Sulfur-driven adaptive succession of denitrifiers enables coordinated autotrophic-heterotrophic deep denitrification

Nitrogen removal is the major driver of energy and chemical demand in municipal wastewater treatment. Herein, deep denitrification was achieved by leveraging sludge-released endogenous carbon together with sulfur-based autotrophic pathways in a sequencing batch reactor. Dynamic electron donor-acceptor switching under introducing micro-electric fields or moderate molecular oxygen achieved nitrate removal of 98.7% and 96.6%, respectively, with sulfate generation limited to 5.8 mg SO₄^2- mg^-1N. Bacterial lysis-derived endogenous dissolved organic matter served as both a carbon source and a potential electron shuttle, contributing to sulfur-carbon-nitrogen redox coupling. Performance enhancement was driven by niche differentiation resulting from regulation of carbon- and sulfur-based metabolisms in heterotrophic and sulfur-autotrophic denitrifiers. The enrichment of autotrophic (Thiobacillus, Sulfurimonas) and mixotrophic (Thermomonas) denitrifiers was positively associated with a 0.8-24.1-fold increase in the abundance of denitrification (nar, nap, nir) and sulfur-redox (sox, dsr) genes, as revealed by quantitative analysis and machine-learning prediction. These shifts strengthened enzyme-substrate coupling, upregulated energy-metabolism and transport functions, and increased intracellular ATP by 1.3-8.2%. The upregulation of genes encoding cytochrome c-mediated electron transfer, alongside increased taxonomic contributions, improved electron transfer efficiency. The change in metabolites further revealed a complex metabolic network reshaped by coupled sulfur/nitrogen/carbon redox regulation. By coordinating carbon- and sulfur-derived electrons toward nitrogen reduction, the system maintained strong microbial adaptability and metabolic resilience under dynamic redox conditions, enabling stable deep denitrification in wastewater treatment.