Thermoanaerobacter Subterraneus Sp. Nov., a Novel Thermophile Isolated from Oilﬁeld Water

International Journal of Systematic and Evolutionary Microbiology (2000), 50, 2141–2149 Printed in Great Britain Thermoanaerobacter subterraneus sp. nov., a novel thermophile isolated from oilfield water Marie-Laure Fardeau,1 Michel Magot,2 Bharat K. C. Patel,3 Pierre Thomas,1 Jean-Louis Garcia1 and Bernard Ollivier1,3 Author for correspondence: Bernard Ollivier. Tel: j33 4 91 82 85 76. Fax: j33491828570. e-mail: ollivier!esil.univ-mrs.fr 1 Laboratoire de A new thermophilic, anaerobic glucose-fermenting, Gram-positive, rod-shaped Microbiologie IRD, bacterium, designated strain SEBR 7858T, was isolated from an oilfield water Universite! s de Provence et de la Me! diterrane! e, ESIL sample. Under optimal conditions on a glucose-containing medium (3% NaCl, case 925, 163 Avenue de 65 SC and pH 75), the generation time was 25 h. No growth occurred at 35 or Luminy, 13288 Marseille 80 SC, nor at pH 55or90. Strain SEBR 7858T possessed lateral flagella. Spores cedex 9, France were undetected but heat-resistant forms were present. Strain SEBR 7858T 2 Sanofi Recherche, Unite! fermented a range of carbohydrates to acetate, L-alanine, lactate, H and CO . de Microbiologie, 2 2 31676 Labe' ge, The isolate reduced thiosulfate and elemental sulfur, but not sulfate or sulfite France to sulfide. In the presence of thiosulfate, the ratio of acetate produced per 3 School of Biomolecular mole of glucose consumed increased, suggesting a shift in the use of electron and Biomedical Sciences, acceptors during carbohydrate metabolism. The DNA GMC content was 41 Faculty of Science, Griffith mol%. Based on 16S rRNA gene sequence analysis, the strain was almost University, Brisbane, Queensland 4111, equidistantly related to all members of the genus Thermoanaerobacter (mean Australia similarity 92%). Based on phenotypic, genomic and phylogenetic characteristics, strain SEBR 7858T was clearly different from all members of the genus Thermoanaerobacter and was therefore designated as a new species, Thermoanaerobacter subterraneus sp. nov. The type strain is SEBR 7858T (l CNCM I-2383T, DSM 13054T). Keywords: thiosulfate reduction, Thermoanaerobacter subterraneus sp. nov., thermophile, phylogeny, oil well INTRODUCTION sugar beet and sugar cane extraction juices, thermal spas, volcanic hot springs, non-volcanic geothermally In the last two decades, intensive research on heated subsurface aquifers, hydrothermal vents and anaerobic, thermophilic, carbohydrate-fermenting oil-producing wells (Cayol et al., 1995; Cook et al., micro-organisms from marine and terrestrial volcanic 1996; Jin et al., 1988; Kozianowski et al., 1997; Larsen hot springs has led to the isolation of several new et al., 1997; Lee et al., 1993; Leigh et al., 1981; genera and species in the domains Bacteria and Slobodkin et al., 1999; Wiegel, 1986; Wynter et al., Archaea. The major rationale for this research stems 1996). Thermoanaerobacter species originating from from the biotechnological potential and the basic oil-producing wells have only recently been studied evolutionary traits of these microbes. In Bacteria, and have been found to be similar to surface-inhabiting particular attention has been paid to members of the Thermoanaerobacter species (Cayol et al., 1995; Magot order Thermotogales and the family Thermoanaerobi- et al., 2000; Wynter et al., 1996). We report here on the aceae. The latter includes the genera Thermoanaero- characterization of a new member of this genus, bacter and Thermoanaerobacterium, whose members Thermoanaerobacter subterraneus sp. nov., which was reduce thiosulfate or elemental sulfur, respectively, to isolated from a French oilfield. sulfide (Lee et al., 1993; Wiegel & Ljungdahl, 1981). Thermoanaerobacter species are thermophilic, hetero- METHODS trophic, saccharolytic anaerobes found in soil, faeces, Collection site. Strain SEBR 7858T was isolated from the ................................................................................................................................................. ‘Lacq Supe! rieur’ oilfield located in south-west France. The The GenBank accession number for the 16S rRNA gene sequence of in situ temperature of the geological formation, at a depth of strain SEBR 7858T is AF195797. 645 m, was 52 mC. A water sample was aseptically collected 01423 # 2000 IUMS 2141 M.-L. Fardeau and others at the wellhead, as described previously (Bernard et al., sequencing of the 16S rRNA gene have been described 1992), transported to the laboratory and stored at 4 mC until previously (Andrews & Patel, 1996). The 16S rRNA gene required. sequence was manually aligned with reference sequences of Medium, enrichment and isolation. Enrichment and routine various members of Bacteria using the editor ae2 (Maidak et growth were performed using a culture medium containing al., 1999). Reference sequences were obtained from the −" Ribosomal Database Project (Maidak et al., 1999), EMBL (l ): NH%Cl, 1n0g;K#HPO%,0n3g;KH#PO%,0n3 g; MgCl# ;6H#O, 1n0 g; CaCl#;2H#O, 0n1 g; NaCl, 10n0 g; KCl, 0n2g; and GenBank databases. Positions of sequence and align- Na#S#O$,3n16 g; glucose, 20 mM; sodium thiosulfate, ment uncertainty were omitted from the analysis. The 20 mM; cysteine\HCl, 0n5 g; yeast extract (Difco), 2n0g; pairwise evolutionary distances based on 1224 unambiguous Bio-trypticase (BioMe! rieux), 2n0 g; trace mineral element nucleotides were computed by using the method of Jukes & solution (Balch et al., 1979), 10 ml; resazurin, 1n0 mg. The Cantor (1969) and dendrograms were constructed from pH was adjusted to 7n0 with 10 M KOH and the medium was these distances by using the neighbour-joining method, both boiled under a stream of O#-free N# gas and cooled to room of which were from part of the suite of programs temperature. Five or 20 ml aliquots were dispensed into (Felsenstein, 1993). A maximum-likelihood approach with Hungate tubes or serum bottles, respectively, under a stream FastDNAml was used as an alternative method for tree of N#\CO# (80:20, v\v) gas and the vessels were autoclaved construction (Olsen et al., 1994). for 45 min at 110 mC. Prior to inoculation, Na#S;9H#O and NaHCO$ were injected from sterile stock solutions to final RESULTS concentrations of 0n04 and 0n2%, respectively. An aliquot of the water sample was inoculated into 20 ml medium and Enrichment and isolation incubated at 70 mC without agitation to initiate an enrichment culture. Several pure cultures were obtained by Enrichment cultures were positive after incubation at picking well isolated colonies that developed in the culture 70 mC for 3 d and H#S was detected, presumably de- medium solidified with 4% (w\v) Phytagel (Sigma; Deming rived from thiosulfate reduction. Microscopic exam- & Baross, 1986) by the repeated use of the Hungate roll- ination revealed the presence of rod-shaped bacteria. tube method (Hungate, 1969). One of these was used for Single, well isolated colonies (3 mm diam.) that de- subsequent studies. veloped in Phytagel roll tubes after 3 d incubation at Characterization studies. pH, temperature and NaCl ranges 70 mC were picked and serially diluted in Phytagel roll for growth were determined using the culture medium. The tubes at least twice before the culture was considered medium in Hungate tubes was adjusted to the desired pH, pure. Several axenic cultures were obtained and the measured at ambient temperature, by injecting 10% (w\v) culture designated strain SEBR 7858T was used for sterile anaerobic stock solutions of NaHCO$ or Na#CO$. further studies. NaCl was weighed directly in the tubes prior to dispensing the culture medium. The strain was subcultured at least once under the same experimental conditions prior to deter- Morphology mination of growth rates. Substrates were tested in culture T medium lacking glucose at a final concentration of 20 mM. Strain SEBR 7858 was a rod-shaped bacterium To test for electron acceptors, sodium thiosulfate, sodium (0n5–0n7i2–8 µm), which occurred singly (Fig. 1a). sulfate, sodium sulfite and elemental sulfur were added to No motility was observed but the cells possessed the culture medium at final concentrations of 20 mM, laterally inserted peritrichous flagella (Fig. 1b). No 20 mM, 2 mM and 2% (w\v), respectively. spores were observed under microscopic examination Light and electron microscopy. Light microscopy was per- (electron and light microscopy), but cultures exposed formed as described by Cayol et al. (1994). Electron at 120 mC for 45 min could be subcultured, suggesting microscopy was performed as described by Fardeau et al. the presence of heat-resistant forms. Electron micro- (1997a). scopy of ultrathin sections revealed a Gram-posi- Analytical techniques. Unless otherwise indicated, duplicate tive cell wall composed of a thin dense surface layer culture tubes were used throughout this study. Growth was separated from a thicker inner layer (Fig. 1c). measured by inserting tubes directly into a model UV-160A spectrophotometer (Shimadzu) and measuring changes in OD&)!. Sulfide was determined photometrically as colloidal Optimum growth conditions CuS by using the method of Cord-Ruwisch (1985). H#,CO#, Strain SEBR 7858 T was strictly anaerobic and did not sugars, alcohols, volatile and non-volatile fatty acids were grow in culture medium containing traces of oxygen, measured as described previously (Fardeau et al., 1996). - Alanine was measured by HPLC as described by Moore et as indicated by the anaerobiosis indicator, resazurin. al. (1958). The strain grew optimally at 65 mC (temperature range between 40 and 75 mC, but not at 80 mC; Fig. 2a) and a Determination of DNA GjC content. The DNA GjC content was determined by the DSMZ (Deutsche Sammlung pH of 7n5 (pH range between 6n0 and 8n5; Fig. 2b). The von Mikroorganismen und Zellkulturen GmbH, Braun- isolate grew in the presence of 0–3% NaCl with an schweig, Germany). DNA was isolated and purified by optimum of 0% at pH 7n0 and 70 mC (Fig. 2c). chromatography on hydroxyapatite and its GjC content was determined by using HPLC as described by Mesbah et Substrates for growth al. (1989). Non-methylated λ DNA (Sigma) was used as the standard. Yeast extract or Bio-trypticase was required for 16S rDNA sequence analysis.

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