To identify the methanogenic pathways present in a deep aquifer associated with an accretionary prism in Southwest Japan, a series of geochemical and microbiological studies of natural gas and groundwater derived from a deep aquifer were performed. Stable carbon isotopic analysis of methane in the natural gas and dissolved inorganic carbon (mainly bicarbonate) in groundwater suggested that the methane was derived from both thermogenic and biogenic processes. Archaeal 16S rRNA gene analysis revealed the dominance of H 2 -using methanogens in the groundwater. Furthermore, the high potential of methane production by H 2 -using methanogens was shown in enrichments using groundwater amended with H 2 and CO 2 . Bacterial 16S rRNA gene analysis showed that fermentative bacteria inhabited the deep aquifer. Anaerobic incubations using groundwater amended with organic substrates and bromoethanesulfonate (a methanogen inhibitor) suggested a high potential of H 2 and CO 2 generation by fermentative bacteria. To confirm whether or not methane is produced by a syntrophic consortium of H 2 -producing fermentative bacteria and H 2 -using methanogens, anaerobic incubations using the groundwater amended with organic substrates were performed. Consequently, H 2 accumulation and rapid methane production were observed in these enrichments incubated at 55 and 65 1C. Thus, our results suggested that past and ongoing syntrophic biodegradation of organic compounds by H 2 -producing fermentative bacteria and H 2 -using methanogens, as well as a thermogenic reaction, contributes to the significant methane reserves in the deep aquifer associated with the accretionary prism in Southwest Japan.

The basic mechanisms of hydrogen and carbon isotope fractionations in CO 2 reduction are not yet fully understood. Less information related to hydrogen isotope fractionation in CO 2 reduction is available than for carbon isotope fractionation. Hydrogen isotope fractionation achieved from a laboratory incubation study of CO 2 reduction with pure cultures (>300‰; Balabane et al., 1987) was greater than those observed in natural environments such as deep-sea sediments in which CO 2 reduction is the dominant pathway in CH 4 production (approximately 160‰; Whiticar et al., 1986). Incubation experiments have never indicated hydrogen isotope fractionation factors equivalent to those observed in natural environments.

Sulfur-turf microbial mats develop in sulfide-containing hot spring water dominated by chemolithoautotrophic sulfur-oxidizing bacteria. The sulfur-turf mat that developed at a source of hot water (72°C, pH 6.8) exhibited a growth rate of 0.48±0.04 h −1 and biomass production of 4.6±1.0 mg of C h −1 . On a per-cell basis, this biomass production was at least an order of magnitude higher than the CO2 uptake rate calculated for a photosynthetic mat dominated by thermophilic Synechococcus spp. at 70°C. The sulfur-turf-associated microbial community likely contributes to carbon fixation and primary production in this geothermal habitat.

Microbial biomass production has been measured to investigate the contribution of planktonic bacteria to fluxations in dissolved organic matter in marine and freshwater environments, but little is known about biomass production of thermophiles inhabiting geothermal and hydrothermal regions. The biomass production of thermophiles inhabiting an 85 degrees C geothermal pool was measured by in situ cultivation using diffusion chambers. The thermophiles' growth rates ranged from 0.43 to 0.82 day(-1), similar to those of planktonic bacteria in marine and freshwater habitats. Biomass production was estimated based on cellular carbon content measured directly from the thermophiles inhabiting the geothermal pool, which ranged from 5.0 to 6.1 microg C l(-1) h(-1). This production was 2-75 times higher than that of planktonic bacteria in other habitats, because the cellular carbon content of the thermophiles was much higher. Quantitative PCR and phylogenetic analysis targeting 16S rRNA genes revealed that thermophilic H2-oxidizing bacteria closely related to Calderobacterium and Geothermobacterium were dominant in the geothermal pool. Chemical analysis showed the presence of H2 in gases bubbling from the bottom of the geothermal pool. These results strongly suggested that H2 plays an important role as a primary energy source of thermophiles in the geothermal pool.

Anaerobic microbial activity has a major influence on the subsurface environment. We investigated the denitrification and methanogenesis in anoxic groundwater at a depth of 140 m in two boreholes drilled in a sedimentary geological setting, where the redox potential fluctuated. The average maximum potential denitrification rates, measured under anaerobic conditions in the two boreholes using an (15) N tracer, were 0.060 and 0.085 nmol (30) N2  mL(-1)  h(-1) . The deduced NirS amino acid sequences obtained from in situ samples were similar to those of isolates belonging to the α-, β-, and γ-Proteobacteria, and the Firmicutes (72-100% similarity). Based on the nirS gene, the same operational taxonomic unit dominated incubated samples from each borehole. Methanogenesis candidates were detected by 16S rRNA gene analysis, but no sequence was detected using primers for the functional methanogenesis gene mcrA. Although the stable isotope signatures suggested that some of the dissolved methane was of biogenic origin, no potential for methane production was evident during the incubations. The groundwater at 140 m depth did not contain oxygen, had an Eh ranging from -144 to 6.8 mV, and was found to be a potential field for denitrification.