Ashley E. Franks

Microbial Electrosynthesis: Feeding Microbes Electricity To Convert Carbon Dioxide and Water to Multicarbon Extracellular Organic Compounds

ABSTRACT The possibility of providing the acetogenic microorganism Sporomusa ovata with electrons delivered directly to the cells with a graphite electrode for the reduction of carbon dioxide to organic compounds was investigated. Biofilms of S. ovata growing on graphite cathode surfaces consumed electrons with the reduction of carbon dioxide to acetate and small amounts of 2-oxobutyrate. Electrons appearing in these products accounted for over 85% of the electrons consumed. These results demonstrate that microbial production of multicarbon organic compounds from carbon dioxide and water with electricity as the energy source is feasible. IMPORTANCE Reducing carbon dioxide to multicarbon organic chemicals and fuels with electricity has been identified as an attractive strategy to convert solar energy that is harvested intermittently with photovoltaic technology and store it as covalent chemical bonds. The organic compounds produced can then be distributed via existing infrastructure. Nonbiological electrochemical reduction of carbon dioxide has proven problematic. The results presented here suggest that microbiological catalysts may be a robust alternative, and when coupled with photovoltaics, current-driven microbial carbon dioxide reduction represents a new form of photosynthesis that might convert solar energy to organic products more effectively than traditional biomass-based strategies. Reducing carbon dioxide to multicarbon organic chemicals and fuels with electricity has been identified as an attractive strategy to convert solar energy that is harvested intermittently with photovoltaic technology and store it as covalent chemical bonds. The organic compounds produced can then be distributed via existing infrastructure. Nonbiological electrochemical reduction of carbon dioxide has proven problematic. The results presented here suggest that microbiological catalysts may be a robust alternative, and when coupled with photovoltaics, current-driven microbial carbon dioxide reduction represents a new form of photosynthesis that might convert solar energy to organic products more effectively than traditional biomass-based strategies.

Direct Exchange of Electrons Within Aggregates of an Evolved Syntrophic Coculture of Anaerobic Bacteria

Wired for Life Syntrophic bacteria live on the metabolic by-products of a partner species. The exchange of the by-products accompanies a flow of electrons in the opposite direction that helps some species grow in conditions that would otherwise be unfavorable. In mixed anaerobic cultures of two related Geobacter species, Summers et al. (p. 1413) observed that one species evolved to promote the transfer of electrons directly to the other, in large aggregated cell clusters, without coupling to common anaerobic by-products such as hydrogen or formate. Selection pressures in nine parallel populations all resulted in a point mutation that truncated a protein involved in the production of small hairlike projections involved in intercellular communication—pili—and indirectly increased the expression of a c-type multiheme cytochrome responsible for extracellular electron transfer. The evolved aggregates were conductive, suggesting that the direct exchange of electrons between partner species is a possible alternative route to anaerobic syntrophy rather than interspecies hydrogen transfer; indeed, deleting a gene that encodes a hydrogenase involved in hydrogen transfer conferred a growth advantage in the cocultures. Direct cell-to-cell electron transfer occurs between two related species of bacteria. Microbial consortia that cooperatively exchange electrons play a key role in the anaerobic processing of organic matter. Interspecies hydrogen transfer is a well-documented strategy for electron exchange in dispersed laboratory cultures, but cooperative partners in natural environments often form multispecies aggregates. We found that laboratory evolution of a coculture of Geobacter metallireducens and Geobacter sulfurreducens metabolizing ethanol favored the formation of aggregates that were electrically conductive. Sequencing aggregate DNA revealed selection for a mutation that enhances the production of a c-type cytochrome involved in extracellular electron transfer and accelerates the formation of aggregates. Aggregate formation was also much faster in mutants that were deficient in interspecies hydrogen transfer, further suggesting direct interspecies electron transfer.

Electrosynthesis of Organic Compounds from Carbon Dioxide Is Catalyzed by a Diversity of Acetogenic Microorganisms

ABSTRACT Microbial electrosynthesis, a process in which microorganisms use electrons derived from electrodes to reduce carbon dioxide to multicarbon, extracellular organic compounds, is a potential strategy for capturing electrical energy in carbon-carbon bonds of readily stored and easily distributed products, such as transportation fuels. To date, only one organism, the acetogen Sporomusa ovata, has been shown to be capable of electrosynthesis. The purpose of this study was to determine if a wider range of microorganisms is capable of this process. Several other acetogenic bacteria, including two other Sporomusa species, Clostridium ljungdahlii, Clostridium aceticum, and Moorella thermoacetica, consumed current with the production of organic acids. In general acetate was the primary product, but 2-oxobutyrate and formate also were formed, with 2-oxobutyrate being the predominant identified product of electrosynthesis by C. aceticum. S. sphaeroides, C. ljungdahlii, and M. thermoacetica had high (>80%) efficiencies of electrons consumed and recovered in identified products. The acetogen Acetobacterium woodii was unable to consume current. These results expand the known range of microorganisms capable of electrosynthesis, providing multiple options for the further optimization of this process.

Anode Biofilm Transcriptomics Reveals Outer Surface Components Essential for High Density Current Production in Geobacter sulfurreducens Fuel Cells

The mechanisms by which Geobacter sulfurreducens transfers electrons through relatively thick (>50 µm) biofilms to electrodes acting as a sole electron acceptor were investigated. Biofilms of Geobacter sulfurreducens were grown either in flow-through systems with graphite anodes as the electron acceptor or on the same graphite surface, but with fumarate as the sole electron acceptor. Fumarate-grown biofilms were not immediately capable of significant current production, suggesting substantial physiological differences from current-producing biofilms. Microarray analysis revealed 13 genes in current-harvesting biofilms that had significantly higher transcript levels. The greatest increases were for pilA, the gene immediately downstream of pilA, and the genes for two outer c-type membrane cytochromes, OmcB and OmcZ. Down-regulated genes included the genes for the outer-membrane c-type cytochromes, OmcS and OmcT. Results of quantitative RT-PCR of gene transcript levels during biofilm growth were consistent with microarray results. OmcZ and the outer-surface c-type cytochrome, OmcE, were more abundant and OmcS was less abundant in current-harvesting cells. Strains in which pilA, the gene immediately downstream from pilA, omcB, omcS, omcE, or omcZ was deleted demonstrated that only deletion of pilA or omcZ severely inhibited current production and biofilm formation in current-harvesting mode. In contrast, these gene deletions had no impact on biofilm formation on graphite surfaces when fumarate served as the electron acceptor. These results suggest that biofilms grown harvesting current are specifically poised for electron transfer to electrodes and that, in addition to pili, OmcZ is a key component in electron transfer through differentiated G. sulfurreducens biofilms to electrodes.

Potential for Direct Interspecies Electron Transfer in Methanogenic Wastewater Digester Aggregates

ABSTRACT Mechanisms for electron transfer within microbial aggregates derived from an upflow anaerobic sludge blanket reactor converting brewery waste to methane were investigated in order to better understand the function of methanogenic consortia. The aggregates were electrically conductive, with conductivities 3-fold higher than the conductivities previously reported for dual-species aggregates of Geobacter species in which the two species appeared to exchange electrons via interspecies electron transfer. The temperature dependence response of the aggregate conductance was characteristic of the organic metallic-like conductance previously described for the conductive pili of Geobacter sulfurreducens and was inconsistent with electron conduction through minerals. Studies in which aggregates were incubated with high concentrations of potential electron donors demonstrated that the aggregates had no significant capacity for conversion of hydrogen to methane. The aggregates converted formate to methane but at rates too low to account for the rates at which that the aggregates syntrophically metabolized ethanol, an important component of the reactor influent. Geobacter species comprised 25% of 16S rRNA gene sequences recovered from the aggregates, suggesting that Geobacter species may have contributed to some but probably not all of the aggregate conductivity. Microorganisms most closely related to the acetate-utilizing Methanosaeta concilii accounted for more than 90% of the sequences that could be assigned to methane producers, consistent with the poor capacity for hydrogen and formate utilization. These results demonstrate for the first time that methanogenic wastewater aggregates can be electrically conductive and suggest that direct interspecies electron transfer could be an important mechanism for electron exchange in some methanogenic systems. IMPORTANCE The conversion of waste organic matter to methane is an important bioenergy strategy, and a similar microbial metabolism of complex organic matter in anaerobic soils and sediments plays an important role in the global carbon cycle. Studies with laboratory cultures have demonstrated that hydrogen or formate can serve as an electron shuttle between the microorganisms degrading organic compounds and methanogens. However, the importance of hydrogen and formate as intermediates in the conversion of organic matter to methane in natural communities is less clear. The possibility that microorganisms within some natural methanogenic aggregates may directly exchange electrons, rather than producing hydrogen or formate as an intermediary electron carrier, is a significant paradigm shift with implications for the modeling and design of anaerobic wastewater reactors and for understanding how methanogenic communities will respond to environmental perturbations. The conversion of waste organic matter to methane is an important bioenergy strategy, and a similar microbial metabolism of complex organic matter in anaerobic soils and sediments plays an important role in the global carbon cycle. Studies with laboratory cultures have demonstrated that hydrogen or formate can serve as an electron shuttle between the microorganisms degrading organic compounds and methanogens. However, the importance of hydrogen and formate as intermediates in the conversion of organic matter to methane in natural communities is less clear. The possibility that microorganisms within some natural methanogenic aggregates may directly exchange electrons, rather than producing hydrogen or formate as an intermediary electron carrier, is a significant paradigm shift with implications for the modeling and design of anaerobic wastewater reactors and for understanding how methanogenic communities will respond to environmental perturbations.

Novel strategy for three-dimensional real-time imaging of microbial fuel cell communities: monitoring the inhibitory effects of proton accumulation within the anode biofilm

Harvesting electricity from the environment, organic wastes, or renewable biomass with microbial fuel cells (MFCs) is an appealing strategy, but the destructive sampling required to investigate the anode-associated biofilms has hampered research designed to better understand and optimize microbe–anode interactions. Therefore, a MFC that permits real-time imaging of the anode biofilm with confocal scanning laser microscopy was developed. In this new MFC Geobacter sulfurreducens, an organism closely related to those often found on MFC anodes and capable of high current densities, produced current comparable to that previously reported with other MFC designs. G. sulfurreducens engineered to produce the fluorescent protein mcherry to facilitate real-time imaging produced current comparable to wild-type cells. Introducing C-SNARF-4, a pH-sensitive fluoroprobe, into the anode chamber revealed strong pH gradients within the anode biofilms. The pH decreased with increased proximity to the anode surface and from the exterior to the interior of biofilm pillars. Near the anode surface pH levels were as low as 6.1 compared to ca. 7 in the external medium. Various controls demonstrated that the proton accumulation was associated with current production. Dropping the pH of culture medium from 7 to 6 severely limited the growth of G. sulfurreducens. These results demonstrate that it is feasible to non-destructively monitor the activity of anode biofilms in real time and suggest that the accumulation of protons that are released from organic matter oxidation within anode biofilms can limit current production.

Stimulating the anaerobic degradation of aromatic hydrocarbons in contaminated sediments by providing an electrode as the electron acceptor.

The possibility that electrodes might serve as an electron acceptor to simulate the degradation of aromatic hydrocarbons in anaerobic contaminated sediments was investigated. Initial studies with Geobacter metallireducens demonstrated that although toluene was rapidly adsorbed onto the graphite electrodes it was rapidly oxidized to carbon dioxide with the electrode serving as the sole electron acceptor. Providing graphite electrodes as an electron acceptor in hydrocarbon-contaminated sediments significantly stimulated the removal of added toluene and benzene. Rates of toluene and benzene removal accelerated with continued additions of toluene and benzene. [(14)C]-Toluene and [(14)C]-benzene were quantitatively recovered as [(14)C]-CO(2), demonstrating that even though the graphite adsorbed toluene and benzene they were degraded. Introducing an electrode as an electron acceptor also accelerated the loss of added naphthalene and [(14)C]-naphthalene was converted to [(14)C]-CO(2). The results suggest that graphite electrodes can serve as an electron acceptor for the degradation of aromatic hydrocarbon contaminants in sediments, co-localizing the contaminants, the degradative organisms and the electron acceptor. Once in position, they provide a permanent, low-maintenance source of electron acceptor. Thus, graphite electrodes may offer an attractive alternative for enhancing contaminant degradation in anoxic environments.

Antifouling activities expressed by marine surface associated Pseudoalteromonas species

Abstract Members of the marine bacterial genus Pseudoalteromonas have been found in association with living surfaces and are suggested to produce bioactive compounds against settlement of algal spores, invertebrate larvae, bacteria and fungi. To determine the extent by which these antifouling activities and the production of bioactive compounds are distributed amongst the members of the genus Pseudoalteromonas, 10 different Pseudoalteromonas species mostly derived from different host organisms were tested in a broad range of biofouling bioassays. These assays included the settlement of larvae of two ubiquitous invertebrates Hydroides elegans and Balanus amphitrite as well as the settlement of spores of the common fouling algae Ulva lactuca and Polysiphonia sp. The growth of bacteria and fungi, which are the initial fouling organisms on marine surfaces, was also assayed in the presence of each of the 10 Pseudoalteromonas species. It was found that most members of this genus produced a variety of bioactive compounds. The broadest range of inhibitory activities was expressed by Pseudoalteromonas tunicata which inhibited all target fouling organisms. Only two species, Pseudoalteromonas haloplanktis and Pseudoalteromonas nigrifaciens, displayed negligible activity in the bioassays. These were also the only two non-pigmented species tested in this study which indicates a correlation between production of bioactive compounds and expression of pigment. Three members, P. tunicata, Pseudoalteromonas citrea and Pseudoalteromonas rubra, were demonstrated to express autoinhibitory activity. It is suggested that most Pseudoalteromonas species are efficient producers of antifouling agents and that the production of inhibitory compounds by surface associated Pseudoalteromonas species may aid the host against colonisation of its surface.

Specific localization of the c‐type cytochrome OmcZ at the anode surface in current‐producing biofilms of Geobacter sulfurreducens

The outer-surface, c-type cytochrome OmcZ is essential for optimal current production with Geobacter sulfurreducens, a genetically tractable, environmentally relevant model microorganism for the production of electricity with microbial fuel cells in a diversity of environments. In order to further investigate the role of OmcZ in current production, its location was investigated with immunogold labelling. OmcZ was dispersed throughout the extracellular matrix surrounding the cells that accumulated at the bottom of the culture tubes of cells grown under standard conditions with fumarate as the electron acceptor. When G. sulfurreducens grew as a biofilm on a graphite electrode that served as an anode and the sole electron acceptor for growth, OmcZ was highly concentrated at the biofilm-electrode interface. Controls in which the biofilm was grown on the same graphite material, but with fumarate as the electron acceptor, did not have accumulations of OmcZ at the anode, corresponding with the reduced capacity for current production in fumarate-grown biofilms. The specific localization of OmcZ at the anode surface under current-producing conditions, coupled with the previously published finding that deleting the gene for OmcZ dramatically increases the resistance of electron exchange between the anode and the biofilm, suggests that OmcZ may serve as an electrochemical gate facilitating electron transfer from G. sulfurreducens biofilms to the anode surface.

Improved cathode materials for microbial electrosynthesis

Microbial electrosynthesis is a promising strategy for the microbial conversion of carbon dioxide to transportation fuels and other organic commodities, but optimization of this process is required for commercialization. Cathodes which enhance electrode–microbe electron transfer might improve rates of product formation. To evaluate this possibility, biofilms of Sporomusa ovata, which are effective in acetate electrosynthesis, were grown on a range of cathode materials and acetate production was monitored over time. Modifications of carbon cloth that resulted in a positive-charge enhanced microbial electrosynthesis. Functionalization with chitosan or cyanuric chloride increased acetate production rates 6–7 fold and modification with 3-aminopropyltriethoxysilane gave rates 3-fold higher than untreated controls. A 3-fold increase in electrosynthesis over untreated carbon cloth cathodes was also achieved with polyaniline cathodes. However, not all strategies to provide positively charged surfaces were successful, as treatment of carbon cloth with melamine or ammonia gas did not stimulate acetate electrosynthesis. Treating carbon cloth with metal, in particular gold, palladium, or nickel nanoparticles, also promoted electrosynthesis, yielding electrosynthesis rates that were 6-, 4.7- or 4.5-fold faster than the untreated control, respectively. Cathodes comprised of cotton or polyester fabric treated with carbon nanotubes yielded cathodes that supported acetate electrosynthesis rates that were ∼3-fold higher than carbon cloth controls. Recovery of electrons consumed in acetate was ∼80% for all materials. The results demonstrate that one approach to increase rates of carbon dioxide reduction in microbial electrosynthesis is to modify cathode surfaces to improve microbe-electrode interactions.