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Item Application of molecular techniques to elucidate the influence of cellulosic waste on the bacterial community structure at a simulated low level waste site(2010-03) Field, E. K.; D'Imperio, Seth; Miller, A. R.; VanEngelen, Michael R.; Gerlach, Robin; Lee, Brady D.; Apel, William A.; Peyton, Brent M.Low-level radioactive waste sites, including those at various U.S. Department of Energy (DOE) sites, frequently contain cellulosic waste in the form of paper towels, cardboard boxes, or wood contaminated with heavy metals and radionuclides such as chromium and uranium. To understand how the soil microbial community is influenced by the presence of cellulosic waste products, multiple soil samples were obtained from a non-radioactive model low-level waste test pit at the Idaho National Laboratory. Samples were analyzed using 16S rRNA gene clone libraries and 16S rRNA gene microarray (PhyloChip) analyses. Both methods revealed changes in the bacterial community structure with depth. In all samples, the PhyloChip detected significantly more Operational Taxonomic Units (OTUs), and therefore relative diversity, than the clone libraries. Diversity indices suggest that diversity is lowest in the Fill (F) and Fill Waste (FW) layers and greater in the Wood Waste (WW) and Waste Clay (WC) layers. Principal coordinates analysis and lineage specific analysis determined that Bacteroidetes and Actinobacteria phyla account for most of the significant differences observed between the layers. The decreased diversity in the FW layer and increased members of families containing known cellulose degrading microorganisms suggests the FW layer is an enrichment environment for these organisms. These results suggest that the presence of the cellulosic material significantly influences the bacterial community structure in a stratified soil system.Item Microbial community signature in Lake Coeur d'Alene: Association of environmental variables and toxic heavy metal phases(2016-03) Moberly, James G.; D'Imperio, Seth; Parker, Albert E.; Peyton, Brent M.The water and sediments of Lake Coeur d'Alene in northern Idaho (USA) have been impacted by decades of mining operations within the Coeur d'Alene mining district. Using a multivariate statistical approach, correlations were explored between the microbial community (via 16S rDNA microarray) in sediment cores and operationally defined heavy metal phases (via continuous sequential extractions). Candidate phyla NC10, OP8 and LD1PA were only detected in metal contaminated cores and diversity doubled among Natronoanaerobium in metal contaminated cores compared to the uncontaminated control site. This may suggest some increased fitness of these phyla in contaminated sediments. In contrast, diversity within the phyla Aquificae, Coprothermobacteria, and Synergistes was at least double in the uncontaminated control site. In linear models composed of two geochemical variables from the presumed sulfate reducing lineages detected in this study, orders Desulfobacterales, Desulfuromonadales, Desulfotomaculum, and Syntrophobacterales were highly correlated with Pb (positive influence) and Zn (negative influence) in the operationally defined residual fraction, and most taxa within orders from Desulfovibrionales. Bdellovibrionales highly correlated with Pb in the exchangeable/carbonate (negative influence) and oxyhydroxide (positive influence) phases. Diversity within families from metal reducing bacterial lineages Shewanellaceae, Geobacteraceae, and Rhodocyclaceae showed high correlation with Pb in the exchangeable/carbonate (negative influence) and oxyhydroxide (positive influence) phases. To our knowledge, this is the first time these techniques have been used in combination to describe a contaminated system. Resulting correlations suggest the diversity of the microbial community was influenced primarily by partitioning of heavy metals into exchangeable Pb over other Pb phases and, to a lesser extent, residual Pb to residual Zn phase partitioning.Item Microbial interactions with arsenite, hydrogen and sulfide in an acid-sulfate-chloride geothermal spring(Montana State University - Bozeman, College of Agriculture, 2008) D'Imperio, Seth; Chairperson, Graduate Committee: Timothy R. McDermott.The work presented in this thesis investigated the importance of hydrogen, sulfide and arsenite in microbial community structure and function within a model Acid-Sulfate- Chloride (ASC) spring in Yellowstone National Park. Previous studies in this spring found that microbial arsenite [As(III)] oxidation is absent in regions of the spring outflow channel where H 2S exceeds ~5 microM. Ex situ assays with microbial mat samples demonstrated immediate As(III) oxidation activity when H 2S was absent or in low concentrations, suggesting the presence of functional As(III) oxidase enzymes in regions of the spring where arsenite oxidation had not been previously observed. Cultivation efforts resulted in the isolation of an As(III)-oxidizing chemolithotroph phylogenetically related to the alpha-proteobacterium Acidicaldus. H 2S concentration appeared to be the most important constraint on spatial distribution of this organism. This was verified with pure culture modeling and kinetic experiments. Additionally, a study is presented that addresses the relative importance of dissolved hydrogen and sulfide for primary production in the same spring. Throughout the outflow channel where these gases could be detected, biological H 2S consumption rates exceeded those of H2 by at least three orders of magnitude. Molecular analysis showed that Hydrogenobaculum-like organisms dominate the microbial community in this region of the spring. Culturing efforts resulted in 30 Hydrogenobaculum isolates belonging to three distinct 16S rRNA gene phylotypes. The isolates varied with respect to electron donor (H 2S, H 2) and oxygen tolerance and requirement. These metabolic physiologies are consistent with in situ geochemical conditions. An isolate representative of the dominant 16S phylotype was used as a model organism for controlled studies to determine whether an organism capable of utilizing either of these substrates demonstrated preference for H 2S or H 2, or whether either electron donor exerted regulatory effects on the other. The organism studied utilized both H 2S and H 2 simultaneously and at rates roughly comparable to those measured in the ex situ field assays. Major conclusions drawn from this study are that phylogeny cannot be relied upon to predict physiology, and that, in ASC springs, H 2S clearly dominates H 2 as an energy source, both in terms of availability and apparent consumption rates.