The acetylene reduction assay for measuring nitrogen fixation in waterlogged soil

Nitrogen fixation in waterlogged, soil–straw, and sand–clay–straw mixtures was measured with the C 2 H 2 reduction assay, the 15 N-tracer technique, and the Kjeldahl method. The reduction of 6 to 15 moles C 2 H 2 corresponded to the fixation of 1 mole N 2 . The theoretical ratio is 3 moles C 2 H 2 to 1 mole N 2 . A ratio of 3 moles C 2 H 2 reduced for each mole of N 2 fixed was obtained when samples of sand–clay–straw were incubated under conditions that minimized effects that were due to gas diffusion through the aqueous phase. Calculations indicated that N 2 at a partial pressure of 0.2 atm above the samples was not sufficient to saturate the nitrogenase enzyme of the organisms in lower layers of soil–straw samples. Thus the concentration of N 2 dissolved in the aqueous phase limited nitrogen fixation. C 2 H 2 is more soluble in water than N 2 ; the C 2 H 2 reduction was not as limited by the C 2 H 2 concentration in the aqueous phase.N 2 was experimentally shown to be limited at depth in a sand–clay–glucose system in that fixation decreased from 128 to 36 μg N/g of sand–clay incubated so that the total sample depth ranged from 0.2 to 3 cm.The C 2 H 2 reduction assay provides a method for measuring the potential nitrogenase activity in the waterlogged soil amended with straw; however, this assay must be calibrated for specific conditions. The data also indicate that where N 2 diffusion rates may limit N fixation, a normal atmosphere (80%) of N 2 should be used in the experiment.