Besides N₂, the nitrogenase enzyme can reduce other substrates such as cyanide, azide and acetylene. Direct measurement of N₂ fixation (reduction) requires a mass spectrometer, instrumentation that is not readily available, but the reduction of acetylene to ethylene can easily be measured by gas chromatography (Dilworth 1966). Acetylene reduction involves two electrons, C₂H₂ + 2e^– + 2 H⁺ → C₂H₄, as opposed to the eight electrons required for the reduction of N₂ and 2 H⁺:

Therefore, reduction of four ethylene molecules correspond to the reduction of one N₂ molecule. The acetylene method has limitations, however: Acetylene blocks gas diffusion into nodules, and rhizosphere microorganisms produce ethylene. For these reasons, the acetylene method is suitable for comparative studies (Dennison et al. 1992), while mass spectroscopy is used for direct measurements of N₂ reduction, which is required for precise quantification of nitrogen fixation.