Comammox Nitrospira are the dominant ammonia oxidizers in the Yangtze estuarine biofilms

Biofilms are indispensable ecological habitats for microbes that have garnered global attention and play a potential role in influencing the biogeochemical cycling of nitrogen. However, the biogeochemical significance of biofilms and the mechanisms by which they regulate nitrogen cycling remain elusive. In this study, we utilized DNA-stable isotope probing (DNA-SIP) labelling techniques in conjunction with metagenomics to reveal a nitrifying ecological niche in biofilms taken from the Yangtze Estuary, with those from sediment and water samples for comparison. Quantitative analysis showed that the amoA gene abundance of comammox Nitrospira (2.3 × 10³ copies ng⁻¹ DNA) was significantly higher than that of ammonia-oxidizing archaea (AOA-amoA, 62.4 copies ng⁻¹ DNA) and ammonia-oxidizing bacteria (AOB-amoA, 218.1 copies ng⁻¹ DNA) in biofilms, and the average abundance of comammox Nitrospira showed the following order: water > biofilm > sediment. Moreover, the NOB nxrB gene was more abundant than the amoA gene of ammonia oxidizers in all three media. DNA-SIP further revealed that the active comammox Nitrospira clade A mediates the nitrification process in biofilms with peak abundance at a buoyant density of 1.715 g mL⁻¹. Active nitrifying bacteria exhibit metabolic diversity in both biofilms and sediments, and occupy unique nitrifying ecological niches. Additionally, the co-occurrence network showed that chlorophyll a, NO₃⁻ and salinity emerged as the predominant physicochemical factors affecting the nitrogen transformation genes in biofilms. Taken together, this study indicates that biofilms constitute an emerging nitrifying ecological niche in estuarine environments and deepens our understanding of the mechanisms by which biofilms function in marine biogeochemistry.