N2 fixation by planktonic heterotrophic diazotrophs is more wide spread than previously thought, including environments considered “unfavorable” for diazotrophy. These environments include a substantial fraction of the aquatic biosphere such as eutrophic estuaries with high ambient nitrogen concentrations and oxidized aphotic water. Different studies suggested that heterotrophic diazotrophs associated with aggregates may promote N2 fixation in such environments. However, this association was never validated directly and relies mainly on indirect relationships and different statistical approaches. Here, we identified, for the first time, a direct link between active heterotrophic diazotrophs and aggregates that comprise polysaccharides. Our new staining method combines fluorescent tagging of active diazotrophs by nitrogenase-immunolabeling, polysaccharides staining by Alcian blue or concanavalin-A, and total bacteria via nucleic-acid staining. Concomitant to N2 fixation rates and bacterial activity, this new method provided specific localization of heterotrophic diazotrophs on artificial and natural aggregates. We postulate that the insights gained by this new visualization approach will have a broad significance for future research on the aquatic nitrogen cycle, including environments in which diazotrophy has traditionally been overlooked.
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