一株红霉素降解菌的筛选、鉴定与降解特性

doi:10.3969/j.issn.1004-1524.2021.01.16

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (1): 131-141.DOI: 10.3969/j.issn.1004-1524.2021.01.16

一株红霉素降解菌的筛选、鉴定与降解特性

许双燕¹(), 张涛¹, 张成¹^,², 林辉³, 水贤磊¹, 郑华宝¹^,²^,^*()

1.浙江农林大学环境与资源学院,浙江杭州 311300
2.浙江省土壤污染生物修复重点实验室,浙江杭州 311300
3.浙江省农业科学院环境资源与土壤肥料研究所,浙江杭州 310021

收稿日期:2020-07-03 出版日期:2021-01-25 发布日期:2021-01-25
通讯作者: 郑华宝
作者简介:*郑华宝,E-mail:zhenghuabao@zafu.edu.cn
许双燕(1995—),女,浙江嘉兴人,硕士研究生,主要从事农业废弃物资源化利用和环境中抗生素的微生物降解研究。E-mail:xu_shuangyan11@163.com
基金资助:
国家重点研发计划(2018YFD0500206);浙江农林大学科研发展基金(2034020081);浙江农林大学大学生科研训练项目(2013200064)

Isolation and identification of an erythromycin degradation bacterium strain and its biodegradation characteristics

XU Shuangyan¹(), ZHANG Tao¹, ZHANG Cheng¹^,², LIN Hui³, SHUI Xianlei¹, ZHENG Huabao¹^,²^,^*()

1. College of Environmental and Resource Sciences, Zhejiang A & F University, Hangzhou 311300, China
2. Key Laboratory of Soil Contamination Bioremediation of Zhejiang Province, Hangzhou 311300, China
3. Institute of Environmental Resources and Soil Fertilizer, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021,China

Received:2020-07-03 Online:2021-01-25 Published:2021-01-25
Contact: ZHENG Huabao

摘要/Abstract

摘要：

优良的菌种资源是污染环境微生物修复技术的核心。为获取红霉素高效降解菌,采用梯度驯化法,以长期堆放鸡粪的有机肥生产车间土壤为对象,开展降解菌筛选鉴定,并研究不同红霉素质量浓度、培养温度、转速、初始pH值,以及外加碳氮源、金属离子对菌株降解红霉素的影响。结果表明,筛选获得一株红霉素高效降解菌株Ery-6。通过菌落形态和16S rDNA序列分析方法,将该菌株鉴定为甲基菌属(Methylobacillus sp.)。Ery-6菌株可以在以红霉素为唯一碳源的无机盐培养基中快速生长,60 h后进入生长稳定期。接种Ery-6菌株可提高红霉素在培养基中的降解速率常数,使其半衰期从88.4 h降低至30.7 h。该菌株在含有100 mg·L^-1红霉素的无机盐培养基中,在温度35 ℃、转速120 r·min^-1、初始pH值7.0、外加50 mg·L^-1蔗糖的条件下,对红霉素的降解效果最佳,48 h降解率达88.68%。菌株可耐受1 000 mg·L^-1高质量浓度的红霉素,在温度35 ℃、转速120 r·min^-1、初始pH值7.0的条件下48 h降解率达31.95%。该菌株对多种金属离子具有良好的耐受性;但Cu²⁺既会抑制Ery-6菌株的生长,也会对其降解红霉素产生一定的影响。本研究首次发现甲基菌属菌株具有降解红霉素的能力,且降解效果较好,为生物降解养殖废弃物与环境中的抗生素污染提供了一种新的微生物资源。

关键词: 红霉素, 降解菌, 筛选, 降解特性

Abstract:

In order to obtain efficient erythromycin degradation bacteria, erythromycin was used as the sole carbon source, and gradient domestication method was used to isolate and screen erythromycin degradation bacteria. It was shown that an erythromycin degradation bacterium strain Ery-6 was isolated from the soil of an organic fertilizer production workshop where chicken manure was stacked for a long time. The Ery-6 strain was identified as Methylobacillus sp. through colony morphology identification and 16S rDNA sequence analysis. Further, high performance liquid chromatography (HPLC) was used to measure the degradation rate of erythromycin, and the degradation conditions of erythromycin by Ery-6 strain were optimized, including different erythromycin concentration, culture temperature, rotation speed, initial pH value, exogenous carbon and nitrogen sources and metal ions. The Ery-6 strain could reach a degradation rate of 88.68% after 48 h in inorganic salt medium containing 100 mg·L^-1 erythromycin under temperature of 35 ℃, rotation speed of 120 r·min^-1, initial pH value of 7.0, and 50 mg·L^-1 sucrose. Meanwhile, the Ery-6 strain could tolerate 1 000 mg·L^-1 high-concentration erythromycin, and exhibited a degradation rate of 31.95% after 48 h under temperature of 35 ℃, rotation speed of 120 r·min^-1, initial pH value of 7.0. The Ery-6 strain had a good tolerance to various metal ions. Cu²⁺ could inhibit its growth, and pose direct effect on the degradation of erythromycin by Ery-6. The present study for the first time reported that the Methylobacillus sp. strain had the ability to degrade erythromycin, and it could tolerate high concentration of erythromycin, and maintain a certain degradation rate, which provided new microorganism for biodegradation of erythromycin in the environment.

Key words: erythromycin, degrading bacteria, isolation, degradation characteristics

中图分类号:

S154.39

许双燕, 张涛, 张成, 林辉, 水贤磊, 郑华宝. 一株红霉素降解菌的筛选、鉴定与降解特性[J]. 浙江农业学报, 2021, 33(1): 131-141.

XU Shuangyan, ZHANG Tao, ZHANG Cheng, LIN Hui, SHUI Xianlei, ZHENG Huabao. Isolation and identification of an erythromycin degradation bacterium strain and its biodegradation characteristics[J]. Acta Agriculturae Zhejiangensis, 2021, 33(1): 131-141.

图/表 10

表1 液相色谱测定红霉素时的梯度洗脱程序

Table 1 Gradient elution procedure for erythromycin determination

t/min	流动相比例Mobile phase ratio/%
t/min	A	B
0	90.0	10.0
1.0	90.0	10.0
2.0	80.0	20.0
3.5	77.0	23.0
5.0	76.0	24.0
8.0	76.5	23.5
9.0	70.0	30.0
16	70.0	30.0
20	50.0	50.0
24	90.0	10.0
25	90.0	10.0

图1 Ery-6的菌落形态(A)、透射电镜图(B)和系统发育树(C)

Fig.1 Colony morphology (A), transmission electron microscopy (B) and phylogenetic tree (C) of Ery-6 strain

图2 Ery-6菌株的生长曲线(A)和对红霉素的生物降解动力学曲线(B) Ct/C0为t时体系中红霉素质量浓度与初始红霉素质量浓度之比。CK为不加菌的对照。

Fig.2 Growth curve (A) and biodegradation kinetic curve (B) of Ery-6 strain Ct/C0 was the ratio of erythromycin concentration at t time to the initial concentrution.CK, Control withow Ery-6 strain inoculation.

表2 红霉素降解的动力学方程与动力参数

Table 2 Biodegradation kinetic equation and kinetic parameter of erythromycin

处理Treatment	一级反应方程First order reaction equation	k/h^-1	t_1/2/h	R²
不接菌Not inoculated	C_t=92.14e^-0.00049^t	0.000 49	88.42	0.928 4
接菌Inoculated	C_t=57.69e^-0.00753^t	0.007 53	30.65	0.925 9

图3 初始红霉素质量浓度对Ery-6菌株降解红霉素的影响

Fig.3 Effect of initial erythromycin concentration on erythromycin degradation by Ery-6 strain

图4 温度对Ery-6菌株降解红霉素的影响

Fig.4 Effect of temperature on erythromycin degradation by Ery-6 strain

图5 转速对Ery-6菌株降解红霉素的影响

Fig.5 Effect of rotation speed on erythromycin degradation by Ery-6 strain

图6 初始pH值对Ery-6菌株降解红霉素的影响

Fig.6 Effect of initial pH on erythromycin degradation by Ery-6 strain

图7 外源添加碳氮源对Ery-6菌株降解红霉素的影响柱上无相同字母的表示处理间差异显著(P<0.05)。下同。对照中不外源添加碳氮源。

Fig.7 Effect of exogenous carbon or nitrogen source on erythromycin degradation by Ery-6 strain Bars marked without the same letters indicated significant difference at P<0.05. The same as below. No exogenous carbon or nitrogen source was added in the control.

图8 外加金属离子对Ery-6菌株降解红霉素的影响对照中不外源添加金属离子。

Fig.8 Effect of exogenous metal ions on erythromycin degradation by Ery-6 strain No exogenous metal ion was added in the control.

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一株红霉素降解菌的筛选、鉴定与降解特性

Isolation and identification of an erythromycin degradation bacterium strain and its biodegradation characteristics

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