Bioaugmentation For Dioxins Polluted Soil With Catabolic Plasmids In Rhodococcus Sp.strain P52

Dioxin refers to polychlorinated dibenzo-p-dioxin/polychlorinated dibenzofuran,(PCDD/DFs),which are stable in chemical properties,difficult to volatilize,easy to be adsorbed on the surface of soil and particles,and highly carcinogenic and teratogenic.The health effects on human body include endocrine interference,autism,feminization and tumor induction.The main sources of dioxins in the environment include the production of pesticides containing chlorophenol,the papermaking process using chlorine bleached wood pulp,the combustion of urban waste and other non-human sources such as compost and forest fire.Compared with the traditional physical and chemical treatment of dioxin contaminated soil,microbial degradation method has good environmental adaptability,economic adaptability and low environmental burden,and the application of biological strengthening combined with bioslurry reactor in soil remediation has certain advantages.In this study,Rhodococcus sp.strain p52,which was isolated from oil contaminated soil by our research group,was found to have two mobile genetic elements---plasmid pDF01(170kb)and pDF02(242kb).There is a gene cluster dfdA1A2A3A4 and dbfA1A2 on pDF01 and pDF02,respectively.They can participate in the initial dihydroxylation of dioxins,mineralization of dioxin carbon chain and degradation of dioxins.Rhodococcus sp.strain p52 was inoculated as donor to the slurry mixed with soil contaminated by dionxins and water in bioslurry reactor for bioaugmentation,and to explore the effect of bioaugmentation on the removal of soil contaminated by dionxins.Screen types and species of transconjugant to confirm whether two catabolic plasmid pDF01 and pDF02 in Rhodococcus sp.strain p52 were able to transfer to the receptors by horizontal gene transfer,and to explore the effect of selection pressure on conjugative transfer;in the experiment of removing dioxin contamination in soil,by the transconjugants carrying degradable plasmids as donor inoculated to reactor,the continuous conjugative transferability of plasmids was verified from two aspects:screening of the 2nd/3rd generation transconjugants and quantitative determination of the ratio of catabolic plasmids to donor bacteria by qPCR.The results showed that the degradation efficiency of DD and DF could be effectively improved by inoculating Rhodococcus sp.p52.The removal efficiency of DD and DF in the experimental group was significantly improved within 5 days compared with the blank group,and the selective pressure promoted horizontal gene transfer.The plasmids pDF01 and pDF02 were able to conjugative transfer between intergenerics,and transfer to Arthrobacter sp.,Pseudomonas putida,Microbacterium sp.,Stenotrophlomonas maltophilia and other receptor bacteria;The transconjugants were able to effectively degrade DF with a concentration of 500 mg/L in the flask degradation experiment,and it was also able to degrade DF in the bioslurry reactor degradation experiment with a slightly lower removal efficiency than Rhodococcus sp.strain.P52,indicating the successful expression of degrading genes in plasmids in transconjugants;the 2nd/3rd generation transconjugants were screened,and next generation transconjugant with degrading plasmids pDF01 or pDF02 were found,including Enterobacteriaceae bacterium X6/SB50,Actinobacteria bacterium and Brachybacterium sp..The ratio of plasmid pDF01 to the donor transconjugant was about 1.16±0.8.It was confirmed that the catabolic plasmids in the primary and the second generation of transconjugant were able to conjugative transfer.The above results show that Rhodococcus sp.strain p52 can not only enhance the removal of dioxin pollution in soil,but also transfer the ability of dioxin degradation to the native microflora,indicating its great potential in the remediation of soil contaminated by dioxins.