Bacteria able to transfer electrons to metals are key agents in biogeochemical metal cycling, subsurface bioremediation, and corrosion processes. More recently, these bacteria have gained attention as the transfer of electrons from the cell surface to conductive materials can be used in multiple applications. In this work, we adapted electrochemical techniques to probe intact biofilms of Shewanella oneidensis MR-1 and Shewanella sp. MR-4 grown by using a poised electrode as an electron acceptor. This approach detected redox-active molecules within biofilms, which were involved in electron transfer to the electrode. A combination of methods identified a mixture of riboflavin and riboflavin-5 -phosphate in supernatants from biofilm reactors, with riboflavin representing the dominant component during sustained incubations (>72 h). Removal of riboflavin from biofilms reduced the rate of electron transfer to electrodes by >70%, consistent with a role as a soluble redox shuttle carrying electrons from the cell surface to external acceptors. Differential pulse voltammetry and cyclic voltammetry revealed a layer of flavins adsorbed to electrodes, even after soluble components were removed, especially in older biofilms. Riboflavin adsorbed quickly to other surfaces of geochemical interest, such as Fe(III) and Mn(IV) oxy(hydr)oxides. This in situ demonstration of flavin production, and sequestration at surfaces, requires the paradigm of soluble redox shuttles in geochemistry to be adjusted to include binding and modification of surfaces. Moreover, the known ability of isoalloxazine rings to act as metal chelators, along with their electron shuttling capacity, suggests that extracellular respiration of minerals by Shewanella is more complex than originally conceived.bioelectrochemistry ͉ biogeochemistry ͉ redox mediator ͉ riboflavin

Previous studies have suggested that members of the Geobacteraceae can use electrodes as electron acceptors for anaerobic respiration. In order to better understand this electron transfer process for energy production, Geobacter sulfurreducens was inoculated into chambers in which a graphite electrode served as the sole electron acceptor and acetate or hydrogen was the electron donor. The electron-accepting electrodes were maintained at oxidizing potentials by connecting them to similar electrodes in oxygenated medium (fuel cells) or to potentiostats that poised electrodes at ؉0.2 V versus an Ag/AgCl reference electrode (poised potential). When a small inoculum of G. sulfurreducens was introduced into electrode-containing chambers, electrical current production was dependent upon oxidation of acetate to carbon dioxide and increased exponentially, indicating for the first time that electrode reduction supported the growth of this organism. When the medium was replaced with an anaerobic buffer lacking nutrients required for growth, acetate-dependent electrical current production was unaffected and cells attached to these electrodes continued to generate electrical current for weeks. This represents the first report of microbial electricity production solely by cells attached to an electrode. Electrode-attached cells completely oxidized acetate to levels below detection (<10 M), and hydrogen was metabolized to a threshold of 3 Pa. The rates of electron transfer to electrodes (0.21 to 1.2 mol of electrons/mg of protein/min) were similar to those observed for respiration with Fe(III) citrate as the electron acceptor (E o ‫73.0؉؍‬ V). The production of current in microbial fuel cell (65 mA/m 2 of electrode surface) or poised-potential (163 to 1,143 mA/m 2 ) mode was greater than what has been reported for other microbial systems, even those that employed higher cell densities and electron-shuttling compounds. Since acetate was completely oxidized, the efficiency of conversion of organic electron donor to electricity was significantly higher than in previously described microbial fuel cells. These results suggest that the effectiveness of microbial fuel cells can be increased with organisms such as G. sulfurreducens that can attach to electrodes and remain viable for long periods of time while completely oxidizing organic substrates with quantitative transfer of electrons to an electrode.

Energy in the form of electricity can be harvested from marine sediments by placing a graphite electrode (the anode) in the anoxic zone and connecting it to a graphite cathode in the overlying aerobic water. We report a specific enrichment of microorganisms of the family Geobacteraceae on energy-harvesting anodes, and we show that these microorganisms can conserve energy to support their growth by oxidizing organic compounds with an electrode serving as the sole electron acceptor. This finding not only provides a method for extracting energy from organic matter, but also suggests a strategy for promoting the bioremediation of organic contaminants in subsurface environments.

It has been demonstrated previously that Geobacter species can transfer electrons directly to electrodes. In order to determine whether electrodes could serve as electron donors for microbial respiration, enrichment cultures were established from a sediment inoculum with a potentiostat-poised graphite electrode as the sole electron donor and nitrate as the electron acceptor. Nitrate was reduced to nitrite with the consumption of electrical current. The stoichiometry of electron and nitrate consumption and nitrite accumulation were consistent with the electrode serving as the sole electron donor for nitrate reduction. Analysis of 16 rRNA gene sequences demonstrated that the electrodes supplied with current were specifically enriched in microorganisms with sequences most closely related to the sequences of known Geobacter species. A pure culture of Geobacter metallireducens was shown to reduce nitrate to nitrite with the electrode as the sole electron donor with the expected stoichiometry of electron consumption. Cells attached to the electrode appeared to be responsible for the nitrate reduction. Attached cells of Geobacter sulfurreducens reduced fumarate to succinate with the electrode as an electron donor. These results demonstrate for the first time that electrodes may serve as a direct electron donor for anaerobic respiration. This finding has implications for the harvesting of electricity from anaerobic sediments and the bioremediation of oxidized contaminants.

In many marine environments, a voltage gradient exists across the water sediment interface resulting from sedimentary microbial activity. Here we show that a fuel cell consisting of an anode embedded in marine sediment and a cathode in overlying seawater can use this voltage gradient to generate electrical power in situ. Fuel cells of this design generated sustained power in a boat basin carved into a salt marsh near Tuckerton, New Jersey, and in the Yaquina Bay Estuary near Newport, Oregon. Retrieval and analysis of the Tuckerton fuel cell indicates that power generation results from at least two anode reactions: oxidation of sediment sulfide (a by-product of microbial oxidation of sedimentary organic carbon) and oxidation of sedimentary organic carbon catalyzed by microorganisms colonizing the anode. These results demonstrate in real marine environments a new form of power generation that uses an immense, renewable energy reservoir (sedimentary organic carbon) and has near-immediate application.

While electrochemical characterization of enzymes immobilized on electrodes has become common, there is still a need for reliable quantitative methods for study of electron transfer between living cells and conductive surfaces. This work describes growth of thin (<20 m) Geobacter sulfurreducens biofilms on polished glassy carbon electrodes, using stirred three-electrode anaerobic bioreactors controlled by potentiostats and nondestructive voltammetry techniques for characterization of viable biofilms. Routine in vivo analysis of electron transfer between bacterial cells and electrodes was performed, providing insight into the main redox-active species participating in electron transfer to electrodes. At low scan rates, cyclic voltammetry revealed catalytic electron transfer between cells and the electrode, similar to what has been observed for pure enzymes attached to electrodes under continuous turnover conditions. Differential pulse voltammetry and electrochemical impedance spectroscopy also revealed features that were consistent with electron transfer being mediated by an adsorbed catalyst. Multiple redox-active species were detected, revealing complexity at the outer surfaces of this bacterium. These techniques provide the basis for cataloging quantifiable, defined electron transfer phenotypes as a function of potential, electrode material, growth phase, and culture conditions and provide a framework for comparisons with other species or communities.

The Mtr respiratory pathway of Shewanella oneidensis strain MR-1 is required to effectively respire both soluble and insoluble forms of oxidized iron. Flavins (riboflavin and flavin mononucleotide) recently have been shown to be excreted by MR-1 and facilitate the reduction of insoluble substrates. Other Shewanella species tested accumulated flavins in supernatants to an extent similar to that of MR-1, suggesting that flavin secretion is a general trait of the species. External flavins have been proposed to act as both a soluble electron shuttle and a metal chelator; however, at biologically relevant concentrations, our results suggest that external flavins primarily act as electron shuttles for MR-1. Using deletion mutants lacking various Mtr-associated proteins, we demonstrate that the Mtr extracellular respiratory pathway is essential for the reduction of flavins and that decaheme cytochromes found on the outer surface of the cell (MtrC and OmcA) are required for the majority of this activity. Given the involvement of external flavins in the reduction of electrodes, we monitored current production by Mtr respiratory pathway mutants in three-electrode bioreactors under controlled flavin concentrations. While mutants lacking MtrC were able to reduce flavins at 50% of the rate of the wild type in cell suspension assays, these strains were unable to grow into productive electrode-reducing biofilms. The analysis of mutants lacking OmcA suggests a role for this protein in both electron transfer to electrodes and attachment to surfaces. The parallel phenotypes of Mtr mutants in flavin and electrode reduction blur the distinction between direct contact and the redox shuttling strategies of insoluble substrate reduction by MR-1.Shewanella oneidensis strain MR-1 (MR-1) is a facultative anaerobe capable of respiring a variety of substrates, including various metals and metal oxides, a phenotype that is important for bioremediation and metal cycling in natural environments (22,53). At near-neutral pH, Fe(III) and Mn(IV) often are present as insoluble oxide minerals. Dissimilatory metal-reducing bacteria such as MR-1 have developed pathways to transfer electrons from the interior of the cell to these external terminal electron acceptors. In some bacteria, these pathways also can transfer electrons to electrodes, which can be harnessed for renewable energy and remote biosensor applications (23,26,27). Beyond increasing our understanding of this unusual process, applying anaerobic microbial extracellular respiration to new technologies requires a thorough understanding of the molecular dynamics and cellular physiology of electron source utilization (substrate oxidation) and the reduction of insoluble terminal electron acceptor(s). There are four proposed mechanisms to explain how insoluble substrates are reduced by Shewanella: (i) direct contact, (ii) electron shuttling, (iii) chelation, and (iv) electrically conductive appendages (reviewed in reference 18). We will focus on the first three strategies here.Flavins recently ...

The microbial communities associated with electrodes from underwater fuel cells harvesting electricity from five different aquatic sediments were investigated. Three fuel cells were constructed with marine, salt-marsh, or freshwater sediments incubated in the laboratory. Fuel cells were also deployed in the field in salt marsh sediments in New Jersey and estuarine sediments in Oregon, USA. All of the sediments produced comparable amounts of power. Analysis of 16S rRNA gene sequences after 3-7 months of incubation demonstrated that all of the energy-harvesting anodes were highly enriched in microorganisms in the delta-Proteobacteria when compared with control electrodes not connected to a cathode. Geobacteraceae accounted for the majority of delta-Proteobacterial sequences or all of the energy-harvesting anodes, except the one deployed at the Oregon estuarine site. Quantitative PCR analysis of 16S rRNA genes and culturing studies indicated that Geobacteraceae were 100-fold more abundant on the marine-deployed anodes versus controls. Sequences most similar to microorganisms in the family Desulfobulbaceae predominated on the anode deployed in the estuarine sediments, and a significant proportion of the sequences recovered from the freshwater anodes were closely related to the Fe(III)-reducing isolate, Geothrix fermentans. There was also a specific enrichment of microorganisms on energy harvesting cathodes, but the enriched populations varied with the sediment/water source. Thus, future studies designed to help optimize the harvesting of electricity from aquatic sediments or waste organic matter should focus on the electrode interactions of these microorganisms which are most competitive in colonizing anodes and cathodes.