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Treating high-strength nitrate wastewater effectively and economically has been a major challenge for some manufacturing industries. In this study, we fabricated a membrane-less two-phase bio-cathode microbial fuel cell (TPBC-MFC) by dividing the bio-cathode into a solid part (S-cathode, above the water surface) and a liquid part (L-cathode, below the water surface), on which performances and mechanisms for the treatment of pickling wastewater containing more than 1000 mg/L NO- 3-N (electron acceptor) by watermelon rind extracts (WMREs, electron donor) were explored. Results showed that the TPBC-MFC presented an excellent performance since the maximum power density, the greatest nitrate removal rate and the highest COD removal efficiency reached 16.6 mW/m2, 780 g/m3·d and 94.8%, respectively. There were identical microorganisms (unclassified- Chitinophagaceae and Comamonadaceae) predominating on all electrodes owing to WMREs as carbon sources. In addition, some special functional species were also enriched, including Anaeroarcus (3.48%, fermentation bacterium) and Desulfovibrio (12.57%, electricigen) for the anode, Thauera (5.02%, denitrifier) for the L-cathode and Chryseolinea (12.13%, denitrifier) for the S-cathode. The detection of denitrifying genes of narG, narK/S, norB and nosZ, as well as function prediction demonstrated that the anode degraded WMREs to release H+ and e− that were subsequently utilized by the cathode to reduce NO- 3-N to N 2. All these results proved that the membrane-less TPBC-MFC has a good capability in electricity generation, nitrate removal and WMREs degradation, most likely because the S-cathode benefits the promotion of mass transfer. [Display omitted] • TPBC-MFC consisting of S-cathode and L-cathode was developed. • Watermelon rinds fueled TPBC-MFC for sustainable pickling wastewater treatment. • S-cathode was vital for TPBC-MFC due to its role in promoting mass transfer. • Each of bio-electrodes possessed its own specific functional microorganisms. • Reduction of nitrate to N 2 was mainly carried out by S-cathode. [ABSTRACT FROM AUTHOR]