Acetylene reduction assay

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The most common way of measuring nitrogen fixation is by an indirect method which uses the ability of the nitrogenase enzyme to reduce triple bounded substrates. The nitrogenase enzyme reduces acetylene gas to ethylene. Both gases can easily be quantified using gas chromatography. The method is called acetylene reduction assay (ARA) [1]. The main benefits of ARA is that it is easy to use, sensitive and has a good reproducibility.

Another way of measuring nitrogen fixation is directly by 15N2 assimilation. The fixation and incorporation of 15N2 is measured by emission spectrophotometry or mass spectrometry. This method is preferred when the rates of nitrogen fixation are high and high sensitivity is not necessary. Another direct method to measure nitrogen fixation is by CHN analyses, Kjeldahl digestion/oxidation, and high temperature catylic combustion techniques measure nitrogen accumulation into particulate, dissolved or total organic matter. This method is best suited when microbial biomass makes up the major portion of the particulate nitrogen and the rates of nitrogen fixation are very high.

Conversion factors/stoichiometry of the acetylene reduction assay: In theory, for every N2 molecule that is fixed three molecules of C2H2 are reduced. Observations of N2 fixation using 15N2 have revealed ratios significantly deviating from theory. In a reaction that accompanies N2 reduction but not acetylene reduction, nitrogenase can produce H2 from H2O, which can affect the reduction stoichiometry. There can also be a problem with relating potential activity to actual in vivo product formation. For example, if the N2 fixing microbes are nutrient limited then the potential activity which is measured using acetylene reduction might not be fully realized. However, a drawback of bottle-based incubation techniques is that delicate N2 fixating associations and aggregates may be disrupted, leading to potential underestimates of rates of nitrogen fixation.

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