有机磷农药草甘膦降解菌的筛选、分离及其鉴定
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摘要
有机磷农药因其经济、高效、使用方便等特点,是目前使用最广泛的农药品种之一。对于防治病虫草害、保证农作物产量起到了巨大的作用。我国是农药生产大国,不仅自身需求量很大,而且还远销东南亚多个国家。其中有机磷农药有200多种,产量占全国农药总量的50%以上,占世界农药总量的30%。
微生物是生物修复的重要生物资源。利用微生物产生的降解酶降解处理环境中有机磷农药的方法,已显示出良好的应用前景,是近年来研究有机磷农药降解的主要方向。因此,本实验的目的就是寻找出对有机磷农药降解效果好的优势菌种,对其进行生理生化分析和鉴定,同时,探索优化降解有机磷农药的最佳条件,为深入研究有机磷降解菌制剂做好前期准备工作。
作者从四川贝尔农药厂污水处理池中采集活性污泥样品,对其进行解磷细菌的分离和筛选。根据解磷圈的大小和菌液浓度值,初筛获得了26株解磷细菌；通过钼锑抗比色法,从降解性能和稳定性能两方面考率,复筛出两株降解有机磷农药草甘膦性能较好且稳定的菌株BR13和BR57,其降解率分别为60.20%和57.67%。
对这两株菌进行生理生化试验和16S rDNA序列分析,结果表明BR13为恶臭假单胞菌,BR57为肠肝菌科。通过对这两株菌降解有机磷农药影响因素的一系列分析,表明菌株BR13和BR57对有机磷农药草甘膦的降解具有遗传稳定性,能较好的降解草甘膦。通过单因素及正交实验,确定菌株BR13降解草甘膦的最佳培养组合为：蔗糖2.5%、草酸铵0.1%、培养温度28℃、摇床转速180r/min。菌株BR57降解草甘膦的最佳培养组合为：蔗糖1%、草酸铵0.1%、培养温度30℃、摇床转速120r/min。
Organophosphate pesticides is the most widely used pesticide varieties because of their economic, efficient, easy to use. For the prevention and control pests and ensure food production has played a very important role. China is a large pesticide production country, not only its own requirements for large, but also exported to many countries in southeast Asia. Which has more than 200 kinds of organophosphorus pesticides, production accounting for more than 50% of the total pesticides, about 30% of the world's total.
microbes feature fast and easy manipulation, and have many varieties, They are important biological resources of bioremediation. Use of degrading enzymes produced by micro-organisms to deal with environmental degradation of organophosphorus pesticides, have shown a good prospect, is the main development direction about the degradation of organophosphorus pesticides in recent years. Therefore, the purpose of this study is to find out the degradation of organophosphorus pesticides good dominant species, to do some physiological and biochemical analysis and identification. At the same time,to find the optimum conditions of optimizing the degradation of organophosphorus pesticides, finish the preparatory work for in-depth study of preparation of organic phosphorus degrading bacteria.
Isolate and identificate the methlparathion-degrading bacteria from activated sludge sample of wastewater treating pond of Sichuan Bei'er pesticide factory. According to a series of mathods, we have high efficient methlparathion-degrading bacteria BR13 and BR57, the degradation rates were 60.20% and 57.67%.
Based on the results of morphology, physiological and biochemical properties and phylogenetic analysis of 16S rDNA sequence, strain BR13 was identified as P. Putida, strain BR57 was identified as Morganella sp. Through analyze factors of organophosphorus pesticide impact at bacterail of degradation. BR13 and BR57 on the degradation of organophosphorus pesticide with the genetic stability can be better to degrade glyphosate. though the single factor and orthogonal experiment. the degradation of glyphosate BR13 best combination of sucrose 2.5%,0.1% oxalate ammonium temperature 28℃, shaking speed 180r/min. Degradation of glyphosate BR57 best combination of 1% sucrose,0.1% oxalate ammonium, temperature 30℃, shaking speed 120r/min.
微生物是生物修复的重要生物资源。利用微生物产生的降解酶降解处理环境中有机磷农药的方法,已显示出良好的应用前景,是近年来研究有机磷农药降解的主要方向。因此,本实验的目的就是寻找出对有机磷农药降解效果好的优势菌种,对其进行生理生化分析和鉴定,同时,探索优化降解有机磷农药的最佳条件,为深入研究有机磷降解菌制剂做好前期准备工作。
作者从四川贝尔农药厂污水处理池中采集活性污泥样品,对其进行解磷细菌的分离和筛选。根据解磷圈的大小和菌液浓度值,初筛获得了26株解磷细菌；通过钼锑抗比色法,从降解性能和稳定性能两方面考率,复筛出两株降解有机磷农药草甘膦性能较好且稳定的菌株BR13和BR57,其降解率分别为60.20%和57.67%。
对这两株菌进行生理生化试验和16S rDNA序列分析,结果表明BR13为恶臭假单胞菌,BR57为肠肝菌科。通过对这两株菌降解有机磷农药影响因素的一系列分析,表明菌株BR13和BR57对有机磷农药草甘膦的降解具有遗传稳定性,能较好的降解草甘膦。通过单因素及正交实验,确定菌株BR13降解草甘膦的最佳培养组合为：蔗糖2.5%、草酸铵0.1%、培养温度28℃、摇床转速180r/min。菌株BR57降解草甘膦的最佳培养组合为：蔗糖1%、草酸铵0.1%、培养温度30℃、摇床转速120r/min。
Organophosphate pesticides is the most widely used pesticide varieties because of their economic, efficient, easy to use. For the prevention and control pests and ensure food production has played a very important role. China is a large pesticide production country, not only its own requirements for large, but also exported to many countries in southeast Asia. Which has more than 200 kinds of organophosphorus pesticides, production accounting for more than 50% of the total pesticides, about 30% of the world's total.
microbes feature fast and easy manipulation, and have many varieties, They are important biological resources of bioremediation. Use of degrading enzymes produced by micro-organisms to deal with environmental degradation of organophosphorus pesticides, have shown a good prospect, is the main development direction about the degradation of organophosphorus pesticides in recent years. Therefore, the purpose of this study is to find out the degradation of organophosphorus pesticides good dominant species, to do some physiological and biochemical analysis and identification. At the same time,to find the optimum conditions of optimizing the degradation of organophosphorus pesticides, finish the preparatory work for in-depth study of preparation of organic phosphorus degrading bacteria.
Isolate and identificate the methlparathion-degrading bacteria from activated sludge sample of wastewater treating pond of Sichuan Bei'er pesticide factory. According to a series of mathods, we have high efficient methlparathion-degrading bacteria BR13 and BR57, the degradation rates were 60.20% and 57.67%.
Based on the results of morphology, physiological and biochemical properties and phylogenetic analysis of 16S rDNA sequence, strain BR13 was identified as P. Putida, strain BR57 was identified as Morganella sp. Through analyze factors of organophosphorus pesticide impact at bacterail of degradation. BR13 and BR57 on the degradation of organophosphorus pesticide with the genetic stability can be better to degrade glyphosate. though the single factor and orthogonal experiment. the degradation of glyphosate BR13 best combination of sucrose 2.5%,0.1% oxalate ammonium temperature 28℃, shaking speed 180r/min. Degradation of glyphosate BR57 best combination of 1% sucrose,0.1% oxalate ammonium, temperature 30℃, shaking speed 120r/min.
引文
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[5]L.Colosio, E Gorsini, W.Bareellini. et al. Immune Parameters in biological monitoring of Pesticide exposure Current knowledge and Perspectives[J]. Toxicol.Lett.,1999,108:285-295
[6]Beate R, Fei Y. Parkinson's disease mortality and pesticide exposure in Californial 1984-1994[J]. International Journal of Epidemiology,2000,29:323-329
[7]王永杰,李顺鹏,沈标,等.有机磷农药广谱活性降解茵的分离及其生理特性研究[J].南京农业大学学报,1999,22(2)：42-45
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[9]戴树桂.环境化学[M].高等教育出版社,1997,212-223
[10]张超,李冀新.微生物降解有机磷农药残留机理及菌种筛选研究进展[J].农药科学与管理,2006,27(4)：29-32
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[12]张素琴.微生物分子生态学[M].北京：科学出版社,2006
[13]王宝军,刘志培,杨惠芳.单甲脒农药的微生物降解代谢研究[J].环境科学学报,1998,18(3)：296-302
[14]虞云龙,樊德方,陈鹤鑫.农药微生物降解的研究现状与繁殖策略[J].环境科学进展,1996,4(3)：28-36
[15]马瑛,张甲耀,管筱武,罗宇煊.原毛平革菌堆肥处理有害废弃物的可行性[J].环境科学,1999,20(6)：67-70
[16]刘云焕,陈东海.微生物降解有机磷农药的研究进展[J].北方环境,2005,30(1)：22-24
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[18]刘斌斌,赵永芳,钞亚鹏,谢裕敏,王银善.鲁氏酵母WY-3降解甲胺磷的性能[J].环境科学,2001,22(4)：40-44
[20]王永杰.污染物生物降解[J].微生物学杂志,1999,19(4)：58-62
[21]郑重.农药的微生物降解[J].环境科学,1990,11(2)：68-72
[22]Barik S. Metabolism of in secticides by microorganisms[A]. R Lal.ed. In:Insecticide microbiology [M]. Springer Berling,1984,87-130
[23]Mae A A, Marits R O, Ausmees N R, Koiv V M, Heinaru A L. Characterization of a new 2,4-dich lorophenoxyacetic acid degarading plasmid PEST4011 [J]. Physical Microbiol,1993,139:3165-3170
[24]芳玲.降解有机氯农药的微生物菌株分离筛选及其应用效果[J].应用生态学报,11(2)：249-252
[25]方晓航,仇荣亮.有机磷农药在十壤环境中的降解转化[J].环境利学与技术,2003,16(2)：57-62
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