Geomicrobiological properties of Tertiary sedimentary rocks from the deep terrestrial subsurface

Geomicrobiological properties of Tertiary sedimentary rocks from the deep terrestrial subsurface

Physics and Chemistry of the Earth 58–60 (2013) 28–33 Contents lists available at SciVerse ScienceDirect Physics and Chemistry of the Earth journal ...

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Physics and Chemistry of the Earth 58–60 (2013) 28–33

Contents lists available at SciVerse ScienceDirect

Physics and Chemistry of the Earth journal homepage:

Geomicrobiological properties of Tertiary sedimentary rocks from the deep terrestrial subsurface Takeshi Suko a,1, Mariko Kouduka a, Akari Fukuda a, Kenji Nanba b, Manabu Takahashi a, Kazumasa Ito a, Yohey Suzuki a,2,⇑ a b

Geological Survey of Japan, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8567, Japan Faculty of Symbiotic System Science, Fukushima University, 1 Kanayagawa, Fukushima 960-1296, Japan

a r t i c l e

i n f o

Article history: Available online 19 April 2013 Keywords: Subsurface microbiology Deep biosphere Miocene marine sediment Porosity Denitrification Groundwater geochemistry

a b s t r a c t Microbial metabolic activity within the deep subsurface can potentially impact radionuclide migration during geological disposal of nuclear waste. To evaluate the geomicrobiological properties of Tertiary sedimentary rocks, which are widely distributed in the repository environment in Japan, aseptic and deoxygenated drilling was conducted with the installation of a multi-packer system to collect cores and groundwater. Integrated results from measurements on potential rates of denitrification and pore-size distributions in drill core samples indicated that in situ microbial activity is constrained by the availability of pore spaces larger than 0.1 lm in radius. Comparison of geochemical profiles of porewater extracted from the core samples and groundwater collected within multi-packer intervals revealed that terminal electron acceptors such as nitrite and sulfate were depleted in groundwater. Microbial community structures based on 16S rRNA gene sequences were represented by phylotypes related to Fe-, Mn-, elemental sulfur- and sulfate-reducing bacteria in groundwater. In addition, a phylotype closely related to denitrifying Acidovorax sp. of the b-proteobacteria was dominant in the lower borehole interval. From our results, it is likely that groundwater microorganisms mediate redox reactions that influence the mobility of radionuclides in the deep subsurface. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Many countries are facing scientific and social challenges with long-term management of nuclear waste, and are considering the practical option of permanent disposal in repositories in deep geological formations. Total system performance assessments have been exercised for repositories in generic and site-specific geological formations. The host rock types, such as granite (Finland and Sweden), tuff (USA), clay stone (France) and salt stone (USA), have been identified as suitable for geological disposal. Although recent studies demonstrated that microorganisms are present in the repository environments, the influences of, and factors constraining, their metabolic activities vary from one repository site to another (Gillow et al., 2000; Hallbeck and Pedersen, 2008; Kieft et al., 1997; Pedersen et al., 2008). The study of microbial populations in host rocks is important because their metabolic activity profoundly

⇑ Corresponding author. Tel.: +81 29 861 3978; fax: +81 29 861 3643. E-mail address: [email protected] (Y. Suzuki). Present address: Radioactive Waste Management and Transport Safety Division, Japan Nuclear Energy Safety Organization, 4-3-20 Toranomon, Minato-ku, Tokyo 1050001, Japan. 2 Present address: Department of Earth and Planetary Science, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. 1

1474-7065/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.

affects the redox state in the repository environment (Hallbeck and Pedersen, 2008; Stroes-Gascoyne et al., 2007). Under reducing conditions, many radionuclides from nuclear wastes are immobile as solubility-limiting solid phases (Langmuir, 1997). In Japan, a candidate site, at which a repository is constructed below a legal depth of 300 m, is being recruited among local governments. Surface exposed rocks are composed of about 60% sedimentary, and the areal occupancy of Tertiary consolidated sedimentary rocks is higher than that of Quaternary unconsolidated sediments within a depth range targeted for geological disposal. To evaluate potential microbial impacts on geological disposal in Tertiary sedimentary rocks, we selected a study site in a geologically and tectonically stable fore-arc region where no volcanic activity or major faults have been observed in the field or geological record (Suzuki et al., 2009). We applied an aseptic and deoxygenated drilling procedure to characterize the Tertiary sedimentary rocks (Suzuki et al., 2009). To constrain factors controlling the activity of subsurface microorganisms in the Tertiary sedimentary rocks, we investigated the correlation between metabolic activity and the distribution of pore sizes. This relationship is known to be determinative for microbial activity in Cretaceous sedimentary rocks (Fredrickson et al., 1997; Stroes-Gascoyne et al., 2007). Geochemical and microbiological properties of groundwater also were investigated by installing a

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multi-packer system into the borehole. The main purpose of this study is to clarify geomicrobiological properties of Tertiary sedimentary rocks by integrating results from core- and groundwaterbased investigations. Collectively, these data reveal the relation of microbial activities to pore space availability and microbial influences on the redox state of groundwater and the solubility of radionuclides in Tertiary sedimentary rocks.

2. Materials and methods 2.1. Drilling and sampling procedures A large amount of groundwater was obtained from a relatively shallow borehole (Kr-2). The depth of the borehole was 90 m below ground level. Drilling fluid was collected at this depth. The shallow groundwater was ultrafiltered to remove microbial cells, and then deoxygenated by two cycles of evacuation and purging with N2 gas. By a multi parameter probe (Horiba Multi Probe W22), the concentration of dissolved O2 was measured to be less than 0.2 mg/L. Sodium sulfite solution (5%) was used for a zeropoint standard. The instrumentation to produce deoxygenated and/or ultrafiltered drilling fluid and quality controls for the drilling procedure have been previously described (Ito et al., 2008; Suzuki et al., 2009), respectively. Near the Kr-2 borehole, a 352m deep borehole (Kr-1) was drilled, and whole-round cores were obtained using a hydraulic rotary method with a wire-line corebarrel. A multi-packer (MP) system (Westbay MP38 system) was installed in the borehole after it was cleaned with the ultrafiltered drilling fluid containing 40 ppm of iodide as a tracer. The drilling fluid was diluted with formation water in the borehole for approximately 1 year after the installation and then sampled from two intervals (at 294–312 m and 330–352 m in depth) (Fig. 1). These intervals were selected to study the repository environment in Japanese near the legal depth of 300 m and due to unsuccessful porewater extraction from an interval at 312–330 m in depth. As groundwater samples from the two intervals were depleted in the iodide trace, it is indicated that the cleaning fluid in the borehole intervals was replaced with groundwater from the surrounding formations during the 1-year procedure. During this period, the porewater pressures of the intervals at 294–312 m and 330–352 m before groundwater sampling were 2.2 and 2.8 MPa, respectively. To collect groundwater from the intervals with minimized exposure to air, 250 mL gas-tight stainless steel bottles were connected to the port of a given interval with an electronic valve. The bottles had been previously rinsed with distilled and deionized water

Fig. 1. Schematic illustration of geological formations and the installed multipacker intervals in deep sedimentary rocks of the study site. Numbers indicate depths in meters below ground level.


(DDW), autoclaved for sterilization and then evacuated with N2 flushing. 2.2. Denitrification activity measurement in core samples Immediately after retrieval from the borehole, core samples were placed in an N2-purged glove bag. The core samples were transported to the laboratory at 4 °C within a day. Upon arrival at the laboratory, the core surfaces were removed aseptically in an anaerobic glove chamber at an atmosphere of 5% H2 and 95% N2. The inner core samples used in this study were taken from depths of 293 (siltstone), 300 (tuffaceous sandstone), 302 (tuffaceous sandstone), 324 (silty sandstone), 340 (silty sandstone), and 351 m (silty sandstone). We adopted the acetylene blockage method for measuring the potential rate of denitrification (Payne, 1991). In the anaerobic chamber described above, 7.5 g of a crushed core sample and 2.9 mL of the ultrafiltered fluid, supplemented with 0.01% (wt/vol) of yeast extract, peptone and casamino acid, 5 mM of Na-lactate, Na-glycerol, Na-succinate and Na-acetate and 10 mM of Na-nitrate, were put into a 67-mL autoclaved glass vial plugged with a butyl rubber stopper. The head space of the plugged vial was replaced with a gas mixture containing 9% acetylene, 5% H2 and 86% N2 and incubated at 25 °C in darkness. The concentration of N2O was measured using a gas chromatograph equipped with a 63Ni electron capture detector (Shimadzu GC2014). All experiments were conducted in duplicate with errors being less than 10%. 2.3. Pore size distribution measurement To determine pore size distribution, uncrushed core samples adjacent to those used for the activity measurement were injected with mercury using a mercury porosimeter (Micromeritics, Autopore). This exercise was conducted to obtain capillary pressure curves. The distribution of pore sizes was calculated from the relationship between mercury injection pressure and pore entry radius (Washburn, 1921; Weiren and Takahashi, 2000):

Ri ¼ 2c cos h=Pc where Ri is the pore radius (micrometers), Pc is the injection pressure (MPa), c is the interfacial tension (480 dynes/cm, air:mercury), and w is the contact angle (141.3°, air:mercury). Median pore throat radii were determined when 50% of the total volume of Hg was injected into the sample. Total porosity is based on the total volume of Hg injected into the sample. 2.4. Geochemical analyses of groundwater The pH, Eh and dissolved O2 (DO) concentrations of unfiltered groundwater samples were measured with the multi parameter probe without being exposed to air. Subsamples were filtered through a 0.2 lm nylon filter with polypropylene housing for subsequent analyses. Aqueous Fe(II), ammonia and hydrogen sulfide concentrations were measured with a spectrophotometer (Hach DE/2010) using the methods of 1,10-phenanthroline, salicylate and methylene blue, respectively (Hach Water Analysis Handbook; Hach, Loveland, CO). Anionic species, including chloride, nitrate, nitrite, sulfate and iodide were analyzed by ion chromatography (Shimadzu PIA-1000). A portion of filtered groundwater was acidified with 6 N HNO3 (the final HNO3 conc. was 0.12 N) and then analyzed with inductively coupled plasma atomic emission spectroscopy (Seiko Instruments SPS1200AR). The Na, K, Ca, Mg, Fe and Mn concentrations were determined with a flame emission spectrometer (Shimadzu AA-680). The dissolved organic carbon (DOC) content was measured by a combustion carbon analyzer (Shimadzu TOC-V). Cationic and anionic species and DOC were


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measured according to Japanese Industrial Standards (JISs) K0133, K0127, K0101 20.2, respectively. The alkalinity of unfiltered groundwater was determined by potentiometric titration with 0.1 N HCl solution using the Glan Plot method (Manheim and Sayles, 1974).

cies and minerals described by Fang et al. (2009) were used for modeling. 3. Results and discussion 3.1. Correlation between microbial activity and porosity

2.5. Microscopic observations of groundwater samples Total cell numbers of the groundwater samples were determined according to a direct count method using SYBR Green I (Takara, Tokyo, Japan). Groundwater samples were fixed with 3.7% formaldehyde at neutral pH. The fixed groundwater samples were filtered with a 0.22 lm pore-size, 25 mm-diameter black polycarbonate filter (Advantec, Tokyo, Japan). For staining, the filter was incubated in 1TAE buffer containing 1SYBR Green I for 5 min. The stained filter was rinsed with 10 mL of DDW and examined under epifluorescence using an Olympus BX51 microscope equipped with a DP70 digital microscope camera (Olympus, Tokyo, Japan). 2.6. DNA extraction, library construction and sequencing for phylogenetic analysis Groundwater was filtrated through a sterilized 0.2 lm polycarbonate filter with sterilized polypropylene housing, and then DNA was extracted from the cells on the filter using a Soil DNA Kit (Mo Bio Lab., Inc., Solana Beach, CA, USA), following the manufacturer’s instructions. Using universal bacteria primer sets of Bact27F and Uni1492R (Lane, 1991), the near-full region of 16S rRNA gene sequences was amplified by polymerase chain reaction (PCR) using LA Taq polymerase (TaKaRa, Tokyo, Japan). Reaction mixtures were prepared in which the concentration of each oligonucleotide primer was 0.1 lM and that of the DNA template was about 0.1 ng/lL. Thermal cycling was performed using a GeneAmp 9700 thermocycler (PE Applied Biosystems, Foster City, CA, USA) with 35 cycles of denaturation at 96 °C for 20 s, annealing at 53 °C for 45 s, and extension at 72 °C for 120 s. PCR-amplified products with expected sizes were purified using a PCR purification kit (Mo Bio Lab., Inc.). The purified PCR product was inserted in the vector pCR2.1 (Invitrogen, Carlsbad, CA, USA) using a DNA Ligation Kit Ver. 2.1 (TaKaRa, Tokyo, Japan) at 16 °C for 6 h, and the inserted vectors were cloned using an Original TA cloning kit. The inserted nucleotide sequences were amplified directly by PCR from randomly selected colonies using the M13 primers treated with exonuclease I and shrimp alkaline phosphatase (Amersham Pharmacia Biotech, Buckinghamshire, UK). The nucleotides were sequenced by a dideoxynucleotide chain-termination method using a BigDye sequencing kit (PE Applied Biosystems) following the manufacturer’s recommendations. The Bac27F primer was used in the partial sequencing analysis. Single-strand sequences more than 500 nucleotides in length were analyzed. The sequence similarity among all of the single-strand sequences was obtained using the BLAST program equipped with a DNASIS Taxon software (Hitachi Software, Tokyo, Japan). Chimeric sequences were also checked by comparing the phylogenetic affiliations of the 50 and 30 halves of each sequence. Accession numbers for sequences used in this study were obtained through DDBJ (AB571102–AB571111).

The pore radius histograms, porosities and median pore throat radii of the intact core samples are shown in Fig. 2 and Table 1. As the pore spaces larger than 0.1 lm in radius are essential for metabolic activity of microorganisms in Cretaceous sedimentary rocks (Fredrickson et al., 1997; Fry et al., 2009; Stroes-Gascoyne et al., 2007), the volumetric ratio of pore spaces with radii larger than 0.1 lm are shown together with the denitrification potential after 1-day incubation (Table 1). The porosities and median pore throat radii of the silty sandstone samples were higher than those of the siltstone sample. The smallest porosity is associated with the tuffaceous sandstone sample, although the median pore throat radius is the highest. The proportions of pore spaces larger than 0.1 lm are small: 3.5% and 19.0% for the siltstone sample and the silty sandstone sample from 324 m, respectively. In these core samples with smaller pore sizes, the potential rates of denitrification were significantly low. Like the Cretaceous sedimentary rocks previously reported for the pore-size constraints (Fredrickson et al., 1997; Fry et al., 2009; Stroes-Gascoyne et al., 2007), microorganisms in Tertiary sedimentary rocks are likely to be constrained in their metabolic activity by a pore throat radius of 0.1 lm.

2.7. Geochemical modeling A mineral saturation index with respect to a uranium mineral (amorphous UO2) and oxidation and reduction potentials (Eh) for redox couples were calculated for groundwater by PHREEQ C (Parkhurst, 1995). Thermodynamic data for uranium aqueous spe-

Fig. 2. Pore-radius histograms as determined by Hg porosimetry in intact cores adjacent to samples used for the denitrification potential measurements. A pore radius of 0.1 lm is indicated by the red line. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


T. Suko et al. / Physics and Chemistry of the Earth 58–60 (2013) 28–33 Table 1 Porosities and median pore throat radii of intact core samples from deep sedimentary rocks. Volumetric ratios of pore spaces larger than 0.1 lm in radius and denitrification potentials measured after 1 day for adjacent core samples are shown.


Depth (m)

Porosity (%)

Median pore throat radius (lm)

Volumetric ration of pore radius > 0.1 lm (%)

Denitrification potential after 1 day (nm/g dry rock/day)

293 309 324 350

32.8 27.9 40.0 37.7

0.016 0.160 0.051 0.082

3.5 46.3 19.0 37.0

<0.1 8.4a 0.3 28.3

Denitrification potential in a tuffaceous sandstone sample from 302 m.

3.2. Comparison of geochemical properties between groundwater and porewater The multi-packer system used in the present study is widely applied to geochemical analyses of deep groundwater (Iwatsuki et al., 2005). However, there are few studies that compare geochemical profiles of pore-water extracted from drilled cores with groundwater collected from the corresponding depth intervals (Sacchi et al., 2001). Geochemical data from the groundwater samples, in addition to those from the corresponding porewater samples and the ultrafiltered fluid for cleaning, are presented in Table 2. The pH values of the groundwater samples were slightly alkaline, while the Eh values were variable (59 mV and 203 mV) for the upper and lower intervals, respectively. The levels of dissolved components were similar between the two intervals, except for ammonia, sodium, chloride and nitrate. Gradual increases in sodium and chloride concentrations with depth also were evident in the groundwater samples. These concentrations have been observed previously for the porewater samples between depths of 302 and 351 m. The similar ranges of sodium and chloride concentrations in the groundwater samples to those of the corresponding porewater samples (and not in the ultrafiltered fluids) indicate the replacement of the ultrafiltered fluid by water accumulation in the sampling intervals. The Eh value of a redox couple of nitrate/nitrite in the lower groundwater was thermodynamically calculated to be 426 mV, while those of Fe(III)/Fe(II) and sulfate/hydrogen sulfides were 56 and 254 mV,

respectively. It is therefore inferred that the positive Eh value of the lower interval may be attributed to the detectable level of nitrate. The main geochemical difference between porewater and groundwater was associated with nitrite and sulfate concentrations, which were below the detection limits in the groundwater samples. A decrease in DOC content was observed with an increase in bicarbonate concentration for the upper interval. Although dissolved organic carbon was not measured for the 351-m deep porewater, due to the limitation of porewater extraction, the HCO 3 concentration of the lower interval also increased. One clear explanation for this geochemical shift is that the oxidation of DOC, coupled with the reduction of nitrite and sulfate, was microbiologically mediated in the borehole intervals.

3.3. Microbial populations colonizing deep groundwater Although we minimized the introduction of contaminant microorganisms into the sampling intervals by cleaning the borehole with the ultrafiltered fluid, it was impossible to thoroughly exclude microbial contamination. In addition, electron donors and acceptors in the ultrafiltered fluid (Table 2) may support the initial growth of microorganisms. Therefore, prolonged incubation (1 year) was needed to enrich microorganisms that depend on nutrients from the in situ chemical fluxes. Table 2 shows that total cell numbers in groundwater from the upper and lower intervals were 3.5  106 and 1.1  106 cells/mL respectively. As 1 mL of the ultrafiltered fluid contained less than 21 cells (Table 2), it is clear that the level of microbial cells significantly increased in the borehole intervals. For the upper and lower intervals, 72 and 84 bacterial 16S rRNA gene sequences were analyzed, respectively. Phylogenetic distributions and affiliations of the bacterial 16S rRNA gene sequences are illustrated in Fig. 3 and listed in Table 3, respectively. Sequences represented by the phylotype KrMPB-1 dominantly occurred in both groundwater samples. A main difference in the microbial community structure between the intervals was the dominant occurrence of KrMPB-2 sequences in the lower interval. Most of the other dominant phylotypes listed in Table 3 occurred in both groundwater samples, except for KrMPB-7 and -10. Many sequences classified within the phylum Firmicutes were only distantly related to known culture species (Table 3). In addi-

Table 2 Geochemical profiles of groundwater and porewater samples from the Kr-1 and Kr-2 boreholes. Ultrafiltered Kr-2 groundwater was used for cleaning the Kr-1 borehole. Depth (m)





90 (Kr-2)

Sample type Rock type pH Eh (mV) DO (mg/L) Fe(II) (lM) H2S (lM) NHþ 4 (lM) Dissolved organic C (mg/L) Na(mM) K (mM) Ca(mM) Mg (mM) Fe(lM) Mn (lM) Cl (mM) HCO 3 (mM) NO 2 (lM)  NO3 (lM)

Groundwater Siltstone and tuffaceous sandstone 9.2 59 >0.1 1.8 0.9 1.1 4.5 4.9 0.1 0.2 <0.1 15.8 0.5 0.9 4.1 <1.5 <1.5 <1.5

Groundwater Silty sandstone 8.5 203 >0.1 2.8 0.6 51.7 5.7 14.4 0.3 0.4 <0.1 7.2 0.3 7.7 6.9 <1.5 16.1 <1.5

Porewater Tuffaceous sandstone 9.0 179 n.m.a n.m. <1.0 n.m. 28.8 4.0 0.1 0.1 <0.1 3.9 0.2 03 3.3 42.8 <1.5 164

Porewater Silty sandstone 9.0 177 n.m. n.m. <1.0 n.m. n.m. 19.7 0.1 0.3 <0.1 10.1 0.4 9.9 5.3 10.5 <1.5 517

Ultrafiltered fluid 8.2 127 n.m. n.m. <1.0 n.m. 3.2 0.3 0.3 0.3 0.14 <0.2 <0.2 0.2 0.5 <1.5 82.8 33.6

3.5  l06

1.1  106



<2.1  l01

SO2 4 (lM) Direct cell count (cells/mL) a

Not measured.


T. Suko et al. / Physics and Chemistry of the Earth 58–60 (2013) 28–33

Fig. 3. The phylum-, class- or species-level composition of microbial communities obtained from groundwater samples from multi-packer intervals of 294–312 and 330–352 m (MP294-312 m and MP330–352 m).

Table 3 The occurrence of 16S rRNA gene sequence types (phylotypes) of bacteria in groundwater samples collected from depth intervals of 294–312 and 330–352 m. Phylotypes with occurrences of more than three times in the entire libraries are listed. Phylotype

KrMPB-1 KrMPB-2 KrMPB-3 KrMPB-4 KrMPB-5 KrMPB-6 KrMPB-7 KrMPB-8 KrMPB-9 KrMPB-10 Total

Depth interval (m) 294312


16 0 10 2 1 4 0 2 2 0 72

12 28 4 5 2 1 3 2 1 3 84

Taxonomy (phylum or class)

Closely related culture species

Identity (%)

Bacteroidetes Betaproteobacteria Firmicutes Betaproteobacteria Firmicutes Firmicutes Deltaproteobacteria Firmicutes Deltaproteobacteria Firmicutes

Y13206 enrichment culture LET-13 Y18616 Acidovorax defluvii EF422412 Dethiobacter alkaliphilus DQ372987 Simplicispira limi DQ117468 Gracilibacter thermotolerans AB274040 Syntrophomonas palmitatica AF126282 Smithella propionica AY949857 Clostridium sp. strain P6 AJ277894 Desulfomicrobium baculatum EF060194 Moorella perchloratireduces

96.798.3 96.799.8 93.695.0 97.298.5 90.390.5 88.489.8 92.992.9 87.693.8 99.399.7 90.290.5

tion, 11% and 5% of the analyzed 16S rRNA gene sequences from the upper and lower intervals, respectively, were not classified within known phyla (Fig. 3). Hence, the physiological and ecological inference is limited to the sequences without known relatives. The phylotype KrMPB-1 related to the enrichment culture LET-13 was obtained from sediment columns where toluene is transformed under Mn(IV)-reducing conditions (Langenhoff et al., 1997). The phylotype KrMPB-2 was closely related to Acidovorax defluvii with a similarity range of 96.7–99.8%. The members of the genus Acidovorax are heterotrophs, which use nitrate as a terminal electron acceptor (Schulze et al., 1999). The third dominant phylotype KrMPB-3 was related to Dethiobacter alkaliphilus of the phylum Firmicutes, which is known as a chemoautotroph and uses H2 as the electron donor, and thiosulfate, elemental sulfur and polysulfide as electron acceptors (Sorokin et al., 2008). 3.4. Geomicrobiological processes in the borehole intervals It is likely that organic matter and oxidants (e.g., O2 and nitrate) introduced in the borehole from the ultrafiltered fluid were initially consumed by the mixtures of indigenous and contaminant microorganisms in the multi-packer intervals. Regardless of whether they are contaminants or not, microorganisms colonizing the borehole intervals became dependent on chemical fluxes from the surrounding formations. Similarly to other deep groundwater habitats, refractory organic matter is suggested to be a primary energy source for microorganisms in the borehole intervals (Krumholz, 2000; Parkes et al., 2000; Pedersen, 2000). The establishment of deep groundwater conditions also is supported by the fact that 16S rRNA gene sequences closely related to KrMPB-1 have been obtained from deep groundwater from geographically distinct Tertiary sedimentary rocks and coal beds (Shimizu et al., 2006, 2007). Additionally, 16S rRNA gene sequences closely related to KrMPB-3

have been detected in ultradeep groundwater from a gold mine in South Africa (Lin et al., 2006). With regard to terminal electron accepting processes in the intervals, integrated geochemical and molecular phylogenetic results suggest that the reduction of Fe(III) is mediated by bacteria related to the enrichment culture LET-13 in both intervals. The presence of Fe(III) was indicated by a difference between Fe and Fe(II). Nitrate reduction is mediated by bacteria related to A. defluvii in the lower interval. As the porewater samples from depths of 302 and 351 m contained sulfate at 164 and 517 lM, respectively, the removal of sulfate in the borehole intervals may have resulted from the activity of sulfate-reducing bacteria related to Desulfomicrobium baculatum. 3.5. Microbiological impacts on radionuclide migration in the repository environment To evaluate how microbial metabolic activities affect radionuclide migration, the solubility of a solubility-limiting solid phase of uranium (amorphous UO2) in groundwater from the two intervals was calculated by geochemical modeling. Although amorphous UO2 was undersaturated at a uranium concentration of 108 M and a pCO2 of 102, the saturation index of the upper interval with Eh = 59 mV was 9.2, which was substantially higher than that of the lower interval (19.8) with Eh = 203. These results clearly show that microbially mediated redox reactions potentially control radionuclide migration in the deep subsurface investigated in this study. 4. Conclusion In the Tertiary sedimentary rocks, microbial metabolic activities were also constrained by the pore availability. Groundwater sam-

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ples collected from the multi-packer intervals were mainly colonized by microorganisms that could mediate the reduction of nitrate, Mn(IV), Fe(III) or sulfur compounds coupled to the oxidation of refractory organic matter or inorganic compounds. Our integrated results indicate that there appears to be a flux of nitrate that sustains the activity of nitrate-reducing microorganisms and relatively oxidizing conditions in the deep Tertiary sedimentary rocks. Acknowledgements This study was supported by grants from the Nuclear and Industrial Safety Agency (NISA) and Japan Nuclear Energy Safety Organization (JNES). We are indebted to Nobuyuki Hirayama from Oyo-Chisitsu Corporation for his management of water sampling. We are grateful to Junichiro Ishibashi for geochemical analyses. We also thank Naoto Takeno, Yoji Seki, Koichi Okuzawa and Daisaku Sato for their assistance. References Fang, Y., Yabusaki, S.B., Morrison, S.J., Amonette, J.P., Long, P.E., 2009. Multicomponent reactive transport modeling of uranium bioremediation field experiments. Geochim. Cosmochim. Acta 73, 6029–6051. Fredrickson, J.K., McKinley, J.P., Bjornstad, B.N., Long, P.E., Ringelberg, D.B., White, D.C., Krumholz, L.R., Suflita, J.M., Colwell, F.S., Lehman, R.M., Phelps, T.J., Onstott, T.C., 1997. Pore-size constraints on the activity and survival of subsurface bacteria in a late Cretaceous shale–sandstone sequence, northwestern New Mexico. Geomicrobiol. J. 14, 183–202. Fry, J.C., Horsfield, B., Sykes, R., Cragg, B.A., Heywood, C., Kim, G.T., Mangelsdorf, K., Mildenhall, D.C., Rinna, J., Vieth, A., 2009. Prokaryotic populations and activities in an interbedded coal deposit, including a previously deeply buried section (1.6–2.3 km) above 150 Ma basement rock. Geomicrobiol. J. 26, 163–178. Gillow, J.B., Dunn, M., Francis, A.J., Lucero, D.A., Papenguth, H.W., 2000. The potential of subterranean microbes in facilitating actinide migration at the Grimsel Test Site and Waste Isolation Pilot Plant. Radiochim. Acta 88, 769–774. Hallbeck, L., Pedersen, K., 2008. Characterization of microbial processes in deep aquifers of the Fennoscandian Shield. Appl. Geochem. 23, 1796–1819. Ito, K.S., Suzuki, Y., Suko, T., Takno, N., 2008. Conceptual groundwater flow modeling from non-disturbing drilling in sedimentary rocks. In: 12th International High Level Nuclear Waste Management Conference, Las Vegas, pp. 165–170. Iwatsuki, T., Furue, R., Mie, H., Ioka, S., Mizuno, T., 2005. Hydrochemical baseline condition of groundwater at the Mizunami underground research laboratory (MIU). Appl. Geochem. 20, 2283–2302. Kieft, T.L., Kovacik, W.P., Ringelberg, D.B., White, D.C., Haldeman, D.L., Amy, P.S., Hersman, L.E., 1997. Factors limiting microbial growth and activity at a proposed high-level nuclear repository, Yucca mountain, Nevada. Appl. Environ. Microbiol. 63, 3128–3133. Krumholz, L.R., 2000. Microbial communities in the deep subsurface. Hydrogeol. J. 8, 4–10. Lane, D.J., 1991. 16S/23S rRNA sequencing. In: Stackebrandt, E., Goodfellow, M. (Eds.), Nucleic Acid Techniques in Bacterial Systematics. John Willy & Sons, New York, pp. 115–175.


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