Compound-specific ¹⁵N stable isotope probing of N assimilation by the soil microbial biomass: a new methodological paradigm in soil N cycling

Abstract. A compound-specific nitrogen-15 stable isotope probing (¹⁵N-SIP) technique is described which allows investigation of the fate of inorganic- or organic-N amendments to soils. The technique uses gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) to determine the δ¹⁵N values of individual amino acids (AAs; determined as N-acetyl, O-isopropyl derivatives) as proxies of biomass protein production. The δ¹⁵N values are used together with AA concentrations to quantify N assimilation of ¹⁵N-labelled substrates by the soil microbial biomass. The utility of the approach is demonstrated through incubation experiments using inorganic ¹⁵N-labelled substrates ammonium (¹⁵NH₄⁺) and nitrate (¹⁵NO₃^-) and an organic ¹⁵N-labelled substrate, glutamic acid (¹⁵N-Glu). Assimilation of all the applied substrates was undetectable based on bulk soil properties, i.e. % total N (% TN), bulk soil N isotope composition and AA concentrations, all of which remained relatively constant throughout the incubation experiments. In contrast, compound-specific AA δ¹⁵N values were highly sensitive to N assimilation, providing qualitative and quantitative insights into the cycling and fate of the applied ¹⁵N-labelled substrates. The utility of this ¹⁵N-AA-SIP technique is considered in relation to other currently available methods for investigating the microbially-mediated assimilation of nitrogenous substrates into the soil organic N pool. This approach will be generally applicable to the study of N cycling in any soil, or indeed, in any complex ecosystem.