Terrestrial rocks, petroleum reservoirs, faults, coal seams, and subseafloor gas hydrates contain an abundance of diverse methanoarchaea. However, reports on the isolation, purification and characterization of methanoarchaea in the subsurface environment are rare. Currently, no studies investigating methanoarchaea within fault environments exist. In this report, we succeeded in obtaining two new methanogen isolates, Methanolobus chelungpuianus St545Mb(T) and Methanobacterium palustre FG694aF, from the Chelungpu fault, which is the fault that caused a devastating earthquake in central Taiwan in 1999. Strain FG694aF was isolated from a fault gouge sample obtained at 694 meter below land surface (mbls) and is an autotrophic, mesophilic, nonmotile, thin, filamentous-rod shaped organism capable of using H2/CO2 and formate as substrates for methanogenesis. The morphological, biochemical and physiological characteristics and 16S rRNA gene sequence analysis revealed that this isolate belongs to Methanobacterium palustre. The mesophilic strain St545Mb(T), isolated from a sandstone sample at 545 mbls is a nonmotile, irregular coccoid organism that uses methanol and trimethylamine as substrates for methanogenesis. The 16S rRNA gene sequence of strain St545Mb(T) was 99.0% similar to Methanolobus psychrophilus strain R15 and was 96-97.5% similar to the other Methanolobus species. However, the optimal growth temperature and total cell protein profile of strain St545Mb(T) were different from M. psychrophilus strain R15, and whole genome DNA-DNA hybridization revealed less than 20% relatedness between these two strains. On the basis of these observations, we propose that strain St545Mb(T) (DSM 19953(T); BBRC AR10030; JCM15190) be named Methanolobus chelungpuianus sp. nov. Moreover, the environmental DNA database survey indicates that both Methanolobus chelungpuianus and Methanobacterium palustre are widespread in the subsurface environment.
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