Denitrification in Agricultural Soils:
Integrated control and Modelling at various scales (DASIM)

Abstract:

Biogeochemical models are essential for the prediction and management of nitrogen (N) cycling in agroecosystems, but the accuracy of the denitrification and decomposition sub-modules is critical. Current models were developed before suitable soil N2 flux data were available, which may have led to inaccuracies in how denitrification was described. New measurement techniques, using gas chromatography and isotope-ratio mass spectrometry (IRMS), have enabled the collection of more robust N2, N2O and CO2 data. We incubated two arable soils – a silt-loam and a sand soil – for 34 and 58?d, respectively, with small field-relevant changes made to control factors during this period. For the silt-loam soil, seven treatments varying in moisture, bulk density and contents were included, with temperature changing during the incubation. The sandy soil was incubated with and without incorporation of litter (ryegrass), with temperature, water content and content changing during the incubation. The denitrification and decomposition sub-modules of DeNi, Coup and DNDC were tested using the data. No systematic calibration of the model parameters was conducted since our intention was to evaluate the general model structure or “default” model runs. Measured fluxes generally responded as expected to control factors. We assessed the direction of modeled responses to control factors using three categories: no response, a response in the same direction as measurements or a response in the opposite direction to measurements. DNDC responses were 14?%, 52?% and 34?%, respectively. Coup responses were 47?%, 19?% and 34?%, respectively. DeNi responses were 0?%, 67?% and 33?%, respectively. The magnitudes of the modeled fluxes were underestimated by Coup and DNDC and overestimated by DeNi for the sandy soil, while there was no general trend for the silt-loam soil. None of the models was able to determine litter-induced decomposition correctly. To conclude, the currently used sub-modules are not able to consistently simulate the denitrification and decomposition processes. For better model evaluation and development, we need to design better experiments, take more frequent measurements, use new or updated measurement techniques, address model complexity, add missing processes to the models, calibrate denitrifier microbial dynamics, and evaluate the anaerobic soil volume concept.

Abstract:

Ntrace is a family of tools for the analysis of 15N tracing data sets, to quantify the simultaneous gross nitrogen (N) transformation rates in terrestrial ecosystems. Starting with the original publication of Ntrace in 2007, a number of specialised tools have been developed that cover a range of applications such as nitrite and gaseous N dynamics. While the capability of the tool has been extended to cover a range of applications, the underlying parameter optimisation algorithm, based on the Metropolis MCMC algorithm, has remained unchanged. However, in more computational demanding applications, this algorithm turned out to be very time consuming, and in some cases it was not even able to identify unambiguous parameter sets. The aim of this study was, to test a new global optimisation algorithm (GlobalSearch), by re-analysing some of the published datasets. In the evaluation, the speed of finding a suitable solution but in particular the accuracy of the new algorithm to obtain an adequate fit to the observations was taken into account. A new uncertainty calculation for the optimised parameters, based on the Levenberg-Marquardt method, has also been implemented. The new Ntrace tool was able to find an adequate fit for all tested datasets, in often a fraction of the time compared to the original MCMC algorithm. For all test cases the accuracy of the fit was at least as good as with the original model. This is partly due to a new algorithm's option to vary the initial pool size. In summary, the new Ntrace tool is quicker in finding suitable parameters for 15N tracing data sets. The fit to measured data is often improved over the original tool. Thus, we recommend the use of the new Ntrace tool for analysing 15N tracing data. The new tool is programmed in MATLAB and can easily be adapted to new applications.

Abstract:

Nitrite (NO2?) is a crucial compound in the N soil cycle. As an intermediate of nearly all N transformations, its isotopic signature may provide precious information on the active pathways and processes. NO2? analyses have already been applied in 15N tracing studies, increasing their interpretation perspectives. Natural abundance NO2? isotope studies in soils were so far not applied and this study aims at testing if such analyses are useful in tracing the soil N cycle. We conducted laboratory soil incubations with parallel natural abundance and 15N treatments, accompanied by isotopic analyses of soil N compounds (NO3?, NO2?, NH4+). The double 15N tracing method was used as a reference method for estimations of N transformation processes based on natural abundance nitrite dynamics. We obtained a very good agreement between the results from nitrite isotope model proposed here and the 15N tracing approach. Natural abundance nitrite isotope studies are a promising tool to our understanding of soil N cycling.

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/?18O mapping approach (SP/?18O 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/?18O 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.

Abstract:

The prediction of nitrous oxide (N2O) and of dinitrogen (N2) emissions formed by biotic denitrification in soil is notoriously difficult due to challenges in capturing co-occurring processes at microscopic scales. N2O production and reduction depend on the spatial extent of anoxic conditions in soil, which in turn are a function of oxygen (O2) supply through diffusion and O2 demand by respiration in the presence of an alternative electron acceptor (e.g. nitrate). This study aimed to explore controlling factors of complete denitrification in terms of N2O and (N2O?+?N2) fluxes in repacked soils by taking micro-environmental conditions directly into account. This was achieved by measuring microscale oxygen saturation and estimating the anaerobic soil volume fraction (ansvf) based on internal air distribution measured with X-ray computed tomography (X-ray CT). O2 supply and demand were explored systemically in a full factorial design with soil organic matter (SOM; 1.2?% and 4.5?%), aggregate size (2–4 and 4–8?mm), and water saturation (70?%, 83?%, and 95?% water-holding capacity, WHC) as factors. CO2 and N2O emissions were monitored with gas chromatography. The 15N gas flux method was used to estimate the N2O reduction to N2. N gas emissions could only be predicted well when explanatory variables for O2 demand and O2 supply were considered jointly. Combining CO2 emission and ansvf as proxies for O2 demand and supply resulted in 83?% explained variability in (N2O?+?N2) emissions and together with the denitrification product ratio [N2O?/ ?(N2O?+?N2)] (pr) 81?% in N2O emissions. O2 concentration measured by microsensors was a poor predictor due to the variability in O2 over small distances combined with the small measurement volume of the microsensors. The substitution of predictors by independent, readily available proxies for O2 demand (SOM) and O2 supply (diffusivity) reduced the predictive power considerably (60?% and 66?% for N2O and (N2O+N2) fluxes, respectively). The new approach of using X-ray CT imaging analysis to directly quantify soil structure in terms of ansvf in combination with N2O and (N2O?+?N2) flux measurements opens up new perspectives to estimate complete denitrification in soil. This will also contribute to improving N2O flux models and can help to develop mitigation strategies for N2O fluxes and improve N use efficiency.

Abstract:

Water-extractable organic carbon (WEOC) is considered as the most important carbon (C) source for denitrifying organisms, but the contribution of individual organic matter (OM) fractions (i.e., particulate (POM) and mineral-associated (MOM)) to its release and, thus, to denitrification remains unresolved. Here we tested short-time effects of POM and MOM on potential denitrification and estimated the contribution of POM- and MOM-derived WEOC to denitrification and CO2 production of three agricultural topsoils. Suspensions of bulk soils with and without addition of soil-derived POM or MOM were incubated for 24 h under anoxic conditions. Acetylene inhibition was used to determine the potential denitrification and respective product ratio at constant nitrate supply. Normalized to added OC, effects of POM on CO2 production, total denitrification, and its product ratios were much stronger than those of MOM. While the addition of OM generally increased the (N2O + N2)-N/CO2-C ratio, the N2O/(N2O + N2) ratio changed differently depending on the soil. Gas emissions and the respective shares of initial WEOC were then used to estimate the contribution of POM and MOM-derived WEOC to total CO2, N2O, and N2O + N2 production. Water-extractable OC derived from POM accounted for 53–85% of total denitrification and WEOC released from MOM accounted for 15–47%. Total gas emissions from bulk soils were partly over- or underestimated, mainly due to nonproportional responses of denitrification to the addition of individual OM fractions. Our findings show that MOM plays a role in providing organic substrates during denitrification but is generally less dominant than POM. We conclude that the denitrification potential of soils is not predictable based on the C distribution over POM and MOM alone. Instead, the source strength of POM and MOM for WEOC plus the WEOC’s quality turned out as the most decisive determinants of potential denitrification.

Abstract:

Denitrification usually takes place under anoxic conditions and over short periods of time, and depends on readily available nitrate and carbon sources. Variations in CO2 and N2O emissions associated with plant residues have mainly been explained by differences in their decomposability. A factor rarely considered so far is water-extractable organic matter (WEOM) released to the soil during residue decomposition. Here, we examined the potential effect of plant residues on denitrification with special emphasis on WEOM. A range of fresh and leached plant residues was characterized by elemental analyses, 13C-NMR spectroscopy, and extraction with ultrapure water. The obtained solutions were analyzed for the concentrations of organic carbon (OC) and organic nitrogen (ON), and by UV-VIS spectroscopy. To test the potential denitrification induced by plant residues or three different OM solutions, these carbon sources were added to soil suspensions and incubated for 24 h at 20 °C in the dark under anoxic conditions; KNO3 was added to ensure unlimited nitrate supply. Evolving N2O and CO2 were analyzed by gas chromatography, and acetylene inhibition was used to determine denitrification and its product ratio. The production of all gases, as well as the molar (N2O + N2)–N/CO2–C ratio, was directly related to the water-extractable OC (WEOC) content of the plant residues, and the WEOC increased with carboxylic/carbonyl C and decreasing OC/ON ratio of the plant residues. Incubation of OM solutions revealed that the molar (N2O + N2)–N/CO2–C ratio and share of N2O are influenced by the WEOM's chemical composition. In conclusion, our results emphasize the potential of WEOM in largely undecomposed plant residues to support short-term denitrification activity in a typical ?hot spot–hot moment? situation.

Abstract:

Content and quality of organic matter (OM) may strongly affect the denitrification potential of soils. In particular, the impact of soil OM fractions of differing bioavailability (soluble, particulate, and mineral-associated OM) on denitrification remains unresolved. We determined the potential N2O and N2 as well as CO2 production for samples of a Haplic Chernozem from six treatment plots (control, mineral N and NP, farmyard manure - FYM, and FYM + mineral N or NP) of the Static Fertilization Experiment Bad Lauchstädt (Germany) as related to OM properties and denitrifier gene abundances. Soil OM was analyzed for bulk chemical composition (13C-CPMAS NMR spectroscopy) as well as water-extractable, particulate, and mineral-associated fractions. Soils receiving FYM had more total OM and larger portions of labile fractions such as particulate and water-extractable OM. Incubations were run under anoxic conditions without nitrate limitation for seven days at 25 °C in the dark to determine the denitrification potential (N2O and N2) using the acetylene inhibition technique. Abundances of nirS, nirK, and nosZ (I + II) genes were analyzed before and after incubation. The denitrification potential, defined as the combined amount of N released as N2O + N2 over the experimental period, was larger for plots receiving FYM (25.9–27.2 mg N kg?1) than pure mineral fertilization (17.1–19.2 mg N kg?1) or no fertilization (12.6 mg N kg?1). The CO2 and N2O production were well related and up to three-fold larger for FYM-receiving soils than under pure mineral fertilization. The N2 production differed significantly only between all manured and non-manured soils. Nitrogenous gas emissions related most closely to water-extractable organic carbon (WEOC), which again related well to free particulate OM. The larger contribution of N2 production in soils without FYM application, and thus, with less readily decomposable OM, coincided with decreasing abundances of nirS genes (NO2? reductase) and increasing abundances of genes indicating complete denitrifying organisms (nosZ I) during anoxic conditions. Limited OM sources, thus, favored a microbial community more efficient in resource use. This study suggests that WEOC, representing readily bioavailable OM, is a straightforward indicator of the denitrification potential of soils. • Largest N2O and CO2 emissions from manured soils rich in labile organic matter • N2 production was less affected by changes in soil organic matter than N2O production. • Denitrification potential and share of N2O related closely to water-extractable OC. • Water-extractable OC likely derived from N-rich free particulate organic matter. • Limited OC availability favored abundances of complete denitrifiers possessing nosZ I.

Abstract:

Returning crop residues to agricultural fields can accelerate nutrient turnover and increase N2O and NO emissions. Increased microbial respiration may lead to formation of local hotspots with anoxic or microoxic conditions promoting denitrification. To investigate the effect of litter quality on CO2, NO, N2O, and N2 emissions, we conducted a laboratory incubation study in a controlled atmosphere (He/O2, or pure He) with different maize litter types (Zea mays L., young leaves and roots, straw). We applied the N2O isotopocule mapping approach to distinguish between N2O emitting processes and partitioned the CO2 efflux into litter- and soil organic matter (SOM)-derived CO2 based on the natural 13C isotope abundances. Maize litter increased total and SOM derived CO2 emissions leading to a positive priming effect. Although C turnover was high, NO and N2O fluxes were low under oxic conditions as high O2 diffusivity limited denitrification. In the first week, nitrification contributed to NO emissions, which increased with increasing net N mineralization. Isotopocule mapping indicated that bacterial processes dominated N2O formation in litter-amended soil in the beginning of the incubation experiment with a subsequent shift towards fungal denitrification. With onset of anoxic incubation conditions after 47 days, N fluxes strongly increased, and heterotrophic bacterial denitrification became the main source of N2O. The N2O/(N2O+N2) ratio decreased with increasing litter C:N ratio and Corg:NO3? ratio in soil, confirming that the ratio of available C:N is a major control of denitrification product stoichiometry.

Abstract:

Background and aims Plant growth affects soil moisture, mineral N and organic C availability in soil, all of which influence denitrification. With increasing plant growth, root exudation may stimulate denitrification, while N uptake restricts nitrate availability. Methods We conducted a double labeling pot experiment with either maize (Zea mays L.) or cup plant (Silphium perfoliatum L.) of the same age but differing in size of their shoot and root systems. The 15N gas flux method was applied to directly quantify N2O and N2 fluxes in situ. To link denitrification with available C in the rhizosphere, 13CO2 pulse labeling was used to trace C translocation from shoots to roots and its release by roots into the soil. Results Plant water and N uptake were the main factors controlling daily N2O?+?N2 fluxes, cumulative N emissions, and N2O production pathways. Accordingly, pool-derived N2O?+?N2 emissions were 30–40 times higher in the treatment with highest soil NO3? content and highest soil moisture. CO2 efflux from soil was positively correlated with root dry matter, but we could not detect any relationship between root-derived C and N2O?+?N2 emissions. Conclusions Root-derived C may stimulate denitrification under small plants, while N and water uptake become the controlling factors with increasing plant and root growth.

Abstract:

The 15N gas-flux method allows for the quantification of N2 flux and tracing soil N transformations. An important requirement for this method is a homogeneous distribution of the 15N tracer added to soil. This is usually achieved through soil homogenization and admixture of the 15N tracer solution or multipoint injection of tracer solution to intact soil. Both methods may create artefacts. We aimed at comparing the N2 flux determined by the gas-flux method using both tracer distribution approaches. Soil incubation experiments with silt loam soil using (i) intact soil cores injected with 15N label solution, (ii) homogenized soil with injected label solution, and (iii) homogenized soil with admixture of label solution were performed. Intact soil cores with injected 15N tracer solution show a larger variability of the results. Homogenized soil shows better agreement between repetitions, but significant differences in 15N enrichment measured in soil nitrate and in emitted gases were observed. For intact soil, the larger variability of measured values results rather from natural diversity of non-homogenized soil cores than from inhomogeneous label distribution. Generally, comparison of the results of intact cores and homogenized soil did not reveal statistically significant differences in N2 flux determination. In both cases, a pronounced dominance of N2 flux over N2O flux was noted. It can be concluded that both methods showed close agreement, and homogenized soil is not necessarily characterized by more homogenous 15N label distribution.

Abstract:

Chemical composition of root and shoot litter controls decomposition and, subsequently, C availability for biological nitrogen transformation processes in soils. While aboveground plant residues have been proven to increase N2O emissions, studies on root litter effects are scarce. This study aimed (1) to evaluate how fresh maize root litter affects N2O emissions compared to fresh maize shoot litter, (2) to assess whether N2O emissions are related to the interaction of C and N mineralization from soil and litter, and (3) to analyze changes in soil microbial community structures related to litter input and N2O emissions. To obtain root and shoot litter, maize plants (Zea mays L.) were cultivated with two N fertilizer levels in a greenhouse and harvested. A two-factorial 22?d laboratory incubation experiment was set up with soil from both N levels (N1, N2) and three litter addition treatments (control, root, root?+?shoot). We measured CO2 and N2O fluxes, analyzed soil mineral N and water-extractable organic C (WEOC) concentrations, and determined quality parameters of maize litter. Bacterial community structures were analyzed using 16S rRNA gene sequencing. Maize litter quality controlled NO?3 and WEOC availability and decomposition-related CO2 emissions. Emissions induced by maize root litter remained low, while high bioavailability of maize shoot litter strongly increased CO2 and N2O emissions when both root and shoot litter were added. We identified a strong positive correlation between cumulative CO2 and N2O emissions, supporting our hypothesis that litter quality affects denitrification by creating plant-litter-associated anaerobic microsites. The interdependency of C and N availability was validated by analyses of regression. Moreover, there was a strong positive interaction between soil NO?3 and WEOC concentration resulting in much higher N2O emissions, when both NO?3 and WEOC were available. A significant correlation was observed between total CO2 and N2O emissions, the soil bacterial community composition, and the litter level, showing a clear separation of root?+?shoot samples of all remaining samples. Bacterial diversity decreased with higher N level and higher input of easily available C. Altogether, changes in bacterial community structure reflected degradability of maize litter with easily degradable C from maize shoot litter favoring fast-growing C-cycling and N-reducing bacteria of the phyla Actinobacteria, Chloroflexi, Firmicutes, and Proteobacteria. In conclusion, litter quality is a major driver of N2O and CO2 emissions from crop residues, especially when soil mineral N is limited.

Abstract:

Soil denitrification is the most important terrestrial process returning reactive nitrogen to the atmosphere, but remains poorly understood. In upland soils, denitrification occurs in hotspots of enhanced microbial activity, even under well-aerated conditions, and causes harmful emissions of nitric (NO) and nitrous oxide (N2O). The timing and magnitude of such emissions are difficult to predict due to the delicate balance of oxygen (O2) consumption and diffusion in soil. To study how spatial distribution of hotspots affects O2 exchange and denitrification, we embedded microbial hotspots composed of porous glass beads saturated with growing cultures of either Agrobacterium tumefaciens (a denitrifier lacking N2O reductase) or Paracoccus denitrificans (a “complete” denitrifier) in different architectures (random vs. layered) in sterile sand that was adjusted to different water saturations (30?%, 60?%, 90?%). Gas kinetics (O2, CO2, NO, N2O and N2) were measured at high temporal resolution in batch mode. Air connectivity, air distance and air tortuosity were determined by X-ray tomography after the experiment. The hotspot architecture exerted strong control on microbial growth and timing of denitrification at low and intermediate saturations, because the separation distance between the microbial hotspots governed local oxygen supply. Electron flow diverted to denitrification in anoxic hotspot centers was low (2?%–7?%) but increased markedly (17?%–27?%) at high water saturation. X-ray analysis revealed that the air phase around most of the hotspots remained connected to the headspace even at 90?% saturation, suggesting that the threshold response of denitrification to soil moisture could be ascribed to increasing tortuosity of air-filled pores and the distance from the saturated hotspots to these air-filled pores. Our findings suggest that denitrification and its gaseous product stoichiometry depend not only on the amount of microbial hotspots in aerated soil, but also on their spatial distribution. We demonstrate that combining measurements of microbial activity with quantitative analysis of diffusion lengths using X-ray tomography provides unprecedented insights into physical constraints regulating soil microbial respiration in general and denitrification in particular. This paves the way to using observable soil structural attributes to predict denitrification and to parameterize models. Further experiments with natural soil structure, carbon substrates and microbial communities are required to devise and parametrize denitrification models explicit for microbial hotspots.

Abstract:

Highlights • An isotope based model to quantify N2O reduction to N2 at field scales are presented. • Model evaluation were conducted based on seven independent studies. • The model performance strongly differed between studies and incubation conditions. • Using soils-specific instead endmember values largely improve model performance. Abstract The last step of denitrification, i.e. the reduction of N2O to N2, has been intensively studied in the laboratory to understand the denitrification process, predict nitrogen fertiliser losses, and to establish mitigation strategies for N2O. However, assessing N2 production via denitrification at large spatial scales is still not possible due to lack of reliable quantitative approaches. Here, we present a novel numerical “mapping approach” model using the ?15Nsp/?18O slope that has been proposed to potentially be used to indirectly quantify N2O reduction to N2 at field or larger spatial scales. We evaluate the model using data obtained from seven independent soil incubation studies conducted under a He–O2 atmosphere. Furthermore, we analyse the contribution of different parameters to the uncertainty of the model. The model performance strongly differed between studies and incubation conditions. Re-evaluation of the previous data set demonstrated that using soils-specific instead of default endmember values could largely improve model performance. Since the uncertainty of modelled N2O reduction was relatively high, further improvements to estimate model parameters to obtain more precise estimations remain an on-going matter, e.g. by determination of soil-specific isotope fractionation factors and isotopocule endmember values of N2O production processes using controlled laboratory incubations. The applicability of the mapping approach model is promising with an increasing availability of real-time and field based analysis of N2O isotope signatures.

Abstract:

Highlights •N2O production rates via denitrification declined with decreasing temperature. •N2O production rates via autotrophic nitrification were significantly and positively correlated with incubation temperature. •N2O production rates via heterotrophic nitrification also showed a significantly positive correlation with temperature. •The results in the field experiments were corresponded to the laboratory results. Nitrous oxide (N2O) is an important greenhouse gas and contributes to stratospheric ozone depletion. Increasing temperature generally exerts a positive effect on soil N2O production. However, not much is known on the temperature influence on individual N2O production pathways. In this study, both laboratory 15N labelling experiments with an incubation temperature gradient (35?°C, 25?°C, 15?°C, 5?°C) and field 15N labelling experiments carried out in different seasons were conducted in Korean pine forest (KF) and Redwood coniferous forest (RF) soils. The results showed that the contribution of denitrification was positively correlated with temperature in KF and negatively correlated with temperature in RF, while their N2O production rates via denitrification (N2Od) all declined with decreasing temperature. The contribution of autotrophic nitrification in KF ranged from 11% to 21%, while the contribution in RF significantly increased with decreasing temperature (P?<?0.05). However, the N2O production rates via autotrophic nitrification process (N2Oa) were significantly and positively correlated with incubation temperature (P?<?0.05). In addition, the contribution of heterotrophic nitrification to N2O production showed a negative and positive relation with increasing temperature in KF and RF, respectively. Whereas, the N2O production rates via heterotrophic nitrification (N2Oh) showed a significantly positive correlation with temperature (P?<?0.05), but a negative relation with gross heterotrophic nitrification rates. The results in the field experiments corresponded to the laboratory results, indicating that the methods applied in field experiments were suitable for the estimation and prediction of in situ N2O production. The response of calculated N2O production rates to seasonal temperature in KF during the year of 2015–2017 also confirmed the suitability of the field research methods. This novel in situ technique to determine N2O production in temperate forest soils should be validated for other ecosystems.

Abstract:

Microbial denitrification is the primary driver of nitrogen losses from the plant-soil system and the key process for the closure of the global N cycle. All major controls of denitrification might be directly or indirectly affected by plants. However, there is a lack of research of the direct effects of plants on soil denitrification and how this effect might be mediated by soil properties. This study assesses the effect of three common crop species and two agricultural soils on denitrification potentials. We conducted a factorial experiment under controlled conditions to analyze the effects of (1) different plant species (barley, wheat or ryegrass), (2) two different soils (texture/ SOC) and (3) two different soil moisture levels on Denitrification Enzyme Activity (DEA) in bulk and rhizosphere soil. The SOC richer clay loam soil showed on average higher DEA (+81%) compared to the SOC poorer silty loam soil. All three plants were found to stimulate denitrification with significant differences between certain species: rye grass (+92%?±?14%)???barley (+75%?±?26%)???wheat (+50%?±?19%). DEA in agricultural soils is interactively controlled by plant species and soil type with an overall stimulating effect of plants on the denitrification potential. Future research should focus on disentangling single mechanisms of plant control on actual denitrification rates and N gas product ratios.

Abstract:

Common methods for measuring soil denitrification in situ include monitoring the accumulation of 15N-labelled N2 and N2O evolved from 15N-labelled soil nitrate pool in closed chambers that are placed on the soil surface. Gas diffusion is considered to be the main transport process in the soil. Because accumulation of gases within the chamber decreases concentration gradients between soil and the chamber over time, the surface efflux of gases decreases as well, and gas production rates are underestimated if calculated from chamber concentrations without consideration of this mechanism. Moreover, concentration gradients to the non-labelled subsoil exist, inevitably causing downward diffusion of 15N-labelled denitrification products. A numerical 3-D model for simulating gas diffusion in soil was used in order to determine the significance of this source of error. Results show that subsoil diffusion of 15N-labelled N2 and N2O – and thus potential underestimation of denitrification derived from chamber fluxes – increases with chamber deployment time as well as with increasing soil gas diffusivity. Simulations based on the range of typical soil gas diffusivities of unsaturated soils showed that the fraction of N2 and N2O evolved from 15N-labelled NO?3 that is not emitted at the soil surface during 1?h chamber closing is always significant, with values up to >50?% of total production. This is due to accumulation in the pore space of the 15N-labelled soil and diffusive flux to the unlabelled subsoil. Empirical coefficients to calculate denitrification from surface fluxes were derived by modelling multiple scenarios with varying soil water content. Modelling several theoretical experimental set-ups showed that the fraction of produced gases that are retained in soil can be lowered by lowering the depth of 15N labelling and/or increasing the length of the confining cylinder. Field experiments with arable silt loam soil for measuring denitrification with the 15N gas flux method were conducted to obtain direct evidence for the incomplete surface emission of gaseous denitrification products. We compared surface fluxes of 15N2 and 15N2O from 15N-labelled micro-plots confined by cylinders using the closed-chamber method with cylinders open or closed at the bottom, finding 37?% higher surface fluxes with the bottom closed. Modelling fluxes of this experiment confirmed this effect, however with a higher increase in surface flux of 89?%. From our model and experimental results we conclude that field surface fluxes of 15N-labelled N2 and N2O severely underestimate denitrification rates if calculated from chamber accumulation only. The extent of this underestimation increases with closure time. Underestimation also occurs during laboratory incubations in closed systems due to pore space accumulation of 15N-labelled N2 and N2O. Due to this bias in past denitrification measurements, denitrification in soils might be more relevant than assumed to date. Corrected denitrification rates can be obtained by estimating subsurface flux and storage with our model. The observed deviation between experimental and modelled subsurface flux revealed the need for refined model evaluation, which must include assessment of the spatial variability in diffusivity and production and the spatial dimension of the chamber.

Abstract:

Highlights • 3D crack dynamics in structured soil during WD cycles observed with X-ray CT • Soil structure dynamics measured via structure labeling with garnet particles • Soil structure dynamics dependent on, bulk density, SOM and clay content • Higher SOM content led to a higher density of cracks with smaller aperture • Soil structure dynamics is negligible due to reactivation of old cracks Soil structure is not static but undergoes continuous changes due to a wide range of biotic and abiotic drivers such as bioturbation and the mechanical disturbance by tillage. This continuous alteration of soil structure beyond the pure swelling and shrinking of some stable structure is what we refer to as soil structure dynamics. It has important consequences for carbon turnover in soil as it controls how quickly soil organic matter gets occluded from or exposed to mineralization. So far there are hardly any direct observations of the rate at which soil pores are formed and destroyed. Here we employ are recently introduced labeling approach for soil structure that measures how quickly the locations of small garnet particles get randomized in soil as a measure for soil structure dynamics. We investigate the effect of desiccation crack dynamics on pore space attributes in general and soils structure turnover in particular using X-ray microtomography for repeated wetting-drying cycles. This is explored for three different soils with a range of soil organic matter content, clay content and different clay mineralogy that were sieved to a certain aggregate size fraction (0.63–2?mm) and repacked at two different bulk density levels. The total magnitude of desiccation crack formation mainly depended on the clay content and clay mineralogy. Higher soil organic matter content led to a denser crack pattern with smaller aperture. Wetting-drying cycles did not only effect visible macroporosity (>8??m), but also unresolved mesoporosity. The changes in macroporosity were higher at lower bulk density. Most importantly, repeated wetting-drying cycles did not lead to a randomization of distances between garnet particles and pores. This demonstrates that former failure zones are reactivated during subsequent drying cycles. Hence, wetting-drying resulted in reversible particle displacement and therefore would not have triggered the exposure of occluded carbon that was not already exposed during the previous drying event.

Abstract:

The microenvironmental conditions in soil exert a major control on many ecosystem functions of soil. Their investigation in intact soil samples is impaired by methodological challenges in the joint investigation of structural heterogeneity that defines pathways for matter fluxes and biogeochemical heterogeneity that governs reaction patterns and microhabitats. Here we demonstrate how these challenges can be overcome with a novel protocol for correlative imaging based on image registration to combine three-dimensional microstructure analysis of X-ray tomography data with biogeochemical microscopic data of various modalities and scales (light microscopy, fluorescence microscopy, electron microscopy, secondary ion mass spectrometry). Correlative imaging of a microcosm study shows that the majority (75%) of bacteria are located in mesopores (<10 ?m). Furthermore, they have a preference to forage near macropore surfaces and near fresh particulate organic matter. Ignoring the structural complexity coming from the third dimension is justified for metrics based on size and distances but leads to a substantial bias for metrics based on continuity. This versatile combination of imaging modalities with freely available software and protocols may open up completely new avenues for the investigation of many important biogeochemical and physical processes in structured soils.

Abstract:

RATIONALE Isotopic signatures of N2O can help distinguish between two sources (fertiliser N, or endogenous soil N) of N2O emissions. The contribution of each source to N2O emissions after N–application is difficult to determine. Here, isotopologue signatures of emitted N2O are used in an improved isotopic model based on Rayleigh type equations. METHODS The effects of a partial (33% of surface area, treatment 1c) or total (100% of surface area, treatment 3c) dispersal of N and C on gaseous emissions from denitrification were measured in a laboratory incubation system (DENIS) allowing simultaneous measurements of NO, N2O, N2 and CO2 over a 12?day incubation period. To determine the source of N2O emissions those results were combined with both the isotope ratio mass spectrometry analysis of the isotopocules of emitted N2O and the 15N?tracing technique. RESULTS The spatial dispersal of N and C significantly affected the quantity, but not the timing of gas fluxes. Cumulative emissions are larger for 3c than 1c. The 15N?enrichment analysis shows that initially ~70% of the emitted N2O derived from the applied amendment followed by a constant decrease. The decrease in contribution of the fertiliser N?pool after an initial increase is sooner and larger for 1c. The Rayleigh type model applied to N2O isotopocules data (?15Nbulk?N2O values) shows poor agreement with the measurements for the original 1?pool model for 1c; the 2?pool models gives better results when using a third order polynomial equation. In contrast, in 3c little difference is observed between the two modelling approaches. CONCLUSIONS The importance of N2O emissions from different N?pools in soil for the interpretation of N2O isotopocules data was demonstrated using a Rayleigh type model. Earlier statements concerning exponential increase of native soil nitrate pool activity highlighted in previous studies should be replaced with a polynomial increase with dependency on both N?pool sizes.