Extracellular Polymeric Substances (EPS) extracted from aerobic granular sludge are a promising source of gelforming biopolymers. Gaining insights on the charge distribution of these polymers was performed through a fractionation using anionic chromatography. A gelling factor (GF) based on the apparent gel volume and the reactivity of polymers to calcium was attributed to each eluted fraction. The GF values of the eluted anionic EPS correlated with their negative charge. A GF of 0.04 ± 0.03 is measured for molecules eluted at NaCl 0.25 M and a GF of 0.32 ± 0.02 for the molecules eluted at NaCl 0.75 M. However, upon reaching a very high negative charge (elution at NaCl 1 M), the gelation capacity decreases. The size distribution of gelling molecules was analyzed by Size Exclusion Chromatography. High Molecular Weight (MW) molecules are predominantly involved in gelation (between 65 % and 100 % of the high molecular weight fraction from each sample) while low MW molecules participation is relatively limited (lower than 40 % of the low molecular weight fraction from each sample). Moreover, infra-red spectroscopy analysis of the molecules before and after calcium addition showed that mostly proteins and polysaccharides contribute to the hydrogel formation. Colorimetric methods showed that around 60 % of the uronic acids present in the samples could form hydrogels whereas proteins' participation varied between 0 % and 100 % depending on the fraction. Thus, modulating EPS extraction and purification methods to ensure the preservation of their molecular weight and the selection of appropriate negative charge could be a strategy for EPS valorization.