We have developed a technique for cultivation of chemolithoautotrophs under high hydrostatic pressures that is successfully applicable to various types of deep-sea chemolithoautotrophs, including methanogens. It is based on a glass-syringe-sealing liquid medium and gas mixture used in conjunction with a butyl rubber piston and a metallic needle stuck into butyl rubber. By using this technique, growth, survival, and methane production of a newly isolated, hyperthermophilic methanogen Methanopyrus kandleri strain 116 are characterized under high temperatures and hydrostatic pressures. Elevated hydrostatic pressures extend the temperature maximum for possible cell proliferation from 116°C at 0.4 MPa to 122°C at 20 MPa, providing the potential for growth even at 122°C under an in situ high pressure. In addition, piezophilic growth significantly affected stable carbon isotope fractionation of methanogenesis from CO 2. Under conventional growth conditions, the isotope fractionation of methanogenesis by M. kandleri strain 116 was similar to values (؊34‰ to؊27‰) previously reported for other hydrogenotrophic methanogens. However, under high hydrostatic pressures, the isotope fractionation effect became much smaller (<؊12‰), and the kinetic isotope effect at 122°C and 40 MPa was ؊9.4‰, which is one of the smallest effects ever reported. This observation will shed light on the sources and production mechanisms of deep-sea methane.carbon isotope fractionation ͉ deep-sea hydrothermal vent ͉ hyperthermophile ͉ methanogenesis ͉ piezophilic M icrobial methanogenesis in the deep sea is a key process in the carbon cycle of Earth. It contributes to the CH 4 pool (free gas and methane hydrate), a potential energy source and alternative to petroleum (1, 2) as well as a strong greenhouse gas with a potential for rapid release (3), in deep-sea and subseafloor sediments. Methanogens are known to have several methanogenic types using different substrates of H 2 , acetate, methanol, CO, and so on. Hyperthermophilic hydrogenotrophic methanogens play a major role in primary production of ecosystems in deep-sea hydrothermal areas in the present Earth (4, 5) and may represent the most ancient type of microorganisms flourishing in the Archean Earth (6-10).Despite the significance of methanogens in the deep-sea and subseafloor ecosystems, the ecophysiological and biogeochemical characteristics of their in situ habitats have been little understood. It has been quite difficult to incorporate high hydrostatic pressures into experiments involving gaseous substrates such as H 2 and CO 2 . If this difficulty can be overcome by any specific apparatus (11,12), the subsequent handling of microbiological experiments under high hydrostatic pressures remains a great technical barrier. Thus, growth characterization of only thermophilic methanogens Methanocaldococcus jannaschii and Methanothermococcus thermolithotrophicus under high pressures has been successfully achieved, and only their piezophilic responses of growth and methane production have been inv...

Geochemical characteristics of hydrothermal fluids in the Iheya North hydrothermal field, mid-Okinawa Trough, was investigated. Twelve-years observation reveals temporal variation of vent fluid chemistry potentially controlled by temporally varying pattern of the phase-separation and -segregation, while the constant Element/Cl ratios among the periods and chimneys indicate the stable chemical composition of the source hydrothermal fluid prior to undergoing phaseseparation. The high K contents in the estimated source fluid are typical in the arc-backarc hydrothermal systems due to the hydrothermal reaction with the K-enriched felsic rocks. The high I, B and NH 4 contents and alkalinity are derived from decomposition of the sedimentary organic matters.Compositional and isotopic properties of gas species, CH 4 , H 2 , CO 2 , and C 2 H 6 , strongly suggest a dominance of biogenic CH 4 associated with the sedimentary organic matter. Based on the carbon mass balance calculation and the multidisciplinary investigations of the Iheya North hydrothermal system since the discovery, we hypothesized that the microbial methanogenesis occurs not only within the Central Valley where hydrothermal vents exist, but also in the spatially abundant and widespread basin-filling sediments surrounding the Iheya North Knoll, and that the microbially produced CH 4 is recharged together with the source fluid into the deep hydrothermal reaction zone. This "Microbial Methanogenesis at Recharge area in hydrothermal circulation" (MMR) model would be an implication for the generation and incorporation of hydrothermal fluid CH 4 in the deep-sea hydrothermal systems but also for those of cold seep CH 4 and for the presently uncertain hydrothermal fluid paths in the subseafloor environments. In the near future, the IODP drilling will be conducted in the Iheya North hydrothermal system, and give an excellent opportunity to testify our MMR model.

Hydrogen-rich hydrothermal areas, such as those in the Indian Ocean, may have had an influence on early evolution of life on Earth and thus have attracted interest because they may be a proxy for ancient ecosystems. The Kairei and Edmond hydrothermal fields in the Indian Ocean are separated by 160 km, but exhibit distinct fluid chemistry: Kairei fluids are hydrogen-rich; Edmond fluids are hydrogen-poor. At this region, the Central Indian Ridge shows an intermediate spreading rate, 48 mm year )1 as full rate, where the hydrothemal fields occur. Kairei field vent fluids show persistently high concentrations of H 2 . The Kairei field seems to be unique among hydrogen-enriched hydrothermal regions: most similar hydrogen-rich hydrothermal activity occurs along slowly spreading ridge, <40 mm year )1 . The geological and tectonic aspects of the Kairei and Edmond hydrothermal fields were studied to try to elucidate geological constraints on hydrogen production. Visual observations of the seafloor near Kairei from a submersible revealed olivine-rich plutonic rocks with olivine gabbro-troctolite-dunite assemblages exposed within 15 km of the vent field, with serpentinized ultramafic mantle rocks on the Oceanic Core Complex (OCC). The OCC area might be a recharge zone of Kairei hydrothermal activity producing H 2 -rich vent fluids. The chaotic seafloor within 30 km of the Kairei field reflects a magma-starved condition persisting there for 1 Myr. Asymmetric geomagnetic and gravity anomalies near the Kairei field can be used to infer that patchy olivine-rich intrusions are scattered within mantle ultramafics, where infiltrated seawater reacts with magma and ultramafic rocks or olivine-rich rocks. The heterogeneous uppermost lithosphere containing shallow olivine-rich rock facies surrounding the Kairei field provides abundant H 2 into the vent fluid through serpentinization. The hydrogen-rich Kairei field is hosted by basalt, with mafic-ultramafic olivine-rich lithology; the ordinary, hydrogen-poor Edmond field is hosted by a normal basaltic lithology. The contrasting geochemical signatures of the two fields reported here can also be found in ancient rocks from a juvenile Earth. This suggests that lithology-controlled generation of hydrogen may have operated for a long time and be relevant to the origin of life on Earth.

The galatheid crab, Shinkaia crosnieri (Decapoda: Galatheidae), forms dense colonies in the Iheya North and Hatoma Knoll deep-sea hydrothermal fields and has numerous setae covered with filamentous epibiotic microorganisms. Molecular phylogenetic analyses revealed that the epibiotic communities in S. crosnieri consisted mainly of yet-uncultivated phylotypes within Epsilonproteobacteria and Gammaproteobacteria in both hydrothermal vent fields. Uptake experiments using 13 C-labeled tracers clearly demonstrated that both H 13 CO3 − and 13 CH4 were assimilated into not only the epibiotic microbial communities associated with the setae, but also the epibiont-free tissue of living S. crosnieri. In addition, the incorporation of H 13 CO3 − into the microbial cells was strongly stimulated by the presence of reduced sulfur compounds but not by H2. In conclusion, the uptake experiments suggested that sulfur-oxidizing chemolithoautotrophic and methanotrophic production by the epibionts provides the nutrition for S. crosnieri.

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Scientific drilling at a submarine mud volcano shows that geofluid migration stimulates methanogenesis in the deep biosphere.

SummaryTo extend knowledge of subseafloor microbial communities within the oceanic crust, the abundance, diversity and composition of microbial communities in crustal fluids at back-arc hydrothermal fields of the Southern Mariana Trough (SMT) were investigated using culture-independent molecular techniques based on 16S rRNA gene sequences. Seafloor drilling was carried out at two hydrothermal fields, on-and off-ridge of the back-arc spreading centre of the SMT. 16S rRNA gene clone libraries for bacterial and archaeal communities were constructed from the fluid samples collected from the boreholes. Phylotypes related to Thiomicrospira in the Gammaproteobacteria (putative sulfide-oxidizers) and Mariprofundus in the Zetaproteobacteria (putative iron-oxidizers) were recovered from the fluid samples. A number of unique archaeal phylotypes were also recovered. Fluorescence in situ hybridization (FISH) analysis indicated the presence of active bacterial and archaeal populations in the fluids. The Zetaproteobacteria accounted for up to 32% of the total prokaryotic cell number as shown by FISH analysis using a specific probe designed in this study. Our results lead to the hypothesis that the Zetaproteobacteria play a role in iron oxidation within the oceanic crust.