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Japan Marine Science and Technology Center Frontier Research System for Extremophiles able community structures and distribution profiles of each member in the chimney structures and substrata of ISCSs were highly fluctuated at each vent site, although geochemical analyses indicated the hydrothermal fluids from each vent had the identical origin. Especially culturable population of methanogen were prominent in two substrata of ISCSs deployed into hydrothermal conduits for two years, suggesting the vent fluids from the vents contained a large amount of viable methanogens and the occurrence of indigenous population of methanogen at subvent biosphere beneath those vent sites. These characteristic distribution profiles of viable microorganisms indicate that microbial population which could be detected above the seafloor of hydrothermal systems probably reflected the microbial populations occurring at subvent biosphere associating with hydrological structure beneath the seafloor. (c) ODP Leg. Subseafloor biosphere in the Peru Margin The subseafloor environment has been proposed to be the largest biosphere on Earth, as based on estimates of cells in marine sediment cores recovered by the Ocean Drilling Program (ODP). However, it is not well-known what kinds of microorganisms are present, how their distributions relate with geological settings, and how their metabolic activities impact the global geochemical cycles. The ODP Leg. took place in the eastern equatorial Pacific and Peru coastal margin in , and was the first expedition in ODP history targeted mainly on microbiology and biogeochemistry in the subseafloor biosphere. In this expedition, we investigated the vertical profile data of the results from culture-independent molecular ecological surveys in ODP sediment core columns collected from the two drilling sites, and , located on the land slope of the accretionary wedge in the Peru Trench. The sediment cores recovered from Site contained high organic carbon and methane, whereas those from Site contained low concentrations of these chemical components. Bulk prokaryotic nucleic acids were extacted and purified from each sediment, and S rRNA genes (rDNA) were amplified by PCR using domain specific primers. The analyses of rDNA sequences of clone libraries, quantitative-PCR for archaeal and bacterial rDNA, and T-RFLP fingerprint analysis revealed the previously unknown vertical distribution and diversity of Archaea and Bacteria in two geologically discrete subseafloor environments. (d) IMAGES Subseafloor Biosphere in the southwestern Sea of Okhotsk Microbial communities from a subseafloor sediment core from the southwestern Sea of Okhotsk were evaluated using both cultivation-dependent and -independent (molecular) analyses. The core, which extended . meters below the seafloor (mbsf), was composed of pelagic clay with several volcanic ash layers containing fine pumice grains. Direct cell counts and quantitative- PCR (qPCR) analysis of archaeal and bacterial S ribosomal RNA gene (rDNA) fragments indicated that the bacterial populations in ash layers were approximately - times higher than those in the clays. Partial sequences of rDNA clones revealed qualitative differences in the microbial communities from the two different layers. Two phylogenetically distinct archaeal assemblages within the Crenarchaeota, MCG (Miscellaneous Crenarchaeotic Group) and DSAG (Deep-Sea Archaeal Group), were the most predominant archaeal rDNA components in ash layers and pelagic clays, respectively. Proteobacterial rDNA within the gamma-Proteobacteria dominated the ash layers, whereas the sequences within the candidate division OP and the green non-sulfur bacteria dominated the pelagic clay environments. Molecular (S rDNA sequence analysis) of isolated colonies revealed a regional proliferation of viable heterotrophic mesophiles in the volcanic ash layers, along with some Gram-positives and Actinobacteria. The porous ash layers, ranging in age from tens to hundreds of thousands of years, thus appear to be discrete microbial habitats within the coastal subseafloor clay sediment, capable of harboring microbial communities very distinct from those seen in the more abundant pelagic clays. 81
JAMSTEC 2002 Annual Report Frontier Research System for Extremophiles (e) Subterrestrial Biosphere in the Hishikari Gold Mine Distribution and phylogenetic diversity of subterranean microbial communities in the Hishikari gold mine, southern part of Kyushu, Japan, were evaluated using the molecular phylogenetic analyses. The examined samples were the drilled cores such as the volcanic rock of andesite (.-.Ma) and the oceanic sedimentary basement rock of Shimanto-Supergroup (Ma), and were the geothermal hot aquifer waters directly collected from two discrete deposit sites, AWsite (.˚C, pH.) and XW-site (.˚C, pH.) at a depth of mbls (meter below land surface). Based on S rDNA clone analysis, the rDNA communities in the drilled cores and the hot aquifer water from XWsite consisted largely of the rDNA sequences closely related with the sequences often found in the marine environments, while the aquifer water from AW-site contained the rDNA sequences representing the members of Aquificales, thermophilic methanotrophs within g-subdivision of Proteobacteria and uncultivated strains within b-subdivision of Proteobacteria. The discrete microbial rDNA community structures might be associated with the physical and geochemical settings of the microbial habitats and the geological history of the formation in the gold mine. In addition, a shift in community structure of microbial mat colonizing along a subsurface hot spring stream in a Japanese gold mine was investigated by culture-independent molecular techniques and geochemical characterization. The stream occurs on a surface of a tunnel bored at m below a land surface and the transition of physical and chemical properties is evident throughout a m distance of stream (e.g. decreasing temperatures; ˚C in the discharging site, ˚C in the middle stream, and ˚C in the lower stream). The discharging hot water is derived from subsurface anaerobic aquifer containing plentiful CO , CH , H gases, and ammonium. In the upstream microbial mat, approximately % of the prokaryotic rDNA population was dominated by Archaea, whereas more than % of the population was bacterial rDNA in the lower microbial mat. The rDNA clone analysis revealed the predominant occurrence of a previously uncultivated crenarchaeotic phylotype, thermophilic methane-oxidizing g-proteobacteria and thermophilic hydrogen- and sulfur-oxidizing Aquificales in the upper mat while prevailing population of bacterial phylotypes closely related to ammonia-oxidizing Nitrosomonas and nitrite-oxidizing Nitrospira members in the middle and lower mat. Whole-cell fluorescent in situ hybridization analysis, quantitative analysis of key enzyme genes, and chemical analysis of interstitial water inside the mat structures strongly suggested the transition of the microbial community structure and the concomitant biogeochemical processes along a subsurface geothermal water stream. 4.2. Isolation of novel subsurface microorganisms Sulfurihydrogenobium subterraneus gen. nov., sp. nov. (Fig. ) A polyphasic taxonomic study was performed on a novel facultatively anaerobic, hydrogen- or sulfur/thiosulfate-oxidizing, thermophilic chemolithoautotroph recently isolated from subsurface hot aquifer water in a Japanese gold mine. The cells were straight to slightly curved rods, with a single polar flagellum. Growth was observed from ˚C to ˚C (optimum: –˚C; -min doubling time) and at pH .–. (optimum: pH .). The isolate was unable to use complex organic compounds, carbohydrates, amino acids and organic acids as the sole energy and carbon sources. The G + C content of the genomic DNA was . mol%. Phylogenetic analysis based on S rDNA sequences indicated that the isolate was closely related to an uncultivated group of microorganisms within Aquificales obtained from Icelandic and Japanese hot spring microbial mats but distantly related to previously identified genera of Aquificales such as Persephonella, Aquifex and Hydrogenobacter. We propose the name Sulfurihydrogenobium subterraneus gen. nov., sp. nov. (type strain: HGMK T ; JCM and ATCC BAA-). 82
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