Screening Of Sulfamonomethoxine Degrading Bacteria And Study On Its Degradation Pathway

A method was developed for the determination of sulfadiazine,sulfamethoxazole and sulfamonomethoxine in animal feces by solid phase extraction(SPE)and high performance liquid chromatography(HPLC).The mixture of methanol:acetonitrile:phosphate buffer(1:1:2,V/V)was selected as the sample extraction solution.Ultrasonic wave was used for 20 min,centrifuged at 4?10000 r/min for 15 min,and repeated twice.The supernatant was combined and diluted,adjusted to p H 4.0.After the HLB column was activated,the eluent was blown to near dryness under weak nitrogen at 40?,and redissolved with 1 ml 0.1%formic acid methanol,and then redissolved with 0.22?m filter membrane.The filtrate was separated by C18column and gradient eluted with 0.1%formic acid methanol as mobile phase.Quantitative analysis by external standard method showed that there was a linear relationship between the three sulfonamides and their corresponding peak areas in a certain concentration range,and the correlation coefficient R~2 was more than 0.999,indicating that the linear relationship was good.The limits of quantitation and detection of the three antibiotics were 20.83?g/kg and 6.25?g/kg,respectively.The relative standard deviations were in the range of 2.19%?13.53%.The recoveries of the optimized HLB-HPLC method ranged from 77.16%to 95.54%,with good repeatability and accuracy.It can be used for the rapid detection of three sulfonamides in laboratory.Strain DT-8 was isolated from chicken manure containing sulfamonomethoxine and identified as Enterobacter hormaechei by morphology and molecular biology.Through the single factor test,four factors which have obvious influence on the degradation performance of degradation bacteria were screened out,which were p H,temperature,inoculum and substrate concentration.Then,the degradation performance test of degradation bacteria was designed by response surface method.Through software analysis,the optimal degradation conditions were p H6.0,temperature 30?,inoculum 3%,substrate concentration 1 mg/L,and the optimized degradation rate could reach 43.51%.In order to further explore the specific way of degradation of sulfamonomethoxine by degrading bacteria,LC/MS-MS was used to explore the changes of metabolites in the process of microbial culture,and two possible degradation pathways were deduced.In these two pathways,sulfamonomethoxine was finally degraded into simple-structuerd carbon chain.