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Electrostatic binding sites of extracellular polymeric substances (EPS) were characterized from titration data using linear programming analysis. Test results for three synthetic solutions of given solutes comprising amino, carboxyl, and phenolic groups indicated that this method was able to identify the electrostatic binding sites. For the six sites with pK_a between 3 and 10, the estimated pK_a deviated 0.11 ± 0.09 from the theoretical values, and the estimated concentrations deviated 3.0% ± 0.9% from the actual concentrations. Two EPS samples were then extracted from a hydrogen‐producing sludge (HPS) and a sulfate‐reducing biofilm (SRB). Analysis of charge excess data in titration from pH 3 to 11 indicated that the EPS of HPS comprised of five electrostatic binding sites with pK_a ranging from 3 to 11. The pK_a values of these binding sites and the possible corresponding functional groups were pK_a 4.8 (carboxyl …