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 genetika klasifikace izolace a purifikace MeSH
- bentonit * chemie MeSH
- DNA bakterií * genetika izolace a purifikace MeSH
- mikrobiota genetika MeSH
- půdní mikrobiologie MeSH
- RNA ribozomální 16S * genetika MeSH
- sekvenční analýza DNA MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
- Názvy látek
- bentonit * MeSH
- DNA bakterií * MeSH
- RNA ribozomální 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.
- Klíčová slova
- Bentonite buffer, Deep geological repository, Elevated temperature, Extremophiles, Gamma radiation, Microbial limiting factors, Radioactive waste disposal,
- MeSH
- Bacteria * klasifikace účinky záření genetika růst a vývoj MeSH
- bentonit * chemie MeSH
- mikrobiální viabilita * účinky záření MeSH
- půdní mikrobiologie MeSH
- radioaktivní odpad analýza MeSH
- vysoká teplota * MeSH
- záření gama * MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- bentonit * MeSH
- radioaktivní odpad MeSH
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.
- Klíčová slova
- Bentonite suspension, Deep geological repositories, Limiting factors, Microbial activity, Microorganism survivability, Radioactive waste disposal,
- MeSH
- bentonit * analýza chemie MeSH
- chemické jevy MeSH
- proliferace buněk MeSH
- radioaktivní odpad * analýza MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- bentonit * MeSH
- radioaktivní odpad * MeSH
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.
- Klíčová slova
- Bentonite, Indigenous microorganisms, Low-pH concrete, Nuclear waste repository safety,
- MeSH
- bentonit MeSH
- koncentrace vodíkových iontů MeSH
- mikrobiota * MeSH
- radioaktivní odpad * analýza MeSH
- RNA ribozomální 16S genetika MeSH
- suspenze MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- bentonit MeSH
- radioaktivní odpad * MeSH
- RNA ribozomální 16S MeSH
- suspenze MeSH
