氯霉素高效降解菌分离鉴定及其特性研究

Study on Isolation,Identification and Degradation Characteristics of Chloramphenicol Degrading Bacteria

本研究筛选可高效降解氯霉素的微生物,并研究其降解特性。选用氯霉素高残留的花甲螺进行培养驯化,使用含氯霉素的营养琼脂平板对目标菌进行筛选分离;采用显微镜对目标菌进行形态学观察,使用全自动微生物分析系统对目标菌株进行生化鉴定,同时利用细菌16S rDNA序列测序的方法对目标菌株进行菌种鉴定;利用高效液相色谱-串联质谱法测定样品和反应物中氯霉素的含量,在不同反应时间、温度、pH、底物浓度、菌悬液含量等条件下,进行降解特性研究。最终,驯化分离一株具有降解氯霉素作用的菌株,代号SA-cd1,经形态特征、生理生化和16S rDNA序列比对结果鉴定为海藻希瓦氏菌(Shewanella algae)。SA-cd1菌降解氯霉素的温度为36~50℃,pH为6.0~9.0,0.1 mg/L氯霉素5 min的降解率为83.8%,降解率随菌悬液浓度增大而升高,随反应时间变长而升高;底物浓度提高,降解率有略微下降,10 mg/L氯霉素1 h内降解率达92.5%;除去菌体的上清液仍然具有较强的降解率,为47.3%。SA-cd1海藻希瓦氏菌分离培养容易,对氯霉素的降解效率高,适宜的温度和pH条件宽泛,可应用于环境中氯霉素的降解清除以及相关降解工程菌、降解酶的研发。

This study focuses on screening out the microorganisms that can degrade chloramphenicol efficiently and analyzing their degradation characteristics.The Paphia undulata with high chloramphenicol residue was cultured and domesticated.The target bacteria was screened and separated by nutrient agar plate containing chloramphenicol.In this research,a microscope was used to observe morphology of the target bacteria;a fully automatic microbial analysis system was adopted to conduct biochemical identification of the target strains;and bacterial 16S rDNA sequencing was used to identify the target strains.The content of chloramphenicol in samples or reactants was determined by high performance liquid chromatography tandem mass spectrometry.The degradation characteristics were studied under different reaction time,temperature,pH,substrate concentration and bacterial suspension content.A strain named SA-cd1,which can degrade chloramphenicol efficiently,was obtained by domestication and isolation.The bacteria was identified as Shewanella algae according to its morphological characteristics,physiological and biochemical characteristics and 16S rDNA sequencing.The optimal temperature and pH for chloramphenicol degradation of SA-cd1 was 36-50℃and 6.0-9.0.The degradation rate of 0.1 mg/L chloramphenicol in 5 min reached 83.8%,and rose with the increase of the concentration of the bacterial suspension or the reaction time.The degradation rate did not decrease significantly with the increase of the substrate concentration,and the degradation rate of 10 mg/L chloramphenicol in 1 h was 92.5%.The supernatant after the removal of bacteria still had a strong degradation rate of 47.3%.SA-cd1 is easy to isolate and culture,with high degradation efficiency to chloramphenicol,as well as a wide range of suitable temperature and pH conditions,thus it can be applied to the degradation and removal of chloramphenicol in the environment,as well as the development of related degradation engineering bacteria and enzymes.