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 1 (toluene o-monooxygenase) genes from Burkholderia cepacia PR123(TOM23C). A transposon integration vector was used to insert tomA 1 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 z mg of protein (initial TCE concentration, 10 mM, 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 z mg of protein in the presence of 10 mM TCE [cf. B. cepacia G4 PR123(TOM23C), which degraded TCE at an initial rate of 2.5 nmol/min z mg of protein]. A constitutive strain, P. fluorescens 2-79TOM, grew (maximum specific growth rate, 0.78 h 21 ) and colonized wheat (3 3 10 6 CFU/cm of root) as well as wild-type P. fluorescens 2-79 (maximum specific growth rate, 0.77 h 21 ; level of colonization, 4 3 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 z 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. The term rhizosphere refers to the environment influenced by plant roots in which elevated bacterial activity is observed (13). Rhizoremediation (the degradation of recalcitrant pollutants by bacteria in the rhizosphere) is an attractive process since plant roots provide a large surface area for a significant population of bacteria and transport the root-colonizing, remediating microorganism to pollutants 10 to 15 m deep in the soil (27, 45). The roots supply nutrients (amino acids, carbohydrates, and organic acids) (1, 3, 29) so no exogenous carbon source must be added, and they may also supply bacteria with cofactors required for the activation of bacterial enzymes involved in the pollutant degradation pathway. Root-colonizing bacteria genetically engineered to degrade pollutants should also retain their competitive advantage in the rhizosphere compared with indigenous non-root-colonizing bacteria. In addition, horizontal transfer can be reduced by incorporating the biodegradation genes into the bacterial chromosome (6); once an area is remediated, harvesting the plants should remove the niche for the specific bacteria. Consequently, rhizoremediation appears to be an aesthetically pleasing, low-cost, minimalmaintenance, in situ treatment for pollutants in surface soils. Rhizoremediation has shown promise based on the use of wild-type bacteria in their native environments to degrade a variety of pollutants. A consortium isolated from the rhizosphere of wheat utilized the herbicide mecoprop as a sole carbon and energy source (28), and bacteria from rhizosphere soils amended with succinic acid and formic acid produced 5 to 10 times more 14 CO2 from mineralization of radiolabelled pyrene than nonsterile control soils produced (44). Soils planted with rice degraded four times more parathion to CO2 than unplanted soils degraded (41). Up to three times more trichloroethylene (TCE) was mineralized to CO2 in rhizosphere soil and plant samples from TCE-contaminated waste sites than in samples from nonvegetated soils (2).