Biodegradation Of Polycyclic Aromatic Hydrocarbons Accumulated In Bioretention Soils Using Genetically Engineered Bacterium

Accumulation of the polycyclic aromatic hydrocarbons(PAHs)in bioretention soils could pose a long-term ecological risk to the environment.Therefore,it is important to establish an adaptive approach to improve the PAHs biodegradability in natural bioretention soils.In this research,the microbial community of an in situ bioretention were characterized firstly,and a predominant indigenous bacterium was isolated for further genetic operation.PAHs degrading gene was introduced into this bacterium by tri-parental mating in order to improve its PAHs degradability.Meanwhile,an active biological containment(ABC)system was constructed within this bacterium to ensure its ecological security.?PAHs(sum of the 16 EPA priority PAHs)concentrations ranged from 0.32±0.11 to 9.76±4.71 mg/kg within the in-situ bioretention cells,and high molecule weight PAHs which have more than four ring numbers(especially for pyrene)were characterized as the typical PAHs within the bioretention soils.From further analysis on the phylogenetic information(based on the genus level),it was found that bacterial sequences affiliated with Thauera,Comamonas and Pseudomonas were the three most abundant genu in bioretention soils.Accordingly,a predominant indigenous bacterium named Pseudomonas sp.GLB3(KT804567)was isolated and prepared for further genetic operation to improve its PAHs degradability.The efficient PAHs degrading gene RHD2,which was cloned from genomic DNA of Mycobacterium vanbaalenii PYR-1(CP000511),was fused with Gfp gene by overlap PCR.This fusion gene was introduced into the Pseudomonas sp.GLB3 genomic DNA by triparental mating.The recombinant strain,which could express the fusion protein of green fluorescent protein and PAH-ring hydroxylating dioxygenases,have been successfully constructed.Moreover,an ABC system,in which the lethal gene nuc was placed downstream of lac promoter and can be induced by the presence of IPTG,was introduced into the recipient bacterium to construct a GEB(Named Pseudomonas sp.GLEKB(CCTCC:13014))that degrades PAHs and commits suicide when required.Over 76.4% pyrene could be degraded by the above GEB in a 6 days shake flask experiment and the expression level of RHD gene showed significant relationship with the pyrene's degradation rate.Additionally,the GEB showed good adaptability in a simulating bioretention cell,and over 70.5% of pyrene could be degraded in a 90 days simulating bioaugmentation by inoculating this GEB.Additionally,this GEB also showed a conditional suicide effect which could induced by IPTG during the bioaugmentation process,suggesting that the new GEB has high ecological security for environmental application and could be a potential tool used for bioaugmentation of PAHs accumulated in the bioretention.