Abstract
Microorganisms in clay barriers could affect the long-term performance of waste containers in future deep geological repositories (DGR) for used nuclear fuel through production of corrosive metabolites (e.g., sulfide), which is why clay materials are highly compacted: to reduce both physical space and access to water for microorganisms to grow. However, the highly compacted nature of clays and the resulting low activity or dormancy of microorganisms complicate the extraction of biomarkers (i.e., PLFA, DNA etc.) from such barriers for predictive analysis of microbial risks. In order to overcome these challenges, we have combined culture- and 16S rRNA gene amplicon sequencing-based approaches to describe the functional diversity of microorganisms in several commercial clay products, including two different samples of Wyoming type MX-80 bentonite (Batch 1 and Batch 2), the reference clay for a future Canadian DGR, and Avonlea type Canaprill, a clay sample for comparison. Microorganisms from as-received bentonites were enriched in anoxic 10% w/v clay microcosms for three months at ambient temperature with addition of 10% hydrogen along with presumable indigenous organics and sulfate in the clay. High-throughput sequencing of 16S rRNA gene fragments indicated a high abundance of Gram-positive bacteria of the phylum Firmicutes (82%) in MX-80 Batch 1 incubations. Bacterial libraries from microcosms with MX-80 Batch 2 were enriched with Firmicutes (53%) and Chloroflexi (43%). Firmicutes also significantly contributed (<15%) to the bacterial community in Canaprill clay microcosm, which was dominated by Gram-negative Proteobacteria (>70%). Sequence analysis revealed presence of the bacterial families Peptostreptococcaceae, Clostridiaceae, Peptococcaceae, Bacillaceae, Enterobacteriaceae, Veillonellaceae, Tissierellaceae and Planococcaceae in MX-80 Batch 1 incubations; Bacillaceae, along with unidentified bacteria of the phylum Chloroflexi, in MX-80 Batch 2 clay microcosms, and Pseudomonadaceae, Hydrogenophilaceae, Bacillaceae, Desulfobacteraceae, Desulfobulbaceae, Peptococcaceae, Pelobacteraceae, Alcaligenaceae, Rhodospirillaceae in Canaprill microcosms. Exploration of potential metabolic pathways in the bacterial communities from the clay microcosms suggested variable patterns of sulfur cycling in the different clays with the possible prevalence of bacterial sulfate-reduction in MX-80 bentonite, and probably successive sulfate-reduction/sulfur-oxidation reactions in Canaprill microcosms. Furthermore, analysis of potential metabolic pathways in the bentonite enrichments suggested that bacteria with acid-producing capabilities (i.e., fermenters and acetogens) together with sulfide-producing prokaryotes might perhaps contribute to corrosion risks in clay systems. However, the low activity or dormancy of microorganisms in highly compacted bentonites as a result of severe environmental constraints (e.g., low water activity and high swelling pressure in the confined bentonite) in situ would be expected to largely inhibit bacterial activity in highly compacted clay-based barriers in a future DGR.