Cosubstrate-dependent modulation of anaerobic azo dye decolorization under conductive and non-conductive conditions

• Joint effect of cosubstrate type and Fe 3 O 4 on decolorization was investigated. • Different cosubstrate types shape distinct dye decolorization pathways. • Extracellular electron transfer (EET) is a crucial mechanism in dye decolorization. • The ethanol-Fe 3 O 4 is the most effective combination for dye decolorization. • Long-term Fe 3 O 4 exposure reshaped microbial communities toward EET-active taxa. Cosubstrate type is a key factor shaping decolorization behavior, while magnetite (Fe 3 O 4 ) is recognized as an electron transfer facilitator in anaerobic dye treatment systems. This study elucidated how cosubstrate type and Fe 3 O 4 jointly influence anaerobic azo dye decolorization through batch and continuous experiments. In batch tests, five cosubstrates were comparatively evaluated for their decolorization performance under Fe 3 O 4 -free and Fe 3 O 4 -amended conditions. Complex cosubstrates (glucose and starch) likely favored enzyme-associated azo reduction, whereas simpler cosubstrates (ethanol, acetate, and propionate) exhibited decolorization patterns consistent with enhanced extracellular electron transfer (EET), particularly in the presence of Fe 3 O 4 . Ethanol achieved 85–95% decolorization efficiency and was identified as the most effective cosubstrate-Fe 3 O 4 combination. In continuous operation, ethanol maintained stable decolorization over 235 days, and Fe 3 O 4 addition further enhanced performance by 15%. A methanogenic activity assay confirmed that the acetoclastic pathway remained stable, while conductive conditions likely enabled additional electron-utilization pathways, such as EET, allowing concurrent methane production and dye reduction. Microbial and predicted functional analyses revealed enrichment of putative EET-associated taxa (e.g., Geobacter, Desulfovibrio, Pseudomonas, Methanosarcina , and Methanobacterium ). This shift was accompanied by increased abundances of EET-related genes (1.5–2.3-fold) and reduced abundances of azoreductase-related genes (1.6-fold), supporting a transition toward EET-associated decolorization under conductive conditions. These findings highlight the synergistic role of cosubstrate selection and conductive materials in regulating decolorization behavior, offering practical insights for optimizing anaerobic dye wastewater treatment.