Assessment of biological nitrogen fixation

The four commonly used methods for measuring biological nitrogen fixation (BNF) in plants are: the total nitrogen difference (TND) method, acetylene reduction assay (ARA) technique, xylem-solute (or ureide production) method and the use of N-15 labelled compounds. The TND method relies on a control non-N-2-fixing plant to estimate the amount of N absorbed by the fixing plant from soil. It is one of the simplest and least expensive methods, but works best under low soil N conditions. The ARA technique measures the rate of acetylene conversion to ethylene by the N-2-fixing enzyme, nitrogenase. The ethylene produced can then be converted into N-2 fixed, using a conversion ratio, originally recommended as 3. Although the method is inexpensive and highly sensitive, its major disadvantages are, the short-term nature of the assays, the doubtful validity of always using a conversion ratio of 3 and the auto-inhibition of acetylene conversion to ethylene. The ARA technique is therefore not a method of choice for measuring BNF. The xylem-solute technique can be used to measure BNF for those species that produce significant quantities of ureide as product of BNF Although simple and relatively inexpensive, it is an instantaneous assay and also needs to be calibrated against a known method. The most serious limitation is, that only a small proportion of N-2-fixing plants examined are ureide exporters, and the method is therefore not widely applicable. The N-15 methods, classified into the isotope dilution and A-value methods, appear to be the most accurate, but also the most expensive. They involve labelling soil with N-15 fertilizer and using a non-N-2-fixing reference plant to measure the N-15/N-14 ratio in the soil. The N-15 isotope dilution approach is both operationally and mathematically simpler than the A-value approach. To limit potential errors in the selection of reference crops, it is recommended to use N-15 labelled compounds or soil labelling methods that result in the slow release of N-15 or the slow decline of N-15/N-14 ratio in the soil. Additionally, the use of several reference plants rather than a single one can improve the accuracy of the results.