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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.

Background Biotechnologies that utilize microorganisms as production hosts for lipid synthesis will enable an efficient and sustainable solution to produce lipids, decreasing reliance on traditional routes for production (either petrochemical or plant-derived) and supporting a circular bioeconomy. To realize this goal, continuous biomanufacturing processes must be developed to maximize productivity and minimize costs compared to traditional batch fermentation processes. Results Here, we utilized biofilms of the marine bacterium, Marinobacter atlanticus , to produce wax esters from succinate (i.e., a non-sugar feedstock) to determine its potential to serve as a production chassis in a continuous flow, biofilm-based biomanufacturing process. To accomplish this, we evaluated growth as a function of protein concentration and wax ester production from M. atlanticus biofilms in a continuously operated 3-D printed fixed bed bioreactor. We determined that exposing M. atlanticus biofilms to alternating nitrogen-rich (1.8 mM NH 4 + ) and nitrogen-poor (0 mM NH 4 + ) conditions in the bioreactor resulted in wax ester production (26 ± 5 mg/L, normalized to reactor volume) at a similar concentration to what is observed from planktonic M. atlanticus cells grown in shake flasks previously in our lab (ca. 25 mg/L cell culture). The wax ester profile was predominated by multiple compounds with 32 carbon chain length (C 32 ; 50–60% of the total). Biomass production in the reactor was positively correlated with dilution rate, as indicated by protein concentration (maximum of 1380 ± 110 mg/L at 0.4 min −1 dilution rate) and oxygen uptake rate (maximum of 4 mmol O 2 /L/h at 0.4 min −1 dilution rate) measurements at different flow rates. Further, we determined the baseline succinate consumption rate for M. atlanticus biofilms to be 0.16 ± 0.03 mmol/L/h, which indicated that oxygen is the limiting reactant in the process. Conclusion The results presented here are the first step toward demonstrating that M. atlanticus biofilms can be used as the basis for development of a continuous flow wax ester biomanufacturing process from non-sugar feedstocks, which will further enable sustainable lipid production in a future circular bioeconomy

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. 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. In this review, we will explore the metabolic and electrochemical potential of the Marinobacter genus, suggesting applications to which these organisms may be best suited.

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.

Natural living conductive biofilms transport electrons between electrodes and cells, as well as among cells fixed within the film, catalyzing an array of reactions from acetate oxidation to CO2 reduction. Synthetic biology offers tools to modify or improve electron transport through biofilms, creating a new class of engineered living conductive materials. Engineered living conductive materials could be used in a range of applications for which traditional conducting polymers are not appropriate, including improved catalytic coatings for microbial fuel-cell electrodes, self-powered sensors for austere environments, and next-generation living components of bioelectronic devices that interact with the human microbiome.

Chemolithotrophic bacteria capable of extracellular electron uptake to drive energy metabolism and CO 2 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. 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 cbb 3 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 CO 2 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.

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 CO 2 fixation. Our results provide insight into developing biocathode technology, such as microbial electrosynthesis, as well as advancing our understanding of chemolithoautotrophy. 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 CO 2 fixation-related genes is 0.27 log 2 -fold higher at 310 mV, indicating that reduced energy availability at 470 mV decreased CO 2 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 CO 2 fixation. Our results provide insight into developing biocathode technology, such as microbial electrosynthesis, as well as advancing our understanding of chemolithoautotrophy.

ObjectivesEvaluate 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).ResultsHere 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.ConclusionsThese 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.