Approaches to DNA extraction play a crucial role in determining the variability of results obtained through 16S rRNA amplicon sequencing. Particularly, clay-rich samples can impede the efficiency of various standard cultivation-independent techniques. We conducted an inter-laboratory comparison study to thoroughly assess the efficacy of two published DNA extraction methods (kit-based and phenol-chloroform-based) specifically designed for bentonite samples. To this end, we spiked Wyoming MX 80 bentonite with two different mock communities and compared the obtained DNA yield and purity, the presence of contaminants and the community profile. Our findings suggest that both methods are equally viable, with the best choice depending on the specific requirements of the downstream analysis. However, it is crucial to maintain consistency in the chosen method, as comparing results becomes challenging, particularly in the presence of bentonite. In summary, our study emphasizes the significance of standardized DNA extraction methods and underscores the importance of validating these methods using appropriate controls when studying microbial communities with 16S rRNA amplicon sequencing, particularly in environments characterized by low biomass and clay-rich compositions. Additionally, slight modifications to one of the extraction methods can substantially enhance its efficiency.

• MeSH
• Bacteria genetics   classification   isolation & purification   MeSH
• Bentonite * chemistry   MeSH
• DNA, Bacterial * genetics   isolation & purification   MeSH
• Microbiota genetics   MeSH
• Soil Microbiology MeSH
• RNA, Ribosomal, 16S * genetics   MeSH
• Sequence Analysis, DNA MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• Bentonite * MeSH
• DNA, Bacterial * MeSH
• RNA, Ribosomal, 16S * MeSH

Bentonite is an integral part of the engineered barrier system (EBS) in deep geological repositories (DGR) for nuclear waste, but its indigenous microorganisms may jeopardize long-term EBS integrity. To predict microbial activity in DGRs, it is essential to understand microbial reactions to the early hot phase of DGR evolution. Two bentonites (BCV and MX-80) with varied bentonite/water ratios and saturation levels (compacted to 1600 kg.m- 3 dry density/powder/suspension), were subjected to heat (90-150 °C) and irradiation (0.4 Gy.h- 1) in the long-term experiments (up to 18 months). Molecular-genetic, microscopic, and cultivation-based techniques assessed microbial survivability. Exposure to 90 °C and 150 °C notably diminished microbial viability, irrespective of bentonite form, with negligible impacts from irradiation or sample type compared to temperature. Bentonite powder samples exhibited microbial recovery after 90 °C heating for up to 6 months but not 12 months in most cases; exposure to 150 °C had an even stronger effect. Further long-term experiments at additional temperatures combined with the mathematical prediction of temperature evolution in DGR are recommended to validate the possible evolution and spatial distribution of microbially depleted zones in bentonite buffer around the waste canisters and refine predictions of microbial effects over time in the DGR.

• Keywords
• Bentonite buffer, Deep geological repository, Elevated temperature, Extremophiles, Gamma radiation, Microbial limiting factors, Radioactive waste disposal,
• MeSH
• Bacteria * classification   radiation effects   genetics   growth & development   MeSH
• Bentonite * chemistry   MeSH
• Microbial Viability * radiation effects   MeSH
• Soil Microbiology MeSH
• Radioactive Waste analysis   MeSH
• Hot Temperature * MeSH
• Gamma Rays * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bentonite * MeSH
• Radioactive Waste MeSH