Inoculum source determines the stress resistance of electroactive functional taxa in biofilms: A metagenomic perspective.

The inoculum has a crucial impact on bioreactor initialization and performance. However, there is currently a lack of guidance on selecting appropriate inocula for applications in environmental biotechnology. In this study, we applied microbial electrolysis cells (MECs) as models to investigate the differences in the functional potential of electroactive microorganisms (EAMs) within anodic biofilms developed from four different inocula (natural or artificial), using shotgun metagenomic techniques. We specifically focused on extracellular electron transfer (EET) function and stress resistance, which affect the performance and stability of MECs. Community profiling revealed that the family Geobacteraceae was the key EAM taxon in all biofilms, with Geobacter as the dominant genus. The c-type cytochrome gene imcH showed universal importance for Geobacteraceae EET and was utilized as a marker gene to evaluate the EET potential of EAMs. Additionally, stress response functional genes were used to assess the stress resistance potential of Geobacter species. Comparative analysis of imcH gene abundance revealed that EAMs with comparable overall EET potential could be enriched from artificial and natural inocula (P > 0.05). However, quantification of stress response gene copy numbers in the genomes demonstrated that EAMs originating from natural inocula possessed superior stress resistance potential (196 vs. 163). Overall, this study provides novel perspectives on the inoculum effect in bioreactors and offers theoretical guidance for selecting inoculum in environmental engineering applications.

Interspecies ecological competition rejuvenates decayed Geobacter electroactive biofilm.

Bioelectrochemical systems (BESs) exploit electroactive biofilms (EABs) for promising applications in biosensing, wastewater treatment, energy production, and chemical biosynthesis. However, during the operation of BESs, EABs inevitably decay. Seeking approaches to rejuvenate decayed EABs is critical for the sustainability and practical application of BESs. Prophage induction has been recognized as the primary reason for EAB decay. Herein, we report that introducing a competitive species of Geobacter uraniireducens suspended prophage induction in Geobacter sulfurreducens and thereby rejuvenated the decayed G. sulfurreducens EAB. The transcriptomic profile of G. sulfurreducens demonstrated that the addition of G. uraniireducens significantly affected the expression of metabolism- and stress response system-related genes and in particular suppressed the induction of phage-related genes. Mechanistic analyses revealed that interspecies ecological competition exerted by G. uraniireducens suppressed prophage induction. Our findings not only reveal a novel strategy to rejuvenate decayed EABs, which is significant for the sustainability of BESs, but also provide new knowledge for understanding phage-host interactions from an ecological perspective, with implications for developing therapies to defend against phage attack.

Deciphering the differentiated performance on electricity generation and COD degradation by Rhodopseudomonas-dominated bioanode in light or dark.

Anoxygenic phototrophic bacteria is a promising catalyst for constructing bioanode, but the mixed culture with non-photosynthetic bacteria is inevitable in an open environment application. In this study, a Rhodopseudomonas-dominated mixed culture with other electrogenic bacteria was investigated for deciphering the differentiated performance on electricity generation in light or dark conditions. The kinetic study showed that reaction rate of OM degradation was 9 times higher than that under dark condition, demonstrating that OM degradation was enhanced by photosynthesis. However, CE under light condition was lower. It indicated that part of OM was used to provide hydrogen donors for the fixation of CO2 or hydrogen production in photosynthesis, decreasing the OM used for electron transfer. In addition, higher COD concentration was not conducive to electricity generation. EIS analysis demonstrated that higher OM concentration would increase Rct to hinder the transfer of electrons from bacteria to the electrode. Indirect and direct electron transfer were revealed by CV analysis for light and dark biofilm, respectively, and nanowires were also observed by SEM graphs, further revealing the differentiate performance. Microbial community analysis demonstrated Rhodopseudomonas was dominated in light and decreased in dark, but Geobacter increased apparently from light to dark, resulting in different power generation performance. The findings revealed the differentiated performance on electricity generation and pollutant removal by mixed culture of phototrophic bacteria in light or dark, which will improve the power generation from photo-microbial fuel cells.

Significance of a minor pilin PilV in biofilm cohesion of Geobacter sulfurreducens.

Bacterial adhesion plays a vital role in forming and shaping the structure of electroactive biofilms that are essential for the performance of bioelectrochemical systems (BESs). Type IV pili are known to mediate cell adhesion in many Gram-negative bacteria, but the mechanism of pili-mediated cell adhesion of Geobacter species on anode surface remains unclear. Herein, a minor pilin PilV2 was found to be essential for cell adhesion ability of Geobacter sulfurreducens since the lack of pilV2 gene depressed the cell adhesion capability by 81.2% in microplate and the anodic biofilm density by 23.1 % at -0.1 V and 37.7 % at -0.3 V in BESs. The less cohesiveness of mutant biofilms increased the charge transfer resistance and biofilm resistance, which correspondingly lowered current generation of the pilV2-deficient strain by up to 63.2 % compared with that of the wild-type strain in BESs. The deletion of pilV2 posed an insignificant effect on the production of extracellular polysaccharides, pili, extracellular cytochromes and electron shuttles that are involved in biofilm formation or extracellular electron transfer (EET) process. This study demonstrated the significance of pilV2 gene in cell adhesion and biofilm formation of G. sulfurreducens, as well as the importance of pili-mediated adhesion for EET of electroactive biofilm.

Reduction of S0 deposited on electroactive biofilm under an oxidative potential.

The inevitable deposition of S0 on the electroactive biofilm (EAB) via anodic sulfide oxidation affects the stability of bioelectrochemical systems (BESs) when an accidental discharge of sulfide occurred, leading to the inhibition of electroacitivity, because the potential of anode (e.g., 0 V versus Ag/AgCl) is ~500 mV more positive than the redox potential of S2-/S0. Here we found that S0 deposited on the EAB can be spontaneously reduced under this oxidative potential independent of microbial community variation, leading to a self-recovery of electroactivity (> 100 % in current density) with biofilm thickening (~210 µm). Transcriptomics of pure culture indicated that Geobacter highly expressed genes involving in S0 metabolism, which had an additional benefit to improve the viability (25 % - 36 %) of bacterial cells in biofilm distant from the anode and the cellular metabolic activity via electron shuttle pair of S0/S2-(Sx2-). Our findings highlighted the importance of spatially heterogeneous metabolism to its stability when EABs encountered with the problem of S0 deposition, and that in turn improved the electroactivity of EABs.

Probing the robustness of Geobacter sulfurreducens against fermentation hydrolysate for uses in bioelectrochemical systems.

In this study, impacts of toxic ions/acids found in real fermentation-hydrolysate on the model exoelectrogenic G. sulfurreducens were investigated. Initially, different concentrations of acetate, butyrate, propionate, Na+, and K+ were tested, individually and in combination, for effects on the planktonic growth, followed by validation with diluted-hydrolysate. Meanwhile, it could be shown that (1) excess Na+ (≥100 mM) causes inhibition that can be reduced by K+ replacement, (2) butyrate (≥10 mM) induces higher toxicity than propionate, and (3) hydrolysate induces synergistic inhibition to G. sulfurreducens where organic constituents contributed more than Na+. Afterwards, compared with impacts on planktonic cells, the pre-enriched anodic biofilm of G. sulfurreducens in BESs showed higher robustness against diluted-hydrolysate, achieving current densities of 1.4-1.7 A/m2 (at up to ∼30 mM butyrate and propionate as well as ∼240 mM Na+). As a conclusion, using G. sulfurreducens in BESs dealing with fermentation-hydrolysate can be regulated for efficacious energy recovery.

Potassium channel mediates electroactive biofilm formation via recruiting planktonic Geobacter cells.

Potassium (K+)-channel-based electrical signaling can coordinate microbial actions at a distance that provides an evolutionary advantage to cell communities. Electroactive cells are usually cultured surrounded by an electric field which provided stronger electrical signaling than the K+-mediated electrical signaling. Whether the K+ signaling also plays a role in coordinating the behavior of electroactive microorganisms has not been accurately demonstrated. Thus, we constructed a K+-channel-deficient strain ΔgsuK of Geobacter sulfurreducens to directly investigate roles of K+ signaling in electroactive biofilm formation for the first time. The ΔgsuK strain exhibited significantly inferior biofilm formation (i.e., biomass, thickness and component) and consequently showed weaker electrical performance (i.e., start-up time, current output, electrochemical catalytic behavior and charge transfer resistance) than the wild-type strain. Individual electric generation capacity and the expression of genes involved in biofilm formation and electrical performance in the single cell did not significantly change with the deletion of gsuK, indicating that K+ signaling indeed influenced the recruiting behavior of planktonic cell but not the functioning of the single cell related to biofilm formation or electric generation. This study is intended to provide an in-depth understanding of electroactive biofilm formation and serve as a basis for optimizing its electrical performance via strengthening the recruitment behavior.

Nutrient limitation regulates the properties of extracellular electron transfer and hydraulic shear resistance of electroactive biofilm.

Understanding the roles of nutrient restriction in extracellular electron transfer (EET) and stability of mixed electroactive biofilm is essential in pollutant degradation and bioenergy production. However, the relevant studies are still limited so far. Herein, the effect of nutrient restriction on the EET pathways and stability of mixed electroactive biofilm was explored. It was found that the electroactive Pseudomonas and Geobacter genera were selectively enriched in the biofilms cultured under total nutrient and P-constrained conditions, and two EET pathways including direct and indirect were found, while Rhodopseudomonas genus was enriched in the N-constrained biofilm, which only had the direct EET pathway. Moreover, multiple analyses including 2D confocal Raman spectra revealed that P-constrained biofilm was rich in extracellular polymeric substances (EPS) especially for polysaccharide, presented a dense and uniform layered distribution, and had better stability than N-constrained biofilm with lower EPS and biofilm with heterostructures cultured under total nutrient conditions.

Adaptive Synthesis of a Rough Lipopolysaccharide in Geobacter sulfurreducens for Metal Reduction and Detoxification.

The ability of some metal-reducing bacteria to produce a rough (no O-antigen) lipopolysaccharide (LPS) could facilitate surface interactions with minerals and metal reduction. Consistent with this, the laboratory model metal reducer Geobacter sulfurreducens PCA produced two rough LPS isoforms (with or without a terminal methyl-quinovosamine sugar) when growing with the soluble electron acceptor fumarate but expressed only the shorter and more hydrophilic variant when reducing iron oxides. We reconstructed from genomic data conserved pathways for the synthesis of the rough LPS and generated heptosyltransferase mutants with partial (ΔrfaQ) or complete (ΔrfaC) truncations in the core oligosaccharide. The stepwise removal of the LPS core sugars reduced the hydrophilicity of the cell and increased outer membrane vesiculation. These changes in surface charge and remodeling did not substantially impact planktonic growth but disrupted the developmental stages and structure of electroactive biofilms. Furthermore, the mutants assembled conductive pili for extracellular mineralization of the toxic uranyl cation but were unable to prevent permeation and mineralization of the radionuclide in the cell envelope. Hence, not only does the rough LPS promote cell-cell and cell-mineral interactions critical to biofilm formation and metal respiration but it also functions as a permeability barrier to toxic metal cations. In doing so, the rough LPS maximizes the extracellular reduction of soluble and insoluble metals and preserves cell envelope functions critical to the environmental survival of Geobacter bacteria in metal-rich environments and their performance in bioremediation and bioenergy applications. IMPORTANCE Some metal-reducing bacteria produce an LPS without the repeating sugars (O-antigen) that decorate the surface of most Gram-negative bacteria, but the biological significance of this adaptive feature was not previously investigated. Using the model representative Geobacter sulfurreducens strain PCA and mutants carrying stepwise truncations in the LPS core sugars, we demonstrate the importance of the rough LPS in the control of cell surface chemistry during the respiration of iron minerals and the formation of electroactive biofilms. Importantly, we describe hitherto overlooked roles for the rough LPS in metal sequestration and outer membrane vesiculation that are critical for the extracellular reduction and detoxification of toxic metals and radionuclides. These results are of interest for the optimization of bioremediation schemes and electricity-harvesting platforms using these bacteria.

Multistep Signaling in Nature: A Close-Up of Geobacter Chemotaxis Sensing.

Environmental changes trigger the continuous adaptation of bacteria to ensure their survival. This is possible through a variety of signal transduction pathways involving chemoreceptors known as methyl-accepting chemotaxis proteins (MCP) that allow the microorganisms to redirect their mobility towards favorable environments. MCP are two-component regulatory (or signal transduction) systems (TCS) formed by a sensor and a response regulator domain. These domains synchronize transient protein phosphorylation and dephosphorylation events to convert the stimuli into an appropriate cellular response. In this review, the variability of TCS domains and the most common signaling mechanisms are highlighted. This is followed by the description of the overall cellular topology, classification and mechanisms of MCP. Finally, the structural and functional properties of a new family of MCP found in Geobacter sulfurreducens are revisited. This bacterium has a diverse repertoire of chemosensory systems, which represents a striking example of a survival mechanism in challenging environments. Two G. sulfurreducens MCP-GSU0582 and GSU0935-are members of a new family of chemotaxis sensor proteins containing a periplasmic PAS-like sensor domain with a c-type heme. Interestingly, the cellular location of this domain opens new routes to the understanding of the redox potential sensing signaling transduction pathways.

Evidence of a Streamlined Extracellular Electron Transfer Pathway from Biofilm Structure, Metabolic Stratification, and Long-Range Electron Transfer Parameters.

A strain of Geobacter sulfurreducens, an organism capable of respiring solid extracellular substrates, lacking four of five outer membrane cytochrome complexes (extABCD+ strain) grows faster and produces greater current density than the wild type grown under identical conditions. To understand cellular and biofilm modifications in the extABCD+ strain responsible for this increased performance, biofilms grown using electrodes as terminal electron acceptors were sectioned and imaged using electron microscopy to determine changes in thickness and cell density, while parallel biofilms incubated in the presence of nitrogen and carbon isotopes were analyzed using NanoSIMS (nanoscale secondary ion mass spectrometry) to quantify and localize anabolic activity. Long-distance electron transfer parameters were measured for wild-type and extABCD+ biofilms spanning 5-µm gaps. Our results reveal that extABCD+ biofilms achieved higher current densities through the additive effects of denser cell packing close to the electrode (based on electron microscopy), combined with higher metabolic rates per cell compared to the wild type (based on increased rates of 15N incorporation). We also observed an increased rate of electron transfer through extABCD+ versus wild-type biofilms, suggesting that denser biofilms resulting from the deletion of unnecessary multiheme cytochromes streamline electron transfer to electrodes. The combination of imaging, physiological, and electrochemical data confirms that engineered electrogenic bacteria are capable of producing more current per cell and, in combination with higher biofilm density and electron diffusion rates, can produce a higher final current density than the wild type. IMPORTANCE Current-producing biofilms in microbial electrochemical systems could potentially sustain technologies ranging from wastewater treatment to bioproduction of electricity if the maximum current produced could be increased and current production start-up times after inoculation could be reduced. Enhancing the current output of microbial electrochemical systems has been mostly approached by engineering physical components of reactors and electrodes. Here, we show that biofilms formed by a Geobacter sulfurreducens strain producing ∼1.4× higher current than the wild type results from a combination of denser cell packing and higher anabolic activity, enabled by an increased rate of electron diffusion through the biofilms. Our results confirm that it is possible to engineer electrode-specific G. sulfurreducens strains with both faster growth on electrodes and streamlined electron transfer pathways for enhanced current production.

Engineering Cooperation in an Anaerobic Coculture.

Over the past century, microbiologists have studied organisms in pure culture, yet it is becoming increasingly apparent that the majority of biological processes rely on multispecies cooperation and interaction. While little is known about how such interactions permit cooperation, even less is known about how cooperation arises. To study the emergence of cooperation in the laboratory, we constructed both a commensal community and an obligate mutualism using the previously noninteracting bacteria Shewanella oneidensis and Geobacter sulfurreducens Incorporation of a glycerol utilization plasmid (pGUT2) enabled S. oneidensis to metabolize glycerol and produce acetate as a carbon source for G. sulfurreducens, establishing a cross-feeding, commensal coculture. In the commensal coculture, both species coupled oxidative metabolism to the respiration of fumarate as the terminal electron acceptor. Deletion of the gene encoding fumarate reductase in the S. oneidensis/pGUT2 strain shifted the coculture with G. sulfurreducens to an obligate mutualism where neither species could grow in the absence of the other. A shift in metabolic strategy from glycerol catabolism to malate metabolism was associated with obligate coculture growth. Further targeted deletions in malate uptake and acetate generation pathways in S. oneidensis significantly inhibited coculture growth with G. sulfurreducens The engineered coculture between S. oneidensis and G. sulfurreducens provides a model laboratory system to study the emergence of cooperation in bacterial communities, and the shift in metabolic strategy observed in the obligate coculture highlights the importance of genetic change in shaping microbial interactions in the environment.IMPORTANCE Microbes seldom live alone in the environment, yet this scenario is approximated in the vast majority of pure-culture laboratory experiments. Here, we develop an anaerobic coculture system to begin understanding microbial physiology in a more complex setting but also to determine how anaerobic microbial communities can form. Using synthetic biology, we generated a coculture system where the facultative anaerobe Shewanella oneidensis consumes glycerol and provides acetate to the strict anaerobe Geobacter sulfurreducens In the commensal system, growth of G. sulfurreducens is dependent on the presence of S. oneidensis To generate an obligate coculture, where each organism requires the other, we eliminated the ability of S. oneidensis to respire fumarate. An unexpected shift in metabolic strategy from glycerol catabolism to malate metabolism was observed in the obligate coculture. Our work highlights how metabolic landscapes can be expanded in multispecies communities and provides a system to evaluate the evolution of cooperation under anaerobic conditions.

The electrode potential determines the yield coefficients of early-stage Geobacter sulfurreducens biofilm anodes.

Geobacter sulfurreducens is the model for electroactive microorganisms (EAM). EAM can use solid state terminal electron acceptors (TEA) including anodes via extracellular electron transfer (EET). Yield coefficients relate the produced cell number or biomass to the oxidized substrate or the reduced TEA. These data are not yet sufficiently available for EAM growing at anodes. Thus, this study provides information about kinetics as well as yield coefficients of early-stage G. sulfurreducens biofilms using anodes as TEA at the potentials of -200 mV, 0 mV and +200 mV (vs. Ag/AgCl sat. KCl). The selected microorganism was therefore cultivated in single and double chamber batch reactors on graphite or AuPd anodes. Interestingly, whereas the lag time and maximum current density within 12 days of growth differed, the anode potential does not influence the coulombic efficiency and the formal potential of the EET, which remains constant for all the experiments at ~ -300 to -350 mV. We demonstrated for the first time that the anode potential has a strong influence on single cell yield coefficients which ranged from 2.69 × 1012 cells mole--1 at -200 mV and 1.48 × 1012 cells mole--1 at 0 mV to 2.58 × 1011 cells mole--1 at +200 mV in single chamber reactors and from 1.15 × 1012 cells mole--1 at -200 mV to 8.98× 1011 cells mole--1 at 0 mV in double chamber reactors. This data can be useful for optimization and scaling-up of primary microbial electrochemical technologies.

Electric field stimulates production of highly conductive microbial OmcZ nanowires.

Multifunctional living materials are attractive due to their powerful ability to self-repair and replicate. However, most natural materials lack electronic functionality. Here we show that an electric field, applied to electricity-producing Geobacter sulfurreducens biofilms, stimulates production of cytochrome OmcZ nanowires with 1,000-fold higher conductivity (30 S cm-1) and threefold higher stiffness (1.5 GPa) than the cytochrome OmcS nanowires that are important in natural environments. Using chemical imaging-based multimodal nanospectroscopy, we correlate protein structure with function and observe pH-induced conformational switching to ß-sheets in individual nanowires, which increases their stiffness and conductivity by 100-fold due to enhanced π-stacking of heme groups; this was further confirmed by computational modeling and bulk spectroscopic studies. These nanowires can transduce mechanical and chemical stimuli into electrical signals to perform sensing, synthesis and energy production. These findings of biologically produced, highly conductive protein nanowires may help to guide the development of seamless, bidirectional interfaces between biological and electronic systems.

A New Approach for Evaluating Electron Transfer Dynamics by Using In Situ Resonance Raman Microscopy and Chronoamperometry in Conjunction with a Dynamic Model.

Geobacter sulfurreducens is a good candidate as a chassis organism due to its ability to form thick, conductive biofilms, enabling long-distance extracellular electron transfer (EET). Due to the complexity of EET pathways in G. sulfurreducens, a dynamic approach is required to study genetically modified EET rates in the biofilm. By coupling online resonance Raman microscopy with chronoamperometry, we were able to observe the dynamic discharge response in the biofilm's cytochromes to an increase in anode voltage. Measuring the heme redox state alongside the current allows for the fitting of a dynamic model using the current response and a subsequent validation of the model via the value of a reduced cytochrome c Raman peak. The modeled reduced cytochromes closely fitted the Raman response data from the G. sulfurreducens wild-type strain, showing the oxidation of heme groups in cytochromes until a new steady state was achieved. Furthermore, the use of a dynamic model also allows for the calculation of internal rates, such as acetate and NADH consumption rates. The Raman response of a mutant lacking OmcS showed a higher initial oxidation rate than predicted, followed by an almost linear decrease of the reduced mediators. The increased initial rate could be attributed to an increase in biofilm conductivity, previously observed in biofilms lacking OmcS. One explanation for this is that OmcS acts as a conduit between cytochromes; therefore, deleting the gene restricts the rate of electron transfer to the extracellular matrix. This could, however, be modeled assuming a linear oxidation rate of intercellular mediators.IMPORTANCE Bioelectrochemical systems can fill a vast array of application niches, due to the control of redox reactions that it offers. Although native microorganisms are preferred for applications such as bioremediation, more control is required for applications such as biosensors or biocomputing. The development of a chassis organism, in which the EET is well defined and readily controllable, is therefore essential. The combined approach in this work offers a unique way of monitoring and describing the reaction kinetics of a G. sulfurreducens biofilm, as well as offering a dynamic model that can be used in conjunction with applications such as biosensors.

In Vivo Molecular Insights into Syntrophic Geobacter Aggregates.

Direct interspecies electron transfer (DIET) has been considered as a novel and highly efficient strategy in both natural anaerobic environments and artificial microbial fuel cells. A syntrophic model consisting of Geobacter metallireducens and Geobacter sulfurreducens was studied in this work. We conducted in vivo molecular mapping of the outer surface of the syntrophic community as the interface of nutrients and energy exchange. System for Analysis at the Liquid Vacuum Interface combined with time-of-flight secondary ion mass spectrometry was employed to capture the molecular distribution of syntrophic Geobacter communities in the living and hydrated state. Principal component analysis with selected peaks revealed that syntrophic Geobacter aggregates were well differentiated from other control samples, including syntrophic planktonic cells, pure cultured planktonic cells, and single population biofilms. Our in vivo imaging indicated that a unique molecular surface was formed. Specifically, aromatic amino acids, phosphatidylethanolamine components, and large water clusters were identified as key components that favored the DIET of syntrophic Geobacter aggregates. Moreover, the molecular changes in depths of the Geobacter aggregates were captured using dynamic depth profiling. Our findings shed new light on the interface components supporting electron transfer in syntrophic communities based on in vivo molecular imaging.

Determining incremental coulombic efficiency and physiological parameters of early stage Geobacter spp. enrichment biofilms.

Geobacter spp. enrichment biofilms were cultivated in batch using one-chamber and two-chamber bioelectrochemical reactors. Time-resolved substrate quantification was performed to derive physiological parameters as well as incremental coulombic efficiency (i.e., coulombic efficiency during one batch cycle, here every 6h) during early stage biofilm development. The results of one-chamber reactors revealed an intermediate acetate increase putatively due to the presence of acetogens. Total coulombic efficiencies of two-chamber reactors were considerable lower (19.6±8.3% and 49.3±13.2% for 1st and 2nd batch cycle, respectively) compared to usually reported values of mature Geobacter spp. enrichment biofilms presumably reflecting energetic requirements for biomass production (i.e., cells and extracellular polymeric substances) during early stages of biofilm development. The incremental coulombic efficiency exhibits considerable changes during batch cycles indicating shifts between phases of maximizing metabolic rates and maximizing biomass yield. Analysis based on Michaelis-Menten kinetics yielded maximum substrate uptake rates (vmax,Ac, vmax,I) and half-saturation concentration coefficients (KM,Ac,KM,I) based on acetate uptake or current production, respectively. The latter is usually reported in literature but neglects energy demands for biofilm growth and maintenance as well as acetate and electron storage. From 1st to 2nd batch cycle, vmax,Ac and KM,Ac, decreased from 0.0042-0.0051 mmol Ac- h-1 cm-2 to 0.0031-0.0037 mmol Ac- h-1 cm-2 and 1.02-2.61 mM Ac- to 0.28-0.42 mM Ac-, respectively. Furthermore, differences between KM,Ac/KM,I and vmax,Ac/vmax,I were observed providing insights into the physiology of Geobacter spp. enrichment biofilms. Notably, KM,I considerably scattered while vmax,Ac/vmax,I and KM,Ac remained rather stable indicating that acetate transport within biofilm only marginally affects reaction rates. The observed data variation mandates the requirement of a more detailed analysis with an improved experimental system, e.g., using flow conditions and a comparison with Geobacter spp. pure cultures.

Stratified microbial structure and activity within anode biofilm during electrochemically assisted brewery wastewater treatment.

In a bioelectrochemical system (BES), microbial community of anode biofilm is crucial to BES performance. In this study, the stratified pattern of community structure and activity of an anode-respiring biofilm in a BES fueled with brewery wastewater was investigated over time. The anode biofilm exhibited a superior performance in the removal of ethanol to that of an open-circuit system. The electrical current density reached a high level of 0.55mA/cm2 with a Coulombic efficiency of 71.4%, but decreased to 0.18mA/cm2 in the late stage of operation. A mature biofilm developed a more active outer layer covering a less active inner core, although the activities of the outer and inner layers of biofilm were similar in the early stage. More Geobacter spp., typical exoelectrogens, were enriched in the outer layer than in the inner layer of biofilm in the early stage, while more Geobacter spp. were distributed in the inner layer than in the outer layer in the late stage. The inactive and Geobacter-occupied inner layer of biofilm might be responsible for the decreased electricity generation from wastewater in the late stage of operation. This study provides better understanding of the effect of anode biofilm structure on BES performance.

Disparity of Cytochrome Utilization in Anodic and Cathodic Extracellular Electron Transfer Pathways of Geobacter sulfurreducens Biofilms.

Extracellular electron transfer (EET) in microorganisms is prevalent in nature and has been utilized in functional bioelectrochemical systems. EET of Geobacter sulfurreducens has been extensively studied and has been revealed to be facilitated through c-type cytochromes, which mediate charge between the electrode and G. sulfurreducens in anodic mode. However, the EET pathway of cathodic conversion of fumarate to succinate is still under debate. Here, we apply a variety of analytical methods, including electrochemistry, UV-vis absorption and resonance Raman spectroscopy, quartz crystal microbalance with dissipation, and electron microscopy, to understand the involvement of cytochromes and other possible electron-mediating species in the switching between anodic and cathodic reaction modes. By switching the applied bias for a G. sulfurreducens biofilm coupled to investigating the quantity and function of cytochromes, as well as the emergence of Fe-containing particles on the cell membrane, we provide evidence of a diminished role of cytochromes in cathodic EET. This work sheds light on the mechanisms of G. sulfurreducens biofilm growth and suggests the possible existence of a nonheme, iron-involving EET process in cathodic mode.

Molecular evidence for the adaptive evolution of Geobacter sulfurreducens to perform dissimilatory iron reduction in natural environments.

The electrically conductive pili (e-pili) of Geobacter species enable extracellular electron transfer to insoluble metallic minerals, electrodes and other microbial species, which confers biogeochemical significance and global prevalence on Geobacter in diverse anaerobic environments. E-pili are constructed by truncated PilA which is considered to have evolved from full-length pilin by gene fission under positive evolutionary selection. However, this hypothesis is based on phylogenetic analysis and has not yet been experimentally confirmed. Here, we reconstructed an ancestral strain of G. sulfurreducens (designated COMB) carrying full-length PilA by combining genes GSU1496 and GSU1497. The results demonstrated that strain COMB expressed and assembled the full-length fused PilA and exhibited an outer membrane c-type cytochrome profile similar to the wild-type strain. Surprisingly, the generated COMB-pili were also conductive, indicating the evolution of truncated PilA did not occur for conductivity. Moreover, strain COMB minimally reduced Fe(III) oxides but maintained its ability to respire electrodes, demonstrating the truncation of pilin enables iron respiration. This study provides the first experimental evidence that the truncation of pilin in Geobacter species confers adaption to Fe(III)-mineral-mediated selective pressures, and suggests an evolutionary event during which the separation of the GSU1497 gene helped Geobacter survive and thrive in natural environments.