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.

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.

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.

Abstract As bentonite hosts a diverse spectrum of indigenous microorganisms with the potential to influence the long-term stability of deep geological repositories, it is essential to understand the factors determining microbial activity under repository conditions. Here, we focus on two such factors, temperature and swelling pressure, using a suspension of Cerny Vrch bentonite to boost microbial activity and visualise microbial response. Suspensions were exposed to differing pressures (10–15 MPa; simulating the effect of swelling pressure) and temperatures (60–90°C), followed by a period of anaerobic incubation at atmospheric pressure/laboratory temperature to assess microbial recovery. Microbial load and community structure were estimated using molecular-genetic methods, with presence of living cells confirmed through microscopic analysis. Pressure application had no influence on overall microbial activity or proliferation, proving that pressure evolution during bentonite swelling is not the key factor responsible for microbial suppression in saturated compacted bentonites. However, pressure treatment did cause significant shifts in microbial community structure. We also demonstrated that microbial activity decreased with increasing temperature, and that heat treatment strongly influenced bentonite microbial community structure, with several thermophilic taxa identified. A temperature of 90°C proved to be limiting for microbial activity and proliferation in all bentonite suspensions.

Bentonite is a clay material with a broad range of applications in construction, industry (food, pharmacy), and civil engineering including water treatment and waste disposal. It has also been proposed as a buffer and backfill material in deep geological repositories (DGRs). However, the presence of metabolically active bacteria in bentonite could compromise the long-term safety of such DGRs, highlighting the need for a method for detection of microorganisms in bentonite materials. Here, we propose a novel protocol for the detection of both living (metabolically active and dead indigenous bacterial cells in bentonite. The extraction protocol requires the addition of a dispersant (2.5 mM sodium pyrophosphate (NaPP)/12.5 mM EDTA solution or 1% methanol) to a bentonite sample, followed by a two-step centrifugation over high-density media (i.e., sucrose and histodenz) to separate and concentrate the cells. The extracted bacterial cells can then be examined by epifluorescence microscopy using LIVE/DEAD staining and using molecular biology methods. Overall, the NaPP-based dispersant afforded a better extraction efficiency (20%) than methanol (6%). The light clay fraction acted as a sieve effectively retaining dispersed cells during centrifugation; this light clay fraction with attached cells being later detected in the final extracts. Importantly, the microbial community composition detected in cell extracts by 16S rRNA sequencing corresponded to that in the original suspension. The protocol has been successfully applied on different bentonite samples in different environments, demonstrating the high potential of this approach for evaluation of microbial activity in bentonite.

As bentonite hosts a diverse spectrum of indigenous microorganisms with the potential to influence the long-term stability of deep geological repositories, it is essential to understand the factors influencing microbial activity under repository conditions. Here, we focus on two factors, i.e., temperature and swelling pressure, using a suspension of Cerny Vrch bentonite to boost microbial activity and evaluate microbial response. Suspensions were exposed either to different pressures (10, 12 and 15 MPa; to simulate the effect of swelling pressure) or elevated temperatures (60, 70, 80 and 90 °C; to simulate the effect of cannister heating) for four weeks. Each treatment was followed by a period of anaerobic incubation at atmospheric pressure/laboratory temperature to assess microbial recovery after treatment. Microbial load and community structure were then estimated using molecular-genetic methods, with presence of living cells confirmed through microscopic analysis. Our study demonstrated that discrete application of pressure did not influence on overall microbial activity or proliferation, implying that pressure evolution during bentonite swelling is not the critical factor responsible for microbial suppression in saturated bentonites. However, pressure treatment caused significant shifts in microbial community structure. We also demonstrated that microbial activity decreased with increasing temperature, and that heat treatment strongly influenced bentonite microbial community structure, with several thermophilic taxa identified. A temperature of 90 °C proved to be limiting for microbial activity and proliferation in all bentonite suspensions. Our study emphasizes the crucial role of a deep understanding of microbial activity under repository-relevant conditions in identifying possible strategies to mitigate the microbial potential within the deep geological repository and increase its long-term stability and safety.

Bentonite buffers at temperatures beyond 100 °C could reduce the amount of high-level radioactive waste in a deep geological repository. However, it is necessary to demonstrate that the buffer surrounding the canisters withstands such elevated temperatures, while maintaining its safety functions (regarding long-term performance). For this reason, an experiment with thermal loading of bentonite powder at 150 °C was arranged. The paper presents changes that the Czech Mg/Ca bentonite underwent during heating for one year. These changes were examined by X-ray diffraction (XRD), thermal analysis with evolved gas analysis (TA-EGA), aqueous leachates, Cs sorption, cation exchange capacity (CEC), specific surface area (SSA), free swelling, saturated hydraulic conductivity, water retention curves (WRC), quantitative polymerase chain reaction (qPCR), and next-generation sequencing (NGS). It was concluded that montmorillonite was partially altered, in terms of the magnitude of the surface charge density of montmorillonite particles, based on the measurement interpretations of CEC, SSA, and Cs sorption. Montmorillonite alteration towards low- or non-swelling clay structures corresponded well to significantly lower swelling ability and water uptake ability, and higher saturated hydraulic conductivity of thermally loaded samples. Microbial survivability decreased with the thermal loading time, but it was not completely diminished, even in samples heated for one year.

Concrete as an important component of an engineered barrier system in deep geological repositories (DGR) for radioactive waste may come into contact with bentonite, or other clays, rich in indigenous microorganisms, with potentially harmful impacts on barrier integrity. Our study aimed to assess the effect of a concrete environment on indigenous bentonite and groundwater microbial communities as these particular conditions will select for the potentially harmful microorganisms to the concrete in the future DGR. The two-month experiment under anoxic conditions consisted of crushed, aged, low-pH concrete, Czech Ca-Mg bentonite, and anoxic groundwater, with control samples without concrete or with sterile groundwater. The microbial diversity and proliferation were estimated by qPCR and 16S rRNA gene amplicon sequencing. The presence of concrete had a strong effect on microbial diversity and reduced the increase in total microbial biomass, though low-pH concrete harbored indigenous bacteria. The growth of sulfate reducers was also limited in concrete samples. Several genera, such as Massilia, Citrifermentans, and Lacunisphaera, dominant in bentonite controls, were suppressed in concrete-containing samples. In contrast, genera such as Bacillus, Dethiobacter and Anaerosolibacter specifically proliferated in the presence of concrete. Genera such as Thermincola or Pseudomonas exhibited high versatility and proliferated well under both conditions. Because several of the detected bacterial genera are known to affect concrete integrity, further long-term studies are needed to estimate the effect of bentonite and groundwater microorganisms on concrete stability in future DGR.

Bentonite is a swelling clay, consisting mainly of montmorillonire, being planned to be used as a backfill material in the nuclear waste repository. It contains indigenous microbial populations that can negatively influence the long-term safety of the geological repository due to their metabolic activity (canister corrosion, illitization of bentonite, gas production, degradation of cementitious materials). However, reliable detection of microorganisms in clayish material is generally very difficult. Although the compactness of bentonite will undoubtedly limit the microbial activity, in the extremely long-time frame of repository lifetime this condition can fail. It is thus crucial to understand the potential of the naturally present microbial community in bentonite to compromise the safety of repository, if not limited by the compactness. Higher metabolic activity can be mainly expected at the interfaces or in the places with a lower density of bentonite. Here we present an optimized cell extraction method enabling direct estimation of bacterial density and viability in bentonite. Indigenous bacterial cells were extracted from bentonite suspensions by an improved step-wise protocol and their viability was detected using live/dead staining and epifluorescence microscopy. We used dispersant (2.5 mM natrium pyrophosphate-based solution or 1% methanol) to partially disintegrate the bentonite and detach the vital and dead microbial cells from its surface. The dispersed material was subsequently stepwise centrifuged over two high-density media (sucrose and Histodenz) to remove most of the heavy bentonite particles while keeping the light bentonite particles and cells in the final extract. We were able to detect and enumerate the cells concentrated at the surface of the light bentonite particles, which served as a sieve to retain all free cells during centrifugation. Different extraction procedures were tested and their efficiency was estimated by comparing live/dead ratios of resulting extracts and was also proved by implementing both NGS and quantitative PCR. The results show that most of the microbial genera present in the original suspension are also present in extracts but as proved by Deseq2 analysis some genera tend to settle down with heavier bentonite particles during the first centrifugation step. To conclude, we present a protocol for extraction and detection of metabolically active cells in clayish material – bentonite. The quality of the extraction procedure was estimated both by a combination of fluorescent microscopy and genetic methods. The protocol was successfully tested on different bentonite types showing general applicability of this approach for clay materials.