Multilayer microbial framework of aerobic granule microecosystems: Integrating community ecology, genetic networks,...

Aerobic granular sludge (AGS), a next-generation wastewater treatment technology, has advanced worldwide. Microbiological mechanisms underlying its formation and stability lack a unified explanation. Based on the concept that AGS construction involves progressive participation and stabilization of microbial organization under environmental selection, this review integrates evidence from community ecology and gene-regulatory networks and proposes a unified conceptual framework. The findings show that AGS formation follows a functional trait-driven self-organization process rather than simple cellular aggregation. Selection pressures shape community structure, promoting enrichment of microorganisms. On this basis, quorum-sensing (QS) system is activated with cell density. Rather than acting independently, QS is associated with the reinforcement of existing aggregates, the strengthening of metabolic synergy, and the stabilization of functional zonation. As a density-dependent coordination mechanism, it is involved in regulating the luxI/luxR pathway and key functional genes involved in extracellular polymeric substance synthesis, nitrogen removal, and phosphorus removal. This regulation primarily targets existing aggregates to enhance metabolic synergy and reinforce C/N/P-coupled functional zonation. Operational stability and system performance are improved, bridging community ecological processes with cross-scale gene-regulatory mechanisms. Within this framework, the rationale and limitations of enhancement strategies are evaluated, including QS regulation, microbial reinforcement, and synthetic biology. In the future, quantitative cross-scale coupling models and AI-driven process control may enable engineering universality and designability of AGS. Overall, the proposed paradigm of multilayer microbial programming provides a unified perspective on AGS formation and stability, supporting predictable and designable applications in next-generation biological wastewater treatment systems.