Stream ecosystems exhibit high degrees of spatial heterogeneity in environmental conditions, communities of organisms, and ecosystem processes at nested scales from landscapes to microhabitats. This heterogeneity may facilitate the co-occurrence of biogeochemical processes that are favored under incompatible environmental conditions, like dinitrogen (N2 gas) fixation and denitrification. We hypothesized that environmental variation at the patch scale (1-10’s m) would facilitate the co-occurrence of N2 fixation and denitrification through the formation of ecosystem control points or patches that show high reaction rates relative to the surrounding area. We measured rates of N2 fixation and denitrification and relative abundances of the genes nifH and nirS (genes that encode for the enzymes nitrogenase and nitrite reductase respectively) in patches determined by channel geomorphic units and substrate type in seven streams encompassing a gradient of N and P concentrations. We found ecosystem control points, where rates of N2 fixation and denitrification were 1 to 4 times higher than reach-average rates (0.2 – 1400 μg m-2 h-1 and 350 – 60000 μg m-2 h-1, respectively), occurred in all study streams. Most N2 fixation control points were in patches with rock substrates, while denitrification rates and relative abundances of nifH and nirS were higher in fine sediment patches. Yet, in two of the streams, rates in the top 25% of all patches for both denitrification and N2 fixation occurred in the same patches, suggesting that variation in conditions at the sub-patch scale can also facilitate co-occurrence of these processes. Across all streams and patches, organic matter and dissolved oxygen concentrations were important predictors of rates of N2 fixation, denitrification, and nifH relative abundance, while P concentration was important to N2 fixation and denitrification. Our results demonstrate that understanding the spatial ecology of microbially-driven nutrient cycling is required to characterize nutrient fluxes more completely in stream ecosystems.

It is typically assumed that dinitrogen (N2) fixation and denitrification are mutually exclusive processes in riverine ecosystems because N2 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 N2 fixation can happen under high N conditions and in sediments, challenging this assumption. We conducted a cross-ecoregion study to test the hypothesis that N2 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 N2 flux using membrane inlet mass spectrometry, N2 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 N2 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 N2 fixation rates and the highest rates measured in sediments. Substrate heterogeneity, and associated variation in environmental conditions, appeared to facilitate the coexistence of N2 fixation and denitrification in the study streams. Rates of denitrification were significantly positively related to streamwater nitrate concentrations (r2 = 0.35), but N2 fixation rates were not, despite the common simplifying assumption that denitrification dominates the N2 flux in streams under high N and N2 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.