A Cross-Ecoregion Evaluation of Nitrogen Fixation and Denitrification in Streams and Rivers of the United States of America

It is typically assumed that dinitrogen (N 2 ) fixation and denitrification are mutually exclusive processes in riverine ecosystems because N 2 fixation is favored in high light, low nitrogen (N) environments but denitrification is favored under anoxic, high N conditions. Yet recent work in marine and lake ecosystems has demonstrated that N 2 fixation can happen under high N conditions and in sediments, challenging this assumption. We conducted a cross-ecoregion study to test the hypothesis that N 2 fixation and denitrification would co-occur in streams and rivers across a range of reactive N concentrations. Between 2017 and 2019, we sampled 30 streams in 13 ecoregions, using chambers to quantify N 2 flux using membrane inlet mass spectrometry, N 2 fixation using acetylene reduction, denitrification using acetylene block, and microbial diversity using 16S gene sequencing. 25 of the study streams were part of the National Ecological Observatory Network or the StreamPULSE network, which provided data on water temperature, light, nutrients, discharge and metabolism. We found that N 2 fixation rates were detectable in half of the streams surveyed, and were most frequently detected on rock, wood, and/or macrophyte substrates. Denitrification potential was detected in all streams, with rates 1-2 orders of magnitude higher than N 2 fixation rates and the highest rates measured in sediments. Substrate heterogeneity, and associated variation in environmental conditions, appeared to facilitate the coexistence of N 2 fixation and denitrification in the study streams. Rates of denitrification were significantly positively related to streamwater nitrate concentrations (r 2 = 0.35), but N 2 fixation rates were not, despite the common simplifying assumption that denitrification dominates the N 2 flux in streams under high N and N 2 fixation only occurs under low N conditions. Additional analyses are exploring reach to watershed characteristics, and metabolic regimes as drivers of cross-ecoregion patterns in processes.