Isolation Of Di-(2-ethyl Hexyl) Phthalate Degrading Bacteria And Research On Its Degradation Mechanism

PAEs(Phthalate acid esters)are a family of refractory organic compounds,and It have endocrine disrupting characteristics.In this paper,di-(2-ehtylehxyl)phthalate(DEHP)was selected as the most broadly typical PAEs.Firstly,a high-efficient microorganism capable of degrading DEHP was isolated and identified,then the characteristics and mechanism of DEHP biodegradation was investigated.The complete genetic information of degrading bacteria was obtained through whole-genome sequencing,and the related degradation genes were obtained by comparative genomics and bioinformatics analysis.The functional verification of the related degradation enzymes was applied by molecular biological methods,as well as the analyzation of the metabolic pathways,which elucidated the metabolic mechanism and metabolic pluripotency.The main conclusion gained from this paper are as follows:(1)A high-efficient DEHP-degrading strain was isolated from marine plastic debris found in coastal saline seawater and identified as Rhodococcus ruber,which could complete degrade 100 mg/L DEHP within 72 hours.Moreover,strain YC-YT1 has the most substrate spectrum among the reports.It could degrade up to thirteen kinds of PAEs and some kinds of aromatic compounds.Furthermore,strain YC-YT1 could metabolize initial concentration of DEHP from 0.5-1000 mg/L.Especially,it could degrade 0.5 mg/L DEHP up to 60%.(2)Different environmental factors were studied by single factor analysis to measure the degradation efficiency of DEHP.The results discovered that YC-YT1 had great tolerance on the temperature(10-50?),pH(4-10)and salinity(0-120 g/L).The inoculum size of YC-YT1 did not affect the degradation significantly,however,mixed with the appropriate amount of glucose could effectually enhance the degradation ability of YC-YT1(?5.0 g/L).The results of the response surface methodology(RSM)showed that the optimum degradation conditions for YC-YT1 were pH 7.5,33? and the addition of 5.0 g/L glucose.(3)According to the HPLC-MS analysis,it is deduced that DEHP was transformed by strain YC-YT1 into phthalate(PA)via mono-(2-ethylehxyl)phthalate(MEHP)and then the mediate PA was used for cell growth.It is revealed that excellent environmental adaptability of strain YC-YT1 is contributed to adjusting its cell surface hydrophobicity(CSH)and it remedied 79.7%-95.9% DEHP-contaminated soils and water.These results demonstrated that R.ruber YC-YT1 has vast potential to bioremediate various DEHP-contaminated environments,especially for the saline environments.(4)The third-generation(Pac Bo sequencing platform)was used to sequencing the whole genome i sequence of the strain YC-YT1,and the sequence data of YC-YT1(CP023712)was submitted to Gen Bank.There is a circular genome(5.66 Mb,GC 70.41%)and two plasmids(plasmid01,159 Kb,GC 67.2%;plasmid02,77 Kb,GC 64.92%)in the strain YC-YT1.In the genome,there are 5,525 coding sequence(CDSs)of which 5,375 CDSs(97%)were predicted and annotated to the public databases.The genes and gene clusters involved in the degradation of phthalate acid esters and aromatic compounds,especially the phthalate dioxygenase genes and ring-cleavage dioxygenase genes,which involved the degradation of diphenyl,protocatechuic acid,benzoate and phthalate were analyzed by comparative genomics analysis.Two new esterase genes,Dehp1199 and Mehp4077,which involving the degradation of DEHP were predicted.The biological functional and enzymatic characteristics of Dehp1199 were analyzed.It found that Dehp1199 has a wider pH and temperature range,better stability tolerance,which provides important materials for the development and transformation of DEHP degradation enzymes.