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• Integrated fixed-film activated sludge tested for shortcut nitrogen removal. • Combining strategies selectively suppressed activity nitrite oxidizing bacteria. • Flocs acted as nitrite source, while carriers were nitrite sinks. • Optimal nitrite source: low, but sufficient aerobic floc retention time (±7 days) • Optimal sink: dissolved oxygen < 0.47 mg O 2 L−1 and N loading rate > 150 mg N L−1 d−1. Implementation of mainstream partial nitritation/anammox (PN/A) can lead to more sustainable and cost-effective sewage treatment. For mainstream PN/A reactor, an integrated fixed-film activated sludge (IFAS) was operated (26 °C). The effects of floccular aerobic sludge retention time (AerSRT floc), a novel aeration strategy, and N-loading rate were tested to optimize the operational strategy. The best performance was observed with a low, but sufficient AerSRT floc (~7d) and continuous aeration with two alternating dissolved oxygen setpoints: 10 min at 0.07–0.13 mg O 2 L−1 and 5 min at 0.27–0.43 mg O 2 L−1. Nitrogen removal rates were 122 ± 23 mg N L−1 d−1, and removal efficiencies 73 ± 13%. These conditions enabled flocs to act as nitrite sources while the carriers were nitrite sinks, with low abundance of nitrite oxidizing bacteria. The operational strategies in the source-sink framework can serve as a guideline for successful operation of mainstream PN/A reactors. [ABSTRACT FROM AUTHOR]