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 (N-2) fixation rates. To address this, we developed a novel compound-specific N-15-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 N-15 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 N-2 fixed in soils. Laboratory incubations using N-15(2) gas were carried out on surface peat over 15 days to obtain quantitative measures of N-2 fixation by the microbial community. Longer incubations with the addition of a glucose energy source significantly increased the level of N-15 enrichment, i.e. N fixed. The enhanced detection limits of N-15-AAs by GC-C-IRMS, compared to bulk soil delta N-15 value determinations, was key to assessments of N-2 fixation. Valuable insights into the assimilation pathway of the applied N-15(2)-substrate were revealed; for peat soils, N-15 incorporation into glutamate dominated over other AAs.