Lack of physiological evidence for cytochrome filaments functioning as conduits for extracellular electron transfer.

Extracellular cytochrome filaments are proposed to serve as conduits for long-range extracellular electron transfer. The primary functional physiological evidence has been the reported inhibition of Geobacter sulfurreducens Fe(III) oxide reduction when the gene for the filament-forming cytochrome OmcS is deleted. Here we report that the OmcS-deficient strain from that original report reduces Fe(III) oxide as well as the wild-type, as does a triple mutant in which the genes for the other known filament-forming cytochromes were also deleted. The triple cytochrome mutant displayed filaments with the same 3 nm diameter morphology and conductance as those produced by Escherichia coli heterologously expressing the G. sulfurreducens PilA pilin gene. Fe(III) oxide reduction was inhibited when the pilin gene in cytochrome-deficient mutants was modified to yield poorly conductive 3 nm diameter filaments. The results are consistent with the concept that 3 nm diameter electrically conductive pili (e-pili) are required for G. sulfurreducens long-range extracellular electron transfer. In contrast, rigorous physiological functional evidence is lacking for cytochrome filaments serving as conduits for long-range electron transport. IMPORTANCE: Unraveling microbial extracellular electron transfer mechanisms has profound implications for environmental processes and advancing biological applications. This study on Geobacter sulfurreducens challenges prevailing beliefs on cytochrome filaments as crucial components thought to facilitate long-range electron transport. The discovery of an OmcS-deficient strain's unexpected effectiveness in Fe(III) oxide reduction prompted a reevaluation of the key conduits for extracellular electron transfer. By exploring the impact of genetic modifications on G. sulfurreducens' performance, this research sheds light on the importance of 3-nm diameter electrically conductive pili in Fe(III) oxide reduction. Reassessing these mechanisms is essential for uncovering the true drivers of extracellular electron transfer in microbial systems, offering insights that could revolutionize applications across diverse fields.

Bioproduction of cerium-bearing magnetite and application to improve carbon-black supported platinum catalysts.

BACKGROUND: Biogeochemical processing of metals including the fabrication of novel nanomaterials from metal contaminated waste streams by microbial cells is an area of intense interest in the environmental sciences. RESULTS: Here we focus on the fate of Ce during the microbial reduction of a suite of Ce-bearing ferrihydrites with between 0.2 and 4.2 mol% Ce. Cerium K-edge X-ray absorption near edge structure (XANES) analyses showed that trivalent and tetravalent cerium co-existed, with a higher proportion of tetravalent cerium observed with increasing Ce-bearing of the ferrihydrite. The subsurface metal-reducing bacterium Geobacter sulfurreducens was used to bioreduce Ce-bearing ferrihydrite, and with 0.2 mol% and 0.5 mol% Ce, an Fe(II)-bearing mineral, magnetite (Fe(II)(III)2O4), formed alongside a small amount of goethite (FeOOH). At higher Ce-doping (1.4 mol% and 4.2 mol%) Fe(III) bioreduction was inhibited and goethite dominated the final products. During microbial Fe(III) reduction Ce was not released to solution, suggesting Ce remained associated with the Fe minerals during redox cycling, even at high Ce loadings. In addition, Fe L2,3 X-ray magnetic circular dichroism (XMCD) analyses suggested that Ce partially incorporated into the Fe(III) crystallographic sites in the magnetite. The use of Ce-bearing biomagnetite prepared in this study was tested for hydrogen fuel cell catalyst applications. Platinum/carbon black electrodes were fabricated, containing 10% biomagnetite with 0.2 mol% Ce in the catalyst. The addition of bioreduced Ce-magnetite improved the electrode durability when compared to a normal Pt/CB catalyst. CONCLUSION: Different concentrations of Ce can inhibit the bioreduction of Fe(III) minerals, resulting in the formation of different bioreduction products. Bioprocessing of Fe-minerals to form Ce-containing magnetite (potentially from waste sources) offers a sustainable route to the production of fuel cell catalysts with improved performance.

Removal and reduction mechanism of Cr (VI) in Leersia hexandra Swartz constructed wetland-microbial fuel cell coupling system.

Cr (VI) is extremely harmful to both the environment and human health, and it can linger in the environment for a very long period. In this research, the Leersia hexandra Swartz constructed wetland-microbial fuel cell (CW-MFC) system was constructed to purify Cr (VI) wastewater. By comparing with the constructed wetland (CW) system, the system electricity generation, pollutants removal, Cr enrichment, and morphological transformation of the system were discussed. The results demonstrated that the L. hexandra CW-MFC system promoted removal of pollutants and production of electricity of the system. The maximum voltage of the system was 499 mV, the COD and Cr (VI) removal efficiency was 93.73% and 97.00%. At the same time, it enhanced the substrate and L. hexandra ability to absorb Cr and change it morphologically transformation. Additionally, the results of XPS and XANES showed that the majority of the Cr in the L. hexandra and substrate was present as Cr (III). In the L. hexandra CW-MFC system, Geobacter also functioned as the primary metal catabolic reducing and electrogenic bacteria. As a result, L. hexandra CW-MFC system possesses the added benefit of removing Cr (VI) while producing energy compared to the traditional CW system.

Widespread extracellular electron transfer pathways for charging microbial cytochrome OmcS nanowires via periplasmic cytochromes PpcABCDE.

Extracellular electron transfer (EET) via microbial nanowires drives globally-important environmental processes and biotechnological applications for bioenergy, bioremediation, and bioelectronics. Due to highly-redundant and complex EET pathways, it is unclear how microbes wire electrons rapidly (>106 s-1) from the inner-membrane through outer-surface nanowires directly to an external environment despite a crowded periplasm and slow (<105 s-1) electron diffusion among periplasmic cytochromes. Here, we show that Geobacter sulfurreducens periplasmic cytochromes PpcABCDE inject electrons directly into OmcS nanowires by binding transiently with differing efficiencies, with the least-abundant cytochrome (PpcC) showing the highest efficiency. Remarkably, this defined nanowire-charging pathway is evolutionarily conserved in phylogenetically-diverse bacteria capable of EET. OmcS heme reduction potentials are within 200 mV of each other, with a midpoint 82 mV-higher than reported previously. This could explain efficient EET over micrometres at ultrafast (<200 fs) rates with negligible energy loss. Engineering this minimal nanowire-charging pathway may yield microbial chassis with improved performance.

Identification of bacterial dissimilatory antimonate reductase AnrA: genes and proteins involved in antimonate respiration and resistance in Geobacter sp. strain SVR.

Geobacter sp. strain SVR uses antimonate [Sb(V)] as a terminal electron acceptor for anaerobic respiration. Here, we visualized a possible key enzyme, periplasmic Sb(V) reductase (Anr), via active staining and non-denaturing gel electrophoresis. Liquid chromatography-tandem mass spectrometry analysis revealed that a novel dimethyl sulfoxide (DMSO) reductase family protein, WP_173201954.1, is involved in Anr. This protein was closely related with AnrA, a protein suggested to be the catalytic subunit of a respiratory Sb(V) reductase in Desulfuribacillus stibiiarsenatis. The anr genes of strain SVR (anrXSRBAD) formed an operon-like structure, and their transcription was upregulated under Sb(V)-respiring conditions. The expression of anrA gene was induced by more than 1 µM of antimonite [Sb(III)]; however, arsenite [As(III)] did not induce the expression of anrA gene. Tandem mass tag-based proteomic analysis revealed that, in addition to Anr proteins, proteins in the following categories were upregulated under Sb(V)-respiring conditions: (i) Sb(III) efflux systems such as Ant and Ars; (ii) antioxidizing proteins such as ferritin, rubredoxin, and thioredoxin; (iii) protein quality control systems such as HspA, HslO, and DnaK; and (iv) DNA repair proteins such as UspA and UvrB. These results suggest that strain SVR copes with antimony stress by modulating pleiotropic processes to resist and actively metabolize antimony. To the best of our knowledge, this is the first report to demonstrate the involvement of AnrA in Sb(V) respiration at the protein level. Furthermore, this is the first example to show high expression of the Ant system proteins in the Sb(V)-respiring bacterium.IMPORTANCEAntimony (Sb) exists mainly as antimonite [Sb(III)] or antimonate [Sb(V)] in the environment, and Sb(III) is more toxic than Sb(V). Recently, microbial involvement in Sb redox reactions has received attention. Although more than 90 Sb(III)-oxidizing bacteria have been reported, information on Sb(V)-reducing bacteria is limited. Especially, the enzyme involved in dissimilatory Sb(V) reduction, or Sb(V) respiration, is unclear, despite this pathway being very important for the circulation of Sb in nature. In this study, we demonstrated that the Sb(V) reductase (Anr) of an Sb(V)-respiring bacterium (Geobacter sp. SVR) is a novel member of the dimethyl sulfoxide (DMSO) reductase family. In addition, we found that strain SVR copes with Sb stress by modulating pleiotropic processes, including the Ant and Ars systems, and upregulating the antioxidant and quality control protein levels. Considering the abundance and diversity of putative anr genes in the environment, Anr may play a significant role in global Sb cycling in both marine and terrestrial environments.

Electron syntrophy between mixed hydrogenogens and Geobacter metallireducens boosted dark hydrogen fermentation: Clarifying roles of electroactive extracellular polymeric substances.

To modulate the electron transfer behavior of hydrogen-producing bacteria (HPB) for enhanced hydrogen production, Geobacter metallireducens culture (GM) was introduced as an electron syntrophy partner and redox balance regulator in dark fermentation systems with hydrogen-producing sludge (HPS) as inoculum. The highest hydrogen yield was 306.5 mL/g-COD at the GM/HPS volatile solids ratio of 0.08, which was 65.2 % higher than the HPS group. The multi-layered extracellular polymeric substances (EPS) of GM played a significant role in promoting hydrogen production, with c-type cytochromes probably serving as electroactive functional components. The addition of GM significantly improved the NADH/NAD+ ratio, electron transport system activity, hydrogenase activity, and electrochemical properties of HPS. Furthermore, the microbial community structure and metabolic functions were optimized due to the potential syntrophic interaction between Clostridium sensu stricto (dominant HPB) and Geobacter, thus promoting hydrogen production. This study provided novel insights into the interactions among exoelectrogens, electroactive EPS, and mixed HPB.

Characterization of the inner membrane cytochrome ImcH from Geobacter reveals its importance for extracellular electron transfer and energy conservation.

Electroactive bacteria combine the oxidation of carbon substrates with an extracellular electron transfer (EET) process that discharges electrons to an electron acceptor outside the cell. This process involves electron transfer through consecutive redox proteins that efficiently connect the inner membrane to the cell exterior. In this study, we isolated and characterized the quinone-interacting membrane cytochrome c ImcH from Geobacter sulfurreducens, which is involved in the EET process to high redox potential acceptors. Spectroscopic and electrochemical studies show that ImcH hemes have low midpoint redox potentials, ranging from -150 to -358 mV, and connect the oxidation of the quinol-pool to EET, transferring electrons to the highly abundant periplasmic cytochrome PpcA with higher affinity than to its homologues. Despite the larger number of hemes and transmembrane helices, the ImcH structural model has similarities with the NapC/NirT/NrfH superfamily, namely the presence of a quinone-binding site on the P-side of the membrane. In addition, the first heme, likely involved on the quinol oxidation, has apparently an unusual His/Gln coordination. Our work suggests that ImcH is electroneutral and transfers electrons and protons to the same side of the membrane, contributing to the maintenance of a proton motive force and playing a central role in recycling the menaquinone pool.

Global transcriptional analysis of Geobacter sulfurreducens gsu1771 mutant biofilm grown on two different support structures.

Electroactive biofilms formation by the metal-reducing bacterium Geobacter sulfurreducens is a step crucial for bioelectricity generation and bioremediation. The transcriptional regulator GSU1771 controls the expression of essential genes involved in electron transfer and biofilm formation in G. sulfurreducens, with GSU1771-deficient producing thicker and more electroactive biofilms. Here, RNA-seq analyses were conducted to compare the global gene expression patterns of wild-type and Δgsu1771 mutant biofilms grown on non-conductive (glass) and conductive (graphite electrode) materials. The Δgsu1771 biofilm grown on the glass surface exhibited 467 differentially expressed (DE) genes (167 upregulated and 300 downregulated) versus the wild-type biofilm. In contrast, the Δgsu1771 biofilm grown on the graphite electrode exhibited 119 DE genes (79 upregulated and 40 downregulated) versus the wild-type biofilm. Among these DE genes, 67 were also differentially expressed in the Δgsu1771 biofilm grown on glass (56 with the same regulation and 11 exhibiting counter-regulation). Among the upregulated genes in the Δgsu1771 biofilms, we identified potential target genes involved in exopolysaccharide synthesis (gsu1961-63, gsu1959, gsu1972-73, gsu1976-77). RT-qPCR analyses were then conducted to confirm the differential expression of a selection of genes of interest. DNA-protein binding assays demonstrated the direct binding of the GSU1771 regulator to the promoter region of pgcA, pulF, relA, and gsu3356. Furthermore, heme-staining and western blotting revealed an increase in c-type cytochromes including OmcS and OmcZ in Δgsu1771 biofilms. Collectively, our findings demonstrated that GSU1771 is a global regulator that controls extracellular electron transfer and exopolysaccharide synthesis in G. sulfurreducens, which is crucial for electroconductive biofilm development.

Acclimation of electroactive biofilms under different operating conditions: comprehensive analysis from architecture, composition, and metabolic activity.

Electroactive biofilms (EABs) have aroused wide concern in waste treatment due to their unique capability of extracellular electron transfer with solid materials. The combined effect of different operating conditions on the formation, microbial architecture, composition, and metabolic activity of EABs is still unknown. In this study, the impact of three different factors (anode electrode, substrate concentration, and resistance) on the acclimation and performance of EABs was investigated. The results showed that the shortest start-up time of 127.3 h and highest power density of 0.84 W m-2 were obtained with carbon brush as electrode, low concentration of substrate (1.0 g L-1), and 1000 Ω external resistance (denoted as N1). The EABs under N1 condition also represented strongest redox capacity, lowest internal resistance, and close arrangement of bacteria. Moreover, the EABs cultured under different conditions both showed similar results, with direct electron transfer (DET) dominated from EABs to anode. Microbial community compositions indicated that EABs under N1 condition have lowest diversity and highest abundance of electroactive bacteria (46.68%). Higher substrate concentration (3.0 g L-1) promoted the proliferation of some other bacteria without electroactivity, which was adverse to EABs. The metabolic analysis showed the difference of genes related to electron transfer (cytochrome C and pili) and biofilm formation (xap) of EABs under different conditions, which further demonstrated the higher electroactivity of EABs under N1. These results provided a comprehensive understanding of the effect of different operating conditions on EABs including biofilm formation and electrochemical activity.

Accelerated Microbial Corrosion by Magnetite and Electrically Conductive Pili through Direct Fe0 -to-Microbe Electron Transfer.

Electrobiocorrosion, the process in which microbes extract electrons from metallic iron (Fe0 ) through direct Fe0 -microbe electrical connections, is thought to contribute to the costly corrosion of iron-containing metals that impacts many industries. However, electrobiocorrosion mechanisms are poorly understood. We report here that electrically conductive pili (e-pili) and the conductive mineral magnetite play an important role in the electron transfer between Fe0 and Geobacter sulfurreducens, the first microbe in which electrobiocorrosion has been rigorously documented. Genetic modification to express poorly conductive pili substantially diminished corrosive pitting and rates of Fe0 -to-microbe electron flux. Magnetite reduced resistance to electron transfer, increasing corrosion currents and intensifying pitting. Studies with mutants suggested that the magnetite promoted electron transfer in a manner similar to the outer-surface c-type cytochrome OmcS. These findings, and the fact that magnetite is a common product of iron corrosion, suggest a potential positive feedback loop of magnetite produced during corrosion further accelerating electrobiocorrosion. The interactions of e-pili, cytochromes, and magnetite demonstrate mechanistic complexities of electrobiocorrosion, but also provide insights into detecting and possibly mitigating this economically damaging process.

Structural Determinants of Redox Conduction Favor Robustness over Tunability in Microbial Cytochrome Nanowires.

Structural determinants of a 103-fold variation in electrical conductivity for helical homopolymers of tetra-, hexa-, and octa-heme cytochromes (named Omc- E, S, and Z, respectively) from Geobacter sulfurreducens are investigated with the Pathways model for electron tunneling, classical molecular dynamics, and hybrid quantum/classical molecular mechanics. Thermally averaged electronic couplings for through-space heme-to-heme electron transfer in the "nanowires" computed with density functional theory are ≤0.015 eV. Pathways analyses also indicate that couplings match within a factor of 5 for all "nanowires", but some alternative tunneling routes are found involving covalent protein backbone bonds (Omc- S and Z) or propionic acid-ligating His H-bonds on adjacent hemes (OmcZ). Reorganization energies computed from electrostatic vertical energy gaps or a version of the Marcus continuum expression parameterized on the total (donor + acceptor) solvent-accessible surface area typically agree within 20% and fall within the range 0.48-0.98 eV. Reaction free energies in all three "nanowires" are ≤|0.28| eV, even though Coulombic interactions primarily tune the site redox energies by 0.7-1.2 eV. Given the conserved energetic parameters, redox conductivity differs by < 103-fold among the cytochrome "nanowires". Redox currents do not exceed 3.0 × 10-3 pA at a physiologically relevant 0.1 V bias, with the slowest electron transfers being on a (µs) timescale much faster than typical (ms) enzymatic turnovers. Thus, the "nanowires" are proposed to be functionally robust to variations in structure that provide a habitat-customized protein interface. The 30 pA to 30 nA variation in conductivity previously reported from atomic force microscopy experiments is not intrinsic to the structures and/or does not result from the physiologically relevant redox conduction mechanism.

Biochar establishing syntrophic partnership between exoelectrogens to facilitate extracellular electron transfer.

Biochar was regarded as a promising accelerator for extracellular electron transfer (EET), while the mechanism of biochar facilitating electricity harvest in bioelectrochemical system (BES) was in debates. In this study, sawdust-based biochar with low conductivity but strong redox-based electron exchange capacity was added into BES with two forms, including a suspended form (S-BC) added in anode chamber and a fixed form closely wrapping up the anode (F-BC). Compared with the control group, S-BC and F-BC addition dramatically increased accumulated electricity output by 2.0 and 5.1 times. However, electrochemical analysis characterized the lowest electrochemical property on anode surface in F-BC modified group. A 2nd period conducted by separating F-BC modified group with "aged F-BC + new anode" group and "single aged anode" group demonstrated that F-BC contributed >95 % to the current generation of F-BC modified group, while the anode almost acted as a conductor to transfer the generated electrons to cathode. Microbial community analysis revealed that both heterotrophic and autotrophic exoelectrogens contributed to current generation. The presence of biochar upregulated functional genes encoding cytochrome-c and type IV pilus, thereby boosting electricity harvest efficiency. Interestingly, the heterotrophic exoelectrogens of Geobacter/Desulfovibrio tended to attach on fixed surfaces of both biochar and anode, and the autotrophic exelectrogen of Hydrogenophaga was selectively enriched on biochar surfaces whatever fixed or suspended form. Consequently, a syntrophic partnership between Geobacter/Desulfovibrio and Hydrogenophaga was potentially establishment on F-BC surface for highly-efficient electricity harvest. In this syntrophic EET model, biochar potentially acted as the redox-active mediator, which temporarily accepted electron released by Geobacter/Desulfovibrio via acetate oxidation, and then donated them to Hydrogenophaga attached on biochar surfaces for autotrophic EET. This was distinct from a regular EET conducted by heterotrophic exoelectrogens. These findings provided new insights to understand the mechanisms of biochar facilitating EET by syntrophic metabolism pathway.

Comparing theoretical and practical biomass yields calls for revisiting thermodynamic growth models for electroactive microorganisms.

Research on electroactive microorganisms (EAM) often focuses either on their physiology and the underlying mechanisms of extracellular electron transfer or on their application in microbial electrochemical technologies (MET). Thermodynamic understanding of energy conversions related to growth and activity of EAM has received only a little attention. In this study, we aimed to prove the hypothesized restricted energy harvest of EAM by determining biomass yields by monitoring growth of acetate-fed biofilms presumably enriched in Geobacter, using optical coherence tomography, at three anode potentials and four acetate concentrations. Experiments were concurrently simulated using a refined thermodynamic model for EAM. Neither clear correlations were observed between biomass yield and anode potential nor acetate concentration, albeit the statistical significances are limited, mainly due to the observed experimental variances. The experimental biomass yield based on acetate consumption (YX/ac = 37 ± 9 mgCODbiomass gCODac-1) was higher than estimated by modeling, indicating limitations of existing growth models to predict yields of EAM. In contrast, the modeled biomass yield based on catabolic energy harvest was higher than the biomass yield from experimental data (YX/cat = 25.9 ± 6.8 mgCODbiomass kJ-1), supporting restricted energy harvest of EAM and indicating a role of not considered energy sinks. This calls for an adjusted growth model for EAM, including, e.g., the microbial electrochemical Peltier heat to improve the understanding and modeling of their energy metabolism. Furthermore, the reported biomass yields are important parameters to design strategies for influencing the interactions between EAM and other microorganisms and allowing more realistic feasibility assessments of MET.

Methane production by Methanothrix thermoacetophila via direct interspecies electron transfer with Geobacter metallireducens.

Methanothrix is widely distributed in natural and artificial anoxic environments and plays a major role in global methane emissions. It is one of only two genera that can form methane from acetate dismutation and through participation in direct interspecies electron transfer (DIET) with exoelectrogens. Although Methanothrix is a significant member of many methanogenic communities, little is known about its physiology. In this study, transcriptomics helped to identify potential routes of electron transfer during DIET between Geobacter metallireducens and Methanothrix thermoacetophila. Additions of magnetite to cultures significantly enhanced growth by acetoclastic methanogenesis and by DIET, while granular activated carbon (GAC) amendments impaired growth. Transcriptomics suggested that the OmaF-OmbF-OmcF porin complex and the octaheme outer membrane c-type cytochrome encoded by Gmet_0930, were important for electron transport across the outer membrane of G. metallireducens during DIET with Mx. thermoacetophila. Clear differences in the metabolism of Mx. thermoacetophila when grown via DIET or acetate dismutation were not apparent. However, genes coding for proteins involved in carbon fixation, the sheath fiber protein MspA, and a surface-associated quinoprotein, SqpA, were highly expressed in all conditions. Expression of gas vesicle genes was significantly lower in DIET- than acetate-grown cells, possibly to facilitate better contact between membrane-associated redox proteins during DIET. These studies reveal potential electron transfer mechanisms utilized by both Geobacter and Methanothrix during DIET and provide important insights into the physiology of Methanothrix in anoxic environments. IMPORTANCE Methanothrix is a significant methane producer in a variety of methanogenic environments including soils and sediments as well as anaerobic digesters. Its abundance in these anoxic environments has mostly been attributed to its high affinity for acetate and its ability to grow by acetoclastic methanogenesis. However, Methanothrix species can also generate methane by directly accepting electrons from exoelectrogenic bacteria through direct interspecies electron transfer (DIET). Methane production through DIET is likely to further increase their contribution to methane production in natural and artificial environments. Therefore, acquiring a better understanding of DIET with Methanothrix will help shed light on ways to (i) minimize microbial methane production in natural terrestrial environments and (ii) maximize biogas formation by anaerobic digesters treating waste.

Mechanisms of magnetic sensing and regulating extracellular electron transfer of electroactive bacteria under magnetic fields.

Electroactive bacteria can display notable plasticity in their response to magnetic field (MF), which prompted bioelectrochemical system as promising candidates for magnetic sensor applications. In this study, we explored the sensing and stimulatory effect of MF on current generation by Geobacter sulfurreducens, and elucidated the related molecular mechanism at the transcriptomic level. MF treatment significantly enhanced electricity generation and overall energy efficiency of G. sulfurreducens by 50 % and 22 %, respectively. The response of current to MFs was instantaneous and reversible. Cyclic voltammetry analysis of the anode biofilm revealed that the redox couples changed from -0.31 to -0.39 V (vs. Ag/AgCl), suggesting that MFs could alter electron transfer related components. Differential gene expression analysis further verified this hypothesis, genes associated with electron transfer were upregulated in G. sulfurreducens under MF treatment relative to the control group, specifically, genes encoding periplasmic c-type cytochromes (ppcA and ppcD), outer membrane cytochrome (omcF, omcZ, omcB), pili (pilA-C, pilM, and pilV2), and ribosome. The enhanced bacterial extracellular electron transfer process was also linked to the overexpression of the NADH dehydrogenase I subunit, the ABC transporter, transcriptional regulation, and ATP synthase. Overall, our findings shed light on the molecular mechanism underlying the effects of magnetic field stimuli on EAB and provide a theoretical basis for its further application in magnetic sensors and other biological system.

Intracytoplasmic membranes develop in Geobacter sulfurreducens under thermodynamically limiting conditions.

Geobacter sulfurreducens is an electroactive bacterium capable of reducing metal oxides in the environment and electrodes in engineered systems1,2. Geobacter sp. are the keystone organisms in electrogenic biofilms, as their respiration consumes fermentation products produced by other organisms and reduces a terminal electron acceptor e.g. iron oxide or an electrode. To respire extracellular electron acceptors with a wide range of redox potentials, G. sulfurreducens has a complex network of respiratory proteins, many of which are membrane-bound3-5. We have identified intracytoplasmic membrane (ICM) structures in G. sulfurreducens. This ICM is an invagination of the inner membrane that has folded and organized by an unknown mechanism, often but not always located near the tip of a cell. Using confocal microscopy, we can identify that at least half of the cells contain an ICM when grown on low potential anode surfaces, whereas cells grown at higher potential anode surfaces or using fumarate as electron acceptor had significantly lower ICM frequency. 3D models developed from cryo-electron tomograms show the ICM to be a continuous extension of the inner membrane in contact with the cytoplasmic and periplasmic space. The differential abundance of ICM in cells grown under different thermodynamic conditions supports the hypothesis that it is an adaptation to limited energy availability, as an increase in membrane-bound respiratory proteins could increase electron flux. Thus, the ICM provides extra inner-membrane surface to increase the abundance of these proteins. G. sulfurreducens is the first Thermodesulfobacterium or metal-oxide reducer found to produce ICMs.

A Biochemical Deconstruction-Based Strategy to Assist the Characterization of Bacterial Electric Conductive Filaments.

Periplasmic nanowires and electric conductive filaments made of the polymeric assembly of c-type cytochromes from Geobacter sulfurreducens bacterium are crucial for electron storage and/or extracellular electron transfer. The elucidation of the redox properties of each heme is fundamental to the understanding of the electron transfer mechanisms in these systems, which first requires the specific assignment of the heme NMR signals. The high number of hemes and the molecular weight of the nanowires dramatically decrease the spectral resolution and make this assignment extremely complex or unattainable. The nanowire cytochrome GSU1996 (~42 kDa) is composed of four domains (A to D) each containing three c-type heme groups. In this work, the individual domains (A to D), bi-domains (AB, CD) and full-length nanowire were separately produced at natural abundance. Sufficient protein expression was obtained for domains C (~11 kDa/three hemes) and D (~10 kDa/three hemes), as well as for bi-domain CD (~21 kDa/six hemes). Using 2D-NMR experiments, the assignment of the heme proton NMR signals for domains C and D was obtained and then used to guide the assignment of the corresponding signals in the hexaheme bi-domain CD. This new biochemical deconstruction-based procedure, using nanowire GSU1996 as a model, establishes a new strategy to functionally characterize large multiheme cytochromes.

The Combined Effect of Hg(II) Speciation, Thiol Metabolism, and Cell Physiology on Methylmercury Formation by Geobacter sulfurreducens.

The chemical and biological factors controlling microbial formation of methylmercury (MeHg) are widely studied separately, but the combined effects of these factors are largely unknown. We examined how the chemical speciation of divalent, inorganic mercury (Hg(II)), as controlled by low-molecular-mass thiols, and cell physiology govern MeHg formation by Geobacter sulfurreducens. We compared MeHg formation with and without addition of exogenous cysteine (Cys) to experimental assays with varying nutrient and bacterial metabolite concentrations. Cysteine additions initially (0-2 h) enhanced MeHg formation by two mechanisms: (i) altering the Hg(II) partitioning from the cellular to the dissolved phase and/or (ii) shifting the chemical speciation of dissolved Hg(II) in favor of the Hg(Cys)2 complex. Nutrient additions increased MeHg formation by enhancing cell metabolism. These two effects were, however, not additive since cysteine was largely metabolized to penicillamine (PEN) over time at a rate that increased with nutrient addition. These processes shifted the speciation of dissolved Hg(II) from complexes with relatively high availability, Hg(Cys)2, to complexes with lower availability, Hg(PEN)2, for methylation. This thiol conversion by the cells thereby contributed to stalled MeHg formation after 2-6 h Hg(II) exposure. Overall, our results showed a complex influence of thiol metabolism on microbial MeHg formation and suggest that the conversion of cysteine to penicillamine may partly suppress MeHg formation in cysteine-rich environments like natural biofilms.

Nitrogen Fixation and Ammonium Assimilation Pathway Expression of Geobacter sulfurreducens Changes in Response to the Anode Potential in Microbial Electrochemical Cells.

Nitrogen gas (N2) fixation in the anode-respiring bacterium Geobacter sulfurreducens occurs through complex, multistep processes. Optimizing ammonium (NH4+) production from this bacterium in microbial electrochemical technologies (METs) requires an understanding of how those processes are regulated in response to electrical driving forces. In this study, we quantified gene expression levels (via RNA sequencing) of G. sulfurreducens growing on anodes fixed at two different potentials (-0.15 V and +0.15 V versus standard hydrogen electrode). The anode potential had a significant impact on the expression levels of N2 fixation genes. At -0.15 V, the expression of nitrogenase genes, such as nifH, nifD, and nifK, significantly increased relative to that at +0.15 V, as well as genes associated with NH4+ uptake and transformation, such as glutamine and glutamate synthetases. Metabolite analysis confirmed that both of these organic compounds were present in significantly higher intracellular concentrations at -0.15 V. N2 fixation rates (estimated using the acetylene reduction assay and normalized to total protein) were significantly larger at -0.15 V. Genes expressing flavin-based electron bifurcation complexes, such as electron-transferring flavoproteins (EtfAB) and the NADH-dependent ferredoxin:NADP reductase (NfnAB), were also significantly upregulated at -0.15 V, suggesting that these mechanisms may be involved in N2 fixation at that potential. Our results show that in energy-constrained situations (i.e., low anode potential), the cells increase per-cell respiration and N2 fixation rates. We hypothesize that at -0.15 V, they increase N2 fixation activity to help maintain redox homeostasis, and they leverage electron bifurcation as a strategy to optimize energy generation and use. IMPORTANCE Biological nitrogen fixation coupled with ammonium recovery provides a sustainable alternative to the carbon-, water-, and energy-intensive Haber-Bosch process. Aerobic biological nitrogen fixation technologies are hindered by oxygen gas inhibition of the nitrogenase enzyme. Electrically driving biological nitrogen fixation in anaerobic microbial electrochemical technologies overcomes this challenge. Using Geobacter sulfurreducens as a model exoelectrogenic diazotroph, we show that the anode potential in microbial electrochemical technologies has a significant impact on nitrogen gas fixation rates, ammonium assimilation pathways, and expression of genes associated with nitrogen gas fixation. These findings have important implications for understanding regulatory pathways of nitrogen gas fixation and will help identify target genes and operational strategies to enhance ammonium production in microbial electrochemical technologies.

Microbial Reduction of Antimony(V)-Bearing Ferrihydrite by Geobacter sulfurreducens.

The reduction of Sb(V)-bearing ferrihydrite by Geobacter sulfurreducens was studied to determine the fate of the metalloid in Fe-rich systems undergoing redox transformations. Sb(V) added at a range of concentrations adsorbed readily to ferrihydrite, and the loadings had a pronounced impact on the rate and extent of Fe(III) reduction and the products formed. Magnetite dominated at low (0.5 and 1 mol%) Sb(V) concentrations, with crystallite sizes decreasing at higher Sb loadings: 37-, 25-, and 17-nm particles for no-Sb, 0.5% Sb, and 1% Sb samples, respectively. In contrast, goethite was the dominant end product for samples with higher antimony loadings (2 and 5 mol%), with increased goethite grain size in the 5% Sb sample. Inductively coupled mass spectrometry (ICP-MS) analysis confirmed that Sb was not released to solution during the bioreduction process, and X-ray photoelectron spectroscopy (XPS) analyses showed that no Sb(III) was formed throughout the experiments, confirming that the Fe(III)-reducing bacterium Geobacter sulfurreducens cannot reduce Sb(V) enzymatically or via biogenic Fe(II). These findings suggest that Fe (bio)minerals have a potential role in limiting antimony pollution in the environment, even when undergoing redox transformations. IMPORTANCE Antimony is an emerging contaminant that shares chemical characteristics with arsenic. Metal-reducing bacteria (such as Geobacter sulfurreducens) can cause the mobilization of arsenic from Fe(III) minerals under anaerobic conditions, causing widespread contamination of aquifers worldwide. This research explores whether metal-reducing bacteria can drive the mobilization of antimony under similar conditions. In this study, we show that G. sulfurreducens cannot reduce Sb(V) directly or cause Sb release during the bioreduction of the Fe(III) mineral ferrihydrite [although the sorbed Sb(V) did alter the Fe(II) mineral end products formed]. Overall, this study highlights the tight associations between Fe and Sb in environmental systems, suggesting that the microbial reduction of Fe(III)/Sb mineral assemblages may not lead to Sb release (in stark contrast to the mobilization of As in iron-rich systems) and offers potential Fe-based remediation options for Sb-contaminated environments.