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This study compares the effects and bacterial community structure of single-chamber microbial fuel cells (MFCs) in the treatment of NH4+-containing wastewater with different chemical oxygen demand (COD)/N ratios, whilst simultaneously conducting stratification research on the cathode biofilm. To this end, five nitrifier pre-enriched single-chamber MFC reactors are established to treat five different COD/N wastewaters, respectively. The results show that MFCs with low COD/N have better NH4+-N removal, electrochemical performance, but the re