Elucidating the aromatic formation of humus via quorum sensing and redox function of thermophilic microbiota in compost.

Quorum sensing (QS) modulates bacterial metabolism to regulate humus formation. However, the functional interaction modes of signal molecules, QS-related proteins, and microbial humification function during composting remain unclear. This study aimed to clarify the role of QS-related gene expression in regulating humification via applying metagenomics and metaproteomics. Combined addition of MnSO4 and Fe0 (CMF) significantly raised humic acid (HA) content (33.53 mg g-1) and polymerization degree (2.60) compared with other treatments. Multi-dimensional results revealed that CMF-derived HA and its products exhibited greater thermal stability, high aromaticity, and practical applicability. CMF increased the relative abundances of most QS-related and humification-related oxidoreductase genes throughout composting, especially during the thermophilic stage. Metaproteomic profiles verified that both QS expression and humification metabolism were hyperactivated during the thermophilic stage of CMF. Correlation analysis showed that QS gene expression potentially and preferentially influenced lignin-protein and polyphenol pathways during humification. Overall, CMF enriched thermophiles (Bacillota, Chloroflexota, and Myxococcota), further up-regulated the expression of major QS-related genes [K02035 (ABC.PE.S), K13075 (ahlD), K14645, K15580 (oppA), and K02055 (ABC.SP.S)], elevated levels of signal molecules, and promoted QS effects. Network analysis, qPCR, and simulated in vitro validation experiments confirmed that the response of key QS modules [K13075 (ahlD) and 3-OXO-C8-HSL] potentially facilitated interspecies communication and cooperation among bacteria, which concurrently enhanced downstream microbial metabolic behaviors (microbial redox activities and the metabolism of carbohydrates and amino acids) that aid in driving HA aromatization. This work provides new insight into the ecological roles of QS-related microbes and their precise regulation of compost humification.