DNA-SIP unveiled niche differentiation of active ammonia oxidizers in stratified sediments of the Three Gorges Reservoir

Comammox bacteria represent a newly discovered group of microorganisms capable of complete ammonia oxidation, playing a crucial role in the nitrogen cycle. Although the understanding of Comammox bacteria remains in its early stages, key aspects of their ecological roles and physiological status are still uncertain, especially regarding their distinction from other nitrifying microorganisms. To address these uncertainties, this study employed DNA-based stable isotope probing (DNA-SIP) to investigate depth-specific microbial communities in sediments and identify active ammonia-oxidizing populations along with their ecological niche differentiation. Results showed that net nitrification rates were significantly higher in the 0–10 cm sediment layer compared to the 10–30 cm layer. Across all depths, the abundance of the ammonia-oxidizing archaea (AOA) gene exceeded that of comammox Nitrospira clade A (ComA) and ammonia-oxidizing bacteria (AOB) genes. During active nitrification, substantial ¹ ³C assimilation by AOA was detected in the 0–10 cm layer, whereas only a minor shift from light to heavy fractions was observed for ComA in the 10–20 cm layer. Phylogenetic analysis of heavy fraction DNA further revealed that Nitrososphaera gargensis dominated active AOA, while clade A.2 represented the active ComA population. These findings provide compelling evidence that both AOA and ComA contribute to nitrification in Three Gorges Reservoir sediments. Notably, the dominant active ammonia oxidizers shifted from AOA in surface layers to ComA at greater depths, highlighting a more significant functional role of sedimentary ComA than previously recognized. The niche differentiation of active ammonia oxidizers in stratified sediments played a critical role in shaping nitrogen cycling processes in aquatic ecosystems. • In stratified sediments, active ammonia oxidizers exhibit clear niche differentiation. • Nitrososphaera gargensis dominated the active AOA community, while clade A.2 represented the active comammox population. • Comammox emerged as the functionally dominant group among active AOMs at 10–20 cm depth sediments.