This Small Business Innovative Research Phase I project involves the development of chlorinated solvent-degrading biocatalysts that express degradative genes in response to environmental stimuli. Chlorinated solvent-degrading genes will be cloned under the control of promoters that are induced (turned on) in the presence of target chemicals, under low pH, and/or under low temperature. The biocatalysts will be applicable to both reactorbase( and in situ biotreatment processes for destroying recalcitrant chlorinated solvents. Chlorinated solvents are the most widespread and abundant contaminants of the Nation's groundwater and soils, and many are directly carcinogenic even in small amounts. Current technologies for remediating soils and/or groundwater contaminated with chlorinated solvents are limited by their high costs or the fact that they often do not destroy the contaminating chemicals. Biological oxidation of chlorinated solvents, however, results in the complete destruction of the contaminant, and degradative microorganisms can be used to destroy contaminants spread over large areas or contaminants trapped in aquifers. Although many bacteria are capable of destroying chlorinated solvents, the process is co-metabolic; i.e. the compounds do not act as growth substrates or gene inducers for the degradative organisms. Degradation is often limited by repression of enzyme synthesis in the absence of toxic inducing substrates such as toluene or phenol. Envirogen, Inc. proposes to use molecular biological techniques to develop biocatalysts that no longer require the presence of toxic inducing substrates for destroying chlorinated solvents, but rather have degradative genes under control of DNA promoters that are induced by environmental conditions (low temperature or low pH) or the presence of the target compounds themselves. Cloned toluene monooxygenase genes will be fused to existing environmentally induced promoters, and to promoters isolated during the proposed project. The resulting biocatalysts will be capable of destroying chlorinated solvents under controlled conditions in bioreactors, and also under specific in situ conditions in aquifers and/or soils.
