Biochar-Associated Free Radicals Reduce Soil Bacterial Diversity: New Insight into Ecoenzymatic Stoichiometry

The toxicity of environmentally persistent free radicals (EPFRs), often generated during biochar production, on soil bacteria is still not truly reflected when considering the conditions in real soil. Herein, the influence of free radicals within biochar on soil bacteria was investigated from the perspectives of enzyme activity, community structure, and ecoenzymatic stoichiometry. Biochar addition enhanced the contents of EPFRs and derived hydroxyl radicals (^•OH) in the soil, while it reduced bacterial alpha diversity by 5.06-35.44%. The results of redundancy analysis and inhibition experiments collectively demonstrated the key role of EPFRs and ^•OH in reducing the bacterial alpha diversity. Specifically, EPFRs and ^•OH increased the stoichiometric imbalance by promoting the release of dissolved organic carbon and ammonium N, thus aggravating the P limitation in soil. This was further confirmed by increased alkaline phosphatase activity from 702 to 874 nmol g^-1 h^-1. The P limitation induced by EPFRs and ^•OH decreased the bacterial alpha diversity, as evidenced by the negative correlation between P limitation and bacterial alpha diversity (r² = -0.931 to -0.979, P < 0.01) and the structural equation model. The obtained results demonstrate a ubiquitous but previously overlooked mechanism for bacterial toxicity of biochar-associated free radicals, providing scientific guidance for safe utilization of biochar.