Appl Microbiol Biotechnol (2005) 68: 808–817 DOI 10.1007/s00253-005-1974-6 ENVIRONMENTAL BIOTECHNOLOGY A. Olav Sliekers . Suzanne C. M. Haaijer . Marit H. Stafsnes . J. Gijs Kuenen . Mike S. M. Jetten Competition and coexistence of aerobic ammonium- and nitrite-oxidizing bacteria at low oxygen concentrations Received: 19 January 2005 / Revised: 16 March 2005 / Accepted: 20 March 2005 / Published online: 8 April 2005 # Springer-Verlag 2005 Abstract In natural and man-made ecosystems nitrifying bacteria experience frequent exposure to oxygen-limited conditions and thus have to compete for oxygen. In several reactor systems (retentostat, chemostat and sequencing batch reactors) it was possible to establish co-cultures of aerobic ammonium- and nitrite-oxidizing bacteria at very low oxygen concentrations (2–8 μM) provided that am- monium was the limiting N compound. When ammonia was in excess of oxygen, the nitrite-oxidizing bacteria were washed out of the reactors, and ammonium was converted to mainly nitrite, nitric oxide and nitrous oxide by Nitrosomo- nas-related bacteria. The situation could be rapidly reversed by adjusting the oxygen to ammonium ratio in the reactor. In batch and continuous tests, no inhibitory effect of am- monium, nitric oxide or nitrous oxide on nitrite-oxidizing bacteria could be detected in our studies. The recently developed oxygen microsensors may be helpful to de- termine the kinetic parameters of the nitrifying bacteria, which are needed to make predictive kinetic models of their competition. Introduction Nitrification is known as an aerobic two-step microbial process in which ammonia is oxidized to nitrate by two groups of chemolithoautotrophic bacteria. Aerobic ammo- nia-oxidizing bacteria (AOB), such as Nitrosospira and Nitrosomonas, oxidize ammonia to nitrite, and aerobic nitrite-oxidizing bacteria (NOB), such as Nitrospira and Nitrobacter, oxidize the nitrite further to nitrate (Bock and Wagner 2001; Koops and Pommerening-Röser 2001). The process of nitrification is an important part of the microbial nitrogen cycle and is most often studied under fully oxic conditions. However, in many natural and man- made ecosystems such as wastewater treatment systems, oxygen-limiting conditions are frequently observed (Jetten et al. 2003; Pynaert et al. 2002a,b, 2003, 2004; Sliekers et al. 2002, 2004, van Loosdrecht and Jetten 1998; van Loosdrecht et al. 2004; Wijffels et al. 2004). In wastewater treatment practice, this oxygen limitation can lead to periodic buildup of nitrite, which causes severe inhibition of the activated sludge biomass. On the other hand, permanent oxygen limitation can be used to intentionally limit or even cir- cumvent nitrite oxidation to nitrate (Abeling and Seyfried 1992; Garrido et al. 1997; Schmidt et al. 2003). This is practiced in processes like completely autotrophic nitrogen removal over nitrite (CANON) and oxygen-limited auto- tropic nitrogen removal (OLAND) processes (Kuai and Verstraete 1998; Helmer-Madhok et al. 2002; Pynaert et al. 2003; Sliekers et al. 2002, 2003; van Dongen et al. 2001). Nitrifying bacteria can be detected in many aerobic environments where sufficient ammonia is available (i.e. from mineralization processes) to support their growth (Bollmann and Laanbroek 2002). Furthermore, nitrifying bacteria are active at the oxic–anoxic interface where the ammonia liberated by anaerobic mineralization meets the oxygen diffusing from the upper layers (Kuypers et al. 2003; Nielsen et al. 2005; Risgaard-Petersen et al. 2004; Thamdrup and Dalsgaard 2002). At such interfaces, the volumetric activity of nitrifying bacteria can be very high (Lorenzen et al. 1998). In laboratory studies, the nitrifying bacteria are usually cultivated under fully oxygenated conditions, but it is likely that most of the nitrifying bac- teria have adapted to the low and limiting amounts of oxygen present at the oxic–anoxic interface. This view is supported by the detection of nitrifying bacteria in systems A. O. Sliekers . J. G. Kuenen . M. S. M. Jetten Department of Biotechnology, Faculty of Applied Science, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands S. C. M. Haaijer (*) . M. S. M. Jetten Department of Microbiology, Institute for Water and Wetland Research, Faculty of Science, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands e-mail: S.Haaijer@science.ru.nl M. H. Stafsnes Department of Biotechnology, Norwegian Technical University Trondheim, Trondheim, Norway