A pilot-scale moving-bed biofilm reactor (MBBR) for biological treatment of micro-polluted raw water was operated over 400 days to investigate the responses of biofilm characteristics and nitrification performance to variations in temperature and NH4+-N loading. The mean removal efficiency of NH4+-N in the MBBR reached 71.4 +/- 26.9%, and batch experiments were performed to study nitrification kinetics for better process understanding. Seven physical-chemical parameters, including volatile solids (VS), polysaccharides (PS) and phospholipids (PL) increased firstly, and then rapidly decreased with increasing temperature and NH4+-N loading, and properly characterized the attached biomass during biofilm development and detachment in the MBBR. The biofilm compositions were described by six ratios, e.g., PS/VS and PL/VS ratios showed different variation trends, indicating different responses of PS and PL to the changes in temperature and NH4+-N loading. Furthermore, fluorescent in situ hybridization (FISH) analysis revealed that increased NH4+-N loadings caused an enrichment of the nitrifying biofilm. (C) 2013 Elsevier Ltd. All rights reserved.