of tethering. Furthermore, sea urchins in both areas are known to be opportunistic and non-selective feeders 35 , preying on a wide range of algal species when available. Thus, while the possibility of an artifact effect from tethering exists, its impact is likely minimal, given the broader context of algal availability and feeding behaviour observed in both protected and unprotected sites. These findings suggest that the increased grazing rates inside the MPA are a reflection of actual preferences and ecological interactions rather than an experimental artifact. Nonetheless, future studies could include direct observations of natural feeding behaviour or additional experiments using untethered kelp to further corroborate these results. Although here, kelp forests are degraded inside the MPA, several studies report the opposite, namely ones representing habitats where sea urchins are the biggest threat to kelp. For ecosystems where sea urchins pose as the primary menace to kelp forests in terms of grazing, the implementation of MPAs with reduced/none fishing policies can more easily have a positive effect on lowering grazing and protecting the habitat and associated fauna, including fish and overall facilitating the recovery of kelp 9 , 17 . However, for ecosystems where herbivorous fish have a more impactful role in kelp grazing, the implementation of MPAs has more complex dynamics associated and can have a critical impact on these habitats. It creates an increase in fish populations, including, inevitably, herbivorous fish. This can lead to a significant downfall of kelp forests 21 , 29 , 36 . The collapse of kelp forests due to herbivorous fish increment in MPAs can be even more accentuated in climatic transition zones, like our case, especially when under climate change effects. In situations like this, species distributions are often very sensitive and prone to shifts occurring due to changes in seawater temperature 26 . It is well known that tropicalisation can lead to increased consumer pressure by herbivorous fish in primary producers like kelp 37 – 39 . This phenomenon is quite known in the Mediterranean Sea, where the protection of specific areas, especially seagrass meadows and other brown canopy-forming seaweeds, promoted the ascension of herbivorous fish populations, compromising the existing primary producers 28 , 40 , 41 . Despite being a relatively common phenomenon in the Mediterranean Sea, it is relatively unknown in the Atlantic. The fact that it is happening, namely in kelp habitats, can indicate a degree of tropicalisation and a changing habitat. A part of this problem in the Mediterranean comes from the rise of the herbivorous fish species Sarpa salpa 42 . In our case, we suggest S. salpa is also the most predominant herbivorous fish, although we could not identify precisely the amount of biomass it consumed. This species is known to be increasing its numbers and distribution across continental Portugal as a consequence of tropicalisation 43 . However, its impacts on grazing and ecosystem dynamics are still quite unknown and should be further investigated. Our results are based on a specific region, but they are supported by other studies’ outcomes, suggesting that they can accurately reflect the reality of ecosystems in transitional temperate zones. Similar examples in the Mediterranean, as mentioned above, as well as other temperate regions, show increased herbivorous fish populations due to MPA implementation. For instance, in New Zealand’s oldest marine reserve, a growing number of herbivorous fish was registered over time inside the reserve 44 . In Australia, studies evaluating the effects of marine reserves on herbivorous fish populations and their grazing activities on temperate reefs also revealed higher numbers of several herbivorous fish and grazing rates inside the reserves as opposed to outside 45 . Perhaps the most relevant example here is the one from the coast of Galicia, Spain, where the kelp forests inside the MPA reached a degraded state due to overgrazing by herbivorous fish inside the reserve, despite the protection status 21 , 29 . Although there are few studies with conditions directly comparable to ours, this does not undermine the validity of our findings. As demonstrated by the studies cited, the impacts of MPAs on herbivorous fish populations and their grazing pressure are context-dependent and well-documented in various regions. While our tethering experiments provided valuable insights into herbivory rates within and outside the MPA, we acknowledge that this approach does not fully capture long-term ecological dynamics or the broader impacts of increased herbivory on kelp forests. The observed increase in herbivorous fish populations within the MPA and their selective grazing on kelp species indicate a potential shift in trophic interactions. However, to comprehensively understand the implications of these interactions, long-term studies are necessary. Future research should focus on monitoring changes in kelp cover, shifts in fish populations, and the overall ecosystem structure over extended periods. This will help to clarify whether increased grazing pressure leads to sustained reductions in kelp abundance or triggers shifts in the ecosystem over time. MPA implementation can facilitate the establishment of herbivorous fish more rapidly 46 , contributing to a more accelerated phase shift and potential collapse of kelp populations. It is essential to be aware that different ecosystem types require different MPA implementation strategies and that inferring implementation success can be misleading and should be made focusing on key groups 10 , 47 , not solely on fish populations. Macroalgae, such as kelp, can be indeed a vital indicator species when assessing MPA implementation success since they are sessile, react very strongly to biotic and abiotic factors, and can increase or reduce biomass and biodiversity very quickly 47 – 49 . In addition, the prevalence and resilience of macroalgae forests, namely kelp, is a very crucial issue nowadays, given their unarguable value in terms of oxygen production 50 , potential carbon sequestration 51 , and habitat for other species. Associated with kelp loss, a myriad of ecosystem services are also lost 13 , 14 . Given this, and since MPAs may not guarantee that every component of the ecosystem will see a benefit 16 , 29 , 52 , there is a clear need for further efforts to a better understanding of ecosystem interactions, namely the interactions of kelp within the food web and abiotic factors. Interactions within the top-down cascades 47 are a critical research point that needs to be addressed. Changes in trophic relationships resulting from the implementation of no-take/ controlled fisheries’ MPAs and the importance of these processes in habitat restoration need to be well comprehended since differing habitats are expected to exhibit differing interactions between primary producers and herbivores according to benthic habitat type and desired habitat goals 47 , 53 , 54 . This highlights the need for MPA-specific management for different habitats and possible outcomes if fisheries are reduced or totally restricted. Moreover, to fully understand the dynamics of the ecosystems that are targeted for restoring an increase in resilience and stability, it is mandatory to have consistent and complete long-term studies and information about these habitats to understand the time frames and successional pathways involved 7 , 9 , 55 . Besides the study of biological interactions and trophic relations, it is also important to address the interaction with abiotic parameters such as depth and seawater temperature, among others, in a way that climate change adaptation can be incorporated into MPA design 56 . In cases where the balanced status is already disturbed, some measures can be taken to mitigate the rise of herbivorous fish and increase the number of primary producers. These measures are quite complex and need to be adapted for each scenario. Methods like fish-deterring devices can protect seaweeds from fish grazers; however, they are expensive and difficult to scale up 20 , 28 , 57 . Transplantations or other reforestation methods, such as green gravel 58 , can also work, but they need to be applied together with other population measures so that new recruitments can thrive and surpass the shift threshold 20 , 57 . Herbivore population control is, to date, the best-suited option to control herbivore impact. This has been widely applied in temperate reefs where sea urchins have decimated kelp forests 22 , 59 , 60 . However, for herbivorous fish, particularly inside an MPA, this strategy is not as efficient and is much more complicated to set in practice. For this case, a good approach could be to promote the commercial value and applications of herbivorous fish, namely S. salpa , within the local population so it could gain value in fish markets 57 . This would increment fisheries of S. salpa instead of only being fished by bycatch and most likely thrown away. In addition, applying fisheries’ restrictions to key herbivore predators like seabass could also be an option to consider. However, this requires a multidisciplinary approach and a proper assessment done beforehand. Here, the observed variations in herbivory rates and herbivore abundance across different sites and depths underline the need for tailored conservation strategies that consider the specific ecological dynamics of each marine habitat. The observed higher numbers of fish populations, mainly herbivorous species, within the MPA boundaries underscores the efficacy of fisheries protection in conserving aquatic life. However, this conservation success story harbours a nuanced ecological dilemma. While MPAs are a very important tool that aims to bolster fish populations by offering refuge from fishing pressures, the resultant surge in herbivorous fish could lead to heightened herbivory, potentially destabilising kelp forest ecosystems. As such, the design and management of MPAs should be informed by understanding these complex interactions to ensure the effective conservation of marine biodiversity and the sustainability of kelp forest ecosystems."
} | 5,319 |
28811302 | PMC5576085 | pmc | 6,422 | {
"abstract": "ABSTRACT Coral bleaching events are predicted to occur more frequently in the coming decades with global warming. The susceptibility of corals to bleaching during thermal stress episodes depends on many factors, including the magnitude of thermal stress and irradiance. The interactions among these two factors, and in particular with ultra-violet radiation (UVR), the most harmful component of light, are more complex than assumed, and are not yet well understood. This paper explores the individual and combined effects of temperature and UVR on the metabolism of Acropora muricata , one of the most abundant coral species worldwide. Particulate and dissolved organic matter (POM/DOM) fluxes and organic matter (OM) degradation by the mucus-associated bacteria were also monitored in all conditions. The results show that UVR exposure exacerbated the temperature-induced bleaching, but did not affect OM fluxes, which were only altered by seawater warming. Temperature increase induced a shift from POM release and DOM uptake in healthy corals to POM uptake and DOM release in stressed ones. POM uptake was linked to a significant grazing of pico- and nanoplankton particles during the incubation, to fulfil the energetic requirements of A. muricata in the absence of autotrophy. Finally, OM degradation by mucus-associated bacterial activity was unaffected by UVR exposure, but significantly increased under high temperature. Altogether, our results demonstrate that seawater warming and UVR not only affect coral physiology, but also the way corals interact with the surrounding seawater, with potential consequences for coral reef biogeochemical cycles and food webs.",
"introduction": "INTRODUCTION Tropical marine ecosystems, including coral reefs, harbor more than 30% of the marine biodiversity ( Doney et al., 2012 ), and provide goods and services to almost one billion people every year ( Moberg and Folke, 1999 ; Wilkinson et al., 1999 ). However, they are currently threatened by climate change-induced increase in sea surface temperature ( Nicholls et al., 2007 ), and in the incident flux of ultra-violet radiation (UVR, 280-400 nm) ( Häder et al., 2007 ). This later increase is due to the effects of global warming on the stratospheric circulation and to a greater water stratification ( Watanabe et al., 2011 ), leading to a deeper penetration of UVR in the water column ( Vodacek et al., 1997 ). The effects of rising sea surface temperature on coral physiology have already been well studied. Since most corals live at or near their threshold of temperature tolerance ( Hoegh-Guldberg, 1999 ), thermal stress induces coral bleaching (i.e. loss of photosynthetic symbionts and/or chlorophyll content) and reduces coral photosynthesis and calcification ( Hoegh-Guldberg, 1999 ). The coral response is, however, species specific, depending on the symbiont clade associated to the coral species ( Wham et al., 2017 ), or the energetic reserves of the host tissue (i.e. lipid and protein content) ( Fitt et al., 2009 ). It is also influenced by a myriad of environmental factors, including the level of UVR received by corals. Although UVR is highly mutagenic and enhances cell oxidative state, especially under elevated temperatures ( Häder et al., 2007 ; Sharma et al., 2012 ), the combined effects of UVR and temperature on coral physiology remain poorly understood because of the complexity of the interactions between these two factors ( Courtial et al., 2017 ; D'Croz and Maté, 2002 ; D'Croz et al., 2001 ; Ferrier-Pagès et al., 2007 ; Fitt and Warner, 1995 ; Lesser and Farrell, 2004 ; Lesser et al., 1990 ). Indeed, while no change was observed on the photosynthetic/autotrophic capacities of Porites lobata or Turbinaria reniformis under the combined stressors ( Courtial et al., 2017 ; D'Croz et al., 2001 ), these capacities were strongly affected in Montastrea annularis and Pocillopora damicornis ( Courtial et al., 2017 ; D'Croz and Maté, 2002 ; Fitt and Warner, 1995 ). The scarcity of experimental studies in this field does not allow good predictions of the combined effects of UVR and temperature on coral physiology. More studies are thus needed to better understand the species-specific response to these factors, and the mechanisms underlying coral susceptibility to thermal stress. Two other underestimated aspects of thermal and UVR stress on coral biology concern the changes in organic matter (OM) fluxes (uptake and/or release of OM by corals) and recycling by the associated bacteria. Under healthy conditions, corals can release half of the photosynthetically fixed carbon and nitrogen into the surrounding reef waters in the form of mucus, i.e. dissolved and particulate carbon (DOC and POC, respectively) and nitrogen (DON and PON, respectively) ( Crossland et al., 1980 ; Davies, 1984 ). OM is then degraded by prokaryotes through their extracellular enzyme activity (EEA), and is used for bacterial growth ( Cunha et al., 2010 ), or it enters into the recycling pathways of carbon and nitrogen ( Wild et al., 2004 ). OM therefore supports pelagic and benthic production, and plays a major role in the nutrient cycles and trophic structure of the whole reef ecosystem ( Bythell and Wild, 2011 ). Elevated temperature, UVR and other stressors can, however, indirectly alter the quality and quantity of OM released by corals ( Niggl et al., 2008 ; Tremblay et al., 2012 ; Wooldridge, 2009 ), and change the associated bacterial diversity ( Ainsworth and Hoegh-Guldberg, 2009 ), likely affecting OM degradation rates. Although few studies have investigated OM fluxes in healthy and thermally stressed corals ( Fonvielle et al., 2015 ; Grottoli et al., 2006 ; Levas et al., 2015 ; Niggl et al., 2008 ; Tremblay et al., 2012 ; Wooldridge, 2009 ), the effects of UVR on these fluxes remain unknown. As far as we know, the impact of elevated temperature and/or UVR on the enzymatic activities of mucus-associated bacteria has also never been investigated in tropical corals. The only knowledge on this subject comes from studies performed on water column bacteria from temperate and cold systems (reviewed in Cunha et al., 2010 ). They showed that bacterial enzymatic activities can be enhanced by temperature and repressed by UVR because of direct enzyme photolysis. Understanding how thermal and UVR stresses alter microbial degradation of coral OM and microbial growth will improve our understanding on future changes of the reef biogeochemical cycling, remineralization pathway and reef trophic structure. The purpose of this study was to address some existing knowledge gaps regarding the effects of thermal stress, UVR and their combination on the quality, quantity and bacterial degradation of OM produced by a scleractinian coral, and to link these changes to coral metabolism. A. muricata was chosen because it belongs to one of the 10 most abundant genera in New Caledonia ( Fenner and Muir, 2008 ) and worldwide ( Veron, 2000 ), and is likely to be one of the major contributors affecting the reef biogeochemical processes. We hypothesize that UVR will exacerbate the effect of thermal stress on coral bleaching and overall metabolism. We also hypothesize that each stressor, alone or in combination, will alter organic carbon and nitrogen fluxes, both in terms of quantity and quality, which will likely change bacterial enzymatic activity in the released mucus. These changes will have a cascading effect on the whole pattern of reef nutrient recycling under global warming scenario.",
"discussion": "DISCUSSION By simultaneously quantifying covariation in coral photosynthesis, calcification, tissue composition, OM fluxes and degradation by mucus-associated bacteria, this study allows deeper understanding of how two major environmental factors, elevated temperature and UVR, alone or in combination, impact the metabolism and close surrounding environment of A. muricata , a dominant coral reef species ( Fenner and Muir, 2008 ). Specifically, our results demonstrate that elevated temperature was the main factor to affect the overall metabolism of A. muricata , as well as OM fluxes and bacterial activity. The results, however, highlight a significant combined effect of UVR and temperature on the bleaching susceptibility and photosynthetic efficiency of this coral species, as well as on the decrease in protein reserves over time. This study also reveals, for the first time, that the shift from auto- to heterotrophy that occurred in the short-term bleached Acropora nubbins led to a change in both the quality of the OM released and the population dynamics of the associated microorganisms. Finally, OM degradation by mucus-associated bacterial activity was unaffected by UVR exposure, but significantly increased under high temperature. Altogether, our results demonstrate that seawater warming not only affects coral physiology, but also the way corals interact with their nearest environment, with potential consequences for coral reef biogeochemical cycles and food webs. A. muricata nubbins did not present any change in their physiology when they were experimentally shaded from UVR and maintained under their normal growth temperature. This lack of UVR effect was likely due to the acclimation to UVR of the colonies used in this experiment, which grew at 2-3 m depth and were therefore likely protected by mycosporine-like amino acids (MAAs), synthesized in most shallow water corals ( Shick et al., 1995 ). This protection was, however, suppressed or reduced under thermal stress ( Fitt and Warner, 1995 ), lowering the coral's capacity to cope with the accumulation of reactive oxygen species and oxidative stress ( Lesser et al., 1990 ). Temperature presented an additive and synergistic effect with UVR on symbiont density and chl a content, respectively. Corals, therefore, bleached and lost 35% of their symbionts under thermal stress alone, and up to 68% under the combined stress, consistent with previous laboratory and field studies which showed greater effects of double than single stress in diverse coral species ( Courtial et al., 2017 ; D'Croz and Maté, 2002 ; Ferrier-Pagès et al., 2007 ; Fitt and Warner, 1995 ). Bleaching was amplified with the stress duration as only 5% of the symbionts remained in nubbins kept for 4 weeks at high temperature under UVR. These results suggest that corals naturally exposed to low UVR could better resist long thermal stress events than UVR-exposed corals. In addition to studying the effects of thermal and UVR stress on coral physiology, we also expanded our measurements to assess the coral-induced changes in seawater biogeochemistry (via mucus release and degradation) with thermal and/or UVR stress. Coral mucus (i.e. dissolved and particulate organic carbon and nitrogen) has several functions, both for corals (defense against external stressors and food source ( Brown and Bythell, 2005 ; Levas et al., 2016 ) and for the reef organisms (energy carrier and particle trap) ( Wild et al., 2004 ); however, changes in mucus quality and quantity under different environmental conditions are still poorly understood ( Niggl et al., 2008 ; Tremblay et al., 2012 ), because few studies have investigated both carbon and nitrogen fluxes, in dissolved or particulate forms, in healthy and stressed coral species ( Bednarz et al., 2012 ; Naumann et al., 2010 ), preventing a comprehensive overview of OM fluxes in corals. Our results first show constant release rates of total organic carbon (TOC) and total organic nitrogen (TON) by A. muricata , irrespective of the stress state. The high TOC/TON ratio (13 to 15) of the released OM, already observed for several coral species of the Red Sea ( Naumann et al., 2010 ), indicates a higher degree of nitrogen retention in coral tissue compared to carbon. Although A. muricata also presents 10 times higher TOC release rates than other species of the Red Sea or the Caribbean ( Levas et al., 2016 ; Naumann et al., 2010 ), these rates are in the range of previously reported values in several Acropora species from Malaysia and Jordan ( Nakajima et al., 2009 , 2010 ; Naumann et al., 2010 ). Overall, Acropora species tend to mainly be a source of energy-rich carbon compounds to the reef food chain. In New Caledonia, this production can partly explain the exceptionally high rates of N 2 fixation in the water column ( Camps et al., 2016 ) compared to other reef systems ( Bednarz et al., 2017 ), since diazotrophs need large amounts of energy-rich photosynthates to perform N 2 fixation ( Bednarz et al., 2017 ). The analysis of the DOM and POM forms shows that the quality of the OM (i.e. particulate or dissolved matter) changes with the environmental conditions under which corals are thriving. A muricata released POM and took up DOM under normal growth conditions, while the reverse was observed in bleached colonies. Although most previous studies show a release of DOC by healthy corals ( Crossland, 1987 ; Wild et al., 2010a , b , 2004a , b , 2005 , 2008 ; Houlbrèque et al., 2004 ; Tanaka et al., 2008 , 2009 ; Haas et al., 2010 ; Naumann et al., 2010 ; Levas et al., 2015 ), some studies show the contrary ( Houlbrèque et al., 2004 ; Naumann et al., 2010 ; Niggl et al., 2008 ). Observations of OM fluxes in bleached or thermally-stressed corals evidenced the same contrasted results: while Porites divaricata , Porites astreoides and Orbicella faveolata , were shown to take up DOC ( Grottoli et al., 2006 , 2014 ; Levas et al., 2013 , 2016 ), Acropora sp., Porites spp. and Stylophora pistillata released it ( Haas et al., 2010 ; Niggl et al., 2008 ; Tremblay et al., 2012 ). Overall, no common pattern can be deduced from these previous observations. Our measurements, which took into account the OM as well as the pico- and nanoplankton concentrations, however, suggest that the changes in POM and DOM fluxes in A. muricata are linked to its heterotrophic activity. POM includes both nonliving material and living particles such as bacteria and small autotrophs contained or grown in the mucus. Pico- and nanoplankton, which multiplied in the incubations with healthy coral colonies, were instead grazed by corals when bleached. A shift thus occurred between low-energy DOM uptake under healthy conditions (maximum of 1.5 µg C and N h −1 cm −2 when all DOM is consumed) to high energy POM uptake under bleached conditions, (maximum of 3.9 µg C and N h −1 cm −2 when all POM is consumed), suggesting a greater need for heterotrophic nutrients, likely to compensate for autotrophic loss and meet metabolic demand. POM uptake contributed 80% of the respiratory needs of the heat-stress colonies, compared to 40% for DOM under healthy conditions. In addition, POM consumption in bleached corals may have enhanced DOC release via sloppy feeding. Although this process was never studied in corals, it is well-known in copepods as a dominant mode of DOM production ( Saba et al., 2011 ). Moreover, our results highlight a positive correlation between the stress level inflicted to the corals and their micro-heterotrophy level: corals shifted from total autotrophy under healthy conditions to partial heterotrophy on prokaryotes alone under thermal stress, and to predation on both prokaryotes and autotrophs when exposed to thermal and UVR stress. These results demonstrate the role of pico/nanoplankton food sources for A. muricata resilience to thermal stress, and for bleached corals in general ( Houlbrèque and Ferrier-Pagès, 2009 ; Tremblay et al., 2012 ). They also clearly indicate that corals can feed on allochtonous aggregates ( Coffroth, 1984 ), and on their own mucus and mucus associated particles, when needed. The ability to shift from autotrophy to heterotrophy is believed to provide significant advantage over species that are unable to do so ( Hughes and Grottoli, 2013 ; Levas et al., 2016 ). In this study, high temperature induced a decrease in the rates of photosynthesis, without any impact on calcification, although the two processes are usually correlated ( Gattuso et al. 1999 ). The shift to heterotrophy at high temperature indeed allowed A. muricata to increase its respiration rates, likely to keep up with energy costs associated with the reparation of damages caused by thermal stress ( Coles and Jokiel, 1977 ; Fitt et al., 2001 ). This increased production of internal CO 2 likely sustained calcification rates, since more than 70% of the CO 2 used in calcification come from internal respiration ( Furla et al., 2000 ). Since bacteria are the first consumers of the carbon-rich compounds (i.e. wax esters, triglycerides, fatty acids) contained in the mucus, to convert them into bacterial biomass ( Ferrier-Pagès et al., 1998 ; Herndl and Velimirov, 1986 ), we quantified in the different temperature and UV conditions, the activity of the two main bacterial enzymes responsible for carbon (α-glucosidase) and nitrogen (aminopeptidase) degradation. Shading nubbins from UVR had little effect on enzymatic activities, which contrasts with previous studies performed in vitro with isolated enzymes, showing a decrease in EEA under UVR because of photolysis ( Espeland and Wetzel, 2001 ). MAAs release by corals in the mucus ( Drollet et al., 1997 ) might have protected the associated bacteria and enzymes from UVR damage. On the other hand, and consistent with observations made on water column bacteria ( Cunha et al., 2010 ; Price and Sowers, 2004 ), high temperature enhanced both aminopeptidase and α-glucosidase EEA, either directly or indirectly through increased bacterial concentration. As a consequence, carbon and nitrogen degradation rates were 20 and 10 times higher, respectively, than at normal temperature. Despite this large increase in OM degradation, the matter degraded by mucus-associated bacteria represented <1% of the carbon and 10% of the nitrogen contained in the excreted mucus. This shows that the recycling of the coral derived-matter is a long-term process, rather performed by bacteria free living in the water column or in reef sediment. Nitrogen was overall 10 times more degraded than carbon, likely because it is one of the major nutrients limiting bacterioplankton growth ( Antia et al., 1991 ; Keil and Kirchman, 1991 ). Overall, our study highlights the major changes in OM fluxes, composition and degradation following A. muricata bleaching. The potential organic carbon and nitrogen pathways expected with healthy (A) and bleached (B) A. muricata are represented in Fig. 5 . Under healthy conditions, A. muricata releases POM, which is poorly degraded by prokaryotes. POM will thus rapidly sediment to the reef bottoms, where it will sustain bacterial growth, and will contribute to the important nutrient recycling pathways observed in reefs ( Muscatine and Porter, 1977 ; Richter et al., 2001 ; Wild et al., 2004 ). During bleaching, A. muricata will release labile DOM, which is more likely to stay in the water column and therefore promote the development of free-living-bacteria ( Ferrier-Pagès et al., 2000 ; Wild et al., 2004 ), including pathogenic communities ( Haas et al., 2013 ; Nelson et al., 2013 ). Unless bacteria enter the microbial loop and higher trophic levels, such stimulation may lead to the ‘microbialization’ of the reef ( Haas et al., 2016 ) with negative consequences for coral health such as the promotion of opportunistic pathogen invasion ( Barott and Rohwer, 2012 ). Our study also emphasizes the importance of considering UVR exposure when predicting long-term coral bleaching. As UVR impact on coral physiology is increased with the stress duration, the effects measured during this short-term experiment could be underestimated on a longer term.\n Fig. 5. Summary of organic matter exchange between coral microbial communities. Uptake (black arrows) and release (grey arrows) fluxes between coral nubbins-associated micro-organisms and near seawater at 26°C (A) and 30°C (B). Blue arrows indicate organisms' growth. Diamond-headed lines (from ‘Prokaryotes/Autotrophs’ to ‘Microbial loop/Open water’) indicate the contribution of mucus-associated microbiome to the open water microbial loop. Dashed lines indicate low fluxes, solid lines indicate enhanced processes. At normal temperature (26°C), coral releases POM and ingests DOM. Prokaryotes and autotrophs grow in the mucus and contribute to the open water microbial loop. The released POM sinks and is then available for the development of the microbial loop inside sediments and in open water. At high temperature (30°C), corals ingest POM and release DOM. They graze on associated microorganisms, which contribute less to the microbial loop of the water column. Overall, DOM is released to the seawater which enhances the development of microorganisms in the water column."
} | 5,224 |
34733822 | PMC8558561 | pmc | 6,423 | {
"abstract": "Currently, stretchable hydrogel has attracted great attention in the field of wearable flexible sensors. However, fabricating flexible hydrogel sensor simultaneously with superstretchability, high mechanical strength, remarkable self-healing ability, excellent anti-freezing and sensing features via a facile method remains a huge challenge. Herein, a fully physically linked poly(hydroxyethyl acrylamide)-gelatin-glycerol-lithium chloride (PHEAA-GE-Gl-LiCl) double network organohydrogel is prepared via a simple one-pot heating-cooling-photopolymerization method. The prepared PHEAA-GE-Gl-LiCl organohydrogel exhibits favorable stretchability (970%) and remarkable self-healing property. Meanwhile, due to the presence of glycerol and LiCl, the PHEAA-GE-Gl-LiCl organohydrogel possesses outstanding anti-freezing capability, it can maintain excellent stretchability (608%) and conductivity (0.102 S/m) even at −40°C. In addition, the PHEAA-GE-Gl-LiCl organohydrogel-based strain sensor is capable of repeatedly and stably detecting and monitoring both large-scale human motions and subtle physiological signals in a wide temperature range (from −40°C to 25°C). More importantly, the PHEAA-GE-Gl-LiCl organohydrogel-based sensor displays excellent strain sensitivity (GF = 13.16 at 500% strain), fast response time (300 ms), and outstanding repeatability. Based on these super characteristics, it is envisioned that PHEAA-GE-Gl-LiCl organohydrogel holds promising potentials as wearable strain sensor.",
"conclusion": "Conclusion In summary, we have successfully developed a fully physically linked PHEAA-GE-Gl-LiCl organohydrogel via a facile one-pot heating-cooling-photopolymerization method. The resulting PHEAA-GE-Gl-LiCl organohydrogel exhibited superstretchability (970%), remarkable self-healing ability (the strain of self-healed organohydrogel was 263%), and a high gauge factor of 13.16. Meanwhile, on account of the presence of both glycerol and LiCl, PHEAA-GE-Gl-LiCl organohydrogel possessed excellent anti-freezing, and ultra-high strain sensitivity features, which could be designed as wearable strain sensors for strain detection in wide temperature ranges (from −40 to 25°C). The PHEAA-GE-Gl-LiCl organohydrogel based-strain sensor was able to accurately monitor and distinguish both large-scale human motions such as joint bending and subtle physiological signals such as swallowing and pronouncing. Owing to these outstanding performances, it was conceivable that the PHEAA-GE-Gl-LiCl organohydrogel would present an unpredictable prospect for use in wearable strain sensors.",
"introduction": "Introduction Flexible and wearable skin-like strain sensors can convert mechanical deformations into detectable electronic signals (e.g., resistance, capacitance, current, etc.), they have attracted more and more attentions due to the promising potential applications in the field of motion detecting and human-machine interaction ( Ge et al., 2018 ; Xu et al., 2019 ; Zeng et al., 2019 ; Zhu et al., 2019 ; Kim et al., 2020 ; Kim and Moon, 2020 ). For example, Qu et al. fabricated a flexible strain sensor based on polydopamine-coated nanocomposites of nitrile rubber and carbon black ( Qu et al., 2020 ). Zhan et al. prepared a high sensitivity of multi-sensing materials based on reduced graphene oxide and natural rubber ( Zhan et al., 2021 ). To meet the requirements of applications, the strain sensors should have desired stretchability, mechanical strength, self-healing ability and strain sensitivity ( Xin et al., 2016 ; Lei et al., 2017 ; Si et al., 2017 ; Wang L. et al., 2019 ; Jing et al., 2019 ; Kweon et al., 2019 ). However, the conventional strain sensors prepared by integrating conductive components (e.g., conductive polymers, metal materials, carbon-based materials, etc.) into elastomer (e.g., polyethylene terephthalate, polyurethane, etc.) show poor stretchability ( Lee et al., 2014 ; Kim et al., 2015 ; Lee et al., 2015 ; Kim et al., 2018 ; Zhou et al., 2018 ). Therefore, it is very important to choose intrinsically highly stretchable conductive materials for fabricating high-performance wearable sensors. Recently, ionic double network (DN) hydrogels by introducing salt ions (e.g., LiCl, NaCl, KCl, Fe 3+ , Ca 2+ , etc.) have drawn special interest to fabricate flexible and stretchable strain sensors, as they are stretchable and can maintain bulk conductivity during large reversible deformation ( Liu and Li, 2017 ; Cheng et al., 2019 ; Hou et al., 2019 ; Yang and Yuan, 2019 ; Liu et al., 2020 ; Zhu et al., 2020 ). For example, Hou et al. fabricated a polyacrylamide (PAAm)-agarose-NaCl hydrogel-based strain sensor with high sensitivity via chemically crosslinked PAAm network and physically crosslinked agarose network ( Hou et al., 2019 ). Liu et al. prepared a stretchable strain sensor based on PAAm-carrageenan-KCl DN hydrogel via chemically crosslinked PAAm network and physically crosslinked carrageenan network, which could monitor and distinguish various human motions ( Liu and Li, 2017 ). Owing to the existence of Na + , K + , Ca 2+ ions in gellan gum (GG), Liu et al. engineered a highly stretchable PAAm-GG hydrogel-based sensor for human motion monitoring via chemically crosslinked PAAm network and physically crosslinked GG network ( Liu et al., 2020 ). However, due to the intrinsic irreversibility of the covalent crosslinking, these reported conductive DN hydrogel had unsatisfactory self-healing ability, leading to low durability as the sensor. Therefore, developing a conductive DN hydrogels crosslinked by physical interactions may be an effective way to prepare the flexible strain sensors ( Liu et al., 2017 ; Liu et al., 2018 ; Chen et al., 2019 ; Xia et al., 2019 ; Zhang et al., 2020 ). Water-based ionic hydrogels will inevitably freeze at subzero temperatures, which further decrease the stretchability and conductivity properties. Recently, some polyol agents (e.g., glycol, glycerol, sorbitol, etc.) has been introduced into hydrogels to improve the freezing-tolerant property of hydrogel ( Liao et al., 2019 ; Tu et al., 2019 ; Wu et al., 2019 ; Pan et al., 2020 ; Peng et al., 2020 ). These anti-freezing agents can effectively reduce the aggregation of water molecules and weaken their internal hydrogen bonding, resulting in preventing the formation of ice crystalline and decreasing the freezing points of hydrogels. Based on this point of view, Liao et al. developed an anti-freezing MXene/PAAm-PVA organohydrogel by immersing hydrogel in glycol solution, the prepared organohydrogel exhibited outstanding anti-freezing property at −40°C ( Liao et al., 2019 ). Nevertheless, the immersion procedure is often involved in this strategy, which is laborious and further limiting their large-scale application. Therefore, constructing anti-freezing hydrogels via a facile “one-pot” method was more desired. Based on this idea, Peng et al. constructed an anti-freezing PVA-PEDOT/PSS DN organohydrogel by directly using glycol-water binary solvent as dispersion medium ( Peng et al., 2020 ). Pan et al. prepared a poly(vinyl alcohol) hydrogel by dissolving poly(vinyl alcohol) in glycerol-water binary solvent, followed by soaking it in saturated NaCl aqueous solution, the obtained organohydrogel-based sensor could maintain good strain-sensitive performance at −20°C ( Pan et al., 2020 ). However, these hydrogels could not heal once damaged. In consequence, it is highly anticipated to find a facile “one-pot” method to realize an ionic conductive hydrogel simultaneously with integrated superstretchability, high mechanical strength, remarkable self-healing ability, excellent anti-freezing behaviors and sensing features to broaden their application range in spite of the great progress in hydrogel sensors. Herein, a fully physically crosslinked poly(hydroxyethyl acrylamide)-gelatin-glycerol-lithium chloride (PHEAA-GE-Gl-LiCl) organohydrogel was constructed by a simple one-pot method. It was expected that it had superstretchability, high mechanical strength, remarkable self-healing ability, excellent anti-freezing behaviors and sensing features. The networks of the organohydrogel were formed via hydrogen bonds. Incorporation of Li + and Cl − ions imparted PHEAA-GE-Gl-LiCl organohydrogel prominent ionic conductivity. Owing to the presence of glycerol and LiCl, the PHEAA-GE-Gl-LiCl organohydrogel possessed excellent anti-freezing and ultra-high strain sensitivity features, which could be designed as wearable sensors for strain detection in wide temperature ranges (from −40°C to 25°C).",
"discussion": "Results and Discussion Formation of PHEAA-GE-Gl-LiCl Organohydrogel \n Figure 1A displayed the fabrication procedure of the PHEAA-GE-Gl-LiCl organohydrogel via a simple one-pot heating-cooling-photopolymerization method. At first, all reactants including UV initiator Irgacure 2959, HEAA, GE, glycerol and LiCl were dispersed in deionized water. Subsequently, the mixture solution was heated to 60°C to dissolve GE, and then was cooled to room temperature. Then, it was exposed to UV light to initiate the polymerization of HEAA monomer and form the physical crosslinked PHEAA network via the hydrogen bonding among hydroxyl groups of PHEAA chains, which interpenetrated with the GE network, and finally formed PHEAA-GE-Gl-LiCl organohydrogel with a milky white color. During this preparation process, no chemical cross-linked structure was introduced. The prepared PHEAA-GE-Gl-LiCl organohydrogel was formed via the reversible noncovalent bonds. FIGURE 1 \n (A) Schematic of a one-pot synthesis of a fully physically cross-linked PHEAA-GE-Gl-LiCl organohydrogel. (B) ATR-FTIR spectroscopy of PHEAA SN hydrogel, GE SN hydrogel, and PHEAA-GE DN hydrogel. (C) ATR-FTIR spectroscopy of PHEAA-GE-LiCl hydrogel and PHEAA-GE-Gl-LiCl organohydrogel. ATR-FTIR spectra were used to investigate the interactions in PHEAA-GE-Gl-LiCl organohydrogel. As shown in Figure 1B , PHEAA hydrogel exhibited the absorption band at 3,271 cm −1 , which was attributed to the stretching vibration of O-H and N-H. Meanwhile, the two characteristic peaks at 1,625 cm −1 and 1,551 cm −1 were assigned to the stretching vibration of C=O and bending vibration of N-H from amide, respectively. For GE hydrogel, the broadband at 3,277 cm −1 was assigned to the N-H stretching vibrations, and the characteristic peaks at 1,627 cm −1 and 1,539 cm −1 were assigned to the stretching vibration of C=O and bending vibration of N-H from amide, respectively. Compared with PHEAA hydrogel and GE hydrogel, few new absorption bands appear in the PHEAA-GE hydrogel, indicating there were no chemical reactions between PHEAA and GE networks. Meanwhile, the stretching vibrations of O-H and N-H, stretching vibration of C=O and bending vibration of N-H from amide shifted to 3,275, 1,626, and 1,549 cm −1 , respectively, suggesting the formation of hydrogen bonds between the PHEAA networks and GE networks. To illustrate the interactions between glycerol and PHEAA-GE-LiCl network, ATR-FTIR of PHEAA-GE-LiCl hydrogel and PHEAA-GE-Gl-LiCl organohydrogel was also analyzed. As presented in Figure 1C , after addition glycerol, the stretching vibration of O-H and N-H (3,268 cm −1 ), stretching vibration of C=O (1,626 cm −1 ), and bending vibration of N-H (1,549 cm −1 ) in PHEAA-GE-LiCl hydrogel shifted to 3,275, 1,629, and 1,557 cm −1 in the PHEAA-GE-Gl-LiCl organohydrogel, respectively, indicating the formation of hydrogen bonds between glycerol molecules and the gel networks owing to the -OH and -NH- groups among PHEAA, GE and glycerol. Mechanical Properties of PHEAA-GE-Gl-LiCl Organohydrogel The mechanical property of PHEAA-GE-Gl-LiCl organohydrogel was primarily dependent on the network composition and structure. Figures 2A,B showed the typical tensile stress-strain curves of different hydrogels/organohydrogels and the corresponding elongation at break. Compared with the PHEAA hydrogel (0.23 MPa), the stress of PHEAA-GE hydrogel remarkably enhanced to 1.44 MPa. After introducing LiCl and glycerol, the stress appeared to decrease slightly. The elasticity modulus of PHEAA hydrogel, PHEAA-GE hydrogel, PHEAA-GE-LiCl hydrogel, and PHEAA-GE-Gl-LiCl organohydrogel was 0.10, 0.29, 0.30, and 0.22 MPa, respectively. Besides, all these hydrogels exhibited high fracture strain in the range of 1,158%–964% ( Figure 2B ). Figures 2C,D visually illustrated PHEAA-GE-Gl-LiCl organohydrogel was flexible and tough, it could be easily stretched to approximately 10 times of its original length without rupture even at twisted state. In addition, the PHEAA-GE-Gl-LiCl organohydrogel was strong enough to sustain a weight of 500 g (625 times of its own weight) without breaking ( Figure 2E ) and was tough enough to resist to the puncture ( Figure 2F ). Supplementary Figure S1 indicated PHEAA-GE-Gl-LiCl organohydrogel displayed a very smooth and flat structure, which was possibly because noncovalent interactions within the PHEAA-GE-Gl-LiCl organohydrogel promoted the connected network. FIGURE 2 The mechanical property of PHEAA-GE-Gl-LiCl DN organohydrogel. (A) The typical tensile stress-strain curves of PHEAA hydrogel (C PHEAA = 40 wt%), PHEAA-GE hydrogel (C PHEAA = 36 wt%, C GE = 4 wt%), PHEAA-GE-LiCl hydrogel (C PHEAA = 36 wt%, C GE = 4 wt%, C LiCl = 1.2 wt%), PHEAA-GE-Gl-LiCl DN organohydrogel [C PHEAA = 36 wt%, C GE = 4 wt%, C LiCl = 1.2 wt%, and Gl:water (w/w) = 1:3], and (B) their corresponding elongations at break. Photos of PHEAA-GE-Gl-LiCl DN organohydrogel at different states of (C) stretching, (D) curly stretching, (E) sustaining a weight of 500 g and (F) puncture resistance. (G) Ten continuous cyclic stretching and releasing process of the PHEAA-GE-Gl-LiCl organohydrogel under 300% tensile strain and (H) the corresponding hysteresis energy in every cycle. To better illustrate the mechanical properties of the prepared organohydrogel, the effects of the solid content of PHEAA, the weight ratios of PHEAA to GE, the amount of LiCl and glycerol on the mechanical properties of hydrogels were investigated by uniaxial tensile tests. As shown in Supplementary Figure S2 , a minimum concentration of HEAA monomers (20 wt%) was required to form PHEAA SN hydrogels. With the enhancement of PHEAA concentration from 25 to 50 wt%, the tensile strength of PHEAA SN hydrogels increased from 0.14 to 0.32 MPa and the elongation at break of all the PHEAA SN hydrogels was higher than 800%, indicating PHEAA network was soft and tough ( Supplementary Figure S3 ). The mechanical property of PHEAA-GE hydrogel was remarkably enhanced compared with PHEAA SN hydrogel ( Figure 2A ). Meanwhile, as the weight ratios of PHEAA/GE decreased from 19:1 to 3:1, the tensile strength of PHEAA-GE hydrogel continuously increased from 0.69 to 2.31 MPa, while both of elongation at break and toughness increased first and then decreased. At the weight ratio of 9:1, the maximum fracture strain (1,158%) and toughness (8.17 MJ/m 3 ) were achieved ( Supplementary Figure S4 ). Owing to the rigid and brittle nature of the GE network, the higher concentration of GE made PHEAA-GE hydrogel stronger but more brittle. Besides, the introduction of LiCl or glycerol can also impact the mechanical property ( Supplementary Figure S5 , Supplementary Figure S6 ). With the enhancement of LiCl concentration from 0.2 to 1.6 wt%, the tensile strength decreased from 1.26 to 0.74 MPa, and the elongation at break decreased from 1,238 to 806%, which was likely due to the destruction of hydrogen bonds between PHEAA networks and/or GE networks. Furthermore, there were large amounts of -C=O and -NH groups in the hydrogel matrix, so the interaction between PHEAA networks and/or GE networks might be influenced when glycerol replaced part of water. As presented in Supplementary Figure S6 , with an increase in the proportion of glycerol/water from 1:4 to 1:2, the tensile stress decreased from 1.37 to 0.76 MPa and the fracture strain increased from 974 to 1,274%, which indicated the introduction of glycerol weakened the interactions between PHEAA networks and/or GE networks and meanwhile enhanced the gel flexibility. PHEAA-GE-Gl-LiCl organohydrogel was formed via the fully reversible non-covalent bonds, so it was expected that the PHEAA-GE-Gl-LiCl organohydrogel had energy dissipation and self-recovery ability. As displayed in Figure 2G , a successive cyclic tensile test at a maximum strain of 300% without resting time between each cycle was conducted. Obviously, the PHEAA-GE-Gl-LiCl organohydrogel exhibited a large hysteresis loop in the first cycle, indicating plenty of energy could be dissipated by rapid dissociation of physical interactions. From the second to 10th cycles, the corresponding dissipated energies remained almost unchanged at around 62 KJ/m 3 ( Figure 2H ), indicating the good fatigue resistance of PHEAA-GE-Gl-LiCl organohydrogel due to the rapid self-recoverability ( Qin et al., 2020 ). Healing Performances of PHEAA-GE-Gl-LiCl Organohydrogel Due to the reversible non-covalent interactions in PHEAA-GE-Gl-LiCl organohydrogel, good self-healing property of PHEAA-GE-Gl-LiCl organohydrogel was expected. As shown in Figure 3A , when two pieces of PHEAA-GE-Gl-LiCl organohydrogel (red and blue) were in contact with each other, the obvious interface could be observed. After storing at room temperature (25°C) for 12 h, the two hydrogels fused together, and the interface became indistinct. Moreover, the healed PHEAA-GE-Gl-LiCl organohydrogel could sustain a load of 50 g (55 times of its own weight), implying PHEAA-GE-Gl-LiCl organohydrogel possessed good self-healing property at room temperature. To quantify the self-healing property, the tensile stress-strain curves of the healed PHEAA-GE-Gl-LiCl organohydrogel were investigated. As shown in Figure 3B , the stress and elongation at break increased with the increase of healing time. The tensile strength and elongation at break of healed PHEAA-GE-Gl-LiCl organohydrogel for 12 h was 0.09 MPa and 139%, respectively. The healing efficiency was 7.02% at 12 h ( Figure 3C ). At 25°C, the self-healing property was attributed to the facile breaking and reforming of hydrogen bonds originated from the self-association of the PHEAA network. This performance was unlike with PAAm-based hydrogels crosslinked via covalent bond, whose self-healing property could not be achieved at 25°C because the fractured chemically cross-linked PAAm network was unable to be reconstructed. Usually, to realize the self-healing ability of PAAm-based hydrogels, additional heat treatment must be needed to promote the movement of polymeric chain in network. However, even so, the healing efficiency was not satisfying. FIGURE 3 The self-healing property of PHEAA-GE-Gl-LiCl organohydrogel. The self-healed PHEAA-GE-Gl-LiCl organohydrogel (A) at 25°C for 12 h and (D) at 60°C for 12 h could withstand a weight of 50 and 100 g, respectively. Stress-strain curve and self-healing efficiency of the self-healed PHEAA-GE-Gl-LiCl organohydrogel at different healing time (B,C) at 25°C and (E,F) at 60°C. The triple-helix bundles of GE can be transformed into coils when the temperature is high than its conformational transition temperature, then the triple-helix bundles can be re-associated when temperature decreases ( Chen et al., 2017 ; He et al., 2018 ; Qin et al., 2020 ). Therefore, the self-healing ability of PHEAA-GE-Gl-LiCl organohydrogel could be enhanced at 60°C. As displayed in Figure 3D , compared with the healed PHEAA-GE-Gl-LiCl organohydrogel at 25°C for 12 h, the healed PHEAA-GE-Gl-LiCl organohydrogel at 60°C for 12 h could sustain a weight of 100 g. Figures 3E,F showed the stress, fracture strain, and the healing efficiency of the healed PHEAA-GE-Gl-LiCl at 60°C was much higher than those at 25°C at the same healing time. Upon the heating treatment at 60°C for 12 h, the tensile stress, fracture strain, and the healing efficiency was 0.47 MPa, 263%, and 37.23%, respectively, which was much higher than those of the healed PHEAA-GE-Gl-LiCl organohydrogel at 25°C. The schematic diagram of the self-healing mechanism was shown in Supplementary Figure S7 . On the whole, due to the collaborative results of two physical crosslinked networks, the healing behaviors of PHEAA-GE-Gl-LiCl organohydrogel is much higher than all the reported self-healed PAAm-based or PHEAA-based hydrogel sensor ( Supplementary Table S1 ). Anti-Freezing Property of PHEAA-GE-Gl-LiCl Organohydrogels Owing to large amounts of water, ionically conductive hydrogels will be frozen and lose stretchable property. Meanwhile, the conductivity also obviously decreases owing to the low movement capacity of the free ions at subzero temperatures ( Chen et al., 2018 ; Rong et al., 2018 ). Therefore, the water-based hydrogel cannot be used under subzero temperature. In this work, the glycerol and LiCl were introduced into the hydrogel network to improve the freezing tolerances. DSC experiment was performed to investigate the ice formation in the organohydrogel. As shown in Figure 4A , a sharp exothermic peak at −19°C appeared in PHEAA-GE hydrogel, indicating large amounts of water was converted to ice at this condition. After addition of LiCl, the exothermic peak reduced to −27°C owing to the colligative property of ionic compounds ( Gao et al., 2019 ), indicating that the introduction of LiCl decreased the freezing point of water. Further, the freezing point was dramatically reduced to −52°C when 20% glycerol was introduced (glycerol/water, 1:4). Surprisingly, the exothermic peak entirely disappeared when the glycerol/water ratio increased to 1:3 and 1:2. These results demonstrated the introduction of glycerol could effectively decrease the freezing point or even completely depress ice generation by the comprehensive effect combined colligative property of LiCl and forming hydrogen bonds between the hydroxyls in the glycerol and water molecules. FIGURE 4 Anti-freezing properties of PHEAA-GE-Gl-LiCl organohydrogels. (A) DSC curves of hydrogel and organohydrogel with different Gl/water ratios (0:1, 1:4, 1:3, and 1:2). (B) Storage modulus (G′) of hydrogel and organohydrogel with different Gl/water ratios (0:1, 1:4, 1:3, and 1:2) at the temperature range of 25°C to −40°C. The (C) stress-strain curves, (D) stress and elongation at break of PHEAA-GE-LiCl hydrogel and PHEAA-GE-Gl-LiCl organohydrogel with different weight ratios of Gl/water at −40°C. The rheological test at temperature ranges from 25°C to −40°C showed a similar phenomenon ( Figure 4B ). The G′ value of the PHEAA-GE abruptly increased when the temperature fell below −13°C. At this temperature, the ice crystals started to grow and PHEAA-GE-LiCl hydrogel began to transform into an ice-like solid. The introduction of LiCl decreased this behavior, and the G′ value of PHEAA-GE-LiCl hydrogel abruptly increased when temperature fell below −20°C. In contrast, the G′ value of PHEAA-GE-Gl-LiCl organohydrogels (1:4, 1:3, and 1:2) showed persistent stability within the test temperature range (from 25°C to −40°C), indicating that the introduction of glycerol was beneficial to the maintenance of the flexible feature of PHEAA-GE-Gl-LiCl organohydrogels at subzero temperature. To further verify this result, the stretching behavior of the different gels was also investigated at −40°C. As shown in Figures 4C,D , compared with the stress (26.78 MPa) and elongation at break (80%) of PHEAA-GE-LiCl hydrogel, all the PHEAA-GE-Gl-LiCl organohydrogel possessed lower stress (1.65–2.91 MPa) and higher fracture strain (585–784%) ( Supplementary Video S1 ). This behavior was caused by the existing glycerol in the organohydrogel, which hindered the crystallization of water and enhanced the anti-freezing property. Meanwhile, as the glycerol content increased, the stress decreased and elongation at break increased. The fracture strain was much higher than most of the previously reported anti-freezing hydrogel-based gel ( Supplementary Table S2 ). On the whole, on account of the presence of both glycerol and LiCl, PHEAA-GE-Gl-LiCl organohydrogel possessed outstanding anti-freezing capability, which could effectively expand its application in low-temperature environments. Conductivity of PHEAA-GE-Gl-LiCl Organohydrogels To improve the conductivity, LiCl was introduced into PHEAA-GE-LiCl hydrogel. As shown in Figure 5A , with the enhancement of LiCl concentration from 0 to 1.6 wt%, the conductivity rapidly increased from 0.012 to 0.576 S/m. The glycerol/water ratio can also modulate the conductivity of PHEAA-GE-Gl-LiCl organohydrogel, the conductivity decreased from 0.454 to 0.124 S/m when the glycerol/water ratios increased from 0:1 to 1:2 ( Figure 5B ). This phenomenon was because the migration velocity of free ions (Li + and Cl − ) in the PHEAA-GE-Gl-LiCl organohydrogel reduced owing to the high viscosity characteristics of glycerol ( Wang Q. et al., 2019 ; Yu et al., 2021 ). In addition, the temperature also impacted the conductivity of PHEAA-GE-Gl-LiCl organohydrogel. As displayed in Figure 5C , when the temperature was above −10°C, the conductivity of PHEAA-GE-Gl-LiCl organohydrogel changed slightly. Once the temperature dropped to −10°C, the conductivity decreased with the decrease of temperature, which was because the free ions were more difficult to move in the network at low temperature ( Yang et al., 2021 ). Even so, PHEAA-GE-Gl-LiCl organohydrogel also possessed good conductivity (0.102 S/m) at −40°C. FIGURE 5 The conductivity of PHEAA-GE-Gl-LiCl organohydrogels. Effect of (A) LiCl concentration, (B) the weight ratios of Gl/water, (C) temperature, and (D) circuit comprises an LED indicator connected by original, cut, self-healed, stretched self-healed PHEAA-GE-Gl-LiCl organohydrogel sheets. The conductive activity of the healed PHEAA-GE-Gl-LiCl organohydrogel was investigated via connecting in the circuit with a blue LED bulb. As shown in Figure 5D , the LED bulb was successfully lit when a voltage of 4.5 V was applied. When PHEAA-GE-Gl-LiCl organohydrogel was cut, the conductive circuit was disconnected, and thus the LED bulb was extinguished. Subsequently, the two fractured surfaces were stained and contacted to heal at 60°C for 12 h, the LED bulb was lit again due to the self-healing capacity of PHEAA-GE-Gl-LiCl organohydrogel. The conductivity of the healed PHEAA-GE-Gl-LiCl organohydrogel recovered to 95.21% of its original state. When the healed PHEAA-GE-Gl-LiCl organohydrogel was pulled to 2.5 times of its original length, the brightness of the LED bulb darkened obviously, signifying that the self-healed PHEAA-GE-Gl-LiCl organohydrogel still possessed outstanding strain-sensitive behaviors. Strain Sensor Based on PHEAA-GE-Gl-LiCl Organohydrogel for Detection of Human Motions Responsivity is one of the most important parameters for sensors. Here, the response and recovery time was defined as the time interval during which relative resistance variation changed from one stable value to another in response to an instantaneous stretching and releasing deformation. As displayed in Figure 6A , the response and recovery time of PHEAA-GE-Gl-LiCl organohydrogel was 300 and 200 ms, respectively, demonstrating that PHEAA-GE-Gl-LiCl organohydrogel-based strain sensor possessed a rapid response and recovery ability. Figure 6B presented PHEAA-GE-Gl-LiCl organohydrogel-based sensor could sensitively detect both small strain (5–20%) and relatively large strain (25–200%) with good stability and repeatability. And the corresponding relative resistance change ( ΔR/R \n \n 0 \n ) monotonously increased as the strain increased from 0 to 500% ( Figure 6C ). The obvious increase in resistance during stretching was due to the narrowing of cross-section, which reduced the transportation efficiency of Li + and Cl − ions. The resistance change of PHEAA-GE-Gl-LiCl organohydrogel was also visually observed in Supplementary Video S2 , an LED bulb in the circuit displayed alternate luminance variation during stretching and releasing PHEAA-GE-Gl-LiCl organohydrogel under 300% tensile strain. The GF values of PHEAA-GE-Gl-LiCl organohydrogel were 2.57, 6.09, and 13.16 in the 0–20%, 20–250%, and 250–500% strain ranges, respectively. When the strain was 500%, the ΔR/R \n \n 0 \n was 13.16%, indicating a high strain sensitivity and broad working range. Notably, the sensitivity of PHEAA-GE-Gl-LiCl organohydrogel-based sensor was much higher than that of previously reported stretchable strain sensors ( Supplementary Table S3 ). Figure 6D displayed PHEAA-GE-Gl-LiCl organohydrogel-based sensor was able to maintain repeatable output signals during 300 consecutive cycles at a strain of 50%. FIGURE 6 Strain sensing behavior of PHEAA-GE-Gl-LiCl organohydrogel. (A) Response time, (B) the relative resistance under different small reciprocating strains (5, 10, 15, and 20%) and large reciprocating strains (25, 50, 75, 100, and 200%), (C) relative resistance changes and corresponding gauge factors variation with successive tensile strain from 0 to 500%, (D) the relative resistance change under cyclic loading of 50% for 300 cycles. Owing to their favorable stretchability, excellent self-healing ability and high strain sensitivity, the PHEAA-GE-Gl-LiCl organohydrogel showed tremendous potential applications in wearable devices. Here, PHEAA-GE-Gl-LiCl organohydrogel was assembled to be a resistance-type strain sensor to detect full-range human activities in real time. First, the strain sensor was attached on human joints (finger, wrist, elbow, and knee joints) to detect the joint motions. As shown in Figure 7A , when the finger was bent from the straightened state to 30, 60, and 90°, the ΔR/R \n \n 0 \n gradually increased to 20.71, 36.91, and 74.36%, respectively. When the finger was held at the same bending angle, the resistance values were consistent. Besides, the resistance value immediately returned to its original level when the finger was straightened. The PHEAA-GE-Gl-LiCl organohydrogel-based strain sensor could also be used to precisely monitor and distinguish the extending/flexing of wrist, elbow, and knee ( Figures 7B,C,D ). The extending/flexing of elbow displayed the largest relative resistance change due to the largest deformation. Meanwhile, the response signals were repeatable during the cyclic extending/flexing process. Apart from the capacity of monitoring large-scale human motions, the PHEAA-GE-Gl-LiCl organohydrogel-based strain sensor could also perceive subtle deformation. As shown in Figure 7E , the PHEAA-GE-Gl-LiCl organohydrogel-based strain sensor can be fixed on the throat of a volunteer to monitor the swallowing motion. The resistance signals were obvious and stable when the volunteer performed periodic swallowing motions. Furthermore, the PHEAA-GE-Gl-LiCl organohydrogel-based strain sensor could also be used to detect and distinguish pronouncing. The characteristic peaks were similar when the tester pronounced the same word repeatedly, while the PHEAA-GE-Gl-LiCl organohydrogel-based strain sensor showed distinguishable characteristic peaks when saying different words, such as “China” and “Hydrogel” ( Figure 7F and Supplementary Figure S8 ). These results demonstrated that the PHEAA-GE-Gl-LiCl organohydrogel-based strain sensor exhibited great potential as a high-performance wearable device to detect full-range human activities. FIGURE 7 Real-time monitoring of human physical activities based on wearable PHEAA-GE-Gl-LiCl organohydrogel sensor. Relative resistance changes ( ΔR/R \n \n 0 \n ) of different human joint motions: (A) finger bending at different angles, (B) wrist joint bending, (C) elbow joint bending, (D) knee joint bending. Detection of subtle motions of (E) swallowing and (F) pronouncing word “China.” Flexible Sensing Behavior at Subzero Temperature The PHEAA-GE-Gl-LiCl organohydrogel can be used as an anti-freezing wearable strain sensor. Firstly, the strain-sensing behaviors of the PHEAA-GE-Gl-LiCl organohydrogel were investigated at −40°C ( Figure 8A ). Similar to that at room temperature, ΔR/R \n \n 0 \n of PHEAA-GE-Gl-LiCl organohydrogel based sensor at −40°C increased rapidly with the increase of strain. When the strain was 280%, the ΔR/R \n \n 0 \n was 3,100%, indicating that PHEAA-GE-Gl-LiCl organohydrogel could work under a broad strain range even at −40°C. Compared with that at room temperature, the PHEAA-GE-Gl-LiCl organohydrogel displayed a higher GF value (2.59 at 0–100% strain, 7.50 at 100–172% strain, and 25.35 at 172–263% strain) at −40°C. The enhancement of GF might be attributed to the following two reasons. First, the PHEAA-GE-Gl-LiCl organohydrogel displayed a tougher network at lower temperature, resulting in restricting the movement of free ions ( Figure 4D ). Second, the free ions were more difficult to move in the gel network at low temperature, so the sensitivity increased more intensely during stretching. However, the deeper mechanism remained to be explored. FIGURE 8 Sensing performance of PHEAA-GE-Gl-LiCl organohydrogel-based strain sensor at −40°C. (A) Relative resistance changes and corresponding gauge factors variation with successive tensile strain from 0 to 280%, (B) the relative resistance under different small reciprocating strains and large reciprocating strains. Relative resistance changes of imitative human joints motions: (C) finger bending, (D) wrist joint bending, (E) elbow joint bending, and (F) knee joint bending at −40°C. Same as at room temperature, the PHEAA-GE-Gl-LiCl organohydrogel-based sensor could sensitively detect both small strain and relatively large strain (5–200%) with good stability and repeatability at −40°C ( Figure 8B ). In addition, PHEAA-GE-Gl-LiCl organohydrogel was designed as a wearable strain sensor to detect human model motions at −40°C. As shown in Figures 8C–F , it could monitor the movement of a human model with stable and repeatable signals at −40°C, including the bending or relaxation states of various joints (e.g., finger, wrist, elbow, knee). Meanwhile, we found the relative resistance variation was positively correlated with the degree of deformation. Owing to the largest deformation, the joint motion of knee produced the highest peaks. All these results suggested the PHEAA-GE-Gl-LiCl organohydrogel-based strain sensor has a bright future in wearable devices to detect full-range human activities under a broad range of temperatures. There were some limitations in this study. In the self-healing experiment, the operation requirement is high. First, the hydrogel should be cut with a sharp scalpel to ensure the cross section smooth and intact to the maximum extent. Second, when manually put the cut hydrogel together, we must carefully observe whether the gel section was complete contact. These operation details would influence the self-healing efficiency of the hydrogel. Further studies will optimize the effect of these experimental details on the self-healing efficiency."
} | 8,799 |
28924511 | PMC5584815 | pmc | 6,425 | {
"abstract": "Premise of the study: Arbuscular mycorrhizal fungi (AMF) are globally important root symbioses that enhance plant growth and nutrition and influence ecosystem structure and function. To better characterize levels of AMF diversity relevant to ecosystem function, deeper sequencing depth in environmental samples is needed. In this study, Illumina barcoded primers and a bioinformatics pipeline were developed and applied to study AMF diversity and community structure in environmental samples. Methods: Libraries of small subunit ribosomal RNA fragment amplicons were amplified from environmental DNA using a single-step PCR reaction with barcoded NS31/AML2 primers. Amplicons were sequenced on an Illumina MiSeq sequencer using version 2, 2 × 250-bp paired-end chemistry, and analyzed using QIIME and RDP Classifier. Results: Sequencing captured 196 to 6416 operational taxonomic units (OTUs; depending on clustering parameters) representing nine AMF genera. Regardless of clustering parameters, ∼20 OTUs dominated AMF communities (78–87% reads) with the remaining reads distributed among other OTUs. Analyses also showed significant biogeographic differences in AMF communities and that community composition could be linked to specific edaphic factors. Discussion: Barcoded NS31/AML2 primers and Illumina MiSeq sequencing provide a powerful approach to address AMF diversity and variations in fungal assemblages across host plants, ecosystems, and responses to environmental drivers including global change.",
"discussion": "DISCUSSION The overarching goal of our study was to determine whether AMF diversity and variations in AMF communities could be adequately captured on the Illumina MiSeq platform, and to determine its potential utility in large-scale surveys of AMF communities. To address this goal, we modified existing 18S primers for barcoding, applied robust protocols with which to undertake 18S amplicon analysis on the Illumina MiSeq platform, and developed bioinformatics pipelines for data processing. Our results clearly demonstrate that the application of barcoded NS31/AML2 primers improves the level of resolution in AMF species identification, diversity, and community composition in complex environmental samples. This primer pair effectively amplified a wide diversity of AMF genera and species, and did not appear to exclude taxa that have been omitted previously due to primer bias (e.g., Archaeosporaceae and Paraglomeraceae; Lee et al., 2008 ). Along with the deeper sequence coverage provide by the Illumina MiSeq, this approach also revealed one of the highest levels of AMF species richness recorded to date ( Öpik et al., 2010 ), ranging from 196 OTUs in the most conservative data set (≥10-ton, 90% threshold) to more than 6000 at the highest levels of OTU clustering similarity and rare OTU inclusivity (all samples, 97% threshold). A large percentage of AMF taxa were also present in extremely low abundance, thereby supporting the capacity of our protocols to capture rare taxa. In contrast, previous studies using 454 pyrosequencing indicated that AMF communities hosted, on average, 70 AMF taxa (e.g., Dumbrell et al., 2010 ), while estimates using morphological methods indicated ∼45 AMF taxa within a community ( Eom et al., 2000 ; Egerton-Warburton et al., 2007 ). We also captured biogeographic differences in AMF communities between the two study sites. Across all data sets, there were consistent and significant differences in OTU richness abundance. For example, La Higuera contained a greater abundance and diversity of Diversispora species and lower levels of Claroideoglomus than El Eden. In addition, the significantly higher pH and levels of NH 4 and P at La Higuera appeared to drive the observed site effect on AMF community composition and structure. These results are in general agreement with spore-based studies of AMF communities in other systems ( Egerton-Warburton et al., 2007 ). Our study was not designed to examine the mechanistic basis of these results. Based on studies elsewhere, however, it is possible that the dry season constrains the AMF community to taxa that are physically or physiologically tolerant of low soil moisture (e.g., Glomus and Diversispora ; Augé, 2001 ) or to high soil P fertility or pH ( Wang et al., 2016 ). Alternatively, these shifts may reflect differences in host plant requirements during the dry season for AMF that increase host drought tolerance (e.g., stomatal control, cytokinin production; Augé, 2001 ) or increase the acquisition of limiting nutrients from carbonate substrates (N, Fe, Zn; Labidi et al., 2012 ). Irrespective, our results indicate a high potential to use our AMF protocol in large-scale sequencing projects to address AMF diversity with sufficient taxonomic precision, to determine the extent to which AMF assemblages vary across host plants and ecosystems, and to resolve AMF species’ responses to edaphic stressors, such as anthropogenic N deposition, in complex environmental samples. Our study also highlighted a number of technical considerations. First, a key finding was OTU inflation, particularly at the 97% clustering threshold, which is the level applied in most mycorrhizal fungal studies. Between the 97% and 90% clustering thresholds, there was a twofold increase in OTU richness at the ≥10-ton level, an 8-fold increase when considering ≥2-ton OTUs, and a 17-fold increase when considering all clustered OTUs ( Table 2 ). In our study, this result was primarily due to the recovery of numerous rare or unique AMF taxa in the most read-rich samples (see Table 4 ; Appendix S1 ), rather than to issues such as uneven number of sequences among samples. To avoid overestimation of AMF community diversity, excluding all OTUs with fewer than 10 constituent sequences (regardless of clustering threshold) will result in levels of taxonomic (OTU) richness consistent with current estimates of AMF species richness ( Öpik et al., 2010 ; Schüßler and Walker, 2010 ). Second, the majority of OTUs could not be assigned to any species-level accession in the Maarj AM database. While this result indicates that novel AMF species likely occur in the Yucatán as they do in other tropical systems ( Chaiyasen et al., 2014 ), it raises questions about our ability to identify AMF species and catalog their diversity in environmental samples. One possibility is that OTU matching was hampered by the limited availability of well-characterized AMF taxa from tropical forests. Alternatively, the clustering of sequences to generate AMF VT ( Öpik et al., 2010 ) may have reduced the potential for OTU matching if sequences of multiple species were lumped into the same OTU ( Bruns and Taylor, 2016 ) or if sequences from a single species were assigned to multiple OTUs ( House et al., 2016 ). Either scenario could mask phylogenetic diversity (and inferences of functionality) or AMF species with large amounts of intraspecific variation. Thus, a more comprehensive reference database of AMF sequences is now needed to improve our capacity to identify AMF to species level and to address within-species sequence variation ( House et al., 2016 ). In particular, more direct assessments that use well-characterized sequences from individual spores or single-spore cultures are needed. Finally, there are limitations to using MiSeq version 2, 2 × 250-bp chemistry with AMF-barcoded samples. Improvements in the MiSeq chemistry with version 3 (2 × 300 bp) is expected to further improve the differentiation of OTUs and the taxonomic resolution of AMF species by allowing consistent assembly of forward and reverse reads into a ∼530-bp sequence fragment. Preliminary analyses of recent 2 × 300-bp sequencing data suggest that OTUs clustered using assembled reads are assigned to species level at approximately two times the rate of OTUs clustered from forward reads alone using similar screening stringency levels (Morgan and Egerton-Warburton, unpublished data). Our results support the continuing development and use of high-throughput sequencing approaches to address the AMF “black box.” As a first step, the tools detailed herein allow the detection of ecologically relevant levels of AMF diversity that shape plant community composition, diversity, and nutrient acquisition in natural and restored communities, including rare and unique species ( Sanders and Rodriguez, 2016 ). While this approach is broadly applicable to most ecosystems, it is especially important in those where major gaps remain in our understanding of AMF species richness and their role in plant community composition and function."
} | 2,155 |
36990448 | PMC10091414 | pmc | 6,427 | {
"abstract": "Spider dragline silk\nis a remarkably tough biomaterial and composed\nprimarily of spidroins MaSp1 and MaSp2. During fiber self-assembly,\nthe spidroin N-terminal domains (NTDs) undergo rapid dimerization\nin response to a pH gradient. However, obtaining a detailed understanding\nof this mechanism has been hampered by a lack of direct evidence regarding\nthe protonation states of key ionic residues. Here, we elucidated\nthe solution structures of MaSp1 and MaSp2 NTDs from Trichonephila clavipes and determined the experimental\np K a values of conserved residues involved in dimerization\nusing NMR. Surprisingly, we found that the Asp40 located on an acidic\ncluster protonates at an unusually high pH (∼6.5–7.1),\nsuggesting the first step in the pH response. Then, protonation of\nGlu119 and Glu79 follows, with p K a s above\ntheir intrinsic values, contributing toward stable dimer formation.\nWe propose that exploiting the atypical p K a values is a strategy to achieve tight spatiotemporal control of\nspider silk self-assembly.",
"conclusion": "Conclusions We propose the mechanism\nof pH-induced NTD dimerization ( Figure 6 b). Naturally, the\ndistribution of acidic and basic residues on NTDs generates the positive\nand negative poles, which allow electrostatic interaction, thereby\nleading to an antiparallel orientation of the NTDs. As the salt concentration\nand pH decrease, dimerization is initiated by the protonation of D40,\nwhich forms an interaction with its basic counterparts (K65 and/or\nR60/K60). This is the initial step that locks the dimer in two positions.\nSubsequently, E79 and E119 are protonated to form a stable dimer since\nboth residues are buried in the hydrophobic interior. Here, we also\ndemonstrate that this molecular mechanism is conserved on MaSp1 and\nMaSp2 NTDs. In this study, we clarify the sequential step of NTD dimerization\nin both MaSp1 and MaSp2 NTDs by showing direct evidence based on experimental\np K a values obtained from individual amino\nacids. This pH-response NTD dimerization is connecting spidroins via intermolecular interactions to form rapid self-assembly\nof hierarchically spider silks and is thereby required for synthesizing\nstrong artificial spider silk. This study also elucidates the molecular\nmechanism and introduces the possibility of making hybrid MaSp1/MaSp2\nspider silks via NTD dimerization, which results\nin artificial spider silks with tunable properties.",
"introduction": "Introduction Spider dragline silk is a natural high-performance\nfiber that is\nwell recognized for its outstanding mechanical properties and biocompatibility.\nDragline silk is a hierarchically structured fiber that is primarily\ncomposed of spider silk proteins (spidroins) MaSp1 and MaSp2. Both\nproteins contain conserved N-terminal and C-terminal domains (NTD\nand CTD) flanked by a long repetitive domain (RD). Despite the predominance\nof repetitive sequences, the self-assembly of soluble spidroins into\nmature silk fibers is controlled via the small globular\nNTD and CTD (which have molecular weights of ∼14 and 10 kDa,\nrespectively). 1 , 2 Inside the ampullate gland, spidroin\nis stored at a high concentration in a storage sac (up to 50% w/v). 3 During the formation of native spider silk, the\nterminal domains are highly responsive to changes in pH and ion composition\nwithin the spinning ducts of the silk glands. 2 , 4 The\nNTD maintains a monomeric structure at higher pH conditions found\nin the silk gland storage sac (∼pH 7), whereas at lower pH\nvalues [as occurs in the spinning duct (∼pH 6–5)], the\nNTD undergoes dimerization with an antiparallel orientation, a response\nalso mediated by the NaCl concentration. 1 , 5 In contrast,\nthe CTD forms a stable dimer at neutral pH and gradually unfolds at\nmore acidic pH values. 2 The repetitive\nregions, on the other hand, maintain a dynamically disordered structure\nin solution, 6 , 7 mainly composed of random coil\nconformations together with small amounts of PPII helices. 8 , 9 The conformation of the isolated RD was not found to depend on the\npH, 9 but the intra- and intermolecular\ninteractions of the RD were affected by the ion composition and concentration. 10 Recently, it was also shown that liquid–liquid\nphase separation in response to anion and pH gradients is essential\nin controlling the self-assembly of spidroins into hierarchical structures. 11 The pH response of the NTD must be tightly\nregulated to ensure\nthe correct timing of the structural changes leading to dimerization\nas the spidroins migrate along the multiple physicochemical gradients.\nPrevious studies have identified an array of conserved anionic and\ncationic residues on the NTD surface that regulate different stages\nof the dimerization process ( Figure 1 ). 12 , 13 Most studies on the structure\nand function of spidroin NTD have been based on MaSp1 from Euprosthenops australis , which contains a globular\nstructure consisting of five α-helices, and a main acidic cluster\nthat includes conserved residues D39, D40 (helix 2), and E79 (helix\n3), which play important roles in dimerization, along with the more\ndistally located E84 and E119 (helix 5). 12 , 14 , 15 There is also a basic cluster that includes\nR60 and K65 on a loop between helices 2 and 3, which, upon antiparallel\ndimerization, are well positioned to form intermolecular interactions\nwith the acidic cluster on the opposite subunit. 1 , 12 Despite\nthe conservation of sequence and overall structure, it is unclear\nwhether the specifics of the dimerization mechanism are the same among\ndifferent species as well as among different spidroin types, as discrepancies\nin the structures and mechanistic contributions of residues have been\nreported. 13 , 15 , 16 To understand\nthe contributions of the different anionic and cationic residues to\nthe NTD dimerization mechanism, tryptophan (Trp) fluorescence spectroscopy,\nin conjunction with mutational analyses, has been used extensively. 12 − 15 In addition, the monomer–dimer equilibrium of the E. australis MaSp1 NTD was investigated using fluorescence\ncross-correlation spectroscopy (FCCS), 12 while the kinetic dimerization on wild-type (WT) and mutant NTDs\nwas studied using steady-state and time-resolved fluorescence experiments. 17 Figure 1 Alignment of MaSp1 (M1) and MaSp2 (M2) NTD amino acid\nsequences.\nTc = Trichonephila clavipes (family:\nAraneidae); Av = Araneus ventricosus (Araneidae) and Argiope argentata (Araneidae); Lh = Latrodectus hesperus (Theridiidae); Ea = E. australis (Pisauridae).\nThe locations of the N-terminal signal sequence (which is omitted\nfrom the constructs), α-helix, and loop elements are indicated\non the top, and the residue numbers are shown on the bottom. The residue\nnumbering for Tc-M2 has been shifted by −1 relative to the\ndeposited structure (PDB ID: 8GS7 ) throughout this paper to facilitate comparisons with\nthe other NTD variant. 12 , 14 Several residues relevant to\nthis study are indicated. While the interpretation based on Trp fluorescence\nspectroscopy\nprovides useful insights, we consider that the results are less straightforward\nfor the NTD dimerization since the technique shows the overall monomer–dimer\nequilibrium of WT and mutant NTDs; in contrast, the pH-dependent dimerization\nof NTD originates from intermolecular interaction between monomers,\nwhich is the result of ionization (protonation or deprotonation) events\nof several key residues, and this event is not well understood. To\ndate, the detailed molecular basis that explains the step-by-step\nmechanism has remained unclear because direct evidence of the relevant\nexperimental p K a values of individual\nkey residues is still missing. Previously, computational methods have\nbeen used to predict the protonation behavior of NTD residues in a\npH gradient. 18 Furthermore, in another\nstudy using a coarse-grained model, the p K a values of individual acidic and basic WTs and mutant NTDs were also\ncalculated. 19 Although computational methods\nfor estimating protein p K a profiles are\nundoubtedly powerful, such approaches sometimes have limitations,\nand in some cases, the predictions may deviate from reality. 20 , 21 Strictly speaking, such theoretical methods are valid only when\nthe three-dimensional protein models (on which the calculations represent\nthe conformational spaces sampled by the protein during its structural\ntransition) are adequate. Furthermore, the theoretical calculations,\nwhich are based on static structures, are further complicated by the\nplasticity and conformational dynamics of the NTD. 14 , 16 , 22 To fabricate artificial spider silk,\nwhich mimics the mechanical\nproperties of native silk, a comprehensive understanding of the spider\nsilk self-assembly mechanism must be obtained, including the universal\ndimerization mechanism of the NTD. Here, we determined the solution\nstructure and dynamics of MaSp1 and MaSp2 NTDs from T. clavipes and investigated the dimer formation\nof both NTDs. To pinpoint the sequential molecular mechanism of NTD\ndimerization, we performed pH titration on the MaSp1 and MaSp2 NTDs\nand followed the chemical shift changes of individual key amino acid\nresidues using NMR spectroscopy, whereby we obtained the relevant\nexperimental p K a value and protonation\nstate of individual amino acid residues. Additionally, we performed\nextensive mutational analysis on the NTDs and dimerization assays\nbased on tryptophan fluorescence, the results of which largely converged\nwith previous findings. 12 , 13 , 15 Altogether, the results of this study provide clear and direct evidence\nof the pH-response dimerization of both MaSp1 and MaSp2 NTDs, leading\nto a better understanding of the mechanism of NTD dimerization, which\nis crucial for spider silk spinning.",
"discussion": "Results and Discussion MaSp1 and MaSp2 NTD Structures from T. clavipes The structures of T. clavipes MaSp1 and MaSp2 NTDs at pH 7.0 were solved\nby solution NMR spectroscopy\n( Figure 2 ; the refinement\nstatistics are summarized in Table S1 ).\nThis is the first report of a MaSp2 NTD structure from any species,\nwhile the MaSp1 NTD model supplements previously published structures\nobtained from different species and under different conditions. 1 , 14 , 15 , 38 The 2D 1 H– 15 N heteronuclear single\nquantum coherence (HSQC) spectra mapping the backbone amide proton\nand amide nitrogen chemical shifts showed some divergence between\nthe MaSp1 and MaSp2 29 data ( Figure 2 a) due to several amino acid\ndifferences on the helix regions ( Figure 1 ), which encouraged to experimentally determine\nthe structure of NTD MaSp2. Figure 2 Determination of the NMR structures of MaSp1\nand MaSp2 monomeric\nNTDs from T. clavipes at pH 7.0. (a) 1 H– 15 N HSQC spectra of MaSp1 NTD (spectral\nresolutions for 1 H and 15 N are 10.1 and 7.1\nHz, respectively) and MaSp2 NTD (spectral resolutions for 1 H and 15 N are 10.1 and 6.0 Hz, respectively) in 10 mM\nphosphate buffer pH 7.0 and 300 mM NaCl. The chemical shift assignments\nfor MaSp1-NTD and MaSp2-NTD were deposited in the BMRB with accession\ncodes 50972 and 50353, respectively. (b,c). Superimposed ensemble\nstructures of MaSp1 NTD (b) and MaSp2 NTD (c) (PDB ID: 7W1O and 8GS7 , respectively).\nLeft, 20 superimposed conformers that are colored according to the\nroot-mean-square deviation (RMSD) of all atoms (green = low; magenta\n= high RMSD). The right portion shows the main acidic cluster region\non the monomer surface, and residue side chains are represented as\nsticks. (d) Superimposed structures of all 20 MaSp1 and 20 MaSp2 NTD\nconformers, showing the Cα positions, with MaSp1 and MaSp2 in\ngreen and blue, respectively. (e) Surface representation of the MaSp2\nNTD core region (residues 8–131) in two views, which is colored\nby the electrostatic surface potential and was calculated using APBS.\nThe relative orientation (rotation) of the structure is 180°\nalong the y -axis. (f) Representative conformer of\nthe MaSp2 NTD is shown in cartoon and surface representations, with\nthe conserved acidic residues depicted as spheres. The two acidic\nclusters are shown, with cluster 1 comprising D36, D39, D40, and E79\nand cluster 2 comprising D17, D46, and D53. (g) Representative conformer\nof the MaSp1 NTD dimer structure from E. australis at pH 5.5 (PDB ID: 2LTH ). The backbone of subunit A is shown in dark blue, and the conserved\nacidic residues are depicted as in (f). The subunit B backbone is\nshown in gray, and the basic residues R60 and K65 that come in close\ncontact with the acidic residues of the subunit in the dimer form\nare indicated. Interestingly, despite some differences\non the fingerprint 2D 1 H– 15 N HSQC spectra\nof both NTD MaSp1 and\nMaSp2, the translation of the backbone chemical shifts into secondary\nstructures of both MaSp1 and MaSp2 demonstrated five helical regions\nconnected by flexible loops ( Figure S1 ).\nThe final models were deposited in the protein data bank (PDB) with\naccession codes 7W1O and 8GS7 for\nMaSp1 NTD and MaSp2 NTD, respectively. The two proteins showed similar\noverall structures due to high sequence similarity (75% sequence identity)\n( Figure 2 b–d),\nas well as similarity to other previously reported NTD structures\n( Figure S2 ), 13 , 14 , 38 which was consistent with a high degree of functional\nconservation across spider taxa and spidroin types. Within the\ncore structure (residues 9–131, excluding the\nflexible loops at either end), the positions of the helices were constant\nfor all conformers, while the highest degree of conformational flexibility\noccurred in two loop regions (between helix 2 and helix 3 and between\nhelix 3 and helix 4). As found in previous studies, a distinct asymmetry\nin the distribution of charged residues was observed in the T. clavipes structures, leading to a pronounced dipole\nmoment within each monomer ( Figure 2 e). The positions of the conserved acidic residues\non the surface of the MaSp2 NTD are shown in Figure 2 f. These acidic residues are grouped mainly\ninto two clusters; acidic cluster 1 contains residue D40 in the center,\nwhich is flanked closely by D39 and D36 on one side and by E79 on\nthe opposite side. Notably, D36 is not present in the well-studied E. australis MaSp1. The E84 residue on the loop between\nhelices 3 and 4 is located close to this cluster in some of the conformers;\nhowever, the varying side chain orientations suggest that E84 does\nnot form an integral part of this cluster. A smaller cluster (acidic\ncluster 2), consisting of D17, D46, and D53, is also apparent. The\npresence of the Asp/Glu residue at position 17 is well conserved among\nMaSp NTDs, while D53 is also conserved (although E.\naustralis MaSp1 has a Gln at the equivalent position)\n( Figure 2 f), and D46\nis found to be conserved within the Araneidae family. Interestingly,\nthe intramolecular “handshake” that was reported between\nD17 and D53 in the intermediate pH 6.5 dimer model of the T. clavipes MaSp1 NTD 38 was not apparent in our pH 7 structures. The conserved E119 residue\non helix 5 is situated on the opposite side of the subunit surface\nand is not part of either acidic cluster. As a comparison, Figure 2 g shows the dimeric\nstructure of the E. australis MaSp1\nNTD elucidated at pH 5.5 (PDB ID: 2LTH ). Residues D40, E79, and E119 are located\nat the dimer interface, and dimerization in the anti-parallel orientation\nshifts cationic groups from the opposite subunit (in this case R60\nand K65) near acidic cluster 1. Notably, for both T.\nclavipes MaSp1 and MaSp2, position 60 is occupied\nby Lys (K60) instead of Arg, and additional basic residues occupy\nnearby positions as well (K54 and R58). Experimental p K a Measurements of\nKey Acidic Residues in WT NTD Using solution-state NMR, we\nmeasured the individual side chain p K a values of conserved acidic residues D40, E79, E84, and E119 for\nboth MaSp1 and MaSp2 WT NTDs (NTD WT ), as these side chains\nare highly conserved and located at the dimer interface. Regarding\nresidue D39, unfortunately, the side chain chemical shift of this\nresidue overlaps with other aspartic acids (D18 and D54) ( Figure S3 ). During the pH titration, we could\nnot observe the chemical shift changes other than D40. Therefore,\nwe consider that D39 does not undergo protonation in the relevant\npH range. Consistently, Cβ chemical shift of residue D39 (BMRB\nID: 18480) of the previously reported E. australis NTD dimer structure (PDB ID 2LTH ) is 41.828 ppm, 12 which is a typical chemical shift of deprotonated aspartic acid. 37 The titration experiments were performed\nover a pH range of 9–5 (with intervals of 0.25 to 0.35 pH units)\nin the absence and presence of 300 mM NaCl, and the resulting chemical\nshift data were fitted against the Henderson–Hasselbalch equation\n(see the experimental section in Supporting Information ). The chemical shifts of 13 Cγ and 13 Cδ were used as reporters for the p K a values of Asp and Glu, respectively. To avoid overlapping signals,\nwe performed 2D NMR experiments that correlated Cγ–Hβ\nresonances for Asp and Cδ−Hγ resonances for Glu 24 , 39 to follow the chemical shift changes as a function of pH ( Figure S3 ). Figure 3 a shows the data and the curve-fitting results,\nand the individual side chain p K a values\nthat were estimated are shown in Table 1 , in which similar trends were observed for MaSp2 and\nMaSp1. The p K a of D40 NTD MaSp1 in the\nabsence of NaCl could not be determined since the D40 side chain chemical\nshift experienced severe exchange broadening due to monomer–dimer\nequilibrium and caused less data points, particularly for the fitting\non the transition region. On the other hand, the side chain chemical\nshifts (Cδ−Hγ) of E79 and E119 are in full protonated\nform, could not be obtained due to the exchange broadening at acidic\npH ( Figure S4 ) and sample instability at\npH less than 5. In this study, upon pH titration, the chemical shift\nchanges (Δδ) of those glutamates were more than 0.7 ppm.\nConsidering, the chemical shift difference in the well-defined baseline\nof the deprotonation region is not more than 0.1 ppm, therefore, the\nchemical shift differences for more than 0.6 ppm in the transition\nregion are sufficient for the fitting and estimate the p K a values with assumption of Δδ as described\npreviously. 37 Figure 3 Experimental p K a values of key MaSp1\nand MaSp2 NTD residues obtained by NMR spectroscopy. (a) Titrations\nwere performed in a pH range of 9–4 and under two different\nsalt conditions (0 and 300 mM NaCl). Curve fitting to the chemical\nshift data was performed using the Henderson–Hasselbalch equation.\nThe conserved residues D40, E79, E84, and E119 were probed, and the\nnonnative residue H2 (remains at the N-terminus after cleavage from\ntag purification) was used as a control. The vertical dotted lines\nshow the intrinsic p K a of the side chains\nof Asp (3.86, red) and Glu (4.34, gray). 37 (b) Comparison of the experimentally measured p K a values with the intrinsic p K a values. Unusually elevated p K a values\nwere detected for D40, E79, and E119 compared to the intrinsic values.\nIn some cases, proper curve fitting was prevented because the line\nof experimental data was excessively broadened in the low pH range,\nwhich is indicated as ND. The data are arranged according to residue\ntype. Statistically significant differences relative to the intrinsic\np K a value of each residue type are indicated\nby asterisks (**** P < 0.0001, *** P < 0.001, ** P < 0.01, * P <\n0.05; ns = not significant). Table 1 Summary of the Estimated Individual\np K a Values of the Conserved Acidic NTD\nMaSp1 and NTD MaSp2 from T. clavipes by NMR Spectroscopy a MaSp1\nNTD WT MaSp2\nNTD WT 0 NaCl 300 mM NaCl 0 NaCl 300 mM NaCl residue p K a est n Hill p K a est n Hill p K a est n Hill p K a est n Hill D40 N.D N.D 6.4 ± 0.1 2.2 ± 0.2 7.1 ± 0.1 1.5 ± 0.2 6.3 ± 0.1 6.1 ± 0.3 E79 6.6 ± 0.1 1.2 ± 0.1 6.0 ± 0.1 1.5 ± 0.1 6.8 ± 0.1 1.0 ± 0.1 5.7 ± 0.1 1.3 ± 0.1 E84 4.4 ± 0.1 0.7 ± 0.1 4.6 ± 0.1 0.9 ± 0.1 4.4 ± 0.1 0.6 ± 0.1 4.5 ± 0.1 0.7 ± 0.1 E119 6.3 ± 0.1 1.3 ± 0.1 6.1 ± 0.1 3.4 ± 0.4 6.2 ± 0.1 1.0 ± 0.1 5.5 ± 0.1 1.2 ± 0.1 H2 6.4 ± 0.1 0.8 ± 0.1 6.5 ± 0.1 0.9 ± 0.1 6.5 ± 0.1 1.0 ± 0.1 6.5 ± 0.1 1.1 ± 0.1 a ND means not determined\ndue to line\nbroadening of NMR signals. Strikingly, residues D40, E79, and E119 displayed\np K a values that were considerably higher\nthan the intrinsic\np K a values (reported as 3.86 and 4.34\nfor Asp and Glu, respectively) ( Figure 3 b). 37 The deviation was\nmost prominent in D40, in which the experimentally measured p K a value was approximately 2.5–3 pH units\nhigher than the intrinsic value (6.4 ± 0.1 for MaSp1 at 300 mM\nNaCl, 6.3 ± 0.1 for MaSp2 at 300 mM NaCl, and 7.1 ± 0.1\nfor MaSp2 at 0 NaCl). This result contrasts with a previous computational\nstudy, in which the p K a of D40 was estimated\nto be approximately 4.1, that is, very close to the intrinsic value\nfor Asp. 18 For E79 and E119, on the other\nhand, the measured p K a values were approximately\n1–2 pH units higher than the intrinsic values, while for E84,\nlocated on the solvent-exposed helix 2–3 loop, the observed\np K a ’ s (4.4–4.6)\nwere very close to the intrinsic values. In agreement with our data,\nthe previously reported Cβ chemical shift of D40 and Cγ\nchemical shifts of E79 and E119 of the E. australis NTD MaSp1 dimer at pH 5.5 are 39.312, 33.080, and 33.423 ppm (BMRB\nID: 18480), 12 respectively, which are typical\nside chain chemical shifts of protonated aspartate and glutamate,\nas mentioned previously. 37 Furthermore,\nwe found that the 15 Nζ K65, K60,\nand 15 Nε R58 chemical shifts of the MaSp2 NTD at\nneutral pH were approximately 33.5, 32.8, and 85.2 ppm, respectively\n( Figure S3 ), which are typical chemical\nshifts for protonated Lys and Arg, respectively. 40 However, the side chain Lys and Arg signals of the MaSp2\nNTD at acidic pH could not be observed because the signals were broadened\nbeyond detection. On the other hand, in the final NTD dimer structure\n( Figure 2 g), the K65\nside chain was in proximity to D40 (the D40–K65 distance is\nless than 4 Å), 12 suggesting that\na salt-bridge interaction formed between the two residues. Probing\npH-Induced Dimerization via Tryptophan\nFluorescence Spectroscopy We carried out Trp fluorescence\nspectroscopy 1 , 14 on NTD WT and on a\nwide range of mutants to monitor the overall monomer-to-dimer equilibrium\nas a function of pH. The present data covered a wider array of residues\nthan that of previous studies 12 , 13 , 15 and under three different salt conditions. Figure 4 a shows the spectra of a subset of MaSp2\nand MaSp1 variants at 300 mM, while the entire set of curves is presented\nin Figure S5 . We calculated the pH transition\nmidpoint (p K , or equilibrium point between monomer\nand dimer forms) by fitting a curve against the ratio of the fluorescence\nemission intensity ratio at 339 versus 351 nm (excitation\n= 280 nm). For WT MaSp1 and MaSp2, we observed a consistent trend\nof decreasing p K with increasing NaCl concentration\n(p K of 6.48 ± 0.03, 6.06 ± 0.02, and 5.82\n± 0.01 for MaSp1 NTD at 7.5, 150, and 300 mM NaCl, respectively,\nand 6.37 ± 0.01, 5.98 ± 0.02, 5.78 ± 0.03 for MaSp2\nNTD at 20, 150, and 300 mM NaCl, respectively), indicating the stabilization\nof the monomer form of NTD with increasing concentrations of NaCl,\nwhich is in line with previous findings. 12 , 41 Figure 4 Investigation\nof pH-dependent dimerization of NTD via tryptophan\nfluorescence spectroscopy. (a) pH response of MaSp1 NTD\nand MaSp2 NTD as determined by changes in the fluorescence emission\nintensity ratios between 339 and 351 nm. The measured inflection points\n(p K ) are provided (±SE), corresponding to the\nmidpoint of the sigmoidal response curves. The results are shown for\nthe WT NTD as well as for key variants (D39N, D40N, K65A, E79Q, E84Q,\nand E119Q) against a background of 150 mM NaCl. The full set of mutants\nand NaCl conditions tested are presented in Figure S4b . (b) Change in the pH-dependent dimerization midpoints\nfor the MaSp2 NTD mutants relative to WT at three NaCl concentrations\n(20, 150, and 300 mM NaCl). The data are plotted as Δp K on the y -axis and are calculated as p K WT – p K mut . For A72R and the D39N/D40N/E79Q/E119Q quadruple mutant, the values\nof some inflection points (and hence of Δp K ) could not be precisely determined, although minimum values can\nbe estimated from the data and are indicated by arrows ( e.g. , for A72R at 150 mM NaCl, the inflection is at least 1 pH unit lower\nthan that for the WT). Statistically significant differences relative\nto the respective WT value at each concentration are indicated by\nasterisks (**** P < 0.0001, *** P < 0.001, ** P < 0.01, * P <\n0.05). In the mutational analysis, for\nthe acidic residues, the carboxylate\nside chains were substituted with their non-titratable amide counterparts\n(Asp to Asn; Glu to Gln), whereas the basic residues (Arg or Lys)\nwere replaced with uncharged Ala. We also analyzed the A72R mutant,\nwhich prevents dimer formation through steric and repulsive effects\nat the interface. 14 Figure 4 b presents the MaSp2 NTD variants in terms\nof Δp K , which is a shift in the pH inflection\npoint introduced by each mutation relative to the WT value. Among\nthe acidic point mutations, D40N produced the largest change in the\npH midpoint value, shifting the p K value by approximately\n−0.4 relative to that of the WT. As shown by the experimental\np K a , D40 undergoes the earliest protonation\nevent in the acidic gradient among the different residues probed ( Figure 3 ). Our results suggest\nthat D40 plays a crucial role in maintaining the NTD dimerization\nresponse at the relatively high pH levels found in the silk spinning\nducts. Interestingly, E79Q produced an overall decrease in the pH\ntransition point (Δp K of approximately −0.24\nand −0.1 for MaSp1 and MaSp2, respectively, at 300 mM NaCl\nconditions), whereas the same mutation was reported to increase the\nmidpoint p K of E. australis MaSp1, 12 which might reflect differences\nin the functions of NTD residues between spider species. Mutations\nat the other sites on the acidic cluster (D36N, D39N, and E84Q) exhibited\nonly mild or negligible effects on the dimerization curves. Mutagenesis\nat the second acidic patch (D17N, D46N, and D53N) caused only slight\nperturbations in the pH response. On the other hand, the E119Q mutation\nproduced a marked positive effect on Δp K (at\nleast +0.3 relative to WT at 300 mM NaCl), consistent with previous\nfindings. 12 E119 is located away from the\nacidic clusters, and upon dimerization, the side chain inserts into\na hydrophobic pocket on its binding partner, which could explain the\nΔp K effects. Among single-point mutations of\nthe basic residues, K60A yielded the most prominent results, with\na Δp K perturbation of approximately −0.2.\nNotably, our K65A results diverged somewhat from those of previous\nreports; we observed a slight increase in Δp K (approximately +0.1), whereas other studies showed a decrease in\nΔp K. 12 , 16 Interestingly, among\nthe single point mutations, A72R produced an almost flat line for\nthe MaSp2 data from pH 7 to 5, suggesting that this variant stabilized\nthe monomer conformation. Below pH 5, a gradual fluorescence shift\nthat was consistent with dimerization was observed, although insufficient\ndata points in the lower pH range were obtained, thus precluding a\nprecise calculation of p K values. A number of double,\ntriple, and quadruple point mutations were also investigated (K60A/K65A,\nD39N/E79Q, D40N/E79Q/E119Q, and D39N/D40N/E79Q/E119Q), which produced\nsignificant downward shifts in Δp K , although\nnone of these mutations produced a perturbation greater than that\nof D40N alone (with the exception of the quadruple mutant), highlighting\nthe important role of D40 in governing the pH-dependent dimerization\nmechanism. Chemical Shift Perturbation and p K a Determination of Selected Mutants of the MaSp2\nNTD by NMR Spectroscopy Since the effects of mutations on\nthe structure and residue-specific\np K a ’ s of key amino acids\nremained unclear, we measured the chemical shift perturbation (CSP)\nat pH 7.0 ( Figure 5 a). We selected five mutants that targeted conserved sites on the\nMaSp2 NTD (D39N, D40N, K65A, A72R, and E119Q) and investigated the\neffect of those mutants on the structures. The CSPs of E119Q and D39N\nwere found to localize around the mutated amino acids, suggesting\nthat no large structural changes or long-range effects were induced\nby these mutations. Interestingly, the CSP in D40N was localized not\nonly around residue 40 but also around residues 59–62, which\nare located in the loop between helices 2 and 3 (and containing conserved\nbasic residues K60), indicating that the D40 side chain is close to\nK60. Figure 5 Probing effects of key NTD mutations on the conformation and p K a of conserved acidic residues using NMR spectroscopy.\n(a) CSPs induced by specific mutations (at pH 7 in the presence of\n300 mM NaCl). Combined chemical shift differences were calculated\nusing eq 4 (see the Supporting Information ). For the D39N and E119Q\nmutants, CSPs (>0.1 ppm) were localized around the mutated residues.\nFor D40N, perturbations were observed around the mutated site, although\nthe highest CSP values were found in the region around residues 59–62.\nFor the K65A and A72R mutants, the effects were distributed across\na wide range of residues that include the mutated site (although in\nthe latter case, data could not be measured at position 72). (b,c)\nExperimental p K a measurement for the conserved\nacidic residues of selected NTD mutants. (b) Experimental p K a values for D40, E79, E84, E119, and H2 were\nmeasured for the MaSp2 NTD variants D39N, D40N, K65A, A72R, and E119Q\nat 300 mM NaCl. Chemical shift measurements against pH titration were\ncarried out, and curve fitting was performed according to the Henderson–Hasselbalch\nequation. (c) Effect of MaSp2 NTD mutations on the experimental p K a values of key acidic residues, which are expressed\nin terms of Δp K a , i.e. , the difference between measured and intrinsic p K a values (p K a exp – p K a int ). For the\nMaSp2 NTD WT residues with deviant p K a values relative to the intrinsic values (notably D40,\nE79, and E119), the introduction of a mutation at a different site\nled to varying degrees of either reversion toward the intrinsic p K a value (seen most prominently in D40 with the\nA72R variant) or a more pronounced deviation from the intrinsic value\n( e.g. , in E79 with the E119Q variant). As a control,\nH2 p K a values are close to the intrinsic\np K a value of His in all variants. Statistically\nsignificant differences relative to the respective WT Δp K a value are indicated (**** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05). Since D40 is not proximal to residues 60 in the\nmonomer 3D structure,\nthe unusual CSP result suggests that the intermolecular contact between\ntwo residues (weak dimer formation) occurs even at pH 7 and in the\npresent of 300 mM NaCl. Conversely, variant K65A was found to affect\na broad range of residues (from residue 44 to 65), although the effect\nis subtle, implying that the K65A mutant causes minor structural rearrangements\nthat are distributed across a wide span of residues. Similarly, the\nA72R mutant caused wider effects, localized around helix 3, where\nresidue 72 is found, as well as at residues 44 and 48; however, the\nlow CSP values suggest only minor structural perturbations occurred. To understand the effect of site-directed mutagenesis on the p K a of key residues, we also measured the experimental\np K a ’ s of ionizable residues\nagainst point mutation backgrounds ( Figure 5 b,c). A previous study showed that neighboring\nacidic residues, which act as clusters, might cause elevated p K a values. 42 For D40,\nwhich exhibits an unusually high p K a in\nWT NTD (∼6.3 for MaSp2 at 300 mM NaCl), we found that the D39N\nmutation at the adjacent site led to a lower p K a (∼5.9) for D40, although the value was still considerably\nhigher than the intrinsic p K a . In contrast,\nthe A72R mutation caused a drastic reduction in the D40 p K a to ∼4.4, close to the intrinsic p K a of Asp (3.86). Contrary to the D40N variant, Trp fluorescence\ndata showed the highest p K value of E119Q compared\nto those of other mutants, which indicates that this variant stabilized\nthe dimer conformation ( Figure S5 ). NMR\ndata showed that the p K a of D40 of the\nMaSp2 E119Q variant NTD is similar to the side chain p K a of the WT D40 MaSp1 NTD, but the side chain p K a of E79 of the E119Q variant is higher than\nthat of the WT. This phenomenon occurs since E119 is surrounded by\nhydrophobic residues and buried in the hydrophobic interior; therefore,\nthe mutation of Glu to Gln at position 119 is more favorable for the\ndimer interaction. Origin of the Elevated p K a Values\nof D40, E79, and E119 of NTD MaSp1 and MaSp2 The A72R mutation,\nwhich prevents dimer interaction, cause drastic reduction on the p K a value of D40 to ∼4.4 (close to the\nintrinsic p K a value of Asp), implies that\nthe unusually high p K a value of D40 around\nneutral pH is originated from the salt bridge interaction between\noxygen from the carboxyl group of residue D40 with proton from the\ncounterpart lysine or arginine that is close in the space (K60/R60/K65).\nA similar phenomenon also occurred in the previously reported study\nin BPTI protein, where the salt bridge caused the p K a at a rather basic pH. 43 Interestingly,\nwe also found weak dimer interaction between D40 and K60 which exists,\neven at pH 7 in the presence of 300 mM NaCl, as shown by CSP ( Figure 5 ). This phenomenon\nalso explains the drastic reduction on p K value of\nD40N since the conversion of the carboxyl group in aspartate to the\ncarbonyl group in asparagine hinders the salt-bridge interaction and\nstabilizes the monomer conformation ( Figure 4 ). Even though the salt-bridge interaction\ncould not be observed in the T. clavipes NTD dimer structure due to the lack of the dimer structure; however,\nthis finding is also supported by the fact that the E. australis NTD MaSp1 dimer structure distance between\nthe side chain oxygen of D40 and the side chain proton (Hζ)\nK65 of the E. australis NTD MaSp1 dimer\n(PDB ID: 2LTH ) is 3.2 Å ( Figure S6 ), which is\nqualified as salt bridge interaction for the distance less than 4\nÅ. 44 Once D40 is protonated\nand a salt bridge is formed with counterpart\nbasic residues, the E79 and E119 residues will be buried at relatively\nhydrophobic interior environment. The subsequent protonation of those\nresidues at elevated pH is required to stabilize the dimer interaction. Our experimental study provides a direct glimpse into the protonation\nstates of key residues in the MaSp1 and MaSp2 NTDs as a function of\npH, which is essential to properly understand the mechanism behind\nthe pH relay system during spider dragline silk self-assembly. Since\nthe p K a values of those conserved acidic\nresidues are relevant to the pH gradient of the spider gland, 4 this finding suggests that D40, E79, and E119\nparticipate in the NTD dimerization mechanism. This study directly\npinpointed the unusual p K a value of D40\n(∼6.5–7.1), suggesting that protonation of D40 is important\nin the initial step to lock the dimer and the protonation of two glutamic\nacid residues, E79 and E119, which have estimated p K a values of ∼5.4–6.2, are required to stabilize\nthe NTD dimer. In contrast to previous studies, our NMR data showed\nthat the p K a value of E84 was ∼4.4–4.6\n( Table 1 , Figure 5 c), which is close\nto the intrinsic p K a value of glutamic\nacid and is not affected by the salt concentration, suggesting that\nE84 is a solvent-exposed residue. This finding is also in line with\nthe solvent exposed of E84 residue of previously reported NMR structure\nof a homologous monomer NTD MaSp1 (PDB ID: 2LPJ ). 14 Interestingly, some striking discrepancies were observed between\nthe experimental measurements and the computational predictions ( Table S2 ). Continuous constant pH molecular dynamics\n(CpHMD) 18 and PROPKA3 45 predicted only small perturbations in the p K a of D40 (within −0.7 to +1.4 pH units relative\nto the intrinsic value) when applied to known NTD dimer structures,\nin contrast to the large shift measured by the NMR titration experiments\n(within +2.4 to +2.6 pH units relative to the intrinsic value). On\nthe other hand, the two computational methods reported positive deviation\np K a values for E79 and E119 relative to\nthe intrinsic p K a value of glutamic acid,\nconsistent with the experimental findings. By integrating the\nresults of the experimental p K a measurements\nand the dimerization assays, we show that\nD40 protonation precedes the midpoint of the fluorescence curves that\nreport on the conformational changes accompanying dimerization in\nresponse to the decreasing pH gradient ( Figure 6 a), therefore suggesting that the protonation\nof D40 represents an initial step in the pH response mechanism. On\nthe other hand, the protonation of E79 and E119, while within the\npH range found in the spinning ducts, is shown to occur after the\nfluorescence inflection point, suggesting that these residues perform\nstabilizing functions toward the formation of the final dimer structures. Figure 6 Proposed\nstep-by-step dimerization mechanism of spidroin NTD. (a)\nSequence of protonation events during MaSp NTD dimerization relative\nto the pH gradient. The x -axis corresponds to pH\n(neutral to acidic from left to right), along which the measured p K a values (under 300 mM NaCl conditions) of key\nconserved acidic residues D40, E79, E84, and E119 are indicated for\nthe MaSp2 NTD variant. Additionally, for each variant, the pH inflection\npoint corresponding to the overall dimerization event as monitored\nby tryptophan fluorescence spectroscopy is indicated with black circles.\nProtonation of D40 along the pH gradient precedes the dimerization\nevent and is likely crucial for the initial step, which locks the\ndimer in two positions. The protonation of E79 and E119 occurs downstream\nof the Trp-associated conformational change, suggesting that these\nresidues stabilize the formation of the dimer structures. E84 undergoes\nprotonation below pH 5 and thus a change in its ionization state is\nunlikely to play a role in the dimerization mechanism. (b) Distribution\nof acidic and basic residues on NTD produces positive and negative\npoles, which cause the NTD to be oriented in an antiparallel manner.\nAs the NaCl concentration and pH decrease, dimerization will be initiated\nby protonation of D40, which possibly forms salt bridge interactions\nwith K65 and/or R60/K60. This process is subsequently followed by\nthe protonation of E79 and E119. The protonation of D40 at the beginning of the\npH gradient seems\nto be necessary for maintaining the relatively high midpoint for dimerization\nof the NTD, whereas in the D40N mutant, the fluorescence assay inflection\npoint shifts drastically lower, to approximately pH 5.5, suggesting\nthat D40N stabilizes the monomer conformation of the NTD. This effect\nof the D40 mutation is consistent with previous reports, 12 , 13 , 17 although the implications have\nbeen relatively elusive in previous models. As previously mentioned,\nthe A72R mutation caused the NTD to stay in monomeric form from pH\n7 to 5; 14 thus, the fact that the A72R\nmutant decreased the p K a of D40 drastically\nto a p K a of D40 ∼ 4.4 highlights\nthe unusually high p K a value of D40 in\nthe WT MaSp2 NTD, originating from the interaction between D40 and\nits counterpart in the dimeric form of the NTD rather than from its\nneighboring acidic residues. This interpretation is also supported\nby the CSP results, where long-range perturbations were observed in\nthe D40N mutant relative to the WT. However, we also found that the\nK65A mutation does not alter the p K a of\nD40, which suggests that D40 might interact with multiple residues\n(such as K60 and possibly also with R58 in the case for T. clavipes MaSp1/MaSp2). In a similar manner, for\nresidue E79, the A72R mutation leads to a large decrease in the p K a (∼5.6 in the WT) to ∼4.2, close\nto the intrinsic value for Glu side chains, and likely reflects the\neffects of intermolecular contacts. Intriguingly, considering\nthe important role of D40, E79, and E119,\nmutations of those residues (D40N–E79Q–E119Q) do not\nabolish dimer formation at pH 5, as shown by Trp fluorescence spectroscopy\ndata ( Figure S5 ). The mutations of D40N\nand E119Q have opposite effects. D40N stabilized the monomeric form,\nwhile E119Q stabilized the dimeric form. Comparison of the 2D 1 H– 15 N HSQC spectra of WT, D40N, E119Q, and\nD40N–E79Q–E119Q at pH 7, 6, and 5 demonstrates that\nthe 2D spectrum of triple mutants D40N–E79Q–E119Q at\npH 6 is still quite similar with 2D spectrum of D40N, suggesting that\nthe resultant effect of the triple mutant is to stabilize the monomeric\nform at pH 6 ( Figure S7 ). However, at pH\n5, this triple mutant experiences exchange broadening due to monomer–dimer\nequilibrium. Furthermore, when quadruple mutations are introduced\non cluster acidic residues (D40N–D39N–E79Q–E119Q),\na greater effect on stabilizing monomer conformation is observed ( Figure S5 ). This result implies that the ability\nof the NTD to form an antiparallel dimer is not limited to one key\nresidue, but the entire acid and basic clusters function to generate\npositive and negative poles, which cause electrostatic interactions\nand lead to the formation of an antiparallel dimer of the NTD. This\nfinding is also supported by the observation of accelerated association\nof NTD that is insensitive to charge screening in case of a dipole–dipole\ninteraction. 17 A decrease in the salt concentration,\nwhich is relevant to the condition in the spinning duct of the spider\ngland, 46 can be interpreted as increasing\nthe probability of the monomers encountering the correct orientation,\nthereby increasing the rate at which the monomer transforms into the\ndimer."
} | 10,515 |
40313389 | PMC12045287 | pmc | 6,428 | {
"abstract": "Background Bacterial laccases play a crucial role in the degradation of lignin and the turnover of soil organic matter. Their advantageous properties make them highly suitable for a wide range of industrial applications. However, the limited identification of these potential enzymes has impeded their full utilization. The straw-amended soil provides materials for the development of bacterial laccases. Methods Metagenomic sequencing of a straw-amended soil was conducted to explore novel bacterial laccases. The putative bacterial laccases were then screened using profile hidden Markov models for further analysis. The most abundant gene, lacS1 , was heterologously expressed in Escherichia coli and the recombinant laccase was purified for enzymatic characterization. Results A total of 322 putative bacterial laccases were identified in the straw-amended soil. Among them, 45 sequences had less than 30% identity to any entries in the Carbohydrate-Active Enzyme database and only 4.66% were more than 75% similar to proteins in the NCBI environmental database, exhibiting their novelty. These enzymes were found across various bacterial orders, demonstrating substantial diversity. Phylogenetic analysis revealed a number of the bacterial laccase sequences clustered with homologs characterized by favorable enzymatic properties. Five full-length representative bacterial laccase genes were obtained by modified thermal asymmetric interlaced PCR. The laccase activity of lacS1 was validated. It was a mesophilic enzyme with alkaline stability and halotolerance, indicating its promise for industrial applications. Implications These findings highlight novel bacterial laccase resources with potential for industrial applications and enzyme engineering.",
"conclusion": "Conclusions In this study, we investigated bacterial laccases in a straw-amended soil using metagenomic sequencing and a pHMM-based search. The identified bacterial laccases were predominantly distributed across the Proteobacteria, Actinobacteria, and Gemmatimonadetes phyla, indicating their capacity for laccase production to synergistically act on the decomposition of lignin. These laccases exhibited substantial novelty and diversity, with many likely possessing promising enzymatic properties. The characterization of recombinant lacS1 revealed good pH stability and chloride tolerance, indicating its potential for application in wastewater treatment. This study provides valuable materials for the development and exploitation of bacterial laccase resources.",
"introduction": "Introduction Plant biomass constitutes the primary source of soil organic matter, playing a crucial role in carbon and nutrient cycling as it decomposes ( Prescott, 2005 ; Poll et al., 2008 ). This decomposition process is predominantly driven by soil microorganisms, which break down plant litter—composed of both labile and recalcitrant compounds ( Paterson et al., 2008 ). Among these compounds, recalcitrant substrates such as aromatic polymers are particularly resistant to microbial degradation. Specific microbial groups thrive only when simpler substrates are made available by other groups that specialize in the degradation of complex compounds, like lignin ( McGuire & Treseder, 2010 ) As the second most abundant component of plant residues, lignin serves as a major source of these recalcitrant natural polymers ( Wong, 2009 ; Theuerl & Buscot, 2010 ). Microorganisms produce several intra- and extracellular enzymes to catalyze the breakdown of these plant polymers, including lignin peroxidases, manganese peroxidases, versatile peroxidases, and laccases ( Pollegioni, Tonin & Rosini, 2015 ). Among these enzymes, laccases are noted for forming the most aromatic-aromatic interactions and appear particularly effective in lignin deconstruction ( De Angelis et al., 2011 ; Chen et al., 2011 ). Laccases (benzendiol: oxygen oxidoreductase, EC 1.10.3.2), members of the multicopper oxidase (MCO) superfamily, are capable of oxidizing a wide array of phenolic compounds and certain non-phenolic substrates, often through the mediation of small molecules ( Zhai et al., 2024 ). During this oxidation process, molecular oxygen serves as the electron acceptor and is reduced to water as the sole by-product. Due to these unique characteristics, laccases hold significant promise as environmentally friendly biocatalysts for various industrial applications, including textile dye decolorization, effluent detoxification, and paper and pulp production ( Sodhi, Bhatia & Batra, 2024 ). Initially discovered in the sap of the Japanese lacquer tree ( Rhus vernicifera ), laccases have since been found to be widely distributed among fungi ( Yoshida, 1883 ; Thurston, 1994 ). To date, most identified and characterized laccases originate from fungal sources, and fungal laccases are extensively used in industrial processes due to their relatively high activity. Recently, however, increasing evidence has shown that laccases are also prevalent in bacteria ( Alexandre & Zhulin, 2000 ; Claus, 2003 ). Compared to their fungal counterparts, bacterial laccases exhibit several advantageous properties, such as high thermal stability, chloride tolerance, and broad pH stability, which are required in harsh industrial environments ( Liu et al., 2017 ; Sharma & Leung, 2021 ; Liu et al., 2023 ). Therefore, they were more suitable for industrial applications. Despite their potential, only a limited number of bacterial laccases have been thoroughly characterized, underscoring the need for further investigation into their properties for novel biotechnological applications. In the context of sustainable agriculture, the incorporation of straw as an amendment is widely regarded as an effective and economical agricultural practice. Long-term straw amendment promotes the sequestration of soil organic carbon, thereby contributing to soil quality maintenance ( Liu et al., 2014 ). Straw is primarily decomposed by soil microorganisms, and its incorporation into the soil can influence microbial community composition. Our previous research demonstrated that the soil bacterial communities were altered after six years of continuous straw return treatment ( Yu et al., 2018 ). Given that the straw-amended soil serves as a unique habitat for lignocellulosic-degrading microbial consortia, it is likely to be rich in bacterial laccase resources. In previous studies, bacterial laccase genes have primarily been identified using pure culture techniques and metagenomic library approaches ( Zhang et al., 2022 ; Fang et al., 2012 ). However, these methods are often time-consuming and inherently biased, limiting their effectiveness for making a general survey of the capacity of bacterial laccase genes. High-throughput sequencing offers a novel and unbiased approach to detect genes in environmental samples and can also assess their abundance and diversity. In this study, we comprehensively investigated the straw-amended soil through metagenomic sequencing to uncover novel laccases with potential for biotechnological applications.",
"discussion": "Discussion In this study, we utilized metagenomic sequencing to explore bacterial laccases in the straw-amended soil. Metagenomics is a powerful approach for discovering novel biocatalysts that are often inaccessible through traditional pure culture methods. By constructing metagenomic libraries and employing sequence- or activity-based screening, numerous bacterial laccases with promising industrial applications have been identified in previous studies ( Ye et al., 2010 ; Ausec et al., 2017 ; Fang et al., 2011 ; Fang et al., 2012 ; Yang et al., 2018 ). However, metagenomic library construction and screening can be labor-intensive and typically yield limited information ( Robinson, Piel & Sunagawa, 2021 ). In contrast, high-throughput sequencing without the need for cloning allows for a more comprehensive analysis of microbial community structure and function, enabling a more thorough investigation of bacterial laccases in environmental samples, such as straw-amended soil. We first analyzed the microbial communities in the straw-amended soil. The results indicated that the dominant bacterial phyla were consistent with those identified in our previous study using 16S rRNA gene amplicon sequencing ( Yu et al., 2018 ). The only notable differences were the detection of Thaumarchaeota and Cyanobacteria as dominant phyla in this study. These discrepancies may arise from the limitations of 16S rRNA gene amplicon sequencing, which is influenced by the variability of 16S rRNA gene sequences and copy numbers in bacterial genomes ( Větrovský & Baldrian, 2013 ). Additionally, the primer pair used in the previous study (original 515F and 806R) has been shown to exhibit a bias against Thaumarchaeota ( Hugerth et al., 2014 ). Based on the metagenomic sequencing data, bacterial laccases were identified using laccase-specific pHMMs, rather than simple BLAST searches, to improve accuracy ( Ausec et al., 2011 ). Laccases belong to the MCO superfamily and are difficult to discriminate from other MCOs. Furthermore, many of the ORFs in our dataset were incomplete, necessitating the use of pHMMs for a more accurate identification. Although metagenomic sequencing and assembly have inherent error rates, PCR amplification and Sanger sequencing validated the authenticity of most of the bacterial laccase genes identified in our dataset. The phylogenetic distribution of laccase-coding genes at the phylum level closely mirrored the overall microbial community structure in the straw-amended soil. This supports the idea that laccase gene composition may be linked to factors that structure microbial communities or that there is a correlation between the functional traits of microbial consortia and their taxonomic profiles ( Zhou et al., 2017 ; Lauber, Sinsabaugh & Zak, 2009 ; Wang et al., 2016 ). At the order level, Gemmatimonadales, which contained the largest number of laccase-coding genes, is part of the Gemmatimonadetes phylum and is believed to play a role in phosphorus removal ( Wang et al., 2009 ). Although Gemmatimonadales has few cultivable representatives, studies suggest that it possesses high sulfur and nitrogen metabolic diversity ( Rasigraf et al., 2020 ). Furthermore, it has been found to be more abundant in soils polluted with aromatic compounds, indicating its potential role in aromatic compound degradation ( Cecotti et al., 2018 ; Thelusmond et al., 2018 ). Therefore, Gemmatimonadales may play a central role in SOM and nutrient cycling, contributing to improved soil fertility and plant growth. In addition to Gemmatimonadales, orders such as Geodermatophilales, Micrococcales, Jiangellales and Rubrobacterales of the Actinobacteria phylum, and Nitrososphaerales of the Thaumarchaeota phylum were also represented. Actinobacteria are usually found as a major component of bacterial consortia with high lignolytic activity ( Wang et al., 2013 ; Moraes et al., 2018 ). Thaumarchaeota include all known ammonia-oxidizing archaea and multiple laccases have been identified in Nitrososphaerales genomes ( Kerou et al., 2016 ). Moreover, several orders within the Proteobacteria phylum, including Burkholderiales, Rhodospirillales, Xanthomonadales, Rhizobiales, Myxococcales, Hydrogenophilales, Pseudomonadales, and Desulfuromonadales, were also observed as major laccase-producers in the straw-amended soil. This is in line with a recent study conducted in coniferous forest soils from across North America, which identified many of these taxa as among the top 10 richest laccase-coding gene-harboring bacteria ( Wilhelm et al., 2019 ). The laccase profile reveals that the predominant Proteobacteria cooperate with Actinobacteria and Gemmatimonadales to synergistically act on the decomposition of lignin. A phylogenetic tree was constructed using the bacterial laccase fragments containing the conserved regions between cbr I and cbr II, along with reference sequences. This analysis further demonstrated the significant diversity of bacterial laccases in the straw-amended soil, many of which may exhibit valuable enzymatic properties. For example, reference sequences in cluster A were derived from three marine bacterial laccases. These marine laccases exhibit strong chloride tolerance, with Lac21 maintaining its original activity in the presence of 250 mM NaCl, and Lac15 and Lac1326 maintaining their original activity even at 1 M NaCl ( Fang et al., 2012 ; Fang et al., 2011 ; Yang et al., 2018 ). The close phylogenetic relationship between the bacterial laccase fragments from the straw-amended soil and these reference sequences suggests the presence of Cl − resistant bacterial laccases. The bacterial laccase fragment in cluster C was closely related to laccases from Thermus thermophilus , Meiothermus ruber , and Sinorhizobium meliloti , which have been demonstrated to possess high thermostability, suggesting similar properties for this enzyme ( Miyazaki, 2005 ; Kalyani et al., 2016 ; Pawlik et al., 2016 ). Furthermore, seven bacterial laccase genes with full-length ORFs were translated into amino acid sequences, and four of these contained TAT signal peptides, indicating bacterial laccases could be involved in extracellular lignin degradation through the twin-arginine translocation system, in addition to various intracellular developmental processes. Characterization of lacS1 revealed it was most active at slightly acidic pH when ABTS was used as the substrate. This behavior is similar to the laccase from Geobacillus yumthangensis ; however, lacS1 demonstrated greater stability under basic conditions, akin to CotA laccases from Bacillus species ( Sharma & Leung, 2021 ; Liu et al., 2023 ). Additionally, lacS1 showed optimal activity at 50 °C but remained stable only up to 40 °C, suggesting it is a mesophilic enzyme. Notably, the addition of copper ions did not affect the enzyme’s activity, likely due to the incorporation of Cu 2+ into the active sites during laccase synthesis under microaerobic fermentation conditions ( Sun et al., 2024 ). Moreover, lacS1 displayed halotolerance, similar to LacM, though weaker than the marine laccases Lac15, Lac21, and Lac1326 ( Fang et al., 2012 ; Ausec et al., 2017 ; Fang et al., 2011 ; Yang et al., 2018 ). These differences in halotolerance may be attributed to the distinct habitats of the laccase-producing bacteria. Overall, these properties suggest that lacS1 is well-suited for applications in wastewater treatment. It is important to highlight that the findings regarding bacterial laccase diversity in this study are limited to the specific sampling location. This is due to the distinct responses of soil microbial communities to straw incorporation, which vary based on factors such as soil taxonomy, soil properties, straw materials, and fertilization practices. In this study, we focused specifically on mining bacterial laccases using the straw-amended soil as material. Moving forward, other cloned bacterial laccase genes will be heterologously expressed to determine their enzymatic properties. Through a combination of mutagenesis experiments and functional studies, their catalytic mechanisms will be elucidated. This will pave the way for the development of novel bacterial laccases with enhanced performance and significant potential for diverse applications."
} | 3,848 |
27812175 | PMC5094794 | pmc | 6,430 | {
"abstract": "The fruit fly optimization algorithm (FOA) is a newly developed bio-inspired algorithm. The continuous variant version of FOA has been proven to be a powerful evolutionary approach to determining the optima of a numerical function on a continuous definition domain. In this study, a discrete FOA (DFOA) is developed and applied to the traveling salesman problem (TSP), a common combinatorial problem. In the DFOA, the TSP tour is represented by an ordering of city indices, and the bio-inspired meta-heuristic search processes are executed with two elaborately designed main procedures: the smelling and tasting processes. In the smelling process, an effective crossover operator is used by the fruit fly group to search for the neighbors of the best-known swarm location. During the tasting process, an edge intersection elimination (EXE) operator is designed to improve the neighbors of the non-optimum food location in order to enhance the exploration performance of the DFOA. In addition, benchmark instances from the TSPLIB are classified in order to test the searching ability of the proposed algorithm. Furthermore, the effectiveness of the proposed DFOA is compared to that of other meta-heuristic algorithms. The results indicate that the proposed DFOA can be effectively used to solve TSPs, especially large-scale problems.",
"conclusion": "Conclusions In this study, a novel discrete fruit fly optimization algorithm was applied to the traveling salesman problem (TSP). An effective crossover operator was developed in order to allow the fruit fly group to search the neighbors of the best-known swarm location. Tasting and smelling processes were introduced into the algorithm. In addition, an edge intersection elimination operator was incorporated into the DFOA in order to improve the neighbors of the non-optimum food location. According to the results of the computational tests and comparisons, the proposed DFOA yielded better answers for all of the cases with less computational effort. This research not only provided a TSP with a powerful solution algorithm, but also realized the application of a DFOA to a discrete field. The proposed DFOA is a population-based parallel algorithm with few required simple search frameworks and control parameters. A number of local search operators or knowledge-based principles can be easily implanted into the framework of the proposed DFOA. Therefore, future work could focus on the development of adaptive algorithms with parameter learning mechanisms and the implementation of other problem-specific features that could improve the performance of the DFOA. In addition, the proposed DFOA could be applied to other variations of the TSP, such as fixed edges are listed that are required to appear in each solution to the problem, Hamiltonian cycle or path problem, Capacitated vehicle routing problem etc. Furthermore, while the proposed encoding schema is hard to carry out crossover, an effective solution representation schema, which is suitable for crossover and inheriting the properties from parental tour, can be designed in a future work. Moreover, new candidate sets generation mechanisms, not only the NN, but a good estimate of the edges’ chances of belonging to an optimal tour, and more effective local search methods can be used in a future work.",
"introduction": "Introduction The traveling salesman problem (TSP), one of the most complex combinatorial optimization problems, has been extensively studied due to its practical applications. This problem can be described as a salesman who wants to travel a series of n cities. Suppose that d ij ( i , j ∈ {1,2,⋯, n }), which denotes the distance between the traveling points i and j , is well known by the salesman. The salesman wants to select the route or tour that includes one stop in all of the cities with the minimum travel distance. The route can begin in any city, but the salesman must return to the city of departure. Other factors, such as time and cost, can be considered as well. In a TSP, if the travel distance or cost from city i to city j equals from j to i , then it is considered to be a symmetric problem, or otherwise, an asymmetric problem. Since any asymmetric Euclidean TSP can be transformed into a symmetric problem, symmetric problems have been more extensively studied. In fact, symmetric problems, particularly Euclidean TSPs involving cities located in a two dimensional plane and in which Euclidean distances are used as a metric, play an important role in practical applications, such as VLSI chip fabrication, X-ray crystallography, flexible flow shop scheduling, and schoolbus routing [ 1 , 2 ]. However, the TSP has been proven to be an NP-hard problem, in which any exact approaches to determining optimal solutions may necessitate the long running times associated with high dimensionality [ 3 ]. As a result, researchers have primarily developed approaches that can only obtain near-optimal solutions in a relatively short running time. However, others have attempted to develop optimization algorithms that function substantially well in practical cases rather than worst-case scenarios. Due to the importance of this problem in both practical applications and academic research, intelligent and knowledge-based algorithms are needed. In the past twenty years, TSP problems have served as benchmarking and initial testing tools for novel algorithms. These algorithms can be classified as exact approaches and approximate approaches or heuristics. Exact approaches are used to enumerate the optimal tours of finite-stage TSPs. However, the running times of exact approaches are comparatively long in power time complexity. Therefore, exact approaches cannot be effectively applied to large-scale problems. In contrast, heuristics can be used to determine good tours in polynomial time complexity, although they do not necessarily yield the optimum tours. Seyed Mohsen Mousavi et al. have developed two parameter-tuned meta-heuristics and two meta-heuristics algorithms to solve a discounted inventory control problem or multi-item multi-period inventory control problem under storage constraints and discounts [ 4 , 5 ], which greatly increased the scope of application for heuristics. The heuristics for TSP can be subdivided into travel cycle construction approaches and improvement approaches. In travel cycle construction approaches, a tour is generated in n steps by gradually adding the indices of different cities. Strategies used to select the city in the next step include the nearest or farthest neighbor criteria, greedy method, Clarke-Wright algorithm [ 6 ], and Christofides algorithm [ 2 ]. In tour improvement approaches, an entire tour is generated. Then, improvement or exchange strategies are employed to improve those tours. These strategies include local search or local optimization, simulated annealing [ 7 ], ant colony optimization [ 8 ], particle swarm optimization (PSO) [ 9 ], and genetic algorithms (GA). Especially, PSO has been improved and used in many fields to solve problems like a multi-product multi-period inventory control under inflation and discount [ 10 ], and the integrated location and inventory control in a two-echelon supply chain network [ 11 ]. However, local search is the most simple and effective approach. Since most of the earlier studies that used GA to solve TSP focused on designing proper encoding representations, reproduction, crossover and mutation operators [ 12 – 14 ], they followed the evolution strategy of simple genetic algorithms. Although these methods can be used as valuable references for other evolutionary optimization algorithms, their performances are lacking compared to local search approaches, such as the two-OPT, three-OPT, and Lin-Kernighan (LK) approaches [ 15 ]. Currently, other new evolutionary optimization algorithms for TSPs exist, such as the discrete bat algorithm [ 16 ], discrete firefly algorithms [ 17 ], and the discrete invasive weed optimization algorithm [ 18 ]. Although these algorithms have yielded good results, they still require further improvements and modification. Usually, tour construction and improvement are combined to allow for the construction of initial tours and later improvement of those tours, respectively. Therefore, a hybrid approach is more applicable. Baraglia et al . developed a hybrid GA with LK local search capabilities [ 19 ], Hung et al . also developed a hybrid GA with LK in order to improve the local search capability of the GA [ 20 ]. Hybrid GAs utilize global optimization capabilities of a GA and local optimization capabilities of other heuristics to overcome premature. However, due to the complexity of genetic operators and local improvement operators, hybrid GA approaches entail high computational loads and long CPU running times. Thus, these approaches are not best applied to large-scale problems. Recently, Pan developed a novel optimization algorithm called the fruit fly optimization algorithm (FOA) based on swarm intelligence by carefully observing the foraging behavior of fruit flies [ 21 ]. The FOA possesses numerous advantages, including a simple structure, few adjustable parameters, and a relatively short CPU running time. The FOA is also easy to program and can be modified to other practical applications. Due to these advantages, the FOA has been used to solve a wide range of optimization problems, including prediction and classification problems [ 22 – 24 ], continuous function optimization problems [ 25 ], the multidimensional knapsack problem [ 26 ], and scheduling problems [ 27 ]. And in the newest research fields, identification of dynamic protein complexes [ 28 ], selecting evolutionary direction intelligently and joint replenishment problems [ 29 , 30 ], also can acquire good results based on fruit fly optimization algorithm. Although the original FOA has primarily been applied to problems on a continuous definition domain, it can also be successfully applied to problems with continuous variables. However, the FOA must be modified in order to effectively manage the discrete variables associated with combinatorial optimization issues, such as the food source representations and effective generation mechanisms of candidate solutions near swarm locations in the TSP, intelligent parallel test sheet generation [ 31 ], and flow shop scheduling problems with intermingling equivalent sublots [ 32 ], optimizing a location allocation-inventory problem in a two-echelon supply chain network [ 33 ], and the homogeneous fuzzy series-parallel redundancy allocation problem [ 34 ]. As stated previously, the TSP is an NP-hard combinatorial optimization issue involving a large search area that cannot be easily solved with traditional algorithms. However, the FOA is a parallel evolutionary algorithm based on smelling and vision processes. In addition, problem-dependent operators can be modified to adapt the smelling process of an FOA to further enhance exploitation. Furthermore, local search methods can be effectively incorporated by sharing information regarding swarm food locations to precipitate exploration. Therefore, the FOA could be modified to solve TSP. Li Heng-yu adopted and adapted step size and mutation strategies in order to solve TSPs [ 35 ]. Wang Ke-fu et al . introduced the radius of local optimum through which whether the fruit fly was in a local optimum area could be judged [ 36 ]. Roulette method is used to initialize the path. At the same time the local search ability and convergence speed up by using C2Opt to optimize the local path [ 37 ]. Yin Lvjiang et al . integrated PSO and GA algorithm into FOA to improve its advantages. IFOA and PSO were compared; for most of the traveling salesman problem, the effect of the IFOA is better than the PSO, different from this paper that it for all [ 38 ]. The smell function took into account randomly generated variant of the fruit fly encoding with Bit Mutation Operator, and the data sets used in the experiment were different from us. The methods have been discussed by Nitin S. Choubey et al . only applied to the instances with a small number of cities [ 39 ]. However, these strategies can be further optimized. Therefore, in this study, a discrete FOA containing a new strategy is used to solve the TSP. Specifically, an ordering of city indices is used to directly represent the solution. Based on the characteristics of the problem, an effective crossover operator is designed for the smelling process of fruit flies. In addition, an effective local search method, the edge intersection elimination method, is used by the fruit fly group to sense the smell of the swarm food location. According to the results, the combination of the crossover operation and edge intersection elimination strategies in the DFOA effectively prevented the occurrence of local optima and low convergence. Furthermore, the effectiveness of the proposed DFOA is demonstrated with computational tests using benchmarking problems and a comparative analysis of other nature-inspired algorithms. The remainder of this paper is organized as follows. In section 2, the mathematical formulation of the TSP is described. In section 3, the original FOA is presented, and the DFOA procedure of the TSP is illustrated in detail. In section 4, numerical testing results obtained using a classified set of benchmarking problems and a comparison of the proposed DFOA and other existing algorithms are provided. The conclusions of this paper and future research opportunities are presented in section 5."
} | 3,395 |
22068594 | null | s2 | 6,431 | {
"abstract": "An important feature of naturally self-assembled systems such as leaves and tissues is that they are curved and have embedded fluidic channels that enable the transport of nutrients to, or removal of waste from, specific three-dimensional regions. Here we report the self-assembly of photopatterned polymers, and consequently microfluidic devices, into curved geometries. We discover that differentially photo-crosslinked SU-8 films spontaneously and reversibly curve on film de-solvation and re-solvation. Photolithographic patterning of the SU-8 films enables the self-assembly of cylinders, cubes and bidirectionally folded sheets. We integrate polydimethylsiloxane microfluidic channels with these SU-8 films to self-assemble curved microfluidic networks."
} | 189 |
32875115 | PMC7438107 | pmc | 6,432 | {
"abstract": "A multifunctional self-powered sensor is developed for pressure, temperature, and material sensing.",
"introduction": "INTRODUCTION Humans can apperceive pressure and temperature and deduce their material properties while applying contact between objects and skin ( 1 ). The development in functional electronics reveals strategies that allow us to realize some tactile functions of human skin ( 2 – 6 ). For example, electronic skins and flexible sensors have been implemented in robotics and wearable health-monitoring devices to detect ambient changes of strain, vibration, and the direction of applied pressure ( 7 – 13 ). In addition, graphene channels were exploited to render stretchable thermistor ( 14 ), and stretchable-gated sensors were used to gather the temperature of the object ( 15 ). Recently, even more fascinating sensors can simultaneously detect pressure and thermal variations in a single device using ferroelectric or organic thermoelectric materials ( 16 – 18 ). An important future to imitate the feature of human skin lies in the development of multifunctional sensing, especially for inferring material properties. Inferring material properties of objects is needed for many industrial and medical conditions ( 19 , 20 ). A number of technologies, including various image pattern recognition methods and machine learning technology, have been implemented in material identification ( 21 – 22 ). For example, Sundaram et al. ( 23 ) recently demonstrated a scalable tactile glove that can perceive individual objects by deep convolution neural networks. Although these approaches have promoted the performance boundaries for identifying objects, their potential to infer smooth materials and further improve their capability may be limited by the use of grasping signals and complex algorithms. It is advantageous for the systems to directly use signals generated from sensors without the complicated data processing. Triboelectric nanogenerator (TENG) is a promising alternative approach to bridging the technological gap ( 24 , 25 ). Recently, the TENG based on the coupling of triboelectrification and electrostatic induction has been investigated for energy harvesting and self-powered mechanical sensing ( 26 – 29 ). For example, Guo et al. ( 30 ) demonstrated a triboelectric auditory sensor for both robotics and human beings. Triboelectrification between different materials can indicate the natural physical property of materials; however, it mainly suffers from the applied pressure and temperature. Integrating a TENG with other sensors may provide a simple solution to realize skin-like sensing systems. Here, we present a multifunctional, tactile self-powered sensor that enables pressure, temperature, and material sensing. The constitution takes the form of a multilayer stack: (i) a hydrophobic polytetrafluoroethylene (PTFE) film as the electrification layer, (ii) two Cu sheets coated with the silver nanowires (Ag NWs) film as electrodes, and (iii) a sponge-like graphene/polydimethylsiloxane (PDMS) composite as the responsive component to piezoresistive and thermoelectric effects. The device characterizes with a high-temperature detection resolution and a pressure-sensing sensitivity of 1 K and 15.22 kPa −1 , respectively. The key concept of our device lies in inferring material properties based on generated electric signals between the PTFE film and objects. We introduce a simple algorithm—lookup table algorithm operated with MATLAB—to analyze the signals on the computer. As proof of concept, we indicate that the device can infer 10 different flat materials. This work opens up new paths for using self-powered sensors in multifunctional tactile sensing.",
"discussion": "DISCUSSION We present material and structural designs that enable a multifunctional sensing mechanism. Incorporating the graphene into the PDMS via a simple template method, sponge-like conducting composites have been achieved. The composite not only has outstanding electrical properties but also exhibits thermoelectric features. This capability makes it a prospective element for temperature-pressure sensing. In addition, we prepared hydrophobic PTFE films with micro-nano porosities. Given that the functional materials are sensitive in the vertical direction, we developed a structural design using the form of a multilayer stack to realize the independent multifunctional sensing. An attractive feature of our devices is the multifunctional sensing functionality, especially for material identification. The devices exhibited a high pressure–sensing sensitivity of 15.22 kPa −1 and an accurate temperature resolution of 1 K based on the piezoresistance and thermoelectric property of the graphene/PDMS composite, respectively. Moreover, our devices were able to infer material properties based on the universal contact electrification. Notably, even with a simple contact-release movement, the devices can discriminate 10 common flat materials. The size of the devices could be further decreased using advanced responsive components, as summarized in fig. S7. Our devices exhibit prominent performances of pressure and temperature sensing by realizing a self-powered material identification (table S3). This is one of few works, to our knowledge, that use three mechanisms (piezoresistive, thermoelectric, and triboelectric effects) in a single device to achieve pressure, temperature, and material sensing. For portable applications, sensing devices should be of low cost and power. We fabricated graphene/PDMS sponges via the template method due to its low cost and simple process with promising large-area production. To improve the performance of devices, we could develop microfluidic methods to create responsive components with a uniform distribution of pore size ( 36 ). On the basis of the sensing mechanism of the thermoelectric and the triboelectric effects, our devices exhibited self-powered performance, enabling their long-term monitoring applications with low consumption. One limitation of the device is the possible electron transfer of the electrification layer when it operates under the very hot or humid environment ( 37 , 38 ). The electron transfer will obstruct the accurate measurement of the output voltage via the electrification mechanism. An important research direction, therefore, might be the development of stable electrification layers and sensing mechanisms for special applications. In summary, we have presented a simple, low-cost method to fabricate a multifunctional sensor using a hydrophobic PTFE film and a sponge-like graphene/PDMS composite. Using the piezoresistive and thermoelectric properties of the composite, our sensor exhibits a pressure sensitivity and self-powered temperature-sensing accuracy of 15.22 kPa −1 and 1 K, respectively. The use of contact electrification enables the material identification of objects. We hope that our methodologies offer an approach to multifunctional sensors with potential applications in wearable electronics and robotics."
} | 1,753 |
38135931 | PMC10740855 | pmc | 6,433 | {
"abstract": "This case study assesses the valorization of industrial wastewater streams for bioenergy generation in an industrial munition facility. On-site pilot-scale demonstrations were performed to investigate the feasibility of algal growth in the target wastewater on a larger outdoor scale. An exploratory field study followed by an optimized one were carried out using two 1000 L open raceway ponds deployed within a greenhouse at an industrial munition facility. An online system allowed for constant monitoring of operational parameters such as temperature, pH, light intensity, and dissolved oxygen within the ponds. The original algal seed evolved into an open-air resilient consortium of green microalgae and cyanobacteria that were identified and characterized successfully. Weekly measurements of the level of nutrients in pond liquors were performed along with the determination of the algal biomass to quantitatively evaluate growth yields. After harvesting algae from the ponds, the biomass was concentrated and evaluated for oil content and biochemical methane potential (BMP) to provide an estimate of the algae-based energy production. Additionally, the correlation among biomass, culturing conditions, oil content, and BMP was evaluated. The higher average areal biomass productivity achieved during the summer months was 23.9 ± 0.9 g/m 2 d, with a BMP of 350 scc/gVS. An oil content of 22 wt.% was observed during operation under low nitrogen loads. Furthermore, a technoeconomic analysis and life cycle assessment demonstrated the viability of the proposed wastewater valorization scenario and aided in optimizing process performance towards further scale-up.",
"conclusion": "4. Conclusions The field pilot tests conducted in this study assessed the feasibility of using nitrogen-rich industrial wastewater streams from a munitions production facility to support on-site production of algal biomass under local climatic conditions for the purpose of extracting bioenergy in the form of oil or biogas. This on-site pilot-scale study demonstrated the practicability of the process and the viability of the applied industrial waste stream valorization strategy, which promotes loop closure of nutrients and water resources within the facility and encourages a circular bioeconomy model. During the optimized run, during which operational conditions were refined based on the experience gained in the first exploratory run, an average areal biomass productivity of 23.9 ± 0.9 g/m 2 d was obtained during the late spring–early summer testing period. Measurements of oil content and BMP of the harvested biomass indicated that changes in culture conditions and harvesting frequency have a direct impact on the amount of algal oil and biogas produced. Moreover, the values obtained, 20% for oil content and 350 scc/gVS for BMP, are comparable to values reported in the literature, and in the case of biogas, 70% of the theoretical amount was produced. In addition, we demonstrated that using a consortium of algae, which is better suited to the local and outdoor conditions and resilient to predators, favored the performance of the process and avoided algae population crashes during the operation. This contributed to improved biomass productivity without vitiating oil or biogas production. Furthermore, LCA and TEA analyses for a 100 ha algae farm producing biogas were successfully performed with the data obtained in this study. The results illustrate that the price of biogas is affordable and competitive with natural fossil gas due to the incentive credits, while, from an environmental perspective, the benefit of producing bioenergy is larger than with natural gas due to the mitigation of CO 2 and the valorization of waste within the facility. Lastly, the encouraging results will lead to more actionable research into scale-up to achieve zero waste and, if implemented, indicate potential opportunities for various industrial sectors to reduce industrial waste and lower process costs.",
"introduction": "1. Introduction Over the last few decades, the pursuit of renewable energy resources has followed different approaches. The conversion of solar into chemical energy, known as photosynthesis, has been explored with the aim of producing biomass with high energy potential. It has been established that, compared to terrestrial plant biomass, algae can produce the highest areal biomass yields, thus making them a preferred substrate for renewable biofuel applications employing a variety of municipal, saline, or industrial wastewater [ 1 , 2 , 3 ]. In addition, photosynthetic microorganisms, like microalgae and cyanobacteria, have two important advantages: (1) They grow fast and (2) they can be cultured in non-arable lands. Hence, they do not compete with food production and could minimize the need for freshwater resources [ 4 ]. Moreover, several types of bioenergy can be produced from algae biomass via chemical, biochemical, and/or thermochemical conversion into biogas, biodiesel, green diesel, sustainable aviation fuel (SAF), hydrogen gas (H 2 ), and other biofuels [ 5 ]. To date, several studies have shown substantial benefits in the integration of wastewater-grown microalgae-based biofuel production [ 6 , 7 , 8 , 9 , 10 ]. The production of algal biomass for food, biofuels, and other commercial products dates from the early 1950s and has been documented in more than 17,000 publications worldwide [ 11 , 12 ]. Among several reactor configurations used for this purpose, open pond systems are the most widely used. The major advantages of open pond systems are the low capital and operating costs as well as the low energy requirements for culture mixing. Conversely, open pond reactors need large areas to scale up and are susceptible to weather conditions and contamination. Yet, sustainability studies show that efforts should be focused on enhancing biomass productivity, valorizing all products obtained from biomass and improving the integration of various industrial nutrient sources from waste streams to lower the cost of operations [ 13 , 14 ]. Diverse options may be considered for maximizing the recovery of bioenergy from algal biomass within the framework of a net-zero conversion process. In this scenario, anaerobic digestion (AD) is one of the most promising and feasible technologies for the valorization of algae biomass cultures both as a product (i.e., raw) and after lipid extraction (i.e., residue post extraction) [ 15 ]. The process is particularly attractive due to the production of biogas, a mixture of methane and carbon dioxide with traces of volatile organics and hydrogen sulfide. Due to the biomethane content and easier compression storage, biogas is characterized by its superiority in energy conservation and emissions reduction. Biomethane represents a renewable fuel source that can be combusted to generate heat and electricity for residential, commercial, industrial, and transportation purposes [ 16 , 17 ]. The biochemical methane potential (BMP), defined as the total methane yield per unit of substrate added, represents a key parameter for assessing the suitability of AD feedstocks [ 18 ]. The incorporation of AD is considered critical for both economics and sustainability reasons since this technology provides a main route for recycling nutrients to the algae cultivation process [ 19 ]. The reuse and recycling of nutrient resources are particularly important for minimizing the environmental footprint of the biofuel production chain. In that respect, the processes considered in this study align with general circular bioeconomy approaches, in which the production of renewable biological resources and their conversion into value-added products is highly promoted [ 20 , 21 ]. For instance, atmospheric nitrogen fixation for ammonia production (via the Haber–Bosch process) and its subsequent conversion to other nitrogen species of interest (i.e., nitrate salts, nitric acid, etc.) consume valuable resources and generate products that require treatment, exerting additional resource demand and further increasing the environmental footprint. By utilizing nitrogen-rich waste streams in the current study, a portion of the nitrogen species can be reclaimed and converted into biomass, generating a valuable resource (i.e., for bioenergy or other uses), along with savings in denitrification treatment costs. In this case study, the technical and economic feasibility of growing algae feedstocks for biofuel production was assessed using two 1000 L open raceway ponds and industrial wastewater in an outdoor setting at an industrial munitions facility located in the continental US. The study falls under the facility’s efforts to reduce its environmental and energy footprint by utilizing nutrient-rich wastewaters to produce useful bio-stocks. The study focuses on the improvement of algal biomass productivity and bioenergy potential. The two most researched pathways to generate energy from algae, oil, and biogas were assessed. Moreover, to complement the assessment of the experimental approach, a techno-economic analysis (TEA) and life cycle assessment (LCA) for a 100 ha algae farm were performed with the data obtained in this study. On one hand, TEA establishes the capital and operating cost profile to determine the potential economic viability of the selected conversion process towards commercial feasibility. On the other hand, LCA evaluates the potential environmental and social impacts associated with a product throughout its life cycle from raw material extraction to disposal [ 22 ]. The global warming potential over 100 years (GWP 100 ) per mega joule of energy produced was used as an impact category. To the best of our knowledge, this is the first on-site study assessing the valorization of this type of industrial nitrogen-rich wastewater for bioenergy generation purposes.",
"discussion": "3. Results and Discussion 3.1. Wastewater Analysis An extensive characterization of the industrial wastewater streams used in this study was first carried out. Table 2 lists some of the characteristics of the two wastewater streams used: industrial wastewater plant influent (IWWPI) and industrially generated ammonium nitrate filtered solution (AN). The latter contains a high concentration of nitrogen (N) in the form of ammonium and nitrate; both ammonia and nitrates are macronutrients that algae can use to grow to a certain threshold. In addition, ammonia can be easily assimilated since microorganisms can use it directly without any reduction or energy demands, as required for nitrate assimilation [ 32 , 33 ]. Table 3 shows the different AN concentrations tested, along with the calculated growth rate (µ) and the % of growth rate inhibition. In these tests, all of the nutrients were added in excess to avoid growth inhibition due to lack of nutrients, with the exception of N provided by AN. Results from tests with different nitrogen concentrations demonstrated that AN can be used as a sole source of nitrogen for growing algae biomass without significant toxicity up to approximately 750 mg N/L ( Figure S1 ). In fact, concentrations of total nitrogen higher than 1000 mg N/L resulted in inhibition of growth, as evident by the absence of cell chlorophyll and green color development. From this assessment, AN solution was used after dilution with IWWPI. The amount of AN added initially, and after every harvest, upon refill of the raceway reactors fell within the first four columns of serial dilutions in Table 3 , resulting in a total nitrogen concentration in the order of 50–200 mg/L (depending on the tested low/high N range, as described in Table 1 ). The level of N was determined based on the toxicity results and the amount of nitrogen remaining in the supernatant liquor after the algae settled. The rationale was based on minimizing the amount of nitrogen that is recycled to the IWWTP for treatment and discharge. Similarly, a high amount of ammonia nitrogen from dairy farm wastewater [ 34 ] and digested piggery wastewater [ 35 ] streams have been valorized as a source of algal growth; however, to date, there is no evidence of the utilization of this nitrogen-rich wastewater from munition facilities. 3.2. Culture Conditions and Areal Biomass Productivity 3.2.1. Seeding the Reactors: Developing a Resilient Consortium For this study, an initial seed of freshwater microalga S. obliquus was acclimated to grow in the selected wastewater mixture under controlled conditions (i.e., 25 °C, continuous 120 rpm agitation, 68 µmol photons/m 2 s with a 14:10 h light:dark photoperiod) in 5 L flasks, which later served as an inoculum for the 100 L open pond reactors. Once the 100 L reactors achieved a high concentration of biomass (~1 g/L), the algal liquor was transported to the industrial facility and used to seed the on-site 1000 L reactors. As stated in our previous laboratory and indoor pilot work [ 23 , 28 , 30 ], S. obliquus strain was originally selected due to its high growth rate, ability to grow in wastewater streams, and improved lipid content when cultured in N-depleted media. However, in this study, the scaling up to an outdoor environment showed a rapid development of various species, opening the possibility of developing a polyculture more adaptable to the local environmental conditions. In open pond systems, the benefits of polycultures (more than 4 or 6 species) over monocultures include improved biomass productivity and resilience to disturbances by predators or adverse environmental conditions [ 36 ]. An assembly of microscopy images that depict the microbial population in the 1000 L outdoor open ponds is shown in Figure 2 . The initial predominant culture of S. obliquus (with characteristic rice shape, Figure 2 a) evolved naturally to a diverse culture of microorganisms. Most of the microorganisms developed were green freshwater microalgae and cyanobacteria. Most likely, the new species were introduced into the system via the wastewater feed streams and the atmosphere. Furthermore, the system’s microbial ecology evolved in response to the prevailing environmental and operational conditions, such as temperature, pH, solar radiation, and feed composition. Clusters of green microalgae developed due to the presence of fibers and other particles present in the wastewater ( Figure 2 b). Additionally, these clusters could have developed because of strong grazing pressure to the initial monoculture [ 37 ]. The development of diverse freshwater green microalgae and cyanobacteria observed included Ankistrodesmus ; pennate diatoms ( Figure 2 c); spiny microalgae with the formation of typical protective eight-celled colonies from the Scenesmaceae family like Desmodesmus , Scenedesmus, Tetradesmus , and Coelastrum ( Figure 2 d,e); and round microalgae from the Chlorellaceae family such as Chrorella , Chlorococcum , Micractinium , and Dictyosphaerium ( Figure 2 d,e). Filamentous algae identified as Stigeoclonium ( Figure 2 f) and Ulothrix ( Figure 2 g) also developed in the ponds, as well as the cyanobacteria Aphanizomenon , Nostoc , and Calothrix , amongst others. A metagenomic analysis confirmed the species identified by microscopy, as shown in Figure S2 . This consortium prevailed in both experimental runs. 3.2.2. Exploratory Run (ER): Operating Conditions and Performance The exploratory run was performed from spring to fall of 2019 using the industrial wastewater streams characterized in Section 3.1 and the autochthonous polyculture developed on site described in Section 3.2.1 . The conditions of this study are described in Table 1 . Over this period, operational parameters were monitored as described in Section 2.1 . In addition, two N concentrations were used to evaluate biomass productivity as well as bioenergy production ( Table 1 ). The water temperature of the ponds varied significantly over the course of the day–night cycle; moreover, the temperature changed from the first month of operation, with the temperature increasing several degrees due to the transition from spring to summer and then gradually decreasing going from summer to fall ( Table 4 ). Overall, the cultures were grown under an average temperature within the optimum range for algae growth (i.e., 20–30 °C) [ 38 ]. Furthermore, DO varied from 0 to 29.6 mg/L and 28.4 mg/L for R1 and R2, respectively. The highest DO values measured corresponded to DO levels during daylight hours when photosynthetic activity was high. Conversely, the low DO values were recorded at nighttime, when photosynthesis was absent and natural reaeration was not capable of satisfying the oxygen demand required by biomass respiration. Figure 3 a shows the pH, T, and DO during the testing of low N concentrations, whereas Figure 3 b shows the same parameters when testing higher N loads. The pH was maintained within 6.50–7.50 by bubbling CO 2 when the pH was higher than 7.50. Conversely, Na 2 CO 3 was added when the pH was lower than 6.50; this approach was needed with the use of higher concentrations of AN (i.e., ER—high N feed, from 8 H to 13 H). In fact, the consumption of ammonia caused a decrease in pH, making it necessary to add alkalinity to maintain satisfactory productivity yields ( Figure 3 b). Eustance et al. [ 39 ] reported similar results at laboratory scale; they observed an extreme shift in pH when the strains were grown at high ammonium concentrations (i.e., 2.94 mM ammonium chloride) in unbuffered medium, which triggered growth inhibition and chlorophyll degradation. Furthermore, the results from our previous work [ 40 ] showed that mineral carbonates such as sodium carbonate (Na 2 CO 3 ) or MgCO 3 .3H 2 O (nesquehonite) can buffer the pH well and maintain it within acceptable algae physiological values. Biomass growth over time was evaluated by measuring fluorescence (fluorescence units, FU) and ash-free dry weight (AFDW, in g/L). Fluorescence measurements were performed as a rapid estimate of healthy biomass accumulation in the ponds since this measurement is linked to chlorophyll. A linear correlation between AFDW and FU was found ( Figure S3 ), suggesting that even though dry biomass is an essential parameter for areal productivity evaluation, the measurement of FU is an additional rapid way to monitor the culture’s growth, particularly on site, when sometimes decisions need to be made in a short amount of time. Figure 4 shows the areal biomass productivity calculated for each reactor and each harvest during the exploratory run. The average areal biomass productivity was 15 and 10 g/m 2 d for R1 and R2, respectively, over the summer period, dropping to 8.16 and 8.04 g/m 2 d for R1 and R2, respectively, during fall. Overall, the areal productivity observed for each reactor per season was reproducible. These values were similar to the ones found in the literature [ 36 , 41 , 42 ]. However, the difference between R1 and R2 for the summer period could be related to the reactor’s orientation (not in parallel due to space constraints) and the light intensity received during the day. The ER aimed to assess process feasibility and identify site-specific operational limitations, biomass productivity, and related seasonal changes. Hence, from the information gathered, required changes were implemented in the optimized run to increase biomass productivity and bioenergy potential. 3.2.3. Optimized Run (OR): Improving Operating Conditions The optimized run was performed in late spring to early summer of 2021 ( Table 1 ), during which lower N amounts were supplied from AN wastewater alone, and the harvesting frequency was reduced to between 3–5 days. For this run, the photosynthetic active radiation or quantum radiation (PAR) was also measured ( Figure 5 ); the PAR measure inside each reactor was similar, although R2 showed higher PAR readings, likely due to the reactor orientation. Additional PAR readings were obtained from the air-exposed sensor, which measured the light intensity reaching the surface of the reactors; the highest intensity measured and calculated from the area under the curve was 1418 µmol/m 2 s, which was threefold higher than the minimum reading of 411 µmol/m 2 s. High PAR readings occurred around harvests 3, 4, and 5, which also matched the highest biomass productivity. Figure 6 shows the areal biomass productivity for the OR after each harvest. The average areal biomass productivity for the duration of this run was 23.3 and 24.5 g/m 2 d for R1 and R2, respectively. The average areal productivity for the entire run was 23.9 ± 0.9 g/m 2 d. Overall, the productivity increased twofold compared to the ER (i.e., an average of 12 g/m 2 d in the same period) by controlling the nutrient level from N species and increasing the harvesting frequency. As this study was only performed during late spring–early summer, areal productivity values should be adjusted, taking into consideration seasonal variations. A predominance of filamentous photosynthetic microorganisms was observed due to the higher frequency of harvests compared to that of the previous run. 3.3. Biomass Harvested from ER and OR Elemental analysis (CHN) results for the ER show that the carbon content in the initial biomass increased from 38% to 47% and 49% at the eighth harvest (8H) for R1 and R2, respectively. Thereafter, the carbon content in the biomass decreased to 38% and 36% for R1 and R2, respectively. This trend correlates well with the increase in N in the feed from harvests 8H to 13H. The observed biomass increase of more than 20% of carbon content was also reflected in the oil content from the same harvests ( Section 3.4 ). Several studies observed increments in the carbon and lipid contents for microalgae cultured under nitrogen limitations [ 28 , 43 , 44 ]. Furthermore, for the OR, a similar trend for carbon content was observed (the carbon content of 38% in the initial biomass increased to 46% and 47% for R1 and R2, respectively). Although a similar C composition was observed in both runs (ER—low N and OR), the higher harvesting frequency adopted in the OR seemed to promote the increase in biomass productivity, to the detriment of the biomass’s oil content ( Section 3.4 ). Such results are consistent with those reported in the literature [ 45 , 46 ]. Finally, the results from solvent biomass extraction demonstrate that no energetic compounds were present in the algae biomass harvested, eliminating any potential interference on the downstream algae processing. Figure S4 shows the mass spectrometry scans for several acetonitrile extracts, in which no peaks were observed for any of the energetic materials of potential concern. 3.4. Oil Content Obtained from ER and OR An additional goal of the current study was to evaluate the oil content within the wastewater-grown biomass to estimate its energy potential. Initially, the oil content of algae grown in the 100 L raceways ponds (seed) was assessed using the extraction method presented in Section 2.4 , with an oil content of S. obliquus of 21.7 ± 0.5%. During the ER, the oil content was extracted from the harvested algae from the reactors ( Figure 7 a). An oil content similar to that of the seed was measured for the biomass obtained from the first harvest (1H). The oil content percentage was similar for harvests 1H to 4H and slightly increased from harvests 5H to 8H. As stated previously, during this period, the biomass was exposed to low N levels and favorable weather conditions; thus, there were no observed effects from the change in biomass population with regards to oil content ( Figure 7 a). Even though the oil content ranged between 18% and 22% during this period, the oil productivity increased due to an increase in areal biomass productivity ( Figure 4 ). From harvests 9H to 13H, the oil content decreased most likely due to the increase in N loads; similarly, the biomass and oil productivity decreased. The oil content from the biomass obtained after settling with FeCl 3 was also evaluated. The results demonstrate that the oil content was not affected by the addition of this coagulant ( Figure S5 ). The only change observed was with respect to the elemental analysis, which contained less carbon compared to the biomass without coagulant. On the other hand, the results from both the ER and OR (with harvests every 1–2 weeks or 3–5 days, respectively) indicate that long harvesting intervals favored the accumulation of lipids, whereas increasing the frequency of harvesting aided the biomass areal productivity but not the oil content recovered [ 45 ] ( Figure 7 b). Furthermore, filamentous algae and cyanobacteria (which predominated in OR, as indicated before in Section 3.2.3 ) had lower lipid content compared to loose microalgae. For instance, [ 47 ] reported a lipid content of between 8% and 13% for cyanobacteria, whereas [ 48 ] found an oil content of 13.8% in Stigeoclonium sp. 3.5. Biochemical Methane Potential (BMP) from ER and OR Biogas production screening tests on the algal samples were performed with the intent of estimating the biomethane potential (BMP) per gram of the volatile solids (VS) of the algae. The initial S. obliquus inoculum was first tested, and a BMP of 105 scc/gVS was obtained. The samples from both runs, ER and OR, were tested for BMP, along with positive (digestion of 1 g of glucose) and negative (digested sludge without any substrate) controls. Theoretically, the biomethane (BMP) that can be produced from the digestion of glucose is 373 scc per gram of substrate, which is similar to the value obtained in this study (i.e., 363.5 scc/g of glucose) ( Figure S6 ). The BMP from the ER, low N feed (1H to 8H), ranged between 150 and 230 scc/gVS, with an evident increase after harvest 8H ( Figure 8 a). For harvests 9H to 13H, the amount of N used for algae cultivation was increased and the BMP obtained from these harvests ranged from 48 to 300 scc/gVS. Overall, the maximum average BMP was realized from harvest 9H (272 ± 38 scc/gVS), whereas the lowest amount of BMP was obtained from harvest 1H (126 ± 44 scc/gVS), averaging the BMPs produced from the algae harvested from R1 and R2 for each harvesting period. The latter value is similar to BMP results from wastewater-grown S. obliquus under similar AD conditions [ 49 ]. Figure 8 b shows the BMP obtained from each sample digested from the OR while indicating the method used to concentrate the algae. The use of FeCl 3 did not have a major effect on the BMP. The BMP achieved from this OR varied between 94 and 398 scc/gVS. The maximum average BMP (i.e., 352 ± 62 scc/gVS) was obtained from the first harvest concentrated without the use of coagulant, whereas the lowest average BMP (i.e., 163 ± 21 scc/gVS) corresponded to the last harvest without coagulant. Although the BMP is related to the biochemical composition of the species, it seems that the shift from S. obliquus to a consortium did not affect the biogas production. In fact, the BMP obtained was slightly higher for the OR, indicating better digestibility of the filamentous algal polyculture. In addition, the values of BMP obtained in this study are in accordance with the ones obtained by [ 15 ] for three single freshwater microalgae: Chlorella sp., Nannochloropsis sp., and Scenedesmus sp. The elemental analysis performed on the harvested algae from the OR yielded the average percentages by mass of C, H, and N of 45.2%, 6.9%, and 7.7%, respectively, whereas the percentage of O was determined by the difference (40.1%). Based on this elemental analysis, the theoretical BMP realizable from the algae generated from this study was 499 scc/gVS. Compared to the maximum average BMP obtained, this corresponds to about 70% of the theoretical value. As reported in [ 50 ], the amount of BMP produced from algae depends on the strain digested, and it typically varies between 24 and 600 scc/gVS. The values of BMP obtained here fall within this range. 3.6. TEA-LCA Considering the encouraging results from the pilot system and the data obtained from the optimized run, a technoeconomic analysis (TEA) and a life cycle assessment (LCA) were performed by MicroBio Engineering using a proprietary Environmental Sustainability and Process Economics model based on the CA-GREET model [ 51 ] for a full-scale hypothetical 100 ha algae cultivation, harvesting, and processing system for this industrial munitions plant, with an emphasis on maximum biomass production for biogas generation. Two scenarios were investigated: (1) The primary product detailed is raw, unpurified biogas, and (2) raw biogas is upgraded to biomethane with membrane separation. As an additional environmental benefit, coproduct accounting of unrecycled digestate for soil amendment through system boundary expansion was considered in both scenarios. The analysis assumes annual average values for 9 months of facility operation per year (i.e., the algae growing season) and does not provide sensitivity analysis for seasonal variability. The model estimates the costs of the facility using “n-th” plant assumptions, in which the technology is mature, and construction and operation reflects know-how developed from prior facilities, excluding the costly first-of-a-kind facilities, which yields more accurate long-term projections [ 52 ]. Furthermore, the construction costs of the ponds and digesters are based on low-cost agricultural engineering practices, recognizing that algae cultivation and digestion are more akin to agricultural rather than manufacturing activity. The assessed algal biomass production facility comprises 100 ha of land, 80% of which would be used for algae cultivation ponds and the remainder for pond berms, buffers, roads, facilities, fencing, etc. The proposed process makes use of CO 2 from coal-fired flue gas, wastewater, and N from ammonium nitrate wastewater that would otherwise be emitted to the atmosphere or disposed of. The biomass is harvested and thickened via gravity settling to a concentration of approximately 3% volatile suspended solids (VSS). The thickened biomass is then anaerobically digested using in-ground, covered, plug-flow digesters, like those used currently in the dairy and swine industries. The digesters are unmixed and unheated; thus, some natural settling may occur. Up to 90% of the digestate is then recycled back into the production ponds to use bioavailable nutrients such as soluble N and P, whereas the remainder is thickened by polymer addition and screw pressing to use for soil application. The raw biogas can then either be upgraded to biomethane or used on site as raw biogas for processes requiring power and/or heat generation. The TEA process boundary ends after the production of biomethane or biogas. As expected, natural gas was less expensive (USD 3.92/MMBTU October 2019) than algae-derived biogas due to the facility’s capital costs. The total cost for raw unpurified biogas was estimated to be USD 43.23/MMBTU. However, the introduction of credits and incentives showed that it may be possible to make this technology competitive if it becomes more heavily incentivized. Although the upgraded biogas scenario involves an additional capital and operating expense for membrane separation, the revenue from a D3 RIN credit (which is not applicable to raw biogas) would considerably the cost, making the estimate decrease to a more affordable and competitive price (USD 5.62/MMBTU). It is important to highlight that this study was performed during the first quarter of 2021 and that nowadays the price of natural gas is higher (USD 8.29/MMBTU in 2022, according to the U.S. Energy Information Agency [ 53 ]); therefore, the results should be even more favorable to producing renewable energy. A life cycle analysis (LCA) was performed for the envisioned algal production facility. The impact category of focus for this LCA was greenhouse gas (GHG) potential, specifically the global warming potential over 100 years (GWP100); thus, a functional unit of grams CO 2 equivalent per megajoule of energy produced (g CO 2 eq/MJ) was chosen. Because the LCA does not fully investigate the “cradle-to-grave” inventory of emissions and resource consumption—it excludes biogas transport, power plant resource use, and facility dismantling—the results herein compare emissions on a “well-to-combustion” basis to natural gas extraction, gathering/boosting, and processing. Transportation and power plant costs were assumed to be identical for biogas and natural gas, making them unnecessary to include in the comparison. Additionally, the combustion of biogas was assumed to be a net-zero process because the CO 2 released is the same CO 2 captured by the algae biomass. Values for GHG impact were taken from published literature, LCAs involving production of biogas through AD of microalgae, and LCA databases such as Ecoinvent. Furthermore, an expansion of system boundaries was performed in accordance with ISO guidelines to account for impacts of resulting coproducts. From an environmental standpoint, the benefits were large; in fact, net global warming potential (GWP) for scenarios 1 and 2 (i.e., 5.4 g CO 2 eq/MJ and 13.1 g CO 2 eq/MJ, respectively) was far more favorable than for the extraction and processing of natural gas (i.e., 104 g CO 2 eq/MJ). This benefit was largely due to CO 2 mitigation and the utilization of waste streams as process inputs. Overall, the production of biogas obtained from algal biomass is more affordable and competitive than natural fossil gas due to incentive credits. In addition, the environmental impact of producing bioenergy is larger than with natural gas due to the mitigation of CO 2 and the valorization of waste within the facility."
} | 8,447 |
28890939 | PMC5584659 | pmc | 6,434 | {
"abstract": "Bacteria gain antibiotic resistance genes by horizontal acquisition of mobile genetic elements (MGE) from other lineages. Newly acquired MGEs are often poorly adapted causing intragenomic conflicts, resolved by compensatory adaptation of the chromosome, the MGE or reciprocal coadaptation. The footprints of such intragenomic coevolution are present in bacterial genomes, suggesting an important role promoting genomic integration of horizontally acquired genes, but direct experimental evidence of the process is limited. Here we show adaptive modulation of tetracycline resistance via intragenomic coevolution between Escherichia coli and the multi-drug resistant (MDR) plasmid RK2. Tetracycline treatments, including monotherapy or combination therapies with ampicillin, favoured de novo chromosomal resistance mutations coupled with mutations on RK2 impairing the plasmid-encoded tetracycline efflux-pump. These mutations together provided increased tetracycline resistance at reduced cost. Additionally, the chromosomal resistance mutations conferred cross-resistance to chloramphenicol. Reciprocal coadaptation was not observed under ampicillin-only or no antibiotic selection. Intragenomic coevolution can create genomes comprised of multiple replicons that together provide high-level, low-cost resistance, but the resulting co-dependence may limit the spread of coadapted MGEs to other lineages.",
"discussion": "Discussion Our current model of bacterial evolution suggests that horizontal acquisition of ARGs accelerates resistance evolution by providing bacteria with ready-made resistance mechanisms, bypassing the requirement for rare de-novo mutations 1 . However, recent population genomic data suggesting that lineages independently acquire and then subsequently coevolve with MDR plasmids 13 , 36 , 37 imply a more dynamic evolutionary process. Consistent with this, here we show here that gaining an ARG can be just the starting point in the evolution of resistance and, due to the costs of expressing horizontally acquired ARGs, does not preclude subsequent de novo evolution of chromosomal resistance. Evolved strains from TET-containing treatments gained chromosomal resistance mutations reducing membrane permeability and enhancing efflux of TET and providing cross-resistance to other antibiotics, shortening lag phase in the presence of TET. These mutations also reduced the need for a fully operational plasmid-encoded tetracycline efflux pump, expression of which is highly costly 35 , allowing plasmid mutations in the TET efflux pump and its regulator which reduced the cost of plasmid-encoded resistance. A consequence of this intragenomic coevolution is that the increased TET resistance of evolved strains from T, AT and A/T treatments required the action of both the chromosomal- and plasmid-encoded resistances, which together acted multiplicatively. Thus intragenomic coevolution can lead to the evolution of bacterial genomes comprised of co-dependent replicons, limiting the potential for onward transmission of the plasmid due to the weaker resistance it now encodes in other lineages."
} | 778 |
31210720 | PMC6562305 | pmc | 6,435 | {
"abstract": "Abstract Mutualistic interactions involve 2 species beneficially cooperating, but it is not clear how these interactions are maintained. In many mutualisms, one species interacts with multiple species, and since partners differ in terms of the commodities they trade, partner identity will directly influence the decisions and behaviors of interacting individuals. Here, we investigated the consequences of within and between-species diversity on a model cleaner–client interaction in a natural environment, by quantifying the behavior of both partners. We found that the predominant Caribbean cleaner fish, the sharknose goby ( Elacatinus evelynae ), shows personality variation as we documented repeatable individual differences in activity, boldness, and exploratory behaviors. Personality variation was associated with cleaner–client interactions: cleaner boldness and activity were significantly related to posing by clients and cleaning, respectively. Cleaner personality variation was also associated with the functional identity (sociality, mobility, body size, and trophic level) of clients posing and being cleaned. We thus demonstrate that partner identity can have consequences on mutualistic outcomes which will contribute to the context-dependency and highly heterogeneous patterns we observe at a population level. We also suggest that within- and between-species differences have consequences on partner choice, a feature that has been previously thought to be absent from these cleaner–client interactions.",
"introduction": "INTRODUCTION Mutualistic interactions, where 2 species beneficially cooperate, are observed in all ecosystems ( Bronstein 2015 ), yet it is still not clear how these interspecific interactions are maintained. Mutualisms often involve food resources (e.g., nectar and ectoparasites) being traded for a beneficial act (e.g., pollination; Landry 2012 , parasite removal; Arnal et al. 2001 ), known as service–resource interactions ( Holland et al. 2005 ), but not all partners are equal in terms of the commodities they trade ( Palmer et al. 2015 ). These interspecific interactions involve 2 individuals directly interacting at any one time, and thus the behaviors and traits of one partner, could directly influence the behaviors and traits of the other ( Wolf and Weissing 2012 ). Partner identity will hence underpin the behavioral responses and decisions of animals during these cooperative interactions, influencing when individuals interact, with whom, and by how much ( McAuliffe and Thornton 2015 ). Currently, our understanding of mutualisms is hypothesized to be context-dependent and highly heterogeneous ( Bronstein 2015 ); so, investigating how individual partners influence mutualism outcomes will help to clarify the dynamics and hence evolution of mutualisms under natural conditions. Within an environment, service providers only make up a small proportion of the biomass but interact with a disproportionately large number of other species ( Sazima et al. 2010 ). As a result, mutualisms are often composed of networks of interacting species, with service providers carrying out ecosystem services, such as pollination ( Landry 2012 ) and health enhancing parasite control ( Clague et al. 2011 ; Waldie et al. 2011 ). Mutualists thus play a pivotal role in the structuring and functioning of ecological communities ( Floeter et al. 2007 ; Sazima et al. 2010 ; Quimbayo et al. 2018 ). An iconic, well-studied service–resource mutualism, the cleaner–client interaction, is observed ubiquitously on coral reefs ( White et al. 2007 ; Leung and Poulin 2008 ). The mutualism involves a cleaner removing ectoparasites and other material from the bodies of many client fish species (up to 132 different species; Grutter and Poulin 1998 ). Cleaning patterns, however, are inconsistent, with the same cleaner species showing preferences for different client types across studies. For example, cleaning gobies from the genus Elacatinus prefer larger clients in some studies (e.g., Whiteman and Côté 2002b ; Grutter et al. 2005 ; Silvano et al. 2012 ), but not in others (e.g., Grutter and Poulin 1998 ; Arnal et al. 2000 ). These, like many other behavioral studies, focus on population patterns, which assume all conspecifics exhibit the same traits, or that variation around an average is random ( Bolnick et al. 2011 ). Individuals within many invertebrate and vertebrate populations vary consistently in their behavior (also known as animal personality variation; Reale et al. 2007 ), and this variation can play a major role in shaping population-level patterns of species interactions and other ecological processes ( Wolf and Weissing 2012 ). There are 5 recognized animal personality traits ( Reale et al. 2007 ), and for many taxonomically distinct species, these traits can affect feeding and foraging behaviors. The personality traits boldness and exploration, for example, which can be broadly defined as an individual’s reaction to a risky (boldness) and new situation (exploration) ( Reale et al. 2007 ), influence both an individual’s food intake and foraging success ( Ioannou et al. 2008 ; David et al. 2011 ). Bolder and more exploratory individuals are expected to have increased metabolic demands since they are at an increased risk (e.g., to predation) and utilize the environment more widely ( Careau et al. 2008 ; Brommer and Class 2017 ). A third personality trait, activity, which quantifies the general activity level of an individual ( Reale et al. 2007 ), may also often predict foraging behaviors ( Pruitt et al. 2012 ) as more active individuals are also expected to have increased energy demands ( Careau et al. 2008 ; Brommer and Class 2017 ). Thus, personality traits, and their correlations with one another (forming a behavioral syndrome; Sih et al. 2012 ) are likely to play a role in food acquisition during mutualistic interactions: dedicated cleaners for example, gain all their nutrition from client derived material ( Vaughan et al. 2017 ). Indeed, bolder cleaner fish ( Labroides dimidiatus ) have been shown to clean less honestly (i.e., cheat more) to acquire a more favorable reward ( Wilson et al. 2014 ), while bolder black-billed magpie cleaner birds ( Pica pica ) interact with clients more frequently, facilitating greater access to protein-rich ticks ( Found 2017 ). However, the dynamics of mutualistic interactions are not just driven by a cleaner’s food dependency ( Lenke 1988 ), because the resource provider’s behavior, engagement, and traits can also regulate outcomes of an interaction ( Bever 2002 ; Bshary and Schäffer 2002 ). In cleaning interactions, clients can choose which cleaners to visit ( Bshary and Schäffer 2002 ), and increase their chances of being cleaned ( Côté et al. 1998 ), by presenting their body to cleaners (termed posing; Feder 1966 ). However, posing does not necessarily guarantee cleaning, and for some clients, they need not pose at all to be cleaned ( Arnal et al. 2001 ; Dunkley et al. 2018 ). The cleaners past behavior towards the client can also influence their interactions with different cleaners: if a client has received a negative response from the cleaner, for example, they are less likely to revisit ( Bshary and Schäffer 2002 ). Cleaners thus adapt their behaviors to ensure client satisfaction ( Grutter and Bshary 2003 ). Partner feedbacks are hence an important component for maintaining positive interspecific interactions ( Frederickson 2013 ), yet their role is largely ignored. Given that feedbacks can reinforce the development of behaviors ( Houston and McNamara 1999 ; Sih et al. 2015 ), it would be expected that the expression of personality variation by cleaners would link with both the actor’s and receiver’s behavior. This prediction however has not yet been tested in a cleaning context, but personality variations have been shown to mediate other interaction types (e.g., predator–prey interactions; Pruitt et al. 2012 , and service–service mutualisms; Schmiege et al. 2017 ). Client species differ in their propensity to engage in cleaning interactions ( Côté et al. 1998 ; Bshary and Schäffer 2002 ), as well as the nutritional content that they represent to cleaners ( Eckes et al. 2015 ). These differences mean that different clients will provide asymmetric benefits to the cleaning interaction. Larger ( Poulin and Rohde 1997 ), group living and sedentary ( Patterson and Ruckstuhl 2013 ) species, for example, are more prone to increased parasite loads. It is unknown whether individual cleaners respond asymmetrically to client identities and vice versa, influencing interaction patterns. Here, to investigate the consequences of within and between-species diversity on the outcome of mutualistic interactions, we quantified both cleaner and client behavior in situ. We observed the cleaning interactions between the predominant Caribbean cleaner fish, the sharknose goby ( Elacatinus evelynae ), and their reef fish clients. These cleaner species rarely cheat by causing damage to client bodies ( Soares et al. 2008 ), and thus their cleaning behavior represents a simpler system for studying cleaner–client interactions compared to the iconic bluestreak wrasse cleaners ( L. dimidiatus , Côté and Soares 2011 ). Previous work has documented personality variation in (noncleaning) goby species (e.g., Magnhagen et al. 2014 ; Moran et al. 2016 ; Vallon et al. 2016 ), and as such, we expected sharknose gobies to show individual variation in major axes of personality traits (activity, boldness, and exploration). As personality traits can influence foraging behaviors, and clients will differ in the food material they host, we then determined whether different personality variations had consequences on cleaning behaviors (frequency, rate and which clients’ cleaners interacted with). Finally, since clients can also regulate mutualistic outcome patterns, we tested whether clients interacted differently with cleaners based on the cleaners’ personality traits (posing frequency, rates, and client functional identity).",
"discussion": "DISCUSSION This field study demonstrates that sharknose goby ( E. evelynae ) cleaners show personality variation with consistent interindividual variation in their activity, boldness and exploration behaviors. Both activity and boldness were linked with cleaner–client interactions: more active cleaners cleaned a lower diversity of clients at a lower rate, while bolder individuals experienced an increased posing frequency by their clients. Personality variation was associated with client functional traits (sociality, mobility, trophic level, and body size), influencing which client species interacted with an individual goby of a given personality type. In summary, we show that within and between-species diversity has consequences on mutualistic outcomes. Personality variation in activity influenced goby cleaner–client interaction dynamics. Due to increased metabolic demands, more active individuals are expected to increase their foraging behavior ( Careau et al. 2008 ; Brommer and Class 2017 ), but here, more active individuals cleaned at a lower rate, and cleaned a lower diversity of clients. For other cleaner species, active behaviors (e.g., dancing; Youngbluth 1968 , clapping; Chapuis and Bshary 2010 , and rocking; Becker and Grutter 2005 ) attracts clients, but here the most active cleaners were not visited more frequently by client fish, suggesting gobies do not use obvious advertising movements. Given that sharknose goby cleaners gain all their nutrition from client derived material ( Vaughan et al. 2017 ), more active gobies are utilizing a more limited resource (reduced cleaning rate and diversity of clients cleaned) for foraging gains. Therefore, they could be more efficient cleaners, or else the trait would not be expected to persist. An increased cleaning efficiency may explain why larger fish posed for more active gobies. Larger bodied fish tend to host more parasites ( Poulin and Rohde 1997 ), and will also gain a greater cost when posing: posing temporarily stops a client from foraging ( Grutter et al. 2002 ) and larger fish have increased energy demands ( Bachiller and Irigoien 2012 ). Clients can learn the identity of specific cleaners from past positive experiences ( Bshary and Schäffer 2002 ) or from observing how other individuals have been treated by the cleaner ( Bshary 2002 ), thus visiting more efficient cleaners could reduce a client’s costs associated with cleaning. Conversely, more active gobies may not need to be efficient since here they interacted with all client types: more active gobies would thus not be restricted in the types of food resources available. A future study comparing the diets (in terms of nutritional gains) between cleaner gobies with contrasting levels of activity would be useful for determining how important these traits are for goby fitness in a foraging context. Boldness influences foraging behaviors across many species ( Reale et al. 2007 ; Biro and Stamps 2008 ; David et al. 2011 ), but here bolder cleaners did not differ in their cleaning behavior (i.e., foraging rates/frequencies) compared to shyer fish (contrasting Wilson et al. 2014 ). Partner choice can facilitate cooperation ( Noë 2001 ), and bolder individuals were visited more frequently by clients compared to shyer individuals. Bolder animals are greater risk takers by definition ( Reale et al. 2007 ); bolder L. dimidiatus cleaners for example, take risks by cheating their clients more frequently than shyer fish ( Wilson et al. 2014 ). Although in other interaction contexts, bolder individuals are more likely to initiate and lead conspecific interactions ( Ioannou and Dall 2016 ), a beneficial trait for posing clients, bolder individuals may risk not interacting with, and appeasing, all clients. Instead, bolder fish may reduce their own energetic costs by only cleaning preferred clients for maximum benefit (facilitated by an increased abundance of client fish posing for them creating choice options). Indeed, bolder individuals only cleaned herbivorous clients which feed intensely on the benthos throughout the day ( Hay 1997 ). Benthic feeding brings potential clients in direct contact with the mobile crustacean ectoparasites which are often consumed during cleaning ( Arnal et al. 2001 ; Grutter 2002 ), thus these clients may host high parasite loads and hence food rewards. Exploration tendency increases how efficiently individuals utilize environments ( Brommer and Class 2017 ; Careau et al. 2008 ), and although exploration did not link with cleaning behavior (contrasting Wilson et al. 2014 ), more exploratory cleaners differed in which clients they cleaned (more exploratory individuals cleaned larger clients and the free-ranging fish). Larger clients are assumed to be prone to increased parasite loads ( Poulin and Rohde 1997 ) and being more exploratory may enable cleaners to quickly find parasites over a larger surface area: exploration is a measure of speed with which an individual moves around a novel environment ( Reale et al. 2007 ). In contrast, free-ranging clients are assumed to host fewer parasites compared to sedentary species ( Patterson and Ruckstuhl 2013 ), and thus being more exploratory may also facilitate cleaners to find and exploit more patchily distributed food sources ( Mathot et al. 2012 ). Mutualisms are maintained by positive interactions between partners, and for clients interacting with a cleaner they pay a cost. Thus clients must be responded to beneficially for them to return ( Bshary and Schäffer 2002 ). Although we found strong feedbacks between posing and cleaning behavior, this was not reflected at an individual level. Cleaning behaviors expressed towards clients by more active, more exploratory or bolder fish did not reflect client posing behavior and vice versa. The identities of clients cleaned versus those posed also did not align, with the exception of herbivorous fish posing to and being cleaned by bolder gobies. Cleaner gobies are thought to rarely cheat by causing damage to client bodies ( Soares et al. 2008 ), but this selective strategy for certain clients, irrespective of who is posing, may represent a subtler form of dishonesty. Overall, through partner identity, choice, and behavior, sharknose gobies with certain personality variations may reduce the maintenance of the mutualism in terms of the positive feedback between cleaning and posing. Mutualisms involve many different asymmetric partners interacting with one another, and here we show for the iconic cleaner–client interaction that within and between-species diversity can influence mutualism outcomes. We demonstrate that there are asymmetries in interaction outcomes between different individuals, which will create heterogeneous patterns at the population level, a common feature across studies of cleaner–client interactions. Here, within-species individual differences (of cleaners) linked with between-species differences (of clients), contributing to who interacts with whom. Sharknose goby cleaning interactions have often been regarded as simple cleaning interactions with cleaner and client behaviors having no consequences on the interacting partner ( Soares et al. 2008 ; Côté and Soares 2011 ). However, through behavioral feedbacks, and the expression of differing traits, we suggest that partner behaviors and identities can strongly influence mutualisms, albeit in a subtler way than those observed for the bluestreak wrasse ( L. dimidiatus ). Ultimately, this work may be applied to aquaculture, where cleaner fish are currently inefficiently deployed to biologically control ectoparasites of farmed fish (see Rae 2002 ). Given that client identity is fixed in these systems, our study suggests that selecting cleaners based on their behavioral traits (as suggested by Powell et al. 2017 ) or altering personality types through training (e.g., Frost et al. 2007 ) may increase the efficiency of deployed cleaners."
} | 4,520 |
24019922 | PMC3760914 | pmc | 6,436 | {
"abstract": "Extracellular electron transfer involving microbes is important as it closely reflects the ability of cells to communicate with the environment. However, there are few reports on electron transfer mechanisms of pure microalgae and a lack of any model alga to study the transfer processes. In the present study, nine green microalgae species were isolated from wastewater and characterized in terms of their ability to transfer electrons between cells and an electrode. One species showed direct electron transfer via membrane-associated proteins and indirect electron transfer via secreted oxygen. The microalga was identified as Desmodesmus sp. based on phylogenetic analysis and electron microscopy. Electrochemical tests demonstrated that Desmodesmus sp. was able to act as a cathodic microorganism. Stable current densities of −0.24, 35.54 and 170 mA m −2 were achieved at potentials of +0.2, −0.2 and −0.4 V, respectively, under illumination. Dissolved oxygen concentration measurement showed gradients within the microalgae biofilm: 18.3 mg L −1 in light decreasing to 4.29 mg L −1 in the dark. This study diversified the exoelectrogen library and provided a potential model microalga to explore the associated mechanism of extracellular electron transfer.",
"conclusion": "Conclusions \n Desmodesmus sp. A8 displayed an oxidation peak in the potential range of +100 to +200 mV in cyclic voltammogram. The results confirmed that Desmodesmus sp. has the ability to transfer electrons to the electrode via electro-active proteins located on the cellular surface or via secreted oxygen. Illumination was shown to affect the current output due to the influence on oxygen generation by A8 cells. Hence, Desmodesmus sp. A8 is able to act as a cathodic microorganism under illumination.",
"introduction": "Introduction Bioelectrochemical systems (BES) are paid increasing attentions because of their ability to provide power and to treat wastewater with the assistance of electroactive microorganisms [1] . Recently, the function of BES has been expanded to generate value-added products [2] . Microalgae, as one of the most abundant microorganisms, are able to access solar energy to split water, providing electrons and oxygen [3] , [4] . Adoption of microalgae in BES can produce organic matter and simultaneously consume carbon dioxide on an electrode surface [5] , [6] . Other functions, such as N, P absorption [7] , biodiesel production [8] and biomass supply [9] increase the potential application of microalgae in renewable energy generation and wastewater treatment. Understanding of extracellular electron transfer will be helpful in optimizing practical applications and developing new functions for BES. The electron transfer mechanism for bacteria has been proposed through analysis of model organisms, e.g. Geobacter and Shewanella genera [10] , [11] . Direct electron transfer is via electroactive proteins, while indirect transfer is with the aid of redox mediators secreted by bacteria. However, electron transfer between electrodes and microalgae has not been addressed [12] , which limits the available information on optimization and extension of the functions of electrode-microalgae interactions. In view of increasing interest in adopting photosynthetic microalgae, a more comprehensive insight into the electron transfer mechanism between microalgae and electrode is of interest. Moreover, a model for pure algae is required to study the mechanism. In the present study, the novel microalgae strain Desmodesmus sp. isolated from wastewater was investigated in terms of electron transfer mechanism and application to enhance the current generation under various conditions.",
"discussion": "Results and Discussion Light Microscopic Observation of the Isolated Microalgae Wastewater was sampled onto agar plates. Green microalgae clones were picked up after a few cycles of agar plate spreading. Nine species were isolated. The morphology of isolated strains was observed with light microscopy ( Figure 1 ). Five species ( Figure 1b,1 e,1 f, 1h and 1i) were spheres with different size, of which the largest diameter is about 30 µm ( Figure 1b ) and the smallest one is about 3 µm ( Figure 1i ). The isolated unicellular alga shown in Figure 1a has a spherical cell body with spiny projections, and a diameter of 18–20 µm, which are the diagnostic characteristics of Golenkinia . From Figure 1d , the cells are deeply divided in the middle by a short isthmus, and the two semi-cells are oval. Microscopic analysis of the samples allowed preliminary identification of this isolate as genus Cosmarium . Two Desmodesmus with different size and form were isolated ( Figure 1c and 1g ); one is a four-cell coenobia which is crescent-shaped, and the other is two-cell coenobia which is the most frequently with oven form and denoted A8. 10.1371/journal.pone.0073442.g001 Figure 1 Light microscope (1000×) pictures of microalgae isolates. Electron Transfer Mechanism To evaluate the redox activity of the nine isolates, CV measurements for the isolated microalgae on glassy carbon were carried out under anaerobic conditions. The supernatant of the microalgae culture solutions were also collected at the end of the batch experiment. As shown in Figure 2 , an oxidation peak was observed in the potential range of +100 to +200 mV for the isolated alga A8 under nitrogen conditions, but no peaks were observed when the supernatant was tested (Figure S1 in File S1 ). The ability of electron transfer at electrode/biofilm interfaces is the characteristic to distinguish exoelectrogens. For bacteria (i.e. Shewanella oneidensis MR-1 [20] and Geobacter sulferreducens \n [21] ), mechanisms including indirect transfer via flavin and direct transfer via proteins were reported [19] ; some cytochromes of terminal reductases are involved in electron transfer processes. For microalga A8, since there was no electrochemical response of the supernatant, a proposed mechanism is that some proteins such as cytochromes on the outer membrane may be involved in direct electron transfer involving A8. After electrochemical testing, the A8 strain was adopted for further study. 10.1371/journal.pone.0073442.g002 Figure 2 Cyclic voltammograms of strain A8 on glassy carbon under anaerobic conditions. Morphological Characterization For the isolated strain A8, the morphological characteristics included the presence of dents at the pole of the coenobia and ribs on the cell surface ( Figure 3a and 3b ), which are characteristic to Desmodesmus \n [22] , [23] . The most striking feature of the cell wall ornamentation is the uninterrupted pattern of ribs and the absence of large warts, which are also in line with the diagnostic characters of Desmodesmus \n [24] ; the presence of spines and the surface morphology of the cell wall are two important diagnostic characteristics to distinguish Scenedesmus and Desmodesmus species [23] . In cellular suspensions with moderate density, two-cell coenobia were the most frequent, but singular cell coenobia was rarely observed (Figure S2 and S3 in File S1 ). 10.1371/journal.pone.0073442.g003 Figure 3 Scanning electron micrographs of A8 cell. A: Magnification×2000; B: Magnification×11000. The strain A8 shows small dents observed at the poles (shown in thin arrow). \n Figure 4a shows the overall image and cross-sections through the cell of A8. The TEM images showed that the A8 is an oval-shaped microalga, ranging from 3 to 5 µm in width and 8 to 12 µm in length ( Figure 4b ). Abundant chloroplasts are located peripherally, with sparse opaque thylakiod lamellae, and occupy almost half of the cell volume. A well-developed pyrenoid surrounded by a ring of starch granules is observed. Next to the starch grains, a nucleus is located, in which a nucleolus can be seen. Dictyosomes occur in a close proximity to the nucleus. Several mitochondrial profiles with different size are also easily seen in the cytoplasm. The outermost cell wall layer surrounding the entire coenobium is observed, and the thick cell wall consists of at least two layers. The inner, thickest layer is the cellulose layer (Cl) ( Figure 4c ). The outer layer, named the “warty layer”, has wall ornamentation consisting of rosettes and spines [25] . 10.1371/journal.pone.0073442.g004 Figure 4 Transmission electron micrographs of A8 cell. A: Transverse section through two-celled coenobia, Bar = 2 µm; B: Transverse section through a cell, Bar = 0.5 µm; C: Part of a cell wall, Bar = 0.5 µm (Abbreviations: Ch = chloroplast; Cl = cellulosic layer; M = mitochondrial profiles; Nu = nucleolus; P = pyrenoid; S = starch; Sp = spine.). Sequences and Phylogenic Analysis The ITS1 and ITS2 sequences are important alternative markers for investigating the phylogenetic relationship within the Scenedesmaceae \n [26] . To assess the molecular diversity of the morphotype represented by strain A8, the 18S rRNA gene–ITS1–5.8S rRNA gene–ITS2 regions were sequenced. The length of the sequence amplied was 714 bp, including 27 bp 18S rRNA gene, 213 bp ITS1, 181 bp 5.8S rRNA gene, 280 bp ITS2, and 13 bp 28S rRNA gene. The sequence in this study has been deposited in the GenBank database under accession number JQ973888. BLAST was applied to find regions of similarity between biological sequences. The results showed that strain A8 should indeed be assigned to the genus Desmodesmus and was most closely related to Desmodesmus hystrix isolate NDem 9/21 T-9W DQ417551 (98% sequence similarity), Desmodesmus brasiliensis FR865708 (96% sequence similarity), and Desmodesmus pannonicus FR865710 (94% sequence similarity). These three strains formed a distinct subcluster in the neighbor-joining, in which the new isolate and Desmodesmus hystrix isolate NDem 9/21 T-9W DQ417551 formed a distinct subline ( Figure 5 ). 10.1371/journal.pone.0073442.g005 Figure 5 Phylogenetic tree of strain A8 and closely related species based on ITS sequences of ribosomal DNA. The tree was constructed using the neighbor-joining method. The numbers at nodes indicate the percentages of occurrence of the branching order in 1000 bootstrapped trees for values greater than 50%. Scale bar = 1% divergence. Contribution of A8 to Current Generation The function of A8 for current generation under illumination was evaluated by holding potentials of −0.4, −0.2, and +0.2 V vs Ag/AgCl. The setup was autoclaved and then filled with sterile BG11 and strain A8 (OD 680 = 0.4) as inoculum. The current generation was clearly related to potential as shown in Figure 6 . The current density at −0.4 V was 170 mA m −2 for Desmodesmus sp. A8, while the control setup generated a current density of 109 mA m −2 (Figure S4 in File S1 ). When the potential was increased to −0.2 V, the current density decreased to 35.9 mA m −2 , which further decreased −0.24 mA m −2 as the potential was shifted to +0.2 V ( Figure 6 ). Wang et al reported the effect of potential on current generation for an aerobic biocathode [27] ; a low potential can generate high current for oxygen reduction reaction. 10.1371/journal.pone.0073442.g006 Figure 6 Current–time responses of strain A8 at potentials of +0.2,−0.2 and −0.4 V versus Ag/AgCl under illumination. Effect of Illumination on Current Generation Light supply is one of the most important factors that can affect the photosynthesis efficiency and metabolic pathways of microalgae [28] . Therefore, light should affect the performance of A8 incubated biocathode. In this study, current generation was compared in light and dark. The current density gradually increased from 17.7 to 164 mA m −2 under illumination in the first cycle. As the light turned off, the current rapidly decreased to 18.3 mA m −2 . For the control, no current change was observed ( Figure 7 ). As previously reported, DO concentration in the cathode is a key factor that can affect the performance of the cathode. Light supplies to the microalgae can accelerate the photosynthesis reaction, produce more oxygen for the cathode as electron acceptor, and thus enhance the current output. The same phenomenon was also observed by other research groups [28] , [29] . 10.1371/journal.pone.0073442.g007 Figure 7 Current responses to light and dark at −0.4 V. The grey shaded zones indicate dark conditions. In the study, DO concentration was also determined using a microelectrode technique to get comprehensive understanding on how illumination affects the activity of algae at the micro-scale. Accompanying with the change in current, the DO rose to 18.3 mg L −1 under illumination, more than twice the saturated DO in deionized water ( Figure 8 ), in agreement with Xiao et al. [30] . While in the dark, the DO concentration dropped to 4.22 mg L −1 , lower than that of the control in light and suggesting that oxygen was consumed. Moreover, the DO concentration changed with the biofilm depth. In light, the DO apparently increased from 17.4 to 18.2 mg L −1 as the biofilm depth increased from −200 to 550 µm, and then decreased to 17.8 mg L −1 as the depth further increased to 800 µm. However, the DO within the biofilm decreased from 4.63 to 4.22 mg L −1 in the dark. For the control under illumination, the DO decreased from 8.75 to 8.52 mg L −1 as depth increased. These results demonstrated that A8 contributed to the DO concentration under illumination, which was dependent on the thickness of the biofilm. Under illumination, light energy was captured by chlorophyll antennae of algae, to split water with oxygen release [31] . Light intensity decreased as the biofilm depth increased, resulting in different amounts of energy captured by microalgae and thus different amounts of oxygen generated. The oxygen produced tended to diffuse out from the biofilm to the bulk solution. Therefore, the DO concentration first displayed an increasing gradient, but as the biofilm depth further increased, insufficient light for photosynthesis and oxygen consumption by microalgae/electrode resulted in the decreasing DO concentration. 10.1371/journal.pone.0073442.g008 Figure 8 Profiles of dissolved oxygen within the A8 biofilm. DO is, in general, a limiting factor for biocathodes using oxygen as electron acceptor. One difference between microalgae and bacteria is the function of oxygen production. The DO gradient was positively related to the current; an anoxic zone near the electrode surface would ultimately be achieved for bacteria system [27] , but would be overcome by microalgal biocathode systems."
} | 3,631 |
34081409 | PMC8313290 | pmc | 6,437 | {
"abstract": "Summary The use of the methylotrophic yeast Pichia pastoris ( Komagataella phaffi ) to produce heterologous proteins has been largely reported. However, investigations addressing the potential of this yeast to produce bulk chemicals are still scarce. In this study, we have studied the use of P. pastoris as a cell factory to produce the commodity chemical 3‐hydroxypropionic acid (3‐HP) from glycerol. 3‐HP is a chemical platform which can be converted into acrylic acid and to other alternatives to petroleum‐based products. To this end, the mcr gene from Chloroflexus aurantiacus was introduced into P. pastoris . This single modification allowed the production of 3‐HP from glycerol through the malonyl‐CoA pathway. Further enzyme and metabolic engineering modifications aimed at increasing cofactor and metabolic precursors availability allowed a 14‐fold increase in the production of 3‐HP compared to the initial strain. The best strain (PpHP6) was tested in a fed‐batch culture, achieving a final concentration of 3‐HP of 24.75 g l −1 , a product yield of 0.13 g g −1 and a volumetric productivity of 0.54 g l −1 h −1 , which, to our knowledge, is the highest volumetric productivity reported in yeast. These results benchmark P. pastoris as a promising platform to produce bulk chemicals for the revalorization of crude glycerol and, in particular, to produce 3‐HP.",
"conclusion": "Conclusions In this study, we successfully introduced the malonyl‐CoA to 3‐HP pathway in P. pastoris for 3‐HP production. The use of pGAP, a strong constitutive promoter, to drive expression of the biosynthetic mcr gene, combined with the use of glycerol as carbon source, proved to be key to obtain higher space‐time yields than in other yeasts. The subsequent combination of protein and metabolic engineering strategies performed in this study have led to a 14‐fold increase in the 3‐HP yield. Moreover, a controlled fed‐batch strategy has shown the ability of P. pastoris to produce up to 24.75 g l −1 of 3‐HP in 45.5 h, achieving an overall yield of 0.13 Cmol Cmol −1 , and a productivity of 0.54 g l −1 h −1 . Overall, we benchmarked P. pastoris for 3‐HP production, demonstrating the potential of this cell factory for platform chemicals bioproduction. In addition, this study serves as a basis for further optimization of this platform through integrated systems metabolic engineering and bioprocess engineering strategies, which are paramount to reach economically attractive metrics for 3‐HP production from crude glycerol in P. pastoris .",
"introduction": "Introduction In 2004, the US Department of Energy (DOE) published the list of the top value‐added bio‐based chemicals to be produced from biomass (Werpy and Petersen, 2004 ). The production of such bio‐based products in a biorefinery is necessary to complement biofuel production, which is a low value‐added product with a high price volatility. Co‐producing biofuels and chemicals in an integrated biorefinery would allow the design of an economically robust and sustainable process, making it an attractive investment option. The DOE report ranked 3‐hydroxypropionic acid (3‐HP) among one of these top value‐added chemicals to be produced in a biorefinery. The largest application of 3‐HP is its conversion into acrylic acid and other products typically derived from petroleum (Della Pina et al ., 2011 ; Kumar et al ., 2013 ). The global market size of acrylic acid is estimated to reach 22 550 M$ in 2022 (Grand View Research, 2016 ). Due to such promising market forecast, Novozymes and Cargill announced a joint agreement to develop a platform to produce acrylic acid from biologically produced 3‐HP in 2008 (Novozymes, 2008 ). Nevertheless, up to date, the process has not been implemented at a large scale, as further development is still required. While the revalorization of lignocellulosic products consisting of glucose and xylose has been largely investigated, there are other waste products that can be used as substrates to produce bulk chemicals. This is the case of crude glycerol, a side‐product obtained during the enzymatic production of biodiesel. It is a mixture made of 60–80% glycerol, 10–20% methanol and 10–20% of soap or other undefined organic matter compounds (Luo et al ., 2016 ). The use of this mixture as substrate is limited by the fact that methanol is toxic to many microorganisms. The methylotrophic yeast Pichia pastoris is a promising microorganism for the revalorization of crude glycerol as it can efficiently grow using both glycerol and methanol as carbon sources. Moreover, P. pastoris can grow at a low pH, and it is reported that the cost of the downstream process is reduced if the fermentation is performed at a pH below the pKa value of the acidic product (van Maris et al ., 2004 ). For all these reasons, in this study, we investigate the production of 3‐HP from glycerol in the yeast P . pastoris . \n Pichia pastoris is widely used in industrial biotechnology as an efficient host for recombinant protein production, and it has received increasing interest as a platform to produce fine and bulk chemicals (Schwarzhans et al ., 2017 ; Peña et al ., 2018 ). Moreover, the efforts of the P. pastoris community have allowed the implementation of the necessary tools allowing its use in metabolic engineering research, including up‐to‐date genetic engineering tools like the GoldenMOCS (Prielhofer et al ., 2017 ) and CRISPR‐Cas9 (Weninger et al ., 2016 ), metabolic genome‐scale models (GSM; Tomàs‐Gamisans et al ., 2016 ), metabolomics and fluxomics protocols (Carnicer et al ., 2012 ; Ferrer and Albiol, 2014 ), and a wide knowledge of its behaviour at the bioreactor scale (Looser et al ., 2014 ; Yang and Zhang, 2018 ). Another interesting trait of P. pastoris is that it is a Crabtree‐negative yeast. This is an interesting feature in metabolic engineering because overflow metabolism to undesired by‐products, such as ethanol or glycerol, can be minimized. The full oxidation of the carbon source leads to a higher energetic yield than in Crabtree‐positive yeasts, like Saccharomyces cerevisiae , thus leading to potentially higher product yields (Dai et al ., 2018 ; Peña et al ., 2018 ). Moreover, the extracellular concentration of some intermediate metabolites of the TCA cycle (i.e. malate or citrate) is lower in P. pastoris than in S. cerevisiae under similar conditions (Carnicer et al ., 2012 ). This trait simplifies the downstream processing of the products of interest. The biological production of 3‐HP has been largely investigated (de Fouchécour et al ., 2018 ). Several pathways have been tested in a number of microorganisms, including the industrial workhorses Escherichia coli and S . cerevisiae . Each pathway to produce 3‐HP is named according to its precursor. The route that has obtained the highest yields and productivities is the coenzyme B12‐dependant glycerol pathway (Raj et al ., 2008 ; Rathnasingh et al ., 2009 ). However, the implementation of this route in P. pastoris is currently unfeasible at an industrial scale due to the high production costs caused by the requirement of coenzyme B12 addition, which is an expensive compound (Chen et al ., 2014 ). The 3‐HP route through β‐alanine has been investigated in S. cerevisiae (Borodina et al ., 2015 ) and E. coli (Song et al ., 2016 ) and the route which uses malonyl‐CoA as a precursor has been implemented in several microorganisms, including E. coli (Rathnasingh et al ., 2012 ; Liu et al ., 2013 , 2016 ), S. cerevisiae (Chen et al ., 2014 ; Kildegaard et al ., 2016 ) and Shizosaccharomyces pombe (Suyama et al ., 2017 ). Using glucose as substrate, the maximum theoretical yield of the β‐alanine to 3‐HP pathway is higher than the route starting from malonyl‐CoA, as more ATP is required for the latter. However, when glycerol is used as a substrate, the maximum theoretical yield for both pathways equals 1, as there is net ATP production in both cases (see Supplementary materials Section Data S1 ). While the β‐alanine pathway would require the expression of 3 heterologous genes (Borodina et al ., 2015 ) to achieve 3‐HP production in P. pastoris , it is reported that the single expression of the bifunctional enzyme malonyl‐CoA reductase from Chloroflexus aurantiacus (MCR Ca ) triggers 3‐HP production in yeast through the malonyl‐CoA route (Chen et al ., 2014 ). This enzyme performs two NADPH‐consuming consecutive reactions sequentially converting malonyl‐CoA to malonate semialdehyde (MSA), and then, MSA is converted to 3‐HP (Fig. 1 ). Fig. 1 Simplified representation of the conversion of glycerol to 3‐HP through the malonyl‐CoA route. The metabolic engineering targets to increase the availability of the precursors of the malonyl‐CoA to 3‐HP pathway are included. Acc, acetyl‐CoA carboxylase; cPos5, cytosolic NADH kinase; MCR (C‐ter), C‐terminal domain of malonyl‐CoA reductase; MCR (N‐ter), N‐terminal domain of malonyl‐CoA reductase. In this study, the production of 3‐HP using glycerol as substrate has been implemented in P. pastoris through the malonyl‐CoA pathway. The expression of mcr Ca \n leads to 3‐HP production in P. pastoris . This base strain has been further modified using two different strategies: protein engineering and metabolic engineering. The independent expression of the two subunits of the malonyl‐CoA reductase has shown higher 3‐HP production in other microorganisms (Liu et al ., 2013 ). This strategy has been further tested in P. pastoris, yielding a substantial improvement compared to the initial strain. Further modifications have been implemented to increase the fluxes producing the substrates of the malonyl‐CoA pathway to 3‐HP (NADPH and malonyl‐CoA). The strain producing the highest 3‐HP titre has been characterized in a fed‐batch culture using glycerol as a substrate. Overall, the potential of P. pastoris as a promising host for 3‐HP production from this renewable feedstock was demonstrated for the first time.",
"discussion": "Results and discussion Expression of mcr Ca \n in P. pastoris leads to 3‐HP production In the present work, we expressed mcr \n Ca gene in P. pastoris under the control of the constitutive and strong GAP promoter (Waterham et al ., 1997 ). Comparison of the resulting strain, PpHP1, to the reference strain (X‐33) in triplicate shake flasks on buffered minimal glycerol (BMG) medium showed that the sole expression of mcr Ca \n resulted in 3‐HP production, while it did not affect cell growth (the µ max of the control and PpHP1 strains was the same within the precision range, 0.24 ± 0.01 h −1 and 0.24 ± 0.01 h −1 respectively). Moreover, no by‐products were detected in any of the two strains (Fig. S2 ). Notably, the PpHP1 strain produced 0.19 ± 0.03 g l −1 of 3‐HP after 24 h of cultivation. Such 3‐HP titre is considerably higher than those achieved in S . pombe (Takayama et al ., 2018 ) and S. cerevisiae (Chen et al ., 2014 ) harbouring a similar genetic construction and using glucose as a substrate (0.016 g l −1 and 0.093 g l −1 respectively). Furthermore, the C‐yield (Cmol of 3‐HP per Cmol of substrate, i.e. glycerol or glucose) for the PpHP1 strain (0.015 ± 0.002 Cmol Cmol −1 ) was remarkably higher than the one observed in other yeasts (0.0003 Cmol Cmol −1 in S . pombe , and 0.0048 Cmol Cmol −1 in S. cerevisiae ). Remarkably, the specific activity of MCR in PpHP1 was 0.30 ± 0.06 U mg −1 of protein, which is remarkably higher than the specific activity reported in S. cerevisiae (0.008 U mg −1 ; Chen et al ., 2014 ). The combined effect of the strength of the P. pastoris GAP promoter and the fact that glycerol is more reduced than glucose, which leads to net production of ATP from the substrate, instead of net ATP consumption (Fig. S1 ), points at P. pastoris as a promising cell factory for the bioproduction of 3‐HP from glycerol. Setting up the screening conditions for 3‐HP producing P. pastoris clones We established two independent analytical methods based on NMR and HPLC‐MS for 3‐HP quantification, which were cross‐validated. The results using the HPLC‐MS method diverge from those obtained using NMR when glycerol is still present in the medium (Fig. S3 ). This is explained by the matrix effect caused by the glycerol co‐eluting with 3‐HP from the column, which affects the ionization efficiency at the ionization source. However, once the glycerol is fully consumed, the HPLC‐MS and the NMR methods yielded statistically identical results when measuring the final 3‐HP concentrations in the PpHP1 shake flask cultures described in Section 3.1 (i.e. 3‐HP final concentrations of 0.18 ± 0.02 g l −1 of 3‐HP and 0.19 ± 0.02 g l −1 of 3‐HP respectively). Consumption of 3‐HP has been reported in several microorganisms (Zhou et al ., 2014 ; Yang et al ., 2017 ). In order to evaluate whether the PpHP1 strain was able to assimilate this compound, this strain was grown in triplicate shake flasks using BMG, BMG medium supplemented with 2.5 g l −1 of 3‐HP (BMG3HP) or buffered minimal medium supplemented with 2.5 g l −1 of 3‐HP as a sole carbon source (BM3HP). Samples were collected after 24, 48 and 72 h. The 3‐HP concentration remained constant throughout the 72 h in the cultures grown on BM3HP (no growth was observed). After 24 h, glycerol was exhausted from the BMG and BMG3HP, while the concentration of 3‐HP remained unaltered between the 24 and the 72 h (data not shown), thereby indicating that P. pastoris does not assimilate 3‐HP as C‐source within the experimental time frame tested. Considering these results, screening of the 3‐HP‐producing strains was performed taking end‐point samples after 48 h of incubation to ensure full consumption of glycerol, followed by 3‐HP quantification using the HPLC‐MS method. Improvement of 3‐HP production by MCR enzyme engineering Recent studies have shown the positive effect of dissecting the MCR enzyme in the two subunits catalysing each of the two reactions converting malonyl‐CoA into 3‐HP (Liu et al ., 2013 ). The presence of three point mutations causing three amino acidic changes (N940V, K1106R and S1114R) had an additional positive impact on 3‐HP production in E. coli (Liu et al ., 2016 ). The same outcome has been demonstrated in S . pombe , where the expression of the N‐terminal and the improved version of the C‐terminal domain of MCR using two independent expression cassettes led to a 30‐fold improvement in the final 3‐HP titre, compared to the starting strain expressing the original sequence of MCR Ca (Takayama et al ., 2018 ). Therefore, we introduced two expression cassettes into P. pastoris allowing the independent expression of the coding DNA sequences for the N‐terminal and the C‐terminal domains of MCR (including the wild‐type and the mutated version of the latter) under the control of the GAP promoter. As shown in Fig. 2 , the dissection of MCR (strain PpHP2) had a positive impact on 3‐HP production, triggering a 12.5‐fold increase in 3‐HP production (1.63 ± 0.09 g l −1 3‐HP). Nevertheless, the introduction of the 3 point mutations in the C‐terminal domain (strain PpHP3) resulted in a non‐producing strain. Similarly, the introduction of these 3 point mutations to a non‐dissected version of MCR resulted in a non‐producing P. pastoris strain (data not shown). These point mutations are far from the reactive site and the NADPH binding site of the enzyme. It remains poorly understood how distant mutations may affect catalytic properties. Moreover, as the structure of the C‐terminal domain of MCR from C . aurantiacus is not available, the exact effect of the point mutations on the protein conformational stability/quality is hard to predict, particularly when the protein is synthetized at high rates (i.e. using a strong promoter). Notably, the 12.5‐fold improvement obtained by dissecting MCR is in the same range as those reported in S . pombe and E. coli . Fig. 2 Production of 3‐HP for each strain in the screening experiments. The genes heterologously expressed in each strain are depicted in the left side of the graph. The grey bars show the average 3‐HP concentration at the end of the culture, the discontinuous line shows the standard deviation, the solid line indicates the SE and the circles show the average result for each clone. The solid circle of a PpHP5 clone shows the result of a clone which was discarded for the calculations, as it had a different behaviour from the rest of the clones of that strain. For PpHP6, only 3 clones could be screened. Despite that more than 50 transformants of PpHP6 from 2 independent transformations were checked using colony PCR, only 3 clones resulted positive. Metabolic engineering of P. pastoris to improve 3‐HP production Metabolic engineering for 3‐HP production through the malonyl‐CoA pathway was aimed at increasing the availability of the two precursors of this route, namely NADPH and malonyl‐CoA. To do so, the ACC Yl \n and cPOS5 Sc \n genes have been heterologously expressed in the PpHP2 strain. The reactions catalysed by the enzymes encoded by these two genes are depicted in Fig. 1 . Acc produces malonyl‐CoA from acetyl‐CoA, which is the precursor of the central carbon metabolism for 3‐HP production. The overexpression of such enzyme has already been performed in E. coli (Rathnasingh et al ., 2012 ), S. cerevisiae (Chen et al ., 2014 ; Kildegaard et al ., 2016 ) and S . pombe (Takayama et al ., 2018 ) to increase the production of 3‐HP. The enzyme cPos5 produces NADPH by means of NADH and ATP consumption, and the overexpression of its gene leads to an increase in the NADPH/NADP ratio (Tomàs‐Gamisans et al ., 2020 ). As shown in Fig. 2 , the overexpression of Acc or cPos5 in the strain PpHP2 – resulting in the strains PpHP4 and PpHP5, respectively – led to a small increase in 3‐HP production. Still, such increases were not statistically significant compared to the parental strain PpHP2 ( P ‐values of 0.27 and 0.15 respectively). However, when both genes were overexpressed at the same time (PpHP6), a significant increase in the final 3‐HP concentration was observed ( P = 0.015). The PpHP6 strain produced 1.81 ± 0.04 g l −1 of 3‐HP, which represents a 14‐fold increase compared to the starting strain (PpHP1), and a 12% increase compared to PpHP2. The C‐yield for PpHP6 was 0.146 ± 0.003 Cmol Cmol −1 . Similar strategies in other microorganisms resulted in similar outcomes. For example, in E. coli , the overexpression of either ACC or PntAB (a transhydrogenase encoding gene) led to a twofold increase, while the co‐expression of both genes led to a threefold increase (Rathnasingh et al ., 2012 ). It is also worth noticing that in S . pombe , enzyme engineering and expression level adjustment of MCR led to a 30‐fold increase in 3‐HP titres, while increasing the availability of acetyl‐CoA and CoA in this strain resulted in a twofold increase. Altogether, these results point at the flux from malonyl‐CoA to 3‐HP as the main limiting factor for 3‐HP production. Further metabolic studies should corroborate this hypothesis. Indeed, the increase of the number of copies of the mcr gene has already been demonstrated to have a positive effect in 3‐HP production in both S. cerevisiae (Kildegaard et al ., 2016 ) and S . pombe (Takayama et al ., 2018 ). Overall, the product yield obtained by the strain PpHP6 during the screening phase (0.146 ± 0.003 Cmol Cmol −1 ) is significantly higher (around 1.8‐fold) to the highest yield observed in yeast under comparable conditions (deep‐well plate culture using defined medium), i.e. using the S. cerevisiae strain 3HP‐M11 producing 3‐HP through the malonyl‐CoA pathway, with a yield on glucose of 0.080 ± 0.008 Cmol Cmol −1 (calculated from data given by Kildegaard et al ., 2016 ). Production of 3‐HP in a fed‐batch culture The strain PpHP6 was further cultivated in a controlled fed‐batch culture. First, a batch experiment was performed in order to determine the µ max of PpHP6, and also the initial biomass concentration ( X \n 0 ) and the biomass to substrate yield ( Y \n X/S ), which are the parameters required to set the exponential feeding rate (see Section 2.4). The µ max was 0.19 ± 0.1 h −1 , the X \n 0 was 18.6 ± 0.2 g l −1 and the Y \n X/S 0.47 ± 0.01 g g −1 . After the initial batch phase (22 h), the exponential feeding rate was set to maintain a growth rate equal to 0.1 h −1 (approximately 50% of the µ max ; Fig. 3A ). After 44 h of cultivation (22 h of feeding phase), glycerol accumulation was observed, and the feeding pump was stopped. Thereafter, the fermentation was terminated after 45.5 h of cultivation, when the pO 2 increased, indicating that all the glycerol had been consumed. Overall, 195 g l −1 of glycerol was added into the reactor, and 24.75 ± 0.54 g l −1 of 3‐HP was produced. The only by‐product detected by NMR was arabitol in the late stages of the batch and the fed‐batch phases. Fig. 3 Fed‐batch culture of PpHP6. A. Biomass and metabolites concentration during cultivation and total glycerol added (per litre) into the reactor is shown. It was calculated considering the volume of feeding added to the reactor and the actual culture volume. Error bars denote SE. B. Growth rate (µ), product yield (Yield 3‐HP/Glyc ), q‐rate of 3‐HP (qP 3‐HP), and q‐rate of arabitol (qP Arabitol) of the strain PpHP6 throughout the feeding phase of the fed‐batch culture at a pre‐set µ of 0.1 h −1 . Error bars show the SE. As already inferred from the evolution of physiological growth parameters over time, growth rate was not maintained during the whole feeding phase. Furthermore, the decrease in the growth rate was accompanied with the accumulation of glycerol and the production of arabitol towards the end of the cultivation. These results differ from previous observations of P. pastoris producing heterologous proteins in fed‐batch cultures operated under analogous conditions (i.e. using a pre‐programmed exponential substrate feeding strategy for controlled specific growth rate at 0.1 h −1 ), where the µ remained constant throughout the feeding phase and no arabitol accumulation was detected, achieving up to 100 g l −1 of dry cell biomass (Garcia‐Ortega et al ., 2013 ). The spline curves fitted to the evolution of the growth rate (µ), product yield ( Y \n P/S ), qP 3‐HP and qP Arabitol throughout the cultivation further reveal that the decrease in the growth rate coincides with the onset of glycerol accumulation and arabitol by‐product excretion (Fig. 3B ). Conversely, product yield and qP 3‐HP followed the opposite trend, i.e. the Y \n P/S is reduced from 0.134 ± 0.010 Cmol Cmol −1 at the first part of the feeding phase to yields close to 0.1 Cmol Cmol −1 coinciding with the highest qP Arabitol values. These results show how during the beginning of the feeding phase the C‐yield was close to the one obtained in the screening phase (0.146 ± 0.003 Cmol Cmol −1 ), even though the average yield of the overall fed‐batch culture was lower (0.130 ± 0.003 Cmol Cmol −1 and 0.127 ± 0.003 g g −1 ). A possible explanation for the observed trends is the increase in the 3‐HP concentration. Accumulation of 3‐HP in the fermentation broth has been reported to be toxic for other microorganisms like E. coli and S. cerevisiae . Such toxicity may be triggered by the conversion of 3‐HP into 3‐hydroxypropanaldehyde (reuterin), which causes oxidative stress to the cells through its interaction with reduced glutathione (Schaefer et al ., 2010 ; Kildegaard et al ., 2014 ). The sequestration of glutathione leads to a decrease in the maximal growth rate of S. cerevisiae at high 3‐HP concentrations (Kildegaard et al ., 2014 ). Another plausible explanation of the decrease in the µ at the final stage of the culture may be the increase in the ATP expenditure for maintenance at high extracellular concentrations of weak organic acid, i.e. 3‐HP. Protonated acid molecules may enter the cytoplasm by diffusion and later on dissociate, leading to an ATP cost for the re‐secretion of the organic acid and the restoration of the intracellular pH. The expression of a 3‐HP exporter in E. coli has proven to be beneficial, as it reduces stress caused by the intracellular dissociation of 3‐HP (Nguyen‐Vo et al ., 2020 ). Moreover, higher extracellular concentrations of 3‐HP have a direct impact on the thermodynamics of its secretion, leading to an increase in ATP expenses for 3‐HP export (van Maris et al ., 2004 ). All these phenomena would ultimately impact on the Y \n X/S in P. pastoris , explaining the decrease in the growth rate and the accumulation of glycerol at the later stage of the fed‐batch culture. Moreover, we observe the production of arabitol at the end of the batch and the fed‐batch phases (Fig. 3A ). Arabitol production in P. pastoris has been related to stress conditions caused by the unfolded protein response (UPR; Tredwell et al ., 2017 ), high osmolarity (Dragosits et al ., 2010 ) or hypoxic conditions, where arabitol production has been proposed to be used as a redox sink (Baumann et al ., 2010 ). Additionally, arabitol by‐product formation has been observed in lactic acid‐producing recombinant P. pastoris growing in batch cultures using glycerol as carbon source (Melo et al ., 2020 ). Therefore, the presence of arabitol points to a redox imbalance, either caused by 3‐HP‐derived toxicity effects, or by overexpression of cPos5. Further metabolomic and fluoxmic studies will help elucidating the cause. Altogether, these results suggest that the pre‐established bioreactor fed‐batch cultivation protocols for heterologous protein production in P. pastoris are not necessarily optimal for the production of weak acids such as 3‐HP, as its accumulation in the extracellular space and/or the toxic effect at high concentrations may lead to physiological changes negatively impacting on the bioprocess parameters such as the µ max or Y \n X/S . Moreover, the current cultivation protocol led to the production of arabitol as a by‐product at the end of the fed‐batch process, which caused a significant decrease in the product yield. Deletion of the arabitol dehydrogenase (ArDH) gene in a lactic acid‐producing strain reduced arabitol production, resulting in a 20% increase in product titres (Melo et al ., 2020 ). The tolerance to 3‐HP should also be addressed in order to increase further the product yield. To this end, both adaptive laboratory evolution (ALE) experiments and rational engineering strategies have proven successful to increase 3‐HP tolerance in other yeast and could be therefore transferred to P. pastoris . Specifically, ALE experiments led to the conclusion that overexpression of the S‐(hydroxymethyl)glutathione dehydrogenase gene ( SFA1 ) in S. cerevisiae restored growth at 50 g l −1 of 3‐HP (Kildegaard et al ., 2014 ). Expression of two mutated versions of SFA1 under the control of the native SFA1 promoter was also able to restore cell growth at 50 g l −1 of 3‐HP. The SFA1 residues which were mutated in S. cerevisiae are conserved in the homologous P. pastoris gene (PAS_chr3_1028). Therefore, a similar approach to avoid 3‐HP toxicity could be tested in P. pastoris . The overall productivity of the fed‐batch culture was 0.54 ± 0.01 g l −1 h −1 . To our knowledge, this is the highest 3‐HP productivity reported in yeast (de Fouchécour et al ., 2018 ; Lis et al ., 2019 ), and it is almost identical to the highest productivity reported using the malonyl‐CoA pathway, which was 0.56 g l −1 h −1 and it was reported in E. coli (Liu et al ., 2016 ). Such promising results can be attributed to the combination of three main factors: (i) the high specific MCR activity due to the strength of pGAP, extensively proven for heterologous protein production in P. pastoris ; (ii) the use of glycerol as a substrate, which delivers more ATP than glucose under aerobic conditions, leading to higher biomass and product yields compared to the use of glucose; and (iii) the use of glycerol as substrate allows for higher growth rates during the feeding phase compared to typical fed‐batch cultivations of Crabtree‐positive yeasts growing on glucose as substrate, thereby supporting higher volumetric productivities, while minimizing by‐product formation."
} | 7,125 |
36970394 | PMC9996702 | pmc | 6,439 | {
"abstract": "Using hydrogen oxidising bacteria to produce protein and other food and feed ingredients is a form of industrial biotechnology that is gaining traction. The technology fixes carbon dioxide into products without the light requirements of agriculture and biotech that rely on primary producers such as plants and algae while promising higher growth rates, drastically less land, fresh water, and mineral requirements. The significant body of scientific knowledge on hydrogen oxidising bacteria continues to grow and genetic engineering tools are well developed for specific species. The scale‐up success of other types of gas‐ fermentation using carbon monoxide or methane has paved the way for scale‐up of a process that uses a mix of hydrogen, oxygen, and carbon dioxide to produce bacteria as a food and feed ingredients in a highly sustainable fashion.",
"conclusion": "5 Conclusion Feeding H 2 , O 2 , and CO 2 to HOB to produce SCP and added value food and feed ingredients is a promising form of biotechnology as is demonstrated by the range of start‐up companies involved. There are still process efficiency and scale‐up challenges to overcome but both industry and academia are highly motivated. Furthermore, the increased need for sustainability and recent success stories in adjacent technologies suggest that this powerful application of HOB technology will be realised in the short‐ to medium‐term.",
"introduction": "1 Introduction Bacteria have been part of the human diet for millennia as components of many traditional foods, such as yoghurt, cheese, fermented vegetables, and fermented fish [ 1 , 2 ]. In the 1960s, initial steps were taken to directly harness bacterial biomass as a source of protein, fats, and vitamins, for which the term single‐cell protein (SCP) was subsequently coined [ 3 , 4 ]. Today multiple factors coincide to drive market interest in SCP, most importantly the increased market demand for protein, and sustainability issues surrounding current protein production. Here, the authors will focus on one of the most sustainable forms of SCP: SCP produced with autotrophic bacteria that fix carbon dioxide (CO 2 ) into their cellular biomass (Fig. 1 ).\n Fig. 1 \nGeneral process overview for SCP production using HOB and gaseous substrates\n The United Nations has forecasted the world population to grow from 7.7 billion today to 9.7 billion by 2050 [ 5 ]. Inevitably, this has prompted concerns for future global food security, particularly as 2 billion individuals already experienced some level of food insecurity in 2019 [ 6 ]. The supply of animal‐derived protein is expected to double by 2050 to meet the persistent global consumption demand for high‐quality protein. Correspondingly, protein feed sources for livestock and aquaculture must also increase, despite land resources already being stretched [ 7 , 8 ]. Agriculture currently utilises ∼43% of the land that is not a desert or covered by ice. This percentage will rise if agriculture is to meet the growing protein demand, which in turn will negatively affect biodiversity. Production of the type of SCP we focus on here requires much less land than agricultural protein (Fig. 2 ). To fix CO 2 energy is required. The basis of our current food supply is formed by plants that use light as their energy source. The energy requirement for the most sustainably produced SCP, as discussed in this review, uses hydrogen (H 2 ). This H 2 can be produced by electrolysis using sustainably produced electricity. If the required electricity is based on solar power it requires 0.18–0.26 m 2 /kg of protein/year, which is considerably less than the 6–16 m 2 of land/kg of protein per year needed for soybean. If wind power is used land‐use decreases to around 0.04 m 2 /kg of protein/year [ 9 ]. Fig. 2 \nIllustrating the advantage of protein production using HOB: reduced land and freshwater use"
} | 963 |
34909360 | PMC8639198 | pmc | 6,441 | {
"abstract": "Active matter refers to systems composed of elements that are self-propelled by the dissipation of energy, in which dynamical patterns emerge, as is the case of flocks of birds and schools of fish. Some researchers in active matter physics seek to identify unified descriptions of such collective motions through interdisciplinary approaches by biologists and physicists. Through such collaborations, experimental studies pertaining to active matter physics have been developing recently, which allow us to verify the proposed mathematical models. Here, we review collective pattern formations and behaviors of animals from the perspective of active matter physics.",
"conclusion": "Conclusion Active matter physics begun with the Vicsek’s theoretical model, which shed light on the pattern formation by local alignment. This review introduced some real systems of pattern formation consisting of traveling animals. These animals showed diverse pattern formations that Vicsek model cannot deal with., e.g., the dynamical network structure by C. elegans . Each pattern can be explained by the complex theoretical model. Getting an agreement of a real system and model require to control the parameters in not only the model but animals. C. elegans is a good model animal that serves a lot of ways to control the inner parameters. Then, active matter physicists expect to be able to unify these models and develop the unified theoretical model that can reproduce a wide range of pattern formations which Vicsek model cannot. However, there are still few experimental systems for controlling the parameters of mathematical models using animals. For such studies, the aforementioned Prof. Iain Couzin and his colleagues recently established a department of collective behaviour at the Max Planck Institute and the University of Konstanz. This department has attracted scientists from a wide range of disciplines, including not only biologists and physicists but also engineers studying the satellite tracking of animal behavior, data scientists with expertise in machine learning, and network scientists. They have focused on issues of the collective behavior and movement patterns of animals. In the domain of biology, ethologists have examined the collective behaviors of animals. The description based on observations has been the main tool in the research area. We anticipate more researchers who have interests in active matter physics will join the field of animal behavior in the future. Collective pattern formation in animals can lead to the emergence of new functions that do not appear at the individual level. Since animals have a nervous system, they possess memory and learning abilities, and it is interesting to discover whether these neural functions are related to the physics of collective pattern formation. To date, it has been noted that collective pattern formation can improve the detection sensitivity of foreign organisms and prey and enhance the ability to make correct decisions [ 23 – 25 ]. C. elegans also has a nervous system consisting of 302 neurons and is known to have the ability to memorize the past cultivation temperature [ 26 ] and to move to a preferable humid place [ 27 ]. Therefore, it is interesting to examine the relationship between these neural functions and collective pattern formation in C. elegans . In conclusion, active matter physics seeks to identify unified descriptions of collective pattern formations and needs more experimental verification of the proposed mathematical models through parameter controls. Additionally, the functional significance of each animal’s collective pattern formation and its mechanical relevance to neural functions have been important open questions. Furthermore, considering the research field of ‘soft robotics’, one of whose purposes is the elaborate control of collectives of robots, we hope that an established algorithm can be applied in controlling the collective motions of soft robots.",
"introduction": "Introduction How do locally interacting elements form a globally ordered pattern? Pattern formation by cells or individuals has fascinated us, and their underlying mechanisms have long been investigated in the field of biology. For example, developmental biologists have aimed to understand how each cell finds its own position to form tissue organs during morphogenesis. In the second half of the 20th century, morphogens, which are molecules that determine positional information by creating concentration gradients through diffusion, were identified, and attempts have been made to investigate morphogen-dependent molecular mechanisms. Furthermore, the mechanisms underlying skin pattern formation in fishes, such as the formation of stripe patterns, in terms of the reaction-diffusion system proposed by Turing, are widely studied in the field of mathematical biology [ 1 ]. In addition to transmitter-mediated pattern formation phenomena, there is a pattern formation phenomenon generated by elements that are self-propelled by the dissipation of energy, as observed in nonliving things, such as colloids and cytoskeletons, and living things such as biofilms of motile bacteria, flocks of birds, schools of fish, and crowds of humans [ 2 ]. Why do randomly moving particles collectively generate patterns without knowing their own positions in the entire structure? The research field that studies such pattern formation mechanisms is known as active matter physics. Active matter refers to systems composed of elements that are self-propelled by the dissipation of energy. The goal of active matter physics is to seek the universal laws underlying collective pattern formation. To achieve this purpose, both theoretical and experimental studies are required; a mathematical model proposed on the basis of theoretical research should be verified by the observation and perturbation of collective motions using living or nonliving things. There are many good reviews summarizing pattern formations from non-living things, such as colloids and molecular motors to unicellular organisms [ 2 – 5 ], but it is in contrast with a smaller number of reviews for animals’ pattern formations. Through the evolution of unicellular to multicellular organisms, animals have acquired complex brain functions such as memory, learning, and cognition. These brain functions enhance the efficiency of social behaviors represented by foraging, escaping from a predatory attack, and collective decision making. Thus, there are more examples of collective behaviors that have functional significance than those of non-living things and unicellular organisms. Therefore, it has been fascinating to consider which mechanisms underlie pattern formation by animal collectives, as observed in flocks of birds and schools of fish. Therefore, we here discuss the studies on animals’ collective pattern formations through their behaviors. Finally, we discuss future directions for this research field."
} | 1,735 |
27566647 | PMC5001958 | pmc | 6,444 | {
"abstract": "Co-fermentation of glucose, xylose and l -arabinose from lignocellulosic biomass by an oleaginous yeast is anticipated as a method for biodiesel production. However, most yeasts ferment glucose first before consuming pentoses, due to glucose repression. This preferential utilization results in delayed fermentation time and lower productivity. Therefore, co-fermentation of lignocellulosic sugars could achieve cost-effective conversion of lignocellulosic biomass to microbial lipid. Comprehensive screening of oleaginous yeasts capable of simultaneously utilizing glucose, xylose, and l -arabinose was performed by measuring the concentration of sugars remaining in the medium and of lipids accumulated in the cells. We found that of 1189 strains tested, 12 had the ability to co-ferment the sugars. The basidiomycete yeast Pseudozyma hubeiensis IPM1-10, which had the highest sugars consumption rate of 94.1 %, was selected by culturing in a batch culture with the mixed-sugar medium. The strain showed (1) simultaneous utilization of all three sugars, and (2) high lipid-accumulating ability. This study suggests that P. hubeiensis IPM1-10 is a promising candidate for second-generation biodiesel production from hydrolysate of lignocellulosic biomass.",
"introduction": "Introduction The lipid produced by microorganisms is considered to have powerful potential for the development of a new kind of energy, and has received significant interest from sustainable energy researchers. Lipid accumulated by oleaginous yeast is viewed as a promising alternative to second-generation biodiesel, since the composition of the fatty acids produced by yeast is suitable for biodiesel production. That is, it contains palmitic (16:0), stearic (18:0), oleic (18:1), and linoleic (18:2) acids at a high ratio, mainly in the form of triacylglycerol (TAG) (Beopoulos et al. 2011 ; Knothe 2009 ; Meng et al. 2009 ; Sitepu et al. 2014 ). Compared to other oleaginous microorganisms, oleaginous yeasts are advantageous due to their rapid growth rate (Li et al. 2008 ), and they are deemed to have the potential to convert various carbon sources, such as cellobiose, xylose and starch, to lipid (Gong et al. 2012 ; Hu et al. 2011 ; Huang et al. 2014 ; Tanimura et al. 2014a ). Second-generation biodiesel is made from non-food sources such as rice straw, wood residue, corncob, and sugarcane bagasse. Lignocellulosic hydrolysates from these feedstocks are composed mainly of glucose, xylose, and l -arabinose (hereafter referred to simply as arabinose) (Huang et al. 2009 ; Kumar et al. 2009 ; Madhavan et al. 2012 ; Roberto et al. 1995 ; Tsigie et al. 2011 ). The ratio of the sugars and their concentration in the hydrolysates vary depending on the feedstock used and pretreatment conditions (Behera et al. 2014 ; Kumar et al. 2009 ). A previous study investigated lipid accumulation using a medium containing 3 % glucose by Vanrija musci JCM 24512 (formally Cryptococcus musci ) (Tanimura et al. 2014b ). The strain showed higher lipid-producing ability from glucose compared to typical oleaginous yeasts such as Lipomyces starkeyi and Rhodosporidium toruloides . Strains like this that can convert glucose to lipid with high productivity are well suited for the production of glucose-rich hydrolysate such as the hydrolysate of starchy biomass. However, because pentoses content ranged from 20 to 40 % of the total released sugars (Sumphanwanich et al. 2008 ; Tanimura et al. 2012 ), glucose utilization alone is insufficient for the conversion of lignocellulosic biomass. In other words, sequential utilization of the sugars extends fermentation times. Therefore, economically feasible production of lipid will require a yeast strain with the ability to co-ferment the lignocellulosic sugars. Research has shown that engineered yeast can be valuable in expanding the substrate range. For example, Tai engineered Yarrowia lipolytica to make it utilize xylose (Tai 2012 ). In that case, the xylose reductase encoding gene ( XYL1 ) and xylitol dehydrogenase encoding gene ( XYL2 ) were transferred from the xylose-fermenting yeast Scheffersomyces stipitis into the strain. The uptake of arabinose has not yet been reported, and therefore, research in this area is expected. In addition, to avoid the problem caused by glucose repression, the quest for novel oleaginous yeasts able to co-ferment glucose, xylose, and arabinose would seem to be an efficient strategy. To the best of our knowledge, there has not yet been a screening study of oleaginous yeasts able to ferment the three sugars. The application of the following new oleaginous yeasts to the conversion of lignocellulosic sugars to lipids has been carried out: Trichosporon fermentans (Huang et al. 2009 , 2014 ), L. starkeyi (Anschau et al. 2014 ), Cryptococcus curvatus (Liang et al. 2014 ), R. toruloides (Wiebe et al. 2012 ) and Y. lipolytica (Tsigie et al. 2011 ). However, in these sugar-consumption profiles, sequential utilization of arabinose was not observed. In this study, exhaustive screening of 1189 isolates was undertaken to identify an oleaginous yeast strain that was able to convert the glucose, xylose, and arabinose in artificial hydrolysate to lipid. We here report the discovery of Pseudozyma hubeiensis IPM1-10, which shows a significant utilization of a mixture of the sugars.",
"discussion": "Discussion As shown in Table 1 , the hydrolysate of lignocellulosic biomass mainly contains 3 kinds of sugars: glucose, xylose, and arabinose. The ratio of the released sugars varies, depending on the raw material types and pretreatment conditions; the ratio of hexoses to pentoses generally ranged from 1.5:1 to 3:1 (Huang et al. 2013 ). The final product contains a not negligible amount of arabinose. In the present study, glucose, xylose, and arabinose concentration were set at 20, 10 and 5 g/L, respectively. These concentrations are within the measured values, and were appropriate as the screening medium. Table 1 Sugar composition of lignocellulosic hydrolysates Material Glucose [g/L] Xylose [g/L] Arabinose [g/L] Total [g/L] References Rice straw 15.5 84.3 17.1 116.9 Huang et al. ( 2009 ) Rice straw 22.6 79.3 13.4 115.3 Roberto et al. ( 1995 ) Rice straw 55 10 3 68 Oberoi et al. ( 2012 ) Rice bran 43 5 2 50 Tsigie et al. ( 2012 ) Sugarcane bagasse 4 14 3 21 Tsigie et al. ( 2011 ) Wheat straw 30 25 5 60 Zhang et al. ( 2014 ) Bagasse 16.8 92.9 11.4 121.1 Huang et al. ( 2012 ) Interestingly, all selected strains belonged to the Ustilaginales species (Table 2 ). Incidentally, IP056 is assumed to be a new species in clade 7 of Wang et al. ( 2015 ), as the sequence of the D1/D2 region of LSU rRNA gene showed a 5-nucleotide difference from that of Macalpinomyces viridans (HQ013125) and a 6-nucleotide difference from that of Macalpinomyces spermophorus (HQ013130), respectively. According to Wang et al., the species in clade 7 was not reclassified due to the taxonomic confusion of teleomorphic genera; consequently we treat this strain as an unidentified yeast strain (Wang et al. 2015 ). Furthermore, the selected strains (except for Moesziomyces aphidis RS041) were isolated from plants (leaf surface) collected on Iriomote Island. As previously reported, Ustilaginales species are generally distributed on the surface of leaves (Wang et al. 2006 ; Yoshida et al. 2014 ). This suggests that the inhabitants of the phyllosphere are associated with the fermentation ability of lignocellulosic sugars. Although this phenomenon is not presently understood, it is likely that the strains assimilate lignocellulose degradation products supplied by themselves or another microorganism. The xylanases-producing ability of the species has actually been reported (Adsul et al. 2009 ). Another feature of Ustilaginales species is their biosurfactant-producing ability (Jaseetha and Das 2013 ; Morita et al. 2010 ); namely, the strain can accumulate lipid intracellularly and/or produce biosurfactant extracellularly. This is the first report of mixed-sugar fermentation and of lipids accumulation using Ustilaginales species. Table 2 Yeast species, source, and JCM number of 12 selected oleaginous yeasts Strain Species Source JCM number IPM1-7 \n Pseudozyma hubeiensis \n Plant, Iriomote Island 24583 IPM1-9 \n Pseudozyma hubeiensis \n Plant, Iriomote Island 24584 IPM1-10 \n Pseudozyma hubeiensis \n Plant, Iriomote Island 24585 RS041 \n Moesziomyces aphidis \n Soil, Rishiri Island 24586 IP068 \n Pseudozyma hubeiensis \n Plant, Iriomote Island 24587 IP045 \n Pseudozyma hubeiensis \n Plant, Iriomote Island 24588 IP037 \n Ustilago siamensis \n Plant, Iriomote Island 24589 IP040 \n Moesziomyces antarctica \n Plant, Iriomote Island 24590 IP026 \n Pseudozyma hubeiensis \n Plant, Iriomote Island 24591 IP004 \n Pseudozyma hubeiensis \n Plant, Iriomote Island 24592 IP056 Unidentified Ustilaginales species Plant, Iriomote Island 24593 IPM46-16 \n Anthracocystis elionuri \n Plant, Iriomote Island 24544 As shown in Fig. 2 , all 12 candidates showed favorable results in terms of the assimilation of pentoses. The lipid concentration of M. aphidis RS041, U. siamensis IP037, M. antarctica IP040, and A. elionuri IPM46-16 were relatively higher from the viewpoint of sugar yield (g of lipid produced per g of sugar consumed). However, their sugar consumption was not comparable to that of P. hubeiensis IPM1-10, which led to the lower lipid productivity (duration of time needed for lipid concentration), because the slow sugar uptake increased cultivation time. Lipid productivity is considered to be the most important parameter. Higher lipid productivity decreases production cost. In the selected strain, P. hubeiensis IPM1-10, the highest lipid concentration and cell mass were achieved with almost complete utilization of the sugars. Similar to the other Ustilaginales species, P. hubeiensis has been recognized as a biosurfactant producer (Konishi et al. 2008 ). P. hubeiensis produces lipases, assimilates oil (soy oil or bovine fat), and secrets biosurfactant (Bussamara et al. 2010 , 2012 ). Since P. hubeiensis can also convert lignocellulosic sugars to lipid, it has great potential for utilization of unused biomass and low-cost raw materials. As shown in Fig. 4 , the lipid-producing ability using arabinose was 30 % lower than those using glucose and xylose, even though the sugar consumption rates were similar (Fig. 3 ). The data suggested that arabinose was a less effective carbon source for P. hubeiensis IPM1-10 in terms of lipid concentration. It seems that the assimilated arabinose converted to lipid and supported cell growth at the same time, because no significant difference was observed in the cell mass between carbon sources (Fig. 4 ). The fatty acid composition of the lipid accumulated in P. hubeiensis IPM1-10 (Table 3 ) was similar to that of plant oil, which consists mainly of C16 and C18. These fatty acids are widely applicable, e.g., for biodiesel, chemicals, and toiletries. Compared to plant oil, lipid from oleaginous yeast is advantageous in terms of elements of economical production, such as reductions in the lifecycle, the amount of land required, and the effects of climate. Table 3 Fatty acid composition of P. hubeiensis IPM1-10 after a 10-day culture Carbon source C12:0 C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C22:0 C24:0 Glucose 2.9 1.5 18.1 0.2 21.4 25.1 18.2 3.5 8.7 Xylose 0.2 1.3 22.8 0.5 16.4 26.7 21.9 3.4 6.9 Arabinose 0.9 1.3 20.4 0.3 19.8 33.6 11.3 3.3 9.1 Glucose, xylose and arabinose 2.7 1.4 19.5 0.3 20.0 26.6 17.6 3.4 8.2 Data are mean of three independent assays When grown in the mixed-sugar medium, P. hubeiensis IPM1-10 required a 10-day culture. There have been several previous reports on lipid production by oleaginous yeast from mixtures of glucose, xylose, and arabinose. Sugar exhaustion was achieved at 11 days from rice straw hydrolysate by T. fermentans (Huang et al. 2009 ), 10 days from a semi-defined medium by T. fermentans (Huang et al. 2014 ), and 7 days from sugarcane bagasse hydrolysate by Y. lipolytica (Tsigie et al. 2011 ). Further consideration is needed to determine how best to improve fermentation conditions. On the other hand, to increase lipid accumulation, continuous or fed-batch culture might be effective (Gong et al. 2012 ; Zhao et al. 2008 ). When the sugar mixtures were used as the carbon source, the lipid concentration was higher than with glucose alone. Increasing the proportion of pentoses in the carbon source increased lipid accumulation. Papanikolaou and Aggelis indicated that xylose affected lipid yield rather than glucose, because oleaginous microorganisms exclusively utilize the phosphoketolase pathway for xylose (Papanikolaou and Aggelis 2011 ). Therefore, P. hubeiensis IPM1-10 provides an efficient process for converting lignocellulosic biomass, such as the glucose, xylose, and arabinose present in hydrolysates, into lipid. Comprehensive screening of oleaginous yeasts capable of simultaneously utilizing glucose, xylose, and l -arabinose was performed. Among the strains tested here, P. hubeiensis IPM1-10 had the best lipid productivity grown on lignocellulosic sugars. The strain may also be useful as a genetic resource for engineering pentoses metabolism in oleaginous microorganisms in order to improve their ability to convert sugar mixtures to lipid. More importantly, the absence of glucose repression could facilitate further study to unravel the unique sugar-assimilation mechanism."
} | 3,392 |
29373572 | PMC5786289 | pmc | 6,445 | {
"abstract": "Nematostella vectensis is a member of the phylum Cnidaria, a lineage that includes anemones, corals, hydras, and jellyfishes. This estuarine anemone is an excellent model system for investigating the evolution of stress tolerance because it is easy to collect in its natural habitat and to culture in the laboratory, and it has a sequenced genome. Additionally, there is evidence of local adaptation to environmental stress in different N . vectensis populations, and abundant protein-coding polymorphisms have been identified, including polymorphisms in proteins that are implicated in stress responses. N . vectensis can tolerate a wide range of environmental parameters, and has recently been shown to have substantial intraspecific variation in temperature preference. We investigated whether different clonal lines of anemones also exhibit differential tolerance to oxidative stress. N . vectensis populations are continually exposed to reactive oxygen species (ROS) generated during cellular metabolism and by other environmental factors. Fifteen clonal lines of N . vectensis collected from four different estuaries were exposed to hydrogen peroxide. Pronounced differences in survival and regeneration were apparent between clonal lines collected from Meadowlands, NJ, Baruch, SC, and Kingsport, NS, as well as among 12 clonal lines collected from a single Cape Cod marsh. To our knowledge, this is the first example of intraspecific variability in oxidative stress resistance in cnidarians or in any marine animal. As oxidative stress often accompanies heat stress in marine organisms, resistance to oxidative stress could strongly influence survival in warming oceans. For example, while elevated temperatures trigger bleaching in corals, oxidative stress is thought to be the proximal trigger of bleaching at the cellular level.",
"introduction": "Introduction The starlet sea anemone, Nematostella vectensis , is a leading laboratory model organism in the phylum Cnidaria due to its ease of culture, amenability to a wide range of experimental protocols, and the availability of abundant genomic resources [ 1 – 19 ]. In its natural habitat—estuaries along the Atlantic coast of North America—this animal experiences dramatic variation in key environmental variables over short temporal and spatial scales [ 20 ]. Furthermore, there is extensive genetic and phenotypic variation between populations of N . vectensis [ 21 – 27 ]. Atlantic coast populations have a high degree of genetic differences and regional population genetic structure that is not correlated to relative geographic distances [ 21 , 25 ]. These differences within local populations are hypothesized to be the result of limited gene flow within and between N . vectensis populations, adaptation to local environmental conditions, and episodic anthropogenic dispersal over large distances. The estuarine habitats inhabited by N . vectensis are often physically isolated, and these habitats can differ dramatically with respect to key environmental parameters, such as temperature, pH, and salinity [ 3 , 20 ]. Moreover, the early stages of the N . vectensis lifecycle exhibit limited dispersal ability, due in part to the egg mass being negatively buoyant [ 3 ]. The combination of diverse environments and limited dispersal will tend to promote adaptive genetic differentiation between populations. For this reason, N . vectensis has been advocated as a useful model for studying adaptive microevolution in a coastal invertebrate beset by rapid environmental change [ 6 , 24 ]. One environmental stressor that varies dramatically in salt marshes and other estuarine habitats is hydrogen peroxide (H 2 O 2 ) concentration. For example, in an intertidal salt-flat off the coast of Germany, H 2 O 2 concentrations in the water were found to vary almost 100-fold, from 0.05 μM to approximately 4.5 μM, with concentrations at the sediment-water boundary layer being 2–4 times higher than concentrations in the adjacent bottom water [ 28 ]. Peroxide levels can accumulate in shallow coastal habitats due to natural inputs such as photochemical production and atmospheric wet deposition as well as anthropogenic inputs such as runoff from industrial processes, e.g., mining and industrial manufacturing. Salt marshes are particularly susceptible to peroxide accumulation because large amounts of organic matter can fuel peroxide production through photo-degradation [ 29 ], and isolated pools may seldom be flushed. N . vectensis is a benthic salt-marsh organism with relatively low mobility, so this species is readily exposed to compounds that accumulate in sediments of estuaries. In its natural habitat, N . vectensis can be exposed to a wide range of exogenously produced reactive oxygen species (ROS) including H 2 O 2 . This anemone must, therefore, be under strong selection to counteract oxidative stress. N . vectensis is known to tolerate a wide range of other highly variable environmental parameters, e.g., salinities from ~0–50 ppt and temperatures from 2–28.5°C [ 3 , 20 ]. The ability to tolerate rapid and dramatic variations in these environmental conditions suggests that individual N . vectensis have wide environmental tolerances. At the same time, the pronounced environmental differences between and within estuaries inhabited by N . vectensis suggest the potential for local adaptation to differing selection pressures. The potential for local adaptation is increased by the fact that N . vectensis ’ natural dispersal ability appears limited, as evidenced by significant genetic structure between and within estuaries [ 21 , 25 ]. Specifically with reference to peroxide tolerance, one might expect that all N . vectensis would exhibit a wide tolerance to peroxide, with no observable differences between or within populations. However, given that a recent study identified substantial differences in temperature-specific growth and regeneration rates in N . vectensis [ 24 ], we sought to determine whether different clonally derived lines of anemones would also exhibit differential peroxide tolerance. Indeed, as elevated temperatures promote oxidative stress (e.g., [ 30 ]), we hypothesized that the same clonal lines that are best adapted to high temperatures would also be most resistant to oxidative stress. In this study, we exposed fifteen clonal lines of N . vectensis collected from four geographically isolated estuaries to different levels of H 2 O 2 . Our study included three clonal lines (one each from Baruch, South Carolina, Meadowlands, New Jersey, and Kingsport, Nova Scotia) known to vary in their growth and/or regeneration rates at 21 and 29°C [ 24 ], as well as 12 previously uncharacterized clonal lines established from founder individuals collected in Great Sippewissett Marsh, Falmouth, MA. We monitored the effects of H 2 O 2 on adult survival and on the ability of animals to regenerate a new “head” following bisection through the body column. We identified substantial differences in the effects of peroxide on survival and regeneration between the SC, NJ, and NS clonal lines and among clonal lines originating from the single estuary in Sippewissett.",
"discussion": "Discussion In this study, we applied exogenous H 2 O 2 to assess the oxidative stress response of the estuarine sea anemone Nematostella vectensis . This is a straightforward and biologically relevant way to challenge the oxidative stress response of a coastal marine invertebrate because organisms in shallow coastal waters regularly encounter elevated H 2 O 2 levels that can trigger oxidative stress (e.g., [ 28 ]). Furthermore, peroxide is the most abundant and long-lived ROS in marine environments, and unlike other ROS, H 2 O 2 is uncharged and can diffuse freely across membranes (reviewed in [ 33 ]). The peroxide levels used in our experiments (82 and 163 μM) are substantially higher than those recorded in marine and freshwater environments ( Table 1 ). Across numerous ocean basins, the peroxide concentration of sea surface waters have been found to range from 10–420 nM [ 34 – 36 ], while peroxide concentrations in the surface waters of lakes have been found to exceed 1 μM ( e . g ., [ 37 ]). At a site more comparable to the habitat of N . vectensis , peroxide levels in a shallow tidal pool located on a sand flat, ~500 m off the coast of Germany were found to vary between 0.5 and 4.5 μM [ 28 ]. However, in contrast to the high marsh habitat in which the anemones used in the current study were collected, this sand flat is inundated by the tides for ~19 h each day [ 28 ]. Of note, the levels of H 2 O 2 in rainwater can be substantially higher than the surface waters of oceans or lakes, with continental rainwater ranging from 0.1–247 μM (reviewed in [ 38 , 39 ]) and oceanic rainwater ranging from 3.5 to 82 μM [ 40 , 41 ]. As a result, precipitation events can substantially increase H 2 O 2 levels in surface waters [ 40 – 42 ]. 10.1371/journal.pone.0188265.t001 Table 1 Hydrogen peroxide levels measured in aquatic environments. Geographic locale Descriptions Max [H 2 O 2 ] Ref. Gulf of Mexico off the western coast of Florida Sea surface waters to 200 m ~0.024 x 10 −6 M [ 43 ] Wadden coast, Germany Shallow tidal pool ~500 m from land ~4.5 x 10 −6 M [ 28 ] King George Isl., Antarctica Offshore surface waters ~1.8 x 10 −6 M [ 68 ] Intertidal pools ~2.0 x 10 −6 M Freshly fallen snow ~13.6 x 10 −6 M Jacks Lake, Ontario, CA Oligotrophic near-shore lake water ~0.8 x 10 −6 M [ 69 ] Continental rain 34 x 10 −6 M Gulf of Mexico Marine rain 82 x 10 −6 M [ 40 ] While H 2 O 2 levels have not yet been reported for any saltmarsh, we suspect that the pools inhabited by N . vectensis regularly experience higher concentrations than those typically found in oceans or lakes. The saltmarsh pools where the anemones in this study were collected are small, shallow bodies of water. As such, they receive proportionately large inputs of H 2 O 2 through precipitation. Snow and rain can both have much higher peroxide concentrations than seawater ( Table 1 ), and in shallow, stratified layers of marine surface waters, rain events have been shown to increase the H 2 O 2 concentration by more than 10-fold [ 40 ]. Furthermore, saltmarsh pools receive abundant direct sunlight and are rich in organic matter. As a result, the production of H 2 O 2 via the photochemical decay of organic matter, which is thought to be the principal source of H 2 O 2 in surface waters [ 29 , 43 ], should be particularly high. Furthermore, saltmarsh pools, particularly those at higher elevation, may only rarely be inundated by the tides, so their H 2 O 2 content would not often be diluted by an influx of seawater. Finally, in the upper layer of the sediment, where the anemones burrow, photosynthetic algae and bacteria can generate locally high levels of oxygen [ 44 ] and concomitantly high levels of ROS. The effects of H 2 O 2 exposure have been studied in a handful of marine invertebrates, including polychaete worms, molluscs, crustaceans, and corals, but the concentrations of peroxide that were used and the effects that were monitored have varied substantially among these studies ( Table 2 ). In whole-organism studies, micromolar concentrations of H 2 O 2 (0.5–20 μM) were shown to decrease oxygen consumption and aerobic metabolism in three intertidal species, a burrowing polychaete [ 45 ], a clam [ 46 ], and a limpet [ 47 ]. Five micromolar H 2 O 2 also caused a substantial drop in water filtration rate in an intertidal clam [ 46 ]. A higher level of H 2 O 2 (50 μM) was used in experiments on the estuarine polychaete, Laeonereis acuta . In this infaunal worm, peroxide exposure caused lipid peroxidation and DNA damage [ 48 ]; there was also a biphasic effect on aerobic metabolism, with O 2 consumption falling after 4 d exposure and rising again after 7 d [ 49 ]. In the tropical reef coral, Galaxea fascicularis , exposure to 0.3 or 3 μM H 2 O 2 was accompanied by an increase in catalase activity, but no increase in superoxide dismutase activity [ 50 ], and this pattern was observed at both 27°C and 31°C [ 51 ]. In comparison to our study, all of these organismal investigations utilized lower levels of H 2 O 2 , and they did not examine tissue integrity, mortality, or a developmental process (such as regeneration). Significantly higher levels of peroxide have been applied to an isolated body-wall preparation obtained from an intertidal polychaete (42–664 μM; [ 52 ]) and to a mitochondrial suspension from a sipunculid (90.9 μM; [ 53 ]). Finally, millimolar levels of H 2 O 2 have been shown to induce synchronous spawning in molluscs in a study that did not investigate subsequent impacts of H 2 O 2 exposure on survivorship or physiological function [ 54 ]. In studies on cultured cells, H 2 O 2 levels comparable to or greater than those employed here have been used to induce “mild” oxidative stress; for example, Huang et al. exposed HeLa cells to 1000 μM H 2 O 2 for 1 h or 100 μM H 2 O 2 for 6 hours to investigate its effects on protein localization [ 55 ]. Of note, our study demonstrates the highest level of survivable peroxide exposure that has been found to date for any marine or estuarine invertebrate. 10.1371/journal.pone.0188265.t002 Table 2 Effects of hydrogen peroxide exposure on marine invertebrates. Organism Habitat / Organismal description Peroxide Level Tested Effect(s) of exposure Ref. Galaxea fascicularis Tropical reef coral (Cnidaria/Anthozoa) 0.3; 3 μM Increased catalase activity [ 50 ] 0.3; 3 μM + 27/31°C Increased catalase activity in coral host and symbiotic zooxanthellae at either temp. [ 51 ] Nereis diversicolor Intertidal burrowing worm (Polychaeta) 0.5; 5 μM Locomotion ceases; O 2 consumption was reduced by ~40% [ 45 ] Cerastoderma edule Intertidal clam (Bivalva) 5 μM Filtration rates reduced by 40% [ 46 ] Nacella concinna Antarctic intertidal limpet (Gastropoda) 5 μM Lysosomal membrane instability; 45% reduction in aerobic metabolism [ 47 ] Crangon crangon Intertidal shrimp (Crustacea) 20 μM O 2 uptake reduced by 25.7%; decrease in muscle intracellular pH [ 70 ] Laeonereis acuta Infaunal estuarine worm (Polychaeta) 10; 50 μM Lipid peroxidation; DNA damage; [ 49 ] Initial fall (at 4 d) and later rise (at 10 d) in O 2 consumption [ 48 ] Sipunculus nudus Intertidal burrowing worm (Sipuncula) mitochondrial suspension. 90.9 μM Did not inhibit ATP synthesis [ 53 ] Arenicola marina Intertidal burrowing worm (Polychaeta); isolated body-wall prep 42 μM 328 μM 664 μM 17% increase in O 2 consumption 17% decrease in O 2 consumption 9% decrease in O 2 consumption [ 52 ] While previous studies on oxidative stress tolerance in marine animals have generally sought to characterize a species’ average or typical response to a given challenge, our study is the first to examine intraspecific variation in the oxidative stress response of a marine animal. Indeed, given their differing thermal preferences, we suspected that these clonal lines might differ in peroxide tolerance, because thermal stress is known to induce oxidative stress and because ROS such as H 2 O 2 can work synergistically with acute temperature changes to cause oxidative stress. For example, in the Antarctic limpet Nacella concina , H 2 O 2 exposure and elevated temperatures contribute additively to oxidative stress [ 47 ]. Similarly, in corals thermal stress has been shown to result in oxidative DNA damage (e.g., [ 56 ]) and to induce antioxidant enzyme activity [ 51 ]. Although our study did not directly determine whether there is a correlation between sensitivity to thermal stress and to oxidative stress, the SC clonal line, which was found to be most tolerant of high temperature (29°C; [ 24 ]), was among the least affected by H 2 O 2 , and the NS clonal line, which was most sensitive to high temperature, was more affected by H 2 O 2 . However, the Sippewisset clone lines varied extensively in H 2 O 2 tolerance, and the previous temperature study did not sample enough individuals from this single estuary to determine whether thermal tolerance varies correspondingly within this population. The most surprising result of our study is the significant differences that we identified in survival and regeneration during H 2 O 2 exposure among clonal lines taken from a single marsh in Sippewissett, MA. The Sippewissett clonal lines used in our experiments were derived from wild-caught individuals that lived within a hundred meters of each other, some collected from the same small pool where they would have presumably encountered similar environmental stressors; i.e., each of the genets collected from the same pool would have been subjected to the same daily and seasonal variation in temperatures. Given these similar temperature exposures, one would not expect that animals from a single pool would differ widely in their temperature preferences. Nevertheless, the differences among clonal lines from this single pool in their ability to tolerate H 2 O 2 exposure actually exceeded the variation exhibited between the SC and NS clonal lines. The clonal lines from Sippewisset were collected at the same time and maintained in the laboratory for an equivalent length of time prior to the conduct of this study. Therefore, the differences we observed cannot be explained by differing lengths of time under laboratory culture. There are multiple possible explanations for the variation we observed within and between pools at Sippewissett. For example, there may be adaptation or long-term acclimation to different microhabitats. Spatially- and temporally-variable factors that can affect peroxide levels include sediment composition and shifting sediment distribution, the amount of organic matter, water depth and tidal action, the amount of pollution and heavy metal contamination, primary production, flooding events (and the associated interconnectedness of tidal pools), aerial exposure, periods of anoxia, solar radiation, and other microbial plant and algal species present [ 33 ]. Comparable fine-scale variation in other environmental variables has been shown to affect organismal physiology. For example, substantial environmental micro-variation in temperature has been shown to exist in tide pool habitats [ 57 ], and mussels originating only 24 m apart were found to differ substantially in their response to thermal variation [ 58 ]. Similarly, changes in vertical gradients of oxygen, H 2 S, temperature, and pH have been shown to effect changes in molecular response to ROS in the burrowing marine polychaete Heteromastus filiformis [ 59 ]. Gradients of pH have also been shown to affect both oxygen consumption and H 2 O 2 levels. Previous exposure to sublethal stressors has also been shown to increase stress resistance in a number of model systems an in response to a number of stressors (reviewed in [ 60 ]). Relatively few studies have investigated intraspecific variation in the response to H 2 O 2 , and the bulk of these studies have been performed on plants and algae. For example, intertidal individuals of the green macroalga Ulva lactuca exhibited a greater ability to scavenge exogenous H 2 O 2 than subtidal individuals of the same species [ 61 ]. To our knowledge, the only experimental study documenting intraspecific variation in H 2 O 2 tolerance in an animal model was conducted on the soil nematode Caenorhabditis remanei [ 62 ]. In this study, multiple strains of C . remanei were exposed to 3.5 mM H 2 O 2 , an oxidative challenge that kills all individuals within 4-8h of exposure. The offspring of different sires were found to differ in their ability to withstand this level of H 2 O 2 exposure, and the heritability of this trait was found to be 2x greater than that for heat stress resistance and 11x greater than that for lifespan [ 62 ]. This study used a qualitatively different challenge than used in the current study, where the anemones were tested at a level of H 2 O 2 exposure they could survive for two weeks. Intraspecific variability in oxidative stress response has been studied in a number of marine invertebrates, including corals and sea anemones. ROS-levels and antioxidant enzyme activities exhibit seasonal variation in the coral Pocillopora capitata [ 63 ]. In a population of the sea anemone Anemonia viridis spanning a natural pCO 2 gradient, levels of the algal secondary metabolite dimethylsulfoniopropionate (DMSP) as well as the activity of the enzyme superoxide dismutase (SOD) were found to be lower where CO 2 concentrations were higher [ 64 ]. The authors suggest that the symbiotic algae inhabiting the sea anemone’s tissues experience greater photosynthetic efficiency at higher CO 2 levels, and they therefore generate fewer ROS. In both of these naturalistic studies, the role of ROS could not be isolated from other correlated environmental factors. Nor could these studies discriminate environmental versus genetic causes of the observed phenotypic changes, because it was not known whether the individuals experiencing different environments were part of the same genet or different genets. Moving forward, an important goal will be to understand the genetic mechanisms (or stably inherited epigenetic mechanisms) that explain the differences in peroxide tolerance observed here. Importantly, according to population genetic studies using AFLPs, the Sippewisset population is genetically distinct from any of the other twenty-three populations that have been studied, including the Baruch, Kingsport, and New Jersey populations [ 21 , 24 ]. All 36 Sippewisset individuals characterized in these studies appear far more similar to each other than to individuals from any other population [ 25 ]. Thus, the most highly resistant clone line from Sippewisset, MA-C2, is much more similar genetically to the least resistant clones line from Sippewissett, MA-A2, than it is to the highly resistant SC clone line. This suggests that differences in oxidative stress tolerance could have evolved independently, and perhaps with a different genetic mechanism, in different populations. The current study illustrates the importance of incorporating organismal assays into studies of oxidative stress. That an organism is experiencing oxidative stress is often inferred from elevated levels of antioxidant enzymes or non-enzymatic antioxidant defense mechanisms. However, resistant individuals may exhibit greater stress tolerance precisely because they express stress-response genes at higher levels, either constitutively or conditionally (i.e., in response to the relevant stressor) [ 65 ]. What ultimately determines the fate of an organism under stress is its ability to maintain physiological function in the face of stressful environmental conditions. If the degree of fine-scale variability in oxidative stress tolerance observed here in N . vectensis is widespread among natural populations, it could have profound importance for the resilience of species and ecological communities in this era of rapidly increasing global temperatures because thermal stress is so often coupled with oxidative stress. This is particularly true for reef-building corals. The loss of photosynthetic endosymbiotic zooxanthellae (“bleaching”) is a leading cause of coral mortality. Bleaching is most often associated with elevated temperatures, but the proximate mechanism underlying bleaching is thought to be oxidative stress [ 66 , 67 ]. As a result, those corals that prove most resistant to thermal bleaching may be those with the most robust anti-oxidant defenses. We suggest that future studies should assess the natural variability in oxidative stress resistance that exists among corals (and other relatively heat-sensitive taxa), and determine if the molecular mechanisms responsible for oxidative stress resistance also confer greater resistance to thermal stress. Such knowledge could prove valuable in developing predictive models for the fate of natural populations in the face of climate change."
} | 6,038 |
35292683 | PMC8924167 | pmc | 6,446 | {
"abstract": "Marine species not only suffer from direct effects of warming oceans but also indirectly via the emergence of novel species interactions. While metabolic adjustments can be crucial to improve resilience to warming, it is largely unknown if this improves performance relative to novel competitors. We aimed to identify if spiny lobsters—inhabiting a global warming and species re-distribution hotspot—align their metabolic performance to improve resilience to both warming and novel species interactions. We measured metabolic and escape capacity of two Australian spiny lobsters, resident Jasus edwardsii and the range-shifting Sagmariasus verreauxi, acclimated to current average—(14.0 °C), current summer—(17.5 °C) and projected future summer—(21.5 °C) habitat temperatures. We found that both species decreased their standard metabolic rate with increased acclimation temperature, while sustaining their scope for aerobic metabolism. However, the resident lobster showed reduced anaerobic escape performance at warmer temperatures and failed to match the metabolic capacity of the range-shifting lobster. We conclude that although resident spiny lobsters optimise metabolism in response to seasonal and future temperature changes, they may be unable to physiologically outperform their range-shifting competitors. This highlights the critical importance of exploring direct as well as indirect effects of temperature changes to understand climate change impacts.",
"conclusion": "Conclusion In this study we showed that resident J. edwardsii increased its resilience to ocean warming by metabolic plasticity, helping to conserve basic energy consumption and sustain scope for aerobic activities at future summer temperatures. However, this did not aid J. edwardsii to overcome the metabolic performance deficits in comparison to the range-shifting spiny lobster S. verreauxi and was further set back by reduced anaerobic escape capacities of J. edwardsii in response to future summer temperatures. We conclude that resident species like Tasmanian spiny lobsters may be able to cope with the direct effects of increasing ocean temperatures but will struggle to endure additional indirect pressures brought by warming such as novel interactions with range-shifting competitors (Fig. 5 ). Trends exhibited for American lobsters, where distributions shifted poleward and offshore in response to warming, shell disease and novel invasive species 33 , 94 may foreshadow J. edwardsii’ s future. Yet, the lack of coastal habitat hinders any poleward shift for J. edwardsii , stressing the importance to ease environmental and fishing pressures, particularly for northern populations being most exposed to warming and novel species interactions. Figure 5 Conceptual diagram, illustrating that metabolic plasticity may aid resident spiny lobsters to resist direct effects of increasing ocean temperatures but not additional indirect pressures brought about by warming such as novel interactions with range-shifting competitors. Image courtesy of Stacey McCormack.",
"introduction": "Introduction By the end of this century our oceans will likely be, on average, 3.5 °C warmer (relative to 1870–1899, RCP8.5 1 ). Local warming can be even more extreme, due to heat waves 2 , changing ocean currents 3 , or cyclic weather patterns 4 . Such warming hotspots show rapid change of ecosystems, characterised by altered species abundance, biodiversity decline and local extinctions 5 , 6 . Species persistence will depend on their ability to acclimate or adapt rapidly 7 , or alternatively by ‘escaping’ to geographically track suitable temperatures poleward 8 – 10 . As a result, species are now re-distributing globally, particularly in our ocean, where species ranges shift up to six times faster than on land (5.9 vs. 1.1 km per year 9 , 11 ). However, due to differences in physiological tolerance, species traits, behaviour, habitat availability, adaptive capacity or access to microclimates, species may shift at different rates leading to disassembly of existing communities or emergence of novel biotic interactions 9 , 10 . Shifting to keep pace with preferred temperatures, or conversely, maintaining presence in an existing part of a specie’s distribution, may be further complicated by changing predation or competition pressures as result of range-shifting species 12 – 14 . For many species, acclimation or adaptation will increase resilience to these challenges and be key for their survival 7 . Niche shifts via physiological adjustments in response to environmental change (i.e. physiological plasticity or acclimation) are a critical and rapid mechanism by individuals to improve resilience to increasing temperatures 15 – 17 and reduce extinction risk 7 . Energy metabolism plays an important role in this context 18 . It powers fundamental processes such as growth, locomotion, or reproduction that require energy in the form of ATP produced either aerobically or anaerobically. Aerobic metabolism is far more efficient (~ 36 ATP/glucose molecule) and consequently the predominant process in most organisms to power sustained activities 19 . Performance declines due to temperature changes are thus frequently compensated by plastic adjustments of aerobic pathways, characterized by shifts in e.g. metabolic rate 20 or mitochondrial function 21 , 22 . On the other hand, anaerobic sources of energy, such as free ATP, muscle phosphocreatine, or ATP derived from glycolysis 23 , 24 , are quickly exhausted but can release energy more rapidly. This supports short, strong bursts of activity, and is critical to survival e.g. when escaping from predators 25 . Consequently, failure to sustain or adjust both aerobic and anaerobic energy metabolism may not only impair an animal`s performance but directly affect their survival in increasingly warmer waters. A largely unexplored aspect has been how such metabolic plasticity shapes outcomes of species interactions, particularly between resident and range-shifting species 8 . Most marine animals are unable to regulate their body temperature (i.e., they are ectothermic) and perform well only within a limited range of temperatures. Temperature, therefore not only limits species’ geographic distribution 26 but also regulates how ectothermic species perform relative to each other, depending on the individual shape and overlap of their thermal windows 18 , 27 . Physiological plasticity can shift thermal niches and consequently the outcomes of direct interactions, such as competition 28 , resulting in the dominance of the resident or the range-shifting species. Given this, it is essential to understand species’ capacity for physiological plasticity to predict their future distributions and outcomes of biotic interactions 8 .",
"discussion": "Discussion Marine organisms are highly vulnerable to climate warming, not only by being exposed to (potentially) critically high temperatures but also indirectly by a global climate-driven redistribution of species that may bring novel prey, predators, and competitors 9 . Phenotypic plasticity is one vital mechanism of species to reduce climate-driven temperature stress 15 and extinction risk 7 , yet whether this improves resilience to novel competitors remains unknown. In this study we found that both a resident and a range-shifting spiny lobster dynamically adjust aerobic metabolism to sustain physiological performance in response to seasonal and forecasted temperature changes. However, at future summer temperatures, resident lobsters lose anaerobic escape capacity and fail to match the metabolic performance of range-shifting lobsters. Resident Tasmanian spiny lobsters are thus mal-equipped to cope with the dual pressures of warming and novel competitors. Metabolic plasticity Plastic adjustments (phenotypic plasticity) of metabolic rate are a common means for organisms to balance performance and thermodynamic increases of energy expenditure when temperatures rise 15 . This is particularly vital for organisms inhabiting ocean warming hotspots, such as South–East Australia, which is heating up to 3–4 times faster than the global average 3 , 40 . We found that resident spiny lobster J. edwardsii can respond to such drastic changes, by dynamic shifts of standard metabolic rate, which reduces its basic energy needs by 29% at projected summer temperatures of 21.5 °C (by 2070) compared to cold-acclimated lobsters (14 °C, Fig. 2 a). Without this crucial adjustment, basic metabolic energy demand would increase and require lobsters to invest more time, energy, and risk to find prey to fuel this extra demand as well as trigger risky behavioural responses to predators 61 . Adjusted standard metabolic rates further aided to sustain aerobic scope (the range between standard- and maximum metabolic rate) in both lobster species at warmer temperatures (Fig. 2 c–d). This is because, given the lack of thermal compensation and a lower thermal increase of maximum metabolic rate (Table 1 ), aerobic scope would decline towards warmer temperatures without the observed reduction of standard metabolic rate. Such an adjustment aids both lobster species, particularly cold-temperate J. edwardsii , to sustain full capacity for other non-maintenance related oxygen-fuelled activities up to 21.5 °C projected summer temperatures. This may include feeding, digestion, migration, social interactions, or highly stressful events like moulting, which can exhaust the full aerobic scope of individual lobsters (personal observation, Supplementary Fig. S1 ). As for the two spiny lobster species in this study, metabolic plasticity is an important means for several other species to cope with temperature changes, particularly for aquatic ectotherms, such as molluscs and fish, but also other decapod crustaceans 15 , 62 . Yet, the underlying patterns are less well understood. Plastic standard metabolic rates but more rigid maximum metabolic rate observed in J. edwardsii and S. verreauxi (Fig. 2 a–b), have been found in European perch too, and was coined as “plastic floors and concrete ceilings” 63 . Although not being a universal strategy 64 , this indicates that, like perch, spiny lobsters prioritise adjustments of standard metabolic rate over maximum metabolic rates, which has the dual benefit of lowering maintenance costs while maintaining scope for aerobic activities. A further increase of aerobic scope at warmer temperatures is either not critical to sustain daily activities or further rate increases of maximum metabolic rate are limited, by exhausted mitochondrial densities or capacities 51 , cardiac performance 65 , ventilatory oxygen extraction, or blood oxygen transport 66 – 68 . On the other hand, alternative mechanisms may cause the observed depression of standard metabolic rate. For example, variation of organ mass can be an effective way to modify basic energy consumption 19 , particularly in case of highly active tissues such as hearts or liver, which can explain up to 38% of standard metabolic rate variation (e.g., European eel 69 ). Changes of mitochondrial activities and densities are additional means to modify energy consumption, as the case for American lobsters where the activity of mitochondria’s key enzyme citrate synthase declined by 35% in tail muscle under combined exposure to high temperatures and CO 2 70 . Temperature induced changes in mitochondrial function, such as differential substrate use or improved efficiency of oxidative phosphorylation can further optimise energy consumption 21 , 71 . Lobsters can also enlarge muscle fibres—at least during development—to lower surface to volume ratio and consequently reduce energetic costs to maintain membrane potential 72 , 73 . This however is limited by larger intracellular diffusion distances limiting oxygen flux 73 . Irrespective of the mechanism at work, the observed plasticity of standard metabolic rate aids resident J. edwardsii to reduce fundamental energetic costs, which increases resilience to future warming trends particularly for marginal populations at the warmer trailing edge 8 . Anaerobic escape performance Spiny lobsters escape from predators or other threats by powerful tail-flips, largely fuelled by anaerobic energy metabolism 74 , 75 . Exhaustive escape trials in this study revealed that anaerobic capacity, measured indirectly as excess post-exercise oxygen consumption rate (EPOC), declined steeply by 59% in warm-acclimated (21.5 °C) J. edwardsii (Fig. 3 a). This was further supported by fewer escape attempts (tail flips), aligning closely with decreased EPOC (Fig. 2 e). Therefore, although J. edwardsii’ s aerobic performance remained stable, anaerobic performance declined considerably, compromising its endurance to escape repeated threats when climate warming or heatwaves increase ocean temperatures to 21.5 °C or higher in South–East Australia. But why would J. edwardsii’ s escape performance decline when ocean temperatures rise? In crustaceans, including spiny lobsters, burst muscle contractions are fuelled anaerobically 75 , particularly in large white muscle fibres 74 . Initial bursts compose rapid tail flips fuelled by intracellular arginine phosphate pools, followed by slower less powerful bursts supported by anaerobic glycogenolysis, generating ATP from stored glycogen 76 , 77 . Initial arginine phosphates stores did not seem limiting in warm-acclimated J. edwardsii , as it performed initial escape bursts at full speed. However, its, by trend, lower numbers of repeated bursts compared to cold-acclimated conspecifics (Fig. 3 e), suggests that instead anaerobic glycogenolysis was limiting. This could be explained by lower glycogen stores or decreased glycolytic enzyme activity induced by chronic warming, yet current evidence is lacking, and abdominal glycogen rather seems to be enhanced in warm-acclimated European lobsters ( Homarus gammarus ) 78 . Alternatively, warmer temperatures may reduce J. edwardsii’ s ability to buffer lactate and protons accumulated during anaerobic (glycolytic) ATP production, as is the case for American lobsters, which have limited capacity to buffer low haemolymph pH with HCO 3 − and ammonia at higher temperature and p CO 2 70 . The resulting drop in intracellular pH would then inhibit glycolytic enzymes and limit anaerobic power supply 79 . Following the anaerobic power phase, lobsters eventually enter a slow oxygen-consuming (aerobic) recovery phase (i.e., EPOC) to return glycogen, pH, and lactate to pre-exercise levels 59 . Here, warm-acclimated J. edwardsii recovered 4.7 h faster than cold-acclimated conspecifics (Fig. 2 c), mirroring the lower oxygen depth accumulated during the limited anaerobic power phase. Yet interestingly, warm-acclimated J. edwardsii tended to have 32.1% lower recovery rates (i.e., oxygen replenished per hour) than cold-acclimated animals, indicating reduced capacity for aerobic recovery, due to e.g., reduced mitochondrial enzymes activity or densities 70 . This may link to the observed reduction in basic energy consumption and would be an interesting path for further investigation together with spiny lobster’s capacity to buffer lactate and pH at future ocean warming scenarios. As a result, if ocean temperatures continue to rise till 21.5 °C, J. edwardsii will sustain its ability to escape from predator attacks, however, only if they occur at low frequency. At higher frequencies, J. edwardsii will quickly exhaust and be highly vulnerable to vigorous predators. Its sensitivity may increase even further in face of additional disturbances, such as high fishing pressure 80 , disease 81 , or lack of shelter in impoverished habitats 82 , 83 . Resident versus range shifting lobsters Although we found that J. edwardsii improves resilience to future ocean warming, by energy-conserving metabolic adjustments, this did not improve physiological performance relative to a novel range-shifting competitor, the eastern rock lobster S. verreauxi . This subtropical species increasingly co-occurs in the resident temperate habitat of J. edwardsii (Fig. 1 ) and will likely compete for shelter and/or food 84 , 85 , particularly in resource impoverished localities 86 , such as recently formed urchin-dominated barren habitats 83 . In addition to the fact that S. verreauxi grows faster and much larger 46 , we found that it consistently matches or exceeds physiological and escape performance of J. edwardsii between 14 and 21 °C, which included higher maximum metabolic rate, aerobic scope, escape frequency and speed (Fig. 4 , Table 2 ). Even at, for S. verreauxi , relatively cold temperatures of 14 °C, which is far below its optimal temperatures for growth (21.2 °C 57 ) or aerobic scope (24.9 °C 34 ), it performed 35% more tail-flips and escaped 20% faster than J. edwardsii (Fig. 4 ). Furthermore, despite their different thermal origins, S. verreauxi’ s standard metabolic rate closely matched that of J. edwardsii between 14 and 21.5 °C, and equally decreased with warm acclimation conserving 36% of metabolic energy (Fig. 2 b). These findings are in line with previous results of higher maximum metabolic rate and aerobic scope but similar standard metabolic rates between 22 and 24 °C for S. verreauxi puerulus larvae and juveniles compared to J. edwardsii 49 . This indicates that various life stages of S. verreauxi will outperform J. edwardsii in situations where physiological capacities become critical, particularly at current and future summer temperatures. For instance, during summer, S. verreauxi would have a larger metabolic scope to support migration, ranging or feeding 87 , which could become a vital factor if both lobsters increasingly share habitat and resources, particularly in resource-poor habitats such as urchin barrens. Moreover, although, S. verreauxi’s escape performance is below or at similar levels of J. edwardsii’ s at larval and juvenile life stages 49 , our study showed that this relationship inverts once S. verreauxi matures. As a result, larger S. verreauxi will be better able to fend-off vigorous predators such as Maori octopus ( Octopus maorum ), which may then preferentially target J. edwardsii instead, benefiting further expansion of S. verreauxi . If such flow-on effects add to negative interactions with other range-shifting species (e.g., Octopus tetricus ), resident J. edwardsii will face increasing risk of range contraction 88 . Metabolic performance differences have shown to influence competitive outcomes in other species. For example, a 3.2-fold higher routine metabolic rate linked to a three times higher feeding rate and a 6.7 times higher attack coefficient in the invasive Chinese mitten crab Eriocheir sinensis, compared to the co-existing native European crayfish Austropotamobius pallipes 89 . Similarly, at 9 °C cold-adapted Arctic staghorn sculpin Gymnocanthus tricuspis had a lower aerobic scope than sculpins from warmer latitudes, which outcompeted Arctic staghorn sculpin in the search for protective shelter 90 . Both examples indicate that a larger metabolic scope for activity supports competitive dominance. However, given that differences of aerobic scope were much reduced at 4 °C among sculpins, it further highlights the modulating role of temperature. For example, warm-acclimated freshwater crayfish Cherax destructor won over cold-acclimated conspecifics, supported by up-regulated mitochondrial ATP production capacity in chelae (pincer) muscle 71 . Further, in case of two co-existing Australian crayfish , temperatures markedly changed tail-flip performance, which peaked at different temperatures for each species, benefiting warm-adapted crayfish to better escape predators when temperatures rise 91 . This is in line with our findings, underlining that range-shifting subtropical S. verreauxi’ s higher aerobic and escape performance, will provide a clear advantage over resident temperate J. edwardsii at current and future summer temperatures. This short-coming was not set-off by J. edwardsii’ s adjustments of standard metabolic rates as range-shifting S. verreauxi mirrored this metabolic plasticity. While this study provided valuable mechanistic insights about spiny lobster’s adaptive capacity to warming and additional impacts by range shifting competitors, it must be noted that inference was drawn from 18 specimens per species, each sampled from a single local population. For example, S. verreauxi was collected from a poleward-edge population, which may consist of pioneering specimens with higher cold-tolerance or ability to cope with novel combinations of abiotic and biotic conditions compared to centre or trailing edge populations (Donelson et al. 2019 8 ). Additional observation may provide advanced insights as to whether the observed patterns are consistent, or if there are additional physiological phenotypes more or less resilient to ocean warming and novel species interactions (Kroeker and Sanford 2021 18 ). Therefore, although, fully factorial physiological experiments like this study are logistically and financially challenging, this study provides a solid basis for future studies to focus efforts on critical traits and assess adaptive potential of spiny lobsters across diverse populations and latitudinal gradients. Further, while this study highlighted critical interactions between physiological performance, temperature, and competition, we stress the need to integrate further important factors driving abundance and species range shift such as fishing pressure 80 , predation 92 , larval recruitment 34 , disease 81 or habitat loss 93 . Conclusion In this study we showed that resident J. edwardsii increased its resilience to ocean warming by metabolic plasticity, helping to conserve basic energy consumption and sustain scope for aerobic activities at future summer temperatures. However, this did not aid J. edwardsii to overcome the metabolic performance deficits in comparison to the range-shifting spiny lobster S. verreauxi and was further set back by reduced anaerobic escape capacities of J. edwardsii in response to future summer temperatures. We conclude that resident species like Tasmanian spiny lobsters may be able to cope with the direct effects of increasing ocean temperatures but will struggle to endure additional indirect pressures brought by warming such as novel interactions with range-shifting competitors (Fig. 5 ). Trends exhibited for American lobsters, where distributions shifted poleward and offshore in response to warming, shell disease and novel invasive species 33 , 94 may foreshadow J. edwardsii’ s future. Yet, the lack of coastal habitat hinders any poleward shift for J. edwardsii , stressing the importance to ease environmental and fishing pressures, particularly for northern populations being most exposed to warming and novel species interactions. Figure 5 Conceptual diagram, illustrating that metabolic plasticity may aid resident spiny lobsters to resist direct effects of increasing ocean temperatures but not additional indirect pressures brought about by warming such as novel interactions with range-shifting competitors. Image courtesy of Stacey McCormack."
} | 5,821 |
37273619 | PMC10233667 | pmc | 6,449 | {
"abstract": "Nacreous architecture has a good combination of toughness\nand modulus,\nwhich can be mimicked at the micron to submicron level using 3D printing\nto resolve the demand in numerous applications such as automobile,\naerospace, and protection equipment. The present study examines the\nfabrication of two nacre structures, a nacre columnar (NC) and a nacre\nsheet (NS), and a pristine structure via fused deposition modeling\n(FDM) and explores their mechanically superior stacking structure,\nmechanism of failure, crack propagation, and energy dissipation. The\nexamination reveals that the nacre structure has significant mechanical\nproperties compared to a neat sample. Additionally, NS has 112.098\nJ/m impact resistance (9.37% improvement), 803.415 MPa elastic modulus\n(11.23% improvement), and 1563 MPa flexural modulus (10.85% improvement),\nwhich are all higher than those of the NC arrangement.",
"conclusion": "5 Conclusions Nacre has remarkable resistance\nto impact load due to the distinctive\nhierarchy created by the two diverse contrasting arrangements: the\nunidirectional tablet stacking columnar and an angular tablet organized\nsheet. By controlling the proportions of the intermediate component\n(soft phase), the augmented resistance of impact, elastic and flexural\nmodulus, and strength of the unique nacre-like compounds could be\nattained. The aragonite phase is surrounded by biopolymeric materials\nand entails a brick-and-mortar microarchitecture in a lamellar manner\nwith tremendous energy dissipation during rupture. The biomineralization\nprocess of nacre occurs in a bottom-up approach, which can be mimicked\nusing a similar bottom-up fabrication tool, such as FDM, a 3D printing\ntechnique. Organic–inorganic bonding, tablet size, interlocking,\ntablet distribution, volumetric percentage of soft and hard phases,\nand intra-layer adhesion are crucial to strengthening fracture resistance\nin an artificial nacre. This study demonstrates a unified approach\nto identifying and validating numerous analytic and simulation models\nof NC and NS. We first utilized dimensionless parameters and analytical\nmodels to illustrate the stacking of geometry, characteristics of\nthe material, and mechanical response of the nacre-like composites\nfor 3D design. The designed models were fabricated via FDM printing\nwith the PC-ABS filament in a brick-and-mortar manner. We envisioned\nthat the NS structure with the same other parameters, excluding stacking,\nwas superior in impact, tensile, and bending properties. The mechanical\nbehavior of NS structures showed improvement of 9.37% in impact resistance,\n11.23% in elastic modulus, and 10.85% in flexural modulus as compared\nto NC, while 36, 29, and 37% improvements in impact, elastic modulus,\nand flexural modulus, respectively, were noticed in contrast to pure\ngeometry. Nacre design structures can be applied as bulk implants,\ncoatings, inflammable films, structural components, or composite components\nwhen merged with ceramics and other polymeric materials.",
"introduction": "1 Introduction Nacre, the iridescent\nhierarchy, yields superior mechanical performance,\nwhich is attributed to their toughening mechanisms at the micro level,\nsuch as the stacking arrangement of a tablet, the aspect ratio of\na tablet, the volume fraction of hard and soft parts, and the interlocking\nangle, as well as at the nano level, such as nanoasperities, mineral\nbridges, organic interlamellar matrixes, and axial growth in the perpendicular\ndirection. 1 Two diverse microarchitectures\nof nacre were found in nature known as bivalves and gastropods. The\nbivalve shell has a nacre sheet (NS), in which the platelets are arranged\nin a “brick wall” design where a mortar phase bridges\nthe boundary among the respective underlying tiles. Columnar nacre\n(NC) is found in shells of gastropods, which have hexagonal platelets\nof almost equivalent size with matching centers that regulate the\noverlying tablets’ nucleation. 2 , 3 The vigorous\nmechanical performance of nacre architecture relies on the accurate\ngeometric sequencing of the tablet at the microscopic level. 4 The inner layer of mollusk shells comprises an\niridescent nacre material, which has the most sought-after natural\nstructure as it demonstrates unusual impact resistance and fracture\ntoughness, although most of the material constitutes brittle ceramic. 5 It comprises 95 wt % brittle aragonite platelets,\ncalcium carbonate (CaCO 3 ) crystal form, and a 5 wt % soft\nphase of polysaccharides and protein, forming a nanostructure of brick-and-mortar\narrangement. The bricks are densely packed with polygonal aragonites\nof 5–8 μm diameter and 20–90 Å thick platelets\nand welded by the mortar matrix of organic interlamellar of 1–5\nÅ thickness. 6 , 7 This distinctive hierarchical\nbuilding could be the basis of protective equipment and body armor\nwith numerous functions, including high strength, lightweight, high\nenergy absorbing capacity, and high stiffness due to its equally 3\ntimes superior energy absorption associated with the fundamental composites. 8 Nacre columnar (NC) comprises almost uniform\ntablets which regulate the nucleation site of the overlying tablets\nas centers are overlapping, whereas in a NS, a “brick wall”\npattern is followed for tablet stacking, and deposition starts over\nmost of the inner surface of the shell. 9 The top view of the columnar nacre has revealed that the adjacent\nlayers intersect and represent polygonal tablets in such a way that\nthe lamellar interfaces are perpendicular to inter-platelet borders,\nwhich form tessellated bands on the other hand in NS geometry; the\ndistributions of inter-tablet boundaries are random. 10 The overlap and core regions are significant in the NC\nbecause the stress experienced in both areas varies. In the NS, the\ncore and overlap regions experience no distinction. 11 , 12 Nacre can achieve remarkable toughness and strength simultaneously,\nemployed in diverse structural applications like aerospace and automotive,\nbased on the loading direction and fracture mechanism. NC has well-defined\ndeformation bands that are perpendicular to the loading direction\nof the columnar structure, and NS forms an unusual network of deformation\nbands at an angle to the main crack. 13 The\nresearch focuses on developing artificial nacre structures with strengthening\nmechanisms, such as crack blunt, deflection, stress delocalization,\ncrack bridging, interfacial strengthening, topological interlocking,\nand aspect ratio 14 , 15 to accomplish the requirements\nof high structural applications. Researchers have studied the fracture\nmodes and gained an understanding of the failure of the nacre using\nsimulation and analytical modeling approaches; nevertheless, there\nhave not been many experimental studies conducted to understand the\nobserved behavior. 16 − 18 Modern manufacturing methods, such as 3D printing,\nopen room to designing bio-inspired artificial nacre materials with\nsystematic control over brick-and-mortar stacking, interlocking, toughening,\nand failure understanding. 19 , 20 Additive manufacturing\n(AM) enables on-demand customized construction of the object by virtue\nof 3D scan’s digital slicing, CAD, or tomography data, where\nsubstances form layer-by-layer, devoid of the demand for machining\nand molds. AM techniques covered numerous categories per ISO/ASTM\n59200:2021 based on the feeding material type or curing mechanisms.\nExisting AM technologies include extrusion (Fused deposition modeling\n(FDM), 3D dispensing, direct ink writing (DIW), 3D plotting, and 3D\nfiber deposition), vat photopolymerization [stereolithography (SLA)\nand digital light processing (DLP)], powder bed fusion [selective\nlaser sintering (SLS) and selective laser melting (SLM)], binder jetting\n(3D bioprinting, aerosol 3D printing, and inkjet), material jetting,\nsheet lamination (LOM), and direct energy deposition (DED). 21 The working principle, pertinent features, advantages,\nand limitations are covered in Table 1 and portrayed in Figure 1 . FDM, fused filament fabrication (FFF),\nDIW, 3D dispensing, and 3D bio plotting fall into the material extrusion\ncategory where feedstock is particularly dispensed in an x – y plane through a heated nozzle with a predefined\ndiameter, as described in Figure 1 . 22 Extrusion-based AM involves\nprinting of thin thread in a predefined first-layer pattern, followed\nby subsequent layer deposition (downward movement of platform in the z -axis) until the desired 3D piece is obtained 23 , 24 ( Figure 1 a,b). Transformation\nof a liquid photopolymer to a solid object by application of a light\nsource falls under the category of vat polymerization, where ultra-high\nmolecular-weight monomer or oligomer material is reticulated using\nultraviolet light. SLA and DLP are two sub-categories of vat polymerization\n( Figure 1 c). First-layer\nadhesion with a platform or solidification of resin initiated at a\nspecific depth with a precise geometrical pattern via a light beam\nfrom laser. After curing of the initial layer, the building stage\nis shifted downward for another liquid filling and second layer reticulation;\nsubsequently, other layers are added until the defined height. 25 In the material jetting process ( Figure 1 d), the photopolymer droplets\nof specific shape are selectively deposited on a platform using one\nor more mobile printing heads. To facilitate a uniform and continuous\nflow of material during the injection process, the viscosity of material\nis reduced using heating and later deposited material is cured through\na UV light beam. The drop deposition method permits accurate aligning\nof the material, providing high tolerance to the body under the building\nand reducing material waste. Powder-based AM begins by conveying a\npowder layer material from the materials feed platform to the printing\nstage using feeding rollers. 26 Fusion (in\nbinder jetting, Figure 1 e) or sintering (in SLS, SLM, and EBM, Figure 1 g) of the powder material to a desired shape\nis accomplished by a programed energy source or binder deposition\nto the platform surface. This step is repeated to obtain the final\n3D geometry, and further post-processing is performed to eliminate\nthe infiltrated or support material. 27 The\nsheet lamination printing technique can be employed to construct three-dimensional\n(3D) pieces from continuous cutting of 2D sections followed by lamination.\nThis AM method can be classified into two, unrolled and rolled procedures\nlinked by a sheet of material piloted by the revolving rollers. Deeding\nand storing of material are simultaneously performed in this system.\nIn addition, sheet lamination has a printing stage (that can execute\nvertical changes), a roller, and a laser 28 ( Figure 1 f). Figure 1 Graphic representation\nof numerous 3D printing processes. (a) extrusion-based\nFDM with its features and proper extrusion, (b) extrusion-based DIW\nprocess, (c) SLA method of liquid resin, (d) photopolymerization in\nPolyjet, (e) 3D modeling via binder jetting, (f) sheet lamination\nprocess (LOM), and (g) method of SLM and SLS. Reused from refs ( 30 ) and ( 31 ) with permission: Copyright\n2017, MDPI, and Copyright 2021, Springer Nature. Table 1 Summary of Various Printing Technologies\nwith Their Features, Merits, and Demerits a type of 3D printing material used solidification\nmechanism feature resolution (μm) pros cons vat polymerization photosensitive resins, acrylates,\nepoxides photopolymerization 25–100 high resolution, precision,\nand surface quality photo processing is necessary; objects\nare vulnerable to weak\nand heat extrusion-based soft\npolymers, thermoplastics, inks, PLA, ABS, PC, composites sequencing layered cooling at moderate and room temperature 100–150 excellent strength component reduces the cost of production,\nversatility in material selection a sluggish process,\nhigh roughness, high processing temperature powder-based fusion PA12, PEEK, ceramics, metal, alloys sintering, melting 50–100 a robust and complex part, less anisotropy rough surfaces; poor Reusability of unsintered powder material or binder jetting dielectric\nstarch, conductive inks, gels crosslinking of polymers,\nroom temperature cooling 10–25 multimaterial, fast printing, low-temperature process low viscous ink needed, limited strength of the part, low surface\nfinish sheet lamination PVC,\npaper, plastic sheet laser cutting and binder curing\nvia laser 200–300 compact desktop printing high anisotropy,\nlow resolution, limited materials DED metal powder pr wire laser sintering or melting 150–200 more complex design printing, any shape building, low material\nwaste expensive, low resolution, surface finish 3D bioprinting thermoplastics, composites,\nphotoresins, hydrogels, biomaterials UV curing, crosslinking\ncuring at an average temperature 10–100 a broad range of materials narrow viscosity\nprocess window a Reused from refs ( 21 ) and ( 29 ) with permission. Copyright\n2017 & 2021, ACS. FDM demonstrates an encouraging route among all 3D-printing\nmanufacturing\ntechniques to facilitate direct incorporation of the intricate, micro-level,\nmulti-material, and porous geometries within the final 3D object. 32 FFF and FDM are widely utilized techniques among\nother 3D printing techniques that permit the adequate printing of\nintricate constructions guided by XYZ movement according to CAD models.\nOver traditional manufacturing, the key advantages of the FDM technique\nare that it enables rapid prototyping and on-demand fabrications.\nMoreover, FDM is at the lead of facilitating restructured production,\nwhich is crucial to diminishing the carbon footprint and empowering\nsmart production approaches in the upcoming market. 23 Though effective in building satisfactory erections, compared\nwith FDM, the existing volumetric AM technologies, SLA, and DLP printing\nhave a low utilization efficiency of wet material (the weight proportion\nof the final fabricated architecture to the original quantity of liquid\nresin in a bath). Other than this, they have less utilization efficiency\nof the remaining material (the weight proportion of the attained dry\nprinted geometry to the initial quantity of liquid resin). 33 Aspects of FDM printing and processing parameters\nsuch as building speed and improving the accuracy, functionality,\nmechanical properties, porosity, surface finish, and stability are\naddressed. 34 Versatile applications establish\nhow FDM-based AM has been developed for energy technology, lightweight\nengineering, optics, architecture, food processing, drug delivery,\ndentistry, and personalized medicine. 35 The collection of polymeric materials used in FDM encompasses materials\nlike polymer blends, thermoplastics, functional polymers, elastomers,\nhydrogels, biological systems, and composites. FDM uses most thermoplastic\nmaterials, such as polylactic acid (PLA), acrylonitrile butadiene\nstyrene (ABS), nylon, polyethylene terephthalate glycol, and their\nblends like polycarbonate–ABS (PC-ABS), nylon carbon fibers\nin the form of the continuous diameter filament. 20 , 36 Apart from conventional materials, filaments are produced with reinforced\ncomposites to enhance thr mechanical properties, non-flammability,\nchemical stability, and high abrasion. Wang et al. evaluated a high-performance\nPEEK composite reinforced with the carbon fiber (CF) and glass fiber\n(GF). The investigation indicated that incorporation of CF and GF\nfrom 5 to 15 wt % improved the thermal properties, mechanical characteristics\n(flexural properties are 5 wt % GF/PEEK (165 MPa) and 5 wt % CF/PEEK\n(94 MPa), with the improvement of 17% and 19% over that of produced\npure PEEK, respectively), and surface porosity. 37 A mechanical attribute of an object significantly differs\nfrom bulk material or input material properties, in which adhesive\nbonding creates each layer from the extruded fiber’s connection.\nLayered fabrication depends on various property-controllable characteristics\nlike direction of the printing layer, raster angle, infill pattern,\nand infill direction, which untimely result in either being brittle\nor ductile depending on the combination of printing parameters. 38 Josef Kiendl investigated that incorporating\na brittle PLA material in FDM-printed objects renders brittle behavior\nin the parallel printing direction to the loading direction, while\nthe inclined direction of printing to loading results in ductile behaviour. 39 Consequently, many of the researchers have established\nnumerous nature-inspired hierarchies, including a mollusk shell layer\nof nacreous material, utilizing filament-based AM and verifying the\nmodification in structural characteristics. Peng and co-workers designed\nfused deposition-modeled biomimetic architecture to mimic Elytrigia\nrepens and examined the resilience and stiffness through mechanical\nanalysis and multiscale finite element analysis (FEA). 40 Jia et al. created numerous nature-inspired\nconstructions, through multi-material FDM printing for the compact\ntension (CT) fracture test. They estimated the toughness for fracture\nemploying a nonlinear elastic J -integral approach\nand evaluated the mechanisms of toughening using fracture theories. 41 Padole et al. fabricated a range of biomimetic\nstructures, including NC and NS, prismatic, complex cross lamellar,\nand foliated structures using FDM-based 3D printing. The result of\ntheir study shows that, owing to the well-defined hierarchical architecture,\nsuperior toughness and extraordinary impact strength were observed\nin the nacreous structure among the produced architectures. 3 Yadav et al., developed nature-inspired materials\nby mimicking the architecture of molluscan shells, such as complex\ncross lamellae, foliated, cross-lamellae, and nacre, using a layer-by-layer\nFDM printing technique. They concluded each constituent affected the\nmechanical and surface frictional properties according to structural\nmanipulation. 42 Ko et al. designed the\nnacre-mimicked architecture to fabricate an optimum impact resistance\nunder three various impact conditions through an integrative utilization\nof bi-material 3D printing, drop-weight impact test, parametric study,\nand FEA. 43 3D printed materials are\nnot limited to static nature but can be\nreformed by shifting distinct characteristics, such as shape, hardness,\ncolor, functions, and transparency, when subjected to heat, magnetic\nor electrical source, water, pH, and light, which is an advance form\nof 3D printing known as 4D printing 44 , 45 ( Figure 2 a). 3D- and 4D-printed\ncomposites cover vital applications in mechanical constructions, in\naeronautical, automotive, consumer goods, and aviation industries,\nand in sports or safety equipment. The prime benefits of 3D and 4D\nprinting technologies are that they have fewer waste materials, require\nless energy during manufacturing, and have extreme flexibility in\nthe fabrication related to conventional techniques; therefore, both\nthe printing techniques have tremendous potential for innovation,\ndesign, and development in various sectors 46 , 47 ( Figure 2 b,c). A\nrecapitulation of 3D/4D printing based on innovation, materials employed,\nand changes in printed objects, printers, and applications is presented\nin Table 2 . Figure 2 (a) Difference\nbetween the 3D and 4D printing processes and output.\nReused from ref 22 with permission. Copyright\n2021, Taylor & Francis, (b) General advantages and disadvantages\nachieved by 3D and 4D printing technology. Reprinted from ref 48 with permission. Copyright 2022, Elsevier. (c)\nSWOT analysis for both techniques. Adapted from ref 46 with permission. Copyright 2023, Elsevier. Table 2 Comparison of 3D and 4D Printing with\nDifferent Materials, Designs, Changes Undergone (if Any), and Applications a 3D printing 4D printing materials thermoplastics,\nmetals, ceramics, biomaterials, or nanomaterials self-assembled\nmaterials, multi-materials designed materials\nexamples: shape memory alloy/polymer/hybrids, self-degradation/deformation materials, temperature/UV-driven materials design 3D digital information 3D\ndigital information for change printer 3D printer examples: SLA, material extrusion, and SLS smart 3D printer examples: modified nozzle, binder, and laser\nmultimaterial 3D printer examples: solid/liquid, solid/solid, gradient materials,\nand nanocomposites static or astatic as printed/static After printing changes in shape, color functions over time applications jewelery, toys, fashion, entertainment, automobiles, aerospace,\ndefense, and bio/medical devices applications involved\na dynamic change in configurations a Reused from 47 with permission. Copyright 2020, Elsevier. The present investigation focuses on an experimental\ncomparison\nof FDM-printed artificial nacre structures concerning different stacking\narrangements, columnar nacre, and sheet nacre. The monolithic Polycarbonate-Acrylonitrile\nButadiene Styrene (PC-ABS) was chosen for the experiment because it\nprovides excellent processibility and heat distortion resistance and\nexhibits good impact resistance, improved shrinkage, and dimensional\nstability. The mechanical response via the Izod impact test, tensile\ntest, and flexural bending testing elucidated NS’s superior\nstress-distributing capability compared to NC erection. Therefore,\nour report comparing the performance of different nacreous structures\nshows the experimental validation and potentially broadens the feasible\nfabrication route of NS and NC architectures.",
"discussion": "4 Results and Discussion The impact resistance\nwas performed as per ASTM D256, where the\nspecimen was designed via Solid Works, followed by fused deposition\n3D printing of PC-ABS. According to the NS and NC, the prepared specimens\nhad different stacking arrangements, tablet interfaces, and tablet\nlocking from each other. Therefore, each sample noticeably comprises\na varied capacity for absorbing impact energy. 43 Figure 3 a delineates identical features of the printed nacre composite, where\nthe impact resistance (IR) and absorb energy (AE) of the NS system\n(AE = 0.7323 J) was thought to be uppermost in addition to the supreme\nenergy absorption of the NC skeleton (AE = 0.6106 J) compared to the\nmonolithic PC-ABS sample (AE = 0.5457 J). The toughness of mollusk\nshells is attributed (deformation and fracture) to various mechanisms\ncomprising void formation, nanoasperities, and their controlled sliding,\ninterlocking of a tablet, and interlayer mineral bridges. Meanwhile,\nthe developed nacre architecture does not encompass\neither interlocking, mineral bridges, or nanohierarchy; consequently,\nthe impact performance could be a function of the tablet’s\ncenter direction and its configurations. 55 Figure 4 a delineated\nthe average value of five sequential experiments for impact resistance\nand absorb energy. Furthermore, tensile testing was conducted as per\nthe ASTM D638 type IV standards for PC-ABS printed samples, which\nboosted the experimental and analytical study on NC and NS. It was\nevident from the survey that crack propagation, while fracture in\nmollusk shell follows numerous methodologies; however, the phenomenon\nof stress whitening or process zone was observed in the energy dissipation\nmechanism of nacre, which is equivalent to the polymeric systems crazing. 56 Following that, Figure 4 b deduces the tensile characteristics of\nadvanced composites, where the initial stress–strain curve\nindicates an almost equivalent nature to yielding or low-strain applications. Figure 4 Mechanical\ntesting results of (a) Izod impact test, (b) tensile\ntest, and (c) flexural bending test. Additionally, promoting further load on structures,\nNS manifests\na noteworthy enhancement in force dissipation up to 33.3897 MPa of\nultimate stress, although NC ends at 31.5986 MPa and the clean sample\nreached 27.9189 MPa. In contrast, Young’s modulus ( E ) of the NS (803.42 MPa) describes a superior value to\nthe NC (746.55 MPa) sequentially compared to the pristine structure\n(623.33). As per the RVE model, a similar behavior shows that under\nthe action of uniaxial force, cracks propagate in the direction that\ngrips low energy; hence, a NC skeleton made of uniform stacking yields\na crack deflection path, while at the same point, breaks encounters\nwith hard bricks in an asymmetrical network, which urges for surpassing\nenergy in NS. Additionally, Figure 4 c also provides the load–displacement\ncurve of dynamic mechanical\nflexural bending flexural results of monolithic, NC, and sheet architecture. 57 As per Figure 4 c, from the initial loading until the fracture, the\nNC structure deforming slightly subordinately corresponds to a non-uniformed\nnacre array, whereas the pristine geometry trails an identical path.\nFor columnar stacking, the crack propagates at the interface of brick,\nwhich can be separated quickly, leading to crazing in the zone, while\nthe sheet also has an equivalent tendency to a generation of crack,\nyet the propagation hinders subsequent bricks in layered position.\nThe bending strengths of 3D-printed immaculate geometry and gastropod\narchitecture achieved the maximum levels of 32.05 and 33.79 MPa, respectively,\nwhereas bivalve shell geometry reached the ultimate power of 43.75\nMPa. However, it has been noticed that total energy absorbance capacity\nwas superior for the NC, attributed to minor interlayer deviation\nand interface tablet cohesion against bending load conditions. 58 The surface unevenness value of the upper\nlayer of PCABS objects\nis calculated, and the mean average of surface roughness (Ra) and\nthe mean maximum profile height (Rz) for each bio-inspired skeleton\nare provided in Figure 5 a,b. As per shown in Figure 5 c for the 2D roughness profiles, it is observed that the roughness\nprofile is higher if the peak-to-valley difference is height, which\nis described by the nacreous sheet; however, it progressively declines\nfor columnar nacre and a neat sample. 59 The new PCABS component comprises an R A value (arithmetic average) of 2.02 μm and an R Z value of 14.336 μm; nonetheless, for the NC structure,\na maximum R A reaches 11.038 μm,\nand a value of R Z observed as 49.408 μm\nand the highest surface roughness R A of\n20.842 μm, and R Z of 103.959 μm\nare obtained for densely packed NC tablets. Printing top layer patterns\nsuch as 0/90, −45/+45, hexagonal, and concentric have voids\nand porosity, which act as unevenness in the outermost layers, increasing\nroughness. Chemical treatments in 3D printing modify the top surface\ndue to dissolving surface material and filling the voids between rasters,\nresulting in a smoother and more uniform surface. Surface texture\nis a surface roughness component that plays a crucial role in defining\nthe composite interaction with other environments such as temperature,\nwater, coatings, and adhesives. Roughness is an excellent gauge of\nthe prospective functioning of a mechanical component owing to irregularities\non the plane that could form a nucleation site for corrosion or cracks. 60 , 61 Figure 5 Representation\nof surface roughness evolution results, (a) surface\nroughness, R A value of three different\narrangements, (b) maximum height of top layer profile, R z value, and (c) surface waviness graphs of pristine,\nNC, and NS geometry. Combining the high toughness and strength of nacre-like\n2D sheets\ncan be achieved by blocking crack propagation along the platelet side\nand the interface in their nanostructure of brick-and-mortar. Although\nthe variation of cracks in the microstructure of brick-and-mortar\nis well detailed, numerous other mechanisms for energy absorption\nin bio-inspired materials can exist. 62 Numerical\nconnections between bulk, micro-, and nanostructures revealed the\nmicroscopic effect on mechanical performance. While a crack propagates\nin locally disordered zones, the crack is pinned and generates stress\nconcentration around that zone. These hurdles to crack propagation\nare either bunches of platelet unevenness or greater-than-mean platelets\nconcerning most of the platelets. Such microstructural divergencies\nare, therefore, crucial to inhibiting crack propagation in nacre-like\ngeometries. 63 , 64 There are various computation\nmodels to predict fracture behavior, toughening mechanisms, and microstructural\nchange during loadings like the cohesive finite-element method, MD\nsimulation, Monte Carlo simulation, DEM, representative volume element,\nFEA, and trans-scale shear-lag model. 65 − 67 The nacre hierarchy\nwas produced as a 3D brick and mortar model for both sheet and columnar\nand fractured evolute using the optical microscopic image and SEM\nto study the surface morphology of fabricated samples, as illustrated\nin Figure 6 . It has\nbeen expansively studied that the glassy polymers’ failure\nprocess largely relies on shear banding or crazing (microscopic) or\nnecking (macroscopic) flow, and it scatters the constrained stress\nvia cavitation, bond rupture, viscoelastic deformation, crack growth,\nand crazing before catastrophic material failure. The SEM analysis\nof ruptured PCABS parts has proved their distinctive fracture mechanics,\nthat is, the interlayer raster delamination, multiple crazing, and\nshear deformation mechanism. 68 , 69 The FDM-printed nacre\ndesign revealed the nonregular contiguity formation between the layers\nof PCABS samples; however, such structural local imperfection does\nnot relinquish any compromise in the impact resistance compared to\nthe neat PCABS sample. As mentioned above, mollusk structure follows\nvarious methodologies for dissipating energy during cracking. Nevertheless,\nBarthelat illuminated that the “process zone” known\nas “stress whitening” was exhibited in nacreous architectures\nduring the fracture process. Such an exterior surface fracture is\nequivalent to crazing in polymeric systems. 42 , 56 Figure 6 Design\nof NC and NS architectures. (a) design of the single tablet,\na 2D sheet of brick-and-mortar phase, CAD model of NC and sheet, and\n3D printed view (expanded top view); (b) fracture surface morphology\nof pristine, columnar, and sheet structure analysis using an optical\nmicroscope and SEM images with a specific region of hard (brick) and\nsoft (mortar) segments. Moreover, FESEM micrographs of microstructure observations\nconcerning\nIzod and cryogenic fracture surfaces are elucidated in Figure 7 . Impact fracture of NS and\nNC articulated architecture is coined as a non-brittle failure phenomenon\nthat exhibits a tearing surface that involves crazing and cavitation\nphenomena of plastic deformation. Additionally, liquid nitrogen-dipped\nfracture of NS and NC, evident interlayer crack propagation, and cracked\ndelocalization result in energy dissipation, presumably without interfacial\ndebonding. 42 Figure 7 FESEM images of NS and\nNC for (a,b) Izod and (c,d) cryogenic fracture. PC/ABS composites are known for their excellent\nmechanical properties,\nthermal stability, extrudability, and poor biodegradation due to inadequate\nPC and ABS links. The structural properties of PC/ABS cannot be altered\nusing mechanical or chemical separation, so the utilization of this\ncomposite is highly beneficial for structural applications. On the\nother hand, the decomposition of the PCABS composite was conducted\nusing pyrolysis, where heating in the presence of various catalysts\nand bromine was generally combined with solid residues or pyrolysis\noil. It is essential to utilize feasible methods to decompose PCABS\ncomponents to obtain di-brominated products without catalysts or additives. 35 , 70 , 71 Li and Xu applied a supercritical\nwater oxidation process for environmentally friendly and efficient\ndecomposition where depolymerization, conjugation of free radicals,\ncarbonization, and generation of free radicals could be the mechanism. 72"
} | 7,928 |
33116516 | PMC7585798 | pmc | 6,450 | {
"abstract": "Background Artificial synaptic behaviors are necessary to investigate and implement since they are considered to be a new computing mechanism for the analysis of complex brain information. However, flexible and transparent artificial synapse devices based on thin-film transistors (TFTs) still need further research. Purpose To study the application of flexible and transparent thin-film transistors with nanometer thickness on artificial synapses. Materials and Methods Here, we report the design and fabrication of flexible and transparent artificial synapse devices based on TFTs with polyethylene terephthalate (PET) as the flexible substrate, indium tin oxide (ITO) as the gate and a polyvinyl alcohol (PVA) grid insulating layer as the gate insulation layer at room temperature. Results The charge and discharge of the carriers in the flexible and transparent thin-film transistors with nanometer thickness can be used for artificial synaptic behavior. Conclusion In summary, flexible and transparent thin-film transistors with nanometer thickness can be used as pressure and temperature sensors. Besides, inherent charge transfer characteristics of indium gallium zinc oxide semiconductors have been employed to study the biological synapse-like behaviors, including synaptic plasticity, excitatory postsynaptic current (EPSC), paired-pulse facilitation (PPF), and long-term memory (LTM). More precisely, the spike rate plasticity (SRDP), one representative synaptic plasticity, has been demonstrated. Such TFTs are interesting for building future neuromorphic systems and provide a possibility to act as fundamental blocks for neuromorphic system applications.",
"conclusion": "Conclusion In summary, in this study, we have fabricated flexible and transparent artificial synapse transistors with PET as the flexible substrate and PVA as the dielectric layer, and its I ON /I OFF ratio was 4.58×10 6 . The TFTs have a thickness on the order of nanometers. They can be used as temperature sensors and shows good spiking-rate dependent plasticity (SRDP). Furthermore, we succeeded in mimicking the biological synapse-like behaviors of EPSC, PPI, and LTM in the biological nervous system with artificial synaptic transistors. Although it cannot be used on a large scale at present, such artificial synaptic transistors provide a possibility to realize synaptic-like information processing and learning, and lay the foundation for the development of artificial intelligence.",
"introduction": "Introduction In recent years, amorphous oxide semiconductor (AOS) devices have attracted much attention. A representative AOS material is In-Ga-Zn-O (IGZO) which has been one of the most popular AOS materials since it was first published in 2004. 1 They have some attractive properties, including transparency, flexibility, relatively low-cost fabrication and low-temperature processing at no higher than 200 °C. TFTs can be used as pressure and temperature sensors. 2 , 3 TFTs are used in many ways and artificial synapse devices based on TFTs have been extensively studied, however, flexible and transparent artificial synapses based on nano TFTs have not been researched as much despite their promise in numerous applications. Synapse is the functional unit in the brain, and it connects the presynaptic and postsynaptic neurons. 4 In biological synapses, action potentials reach the pre-synapse, promote the release of the neurotransmitter, and cause excitatory postsynaptic potential (EPSP). 5 In the artificial synaptic device mentioned in the manuscript, a voltage is applied to the gate electrode to simulate the action potential, and the change of the channel current is to simulate the excitatory postsynaptic current. Therefore, the presynaptic stimulation is the pulse applied to the gate electrode. 6 , 7 For example, neurosynaptic behaviors are the basis of our learning, memory, and information processing. Artificial synaptic devices based on flexible TFTs can mimic biological synaptic behaviors like our brain and be useful for a number of applications. 8 Therefore, artificial synapse devices are worthy of further study. The biological synapse-like behaviors include information memory and possessing, which are triggered by the inflow dynamics of ion transport. Artificial flexible organic synaptic transistors capable of concurrently exhibiting signal transmission and learning functions were verified using a C 60 /poly (methyl methacrylate) (PMMA) hybrid layer. 9 Photonic non-volatile memory devices with hybrid polymer/UC nanocrystal composite materials as the active layer have been confirmed. 10 In this work, the flexible and transparent artificial synapse devices based on TFTs with PET as the flexible substrate, ITO as the gate, and PVA as the gate insulation layer were made and showed some representative synaptic characteristics. The device shows good synaptic plasticity. Some biological synapse-like behaviors are mimicked in this paper, for instance, excitatory postsynaptic current (EPSC), 11 – 13 paired-pulse facilitation (PPF) and long-term memory (LTM) demonstrating much promise for these materials in many fields. 14 – 17 As far as we know, few previous publications are on such a standard thin-film transistor structure, which is based on PVA insulators and IGZO semiconductors. At the same time, the design here has a combination of artificial synapse functions and temperature sensor characteristics.",
"discussion": "Results and Discussion Figure 2A shows the transfer characteristic curve, where V DS is 1.1 V, the gate voltage V GS is scanned from \\documentclass[12pt]{minimal}\n\\usepackage{wasysym}\n\\usepackage[substack]{amsmath}\n\\usepackage{amsfonts}\n\\usepackage{amssymb}\n\\usepackage{amsbsy}\n\\usepackage[mathscr]{eucal}\n\\usepackage{mathrsfs}\n\\DeclareFontFamily{T1}{linotext}{}\n\\DeclareFontShape{T1}{linotext}{m}{n} {linotext }{}\n\\DeclareSymbolFont{linotext}{T1}{linotext}{m}{n}\n\\DeclareSymbolFontAlphabet{\\mathLINOTEXT}{linotext}\n\\begin{document}\n$$ - $$\n\\end{document} 2 V to 3 V, and the voltage sweeping rate is 0.1 V. It can be calculated that I ON /I OFF = 4.58×10 6 from the data in Figure 2A . The carrier charges gradually increase on the interface between the dielectrics and IGZO semiconductor channel as the voltage increases, and then the current is generated between the source and the drain electrodes. As shown in the Figure 2B , the V DS sweeps from 0 V to 2 V, and the gate voltage V GS increases from 0.35 V to 2 V with a step of 0.55 V. When V DS is consistent, the value of I DS increases when V GS increases, so the black line at the bottom represents V GS = 0.35 V. As can be seen from the figure, when the V DS is low, the device has an obvious linear region. When the V DS is high, the device shows good saturation current characteristics. The device can be used as temperature sensors, too. In Figure 2C , the transfer curves are measured when the drain-source voltage V DS = 1 V. When the temperature increases, the curves have a positive shift, this is the key to prove the device can work as a temperature sensor. The continuous modulation of the synaptic weight can be defined as synaptic plasticity. 18 Therefore, the synapse can perform learning and memory functions. Spiking-rate dependent plasticity (SRDP) belongs to the class of synaptic plasticity. It is important to realize this in artificial synapses. Figure 2D shows the experiment of the spiking-rate dependent plasticity (SRDP). The drain-source voltage is 1.5 V and the gate-source voltage is 2 V. Here, the pulse width and pulse interval remain the same. When the pulse interval is 0.02 s, 0.03 s, and 0.04 s, the corresponding amplitudes of the drain current are 12 µA, 8 µA, and 6 µA, respectively, which decrease sequentially. The synaptic response is related to the frequency and can prove the learning rule of SRDP. Due to the change of the frequency, the synapse weight between neurons changes. In other words, presynaptic spikes with high frequency will lead to potentiation, whereas presynaptic spikes with low frequency will result in depression. Figure 2 The biological synapse-like transfer and output characteristic curves of the thin film transistors (TFTs). ( A ) Transfer characteristic curves. ( B ) Output characteristic curves. ( C ) Transfer curves measured by different temperatures and ( D ) The spiking-rate-dependent plasticity experiment. Figure 3A shows a simplified biological dendritic synapse. A synapse is a connection between two neurons in the brain which allow neurons to transmit electrical or chemical signals to another neuron. 19 The local magnification shows that synaptic plasticity is regulated by the migration of neurotransmitters, which are triggered by ion exchange at the membrane and synaptic junctions when the stimulation signal arrives. 20 , 21 When the nerve impulse reaches the presynaptic region, the voltage-gated ion channel is opened, and then the ions migrate through the channel, causing the neurotransmitter to release into the synaptic spaces. The neurotransmitters then bind to the postsynaptic receptors to complete transmission of the information. Therefore, the ion transport plays a key role in regulating synaptic weight and transport information. As shown in Figure 3B , the gate electrode of the flexible and transparent TFTs corresponds to the presynaptic region, the source/drain electrode as the postsynaptic region, the IGZO channel as the synaptic cleft, and the carriers in the channel as the ions in the neural synapse. Figure 3 ( A ) Structure of synapses, including the synaptic cleft; presynaptic and postsynaptic neuron parts and ( B ) Corresponding relationship between biological synapses and TFTs. Figure 4A shows the measurement condition for the Keithley 4200 with probe pulses. One or more pulses are applied to the gate electrode and then the current at the drain is read. Figure 4B shows the EPSC of the artificial synaptic transistor and during the EPSC measurement, a constant drain voltage of 1.5 V was applied. The EPSC was triggered by a presynaptic spike (9.0 V, 1.0 s) on the ITO bottom gate electrode. The presynaptic spike triggers an EPSC current with a peak value of ~2.5×10 −6 A and at the end of the spike, it gradually reduced to the initial current. When the voltage increases and the transistor is in the over-threshold region, the channel can be seen as a capacitor. Voltage pulses with amplitudes 915 V can be regarded as the maximum voltage at which the capacitor discharges. When a positive presynaptic spike is applied on the bottom ITO gate electrode, the mobile carriers will migrate and accumulate at the interface between the dielectrics and the IGZO semiconductor channel. When the spike ends for a while, the accumulated carriers will gradually migrate back to their initial equilibrium position due to the concentration gradient. 22 The postsynaptic parameters can influence the EPSC, and it can be found in our previous publications. 11 The drain-source voltages can provide a range of post-spikes and influence the output current. The equation can be seen as follows:\n (1) \\documentclass[12pt]{minimal}\n\\usepackage{wasysym}\n\\usepackage[substack]{amsmath}\n\\usepackage{amsfonts}\n\\usepackage{amssymb}\n\\usepackage{amsbsy}\n\\usepackage[mathscr]{eucal}\n\\usepackage{mathrsfs}\n\\DeclareFontFamily{T1}{linotext}{}\n\\DeclareFontShape{T1}{linotext}{m}{n} {linotext }{}\n\\DeclareSymbolFont{linotext}{T1}{linotext}{m}{n}\n\\DeclareSymbolFontAlphabet{\\mathLINOTEXT}{linotext}\n\\begin{document}\n$$I = \\left({{I_0}-{I_\\infty }} \\right){\\rm{ }}exp\\left[{-\\left({{{t - {t_0}} \\over {b - aVd{\\tau _0}}}} \\right)\\beta } \\right] + {\\rm{ }}{I_\\infty }$$\n\\end{document} \n where τ is the retention time, t 0 is the time when the presynaptic spike finishes, I 0 is the triggered maximum EPSC, and I ∞ is the EPSC at the end of the presynaptic spike. τ is found to be voltage-dependent. When the presynaptic neurons are stimulated by two consecutive pulses, the second peak triggered by the pulses is larger than the first peak. This phenomenon is called a paired-pulse boost (PPF), which plays an important role in visual and auditory signal processing. 23 Here, we can imitate the phenomenon of the PPF in the artificial synaptic transistor. Figure 4C shows a PPF response in the flexible and transparent artificial synaptic transistor. Two successive identical pulses (15.0 V, 1.0 s) with an interval time of 2.8 s are applied to the ITO gate with a constant V D of 1.5 V. The values of the first EPSC (I 1 ) and the second EPSC (I 2 ) are respectively ~6.9×10 −7 A and ~4.5×10 −6 A, meanwhile, an EPSC gain (I 2 /I 1 ) of ~6.5 times is obtained. At the end of the first spike, some of the activated carriers remain on the interface between the IGZO channel and the insulating layer before they return to their original position. 17 Therefore, when the second peak is applied to the gate after the first spike with a small inter-spike interval, the values of the second EPSC (I 2 ) is larger than the first EPSC (I 1 ). In the biological nervous system, some of the changes in synaptic connections can last for hours or even days. This phenomenon is known as the long-term memory (LTM), which is the basis for learning, memory and information processing. 24 As shown in Figure 4D , ten sequential identical pulses (15.0 V, 1.0 s) with an interval time of 2.0 s are applied to the ITO gate with a constant V D of 1.5 V. Figure 4C shows the peaks progressively increase by the successive application of gate pulses and it lasts a long time after the end of the pulse. The LTM process is due to some of the carriers of the IGZO channel changing irreversibly under the continuous high gate pulses. 25 Figure 4 ( A ) The pulse measurement schematic diagram based on IGZO transistors. ( B ) The excitatory postsynaptic current (EPSC) plotted versus time. ( C ) A PPF response for the artificial synaptic transistor and ( D ) The EPSC response triggered by ten sequential gate pulses. Artificial synaptic behaviors are caused by charging and discharging of the moving carriers stimulated by the gate pulse as shown in Figure 5 . Figure 5A shows that the carrier transport behavior during a period of the pulse could be divided into five stages: the basic voltage V 1 , the instantaneous rising voltage V 2 , the higher voltage V 3 , one instantaneous dropping voltage V 4, and the last basic voltage V 5 . As shown in Figure 5B , at stage ①, when the V 1 is applied, the mobile carriers are not attracted to the interface between the dielectrics and the IGZO semiconductor channel. At stage ②, when the V 2 is applied to the gate, it increases instantaneously, and the mobile carriers at the interface between the IGZO channel and the insulating layer PVA gradually increase. Meanwhile, the functional groups such as In-O, Ga-O, and Zn-O can interact with metal ions and persist in the channel. At stage ③, when the gate voltage is kept at a higher voltage V 3 , more carriers remain at the interface between the dielectrics and the IGZO semiconductor channel. Stage ④ is just the opposite of stage ②. The accumulated mobile carriers at the interface between the dielectrics and the IGZO semiconductor channel are driven back into the source and the drain electrodes. The migration rate in stage ④ is slower than in stage ②, because, at stage ②, the free carriers move faster under pulse pressure, while at stage ④, free carriers diffuse slower back to the original position due to the concentration gradient. 26 Stage ⑤ is the same as stage ①. When the gate voltage drops to V 5 , there are no carriers at the interface between the IGZO channel and the insulating layer and then the device returns to its initial state. 27 , 28 Figure 5 ( A ) Schematic diagram of the pre-synaptic spike applied to the gate electrode and ( B ) Overview of the voltage pulse profile and the corresponding discharge current in the TFTs with the IGZO channel."
} | 3,992 |
28688575 | null | s2 | 6,451 | {
"abstract": "Cell-cell communication enables bacteria to coordinate their behavior through the production, recognition, and response to chemical signals produced by their microbial neighbors. An important example of coordinated behavior in bacteria is biofilm formation, where individual cells organize into highly complex, matrix-encased communities that differentiate into distinct cell types and divide labor among individual cells. Bacteria rely on environmental cues to influence biofilm development, including chemical cues produced by other microbes. A multitude of recent studies have demonstrated that natural-product antibiotics at subinhibitory concentrations can impact biofilm formation in neighboring microbes, supporting the hypothesis that these compounds may have evolved as signaling molecules that mediate cell-cell interactions. In this review we discuss the role of antibiotics in modulating biofilm formation and interspecies communication in bacteria."
} | 240 |
24141053 | null | s2 | 6,452 | {
"abstract": "Metabolic engineering offers the opportunity to produce a wide range of commodity chemicals that are currently derived from petroleum or other non-renewable resources. Microbial synthesis of fatty alcohols is an attractive process because it can control the distribution of chain lengths and utilize low cost fermentation substrates. Specifically, primary alcohols with chain lengths of 12 to 14 carbons have many uses in the production of detergents, surfactants, and personal care products. The current challenge is to produce these compounds at titers and yields that would make them economically competitive. Here, we demonstrate a metabolic engineering strategy for producing fatty alcohols from glucose. To produce a high level of 1-dodecanol and 1-tetradecanol, an acyl-ACP thioesterase (BTE), an acyl-CoA ligase (FadD), and an acyl-CoA/aldehyde reductase (MAACR) were overexpressed in an engineered strain of Escherichia coli. Yields were improved by balancing expression levels of each gene, using a fed-batch cultivation strategy, and adding a solvent to the culture for extracting the product from cells. Using these strategies, a titer of over 1.6 g/L fatty alcohol with a yield of over 0.13 g fatty alcohol/g carbon source was achieved. These are the highest reported yield of fatty alcohols produced from glucose in E. coli."
} | 334 |
27148237 | PMC4838625 | pmc | 6,453 | {
"abstract": "Nitrogen starvation is an efficient environmental pressure for increasing lipid accumulation in microalgae, but it could also significantly lower the biomass productivity, resulting in lower lipid productivity. In this study, green alga Chlorella sp. A2 was cultivated by using a minimal nitrogen supply strategy under both laboratory and outdoor cultivation conditions to evaluate biomass accumulation and lipid production. Results showed that minimal nitrogen supply could promote neutral lipid accumulation of Chlorella sp. A2 without a significant negative effect on cell growth. In laboratory cultivation mode, alga cells cultured with 18 mg L −1 d −1 urea addition could generate 74 and 416% (w/w) more neutral lipid productivity than cells cultured with regular BG11 and nitrogen starvation media, respectively. In outdoor cultivation mode, lipid productivity of cells cultured with 18 mg L −1 d −1 urea addition is approximately 10 and 88% higher than the one with regular BG11 and nitrogen starvation media, respectively. Notably, the results of photosynthetic analysis clarified that minimal nitrogen supply reduced the loss of photosynthetic capacity to keep CO 2 fixation during photosynthesis for biomass production. The minimal nitrogen supply strategy for microalgae cultivation could promote neutral lipid accumulation without a significant negative effect on cell growth, resulting in a significant improvement in the lipid productivity.",
"introduction": "Introduction In the past few decades, the extensive utilization and irreversible depletion of fossil fuels has led to global climate change, environmental pollution, health problems, and an energy crisis (Amaro et al., 2011 ; Chen et al., 2016 ). Biofuel, as a new, alternative, clean, and sustainable energy source, has attracted great interest from researchers, local governments, and international traders (Lam and Lee, 2012 ). A variety of biolipids can be used to produce biodiesel, which is one of the most commonly used biofuel, and vegetable oils, such as soybean, sunflower, rapeseed, and palm oil, are renewable and potentially inexhaustible sources of energy with an energy content close to that of diesel fuel (Demirbas, 2011 ; Zhang et al., 2014 ). However, extensive use of vegetable oils may cause significant problems, for example, abundance of resource applied to produce fuel will cause starvation in developing countries, and it is important that productive and cultivated land should be used for food instead of fuel production (Paiva and Wolde-Georgis, 2010 ). One possible alternative, non-food, source of biological material for biofuel production is microalgae (Day et al., 1999 ; Qiao et al., 2015 ), which can be feedstock for biofuel production via photosynthesis by harvesting solar energy and fixing CO 2 and convert it into biolipids (Razeghifard, 2013 ; Chen et al., 2015b ). Moreover, microalgae are capable of rapid growth under a wide range of culture conditions and more photosynthetically efficient than higher plants (John Pirt, 1986 ). Thus, microalgae are now considered to be one of the most promising types of feedstock for making biodiesel (Mata et al., 2010 ; Chen et al., 2014 ). The green microalga Chlorella (Chlorophyta) can grow photoautotrophically, mixotrophically, or heterotrophically under various culture conditions, with high biomass yield. The oil content in some species of Chlorella varies from about 14 to 63% of dry weight, and the fatty acid composition has been reported to range from C-14:0 to C-20:0 (O'Grady and Morgan, 2011 ). Considering the above advantages, Chlorella would appear to be a good material for biodiesel production. Microalgal lipid accumulation is influenced by culture conditions. Previous studies have indicated that the lipid content of some microalgae can be increased by means of nitrogen (N) starvation (Illman et al., 2000 ; Takagi et al., 2000 ; Rodolfi et al., 2009 ; Chen et al., 2015a ). The general theory is that when there is insufficient N for the protein synthesis required for growth, excess carbon from photosynthesis is channeled into storage molecules e.g., triglycerides or starch (Scott et al., 2010 ). However, although N starvation can increase the lipid content, it is not conducive for biomass accumulation or lipid productivity of microalgae (Vona et al., 1999 ). In our previous study (Zhang et al., 2013 ), it was reported that N starvation resulted in significant neutral lipid accumulation in Chlorella cells, but in the meantime decreased photosynthetic rate, oxygen evolution, respiration rate, and photochemistry efficiency, as well as increasing damage to the Photosystem II (PSII), were observed. Therefore, the lipid productivity of microalgae was not significantly enhanced under N starvation condition. In this study, a minimal nitrogen supply strategy was used in the cultivation of Chlorella sp. A2 (Hu et al., 2008 ) (an oil-producing microalgae isolated from the wild) for improving neutral lipid productivity. Results demonstrated that the minimal nitrogen supply strategy for microalgae cultivation could promote neutral lipid accumulation without a significant negative effect on cell growth, resulting in a significant improvement in the neutral lipid productivity.",
"discussion": "Discussion In the course of microalgae biodiesel research, numerous reports have revealed that nitrogen deficiency and limitation could increase the lipid content (Jiang et al., 2012 ; Liu et al., 2012 ). Stress can increase the lipid content and can also decrease the biomass, because high lipid content accumulation is often accompanied by weak or inhibited cell division (Courchesne et al., 2009 ; Gouveia and Oliveira, 2009 ; Widjaja et al., 2009 ; Ahmad et al., 2011 ). While the economic feasibility of microalgae as feedstock for biofuel production depends on three main key factors: biomass accumulation, lipid content, and lipid productivity. Furthermore, biomass and lipid content determine the total lipid productivity. Our strategy did not affect the biomass growth significantly (Table 1 ). The minimal nitrogen supply strategy seems to induce the algae, at every daily minimal urea addition, to enter a transient state of lipid accumulation. This may be fundamentally different to the processes occurring in the algal cells under traditional nitrogen-deficiency stress. In the early period of a cycle of 24 h, microalgae tend to undergo cell division and proliferation with the inadequate urea; in the later period, microalgae begin to accumulate more lipids due to the depletion of urea as a result of the nutritional stress. There are two ways that nitrogen-deficiency stress can promote lipid synthesis: one is to compel some carbon fixed during photosynthesis to be channeled into the lipid synthesis pathway (Suen et al., 1987 ), and another is to cause the carbohydrates to flow into the lipid synthesis pathway directly (Wang et al., 2009 ; Xin et al., 2010 ). In addition, in the present study, the photosynthetic capacity was not adversely affected by minimal nitrogen supply. Under conditions of nutritional stress, including nitrogen deficiency, when photosynthesis of algae cells is not interrupted and carbon assimilation is still occurring, carbon flow has been shown to shift from protein synthesis to lipid synthesis (Sheehan et al., 1998 ). To lessen the decrease in biomass under conditions of nitrogen deficiency, some researchers invented a two-stage cultivation strategy to improve lipid productivity (Widjaja et al., 2009 ; Ho et al., 2010 ), obtaining maximum biomass accumulation in nitrogen-sufficient media and then changing the microalgae conditions to a lipid-accumulation environment. However, the two-stage cultivation strategy is difficult to practice for large-scale cultivation. One approach has also been shown that urea limitation could enhance oil production in other Chlorella sp. (Hsieh and Wu, 2009 ). In the present study, addition of smaller amounts of urea was demonstrated to promote the lipid content and to maintain high biomass accumulation. It appears that, under conditions of low levels of urea addition, a balance between the growth rate and the lipid content accumulation is thus obtained. Compared with the cultivation in regular BG11, this strategy not only unaffected the biomass accumulation of the microalgae, but also promoted the lipid content, and the total lipid productivity was improved (Figure 5A , Table 1 ). In contrast to the two-stage cultivation strategy, the minimal nitrogen supply strategy can greatly simplify the cultivation process, which has significant advantages in terms of utilizing a single, straightforward cultivation procedure. In a study by Pirastru et al. ( 2012 ), it was found that both PSI and PSII activities were inhibited by nitrogen limitation, and the activity of the PSI declines much more rapidly than PSII. In our previous studies, it was also found that increased damage to PSII was observed in Chlorella cells under nitrogen starvation (Zhang et al., 2013 ; Chen et al., 2015a ). However, with increased proportion and energy distribution to PSI in the meantime, the Ca 2+ -regulated cyclic electronic flow increased to produce more ATP for nitrogen starvation-induced lipid synthesis (Zhang et al., 2013 ; Chen et al., 2014 , 2015a ). In the present study, only minor differences in Fv/Fm, Φ II , and NPQ were detected between the regular BG11-grown and the minimal urea treatments (Figures 6A–E ). It is believed that Φ II is well correlated with CO 2 fixation in the Calvin cycle (Edwards and Baker, 1993 ). The results indicated that CO 2 fixation in cells with scant urea treatments is still running regularly, thus providing organic carbon source for constant biomass production (Figures 1 , 5 , Tables 1 , 2 ). The application of microalgae in biodiesel production is currently in the laboratory scale and will be expected to just stay in the pilot or exemplary scale in a long time (Chen et al., 2015b ). In the present study, cells cultured with addition of minimal urea accumulated 34–44% lower lipids content than in N− medium (Table 1 ), which means more cells should be harvested and treated for extracting the same lipid yield, and average energy consumption per unit mass of the oil may be higher and extracting lipids from a lower lipid content culture can be more laborious and more costs than extracting from a lipid rich culture. However, the 72–99% increased lipid productivity (Table 1 ) in those cultures should be more than enough to cover the labor and the energy, and moreover, the lowered lipid content (22%) of cells cultured with addition of minimal urea than in N− medium (38%) is not enough to change the extraction process, i.e., the impact factor of decreased oil content is relatively small in the total cost. In summary, this study suggests a minimal urea addition strategy for microalgae cultivation for biofuel feedstock production that could promote neutral lipid accumulation without a significant negative effect on cell growth, which is also a single step process and is applicable to outdoor cultivation condition. For achieving excellent biomass growth and lipid productivity, appropriate urea consumption is ~65–100 mg L −1 \n OD 680 − 1 for the algae cultivation both in laboratory and outdoor conditions. Notably, our research has clarified the mechanism that minimal urea addition reduced the loss of photosynthetic capacity to keep CO 2 fixation during photosynthesis for biomass production, and nitrogen limitation promoted the accumulation of neutral lipids, resulting in a significant improvement in the productivity of neutral lipids."
} | 2,930 |
30349546 | PMC6187967 | pmc | 6,458 | {
"abstract": "Root nodule symbiosis (RNS) is a symbiotic interaction established between angiosperm hosts and nitrogen-fixing soil bacteria in specialized organs called root nodules. The host plants provide photosynthate and the microsymbionts supply fixed nitrogen. The origin of RNS represents a major evolutionary event in the angiosperms, and understanding the genetic underpinnings of this event is of major economic and agricultural importance. Plants that engage in RNS are restricted to a single angiosperm clade known as the nitrogen-fixing clade (NFC), yet occur in multiple lineages scattered within the NFC. It has been postulated that RNS evolved in two steps: a gain-of-predisposition event occurring at the base of the NFC, followed by a gain-of-function event in each host plant lineage. Here, we first explore the premise that RNS has evolved from a single common background, and then we explore whether a two-step process better explains the evolutionary origin of RNS than either a single-step process, or multiple origins. We assembled the transcriptomes of root and nodule of two actinorhizal plants, Ceanothus \n thyrsiflorus and Datisca \n glomerata . Together with the corresponding published transcriptomes of the model legume Medicago \n truncatula , the gene expression patterns in roots and nodules were compared across the three lineages. We found that orthologs of many genes essential for RNS in the model legumes are expressed in all three lineages, and that the overall nodule gene expression patterns were more similar to each other than expected by random chance, a finding that supports a common evolutionary background for RNS shared by the three lineages. Moreover, phylogenetic analyses suggested that a substantial portion of the genes experiencing selection pressure changes at the base of the NFC also experienced additional changes at the base of each host plant lineage. Our results (1) support the occurrence of an event that led to RNS at the base of the NFC, and (2) suggest a subsequent change in each lineage, most consistent with a two-step origin of RNS. Among several conserved functions identified, strigolactone-related genes were down-regulated in nodules of all three species, suggesting a shared function similar to that shown for arbuscular mycorrhizal symbioses.",
"conclusion": "Conclusion The evolution of RNS represents a major event in the biology of plant-microbe interactions ( Doyle, 2016 ), and different explanations of the evolutionary origins have been proposed. We have demonstrated the genetic homology of RNS in the three lineages based on the presence of RNS pathway orthologs and the high similarity of gene expression patterns across the three species, thus demonstrating that RNS shares a common evolutionary event at the base of the NFC. At the same time, we show that most genes (regardless of whether the gene is involved in the process of RNS or not) that experience change in selection pressure at the base of the NFC also experienced subsequent changes in selection pressure at the base of each RNS host lineage. Taken together, our results are most consistent with the two-step hypothesis of the origin of RNS. The work of Werner et al. (2014) supported the two-step hypothesis, but was based on a single trait (capability to establish RNS) and had been criticized for being based on a flawed phylogenetic tree ( Doyle, 2016 ; LPWG, 2017 ). Our findings provide additional support for the two-step hypothesis. On the other hand, two recent papers suggest a more ancient origin of functional RNS within the NFC followed by multiple losses ( Griesmann et al., 2018 ; van Velzen et al., 2018 ). In Cannabaceae (Rosales), Parasponia retains the capability for RNS, whereas closely related Trema has lost it ( van Velzen et al., 2018 ). Within a single genus, Dryas \n octopetala (Rosaceae) apparently does not form root nodules, whereas other Dryas species retain this trait ( Becking, 1984 ). In a larger scale, two recent studies found that NIN and RPG have been lost among plants within the NFC that are not RNS hosts multiple times ( Griesmann et al., 2018 ; van Velzen et al., 2018 ). Since the known functions of these genes are specific to RNS, multiple losses are difficult to explain under two-step hypothesis where these genes would be maintained for millions of years after the gain-of-predisposition event until the gain-of-function event. Based on the known phylogenetic distribution of RNS hosts, the gain-of-predisposition at the MRCA of the NFC followed by a gain-of-function has been postulated as a parsimonious hypothesis since the discovery of the NFC ( Soltis et al., 1995 ). What is the genetic nature of the predisposition assumed in the two-step hypothesis? Natural selection can only operate on a “predisposition” if the predisposition has a function of its own. Otherwise, the propensity for a gain-of-function could not have been conserved for tens of millions of years in multiple lineages ( Doyle, 2011 ; Werner et al., 2014 ; Li et al., 2015 ). Likewise, a single-origin hypothesis needs an explanation for its apparently unparsimonious distribution of RNS hosts within the NFC. The high cost of RNS might be an explanation ( Griesmann et al., 2018 ), but no direct evidence is available yet. In either case, the key to answer this question depends on an understanding of the genetic underpinnings that led to RNS, which still remains incomplete.",
"introduction": "Introduction Root nodule symbiosis is a symbiotic interaction established between certain groups of angiosperm hosts and nitrogen-fixing soil bacteria that are housed in specialized organs called root nodules. The host plants provide photosynthate to their microsymbionts, and in turn the microsymbionts provide fixed nitrogen to their host plants. This symbiotic relationship enables host species to thrive in nutrient-poor soils, and thus these RNS hosts play a major role in terrestrial ecosystems as pioneer plants ( Chapin et al., 1994 ). Moreover, legumes play key roles in agriculture, where plant-based biological nitrogen fixation accounts for as much as 10% of the total nitrogen fixed in the world ( Herridge et al., 2008 ; Fowler et al., 2013 ). Thus, understanding the genetic underpinnings of the origin of RNS not only provides insight into a major biological event in the evolution of angiosperms, but is also of major economic and agricultural importance. RNS occurs in ten families of angiosperms within four orders: Fabales, Rosales, Cucurbitales, and Fagales. Molecular phylogenetic studies have revealed that these four orders, which were previously considered to be distantly related within the angiosperms ( Cronquist, 1988 ), together form a clade known as the nitrogen-fixing clade (NFC) ( Soltis et al., 1995 ). Within each of the four orders, RNS occurs in a subset of the families, which are phylogenetically scattered within each order ( Swensen, 1996 ), and within each family, RNS is restricted to a subset of the genera. There are several possible hypotheses regarding the evolutionary origin of RNS that can explain this restricted (found only among orders of the NFC) yet scattered (found only in some families and genera of the NFC) distribution of RNS hosts ( Doyle, 2011 ). The single-origin hypothesis proposes that the capability of forming nitrogen-fixing root nodules evolved once in the MRCA of the NFC and was subsequently lost multiple times independently in the currently non-fixing lineages. The multiple-origin hypothesis proposes that the evolution of RNS occurred independently at least six and as many as ten times ( Doyle, 2011 ). The two-step hypothesis postulates that a predisposition for, i.e., propensity to subsequently gain, RNS was first gained at the base of the NFC, which was then followed by a gain of function that occurred independently in the aforementioned six to ten different lineages ( Soltis et al., 1995 ; Swensen, 1996 ; Werner et al., 2014 ). The two-step hypothesis has been supported by phylogenetic analysis based on the distribution of RNS hosts within the NFC ( Werner et al., 2014 ). The hypothesis can parsimoniously explain the restricted yet scattered phylogenetic distribution of RNS hosts, but raises the question, what was the genetic basis of the “predisposition” to RNS? In addition to the phylogenetic evidence, several shared cellular, molecular, and genetic characteristics of RNS hosts in different lineages are consistent with a common evolutionary predisposition to RNS: (1) all RNSs result in a stable accommodation of the microsymbiont within the host cells ( Pawlowski and Demchenko, 2012 ); (2) homologs of many essential genes required for RNS in the model legumes Medicago \n truncatula and Lotus \n japonicus have been shown to be expressed in the nodules of non-legume RNS hosts ( Hocher et al., 2011 ; Demina et al., 2013 ); (3) calcium oscillation, an early host physiological response, is induced during initiation of RNS in both legume and non-legume hosts ( Granqvist et al., 2015 ). Multiple lineages sharing these aforementioned series of characters support the common descent of RNS across multiple lineages (i.e., supports single-origin and two-step hypotheses over multiple-origin hypothesis), but some morphological, cellular, and molecular characteristics are clearly distinct in different lineages of RNS hosts ( Pawlowski and Demchenko, 2012 ), which could favor a multiple-origin hypothesis. Most notably, RNS hosts in two families (Fabaceae and Cannabaceae) associate with rhizobia as their microsymbiont, while the hosts in the remaining eight families associate with members of the actinobacterial genus Frankia . These eight families are collectively called the actinorhizal plants. Most Frankia genomes lack the genes coding for Nod factor, the signaling molecule responsible for the initiation of RNS in the model legumes ( Oldroyd, 2013 ), but homologs for the nodABC genes have been identified in some groups of Frankia ( Persson et al., 2015 ; Nguyen et al., 2016 ). Similarly, some legume hosts can be nodulated by rhizobia without the Nod factor signaling pathway ( Okazaki et al., 2015 ). Therefore, a range of different mechanisms for initiating RNS must exist among the legumes and the actinorhizal plants. Transcriptomes generated via RNA-seq represent a powerful source of data that can provide a comprehensive set of characters. In the present study, we have used evidence from comparative transcriptomics and molecular evolutionary analyses to test the competing hypotheses for the evolutionary origin(s) of RNS. To this end, we assembled the root nodule and root transcriptomes of two actinorhizal plant species, Ceanothus \n thyrsiflorus (Rhamnaceae, Rosales) and Datisca \n glomerata (Datiscaceae, Cucurbitales) and compared them to published transcriptomes of M. \n truncatula (Fabaceae, Fabales) ( Roux et al., 2014 ). We conducted differential gene expression analysis between nodules and roots to determine a set of genes that are root- or nodule-enhanced for each species. Then, to allow interspecific comparisons, phylogeny-based orthology prediction was conducted across the three species and other taxa that are either members or close outgroups of the NFC. We first explored if RNS has evolved from a single common evolutionary event that occurred at the base of the NFC, regardless of whether this event gave rise to the function or the predisposition of RNS. To test the homology of RNS in the three species, we first focused on the presence/absence of orthologs in C. \n thyrsiflorus and D. \n glomerata for 19 genes required for the initiation and development of root nodules in the model legumes M. \n truncatula or L. \n japonicus , because both the single-origin hypothesis and the two-step hypothesis would require that at least some of the orthologs of genes required for nodulation would be shared among all RNS hosts. Determining orthology is an improvement with respect to previous studies that have identified homologs of genes required for RNS in legumes in several actinorhizal plants ( Hocher et al., 2011 ; Demina et al., 2013 ), as orthologs are a subset of homologs that are most likely to be functionally equivalent according to ortholog conjecture ( Koonin, 2005 ; Altenhoff et al., 2012 ). A recent study has taken a similar approach, comparing nodule gene expression in Parasponia spp. the only non-legume host that can establish RNS with rhizobia, to M. \n truncatula ( van Velzen et al., 2018 ). Second, we compared the overall expression patterns of orthologous genes across the three species between roots and nodules. Our assumption was that a significant degree of similarity across the three species, which are known to belong to three different lineages of RNS hosts ( Doyle, 2011 ), is not expected if they had completely independent origins of RNS. Thus, sharing a significant degree of similarity would refute the multiple-origin hypothesis, and indicate a single common ancestor for RNS. Gene expression analysis and orthology predictions allowed us to identify a set of genes that showed a consistent pattern of differential expression across the three species, which we designated as the core set of genes for RNS. Then, we have further explored which of the three competing hypotheses best explains the origin of RNS, particularly whether a single-origin or a two-step process better explains the evolutionary basis of RNS. For this, we employed a model-based phylogenetic test. We focused on the fact that the three hypotheses each assume different timing(s) for the gain-of-predisposition or gain-of-function event leading to RNS in the evolutionary history of the NFC. We assumed that gain of a new function would result in a change in selection pressure on that gene, which should be reflected in the average ratio of non-synonymous to synonymous mutations ( dN / dS ) ( Hurst, 2002 ) in the coding regions of the gene. Thus, for each set of orthologs, we tested when (if ever) each member gained a new function by calculating dN / dS on key branches of their respective phylogenetic trees.",
"discussion": "Discussion Newly Assembled Transcriptomes Are of High Quality Both C. \n thyrsiflorus and D. \n glomerata root + nodule transcriptomes scored well in multiple measures of quality (e.g., proper insert size, long N50, and high % fragment mapped) throughout the assembly process. After the assembly, KEGG annotation found the two transcriptomes to have similar numbers of ECs to the M. \n truncatula transcriptome (and even to the M. \n truncatula genome) for most of the pathways ( Table 1 and Supplementary Table S4 ). Both transcriptomes were annotated (by KO, EC, and/or GO) for >85% of the transcripts. Moreover, BUSCO found 62.4%, 82.2% of the plant-universal orthologs for C. \n thyrsiflorus and D. \n glomerata , respectively. Furthermore, all the qPCR validated the RNA-seq results except for one gene ( Supplementary Table S7 ). This all together indicate that the root + nodule transcriptomes of C. \n thyrsiflorus and D. \n glomerata are of high quality. Nodule Gene Expression Patterns in C. thyrsiflorus, D. glomerata , and M. truncatula Are More Similar to Each Other Than Would be Expected by Random Chance Our analysis supports the homology (shared by common descent) of RNS among the three plant species based on multiple lines of evidence. First, orthologs for most of the 19 RNS pathway genes required for the proper nodulation in M. \n truncatula were present and expressed in the nodules of C. \n thyrsiflorus (17 out of 19 found) and D. \n glomerata (18 out of 19 found) ( Figure 1 ). Transcripts with high sequence similarity were found, even for the few genes that did not have an ortholog predicted ( Figure 1 and Table 2 ). Results of this orthology-based analysis strengthens previous homology-based reports (inferred based on high scores in BLAST searches) throughout the RNS pathway in D. \n glomerata, Alnus \n glutinosa (Betulaceae, Fagales) and Casuarina \n glauca (Casuarinaceae, Fagales) ( Hocher et al., 2011 ; Demina et al., 2013 ); and are consistent with reports demonstrating functional equivalents (presumed orthologs) of a specific member of the RNS pathway, such as SYMRK ( Markmann et al., 2008 ), CCaMK ( Svistoonoff et al., 2013 ), and NIN ( Clavijo et al., 2015 ), across multiple lineages. This is the first time, to our knowledge, that the entire pathway has been detected comprehensively in the context of a phylogenetically based orthology framework. It is also important to emphasize that the orthologs identified in our analysis included NFR1, NSP1, ERN1 , and NIN , genes not shared with the more ancient AM symbiosis ( Oldroyd, 2013 ). The presence and the expression of orthologs across these three species indicates that their existence predates the NFC, which is required for the RNS to share a common function and common origin across them. In all pairwise comparisons of gene expression patterns between two species, we found a moderate to strong positive correlation for genes that were significantly enhanced in the nodules ( r = 0.43–0.60); and there was a weak to moderate positive correlation for genes that were significantly enhanced in the roots ( r = 0.25–0.47). The correlation was much weaker when all genes (including genes that are not necessarily presumed to be involved in RNS) were compared at once across two transcriptomes ( r = 0.18–0.38) ( Figure 2 ). While it is common to describe Pearson correlation coefficients of r = 0.20–0.39, r = 0.40–0.59, r = 0.6–0.79, as indicating weak, moderate, and strong correlation, respectively, meaningful interpretations of particular values depend on the context in which they were obtained, in this case, comparisons of transcriptomes across a considerable phylogenetic distance. For example, the Pearson correlation coefficient between the gene expression patterns between two sets of 20 plants in one species, Arabidopsis \n thaliana , was 0.81 ( Kempema et al., 2007 ). By contrast, the comparisons made in our study are between pairs of different plant species that have been diverging for nearly 100 million years ( Bell et al., 2010 ); thus, we consider that the values of r = 0.43–0.60 found for nodule-enhanced genes indicate a high degree of similarity and conservation, compared with 0.18–0.38 for all genes. Finally, permutation tests based on the dissonance scores indicated that the overall gene expression patterns of the nodules in the three RNS hosts tested are more similar to each other than expected by random chance ( p < 0.0001). These results strongly support the homology of RNS in all three lineages, i.e., that their similarity is due to common descent. It is possible that other factors, such as similarity in the age of these tissues across the three transcriptomes, may have contributed to the similarity of the gene expression patterns. An increased spatiotemporal resolution for C. \n thyrsiflorus and D. \n glomerata , as obtained for M. truncatula through a time course transcriptome ( Larrainzar et al., 2015 ) or tissue-specific transcriptome ( Roux et al., 2014 ) would provide further clarity. Features of RNS Conserved Among the Three Lineages Orthologs of 17 or 18 of the 19 RNS pathway genes were expressed in the root + nodule transcriptomes of C. \n thyrsiflorus and D. \n glomerata , respectively, and three of them ( SYMREM, NIN , and RPG ) were universally nodule-enhanced ( Table 2 ). The up-regulation of SYMREM, NIN , and RPG in the nodules was also found in A. \n glutinosa , and C. \n glauca ( Hocher et al., 2011 ), and is consistent with what was found for NIN in D. \n glomerata ( Demina et al., 2013 ). With the inclusion of C. \n thyrsiflorus , we now show that an up-regulation of SYMREM, NIN , and RPG in the nodules of RNS hosts is found in all four orders within the NFC. Because the initiation and establishment of RNS consists of multiple developmental stages ( Pawlowski and Demchenko, 2012 ; Roux et al., 2014 ), a high degree of spatiotemporal resolution is crucial to accurately trace the expression pattern of a gene ( Roux et al., 2014 ; Larrainzar et al., 2015 ): Genes up-regulated only at a specific stage of nodule development may not be up-regulated within a transcriptome that is inclusive of the entire nodule. Thus, genes that are up-regulated (relative to root) in the whole nodule are expected either (1) to be so strongly nodule-enhanced in a given stage that they are detected as up-regulated even after averaging expression values over the whole nodule, or (2) to be nodule-enhanced throughout the process of nodulation. The latter expression pattern has been well documented in the case of NIN ( Schauser et al., 1999 ). For the remaining genes, whose orthologs were not universally up-regulated in either nodules or roots (some were up-regulated in two hosts and non-significant in the third), a higher resolution of space and time would be helpful for accurate comparisons across species. Among the processes found to be universally enriched in either nodules or in roots among the core genes, we focused on the following four processes based on the potential relevance to RNS. Nitrate Transporter Orthologs of NRT1.8 , a low-affinity nitrate transporter, were significantly up-regulated in the nodules of all three species of RNS hosts. In A. thaliana, NRT1.8 is up-regulated by nitrate, and is hypothesized to export nitrate from xylem conducting cells to xylem parenchyma ( Li et al., 2010 ). In M. \n truncatula , 50% of the expression of NRT1.8 ortholog was located in Zone I ( Roux et al., 2014 ), which corresponds to the nodule meristem. However, there are no functional conducting xylem elements in root meristems of higher plants, which would be the tissue equivalent of Zone I of the root nodule. Moreover, nitrate is known to suppress nodulation in Ceanothus ( Thomas and Berry, 1989 ) and in L. \n japonicus ( Soyano et al., 2014 ). In L. \n japonicus , nitrate and NIN work antagonistically against each other ( Soyano et al., 2014 ): NIN expression suppresses the activation of nitrate-induced genes while nitrate suppresses the activation of NF-YA1 and NF-YB1 , the known targets of NIN . Taken together, these lines of evidence suggest that the function of NRT1.8 in the root nodules is not related to nitrate. While the understanding of NRT1.8 is still limited, the NRT1 family proteins are also capable of transporting signaling compounds and phytohormones: In A. \n thaliana, NRT1.1, NRT1.2 , and NRT1.10 are involved in the transport of IAA, ABA, and glucosinolates, respectively ( Chiba et al., 2015 ). Strigolactone Biosynthesis The genetic dissection of RNS has revealed that the pathway for its establishment shares many of its genes with the more ancient pathway to form AM symbiosis ( Oldroyd, 2013 ). It is thus of substantial importance that D27 and MAX4 , genes coding for the enzymes of the first and the third steps of strigolactone biosynthesis ( Ruyter-Spira et al., 2013 ) are apparently suppressed in the nodules of all three nitrogen-fixing hosts ( Table 3B ). Strigolactone has a number of functions in plants ( Besserer et al., 2006 ; Gomez-Roldan et al., 2008 ), particularly as a key regulator in root development, with possible regulatory feedback interactions with auxin transport and metabolism ( Kapulnik and Koltai, 2014 ). Strigolactone is also a signaling molecule that initiates AM symbiosis by stimulating the branching and growth of hyphae of the AM fungi ( Besserer et al., 2006 ). Within the mature AM symbiotic tissue, however, strigolactone is down-regulated ( Kapulnik and Koltai, 2014 ). A similar pattern may be inferred for RNS. Strigolactone has been shown to play a role in promoting nodulation in Pisum \n sativum , in that a strigolactone mutant formed fewer nodules than wild-type ( Foo and Davies, 2011 ), although, strigolactone is not required for nodulation, since nodulation does occur in the mutant. The influence of strigolactone on nodulation in P. sativum seems to be limited to the early stage of infection thread formation ( McAdam et al., 2017 ). Thus, the down-regulation of strigolactone biosynthesis in the nodule tissue of three phylogenetically distinct hosts that was observed in this study could be a function derived from AM symbiosis, to inhibit a portion of the root system from forming further infection sites, or to limit a stage of root development from further growth, allowing for nodule development or maintenance. The enrichment of the GO term related to secondary shoot formation in the root transcriptome is likely related to the pattern of strigolactone gene expression observed, since MAX4 plays a role in regulating branching in the shoot ( Sorefan et al., 2003 ). Cytokinin Biosynthesis Up-regulation of genes associated with cytokinin biosynthesis and/or metabolism during the establishment of RNS is well documented in L. \n japonicus and M. \n truncatula ( Tirichine et al., 2007 ; van Zeijl et al., 2015b ; Gamas et al., 2017 ). Cytokinin in legume RNS is a major signal to the cortical cells to initiate nodule organogenesis by induction of NIN through the activation of a cytokinin receptor LHK1 ( Tirichine et al., 2007 ). Since the up-regulation of NIN has been found in the nodules of A. \n glutinosa, C. \n glauca , and D. \n glomerata ( Hocher et al., 2011 ; Demina et al., 2013 ), NIN is considered to play a major role in RNS in actinorhizal plants as well as in the legumes. We found a universal up-regulation of CYP735A , a gene coding for the biosynthesis of trans-zeatin. IPT was also up-regulated in the nodules in C. \n thyrsiflorus and D. \n glomerata . Although IPT was down-regulated overall in M. \n truncatula , this is likely explained by the fact that >98% of M. \n truncatula \n IPT ortholog (Medtr2g022140) expression was restricted to the pre-infected cells ( Roux et al., 2014 ). Nitric Oxide Response The cellular response to NO was universally down-regulated in the nodules of the three host plants in this study. Two sets of orthologs responsible for this pattern were detected, but only one had an assigned name: AHK5 , a histidine kinase originally identified in A. \n thaliana as a regulator of stress response in guard cells ( Desikan et al., 2008 ). In M. \n truncatula , NO was found to be a regulator of nodule senescence: increased and decreased levels of NO led to quickening and delay of nodule senescence, respectively ( Cam et al., 2012 ). Since the nodules used in this study were all in relatively early stages of development, NO production in the nodules would not be expected to be high. Moreover, NO binds to and reduces activity of glutamine synthetase ( Melo et al., 2011 ), the key enzyme in primary N assimilation. Thus, the down-regulation of AHK5 would be important to maintain low concentrations of NO in the nodules. Alternatively, in Arabidopsis, AHK5 is known to confer resistance to pathogens such as Pseudomonas \n syringae and Botrytis \n cinerea ( Pham et al., 2012 ). Moreover, AHK5 was most highly expressed in the roots of A. thaliana ( Desikan et al., 2008 ). An inverse relationship exists between the host plant immune response and symbiotic processes established in the root nodules ( Toth and Stacey, 2015 ). Down-regulation of AHK5 could be part of the mechanism that enables the harboring of bacterial cells within the plant cells. dN/dS Analysis Disfavored the Single-Origin Hypothesis Of the 3,894 MergedOrthoGroups, 31.5% (1,226) rejected the NULL scenario, which assumes a single rate of dN / dS throughout the tree. Of these 1,226 MergedOrthoGroups, 95.1% (1,166) supported a change in selection pressure at the base of the NFC. Among these, only 0.5% (6) rejected the TWOSTEP scenario while 68.3% (796) rejected the SINGLE scenario. The 58 core genes (MergedOrthoGroups), which should be strong candidates for playing key roles in the evolutionary origin of RNS, showed the same general pattern: 32.8% (19) rejected the NULL scenario, of which 89.5% (17) supported a change in selection pressure at the base of the NFC. Among these, 76.5% (13) rejected the SINGLE scenario, but none rejected the TWOSTEP scenario. We did not determine how many, if any, of the MergedOrthoGroups (i.e., sets of orthologous genes) are in fact the gene(s) that gave rise to RNS; thus our findings do not reject the single-origin hypothesis. However, the results of analyzing nearly 4,000 genes clearly disfavor the single-origin hypothesis. Even the MergedOrthoGroups that rejected the TWOSTEP scenario are not in conflict with the two-step hypothesis, because the two-step hypothesis does not require the same gene to be responsible for both the gain-of-predisposition and the subsequent gain-of-function."
} | 7,235 |
38064042 | PMC10787697 | pmc | 6,459 | {
"abstract": "d -Glucaric acid is a potential biobased platform chemical. Previously mainly Escherichia coli, but also the yeast Saccharomyces cerevisiae, and Pichia pastoris, have been engineered for conversion of d -glucose to d -glucaric acid via myo-inositol. One reason for low yields from the yeast strains is the strong flux towards glycolysis. Thus, to decrease the flux of d -glucose to biomass, and to increase d -glucaric acid yield, the four step d -glucaric acid pathway was introduced into a phosphoglucose isomerase deficient (Pgi1p-deficient) Saccharomyces cerevisiae strain. High d -glucose concentrations are toxic to the Pgi1p-deficient strains, so various feeding strategies and use of polymeric substrates were studied. Uniformly labelled 13 C-glucose confirmed conversion of d -glucose to d -glucaric acid. In batch bioreactor cultures with pulsed d -fructose and ethanol provision 1.3 g d -glucaric acid L −1 was produced. The d -glucaric acid titer (0.71 g d -glucaric acid L −1 ) was lower in nitrogen limited conditions, but the yield, 0.23 g d -glucaric acid [g d -glucose consumed] −1 , was among the highest that has so far been reported from yeast. Accumulation of myo-inositol indicated that myo-inositol oxygenase activity was limiting, and that there would be potential to even higher yield. The Pgi1p-deficiency in S. cerevisiae provides an approach that in combination with other reported modifications and bioprocess strategies would promote the development of high yield d -glucaric acid yeast strains.",
"introduction": "Introduction d -Glucaric acid ( d -saccharic acid) is a di-carboxylic acid that can be used for example to produce furan dicarboxylic acid (van Strien et al. 2020 ) or various polyamides, and polyesters (Sakuta and Nakamura 2019 ). Biotechnical conversion of d -glucose to d -glucaric acid (or to the conjugate salt d -glucarate) can provide a selective and less energy intensive alternative to chemical production processes (Zhang et al. 2021 ). Moon et al. ( 2009 ) were the first to engineer Escherichia coli for production of d -glucaric acid. They introduced activities for myo-inositol-1-phosphate synthase, myo-inositol oxygenase and uronate dehydrogenase into E. coli for conversion of D-glucose via glucose-6-phosphate and 1L-myo-inositol-1-phosphate (1 d -myo-inositol 3-phosphate) to myo-inositol, d -glucuronate and finally to d -glucaric acid, resulting in production of 1.1 g L −1 of d -glucaric acid (Table 1 ). By introducing a polypeptide scaffold to co-localize the pathway enzymes d -glucaric acid concentration was increased to ~ 2.5 g L −1 (Moon et al. 2010 ). The myo-inositol oxygenase (MIOX) with its di-iron center and low activity was suggested to be the rate-limiting step (Moon et al. 2010 ). The MIOX activity was subsequently improved by using an N -terminal fusion of small ubiquitin-related modifier (SUMO) to MIOX, showing 75% increase in myo-inositol to d -glucaric acid conversion (Shiue and Prather 2014 ). Overexpression of myo-inositol-1-phosphate phosphatase from E. coli was tested for further enhancement of the process and the flux of d -glucose from catabolism towards myo-inositol-1-phosphate was redirected by deletion of the phosphoglucose isomerase (Pgi) and glucose 6-phosphate dehydrogenase (Zwf) encoding genes in E. coli (Shiue et al. 2015 ). This resulted in an increased yield of d -glucaric acid from d -glucose (yield 0.73 g g −1 with titer of 1.19 g L −1 , d -xylose as supplementing carbon source). Another approach used to decrease the flux to glycolysis was altering Pfk activity (Brockman and Prather 2015 ; Gupta et al. 2017 ; Hou et al. 2020 ). The d -glucaric acid pathway has also been used as a demonstration pathway for different synthetic biology approaches including use of MAGE (Raman et al. 2014 ), and small molecule reporter (Rogers and Church 2016 ). Dynamic pathway regulation with a quorum sensing based system or myo-inositol biosensor (Doong et al. 2018 ; Verma et al. 2022 ), regulation of Pgi translation by a d -fructose dependent control system (Qu et al. 2018 ), and NAD + regeneration system (Su et al. 2020 ) have been applied for d -glucaric acid production in E. coli . d -Glucaric acid production has also been demonstrated by in vitro conversion (Lee et al. 2016 ; Petroll et al. 2020 ; Su et al. 2019 ). Without myo-inositol addition, volumetric titers have remained between 1 and 2.5 g L −1 , although a recent study reported 5.35 g L −1 for intra plus extracellular titer in E. coli (Su et al. 2020 ) (reviewed by (Chen et al. 2020 )). Table 1 Production of d -glucaric acid by E. coli , yeast S. cerevisiae or P. pastoris in vivo. The highest extracellular volumetric titer and corresponding yield of a study are presented. Partly adapted from (Chen et al. 2020 ) Organism Year Pgip modified (yes/no) Culture type Carbon source Titer (g L −1 ) Yield (g g −1 \n d -glucose) References E. coli 2009 No Batch (flask) d -glucose 1.13 0.151 a Moon et al. ( 2009 ) E. coli 2009 No Batch (flask) d -glucose 1.7 – Dueber et al. ( 2009 ) E. coli 2010 No Batch (flask) d -glucose 2.50 0.250 Moon et al. ( 2010 ) E. coli 2014 No Batch (flask) Myo-inositol 4.85 0.449 Shiue and Prather ( 2014 ) E. coli 2015 Yes Batch (flask) d -glucose 1.19 0.73 Shiue et al. ( 2015 ) E. coli 2015 No Simulated fed-batch, BioLector (starch release) d -glucose/starch 1.56 0.124 Reizman et al. ( 2015 ) E. coli 2017 No Batch (flask) d -glucose ~ 0.85 – Gupta et al. ( 2017 ) E. coli 2018 No Batch (bioreactor) d -glucose 1.98 0.229 a Doong et al. ( 2018 ) E. coli 2018 Yes Batch/sucrose (flask) Sucrose ~ 1.42 0.27 Qu et al. ( 2018 ) E. coli 2020 Yes Batch (flask) d -glucose 3.91 0.514 a Su et al. ( 2020 ) E. coli 2020 No Batch (bioreactor) d -glucose 1.56 – Hou et al. ( 2020 ) P. pastoris 2016 No Fed-batch (bioreactor) d -glucose, myo-inositol 6.61 – Liu et al. ( 2016 ) S. cerevisiae 2016 No Batch (flask) d -glucose, myo-inositol 1.6 – Gupta et al. ( 2016 ) S. cerevisiae 2016 No Batch with spiking glucose (flask) d -glucose 0.98 0.033 Gupta et al. ( 2016 ) S. cerevisiae 2018 No Fed-batch (bioreactor) d -glucose, myo-inositol 6.0 – Chen et al. ( 2018 ) S. cerevisiae 2020 No Fed-batch (bioreactor) d -glucose 5.23 – Zhang et al. ( 2020 ) S. cerevisiae 2020 No Batch (flask) d -glucose, myo-inositol 1.76 – Marques et al. ( 2020 ) S. cerevisiae 2021 No Batch with spiking glucose (bioreactor) d -glucose, myo-inositol 10.6 – Zhao et al. ( 2021 ) S. cerevisiae 2021 No Fed-batch (flask) d -glucose, myo-inositol 11.21 – Li et al. ( 2021 ) S. cerevisiae 2021 No Fed-batch (flask) d -glucose 4.52 – Li et (al. ( 2021 ) S. cerevisiae, T. reesei 2021 No CBP (flask) Cellulose (Avicel) 0.54 0.036 Li et al. ( 2021 ) Corn stover 0.45 0.03 S. cerevisiae 2021 No Batch (flask) Galactose, myo-inositol 0.142 – Cheah et al. ( 2021 ) S. cerevisiae 2022 No Fed-batch (flask) d -glucose, myo-inositol 12.96 – Fang et al. ( 2022 ) S. cerevisiae 2022 No Fed-batch (flask) d -glucose 6.94 – Fang et al. ( 2022 ) S. cerevisiae, T. reesei 2022 No CBP (flask) Corn stover 6.42 – b Fang et al. ( 2022 ) S. cerevisiae 2022 No Fed-batch (bioreactor) d -glucose 9.5 0.216 Guo et al. ( 2022 ) S. cerevisiae, T. reesei 2023 No CBP (bioreactor) Corn stover 10.03 – Fang et al. ( 2023 ) Wheat straw 9.53 Rice straw 8.87 Switchgrass 10.66 a Originally reported in mol/mol b Reported only per steam exploded corn stover Yeast are considered advantageous for organic acid production because of their low pH tolerance and robustness (Abbott et al. 2009 ). Yeasts have also been engineered for production of d -glucaric acid, although more recently than E. coli (Table 1 ). In 2016 Gupta et al. first engineered Saccharomyces cerevisiae for production of d -glucaric acid by expressing the d -glucaric acid pathway genes coding for inositol monophosphatase, myo-inositol-1-phosphate synthase, myo-inositol oxygenase either from Mus musculus or from Arabidopsis thaliana and uronate dehydrogenase in a opi1 deletion background (Gupta et al. 2016 ). The engineered yeast produced a maximum titer of 0.56 g L −1 in batch culture, and 0.98 g L −1 in fed-batch. Liu et al. ( 2016 ) introduced the pathway into Pichia pastoris and produced 0.107 g L −1 \n d -glucaric acid from d -glucose. Since these pioneering studies further pathway engineering approaches by e.g. enzyme and expression optimization, use of scaffolds, or by improving viability, have increased d -glucaric acid titers up to 9.5 g L −1 (Fang et al. 2022 , 2023 ; Li et al. 2021 ; Zhang et al. 2020 ). In general, co-feeding of myo-inositol has resulted in higher final d -glucaric acid titers, up to 12.96 g L −1 in S. cerevisiae (Chen et al. 2018 ; Fang et al. 2022 ; Guo et al. 2022 ; Gupta et al. 2016 ; Li et al. 2021 ; Marques et al. 2020 ; Zhao et al. 2021 ), and 6.61 g L −1 in P. pastoris (Liu et al. 2016 ). The reported yields on d -glucose using yeast are scarce, and typically below 0.1 g g −1 , although recently a yield of 0.216 g g −1 (Guo et al. 2022 ) was reported (Table 1 ). In E. coli deletion of the phosphoglucose isomerase (Pgi) and glucose 6-phosphate dehydrogenase (Zwf) encoding genes resulted in 2.9-fold (Qu et al. 2018 ), or nearly 18-fold higher yield on d -glucose (Shiue et al. 2015 ), compared to when the genes were present. The phosphoglucose isomerase encoding gene has not been deleted from S. cerevisiae or P. pastoris strains engineered for d -glucaric acid production. The phosphoglucose isomerase (Pgi1p) -deficient S. cerevisiae strains metabolize d -glucose only poorly, whereas the Pgi-deficient strains of E. coli are able to grow on d -glucose (Vinopal et al. 1975 ). Even relatively low (< 2 g L −1 ) d -glucose concentrations inhibit or reduce the growth of S. cerevisiae Pgi1p-deficient strains, possibly because of ATP depletion or glucose-6-phosphate accumulation (Maitra 1971 ). The accumulation of glucose-6-phosphate, the first metabolite in the d -glucaric acid pathway, could be advantageous for directing d -glucose flux from glycolysis to d -glucaric acid and thus improve the yield of d -glucaric acid on d -glucose. We introduced the d -glucaric acid pathway into a Pgi1p-deficient S. cerevisiae strain (Fig. 1 ) and studied d -glucaric acid production from monomeric and polymeric d -glucose substrates in shaken flasks and controlled bioreactor conditions with varying nitrogen concentrations. The formation of myo-inositol and d -glucaric acid, and the absence of d -glucuronate was confirmed by using 13 C-labelled d -glucose and GC–MS analysis. Fig. 1 Schematic outline for conversion of d -glucose to d -glucaric acid with Pgi1p-deficient S. cerevisiae . In the Pgi1p-deficient strain, d -glucose should be channeled to myo-inositol, while d -fructose and ethanol would allow growth and support the production of biomass. Glucose-6-phosphate can also be channeled to pentose phosphate pathway or to storage carbohydrates",
"discussion": "Discussion In S. cerevisiae the major flux of d -glucose is through glycolysis, and only a small fraction, approximately 1–4%, is channelled to the PPP, depending on strain and culture conditions (Blank et al. 2005 ; Fiaux et al. 2003 ; Gancedo and Lagunas 1973 ; Maaheimo et al. 2001 ; Nidelet et al. 2016 ). In the pgi1 -deficient S. cerevisiae strains, the block to glycolysis leads to accumulation of glucose-6-phosphate (e.g. Ciriacy and Breitenbach 1979 ; Heux et al. 2008 ; Maitra 1971 ). Possibly, this glucose-6-phosphate accumulation could increase the flux of glucose-6-phosphate to myo-inositol and further to d -glucaric acid. Indeed, the yield of d -glucaric acid on d -glucose (0.12 g g −1 ) in the bioreactor with pulsed d -glucose was four-fold higher in our pgi1 -deficient strains compared to the value in a strain with intact Pgi1p reported by Gupta et al. ( 2016 ) and was further increased to 0.23 g g −1 (seven fold improvement over Gupta et al. ( 2016 )) by restricting the nitrogen supply. Recently, Guo et al. reported a yield of 0.216 g g −1 (Guo et al. 2022 ), close to our best yield. However, considering that our strains accumulated inositol at 12–17% of the d -glucaric acid concentration, it is clear that the yield could be even higher if all myo-inositol would be converted to d -glucaric acid. In S. cerevisiae , the PGI1 deletion alone increased the yield, compared to E. coli where the zwf deletion was also needed (Shiue et al. 2015 ). In yeast, deletion or downregulation of both PGI1 and glucose-6-phosphate dehydrogenase encoding gene ZWF1 has not been reported, but during preparation of this work Zhao et al. ( 2023 ) reported that downregulation of ZWF1 in S. cerevisiae improved d -glucaric acid titer by 22.4%. In volumetric terms our pgi1 -deficient strains produced about half of the d -glucaric acid concentration reported by Gupta et al. ( 2016 ) in shake flasks (average 0.26 compared to 0.54 g L −1 ), as confirmed by using 13 C-labelled d -glucose. The titer was improved to 1.3 g L −1 in the bioreactor by providing the d -glucose in pulses, which was higher than reported by Gupta et al. ( 2016 ), but lower compared to recently reported volumetric titers of up to 9.5 g L −1 (Table 1 ). Also, the highest yield was observed with restricted nitrogen supply, and in this condition the volumetric concentration was only 0.71 g L −1 . Interestingly, our parent strains with intact Pgi1p produced only ~ 30 mg L −1 of d -glucaric acid as measured with GC–MS. This in line with the results of Liu et al. ( 2016 ) who found that P. pastoris produced 108 mg L −1 \n d -glucaric acid on d -glucose, but much lower compared to other studies (Table 1 ). In our strains the OPI1 gene was intact. We hypothesized that expression of INO1 under a constitutive promoter is comparable to the effect of OPI1 deletion because Opi1p is reported to regulate INO1 at the transcriptional level (Henry et al. 2014 ; Ye et al. 2013 ). However, possibly the OPI1 deletion could improve d -glucaric acid production also in the pgi1 -deficient strains by a still unknown mechanism. In addition, differences in the strain background, pathway genes, and culture conditions, e.g. aeration and medium composition, may contribute to the differences in D-glucaric acid amounts produced. The S. cerevisiae pgi1 -deficient strains do not tolerate d -glucose concentration above 2 g L −1 , potentially decreasing their viability, and suitability to larger scale processes. Our fed-batch cultures did not drastically improve viability and more optimised feeding strategies would be needed. The viability was better on polymeric substrate α-cellulose where hydrolytic enzymes were used to release d -glucose, but the rate of d -glucose release would require further optimization to increase d -glucaric acid production. Recently, consolidated bioprocess (CBP) with concomitant sugar release and conversion to products has been developed for d -glucaric acid production (Fang et al. 2022 , 2023 ; Li et al. 2021 ). This could be an option for the pgi1 -deficient strains. d -Glucaric acid production with S. cerevisiae has developed rapidly during recent years, especially considering volumetric titers (Table 1 ). New myo-inositol oxygenases (Marques et al. 2020 ), and ways for improved viability (Guo et al. 2022 ) would be highly interesting to test in the pgi1 -deficient S. cerevisiae . Also, uncoupling growth and production, or regulating the Pgi1p amount by synthetic promoters, gene switches, and/or degradation approaches like those implemented in E. coli (Brockman and Prather 2015 ; Gupta et al. 2017 ; Hou et al. 2020 ; Qu et al. 2018 ) could improve d -glucaric acid production and viability in the pgi1 -deficient S. cerevisiae ."
} | 3,985 |
34735796 | null | s2 | 6,460 | {
"abstract": "Biofilms are community architectures adopted by bacteria inclusive of a self-formed extracellular matrix that protects resident bacteria from diverse environmental stresses and, in many species, incorporates extracellular DNA (eDNA) and DNABII proteins for structural integrity throughout biofilm development. Here, we present evidence that this eDNA-based architecture relies on the rare Z-form. Z-form DNA accumulates as biofilms mature and, through stabilization by the DNABII proteins, confers structural integrity to the biofilm matrix. Indeed, substances known to drive B-DNA into Z-DNA promoted biofilm formation whereas those that drive Z-DNA into B-DNA disrupted extant biofilms. Importantly, we demonstrated that the universal bacterial DNABII family of proteins stabilizes both bacterial- and host-eDNA in the Z-form in situ. A model is proposed that incorporates the role of Z-DNA in biofilm pathogenesis, innate immune response, and immune evasion."
} | 240 |
28773390 | PMC5502930 | pmc | 6,461 | {
"abstract": "Fibers and fibrous structures are used extensively in various fields due to their many advantages. Microfibers, as well as nanofibers, are considered to be some of the most valuable forms of advanced materials. Accordingly, various methods for fabricating microfibers have been developed. Electrospinning is a useful fabrication method for continuous polymeric nano- and microfibers with attractive merits. However, this technique has limitations in its ability to control the geometry of fibrous structures. Herein, advanced electrospinning with direct-writing functionality was used to fabricate microfiber patterns with ivy shoot-like geometries after experimentally investigating the effects of the process conditions on the fiber formation. The surface properties of the fibers were also modified by introducing nanoscale pores through the use of higher levels of humidity during the fabrication process.",
"conclusion": "4. Conclusions We demonstrated that microfibrous patterns with fractal geometries that looked like ivy shoots on a wall could be successfully fabricated using an advanced electrospinning method with specific process conditions. A direct-write electrospinning method consisting of an additional cylindrical side-wall electrode, a dielectric thin collector plate with planar motion, and a sharp-pin grounded electrode, as well as a conventional electrospinning apparatus, was employed in the fabrication to introduce focusing and scanning functionalities to the electrospinning jet. As a result, fibrous patterns with regular shapes were successfully fabricated. The solution flow rate, scan speed of the collector, and relative humidity were considered as key parameters in the fabrication of the microfibrous patterns with fractal geometries. The solution flow rate directly influenced the fiber diameter; microscale fibers were obtained using flow rates greater than 0.3 mL/h. The fractal geometries were introduced to the fibrous patterns by using collector scan speeds that were less than 100 mm/s with a flow rate that was greater than 0.6 mL/h. The scan speed strongly influenced the dimensions and complexity of the fractal geometries. Higher levels of humidity introduced nanoscale pores on the surface of the microfibers, which could offer a variety of functionalities. The ivy shoot-like pattern presented in this study can be used in various applications related to tissue engineering, biochip application, and drug delivery. Its more-natural geometry can give cells in culture a positive effect on guidance toward more realistic engineered tissues. It can be used as a mold master for microfluidic biochip which can introduce more realistic vascular network or surface properties. Also, it would be a good drug carrier because of its area filling property, which is a well-known feature of fractal geometry.",
"introduction": "1. Introduction Various fibers and fibrous structures are attracting significant attention in many different fields, such as cosmetics, clothing, electronics, environmental, chemical, and nano- and bioengineering [ 1 ]. Well-known nanofibers are a promising advanced material, but microfibers, a fiber form having a different order of scale, are also used widely in various areas, such as tissue engineering [ 2 , 3 , 4 ], drug delivery [ 5 , 6 ], filtration [ 7 , 8 ], and composite reinforcement [ 9 ], owing to their numerous advantages, including high porosity, large pore size, large surface area-to-volume ratio, high flexibility, and similarity in structural form with the human body’s extracellular matrices. In particular, microfibers have yielded better quality results when compared to nanofibers; e.g., microfiber and multilayered scaffolds have improved quality and perform better in terms of initial cell attachments and cell infiltration processes compared with nanofibrous scaffolds in tissue engineering [ 3 ]. To date, various methods have been developed to fabricate microfibers. In general, spinning methods such as wet-, dry-, and gel-spinning processes have been used successfully to fabricate microfibers for various applications [ 10 , 11 , 12 , 13 , 14 ]. Various types of microfluidic chips have also been used as nozzles for extrusion with versatile functionalization, such as Janus structures with multiple materials and bubbling [ 2 , 4 , 15 , 16 ]. In particular, the electrohydrodynamic hot-jet plotting process has been introduced to microfiber fabrication, providing better controllability and fiber diameters as small as 5 μm [ 17 ]. A slit die extrusion and heat stretching process for fabricating microfibers has also been demonstrated [ 18 ]. Electrospinning is one of the most attractive methods for fabricating continuous polymeric fibers with diameters ranging from micro- to nanometer scales because this technique does not pose serious limitations on the material selection and requires simple fabrication equipment with relatively easy process operations [ 1 ]. Therefore, various fibers and fibrous structures with different sizes, geometries, and materials have been fabricated using electrospinning and applied to a variety of fields. Especially, Pham et al. reported that the diameter of electrospun microfibers could be accurately controlled through the proper selection of process parameters, such as electric field, concentration, and flow rate [ 3 ]. Fridrikh et al. identified the relationship between fiber diameter, surface tension, flow rate, and electric current in the jet [ 19 ]. McKee et al. investigated the effects of concentration/viscosity on electrospun fiber diameter [ 20 ]. However, the conventional electrospinning process has limitations in terms of the geometric control of fibrous structures owing to the whipping motion that results from the bending instability of the electrospinning jet [ 21 ]. In this regard, conventional electrospinning is only suitable for fabricating randomly deposited nonwoven fibrous meshes, even though uniaxially aligned fibrous mats can be fabricated using electrospinning with a drum collector [ 22 ]. Many approaches have been tried to address this problem and obtain fibrous structures having regular shapes [ 23 , 24 , 25 , 26 , 27 , 28 ]. In the current study, microfibrous patterns with ivy shoot-like geometries were fabricated using an improved electrospinning process, known as direct-write electrospinning (DWES), which was developed in our previous study [ 29 ]. Since DWES improves the geometric regularity of electrospun fibers in a controllable manner, it was used to fabricate electropsun microfiber patterns to overcome their geometric control limitation. The effects of the experimental conditions, such as flow rate, scan speed, and humidity, on the microfiber formation were investigated using Euclidean and fractal analyses. As a result, we demonstrated that controllable microfiber patterns could be fabricated using the proposed electrospinning process, yielding complex and random ivy shoot-like shapes in the microfiber patterns. In addition, the surface properties could be modified by using higher levels of humidity in the fabrication process.",
"discussion": "2. Results and Discussion 2.1. Influence of the Solution Flow Rate on the Microfibers Polymeric fibers were electrospun under various solution flow rate conditions because this parameter was considered to be one of the dominant factors that influence the electrospun microfiber diameter [ 30 ]. In the experiments, the fabricated pattern had a lattice shape with a grid size of 500 μm, and the flow rates of the polycaprolactone (PCL) solution from the nozzle were 0.1, 0.3, 0.6 and 0.8 mL/h. Other experimental conditions were kept nearly constant, as indicated in Table 1 . The tip-to-collector distance (TCD) and voltage for electrospinning were 60.0 mm and 21–24 kV, respectively. Figure 1 shows the fibrous lattice patterns fabricated under various flow rates, and their fiber diameter distributions. The fibrous pattern fabricated with a flow rate of 0.1 mL/h is depicted in Figure 1 a,b. It contained many fibers with diameters ranging from 150 nm to 5 μm. However, although plenty of microfibers are shown in Figure 1 b, the nanometer-scale fibers still dominated the pattern. A well-defined lattice pattern was obtained under these conditions. The most common fiber diameter was 425 nm, as depicted in Figure 1 c. Figure 1 d–f show the pattern and fiber diameter distribution obtained using a flow rate of 0.3 mL/h. The fibrous line pattern width of the lattice pattern increased because of the higher flow rate, and most of the fibers had microscale diameters. The most common fiber diameter was 2 μm, and the minimum diameter was 600 nm. The pattern still exhibited a regular lattice shape. Thus, the higher flow rate provided larger-diameter fibers. The fiber diameter continued to increase with the flow rate. A unique morphology of the fibrous pattern was observed at a flow rate greater than 0.6 mL/h. Figure 1 g,h show the lattice pattern obtained at a flow rate of 0.6 mL/h. In Figure 1 g, the pattern had a unique geometry that looked like ivy shoots on a wall. The stems of the ivy shoot-like pattern (central lines) still maintained the lattice shape. However, tendril-like microfibers stretched radially from the stem lines and caused the pattern to resemble ivy shoots. The fibers had larger diameters and were fused together to create the complex morphology shown in Figure 1 h, likely because a significant amount of the solvent remained in the electrospun fibers after they reached the collector plate. Therefore, the pattern had better interconnectivity among fibers compared with Figure 1 b,e. Also, the width of the line pattern became narrower even though the solution flow rate was higher, and a few pores were exposed on the surface of the fibers due to incomplete evaporation of solvent. As shown in Figure 1 i, the most common fiber diameter was between 2.2 and 3.2 μm. The fiber interconnectivity continued to improve when the solution flow rate was increased to 0.8 mL/h due to a higher degree of fiber fusion, as shown in Figure 1 j,k. However, the ivy shoot-like geometric features became weaker, as depicted in Figure 1 k. As expected, the porosity of the fiber surfaces increased significantly. From Figure 1 l, the most common microfiber diameter was 3.35 μm. From the results shown in Figure 1 , the fabricated nano- and microfibrous patterns had well-defined lattice shapes with various line patterns, depending on the solution flow rate used. Especially, it could be seen that the clearest ivy shoot pattern could be obtained at a flow rate of 0.6 mL/h, as shown in Figure 1 g. However, at flow rates greater than 0.9 mL/h, it was difficult to obtain the ivy shoot pattern from the electrospun fibers. The relationship between the solution flow rate and the fiber diameter of the fabricated lattice patterns shown in Figure 1 was obtained via statistical analysis. As illustrated in Figure 2 , the average fiber diameter of the fabricated patterns increased with the solution flow rate. Thus, the average diameter of the electrospun fibers could be controlled by modifying the solution flow rate. However, the higher flow rates resulted in larger standard deviations among fiber diameters. 2.2. Influence of the Scan Speed of the Collector In the experiments described in the previous section, we observed that the lattice patterns consistently possessed ivy shoot-like geometries when the solution flow rate was between 0.6 and 0.8 mL/h. Next, we modified the scan speed of the collector to adjust the degree of complexity of the ivy shoot-like geometries in the microfibrous patterns, because extending the length of time that the collector remained at one point could increase the possibility for a tendril to form. Thus, parallel-line patterns were fabricated using various collector scan speeds from 10 to 450 mm/s, as shown in Figure 3 . The other conditions were fixed as listed in Table 2 . The scan path used to fabricate the parallel-line pattern had a pitch of 500 μm at all scan speeds except for 450 mm/s, for which the pitch was 50 μm. Figure 3 a shows the parallel-line pattern fabricated at a scan speed of 10 mm/s. The central lines corresponding to the stem in an ivy shoot followed the scan path well with a regular pitch. Moreover, the pattern clearly showed complex tendril formation. The fabricated microfibrous patterns obtained at scan speeds of 20, 50 and 75 mm/s are presented in Figure 3 b–d, respectively. The formation of tendrils in the microfibrous line patterns decreased gradually as the scan speed increased. At the same time, the width of the line patterns narrowed. In Figure 3 e, which shows the pattern fabricated at a scan speed of 150 mm/s, it is difficult to identify any clear tendrils along the line patterns. Moreover, we obtained a straight-line pattern without tendrils at a scan speed of 450 mm/s, as shown in Figure 3 f, with highly arranged entanglements. Thus, the degree of complexity obtained using the proposed electrospinning process could be controlled via changes in the scan speed. The inferred explanation for this result is that the longer durations spent by the collector at a given point due to the slower scan speeds introduced a greater possibility for the microfibrous pattern to extend tendrils (fingers). The effect of the scan speed on the tendril formation in the fabricated microfibrous patterns was evaluated by the well-defined fractal dimension, which could describe complexity or area filling of chaotic geometries in nature [ 31 , 32 ]. We calculated fractal dimensions of the patterns for various scan speed conditions. Figure 4 shows fractal dimension of line patterns fabricated under the conditions mentioned in Table 2 and Figure 3 . The fractal dimension of the line pattern fabricated at a scan speed of 10 mm/s was about 1.612 with a standard deviation of about 0.024. The dimension decreased as the scan speed increased, thus it became down to 1.205 ± 0.019 when the scan speed was 150 mm/s. Especially, the fractal dimension of the pattern fabricated at a scan speed of 450 mm/s was 1.059 ± 0.011, which was close to 1.0, corresponding to the typical one-dimensional geometry ( i.e ., simple line). In this regard, it could be concluded from the figure that the fractal dimension of the microfibrous patterns at flow rate of 0.6 mL/h ranged between 2.0 and 1.0 and the slower scan speed led the higher fractal dimension. The characteristics of the ivy shoot-like geometries in the fabricated microfibrous patterns were analyzed to more clearly identify the effect of scan speed, as shown in Figure 5 . First, we established a simple measure of the degree of complexity of a fractal geometry in the microfibrous line patterns. The shapes in Figure 5 a, which presents a typical fractal geometry in a fabricated microfibrous pattern, resemble ivy shoots on a wall, and thus the geometric characteristics were defined according to the geometry of ivy shoots. The line patterns of microfibers with fractal geometries were therefore identified as shoots in this study. The main line patterns corresponding to the scan path were identified as stems, and the branches from the stems were identified as tendrils. Therefore, a shoot consisted of a stem and many tendrils. Figure 5 b illustrates the relationship between the stem width and the scan speed. The width narrowed with increasing scan speed. The line width ranged between 20 and 30 μm at scan speeds greater than 100 mm/s; in particular, the width could be reduced to 20 μm when the scan speed was 450 mm/s. Also, the line widths tended to have larger standard deviations at lower scan speeds because the instability of the deposition increased. The effects of the scan speed on the length and number of tendrils showed trends similar to the stem width, as shown in Figure 5 c,d. The tendrils extended to about 90 μm on average; however, no tendrils were identified in the samples fabricated with scan speeds greater than 200 mm/s. Consequently, the complexity of the fractal geometries in a fabricated microfibrous mat, which could be characterized by the average width of the stems, and the number and average length of the tendrils, could be controlled via the scan speed. 2.3. Influence of the Relative Humidity Porosity can be introduced to the surface of electrospun micro- or nanofibers by controlling the humidity. This is because the humidity can influence the evaporation of solvent from electrospun fibers. Nanopores introduced to microfibers can affect various surface properties. Thus, parallel-line patterns with a line pitch of 500 μm were fabricated at two different levels of relative humidity, 51% and 60%, which was selected from preliminary tests and the experiments mentioned at Table 1 and Table 2 . The higher humidity led to more pores on the microfiber surface under the flow rate fixed at 0.6 mL/h. However, clear pores could not be observed at humidity lower than 60 percent relative humidity (RH%). When humidity was 60 RH%, clear pores covering the surface could be obtained. However, the amount of electrospun fibers significantly decreased when the humidity was higher than 60 RH%. The experimental conditions are listed in Table 3 . All patterns exhibited fractal geometries, regardless of the relative humidity. However, the pattern fabricated at the lower humidity that had no pores on its surface ( Figure 6 a–c), unlike the pattern fabricated at higher humidity was covered by micro- and nanopores ( Figure 6 d–f). The diameter distribution of the pores covering the surface of the pattern shown in Figure 6 f is given in Figure 7 . Most of the pores had diameters ranging between 200 and 800 nm, and the most common pore diameter was about 550 nm. The average pore diameter was 599.3 nm."
} | 4,460 |
27211860 | null | s2 | 6,463 | {
"abstract": "Understanding the complex interactions that occur between heterologous and native biochemical pathways represents a major challenge in metabolic engineering and synthetic biology. We present a workflow that integrates metabolomics, proteomics, and genome-scale models of Escherichia coli metabolism to study the effects of introducing a heterologous pathway into a microbial host. This workflow incorporates complementary approaches from computational systems biology, metabolic engineering, and synthetic biology; provides molecular insight into how the host organism microenvironment changes due to pathway engineering; and demonstrates how biological mechanisms underlying strain variation can be exploited as an engineering strategy to increase product yield. As a proof of concept, we present the analysis of eight engineered strains producing three biofuels: isopentenol, limonene, and bisabolene. Application of this workflow identified the roles of candidate genes, pathways, and biochemical reactions in observed experimental phenomena and facilitated the construction of a mutant strain with improved productivity. The contributed workflow is available as an open-source tool in the form of iPython notebooks."
} | 304 |
21682348 | null | s2 | 6,464 | {
"abstract": "Structural DNA nanotechnology utilizes DNA molecules as programmable information-coding polymers to create higher order structures at the nanometer scale. An important milestone in structural DNA nanotechnology was the development of scaffolded DNA origami in which a long single-stranded viral genome (scaffold strand) is folded into arbitrary shapes by hundreds of short synthetic oligonucleotides (staple strands). The achievable dimensions of the DNA origami tile units are currently limited by the length of the scaffold strand. Here we demonstrate a strategy referred to as \"superorigami\" or \"origami of origami\" to scale up DNA origami technology. First, this method uses a collection of bridge strands to prefold a single-stranded DNA scaffold into a loose framework. Subsequently, preformed individual DNA origami tiles are directed onto the loose framework so that each origami tile serves as a large staple. Using this strategy, we demonstrate the ability to organize DNA origami nanostructures into larger spatially addressable architectures."
} | 263 |
28347074 | PMC5304633 | pmc | 6,465 | {
"abstract": "The effect of laser irradiation on surface wettability of cyclic olefin polymer (COP) was investigated. Under different laser parameters, a superhydrophilic or a superhydrophobic COP surface with a water contact angle (WCA) of almost 0° or 163°, respectively, could be achieved by direct femtosecond laser irradiation. The laser power deposition rate (PDR) was found to be a key factor on the wettability of the laser-treated COP surface. The surface roughness and surface chemistry of the laser-irradiated samples were characterized by surface profilometer and X-ray photoelectron spectroscopy, respectively; they were found to be responsible for the changes of the laser-induced surface wettability. The mechanisms involved in the laser surface wettability modification process were discussed.",
"conclusion": "4. Conclusions The effects of laser irradiation on surface wettability of COP were investigated. It was demonstrated that a superhydrophilic COP surface with a water contact angle of almost 0° and a superhydrophobic COP surface with a water contact angle of 163° could be achieved by direct fs laser irradiation with different laser parameters. The laser power deposition rate was found to be a key factor in determining the wettability of the laser-treated COP surface. The surface roughness of the laser-irradiated samples was found to be responsible for the changes of the laser-induced surface wettability at a high laser power deposition rate; in contrast, the laser-induced change in surface chemistry was a key factor in determining the wettability change for the COP substrates treated at low laser power deposition rates. The developed process could be applied for the controlled modification of localized surface wettability of microfluidic devices or the formation of high wettability contrast surfaces for inkjet printing.",
"introduction": "1. Introduction The controlled modification of polymer surface wettability to either hydrophilic or hydrophobic is highly desirable for various applications. For example, super-hydrophobic surfaces are well suited for the self-cleaning of contaminants. Surface wettability also determines the reagent flow behavior in microfluidic channels, and the ease of adhesion of cells. A highly hydrophilic surface can improve surface adhesion and wettability, a desirable characteristic for coating and joining applications. High wettability contrast surfaces could confine inkjet droplets to the hydrophilic region and avoid the hydrophobic region, with the potential of a printed line-width less than the droplet diameter. Indeed, the surface modification of polymer materials has been widely investigated recently [ 1 , 2 , 3 , 4 ]. Cyclic olefin polymer (COP) is one of the widely used polymers in microfluidics and biotechnologies. The controlled modification of COP surface wettability is highly desirable for the fabrication of COP-based microfluidic devices. Different techniques can be employed to modify polymer surface wettability which include wet-chemical etching [ 5 ], corona discharges [ 6 ], plasma treatment [ 7 , 8 ], ion beam treatment [ 9 , 10 ], electron beam irradiation [ 11 ], and laser irradiation. Compared with other methods, laser irradiation, being a fast and clean process, has a number of unique advantages. It is a non-contact and highly selective process. It can provide localized treatment and precise control for producing complex features with minimal thermal effect on the bulk material. Laser irradiation has been applied for surface modification of metals, polymers, and semiconductors [ 4 , 12 , 13 , 14 , 15 , 16 , 17 ]. Ultrashort pulsed lasers are increasingly used as a tool for surface treatment. In contrast to long pulsed lasers, ultrashort pulsed lasers can induce nonlinear absorption of photons and have minimum thermal effect. In addition, the use of ultrashort femtosecond laser pulses allows treatment of features with micrometric precision, as the laser beam can be accurately positioned and tightly focused into a spot size of several microns. In this investigation, we demonstrated that an infrared femtosecond laser can induce both superhydrophobicity (WCA of 163°) and superhydrophilicity (WCA of almost 0°) on COP material surface through direct laser irradiation under different laser parameters. The effects of laser fluence, power deposition rate, and surface roughness on the COP surface's wettability were investigated. This developed process has potential applications for the fabrication of polymer microfluidic devices or to generate high wettability contrast surfaces for inkjet printing.",
"discussion": "3. Experimental Results and Discussion To investigate the wettability change induced by fs laser treatment under different laser conditions, the COP substrates were treated with a number of different laser fluences from 17.6 to 30.79 J/cm 2 . Fluence is the average energy deposited per unit area on the sample surface by a single pulse [ 18 ], which is calculated as:\n (1) F = E p u l s e A \nwhere E p u l s e is the pulse energy and A is the spot area. For a line scan of the laser beam, the power deposition rate (PDR) is an appropriate parameter to be considered, which is defined as the energy deposited on the sample surface per unit time per unit area [ 18 ]. PDR can be calculated as:\n (2) PDR = E p u l s e · R R D · S S \nwhere RR is the laser repetition rate (fixed at 1 kHz in this investigation), D is the scanning width equal to the laser spot diameter on the surface, and SS represents the scanning speed. Overlapped multiple scans are required to modify the wettability of an area [ 19 ]. In this investigation, the shifting pitch was fixed at 0.02 mm. It was found that the WCA varies with the fluence and power deposition rate. With the appropriate laser conditions, the surface wettability of COP could be tuned from superhydropobic to superhydrophilic as shown in Figure 1 . When the COP surface was treated at a high fluence of 30.79 J/cm 2 and a high PDR of 6.5 W/mm 2 , the WCA was increased to around 163° from the original 90°, as shown in Figure 1 b. In contrast, when the COP surface was treated at a low fluence of 17.60 J/cm 2 and a low PDR of 0.62 W/mm 2 , the WCA was decreased from the original 90° to almost zero degrees, as shown in Figure 1 c. Furthermore, as shown in Figure 2 , when the laser fluence was fixed at a constant fluence, with decreasing PDR, the WCA decreased to less than 40° from the original 90°. These findings indicated that the power deposition rate is a key factor in determining the wettability of the laser-treated COP surface. The WCA could be tuned from superhydrophic to superhydrophilic by adjusting PDR via controlling the laser pulse energy and the beam scanning speed. Figure 1 Water contact angle of ( a ) pristine cyclic olefin polymer (COP) surface; ( b ) COP surface treated with fluence of 30.79 J/cm 2 and power deposition rate ( PDR ) of 6.5 W/mm 2 ; and ( c ) COP surface treated with fluence of 17.60 J/cm 2 and PDR of 0.62 W/mm 2 . Figure 2 Effect of power deposition rate on water contact angle at different fluence. It is well known that surface wettability is governed by surface morphology and surface chemistry [ 13 ]. As such, the laser-induced surface wettability changes may be due to changes in surface roughness and/or surface chemistry. There are two existing theories for describing the effect of surface roughness on wettability. The first theory by Wenzel [ 20 ] assumes that liquid fills the whole area of the rough surface. The relationship between the macroscopic contact angle and the equilibrium contact angle may be written as:\n (3) cos θ m = r . cos θ s \nwhere θ m and θ s are, respectively, the measured contact angle on a structured surface and the equilibrium contact angle on an ideally smooth surface; the roughness factor r is the ratio of the actual rough surface area to the geometrically projected area. Assume that the contact angle is more than 90° on a smooth surface. If a micro- or nano-pattern is fabricated on this smooth surface, its hydrophobic characteristic will be enhanced. The same analogy applies to a hydrophilic surface. For example, for a smooth surface with a contact angle less than 90°, a micro- or nano-pattern fabricated on this smooth surface will enhance its hydrophilic characteristic. r should increase closer to 1, and θ m should increase for a hydrophobic surface and decrease for a hydrophilic surface. The second theory by Cassie and Baxter [ 21 ] assumes that the liquid does not completely wet the roughened substrate. The relationship between the macroscopic contact angle and the equilibrium contact angle may be written as: (4) cos θ m = φ . cos θ s + φ − 1 \nwhere φ is the fraction of wetted surface area divided by its projected area. If a surface is rough enough so that air may be entrapped between the liquid and the solid, the interface becomes composite and the contact angle increases with the roughness even if the surface chemistry is intrinsically hydrophilic. Figure 3 shows the measured surface morphologies of the laser-treated and pristine COP surfaces at different PDRs with a fixed fluence of 26.39 J/cm 2 . Ablation occurred on all samples. When the COP substrate was treated at a high PDR of 5.57 W/mm 2 , a superhydrophobic surface was obtained. The surface became rougher and micro-groove patterns were formed on the surface, as in Figure 3 b. In contrast, when the COP surface was treated at a low PDR such as 0.93 W/mm 2 , a hydrophilic surface was obtained. The surface became slightly rougher but was still quite consistently smooth with no grooved patterns formed, as shown in Figure 3 e. Figure 4 shows the change of the measured water contact angles of laser-treated COP surfaces as a function of surface roughness. Here, the laser treatment was conducted at fluences of 30.79 J/cm 2 , 26.39 J/cm 2 , 21.99 J/cm 2 , and 17.60 J/cm 2 with different PDRs from 0.31 W/mm 2 to 6.5 W/mm 2 , as shown in Figure 2 . The surface roughness ( Ra ) was measured by a one-dimensional roughness scan perpendicular to the laser scanning direction. The scanning length was 5 mm. It can be observed that the water contact angle increased from about 90° for the pristine COP surface to almost 150° with an increase in the surface roughness. In contrast, when the substrate was treated at a lower PDR, the induced surface roughness was in the range of 1.0 to 2.5 µm, and the water contact angle decreased from 90° for the pristine COP surface to almost 0° for laser-treated surfaces. The effect of PDR on surface roughness is depicted in Figure 5 . Figure 3 Scanning electron microscopy (SEM) images of surface morphologies at different PDRs with laser fluence fixed at 26.39 J/cm 2 . ( a ) Pristine COP surface; ( b ) PDR: 5.57 W/mm 2 ; ( c ) PDR: 2.79 W/mm 2 ; ( d ) PDR: 1.39 W/mm 2 ; ( e ) PDR: 0.93 W/mm 2 ; ( f ) PDR: 0.7 W/mm 2 . Figure 4 Water contact angle as a function of surface roughness of pristine surface and laser-treated surfaces with various different roughnesses. Here, the laser treatment was conducted at fluences of 30.79 J/cm 2 , 26.39 J/cm 2 , 21.99 J/cm 2 , and 17.60 J/cm 2 with different PDRs from 0.31 W/mm 2 to 6.5 W/mm 2 as shown in Figure 2 . Figure 5 Effect of PDR on surface roughness at a fixed laser fluence of 26.39 J/cm 2 . Based on the experimental observations and Wenzel and Cassie and Baxter theories, the formation of the superhydrophobic surfaces treated with higher laser PDR are believed to be mainly due to the increase of the surface roughness, leading air to become entrapped between the liquid and the solid; the interface becomes composite and the contact angle increases with roughness. For COP substrates treated under lower laser PDR, the formation of the hydrophilic surfaces is most possibly due to the laser-induced change of the surface chemistry instead of the surface roughness. It is known that the water contact angle of pristine COP is around 90°. Based on the Wenzel or Cassie and Baxter theories, the water contact angle will increase with an increase of surface roughness for this inherently hydrophobic material. However, when the surface roughness was increased from that of the original surface ( Ra = 7.98 nm) to an Ra of 2.5 µm, almost 0° superhydrophilic surface was achieved as shown in Figure 1 c. Thus, the laser-induced change in surface chemistry must play a key role in the formation of the superhydrophilic COP surfaces. It is known that oxygen-containing groups on a polymer surface, such as C–O, C=O, and O–C=O, are responsible for the change of surface hydrophilicity [ 22 ]. As the polarity of the C=O bond is more intense than that of the C–O bond, the C=O double bond is most important for the hydrophilicity of polymer surfaces. To fully understand the changes in surface wettability induced by laser irradiation, X-ray photoelectron spectroscopy (XPS) analysis using a VG ESCALAB 220i-XL was employed to identify the chemical bonds. Monochromatic Al Ka X-ray (hν = 1486.7 eV) was employed for XPS analysis with a photoelectron take-off angle of 90° with respect to the surface plane. The analysis area was approximately 700 μm in diameter with the maximum analysis depth in the range of 4–8 nm. The resolution of binding energy is estimated to be within ±0.2 eV. The measured C1s spectra of pristine and laser-treated samples at a PDR of 2.79 W/mm 2 were shown in Figure 6 . The main peak at around 285.0 eV was the C–H or C–C bond, while the sub-peaks at 286.3 eV and 288.6 eV were attributed to the C–O bond and C=O bond, respectively [ 7 ]. In the XPS measurements, the analysis area is approximately 700 μm in diameter, which is much larger than the laser-induced micro-feature dimensions of around 50 µm. The XPS signal is an average value for the whole analyzed surface area, which should be reliable to illustrate the changes between the original surface and the laser-treated surfaces under different laser parameters, even if the surface roughness (several micrometers) is much larger than the XPS information depth of about a few nm [ 23 , 24 ]. Oxygen concentration or polar groups increased on the surface after laser treatment as shown in Table 1 . This increase in oxygen polar groups on the COP surface led to an increase in wettability; this could result in a highly hydrophilic surface. For a laser-induced superhydrophobic surface treated with a fluence of 26.39 J/cm 2 and a PDR of 5.57 W/mm 2 , although the oxygen content also increased slightly, the roughness of the surface increased significantly ( Ra of 25.6 µm) with the formation of micro-ripple structures as shown in Figure 3 b. Thus, air might be entrapped between the liquid and the solid; the interface became composite and the contact angle increased to more than 150° even if the surface had some oxygen polar groups. The WCA decreased to hydrophilic at lower a PDR compared with pristine COP. The WCA is significantly influenced by surface structure when the surface is rough. In contrast, the influence of surface chemistry on the WCA is significant when the surface is relatively smooth ( Ra less than 1.76 µm). The time dependency of the WCA of a laser-modified surface, especially for a laser-induced hydrophilic surface, was observed. The laser-modified hydrophilic or even superhydrophilic surface is not stable, in that the surface could recover to hydrophobic after a period of time subsequent to laser treatment. This recovery of the WCA over time is currently being investigated. Figure 6 X-ray photoelectron spectroscopy (XPS) spectra of ( a ) pristine COP surface and ( b ) COP surface treated with a PDR of 2.79 W/mm 2 at a fixed fluence of 26.39 J/cm 2 . nanomaterials-05-01442-t001_Table 1 Table 1 Polar and non-polar groups formed on the COP surface before and after laser treatment at a fixed laser fluence of 26.39 J/cm 2 . PDR (W /mm 2 ) Non-polar Groups C–C/C–H (at. %) Polar Group C–O (at. %) Polar Group C=O (at. %) Sum of Polar Groups (at. %) Overall Ratio of Polar and Non-Polar Groups Water Contact Angle 0 98.35 - 1.65 1.65 0.02 90.0° 5.57 75.03 19.11 5.85 24.96 0.33 150° 2.79 71.05 23.22 5.72 28.94 0.41 147.55° 1.39 62.33 31.52 6.14 37.66 0.60 90.8° 0.93 70.96 20.98 8.04 29.02 0.41 39.2° 0.70 59.32 34.71 5.95 40.66 0.69 19.9° 0.56 71.44 20.37 8.17 28.54 0.40 36.6°"
} | 4,084 |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.