Enhanced heterotrophic denitrification in clay media: The role of mineral electron donors

- The presence of clayey rock enhances nitrifier bacterial growth and activity.
- Denitrification occurs in clay medium but its rate decreases with time.
- The presence of solid COx particles affects the (bio)geochemical reactions.
- Nitrite accumulates temporarily in solution and reacts with solid COx particles.
- Accumulated nitrite can oxidize solid COx particles, even where bacteria are excluded.

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 enhanced 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 electron donor 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 interface of the clayey host rock after closure, resaturation and release of waste components in a nuclear waste repository. 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 abiotic reactivity which may occur within the compact clay formation despite a lack of space for bacterial activity.