厌氧氨氧化污泥的细菌多态性分析-环境工程学报.doc

一种未见报道的厌氧氨氧化菌分子生物学鉴定* 任宏洋1，张代钧2**，丛丽影1，蔡庆1 （1重庆大学；2西南资源开发及环境灾害控制工程教育部重点实验室重庆 400030）(NH4)2SO4和NaNO2作为基质，16SrDNA进行PCR扩增。扩增产物连接到pMD19-T载体，将载体转化到感受态细胞大肠杆菌JM109中，并对其16SrDNA基因进行测序。将测序结果进行系统发育树分析发现Candidatus Anammoxoglobus propionicus 进化关系比较接近关键词: 16SrDNA, 鉴定 中图分类号 X703 文献标识码（A） 文章编号: Identification of an unknown ANAMMOX bacterium by molecular biology * REN Hongyang1, ZHANG Daijun1,2**, CONG Liying1, CAI Qing1 (1.Department of Environmental Science, Chongqing University, Chongqing 400030, China; 2 Key Laboratory of Southwest-China Resources Exploitation Environmental Disaster Control Engineering, Ministry of Education, Chongqing. 400030, China) Abstract: Anaerobic ammonium oxidation (ANAMMOX ) sludge was enriched with (NH4)2SO4 and NaNO2. Crude DNA of the total bacteria in cultivated anaerobic ammonium-oxidizing sludge was extracted and purified. Then partial 16SrDNA sequence of ANAMMOX bacterium was amplified by Polymerase Chain Reaction(PCR) with a pair of specific primers. The PCR product was cloned into the vector pMD19-T and was transfected into competent cells E.coil JM109, then 16SrDNA sequence was determined. Phylogenetic analysis indicates that the cultivated ANAMMOX bacterium closes to Candidatus Anammoxoglobus propionicus, and has never been found. Key words：Anaerobic ammonium-oxidizing bacteria 16SrDNA, identification 引言 厌氧氨氧化菌能直接以NO2-为电子受体，NH4+为电子供体在厌氧条件将同时转化为氮气与传统的硝化、反硝化脱氮过程相比，厌氧氨氧化能节省大量的氧气和有机碳源，具有广泛的应用前景。菌生长十分缓慢[]，并且在较高细菌浓度条件下才厌氧氨氧化，采用传统的微生物纯化培养方法[3]。Schmid等[4]采用特异引物Pla46/1037R对取自生物滤池中的厌氧氨氧化污泥样品进行分子生物学鉴定，最先测定了一种厌氧氨氧化细菌的16SrDNA序列，并确定了其在细菌系统进化树中的位置，属于分支较深的浮霉细菌（Planctomycetales），命名为Candidatus Brocadia anammoxidans。Schmid[5]设国Kartal等本研究采用分子生物学手段对实验室的厌氧氨氧化进行。1 材料与方法在EGSB膨胀颗粒污泥床Expanded Granular Sludge Bed）中接种实验室前期培养反应器有效容积2.5L，反应区1.5L，高度1.0m。(NH4)2SO4和NaNO2作为基质NH4+-N和NO2--N浓度为mg/L和65mg/L，1L合成废水加入1mL微量元素液，主要组分：ZnSO4·7H2O，2.2 g/L；CoCl2·6H2O，1.6 g/L；FeSO4·7H2O，5.0 g/L；CaCl2·2H2O，5.5 g/L； MnCl2·4H2O，5.0 g/L；CuSO4·5H2O，1.6 g/L；MgSO4·7H2O，5.0 g/L；(NH4)6Mo