Pangenome-driven insights into nitrogen metabolic characteristics of Citrobacter portucalensis strain AAK_AS5 associated with wastewater nitrogen removal

Nitrogen metabolism in the genus Citrobacter is very poorly studied despite its several implications in wastewater treatment. In the current study, Citrobacter portucalensis strain AAK_AS5 was assessed for remediation of simulated wastewater supplemented with different inorganic nitrogen sources. Combination of (NH 4 ) 2 SO 4 with KNO 3 was the most preferred for achieving high growth density followed by (NH 4 ) 2 SO 4 and KNO 3 alone. This was in agreement with highest ammonical nitrogen removal of 92.9% in the presence of combined nitrogen sources and the corresponding nitrate nitrogen removal of 93% in the presence of KNO 3 . Furthermore, these removal capacities were validated by investigating the uniqueness and the spread of metabolic features through pan-genomic approach that revealed the largest number of unique genes (2097) and accessory genes (705) in strain AAK_AS5. Of the total 44 different types of nitrogen metabolism-related genes, 39 genes were associated with the core genome, while 5 genes such as gltI , nasA , nasR , nrtA , and ntrC uniquely belonged to the accessory genome. Strain AAK_AS5 possessed three major nitrate removal pathways viz., assimilatory and dissimilatory nitrate reduction to ammonia (ANRA & DNRA), and denitrification; however, the absence of nitrification was compensated by ammonia assimilation catalyzed by gene products of the GDH and GS-GOGAT pathways. narGHIJ encoding the respiratory nitrate reductase was commonly identified in all the studied genomes, while genes such as nirK , norB , and nosZ were uniquely present in the strain AAK_AS5 only. A markedly different genetic content and metabolic diversity between the strains reflected their adaptive evolution in the environment thus highlighting the significance of C. portucalensis AAK_AS5 for potential application in nitrogen removal from wastewater.