Screening Of Phthalate Esters Degrading Microorganisms And Soil Remediation Combined With Forages

Phthalic acid esters(PAEs)are a class of persistent organic compounds that are widely used and durable in the environment.PAEs affect plant growth;and the quality of agricultural products.With the enrichment and accumulation in the biological chain,PAEs pose a potential risk to the ecosystem and human health.With the development of industry and wide application of plastics and films,studies on the remediation of phthalate esters in the environment many are the current focus of scholars.This study isolated several fungi and bacteria with efficiently PAEs degrading capacity revealed their degradation mechanism,and then studied their combined remediation effects with forage on soil PAEs pollution.(1)Three fungal strains with efficient degradation of PAEs were screened by a gradient concentration acclimation method,and their degradation characteristics on PAEs were studied.The degradation mechanism is speculated Phthalic acid esters?monoester?Phthalic acid?Protocatechuic acid?CO₂+H₂O.The three strains had good degradation effects on DBP(Dibutyl phthalate)and DEHP(Dioctyl phthalate)at different concentrations,the degradation efficiency of DBP and DEHP was 41.5%-68.0%and 22.3%-39.4%,respectively.In addition,the three strains had good remediation effects on the low concentrations of DBP and DEHP in the soil,and 47.2%-70.6%of DBP and 54.1%-73.4%of DEHP were degraded in soil after 21 d.(2)Twenty-seven strains of highly degradable bacteria could tolerate high concentrations of PAEs were screened and isolated,and degradation characteristics on PAEs were studied.The results showed that 23 strains were Bacillus subtilis,and the degradation efficiency of DBP was more than 75%,and 5 of them even exceeded 90%.Artificial acclimation experiments showed that the content of Bacillus subtilis in soil was positively proportional to DBP.It was found that all 11 Bacillus subtilis strains,which were notdomesticated by PAEs,could degrade DBP with a degradation rate of75%-97%.Moreover,the functional degradation enzyme was the intracellular enzyme of Bacillus subtilis.(3)A key esterase gene EST-BS was screened from the genome of strain Bacillus subtilis BS168 by bioinformatics analysis,which has two key functional domains.The gene was cloned in vitro and expressed in prokaryotic expression and verify its degradability to DBP after obtaining a crude enzyme solution.The results showed that the degradation rates of EST-BS and the crude enzyme solution of EST-BS300 and EST-BS800 were 81.6%,49.3%and 54.1%.EST-BS gene was transformed into Saccharomyces cereviscera strain for eukaryotic expression.The results showed that EST-BS mutant strain could tolerate high concentration of DBP and various PAEs,and could grow on the medium supplemented with 0-800 mg kg^-1 DBP or various PAEs.(4)Alfalfa and perennial ryegrass was chosen as the model to understand the remediation effect on DBP and DEHP pollution in soil and the enhancement effect of Bacillus subtilis with them.The results showed that alfalfa and ryegrass had a good removal effect on DBP and DEHP in soil,and the removal rate of both plants on DEHP was higher than that of DBP.Within 21 days,the DBP and DEHP removal rates of alfalfa were 55.2%and 69.6%,and removal rates of ryegrass were 48.66%and 60.3%.In addition,inoculation with Bacillus subtilis BS168 significantly enhanced the restoration of DBP and DEHP by the two forages.After inoculation with Bacillus subtilis BS168 for 7 days,the DBP removal rate of alfalfa increased to 74.5%and DEHP removal rate reached 82.2%.The removal rate of DBP and DEHP by ryegrass was 68.68%and 74.1%respectively,indicating that Bacillus subtilis and forage had a bioremediation combined enhancement effect on PAEs contamination.