AIMS: This study evaluates the effectiveness of two standard sterilization methods on microorganisms in bentonite, which is proposed as a buffer around metal canisters containing long-lived radioactive waste. Bentonite, as a natural clay, contains microorganisms with enhanced resistance to harsh conditions and the ability to reactivate upon decompaction. Sterile controls are crucial in experiments estimating the impact of microorganisms on nuclear waste repositories. Yet, the effectiveness of common sterilization methods on bentonite microorganisms has not been fully evaluated. METHODS AND RESULTS: Two methods were compared: dry heat (nine cycles at 121°C for 4 h) and gamma irradiation (10-140 kGy at 147 Gy·min-1). Molecular-genetic, microscopic, and cultivation techniques were used to assess sterilization. Heat sterilization did not eliminate heat-resistant microorganisms, such as Bacillus, Paenibacillus, and Terribacillus, from bentonite powder even after nine heat cycles. However, bentonite suspended in deionized water was sterile after four heat cycles. In contrast, gamma irradiation effectively reduced microbial survivability above a dose of 10 kGy, with the highest doses (100-140 kGy) potentially degrading DNA. CONCLUSIONS: Gamma irradiation at 30 kGy effectively sterilized bentonite powder. The findings of our experiments emphasize the importance of using appropriate sterilization methods to maintain sterile controls in experiments that evaluate the microbial impacts in nuclear waste repositories. However, further assessment is needed to determine the effects of potential alterations induced by gamma radiation on bentonite properties.

Disposal Processes and Safety ÚJV Řež a s Hlavní 130 250 68 Husinec Czech Republic

Faculty of Nuclear Sciences and Physical Engineering Czech Technical University Prague Břehová 78 7 115 19 Prague Czech Republic

Institute for Nanomaterials Advanced Technologies and Innovation Technical University of Liberec Bendlova 7 460 01 Liberec Czech Republic

Research and Development Department UJP PRAHA a s Nad Kamínkou 1345 156 00 Prague Czech Republic

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