Recovery of crushed-dried aerobic granules via acyl-homoserine lactone (AHL)-producing Citrobacter bioaugmentation: 5-day rapid granulation with high protein

AHL-mediated quorum sensing (QS) critically regulates protein (PN) synthesis during aerobic granular sludge (AGS) reactivation. This study proposes Citrobacter bioaugmentation to accelerate stored AGS recovery by enhancing PN secretion. Reactivation of refrigerated, crushed-refrigerated, and crushed-dried granules with supplementing Citrobacter revealed that crushed-dried AGS exhibits superior recovery with microbial community restructuring amplifying Citrobacter -driven QS effects. The effects could reduce denitrifiers ( Arenimonas , Thauera , and Thermomona ) by 88.4 % while enriching PN-producing genera. Citrobacter bioaugmentation selectively stimulates Leucobacter , Sphingobacterium , Brachymonas and Glutamicibacter to boost ATP-dependent PN synthesis, achieving 84.4 % NH₄⁺-N removal and a 57.9 % PN increase, enhancing hydrophobicity and enabling rapid granulation within 5 days. PN accumulation, however, could reach the maximum 5.94 mg/g, shifting granule metabolism from aerobic to electron transfer of PN for anaerobic metabolism. This could reduce 42.6 % SOUR and 10.5 % COD removal, demonstrating a critical threshold effect for PN accumulation. Dry storage with Citrobacter bioaugmentation successfully transforms crushed-dried stored AGS into PN-rich AGS. This study significantly enhances the understanding of EPS and microbial community in stored aerobic granules, emphasizing the significant role of storage conditions in determining QS bioaugmentation. • Denitrifiers hinder biofilm recovery. • 1 g Citrobacter inoculum best boosts C8-HSL and PN production. • Crushed-dried granules become PN-rich via Citrobacter bioaugmentation. • PN-rich granules fully recover structure in 5 days. • PN accumulation alters metabolism, lowering aerobic activity and COD degradation.