| The purpose of this thesis is to assess and determine the application of molecular tools in hazardous waste bioremediation. If useful, some these applications may result in more predictability for hazardous waste management in the environmental engineering field. Isolation of DNA from contaminated soil may become a useful tool to study function of microbial systems. Different methods (Ogram et al., 1987; Holben et al., 1988; Tsai et al.,1991) for the extraction and purification of DNA from contaminated soil were evaluated: a Mechanical/Hydroxyapatite method with and without PVPP(polyvinylpolypyrrolidone) and a Enzymatic/Elutip-d method for direct DNA extraction from soil. Some researchers (Holben et al., 1988) indicate humic compounds can be removed by using PVPP, thus the effect of PVPP with the M/H method was tested. Improvement in the purity of DNA was observed. Subsequent studies were done with the E/E method (Tsai et al., 1991).; During DNA isolation, it was found that the incubation time with lysozyme and SDS (sodium dodecyl sulfate) is critical with regard to cell cleavage. It is necessary to standardize this procedure if it is used to be for cell lysis in determining the structure and function of microbial communities in contaminated soils by DNA methods. This study produces clear evidence that cells in soil can be completely broken down with lysozyme and SDS treatment. It was found that the published 2 or 3 hr treatment was inadequate for cell cleavage in these soil systems. Studies have been made to correlate DNA yield with these treatments. In order to extend the study of the effectiveness of DNA extraction from contaminated soils, extracted DNA from these systems was analyzed by gel electrophoresis to give evidence of the quality of the extracted DNA.; These DNA studies were correlated with the biodegradability of BTEX in contaminated soils in an attempt to relate structure and function. Biodegradation rate coefficients were measured to correlate structure and function. In this system, soil exposed to BTEX showed a smaller quantity of cells and better biodegradation rates of BTEX than unexposed soils. |
