Cover crop residue decomposition triggered soil oxygen depletion and promoted nitrous oxide emissions

Cover cropping is a promising strategy to improve soil health, but it may also trigger greenhouse gas emissions, especially nitrous oxide (N2O). Beyond nitrogen (N) availability, cover crop residue decomposition may accelerate heterotrophic respiration to limit soil O2 availability, hence promote N2O emissions from denitrification under sub-optimal water-filled pore space (WFPS) conditions that are typically not conducive to large N2O production. We conducted a 21-day incubation experiment to examine the effects of contrasting cover crop residue (grass vs legume) decomposition on soil O2 and biogeochemical changes to influence N2O and CO2 emissions from 15N labeled fertilized soils under 50% and 80% WFPS levels. Irrespective of cover crop type, mixing cover crop residue with N fertilizer resulted in high cumulative N2O emissions under both WFPS conditions. In the absence of cover crop residues, the N fertilizer effect of N2O was only realized under 80% WFPS, whereas it was comparable to the control under 50% WFPS. The N2O peaks under 50% WFPS coincided with soil O2 depletion and concomitant high CO2 emissions when cover crop residues were mixed with N fertilizer. While N fertilizer largely contributed to the total N2O emissions from the cover crop treatments, soil organic matter and/or cover crop residue derived N2O had a greater contribution under 50% than 80% WFPS. Our results underscore the importance of N2O emissions from cover crop-based fertilized systems under relatively lower WFPS via a mechanism of respiration-induced anoxia and highlight potential risks of underestimating N2O emissions under sole reliance on WFPS.

Global agriculture annually generates approximately 3.8 billion Mg of crop residues, of which 4.8 million Mg is contributed by the U.S. 1 .Increasing adoption of winter cover crops as a soil health practice will continue generating organic residues.For example, U.S. cover crop acres in 2017 (15.4 million acres) was 50% higher than that in 2012 (10.3 million acres) 2 .When cover and cash crop residues are returned back to the soils, they can provide diverse ecosystem services including soil carbon (C) sequestration 3,4 and overall improvement of soil health [5][6][7][8] .However, decomposing fresh residues can influence coupled soil C and nitrogen (N) cycling, which could be important with regards to the emission of nitrous oxide (N 2 O) [9][10][11][12][13] ; a long-lived potent greenhouse gas (GHG) largely emitted from global agricultural soils 14 .This process has the potential to be amplified when winter cover crops are used, as their decomposition upon termination often coincides with N fertilization before summer crop planting in many agricultural production systems.
Cover crop residue quality determines N release during decomposition, with high-quality residues (low C:N ratio, e.g., legumes) often exhibit faster mineralization and N release than high C:N ratio non-legume residues 15,16 .The cover crop residue influence on N 2 O emissions is far from straightforward and further depends on management practices (termination method, N fertilization source and rate) and environmental conditions 9,17 .Beyond N supply, simultaneous increase in C availability during residue decomposition can trigger heterotrophic microbial respiration, leading to rapid soil oxygen (O 2 ) consumption.Under such conditions, water induced O 2 diffusion limitation may not be required to prevail anoxic conditions and N 2 O emissions 18 .While N 2 O emissions in response to residue addition have been widely attributed to altered C and N availability 19 , wetness independent anoxia during residue decomposition as a possible mechanism of N 2 O production has only been postulated with

Cover crop residue and N fertilization impacts on temporal N 2 O and CO 2 emissions
The combination of cover crop residue and N fertilizer addition exhibited higher peak N 2 O emissions than the N fertilized without cover crop and control treatments under both 50% and 80% WFPS conditions (Fig. 1a,b).Temporal N 2 O emissions showed different trends across cover crop treatments under 50% and 80% WFPS.First, cover crop treatments under 80% WFPS immediately showed high daily emissions ranging from 674 to 2728 µg N 2 O-N kg soil −1 day −1 during day 0-4.Whereas peak N 2 O emissions were slightly delayed under 50% WFPS ranging from 529 to 2824 µg N 2 O-N kg soil −1 day −1 , the same or slightly higher in magnitude than that under 80% WFPS.Second, N 2 O emissions from the cover crop treatments sharply decreased following the peak emissions under 80% WFPS, with identical emissions to the control treatment after day 7.In contrast, the decline in emissions were more gradual under 50% WFPS where moderate daily N 2 O emissions (ranging from 88 to 607 µg N 2 O-N kg soil −1 day −1 ), significantly higher than the control treatment (p < 0.05), were observed until the end of the incubation experiment.Third, in the absence of cover crop residues, the N fertilized treatment produced very little N 2 O emissions under 50% WFPS, about the same as the control treatment.In contrast, N fertilization under 80% WFPS had significantly higher N 2 O emissions than the control treatment from day 1 to day 16; however, the peak emissions were much lower in magnitude (ranging from 44 to 859 µg N 2 O-N kg soil −1 day −1 ) and showed a slightly different emission patterns than the cover crop treatments.Lastly, residue type (legume vs grass) effect on temporal N 2 O emissions was not consistent across the WFPS treatments, with higher peak emissions from hairy vetch than winter wheat residue addition with N fertilizer under 50% WFPS (p > 0.05).
The temporal variability of N 2 O emissions derived from N fertilizer showed different patterns across WFPS and cover crop treatments that were consistent with the temporal N 2 O emission trends (Fig. 1c, d).Firstly, in the cover crops treatments under 80% WFPS, a high proportion of N 2 O emissions was derived from N fertilizer, ranging from 89 to 97% during day 0 to 4, coinciding with peak N 2 O emissions.The contribution sharply decreased after day 5, concomitant with the decline in total N 2 O emissions.Similarly, in the same treatments under 50% WFPS, N 2 O emissions derived from N fertilizer closely followed the daily N 2 O emission pattern, accounting for 79 to 91% of total N 2 O emissions during day 2 to 5, and gradually decreased to 33% by the end of the incubation experiment.Secondly, in the absence of cover crop residues under 50% WFPS, around one-third of the total N 2 O emissions was derived from N fertilizer after day 2, and it remained relatively constant until the end of the experiment.Nitrogen fertilizer derived N 2 O under 80% WFPS from only N fertilized treatment comprised a much greater proportion (50-80%) to the total emission from day 1 to 16.

Cumulative N 2 O and CO 2 emissions
Cumulative N 2 O and CO 2 emissions reflected their daily emission patterns (Fig. 3 and Table 1).Irrespective of cover crop type, residue addition with N fertilizer under 50% WFPS produced statistically similar N 2 O emissions to 80% WFPS (Fig. 3, p > 0.05), known to facilitate large N 2 O emissions.However, 50% WFPS treatments with cover crop exhibited a greater variability of total N 2 O emissions compared to the corresponding treatments under 80% WFPS.Contrastingly, sole N fertilizer application increased N 2 O emissions only under 80% WFPS, the total emission being similar to the cover crop treatments (p > 0.05).Whereas N fertilization alone under 50% WFPS resulted in cumulative N 2 O emissions comparable to the control treatment (p > 0.05) and six times less than that in 80% WFPS (p < 0.05).Cover crop residue addition effect on CO 2 emissions was more pronounced   www.nature.com/scientificreports/under 50% WFPS content and exhibited approximately twice as high cumulative CO 2 emissions compared to the only N fertilizer and control treatments (p < 0.05) (Table 1).CO 2 emissions were suppressed in all the treatments under 80% WFPS (p > 0.05).
Fertilizer N was the main source of N 2 O across WFPS conditions, except for the only N fertilized treatment under 50% WFPS (Fig. 3).Under 80% WFPS, cover crop addition had little or no effect on N 2 O emissions derived from non-fertilizer sources such as soil organic matter (SOM) and/or cover crop.Cover crop treatments exhibited around 1.7 times higher fertilizer derived N 2 O emissions compared to the only N fertilized treatments under 80% WFPS.Cover crop addition had significantly (p < 0.05) greater N 2 O emission contribution from non-fertilizer (SOM and/or cover crop) sources under 50% than 80% WFPS.Under 50% WFPS, cover crop addition resulted in 23× and 3× higher N 2 O emissions derived from fertilizer and SOM and/or cover crop, respectively, than the N fertilized treatments.In presence of cover crop under 50% WFPS, N 2 O emissions derived from SOM and/or cover crop was 2× higher than that in the control treatment.

Soil O 2 , C, and N availability
Soil O 2 concentration in the top 3-cm soil layer remained at anoxic levels during the whole incubation period for 80% WFPS treatments (Fig. 4b and Supplementary Fig. S2c).The 50% WFPS treatments with hairy vetch and winter wheat residues exhibited a sharp drop in O 2 concentration by day 1 (mean air saturation of 20% and 27%, respectively), reaching anoxic soil O 2 levels in some replicates (Fig. 5A and Supplementary Fig. S2a).Such a drop in soil O 2 following cover crop residue incorporation under 50% WFPS also coincided with high CO 2 emissions and concomitant onset of peak N 2 O emissions (Figs.1a, 2a).Soil remained oxic throughout the incubation period under the N fertilized treatment without cover crop under 50% WFPS (Figs. 4a, 5B).Divergence in soil O 2 between the 50% WFPS treatments with and without cover crops remained noticeable until around day 11 of the incubation experiment, and thereafter became comparable, with a much shorter duration for hairy vetch than winter wheat (Figs.4a, 5, Supplementary Fig. S2a, b).www.nature.com/scientificreports/Prior to N fertilizer addition, all treatments had very low initial soil NO − 3 and NH + 4 concentrations (0.76 and 7.02 mg N kg −1 , respectively).As expected, on day 2 after N addition, all N fertilized treatments showed significantly higher NO − 3 levels than the controls under each WFPS (p < 0.05), except for the 50% WFPS treatment with vetch (Table 2).In general, NO − 3 concentrations consistently decreased over time in all fertilized treatments under 80% WFPS, with only N fertilization maintained slightly higher NO − 3 levels for most of the sampling days.By day 7, 80% WFPS treatments with cover crop addition had NO − 3 concentrations comparable to the 80% WFPS control.In contrast, NO − 3 concentrations under 50% WFPS treatments with N addition either remained constant or slightly decreased over time and remained higher than that under 80% WFPS.Unlike NO − 3 , soil NH + 4 concentrations decreased over time under 50% WFPS and the hairy vetch treatment had notably higher NH + 4 concentrations than the other treatments.www.nature.com/scientificreports/ In general, soil POXC concentrations were higher in the 80% WFPS than the 50% WFPS treatments (mean value: 735 vs 677 mg kg soil −1 ), and the differences were only significant under the cover crop treatments on days 7, 11 and 21, and on day 2 in the hairy vetch treatment (Table 3).Cover crop residue addition had no significant effect on POXC concentrations when compared to the only N fertilized and control treatments under each WFPS condition.

Drivers of N 2 O emissions
The Random Forest model explained daily N 2 O emission variations on the test data for 50% (R 2 = 0.67, RMSE = 21.3) and 80% (R 2 = 0.62, RMSE = 19.3)WFPS conditions (Table 4 and Supplementary Fig. S3).For 50% WFPS treatments, the model identified NH + 4 and O 2 as the most influential variables impacting N 2 O emissions (Table 4).These variables collectively accounted for nearly 75% of the model's performance, explaining 67% of the variance in N 2 O emissions on the test data set (Table 4).Conversely, at 80% WFPS, NO − 3 was the main driver of N 2 O emissions, and the model accounted for 62% of the N 2 O emissions variance on the test data set.www.nature.com/scientificreports/

Discussion
Our findings revealed that even under suboptimal WFPS levels for denitrification (i.e., 50% WFPS in this study), N fertilized soils with cover crop residue addition exhibited N 2 O emissions of similar or higher magnitude than soils experiencing water-induced anoxia at 80% WFPS, widely reported to promote N 2 O emissions from denitrification 34,44,45 .This is in line with previous studies contradicting the conventional understanding that full pore saturation is a prerequisite for denitrification 37 .These findings carry notable implications for managing agricultural systems incorporating cover crops in the rotation to improve soil health through soil C sequestration.While elevated N 2 O emissions can offset the soil C sequestration benefits, our study underscores the importance of understanding the fundamental mechanisms of water-independent soil anoxia and controls of N 2 O emissions in response to cover crop management practices through the lens of high-resolution soil O 2 measurements, a proximal driver of N 2 O emissions.This understanding is critical for accurate assessment of the net GHG mitigation potential of cover crops.

Respiration induced anoxia during cover crop residue decomposition decouples WFPS control on N 2 O emissions
Mixing cover crop residues with N fertilizer created anoxia conducive for N 2 O production under 50% WFPS, which is otherwise well-aerated to limit large N 2 O emissions, especially from denitrification 21 .This led to high cumulative N 2 O emissions comparable with the respective treatments under 80% WFPS conditions (Fig. 3).On the other hand, the no cover crop treatments that received only N fertilizer under 50% WFPS produced N 2 O emissions as low as the control treatment and six times lower than that under primarily water-induced anoxia at 80% WFPS.These findings align with our first hypothesis which postulated that respiration-induced anoxia caused by decomposing cover crop residues can promote N 2 O emissions, even under sub-optimal WFPS conditions for denitrification.There are several lines of evidence that support this hypothesis.First, under 50% WFPS condition, cover crop residue addition exhibited a two-fold increase in cumulative CO 2 production compared to the no cover crop and control treatments (Table 1).This indicates higher heterotrophic respiration from residue C mineralization in well-aerated conditions under moderate WFPS.Increased C availability, acting as energy source for the denitrifiers 24 , during cover crop residue decomposition was previously found to increase N 2 O emissions 28 .Such an effect was limited under the water-induced anoxia environment at 80% WFPS due to decreased overall residue decomposition.The divergence in temporal N 2 O emissions from the cover crop treatments under 50% and 80% WFPS conditions was evident up to day 5 of the incubation (Fig. 1a, b) indicating an accelerated phase of decomposition.Second, under 50% WFPS, peak N 2 O and CO 2 emissions from the cover crop treatments coincided with depletion of soil O 2 concentration two days following N fertilization (Figs.1a, 2a, 4a).As previously reported in other studies 46,47 , the use of optode technology enables the visualization of highly-resolved spatial soil O 2 dynamics following exogenous C incorporation.In the present study, the O 2 images clearly demonstrated that within 24 h following N fertilization, the top 3 cm of soil experienced significant O 2 depletion in treatments with cover crops and 50% WFPS (Figs. 4a, 5a), leading to the development of hypoxic or even anoxic conditions in certain replications.Such a mechanism to produce anoxia with a simultaneous increase in N 2 O will remain unexplained when WFPS alone is used as surrogate of soil O 2 availability.Therefore, these findings further highlight the limitations of relying solely on WFPS in interpreting and predicting N 2 O fluxes, which can only account for biophysical mechanisms of anoxia resulting from O 2 diffusion limitation in wet soils 48,49 , while neglecting anoxia caused by microbial O 2 consumption during residue decomposition 20 .
Cover crop incorporation led to high N 2 O emissions in 50% WFPS soils, which can pose a greater environmental risk compared to the same emissions in soils under water-induced anoxia.This is due to the higher relative gas diffusivity of N 2 O in soil versus air under 50% WFPS, with air-filled macropores, compared to 80% WFPS.The higher diffusivity would facilitate rapid escape of N 2 O from the soil 49,50 , reducing the chances of biological N 2 O reduction to N 2 .Under 80% WFPS, a greater potential exists for further reduction of N 2 O to N 2 before the gas escapes from the soil into the atmosphere 51 .This scenario is particularly plausible in our study due to the depletion of substrates with a higher redox reaction energy yield, such as soil NO − 3 supply under 80% WFPS (Table 2).In response, it is conceivable that microbes resort to using a redox couple with a lower energy yield, forcing greater N 2 O reduction to N 2 24 .Irrespective of the WFPS content, there was no significant difference in cumulative N 2 O production between legume and non-legume cover crops (hairy vetch vs winter wheat, Fig. 3).This finding does not support our second hypothesis, which proposed that co-locating mineral N fertilizer and high-quality vetch cover crop residues would deplete soil O 2 at a faster rate compared to grass wheat cover crop residues, resulting in greater N 2 O emissions.This finding contrasts with several studies that have demonstrated higher N 2 O emissions via denitrification from legume residues compared to grass residues, across different WFPS content 10,54,61 .This discrepancy between studies can be attributed to the following reasons: First, due to the mixing of residues with high level of N fertilizer, NO − 3 levels were never limiting throughout the entire incubation period under 50% WFPS (Table 2).Thus, the NH + 4 supply from vetch residue decomposition and the subsequent N 2 O emissions derived from nitrification were not sufficient to significantly differentiate cumulative N 2 O emissions between treatments with hairy vetch and winter wheat.Second, soil O 2 levels during the incubation period exhibited similar patterns between the different cover crop types under 50% WFPS content (Fig. 4a), suggesting that the easily decomposable C fraction did not differ significantly between treatments.Third, winter wheat C:N ratio (24:1) was not high enough to induce net N immobilization and reduce soil N 2 O emissions 20,62,63 .

Conclusions
Our study illustrates the biogeochemical mechanism of anoxia formation by accelerated microbial respiration following cover crop residue and N fertilizer addition to influence N 2 O emissions.This was achieved by using a novel planar optode sensing technology, enabling high-resolution measurement of soil profile O 2 dynamics in response to residue addition.Under suboptimal WFPS levels for denitrification, N 2 O emissions can be triggered by cover crop residues to a similar or even higher level than in soils experiencing water-induced anoxia that typically promotes large N 2 O emissions from denitrification.Under water induced anaerobic conditions (80% WFPS), cover crops controlled N 2 O emissions via altering labile C availability and had little effect on mineral N availability.Whereas under relatively aerobic conditions (50% WFPS), cover crop residue decomposition consumed soil O 2 to promote anoxia that led to increased N 2 O emissions.This scenario poses a greater environmental risk compared to soils under water-induced anoxia as it enables the rapid escape of N 2 O from soil due to higher diffusivity and reduces the likelihood of biological N 2 O reduction to N 2 .These findings hold crucial implications for managing agricultural systems using cover crops.The respiration-induced anoxia mechanism observed in this study along with cover crop's role in altering coupled soil C, N, and water cycling will drive net soil N 2 O emissions which should be accounted for within the broader context of assessing cover crop impacts on soil health.Elevated N 2 O emissions can offset the benefits of soil C sequestration, often intended when using cover crops.This further highlights the importance of accurately assessing the C footprint of cover crops by quantifying their impacts on N 2 O emissions.
The decoupling of WFPS controls on soil O 2 can be prominent as decomposition rate increases.This poses a formidable challenge in accurately predicting N 2 O emissions, particularly in the context of growing adoption of cover cropping for soil health.Our study suggests that the occurrence of respiration-induced anoxia during cover crop residue decomposition in fertilized soils can disrupt the traditional WFPS controls on N 2 O emissions.Relying solely on WFPS, an imperfect proxy for diffusion-induced O 2 limitation, may lead to the underestimation and inaccurate prediction of potential risks associated with N 2 O emissions, especially in cover crop based fertilized agricultural systems.Therefore, models should incorporate a more comprehensive understanding of these dynamics to enhance predictive accuracy and better capture the complexities of N 2 O emissions in such agroecosystems.

Experimental site and soil sampling
Surface soil samples (0-10 cm depth) were collected from a no-till corn (Zea mays) field at the University of Tennessee's West Tennessee Research and Education Center in Jackson, Tennessee (35° 37′ 22″ N, 88° 50′ 47″ W; elevation 125 m), United States, in August 2022.A total of 50 kg of dry soil were randomly collected from four replicated plots maintained without N fertilization for two years to achieve a low background soil N level.The study site soil is classified as a Lexington silt loam (fine-silty, mixed, thermic Ultic Hapludalfs), organic matter was 15.5 g kg −1 , total N was 0.85 g kg −1 , and pH (H 2 O) was 6.3.The moist soil was thoroughly mixed, air dried, sieved (< 6 mm), and stored at 21 °C until the experiment started.At the start of the incubation experiment, the soil contained 0.6 mg kg −1 NO − 3 and 7.1 mg kg −1 ammonium-N NH + 4 .

Cover crop biomass sampling
Two cover crops were included in this study, (1) hairy vetch (Vicia villosa), which is a legume with a low C/N ratio (10:1), and (2) winter wheat (Triticum aestivum L.), which is a cereal with a relatively higher C/N ratio (24:1).Above ground cover crop biomass was sampled at approximately 2 cm above the soil surface in April 2022, just before cover crop termination.Oven dried (60 °C for 48 h) samples were cut into 5 mm pieces and stored until the start of the incubation experiment.Total C and N concentrations of ground residue subsamples were determined using an Elementar vario Max cube CN analyzer (Elementar, Hanau, Germany).

Incubation experimental set up
A three-week long incubation experiment was established in a randomized complete block design with three factors: two levels of N addition (control 0 N and equivalent rate of 160 kg N ha −1 as K 15 NO 3 at 10 atom % excess), three levels of cover crop residue addition (hairy vetch, winter wheat, and no cover crop) and two levels of WFPS (50% and 80%).The experimental design resulted in eight treatments as follows: T1: 50% WFPS + hairy www.nature.com/scientificreports/vetch + 160 kg N ha −1 , T2: 50% WFPS + wheat + 160 kg N ha −1 , T3: 50% WFPS + no cover crop + 160 kg N ha −1 , T4: 50% WFPS (0 N and no cover crop addition as Control), T5: 80% WFPS + hairy vetch + 160 kg N ha −1 , T6: 80% WFPS + wheat + 160 kg N ha −1 , T7: 80% WFPS + no cover crop + 160 kg N ha −1 , and T8: 80% WFPS (0 N and no cover crop addition as Control).Cover crop residues were added at an equivalent rate of 3 Mg dry matter (DM) ha −1 , a typical biomass production for spring cover crops in Tennessee under desirable weather conditions.Four replicates were prepared for each of the 8 treatments and four additional sets of samples were included for five-time points destructive soil samplings, with a total of 160 (32 for gas sampling + 128 for destructive soil sampling) experimental units.Soil cores (5 cm w × 5 cm l × 10 cm h) were packed at a bulk density of 1.2 g cm −3 in 15 cm long rectangular transparent acrylic liners.The soil and cover crops residues were weighed (300 g dry soil and 0.750 g DM residue equivalent to 3 Mg DM ha −1 for treatments receiving cover crops), mixed, and added to each acrylic liner.Experimental units were pre-incubated in the dark for 72 h with a soil water content amended to achieve the targeted 50% and 80% WFPS, saving 10 mL of water to dissolve the N fertilizer.Water was added from the bottom end of the cores, which were sealed with parafilm with small holes to prevent soil leakage but allows wetting through capillary rise.The top end of plastic liners was covered with perforated parafilm to minimize evaporation, which was removed 1 h before gas sampling.
At the start of incubation, 0.289 g K 15 NO 3 (10 atom % excess 15 N), equivalent to the recommended 160 kg N ha −1 , was dissolved in 10 mL de-ionized water and applied to the fertilized treatments with a syringe from the top (133 mg N kg −1 soil was added as KNO 3 ).We used K 15 NO 3 as fertilizer source to facilitate testing our hypothesis that anoxia from residue decomposition would promote N 2 O production from denitrification (i.e., reduction of NO − 3 ) independent of WFPS conditions.All experimental units were incubated in the dark at 21 °C until gas sampling for 21 consecutive days and kept at a constant %WFPS level for the duration of the incubation experiment by adding water based on daily weight losses of the cores.

Gas sampling, analysis, and N 2 O source calculations
Gas samples were taken on days 0 (12 h after N fertilizer addition), 1, 2, 3, 4, 5, 7, 9, 11, 13, 16, and 21.Each core was transferred to a 2 L jar and closed with a lid that contains a septum for gas sampling.Headspace samples (120-mL) were withdrawn from the jars at 60 and 120 min after closure.Three additional samples of lab air were taken each sampling day.The 60 mL syringe was plunged 3 times to mix the gas in the 2L chamber before final collection.The collected gas samples were injected into pre-evacuated 100 mL crimp top clear serum vials and analyzed for CO 2 and N 2 O on a Delta + XL mass spectrometer (Thermo Finnigan) coupled with a Precon and Gasbench II (ThermoScientific).Overpressure in the vials allowed for sequential sampling of the gas sample for the two gases.CO 2 was measured and then the overpressure in the jar was vented.The entire volume of the vial gas was then transferred to a liquid N cooled trap for N 2 O measurement.
The fluxes of CO 2 and N 2 O were calculated from the increasing gas concentrations during the 120-min headspace closure.The cumulative gas emissions during the 21-day experiment were calculated by linear interpolation of daily emissions.
The 15 N abundance in N 2 O was determined using the same headspace samples.The relative contributions of N 2 O by fertilizer-N (f N2O_fertilizer ) and other-N sources (SOM + cover crop residues, f N2O_Other ) were determined with a mixing model as follows: where δ 15 N Treatment is the measured δ 15 N of the total N 2 O, δ 15 N fertilizer is the δ 15 N of KNO 3 (10 atom % excess 15 N), and δ 15 N Other is the δ 15 N in the N 2 O produced by the SOM and/or cover crop residues.Since the natural enrichment in 15 N between soil and cover crop residues is small compared to the 15 N enrichment in the labelled N fertilizer, the isotopic composition of the N 2 O produced by the other sources was assumed to be the value measured in the control treatments (T4 and T8).

Soil O 2 measurement
Spatially resolved soil O 2 dynamics, measured as % air saturation, was monitored using planar optode technology of VisiSensTM A1 system (PreSens GmbH, Germany) with a portable detector unit DU01 containing a Universal Serial Bus (USB) microscope [64][65][66] .Each incubation core for gas sampling had an O 2 sensor foil (SF-RPSu4) attached to the inner side of the acrylic liner wall throughout the length of the 10-cm soil column that allowed the high-resolution 2-D imaging of soil O 2 saturation.Briefly, the O 2 sensor foil contains fluorescent dyes sensitive to soil O 2 concentration that, when exposed to the LED light from the detector unit, emit fluorescence of specific wavelengths that are captured by the microscope, which translates the data into color images.The images were taken immediately after gas sampling and under dark conditions.Prior to starting the measurements, calibration was performed with identical ambient and temperature conditions as experimental readings.Calibration was performed using a two-point calibration method as recommended by the manufacture's instruction manual, where a solution of oxygen-free water (0% air saturation) was used as first calibration point and ambient air (100% air saturation) was used as second calibration point.Three images were captured in each acrylic liner by placing the detector in the sensor foil at 0-3, 3-6, and 6-9 cm depths to ensure the measurement of real-time high resolution spatial distribution of O 2 along the soil profile.Image processing was performed using ViSiens Imaging System Software (version VA1.12).A graphical description of the measurement setup is provided in Supplementary Fig S1 .For a more detailed description, refer to Keiluweit et al. 59 .

Figure 1 .
Figure 1.Daily nitrous oxide (N 2 O) emissions (a, b) and N 2 O emissions derived from N fertilizer (c, d) over the incubation period from four cover crops treatments at 50% WFPS (a, c), and 80% WFPS (b, d).Nitrogen fertilizer was added at the beginning of the incubation experiment as indicated by black arrow in panels (a) and (b).Bars in panels (a) and (b) indicate mean standard error.HV, Hairy vetch; WW, Winter wheat; No cover, No cover crop; No cover + 0 N, Control.

Figure 3 .
Figure 3. Cumulative N 2 O emissions over the experiment incubation period derived from the fertilizer (yellow) and soil organic matter and/or cover crops (brown) for four cover crops treatments at 50% and 80% WFPS.The error bars represent the mean standard errors for the total N 2 O emissions, and the percentage shown in the columns represents the proportion of cumulative total N 2 O emissions from each source.Uppercase letters indicate significant differences (p < 0.05) in cumulative N 2 O emissions and N 2 O emissions derived from N fertilizer among the treatments, while lowercase letters indicate differences in N 2 O emissions derived from SOM and/or cover crops among the treatments.HV, Hairy vetch; WW, Winter wheat; NC, No cover crop; NC + 0 N, Control.

Figure 4 .
Figure 4. Average soil O 2 content expressed as percentage of air saturation at 0 to 3 cm depth over the incubation period from four cover crops treatments at (a) 50% WFPS and (b) 80% WFPS.Bars represent the mean standard error.HV, Hairy vetch; WW, Winter wheat; No Cover, No cover crop; No cover + 0 N, Control.

Figure 5 .
Figure 5. Selected images of O 2 content expressed as percentage of air saturation in 0-3 cm soil depth over the incubation period from two treatments: (A) 50% WFPS with winter wheat + 160 N, (B) 50% WFPS with no cover crop + 160 N. Images (one of the four replicates) illustrate the maximum achieved O 2 depletion effect resulting from cover crop addition.

Table 1 .
Cumulative CO 2 emissions from all eight treatments after 21-day incubation.*Values are means and standard errors in parenthesis.Lowercase letters indicate significant differences among the treatments (p < 0.05).

Table 2 .
Nitrate ( NO − 3 ) and ammonium ( NH + 4 ) concentration over the incubation period for four cover crop treatments at 50% and 80% WFPS.HV, Hairy vetch; WW, Winter wheat; NC, No cover crop; NC + 0 N, Control.*Values are means and standard errors in parenthesis.Lowercase letters indicate significant differences among the treatments (p < 0.05) within a column.

Table 4 .
Features importance to predict N 2 O emissions under 50% and 80% WFPS conditions as predicted by the Random Forest model.Model R 2 value indicates variability explained on the test data set.*Values in the table rank the relevance of features in the model based on the Gini importance or mean decrease impurity.