| As we all know,my country is both a major producer of antibiotics and a major country in the use of antibiotics.Animal husbandry and aquaculture,hospitals and antibiotic factories,as well as landfills and sewage treatment plants,discharge of wastewater and wastes,all contribute to environmental antibiotic pollution to varying degrees.Moreover,most antibiotics cannot be effectively removed under the existing process,resulting in rivers and lakes becoming antibiotics and drug resistance gene pools.Authoritative experts called for the threat of antibiotics to be included in the national security monitoring system as soon as possible,and the solution to the problem of antibiotic residues in the environment is urgent.The use of microorganisms to degrade antibiotics in the environment can not only effectively solve its residual problems in the environment,but also avoid secondary harm to the environment.Therefore,this article first isolates and screens highly efficient degrading strains of tetracycline,oxytetracycline and sulfadiazine.Through the isolation and purification of the metabolites of the strain,the types and properties of the substances that play the role of antibiotic degradation were analyzed.The molecular structure of the degradation products was detected by HPLC and ESI-MS,and the degradation pathway was deduced accordingly,and the safety of antibiotic degradation products was tested.The results as follow:1.By means of enrichment culture,plate preliminary screening and HPLC quantitative determination,a strain named "N2-13" was isolated and screened from feces.sewage,soil and other samples of farm.The degradation rate of tetracycline was 73.88%.After morphological observation,physiological and biochemical tests,and 16S rRNA gene detection,it was identified as Bacillus amylolytic.A strain named "N2-18" was screened.the degradation rate of oxytetracycline was 60.47%.and it was identified as Bacillus subtilis.A strain named "T-14" was selected and the degradation rate of sulfadiazine was 63.72%.which was identified as Bacillus subtilis.Conduct follow-up research on the above three strains.2.The degradation characteristics and enzyme production conditions of the strain were studied.The degradation characteristics and enzyme production conditions of the strain were studied by single factor test,and the effects of pH,inorganic salt,treatment time,bacterial content and dissolved oxygen on the degradation of tetracycline,oxytetracycline and sulfadiazine strains were investigated.The result shows:After culturing for 3 days at pH 7.0,100 mL of liquid,0.03%FeSO4,2%inoculation,and 37?,the degradation rate of tetracycline by N2-13 strain can reach 97.27%.After being cultured for 3 days under the conditions of pH 7.0,100 mL of liquid,0.03%FeSO4,inoculation volume of 5%and 37?,the degradation rate of N2-1 8 strain to oxytetracycline can reach 94.4%.Under the conditions of pH 7.0,100 mL of liquid.0.03%FeSO4,3%inoculation,and 37? for 3 days,the degradation rate of sulfadiazine by T-14 strain can reach 72.35%.3.The intermediate metabolites of the degrading strain were analyzed and the degrading enzymes were isolated and purified.The degradation enzyme produced by the strain was separated and purified by ammonium sulfate fractionation precipitation test and dextran gel G-200.The result shows:The crude enzyme for tetracycline degradation produced by strain N2-13 has a protein size range of 14 kDa-22 kDa and contains 6-7 bands.The crude oxytetracycline-degrading enzyme produced by strain N2-18 has a protein size range of 31 kDa-43 kDa and contains 4 bands.The sulfadiazine-degrading enzyme produced by the T-14 strain has a protein size of about 33 kDa and 1 band.4.Identify the molecular structure of tetracycline,oxytetracycline,and sulfadiazine degradation products,deduce their degradation pathways,and test the antibacterial ability of the products.After the degradation enzymes obtained by the above purification are reacted with corresponding antibiotics,the degradation products are collected by HPLC,and the molecular structure is detected by ESI-MS and the degradation pathway is deduced.The results show that:The tetracycline degrading enzyme can produce 3 degradation products,which are M/Z415,M/Z369 and M/Z3 12,According to this,it is preliminarily inferred that there are 3 degradation pathways and 6 degradation steps of tetracycline:demethylation,deamination,oxidation,demethylation,deamidation and dehydration.The oxytetracycline degrading enzyme can produce three kinds of degradation products,which are m/z543,m/z369 and m/z431.Three degradation pathways and seven degradation steps of oxytetracycline are preliminarily inferred:hydroxylation.demethylation and dehydration,Deamination,decarbonylation.secondary alcohol oxidation,benzene ring cracking.The sulfadiazine degradation enzyme can produce two kinds of degradation products,m/z143 and m/z283.It is preliminarily deduced that sulfadiazine has two degradation pathways and four degradation steps:S-N bond hydrolysis,dehydrogenation,hydroxylation,and deamination.5.The drug sensitivity of the strains degrading tetracycline,oxytetracycline and sulfadiazine and the safety of the degradation products were proved.Using the bacteriostatic circle method to detect the antibacterial ability of the degradation products obtained by the degradation of tetracycline,oxytetracycline and sulfadiazine against Escherichia coli,Salmonella,Staphylococcus aureus and Shigella.The results show that:Tetracycline degradation products m/z369,m/z415,m/z312 significantly reduced the inhibitory ability of the above four pathogens.Oxytetracycline degradation product m/z 369,product m/z 543,product m/z 431 significantly reduced the ability to inhibit the above four pathogens.Sulfadiazine degradation product m/z283,product m/z 143 significantly reduced the ability to inhibit the above four pathogens.In other words,the bacteriostatic ability of the products of the three antibiotics after degradation was significantly lower than that before degradation.It reduces the potential biosecurity risks of the degradation products of the three antibiotics to the environment.Six antibiotics,namely ampicillin,doxycycline,ciprofloxacin,streptomycin sulfate,compound sulfamethoxazole,and cefazolin ammonium,were selected according to the method specified in THE MINISTRY of Agriculture standard NY1109-2017 to test their inhibitory ability on the degradation strains of tetracycline,oxytetracycline and sulfadiazine.The results showed that tetracycline degrading bacteria N2-13,oxytetracycline degrading bacteria N2-18 and sulfadiazine degrading bacteria T-14 all met the relevant safety regulations of the Ministry of Agriculture and could be used in agricultural production.This result determined the drug sensitivity of the tetracycline degrading bacteria N2-13,oxytetracycline degrading bacteria N2-18,and sulfadiazine degrading bacteria T-14.6.Research on the practical application of strains.Scientifically mix the degrading bacteria agent with the decomposed bacteria agent to make a compound bacteria.In order to investigate the application potential of three biodegradable strains in manure composting technology,the artificial simulated mixed composting was conducted by using compound bacteria in the anti-fecal material.The results show that:After the composting,the compound fungus composed of tetracycline degrading bacteria N2-13 has a degradation rate of 94.51%for tetracycline,which can effectively promote the temperature of the pile to reach a high temperature period and accelerate the rot of the pile.At the end of the compost,the total nitrogen in the pile Increased by 1.14%.organic matter content reduced to 45.42%,seed germination index reached more than 80%,in line with national harmless organic fertilizer requirements.Oxytetracycline-degrading bacteria N2-18 has a deg,radation rate of 86.22%for oxytetracycline after composting,which can effectively promote the temperature of the pile to reach a high temperature period and accelerate the maturation of the pile.At the end of the compost,the total nitrogen Increased by 1.06%.organic matter content reduced to 43.83%,seed germination index reached more than 80%.in line with national harmless organic fertilizer requirements.Sulfadiazine degrading bacteria T-14 at the end of the composting,the degradation rate of 89.55%of sulfadiazine,can effectively promote the pile body temperature to high temperature period in advance and to accelerate the pile of rotten body,in the late compost,pile body increased 1.07%amount of total nitrogen and organic matter content down to 45.21%,seed germination index above 80%,conform to the requirements of the national harmless organic fertilizer.In short,the three degrading strains not only have good actual degradability,but the composite bacterial agent composed of them can also promote the harmless treatment of chicken manure.To sum up:This study obtained can be efficient degradation of tetracycline,oxytetracycline,sulfadiazine strain of three kinds of antibiotics,explores the reason the degradation mechanism of degradation strains to the appropriate antibiotics,the three antibiotics degradation pathway is deduced,verified the degradation bacterium and antibiotics degradation products of safety,finally proved the degradation characteristics,and investigates the feasibility of applying degradation strains to manure composting technology and application effect,tetracycline,oxytetracycline,sulfadiazine biodegradation laid a theoretical foundation and provides the data for the research of practical production. |
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