Survival of a GFP-Labeled Polychlorinated Biphenyl Degrading Psychrotolerant Pseudomonas spp. in 4 and 22°C Soil Microcosms

The green fluorescent protein gene (gfp) was inserted into the chromosome of Pseudomonas spp. Cam-1 and Sag-50G, two psychrotolerant polychlorinated biphenyl (PCB)-degrading bacteria. The gfp-transformed microorganisms, designated Cam-1-gfp1, Cam-1-gfp2, Sag-50G-gfp1, and Sag-50G-gfp2, exhibited green fluorescence under an epifluorescent microscope. The gfp was inserted into the chromosome of each psychrotolerant strain and was stable with no apparent adverse affects on the metabolism and growth of each organism. Activity of gfp-transformed microorganisms against biphenyl and 2,3-dichlorobiphenyl was determined by assaying for BphC activity and by resting cell assays. The patterns of BphC activity at two different growth temperatures in batch cultures were similar for each of the gfp-transformed microorganisms. Resting cell assays of both the parent strains (Cam-1, Sag-50G) and the gfp-transformed strains (Cam-1-gfp1, Cam-1-gfp2, Sag-50G-gfp1, Sag-50G-gfp2), grown on glycerol or glucose, exhibited BphC activity to a lesser extent and at a slower rate than those observed for biphenyl grown cells. In addition, all gfp-transformed microorganisms degraded 2,3-dichlorobiphenyl (2,3-DCB) in broth to the same extent as the parent strains. When Cam-1-gfp1 and Sag-50G-gfp1 were used as a bioremediation amendment in soil microcosms spiked with 2,3-DCB, both strains survived in high numbers (5.6 to 7.9 log cfu g?1 and 5.6 to 8.0 log cfu g?1) when inoculated into nonsterilized soil over 16 weeks at 22°C and 18 weeks at 4°C, respectively. Biodegradation of 2,3-DCB was enhanced with the microbial amendment; however, the addition of sunflower oil did not help the PCB degrading bacteria and may have enhanced the growth of the indigenous population, thereby decreasing the amended PCB-degrading population.