Indigenous bacterial adaptation and survival: Exploring the shifts in highly compacted bentonite over a 5-year long-term study for nuclear repository purposes

Compacted bentonite is one of the most promising engineered barrier materials used in Deep Geological Repositories (DGR) of high-level radioactive waste encapsulated in metal canisters. Determining bentonite compaction density threshold for bacterial presence and activity has been a long-standing objective, due to their implications for canisters' durability and, therefore, in the safety performance of DGR. This study provided new insights into the effect of dry density (1.5 and 1.7 g cm⁻³), acetate amendment, and long-term incubation (5 years) on the bentonite mineralogy as well as their bacterial community distribution and survival. Through Illumina sequencing, we demonstrated that higher dry density reduces the bacterial diversity with spore-forming bacteria such as Nocardioides, and Promicromonospora being predominant. Interestingly, Paracoccus and Pseudomonas were enriched in acetate-treated samples, suggesting the utilization of this carbon source and, consequently, supporting their viability and survival. In addition, spore-forming (e.g., Bacillus) and desiccation-resistant (e.g., Arthrobacter) microorganisms were isolated. X-ray diffraction and scanning electron microscopy analyses showed the stability of bentonite while indicating the probable formation of iron sulfides. These findings confirm the influence of bentonite compaction degree and long-term incubation on microbial viability and activity, highlighting their potential impact on the integrity and safety of future DGR.