Humification Pathways of Crop Residues Under Ammonification–Steam Explosion Pretreatment and Multi-Fungal Inoculation

The pathways governing the transformation of crop residues into humic acid (HA) remain incompletely understood because multiple biochemical routes may operate simultaneously during composting-like humification. In this study, a 30-day solid-state humification experiment was conducted by integrating physicochemical pretreatments, including steam explosion (SE) and ammonification coupled with steam explosion (SE-N), with a multi-fungal inoculation strategy involving Aspergillus niger, Candida spp., and Phanerochaete chrysosporium. Across three representative substrate–pretreatment systems and 81 experimental groups, the contents of lignocellulosic fractions, reducing sugars (RS), a UV-280-based soluble nitrogen-containing precursor index (operationally denoted as SNP), fulvic acid (FA), and HA were compared. The results showed that neither physicochemical pretreatment alone nor single-strain inoculation was sufficient to achieve substantial HA formation. SE mainly improved substrate accessibility and promoted carbon release, whereas ammonification provided essential nitrogen preloading for subsequent precursor coupling. In the saccharification-dominant treatment, RS reached 27.5%, but HA remained negligible. In the Candida-only treatment, the soluble nitrogen-containing precursor index increased markedly, yet HA formation was still minimal. By contrast, the highest HA yield (13.7%) was obtained under multi-fungal co-inoculation, particularly when nitrogen preloading by ammonification was combined with concurrent accumulation of carbon and aromatic precursors. The data suggest that lignin-targeting activity by P. chrysosporium was associated with the likely generation of phenolic and quinone-like intermediates that bridged the condensation of sugar- and nitrogen-derived compounds. Overall, the findings support a synergistic humification framework in which polysaccharide depolymerization, microbial nitrogen transformation, and lignin-derived aromatic precursor formation jointly contribute to HA accumulation, rather than a single linear pathway dominating the process.