Sarah M. Strycharz-Glaven

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.

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.

Electrochemical Investigation of a Microbial Solar Cell Reveals a Nonphotosynthetic Biocathode Catalyst

ABSTRACT Microbial solar cells (MSCs) are microbial fuel cells (MFCs) that generate their own oxidant and/or fuel through photosynthetic reactions. Here, we present electrochemical analyses and biofilm 16S rRNA gene profiling of biocathodes of sediment/seawater-based MSCs inoculated from the biocathode of a previously described sediment/seawater-based MSC. Electrochemical analyses indicate that for these second-generation MSC biocathodes, catalytic activity diminishes over time if illumination is provided during growth, whereas it remains relatively stable if growth occurs in the dark. For both illuminated and dark MSC biocathodes, cyclic voltammetry reveals a catalytic-current–potential dependency consistent with heterogeneous electron transfer mediated by an insoluble microbial redox cofactor, which was conserved following enrichment of the dark MSC biocathode using a three-electrode configuration. 16S rRNA gene profiling showed Gammaproteobacteria, most closely related to Marinobacter spp., predominated in the enriched biocathode. The enriched biocathode biofilm is easily cultured on graphite cathodes, forms a multimicrobe-thick biofilm (up to 8.2 μm), and does not lose catalytic activity after exchanges of the reactor medium. Moreover, the consortium can be grown on cathodes with only inorganic carbon provided as the carbon source, which may be exploited for proposed bioelectrochemical systems for electrosynthesis of organic carbon from carbon dioxide. These results support a scheme where two distinct communities of organisms develop within MSC biocathodes: one that is photosynthetically active and one that catalyzes reduction of O2 by the cathode, where the former partially inhibits the latter. The relationship between the two communities must be further explored to fully realize the potential for MSC applications.

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.

A Previously Uncharacterized, Nonphotosynthetic Member of the Chromatiaceae Is the Primary CO2-Fixing Constituent in a Self-Regenerating Biocathode

ABSTRACT Biocathode extracellular electron transfer (EET) may be exploited for biotechnology applications, including microbially mediated O2 reduction in microbial fuel cells and microbial electrosynthesis. However, biocathode mechanistic studies needed to improve or engineer functionality have been limited to a few select species that form sparse, homogeneous biofilms characterized by little or no growth. Attempts to cultivate isolates from biocathode environmental enrichments often fail due to a lack of some advantage provided by life in a consortium, highlighting the need to study and understand biocathode consortia in situ. Here, we present metagenomic and metaproteomic characterization of a previously described biocathode biofilm (+310 mV versus a standard hydrogen electrode [SHE]) enriched from seawater, reducing O2, and presumably fixing CO2 for biomass generation. Metagenomics identified 16 distinct cluster genomes, 15 of which could be assigned at the family or genus level and whose abundance was roughly divided between Alpha- and Gammaproteobacteria. A total of 644 proteins were identified from shotgun metaproteomics and have been deposited in the the ProteomeXchange with identifier PXD001045. Cluster genomes were used to assign the taxonomic identities of 599 proteins, with Marinobacter, Chromatiaceae, and Labrenzia the most represented. RubisCO and phosphoribulokinase, along with 9 other Calvin-Benson-Bassham cycle proteins, were identified from Chromatiaceae. In addition, proteins similar to those predicted for iron oxidation pathways of known iron-oxidizing bacteria were observed for Chromatiaceae. These findings represent the first description of putative EET and CO2 fixation mechanisms for a self-regenerating, self-sustaining multispecies biocathode, providing potential targets for functional engineering, as well as new insights into biocathode EET pathways using proteomics.

Spatially Resolved Confocal Resonant Raman Microscopic Analysis of Anode-Grown Geobacter sulfurreducens Biofilms

When grown on the surface of an anode electrode, Geobacter sulfurreducens forms a multi-cell thick biofilm in which all cells appear to couple the oxidation of acetate with electron transport to the anode, which serves as the terminal metabolic electron acceptor. Just how electrons are transported through such a biofilm from cells to the underlying anode surface over distances that can exceed 20 microns remains unresolved. Current evidence suggests it may occur by electron hopping through a proposed network of redox cofactors composed of immobile outer membrane and/or extracellular multi-heme c-type cytochromes. In the present work, we perform a spatially resolved confocal resonant Raman (CRR) microscopic analysis to investigate anode-grown Geobacter biofilms. The results confirm the presence of an intra-biofilm redox gradient whereby the probability that a heme is in the reduced state increases with increasing distance from the anode surface. Such a gradient is required to drive electron transport toward the anode surface by electron hopping via cytochromes. The results also indicate that at open circuit, when electrons are expected to accumulate in redox cofactors involved in electron transport due to the inability of the anode to accept electrons, nearly all c-type cytochrome hemes detected in the biofilm are oxidized. The same outcome occurs when a comparable potential to that measured at open circuit (-0.30 V vs. SHE) is applied to the anode, whereas nearly all hemes are reduced when an exceedingly negative potential (-0.50 V vs. SHE) is applied to the anode. These results suggest that nearly all c-type cytochrome hemes detected in the biofilm can be electrochemically accessed by the electrode, but most have oxidation potentials too negative to transport electrons originating from acetate metabolism. The results also reveal a lateral heterogeneity (x-y dimensions) in the type of c-type cytochromes within the biofilm that may affect electron transport to the electrode.

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: