Kazem Kashefi

Microbiological Evidence for Fe(III) Reduction on Early Earth

It is generally considered that sulphur reduction was one of the earliest forms of microbial respiration, because the known microorganisms that are most closely related to the last common ancestor of modern life are primarily anaerobic, sulphur-reducing hyperthermophiles. However, geochemical evidence indicates that Fe(III) is more likely than sulphur to have been the first external electron acceptor of global significance in microbial metabolism. Here we show that Archaea and Bacteria that are most closely related to the last common ancestor can reduce Fe(III) to Fe(II) and conserve energy to support growth from this respiration. Surprisingly, even Thermotoga maritima, previously considered to have only a fermentative metabolism, could grow as a respiratory organism when Fe(III) was provided as an electron acceptor. These results provide microbiological evidence that Fe(III) reduction could have been an important process on early Earth and suggest that microorganisms might contribute to Fe(III) reduction in modern hot biospheres. Furthermore, our discovery that hyperthermophiles that had previously been thought to require sulphur for cultivation can instead be grown without the production of toxic and corrosive sulphide, should aid biochemical investigations of these poorly understood organisms.

Reduction of Fe(III), Mn(IV), and Toxic Metals at 100°C by Pyrobaculum islandicum

ABSTRACT It has recently been noted that a diversity of hyperthermophilic microorganisms have the ability to reduce Fe(III) with hydrogen as the electron donor, but the reduction of Fe(III) or other metals by these organisms has not been previously examined in detail. WhenPyrobaculum islandicum was grown at 100°C in a medium with hydrogen as the electron donor and Fe(III)-citrate as the electron acceptor, the increase in cell numbers of P. islandicum per mole of Fe(III) reduced was found to be ca. 10-fold higher than previously reported. Poorly crystalline Fe(III) oxide could also serve as the electron acceptor for growth on hydrogen. The stoichiometry of hydrogen uptake and Fe(III) oxide reduction was consistent with the oxidation of 1 mol of hydrogen resulting in the reduction of 2 mol of Fe(III). The poorly crystalline Fe(III) oxide was reduced to extracellular magnetite. P. islandicum could not effectively reduce the crystalline Fe(III) oxide minerals goethite and hematite. In addition to using hydrogen as an electron donor for Fe(III) reduction, P. islandicum grew via Fe(III) reduction in media in which peptone and yeast extract served as potential electron donors. The closely related species P. aerophilumgrew via Fe(III) reduction in a similar complex medium. Cell suspensions of P. islandicum reduced the following metals with hydrogen as the electron donor: U(VI), Tc(VII), Cr(VI), Co(III), and Mn(IV). The reduction of these metals was dependent upon the presence of cells and hydrogen. The metalloids arsenate and selenate were not reduced. U(VI) was reduced to the insoluble U(IV) mineral uraninite, which was extracellular. Tc(VII) was reduced to insoluble Tc(IV) or Tc(V). Cr(VI) was reduced to the less toxic, less soluble Cr(III). Co(III) was reduced to Co(II). Mn(IV) was reduced to Mn(II) with the formation of manganese carbonate. These results demonstrate that biological reduction may contribute to the speciation of metals in hydrothermal environments and could account for such phenomena as magnetite accumulation and the formation of uranium deposits at ca. 100°C. Reduction of toxic metals with hyperthermophilic microorganisms or their enzymes might be applied to the remediation of metal-contaminated waters or waste streams.

Reductive precipitation of gold by dissimilatory Fe(III)-reducing bacteria and archaea.

ABSTRACT Studies with a diversity of hyperthermophilic and mesophilic dissimilatory Fe(III)-reducing Bacteria andArchaea demonstrated that some of these organisms are capable of precipitating gold by reducing Au(III) to Au(0) with hydrogen as the electron donor. These studies suggest that models for the formation of gold deposits in both hydrothermal and cooler environments should consider the possibility that dissimilatory metal-reducing microorganisms can reductively precipitate gold from solution.

Geoglobus Ahangari gen. nov., sp. nov., a Novel Hyperthermophilic Archaeon Capable of Oxidizing Organic Acids and Growing Autotrophically on Hydrogen with Fe(III) Serving as the Sole Electron Acceptor

A novel, regular to irregular, coccoid-shaped, anaerobic, Fe(III)-reducing microorganism was isolated from the Guaymas Basin hydrothermal system at a depth of 2000 m. Isolation was carried out with a new technique using Fe(III) oxide as the electron acceptor for the recovery of colonies on solid medium. The isolate, designated strain 234T, was strictly anaerobic and exhibited a tumbling motility. The cells had a single flagellum. Strain 234T grew at temperatures between 65 and 90 degrees C, with an optimum at about 88 degrees C. The optimal salt concentration for growth was around 19 g l(-1). The isolate was capable of growth with H2 as the sole electron donor coupled to the reduction of Fe(III) without the need for an organic carbon source. This is the first example of a dissimilatory Fe(III)-reducing micro-organism capable of growing autotrophically on hydrogen. In addition to molecular hydrogen, strain 234T oxidizes pyruvate, acetate, malate, succinate, peptone, formate, fumarate, yeast extract, glycerol, isoleucine, arginine, serine, glutamine, asparagine, stearate, palmitate, valerate, butyrate and propionate with the reduction of Fe(III). This isolate is the first example of a hyperthermophile capable of oxidizing long-chain fatty acids anaerobically. Isolate 234T grew exclusively with Fe(III) as the sole electron acceptor. The G+C content was 58.7 mol%. Based on detailed analysis of its 16S rDNA sequence, G+C content, distinguishing physiological features and metabolism, strain 234T is proposed to represent a novel genus within the Archaeoglobales. The name proposed for strain 234T is Geoglobus ahangari gen. nov., sp. nov..

Use of Fe(III) as an Electron Acceptor To Recover Previously Uncultured Hyperthermophiles: Isolation and Characterization of Geothermobacterium ferrireducens gen. nov., sp. nov.

ABSTRACT It has recently been recognized that the ability to use Fe(III) as a terminal electron acceptor is a highly conserved characteristic in hyperthermophilic microorganisms. This suggests that it may be possible to recover as-yet-uncultured hyperthermophiles in pure culture if Fe(III) is used as an electron acceptor. As part of a study of the microbial diversity of the Obsidian Pool area in Yellowstone National Park, Wyo., hot sediment samples were used as the inoculum for enrichment cultures in media containing hydrogen as the sole electron donor and poorly crystalline Fe(III) oxide as the electron acceptor. A pure culture was recovered on solidified, Fe(III) oxide medium. The isolate, designated FW-1a, is a hyperthermophilic anaerobe that grows exclusively by coupling hydrogen oxidation to the reduction of poorly crystalline Fe(III) oxide. Organic carbon is not required for growth. Magnetite is the end product of Fe(III) oxide reduction under the culture conditions evaluated. The cells are rod shaped, about 0.5 μm by 1.0 to 1.2 μm, and motile and have a single flagellum. Strain FW-1a grows at circumneutral pH, at freshwater salinities, and at temperatures of between 65 and 100°C with an optimum of 85 to 90°C. To our knowledge this is the highest temperature optimum of any organism in the Bacteria. Analysis of the 16S ribosomal DNA (rDNA) sequence of strain FW-1a places it within the Bacteria, most closely related to abundant but uncultured microorganisms whose 16S rDNA sequences have been previously recovered from Obsidian Pool and a terrestrial hot spring in Iceland. While previous studies inferred that the uncultured microorganisms with these 16S rDNA sequences were sulfate-reducing organisms, the physiology of the strain FW-1a, which does not reduce sulfate, indicates that these organisms are just as likely to be Fe(III) reducers. These results further demonstrate that Fe(III) may be helpful for recovering as-yet-uncultured microorganisms from hydrothermal environments and illustrate that caution must be used in inferring the physiological characteristics of at least some thermophilic microorganisms solely from 16S rDNA sequences. Based on both its 16S rDNA sequence and physiological characteristics, strain FW-1a represents a new genus among the Bacteria. The name Geothermobacterium ferrireducens gen. nov., sp. nov., is proposed (ATCC BAA-426).

Reduction of humic substances and Fe III by hyperthermophilic microorganisms

Abstract The ability of hyperthermophilic microorganisms to transfer electrons to humic substances (humics) and other extracellular quinones was evaluated. When H 2 was provided as the electron donor, the hyperthermophile, Pyrobaculum islandicum , transferred electrons to highly purified humics and the humics analog, anthraquinone-2,6-disulfonate (AQDS). A diversity of other hyperthermophilic Archaea including: Pyrodictium abyssi , Pyrococcus furiosus , Archaeoglobus fulgidus , Thermococcus celer , Methanopyrus kandleri , as well as the thermophiles Methanococcus thermolithitrophicus and Methanobacterium thermoautotrophicum , exhibited H 2 -dependent AQDS reduction as did the hyperthermophilic bacterium Thermotoga maritima . AQDS acted as an electron shuttle between P. islandicum and poorly crystalline Fe(III) oxide and greatly accelerated rates of Fe(III) reduction. Electron shuttling by AQDS also promoted the reduction of the crystalline Fe(III) oxide forms, goethite and hematite. These results have implications for the potential mechanisms of Fe(III) reduction in various hot Fe(III)-containing environments such as near hydrothermal marine vents, terrestrial hot springs, and the deep terrestrial subsurface. The finding that the ability to reduce extracellular quinones is a characteristic of all of the hyperthermophiles evaluated and the fact that these hyperthermophiles are the organisms most closely related to the last common ancestor of extant organisms suggests that the last common ancestor had the ability to reduce humics. In combination with plausible geochemical scenarios, these results suggest that electron transfer to extracellular quinones and Fe(III) were initial steps in the eventual evolution of intracellular electron transport chains that employ quinones and iron-containing proteins.

Acetate Oxidation Coupled to Fe(III) Reduction in Hyperthermophilic Microorganisms

ABSTRACT No hyperthermophilic microorganisms have previously been shown to anaerobically oxidize acetate, the key extracellular intermediate in the anaerobic oxidation of organic matter. Here we report that two hyperthermophiles, Ferroglobus placidus and “Geoglobus ahangari,” grow at 85°C by oxidizing acetate to carbon dioxide, with Fe(III) serving as the electron acceptor. These results demonstrate that acetate could potentially be metabolized within the hot microbial ecosystems in which hyperthermophiles predominate, rather than diffusing to cooler environments prior to degradation as has been previously proposed.

Thermophily in the Geobacteraceae: Geothermobacter ehrlichii gen. nov., sp. nov., a Novel Thermophilic Member of the Geobacteraceae from the “Bag City” Hydrothermal Vent

ABSTRACT Little is known about the microbiology of the “Bag City” hydrothermal vent, which is part of a new eruption site on the Juan de Fuca Ridge and which is notable for its accumulation of polysaccharide on the sediment surface. A pure culture, designated strain SS015, was recovered from a vent fluid sample from the Bag City site through serial dilution in liquid medium with malate as the electron donor and Fe(III) oxide as the electron acceptor and then isolation of single colonies on solid Fe(III) oxide medium. The cells were gram-negative rods, about 0.5 μm by 1.2 to 1.5 μm, and motile and contained c-type cytochromes. Analysis of the 16S ribosomal DNA (rDNA) sequence of strain SS015 placed it in the family Geobacteraceae in the delta subclass of the Proteobacteria. Unlike previously described members of the Geobacteraceae, which are mesophiles, strain SS015 was a thermophile and grew at temperatures of between 35 and 65°C, with an optimum temperature of 55°C. Like many previously described members of the Geobacteraceae, strain SS015 grew with organic acids as the electron donors and Fe(III) or nitrate as the electron acceptor, with nitrate being reduced to ammonia. Strain SS015 was unique among the Geobacteraceae in its ability to use sugars, starch, or amino acids as electron donors for Fe(III) reduction. Under stress conditions, strain SS015 produced copious quantities of extracellular polysaccharide, providing a model for the microbial production of the polysaccharide accumulation at the Bag City site. The 16S rDNA sequence of strain SS015 was less than 94% similar to the sequences of previously described members of the Geobacteraceae; this fact, coupled with its unique physiological properties, suggests that strain SS015 represents a new genus in the family Geobacteraceae. The name Geothermobacter ehrlichii gen. nov., sp. nov., is proposed (ATCC BAA-635 and DSM 15274). Although strains of Geobacteraceae are known to be the predominant Fe(III)-reducing microorganisms in a variety of Fe(III)-reducing environments at moderate temperatures, strain SS015 represents the first described thermophilic member of the Geobacteraceae and thus extends the known environmental range of this family to hydrothermal environments.

Potential Importance of Dissimilatory Fe(III)‐Reducing Microorganisms in Hot Sedimentary Environments

Fe(III) is available as an electron acceptor in many modern hot (80-110°C) sedimentary environments which hyperthermophilic microorganisms inhabit and Fe(III) may have been an important electron acceptor as microbial life evolved on hot, ancient Earth. Recent studies have demonstrated that the ability to reduce Fe(III) is a highly conserved characteristic of hyperthermophiles and that the metabolic capabilities of some hyperthermophiles are greatly expanded when Fe(III) is available as an electron acceptor. An increasing diversity of novel hyperthermophiles, including some that are known to be important in the environment from molecular studies, are being recovered from hot environments when Fe(III) oxide is used as the electron acceptor for enrichment and isolation. These include the first hyperthermophiles documented to anaerobically oxidize acetate, a key intermediate in anaerobic carbon and electron flow, as well aromatic compounds and long-chain fatty acids. This suggests that complex organic matter can be oxidized to carbon dioxide with Fe(III) serving as the sole electron acceptor in hot microbial ecosystems. In addition to reducing Fe(III), some hyperthermophiles can reduce a variety of other metals, including U(VI) and Au(III), providing a potential explanation for the deposition of metals in some hot environments. Although the study of Fe(III) reduction in hyperthermophiles is still in its infancy, it is clear that Fe(III) reduction is central to the metabolism of many hyperthermophiles and that when Fe(III) is available in hot, microbial ecosystems, it has the potential to be an important electron acceptor for the anaerobic oxidation of hydrogen and organic matter.