Occurrence and potential activity of denitrifiers and methanogens in groundwater at 140 m depth in Pliocene diatomaceous mudstone of northern Japan.

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-, ß-, 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.

Population dynamics of Crenarchaeota and Euryarchaeota in the mixing front of river and marine waters.

A transect from the Tomoe River Mouth through Shimizu Port to Suruga Bay, Japan, was examined between 2005 and 2009 to reveal the population dynamics of Crenarchaeota and Euryarchaeota in an estuary environment. Crenarchaeota tended to increase in abundance in waters deeper than 100 m compared with Euryarchaeota, and comprised 11% of total direct counts. Archaeal abundance was highest in the Tomoe River Mouth, with a strong negative correlation between surface euryarchaeal abundance and salinity (P<0.001). The diversity index for the phylotypic archaeal community in the mouth was three times higher than that at sites St1-1m and St1-10m in the estuary, and OTUs represented most of the OTU groups at the sites. Three of the seven total OTUs, which comprised 83.6% of the 140 sequenced clones in the estuary, were related to the OTUs in the mouth with similarities higher than 97%. A significant proportion of the archaeal community appears to be derived from the Tomoe River. The two dominant phylotypes of the archaeal community in Shimizu Port, belonging to MGI and MGII, occurred ubiquitously.

Unique distribution of deep groundwater bacteria constrained by geological setting.

We collected groundwater samples at depths of up to 482 m from three boreholes drilled into sedimentary rock within two formations in Hokkaido, Japan. The prokaryotic community in each subsurface groundwater sample was analysed by microscopic counts and cloning-sequencing the 16S rRNA genes. On total direct counts, there were between 4.61 × 10(4) and 5.06 × 10(6) prokaryote cells ml(-1) in the samples, which is similar to the numbers observed at the marine subsurface. However, the vertical distribution of the prokaryotes did not show a simple decrease in abundance with increasing depth. A high abundance of cells with significant amounts of RNA was identified in the domain Bacteria using fluorescence in situ hybridization, with a high frequency of dividing cells at the transition zone between the two sedimentary rock formations. Cloning-sequencing analysis showed the predominance of γ-Proteobacteria at this transition zone at 281-312 m. The horizontal heterogeneity of the microbial distribution in the subsurface environment was also demonstrated by a relatively high density of members of the domain Archaea in borehole HDB-4, drilled only 1.5 km northeast of HDB-6 and in the same formation.

Denitrification activity and relevant bacteria revealed by nitrite reductase gene fragments in soil of temperate mixed forest.

Denitrification activity and bacterial community constituents were investigated in both well-drained and poorly drained soils of a temperate forest in central Japan by (15)N tracer experiments and a cloning-sequencing approach. Denitrification activity was much higher in wet soil than in dry soil, based on (15)N(15)N ((30)N(2)) and (15)N(15)NO ((46)N(2)O) production. Labeled nitrate ((15)NO(3)(-)) was immediately reduced to (30)N(2) in wet soil, whereas it was only reduced to (46)N(2)O in dry soil. Thus, the wet soil at the lower end of the catchment is a functional site for the scavenging for NO(3)(-) and N(2)O. Nitrite reductase gene (nirK and nirS) fragments from these soils were PCR amplified, cloned, and sequenced. Both nirK and nirS fragments were detected in the wet soil, whereas only nirK fragments were detected in the dry soil. All the nirK and nirS clones showed less than 90% similarity to known clones. Numerous operational taxonomic units for nirK and nirS were found in the wet soil. Considerable diversification within the largest clade on the nirK phylogenetic tree, which contained no known sequence, was observed in wet soil. Thus, a wet soil environment can provide both the habitat and conditions for the expression of denitrification activity.