Acetylene Reduction |
Acetylene reduction is a simple method to measure the activity of nitrogenase enzyme activity in nodules. When nodule nitrogenase is exposed to acetylene electron transfer to N2 in the nodule is interrupted and the acetylene is converted to ethylene:
C2H2 + 2e- + 2H+ --> C2H4
The reaction with acetylene mimicks the nitrogenase enzyme reaction with N2:
N2 + 6e- + 6H --> 2NH3
At one time scientists believed acetylene reduction would be useful to quantify BNF. There are, however, problems converting ethylene production measured in the assay into absolute amounts of N2 fixed by the crop over time. The most significant problem using the method to estimate BNF is the short incubation of nodules in acetylene (usually 30-90 minutes). Since nodule activity is very dependent upon current photosynthate availability and plant growth rate short term environmental factors that affect growth, such as reduced solar radiation during periodic cloud cover or temporary soil moisture deficit, immediately affects nodule activity. This fact makes it problematic to integrate short term nitrogenase activity estimates to quantify BNF over a longer crop duration.
Total crop N and dry weight measures (especially in conditions of low mineral N availability) or isotope dilution methods with appropriate non-fixing reference plants are preferred methods to quantify treatment differences (e.g. strain differences, BNF increase due to management etc.) or measure BNF. Contact the Joint FFAO/IAEA Program in Agriculture at
for more information on isotope dilution methods to estimate BNF.
For more detail on the acetylene reduction method see Somasegaran and Hoben's Methods in the Legume-Rhizobium Symbiosis.
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The Acetylene Reduction Method:
Nodules can be sampled from a variety legumes in any number of growing conditions. |
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| Supply containers are filled with acetylene. These containers are frequently collapsible bags, ball bladders or constant head devices that can deliver a constant amount of acetylene when sampled with a syringe. |
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| In most experiments plant shoots are harvested first to determine total dry weight and N accumulated by the plant. |
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| Plant shoots are usually put into bags for drying in an oven at 70 C prior to weighing and tissue analysis. |
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| Roots are removed from the growing medium |
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| Roots are placed in a gas tight vessel with a rubber septum. |
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| A volume of acetylene is removed from the supply vessel that will provide 1%-5% acetylene when introduced into the sample chamber. Usually the same volume of air is removed from the sample chamber prior to introducing the acetylene. |
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| Then acetylene is injected into the sample chamber holding the plant root systems and usually incubated for 30 - 90 minutes. Contents of the vessel are usually mixed briefly to ensure rapid exposure of the nitrogenase system to the substrate gas. |
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| After the incubation period is finished a sample of the gas is removed from the sample vessel with a syringe or an evacuated blood sample tube to determine ethylene production by gas chromatography. |
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