15N Site Research Articles

Unveiling riverine N2O dynamics along urbanization gradients by integrating hydrological, biogeochemical and microbial processes

Human-disturbed rivers are globally significant sources of atmospheric nitrous oxide (N2O). Yet, the underlying mechanisms of urbanization impact on riverine N2O dynamics are not well understood. This study unveiled the effects of urbanization on N2O dynamics by integrating hydrological, biogeochemical and microbial processes in a river with various urbanization intensities. Riverine NO3− concentration enhanced with increasing urban land percentage, primarily because of the increased proportional contribution of sewage & manure source. The 15N site preference and relevant isotopic evidences revealed that the proportion of denitrification derived N2O increased from 60% to 76%, with the urban land percentage increasing from < 5% to > 22%, which was caused by decreases in flow velocity and dissolved oxygen saturation, increases in NO3− concentration and N2O-denitrifying genes. The non-negligible contribution of nitrification to N2O production (∼ 40%) in lower-urbanized river stretches may be attributed to aerobic conditions and lower impermeable riparian zone facilitating the occurrence of in-river nitrification and the access of in-soil nitrification to river. Urbanization-mediated decreases in flow velocity and dissolved oxygen and increases in nitrogen availability and denitrification process resulted in an increase in N2O concentration and flux, with N2O concentration approximately four times higher in higher-urbanized river reaches (50.7 ± 26.3 nmol/L) than in lower-urbanized river reaches (14.4 ± 2.5 nmol/L). In addition, increased proportional contribution of sewage & manure source also provides the possibility for exogenous N2O inputs with urban expansion. These findings contribute to deepening our understanding of how urbanization drives N2O dynamics in river systems.

15N Hyperpolarization of Metronidazole Antibiotic in Aqueous Media Using Phase-Separated Signal Amplification by Reversible Exchange with Parahydrogen.

Metronidazole is a prospective hyperpolarized MRI contrast agent with potential hypoxia sensing utility for applications in cancer, stroke, neurodegenerative diseases, etc. We demonstrate a pilot procedure for production of ∼30 mM hyperpolarized [15N3]metronidazole in aqueous media by using a phase-separated SABRE-SHEATH hyperpolarization method, with nitrogen-15 polarization exceeding 2.2% on all three 15N sites achieved in less than 2 min. The 15N polarization T1 of ∼12 min is reported for the 15NO2 group at the clinically relevant field of 1.4 T in the aqueous phase, demonstrating a remarkably long lifetime of the hyperpolarized state. The produced aqueous solution of [15N3]metronidazole that contained only ∼100 μM of residual Ir was deemed biocompatible via validation through the MTT colorimetric test for assessing cell metabolic activity using human embryotic kidney HEK293T cells. This low-cost and ultrafast hyperpolarization procedure represents a major advance for the production of a biocompatible HP [15N3]metronidazole (and potentially other hyperpolarized drugs) formulation for MRI sensing applications.

Increasing the accuracy of exchange parameters reporting on slow dynamics by performing CEST experiments with ‘high’ B1 fields

Over the last decade chemical exchange saturation transfer (CEST) NMR methods have emerged as powerful tools to characterize biomolecular conformational dynamics occurring between a visible major state and ‘invisible’ minor states. The ability of the CEST experiment to detect these minor states, and provide precise exchange parameters, hinges on using appropriate B1 field strengths during the saturation period. Typically, a pair of B1 fields with ω1 (=2πB1) values around the exchange rate kex are chosen. Here we show that the transverse relaxation rate of the minor state resonance (R2,B) also plays a crucial role in determining the B1 fields that lead to the most informative datasets. Using K=kexkex+R2,B12 ≥ kex, to guide the choice of B1, instead of kex, leads to data wherefrom substantially more accurate exchange parameters can be derived. The need for higher B1 fields, guided by K, is demonstrated by studying the conformational exchange in two mutants of the 71 residue FF domain with kex ∼ 11 s−1 and ∼ 72 s−1, respectively. In both cases analysis of CEST datasets recorded using B1 field values guided by kex lead to imprecise exchange parameters, whereas using B1 values guided by K resulted in precise site-specific exchange parameters. The conclusions presented here will be valuable while using CEST to study slow processes at sites with large intrinsic relaxation rates, including carbonyl sites in small to medium sized proteins, amide 15N sites in large proteins and when the minor state dips are broadened due to exchange among the minor states.

Mixed Archaeal Production and Nitrifier Denitrification Dominate N2O Production in the East China Sea: Insights From Isotopocule and Hydroxylamine Analyses

AbstractOceans are identified as potent sources of atmospheric nitrous oxide (N2O), while the magnitude of its flux and microbial production mechanisms remain uncertain in highly perturbed coastal zones. Here, the first analyses of N2O isotopocule signatures in the East China Sea (ECS) are presented, along with hydroxylamine (NH2OH) and N2O concentrations, to clarify the dominant N2O production processes in coastal water. In the ECS in October 2015, N2O ranged from 6.3 to 33.1 nmol L−1, equivalent to 99%–251% saturation, leading to air‐sea fluxes of 1.6–10.5 μmol m−2 d−1 (4.8 ± 2.5 μmol m−2 d−1) using the W2014 formula. The coexistence of high levels of NH4+, NH2OH, and NO2− indicated the potential for nitrification and/or hybrid N2O formation. In the shallow water (&lt;300 m), the concentration (∼9.3 nmol L−1), δ15Nbulk–N2O (∼6.8‰), δ18O–N2O (∼45.1‰), and 15N site preference (SP, ∼14.8‰) of N2O were close to the isotopic signatures in atmospheric N2O, whereas values in the deep water increased with depth, with N2O reaching maxima of 33.1 nmol L−1, 8.6‰, 54.7‰, and 18.7‰, respectively. From the dual N2O isotopocule mapping approach, almost equal contributions of archaeal N2O production (archaeal nitrification and/or hybrid mechanism, ∼47%) and nitrifier denitrification (or denitrification) (∼53%) to total in situ N2O production were identified for the shallow water, but archaeal nitrification was responsible for ∼83% of the deeper N2O production. Moreover, the far‐field lateral advection from other areas served as a potential physical supply of deeper N2O. Our findings enhance the understanding of N2O dynamics in coastal waters.

Nitrogen-15 and Fluorine-19 Relaxation Dynamics and Spin-Relayed SABRE-SHEATH Hyperpolarization of Fluoro-[15N3]metronidazole.

Efficient 15N-hyperpolarization of [15N3]metronidazole was reported previously using the Signal Amplification By Reversible Exchange in SHield Enabled Alignment Transfer (SABRE-SHEATH) technique. This hyperpolarized FDA-approved antibiotic is a potential contrast agent because it can be administered in a large dose and because previous studies revealed long-lasting HP states with exponential decay constant T1 values of up to 10 min. Possible hypoxia-sensing applications have been proposed using hyperpolarized [15N3]metronidazole. In this work, we report on the functionalization of [15N3]metronidazole with a fluorine-19 moiety via a one-step reaction to substitute the -OH group. SABRE-SHEATH hyperpolarization studies of fluoro-[15N3]metronidazole revealed efficient hyperpolarization of all three 15N sites with maximum %P15N values ranging from 4.2 to 6.2%, indicating efficient spin-relayed polarization transfer in microtesla fields via the network formed by 2J15N-15N. The corresponding 15N to 19F spin-relayed polarization transfer was found to be far less efficient with %P19F of 0.16%, i.e., more than an order of magnitude lower than that of 15N. Relaxation dynamics studies in microtesla fields support a spin-relayed polarization transfer mechanism because all 15N and 19F spins share the same T1 value of ca. 16-20 s and the same magnetic field profile for the SABRE-SHEATH polarization process. We envision the use of fluoro-[15N3]metronidazole as a potential hypoxia sensor. It is anticipated that under hypoxic conditions, the nitro group of fluoro-[15N3]metronidazole undergoes electronic stepwise reduction to an amino derivative. Ab initio calculations of 15N and 19F chemical shifts of fluoro-[15N3]metronidazole and its putative hypoxia-induced metabolites clearly indicate that the chemical shift dispersions of all three 15N sites and the 19F site are large enough to enable the envisioned hypoxia-sensing approaches.

Parahydrogen in Reversible Exchange Induces Long-Lived 15N Hyperpolarization of Anticancer Drugs Anastrozole and Letrozole.

Hyperpolarization modalities overcome the sensitivity limitations of NMR and unlock new applications. Signal amplification by reversible exchange (SABRE) is a particularly cheap, quick, and robust hyperpolarization modality. Here, we employ SABRE for simultaneous chemical exchange of parahydrogen and nitrile-containing anticancer drugs (letrozole or anastrozole) to enhance 15N polarization. Distinct substrates require unique optimal parameter sets, including temperature, magnetic field, or a shaped magnetic field profile. The fine tuning of these parameters for individual substrates is demonstrated here to maximize 15N polarization. After optimization, including the usage of pulsed μT fields, the 15N nuclei on common anticancer drugs, letrozole and anastrozole, can be polarized within 1-2 min. The hyperpolarization can exceed 10%, corresponding to 15N signal enhancement of over 280,000-fold at a clinically relevant magnetic field of 1 T. This sensitivity gain enables polarization studies at naturally abundant 15N enrichment level (0.4%). Moreover, the nitrile 15N sites enable long-lasting polarization storage with [15N]T1 over 9 min, enabling signal detection from a single hyperpolarization cycle for over 30 min.

Quantitative discrimination of algae multi-impacts on N2O emissions in eutrophic lakes: Implications for N2O budgets and mitigation

It is generally accepted that eutrophic lakes significantly contribute to nitrous oxide (N2O) emissions. However, how these emissions are affected by the formation, disappearance, and mechanisms of algal blooms in these lakes has not been systematically investigated. This study examined and determined the relative contribution of spatiotemporal N2O production pathways in hypereutrophic Lake Taihu. Synchronously, the multi-impacts of algae on N2O production and release potential were measured in the field and in microcosms using isotope ratios of oxygen (δ18O) and bulk nitrogen (δ15N) to N2O and to intramolecular 15N site preference (SP). Results showed that N2O production in Lake Taihu was derived from microbial effects (nitrification and incomplete denitrification) and water air exchanges. N2O production was also affected by the N2O reduction process. The mean dissolved N2O concentrations in the water column during the pre-outbreak, outbreak, and decay stages of algae accumulation were almost the same (0.05 μmol·L–1), which was 2–10 times higher than in lake areas algae was not accumulating. However, except for the central lake area, all surveyed areas (with and without accumulated algae) displayed strong release potential and acted as the emission source because of dissolved N2O supersaturation in the water column. The mean N2O release fluxes during the pre-outbreak, outbreak, and decay stages of algae accumulation areas were 17.95, 26.36, and 79.32 μmol·m–2·d–1, respectively, which were 2.0–7.5 times higher than the values in the non-algae accumulation areas. In addition, the decay and decomposition of algae released large amounts of nutrients and changed the physiochemical properties of the water column. Additionally, the increased algae biomass promoted N2O release and improved the proportion of N2O produced via denitrification process to being 9.8–20.4% microbial-derived N2O. This proportion became higher when the N2O consumption during denitrification was considered as evidenced by isotopic data. However, when the algae biomass was excessive in hypereutrophic state, the algae decomposition also consumed a large amount of oxygen, thus limiting the N2O production due to complete denitrification as well as due to the limited substrate supply of nitrate by nitrification in hypoxic or anoxic conditions. Further, the excessive algae accumulation on the water surface reduced N2O release fluxes via hindering the migration of the dissolved N2O into the atmosphere. These findings provide a new perspective and understanding for accurately evaluating N2O release fluxes driven by algae processes in eutrophic lakes.

Sources of nitrous oxide emissions from hydroponic tomato cultivation: Evidence from stable isotope analyses.

Hydroponic vegetable cultivation is characterized by high intensity and frequent nitrogen fertilizer application, which is related to greenhouse gas emissions, especially in the form of nitrous oxide (N2O). So far, there is little knowledge about the sources of N2O emissions from hydroponic systems, with the few studies indicating that denitrification could play a major role. Here, we use evidence from an experiment with tomato plants (Solanum lycopersicum) grown in a hydroponic greenhouse setup to further shed light into the process of N2O production based on the N2O isotopocule method and the 15N tracing approach. Gas samples from the headspace of rock wool substrate were collected prior to and after 15N labeling at two occasions using the closed chamber method and analyzed by gas chromatography and stable isotope ratio mass spectrometry. The isotopocule analyses revealed that either heterotrophic bacterial denitrification (bD) or nitrifier denitrification (nD) was the major source of N2O emissions, when a typical nutrient solution with a low ammonium concentration (1-6 mg L-1) was applied. Furthermore, the isotopic shift in 15N site preference and in δ18O values indicated that approximately 80-90% of the N2O produced were already reduced to N2 by denitrifiers inside the rock wool substrate. Despite higher concentrations of ammonium present during the 15N labeling (30-60 mg L-1), results from the 15N tracing approach showed that N2O mainly originated from bD. Both, 15N label supplied in the form of ammonium and 15N label supplied in the form of nitrate, increased the 15N enrichment of N2O. This pointed to the contribution of other processes than bD. Nitrification activity was indicated by the conversion of small amounts of 15N-labeled ammonium into nitrate. Comparing the results from N2O isotopocule analyses and the 15N tracing approach, likely a combination of bD, nD, and coupled nitrification and denitrification (cND) was responsible for the vast part of N2O emissions observed in this study. Overall, our findings help to better understand the processes underlying N2O and N2 emissions from hydroponic tomato cultivation, and thereby facilitate the development of targeted N2O mitigation measures.

Slice & Dice: nested spin-lattice relaxation measurements.

Spin-lattice relaxation rate (R1) measurements are commonly used to characterize protein dynamics. However, the time needed to collect the data can be quite long due to long relaxation times of the low-gamma nuclei, especially in the solid state. We present a method to collect backbone heavy atom relaxation data by nesting the collection of datasets in the solid state. This method results in a factor of 2 to 2.5 times faster data acquisition for backbone R1 relaxation data for the 13C and 15N sites of proteins.

Nitrification Regulates the Spatiotemporal Variability of N2O Emissions in a Eutrophic Lake.

Nitrous oxide (N2O) emissions from lakes exhibit significant spatiotemporal heterogeneity, and quantitative identification of the different N2O production processes is greatly limited, causing the role of nitrification to be undervalued or ignored in models of a lake's N2O emissions. Here, the contributions of nitrification and denitrification to N2O production were quantitatively assessed in the eutrophic Lake Taihu using molecular biology and isotope mapping techniques. The N2O fluxes ranged from -41.48 to 28.84 μmol m-2 d-1 in the lake, with lower N2O concentrations being observed in spring and summer and significantly higher N2O emissions being observed in autumn and winter. The 15N site preference and relevant isotopic evidence demonstrated that denitrification contributed approximately 90% of the lake's gross N2O production during summer and autumn, 27-83% of which was simultaneously eliminated via N2O reduction. Surprisingly, nitrification seemed to act as a key process promoting N2O production and contributing to the lake as a source of N2O emissions. A combination of N2O isotopocule-based approaches and molecular techniques can be used to determine the precise characteristics of microbial N2O production and consumption in eutrophic lakes. The results of this study provide a basis for accurately assessing N2O emissions from lakes at the regional and global scales.

Clumped isotope signatures of nitrous oxide formed by bacterial denitrification

Multiply substituted isotopic species of nitrous oxide (N2O), referred to as clumped isotopes, represent a promising new tool for distinguishing production pathways of this potent greenhouse gas. This work presents the first determination of enrichment factors of N2O clumped isotopes during bacterial denitrification. Samples of N2O obtained after 1-, 3-, and 7-day incubations of a pure culture of the denitrifier Pseudomonas aureofaciens at 20 °C and 30 °C were analysed by the recently developed quantum cascade laser absorption spectroscopy (QCLAS) method. Enrichment factors εp/s of the cumulative product (p) relative to the substrate (s) were determined using a Rayleigh model for the seven most abundant isotopically substituted molecules (isotopocules) of N2O. Values of the enrichment factors εp/s (with uncertainty expressed as expanded standard uncertainty at the 95% confidence interval) at the two incubation temperatures (20 °C/30 °C) are:14N15N16O (456): ε456 = (−40.3 ± 2.6)‰/(−35.1 ± 0.7)‰15N14N16O (546): ε546 = (−38.1 ± 3.4)‰/(−31.2 ± 0.6)‰14N14N17O (447): ε447 = (21.3 ± 1.2)‰/(24.5 ± 0.5)‰14N14N18O (448): ε448 = (38.8 ± 1.5)‰/(46.4 ± 1.2)‰14N15N18O (458): ε458 = (−8.9 ± 2.0)‰/(−11.7 ± 0.6)‰15N14N18O (548): ε548 = (−3.4 ± 1.1)‰/(−1.8 ± 0.5)‰15N15N16O (556): ε556 = (−85.9 ± 1.5)‰/(−63.9 ± 1.4)‰Temporal evolutions of the abundances of singly substituted N2O isotopocules during nitrate reduction agree with previously published experiments: there is normal isotope effect associated with the production of 14N15N16O and 15N14N16O; i.e., intermediates leading to 14N14N16O react faster than intermediates leading to 14N15N16O and 15N14N16O. However, the production of 14N14N17O and 14N14N18O is associated with inverse isotope effect; i.e., intermediates leading to 14N14N16O react slower than intermediates leading to 14N14N17O and 14N14N18O due to preferential cleavage of 16O during nitrate reduction to N2O. Isotopic fractionation at the incubation temperature of 30 °C was significantly lower compared to 20 °C. We observed a large kinetic isotope effect of the 15N site preference (SP) and the 15N–18O site preference (SP18) at the onset of the reaction. SP18 was found to be closer to 0‰ than SP, which is thought to arise from similar rates of breakage of the 15N–O and 14N–O bonds in the reaction intermediates. The 15N–18O clumped isotope anomalies in two isotopic isomers (isotopomers) 14N15N18O and 15N14N18O (Δ458+548avg) follow a temporal trend similar to those of SP and SP18. The 15N–15N clumped isotope anomalies in 15N15N16O are greater than 0‰ and show no clear temporal trend or influence of incubation temperature, suggesting no strong combinatorial effects involved during the N–N bond formation. Overall, our data illustrate that clumped N2O isotopes may be used as independent tracers for reaction mechanisms of N2O conversion and may establish themselves as a worthwhile tool to study the biogeochemical cycle of N2O.

Isotopically characterised N2 O reference materials for use as community standards.

RationaleInformation on the isotopic composition of nitrous oxide (N2O) at natural abundance supports the identification of its source and sink processes. In recent years, a number of mass spectrometric and laser spectroscopic techniques have been developed and are increasingly used by the research community. Advances in this active research area, however, critically depend on the availability of suitable N2O isotope Reference Materials (RMs).MethodsWithin the project Metrology for Stable Isotope Reference Standards (SIRS), seven pure N2O isotope RMs have been developed and their 15N/14N, 18O/16O, 17O/16O ratios and 15N site preference (SP) have been analysed by specialised laboratories against isotope reference materials. A particular focus was on the 15N site‐specific isotopic composition, as this measurand is both highly diagnostic for source appointment and challenging to analyse and link to existing scales.ResultsThe established N2O isotope RMs offer a wide spread in delta (δ) values: δ 15N: 0 to +104‰, δ 18O: +39 to +155‰, and δ 15NSP: −4 to +20‰. Conversion and uncertainty propagation of δ 15N and δ 18O to the Air‐N2 and VSMOW scales, respectively, provides robust estimates for δ 15N(N2O) and δ 18O(N2O), with overall uncertainties of about 0.05‰ and 0.15‰, respectively. For δ 15NSP, an offset of >1.5‰ compared with earlier calibration approaches was detected, which should be revisited in the future.ConclusionsA set of seven N2O isotope RMs anchored to the international isotope‐ratio scales was developed that will promote the implementation of the recommended two‐point calibration approach. Particularly, the availability of δ 17O data for N2O RMs is expected to improve data quality/correction algorithms with respect to δ 15NSP and δ 15N analysis by mass spectrometry. We anticipate that the N2O isotope RMs will enhance compatibility between laboratories and accelerate research progress in this emerging field.

Isotopic assessment of soil N2O emission from a sub-tropical agricultural soil under varying N-inputs

Nitrogen fertilizers result in high crop productivity but also enhance the emission of N2O, an environmentally harmful greenhouse gas. Only approximately a half of the applied nitrogen is utilized by crops and the rest is either vaporized, leached, or lost as NO, N2O and N2 via soil microbial activity. Thus, improving the nitrogen use efficiency of cropping systems has become a global concern. Factors such as types and rates of fertilizer application, soil texture, moisture level, pH, and microbial activity/diversity play important roles in N2O production. Here, we report the results of N2O production from a set of chamber experiments on an acidic sandy-loam agricultural soil under varying levels of an inorganic N-fertilizer, urea. Stable isotope technique was employed to determine the effect of increasing N-fertilizer levels on N2O emissions and identify the microbial processes involved in fertilizer N-transformation that give rise to N2O. We monitored the isotopic changes in both substrate (ammonium and nitrate) and the product N2O during the entire course of the incubation experiments. Peak N2O emissions of 122 ± 98 μg N2O-N m−2 h−1, 338 ± 49 μg N2O-N m−2 h−1 and 739 ± 296 μg N2O-N m−2 h−1 were observed for urea application rate of 40, 80, and 120 μg N g−1. The duration of emissions also increased with urea levels. The concentration and isotopic compositions of the substrates and product showed time-bound variation. Combining the observations of isotopic effects in δ15N, δ18O, and 15N site preference, we inferred co-occurrence of several microbial N2O production pathways with nitrification and/or fungal denitrification as the dominant processes responsible for N2O emissions. Besides this, dominant signatures of bacterial denitrification were observed in a second N2O emission pulse in intermediate urea-N levels. Signature of N2O consumption by reduction could be traced during declining emissions in treatment with high urea level.

Versatile soil gas concentration and isotope monitoring: optimization and integration of novel soil gas probes with online trace gas detection

Abstract. Gas concentrations and isotopic signatures can unveil microbial metabolisms and their responses to environmental changes in soil. Currently, few methods measure in situ soil trace gases such as the products of nitrogen and carbon cycling or volatile organic compounds (VOCs) that constrain microbial biochemical processes like nitrification, methanogenesis, respiration, and microbial communication. Versatile trace gas sampling systems that integrate soil probes with sensitive trace gas analyzers could fill this gap with in situ soil gas measurements that resolve spatial (centimeters) and temporal (minutes) patterns. We developed a system that integrates new porous and hydrophobic sintered polytetrafluoroethylene (sPTFE) diffusive soil gas probes that non-disruptively collect soil gas samples with a transfer system to direct gas from multiple probes to one or more central gas analyzer(s) such as laser and mass spectrometers. Here, we demonstrate the feasibility and versatility of this automated multiprobe system for soil gas measurements of isotopic ratios of nitrous oxide (δ18O, δ15N, and the 15N site preference of N2O), methane, carbon dioxide (δ13C), and VOCs. First, we used an inert silica matrix to challenge probe measurements under controlled gas conditions. By changing and controlling system flow parameters, including the probe flow rate, we optimized recovery of representative soil gas samples while reducing sampling artifacts on subsurface concentrations. Second, we used this system to provide a real-time window into the impact of environmental manipulation of irrigation and soil redox conditions on in situ N2O and VOC concentrations. Moreover, to reveal the dynamics in the stable isotope ratios of N2O (i.e., 14N14N16O, 14N15N16O, 15N14N16O, and 14N14N18O), we developed a new high-precision laser spectrometer with a reduced sample volume demand. Our integrated system – a tunable infrared laser direct absorption spectrometry (TILDAS) in parallel with Vocus proton transfer reaction mass spectrometry (PTR-MS), in line with sPTFE soil gas probes – successfully quantified isotopic signatures for N2O, CO2, and VOCs in real time as responses to changes in the dry–wetting cycle and redox conditions. Broadening the collection of trace gases that can be monitored in the subsurface is critical for monitoring biogeochemical cycles, ecosystem health, and management practices at scales relevant to the soil system.

Land use conversion and soil moisture affect the magnitude and pattern of soil-borne N2, NO, and N2O emissions

In this study, soil-borne N2, NO, and N2O emissions induced by land use conversion and water management were investigated in intact soil cores under a helium/oxygen atmosphere by a robotized incubation system in combination with the N2O 15N site preference signature and molecular-based microbial analysis. The experiment consisted of five treatments: i) paddy-flooded (PF); ii) orchard-wet (OW, 70% WFPS); iii) orchard-dry (OD, 43% WFPS); iv) vegetable-wet (VW, 70% WFPS); and v) vegetable-dry (VD, 43% WFPS). The vessels of each treatment received 200 mg urea-nitrogen (N) (equivalent to 210 kg of urea-N ha−1), and soil moisture in the OW and VW treatments was adjusted to a higher constant moisture to simulate a scenario after irrigation or rainfall. The results showed that total gaseous N losses during the incubation period were 25.33 ± 0.33 kg N ha−1 in the PF treatment, whereas smaller losses were recorded in the OD and VD treatments (4.28 ± 2.04 and 9.75 ± 3.75 kg N ha−1, respectively). The potential contribution of bacterial denitrification to N2O emissions in the OD and VD treatments was 11.1% and 15.4% higher, respectively, than that in the PF treatment (58.8% ± 0.5%). Furthermore, the corresponding N2O/(N2O + N2) ratio in the OD and VD treatments decreased by 50% and 73.8%, respectively, relative to the ratio in the PF treatment (0.42 ± 0.01). Such changes indicated the crucial role of altered soil properties caused by land use conversion in regulating the production and consumption of N2O. Relative to the normal moisture (dry) condition, enhanced soil moisture increased total gaseous N losses in the orchard and vegetable soils by 386.9% and 67.4%, accompanied by a higher N2O/(N2O + N2) product ratio, but decreased the share of bacterial N2O by 11.1% and 15.4%, respectively. The changes in the abundance and community composition of soil denitrifiers caused by land use conversion from rice paddies to orchards and vegetable fields could partly explain the differences in gaseous N loss therein. These findings highlight the influence of land use conversion on soil gaseous N emissions and demonstrate that increased moisture in upland soils reduced the dominance of bacterial denitrification in N2O production.

Plants are a natural source of nitrous oxide even in field conditions as explained by 15N site preference

Plants are either recognized to produce nitrous oxide (N2O) or considered as a medium to transport soil-produced N2O. To date, it is not clear whether in their habitat plants conduit N2O produced in soil or are a natural source. We aimed to understand role of plants in N2O emissions in field conditions. Therefore, rubber plants (Ficus elastica) were planted in the field; then plant and soil chambers were deployed simultaneously to collect gas samples, and 15N site preference (SP) of N2O was evaluated. The mean SP values of plant and soil emitted N2O were −20.85 ± 2.8‰ and −8.85 ± 1.08‰, respectively, and were significantly different (p < 0.0001); while bulk 15N of plant and soil emitted N2O were −10.83 ± 3.33‰ and −22.56 ± 3.37‰, respectively and were similar (p = 0.06). In the current study, soil always acted as a source of N2O, while plants were both source and sink. Plant and soil N2O fluxes had significant positive exponential relationship with both soil and air temperature. Soil water-filled pore space (WFPS) had significant negative linear relationship with only soil N2O fluxes. Plant N2O fluxes had significant positive linear relationship with plant respiration rates and negative linear relationship with plant surface areas. Based on the relationship between plant respiration rates and N2O fluxes, we suggest that mitochondria are the possible sites of N2O formation in plant cells while the relationship between plant surface areas and N2O fluxes suggests that roots are the parts of its formation in natural and field conditions. Our results suggest that plants are a natural source of N2O even at field conditions and challenge a view that plants are a medium to transport soil-produced N2O into the atmosphere.

Comparing modified substrate-induced respiration with selective inhibition (SIRIN) and N&amp;lt;sub&amp;gt;2&amp;lt;/sub&amp;gt;O isotope approaches to estimate fungal contribution to denitrification in three arable soils under anoxic conditions

Abstract. The coexistence of many N2O production pathways in soil hampers differentiation of microbial pathways. The question of whether fungi are significant contributors to soil emissions of the greenhouse gas nitrous oxide (N2O) from denitrification has not yet been resolved. Here, three approaches to independently investigate the fungal fraction contributing to N2O from denitrification were used simultaneously for, as far as we know, the first time (modified substrate-induced respiration with selective inhibition (SIRIN) approach and two isotopic approaches, i.e. end-member mixing approach (IEM) using the 15N site preference of N2O produced (SPN2O) and the SP/δ18O mapping approach (SP/δ18O Map)). This enabled a comparison of methods and a quantification of the importance of fungal denitrification in soil. Three soils were incubated in four treatments of the SIRIN approach under anaerobic conditions to promote denitrification. While one treatment without microbial inhibition served as a control, the other three treatments were amended with inhibitors to selectively inhibit bacterial, fungal, or bacterial and fungal growth. These treatments were performed in three variants. In one variant, the 15N tracer technique was used to estimate the effect of N2O reduction on the N2O produced, while two other variants were performed under natural isotopic conditions with and without acetylene. All three approaches revealed a small contribution of fungal denitrification to N2O fluxes (fFD) under anaerobic conditions in the soils tested. Quantifying the fungal fraction with modified SIRIN was not successful due to large amounts of uninhibited N2O production. In only one soil could fFD be estimated using modified SIRIN, and this resulted in 28 ± 9 %, which was possibly an overestimation, since results obtained by IEM and SP/δ18O Map for this soil resulted in fFD of below 15 % and 20 %, respectively. As a consequence of the unsuccessful SIRIN approach, estimation of fungal SPN2O values was impossible. While all successful methods consistently suggested a small or missing fungal contribution, further studies with stimulated fungal N2O fluxes by adding fungal C substrates and an improved modified SIRIN approach, including alternative inhibitors, are needed to better cross-validate the methods.

Nitrogen and oxygen isotopomeric constraints on the sources of nitrous oxide and the role of submarine groundwater discharge in a temperate eutrophic salt‐wedge estuary

AbstractEstuaries have been identified as sources of nitrous oxide (N2O) emissions to the atmosphere but questions remain as to which production pathway(s) govern the oversaturation of N2O observed in most estuaries worldwide. Here, we use a suite of nitrate and N2O isotopes, as well as the 15N site preference signatures of N2O to assess (1) the relative importance of different N2O production pathways in a eutrophic groundwater‐impacted salt‐wedge estuary and the aquifers underlying the estuary and (2) the influence of groundwater input on the overall N2O saturation in the estuary. This is one of the few studies to examine the effect of groundwater–surface water interaction on N2O cycling using N2O isotopes. The site preference values of N2O in the deep aquifer below the estuary were distinctive (83‰ ± 25‰) and were much higher than in either surface water (21‰ ± 6‰) or shallow groundwater (44‰ ± 8‰), suggesting the influence of multiple biotic and/or abiotic processes which proceed through multiple cycles, and/or the occurrence of a yet unidentified N2O production pathway. Isotope endmember considerations revealed that nitrifier‐denitrification was the major N2O production pathway within the shallow aquifer whereas within the estuarine water column, N2O saturation was governed by chemodenitrification and discharge of N2O‐laden submarine groundwater. Our study not only emphasizes the substantial, yet often underappreciated role of submarine groundwater discharge in estuarine N2O budgets, but also highlights the need to reevaluate the importance of the noncanonical denitrification pathways (i.e., chemodenitrification and nitrifier‐denitrification) in controlling the overall N2O production from estuarine and groundwater environments.

15N NMR Hyperpolarization of Radiosensitizing Antibiotic Nimorazole by Reversible Parahydrogen Exchange in Microtesla Magnetic Fields

AbstractNimorazole belongs to the imidazole‐based family of antibiotics to fight against anaerobic bacteria. Moreover, nimorazole is now in Phase 3 clinical trial in Europe for potential use as a hypoxia radiosensitizer for treatment of head and neck cancers. We envision the use of [15N3]nimorazole as a theragnostic hypoxia contrast agent that can be potentially deployed in the next‐generation MRI‐LINAC systems. Herein, we report the first steps to create long‐lasting (for tens of minutes) hyperpolarized state on three 15N sites of [15N3]nimorazole with T1 of up to ca. 6 minutes. The nuclear spin polarization was boosted by ca. 67000‐fold at 1.4 T (corresponding to P15N of 3.2 %) by 15N−15N spin‐relayed SABRE‐SHEATH hyperpolarization technique, relying on simultaneous exchange of [15N3]nimorazole and parahydrogen on polarization transfer Ir‐IMes catalyst. The presented results pave the way to efficient spin‐relayed SABRE‐SHEATH hyperpolarization of a wide range of imidazole‐based antibiotics and chemotherapeutics.

Nitrite induced transcription of p450nor during denitrification by Fusarium oxysporum correlates with the production of N2O with a high 15N site preference

The greenhouse gas nitrous oxide (N2O) is produced in soil as a consequence of complex co-occurring processes conducted by diverse microbial species, including fungi. The fungal p450nor gene encodes a nitric oxide reductase associated with fungal denitrification. We thus hypothesized that p450nor gene expression is a marker for ongoing fungal denitrification. Specific PCR primers and quantitative PCR (qPCR) assays were developed targeting p450nor genes and transcripts. The novel PCR primers successfully amplified p450nor from pure cultures, and were used in an mRNA targeted qPCR to quantify p450nor gene transcription (i.e., gene expression) during denitrification activity in cultures of the fungal model denitrifier Fusarium oxysporum. Gene expression was induced by high (5 mM) and low (0.25 mM) nitrite concentrations. Nitrite stimulated N2O production rates by F. oxysporum, which correlated well with an up to 70-fold increase in p450nor gene expression during the first 12–24 h of anoxic incubation. The relative p450nor gene peak expression and peak N2O production rates declined 20- and 2-fold on average, respectively, towards the later phase of incubation (48–120 h). The 15N site preference of N2O (SP(N2O)) was high for F. oxysporum and independent of reaction progress, confirming the fungal origin of N2O produced. In conclusion, the developed fungal p450nor gene expression assay together with the analysis of SP(N2O) values provide a basis to improve current tools for the identification of fungal denitrification and/or N2O production in natural systems like soils.