Effect of Agitation Speed on ANAMMOX Granule Formation and Nitrogen Removal Efficiency in a Continuous Stirred Tank Reactor

Anaerobic ammonium oxidation (ANAMMOX) is a process mediated by autotrophic bacteria. Its unique metabolism, characterized by oxygen-independent energy production and carbon self-sufficiency, offers significant advantages for energy-efficient nitrogen removal in wastewater treatment, eliminating the need for aeration and external carbon sources. However, practical application is hindered by the inherently low growth rate and biomass washout under low nitrogen loading conditions. To address these challenges, this research explored granule formation under a low-intensity agitation condition of 55 revolutions per minute in a continuous stirred tank reactor seeded with return activated sludge. Slow mechanical mixing acted as both a selective pressure and physical driver for granule formation. Over 500 days of operation, nitrogen removal efficiency increased markedly, reaching complete substrate conversion with stoichiometric ratios consistent with ANAMMOX metabolism. The formed granules exhibited vertical stratification, with the largest particles settling in the reactor bottom. Cell activity assays using granules collected from three distinct layers revealed uniform ammonium and nitrite removal performance across all layers, confirming homogeneous distribution of ANAMMOX activity throughout the sludge bed. These results suggest that slow mechanical mixing supports not only granule formation and retention, but also uniform microbial activity - key for stable and scalable ANAMMOX application in mainstream wastewater treatment.