Low bentonite biomass leads to inconsistent culture-based estimates of microbial abundances.

Bentonite is an important component of deep geological repositories for long-term storage of used nuclear fuel. Studying the microbiology of bentonite exposed to various conditions is relevant because certain microorganisms (e.g. those that produce corrosive sulfide or gaseous metabolites) could lead to deterioration of engineered barrier components of the repository. In previous research, a high degree of variability in the abundance of culturable microorganisms among replicate samples has been observed. The purpose of this study was to test whether experimental technique (e.g. inadequate mixing of bentonite) or extremely low biomass represent mechanisms to explain such variability. Using a combination of cultivation- and DNA-based techniques to study six replicate hydrated bentonite microcosms, as well as six replicate bentonite aliquots originating from the same hydrated bentonite microcosm, the results of this study demonstrate that observed heterogeneity is likely not due to inadequate bentonite mixing. Instead, the data indicate that low biomass of as-received bentonite leads to unique populations of culturable bacteria associating with each sample, or to a lesser degree within different areas of a single bentonite sample used to establish a microcosm. Because some microorganisms that grow in bentonite are culturable under commonly used cultivation conditions and others are not, this can lead to differences in culture-based abundance estimates among replicate samples. Although cultivation is a useful technique to demonstrate viability of microorganisms in bentonite, the results of this study highlight the importance of a multifaceted experimental approach (i.e. coupling cultivation to DNA-based analysis) and careful analysis of replicates when working with such low biomass samples.