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© Thünen-Institut/AK
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Institute of

AK Climate-Smart Agriculture


Quantification of denitrification using stable isotopes

Combined application of the natural abundance stable isotope and 15N tracing methods to quantify denitrification in agricultural soils in field studies

The nitrogen losses due to microbial denitrification, namely the conversion of nitrate and nitrite to molecular nitrogen (N2), are important component of soil N cycling. But these cannot be directly quantified in the field studies. Can the isotopic analyses of the intermediate denitrification product, N2O, be helpful to estimate soil N2-fluxes?

Background and Objective

Denitrification is an anaerobic microbial process of successive reduction of nitrate (NO3-) and nitrite (NO2-) to N2 through the following reaction steps: NO3- - NO2- - NO - N2O - N2. The intermediate product, nitrous oxide (N2O), is partially not further reduced to N2. The resulting emission to the atmosphere constitutes the main emission source of this greenhouse gas from agricultural soils.

Quantification of soil denitrification is crucial for mitigating nitrogen fertiliser loss as well as for reducing N2O emission. Commonly applied analytical techniques in field studies enable us to analyse only N2O. However, the measured N2O-fluxes alone do not provide information on the entire N loss by soil denitrification because (i) the emissions due to other microbial processes (nitrification, co-denitrification) are indistinguishable, and (ii) the contribution of N2O reduction to N2 is unknown, since it is difficult to directly measure N2 emissions due to the high atmospheric background.

To overcome this problem, the 15N gas flux method can be applied, which includes stable isotope analyses of gas fluxes after the addition of 15N-labelled NO3-. This method has been applied in numerous laboratory but few field studies, showing that the fraction of N2O reduction to N2 (i.e. the ‘product ratio’: N2O/(N2O+N2)) during denitrification ranging from 0 to 1 is extremely variable. So far, due to technical limitations, no comprehensive data sets from field-based measurements of soil N2 emissions are available. This reinforces the necessity of more in situ studies.

Isotopic analyses of N2O is altered during the partial N2O reduction to N2. The magnitude of the observed change depends largely on the product ratio. Hence, the measurement of isotopic signatures of the emitted N2O hasa great potential to determine the product ratio under field conditions. However, to enable this determination, the characteristic isotopic signatures for produced N2O and the characteristic fractionation factors associated with N2O reduction must be known. The reported ranges of apparent isotopic fractionation factors are very wide and an explanation of these large variations is still ambiguous. Hence, in this project N2O isotopic measurements are coupled with 15N tracing as a reference method. The aim is to ultimately evaluate the N2O isotopic method as a robust quantification method for field studies.

The advantage of the N2O isotopic methods over the 15N tracing method lies in its much lower costs, easier application and avoidance of various artefacts associated with the 15N tracing method. The N2O isotopic method has thus the potential for more widespread use.


To precisely determine the isotopic fractionation factors associated with denitrification and possible accompanying processes valid for field conditions, detailed microcosm experiments under controlled conditions with 15N-tracing as a reference method will be conducted. The fractionation factors associated with N2O reduction will be validated for field conditions through direct determination of the product ratio (N2O/(N2+N2O)) of denitrification from 15N tracing results obtained parallel to N2O isotopic analyses. Moreover, a 15N-tracer model will be used to estimate the extent of the possibly co-occurring N-transformation processes (nitrification, ammonification, immobilisation and ammonia oxidation) and their impact on isotopic values of emitted N2O. Natural abundance studies of soil N compounds carried out in parallel will aim to determine the isotopic fractionation characteristic for these processes. Moreover, the analysis of natural abundance isotopic signatures of soil nitrite will be conducted for the first time in order to better assess the process dynamics of this crucial compound in N-cycling.

As an ultimate result of this project, a complex isotopic model will be developed, which should provide an approach to quantify the whole denitrification process in agricultural soils under field conditions. In perspective, this model can be combined with the process-based modelling of N2O emissions to help in better prediction of N2O reduction in soils.

Our Research Questions

Is the isotopic fractionation during denitrification stable and predictable? Which factors control the magnitude of the respective isotope fractionation factors?

Do other processes beside denitrification significantly influence the isotopic signatures of soil NO3- and of the emitted N2O? Is this a limitation for the field application of the N2O isotopic method?

Can we quantify N2O reduction to N2 based on N2O isotopic signatures? Which requirements have to be taken into account and how exact can this quantification be?


Involved external Thünen-Partners

  • University of Gothenburg
    (Gothenburg, Schweden)

Funding Body

  • Deutsche Forschungsgemeinschaft (DFG)
    (national, öffentlich)


4.2015 - 3.2018

More Information

Project funding number: LE 3367/1-1
Project status: finished


  1. 0

    Cárdenas LM, Bol R, Lewicka-Szczebak D, Gregory AS, Matthews GP, Whalley WR, Misselbrook TH, Scholefield D, Well R (2017) Effect of soil saturation on denitrification in a grassland soil. Biogeosciences 14(20):4691-4710, DOI:10.5194/bg-14-4691-2017

  2. 1

    Denk TR, Mohn J, Decock C, Lewicka-Szczebak D, Harris E, Butterbach-Bahl K, Kiese R, Wolf B (2017) The nitrogen cycle: a review of isotope effects and isotope modeling approaches. Soil Biol Biochem 105:121-137, DOI:10.1016/j.soilbio.2016.11.015

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