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 conceptual 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 ¹⁵N-ammonium, ¹⁵N-nitrate and ¹⁵N-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 nitrogen-15 stable isotope probing (¹⁵N-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 ¹⁵N-labelled substrate by the soil microbial biomass and an estimate of newly synthesized soil protein.

RESULTS: The cycling of agriculturally relevant N additions is undetectable via bulk soil N content and δ¹⁵N measurements and AA concentrations. The assimilation pathways of the three substrates were revealed via patterns in AA δ¹⁵N 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 ¹⁵N-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 more widely applicable to the study of N cycling in any soil, or indeed, in any complex ecosystem.