Spatio-temporal variability of nitrogen-cycling potentials in particle-attached and free-living microbial communities in the Yangtze River estuary and adjacent regions

Particle-attached (PA) and free-living (FL) microorganisms regulate coastal biogeochemical cycles, yet their roles in nitrogen transformation remain unclear. To address this knowledge gap, we seasonally sampled PA and FL from seawater along salinity gradients in the Yangtze River estuary (YRE) and adjacent regions to investigate the spatio-temporal variability of microbial communities, abundances of nitrogen-cycling genes, and key microbial groups affiliated with the nitrogen cycle in PA and FL. Compared to FL, the composition, structure and diversity of PA exhibited more pronounced variations in response to salinity and [NO₃^-]. Metagenomic analyses indicated a predominant role of denitrification in both PA and FL, with greater abundances of genes involved in most nitrogen transformation processes observed in the estuarine region. The potential for the nitrogen cycle in PA was relatively lower in May, while greater in FL, potentially due to competition for nitrogen substrates between PA and phytoplankton during spring. PERMANOVA and Mantel tests showed that gene abundances exhibited spatio-temporal dynamics and were associated with species and environmental factors. Gene-affiliated taxa identification and the Weighted Correlation Network Analysis revealed that the differences in environmental factors and taxa responsible for the nitrogen transformation drove spatio-temporal variations of the nitrogen cycle between PA and FL, and implied the significance of their interaction in nitrogen fates in coastal ecosystem. Gammaproteobacteria and Betaproteobacteria were highly affiliated with nitrogen-cycling genes, while Nitrososphaeria played an important role in nitrification and denitrification. This study offered practical insights for mitigating eutrophication through targeted regulation of microbial-mediated nitrogen fluxes.