Microbial diversity and metabolic pathways driving humic acid formation in two-stage insect bioconversion system

A two-stage insect-assisted system using black soldier fly larvae (BSFL) followed by Protaetia brevitarsis larvae (PBL) was developed to enhance humification of chicken manure. BSFL treatment reduced electrical conductivity and labile organic matter, indicating efficient hydrolysis and detoxification. A dry-weight-normalized mass balance revealed that humic acid (HA) decreased during the BSFL stage but increased significantly after PBL processing, confirming true humification enhancement rather than a concentration effect. Fluorescence analysis further indicated increased structural complexity of humic substances in the PBL stage, while seed germination assays demonstrated reduced phytotoxicity and improved agronomic quality. Multi-omics analyses showed a functional shift from GH-dominated depolymerization to AA-associated oxidative metabolism , accompanied by microbial succession toward Actinobacteriota and enrichment of aromatic-related pathways. Correlation analyses suggested that BSFL-derived intermediates may shape subsequent microbial assembly, although causality remains to be validated. Overall, the BSFL-PBL relay system promotes humus stabilization through coordinated hydrolytic and oxidative processes, providing a mechanistic basis for sustainable organic waste valorization.