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

Bentonite is a clay material with a broad range of applications in construction, industry (food, pharmacy), and civil engineering including water treatment and waste disposal. It has also been proposed as a buffer and backfill material in deep geological repositories (DGRs). However, the presence of metabolically active bacteria in bentonite could compromise the long-term safety of such DGRs, highlighting the need for a method for detection of microorganisms in bentonite materials. Here, we propose a novel protocol for the detection of both living (metabolically active and dead indigenous bacterial cells in bentonite. The extraction protocol requires the addition of a dispersant (2.5 mM sodium pyrophosphate (NaPP)/12.5 mM EDTA solution or 1% methanol) to a bentonite sample, followed by a two-step centrifugation over high-density media (i.e., sucrose and histodenz) to separate and concentrate the cells. The extracted bacterial cells can then be examined by epifluorescence microscopy using LIVE/DEAD staining and using molecular biology methods. Overall, the NaPP-based dispersant afforded a better extraction efficiency (20%) than methanol (6%). The light clay fraction acted as a sieve effectively retaining dispersed cells during centrifugation; this light clay fraction with attached cells being later detected in the final extracts. Importantly, the microbial community composition detected in cell extracts by 16S rRNA sequencing corresponded to that in the original suspension. The protocol has been successfully applied on different bentonite samples in different environments, demonstrating the high potential of this approach for evaluation of microbial activity in bentonite.