一株红霉素降解菌Penicillium sp. ERY-1的筛选、降解特性及其应用研究

doi:10.16258/j.cnki.1674-5906.2024.04.011

生态环境学报 ›› 2024, Vol. 33 ›› Issue (4): 607-616.DOI: 10.16258/j.cnki.1674-5906.2024.04.011

• 研究论文【环境科学】 • 上一篇下一篇

一株红霉素降解菌Penicillium sp. ERY-1的筛选、降解特性及其应用研究

刘菁华(), 张玉平, 陈雪婷^*()

上海市水产研究所（上海市水产技术推广站），上海 200433

收稿日期:2024-01-11 出版日期:2024-04-18 发布日期:2024-05-31
通讯作者: *陈雪婷。E-mail: cxtsnow@126.com
作者简介:刘菁华（1986年生），女，工程师，硕士，主要从事渔业环境有机污染微生物修复方面的研究。E-mail: lingqitutu@126.com
基金资助:
上海市科技兴农重点攻关项目(2020-02-08-00-07-F01483);国家自然科学基金项目(31900097)

Isolation, Characterization and Application of an Erythromycin-degrading Fungus Penicillium sp. ERY-1

LIU Jinghua(), ZHANG Yuping, CHEN Xueting^*()

Shanghai Fisheries Research Institute (Shanghai Fisheries Technical Extension Station), Shanghai 200433, P. R. China

Received:2024-01-11 Online:2024-04-18 Published:2024-05-31

摘要/Abstract

摘要：

在渔业养殖过程中，使用绿色环保、高效低廉的微生物菌剂解决渔业废水中的抗生素残留，对保障水产品质量安全和生态环境有重要意义。为获得可应用于渔业养殖废水的红霉素降解菌剂，采用梯度富集驯化的方法从养殖污染底泥样品中分离筛选红霉素降解菌株，通过形态学特征和ITS（Internal Transcribed Spaces）序列分析进行微生物分类鉴定；采用单因素试验确定最适降解温度和pH；利用超高效液相色谱串联高分辨率电喷雾电离质谱仪鉴定降解产物，并据此推测降解途径，通过斑马鱼攻毒试验初步评估菌株的生物安全性；优化菌株固态发酵参数，制备红霉素降解菌剂，并初探降解菌剂对红霉素污染的养殖废水的修复效果。结果显示，获得一株红霉素降解菌株ERY-1并鉴定其为青霉菌属（Penicillium sp.）；菌株ERY-1在温度20－35 ℃、pH 5－7的环境中具有较好的降解率，在该条件下，菌株对质量浓度为1.0 mg·L^-1的红霉素的降解率在97%以上；鉴定了5种降解中间产物，并据此推测菌株ERY-1通过脱水、脱红霉支糖和开环逐渐使红霉素失去抗菌活性；斑马鱼攻毒试验初步表明菌株ERY-1对水生生物具有较高的安全性；以稻壳：麦麸：玉米粉质量比（m꞉m꞉m）为1꞉1꞉1作为固态培养基配方，初始料水比为1 g:1 L，接种量为0.75%，在温度为25 ℃，发酵13 d后获得产孢量约9.14×10⁹ cfu·g^-1的降解菌剂。将制备的降解菌剂投撒到有红霉素残留的养殖废水中，3 d后水中的红霉素未达到方法的检出质量浓度（0.2 ng·L^-1）。结果表明，菌株ERY-1可有效的解决渔业养殖废水中的红霉素残留，由其制备的降解菌剂具有良好的应用和推广价值。

关键词: 红霉素, 微生物降解, 降解特性, 代谢产物, 固态发酵, 菌剂应用

Abstract:

The use of green, efficient, and inexpensive microbial agents to eliminate antibiotic residues in fishery wastewater is important for ensuring the quality and ecological environment of aquatic products. To obtain erythromycin-degrading microbes that can be applied to fishery aquaculture wastewater, an erythromycin-degrading fungus was isolated using traditional methods and identified by morphological observation and internal transcribed spacer (ITS) sequence analysis. The optimal temperature and pH for erythromycin degradation were determined using single-factor tests, with the degradation rate as the indicator. The degradation metabolites of erythromycin were identified using ultra-high-performance liquid chromatography-tandem high-resolution electrospray ionization mass spectrometry. A zebrafish challenge test was performed to evaluate the biological safety of the strain. An ecological agent capable of degrading erythromycin was prepared through solid-state fermentation using the optimized parameters. This agent has been used to remediate erythromycin residues in aquaculture wastewaters. The results showed that strain ERY-1 is a member of Penicillium. The optimal conditions for erythromycin degradation by the strain ERY-1 were 20-35 ℃ and pH 5-7. Under these conditions, over 97% of erythromycin with an initial concentration of 1.0 mg·L^-1 was degraded. Five potential erythromycin degradation products were identified in this study. Erythromycin gradually loses its antibacterial activity due to dehydration, cladinose removal, and ring cleavage. Strain ERY-1 is relatively safe for aquatic organisms. The mass ratio of rice husk, wheat bran, and corn flour (m꞉m꞉m) was 1꞉1꞉1, which was used as the substrate for fermentation. The ratio of material to water was 1 g:1 L, and the initial inoculation ratio was 0.75%. Under such circumstances, a microbial ecological agent with a spore yield of approximately 9.14 × 10⁹ cfu·g^-1 was obtained after 13 days of fermentation at 25 ℃. The concentration of residual erythromycin is undetectable (below the detection limit of 0.2 ng·L^-1) after 3 days since application of this microbiological ecological agent into the erythromycin-contaminated aquaculture wastewater. The results showed that strain ERY-1 could effectively remediate erythromycin-contaminated aquaculture wastewater, and that the microbial ecological agent derived from this strain showed good application and promotion value.

Key words: erythromycin, microbial degradation, degradation characteristics, metabolites, solid-state fermentation, microbiol ecological agent application

中图分类号:

X172

刘菁华, 张玉平, 陈雪婷. 一株红霉素降解菌Penicillium sp. ERY-1的筛选、降解特性及其应用研究[J]. 生态环境学报, 2024, 33(4): 607-616.

LIU Jinghua, ZHANG Yuping, CHEN Xueting. Isolation, Characterization and Application of an Erythromycin-degrading Fungus Penicillium sp. ERY-1[J]. Ecology and Environment, 2024, 33(4): 607-616.

图/表 11

表1 超高效液相色谱串联高分辨率电喷雾电离质谱仪（UPLC-HRMS）流动相梯度洗脱程序

Table 1 Mobile phase gradient elution program of ultra high performance liquid chromatography tandem high-resolution electrospray ionization mass spectrometry (UPLC-HRMS)

时间/min	流动相A	流动相B
0.1	95%	5%
2.0	95%	5%
25.0	5%	95%
35.0	5%	95%
40.0	95%	5%

表2 养殖废水理化性质及红霉素检出质量浓度

Table 2 Physico-chemical properties and erythromycin concentration of aquaculture wastewater

样品	水温	盐度	COD_Mn质量浓度	DO	pH	红霉素检出质量浓度
渔业养殖废水	29.6 ℃	0.7‰	39.08 mg·L^-1	13.02 mg·L^-1	8.96	0.47 μg·mL^-1

图1 ERY-1在PDA培养基上生长的菌落和细胞形态（a）菌落的正面；（b）菌落的反面；（c）菌丝及孢子梗的光学显微图；（d）孢子的光学显微图

Figure 1 Cell morphology of ERY-1 grown on PDA medium

图2 菌株ERY-1基于ITS序列构建的系统进化树

Figure 2 Phylogenetic tree of strain ERY-1 built based on the ITS sequence

图3 温度和pH对ERY-1降解效率影响不同小写字母表示不同处理间存在显著差异（p<0.05），n=3，下同

Figure 3 Effect of temperature and pH on degradation efficiency of ERY-1

表3 红霉素及其代谢产物的超高效液相色谱串联高分辨率电喷雾电离质谱（UPLC-HRMS）数据

Table 3 Spectral data for Erythromycin and postulated biodegradation metabolites determined from UPLC-HRMS

化合物	一级质谱 [M±H]^±(m/z)	化学式	理论质核比 [M+H]⁺(m/z)	误差 (×10^-6)
ERY	734.4679	C₃₇H₆₈O₁₃N⁺	734.46852	-0.85
C1	716.4594	C₃₇H₆₆O₁₂N⁺	716.45795	2.04
C2	558.3651	C₂₉H₅₂O₉N⁺	558.36366	2.49
C3	576.3754	C₂₉H₅₄O₁₀N⁺	576.37422	1.98
C4	435.2591	C₂₁H₃₉O₉^-	435.25886	0.47
C5	176.1274	C₈H₁₈O₃N⁺	176.12812	-4.03

图4 红霉素及其降解产物质谱图

Figure 4 Spectra of Erythromycin and its degradation products

图5 菌株ERY-1降解红霉素的代谢途径

Figure 5 The proposed erythromycin degradation pathway by strain ERY-1

图6 菌株ERY-1对斑马鱼生物安全试验

Figure 6 Biosafety test of strain ERY-1 to zebrafish

图7 不同基质配比对ERY-1菌株产孢量的影响

Figure 7 Effect of different substrate ratio on sporulation of ERY-1 strain

图8 不同发酵条件对ERY-1产孢量的影响

Figure 8 Effects of different fermentation conditions on sporulation of ERY-1

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一株红霉素降解菌Penicillium sp. ERY-1的筛选、降解特性及其应用研究

Isolation, Characterization and Application of an Erythromycin-degrading Fungus Penicillium sp. ERY-1

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