Temperature affects the ratio of ethylene produced to N₂-fixed more than immediate release of fixed N in streams

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.