Matthew D. Yates

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.

Measuring conductivity of living Geobacter sulfurreducens biofilms

To the Editor — Certain microorganisms can use an electrode as a metabolic electron acceptor or donor by means of extracellular electron transport (EET) processes1,2. Such microorganisms are studied as potential catalysts for electrode reactions such as the electrosynthesis of fuel precursors from reduction of CO2 using renewable sources of electricity3. The appeal of microbial electrode catalysts is that they self-assemble and self-heal, and the prospect of optimizing their catalytic properties (for example, reaction product and yield) through molecular engineering. In addition to enabling electron transport across a microbial/electrode interface, EET processes can often facilitate long-distance electron transport, resulting in the formation of multi-cell-thick electrode-grown biofilms, which are electrically conductive and can exceed 100 μm thickness. Such biofilms challenge the notion that biological electron transport is limited to molecular length scales. The fundamental mechanism of EET underlying biofilm conductivity has implications across many disciplines and is yet unresolved. Malvankar et al. reported that living electrode-grown biofilms comprising Geobacter sulfurreducens, a well-studied long-distance EET-capable microorganism, possess metallic-like conductivity similar to that of organic semiconductors4, a property that would make these biofilms unique among all biological materials. Electrochemical gating measurements were performed in a manner similar to that used to study conducting polymer films in electrolytic solutions5. Based on the resulting conductivity versus gate potential response, the authors proposed that living electrodegrown G. sulfurreducens biofilms are metallic-like conductors. When performing our own electrical electrochemical gating measurements of living electrode-grown G. sulfurreducens biofilms, we obtained a distinctly different conductivity versus gate potential response — one consistent with redox conductivity, similar to that of redox polymers6 and not consistent with metalliclike conductivity (Fig. 1 and Supplementary Fig. 1)5. Furthermore, it was recently demonstrated that conductivity of living electrode-grown G. sulfurreducens biofilms decreases with decreasing temperature in a manner that is also consistent with redox conductivity and not with metallic-like conductivity1. And it was also recently demonstrated that conductivity of these biofilms examined in air increases with decreasing temperature when the ambient water content is kept constant, and decreases with decreasing temperature when the ambient relative humidity is kept constant2. Again, both dependencies are consistent with redox conductivity and not with metallic-like conductivity2. Redox conductivity is ubiquitous in biological systems at molecular length scales, but is without precedence for distances over which electron transport appears to occur through electrode-grown G. sulfurreducens biofilms. The different conductivity versus gate potential response we obtained for living electrode-grown G. sulfurreducens biofilms prompted us to undertake a direct comparison of electrochemical gating measurements performed using our methods and measurements we replicated using the methods of Malvankar and colleagues. In this comparison, electrochemical gating measurements were performed on living G. sulfurreducens biofilms (Fig. 1 and Supplementary Fig. 1) as well as on two well-known conducting polymers: electropolymerized polyaniline, a known organic semiconductor5 (referred to here as PANI) (Fig. 2 and Supplementary Fig. 2); and poly(Nvinylimidazole [Os(bipyridine)2Cl]), a known redox conductor7 (referred to here as PVI-Os(bipy)2Cl) (Fig. 2 and Supplementary Fig. 3). Following Malvankar and colleagues’ approach, our biofilm electrochemical gating measurements were performed under physiologically relevant conditions in an aqueous electrolyte medium using gold source and drain electrodes patterned on a glass surface. Biofilms were grown that extended across the gap separating the electrodes, electrically connecting the source and drain. Different potentials were applied to the electrodes (ES and ED), generating a source–drain current (ISD) through the biofilm between the electrodes. In the limit of sufficiently small source–drain voltage, VSD = ED – ES ≤ 0.05 V (ref. 1), Ohm’s law applies such that:

Toward understanding long-distance extracellular electron transport in an electroautotrophic microbial community

Microbial electrosynthesis (ME) seeks to use electroautotrophy (the reduction of CO2 by microbial electrode catalysts) to generate useful multi-carbon compounds. It combines the utility of electrosynthesis with the durability of microorganisms and potential to engineer microbial metabolic processes. Central to achieving efficient ME is understanding the extracellular electron transport (EET) processes that enable certain microorganisms to utilize electrodes as metabolic electron donors. The Marinobacter-Chromatiaceae-Labrenzia (MCL) biocathode is an electroautotrophic biofilm-forming microbial community enriched from seawater that grows aerobically on gold or graphite cathodes, which we study to understand the mechanisms underpinning electroautotrophy. Evidence suggests that MCL reduces O2 using the cathode as its sole electron donor, directing a portion of the acquired electrons and energy to fix CO2 for biomass. A key feature of MCL is that it grows at +310 mV vs. SHE. Here, we apply electrochemical gating measurements, originally developed to study electron transport through polymer films, to study EET through living MCL biofilms. The results indicate that MCL biofilms employ a redox conduction mechanism to transport electrons across the biofilm/electrode interface and into the biofilm over multiple cell lengths (at least 5 μm) away from the electrode surface. In addition to making living MCL biofilms electrically conductive (60 μS cm−1 at 30 °C – more than 10 times greater conductivity than any other living microbial biofilm for which reliable measurements have been made), it enables electron uptake by cells not in direct contact with the electrode surface, which has not been previously reported for any biocathode. Confocal resonance Raman microscopy confirms the presence of c-type cytochromes as the putative redox cofactors involved in LD-EET, consistent with the activation energy for LD-EET obtained from the temperature dependency of the electrochemical gating measurements. These results provide the first report and mechanistic characterization of long-distance EET occurring within a multi-cell thick electroautotrophic biofilm – key milestones toward rational design and optimization of viable ME systems.

Redox-gradient driven electron transport in a mixed community anodic biofilm

Here, we describe the long-distance (multi-cell-length) extracellular electron transport (LD-EET) that occurs in an anode-grown mixed community biofilm (MCB) enriched from river sediment that contains 3%-45% Geobacter spp. High signal-to-noise temperature-dependent electrochemical gating measurements (EGM) using interdigitated microelectrode arrays reveal a peak-shaped electrical conductivity vs. potential dependency, indicating MCB acts as a redox conductor, similar to pure culture anode-grown Geobacter sulfurreducens biofilms (GSB). EGM also reveal that the maximum sustained rate of LD-EET in MCB is comparable to GSB, and the same whether under acetate-oxidizing or acetate-free conditions. Voltammetry indicated that MCB possesses 3- to 5-fold less electrode-accessible redox cofactors than GSB, suggesting that MCB may be more efficiently organized than GSB for LD-EET or that a small portion of electrode accessible redox cofactors of GSB are involved in LD-EET. The activation energy for LD-EET (0.11 ± 0.01 eV) was comparable to GSB, consistent with the possible role of c-type cytochromes as LD-EET cofactors, detected in abundance by confocal resonance Raman microscopy. Taken together, the results demonstrate LD-EET for a mixed community anode-grown microbial biofilm that is remarkably similar to GSB even though it contains many different types of microorganisms and appears to utilize far fewer EET redox cofactors.

On the relationship between long-distance and heterogeneous electron transfer in electrode-grown Geobacter sulfurreducens biofilms

The ability of certain microorganisms to live in a multi-cell thick, electrode-grown biofilm by utilizing the electrode as a metabolic electron acceptor or donor requires electron transfer across cell membranes, through the biofilm, and across the biofilm/electrode interface. Even for the most studied system, anode-grown Geobacter sulfurreducens, the mechanisms underpinning each process and how they connect is largely unresolved. Here we report on G. sulfurreducens biofilms grown across the gap separating two electrodes by maintaining one electrode at 0.300V vs. Ag/AgCl (0.510V vs. SHE) to act as a sustained metabolic electron acceptor while the second electrode was at open circuit. The poised electrode exhibited the characteristic current-time profile for electrode-dependent G. sulfurreducens biofilm growth. The open circuit potential (OCP) of the second electrode however increased after initially decreasing for 1.5-2days. The increase in OCP is taken to indicate the point at which the growing biofilm bridged the gap between the electrodes, enabling cells in contact with the open circuit electrode to utilize the poised electrode as an electron acceptor. After but not prior to reaching this point, the second electrode was able to act as a sustainable electron acceptor immediately after being placed under potential control without requiring further time to develop. These results indicate that heterogeneous ET (H-ET) across the biofilm/electrode interface and long-distance ET (LD-ET) through the biofilm are highly correlated, if not inseparable, and may share many common components.

Characterizing Electron Transport through Living Biofilms

Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under physiologically relevant conditions.1 These measurements are performed on living biofilms in aqueous medium using source and drain electrodes patterned on a glass surface in a specialized configuration referred to as an interdigitated electrode (IDA) array. A biofilm is grown that extends across the gap connecting the source and drain. Potentials are applied to the electrodes (ES and ED) generating a source-drain current (ISD) through the biofilm between the electrodes. The dependency of electrical conductivity on gate potential (the average of the source and drain potentials, EG = [ED + ES]/2) is determined by systematically changing the gate potential and measuring the resulting source-drain current. The dependency of conductivity on gate potential provides mechanistic information about the extracellular electron transport process underlying the electrical conductivity of the specific biofilm under investigation. The electrochemical gating measurement method described here is based directly on that used by M. S. Wrighton2,3 and colleagues and R. W. Murray4,5,6 and colleagues in the 1980s to investigate thin film conductive polymers.

Internal Redox Polarity of an Individual G. sulfurreducens Bacterial Cell Attached to an Inorganic Substrate

Bacterial cell polarity is an internal asymmetric distribution of subcellular components, including proteins, lipids, and other molecules that correlates with the cell ability to sense energy and metabolite sources, chemical signals, quorum signals, toxins, and movement in the desired directions. This ability also plays central role in cell attachment to various surfaces and biofilm formation. Mechanisms and factors controlling formation of this cell internal asymmetry are not completely understood. As a step in this direction, in the present work, we develop an approach for analyzing how information about inorganic substrate can be non-genetically coded inside an individual bacterial cell. As a model system, we use G. sulfurreducens cells attached to an inorganic mineral, mica. The approach utilizes confocal Raman microscopy, Gaussian deconvolution, and Principal Component Analysis (PCA) and allows for quick label-free identification of the molecular signature of cytochrome intracellular location and the cell to substrate binding down to the level of individual bacterial cells. Our results describe a spectroscopic signature of cell adhesion and how the information about cell adhesion can be coded inside individual bacterial cells.