Membrane-covered composting systems with microbial inoculation enhance humification by shifting regulation toward microbially mediated transformation.

Membrane-covered composting systems have been increasingly used to enhance humification, yet the microbial-metabolic mechanisms of humic substance (HS) transformation under inoculated conditions remain unclear. This study compared membrane-covered and uncovered composting systems under identical microbial inoculation conditions by integrating excitation-emission matrix fluorescence spectroscopy (EEM-PARAFAC), untargeted metabolomics, and high-throughput microbial community profiling. Under microbial inoculation conditions, membrane covering increased humic-like components by 7.8% while reducing protein-like components by 12.7%, indicating a higher degree of humification. Concurrently, lignin degradation showed a relative increase of 85.9%, suggesting stronger coupling between lignocellulose degradation and HS formation. Metabolomic analysis revealed that C1- and C2-associated metabolites in the uncovered system were mainly lignin-derived aromatic compounds, reflecting a substrate-associated humification pathway. In contrast, membrane covering shifted the HS-associated metabolite profile toward carbohydrates, nucleosides, and organic oxygen compounds, suggesting a potential association with higher microbial-metabolic involvement in HS transformation. KEGG enrichment further showed that aromatic amino acid biosynthesis and benzoate-derivative degradation were enhanced under membrane covering, potentially supplying aromatic intermediates and reactive functional groups for condensation with lignin-derived polyphenols. Moreover, co-occurrence network analysis suggested that membrane covering shifted the HS-associated bacterial-metabolite-lignocellulose degradation network from a single connected network to two distinct modules, indicating a more compartmentalized microbial-metabolic association pattern. Overall, membrane covering reorganized humification pathways under microbial inoculation conditions, shifting HS transformation toward greater microbial-metabolic involvement and providing mechanistic insights into HS transformation and composting optimization.