Upflow Velocity-Regulated Partial Granulation in a Continuous Flow Anaerobic Anoxic Oxic System: Mechanistic Insights from Sludge Morphology, Carbon Utilization, and Microbial Succession

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Cultivating aerobic granular sludge (AGS) in continuous flow reactors remains challenging due to the lack of selection pressure compared with sequencing batch reactors. This study used a upflow velocity-regulated selection strategy to cultivate AGS in a continuous flow anaerobic anoxic oxic (AAO) reactor with an upflow selection tank by adjusting internal reflow ratios. The treatment performance, sludge characteristics, carbon utilization pathways, microbial communities, and functional gene profiles were analyzed. Results showed that intensified upflow velocity (3 m·h–1) effectively promoted partial granulation. The average particle size increased from 55.5 to 130.5 μm on the 120th day, with 23.8% of granules (>200 μm). Stable chemical oxygen demand (COD) (>88%) and NH4+-N removal performances (>97%) were maintained throughout. Batch phosphorus (P) experiments exhibited improved biological phosphorus activity with granulation (P release rate increased by 1.15 mg P·(g MLSS·h)−1) and enhanced anoxic P uptake, with the denitrifying phosphorus accumulating organisms (DPAO)/phosphorus accumulating organisms (PAO) ratio increasing from 20.20 to 57.54%. High sludge reflow carried more NO3–-N into the anaerobic tank, suppressing PAO despite the predicted enzymatic potential being maintained. The increased extracellular polymeric substance (EPS), intensified feast-famine extent, and strengthened internal metabolic cycling were associated with granule densification. Overall, this study provided insights into AGS formation in continuous flow systems and offered a potential engineering-feasible pathway for upgrading conventional AAO processes.