Changes in the hydrochemical conditions of groundwater were evaluated following the construction of a large-scale underground facility at the Mizunami Underground Research Laboratory (MIU), Japan. The facility was constructed to a depth of 500 m in sedimentary and granitic rocks. Drawdown of the groundwater level in the range of several tens to hundreds of meters was observed up to hundreds of meters away from the shafts during the first ten years of facility construction and operation. Subsequent changes in groundwater chemistry occurred due to upconing of high-salinity groundwater from the deepest part of the shaft and the infiltration of low-salinity shallow groundwater. We predict that future deep groundwater chemistry in the vicinity of the MIU facility will resemble that of the present-day shallow groundwater. Multivariate statistical analysis provides fundamental insights into such a site. We found that the extent of hydrochemical variability related to MIU construction and operation was dependent on the distance from the facility shafts and galleries and on hydrogeological compartmentalization resulting from lithological boundaries (such as permeable conglomerates vs. more compact lithological units) and other features (such as faults or clay layers). We conclude that hydrochemical impact assessment of groundwater in low-permeability rock is essential prior to the construction of such a facility. This should include characterization of hydrogeological structures and compartments to propose suitable location of shafts and galleries.

In contrast to the deep subseafloor biosphere, a volumetrically vast and stable habitat for microbial life in the terrestrial crust remains poorly explored. For the long-term sustainability of a crustal biome, high-energy fluxes derived from hydrothermal circulation and water radiolysis in uranium-enriched rocks are seemingly essential. However, the crustal habitability depending on a low supply of energy is unknown. We present multi-isotopic evidence of microbially mediated sulfate reduction in a granitic aquifer, a representative of the terrestrial crust habitat. Deep meteoric groundwater was collected from underground boreholes drilled into Cretaceous Toki granite (central Japan). A large sulfur isotopic fractionation of 20–60‰ diagnostic to microbial sulfate reduction is associated with the investigated groundwater containing sulfate below 0.2 mM. In contrast, a small carbon isotopic fractionation (<30‰) is not indicative of methanogenesis. Except for 2011, the concentrations of H2 ranged mostly from 1 to 5 nM, which is also consistent with an aquifer where a terminal electron accepting process is dominantly controlled by ongoing sulfate reduction. High isotopic ratios of mantle-derived 3He relative to radiogenic 4He in groundwater and the flux of H2 along adjacent faults suggest that, in addition to low concentrations of organic matter (<70 µM), H2 from deeper sources might partly fuel metabolic activities. Our results demonstrate that the deep biosphere in the terrestrial crust is metabolically active and playing a crucial role in the formation of reducing groundwater even under low-energy fluxes.

Although deep subterranean crystalline rocks are known to harbor microbial ecosystems, geochemical factors that constrain the biomass, diversity, and metabolic activities of microorganisms remain to be clearly defined. To better understand the geochemical and microbiological relationships, we characterized granitic groundwater collected from a 1,148- to 1,169-m-deep borehole interval at the Mizunami Underground Research Laboratory site, Japan, in 2005 and 2008. Geochemical analyses of the groundwater samples indicated that major electron acceptors, such as NO(3)(-) and SO(4)(2-), were not abundant, while dissolved organic carbon (not including organic acids), CH(4) and H(2), was moderately rich in the groundwater sample collected in 2008. The total number of acridine orange-stained cells in groundwater samples collected in 2005 and 2008 were 1.1 x 10(4) and 5.2 x 10(4) cells/mL, respectively. In 2005 and 2008, the most common phylotypes determined by 16S rRNA gene sequence analysis were both related to Thauera spp., the cultivated members of which can utilize minor electron donors, such as aromatic and aliphatic hydrocarbons. After a 3-5-week incubation period with potential electron donors (organic acids or CH(4) + H(2)) and with/without electron acceptors (O(2) or NO(3)(-)), dominant microbial populations shifted to Brevundimonas spp. These geomicrobiological results suggest that deep granitic groundwater has been stably colonized by Thauera spp. probably owing to the limitation of O(2), NO(3)(-), and organic acids.

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