This paper reviews a growing body of literature on the use of variations in the natural abundance of 15N to estimate the fractional contribution of N2-fixation to N2-fixing systems. This method is based on the small difference in 15NN abundance which frequently occurs between N derived from N2-fixation and N derived from other sources. The requirement of the method is that this difference be significant. Whether this requirement is met is site specific and must be empirically established at each site of interest. Advantages and disadvantages of this method are compared with those of more conventional methods. Sources of error, including heterogeneity of 15NN abundance of non-atmospheric N sources are considered. Tests of the method, under both greenhouse and field conditions, are described. Estimates based on this method compare favourably with other methods for field evaluation of N2-fixation, provided that the site and the sampling strategy are appropriate for application of the method. Applications of the method in several ecosystems are described.

N2-Fixation in Field Settings: Estimations Based on Natural 15N Abundance

A comprehensive report of the acetylene reduction assay for measurement of N2 fixation is presented. The objective is to facilitate the effective use and identify some potential limitations of the method. The report is based on more than 200 accounts of the use of this technique in 15 countries during the last 5 years. Methods of measurement of N2 fixation are compared. Nomenclature, e.g., N2[C2H2] fixed, is introduced to identify values of N2 fixation determined by C2H2-C2H2 assay. The biochemical basis of the assay is described along with relevant characteristics including Km, C2H2/N2 conversion factor, and specific N2[C2H2]-fixing activities obtained with various systems. Effects of combined nitrogen, temperature, light, pO2, N2, pC2H2 and water on activity are summarized. Available methods for sample preparation, assay chamber, gas phase, assay condition, termination of reaction, C2H4 analysis and expression of results are compared. The many uses of the C2H2-C2H4 assay for investigations of the biochemistry of nitrogenase and physiology of N2-fixing organisms, definition of N2-fixing organisms and measurement of field N2 fixation by legume, non-legume, soil, marine, rhizosphere, phylloplane and mammalian samples are tabulated.

Applications of the acetylene-ethylene assay for measurement of nitrogen fixation

Oxygen relations of nitrogen fixation in cyanobacteria.

Measuring plant-associated nitrogen fixation in agricultural systems

Observations on both attached nodulated roots and detached nodules have revealed that nitrogenase activity in many legume species declined rapidly in the presence of acetylene, with a concurrent reduction in respiration. The reduction began within a few minutes of exposure to acetylene and continued for 30^60 min before a new steady-state was attained. A similar decline in H2 evolution and respiration was observed when N2 was replaced with argon or helium. This suggests that the decrease is linked to the cessation of ammonia production. Measurements of 15N2 uptake demonstrated that it is the pre-decline rather than final rate of ethylene production which represents the real rate of nitrogenase activity. The implications of these findings for the interpretation of acetylene reduction and hydrogen evolution data are considered.

A Major Error in the Acetylene Reduction Assay: Decreases in Nodular Nitrogenase Activity Under Assay Conditions

Summary
Acetylene reduction by soybean nodules started without a lag and remained linear with time for about 80 minutes. There was no apparent injury to nodules from evacuation. Rates of reduction of acetylene increased with increasing pO2 to 1 atm; respiration did not seriously deplete the available O2 from bottles in which nodules occupied less than 20% of the volume of the bottles. Reduction of acetylene decreased markedly in nodules soaked in water. About 0.2 atm acetylene supported the most rapid reduction of acetylene, and nodules 3–5 mm in diameter were most active. Intact soybean plants, nodulated roots and detached nodules fixed nitrogen at relative rates of approximately 100/46/23. The soybean plants exhibited a diurnal cycle of acetylene reduction dependent upon light intensity and air temperature. The observed ratio of acetylene reduced to N2 reduced was approximately 2.0 rather than the often assumed theoretical value of 3.0. There was a dramatic difference among soybean varieties in the date of onset and termination of acetylene reduction. Acetylene reduction was most vigorous in plants inoculated with a mixture of effective strains of Rhizobium japonicum.

https://nph.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1469-8137.1972.tb04074.x

Reduction of acetylene and nitrogen by field-grown soybeans

Summary
Nitrogen fixation in various Wisconsin lakes was compared using the C2H2 reduction technique. Rates of C2H2 reduction were unaffected by N2 when sufficient C2H2 was added to the gas phase. Thus in field studies the natural gas phase need not be removed before the addition of C2H2. C2H2 was reduced rapidly in eutrophic waters (Little Arbor Vitae Lake, Lake Mendota and southern Green Bay) but more slowly in oligotrophic waters (Crystal Lake, Trout Lake, Lake Mary and northern Green Bay). C2H2 reduction was greatest in the surface waters, was largely light-dependent and showed a marked diurnal variation. There was also significant variation with area and with time, and in general acetylene reduction was detected only when heterocystous algae were present. The rate of nitrogen fixation in Lake Mendota, calculated on the basis of weekly measurements taken during most of the ice-free season, was approximately 2.4 kg/ha/year.

https://nph.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1469-8137.1971.tb02551.x

T. H. Mague

Papers

Reduction of acetylene and nitrogen by field-grown soybeans

Nitrogenase activity in wisconsin lakes of differing degrees of eutrophication