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An H2/CO2 gas mixture (4:1, v/v) did not support growth. No growth was ... were straight polimorphic rods (Fig.5). ... Fig.3 Growth of Carboxydocella therma- ... Ljungdahl, L.G. (1994) The acetyl-CoA pathway and the chemiosmotic generation.
ANAEROBIC CO-OXIDIZING, H2-PRODUCING PROKARYOTES FROM VOLCANIC HABITATS T.G.Sokolova,1 J.González,2 N.A.Kostrikina,1 N.A.Chernyh,1 T.P.Tourova,1 E.A.Bonch-Osmolovskaya,1 F.Robb2 1 Institute of Microbiology, Russian Academy of Sciences, Prospect 60 Let Oktyabrya, 7/2, 117811, Moscow, Russia. 2 COMB, Columbus Center, 701 E Pratt St., Baltimore MD 21202, USA

Carbon monoxide is regarded as the common component of volcanic gases both in terrestrial and deep-sea environments. A variety of diverse bacteria possess CO-oxidizing enzymes termed CO dehydrogenases which allow them to utilise CO as a sole source of carbon and energy. Oxygen-dependent CO oxidation is a well known process carried out by a voluminous group of aerobic carboxydobacteria described by Zavarzin and Nozhevnikova (1977) and by Meyer et al. (1986). In anoxic environments CO could be oxidized by acetogenic bacteria with acetate production (Wood and Ljungdahl, 1991; Ljungdahl, 1994; Drake, 1994), or by methanogenic bacteria with methane production (Deppenmeier et al., 1996; Ferry, 1999). At the beginning of nineties a process of anaerobic CO oxidation resulting in production of hydrogen and carbon dioxide according to the reaction

CO + H2O = CO2 + H2 was found in thermophilic bacteria (Svetlichny et al., 1991). The aim of our work was the description of anaerobic thermophilic CO-oxidizing H2-producing microorganisms from volcanic environments. From terrestrial hot springs of Kamchatka, Yellowstone, Kuril and Kermadec Islands and Baikal region, deep-sea hot vents of West and East Pacific oceans samples of hot water and mud were taken anaerobically and stored in tightly stoppered bottles. The samples were inoculated in anaerobically prepared medium under atmosphere of CO at temperatures 60, 70, 80, and 85 oC. Freshwater (Svetlichny et al., 1991) or marine (Sokolova et al., 2001) medium were used. Gaseous and liquid fermentation products were detected by GLC. Phylogenetic position was determined by 16S rDNA sequencing (Sokolova et al., 2001). Workshop «Astrobiology in Russia» March 25-29, 2002, St.Petersburg, Russia

Carboxydothermus hydrogenoformans isolated from terrestrial hot springs of Kuril Islands was the first taxonomically described representative (Svetlichny et al., 1991). It was found to be a Gram-positive bacterium; its cells were short, slihgtly curved rods with lateral flagella (Fig.1). Ultrathin sections revealed a globular S-layer. Temperature of growth was from 40 to 78, optimum 70-72 oC and pH range of growth from 6.6 to 8.0, optimum 7.0. It grew anaerobically in the carbon monoxide athmosphere producing equimolar quantities of hydrogen and carbon dioxide. Except CO, the only substrate utilized by this organism was pyruvate. Generation time under optimal conditions for chemolithotrophic growth was 2.0 h-1. The DNA G+C content is 39+1 mol %. Growth and CO utilization was completely inhibited by penicillin, vancomycin, erythromycin, streptomycin and chloramphenicol. From a terrestrial hot vent at Kamchatka Peninsula located in Geyzer Valley a novel anaerobic, thermophilic, CO-utilizing H2-producing bacterium was isolated (Sokolova et al., 2002). Cells of this isolate were straight rods with a various length from 1 mm to 3 mm and a width about 0.4-0.5 mm (Fig.2), arranged singly or in short chains of three to five bacteria. Cells were motile due to lateral flagella. Electron microscopy of ultrathin sections revealed that cell envelope was of Gram positive type. Growth of strain occurred within a temperature range 40 and 68 oC with an optimum 58 oC. No growth was observed at 37 oC and 70 oC. The isolate grew in a pH range of 6.5-7.6, with an optimum at 7.0, while no growth was detected at pH 6.2 and 7.8. It grew in the atmosphere of 100% CO (0.23 mmoles CO in the gas phase per 1 ml of medium) on the mineral medium without yeast extract or other organic compounds except vitamins. CO oxidation was coupled with H2 and CO2 formation in equimolar quantities according to the equation: CO + H2O M CO2 + H2 (Fig.3). Methane, acetate, or any other metabolic products were not detected. The generation time of the strain under optimal conditions was 1.1 h. The isolate grew only chemolithoautotrophically on CO, producing equimolar quantities of H2 and CO2. It did not grow on peptone, yeast extract, starch, cellobiose, sucrose, lactose, glucose, maltose, galactose, arabinose, fructose, acetate, formate, pyruvate, ethanol, or methanol. An H2/CO2 gas mixture (4:1, v/v) did not support growth. No growth was observed on acetate, ethanol or lactate in the presence or absence of elemental sulfur or sulfate. Sulfur or sulfate were not reduced during growth with CO. No growth or H2S production occurred in the medium with elemental sulfur or sulfate and H2 in the gas phase. Penicillin, ampicillin, streptomycin, kanamycin, and neomycin completely inhibited CO utilization and the growth of the strain. DNA G+C content in strain was 46+1 mol %. Based on physiological and phylogenetic features of the strain it was assigned to new genus, Carboxydocella thermautotrophica (Sokolova et al., 2002). 157

Fig.1 Electron micrographs of cells of Carboxydothermus hydrogenoformans. Negative staining.

Fig.2 Electron micrographs of cells of Carboxydocella thermautotrophica strain 41. Negative staining.

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Another organism was isolated from terrestrial hot vent of Raoul Island (Kermadek Archipelago, New Zealand). It was found to be a Gram-positive bacterium, its cells being short straight motile rods with peritrichous flagella (Fig.4). The isolate grew only chemolithotrophically on CO, producing equimolar quantities of H2 and CO2 (according to the equation CO + H2O M CO2 + H2). Growth was observed in the temperature range from 45 Fig.3 Growth of Carboxydocella thermato 82 oC with an optimum at utotrophica strain 41 at 60 oC in mineral medium uno 70 C and in the pH range der a CO atmosphere (filled circle), CO consumption from 6.6 to 8.0 with an opti(filled quadrant) and H2 production (filled triangle), mum at 7.0. Generation time and CO2 production (blank triangle). under optimal conditions for chemolithotrophic growth was 8.3 h-1. The DNA G+C content is 50+1 mol %. Growth CO utilization was completely inhibited by penicillin, vancomycin, erythromycin and chloramphenicol. The isolate was not sensitive to streptomycin, rifampicin, and tetracycline (100 µg/ml). On the basis of phenotypic and phylogenetic features, we propose this isolate to represent a new species of the new genus with proposed name Thermolithotrophum carboxydophilum. From a hot spring of Baikal Lake region a first moderately alkaliphilic hydrogen-producing anaerobic carboxydotrophic bacterium was isolated. The isolate appeared to be a Gram-positive motile bacterium with an S-layer; cells were straight polimorphic rods (Fig.5). Aggregates of 3 to 15 cells were often observed. The isolate grew only chemolithotrophically on CO, requiering acetate or yeast extract. Growth was observed from 37 to 70 oC with an optimum at 55 oC. Optimum pH was 8.5. Generation time under optimal conditions was 10.3 h-1. The DNA G+C content is 48 mol %. Penicillin, erythromycin, streptomycin, rifampicin, vancomycin, and tetracycline completely inhibited both growth and CO utilization. On the basis of phenotypic and phylogenetic features, we proposed this isolate to represent a new genus named Thermaecola, type species T. carboxydotropha. 159

Fig.4 Electron micrographs of cells of Thermolithotrophum carboxydophilum strain R1. Negative staining.

Fig.5 Electron micrographs of cells of Thermaecola carboxydotropha strain 2204. Negative staining.

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Next isolate was obtained from the sample of a hot spring in Norris Basin in the Yellowstone National Park. This strain being capable of chemolithotrophic growth on CO as previously mentioned microorganisms showed additional ability to reduce ferric iron during the growth on CO. During growth of the culture, CO in the gas phase was changed to CO2 and H2, and Fe(II) was produced (Fig.6). The strain grew with 100% CO and Fe(III) given as Fe(III) citrate or hydromorphic ferric oxide. Growth products were Fe(II), H2 and CO2. It was able to grow on CO in the absence of Fe(III). Strain Nor1 did not grow in the presence of 2% or 10% of oxygen in the gas phase. Yeast extract (100 mg l-1) was obligately required for growth, but did not support the growth in the absence of CO. Cells of strain Nor1 are Gram positive, curved, motile rods 0.6x2.0 µm (Fig.7). Phylogenetic analysis revealed that the isolate should be assighned to new genus with proposed name Thermosinus as type species T. carboxydophilus. Thus, these isolates were obtained from terrestrial hot springs. First three isolates were able to grow chemolithoautotrophically on CO, the two others required acetate or yeast extract for growth. Phylogenetic analyses showed that isolates from terrestrial inhabitats should be assigned to different new genera. They all belong to Gram-positive branch but do not form single phylogenetic group. From a submarine hot vent in Okinawa Trough an anaerobic thermophilic, CO-utilizing H2-producing marine bacterium was isolated. Cells of the isolate were non-motile thin straight rods, sometimesbranching, with a cell wall of gram-positive type, surrounded with an S-layer (Fig.8). Chains of three to five cells were often observed. The isolate grew chemolithotrophically on CO, producing equimolar quantities of H2 and CO2 and organotrophically on several fermentable substrates producing hydrogen, acetate and CO2. Growth was observed from 50 to 80 oC with an optimum at 70 oC. The optimum pH was 6.8-7.1. The optimum concentration of sea salts in the medium was 20.5-25.5 g l-1. The DNA content was 33 mol %. Growth of the isolate was not inhibited by penicillin, but ampicillin, streptomycin, kanamycin, and neomycin completely inhibited the growth. Based on physiological and phylogenetic features of the isolate it was assigned to new genus, Carboxydobrachium as type species C. pacificum (Sokolova et al., 2001). From the samples of mud and Alvinella tubes from a deep-sea hot environment of East Pacific Rise several strains were isolated that grew under CO atmosphere producing equimolar quantities of hydrogen and carbon dioxide. Strain AM-4 was characterized in details. Cells were cocci with polar tuft of flagella. The isolate was extreme thermophile growing optimally at 82 oC. Yeast extract stimulated growth rate and cell yield during the growth on CO, but did not support the growth in the absence of CO. Phylogenetic analysis revealed that the isolate belonged to genus Thermococcus. 161

Fig.7 Electron micrographs of cells of Thermosinus carboxydophilus strain Nor1. Negative staining.

Fig.8 Electron micrographs of cells of Carboxydobrachium pacificum strain JM. Negative staining.

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Thus, our data show that diverse thermal environments are inhabited by thermophilic CO-oxidizing anaerobic prokaryotes capable of growth without sun light and oxygen on volcanic sources of energy and carbon. REFERENCES Deppenmeier, U., Müller, V. and Gottschalk, G. (1996) Pathways of energy conservation in methanogenic archaea. Arch.Microbiol., 165, 149-163. Drake, H.L. (1994) Acetogenesis, acetogenic bacetria, and the acetyl-CoA “Wood/ Ljungdahl” pathway: past and current perspectives. In: Acetogenesis (H.L.Drake, ed.), New York: Chapman and Hall, 1-15. Ferry, J.G. (1999) Enzymology of one-carbon metabolism in methanogenic pathways. FEMS Microbiol.Rev., 23, 13-38. Ljungdahl, L.G. (1994) The acetyl-CoA pathway and the chemiosmotic generation of ATP during acetogenesis. In: Acetogenesis (H.L.Drake, ed.), New York: Chapman and Hall, 65-87. Meyer, O., Jacobitz, S. and Kruger, B. (1986) Biochemistry and physiology of aerobic carbon monoxide-utilizing bacteria. FEMS Microbiol.Rev., 39, 161-179. Sokolova, T.G., González, J.M., Kostrikina, N.A., Chernyh, N.A., Tourova, T.P., Kato, C., Bonch-Osmolovskaya, E.A. and Robb, F.T. (2001) Carboxydobrachium pacificum gen.nov., sp.nov., a new anaerobic, thermophilic, COutilizing marine bacterium from Okinawa Trough. Int.J.Syst.Evol.Microbiol., 51, 141-149. Sokolova, T.G., Kostrikina, N.A., Chernyh, N.A., Tourova, T.P., Kolganova T.V. and Bonch-Osmolovskaya, E.A. (2002) Carboxydocella thermautotrophica gen.nov., sp.nov., a novel anaerobic CO-utilizing thermophile from Kamchatka hot spring. Int.J.Syst.Evol.Microbiol., in press. Svetlichny, V.A., Sokolova T.G., Gerhardt M., Ringpfeil M., Kostrikina N.A. and Zavarzin G.A. (1991) Carboxydothermus hydrogenoformans gen.nov., sp.nov., a CO-utilizing thermophilic anaerobic bacterium from hydrothermal environments of Kunashir Island. System.Appl.Microbiol., 14, 254-260. Wood, H.G. and Ljungdahl, L.G. (1991) Autotrophic character of acetogenic bacteria. In: Variations in Autotrophic Life (J.M.Shively and L.L.Barton, eds), San Diego: Academic Press, 201-255. Zavarzin, G.A. and Nozhevnikova, A.N. (1977) Aerobic carboxydobacteria. Microbial.Ecol., 3, 305-326.

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