bluuebunny/crossref_metadata_2025 huggingface dataset:
Online Analysis of CO2 Production in Electroactive Biofilms by Differential Electrochemical Mass Spectrometry
Authors: Kubannek Fabian , Schröder Uwe , Krewer Ulrike
Published: July 2020

Electroactive biofilms are routinely characterized in-operando by dynamic electrochemical measurement techniques such as cyclic voltammetry or electrochemical impedance spectroscopy. Since electrical signals can be recorded and processed very quickly, these techniques allow to investigate slow and fast electron transfer processes.

 

In contrast, the dynamics of species production rates are usually not addressed because standard measurement techniques for the quantification of reaction products such as gas chromatography are slow. Instead it is often assumed that species production rates are either directly proportional to the current - under so called turnover conditions - or equal zero - under so called non-turnover conditions.

 

To challenge this assumption, we measured species production rates of a biofilm electrode with a high time resolution by differential electrochemical mass spectrometry (DEMS). An acetate oxidizing biofilm electrode was placed just micrometers away from the mass spectrometer inlet in which enabled us to observe CO2 production directly at the electrode during cyclic voltammetry (CV) and potential steps.

 

The measurement results showed that the CO2 production deviates significantly from the expected value calculated from the current by Faraday’s law under certain operating conditions. We analyze this effect in detail and show that it can be explained with biofilm storage capacities for charge and substrate. These capacities are quantified by deconvoluting the faradaic and non-faradaic currents. [1]

 

Also, the onset of the complete oxidation of acetate to CO2 during CVs was determined to be just 22 mV above the standard potential for acetate oxidation. Determining this value by directly measuring CO2 instead of current is advantageous because capacitive effects can be excluded. [1]

 

In conclusion, we demonstrate that electrical current and CO2 production can be partly decoupled in biofilm electrodes and that DEMS is a valuable technique for analyzing processes in such electrodes.

 

[1] Kubannek, F., Schröder, U., Krewer, U. (2018). Revealing metabolic storage processes in electrode respiring bacteria by differential electrochemical mass spectrometry. Bioelectrochemistry, 121, 160–168, doi: 10.1016/j.bioelechem.2018.01.014
Impact of estradiol on biofilm formation of Pseudomonas aeruginosa clinical isolates from cystic fibrosis patients
Authors: Afzal Faria , Le Gall Tony , Montier Tristan , Müller Mareike
Published: July 2020

Women with Cystic Fibrosis (CF) have a significantly lower life expectancy compared to men, which is indicated by an earlier impairment of lung function due to chronic colonization of pathogenic bacteria like Pseudomonas aeruginosa (PA). Reasons for this “CF gender gap” until now have not yet been fully clarified and are assumed to be multifactorial.

This study aims to shed light on the contribution of sex hormones to the CF-Gender gap considering microbial endocrinology. Therefor the study investigates whether the sex hormone estradiol, whose blood serum concentrations are significantly fluctuating within the female cycle and during pregnancy, has a regulatory influence on the development of PA biofilms in the context of CF.

For that purpose, biofilms of PA isolates from CF-patients are treated in vitro with various estradiol concentrations and are examined in a comparative study quantitatively regarding the total biomass, e.g. via crystal violet staining, and qualitatively, e.g. via scanning electron microscopy, to characterize the ultrastructure of the biofilm.

We observed that estradiol induces biofilm-forming capacity of CF-PA-isolates with respect to the total biomass and modulates the biofilm structure especially concerning the distribution and clustering of bacteria.

The observed in vitro correlation between estradiol concentration and extent of biofilm growth provides a possible microbiological explanation for gender differences in CF disease progression.

These insights and further research on possible underlying mechanisms might be relevant in the long-term for new approaches in personalized treatment for female CF patients.

Acknowledgement

This work is supported by a financial grant from Mukoviszidose Institut gGmbH, Bonn, the research and development arm of the German Cystic Fibrosis Association Mukoviszidose e.V., the Christiane Herzog foundation. We further thank the Equal Opportunities Office of the University Siegen as well as the DAAD PPP Frankreich (Project-ID 55976814) for financial support.

 
Cellular and environmental factors influencing biofilm formation and colonization of plant tissue by a beneficial strain of bacteria, Pseudomonas donghuensis P482.
Authors: Rajewska Magdalena , Matuszewska Marta , Jafra Sylwia
Published: July 2020

The ability to colonize different environmental niches by bacteria is most often determined by the ability to form biofilms - complex, multicellular communities. This, in turn, depends on both cellular and extracellular factors such as genetic background of the strain, type of surface (biotic or abiotic) to which bacteria attach, availability of nutrients, temperature, etc. Pseudomonas donghuensis P482 strain is a little-known isolate from tomato rhizosphere, exhibiting antimicrobial activity towards bacterial and fungal plant pathogens. Studies have shown that it efficiently colonizes plant rhizosphere and forms biofilm on artificial surfaces. Which genetic or environmental factors underlie the mechanism of biofilm formation were yet to be elucidated. The presented research aimed at identifying those factors. Basing on the analysis of genome, knock-out mutants of the P482 strain were constructed in the genes potentially involved in biofilm formation and further analyzed for motility, colony morphology, attachment to artificial surfaces in different culture conditions, and colonization of maize and tomato rhizosphere.
Tailoring nanoparticle-biofilm interactions to increase efficacy of antimicrobial agents against Staphylococcus aureus
Authors: Devlin Henry , Fulaz Stephanie , Vitale Stefania , Quinn Laura , O'Gara James , Casey Eoin
Published: July 2020

Considering the timeline required for the development of novel antimicrobial drugs, increased attention should be given to repurposing existing drugs and improving their antimicrobial efficacy, particularly for chronic infections associated with biofilms. Methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) are common causes of biofilm-associated infections however each species has a distinct biofilm phenotype resulting in different biofilm matrix characteristics.. Nanoparticles (NPs) have the potential to significantly enhance the delivery of antimicrobial agents into biofilms, however the physicochemical properties which influence these interactions between NPs and the biofilm are not fully understood. The influence of NP surface chemistry on interactions with MRSA and MSSA biofilms was explored in this study. Mesoporous silica nanoparticles (MSNs) with different surface functionalizations (bare-B, amine-D, carboxyl-C, aromatic-A) were synthesised. Following interaction studies, MSNs were loaded with vancomycin (VAN) to observe biofilm eradication. The two negatively charged MSNs (MSN-B and MSN-C) showed a higher VAN loading in comparison to the positively charged MSNs (MSN-D and MSN-A). Cellular binding with MSN suspensions (0.25 mg mL-1) correlated with reduced viability of both MSSA and MRSA biofilm cells. MSNs were shown to be efficient carriers of vancomycin while also displaying significantly improved efficiency compared to free VAN. This allowed the administration of low MSNs concentrations, while maintaining a high local concentration of the antibiotic surrounding the bacterial cells, indicating a promising novel therapeutic approach for S. aureus biofilm infections.
In situ probing and evaluation of two different electrode materials in bio electrochemical systems by means of Optical Coherence Tomography on automated robotic platform
Authors: Liu Jinpeng , Horn Harald , Wagner Michael
Published: July 2020

Carbon-based and stainless steel-based materials are widely utilized as anode/cathode electrodes in bio electrochemical systems (BESs) due to its low capital cost, high conductivity and large specific surface area. Carbon-based materials such as carbon veil are mostly applied in lab-scale reactors because of its versatile shape and configuration. Moreover, stainless steel type materials show higher strength and are easier to incorporate within flow field. Optical coherence tomography (OCT) as an image technique is a suitable method to monitor biofilm growth and fluid-structure interactions at the meso-scale. In BESs, investigating bulk-biofilm interface (fluid-structure interactions) is of particular interest to optimize the mass transfer under suitable hydrodynamic condition and enhances the overall effectivity of BESs systems. To extend the knowledge about the influence of different anode electrodes as substratum on OCT monitoring and quantification, the biofilm structural properties analyzed by OCT image processing and bioelectrochemical systems performance were compared.  

A custom-designed dual-chamber setup was constructed by two transparent optical flow cells and fixed in the automated monitoring platform (Evobot). Herein, we applied OCT to in-situ characterize and quantify the biofilm structure properties on two different anode electrodes (carbon veil-CV and porous stainless steel-SS) as substratum in microbial fuel cell (MFC) mode.  3D OCT dataset analysis presented 3 structural parameters for biofilm-carbon veil interface and 5 structural parameters for biofilm-stainless steel interface, separately. Biofilm volume (BioV) was calculated to compare CV and SS anode electrodes.

In this study, a time-series of biofilm development was performed on both CV and SS materials. At the fourth day, the biofilm almost covered the entire anode surface and achieved 97% substratum coverage. Afterwards the biofilm grew mostly in vertical direction. With the further biofilm growth along height the electric resistance increased and power production gradually reached the equilibrium. Nevertheless, both materials did not show predominant advantage on power production. Furthermore, a relatively small error appeared on quantitative analysis of Biofilm volume using stainless steel. Whereas, the predictability of biofilm volume on the carbon veil anodes was hindered by the appearance of shadowing effects. Thus, it can be concluded that stainless steel flat plate electrode is preferable as anode material to investigate the interaction between biofilm structure, hydrodynamic conditions and mass transfer in BESs via OCT.
Effects of temperature gradients on AOB/NOB competition in MABR biofilms
Authors: Perez Patricia , Clements Emily , Picioreanu Cristian , nerenberg Robert
Published: July 2020

The membrane aerated biofilm reactor (MABR) is an emerging wastewater treatment technology that can greatly decrease energy requirements for wastewater treatment. It consists of cassettes of air-supplying, hollow-fiber membranes that can retrofit existing activated sludge processes. MABR behavior differs from conventional biofilm processes due to the counter-diffusion of the electron donor (ammonia) and acceptor (oxygen).

 

Partial nitrification (PN), or partial nitrification Anammox (PNA), can further improve MABR energy efficiency and cost effectiveness.  To achieve this, ammonia oxidizing bacteria (AOB) must outcompete nitrite-oxidizing bacteria (NOB).  High temperatures favor AOB, but it is not feasible to heat the wastewater influent.  However, high-temperature compressed air can be supplied to the membrane lumen, increasing temperatures inside the biofilm without increasing the bulk temperatures. No previous research has addressed temperature gradients in biofilms, which can lead to gradients in  biodegradation kinetics, diffusivities, and O2 solubility.

 

The objective of this research was to explore the effect of temperature gradients in MABR biofilms, especially with respect to PN. We used a one-dimensional multi-species biofilm model, which considers the MABR physical and biochemical behavior, especially with respect to temperature. The model was implemented using COMSOL Multiphysics. We also used bench-scale experiments to explore the effect of biofilm temperature gradients on MABR nitrification and PN performances and microbial community structure.

 

Model simulations showed that MABR biofilms exposed to a temperature gradient from 20 ºC (biofilm interior) to 10 ºC (bulk liquid) had a 60% increase in nitrification rates compared with biofilms at 10 ºC. More importantly, the model predicted a complete out competition of NOBs within the biofilm.

 

Preliminary experimental results confirm a significant (105%) increase in nitrification fluxes with a temperature of 30ºC compared to ambient temperatures (20ºC). Future experiments will validate the model predicted effects of biofilm temperature gradients on nitrification fluxes and microbial community structure.
Seeing is believing: Can biofilm diagnostics guide individualized therapy in endocarditis?
Authors: Kikhney Judith , Kursawe Laura , Eichinger Swb , Eichinger Walter , Schmidt Julia , Wiessner Alexandra , Moter Annette
Published: July 2020

Introduction

In Infective Endocarditis (IE), early diagnosis of the causative microorganism is crucial for correct antibiotic therapy, which improves the patients’ outcome.

Objectives

We studies the impact of biofilm formation in IE samples.

Materials & methods

We used Fluorescence in situ Hybridization (FISH) combined with 16S rRNA-gene PCR and sequencing to visualize and identify the infectious agents in native as well as prosthetic valves and to study any biofilm formation. The signal intensity of the fluorescence-labelled FISH probes correlates to a high ribosome content of the bacteria indicating metabolic activity at the time point of surgery. We developed a spacer FISH assay for the detection of the 16S-23S intergenic spacer region that is only present in actively transcribing cells to detect the activity of bacterial cells more precisely on a single cell level.

Results

FISH visualized bacteria in the heart valves ranging from single cells to highly organized biofilms. Interestingly, we found FISH positive bacteria in culture negative samples and samples from patients under antibiotic therapy. Using the spacer FISH, we visualized positive microbial cells in heart valves of patients under adequate therapy. Preliminary data point to a correlation between the biofilm state and treatment inefficiency.

Conclusion

FISH/PCR not only allows timely identification of the pathogens in IE, but also biofilm-staging and visualization of the effect of antimicrobial therapy at time of surgery. The technique provides crucial information for successful targeted antibiotic therapy, and it might guide therapeutical decisions in relation to biofilm state in the future.
Functionality and composition of marine biofilms on antifouling coatings.
Authors: Papadatou Maria , Robson Samuel , Watts Joy , Dobretsov Sergey , Salta Maria
Published: July 2020

Marine biofilms are assemblages of microbial cells irreversibly attached to living or non-living surfaces, embedded in a self-produced matrix of hydrated extracellular polymeric substances (EPS). The phenomenon of biofouling occurs upon the adhesion and accumulation of biofilms, composing the primary colonizers that are capable of EPS production, followed by the sequential growth of secondary colonizers on submerged structures. Biofouling constitutes a significant issue in marine industries (e.g. maritime transportation) and problems related to biofilm fouling include an increase in drag force, modification of surface properties (e.g. metal corrosion) and production of chemical compounds with inhibition effects to other foulers. The use of powerful biocides exhibits a good performance against biofouling, however, often their efficacy is evident to a lesser degree against biofilms. These chemically active compounds have been found to have toxicity effects for marine life and there is a need to discover high-performance environmentally acceptable products.

The aim of the present study was to investigate the biofilm community composition and gene-expression on commercial antifouling (AF) coatings employing next-generation sequencing approaches. Natural mixed-species biofilms were examined after a four-month immersion of two commercial AF coatings, including a biocidal (BAF) and a fouling release (FR), and a control non-treated surface in Langstone Harbour UK. Replicated biofilm samples were used for nucleic acid extraction and sequenced targeting the 16S rRNA gene and metatranscriptome.

We uncovered distinct biofilm community profiles between the two coatings; the BAF samples were dominated by Bacillariophyceae (diatoms), contrary to the FR and control samples where Oscillatoriophycideae (phylum Cyanobacteria) were prevailing. Alphaproteobacteria and Gammaproteobacteria contributed to a high abundance in all samples. Biofilms on BAF samples exhibited a lower species diversity compared to the FR. Here, we introduce a set of functional genes present across all biofilm-associated communities and highlight the differing gene transcriptional profiles in biocidal treatments. The gene transcriptional analysis uncovered highly enriched genes coding for proteins involved in biofilm regulation and formation. We demonstrated that biocidal-associated biofilms harbor genes that regulate defense mechanisms. Overall, the findings highlight links between differentially expressed protein functions and effects of AF coating type during biofilm development. We anticipate these results to contribute towards further development of antibiofilm strategies and fill gaps related to marine biofilm functions.
The bacterial lifecycle in cotton and polyester textiles
Authors: Moellebjerg Andreas , Meyer Rikke
Published: July 2020

Colonization of textiles and subsequent metabolic degradation of sweat and sebum components by axillary skin bacteria cause the characteristic sweat malodor and discoloring of dirty clothes. Once inside the textile, the bacteria can form biofilms that are hard to remove by conventional washing. When the biofilm persists after washing, the textiles retain the sweat odor. In addition to posing a huge industrial problem, textile biofilms constitute an interesting case study of bacterial behavior in periodically wetted and dried substrates with varying surface hydrophobicity. Here we aim to study the bacterial behavior in each of the four stages of the bacterial lifecycle in textiles: adhesion, growth, drying and washing. To accomplish this, we designed a novel in vitro model to mimic physiological sweating while wearing cotton and polyester textiles. The hydrophobic polyester adhered bacteria more strongly and absorbed more sebum, the bacteria’s primary nutrient source. Bacteria were therefore initially more active in polyester textiles than in cotton. However, polyester did not bind water as well as cotton. The increased water content of cotton allowed the bacteria to retain a higher activity after the textile had dried. However, neither of the textiles retained enough water upon drying to prevent the bacteria from irreversibly adhering to the textile fibers by capillary action. This demonstrates that bacterial colonization depends on the hydrophobic and hygroscopic properties of the colonized material while highlighting the possibility of controlling bacterial behavior by either changing the surface properties or the surrounding environment.
Study of the ability to form biofilms of microorganisms isolated from the milk industry in Canada
Authors: Goetz Coralie , Niboucha Nissa , Jean Julie
Published: July 2020

The ability of microorganisms to form biofilms has become a major problem in the dairy industry in Canada, notably by affecting the quality and the safety of the by-products. Established biofilms are difficult to remove during the CIP cleaning system and may become resistant to sanitizers. Therefore, it is important to identify and characterize the microorganisms associated to biofilm in the Canadian dairy industry, allowing to develop improvement strategies of biofilm control. The purpose of this study is to evaluate the ability to form biofilms by spoilage microorganisms isolated in processing plants in Canada. For this purpose, 19 strains were isolated from problems associated with the formation of biofilms in the dairy industry and identified using a MALDI-TOF mass spectrometer. The single species biofilm production of these isolates was then measured after a crystal violet coloration using 96-well microplates. The results revealed different biofilm formation profiles depending of the isolates in culture medium. Indeed, 7/19 isolates are moderate or strong biofilm producers and 12/19 isolates are negative or weak biofilm producers. Furthermore, enzymatic treatments revealed that the composition of the biofilms was different depending of the species but also the isolates. In conclusion, the results suggest that some of the isolates collected in the dairy industry have the ability to produce moderate or strong biofilms and thus, to facilitate the persistence of other spoilage microorganisms but also potential pathogenic microorganisms such as Listeria monocytogenes. The characterization of those biofilms will be helpful to the development of an effective approach allowing a better control of the biofilms in the dairy industry.
Electrode assisted production of platform chemicals in Rhodobacter sphaeroides.
Authors: Schmid Ferdinand , Gescher Johannes
Published: July 2020

The aim of this study was to establish cathodic biofilms of the photosynthetic non sulfur purple bacterium Rhodobacter sphaeroides as biocatalyst for the production of platform chemicals from carbon dioxide as carbon source and an electrical current as energy and electron source.  Therefore, R. sphaeroides was cultivated in a bioelectrical system (BES) in which light, CO2 and a stable current were provided. Chronopotentiometric measurements revealed the cathode potential necessary to maintain the applied current of I = 22,2 µA/cm². Interestingly, exposure of R. sphaeroides to the antibiotic kanamycin lead to increased biofilm production on the cathode although the organism expressed the necessary resistance marker. This enhanced biofilm production raised the potential by 170 mV to E = -1 V compared to the wildtype (E = -1,17 V) and hence increased the efficiency of the process. To date, the molecular basis of this effect remains unclear and is under investigation using a proteomic approach. To elucidate, if the productivity of R. sphaeroides as a production strain is also enhanced, the production of acetoin was established as proof of principle. After the confirmation of the acetoin production under autotrophic conditions, various approaches to increase the space-time yields of the process were conducted and their effect will be presented.  
Wetting properties of biofilm-coated surfaces produced at controlled shear flow conditions
Authors: Recupido Federica , Petala Maria , Kostoglou Margaritis , Caserta Sergio , Guido Stefano , Karapantsios Thodoris D.
Published: July 2020

Biofilms prevention and removal are crucial in many industrial and medical applications. Their complex and cohesive structure provides resistance to cleaning even to strong disinfectants. A key factor for their behavior is the wetting properties of their surfaces.

The main goal of this work is to study the wetting properties of biofilms produced by bacteria Pseudomonas fluorescens. Biofilms are obtained on glass coupons under well controlled flow conditions, using custom-made flow cell devices. Different nutrient concentration and shear flow conditions are investigated.

Biofilm wetting properties are examined under imposed external body forces (forced wetting) through a specialized device, named Kerberos®. Kerberos® is capable of subjecting sessile droplets to varying tilting angles and centrifugal forces while monitoring the variation of the droplet shape in X, Y and Z-directions through three Wi-Fi cameras. Wetting experiments are carried out using water-based solution (dye solution) droplets on biofilm-coated glass coupons. In this work, spreading/sliding behaviour of droplets are investigated only on horizontal substrates (no tilting) under the action of centrifugal forces. Apart from wetting properties, biofilm growth kinetics and surface morphology at different nutrient and shear flow conditions are also assessed.

Results show that, according to the different growth conditions, biofilms present different wetting properties. At lower nutrient concentration and shear flow conditions, spreading and sliding behaviour are similar to that observed in glass coupons in the absence of biofilm. At higher nutrient and shear flow conditions, spontaneous wicking of the biofilm occurs the moment of droplet deposition on the biofilm leading to irregular and jagged shapes of droplets, while on the contrary water droplets look like smooth spherical sections on pure glass. The spontaneous wicking affects the droplet initial shape and so the wetting behaviour during the subsequent rotation tests. In each examined condition, biofilms show hydrophilic properties.

 
The influence of the potential on single cell yield coefficients of early stage anode biofilms of Geobacter sulfurreducens
Authors: Scarabotti Francesco , Korth Benjamin , Rago Laura , Bühler Katja , Harnisch Falk
Published: July 2020

Geobacter sulfurreducens is a gram- rod shaped microorganism and the model system for direct extracellular electron transfer (EET). Although several details of the molecular mechanisms of EET are deciphered [1, 2], there is still a significant lack of knowledge, for instance on kinetics or cell growth. It is the aim of this study to provide information about the influence of the anode potential on yield coefficients on the single cell level, i.e. the moles of substrate consumed or moles of electrons transferred to the anode per cell. This information is highly relevant for physiological as well as technical considerations and for further improvement of bioelectrochemical systems (BES). Therefore, single and double chamber bioelectrochemical reactors were operated in batch mode using either the “standard” graphite rods or a microscopy slide sputter-coated with 10 nm of Cr and 25 nm of AuPd as anode material for allowing analysis of the biofilms with CLSM, as a tool for cell number determination.
We demonstrate that the anode potential and the electrode material has a strong influence on the kinetics of initial growth as well as on the yield coefficients of early stage anode biofilms of Geobacter sulfurreducens, but does not influence other parameters like coulombic efficiency. For instance, in single chamber reactors, the observed lag time, expressed as the time when the biofilm delivered a current density of 1 µA cm-2, was of 1.76±1.02, 6.99±2.26 days and 5.32±1.82 days at -200mV, 0 mV and +200mV (vs. Ag/AgCl sat. KCl) respectively with the sputter-coated glass and of 2.44±0.48 days, 1.38±0.46 days and 1.73±1.45 at -200 mV, 0 mV and +200 mV (vs. Ag/AgCl sat. KCl) respectively using graphite rods.

References

[1] Annu Rev Microbiol. 2012; 66:391-409

[2] Microbial Electrochemical Technology Sustainable Platform for Fuels, Chemicals and Remediation Biomass, Biofuels and Biochemicals 2019, Pages 339-351
Evaluation of biological risks related to the use of different kind of road surfaces
Authors: Świeczko-Żurek Beata , Ejsmont Jerzy , Pałubicka Anna
Published: July 2020

The paper presents the biological degradation of different road surfaces. Microorganisms are able to damage the structure and function of synthetic polymers. Some of road pavements, that are still in developing stage are porous and contain high amount of polyurethane (up to 10% by weight) or polymer modified asphalts. The same fungal enzymes can break down the polyurethanes or metabolize the plasticizers in various polymers, resulting in embrittlement and loss of strength. Many sources, indicate that enzymes produced by fungi (so also produced by lichens) have adverse effect on polyurethane resins. According to literature, bacteria and lichens may deteriorate polyurethane resin resulting in loss of bonds between particles (rubber and stone chippings). It may be possible to decrease sensitivity of the road pavements to attack of bacteria and lichens by adding certain substances (inhibitors) to the mix. The paper presents pilot study related to biological interactions with poroelastic road pavements containing rubber and polyurethane resin.
A multimeric matrix-associated lectin (RapD) affects proper exopolysaccharide processing in Rhizobium leguminosarum
Authors: Tarsitano Julian , Russo Daniela Marta , Alonso Leonardo , Zorreguieta Angeles
Published: July 2020

Rhizobium leguminosarum synthesizes an acidic polysaccharide formed by the polymerization of octasaccharide repeating units containing glucose (Glc), glucuronic acid (GlcA) and galactose (Gal) in a 5:2:1 ratio with particular substitutions; most of it is secreted to the extracellular medium (EPS) and part of it is retained on the bacterial surface as a capsular polysaccharide (CPS). Rap proteins, substrates of the PrsDE type I secretion system (TISS) share at least one Ra/CDHL (cadherin-like) domain and are involved in biofilm and matrix development either by cleaving the polysaccharide (Ply glycanases) or by altering the bacterial adhesive properties. Previous studies have shown that RapA2 is a monomeric calcium-binding lectin capable of binding specifically the R. leguminosarum CPS through a Ra/CDHL domain. It was shown that the absence or excess of RapA2 in the extracellular medium alters the biofilm matrix’s properties.

In this work we identified a new Rap protein (RapD), which comprises an N-terminal Ra/CDHL domain and a C-terminal domain of unknown function. By Western blot analysis using specific polyclonal antibodies we showed that in planktonic cultures RapD is co-secreted with the other Rap proteins in a PrsDE-dependent manner. Furthermore, under conditions that favor EPS production, a prominent RapD secretion was observed. In addition, colony blot assays indicated that RapD is associated with the biofilm matrix.  Interestingly, size exclusion chromatography of the EPS produced by the ΔrapA2 ΔrapD double mutant showed differences in the EPS profiles compared with those of the single mutants and the wild type strain, thus suggesting a functional interaction between the RapA2 and RapD proteins.

 Biophysical studies showed that calcium triggers proper folding and multimerization of recombinant RapD. Besides, further RapD conformation changes were observed in the presence of EPS.

ELISA and BIA (binding inhibition assay) assays showed that in the presence of calcium, RapD specifically binds the EPS and that galactose residues would be involved in this interaction.

In conclusion, RapD is a multimeric calcium-dependent EPS lectin that is co-secreted with the other Rap proteins via TISS PrsDE. Unlike RapA2, RapD is not retained on the bacterial surface but would rather interact with the released EPS. Finally, our results suggest that the interaction of RapA2 and RapD with the CPS or the EPS somehow affects the polysaccharide processing and therefore the biofilm matrix.
A thin line between plankton and biofilm
Authors: Dogsa Iztok , Kostanjsek Rok , Stopar David
Published: July 2020

Planktonic bacterial cells are by definition not aggregated. However, our previous work, where we have demonstrated the invisible mechanical connections between bacterial cells in dilute planktonic suspensions, challenged this assumption. Here we provide an experimental evidence using autocorrelation analysis of micrographs that in planktonic suspensions of B. subtilis a size continuum of aggregated structures is formed. In the microbial aggregates viable cells were embedded in the nucleic acid network. The eDNA was released during regular cell lysis events. To determine the size distribution of planktonic bacterial aggregates a pair-wise spatial correlations of bacterial cells in microscopic images were calculated. The monotonously decreasing shape of the autocorrelation function indicated a continuous distribution of bacterial aggregate sizes from monomer to multimers. Soft bacterial aggregates in dilute suspensions provide a missing link in a continuum of organic matter in aqueous environments and can significantly improve our understanding how non-attached biofilms form during planktonic growth.
Drinking Water Biofilm Management and Monitoring
Authors: Pick Frances , Fish Katherine , Husband Stewart , Boxall Joby
Published: July 2020

Biofilms within drinking water distribution systems can pose risks to consumers, especially when mobilised, as high concentrations of microorganisms and associated material can be released leading to degradation of water quality. Access and sampling of biofilms within drinking water pipelines can be difficult without disrupting supply in these extensive and buried systems. A novel biofilm monitoring device was developed to determine if biofilm formation rates can be used to assess microbiological water quality, track fouling rates and ultimately indicate distribution system performance. The device comprises a sample-line pipe with multiple, independent removable sections (allowing for biofilm sampling) that can be easily connected to sampling points in the distribution system. Biofilm is removed from the device and flow cytometry used to determine total and intact cell concentrations. The biomonitoring device was tested in a series of laboratory trials, to establish the impact of different flow rates and orientations on biofilm formation and to determine the optimum configuration that achieves accurate and repeatable results. Subsequently, these devices were installed in two operational systems, with different water qualities, and biofilms were sampled for two months to obtain biofilm growth rates. The results provide the first direct evidence of different biofilm formation rates in distribution systems with different water qualities. This evidence is now being used to investigate fouling rates via risk analysis and modelling. The use of the device has potential to improve understanding of biofilm behaviour and help inform biofilm and asset management to safeguard the quality of delivered drinking water.
Discovery and characterization of NO-responsive hemoproteins that regulate bacterial biofilms
Authors: Boon Elizabeth
Published: July 2020

Bacteria colonize most surfaces, forming multicellular, antibiotic-resistant, communities known as biofilms. Biofilms cause chronic infections and persistent biofouling of medical implants, marine vessels, and environmental sensors. Biofilm dispersal by nanomolar nitric oxide (NO) appears to be a general phenomenon, but fundamental questions remain concerning the identity of the NO sensor and mechanism of signal transduction. NO has been reported to disperse bacterial biofilms through regulation of intracellular cyclic-di-guanosine monophosphate concentrations. C-di-GMP is a tightly regulated second messenger-signaling molecule that is tightly correlated with biofilm formation. H-NOX proteins are well known NO sensors conserved in many bacteria. Indeed, we have shown that NO/H-NOX signaling disperses bacterial biofilms through a mechanism consistent with c-di-GMP signaling. However, H-NOX proteins are not conserved in most human pathogens. Therefore, an alternate NO sensor must also exist. We have identified a potential alternate NO sensor, a novel hemoprotein we named NosP (nitric oxide sensing protein). NosP domains are conserved in many bacterial genomes, they bind NO, but not molecular oxygen, as expected for a NO-specific sensor, and they are encoded as fusions with, or in close chromosomal proximity to, proteins annotated as c-di-GMP synthesis or hydrolysis enyzmes. We hypothesize that NO generally disperses bacterial biofilms through regulation of intracellular c-di-GMP concentrations, but the sensor varies; both NosP and H-NOX can fill this role.
Biofilm Architectural Breakdown in Response to Antibiotics Facilitates Community Invasion
Authors: Diaz-Pascual Francisco , Drescher Knut
Published: July 2020

Bacterial cells are often exposed to stress by changes in their environment. During the last decades the response of isolated cells to stress has been investigated in great detail. By contrast, little is known about the emergent multicellular level responses to stress, such as antibiotic exposure. Studying responses at the community level is key to understand the structure and function of the most common bacterial state: the multicellular communities termed biofilms. Here, by analysing Vibrio cholerae biofilms exposed to all different classes of antibiotics with single-cell resolution, we found that inhibition of protein synthesis cause striking changes in cell volume and biofilm architecture. The observed changes in cell volume are a single-cell level response driven by metabolic effects of the translational inhibition. The multicellular-level responses result from changes in matrix composition, matrix-cell dissociation and mechanical properties of the biofilms. We observed that these antibiotic-induced changes in biofilm architecture have strong consequences on the ecological dynamics of biofilms by making biofilms prone to invasion by bacteriophages and other bacterial cells. These mechanistic and ecological consequences of the emergent group-level architectural response to antibiotics are important to fully understand the ecological succession of biofilms and the implications of antibiotic therapy.
Bacteria and biofilms under the influence of shear
Authors: Müller-Renno Christine , Seehase Jürgen , Huttenlochner Katharina , Chodorski Jonas , Ulber Roland , Ziegler Christiane
Published: July 2020

In our attempts to improve the biofilm formation for productive bacteria (here the gram-negative seawater bacterium Paracoccus seriniphilus), we focus on the attachment of single bacteria to a solid surface as the first step of the biofilm formation process. Beside adhesion forces and elasticity of the bacteria, we investigate the minimal detachment forces due to lateral shear forces.

In order to investigate the influence of shear forces on already adhered bacteria in the laboratory, the Lateral Force Microscopy(LFM) was used first.The tip is moved laterally towards the adherent cell with different lateral forces until the cell detaches and thus the force required to shear the cell is determined.

By applying LFM, we found a correlation between the applied force and the number of moved bacteria as well as between the number of detached bacteria and the surface energy of the substrate. Further, any structuring of the substrate hinders the detachment substantially [1]. In agreement with the vertical adhesion forces, the bacteria are harder to detach at pH 4 than at pH 7.

In order to get closer to reality, the next step is to examine the (lateral) scanning force microscopic measurements under the influence of a flowing liquid and compare them with the LFM measurements. In combination with digital holography and proteome analysis, a better understanding of biofilm formation under the influence of a flowing liquid is to be achieved.

[1] K. Huttenlochner, N. Davoudi, C. Schlegel, M. Bohley, C. Müller-Renno, J. C. Aurich, R. Ulber, and C. Ziegle "Paracoccus seriniphilus adhered on surfaces: Resistance of a seawater bacterium against shear forces under the influence of roughness, surface energy, and zeta potential of the surfaces." Biointerphases 13.5 (2018)
Driving factors for bioclogging of pores and porous media
Authors: Kurz Dorothee , Secchi Eleonora , Stocker Roman , Jimenez-Martinez Joaquin
Published: July 2020

Understanding the interplay between hydrodynamics and biogeochemical processes is of growing importance in environmental applications and studies, especially in the fields of bioremediation and ecology. The majority of the microbial communities living in soil have a surface-attached lifestyle, allowing them to form biofilms. The biofilm growth influences pore geometries by clogging them and thus redirecting the flow, which in return affects biofilm development and local mass transport. After initially clogging single pores, the biofilm structure expands to larger clusters before eventually clogging the porous medium entirely. We study these processes with a soil-born microorganism, Bacillus subtilis, in microfluidic devices mimicking porous media to get a mechanistic understanding of the driving factors of bioclogging of porous media on different scales.

Carefully designed porous geometries were used for the experiments to study biofilm growth under different flow conditions. After being seeded with bacteria, devices were exposed to a continuous nutrient flow during several days. Continuously monitoring the pressure evaluation and imaging the biofilm growth using Brightfield microscopy allowed a high temporal resolution of biofilm growth processes.

An interplay of hydraulic parameters and geometric features of the porous medium as well as the mass flow rate of nutrients drive the speed of pore clogging. Besides the pore scale clogging, the initiation of biofilm formation as well as the speed of clogging of the entire medium are influenced by the before mentioned parameters. Furthermore, the size and number of the biofilm clusters formed seem to drive the medium scale clogging. This leads to inverting trends concerning the clogging rate in one pore when compared to the porous medium scale for different pore sizes. These results shed light on the pore-scale mechanism as well as driving parameters of biofilm formation and bioclogging and their transferability to the next larger scale, e.g. a porous medium.
Genomic screening for novel peptide antibiotics in biofilm cyanobacteria by in-silico analysis and PCR
Authors: Lenz Selina
Published: July 2020

Cyanobacteria are a group of phototrophic prokaryotes commonly known as blue-green algae. They grow embedded as biofilms in a thick matrix of extracellular polymeric substances (EPS) and can produce a highly diverse range of secondary metabolites, which are interesting in terms of their antimicrobial activity. Among these components, polyketide and polypeptide molecules are dominating. Antimicrobial polypeptide molecules are usually post-translational-modified or synthesised by non-ribosomal peptide synthetase (NRPS). Standard screening for antibiotics by inhibition tests is very time consuming and expression of antimicrobic activity highly depend on cultivation conditions. Therefore, they can vary between different cultivations. On a genomic level existing, but in this cultivation not synthesized, antibiotics are completely neglected. Due to the increasing amount of available genomic sequence data, screening for novel antibiotics can also be done in-silico. Highly homologous sequences to known antibiotic gen clusters can be determined in cyanobacterial genomes and eventually be detected in-vivo through PCR analysis. Compared to inhibition tests, a major advantage of PCR is the little amount of biomass needed. As the growth of cyanobacteria is slow, e.g. Trichocoleus sociatus (0.44 d-1) compared to bacteria like Escherichia coli (2.08 h-1), this leads to significant shorter cultivation and screening time. In addition, qPCR can be used to determine gene expression quantity of the considered genes. PCR with degenerated primers for specific gen cluster like NRPS, polyketide synthetases, lanthipeptides etc. can also be used to screen non-sequenced cyanobacteria for the possible origin of an unidentified antibiotic.

The following work is part of the iProcess project, whose overall scientific goal is to develop the process engineering fundamentals for using fungi and cyanobacteria as production organisms for pharmaceutically active substances. As part of the iProcess project, a semi-continuous process for the production of antibiotics from cyanobacteria biofilms in aerosol reactors shall be developed. Aim of the following work is the in-silico search for new polypeptide antibiotics, as well as the subsequent in-vivo detection to discover promising cyanobacteria as production strains. In the first instance, the screening is focusing on the intern cyanobacteria strain collection of the TU Kaiserslautern. Subsequently the new strains will be cultivated as biofilms in an aerosol reactor and the resulting extracellular polymeric substances can be analysed for their antimicrobial activity.

 

This project is financially supported by Ministry of Science, Further Education and Culture of Rhineland-Palatinate (mwwk.rlp) (iProcess intelligent process development – from modelling to product).
Climate-friendly production of renewable resources by the innovative use of diazotrophic, terrestrial cyanobacteria
Authors: Strieth Dorina , Stiefelmaier Judith , Di Nonno Sarah , Kronenberger Daniel , Ulber Roland
Published: July 2020

Renewable raw materials from agriculture help to reduce greenhouse gases, as they expand the CO2 cycle and release less greenhouse gases than fossil raw materials when used for energy purposes. However, since high levels of nitrous oxide are released, especially by organic fertilization, no emission-free production of renewable raw materials is possible so far. Accordingly, there is an urgent need to replace organic and mineral fertilizers with nitrogen-binding systems. Here, diazotrophic (atmospheric nitrogen fixing), terrestrial cyanobacteria provide a way to fix atmospheric nitrogen and pass it on to plants for the production of renewable raw materials. Especially terrestrial cyanobacteria grow embedded in a thick matrix of extracellular polymeric substances which can contribute to a desirable soil stabilization and thus protection against soil erosion and to promoting water retention in the soil.

The main goal of this research is the sustainable establishment of nitrogen-fixing terrestrial cyanobacteria, which are immobilized on biodegradable carriers, in the ground in agriculture. Based on the aerosol-based photobioreactors constructed and established at the chair of bioprocess engineering, a new reactor system for the cultivation of cyanobacteria on carriers was developed and characterized. In addition, a screening for potential diazotrophic terrestrial cyanobacteria including the formation from vegetative cells to heterocysts (nitrogen fixing cells), nitrogen uptake and release rates for the use in the German agricultural economy was performed. A proof-of-principle to prove the use of terrestrial cyanobacteria as fertilizer for a climate-friendly production of renewable resources was tested by sowing seeds of Arabidopsis thaliana on agar plates with and without nitrogen as well as with and without a diazotrophic phototrophic biofilm.

This project is financially supported by the def DFG (UL 170/16-1) and the “Landesforschungsschwerpunkt NanoKat”.