Mechanisms Underlying The Degradation Of Herbicide Dichlorprop By A Microbial Consortium

Rac-dichlorprop（Rac-2-（2,4-dichlorophenoxy）propanoic acid,Rac-DCPP）,an important phenoxyalkanoic acid herbicide,is widely used worldwide to control the great majority of annual and perennial broad leaf weeds in wheat,rice,maize,barley,sorghum,and other gramineous crops production.Rac-DCPP is a typical chiral phenoxyalkanoic acid herbicide including（R）-DCPP and（S）-DCPP.Due to its unreasonable and excessive use and relatively high solubility in water,Rac-DCPP was persistently residual in diverse environments,including soil and groundwater,posing threats to environmental safety and human health.Degradation of Rac-DCPP in the natural environment is mainly mediated by microorganism activity.Compared to a single strain,microbial consortia show much better environmental adaptability and degradation efficiency,causing widespread concern about their applications in the bioremediation of polluted environments.Understanding of the key microbial consortia involved in Rac-DCPP degradation is pivotal for the design of synergistic consortia used for enhanced bioremediation of Rac-DCPP-contaminated sites.However,studies on the microbial degradation of Rac-DCPP were mainly focused on the isolation of pure degrading strains and the degradation mechanism at the molecular level.The degradation mechanism of the microbial consortia involved in Rac-dichlorprop degradation remains largely unknown.Consequently,it’s high time that we should focus on the research on the microbial degradation mechanism of Rac-DCPP at the microbial community level,which is useful to obtain optimized consortia for bioremediation of Rac-DCPP-contaminated environments.In this study,based on a Rac-DCPP-degrading enrichment obtained previously in our lab,DNA-stable isotope probing and metagenomics analysis were integrated to reveal the key microbial consortium responsible for Rac-DCPP degradation in the enrichment.In addition,Rac-DCPP-degrading strains were isolated from the enrichment on the basis of metagenomic antibiotics resistance gene analysis.On this basis,a highly efficient degrading microbial consortium was constructed for the degradation of Rac-DCPP.Subsequently,the ecological study of the bioremediation of Rac-DCPP,（R）-and（S）-DCPP contaminated soils by the microbial consortium was carried out.The main results of this study are as follows:1.Degradation characteristics of enrichment culture capable of degrading Rac-DCPPAn enrichment culture capable of degrading Rac-DCPP obtained previously in our lab was used as the material to explore the degradation characteristics of the enrichment.In this study,the effects of environmental factors on the degradation of Rac-DCPP,（R）-DCPP,and（S）-DCPP were investigated.There was a small difference in the degradation of Rac-DCPP,（R）-DCPP,and（S）-DCPP by the enrichment,respectively.The optimum temperature was30℃,the optimum initial p H of the medium was 6.0-7.0,and the optimum Na Cl concentration was 0.1-1.5%for the degradation of Rac-DCPP,（R）-DCPP,and（S）-DCPP.The key functional genes rdpA and sdpA for the first step degradation of Rac-DCPP were identified,and the microbial community was analyzed from the enrichment.At the phylum level,Proteobacteria（99.10%）was dominated in the enrichment,and Methylobacillus（72.70%）,Dyella（15.07%）,Hyphomicrobium（4.07%）,Pseudomonas（2.43%）,Labrys（1.62%）,Achromobacter（1.60%）,Dokdonella（0.65%）,Sphingobium（0.10%）,Rhodanobacter（0.09%）,Sphingopyxis（0.02%）were dominated at the genus level.2.key microbial consortium responsible for Rac-DCPP degradation in the Rac-DCPP degrading enrichmentDNA-stable isotope probing and metagenomics analysis were integrated to reveal the key microbial consortium responsible for Rac-DCPP degradation in the Rac-DCPP-degrading enrichment.The results showed that OTU340（Sphingobium sp.）and OTU348（Sphingopyxis sp.）were key microorganisms involved in Rac-DCPP degradation in the Rac-DCPP-degrading enrichment.As revealed by functional profiling of the metagenomes of the heavy DNA,the genes rdpA and sdpA,involved in the initial degradation of the（R）-DCPP and（S）-DCPP,respectively,were mostly taxonomically assigned to Sphingobium species,while neither rdpA nor sdpA was assigned to the Sphingopyxis species,indicating that Sphingopyxis species might harbor novel Rac-DCPP-degrading genes.Metagenomics analysis further showed that Dyella,Sphingomonas,Pseudomonas,and Achromobacter were presumed to synergistically cooperate with the key degraders Sphingobium/Sphingopyxis for enhanced degradation of Rac-DCPP.The mechanism of the synergistic degradation of Rac-DCPP in the enrichment was that Dyella,Sphingomonas,Pseudomonas,and Achromobacter reduce the inhibition effect of 2,4-dichlorophenol on the key degraders such as Sphingobium and Sphingopyxis.In addition,the cross-feeding between Sphingobium/Sphingopyxi and Dyella,Sphingomonas,Pseudomonas,and Achromobacter members also promotes the cell growth of functional microorganisms,thereby enhancing the degradation of Rac-DCPP.On the basis of DNA-SIP and metagenomics analysis,it was found that Rac-DCPP-degrading strains were closely related to fluoroquinolone resistance genes.A Rac-DCPP degrader,Sphingobium sp.L3,was isolated from the enrichment by the traditional enrichment method but with additional supplementation of the antibiotic ciprofloxacin.The degradation efficiency of Sphingobium sp.L3 was proved to be superior than that of Sphingopyxis sp.DBS4,Rac-DCPP degrader isolated from the enrichment previously in our lab.Sphingobium sp.L3 preferred to degrade（R）-DCPP over（S）-DCPP.In addition,a bacterial strain,Achromobacter sp.D1,was isolated from the enrichment using 2,4-dichlorophenol as the enriching substrate.On this basis,a highly efficient degrading microbial artificial consortium（Sphingobium sp.L3 and Achromobacter sp.D1）was constructed for the degradation of Rac-DCPP.The optimum temperature was 30℃,the optimum initial p H of the medium was 7.0,and the optimum Na Cl concentration was 0.1-1.0%for the degradation of Rac-DCPP by the microbial artificial consortium.1 m M Co²⁺,Hg²⁺,Ni²⁺and Cu²⁺could significantly inhibit the degrading activity,and 1 m M Zn²⁺,Ca²⁺and Li⁺could slightly inhibit the degrading activity,while 1 m M Fe³⁺had no obvious effect on the Rac-DCPP degradation.In addition,the degradation efficiency of artificial consortium（Sphingobium sp.L3 and Achromobacter sp.D1）was proved to be superior than that of Sphingobium sp.L3,and the degradation rate of Rac-DCPP was increased by 10.11%.3.The bioaugmentation of Rac-DCPP,（R）-and（S）-DCPP contaminated soils by a microbial artificial consortiumUsing the microbial artificial consortium（Sphingobium sp.L3 and Achromobacter sp.D1）with a high-efficiency degrading activity for Rac-DCPP as a material to explore the bioaugmentation process of the soils contaminated with Rac-DCPP,（R）-and（S）-DCPP.The addition of（R）-DCPP and（S）-DCPP can change the bacterial community structure but had no significant impact on the function of the bacterial community in the soils.The structure of the indigenous bacterial community was considerably changed by high concentrations of（R）-or（S）-DCPP,whereas the influences of the different enantiomers of dichlorprop were limited.A low concentration of dichlorprop had little effect on the soil bacterial community structure,and a high concentration of dichlorprop had a great influence on the structure of the soil bacterial community.The natural degradation of Rac-DCPP,（R）-and（S）-DCPP was mediated by indigenous microorganisms in the contaminated soils.Using one-way ANOVA,Illumina Mi Seq sequencing data demonstrated that the Sphingobium and Methylobacillus were key functional indigenous microorganisms for the degradation of Rac-DCPP in the contaminated soils,and microbial artificial consortium can synergistically cooperate with indigenous microorganisms in the contaminated soils to promote Rac-DCPP degradation.In addition,the remediation efficiency of artificial consortium（Sphingobium sp.L3 and Achromobacter sp.D1）was proved to be superior than that of Sphingobium sp.L3.Compared with the Sphingobium sp.L3,the removal rate was increased by 28.65%and 13.58%by the artificial consortium in applied soils with 10 mg kg^-1 and 30 mg kg^-1 of Rac-DCPP,respectively.In addition,q PCR analysis revealed that the inoculated artificial consortium survived well and maintained a relatively high abundance in the soils.These data demonstrate that the artificial consortium（Sphingobium sp.L3 and Achromobacter sp.D1）can be used as an efficient consortium for the in-situ remediation of Rac-DCPP contaminated soils.In summary,DNA-stable isotope probing and metagenomics analysis were integrated to reveal the key microbial consortium responsible for Rac-DCPP degradation in a Rac-DCPP-degrading enrichment at the community level.An artificial consortium（Sphingobium sp.L3 and Achromobacter sp.D1）for enhanced bioremediation of Rac-DCPP was successfully constructed,and the microbial artificial consortium was proved to be a potential resource for in-situ bioremediation of Rac-DCPP contaminated soils.These results have important guiding significance and application value for enhanced microbial remediation of Rac-DCPP contaminated soils.