Spatial configuration governs the formation of a functional root–packing interface in sulfur-based autotrophic denitrification

Plant-integrated sulfur-based autotrophic denitrification (SAD) has been widely explored as a low-carbon strategy for nitrogen removal, yet reported performances vary substantially, raising the question of whether synergy arises from plant–microbe coexistence itself or from specific process-level controls. Using three systems with identical influent conditions and hydraulic retention times but distinct spatial arrangements, this study shows that system behavior is governed by the formation of a functional reaction interface rather than by reactor configuration alone. Compared to spatially separated (Ex-situ) configurations, the In-situ integrated system achieved superior and stable volumetric removal loads for nitrate (356.1 g-N/m3·d), ammonium (33.2 g-N/m3·d), total phosphorus (4.8 g-P/m3·d) and higher nutrient uptake, while maintaining robust plant growth under low-oxygen conditions. Direct measurements revealed that interface formation supported a stable redox and nutrients microenvironment, with dissolved oxygen maintained at approximately 0.1 mg/L within the packing layer and gradually increasing to ∼ 0.4 mg/L toward the water surface, while spatial separation induced redox discontinuity through re-aeration. Consistent with these patterns, plants in the In-situ system maintained stable antioxidant enzyme activities and accumulated biomass, whereas Ex-situ plants exhibited stress responses and biomass loss. Microbial communities on sulfur-based packing further shifted from Sulfurimonas dominance toward a more diverse assemblage under interface-coupled conditions, indicating progressive stabilization of interfacial processes. By identifying the root–packing interface as the key determinant of system performance, this work provides a transferable framework for advancing the design and operational stability of plant-integrated SAD and other biofilm-based technologies.