Measurement of Nitrogenase Activity

There are various methods to find out whether an organism is a N₂-fixer or not. If an organism can grow in normal atmosphere without any provision of a nitrogen source like nitrate or ammonia, one infers the organism to be a N₂-fixer. It is important to mention here that N₂-fixer heterocysts cyanobacteria or Rhizobium-legume system are known to grow without any supply of fixed nitrogen. The other more reliable method is the incorporation of ¹⁵N isotope into cellular nitrogen containing compound which can be estimated in a mass spectrometer. This method is very costly and is, therefore, not easily accessible for day-to-day measurement of nitrogenase activity and N,-fixation. The most extensively used method for measuring nitrogenase activity is the acetylene reduction method. The method consists of incubating the N₂-fixing system with about 10% acetylene for a period of 10 to 30 min during which nitrogenase converts acetylene into ethylene depending on its efficiency.

Gas samples are then taken out by syringe from the reaction sample and the acetylene reduced is assayed by injecting the gas sample into a gas chromatograph which separates quantitatively acetylene and ethylene and thus provides easy quantitative assay of ethylene. Nitrogenase catalysed reduction of acetylene to ethylene is not accompanied by reduction of proton to molecular hydrogen as happens in the reduction of N₂ to NH_­3. The reduction of acetylene to ethylene involves two electrons and if one wants to equate the amount of ethylene produced with the amount of N₂ fixed then the amount of ethylene has to be divided by a factor of 4 to find out the optimum level of N₂-fixing activity of nitrogenase.