Microbial biomass stoichiometry and proportion of Fe organic complexes separately shape the heterogeneity of mixotrophic denitrification and net NO sinks in iron-carbon amended ecological ditch.

Coupling of iron-carbon can form a mixotrophic denitrification and is regarded as a promising solution for purifying nitrate-rich agricultural runoff. However, its prevalence and efficacy of the synergistic augmentation of nitrogen elimination and net NO sinks remain crucial knowledge gaps in ecological ditches (eco-ditches). Here, we investigated the underlying variability mechanisms by implementing sponge iron (sFe)-coupled Iris hexagonus (IH)- or Myriophyllum aquaticum (MA)-derived biochar produced via microwave-assisted (MW) pyrolysis and conventional pyrolysis. Surprisingly, unamened eco-ditch became net NO sink while exhibiting a significant increase in total nitrogen (TN) removal rate of 319 % (P < 0.001) compared to soil ditch. The integration of MW pyrolyzed IH-derived biochar with sFe to amend eco-ditch achieved synchronous enhancement in net NO sinks (P < 0.01) and TN removal rate (P < 0.001), whereas the remaining amended eco-ditches that significantly intensified TN removal performance, were NO emitters. Such heterogeneity primarily depends on Fe organic complexes (Fep) / the total reactive Fe oxides (Fed) ratio, rather than the prevailing nosZ gene, underscoring that low density metastable reactive iron plays a more important role than biological reactions during the mixotrophic denitrification process. As such, iron oxides are not necessarily a bottleneck for denitrification and contribute to NO sinks. Conversely, microbial biomass C:(C + N), together with nirK and nosZ genes, mainly explain the TN removal heterogeneity of sFe-biochar eco-ditch. This study revisits the discrepant resilience of iron-carbon coupling to N abatement and NO sink-induced cooling and has significant practical implications for better understanding the cascading effects of mixotrophic denitrification driven by iron-carbon interactions.