Cultivation of Small Granular Flocs with Predominant Aerobic Denitrifiers and Formation of Densified Activated Sludge in a Sequencing Batch Reactor

Efficient simultaneous nitrification, denitrification, and phosphorus removal typically requires either separate tanks with oxic and anoxic conditions or aerobic granular sludge (AGS) with granules larger than 0.2 mm. In this study, we cultivated small granular flocs (50-100 µm) capable of simultaneous heterotrophic nitrification and aerobic denitrification (HN-AD) in a sequencing batch reactor. The bioreactor was seeded with biomass from a municipal wastewater treatment plant and fed acetate-based synthetic wastewater for 114 days. The small granular flocs formed densified activated sludge (DAS), with biomass accumulating to approximately 12 g/L mixed liquor suspended solids. DAS was cultivated under selection pressures commonly used for AGS formation, including anoxic feeding, extended anoxic mixing, elevated hydrodynamic shear, and a gradual reduction in settling time. The sludge volume index (SVI) improved to 30 mL/g, comparable to that of granular sludge, with a moderate SVI₃₀/SVI₅ ratio of 72%. The relative abundance of Paracoccus reached 72.9%, indicating the metabolic capability of these small granular flocs to carry out HN-AD. Overall, these findings show that efficient simultaneous nutrient removal does not strictly depend on large granular structures. Achieving DAS and HN-AD offers a scalable approach to intensify biological nutrient removal while reducing reliance on distinct anoxic zones.