Comparison of acetylene-reduction and nitrogen-15 techniques for the determination of nitrogen fixation by field bean (Vicia faba) nodules

Field bean (Vicia faba L.) cv. Maris Bead seeds were inoculated with Rhizobium Catalogue No. 1001, supplied by Rothamsted Experimental Station, and grown in sand culture supplied with a complete nutrient solution which included nitrate at either 1.5 or 6.0 mM. Nodules were detached from the roots at intervals during plant development and their rates of nitrogen fixation estimated by both acetylene reduction and ¹⁵N gas technique. There was a constant relationship, independent of nitrate supply, between the results obtained by these two methods at all samplings. The amounts of acetylene reduced divided by a factor of 5.75 gave the amount of true nitrogen fixation; this factor is about twice the theoretical value. It is suggested that this discrepancy arose because, with acetylene, all the electrons available to the nitrogenase were used to form ethylene, whereas during normal fixation only about half the electron supply was used to fix nitrogen, the remainder having been consumed in the production of hydrogen gas.