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 theorised and modelled as refuges where low-oxygen and labile carbon conditions meet, favouring NCDs. However, empirical proof is lacking, hindering our understanding of NCDs’ role in nitrogen availability in aquatic ecosystems. We tested particle-NCD associations in a controlled model where Vibrio diazotrophicus colonized laboratory-generated diatom aggregates. Using nitrogenase (NifH) immunolabeling and a respiration probe, we mapped cell activity across particle interiors. In parallel, bulk ¹⁵ N₂ tracing quantified N₂ fixation and membrane-inlet mass spectrometry O₂/Ar tracked respiration. We found a clear metabolic succession: respiring cells exceeded 50% at 12 h, followed at 24 h by an increase in NifH-positive cells (30%) concentrated in particle interiors. NCDs were up to 1.5 × 10³ more concentrated in particles than in surrounding seawater, with nitrogenase-expressing cells 1.5× denser in cores than peripheries in nitrate-free treatments by 24 h. In this system, the spatial arrangement of V.diazotrophicus within aggregates was associated with enhanced N₂ fixation in particle interiors, supporting the view that particles can act as micro-niches for heterotrophic diazotrophy. Our results provide empirical evidence supporting previous theories and modeling predictions that the spatial distribution of NCDs within particles influences their nitrogen fixation potential.