Biochar has been extensively demonstrated as an abundant, cost-effective, and highly efficient adsorbent material for removing ammonium (NH4+) from non-point source-polluted water. The new technology of biochar-loaded NH4+-N (BLN) is urgently needed. However, the underlying mechanisms of BLN in regulating N2O emissions by substituting chemical fertilizer nitrogen (CFN) were poorly understood. Based on the substitution rate of CFN with BLN, a soil column experiment was designed with six treatments: CFN-only (BLN0), 20% BLN plus 80% CFN (BLN20), 40% BLN plus 60% CFN (BLN40), 60% BLN plus 40% CFN (BLN60), 80% BLN plus 20% CFN (BLN80), and BLN only (BLN100). BLN application significantly decreased cumulative N2O emissions by 20.50-40.78% compared to the BLN0 treatment. The optimal ratio of CFN substitution was 66.10%, which was supported by the lowest N2O emissions in the BLN60 treatment. In BLNtreated soil, the abundance of nosZ played a dominant role in N transformation, followed by ammonia-oxidizing bacteria (AOB). Particularly, BLN60 significantly increased nosZ abundance. The high substitution ratio (BLN60-100) promoted N2O reduction by increasing the overall abundance of nosZ-harboring communities, however, low substitution ratio (BLN0-40) promoted it by increasing nosZ cluster IX abundance and the connectivity of operational taxonomic units within the module. Soil pH and C/N ratio were critical influencing factors to alter the nosZ community structure. These findings highlighted that BLN substitution treatments reduced N2O emissions, while the microbiological mechanism of emission reduction varied with the proportion
