6. Biodegradation
In many biodegradation studies in the laboratory, bacterial cells which are metabolically capable of degrading or mineralizing a pollutant are added to contaminated environmental samples to determine biodegradation
of target compound(s) in the environment. Cell counts, survival, metabolic activity, spatial location and degradative capability are all important factors that provide valuable information to advance our understanding of the processes occur ring. The use of the GFP as a marker has allowed researchers to detect the added cells, and determine more precise data about cell counts, survival and spatial location than has previously been possible. In our laboratory, the GFP marker was employed to study the survival of a p-nitrophenol
(PNP)-degrading Moraxella strain in soil. The chromosomally gfp-labelled Moraxella sp. strain G21 had been constructed using a mini-Tn5-gfp suicidal plasmid (see Fig. 2) (Tresse et al., 1998). The labeled bacteria degraded up to 1440 pM PNP, both in broth and soil, to the same extent as the parent strain. Survival studies indicated that individual green fluorescent colonies of G21 were detected up to 2 weeks after inoculation in soil samples. It is noteworthy that although only 0.1% of the original inoculum was detected by plating, 58% of the radiolabelled PNP was mineralized. This suggests that not all cells were culturable, or that there may have been an indigenous population of PNP-degrading microorganisms present in the soil. Errampalli et al. (1998) detected GFP-marked Pseudomonas sp. UGI4Gr up to 13 months after inoculation into creosote contaminated soil. By inserting the mini Tn5-gfp into the chromosome of a phenanthrenedegrading bacterium, a green fluorescent mutant, Pseudomonas sp. UGI4Gr, was obtained. The gfp was stable in UGi4Gr and did not affect mineralization of C-labelled phenanthrene which verified the gfp insertion had not interfered with phenan threne-degradation genes. Colony forming units were used to enumerate GFP-marked cells from soil samples and UG14Gr colonies on solid agar plates were detected using a hand-held UV lamp. Detection of GFP-marked cells for more than a year after inoculation into soil microcosms demonstrated the GFP marker can be useful for long-term bacterial survival in soil biodegradation or studies.