Hydrodynamic control of oxygen intrusion and shear stabilizes functional zonation for nitrogen removal in an integrated UASB.

Aeration-reflux coupling can establish functional zonation in integrated upflow anaerobic sludge blanket (UASB) reactors, but the hydrodynamic basis remains insufficiently quantified. This study integrated computational fluid dynamics (CFD) with metagenome-derived KO-genus profiling based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) from vertically resolved samples to link hydrodynamic characteristics with microbial functions. CFD analysis showed that aeration established an oxygen-exposed zone in the upper reactor, whereas internal reflux regulated downward bubble entrainment and generated shear hotspots. These hydrodynamic features shaped mixing patterns, maintained stable functional zoning, and preserved a micro-oxic niche for simultaneous anammox and denitrification (SAD) granules. Metagenomic and gene-network analyses revealed distinct vertical stratification of ammonia-oxidizing bacteria (AOB) and anaerobic ammonium-oxidizing bacteria (AnAOB) along the reactor height. They also identified a coupled NO2- supply-sink loop involving Nitrosomonas, Ca. Kuenenia, and denitrifying bacterium, supporting functional partitioning within the UASB. Operationally, a reflux ratio of 15 achieved the highest and most stable total nitrogen removal efficiency 90% within the optimal aeration, corresponding to a dissolved oxygen concentration of approximately 2 mg/L. In addition to nitrogen-transformation pathways, the vertically resolved metagenomes revealed cofactor-related functional potential, including molybdenum-cofactor and folate-associated metabolism, within the spatially structured microbial community. Together, these results demonstrate that aeration-reflux design can mechanistically sustain functional partitioning, granulation, and efficient nitrogen removal in integrated UASB systems by jointly regulating the oxygen and shear threshold. This strategy provides practical guidance for treating low-carbon, ammonia-rich side streams.