Biodegradation Of An Endocrine-disrupting Chemical Di-n-butyl Phthalate By Immobilized Camelimonas Sp.and Enzymatic Properties Of Its Hydrolase

Phthalic acid esters(PAEs)are global toxic organic pollutants,belonging to typical persistent organic pollutants(POPs),they are endocrine-disrupting chemicals.Phthalic acid esters(PAEs),a class of refractory organic compounds which is widely used in the industries,have received extensive environmental concerns in recent years due to their release into the environment and their toxicity to human beings and other organisms.The metabolic breakdown of PAEs by microorganisms is considered to be one of the major routes of environmental degradation for this widespread pollutant since the rate of hydrolysis and photolysis is relatively low.In this study,Di-n-butyl phthalate(DBP)was selected as a representative PAEs.Firstly,a high-efficient microorganism capable of degrading DBP was isolated and identified.Then the characteristics and mechanism of DBP biodegradation was investigated,the properties of DBP hydrolase was explored from the angle of molecular biology.Finally,the microbial immobilization method using sodium alginate(SA)as carrier was optimized.This study focused on the isolation of high efficiency DBP-degrading microorganisms to provide the theoretic and technical basis for bioremediating DBP-containing wastewater and soil.The results have an important theoretic and practical significance for the removal of recalcitrant pollutants from wastewater and soilThe main conclusions obtained from this study are as follows:1.An efficient DBP-degrading bacteria M11 was isolated from crude PAEs-contaminated soils.It was identified as Camelimonas sp.,according to its morphological,physiological biochemical characteristics sequences analysis of 16SrDNA.2.M11 showed a wide range degradation capacity of various dialkyl PAEs.The degradation of DBP was best fitted by first-order kinetic equation at the optimal degradation conditions.The results of high Performance liquid chromatography-mass spectrometry(HPLC-MS)analysis indicated the pathway of DBP degradation was as follows:DBP was hydrolyzed to mono-butyl phthalate,but not to phthalic acid.3.In this paper,the plasmid vector was selected to construct DBP-degradation strains M11 genomic library,through screening function degradation related genes to obtain M11 hydrolase gene,which was 1032 bp.4.Optimized IPTG inducing conditions,a high-efficiency DBP hydrolase was obtained,and that DBP hydrolase also has a wide range degradation of PAEs,the substrate specificity of the enzyme was tested with various dialkyl PAEs and monoalkyl PAEs as the substrates.The recombinant DBP hydrolase was thermostable at temperature below 50℃,and retained high residual activity in the pH range from 5.0 to 8.0.The behavior of the enzyme toward various inhibitors was also examined under optimum conditions.5.The microbial cells of Camelimonas sp.were entrapped by SA carrier,through optimized the immobilization conditions,we obtained high-mechanical strength and biodegradation immobilized cells.The results indicated that after immobilization,the immobilized cells showed wide range of pH,temperature and various chemical reagents in comparison with free cells,suggesting the advantages of immobilized microbial cells.The DBP degradation process by immobilized Camelimonas sp.could be also described by the first-order kinetic equation.The different methods for storage of the immobilized cells were studied,then we obtained a suitable storage method.The relative biological activity of the immobilized beads was obtained 70% after 30 days application.The physical characteristics and microbial activity of beads changed with time,and stabilized finally.