Evaluation of isotopic dilution method for measuring N2fixation in Azolla: Comparison with other methods1

Abstract

Abstract 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 a...