Evaluation of the Nitrogen Removal Performance and Study on Nitrogen Transformation Mechanism of Bacillus mojavensis

To explore the nitrogen removal performance and mechanism of a strain of Bacillus mojavensis, C3, isolated from the sediment of the Yellow Sea cold water mass, we investigated its nitrogen removal performance and mechanism. The results showed that C3 effectively removed NH₄⁺-N, NO₂^--N and NO₃^--N from sole nitrogen sources media with the initial inorganic nitrogen concentration of 21 mg/L, with the removal rate of 85.95%, 75.89% and 96.29%, respectively, and the maximum nitrogen removal rate, 0.36 mg/(L∙h), 0.36 mg/(L∙h), and 0.50 mg/(L∙h), correspondingly. Nitrogen balance analysis revealed that inorganic nitrogen in the system was removed through assimilation and heterotrophic nitrification-aerobic denitrification (HN-AD). Among these, nitrogen assimilation dominated the strain's inorganic nitrogen metabolism transformation while nitrogen released into the atmosphere through heterotrophic nitrification-aerobic denitrification varied between 13.29% and 16.83% of the total. The results of ¹⁵N isotope tracing showed that it only produced ¹⁵N₂O but not ¹⁵N₂ when strain C3 used ¹⁵NH₄⁺-N, ¹⁵NO₂^--N and ¹⁵NO₃^--N as the sole nitrogen source, indicating that N₂O was the primary gaseous product of denitrification of C3. Further exploration of the nitrification pathway using ammonia oxidation inhibitors revealed that adding different concentrations of allylthiourea (ATU) had no significant effect on ammonia removal rate or nitrogen loss, suggesting that C3 may possess a novel ammonia monooxygenase with a unique enzymatic structure, being capable of converting NH₄⁺-N to NH₂OH. Whole-genome sequencing analysis revealed that nitrogen utilization during assimilation mainly occurs through the glutamate dehydrogenase (GDH) pathway (extracellular NO₃^-/NO₂^-/NH₄⁺→intracellular NH₄⁺→Glutamate→Bacterial protein) and the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway (extracellular NO₃^-/NO₂^-/NH₄⁺→intracellular NH₄⁺→Glutamine→Glutamate→Bacterial protein). The results indicated that C3 achieves nitrogen dissimilation through the heterotrophic nitrification-aerobic denitrification (HN-AD) pathway which includes heterotrophic nitrification (NH₄⁺→NH₂OH→NO→N₂O) and aerobic denitrification (NO₃^-→NO₂^-→NO→N₂O). These findings provided important insights into the nitrogen transformation metabolism mechanism of B. mojavensis and offered a scientific basis for the potential application of C3 in the treatment of wastewater from marine aquaculture.