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Thermotoga Petrophila Sp. Nov. and Thermotoga Naphthophila Sp. Nov., Two Hyperthermophilic Bacteria from the Kubiki Oil Reservoir in Niigata, Japan

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Thermotoga Petrophila Sp. Nov. and Thermotoga Naphthophila Sp. Nov., Two Hyperthermophilic Bacteria from the Kubiki Oil Reservoir in Niigata, Japan International Journal of Systematic and Evolutionary Microbiology (2001), 51, 1901–1909 Printed in Great Britain Thermotoga petrophila sp. nov. and Thermotoga naphthophila sp. nov., two hyperthermophilic bacteria from the Kubiki oil reservoir in Niigata, Japan 1 Marine Biotechnology Yoh Takahata,1† Miyuki Nishijima,2 Toshihiro Hoaki3 Institute, Kamaishi 1 Laboratories, 3-75-1 Heita, and Tadashi Maruyama Kamaishi City, Iwate 026-0001, Japan Author for correspondence: Yoh Takahata. Tel: 81458147226. Fax: 81458147257. 2 j j Marine Biotechnology e-mail: yoh.takahata!sakura.taisei.co.jp Institute, Shimizu Laboratories, 1900 Sodeshi, Shimizu City, Shizuoka T T 424-0037, Japan Two hyperthermophilic bacteria, strains RKU-1 and RKU-10 , which grew optimally at 80 SC, were isolated from the production fluid of the Kubiki oil 3 Taisei Corporation reservoir in Niigata, Japan. They were strictly anaerobic, rod-shaped Technology Center, 344-1 Nase-cho Totsuka-ku, fermentative heterotrophs. Based on the presence of an outer sheath-like Yokohama, Kanagawa structure (toga) and 16S rDNA sequences, they were shown to belong to the 245-0051, Japan genus Thermotoga. Cells of strain RKU-1T were 2–7 µmby07–10 µm, with flagella. They grew at 47–88 SC on yeast extract, peptone, glucose, fructose, ribose, arabinose, sucrose, lactose, maltose, starch and cellulose as sole carbon sources. Cells of strain RKU-10T were 2–7 µmby08–12 µm, with flagella. They grew at 48–86 SC on yeast extract, peptone, glucose, galactose, fructose, mannitol, ribose, arabinose, sucrose, lactose, maltose and starch as sole carbon sources. While strains RKU-1T and RKU-10T reduced elemental sulfur to hydrogen sulfide, their final cell yields and specific growth rates decreased in the presence of elemental sulfur. Thiosulfate also inhibited growth of strain RKU-1T but not strain RKU-10T. The GMC contents of the DNA from strains RKU-1T and RKU-10T were 468 and 461 mol%. Phenotypic characteristics and 16S rDNA sequences of the isolates were similar to those of Thermotoga maritima and Thermotoga neapolitana, both being hyperthermophilic bacteria isolated from hydrothermal fields. However, the isolates differed from these species in their minimum growth temperatures, utilization of some sugars, sensitivity to rifampicin and the effects of elemental sulfur and thiosulfate on growth. The low levels (less than 31%) of DNA reassociation between any two of these hyperthermophilic Thermotoga strains indicated that the isolates were novel species. Analysis of the gyrB gene sequences supported the view that the isolates were genotypically different from these reference species. The isolates were named Thermotoga petrophila sp. nov., with type strain RKU-1T (l DSM 13995T l JCM 10881T), and Thermotoga naphthophila sp. nov., with type strain RKU-10T ( l DSM 13996T l JCM 10882T). Keywords: Thermotoga petrophila, Thermotoga naphthophila, hyperthermophilic bacteria, oil reservoir INTRODUCTION growth temperatures higher than 80 mC have been isolated from various hot environments (Stetter, 1996). A large number of hyperthermophiles with optimum While most of them are members of the domain ................................................................................................................................................................................................................................................................................................................. † Present address: Taisei Corporation Technology Center, 344-1 Nase-cho Totsuka-ku, Yokohama, Kanagawa 245-0051, Japan. Abbreviation: ASW, artificial sea water. The GenBank/EMBL/DDBJ accession numbers for the 16S rDNA sequences of Thermotoga petrophila RKU-1T, Thermotoga naphthophila RKU-10T, Thermotoga neapolitana DSM 4359T and Thermotoga thermarum DSM 5069T are AB027016, AB027017, AB039768 and AB039769. The accession numbers for the gyrB gene sequences of T. petrophila RKU-1T, T. naphthophila RKU-10T, Thermotoga maritima DSM 3109T and T. neapolitana DSM 4359T are AB039770–AB039773. 01704 # 2001 IUMS 1901 Y. Takahata and others Archaea, some hyperthermophiles belong to the genera tightly with sterile butyl-rubber stoppers. Sodium sulfide, Aquifex and Thermotoga in the domain Bacteria which had been sterilized by filtration (disk capsule of (Blo$ chl et al., 1995). According to 16S rRNA gene 0n2 µm pore size; Fuji), was added to a final concentration of sequencing, they represent the deepest phylogenetic approximately 400 µM. After autoclaving, flushing with branches within the domain Bacteria (Winker & nitrogen and adding sodium sulfide, the final pH in the medium was between 6n9 and 7n1 at room temperature. One Woese, 1991). The members of the genus Thermotoga, millilitre of the formation water in the production fluid was in the order Thermotogales, include Thermotoga inoculated into 9 ml of YE medium in a Hungate tube and maritima (Huber et al., 1986), Thermotoga neapolitana incubated at 85 mC for 1–7 d. Hyperthermophiles were (Belkin et al., 1986; Jannasch et al., 1988), Thermotoga purified by the Gelrite plating method as described pre- thermarum (Windberger et al., 1989), Thermotoga elfii viously (Takahata et al., 2000). (Ravot et al., 1995a), Thermotoga subterranea Electron microscopy. Cell morphology and flagellation were (Jeanthon et al., 1995) and Thermotoga hypogea observed with a transmission electron microscope (H-7000; (Fardeau et al., 1997). T. maritima and T. neapolitana Hitachi) as described previously (Takahata et al., 2000). The grow optimally at 80 mC and are hyperthermophilic cells were fixed with 2n5% glutaraldehyde and post-fixed species. In the last decade, hyperthermophilic archaea with 1% osmium tetroxide. Ultrathin sections of the cells (Beeder et al., 1994; Grassia et al., 1996; L’Haridon et embedded in Epon 812 resin were cut with a Reichelt-Nissei al., 1995; Orphan et al., 2000; Stetter et al., 1993; Ultracut-N ultramicrotome and doubly stained with uranyl Takahata et al., 2000) and thermophilic bacteria with acetate and lead citrate (Reynolds, 1963). After fixation with an optimal temperature below 80 C (Beeder et al., 2n5% glutaraldehyde, whole mounted cells were negatively m et al 1995; Cayol et al., 1995; Greene et al., 1997; Magot stained with 4% (w\v) uranyl acetate (Kurr ., 1991). et al., 1997; Nilsen et al., 1996; Rees et al., 1995), Determination of cell numbers. Cell growth was monitored including T. elfii, T. subterranea and T. hypogea, have by the acridine orange direct-counting method (Hobbie et been isolated from the production fluids of oil al., 1977) under an epifluorescence microscope (model BX- reservoirs. Thermotoga-like bacteria with togas, grow- 60; Olympus). ing at 65–85 mC, have also been reported from oil Growth characteristics. The effect of temperature on mi- reservoirs in Alaska and California (Orphan et al., crobial growth was examined in YE medium with a mineral 2000; Stetter et al., 1993). We describe two novel oil bath (model OH-16; Taitec). The effects of pH and NaCl hyperthermophilic Thermotoga species, Thermotoga on growth were examined in a YE-based medium incubated at 80 mC. The pH dependency of growth was determined petrophila sp. nov. and Thermotoga naphthophila sp. with various buffer systems, as described previously (Hoaki nov., isolated from the Kubiki oil reservoir. et al., 1994). The pH of the medium was checked and readjusted at room temperature after autoclaving, flushing METHODS with nitrogen and adding sodium sulfide. The effects of various gaseous phases on microbial growth were examined Reference micro-organisms. T. maritima DSM 3109T, T. by using ASW for autotrophic growth or YE medium for neapolitana DSM 4359T and T. thermarum DSM 5069T were heterotrophic growth. The gaseous phase in a Hungate tube obtained from the DSMZ (Deutsche Sammlung von Mikro- was filled with CO#,H#, air or a mixture of H# and CO# (4: organismen und Zellkulturen), Braunschweig, Germany. 1). To determine the nutritional requirements of the isolates, Collection of the sample. The production fluid was taken their growth was examined in 10 ml ASW medium (pH 7n0) from the no. 3 storage tank of the Kubiki oil reservoir in without shaking at 80 mC with one of the following carbon Niigata, Japan. The geological structure and physical sources at a final concentration of 0n1%(w\v): yeast extract, properties of the Kubiki oil reservoir have been described peptone, casein, albumin, Casamino acids, a mixture of 20 previously (Takahata et al., 2000). The production fluid was amino acids, glucose, galactose, fructose, mannitol, ribose, collected in sterile glass bottles, which were then sealed with arabinose, xylose, sucrose, lactose, maltose, starch, acetate, sterile silicon stoppers and plastic screw caps. They were lactate, formate, propionate, maleic and citric acids, meth- chilled in a cooler box with ice and transported to the anol and ethanol. Growth was also examined in the presence laboratory. of 1% (v\v) cellulose, chitin, kerosene, light oil, A-heavy oil (Nippon Mitsubishi Oil) or crude oil in 30 ml ASW medium Isolation and cultivation. Anaerobic, heterotrophic hyper- (pH 7n0) with shaking. Crude oil was obtained from the thermophiles were grown on YE medium (Takahata et al., Kubiki oil reservoir. To confirm growth on any of the 2000) containing 0n2% (w\v) yeast extract in artificial sea substrates, the isolate was subcultured twice in the same water (ASW; Jannasch et al., 1995). One litre of ASW medium. contained 20 g NaCl, 3 g MgCl#.6H#O, 6 g MgSO%.7H#O, Metabolic products. Metabolic products
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