Abstract:Stringent nitrogen discharge standards necessitate advanced treatment technologies for secondary effluent from municipal wastewater treatment plants (WWTPs). Conventional heterotrophic denitrification faces limitations, such as dependence on organic carbon and potential emissions of nitrous oxide (N2O), a potent greenhouse gas. To address these challenges, this study established and systematically evaluated a novel staged denitrification system that integrates sulfur autotrophic and heterotrophic processes. The primary objective was to investigate the effects of different coupling patterns, i.e., sulfur autotrophic followed by heterotrophic (R1) versus heterotrophic followed by sulfur autotrophic (R2), on overall nitrogen removal efficiency, N2O emission dynamics, and the underlying electron distribution patterns within the microbial consortia. Results demonstrated that the R1 configuration exhibited superior nitrogen removal performance. In the initial sulfur autotrophic stage, high nitrate removal efficiency (>75%) was achieved, accompanied by consistent nitrite accumulation (6–8 mg/L), indicating incomplete denitrification. This intermediate was subsequently utilized in the downstream heterotrophic stage, facilitating the near-complete removal of residual nitrogen substrates. Consequently, the R1 system produced a final effluent with a remarkably low total nitrogen (TN) concentration of 4−5 mg/L and a TN removal efficiency of 75.5%. In contrast, the R2 system, initiating with heterotrophic denitrification, demonstrated negligible accumulation of denitrification intermediates but achieved lower overall TN removal. Critically, the R1 configuration demonstrated a significantly lower N2O emission factor (0.12% of the removed TN) compared to the R2 system (0.64%), highlighting its environmental advantage in mitigating greenhouse gas emissions. To elucidate the mechanistic basis for these performance differences, targeted batch experiments were conducted to quantify electron flux distribution among key denitrifying enzymes. Results indicated that in the sulfur autotrophic stage of R1, electrons were preferentially channeled towards nitrate reductase (NAR), accounting for 45.36% of the total electron flux. Conversely, in the subsequent heterotrophic stage, a substantially larger proportion of electrons was allocated to the downstream enzymes nitrite reductase (NIR), nitric oxide reductase (NOR), and nitrous oxide reductase (NOS). This electron allocation pattern in the heterotrophic phase likely promoted the efficient reduction and removal of N2O, contributing to the lower emissions observed in the R1 system. Microbial community analysis revealed structural similarities between the systems, with the sulfur autotrophic stage dominated by the genus Thiobacillus (54.25%−69.00%), renowned for its sulfur-oxidizing denitrification capability. The heterotrophic stages were primarily colonized by members of the family Burkholderiaceae (15.82%−24.21%) and the genus Thauera (5.87%−16.45%). This study provides compelling evidence that the staged sulfur autotrophic-heterotrophic denitrification system, particularly with autotrophy preceding heterotrophy (R1), is a highly effective and sustainable strategy. It simultaneously achieves advanced nitrogen removal and significantly reduces N2O emissions, offering a promising solution for enhancing the environmental sustainability of municipal WWTPs. Close-