Although amino sugars represent a major component of soil organic nitrogen (ON), the assimilation of nitrate (NO3 −) and ammonium (NH4 +) into amino sugars (AS) by soil bacteria and fungi represents a neglected aspect of the global N cycle. A deeper knowledge of AS responses to N fertiliser addition may help enhance N use efficiency (NUE) within agricultural systems. Our aim was to extend a sensitive compound-specific 15 N-stable isotope probing (SIP) approach developed for amino acids to investigate the immobilization of inorganic N into a range of amino sugars (muramic acid, glucosamine, galactosamine, mannosamine). Laboratory incubations using 15 N-ammonium and 15 N-nitrate applied at agriculturally relevant rates (190 and 100 kg N ha −1 for 15 NH4 + and 15 NO3 − , respectively) were carried out to obtain quantitative measures of N-assimilation into the AS pool of a grassland soil over a 32-d period. Using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) we found that δ 15 N values for individual AS reflected differences in routing of the applied ammonium and nitrate. The contrasting N-assimilation dynamics of bacterial and fungal communities were demonstrated through determinations of percentage 15 N incorporation into diagnostic AS. Nassimilation dynamics of the bacterial community were altered with the applied substrate whilst fungal N-assimilation dynamics were unaffected. Rates and fluxes of the applied N-substrates into the bacterial AS pool reflected known biosynthetic pathways for AS, with fungal glucosamine appearing to be biosynthetically further from the applied substrates than bacterial glucosamine due to different turnover rates. This sensitive and specific compound-specific 15 N-SIP approach using AS, building on existing approaches with amino acids (AA), enables differentiation of N-assimilation dynamics within the microbial community and assessment of microbial NUE with agriculturally relevant fertilisation rates.
Development of alditol acetate derivatives for the determination of 15 Nenriched amino sugars by gas chromatography-combustion-isotope ratio mass spectrometry
Urea represents a common form of organic nitrogen (N) which is added to agricultural soils in large quantities in both cropping (e.g. fertiliser) and livestock (e.g. urine) systems. In addition, there is a small, dynamic ambient pool of urea in soil associated with metabolic functioning in the microbial community. The diacetyl monoxime (DAM) colorimetric method is routinely used to quantify urea in soil, however, it lacks specificity due to the potential to react with the ureido group (R1NHCONHR2), a common structural moiety in soil organic matter. The aim of this study was therefore to critically evaluate the accuracy of this method for urea determination in soil. Using the DAM assay, we demonstrated significant cross-reactivity with a range of ureido compounds, many of which are ubiquitous in soil. We conclude therefore that the DAM assay is highly likely to overestimate urea concentrations in environmental materials. Such overestimation was confirmed using high resolution HPLC-Orbitrap MS to quantify urea in grassland soils using standard addition and the concentrations compared with those of the DAM assay. The results obtained show the DAM colorimetric method overestimated urea concentration by between 7.2 and 58 times for the sites studied. This significant overestimation of urea emphasises the need to validate the colorimetric method with reference to the LC-MS assay to ensure the robustness of measured urea concentrations. On this basis we recommend that reporting of the results from the DAM colorimetric method as "urea" concentration be curtailed and reported as "ureido-N" to recognise the contribution of unknown and variable contributions from other compounds. Indeed, given the problems with quantitatively assessing the latter contributions we would recommend the DAM method is now avoided in surveys of urea concentrations in soil and the wider environment.
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