Leonard M. Tender

Harnessing microbially generated power on the seafloor

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.

Cyclic Voltammetry of Biofilms of Wild Type and Mutant Geobacter Sulfurreducens on Fuel Cell Anodes Indicates Possible Roles of OmcB, OmcZ, type IV Pili, and Protons in Extracellular Electron Transfer

Geobacteracea are distinct for their ability to reduce insoluble oxidants including minerals and electrodes without apparent reliance on soluble extracellular electron transfer (ET) mediators. This property makes them important anode catalysts in new generation microbial fuel cells (MFCs) because it obviates the need to replenish ET mediators otherwise necessary to sustain power. Here we report cyclic voltammetry (CV) of biofilms of wild type (WT) and mutant G. sulfurreducens strains grown on graphite cloth anodes acting as electron acceptors with acetate as the electron donor. Our analysis indicates that WT biofilms contain a conductive network of bound ET mediators in which OmcZ (outer membranec-type cytochrome Z) participates in homogeneous ET (through the biofilm bulk) while OmcB mediates heterogeneous ET (across the biofilm/electrode interface); that type IV pili are important in both reactions; that OmcS plays a secondary role in homogenous ET; that OmcE, important in Fe(III) oxide reduction, is not involved in either reaction; that catalytic current is limited overall by the rate of microbial uptake of acetate; that protons generated from acetate oxidation act as charge compensating ions in homogenous ET; and that homogenous ET, when accelerated by fast voltammetric scan rates, is limited by diffusion of protons within the biofilm. These results provide the first direct electrochemical evidence substantiating utilization of bound ET mediators by Geobacter biofilms and the distinct roles of OmcB and OmcZ in the extracellular ET properties of anode-reducing G. sulfurreducens.

Selection of a variant of Geobacter sulfurreducens with enhanced capacity for current production in microbial fuel cells

Geobacter sulfurreducens produces current densities in microbial fuel cells that are among the highest known for pure cultures. The possibility of adapting this organism to produce even higher current densities was evaluated. A system in which a graphite anode was poised at -400 mV (versus Ag/AgCl) was inoculated with the wild-type strain of G. sulfurreducens, strain DL-1. An isolate, designated strain KN400, was recovered from the biofilm after 5 months of growth on the electrode. KN400 was much more effective in current production than strain DL-1. This was apparent with anodes poised at -400 mV, as well as in systems run in true fuel cell mode. KN400 had current (7.6A/m(2)) and power (3.9 W/m(2)) densities that respectively were substantially higher than those of DL1 (1.4A/m(2) and 0.5 W/m(2)). On a per cell basis KN400 was more effective in current production than DL1, requiring thinner biofilms to make equivalent current. The enhanced capacity for current production in KN400 was associated with a greater abundance of electrically conductive microbial nanowires than DL1 and lower internal resistance (0.015 versus 0.130 Omega/m(2)) and mass transfer limitation in KN400 fuel cells. KN400 produced flagella, whereas DL1 does not. Surprisingly, KN400 had much less outer-surface c-type cytochromes than DL1. KN400 also had a greater propensity to form biofilms on glass or graphite than DL1, even when growing with the soluble electron acceptor, fumarate. These results demonstrate that it is possible to enhance the ability of microorganisms to electrochemically interact with electrodes with the appropriate selective pressure and that improved current production is associated with clear differences in the properties of the outer surface of the cell that may provide insights into the mechanisms for microbe-electrode interactions.

On the electrical conductivity of microbial nanowires and biofilms

Dissimilatory metal-reducing bacteria (DMRB), such as Geobacter and Shewanella spp., occupy a distinct metabolic niche in which they acquire energy by coupling oxidation of organic fuels with reduction of insoluble extracellular electron acceptors (i.e., minerals). Their unique extracellular electron transfer (EET) capabilities extend to reduction of anodes (electrodes maintained at sufficiently positive potentials) on which they form persistent, electric current generating biofilms. One hypothesis describing the mechanism of EET by Geobacter and Shewanella spp. involves superexchange in which electrons are conducted by a succession of electron transfer reactions among redox proteins associated with the outer cell membranes, aligned along pilus-like filaments (e.g.pili), and/or throughout the extracellular matrix. Here we present theory, previously developed to describe superexchange within abiotic redox polymers, to describe superexchange within DMRB biofilms grown on anodes. We show that this theory appears to apply to recent ex situ measurements of electrical conductivity by individual pilus-like filaments of S. oneidensis MR-1 and G. sulfurreducensDL1, referred to as microbial nanowires. Microbial nanowires have received much recent attention because they are thought by some to impart electrical conductivity to DMRB biofilms and because of the prospect of microbe-produced conductive nanomaterials. We also show that this theory appears to apply to preliminary in situ demonstration of electrical conductivity of an anode-grown G. sulfurreducensDL1 biofilm. Based on these results we suggest a role for nanowires of S. oneidensis and G. sulfurreducens in biofilm conductivity.

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.

Application of cyclic voltammetry to investigate enhanced catalytic current generation by biofilm-modified anodes of Geobacter sulfurreducens strain DL1 vs. variant strain KN400

A biofilm of Geobacter sulfurreducens will grow on an anode surface and catalyze the generation of an electrical current by oxidizing acetate and utilizing the anode as its metabolic terminal electron acceptor. Here we report qualitative analysis of cyclic voltammetry of anodes modified with biofilms of G. sulfurreducens strains DL1 and KN400 to predict possible rate-limiting steps in current generation. Strain KN400 generates approximately 2 to 8-fold greater current than strain DL1 depending upon the electrode material, enabling comparative electrochemical analysis to study the mechanism of current generation. This analysis is based on our recently reported electrochemical model for biofilm-catalyzed current generation expanded here to a five step model; Step 1 is mass transport of acetate, carbon dioxide and protons into and out of the biofilm, Step 2 is microbial turnover of acetate to carbon dioxide and protons, Step 3 is the non-concerted, 1-electron reduction of 8 equivalents of electron transfer (ET) mediator, Step 4 is extracellular electron transfer (EET) through the biofilm to the electrode surface, and Step 5 is the reversible oxidation of reduced mediator by the electrode. Five idealized voltammetric current vs. potential dependencies (voltammograms) are derived, one for when each step in the model is assumed to limit catalytic current. Comparison to experimental voltammetry of DL1 and KN400 biofilm-modified anodes suggests that for both strains, the microbial oxidation of acetate (Step 2) is fast compared to microbial reduction of ET mediator (Step 3), and either Step 3 or EET through the biofilm (Step 4) limits catalytic current generation. The possible limitation of catalytic current by Step 4 is consistent with proton concentration gradients observed within these biofilms and finite thicknesses achieved by these biofilms. The model presented here has been universally designed for application to biofilms other than G. sulfurreducens and could serve as a platform for future quantitative voltammetric analysis of non-corrosive anode and cathode reactions catalyzed by microorganisms.

On Electron Transport through Geobacter Biofilms

Geobacter spp. can form a biofilm that is more than 20 μm thick on an anode surface by utilizing the anode as a terminal respiratory electron acceptor. Just how microbes transport electrons through a thick biofilm and across the biofilm/anode interface, and what determines the upper limit to biofilm thickness and catalytic activity (i.e., current, the rate at which electrons are transferred to the anode), are fundamental questions attracting substantial attention. A significant body of experimental evidence suggests that electrons are transferred from individual cells through a network of cytochromes associated with cell outer membranes, within extracellular polymeric substances, and along pili. Here, we describe what is known about this extracellular electron transfer process, referred to as electron superexchange, and its proposed role in biofilm anode respiration. Superexchange is able to account for many different types of experimental results, as well as for the upper limit to biofilm thickness and catalytic activity that Geobacter biofilm anodes can achieve.

Reply to the ‘Comment on “On electrical conductivity of microbial nanowires and biofilms”’ by N. S. Malvankar, M. T. Tuominen and D. R. Lovley, Energy Environ. Sci., 2012, 5, DOI: 10.1039/c2ee02613a

Geobacter sulfurreducens can acquire energy by coupling oxidation of acetate with extracellular electron transfer to an anode, forming an electrically conductive biofilm extending many cell lengths away from the anode surface. Owing to their conductivity, such biofilms may play important roles in emerging technologies referred to as bioelectrochemical systems (BES). In these systems, microbes are used to catalyze anode processes for which abiotic catalysts do not exist, such as wastewater treatment and energy generation from biomass by fuel cells. Two models describing the conductive nature of G. sulfurreducens biofilms grown on anodes (biofilm anodes) have recently been put forth; superexchange proposed by our group, recently published in Energy and Environmental Science, which invokes electron-transfer among a network of cytochromes, and metallic-like conductivity proposed by Malvankar et al., recently published in Nature Nanotechnology, which invokes intrinsic conductivity of certain secreted microbial filaments referred to as nanowires. Here, we respond to criticisms raised by Malvankar et al. in the preceding commentary concerning superexchange.

Study of the Mechanism of Catalytic Activity of G. Sulfurreducens Biofilm Anodes during Biofilm Growth

The number of investigations involving bioelectrochemical systems (BES), processes in which microorganisms catalyze electrode reactions, is increasing while their mechanisms remain unresolved. Geobacter sulfurreducens strain DL1 is a model electrode catalyst that forms multimicrobe-thick biofilms on anodes that catalyze the oxidation of acetate to result in an electric current. Here, we report the characterization by cyclic voltammetry (CV) of DL1 biofilm-modified anodes (biofilm anodes) performed during biofilm development. This characterization, based on our recently reported model of biofilm anode catalytic activity, indicates the following. 1) As a biofilm grows, catalytic activity scales linearly with the amount of anode-accessible redox cofactor in the biofilm. This observation is consistent with a catalytic activity that is limited during biofilm growth by electron transport from within cells to the extracellular redox cofactor. 2) Distinct voltammetric features are exhibited that reflect the presence of a redox cofactor expressed by cells that initially colonize an anode that is not involved in catalytic current generation.

A self-assembling self-repairing microbial photoelectrochemical solar cell

Biologically-based approaches to large-scale solar power generation promise low cost durable technologies that will exhibit the self-repairing capabilities of photosynthetic organisms1–3 (Basic Research Needs for Solar Energy Utilization, U.S. Department of Energy, Washington DC, 2005; J. Barber and B. Andersson, Trends Biochem. Sci., 1992, 17, 61; A. Huijser et al., J. Phys. Chem. C, 2007, 111, 11726). Most proposed approaches however utilize photosynthetic proteins extracted from organisms4–6 (S. A. Trammell et al., J. Phys. Chem. C, 2007, 111, 17122; R. Das et al., Nano Lett., 2004, 4, 1079; E. Greenbaum, Science, 1985, 230, 1375) and forgo the self-repair capabilities of organisms resulting in short-lived power generation. Beginning with two non-descript graphite electrodes and marine sediment and seawater, we report here a proof-of-concept demonstration of a self-assembling and self-repairing microbial photoelectrochemical solar cell that generates electricity from sunlight7 (S. A. Licht, Nature, 1987, 330, 148). Time records of voltage and current generated by this solar cell reveal a circadian rhythm consistent with a photosynthetic nature. This result supports the interpretation that the electrode reactions are catalyzed by self-maintaining biofilms spontaneously formed on each electrode surface, and that the electrode reactants are photosynthetically regenerated from the electrode products by a self-maintaining spontaneously formed photosynthetic consortium. Our finding suggests a strait-forward approach toward durable biologically-based solar power generation.