Biological nitrogen (N) fixation, the microbial conversion of N2 gas to ammonia, makes N available to food webs. Low-N streams often have a high relative abundance of N-fixing taxa, suggesting that N fixation is an important N source in these systems. Despite this potential, stream N fixation has not been well-characterised, particularly compared to lakes and marine environments. One unknown is the relative contributions of various N-fixing organisms, particularly heterotrophic microbes. In low-N streams in the Cascade Mountains (Washington, USA), three groups of N-fixers predominate: cyanobacteria (Nostoc paramelioides) colonies that house a midge symbiont (Cricotopus spp.), cyanobacteria without a midge symbiont, and heterotrophic sediment microbes. In seven streams, we measured N fixation rates in each group with the acetylene reduction assay and a 15N2 calibration. Cyanobacteria N fixation rates were relatively low (7.9 ± 8.9 μg N m−2 hr−1, mean ± SD) compared to other mountain streams. Although rates were comparable among types of N-fixers, our sediment conversion ratio (moles of ethylene produced:moles of N fixed) was 0.16:1, much lower than our cyanobacteria conversion ratio of 1.72:1 and the commonly used theoretical ratio of 3:1. Sediment N fixation rates (5.7 ± 4.0 μg N m−2 hr−1) were higher than previously reported rates measured only with acetylene reduction. The midge symbiosis did not greatly impact N fixation rates; however, owing to their prevalence, colonies with the midge probably contributed more total N to streams than colonies without the midge. Additionally, N fixation by sediment heterotrophs was comparable to that of cyanobacteria colonies on an areal basis. Our study demonstrated that the contribution of sediment heterotrophs previously may have been underestimated in streams, especially considering that sediment heterotrophs are probably present for a longer portion of the growing season than cyanobacteria in temperate and boreal ecosystems
