Isolation of Exiguobacterium sp. H-1 from the sulfamethazine wastewater treatment system and its environmental adaptation characteristics
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摘要:
磺胺二甲嘧啶（SMZ）是一种难降解的广谱抗生素，其广泛存在已对水环境构成严重的威胁。微生物是环境中抗生素降解转化的主要驱动者，但高效降解SMZ的微生物资源匮乏。以SMZ废水处理系统的活性污泥为原料，采用纯培养技术从中分离筛选出一株SMZ降解菌H-1。经形态学观察、生理生化特征、16S rRNA基因序列分析，H-1归属于微小杆菌属（Exiguobacterium sp.）。通过单因素试验研究初始SMZ浓度、接种量、pH和温度对菌株H-1降解SMZ效果的影响。结果表明，接种量、pH和温度对该菌株降解SMZ的影响较大。进一步采用响应面法优化菌株Exiguobacterium sp. H-1降解SMZ的最佳条件，得出pH为7.21，温度为28.86 ℃，接种量为4.40%时，其对5 mg/L SMZ降解率为10.54%。本研究发现微小杆菌Exiguobacterium sp. H-1具有降解SMZ的能力，其降解SMZ的独特之处是能够将SMZ脱去SO2，生成嘧啶环和苯胺环，经过耦合生成N-（4,6-二甲基嘧啶-2基）-1,4-二苯胺，然后进行脱氨反应，生成2-苯-4,6-二甲基嘧啶，随后去甲基化生成产物6（2-苯基-1,2-二氢嘧啶，m/z为159.97）。此外，菌株H-1在0~10% NaCl的广泛范围内也表现出很强的耐盐性，能够为SMZ污染水体的生物修复提供耐盐能力强的微生物菌株资源。
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关键词:
- 磺胺二甲嘧啶 /
- Exiguobacterium /
- 生物降解 /
- 环境适应性 /
- 生物修复
Abstract:Sulfamethazine (SMZ) is a broad-spectrum antibiotic that is difficult to degrade, and its widespread presence poses a serious threat to the aquatic environment. Microorganisms are the main drivers of antibiotic degradation and transformation in the environment, but microbial resources for efficient degradation of SMZ are limited. Therefore, a strain of SMZ-degrading bacteria H-1 was isolated and screened from the activated sludge of SMZ wastewater treatment system by the pure culture technique. The strain H-1 was classified as the genus of Exiguobacterium sp. based on its morphological observation, physiological and biochemical characteristics, and 16S rRNA gene sequence analysis. The effects of initial SMZ concentration, inoculum, pH and temperature on the degradation of SMZ by strain H-1 were investigated by a single-factor test, and it indicated that the inoculum, pH and temperature affected greater on the degradation of SMZ by strain H-1. The optimal conditions for the degradation of SMZ by strain Exiguobacterium sp. H-1 were further optimized by response surface methodology: pH 7.21, temperature 28.86 ℃, inoculum level 4.40%, and its removal efficiency of 5 mg/L SMZ was 10.54%. It is found for the first time that Exiguobacterium sp. H-1 has the ability to degrade SMZ, and it is able to remove SMZ from SO2 first to produce pyrimidine and aniline rings, which are coupled to generate N-4,6-dimethylpyrimidin-2-yl)-1,4-diphenylamine, and then the deamination reaction proceeds to produce 2-phenyl-4,6-dimethylpyrimidine, which is subsequently demethylated to produce product 6 (m/z=159.97). In addition, strain H-1 also shows a strong salt tolerance within a wide range of 0-10% NaCl, providing a resource of salt-tolerant microbial strains for the bioremediation of SMZ-polluted waters.
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Key words:
- sulfamethazine /
- Exiguobacterium /
- biodegradation /
- environmental adaptability /
- bioremediation
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图 2 菌株H-1基于16S rRNA基因序列构建的系统发育树
注：括号内为GenBank中的登录号；分支处数值是以百分数表示的Bootstrap值；标尺表示进化距离。
Figure 2. Phylogenetic tree of strain H-1 based on 16S rRNA gene sequence
图 4 初始SMZ浓度、接种量、pH和温度对菌株H-1环境适应性的影响
注：图中相同标记字母为差异不显著，不同标记字母为差异显著。
Figure 4. Effect of initial sulfamethazine concentration, inoculum, pH and temperature on the environmental adaptation of strain H-1
图 5 接种量与pH对菌株H-1在SMZ废水中环境适应性的响应面及等高线
Figure 5. Response surface plots and contour plots of inoculum and pH on the environmental adaptation of strain H-1 in SMZ wastewater
图 6 接种量与温度对菌株H-1在SMZ废水中环境适应性的响应面及等高线
Figure 6. Response surface plots and contour plots of inoculum and temperature on the environmental adaptation of strain H-1 in SMZ wastewater
图 7 pH及温度对菌株H-1在SMZ废水中环境适应性的响应面及等高线
Figure 7. Response surface plots and contour plots of pH and temperature on the environmental adaptation of strain H-1 in SMZ wastewater
表 1 响应面分析因素和水平
Table 1. Factors and levels of response surface analysis
代码 因素 编码水平 −1 0 +1 A 接种量 2 4 6 B pH 6.0 7.0 8.0 C 温度 24 28 32 
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表 2 菌株H-1的部分生理生化特性
Table 2. Partial physiological and biochemical properties of strain H-1
项目 结果 项目 结果 革兰氏染色 + 淀粉水解试验 − 氧化酶试验 + 荧光色素试验 − 接触酶试验 + 耐盐性（氯化钠）试验 10% 甲基红试验 − 硝酸盐还原试验 + 注：−表示阴性；+表示阳性。
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表 3 菌株H-1的回归方程的方差分析
Table 3. Variance analysis of the regression equation for strain H-1
来源 平方和 自由度 均方 F P 模型 73.16 9 8.13 4.88 0.0242* A （接种量） 4.25 1 4.25 2.55 0.1540 B（pH） 17.22 1 17.22 10.34 0.0148* C（温度） 0.4281 1 0.4281 0.2571 0.6277 AB 0.1026 1 0.1026 0.0616 0.8111 AC 0.4226 1 0.4226 0.2538 0.6299 BC 0.1839 1 0.1839 0.1104 0.7494 A2 0.0436 1 0.0436 0.0262 0.8761 B2 43.47 1 43.47 26.10 0.0014** C2 5.27 1 5.27 3.16 0.1186 残差 11.66 7 1.67 失拟项 9.39 3 3.13 5.51 0.0664 纯差 2.27 4 0.5680 总和 84.82 16 注：**表示P小于0.01，为差异高度显著；*表示P小于0.05，为差异显著；总和为模型平方和与残差平方和之和，用以量化数据总变异量。
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表 4 已报道的部分磺胺二甲嘧啶降解菌的降解效果
Table 4. Degradation effect of some reported sulfamethazine degrading bacteria
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[1] 车琦, 王继华, 崔红, 等. 一株磺胺二甲嘧啶抗性菌的筛选及抗性机制研究[J]. 环境科学研究,2021,34(10):2405-2411. doi: 10.13198/j.issn.1001-6929.2021.06.13CHE Q, WANG J H, CUI H, et al. Screening and resistance mechanism of sulfamethazine-resistant strain[J]. Research of Environmental Sciences,2021,34(10):2405-2411. doi: 10.13198/j.issn.1001-6929.2021.06.13 [2] 孙爱荣. 噁喹酸、环丙沙星和磺胺二甲嘧啶在大菱鲆体内的药代动力学及其代谢酶基因CYP3A的克隆与表达分析[D]. 上海: 上海海洋大学, 2012. [3] BELAKHOV V V. Heptaene macrolide antibiotic perimycin: preparation, physicochemical properties, structure, biological activity, and application in agriculture as an eco-friendly fungicide (a review)[J]. Russian Journal of General Chemistry,2021,91(13):2943-2952. doi: 10.1134/S1070363221130235 [4] BELAKHOV V V. Ecological aspects of application of tetraene macrolide antibiotic tetramycin in agriculture and food industry : a review[J]. Russian Journal of General Chemistry, 2021, 91(13): 2858-2880. [5] 许振山, 张伟丽, 孙晓彤, 等. 磺胺二甲嘧啶在黄河鲤体内的代谢动力学研究[J]. 河南农业科学,2021,50(9):149-156. doi: 10.15933/j.cnki.1004-3268.2021.09.018XU Z S, ZHANG W L, SUN X T, et al. Pharmacokinetics study of sulfamethazine in Cyprinus carpio[J]. Journal of Henan Agricultural Sciences,2021,50(9):149-156. doi: 10.15933/j.cnki.1004-3268.2021.09.018 [6] 逯延军, 董艳慧, 赵平歌, 等. 复合型纳米铁还原法处理抗生素废水研究[J]. 应用化工,2021,50(7):1847-1849. doi: 10.3969/j.issn.1671-3206.2021.07.023 [7] 徐森, 胡晓东, 郑秋辉. 生物组合工艺处理抗生素废水现状及展望[J]. 工业水处理,2011,31(2):5-8. doi: 10.3969/j.issn.1005-829X.2011.02.002XU S, HU X D, ZHENG Q H. Present situation and forecast of the treatment of antibiotic wastewater by biological combined processes[J]. Industrial Water Treatment,2011,31(2):5-8. doi: 10.3969/j.issn.1005-829X.2011.02.002 [8] 赵慈, 张茹, 李思琦,等. 一株金霉素降解新菌株的分离鉴定及降解条件优化[J]. 环境工程技术学报,2022,12(6):2082-2088. doi: 10.12153/j.issn.1674-991X.20210430ZHAO C, ZHANG R, LI S Q, et al. Isolation, identification and degradation conditions optimization of a new bacterial strain degrading chlortetracycline[J]. Journal of Environmental Engineering Technology,2022,12(6):2082-2088. doi: 10.12153/j.issn.1674-991X.20210430 [9] 张珈瑜, 彭星星, 贾晓珊. 磺胺二甲基嘧啶(SM2)高效降解菌J2的分离筛选及降解特性研究[J]. 环境科学学报,2019,39(9):2919-2927. doi: 10.13671/j.hjkxxb.2019.0096ZHANG J Y, PENG X X, JIA X S. Isolation and characterization of highly efficient sulfamethazine-degrading bacterium strain J2[J]. Acta Scientiae Circumstantiae,2019,39(9):2919-2927. doi: 10.13671/j.hjkxxb.2019.0096 [10] 车琦. 地下水中磺胺类降解菌的筛选、鉴定及代谢途径的研究[D]. 哈尔滨: 哈尔滨师范大学, 2021. [11] PAN L, TANG X, LI C, et al. Biodegradation of sulfamethazine by an isolated thermophile– Geobacillus sp. S-07[J]. World Journal of Microbiology and Biotechnology,2017,33:1-8. doi: 10.1007/s11274-016-2144-y [12] HUANG M H, TIAN S X, CHEN D H, et al. Removal of sulfamethazine antibiotics by aerobic sludge and an isolated Achromobacter sp. S-3[J]. Journal of Environmental Sciences,2012,24(09):1594-1599. doi: 10.1016/S1001-0742(11)60973-X [13] 刘文举, 姚甜甜, 申艳敏, 等. 磺胺二甲嘧啶-对羟基苯甲酸共晶在乙腈溶剂中的三元相图测定[J]. 精细化工,2020,37(9):1870-1876. doi: 10.13550/j.jxhg.20200166 [14] 张淑红, 杜林南, 刘婷婷, 等. 河南省四城市饮用水中细菌对四环素和磺胺二甲嘧啶的耐药性[J]. 环境与健康杂志,2020,37(4):326-329. doi: 10.16241/j.cnki.1001-5914.2020.04.011 [15] YU B, ZHOU Y, HUANG Z W, et al. Effect of hydraulic retention time on pollutant removal performance of biological contact oxidation process treating hospital wastewater[J]. Applied Mechanics and Materials,2014,507:725-729. doi: 10.4028/www.scientific.net/AMM.507.725 [16] SHI Y J, HUANG C K, GAMAL El-D M, et al. Optimization of moving bed biofilm reactors for oil sands process-affected water treatment: the effect of HRT and ammonia concentrations[J]. Science of the Total Environment,2017,598:690-696. doi: 10.1016/j.scitotenv.2017.04.144 [17] YANG X, CHEN L, WANG C Q. Advance in application of 16S rRNA gene in bacteriology[J]. Journal of Northwest A & F University:Natural Science Edition,2008,36(2):55-60. [18] 王丽娟, 钱子雯, 沈海波, 等. 一株耐盐菌的分离及其降解特性[J]. 化工进展,2017,36(3):1047-1051. doi: 10.16085/j.issn.1000-6613.2017.03.037 [19] 付袁芝. 磺胺二甲基嘧啶降解菌的筛选及降解特性研究[D]. 哈尔滨: 哈尔滨工业大学, 2018. [20] KLEIN E Y, BOECKELTP V, MARTINEZ E M, et al. Global increase and geographic convergencein antibiotic consumption between 2000 and 2015[J]. Proceedings of the National Academy of Sciences,2018,115:3463-3470. [21] GARCLA M J, GARCFA G, DAY J W, et al. A review of emerging organic contaminants (EOCs), antibiotie resistant bacteria (ARB), and antibiotic resistance genes (ARGs) in the environment: increasing removal with wetlands and reducing environmental impacts[J]. Bioresource Technology, 2020, 307, 123228. [22] 陈智明, 刘国光, 马京帅, 等. Fe2+催化过碳酸钠降解水体中的磺胺二甲嘧啶[J]. 环境科学学报,2017,37(1):234-242.CHEN Z M, LIU G G, MA J S, et al. Degradation of sulfamethazine with Fe2+-catalyzed sodium percarbonate system[J]. Acta Sctentiae Circumstantiae,2017,37(1):234-242. [23] 杨振兴, 李烨莹, 叶芳芳, 等. 抗生素废水处理技术研究进展[J]. 现代化工,2021,41(1):57-61. doi: 10.16606/j.cnki.issn0253-4320.2021.01.012 [24] YANG C W, HSIAO W C, CHANG B V. Biodegradation of sulfonamide antibiotics in sludge[J]. Chemosphere,2016,150:559-565. doi: 10.1016/j.chemosphere.2016.02.064 [25] 冒学宇, 李勇, 吴丛, 等. 石油烃降解菌的筛选及其降解特性研究[J]. 环境工程技术学报,2022,2(3):886-892. doi: 10.12153/j.issn.1674-991X.20210203MAO X Y, LI Y, WU C, et al. Research on the screening of petroleum hydrocarbon degrading bacteria and their degradation characteristics[J]. Journal of Environmental Engineering Technology,2022,2(3):886-892. doi: 10.12153/j.issn.1674-991X.20210203 [26] 王强锋, 朱彭玲, 夏中梅, 等. 三种农用抗生素降解真菌的筛选及其降解性能[J]. 农业资源与环境学报, 2018, 35(6): 533-539.WANG Q F, ZHU P L, XIA Z M, et al. Screening and degradation properties of three kinds of agricultural antibiotics degrading fungi[J]. Journal of Agricultural Resources and Environment, 2018, 35(6): 533-539. [27] MAO F, LIU X, WU K, et al. Biodegradation of sulfonamides by Shewanella oneidensis MR-1 and Shewanella sp. strain MR-4[J]. Biodegradation,2018,29:129-140. doi: 10.1007/s10532-017-9818-5 [28] 李晨钰. 磺胺二甲基嘧啶高效降解菌的筛选及降解机理研究[D]. 泰安: 山东农业大学, 2021. [29] CAO L, ZHANG J, ZHAO R, et al. Genomic characterization, kinetics, and pathways of sulfamethazine biodegradation by Paenarthrobacter sp. A01[J]. Environment International,2019,131:104961. doi: 10.1016/j.envint.2019.104961 [30] 赵芮, 李登辉, 李学恭, 等. 微小杆菌属 ( Exiguobacterium)细菌的能量代谢途径分析[J]. 微生物学报,2023,63(6):2078-2093.ZHAO R, LI D H, LI X G, et al. Energy metabolism pathways in Exiguobacterium[J]. Acta Microbiologica Sinica,2023,63(6):2078-2093. [31] 胡江. 阿特拉津降解菌株BTAH1的分离鉴定、降解特性及应用的研究[D]. 南京: 南京农业大学, 2004. [32] OKEKE B C. Bioremoval of hexavalent chromium from water by a salt tolerant bacterium, Exiguobacterium sp. GS1[J]. Journal of Industrial Microbiology and Biotechnology,2008,35(12):1571-1579. doi: 10.1007/s10295-008-0399-5 [33] YUMOTO I, HISHINUMA-NARISAWA M, HIROTA K, et al. Exiguobacterium oxidotolerans sp. nov., a novel alkaliphile exhibiting high catalase activity[J]. International Journal of Systematic and Evolutionary Microbiology, 2004, 54(6): 2013-2017. [34] 杨锐. 磺胺二甲嘧啶降解菌的固定化及其在修复磺胺二甲嘧啶污染土壤中的应用[D]. 泰安: 山东农业大学, 2021. [35] 李鸿炫. 复苏促进因子强化多氯联苯污染土壤微生物修复机制研究[D]. 杭州: 浙江大学, 2018. ⊕ -
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