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

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 N-ammonium, N-nitrate and N-glutamate were carried out and the high sensitivity and selectivity of gas chromatography/combustion/isotope ratiomass spectrometry (GC/C/IRMS) exploited for compound-specific N stable isotope probing (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 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 δN values and AA concentrations. The assimilation pathways of the three substrates were revealed via patterns in AA δN values with time, reflecting knownbiosynthetic pathways (e.g. ammoniumuptake occursfirst via glutamate) and these datawere 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 N-SIP approach using GC/C/IRMS offers a number of insights, inaccessible via existing techniques, into the fate of applied 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.