Compound-specific 15 N 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 ( 15 N-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 δ 15 N values of individual amino acids (AAs; determined as N -acetyl, O -isopropyl derivatives) as proxies of biomass protein production. The δ 15 N values are used together with AA concentrations to quantify N assimilation of 15 N-labelled substrates by the soil microbial biomass. The utility of the approach is demonstrated through incubation experiments using inorganic 15 N-labelled substrates ammonium ( 15 NH 4 +) and nitrate ( 15 NO 3 -) and an organic 15 N-labelled substrate, glutamic acid ( 15 N-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 δ 15 N values were highly sensitive to N assimilation, providing qualitative and quantitative insights into the cycling and fate of the applied 15 N-labelled substrates. The utility of this 15 N-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.