Polychlorinated biphenyl (PCB) degradation and persistence of a gfp-marked Ralstonia eutropha H850 in...

Ralstonia eutropha H850 was labelled chromosomally with a gfp marker gene encoding for the green fluorescent protein, and designated R. eutropha H850g13. Visual observation of green fluorescent cells under an epifluorescence microscope, and PCR amplification products, confirmed that the bacterium was labelled with gfp. Southern blot hybridization products further confirmed the gfp was chromosomally labelled. Using resting cell assays, it was determined that insertion of the gfp gene decreased the microorganisms' ability to degrade biphenyl compared to the parent strain. However, this marker facilitated the identification and monitoring of R. eutropha H850g13 survival in soil microcosm experiments. Survival and polychlorinated biphenyl degradation by R. eutropha H850g13 was analysed in soil microcosms spiked with 2,2',5,5'-tetrachlorobiphenyl (TeCB). R. eutropha H850g13 was detected by viable plate counts and most-probable-number/PCR after 102 days in TeCB-contaminated soil microcosms, and was likely outcompeted by indigenous soil microorganisms in microcosms amended with oil and Daramend (an organic amendment, http://www.adventusremediation.com). R. eutropha H850g13 did not degrade TeCB in any of the soil microcosms. This research confirmed that gfp was useful as a marker to distinguish R. eutropha H850g13 from indigenous soil microorganisms over a 102 day period and that, under the experimental conditions used, R. eutropha H850g13 did not degrade TeCB.