Residual　ammonium　is　a　critical　parameter　affecting　the　stability　of　mainstream　partial　nitritation/anammox　(PN/A),　but　the　underlying　mechanism　remains　unclear.　In　this　study,　mainstream　PN/A　was　established　and　operated　with　progressively　decreasing　residual　ammonium.　PN/A　deteriorated　as　the　residual　ammonium　decreased　to　below　5　mg/L,　and　this　was　paralleled　by　a　significant　loss　in　anammox　activity　in　situ　and　an　increasing　nitrite　oxidation　rate.　Further　analysis　revealed　that　the　low-ammonium　condition　directly　decreased　anammox　activity　in　situ　via　two　distinct　mechanisms.　First,　anammox　bacteria　were　located　in　the　inner　layer　of　the　granular　sludge,　and　thus　were　disadvantageous　when　competing　for　ammonium　with　ammonium-oxidizing　bacteria　(AOB)　in　the　outer　layer.　Second,　the　complete　ammonia　oxidizer　(comammox)　was　enriched　at　low　residual　ammonium　concentrations　because　of　its　high　ammonium　affinity.　Both　AOB　and　comammox　presented　kinetic　advantages　over　anammox　bacteria.　At　high　residual　ammonium　concentrations,　nitrite-oxidizing　bacteria　(NOB)　were　effectively　suppressed,　even　when　their　maximum　activity　was　high　due　to　competition　for　nitrite　with　anammox　bacteria.　At　low　residual　ammonium　concentrations,　the　decrease　in　anammox　activity　in　situ　led　to　an　increase　in　nitrite　availability　for　nitrite　oxidation,　facilitating　the　activation　of　NOB　despite　the　dissolved　oxygen　limitation　(0.15–0.35　mg/L)　for　NOB　persisting　throughout　the　operation.　Therefore,　the　deterioration　of　mainstream　PN/A　at　low　residual　ammonium　was　primarily　triggered　by　a　decline　in　anammox　activity　in　situ.　This　study　provides　novel　insights　into　the　optimized　design　of　mainstream　PN/As　in　engineering　applications.　©　2022