This Phase II SBIR project involves the development and testing of biosensors to measure trichloroethylene (TCE) in groundwater. During Phase I, TCE reporter strains were constructed by fusing a TCE-inducible transcriptional promoter from Pseudomonas mendocina KRl to a bioluminescence gene cassette. The reporter strains produced light in the presence of TCE, and the amount of light produced was proportional to the amount of TCE present. During Phase II we will determine and validate the performance characteristics of the biosensors by measuring TCE in a wide variety of TCE-contaminated groundwaters. We also will develop methods for applying the reporter strains in TCE test kits, including the use of freeze-dried cells. Furthermore, we will use the reporter strains to develop on-line biosensor probes that can be used for continuous monitoring of TCE in bioreactors and/or down hole monitoring of TCE in groundwater monitoring wells. In addition to TCE biosensors, new biosensors will be constructed to measure gasoline (BTEX) contamination in groundwater. The BTEX sensors will also be used as QA/QC check standards to evaluate the accuracy of TCE measurements made with the TCE biosensors. A new series of biosensors for both TCE and BTEX will be constructed by replacing the bioluminescence reporter cassettes with genes encoding the production of green fluorescent protein, thereby allowing microscopic examination of reporter stains used for modeling the migration and performance of degradative organisms in aquifers. The biosensors and biosensor probes will be used commercially to measure TCE in groundwater, improve process control in bioreactors, and reduce the cost of in situ remediation technologies.
