一株孔雀石绿降解菌<i>Citrobacter</i> sp. D3的分离鉴定及降解特性

刘菁华; 孙振中; 陈琳

doi:10.19303/j.issn.1008-0384.2020.06.012

一株孔雀石绿降解菌Citrobacter sp. D3的分离鉴定及降解特性

doi: 10.19303/j.issn.1008-0384.2020.06.012

上海市水产研究所，上海　200433

基金项目: 上海市水产研究所青年人才成长计划（沪农青字[2018]第3-6号）

详细信息

作者简介:
刘菁华（1986−），女，硕士，工程师，研究方向：环境药物残留检测分析及微生物降解修复研究（E - mail：lingqitutu@126.com）

中图分类号: X 172
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出版历程
- 收稿日期: 2019-12-10
- 修回日期: 2020-04-03
- 刊出日期: 2020-08-10

Identification and Malachite Green-Degrading Ability of Citrobacter Sp., D3

Shanghai Fisheries Research Institute, Shanghai　200433, China

摘要

摘要: 目的筛选获得可高效降解孔雀石绿的菌株。方法采用富集驯化的方法对渔业养殖环境中的土著微生物进行分离筛选，获得的降解菌株通过生理生化、扫描电镜和16S rDNA分析进行鉴定，采用单因素试验研究温度、pH及药物初始浓度对菌株降解效率的影响，并对其降解动力学参数进行分析。通过发光细菌法结合高分辨质谱仪对其降解产物的综合毒性和降解途径进行分析。结果从上海某渔业养殖池塘中分离到一株孔雀石绿降解菌D3，初步鉴定其为柠檬酸杆菌Citrobacter sp.。菌株D3在pH7～8、温度30～35 ℃的环境中具有较好的生长和降解率，在该条件下，菌株对质量浓度为2 mg·L⁻¹的孔雀石绿的降解率为96.32%，半衰期为0.563 0 d，且其代谢产物隐性孔雀石绿无明显累积；孔雀石绿质量浓度超过30 mg·L⁻¹，菌株生长及降解受到明显抑制。菌体降解孔雀石绿可以用一级反应动力学方程描述，拟合曲线的相关系数在0.916 9～0.963 5。菌株对孔雀石绿的降解产物综合毒性降低明显，72 h降解产物对发光细菌的抑制率降低50%以上。从降解产物中解析获得3种特征降解中间产物，分别为4-二甲氨基二苯基甲酮（m/z=226.12）、N,N-二甲基苯胺（m/z=122.10）和4-二甲氨基-苯酚（m/z=138.09），推测D3菌株对孔雀石绿的降解过程为逐步脱掉苯环获得次级代谢产物。结论 D3菌株对孔雀石绿具有很好的降解效果，对解决渔业生态中的孔雀石绿残留具有较高的应用价值。
- 显性孔雀石绿 /
- 隐性孔雀石绿 /
- 微生物降解
Abstract: Objective A microorganism capable of decomposing malachite green (MG) was identified and its degradation ability studied. Method Indigenous microbes in water samples from aquaculture farms were enriched and domesticated in laboratory to screen for the species that could degrade the organic germicide, MG, a pollutant in aquaculture ponds. Strains showing such capability by physiological and biochemical tests were examined under scanning electron microscopy and identified by a 16S rDNA analysis. A single factor experiment was conducted to determine the effects of temperature, pH, and initial MG concentration on the degradation kinetics of the isolated bacterium. The inoculated culture medium was monitored continuously for the luminescent bacteria toxicity determination and sampled for the high-resolution mass spectrometry analysis to reveal the decomposition pathway. Result An MG-degrading bacterium isolated from the specimens collected at a fish pond in Shanghai was identified as D3 belonging to Citrobacter sp. It exhibited a high rate of growth and MG-degradation on the medium at the pH ranging from 7 to 8 and temperature between 30℃ and 35℃. Its degradation rate under MG 2 mg·L⁻¹ was 96.32% and a half-life of 0.563 0 d with no significant accumulation of leucomalachite green (LMG). Once the MG concentration exceeded 30 mg·L⁻¹, the bacterial growth and degradation effect were significantly hindered. The first-order kinetic degradation function of D3 had a correlation coefficient of 0.916 9–0.963 5. After inoculation for 72 h, the overall toxicity shown on the medium was significantly decreased with more than 50% reduction on the inhibition of the luminescent bacterial growth. Three compounds, 4-dimethylaminodiphenyl ketone (m/z=226.12), N, N-dimethylaniline (m/z=122.10), and 4-dimethylamino-phenol (m/z=138.09), in the metabolites were the degradation intermediates. It appeared that D3 removed the benzene rings in MG step by step to result in those degraded secondary metabolites. Conclusion D3 of Citrobacter sp. might be applied to decontaminate MG in aquaculture ponds.
- Malachite green /
- leucomalachite green /
- microbial degradation

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图 1 D3菌株扫描电镜图

Figure 1. Scanning electron microscopy of D3 strain

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图 2 D3菌株在不同温度条件下对MG和LMG的降解率

Figure 2. Degradation rates of MG and LMG by D3 at different temperatures

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图 3 D3菌株在不同pH条件下对MG和LMG的降解率

Figure 3. Degradation rates of MG and LMG by D3 at varied pH

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图 4 不同初始浓度条件下D3对MG降解的影响

Figure 4. Effects of D3 on degradation of MG with varied initial concentrations

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图 5 3种降解产物的分子结构和质谱图

Figure 5. Molecular structures and mass spectrograms of 3 metabolites

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图 6 MG降解途径推测

Figure 6. Proposed metabolic pathway of MG-degradation by D3

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表 1 D3菌株在不同初始浓度MG条件下的降解动力学参数

Table 1. Parameters on D3 degradation kinetics of varied starting MG concentrations

初始质量浓度 Initial concentration/（µg·mL⁻¹）	降解动力学方程 Degradation kinetics equation	相关系数R²	降解半衰期t_1/2/d	降解率 Degradation/%
2	C_t=1.485 8e^{−1.231 1t}	0.936 0	0.563 0	96.32
5	C_t=4.192 7e^{−0.636 5t}	0.918 1	1.089	92.49
10	C_t=8.977 2e^{−0.230 6t}	0.916 9	3.005 8	85.80
15	C_t=11.712 4e^{−0.113 6t}	0.922 5	6.101 6	72.81
20	C_t=21.289 9e^{−0.095 9t}	0.963 5	7.227 8	66.70
30	C_t=28.962 4e^{−0.028 6t}	0.917 9	24.235 9	25.70

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表 2 D3菌株对MG降解产物的综合毒性

Table 2. Toxicity of MG degraded by D3 strain

样品 Sample	相对发光值 RL₀	相对发光值 RL（24 h）	抑制率X （24 h）/%	相对发光值 RL（72 h）	抑制率X （72 h）/%
D3	100	99.7	0.2	99.4	0.5
MG	100	49.9	50.1	49.2	50.8
MG+D3	100	60.8	39.2	78.1	21.9

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