The overall objective of this study is to develop non-culture based tools for evaluating the progress of in-situ bioremediation of chlorinated solvents in the field. Two specific approaches will be used to achieve these objective, the first based upon stable isotopic analyses and the second based upon application of molecular biological tools. The hypothesis that we propose to test in the first approach is as follows: stable isotope ratios of chlorine and carbon components of reactants, products and microbial cells involved in the biological degradation of chlorinated solvents can be analyzed to determine the dominant metabolic pathways and redox conditions of the biodegradation reaction. The specific goal is to broaden our understanding of the fractioning of chlorine and carbon stable isotopes during the metabolic, co-metabolic, and abiotic degradation of chlorinated solvents such as perchloroethylene (PCE), trichloroethylene (TCE), and carbon tetrachloride (CT) under aerobic and anaerobic conditions. Although a number of studies have been performed to measure the fractionation of stable carbon isotopes during the aerobic and methanogenic degradation of organics, only a few studies have been performed to evaluate carbon fractioning during the degradation of chlorinated solvents under anaerobic conditions only, and chlorine isotope fractionation has not been evaluated under any biodegradation scenario. Also, there is no published information on either carbon or chlorine isotope fractionation during abiotic degradation of chlorinated solvents. The hypothesis that we propose to test in the second approach is as follows: tools based upon molecular biology and direct microscopy and direct microscopy can be developed for application to subsurface environments in order to quantify microbial community activity and structure during in situ bioremediation. The specific goal is to adapt tools such as fluorescence in-situ hybridization (FISH) techniques for use in evaluating the relative contribution of different physiological groups of bacteria to the degradation of chlorinated solvents inc contaminated subsurface soils. FISH techniques using a series of rRNA- targeted oligonucleotides probes specific for chlorinated solvent- degrading microorganisms will be developed and applied. While several previous studies have used FISH techniques for characterizing subsurface soil community structure, they have not been used to evaluate microbial populations associated with degradations of chlorinated solvents in contaminated soils.
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