Link between capacity for current production and syntrophic growth in Geobacter species.
ABSTRACT: Electrodes are unnatural electron acceptors, and it is yet unknown how some Geobacter species evolved to use electrodes as terminal electron acceptors. Analysis of different Geobacter species revealed that they varied in their capacity for current production. Geobacter metallireducens and G. hydrogenophilus generated high current densities (ca. 0.2 mA/cm(2)), comparable to G. sulfurreducens. G. bremensis, G. chapellei, G. humireducens, and G. uraniireducens, produced much lower currents (ca. 0.05 mA/cm(2)) and G. bemidjiensis was previously found to not produce current. There was no correspondence between the effectiveness of current generation and Fe(III) oxide reduction rates. Some high-current-density strains (G. metallireducens and G. hydrogenophilus) reduced Fe(III)-oxides as fast as some low-current-density strains (G. bremensis, G. humireducens, and G. uraniireducens) whereas other low-current-density strains (G. bemidjiensis and G. chapellei) reduced Fe(III) oxide as slowly as G. sulfurreducens, a high-current-density strain. However, there was a correspondence between the ability to produce higher currents and the ability to grow syntrophically. G. hydrogenophilus was found to grow in co-culture with Methanosarcina barkeri, which is capable of direct interspecies electron transfer (DIET), but not with Methanospirillum hungatei capable only of H2 or formate transfer. Conductive granular activated carbon (GAC) stimulated metabolism of the G. hydrogenophilus - M. barkeri co-culture, consistent with electron exchange via DIET. These findings, coupled with the previous finding that G. metallireducens and G. sulfurreducens are also capable of DIET, suggest that evolution to optimize DIET has fortuitously conferred the capability for high-density current production to some Geobacter species.
Project description:Background:Magnetite-mediated direct interspecies electron transfer (DIET) between Geobacter and Methanosarcina species is increasingly being invoked to explain magnetite stimulation of methane production in anaerobic soils and sediments. Although magnetite-mediated DIET has been documented in defined co-cultures reducing fumarate or nitrate as the electron acceptor, the effects of magnetite have only been inferred in methanogenic systems. Methods:Concentrations of methane and organic acid were analysed with a gas chromatograph and high-performance liquid chromatography, respectively. The concentration of HCl-extractable Fe(II) was determined by the ferrozine method. The association of the defined co-cultures of G. metallireducens and M. barkeri with magnetite was observed with transmission electron micrographs. Results:Magnetite stimulated ethanol metabolism and methane production in defined co-cultures of G. metallireducens and M. barkeri; however, magnetite did not promote methane production in co-cultures initiated with a culture of G. metallireducens that could not produce electrically conductive pili (e-pili), unlike the conductive carbon materials that facilitate DIET in the absence of e-pili. Transmission electron microscopy revealed that G. metallireducens and M. barkeri were closely associated when magnetite was present, as previously observed in G. metallireducens/G. sulfurreducens co-cultures. These results show that magnetite can promote DIET between Geobacter and Methanosarcina species, but not as a substitute for e-pili, and probably functions to facilitate electron transfer from the e-pili to Methanosarcina. Conclusion:In summary, the e-pili are necessary for the stimulation of not only G. metallireducens/G. sulfurreducens, but also methanogenic G. metallireducens/M. barkeri co-cultures with magnetite.
Project description:Studies on the mechanisms for extracellular electron transfer in Geobacter species have primarily focused on Geobacter sulfurreducens, but the poor conservation of genes for some electron transfer components within the Geobacter genus suggests that there may be a diversity of extracellular electron transport strategies among Geobacter species. Examination of the gene sequences for PilA, the type IV pilus monomer, in Geobacter species revealed that the PilA sequence of Geobacter uraniireducens was much longer than that of G. sulfurreducens. This is of interest because it has been proposed that the relatively short PilA sequence of G. sulfurreducens is an important feature conferring conductivity to G. sulfurreducens pili. In order to investigate the properties of the G. uraniireducens pili in more detail, a strain of G. sulfurreducens that expressed pili comprised the PilA of G. uraniireducens was constructed. This strain, designated strain GUP, produced abundant pili, but generated low current densities and reduced Fe(III) very poorly. At pH 7, the conductivity of the G. uraniireducens pili was 3 × 10(-4) S/cm, much lower than the previously reported 5 × 10(-2) S/cm conductivity of G. sulfurreducens pili at the same pH. Consideration of the likely voltage difference across pili during Fe(III) oxide reduction suggested that G. sulfurreducens pili can readily accommodate maximum reported rates of respiration, but that G. uraniireducens pili are not sufficiently conductive to be an effective mediator of long-range electron transfer. In contrast to G. sulfurreducens and G. metallireducens, which require direct contact with Fe(III) oxides in order to reduce them, G. uraniireducens reduced Fe(III) oxides occluded within microporous beads, demonstrating that G. uraniireducens produces a soluble electron shuttle to facilitate Fe(III) oxide reduction. The results demonstrate that Geobacter species may differ substantially in their mechanisms for long-range electron transport and that it is important to have information beyond a phylogenetic affiliation in order to make conclusions about the mechanisms by which Geobacter species are transferring electrons to extracellular electron acceptors.
Project description:Direct interspecies electron transfer (DIET) has been identified as an efficient metabolism between symbiotically interacting organisms. One method of DIET uses conductive materials (e.g., granular activated carbon (GAC)) as a medium to shuttle electrons from electron donating organisms (eg., Geobacter metallireducens) to electron accepting organisms (e.g., Geobacter sulfurreducens and Methanosarcina barkeri). Conductive materials such as GAC, become negatively charged in DIET processes due to reduction by electron donating organisms. This high excess electron density in GAC leads to quantum tunnelling of electrons being a significant electron transfer mechanism for DIET. Thus, a theoretical model obeying the Wentzel-Kramers-Brillouin (WKB) approximation and Fermi-Dirac statistics was developed and simulated. In the model, the electron tunnelling transfer barrier was described by an effective rectangular barrier. The result of our 1D tunnelling simulations indicates that within 29.4 nm of the GAC, tunnelling can sufficiently supply electrons from GAC to G. sulfurreducens and M. barkeri. The phenomenon of tunnelling may also have significance as a stimulant of chemotaxis for G. sulfurreducens and other electron accepting microbes when attempting to adsorb onto GAC. This study sheds light on quantum tunnelling's significant potential in both bacterium and archaeon DIET-centric processes.
Project description:Direct interspecies electron transfer (DIET) has been recognized as an alternative to interspecies H2 transfer as a mechanism for syntrophic growth, but previous studies on DIET with defined co-cultures have only documented DIET with ethanol as the electron donor in the absence of conductive materials. Co-cultures of Geobacter metallireducens and Geobacter sulfurreducens metabolized propanol, butanol, propionate, and butyrate with the reduction of fumarate to succinate. G. metallireducens utilized each of these substrates whereas only electrons available from DIET supported G. sulfurreducens respiration. A co-culture of G. metallireducens and a strain of G. sulfurreducens that could not metabolize acetate oxidized acetate with fumarate as the electron acceptor, demonstrating that acetate can also be syntrophically metabolized via DIET. A co-culture of G. metallireducens and Methanosaeta harundinacea previously shown to syntrophically convert ethanol to methane via DIET metabolized propanol or butanol as the sole electron donor, but not propionate or butyrate. The stoichiometric accumulation of propionate or butyrate in the propanol- or butanol-fed cultures demonstrated that M. harundinaceae could conserve energy to support growth solely from electrons derived from DIET. Co-cultures of G. metallireducens and Methanosarcina barkeri could also incompletely metabolize propanol and butanol and did not metabolize propionate or butyrate as sole electron donors. These results expand the range of substrates that are known to be syntrophically metabolized through DIET, but suggest that claims of propionate and butyrate metabolism via DIET in mixed microbial communities warrant further validation.
Project description:Biochar, a charcoal-like product of the incomplete combustion of organic materials, is an increasingly popular soil amendment designed to improve soil fertility. We investigated the possibility that biochar could promote direct interspecies electron transfer (DIET) in a manner similar to that previously reported for granular activated carbon (GAC). Although the biochars investigated were 1000 times less conductive than GAC, they stimulated DIET in co-cultures of Geobacter metallireducens with Geobacter sulfurreducens or Methanosarcina barkeri in which ethanol was the electron donor. Cells were attached to the biochar, yet not in close contact, suggesting that electrons were likely conducted through the biochar, rather than biological electrical connections. The finding that biochar can stimulate DIET may be an important consideration when amending soils with biochar and can help explain why biochar may enhance methane production from organic wastes under anaerobic conditions.
Project description:A hydrogen utilizing exoelectrogenic bacterium (Geobacter sulfurreducens) was compared to both a nonhydrogen oxidizer (Geobacter metallireducens) and a mixed consortium in order to compare the hydrogen production rates and hydrogen recoveries of pure and mixed cultures in microbial electrolysis cells (MECs). At an applied voltage of 0.7 V, both G. sulfurreducens and the mixed culture generated similar current densities (ca. 160 A/m3), resulting in hydrogen production rates of ca. 1.9 m(3) H2/m3/day, whereas G. metallireducens exhibited lower current densities and production rates of 110 +/- 7 A/m3 and 1.3 +/- 0.1 m3 H2/m3/day, respectively. Before methane was detected in the mixed-culture MEC, the mixed consortium achieved the highest overall energy recovery (relative to both electricity and substrate energy inputs) of 82% +/- 8% compared to G. sulfurreducens (77% +/- 2%) and G. metallireducens (78% +/- 5%), due to the higher coulombic efficiency of the mixed consortium. At an applied voltage of 0.4 V, methane production increased in the mixed-culture MEC and, as a result, the hydrogen recovery decreased and the overall energy recovery dropped to 38% +/- 16% compared to 80% +/- 5% for G. sulfurreducens and 76% +/- 0% for G. metallireducens. Internal hydrogen recycling was confirmed since the mixed culture generated a stable current density of 31 +/- 0 A/m3 when fed hydrogen gas, whereas G. sulfurreducens exhibited a steady decrease in current production. Community analysis suggested that G. sulfurreducens was predominant in the mixed-culture MEC (72% of clones) despite its relative absence in the mixed-culture inoculum obtained from a microbial fuel cell reactor (2% of clones). These results demonstrate that Geobacter species are capable of obtaining similar hydrogen production rates and energy recoveries as mixed cultures in an MEC and that high coulombic efficiencies in mixed culture MECs can be attributed in part to the recycling of hydrogen into current.
Project description:The possibility that metatranscriptomic analysis could distinguish between direct interspecies electron transfer (DIET) and H2 interspecies transfer (HIT) in anaerobic communities was investigated by comparing gene transcript abundance in cocultures in which Geobacter sulfurreducens was the electron-accepting partner for either Geobacter metallireducens, which performs DIET, or Pelobacter carbinolicus, which relies on HIT. Transcript abundance for G. sulfurreducens uptake hydrogenase genes was 7-fold lower in cocultures with G. metallireducens than in cocultures with P. carbinolicus, consistent with DIET and HIT, respectively, in the two cocultures. Transcript abundance for the pilus-associated cytochrome OmcS, which is essential for DIET but not for HIT, was 240-fold higher in the cocultures with G. metallireducens than in cocultures with P. carbinolicus. The pilin gene pilA was moderately expressed despite a mutation that might be expected to repress pilA expression. Lower transcript abundance for G. sulfurreducens genes associated with acetate metabolism in the cocultures with P. carbinolicus was consistent with the repression of these genes by H2 during HIT. Genes for the biogenesis of pili and flagella and several c-type cytochrome genes were among the most highly expressed in G. metallireducens. Mutant strains that lacked the ability to produce pili, flagella, or the outer surface c-type cytochrome encoded by Gmet_2896 were not able to form cocultures with G. sulfurreducens. These results demonstrate that there are unique gene expression patterns that distinguish DIET from HIT and suggest that metatranscriptomics may be a promising route to investigate interspecies electron transfer pathways in more-complex environments.
Project description:Direct interspecies electron transfer (DIET) is an alternative to interspecies H(2)/formate transfer as a mechanism for microbial species to cooperatively exchange electrons during syntrophic metabolism. To understand what specific properties contribute to DIET, studies were conducted with Pelobacter carbinolicus, a close relative of Geobacter metallireducens, which is capable of DIET. P. carbinolicus grew in coculture with Geobacter sulfurreducens with ethanol as the electron donor and fumarate as the electron acceptor, conditions under which G. sulfurreducens formed direct electrical connections with G. metallireducens. In contrast to the cell aggregation associated with DIET, P. carbinolicus and G. sulfurreducens did not aggregate. Attempts to initiate cocultures with a genetically modified strain of G. sulfurreducens incapable of both H(2) and formate utilization were unsuccessful, whereas cocultures readily grew with mutant strains capable of formate but not H(2) uptake or vice versa. The hydrogenase mutant of G. sulfurreducens compensated, in cocultures, with significantly increased formate dehydrogenase gene expression. In contrast, the transcript abundance of a hydrogenase gene was comparable in cocultures with that for the formate dehydrogenase mutant of G. sulfurreducens or the wild type, suggesting that H(2) was the primary electron carrier in the wild-type cocultures. Cocultures were also initiated with strains of G. sulfurreducens that could not produce pili or OmcS, two essential components for DIET. The finding that P. carbinolicus exchanged electrons with G. sulfurreducens via interspecies transfer of H(2)/formate rather than DIET demonstrates that not all microorganisms that can grow syntrophically are capable of DIET and that closely related microorganisms may use significantly different strategies for interspecies electron exchange.
Project description:Cytochrome-to-cytochrome electron transfer and electron transfer along conduits of multiple extracellular magnetite grains are often proposed as strategies for direct interspecies electron transfer (DIET) that do not require electrically conductive pili (e-pili). However, physical evidence for these proposed DIET mechanisms has been lacking. To investigate these possibilities further, we constructed Geobacter metallireducens strain Aro-5, in which the wild-type pilin gene was replaced with the aro-5 pilin gene that was previously shown to yield poorly conductive pili in Geobacter sulfurreducens strain Aro-5. G. metallireducens strain Aro-5 did not reduce Fe(III) oxide and produced only low current densities, phenotypes consistent with expression of poorly conductive pili. Like G. sulfurreducens strain Aro-5, G. metallireducens strain Aro-5 displayed abundant outer surface cytochromes. Cocultures initiated with wild-type G. metallireducens as the electron-donating strain and G. sulfurreducens strain Aro-5 as the electron-accepting strain grew via DIET. However, G. metallireducens Aro-5/G. sulfurreducens wild-type cocultures did not. Cocultures initiated with the Aro-5 strains of both species grew only when amended with granular activated carbon (GAC), a conductive material known to be a conduit for DIET. Magnetite could not substitute for GAC. The inability of the two Aro-5 strains to adapt for DIET in the absence of GAC suggests that there are physical constraints on establishing DIET solely through cytochrome-to-cytochrome electron transfer or along chains of magnetite. The finding that DIET is possible with electron-accepting partners that lack highly conductive pili greatly expands the range of potential electron-accepting partners that might participate in DIET.IMPORTANCE DIET is thought to be an important mechanism for interspecies electron exchange in natural anaerobic soils and sediments in which methane is either produced or consumed, as well as in some photosynthetic mats and anaerobic digesters converting organic wastes to methane. Understanding the potential mechanisms for DIET will not only aid in modeling carbon and electron flow in these geochemically significant environments but will also be helpful for interpreting meta-omic data from as-yet-uncultured microbes in DIET-based communities and for designing strategies to promote DIET in anaerobic digesters. The results demonstrate the need to develop a better understanding of the diversity of types of e-pili in the microbial world to identify potential electron-donating partners for DIET. Novel methods for recovering as-yet-uncultivated microorganisms capable of DIET in culture will be needed to further evaluate whether DIET is possible without e-pili in the absence of conductive materials such as GAC.
Project description:<i>Geobacter</i> species are capable of utilizing solid-state compounds, including anodic electrodes, as electron acceptors of respiration via extracellular electron transfer (EET) and have attracted considerable attention for their crucial role as biocatalysts of bioelectrochemical systems (BES's). Recent studies disclosed that anode potentials affect power output and anodic microbial communities, including selection of dominant <i>Geobacter</i> species, in various BES's. However, the details in current-generating properties and responses to anode potentials have been investigated only for a model species, namely <i>Geobacter sulfurreducens</i>. In this study, the effects of anode potentials on the current generation and the EET paths were investigated by cultivating six <i>Geobacter</i> species with different anode potentials, followed by electrochemical analyses. The electrochemical cultivation demonstrated that the <i>G. metallireducens</i> clade species (<i>G. sulfurreducens</i> and <i>G. metallireducens</i>) constantly generate high current densities at a wide range of anode potentials (?-0.3 or -0.2 V vs. Ag/AgCl), while the subsurface clades species (<i>G. daltonii</i>, <i>G. bemidjensis</i>, <i>G. chapellei</i>, and <i>G. pelophilus</i>) generate a relatively large current only at limited potential regions (-0.1 to -0.3 V vs. Ag/AgCl). The linear sweep voltammetry analyses indicated that the <i>G. metallireducens</i> clade species utilize only one EET path irrespective of the anode potentials, while the subsurface clades species utilize multiple EET paths, which can be optimized depending on the anode potentials. These results clearly demonstrate that the response features to anode potentials are divergent among species (or clades) of <i>Geobacter</i>.