Sealed-tube combustion for natural 15N abundance estimation of N2 fixation and application to supernodulating soybean mutants

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Abstract
Measurement of biological N2 fixation by the natural 15N abundance technique is based on the fact that soil N is usually enriched in 15N 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 °C in Vycor tubes with Cu, CuO, CaO, and Ag foil, and N2 gas was admitted directly into the mass spectrometer. A δ15N 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 15N abundance method consistently gave estimates of N2 fixation that were 60–90 kg N ha−1 higher than the N difference method. Key words: sealed-tube combustion, natural 15N abundance, supernodulating soybean mutants, biological N2 fixation.

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