Characterization of microbial isolates from the Idaho National Engineering and Environmental Laboratory Test Area North aquifer: Identifying potential enzymatic pathways for toluene oxidation

We obtained 45 microbial isolates from the Test Area North (TAN) aquifer of the Idaho National Engineering and Environmental Laboratory by sterile filtration of groundwater from well 35 within the trichloroethylene (TCE) contamination plume. In addition to standard morphological and biochemical screening, isolates were analyzed for the expression of various enzyme pathways responsible for toluene oxidation. Many of the known toluene oxidation enzyme systems are capable of cometabolically degrading TCE, and preliminary enrichment experiments have demonstrated the presence of toluene-degrading organisms within this aquifer. Isolates were tested for reaction with phenylacetylene, an enzyme-activity-dependent probe that, when used as a chromogenic substrate (yields colored product), can identify toluenedegradative pathways that include toluene 2,3-dioxygenase or toluene 2-monooxygenase. Isolates were also screened for growth and pigment production on indole plates. Another indicator test was inhibition of growth on toluene during exposure to 1- pentyne, a selective growth inhibitor for bacteria that express toluene 2-monooxygenase or toluene 3-monooxygenase. Results for the TAN isolates were compared with those obtained for control organisms known to exhibit various toluene oxidation pathways. Isolates were also subjected to analysis with Biolog GN plates. Principal components analysis was used to distinguish groups of isolates and to compare them with various control species based on their ability to oxidize individual carbon sources. Results of this study suggest the presence of known toluene oxidation pathways within the aerobic microbial community at this site. Results also indicate that the 45 isolates represent fewer than 10 distinct bacterial species.