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Separator plays an important role in microbial fuel cells (MFCs). Despite of the rapid development of separators in recent years, there are remaining barriers such as proton transfer limitation and oxygen leakage, which increase the internal resistance and decrease the MFC performance, and thus limit the practical application of MFCs. In this review, various separator materials, including cation exchange membrane, anion exchange membrane, bipolar membrane, microfiltration membrane, ultrafiltration membranes, porous fabrics, glass fibers, J-Cloth and salt bridge, are systematically compared. In addition, recent progresses in separator configuration, especially the development of separator electrode assemblies, are summarized. The advances in separator materials and configurations have opened up new promises to overcome these limitations, but challenges remain for the practical application. Here, an …