Browsing by Subject "microbiology"
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Item An Investigation of Linked Physical And Biogeochemical Processes In Heterogeneous Soils In The Vadose Zone(2012-10-19) Hansen, David JosephChemical dynamics in the vadose zone are poorly understood due to the transient nature of chemical and hydrologic conditions, but are nonetheless critical to understanding contaminant fate and transport. This work explored the effects of soil structure (i.e. layers, lenses) on linked geochemical, hydrological, and microbiological processes under changing hydrologic conditions (e.g. rainfall, introduction of groundwater, and fluctuating water table heights). A homogenized medium-grained sand, homogenized organic-rich loam and a sand-over-loam layered column were constructed for the first series of experiments. The second series of experiments employed two soil columns with lenses that were packed identically with sterilized and untreated sediments. Each consisted of two lenses of organic-rich loam in a medium-grained sand matrix. Lenses were located at different vertical depths and were horizontally offset. In-situ collocated probes collected soil hydrologic and chemical data. In the layered column, enhanced biogeochemical cycling was observed over the texturally homogeneous soil columns. Enumerations of Fe(III) and SO42- reducing microorganisms also show 1-2 orders of magnitude greater community numbers in the layered column. The greatest concentrations of aqueous FeS clusters (FeSaq) were observed in close proximity to the soil interface. To our knowledge, this was the first documentation of FeSaq in partially saturated sediments. Mineral and soil aggregate composite layers were also most abundant near the soil layer interface; the presence of which, likely contributed to an order of magnitude decrease of hydraulic conductivity. In the live lens column, Fe-oxide bands formed at the fringes of the lenses that retarded water flow rates by an order of magnitude compared to the sterilized column. Microbial activity also produced insoluble gases and that led to the creation of a separate gas phase that reduced hydraulic conductivity. This limited the interaction between groundwater with soil-pore waters that led to the formation of geochemically distinct water masses in relatively close proximity to one another. No such changes were observed in the sterilized column. When compared to homogenous columns, the presence of soil heterogeneities altered biogeochemical and hydrologic processes considerably which highlights the need to consider soil heterogeneity in contaminant fate and transport models. These findings suggest that quantifying coupled hydrologic-biogeochemical processes occurring at small scale soil interfaces is critical to accurately describing and predicting chemical changes at the larger system scale.Item Engineering pH tolerant mutants of a cyanide dihydratase of Bacillus pumilus C1 and identifying constraints on substrate specificity in nitrilases(2009-05-15) Wang, LanThis study generated two cyanide dihydratase (CynD) mutants of Bacillus pumilus C1 with improved activity at higher pH by random mutagenesis. The purpose of this study was to create enzyme variants better suited to degrade cyanide under the harsh conditions of industrial applications. We employed error-prone PCR to construct a library of CynD mutants. A high throughput screening system was developed to screen the library for improved activity. Two mutants were identified that could degrade cyanide at pH10 whereas the wild-type enzyme was inactive at pH9 or higher. The mutants each had three amino acid substitutions compared to the wild-type enzyme. The mutants were also more stable than the wild-type enzyme at 42oC. E327G was identified as one of the key amino acids that are responsible for the improved activity. The goal of the second project was to convert substrate specificity of the Bacillus sp. OxB-1 nitrilase to that of a cyanidase by mutagenesis or construction of hybrid genes. The OxB-1 nitrilase of Bacillus sp. shows a high level of identity with the cyanide dihydratases from B. pumilus C1 and P. stutzeri AK61 but utilizes different substrate. This provides a valuable resource to study the substrate specificity determinants of cyanide degrading enzymes. One deletion mutant and four hybrid proteins were constructed based on the alignment information. The constructed proteins were all unable to degrade cyanide.