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In this study, two vertical constructed wetland using natural pyrite (P-VFCW) and quartz sand (C-VFCW) as substrate were constructed, and the enhanced nitrate removal mechanism by pyrite was further investigated. Results showed that the nitrate removal efficiency (NRE) of P-VFCW was 4% higher than that of C-VFCW with a C/N of 0. Interestingly, the difference on NRE between CWs markedly enlarged with C/N and hydraulic retention time (HRT) simultaneously increasing. At a COD/N of 6 and HRT of 24 h, the effluent average NO3--N and NO2--N co