Fatty acid cycling drives metabolic uncoupling and simultaneous nitrogen removal in a low-aeration and no-recirculation activated sludge system

Metabolic uncoupling represents a fundamental strategy for in-situ sludge reduction, yet its underlying metabolic mechanisms under low-aeration conditions remain poorly understood. Here, we investigated activated sludge performance in a reactor with a depth-to-diameter ratio of 4:1 operated without recirculation under low-aeration condition, achieving simultaneous nitrogen removal and sludge reduction. Integrating metagenomic and metaproteomic analyses, we discovered a previously unrecognized fatty acid cycling mechanism that drives metabolic uncoupling. Specifically, the concurrent upregulation of acetyl-CoA carboxylase (accA) for fatty acid biosynthesis and acyl-CoA dehydrogenase (ACADM/acd) for β-oxidation established an energy-dissipating futile cycle that consumed ATP without contributing to biomass synthesis. This futile cycling, rather than the conventionally assumed polyhydroxybutyrate (PHB) accumulation, constituted the primary carbon sink responsible for sludge reduction. Concurrently, carbon flux was partitioned toward nitrogen removal, with metagenomic evidence revealing the co-occurrence of heterotrophic denitrification and sulfur-driven autotrophic denitrification pathways. The decline of filamentous bacterium Sphaerotilus further suppressed sludge bulking, contributing to stable sludge volume maintenance in the absence of recirculation. Collectively, our findings reveal a dual role of carbon flux in simultaneously driving nitrogen removal and dissipating energy through fatty acid futile cycling. These results establish a new mechanistic framework for understanding metabolic uncoupling-mediated sludge reduction under low-aeration, no-recirculation conditions.