Microbial Ecology

, Volume 60, Issue 1, pp 214–225 | Cite as

Geomicrobiological Properties of Ultra-Deep Granitic Groundwater from the Mizunami Underground Research Laboratory (MIU), Central Japan

  • Akari Fukuda
  • Hiroki Hagiwara
  • Toyoho Ishimura
  • Mariko Kouduka
  • Seiichiro Ioka
  • Yuki Amano
  • Urumu Tsunogai
  • Yohey Suzuki
  • Takashi Mizuno
Environmental Microbiology


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), CH4 and H2, 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 × 104 and 5.2 × 104 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 CH4 + H2) and with/without electron acceptors (O2 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 O2, NO 3 , and organic acids.


Groundwater Sample Denitrification Potential Thauera Abundant Phylotype Japan Industrial Standard 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We would like to thank Katsuhiro Hama and Kazumasa Ito for their management of this investigation. Comments from three anonymous reviewers significantly improved the quality of our manuscript. A part of this study was supported by grants from the Nuclear and Industrial Safety Agency (NISA) for AIST.


  1. 1.
    Abraham WR, Strompl C, Meyer H, Lindholst S, Moore ERB, Christ R, Vancanneyt M, Tindall BJ, Bennasar A, Smit J, Tesar M (1999) Phylogeny and polyphasic taxonomy of Caulobacter species. Proposal of Maricaulis gen. nov with Maricaulis maris (Poindexter) comb. nov as the type species, and emended description of the genera Brevundimonas and Caulobacter. Int J Syst Bacteriol 49:1053–1073CrossRefPubMedGoogle Scholar
  2. 2.
    Baker BJ, Moser DP, MacGregor BJ, Fishbain S, Wagner M, Fry NK, Jackson B, Speolstra N, Loos S, Takai K, Lollar BS, Fredrickson J, Balkwill D, Onstott TC, Wimpee CF, Stahl DA (2003) Related assemblages of sulphate-reducing bacteria associated with ultradeep gold mines of South Africa and deep basalt aquifers of Washington State. Environ Microbiol 5:267–277CrossRefPubMedGoogle Scholar
  3. 3.
    Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Sayers EW (2009) GenBank. Nucleic Acids Res 37:D26–D31CrossRefPubMedGoogle Scholar
  4. 4.
    Chapelle FH, O’Neill K, Bradley PM, Methe BA, Ciufo SA, Knobel LL, Lovley DR (2002) A hydrogen-based subsurface microbial community dominated by methanogens. Nature 415:312–315CrossRefPubMedGoogle Scholar
  5. 5.
    Chivian D, Brodie EL, Alm EJ, Culley DE, Dehal PS, DeSantis TZ, Gihring TM, Lapidus A, Lin LH, Lowry SR, Moser DP, Richardson PM, Southam G, Wanger G, Pratt LM, Andersen GL, Hazen TC, Brockman FJ, Arkin AP, Onstott TC (2008) Environmental genomics reveals a single-species ecosystem deep within earth. Science 322:275–278CrossRefPubMedGoogle Scholar
  6. 6.
    Chung BS, Ryu SH, Park M, Jeon Y, Chung YR, Jeon CO (2007) Hydrogenophaga caeni sp nov., isolated from activated sludge. Int J Syst Evol Microbiol 57:1126–1130CrossRefPubMedGoogle Scholar
  7. 7.
    Delong EF (1992) Archaea in coastal marine environments. Proc Natl Acad Sci USA 89:5685–5689CrossRefPubMedGoogle Scholar
  8. 8.
    Demolldecker H, Macy JM (1993) The periplasmic nitrite reductase of Thauera selenatis may catalyze the reduction of selenite to elemental selenium. Arch Microbiol 160:241–247Google Scholar
  9. 9.
    Fredrickson JK, Balkwill DL (2006) Geomicrobial processes and biodiversity in the deep terrestrial subsurface. Geomicrobiol J 23:345–356CrossRefGoogle Scholar
  10. 10.
    Fritz I, Strompl C, Nikitin DI, Lysenko AM, Abraham WR (2005) Brevundimonas mediterranea sp nov., a non-stalked species from the Mediterranean Sea. Int J Syst Evol Microbiol 55:479–486CrossRefPubMedGoogle Scholar
  11. 11.
    Haveman SA, Pedersen K, Ruotsalainen P (1999) Distribution and metabolic diversity of microorganisms in deep igneous rock aquifers of Finland. Geomicrobiol J 16:277–294CrossRefGoogle Scholar
  12. 12.
    Heider J, Spormann AM, Beller HR, Widdel F (1998) Anaerobic bacterial metabolism of hydrocarbons. Fems Microbiol Rev 22:459–473CrossRefGoogle Scholar
  13. 13.
    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 20:2283–2302CrossRefGoogle Scholar
  14. 14.
    Kämpfer P, Schulze R, Jackel U, Malik KA, Amann R, Spring S (2005) Hydrogenophaga defluvii sp nov and Hydrogenophaga atypica sp nov., isolated from activated sludge. Int J Syst Evol Microbiol 55:341–344CrossRefPubMedGoogle Scholar
  15. 15.
    Konno U, Tsunogai U, Nakagawa F, Nakaseama M, Ishibashi JI, Nunoura T, Nakamura KI (2006) Liquid CO2 venting on the seafloor: Yonaguni Knoll IV hydrothermal system, Okinawa Trough. Geophys Res Lett 33:1–5CrossRefGoogle Scholar
  16. 16.
    Kotelnikova S, Pedersen K (1998) Distribution and activity of methanogens and homoacetogens in deep granitic aquifers at Äspö Hard Rock Laboratory, Sweden. FEMS Microbiol Ecol 26:121–134Google Scholar
  17. 17.
    Lin LH, Hall J, Lippmann-Pipke J, Ward JA, Lollar BS, DeFlaun M, Rothmel R, Moser D, Gihring TM, Mislowack B, Onstott TC (2005) Radiolytic H2 in continental crust: nuclear power for deep subsurface microbial communities. Geochemistry Geophysics Geosystems 6Google Scholar
  18. 18.
    Lin LH, Wang PL, Rumble D, Lippmann-Pipke J, Boice E, Pratt LM, Lollar BS, Brodie EL, Hazen TC, Andersen GL, DeSantis TZ, Moser DP, Kershaw D, Onstott TC (2006) Long-term sustainability of a high-energy, low-diversity crustal biome. Science 314:479–482CrossRefPubMedGoogle Scholar
  19. 19.
    Lovley DR, Chapelle FH (1995) Deep subsurface microbial processes. Rev Geophys 33:365–381CrossRefGoogle Scholar
  20. 20.
    Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar BA, Lai T, Steppi S, Jobb G, Forster W, Brettske I, Gerber S, Ginhart AW, Gross O, Grumann S, Hermann S, Jost R, Konig A, Liss T, Lussmann R, May M, Nonhoff B, Reichel B, Strehlow R, Stamatakis A, Stuckmann N, Vilbig A, Lenke M, Ludwig T, Bode A, Schleifer KH (2004) ARB: a software environment for sequence data. Nucleic Acids Res 32:1363–1371CrossRefPubMedGoogle Scholar
  21. 21.
    Murakami Y, Fujita Y, Naganuma T, Iwatsuki T (2002) Abundance and viability of the groundwater microbial communities from a borehole in the Tono uranium deposit area, central Japan. Microbes and Environ 17:63–74CrossRefGoogle Scholar
  22. 22.
    Orphan VJ, Hinrichs KU, Ussler W, Paull CK, Taylor LT, Sylva SP, Hayes JM, Delong EF (2001) Comparative analysis of methane-oxidizing archaea and sulfate-reducing bacteria in anoxic marine sediments. Appl Environ Microbiol 67:1922–1934CrossRefPubMedGoogle Scholar
  23. 23.
    Payne WJ (1991) A review of methods for field measurement of denitrification. Forest Ecol Manage 44:5–14CrossRefGoogle Scholar
  24. 24.
    Pedersen K (1996) Investigations of subterranean bacteria in deep crystalline bedrock and their importance for the disposal of nuclear waste. Can J Microbiol 42:382–391CrossRefGoogle Scholar
  25. 25.
    Pedersen K (1997) Microbial life in deep granitic rock. Fems Microbiol Rev 20:399–414CrossRefGoogle Scholar
  26. 26.
    Pedersen K, Arlinger J, Ekendahl S, Hallbeck L (1996) 16S rRNA gene diversity of attached and unattached bacteria in boreholes along the access tunnel to the Äspö hard rock laboratory, Sweden. FEMS Microbiol Ecol 19:249–262Google Scholar
  27. 27.
    Pedersen K, Ekendahl S (1990) Distribution and activity of bacteria in deep granitic groundwaters of southeastern Sweden. Microb Ecol 20:37–52CrossRefGoogle Scholar
  28. 28.
    Pedersen K, Ekendahl S (1992) Assimilation of CO2 and Introduced organic-compounds by bacterial communities in groundwater from southeastern Sweden deep crystalline bedrock. Microb Ecol 23:1–14CrossRefGoogle Scholar
  29. 29.
    Pedersen K, Hallbeck L, Arlinger J, Erlandson AC, Jahromi N (1997) Investigation of the potential for microbial contamination of deep granitic aquifers during drilling using 16S rRNA gene sequencing and culturing methods. J Microbiol Methods 30:179–192CrossRefGoogle Scholar
  30. 30.
    Reinhold-Hurek B, Hurek T (2000) Reassessment of the taxonomic structure of the diazotrophic genus Azoarcus sensu lato and description of three new genera and new species, Azovibrio restrictus gen. nov., sp nov., Azospira oryzae gen. nov., sp nov and Azonexus fungiphilus gen. nov., sp nov. Int J Syst Evol Microbiol 50:649–659PubMedGoogle Scholar
  31. 31.
    Sahl JW, Schmidt RH, Swanner ED, Mandernack KW, Templeton AS, Kieft TL, Smith RL, Sanford WE, Callaghan RL, Mitton JB, Spear JR (2008) Subsurface microbial diversity in deep-granitic-fracture water in Colorado. Appl Environ Microbiol 74:143–152CrossRefPubMedGoogle Scholar
  32. 32.
    Sasao E, Ota K, Iwatsuki T, Niizato T, Arthur RC, Stenhouse MJ, Zhou W, Metcalfe R, Takase H, Mackenzie AB (2006) An overview of a natural analogue study of the Tono Uranium Deposit, central Japan. Geochem-Explor Environ Anal 6:5–12CrossRefGoogle Scholar
  33. 33.
    Sayers EW, Barrett T, Benson DA, Bryant SH, Canese K, Chetvernin V, Church DM, DiCuccio M, Edgar R, Federhen S, Feolo M, Geer LY, Helmberg W, Kapustin Y, Landsman D, Lipman DJ, Madden TL, Maglott DR, Miller V, Mizrachi I, Ostell J, Pruitt KD, Schuler GD, Sequeira E, Sherry ST, Shumway M, Sirotkin K, Souvorov A, Starchenko G, Tatusova TA, Wagner L, Yaschenko E, Ye J (2009) Database resources of the National Center for Biotechnology Information. Nucleic Acids Res 37:D5–15CrossRefPubMedGoogle Scholar
  34. 34.
    Scholten E, Lukow T, Auling G, Kroppenstedt RM, Rainey FA, Diekmann H (1999) Thauera mechernichensis sp nov., an aerobic denitrifier from a leachate treatment plant. Int J Syst Bacteriol 49:1045–1051CrossRefPubMedGoogle Scholar
  35. 35.
    Stevens TO, McKinley JP (1995) Lithoautotrophic microbial ecosystems in deep basalt aquifers. Science 270:450–454CrossRefGoogle Scholar
  36. 36.
    Tsunogai U, Nakagawa F, Hachisu Y, Yoshida N (2000) Stable carbon and oxygen isotopic analysis of carbon monoxide in natural waters. Rapid Commun Mass Spectrom 14:1507–1512CrossRefPubMedGoogle Scholar
  37. 37.
    Tsunogai U, Yoshida N, Ishibashi J, Gamo T (2000) Carbon isotopic distribution of methane in deep-sea hydrothermal plume, Myojin Knoll Caldera, Izu-Bonin arc: implications for microbial methane oxidation in the oceans and applications to heat flux estimation. Geochimica Et Cosmochimica Acta 64:2439–2452CrossRefGoogle Scholar
  38. 38.
    Valentine DL (2002) Biogeochemistry and microbial ecology of methane oxidation in anoxic environments: a review. Antonie Van Leeuwenhoek 81:271–282CrossRefPubMedGoogle Scholar
  39. 39.
    Wagner M, Roger AJ, Flax JL, Brusseau GA, Stahl DA (1998) Phylogeny of dissimilatory sulfite reductases supports an early origin of sulfate respiration. J Bacteriol 180:2975–2982PubMedGoogle Scholar
  40. 40.
    Whiticar MJ (2002) Diagenetic relationships of methanogenesis, nutrients, acoustic turbidity, pockmarks and freshwater seepages in Eckernförde Bay. Mar Geol 182:29–53CrossRefGoogle Scholar
  41. 41.
    Willems A, Busse J, Goor M, Pot B, Falsen E, Jantzen E, Hoste B, Gillis M, Kersters K, Auling G, Deley J (1989) Hydrogenophaga, a new genus of hydrogen-oxidizing bacteria that includes Hydrogenophaga flava comb. nov. (formerly Pseudomonas flava), Hydrogenophaga palleronii (formerly Pseudomonas palleronii), Hydrogenophaga pseudoflava (formerly Pseudomonas pseudoflava and Pseudomonas carboxydoflava), and Hydrogenophaga taeniospiralis (formerly Pseudomonas taeniospiralis). Int J Syst Bacteriol 39:319–333CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Akari Fukuda
    • 1
    • 5
  • Hiroki Hagiwara
    • 2
  • Toyoho Ishimura
    • 3
  • Mariko Kouduka
    • 1
  • Seiichiro Ioka
    • 4
  • Yuki Amano
    • 2
  • Urumu Tsunogai
    • 3
  • Yohey Suzuki
    • 1
  • Takashi Mizuno
    • 2
  1. 1.Institute for Geo-Resources and EnvironmentNational Institute of Advanced Industrial Science and Technology (AIST)TsukubaJapan
  2. 2.Japan Atomic Energy AgencyMizunamiJapan
  3. 3.Earth and Planetary System Science, Faculty of ScienceHokkaido UniversitySapporoJapan
  4. 4.Horonobe Research Institute for the Subsurface EnvironmentNorthern Advancement Center for Science & TechnologyTeshio-gunJapan
  5. 5.Mizunami Underground Research Laboratory, Geological Isolation Research and Development DirectorateJapan Atomic Energy AgencyMizunamiJapan

Personalised recommendations