Compound-specific amino acid 15N-stable isotope probing for the quantification of biological nitrogen fixation in soils

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.