Impact of the electron donor on in situ microbial nitrate reduction in Opalinus Clay: results from the Mont Terri rock laboratory (Switzerland)

  • Nele Bleyen
  • Steven Smets
  • Joe Small
  • Hugo Moors
  • Natalie Leys
  • Achim Albrecht
  • Pierre De Cannière
  • Bernhard Schwyn
  • Charles Wittebroodt
  • Elie Valcke
Part of the Swiss Journal of Geosciences Supplement book series (SWISSGEO, volume 5)


At the MontTerri rock laboratory (Switzerland), an in situ experiment is being carried out to examine the fate of nitrate leaching from nitrate-containing bituminized radioactive waste, in a clay host rock for geological disposal. Such a release of nitrate may cause a geochemical perturbation of the clay, possibly affecting some of the favorable characteristics of the host rock. In this in situ experiment, combined transport and reactivity of nitrate is studied inside anoxic and water-saturated chambers in a borehole in the Opalinus Clay.Continuous circulation of the solution from the borehole to the surface equipment allows a regular sampling and online monitoring of its chemical composition. In this paper, in situ microbial nitrate reduction in the Opalinus Clay is discussed, in the presence or absence of additional electron donors relevant for the disposal concept and likely to be released from nitrate-containing bituminized radioactive waste: acetate (simulating bitumen degradation products) and H2 (originating from radiolysis and corrosion in the repository). The results of these tests indicate that—in case microorganisms would be active in the repository or the surrounding clay—microbial nitrate reduction can occur using electron donors naturally present in the clay (e.g. pyrite, dissolved organic matter). Nevertheless, non-reactive transport of nitrate in the clay is expected to be the main process. In contrast, when easily oxidizable electron donors would be available (e.g. acetate and H2), the microbial activity will be strongly stimulated. Both in the presence of H2 and acetate, nitrite and nitrogenous gases are predominantly produced, although some ammonium can also be formed when H2 is present. The reduction of nitrate in the clay could have an impact on the redox conditions in the pore-water and might also lead to a gas-related perturbation of the host rock, depending on the electron donor used during denitrification.


Nitrite Redox Clay Acetate Hydrogen Microorganisms Nuclear waste disposal 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.



This work is undertaken in close co-operation with Swisstopo, the operator of the rock laboratory and the project management team at Mont Terri, namely Christophe Nussbaum and Thierry Theurillat. Financial support was provided by the Mont Terri Consortium. Joe Small acknowledges funding by the U.K. Natural Environment Research Council (NERC) BIGRAD consortium through Grant No. NE/H007768/1 and the National Nuclear Laboratory. Gesine Lorenz (Hydroisotop GmbH) and Elke Jacops (SCK•CEN) are acknowledged for performing the gas analyses. In addition, the technical assistance of Wim Verwimp and Patrick Boven (SCK•CEN) is greatly appreciated. Finally, we thank Dr. Marc Parmentier (French Geological Survey BRGM, Orléans, France) and Dr. Alexandre Bagnoud (Stream Biofilm and Ecosystem Research Laboratory at EPFL, Lausanne, Switzerland) for their constructive comments and suggestions.


  1. Almeida, J., Reis, M., & Carrondo, M. (1995). Competition between nitrate and nitrite reduction in denitrification by Pseudomonas fluorescens. Biotechnology and Bioengineering, 46, 476–484.Google Scholar
  2. Alt-Epping, P., Gimmi, T., & Waber, N. (2008). Porewater chemistry (PC) experiment: Reactive transport simulations. Mont Terri Technical Report, TR 07-03, Federal Office of Topography (swisstopo), Wabern, Switzerland.
  3. ANDRA (2005). Dossier 2005. ANDRA research on the geological disposal of high-level long-lived radioactive waste. Results and perspectives. ANDRA report, ANDRA, Châtenay-Malabry, France.Google Scholar
  4. Bagnoud, A., de Bruijn, I., Andersson, A. F., Diomidis, N., Leupin, O. X., Schwyn, B., et al. (2016). A minimalistic microbial food web in an excavated deep subsurface clay rock. FEMS Microbiology Ecology, 92, fiv138.Google Scholar
  5. Bertani, G. (1951). STUDIES ON LYSOGENESIS I: The mode of phage liberation by lysogenic Escherichia coli. Journal of Bacteriology, 62, 293–300.Google Scholar
  6. Bleyen, N., Smets, S., Albrecht, A., De Cannière, P., Schwyn, B., Witebroodt, C., & Valcke, E. (in preparation) Use of non-destructive on-line spectrophotometric and pH monitoring to assess in situ microbial nitrate and nitrite reduction. International Journal of Environmental Analytical Chemistry.Google Scholar
  7. Bleyen, N., Smets, S., & Valcke, E. (2011). BN Experiment: Status and raw data report of phases 15 and 16. Mont Terri Technical Note, TN 2009-49, Federal Office of Topography (swisstopo), Wabern, Switzerland.
  8. Bleyen, N., Vasile, M., Bruggeman, C., & Valcke, E. (2015). Abiotic and biotic nitrate and nitrite reduction by pyrite. In: Proceedings of the Clays in Natural and Engineered Barriers for Radioactive Waste Confinement Conference, P-06-16.Google Scholar
  9. Bleyen, N., Vasile, M., Marien, A., Bruggeman, C., & Valcke, E. (2016). Assessing the oxidising effect of NaNO3 and NaNO2 from disposed EUROBITUM bituminised waste on the dissolved organic matter in Boom Clay. Applied Geochemistry, 68, 29–38.Google Scholar
  10. Bohn, H. L. (1971). Redox potentials. Soil Science, 112, 39–45.Google Scholar
  11. Bossart, P., Bernier, F., Birkholzer, J., Bruggeman, C., Connolly, P., Dewonck, S., Fukaya, M., Herfort, M., Jensen, M., Matray, J-M., Mayor, J. C., Moeri, A., Oyama, T., Schuster, K., Shigeta, N., Vietor, T., & Wieczorek, K. (2017). Mont Terri rock laboratory, 20 years: introduction, geology and overview of papers included in the Special Issue. Swiss Journal of Geosciences, 110. (this issue).
  12. Chang, C. C., Tseng, S. K., & Huang, H. K. (1999). Hydrogenotrophic denitrification with immobilized Alcaligenes eutrophus for drinking water treatment. Bioresource technology, 69, 53–58.Google Scholar
  13. Courdouan, A., Christl, I., Meylan, S., Wersin, P., & Kretzschmar, R. (2007). Characterization of dissolved organic matter in anoxic rock extracts and in situ pore water of the Opalinus Clay. Applied Geochemistry, 22, 2926–2939.Google Scholar
  14. De Canniere, P., Schwarzbauer, J., & Van Geet, M. (2008). Leaching experiments and analyses of organic matter released by the materials used for the construction of Porewater Chemistry experiment at Mont Terri Rock Laboratory. Mont Terri Technical Note, TN 2005-12. Federal Office of Topography (swisstopo), Wabern, Switzerland.
  15. De Cannière, P., Maes, A., Williams, S., Bruggeman, C., Beauwens, T., Maes, N., Cowper, M. (2010). Behaviour of selenium in Boom Clay. External Report, SCK•CEN-ER-120, 328 pp. SCK•CEN, Boeretang 200, 2400 Mol, Belgium.Google Scholar
  16. De Cannière, P., Schwarzbauer, J., Höhener, P., Lorenz, G., Salah, S., Leupin, O., et al. (2011). Biogeochemical processes in a clay formation in situ experiment: part C—organic contamination and leaching data. Applied Geochemistry, 26, 967–979.Google Scholar
  17. De Craen, M., Wang, L., Van Geet, M., & Moors, H. (2004). Geochemistry of Boom Clay pore water at the Mol site. Status 2004. Scientific report, SCK•CEN-BLG-990, 179 pp. SCK•CEN, Mol, Belgium.Google Scholar
  18. DeSantis, T. Z., Hugenholtz, P., Larsen, N., Rojas, M., Brodie, E. L., Keller, K., et al. (2006). Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Applied and Environmental Microbiology, 72, 5069–5072.Google Scholar
  19. Devlin, J., Eedy, R., & Butler, B. (2000). The effects of electron donor and granular iron on nitrate transformation rates in sediments from a municipal water supply aquifer. Journal of Contaminant Hydrology, 46, 81–97.Google Scholar
  20. Eichinger, F., Lorenz, G., & Eichinger, L. (2011). WS-H Experiment: Water sampling from borehole BWS-H2—Report on physicalchemical and isotopic analyses. Mont Terri Technical Note, TN 2010-49. Federal Office of Topography (swisstopo), Wabern, Switzerland.
  21. Garthright, W., & Blodgett, R. (2003). FDA’s preferred MPN methods for standard, large or unusual tests, with a spreadsheet. Food Microbiology, 20, 439–445.Google Scholar
  22. Harrington, J., Milodowski, A., Graham, C., Rushton, J., & Cuss, R. (2012). Evidence for gas-induced pathways in clay using a nanoparticle injection technique. Mineralogical Magazine, 76, 3327–3336.Google Scholar
  23. Hauck, S., Benz, M., Brune, A., & Schink, B. (2001). Ferrous iron oxidation by denitrifying bacteria in profundal sediments of a deep lake (Lake Constance). FEMS Microbiology Ecology, 37, 127–134.Google Scholar
  24. Haugen, K., Semmens, M., & Novak, P. (2002). A novel in situ technology for the treatment of nitrate contaminated groundwater. Water Research, 36, 3497–3506.Google Scholar
  25. Heylen, K., Vanparys, B., Wittebolle, L., Verstraete, W., Boon, N., & De Vos, P. (2006). Cultivation of denitrifying bacteria: optimization of isolation conditions and diversity study. Applied and Environmental Microbiology, 72, 2637–2643.Google Scholar
  26. Jørgensen, C. J., Jacobsen, O. S., Elberling, B., & Aamand, J. (2009). Microbial oxidation of pyrite coupled to nitrate reduction in anoxic groundwater sediment. Environmental Science and Technology, 43, 4851–4857.Google Scholar
  27. Karanasios, K., Michailides, M., Vasiliadou, I., Pavlou, S., & Vayenas, D. (2011). Potable water hydrogenotrophic denitrification in packed-bed bioreactors coupled with a solar-electrolysis hydrogen production system. Desalination and Water Treatment, 33, 86–96.Google Scholar
  28. Klindworth, A., Pruesse, E., Schweer, T., Peplies, J., Quast, C., Horn, M., et al. (2012). Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Research, 2012, 1–11.Google Scholar
  29. Libert, M., Bildstein, O., Esnault, L., Jullien, M., & Sellier, R. (2011). Molecular hydrogen: An abundant energy source for bacterial activity in nuclear waste repositories. Physics and Chemistry of the Earth, Parts A/B/C, 36, 1616–1623.Google Scholar
  30. Lide, D. R., & Frederikse, H. P. R. (1995). CRC handbook of chemistry and physics. Boca Raton: CRC Press.Google Scholar
  31. Madigan, M. T., Martinko, J. M., & Parker, J. (2000). Brock biology of microorganisms. Upper Saddle River: Prentice-Hall.Google Scholar
  32. Mallants, D., Jacques, D., & Perko, J. (2007). Modelling multi-phase flow phenomena in concrete barriers used for geological disposal of radioactive waste. Proceedings of the 11th International Conference on Environmental Remediation and Radioactive Waste Management (pp. 741–749). American Society of Mechanical Engineers.Google Scholar
  33. Mariën, A., Bleyen, N., Aerts, S., & Valcke, E. (2011). The study of abiotic reduction of nitrate and nitrite in Boom Clay. Physics and Chemistry of the Earth, 36, 1639–1647.Google Scholar
  34. Moors, H., Geissler, A., Boven, P., Selenska-Pobell, S., & Leys, N. (2012). BN experiment: intermediate results of the microbiological analyses. Mont Terri Technical Note, TN 2011-39. Federal Office of Topography (swisstopo), Wabern, Switzerland.
  35. Moors, H., Bleyen, N., Ahmed, M., Boven, P., Leys, N., Valcke, E., Cherkouk, A., Stroes-Gascoyne, S., Nussbaum, C., Schwyn, B., Albrecht, A., Wittebroodt, C., Small, J., & De Cannie`re, P. (2015a). The Bitumen-Nitrate-Clay Interaction Experiment at the Mont Terri Rock Laboratory: Response of microbial communities to additions of nitrate and acetate. Proceedings to the Clays in Natural and Engineered Barriers for Radioactive Waste Confinement Conference, O-06A-03.Google Scholar
  36. Moors, H., Mysara, M., Bleyen, N., Cherkouk, A., Boven, P., & Leys, N. (2015b). BN Experiment: Results of the microbiological analyses obtained during phase 19 & 20. Mont Terri Technical Note, TN 2015-72. Federal Office of Topography (swisstopo), Wabern, Switzerland.
  37. Mysara, M., Leys, N., Raes, J., & Monsieurs, P. (2015a). NoDe: a fast error-correction algorithm for pyrosequencing amplicon reads. BMC Bioinformatics, 16, 1–10.Google Scholar
  38. Mysara, M., Saeys, Y., Leys, N., Raes, J., & Monsieurs, P. (2015b). CATCh, an ensemble classifier for chimera detection in 16S rRNA sequencing studies. Applied and Environmental Microbiology, 81, 1573–1584.Google Scholar
  39. Mysara, M., Leys, N., Raes, J., & Monsieurs, P. (2016). IPED: a highly efficient denoising tool for Illumina MiSeq Paired-end 16S rRNA gene amplicon sequencing data. BMC Bioinformatics, 17, 1–11.Google Scholar
  40. Neidhart, F. (1996). Escherichia coli and Salmonella typhimurium: Cellular and molecular biology. Washington: ASM Press.Google Scholar
  41. Nussbaum, C., Kloppenburg, A., Caër, T., & Bossart, P. (2017). Tectonic evolution around the Mont Terri rock laboratory, northwestern Swiss Jura: constraints from kinematic forward modelling. Swiss Journal of Geosciences, 110. (this issue).
  42. Oh, J., & Silverstein, J. (1999). Acetate limitation and nitrite accumulation during denitrification. Journal of Environmental Engineering, 125, 234–242.Google Scholar
  43. Oremland, R. S., Blum, J. S., Bindi, A. B., Dowdle, P. R., Herbel, M., & Stolz, J. F. (1999). Simultaneous reduction of nitrate and selenate by cell suspensions of selenium-respiring bacteria. Applied and Environmental Microbiology, 65, 4385–4392.Google Scholar
  44. Parmentier, M., Ollivier, P., Joulian, C., Albrecht, A., Hadi, J., Greneche, J.-M., et al. (2014). Enhanced heterotrophic denitrification in clay media: the role of mineral electron donors. Chemical geology, 390, 87–99.Google Scholar
  45. Pearson, F. J. (1999). WS-A experiment: artificial waters for use in laboratory and field experiments with Opalinus Clay. Status June 1998. Mont Terri Technical Note, TN 1999-31. Federal Office of Topography (swisstopo), Wabern, Switzerland.
  46. Pearson, F. J., Arcos, D., Bath, A., Boisson, J. Y., Fernández, A. M., Gabler, H. E., Gaucher, E., Gautschi, A., Griffault, L., Hernán, P., & Waber, H. N. (2003). Mont Terri Project - Geochemistry of water in the Opalinus Clay formation at the Mont Terri Rock Laboratory. Federal Office for Water and Geology Geology Series, No. 5. Federal Office of Topography (swisstopo), Wabern, Switzerland.
  47. Percheron, G., Michaud, S., Bernet, N., & Moletta, R. (1998). Nitrate and nitrite reduction of a sulphide-rich environment. Journal of Chemical Technology and Biotechnology, 72, 213–220.Google Scholar
  48. Phister, A., Jaeggi, D., & Nussbaum, C. (2010). Drilling campaign of phase 15: drilling data, photo documentation and drill core documentation. Mont Terri Technical Note, TN 2010-38. Federal Office of Topography (swisstopo), Wabern, Switzerland.
  49. Reasoner, D., & Geldreich, E. (1985). A new medium for the enumeration and subculture of bacteria from potable water. Applied and Environmental Microbiology, 49, 1–7.Google Scholar
  50. Schloss, P. D., & Westcott, S. L. (2011). Assessing and improving methods used in operational taxonomic unit-based approaches for 16S rRNA gene sequence analysis. Applied and Environmental Microbiology, 77, 3219–3226.Google Scholar
  51. Small, J. (2015). BN Experiment: GRM biogeochemical modelling during Phase 18 and Phase 19 of the Bitumen-Nitrate-Clay interaction experiment. Mont Terri Technical Note, TN 2013-44. Federal Office of Topography (swisstopo), Wabern, Switzerland.
  52. Small, J., Nykyri, M., Helin, M., Hovi, U., Sarlin, T., & Ita¨vaara, M. (2008). Experimental and modelling investigations of the biogeochemistry of gas production from low and intermediate level radioactive waste. Applied Geochemistry, 23, 1383–1418.Google Scholar
  53. Smith, P., Cornélis, B., Capouet, M., Depaus, C., & Van Geet, M. (2009). The long-term safety assessment methodology for the geological disposal of radioactive waste. SFC1 level 4 report, NIROND-TR 2009-14 E, ONDRAF/NIRAS, Brussels, Belgium.Google Scholar
  54. Thury, M., & Bossart, P. (1999). The Mont Terri Rock Laboratory, a new international research project in a Mesozoic shale formation in Switzerland. Engineering Geology, 52, 347–359.Google Scholar
  55. Tournassat, C., Alt-Epping, P., Gaucher, E. C., Gimmi, T., Leupin, O. X., & Wersin, P. (2011). Biogeochemical processes in a clay formation in situ experiment: Part F—reactive transport modelling. Applied Geochemistry, 26, 1009–1022.Google Scholar
  56. Valcke, E., Sneyers, A., & Van Iseghem, P. (2000a). The effect of radiolytic degradation products of Eurobitum on the solubility and sorption of Pu and Am in Boom Clay. Proceedings to the Materials Research Society Symposium, volume 663 (pp. 141–149). Cambridge University Press.Google Scholar
  57. Valcke, E., Sneyers, A., & Van Iseghem, P. (2000b). The long-term behavior of bituminized waste in a deep clay formation. Proceedings to the Safewaste Conference, volume 2 (pp. 562–573). Société française d’énergie nucléaire.Google Scholar
  58. Valcke, E., Marien, A., & Van Geet, M. (2009). The methodology followed in Belgium to investigate the compatibility with geological disposal of Eurobitum bituminized intermediate level radioactive waste. Proceedings of the Materials Research Society Symposium, volume 1193 (pp. 105–116). Cambridge University Press.Google Scholar
  59. Van Loon, L. R., Soler, J. M., Müller, W., & Bradbury, M. H. (2004a). Anisotropic diffusion in layered argillaceous rocks: a case study with Opalinus Clay. Environmental Science & Technology, 38, 5721–5728.Google Scholar
  60. Van Loon, L. R., Wersin, P., Soler, J., Eikenberg, J., Gimmi, T., Hernán, P., Dewonck, S., & Savoye, S. (2004b). In-situ diffusion of HTO, 22Na+, Cs+ and I- in Opalinus Clay at the Mont Terri underground rock laboratory. Radiochimica Acta/International Journal for Chemical Aspects of Nuclear Science and Technology, 92, 757–763.Google Scholar
  61. Wersin, P., Soler, J., Van Loon, L., Eikenberg, J., Baeyens, B., Grolimund, D., et al. (2008). Diffusion of HTO, Br, I, Cs+, 85Sr2+ and 60Co2+ in a clay formation: results and modelling from an in situ experiment in Opalinus Clay. Applied Geochemistry, 23, 678–691.Google Scholar
  62. Wilhelm, E., Battino, R., & Wilcock, R. J. (1977). Low-pressure solubility of gases in liquid water. Chemical Reviews, 77, 219–262.Google Scholar
  63. Wouters, K., Moors, H., Boven, P., & Leys, N. (2013). Evidence and characteristics of a diverse and metabolically active microbial community in deep subsurface clay borehole water. FEMS Microbiology Ecology, 86, 458–473.Google Scholar
  64. Xiong, Z., Xing, G., & Zhu, Z. (2006). Water dissolved nitrous oxide from paddy agroecosystem in China. Geoderma, 136, 524–532.Google Scholar
  65. Zhang, Y.-C., Slomp, C. P., Broers, H. P., Bostick, B., Passier, H. F., Bottcher, M. E., et al. (2012). Isotopic and microbiological signatures of pyrite-driven denitrification in a sandy aquifer. Chemical Geology, 300, 123–132.Google Scholar
  66. Zhang, J., Dong, H., Liu, D., & Agrawal, A. (2013). Microbial reduction of Fe(III) in smectite minerals by thermophilic methanogen Methanothermobacter thermautotrophicus. Geochimica et Cosmochimica Acta, 106, 203–215.Google Scholar

Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  • Nele Bleyen
    • 1
  • Steven Smets
    • 1
  • Joe Small
    • 2
  • Hugo Moors
    • 1
  • Natalie Leys
    • 1
  • Achim Albrecht
    • 3
  • Pierre De Cannière
    • 4
  • Bernhard Schwyn
    • 5
  • Charles Wittebroodt
    • 6
  • Elie Valcke
    • 1
  1. 1.Belgian Nuclear Research Centre SCK•CENMolBelgium
  2. 2.National Nuclear Laboratory NLLWarringtonUK
  3. 3.Agence Nationale pour la Gestion des Déchets Radioactifs AndraChâtenay-Malabry CedexFrance
  4. 4.Federal Agency for Nuclear Control FANCBrusselsBelgium
  5. 5.National Cooperative for the Disposal of Radioactive Waste NAGRAWettingenSwitzerland
  6. 6.Institut de Radioprotection et de Sûreté Nucléaire IRSNFontenay-Aux-RosesFrance

Personalised recommendations