Bacterial and Structural Dynamics During the Bioaggregation of Aerobic Granular Biofilms

Granular sludgeGranular sludge forms by microbial self-aggregation into mobileMobile biofilmBiofilm particlesParticle with a gel-like consistence. Bacterial and structural dynamicsStructural dynamics from flocsFlocs to granulesGranule were examined in anaerobicAnaerobic-aerobicAerobic sequencing batch reactorsSequencing batch reactor (SBRsSBR) by microbiomeMicrobiome profilingProfiling, fluorescence in situ hybridizationFluorescence in situ hybridization, fluorescence lectinLectin-binding analysis, and confocal laser scanning microscopyConfocal laser scanning microscopy. First, in a bubble-column SBRSBR operated under wash-outWash-out, granulesGranule developed rapidly in 2 weeks by swelling of ZoogloeaZoogloea coloniesColonies around flocsFlocs, forming smooth biofilmsBiofilm. However, nutrient removalNutrient removal deteriorated under ZoogloeaZoogloea predominancePredominance (37–79%). NitrificationNitrification (80–100%), N-removalN-removal (43–83%), and P-removalP-removal (75–100%) improved during granulesGranule maturation. Full anaerobicAnaerobic uptakeUptake of volatile fatty acidsVolatile fatty acid selected for PAOsPAO and P-removalP-removal. ProliferationProliferation of denseDense microcoloniesMicrocolony of nitrifying (NitrosomonasNitrosomonas), polyphosphatePolyphosphate—(PAOsPAO; “Candidatus Accumulibacter“Candidatus Accumulibacter””) and glycogenGlycogen-accumulating organisms (GAOsGAO; “Ca. CompetibacterCompetibacter”) from the granuleGranule cores outwards resulted in heterogeneousHeterogeneous granulesGranule. Second, biomassBiomass granulated spontaneously in 2 months in a PAOPAO enrichmentEnrichment (“Ca. AccumulibacterAccumulibacter”, 56 ± 10%) and after 480 days in a GAOGAO enrichmentEnrichment (“Ca. CompetibacterCompetibacter”, 37 ± 11%) during a period of over-aerationAeration caused by biofoulingBiofouling of oxygen sensorSensor. PAOsPAO and GAOsGAO aggregated as denseDense, heterogeneousHeterogeneous clustersCluster. ZoogloeaZoogloea was never abundant in both enrichmentEnrichment culturesCulture. GlycoconjugatesGlycoconjugate of extracellular polymeric substancesExtracellular polymeric substances were detected with fluorescent lectinsLectin in the granular biofilmGranular biofilm continuumContinuum (N-acetyl-glucosamineN-acetyl-glucosamine multimersMultimer by STA lectinLectin), in matricesMatrix surrounding microcoloniesMicrocolony (N-acetyl-glucosamineN-acetyl-glucosamine monomersMonomer by LEA, WGA), around bacterial clustersCluster (N-acetyl-galactosamineN-acetyl-galactosamine by HAA, SBA), and at cell surfacesSurface (α-glucoseGlucose and α-mannoseMannose by Con A; fucoseFucose by AAL; sialic acidsSialic acids by WGA; complex sugarsSugar by PHA-L). GranulationGranulation mechanisms rely on predominant populationsPopulation and their physiology: fast-growingFast-growing ones form smooth granulesSmooth granule; slower-growing form denseDense, heterogeneousHeterogeneous conglomeratesConglomerate. GranulesGranule unveil complex functional exopolymersExopolymer.