In-Situ sulfur implantation efficiently promoting nitrogen removal in low-carbon anoxic-oxic systems

Efficient total nitrogen (TN) removal from low-carbon wastewater remains challenging due to electron donor scarcity, often causing incomplete denitrification and nitrite accumulation. To address this, an in-situ sulfur-enhanced anoxic/oxic (HS⁰AD-A/O) system was established, enabling S⁰-driven electron redistribution for enhanced TN removal without additional carbon input or process restructuring. Under decreasing influent C/N ratios (4 to 2), HS⁰AD-A/O outperformed conventional HD-A/O by 32.76-111.16% of TN removal efficiency. Electron balance showed S⁰ oxidation contributed 9.21-27.59% of total electron flux, compensating for carbon deficiency. Increasing the S⁰ implantation ratio to 28% shifted the dominant pathway toward S⁰-based autotrophic denitrification, where S⁰-derived electrons surpassed those from COD (58.27% vs. 41.73%). Kinetic assays revealed that S⁰-driven denitrification preferentially reduced NO₂⁻ over NO₃⁻, thereby minimizing NO₂⁻ accumulation and yielding a distinct S⁰‑saving effect (1.14-1.57 g-S⁰/g-N here). Microbial and transcriptional analyses further elucidated a synergistic division of labor: heterotrophic denitrifiers (e.g., Hydrogenophaga, Rhodocyclaceae) in sludge primarily reduced NO₃⁻-N but tended to cause partial denitrification, whereas S⁰-attached autotrophs (e.g., Thiobacillus, up to 46.10% in biofilms) specialize in complete denitrification and efficiently converted NO₂⁻-N to N₂, accompanied by marked upregulation of nirKS and nosZ genes. Overall, in-situ S⁰ implantation restructured electron transfer networks, enabling stable, efficient, and dual-saving (carbon and S⁰) TN removal while providing mechanistic insight for scalable applications.