好氧氯苯降解菌的分离鉴定

Identification and characterization of an aerobic bacterium degrading chlorobenzene

摘要:

摘要：【目的】分离好氧氯苯降解菌，并通过研究降解特性为应用提供理论依据。【方法】利用富集培养技术分离菌株，通过形态、生理生化反应特征及16S rRNA基因序列分析鉴定菌株，测定培养液中氯苯、其它氯苯类化合物和氯离子的浓度以及菌体细胞的密度和菌体细胞粗提液中邻苯二酚双加氧酶的活性，研究菌株的降解特性。【结果】16S rRNA基因序列相似性比较表明，分离出的菌株与乙酸钙不动杆菌（Acinetobacter calcoaceticus）的相似性高达98.5％。以初始浓度为50 mg/L的氯苯为唯一碳源和能源时，120 h内菌株对氯苯的降解率高达98.2％，氯离子净释放量和氯苯降解量的摩尔比范围为1:1.85－1:1.39，菌体细胞粗提液中邻苯二酚1,2-双加氧酶的平均活性为0.538 U/mg蛋白质。加入葡萄糖后，菌体细胞数量和氯离子浓度明显增加，但单位细胞的氯苯降解能力明显下降。在二氯苯和三氯苯共存时，菌株对氯苯的降解能力受到明显的抑制作用，但对二氯苯有一定的降解作用，降解能力大小顺序为：1,3-二氯苯>1,2-二氯苯>1,4-二氯苯。【结论】分离出的好氧氯苯降解菌属于Acinetobacter 属菌株，该菌株对氯苯和二氯苯均具有降解作用，可能通过邻位裂环途径降解氯苯，氯苯对菌株的降解能力和邻苯二酚1,2-双加氧酶的活性具有明显的增强作用。

Abstract:

Abstract: [Objective] To isolate and characterize aerobic bacteria to degrade effectively chlorobenzene (CB). [Methods] We used enrichment culture to isolate and characterized bacterial strain through the observation of morphological and biochemical characters and analysis of 16S rRNA gene sequences. We determined the concentration of CB, other chlorobenzenes and released Cl-, densities of strain cells and activities of catechol dioxygenase in crude extracts from cells in pure culture liquid. [Results] We isolated a bacterium able to effectively degrade CB and assigned as strain CB001.Phylogenetic analysis based on 16S rRNA gene showed the similarity of 98.5% between strain CB001 and Acinetobacter calcoaceticus. In pure culture with CB as the sole carbon and energy source, 98.2% of CB at initial concentration of 50 mg/L was degraded; the molar ratio of the amount of net released Cl- to the amount of CB degraded by strain CB001 ranged from 1:1.85 to 1:1.39; the average activity of catechol 1,2-dioxygenase in crude extracts was 0.538 U/mg protein. The addition of glucose increased significantly the density of cells and the concentration of net released Cl-, but decreased obviously the ability of per cell to degrade CB. The capacity of strain CB001 to degrade CB was inhibited in di-chlorobenzenes and tri-chlorobenzenes coexisting culture system. The order in which strain CB001 readily degraded di-chlorobenzenes was 1,3-dichlorobenzene >1,2-dichlorobenzene >1,4-dichlorobenzene. [Conclusion] The results showed that strain CB001 belonged to the genus Acinetobacter, which showed capacity to degrade CB, di-chlorobenzenes. Strain CB001 possibly degraded CB through the pathway of meta ring cleavage. The addition of CB in the culture liquid increased significantly the ability of strain CB001 to degrade CB and the activity of catechol 1,2-dioxygenase.