Inorganic carbon is an important carbon source of autotrophic bacteria, e.g., ammonia-oxidizing bacteria. Ammonia-oxidizing bacteria are chemoautotrophic bacteria with carbon sequestration capacity. Experiments were performed on partial nitrification sludge with different influent matrices, and optimal experimental operational conditions were established. The carbon fixation pathway of ammonia-oxidizing sludge was determined via C-13 isotope tracers and qPCR. The denitrification effect was better when the NH4+-N, HCO3-, - Ca2+, Mg2+, and microbial accelerant concentrations were 15, 250, 113, 100 and 1 mL/L, respectively. The nitrite accumulation rate reached 96.95%. C-13 isotope tracing showed that C-13 abundance in sludge increased significantly. The results showed that IC added into the influent participated in the carbon metabolism of microorganisms. The functional gene cbbL, which follows the Calvin cycle carbon sequestration pathway, was identified in the ammonia-oxidizing bacteria, and the effect of influent NH4+-N on the gene abundance was greater than that of other substrates.