Restoring partial denitrification under dual cadmium-salinity stress using betaine: Insights into granule stability, microbial adaptation, and size-dependent functionality.

Partial denitrification (PD) is a promising strategy for sustainable nitrite supply to anammox systems, yet its practical application in industrial wastewater treatment is often constrained by simultaneous heavy metal and salinity stress. This study investigated the long-term role of betaine as an osmoprotective regulator in restoring PD granular sludge exposed to Cd(II) (80 mg/L) and elevated salinity (20-60 g/L NaCl). Under initial stress without betaine, nitrate reduction was severely inhibited and nitrite accumulation was below the detection limit. Betaine supplementation substantially restored reactor performance, increasing the specific nitrite production rate from 5.35 ± 0.51-73.65 ± 2.92 mg N/g VSS/h and promoting stable nitrite accumulation under moderate salinity (20-40 g/L NaCl). Further studies showed that betaine may have stimulated extracellular polymeric substance (EPS) secretion, particularly proteins, potentially enhancing granule aggregation and structural stability. It also reshaped microbial communities by enriching stress-tolerant taxa while maintaining the core PD-related genus (Thauera). However, under extreme salinity (60 g/L NaCl), microbial activity, EPS, and granule size declined markedly, and the recovery efficiency of betaine was relatively low. Granule size further governed system resilience: due to stable spatial frameworks, large granules (d > 0.5 mm) are able to withstand combined stress shocks, making it easier to maintain stable performance when betaine is added. In contrast, small granules (d < 0.5 mm) have a simple structure and exhibit impaired performance. These findings demonstrate that betaine can effectively enhance PD robustness under combined Cd(II) and salinity stress, while maintaining large and structurally intact granules is critical for maximizing its protective effects.