• 中文核心期刊
  • CSCD来源期刊
  • 中国科技核心期刊
  • CA、CABI、ZR收录期刊

一株孔雀石绿降解菌Citrobacter sp. D3的分离鉴定及降解特性

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

  • 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。

     

    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.

     

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