Previous studies have shown that membrane-aerated biofilm (MAB) reactors can simultaneously remove carbonaceous and nitrogenous pollutants from wastewater in a single reactor. Oxygen is provided to MABs through gas-permeable membranes such that the region nearest the membrane is rich in oxygen but low in organic carbon, whereas the outer region of the biofilm is void of oxygen but rich in organic carbon. In this study, MABs were grown under similar conditions but at two different fluid velocities (2 and 14 cm s ؊1 ) across the biofilm. MABs were analyzed for changes in biomass density, respiratory activity, and bacterial community structure as functions of biofilm depth. Biomass density was generally highest near the membrane and declined with distance from the membrane. Respiratory activity exhibited a hump-shaped profile, with the highest activity occurring in the middle of the biofilm. Community analysis by PCR cloning and PCR-denaturing gradient gel electrophoresis of 16S rRNA genes demonstrated substantial stratification of the community structure across the biofilm. Population profiles were also generated by competitive quantitative PCR of gene fragments specific for ammonia-oxidizing bacteria (AOB) (amoA) and denitrifying bacteria (nirK and nirS). At a flow velocity of 14 cm s ؊1 , AOB were found only near the membrane, whereas denitrifying bacteria proliferated in the anoxic outer regions of the biofilm. In contrast, at a flow velocity of 2 cm s ؊1 , AOB were either not detected or detected at a concentration near the detection limit. This study suggests that, under the appropriate conditions, both AOB and denitrifying bacteria can coexist within an MAB.The aeration of bioreactors is one of the most significant operating costs incurred during the treatment of municipal and industrial wastewaters. One technological alternative to conventional coarse and fine bubble diffusers is the use of microporous membranes that contain small (Ͻ0.1 m) hydrophobic pores for bubbleless oxygen transfer to the wastewater (1). Biofilms actively grow on these membranes, such that membrane-aerated biofilm (MAB) reactors can treat wastewater at rates comparable to conventional suspended-growth processes (6,33,37). MAB reactors have lower operating costs, because oxygen transfer efficiency approaches 100% (5, 26) and because expensive air compression is no longer required (9). In addition, MAB reactors generate fewer odors, because volatile pollutants are not released into the atmosphere (5, 6).MABs are unique compared to biofilms grown on inert substrata. Nutrient concentrations in MABs are highest at the biofilm-liquid boundary and decrease with depth into the biofilm; in contrast, oxygen concentrations are highest at the membrane and decrease toward the outside of the biofilm (Fig. 1). In thick MABs, oxygen partially penetrates the biofilm, generating an anoxic zone at the biofilm-liquid boundary (6). In such biofilms, the region of greatest microbial activity is variable and depends on the relative availability of oxygen a...