The biofilm granulation mechanisms depend on the predominant bacterial populations involved

Introduction Aerobic granular sludge used for intensified biological wastewater treatment is based on fast-settling mobile biofilms with a gel-like consistence, called “granules”, formed from activated sludge flocs (Seviour et al. 2012). Fundamental aspects of the mechanisms of microbial selection and self-immobilisation as spherical biofilms have recently been studied in detail (Weissbrodt et al. 2013). In an approach combining biofilm engineering, molecular microbial ecology, and laser scanning microscopy, we provide insights in the relationships between bacterial selection mechanisms, predominant populations involved, and resulting biofilm architectures in the granular sludge microbiome. Methods Biofilm granulation mechanisms were followed in anaerobic-aerobic sequencing batch reactors inoculated with activated sludge and operated under conditions that selected for populations displaying distinct physiologies. Bacterial population dynamics were analyzed using the PyroTRF-ID molecular workflow combining community fingerprinting by terminal-restriction fragment length polymorphism and amplicon-pyrosequencing targeting the v1-v3 region of the 16S rRNA gene pool (Weissbrodt et al. 2012). Microbial structures were followed over time by laser scanning microscopy (Neu et al. 2010) combined with overall biomass staining using Rhodamine 6G, fluorescence lectin-binding analysis of glycoconjugate matrices, and 16S rRNA based fluorescence in situ hybridization targeting predominant phylotypes. Results Under wash-out conditions selecting for a fast-settling biomass and non-limiting amount of biodegradable organic substrate in the aeration phase, fast-growing heterotrophs affiliating with Zoogloea spp. proliferated and formed smooth homogeneous granular biofilms by swelling and outgrowth of microbial colonies around flocs. Under mid-mesophilic temperature and low-aeration shear stress, filamentous structures of Burkholderiales relatives penetrated outside aggregates and resulted in unfavorable slow-settling fluffy architectures. As soon as granular sludge accumulated in the system and organic loads were fully removed anaerobically prior to aeration, slower-growing populations of nitrifiers and of polyphosphate- (“Ca. Accumulibacter”) and glycogen-accumulating organisms (“Ca. Competibacter”) proliferated from granule cores outwards as heterogeneous compact colonies inside the zoogloeal continuous matrices. Enrichment cultures of the two latter guilds validated the mechanism of development of granular conglomerates by proliferation as compact clusters in the floc structure. Mature granular biofilms that removed all nutrients biologically exhibited a relatively high bacterial diversity (H’>3) comprising side populations with tentative metabolisms of exopolysaccharide production and consumption related to Xanthomonadaceae, Sphingomonadales, Rhizobiales, and Sphingobacteriales, respectively. The structural gelling agent of these multi-species biofilms was compos