Pre-fermentation retains flavonoid-like precursors and enhances lignin humification during straw-manure...

The directional conversion of lignin into humic substances (HS) was hindered by the asynchrony between its depolymerization and reassembly, resulting in substantial carbon loss and limited environmental benefit. This study developed a pre-fermentation/co-fermentation strategy clarifying straw lignin conversion into HS structures. Rice straw was pre-fermented for 0, 3, 8, and 15 days, followed by co-fermentation with chicken manure. The 8-day pre-fermentation (PF) stage was identified as the window for precursor retention. At this stage, total phenolic content reached 7.13 mg/g, laccase activity reached 67.12 U/g, and lignin-like condensed aromatic compounds accounted for 72.52% of the detected molecular pool. The relative abundances of the laccase gene, manganese peroxidase gene, and lignin peroxidase gene were higher, whereas the lignin-derived aromatic catabolism gene was lower (0.08-fold). This shift promoted the accumulation of phenolic and flavonoid-like precursors. Among them, 6-methoxyluteolin was the dominant flavonoid, accounting for 20.18% of detected phenolics. During co-fermentation, this flavonoid-enriched precursor pool was associated with the rapid accumulation of epicatechin gallate (HS-3), a dominant polyphenolic HS component. HS-3 reached 7.68 mg/g at the beginning of co-fermentation, exceeding the delayed peak in the control system. Manure nitrogen (N) was associated with polar N-containing HS enrichment and molecular maturation. Consequently, HS yield increased by 38.19%, and the aromaticity index increased by up to 75.38%. Microbial analysis identified lignin-degrading genera, including Pseudomonas and Paenibacillus, and nitrogen-transformation-related genera, including Nocardiopsis and Devosia, as putative functional contributors associated with HS assembly. This strategy provided a mechanistic basis for upgrading lignocellulosic waste into humified organic products.