Degradation of trichloroethylene by a growth-arrestedPseudomonas putida

Abstract

A toluene-oxidizing strain ofPseudomonas mendocina KR1 containing toluene-4-mono-oxygenase (TMO) completely degrades TCE with the addition of toluene as a co-substrate in aerobic condition. In order to constructin situ bioremediation system for TCE degradation without any growth-stimulating nutrients or toxic inducers such as toluene, we used the carbon-starvation promoter ofPseudomonas putida MK1 (Kim, Y.et al., J. bacteriol., 1995). Upon entry into the stationary phase due to the deprivation of nutrients, this promoter is strongly induced without further cell growth. The TMO gene cluster (4.5 kb) was spliced downstream of the carbon starvation promoter ofPseudomona putida MK1, already cloned in pUC19. TMO under the carbon starvation promoter was not expressed inE. coli cells either in stationary phase or exponential phase. For TMO expression inPseudomonas strains,tmo and carbon starvation promoter region were recloned into a modified broad-host range vector pMMB67HES which was made from pMMB67HE (8.9 kb) by deletion oftac promoter andlacI ^q (about 1.5 kb). Indigo was produced by TMO under the carbon starvation promoter in aPseudomonas strain of post-exponential phase on M9 (0.2% glucose and 1mM indole) or LB. 18% of TCE was degraded in 14 hours after entering the stationary phase at the initial concentration of 6.6μ M in liquid phase.