Application of Molecular Biology Techniques for Isolating and Monitoring Pollutant-Degrading Bacteria

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Abstract
The characterization of genes from bacterial organic pollutant degradation pathways has enabled the development and application of molecular techniques to study the microbial ecology of contaminated environments. The techniques include the use of catabolic gene probes, nucleic acid hybridization techniques, and use of the polymerase chain reaction (PCR) to amplify specific target regions of nucleic acid. Gene probes encoding enzymes involved in the dissimilation of chlorobiphenyls (bphC) and chlorobenzoic acids (clcA) have been used to isolate bacteria from polychlorinated biphenyl (PCB) contaminated soils. The xylE (toluene, xylene degradation), ndoB (naphthalene degradation) and alkB (C6 to C12 n-paraffin degradation) gene probes have been used to isolate indigenous bacteria from petroleum hydrocarbon-contaminated soils and to enrich bacteria expressing these genotypes for subsequent bioaugmentation of the soils. A reporter gene system has been developed to study the fate of a 2,4-dichlorophenoxyacetate-degrading pseudomonad. The genes for lactose utilization (lacYZ) and for bioluminescence (luxAB) were integrated into the chromosome, providing genetically stable, non-transferable markers. This strain can be unequivocally differentiated from indigenous bacteria and is detectable to 10 viable cells per gram of soil.

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