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[Display omitted] • AA-TFCW accelerated NH 4 +-N transformation with 6.68 mg/(L·h) in start-up phases. • Heterotroph and autotroph synergistically removed 74.2% TN at low temperature. • HN-AD bacteria achieved direct NH 4 +-N removal by nitrate assimilation pathway. • Ferribacterium and Nitrosomonas carried high-weight key genes for N removal. This study explored the quantitative mechanisms of heterotrophic nitrification-aerobic denitrification (HN-AD) in a pilot-scale two-stage tidal flow constructed wetland (TFCW). The TFCW packed shale ceramsite (SC) and activated alumina (AA) at each stage, respectively, and aimed to improve decentralized wastewater treatment efficiency. In start-up phases, AA-TFCW accelerated NH 4 +-N decline, reaching transformation rates of 6.68 mg NH 4 +-N/(L·h). In stable phases, SC-AA-TFCW resisted low-temperatures (<13 °C), achieving stable NH 4 +-N and TN removal with effluents ranging 6.36–8.13 mg/L and 9.43–14.7 mg/L, respectively. The dominant genus, Ferribacterium, was the core of HN-AD bacteria, simultaneously removing NH 4 +-N and NO 3 –-N by nitrate assimilation and complete denitrification (NO 3 –-N → N 2), respectively. The quantitative associations highlighted importance of nitrification, nitrate assimilation, and denitrification in nitrogen removal. HN-AD bacteria (e.g., Lactococcus , Thauera , and Aeromonas) carried high-weight genes in quantitative associations, including nap AB, nas A and glt BD, implying that HN-AD bacteria have multiple roles in SC-AA-TFCW operation. [ABSTRACT FROM AUTHOR]