Cross-layer functional decoupling marks the transition toward structural deterioration in aerobic granular sludge

Identifying aerobic granular sludge (AGS) that is progressing toward instability before visible structural damage occurs remains a key challenge. Existing studies mostly compare stable and disintegrated granules at discrete endpoints, and conventional omics analyses focus on what changed rather than whether different molecular layers remain coordinated. We hypothesised that the transition toward granule instability is associated with progressive loss of functional coordination across genomic, proteomic, and metabolic layers. Aerobic granules were collected at five size classes (0.5, 1.0, 2.0, 3.5, and 5.0 mm) and analysed by morphology, SEM, reactor performance monitoring, metagenomics, metaproteomics, and metabolomics. Module-level coordination analysis and an inter-module coordination index (IMCI) were applied across the size gradient. At 3.5 mm, granules retained largely intact surface architecture and sustained bulk COD removal above 90%, yet metabolomic IMCI had declined continuously from 0.75 to 0.15 and proteomic IMCI had peaked at 2.0 mm before decreasing, revealing asynchronous coordination loss across molecular layers. A candidate pre-destabilisation window (2.0-3.5 mm) was identified where functional coherence had weakened but pronounced structural deterioration had not yet developed. Based on these findings, we propose hierarchical functional decoupling as a mechanistic framework in which cross-layer coordination loss marks the transition toward structural deterioration, providing an early molecular signature of incipient instability prior to macroscopic failure.