Nitrogen fixation can be a substantial flux of new biologically available nitrogen (N) into some aquatic ecosystems. The most commonly used method for measuring N₂-fixation is the acetylene reduction method, which measures nitrogenase activity. This method requires the assumption of a theoretical ratio of ethylene produced to N₂-fixed unless the estimates are calibrated using ¹⁵N₂ as a tracer. We measured N₂-fixation using both the acetylene reduction method and ¹⁵N₂ uptake methods to examine the ratio of ethylene produced to N₂-fixed (C₂H₄:N₂), estimate controls on variation in this ratio, and quantify the immediate release of N₂-fixed as dissolved N. We estimated N₂-fixation for benthic biofilms and separated Nostoc in four streams in Wyoming, Little Laramie River, Ditch Creek, Spread Creek, and Polecat Creek. Estimates of the C₂H₄:N₂ ratio for any one stream and date ranged from 1.6 to 7.1 with a group-level mean of 3.8. Immediate release of N₂-fixed as dissolved organic and inorganic N was < 1% of N₂-fixation. Ethylene production increased 1.8 times faster than N₂-fixation as temperature increased showing strong control of temperature in the C₂H₄:N₂ ratio. Temperature contributed substantially to the variability in ratios of ethylene produced to N₂-fixed. Slight differences in the C₂H₄:N₂ ratio can considerably alter N₂-fixation estimates particularly in N₂-fixation dominated stream biofilms, thus when calibrating using a range of temperatures may be essential.

固氮可以使新的生物可用氮（N）大量流入某些水生生态系统。测量N ₂固定最常用的方法是乙炔还原法，其测量固氮酶活性。该方法需要假设所产生的乙烯与N ₂固定的理论比率，除非使用¹⁵ N ₂作为示踪剂来校准估计值。我们使用乙炔还原法和¹⁵ N _{2吸收法测量 N 2固定，以检查产生的乙烯与 N 2}固定的比率（C ₂ H ₄ :N ₂），估计对该比率变化的控制，并量化 N ₂ -固定为溶解 N 的立即释放。我们估计了怀俄明州、小拉勒米河、Ditch Creek、Spread 的四个溪流中底栖生物膜和分离发菜的 N _{2 -}固定小溪和臭猫溪。任何一个流和日期的 C ₂ H ₄ :N ₂比率的估计值范围为 1.6 至 7.1，组级平均值为 3.8。_{作为溶解的有机和无机氮的固定N 2}的立即释放小于固定N ₂的1% 。_{乙烯产量增长速度是N 2}的1.8倍-随着温度升高的固定，显示C ₂ H ₄ :N ₂比率对温度的强烈控制。_{温度对产生的乙烯与固定的N 2}的比率的变化有很大影响。_{C 2} H ₄ :N ₂比率的微小差异可以显着改变N ₂固定估计，特别是在N ₂固定占主导地位的流生物膜中，因此当使用一系列温度进行校准时可能是必要的。