Responses of methane oxidation to temperature and water content in cover soil of a boreal landfill

ARTICLE IN PRESS

Soil Biology & Biochemistry 39 (2007) 1156–1164 www.elsevier.com/locate/soilbio

Responses of methane oxidation to temperature and water content in cover soil of a boreal landfill Juha-Kalle M. Einolaa,, Riitta H. Kettunena,b, Jukka A. Rintalaa a

Department of Biological and Environmental Sciences, University of Jyva¨skyla¨, P.O. Box 35, FIN-40014, Finland b Tritonet Ltd., Pinninkatu 52 C, FIN-33100 Tampere, Finland Received 3 August 2006; received in revised form 2 December 2006; accepted 7 December 2006 Available online 11 January 2007

Abstract Methane oxidation in a cover soil of a landfill located in a boreal climate was studied at temperatures ranging from 1–19 1C and with water content of 7–34% of dry weight (dw), corresponding to 17–81% of water-holding capacity (WHC) in order to better understand the factors regulating CH4 oxidation at low temperatures. CH4 consumption was detected at all the temperatures studied (1–19 1C) and an increase in CH4 consumption rate in consecutive incubations was obtained even at 1 1C, indicating activation or increase in enzymes and/or microorganisms responsible for CH4 oxidation. CH4 consumption was reduced with low water content (17%WHC) at all temperatures. The response of CH4 consumption to temperature was high with Q10 values from 6.5 to 8.4 and dependent on water content: at 33%WHC or more an increase in water content was accompanied by a decrease in Q10 values. The responses of CH4 consumption to water content varied at different temperatures so that at 1–6 1C, CH4 consumption increased along with water content (33–67%WHC) while at 12–19 1C the response was curvilinear, peaking at 50%WHC. CH4 consumption was less tolerant (higher Q10 values; 6.5–8.4) of low temperatures compared to basal respiration (Q10 values for CO2 production and O2 consumption 3.2–4.0). Overall, the present results demonstrate the presence of CH4-oxidizing microorganisms, which are able to consume CH4 and to be activated or grow at low temperatures, suggesting that CH4 oxidation can reduce atmospheric CH4 emissions from methanogenic environments even in cold climates. r 2007 Elsevier Ltd. All rights reserved. Keywords: Carbon dioxide; Greenhouse gases; Landfill cover soil; Low temperature; Methane oxidation; Methanotroph; Moisture; Oxygen; Soil respiration; Water content

1. Introduction Many important sources of atmospheric methane (CH4), such as wetlands and landfills, are located in seasonally or permanently cold environments. CH4 production in the deeper anaerobic layers of these environments continues throughout the year despite the low ambient temperatures, as indicated by wintertime CH4 emissions from wetlands (Alm et al., 1999; Zhang et al., 2005) and landfills (Bo¨rjesson et al., 2001). Methanotrophic bacteria exist in the upper layers of these environments, wherever CH4 and oxygen (O2) are simultaneously available, and oxidize CH4 to carbon dioxide (CO2), thereby reducing the global Corresponding author. Tel.: +358 14 260 4218; fax: +358 14 260 2321.

E-mail address: [email protected].fi (J.-K.M. Einola). 0038-0717/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2006.12.022

warming potential of the gas emitting to the atmosphere. CH4 oxidation at low temperatures (down to 0–10 1C) has been detected in many environmental samples but the rates are greatly reduced compared to those at the optimum temperatures which have ranged from 20 to 38 1C, as determined in batch assays (Whalen et al., 1990; Dunfield et al., 1993; Gebert et al., 2003; Scheutz and Kjeldsen, 2004). More information about the factors regulating CH4 oxidation at low temperatures is needed in order to estimate how changes in climatic conditions may affect CH4 oxidation in soils or, on the other hand, how soils, especially in man-made methanogenic environments such as landfills, can be managed to promote CH4 oxidation. The effect of water content on CH4 consumption in soils typically follows a parabolic curve, with reduced rates in conditions of low as well as at high water content (Czepiel

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et al., 1996; Whalen and Reeburgh, 1996; Christophersen et al., 2000; Scheutz and Kjeldsen, 2004; Park et al., 2005). Many studies have determined the responses of CH4 consumption to changes in temperature with a constant level of water content, and responses to changes in water content at a constant temperature, usually close to the optimum range, while studies on the effect of changes in water content on CH4 consumption at low temperatures are scarce. The results presented by Whalen and Reeburgh (1996) suggest a systematic interactive effect of temperature and water content on CH4 consumption in a bog soil incubated at 1000 ml l1 CH4, i.e. the response of CH4 consumption to water content across the range studied was more symmetrical towards lower temperatures and increasing the water content had a more negative effect on CH4 consumption at higher as opposed to lower temperatures (ranges studied 1–15 1C and 41–77%WHC), suggesting that the response to temperature decreases as water content rises. As the responses of CH4 oxidation to temperature and water content appear to depend on a CH4 concentration within the range of 2–10,000 ml l1 (King and Adamsen, 1992; Whalen and Reeburgh, 1996), more studies are needed to establish whether and how temperature and water content interactively regulate CH4 consumption in soils, especially in those with gas-phase CH4 concentrations in the percentage range, which are encountered in methanogenic environments (Nozhevnikova et al., 1993; Tokida et al., 2005). The main question addressed in the present study was whether CH4 consumption occurs at low temperatures in soils exposed to gas with a high CH4 content (such as landfill gas) and how it is regulated by temperature and water content. We also sought to answer whether the soil CH4 consumption rate is able to increase at low temperatures and whether the response of CH4 consumption to temperature differs from that of basal soil respiration. To investigate these questions, we determined the effects of temperature (1–19 1C) and water content (7–28%dw, i.e., 17–67% of WHC) on CH4 consumption at a percentage-level CH4 concentration, on the associated O2 consumption and CO2 production, and on basal respiration (O2 consumption and CO2 production without external substrates) in laboratory batch assays with a landfill cover soil previously found to show a high rate of CH4 consumption. 2. Materials and methods 2.1. Landfill cover soil studied The landfill cover soil studied was originally a composted mixture (1:2 v/v) of municipal sewage sludge, which was anaerobically stabilized prior to composting, and chemical sludge from the treatment of food-board factory effluents. The material was spread on Tarastenja¨rvi municipal solid waste landfill (Tampere, Finland; 61.2N, 23.5E, annual mean, coldest and warmest month mean

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temperatures 4.1, 9.4 and 16.2 1C, respectively) 4–5 years before the present experiment. The landfill was established in 1977, covers an area of 30 ha, and contains around 3.4 million m3 of deposited waste. This soil was selected for the study as it showed the highest CH4 consumption out of seven screened cover soils from different landfills (J. Einola, 2002. M.Sc. Thesis, University of Jyva¨skyla¨) and as it represents the utilization of waste-based materials, instead of natural soils, in landfill construction. Five samples (a´ 2 l) for the present study were taken by a shovel from the corners and center of a randomly selected 5 m  5 m square from a depth of 10–30 cm. The samples were taken in December 2000 at an ambient temperature 3 1C and soil temperature of 5 1C, and combined in the laboratory. Samples were stored at 5 1C, with no added CH4, for 13–20 weeks before the actual experiments. This storage time was chosen due to practical reasons, including preliminary experiments and analyses to determine the relevant water content range for the study. Despite the relatively long storage time, the activity of the microbial community responsible for CH4 oxidation did not appear to be significantly affected. This is indicated by the facts that CH4 consumption rate after the storage did not significantly differ from the value observed in the experiments (at 19 1C) conducted immediately after sampling (data not shown) and that lag phases did not occur in CH4 consumption experiments. The soil had a 33.9% field water content, loss on ignition of 7.3% and a water-holding capacity (WHC) of 42% of dw. The proportion of fine particles (o0.06 mm) in the soil was less than 10%, that of large particles (46.0 mm) less than 15%, and the mean particle size (d50) was 0.5 mm, thus its particle size distribution resembled that of sandy moraine (GSF, 2005). pH of the soil was 6.7 and conductivity was 670 mS cm1 The total and water-soluble element concentrations, as well as concentrations of watersoluble nitrate, sulphate, and phosphate, are described in Supplemental Table 1 (online supplementary material). The total concentrations of arsenic, cadmium, copper, nickel, lead, and zinc, were within the range defined as noncontaminated soil according to the Finnish reference values

Table 1 Q10 values describing the temperature responses of CH4 consumption rate and CO2 production and O2 consumption in the CH4 assays and in the basal respiration assays in landfill cover soil at different water contents Water content (%dw)

14 21 28 a

CH4 assaysa

Basal assaysb

CH4

CO2

O2

CO2

O2

8.4 7.7 6.5

5.7 5.7 5.8

6.6 6.4 5.8

NDc 3.2 3.5

NDc 3.3 4.0

Temperature range 1–19 1C. Temperature range 1–12 1C. c Not determined (Q10 values for basal respiration at 14%dw were not calculated as temperature response did not follow an exponential curve). b

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whereas those of boron and chromium were above this range. 2.2. Soil incubations to determine gas consumption and production rates Rates of gas consumption (CH4, O2) and production (CO2) of soil samples were determined in duplicate batch assays at 1, 6, 12 and 19 1C in water contents of 7%dw, 14%dw, 21%dw, and 28%dw (for 19 1C also 34%dw was assayed). Soil aliquots were air-dried at 30 1C to a water content of 7%dw. Deionised water was added to the dried samples to reach the desired water contents. After overnight storage (5 1C), soil amounts corresponding to 14 g dw were placed to 120 ml serum bottles, which were then closed in ambient air with butyl stoppers and sealed with aluminium crimps. In the CH4 assays, 10 ml air was removed and 10 ml of CH4 (99.5%, Aga Ltd., Finland) was added to the bottles. In the basal respiration assays, no air was removed and no CH4 was added. The initial partial pressures in the CH4 assays were 8–9% for CH4 and 18–19% for O2. The samples were pre-incubated for 1 day in assay conditions (temperature, water content, initial gas phase), i.e., samples for CH4 assays were pre-incubated with CH4 while those for basal assays were pre-incubated without CH4. After pre-incubation, the bottles were opened for 30 min for aeration and the actual assays were started as described above. Incubations were performed in a temperature-controlled (accuracy 0.1 1C) water bath. Gas samples (0.1 ml) were taken periodically with a pressurelock syringe (Vici Precision Sampling Inc., US) and analysed immediately. The gas sampling interval and the duration of the experiments were chosen according to the reaction rates and ranged from 2 to 24 h and 1–13 d, respectively, from 19 to 1 1C. The rates of gas consumption (CH4, O2) and production (CO2) were defined by the slopes of linear regressions from data presenting total (gas and liquid phases) molar amounts versus time. Consumption of CH4 and O2, as well as production of CO2, started immediately, with the exception of undetectable CH4 consumption at the lowest water content studied (7%dw). The rates were defined from the initial period when all the rates followed zero-order kinetics: examples of some of the assays are shown in Fig. 1. Decreased rates were seen in some of the CH4 assays when CH4 and O2 concentrations fell below the apparent threshold level; these data points were excluded from the rate calculations. In the linear regression calculations N was 4–10 and R2 was 40.95. The results presented are mean (7standard error of mean; SEM) rates of duplicate assays. 2.3. Analyses Dry matter content was analysed by heating the samples at 105 1C while organic matter was determined as loss on ignition at 550 1C (Clesceri et al., 1998). Particle size was

60 Gas amount (µmol gdw-1)

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O2

O2

40 CH4

CH4

CO2

CO2

20

0 0

100 200 Time (h)

300 0

10 20 Time (h)

30

Fig. 1. An example of changes in total amounts (gas phase+liquid phase) of CH4, CO2, and O2 in bottles over time in batch assays with landfill cover soil at 1 1C (left) and 12 1C (right) at water content of 21%dw. Initial CH4 concentration was 9%. Symbols and parallel regression lines present the duplicate samples.

determined using a standard set of sieves (SFS, 1998). WHC was determined by a pressure-free method adapted from The US Composting Council (1997): Water was poured slowly to saturate a soil aliquot of 1.1 kg (field water content) with water. Soil was allowed to drain for 30 min through perforations in the bottom of the container. The saturation-draining cycle was repeated four times and the final draining was continued for 4 h. WHC was calculated from the water content per dw after the final 4-h draining. Soil pH was measured with a PHM 210 meter (Radiometer Analytical, France) from suspensions of airdried (o40 1C) soil in 0.01 M CaCl2 (1:5 vsoil/vsolution) after shaking for 5 min in an orbital shaker (200 rev min1), overnight settling and manual shaking immediately prior to measurement. Conductivity suspensions were prepared and treated similarly, except that distilled water in ratio 1:2.5 (v/v) was used, and measured with a CDM 210 meter (Radiometer Analytical, France). CH4, CO2, and O2 concentrations in the gas samples were analysed using a Perkin Elmer Autosystem XL gas chromatograph with a Supelco Carboxen 1010-PLOT molecular sieve column (diameter 0.53 mm, length 30 m) and a TCD; temperatures: column 35 1C, injector 230 1C and detector 230 1C. Carrier (He) and reference gas (He) flow rates were 7 ml min1. Gas concentrations in the water phase were calculated from the partial pressures in the gas phase according to the Henry’s law constants (KH) 1.4  102, 3.4  102 and 1.3  102 M atm1 (at 298.15 K) and temperature dependence coefficients of KH of 1600, 2400 and 1500 K for CH4, CO2 and O2, respectively (Sander, 1999). 2.4. Definitions and calculations The terms CH4-C metabolism and CH4-C-associated O2 consumption or CO2 production are used with reference to O2 consumption and CO2 production linked to the oxidation of CH4, or compounds derived from CH4 added in the experiment. CH4-C-associated consumption or

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production of gases was calculated by subtracting the values in the basal assays from the corresponding values in the CH4 assays. The term basal respiration is used for O2 consumption and CO2 production due to the metabolism of substrates originally present in the soil as determined in the assays with no added substrates. 2.5. Increase in CH4 consumption rate The increase in CH4 consumption rate was calculated from the difference in the CH4 consumption rates between two consecutive CH4 feeding cycles at 1 and 12 1C at water content 21% of dw. When the initial experiment, i.e., the first feeding cycle, was completed, the bottles were opened for 30 min and the second feeding cycle was started similarly as the first one (Section 2.1). The difference in the CH4 consumption rates between the two feeding cycles was divided by the rate in the first cycle to obtain the specific growth of CH4 consumption rate, which was then divided by the duration of the first feeding cycle (13 d at 1 1C and 1.0 d at 12 1C) to obtain the specific growth rate of CH4 consumption. 2.6. Statistical analysis For the statistical analysis, natural logarithm transformations for the gas consumption and production rates in the CH4 and basal assays were done. The responses of these parameters to temperature within each of the studied water contents, and the responses to water content within each of the studied temperatures, were analysed using the results from each of the duplicate samples as individual data points (n ¼ 628 for each regression analysis). Q10 values were calculated from linear regressions (R2 X0:97) between the ln-transformed gas consumption and production rates and temperature. The statistical analyses were performed using software SPSS 12.0.1 for Windows. 3. Results The effects of temperature and water content on the metabolism of CH4-C (CH4 consumption and the associated O2 consumption and CO2 production) and on basal respiration (O2 consumption and CO2 production) in a landfill cover soil were studied in batch assays with and without CH4 amendment, respectively (Fig. 2a–h). When water content was between 14%dw and 28%dw (33–67%WHC), CH4 consumption, O2 consumption, and CO2 production in the CH4 assays were detectable at all studied temperatures and showed ln-linear increases with temperature (Fig. 2a, 2c). Q10 values for CH4 consumption varied between 6.5 and 8.4 (Table 1). When water content was 7%dw, the rates of gas consumption and production attained their minimum values within each of the studied temperatures (Fig. 2a, b). There was a tendency towards a decreased temperature effect of CH4 consumption with increasing water content at 14–28%dw, as shown by the

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diminishing Q10 values (Table 1). At 1–6 1C CH4 consumption increased up to the highest water content studied (28%dw) (po0:05 for both linear and quadratic terms in polynomial curves) while at 12 1C CH4 consumption peaked at 21%dw, and thus the water content response was curvilinear with a maximum (po0:05 for the quadratic term) but no overall trend (p40:10 for linear term) (Figs. 2b, d). At 19 1C a curvilinear response to water content was apparent in the water content range 14–34%dw (33–81%WHC) (Fig. 2b, d), although statistically insignificant (p40:10 for linear and quadratic terms). Basal CO2 production (Fig. 2e–h) and O2 consumption (data not shown) increased ln-linearly with increasing temperature at water contents 21%dw and 28%dw (R2 40:97) (Fig. 2e, g). In contrast, at 14%dw, basal O2 consumption showed no ln-linear behaviour (Fig. 2e, g) and thus Q10 values were only calculated for 21%dw and 28%dw (Table 1). Response of basal respiration to temperature (Q10 values 3.2–4.0) was lower than that for CH4 consumption, or for CO2 consumption and O2 production in the CH4 assays (Table 1). At the lowest water content (7%dw) basal respiration was negligible or very low (Fig. 2e–h) compared to higher water contents. Basal respiration increased with increasing water content at 1 and 12 1C (Fig. 2f, 2h). CH4-C metabolism accounted for 55–83% of total O2 consumption, the residual O2 consumption owing to basal respiration. The proportion of CH4-C metabolism over O2 consumption increased with temperature at 1–12 1C, the mean proportions being 55–64% at 1 1C and 78–83% at 12 1C, while no dependence on water content was indicated (data not shown). The dominance of CH4-C metabolism was also indicated by the 2–6-fold higher O2 consumption in the CH4-amended assays compared to the basal assays (data not shown), as well as by the ratios (mol/mol) of CO2 production (range 0.49–0.93) and O2 consumption (range 1.40–2.30) in relation to CH4 consumption, which were close to values reported for methanotrophs (Whittenbury et al., 1970; Megraw and Knowles, 1987) or to values indicated by the net equation of the complete oxidation of CH4 to CO2 (2 mol O2 consumed and 1 mol CO2 produced per 1 mol CH4 consumed; Anthony, 1982). The ability of the soil CH4 oxidizers to be activated or grow in the presence of CH4 at 1 and 12 1C was studied by determining the specific growth rate of CH4 consumption from the increase in CH4 consumption in consecutive CH4 feeding cycles at water content 21%dw. The rates of CH4 and O2 consumption, as well as CO2 production recorded in the second feeding cycle (Table 2) were higher compared to the first one, corresponding to a specific growth rate (m) of 0.1070.001 d1 for CH4 consumption at 1 1C, which was 15% of the value at 12 1C (0.7070.006 d1). 4. Discussion Understanding the factors regulating CH4 oxidation at low temperatures in CH4 sources such as landfills and

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CH4 consumption (µmolCH4 gdw-1 h-1)

3

a

2.5 2

Water content

b

17%WHC (7%dw)

Temperature (°C)

12

50%WHC (21%dw)

1.5

1 6

33%WHC (14%dw)

19

67%WHC (28 %dw)

1 0.5

CH4 consumption ln (µmolCH4 gdw-1h-1)

0 1

c

d

e

f

g

h

0 -1 -2 -3

0.12 0.1 0.08 0.06 0.04 0.02

Basal CO2 production ln (µmolCO2 gdw-1h-1)

Basal CO2 production (µmolCO2 gdw-1h-1)

-4

0 -2 -3 -4 -5 -6 0

5

10

15

Temperature (°C)

0

20

40

60

80 (%WHC)

0

8

17

25

34 (%dw)

Water content Fig. 2. Rates (direct and ln-transformed) of CH4 consumption (a–d) and basal CO2 production (e–h) in the studied landfill cover soil at different water contents as a function of temperature (left) and at different temperatures as a function of water content (right). Results are mean values of duplicate samples. Error bars present 71 SEM and are smaller than the symbols when not apparent. Assays at 34%dw (81%WHC) were only performed for 19 1C.

Table 2 CH4 consumption rates and the molar ratios of CO2 production and O2 consumption in relation to CH4 consumption, duration of CH4 feeding cycles, and specific growth rates (m) for CH4 consumption rates in consecutive batch assays with landfill cover soil samples at 1 and 12 1C at water content of 21%dw; the results are means (7SEM) of duplicate samples CH4 feeding cycle 1 1C

1 CH4 consumption (mmolCH4 g1 dw h ) CO2/CH4 (mol/mol) O2/CH4 (mol/mol)

Duration of 1st feeding cycle (d) Specific growth rate m (d1) Generation time tg (d)

12 1C

1st

2nd

1st

2nd

0.05970.005 0.9270.042 2.0470.092

0.1370.012 0.4570.042 1.1970.198

0.7070.049 0.4970.014 1.4170.028

1.4970.042 0.3970.071 1.1770.021

1 1C

12 1C

13 0.09670.001 10.4

1.6 0.7070.006 1.43

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wetlands is important for the prediction of CH4 emissions in changing climatic conditions and for finding appropriate measures of soil management to promote oxidation. The present results suggest that microbial oxidation can reduce CH4 emissions from methanogenic environments even at temperatures near freezing point. A novel finding of the present study is that the CH4 consumption rate per gram of soil can increase even at 1 1C, as shown by the consecutive incubation assays. Increases in the CH4 consumption rates in batch assays have been previously reported only at 20–25 1C (Bo¨rjesson et al., 1998; De Visscher et al., 1999). The increase in consumption rate is probably due to an increase in the number of enzymes catalyzing CH4 oxidation reactions. This in turn may be due to an increase in cell numbers of CH4 oxidizers, as in the present study; the release of CO2-C was smaller than the consumption of CH4-C, suggesting that CH4-C was assimilated in biomass. Activation of resting stages which can be produced by methanotrophs (Hanson and Hanson, 1996) may also have contributed to the increase in the CH4 consumption rate. The increase in CH4 consumption rates indicates that CH4 oxidizers in the present soil were rapidly activated when they were subjected to a high O2 and CH4 concentration in the assays. The time needed for the CH4 consumption rate to double (10–1.4 d at 1–12 1C) is probably fast enough to allow the CH4-oxidizing community to respond to the decrease in temperatures during the cold season in temperate or boreal areas, either by the activation of dormant forms, or by growth. This is in line with previous laboratory studies which have shown that temperature decrease may induce changes in vertical distribution of CH4 oxidation (Kettunen et al., 2006) or species composition of the CH4-oxidizing community (Gebert et al., 2003; Bo¨rjesson et al., 2004). The fact that the CH4 consumption rate increased even at near-freezing temperatures corroborates the finding obtained in the laboratory column study by Kettunen et al. (2006). They concluded that the broadening of the active CH4-oxidizing zone along the soil vertical profile, in response to increased O2 levels, could mitigate the effect of temperature decrease on CH4 oxidation. A prerequisite for this effect is that the rate of CH4 consumption of CH4 oxidizers per gram of soil is able to increase at low temperatures, as was shown by the present batch assays. For comparison, the lowest temperature at which the growth of CH4-oxidizing microorganisms in boreal or warmer environments has been reported is 6 1C (temperatures o6 1C not studied). The growth at this temperature was observed with enrichments from landfill cover soil and sludge checks in northern Europe (Nozhevnikova et al., 1993 and 2001). In permanently cold environments, psychrophilic methanotrophs with optimum growth temperatures of 6–10 1C are known (Trotsenko and Khmelenina, 2005). Many authors have detected CH4 consumption at low temperatures (down to 0–5 1C) in batch assays with various soils. The rates recorded in landfill soils (Whalen et al., 1990; Christophersen et al., 2000; Bo¨rjesson et al., 2004;

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Scheutz and Kjeldsen, 2004) and in plant roots and rhizomes (King, 1994) are fairly similar to those found in the present study at 1–6 1C (0.03–0.23 compared to 1 0.01–0.27 mmolCH4 g1 dw h ). The CH4 consumption rates observed in slurries of temperate and boreal peat (Dunfield 1 et al., 1993) (p0.05 mmolCH4 g1 at 0–5 1C) are wet weight h lower than those in the soils presented above on a wet weight basis, which may partially be explained by the typically high water content of peat. Moreover, the addition of water to obtain a slurry decreased the rates (Dunfield et al., 1993). Markedly lower CH4 consumption 1 rates (p0.004–0.007 mmol CH4 g1 at 1–5 1C) wet weight h were reported for tundra soils (Wagner et al., 2003). The response of CH4 consumption rate to temperature when water content was adequate (14–28%dw) was similar to that generally observed at temperature ranges typical of temperate or boreal environments, as CH4 consumption increases along with temperature (Whalen et al., 1990, Czepiel et al., 1996, Gebert et al., 2003, Bo¨rjesson et al., 2004, Park et al., 2005) when CH4 concentration or water content are not themselves limiting factors. CH4 consumption, defined as the rate of CH4 disappearance—and its response to temperature—are probably regulated by the rate of turnover of the first enzyme reaction in the CH4 oxidation chain, the hydroxylation of CH4 to CH3OH by CH4 monooxygenase, which removes the dissolved CH4 from the soil water. The turnover rate of this reaction in soil is affected by the abundance and activity of methane monooxygenase enzymes, as well as by the rate of substrate (CH4 and O2) supply. As in the present study (9% initial CH4 conc.), pronounced responses of CH4 consumption to temperature were observed in soils and methanotroph cultures which were incubated at CH4 concentrations of 1000–10,000 ml l1 (0.1–1%), as CH4 consumption depended on the potential enzyme activity, while less pronounced responses were seen at low initial CH4 concentration (2–100 ml l1) because CH4 consumption was restricted by the supply of CH4 (King and Adamsen, 1992; Whalen and Reeburgh, 1996). Accordingly, Boeckx et al. (1996) observed low responses of CH4 consumption to temperature (average Q10 1.9) in landfill cover soil incubated at 10 ml l1 in contrast to the higher Q10 values (1.9–7.26) in previous studies at 41% CH4 concentrations (Czepiel et al., 1996; Christophersen et al., 2000; De Visscher et al., 2001; Bo¨rjesson et al., 2004) as well as in the present study (Q10 6.5–8.4). The response of CH4 consumption to water content followed the typical curvilinear pattern (Whalen et al., 1990; Boeckx et al., 1996; Czepiel et al., 1996; Whalen and Reeburgh, 1996; Christophersen et al., 2000; Scheutz and Kjeldsen, 2004; Park et al., 2005) at temperatures 12–19 1C in the present study. The present water content regime, which produced high CH4 consumption (38–67% WHC), is in line with previous results where optimum water content has ranged from 40% to 80% WHC (Figueroa, 1993; Boeckx et al., 1996; Whalen and Reeburgh, 1996), when compared on the basis of %WHC. The decrease in

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CH4 consumption to low levels when soil water content falls to an unfavourable level, i.e., from 33%WHC to 17%WHC (14–7%dw) in the present study at all temperatures, is apparently due to the decrease in water potential, i.e., increase in water stress (Brown, 1976). Correspondingly, the increase in CH4 consumption along with the rise in water content from dry conditions, e.g. in the present study from 17%WHC to 50%WHC (14–21%dw) (at 12 1C) or up to 67 WHC (28%dw), the highest water content studied (at 1–6 1C), is apparently due to the increased water potential. Once optimal water content is reached, CH4 consumption starts to decrease if water content is further increased, as observed in the present study (12–19 1C), owing to the reduced supply of CH4 and/ or O2, as these gases are poorly water soluble and their liquid-phase diffusion is slow. Although in the present study at 1–6 1C CH4 consumption did not decrease with increasing water content within the water content range studied, it is likely that a decrease in water contents above this range (467% WHC, i.e., 428%dw) would also occur at 1–6 1C (Christophersen et al., 2000; Whalen and Reeburgh, 1996). The present results showed that the response of CH4 consumption to water content was dependent on temperature: an increase in water content was accompanied by a decrease in the response to temperature, and optimum water content for CH4 consumption was higher at low temperatures (1–6 1C) as opposed to higher temperatures (12–19 1C). This can be explained by the fact that, at higher temperatures, as potential methane monooxygenase activity increases, CH4 becomes more limited by the supply of O2 and CH4, and, therefore, the optimum water content is lower, compared to the situation at low temperatures. Similar CH4 consumption behaviour can also be seen in the data presented by Whalen and Reeburgh (1996) for a bog soil where the response curve of CH4 consumption to water content was more symmetrical towards lower temperatures and increasing water content had a more negative effect on CH4 consumption at higher as opposed to lower temperatures (ranges studied 1–15 1C and 41–77%WHC). The nonsystematic variation between temperature and water content responses in landfill soils reported by Christophersen et al. (2000) may be explained by the fact that CH4 consumption begun with lag phases, and therefore the results may reflect growth or activation of CH4 oxidizers in the assays and not just their initial activity. The interdependence between temperature and water content effects on CH4 consumption indicates that both temperature and water content should be considered when estimating the rates of CH4 oxidation and thus CH4 emissions in boreal soils in different climatic conditions, as well as in management of soils to promote oxidation. This also has implications for the choice of incubation methods in studies of CH4 consumption rate: addition of water to soil samples to obtain a slurry will likely lead, not only to a fall in CH4 consumption, as reported by Dunfield et al. (1993), but also to a muted response to temperature, compared to

soil with the original water content, unless mass-transfer is optimized by shaking. CH4 consumption was less tolerant (higher Q10 values) to low temperatures compared to basal respiration. A similar observation can also be deduced from the data obtained from soils sampled from CH4-sparged laboratory columns (Kettunen et al., 2006). However, we are not aware of previous publications comparing the responses of both of processes to temperature within soils sampled from an actual environment. In studies which have determined either one of the processes, CH4 consumption has been more responsive to temperature (Q10 1.9–7.26; references above) than basal respiration (Q10 1.9–4.3; Howard and Howard, 1993; King and Adamsen, 1992 and references therein). The higher response of CH4 consumption to temperature may partially be explained by high initial CH4 concentrations and thus high substrate availability to CH4 oxidizers, and lower substrate concentrations for microbes contributing to basal respiration. The present results show that part of the CH4-C consumed by CH4 oxidizers may accumulate in soil, as shown by the fact that less CO2 was produced than CH4 consumed at all studied temperatures and water contents, as also observed previously (Megraw and Knowles, 1987; Whalen et al., 1990; Bo¨rjesson et al., 1998, 2001). Thus, CH4-oxidizing soils not only reduce the global warming potential of methanogenic environments by converting CH4 to CO2, but may in fact capture part of the CH4-C produced, thus reducing the fluxes of greenhouse gases and C entering the atmosphere. The C balance of CH4oxidizing soils is affected, besides of the accumulation of the CH4-C oxidized, also by CO2 production from soil organic matter. In fact, CO2 production from soil organic matter (as determined by basal respiration assays) may partially represent a delayed oxidation of CH4-C to CO2-C, as part of the CO2 may originate from organic compounds derived from CH4-C previously accumulated in soil. The higher temperature response of CH4 consumption than basal CO2 production shows that, if C is accumulating in soil, i.e., if the rate of CH4-C accumulation is higher than that of CO2 production from soil organic matter, then the amount of C stored will positively correlate with temperature (assuming that CH4-C accumulation has the same temperature response as CH4 consumption). Further studies are needed to find out how other factors, such as the plant cover, which affects CH4 flux and oxidation (Watson et al., 1997), regulate the C storage in CH4-oxidizing soils of methanogenic environments and whether the accumulated CH4-C can be stored for a meaningful period of time considering climate change mitigation. 5. Conclusions Microorganisms in the cover soil of a boreal landfill were able to consume CH4 at an increasing rate even at temperatures below 10 1C and close to freezing point. The

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increase in consumption rates is due to growth or activation of CH4 oxidizers. The doubling time of CH4 consumption rate (1.4–10 d) is probably fast enough to allow the CH4 oxidizers to respond to a decrease in temperature by changes in their vertical distribution or species composition during the cold season. In dry soil (17%WHC) CH4 consumption was negligible throughout the temperature range studied (1–19 1C). When the water content was more favorable (33 to 67%WHC), the response of CH4 consumption to temperature decreased along with increasing water content and, accordingly, the response of CH4 consumption to water content varied with temperature. This interdependence of the effects of temperature and water content on CH4 oxidation indicates that both factors must be taken into account when estimating the effects of temperature and water content on CH4 oxidation. CH4 consumption was less tolerant (higher Q10 values; 6.5–8.4) of low temperatures compared to basal respiration (Q10 3.2–4.0). This was probably partially due to the higher availability of substrate to CH4oxidizing microorganisms while basal respiration was more substrate-limited, a situation that may prevail in many methanogenic environments. The present finding of increasing CH4 consumption rate in batch assays at low temperatures corroborates the results from a previous soil column study which showed that a decrease in total O2 consumption per gram of soil with temperature decrease may allow the active CH4 oxidation layer to broaden along the soil depth profile. Moreover, less CO2-C was released than CH4-C consumed at all temperatures and water contents studied, suggesting that CH4-oxidizing soils have the potential to accumulate CH4-C and reduce the amount of C entering the atmosphere. Overall, these results from laboratory batch assays suggest that CH4 oxidation can reduce global warming potential of methanogenic environments even at low ambient temperatures. Acknowledgements This study was financially supported by the Academy of Finland (project ‘Control of CH4 emissions from waste by methanotrophs and effect of low temperature on CH4 oxidation’) and by the Finnish Graduate School in Environmental Science and Technology. The authors wish to thank Dr. P. Aphalo for statistical advice. Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version at doi:10.1016/j.soilbio.2006. 12.022 References Alm, J., Saarnio, S., Nyka¨nen, H., Silvola, J., Martikainen, P.J., 1999. Winter CO2, CH4 and N2O fluxes on some natural and drained boreal peatlands. Biogeochemistry 44, 163–186.

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