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 C2H2 reduction assay, the 15N-tracer technique, and the Kjeldahl method. The reduction of 6 to 15 moles C2H2 corresponded to the fixation of 1 mole N2. The theoretical ratio is 3 moles C2H2 to 1 mole N2. A ratio of 3 moles C2H2 reduced for each mole of N2 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 N2 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 N2 dissolved in the aqueous phase limited nitrogen fixation. C2H2 is more soluble in water than N2; the C2H2 reduction was not as limited by the C2H2 concentration in the aqueous phase.N2 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 C2H2 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 N2 diffusion rates may limit N fixation, a normal atmosphere (80%) of N2 should be used in the experiment.