Rates of N₂ fixation can be measured by a number of techniques to address questions of nodule efficiency and nitrogen cycling in agricultural and natural plant systems. Nitrogenase is pivotal for initial reduction of N₂ but this same enzyme will also reduce acetylene (C₂H₂) to ethylene (C₂H₄). Acetylene is an effective competitor with N₂ for nitrogenase so the rate of C₂H₄ synthesis is proportional to nitrogenase activity. Acetylene reduction gives an instantaneous estimate of the N₂ fixation rate. Another instantaneous technique requires flushing nodulated roots with an argon : oxygen gas mixture (79:21) to displace all N₂. All electron flux through nitrogenase is then diverted to the reduction of protons to H₂ rather than N₂ to NH₄⁺ (Equation 2). The rate of H₂ evolution by roots can thus be used to estimate nitrogenase activity.

Alternative approaches to ‘instantaneous’ estimates of N₂ fixation provide an integrated rate of fixation over periods of hours or days. The proportions of inorganic and organic nitrogen compounds in xylem sap are affected by the ratio of inorganic nitrogen taken up to symbiotic N₂ fixation; this can be exploited in genera of legumes in which amides and ureides are major products of N₂ fixation. Soybean, for example, exports less than 10% of nitrogen to shoots in the form of ureides when supplied nitrate but more than 80% when all nitrogen is biologically fixed. Thus, relative ureide levels in sap give an estimate of N₂ fixation.

Many experiments now rely on ¹⁵N-based techniques to obtain an integral of fixation over the life of a plant. These techniques rely on a difference in ratio of the stable isotopes of nitrogen (¹⁵N and ¹⁴N) in soil and atmosphere (Figure 4.50). The soil must be enriched in ¹⁵N relative to the atmosphere — either naturally (the process of denitrification causes a fractionation of the two isotopes, leaving the soil enriched in ¹⁵N) or by artificial ¹⁵N addition. The N₂-fixing plant of interest is sampled, together with an adjacent non-N₂-fixing plant (e.g. grass) whose ¹⁵N enrichment represents that of soil nitrogen. ¹⁵N enrichment in digested plant material and soil is analysed isotopically in a mass spectrometer and contribution of biological N₂ fixation calculated.

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Figure 4.50  Basis of the natural abundance method for assessing the contribution of N₂ fixation to legume nutrition. This method entails measuring plant ¹⁵N/¹⁴N ratio by mass spectrometry. Natural differences in ¹⁵N/¹⁴N ratio between soil and atmospheric nitrogen are exploited. Legumes to the left and right of the figure each have a unique source of nitrogen, while a test plant in the middle relies on both fixed nitrogen and soil inorganic nitrogen. Plants (left) denied a source of inorganic nitrogen (e.g. nitrate) fix atmospheric nitrogen and therefore have low ¹⁵N/¹⁴N ratios. Plants without nodules (right) take up only soil-derived nitrogen and are enriched with ¹⁵N (high ¹⁵N/¹⁴N ratios). ¹⁵N 'signatures' of these two sets of plants can be used to estimate the relative contributions of soil and atmospheric nitrogen as nitrogen sources in the test plant, and therefore to assess the significance of N₂ fixation. (Based on Peoples et al. 1989; reproduced with permission of ACIAR)

A typical ‘good’ rate of fixation for a (non-irrigated) field of subtropical legumes in northern Australia is c. 60–100 kg N ha^–1 year^–1. About the same amount of nitrogen is harvested as seed from a crop of cowpea, soybean or chickpea, so growing these legumes does not add net nitrogen to the soil; it does, however, spare nitrogen which would otherwise be removed at harvest. Irrigated legume-based pastures in temperate Australia or New Zealand fix 250–300 kg N ha^–1 year^–1 and make a substantial contribution to the low energy costs of agriculture in these regions. Selection of appropriate biological N₂ fixers could greatly improve N₂ fixation in tropical legume crops.