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Methane oxidation by soils of an N limited and N fertilized spruce forest

in the Black Forest, Germany

R. Steinkamp, K. Butterbach-Bahl, H. Papen*

Department of Soil Microbiology, Fraunhofer Institute for Atmospheric Environmental Research (IFU), Kreuzeckbahnstraûe 19, D-82467 Garmisch-Partenkirchen, Germany

Received 18 June 1999; received in revised form 31 January 2000; accepted 8 June 2000

Abstract

A long-term experiment was performed at two sites in the Black Forest (Germany), in which methane oxidation rates of soils of an unfertilized spruce site and of a spruce site that had been fertilized with 150 kg of N ha21

(as (NH4)2SO4) were followed seasonally over approximately three years (1994±1996). Throughout the observation period, the soil at both sites functioned exclusively as a sink for atmospheric CH4. Mean CH4 oxidation rates at both sites were almost identical in magnitude (82.2^34.6mg CH4m22h21 for the unfertilized site, and 84.2^31.8mg CH4m22_h21 _{for the N fertilized site) during the observation period. Results from an additional} small-scale N fertilization experiment indicate that high N applications to the soil of this N-limited forest resulted only in a small reduction of CH4oxidation: less than 30% for less than 72 d. The results indicate that the atmospheric CH4 uptake activity of the soils of forest ecosystems characterized by N limitation has the capacity to recover rapidly from the inhibitory effects of high inorganic N inputs. CH4 oxidation rates at both sites showed no signi®cant diurnal variation. However, there were signi®cant seasonal differences in the magnitude of CH4oxidation rates at both experimental sites with high rates during summer, relative low rates during winter and intermediate rates during spring and autumn. Correlation analysis revealed that CH4oxidation rates were positively correlated with soil temperature and negatively with soil moisture. However, at low soil temperatures (,108C), temperature was a stronger modulator of CH4oxidation than soil moisture. Process studies on soil samples in the laboratory con®rmed a pronounced positive response of CH4oxidation to changes in temperature, only within a range of 0±108C. At both experimental sites, the highest CH4oxidation activity was observed in the Ahlayer (0±120 mm soil depth). Exposure of this layer to the atmosphere, as a result of the removal of the organic layer, resulted in a signi®cant increase of CH4oxidation rates. Apparently the organic layer functions as a diffusive barrier for atmospheric CH4or O2to CH4oxidizing sites.q_{2001 Elsevier Science}

Keywords: Methane oxidation; NH41inhibition of CH4oxidation; Forest soils; Soil temperature; Seasonal variations

1. Introduction

Next to CO2, CH4is the most important greenhouse gas in

the atmosphere. Its atmospheric concentration has increased during the last 300 years, from about 0.75 to 1.7ml l21 (Lelieveld et al., 1993). In the last 20 years atmospheric CH4 concentration increased, on an average, at a rate of

0.8% y21; at present the rate of increase has slowed down to less than 0.3% y21(Prinn, 1995). The main sink for atmo-spheric CH4is photochemical oxidation with hydroxyl

radi-cals in the troposphere. In addition, microbial oxidation in soils has been identi®ed as a signi®cant sink for atmospheric CH4. The global uptake rate of atmospheric CH4by

micro-bial activity in soils is estimated to be in a range of 15±

45 Tg CH4y2 1

(Watson et al., 1992; IPCC, 1996) and, thus, in the same magnitude as the annual increase in atmospheric CH4. Therefore, environmental changes (e.g. N input, land

use change) that can provide feed back on the capacity of soils to oxidize atmospheric CH4, may have signi®cant

consequences on the global atmospheric CH4 budget.

Well-aerated forest soils of the temperate zone and other regions as well are known to be signi®cant sinks for atmo-spheric CH4(Steudler et al., 1989; DoÈrr et al., 1993; Castro

et al., 1993; Sitaula et al., 1995; Butterbach-Bahl et al., 1997). In recent decades, these ecosystems have received increasing inputs of nitrogen by atmospheric N deposition. As a consequence of excessive N deposition the N status of forest ecosystems may have shifted from N limited towards N saturated (Aber et al., 1989; ZoÈttl, 1990; Aber, 1992; Dise and Wright, 1995). To study the effect of N deposition on CH4oxidation of forest soils, several N fertilization

experi-ments have been carried out in the past. Most of these

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* Corresponding author. Tel.: 149-8821-183130; fax: 1 49-8821-183294.

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studies have found that the N fertilization of forest soils had an inhibitory effect on CH4oxidation rates (Steudler et al.,

1989; Adamsen and King, 1993; Sitaula et al., 1995; Castro et al., 1995; Macdonald et al., 1996). However, an inhibitory effect of N application on CH4oxidation rates of forest soils

was not observed by Castro et al. (1993), Goldman et al. (1995), and Gulledge et al. (1997). The reason for these contradictory results remain unclear, but it can be hypothe-sized that the consequences of N deposition for CH4

oxida-tion rates of forest soils may depend on the N status of ecosystems, i.e. CH4oxidation in soils of N saturated forest

ecosystems may be more sensitive to additional N input from the atmosphere than CH4 oxidation in soils of N

limited forest ecosystems. We have compared measure-ments of CH4oxidation from an unfertilized and N fertilized

site. In addition, seasonal variations of CH4oxidation rates

and their dependency on basic environmental variables, such as soil temperature and moisture, were investigated.

2. Materials and methods

2.1. Study site

Experiments were carried out in a coniferous forest ecosystem (810±920 m a.s.l.) in the Black Forest near the town of Villingen, Germany (488030_{N, 8}₈₂₂0_{E) at ®eld sites}

which were also used within the ARINUS research project (Armbruster and Feger, 1998). The stand is dominated by Picea abies(approximately 110 years old) interspersed with Abies alba and Pinus sylvestris. The mean annual air temperature is 68C; the mean annual precipitation is 1200 mm (Feger, 1993). The soil is an acid brown soil with a pH (CaCl2) of 2.4±3.3 in the organic layer (L, Oh,

Of) and 3.2±3.7 in the uppermost mineral soil layers (Ah, AhBv). Atmospheric N input by wet deposition is about 10 kg N ha21y21(Armbruster et al., 1998). Until the middle of the 20th century, this forest was used as a pasture site and as a site for litter and ®rewood collection, which in conse-quence has led to a considerable degradation of the soils and to severe N limitation of the ecosystem. Such an ecosystem is characterized by high C-to-N ratios (.35) in the organic horizons, the net nitri®cation rates are close to zero, there is limited N supply of trees and marginal N-losses to the groundwater (ZoÈttl, 1990; Armbruster and Feger, 1998; Feger and ZoÈttl, 1998).

In our study, two sites within this forest ecosystem were investigated: one site (48 ha) had received high doses of N application in June 1988, May 1991 and ®nally in May 1994 by surface application of 150 kg N ha21as (NH4)2SO4. The

other site (46 ha) remained unfertilized. An additional small scale N application experiment at two plots (3£3 m sample area) was performed in May 1996 on the site that had already received several doses of 150 kg N ha21 in the past. One of these plots received another 150 kg N ha21 (as (NH4)2SO4) by spreading an aqueous solution

(equivalent to a 4 mm rainfall event) onto the soil, whereas the other plot received the same amount of distilled water and was used as a control.

2.2. Measurement of CH4oxidation rates

Measurements of CH4 oxidation rates at the two forest

sites were carried out during periods lasting 2±3 weeks each. Measurements were performed at different seasons in the years 1994±1996 (July 94, October/November 94, February/March 95, May 95, July/August 95, October/ November 95, May 96, July 96, October/November 96). Measurements in the ®eld were carried out using the static chamber method, i.e. by following the time-dependent decrease of CH4 concentrations in the atmosphere of a

closed chamber. For these measurements a fully-automated measuring system was used. The system consisted of: eight chambers that were placed on each site and could be opened and closed automatically by the use of pneumatic pistons and clamps; an automated sampling device; a pump; a ¯ow-controller; and a gas chromatograph equipped with a ¯ame ionization detector (FID) for CH4analysis. The design of the

chambers (0.5£0.5£0.15 m) has been described in detail by Bahl et al. (1997) and Papen and Butterbach-Bahl (1999). The chambers were ®xed on stainless steel frames (25 mm), which were driven 20 mm into the soil. Four steel frames were permanently installed, whereas the other four steel frames were distributed at random in the ®eld at the beginning of each measurement period. A measuring interval lasted for 2 h in which two sets of four chambers were closed and reopened for 1 h each in an alter-nating way. When the chambers were closed, air samples were removed automatically from the chamber-atmosphere (Butterbach-Bahl et al., 1997) and analyzed for CH4by gas

chromatography (Shimadzu GC-8AIF, equipped with FID; column: 1.5 m stainless steel 6 mm ®lled with molecular sieve 60±80 mesh, carrier gas: 70 ml of N25.0 min21;

burn-ing gas: 40 ml of H2 5.0 min21; burning air: synthetic air

350 ml min21; detector temperature: 1008C; column temperature: 808C; all gases were supplied by Messer Griesheim, Germany). After 2 h the g.c. was automatically re-calibrated using standard gas (4.0ml l21CH4in synthetic

air, Messer Griesheim, Germany). CH4oxidation rates were

calculated from ®ve CH4 concentration measurements

obtained for each chamber. Since the oxidation of atmo-spheric CH4of soils follows a ®rst-order decay function, an

exponential regression was used to calculate CH4oxidation

rates. For further small scale N application experiments two additional chambers on each plot were installed in the ®eld. For this experiment, gas samples were taken manually by gas-tight syringes. Sample air was directly injected onto the column of the g.c. and analyzed for CH4, as described above.

2.3. Distribution of CH4oxidation activity in the soil pro®le and measurements of soil temperature and moisture

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the soil pro®le was determined sporadically at selected chambers by removing stepwise the uppermost soil layers. From the comparison of CH4oxidation rates before and after

removing the different soil layers, the CH4oxidation activity

of selected soil horizons was calculated (Table 1). Soil temperature was measured with on-line semiconductor sensors (Analog Devices Norwood, USA) at 20, 50 and 100 mm soil depth with a time resolution of 15 min. The volumetric soil water content at 20, 50 and 100 mm soil depth was measured daily by the use of a TDR (time domain re¯ectometry) probe (Imko, Ettlingen).

2.4. Determination of NH41NO32and NO22concentrations in the soil

Ammonium, NO32 and NO22 concentrations in the soil

were determined for different soil horizons (organic layer, 0±50 and 50±100 mm of the mineral soil): 5 g of soil were extracted with 50 ml of a 0.01 N KAl(SO4)2 solution by

vigorous shaking for 20 min. The suspension was centri-fuged (Centrifuge J2-21; Beckmann, MuÈnchen) for 15 min at 15 000 rev min21, the supernatant was decanted, ®ltered (Becton-Dickinson, Ireland, 5 and 2mm mesh) and then stored frozen until analysis. Ammonium, NO32 and NO22

in the soil extracts were determined by ion chromatography (DX-500, Fa. Dionex GmbH, Idstein, Germany). For NH41

separation, a Ionpac CS12 column and a Ag4A-SC-pre-column, with 20 mM methanesulfonic acid (¯ow rate of 1 ml min21

) as eluent was used. NO32and NO22were

sepa-rated by an IonPac AS4A-SC column equipped with a IonPac Ng1 and Cg12 pre-column (supplier of analytical columns: Fa. Dionex GmbH, Idstein, Germany). The eluent (¯ow rate: 2 ml min21) was a 1:1 mixture of an 1.7 mmol NaHCO3l21and an 1.8 mmol Na2CO3l21solution.

2.5. Process level studies on the response of CH4oxidation to soil temperature

The effects of soil temperature on consumption of atmo-spheric CH4 was determined on fresh soil samples taken

from the upper 50 mm of the mineral soil, which showed the highest CH4 oxidation activity in the ®eld. For these

experiments, 40 g of sieved (3 mm mesh) soil samples were added to 320 ml glass ¯asks. At the beginning of each experiment, soil samples were equilibrated to a

temperature of 258C for 3 days. Thereafter, the incubation temperature was decreased every second day stepwise by 4± 58C. As a control, soil samples were incubated at constant temperature (258C) and were checked for stability of CH4

oxidation activity. CH4 oxidation activity remained stable

over a period .14 days. To avoid CH4 deprivation and

moisture loss of the soil samples during the incubation, the ¯asks were ¯ushed continuously with humidi®ed ambi-ent air (CH4concentration: 1.8ml l21). For determination of

CH4oxidation activity the ¯asks were closed gas tight. The

kinetics of decrease of CH4concentrations in the headspace

of the ¯asks were determined by removing gas samples with a gas-tight syringe and injecting them on a g.c. equipped with FID (for details see above). CH4oxidation rates were

calculated from the time-dependent exponential decrease in CH4concentration in the headspace of the ¯asks. The dry

weight of the soil samples was determined gravimetrically at the beginning and at the end of the experiment.

2.6. Statistical analysis

Data sets were tested for normal distribution by the Kolmogorov±Smirnov test. For identifying signi®cant differences between different data-sets either the t-test (for normal distributed data-sets) or the U-test by Mann and Whitney (for non-normal distributed data-sets) was used. Relations between CH4 oxidation rates and soil moisture

and soil temperature were investigated by linear, partial and multiple regression analysis. In order to exclude the in¯uence of spatial variation, for the correlation analysis between CH4oxidation rates and soil temperature and soil

moisture, respectively, we used only CH4 oxidation rates

obtained from the four chambers with permanent positions. For statistical analysis SPSS version 6.1.2 (SPSS Inc., USA) was used.

3. Results

3.1. Methane oxidation rates at the unfertilized and (NH4)2SO4fertilized sites

Measurement of CH4exchange between the soil and the

atmosphere showed that the soil of the spruce forest sites near Villingen functioned exclusively as a sink for

R. Steinkamp et al. / Soil Biology & Biochemistry 33 (2001) 145±153

Table 1

Main characteristics of the organic soil horizons (L, Of, Oh) and the underlying mineral soil horizons (Ah, AhBv, Bv), of the spruce control site; data from Armbruster and Feger (1998) (ND, no determination)

Depth (mm) Horizon Bulk density (g cm-3) pH (CaCl2) C (mg C g21soil) N (mg N g21soil) C to N ratio

120 L 0.13 3.3 531 10.1 52.6

130 Of 0.12 2.9 522 14.3 36.5

110 Oh 0.27 2.4 500 12.6 39.7

0±120 Ah 1.07 3.2 36 2.1 17.1

120±200 AhBv ND 3.7 16 1.2 13.3

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atmospheric CH4 (Fig. 1). Even during a wet period in

February±March 1994, when mean soil moisture at 50 mm soil depth reached values of up to 35% (v/v), no net emission of CH4 from the soils into the atmosphere

was recorded (Fig. 1). CH4oxidation rates exhibited a strong

seasonal pattern with highest CH4oxidation rates in summer

(81.3±116.6mg CH4m22h21), low oxidation rates in

winter (40.3±41.2mg CH4m2 2

h21

) and intermediate rates (60.0±106.1mg CH4m2

2

h21) in spring and autumn (Fig. 1). In general, oxidation rates of atmospheric CH4 were

higher in autumn (up to 38%) than in spring. In contrast to the strong seasonal changes in CH4oxidation rates,

diur-nal variations in CH4oxidation rates could not be

demon-strated with certainty (P.0.05) and were small at best (,7%). A representative example for the low diurnal varia-tions in CH4oxidation rates is given in Fig. 2. Mean CH4

oxidation rates calculated for the entire observation period

(July 1994±November 1996) were 82.2^34.6mg

CH4m22h21 for the unfertilized site and 84.2^31.8mg

CH4m22 h21 for the N fertilized site, respectively, and

were statistically not different (P_.0.05). Thus, long-term positive or negative effects of N fertilization on the magni-tude of CH4oxidation rates could not be detected.

However, this does not exclude the possibility that short-term effects of N fertilization might exist. In an additional small scale fertilization experiment carried out in May 1996, an additional dose equivalent to 150 kg N ha21in the form

Fig. 1. (A) Seasonal course of CH4oxidation rates, (B) volumetric soil moisture, (C) soil temperature, and (D) soil NH41-N concentration of the spruce control site (open symbols) and the N fertilized spruce site (closed symbols); data represent the mean (_^SD) of more than 300 single CH4 oxidation rates obtained during 2±3 weeks of ®eld measurements, and the mean soil temperature and soil moisture in 50 mm soil depth for this period of time. Values of NH41concentration are the mean of six replicates taken from the organic layer.

Fig. 2. Time course of CH4oxidation rates and soil temperature (at different soil depth) at the control site during 24 July and 31 July, 1996; CH4 oxida-tion rates represent 2-hourly mean rates (^SD) calculated from eight measuring chambers.

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of (NH4)2SO4 was applied to a small area of the 1994 N

fertilized site. The results obtained from this experiment are shown in Fig. 3. Before fertilizer application (d 0) no obvious differences in CH4oxidation rates between the ®eld

plot, which was designated for additional fertilization (55.5_^5.4mg CH4m22h21), and the control plot were

found (50.2_^8.6mg CH4m22h21). Five and 11 days,

respectively, after the application of the additional dose of 150 kg N ha21, CH4oxidation rates of the newly fertilized

site were clearly lower (P,0.01) (28.8 and 25.9%, respec-tively) than CH4oxidation rates of the control site (Fig. 3).

Fertilization had led to a strong increase in NH41

concentra-tion in the organic layer (control: 1.2^0.4mmol NH41

-N g21dw; N fertilized: 55.8^4.9mmol NH41-N g21dw),

as well as at 50 mm depth in the mineral soil (control: 0.18^0.1mmol NH41-N g21dw; fertilized:

9.7^2.4mmol NH41-N g21dw), whereas at 100±150 mm

soil depth the NH41concentrations were,0.6mmol NH41

-N g21

dw in both plots. Seventy-two days after fertilization, when NH41 concentrations of the N fertilized plot had

decreased in the organic layer to 24.1_^6.2mmol NH41

-N g21dw and to 7.4_^2.1mmol NH41-N g21dw in the

upper 50 mm of the mineral soil, CH4 oxidation rates in

the fertilized plot (56.9^4.9mg CH4m22h21) were

again almost identical to rates in the control plot (54.3^9.3mg CH4m22h21). The same statements hold

for measurements performed approximately half a year after fertilization (Fig. 3). The initial reduction of CH4

oxidation rates of the soil of the N fertilized plot was most likely due to the highly increased NH41-concentrations

(Fig. 3), since at all sampling dates soil pH-values and NO32

-concentrations in the organic layer and the mineral soil were not affected by fertilization (NO32concentration: fertilized

site: 0.16_^0.1mmol NO23-N g21dw, control site 0.22_^0.04mmol NO32-N g21dw). Inhibition of CH4

oxidation by nitrite could also be excluded, since nitrite was never detected in any soil sample.

3.2. Effect of temperature and soil moisture on CH4 oxidation rates

CH4oxidation rates in the ®eld showed a strong

depen-dency upon changes in soil temperature and moisture (Fig. 1). Highest CH4oxidation rates at the ®eld sites were always

observed during periods of high soil temperature. Linear regression analysis, using mean daily CH4oxidation rates

and mean daily temperature values at 50 mm soil depth for the entire observation period, revealed a strong positive correlation (control:r0.87; fertilized:r0.86) between soil temperature and CH4oxidation rates (Fig. 4, Table 2). In

contrast, soil moisture (measured at 50 mm soil depth) and CH4 oxidation rates were negatively correlated (control:

r20.83; fertilized:r20.82) (Fig. 5, Table 2), i.e. CH4

oxidation rates decreased with increasing soil moisture. If a partial regression analysis is used, i.e. the interference of either soil moisture or soil temperature is taken into account, the regression coef®cients are strongly reduced as compared to the normal linear regression (Table 2). Since the partial regression coef®cents are lower for soil moisture than for soil temperature, one can conclude that at our sites the soil temperature was a stronger controller of CH4 oxidation

rates than soil moisture. This interpretation is supported further by multiple regression analysis. Compared to the linear regression, a multiple regression analysis using both

Fig. 4. Relation between daily mean values of CH4oxidation rates and soil temperature at 50 mm soil depth for the control site (± ±A± ±) and the N fertilized site (ÐBÐ).

Table 2

Results of normal and partial linear regression analysis between mean daily values of soil temperature, soil moisture and CH4oxidation rates (r, correlation coef®cient; level of signi®cance, *P,0.05; **P,0.01; ***P,0.001.)

Control site (r) Fertilized site (r)

Entire data set

Normal lin. regression Soil temperature 0.870*** 0.860***

Partial lin. Regression 0.554*** 0.483**

Normal lin. regression Soil moisture 20.831*** 20.816***

Partial lin. Regression 20.055 20.338*

Soil temperature,108C Soil temperature 0.755*** 0.81*** Soil moisture 20.692*** 20.59**

Soil temperature.108C Soil temperature 0.067 0.66**

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soil moisture and soil temperature as predictor variables of CH4oxidation rates, led only to a slight increase of the

corre-lation coef®cients (control:r0.89; fertilized:r0.87). A more detailed insight into the effects of changes in soil moisture and soil temperature on CH4oxidation rates in the

®eld can be obtained, if data were strati®ed: (a) for CH4

oxidation rates observed at soil temperatures ,108C; and (b) for CH4 oxidation rates observed at soil temperatures

.108C (Table 2). A common linear correlation analysis showed that for case (a) temperature was the dominating factor modulating CH4 oxidation rates. This dominance

was more pronounced at the fertilized site. In case (b) soil moisture was the more important factor for modulation of CH4oxidation at the fertilized site (Table 2). However, at

the control site no signi®cant correlation between CH4

oxidation rates and soil moisture could be demonstrated (Table 2).

These ®ndings indicate that soil temperature rather than soil moisture was the more important modulator of CH4

oxidation rates at our study sites at low soil temperatures, i.e. during winter and spring. The effect of changes in temperature on soil CH4oxidation activity was also

inves-tigated in the laboratory using soil samples taken in the ®eld

(control site) from the mineral soil at 0±40 mm soil depth (Fig. 6). As found for the ®eld study, CH4oxidation rates

were positively correlated with temperature. The tempera-ture effect on soil CH4oxidation rates was more pronounced

in the temperature range 0±108C. Further increases in temperature (up to 258C) were followed only by slight increases in CH4 oxidation rates (Fig. 6). The more

pronounced temperature dependency of CH4 oxidation at

lower temperatures became evident by calculating the apparentQ10values for the temperature ranges 2.5±12.58C

and 12.5±22.58C, respectively. TheQ10values for the lower

temperature range (Q101.722.9) was signi®cantly

higher (P_,0.001) than the Q10 values for the higher

temperature range (Q10,1.2). It should be noted that the

calculatedQ10values refer to the CH4oxidation activity of

individual soil samples used in the laboratory experiment.

3.3. Vertical distribution of CH4oxidation activity

In May and July, 1995, additional experiments were carried out to identify the soil horizon that is most active with regard to the oxidation of atmospheric CH4. For these

experiments, CH4oxidation rates of the soil were compared

after different soil layers had been removed one after the other. As a representative example the results of the experi-ments in May 1995 are given in Fig. 7. The highest CH4

oxidation rates were always observed after removal of the organic layer, i.e. exposure of the uppermost mineral soil layer (Ah horizon) to the atmosphere (1.4±to 2.5-fold higher than CH4oxidation rates of the undisturbed site) (Fig. 7)

indicating that the Ah horizon is the most important for atmospheric CH4oxidation and that CH4oxidation in the

uppermost mineral layer is limited by gas diffusion. The observed differences for the increase in CH4 oxidation

rates after removal of the organic layer may re¯ect the differences in the thickness of the organic layer, which were non-typically high at the selected areas. If further

Fig. 5. Relation between daily mean values of CH4oxidation rates and soil moisture at 50 mm soil depth for the control site (± ±A± ±) and the N fertilized site (Ð_BÐ).

Fig. 6. Temperature dependency of CH4oxidation in six independently incubated soil samples (_BD_XAWV), which were taken from the uppermost mineral soil layer.

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mineral soil layers were removed, the CH4oxidation rates of

the soil would decrease signi®cantly (Fig. 7).

4. Discussion

4.1. Methane oxidation rates at the Black Forest sites as compared to other studies in temperate forest ecosystems

Throughout the entire 2.5 year observation period, the soil of the N-limited coniferous forest ecosystem was a signi®cant sink for atmospheric CH4. The mean CH4

oxida-tion rate of the control site was 82.2mg CH4m22h21.

Annual mean CH4 oxidation rate during 1995, the only

year in which CH4 oxidation was measured in all of the

four seasons, was 76.5mg CH4m2 2

h21

. These values are in the same range as values for CH4 oxidation by soils

obtained for other temperate forest ecosystems (e.g. Steu-dler et al., 1989; Castro et al., 1995; Sitaula et al., 1995; Butterbach-Bahl et al., 1998).

4.2. Diurnal and seasonal variations of CH4oxidation rates at the Black Forest sites

Though we used an automatic measurement system, we did not record any signi®cant diurnal variations of CH4¯ux

rates. This could be due to: (a) the fact that the amplitude of changes in daily soil temperature (maximum 3.98C at 50 mm soil depth) was too low to cause signi®cant varia-tions in CH4oxidation rates; or (b) to CH4diffusion through

the forest ¯oor was limited to sites of methanotrophic activ-ity in the uppermost mineral soil. In contrast, seasonal varia-tions of CH4oxidation rates in the ®eld were signi®cant with

low rates in winter and high rates in summer. Seasonal variations of CH4 oxidation activity in soils have been

recorded in other studies (e.g DoÈrr et al., 1993). In our investigation CH4 oxidation rates were lowest at soil

temperatures close to 08C (e.g. February/March 1995). During this time of the year CH4oxidation was

approxi-mately 40mg CH4m22h21, demonstrating that soils at the

Villingen spruce forest site act in winter also as an important net sink for atmospheric CH4, that microbes responsible for

atmospheric CH4 oxidation were well adapted to low

temperatures, and that higher moisture contents were not severely affecting CH4 uptake. Compared to winter, CH4

oxidation rates were 2- to 3-fold higher in summer, which can be interpreted as a combined effect of increased temperatures and reduced soil moisture (increased gas diffu-sibility) on CH4uptake. As shown by multiple and partial

linear regression analysis the main regulating factor of CH4

oxidation in the ®eld was soil temperature and not soil moisture. The strati®cation of the entire data set for obser-vations of CH4oxidation rates at soil temperatures,108C

and for observations at soil temperatures.108C revealed that the response of CH4oxidation rates to changes in soil

temperature was strongest at low temperatures. At soil temperatures.108C soil moisture was demonstrated to be

the dominant factor controlling CH4oxidation at the

ferti-lized site, but not at the control site. At present, it cannot be decided whether at soil temperatures .108C soil moisture becomes in general the dominant factor controlling CH4

oxidation in the ®eld. Our results, derived from correlation analysis, about the temperature response of CH4oxidation

rates to soil temperature_,10₈C are in agreement with ®nd-ings by Castro et al. (1995). These authors reported that for soils of a pine and a deciduous forest ecosystem in Massa-chusetts, USA, temperature is an important controller of CH4 oxidation in winter, early spring and late autumn

when soil temperature was in the range of25±108C; during the warmer seasons when soil temperature was in the range of 10±208C, CH4oxidation rates became independent from

soil temperature. Also Crill (1991), described a strong posi-tive correlation between soil temperature and CH4oxidation

rates in spring and early summer and a lack of such a corre-lation during July±October. The authors hypothesized that the atmospheric CH4 oxidizers reached their optimum

temperature in late spring so that other factors, such as soil moisture, became the most important controller of CH4oxidation. Most of the published investigations on the

effects of changes of soil moisture or soil temperature on CH4oxidation rates did not consider the fact that in the ®eld

a temperature threshold may exist, below which soil temperature becomes more important for CH4 oxidation

than soil moisture. Therefore, results obtained by others, such as DoÈrr et al. (1993), who found that soil temperature is of minor importance for the regulation of CH4oxidation

by soils, and that changes in soil gas permeability due to changes in soil moisture is the main factor in¯uencing the magnitude of CH4oxidation, should be re-considered. The

importance of soil temperature in regulating CH4oxidation

in soils at lower temperatures was also con®rmed in our laboratory studies. These experiments show a sensitivity threshold for temperature, which is close to 108C.

4.3. Methane oxidation rates at the unfertilized and (NH4)2SO4fertilized sites

Increased atmospheric N input may have signi®cant effects on oxidation rates of atmospheric CH4 by soils,

due to the sensitivity of microbial CH4 oxidation to

increased NH41 concentration. A direct negative effect of

increased N input by wet deposition on CH4 oxidation

rates was demonstrated by Butterbach-Bahl et al. (1997) under in situ conditions. In several earlier studies the effect of increasing atmospheric N input on CH4oxidation in soils

was investigated by N fertilization experiments with NH41

-containing fertilizers. These experiments showed that ferti-lization of soils with NH41 can inhibit atmospheric CH4

oxidation (Steudler et al., 1989; Sitaula et al., 1995; Castro et al., 1995; Macdonald et al., 1996).

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CH4¯uxes obtained for the unfertilized and the N fertilized

site in the years 1994±1996, showed that N fertilization did not lead to obvious changes in the CH4oxidation activity of

the soil. Results obtained from the additional experiments in which the short-term effect of N fertilization on CH4

oxida-tion was studied, indicate that N fertilizaoxida-tion exhibited a short-term reduction of CH4oxidation (,72 days) by 30%

at most.

Earlier N fertilization experiments in forest ecosystems (Steudler et al., 1989; Sitaula et al., 1995; Castro et al., 1995; Macdonald et al., 1996), revealed a stronger inhibi-tory effect on CH4oxidation rates, as was found in our study

also. Further, the persistence of the inhibition of CH4

oxida-tion rates by N fertilizaoxida-tion was more pronounced in other studies. A signi®cant reduction of CH4 oxidation rates by

33% after application of 120 kg N ha21y21(as NH4NO3)

was reported by Steudler et al. (1989). The observed reduc-tion persisted for more than 6 months. For a Scot pine Forest in Norway Sitaula et al. (1995) reported a reduction of CH4

oxidation rates by 38%, as a result of fertilization with 90 kg N ha21y21 (as NH4NO3) in the two following years. A

persistent inhibition of CH4oxidation rates for many years

after N fertilizer application, has been demonstrated in Colorado grassland soils (Mosier et al., 1991) and agricul-tural soils (HuÈtsch et al., 1993; HuÈtsch, 1996). The reasons why the inhibitory effect of (NH4)2SO4fertilization on CH4

oxidation in our study was not as persistent as in other studies, remain uncertain. However, Gulledge et al. (1997) also could not detect a clear inhibitory effect of N fertiliza-tion on CH4 oxidation for Alaskan spruce soils. These

authors suggested that this result may be related to a higher N immobilization capacity of the soil studied, which could protect CH4oxidizers from exposure to NH41.

4.4. Vertical distribution of CH4oxidation activity

Atmospheric CH4oxidation activity at both of our

experi-mental sites showed a distinct vertical strati®cation within the soil pro®le. Maximum CH4oxidation activity was

loca-lized in the uppermost mineral soil layer (Ah layer), and markedly decreased with increasing soil depth (see also: Crill, 1991; Koschorrek and Conrad, 1993; Bender and Conrad, 1994; Schnell and King, 1994; Kruse et al., 1996; Czepiel et al., 1995). Decreasing CH4 oxidation activity

with increasing soil depth may result from the limitation of CH4-diffusion within the soil pro®le, which in

conse-quence could lead to a substrate limitation (Koschorrek and Conrad, 1993). Our results show that the removal of the organic layer caused a signi®cant increase of CH4

oxida-tion rates by a factor of 1.4±2.5. From this observaoxida-tion, it is concluded that none or only minor CH4oxidation activity is

present in the organic layer, but that the organic layer appar-ently acts as a diffusion barrier for gases (e.g. CH4, O2) at

least over short periods. The phenomenon of increasing CH4

oxidation rates after exposure of the uppermost mineral soil layer to the atmosphere was also described by Borken and

Brumme (1997). These authors observed an increase in CH4

oxidation rates of up to 2.71-fold after removal of the organic layer from water-saturated soil cores taken from deciduous and spruce forests. Comparable results were also obtained by Saari et al. (1998) for soils of a boreal Scots pine forest in Finland. These authors showed that CH4oxidation rates increased 1.5-fold after removal of a

thin O horizon (20±30 mm). The reasons for the observation that the main CH4oxidation activity of soils is located in the

mineral soil layer and not in the organic layer, which is directly exposed to the atmosphere, are still uncertain. Some authors assumed that the higher NH41content in the

organic layer as compared to the uppermost mineral layer, may be responsible for the inhibition of CH4 oxidation

(Bender and Conrad, 1994; Schnell and King, 1994; Conrad, 1996). Furthermore, the organic layer may contain other compounds that can inhibit CH4oxidation activity (Amaral

and Knowles, 1998) such as monoterpenes. Moreover, since methanotrophic activity in soils might be reduced due to water stress (Schnell and King, 1996; Nesbit and Breiten-beck, 1992), the mineral soil may offer a more stable ecolo-gical niche for CH4oxidizers than the organic layer.

Acknowledgements

The authors are indebted to Elisabeth Zumbusch for expert technical assistance. The authors thank Professor Dr Heinz Rennenberg, Chair of Tree Physiology of the University of Freiburg, Germany, for valuable comments on the manuscript. They also thank the Bundesministerium fuÈr Bildung, Wissenschaft, Forschung und Technologie (BMBF), Bonn, for funding this work within the German Climate Research Programme ªSpurenstoffkreislaÈufeº.

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