Screening Of Sulfamethazine High-efficiency Degradation Bacteria And Study On Degradation Mechanism

Sulfonamide antibiotics are widely used in clinical treatment,livestock and poultry breeding and other fields.However,in recent years,excessive residue levels have been continuously detected in water,soil,and even food.The environmental pollution caused by them and the induction of bacterial resistance have gradually become prominent.Bringing certain hidden dangers and hazards to ecological balance and human health,it is imminent to study efficient and environmentally friendly sulfonamide antibiotic removal methods.Microbial remediation methods can degrade or convert pollutants in the environment into simple compounds without pollution,and have the advantages of good effect,low cost,and environmental friendliness,and have become a research hotspot in the field of pollution remediation.Therefore,screening and isolating microorganisms that can effectively remove sulfonamides antibiotics and studying their degradation characteristics and related mechanisms have practical significance for the pollution control of sulfonamides antibiotics in the environment,and provide a certain scientific basis for the practical application of efficient degrading bacteria from the degradation mechanism level.In this study,sulfamethazine(SM2)was selected as the representative sulfa antibiotics,and the efficient degrading microorganisms of SM2 were screened and identified,and then the degradation characteristics,degradation enzymes,degradation pathways and functional genes of the strains were studied and analyzed.The specific research conclusions were as follows:(1)Screen out and identify microorganisms with high efficiency of SM2 degradation.A high-efficiency degrading bacterium that can degrade SM2 was isolated and screened from the soil,numbered H38.The bacterium can completely degrade SM2 within 72 hours.After morphological observation,scanning electron microscopy analysis,16S r RNA gene sequencing,and phylogenetic tree Genetic relationship analysis identified the strain as Bacillus thuringiensis.(2)Research and determine the degradation characteristics of strain H38.The OD₆₀₀growth curve and degradation curve of the strain and the optimal growth and degradation environmental conditions of the strain are initially obtained:the optimal temperature is 25?,the optimal p H of the culture solution is 7.0,the optimal initial concentration of SM2 is 5mg/L,and the optimum amount of inoculation is 5%,the shaker speed is 150 r/min,the optimal culture time is 9 h,and the complete degradation time is 72 h.(3)Research and analyze the degradation enzymes and degradation pathways of strain H38.(1)Positioning of degrading enzymes:The degrading enzymes of the bacteria that efficiently degrade SM2 mainly play a role in the cell and belong to intracellular enzymes.(2)Analysis of degradation products and degradation pathways:using liquid quality to analyze the pathways and intermediate products of strain H38 degrading SM2,it was detected that 2-amino-4,6-dimethylpyrimidine and N-(4,6-Dimethylpyrimidin-2yl)-1,4-diphenylamine,N-(3,5-dimethylpyrimidine)-benzenesulfonamide and other three main degradation products.Through qualitative and quantitative analysis of the product,two possible ways of degradation of SM2 by this strain were analyzed.(4)Functional genes were analyzed by complete genome sequencing of strain H38.(1)The basic information of H38 genome was obtained:the total length of gene sequence was5477631 bp,the total number of scaffold was 72,and the GC content was 35.21%.(2)The pathway of SM2 participating in nitrogen and sulfur metabolism of strain H38 was analyzed.The structure of dimethyl sulfone in SM2 was transferred to sulfite through dimethyl sulfone monooxygenase and alkane sulfonate monooxygenase,and then assimilation reaction was initiated by assimilation sulfite reductase to synthesize amino acids,succinate and acetate,etc.,to maintain the life activity of strain H38.(3)Analyze the functional gene information annotated by the strain:it was found that the plasmid of strain H38 carried the gene sul4related to sulfa antibiotic resistance,which made the strain H38 tolerate antibiotic pollution and could grow and metabolize normally and exert degradation effects.In this study,strain H38 with good degradation effect on sulfadimethylpyrimidine was obtained,the degradation characteristics of this degradant were clarified,the degradation mechanism of the degradant was elucidated at multiple angles,which provided material accumulation and theoretical basis for the application of degradant bacteria to the remediation of antibiotic polluted environment.