Performance of a combined electrotrophic and electrogenic biofilm operated under long-term, continuous cycling.

OBJECTIVES: Evaluate electrochemically active biofilms as high energy density rechargeable microbial batteries toward providing persistent power in applications where traditional battery technology is limiting (, remote monitoring applications). RESULTS: Here we demonstrated that an electrochemically active biofilm was able to store and release electrical charge for alternating charge/discharge cycles of up to 24 h periodicity (50% duty cycle) with no significant decrease in average current density (0.16 ± 0.04 A/m2) for over 600 days. However, operation at 24 h periodicity for > 50 days resulted in a sharp decrease in the current to nearly zero. This current crash was recoverable by decreasing the periodicity. Overall, the coulombic efficiency remained near unity within experimental error (102 ± 3%) for all of the tested cycling periods. Electrochemical characterization here suggests that electron transfer occurs through multiple routes, likely a mixture of direct and mediated mechanisms. CONCLUSIONS: These results indicate that bidirectional electrogenic/electrotrophic biofilms are capable of efficient charge storage/release over a wide range of cycling frequency and may eventually enable development of sustainable, high energy density rechargeable batteries.

Pyocyanin-dependent electrochemical inhibition of Pseudomonas aeruginosa biofilms is synergistic with antibiotic treatment.

Pseudomonas aeruginosa biofilms are common in chronic wound infections and recalcitrant to treatment. Survival of cells within oxygen-limited regions in these biofilms is enabled by extracellular electron transfer (EET), whereby small redox active molecules act as electron shuttles to access distal oxidants. Here, we report that electrochemically controlling the redox state of these electron shuttles, specifically pyocyanin (PYO), can impact cell survival within anaerobic P. aeruginosa biofilms and can act synergistically with antibiotic treatment. Prior results demonstrated that under anoxic conditions, an electrode poised at sufficiently oxidizing potential (+100 mV vs Ag/AgCl) promotes EET within P. aeruginosa biofilms by re-oxidizing PYO for reuse by the cells. Here, when a reducing potential (-400 mV vs Ag/AgCl) was used to disrupt PYO redox cycling by maintaining PYO in the reduced state, we observed a 100-fold decrease in colony forming units within these biofilms compared with those exposed to electrodes poised at +100 mV vs Ag/AgCl. Phenazine-deficient Δphz* biofilms were unaffected by the potential applied to the electrode but were re-sensitized by adding PYO. The effect at -400 mV was exacerbated when biofilms were treated with sub-MICs of a range of antibiotics. Most notably, addition of the aminoglycoside gentamicin in a reductive environment almost completely eradicated wild-type biofilms but had no effect on the survival of Δphz* biofilms in the absence of phenazines. These data suggest that antibiotic treatment combined with the electrochemical disruption of PYO redox cycling, either through the toxicity of accumulated reduced PYO or the disruption of EET, or both, can lead to extensive killing. IMPORTANCE Biofilms provide a protective environment but also present challenges to the cells living within them, such as overcoming nutrient and oxygen diffusion limitations. Pseudomonas aeruginosa overcomes oxygen limitation by secreting soluble redox active phenazines, which act as electron shuttles to distal oxygen. Here, we show that electrochemically blocking the re-oxidation of one of these electron shuttles, pyocyanin, decreases cell survival within biofilms and acts synergistically with gentamicin to kill cells. Our results highlight the importance of the role that the redox cycling of electron shuttles fulfills within P. aeruginosa biofilms.

Marine Biofilm Engineered to Produce Current in Response to Small Molecules.

Engineered electroactive bacteria have potential applications ranging from sensing to biosynthesis. In order to advance the use of engineered electroactive bacteria, it is important to demonstrate functional expression of electron transfer modules in chassis adapted to operationally relevant conditions, such as non-freshwater environments. Here, we use the Shewanella oneidensis electron transfer pathway to induce current production in a marine bacterium, Marinobacter atlanticus, during biofilm growth in artificial seawater. Genetically encoded sensors optimized for use in Escherichia coli were used to control protein expression in planktonic and biofilm attached cells. Significant current production required the addition of menaquinone, which M. atlanticus does not produce, for electron transfer from the inner membrane to the expressed electron transfer pathway. Current through the S. oneidensis pathway in M. atlanticus was observed when inducing molecules were present during biofilm formation. Electron transfer was also reversible, indicating that electron transfer into M. atlanticus could be controlled. These results show that an operationally relevant marine bacterium can be genetically engineered for environmental sensing and response using an electrical signal.

Metagenomic and Metatranscriptomic Characterization of a Microbial Community That Catalyzes Both Energy-Generating and Energy-Storing Electrode Reactions.

Electroactive bacteria are living catalysts, mediating energy-generating reactions at anodes or energy storage reactions at cathodes via extracellular electron transfer (EET). The Cathode-ANode (CANode) biofilm community was recently shown to facilitate both reactions; however, the identities of the primary constituents and underlying molecular mechanisms remain unknown. Here, we used metagenomics and metatranscriptomics to characterize the CANode biofilm. We show that a previously uncharacterized member of the family Desulfobulbaceae, Desulfobulbaceae-2, which had <1% relative abundance, had the highest relative gene expression and accounted for over 60% of all differentially expressed genes. At the anode potential, differential expression of genes for a conserved flavin oxidoreductase (Flx) and heterodisulfide reductase (Hdr) known to be involved in ethanol oxidation suggests a source of electrons for the energy-generating reaction. Genes for sulfate and carbon dioxide reduction pathways were expressed by Desulfobulbaceae-2 at both potentials and are the proposed energy storage reactions. Reduction reactions may be mediated by direct electron uptake from the electrode or from hydrogen generated at the cathode potential. The Desulfobulbaceae-2 genome is predicted to encode at least 85 multiheme (≥3 hemes) c-type cytochromes, some with as many as 26 heme-binding domains, that could facilitate reversible electron transfer with the electrode. Gene expression in other CANode biofilm species was also affected by the electrode potential, although to a lesser extent, and we cannot rule out their contribution to observed current. Results provide evidence of gene expression linked to energy storage and energy-generating reactions and will enable development of the CANode biofilm as a microbially driven rechargeable battery. IMPORTANCE Microbial electrochemical technologies (METs) rely on electroactive bacteria to catalyze energy-generating and energy storage reactions at electrodes. Known electroactive bacteria are not equally capable of both reactions, and METs are typically configured to be unidirectional. Here, we report on genomic and transcriptomic characterization of a recently described microbial electrode community called the Cathode-ANode (CANode). The CANode community is able to generate or store electrical current based on the electrode potential. During periods where energy is not needed, electrons generated from a renewable source, such as solar power, could be converted into energy storage compounds to later be reversibly oxidized by the same microbial catalyst. Thus, the CANode system can be thought of as a living "rechargeable battery." Results show that a single organism may be responsible for both reactions demonstrating a new paradigm for electroactive bacteria.

Microbial survival and growth on non-corrodible conductive materials.

Biofilms growing aerobically on conductive substrates are often correlated with a positive, sustained shift in their redox potential. This phenomenon has a beneficial impact on microbial fuel cells by increasing their overall power output but can be detrimental when occurring on stainless steel by enhancing corrosion. The biological mechanism behind this potential shift is unresolved and a metabolic benefit to cells has not been demonstrated. Here, biofilms containing the electroautotroph 'Candidatus Tenderia electrophaga' catalysed a shift in the open circuit potential of graphite and indium tin oxide electrodes by >100 mV. Biofilms on open circuit electrodes had increased biomass and a significantly higher proportion of 'Ca. Tenderia electrophaga' compared to those on plain glass. Addition of metabolic inhibitors showed that living cells were required to maintain the more positive potential. We propose a model to describe these observations, in which 'Ca. Tenderia electrophaga' drives the shift in open circuit potential through electron uptake for oxygen reduction and CO2 fixation. We further propose that the electrode is continuously recharged by oxidation of trace redox-active molecules in the medium at the more positive potential. A similar phenomenon is possible on natural conductive substrates in the environment.

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.

Activation of Protein Expression in Electroactive Biofilms.

Microbes that form biofilms on electrodes and generate electrical current responses could be integrated into devices to perform sensing, conduct signals, or act as living microprocessors. A challenge in working with these species is the ability to visualize biofilm formation and protein expression in real-time while also measuring current, which is not possible with typical bio-electrochemical reactors. Here, we present a three-dimensional-printed flow cell for simultaneous electrochemistry and fluorescence imaging. Current-producing biofilms of Marinobacter atlanticus constitutively expressing green fluorescent protein were grown on the flow cell working electrode. Increasing current corresponded with increasing surface coverage and was comparable to biofilms grown in typical stirred-batch reactors. An isopropyl ß-d-1-thiogalactopyranoside (IPTG) inducible system driving yellow fluorescent protein was used to assess the spatiotemporal activation of protein expression within the biofilm at different stages of growth and induction dynamics. The response time ranged from 30 min to 5 h, depending on the conditions. These data demonstrate that the electrochemical flow cell can evaluate the performance of an electrically active environmental bacterium under conditions relevant for development as a living electronic sensor.

Extracellular DNA Promotes Efficient Extracellular Electron Transfer by Pyocyanin in Pseudomonas aeruginosa Biofilms.

Redox cycling of extracellular electron shuttles can enable the metabolic activity of subpopulations within multicellular bacterial biofilms that lack direct access to electron acceptors or donors. How these shuttles catalyze extracellular electron transfer (EET) within biofilms without being lost to the environment has been a long-standing question. Here, we show that phenazines mediate efficient EET through interactions with extracellular DNA (eDNA) in Pseudomonas aeruginosa biofilms. Retention of pyocyanin (PYO) and phenazine carboxamide in the biofilm matrix is facilitated by eDNA binding. In vitro, different phenazines can exchange electrons in the presence or absence of DNA and can participate directly in redox reactions through DNA. In vivo, biofilm eDNA can also support rapid electron transfer between redox active intercalators. Together, these results establish that PYO:eDNA interactions support an efficient redox cycle with rapid EET that is faster than the rate of PYO loss from the biofilm.

Multiheme Cytochrome Mediated Redox Conduction through Shewanella oneidensis MR-1 Cells.

Multiheme cytochromes function as extracellular electron transfer (EET) conduits that extend the metabolic reach of microorganisms to external solid surfaces. These conduits are also proposed to facilitate long-distance electron transport along cellular membranes and across multiple cells. Here we report electrochemical gating measurements of Shewanella oneidensis MR-1 cells linking interdigitated electrodes. The dependence of the source-drain current on gate potential demonstrates a redox conduction mechanism, which we link to the presence of multiheme cytochromes of the Mtr pathway. We also find that the measured thermal activation energy of 0.29 ± 0.03 eV is consistent with these obtained from electron hopping calculations through the S. oneidensis Mtr outer-membrane decaheme cytochromes. Our measurements and calculations have implications for understanding and controlling micrometer-scale electron transport in microbial systems.

Internal Redox Polarity of an Individual G. sulfurreducens Bacterial Cell Attached to an Inorganic Substrate.

Bacterial cell polarity is an internal asymmetric distribution of subcellular components, including proteins, lipids, and other molecules that correlates with the cell ability to sense energy and metabolite sources, chemical signals, quorum signals, toxins, and movement in the desired directions. This ability also plays central role in cell attachment to various surfaces and biofilm formation. Mechanisms and factors controlling formation of this cell internal asymmetry are not completely understood. As a step in this direction, in the present work, we develop an approach for analyzing how information about inorganic substrate can be non-genetically coded inside an individual bacterial cell. As a model system, we use G. sulfurreducens cells attached to an inorganic mineral, mica. The approach utilizes confocal Raman microscopy, Gaussian deconvolution, and Principal Component Analysis (PCA) and allows for quick label-free identification of the molecular signature of cytochrome intracellular location and the cell to substrate binding down to the level of individual bacterial cells. Our results describe a spectroscopic signature of cell adhesion and how the information about cell adhesion can be coded inside individual bacterial cells.

Redox-gradient driven electron transport in a mixed community anodic biofilm.

Here, we describe the long-distance (multi-cell-length) extracellular electron transport (LD-EET) that occurs in an anode-grown mixed community biofilm (MCB) enriched from river sediment that contains 3%-45% Geobacter spp. High signal-to-noise temperature-dependent electrochemical gating measurements (EGM) using interdigitated microelectrode arrays reveal a peak-shaped electrical conductivity vs. potential dependency, indicating MCB acts as a redox conductor, similar to pure culture anode-grown Geobacter sulfurreducens biofilms (GSB). EGM also reveal that the maximum sustained rate of LD-EET in MCB is comparable to GSB, and the same whether under acetate-oxidizing or acetate-free conditions. Voltammetry indicated that MCB possesses 3- to 5-fold less electrode-accessible redox cofactors than GSB, suggesting that MCB may be more efficiently organized than GSB for LD-EET or that a small portion of electrode accessible redox cofactors of GSB are involved in LD-EET. The activation energy for LD-EET (0.11 ± 0.01 eV) was comparable to GSB, consistent with the possible role of c-type cytochromes as LD-EET cofactors, detected in abundance by confocal resonance Raman microscopy. Taken together, the results demonstrate LD-EET for a mixed community anode-grown microbial biofilm that is remarkably similar to GSB even though it contains many different types of microorganisms and appears to utilize far fewer EET redox cofactors.

Effect of iron doping on protein molecular conductance.

Protein molecular conductance has attracted attention from researchers for the possibility of constructing innovative flexible biocompatible nanoscale electronic devices and smart hybrid materials. Due to protein complexity, most evaluations of protein conductivity are based on the simple estimation of protein's molecular orbital energy levels and spatial distributions without analysing its protein interaction with electrodes and the calculation of the rates of electron transfer (ET). In the present work, we included in our density functional theory (DFT) analysis an approach based on the non-equilibrium Green's function (NEGF) allowing for calculation from the first principles the molecular interaction with electrodes and thus the role of electrode materials, Fermi level, the thermal distribution of electronic energy levels, and the coupling efficiency between the molecule and the electrodes. Compared to proteins studied so far, mainly artificial peptides, heme-containing cytochromes, and bacterial pili, we choose rubredoxin for our calculation. Rubredoxin contains a non-heme iron that, as we have discovered recently, can be involved in extracellular ET in electroactive bacterial biofilms (Yates et al., Energy Environ. Sci., 2016, 9, 3544-3558). Our calculations show that an iron atom incorporated into the protein structure as an iron-sulfur cluster opens a transmission path at the energy corresponding to the Fermi level of the electrodes. This allows the protein to become an extremely efficient conductor at very low bias voltages (<±350 mV). Calculation of the role of protein amino acids based on the local density of states and electron transfer paths reveals that neither aromatic amino acid Tyr nor Phe at any ring orientation participates in coherent ET through the FeS cluster of the protein. Moreover, direct ET through surrounding amino acids, bypassing FeS, is possible only at biases ±1.5 to ±2 V. The polar amino acid Asn might participate in ET at these bias voltages. The conductivity of the protein core substantially depends on the polarity of the applied electric field, allowing for unidirectional ET and operation of the protein as a molecular rectifier. These results can be used for a wise de novo design of proteins for molecular electronics and cellular energy converting devices, particularly for utilization of iron doping in the construction of conductive protein wires.

On the relationship between long-distance and heterogeneous electron transfer in electrode-grown Geobacter sulfurreducens biofilms.

The ability of certain microorganisms to live in a multi-cell thick, electrode-grown biofilm by utilizing the electrode as a metabolic electron acceptor or donor requires electron transfer across cell membranes, through the biofilm, and across the biofilm/electrode interface. Even for the most studied system, anode-grown Geobacter sulfurreducens, the mechanisms underpinning each process and how they connect is largely unresolved. Here we report on G. sulfurreducens biofilms grown across the gap separating two electrodes by maintaining one electrode at 0.300V vs. Ag/AgCl (0.510V vs. SHE) to act as a sustained metabolic electron acceptor while the second electrode was at open circuit. The poised electrode exhibited the characteristic current-time profile for electrode-dependent G. sulfurreducens biofilm growth. The open circuit potential (OCP) of the second electrode however increased after initially decreasing for 1.5-2days. The increase in OCP is taken to indicate the point at which the growing biofilm bridged the gap between the electrodes, enabling cells in contact with the open circuit electrode to utilize the poised electrode as an electron acceptor. After but not prior to reaching this point, the second electrode was able to act as a sustainable electron acceptor immediately after being placed under potential control without requiring further time to develop. These results indicate that heterogeneous ET (H-ET) across the biofilm/electrode interface and long-distance ET (LD-ET) through the biofilm are highly correlated, if not inseparable, and may share many common components.

Metatranscriptomics Supports the Mechanism for Biocathode Electroautotrophy by "Candidatus Tenderia electrophaga".

Biocathodes provide a stable electron source to drive reduction reactions in electrotrophic microbial electrochemical systems. Electroautotrophic biocathode communities may be more robust than monocultures in environmentally relevant settings, but some members are not easily cultivated outside the electrode environment. We previously used metagenomics and metaproteomics to propose a pathway for coupling extracellular electron transfer (EET) to carbon fixation in "Candidatus Tenderia electrophaga," an uncultivated but dominant member of an electroautotrophic biocathode community. Here we validate and refine this proposed pathway using metatranscriptomics of replicate aerobic biocathodes poised at the growth potential level of 310 mV and the suboptimal 470 mV (versus the standard hydrogen electrode). At both potentials, transcripts were more abundant from "Ca. Tenderia electrophaga" than from any other constituent, and its relative activity was positively correlated with current. Several genes encoding key components of the proposed "Ca. Tenderia electrophaga" EET pathway were more highly expressed at 470 mV, consistent with a need for cells to acquire more electrons to obtain the same amount of energy as at 310 mV. These included cyc2, encoding a homolog of a protein known to be involved in iron oxidation. Mean expression of all CO2 fixation-related genes is 0.27 log2-fold higher at 310 mV, indicating that reduced energy availability at 470 mV decreased CO2 fixation. Our results substantiate the claim that "Ca. Tenderia electrophaga" is the key electroautotroph, which will help guide further development of this community for microbial electrosynthesis. IMPORTANCE Bacteria that directly use electrodes as metabolic electron donors (biocathodes) have been proposed for applications ranging from microbial electrosynthesis to advanced bioelectronics for cellular communication with machines. However, just as we understand very little about oxidation of analogous natural insoluble electron donors, such as iron oxide, the organisms and extracellular electron transfer (EET) pathways underlying the electrode-cell direct electron transfer processes are almost completely unknown. Biocathodes are a stable biofilm cultivation platform to interrogate both the rate and mechanism of EET using electrochemistry and to study the electroautotrophic organisms that catalyze these reactions. Here we provide new evidence supporting the hypothesis that the uncultured bacterium "Candidatus Tenderia electrophaga" directly couples extracellular electron transfer to CO2 fixation. Our results provide insight into developing biocathode technology, such as microbial electrosynthesis, as well as advancing our understanding of chemolithoautotrophy.

Biofilm as a redox conductor: a systematic study of the moisture and temperature dependence of its electrical properties.

Some microbial biofilms are electrically conductive. However, the mechanism of electron transport remains unclear. Here, we show that µm-scale long-distance electron transport through electrode-grown Geobacter sulfurreducens biofilms occurs via redox conduction, as determined by electrical measurements performed under varied hydration states and temperatures.

Imaging Active Surface Processes in Barnacle Adhesive Interfaces.

Surface plasmon resonance imaging (SPRI) and voltammetry were used simultaneously to monitor Amphibalanus (=Balanus) amphitrite barnacles reattached and grown on gold-coated glass slides in artificial seawater. Upon reattachment, SPRI revealed rapid surface adsorption of material with a higher refractive index than seawater at the barnacle/gold interface. Over longer time periods, SPRI also revealed secretory activity around the perimeter of the barnacle along the seawater/gold interface extending many millimeters beyond the barnacle and varying in shape and region with time. Ex situ experiments using attenuated total reflectance infrared (ATR-IR) spectroscopy confirmed that reattachment of barnacles was accompanied by adsorption of protein to surfaces on similar time scales as those in the SPRI experiments. Barnacles were grown through multiple molting cycles. While the initial reattachment region remained largely unchanged, SPRI revealed the formation of sets of paired concentric rings having alternately darker/lighter appearance (corresponding to lower and higher refractive indices, respectively) at the barnacle/gold interface beneath the region of new growth. Ex situ experiments coupling the SPRI imaging with optical and FTIR microscopy revealed that the paired rings coincide with molt cycles, with the brighter rings associated with regions enriched in amide moieties. The brighter rings were located just beyond orifices of cement ducts, consistent with delivery of amide-rich chemistry from the ducts. The darker rings were associated with newly expanded cuticle. In situ voltammetry using the SPRI gold substrate as the working electrode revealed presence of redox active compounds (oxidation potential approx 0.2 V vs Ag/AgCl) after barnacles were reattached on surfaces. Redox activity persisted during the reattachment period. The results reveal surface adsorption processes coupled to the complex secretory and chemical activity under barnacles as they construct their adhesive interfaces.

Thermally activated long range electron transport in living biofilms.

Microbial biofilms grown utilizing electrodes as metabolic electron acceptors or donors are a new class of biomaterials with distinct electronic properties. Here we report that electron transport through living electrode-grown Geobacter sulfurreducens biofilms is a thermally activated process with incoherent redox conductivity. The temperature dependency of this process is consistent with electron-transfer reactions involving hemes of c-type cytochromes known to play important roles in G. sulfurreducens extracellular electron transport. While incoherent redox conductivity is ubiquitous in biological systems at molecular-length scales, it is unprecedented over distances it appears to occur through living G. sulfurreducens biofilms, which can exceed 100 microns in thickness.

Metaproteomic evidence of changes in protein expression following a change in electrode potential in a robust biocathode microbiome.

Microorganisms that respire electrodes may be exploited for biotechnology applications if key pathways for extracellular electron transfer can be identified and manipulated through bioengineering. To determine whether expression of proposed Biocathode-MCL extracellular electron transfer proteins are changed by modulating electrode potential without disrupting the relative distribution of microbial constituents, metaproteomic and 16S rRNA gene expression analyses were performed after switching from an optimal to suboptimal potential based on an expected decrease in electrode respiration. Five hundred and seventy-nine unique proteins were identified across both potentials, the majority of which were assigned to three previously defined Biocathode-MCL metagenomic clusters: a Marinobacter sp., a member of the family Chromatiaceae, and a Labrenzia sp (abbreviated as MCL). Statistical analysis of spectral counts using the Fisher's exact test identified 16 proteins associated with the optimal potential, five of which are predicted electron transfer proteins. The majority of proteins associated with the suboptimal potential were involved in protein turnover/synthesis, motility, and membrane transport. Unipept and 16S rRNA gene expression analyses indicated that the taxonomic profile of the microbiome did not change after 52 h at the suboptimal potential. These findings show that protein expression is sensitive to the electrode potential without inducing shifts in community composition, a feature that may be exploited for engineering Biocathode-MCL. All MS data have been deposited in the ProteomeXchange with identifier PXD001590 (http://proteomecentral.proteomexchange.org/dataset/PXD001590).

A previously uncharacterized, nonphotosynthetic member of the Chromatiaceae is the primary CO2-fixing constituent in a self-regenerating biocathode.

Biocathode extracellular electron transfer (EET) may be exploited for biotechnology applications, including microbially mediated O2 reduction in microbial fuel cells and microbial electrosynthesis. However, biocathode mechanistic studies needed to improve or engineer functionality have been limited to a few select species that form sparse, homogeneous biofilms characterized by little or no growth. Attempts to cultivate isolates from biocathode environmental enrichments often fail due to a lack of some advantage provided by life in a consortium, highlighting the need to study and understand biocathode consortia in situ. Here, we present metagenomic and metaproteomic characterization of a previously described biocathode biofilm (+310 mV versus a standard hydrogen electrode [SHE]) enriched from seawater, reducing O2, and presumably fixing CO2 for biomass generation. Metagenomics identified 16 distinct cluster genomes, 15 of which could be assigned at the family or genus level and whose abundance was roughly divided between Alpha- and Gammaproteobacteria. A total of 644 proteins were identified from shotgun metaproteomics and have been deposited in the the ProteomeXchange with identifier PXD001045. Cluster genomes were used to assign the taxonomic identities of 599 proteins, with Marinobacter, Chromatiaceae, and Labrenzia the most represented. RubisCO and phosphoribulokinase, along with 9 other Calvin-Benson-Bassham cycle proteins, were identified from Chromatiaceae. In addition, proteins similar to those predicted for iron oxidation pathways of known iron-oxidizing bacteria were observed for Chromatiaceae. These findings represent the first description of putative EET and CO2 fixation mechanisms for a self-regenerating, self-sustaining multispecies biocathode, providing potential targets for functional engineering, as well as new insights into biocathode EET pathways using proteomics.