Isolation Of Di(2-Ethylhexyl) Phthalate Degrading Bacteria And Its Combined Remediation With Maize For Contaminated Soil

Phthalic acid esters(PAEs)are widely used as a plastic plasticizer,pesticide carrier and other industrial organic compounds.But PAEs have reproductive toxicity,neurotoxicity,etc.,and is a class of typical environmental endocrine disruptors.The widely use of PAEs in the industrial and agricultural production as well as the daily living,especially the use in plastic film mulching and plastic greenhouses,has led to PAE contamination of agricultural soil,which seriously affected soil quality and agricultural product safety.Research and development of remediation technology for soil contaminated by PAEs caught great of concern.At present,microbial remediation is regarded as the main degradation pathway of PAEs in the environment,and phytoremediation is an environment-friendly and economical technology.Combining the advantages of both microbial remediation and phytoremediation can form an effective bioremediation technique.In this study,di(2-ethylhexyl)phthalate(DEHP),a compound with high detection rate and concentration in the soil,was selected as the target contaminant,which was analyzed by gas chromatography-mass spectrometry(GC-MS)and high performance liquid chromatography time-of-flight mass spectrometry(LC-TOF-MS/MS).DEHP-degrading bacteria were isolated from the activated sludge and identified.The degradation efficiency of DEHP was determined under single factor condition,and the optimal conditions of DEHP degradation were optimized by response surface method.The degradation products of DEHP were identified and the key degrading enzyme genes of DEHP were tested by real-time fluorescence quantitative PCR to speculate comprehensively the degradation pathway of DEHP.Then,a soil potting test of maize(Zea mays)was conducted to investigate the remediation effects of maize inoculating with high-efficiency degrading bacteria on DEHP contaminated soil and effects of maize growth under DEHP stress,which would provide a scientific basis for remediation of soil contaminated by PAEs and ensure the safety of agricultural products.In the experiment of screening and identification of degrading bacteria,a mixture of six PAE compounds was used as the contaminant,and a strain capable degrading DEHP was isolated from the activated sludge by concentration gradient acclimation method.According to the morphological characteristics,physiological and biochemical characteristics of bacteria and the results of 16 S rDNA sequence homology analysis,the strain was identified as Rhodococcus pyridinivorans XB.The effects of temperature,pH and the amount of bacteria on the degradation of DEHP were studied by single factor degradation test.The results showed that the temperature between 25 and 35?,pH between 6 and 8 were corresponding optimal degradation conditions for DEHP,while there was no significant difference in the inoculation amount of bacteria for DEHP degration.Following the response surface methodology,the optimal degradation conditions of DEHP were as follows: temperature was 30.4 ?,pH was 7.08,the amount of inoculation was 1.0(OD600).Under the optimal condition,the predicted degradation rate of DEHP was 98.5%,and the measured degradation rate was 99.1%.The strain XB could degrade completely DEHP at the initial concentrations from 50 to 400 mg·L-1)in 120 h,and the degradation rate of DEHP was 88.3% at the concentration of 800 mg·L-1 in 72 h.The half-life of DEHP degradation at different concentrations varied from 5.44 to 23.48 h.The degradation intermediate products of DEHP was analyzed by LC-TOF-MS/MS,and the degrading enzyme genes of strain XB were verified by real-time fluorescence quantitative PCR to deduce its degradation pathway.First,the strain XB produced the carboxylic-ester hydrolases to break the DEHP ester bonds on both sides.DEHP was converted to monoethylhexyl phthalate(MEHP),MEHP was then converted to phthalic acid(PA).PA was degraded to 4,5-dihydroxy phthalic acid by the phthalic acid 4,5-dioxygenase,and then 4,5-dihydroxyphthalic acid was converted to protocatechuic acid(PCA)by the decarboxylation reaction by strain XB.The expression of 3,4-? subunit dioxygenase gene and protocatechuic acid 3,4-? subunit dioxygenase gene of strain XB were transformed PCA into small molecule organic acids,and finally through tricarboxylic acid cyclic decomposition into CO2 and H2 O.The degradation results of DEHP(100 mg·kg-1)in soil by strain XB showed that,compared with the non-inoculation treatments,the residual concentrations of DEHP in contaminated soil inoculated with strain XB(sterilized and non-sterilizedn)sharply decreased with.52.2% and 59.7% of DEHP removal rates at the 10 th day,and the half-life of DEHP in contaminated soil were 8.78 and 7.30 d,respectively,beinf far shorter than those of the non-inoculation treatments.The results of a pot experiment with soil(the initial DEHP concentration is 20 mg·kg-1 and 100mg·kg-1 planted maize showed that,compared with the control(non-inoculation and without plant),the treatment of inoculating degrading strain XB and planting maize declined the DEHP residual in soil and promoted the degradation of DEHP.The DEHP removal rates of the two treatment in the 25 th day(50th day)varied from 50.50% to 59.91%(71.77%~78.23%).Especially the treatment planting maize inoculating with strain XB,its removal rate of DEHP was up to 85.41%,indicating the combination of degrading strain XB and maize could form a good synergistic effect to degrade effectively DEHP in soil.Compared with the non-inoculation treatment,inoculating strain XB in the maize could significantly increase the activities of superoxide dismutase(3%~154%)and catalase(11%~58%)as well as polyphenol oxidase(85%~288%)of maize roots,stems and leaves,significantly reduced the content of malondialdehyde(11%~60%),increased maize biomass(5%~86%),and increased accumulation of DEHP(7.5%~12.6%)in the maize root while decreased the DEHP content in the stems and levaves under high concentration treatment.These results demonstrated that the combination of maize and strain XB could effectively remediate DEHP contaminated soil and ensure the safety of agricultural products,showing high potential for application.