Anaerobic-aerobic-anoxic (AOA) process is a promising configuration to retrofit current wastewater treatment plants with intensified carbon utilization and nutrient removal, but lacks process optimization for scaling-up in real wastewater scenarios. Solids retention time (SRT) is a fundamental parameter of activated sludge process, but its roles in the AOA process remain vague. Here, we established a pilot-scale AOA process at different SRTs (10, 20, 30 d) to investigate the comprehensive responses and potential mechanisms. The results revealed that proper SRT extension in S20 (20 d) achieved the highest nutrient removal via enhanced nitrification, denitrification, denitrifying phosphate removal (DPR), and expanded phosphorus reservoir. Simultaneously, S20 garnered the optimized carbon conservation and allocation via efficient intracellular carbon transformation, consolidating energy foundation for nutrient removal. In contrast, excessive SRT in S30 (30 d) escalated cellular expenditure for maintenance, stimulated sludge decay with starvation stresses, triggered passive ammonia/phosphate release, and ultimately deteriorated carbon allocation and nutrient removal. Furthermore, microbial insights demonstrated that S20 has tailored habitats for autotrophic nitrifiers, and specialized denitrifying phosphate accumulating organisms (Dechloromonas) and denitrifying glycogen accumulating organisms (Thauera) featuring high carbon priority, favoring nutrient removal
while S30 accelerated exclusion of functional guilds, propagated surplus generalized ordinary heterotrophic and fermentative organisms (Saccharimonadales, Ferruginibacter, Tetrasphaera), impairing the microbial functionality. Functional analysis further corroborated the enhanced nutrient metabolism in S20, and the exacerbated sludge decay and activity attenuation in S30. These findings can advance our understanding of the interactions between SRT and C-N-P cycles in the AOA process, and underscore its significance in practical application.