Study On Degradation Of Nicosulfuron By Bacillus Subtilis YB1 And Aspergillus Niger YF1

Sulfonylurea herbicides are a class of herbicides containing a sulfonylurea bridge structure.They are mainly used for the control of various weeds in crop fields.Nicosulfuron is one of the sulfonylurea herbicides,which has the advantages of high efficiency,low toxicity,etc..But because its long-term use and unreasonable abuse,much nicosulfuron were accumulated in the soil,which could cause the phytotoxicity of the sensitive crops,including production reduction and unharvest.Therefore,the phytotoxicity of nicosulfuron residual has begun to attract the attention of researchers.The microbial remediation approach is an effective measure to solve this problem in the current situation.Related studies have shown that the method of microbial remediation is safe,efficient,and low-cost,and is one of the effective ways to solve the problem of herbicide residues in soil.In the early study,two strains with high activity for degrading nicosulfuron were isolated from the sewage of nicosulfuron production factory,which were Bacillus subtilis YB1 and Aspergillus niger YF1,respectively.YB1 strain degraded nicosulfuron mainly by secreting extracellular enzymes.Three degrading enzymes were isolated from the extracellular enzyme of YB1 strain,namely manganese ABC transporter,vegetative catalase 1 and acetoin dehydrogenase E1.The YF1 strain achieved the degradation of nicosulfuron by a combination of chemical hydrolysis and biodegradation.On the one hand,YF1 strain could secret oxalic acid to hydrolyze nicosulfuron,and on the other hand,it could also secret extracellular enzymes to degrade nicosulfuron.1.In this study,the genomic information of YB1 strain and YF1 strain was obtained by Illumina HiSeq PE150 sequencing platform.The YB1 genome size was 3.30 Mb,the coding gene number was 3882,and the coding region length was 3,698,092 bp.The YF1 genome had a size of 9.19 Mb,the number of coding genes was 6866,and the length of the coding region was 35,215,337 bp.Based the genome information and some prediction of some related software,some genes associated with the degradation of nicosulfuron were predicted and there were 4 possible esterase gene and 9 possilble hydrolase genes in YB1 genome,while threr were 7 possible hydrolase genes in YF1 genome.These genes related the nicosulfuron degradation are potential to further studied.2.Based on the amino acid sequences of the three degrading enzymes obtained in the previous study,the enzyme solutions of the three degrading enzymes were obtained by heterologous expression.Then we detected the degradation activities of three degradation enzymes to four kinds of sulfonylurea herbicides,including nocosulfuron,sulfometuron-methyl,bensulfuron methyl and tribenuron-methyl.The results showed three degradation enzymes only degraded nicosulfuron.To further studied the special degradation activities of three degradation enzymes to nicosulfuron,the interaction between three degrading enzymes and four kinds of sulfonylurea herbicides were studied by computer molecular simulation and surface plasmon resonance technology.It was proved that there were indeed interaction between the three degrading enzymes and nicosulfuron and bensulfuron methyl,while no interactions between three degrading enzymes and sulfometuron-methyl and tribenuron-methyl.The results of computer molecular simulation showed that three degrading enzymes all had binding with four kinds of sulfonylurea herbicides.The results measured by computer molecular simulation and surface plasmon resonance technology were not completely consistent with the actual test results.But the molecular simulation docking results visually show the binding state between the degrading enzyme and the herbicide molecule,and have a clear classification and display of the existing interaction forces.In response to the degradation of nicosulfuron by degrading enzymes,we also detected the degradation products of nicosulfuron by liquid chromatography-mass spectrometry,and determined some products' toxicity to enviromrntal organisiams and predicted the toxicity of all possible degradation products of nicosulfuron using ECOSAR and TEST prediction software.The predicted and actual toxicity results provide a reference for the later remediation of nicosulfuron.Both of the technologies had verified the interaction between the degrading enzyme and the herbicide molecule,but the specific degradation mechanism needs further study.For the reaction of the degradation enzyme to catalyze the degradation of nicosulfuron,this study used liquid chromatography-mass spectrometry to detect the products of the YB1 strain and degradation enzyme to degrade nicosulfuron,and obtained different degradation solutions.Then we found that ECOSAR software could be used to predict acute toxicity to aquatic and terrestrial organisms from nicosulfuron and its degradation products.The acute toxicity results from ECOSAR and actual detected results were highly consistent,which will greatly reduce the determination of pesticide molecules' toxicity to environmental organisms and provide guidance for environmental risk assessment of other pesticide molecules.3.In order to further study the remediation effect of YB1 strain on nicosulfuron,YB1 strain was fermented to prepare granules,and the indoor potting experiments was conducted to simulated the remediation of the nicosulfuron in the field.The results showed YB1 granules had a good remediaton effect to the nicosulfuron residues in soil.And then the microbial diversity method was used to determine the changes of microbial diversity in the process of remediating nicosulfuron residues in soil by YB1 granules.The result indicated the YB1 granules was safety to plants and had no significance effect to the original microbial environment in the soil4.By studying the degradation of nicosulfuron by YF1 strain,it was clarified that YF1 strain degraded nicosulfuron by collective methods of hydrolysis and microbial degradation.Two common enzymes,glucoamylase and lipase 2,were isolated from the extracellular enzyme of YF1 strain,and large amount of glucoamylase and lipase 2 were obtained by the method of induced culture,including medium types,temperatures and time.The degradation characteristics of the crude enzyme solution of the two enzymes to nicosulfuron were studied,which further confirmed the chaotic catalytic function of the enzyme.In conclusion,this study had initially revealed the mechanism of YB1 and YF1 strains degrading nicosulfuron,and had obtained nicosulfuron-degrading enzymes,which had enriched the resources of nicosulfuron-degrading enzymes.The interaction between degrading enzyme and nicosulfuron was studied,which provided a reference for studying the interaction between other degrading enzymes and herbicides.At the same time,this study explored the application of nicosulfuron-degrading strains,clarified the degradation effect of YB1 strain granules and environmentally friendly characteristics,and provided ideas for solving the problem of soil remediation by nicosulfuron in the environment...