Micro-scale organization of non-cyanobacterial diazotrophs drives nitrogen fixation in marine particles

Dinitrogen (N₂) fixation supplies reactive nitrogen to the ocean, and non-cyanobacterial diazotrophs (NCDs) are increasingly recognized as contributors. Marine particles have been theorized as hotspots where low oxygen and labile carbon favor N₂ fixation by NCDs, but empirical proof is lacking. Here, we show that particle-NCD associations in a controlled model where Vibrio diazotrophicus colonizes laboratory-generated diatom aggregates. Using nitrogenase (NifH) immunolabeling and a respiration probe, we map cell activity across particles, while bulk 15 N₂ tracing quantifies N₂ fixation and membrane-inlet mass spectrometry O₂/Ar tracks respiration. We find a clear metabolic succession: respiring cells represented 50% of total cells at 12 h, followed by an increase at 24 h in NifH-positive cells (30%), with higher relative abundances observed toward particle interiors. NCDs are up to 1500 times more concentrated in particles than in the surrounding seawater, with nitrogenase-expressing cells showing a 1.5-fold higher relative abundance in particle cores compared with the peripheries after 24 h without bioavailable nitrogen. The spatial arrangement of V.diazotrophicus within aggregates is associated with conditions favourable for N₂ fixation in particle interiors, supporting the view that particles act as micro-niches for heterotrophic diazotrophy. Our results provide empirical evidence that the spatial distribution of NCDs within particles influences their nitrogen fixation potential.