Microbial processes and the site of N2O production in a temperate grassland soil

Soil Biology & Biochemistry 36 (2004) 453–461 www.elsevier.com/locate/soilbio

Microbial processes and the site of N2O production in a temperate grassland soil Christoph Mu¨llera,*, R.J. Stevensb, R.J. Laughlinb, H.-J. Ja¨gera b

a Department of Plant Ecology, University Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany Agriculture, Food and Environmental Science Division, Department of Agriculture and Rural Development, Newforge Lane, Belfast BT9 5PX, UK

Received 6 March 2003; received in revised form 14 July 2003; accepted 15 August 2003

Abstract To understand nitrous oxide (N2O) emissions from terrestrial ecosystems it is necessary to understand the processes leading to N2O production. Here, for the first time, results are presented which identify in situ the processes of N2O production in a temperate grassland soil. A small portion of the nitrogen (N) applied in the summer to the grassland soil was rapidly transported below the main rooting zone (.20 cm) and resulted in large N2O productions at depths of 20 – 50 cm. Preferential pathways must have been responsible for this movement because the soil conditions were not conducive to leaching by piston flow. The N2O was entirely produced by nitrate (NO2 3) reduction which was surprising because the bulk soil was aerobic. Therefore, reduction processes can operate during times of the year when it is least expected and cause large N2O concentrations deep in the soil profile. q 2004 Elsevier Ltd. All rights reserved. Keywords: 15N; Nitrous oxide (N2O); Nitrification; Denitrification; Root zone

1. Introduction There is a growing concern about the increasing emissions of greenhouse and ozone-depleting gases such as nitrous oxide (N2O). Atmospheric N2O concentrations have been increasing over the last decades through anthropogenic influence (Sowers, 2001). Microbial trans2 formations of ammonium (NHþ 4 ) and nitrate (NO3 ) (Bouwman, 1998), in particular nitrifier-denitrification (Lipschultz et al., 1981; Wrage et al., 2001) and nitrate reduction, but also chemodenitrification (van Cleemput, 1998) and fungal transformations (Shoun et al., 1992; Laughlin and Stevens, 2002), are considered to be the main processes producing N2O in terrestrial ecosystems. N2O emitted from the soil surface via diffusion originates, therefore, most likely from a mixture of N2O produced by a range of different microbial processes. Since the responsible microorganisms operate under various optimum conditions it is generally assumed that nitrification is the predominant N2O-producing process * Corresponding author. Tel.: þ49-641-993-5315; fax: þ 49-641-9935309. E-mail address: [email protected] (C. Mu¨ller). 0038-0717/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2003.08.027

under moderately moist and warm conditions and that denitrification is the predominant process under wet 2 (anaerobic) conditions when NHþ 4 and NO3 are available in soil (Conrad, 1996; Bouwman, 1998). Techniques which selectively inhibit nitrification (Klemedtsson et al., 1988) and 15N labeling (Stevens et al., 1998) have been used to identify the N2O production processes. The 15N gas-flux method (Stevens et al., 1998) has the advantage that it can be used in situ without soil destruction. After fertilizer application in the field it is generally assumed that the observed N2O flux is derived from microbial processes in the top soil layer where most of the applied mineral N remains (Granli and Bøckman, 1994). However, to fully understand the dynamics of N2O emissions into the atmosphere it is necessary to identify the mechanisms responsible for N2O production within the entire soil profile. Subsoil denitrification with N2O production and entrapment after N fertilization has been identified as a potential process capable of large rates of N2O production (Clough et al., 2001; Well et al., 2001). From previous research it was hypothesized that the predominant N2O-producing mechanism is autotrophic nitrification during summer periods and that the main production site is situated within the top soil horizon.

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Therefore, the objectives of this study were: (a) to identify the N2O production processes in the entire soil profile of an old temperate grassland soil during a summer– early winter period after N application and (b) to isolate the production site for N2O within the soil profile. Measurements of gas emissions were combined with detailed measurements of gas concentrations and nitrate 15 (NO2 N signatures 3 ) concentrations in the soil profile. The in the N2O emissions, in the N2O within the soil profile and in the NO2 3 were used to identify the mechanisms and the site of N2O production in the soil profile.

2.2. Soil description

2. Materials and methods

The soil is a Fluvic Gleysol with a texture of sandy clay loam over a clay layer (FAO classification) (Gru¨nhage et al , 2000). The soil texture, bulk density and a detailed description of the soil profile are presented in Table 1. The A-horizon is characterized by high biological activity with a high number of soil animals which create channels in the soil and may support preferential movement. A rapid water movement is further supported by the high saturated hydraulic conductivity ðKsat Þ in the top 50 cm (Table 1). During summer the groundwater table is at a depth of approximately 120 cm while it can reach the soil surface during the winter period.

2.1. Experimental setup

2.3. Gas and soil measurements and 15N analysis

The experiment was carried out on a temperate grassland soil between 30 July 2001 and 10 January 2002 (summer – early winter period) near Giessen/Germany. Nine plots were installed in early May 2001 (three 15N labeling treatments each with three repetitions). Each plot (1 m2) was divided into a gas measuring plot (0.16 m2) and a soil measuring plot (0.84 m2). The gas measuring plots were set up in such a way that the soil gas concentrations could be monitored at eight depths (i.e. 5, 10, 15, 20, 25, 32.5, 40 and 50 cm) beneath the flux measuring sites. The soil profile was monitored at six depths (i.e. 0 –5, 5– 10, 10 –15, 15 – 25, 25 –35 and 35– 50 cm). Ammonium nitrate solutions, where either the ammonium (15NH4NO3), nitrate (NH15 4 NO3), or 15 both moieties (15NH15 N 4 NO3) were labelled at 60 at. % 21 excess, were applied at a rate of 50 kg N ha on 30 July 2001. The liquid applied corresponded to 12.5 mm of rainfall. The N application follows the general management procedures in this grassland soil.

Gas measurements were carried out throughout the entire observation period from 30 July 2001 until 10 January 2002. Trace gas emissions were quantified with the closed chamber technique (Hutchinson and Mosier, 1981) 5 days before and 2 h and 1, 2, 6, 7, 8, 9, 10, 15, 21, 28, 35 and 49 days after N application. Gas concentrations in the soil profile were monitored with silicone gas probes (Kammann et al., 2001) at 18 and 5 days before, 2 h after and 1, 2, 4, 7, 10, 15, 21, 28, 35, 40, 44, 49, 59, 65, 79, 94, 99, 108, 120, 133 and 162 days after N application. Gas samples (emissions and soil profile samples) were analyzed for oxygen (O2) and N2O on a gas chromatograph equipped with an ECD (N2O) and FID (O2) (Mosier and Mack, 1980). The 15N excess in N2O was determined in separate samples by isotope-ratio mass-spectrometry (Stevens et al., 1993). Soil samples from the various soil depths were taken with an auger that allows the sampling of undisturbed soil cores in 250 cm3 stainless steel rings (Eijkelkamp, Inc.) at 12 days

Table 1 Soil texture, bulk density, particle density, saturated hydraulic conductivity ðKsat Þ and organic carbon in the entire soil profile of the old grassland soil used in this study Horizon Depth (cm)

Soil texture (%) Particle Ksat Sand Silt Clay Soil texture Bulk (m s21) density (2000–63 mm) (63–2 mm) (,2 mm) density (g cm23) (g cm23)

Ah

0–12

10

58

32

M

12– 20

10

56

35

MSw

20– 50

15

51

35

IISw

50– 85

21

43

36

IIISd1

85– 110

33

30

37

IIISd2

110 –150 67

17

17

*nd ¼ not determined.

Silty loam (Ls) Silty loam (Ls) Silty loam (Ls) Sandy clay loam (Lcs) Loamy clay (Cl) Loamy clay (Cl)

Organic C Description (%)

0.89

2.51

1.60 £ 1023 6.59

1.24

2.64

3.25 £ 1023 3.47

1.44

2.67

9.57 £ 1024 1.11

nd*

2.69

nd

1.49

2.68

4.00 £ 1025 0.39

nd

2.69

nd

0.64

0.12

Dark brown, humous, biologically very active dark greyish-brown, humous, biologically active Brown, iron congretions Brownish-grey, iron-congretions Light grey, iron-manganese congretions Dark grey, iron –manganes congretions

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before, 2 hours after and 1, 2, 4, 7, 10, 15, 21, 28, 35, 49, 79, 99 and 133 days after N application. The auger equipment was cleaned after each sample to avoid cross contamination. The soil in each jar was extracted by the blending procedure of Stevens and Laughlin (1995). Nitrate was analyzed using a Technicon continuous flow autoanalyzer (Braan and Luebb Co., Germany). The 15N contents of the NO2 3 in the extracts were determined by a method based on their conversion to N2O (Stevens and Laughlin, 1994). The analysis of the 15N excess of N2O was carried out according to the procedures developed by Stevens et al. (1993). 2.4. Soil moisture and temperature Soil temperature and soil moisture were monitored at seven depths (i.e. 5, 10, 15, 20, 25, 32.5 and 50 cm) with specific probes (CS615, 107Temperature probe, Campbell, Scientific Inc.) and logged as half hourly averages on a datalogger (CR10x, Campbell Scientific Inc.).

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decreasing O2 concentrations (Fig. 2) which generated near to anoxic conditions in the deeper soil layers. 3.2. Nitrous oxide emissions and soil profile concentrations The N2O flux and soil profile concentrations determined before the N application are representative of this grassland soil for most of the year except for a period of ca. 20 days after fertilizer application (Kammann, 2001). The largest N2O emissions were observed immediately after fertilizer application (Fig. 3a). Thereafter, emissions declined rapidly and reached background levels within 10 days. N2O concentrations significantly ðP , 0:05Þ higher than background levels as determined before N application (i.e. . 0.4 ppm) occurred in the soil profile only below the main rooting zone (. 20 cm depth) (Fig. 3a). The highest N2O concentrations of approximately 6 mLL21 were observed after 3 days at 50 cm depth (Fig. 3a). N2O emissions and N2O soil profile concentrations from the three treatments were not significantly different from each other.

2.5. Presentation of results 3.3. Values are presented as averages of the replicate treatment blocks ^ standard deviations (AVG ^ SD). On the last observation day (10 January 2002) the 15N enrichments in the N2O were below the detection limit of the mass spectrometer and are therefore not presented.

3. Results 3.1. Soil moisture, temperature and oxygen concentrations The soil moisture and soil temperature during the observation period (Fig. 1) were typical of conditions during summer –early winter in this soil. The application of the fertilizer (arrow in Fig. 1) caused a measurable increase in soil water content in the top 5 cm but not below. Thereafter, the soil volumetric water in the main rooting zone (top 10 cm) stayed at approximately 0.3 cm3 cm23 during the main growth period and increased below the rooting zone to about 0.6 cm3 cm23 at 50 cm (Fig. 1). From the start of the autumn period (95 days after fertilization) onwards, the soil moisture increased steadily in the top 20 cm and reached values near saturation towards the end of the observation period (Fig. 1). The soil temperature in the top 10 cm fluctuated between 20 and 25 8C but decreased towards the end of the observation period to temperatures close to freezing. The fluctuations were less pronounced with soil depth. The O2 concentrations in the entire soil profile stayed between 15 and 21% throughout most of the observation period (Fig. 2), i.e. close to atmospheric concentrations. Towards the end of the observation period (. 95 days after fertilizer application), decreasing soil temperatures coincided with increasing soil water contents (Fig. 1) and

15

N enrichment of the N2O

The 15N enrichment of the N2O in the soil profile increased one day after fertilizer application together with the increase in N2O concentrations (Fig. 3a and b). In the week after fertilizer application, the enrichment of the N2O below 20 cm was close to the enrichment in the applied N, indicating that the observed N2O originated from the applied fertilizer. Furthermore, the enrichment of the surface-emitted N2O corresponded to the enrichment of the N2O in soil layers below 20 cm rather than the top soil (Fig. 3b). Comparing the 15N enrichments in the N2O from the three 15N-treatments showed that the N2O produced was only 15N enriched when the nitrate pool had been labeled 15 15 (Fig. 3b, NH15 NH15 N 4 NO3 or 4 NO3 treatments). The 2 enrichment of the N2O in the treatments where NO3 was labelled were not significantly different. There was a significant difference between the two NO2 3 labelled treatments and the 15NH4NO3 treatment ðP , 0:05Þ in the enrichment of surface-emitted N2O and of N2O in the profile (Fig. 3b). Differences were most pronounced within the first 10 days after N application in the top 25 cm and until 20 days after N application deeper in the profile. The surfaceemitted N2O during the first 10 days was enriched to 5– 10 at.% from plots where the NHþ 4 -pool had been labelled (15NH4NO3 treatment) (Fig. 3b). 3.4. Nitrate concentration and 15N enrichment profiles The largest NO2 3 concentration was observed in the top 5 cm of soil and decreased to 50 cm depth where only a tiny 21 NO 2 ) was 3 concentration increase (ca. 0.4 mg N g 15 detected (Fig. 4a). The N enrichment of the NO2 3 in the entire soil profile showed that within 2 h after fertilizer 15 15 15 application the NO2 3 in NH4 NO3 or NH4 NO3 treatments

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Fig. 1. Soil volumetric water content (solid lines) and soil temperature (dotted lines) in the soil profile during the experimental period (The arrow indicates the time of fertilizer application).

was enriched to values higher than 50 at.% 15N. While the 15 N enrichments in the two NO2 3 enriched treatments 15 15 (NH15 NO and NH NO ) were not significantly different, 4 3 4 3 these treatments had a significantly different NO2 3 enrichment compared to the 15NH4NO3 treatment within the first 10 days after fertilizer application ðP , 0:05Þ: This shows

that the NO2 3 in the entire profile originates predominantly from the applied N. Rapid autotrophic nitrification (con2 version from NHþ 4 to NO3 ) occurred within 5 days after N application in 5– 25 cm depth as indicated by the enrichment of the NO2 3 pool to approximately 20 at.% values 15 when only the NHþ 4 pool was labeled ( NH4NO3 treatment)

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Fig. 2. Oxygen concentrations (AVG ^ SD) in the soil profile of a temperate grassland soil during the experimental period (30 July 2001–8 January 2002).

(Fig. 4b). Therefore, the 5– 10 at.% enrichment in the N2O emissions from this treatment (Fig. 3b) was most likely due to reduction of NO2 3 rather than direct nitrification-related N2O production.

4. Discussion 4.1. N2O emissions and soil profile concentrations The observation that the N2O emissions declined to background levels within 2 weeks of application is in line with numerous studies where the effect of N fertilization on

N2O emissions has been studied (Granli and Bøckman, 1994). The observation that the main N2O production occurred deeper in the soil profile was surprising because it indicated that part of the applied N was rapidly transported into deeper soil layers under conditions when no such transport by piston flow would be expected (Fig. 1). After fertilizer application the soil moisture sensors indicated a measurable increase in soil water content only in the top 5 cm but not deeper in the soil profile (arrow Fig. 1). Proof that a rapid transport of mineral N with subsequent N2O production occurred was provided by the NO2 3 concentration in the soil profile and the 15N enrichment of NO2 3 and N2O (Figs. 3b and 4b).

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Fig. 3. Nitrous oxide emission and concentrations (AVG ^ SD) in the profile of a temperate grassland soil (a) and the N2O enrichments (AVG ^ SD) in 15 15 15 15 treatments where the nitrate pool (NH15 4 NO3), the ammonium pool ( NH4NO3) or both moieties ( NH4 NO3) were labeled with N at 60 atom% excess (30 July–14 December 2001) (b).

The soil used in this study (Table 1) is biologically very active and supports an abundance of soil animals including earthworms. The soil horizons are characterized by high saturated conductivities (Table 1) which are partly due to the flow in large pores such as earthworm channels (Hagedorn and Bundt, 2002). Some of the applied NO2 3 must have trickled down the walls of larger channels under unsaturated conditions causing NO2 3 movement into deeper soil horizons (Beven and Germann, 1982). These translocations occurred within one day of application and were possibly further encouraged by relatively high soil temperatures (Fig. 1). The understanding of preferential water movement in soil is therefore crucial for the prediction of the N2O production site in the soil profile and hence for the prediction of N2O emissions to the atmosphere. The NO2 3 data show that only a very small amount of NO2 was transported to 50 cm (Fig. 4a). This movement is 3

indicated by the slight NO2 3 concentration increase but most of all by the high 15N enrichment of the NO2 3 extracted from soil deeper in the profile (Fig. 4). The 15N enrichments of the NO2 3 in the 35 – 50 cm layer of more than 50 at.% in the 15 NO2 N label 3 labeled treatments were close to the applied 2 (60 at.%). This proves that the extracted NO3 originated from the applied fertilizer (Barraclough et al., 1984). The small amount of NO2 3 leached was sufficient to create a large N2O production in deeper soil layers. This highlights the fact that high NO2 3 concentrations are not a prerequisite for high N2O productions but rather the conditions for denitrification. This observation corresponds to work by Dendooven and colleagues who found that N2O production in a grassland soil was not NO2 3 concentration dependent (Dendooven and Anderson, 1994; Ellis et al., 1996). They showed that the denitrification kinetics with specific affinities for N2O and nitrite (NO2 2 ) determined the amount of N2O produced (Dendooven and Anderson, 1994).

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15 Fig. 4. Nitrate concentrations (a) and their 15N enrichments (b) in treatments where the nitrate pool (NH15 4 NO3), the ammonium pool ( NH4NO3) or both 15 NO ) were labeled with N at 60 atom% excess (30 July–14 December 2001). moieties (15NH15 4 3

Almost all N2O produced in the soil in the first 10 days after N application must have originated from the applied N because the enrichment in the N2O produced was close to the enrichment of the applied fertilizer of 60 at.% (Fig. 3b). Therefore, the N2O produced originated from N that became recently available (i.e. in this case through N fertilization). This is in line with the general trend that N2O emissions are correlated to the soil mineral N concentrations. However, this is also true for conditions which create an imbalance in N producing and N consuming processes as observed under extreme events such as freezing –thawing (Mu¨ller et al., 2003) or wetting – drying (de Siqueira Pinto et al., 2002) which promote mineral N built up in the soil causing high N2O productions. N2O produced deeper in the soil profile may partly diffuse to the atmosphere or dissolve in soil water, enter the groundwater and be released elsewhere (van Cleemput, 1998; Well et al., 2001). The emitted N2O must have been derived from N2O produced deeper in the soil profile because the 15N signature in the emitted N2O from the NO2 3 15 labelled treatments (NH15 NH15 4 NO3 and 4 NO3) is more related to the N2O signature in depths below 20 cm (Fig. 3b). This highlights that during summer periods N2O production deep in the soil profile can contribute to N2O emissions as

observed in other studies on the production of N2O in deeper soil horizons (Clough et al., 1999; Clough et al., 2001). 4.2. Processes of N2O production The observation that N2O in the soil profile was only enriched with 15N when the NO2 3 pool had been labelled (Fig. 3b) demonstrates that the N2O produced in the soil profile, and which was partly emitted subsequently into the atmosphere, originated from NO2 3 reduction processes. Such a result is surprising because of the aerobic nature of the bulk soil during the observation period (Fig. 2). The soil moisture-temperature conditions were conducive for nitrification rather than denitrification. A high nitrification activity was detected in 5– 25 cm depth as indicated by the rapid increase of the NO2 3 enrichment when only NHþ was labelled (Fig. 4b). The small enrichment in the 4 surface-emitted N2O from treatments where the NHþ 4 -pool had been labelled (Fig. 3b) is most likely a result of rapid nitrification combined with subsequent NO2 3 -reduction rather than a direct nitrification-related N2O production. The lack of nitrification-related N2O production is most likely caused by high NO2 2 oxidation activities in the soil which prevented an accumulation of nitrification-related

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NO2 2 (data not shown). Nitrite is a precursor for nitrifierdenitrification (Wrage et al., 2001) and therefore N2O emissions via this pathway could not occur. Methods which selectively inhibit ammonium mono-oxygenase (AMO) by applying small concentrations of acetylene (5 – 10 Pa, Klemedtsson and Hansson, 1990) to identify the proportions of N2O produced via nitrification and denitrification may therefore generate false results during times when high nitrification rates prevail. Acetylene (C2H2) blocks the build-up of NO2 3 and the subsequent N2O production via denitrification. Thus, N2O attributed to nitrification via these inhibition methods may in reality have been produced by denitrification. Therefore, results from process studies which have used the C2H2 method such as the one by Ambus (1998) should be regarded with the necessary care. Azam et al. (2002) even found that the application of NHþ 4 may have stimulated denitrifiers and caused higher N2O production after subsequent NO2 3 applications through denitrification. The N2O in the present grassland soil was produced either in anaerobic niches by bacterial reduction processes (Conrad, 1996) or via NO2 3 reduction under aerobic conditions. Recently, Laughlin and Stevens (2002) have provided evidence that nearly 90% of the N2O in a grassland soil was produced by fungi. They attributed this finding to the process of NO2 3 reduction which is carried out by fungi and which can procede under aerobic conditions (Shoun et al., 1992; Zhou et al., 2001; Watsuji et al., 2003). The processes identified during the summer– early winter period in this study correspond to the findings identified during freezing– thawing events of the same soil, where reduction of NO2 3 was also identified as the predominant N2O producing process (Mu¨ller et al., 2002). Therefore, it appears that there may only be a negligible contribution of nitrification to the observed N2O emissions from the temperate grassland for most of the year. A similar result was obtained by Wolf and Brumme (2002) who also identified the process of NO2 3 reduction to be the main process for the observed N2O emissions between late spring and autum (May – October) in an acid forest soil. Therefore, terrestrial ecosystems which are characterised by a tight N cycle and high internal N cycling rates where rapid gross nitrification rates (Stark and Stephen, 1997) do not allow the build-up of nitrification-derived NO2 2 , may be more conducive to N2O losses via denitrification. 4.3. Conclusions To understand N2O emissions and the underlying mechanisms we conclude that it is not enough to just measure the total N2O emissions and relate them to the observed influencing factors such as soil moisture, soil temperature and mineral N content as done in numerous studies (Granli and Bøckman, 1994). Complex relationships exist between physical and biological soil properties, which support transport processes and microbial activities in

the soil profile. Even under aerobic soil conditions, we found that the process of NO2 3 reduction is the predominant N2O producing mechanism. This contradicts conclusions drawn by researchers who developed theoretical relationships between soil moisture and other factors and N2O production (Bouwman, 1998). Even during the summer when low N2O emissions are observed, applied N fertilizers may partly be transported below the main rooting zone by rainfall and stimulate the build-up of large N2O concentrations in deeper soil layers. Gas produced in deeper soil horizons cannot only be emitted from the soil but may also be dissolved in soil water and transported via the groundwater to cause N2O emissions elsewhere (Sotomayer and Rice, 1996; van Cleemput, 1998; Well et al., 2001). To elucidate the N2O producing processes in soil we recommend that measurements of the N2O emissions and soil profile concentrations should be combined with isotopic signatures of the N2O in 15N labeling studies.

Acknowledgements This study was financially supported by the German Science Foundation.

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