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Microbial attachment to mineral surfaces is a fundamental process for initiating a broad range of biochemical and geological events in a natural environment.[1] The genus Shewanella, [2] which consists of dissimilatory metal-reducing bacteria often found in subsurface sediments, has the ability to recognize the surface of iron(III) oxides [3] and initiate extracellular electron transfer (ET) [4][5][6][7][8] to the attached iron oxides as a terminal process in its metabolism. This is an important process for its influence on the biogeochemical cycling of iron, [9] and it has also gained attention not only for a new aspect of the metabolic strategy of microorganisms, [5][6][7][8] but also for its applicability in microbial fuel cells.[10]The outer-membrane (OM) redox proteins, c-type decaheme cytochromes (c-Cyt), play a crucial role in mediating ET from the cell to iron(III) oxides. [4][5][6][7] 11] A great deal of research has been focused on the electrochemical and spectroscopic investigation of the purified OM proteins. [11] However, few studies have been performed by directly monitoring the ET process of intact cells, and therefore the mechanism of this process has largely remained unsolved. Herein, we report the ability of S. loihica PV-4 to selfassemble into an electrically conductive network in the presence of iron(III) oxides, and demonstrate the role of semiconductive nanominerals in promoting a long-distance extracellular ET process in the bacterial network.To probe the extracellular ET of intact cells of S. loihica PV-4, we used a single-chamber, three-electrode system, with lactate as a carbon source and an electron donor. An optically transparent conductive-glass, tin-doped In 2 O 3 (ITO) electrode, with a surface area of 1.8 cm 2 , was used as the working electrode, and placed on the bottom surface of the reactor. A current was generated immediately after adding the cells into the reactor (Figure 1 a), and reached a constant value 0.4-0.6 mA. Current generation is a consequence of electrical connections from the cells to the electrode, followed by the injection of electrons from the OM c-Cyts to the ITO electrode, which is suggested by the absence of redox species in the cell-free supernatant solution. In addition, the current showed essentially no dependence on the optical cell density OD at 600 nm over the OD 600 range of 0.1-4.0 (Figure 1 a, inset). This result implies that the current generation from S. loihica is dominated by the cells attached directly to the electrode surface. In other words, individual cells are electrically insulated from the others, and thus the long-distance ET process, even if it is present, is much less efficient than the cCyt-mediated ET to the electrode surface. Figure 1. a) Current I versus time t measurements of microbial current generation for S. loihica on an ITO electrode. Inset: the dependence of the steady-state current on OD 600 . b) I versus t curves in the presence (trace 1) and absence (trace 2) of a-Fe 2 O 3 colloids (7.5 mm). The effect of iron citra...

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Little is known about the importance and/or mechanisms of biological mineral oxidation in sediments, partially due to the difficulties associated with culturing mineral-oxidizing microbes. We demonstrate that electrochemical enrichment is a feasible approach for isolation of microbes capable of gaining electrons from insoluble minerals. To this end we constructed sediment microcosms and incubated electrodes at various controlled redox potentials. Negative current production was observed in incubations and increased as redox potential decreased (tested −50 to −400 mV vs. Ag/AgCl). Electrode-associated biomass responded to the addition of nitrate and ferric iron as terminal electron acceptors in secondary sediment-free enrichments. Elemental sulfur, elemental iron and amorphous iron sulfide enrichments derived from electrode biomass demonstrated products indicative of sulfur or iron oxidation. The microbes isolated from these enrichments belong to the genera Halomonas, Idiomarina, Marinobacter, and Pseudomonas of the Gammaproteobacteria, and Thalassospira and Thioclava from the Alphaproteobacteria. Chronoamperometry data demonstrates sustained electrode oxidation from these isolates in the absence of alternate electron sources. Cyclic voltammetry demonstrated the variability in dominant electron transfer modes or interactions with electrodes (i.e., biofilm, planktonic or mediator facilitated) and the wide range of midpoint potentials observed for each microbe (from 8 to −295 mV vs. Ag/AgCl). The diversity of extracellular electron transfer mechanisms observed in one sediment and one redox condition, illustrates the potential importance and abundance of these interactions. This approach has promise for increasing our understanding the extent and diversity of microbe mineral interactions, as well as increasing the repository of microbes available for electrochemical applications.

While typically investigated as a microorganism capable of extracellular electron transfer to minerals or anodes, Shewanella oneidensis MR-1 can also facilitate electron flow from a cathode to terminal electron acceptors, such as fumarate or oxygen, thereby providing a model system for a process that has significant environmental and technological implications. This work demonstrates that cathodic electrons enter the electron transport chain of S. oneidensis when oxygen is used as the terminal electron acceptor. The effect of electron transport chain inhibitors suggested that a proton gradient is generated during cathode oxidation, consistent with the higher cellular ATP levels measured in cathode-respiring cells than in controls. Cathode oxidation also correlated with an increase in the cellular redox (NADH/FMNH2) pool determined with a bioluminescence assay, a proton uncoupler, and a mutant of proton-pumping NADH oxidase complex I. This work suggested that the generation of NADH/FMNH2 under cathodic conditions was linked to reverse electron flow mediated by complex I. A decrease in cathodic electron uptake was observed in various mutant strains, including those lacking the extracellular electron transfer components necessary for anodic-current generation. While no cell growth was observed under these conditions, here we show that cathode oxidation is linked to cellular energy acquisition, resulting in a quantifiable reduction in the cellular decay rate. This work highlights a potential mechanism for cell survival and/or persistence on cathodes, which might extend to environments where growth and division are severely limited.

Geobacter cells utilize self-secreted riboflavin as a bound-cofactor in outer-membrane c-type cytochromes to enhance the rate of bacterial electron transport.

Widely distributed outer-membrane cytochromes enable sulfur-reducing bacteria to obtain energy from solid electron donors.

The variety of solid surfaces to and from which microbes can deliver electrons by extracellular electron transport (EET) processes via outer-membrane c-type cytochromes (OM c-Cyts) expands the importance of microbial respiration in natural environments and industrial applications. Here, we demonstrate that the bifurcated EET pathway of OM c-Cyts sustains the diversity of the EET surface in Shewanella oneidensis MR-1 via specific binding with cell-secreted flavin mononucleotide (FMN) and riboflavin (RF). Microbial current production and whole-cell differential pulse voltammetry revealed that RF and FMN enhance EET as bound cofactors in a similar manner. Conversely, FMN and RF were clearly differentiated in the EET enhancement by gene-deletion of OM c-Cyts and the dependency of the electrode potential and pH. These results indicate that RF and FMN have specific binding sites in OM c-Cyts and highlight the potential roles of these flavin-cytochrome complexes in controlling the rate of electron transfer to surfaces with diverse potential and pH.