A partial nitritation-anammox continuous flow reactor (CFR) was operated for eight months demonstrating that a mixture of large anammox-supported aerobic granules (ASAGs) and small conventional aerobic granules (CAGs) can be maintained stably for extended periods of time. The influent NH4+ was kept at 50 - 60 mg N L-1 to verify that the upper range of total ammonia nitrogen (TAN) for domestic wastewater can supply an inhibitory level of free ammonia (FA) for nitrite oxidizing bacteria (NOB) suppression in CFRs at pH around 7.8. The ammonia oxidizing bacteria (AOB):NOB activity ratio was determined for a series of granule sizes to understand the impact of mass diffusion limitation on the FA inhibition of NOB. When dissolved oxygen (DO) limitation is the only mechanism for NOB suppression, the AOB:NOB ratio was usually found in previous studies to increase with the granule size. However, the trend is reversed when FA has an inhibitory effect on NOB, as was observed in this study. The decrease in AOB:NOB ratio indicates that the resistance to the diffusion of FA along the granule radius limited its ability to inhibit NOB. This means smaller granules, e.g. diameter < 150 microns, are preferred for nitrite accumulation when high FA is present, e.g. in the partial nitritation-anammox process. The trend was further verified by observing the increase in the apparent inhibition coefficient, KI,FAapp, as granule size increased. This study for the first time quantified the effect of diffusion limitation on the KI,FAapp of NOB in granules and biofilms. A mathematical model was then utilized to interpret the observed suppression of NOB. The model predicted that NOB suppression was only complete at the granule surface. The NOB that did survive in larger granules was forced to dwell within the granule interior, where the FA concentration was lower than that in the bulk solution. This means FA inhibition can be taken advantage of as an effective means for NOB suppression in small granules and thin biofilms. Further, FA and DO were found to be both required for the stratification of AOB and NOB in partial nitritation-anammox CFRs. The structural stratification commonly observed in granules is then concluded to be a consequence but not a cause of the NOB suppression. / MS / A partial nitritation-anammox continuous flow reactor (CFR) was operated for eight months demonstrating that granular sludge can be maintained stably for extended periods of time. In this approach, NH3 is only partially converted to NO2 - (partial nitritation), and the conversion to NO3 - is prevented by the suppression of nitrite oxidizing bacteria (NOB). NH3 and NO2 - are then utilized by anammox bacteria to create N2 gas. The influent NH4 + fed to the reactor was kept at 50 to 60 mg N L-1 to verify that the upper range of total ammonia nitrogen (TAN) for domestic wastewater can supply a sufficiently high level of free ammonia (FA) to inhibit NOB growth in CFRs at a pH around 7.8. It is expected that the penetration of a substrate into granule sludge will experience diffusional resistance as it moves from water to denser solid material and is consumed by bacteria. The ammonia oxidizing bacteria (AOB):NOB activity ratio was determined for a series of granule sizes to understand the impact of mass diffusion limitation on the FA inhibition of NOB. When dissolved oxygen (DO) limitation is the only mechanism for NOB suppression, the AOB:NOB ratio was usually found in previous studies to increase with the granule size. However, the trend is reversed when FA has an inhibitory effect on NOB, as was observed in this study. The decrease in AOB:NOB ratio indicates that the resistance to the diffusion of FA, which increases with increasing granule size, along the granule radius limited its ability to inhibit NOB. This means smaller granules, e.g. diameter < 150 µm, are preferred for NO2 - accumulation when high FA is present. The trend was further verified by observing the increase in the apparent inhibition coefficient, KI,FAapp, as granule size increased. This coefficient quantifies the effectiveness of an inhibitor, with larger values indicating weaker inhibition. This study for the first time quantified the effect of diffusion limitation on the KI,FAapp of NOB in granules and biofilms. A mathematical model was then utilized to interpret the observed suppression of NOB. The model predicted that NOB suppression was only complete at the granule surface. The NOB that did survive in larger granules was forced to dwell within the granule interior, where the FA concentration was lower than that in the bulk solution. This means FA inhibition can be taken advantage of as an effective means for NOB suppression in small granules and thin biofilms. Further, FA and DO were found to be both required for the stratification of a layer of AOB at the surface over a layer of NOB in partial nitritation-anammox CFRs. The structural stratification commonly observed in granules is then concluded to be a consequence but not a cause of the NOB suppression.