活性污泥中硝化细菌16S rDNA鉴定方法研究

Identification of Ammonia Oxidizer and Nitrite Oxidizer in Activated Sludge by 16S rDNA Sequencing

目的 以活性污泥为材料 ,研究其中硝化细菌的分离及 16SrDNA鉴定方法。方法 采用氨氧化细菌与亚硝酸氧化细菌富集、分离技术从武汉某啤酒厂曝气池活性污泥中分离得到 2株纯培养菌株N1和N2 。分别提取 2株细菌的总DNA ,利用氨氧化细菌与亚硝酸氧化细菌的 16SrDNA特异性引物进行多聚酶链反应 (PCR)扩增 ;扩增产物经 2 %琼脂糖凝胶电泳和Sanger末端终止法测序分析 ,用NCBI Blast软件将测序结果在Genbank等数据库中进行同源性检索。结果 N1和N2 的DNA扩增产物片断大小分别为 5 2 6bp和 391bp ,测序结果经检索证实N1、N2 分别与标准菌株Nitrosomonassp DYS317和Nitrobactersp R6的保守性片段有 99%、 10 0 %的同源性。 结论 可以判定所分离得到的N1和N2 菌株分别为亚硝化单胞菌属和硝化杆菌属。

Objective To study the isolation of the nitrifying bacteria and identification by 16S rDNA in activated sludge.Methods Two pure cultures, N 1 and N 2, were enriched and isolated based on the procedures for ammonia oxidizer and nitrite oxidizer from activated sludge of a aeration tank in a brewery plant in Wuhan. Total DNA of pure cultures was extracted and specific primers for the 16S rDNA of ammonia oxidizer and nitrite oxidizer were used for polymerase chain reaction (PCR). The amplified products were analyzed by 2 % agarose gel electrophoresis and Sanger sequencing. Homology analysis was made by searching in Genbank with NCBI-Blast. Results The amplified fragments were 526 bp and 391 bp for N 1 and N 2 respectively. 99 % of N 1 nucleotides were identical with the conserved fragment of Nitrosomonas sp. DYS317, while N 2 nucleotides were 100 % homologous with Nitrobacter sp. R6. Conclusion N 1 and N 2 phylogenetically belonged to Nitrosomonas and Nitrobacter respectively.