Extensive research on the anammox-based processes under mainstream conditions is currently in progress. Most studies have used a long acclimation period for the partial nitritation-anammox (PN-An) sludge at a low temperature and ammonium concentration. However, in this study, the results demonstrated that PN-An granular biomass produced under sidestream conditions (30 °C and 1000 mg NH4+-N/L) can operate at 15 °C and 50 mg NH4+-N/L without acclimation. The nitrogen removal efficiency was 70% and was stable for 60 days. The long-term operation of the system with progressive adaptation provided important information for process optimization. Control of the dissolved oxygen (DO) concentration was crucial to maintain the balance between ammonia oxidizing bacteria (AOB) and anammox bacteria activities. A calculation of the oxygen penetration depth inside the granules is proposed to estimate an adequate DO level, which allows for the definition of the aerobic and anoxic zones that depend on the temperature, the size distribution and the granule density. However, the development of NOB was difficult to avoid with DO control alone. The selective washing-out of the floccular biomass, which contains mainly NOB, is proposed, leaving the granular fraction with the AOB and anammox bacteria in the system.