| Dibutyl phthalate(DBP),plastic industry is widely used in plasticizers.DBP has environmental hormones and biological toxicity.It is also difficult to natural degradation in the environment.Complex physical,chemical and biological reactions will occur at the cell surface when microorganisms contact with pollutants.Pollutants must through the cell membrane before it comes into the cells and utilization by microbial.Understanding of cell surface behavior,growth and metabolism process of functional strains is of great significance in developing the efficient repair technology.It is important to understand the behavior of cell interface and the growth and metabolism of DBP in the process of degrading DBP,which is important for the degradation and development of DBP in environment.In this study,In this study,a DBP-degrading bacterium DNB-S2 was screened in the soil for the long-term use of plastic film.The effects of the addition of anthraquinone-2,6-sodium sulfonate(AQDS)on the removal of pollutants from DBP were studied.The DBP-degrading bacteria DNB-S2 were cultured in LB liquid culture medium or the medium contain(1)DBP 50 mg · L-1,(2)DBP 50 mg · L-1 and adding AQDS100 or 500 umol · L-1,(3)DBP 50 mg · L-1 and adding humic acid 250 or 500 mg· L-1.In order to study the effects of exogenous substances on the molecular mechanism of bacterial cell reaction and response to stress during the degradation of DBP in the process of exogenous regulation,the transcriptomics technique of modern molecular biotechnology was used to study the effects of exogenous substances.The effects of exogenous substances on the expression of functional genes in degraded strains were investigated.The molecular mechanism of DBP degradation under exogenous regulation was revealed at the gene level.The main results of the study are as follows:The degrading strain DNB-S2 was identified as Enterobacterium sp.By 16 S rDNA identification and nucleic acid data in Gen Bank,and obtained accession number KY469198.The strain was able to degrade DBP at the concentration of 50,100,200,500 and 1000 mg · L-1 to95.46%,93.91%,95.81%,72.31% and 70.2% within 72 hours.In order to investigate the process of bacterial cell interface interaction with contaminant DBP.The contents of polysaccharides and protein in the extracellular polymer(EPS)of different growth stages were determined.Functional group contained in EPS were analyzed.The changes of surface hydrophobicity(CSH)and Zeta potential were also determined.The results showed that the strain will increase its hydrophobicity to capture DBP.The addition of exogenous AQDS and humic acidresulted in the increase of polysaccharide content in EPS,the decrease of protein content and the significant increase of CSH.But has little effect on Zeta potential.The results showed that the exogenous AQDS and humic acid promoted the biodegradation of DBP by increasing the CSH of the strain DNB-S2 to promote its close contact with the hydrophobic organic pollutant DBP,which laid a foundation for the biodegradation of DBP.The cell structure and cell membrane integrity of DNB-S2 cells under different culture conditions were studied by flow cytometry.The composition and change of fatty acids in DNB-S2 cell membrane were determined by gas chromatography-mass spectrometry(GC-MS).The fluidity of the cell membrane under different culture conditions was studied by analyzing the saturation of fatty acids.The results showed that DBP could lead to the change of DNB-S2 cell size and intracellular particle density,and destroyed the integrity of DNB-S2 cell membrane.The addition of exogenous AQDS and humic acid reduced the number of cells damaged by the cell membrane in the culture system and protected the integrity of the cell membrane.GC-MS detected 17 fatty acids on the DNB-S2 cell membrane.DBP resulted in a decrease in the total amount of fatty acids in the growth retardation and logarithmic growth stages.Exogenous AQDS and humic acid can increase the unsaturated fatty acid content in the strain to make the cell membrane structure become loose,increase the cell membrane fluidity,and increase the adaptability of strain DNB-S2 to environmental stress.The results showed that exogenous AQDS could increase the antioxidant level of DNB-S2 in different culture conditions.AQDS has no significant effect on the ability of intracellular enzyme to remove O2-·,but can improve the ability to remove ·OH and H2O2,which will reduce H2O2 and O2-· reaction to produce more toxic ·OH.Reduce the oxidative stress produced by ·OH radicals that are more toxic to the strain and increase the oxidative capacity of the strain.The results showed that exogenous AQDS could increase the antioxidant level of DNB-S2 in different culture conditions.(ROS),including ·OH,O2-· and H2O2,were used to isolate the antioxidant capacity of the strain.DBP reduced ROS removal capability of strain DNB-S2,indicating that DNB-S2 was able to biodegrade DBP but was still oxidized by DBP.The results of transmission electron microscopy(TEM)showed that DBP could decrease the number of cytoplasm,the extracellular secretions,the cytoplasmic blight,the cell wall fragmentation,the decrease of intracellular mass and even the vacuoles,the microbial morphological changes.Samples were taken in logarithmic growth phase,and three treatments were repeated for each treatment.Twelve samples were sequenced.The average yield of each sample was 1.30 Gb,and the average comparison with the reference genome was 94.63%.Compared with the control group,DBP stress-treated strains showed 10 up-regulated genes and 18 genes were down-regulated.The addition of exogenous AQDS-treated strains showed up-regulated genes 53 and down-regulated genes 95.The addition of exogenous humic acid treated strains showed up-regulated genes 113 and down-regulated genes of 300.The results showed that DBP changed the cell function and metabolic pathways of the strain DNB-S2,especially in the cell membrane and cell membranecomposition and glutathione which related to glutathione metabolism appeared down-regulated genes.Exogenous AQDS and humic acid enhanced the ability of DNB-S2 to bind to DBP by increasing the expression of chemotactic gene.By increasing the cell membrane immobilization component gene expression to reduce DBP through the cell membrane when the cell membrane damage.Increased degradation of DBP intermediate metabolites by increasing the degradation of phenolic acids by improving the degradation of the phenolic acids.While maintaining the stability of the glutathione-metabolizing genes to maintain the stability of the antioxidant system and further promoting the strain DNB-S2 to play a biological function in response to DBP stress in the environment to improve the degradation capacity. |
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