Enhanced heterotrophic denitrification in clay media: The role of mineral electron donors Marc Parmentier a, ⁎, Patrick Ollivier a , Catherine Joulian a , Achim Albrecht b , Jebril Hadi a , Jean-Marc Greneche c , Hélène Pauwels a a BRGM, 3 Avenue C. Guillemin, BP 36009, 45060 Orléans, France b Andra, 1-7 rue Jean-Monnet, 92298 Châtenay-Malabry, France c IMMM, UMR CNRS 6283, Université du Maine, Avenue Olivier Messiaen, 72085 Le Mans, France abstract article info Article history: Received 9 November 2013 Received in revised form 16 October 2014 Accepted 17 October 2014 Available online 28 October 2014 Editor by: Dr. J. Fein Keywords: Denitrification Bacterial growth and activity Pseudomonas mandelii Callovian–Oxfordian Nuclear waste repository Clayey formation Denitrification is expected to occur at and near the interface with clayey formations dedicated to radioactive waste disposal, due to the nitrate content in some waste canisters and the high probability of introducing denitrifiers during the operational phase. Nitrate reduction to nitrous-oxide gas by pure-strain Pseudomonas mandelii, in the presence or absence of sterilized Callovian–Oxfordian (COx) clay rock, was studied over a period of ~41.5 months (1267 days) by means of batch experiments. A culture medium with a similar porewater chemistry to that of COx rocks was used, supplemented with acetate and nitrate. Bacterial growth was monitored by genomic-DNA and narG-gene quantification. Nitrite accumulated in solution concomitantly with a decrease in nitrate content and the weak generation of nitrous oxide, but denitrification rates drastically decreased over the study period. Acetate was both oxidized to inorganic carbon and incorporated into biomass. The presence of solid COx particles significantly affected the geochemical reactions and particularly caused an en- hanced nitrate reduction, a higher bacterial growth and the precipitation of calcium carbonate. Moreover, in the presence of COx a delay of several weeks was observed before the accumulation of nitrite in solution, leading to an imbalance between nitrate consumption and the production of nitrite, nitrous oxide and ammonium. Chemical oxidation of clay along with nitrite reduction to dinitrogen is expected to occur, explaining both the delay in nitrite accumulation and the apparent imbalance in nitrogen species. Although the electrondonor of the COx oxidation was not identified, several hypotheses may be advanced, and we provide new insight into the biogeochemical and geochemical processes that may occur concomitantly at the excavation damaged inter- face of the clayey host rock after closure, resaturation and release of waste components in a nuclear waste repos- itory. Because of denitrification, the oxidative impact of nitrate released from waste will diminish. However, nitrites produced through denitrification and diffusing into the rock will likely play in redox reaction via their abi- otic reactivity which may occur within the compact clay formation despite a lack of space for bacterial activity. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Clay-rich sedimentary formations are being considered in several countries as potential host rocks for the deep geological disposal of radioactive waste, because of their low permeability and high sorption capacity (Rousseau-Gueutin et al., 2008; Koroleva et al., 2011; Landais and Aranyossy, 2011). In France, the Callovian–Oxfordian (COx) clay rock is studied as a reference host rock for the disposal of radioactive waste (Gaucher et al., 2004; Landais, 2006; Tournassat et al., 2008; Grasset et al., 2010; Lerouge et al., 2011), and specifically of intermediate-level long-lived waste (ILW-LL) and high-level waste (HLW). Some ILW-LL contains significant amounts of sodium nitrate (NaNO 3 ) and organic compounds. For several thousand years after the operational phase (i.e. waste-cell closure), water will progressively fill the disposal cell, coming into contact with the waste packages and dissolving the contained salts. The solutes will then migrate into the surrounding environment (Koroleva et al., 2011). The behavior of sodium nitrate is of particular interest because nitrate has a direct im- pact on the redox conditions of radionuclides. Tc-99, Se-79 and U-238, for example, are known to be redox-sensitive, and to have enhanced mobility under oxidizing conditions, i.e. in the presence of nitrate. Se(VI) reduction is known to be inhibited at a 0.1 mM nitrate concentra- tion (Oremland et al., 1999), U(VI) at 1 mM (Senko et al., 2002; Istok et al., 2004) and Tc-99 at 100 mM (Li and Krumholz, 2008a). These three systems have been studied in a variety of contaminated natural sites such as at Hanford (Fredrickson et al., 2004) or Sellafield (Thorpe et al., 2012a; 2012b). More detailed knowledge on the reduction Chemical Geology 390 (2014) xxx–xxx ⁎ Corresponding author at: BRGM, 6ter rue Pierre et Marie Curie, 59260 Lezennes, France. Tel.: +33 3 20 19 15 43. E-mail address: m.parmentier@brgm.fr (M. Parmentier). CHEMGE-17379; No of Pages 13 http://dx.doi.org/10.1016/j.chemgeo.2014.10.014 0009-2541/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Chemical Geology journal homepage: www.elsevier.com/locate/chemgeo Please cite this article as: Parmentier, M., et al., Enhanced heterotrophic denitrification in clay media: The role of mineral electron donors, Chem. Geol. (2014), http://dx.doi.org/10.1016/j.chemgeo.2014.10.014