烟嘧磺隆降解菌Chryseobacterium sp. LAM-M5的分离、鉴定及其降解机理研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-0243

摘要/Abstract

摘要：

为获得更丰富的烟嘧磺隆降解菌资源,以合肥某烟嘧磺隆生产厂活性污泥为研究对象,从中分离并筛选得到一株能以葡萄糖为唯一碳源、烟嘧磺隆为唯一氮源生长的菌株,根据其表型特征、16S rRNA序列相似性、DNA-DNA杂交值（DDH）和平均核苷酸一致性（ANI）的分析结果,将其鉴定为Chryseobacterium lacus LAM-M5。该菌株可在7 d内将初始浓度为50 mg/L的烟嘧磺隆降解92.39%。利用液相色谱-质谱联用（LC-MS）对菌株LAM-M5与烟嘧磺隆混合培养后的代谢产物进行检测与鉴定,共有5种主要物质被检测到,根据烟嘧磺隆的化学结构式和中间代谢产物特征,初步推测出菌株Chryseobacterium sp. LAM-M5降解烟嘧磺隆的可能降解途径。利用高效液相色谱（HPLC）对接菌后降解体系内代谢产生的酸类物质进行分离与鉴定,结果表明产物主要为L-苹果酸。推测菌株LAM-M5受高浓度烟嘧磺隆胁迫,利用葡萄糖产生L-苹果酸,通过降低环境 pH使烟嘧磺隆发生水解,从而解除其胁迫。通过对菌株全基因组信息进行分析,预测了7种可能与烟嘧磺隆降解相关的基因。结果表明,菌株LAM-M5 具有较强的烟嘧磺隆降解能力,在烟嘧磺隆污染环境的生物修复上具有较好的应用潜力。

关键词: 烟嘧磺隆, 金黄杆菌属, 微生物降解, 代谢途径

Abstract:

In order to obtain more nicosulfuron-degrading bacterial resources,the activated sludge samples from a nicosulfuron manufacturer in Hefei city were used for study object,from which an isolated and screened strain grew well with glucose as sole carbon source and nicosulfuron as sole nitrogen source. Based on its phenotypic characteristics,the 16S rRNA gene sequence similarity,the values of DNA-DNA hybridization and average nucleotide identity,it was identified as Chryseobacterium lacus LAM-M5. This strain degraded 92.39% of 50 mg/L nicosulfuron within 7 d. Liquid chromatography mass spectrometry was used to detect and identify the metabolites after mixing culture of LAM-M5 and nicosulfuron,and 5 major substances were detected. The metabolic pathway of LAM-M5 degrading nicosulfuron was primarily speculated based on the chemical structure and characteristics of intermediates. High performance liquid chromatography was employed to isolate and identify the acids generated during the degradation of nicosulfuron by strain LAM-M5,and the results showed that L-malic acid was predominant. It was deduced that strain LAM-M5 metabolized the glucose into L-malic acid when its growth was stressed by the nicosulfuron,from which the environmental pH decreased and the nicosulfuron hydrolyzed,thus the stress was released. Through the analysis of the whole genome sequence of strain LAM-M5,7 annotated genes that may contribute to the degradation of nicosulfuron were predicted. The results reveal that strain LAM-M5 has excellent ability of degrading nicosulfuron and may have promising application potential in the microbial remediation of nicosulfuron -polluted environment.

Key words: nicosulfuron, Chrysobacterium, microbial degradation, metabolic pathway

马青云, 江旭, 李情情, 宋金龙, 周义清, 阮志勇. 烟嘧磺隆降解菌Chryseobacterium sp. LAM-M5的分离、鉴定及其降解机理研究[J]. 生物技术通报, 2022, 38(2): 113-122.

MA Qing-yun, JIANG Xu, LI Qing-qing, SONG Jin-long, ZHOU Yi-qing, RUAN Zhi-yong. Isolation and Identification of Nicosulfuron Degrading Strain Chryseobacterium sp. LAM-M5 and Study on Its Degradation Pathway[J]. Biotechnology Bulletin, 2022, 38(2): 113-122.

图/表 8

图1 烟嘧磺隆化学结构式[3]

Fig.1 Chemical structural formula of nicosulfuron

图2 菌株LAM-M5在牛肉膏蛋白胨培养基平板上的菌落形态

Fig.2 Colony morphology of strain LAM-M5 on the beef extract peptone medium plate

图3 基于16S rRNA基因序列构建的NJ系统发育树

Fig.3 Neighbour-joining phylogenetic tree based on the 16S rRNA gene sequences

图4 不同pH值（A）、温度（B）、接种量（C）对菌株LAM-M5 降解烟嘧磺隆降解率的影响图中误差线代表标准偏差,下同

Fig.4 Effects of different pH（A）,temperature（B）and initial inoculum ratio（C）on the degradation of nicosulfuron by strain LAM-M5 The error line in the figurebars refers to standard errors. The same below

图5 LAM-M5降解烟嘧磺隆过程中pH值的变化

Fig.5 Change of pH value during the nicosulfuron degra-dation by strain LAM-M5

图6 菌株LAM-M5降解烟嘧磺隆的代谢途径

Fig.6 Proposed metabolic pathways of nicosulfuron degra-dation by strain LAM-M5

表1 LAM-M5的基因组组分结果统计

Table 1 Genomic characteristics of strain LAM-M5

基因组组分 Characteristics	菌株LAM-M5 Chryseobacterium lacus LAM-M5
Genome size/bp	6 311 696
Gene number	6 366
Gene length/bp	5 704 104
GC content/%	43.84
genes of genome/%	90.37
Gene average length/bp	896
Gene internal length/bp	607 592
Gene internal GC content/%	36.52
Internal of genome/%	9.63

表2 菌株LAM-M5 基因组中7个降解酶相关的基因

Table 2 7 genes related to degradation enzymes in the genome of strain LAM-M5

编号 No.	预测的降解酶基因 Predicted degeration genes	大小 Size/bp
1	Putative phosphoesterase	172
2	Acyl-CoA thioesterase YneP	264
3	Carboxylesterase	254
4	Catalase X	535
5	Probable manganese catalase	274
6	Hydrolase YhcX	513
7	Putative sporulation hydrolase CotR	317

参考文献 39

[1]	Zhang C, Hu R, Shi G, et al. Overuse or underuse? An observation of pesticide use in China[J]. Sci Total Environ, 2015, 538:1-6. doi: 10.1016/j.scitotenv.2015.08.031 URL
[2]	陆涛, 李燕, 傅正伟, 等. 农药对根际微生物群落的影响及潜在风险[J]. 农药学学报, 2019, 21(Z1):865-870.
	Lu T, Li Y, Fu ZW, et al. Effects of pesticides on the rhizosphere microbial community and potential risks[J]. Chin J Pestic Sci, 2019, 21(Z1):865-870.
[3]	赵卫松, 邱立红, 郭庆港, 等. 烟嘧磺隆的微生物降解研究进展[J]. 农药学学报, 2016, 18(6):676-685.
	Zhao WS, Qiu LH, Guo QG, et al. Research progress on microbial degradation of nicosulfuron[J]. Chin J Pestic Sci, 2016, 18(6):676-685.
[4]	磺酰脲类和咪唑啉酮类除草剂全球市场概述[EB/OL]. [2020-02-12]. http://www.agrichem.cn/news/2014/1/15/201411510334746965.shtml .
	Overview of the global market for sulfonylurea and imidazolinone herbicides[EB/OL]. [2020-02-12]. http://www.agrichem.cn/news/2020/2/12/202021210334746965.shtml .
[5]	Bouri M, Gurau M, Salghi R, et al. Ionic liquids supported on magnetic nanoparticles as a sorbent preconcentration material for sulfonylurea herbicides prior to their determination by capillary liquid chromatography[J]. Anal Bioanal Chem, 2012, 404(5):1529-1538. doi: 10.1007/s00216-012-6221-2 URL
[6]	Trigo C, Spokas KA, Cox L, et al. Influence of soil biochar aging on sorption of the herbicides MCPA, nicosulfuron, terbuthylazine, indaziflam, and fluoroethyldiaminotriazine[J]. J Agric Food Chem, 2014, 62(45):10855-10860. doi: 10.1021/jf5034398 URL
[7]	张伟, 王进军, 张忠明, 等. 烟嘧磺隆在土壤中的吸附及与土壤性质的相关性研究[J]. 农药学学报, 2006, 8(3):265-271.
	Zhang W, Wang JJ, Zhang ZM, et al. Adsorption of nicosulfuron on soils and its correlation with soil properties[J]. Chin J Pestic Sci, 2006, 8(3):265-271.
[8]	Regitano JB, Koskinen WC. Characterization of nicosulfuron availability in aged soils[J]. J Agric Food Chem, 2008, 56(14):5801-5805. doi: 10.1021/jf800753p URL
[9]	Bottaro M, Frascarolo P, Gosetti F, et al. Hydrolytic and photoinduced degradation of tribenuron methyl studied by HPLC-DAD-MS/MS[J]. J Am Soc Mass Spectrom, 2008, 19(8):1221-1229. doi: 10.1016/j.jasms.2008.05.009 URL
[10]	Li-feng G, Jian-dong J, Xiao-hui L, et al. Biodegradation of ethametsulfuron-methyl by Pseudomonas sp. SW₄ isolated from contaminated soil[J]. Curr Microbiol, 2007, 55(5):420-426. pmid: 17713813
[11]	Ma JP, Wang Z, Lu P, et al. Biodegradation of the sulfonylurea herbicide chlorimuron-ethyl by the strain Pseudomonas sp. LW3[J]. FEMS Microbiol Lett, 2009, 296(2):203-209. doi: 10.1111/fml.2009.296.issue-2 URL
[12]	Zhao W, Wang C, Xu L, et al. Biodegradation of nicosulfuron by a novel Alcaligenes faecalis strain ZWS11[J]. J Environ Sci:China, 2015, 35:151-162. doi: 10.1016/j.jes.2015.03.022 URL
[13]	代鹏飞, 蔡天明, 汪林, 等. 一株烟嘧磺隆降解菌的分离鉴定及其降解特性与途径[J]. 环境科技, 2015, 28(4):12-17, 21.
	Dai PF, Cai TM, Wang L, et al. Isolation, identification, degradation characteristics and pathway of A nicosulfuron-degrading bacteria[J]. Environ Sci Technol, 2015, 28(4):12-17, 21.
[14]	张国民, 张松柏, 刘勇, 等. 一株降解烟嘧磺隆光合细菌的分离鉴定及降解特性研究[J]. 环境污染与防治, 2011, 33(5):18-21, 26.
	Zhang GM, Zhang SB, Liu Y, et al. Isolation and identification of a nicosulfuron-degrading strain J5-2 and its degradation characteristics[J]. Environ Pollut Control, 2011, 33(5):18-21, 26.
[15]	Wang L, Zhang X, Li Y. Degradation of nicosulfuron by a novel isolated bacterial strain Klebsiella sp. Y1:condition optimization, kinetics and degradation pathway[J]. Water Sci Technol, 2016, 73(12):2896-2903. doi: 10.2166/wst.2016.140 URL
[16]	Carles L, Joly M, Bonnemoy F, et al. Identification of sulfonylurea biodegradation pathways enabled by a novel nicosulfuron-transforming strain Pseudomonas fluorescens SG-1:Toxicity assessment and effect of formulation[J]. J Hazard Mater, 2017, 324(pt b):184-193. doi: S0304-3894(16)30970-0 pmid: 28340990
[17]	Feng WM, Wei Z, Song JL, et al. Hydrolysis of nicosulfuron under acidic environment caused by oxalate secretion of a novel Penicillium oxalicum strain YC-WM1[J]. Sci Rep, 2017, 7:647. doi: 10.1038/s41598-017-00228-2 URL
[18]	李顺鹏, 何健, 杭宝建, 等. 噻吩磺隆水解酶基因tsmE及其应用:CN102286501 B[P], 2011-12-21.
	Li SP, He J, Hang BJ, et al. Acta Tabacaria Sin[P], 2011-12-21.
[19]	Ruan Z, Zhou S, Jiang S, et al. Isolation and characterization of a novel cinosulfuron degrading Kurthia sp. from a methanogenic microbial consortium[J]. Bioresour Technol, 2013, 147:477-483. doi: 10.1016/j.biortech.2013.08.017 URL
[20]	宋金龙. 烟嘧磺隆降解菌黄篮状菌(Talaromyces flavus)的分离鉴定及降解机理研究[D]. 北京:中国农业科学院, 2013.
	Song JL. Isolation and identification of nicosulfuron-degradtive fungus(Talaromyces flavus)and study of degradation mechanism[D]. Beijing:Chinese Academy of Agricultural Sciences, 2013.
[21]	Yoon SH, Ha SM, Kwon S, et al. Introducing EzBioCloud:a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies[J]. Int J Syst Evol Microbiol, 2017, 67(5):1613-1617. doi: 10.1099/ijsem.0.001755 URL
[22]	Saitou N, Nei M. The neighbor-joining method:a new method for reconstructing phylogenetic trees[J]. Mol Biol Evol, 1987, 4(4):406-425. doi: 10.1093/oxfordjournals.molbev.a040454 pmid: 3447015
[23]	Sakamoto M. Reclassification of Bacteroides forsythus(Tanner et al, 1986)as Tannerella forsythensis corrig., gen. nov., comb. nov[J]. International J Systematic Evolutionary Microbiology, 2002, 52(3):841-849.
[24]	Tamura K, Dudley J, Nei M, et al. MEGA4:Molecular Evolutionary Genetics Analysis(MEGA)software version 4. 0[J]. Mol Biol Evol, 2007, 24(8):1596-1599. doi: 10.1093/molbev/msm092 URL
[25]	Chen S, Hu Q, Hu M, et al. Isolation and characterization of a fungus able to degrade pyrethroids and 3-phenoxybenzaldehyde[J]. Bioresour Technol, 2011, 102(17):8110-8116. doi: 10.1016/j.biortech.2011.06.055 URL
[26]	齐萌, 敖兰, 商爱萍, 等. 烟嘧磺隆高效降解菌株的筛选及其降解特性研究[J]. 河北农业大学学报, 2014, 37(4):74-79.
	Qi M, Ao L, Shang AP, et al. The screening of the nicosulfuron-degradated strain and preliminary study on its degradation characteristics[J]. J Agric Univ Hebei, 2014, 37(4):74-79.
[27]	Dong FS, Liu XG, Li J, et al. Determination of 4-chloro-2-methylphenoxyacetic acid residues in wheat and soil by ultra-performance liquid chromatography/tandem mass spectrometry[J]. J AOAC Int, 2010, 93(3):1013-1019.
[28]	Li R, Dong F, Xu J, et al. Enantioseparation of imazalil and monitoring of its enantioselective degradation in apples and soils using ultrahigh-performance liquid chromatography-tandem mass spectrometry[J]. J Agric Food Chem, 2017, 65(16):3259-3267. doi: 10.1021/acs.jafc.7b00258 URL
[29]	Li R, Zhu H, Ruan J, et al. De novo assembly of human genomes with massively parallel short read sequencing[J]. Genome Res, 2010, 20(2):265-272. doi: 10.1101/gr.097261.109 URL
[30]	Chun J, Oren A, Ventosa A, et al. Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes[J]. Int J Syst Evol Microbiol, 2018, 68(1):461-466. doi: 10.1099/ijsem.0.002516 URL
[31]	Song J, Gu J, Zhai Y, et al. Biodegradation of nicosulfuron by a Talaromyces flavus LZM1[J]. Bioresour Technol, 2013, 140:243-248. doi: 10.1016/j.biortech.2013.02.086 URL
[32]	党晶晶, 杨冬臣, 张哲, 等. 蜡状芽孢杆菌对烟嘧磺隆的降解及降解产物2-氨基-4, 6-二甲氧基嘧啶的环境生物毒性[J]. 农药学学报, 2018, 20(5):652-660.
	Dang JJ, Yang DC, Zhang Z, et al. Degradation of nicosulfuron by Bacillus cereus and toxicity of the degradation product 2-amino-4, 6-dimethoxy pyrimidine to environmental organisms[J]. Chin J Pestic Sci, 2018, 20(5):652-660.
[33]	褚翠伟, 阮志勇, 姚利, 等. 除草剂的微生物降解研究进展[J]. 生物资源, 2018, 40(2):93-100.
	Chu CW, Ruan ZY, Yao L, et al. Research progress of microbial degradation of herbicides[J]. Biotic Resour, 2018, 40(2):93-100.
[34]	Li MM, Song JL, Ma QY, et al. Insight into the characteristics and new mechanism of nicosulfuron biodegradation by a Pseudomonas sp. LAM1902[J]. J Agric Food Chem, 2020, 68(3):826-837. doi: 10.1021/acs.jafc.9b06897 URL
[35]	Lagier JC, Khelaifia S, Alou MT, et al. Culture of previously uncultured members of the human gut microbiota by culturomics[J]. Nat Microbiol, 2016, 1:16203. doi: 10.1038/nmicrobiol.2016.203 URL
[36]	Zhang H, Mu W, Hou Z, et al. Biodegradation of nicosulfuron by the bacterium Serratia marcescens N₈₀[J]. J Environ Sci Health B, 2012, 47(3):153-160. doi: 10.1080/03601234.2012.632249 URL
[37]	Bhattacharjee AK, Dureja P. Light-induced transformation of tribenuron-methyl on glass, soil, and plant surface[J]. J Environ Sci Health B, 2002, 37(2):131-140. doi: 10.1081/PFC-120002985 URL
[38]	Omer CA, Lenstra R, Litle PJ, et al. Genes for two herbicide-inducible cytochromes P-450 from Streptomyces griseolus[J]. J Bacteriol, 1990, 172(6):3335-3345. pmid: 2345149
[39]	Hang BJ, Hong Q, Xie XT, et al. SulE, a sulfonylurea herbicide de-esterification esterase from Hansschlegelia zhihuaiae S113[J]. Appl Environ Microbiol, 2012, 78(6):1962-1968. doi: 10.1128/AEM.07440-11 URL