Deep nitrogen removal in biochar-coupled pyrite constructed wetlands driven by targeted microbial succession:...

To overcome carbon limitations and substrate passivation in constructed wetlands (CWs) treating low carbon-to-nitrogen (C/N) wastewater, a novel vertical subsurface flow CW augmented with a pyrite-coupled biochar matrix (S4 system) was developed. Under optimal operating boundaries (HRT = 24 h, 20°C, and influent TN = 15 mg/L), the S4 system achieved outstanding simultaneous removal efficiencies of 89.74% for NH₄⁺-N and 90.36% for NO₃^--N without nitrite accumulation. Interfacial characterization via SEM-EDS and 3D-EEM demonstrated that the introduction of biochar effectively mitigated pyrite surface passivation by buffering localized pH fluctuations and enhancing the mass transfer of reaction byproducts. Furthermore, biochar functioned as an efficient electron mediator, facilitating direct interspecies electron transfer (DIET) and accelerating autotrophic denitrification driven by the slow-release iron-sulfur minerals. High-throughput sequencing and qPCR analyses revealed a targeted restructuring of the micro-ecosystem, where the chemolithoautotrophic Thiobacillus became the absolute dominant genus. This microbial succession was accompanied by a dramatic upregulation of core denitrification functional genes, with narG and nirS absolute abundances increasing by 2.7- and 4.5-fold, respectively, establishing an efficient enzymatic cascade. Overall, this study clarifies the synergistic biogeochemical mechanisms of the pyrite-biochar complex, offering a high-performance, anti-passivation, and sustainable ecological engineering strategy for the deep purification of low-strength wastewater.