Humidity and temperature boundaries for biofilm formation in Yucca Mountain

To determine the long-term success of the recommended Yucca Mountain high-level nuclear waste repository, studies of bacterial colonization and biofilm development are needed. Bacteria involved in microbially-influenced corrosion and degradation are known to form biofilms with the potential to impact the integrity of repository packaging and structural materials. Temperature and humidity are environmental factors that can greatly affect biofilm formation. Therefore, it is necessary to determine the temperature and humidity conditions that affect biofilm formation. Microcosms, which simulated the repository environment of Yucca Mountain, were placed at temperatures ranging from 30° C to 70° C and in relative humidities ranging from 100% to 32%. The microcosms contained titanium, C22 nickel alloy, and N316 stainless steel coupons buried in crushed Yucca Mountain muckpile rock. The uniform-sized metal coupons were sacrificed at the following timepoints: day 0, 1 day, 1 month, 5 months,! year, and 18 months. The average number of culturable bacteria harvested from the entire surface of each of the three coupon types, incubated at 100% relative humidity and 30° C, increased from approximately 1x102 CPU at day 0 to a range of 4-7x104 CPU at 5 months, followed by a decrease to 5-8x102 CPU after 18 months of incubation. The average number of culturable bacteria harvested from the surfaces of the three metals, incubated at 84%, 70.5%, and 32% relative humidity and 30° C, were unchanged at 102 CPU from day 0 to 18 months or decreased to numbers below the level of detection. Culturable bacterial counts, from the three candidate metals incubated at 60 and 70° C and 100% relative humidity, showed a decrease from 102 CPU at day 0 to numbers below the level of detection at 18 months. Confocal laser microscopy 6 showed minimal differences in the extent of microbial colonization on the three metal surfaces in all but optimum conditions, 100% RH and 30° C, at each timepoint after day zero. These data indicate that a decrease in relative humidity level or an increase in temperature severely affects biofilm formation on the three candidate metals being considered for repository packaging at the recommended Yucca Mountain nuclear waste repository