Assessing microbial activity and survivability in heated and irradiated bentonite samples under deep geological repository relevant conditions

Bentonite is a crucial part of the engineered barrier system (EBS) of the deep geological repositories (DGR) for nuclear waste. However, as a natural material, it harbors diverse indigenous microorganisms whose metabolic activity might compromise the long-term integrity of EBS. One of the critical questions for microbial activity prediction in DGR is the microbial reaction to the early hot phase of nuclear waste repository evolution. Our study investigates the impact of gamma radiation and heat on microbial survivability in bentonite (Czech bentonite BCV and reference bentonite MX-80), considering variations in compaction, temperature, and saturation levels as these factors are known to influence microbial reaction to extreme conditions. Previous results suggested average radiation tolerance and high heat tolerance of indigenous bentonite microorganisms. We aimed to confirm these findings during the medium-term experiment (18 months) at the repository simulating conditions. Four experimental setups were conducted using compacted BCV and MX-80 bentonites under anoxic conditions differing in initial saturation level (15-20 wt. %), heating temperature ( 90 or 150°C), and irradiation (0.4 Gy/h). An additional set of bentonite powder samples heated at 90°C or 150°C for 1, 3, and 6 months was further included to unravel the time effect of heat exposure on microbial survivability. Microbial community analysis, involving natural incubations in the form of bentonite suspension, enrichment cultures, microscopy, and molecular methods, was conducted for each sample to estimate microorganism survival following exposure to extreme conditions.

Contrary to expectations, no microbial growth and recovery were detected in any treated samples except for the additional set. Fresh samples, suspension incubations, and enrichment cultures were checked negative for microbial presence in almost all the cases in the four main experimental sets except for the positive controls. On the other hand, the samples from the additional set showed microbial recovery after heating at 90°C for 1, 3, and 6 months. However, exposure to 150°C for only 1 month resulted in bentonite sterilization. These medium-term data pointed out the high tolerance of indigenous bentonite microorganisms to heat but also indicated that developing a sterile bentonite layer in the proximity of hot and radiation-emitting metal canister is highly likely at the early stage of the DGR evolution. However, the particular development will depend upon the DGR design and actual levels of temperatures and irradiation. To enhance our understanding and predictability of microbial processes in the bentonite sealing layer of DGRs for nuclear waste, further microbiological experiments simulating conditions in distant bentonite layers subjected to lower temperature and studying potential microorganism penetration from non-sterile to sterilized zones are needed.