We investigated microbial methanogenesis and community structure based on 16S rRNA gene sequences from a coal seam aquifer located 843–907 m below ground level in northern Japan; additionally, we studied the δ13C and δ2H (δD) of coal‐bed gases and other physicochemical parameters. Although isotopic analysis suggested a thermocatalytic origin for the gases, the microbial activity and community structure strongly implied the existence of methanogenic microbial communities in situ. Methane was generated in the enrichment cultures of the hydrogenotrophic and methylotrophic microorganisms obtained from coal seam groundwater. Methanogen clones dominated the archaeal 16S rRNA gene libraries and were mostly related to the hydrogenotrophic genus Methanoculleus and the methylotrophic genus Methanolobus. Bacterial 16S rRNA gene libraries were dominated by the clones related to the genera Acetobacterium and Syntrophus which have a symbiotic association with methanogens. LIBSHUFF analysis revealed that N2 gas injected into the coal seam (for enhanced methane production) does not affect the coverage of archaeal and bacterial populations. However, amova analysis does provide evidence for a change in the genetic diversity of archaeal populations that are dominated by methanogens. Therefore, N2 injection into the coal seam might affect the cycling of matter by methanogens in situ.

A methanogenic organism, designated strain HB-1 T , from the domain Archaea was isolated from groundwater sampled from a subsurface Miocene formation located in Horonobe, Hokkaido, Japan. The strain grew on methanol, dimethylamine, trimethylamine, dimethylsulfide and acetate but not on monomethylamine, H 2 /CO 2 , formate, 2-propanol, 2-butanol or cyclopentanol. Cells were Gram-reaction-negative, non-motile, irregular cocci that were 1.4-2.9 mm in diameter and occurred singly or in pairs. The strain grew at 20-42 6C (optimum 37 6C), at pH 6.0-7.75 (optimum pH 7.0-7.25) and in 0-0.35 M NaCl (optimum 0.1 M). The G+C content of the genomic DNA was 41.4 mol%. 16S rRNA gene sequencing revealed that the strain was a member of the genus Methanosarcina but that it clearly differed from all recognized species of this genus (93.1-97.9 % sequence similarity). The phenotypic and phylogenetic features of strain HB-1 T indicate that it represents a novel species of the genus Methanosarcina, for which the name Methanosarcina horonobensis sp. nov. is proposed. The type strain is HB-1 T (5DSM 21571 T 5JCM 15518 T 5NBRC 102577 T ).

We investigated the diversity and distribution of archaeal and bacterial 16S rRNA gene sequences in deep aquifers of mid-to late Miocene hard shale located in the northernmost region of the Japanese archipelago. A major fault in the north-west-south-east (NW-SE) direction runs across the studied area. We collected three groundwater samples from boreholes on the south-west (SW) side of the fault at depths of 296, 374 and 625 m below ground level (m.b.g.l.) and one sample from the north-east (NE) side of the fault at a depth of 458 m.b.g.l. The groundwater samples were observed to be neutral and weakly saline. The total microbial counts after staining with acridine orange were in the order 10 5 − 10 6 cells mL − 1 and 10 3 cells mL − 1 in the aquifers to the SW and to the NE of the fault, respectively. A total of 407 archaeal and bacterial 16S rRNA gene sequences (204 and 203 sequences, respectively) were determined for clone libraries constructed from all groundwater samples. Phylogenetic analyses showed that the libraries constructed from the SW aquifers were generally coherent but considerably different from those constructed from the NE aquifer. All of the archaeal clone libraries from the SW aquifers were predominated by a single sequence closely related to the archaeon Methanoculleus chikugoensis , and the corresponding bacterial libraries were mostly predominated by the sequences related to Bacteroidetes, Firmicutes and δ -Proteobacteria. In contrast, the libraries from the NE aquifer were dominated by uncultured environmental archaeal clones with no methanogen sequences and by β -proteobacterial clones with no sequences related to Bacteroidetes and δ -Proteobacteria. Hence, the possible coexistence of methanogens and sulphate reducers in Horonobe deep borehole (HDB) on the SW side is suggested, particularly in . Moreover, these organisms might play an important geochemical role in the groundwater obtained from the aquifers. permeability (Fukusawa, 1987;Yamamoto et al ., 2004).An underground research laboratory (URL) is being constructed in the unique sediment by the Japan Atomic Energy Agency (JAEA), and the survey boreholes created for the URL construction are used for geophysical, geological, hydrological 204 S. SHIMIZU et al.

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The objective of this research is to clarify the mechanical properties and self-healing ability of ExcavationDamaged Zone (EDZ)

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Uniaxial compression, triaxial compression and Brazialian tests were conducted on several kinds of rock, with particular attention directed to the principal tensile strain. In this paper we aim to clarify the effects of the experimental environment -such as confining pressure, loading rate, water content and anisotropy-on the critical tensile strain, i.e., the measured principal tensile strain at peak load.It was determined that the chain-type extensometer is a most suitable method for measuring the critical tensile strain in uniaxial compression tests. It is also shown that the paper-based strain gage, whose effective length is less than or equal to a tenth of the specimen's diameter and glued on with a rubber-type adhesive, can be effectively used in the Brazilian tests.The effect of confining pressure P C on the critical tensile strain m TC in the brittle failure region was between −0.02× 10 −10 Pa −1 and 0.77 × 10 −10 Pa −1 . This pressure sensitivity is small compared to the critical tensile strain values of around −0.5×10 −2 . The strain rate sensitivities (m TC /({log(dm/dt)} were observed in the same way as the strength constants in other failure criteria. They were found to be from −0.10× 10 −3 to −0.52× 10 −3 per order of magnitude in strain rate in the triaxial tests. The average magnitude of the critical tensile strain m TC increased due to the presence of water by 4% to 20% for some rocks, and decreased by 22% for sandstone. It can at least be said that the critical tensile strain is less sensitive to water content than the uniaxial compressive strength under the experimental conditions reported here. An obvious anisotropy was observed in the P-wave velocity and in the uniaxial compressive strength of Pombetsu sandstone. It was not observed, however, in the critical tensile strain, although the data do show some variation.A ''tensile strain criterion'' was proposed, based on the above experimental results. This criterion signifies that stress begins to drop when the principal tensile strain reaches the critical tensile strain. The criterion is limited to use within the brittle failure region. The critical tensile strain contains an inelastic strain component as well as an elastic one. It is affected by the strain rate, however, it is relatively insensitive to the confining pressure, the presence of water and anisotropy.