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1 Institute of Environmental Engineering and Biotechnology, Tampere University of Technology, Tampere, Finland
2 DSMZ – German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
Correspondence
Anna H. Kaksonen
anna.kaksonen{at}tut.fi
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ABSTRACT |
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MAIN TEXT |
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; Beeder et al., 1994, 1995), hot springs (Zeikus et al., 1983; Nazina et al., 1989; Karnauchow et al., 1992; Henry et al., 1994) and geothermal groundwaters (Daumas et al., 1988; Love et al., 1993). Thermophilic sulfate-reducers include Gram-positive bacteria, such as Desulfotomaculum (Campbell & Postgate, 1965) and Thermodesulfobium (Mori et al., 2003) within the class ‘Clostridia’. Gram-negative sulfate-reducers have been found in genera such as Thermodesulforhabdus (Beeder et al., 1995) and Desulfacinum (Rees et al., 1995) within the class Deltaproteobacteria, Thermodesulfobacterium (Zeikus et al., 1983) and Thermodesulfatator (Moussard et al., 2004) within the class Thermodesulfobacteria and Thermodesulfovibrio (Henry et al., 1994) within the class ‘Nitrospira’. Additionally, one archaeal sulfate-reducing genus, Archaeoglobus (Beeder et al., 1994), has been described. Recently, we have reported the enrichment and isolation of thermophilic sulfate-reducers from a geothermal underground mine in Japan (Kaksonen et al., 2006a, b) and in this paper we describe a novel thermophilic sulfate-reducing bacterium, strain RL80JIVT, isolated from the same mine.
Strain RL80JIVT was isolated from an underground mine 250 m below ground, from a black sediment layer beneath a thin red layer on a tunnel wall that was covered with ferric iron. The in situ temperature of the habitat was 70–80 °C. Enrichment and isolation of strain RL80JIVT were performed at 80 °C using modified Postgate growth medium (pH 7.0–7.5) (Kaksonen et al., 2006a) with lactate as electron donor. Anaerobic roll-tubes solidified with 1.5 % Gelrite gellan gum were used for isolation. For chemotaxonomic analysis and DNA isolation the strain was cultured at 70 °C in modified DSM medium 641 containing pyruvate as electron donor. The medium was supplemented with 1 ml selenate/tungstate solution l–1 (DSM medium 385), and sodium dithionate (25 mg l–1) was used as the reducing agent instead of Na2S.
The isolate was examined via phase-contrast microscopy (Zeiss Axioskop 2). Flagellum staining was performed as described by Heimbrook et al. (1989). Spore formation by the strain was determined microscopically, and by testing for growth after heat treatment (95 °C for 25 min). Gram type of the cells was determined by both Gram staining and the KOH test (Gregersen, 1978).
The effects of temperature, pH and NaCl concentration on growth were determined as previously described (Kaksonen et al., 2006a). The ability of the strain to utilize various electron donors (1–20 mM) was tested in a medium containing 20 mM sulfate. The utilization of various electron acceptors (10 mM) was studied using lactate (10 mM) as the electron donor. Amorphous Fe(III) oxyhydroxide was formed by neutralizing FeCl3 solution to a pH of 7 with NaOH. Cultures were incubated for 1–2 weeks. Electron donor utilization was determined by measuring growth (optical density at 660 nm; Shimadzu UV-1601 spectrophotometer or Ultrospec II LKB Biochrom 4050 UV/visible spectrophotometer), or based on hydrogen sulfide production or substrate conversion as described previously (Kaksonen et al., 2004). Ferrous iron was determined colorimetrically (Shimadzu UV-1601) with ferrozine (Stookey, 1970). Concentrations of sulfate, sulfite, thiosulfate, nitrate and nitrite were determined by using ion chromatography (Dionex DX-120).
Diaminopimelic acid isomers were detected in cell-wall hydrolysates by using TLC as described by Rhuland et al. (1955) and Kaksonen et al. (2006b). Respiratory isoprenoid quinones were extracted and analysed according to the methods described by Collins & Jones (1981), Monciardini et al. (2003) and Groth et al. (1996) by using HPLC and GC-MS (Kaksonen et al., 2006b).
Fatty acid methyl esters of cellular fatty acids were obtained by saponification, methylation, extraction and base wash, as described by Kämpfer & Kroppenstedt (1996), Kroppenstedt (1985) and Miller (1982). The fatty acid methyl mixtures were separated on a gas chromatograph (Hewlett Packard 5890 Series II) as described previously (Kaksonen et al., 2006b) and analysed by using the Microbial Identification Standard software package Sherlock version 4.5 (Sasser, 1990).
Methods for the amplification, sequencing and phylogenetic analysis of the 16S rRNA gene were performed as described previously (Kaksonen et al., 2006a). Genomic DNA for G+C content determination was released by rupturing cells using a French pressure cell (Thermo Spectronic) and was purified by chromatography on hydroxyapatite (Cashion et al., 1977). The DNA was hydrolysed with P1 nuclease and the nucleotides were dephosphorylated with bovine alkaline phosphatase (Mesbah et al., 1989). The G+C content of the resulting deoxyribonucleosides was determined by using reversed-phase HPLC (Shimadzu Corp.) and calculated from the ratio of deoxyguanosine (dG) and thymidine (dT) (Tamaoka & Komagata, 1984; Mesbah et al., 1989).
Cells of strain RL80JIVT were straight or slightly curved rods, 0.8–1 µm in diameter and 2–6 µm in length. The strain formed spherical spores which germinated after a heat shock treatment at 95 °C for 25 min. The spores were located centrally or subterminally and sporulation caused swelling of the cells, creating a lemon-shaped appearance. Cells were motile with two or more flagella and Gram-positive as determined by both Gram staining and the KOH test. Data for temperature, pH and NaCl range for growth of strain RL80JIVT are given in Table 1. The temperature at the sampling point in the mine was 70–80 °C, which is also within the growth range of strain RL80JIVT. Strain RL80JIVT was able to use sulfate, sulfite, thiosulfate and elemental sulfur as electron acceptors. RL80JIVT utilized H2 in the presence of CO2 and various carboxylic acids or their sodium salts as electron donors (Table 1). A number of electron donors (e.g. lactate, pyruvate, crotonate, pentanoate and hexanoate) were oxidized to acetate, whereas no acetate accumulated during the oxidation of others (e.g. fumarate and glutamate). The strain fermented lactate and pyruvate.
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. Significant proportions of saturated fatty acids were found; the major fatty acids were iso-C15 : 0, iso-C17 : 0 and C16 : 0 (Table 2).
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) revealed that strain RL80JIVT clustered with the thiosulfate-reducing bacterium Thermanaeromonas toyohensis DSM 14490T (90.9 % 16S rRNA gene sequence similarity), an isolate from a similar geothermal mine environment (Mori et al., 2002), and with the sulfate-reducer Desulfotomaculum thermocisternum DSM 10259T (90.0 % sequence similarity). The G+C content of the total DNA of strain RL80JIVT (60.1 mol%) (Table 1
) was higher than that of T. toyohensis (49.6 mol%) and those reported for thermophilic Desulfotomaculum species (47–57 mol%) (Castro et al., 2000; Goorissen et al., 2003; Liu et al., 1997), thereby differentiating strain RL80JIVT from T. toyohensis and species of the genus Desulfotomaculum.
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Description of Desulfovirgula gen. nov.
Desulfovirgula (De.sul.fo.vir'gu.la. L. pref. de from; L. n. sulfur sulfur; L. fem. n. virgula twig or rod, N.L. fem. n. Desulfovirgula a rod that reduces sulfur compounds).
Cells are rod-shaped, spore-forming, Gram-positive and thermophilic. Sulfate and other sulfur compounds are used as electron acceptors. Optimal growth occurs at neutral pH. The cell wall contains meso-diaminopimelic acid and MK-7 is the major menaquinone. Major cellular fatty acids are iso-C15 : 0, iso-C17 : 0 and C16 : 0. The genomic DNA G+C content of the type species is 60.1 mol% (as determined by HPLC). Phylogenetic position based on 16S rRNA gene sequence analysis is within the Gram-positive bacteria. The type species is Desulfovirgula thermocuniculi.
Description of Desulfovirgula thermocuniculi sp. nov.
Desulfovirgula thermocuniculi (ther.mo.cu.ni'cu.li. Gr. adj. thermos hot; L. n. cuniculus mine; N.L. gen. n. thermocuniculi of a hot mine).
Has the following properties in addition to those given in the description of the genus. Cells (0.8–1x2–6 µm) are motile. Growth occurs at 61–80 °C (optimum 69–72 °C), pH 6.4–7.9 (optimum pH 6.8–7.3) and NaCl concentrations of 0–0.5 % (optimum 0 % NaCl). Sulfate, sulfite, thiosulfate and elemental sulfur are used as electron acceptors, but Fe(III), nitrate and nitrite are not. H2 in the presence of CO2 and various carboxylic acids or their sodium salts (Table 1) are used as electron donors. Fermentative growth occurs on lactate and pyruvate.
The type strain, RL80JIVT (=DSM 16036T=JCM 13928T), was isolated from a geothermally active underground mine in Japan.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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