SEALED-TUBE COMBUSTION FOR NATURAL N-15 ABUNDANCE ESTIMATION OF N2 FIXATION AND APPLICATION TO SUPERNODULATING SOYBEAN MUTANTS

Measurement of biological N2 fixation by the natural N-15 abundance technique is based on the fact that soil N is usually enriched in N-15 compared with atmospheric N2. The technique has been limited by the care required to prevent isotope fractionation in the Kjeldahl method. We describe a sealed-tube combustion technique that reduces isotope fractionation problems. Plant samples were combusted at 850-degrees-C in Vycor tubes with Cu, CuO, CaO, and Ag foil, and N2 gas was admitted directly into the mass spectrometer. A delta-N-15 value of 5.43 +/- 0.01 (SE, n = 11) was obtained for a nonnodulating soybean seed sample. This method was used to estimate N2 fixation by soybean cv. Bragg and three supernodulating mutants, nts246, nts382, and nts1007, in the field. Average percent N derived from the atmosphere was 70 +/- 4% in vegetative parts at physiological maturity and 72 +/- 2% in the grain at harvest. No significant differences were found. The N harvest index averaged 77%; thus, despite the high percent N derived from the atmosphere, these soybean crops would have been net exporters of soil N. The natural N-15 abundance method consistently gave estimates of N2 fixation that were 60-90 kg N ha-1 higher than the N difference method.