Electrolytic extraction drives volatile fatty acid chain elongation through lactic acid and replaces chemical pH control in thin stillage fermentation

BackgroundVolatile fatty acids (VFA) are building blocks for the chemical industry. Sustainable, biological production is constrained by production and recovery costs, including the need for intensive pH correction. Membrane electrolysis has been developed as an in situ extraction technology tailored to the direct recovery of VFA from fermentation while stabilizing acidogenesis without caustic addition. A current applied across an anion exchange membrane reduces the fermentation broth (catholyte, water reduction: H2O + e− → ½ H2 + OH−) and drives carboxylate ions into a clean, concentrated VFA stream (anolyte, water oxidation: H2O → 2e− + 2 H+ + O2).ResultsIn this study, we fermented thin stillage to generate a mixed VFA extract without chemical pH control. Membrane electrolysis (0.1 A, 3.22 ± 0.60 V) extracted 28 ± 6 % of carboxylates generated per day (on a carbon basis) and completely replaced caustic control of pH, with no impact on the total carboxylate production amount or rate. Hydrogen generated from the applied current shifted the fermentation outcome from predominantly C2 and C3 VFA (64 ± 3 % of the total VFA present in the control) to majority of C4 to C6 (70 ± 12 % in the experiment), with identical proportions in the VFA acid extract. A strain related to Megasphaera elsdenii (maximum abundance of 57 %), a bacteria capable of producing mid-chain VFA at a high rate, was enriched by the applied current, alongside a stable community of Lactobacillus spp. (10 %), enabling chain elongation of VFA through lactic acid. A conversion of 30 ± 5 % VFA produced per sCOD fed (60 ± 10 % of the reactive fraction) was achieved, with a 50 ± 6 % reduction in suspended solids likely by electro-coagulation.ConclusionsVFA can be extracted directly from a fermentation broth by membrane electrolysis. The electrolytic water reduction products are utilized in the fermentation: OH− is used for pH control without added chemicals, and H2 is metabolized by species such as Megasphaera elsdenii to produce greater value, more reduced VFA. Electro-fermentation displays promise for generating added value chemical co-products from biorefinery sidestreams and wastes.

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title: Towards integrated biorefinery from dried distillers grains: Evaluation of feed application for Co-products
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title: Carbon and electron fluxes during the electricity driven 1,3-propanediol biosynthesis from glycerol.
abstract: 1,3-Propanediol (1,3-PDO) can be produced biologically through glycerol fermentation. While such a process typically involves a pure culture system, particularly for crude glycerol, there would be operational advantages if a mixed population could be used. However, in the latter case the yield is typically low. Here, we use electrical current as the driving force for a mixed population fermenting glycerol in the cathode of a microbial bioelectrochemical system (BES). The carbon and electron flows were monitored by a titration and off-gas analysis (TOGA) sensor, and the syntrophic interactions in the BES were also investigated. Results show that on a carbon yield basis, current enhanced 1,3-PDO production from 24.8% (without current) to 50.1% (with a polarized biocathode at -0.9 V versus standard hydrogen electrode, SHE). Flux analysis indicated that the reductive current can be integrated into glycerol metabolism to enhance 1,3-PDO yield and that glycerol metabolism was redirected from propionate fermentation to 1,3-PDO production. A polarization of -0.6 V (vs SHE) resulted in more fermentative hydrogen production (from 2.7% to 8.0% on electron basis). 1,3-PDO production was also enhanced with hydrogen supply (37.7% on carbon basis), by suppressing hydrogen fermentation. Moreover, interspecies hydrogen transfer encouraged hydrogenotrophic methanogenesis, which was also accelerated by the cathodic polarization.

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abstract: Seventeen anaerobic, Gram-negative, tiny coccobacilli were collected in France from various human clinical samples. Biochemical analyses as well as molecular studies, including 16S rRNA and dnaK gene sequencing, affiliated all the isolates to the genus Dialister. However, 16S rRNA and dnaK gene sequence similarities were below 95.2 and 79.7 %, respectively, when comparisons were performed with the currently described species Dialister pneumosintes and Dialister invisus. Two clusters consisting of 13 and four isolates could be differentiated. 16S rRNA- and dnaK-based phylogeny confirmed that these two clusters represent two novel and distinct lineages within the genus Dialister. Finally, phenotypic, genotypic and phylogenetic data supported the proposal of the two novel species Dialister micraerophilus sp. nov. (type strain ADV 04.01T=AIP 25.04T=CIP 108278T=CCUG 48837T) and Dialister propionicifaciens sp. nov. (type strain ADV 1053.03T=AIP 26.04T=CIP 108336T=CCUG 49291T). The G+C content of the DNA of the D. micraerophilus type strain is 36.3 mol%. On the basis of 16S rRNA gene sequence analysis, 11 isolates originating from Canada could also be affiliated to D. micraerophilus sp. nov., and were included in the species description.

title: Prevotella, a new genus to include Bacteroides melaninogenicus and related species formerly classified in the genus Bacteroides.
abstract: It was recently proposed that the genus Bacteroides should be restricted to Bacteroides fragilis (the type species) and closely related organisms (viz., B. caccae, B. distasonis, B. eggerthii, B. merdae, B. ovatus, B. stercoris, B. thetaiotaomicron, B. uniformis, and B. vulgatus). By contrast, the moderately saccharolytic, predominantly oral Bacteroides species, which include B. melaninogenicus, B. oralis, and related species, form a phenotypically and phylogenetically coherent group of species which differ so significantly from the emended description of the genus Bacteroides that they should not be classified in the same genus. Therefore, we formally propose that these species be reclassified in a new genus, Prevotella. The type species is Prevotella melaninogenica.

title: Pectinatus, a New Genus of the Family Bacteroidaceae
abstract: The properties of an anaerobic, rod-shaped bacterium having an unusual flagellar arrangement are described. The flagella emanate from only one side of the cell body and are not limited to the center portion as they are with selenomonads. The organism is a gram-negative, nonsporeforming mesophile producing acetic, propionic, and succinic acids and traces of lactic acid from glucose fermentation. The guanine-plus-cytosine content of the deoxyribonuleic acid is 39.8 mol%. The characteristics of this organism indicate that it belongs in none of the presently described genera. For this organism, we propose a new genus, Pectinatus, and a new species, Pectinatus cerevisiiphilus, in the family Bacteroidaceae. The type strain, CCC B-1022, has been deposited in the American Type Culture Collection under the number 29359.

title: Pectinatus sottacetonis sp. nov., isolated from a commercial pickle spoilage tank.
abstract: A strictly anaerobic, Gram-stain-negative, non-spore-forming, motile bacterium, designated strain FSRU B0405(T), was isolated from a commercial pickle spoilage tank and characterized by biochemical, physiological and molecular biological methods. Analyses of the 16S rRNA gene sequence of strain FSRU B0405(T) showed affiliation to the class Negativicutes in the phylum Firmicutes, with the closest relatives being the type strains of Pectinatus haikarae (96 %) and Pectinatus brassicae (95 %). In maximum-likelihood and neighbour-joining phylogenetic trees, strain FSRU B0405(T) clustered definitively (in 100 % of bootstrapped trees) within the genus Pectinatus, but not specifically with any characterized species within this genus. Strain FSRU B0405(T) was a slightly curved rod, varying from 3 to 30 µm in length, motile with a distinctive X-wise movement, having flagella only on the concave side of the cell. The isolate produced acetate and propionate from fructose and glucose as major metabolites similar to type strains of species of the genus Pectinatus. The major fatty acids were C11 : 0, C13 : 0, C15 : 0, C13 : 0 3-OH, C17 : 1 and C18 : 1ω11t. Strain FSRU B0405(T) differed from the pickle wastewater strain, Pectinatus brassicae TY(T), due to its lack of susceptibility to vancomycin, acetoin production, growth temperature range, acid production from adonitol, erythritol, glycerol, inositol, lactose, maltose, mannose, ribose, salicin, sorbitol, trehalose and xylitol and lack of hydrolysis of milk. Strain FSRU B0405(T) could be differentiated from other species of the genus Pectinatus both phenotypically and genetically. The results indicate that strain FSRU B0405(T) represents a novel species of the genus Pectinatus, for which the name Pectinatus sottacetonis sp. nov. is proposed. The type strain is FSRU B0405(T) ( = ATCC BAA-2501(T) = VTT E-113163(T)). An emended description of the genus Pectinatus is also provided.

title: Dynamics of Cathode-Associated Microbial Communities and Metabolite Profiles in a Glycerol-Fed Bioelectrochemical System
abstract: Electrical current can be used to supply reducing power to microbial metabolism. This phenomenon is typically studied in pure cultures with added redox mediators to transfer charge. Here, we investigate the development of a current-fed mixed microbial community fermenting glycerol at the cathode of a bioelectrochemical system in the absence of added mediators and identify correlations between microbial diversity and the respective product outcomes. Within 1 week of inoculation, a Citrobacter population represented 95 to 99% of the community and the metabolite profiles were dominated by 1,3-propanediol and ethanol. Over time, the Citrobacter population decreased in abundance while that of a Pectinatus population and the formation of propionate increased. After 6 weeks, several Clostridium populations and the production of valerate increased, which suggests that chain elongation was being performed. Current supply was stopped after 9 weeks and was associated with a decrease in glycerol degradation and alcohol formation. This decrease was reversed by resuming current supply; however, when hydrogen gas was bubbled through the reactor during open-circuit operation (open-circuit potential) as an alternative source of reducing power, glycerol degradation and metabolite production were unaffected. Cyclic voltammetry revealed that the community appeared to catalyze the hydrogen evolution reaction, leading to a +400-mV shift in its onset potential. Our results clearly demonstrate that current supply can alter fermentation profiles; however, further work is needed to determine the mechanisms behind this effect. In addition, operational conditions must be refined to gain greater control over community composition and metabolic outcomes.

title: [NiFe] hydrogenases: a common active site for hydrogen metabolism under diverse conditions.
abstract: Hydrogenase proteins catalyze the reversible conversion of molecular hydrogen to protons and electrons. The most abundant hydrogenases contain a [NiFe] active site; these proteins are generally biased towards hydrogen oxidation activity and are reversibly inhibited by oxygen. However, there are [NiFe] hydrogenase that exhibit unique properties, including aerobic hydrogen oxidation and preferential hydrogen production activity; these proteins are highly relevant in the context of biotechnological devices. This review describes four classes of these "nonstandard" [NiFe] hydrogenases and discusses the electrochemical, spectroscopic, and structural studies that have been used to understand the mechanisms behind this exceptional behavior. A revised classification protocol is suggested in the conclusions, particularly with respect to the term "oxygen-tolerance". This article is part of a special issue entitled: metals in bioenergetics and biomimetics systems.

title: Constructing and testing the thermodynamic limits of synthetic NAD(P)H:H2 pathways
abstract: NAD(P)H:H2 pathways are theoretically predicted to reach equilibrium at very low partial headspace H2 pressure. An evaluation of the directionality of such near‐equilibrium pathways in vivo, using a defined experimental system, is therefore important in order to determine its potential for application. Many anaerobic microorganisms have evolved NAD(P)H:H2 pathways; however, they are either not genetically tractable, and/or contain multiple H2 synthesis/consumption pathways linked with other more thermodynamically favourable substrates, such as pyruvate. We therefore constructed a synthetic ferredoxin‐dependent NAD(P)H:H2 pathway model system in Escherichia coli BL21(DE3) and experimentally evaluated the thermodynamic limitations of nucleotide pyridine‐dependent H2 synthesis under closed batch conditions. NADPH‐dependent H2 accumulation was observed with a maximum partial H2 pressure equivalent to a biochemically effective intracellular NADPH/NADP+ ratio of 13:1. The molar yield of the NADPH:H2 pathway was restricted by thermodynamic limitations as it was strongly dependent on the headspace : liquid ratio of the culture vessels. When the substrate specificity was extended to NADH, only the reverse pathway directionality, H2 consumption, was observed above a partial H2 pressure of 40 Pa. Substitution of NADH with NADPH or other intermediates, as the main electron acceptor/donor of glucose catabolism and precursor of H2, is more likely to be applicable for H2 production.

title: Identifying target processes for microbial electrosynthesis by elementary mode analysis
abstract: BackgroundMicrobial electrosynthesis and electro fermentation are techniques that aim to optimize microbial production of chemicals and fuels by regulating the cellular redox balance via interaction with electrodes. While the concept is known for decades major knowledge gaps remain, which make it hard to evaluate its biotechnological potential. Here we present an in silico approach to identify beneficial production processes for electro fermentation by elementary mode analysis. Since the fundamentals of electron transport between electrodes and microbes have not been fully uncovered yet, we propose different options and discuss their impact on biomass and product yields.ResultsFor the first time 20 different valuable products were screened for their potential to show increased yields during anaerobic electrically enhanced fermentation. Surprisingly we found that an increase in product formation by electrical enhancement is not necessarily dependent on the degree of reduction of the product but rather the metabolic pathway it is derived from. We present a variety of beneficial processes with product yield increases of maximal 36% in reductive and 84% in oxidative fermentations and final theoretical product yields up to 100%. This includes compounds that are already produced at industrial scale such as succinic acid, lysine and diaminopentane as well as potential novel bio-commodities such as isoprene, para-hydroxybenzoic acid and para-aminobenzoic acid. Furthermore, it is shown that the way of electron transport has major impact on achievable biomass and product yields. The coupling of electron transport to energy conservation could be identified as crucial for most processes.ConclusionsThis study introduces a powerful tool to determine beneficial substrate and product combinations for electro-fermentation. It also highlights that the maximal yield achievable by bio electrochemical techniques depends strongly on the actual electron transport mechanisms. Therefore it is of great importance to reveal the involved fundamental processes to be able to optimize and advance electro fermentations beyond the level of lab-scale studies.

title: The synthesis of n-caproate from lactate: a new efficient process for medium-chain carboxylates production
abstract: A unique microbiome that metabolizes lactate rather than ethanol for n-caproate production was obtained from a fermentation pit used for the production of Chinese strong-flavour liquor (CSFL). The microbiome was able to produce n-caproate at concentrations as high as 23.41 g/L at a maximum rate of 2.97 g/L/d in batch trials without in-line extraction. Compared with previous work using ethanol as the electron donor, the n-caproate concentration increased by 82.89%. High-throughput sequencing analysis showed that the microbiome was dominated by a Clostridium cluster IV, which accounted for 79.07% of total reads. A new process for n-caproate production was proposed, lactate oxidation coupled to chain elongation, which revealed new insight into the well-studied lactate conversion and carbon chain elongation. In addition, these findings indicated a new synthesis mechanism of n-caproate in CSFL. We believe that this efficient process will provide a promising opportunity for the innovation of waste recovery as well as for n-caproate biosynthesis.

title: Temperature and solids retention time control microbial population dynamics and volatile fatty acid production in replicated anaerobic digesters
abstract: Anaerobic digestion is a widely used technology for waste stabilization and generation of biogas, and has recently emerged as a potentially important process for the production of high value volatile fatty acids (VFAs) and alcohols. Here, three reactors were seeded with inoculum from a stably performing methanogenic digester, and selective operating conditions (37°C and 55°C; 12 day and 4 day solids retention time) were applied to restrict methanogenesis while maintaining hydrolysis and fermentation. Replicated experiments performed at each set of operating conditions led to reproducible VFA production profiles which could be correlated with specific changes in microbial community composition. The mesophilic reactor at short solids retention time showed accumulation of propionate and acetate (42 ± 2% and 15 ± 6% of CODhydrolyzed, respectively), and dominance of Fibrobacter and Bacteroidales. Acetate accumulation (>50% of CODhydrolyzed) was also observed in the thermophilic reactors, which were dominated by Clostridium. Under all tested conditions, there was a shift from acetoclastic to hydrogenotrophic methanogenesis, and a reduction in methane production by >50% of CODhydrolyzed. Our results demonstrate that shortening the SRT and increasing the temperature are effective strategies for driving microbial communities towards controlled production of high levels of specific volatile fatty acids.

title: A biomolecular isolation framework for eco-systems biology
abstract: Mixed microbial communities are complex, dynamic and heterogeneous. It is therefore essential that biomolecular fractions obtained for high-throughput omic analyses are representative of single samples to facilitate meaningful data integration, analysis and modeling. We have developed a new methodological framework for the reproducible isolation of high-quality genomic DNA, large and small RNA, proteins, and polar and non-polar metabolites from single unique mixed microbial community samples. The methodology is based around reproducible cryogenic sample preservation and cell lysis. Metabolites are extracted first using organic solvents, followed by the sequential isolation of nucleic acids and proteins using chromatographic spin columns. The methodology was validated by comparison to traditional dedicated and simultaneous biomolecular isolation methods. To prove the broad applicability of the methodology, we applied it to microbial consortia of biotechnological, environmental and biomedical research interest. The developed methodological framework lays the foundation for standardized molecular eco-systematic studies on a range of different microbial communities in the future.

title: Collateral damage from oral ciprofloxacin versus nitrofurantoin in outpatients with urinary tract infections: a culture-free analysis of gut microbiota.
abstract: Recent treatment guidelines for uncomplicated urinary tract infections (UTIs) discourage fluoroquinolone prescription because of collateral damage to commensal microbiota, but the ecologic impact of alternative agents has not been evaluated by culture-free techniques. We prospectively collected faecal samples at three time points from ambulatory patients with UTIs treated with ciprofloxacin or nitrofurantoin, patients not requiring antibiotics and household contacts of ciprofloxacin-treated patients. We described changes in gut microbiota using a culture-independent approach based on pyrosequencing of the V3-V4 region of the bacterial 16S rRNA gene. All groups were similar at baseline. Ciprofloxacin had a significant global impact on the gut microbiota whereas nitrofurantoin did not. The end of ciprofloxacin treatment correlated with a reduced proportion of Bifidobacterium (Actinobacteria), Alistipes (Bacteroidetes) and four genera from the phylum Firmicutes (Faecalibacterium, Oscillospira, Ruminococcus and Dialister) and an increased relative abundance of Bacteroides (Bacteroidetes) and the Firmicutes genera Blautia, Eubacterium and Roseburia. Substantial recovery had occurred 4 weeks later. Nitrofurantoin treatment correlated with a reduced relative proportion of the genus Clostridium and an increased proportion of the genus Faecalibacterium. This study supports use of nitrofurantoin over fluoroquinolones for treatment of uncomplicated UTIs to minimize perturbation of intestinal microbiota.

title: Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities
abstract: mothur aims to be a comprehensive software package that allows users to use a single piece of software to analyze community sequence data. It builds upon previous tools to provide a flexible and powerful software package for analyzing sequencing data. As a case study, we used mothur to trim, screen, and align sequences; calculate distances; assign sequences to operational taxonomic units; and describe the α and β diversity of eight marine samples previously characterized by pyrosequencing of 16S rRNA gene fragments. This analysis of more than 222,000 sequences was completed in less than 2 h with a laptop computer.

title: UCHIME improves sensitivity and speed of chimera detection
abstract: Chimeric DNA sequences often form during polymerase chain reaction amplification, especially when sequencing single regions (e.g. 16S rRNA or fungal Internal Transcribed Spacer) to assess diversity or compare populations. Undetected chimeras may be misinterpreted as novel species, causing inflated estimates of diversity and spurious inferences of differences between populations. Detection and removal of chimeras is therefore of critical importance in such experiments.  We describe UCHIME, a new program that detects chimeric sequences with two or more segments. UCHIME either uses a database of chimera-free sequences or detects chimeras de novo by exploiting abundance data. UCHIME has better sensitivity than ChimeraSlayer (previously the most sensitive database method), especially with short, noisy sequences. In testing on artificial bacterial communities with known composition, UCHIME de novo sensitivity is shown to be comparable to Perseus. UCHIME is >100× faster than Perseus and >1000× faster than ChimeraSlayer.  Source, binaries and data: http://drive5.com/uchime.

title: Fluorescence in situ hybridization analysis of the prokaryotic community inhabiting crystallizer ponds.
abstract: A fluorescence in situ hybridization (FISH) protocol suitable for the identification of prokaryotes inhabiting hypersaline environments was developed and applied to several crystallizer ponds with salinities above 36% from a multipond solar saltern in Alicante, Spain. Two morphotypes were abundant in these environments: rods and square or square-like prokaryotes that could be affiliated to Bacteria and Archaea, respectively, by FISH with domain-specific probes. FISH with a newly designed probe proved that the archaeal 16S rDNA sequence most frequently recovered from the crystallizers, SPhT, originated from the dominant square-like prokaryotes. These uncultured prokaryotes have the morphology of Walsby's square bacteria. Additionally, FISH with a probe targeted to the genus Haloarcula, members of which are frequently isolated from this environment, indicated that this genus accounts for less than 0.1% of the total prokaryotic community.