1 Introduction

Drainage of peatlands started a few hundred years ago for activities like agriculture, peat extraction and forestry (Joosten and Couwenberg 2001). Drainage generally leads to aerobic decomposition and thus, to greenhouse gas emissions (Canadell et al. 2007; Page et al. 2002; Wösten et al. 1997).

In Europe, peatlands account for about 5 to 6% of the land area, and more than 60% of them are drained (Drösler et al. 2008). In Germany, even 95% of peatlands are drained, causing 5% of Germany’s total anthropogenic greenhouse gas emissions (46 million tons of carbon dioxide equivalents per year) (Hahn-Schöfl 2015). Of these peatland emissions, 80% originate from fens (Höper 2007): their larger nutrient contents compared to bogs made them preferred drainage targets (Timmermann et al. 2016).

Peat mineralization leads to the release of carbon dioxide and nitrous oxide (N2O) (Gelbrecht et al. 2008). N2O is a long-lived greenhouse gas with an average concentration of about 331 ppb in the atmosphere (Tian et al. 2020). In the stratosphere, its decomposition products are involved in ozone destruction (Crutzen 1991; Ravishankara et al. 2009).

There is a range of processes and pathways producing N2O in soils (Butterbach-Bahl et al. 2013). They can take place simultaneously in different soil microsites, making them difficult to distinguish (Heil et al. 2015; Stein 2019; Wrage-Mönnig et al. 2018) and important to understand in order to develop N2O mitigation strategies. In wet fens, denitrification (Fig. 1) is usually considered to be the main source of N2O (Augustin et al. 2001; Lohila et al. 2010). However, especially under drained conditions, also nitrification (Fig. 1) can contribute to N2O production (Martikainen et al. 1993; Regina et al. 1996). Another important pathway is nitrifier denitrification, where NO2− is reduced to N2O and potentially N2 as in denitrification, but by autotrophic ammonia oxidizers (Kool et al. 2007; Wrage et al. 2004). Furthermore, there are various other pathways producing N2O, like heterotrophic nitrification, co-denitrification or fungal denitrification. Various methods exist to distinguish among these sources, but none covers all processes and pathways.

Fig. 1
figure 1

Major pathways of N2O production: nitrifier nitrification, nitrifier denitrification, fertilizer denitrification and nitrification-coupled denitrification. The difference between fertilizer denitrification and nitrification-coupled denitrification is the different source of the nitrate used from either external sources or nitrification

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So far, the effect of rewetting on overall N2O emissions and on soil sources of N2O is not well understood. Research suggests that rewetting causes an overall reduction in N2O emissions (Jordan et al. 2016; Wilson et al. 2016). Thus, direct comparisons of drained and rewetted peatlands demonstrated that drained sites showed larger N2O emissions (Davidsson et al. 2002; Vybornova et al. 2019). However, Gelbrecht et al. (2008) observed that while rewetting of drained fens to a water table level of 0.3 – 0.8 m above ground strongly decreased N2O emissions, a fluctuating groundwater level (-0.3 m ± 0.3 m above ground level) led to their increase (Berendt et al. 2022). Studies systematically investigating sources of N2O from (rewetted) fens under controlled conditions are missing.

Therefore, the aim of this study was to improve our understanding of the influence of fen rewetting on N2O production and its sources under controlled laboratory conditions. We incubated soil of pairs of drained and rewetted sites of three different fen types, using the dual-isotope method according to Kool et al. (2011). With this method, it is possible to distinguish among nitrification, nitrifier denitrification and denitrification as sources of N2O. We chose this method as we suspected that nitrifier denitrification might be important under the conditions encountered. We hypothesized that a) peat from rewetted sites would show smaller N2O fluxes than from drained ones, b) that the average water table height in the field would be the main influencing factor for N2O emissions as it determines both peat mineralization (and thus substrate availability) and microbial community composition, and c) that denitrification would be a larger source of N2O on rewetted sites than on drained ones.

2 Material and Methods

2.1 Material

Soil (0 – 20 cm) was collected from the six study sites (pairs of drained (D) and rewetted (W) sites on a coastal fen (C), percolation fen (P) and alder forest (A)) of the WETSCAPES project (Jurasinski et al. 2020) and stored cool (8—10 °C) until the start of the experiment. One week after soil sampling – which was used for preliminary tests to determine the water content and water-holding capacity (WHC) – the preincubation started. For more information about the study sites and the soil properties, see Supplementary Material and Jurasinksi et al. (2020).

2.2 Methods

Incubation Experiment

The WHC was determined for each soil according to Vengadaramana and Thairiyanathan (2012) using a funnel with filter paper (Whatman No. 1) instead of a perforated tin box.

After a two-day preincubation with 50 g soil (dry mass) in 750 ml Weck jars (n = 5) at room temperature (between 20 and 22 °C) and with a water content of 85% WHC, the main incubation was started by adding isotopic tracers dissolved in distilled water to reach 95% WHC and mixing the dissolved tracers into the soil with a glass rod. All treatments received equal amounts of mineral N in form of 7.14 mg of ammonium nitrate (NH4NO3). These conditions were chosen as a compromise between creating comparable conditions for all sites and not changing site conditions too much, while being able to add isotopic tracers. Incubations were carried out according to the dual-isotope method (Kool et al. 2011). In brief, the method used treatments (TR) with the following isotopic tracers: H218O (TR1), N18O3− (TR2), 15NO3− (TR3) and 15NH4+ (TR4), with the ammonium and nitrate tracers enriched at 10 at% and H2O enriched at 1 at%. In contrast to the initial method, the soil samples were not homogenized or dried in order not to destroy the peat properties, unless the peat was too wet initially: soil that had a larger water content was dried to approximately 85% WHC at room temperature before the start of the pre-incubation. This was the case in three soils: AW, PW and PD. Especially the site AW was completely flooded at sampling. The additional water was included in the calculation of water content, resulting in AW having a calculated water content of 120% WHC. The jars were closed directly after tracer addition with air-tight lids containing a septum.

Gas Measurements

At 3 h, 6 h and 24 h after tracer application, gas samples were taken with a 20 ml syringe and transferred into evacuated exetainer vials for analyses of N2O concentration and its isotopic enrichments. The gas samples were analyzed with a TraceGaspreconcentrator (Elementar, Langenselbold, Germany) coupled to an isotope ratio mass spectrometer (IRMS, IsoPrime 100, Elementar, Langenselbold, Germany). For calibration, we used two working standards (0.9 and 1.8 ppm N2O in synthetic air, δ15N 0.15 and 0.02‰, δ18O 40.66 and 40.32‰, respectively) calibrated against the standards of the laboratory of the Department of Environmental System Science, ETH Zürich (Verhoeven et al. 2019). At the time these experiments were carried out and samples were measured, no official reference materials existed for N2O (Mohn et al. 2022) and also no N2O with known enrichment in 15N in the atom% range expected with tracer addition was available. We regularly measured isotopically enriched as well as natural abundance 15N in solids (see below), finding the IRMS linear over this range. Therefore, we assumed linearity also for N2O. The working standards were run at the start and end of each run and in duplicate every 20 samples. For calibration of the sample peak ratios, an N2O reference gas (100% N2O, Air Liquide, Germany) was run with every sample. Afterwards, the ratios were corrected for drift and span via the working standards. Stability (≤ 0.01‰) and linearity (≤ 0.02‰) of the IRMS were measured by injection of 10 N2O reference gas pulses of similar or varying amount, respectively. Determination of external precision for 15N in N2O was done using at least four samples of our 1.8 ppm N2O working standard per run and was on average 0.22‰.

Soil Extractions

After 24 h, soil KCl extractions (150 ml 1 M KCl per 40 g soil, 1 h shaking, filtration over Whatman No. 1 filter paper) were carried out and extracts prepared for 15N isotopic analyses of NH4+and NO3− using microdiffusion (Brooks et al. 1989).The samples were then measured on an elemental analyzer (vario PYRO cube, Elementar, Germany) coupled to the above IRMS. The external precision for 15N in solid samples, determined as the standard deviation of 7 to 20 natural abundance samples of sulfanilamide during one run with samples intermixed was on average over the lifetime of the used source 0.16‰. As internal standards, we used sulfanilamide and wheat flour. These were calibrated against IAEA-600 and IAEA-NO-3 for 15N, as well as IAEA-311 for samples enriched in 15N. Isotopic values are reported in at% excess for the tracer study.

Calculations and Statistics

N2O fluxes were calculated based on linear regressions of the gas concentrations over time. Calculation of sources was done according to Kool et al. (2011). According to this method, N2O produced from NH4+ is divided into nitrification-coupled denitrification (NCD), nitrifier nitrification (NN) and nitrifier denitrification (ND) using 15N and 18O as tracers. The frequently used 15N tracer method was not able to differentiate between pathways related to nitrification (nitrifier nitrification, nitrification- coupled denitrification and nitrifier denitrification). Here, we also used 18O as a tracer to quantify the O exchange in the different pathways. The method yields maximum and minimum values per pathway. In the results, we present the maximally possible amounts of these production pathways for reasons of clarity. A one-way ANOVA was used to check for differences in variables among sites (α ≤ 0.05). Data were tested for normality using the Shapiro–Wilk-Test and for equal variances with the Brown-Forsythe-Test. If the requirements for ANOVA were not fulfilled, the Kruskal–Wallis-Test was performed. The Tukey- or Holm-Sidák-Test were used as post-hoc tests. Statistical analyses were performed with SigmaPlot 13.0.

3 Results

3.1 NH4 + and NO3 − Concentrations

In all soils, there was less NH4+ at the end of incubations than NO3− (Fig. 2). With the exception of the coastal wetland, drained sites contained significantly more NO3− and less NH4+ than the rewetted one (p ≤ 0.001). CD also had the largest NH4+ concentration of all sites, 25.8 mg NH4+-N kg−1 (p ≤ 0.001), and CW the significantly largest NO3− concentration (Fig. 2), 67.5 mg NO3−-N kg−1 (p ≤ 0.001).

Fig. 2
figure 2

NH4+ and NO3− concentrations (mg NH4+-/NO3−-N kg−1) in soil samples of the rewetted (CW) and drained (CD) coastal fen, rewetted (PW) and drained (PD) percolation fen and rewetted (AW) and drained (AD) alder forest at the end of the incubation experiment. Shown are means and standard deviations

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As expected, in TR3 and TR4, considerable 15N enrichments in mineral nitrogen were measured at the end of the incubations (data in the supplement). In TR3, enrichments in 15N-NO3− ranged from 1.4 to 3.0 at%. There were no enrichments of NH4+ in this treatment. TR4 showed smaller enrichments of 0.6 – 1.4 at% for 15N-NH4+. Furthermore, enrichments in 15N-NO3− of between 0.5 and 1.1 at% were also detected in this treatment (data in the supplement).

3.2 Gas Fluxes

Incubations of soil from all sites showed N2O production (Fig. 3). Generally, fluxes were larger in rewetted than in the respective drained sites (p ≤ 0.001). The largest flux with 516.1 ± 390.8 µg N2O-N kg−1 h−1 occurred in AW, followed by PW with 323.2 ± 205.6 µg N2O-N kg−1 h−1 (p = 0.099). N2O production from incubations of CW, CD and AD was small (17.8 ± 21.6 µg N2O-N h−1 kg−1; 0.8 ± 6.2 µg N2O-N h−1 kg−1; 6.5 ± 18.6 µg N2O-N h−1 kg−1, respectively), and significantly different from that of AW (p ≤ 0.001). PD, however, showed a slightly larger N2O flux than the other drained sites with 38.9 ± 53.2 µg N2O-N h−1 kg−1, significantly different from all sites except of CW (p ≤ 0.001 – p = 0.028, p = 0.534 for CW).

Fig. 3
figure 3

N2O-N fluxes (μg kg−1 h−1) of incubated soil samples of the rewetted (CW) and drained (CD) coastal fen, the rewetted (PW) and drained (PD) percolation fen and the rewetted (AW) and drained (AD) alder forest. Shown are means and standard deviations

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3.3 N2O Source Determination

All sites produced at least half of the N2O from labelled NO3−, i.e. from denitrification (Fig. 4). Interestingly, rewetting produced no clear patterns concerning the contribution of the different sources to N2O production. AD and CW produced the smallest amount of N2O from NO3− (with 52.3 ± 17.1% and 56.1 ± 14.0%, respectively) compared to the other sites (p = 0.001 – 0.008). The contribution of denitrification to N2O production was almost identical for both sites of the percolation fen (84.4 ± 11.4% in PW; 85.8 ± 4.9% in PD, respectively) and AW (81.0 ± 7.4%). These values were not significantly different (p = 0.841 between PW and PD; p = 0.590 between PW and AW, and p = 0.269 between PD and AW). In CD, N2O was formed almost entirely from NO3− under the conditions tested, representing with 90.2 ± 5.2% the largest contribution and showing significant differences to CW and AD (p = 0.001), but not to the other sites (p = 0.054 – 0.690).

Fig. 4
figure 4

N2O produced from 15N-NO3− (%) in soil incubations of the rewetted (CW) and drained (CD) coastal fen, the rewetted (PW) and drained (PD) percolation fen and the rewetted (AW) and drained (AD) alder forest. Shown are means and standard deviations

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The largest maximum contributions of ND to the production of total N2O were estimated for CW (43.9 ± 14.0%) and AD (47.7 ± 17.1%) (p = 0.710, Fig. 5). At the remaining four sites, the maximum amounts of ND were between 10 – 20% (p = 0.054 – 0.841), with significant differences to CW and AD (p ≤ 0.001 to p = 0.009).

Fig. 5
figure 5

Maximum N2O production (%) from nitrification-coupled denitrification (NCDmax), nitrifier nitrification (NNmax) and nitrifier denitrification (NDmax) of the rewetted (CW) and drained (CD) coastal fen, the rewetted (PW) and drained (PD) percolation fen and the rewetted (AW) and drained (AD) alder forest. Shown are means and standard deviations. For further information on calculations, see text

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At CW, the maximal contribution of NCD was equal to that of ND and significantly larger than that of all other study sites (p ≤ 0.001 to p = 0.008). The smallest maximal contributions of NCD were calculated for CD and AD (4.4 ± 7.6%, 7.6 ± 10.8%, respectively), showing a significant difference between CD and PD (p = 0.042), with PW, PD and AW having intermediate values for the maximal contribution of NCD (Fig. 5).

NN did not contribute to N2O production from CW, PW and PD under the conditions tested (Fig. 5). For the other sites, the maximally possible contributions from NN were also small (5.4 ± 3.6%, 3.8 ± 5.6%, 7.6 ± 10.8%, respectively), showing no significant differences among sites (p = 0.503 – 0.841).

4 Discussion

In contrast to the first hypothesis, N2O fluxes were larger from the rewetted sites than from the respective drained ones (Fig. 3). This was remarkable, as considerably larger fluxes are normally expected from drained peatlands than from wet ones (Augustin et al. 1998). In this experiment, however, all soils were incubated at the same water content, making adaptations in water content necessary at the beginning of the incubation. When comparing the change in water content between field conditions at sampling and the start of the incubation, it is striking that the sites that had to be dried before the incubation all showed substantive N2O emissions (Fig. 7a). This reinforces that drainage increases N2O fluxes, even if some water was added again to start the incubation. The N2O fluxes of the other sites, where hardly any drying was required or even a considerable amount of water had to be added, were almost negligible. This indicates that further wetting of the soils did not lead to larger N2O fluxes, but drying of the soils just before the addition of water, i.e. quick reduction in water content, did. This is in line with studies showing increasing N2O emissions with fluctuating water regimes (Gelbrecht et al. 2008; Jørgensen and Elberling 2012) and suggests that drying causes the onset of emissions, even lasting into concurrent wetter conditions. This is important for the management of rewetted sites, where fluctuating water regimes are more usual than in pristine fen peatlands (Kreyling et al. 2021).

When regarding the water table level of the sites previous to sampling (Fig. 6), it was evident that although rewetted sites usually had a higher water table level than drained ones, seasonal fluctuations were large, in line with other findings on fens (Kreyling et al. 2021). The largest fluctuations in the water table level were found on AD, where it dropped to more than -2.5 m in summer 2019 (Fig. 6), reflecting the drought conditions in that year. However, other sites also showed large variations in water table level among the seasons, fluctuating up to 1 m. Even on all rewetted sites, the water table level was more than 0.25 m below the surface in summer. Based on the findings of this current incubation study, such drying could cause increased fluxes of N2O. In field measurements at those sites, larger N2O emissions were measured particularly at PW and AW in summer 2018 (Berendt et al. 2022). At that time, the water table level for AW was more than 60 cm below surface, resulting in large N2O emission during that season.

Fig. 6
figure 6

Water table level of all six sites (rewetted (CW) and drained (CD) coastal fen, rewetted (PW) and drained (PD) percolation fen, rewetted (AW) and drained (AD) alder forest) from October 2017 to June 2020. The red dot marks the time of soil sampling for the incubation experiment. The interruptions in the shown water table levels were due to failures of the measuring instruments

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Despite the low water level in the field and rewetting of the soil before incubation, emissions from AD and CW (as well as CD, were no change in water content had to be carried out) were very small. This is remarkable, as many studies showed large emissions from drained sites (Augustin et al. 1998; Merbach et al. 2001). Nevertheless, there are also some studies that reported small fluxes from drained alder sites (Eickenscheidt et al. 2014). Based on the results seen here, short-term decreases in water content seem to be more important for N2O emission events (Dinsmore et al. 2009; Jørgensen and Elberling 2012) than long-term site conditions, even if substantial overall changes in water content occur over time.

In order to incubate all soils under the same conditions, we used a moisture intermediate between all soils, meaning some soils had to be air-dried and others wetted for preincubation. As our results show, the soils that had to be dried the most showed the largest N2O emissions. It is likely that the soils would have produced considerably smaller N2O emissions without prior drying. This is a methodological effect, but it also shows the large impact of short-term drying of fens on N2O production.

Here, we only concentrated on some production pathways of N2O (denitrification, nitrifier nitrification, nitrifier denitrification and nitrification-coupled denitrification) based on the dual isotope method chosen. Nevertheless, there are many other pathways that can produce N2O. So far, these are not captured by the present methods and efforts should be taken to find a method that differentiates all known major sources of N2O, potentially in a combination of isotope approaches.

Denitrification was an important source of N2O, but not in all cases more important in rewetted than in drained sites (Fig. 4). In contrast to our second hypothesis, the largest contribution of denitrification with more than 90% was found for the drained site CD. When comparing the change in water content before the start of the incubation with the amount of N2O from denitrification (Fig. 7b), it was noticeable that the sites that had to be dried before incubation (AW, PW and PD) showed a larger amount of denitrification. Since these sites were also very moist before incubation, they were probably tending towards denitrification (Lohila et al. 2010). In contrast, soil samples of the sites CW and AD were drier than 85% WHC before the start of the preincubation. These two sites produced smaller amounts of N2O from denitrification. Thus, the soils seem conservative in the main source of N2O, despite short-term changes in conditions before the incubation. This is in line with results from an acidic fen experimentally dried or flooded, which did not show large reactions to experimental conditions in terms of N2O production or denitrifier community structure (Palmer et al. 2016). Another recent study showed that the predominant N2O production pathway of a (mineral) soil determined the effect of biochar on N2O production (Ji et al. 2020). Thus, a (molecular) fingerprint of the dominant N2O production pathway(s) of a soil might help to better understand its behavior in changing conditions.

Fig. 7
figure 7

Change of water contents between sampling time and start of the incubation experiment: shown are the changes in % water-holding capacity (WHC) against the N2O flux (µg h−1 kg.−1) (a) and changes in % WHC against the contribution of denitrification (%) (b)

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The sites CW and AD showed small NH4+ concentrations in relation to NO3−, with almost half of the N2O produced from NH4+. The small NH4+ concentrations indicate that nitrification was fast here in relation to mineralization, and also to denitrification, as large NO3− concentrations as well as 15N enrichments of NO3− in incubations with added 15NH4+ suggest. Since N2O emissions were relatively small in CW and AD, either little N2O was produced or the N2O produced was largely reduced to N2. For CW, N2O produced from NH4+ could originate from either ND or NCD. In contrast, up to 50% of the N2O produced in AD from NH4+ originated from ND. These results were very surprising since we expected that most of the N2O in peatlands would be produced via denitrification at a WHC of 95%. Interestingly, there are other studies reporting a remarkable contribution of nitrification at 80% water-filled pore space (Pihlatie et al. 2004), but most of the studies showed that denitrification was the dominant process of N transformation in the soil under water-saturated conditions (Wolf 2000). Again, the predominant conditions and thus predominant microbial pathways might play a role here. Probably, the dry conditions in the field prior to incubations led to this large contribution of nitrification processes, even after over 24 h at wetter conditions. Even at water contents of 95% WHC, peat soils can still have dry pores, as pores can be very large, draining quickly, making peat a dual-porosity medium (Rezanezhad et al. 2016). Large contributions of nitrifiers to N2O production in rewetted fens were also shown by Masta et al. (2022). In all soils studied here, either ND or NCD could explain N2O production from NH4+, with negligible potential contributions of NN as these showed no or extremely small contributions (smaller than 10%) to the production of N2O (Fig. 5). Thus, pure nitrification does not seem to play a large role for N2O production in these soils.

5 Conclusion

Our results suggest that contrary to our hypothesis, a categorization into drained and rewetted fen sites cannot be used as an indicator for the microbial production pathways of N2O: as largest contributions of denitrification to N2O production were observed on a drained site. Short-term reductions in water content immediately prior to incubation resulted in largest N2O emissions, not rewetting of soil that had been comparatively dry in the field for a longer time. Thus, such quick drainage appears to stimulate N2O production more than lower long-term water table levels. Interestingly, all sites showed contributions to N2O production from both nitrification and denitrification processes, with water addition to field-dry peat soils leading to large contributions of nitrification pathways to N2O emissions.

Interestingly, although short-term changes in water content overruled longer-term conditions in the field in terms of N2O fluxes, its sources were determined by longer term conditions and predominant microbial communities.

This is interesting for the management of rewetted peatlands: It could enable a fingerprint of microbial communities to help predict N2O dynamics and develop an informed management of rewetted peatlands. For this, the stability of such communities over time needs to be investigated. Furthermore, the results underline that short-term changes in water content of rewetted peatlands need to be reduced to minimize N2O emissions.