Impact of dry density and incomplete saturation on microbial growth in bentonite clay for nuclear waste storage.

AIMS: Many countries are in the process of designing a deep geological repository (DGR) for long-term storage of used nuclear fuel. For several designs, used fuel containers will be placed belowground, with emplacement tunnels being backfilled using a combination of highly compacted powdered bentonite clay buffer boxes surrounded by a granulated "gapfill" bentonite. To limit the potential for microbiologically influenced corrosion of used fuel containers, identifying conditions that suppress microbial growth is critical for sustainable DGR design. This study investigated microbial communities in powdered and gapfill bentonite clay incubated in oxic pressure vessels at dry densities between 1.1 g cm-3 (i.e. below repository target) and 1.6 g cm-3 (i.e. at or above repository target) as a 1-year time series. RESULTS: Our results showed an initial (i.e. 1 month) increase in the abundance of culturable heterotrophs associated with all dry densities <1.6 g cm-3, which reveals growth during transient low-pressure conditions associated with the bentonite saturation process. Following saturation, culturable heterotroph abundances decreased to those of starting material by the 6-month time point for all 1.4 and 1.6 g cm-3 pressure vessels, and the most probable numbers of culturable sulfate-reducing bacteria (SRB) remained constant for all vessels and time points. The 16S rRNA gene sequencing results showed a change in microbial community composition from the starting material to the 1-month time point, after which time most samples were dominated by sequences associated with Pseudomonas, Bacillus, Cupriavidus, and Streptomyces. Similar taxa were identified as dominant members of the culture-based community composition, demonstrating that the dominant members of the clay microbial communities are viable. Members of the spore-forming Desulfosporosinus genus were the dominant SRB for both clay and culture profiles. CONCLUSIONS: After initial microbial growth while bentonite was below target pressure in the early phases of saturation, microbial growth in pressure vessels with dry densities of at least 1.4 g cm-3 was eventually suppressed as bentonite neared saturation.

Microbiology of barrier component analogues of a deep geological repository.

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, it is important to examine 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 phylum Actinobacteria, whereas five of the Tsukinuno bentonite samples were dominated by sequences associated with putative acidophilic chemolithoautotrophs capable of sulfur reduction. The remaining Tsukinuno bentonite samples, the Northern Ontario rock samples, and the Opalinus Clay samples generated inconsistent replicate 16S rRNA gene profiles and were associated primarily with contaminant sequences, suggesting that the microbial profiles detected were not sample-specific but spurious. Culturable aerobic heterotroph abundances were relatively low for all Tsukinuno bentonite samples, culturable anaerobic heterotrophs were only detected in half of the Tsukinuno samples, and sulfate-reducing bacteria (SRB) were only detected in one Tsukinuno sample by cultivation. Culture-specific 16S rRNA gene profiles from Tsukinuno clay samples demonstrated the presence of phyla Bacteroidetes, Proteobacteria, Actinobacteria, and Firmicutes among aerobic heterotroph cultures and additional bacteria from the phyla Actinobacteria and Firmicutes from anaerobic heterotroph plate incubations. Only one nucleic acid sequence detected from a culture was also associated with its corresponding clay sample profile, suggesting that nucleic acids from culturable bacteria were relatively rare within the clay samples. Sequencing of DNA extracted from the SRB culture revealed that the taxon present in the culture was affiliated with the genus Desulfosporosinus, which has been found in related bentonite clay analyses. Although the crystalline rock and Opalinus Clay samples were associated with inconsistent, likely spurious 16S rRNA gene profiles, we show evidence for viable and detectable microorganisms within several Tsukinuno natural analogue bentonite samples.

Microbial ecology of the deep terrestrial subsurface.

The terrestrial subsurface hosts microbial communities that, collectively, are predicted to comprise as many microbial cells as global surface soils. Although initially thought to be associated with deposited organic matter, deep subsurface microbial communities are supported by chemolithoautotrophic primary production, with hydrogen serving as an important source of electrons. Despite recent progress, relatively little is known about the deep terrestrial subsurface compared to more commonly studied environments. Understanding the composition of deep terrestrial subsurface microbial communities and the factors that influence them is of importance because of human-associated activities including long-term storage of used nuclear fuel, carbon capture, and storage of hydrogen for use as an energy vector. In addition to identifying deep subsurface microorganisms, recent research focuses on identifying the roles of microorganisms in subsurface communities, as well as elucidating myriad interactions-syntrophic, episymbiotic, and viral-that occur among community members. In recent years, entirely new groups of microorganisms (i.e. candidate phyla radiation bacteria and Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoloarchaeota, Nanoarchaeota archaea) have been discovered in deep terrestrial subsurface environments, suggesting that much remains unknown about this biosphere. This review explores the historical context for deep terrestrial subsurface microbial ecology and highlights recent discoveries that shape current ecological understanding of this poorly explored microbial habitat. Additionally, we highlight the need for multifaceted experimental approaches to observe phenomena such as cryptic cycles, complex interactions, and episymbiosis, which may not be apparent when using single approaches in isolation, but are nonetheless critical to advancing our understanding of this deep biosphere.

Fifteen shades of clay: distinct microbial community profiles obtained from bentonite samples by cultivation and direct nucleic acid extraction.

Characterizing the microbiology of swelling bentonite clays can help predict the long-term behaviour of deep geological repositories (DGRs), which are proposed as a solution for the management of used nuclear fuel worldwide. Such swelling clays represent an important component of several proposed engineered barrier system designs and, although cultivation-based assessments of bentonite clay are routinely conducted, direct nucleic acid detection from these materials has been difficult due to technical challenges. In this study, we generated direct comparisons of microbial abundance and diversity captured by cultivation and direct nucleic acid analyses using 15 reference bentonite clay samples. Regardless of clay starting material, the corresponding profiles from cultivation-based approaches were consistently associated with phylogenetically similar sulfate-reducing bacteria, denitrifiers, aerobic heterotrophs, and fermenters, demonstrating that any DGR-associated growth may be consistent, regardless of the specific bentonite clay starting material selected for its construction. Furthermore, dominant nucleic acid sequences in the as-received clay microbial profiles did not correspond with the bacteria that were enriched or isolated in culture. Few core taxa were shared among cultivation and direct nucleic acid analysis profiles, yet those in common were primarily affiliated with Streptomyces, Micrococcaceae, Bacillus, and Desulfosporosinus genera. These putative desiccation-resistant bacteria associated with diverse bentonite clay samples can serve as targets for experiments that evaluate microbial viability and growth within DGR-relevant conditions. Our data will be important for global nuclear waste management organizations, demonstrating that identifying appropriate design conditions with suitable clay swelling properties will prevent growth of the same subset of clay-associated bacteria, regardless of clay origin or processing conditions.