Abstract: The roles of suspended sludge and fixed sludge at electrode in combined with sulfide-based autotrophic denitrification by progressively decreasing suspended sludge at a fixed nitrate concentration (50 mg/L) in microbial fuel cell (MFC) was investigated. The suspended sludge is involved in nitrate reduction and sulfate conversion; however, the contributions of fixed sludge at electrode are greater than those of suspended sludge in substrate transformations. The electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) analyses suggested that suspended sludge helped to improve the electron transport capacity and reduced the internal resistance. The best performance in terms of electricity generation was demonstrated with fixed sludge at electrode only. The dominant microbial genera in fixed sludge at electrode include Acidithiobacillus, Sulfurimonas, Sulfurovum and Halothiobacillus, while latter three genera are dominant in suspended sludge, with relative high abundance of denitrifying species (Thauera and Rhodobacteraceae).