Microbial extracellular polymeric substances (EPS) with a high polysaccharides content contribute to membrane fouling in membrane bioreactors. Sodium alginate (Alg) has been widely used as a substitute for polysaccharides in EPS to study membrane fouling; however, the rational for such a substitute is unclear and remains to be verified. In this work, the differences in the colloid properties of Alg-Ca2+ and EPS-Ca2+ were explored by integrating rheometry and dynamic light scattering with regard to distinct membrane fouling behaviors. The Alg-Ca2+ exhibited a larger hydrodynamic diameter and higher colloidal stability, which were attributed to its more solidified complexation compared with the electrostatic interactions in EPS-Ca2+. As a result, the gel layer of Alg-Ca2+ that formed on the membrane surface was denser and more impermeable, leading to a faster flux decline in the membrane filtration process. These results indicate that it is questionable to substitute polysaccharides in EPS with Alg for membrane fouling studies. Considering the substantial differences in the colloid properties, it would be better to modify Alg or find a more appropriate substitute for membrane fouling studies. (C) 2018 Elsevier Inc. All rights reserved.