厌氧丁草胺降解菌BAD-20的分离鉴定及降解特性研究

Isolation,identification and degradation characteristics of an anaerobic butachlor-degrading bacterium BAD-20

【目的】分离并鉴定能够降解除草剂丁草胺的厌氧微生物菌株,研究其厌氧降解丁草胺的特性和代谢途径,为深入研究丁草胺厌氧降解机制提供依据。【方法】以丁草胺为碳源作为选择压力从水稻田土壤中富集驯化丁草胺降解菌,利用16S rRNA基因系统发育分析结合菌株培养特征对降解菌株进行初步鉴定,利用液相色谱-时间飞行质谱(LC-TOF-MS)检测菌株降解丁草胺的代谢产物。【结果】筛选到一株降解丁草胺的厌氧细菌,命名为BAD-20,初步鉴定为嗜蛋白质菌属(Proteiniphilum),菌株BAD-20降解丁草胺的最适条件为温度30–35℃、pH 7.5–8.0和0–0.5%NaCl,在有氧条件下该菌不能降解丁草胺。最适条件下,菌株BAD-20在10 d降解90%的20 mg/L丁草胺。菌株BAD-20还能降解甲草胺、乙草胺、丙草胺,降解效率从高到低依次为甲草胺>乙草胺>丙草胺>丁草胺,对这些氯乙酰胺除草剂的降解动力学符合一级动力学方程。鉴定到2个丁草胺降解代谢产物,分别是N-(2,6-二乙基苯基)-N-(丁氧甲基)乙酰胺(DEPBMA)和N-(2,6-二乙基苯基)乙酰胺(DEPA),表明菌株BAD-20降解丁草胺的起始步骤为脱氯,随后脱去N-丁氧甲基。【结论】本研究富集分离到一株降解丁草胺的厌氧细菌嗜蛋白质菌属(Proteiniphilum)BAD-20,为深入研究丁草胺厌氧降解机制及研发含丁草胺废水厌氧生物处理技术提供依据。

[Objective]The aim of this study was to isolate anaerobic bacteria capable of degrading butachlor.[Methods]By enrichment and acclimation method using butachlor as carbon source for enrichment,we screened an anaerobic butachlor-degrading bacterium from paddy soil.The isolated strain was preliminarily identified based on morphological and biochemical characteristics as well as 16S rRNA phylogenetic analysis,and the metabolites of butachlor degradation were identified by liquid chromatography-time of flight mass spectrometry.[Results]An anaerobic bacterium designated as BAD-20 was screened and identified as Proteiniphilum.The optimum conditions for butachlor degradation by strain BAD-20 were 30–35℃,pH 7.5–8.0 and 0–0.5%NaCl.Under the optimal conditions,90%butachlor was degraded within 10 days by Proteiniphilum sp.BAD-20.Under aerobic conditions,the stain lost the ability to degrade butachlor.Proteiniphilum sp.BAD-20 could also degrade alachlor,acetochlor and propionate with the degradation efficiency following the order:alachlor>acetochlor>propranolol>butachlor.The degradation kinetics to these chloroacetamide herbicides fit to a first-order kinetic equation.Two metabolites,N-(2,6-diethylphenyl)-N-(butoxymethyl)acetanilide and N-(2,6-diethylphenyl)acetamide,were identified,indicating that the initial two steps of butachlor degradation are dechlorination and dealkylation.[Conclusion]An anaerobic butachlor-degrading bacterium BAD-20 was enriched and isolated from paddy soil and identified as Proteiniphilum.This study provides a basis for further study on the anaerobic catabolism of butachlor and the development of anaerobic biological treatment technology for butachlor-containing wastewater.