The　high　frequency　of　acetaminophen　(APAP)　detection　in　water　bodies　around　the　world　increases　the　risk　to　the　global　environment.　A　lot　of　work　has　concentrated　on　isolating　and　identifying　the　culturable　APAP　degraders　for　understanding　of　the　microbiota　and　their　ecophysiology,　which　greatly　underrated　the　diversity　of　organisms.　Here,　13C-DNA　stable　isotope　probing　(DNA-SIP)　was　applied　for　the　first　time　to　identify　microorganisms　involved　in　APAP　metabolism　under　conditions　close　to　in　situ　from　three　mixed　microbial　communities　collected　in　lab-scale　nitrification　systems.　Based　on　DNA-SIP,　Achromobacter　(oligotype　ATAA),　Alicycliphilus　(oligotype　GTTCG)　and　Thauera　(oligotype　GAAACTTTCA)　were　identified　as　the　key　APAP　degrading　bacteria　in　the　floc　sludge,　granular　sludge　and　biofilm　reactors,　respectively,　with　the　relative　abundance　of　43.20%,　10.12%　and　12.60%.　Moreover,　Leucobacter,　Achromobacter,　Alicycliphilus,　Thauera,　Microbacterium　and　Lactobacillus　were　found,　for　the　first　time,　to　be　directly　responsible　for　APAP　biodegradation.　However,　Achromobacter,　Lactobacillus　and　Alicycliphilus　were　linked　to　spread　resistance　genes　(intI1,　sul2,　qacEdta1-02　and　qacH-01).　The　results　highlight　that　Acinetobacter,　Leucobacter,　Thauera　and　Microbacterium　could　be　recommended　as　bioaugmentation　strains　for　the　treatment　of　wastewater　contaminated　with　APAP.　Ultimately,　the　information　acquired　in　this　study　could　add　to　the　current　knowledge　on　microorganisms　degrading　APAP　and　accelerate　the　implementation　of　biofortification　processes　to　achieve　APAP　removal.　©　2024　Elsevier　B.V.