Development and application of methods for bioremediation site assessment and monitoring involving toluene oxygenase pathways

In order for bioremediation or natural attenuation to be used to clean up a contaminated site it is necessary to demonstrate that the contaminant-degrading microbes are present at the site and that conditions at the site are appropriate for the degradation to occur. It is also necessary to determine if the degradation is actually occurring. A variety of methods exist to address these questions. Determining if degradation is occurring at a site is usually the most difficult task in monitoring bioremediation, partly because of the lack of good methods to establish this. The use of enzyme activity-dependent probes and stable isotopic analysis are two new techniques that show great promise for filling this gap in site assessment and monitoring methods. This work describes the development of these two techniques for use with toluene oxygenase pathways which are capable of aerobically degrading simple aromatic compounds and cometabolically degrading chlorinated ethenes, which are common groundwater contaminants. These techniques are validated in laboratory studies with known bacterial cultures. They are then applied, along with a variety of other techniques, to assess a hydrocarbon- and chlorinated ethene-contaminated site for the potential of using bioremediation to clean up the site. The new techniques provided information that was complementary to the other techniques used. All of the data together allowed for a recommendation that monitored natural attenuation be considered as a remediation option for the site.