ARTICLE Effect of Oxygen Gradients on the Activity and Microbial Community Structure of a Nitrifying, Membrane-Aerated Biofilm Leon S. Downing, Robert Nerenberg Department of Civil Engineering and Geological Sciences, 156 Fitzpatrick Hall, University of Notre Dame, Notre Dame, Indiana 46556; telephone: þ1-574-631-4098; fax: þ1-574-631-9236; e-mail: nerenberg.1@nd.edu Received 19 March 2008; revision received 21 May 2008; accepted 2 June 2008 Published online 11 June 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.22018 ABSTRACT: Shortcut nitrogen removal, that is, removal via formation and reduction of nitrite rather than nitrate, has been observed in membrane-aerated biofilms (MABs), but the extent, the controlling factors, and the kinetics of nitrite formation in MABs are poorly understood. We used a special MAB reactor to systematically study the effects of the dissolved oxygen (DO) concentration at the membrane surface, which is the biofilm base, on nitrification rates, extent of shortcut nitrification, and microbial community structure. The focus was on anoxic bulk liquids, which is typical in MAB used for total nitrogen (TN) removal, although aerobic bulk liquids were also studied. Nitrifying MABs were grown on a hollow-fiber membrane exposed to 3 mg N/L ammonium. The MAB intra-membrane air pressure was varied to achieve different DO concentrations at the biofilm base, and the bulk liquid was anoxic or with 2 g m 3 DO. With 2.2 and 3.5 g m 3 DO at the biofilm base, and with an anoxic bulk-liquid, the ammonium fluxes were 0.75 and 1.0 gNm 2 day 1 , respectively, and nitrite was the main oxidized nitrogen product. However, with membrane DO of 5.5 g m 3 , and either zero or 2 g m 3 DO in the bulk, the ammonium flux was around 1.3 g N m 2 day 1 , and nitrate flux increased significantly. For all experiments, the cell density of ammonium oxidizing bacteria (AOB) was rela- tively uniform throughout the biofilm, but the density of nitrite oxidizing bacteria (NOB) decreased with decreasing biofilm DO. Among NOB, Nitrobacter spp. were dominant in biofilm regions with 2 g m 3 DO or greater, while Nitrospira spp. were dominant in regions with less than 2gm 3 DO. A biofilm model, including AOB, Nitrobacter spp., and Nitrospira spp., was developed and calibrated with the experimental results. The model predicted the greatest extent of nitrite formation (95%) and the lowest ammonium oxidation flux (0.91 g N m 2 day 1 ) when the membrane DO was 2 g m 3 and the bulk liquid was anoxic. Conversely, the model predicted the lowest extent of nitrite formation (40%) and the highest ammonium oxidation flux (1.5 g N m 2 day 1 ) when the membrane-DO and bulk-DO were 8 g m 3 and 2 gm 3 , respectively. The estimated kinetic parameters for Nitrospira spp., revealed a high affinity for nitrite and oxygen. This explains the dominance of Nitrospira spp. over Nitrobacter spp. in regions with low nitrite and oxygen concentrations. Our results suggest that shortcut nitrification can effectively be controlled by manip- ulating the DO at the membrane surface. A tradeoff is made between increased nitrite accumulation at lower DO, and higher nitrification rates at higher DO. Biotechnol. Bioeng. 2008;xxx: xxx–xxx. ß 2008 Wiley Periodicals, Inc. KEYWORDS: nitrification; shortcut; membrane-aerated biofilm reactor; membrane biofilm reactor; kinetics; Nitrospira spp.; Nitrobacter spp. Introduction Total nitrogen (TN) removal from wastewater is an increasingly important treatment objective, and cost- effective TN removal methods are needed. Shortcut nitrogen removal can enhance the cost-effectiveness of any TN removal process. In shortcut nitrogen removal, ammonium is oxidized to nitrite by ammonia oxidizing bacteria (AOB) (‘‘shortcut nitrification’’), and then reduced to nitrogen gas by denitrifying bacteria (DNB) (‘‘shortcut denitrification’’). Shortcut nitrogen removal avoids formation of nitrate (van Loosdrecht and Jetten, 1998), reducing oxygen requirements by up to 25% and electron donor requirements by up to 40% (Hellinga et al., 1998). In order to achieve shortcut nitrogen removal, nitrite oxidizing bacteria (NOB) must be inhibited. This can be achieved by selective washout of NOB at high temperatures (Hellinga et al., 1998), high free ammonia (FA) concentra- tions (Anthonisen et al., 1976), high pH values (Villaverde et al., 1997), or low dissolved oxygen (DO) concentrations Correspondence to: R. Nerenberg Contract grant sponsor: Cooperative Institute for Coastal and Estuarine Environmental Technology (CICEET) Contract grant sponsor: National Science Foundation (NSF) ß 2008 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. xxx, No. xxx, 2008 1