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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