Compound-specific amino acid ¹⁵N stable isotope probing of nitrogen assimilation by the soil microbial biomass using gas chromatography/combustion/isotope ratio mass spectrometry

Abstract

RATIONALE: Organic nitrogen (N) greatly exceeds inorganic N in soils, but the complexity and heterogeneity of this important soil N pool make investigations into the fate of N‐containing additions and soil organic N cycling challenging. This paper details a novel approach to investigate the fate of applied N in soils, generating quantitative measures of microbial assimilation and of newly synthesized soil protein. METHODS: Laboratory incubation experiments applying (15)N‐ammonium, (15)N‐nitrate and (15)N‐glutamate were carried out and the high sensitivity and selectivity of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) exploited for compound‐specific (15)N stable isotope probing ((15)N‐SIP) of extracted incubation soil amino acids (AAs; as N‐acetyl, O‐isopropyl derivatives). We then describe the interpretation of these data to obtain a measure of the assimilation of the applied (15)N‐labelled substrate by the soil microbial biomass and an estimate of newly synthesised soil protein. RESULTS: The cycling of agriculturally relevant N additions is undetectable via bulk soil N content and δ (15)N values and AA concentrations. The assimilation pathways of the three substrates were revealed via patterns in AA δ (15)N values with time, reflecting known biosynthetic pathways (e.g. ammonium uptake occurs first via glutamate) and these data were used to expose differences in the rates and fluxes of the applied N substrates into the soil protein pool (glutamate > ammonium > nitrate). CONCLUSIONS: Our compound‐specific (15)N‐SIP approach using GC/C/IRMS offers a number of insights, inaccessible via existing techniques, into the fate of applied (15)N in soils and is potentially widely applicable to the study of N cycling in any soil, or indeed, in any complex ecosystem. © 2016 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd