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PubMed1999

Purification and characterization of triheme cytochrome c7 from the metal-reducing bacterium, Geobacter metallireducens.

A soluble c-type cytochrome was first purified from Geobacter metallireducens to an electrophoretically homogeneous state. The purified cytochrome c showed absorption peaks at 530 and 409 nm in the oxidized form and 552, 522, and 418 nm in the reduced form. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate allowed us to calculate the molecular mass at 9.5 kDa. It contained 3 mol of heme c per molecule of the protein on the basis of heme c and protein concentration. The mid-point redox potential at pH 7.0 was determined to be -190 mV. Although the N-terminal amino acid sequence of the first 17 residues was similar to that of Desulfuromonas acetoxidans cytochrome c7, G. metallireducens cytochrome c did not show Fe(III)-reducing activity.

Amino Acid Sequence↗
PubMed2000

Characterization of a membrane-bound NADH-dependent Fe(3+) reductase from the dissimilatory Fe(3+)-reducing bacterium Geobacter sulfurreducens.

Geobacter sulfurreducens produces a single, membrane-associated Fe(3+) reductase activity when grown on fumarate or Fe(3+). The activity was initially isolated by solubilization of membranes with the non-ionic detergent dodecyl-beta-D-maltoside, and partially purified by a combination of ion exchange chromatography and preparative non-denaturing gel electrophoresis. Molecular mass of the reductase, as determined by gel filtration chromatography, was approximately 300 kDa. Cofactor analysis of the purified reductase demonstrates that it contains a hemoprotein and flavin adenine dinucleotide. Kinetic and inhibitor studies show that the reductase is specific for NADH as electron donor, and confirm that the reductase enzymatically reduces Fe(3+). The cytochrome associated with the complex undergoes a reoxidation upon addition of Fe(3+) compounds, indicating an ability to pass reducing equivalents to Fe(3+). This is the first description of a purified NADH-dependent Fe(3+) reductase from a microorganism capable of coupling Fe(3+) reduction to growth.

Cell Membrane↗
PubMed1998

Growth of geobacter sulfurreducens with acetate in syntrophic cooperation with hydrogen-oxidizing anaerobic partners

Pure cultures of Geobacter sulfurreducens and other Fe(III)-reducing bacteria accumulated hydrogen to partial pressures of 5 to 70 Pa with acetate, butyrate, benzoate, ethanol, lactate, or glucose as the electron donor if electron release to an acceptor was limiting. G. sulfurreducens coupled acetate oxidation with electron transfer to an anaerobic partner bacterium in the absence of ferric iron or other electron acceptors. Cocultures of G. sulfurreducens and Wolinella succinogenes with nitrate as the electron acceptor degraded acetate efficiently and grew with doubling times of 6 to 8 h. The hydrogen partial pressures in these acetate-degrading cocultures were considerably lower, in the range of 0.02 to 0.04 Pa. From these values and the concentrations of the other reactants, it was calculated that in this cooperation the free energy change available to G. sulfurreducens should be about -53 kJ per mol of acetate oxidized, assuming complete conversion of acetate to CO2 and H2. However, growth yields (18.5 g of dry mass per mol of acetate for the coculture, about 14 g for G. sulfurreducens) indicated considerably higher energy gains. These yield data, measurement of hydrogen production rates, and calculation of the diffusive hydrogen flux indicated that electron transfer in these cocultures may not proceed exclusively via interspecies hydrogen transfer but may also proceed through an alternative carrier system with higher redox potential, e.g., a c-type cytochrome that was found to be excreted by G. sulfurreducens into the culture fluid. Syntrophic acetate degradation was also possible with G. sulfurreducens and Desulfovibrio desulfuricans CSN but only with nitrate as electron acceptor. These cultures produced cell yields of 4.5 g of dry mass per mol of acetate, to which both partners contributed at about equal rates. These results demonstrate that some Fe(III)-reducing bacteria can oxidize organic compounds under Fe(III) limitation with the production of hydrogen, and they provide the first example of rapid acetate oxidation via interspecies electron transfer at moderate temperature.

Journal Article↗
PubMed1998

Localization and solubilization of the Iron(III) reductase of geobacter sulfurreducens

The iron(III) reductase activity of Geobacter sulfurreducens was determined with the electron donor NADH and the artificial electron donor horse heart cytochrome c. The highest reduction rates were obtained with Fe(III) complexed by nitrilotriacetic acid as an electron acceptor. Fractionation experiments indicated that no iron(III) reductase activity was present in the cytoplasm, that approximately one-third was found in the periplasmic fraction, and that two-thirds were associated with the membrane fraction. Sucrose gradient separation of the outer and cytoplasmic membranes showed that about 80% of the iron(III) reductase was present in the outer membrane. The iron(III) reductase could be solubilized from the membrane fraction with 0.5 M KCl showing that the iron(III) reductase was weakly bound to the membranes. In addition, solubilization of the iron(III) reductase from whole cells with 0.5 M KCl, without disruption of cells, indicated that the iron(III) reductase is a peripheral protein on the outside of the outer membrane. Redox difference spectra of KCl extracts showed the presence of c-type cytochromes which could be oxidized by ferrihydrite. Only one activity band was observed in native polyacrylamide gels stained for the iron(III) reductase activity. Excision of the active band from a preparative gel followed by extraction of the proteins and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of high-molecular-mass, cytochrome-containing proteins in this iron(III) reductase activity band. From these experimental data it can be hypothesized that the iron(III) reductase of G. sulfurreducens is a peripheral outer membrane protein that might contain a c-type cytochrome.

Journal Article↗
PubMed2000

Lack of production of electron-shuttling compounds or solubilization of Fe(III) during reduction of insoluble Fe(III) oxide by Geobacter metallireducens.

Studies with the dissimilatory Fe(III)-reducing microorganism Geobacter metallireducens demonstrated that the common technique of separating Fe(III)-reducing microorganisms and Fe(III) oxides with semipermeable membranes in order to determine whether the Fe(III) reducers release electron-shuttling compounds and/or Fe(III) chelators is invalid. This raised doubts about the mechanisms for Fe(III) oxide reduction by this organism. However, several experimental approaches indicated that G. metallireducens does not release electron-shuttling compounds and does not significantly solubilize Fe(III) during Fe(III) oxide reduction. These results suggest that G. metallireducens directly reduces insoluble Fe(III) oxide.

Deltaproteobacteria↗
PubMed2001

Evidence for iron-dependent nitrate respiration in the dissimilatory iron-reducing bacterium Geobacter metallireducens.

The dissimilatory iron-reducing bacterium Geobacter metallireducens was found to require iron at a concentration in excess of 50 microM for continuous cultivation on nitrate. Growth yield (approximately 3-fold), cytochrome c content (approximately 7-fold), and nitrate (approximately 4.5-fold) and nitrite (approximately 70-fold) reductase activities were all increased significantly when the growth medium was amended with 500 microM iron.

Culture Media↗
PubMed2003

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 micro M), and hydrogen was metabolized to a threshold of 3 Pa. The rates of electron transfer to electrodes (0.21 to 1.2 micro mol 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/m(2) of electrode surface) or poised-potential (163 to 1,143 mA/m(2)) 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.

Culture Media↗
PubMed1998

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.

Cytochrome c Group↗
PubMed1999

The periplasmic 9.6-kilodalton c-type cytochrome of Geobacter sulfurreducens is not an electron shuttle to Fe(III).

Geobacter sulfurreducens contains a 9.6-kDa c-type cytochrome that was previously proposed to serve as an extracellular electron shuttle to insoluble Fe(III) oxides. However, when the cytochrome was added to washed-cell suspensions of G. sulfurreducens it did not enhance Fe(III) oxide reduction, whereas similar concentrations of the known electron shuttle, anthraquinone-2,6-disulfonate, greatly stimulated Fe(III) oxide reduction. Furthermore, analysis of the extracellular c-type cytochromes in cultures of G. sulfurreducens demonstrated that the dominant c-type cytochrome was not the 9.6-kDa cytochrome, but rather a 41-kDa cytochrome. These results and other considerations suggest that the 9.6-kDa cytochrome is not an important extracellular electron shuttle to Fe(III) oxides.

Cytochrome c Group↗
PubMed2003

OmcB, a c-type polyheme cytochrome, involved in Fe(III) reduction in Geobacter sulfurreducens.

Microorganisms in the family Geobacteraceae are the predominant Fe(III)-reducing microorganisms in a variety of subsurface environments in which Fe(III) reduction is an important process, but little is known about the mechanisms for electron transport to Fe(III) in these organisms. The Geobacter sulfurreducens genome was found to contain a 10-kb chromosomal duplication consisting of two tandem three-gene clusters. The last genes of the two clusters, designated omcB and omcC, encode putative outer membrane polyheme c-type cytochromes which are 79% identical. The role of the omcB and omcC genes in Fe(III) reduction in G. sulfurreducens was investigated. OmcB and OmcC were both expressed during growth with acetate as the electron donor and either fumarate or Fe(III) as the electron acceptor. OmcB was ca. twofold more abundant under both conditions. Disrupting omcB or omcC by gene replacement had no impact on growth with fumarate. However, the OmcB-deficient mutant was greatly impaired in its ability to reduce Fe(III) both in cell suspensions and under growth conditions. In contrast, the ability of the OmcC-deficient mutant to reduce Fe(III) was similar to that of the wild type. When omcB was reintroduced into the OmcB-deficient mutant, the capacity for Fe(III) reduction was restored in proportion to the level of OmcB production. These results indicate that OmcB, but not OmcC, has a major role in electron transport to Fe(III) and suggest that electron transport to the outer membrane is an important feature in Fe(III) reduction in this organism.

Amino Acid Sequence↗
PubMed2004

MacA, a diheme c-type cytochrome involved in Fe(III) reduction by Geobacter sulfurreducens.

A 36-kDa diheme c-type cytochrome abundant in Fe(III)-respiring Geobacter sulfurreducens, designated MacA, was more highly expressed during growth with Fe(III) as the electron acceptor than with fumarate. Although MacA has homology to proteins with in vitro peroxidase activity, deletion of macA had no impact on response to oxidative stress. However, the capacity for Fe(III) reduction was greatly diminished, indicating that MacA, which is predicted to be localized in the periplasm, is a key intermediate in electron transfer to Fe(III).

Amino Acid Sequence↗
PubMed2004

Dissimilatory Fe(III) and Mn(IV) reduction.

Dissimilatory Fe(III) and Mn(IV) reduction has an important influence on the geochemistry of modern environments, and Fe(III)-reducing microorganisms, most notably those in the Geobacteraceae family, can play an important role in the bioremediation of subsurface environments contaminated with organic or metal contaminants. Microorganisms with the capacity to conserve energy from Fe(III) and Mn(IV) reduction are phylogenetically dispersed throughout the Bacteria and Archaea. The ability to oxidize hydrogen with the reduction of Fe(III) is a highly conserved characteristic of hyperthermophilic microorganisms and one Fe(III)-reducing Archaea grows at the highest temperature yet recorded for any organism. Fe(III)- and Mn(IV)-reducing microorganisms have the ability to oxidize a wide variety of organic compounds, often completely to carbon dioxide. Typical alternative electron acceptors for Fe(III) reducers include oxygen, nitrate, U(VI) and electrodes. Unlike other commonly considered electron acceptors, Fe(III) and Mn(IV) oxides, the most prevalent form of Fe(III) and Mn(IV) in most environments, are insoluble. Thus, Fe(III)- and Mn(IV)-reducing microorganisms face the dilemma of how to transfer electrons derived from central metabolism onto an insoluble, extracellular electron acceptor. Although microbiological and geochemical evidence suggests that Fe(III) reduction may have been the first form of microbial respiration, the capacity for Fe(III) reduction appears to have evolved several times as phylogenetically distinct Fe(III) reducers have different mechanisms for Fe(III) reduction. Geobacter species, which are representative of the family of Fe(III) reducers that predominate in a wide diversity of sedimentary environments, require direct contact with Fe(III) oxides in order to reduce them. In contrast, Shewanella and Geothrix species produce chelators that solubilize Fe(III) and release electron-shuttling compounds that transfer electrons from the cell surface to the surface of Fe(III) oxides not in direct contact with the cells. Electron transfer from the inner membrane to the outer membrane in Geobacter and Shewanella species appears to involve an electron transport chain of inner-membrane, periplasmic, and outer-membrane c-type cytochromes, but the cytochromes involved in these processes in the two organisms are different. In addition, Geobacter species specifically express flagella and pili during growth on Fe(III) and Mn(IV) oxides and are chemotactic to Fe(II) and Mn(II), which may lead Geobacter species to the oxides under anoxic conditions. The physiological characteristics of Geobacter species appear to explain why they have consistently been found to be the predominant Fe(III)- and Mn(IV)-reducing microorganisms in a variety of sedimentary environments. In comparison with other respiratory processes, the study of Fe(III) and Mn(IV) reduction is in its infancy, but genome-enabled approaches are rapidly advancing our understanding of this environmentally significant physiology.

Archaea↗
PubMed2005

Biological control of hog waste odor through stimulated microbial Fe(III) reduction.

Odor control and disposal of swine waste have inhibited expansion of swine production facilities throughout the United States. Swine waste odor is associated primarily with high concentrations of volatile fatty acids (VFAs). Here, we demonstrate that stimulated Fe(III) reduction in hog manure can rapidly remove the malodorous compounds and enhance methane production by 200%. As part of these studies, we enumerated the indigenous Fe(III)-reducing population in swine waste and identified members of the family Geobacteraceae as the dominant species. These organisms were present at concentrations as high as 2 x 10(5) cells g(-1). Several pure cultures of Fe(III) reducers, including Geobacter metallireducens, Geobacter humireducens, Geobacter sulfurreducens, Geobacter grbiciae, Geothrix fermentans, and Geovibrio ferrireducens, readily degraded some or all of the malodorous VFAs found in swine manure. In contrast, Shewanella algae did not degrade any of these compounds. We isolated an Fe(III) reducer, Geobacter strain NU, from materials collected from primary swine waste lagoons. This organism degraded all of the malodorous VFAs tested and readily grew in swine waste amended with Fe(III). When raw waste amended with Fe(III) was inoculated with strain NU, the VFA content rapidly decreased, corresponding with an almost complete removal of the odor. In contrast, the raw waste without Fe(III) or strain NU showed a marked increase in VFA content and a rapid pH drop. This study showed that Fe(III) supplementation combined with appropriate bioaugmentation provides a simple, cost-effective approach to deodorize and treat swine waste, removing a significant impediment to the expansion of pork production facilities.

Animals↗
PubMed2004

Graphite electrodes as electron donors for anaerobic respiration.

It has been demonstrated previously that Geobacter species can transfer electrons directly to electrodes. In order to determine whether electrodes could serve as electron donors for microbial respiration, enrichment cultures were established from a sediment inoculum with a potentiostat-poised graphite electrode as the sole electron donor and nitrate as the electron acceptor. Nitrate was reduced to nitrite with the consumption of electrical current. The stoichiometry of electron and nitrate consumption and nitrite accumulation were consistent with the electrode serving as the sole electron donor for nitrate reduction. Analysis of 16 rRNA gene sequences demonstrated that the electrodes supplied with current were specifically enriched in microorganisms with sequences most closely related to the sequences of known Geobacter species. A pure culture of Geobacter metallireducens was shown to reduce nitrate to nitrite with the electrode as the sole electron donor with the expected stoichiometry of electron consumption. Cells attached to the electrode appeared to be responsible for the nitrate reduction. Attached cells of Geobacter sulfurreducens reduced fumarate to succinate with the electrode as an electron donor. These results demonstrate for the first time that electrodes may serve as a direct electron donor for anaerobic respiration. This finding has implications for the harvesting of electricity from anaerobic sediments and the bioremediation of oxidized contaminants.

Anaerobiosis↗
PubMed1998

The use of biologically produced ferrihydrite for the isolation of novel iron-reducing bacteria.

Ferric iron was produced anaerobically from ferrous iron through the metabolic activity of recently described ferrous iron-oxidizing, nitrate-reducing bacteria. It was identified as poorly crystallized 2-line ferrihydrite with a particle size of 1-2 nm. This biologically produced ferrihydrite was shown to be a suitable electron acceptor for dissimilatory ferric iron-reducing bacteria in freshwater enrichment cultures, and was completely reduced to the ferrous state; no magnetite formation occurred. Geobacter metallireducens was also able to completely reduce the biologically produced ferrihydrite. These results indicate the possibility of an anaerobic, microbial cycling of iron. Using the biologically produced ferric iron, two isolates of obligately anaerobic, dissimilatory ferric iron-reducing bacteria, strains Dfr1 and Dfr2, were obtained from freshwater enrichment cultures. Analysis of 16S rRNA gene sequences revealed an affiliation with the Geobacter cluster within the family Geobacteraceae. The sequence similarity between strains Dfr1 and Dfr2 is 92.5%. The closest relative of strain Dfr1 is Geobacter sulfurreducens with 92.9%, and of strain Dfr2 Geobacter chapelleii with 93.7% sequence similarity. In addition, strains Dfr1 and Dfr2 are both able to grow by dissimilatory reduction of Mn(IV), S degree, and fumarate. Furthermore, strain Dfr2 is able to reduce akaganeite (beta-FeOOH), a more crystallized type of ferric iron oxide.

Base Composition↗
PubMed2005

Correlations between arsenic in Maine groundwater and microbial populations as determined by fluorescence in situ hybridization.

Arsenic is known to cause serious health effects when consumed in drinking water. In the state of Maine, approximately half of the population relies on private groundwater wells for their drinking water. Of those wells, as many as 13% may contain arsenic levels above the current EPA maximum contaminant level of 10 microgl(-1). Microorganisms can potentially contribute to arsenic release into groundwater through several mechanisms. Some can reduce arsenate to arsenite, which is more toxic and may be more mobile. Sulfurospirillum species NP4, which was isolated from well water, respires arsenate and could act in this way. Microorganisms can also act indirectly by reducing bedrock surface coatings, such as iron oxyhydroxides, that adsorb arsenic in the groundwater environment. The genus Geobacter contains many species that are capable of iron reduction that could play a role in the indirect release of arsenic into groundwater. Water samples from Northport, ME and the Branch Lake region of Ellsworth, ME, which both have elevated groundwater arsenic levels, have been probed using fluorescence in situ hybridization (FISH), to determine the percentage of the population that is NP4 and the percentage that are Geobacter species. Geobacter abundance correlates well with the total arsenic concentration indicating that indirect mechanisms could be important in releasing arsenic. NP4 appears to be reducing arsenate since its prevalence correlates well with arsenite, the end product of arsenate respiration.

Arsenic↗
PubMed2006

Mercury methylation by dissimilatory iron-reducing bacteria.

The Hg-methylating ability of dissimilatory iron-reducing bacteria in the genera Geobacter, Desulfuromonas, and Shewanella was examined. All of the Geobacter and Desulfuromonas strains tested methylated mercury while reducing Fe(III), nitrate, or fumarate. In contrast, none of the Shewanella strains produced methylmercury at higher levels than abiotic controls under similar culture conditions. Geobacter and Desulfuromonas are closely related to known Hg-methylating sulfate-reducing bacteria within the Deltaproteobacteria.

Desulfuromonas↗