The main objectives of the present study were to investigate the interactions of divalent metals (Ca2+ and Mg2+) with protein and polysaccharide components of extracellular polymeric substances (EPS) and to deduce their role in imparting different physicochemical characteristics to aerobic granules in sequencing batch reactor (SBR) system. Three reactors were run for 60 days; R-1 functioned as a control reactor, whereas R-2 and R-3 were operated by dosing 25 mg/L of Ca2+ and Mg2+, respectively, and the other operating conditions were similar in all of the SBRs. The granulation rate, particle size and sludge settleability were rapidly increased by Ca2+ addition, whereas the sludge dewaterability was significantly enhanced by Mg2+ augmentation. During the investigations, R-1, R-2 and R-3 had 1.31, 2.98 and 1.58 times higher quantities of polysaccharides, whereas proteins were 1.28, 1.74 and 2.41 times, respectively, more abundant than seed sludge. The divalent metals profoundly changed the composition of EPS, but their total contents were almost similar. XPS and FTIR results showed that Ca2+ bonded strongly with hydroxyl groups of polysaccharides due to there being less steric hindrance than proteins, and Mg2+ preferably bonded with amide groups of proteins and found no such hindrance due to its smaller size and thus changed the composition of EPS, which eventually altered the granular sludge characteristics. (C) 2015 Elsevier Ltd. All rights reserved.