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.

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.

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.

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.

Molecular Mechanisms Contributing to the Growth and Physiology of an Extremophile Cultured with Dielectric Heating.

The effect of microwave frequency electromagnetic fields on living microorganisms is an active and highly contested area of research. One of the major drawbacks to using mesophilic organisms to study microwave radiation effects is the unavoidable heating of the organism, which has limited the scale (<5 ml) and duration (<1 h) of experiments. However, the negative effects of heating a mesophile can be mitigated by employing thermophiles (organisms able to grow at temperatures of >60°C). This study identified changes in global gene expression profiles during the growth of Thermus scotoductus SA-01 at 65°C using dielectric (2.45 GHz, i.e., microwave) heating. RNA sequencing was performed on cultures at 8, 14, and 24 h after inoculation to determine the molecular mechanisms contributing to long-term cellular growth and survival under microwave heating conditions. Over the course of growth, genes associated with amino acid metabolism, carbohydrate metabolism, and defense mechanisms were upregulated; the number of repressed genes with unknown function increased; and at all time points, transposases were upregulated. Genes involved in cell wall biogenesis and elongation were also upregulated, consistent with the distinct elongated cell morphology observed after 24 h using microwave heating. Analysis of the global differential gene expression data enabled the identification of molecular processes specific to the response of T. scotoductus SA-01 to dielectric heating during growth. IMPORTANCE: The residual heating of living organisms in the microwave region of the electromagnetic spectrum has complicated the identification of radiation-only effects using microorganisms for 50 years. A majority of the previous experiments used either mature cells or short exposure times with low-energy high-frequency radiation. Using global differential gene expression data, we identified molecular processes unique to dielectric heating using Thermus scotoductus SA-01 cultured over 30 h in a commercial microwave digestor. Genes associated with amino acid metabolism, carbohydrate metabolism, and defense mechanisms were upregulated; the number of repressed genes with unknown function increased; and at all time points, transposases were upregulated. These findings serve as a platform for future studies with mesophiles in order to better understand the response of microorganisms to microwave radiation.

'Candidatus Tenderia electrophaga', an uncultivated electroautotroph from a biocathode enrichment.

Biocathode communities are of interest for a variety of applications, including electrosynthesis, bioremediation, and biosensors, yet much remains to be understood about the biological processes that occur to enable these communities to grow. One major difficulty in understanding these communities is that the critical autotrophic organisms are difficult to cultivate. An uncultivated, electroautotrophic bacterium previously identified as an uncultivated member of the family Chromatiaceae appears to be a key organism in an autotrophic biocathode microbial community. Metagenomic, metaproteomic and metatranscriptomic characterization of this community indicates that there is likely a single organism that utilizes electrons from the cathode to fix CO2, yet this organism has not been obtained in pure culture. Fluorescence in situ hybridization reveals that the organism grows as rod-shaped cells approximately 1.8 × 0.6 µm, and forms large clumps on the cathode. The genomic DNA G+C content was 59.2 mol%. Here we identify the key features of this organism and propose 'Candidatus Tenderia electrophaga', within the Gammaproteobacteria on the basis of low nucleotide and predicted protein sequence identity to known members of the orders Chromatiales and Thiotrichales.

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.

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.

Long-range electron transport in Geobacter sulfurreducens biofilms is redox gradient-driven.

Geobacter spp. can acquire energy by coupling intracellular oxidation of organic matter with extracellular electron transfer to an anode (an electrode poised at a metabolically oxidizing potential), forming a biofilm extending many cell lengths away from the anode surface. It has been proposed that long-range electron transport in such biofilms occurs through a network of bound redox cofactors, thought to involve extracellular matrix c-type cytochromes, as occurs for polymers containing discrete redox moieties. Here, we report measurements of electron transport in actively respiring Geobacter sulfurreducens wild type biofilms using interdigitated microelectrode arrays. Measurements when one electrode is used as an anode and the other electrode is used to monitor redox status of the biofilm 15 µm away indicate the presence of an intrabiofilm redox gradient, in which the concentration of electrons residing within the proposed redox cofactor network is higher farther from the anode surface. The magnitude of the redox gradient seems to correlate with current, which is consistent with electron transport from cells in the biofilm to the anode, where electrons effectively diffuse from areas of high to low concentration, hopping between redox cofactors. Comparison with gate measurements, when one electrode is used as an electron source and the other electrode is used as an electron drain, suggests that there are multiple types of redox cofactors in Geobacter biofilms spanning a range in oxidation potential that can engage in electron transport. The majority of these redox cofactors, however, seem to have oxidation potentials too negative to be involved in electron transport when acetate is the electron source.

Spatially resolved confocal resonant Raman microscopic analysis of anode-grown Geobacter sulfurreducens biofilms.

When grown on the surface of an anode electrode, Geobacter sulfurreducens forms a multi-cell thick biofilm in which all cells appear to couple the oxidation of acetate with electron transport to the anode, which serves as the terminal metabolic electron acceptor. Just how electrons are transported through such a biofilm from cells to the underlying anode surface over distances that can exceed 20 microns remains unresolved. Current evidence suggests it may occur by electron hopping through a proposed network of redox cofactors composed of immobile outer membrane and/or extracellular multi-heme c-type cytochromes. In the present work, we perform a spatially resolved confocal resonant Raman (CRR) microscopic analysis to investigate anode-grown Geobacter biofilms. The results confirm the presence of an intra-biofilm redox gradient whereby the probability that a heme is in the reduced state increases with increasing distance from the anode surface. Such a gradient is required to drive electron transport toward the anode surface by electron hopping via cytochromes. The results also indicate that at open circuit, when electrons are expected to accumulate in redox cofactors involved in electron transport due to the inability of the anode to accept electrons, nearly all c-type cytochrome hemes detected in the biofilm are oxidized. The same outcome occurs when a comparable potential to that measured at open circuit (-0.30 V vs. SHE) is applied to the anode, whereas nearly all hemes are reduced when an exceedingly negative potential (-0.50 V vs. SHE) is applied to the anode. These results suggest that nearly all c-type cytochrome hemes detected in the biofilm can be electrochemically accessed by the electrode, but most have oxidation potentials too negative to transport electrons originating from acetate metabolism. The results also reveal a lateral heterogeneity (x-y dimensions) in the type of c-type cytochromes within the biofilm that may affect electron transport to the electrode.

Electrochemical investigation of a microbial solar cell reveals a nonphotosynthetic biocathode catalyst.

Microbial solar cells (MSCs) are microbial fuel cells (MFCs) that generate their own oxidant and/or fuel through photosynthetic reactions. Here, we present electrochemical analyses and biofilm 16S rRNA gene profiling of biocathodes of sediment/seawater-based MSCs inoculated from the biocathode of a previously described sediment/seawater-based MSC. Electrochemical analyses indicate that for these second-generation MSC biocathodes, catalytic activity diminishes over time if illumination is provided during growth, whereas it remains relatively stable if growth occurs in the dark. For both illuminated and dark MSC biocathodes, cyclic voltammetry reveals a catalytic-current-potential dependency consistent with heterogeneous electron transfer mediated by an insoluble microbial redox cofactor, which was conserved following enrichment of the dark MSC biocathode using a three-electrode configuration. 16S rRNA gene profiling showed Gammaproteobacteria, most closely related to Marinobacter spp., predominated in the enriched biocathode. The enriched biocathode biofilm is easily cultured on graphite cathodes, forms a multimicrobe-thick biofilm (up to 8.2 µm), and does not lose catalytic activity after exchanges of the reactor medium. Moreover, the consortium can be grown on cathodes with only inorganic carbon provided as the carbon source, which may be exploited for proposed bioelectrochemical systems for electrosynthesis of organic carbon from carbon dioxide. These results support a scheme where two distinct communities of organisms develop within MSC biocathodes: one that is photosynthetically active and one that catalyzes reduction of O2 by the cathode, where the former partially inhibits the latter. The relationship between the two communities must be further explored to fully realize the potential for MSC applications.

Marinobacter: A case study in bioelectrochemical chassis evaluation.

The junction of bioelectrochemical systems and synthetic biology opens the door to many potentially groundbreaking technologies. When developing these possibilities, choosing the correct chassis organism can save a great deal of engineering effort and, indeed, can mean the difference between success and failure. Choosing the correct chassis for a specific application requires a knowledge of the metabolic potential of the candidate organisms, as well as a clear delineation of the traits, required in the application. In this review, we will explore the metabolic and electrochemical potential of a single genus, Marinobacter. We will cover its strengths, (salt tolerance, biofilm formation and electrochemical potential) and weaknesses (insufficient characterization of many strains and a less developed toolbox for genetic manipulation) in potential synthetic electromicrobiology applications. In doing so, we will provide a roadmap for choosing a chassis organism for bioelectrochemical systems.

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.

Conservation of Energetic Pathways for Electroautotrophy in the Uncultivated Candidate Order Tenderiales.

Electromicrobiology can be used to understand extracellular electron uptake in previously undescribed chemolithotrophs. Enrichment and characterization of the uncultivated electroautotroph "Candidatus Tenderia electrophaga" using electromicrobiology led to the designation of the order Tenderiales. Representative Tenderiales metagenome-assembled genomes (MAGs) have been identified in a number of environmental surveys, yet a comprehensive characterization of conserved genes for extracellular electron uptake has thus far not been conducted. Using comparative genomics, we identified conserved orthologous genes within the Tenderiales and nearest-neighbor orders important for extracellular electron uptake based on a previously proposed pathway from "Ca. Tenderia electrophaga." The Tenderiales contained a conserved cluster we designated uetABCDEFGHIJ, which encodes proteins containing features that would enable transport of extracellular electrons to cytoplasmic membrane-bound energy-transducing complexes such as two conserved cytochrome cbb3 oxidases. For example, UetJ is predicted to be an extracellular undecaheme c-type cytochrome that forms a heme wire. We also identified clusters of genes predicted to facilitate assembly and maturation of electron transport proteins, as well as cellular attachment to surfaces. Autotrophy among the Tenderiales is supported by the presence of carbon fixation and stress response pathways that could allow cellular growth by extracellular electron uptake. Key differences between the Tenderiales and other known neutrophilic iron oxidizers were revealed, including very few Cyc2 genes in the Tenderiales. Our results reveal a possible conserved pathway for extracellular electron uptake and suggest that the Tenderiales have an ecological role in coupling metal or mineral redox chemistry and the carbon cycle in marine and brackish sediments. IMPORTANCE Chemolithotrophic bacteria capable of extracellular electron uptake to drive energy metabolism and CO2 fixation are known as electroautotrophs. The recently described order Tenderiales contains the uncultivated electroautotroph "Ca. Tenderia electrophaga." The "Ca. Tenderia electrophaga" genome contains genes proposed to make up a previously undescribed extracellular electron uptake pathway. Here, we use comparative genomics to show that this pathway is well conserved among Tenderiales spp. recovered by metagenome-assembled genomes. This conservation extends to near neighbors of the Tenderiales but not to other well-studied chemolithotrophs, including iron and sulfur oxidizers, indicating that these genes may be useful markers of growth using insoluble extracellular electron donors. Our findings suggest that extracellular electron uptake and electroautotrophy may be pervasive among the Tenderiales, and the geographic locations from which metagenome-assembled genomes were recovered offer clues to their natural ecological niche.

Marinobacter atlanticus electrode biofilms differentially regulate gene expression depending on electrode potential and lifestyle.

Marinobacter spp. are opportunitrophs with a broad metabolic range including interactions with metals and electrodes. Marinobacter atlanticus strain CP1 was previously isolated from a cathode biofilm microbial community enriched from a sediment microbial fuel cell. Like other Marinobacter spp., M. atlanticus generates small amounts of electrical current when grown as a biofilm on an electrode, which is enhanced by the addition of redox mediators. However, the molecular mechanism resulting in extracellular electron transfer is unknown. Here, RNA-sequencing was used to determine changes in gene expression in electrode-attached and planktonic cells of M. atlanticus when grown at electrode potentials that enable current production (310 and 510 mV vs. SHE) compared to a potential that enables electron uptake (160 mV). Cells grown at current-producing potentials had increased expression of genes for molybdate transport, regardless of planktonic or attached lifestyle. Electrode-attached cells at current-producing potentials showed increased expression of the major export protein for the type VI secretion system. Growth at 160 mV resulted in an increase in expression of genes related to stress response and DNA repair including both RecBCD and the LexA/RecA regulatory network, as well as genes for copper homeostasis. Changes in expression of proteins with PEP C-terminal extracellular export motifs suggests that M. atlanticus is remodeling the biofilm matrix in response to electrode potential. These results improve our understanding of the physiological adaptations required for M. atlanticus growth on electrodes, and suggest a role for metal acquisition, either as a requirement for metal cofactors of redox proteins or as a possible electron shuttling mechanism.

Nanoliter scale electrochemistry of natural and engineered electroactive bacteria.

Bacterial extracellular electron transfer (EET) is envisioned for use in applied biotechnologies, necessitating electrochemical characterization of natural and engineered electroactive biofilms under conditions similar to the target application, including small-scale biosensing or biosynthesis platforms, which is often distinct from standard 100 mL-scale stirred-batch bioelectrochemical test platforms used in the laboratory. Here, we adapted an eight chamber, nanoliter volume (500 nL) electrochemical flow cell to grow biofilms of both natural (Biocathode MCL community, Marinobacter atlanticus, and Shewanella oneidensis MR1) or genetically modified (S. oneidensis ΔMtr and S. oneidensis ΔMtr + pLB2) electroactive bacteria on electrodes held at a constant potential. Maximum current density achieved by unmodified strains was similar between the nano- and milliliter-scale reactors. However, S. oneidensis biofilms engineered to activate EET upon exposure to 2,4-diacetylphloroglucinol (DAPG) produced current at wild-type levels in the stirred-batch reactor, but not in the nanoliter flow cell. We hypothesize this was due to differences in mass transport of DAPG, naturally-produced soluble redox mediators, and oxygen between the two reactor types. Results presented here demonstrate, for the first time, nanoliter scale chronoamperometry and cyclic voltammetry of a range of electroactive bacteria in a three-electrode reactor system towards development of miniaturized, and potentially high throughput, bioelectrochemical platforms.

Engineering Wired Life: Synthetic Biology for Electroactive Bacteria.

Electroactive bacteria produce or consume electrical current by moving electrons to and from extracellular acceptors and donors. This specialized process, known as extracellular electron transfer, relies on pathways composed of redox active proteins and biomolecules and has enabled technologies ranging from harvesting energy on the sea floor, to chemical sensing, to carbon capture. Harnessing and controlling extracellular electron transfer pathways using bioengineering and synthetic biology promises to heighten the limits of established technologies and open doors to new possibilities. In this review, we provide an overview of recent advancements in genetic tools for manipulating native electroactive bacteria to control extracellular electron transfer. After reviewing electron transfer pathways in natively electroactive organisms, we examine lessons learned from the introduction of extracellular electron transfer pathways into Escherichia coli. We conclude by presenting challenges to future efforts and give examples of opportunities to bioengineer microbes for electrochemical applications.

A bacterial membrane sculpting protein with BAR domain-like activity.

Bin/Amphiphysin/RVS (BAR) domain proteins belong to a superfamily of coiled-coil proteins influencing membrane curvature in eukaryotes and are associated with vesicle biogenesis, vesicle-mediated protein trafficking, and intracellular signaling. Here, we report a bacterial protein with BAR domain-like activity, BdpA, from Shewanella oneidensis MR-1, known to produce redox-active membrane vesicles and micrometer-scale outer membrane extensions (OMEs). BdpA is required for uniform size distribution of membrane vesicles and influences scaffolding of OMEs into a consistent diameter and curvature. Cryo-TEM reveals that a strain lacking BdpA produces lobed, disordered OMEs rather than membrane tubules or narrow chains produced by the wild-type strain. Overexpression of BdpA promotes OME formation during planktonic growth of S. oneidensis where they are not typically observed. Heterologous expression results in OME production in Marinobacter atlanticus and Escherichia coli. Based on the ability of BdpA to alter membrane architecture in vivo, we propose that BdpA and its homologs comprise a newly identified class of bacterial BAR domain-like proteins.

Redox Characterization of Electrode-Immobilized Bacterial Microcompartment Shell Proteins Engineered To Bind Metal Centers.

Bacterial microcompartment (BMC) shells are modular, selectively permeable, nanoscale protein shells that self-assemble from hexagonal and pentagonal building blocks in vivo or in vitro. Natural and engineered BMC shells colocalize and concentrate catalysts and metabolites in their lumen, increasing reaction kinetics. Here, we describe the design and characterization of a shell protein (pseudohexameric/trimeric BMC-T1HO protein) engineered to coordinate a Cu ion in its pore. Several designs, each varying the position of an introduced coordinating histidine residue, were shown to maintain their trimeric oligomerization state upon Cu coordination via chemical denaturation experiments. We measured reversible redox activity from electrode-bound Cu-3His BMC-T1HO variants, with formal potential(s) that were dependent on the Cu coordination site within the discoidal shaped trimer (E°' = +208 to +265 mV vs SHE). These results represent important steps toward expanding the functionality (Cu coordination) and applicability (redox activity on an electrode surface) of engineered BMC reactor architectures.