Evaluation of isotopic dilution method for measuring N2 fixation in Azolla: Comparison with other methods 1

An isotopic dilution method that overcomes the drawbacks of commonly used methods for measuring N₂ fixation by aquatic N‐fixers such as Azolla pinnata‐Anabaena azollae association (Azolla) is presented. The method was compared with ¹⁵N₂ gas (while maintaining CO₂) and the difference methods of measuring N₂ fixation. The isotopic dilution method was used for two conditions: a. For ¹⁵N‐free growth medium, Azolla was pre‐enriched with ¹⁵N, and N₂ fixation was determined by measuring the dilution of ¹⁵N in the tissue. b. For the growth medium containing N, N₂ fixation was determined by providing ¹⁵N enriched ammonium sulfate in the growth medium and measuring ¹⁵N to ¹⁴N ratio in the tissue. An airtight chamber, necessary for ¹⁵N₂ gas and acetylene reduction methods, was not representative of the growing environment of Azolla. Temperature in the airtight chamber was far from uniform and CO₂ was rapidly depleted. The isotopic dilution method is simpler, relatively inexpensive, subject to fewer errors and applicable to more diverse conditions, and yet was as accurate as ¹⁵N₂‐gas method.