| Chlorpyrifos as a broad spectrum organophosphorus insecticide is widely used in world due to its high effectiveness, low toxicity to non-target organisms and easy biodegradability and caused great pollution in the environment. The use of chlorpyrifos-degrading bacteria for bioremediation of chlorpyrifos-contaminated sites has been proved to be the most potential clean-up method. In our earlier experiments, a bacterial strain capable of degrading chlorpyrifos, Bacillus laterosporus DSP, was isolated from the soil for the biodegradation and/or detoxification of chlorpyrifos, and the pDOC plasmid in the B. laterosporus DSP strain was found to be responsible for the degradation of chlorpyrifos. In the present study, the development of chlorpyrifos degradation capacity in soil into which a plasmid had been introduced was monitored using the degradation rate and the MPN of the chlorpyrifos degraders as the endpoints of the transfer. The transfer event was monitored using a reporter gene (gfp) combined with microscopic observations and the imaging of GFP expressed in the recipients. The main goal of this study was to develop a plasmid-mediated bioaugmentation method that could result in the persistent capacity for the degradation of chlorpyrifos in the soil. The results were summarized as follows:(1) The insertion of gfp into the plasmid pDOC 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 Bacillus laterosporus DSP. The B. laterosporus DSP strain (pDOC-gfp) was selected based on its resistance to antibiotics, its ability to utilize chlorpyrifos as sole carbon and energy source, and the observation of GFP expression by microscopy.(2) The soil used in this study was collected from Hangzhou, Zhejiang province, China, and contained no detectable amount of residual chlorpyrifos. To explore the development of the chlorpyrifos degradation capacity in the soil by plasmid transfer, the gfp-tagged plasmid was applied to study the bioremediation in soil. Four of GFP-positive strains, ZQ1, ZQ2, ZQ3, and ZQ4, were isolated from the pDOC-gfp-treated soil30days after treatment, which were capable to chlorpyrifos degradation. The isolates were identified as different species due to their morphological characteristics, physiological and biochemical characteristics and16S rDNA sequences. Moreover, by comparing the relative mobilities of the plasmids, all were found to be identical to that in the initial strain, Bacillus laterosporus DSP, suggesting that new chlorpyrifos degraders were formed by the transfer of pDOC.(3) To investigate the persistence of bioremediation in chlorpyrifos-polluted soil by plasmid donor strains B. latersporus DSP (pDOC-gfp) and its impact on soil microbial functional diversity, the BIOLOG technique and soil respiration were used to understand microbial community structure and function. The results indicated that the degradation efficiency of chlorpyrifos in soil was always greatly enhanced by inoculation with strain B. latersporus DSP (pDOC-gfp) after repeated application. Soil microbial functional activity and its activity in inoculated soil was not affected and damaged.(4) By the infiltration test of soil column, the dispersal of plasmid donor strain B. latersporus DSP (pDOC-gfp) in unsaturated and saturated chlorpyrifos contaminated soil was studied. It has been found that chlorpyrifos degraders were throughout the entire length of the column under unsaturated or saturated flow conditions in chlorpyrifos contaminated soil. The enhanced the degradation of chlorpyrifos in soil column may due to the transport of donor strain and its degradative plasmid transfer.(5) The effect of the plasmid donor, temperature, moisture, and soil type on plasmid transfer was studied. The results indicated that the degradation efficiency of chlorpyrifos in soil was greatly enhanced by inoculation with donor strains. The degradation of chlorpyrifos in the E. coli JM109(pDOC-gfp)-treated soils appeared to be more rapid than the degradation in other donor strain inoculated soils. Therefore, the E. coli JM109(pDOC-gfp) inoculated appeared to be more suitable for bioaugmentation. Strain E. coli JM109(pDOC-gfp) was used to study the effect of various factors, such as temperatures, humidity and soil types, which effect the bioremediation of chlorpyrifos in soil. The results showed that the chlorpyrifos degradation efficiency under different conditions were as follows:10mg kg-1≈1mg kg-1>50mg kg-1>100mg kg-1,30℃>40℃>20℃,60%WHC (water-holding capacity)>40%WHC>80%WHC, and HZ soil (Hangzhou soil)> JX soil (Jiaxing soil)> XS soil (Xiaoshan soil)> JH soil (Jinhua soil). |
