A protocol for the extraction of viable bacteria for identification of bacterial communities in bentonite

Hlavackova, Veronika; Shrestha, Rojina; Hofmanová, Eva; Kejzlar, Pavel; Říha, Jakub; Bartak, Deepa; Ševců, Alena; Černá, Kateřina

doi:10.1016/j.clay.2022.106809

Cited by 5 publications

(4 citation statements)

References 49 publications

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“…In a repository environment, bentonite temperatures may become temporarily high, though they are unlikely to exceed 100 °C (Johnson et al 2002 ; Bennett and Gens 2008 ). Consequently, the saturation level will gradually evolve depending on the original water content of the compacted bentonite upon deposition, temperature evolution, and the bentonite’s dry density (Hökmark 2004 ). Our experiment demonstrated that survivability of microorganisms is severally hindered at elevated temperatures in a fully saturated environment; thus, evolution of bentonite water saturation close to the canister during the initial hot stage in the repository may prove to be a crucial parameter.…”

Section: Discussionmentioning

confidence: 99%

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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Section: Discussionmentioning

confidence: 99%

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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“…Cell extraction followed by LIVE/DEAD (L/D) staining was performed on 30D natural incubations using 1 mL of suspension, as described in (Hlavackova et al 2023). L/D staining was also applied to detect the presence of living and dead cells in culture samples, using 8 µL from each culture sample mixed with 4 µL of L/D BacLight ™ Bacterial Viability Kit uorescent dye (Thermo Fischer Scienti c, USA).…”

Section: Cell Extraction and Microscopy Analysismentioning

confidence: 99%

Dramatic loss of microbial viability in bentonite exposed to heat and gamma radiation: Implications for deep geological repository

Bartak,

Šachlová,

Kašpar

et al. 2024

Preprint

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Bentonite is an integral part of the engineered barrier system (EBS) in deep geological repositories (DGRs) 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°C or 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 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 waste canister and refine predictions of microbial effects over time in the DGR.

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“…Cell extraction using 5 mL of fresh suspension was performed on selected samples as described by [37]. To detect the presence of living and dead cells, we applied LIVE/DEAD (L/D) staining, using 8 µL of extracts from each sample mixed with 4 µL of LIVE/DEAD BacLight ™ Bacterial Viability Kit uorescent dye (Thermo Fischer Scienti c, USA).…”

Section: Cell Extraction and Microscopic Analysismentioning

confidence: 99%

“…The stained sample was incubated in the dark for 15 min before observing under a Zeiss Axio Imager M2 epi uorescence microscope (Carl Zeiss, Germany) by means of AxioVision (AxioVs40x69V v.4.9.1.0) imaging software program (Carl Zeiss, Germany). Cell extraction was only used to detect the presence of living cells in the samples; direct cell counts being impossible due to the presence of dense particles formed during the extraction protocol [37].…”

Section: Cell Extraction and Microscopic Analysismentioning

confidence: 99%

Effect of pressure and temperature on survivability and proliferation of microorganisms in bentonite

Bartak,

Bedrníková,

Kašpar

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

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.

show abstract

A protocol for the extraction of viable bacteria for identification of bacterial communities in bentonite

Cited by 5 publications

References 49 publications

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

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

Dramatic loss of microbial viability in bentonite exposed to heat and gamma radiation: Implications for deep geological repository

Effect of pressure and temperature on survivability and proliferation of microorganisms in bentonite

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