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Rationale: Plastic mulch film manages weed growth and moisture loss on the surface of cropping beds. The chemical components of such plastics include polymer(s), additives and non‐intentionally added substances (NIASs). The unknown chemical nature and behaviours of these constituents require investigation due to their potential to add to the anthropogenic chemical burden in the agrifood system. Methods: Solvent extracts of a commercial 15% polylactic acid (PLA)/85% poly(butylene adipate‐co‐terephthalate) mulch film were investigated using gas chromatography–mass spectrometry (GC–MS) with electron ionisation to characterise the additive and NIAS components. The obscurity of some of the NIASs meant their identification was not readily achieved through routine MS library comparisons. As such, the identification of several polymer‐derived compounds required interpretation of the MS data and re‐application of the derived fragmentation patterns with reference to the wider literature. Unknowns were confirmed using commercially available compounds. Results: Unknown NIASs were identified as cyclic oligoesters comprised of the monomeric building blocks of the polymer system. Cyclic structures derived from the monomers of polybutylene adipate (PBA) and polybutylene terephthalate (PBT) fragmented through a primary pathway involving 1,5‐ and 1,3‐H transfers at ester linkages. Characteristic ions at m/z 111, 129, 183 and 201 for PBA‐derived cyclic oligoesters and m/z 104, 132, 149 and 221 for PBT‐derived cyclic oligoesters were assigned in the mass spectra of unknowns. Cyclic oligoesters containing sebacate moieties were also identified, indicating the presence of polybutylene sebacate as an unexpected component of the mulch. Conclusions: Systematic analyses of the sort reported here are valuable for providing alternative approaches for the identification of plastic‐related chemicals. Open publication of MS spectral data is required to build a greater understanding of the mulch film chemical components contributing to the environmental chemical load introduced to agroecosystems. [ABSTRACT FROM AUTHOR]

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 small, with 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 ryegrass. [ABSTRACT FROM AUTHOR]

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 (R 1 NHCONHR 2), 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. Image 1 • Diacetyl monoxime colorimetric method for urea detection is non-specific. • Ureido compounds cross-react and overestimate urea concentration in soil extracts. • Overestimation confirmed using LC-MS, a direct method to detect urea. • Urea concentrations from DAM method should be reported as ureido-N. [ABSTRACT FROM AUTHOR]

Urine patches in grazed systems are hotspots for nitrogen (N) cycling and losses to the wider environment. Retention and subsequent recycling of urinary-N is key to minimise losses and increase ecosystem nitrogen use efficiency. Biosynthesis into the microbial organic N pool is an important N pathway but this has not been directly quantified in a urine patch. Herein, we present the results of a time course experiment using soil mesocosms sown with perennial ryegrass (Lolium perenne L.) and treated with 15N-labelled sheep urine to determine partitioning of the applied N between plant, soil biomass pools and leaching losses following simulated rainfall events. 15N-tracing used bulk and compound-specific 15N-stable isotope probing (SIP) to determine the fate of urinary N. Initial high leaching losses (233 kg N ha−1) were comprised of native soil N, ammonium and nitrate derived from urine by urea hydrolysis and nitrification, respectively. Leaching subsequently decreased whilst uptake into plant biomass and microbial biosynthesis increased during periods of low rainfall. Uptake into above and belowground plant biomass was the largest fate of urinary-15N after 94 d (42%), although assimilation into microbial biomass dominated for ca. 1 month after urine deposition (34%). Compound-specific 15N–SIP of amino acids and amino sugars revealed immobilisation of urinary-N following mineralisation was the dominant pathway for biosynthesis, with incorporation into bacterial organic N pools more rapid than into the fungal biomass. There was also intact utilisation of glycine derived from urine. This study provides clear evidence that direct assimilation of urine-derived N into microbial organic N pools is an important process for retaining N in a urine patch, which will subsequently support plant N supply during microbial turnover. [Display omitted] • 15N-labelled urine was traced into plant, microbial and leaching pools in a grassland mesocosm. • Leaching losses were reduced, and plant uptake increased during periods of low rainfall. • Microbial uptake of urinary-N occurred via mineralisation-immobilisation and intact pathways. • Microbial assimilation was dominated by bacteria over fungi. [ABSTRACT FROM AUTHOR]

Although amino sugars represent a major component of soil organic nitrogen (ON), the assimilation of nitrate (NO 3 −) and ammonium (NH 4 +) 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 15NH 4 + and 15NO 3 −, 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. Image 101300 • 15N-SIP using agriculturally relevant N rates and low 15N-enrichments did not perturb the AS pool. • δ15N values of AS reflected fundamental biochemical differences in assimilation of N-fertilisers. • 15N-incorporated into bacterial AS reflected known fundamental biosynthetic pathways and proximity to 15N- fertilisers. • Bacterial and fungal AS 15N-assimilation trends showed different temporal responses within the SMC. [ABSTRACT FROM AUTHOR]

Biological nitrogen fixation (BNF) performed by diazotrophs is vital to our understanding of ecosystem functions, as plant nitrogen (N) is commonly a limiting nutrient for primary productivity. However, significant limitations have remained in our knowledge of the controls and rates of this process, due to technical difficulties in directly quantifying nitrogen (N 2) fixation rates. To address this, we developed a novel compound-specific 15N-stable isotope probing method involving analysis of acid hydrolysable soil amino acids (AAs) by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) for the quantification of BNF in soils. By analysing 15N-enriched AAs (as N -acetyl, O -isopropyl derivatives), this new approach aimed to provide greater specificity compared to existing methods, and to contribute previously unobtainable quantitative information on the capture and flow of N 2 fixed in soils. Laboratory incubations using 15N 2 gas were carried out on surface peat over 15 days to obtain quantitative measures of N 2 fixation by the microbial community. Longer incubations with the addition of a glucose energy source significantly increased the level of 15N enrichment, i.e. N fixed. The enhanced detection limits of 15N-AAs by GC-C-IRMS, compared to bulk soil δ15N value determinations, was key to assessments of N 2 fixation. Valuable insights into the assimilation pathway of the applied 15N 2 -substrate were revealed; for peat soils, 15N incorporation into glutamate dominated over other AAs. [Display omitted] • Biological N 2 -fixation assayed by 15N–SIP of soil protein amino acids. • Higher specificity achievable compared to bulk 15N methods. • Insights gained into pathways of N 2 assimilation into soil protein pool. • N 2 -fixation assay potentially applicable to all soil types and land-uses. • Potential to link N 2 -fixation rates with gene abundances to inform functionality. [ABSTRACT FROM AUTHOR]

The differential soil microbial assimilation of common nitrogen (N) fertilizer compounds into the soil organic N pool is revealed using novel compound-specific amino acid (AA) 15N-stable isotope probing. The incorporation of fertilizer 15N into individual AAs reflected the known biochemistry of N assimilation-e.g. 15N-labelled ammonium (15NH4+) was assimilated most quickly and to the greatest extent into glutamate. A maximum of 12.9% of applied 15NH4+, or 11.7% of 'retained' 15NH4+ (remaining in the soil) was assimilated into the total hydrolysable AA pool in the Rowden Moor soil. Incorporation was lowest in the Rowden Moor 15N-labelled nitrate (15NO3-) treatment, at 1.7% of applied 15N or 1.6% of retained 15N. Incorporation in the 15NH4+ and 15NO3- treatments in the Winterbourne Abbas soil, and the 15N-urea treatment in both soils was between 4.4% and 6.5% of applied 15N or 5.2% and 6.4% of retained 15N. This represents a key step in greater comprehension of the microbially mediated transformations of fertilizer N to organic N and contributes to a more complete picture of soil N-cycling. The approach also mechanistically links theoretical/pure culture derived biochemical expectations and bulk level fertilizer immobilization studies, bridging these different scales of understanding., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

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 15 N-enriched amino sugars as alditol acetate derivatives prior to application of a novel 15 N 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 δ 15 N value determinations up to δ 15 N t of 469 ± 3.1‰ (where δ 15 N t is the independently measured δ 15 N value) was confirmed when 30 nmol N was injected on-column, with the direction of deviation from δ 15 N t at low sample amount dependent on the 15 N abundance of the analyte. Observed between- and within-run memory effects were significant ( P < 0.007) when a highly enriched standard (469 ± 3.1‰) was run; therefore, analytical run order and variation in 15 N 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 15 N-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.