FEMS Microbiology Ecology 48 (2004) 305–312
www.fems-microbiology.org
Microbial oxidation of CH4 at different temperatures in landfill
cover soils
Gunnar B€
orjesson
a,*
, Ingvar Sundh b, Bo Svensson
a
a
b
Department of Water and Environmental Studies, Link€oping University, SE-581 83 Link€oping, Sweden
Department of Microbiology, Swedish University of Agricultural Sciences, P.O. Box 7025, SE-750 07 Uppsala, Sweden
Received 14 October 2003; received in revised form 10 February 2004; accepted 11 February 2004
First published online 9 March 2004
Abstract
Biological oxidation of CH4 is an important constraint on the emission of this gas from areas, such as landfills to the atmosphere.
We studied the effect of temperature on methanotrophic bacteria in three different landfill cover soils, incubated in the laboratory. In
samples of a young cover, consisting of wood chips and sewage sludge, the phospholipid fatty acids (PLFAs), regarded as biomarkers for type I methanotrophs (16:1x5t, 16:1x6c, 16:1x8c), primarily increased at low temperatures (5–10 °C). On the other
hand, the PLFA marker for type II methanotrophs (18:1x8c) was highly elevated only at 20 °C. These results suggest that temperature can determine the selection of methanotroph populations.
Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Methane oxidation; Methanotroph; Landfill gas; Phospholipid fatty acid
1. Introduction
Methanotrophs are Gram-negative bacteria that use
CH4 to gain energy and carbon for their growth [1].
These bacteria are important regulators of CH4 fluxes
from the biosphere to the atmosphere, for example from
landfills, where CH4 , one of the most important greenhouse gases, is produced in large quantities [2].
There are two main groups of methanotrophic bacteria, which are designated type I and type II [1,3]. Type
I methanotrophs (including two genera named type X)
utilise the ribulose monophosphate pathway as the primary pathway for carbon assimilation, while type II
methanothrophs use the serine pathway. They also differ
in morphology. Phylogenetic studies of 5S rRNA and
16S rRNA have confirmed the distinction between type
I and type II methanotrophs and have placed them,
respectively, in the c and a subdivisions of the Proteobacteria [3,4]. New genera, that are phylogenetically
*
Corresponding author. Tel.: +46-13-28-22-92; fax: +46-13-13-36-
30.
E-mail address: [email protected] (G. B€
orjesson).
related to type II, have recently been discovered in an
acidic peat bog [5], but it is not yet known how common
this group of bacteria is in non-acidic environments.
It has been observed that shifts in the methanotroph
populations in soils can occur in response to environmental stimuli such as changes in concentrations of CH4
and O2 , temperature, pH and nitrogen sources [1].
Furthermore, several studies have indicated that type I
and type II methanotrophs seem to occupy different
niches. For instance, type I strains are likely to dominate
in nutrient-rich environments [6–9], which agrees with
the finding that nitrogen fixation is more common in
type II methanotrophs [10].
Our primary objective was to determine whether the
two types of methanotrophs could be linked to activities
at specific temperatures. Temperature is of the utmost
importance for the ability of the methanotroph community to oxidise CH4 , and a better understanding of the
optimal conditions for oxidation would improve predictions of oxidation rates and would also help in the construction of better landfill covers, biofilters, etc. In this
respect, it has been shown that the analysis of phospholipid fatty acids (PLFAs) can provide a quantitative
0168-6496/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsec.2004.02.006
306
G. B€orjesson et al. / FEMS Microbiology Ecology 48 (2004) 305–312
measure of the bacterial and eukaryotic biomass in environmental samples [11]. Many strains of the two main
types of methanotrophs have been found to produce
large amounts of unusual fatty acids [3,9], hence these
fatty acids can be used as biomarkers. The significance of
the methanotrophic PLFA biomarkers has been demonstrated in several studies. Bowman et al. [12] thoroughly investigated 48 isolates and noted that 16:1x5t
was one of the major PLFAs in type I methanotrophs.
For example, this fatty acid constituted up to 28% of the
PLFAs found in a species of Methylomonas. They also
reported that type I methanotrophs contained large
amounts of 16:1x6c and 16:1x8c (up to 14% and 41%),
whereas the PLFA 18:1x8c was present only in type II
methanotrophs (53–74%). The PLFA 18:1x8c has been
detected almost exclusively in the type II methanotroph genera Methylocystis and Methylosinus [3]. The
methanotrophic PLFAs have been used successfully
as biomarkers in analyses of samples from CH4 -rich
environments [8,13–20].
2. Materials and methods
2.1. Soils
Cover soils were collected from three landfills, which
represented different cover types, ages and climatic
conditions: Filborna in Helsingborg (56°04.20 N,
12°46.20 E), Sundsvall (62°22.50 N, 17°11.20 E) and Visby
(57°38.50 N, 18°20.90 E). All three of these landfill sites
have gas extraction equipment, converting the energy in
CH4 into heat.
All three sites are in active use, so their covering was
not finalized at the time of this study and their soil
covers (approx. 1 m deep) were partly vegetated with
small plants. The cover at the Sundsvall site was 4 years
old and consisted of sewage sludge; the cover at Filborna was 1-year old and comprised a mixture of wood
chips and sewage sludge; the cover of Visby was 5 years
old and was made up mainly of mineral soil. The
physical and chemical characteristics of these landfill
covers are given in Table 1. At Filborna, the temperature in the soil was as high as 19 °C at a depth of 55 cm,
despite the low temperature of the ambient air (3–6 °C),
indicating an ongoing composting process or substantial
venting of landfill gas.
The soils were collected on the following dates:
Filborna, November 28, 2001; Sundsvall, March 6,
2002; Visby, June 7, 2002. At Filborna, two profiles were
sampled at 0.1-m intervals down to a depth of 0.4 m.
Since no obvious trend was found in the methane oxidation capacity and since describing the profile was not
the main purpose of the study, a simplified strategy was
used for the other two landfills, where four composite
samples were collected at depths of 0–0.3 m in different
parts of the landfills. The wet weight (ww) of each
sample was approximately 1 kg and all samples were
treated separately throughout the experiments.
2.2. Incubations
The soil samples were sieved (4 mm mesh) and stored
cold (+3 °C) for a maximum of five days before incubation. At the beginning of the experiment, four aliquots (50–100 g ww) of each soil sample were transferred
to 1.1-L glass flasks. The flasks were sealed with gastight screw caps and were then allowed to stand for 1 h
at different incubation temperature: 3, 10, 15 or 20 °C
(for Filborna 5 °C was used instead of 3 °C). Fifty
millilitres of ambient air was then inserted and shortly
thereafter (at time zero), 60 ml CH4 was added. Thus the
initial partial pressure of CH4 was 5.0%. Zero time
samples were taken immediately after the addition of
CH4 , after which the flasks were returned to their respective temperatures.
Incubation was terminated when the CH4 content
was less than 0.5% of total gas pressure in the flasks and
a portion (approx. 1 g ww) of each soil sample was
immediately frozen in a 50 ml extraction tube for subsequent analysis of PLFAs.
Aliquots of the corresponding unincubated (original)
soil samples were transferred to extraction tubes at the
start of the experiment and these portions were stored
frozen until analysis.
2.3. CH4 analyses
During incubation, three 0.3-ml samples were withdrawn from the headspace of each flask for analysis of the
Table 1
Physical characteristics and pH of the soil samples
Landfill site
Filborna, Helsingborg
Sundsvall
Visby
Solid fractions (% wt/dry wt standard deviation)
Clay < 2 lm
Silt 2–20 lm
Sand 20 lm–2 mm
Loss on ignition 550 °C
14.6 1.1
11.9 4.8
19.8 6.8
9.3 1.4
14.4 4.1
12.6 4.2
42.9 1.3
48.8 12.5
56.7 6.0
33.3 3.2
25.3 9.5
7.5 5.5
Water content
(% water of wet wt)
pH (water)
64.0 6.2
39.8 6.4
25.1 8.3
5.8–6.2
6.7–7.2
7.4–7.6
Number of samples analysed: Filborna n ¼ 8 for soil fractions and n ¼ 2 for pH; Sundsvall and Visby n ¼ 4.
G. B€orjesson et al. / FEMS Microbiology Ecology 48 (2004) 305–312
CH4 content; this was done at time intervals ranging from
one hour up to 3–4 days, as previously described [21]. CH4
was analysed on a Packard model 428 gas chromatograph
(GC) with a flame ionisation detector (FID) and a Porapak T column (2 m 2 mm), operated at 125 °C.
The CH4 concentrations in the flasks were plotted as a
function of time, and initial consumption rates were determined from the best fits of lines (zeroth-order kinetics;
cf. Fig. 1(a)) or from second-degree functions (tangents at
time zero) for curves with accelerating consumption. For
the Filborna samples, lines were fitted to the first five
points (Fig. 1(b)). All CH4 consumption rates were also
plotted as a function of temperature, and exponential
lines were fitted, from which Q10 values were calculated.
2.4. PLFA analyses
PLFA analyses were carried out on all the original
(unincubated) samples and on all the samples from 3 to
6
CH4 concentration (vol-%)
5
4
3
2
1
0
0
2
4
6
8
10
12
14
Time (h)
(a)
CH4 concentration (vol-%)
6
5 and 20 °C incubations. For the Filborna samples, the
PLFA content differed significantly between 5 and
20 °C, and therefore PLFAs were also analysed in the
samples that had been incubated at 10 and 15 °C.
Extraction and methylation of PLFAs and subsequent derivatisations with dimethyl-disulphide
(DMDS) were performed according to the methods
described by B€
orjesson et al. [17]. The methylated
PLFAs were quantified by analysis on a Hewlett–
Packard model 6890 GC, equipped with a 30 m 0.32
mm fused silica capillary column and a FID. Samples (1
ll) were injected in the splitless mode. Methyl nonadecanoate (19:0, Larodan, Malm€
o, Sweden) was used as
internal standard. The oven temperature was raised
from 50 to 110 °C at a rate of 30° min1 , from 110 to 190
°C at 1° min1 and from 190 to 300 °C at 31.4° min1 .
Identification of the fatty acids was achieved by comparison with retention times for defined mixtures of
external standards and by GC–MS analysis, using the
same chromatographic conditions, on a HP 6890
equipped with a HP 5973 mass selective detector. Confirmation of double-bond position and quantification of
mono-unsaturated fatty acids was achieved by analysing
the DMDS derivatives with GC–MS, as described by
Steger et al. [22].
Fatty acids are designated in terms of ‘‘the total
number of carbon atoms: the number of double bonds’’,
followed by the position of the first double bond from
the aliphatic end of the molecule. The suffixes ‘‘c’’ and
‘‘t’’ denote cis and trans conformations. The prefixes
‘‘i’’, ‘‘a’’ and ‘‘10Me’’ denote methyl branching in iso
and anteiso positions, and on the 10th carbon atom,
respectively. The prefix ‘‘cy’’ denotes cyclopropane
branching.
The detection limit was approximately 0.15 nmol (g
dw soil)1 , and in summaries, lower values were set to
zero. PLFAs missing in more than two samples were
omitted.
5
2.5. Particle size distribution
4
The particle size distribution in the soil samples
from Filborna (n ¼ 8), Sundsvall (n ¼ 4) and Visby
(n ¼ 4) was determined according to the methods described by Ljung [23]. The samples were dried and
loss on ignition at 550 °C was determined. After exclusion of particles >2 mm in diameter the particle
size distribution was determined. The size classes included clay (<2 lm), silt (2–20 lm), fine sand (20–200
lm) and coarse sand (0.2–2 mm).
3
2
1
0
(b)
307
0
100
200
300
400
500
600
Time (h)
Fig. 1. Changes in methane consumption with time in two soil samples.
Two examples are illustrated: diagram (a) shows zeroth order kinetics
(Visby 20 °C), and diagram (b) represents growth (Filborna 15 °C).
Error bars are standard deviation for the gas injections (n ¼ 3).
2.6. Statistical tests
ANOVAs (analyses of variance), as well as other
correlations and regressions were performed with JMP
version 3 (SAS Institute Inc., Cary, NC, U.S.A.).
308
G. B€orjesson et al. / FEMS Microbiology Ecology 48 (2004) 305–312
3. Results
3.1. CH4 oxidation capacities
The initial CH4 consumption rates varied between the
five soils. During incubation, the kinetics of CH4 consumption were close to the zeroth order for all the
Sundsvall and Visby samples (cf. Fig. 1(a)), whereas
there was an increase in consumption (i.e., obvious microbial growth) in all of the Filborna samples
(Fig. 1(b)).
CH4 consumption rates increased with increasing
temperature in all samples and one of the samples from
Sundsvall showed the highest individual rate (2.8 lmol
CH4 [g dw soil]1 h1 at 20 °C). At 20 °C, Visby had
significantly higher values than Filborna and Hagby,
whereas there were no significant differences between
samples from the five sites at 15 °C (Table 2). At 10 °C,
the values representing Filborna were significantly lower
than those for Sundvall and Visby. Furthermore, the
CH4 consumption rates were significantly higher for the
Sundsvall samples incubated at 3 °C than for the Filborna samples incubated at 5 °C (cf. mean values in Table
2). The effect of temperature could also be described by
the Q10 -values, which were 3.48 for Hagby, 4.14 for
H€
ogbytorp, 3.17 for Sundsvall and 4.03 for the Visby
samples. Q10 -values were not determined for the Filborna samples, because several of the initial rates were
uncertain.
3.2. PLFA contents
Initially, 40 PLFAs were identified. However,
16:1x8t, 18:1x11, 18:1x10t, 18:1x9t and 18:1x8t were
excluded, because they could be quantified in only one
sample each, and 18:1x7t was also excluded, since it was
detected in very few samples. Accordingly, a total of 34
PLFAs was used in the data matrices. The total amount
of PLFAs in the samples ranged from 266 to 1382 nmol
(g dw soil)1 , but there were no significant differences
between the three sites.
Table 2
Initial CH4 consumption rates (lmol CH4 [g dw soil]1 h1 SD) in samples of five different landfill cover soils
Soil
3–5 °C
10 °C
15 °C
20 °C
Filborna (n ¼ 8)
Sundsvall (n ¼ 4)
Visby (n ¼ 4)
0.026 0.027
0.17 0.17
0.14 0.073
0.11 0.12
0.55 0.50
0.46 0.25
0.23 0.21
0.79 0.83
0.64 0.37
0.64 0.42
1.17 1.14
1.57 0.73
3000
Methane oxidation capacity (nmol CH4 gdw-1 h-1)
y = 327.48 + 49.603x (r2=0.86; p<0.0001)
2500
2000
1500
1000
Filborna
Sundsvall
Visby
500
0
0
10
20
30
40
50
18:1ω8c (nmol PLFA gdw-1)
Fig. 2. Correlation between the PLFA 18:1x8c in unincubated soil samples and methane oxidation capacity at 20 °C.
G. B€orjesson et al. / FEMS Microbiology Ecology 48 (2004) 305–312
309
the total PLFAs) was detected in one of the Sundsvall
samples. The four ‘‘methanotrophic’’ PLFAs in the
unincubated samples were strongly correlated to the
CH4 consumption rates. The best correlation was between PLFA 18:1x8c and the methane consumption
rates at 20 °C (Fig. 2), but the correlation for 16:1x5t
was also good, especially at 15 °C (r2 ¼ 0:76;
p < 0:0001).
After incubation, only the Filborna samples exhibited
significant changes in the levels of the specific PLFAs
(Fig. 3), a result that correlated with the CH4 consumption kinetics, which indicated that growth had
occurred in the samples from that site (cf. Figs. 1(a) and
(b)). In a comparison of individual PLFAs before and
after incubation (Table 3), the methanotroph-specific
PLFAs (16:1x5t, 16:1x6c, 16:1x8c and 18:1x8c) showed
the most marked changes, and there were also clear
differences between types I and II PLFAs. Furthermore,
there were significant increases in all of the mono-unsaturated 16-C PLFAs, except 16:1x7t and 16:1x5c, and
this was especially pronounced at low temperatures (5,
10 and 15 °C). After the 20 °C incubation, the only
significant change in the PLFAs was an increase in
18:1x8c.
3.3. Particle size distribution
The rates of CH4 consumption were more strongly
correlated with the content of sand in the samples than
with other parameters. The content of coarse sand
(particles 0.2–2 mm) correlated with CH4 consumption
at 10 °C (r2 ¼ 0:46, p ¼ 0:0037), and this was also observed at 3–5 °C (p ¼ 0:022), and 15 °C (p ¼ 0:0093). At
20 °C, the best correlation was found between CH4
consumption rates and the finer part of the coarse sand
(0.2–0.6 mm); r2 ¼ 0:39, p ¼ 0:010. However, since these
correlations draw heavily upon samples from the
Sundsvall site, possible effects of grain size fractions will
not be further discussed.
Fig. 3. Contents of certain phospholipid fatty acids in unincubated soil
samples and in samples of the same soils after incubation at different
temperatures. (a) Filborna, (b) Sundsvall and (c) Visby.
4. Discussion
Regarding the original (unincubated) soil samples,
those collected at Filborna generally contained low
levels of the PLFAs associated with methanotrophs
(16:1x5t, 16:1x6c, 16:1x8c and 18:1x8c). With the exception of 14.7 nmol 18:1x8c in one of the topsoil (0–
0.10 m) samples, none of the soil samples had amounts
higher than 4.1 nmol (g dw soil)1 of these four fatty
acids. Nearly all the original samples from the two older
landfill covers (Sundsvall and Visby) contained higher
amounts of these four PLFAs and the highest concentration (50.0 nmol 18:1x8c, corresponding to 7.6% of
The CH4 oxidation capacity was slightly different
among the three soils used in this study. The highest
mean rates of CH4 oxidation were 1.17 and 1.57 lmol
CH4 (g dw soil)1 h1 , which were observed in the
Sundsvall and Visby samples incubated at 20 °C (Table
2). The capacities of these two soils are similar to previously reported values for landfill covers consisting of
mineral soils (cf. 0.998 [24], 0.93 [25], 1.0 [18], and 1.62
lmol CH4 [g dw soil]1 h1 [26]), whereas cover soils
rich in organic matter are known to have higher CH4 oxidising capacities (e.g., 8.0 [27], 10.8 [17], and 25 lmol
CH4 [g dw soil]1 h1 [28]). The lower CH4 oxidation
capacity of the Filborna soil is probably due to the fact
310
G. B€orjesson et al. / FEMS Microbiology Ecology 48 (2004) 305–312
Table 3
Mean values (n ¼ 3) of phospholipid fatty acids (nmol PLFA [g dw soil]
cubation with CH4 at different temperatures
PLFA
i14:0
14:0
i15:0
a15:0
15:0
i16:1
16:1x9c
16:1x8c
16:1x7c
16:1x7t
16:1x6c
16:1x6t
16:1x5c
16:1x5t
16:0
i17:1
10Me16:0
i17:0
a17:0
cy17:0
17:0
10Me17:0
18:2
18:1x9c
18:1x8c
18:1x7c
18:1x5c
18:0
10Me18:0
i19:0
a19:0
cy19:0
20:0
Sum
1
) in soil samples from the Filborna landfill cover, before and after in-
Unincubated
samples
Incubated samples
5 °C
10 °C
15 °C
20 °C
5.15
13.0
46.8
25.8
8.27
28.3
1.08 a
0.82 a
78.3 a
5.60
1.13 a
0.14 ab
22.3
2.09 a
209.5
8.23
31.4
22.8
15.3
100.8
8.46
11.8
13.3
27.5
1.26 a
34.2
1.59
17.1
8.50 a
3.29
1.88
33.4
3.58
800.4
4.52
13.9
36.9
20.8
7.16
24.2
0.82 a
16.4 bc
99.2 ab
7.08
4.93 ab
0a
22.3
15.1 b
193.3
7.87
25.4
21.3
19.6
97.4
7.14
10.1
12.6
24.7
1.12 a
41.8
1.37
15.2
7.32 ab
2.86
1.65
29.2
5.69
804.7
5.94
15.9
51.1
25.2
8.63
29.8
1.70 b
21.9 cd
128.3 b
9.18
8.00 b
1.98 b
31.6
18.8 b
237.1
12.1
34.6
26.4
23.1
115.8
7.50
12.0
11.2
25.0
1.65 a
44.9
1.40
17.3
7.57 ab
3.14
2.45
37.8
4.66
992.4
4.72
13.2
44.9
21.8
7.06
23.5
1.35 ab
9.01 ab
111.9 ab
6.61
6.71 b
0.80 ab
22.9
18.0 b
185.3
10.6
28.9
21.8
19.8
90.4
6.53
10.6
10.7
24.4
3.59 ab
22.9
1.30
14.9
6.25 b
2.63
1.19
31.6
6.80
822.4
4.83
12.4
43.3
24.0
7.52
24.7
1.06 a
3.52 a
95.4 ab
6.71
3.25 ab
0.57 ab
22.3
8.83 ab
183.9
8.96
26.9
22.2
20.3
86.8
7.31
10.7
12.9
25.8
5.60 b
45.7
1.45
15.6
7.06 ab
3.07
1.86
30.3
6.57
788.5
ANOVA Prob
>F
0.91
0.72
0.39
0.97
0.57
0.50
0.071
0.011*
0.096
0.45
0.087
0.22
0.35
0.019*
0.88
0.46
0.81
0.92
0.64
0.97
0.84
0.94
0.98
0.87
0.032*
0.60
0.98
0.13
0.24
0.81
0.80
0.45
0.56
0.36
Within horizontal rows, values with no letters or with the same letter(s) were not significantly different at a ¼ 0:5 (StudentÕs t). * indicates that at
least one of the incubation temperatures had a significant effect.
that it was recently applied; earlier observations indicate
that the establishment of a CH4 -oxidizing population is
a rather slow process [17].
The CH4 consumption rates were markedly affected
by shifts in temperature, as indicated by Q10 -values of
3.17–4.14. Comparable results have been obtained in
many other studies of landfill cover soils, for example
Czepiel et al. [25] reported a Q10 -value of 2.4 for temperatures in the range of 20–30 °C, and Whalen et al.
[29] noted a Q10 -value of 1.9 for 5–26 °C. However,
Christophersen and colleagues [30] have reported that
Q10 -values for CH4 oxidation in a landfill cover soil can
be as high as 4.10–7.26. As discussed by King and Adamsen [31], it can be expected that the Q10 -values for
CH4 oxidation are elevated (P3) when enzyme systems
are saturated with substrate, whereas lower Q10 -values
often indicate a limited supply of substrate.
The results of the PLFA analyses indicated that only
type I methanotrophs grow at low temperatures (3–
10 °C), but both types grow at 20 °C. Several other investigators have observed similar effects of temperature.
For instance, Vecherskaya et al. [32] found that type I
methanotrophs (Methylobacter and Methylomonas)
were more prevalent than type II species (Methylocystis)
in samples of tundra soils, and from areas where temperatures generally do not exceed +9.0 °C, whereas
Horz et al. [33] noted that type II methanotrophs
dominated in samples of meadow soil incubated at
25 °C. Similar results were also achieved by Gebert et al.
[34], who reported that enrichments and isolation of
methanotrophs from a biofilter yielded a Methylosinus
sp. strain (type II) at 28 °C and a Methylobacter sp.
strain at 10 °C.
It should be mentioned also that all psychrophilic
methanotrophs isolated so far have been type I strains.
For instance, Omelchenko et al. [35] isolated a methanotroph (Methylococcus sp.) from tundra soil at 6 °C,
and found that this strain was most active at 3.5 °C. In
G. B€orjesson et al. / FEMS Microbiology Ecology 48 (2004) 305–312
addition, Pacheco-Oliver et al. [36] isolated several
strains of psychrophilic methanotrophs from an Arctic
tundra soil in Canada and noted that they were all of
type I. Other examples of psychrophilic type I methanotrophs include Methylosphaera hansonii, which shows
optimal growth at 10–13 °C [37], and methanotrophic
strains isolated from deep igneous rock [38]. Taken together, previously reported isolations of methanotrophs
from low temperature environments and our data, indicating temperature-selective growth of methanotrophs,
suggest that temperature has a selective effect in determining which of the two main types of methanotrophs
will predominate in a given environmental system, where
low temperatures seem to favour the development of
type I methanotrophs.
Several studies have shown that temperature can
influence the lipid composition in bacteria, and this
has also been observed in a pure culture of the methanotroph M. capsulatus [39]. This effect was seen
mainly as an increase in the ratio of the mono-unsaturated to saturated PLFAs (e.g., 16:1/16:0), from
0.4 at 30 °C to 1.7 at 50 °C. We also observed such a
trend in our samples, but there were no significant
differences at a ¼ 0:05. Nevertheless, it should be noted that some reports have indicated that several
bacterial strains do not display this shift [40,41], and
that other changes, e.g., from 16:1x7c to cy17:0 may
also be possible [42]. Accordingly, even though it is
known that methanotrophs, like other bacteria, can
alter the composition of their membranes in response
to changes in temperature, the increases in the methanotroph-associated PLFAs in our samples were
probably caused by a rise in biomass, since the kinetics of CH4 consumption were consistent with the
growth of CH4 oxidisers.
One might also discuss the influence of the sampling
period on the unincubated samples. One way to make a
comparison is to calculate the ratio between the biomarker PLFAs in the samples, for example between the
PLFAs 18:1x8 and 16:1x8. Such a comparison gives the
ratio of 3.03 for the Visby samples (June ¼ warm), 1.54
for Filborna (November ¼ cold) and 1.75 for Sundsvall
(March ¼ cold). Earlier studies of PLFAs in landfill
cover soils [17], also show a tendency for 18:1x8 to be
more frequent than other methanotroph biomarkers
during warm periods, but attempts to calculate ratios
between these biomarker PLFAs show a high degree of
variation. Furthermore, deep down in the profile, methanotrophs can survive without being active due to
absence of oxygen. Therefore, it is important to take
samples in shallow, aerated soils where methanotrophs
are active.
If the selective influence of temperature that we observed in this study, with a strongly reduced activity of
Type II methanotrophs at low temperatures, is of general
significance, we could also expect effects on the nitrogen
311
cycle, since uptake of atmospheric nitrogen is more or
less restricted to Type II genera. Hopefully, samples from
other environments taken at different seasons will increase our knowledge about these processes.
Acknowledgements
This experiment was part of the project ‘‘Methane
From Landfills in Sweden’’, funded by the Swedish National Energy Administration (P-10856). We are grateful
to Dan Lindmark for help with PLFA extractions, Elisabet B€
orjesson and Kristin Steger for technical assistance with gas chromatographic analyses of PLFAs, and
€
Christina Ohman
at the Department of Soil Sciences,
SLU, for carrying out the particle size analyses.
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