title: Predominance of Viable Spore-Forming Piezophilic Bacteria in High-Pressure Enrichment Cultures from ~1.5 to 2.4 km-Deep Coal-Bearing Sediments below the Ocean Floor.
abstract: Phylogenetically diverse microorganisms have been observed in marine subsurface sediments down to ~2.5 km below the seafloor (kmbsf). However, very little is known about the pressure-adapted and/or pressure-loving microorganisms, the so called piezophiles, in the deep subseafloor biosphere, despite that pressure directly affects microbial physiology, metabolism, and biogeochemical processes of carbon and other elementsin situ. In this study, we studied taxonomic compositions of microbial communities in high-pressure incubated sediment, obtained during the Integrated Ocean Drilling Program (IODP) Expedition 337 off the Shimokita Peninsula, Japan. Analysis of 16S rRNA gene-tagged sequences showed that members of spore-forming bacteria within Firmicutes and Actinobacteria were predominantly detected in all enrichment cultures from ~1.5 to 2.4 km-deep sediment samples, followed by members of Proteobacteria, Acidobacteria, and Bacteroidetes according to the sequence frequency. To further study the physiology of the deep subseafloor sedimentary piezophilic bacteria, we isolated and characterized two bacterial strains, 19R1-5 and 29R7-12, from 1.9 and 2.4 km-deep sediment samples, respectively. The isolates were both low G+C content, gram-positive, endospore-forming and facultative anaerobic piezophilic bacteria, closely related toVirgibacillus pantothenticusandBacillus subtiliswithin the phylum Firmicutes, respectively. The optimal pressure and temperature conditions for growth were 20 MPa and 42°C for strain 19R1-5, and 10 MPa and 43°C for strain 29R7-12. Bacterial (endo)spores were observed in both the enrichment and pure cultures examined, suggesting that these piezophilic members were derived from microbial communities buried in the ~20 million-year-old coal-bearing sediments after the long-term survival as spores and that the deep biosphere may host more abundant gram-positive spore-forming bacteria and their spores than hitherto recognized.
authors.affiliation: Hadal Science and Technology Research Center, Shanghai Ocean UniversityShanghai, China; Department of Natural Sciences, Hawaii Pacific University, HonoluluHI, USA.
authors.forename: Jiasong
authors.initials: J
authors.lastname: Fang
authors.affiliation: Department of Marine Biodiversity Research, Japan Agency for Marine-Earth Science and Technology Yokosuka, Japan.
authors.forename: Chiaki
authors.initials: C
authors.lastname: Kato
authors.affiliation: Department of Natural Sciences, Hawaii Pacific University, Honolulu HI, USA.
authors.forename: Gabriella M
authors.initials: GM
authors.lastname: Runko
authors.affiliation: Department of Marine Biodiversity Research, Japan Agency for Marine-Earth Science and Technology Yokosuka, Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Nogi
authors.affiliation: Environmental Management Research Institute, National Institute of Advanced Industrial Science and Technology Ibaraki, Japan.
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Hori
authors.affiliation: State Key Laboratory of Marine Geology, Tongji University Shanghai, China.
authors.forename: Jiangtao
authors.initials: J
authors.lastname: Li
authors.affiliation: Kochi Institute for Core Sample Research, Japan Agency for Marine-Earth Science and Technology Kochi, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Morono
authors.affiliation: Kochi Institute for Core Sample Research, Japan Agency for Marine-Earth Science and TechnologyKochi, Japan; Research and Development Center for Ocean Drilling Science, Japan Agency for Marine-Earth Science and TechnologyYokohama, Japan; Research and Development Center for Submarine Resources, Japan Agency for Marine-Earth Science and TechnologyYokosuka, Japan.
authors.forename: Fumio
authors.initials: F
authors.lastname: Inagaki
keywords: coal beds; deep biosphere; endospore; high pressure; marine sediment; piezophiles