FEMS Microbiology Ecology 26 (1998) 207^217
Microbial oxidation of CH4 at high partial pressures in an organic
land¢ll cover soil under di¡erent moisture regimes
î sa Frostegaîrd b ,
Gunnar Boërjesson a; *, Ingvar Sundh a , Anders Tunlid b , A
c
Bo H. Svensson
a
Department of Microbiology, Swedish University of Agricultural Sciences, P.O. Box 7025, S-750 07 Uppsala, Sweden
b
Department of Microbial Ecology, Lund University, S-223 62 Lund, Sweden
c
Department of Water and Environmental Studies, Linkoëping University, S-583 81 Linkoëping, Sweden
Received 17 October 1997; revised 21 April 1998; accepted 24 April 1998
Abstract
The uptake and utilization of CH4 at high concentrations (5^18% vol.) and different soil moistures were followed for samples
from a landfill cover soil with a high organic matter content. Measurements of the utilization of CH4 and O2 , and production
of CO2 indicated that the activity of methanotrophic organisms accounted for most of the O2 respiration. At water saturation,
CH4 oxidation rates decreased with time, probably because NH‡
4 accumulated. Denitrification rates, estimated based on both
N2 and N2 O production, were positively related to soil moisture and only slightly influenced by the extent of CH4 oxidation.
Total phospholipid fatty acid concentrations increased, and concentrations of phospholipid fatty acids, typical for obligate
methanotrophic bacteria (e.g. 16:1g8 and 18:1g8), increased in the CH4 -amended samples, indicating growth of both type I
and type II methanotrophs. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
All rights reserved.
Keywords : Ammonium ; Denitri¢cation; Methanotroph; Methane oxidation ; Nitrogen ; Nitrous oxide; Phospholipid fatty acid
1. Introduction
In the greenhouse gas budget, biological methane
oxidation mediated by methanotrophic bacteria is an
important process mitigating CH4 £uxes to the atmosphere [1]. Approximately half of the CH4 produced or seeped from all sources is estimated to be
detained in this way [2]. Biological CH4 oxidation
has been found to restrict the £uxes of CH4 produced in lakes and rice paddies by up to almost
* Corresponding author. Tel.: +46 (18) 67 32 11; Fax:
+46 (18) 67 33 92; E-mail: [email protected]
100% [3,4], while the corresponding values for land¢lls have been estimated to range from 10 to 70% [5^
9]. Large quantities of CH4 are produced in land¢lls,
and CH4 often appears at biogas concentrations
(55% vol. [10]) in the surface of land¢ll cover soils.
At such high partial pressures, O2 is absent, and CH4
oxidation therefore cannot be expected [9]. CH4 oxidation rates are also dependent on soil moisture, as
demonstrated in laboratory incubations of land¢ll
cover soil samples, where CH4 oxidation rates were
much higher under moderate moist conditions compared with under water-logged conditions [6,10].
Thus, the di¡usion of CH4 and O2 through water
0168-6496 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 6 4 9 6 ( 9 8 ) 0 0 0 3 6 - 1
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may be rate-limiting for methane oxidation in soils
[11]. The processes leading to oxidation of CH4 and
NH‡
4 may also interact, since these compounds are
competitive substrates for their respective enzymes
[12], but it has also been shown that both nitri¢cation and denitri¢cation were enhanced by CH4 additions to sediment samples [13]. Interactions between
CH4 and N cycling in land¢ll cover soils have not
been reported previously.
Field observations at land¢lls have demonstrated
that organic cover soils have a high capacity to mitigate CH4 emissions [14], which is also supported by
results from laboratory studies [15]. Furthermore,
the CH4 oxidation potential in mineral soils can be
enhanced by adding organic material, e.g. sewage
sludge [8].
Methanotrophic bacteria seem to oxidize CH4
most e¤ciently when they occur in consortia among
other bacteria, where they may constitute ca. 90% of
the microbial population [16]. In a methane-oxidizing consortium isolated from a humisol, the uptake
of excess methanol, nitrite and hydroxylamine by
accompanying organisms was of great importance
for methanotrophic activity [17,18].
Through the use of phospholipid fatty acid
(PLFA) analysis, the microbial community structure
in soil samples can be determined without cultivation
[19]. Many isolated strains of obligate methanotrophic bacteria contain substantial amounts of the
unusual PLFAs 16:1g8 and 18:1g8 [20^24]. The
quantitation of these two PLFAs has been used to
estimate the abundance of methanotrophic bacteria
in various types of environmental samples, e.g. in
natural gas-enriched soil columns [25], peatlands
[26], land¢ll covers [14], and halocarbon-degrading
methanotrophic mixed cultures [27]. In these studies
the PLFAs suggested as biomarkers for methanotrophs, especially 18:1g8, were often strongly linked
to CH4 oxidation.
The aims of this experiment were to determine (1)
the proportion of the total O2 respiration in an organic land¢ll cover soil due to CH4 oxidation; (2)
which N transformations (e.g. denitri¢cation) are affected by CH4 oxidation at di¡erent levels of soil
moisture, and (3) which methanotrophs contribute
most to CH4 oxidation. A land¢ll cover soil with a
high organic matter content and CH4 oxidation potential was incubated at various CH4 concentrations
and under di¡erent moisture conditions. Because argon was used instead of ambient air in incubation
£asks, it was possible to estimate denitri¢cation rates
based on N2 production. Changes in the microbial
community were estimated based on changes in
PLFA concentrations.
2. Materials and methods
2.1. Soil
Soil was collected from the cover of the
Hoëgbytorp land¢ll on 8 February 1994. Previous experiments have shown that this soil has a high capacity for methane consumption [14]. It had a pH of
7.3 (4 g soil in 10 ml 0.01 M CaCl2 ), a loss on
ignition at 550³C of 29.7%, and of the remaining
mineral part 13.3^13.5% was clay, 36.5^27.8% silt
and 48.7^49.2% sand. Total Kjeldahl N was 0.96^
0.99% (n = 2). The water-holding capacity (WHC)
was 62.1% w/ww (water of wet weight; or 164%
water of dry weight soil). The soil was sieved (4
mm), and after air-drying at 24³C a water content
of 44.5% w/ww (72% of WHC) was achieved. The
soil was mixed and stored dark at 4³C.
Prior to the start of the experiment, the soil was
split into four 300-g portions. To three of these, distilled water was added to give moisture contents of
80, 90 and 100% WHC. After storage in plastic bags
overnight (10 h), to obtain an even distribution of
moisture, each of the four soils was divided into
¢fteen 10-g d.w. samples and transferred to 134-ml
glass £asks, which were then closed with gas-tight
screw caps [10]. Thus, the gas headspace volume in
the £asks ranged from 114 to 122 ml, depending on
the water content used.
2.2. Experimental design
Immediately prior to the experimental start, the
gas phase of the incubation £asks was removed
and replaced with Ar three times, leaving only trace
amounts of N2 in soil water. (A test with sterilized
soil showed that a minimum of 90% of the N2 that
accumulated in the £asks during the course of the
experiment was biologically produced.) At this stage,
one triplicate of each soil moisture level (`time zero'
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G. Boërjesson et al. / FEMS Microbiology Ecology 26 (1998) 207^217
samples) was processed for analyses of PLFAs and
inorganic nitrogen (see below). To the other samples,
30 ml pure O2 was added and the over-pressure obtained was released to achieve atmospheric pressure.
Thereafter, 30 ml of mixtures of CH4 +Ar in di¡erent
ratios were added, giving ¢nal concentrations of 0, 5,
9.4 and 18.8% CH4 in gas headspaces together with
16% O2 in all £asks. All additions were made within
5 min after the change to Ar and repeated in triplicates for each CH4 level and each soil moisture level.
The £asks were incubated at 25³C, and the duration
of the experiment was set at 24 h for all samples.
This duration was based on the results from pre-experiments showing that neither O2 nor CH4 would
become limiting at this time, although both gases
were consumed to a great extent. In a parallel experiment including six samples each of Ar+O2 and ambient air atmospheres, no e¡ect of Ar on CH4 oxidation rates was noted (P = 0.51 in t-test).
2.3. Gas analyses
Samples of 1.5 ml were withdrawn from the gas
headspace of the incubation £asks with an Ar£ushed syringe immediately after starting the experiment (within 5 min after O2 and CH4 addition, respectively), and every third hour thereafter. Concentrations of O2 , N2 and CH4 were immediately
determined on a gas chromatograph with a thermal
conductivity detector according to methods previously described [28], as modi¢ed by Boërjesson and
Svensson [10]. Simultaneously, 1-ml samples were
withdrawn and transferred to 26-ml tubes with Ar
as the gaseous phase for later analysis of CO2 [10]
and N2 O [29].
Amounts of CO2 and N2 O were adjusted for solubility in the water phase according to Henry's constants kP = 31.7 and 43.1 atm mol31 kg H2 O, respectively [30]. The solubility proportions of the other
analyzed gases were treated as non-signi¢cant. All
gases used for the experimental additions and calibration mixtures were obtained from Air Liquide
(Kungsaëngen, Sweden).
Estimates of the consumption and production of
gases and inorganic nitrogen, given in ¢gures and
tables, were calculated as the di¡erence between initial and 24-h concentrations, while initial rates of
consumption and production were calculated from
209
second-degree functions for the ¢rst 8 h. Estimates
of growth were calculated from third-degree functions ¢tted to the changes in time-dependent methane consumption during the whole experimental period (24 h).
2.4. PLFA analyses
At the end of the experiment, 1 g of soil was removed from each incubation £ask and frozen. From
samples incubated at 80, 90 and 100% WHC, PLFAs
were extracted, methylated and derivatized according
to methods previously described [26]. GC and GCMS analyses were made according to Frostegaîrd et
al. [31], as modi¢ed by Boërjesson et al. [14].
Fatty acids are designated in terms of the total
number of carbon atoms: number of double bonds,
followed by the position of the double bond in relation to the g (methyl) end of the molecule. Cis and
trans con¢gurations are indicated by `c' and `t', respectively. The pre¢xes `a' and `i' indicate anteiso
and iso branching; `br' indicates unknown methyl
branching position; `10Me' indicates a methyl group
on the tenth carbon atom from the carboxyl end of
the molecule; and `cy' refers to cyclopropane fatty
acids.
2.5. Inorganic nitrogen fractions
After removing samples for PLFA analyses, the
remaining soil in the incubation £asks was immediately extracted with 50 ml 2 M KCl on a rotary
shaker at 150 rpm for 1 h. After centrifugation at
3000Ug, a 10-ml portion of the supernatant was
frozen and stored at 320³C. Concentrations of inorganic N were later analyzed for NO3
2 with £ow
injection analysis (FIA; Techator, Hoëganaës, Sweden; application note ASN 51-01/84), while NO3
3
and NH‡
4 were determined colorimetrically on a
TRAACS auto-analyzer (Bran and Luebke, Germany).
3. Results
3.1. CH4 utilization
Initial CH4 consumption rates were between 1 and
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G. Boërjesson et al. / FEMS Microbiology Ecology 26 (1998) 207^217
2 Wmol CH4 g d.w.31 h31 in all samples. A small
increase in CH4 oxidation rates occurred during
the last part of the experimental period in samples
incubated at 90% WHC (Fig. 1), while the CH4 oxidation rates in samples incubated at 100% WHC
constantly decreased (Fig. 2), although only a small
amount of oxygen had been consumed. For the former, a doubling time of tg V20 h (speci¢c growth
rate W = 0.035) for the last 6-h period of incubation
was calculated from third-degree functions (Fig. 1;
9.4% and 18.8% CH4 ). In samples incubated at 100%
WHC, only around half of the CH4 was oxidized
compared with the drier samples (Fig. 3A,B). At
lower CH4 levels, the amount of O2 consumed in
the samples exceeded the consumption predicted by
the 1:1 relationship between CH4 and O2 expected
for stoichiometric methanotrophic growth (Fig. 3C).
Likewise, the relative CO2 production (Fig. 3D)
Fig. 2. Cumulative consumption of CH4 for two of the samples
incubated at 100% WHC soil moisture. Plotted lines are thirddegree functions. Initially 5.0% CH4 in gas headspace: CCH4
(Wmol) = 0.00389 t3 30.272 t2 +10.26 t+2.82. Initially 9.4% CH4 in
gas headspace: CCH4 (Wmol) = 30.0051 t3 30.128 t2 +15.93 t+0.85.
was higher at the lowest CH4 levels, probably because O2 was utilized by organisms other than methanotrophs.
3.2. Nitrogen fractions
Fig. 1. Cumulative consumption of O2 and CH4 and production
of CO2 for two of the samples incubated at 90% WHC soil
moisture. Plotted lines are third-degree functions. A: Initially
9.4% CH4 in gas headspace: CCH4 (Wmol) = 0.0087666 t3 30.16041
t2 +16.676 t32.8008; which gives W = 0.0350 and tg = 19.8 for the
last 6 h. B: Initially 18.8% CH4 in gas headspace : CCH4 (Wmol) =
0.011562 t3 30.28061 t2 +21.995 t31.1376; which gives W = 0.0349
and tg = 19.8 for the last 6 h.
Concentrations of ammonium at time zero ranged
31
(Fig. 4a;
between 0.23 and 0.40 Wmol NH‡
4 g d.w.
0.21^0.32 mM in solution). At 70, 80 and 90%
WHC, the NH‡
4 concentration decreased in all incu31
bated samples to 0.004^0.064 Wmol NH‡
4 g d.w. .
‡
At 100% WHC, a signi¢cant increase in NH4 occurred in all incubated samples, up to concentrations
as high as 1.28 Wmol g d.w.31 measured in samples
from the highest CH4 level. This value was signi¢cantly higher than those obtained in samples incubated in 0 and 5% CH4 (0.76 and 0.88 Wmol NH‡
4 g
d.w.31 ).
For no obvious reason, time-zero nitrite concentrations were higher, i.e. 12.5 and 32.0 nmol g d.w.31
NO3
2 (0.016 and 0.020 mM) at 72 and 100% WHC,
respectively, compared with values for the samples at
intermediate moisture levels (Fig. 4b). During the
decreased in all samples
24-h incubation NO3
2
down to concentrations close to the detection limit
(ca. 1 WM).
Time-zero concentrations of nitrate ranged be31
(Fig. 4c;
tween 3.36 and 4.17 Wmol NO3
3 g d.w.
2.6^4.9 mM). A signi¢cant decrease in NO3
3 concentrations occurred in samples incubated with CH4 (all
levels) at 100% WHC (down to 2.42^2.69 Wmol NO3
3
FEMSEC 922 2-7-98
G. Boërjesson et al. / FEMS Microbiology Ecology 26 (1998) 207^217
211
Fig. 3. Comparison of CH4 and O2 utilization and CO2 production between samples incubated for 24 h with di¡erent levels of initial
CH4 : 5.0% CH4 (white bars), 9.4% CH4 (diagonally striped bars), 18.8% CH4 (gray bars). A: Amounts of CH4 consumed. B: CH4 utilized of available amounts. C: Ratio between O2 and CH4 utilization. D: Ratio between CH4 utilization and CO2 production. Error bars
are standard deviations for n = 3.
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G. Boërjesson et al. / FEMS Microbiology Ecology 26 (1998) 207^217
Fig. 4. Amounts of inorganic nitrogen in soil samples at di¡erent moisture levels, including non-incubated (time-zero) samples (white
bars), and 24-h incubated samples with 0% CH4 (light gray bars), 5.0% CH4 (horizontally striped bars), 9.4% CH4 (diagonally striped
bars) and 18.8% CH4 (dark gray bars). Error bars are standard deviations for n = 3.
FEMSEC 922 2-7-98
G. Boërjesson et al. / FEMS Microbiology Ecology 26 (1998) 207^217
g d.w.31 ) and with the highest initial CH4 level at
31
90% WHC (down to 2.54 Wmol NO3
3 g d.w. ).
The formation of N2 O and N2 was primarily dependent on the moisture content, with signi¢cant increase between all four soil moisture levels in increas-
213
ing order (Fig. 4d,e). The e¡ect of CH4 level was
smaller: Higher levels of initial CH4 resulted in increased N2 O rates at lower soil moistures (72 and
80% WHC), whereas the reverse tended to be true
at 100% WHC (Fig. 4d). By contrast, N2 production
Table 1
PLFAs (nmol g d.w.31 ) for di¡erent treatments, in order after mean percentual increase in CH4 -treated samples compared to untreated
(0% CH4 ) samples
Soil moisture/PLFA
Time zero
After 24-h incubation
0% CH4
5% CH4
9% CH4
18% CH4
Increase
(%)
80% WHC
18:1g8
16:1g6
16:1g8
16:1g5
18:1g7
14:0
cy19:0
18:2g6
19:1a
a17:0
18:1g9
16:0
a15:0
16:1g7
Total PLFAs
6.94 ab
7.26 a
8.63 ab
31.3 ab
43.8 ab
16.8 a
10.1 ab
16.1 abc
2.05 a
7.65 a
53.1 ab
77.9 a
38.8 a
70.8 a
548 a
5.53 a
6.53 a
8.09 a
25.7 a
40.9 a
17.3 ab
8.95 a
14.5 a
2.14 ab
7.99 a
50.8 a
84.9 ab
41.1 a
77.1 ab
562 ab
7.87 b
8.31 ab
9.12 ab
31.5 ab
50.0 c
20.0 abc
9.86 ab
16.6 bc
2.28 ab
9.00 b
56.1 b
90.4 b
44.7 a
75.7 a
609 bc
10.1 b
10.6 b
14.2 b
37.9 b
52.6 c
22.4 c
11.9 b
17.1 c
2.58 b
8.46 ab
56.5 b
91.8 b
42.4 a
87.3 b
645 c
7.96 b
7.96 ab
8.38 ab
29.1 ab
48.0 bc
20.5 bc
10.0 ab
15.1 ab
2.24 ab
8.95 b
52.5 ab
85.9 b
44.0 a
74.7 a
585 ab
3.12
2.44
2.47
7.16
9.28
3.66
1.66
1.72
0.23
0.82
4.30
4.49
2.61
2.12
23.2
(56)
(37)
(31)
(28)
(23)
(21)
(19)
(12)
(11)
(10)
( 8)
( 6)
( 5)
( 3)
( 9)
90% WHC
18:1g8
16:1g5
16:1g7
i16:1
18:1g7
14:0
18:2g6
cy19:0
16:1g8
18:1g9
19:1a
16:0
17:1g8
18:0
i17:1
15:0
i17:0
cy17:0
10Me18 :0
17:0
10Me17 :0
i16:0
a15:0
i15:1
i15:0
Total PLFAs
7.19 b
24.7 b
85.0 ab
1.02 a
46.9 b
15.5 a
15.9 b
10.4 b
7.45 a
54.7 b
2.02 ab
82.5 ab
5.11 b
10.5 b
9.24 a
6.40 a
7.02 a
13.7 a
6.11 a
3.10 ab
2.38 a
14.9 ab
37.1 a
8.32 a
49.4 a
563 ab
4.76 a
15.7 a
68.1 a
0.76 a
37.1 a
16.7 a
12.4 a
7.85 a
9.65 ab
44.5 a
1.92 a
79.0 a
4.16 a
9.54 a
9.23 a
6.38 a
7.10 a
13.7 a
5.89 a
2.89 a
2.44 ab
14.4 a
36.5 a
8.45 a
55.2 b
516 a
8.15 bc
23.1 b
94.3 bc
0.96 a
47.1 b
20.1 b
15.4 ab
9.59 b
10.9 ab
50.7 b
2.33 c
88.9 bc
4.52 ac
10.5 ab
10.3 b
6.93 ab
7.73 b
15.0 b
6.32 ab
3.03 ab
2.49 ab
15.1 abc
38.3 a
9.11 b
57.5 bc
597 bc
8.09 bc
24.1 b
95.6 bc
1.02 a
48.8 b
20.2 b
15.6 ab
9.74 b
10.4 ab
52.9 a
2.23 c
91.5 c
4.76 bc
10.8 b
10.2 b
7.21 b
7.92 b
15.0 b
6.52 b
3.21 b
2.64 b
15.8 bc
39.8 a
8.89 ab
57.2 bc
608 cd
8.96 c
28.9 b
108 c
1.14 a
49.8 b
23.3 c
15.9 ab
10.2 b
14.9 b
55.0 a
2.22 bc
95.8 c
5.09 b
11.3 b
10.5 b
7.25 b
8.10 b
15.7 b
6.74 b
3.32 b
2.91 c
16.3 c
40.3 a
8.92 ab
59.1 c
652 d
3.64
9.62
31.4
0.28
11.4
4.52
3.24
2.00
2.41
8.41
0.34
13.1
0.63
1.33
1.13
0.75
0.82
1.50
0.64
0.30
0.24
1.32
3.00
0.52
2.78
103
(76)
(61)
(46)
(37)
(31)
(27)
(26)
(25)
(25)
(19)
(17)
(17)
(15)
(14)
(12)
(12)
(11)
(11)
(11)
(10)
(10)
(10)
( 8)
( 6)
( 5)
(20)
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214
G. Boërjesson et al. / FEMS Microbiology Ecology 26 (1998) 207^217
Table 1 (Continued).
PLFAs (nmol g d.w.31 ) for di¡erent treatments, in order after mean percentual increase in CH4 -treated samples compared to untreated
(0% CH4 ) samples
Soil moisture/PLFA
100% WHC
16:1g8
18:1g8
16:1g5
16:1g6
br18:1
16:1g9
14:0
cy17:0
16:1g7
i17:1
i15:0
i15:1
16:0
Total PLFAs
Time zero
7.31 a
6.16 ab
25.9 a
6.11 a
1.91 a
5.11 a
15.5 a
12.3 a
70.2 a
8.77 a
49.3 a
8.86 a
74.9 a
527 a
After 24-h incubation
0% CH4
5% CH4
9% CH4
18% CH4
6.38 a
5.82 a
26.8 a
7.18 ab
1.72 a
4.86 a
18.2 ab
13.5 ab
70.9 a
9.19 a
54.8 ab
9.15 a
86.6 ab
580 ab
9.22 ab
7.89 ab
32.6 ab
8.69 bc
2.10 a
5.66 a
21.2 b
14.9 ab
79.3 a
10.8 b
61.5 b
10.1 a
95.9 b
648 b
12.2 b
8.47 b
36.5 b
9.25 c
2.09 a
5.68 a
21.5 b
15.6 b
82.9 a
10.3 ab
63.8 b
10.0 a
94.3 ab
638 ab
11.1 b
6.89 ab
34.4 b
8.52 bc
1.94 a
5.48 a
18.4 ab
14.5 ab
73.3 a
9.25 ab
55.7 ab
9.26 a
84.3 ab
575 ab
Increase
(%)
4.67
1.94
7.74
1.64
0.32
0.75
2.13
1.47
7.58
0.92
5.50
0.64
4.90
40.5
(70)
(46)
(29)
(23)
(19)
(15)
(12)
(11)
(11)
(10)
(10)
( 7)
( 6)
( 7)
Values with letters in common in horizontal lines are not signi¢cantly di¡erent (K s 0.05). The Table accounts only for PLFAs where at least
one of the CH4 treatments has signi¢cantly higher amounts than samples incubated without CH4 , where the increase is above 2 nmol g
d.w.31 , or where their proportional increase is above average. Values are means for n = 3.
was signi¢cantly higher in CH4 -amended soil samples only at 90% WHC (Fig. 4e). Accumulated
amounts of N2 O and N2 were correlated, with N2 N g d.w.31 = 18.8 N2 O-N g d.w.31 +1.243 (r2 = 0.73,
P 6 0.0001).
3.3. PLFA contents
Most PLFAs occurred in signi¢cantly higher
amounts in CH4 -treated samples compared with
non-CH4 samples (0% CH4 ). These PLFAs are listed
in Table 1, together with PLFAs that increased proportionally more than the sum of PLFAs. Among
the 32 di¡erent PLFAs quanti¢ed, only the PLFAs
i14:0, a15:0, 10Me16:0, and br18:1 never increased
signi¢cantly at any water content. The total content
of PLFAs increased in most of the samples to which
CH4 had been added: (i) the increase in total PLFA
content for these samples was 71.6 þ 33.4 nmol
PLFA g d.w.31 , which was 13 þ 6% of the PLFA
content in non-incubated time-zero samples (t-test;
K = 0.05); and (ii) the CH4 -incubated samples also
had a higher PLFA content than the 0% CH4 -incubated samples, with 60.6 þ 34.9 nmol PLFA g
d.w.31 , or 11 þ 6% di¡erence. The PLFAs 18:1g7
and 18:1g8, the most common PLFAs in type II
methanotrophs [23], were well correlated with each
other (r2 = 0.61, P 6 0.0001), and the content of both
decreased in samples incubated for 24 h without CH4
(18:1g8 from 6.76 to 5.44 nmol g d.w.31 , P = 0.0007
for n = 9+9). Amounts of PLFAs typical for methanotrophs were also those that increased their proportions most in CH4 -incubated samples. For example,
at 90% WHC the amount of 18:1g8 at 18% CH4 was
1.88 times higher than at 0% CH4 . Corresponding
increases for 16:1g8 were 1.91 times at 100%
WHC, 9% CH4 , and 1.76 times at 80% WHC, 9%
CH4 .
4. Discussion
The addition of water to the soil used in this experiment created two very di¡erent systems regarding CH4 oxidation rates and nitrogen transformations. In the wettest soil treatment (100% WHC)
CH4 oxidation rates were lowered, most likely owing
to decreased rates of CH4 and O2 di¡usion through
‡
water, and NH‡
4 accumulated, whereas NH4 was
consumed in the drier soils.
Ratios between O2 and CH4 consumption (ranging from 1.00 to 1.88; Fig. 3C) indicate that meth-
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G. Boërjesson et al. / FEMS Microbiology Ecology 26 (1998) 207^217
anotrophs dominated the consumption of O2 at the
higher CH4 levels. Moreover, the ratios between CO2
production and CH4 consumption, ranging from
0.17 to 0.36 (Fig. 3D), indicate that most of the
carbon was incorporated into biomass. Utilization
of the gaseous substrates was governed by the initial
amounts of CH4 ; thus the ratio between utilized
amounts of O2 and utilized amounts of CH4 was
lower at high CH4 levels. These results are consistent
with the ¢ndings in another study of CH4 oxidation
in an organic soil. Namely, Megraw and Knowles
[32] reported a ratio of 1.0 mol O2 utilized per mol
CH4 for a humisol under concurrent production of
0.27 mol CO2 [32]. Similarly, pure cultures used by
Whittenbury et al. [33] had a O2 /CH4 of 1.0^1.1 and
a CO2 /CH4 ratio of 0.2^0.3. In mixed methanotrophic continuous cultures, the corresponding ratios
were 1.24 and 0.40 when oxygen was limited [34],
while it was estimated that up to 84% of added
CH4 was used for producing cell biomass when
CH4 was limited [35]. This is close to the lowest
theoretically possible CO2 /CH4 ratio, which is 0.12
according to Gommers et al. [36].
The sum of the analyzed N fractions (in Fig. 4)
was much higher for the wettest soil conditions
(100% WHC; 13.4^18.3 Wmol N g d.w.31 ) than for
the drier conditions (3.04^6.56 Wmol N g d.w.31 ).
Most of this di¡erence was likely due to higher N
uptake due to higher growth of methanotrophs during dry conditions, since 1 mol N is assimilated concomitantly with the assimilation of 4 mol C in methanotrophs [37].
The stagnation of CH4 oxidation rates under
water-saturated conditions (100% WHC) may have
been due to the accumulation of NH‡
4 in these samples. However, the concentration (max. 1.5 Wmol
31
or 0.91 mM) is within the range of
NH‡
4 g d.w.
Ki values for pure cultures of methanotrophs reported by O'Neill and Wilkinson [38]: 17.5 mM
‡
NH‡
4 at pH 6.0 and 0.2 mM NH4 at pH 8.0. Thus,
there are good reasons to believe that NH‡
4 had an
inhibitory e¡ect in this soil, if the original pH of 7.3
did not change considerably during the incubation.
The increases in N2 O and N2 formation observed
with increasing moisture content were expected, since
the activity of denitri¢ers increases as a result of the
decrease in the oxygen supply that occurs as soils
become wetter [39,40]. N2 O formation coupled to
215
autotrophic nitri¢cation may also contribute to the
pattern observed. The positive in£uence of increasing
CH4 levels on net N2 O production at the low moisture contents in the experiment may have also been
due to oxygen limitation. The increase in oxygen
consumption caused by the methanotroph activity
at higher mixing ratios of methane probably decreased the availability of oxygen to the microbial
population in general. The same argument is valid
for denitri¢cation as expressed in N2 formed at 90%
WHC. There was a tendency for larger amounts of
N to be denitri¢ed in the presence of higher amounts
of methane. At 100% WHC the e¡ect of methane as
a factor regulating O2 availability seemed to have
been negligible, since denitri¢cation was about equal
at all initial CH4 levels. However, the possibility that
some of the N2 O formed was due to the activity by
the methanotrophs at low moisture levels cannot be
ruled out, since pure cultures of methanotrophs have
been shown to be able to form N2 O from NH‡
4
[41,42].
The proportion of consumed CH4 that was used
for biomass in the studied samples can be estimated
based on changes in concentrations of PLFA and
CH4 utilization. The average increase in total PLFAs
for the CH4 -treated samples (n = 27) was 61 þ 35
nmol compared with non-CH4 samples (n = 9; Section 3.3, Table 1). If 100 Wmol of PLFA corresponds
to 1 g dry weight of methanotroph cells [43,44], and
carbon accounts for 47% of the dry weight in methanotrophic bacteria [33], then 285 þ 164 Wg C was
assimilated in biomass. This is similar to the di¡erence between utilized CH4 and produced CO2 (data
in Fig. 3), which was 340 þ 173 Wg C (28.3 þ 14.4
Wmol of C) in the corresponding samples.
Results of the PLFA analysis indicated that obligate methanotrophic bacteria had grown, as re£ected
in the increase in PLFAs speci¢c for methanotrophs
in most of the CH4 -treated samples. These results are
consistent with the ¢ndings of Nichols et al. [45] that
the PLFAs 16:1g6, 16:1g8 and 18:1g8 were enriched in soil columns exposed to natural gas. In
addition to the more generic-speci¢c PLFA 18:1g8,
type II methanotrophs also have signi¢cant proportions (up to 89%) of 18:1g7 [22]. Although 18:1g7 is
ubiquitous in bacteria [25] its increase in almost all
CH4 -amended soil samples further substantiates that
growth of type II methanotrophs occurred. Apart
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216
G. Boërjesson et al. / FEMS Microbiology Ecology 26 (1998) 207^217
from the obvious e¡ects on 16:1g8 and other monounsaturated 16-C PLFAs, among the other PLFAs
that increased owing to the oxidation of CH4 , several
saturated PLFAs have also been documented in type
I methanotrophs. Thus, 14:0 (up to 24.6% of the
total PLFAs), 15:0 (up to 12.7%) and 16:0 (up to
56.0%) were all common in type I strains reported by
Bowman et al. [24]. An interesting observation is
that 18:1g8 had the highest percentual increase at
80 and 90% WHC, while 16:1g8 showed the highest
increase at 100% WHC. This is in line with the suggestion of O'Neill and Wilkinson [38], that type I
methanotrophs are more tolerant to ammonium
than type II.
An important conclusion of this experiment is that
under water-saturated conditions in an organic soil
CH4 oxidation is restricted not only by the di¡usion
barrier, but possibly also by the accumulation of
NH‡
4 under the reduced conditions. This has implications for the use of this type of soil for bioremediation, covering of land¢lls etc. In the land¢ll case,
the stoichiometrically unfavorable situation for CH4
oxidation (with 55% CH4 meeting 20% O2 ) could be
counteracted by a prolonged retention time for CH4 ,
thereby extending the period during which CH4 is
exposed to methanotrophs. Land¢ll covering with
organic soils, which are nutrient-rich and have a
high water-holding capacity, could be one way to
achieve this.
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
Acknowledgments
John Stenstroëm suggested the use of argon in the
experimental setup. Rose-Marie Ericsson (Department of Soil Sciences) and Lena Funke carried out
the N analyses. Elisabet Wennberg and Maria Eriksson provided additional laboratory assistance. This
report was part of a project funded by NUTEK
(Swedish National Board for Industrial and Technical Development) under Contract 706 005-1.
[12]
[13]
[14]
[15]
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