Enrichment of comammox Nitrospira with Urea

Urea is a ubiquitous nitrogenous compound pivotal in shaping microbial community dynamics and ecological functions within natural and engineered ecosystems. Understanding the differential response mechanisms of nitrifying microorganisms to urea utilization is crucial for deepening the understanding of the biological nitrogen cycle and optimizing nitrogen removal processes in wastewater treatment systems. In this study, a stable urea nitrification microbial community was successfully established in a sequencing batch reactor (SBR) over prolonged cultivation. Microbial community analysis, combining 16S rRNA gene amplicon sequencing and quantitative PCR, indicated the enrichment of ammonia-oxidizing microorganisms (AOMs), particularly comammox Nitrospira, which significantly outcompeted ammonia-oxidizing bacteria (AOB) in terms of abundance following urea addition. Further metagenomic sequencing and comparative genomic analyses revealed that comammox Nitrospira utilized a highly conserved ABC-type active transport system combined with a urease-dependent pathway characterized by slow substrate turnover. In contrast, Nitrosomonas primarily employed passive urea transporters and exhibited dual ureolytic mechanisms, encompassing both urease and the ATP-dependent urea carboxylase pathways, enabling rapid urea metabolism. These distinct genetic and transcriptional features highlight an ecological strategy differentiation where comammox Nitrospira adopts a high-affinity but energetically costly urea uptake for adaptation in oligotrophic niches, whereas Nitrosomonas favors quick urea utilization in eutrophic conditions. This research elucidates the genetic and ecological basis for selective enrichment of comammox Nitrospira under urea-fed oligotrophic conditions, providing fundamental insights for optimizing nitrogen cycling and management strategies in engineered wastewater treatment systems.