¹⁵N-amino sugar stable isotope probing (¹⁵N-SIP) to trace the assimilation of fertiliser-N by soil bacterial and fungal communities

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 15N-stable isotope probing (SIP) approach developed for amino acids (AAs) to investigate the immobilization of inorganic N into a range of amino sugars (muramic acid, glucosamine, galactosamine, mannosamine). Laboratory incubations using 15N-ammonium and 15N-nitrate applied at agriculturally relevant rates (190 and 100 kg N ha−1 for 15NH4+ and 15NO3−, 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 δ15N 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 15N incorporation into diagnostic AS. N-assimilation 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 15N-SIP approach using AS, building on existing approaches with AAs, 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 15N-Enriched Amino Sugars by Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry

Amino sugars can be used as indices to evaluate the role of soil microorganisms in active nitrogen (N) cycling in soil. This paper details the assessment of the suitability of gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) for the analysis of ¹⁵N-enriched amino sugars as alditol acetate derivatives prior to application of a novel ¹⁵N stable isotope probing (SIP) approach to amino sugars. The efficient derivatization and cleanup of alditol acetate derivatives for GC was achieved using commercially available amino sugars, including glucosamine, mannosamine, galactosamine, and muramic acid, as laboratory standards. A VF-23ms stationary phase was found to produce optimal separations of all four compounds. The structure of the alditol acetate derivatives was confirmed using gas chromatography/mass spectrometry (GC/MS). For GC–C–IRMS determinations, implementation of a two-point normalization confirmed the optimal carrier gas flow rate to be 1.7 mL min^–1. Linearity of δ¹⁵N value determinations up to δ¹⁵N_t of 469 ± 3.1‰ (where δ¹⁵N_t is the independently measured δ¹⁵N value) was confirmed when 30 nmol N was injected on-column, with the direction of deviation from δ¹⁵N_t at low sample amount dependent on the ¹⁵N abundance of the analyte. Observed between- and within-run memory effects were significant (<i>P</i> < 0.007) when a highly enriched standard (469 ± 3.1‰) was run; therefore, analytical run order and variation in ¹⁵N enrichment of analytes within the same sample must be considered. The investigated parameters have confirmed the isotopic robustness of alditol acetate derivatives of amino sugars for the GC–C–IRMS analysis of ¹⁵N-enriched amino sugars in terms of linearity over an enrichment range (natural abundance to 469 ± 3.1‰) with on-column analyte amount over 30 nmol N

Tracing carbon and nitrogen microbial assimilation in suspended particles in freshwaters

The dynamic interactions between dissolved organic matter (DOM) and particulate organic matter (POM) are central in nutrient cycling in freshwater ecosystems. However, the molecular-level mechanisms of such interactions are still poorly defined. Here, we study spatial differences in the chemical (i.e., individual proteinaceous amino acids) and microbial (i.e., 16S rRNA) composition of suspended sediments in the River Chew, UK. We then applied a compound-specific stable isotope probing (SIP) approach to test the potential assimilation of 13C,15N-glutamate (Glu) and 15N-NO3− into proteinaceous biomass by particle-associated microbial communities over a 72-h period. Our results demonstrate that the composition of suspended particles is strongly influenced by the effluent of sewage treatment works. Fluxes and percentages of assimilation of both isotopically labelled substrates into individual proteinaceous amino acids showed contrasting dynamics in processing at each site linked to primary biosynthetic metabolic pathways. Preferential assimilation of the organic molecule glutamate and evidence of its direct assimilation into newly synthesised biomass was obtained. Our approach provides quantitative molecular information on the mechanisms by which low molecular weight DOM is mineralised in the water column compared to an inorganic substrate. This is paramount for better understanding the processing and fate of organic matter in aquatic ecosystems

Mechanisms of nitrogen transfer in a model clover-ryegrass pasture: a 15N-tracer approach

Purpose Nitrogen (N) transfer from white clover (Trifolium repens cv.) to ryegrass (Lolium perenne cv.) has the potential to meet ryegrass N requirements. This study aimed to quantify N transfer in a mixed pasture and investigate the influence of the microbial community and land management on N transfer. Methods Split root 15N-labelling of clover quantified N transfer to ryegrass via exudation, microbial assimilation, decomposition, defoliation and soil biota. Incorporation into the microbial protein pool was determined using compound-specific 15N-stable isotope probing approaches. Results N transfer to ryegrass and soil microbial protein in the model system was relatively smallwith one-third arising from root exudation. N transfer to ryegrass increased with no microbial competition but soil microbes also increased N transfer via shoot decomposition. Addition of mycorrhizal fungi did not alter N transfer, due to the source-sink nature of this pathway, whilst weevil grazing on roots decreased microbial N transfer. N transfer was bidirectional, and comparable on a short-term scale. Conclusions N transfer was low in a model young pasture established from soil from a permanent grassland with long-term N fertilisation. Root exudation and decomposition were major N transfer pathways. N transfer was influenced by soil biota (weevils, mycorrhizae) and land management (e.g. grazing). Previous land management and the role of the microbial community in N transfer must be considered when determining the potential for N transfer to ryegras