Molecular Mechanism Of Pyrazosulfuron-ethyl Degradation By Chenggangzhangella Sp. BI-1 And The Enhancement Bioremediation Of Contaminated Soil With Rhamnolipid

Pyrazosulfuron-ethyl,one of the representative varieties of sulfonylurea herbicides,had good control effect on broad leaf and sedge weeds and was commonly used in rice and other crops.Because of its stable structure and long half-life in soil,pyrazosulfuron-ethyl is easy to produce drug damage to sensitive crops in later crops.It is also found in water bodies and animals.It is an urgent problem to remove the residues of pyrazosulfuron-ethyl in farmland soil.Microbial degradation of organic pollutants has attracted wide attention due to its high efficiency and environmental friendliness.However,currently,the degradation strains of pyrazosulfuron-ethyl are scarce,the degradation mechanism is not clear,and the lack of theoretical and technical support for soil remediation limits the application of microbial remediation of pyrazosulfuron-ethyl contaminated soil.Therefore,it is of great practical significance to isolate and clarify the molecular mechanism of pyrazosulfuron-ethyl degradation by strains,and analyze its potential for remediation of contaminated soil.1.In this study,a pyrazosulfuron-ethyl degrading strain BI-1 was isolated from the soil of Yancheng City,Jiangsu Province by continuous enrichment culture.The strain could completely degrade 50 mg·L^-1pyrazosulfuron-ethyl within 72 h.Based on the morphological,physiological and biochemical characteristics and 16S r RNA gene sequence analysis,the strain BI-1 was preliminarily identified as Chenggangzhangella sp.,which was the first reported to have the ability to degrade pyrazosulfuron-ethyl in this genus.The growth characteristics showed that the strain BI-1 grew well under the conditions of pH 6.0～8.0,Na Cl concentration less than 3%and 15～40℃.The optimal growth temperature was 30℃,pH 7.0 and Na Cl concentration was 2%.The degradation characteristics showed that strain BI-1 had high degradation ability at 25～40℃and pH 6.0～9.0,and the optimum degradation temperature was 30℃and pH was 7.0.The degradation rate of the strain was positively correlated with the inoculated amount in the range of 0.5-2.0%,and the degradation capacity did not change significantly when inoculated amount exceeded 2.0%.The degradation rate of the strain decreased with the increase of pyrazosulfuron-ethyl concentration.When the concentration of pyrazosulfuron-ethyl was lower than 100 mg·L^-1,the degradation rate of the strain BI-1 was more than 90%,and when the concentration was increased to 200 mg·L^-1,the degradation rate of the strain was reduced to 47.5%.2.The mutant strain△BI-1 was screened by continuous plate culture（without pyrazosulfuron-ethyl）,and the degradation ability of pyrazosulfuron-ethyl was deficient.The genomes of strain BI-1 and△BI-1 were compared to find the missing gene in the mutant strain,and the NCBI database was used to compare the genes,an Open Reading Frame had high homology（98.5%）with sul E,the hydrolase gene in Hansschlegelia zhihuaiae S113.Through gene knockout and callback operation,this segment was determined to be the degradation gene of pyrazosulfuron-ethyl in strain BI-1,and it was named pyf E,the first reported degradation gene of pyrazosulfuron-ethyl.The pyf E was hetero-expressed in E.coli and purified to obtain enzyme PyfE.Combined with HPLC-MS/MS analysis,the degradation pathway of pyrazosulfuron-ethyl was deduced:the enzyme PyfE broke the ester bond of the side chain of pyrazosulfuron-ethyl benzene ring through deesterification to produce intermediate pyrazosulfuron-ethyl sulfonic acid.3.The results of enzymatic characteristics showed that PyfE had high stability（degradation rate remained above 70%）and strong enzyme activity in the range of 35～50℃and pH 7.0～8.0.The optimal degradation temperature was 40℃and pH was 7.5.Ag⁺,Cd²⁺,Zn²⁺,SDS,methamidos,PMSF,DEPC,p CMB and iodoacetamide significantly inhibited PyfE activity,while Ni²⁺,EDTA and Tween 80 weakly inhibited PyfE activity.Kinetics analysis showed that the K_mof pyrazosulfuron-ethyl on enzyme PyfE was 17.95μM,and the catalytic efficiency k_cat/K_mwas 0.041 s^-1μm^-1.4.As a hydrophobic organic matter,pyrazosulfuron-ethyl has low bioavailability in soil.In this study,the biosurfactant rhamnolipid（RL）was used to improve the elution efficiency of pyrazosulfuron-ethyl,so as to enhance the degradation ability of strain BI-1 to pyrazosulfuron-ethyl in soil.The results showed that when 2000 mg·L^-1rhamnoolipid solution was added to 5 mg·kg^-1pyrazosulfuron-ethyl polluted soil（soil to water ratio 4:1）,the elution efficiency of pyrazosulfuron-ethyl was the highest,reaching 15%.In the simulated field experiment,the natural degradation rate of 5 mg·kg^-1pyrazosulfuron-ethyl contaminated soil was 4.7%after standing for 10 days,and the natural degradation rate of pyrazosulfuron-ethyl increased to 15.5%when the concentration of RL was 100 mg·kg^-1,indicating that RL solution could promote the natural degradation of residual pyrazosulfuron-ethyl in soil.Adding strain BI-1 to 5 mg·kg^-1pyrazosulfuron-ethyl to contaminated soil,and adding 100 mg·kg^-1RL for 10 days,degradation rate of pyrazosulfuron-ethyl in soil reached 56.6%,which was 1.68 times higher than that in the treatment group without adding RL.The results indicated that RL could enhance the degradation of residual pyrazosulfuron-ethyl in soil by strain BI-1,and provide theoretical and technical support for the remediation of soil contaminated by pyrazosulfuron-ethyl.