Table 6.

Different methods used to study BNF by N₂-fixing systems under controlled-conditions or in the field^a.

Method	Principle	Advantages	Disadvantages	Extent of use
15N2 feeding	Intact or detached plants roots and/or nodules, or soil placed in a chamber with an atmosphere enriched in 15N2. The amount of 15N accumulated at the end of a period of incubation provides a direct measure of the rate of N2 fixation	Direct measure of N2 fixation The only technique apart from growing plants in N-free medium under controlled-conditions to unequivocally prove active N2 fixation	High cost of 15N2 gas Requires high-level technical skills Measurements reflect nitrogenase activity only for the duration of assay Can’t distinguish between N2 fixed by free-living diazotrophs in the soil, or on the external surface of plants, from that occurring within the plant Can be difficulties keeping incubation systems completely sealed while maintaining suitable environmental conditions (e.g. temperature and O2 levels) inside the chamber Difficult to use under field conditions Errors can arise due to the contamination of15N2 gas with traces of other 15N-compounds that can be assimilated by microbes or plants Not suitable for long-term determinations of BNF	Limited use because of the logistical difficulties
Acetylene reduction	The enzyme nitrogenase, which reduces N2 to NH3 is also capable of reducing acetylene (C2H2) to ethylene (C2H4). If roots, nodules, or soil are placed in an airtight vessel or contained within a cuvette connected to a flowing gas-stream, and then exposed to a C2H2 enriched atmosphere, the accumulation of C2H4 over a period of assay is used to provide an indirect assessment of the rate of BNF	Sensitive diagnostic tool for detecting nitrogenase activity Simple, rapid, and relatively inexpensive, and many measurements can be undertaken daily	Requires a gas chromatograph to quantify the concentration of C2H4 in gas samples C2H2 is explosive and poses a possible hazard Establishment of flow-through gas exchange to monitor intact systems is extremely difficult Difficult to use under field conditions Commonly applied to detached roots (or nodules) rather than whole root systems, so total BNF will be underestimated Errors due to changes in gas exchange induced by disturbance of the N2-fixing system for assay Uncertainties about the appropriate conversion ratio to apply to calculate the amount of N fixed from C2H2 reduction data. Ideally should be calibrated with15N2 Provides only a short-term estimate of BNF. Multiple, repeated measurements are required to monitor BNF over a growing season The underlying assumptions that substituting C2H2 for N2 does not affect nitrogenase activity, and that measures obtained under assay conditions are related to BNF rates in situ do not hold for legume nodules	Considered unreliable for nodulated legumes, but still used to assess BNF by non-symbiotic systems and free-living diazotrophs
Hydrogen evolution	Hydrogen (H2) gas is an obligate by-product of BNF in legume nodules. An indirect measure of nitrogenase activity can be obtained by placing a nodulated legume root system in a cuvetter and monitoring the increase in H2 concentration in a gas-stream	Sensitive diagnostic tool for detecting nitrogenase activity Measurements of H2 evolution in air do not inhibit nitrogenase activity so repeated assays can be performed on the same plant material Simple, rapid, and inexpensive	Requires a gas chromatograph or H2-electrode to quantify the concentration of H2 in gas samples H2 evolution in air measures only represents a portion of total electron flux through nitrogenase Some rhizobial strains form symbioses that have an active hydrogenase uptake enzyme that recycles H2 within the nodule, so no H2 will be detected despite BNF occurring To measure total nitrogenase activity it is necessary to incubate nodulated roots in the absence of N2 (e.g. argon:oxygen) rather than in air Difficulties in establishing flow-through gas exchange systems to monitor roots of intact plants Difficult to use under field conditions Commonly applied to detached nodulated roots (or nodules) rather than whole root systems, so total BNF will be underestimated	Predominantly applied to nodulated legumes in the laboratory or in controlled-environment experiments. Potential use in non-legume systems largely unexplored
Sap-ureide	The forms of N transported in the xylem stream from N2-fixing nodules (ureides) differs from soil N assimilated by roots (amino acids and NO3) in some legume species. Consequently, analysis of the N-solute composition in xylem sap (or plant tissue) can be used to assess the percentage of plant N derived from atmospheric N2 (%Ndfa)	Rapid and involves simple colorimetric assay of either xylem sap or tissue extract in a test tube Not technically difficult No special experimental design required Suitable for well-watered experimental and farmers’ legume crops	Restricted to ureide-exporting legume crops including soybean, pigeon pea, Vigna and Phaseolus species Requires calibration with another method such as15N-isotope dilution Calibration relationships are crop specific and may change with growth stage Provides only a short-term estimate of %Ndfa, so multiple, repeated measurements usually required over the duration of the growing season	Can only be applied to certain sub-tropical legume species Not suitable for temperate legumes
15N isotope-dilution	If the15N concentration in atmospheric N2 differs significantly from that of plant-available soil N, %Ndfa can be calculated from a comparison of15N composition of a N2-fixing plant-based system with a non-N2-fixing reference plant(s) Assumes reference plant provides a surrogate measure of the15N signature of the same plant-available soil N pool used by N2-fixing plant	Provides a “time-integrated” estimate of %Ndfa over the period of growth Amounts of N2 fixed can be estimated from a single analysis of plant material for15N and %N contents If15N natural abundance of soil N is sufficiently high and uniform, can be applied to both experimental and farmers’ crops	Requires non-N2 fixing reference plants ideally with similar rooting depths and patterns of N uptake to that of N2-fixing plant Prone to errors if15N composition of plant-available soil N changes markedly with soil depth or with time during the growing season High cost of15N-enriched materials if they are used to expand the difference between the15N composition of soil mineral N and atmospheric N2 With15N natural abundance there is a need to account for isotopic-fractionation that results in a slight depletion of15N in shoots of legumes fully dependent upon BNF for growth when calculating %Ndfa. 15N natural abundance cannot be used to estimat%Ndfa of nodulated roots as isotopic-fractionation results in15N accumulation in nodules	Widely used in both non-symbiotic and symbiotic N2-fixing systems
N-difference	Nitrogen difference compares legume accumulation of N with that of a neighboring non N2-fixing crop or plant over a single growing season. The difference in N between the two is assumed to be due to N2 fixation	Simple, low-cost method that can be applied when facilities for total N analyses are available	Calculations are highly dependent on the accuracy of sampling for the determination of plant biomass and sub-sampling for %N analysis Errors can arise if the amount of soil mineral N accumulated by the non N2-fixing control plant differs markedly from that of the N2 -fixing plant Most reliable in low soil N fertility soils when BNF is high Not suitable for measurement of non-symbiotic BNF because of difficulties in quantifying low levels of N2 fixation	Widely used in legume systems
N-accretion or N balance	All possible external inputs (fertilizer, manures, wet and dry deposition, N in irrigation water, ammonia absorportion by leaves) and outputs of N (N removed in plant or animal products, leaching, run-off and erosion, volatilization, denitrification) need to be accounted for, and incremental changes in soil N quantified in the system under study. If a net positive total N balance is calculated to occur between two points in time, then the N gain this is attributed to inputs of fixed N2	Can potentially be applied to experiments and farmers’ fields	N outputs through various loss processes are difficult and complex to quantify and often rely on assumptions rather than actual measurements Quantification of some N inputs (e.g. atmospheric deposition of N) can be challenging Quantification of changes in soil N pool is subject to large errors, substantial inputs from BNF are necessary to reliably quantify any increase in soil N As the method relies on many independent and unrelated measurements, each made with differing degrees of accuracy, the confidence in the final estimate of BNF can be low	Use is limited to long-term studies for both non-symbiotic and symbitoic N2-fixing systems

^aAdapted and updated from information provided by Unkvoich et al. (2008); Peoples et al. (2009b); Chalk et al. (2017); Soper et al. (2021).