This study aims to clarify the biochemical nature and interactions of Extracellular Polymeric Substances (EPS) involved in the structure and cohesive properties of aerobic granules. Granules were incubated with selective hydrolytic enzymes or with chemicals and the resistance of digested granules to shear stress was evaluated. After alpha-amylase digestion, the hydrodynamic stress released macro-particles (>315 mu m) while soluble molecules (<1.5 mu m) and micro-particles (1.5-315 mu m) where mainly recovered after savinase and EDTA treatments. These data show that alpha (1-4) glucans and proteins are key polymers for granule cohesion and that divalent cationic bridging is a major aggregative mechanism. On the basis of these experiments and microscopy observations, a model is proposed for the spatial organization of EPS in the granular structure, in which a glucans are arranged in a capsular layer surrounding bacterial clusters while anionic proteins constitute the intercellular cement that may reinforce cohesion inside the bacterial clusters. (C) 2014 Elsevier Ltd. All rights reserved.