The multiple anoxic and aerobic (AO) process is an advanced biological nitrogen-removal process, and nitrous oxide (NO) emission might affect its sustainable application. Nitrogen removal and NO emission in a step-feeding multiple AO sequencing batch reactor (SBR) was examined, in comparison with a one-feeding sequencing batch reactor (SBR). Nitrogen removal was enhanced by 12.6% in SBR compared to the removal percentage of 75.8% in SBR. Activated sludge in SBRs possessed a higher NO emission factor during nitrification, denitrification and simultaneous nitrification and denitrification (SND) than in SBR. A high NO emission factor was observed during SND in both reactors, with the emission factor of 4.38% in SBR and 4.66% in SBR. More NO emission occurred in the presence of nitrite. Bacteroidetes and Proteobacteria dominated in both SBR and SBR. A similar abundance of Thauera, Dechloromonas and Zoogloea possible for denitrification was observed in SBR and SBR. Moreover, nosZ from Proteobacteria dominated in both SBR and SBR, with dominating genus of Acidovorax, Ralstonia, Thauera and Marinobacter.

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Two sequencing batch reactors, one with the conventional anoxic and aerobic (AO) process and the other with the multiple AO process, were operated to examine characteristics of biological nitrogen removal, especially of the multiple AO process. The long-term operation showed that the total nitrogen removal percentage of the multiple AO reactor was 38.7% higher than that of the AO reactor. In the multiple AO reactor, at the initial SBR cycle stage, due to the occurrence of simultaneous nitrification and denitrification, no nitrite and/or nitrate were accumulated. In the multiple AO reactor, activities of nitrite oxidizing bacteria were inhibited due to the multiple AO operating mode applied, resulting in the partial nitrification. Denitrifiers in the multiple AO reactor mainly utilized internal organic carbon for denitrification, and their activities were lower than those of denitrifiers in the AO reactor utilizing external organic carbon.