This　study　is　the　first　to　reveal　the　important　roles　of　c-di-GMP　on　the　formation　of　autotrophic　granular　sludge　(AUGS)　and　autotrophic　biofilm　(AUB)　in　integrated　fixed-film　activated　sludge　(IFAS)　system.　The　results　revealed　that　the　partial　nitrification/anammox　(PN/A)　performance　was　successfully　developed　in　the　IFAS　system　and　high　nitrogen　removal　efficiency　was　obtained.　Notably,　it　was　clearly　indicated　that　the　intracellular　c-di-GMP　content　was　highly　linearly　correlated　with　the　concentration　of　protein　(PN)　in　tightly　bound　extracellular　polymeric　substances　(TB-EPS)　during　AUGS　formation.　It　was　also　found　that　the　c-di-GMP　content　was　highly　linearly　correlated　with　the　PN　concentration　during　AUB　formation,　and　also　had　a　good　linear　correlation　with　the　polysaccharide　(PS)　content　in　TB-EPS.　This　proves　that　intracellular　c-di-GMP　played　a　vital　role　in　regulating　the　TB-EPS　secretion,　thereby　further　affecting　the　aggregation　ability　of　AUGS　and　AUB.　In　addition,　when　the　c-di-GMP　regulates　the　content　of　PN　in　TB-EPS,　the　secondary　structure　of　the　PN　also　changes　significantly　correspondingly,　affecting　the　hydrophobicity　of　AUGS　and　AUB.　In　long-term　operation,　Candidatus_Kuenenia,　Nitrosomonas　and　Flavobacterium　gradually　became　dominant　species,　which　were　considered　to　favor　the　formation　of　AUGS　and　AUB　by　promoting　the　production　of　c-di-GMP　and　TB-EPS.　(C)　2022　The　Author(s).　Published　by　Elsevier　B.V.