Reduction of humic substances and Fe(III) by hyperthermophilic microorganisms

Chemical Geology 169 Ž2000. 289–298 www.elsevier.comrlocaterchemgeo

Reduction of humic substances and Fe žIII/ by hyperthermophilic microorganisms Derek R. Lovley ) , Kazem Kashefi, Madelline Vargas 1, Jason M. Tor, Elizabeth L. Blunt-Harris Department of Microbiology, Morrill Science Center IV, UniÕersity of Massachusetts, Amherst, MA 01003, USA Accepted 13 September 1999

Abstract The ability of hyperthermophilic microorganisms to transfer electrons to humic substances Žhumics. 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 ŽAQDS.. 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 FeŽIII. oxide and greatly accelerated rates of FeŽIII. reduction. Electron shuttling by AQDS also promoted the reduction of the crystalline FeŽIII. oxide forms, goethite and hematite. These results have implications for the potential mechanisms of FeŽIII. reduction in various hot FeŽIII.-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 FeŽIII. were initial steps in the eventual evolution of intracellular electron transport chains that employ quinones and iron-containing proteins. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Humics; FeŽIII.; Hyperthermophilic microorganisms

1. Introduction )

Corresponding author. Tel.: q1-413-545-9651; fax: q1-413545-1578. E-mail address: [email protected] ŽD.R. Lovley.. 1 Current address: Department of Biology, College of the Holy Cross, Worcester, MA 01610.

The metabolism of hyperthermophilic microorganisms is of interest because these organisms may have a significant influence on the geochemistry of

0009-2541r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 2 5 4 1 Ž 0 0 . 0 0 2 0 9 - 6

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hot environments on modern Earth and because understanding the activities of modern hyperthermophiles may provide insights into biogeochemical processes on early Earth and other planets ŽBaross and Hoffman, 1985; Pace, 1991; Adams, 1994; Bock and Goode, 1996; Stetter, 1996a.. It is now recognized that some hyperthermophilic microorganisms may grow at temperatures of over 1008C ŽStetter, 1996b.. This means that microorganisms can play an important role in the geochemistry of environments in which, not long ago, it would have been assumed that abiological processes predominated. Furthermore, it has been suggested that since hyperthermophilic microorganisms appear to be the closest living relatives to the microorganisms that gave rise to all modern life, the metabolism of hyperthermophiles provides a window to glimpse what early life on Earth may have been like ŽPace, 1991; Stetter, 1996a.. Until recently, the most predominant forms of respiration in hyperthermophiles were considered to be reduction of sulfur compounds or methane production ŽAdams, 1994; Stetter, 1996b.. However, investigations into the possibility that hyperthermophiles could use FeŽIII. as a terminal electron acceptor revealed that all of the hyperthermophiles evaluated could reduce FeŽIII. to FeŽII. with H 2 serving as the electron donor ŽVargas et al., 1998.. A hyperthermophilic member of the Bacteria, Thermotoga maritima, and a hyperthermophilic Archaea, Pyrobaculum islandicum, that were studied in more detail were found to conserve energy to support growth from FeŽIII. reduction ŽVargas et al., 1998; Kashefi and Lovley, 2000.. The ability of T. maritima to grow via FeŽIII. respiration was especially surprising as this organism had previously been considered to only conserve energy for growth via fermentation ŽStetter, 1996b.. The finding that so many organisms closely related to the last common ancestor of modern microorganisms reduce FeŽIII. suggests that the last common ancestor had the ability to reduce FeŽIII. and that FeŽIII. reduction was one of the earliest forms of microbial respiration ŽVargas et al., 1998.. This conclusion is consistent with geological studies that suggest that FeŽIII. was abundant on prebiotic Earth ŽDe Ronde et al., 1984; Cairns-Smith et al., 1992; de Duve, 1995. and evidence from banded iron formations that indicates that FeŽIII.

reduction was an early mechanism for oxidation of organic matter ŽWalker, 1987.. In addition to the hyperthermophilic FeŽIII.-reducing microorganisms, there is also a wide diversity of mesophilic microorganisms that can conserve energy to support growth from FeŽIII. reduction at temperatures of ca. 15–408C ŽLovley et al., 1997.. An additional physiological characteristic of all of these mesophilic FeŽIII.-reducing microorganisms is the ability to use humic substances Žhumics. as an electron acceptor ŽLovley et al., 1996, 1998; Coates et al., 1998.. Electron spin resonance studies have indicated that quinone moieties in humics serve as the electron accepting groups ŽScott et al., 1998.. The ability of FeŽIII.-reducing microorganisms to reduce extracellular quinones was confirmed in studies that demonstrated that all humics-reducing microorganisms can also reduce the humics analog, anthraquinone-2,6-disulfonate ŽAQDS., to anthrahydroquinone-2,6-disulfonate ŽAHQDS. ŽLovley et al., 1996, 1998; Coates et al., 1998.. An important geochemical consequence of microbial humics reduction is that it can greatly accelerate the rate of FeŽIII. oxide reduction in soils and sediments ŽLovley et al., 1996, 1998; Nevin and Lovley, 2000.. Once the quinone moieties in the humics are reduced, the hydroquinones that are generated can abiotically transfer electrons to FeŽIII., reoxidizing the humics and permitting them to again serve as an electron acceptor for humics-reducing microorganisms. In this manner, even very low concentrations of soluble humics can catalyze a significant amount of FeŽIII. reduction ŽLovley et al., 1996, 1998; Lloyd et al., 1999; Nevin and Lovley, 2000.. Reduction of FeŽIII. oxides is faster in the presence of humics than it is in their absence because the humics alleviate the need of the FeŽIII.-reducing microorganisms to establish contact with the FeŽIII. oxides in order to reduce them ŽLovley, 1997; Lovley et al., 1998.. The finding that all mesophilic FeŽIII.-reducing microorganisms that have been evaluated have the ability to reduce humics suggested that FeŽIII.-reducing hyperthermophilic microorganisms might also have this capacity. Therefore, the ability of hyperthermophiles to reduce humics was assessed. The results demonstrate that all of the hyperthermophilic microorganisms that were previously found to reduce FeŽIII. can also reduce humics or the humics analog,

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AQDS, and that electron shuttling via extracellular quinones can greatly accelerate the reduction of FeŽIII. oxides at hyperthermophilic temperatures. 2. Materials and methods 2.1. Cultures P. islandicum ŽDSM 4184., Pyrodictium abyssi ŽDSM 6158., Pyrococcus furiosus ŽDSM 3638., Archaeoglobus fulgidus ŽDSM 4304., Thermococcus celer ŽDSM 2476., Methanopyrus kandleri ŽDSM 6324., and T. maritima ŽDSM 3109. were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen ŽDSMZ.. Methanococcus thermolithotrophicus ŽOCM 138. and Methanobacterium thermoautotrophicum ŽOCM 142. were obtained from the Oregon Collection of Methanogens. 2.2. Cell suspensions Strict anaerobic techniques ŽMiller and Wolin, 1974; Balch et al., 1979. were used throughout. For studies on the reduction of humics and AQDS with cell suspensions, cells were grown as previously described ŽVargas et al., 1998.. Cells were harvested by centrifugation under N2 –CO 2 Ž80:20., and washed cell suspensions were prepared in anaerobic bicarbonate buffer Ž30 mM; pH 6.8. to provide ca. 0.2 mg of cell protein in 10 ml bicarbonate buffer under N2 –CO 2 Ž80:20. or H 2 –CO 2 Ž80:20. ŽVargas et al., 1998.. Highly purified soil humic acids obtained from the International Humic Substances Society Ž2 grl. or AQDS Ž3 mM for studies with methanogens; 5 mM for all other organisms. were added as potential electron acceptors and the reduction of the humics and AQDS were monitored over time as described below. For studies on the effect of AQDS on the reduction of FeŽIII. oxides by P. islandicum, the organism was grown with H 2 as the electron donor and FeŽIII. citrate Ž20 mM. as the electron acceptor, as previously described ŽVargas et al., 1998.. Cells were anaerobically harvested as described above and resuspended in growth medium Ž10 ml. in which the FeŽIII. citrate had been omitted and replaced with poorly crystalline FeŽIII. oxide Ž10 mmolrl.. The poorly crystalline FeŽIII. oxide was synthesized as

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previously described ŽLovley and Phillips, 1986.. H 2 Ž101 kPa. was provided as the electron donor. AQDS was added from an anaerobic stock Ž10 mM. to provide AQDS concentrations over a range from 0 to 500 mM. FeŽII. concentrations were monitored over time as described below. For studies on the reduction of more crystalline FeŽIII. oxide forms, goethite and hematite were prepared as previously described ŽLovley and Phillips, 1986. and added at concentrations of 10 mmolrl. AQDS was added at 200 mM when noted. 2.3. Analytical techniques Reduction of FeŽIII. to FeŽII. was monitored by measuring the accumulation of HCl-soluble FeŽII. over time. FeŽII. soluble in 0.5N HCl was quantified with ferrozine as previously described ŽLovley and Phillips, 1988.. Reduction of AQDS was monitored by measuring the accumulation of AHQDS that was detected by measuring the increase in absorbance at 450 nm as described previously ŽLovley et al., 1996.. Electrons transferred to humics were determined with FeŽIII.-citrate as previously described ŽLovley et al., 1996; Coates et al., 1998.. Growth was determined with direct cell counts via epifluorescence microscopy ŽHobbie et al., 1977., with the modification that iron forms were first dissolved with oxalate, as previously described ŽLovley and Phillips, 1988.. Total protein was determined with the bicinchoninic acid method ŽSmith et al., 1985. with bovine serum albumin as a standard. 3. Results The capacity for hyperthermophiles to reduce humics was first evaluated with P. islandicum, the hyperthermophile in the Archaea in which FeŽIII. reduction has been studied most intensively ŽVargas et al., 1998; Kashefi and Lovley, 2000.. When cell suspensions of P. islandicum were provided with H 2 as the electron donor and highly purified humic acids as the electron acceptor, electrons were transferred to the humic acids ŽFig. 1.. There was no reduction of the humic acids in the absence of H 2 ŽFig. 1.. Most investigations of humics reduction have been conducted with the humics analog, AQDS ŽLovley et al., 1996, 1998, 1999; Coates et al., 1998. because of

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Fig. 1. H 2 -dependent reduction of humics by P. islandicum. The incubation temperature was 1008C. The concentration of cell protein was 0.02 mgrml. The results are the means of duplicate incubations.

Fig. 3. H 2 -dependent reduction of AQDS by Pyr. abyssi. The incubation temperature was 908C. The concentration of cell protein was 0.02 mgrml. The results are the means of duplicate incubations.

the cost of highly purified humics and the technical difficulties of analyzing microbial humics reduction. All microorganisms that have been found to have the ability to reduce humics can reduce AQDS and

organisms that have been first recovered as AQDSreducing microorganisms have the ability to reduce humics. P. islandicum readily reduced AQDS ŽFig. 2.. As with humics reduction, reduction of AQDS

Fig. 2. H 2-dependent reduction of AQDS by P. islandicum. The incubation temperature was 1008C. The concentration of cell protein was 0.02 mgrml. The results are the means of duplicate incubations.

Fig. 4. H 2-dependent reduction of AQDS by The. celer. The incubation temperature was 808C. The concentration of cell protein was 0.02 mgrml. The results are the means of duplicate incubations.

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Fig. 5. Lactate-dependent reduction of AQDS by A. fulgidus. The incubation temperature was 808C. The concentration of cell protein was 0.02 mgrml. The results are the means of duplicate incubations.

was H 2-dependent. Furthermore, H 2 did not abiotically reduce AQDS in the absence of cells Ždata not shown..

Fig. 6. H 2 -dependent reduction of AQDS by M. kandleri. The incubation temperature was 1008C. The concentration of cell protein was 0.2 mgrml. The results are the means of duplicate incubations.

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Fig. 7. H 2-dependent reduction of AQDS by T. maritima. The incubation temperature was 808C. The concentration of cell protein was 0.02 mgrml. The results are the means of duplicate incubations.

All of the other hyperthermophilic Archaea that were evaluated were able to transfer electrons to AQDS. These covered a wide phylogenetic and

Fig. 8. Effect of various concentrations of AQDS on the rate that P. islandicum reduces poorly crystalline FeŽIII. oxide. The incubation temperature was 1008C. The concentration of cell protein was 0.2 mgrml. The results are the means of duplicate incubations.

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Fig. 9. AQDS stimulation of reduction of goethite and hematite by P. islandicum. Note that the y-axis scales are different for each FeŽIII. oxide. AQDS was added to provide 200 mM final concentration. The incubation temperature was 1008C. The concentration of cell protein was 0.2 mgrml. The results are the means of duplicate incubations.

physiological diversity such as the respiratory hyperthermophile, Pyr. abyssi ŽFig. 3., and hyperthermophiles with a fermentative metabolism such as The. celer ŽFig. 4. and Pyrococcus furiosus Ždata not shown. that each exhibited H 2-dependent AQDS reduction. A. fulgidus did not reduce AQDS with H 2 as the electron donor, but did reduce AQDS when lactate was provided ŽFig. 5.. A phylogenetic diversity of methanogenic Archaea also had the ability to reduce AQDS. These included the hyperthermophile, M. kandleri ŽFig. 6., as well as the thermophiles Met. thermolithitrophicus and Methanobacterium thermoautotrophicum that reduced AQDS with H 2 as the electron donor at 658C Ždata not shown.. H 2-dependent AQDS reduction was also observed in the hyperthermophile T. maritima ŽFig. 7.. This demonstrates that the ability to reduce extracellular quinones at high temperature can also be found in the Bacteria. The rates of AQDS reduction varied with the different organisms, but the relative rates of AQDS reduction exhibited a pattern similar to that previously observed for FeŽIII. reduction ŽVargas et al., 1998. with T. maritima reducing both electron acceptors faster than any of the Archaea. In order to determine if extracellular quinones might function as electron shuttles to stimulate FeŽIII. reduction, the effect of adding various concentrations of AQDS to cell suspensions of P. islandicum that contained H 2 as the electron donor and poorly crystalline FeŽIII. oxide as the electron acceptor was examined. Even low concentrations of AQDS Ž50 mM. stimulated FeŽIII. reduction over that observed in controls without added AQDS ŽFig. 8.. Addition

of AQDS not only stimulated the reduction of poorly crystalline FeŽIII. oxide, but also the reduction of the more crystalline FeŽIII. oxide forms, goethite and hematite ŽFig. 9..

4. Discussion The results demonstrate that hyperthermophilic microorganisms have the ability to transfer electrons to humic substances and other extracellular quinones. This significantly increases the known temperature range of microbial reduction of extracellular quinones because the highest temperature previously shown for this form of electron transfer was 308C ŽLovley et al., 1996, 1998; Coates et al., 1998; Scott et al., 1998.. The ability of such a wide phylogenetic diversity of hyperthermophiles to reduce extracellular quinones contrasts with previous studies that have indicated that the ability of mesophilic microorganisms to reduce extracellular quinones is limited to small, discrete phylogenetic clusters in the Bacteria ŽLovley et al., 1998.. For example, all of the microorganisms in the Geobacteraceae family that were evaluated had the ability to reduce extracellular quinones, but the closely related Desulfomonile tiedjei did not. The ability to reduce extracellular quinones in mesophilic microorganisms correlates with the ability to reduce FeŽIII. ŽLovley et al., 1998.. Thus, it might not be surprising that all of the hyperthermophiles tested were able to reduce extracellular quinones because they also all reduce FeŽIII.. Studies

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are underway to determine if the reductance for FeŽIII. and extracellular quinones are the same or similar. In both instances, the microorganisms need to have the ability to transfer electrons to an electron acceptor outside the cell. This is in contrast to other common electron acceptors for microorganisms such as oxygen, nitrate, sulfate, or carbon dioxide that can be reduced intracellularly. As discussed in detail below, the ability of hyperthermophilic microorganisms to reduce extracellular quinones may impact on the geochemistry of hot, modern environments. Furthermore, these results provide a potential model for the evolution of early electron transport chains in microorganisms. 4.1. Fe(III) and humics reduction in hot enÕironments To date, studies on the biogeochemistry of modern hot environments such as hydrothermal vents, hot terrestrial springs, or the deep subsurface have not provided much information on the rates of anaerobic microbial processes in general, and no data are available on rates of microbial reduction of FeŽIII. and extracellular quinones. However, it has been speculated that FeŽIII. reduction may be one of the most important forms of anaerobic respiration in many of these hot environments. For example, deep sea hydrothermal vents emit high concentrations of dissolved FeŽII. that is oxidized to FeŽIII. upon contact with cooler, oxygen-containing waters. The FeŽIII. formed in this manner precipitates to the nearby sediments that are often hot because of thermal waters seeping through these zones. Thus, it has been proposed that FeŽIII. reduction could be a significant form of microbial metabolism associated with hydrothermal vents ŽJannasch 1995; Karl, 1995.. A similar formation of FeŽIII. oxides ŽBrock et al., 1976. near the source of terrestrial hot springs could lead to FeŽIII. reduction being important in these environments as well. The recovery of large quantities of ultrafine-grained magnetite at depths of over 6 km in the terrestrial subsurface has been suggested to provide evidence that FeŽIII.-reducing microorganisms are important components of the hot, deep subsurface biosphere ŽGold, 1992.. This is because it is well known that, during FeŽIII. reduction, FeŽIII.-

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reducing microorganisms can convert FeŽIII. oxides to magnetite ŽLovley, 1990; Lovley et al., 1987.. There has been less discussion of the possibility for microbial reduction of humics and other extracellular quinones in hot environments. This may be due in part to the fact that microbial reduction of extracellular quinones has only recently been recognized as a form of microbial respiration ŽLovley et al., 1996. and there have been few studies on this process ŽCoates et al., 1998; Lovley et al., 1998; Scott et al., 1998.. However, it is possible that extracellular quinones are present in hot environments. Humic-like materials may be formed from the condensation of simpler organics at hydrothermal temperatures ŽNissenbaum et al., 1975. and the formation of a variety of simpler organic compounds, including possibly quinones, is thermodynamically favorable in such environments ŽShock, 1996.. Although much of the study of humics has focused on humics that are derived from terrestrial plant material, humics can also be generated from purely microbial sources and microbially derived humics have previously been shown to serve as electron acceptors for humics-reducing microorganisms ŽScott et al., 1998.. The high biological productivity associated with many hydrothermal systems might lead to the formation of microbially based humics-like material in these environments. Only low concentrations of humics or other extracellular quinones would be necessary in order for these extracellular quinones to play an important role in microbial respiration in hot environments. This is because of the ability of extracellular quinones to shuttle electrons to FeŽIII. oxides. As shown here, as little as 50 mM AQDS, the lowest concentration tested, greatly stimulated the reduction of synthetic poorly crystalline FeŽIII. oxides by acting as an electron shuttle between P. islandicum and the FeŽIII. oxide. As long as FeŽIII. is available, each molecule of extracellular quinone can be continually recycled so that the total electron transport through a small amount of extracellular quinones can be substantial. Therefore, if as described above, there are significant quantities of FeŽIII. oxides in hot environments, then there could be considerable electron flow through extracellular quinones. The results demonstrate that electron shuttling via extracellular quinones may increase both the rate and

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extent of FeŽIII. oxide reduction. Rates of reduction of poorly crystalline FeŽIII. oxide by P. islandicum were significantly higher in the presence of AQDS than in its absence. Stimulation of FeŽIII. oxide reduction by mesophilic microorganisms in the presence of extracellular quinones has been attributed to the electron shuttling capability of the quinones ŽLovley et al., 1996, 1998.. The necessity of FeŽIII. reducers to directly contact insoluble FeŽIII. oxides in order to reduce them is considered to be a ratelimiting step in microbial FeŽIII. reduction because it is commonly observed that reduction of insoluble FeŽIII. oxides is much slower than the reduction of soluble FeŽIII. forms ŽLovley, 1991.. Soluble extracellular quinones are also likely to be more accessible for microbial reduction than insoluble FeŽIII. oxides. Furthermore, once extracellular quinones are reduced, they are likely to be able to access insoluble FeŽIII. oxides better than the FeŽIII.-reducing microorganisms. Thus, the kinetics of FeŽIII. oxide reduction can be enhanced with extracellular quinones. Extracellular quinones may also increase the extent of FeŽIII. oxide reduction in hot environments. The initial oxidation of FeŽII. to FeŽIII. in such environments is likely to lead to the formation of poorly crystalline FeŽIII. oxide. However, these poorly FeŽIII. oxides are likely to become more crystalline over time. As shown here in cell suspensions of P. islandicum, as elsewhere in growth studies ŽKashefi and Lovley, 2000., like mesophilic FeŽIII. reducers ŽLovley, 1991., hyperthermophilic FeŽIII. reducers can readily reduce poorly crystalline FeŽIII. oxides, but may only slowly reduce more crystalline FeŽIII. forms, if at all. However, in the presence of low concentrations of an extracellular quinone, the rate of crystalline FeŽIII. oxide reduction was substantially increased. Thus, crystalline FeŽIII. oxides that might not be readily available for microbial reduction in the absence of extracellular quinones may be reduced if low concentrations of extracellular quinones are available. 4.2. Implications for early forms of microbial respiration There are numerous models for the geochemical and biological circumstances of early life. One model that has received significant attention is the concept

that the earliest forms of microbial life lived in hot, anaerobic environments ŽBaross and Hoffman, 1985; Pace, 1991; Holm, 1992; Bock and Goode, 1996.. This model is based on plausible geological scenarios and the finding that phylogenetic analysis based on 16S rRNA sequences has indicated that the microorganisms most closely related to the last common ancestor of extant organisms are hyperthermophilic Bacteria and Archaea. If early life did begin in hot environments and extant hyperthermophiles are most closely related to the last common ancestor, then the physiological properties of hyperthermophilic microorganisms may provide insights into the physiology of earlier microorganisms ŽPace, 1991.. Thus, the finding that all of the hyperthermophilic microorganisms that were evaluated had the ability to transfer electrons to extracellular quinones suggests that the last common ancestor had the ability to reduce extracellular quinones. There are several potential mechanisms by which quinones could have accumulated on prebiotic Earth and thus been available, along with FeŽIII., as potential electron acceptors in early forms of respiration. For example, it has been suggested that there was large-scale condensation of dissolved organic matter to form humic-like substances in the prebiotic hydrosphere ŽNissenbaum et al., 1975.. Quinone-moieties in these humic-like substances may have been an abundant potential electron acceptor for early life. Another potential source of quinones on prebiotic Earth was oxidation of polycyclic aromatic hydrocarbons ŽPAHs. with ultraviolet ŽUV. radiation ŽBernstein et al., 1999; McKinney et al., 1999.. It has been suggested that UV oxidation of PAHs in space may have provided an extraterrestrial source of quinones and other oxidized aromatic compounds prior to the start of life on Earth ŽBernstein et al., 1999; Ehrenfreund, 1999.. Furthermore, UV oxidation of PAHs to quinones could have taken place on the Earth’s surface ŽMcKinney et al., 1999.. Thus, the high levels of UV radiation on prebiotic Earth that are considered to have led to the accumulation of FeŽIII. as the result of UV oxidation of FeŽII. to FeŽIII. ŽCairns-Smith et al., 1992 and references therein. may also have resulted in the accumulation of quinones. The suggestion that the last common ancestor had the ability to reduce extracellular quinones and FeŽIII.

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may provide some insight into even earlier evolution of microbial respiration. Although the last common ancestor was likely to have been a metabolically sophisticated respiratory organism ŽPace, 1991; de Duve, 1995; Woese, 1998., earlier, more primitive microorganisms could also have had the capacity to transfer electrons to extracellular FeŽIII. andror quinones. The development of the ability to reduce these external electron acceptors could have been a major event in the evolution of the biosphere because it would have permitted for the first time the complete oxidation of organic compounds to carbon dioxide and provided the possibility of conserving energy from the oxidation of H 2 . A common feature of modern microbial electron transport chains are iron-containing electron transport proteins and lipophilic quinones that serve as electron shuttles between electron transport proteins ŽMadigan et al., 1997.. Thus, early electron transport to extracellular quinones, coupled with electron shuttling by the extracellular quinones to FeŽIII., may have been developed into a more sophisticated intracellular incorporation of quinones and iron-containing proteins as the mechanisms for respiration became more advanced and other electron acceptors such as sulfate, oxygen, and nitrate became more plentiful. Regardless of whether these speculations on early respiration are correct, the results clearly demonstrate for the first time that hyperthermophilic microorganisms are capable of transferring electrons to humics and other extracellular quinones. This indicates that electron shuttling between microorganisms and FeŽIII. oxides via extracellular quinones is possible in hot environments and should be considered as a potentially important mechanism for the reduction of FeŽIII. in hot spots such as around hydrothermal vents, terrestrial hot springs, and the deep terrestrial subsurface.

Acknowledgements This research was supported by grant DEB9714825 from the National Science Foundation and grant N00014-96-1-0382 from the Office of Naval Research.

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