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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]

Soil is an extremely complex and dynamic matrix, in part, due to the wide diversity of organisms living within it. Soil organic matter (SOM) is the fundamental substrate on which the delivery of ecosystem services depends, providing the metabolic fuel to drive soil function. As such, studying the soil metabolome (the diversity and concentration of low molecular weight metabolites), as a subset of SOM, holds the potential to greatly expand our understanding of the behaviour, fate, interaction and functional significance of small organic molecules in soil. Encompassing a wide range of chemical classes (including amino acids, peptides, lipids and carbohydrates) and a large number of individual molecules (ca. n = 105 to 106), the metabolome is a resultant (indirect) output of several layers of a biological hierarchy, namely the metagenome, metatranscriptome and metaproteome. As such, it may also provide support and validation for these "multi-omics" datasets. We present a case for the increased use of untargeted metabolomics in soil biochemistry, particularly for furthering our fundamental understanding of the functions driving SOM composition and biogeochemical cycling. Further, we discuss the scale of the challenge in terms of metabolite extraction, analysis and interpretation in complex plant-soil-microbial systems. Lastly, we highlight key knowledge gaps which currently limit our use of metabolomic approaches to better understand soil processes, including: (i) interpretation of large untargeted metabolomic datasets; (ii) the source, emission and fate of soil-derived volatile organic compounds (VOCs), (iii) assessing temporal fluxes of metabolites, and (iv) monitoring ecological interactions in the rhizosphere. While the application of metabolomics in ecosystem science is still in its relative infancy, its importance in understanding the biochemical system in relation to regulation, management and underpinning the delivery of ecosystem services is key to further elucidating the complex links between organisms, as well as the fundamental ability of the biological community to process and cycle key nutrients. • Soil organic matter is the metabolic fuel that drives soil function. • Untargeted metabolomics offers rich and functional biochemical data. • Discussion of challenges in extraction and analysis of complex substrates. • Emphasis of key knowledge gaps in interpretation and monitoring of soil. [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]

Incorporation of nitrogen (N) into soil microbial protein is central to the soil N cycle to mitigate N losses and support plant N supply. However, the effect of factors, such as water filled pore space (WFPS), which influence inorganic N transformations and losses, and thus microbial incorporation, are only poorly understood. This work aimed to bridge this gap, using compound-specific 15N-stable isotope probing to quantify microbial assimilation into the largest defined soil organic N pool, protein-N. This approach applied differentially 15N-labelled ammonium nitrate (NH 4 NO 3) to an unfertilised UK grassland in a soil mesocosm study over 10 days. The soil microbial community showed a strong preference for NH 4 + over NO 3 −, which varied with WFPS (85% > 55% > 70%). This preference decreased for amino acids further in biosynthetic proximity to the transamination step in amino acid biosynthesis. Combined incorporation of NH 4 + and NO 3 − increased total hydrolysable amino acid-N concentration linked to WFPS (55% ∼ 85% > 70%). Incorporation rates of applied 15N showed the same trend as NH 4 + preference with WFPS (85% > 55% > 70%), which is related to microbial activity and nutrient mobility. Despite differences in incorporation, when normalised to soil available N, incorporation was comparable in the short-term. Mechanistic control of WFPS via assimilation into the largest soil organic N pool is important to mitigate potential positive feedbacks to N losses and support N supply to plants. • Total hydrolysable amino acid concentration increased at 55% and 85% WFPS. • Incorporation into the soil microbial protein pool showed a preference for NH 4 + over NO 3 −. • Preference of amino acids depended on biosynthetic proximity to transamination. • 15N incorporation into soil microbial protein was influenced by WFPS (85% > 55%>70%). [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]

Efficient fertiliser nitrogen (N) management is critical to global food production and ecosystem health. Considering sheep grazing systems as whole ecosystems, and quantifying key ecosystem services provided by the soil microbial community, including plant N supply and N pollution mitigation, is essential in assessments of N use efficiency (NUE). Using a systems approach, we disassembled a low-intensity sheep (>5 ewe ha−1) grazed grassland, dominated by Lolium perenne , into a series of interlinked 15N-tracer experiments in North Wales during a summer growing season to assess fertiliser-N partitioning. 15N was traced into soil microbial protein-N via compound-specific amino acid 15N-stable isotope probing, with subsequent integration to provide a whole-system perspective. Retention of feed-N into sheep was low (11 %), despite high grass 15N-fertiliser uptake (58 %). The majority of grazed-N re-entered the soil N-cycle as excreta (47 % of total 15N) during the peak growing season. Quantifying 15N-assimilation into soil microbial protein (0–15 cm) demonstrated the central role soil microbes occupy in capturing excess fertiliser (16 %) and urinary-N (8 %) of the total 15N-fertiliser applied, thereby reducing N losses and subsequently supporting plant N supply. This approach emphasises how future management of moderate intensity grazing systems should target sheep NUE, alongside the role of the soil microbial community to retain, and later recycle N, for plant supply, optimising essential ecosystem service provisioning. • Interlinked whole ecosystem 15N tracing study for holistic approach • Mass balance of N cycle through grazing system revealed importance of soil retention to reduce losses. • Microbial assimilation of fertiliser and urine is a major fate of applied 15N. [ABSTRACT FROM AUTHOR]

Micro and macroplastics are emerging contaminants in agricultural settings, yet their impact on nitrogen (N) cycling and partitioning in plant-soil-microbial systems is poorly understood. In this mesocosm-scale study, spring barley (Hordeum vulgare L.) was exposed to macro or microplastic produced from low density polyethylene (LDPE) or biodegradable plastic at concentrations equivalent to 1, 10 and 20 years of plastic mulch film use. Partitioning of 15N-labelled fertiliser into plant biomass, soil and leachate yielded a partial mass balance. Soil N partitioning was probed via compound-specific 15N-stable isotope analyses of soil microbial protein. Concentration-dependent decreases in plant 15N uptake occurred with increased leached nitrogen for LDPE microplastic. Assimilation into soil microbial protein was higher for biodegradable plastics, which we associate with early-stage biodegradable plastic degradation. Partitioning of 15N into inorganic soil N pools was affected by LDPE size, with lower assimilation into the microbial protein pool. While microplastics and macroplastics altered soil N cycling, the limited impacts on plant health indicated the threshold for negative effects was not reached at agriculturally relevant concentrations. This study highlights the difference between conventional and biodegradable plastics, and emphasises that the interplay of micro and macroplastics on soil N cycling must be considered in future studies. [Display omitted] • Barley was exposed to biodegradable and LDPE macro and microplastic. • 15N-tracing indicated LDPE microplastic reduced plant 15N uptake due to N losses. • LDPE plastics altered partitioning of 15N within soil N pools. • Biodegradable macro and micro plastic increased microbial N uptake. • Biodegradable and LDPE plastics had differing impacts on plant-soil N partitioning. [ABSTRACT FROM AUTHOR]

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. [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]

Over the last 50 years, the intense use of agricultural plastic in the form of mulch films has led to an accumulation of plastic in soil, creating a legacy of plastic in agricultural fields. Plastic often contains additives, however it is still largely unknown how these compounds affect soil properties, potentially influencing or masking effects of the plastic itself. Therefore, the aim of this study was to investigate the effects of pure plastics of varying sizes and concentrations, to improve our understanding of plastic-only interactions within soil-plant mesocosms. Maize (Zea mays L.) was grown over eight weeks following the addition of micro and macro low-density polyethylene and polypropylene at increasing concentrations (equivalent to 1, 10, 25, and 50 years mulch film use) and the effects of plastic on key soil and plant properties were measured. We found the effect of both macro and microplastic on soil and plant health is negligible in the short-term (1 to <10 years). However, ≥ 10 years of plastic application for both plastic types and sizes resulted in a clear negative effect on plant growth and microbial biomass. This study provides vital insight into the effect of both macro and microplastics on soil and plant properties. [Display omitted] • Crop height and nutrient content decreased with increasing plastic concentration. • Soil moisture content severely reduced with increasing plastic concentration. • Changes in soil & plant properties were more pronounced in microplastic treatments. • Effects of plastic addition are negligible in the short term for low concentrations. • Microbial biomass decreased with increasing plastic concentration. [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]