Compound-specific amino acid 15N-stable isotope probing for the quantification of biological nitrogen fixation in soils
2022
Chiewattanakul, Mashita | McAleer, Adam D.A. | Reay, Michaela K. | Griffiths, Robert I. | Buss, Heather L. | Evershed, Richard P.
Biological nitrogen fixation (BNF) performed by diazotrophs is vital to our understanding of ecosystem functions, as plant nitrogen (N) is commonly a limiting nutrient for primary productivity. However, significant limitations have remained in our knowledge of the controls and rates of this process, due to technical difficulties in directly quantifying nitrogen (N₂) fixation rates. To address this, we developed a novel compound-specific ¹⁵N-stable isotope probing method involving analysis of acid hydrolysable soil amino acids (AAs) by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) for the quantification of BNF in soils. By analysing ¹⁵N-enriched AAs (as N-acetyl, O-isopropyl derivatives), this new approach aimed to provide greater specificity compared to existing methods, and to contribute previously unobtainable quantitative information on the capture and flow of N₂ fixed in soils. Laboratory incubations using ¹⁵N₂ gas were carried out on surface peat over 15 days to obtain quantitative measures of N₂ fixation by the microbial community. Longer incubations with the addition of a glucose energy source significantly increased the level of ¹⁵N enrichment, i.e. N fixed. The enhanced detection limits of ¹⁵N-AAs by GC-C-IRMS, compared to bulk soil δ¹⁵N value determinations, was key to assessments of N₂ fixation. Valuable insights into the assimilation pathway of the applied ¹⁵N₂-substrate were revealed; for peat soils, ¹⁵N incorporation into glutamate dominated over other AAs.
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