Browsing by Author "Michael R. Hyman, Committee Member"

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    Characterization of diazotrophs containing Mo-independent nitrogenases from diverse natural environments
    (2003-02-11) Betancourt, Doris Alicia; Paul E. Bishop, Committee Chair; Michael R. Hyman, Committee Member; James W. Brown, Committee Member; Robert G. Upchurch, Committee Member
    Our laboratory was the first to identify the presence of Mo-independent nitrogenases in Azotobacter vinelandii and in seven isolates from aquatic environments. In this study we extended our search to other environments such as wood chip mulch, soil, and sediments from mangrove swamps. We were able to isolate and characterize 29 diazotrophs with Mo - independent nitrogenases. Phylogenetic analysis of 16S rDNA gene sequences indicated that most of the isolates are members of the gamma subdivision of the class Proteobacteria and they appear to be closely related to the fluorescent pseudomonads. The presence of Mo-independent nitrogenases in all isolates was detected using PCR and/ or Southern Blot hybridization. Of particular interest are isolates, AN1, BJM and LPF4 that are closely related to Citrobacter sp., Klebsiella sp. and Paenibacillus sp., respectively. We were able to clone and sequence Mo-independent nitrogenase genes from these strains. This study also includes a phylogenetic analysis of AnfG and VnfG. In conclusion, we have demonstrated that diazotrophs with Mo-independent nitrogenases can be easily isolated from the environment using Mo-deficient, N-free enrichment media. These results add to our knowledge of the distribution of Mo-independent nitrogenases in natural environments , and may provide clues as to the importance of these enzyme complexes in the nitrogen cycle.
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    Environmental and Spatial Factors Affecting Microbial Ecology and Metabolic Activity During the Initiation of Methanogenesis in Solid Waste
    (2009-08-10) Staley, Bryan Fleet; Morton A. Barlaz, Committee Co-Chair; Neal E. Blair, Committee Member; Michael R. Hyman, Committee Member; Francis L. de los Reyes, III, Committee Co-Chair
    Anaerobic decomposition of organic matter occurs in both natural (e.g., soil, peat bogs, digestive tracts) and engineered (e.g. landfills, anaerobic digesters) ecosystems. The primary end-products of anaerobic decomposition are methane (CH4) and carbon dioxide (CO2). Upon landfilling, rapidly degradable materials within the refuse anaerobically decompose resulting in an accumulation of volatile fatty acids (VFAs) and a commensurate drop in pH to a minimum ranging between 5.5 and 6. These low pH, high carboxylic acid conditions have been shown as inhibitory to methanogenic Archaea in analogous ecosystems such as peat and the rumen. In contrast to these findings, methanogenesis initiation occurs under these conditions, indicating the mechanism by which methane production begins in refuse is poorly understood. There are two theories for how methane production initiates in landfills. One is that methanogenic Archaea (i.e. methanogens) tolerant to the low pH, high VFA conditions consume acids until the bulk pH is suitable for the establishment of methanogens that grow under pH-neutral conditions. The second theory is that spatially isolated areas of neutral pH exist while bulk pH is acidic and these localized regions of neutral pH act as initiation centers for methanogenesis. The goal of this study was to test these two theories and validate their importance relative to methanogenesis initiation in refuse. To evaluate methanogen acid tolerance in decomposing refuse, three liquid inocula were derived: (1) refuse just entering active decomposition, (2) well-decomposed refuse and, (3) peat. Under high VFA concentrations, results showed methanogenesis initiation occurred at pH minima of 6.25, 5.75 and 5 for actively decomposing refuse, well-decomposed refuse and peat, respectively. The hydrogenotrophic Methanoculleus genus facilitated methane initiation in actively decomposing refuse (pH 6.25) while Methanosarcina triggered methane production in well-decompose refuse (pH 5.75). In peat, methanogenesis was facilitated by an uncultured Methanosarcinales. This is the first study to fully characterize methanogens responsible for methane initiation under low pH, high VFA conditions and suggests acid tolerance (pH 5 – 6.25) is relatively common provided sufficient acclimation time. However, methane production rates at lower pH were found to be 3 to 6 fold lower than those at neutral pH. To evaluate the spatial influences on methanogenesis initiation, fresh refuse was placed into triplicate laboratory scale reactors, decomposed to the anaerobic acid phase, and destructively sampled when methanogenesis initiated. Large differences were observed spatially in refuse pH, moisture content and VFA concentration. No pH neutral niches were observed in reactors prior to methanogenesis. RNA clone library results showed most bacterial activity was attributed to the Clostridiales order. Methanogenic Archaea activity at low pH was catalyzed by Methanosarcina barkeri. After methanogenesis, pH neutral conditions developed in high moisture content areas containing substantial populations of M. barkeri. These areas expanded with increasing methane production, forming a unified reaction front that advanced into low pH areas. In the absence of pH neutral niches, this study suggests methanogens tolerant to low pH, such as M. barkeri, are required to overcome the low pH, high VFA conditions typically present during the anaerobic acid phase of refuse decomposition.
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    Evaluation of Bench-Scale Sequencing Batch Reactor Swine Waste Treatment Under Continuous and Cyclic Aeration
    (2007-05-03) Bennett, Todd Alan; Sarah K. Liehr, Committee Member; John J. Classen, Committee Chair; Michael R. Hyman, Committee Member
    The objectives of this project were to develop operating conditions for a bench-scale sequencing batch reactor to match the design of a full-scale sequencing batch reactor system for treating swine waste and to determine the effects of continuous, low oxygen versus cyclic aeration schemes on sequencing batch reactor system performance. The low aeration technique was intended to develop conditions for low oxygen nitrification and simultaneous nitrification and denitrification so that a comparison could be made to a typical cyclic aeration reactor for biological nitrogen and phosphorus removal. The performance of the two reactor configurations was measured by the settling efficiency, mass removal efficiency, and accumulation of chemical oxygen demand (COD), suspended solids (SS), total Kjeldahl nitrogen (TKN), and total phosphorus (TP). The performance of the reactors did not meet expectations due to excessive loading and source inconsistency. Operational changes to the solids wasting mechanism and to the cyclic aeration system were made during the experiment in an attempt to stimulate reactor performance, which provided insight into the responses of the two types of reactors to these changes. The performance of the continuous aeration reactors met or exceeded the performance of the cyclic aeration reactors, while receiving a 73% lower supply of oxygen. The results support the potential for equipment and energy savings by utilizing low-oxygen continuous aeration for the treatment of swine waste with sequencing batch reactors.