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Nitrate (NO3−-N) and nitrites (NO2−-N) are common pollutants in various water bodies causing serious threats not only to aquatic, but also to animals and human beings. In this study, we developed a strategy for efficiently reducing nitrates in microbial fuel cells (MFCs) powered by a granular activated carbon (GAC)-biocathode. GAC was developed by acclimatizing and enriching denitrifying bacteria under a redox potential (0.3 V) generated from MFCs. Thus, using the formed GAC-biocathode we continued to study their effect on denitrification with different cathode materials and circulation speeds in MFCs. The GAC-biocathode with its excellent capacitive property can actively reduce nitrate for over thirty days irrespective of the cathode material used. The stirring speed of GAC in the cathode showed a steady growth in potential generation from 0.25 V to 0.33 V. A rapid lag phase was observed when a new carbon cathode was used with enriched GAC. While a slow lag phase was seen when a stainless-steel cathode was replaced. These observations showed that effective storage and supply of electrons to the GAC plays a crucial role in the reduction process in MFCs. Electrochemical analysis of the GAC properties studied using electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and zeta potential showed distinct properties with different abiotic and biocathode conditions. We found that the enrichment of electrotrophic bacteria on GAC facilitates the direct electron transfer in the cathode chamber for reducing NO3−-N in MFCs as observed by scanning electron microscopy. [ABSTRACT FROM AUTHOR]