多菌灵降解菌的生物学特性及降解能力研究

doi:10.13560/j.cnki.biotech.bull.1985.2017-0675

摘要/Abstract

摘要： 农药污染已成为影响人身健康及环境的主要原因。在污染土壤中引入降解特定农药的微生物可有效降低该农药的浓度,并且该方法绿色环保、无二次污染。为降低棚室土壤中的残留农药多菌灵,通过唯一碳源平板筛选法,从长期使用多菌灵的棚室土壤中分离筛选出一株分解利用多菌灵的微生物菌株WNP-3,该菌株可在含200 mg/L的多菌灵LB培养基中正常生长。经形态学、理化、Biolog及16S rDNA鉴定,确定该菌为小麦苍白杆菌（Ochrobactrum tritici）。该菌表现出宽泛的生长范围：在pH4-9,盐含量1%-3%条件下正常生长,对多种农药具有耐受性。经紫外分光光度法及HPLC验证WNP-3在无机盐培养基、28℃、180r/min摇瓶培养120 h的条件下,对100 mg/L多菌灵的降解率为61%。

关键词: 细菌, 多菌灵, 小麦苍白杆菌, 生物降解

Abstract: Pesticide pollution becomes major cause to environment safety and human health. Introducing microorganisms of degrading specific pesticide into polluted soil may efficiently reduce the concentration of this pesticide;moreover,this is a green technology and causes no secondary pollution. In order to reduce the residual carbendazim in the soil of a greenhouse,a strain WNP-3 decomposing and utilizing carbendazim was isolated via sole carbon source plate screening method from the greenhouse soil where carbendazim has been used for a long time. This strain normally grew in LB medium with carbendazim concentration of 200 mg/L. According to the results of morphology,physiological and biochemical property,Biolog identification,and 16S rDNA sequence analysis,WNP-3 was identified as Ochrobactrum tritici. The WNP-3 had broad growth range,i.e.,growing well at pH 4 - 9 in culture media containing 1%-3% NaCl,and it was resistant to several pesticides. Confirmed by UV spectrophotometry and HPLC,the carbendazim at concentration of 100 mg/L degraded 61% in mineral medium in 180 r/min flask for 120 h at 28℃.

Key words: bacteria, carbendazim, Ochrobactrum tritici, biodegradation

陈锐,孙晓宇,邓媛,路鹏鹏,赵玲侠,瞿佳,沈卫荣. 多菌灵降解菌的生物学特性及降解能力研究[J]. 生物技术通报, 2018, 34(5): 187-194.

CHEN Rui, SUN Xiao-yu, DENG Yuan, LU Peng-peng, ZHAO Ling-xia, QU Jia, SHEN Wei-rong. Biological Characteristic and Biodegradation Capacity of Ochrobactrum tritici WNP-3 for Carbendazim[J]. Biotechnology Bulletin, 2018, 34(5): 187-194.

参考文献

[1] Wang L, Jia H, Liu X, et al.Historical contamination and ecological risk of organochlorine pesticides in sediment core in northeastern Chinese river[J]. Ecotoxicology and Environmental Safety, 2013, 93:112-120.
[2] Cheng HX, Li XH, Wang YP, et al.Profile of organochlorine pesticides in soil cores from some hotspot areas of Beijing, China[J]. Bulletin of Environmental Contamination and Toxicology, 2011, 87(2):175-179.
[3] Jänsch S, Frampton GK, Römbke J, et al.Effects of pesticides on soil invertebrates in model ecosystem and field studies:A review and comparison with laboratory toxicity data[J]. Environmental Toxicology and Chemistry, 2006, 25(9):2490-2501.
[4] Damalas CA, Eleftherohorinos IG.Pesticide exposure, safety issues, and risk assessment indicators[J]. International Journal of Environmental Research and Public Health, 2011, 8(5):1402-1419.
[5] Yan H, Wang D, Dong B, et al.Dissipation of carbendazim and chloramphenicol alone and in combination and their effects on soil fungal:bacterial ratios and soil enzyme activities[J]. Chemosphere, 2011, 84(5):634-641.
[6] Van Gestel CAM.Validation of earthworm toxicity tests by comparison with field studies:A review of benomyl, carbendazim, carbofuran, and carbaryl[J]. Ecotoxicology and Environmental Safety, 1992, 23(2):221-236.
[7] Zhou J, Xiong K, Yang Y, et al.Deleterious effects of benomyl and carbendazim on human placental trophoblast cells[J]. Reproductive Toxicology, 2015, 51:64-71.
[8] Uludag B, Tarlaci S, Yuceyar N, et al.A transient dysfunction of the neuromuscular junction due to carbendazim intoxication[J]. Journal of Neurology, Neurosurgery & Psychiatry, 2001, 70(4):563.
[9] Yu H, Tang H, Zhu X, et al.Molecular mechanism of nicotine degradation by a newly isolated strain, Ochrobactrum sp. strain SJY1[J]. Applied and Environmental Microbiology, 2015, 81(1):272-281.
[10] Hu G, Zhao Y, Liu B, et al.Isolation of an indigenous imidacloprid-degrading bacterium and imidacloprid bioremediation under simulated in situ and ex situ conditions[J]. Journal of Microbiology & Biotechnology, 2013, 23(11):1617-1626.
[11] Aparicio J, Solá MZ, Benimeli CS, et al.Versatility of Streptomyces sp. M7 to bioremediate soils co-contaminated with Cr(VI)and lindane[J]. Ecotoxicology and Environmental Safety, 2015, 116:34-39.
[12] 赵春, 李水清. 湖北太湖港农场主要农产品中甲基硫菌灵及多菌灵的残留分析[J]. 长江大学学报:自然科学版, 2015, 21:43-46.
[13] Goodfellow M, Kämpfer P, Busse HJ, et al.Bergey's Manual of Systematic Bacteriology[M]. New York:Springer, 2012.
[14] 卜伟. 二氯甲烷萃取-HPLC法测定水中的多菌灵[J]. 环境与技术, 2005, 28(12):149-150.
[15] Alvarez A, Saez JM, Davila Costa JS, et al.Actinobacteria:Current research and perspectives for bioremediation of pesticides and heavy metals[J]. Chemosphere, 2017, 166:41-62.
[16] Medo J, Maková J, Kovácsová S, et al.Effect of Dursban 480 EC(chlorpyrifos)and Talstar 10 EC(bifenthrin)on the physiological and genetic diversity of microorganisms in soil[J]. Journal of Environmental Science and Health, Part B, 2015, 50(12):871-883.
[17] Raju MN, Venkateswarlu K.Effect of repeated applications of buprofezin and acephate on soil cellulases, amylase, and invertase[J]. Environmental Monitoring and Assessment, 2014, 186(10):6319-6325.
[18] Srinivasulu M, Jaffer Mohiddin G, Subramanyam K, et al.Effect of insecticides alone and in combination with fungicides on nitrification and phosphatase activity in two groundnut(Arachis hypogeae L. )soils[J]. Environmental Geochemistry and Health, 2012, 34(3):365-374.
[19] 王少云. 多菌灵、百菌清、毒死蜱在大棚黄瓜和土壤中的残留特征及其对土壤遗传毒性的影响[D]. 杭州:浙江大学, 2015.
[20] 张桂山. 多菌灵、吠喃丹对湖南红壤土微生物和酶活性的效应及多菌灵降解细菌的分离鉴定与降解性研究[D]. 杭州:浙江大学, 2004.
[21] 冯燕燕, 蔡继红, 侯振安, 等. 多菌灵对新疆灰漠土壤中六种酶活性的影响[J]. 石河子大学学报:自然科学版, 2009, 04:413-418.
[22] Chowdhury A, Pradhan S, Saha M, et al.Impact of pesticides on soil microbiological parameters and possible bioremediation strategies[J]. Indian Journal of Microbiology, 2008, 48(1):114-127.
[23] Cycoń M, Mrozik A, Piotrowska-Seget Z.Bioaugmentation as a strategy for the remediation of pesticide-polluted soil:A review[J]. Chemosphere, 2017, 172:52-71.
[24] Chen S, Yang L, Hu M, et al.Biodegradation of fenvalerate and 3-phenoxybenzoic acid by a novel Stenotrophomonas sp. strain ZS-S-01 and its use in bioremediation of contaminated soils[J]. Applied Microbiology and Biotechnology, 2011, 90(2):755-767.
[25] 刘双双, 杨仁斌, 陈海平, 等. 多菌灵在水稻及土壤中的消解动态和残留规律研究[J]. 农业环境科学学报, 2012, 02:357-361.
[26] Cycoń M, Wójcik M, Piotrowska-Seget Z.Biodegradation kinetics of the benzimidazole fungicide thiophanate-methyl by bacteria isolated from loamy sand soil[J]. Biodegradation, 2011, 22(3):573-583.
[27] Xiao W, Wang H, Li T, et al.Bioremediation of Cd and carbendazim co-contaminated soil by Cd-hyperaccumulator Sedum alfredii associated with carbendazim-degrading bacterial strains[J]. Environ Sci Pollut Res Int, 2013, 20(1):380-389.
[28] Zhang Y, Wang H, Wang X, et al.Identification of the key amino acid sites of the carbendazim hydrolase(MheI)from a novel carbendazim-degrading strain Mycobacterium sp. SD-4[J]. J Hazard Mater, 2017, 5(331):55-62.
[29] 田连生, 陈菲. 多菌灵降解菌T8-2的分离及其降解条件研究[J]. 江苏农业科学, 2008(6):271-274.
[30] Arya R, Sharma AK.Bioremediation of carbendazim, a benzimida-zole fungicide using Brevibacillus borstelensis and Streptomyces albogriseolus together[J]. Curr Pharm Biotechnol, 2015;17(2):185-189.
[31] Salunkhe VP, Sawant IS, Banerjee K, et al.Kinetics of degradation of carbendazim by B. subtilis strains:possibility of in situ detoxification[J]. Environ Monit Assess . 2014, 186(12):8599-8610.
[32] Fang H, Wang Y, Gao C, et al.Isolation and characterization of Pseudomonas sp. CBW capable of degrading carbendazim[J]. Biodegradation, 2010, 21:939-946.
[33] Zhang X, Huang Y, Harvey PR, et al.Isolation and characterization of carbendazim-degrading Rhodococcus erythropolis djl-11[J]. PLoS One, 2013, 8(10):e74810.
[34] Pandey G, Dorrian SJ, et al.Cloning and biochemical characterization of a novel carbendazim(methyl-1H-benzimidazol-2-ylcarbamate)-hydrolyzing esterase from the newly isolated Nocardioides sp. strain SG-4G and its potential for use in enzymatic bioremediation[J]. Applied and Environmental Microbiology, 2010, 76(9):2940-2945.
[35] Zhang GS, Jia X M, Cheng TF, et al.Isolation and characterization of a new carbendazim-degrading Ralstonia sp. strain[J]. World Journal of Microbiology & Biotechnology, 2005, 21(3):265-269.
[36] Zhang L, Qiao X, Ma L.Influence of environmental factors on degradation of carbendazim by Bacillus pumilus strain NY97-1[J]. International Journal of Environment and Pollution, 2009, 38:309-317.
[37] Alvarado-Gutiérrez ML, Ruiz-Ordaz N, Galíndez-Mayer J, et al.Kinetics of carbendazim degradation in a horizontal tubular biofilm reactor[J]. Bioprocess Biosyst Eng, 2017, 40(4):519-528.
[38] 林秀, 侯振安, 赵思峰, 等. 多菌灵降解菌XJ-D的分离鉴定及特性研究[J]. 中国农学通报, 2011, 27(16):220-226.
[39] 龚芬芬. 多菌灵降解菌的分离鉴定、生物学特性及多菌灵水解酶基因的克隆和表达研究[D]. 南京:南京农业大学, 2010:41-50.
[40] 王一奇. 多菌灵降解菌Pseudomonas sp. CBW的分离、鉴定及其降解特性与降解机理[D].杭州:浙江大学.