Molecular analysis of microbial diversity within Yucca Mountain

Yucca Mountain is the proposed site for a high-level nuclear waste repository. As part of the site characterization project, an estimation of microbial diversity was conducted. In this study, the 16S ribosomal DNA sequences of culturable isolates were determined and used for tentatively identification of bacterial strains. The results of identification by 16S rDNA sequence data were also compared to MIDI analysis, an independent phenotypic approach for bacterial identification; It is accepted that culturable strains only represent a small portion of microbial community in the environment. In order to eliminate bias associated with cultivation, total DNA was extracted from rock samples and subjected to 16S rDNA analysis. 16S rDNAs were amplified from the total DNA pool and cloned. Each cloned fragment in the constructed clone library was then subjected to sequence determination. Microbial diversity derived from total extracted DNA demonstrated a different vision of diversity from culturable isolates within Yucca Mountain; Two sample sites with different geology and geochemistry were selected for analysis. While culturable isolates contained mostly gram positive bacteria, gram negative bacteria in this environment were more dominant by DNA analysis. One study site, YM9, contained 100% of gram negative bacteria by total DNA analysis, with 89% being Pseudomonas species; In order to estimate the reliability of bacterial diversity derived from a series of molecular analyses as a whole, 10 common soil bacteria were seeded into sterile rock and subjected to the same process of sample analysis. The process showed no significant preference when interpreting the major group of gram positive and gram negative bacteria. Pseudomonas, represented by three species in the seeded sample, was not favored by the process. The presence of Pseudomonas species in this environment may be even higher than we detected; The detection of genetic diversity within a natural environment can be considered the very first step towards understanding the role that bacteria play in an ecosystem. The identity of major bacterial populations in Yucca Mountain will be important for development of strategies for storage of high-level nuclear waste and the maintenance of the repository integrity