Reactive oxygen species generation in earthworm burrows and their impact in drilosphere organic carbon mineralization

• Earthworm burrows is the hotspots of reactive oxygen species (ROS) • ROS content in drilosphere (312.03 μmol/kg) was 3.9 times higher than in bulk soil. • Earthworms promote ROS production by mediating microbes and Fe(II) • ROS accelerates the mineralization of SOC via enhancing enzyme activity. The drilosphere is a hotspot of carbon cycling in terrestrial ecosystems, yet the soil organic carbon (SOC) mineralization within this zone associated with reactive oxygen species (ROS) remains underexplored. Herein, we investigated the spatiotemporal variations of ROS in the drilosphere and evaluated their contributions to SOC mineralization. Fluorescence imaging revealed concentrated ROS hotspots within approximately 2.8 mm of soil surrounding earthworm ( Metaphire guillelmi ) burrows. Spatially, hydroxyl radicals ( • OH) and hydrogen peroxide (H 2 O 2 ) contents in drilosphere soils were 2218.65 nmol kg −1 and 309.03 μmol kg −1 , respectively, significantly higher than those in the background soil (883.92 nmol kg −1 and 138.09 μmol kg −1 , p < 0.01). Temporally, • OH and H 2 O 2 contents increased in drilosphere during the first 14 days of incubation, but declined following the removal of earthworms. In contrast, ROS levels in background soils remained stable throughout the incubation period. Correlation analysis revealed that earthworm activity significantly influenced ROS dynamics by altering microbial diversity, soil pH, dissolved organic carbon, and the Fe(II)/Fe(III) cycle (r = 0.60–0.90, p < 0.05). Among the ROS, • OH was an important contributor to SOC mineralization. Inhibiting • OH production decreased CO 2 emissions by 34.9 % and laccase activity by 21.6 %. The addition of exogenous • OH increased SOC mineralization rate from 25.76 to 50.36 μg C g −1 soil d -1 and laccase activity from 2.46 to 9.50 U g −1 , indicating that • OH stimulates CO 2 emissions by increasing enzyme activity. This study identifies earthworm burrows as hotspots for ROS production and provides detailed insights into their ecological role in SOC cycling.