Simultaneous partial nitrification, anammox and denitrification (SNAD) process was started-up in a 2.5 L sequencing batch biofilm reactor (SBBR) using novel biomass carriers. The SNAD process took only 51 d for start-up at nitrogen loading rate (NLR) and organic loading rate (OLR) of 120 and 60 g/m(3)-d, respectively. Long-term stable operation of SNAD process was observed at NLR and OLR of 360 and 180 g/m(3)-d with average total nitrogen and COD removal efficiencies of >88% and >90%, respectively. The values of conversion ratio Y(NO2-+NO3-)/NH4+ remained below 0.11 after the start-up period, which further confirmed the long-term stability of SNAD process. Results of polymerase chain reaction (PCR), qualitative PCR, and scanning electron microscopic (SEM) analysis of sludge samples confirmed the co-existence and enrichment of AOB, anammox bacteria and denitrifying bacteria in the reactor and biofilm formation on to the carriers. (C) 2015 Elsevier Ltd. All rights reserved.