Nitrogen fixation may not alleviate stoichiometric imbalances that limit primary production in eutrophic lake ecosystems.

Ecosystem-scale primary production may be proximately limited by nitrogen (N) but ultimately limited by phosphorus (P) because N fixation contributes new N that accumulates relative to P at ecosystem scales. However, the duration needed to transition between proximate N limitation and ultimate P limitation remains unknown for most ecosystems, including lakes. Here we present the results of a fully replicated, multi-annual lake mesocosm experiment that permitted full air-water-sediment interactions that mimicked lake ecosystem ecology. We manipulated N supply relative to P to achieve a gradient of N:P stoichiometry. Despite N fixation contributing as much as 80% of reactive N in the low N treatments, phytoplankton biomass in these treatments was not different from the unfertilized controls. This suggests that primary production remained N limited in the lowest N treatments, even when N fixation was substantial. Although fixed N inputs reduced the N imbalance relative to P in the low N treatments seasonally, fixed N did not accumulate over multiple years. Additionally, reactive N did not readily accumulate in the high N treatments. Instead, water column stoichiometry was proportional to the experimental N and P additions, suggesting a strong influence from external nutrient loading. Thus, we found no evidence that N accumulation from N fixation was sufficient to trigger a transition to ultimate P limitation seasonally or across our 3-year experiment. Rather, our results indicate that proximate N limitation perpetuates in eutrophic lakes, likely due to N export being proportional to its inputs. These findings offer new insight regarding the biogeochemical controls on ecosystem stoichiometry and their influence on the timeframe for proximate N limitation and ultimate P limitation in freshwater, marine, and terrestrial ecosystems.