Enabling nitrification under deliberately high nitrate accumulation by adapting a nitrifying community from municipal sewage sludge

Efficient conversion of ammonia into less toxic nitrate is crucial for sustainable nitrogen management in agricultural and industrial systems. Stable nitrification under deliberately accumulated nitrate is required to convert ammonia emissions into concentrated nitrate solution that can be stored and transported efficiently, but nitrification performance under such nitrate-salt-driven high osmolarity remains poorly understood. In this study, we evaluated nitrification under deliberately high nitrate accumulation. Two types of inoculum were compared: nitrifying sludge from a freshwater wastewater treatment plant (WWTP) and the same sludge after prolonged exposure to high-nitrate conditions. Semi-continuous supply of aqueous ammonia, used both as substrate and alkaline agent, enabled stable nitrification at nitrate concentrations of approximately 7,000–8,000 mg-N/L, corresponding to osmolarities of 0.9–1.1 osmol/L. 16S rRNA sequencing revealed key microbial shifts, highlighting the adaptability of nitrifiers to extreme nitrate concentrations. The ammonia-oxidizing bacterium (AOB) Nitrosomonas and two nitrite-oxidizing bacteria (NOB) closely related to Nitrobacter winogradskyi and Nitrobacter hamburgensis dominated as nitrate concentrations increased, whereas the common NOB Nitrospira failed to adapt and were eliminated. These results demonstrate that nitrifying consortia originating from freshwater WWTP sludge can adapt to nitrate-driven high osmolarity conditions, extending the upper limit of reported nitrate accumulation in nitrification systems. • Extended reported nitrate accumulation under nitrate-salt-driven high osmolarity • Achieved 7,000–8,000 mg-N/L nitrate by acclimating freshwater WWTP sludge • 16S rRNA sequencing showed enrichment of Nitrosomonas (AOB) and Nitrobacter (NOB) • Nitrospira (NOB) declined as nitrate accumulated, consistent with lower tolerance • Heterotrophic denitrifiers increased but likely had minimal impact on N dynamics