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Masters Degrees (Microbiology and Infection Control)

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Browsing Masters Degrees (Microbiology and Infection Control) by Author "Hunter, Charles Haig."

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    An assessment of synthetic landfill leachate attenuation in soil and the spatial and temporal implications of the leachate on bacterial community diversity.
    (2008) Govender, Kamenthren.; Hunter, Charles Haig.
    The temporal fate of selected parameters, including redox potential; pH; phenol; nitrates; sulphates; copper and zinc, of a young synthetic acetogenic phase landfill leachate was assessed by perfusing a series of sequential soil (Hutton) microcosms (arrays) at two hydraulic loading rates (HLR). We chose HLRs that were representative of areas in South Africa with typically elevated rainfall (Pietermaritzburg – HLRh) and one with relatively low rainfall (Kimberley – HLRl). Preliminary phenol, copper, and zinc adsorption investigations on gamma radiation sterilized soil and unsterilized soil revealed superior adsorption rates for each compound in the unsterilized soil. This revealed the importance of the biological component of soil in phenol, copper, and zinc attenuation in soil. The results presented in this thesis suggest that the HLR of leachate into soil arrays contributes to significant differences in the fate of the landfill leachate parameters mentioned earlier. In addition, we assessed the temporal and spatial succession of bacterial community diversity in each of the soil arrays by polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). Prior to this, we compared two soil DNA isolation techniques, the modified method of Duarte et al. (1998) (Bead Beat) and the commercial Mo-Bio UltraClean™ Soil DNA isolation kit (Kit). The DNA isolated by the Kit method was significantly superior regarding purity and absence of DNA fragmentation. However, the Bead Beat method produced a significantly higher yield per reaction before further purification with Wizard™ Clean-Up columns produced DNA extracts of similar purity at the cost of a significant reduction in DNA yield. The Kit method was chosen for future DNA isolation and PCR-DGGE based on the quality of the PCR amplicons generated from the Kit isolated DNA. PCR-DGGE was further optimized by comparing the efficiency and sensitivity of a silver stain against ethidium bromide. Silver stain generated DGGE gels with greater number of bands (species richness – S) and stronger band signal intensities. Captured DGGE fingerprints generated data that were subjected to the Shannon-Weaver Index (H’) and the associated Shannon-Weaver Evenness Index (EH) to measure the change in spatial and temporal bacterial diversity. There was a significant shift in S and H’ for both HLRs but a significant change in EH was only observed for HLRh. Furthermore, a temporal comparison of S and H’ between both HLRs revealed significant differences throughout the investigation. Canonical Correspondence Analysis (CCA) revealed spatial distribution of bacterial community diversity with depth. Effects of phenol concentration, redox potential, and pH of the effluent leachate on bacterial community diversity was tentatively assessed by three-dimensional graphical representation on PlotIT 3.2 software. Bacterial community diversity showed a decrease with elevated pH and phenol concentration along with decreasing redox potentials for both HLRs. While this study reveals the spatial and temporal dynamics of bacterial community diversity in situ, it provides important evidence with respect to: (i) the effects of rainfall / leaching rates (HLR) on spatial and temporal bacterial community succession; (ii) the importance of the biological component in natural attenuation; (iii) the ability of soil, previously unexposed to landfill leachate, to initiate natural attenuation of phenol and other leachate constituents; (iv) the capacity of PCRDGGE to fingerprint successional changes in bacterial community diversity, (v) and the potential to clone and sequence selected members of bacterial associations for future reference in environmental remediation strategies.