S⁰-S²⁻ co-substrate system achieves efficient nitrite accumulation under high alkalinity and ultra-short HRT:...

Sulfur autotrophic denitrification under highly alkaline conditions provides a novel strategy for nitrite supply, but low nitrate conversion flux often limits nitrite accumulation rate. This study proposes an element sulfur-sulfide (S⁰-S²^-) co-substrate enhancement strategy based on an alkaline environment. Under high pH (10) with an ultra-short hydraulic retention time (0.65 h) and low S²^- feeding (S²⁻/NO₃⁻-N ratio of 0.17), the nitrate conversion efficiency (84.3%) was nearly 20% higher than that only at high pH conditions. Moreover, the strategy achieved an excellent nitrite accumulation efficiency of 60.6% and a rate of 1.54 kg N·m⁻³· d⁻¹. Microbial physiological responses revealed that reactive oxygen species accumulation significantly inhibited microbial activity and disrupted nitrogen-sulfur metabolism at high pH. However, supplemental S²⁻ promoted the ring-opening activation of S⁰ to form high bioavailability polysulfide, and restored electron transfer system activity and energy metabolism. The abundance of sulfur-oxidizing genes Sox and fccAB increased by 15.7%-95.6%, ensuring robust electron flux. Under this strategy, the system exhibited an optimized metabolic trade-off that prioritized survival advantages over complete denitrification. This was achieved by prioritizing energy allocation to Na⁺(K⁺)/H⁺ antiporters (Mrp/Pha/Kef/Trk) and glutamate synthesis to maintain cellular homeostasis. Concurrently, the increased narGHI/napAB abundance (14.6%-58.9%) and reduced nirS abundance (25.6%-41.3%) ensured a higher nitrite accumulation rate. Functional annotation further revealed that Thiobacillus (40.2%) and Pseudoxanthomonas (3.9%) served as key genera driving nitrite accumulation. This study not only proposes an efficient nitrite supply strategy for Anammox but also reveals the underlying microbial response mechanisms under high alkalinity.