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Appl Environ Microbiol, January 1998, p. 112-118, Vol. 64, No. 1
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Rhizoremediation of Trichloroethylene by a Recombinant, Root-Colonizing Pseudomonas fluorescens Strain Expressing Toluene ortho-Monooxygenase Constitutively

Dennis C. Yee, Jennifer A. Maynard,dagger and Thomas K. Wood*

Department of Chemical and Biochemical Engineering, University of California, Irvine, Irvine, California 92697-2575

Received 11 July 1997/Accepted 2 October 1997

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 µM, 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 µM 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 × 106 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 × 106 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 by negative controls consisting of microcosms containing wild-type P. fluorescens 2-79-inoculated wheat, uninoculated wheat, or sterile soil.

* Corresponding author. Mailing address: Department of Chemical and Biochemical Engineering, University of California, Irvine, Irvine, CA 92697-2575. Phone: (714) 824-3147. Fax: (714) 824-2541. E-mail: tkwood{at}uci.edu.

dagger Present address: Department of Chemical Engineering, University of Texas at Austin, Austin, TX 78712-1062.




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