In Situ Sustained Release of High-Value Microbial Products from Waste Activated Sludge for Enhanced Nitrogen Removal in Anammox Coupled with Heterotrophic Denitrification Processes

Integrating heterotrophic denitrification into anammox systems mitigates nitrate accumulation but requires a delicate balance between anaerobic ammonium-oxidizing bacteria (AnAOB) and heterotrophic denitrifiers. This study investigated the feasibility of using fresh waste-activated sludge (WAS) as both a carbon source and a functional microbial inoculum to enhance anammox-coupled heterotrophic denitrification. A 200-day reactor operation revealed that dosing fresh WAS immediately increased total nitrogen removal efficiency to 94.1%, significantly outperforming the bioreactor without WAS addition (86.6%). Initial microbial products (MP) from WAS, rich in unsaturated, reduced CHON and CHONS compounds, acted as preferred electron donors for denitrification. Removing the initial MP promoted the sustained release of biopolymers and low-molecular-weight neutrals, providing a continuous electron donor supply for denitrification. Supplementing with WAS increased the relative abundance of partial denitrifiers (Limniobacter) from 15.9 to 22.2%, which in turn promoted the growth of AnAOB (Candidatus Kuenenia). Functional gene analysis revealed enriched pathways for polymeric and aromatic compound degradation and amino acid metabolism, potentially supporting denitrification. An elevation in nitrate reductase genes (e.g., napAB and narGHI) was observed, probably promoting partial denitrification, and thereby sustaining AnAOB-denitrifier synergy. This study demonstrates a novel strategy for using fresh WAS to mediate the anammox-coupled denitrification process for efficient nitrogen removal.