Comparative proteomics of Geobacter sulfurreducens PCAT in response to acetate,formate and/or hydrogen as electron donor

Geobacter sulfurreducens is a model bacterium to study the degradation of organic compounds coupled to the reduction of Fe(III). The response of G. sulfurreducens to the electron donors acetate, formate, hydrogen and a mixture of all three with Fe(III) citrate as electron acceptor was studied using comparative physiological and proteomic approaches. Variations in the supplied electron donors resulted in differential abundance of proteins involved in the citric acid cycle (CAC), gluconeogenesis, electron transport, and hydrogenases and formate dehydrogenase. Our results provided new insights into the electron donor metabolism of G. sulfurreducens. Remarkably, formate was the preferred electron donor compared to acetate, hydrogen, or acetate plus hydrogen. When hydrogen was the electron donor, formate was formed, which was associated with a high abundance of formate dehydrogenase. Notably, abundant proteins of two CO₂ fixation pathways (acetyl-CoA pathway and the reversed oxidative CAC) corroborated chemolithoautotrophic growth of G. sulfurreducens with formate or hydrogen and CO₂, and provided novel insight into chemolithoautotrophic growth of G. sulfurreducens.     

A Periplasmic and Extracellular c-Type Cytochrome of Geobacter sulfurreducens Acts as a Ferric Iron Reductase and as an Electron Carrier to Other Acceptors or to Partner Bacteria

An extracellular electron carrier excreted into the growth medium by cells of Geobacter sulfurreducens was identified as a c-type cytochrome. The cytochrome was found to be distributed in about equal amounts in the membrane fraction, the periplasmic space, and the surrounding medium during all phases of growth with acetate plus fumarate. It was isolated from periplasmic preparations and purified to homogeneity by cation-exchange chromatography, gel filtration, and hydrophobic interaction chromatography. The electrophoretically homogeneous cytochrome had a molecular mass of 9.57 ± 0.02 kDa and exhibited in its reduced state absorption maxima at wavelengths of 552, 522, and 419 nm. The midpoint redox potential determined by redox titration was −0.167 V. With respect to molecular mass, redox properties, and molecular features, this cytochrome exhibited its highest similarity to the cytochromes c of Desulfovibrio salexigens and Desulfuromonas acetoxidans. The G. sulfurreducens cytochrome c reduced ferrihydrite (Fe(OH)₃), Fe(III) nitrilotriacetic acid, Fe(III) citrate, and manganese dioxide at high rates. Elemental sulfur, anthraquinone disulfonate, and humic acids were reduced more slowly. G. sulfurreducens reduced the cytochrome with acetate as an electron donor and oxidized it with fumarate. Wolinella succinogenes was able to reduce externally provided cytochrome c of G. sulfurreducens with molecular hydrogen or formate as an electron donor and oxidized it with fumarate or nitrate as an electron acceptor. A coculture could be established in which G. sulfurreducens reduced the cytochrome with acetate, and the reduced cytochrome was reoxidized by W. succinogenes in the presence of nitrate. We conclude that this cytochrome can act as iron(III) reductase for electron transfer to insoluble iron hydroxides or to sulfur, manganese dioxide, or other oxidized compounds, and it can transfer electrons to partner bacteria.     

Identification of an uptake hydrogenase required for hydrogen-dependent reduction of Fe(III) and other electron acceptors by Geobacter sulfurreducens

Geobacter sulfurreducens, a representative of the family Geobacteraceae that predominates in Fe(III)-reducing subsurface environments, can grow by coupling the oxidation of hydrogen to the reduction of a variety of electron acceptors, including Fe(III), fumarate, and quinones. An examination of the G. sulfurreducens genome revealed two operons, hya and hyb, which appeared to encode periplasmically oriented respiratory uptake hydrogenases. In order to assess the roles of these two enzymes in hydrogen-dependent growth, Hya- and Hyb-deficient mutants were generated by gene replacement. Hyb was found to be required for hydrogen-dependent reduction of Fe(III), anthraquinone-2,6-disulfonate, and fumarate by resting cell suspensions and to be essential for growth with hydrogen and these three electron acceptors. Hya, in contrast, was not. These findings suggest that Hyb is an essential respiratory hydrogenase in G. sulfurreducens.     

Lack of Electricity Production by Pelobacter carbinolicus Indicates that the Capacity for Fe(III) Oxide Reduction Does Not Necessarily Confer Electron Transfer Ability to Fuel Cell Anodes

The ability of Pelobacter carbinolicus to oxidize electron donors with electron transfer to the anodes of microbial fuel cells was evaluated because microorganisms closely related to Pelobacter species are generally abundant on the anodes of microbial fuel cells harvesting electricity from aquatic sediments. P. carbinolicus could not produce current in a microbial fuel cell with electron donors which support Fe(III) oxide reduction by this organism. Current was produced using a coculture of P. carbinolicus and Geobacter sulfurreducens with ethanol as the fuel. Ethanol consumption was associated with the transitory accumulation of acetate and hydrogen. G. sulfurreducens alone could not metabolize ethanol, suggesting that P. carbinolicus grew in the fuel cell by converting ethanol to hydrogen and acetate, which G. sulfurreducens oxidized with electron transfer to the anode. Up to 83% of the electrons available in ethanol were recovered as electricity and in the metabolic intermediate acetate. Hydrogen consumption by G. sulfurreducens was important for ethanol metabolism by P. carbinolicus. Confocal microscopy and analysis of 16S rRNA genes revealed that half of the cells growing on the anode surface were P. carbinolicus, but there was a nearly equal number of planktonic cells of P. carbinolicus. In contrast, G. sulfurreducens was primarily attached to the anode. P. carbinolicus represents the first Fe(III) oxide-reducing microorganism found to be unable to produce current in a microbial fuel cell, providing the first suggestion that the mechanisms for extracellular electron transfer to Fe(III) oxides and fuel cell anodes may be different.     

Direct and Fe(II)-Mediated Reduction of Technetium by Fe(III)-Reducing Bacteria

The dissimilatory Fe(III)-reducing bacterium Geobacter sulfurreducens reduced and precipitated Tc(VII) by two mechanisms. Washed cell suspensions coupled the oxidation of hydrogen to enzymatic reduction of Tc(VII) to Tc(IV), leading to the precipitation of TcO₂ at the periphery of the cell. An indirect, Fe(II)-mediated mechanism was also identified. Acetate, although not utilized efficiently as an electron donor for direct cell-mediated reduction of technetium, supported the reduction of Fe(III), and the Fe(II) formed was able to transfer electrons abiotically to Tc(VII). Tc(VII) reduction was comparatively inefficient via this indirect mechanism when soluble Fe(III) citrate was supplied to the cultures but was enhanced in the presence of solid Fe(III) oxide. The rate of Tc(VII) reduction was optimal, however, when Fe(III) oxide reduction was stimulated by the addition of the humic analog and electron shuttle anthaquinone-2,6-disulfonate, leading to the rapid formation of the Fe(II)-bearing mineral magnetite. Under these conditions, Tc(VII) was reduced and precipitated abiotically on the nanocrystals of biogenic magnetite as TcO₂ and was removed from solution to concentrations below the limit of detection by scintillation counting. Cultures of Fe(III)-reducing bacteria enriched from radionuclide-contaminated sediment using Fe(III) oxide as an electron acceptor in the presence of 25 μM Tc(VII) contained a single Geobacter sp. detected by 16S ribosomal DNA analysis and were also able to reduce and precipitate the radionuclide via biogenic magnetite. Fe(III) reduction was stimulated in aquifer material, resulting in the formation of Fe(II)-containing minerals that were able to reduce and precipitate Tc(VII). These results suggest that Fe(III)-reducing bacteria may play an important role in immobilizing technetium in sediments via direct and indirect mechanisms.     

Metabolic Efficiency of Geobacter sulfurreducens Growing on Anodes with Different Redox Potentials

Microorganisms respiring Fe(III) in the environment face a range of redox potentials of the prospective terminal ferric electron acceptors, because Fe(III) can be present in different minerals or organic complexes. We investigated the adaptation of Geobacter sulfurreducens to this range by exposing the bacteria to different redox potentials between the electron donor acetate and solid, extracellular anodes in a microbial fuel-cell set-up. Over a range of anode potentials from ?0.105 to +0.645 V versus standard hydrogen electrode, G. sulfurreducens produced identical amounts of biomass per electron respired. This indicated that the organism cannot utilize higher available energies for energy conservation to ATP, and confirmed recent studies. Either the high potentials cannot be used due to physiological limitations, or G. sulfurreducens decreased its metabolic efficiency, and less biomass per unit of energy was produced. In this case, G. sulfurreducens “wasted” energy at high-potential differences, most likely as heat to fuel growth kinetics.     

Electricity Production by Geobacter sulfurreducens Attached to Electrodes

Previous studies have suggested that members of the Geobacteraceae can use electrodes as electron acceptors for anaerobic respiration. In order to better understand this electron transfer process for energy production, Geobacter sulfurreducens was inoculated into chambers in which a graphite electrode served as the sole electron acceptor and acetate or hydrogen was the electron donor. The electron-accepting electrodes were maintained at oxidizing potentials by connecting them to similar electrodes in oxygenated medium (fuel cells) or to potentiostats that poised electrodes at +0.2 V versus an Ag/AgCl reference electrode (poised potential). When a small inoculum of G. sulfurreducens was introduced into electrode-containing chambers, electrical current production was dependent upon oxidation of acetate to carbon dioxide and increased exponentially, indicating for the first time that electrode reduction supported the growth of this organism. When the medium was replaced with an anaerobic buffer lacking nutrients required for growth, acetate-dependent electrical current production was unaffected and cells attached to these electrodes continued to generate electrical current for weeks. This represents the first report of microbial electricity production solely by cells attached to an electrode. Electrode-attached cells completely oxidized acetate to levels below detection (<10 μM), and hydrogen was metabolized to a threshold of 3 Pa. The rates of electron transfer to electrodes (0.21 to 1.2 μmol of electrons/mg of protein/min) were similar to those observed for respiration with Fe(III) citrate as the electron acceptor (E^o′ =+0.37 V). The production of current in microbial fuel cell (65 mA/m² of electrode surface) or poised-potential (163 to 1,143 mA/m²) mode was greater than what has been reported for other microbial systems, even those that employed higher cell densities and electron-shuttling compounds. Since acetate was completely oxidized, the efficiency of conversion of organic electron donor to electricity was significantly higher than in previously described microbial fuel cells. These results suggest that the effectiveness of microbial fuel cells can be increased with organisms such as G. sulfurreducens that can attach to electrodes and remain viable for long periods of time while completely oxidizing organic substrates with quantitative transfer of electrons to an electrode.     

Acetate oxidation by syntrophic association between Geobacter sulfurreducens and a hydrogen-utilizing exoelectrogen

Anodic microbial communities in acetate-fed microbial fuel cells (MFCs) were analyzed using stable-isotope probing of 16S rRNA genes followed by denaturing gradient gel electrophoresis. The results revealed that Geobacter sulfurreducens and Hydrogenophaga sp. predominated in the anodic biofilm. Although the predominance of Geobacter sp. as acetoclastic exoelectrogens in acetate-fed MFC systems has been often reported, the ecophysiological role of Hydrogenophaga sp. is unknown. Therefore, we isolated and characterized a bacterium closely related to Hydrogenophaga sp. (designated strain AR20). The newly isolated strain AR20 could use molecular hydrogen (H₂), but not acetate, with carbon electrode as the electron acceptor, indicating that the strain AR20 was a hydrogenotrophic exoelectrogen. This evidence raises a hypothesis that acetate was oxidized by G. sulfurreducens in syntrophic cooperation with the strain AR20 as a hydrogen-consuming partner in the acetate-fed MFC. To prove this hypothesis, G. sulfurreducens strain PCA was cocultivated with the strain AR20 in the acetate-fed MFC without any dissolved electron acceptors. In the coculture MFC of G. sulfurreducens and strain AR20, current generation and acetate degradation were the highest, and the growth of strain AR20 was observed. No current generation, acetate degradation and cell growth occurred in the strain AR20 pure culture MFC. These results show for the first time that G. sulfurreducens can oxidize acetate in syntrophic cooperation with the isolated Hydrogenophaga sp. strain AR20, with electrode as the electron acceptor.     

异化铁还原细菌Clostridium sp.LQ25分离及其产氢与铁还原特性

[背景] 一些异化铁还原细菌兼具铁还原和发酵产氢能力,可作为发酵型异化铁还原细菌还原机制研究的对象。[目的] 筛选出一株发酵型异化铁还原细菌。在异化铁还原细菌培养体系中,设置不同电子供体并分析电子供体。[方法] 通过三层平板法从海洋沉积物中筛选纯菌株,基于16S rRNA基因序列进行菌株鉴定。通过测定细菌培养液Fe （II）浓度及发酵产氢量分析菌株异化铁还原和产氢性质。[结果] 菌株LQ25与Clostridium butyricum的16S rRNA基因序列相似性达到100%,结合电镜形态观察,菌株命名为Clostridium sp.LQ25。在氢氧化铁为电子受体培养条件下,菌株生长较对照组（未添加氢氧化铁）显著提高。菌株LQ25能够利用丙酮酸钠、葡萄糖和乳酸钠进行生长。丙酮酸钠为电子供体时,菌株LQ25细胞生长和异化铁还原效率最高,菌体蛋白质含量是（78.88±3.40） mg/L,累积产生Fe （II）浓度为（8.27±0.23） mg/L。以葡萄糖为电子供体时,菌株LQ25发酵产氢量最高,达（475.2±14.4） mL/L,相比对照组（未添加氢氧化铁）产氢量提高87.7%。[结论] 筛选到一株具有异化铁还原和发酵产氢能力的菌株Clostridium sp.LQ25,为探究发酵型异化铁还原细菌胞外电子传递机制提供了新的实验材料。     

A Hydrogen-Oxidizing, Fe(III)-Reducing Microorganism from the Great Bay Estuary, New Hampshire

A dissimilatory Fe(III)- and Mn(IV)-reducing bacterium was isolated from bottom sediments of the Great Bay estuary, New Hampshire. The isolate was a facultatively anaerobic gram-negative rod which did not appear to fit into any previously described genus. It was temporarily designated strain BrY. BrY grew anaerobically in a defined medium with hydrogen or lactate as the electron donor and Fe(III) as the electron acceptor. BrY required citrate, fumarate, or malate as a carbon source for growth on H₂ and Fe(III). With Fe(III) as the sole electron acceptor, BrY metabolized hydrogen to a minimum threshold at least 60-fold lower than the threshold reported for pure cultures of sulfate reducers. This finding supports the hypothesis that when Fe(III) is available, Fe(III) reducers can outcompete sulfate reducers for electron donors. Lactate was incompletely oxidized to acetate and carbon dioxide with Fe(III) as the electron acceptor. Lactate oxidation was also coupled to the reduction of Mn(IV), U(VI), fumarate, thiosulfate, or trimethylamine n-oxide under anaerobic conditions. BrY provides a model for how enzymatic metal reduction by respiratory metal-reducing microorganisms has the potential to contribute to the mobilization of iron and trace metals and to the immobilization of uranium in sediments of Great Bay Estuary.     

Characterization of Metabolism in the Fe(III)-Reducing Organism Geobacter sulfurreducens by Constraint-Based Modeling

Geobacter sulfurreducens is a well-studied representative of the Geobacteraceae, which play a critical role in organic matter oxidation coupled to Fe(III) reduction, bioremediation of groundwater contaminated with organics or metals, and electricity production from waste organic matter. In order to investigate G. sulfurreducens central metabolism and electron transport, a metabolic model which integrated genome-based predictions with available genetic and physiological data was developed via the constraint-based modeling approach. Evaluation of the rates of proton production and consumption in the extracellular and cytoplasmic compartments revealed that energy conservation with extracellular electron acceptors, such as Fe(III), was limited relative to that associated with intracellular acceptors. This limitation was attributed to lack of cytoplasmic proton consumption during reduction of extracellular electron acceptors. Model-based analysis of the metabolic cost of producing an extracellular electron shuttle to promote electron transfer to insoluble Fe(III) oxides demonstrated why Geobacter species, which do not produce shuttles, have an energetic advantage over shuttle-producing Fe(III) reducers in subsurface environments. In silico analysis also revealed that the metabolic network of G. sulfurreducens could synthesize amino acids more efficiently than that of Escherichia coli due to the presence of a pyruvate-ferredoxin oxidoreductase, which catalyzes synthesis of pyruvate from acetate and carbon dioxide in a single step. In silico phenotypic analysis of deletion mutants demonstrated the capability of the model to explore the flexibility of G. sulfurreducens central metabolism and correctly predict mutant phenotypes. These results demonstrate that iterative modeling coupled with experimentation can accelerate the understanding of the physiology of poorly studied but environmentally relevant organisms and may help optimize their practical applications.     

A Geobacter sulfurreducens Strain Expressing Pseudomonas aeruginosa Type IV Pili Localizes OmcS on Pili but Is Deficient in Fe(III) Oxide Reduction and Current Production

The conductive pili of Geobacter species play an important role in electron transfer to Fe(III) oxides, in long-range electron transport through current-producing biofilms, and in direct interspecies electron transfer. Although multiple lines of evidence have indicated that the pili of Geobacter sulfurreducens have a metal-like conductivity, independent of the presence of c-type cytochromes, this claim is still controversial. In order to further investigate this phenomenon, a strain of G. sulfurreducens, designated strain PA, was constructed in which the gene for the native PilA, the structural pilin protein, was replaced with the PilA gene of Pseudomonas aeruginosa PAO1. Strain PA expressed and properly assembled P. aeruginosa PilA subunits into pili and exhibited a profile of outer surface c-type cytochromes similar to that of a control strain expressing the G. sulfurreducens PilA. Surprisingly, the strain PA pili were decorated with the c-type cytochrome OmcS in a manner similar to the control strain. However, the strain PA pili were 14-fold less conductive than the pili of the control strain, and strain PA was severely impaired in Fe(III) oxide reduction and current production. These results demonstrate that the presence of OmcS on pili is not sufficient to confer conductivity to pili and suggest that there are unique structural features of the G. sulfurreducens PilA that are necessary for conductivity.     

Novel Mode of Microbial Energy Metabolism: Organic Carbon Oxidation Coupled to Dissimilatory Reduction of Iron or Manganese

A dissimilatory Fe(III)- and Mn(IV)-reducing microorganism was isolated from freshwater sediments of the Potomac River, Maryland. The isolate, designated GS-15, grew in defined anaerobic medium with acetate as the sole electron donor and Fe(III), Mn(IV), or nitrate as the sole electron acceptor. GS-15 oxidized acetate to carbon dioxide with the concomitant reduction of amorphic Fe(III) oxide to magnetite (Fe₃O₄). When Fe(III) citrate replaced amorphic Fe(III) oxide as the electron acceptor, GS-15 grew faster and reduced all of the added Fe(III) to Fe(II). GS-15 reduced a natural amorphic Fe(III) oxide but did not significantly reduce highly crystalline Fe(III) forms. Fe(III) was reduced optimally at pH 6.7 to 7 and at 30 to 35°C. Ethanol, butyrate, and propionate could also serve as electron donors for Fe(III) reduction. A variety of other organic compounds and hydrogen could not. MnO₂ was completely reduced to Mn(II), which precipitated as rhodochrosite (MnCO₃). Nitrate was reduced to ammonia. Oxygen could not serve as an electron acceptor, and it inhibited growth with the other electron acceptors. This is the first demonstration that microorganisms can completely oxidize organic compounds with Fe(III) or Mn(IV) as the sole electron acceptor and that oxidation of organic matter coupled to dissimilatory Fe(III) or Mn(IV) reduction can yield energy for microbial growth. GS-15 provides a model for how enzymatically catalyzed reactions can be quantitatively significant mechanisms for the reduction of iron and manganese in anaerobic environments.     

Mechanisms for Accessing Insoluble Fe(III) Oxide during Dissimilatory Fe(III) Reduction by Geothrix fermentans

Mechanisms for Fe(III) oxide reduction were investigated in Geothrix fermentans, a dissimilatory Fe(III)-reducing microorganism found within the Fe(III) reduction zone of subsurface environments. Culture filtrates of G. fermentans stimulated the reduction of poorly crystalline Fe(III) oxide by washed cell suspensions, suggesting that G. fermentans released one or more extracellular compounds that promoted Fe(III) oxide reduction. In order to determine if G. fermentans released electron-shuttling compounds, poorly crystalline Fe(III) oxide was incorporated into microporous alginate beads, which prevented contact between G. fermentans and the Fe(III) oxide. G. fermentans reduced the Fe(III) within the beads, suggesting that one of the compounds that G. fermentans releases is an electron-shuttling compound that can transfer electrons from the cell to Fe(III) oxide that is not in contact with the organism. Analysis of culture filtrates by thin-layer chromatography suggested that the electron shuttle has characteristics similar to those of a water-soluble quinone. Analysis of filtrates by ion chromatography demonstrated that there was as much as 250 μM dissolved Fe(III) in cultures of G. fermentans growing with Fe(III) oxide as the electron acceptor, suggesting that G. fermentans released one or more compounds capable of chelating and solubilizing Fe(III). Solubilizing Fe(III) is another strategy for alleviating the need for contact between cells and Fe(III) oxide for Fe(III) reduction. This is the first demonstration of a microorganism that, in defined medium without added electron shuttles or chelators, can reduce Fe(III) derived from Fe(III) oxide without directly contacting the Fe(III) oxide. These results are in marked contrast to those with Geobacter metallireducens, which does not produce electron shuttles or Fe(III) chelators. These results demonstrate that phylogenetically distinct Fe(III)-reducing microorganisms may use significantly different strategies for Fe(III) reduction. Thus, it is important to know which Fe(III)-reducing microorganisms predominate in a given environment in order to understand the mechanisms for Fe(III) reduction in the environment of interest.     

Hydrogen and Formate Oxidation Coupled to Dissimilatory Reduction of Iron or Manganese by Alteromonas putrefaciens

The ability of Alteromonas putrefaciens to obtain energy for growth by coupling the oxidation of various electron donors to dissimilatory Fe(III) or Mn(IV) reduction was investigated. A. putrefaciens grew with hydrogen, formate, lactate, or pyruvate as the sole electron donor and Fe(III) as the sole electron acceptor. Lactate and pyruvate were oxidized to acetate, which was not metabolized further. With Fe(III) as the electron acceptor, A. putrefaciens had a high affinity for hydrogen and formate and metabolized hydrogen at partial pressures that were 25-fold lower than those of hydrogen that can be metabolized by pure cultures of sulfate reducers or methanogens. The electron donors for Fe(III) reduction also supported Mn(IV) reduction. The electron donors for Fe(III) and Mn(IV) reduction and the inability of A. putrefaciens to completely oxidize multicarbon substrates to carbon dioxide distinguish A. putrefaciens from GS-15, the only other organism that is known to obtain energy for growth by coupling the oxidation of organic compounds to the reduction of Fe(III) or Mn(IV). The ability of A. putrefaciens to reduce large quantities of Fe(III) and to grow in a defined medium distinguishes it from a Pseudomonas sp., which is the only other known hydrogen-oxidizing, Fe(III)-reducing microorganism. Furthermore, A. putrefaciens is the first organism that is known to grow with hydrogen as the electron donor and Mn(IV) as the electron acceptor and is the first organism that is known to couple the oxidation of formate to the reduction of Fe(III) or Mn(IV). Thus, A. putrefaciens provides a much needed microbial model for key reactions in the oxidation of sediment organic matter coupled to Fe(III) and Mn(IV) reduction.     

Degradation of Acetaldehyde and Its Precursors by Pelobacter carbinolicus and P. acetylenicus

Pelobacter carbinolicus and P. acetylenicus oxidize ethanol in syntrophic cooperation with methanogens. Cocultures with Methanospirillum hungatei served as model systems for the elucidation of syntrophic ethanol oxidation previously done with the lost “Methanobacillus omelianskii” coculture. During growth on ethanol, both Pelobacter species exhibited NAD⁺-dependent alcohol dehydrogenase activity. Two different acetaldehyde-oxidizing activities were found: a benzyl viologen-reducing enzyme forming acetate, and a NAD⁺-reducing enzyme forming acetyl-CoA. Both species synthesized ATP from acetyl-CoA via acetyl phosphate. Comparative 2D-PAGE of ethanol-grown P. carbinolicus revealed enhanced expression of tungsten-dependent acetaldehyde: ferredoxin oxidoreductases and formate dehydrogenase. Tungsten limitation resulted in slower growth and the expression of a molybdenum-dependent isoenzyme. Putative comproportionating hydrogenases and formate dehydrogenase were expressed constitutively and are probably involved in interspecies electron transfer. In ethanol-grown cocultures, the maximum hydrogen partial pressure was about 1,000 Pa (1 mM) while 2 mM formate was produced. The redox potentials of hydrogen and formate released during ethanol oxidation were calculated to be E_H2 = -358±12 mV and E_HCOOH = -366±19 mV, respectively. Hydrogen and formate formation and degradation further proved that both carriers contributed to interspecies electron transfer. The maximum Gibbs free energy that the Pelobacter species could exploit during growth on ethanol was −35 to −28 kJ per mol ethanol. Both species could be cultivated axenically on acetaldehyde, yielding energy from its disproportionation to ethanol and acetate. Syntrophic cocultures grown on acetoin revealed a two-phase degradation: first acetoin degradation to acetate and ethanol without involvement of the methanogenic partner, and subsequent syntrophic ethanol oxidation. Protein expression and activity patterns of both Pelobacter spp. grown with the named substrates were highly similar suggesting that both share the same steps in ethanol and acetalydehyde metabolism. The early assumption that acetaldehyde is a central intermediate in Pelobacter metabolism was now proven biochemically.     

Going Wireless: Fe(III) Oxide Reduction without Pili by Geobacter sulfurreducens Strain JS-1

Previous studies have suggested that the conductive pili of Geobacter sulfurreducens are essential for extracellular electron transfer to Fe(III) oxides and for optimal long-range electron transport through current-producing biofilms. The KN400 strain of G. sulfurreducens reduces poorly crystalline Fe(III) oxide more rapidly than the more extensively studied DL-1 strain. Deletion of the gene encoding PilA, the structural pilin protein, in strain KN400 inhibited Fe(III) oxide reduction. However, low rates of Fe(III) reduction were detected after extended incubation (>30 days) in the presence of Fe(III) oxide. After seven consecutive transfers, the PilA-deficient strain adapted to reduce Fe(III) oxide as fast as the wild type. Microarray, whole-genome resequencing, proteomic, and gene deletion studies indicated that this adaptation was associated with the production of larger amounts of the c-type cytochrome PgcA, which was released into the culture medium. It is proposed that the extracellular cytochrome acts as an electron shuttle, promoting electron transfer from the outer cell surface to Fe(III) oxides. The adapted PilA-deficient strain competed well with the wild-type strain when both were grown together on Fe(III) oxide. However, when 50% of the culture medium was replaced with fresh medium every 3 days, the wild-type strain outcompeted the adapted strain. A possible explanation for this is that the necessity to produce additional PgcA, to replace the PgcA being continually removed, put the adapted strain at a competitive disadvantage, similar to the apparent selection against electron shuttle-producing Fe(III) reducers in many anaerobic soils and sediments. Despite increased extracellular cytochrome production, the adapted PilA-deficient strain produced low levels of current, consistent with the concept that long-range electron transport through G. sulfurreducens biofilms is more effective via pili.     

Metabolic response of <Emphasis Type="Italic">Geobacter sulfurreducens</Emphasis> towards electron donor/acceptor variation

Background  

Geobacter sulfurreducens is capable of coupling the complete oxidation of organic compounds to iron reduction. The metabolic response of G. sulfurreducens towards variations in electron donors (acetate, hydrogen) and acceptors (Fe(III), fumarate) was investigated via ¹³C-based metabolic flux analysis. We examined the ¹³C-labeling patterns of proteinogenic amino acids obtained from G. sulfurreducens cultured with ¹³C-acetate.