异菌脲降解菌YJN-G的分离、鉴定及降解特性
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摘要
为去除环境中异菌脲残留,从某农药厂废水处理系统的活性污泥中分离到一株异菌脲降解菌YJN-G,对其进行鉴定和降解特性分析.通过形态特征、生理生化特性和16S rRNA基因序列相似性分析,将其初步鉴定为微杆菌属(Microbacterium sp.).当接种量为5%时,菌株YJN-G在24 h内能够降解100 mg/L的异菌脲.菌株YJN-G降解异菌脲的最适pH是7.0,最适温度为30-37℃.通过对其降解异菌脲产物的质谱分析,确定其代谢产物为N-(3,5-二氯苯基)-2,4-二氧代咪唑烷和异丙基氨基甲酸;菌株能够利用异丙基氨基甲酸生长,但是不能进一步降解N-(3,5-二氯苯基)-2,4-二氧代咪唑烷.菌株降解异菌脲的水解酶属于胞内酶.本研究结果可为异菌脲污染环境的生物修复提供菌株资源和理论依据.
To clean up iprodione residues in the environment, a bacterial strain YJN-G capable of degrading iprodione was isolated from the activated sludge in the wastewater treatment system of a pesticide factory. Based on its physiological & biochemical characters and the similarity analysis of 16 S r RNA gene sequence, strain YJN-G was primarily identified as Microbacterium sp. With the inoculation amount of 5%, strain YJN-G was able to degrade 100 mg L~(-1) iprodione within 24 hours, with optimum temperature of 30-37 °C and pH of 7.0. The metabolites were analyzed by mass spectrometry and identified as N-(3,5-dichlorophenyl)-2,4-dioxo-imidazolidin and isopropyl carbamic acid. Strain YJN-G could use isopropyl carbamic acid as the carbon source for growth, but could not further degrade N-(3,5-dichlorophenyl)-2,4-dioxo-imidazolidin. The hydrolase responsible for the degradation of iprodione was an endoenzyme. The result of this study provides strain resource and theoretical basis for bioremediation of iprodione-contaminated environment.
To clean up iprodione residues in the environment, a bacterial strain YJN-G capable of degrading iprodione was isolated from the activated sludge in the wastewater treatment system of a pesticide factory. Based on its physiological & biochemical characters and the similarity analysis of 16 S r RNA gene sequence, strain YJN-G was primarily identified as Microbacterium sp. With the inoculation amount of 5%, strain YJN-G was able to degrade 100 mg L~(-1) iprodione within 24 hours, with optimum temperature of 30-37 °C and pH of 7.0. The metabolites were analyzed by mass spectrometry and identified as N-(3,5-dichlorophenyl)-2,4-dioxo-imidazolidin and isopropyl carbamic acid. Strain YJN-G could use isopropyl carbamic acid as the carbon source for growth, but could not further degrade N-(3,5-dichlorophenyl)-2,4-dioxo-imidazolidin. The hydrolase responsible for the degradation of iprodione was an endoenzyme. The result of this study provides strain resource and theoretical basis for bioremediation of iprodione-contaminated environment.
引文
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3赵子丹,牛艳,姜瑞,葛谦,王晓菁.黄瓜及土壤中异菌脲残留量的气相色谱分析[J].湖北农业科学,2015,54(10):2483-2485[Zhao ZD,Niu Y,Jiang R,Ge Q,Wang XJ.Detection of iprodione residue in cucumber and soil by GC[J].Hubei Agric Sci,2015,54(10):2483-2485]
4曹彦卫.异菌脲在梨和土壤中残留量的测定方法[J].河北工业科技,2012,29(5):321-322[Cao YW.Determination of iprodione residue in pear and soil[J].Hebei J Ind Sci Technol,2012,29(5):321-322]
5 Loutfy N,Malhat F,Kamel E,Saber A.Residual pattern and dietary intake of iprodione on grapes under egyptian field conditions:a prelude to risk assessment profile[J].Human Ecol Risk Assess,2015,21(1):265-279
6 Costa AIG,Queiroz MELR,Neves AA,de Sousa FAD,Zambolim L.Determination of pesticides in lettuce using solid-liquid extraction with low temperature partitioning[J].Food Chem,2015,181:64-71
7 Mamiev M,Korolev N,Elad Y.Resistance to polyoxin AL and other fungicides in Botrytis cinerea collected from sweet basil crops in Israel[J].Eur J Plant Pathol,2013,137(1):79-91
8 Athiel P,Alfizarmercadier C,Vega D,Bastide J,Davet P,Brunel B,Cleyet MJ.Degradation of iprodione by a soil Arthrobacter-like strain[J].Appl Environ Microbiol,1995,61(9):3216-3220
9 Mercadier C,Vega D,Bastide J.Iprodione degradation by isolated soil microorganisms[J].FEMS Microbiol Ecol,1997,23(3):207-215
10 Claudia Z,Gianluigi C,Laura C,Fabrizio F,Cesare M.Biodegradation of the fungicide iprodione by Zygosaccharomyces rouxii strain DBVPG639 9[J].J Agric Food Chem,2006,54:4734-4739
11徐剑宏,洪青,洪源范,李顺鹏.呋喃丹降解菌C F DS-1的筛选鉴定及降解特性[J].应用与环境生物学报,2011,17(2):237-242[Xu JH,Hong Q,Hong YF.Isolation,identif ication and degradation of carbofuran-degrading strain CFDS-1[J].Chin J Appl Environ Biol,2011,17(2):237-242]
12 Holt JG,K rieg NR,Sneath PH.Bergey’s Manual of Determinative Bacteriology[M].Baltimore:MD Lippincott Williams and Wilkins,1994
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14 Marchesi JR,Sato T,Weightman AJ,Martin TA,Fry JC.Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S r RNA[J].Appl Environ Microbiol,1998,64(2):795-799
15 Sambrook J E,Fritsch EFM,Maniatis TE.Molecular Cloning:ALaboratory Manual[M].New York:Cold Spring Harbor,1989
16沈萍,范秀荣,李广武.微生物学实验[M].北京:高等教育出版社,1999[Shen P,Fan XR,Li GW.Experiment of microbiology[M].Beijing:China Higher Education Press,1999]
17吴春红,聂继云,李静,李海飞,徐国峰.基质分散固相萃取-高效液相色谱法测定水果中的异菌脲残留量[J].食品科学,2009,14:188-190[Wu CH,Nie J Y,Li J,Li HF,Xu GF.Combination of matrix solid phase dispersion extraction with high performance liquid chromatography for the determination of iprodione residue in fruits[J].Food Sci,2009,14:188-190]
18许敬亮,王志春,王堃,洪青,李顺鹏.降解菌djl-6多菌灵水解酶的提取及性质[J].应用与环境生物学报,2012,18(2):272-275[Xu JL,Wang ZC,Wang K.Extraction and characters of carbendazim hydrolase from strain djl-6[J].Chin J Appl Environ Biol,2012,18(2):272-275]
2梁林,张爱娟,潘金菊,翟淑华,刘伟.超高效液相色谱-串联质谱法测黄瓜及番茄中异菌脲残留[J].农药科学与管理,2014,35(4):45-48[Liang L,Zhang AJ,Pan JJ,Zhai SH,Liu W.Determination of iprodione residue in cucumber and tomato by UPLC-MS/MS[J].Pest Sci Admin,2014,35(4):45-48]
3赵子丹,牛艳,姜瑞,葛谦,王晓菁.黄瓜及土壤中异菌脲残留量的气相色谱分析[J].湖北农业科学,2015,54(10):2483-2485[Zhao ZD,Niu Y,Jiang R,Ge Q,Wang XJ.Detection of iprodione residue in cucumber and soil by GC[J].Hubei Agric Sci,2015,54(10):2483-2485]
4曹彦卫.异菌脲在梨和土壤中残留量的测定方法[J].河北工业科技,2012,29(5):321-322[Cao YW.Determination of iprodione residue in pear and soil[J].Hebei J Ind Sci Technol,2012,29(5):321-322]
5 Loutfy N,Malhat F,Kamel E,Saber A.Residual pattern and dietary intake of iprodione on grapes under egyptian field conditions:a prelude to risk assessment profile[J].Human Ecol Risk Assess,2015,21(1):265-279
6 Costa AIG,Queiroz MELR,Neves AA,de Sousa FAD,Zambolim L.Determination of pesticides in lettuce using solid-liquid extraction with low temperature partitioning[J].Food Chem,2015,181:64-71
7 Mamiev M,Korolev N,Elad Y.Resistance to polyoxin AL and other fungicides in Botrytis cinerea collected from sweet basil crops in Israel[J].Eur J Plant Pathol,2013,137(1):79-91
8 Athiel P,Alfizarmercadier C,Vega D,Bastide J,Davet P,Brunel B,Cleyet MJ.Degradation of iprodione by a soil Arthrobacter-like strain[J].Appl Environ Microbiol,1995,61(9):3216-3220
9 Mercadier C,Vega D,Bastide J.Iprodione degradation by isolated soil microorganisms[J].FEMS Microbiol Ecol,1997,23(3):207-215
10 Claudia Z,Gianluigi C,Laura C,Fabrizio F,Cesare M.Biodegradation of the fungicide iprodione by Zygosaccharomyces rouxii strain DBVPG639 9[J].J Agric Food Chem,2006,54:4734-4739
11徐剑宏,洪青,洪源范,李顺鹏.呋喃丹降解菌C F DS-1的筛选鉴定及降解特性[J].应用与环境生物学报,2011,17(2):237-242[Xu JH,Hong Q,Hong YF.Isolation,identif ication and degradation of carbofuran-degrading strain CFDS-1[J].Chin J Appl Environ Biol,2011,17(2):237-242]
12 Holt JG,K rieg NR,Sneath PH.Bergey’s Manual of Determinative Bacteriology[M].Baltimore:MD Lippincott Williams and Wilkins,1994
13东秀珠,蔡妙英,刘彭涛.常见细菌系统鉴定手册[M].北京:科学出版社,2001[Dong XZ,Cai MY,Liu PT.Manual of Systemaic Common Bacteriology[M].Beijing:Science Press,2001]
14 Marchesi JR,Sato T,Weightman AJ,Martin TA,Fry JC.Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S r RNA[J].Appl Environ Microbiol,1998,64(2):795-799
15 Sambrook J E,Fritsch EFM,Maniatis TE.Molecular Cloning:ALaboratory Manual[M].New York:Cold Spring Harbor,1989
16沈萍,范秀荣,李广武.微生物学实验[M].北京:高等教育出版社,1999[Shen P,Fan XR,Li GW.Experiment of microbiology[M].Beijing:China Higher Education Press,1999]
17吴春红,聂继云,李静,李海飞,徐国峰.基质分散固相萃取-高效液相色谱法测定水果中的异菌脲残留量[J].食品科学,2009,14:188-190[Wu CH,Nie J Y,Li J,Li HF,Xu GF.Combination of matrix solid phase dispersion extraction with high performance liquid chromatography for the determination of iprodione residue in fruits[J].Food Sci,2009,14:188-190]
18许敬亮,王志春,王堃,洪青,李顺鹏.降解菌djl-6多菌灵水解酶的提取及性质[J].应用与环境生物学报,2012,18(2):272-275[Xu JL,Wang ZC,Wang K.Extraction and characters of carbendazim hydrolase from strain djl-6[J].Chin J Appl Environ Biol,2012,18(2):272-275]