Construction Of The Artificial Microbial Consortium Degrading Phenanthrene And Remediation Of Contaminated Soil Under Optimized Degradation Conditions

Polycyclic aromatic hydrocarbons(PAHs)are typically persistent and highly toxic organic pollutants widely distributed in the natural environment.Phenanthrene is one of the tricyclic organic pollutants and is also a model compound for the study of PAHs.With the continuous improvement of human awareness of ecological environment protection,the environmental control of PAHs pollution has become a focus issue.Microbial remediation,due to its advantages of low cost,high efficiency,low consumption,no secondary pollution and thorough remediation,has become one of the main means to remove PAHs from the environment and is of great significance for the remediation of contaminated sites.In this study,firstly,on the basis of the constructed Escherichia coli BL21(DE3)as the host,the downstream pathway of phenanthrene degradation-catechol metabolism module was reconstructed,and a fully artificial degrading-phenanthrene bacterium with complete metabolic pathway was obtained.In the MSM with phenanthrene as the only carbon source,the degradation rate of 100 mg/L phenanthrene by the artificial bacteria was 61.15% after 7 days,and the degradation rate was further improved after adding the biosurfactant rhamnolipid.Then the rhl ABRI operator was cloned,and the recombinant plasmid p2-rhl ABRI containing the original promoter was constructed,and the recombinant plasmids p2-rhl AB-Tac and p2-rhl ABRI-Tac of Tac promoter were constructed.These plasmids were transformed into P.putida KT2440 to obtain the engineered strains P.putida KT-159,P.putida KT-AB,and P.putida KT-ABRI.The results showed that P.putida KT-ABRI could produce 601.7 mg/L rhamnolipid with 3% glycerol as the carbon source.E.coli M123 and P.putida KT-ABRI were selected as the main strains to form an artificial mixture system for degradation optimization.The concentration of IPTG inducer,the amount of substrate glycerol and the sequence of inoculation were optimized in the mixed bacteria system.The optimal IPTG concentration of the mixed bacterial system was 0.8m M,the optimal glycerol dosage was 1%,and the best sequence was to inoculate P.putida first and E.coli 24 h later.Under the optimal conditions,the maximum degradation rate of 100mg/L phenanthrene in 7 days was85.73%.Under the guidance of optimal degradation conditions,two concentrations of phenanthrene pollution and five remediation conditions were set for the remediation of contaminated soil(100 mg/kg,300 mg/kg).Finally,the removal efficiency of 100mg/kg and 300 mg/kg phenanthrene contaminated soil under S4 condition could reach86.52% and 80.67%,which was significantly higher than other remediation conditions.These results laid a foundation for the application of engineered bacteria in the future bioremediation of PAHs.