Rhizoremediation of trichloroethylene by a recombinant, root-colonizing Pseudomonas fluorescens strain expressing toluene ortho-monooxygenase constitutively

Trichloroethylene (TCE) was removed from soils by using a wheat rhizosphere established by coating seeds with a recombinant, TCE-degrading Pseudomonas fluorescens strain that expresses the tomA(+) (toluene o-monooxygenase) genes from Burkholderia cepacia PR123, (TOM23C). A transposon integration vector was used to insert tomA(+) into the chromosome of P. fluorescens 2-79, producing a stable strain that expressed constitutively the monooxygenase at a level of 1.1 nmol/min.mg of protein (initial TCE concentration, 10 micromolar, assuming that all of the TCE was in the liquid) for more than 280 cell generations (36 days). We also constructed a salicylate-inducible P. fluorescens strain that degraded TCE at an initial rate of 2.6 nmol/min.mg of protein in the presence of 10 micromolar TCE [cf. B. cepacia G4 PR123 (TOM23C), which degraded TCE at an initial rate of 2.5 nmol/min.mg of protein]. A constitutive strain P. fluorescens 2-79TOM, grew (maximum specific growth rate, 0.78 h-1) and colonized wheat (3 X 10(6)) CFU/cm of root as well as wild-type P. fluorescens 2-79 (maximum specific growth rate, 0.77 h-1; level of colonization, 4 X 10(6) CFU/cm of root). Rhizoremediation of TCE was demonstrated by using microcosms containing the constitutive monooxygenase expressing microorganism, soil, and wheat. These closed microcosms degraded an average of 63% of the initial TCE in 4 days (20.6 nmol of TCE/day.plant), compared to the 9% of the initial TCE removed bv negative controls consisting of microcosms containing wild-type P. fluorescens 2-79 inoculated wheat, uninoculated wheat, or sterile soil