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Soil Biology & Biochemistry 33 (2001) 145±153
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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 ha 21 (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 ^ 34.6 mg CH4 m 22 h 21 for the
unfertilized site, and 84.2 ^ 31.8 mg CH4 m 22 h 21 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 CH4 oxidation: 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
CH4 oxidation 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 CH4 oxidation rates were positively correlated with soil temperature and negatively
with soil moisture. However, at low soil temperatures (,108C), temperature was a stronger modulator of CH4 oxidation than soil moisture.
Process studies on soil samples in the laboratory con®rmed a pronounced positive response of CH4 oxidation to changes in temperature, only
within a range of 0±108C. At both experimental sites, the highest CH4 oxidation activity was observed in the Ah layer (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 CH4 oxidation
rates. Apparently the organic layer functions as a diffusive barrier for atmospheric CH4 or O2 to CH4 oxidizing sites. q 2001 Elsevier Science
Ltd. All rights reserved.
Keywords: Methane oxidation; NH41 inhibition of CH4 oxidation; Forest soils; Soil temperature; Seasonal variations

1. Introduction
Next to CO2, CH4 is the most important greenhouse gas in
the atmosphere. Its atmospheric concentration has increased
during the last 300 years, from about 0.75 to 1.7 ml l 21
(Lelieveld et al., 1993). In the last 20 years atmospheric
CH4 concentration increased, on an average, at a rate of
0.8% y 21; at present the rate of increase has slowed down
to less than 0.3% y 21 (Prinn, 1995). The main sink for atmospheric CH4 is photochemical oxidation with hydroxyl radicals 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 CH4 by microbial activity in soils is estimated to be in a range of 15±
* Corresponding author. Tel.: 149-8821-183130; fax: 149-8821183294.

E-mail address: papen@ifu.fhg.de (H. Papen).

45 Tg CH4 y 21 (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 atmospheric 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
CH4 oxidation of forest soils, several N fertilization experiments have been carried out in the past. Most of these

0038-0717/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.

PII: S 0038-071 7(00)00124-3

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

studies have found that the N fertilization of forest soils had
an inhibitory effect on CH4 oxidation 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 CH4 oxidation 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 hypothesized that the consequences of N deposition for CH4 oxidation rates of forest soils may depend on the N status of
ecosystems, i.e. CH4 oxidation 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 measurements of CH4 oxidation from an unfertilized and N fertilized
site. In addition, seasonal variations of CH4 oxidation 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 (488 03 0 N, 88 22 0 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 ha 21y 21 (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 consequence 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 ha 21 as (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 ha 21 in the
past. One of these plots received another 150 kg N ha 21
(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 CH4 oxidation 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 ¯owcontroller; and a gas chromatograph equipped with a ¯ame
ionization detector (FID) for CH4 analysis. The design of the
chambers (0.5 £ 0.5 £ 0.15 m) has been described in detail
by Butterbach-Bahl et al. (1997) and Papen and ButterbachBahl (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 alternating way. When the chambers were closed, air samples
were removed automatically from the chamber-atmosphere
(Butterbach-Bahl et al., 1997) and analyzed for CH4 by 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 N2 5.0 min 21; burning gas: 40 ml of H2 5.0 min 21; burning air: synthetic air
350 ml min 21; 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 (4.0 ml l 21 CH4 in synthetic
air, Messer Griesheim, Germany). CH4 oxidation rates were
calculated from ®ve CH4 concentration measurements
obtained for each chamber. Since the oxidation of atmospheric CH4 of soils follows a ®rst-order decay function, an
exponential regression was used to calculate CH4 oxidation
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 gastight syringes. Sample air was directly injected onto the
column of the g.c. and analyzed for CH4, as described above.

2.3. Distribution of CH4 oxidation activity in the soil pro®le
and measurements of soil temperature and moisture
The vertical distribution of CH4 oxidation activity within

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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 g 21 soil)

N (mg N g 21 soil)

C to N ratio

1 20
1 30
1 10
0±120
120±200
200±280

L
Of
Oh
Ah
AhBv
Bv

0.13

0.12
0.27
1.07
ND
1.39

3.3
2.9
2.4
3.2
3.7
4.0

531
522
500
36
16
11

10.1
14.3
12.6
2.1
1.2
1.0

52.6
36.5
39.7
17.1
13.3
11

the soil pro®le was determined sporadically at selected
chambers by removing stepwise the uppermost soil layers.
From the comparison of CH4 oxidation rates before and after
removing the different soil layers, the CH4 oxidation 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 NH41 NO32 and NO22 concentrations 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 centrifuged (Centrifuge J2-21; Beckmann, MuÈnchen) for 15 min
at 15 000 rev min 21, the supernatant was decanted, ®ltered
(Becton-Dickinson, Ireland, 5 and 2 mm 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-precolumn, with 20 mM methanesulfonic acid (¯ow rate of
1 ml min 21) as eluent was used. NO32 and NO22 were separated 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 min 21) was a 1:1 mixture of an 1.7 mmol
NaHCO3 l 21 and an 1.8 mmol Na2CO3 l 21solution.
2.5. Process level studies on the response of CH4 oxidation
to soil temperature

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 ambient air (CH4 concentration: 1.8 ml l 21). For determination of
CH4 oxidation activity the ¯asks were closed gas tight. The
kinetics of decrease of CH4 concentrations 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). CH4 oxidation rates were
calculated from the time-dependent exponential decrease in
CH4 concentration 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 CH4 oxidation 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

The effects of soil temperature on consumption of atmospheric 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

3.1. Methane oxidation rates at the unfertilized and
(NH4)2SO4 fertilized sites
Measurement of CH4 exchange between the soil and the
atmosphere showed that the soil of the spruce forest sites
near Villingen functioned exclusively as a sink for

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

Fig. 1. (A) Seasonal course of CH4 oxidation 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 NH41 concentration are the mean of six replicates taken
from the organic layer.

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

Fig. 3. CH4 oxidation rates and soil NH41-N concentrations before and after
additional fertilization with (NH4)2SO4. CH4 oxidation rates are means
(^SD) (N ˆ 8 2 10) calculated for the control plot and the newly fertilized
plot (1150 kg N ha 21 as (NH4)2SO4) on different days after fertilizer application; each NH41-N concentration (-A-control site; -B- fertilized site) is
the mean of four replicate determinations.

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). CH4 oxidation rates exhibited a strong
seasonal pattern with highest CH4 oxidation rates in summer
(81.3±116.6 mg CH4 m 22 h 21), low oxidation rates in
winter (40.3±41.2 mg CH4 m 22 h 21) and intermediate rates
(60.0±106.1 mg CH4 m 22 h 21) 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 CH4 oxidation rates, diurnal variations in CH4 oxidation rates could not be demonstrated with certainty (P . 0.05) and were small at best
(,7%). A representative example for the low diurnal variations in CH4 oxidation rates is given in Fig. 2. Mean CH4
oxidation rates calculated for the entire observation period
(July 1994±November 1996) were 82.2 ^ 34.6 mg
CH4 m 22 h 21 for the unfertilized site and 84.2 ^ 31.8 mg
CH4 m 22 h 21 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 magnitude of CH4 oxidation rates could not be detected.
However, this does not exclude the possibility that shortterm 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 ha 21 in the form

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

Fig. 4. Relation between daily mean values of CH4 oxidation rates and soil
temperature at 50 mm soil depth for the control site (± ±A± ±) and the N
fertilized site (ÐBÐ).

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 CH4 oxidation rates between the ®eld
plot, which was designated for additional fertilization
(55.5 ^ 5.4 mg CH4 m 22 h 21), and the control plot were
found (50.2 ^ 8.6 mg CH4 m 22 h 21). Five and 11 days,
respectively, after the application of the additional dose of
150 kg N ha 21, CH4 oxidation rates of the newly fertilized
site were clearly lower (P , 0.01) (28.8 and 25.9%, respectively) than CH4 oxidation rates of the control site (Fig. 3).
Fertilization had led to a strong increase in NH41 concentration in the organic layer (control: 1.2 ^ 0.4 mmol NH41N g 21 dw; N fertilized: 55.8 ^ 4.9 mmol NH41-N g 21 dw),
as well as at 50 mm depth in the mineral soil (control:
fertilized:
0.18 ^ 0.1 mmol
NH41-N g 21 dw;
1
9.7 ^ 2.4 mmol NH4 -N g 21 dw), whereas at 100±150 mm
soil depth the NH41 concentrations were ,0.6 mmol NH41N g 21 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 ^ 6.2 mmol NH41N g 21 dw and to 7.4 ^ 2.1 mmol NH41-N g 21 dw in the
upper 50 mm of the mineral soil, CH4 oxidation rates in

149

the fertilized plot (56.9 ^ 4.9 mg CH4 m 22 h 21) were
again almost identical to rates in the control plot
(54.3 ^ 9.3 mg CH4 m 22 h 21). 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 NO32concentrations in the organic layer and the mineral soil were
not affected by fertilization (NO32 concentration: fertilized
21
dw, control site
site: 0.16 ^ 0.1 mmol NO2
3 -N g
2
21
0.22 ^ 0.04 mmol NO3 -N g dw). 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
CH4 oxidation rates in the ®eld showed a strong dependency upon changes in soil temperature and moisture (Fig. 1).
Highest CH4 oxidation rates at the ®eld sites were always
observed during periods of high soil temperature. Linear
regression analysis, using mean daily CH4 oxidation rates
and mean daily temperature values at 50 mm soil depth for
the entire observation period, revealed a strong positive
correlation (control: r ˆ 0.87; fertilized: r ˆ 0.86) between
soil temperature and CH4 oxidation rates (Fig. 4, Table 2). In
contrast, soil moisture (measured at 50 mm soil depth) and
CH4 oxidation rates were negatively correlated (control:
r ˆ 20.83; fertilized: r ˆ 20.82) (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

Table 2
Results of normal and partial linear regression analysis between mean daily values of soil temperature, soil moisture and CH4 oxidation rates (r, correlation
coef®cient; level of signi®cance, *P , 0.05; **P , 0.01; ***P , 0.001.)

Entire data set
Normal lin. regression
Partial lin. Regression
Normal lin. regression
Partial lin. Regression

Soil temperature
Soil moisture

Control site (r)

Fertilized site (r)

0.870***
0.554***
2 0.831***
2 0.055

0.860***
0.483**
2 0.816***
2 0.338*

Soil temperature ,108C

Soil temperature
Soil moisture

0.755***
2 0.692***

0.81***
2 0.59**

Soil temperature .108C

Soil temperature
Soil moisture

0.067
2 0.200

0.66**
2 0.924***

150

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

Fig. 5. Relation between daily mean values of CH4 oxidation rates and soil
moisture at 50 mm soil depth for the control site (± ±A± ±) and the N
fertilized site (ÐBÐ).

soil moisture and soil temperature as predictor variables of
CH4 oxidation rates, led only to a slight increase of the correlation coef®cients (control: r ˆ 0.89; fertilized: r ˆ 0.87).
A more detailed insight into the effects of changes in soil
moisture and soil temperature on CH4 oxidation 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
CH4 oxidation 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 CH4 oxidation activity was also investigated in the laboratory using soil samples taken in the ®eld

Fig. 6. Temperature dependency of CH4 oxidation in six independently
incubated soil samples (BDXAWV), which were taken from the uppermost
mineral soil layer.

Fig. 7. Pro®le of CH4 oxidation activity in soils of the control (open bars)
and the fertilized site (closed bars); given CH4 oxidation rates (results of the
experiments in May 1995) are mean values (^SD).

(control site) from the mineral soil at 0±40 mm soil depth
(Fig. 6). As found for the ®eld study, CH4 oxidation rates
were positively correlated with temperature. The temperature effect on soil CH4 oxidation 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
apparent Q10 values for the temperature ranges 2.5±12.58C
and 12.5±22.58C, respectively. The Q10 values for the lower
temperature range (Q10 ˆ 1.7 2 2.9) 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
calculated Q10 values refer to the CH4 oxidation activity of
individual soil samples used in the laboratory experiment.
3.3. Vertical distribution of CH4 oxidation 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, CH4 oxidation 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 experiments 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 CH4 oxidation rates of the undisturbed site) (Fig. 7)
indicating that the Ah horizon is the most important for
atmospheric CH4 oxidation and that CH4 oxidation 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

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

mineral soil layers were removed, the CH4 oxidation 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 oxidation rate of the control site was 82.2 mg CH4 m 22 h 21.
Annual mean CH4 oxidation rate during 1995, the only
year in which CH4 oxidation was measured in all of the
four seasons, was 76.5 mg CH4 m 22 h 21. These values are
in the same range as values for CH4 oxidation by soils
obtained for other temperate forest ecosystems (e.g. Steudler et al., 1989; Castro et al., 1995; Sitaula et al., 1995;
Butterbach-Bahl et al., 1998).
4.2. Diurnal and seasonal variations of CH4 oxidation 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 variations in CH4 oxidation rates; or (b) to CH4 diffusion through
the forest ¯oor was limited to sites of methanotrophic activity in the uppermost mineral soil. In contrast, seasonal variations of CH4 oxidation 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 CH4 oxidation was approximately 40 mg CH4 m 22 h 21, 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 diffusibility) on CH4 uptake. 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 observations of CH4 oxidation rates at soil temperatures ,108C
and for observations at soil temperatures .108C revealed
that the response of CH4 oxidation rates to changes in soil
temperature was strongest at low temperatures. At soil
temperatures .108C soil moisture was demonstrated to be

151

the dominant factor controlling CH4 oxidation at the fertilized 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 CH4 oxidation
rates to soil temperature ,108C are in agreement with ®ndings by Castro et al. (1995). These authors reported that for
soils of a pine and a deciduous forest ecosystem in Massachusetts, USA, temperature is an important controller of
CH4 oxidation in winter, early spring and late autumn
when soil temperature was in the range of 25±108C; during
the warmer seasons when soil temperature was in the range
of 10±208C, CH4 oxidation rates became independent from
soil temperature. Also Crill (1991), described a strong positive correlation between soil temperature and CH4 oxidation
rates in spring and early summer and a lack of such a correlation 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
CH4 oxidation. Most of the published investigations on the
effects of changes of soil moisture or soil temperature on
CH4 oxidation 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 CH4 oxidation
by soils, and that changes in soil gas permeability due to
changes in soil moisture is the main factor in¯uencing the
magnitude of CH4 oxidation, should be re-considered. The
importance of soil temperature in regulating CH4 oxidation
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)2SO4 fertilized 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 CH4 oxidation in soils
was investigated by N fertilization experiments with NH41containing fertilizers. These experiments showed that fertilization 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).
In our study, increased atmospheric N deposition was
simulated by the surface application of 150 kg N ha 21 (as
(NH4)2SO4) to the forest ¯oor in May 1994. Comparison of

152

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

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 CH4 oxidation activity of
the soil. Results obtained from the additional experiments in
which the short-term effect of N fertilization on CH4 oxidation was studied, indicate that N fertilization exhibited a
short-term reduction of CH4 oxidation (,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 inhibitory effect on CH4 oxidation rates, as was found in our study
also. Further, the persistence of the inhibition of CH4 oxidation rates by N fertilization was more pronounced in other
studies. A signi®cant reduction of CH4 oxidation rates by
33% after application of 120 kg N ha 21 y 21 (as NH4NO3)
was reported by Steudler et al. (1989). The observed reduction 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 ha 21 y 21 (as NH4NO3) in the two following years. A
persistent inhibition of CH4 oxidation rates for many years
after N fertilizer application, has been demonstrated in
Colorado grassland soils (Mosier et al., 1991) and agricultural soils (HuÈtsch et al., 1993; HuÈtsch, 1996). The reasons
why the inhibitory effect of (NH4)2SO4 fertilization 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 fertilization 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 CH4 oxidizers from exposure to NH41.

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
CH4 oxidation rates increased 1.5-fold after removal of a
thin O horizon (20±30 mm). The reasons for the observation
that the main CH4 oxidation 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 NH41 content 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 CH4 oxidation 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 Breitenbeck, 1992), the mineral soil may offer a more stable ecological niche for CH4 oxidizers 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.

4.4. Vertical distribution of CH4 oxidation activity
Atmospheric CH4 oxidation activity at both of our experimental sites showed a distinct vertical strati®cation within
the soil pro®le. Maximum CH4 oxidation activity was localized 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 consequence 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 oxidation rates by a factor of 1.4±2.5. From this observation, it is
concluded that none or only minor CH4 oxidation activity is
present in the organic layer, but that the organic layer apparently 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

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