Ultra-rapid start-up biological nitrification for nutrient recovery from source-separated urine

Biological nitrification presents a sustainable approach for urine resource recovery. However, high salinity and ammonium concentrations in urine inhibit or even damage microorganisms, causing delayed start-up and unstable. This study first introduces betaine (150 mg·L⁻¹) to enhance urine nitrification by improving microbial salt tolerance and metabolic. Compared with the conventional typical process without betaine addition, introducing betaine shortened start-up time from 98 to 36 days, increased nitrification rate from 313.9 to 563.7 mg N·L⁻¹·d⁻¹, reduced nitrite accumulation, and improved resilience to water quality fluctuations. It also upregulated expression of nitrifying bacteria and related functional genes. Mechanistically, betaine stimulated extracellular polymeric substances production and regulated tryptophan and tyrosine metabolism genes, improving sludge aggregation and microbial stability. Betaine modulated genes for K⁺ uptake and Na⁺ extrusion to maintain initial osmotic balance. Subsequently, betaine promoted the uptake/synthesis of osmoprotectants (e.g., betaine and trehalose), upregulated electron transport chain genes and optimized energy metabolism. Notably, Betaine-induced multiple salt-tolerance mechanisms showed synergistic effects, with Rubrivivax sp., Paracoccus aminovorans, and Nitrobacter sp. identified as core salt-tolerant species. Even after betaine discontinuation (at day 40), high nitrification activity and salt tolerance persisted, though reduced amoABC gene abundance may constrain long-term performance. Furthermore, betaine-enhanced urine fertilizers demonstrated high nutrient recovery efficiency and reduced phytotoxicity, indicating strong potential for agricultural reuse. Overall, this study provides novel theoretical and practical insights, establishing betaine as an effective strategy for accelerating and stabilizing biological nitrification in high-salinity wastewater systems such as urine, with broad implications for sustainable treatment and resource recovery.