Dialysis-controlled sulfur substrate delivery enhances Sulfur-Autotrophic denitrification under oxygen stress

Sulfur autotrophic denitrification (SAD) is a low-carbon nitrogen removal process using reduced sulfur compounds as electron donors. However, dissolved oxygen (DO) disrupts SAD by promoting unproductive sulfur oxidation and electron loss. Here, dialysis membranes (1000 and 100 Da) were applied to regulate thiosulfate release and establish controlled substrate gradients under engineering-relevant inhibitory DO conditions (0.5-3.5 mg L^-1). Compared with direct dosing (R_ck), the 100 Da reactor (R₁₀₀) achieved 19% higher nitrate removal efficiency and greater fraction of electrons allocated to denitrification (EDU_den ≈ 76%), accompanied by pronounced zero-valent sulfur accumulation, indicating pathway-level reallocation. Microbial analyzes revealed enrichment of Thiobacillus-like sulfur-oxidizing denitrifiers and increased prevalence of oxygen tolerant nitrate reductase (napA), confirmed by metagenomic and qPCR. These results demonstrate that controlled sulfur release creates an electron-buffered microenvironment that enhances SAD resilience to DO, offering a donor-efficient strategy for nitrogen removal in oxygen-fluctuating wastewater systems.