Fluorescent Marker Tracing Of Highefficiency Degrading Bacteria DNB-S1 And Remediation Of DBP Contaminated Soil By Immobilized Bacteria

Dibutyl phthalate(DBP),as one of the most commonly used plasticizers in the world,is widely used in industry and agricultural products,often detected in soil,water,atmosphere and other environmental media.In recent years,due to the rapid development of industry and agriculture leading to the rapid use of plastic products,soil pollution caused by DBP has increased dramatically.It has become a ubiquitous soil pollutant,causing serious pollution and even threatening human health and ecological safety.Environmental hazards have caused people's attention.The study found that microbial degradation is the main means of removing DBP from agricultural soils.In this study,the DBP high-efficiency degrading bacteria Pseudomonas sp.DNB-S1 screened in the early stage of the laboratory was used as the research object.The GFP fluorescent labeling degrading bacteria GFP-DNB-S1 was constructed by the three-parent transfer method.The activity and dynamic of GFP-DNB-S1 were observed in real time by the expression of its green fluorescent protein.Using sodium alginate(SA)and nano-hydroxyapatite(n-HAP)as a carrier material and Ca Cl₂ as a cross-linking agent,the immobilized microbial agent was prepared by embedding cross-linking and immobilization technology.Then test the remediation effect of this product on DBP contaminated soil.The main research contents results of this topic are as follows:(1)GFP fluorescently labeled degrading bacteria GFP-DNB-S1 was constructed by three-parent zygosity transfer method.Through the introduction of GFP plasmid into the strain DNB-S1using the three-parent conjugate transfer method with the aid of p ET28-EGFP and auxiliary strain S17 carrying green fluorescent protein particles,the fluorescent marker degrading bacteria GFP-DNB-S1 was successfully constructed.The target strain has both kanamycin and ampicillin resistance,and can express green fluorescence under the blue excitation light,which fully proves that the imported GFP plasmid has been fluorescently labeled with the degradation bacteria GFP-DNB-S1 is inherited.(2)The SA and n-HAP/SA immobilized bacteria agents was prepared and characterized by scanning electron microscopy and infrared spectroscopy.In order to strengthen the biological performance of DBP degrading bacteria,the immobilized microbial technology was adopted by embedding-crosslinking immobilized microorganisms,and SA and n-HAP/SA were used as carrier materials to prepare immobilized bacterial agents.The results show that when the carrier material SA and n-HAP ratio is 3:2,the n-HAP/SA immobilized bacterial agent is easier to form.Compared with SA immobilized bacterial agent,the water-soluble swelling rate and breaking rate are reduced by 8.61%,15.88%,higher mechanical properties and chemical stability.In addition,the n-HAP component in the n-HAP/SA immobilized bacterial agent increases the pore structure inside microspheres,and the surface of microspheres becomes rough.When Ca Cl₂ and SA are cross-linked and immobilized,Ca²⁺of n-HAP participates in the coordination reaction,and there is an interaction between SA and n-HAP molecules contained in the n-HAP/SA immobilized bacterial agent.(3)By using DBP as the sole carbon source to cultivate immobilized bacteria,the effect of immobilized bacteria on DBP degradation efficiency was explored.The growth curve,degradation curve of the degrading bacteria and the p H value were measured,and the strain was observed morphologically.The degradation rate of DBP by n-HAP/SA immobilized bacteria can reach 95.1%,it is better than SA immobilized bacteria and free bacteria.The p H value of the culture solution showed a downward trend after 48 h.The cells of free bacteria and SA-immobilized bacteria were damaged by scanning electron microscopy,which affected the activity of degrading bacteria;while the culture medium of n-HAP/SA-immobilized bacteria was maintained at the p H 6.08 avoids acidic environmental stress,reduces cell damage,and promotes the efficient degradation of DBP.In addition,through fluorescent tracing of the degrading bacteria in the immobilized bacteria agent,it was found that GFP-DNB-S1 was evenly distributed on the surface and internal structure of the immobilized bacteria agent,and GFP-DNB-S1 were fixed from fixed when cultured in MSM.The particles are released and evenly dispersed and grown in the culture medium.(4)The n-HAP/SA immobilized bacteria agent can effectively remove DBP in the soil and improve the physical and chemical properties of the soil.Compared with the free bacteria GFP-DNB-S1,it was found that when n-HAP/SA immobilized bacteria was applied to the contaminated soil with an initial DBP concentration of 20 mg·kg^-1,the DBP degradation rate could reach 93.34%,which was significantly higher than that of the free bacteria treatment group.78.72%,indicating that the effect of immobilized bacteria on DBP removal in contaminated soil is more obvious and thorough than that of free bacteria.In addition,the carrier material of the n-HAP/SA immobilized bacteria agent can stabilize the soil p H,increase the content of organic carbon and available phosphorus in the soil,and thus increase the soil fertility.