Nitrogen fixation associated with Microcystis colonies promotes harmful cyanobacterial blooms across North American lakes

Microcystis forms cyanobacterial harmful algal blooms around the world including regions where inorganic nitrogen levels are low. Here, we measured N2 fixation rates and nutrient uptake rates of isolated Microcystis colonies, free-living plankton, and the whole plankton community during Microcystis blooms over a three-year period in six diverse lakes across eastern North America including Lake Erie. We additionally characterized the nifH-containing microbial community and the diversity of prokaryotes (16S rRNA gene) in the same plankton fractions. Across all lakes, there were dense Microcystis blooms and measurable rates of N2 fixation associated with Microcystis colonies. For five of six lakes, rates within the isolated colony fraction were significantly greater than rates measured for free-living plankton (p<0.05). N2 fixation rates were inversely correlated with rates of ammonium uptake by the colony fraction (p<0.05) and in experiments, ammonium additions consistently and significantly reduced N2 fixation rates within the colony fraction by 50-85% (p<0.001). In several lakes, the del15N of isolated colonies were significantly lower (p<0.05) than that of free-living plankton presumably due to the preferential use of 14N2 for diazotrophy by colony-associated plankton. Cyanobacterial and non-cyanobacterial nifH sequences were associated with the Microcystis colonies with the nifH-based diversity of both groups being lower in colonies compared to free-living bacteria. The beta diversity of nifH taxa was significantly higher in smaller, more eutrophic systems compared to larger, less eutrophic systems where the relative abundance of non-cyanobacterial N2-fixers was significantly lower (p<0.05 for both). Compared to nitrate, ammonium, and urea uptake, N2 fixation rates accounted for up to 76% of total N assimilation by the Microcystis colony fraction and averaged 18% across systems, suggesting this process could promote the proliferation of Microcystis blooms.