Study On The Degradation Mechanism Of Prometryne-degrading Strain Pseudomonas Sp. DY-1 And Its Application In Contaminated Soil Remediation

As one of the typical triazine herbicides,prometryn is widely used in agricultural production because of its wide herbicidal spectrum,high efficiency,and lasting efficacy.However,even a small amount of prometryn residues in farmland will cause different degrees of phytotoxicity to succeeding crops,inhibit crop growth,and eventually lead to yield reduction,even lead to no harvest in severe cases.Therefore,excessive application of prometryn will have a certain negative impact on agricultural production,degree of which depends on the sensitivity of succeeding crops to prometryn.In addition,the residual prometryn in farmland soil will a lso pose a serious threat to the ecosystem and human health.These series of problems caused by prometryn residues have become a research hotspot in recent years.Bioaugmentation is a technology with the growth and metabolism of microorganisms as its core,which has gradually become one of the important means to control environmental pollution due to its advantages of high efficiency,no pollution and low energy consumption.At present,some studies have reported some microorganisms in nature can degrade prometryn,but these studies mainly focus on the screening of degradation strains and the analysis of degradation pathways.Only the gene encoding atrazine dichlorination hydrolase(Trz N)has been reported about the functional genes related to prometryn degradation in microorganisms,which shows that there are still some limitations in this field.In addition,genome-wide sequencing and bioaugmentation technologies have not been used to study prometryn-degrading strains.Clarifying the metabolic mechanism of prometryn in microorganisms,identifying the intermediate products in the degradation process,mining the functional genes in the genome of degradation strains.Besides,analyzing the changes in the community structure of indigenous microorga nisms in the process of bioaugmentation can provide theoretical support for bioremediation of prometryn contaminated sites,and can also provide reference basis for studying the degradation mechanism of other triazine herbicides,which has great research significance.In this study,Pseudomonas sp.DY-1,a high-efficiency prometryn-degrading strain isolated in the early stage of laboratory,was used as the material.The optimum degradation conditions of strain DY-1 were studied and optimized by single factor experiment and response surface methodology.The results showed that the optimum degradation conditions of strain DY-1 were 32.6?,pH 7.9,and the optimum inoculation amount was 5.8%.Under the optimized conditions,strain DY-1 could completely degrade prometryn with a concentration of 50 mg/L within 40 h.In addition,the study on the degradation characteristics of strain DY-1 showed that the strain could not only tolerate and degrade prometryn with a concentration as high as 500 mg/L,but also had degradation activity under a high NaCl concentration of 1000 mg/L.In the degradation spectrum analysis of strain DY-1 using other triazine herbicides as substrates,it was observed that strain DY-1 had good degradation effects on simetryn,desmetryn,ametryn,and metribuzin.High-performance liquid chromatography/mass spectrometry(HPLC/MS)was used to identify the intermediate products in the degradation of prometryn by strain DY-1.Three peaks with retention times of 8.07 min,12.93 min and 8.4 min were detected,and the mass-to-charge ratios were 258 m/z,274 m/z and 212 m/z,respectively.According to the molecular structure characteristics of prometryn and the characteristic fragment ion peaks of each compound,these three products were identified as sulfoxide,sulfone,and hydroxyl derivativ es of prometryn,and the main ways of degradation of prometryn by strain DY-1 were analyzed.In the degradation process,prometryn was first oxidized to sulfoxide prometryn,and then sulfone prometryn was produced by the second oxidation,and finally hydrolyzed to produce the final product 2-hydroxy prometryn.PacBio RS II sequencing platform was used to sequence the whole genome of strain DY-1.After the data were processed accordingly,the whole genome sequence of strain DY-1 was annotated and analyzed.The genome size is about 5.89 M,which contains a circular chromosome and a plasmid.The total GC content of the genome is 62.94%,which contains 5543 genes with an average length of 942 bp,it contains 71 tRNA,16 rRNA and 4 ncRNA as well.The application potential of strain DY-1 was analyzed by whole genome mining.The results showed that the chromosome of strain DY-1 contained a variety of genes involved in environmental stress response and toxic substance resistance,which were beneficial to the strain to adapt to the external environment,and these genes played a vital role in the survival of bacteria in harsh environment.Based on the annotated information of DY-1 genome,the possible degradation gene of prometryn were predicted.The target gene is 1530 bp in length and encodes 509 amino acids.Multiple sequence comparison showed that the expression product of the gene was a type?Baeyer-Villiger monooxygenase(BVMO),and shared 52.67%homology with the ethiamide monooxygenase(Eth A)of Mycobacterium Tuberculosis H37Rv.The full-length target gene was amplified and cloned into the corresponding expression vector using DY-1 genome as a template with the specific primers.The recombinant strain was induced by IPTG and purified by Ni-NTA column.SDS-PAGE showed that the specific band size was about 60 k Da,which was consistent with the expected results.The purified protein and 300?M prometryn solution were used to prepare the reaction system.The results of high-performance liquid chromatography(HPLC)showed that the degradation rate of prometryn could reach about 80%after 1 h of reaction.The gene knockout experiment with?-Red recombinase system showed that the mutant strain could hardly detect the degradation activity within 10 h after the reaction began,which indicated that this gene played an important role in the degradation of prometryn by strain DY-1.This gene could be identified as a new coding gene of prometryn oxidase.Strain DY-1 was added to the contaminated soil containing 50 mg/kg prometryn to construct a bioaugmentation system.After 21 days,the degradation rate of prometryn in the soil was 78.1%.The application characteristics of the strain showed that the degradation rate was affected by soil temperature,soil water content and inoculation amount.Strain DY-1 could achieve its best degradation effect under the conditions of temperature 30?,soil water content 20%and inoculation amount 1×10~8 CFU/m L.In addition,the addition of exogenous nutrients can also promote the degradation of prometryn in soil.The culture solution of strain DY-1 was used to irrigate the roots of corn seedlings damaged by prometryn.After 10 days of treatment,it was observed that the growth of corn seedlings returned to normal level,which indicated that this strain could obviously relieve the phytotoxicity of prometryn.The Miseq high-throughput sequencing technology was used to dynamically monitor the microbial community structure of the bioaugmentation system,and the relationship between microbial community structure and function was established.The results showed that the addition of strain DY-1 promoted the succession of microbial community in the soil polluted by prometryn,and the genus Pseudomonas,which the added strain belongs to,became the dominant population in the system,which increased the tolerance and degradation ability of soil microorganisms to prometryn.