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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.