Direct Raman Spectroscopic Measurements of Biological Nitrogen Fixation under Natural Conditions: An Analytical Approach for Studying Nitrogenase Activity

Biological N 2 fixation is a major input of bioavailable nitrogen, which represents the most frequent factor limiting the agricultural production throughout the world. Especially, the symbiotic association between legumes and Rhizobium bacteria can provide substantial amounts of nitrogen (N) and reduce the need for industrial fertilizers. Despite its importance in the global N cycle, rates of biological nitrogen fixation have proven difficult to quantify. In this work, we propose and demonstrate a simple analytical approach to measure biological N 2 fixation rates directly without a proxy or isotopic labeling. We determined a mean N 2 fixation rate of 78 ± 5 μmol N 2 (g dry weight nodule) -1 h -1 of a Medicago sativa-Rhizobium consortium by continuously analyzing the amount of atmospheric N 2 in static environmental chambers with Raman gas spectroscopy. By simultaneously analyzing the CO 2 uptake and photosynthetic plant activity, we think that a minimum CO 2 mixing ratio might be needed for natural N 2 fixation and only used the time interval above this minimum CO 2 mixing ratio for N 2 fixation rate calculations. The proposed approach relies only on noninvasive measurements of the gas phase and, given its simplicity, indicates the potential to estimate biological nitrogen fixation of legume symbioses not only in laboratory experiments. The same methods can presumably also be used to detect N 2 fluxes by denitrification from ecosystems to the atmosphere.