The subject of this proposal is the biological removal of chlorine groups from halogenated compounds that are of public health concern. Through complete dechlorination of chlorinated aliphatic compounds, the toxicity of these compounds is eliminated. The work proposed here involves investigation of a dechlorination reaction identified in anaerobic bacteria from an aquifer contaminated with halogenated compounds. The isolation and characterization of the apparently novel dechlorinating bacterium is proposed, as well as the purification of the catalyst that is responsible for chlorine removal from dechlorinating bacterium is proposed for chlorine removal from trichlorofluoromethane (CFC-11). The hypothesized dechlorinating bacterium is present in a co-culture of two other aquifer bacteria and has resisted traditional isolation attempts because of its inability to grow on agar. We propose to isolate this bacterium by using a robotics microscope in which bacterial cells can be physically isolated through the use of laser tweezers. With the bacterium in pure culture, we propose to purify the dechlorination catalyst using medium pressure liquid chromatography. The hypothesis that the dechlorination catalyst is one of the enzymes of a novel sulfate reduction pathway will guide the purification steps. The goal of this part of the work is to identify and characterize a novel bacterium with a unique ability to simultaneously use two electron acceptors, one physiological, the other a synthetic contaminant (CFC-11). It is also proposed to do dechlorination work at the microbial community level using samples from an aquifer and a wastewater digester. We have documented dechlorination activity in both these samples to propose to investigate the role which the dechlorination reaction plays within the microbial community present in these anaerobic samples. Short-term batch incubations and long-term column flow-through systems will be used to test the effect of pollutant exposure. Metabolic activities will be assayed by liquid and gas analyses for pollutant and metabolites, and pollutant effects on specific populations will be assayed using a combination of Most-Probable-Number techniques with molecular analyses. One molecular analysis will use 16S rRNA probes specific for methanogenic and sulfate-reducer populations. Another method will target these groups as well as other, less defined populations through the use of PCR-amplification of bacterial and archaeal conserved 16S targets, followed by denaturing gradient gel electrophoresis (DDGE) analysis of amplified populations. To resolve some of the microbial diversity present in these types of types of samples, extracted DNA will be fractionated using bis-benzamide gradients before PCR amplification. The goal of this environmental level work is to understand the role which dechlorination plays within the environment that is exposed to halogenated pollutants.
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