Nonrecombinant Genetic Modification of Aquifer Bacteria to Achieve Constitutive Degradation of Trichloroethylene.
Final rept. Jun 94-Aug 95,
UNIVERSITY OF WEST FLORIDA PENSACOLA DEPT OF BIOLOGY
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Aquifer bacteria were isolated for their capacity to predominate following nutrient enrichment field application vectors FAVs, and screened as hosts for the Tn5 containing, constitutive toluene ortho-monoxygenase Tom expressing plasmid TOM31c which causes the cooxidation of trichloroethylene TcE. Tom expression during positive selection in native aquifer sediments contaminated with TCE was determined. Three such FAVs were constructed NFG-2 TOM3ic, MFI-l TOM3ic, and MFG-2 TOM3ic. All stably maintained TOM3ic and constitutively degraded TCE. Like the original TOM31c mutant strain Burkholderia cepacia G4 PR131, MFG-2 TOM31c was unable to significantly degrade TCE in native sediments despite inoculation to high levels - 1 x 108 cellsgram, and did not apparently survive well in glucose amended material. NFG-2 TOM3ic and MFI-l TOM3ic did significantly degrade TCE in glucose and IGEPAL respectively amended aquifer sediments. Like PRl31 TOM31c, Tn5 gene probes indicated that these FAVs also remained above 106 cells per cm3 of sediment during a 20 day feeding and TCE degradation experiment. TOM31c was transferable to these selectable FAVs by non-recombinant mating techniques, and once there encoded constitutive TCE degradation in native sediments untreated save for the addition of a specific carbon and energy sources.