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1 微生物学通报 Microbiology China Apr. 20, 204, 4(4): by Institute of Microbiology, CAS DOI: /j.microbiol.china 简报一株泰乐菌素高效降解菌的分离鉴定及其降解特性孙瑞珠马玉龙,2* 张娟孙永刚王艳王敏 ( ) ( ) 牛海亚摘要目的从长期堆放泰乐菌素药渣附近的土壤中分离出泰乐菌素降解菌, 并考察其对泰乐菌素的降解特性方法采用梯度驯化划线分离法筛选出泰乐菌素优势降解菌, 通过形态观察生理生化特征和 6S rrna 基因序列分析方法对其进行系统发育分析及菌种鉴定, 并考察菌株对泰乐菌素的降解特性结果从长期堆放泰乐菌素药渣的土壤中分离得到株泰乐菌素高效降解菌, 命名为 TS, 其为革兰氏阴性杆菌, 菌落形态呈圆形, 乳白色, 表面光滑, 不透明, 边缘整齐, 鉴定为越南伯克霍尔德氏菌 (Burkholderia vietnamiensis) 该菌株在温度 35 C ph 7.0 的条件下培养 72 h, 对初始浓度为 300 mg/l 泰乐菌素的降解率可达 99% 以上结论说明菌株 TS 对泰乐菌素具有良好的降解特性, 可用于生物修复被泰乐菌素废渣废水污染的生态环境关键词泰乐菌素降解菌,6S rrna, 系统发育分析, 降解特性 Isolation and identification of a tylosin-degrading strain and its degradation characteristics SUN Rui-Zhu MA Yu-Long,2* ZHANG Juan SUN Yong-Gang WANG Yan WANG Min NIU Hai-Ya (. College of Chemistry and Chemical Engineering, Ningxia University, Yinchuan, Ningxia 75002, China) (2. Ningxia Engineering Research Center for Natural Medicine, Yinchuan, Ningxia 75002, China) Abstract: [Objective] This study was to isolate a tylosin-degrading strain from the soil deposited by tylosin pharmaceutical waste and investigated its degradation characteristics. [Methods] The strain was identified based on the morphology observation, experimental results of physiology and biochemsitry, and then the identification and phylogenetic analysis of the isolated strain was performed by 6S rrna gene sequence, also, the characteristics of the strain in degradation of tylosin was investigated. [Results] A strian named TS was newly isolated from the soil deposited by tylosin pharmaceutical waste. The cells of the isolate were Gram-negative rods and the colony morphology was round, oyster white, opaque, and smooth with regular edge. The isolate was identified as Burkholderia vietnamiensis. This strain was capable of degrading 99% of tylosin in medium with an initial concentration of 300 mg/l after 72 h of incubation under conditions of the initial ph 7.0 and 基金项目 :(No ) * 通讯作者 :Tel nxylma@63.com 收稿日期 : 接受日期 : 优先数字出版日期 (

2 682 微生物学通报 Microbiol. China 204, Vol.4, No.4 35 C. [Conclusion] The isolated strain has a high ability to degrade tylosin, and may be used for bioremediation of environment contaminated by solid or liquid waste containing tylosin residue. Keywords: Tylosin-degrading strain, 6S rrna, Phylogenetic analysis, Bioderadation characteristics [] t [2] [3] Hamill [4] t [5] [6] [7-4] Kolz [5] 30 mg/l Zuccato [6] HPLC ng/l ng/kg Dolliver [7].2 μg/l 6.0 μg/l Kolpin [8] 280 μg/l [9-20] (WWTPs) [2-23] [24] (Citrobacter amalonaticus JF27264) 48 h 50 mg/l 95.2% [25] 300 μg/g 材料与方法. 实验材料 Sigma (.26%.4% 32.89%35.35% 2.98%3.78% 0.67%0.7% 0.73%0.79% ) (YPD) g 2 g 2 g 00 ml (.2 g) 0 5 Pa 20 min :0 ( ) 24 h

3 : mol/l K 2 HPO 4 65 ml KH 2 PO ml 0. mol/l ph 7.0 L [26].2 菌种 TS 的筛选驯化与分离 0.0 g 90 ml ph g 2.0 g.0 g 35 C 30 r/min 7 0 ml 90 ml 00 mg/l 500 mg/l YPD 0 ml 300 mg/l YPD 4 C.3 菌种鉴定.3. 形态观察及生理生化特征的鉴定 : TS YPD 35 C 72 h [27] ( 9 ) [28] TS V.P. TS.3.2 抗逆性测定 : TS ph YPD 35 C 30 r/min YPD 7 d S rrna 基因序列扩增及系统发育分析 : DNA (MK047-3) DNA PCR 6S rrna 8f 5 -CACGGATCCA GAGTTTGAT(C/T)(A/C)TGGCTCAG-3 50r 5 -GTGAAGTTACGG(C/T)TACCTTGTTACGAC TT-3 [29] (50 μl) 5 μl 0 mmol/l dntps 0.5 μl 25 mmol/l MgCl 2 5 μl EX Taq DNA.25 μl 0 μg/l μl 0 μmol/l μl94 C min 94 C 20 s 52 C 20 s 68 C 40 s C 0 min 4 C [30] PCR () 6S rrna NCBI Bankit GenBank NCBI BLAST MEGA 5.0 [3].4 检测方法.4. 泰乐菌素浓度的测定 : (LC-20AT ) Agilent HC-C 8 (5 μm 250 mm 4.6 mm) :KH 2 PO 4 (0.02 mol/l ph 2.5)=30:70 ( ) ml/min25 C 20 μl

型紫外分光光度计测定 500 nm 处的光密度 (OD500) 细菌浓度计算公式为 Ccell=34.

7.07.2 接入 0 ml 处于对数生长图分离得到 3 株单一菌的菌落特征 Figure Colony characteristic of the three single strains 期的降解菌

2 The colony characteristic of strain TS 降解效果最大的菌株 TS 其对初始浓度为 300 mg/l 的泰乐菌素在 3 d 内的降解率可达 99%

4 微生物学通报 Microbiol. China , Vol.4, No.4 测波长 286 nm 待测物过 0.45 μm 滤膜供液相色谱测定.4.2 菌株 TS 生长及其对泰乐菌素降解性能降解菌浓度的测定每间隔一定时间取培养液用 UV-200 型紫外分光光度计测定 500 nm 处的光密度 (OD500) 细菌浓度计算公式为 Ccell=34.5 OD500 [32] 绘制吸光度与时间关系曲线即得细菌生长曲线在 250 ml 容量瓶中加入 YPD 培养基和一定量的泰乐菌素粉末调节泰乐菌素初始浓度为 300 mg/l ph 接入 0 ml 处于对数生长图分离得到 3 株单一菌的菌落特征 Figure Colony characteristic of the three single strains 期的降解菌 TS ( 菌液浓度为 08 cells/ml) 于 35 C 30 r/min 的恒温振荡摇床中培养(同时以不接入降解菌为空白对照) 每隔 2 h 取样测定培养液中泰乐菌素的浓度绘制剩余泰乐菌素浓度与时间关系曲线即得泰乐菌素降解曲线以上每个处理设置 3 个平行 2 2. 结果与分析菌种筛选经过初步筛选分离得到 3 株单一菌命名为 TS TS2 和 TS3 (图 ) 通过 3 株菌对泰乐菌素的降解能力进行复筛保留了一株对泰乐菌素图2 菌株 TS 的菌落特征 Figure 2 The colony characteristic of strain TS 降解效果最大的菌株 TS 其对初始浓度为 300 mg/l 的泰乐菌素在 3 d 内的降解率可达 99% 以上而菌株在前期筛选过程中对 300 mg/l 的药渣泰乐菌素降解 99%则需要 7 d[33] 可见经过驯化一段时间后降解菌 TS 对泰乐菌素的降解能力明显提高 2.2 形态观察及生理生化特征的鉴定菌株 TS 在 YPD 固体培养基上呈圆形中间凸起乳白色表面光滑不透明边缘整齐菌落直径约为 23 mm (图 2) 菌株 TS 经革兰氏染色确定为革兰氏阴性菌在电子扫描显微镜下观察菌株为杆状(图 3) 菌株 TS 生理生化特征见表图3 扫描电子显微镜下菌株 TS 的形态(5 000 ) Figure 3 (5 000 ) The morphous of strain TS obtained by SEM

5 : 685 表菌株 TS 的生理生化特征 Table Physiological and biochemical characteristics of strain TS Items Results Items Results Items Cell morphology Gram-staining Catalase β- + Oxidase β- Galactose enzyme Arginine double enzyme hydrolysis Lysine decarboxylase Ornithine decarboxylase Urease + Tryptophan deaminase Gelatin liquefaction Indole + + V.P. Nitrate reduction Citrate Diacetyl D- + D- D-Galatose D-Tagatose Melibiose + D- + Glycerol D-Glucose Gluconate D- + N-- Erythritol D-Fructose N-Acetyl glucosamine D- + D- + D-Arabinose D-Mannose Amygdalin L- + L- L-Arabinose L-Sorbose Arbutin D- + L- D-Ribose L-Rhamnose Esculin D- + + D-Xylose Dulcite Salicin L- + L-Xylose Inositol Cellobiose + Adonitol Mannitol Maltose β--d- + β-methyl-d-xylose Sorbitol Lactose α--d- D- D- α-methyl-d-mannose D-Lyxose D-Fucose α--d- D- + L- α-methyl-d-glucose D-Arabitol L-Arabitol 2-- L- 2-Keto-gluconate L-Fucose Xylitol D- D-Turanose Synanthrin Starch + Sucrose Matsuyama sugar Glycogen Mycose Raffinose Gentiobiose +. Note: +: Positive; : Negative. Results

6 686 微生物学通报 Microbiol. China 204, Vol.4, No 菌株 TS 抗逆性 2 TS (ph 56) ph 7 TS NaCl 3% 2050 C 2.4 菌株 TS 序列测定及系统发育分析 TS 436 bp GenBank KC CAGCTTACCAT GCAGTCGAACGGCAGCACGGGGTGCTTGCAC CTGGTGGCGAGTGGCGAACGGGTGAGTAATAC ATCGGAACATGTCCTGTAGTGGGGGATAGCCCG GCGAAAGCCGGATTAATACCGCATACGATCTAC GGATGAAAGCGGGGGACCTTCGGGCCTCGCGC TATAGGGTTGGCCGATGGCTGATTAGCTAGTTG GTGGGGTAAAGGCCTACCAAGGCGACGATCAG TAGCTGGTCTGAGAGGACGACCAGCCACACTG GGACTGAGACACGGCCCAGACTCCTACGGGAG GCAGCAGTGGGGAATTTTGGACAATGGGCGAA AGCCTGATCCAGCAATGCCGCGTGTGTGAAGA AGGCCTTCGGGTTGTAAAGCACTTTTGTCCGGA AAGAAATCCTTG-3 TS 6S rrna GenBank BLAST TS 6S rrna 99% MEGA 5.0 TS GenBank ( 4) TS Burkholderia sp. 00% Bootstrap ( NR U96928 EF667353) 6S rrna TS (Burkholderia vietnamiensis) Table 2 表 2 ph 值盐和温度对菌株 TS 生长性能的影响 Effect of ph, salt and temperature on growth performance of strain TS ph Growth performance NaCl NaCl concentration (%) Growth performance Temperature ( C) Growth performance Note: +++: Growing eugonic; ++: Well-grown; +: Growth; : No growth.

7 : 687 图 4 菌株 TS 系统发育树 Figure 4 The phylogenetic tree of strain TS GenBank 000 %. Note: Numbers in parentheses represent the sequences accession number in GenBank. Numbers at the nodes indicate the percentage of occurrence in 000 boot-straped trees. Bar 0.0 represent sequence divergence. 2.5 菌株 TS 生长及其对泰乐菌素的降解特性 TS ( 0 8 cells/ml) 0% 300 mg/l YPD 35 C ph r/min 72 h TS h 248 h 4872 h 72 h 99% mg/l 5.75 min ( 6A) TS 72 h ( 6B) min TS 图 5 菌株 TS 生长情况及泰乐菌素的降解效果 Figure 5 Strain TS growth and degradation to tylosin

3 讨论 Holly [34] Loke [35] Teeter [36] 2035 d TS 6S rrna (Burkholderia vietnamiensis) (B. vietnamiensis) B48 [37] [38] TS ph ph 7.08.0 2050 C TS 35 C ph 7.

8 688 微生物学通报 Microbiol. China 204, Vol.4, No.4 图 6 泰乐菌素的 HPLC 图谱 Figure 6 HPLC spectra of tylosin A B TS. Note: A: The blank sample of tylosin; B: Tylosin degraded by strain TS after 72 h. 3 讨论 Holly [34] Loke [35] Teeter [36] 2035 d TS 6S rrna (Burkholderia vietnamiensis) (B. vietnamiensis) B48 [37] [38] TS ph ph C TS 35 C ph 7.0 0% 30 r/min 72 h 300 mg/l 99% TS 参考文献 [] Pal S. A journey across the sequential development of macrolides and ketolides related to erythromycin[j]. Tetrahedron, 2006, 62(4): [2] Wise R. Antimicrobial resistance: priorties for action[j]. Journal of Antimicrobial Chemotherapy, 2002, 49(4):

9 : [3] Thiele-Bruhn S. Pharmaceutical antibiotic compounds in soils-a review[j]. Journal of Plant Nutrition, 2003, 66(2): [4] Hamill RL. Tylosin, a new antibiotic[j]. Antibiotics & Chemotheropy, 960, : [5],,,. [J]., 200, 23(8): [6] Nie XP, Liu BY, Yu HJ, et al. Toxic effects of erythromycin, ciprofloxacin and sulfamethoxazole exposure to the antioxidant system in pseudokirchneriella subcapitata[j]. Environment Pollution, 203, 72: [7] Daugton CG, Ruhoy IS. Environment footprint of pharmaceuticals: the significance of factor beyond direct excretion to sewers[j]. Environmental Toxicology and Chemistry, 2009, 28(2): [8] Gracia-Lor E, Sancho JV, Serrano R, et al. Occurrence and removal of pharmaceuticals in wastewater treatment plants at the Spanish Mediterranean area of valencia[j]. Chemosphere, 202, 87(5): [9] Gros M, Petrovic M, Ginebreda A, et al. Removal of pharmaceuticals during wastewater treatment and environment risk assessment using hazard indexes[j]. Environment International, 200, 36(): [0] Karthikeyan KG, Meyer MT. Occurrence of antibiotics in wastewater treatment facilities in Wisconsin, USA[J]. Science of the Total Environment, 2006, 36(/3): [] Miao XS, Bishay F, Chen M. Occurrence of antimicrobials in the final effluents of waste- water treatment plants in Canada[J]. Environmental Science and Technology, 2004, 38(3): [2] Watkinson AJ, Murby EJ, Kolpin DW, et al. The occurrence of antibiotics in an urban water-shed: from wastewater to drinking water[j]. Science of the Total Environment, 2009, 407(8): [3] Xu W, Zhang G, Li X, et al. Occurrence and elimination of antibiotics at four sewage treatment plants in the Pearl River Delta(PRD), South China[J]. Water Research, 2007, 4(9): [4] Zuccato E, Castiglioni S, Bagnati R, et al. Occurrence and fate of antibiotics in the Italian aquatic environment[j]. Journal of Hazardous Materials, 200, 79(/3): [5] Kolz AC, Ong SK, Mooman TB. Sorption of tylosin onto swine manure[j]. Chemosphere, 2005, 60: [6] Zuccato E, Calamari D, Natangelo M, et al. Presence of therapeutic drugs in the environment[j]. The Lancet, 2000, 355: [7] Dolliver H, Gupta S. Antibiotic losses in leaching and surface runoof from manureamended agricultural land[j]. Environmental Quality, 2008, 37(3): [8] Kolpin DW, Furlong ET, Meyer MT, et al. Pharmaceuticals, bormones, and others orgnic wastewater contaminants in US streams, : a national reconnaissance[j]. Environmental Science and Technology, 2002, 36(6): [9] Underwood JC, Harvey RW, Metge Dw, et al. Effects of the antimicrobial sulfamethoxazole on groundwater bacterial enrichment[j]. Environmental Science Technology, 20, 45(7): [20] Pruden A, Pei RT, Storteboom H, et al. Antibiotic resistance genes as emerging contaminant s: studies in northern Colorado[J]. Environmental Science Technology, 2006, 40(23): [2] Castiglioni S, Bagnati R, Fanelli R, et al. Removal of pharmaceuticals in sewage treatment plants in Italy[J]. Environmental Science and Technolngy, 2006, 40(): [22] Watkinson AJ, Murby EJ, Costanzo SD, et al. Removal of antibiotics in conventional and advanced wastewater treatment implications for enviromental discharge and wastewater recycling[j]. Water Research, 2007, 4(8): [23] Kasprzyk-Hordern B, D insdale RM, Guwy AJ, et al. The removal of pharmaceuticals, personal care products, endocrine disruptors and illicit drugs during wastewater treatment and its impact on the quality of receiving waters[j]. Water Research, 2009, 43(2): [24] Ho YB, Zakaria MP, Latif PA, et al. Degradation of veterinary antibiotics and hormone during broiler manure composting[j]. Bioresource Technology, 203, 3: [25],,,. [J]., 20, 30(5): [26],. [M]. :, 200: [27],,,. [M]. :, 200: 302. [28] Holt JG, Krieg NR, Sneath PHA, et al. Bergey s manual of deteminative bacteriology. 9th ed[j]. Maryland: Williams &Witkins Baltimore, 994: [29] Amann RI, Ludwig W, Schleifer KH. Phylogenetic identification and in situ detection of individual microbial cells without cultivation[j]. Microbiology, 995, 59:

10 690 微生物学通报 Microbiol. China 204, Vol.4, No.4 [30],,. 6S rrna [J]., 2005, 45(5): [3],,,. [] [J]., 2009, 36(4): [32] Li Y, Loh KC. Activated carbon impregnated polysulfone hollow fiber membrance for cell immobilization and cometabolic biotransformation of 4-chlorophenol in the presence of phenol[j]. Journal of Membrance Science, 2006, 276: [33],,,. [J]., 203, 33(3): [34] Holly D, Satish G, Sally N. Antibiotic degradation during manure composting[j]. Environmental Quality, 2008, 37(3): [35] Loke ML, Lngerslev F, Halling-Sansen B, et al. Stability of tylosin A in manure containing test systems by high performance liquid chromatograhy[j]. Chemosphere, 2000, 40(7): [36] Teeter JS, Meyerhoff R. Aerobic degradation of tylosin in cattle, chicken, and swine excreta[j]. Environmental Research, 2003, 93: [37],,,. B48-37 [J]., 2008, 7: [38],,,. T32 [J]., 202, 39(9): 稿件书写规范高校教改纵横栏目简介及撰稿要求

: 307, [], [2],,,, [3] (Response Surface Methodology, RSA),,, [4,5] Design-Expert 6.0,,,, [6] VPJ33 ph 3,, ph, OD, Design-Expert 6.0 Box-Behnken, VPJ3

: 307, [], [2],,,, [3] (Response Surface Methodology, RSA),,, [4,5] Design-Expert 6.0,,,, [6] VPJ33 ph 3,, ph, OD, Design-Expert 6.0 Box-Behnken, VPJ3 微生物学通报 FEB 20, 2008, 35(2) 306~30 Microbiology 2008 by Institute of Microbiology, CAS tongbao@im.ac.cn 生物实验室响应面分析法优化副溶血性弧菌生长条件刘代新宁喜斌 * 张继伦 2 (. 200090) (2. 20202) 摘要