A lab-scale membrane bioreactor (MBR) was continuously operated to investigate the membrane fouling. A new membrane fouling mechanism: osmotic pressure mechanism in cake layer filtration process was identified. Osmotic pressure was proposed to stem from the retention of counter-ions in the matrix of biopolymers in cake layer. Through Fourier transform infrared (FT-IR) spectroscopy and X-ray photoelectron spectroscopy (XPS) analyzes, it was found that functional groups were abundant in the surface of cake layer. Batch filtration tests showed that soluble microbial products (SMP) and biopolymer clusters (BPC) in the supernatant played key roles in osmotic pressure mechanism, and were thus largely responsible for the high cake resistance. The chemical potential of water varied along with cake depth. The formed cake layer was found to be much hydrated and elastic. These findings provided the direct evidence for the existence of osmotic pressure mechanism. (C) 2014 Elsevier Ltd. All rights reserved.