Strengthening　the　activity　competitiveness　of　anaerobic　ammonium　oxidation　(anammox)　bacteria　(AnAOB)　under　low　nitrogen　conditions　is　indispensable　for　mainstream　anammox　application.　This　study　demonstrates　that　sponge　iron　addition　(42.8　g/L)　effectively　increased　apparent　AnAOB　activity　and　extracellular　polymeric　substance　(EPS)　production　of　low　load　anammox　biofilms　cultivated　under　low　(influent　of　60　mg　N/L)　and　even　ultra-low　(influent　of　10　mg　N/L)　nitrogen　conditions.　In-situ　batch　tests　showed　that　after　sponge　iron　addition　the　specific　AnAOB　activity　in　the　low　and　ultra-low　nitrogen　systems　further　increased　to　1.18　and　0.47　mmol/g　VSS/h,　respectively,　with　an　apparent　growth　rate　for　AnAOB　of　0.011　±　0.001　d−1　and　0.004　±　0.001　d−1,　respectively.　The　averaged　EPS　concentration　of　anammox　biofilm　in　both　low　(from　35.84　to　71.05　mg/g　VSS)　and　ultra-low　(from　44.14　to　57.59　mg/g　VSS)　nitrogen　systems　increased　significantly,　while　a　higher　EPS　protein/polysaccharide　ratio,　which　was　positively　correlated　with　AnAOB　activity,　was　observed　in　the　low　nitrogen　system　(3.54　±　0.34)　than　that　in　the　ultra-low　nitrogen　system　(1.82　±　0.10).　In　addition,　Candidatus　Brocadia　was　detected　as　dominant　AnAOB　in　the　anammox　biofilm　under　the　low　(12.2　%)　and　ultra-low　(24.7　%)　nitrogen　condition.　Notably,　the　genus　Streptomyces　(26.3　%),　capable　for　funge-like　codenitrification,　increased　unexpectedly　in　the　low　nitrogen　system,　but　not　affecting　the　nitrogen　removal　performance.　Therefore,　using　sponge　iron　to　strengthen　AnAOB　activity　under　low　nitrogen　conditions　is　feasible,　providing　support　for　mainstream　anammox　applications.　©　2024