¹⁵N methodologies for quantifying the response of N₂-fixing associations to elevated [CO₂]: A review

Methodologies based on ¹⁵N enrichment (E) and ¹⁵N natural abundance (NA) have been used to obtain quantitative estimates of the response of biological N₂ fixation (BNF) of legumes (woody, grain and forage) and actinorhizal plants grown in artificial media or in soil exposed to elevated atmospheric concentrations of carbon dioxide e[CO₂] for extended periods of time, in growth rooms, greenhouses, open top chambers or free-air CO₂ enrichment (FACE) facilities. ¹⁵N₂ has also been used to quantify the response of endophytic and free-living diazotrophs to e[CO₂]. The primary criterion of response was the proportional dependence of the N₂-fixing system on the atmosphere as a source of N. i.e. the symbiotic dependence (P_atm). The unique feature of ¹⁵N-based methods is their ability to provide time-integrated and yield-independent estimates of P_atm. In studies conducted in artificial media or in soil using the E methodology there was either no response or a positive response of P_atm to e[CO₂]. The interpretation of results obtained in artificial media or with ¹⁵N₂ is straight forward, not being subject to the assumptions on which the E and NA soil-cultured methods are based. A variety of methods have been used to estimate isotopic fractionation attendant on the NA technique, the so-called 'B value', which attaches a degree of uncertainty to the results obtained. Using the NA technique, a suite of responses of P_atm to e[CO₂] has been published, from positive to neutral to sometimes negative effects. Several factors which interact with the response of N₂-fixing species to e[CO₂] were identified.