Temperature affects the ratio of ethylene produced to N 2 -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 N 2 -fixation is the acetylene reduction method, which measures nitrogenase activity. This method requires the assumption of a theoretical ratio of ethylene produced to N 2 -fixed unless the estimates are calibrated using 15 N 2 as a tracer. We measured N 2 -fixation using both the acetylene reduction method and 15 N 2 uptake methods to examine the ratio of ethylene produced to N 2 -fixed (C 2 H 4 :N 2 ), estimate controls on variation in this ratio, and quantify the immediate release of N 2 -fixed as dissolved N. We estimated N 2 -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 C 2 H 4 :N 2 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 N 2 -fixed as dissolved organic and inorganic N was < 1% of N 2 -fixation. Ethylene production increased 1.8 times faster than N 2 -fixation as temperature increased showing strong control of temperature in the C 2 H 4 :N 2 ratio. Temperature contributed substantially to the variability in ratios of ethylene produced to N 2 -fixed. Slight differences in the C 2 H 4 :N 2 ratio can considerably alter N 2 -fixation estimates particularly in N 2 -fixation dominated stream biofilms, thus when calibrating using a range of temperatures may be essential.