Development of protocols for sampling and assessment of bentonite and environmental samples associated with in situ proof tests of engineered barrier systems

The Nuclear Waste Management Organization (NWMO) is responsible for implementing Adaptive Phased Management (APM), Canada's plan for the long-term care of used nuclear fuel produced by Canada's nuclear reactors. In preparation for the Canadian work, NWMO is participating in the Grimsel Underground Research Laboratory (URL) Materials Corrosion Test (MaCoTe) in Switzerland. Test modules containing metal coupons surrounded by highly compacted MX-80 bentonite were retrieved from ca. 9 m deep boreholes at the URL MaCoTe site after 394 days of storage. We developed protocols for sampling of bentonite and environmental samples associated with the engineered barrier systems (EBS) and assessed microbial communities and their distributions using DNA-based methods. Swab and bentonite samples were taken from various surfaces and materials of borehole modules 1a and 2a. Genomic DNA was extracted from 77 borehole module samples and 2 Sterivex filters using the PowerSoil or PowerMax DNA Isolation Kit (MO BIO Laboratories). Only 18 DNA extractions yielded sufficient genomic DNA to be quantified using the Qubit fluorometer. Only 55% of samples yielded an amplicon in a conventional 16S rRNA gene PCR with 35 cycles of amplification. A “nested” PCR protocol increased the proportion of successful amplification to 75%, but many amplicons were still weak. A total of 116 borehole module samples (including replicates), 7 DNA Isolation kit controls, and 18 no-template controls were sequenced on a MiSeq instrument (Illumina) and assembled sequences were grouped into 1,612 operational taxonomic units (OTUs).