characterizing-electron-transport-through-living-biofilms

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 ...

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.

Harnessing microbially generated power on the seafloor.

Electrode-immobilized microperoxidase-11 exhibited a titratable potentiometric response to imidazole, demonstrating both molecular recognition and the capability for "switchable" changes in the affinity of an immobilized redox-receptor for a target ligand.

A model recognition switch. Electrochemical control and transduction of imidazole binding by electrode-immobilized microperoxidase-11.

Hydrophilic gold nanoclusters were immobilized onto monolayer-modified gold electrodes and PF6-(-)induced rectification and stepwise capacitance charging was studied in aqueous supporting electrolyte by cyclic voltammetry and ac voltammetry.

Ion-induced discrete charging of immobilized water-soluble gold nanoclusters.

Apo-myoglobin, labeled with the environmentally sensitive redox probe RuII(NH3)4(1,10-phenanthroline-5-maleimide)2+, was immobilized onto gold electrodes modified with 11-mercaptoundecanoic acid and subsequently labeled with biotin; avidin binding to the immobilized biotin was specifically and quantitatively detected by a change in cyclic voltammetry of the co-immobilized probe.

A reagentless electrochemical biosensor based on a protein scaffold.

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.

Study of the mechanism of catalytic activity of G. sulfurreducens biofilm anodes during biofilm growth.

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.

Long-range electron transport in Geobacter sulfurreducens biofilms is redox gradient-driven.

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.

Electrochemical investigation of a microbial solar cell reveals a nonphotosynthetic biocathode catalyst.

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. 

Spatially resolved confocal resonant Raman microscopic analysis of anode-grown Geobacter sulfurreducens biofilms.

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. 

A previously uncharacterized, nonphotosynthetic member of the Chromatiaceae is the primary CO2-fixing constituent in a self-regenerating biocathode.

Microorganisms that respire electrodes may be exploited for biotechnology applications if key pathways for extracellular electron transfer can be identified and manipulated through bioengineering. To determine whether expression of proposed Biocathode-MCL extracellular electron transfer proteins are changed by modulating electrode potential without disrupting the relative distribution of microbial constituents, metaproteomic and 16S rRNA gene expression analyses were performed after switching from an optimal to suboptimal potential based on an expected decrease in electrode respiration. Five hundred and seventy-nine unique proteins were identified across both potentials, the majority of which were assigned to three previously defined Biocathode-MCL metagenomic clusters: a Marinobacter sp., a member of the family Chromatiaceae, and a Labrenzia sp (abbreviated as MCL). Statistical analysis of spectral counts using the Fisher's exact test identified 16 proteins associated with the optimal potential, five of which are predicted electron transfer proteins. The majority of proteins associated with the suboptimal potential were involved in protein turnover/synthesis, motility, and membrane transport. Unipept and 16S rRNA gene expression analyses indicated that the taxonomic profile of the microbiome did not change after 52 h at the suboptimal potential. These findings show that protein expression is sensitive to the electrode potential without inducing shifts in community composition, a feature that may be exploited for engineering Biocathode-MCL. All MS data have been deposited in the ProteomeXchange with identifier PXD001590 (http://proteomecentral.proteomexchange.org/dataset/PXD001590). 

Metaproteomic evidence of changes in protein expression following a change in electrode potential in a robust biocathode microbiome.

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. 

Thermally activated long range electron transport in living biofilms.

Measuring conductivity of living Geobacter sulfurreducens biofilms.

Biocathodes provide a stable electron source to drive reduction reactions in electrotrophic microbial electrochemical systems. Electroautotrophic biocathode communities may be more robust than monocultures in environmentally relevant settings, but some members are not easily cultivated outside the electrode environment. We previously used metagenomics and metaproteomics to propose a pathway for coupling extracellular electron transfer (EET) to carbon fixation in " Tenderia electrophaga," an uncultivated but dominant member of an electroautotrophic biocathode community. Here we validate and refine this proposed pathway using metatranscriptomics of replicate aerobic biocathodes poised at the growth potential level of 310 mV and the suboptimal 470 mV (versus the standard hydrogen electrode). At both potentials, transcripts were more abundant from " Tenderia electrophaga" than from any other constituent, and its relative activity was positively correlated with current. Several genes encoding key components of the proposed " Tenderia electrophaga" EET pathway were more highly expressed at 470 mV, consistent with a need for cells to acquire more electrons to obtain the same amount of energy as at 310 mV. These included , encoding a homolog of a protein known to be involved in iron oxidation. Mean expression of all CO fixation-related genes is 0.27 log-fold higher at 310 mV, indicating that reduced energy availability at 470 mV decreased CO fixation. Our results substantiate the claim that " Tenderia electrophaga" is the key electroautotroph, which will help guide further development of this community for microbial electrosynthesis.  Bacteria that directly use electrodes as metabolic electron donors (biocathodes) have been proposed for applications ranging from microbial electrosynthesis to advanced bioelectronics for cellular communication with machines. However, just as we understand very little about oxidation of analogous natural insoluble electron donors, such as iron oxide, the organisms and extracellular electron transfer (EET) pathways underlying the electrode-cell direct electron transfer processes are almost completely unknown. Biocathodes are a stable biofilm cultivation platform to interrogate both the rate and mechanism of EET using electrochemistry and to study the electroautotrophic organisms that catalyze these reactions. Here we provide new evidence supporting the hypothesis that the uncultured bacterium " Tenderia electrophaga" directly couples extracellular electron transfer to CO fixation. Our results provide insight into developing biocathode technology, such as microbial electrosynthesis, as well as advancing our understanding of chemolithoautotrophy.

Metatranscriptomics Supports the Mechanism for Biocathode Electroautotrophy by " Tenderia electrophaga".

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.

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

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.

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

Protein molecular conductance has attracted attention from researchers for the possibility of constructing innovative flexible biocompatible nanoscale electronic devices and smart hybrid materials. Due to protein complexity, most evaluations of protein conductivity are based on the simple estimation of protein's molecular orbital energy levels and spatial distributions without analysing its protein interaction with electrodes and the calculation of the rates of electron transfer (ET). In the present work, we included in our density functional theory (DFT) analysis an approach based on the non-equilibrium Green's function (NEGF) allowing for calculation from the first principles the molecular interaction with electrodes and thus the role of electrode materials, Fermi level, the thermal distribution of electronic energy levels, and the coupling efficiency between the molecule and the electrodes. Compared to proteins studied so far, mainly artificial peptides, heme-containing cytochromes, and bacterial pili, we choose rubredoxin for our calculation. Rubredoxin contains a non-heme iron that, as we have discovered recently, can be involved in extracellular ET in electroactive bacterial biofilms (Yates et al., Energy Environ. Sci., 2016, 9, 3544-3558). Our calculations show that an iron atom incorporated into the protein structure as an iron-sulfur cluster opens a transmission path at the energy corresponding to the Fermi level of the electrodes. This allows the protein to become an extremely efficient conductor at very low bias voltages (<±350 mV). Calculation of the role of protein amino acids based on the local density of states and electron transfer paths reveals that neither aromatic amino acid Tyr nor Phe at any ring orientation participates in coherent ET through the FeS cluster of the protein. Moreover, direct ET through surrounding amino acids, bypassing FeS, is possible only at biases ±1.5 to ±2 V. The polar amino acid Asn might participate in ET at these bias voltages. The conductivity of the protein core substantially depends on the polarity of the applied electric field, allowing for unidirectional ET and operation of the protein as a molecular rectifier. These results can be used for a wise de novo design of proteins for molecular electronics and cellular energy converting devices, particularly for utilization of iron doping in the construction of conductive protein wires.

Article	Year
Harnessing microbially generated power on the seafloor. Nature biotechnology\| PubMed ID: 12091916	2002
A model recognition switch. Electrochemical control and transduction of imidazole binding by electrode-immobilized microperoxidase-11. Chemical communications (Cambridge, England)\| PubMed ID: 12120519	2002
Ion-induced discrete charging of immobilized water-soluble gold nanoclusters. Chemical communications (Cambridge, England)\| PubMed ID: 12189885	2002
A reagentless electrochemical biosensor based on a protein scaffold. Chemical communications (Cambridge, England)\| PubMed ID: 12613601	2003
Study of the mechanism of catalytic activity of G. sulfurreducens biofilm anodes during biofilm growth. ChemSusChem\| PubMed ID: 22581467	2012
Long-range electron transport in Geobacter sulfurreducens biofilms is redox gradient-driven. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 22955881	2012
Electrochemical investigation of a microbial solar cell reveals a nonphotosynthetic biocathode catalyst. Applied and environmental microbiology\| PubMed ID: 23603672	2013
Spatially resolved confocal resonant Raman microscopic analysis of anode-grown Geobacter sulfurreducens biofilms. Chemphyschem : a European journal of chemical physics and physical chemistry\| PubMed ID: 24402861	2014
A previously uncharacterized, nonphotosynthetic member of the Chromatiaceae is the primary CO2-fixing constituent in a self-regenerating biocathode. Applied and environmental microbiology\| PubMed ID: 25398855	2015
Metaproteomic evidence of changes in protein expression following a change in electrode potential in a robust biocathode microbiome. Proteomics\| PubMed ID: 26260905	2015
Thermally activated long range electron transport in living biofilms. Physical chemistry chemical physics : PCCP\| PubMed ID: 26611733	2015
Measuring conductivity of living Geobacter sulfurreducens biofilms. Nature nanotechnology\| PubMed ID: 27821847	2016
Metatranscriptomics Supports the Mechanism for Biocathode Electroautotrophy by " Tenderia electrophaga". mSystems\| PubMed ID: 28382330	2017
On the relationship between long-distance and heterogeneous electron transfer in electrode-grown Geobacter sulfurreducens biofilms. Bioelectrochemistry (Amsterdam, Netherlands)\| PubMed ID: 28963994	2018
Redox-gradient driven electron transport in a mixed community anodic biofilm. FEMS microbiology ecology\| PubMed ID: 29722806	2018
Effect of iron doping on protein molecular conductance. Physical chemistry chemical physics : PCCP\| PubMed ID: 29748677	2018

Leonard Tender

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