Molecular characterization of microbial communities in fault‐bordered aquifers in the Miocene formation of northernmost Japan

Abstract

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.

Monitoring of the microbial community composition in saline aquifers during CO2 storage by fluorescence in situ hybridisation

“…For example, Basso and colleagues (2009) showed that the microbial community of a deep subsurface gas storage aquifer was primarily composed of Firmicutes and δ-Proteobacteria. Other studies reported that the microbial community in groundwater collected in Japan at a depth of 900 m was composed of 40 % Firmicutes and 27 % δ-Proteobacteria (Shimizu et al, 2007), in the same range as the deep aquifer in a Miocene formation (Shimizu et al, 2006). In addition to the possible contribution by Proteobacteria and Firmicutes, we used more specific probes that targeted phylogenetic groups of the SRB within the δ-subclass of Proteobacteria.…”

Methanogenic Archaea Isolated from Taiwan's Chelungpu Fault

“…Interestingly, surveys of 16S rRNA genes in environmental DNA have found sequences with 98 to 99% sequence similarity (Fig. 3) in clone YWA05, from a coal seam aquifer located 843 to 907 mbls in northern Japan (48), clone HDBW-WA06, from a fault-bordered aquifer located 374 mbls in the Miocene formation of northern Japan (49), and clones PL-10A1 and 10A6, from production waters of a low-temperature biodegraded oil reservoir in Canada (14). The phylotypes with high sequence similarity within the clade of Methanolobus chelungpuianus and M. psychrophilus may be widespread in terrestrial sediment and the subsurface.…”

“…Investigations of microbial communities have used 16S rRNA gene sequence analysis to characterize archaea in the deep subsurface such as in terrestrial rocks (45,52), petroleum reservoirs (8,9,18,39,40,42,58), faults (37,49,57), coal seams (11,48), gas fields (36), and subseafloor gas hydrates (17,33,35). These studies have demonstrated the abundance and diversity of methanoarchaea.…”

Methanosarcina horonobensis sp. nov., a methanogenic archaeon isolated from a deep subsurface Miocene formation

“…The amplified PCR product was sequenced as previously described (Shimizu et al, 2006), using the following sequencing primers: A25F (as above), U533F (59-GTG-CCAGCMGCCGCGGTAA-39, where M5A or C; Dojka et al, 1998), 0922F (59-GAAACTTAAAKGAATTG-39, where K5G or T; Achenbach & Woese, 1995), 1100F (59-GGCAACGAGCGMGACCC-39; Lane, 1991), U1492R (as above), 1098Ra (59-GGGTCTCGCTCGTTSCC-39, where S5C or G; Mikucki et al, 2003), U907R (59-CCGTCA-ATTCCTTTRAGTTT-39; Dojka et al, 1998), 536-519r (59-GWATTACCGCGGCKGCTG-39, where W5A or T; Lane et al, 1985), 368-353r (59-AGKTTTCGCGCCTGCT-39; Kotelnikova et al, 1998) and 0112aR (59-CCACGTG-TTACTSAGC-39; Achenbach & Woese, 1995 T was most similar (99.9 %) to that of uncultured clone HDBW-WA05 (accession no. AB237738), which had previously been obtained from deep subsurface groundwater of the Wakkanai Formation in the Horonobe area (Shimizu et al, 2006). Phylogenetic trees reconstructed using the neighbour-joining, maximum-parsimony and minimum evolution methods showed that strain HB-1 T clustered with members of the genus Methanosarcina and was most closely related to M. mazei S-6 T and M. frisia C16 T ( Fig.…”