Abstract: Biological nitrogen fixation (BNF) is an important nitrogen source in terrestrial ecosystems. Accurate estimation of BNF rate is essential to accurately quantify atmospheric nitrogen input to natural ecosystems. ¹⁵N natural abundance is commonly used to measure the BNF in symbiotic and associative nitrogen fixing plants, but are highly dependent on the choice of the reference plants. In contrast, the ¹⁵N isotope labeling technique allows precise determination of BNF rates of symbiotic, free-living, and associative N-fixing types, and surpasses the previous methods in studying plant nitrogen fixation strategies, nitrogen transfer processes, and carbon-nitrogen trading between nodules and hosts. The ¹⁵N isotope dilution method is mainly used for plant nitrogen fixation research. Although the ¹⁵N stable isotope probe technique is technically challenging and expensive, it enables the detection and study of N-fixing microorganisms by labeling DNA or RNA, and provides an effective way for assessing asymbiotic microorganism nitrogen fixation rates. The development of ¹⁵N stable isotope technique provides a strong technical guarantee for biological nitrogen fixation research.

Key words: ¹⁵N isotope, diazotrophs, labeling, microorganism, natural abundance method