A two-chambered microbial fuel cell (MFC), which can function on the self-driven bioelectrogenic activity operated on anaerobically digested distillery waste (ADDW) i.e. wastewater post anaerobic digestion was designed and fabricated in the laboratory. MFC was evaluated for production of bioelectricity with a simultaneous reduction in the carbon content. Using a surface response methodology with a Box-Behnken design (BBD), operating conditions such as the concentration of antifoam, pH, and resistance were optimized and it was found that the pH and resistance were optimum at 8.3 and 1000 0, respectively with no antifoam in the system. Under optimum conditions, 31.49 Wm-3 was generated, and 60.78 +/- 0.95% total organic carbon was degraded. We revealed that the fermentative bacteria generated organic acids mainly acetate from dextrose present in ADDW and electrogenic bacteria oxidized acetate in a successive manner to generate electrons, which was confirmed by gas chromatography. The development of biofilm analyzed by scanning electron microscope (SEM) was found to be crucial in the transfer of electrons directly to the anode and was confirmed by cyclic voltammetry experiments. Identification of bacteria from biofilm by both culture and denaturing gradient gel electrophoresis methods found bacteria belonging to phylum Firmicutes and gamma-proteobacteria. The study of successive nature of bacterial metabolism to generate electricity could play an important role in the production of electricity in a continuous mode of operation using MFCs fed with ADDW for further reduction of carbon content post anaerobic digestion for the benefit for the environment. Thus MFC can be used as a complementary technology to anaerobic digestion. (C) 2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.