Brief report Diversity of ammonium-oxidizing bacteria in a granular sludge anaerobic ammonium-oxidizing (anammox) reactor Zhe-Xue Quan, 1 Sung-Keun Rhee, 2 Jian-E Zuo, 3 Yang Yang, 3 Jin-Woo Bae, 4 Ja Ryeong Park, 4 Sung-Taik Lee 5 and Yong-Ha Park 4,6 * 1 Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China. 2 Department of Microbiology and Biotechnology Research Institute, Chungbuk National University, Cheongju 361-763, Korea. 3 Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084, China. 4 Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Korea. 5 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea. 6 Department of Applied Microbiology, Yeungnam University, Gyeongsan 712-749, Korea. Summary The ammonium-oxidizing microbial community was investigated in a granular sludge anaerobic ammonium-oxidizing (anammox) reactor that was operated for about 1 year with high anaerobic ammo- nium oxidation activity (up to 0.8 kg NH4 + -N m -3 day -1 ). A Planctomycetales-specific 16S rRNA gene library was constructed to analyse the diversity of the anaerobic ammonium-oxidizing bacteria (AnAOB). Most of the specifically amplified sequences (15/16) were similar to each other (> 99%) but were distantly related to all of the previously recognized sequences (< 94%), with the exception of an unclassified anammox-related clone, KSU-1 (98%). An ammonia monooxygenase (amoA) gene library was also analysed to investigate the diversity of ‘aerobic’ ammonium-oxidizing bacteria (AAOB) from the b-Proteobacteria. Most of the amoA gene fragments (53/55) clustered in the Nitrosomonas europaea– Nitrosococcus mobilis group which has been reported to prevail under oxygen-limiting conditions. The quantitative results from real-time polymerase chain reaction (PCR) amplification showed that the dominant AnAOB comprised approximately 50% of the total bacterial 16S rRNA genes in the reactor, whereas the AAOB of b-Proteobacteria represented only about 3%. A large fragment (4008 bp) of the rRNA gene cluster of the dominant AnAOB (AS-1) in this reactor sludge was sequenced and compared with sequences of other Planctomycetales including four anammox-related candidate genera. The partial sequence of hydrazine-oxidizing enzyme (hzo) of dominant AnAOB was also identified using new designed primers. Based on this analysis, we propose to tentatively name this new AnAOB Candidatus ‘Jettenia asiatica’. Introduction Ammonia removal under anoxic conditions was discov- ered in a laboratory-scale denitrification process and was confirmed to be a biologically mediated process (van de Graaf et al., 1995). Application of the anaerobic ammonium-oxidizing (anammox) process is especially interesting for the biological nitrogen removal from waste- water containing high nitrogen to carbon ratios. Anammox activity has been documented in many different reactor systems including a rotating biological contactor (RBC) (Helmer et al., 2001), a trickling filter (Schmid et al., 2000), an airlift completely autotrophic nitrogen removal over nitrite (CANON) reactor (Third et al., 2001), a sequential batch reactor (SBR) (Strous et al., 1998) and the first full-scale granular sludge anammox process (van der Star et al., 2007). Furthermore, the role of anaerobic ammonium-oxidizing bacteria (AnAOB) in the marine nitrogen cycle has gained increased attention because Received 7 September, 2007; accepted 19 March, 2008. *For correspondence. E-mail peter@yumail.ac.kr; Tel. (+82) 11 9968 4620; Fax (+82) 53 813 4620. Environmental Microbiology (2008) 10(11), 3130–3139 doi:10.1111/j.1462-2920.2008.01642.x © 2008 The Authors Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd