刘长发^1，2，姚敬元²，袁 瑗²，刘卫东³
（1 近岸海洋环境科学与技术辽宁省高校重点实验室，大连海洋大学，大连 116023；
2 大连海洋大学海洋科技与环境学院，大连 116023；
3 辽宁省海洋水产科学研究院，大连 116023）

摘要：研究循环水养殖硝化滤器载体上附着生物膜的微生物群落结构可以为提高其处理速率和效率，并为特异性工程菌构建提供依据。采用改良的AFLP方法分析了循环水养殖硝化滤器载体上附着的氨氧化细菌16S rRNA基因和氨单加氧酶amoA基因片段及其系统发育情况。结果表明：分析16S rRNA基因得到的序列片段比分析amoA基因片段得到了更多信息，准确度较高，可作为分析循环水养殖硝化滤器氨氧化菌群组成的有效方法。克隆测序所得序列与网上公布数据比对，可见存在于循环水养殖硝化滤器载体上的氨氧化细菌与Nitrosomonas cryotolerans、Nitrosomonas oligotropha、Nitrosospira tenuis、Nitrosomonas marina相似度达100%，与Nitrosomonas aestuarii相似度为87%。大部分属于亚硝化单胞菌属（Nitrosomonas），仅少数序列属于亚硝化螺菌属（Nitrosospira）。采用16S rRNA基因和amoA片段分析方法得到的附着于封闭循环海水养殖硝化滤器载体上的氨氧化细菌主要为变形菌（Proteobacteria）的-亚类的亚硝化单胞菌属（Nitrosomonas）和少量的亚硝化螺菌属（Nitrosospira）氨氧化细菌，以及一定数量的-亚类氨氧化细菌。

关键词：氨氧化细菌；硝化作用滤器；循环海水养殖系统；16S rRNA；amoA

Ammonia-oxidizing bacterial community composition in the nitrifying biofilter of recirculating aquaculture system
LIU Chang-fa ^{1, 2}, YAO Jing-yuan ², Yuan Yuan ², LIU Wei-dong ³
( 1 Key Laboratory of Offshore Marine Environmental Research of Liaoning High Education, Dalian 116023, China; 2 College of Marine Sci-Tech and Environment, Dalian Ocean University, Dalian 116023, China;
3 Liaoning Ocean and Fisheries Science Research Institute, Dalian 116023, China )

Abstract: Ammonia-oxidizing bacteria (AOB) play an important role in transformation from ammonia nitrogen to nitrite nitrogen in the nitrifying biofilter. Studying community structure of nitrifying organisms adhered on the media of nitrification biofilter in the recirculating aquaculture system (RAS) could provide a basis of improving removal rate and performance of biofilter, and construction of nitrifying engineering bacteria for removal of ammonia from RAS. The PCR-based gene cloning and mapping of 16S rRNA gene and betaproteobacteria ammonia monooxygenase subunit A (amoA) gene of ammonia-oxidizing bacteria isolated from nitrification biofilter were surveyed by using the method of improved amplified fragment length polymorphism (AFLP) analysis. The results showed that the information of AOB using 16S rRNA gene cloning is more than using amoA gene cloning. We used GenBank BLAST to search for best matches for the obtained sequences, the AOB in the nitrification biofilter were much closed to Nitrosomonas cryotolerans, Nitrosomonas oligotropha, Nitrosospira tenuis, Nitrosomonas marina with 100% of similarity and Nitrosomonas aestuarii with 87% of similarity. The AOB in the nitrification biofilter of RAS belong to the -subclass of Proteobacteria, Nitrosomonas and Nitrosospira. Nitrosospira-like sequences were detected.

Key words: ammonia-oxidizing bacteria; nitrifying biofilter; Recirculating Aquaculture System (RAS); 16S rRNA; ammonia monooxygenase (amoA) gene

基金项目：国家“863”高技术研发计划项目（2007AA10Z410）
作者简介：刘长发（1964—），男，教授，博士，主要从事污染物生物环境地球化学过程与控制研究。E-mail: liucf@dlou.edu.cn

（来源：《渔业现代化》2012,39(3):1-6,12）