Bentonite sterilization methods in relation to radioactive waste disposal: Efficiency of dry heat and gamma radiation

Highly compacted bentonite will be used as a buffer around metal canisters containing long-lived radioactive waste. However, it harbors indigenous microorganisms that can endure harsh conditions and reactivate upon decompaction. Although sterile bentonite samples are crucial to understanding the impacts of these microorganisms on the repository environment and their potential influence on its long-term performance, the effectivity of various sterilization methods on bentonite has not yet been estimated. This study compared the effectiveness of dry heat (nine repeated applications at 121C for 4 hours) and gamma irradiation (doses of 10, 30, 70, 100, and 140 kGy), the two fundamental microbiological sterilization techniques. A comparative investigation, including molecular-genetic, microscopic, and cultivation-based techniques, was conducted to evaluate the sterilization efficiency of these methods. The heat sterilization experiment demonstrated that repeated dry heat applications on bentonite powder failed to eliminate certain heat-resistant microorganisms like Bacillus, Paenibacillus, or Terribacillus, even after nine sterilization cycles. However, bentonite in a suspended state required only four repeated sterilizations to eradicate microorganisms, including heat-tolerant ones. Gamma radiation resulted in an apparent decline in microbial survivability and cultivability in bentonite exposed to all doses above 10 kGy, irrespective of incubation time/media type. The highest doses (100-140 kGy) possibly caused DNA degradation. Overall, our results suggest that an applied dose of 30 kGy is sufficient to sterilize bentonite powder samples; gamma irradiation is thus considered a highly effective method compared to heat sterilization for obtaining sterile abiotic bentonite samples.