Genome-resolved taxonomy, global distribution, and EPS potential of Zoogloea , the canonical floc-former in wastewater treatment systems

ABSTRACT The bacterial genus Zoogloea is the fourth most abundant genus in wastewater treatment plants (WWTPs) globally, where it plays key roles in floc formation and nutrient removal but can also contribute to viscous bulking when highly abundant. The taxonomic assignment of Zoogloea has historically been inconsistent, as several taxa were classified based on phenotypic characteristics and later reassigned to other families or orders. This has obscured both the identity and distribution of Zoogloea and hindered interpretation of its floc-forming mechanisms, which have been attributed to polysaccharides that may not originate from phylogenetically defined Zoogloea . Using our collection of more than 22,000 high-quality genomes from global activated sludge systems (MiDAS global genome catalog), we identified 47 genomes affiliated with the phylogenetically defined Zoogloea . In combination with abundance data, this enabled resolution of the genus’ taxonomy, biogeographical distribution, and metabolic diversity. Building on this framework, we mapped the phylogenetic distribution of a recently identified Zoogloea -associated floc-forming polysaccharide biosynthesis gene cluster by screening the MiDAS global genome catalog and GTDB. The gene cluster was identified across multiple genera within the Burkholderiales order, including Zoogloea , Thauera , Azoarcus , and Ca . Accumulibacter, as well as several previously undescribed taxa. The gene cluster is broadly conserved, with minor variations in gene composition and synteny, suggesting corresponding differences in polysaccharide structure. Genomes encoding this cluster also consistently contained co-occurring floc-regulating genes, indicating a common and widespread mechanism underlying floc formation in activated sludge systems. IMPORTANCE Operation of activated sludge systems depends on stable microbial floc formation for the separation of biomass from treated effluent. Floc formation is mediated by extracellular polymeric substances (EPS) produced by microorganisms, yet the identity, distribution, and biosynthesis of these polymers remain poorly characterized. Zoogloea is widely regarded as a canonical and model floc-forming genus associated with EPS production in wastewater treatment systems. However, its taxonomic classification has historically been problematic, as organisms were often assigned to Zoogloea based on morphology, leading to widespread misclassifications. This has blurred the true origin of polysaccharides attributed to the genus as well as their wider phylogenetic distribution, which has not been systematically resolved. Here, we map the distribution of the Zoogloea -associated polysaccharide biosynthesis gene cluster across a global genome data set, providing a framework to link phylogenetically defined taxa with floc-forming potential. This improves our understanding of EPS production in activated sludge systems and may support strategies for enhanced resource recovery.