Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Fri, 30 Nov 2018 18:02:50 Methanolobus profundi sp. nov., a methylotrophic methanogen isolated from deep subsurface sediments in a natural gas field Hanako Mochimaru, 1,2 Hideyuki Tamaki, 2 Satoshi Hanada, 2 Hiroyuki Imachi, 3 Kohei Nakamura, 2,4 Susumu Sakata 1 and Yoichi Kamagata 2,5 Correspondence Yoichi Kamagata y.kamagata@aist.go.jp 1 Institute for Geo-Resources and Environment, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8567, Japan 2 Institute for Biological Resources and Functions, AIST, Tsukuba, Ibaraki 305-8566, Japan 3 Subground Animalcule Retrieval Program, Extremobiosphere Research Center, Japan Agency for Marine-Earth Science and Technology, Yokosuka, Kanagawa 237-0061, Japan 4 Faculty of Applied Biological Sciences, Gifu University, Yanagido, Gifu 501-1193, Japan 5 Research Institute of Genome-based Biofactory, AIST, Sapporo, Hokkaido 062-8517, Japan A mesophilic, methylotrophic methanogen, strain MobM T , was isolated from a natural gas field in Japan. Strain MobM T grew on methanol and methylamines, but not on H 2 /CO 2 , formate, acetate or dimethyl sulfide. The cells were motile, irregular cocci (diameter, 0.9–1.2 mm) and occurred singly, in pairs, as tetracocci or (occasionally) as aggregates. Strain MobM T grew at 9–37 6C (optimally at 30 6C) and at pH 6.1–7.8 (optimally at pH 6.5). Sodium and magnesium were required for growth, at 0.1–1.0 M Na + (optimally at 0.35 M) and 10–400 mM Mg 2+ (optimally at 15–25 mM). The G+C content of the genomic DNA was 42.4 mol%. 16S rRNA gene sequencing revealed that the isolate is a member of the genus Methanolobus, but distinct from its closest neighbours, Methanolobus tindarius DSM 2278 T (sequence similarity, 98.0 %) and Methanolobus vulcani DSM 3029 T (98.1 %). On the basis of phenotypic and phylogenetic features of MobM T , it is clear that this strain represents a novel species of the genus Methanolobus, for which the name Methanolobus profundi sp. nov. is proposed. The type strain is MobM T (5DSM 21213 T 5NBRC 104158 T ). The genus Methanolobus comprises coccoid, methylo- trophic methanogens that grow optimally in media containing approximately 0.5 M NaCl (Boone et al., 2001; Table 1). To date, five species have been described; all have been found in various saline environments. Methanolobus bombayensis, Methanolobus oregonensis and Methanolobus taylorii can use dimethyl sulfide as a substrate for methanogenesis (Kadam et al., 1994; Liu et al., 1990; Oremland & Boone, 1994), unlike the other two species, Methanolobus tindarius and Methanolobus vulcani (Konig & Stetter, 1982; Kadam & Boone, 1995). In this study, a slightly halophilic, methylotrophic methano- gen, designated strain MobM T , was isolated from subsur- face sediments below 350 m in the Minami-Kanto Gas Field (Mobara, Chiba prefecture, Japan). This natural gas field is a dissolved-in-water type, and analyses of the stable carbon ( 13 C/ 12 C) and deuterium/hydrogen (D/H) isotopic composition of the methane and the ratio of methane to ethane and propane suggest that the methane is biogenic in origin (Igari & Sakata, 1989). The reservoir rocks are turbidite sandstones deposited around 1 Ma (million years ago) in a bathyal environment, being filled with ancient seawater (Sudo, 1967; Kunisue et al., 2002). The chemical composition of the formation water was conspicuously different from that of common seawater, i.e. it contained large amounts of iodine, bicarbonate and ammonia along with negligible amounts of sulfate. Culture-independent analysis of archaeal 16S rRNA gene sequences revealed that the methanogenic community residing in the formation water is diverse and includes close relatives of members of the genera Methanolobus, Methanohalophilus, Methanosaeta, Methanocalculus, Methanobacterium and Methanococcus (Mochimaru et al., 2007). Using a culture- The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain MobM T is AB370245. A supplementary figure showing the effects of variations in temperature and salinity on the specific growth rate of strain MobM T is available with the online version of this paper. International Journal of Systematic and Evolutionary Microbiology (2009), 59, 714–718 DOI 10.1099/ijs.0.001677-0 714 001677 G 2009 IUMS Printed in Great Britain