Nitrogen fixation continuously converts atmospheric nitrogen (N₂) into biologically available forms in the oceans, playing a crucial role in sustaining marine new production, particularly in oligotrophic ecosystems. Recently, heterotrophic diazotrophs were found to be widespread and potentially important for nitrogen fixation in the South China Sea (SCS). However, direct measurements of their contribution to total nitrogen fixation in this region remain lacking.To address this gap, We measured the total heterotrophic nitrogen fixation rates（NFRs） and size-fractioned heterotrophic NFRs using the isotope ¹⁵N tracer technique (revised ¹⁵N₂ gas bubble method), alongside diazotroph community structure analysis based on molecular detection methods. Our results reveal that diazotroph communities in the SCS are predominantly composed of heterotrophic diazotrophs and filamentous cyanobacteria. Notably, although heterotrophic NFRs within the euphotic zone were an order of magnitude lower than total NFRs, their contribution to overall nitrogen fixation was, nonetheless, comparable to that of autotrophic diazotrophs. Furthermore, size-fractionated data indicated that the <10 μm fraction was the major contributor to heterotrophic NFRs throughout the water column. Additionally, we found that heterotrophic N₂ fixation was primarily associated with aggregates. Overall, our study provides the first direct measurements of heterotrophic NFRs in the SCS and underscores the potential importance of non-cyanobacteria in marine nitrogen fixation. This highlights the need for further research to fully understand the ecological role and dynamics of heterotrophic diazotrophs in marine nitrogen cycling.
 

N2 fixation, heterotrophic diazotrophs, heterotrophic nitrogen fixation, South China Sea, aggregates