Abstract

Anaerobic ammonium oxidation (anammox) is a sustainable and energy-efficient technology for the treatment of wastewater with high ammonia and low C/N ratios, but its performance is often challenged by low temperatures, especially under fluctuating conditions commonly encountered in practice. Understanding the adaptation mechanisms of anammox granular sludge to such temperature stress is crucial for the robust application of the anammox process, particularly concerning the interplay between sludge size, microbial community dynamics, and functional genes including those involved in nitrogen-removal and cold resistance. This study investigated two anammox reactors operated at a high temperature (28.3 °C, R 1 ) and a fluctuating low temperature (11–23 °C, R 2 ) to elucidate these mechanisms. Results demonstrated that elevated free nitrous acid (FNA) levels (> 0.5 μg L −1 ), rather than free ammonia (FA), were strongly correlated with inhibited anammox activity at low temperatures, reducing the nitrogen removal efficiency by 16% at 15 ℃ compared with that in the high-temperature reactor R 1 . Apparent temperature coefficient ( Q 10 ) ranged from 0.90 to 1.27, indicating typical temperature-dependent bacterial activity. Low temperatures increased the extracellular polymeric substance (EPS) structural vulnerability, weakened granular strength and increased the proportion of small particles (< 0.25 mm) by 32% relative to R 1 while decreasing biomass content (volatile suspended solids (VSS)/suspended solids (SS) to 27.9%). Our findings demonstrated that the system does not merely undergo inhibition but orchestrates a sophisticated adaptive response: larger granules (> 2 mm) serve as stable reservoirs for core anammox bacteria (notably Candidatus Jettenia caeni ), ensuring biomass retention, while smaller granules (< 0.25 mm), generated through low-temperature-induced fragmentation, function as specialized pioneers. These small granules uniquely enrich a suite of cold-adap