| Chlorobenzene（CB）is one of the most widely used products of the chlorinated aromatic group and has become a persistent organic pollutant prevalent in industrial wastewaters such as pharmaceutical,dyeing and organic synthesis.Chlorobenzene is highly toxic,difficult to degrade and easy to bioaccumulate,which seriously endangers human health and ecological safety.Bioremediation is one of the most effective means of CB removal from the environment.Currently,only three dozen strains of CB-degrading bacteria have been isolated,and the low environmental tolerance makes it difficult to be widely used in engineering.Based on this,this study screened CB degrading strains with high tolerance from contaminated soil by means of enrichment,domestication and purification;Conducting studies on their degradation characteristics and clarifying the metabolic properties of the strains;Systematic resolution of the gene sequence potential of degradative bacteria and construction of CB metabolic pathways based on whole genome sequencing technology;Finally,the strain was used as a bioagent to simulate in situ remediation of CB simulated contaminated soil by means of biostimulation and biofortification.The research results are intended to provide a theoretical and practical basis for in situ bioremediation of chlorinated aromatic hydrocarbons.The main conclusions are as follows:（1）A strain capable of using CB as the sole carbon and energy source,Serratia sp TF-1,was isolated and purified.TF-1 suitable growth temperature range is20℃～35℃,the optimum temperature is 30℃,TF-1 can tolerate high acidic and alkaline environment,the suitable growth p H value is 5～9,the optimum p H value is 7.The maximum tolerated concentration of TF-1 for CB was 200 mg/L,with an average degradation rate of 0.153～0.662 mg/（L·h）.Maximum specific growth rates of0.479～0.032 h-1;The maximum specific growth rate was 4.38 h-1 and the maximum specific degradation rate was 3.99 h-1 based on the Halane equation fit.This strain is the first obtained Serratia marcescens with CB degradation.（2）To clarify the co-metabolic degradation properties of TF-1 on CB.TF-1 can co-metabolically degrade CB with sodium succinate and sodium citrate as substrates with the highest rate of chlorobenzene degradation(VCB)of 0.89 mg/（L·d）and 0.91mg/（L·d）,Theμmax of sodium citrate as substrate was greater than that of sodium succinate when the concentration of chlorobenzene(c CB)was<80 mg/L,and theμmax was up to 0.87 h-1.Sodium citrate is more favorable to promote the growth of microorganisms,VCB（sodium citrate）（8.31～13.91 mg/（L·h））>VCB（sodium succinate）（8.14～13.19mg/（L·h））when c CB>80 mg/L and sodium citrate was used as substrate.（3）Probing the degradation characteristics of CB for composite contaminated environments.The degradation rates of CB by TF-1 were 40%,35%and 30%when typical dichloromethane,dichloromethane and dichloroethane were used as inhibitors,respectively,The CB residual rates corresponding to trichloroethylene and tetrachloroethylene are 40%to 70%and 60%to 70%,The dissociation constants of chlorinated alkanes and chlorinated olefins varied from 1.470～0.206 mg/L and1.772～0.542 mg/L,indicating a stronger inhibitory effect of chlorinated alkanes.（4）TF-1 was subjected to whole genome sequencing and construction of degradation pathways for CB.The genome size of strain Serratia sp.Strain TF-1 was found to be 8830277 bp.The genome contained 87 protein-coding genes with an average gene length of 7692.12 bp and a GC content of 66.79%of the coding gene region,a total of 8,807 functional genes were predicted for functional information.3,643 metabolic maps were obtained from the KEGG database analysis.Based on the annotated information and metabolites,the CB degradation pathway was constructed as chlorobenzene→3-chlorocyclohexane-3,5-diene-1,2-diol→3-chloroethanol→2-chloro-maleic acid salt→4-oxohex-2-enoate→3-oxoadipic acid→TCA.（5）TF-1 enhanced the removal of chlorinated aromatic hydrocarbons from simulated contaminated soil.The maximum degradation rates of simulated contaminated soil were enhanced by 13.03-fold and 11.52-fold with TF-1 and sodium citrate,respectively,compared to native microbial CB.The maximum degradation rate was 0.2 mg/（m3-h）with the simultaneous addition of TF-1 and sodium citrate,which was increased by 3.9 times;meanwhile,the degradation rates of 1,2-DCB were increased by 1.21,1.03 and 1.64 times,respectively,indicating that biostimulation and bioaugmentation could achieve rapid removal of 1,2-DCB.The degradation rates of CB were increased by 11.3,5.2 and 3.7 times for TF-1 and the mixture of TF-1 and sodium succinate,respectively,and by 2.79,6.9 and 4.9 times for 1,2-DCB,respectively,when sodium succinate was added to the simulated contaminated soil.Sodium succinate was more effective as a biostimulating agent. |
