Validating DNA Extraction Protocols for Bentonite Clay

Engel, Katja; Coyotzi, Sara; Vachon, Melody A.; McKelvie, Jennifer; Neufeld, Josh D.

doi:10.1128/msphere.00334-19

Cited by 14 publications

(24 citation statements)

References 88 publications

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“…The 16S rRNA gene copy numbers of the Opalinus clay and NOCR samples were all lower than the range of previously measured 16S rRNA gene copy numbers in MX-80 bentonite [81,95]. The number of 16S rRNA gene copies detected in the NOCR samples are more similar to the estimated microbial abundances of clay-dominated aquifer layers of 10 3 cells/gdw [21].…”

Section: Microbial Abundancementioning

confidence: 48%

“…The use of DNA extraction and subsequent sequencing for clay has only been successfully reported in a few cases [78][79][80][81]. In one study, DNA was extracted from five bentonite clay samples in Almeria (Spain) and the V5-V6 hypervariable regions of the 16S ribosomal RNA (rRNA) gene was amplified and sequenced [79].…”

Section: Methods For Characterizing the Microbiology Of Bentonite Claymentioning

confidence: 99%

“…In previous research quantifying the 16S rRNA gene copies in compacted and dry MX-80 bentonite clay, medians of 4.1 x 10 4 copies/g [95] and 1.7 x 10 7 copies/gdw [81] were detected, respectively. Four of the ten Tsukinuno bentonite sample had 16S rRNA gene copies/gdw within the range of 10 4 and 10 7 , one of the samples was slightly higher than this range, and the remaining four were lower.…”

Section: Microbial Abundancementioning

confidence: 99%

“…A kit control containing no sample was included for each batch of extractions. The DNA extraction method used for clay and rock samples was validated previously [81].…”

Section: Dna Extractionmentioning

confidence: 99%

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Microbiology of barrier component analogues of a deep geological repository

et al. 2022

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Canada is currently implementing a site selection process to identify a location for a deep geological repository (DGR) for the long-term storage of Canada’s used nuclear fuel, wherein used nuclear fuel bundles will be sealed inside copper-coated carbon steel containers, encased in highly compacted bentonite clay buffer boxes and sealed deep underground in a stable geosphere. Because a DGR must remain functional for a million years, there is value to examining ancient natural systems that serve as analogues for planned DGR components. Specifically, studying the microbiology of natural analogue components of a DGR is important for developing an understanding of the types of microorganisms that may be able to grow and influence the long-term stability of a DGR. This study explored the abundance, viability, and composition of microorganisms in several ancient natural analogues using a combination of cultivation and cultivation-independent approaches. Samples were obtained from the Tsukinuno bentonite deposit (Japan) that formed ~10 mya, the Opalinus Clay formation (Switzerland) that formed ~174 mya, and Canadian shield crystalline rock from Northern Ontario that formed ~2.7 bya. Analysis of 16S rRNA gene amplicons revealed that three of the ten Tsukinuno bentonite samples analyzed were dominated by putative aerobic heterotrophs and fermenting bacteria from the Actinobacteria phylum, whereas five of the Tsukinuno bentonite samples were dominated by sequences associated with putative acidophilic chemolithoautotrophs capable of sulfur reduction.

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Section: Microbial Abundancementioning

confidence: 48%

Section: Methods For Characterizing the Microbiology Of Bentonite Claymentioning

confidence: 99%

Section: Microbial Abundancementioning

confidence: 99%

“…A kit control containing no sample was included for each batch of extractions. The DNA extraction method used for clay and rock samples was validated previously [81].…”

Section: Dna Extractionmentioning

confidence: 99%

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Microbiology of barrier component analogues of a deep geological repository

et al. 2022

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“…In addition to the borehole module samples, we also analyzed pellets and powdered Wyoming MX-80 bentonite that was used to load the modules before exposure in the borehole. Depending on sample type, the PowerSoil or PowerMax DNA isolation kits were used, as previously validated for clay samples (10). Although only 18 DNA extractions yielded sufficient amounts of genomic DNA to be quantified using the Qubit fluorometer, 42 samples yielded an amplicon in nested 16S rRNA gene PCR (Table S1); however, many PCR products were only weakly visible by agarose gel electrophoresis (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Stability of Microbial Community Profiles Associated with Compacted Bentonite from the Grimsel Underground Research Laboratory

Engel

Ford

Coyotzi

et al. 2019

mSphere

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The Materials Corrosion Test in Grimsel Underground Research Laboratory, Switzerland, enables an evaluation of microbiological implications of bentonite clay at densities relevant for a deep geological repository. Our research demonstrates that after 13 months of exposure within a granitic host rock, the microbial 16S rRNA gene signatures of saturated bentonite clay within the modules were consistent with the profiles in the original clay used to pack the modules. Such results provide evidence that densities chosen for this emplacement test are refractory to microbial activity, at least on the relatively short time frame leading to the first time point sampling event, which will help inform in situ engineered barrier system science. This study has important implications for the design of deep geological repository sites under consideration for the Canadian Shield.

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Survivability and proliferation of microorganisms in bentonite with implication to radioactive waste geological disposal: strong effect of temperature and negligible effect of pressure

Bartak,

Bedrníková,

Kašpar

et al. 2023

World J Microbiol Biotechnol

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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.

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Validating DNA Extraction Protocols for Bentonite Clay

Cited by 14 publications

References 88 publications

Microbiology of barrier component analogues of a deep geological repository

Microbiology of barrier component analogues of a deep geological repository

Stability of Microbial Community Profiles Associated with Compacted Bentonite from the Grimsel Underground Research Laboratory

Survivability and proliferation of microorganisms in bentonite with implication to radioactive waste geological disposal: strong effect of temperature and negligible effect of pressure

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