Substrate as architect: lignocellulose structures directed assembly lines for humic acid stability in composting

This study established that lignocellulose source dictated distinct humic acid (HA) assembly pathways during composting. We employed an integrated approach combining multi-omics, multispectral analysis, enzyme activity assays, and microbial co-occurrence network modeling to elucidate the humification processes of pine branches (PB), rice straw (RS), and willow branches (WB). A novel metric, the Aromatic Condensation Index (ACI), was developed to quantify the degree of fused aromatic ring structures in HA. Our results revealed divergent humification mechanisms: PB-derived HA exhibited the highest aromatic condensation (ACI = 0.82), facilitated by a tightly clustered microbial community that enabled simultaneous degradation and aromatization. This synergy facilitated biotic (laccase/peroxidase-driven β-O-4 cleavage liberating radicals for CC coupling) and abiotic (Schiff base polymerization of lignin-derived aldehydes and amino acids) pathways. RS composting achieved rapid humification but had low aromatization (ACI = 0.51), attributed to its compartmentalized microbial community decoupling biodegradation from abiotic assembly. Structurally simple RS favored early carbohydrate depolymerization followed by abiotic Maillard reactions. WB composting showed delayed humification and minimal aromatization (ACI = 0.38), with a fragmented microbial network indicating bottlenecks. The recalcitrant, sulfur-rich lignin in WB restricted enzymatic access, necessitating late-stage MnP-mediated demethoxylation; HA formation relied primarily on abiotic "polysaccharides → organic acids → alkyl-HA" assembly. This framework, highlighting lignocellulose structure's role in HA assembly, offers insights into lignin macromolecule degradation and transformation. It provides strategies for efficient lignin utilization, converting it into valuable HA, advancing lignin - based research.