In this study, the minimum sludge age required to allow nitrification in an activated sludge-based reactor was assessed. For this purpose, the sludge retention time (SRT) was gradually decreased and the organic matter and nitrogen conversions were monitored (1st stage). Once the critical SRT for nitrification was reached, the reactor was upgraded to a moving-bed biofilm reactor (MBBR) by the gradual addition of carrier media (2nd stage). The results showed that, for SRTs lower than 2.5 d, a reduction in ammonium removal was observed. Moreover, nitrite accumulation resulting from partial nitrification due to selective nitrite-oxidizing bacteria (NOB) washout began at SRT of 1.5 d and reached its peak at 0.5 d, accounting for almost the entire oxidized nitrogen forms (NOx−). When the SRT was reduced to 0.2 d, nitrification was suppressed and both ammonium-oxidizing bacteria (AOB) and NOB were no longer detected. By upgrading the bioreactor to support biofilm growth (2nd Stage), nitrifying activity was gradually recovered. The biofilm was found to play a major role in nitrification, as it predominated over the suspended solids at higher media filling fractions. Despite boosting nitrification, full ammonium removal was no longer achieved, suggesting an irreversible effect of the selective washout of nitrifiers within the 1st stage. A maximum ammonium removal rate of 0.18 kg N/(m3.d) was observed at 50 % filling ratio at an applied rate of 0.27 kg N/(m3.d).