DDT Degradation Plasmid PDOD And Its Use In Bioaugmentation Of Residual DDTs Degradation In Soil

DDT, a typical persistent organic pollutant as an efficient organochlorine pesticide (OCPs), was widely used for control of agricultural pest and public hygiene in China once. Due to its stabilized chemical structure, DDT could be detected in many areas in China though it has been banned since 1983.The residues of DDTs and their origin in a certain place in Zhejiang were investigated. Genetic studies based on plasmid curing and heat treatment transformation were performed on a DDT degrading bacterium Sphingobacterium sp. DDT-6, which indicated that the plasmid pDOD isolated from strain Sphingobacterium sp. DDT-6 was responsible for degradation. To monitor the survival of degraders and plasmid transfer (emergence of transconjugants), the strain was chromosomally marked with the gfp gene. We applied the gfp-tagged plasmid and its donors to study the bioremediation in soil, setting up a novel method for bioremediation. The results were summarized as follows:Sixty-one soil samples were collected from a certain place in Zhejiang, and∑DDTs was detected. The percentages of p,p'-DDT, p,p'-DDE, o,p'-DDT, p,p'-DDD in∑DTs were 56.1%,34.4%,6.88% and 2.41%, respectively. The ratio of o,p'-DDT to p,p'-DDT (Rs) was used to identify the old or new DDT sources in the environment, the results suggested that the residue of DDT was predominant old source of DDT in regions.When strain Sphingobacterium sp. DDT-6 was treated with acridine orange, it became unable to utilize DDT metabolically. Further, heat treatment transformation was carried out to transform the plasmid pDOD to Sphingobacterium sp. DDT-6' competent recipient cells, and the curing strain Sphingobacterium sp. DDT-6' were determined for its utilization capacity of DDT. Moreover, gel electrophoresis clearly showed that plasmid could not be extracted from the acridine orange treated bacterial strain Sphingobacterium sp. DDT-6', while was visible in the uncured cells. The molecular weight determined using standard ladder indicated the plasmid to be about 20kbp, lying approximately between 9416bp and 23130bp. These results clearly indicated that the plasmid pDOD of strain Sphingobacterium sp. DDT-6 is responsible for the degradation of DDT.The insertion of gfp into the plasmid pDOD was performed by triparental mating in which the helper strain E. coli HB101 (pRK2013) was used to mobilize gfp segment from the donor strain E. coli DH5a (pZP201-gfp) into the recipient Sphingobacterium sp. DDT-6. Isolated native bacteria Klebsiella sp. TZ and E. coli TG I were transformed with gfp-tagged plasmid as-tagged plasmid donors. The gfp-tagged plasmid and its donors were used to bioremediation in soil. The results showed the successful in bioremediation possibly depend on DDT degraders as well as transfer of the plasmids from inoculants to the indigenous bacteria in soil. It also showed positive correlation between the enhanced DDT degraders and accelerated biodegradation. Strain Cellulomonas sp. ZYG2 having DDT degrading ability and fluorescence was isolated from bioremediation of DDT contaminated soil with pDOD, which were also identified to have gfp-tagged degradative plasmid. It further indicated that the degradative plasmid may transfer to the indigenous bacteria enhanced the sizes of DDT-degrading bacteria in soil, and resulted in the successful of bioremediation.Strain E. coli TG I (pDOD-gfp) was used to study the effect of various factors, such as temperatures, humidity and soil types, which effected the bioremediation of DDT in soil. In general, an optimal temperature for bioremediation was 30℃. It revealed that soil moisture had no significant effect on the bioremediation. There was little difference in rate of degradation among Huajiachi, Jinhua, Jiaxing and Xiaoshan soil. However, it was observed that biodegradation was slower in Jinhua soil because of acid pH. These results indicated that bioremediation of DDT was feasible in various environmental conditions, and the success of bioremediation could be attributed to no striking influence of environmental factors on plasmid transfer.Strains Sphingobacterium sp. DDT-6, Sphingobacterium sp. DDT-6 (pDOD-gfp) and E. coli TG I (pDOD-gfp) as plasmid donors were used for bioremediation of DDT in a certain place of Zhejiang. The results showed that after 210d of incubation, 46.5%,52.0% and 50.7% of the initial dose DDT were degraded by strain Sphingobacterium sp. DDT-6, Sphingobacterium sp. DDT-6 (pDOD-gfp) and E. coli TG I (pDOD-gfp), respectively. In control, the hydrolysis percentage of DDT was about 16.2%. Simultaneously, TGGE and partial sequence analysis of PCR-amplified 16S rDNA genes were applied to determine the variation of bacterial community structure. The results showed that bacterial community structure was not significantly changed by the inoculation with donors, but was altered a little after 180d-210d of incubation with Sphingobacterium sp. DDT-6 (pDOD-gfp). It indicated an increased of microorganisms for growth and survival in the soil environment.