Study On Uptake And Degradation Behavior Of A DMP-degrading Bacterium Ensifer Adhaerens DNM-S1

At present,Dimethyl phthalate(DMP)has caused serious pollution to the natural environment.Due to the environmental estrogen effect of DMP,the harm of DMP pollution to humans cannot be ignored.According to the climatic characteristics of the black soil region in Northeast China,this study isolated a degrading bacterium Ensifer adhaerens DNM-S1 which can remove DMP pollution at low temperature from a vegetable greenhouse in the suburb of Harbin.Taking Ensifer adhaerens DNM-S1 as the research object and DMP as the target pollutant,we deeply studied the ingestion mechanism of DNM-S1,the key step in the process of removing DMP pollution by DNM-S1,verified the response of DNM-S1 degradation to environmental factors,and deeply explored of the response of degradation intermediates to temperature and types of PAEs.The results show:(1)Ensifer adhaerens DNM-S1 was isolated and domesticated.DNM-S1 belongs to Proteobacteria,Alphapoteobacteria,Rhizobiales,Rhizobiaceae,Ensifer,Ensifer adhaerens.Combined with the morphological,physiological and biochemical properties of DNM-S1,DNM-S1 was named Ensifer adhaerens DNM-S1.The suitable growth pH of Ensifer adhaerens DNM-S1 is 6-10,and the temperature is 15-35?.Response surface method was used to optimize the culture conditions of DNM-S1:DMP addition amount was 1475 mg·L-1,pH was 7.3,and temperature was 34.2?.The weight of the influence of environmental factors on the growth of DNM-S1 is:DMP addition amount>pH>temperature.When DMP,DEP,DBP,and DEHP are used as carbon sources,the growth of DNM-S1 conforms to the description of Logistic model.(2)The uptake mechanism of Ensifer adhaerens DNM-S1 to DMP was explained.Studies on the hydrophobicity of the cell surface show that Ensifer adhaerens DNM-S1 has a highly hydrophobic surface(more than 80%),and its effect on DNM-S1 CSH is not significant regardless of changing the type of PAEs or changing the carbon source affinity/hydrophobic type.Infrared spectroscopy studies show that the absorption peak of DNM-S1 at 2929 cm-1 indicates that its outer membrane contains a large number of hydrophobic functional groups,which provide a binding site for DNM-S1 to take up DMP.The study of Raman spectroscopy shows that under the induction of DMP,the relative peak intensity of DNM-S1 at 1172 cm-1 increases,indicating that the content of lipopolysaccharide in DNM-S1 cell membrane increases.The research results of DNM-S1 cell membrane composition are consistent with the Raman spectroscopy research.These results indicate that the hydrophobic functional group comes from the fatty acid in the lipopolysaccharide of DNM-S1 cell wall,and the ester bond in the fatty acid relies on the hydrophobic-hydrophobic interaction to form a hydrogen bond with DMP,adsorbing it to the outer membrane of DNM-S1.DNM-S1's adsorption of DMP conforms to the description of Freundlich equation,and DNM-S1's adsorption of DMP conforms to the description of Langmuir equation.After adding the ATPase inhibitor sodium vanadate to the adsorption system,DMP-S1's adsorption of DMP and other isotherms become more in line with the description of Langmuir equation,indicating that DMP needs energy to enter the protoplasm from the outer membrane of DNM-S1.Therefore,the way DMP crosses membrane to enter the protoplasm of DNM-S1 belongs to active transportation.DNM-S1 morphology studies show that DMP does not affect the membrane structural integrity of DNM-S1,but adding DMP to MSM medium and LB medium will induce DNM-S 1 cells to become larger.The analysis of secondary metabolites shows that DNM-S 1 will produce carotenoids,which can offset the oxidative stress caused by DMP to DNM-S1,indicating that DNM-S 1 has a special survival strategy to deal with DMP oxidative stress,thus ensuring that DNM-S 1 has a complete membrane during the DMP degradation process,allowing the degradation reaction to proceed smoothly.(3)Response of Ensifer adhaerens DNM-S 1 degradation behavior to environmental factors.Ensifer adhaerens DNM-S 1 degrades DMP at different initial concentrations in line with the first-order reaction kinetics equation.The degradation rate of DMP treated with 2000 mg·L-1 is 84.89%;the degradation rate of DMP treated with 1500 mg·L-1 is 96.6%;the degradation rate of DMP treated with 1000 mg·L-1 was 98.12%;the degradation rate of DMP Treated with 500 mg·L-1 was 99.73%;the degradation rate of DMP treated with 100 mg·L-1 was 100%within 6 hours;the degradation rate of DMP treated with 50 mg·L-1 was 100%within 3 h.DNM-S1 has a good degradation effect on low concentration(50 mg·L-1)and high concentration(2000 mg·L-1)DMP.The degradation of DEP,DBP and DEHP by DNM-S 1 also accords with the description of the first-order reaction kinetic equations,but with the extension of the side chains of PAEs,the half-life also increases.The degradation of DMP by Ensifer adhaerens DNM-S 1 at 10,15,25 and 35? conforms to the description of the first-order reaction kinetic equation.After 5 days of treatment,the DMP content decreased from the initial 1500 mg·L-1 to 0 mg·L-1,and the degradation rate was 100%at 35?;the DMP content decreased from the initial 1500 mg·L-1 to 200.6 mg·L-1,the degradation rate was 86.62%at 25?;the DMP content decreased from the initial 1500 mg·L-1 to 525.9 mg·L-1,and the degradation rate was 64.94%at 15?;the DMP content decreased from the initial 1500 mg·L-1 to 1115 mg·L-1,and the degradation rate was 25.73%at 10?.The degradation rate of DNM-S 1 decreased with the decrease of temperature.(4)Response of Ensifer adhaerens DNM-S1 degradation intermediate to environmental factors.GC-MS research showed that DMP treatment detected two target molecules,PA and PCA,indicating that DNM-S1 can degrade DMP to PA,and then further degrade PA to the downstream product PCA.Three kinds of target molecules were detected in DEP,DBP and DEHP treatments,namely:DMP.PA and PCA,indicating that DMP,PA and PCA will be generated during the degradation process of DEP,DBP and DEHP.In different treatments,the monoesters corresponding to PAEs were not detected,but DMP was detected,indicating that the degradation of long-chain PAEs will first generate short-chain PAEs-DMP,and then be degraded by PA?PCA.Since PCA is one of the common upstream organics that can enter the tricarboxylic acid cycle,it indicates that the degradation of DMP,DEP,DBP and DEHP by DNM-S1 is complete degradation.Three kinds of metabolites were detected during the treatment at 25 and 35?,namely DMP,phthalic acid and protocatechuic acid.This shows that at 25 and 35 ?,.Ensifer adhaerens DNM-S1 will produce phthalic acid and protocatechuic acid during the degradation of DMP,which can completely degrade DMP.However,in the treatment at 10 and 15?,the metabolite of DNM-S1 degrading DMP can only detect phthalic acid,indicating that DNM-S1 is incomplete degradation of DMP under low temperature conditions,and the temperature threshold of incomplete degradation is 15?.(5)The remediation effect of Ensifer adhaerens DNM-S1 on soil contaminated by DMP.The degradation behavior of DMP in the soil treated at 35,25,15 and 10? is in accordance with the description of the first-order reaction kinetic equation.The degradation half-lives of DMP in the CK group were 72.49,90.03,197.4 and 235.9 d,respectively.These results indicate that the ecological risk of PAEs is higher in low temperature areas.In the DNM-S1 groups,the degradation half-lives of DMP were 17.37,22.62,38.82 and 55.08 d,respectively.The introduction of free DNM-S1 promotes the degradation of DMP in soil.Compared with the unrepaired soil CK,the half-life of DMP in soil inoculated with strain DNM-S1 is reduced,indicating that DNM-S1 driven degradation is an effective strategy to reduce DMP pollution.After adding free bacteria DNM-S1 to the soil contaminated by DMP,the urease activity returns to the natural soil level.Dehydrogenase is inhibited within 7 days,and returns to the level of natural soil after 14 days.Invertase activity does not recover and is inhibited within 7-14 days,but returns to natural soil levels after 28 days.It shows that DNM-S1 can more easily activate the activities of soil urease and dehydrogenase,and accelerate the metabolism of DMP in soil.In addition,DNM-S1 has a certain degree of increase in the total nitrogen,alkali-decomposed nitrogen,available phosphorus and available potassium content of the soil,which effectively improves the soil's fertilizer supply capacity and is beneficial to the growth of crops.The above research results indicate that Ensifer adhaerens DNM-S1 is a strain of DMP degrading bacteria with extremely high application potential,which is suitable for the restoration of DMP pollution in the black soil area of Northeast China.The results of this study fill the gap in the key mechanism of DMP pollution remediation,uptake mechanism,enrich the microbial resources degraded by DMP,and have important guiding significance for the remediation of DMP-polluted environment in the black soil area of Northeast China.