DEVELOPMENT OF FIELD APPLICATION VECTORS FOR BIOREMEDIATION OF SOILS CONTAMINATED WITH POLYCHLORINATED-BIPHENYLS

Field application vectors (FAVs), which are a combination of a selective substrate, a host, and a cloning vector, have been developed for the purpose of expressing foreign genes in nonsterile, competitive environments in which the gene products provide no advantage to the host. Such gene products are exemplified by the enzymes for the cometabolism of polychlorinated biphenyls (PCBs) through the biphenyl degradation pathway. Attempts to use highly competent PCB-cometabolizing strains in the environment in the absence of biphenyl have not been successful, while the addition of biphenyl is limited by its human toxicity and low water solubility. Broad-substrate-specificity PCB-degradative genes (bphABC) were cloned from a naturally occurring isolate, Pseudomonas sp. strain ENV307, into broad-host-range plasmid pRK293. The resulting PCB-degrading plasmids were transferred to the FAV host Pseudomonas paucimobilis 1IGP4, which utilizes the nontoxic, water-soluble, nonionic surfactant Igepal CO-720 as a selective growth substrate. Plasmid stability in the recombinant strains was determined in the absence of antibiotic selection. PCB-degrading activity was determined by resting cell assays. Treatment of contaminated soil (10, 100, or 1,000 ppm of Aroclor 1242) by surfactant amendment (1.0% [wt/wt]Igepal CO-720 in wet soil) and inoculation with recombinant isolates of strain 1IGP4 (approximately 4 x 10(6) cells per g of soil) resulted in degradation of many of the individual PCB congeners in the absence of biphenyl. Further improvements, including the use of non-antibiotic-resistance cloning vectors, addition of the bphD gene, and chromosomal integration of the PCB-degradative genes, may ultimately result in FAVs useful for both reactor-contained and in situ treatment of the partially dechlorinated PCBs often found in contaminated soils and sediments.