Single-well, push-pull tests were conducted in a contaminated aquifer to evaluate the ability of toluene-oxidizing microorganisms to cometabolize chlorinated aliphatic hydrocarbons (CAHs), such as trichloroethene (TCE). Test solutions were injected into the aquifer using a standard monitoring well, and then were transported under natural-gradient conditions. Transport tests demonstrated similar transport characteristics of the conservative tracer and the reactive solutes. Biostimulation tests were then performed by injecting a test solution containing dissolved toluene substrate, hydrogen peroxide, bromide, and nitrate in order to increase the biomass of toluene-utilizing microorganisms. Decreases in toluene concentration and the production of o-cresol as an intermediate oxidation product indicated the simulation of toluene-utilizing microorganisms containing an ortho-monooxygenase enzyme. Transformation tests demonstrated that indigenous microorganisms had the capability to transform the surrogate compounds (e.g. isobutene) and both cis-dichloroethene (cis-DCE) and trans-dichloroethene (trans-DCE). Isobutene was transformed to isobutene oxide, indicating transformation by a toluene ortho-monooxygenase, and both cis-DCE and trans-DCE were transformed. In a final test, the utilization of toluene, and the transformation of isobutene, cis-DCE, and trans-DCE were all inhibited in the presence of 1-butyne, a known inhibitor of the toluene ortho-monooxygenase enzyme. The method assessed the activity of attached microorganisms under in situ conditions of bioremediation.

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