Linking Protist-Bacteria Trophic Interactions to Organic Matter Chemodiversity: Insights from a Coal Mine Ecological Restoration Chronosequence

Abstract Restoring soil biota and dissolved organic matter (DOM) is essential for sustainable ecological restoration of coal mine sites. Protists, as key microbial predators, are recognized as regulators of multiple belowground processes, yet their roles in shaping DOM during ecological restoration remain poorly understood. Here, we investigated a field-scale coal mine ecological restoration chronosequence using vegetation surveys, ultrahigh-resolution Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), and high-throughput Illumina sequencing. Restoration increased plant diversity, coverage, and aboveground biomass, which are key resources for soil organic matter inputs. Soil DOM shifted toward more thermodynamically stable compounds, with enhanced chemodiversity during the restoration process. Protistan diversity, particularly of phagotrophic taxa, increased with restoration age, potentially exerting top-down control that increased bacterial diversity but suppressed fungal diversity. Co-occurrence network analysis and random forest modeling revealed that plant biomass was positively linked with phagotrophic diversity, which might influence DOM composition through direct transformation of organic matter (OM) or indirect regulation of bacterial transformation of OM, contributing to the accumulation of recalcitrant molecules with high stability. Together, these findings uncover a previously unrecognized trophic cascade linking protists to DOM chemodiversity at the molecular level, highlighting the importance of integrating protistan regulation into soil ecological restoration strategies for mine sites.