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Graphical abstract Highlights • A biofilm-CANON system was kept robust by adjusting aeration time and frequency. • NO 2 − accumulation at the end of each non-aeration stage should be <6 mg N/L. • DO microelectrode measurements provided a basis for regulating aeration. • Decrease in temperature and NH 4 + concentration stimulated DO diffusion into biofilm. • NOB were successfully controlled (<0.25%) during the entire operational period. Abstract Dissolved oxygen (DO) is an important parameter for regulation of "completely autotrophic nitrogen removal over nitrite" (CANON) systems, but effective strategies for regulating DO and the mechanisms of its effects remain unclear. Here, DO microelectrode and nitrite accumulation monitoring were to develop an intermittent aeration regime for a biofilm-CANON system with decreasing influent ammonia and temperature. With high ammonium concentration (185 ± 31 mg N/L in the bioreactor) at 35 °C (Phase I) and 20–23 °C (Phase II), three repeats of 40/80 min aeration/non-aeration per reaction cycle enabled the biofilm-CANON to achieve optimal total nitrogen removal rates (TNRR) of 0.35 ± 0.03 and 0.29 ± 0.04 kg N/m3/d, respectively. The nitrite accumulation threshold at the end of each non-aeration period was 6 mg N/L. When the ammonium concentration was decreased to 35.6 ± 4.5 mg N/L (in the bioreactor) at 20–23 °C (Phase III), five repeats of 8/32 min aeration/non-aeration per reaction cycle ensured a stable TNRR of 0.18 ± 0.02 kg N/m3/d. Micro-profile measurements showed that the penetration of DO into the biofilms increased with decreasing temperature and ammonia, to 400, 600 and 1300 μm in Phases I, II and III, respectively, at 0.6 mg O 2 /L, confirming the need to reduce aeration time when temperature and influent ammonia decreased. High-throughput sequencing showed that nitrite oxidizing bacteria were <0.25% of total bacteria during the whole operation. Nitrosomonas -affiliated ammonium oxidizing bacteria (inhabiting both flocs and biofilms) and anammox bacteria Candidatus Kuenenia (mainly inhabiting the biofilms) were consistently the dominant functional bacteria. DO microelectrode provided comprehensive insights into the effects of decreasing temperature and influent ammonia on DO penetration and was valuable in the development of more sophisticated aeration regulation of biofilm-CANON systems. [ABSTRACT FROM AUTHOR]