Data-Driven Identification of Favorable Multi-Fungal Inoculation Timing for Enhanced Humic Acid Recovery from Pretreated Crop Straws

Humic acid (HA) production from crop straw is often limited by lignocellulosic recalcitrance and insufficient coordination among functional microorganisms. In this study, a data-driven strategy was developed to evaluate multi-fungal inoculation timing for HA recovery from pretreated straws. Three substrate platforms, namely raw wheat straw (SW), steam-exploded corn straw (SC-SE), and ammoniated steam-exploded rice straw (SR-SE-N), were comparatively evaluated across an 81-run experimental matrix. Pretreatment markedly improved lignocellulose degradation and precursor turnover, with SR-SE-N showing the best humification performance. Based on the selected substrate, a two-factor interaction (2FI) model was established to describe the effects of inoculation timing on HA yield. The model was significant for HA prediction (R2 = 0.8768, adjusted R2 = 0.8398, predicted R2 = 0.7795). Inoculation timing strongly affected HA formation, and within the investigated timing range, the highest HA yield was obtained under simultaneous inoculation of Aspergillus niger, Phanerochaete chrysosporium, and Candida sp. Predicted and experimental HA yields were in close agreement, supporting the reliability of the model. These results indicate that favorable fungal inoculation timing is substrate-dependent and can be effectively identified through data-driven analysis within a bounded experimental range. The study provides a practical basis for improving HA biomanufacturing from pretreated agricultural residues.