BACKGROUND The main problem for the formation of anaerobic granule in a short period is synchronization between good physicochemical and microbial characteristics. This study aims to investigate the formation of anaerobic granule with good physicochemical and microbial characteristics using simultaneous granulation and microbial adaptation to syntroph-specific substrate. RESULTS Simultaneous microbial adaptation to propionate along with granulation successfully enhanced the activities and the populations of syntroph-related microbial groups, such as propionate-utilizing microorganism and hydrogenotrophic methanogens (HTMs). The development of granules in chitosan-added reactor (R2) was accelerated in shorter period, and larger granules (>1000 mu m) were seen in a higher percentage than those in control reactor (R1). In addition, the granules in R2 exhibited better physicochemical characteristics: granule percentage of 52.8%, mean particle diameter of 535 mu m, and sludge volume index of 23 mL g(-1) suspended solids. During early granulation, protein-extracellular polymeric substance (EPS) and chitosan dominated granule formation in R1 and R2, respectively. However, the increase of polysaccharide-EPS at high organic loading rate improved the morphology and stability of granule in both the reactors. A remarkable result was seen for the granules in R2, showing a multi layered-structure that consisted of Methanosarcinales and Smithella sp. at the inner and middle/outer layers, respectively. While HTMs were spatially distributed nearby with acetogens. CONCLUSION Anaerobic granulation using the combined effect of EPS and chitosan during simultaneous granulation and adaptation on the syntroph-specific substrate did not only lead to the development of large and dense granules but also granules with high syntroph activity, high settling ability, and multilayered structure. (c) 2019 Society of Chemical Industry