Anaerobic ammonium oxidation by Nitrosomonas spp. and anammox bacteria in a sequencing batch reactor Pongsak (Lek) Noophan a, * , Siriporn Sripiboon b , Mongkol Damrongsri b , Junko Munakata-Marr c a Department of Environmental Science, Faculty of Science, Silpakorn University, Nakhon-pathom Province 73000, Thailand b Department of Environmental Engineering, Faculty of Engineering, Kasetsart University, Bangkok 10900, Thailand c Environmental Science and Engineering Division, Colorado School of Mines, Golden, CO 80401, USA article info Article history: Received 19 June 2007 Received in revised form 16 November 2007 Accepted 8 March 2008 Keywords: Biological nitrogen removal Anaerobic ammonium oxidation Anammox abstract A sequencing batch reactor (SBR) was inoculated with mixed nitrifying bacteria from an anoxic tank at the conventional activated sludge wastewater treatment plant in Nongkhaem, Bangkok, Thailand. This enriched nitrifying culture was maintained under anaerobic conditions using ammonium (NH 4 þ ) as an electron donor and nitrite (NO 2  ) as an electron acceptor. Autotrophic ammonium oxidizing bacteria survived under these conditions. The enrichment period for anammox culture was over 100 days. Both ammonium and nitrite conversion rates were proportional to the biomass of ammonium oxidizing bacteria; rates were 0.08 g N/g VSS/d and 0.05 g N/g VSS/d for ammonium and nitrite, respectively, in a culture maintained for 3 months at 42 mg N/L ammonium. The nitrogen transformation rate at a ratio of NH 4 þ –N to NO 2  –N of 1:1.38 was faster, and effluent nitrogen levels were lower, than at ratios of 1:0.671, 1:2.18, and 1:3.05. Fluorescent in situ hybridization (FISH) was used to identify specific auto- trophic ammonium oxidizing bacteria (Nitrosomonas spp., Candidatus Brocadia anammoxidans, and Candidatus Kuenenia stuttgartiensis). The ammonium oxidizing culture maintained at 42 mg N/L ammo- nium was enriched for Nitrosomonas spp. (30%) over Candidati B. anammoxidans and K. stuttgartiensis (2.1%) while the culture maintained at 210 mg N/L ammonium was dominated by Candidati B. anam- moxidans and K. stuttgartiensis (85.6%). The specific nitrogen removal rate of anammox bacteria (0.6 g N/g anammox VSS/d) was significantly higher than that of ammonium oxidizing bacteria (0.4 g N/g Nitro- somonas VSS/d). Anammox bacteria removed up to 979 mg N/L/d of total nitrogen (ammonium:nitrite concentrations, 397:582 mg N/L). These results suggest significant promise of this approach for appli- cation to wastewater with high nitrogen but low carbon content, such as that found in Bangkok. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Conventionally, nitrogen removal from wastewater by biological processes involves well-known aerobic nitrification (ammonium (NH 4 þ ) as the electron donor and oxygen as the electron acceptor) followed by anoxic denitrification (organic matter or carbon source as the electron donor and nitrite (NO 2  ) or nitrate (NO 3  ) as the electron acceptor). Biological nitrogen removal has become com- mon because of its low cost and high efficiency as compared to physical and chemical treatment (van Dongen et al., 2001a,b). However, nitrogen removal by biological processes is seldom used in wastewater with high NH 4 þ but low carbon content. For example, the carbon content in typical Thai domestic wastewater is about half that recommended for conventional biological nitrogen removal (Noophan et al., 2007). Because the available carbon in these wastewaters is insufficient for the denitrification process, an external carbon source such as acetate, glucose, ethanol, methanol or methane gas must be added. All these external carbon sources are expensive and substantially increase the cost of operation. Recently, approaches for biological nitrogen removal have been proposed which bypass the formation of NO 3  and convert NO 2  to N 2 gas with NH 4 þ as the electron donor and NO 2  as the electron acceptor under anaerobic conditions (ANaerobic AMMonium Oxi- dation or anammox). Bock et al. (1995) and Strous et al. (1997) reported that both pure and mixed ammonium oxidizing bacteria and anammox bacteria under anaerobic conditions were able to use nitrite as an electron acceptor and ammonium as an electron donor. The stoichiometric conversion of NO 2  and NH 4 þ to N 2 gas without production of cell material and nitrate is shown below. 1NH D 4 D 1:32NO L 2 D 0:066HCO L 3 D 0:13H D /1:02N 2 D 0:26NO L 3 D 0:066CH 2 O 0:5 N 0:15 D 2:03H 2 O (1) * Corresponding author. Tel.: þ66 34 219 146; fax: þ66 34 273 047. E-mail addresses: pongsak@su.ac.th (P. (Lek) Noophan), siripornsr@yahoo.com (S. Sripiboon), fengmkd@ku.ac.th (M. Damrongsri), junko@mines.edu (J. Munakata-Marr). Contents lists available at ScienceDirect Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman 0301-4797/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jenvman.2008.03.003 Journal of Environmental Management 90 (2009) 967–972