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Short- and medium-term effects of NH4+ on CH4 and N2O fluxes in arable soils with a different texture
Soil Biology & Biochemistry 34 (2002) 669±678
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Short- and medium-term effects of NH41 on CH4 and N2O ¯uxes in arable soils with a different texture I. Kravchenko a,1, P. Boeckx b,*, V. Galchenko a,1, O. Van Cleemput b a
Laboratory of Unique Micro-organisms Classi®cation and Storage, Institute of Microbiology, Russian Academy of Sciences, Prospect 60-let Octyabrya 7/2, 117811 Moscow, Russian Federation b Laboratory of Applied Physical Chemistry, Faculty of Agricultural and Applied Biological Sciences, Gent University, Coupure 653, B-9000 Gent, Belgium Received 13 October 2001; received in revised form 30 October 2001; accepted 9 November 2001
Abstract The short- (24 h) and medium-term (30 d) effect of NH41 on the CH4 and N2O ¯uxes from two arable soils with different textures have been tested. Soil A had a medium (loam) texture and a cation exchange capacity (CEC) of 8.4 cmol(1) kg 21. Soil B had a coarse (loamy sand) texture and a CEC of 4.7 cmol(1) kg 21. During the short-term experiment, both soils showed a decreasing CH4-oxidising capacity upon increasing NH41 application rates. These CH4 oxidation rates were inversely related to the N2O emissions. The two soils showed slightly different CH4 oxidation kinetics. The ki value has been determined as the NH41 concentration of the soil resulting in a reduction by 50% of the initial (no NH41 added) CH4 oxidation rate constant (k1). The ki value in soil A (22.2 mg NH41 ±N kg 21) was twice as high as that of soil B (12.4 mg NH41 ±N kg 21). Similarly, the CEC of soil A was about twice as high as the CEC of soil B. This could, however, not explain the different N2O ¯uxes in soil A and B. Therefore, it was suggested that differences in the microbial community in both soils might be even more important in controlling the CH4 and N2O ¯uxes. This suggestion could also be made from the results obtained in the medium-term experiment. Herein, soil A showed a transient recovery of the CH4 oxidation rate upon NH41 addition and a peak emission of N2O when the CH4 oxidation was inhibited. Soil B, however, gave a persistent inhibition of the CH4 oxidation upon NH41 addition, a low N2O ¯ux and a lag phase of 4 d of the nitri®cation activity. It is hypothesised that the microbial communities of each soil responded or developed differently to the CH4, NH41, NO32 or NO22 concentrations. Therefore, it is suggested that identi®cation of the microbial structure, response or diversity might be as important as soil physical-chemical parameters in explaining CH4 and N2O ¯uxes from aerobic soils. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Methane oxidation; Nitrous oxide emission; Ammonium addition; Soil texture
1. Introduction Methane (CH4) and nitrous oxide (N2O) are important atmospheric trace gases in¯uencing radiative forcing and atmospheric chemistry (WMO, 1995). The atmospheric concentration of CH4 and N2O has been increasing considerably since pre-industrial times (IPCC, 2001). Exchange of CH4 and N2O between terrestrial ecosystems and the atmosphere plays a signi®cant role in the global budgets of these trace gases. The sink strength of aerobic soils for atmospheric CH4 has been estimated at 29 (uncertainty range is 7± . 100) Tg CH4 yr 21 (Smith et al., 2000). On the other hand, natural and agricultural soils are the most * Corresponding author. Tel.: 132-9-264-60-00; fax: 132-9-264-62-30. E-mail addresses: [email protected] (I. Kravchenko), [email protected] (P. Boeckx). 1 Tel.: 17-095-135-01-80; fax 17-095-135-65-30.
important global source of N2O (6.0 and 4.2 Tg N2O± N yr 21, respectively) (IPCC, 2001). It has been demonstrated that the sink and source functions of aerobic soils for CH4 and N2O depend largely on land use, management and climate. In general, conversion of natural soils to agricultural use reduces their sink strength for CH4 and enhances their N2O emission (Boeckx et al., 1998; Smith et al., 2000). The processes of CH4 oxidation and N2O production are strongly interconnected via soil N transformations. Ammonium is an important inhibitor of CH4 oxidation (e.g. Bender and Conrad, 1994; King and Schnell, 1994; Boeckx and Van Cleemput, 1996; HuÈtsch et al., 1996). However, in some cases, no immediate effect was observed upon addition of NH41 (Dobbie and Smith, 1996; Delgado and Mosier, 1996; Gulledge et al., 1997). The mechanism of NH41 inhibition is complex and thought to include both a competitive inhibition of the CH4 mono-oxygenase (MMO) enzyme, as well as a non-competitive (toxic) inhibition by hydroxylamine
0038-0717/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0038-071 7(01)00232-2
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Table 1). Soil A had a medium (loam) texture and a CEC of 8.4 cmol(1) kg 21 and soil B had a coarse (loamy sand) texture and a CEC of 4.7 cmol(1) kg 21.
Table 1 Characteristics of the investigated soils
General Location Land use Soil Texture Clay (%) Sand (%) Loam (%) pH-H2O NH41 (mg N kg 21) NO32 (mg N kg 21) FC (%) CEC (cmol(1) kg 21) CaCO3 (%) Organic matter (%) a b
Soil A
Soil B
51810 0 N, 2855 0 E Arable
5185 0 N, 3831 0 E Arable
a
Loam , medium 20 47 33 7.6 0.31 0.23 26.4 8.4 1.03 2.54
b
a
Loamy sand , coarse 4 87 9 6.7 0.44 1.49 23.4 4.7 0.26 2.92
2.2. Soil conditioning
b
US Department of Agriculture textural classes. FAO textural classes.
(NH2OH) or nitrite (NO22) produced via NH41 oxidation (King and Schnell, 1994). Moreover, additional noncompetitive inhibition resulting from a general salt effect has also been reported (MacDonald et al., 1997; Gulledge and Schimel, 1998). De Visscher et al. (1998) demonstrated that the amount of exchangeable NH41 could be another important factor controlling the inhibitory effect of NH41. The effect of NO32, another product of NH41 oxidation, on CH4 oxidation is ambiguous. Some authors did not observe an effect of NO32 (Boeckx and Van Cleemput, 1996), while others observed the contrary (Whalen, 2000). It has also been suggested that N-turnover rates (mineralisation and gross nitri®cation) (Mosier et al., 1991; Steudler et al., 1996) in¯uence CH4 uptake rather than the actual soil mineral N (NH41, NO32 or NO22) content. We studied the relationship between, and controlling factors of, CH4 oxidation and N2O emission by aerobic soils receiving NH41. Firstly, the short-term (24 h) effect of NH41 applications, ranging from 5 to 100 mg NH41 ± N kg 21, on CH4 oxidation and N2O emission by two different arable soils was investigated. Secondly, the mediumterm (30 d) effect of addition of 100 mg NH41 ±N kg 21 on CH4 oxidation, N2O emission and nitri®cation was tested in the same arable soils.
2. Materials and methods 2.1. Sites and soils The CH4-oxidising capacity and N2O emission were investigated for two arable soils (soil A: 51810 0 N, 2855 0 E and soil B: 5185 0 N, 3831 0 E). The sites were comparable in terms of climatic conditions (temperate), land use (arable soil), pH, organic matter and inorganic N content, but had a different texture and cation exchange capacity (CEC) (see
The soil samples were taken from the surface (0±15 cm) and were air-dried, ground and sieved (2 mm) before use. The effect of NH41 on CH4 and N2O ¯uxes was investigated using batch incubation experiments. To stimulate the methanotrophic community, the soils were re-wetted up to 75% ®eld capacity (FC), being optimal for CH4 oxidation (Boeckx and Van Cleemput, 1996). Thereafter, the soils were placed in Plexiglas cylinders and were continuously fed with pure CH4 from the base. Methane was introduced at a rate of 5 ml min 21 to obtain a CH4 input of 10 mol CH4 m 22 d 21. The headspace was continuously ¯ushed with moist air (11 min 21) to prevent drying of the soil. More details concerning the set-up can be found in De Visscher et al. (1999). At regular intervals gas samples (100 ml) were taken from the outlet of the cylinders during the 10 d of conditioning and analysed for CH4 in order to estimate the CH4-oxidising activity of the soils. A similar conditioning experiment was conducted using ambient CH4 concentrations. 2.3. NH41 ®xation The short-term NH41 ®xation capacity of both soils was estimated. Ammonium sulphate ((NH4)2SO4) solutions (0.5 ml) were added to the soil samples (equal to 30 g dry weight) using the same N concentration range as used in the short-term incubation experiment (5±100 mg NH41 ± N kg 21, see later). The samples were mixed carefully and equilibrated for 2 h at room temperature. Then, the soil samples were extracted with 2 M KCl and analysed for NH41 ±N. 2.4. Experimental design The following experimental design was used to determine the short-term effect of NH41 on CH4 and N2O ¯uxes. Soil samples (equal to 30 g dry weight) were transferred from the conditioning cylinders into 200 ml bottles. Ammonium sulphate was dissolved in distilled water and added to the soil in 0.5 ml and carefully mixed to ensure homogeneous distribution of NH41. The following concentrations were used: 5, 10, 25, 50 and 100 mg NH41 ±N kg 21 soil. Addition of 0.5 ml of distilled water served as a control treatment. If necessary, additional water was added to maintain 75% FC. All experiments were carried out in triplicate and at room temperature (22±23 8C). The bottles were sealed with rubber stoppers and CH4 was added up to an initial concentration of 10 nl CH4 ml 21 in the headspace. Methane uptake and N2O emission was determined simultaneously by analysing the headspace of the bottles 0, 2, 4, 6, 8 and 24 h after CH4 addition. Zero time samples were taken
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5 min after CH4 addition to allow uniform diffusion of the added CH4. A separate set of samples was prepared to evaluate the medium-term (30 d) effect of NH41 (100 mg NH41 ±N kg 21) on CH4 oxidation, N2O emission and nitri®cation. Gas analysis (0, 2, 4 and 6 h after CH4 addition) and analysis of the mineral N content (NH41, NO32 1 NO22 and NO22) of the soil were performed daily during the ®rst week of the incubation and once or twice a week later on. Triplicate treatments were used for soil A and B. Changes in the water content were determined gravimetrically during the incubation and losses were adjusted when necessary. During the short-term experiment, CH4 oxidation followed ®rst order kinetics: dCH4 =dt k1 £ CH4 : From the slope of the log-transformed CH4 concentrations as a function of time, the CH4 oxidation rate constants (k1, h 21) could be calculated via linear regression. The CH4 oxidation rates were calculated by multiplying k1 with the CH4 concentration at time zero. A t-test a 0:01 was used to identify signi®cantly different oxidation rates between the different treatments. The NH41 inhibition constant (ki) was de®ned as the NH41 concentration, that reduced the initial oxidation rate constant (k1) (no NH41 added) by 50%. The ki values were calculated using an optimisation procedure in Solver in Microsoft Excel, using the measured CH4 oxidation rate constants as a function of the applied NH41 concentrations. During the medium-term experiment the decrease of the CH4 concentration was linear during each gas sampling period of 6 h. Thus, zero order kinetics dCH4 =dt k0 were applied to calculate the CH4 oxidation rates. A t-test a 0:01 was used to identify signi®cantly different oxidation rates between the different treatments. The increase of N2O concentration was linear (R 2 $ 0.95) during the ®rst 6±8 h of the incubation in the short- and medium-term experiment. Nitrous oxide ¯uxes were expressed using zero order kinetics: dN2 O=dt k0 and N2O ¯uxes were estimated via linear regression analysis. A t-test a 0:01 was used to identify signi®cantly different oxidation rates between the different treatments. 2.5. Measurements The CH4 concentrations were measured using a Chrompack CP9000 gas chromatograph (Chrompack, The Netherlands) equipped with a ¯ame ionisation detector and a NaI aluminium column. Helium was the carrier gas (37 ml min 21) and the settings were as follows: injector temperature 65 8C, oven temperature 55 8C and detector temperature 200 8C. Nitrous oxide was determined using a Chrompack 437A gas chromatograph (Chrompack, The Netherlands) with a stainless steel packed column (Chromosorb 102) and a 63Ni electron capture detector. The following conditions were used: injector temperature 90 8C, oven temperature 90 8C and detector temperature 300 8C. Dinitrogen was the carrier gas (32 ml min 21). The concen-
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trations of CH4 and N2O were calculated from the peak area in the chromatograms, which were registered and analysed using the WOW software package (Thermo Separation Products, USA). Soil analyses (see Table 1) were carried out after the soil conditioning procedure. The soil moisture content was determined gravimetrically (drying at 105 8C). Soil pH was determined in soil/water extractions (1/5 w/w) using a combined pH electrode connected to a C832 multichannel analyser (Consort, Belgium). Inorganic N compounds were extracted from the soil samples by adding 2 M KCl (soil/KCl ratio 1:2). The extracts were analysed for NH41 and (NO32 1 NO22) via steam distillation of the extract and subsequent titration (Keeney and Nelson, 1982). Nitrite was determined separately using the diazotisation method (HACH, 2000). 3. Results 3.1. Effect of elevated CH4 concentrations During the conditioning incubation at elevated CH4 concentrations, soils A and B showed a substantial increase of the CH4 uptake. Methane uptake in the soil cylinders stabilised after 5±7 d at about 3±4 mol CH4 m 22 d 21 for both soils. The CH4 uptake rates were signi®cantly higher compared to soils conditioned under ambient CH4 concentrations (P , 0.01). In a short-term batch experiment (identical set-up as in the short-term experiments assessing the effect of NH41, except that no NH41 was applied) the headspace CH4 concentrations decreased from about 10 nl CH4 ml 21 to 2.1±2.3 nl CH4 ml 21 and to 7 nl CH4 ml 21 in activated and non-activated soils, respectively (Fig. 1). 3.2. Short-term effect of NH41 on CH4 oxidation and N2O emission From Fig. 2 it is clear that addition of NH41 caused a reduction of the CH4 oxidation rates of soil A and B for all tested NH41 concentrations (5±100 mg NH41 ±N kg 21). The extent of the inhibitory effect of NH41 depended on its concentration. In soil A, treatments with 10±100 mg NH41 ± N kg 21 showed signi®cantly (P , 0.01) lower CH4 oxidation rates than the control (no NH41 added). This effect occurred for all amounts of NH41 added to soil B. Soil A and B showed clear differences in N2O emission rates as a function of the NH41 application (Fig. 2). The N2O ¯uxes increased upon increasing NH41 addition. Soil A showed the highest N2O ¯uxes. Addition of 100 mg NH41 ±N kg 21 gave a N2O ¯ux of 17.6 ^ 2.4 ng N2O g 21 h 21. In soil B, addition of the same amount of NH41 resulted in a much lower N2O emission, 2.8 ^ 0.3 ng N2O g 21 h 21. Moreover, soil A showed slightly different CH4 oxidation kinetics as a function of the NH41 content compared to soil B. In Fig. 3 the k1 and ki values of soil A and B are shown. The ki value was higher in soil A (22.2 mg NH41 ±N kg 21) than in soil B (12.4 mg NH41 ±N kg 21). Similarly, the CEC
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Fig. 1. Change in headspace CH4 concentrations during the incubation of soil A and B. Samples were conditioned at elevated or at ambient CH4 concentrations. Error bars represent standard deviations n 3:
of soil A is a factor twofold than the CEC of soil B (Table 1). Fig. 4 shows the degree of extractable NH41 of soil A and B. In soil A, only 54±99%, depending on the initial NH41 concentration, of the added NH41 could be extracted 2 h after NH41 addition. In contrast, in soil B the added NH41 was extracted completely for all concentrations. In Fig. 5 the relation between the CH4 oxidation rates and the N2O emission rates in soil A and B is shown. A signi®cant (P , 0.01) linear correlation (R2 0:73 and 0.68 for soil A and B, respectively) between CH4 and N2O ¯uxes could be observed. In both cases the slope was signi®cantly different from zero (P , 0.01). 3.3. Medium-term of NH41 on CH4 oxidation, N2O emission and nitri®cation The effect of NH41 supply on CH4 oxidation, N2O emission and nitri®cation dynamics were investigated in a medium-term incubation experiment (30 d). In soil A, NH41 addition caused a strong inhibition of the CH4
oxidation until d 1. Thereafter, the CH4 oxidation increased again and was practically restored between d 3 and 8, reaching comparable CH4 oxidation values as in the control soil (Fig. 6A). Both in the control and the NH41 treatment, the CH4-oxidising capacity started to decrease after d 8. In soil A, the N2O ¯ux was maximum at d 1 (42.8 ng N2O g 21 h 21) and declined sharply thereafter (Fig. 6B). In the control soil, the N2O emission remained low throughout the entire 30 d of the incubation (Fig. 6B). Nitri®cation of the added NH41 resulted in a signi®cant increase in NO32 1 NO22 concentrations during the ®rst 10 d (P , 0.01) of the incubation (Fig. 6C and D). In soil A, the NO22 concentrations were always much lower than the NO32 concentrations (Table 2). The highest NO22 content was observed on d 1. Soil B behaved completely different from soil A. A strong inhibition of the CH4 oxidation (about 90%) was observed during the entire medium-term incubation, despite the fact that more than 92% of the added NH41 was nitri®ed after 14 d (Fig. 7A and C). In the control treatment, the
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Fig. 2. The in¯uence of NH41 addition on the CH4 oxidation rates and the N2O emission in soil A and B. Each data point represents the average ^ standard deviations n 3:
CH4-oxidising capacity started to decrease after d 8, as was the case for soil A. The N2O emission remained low in the NH41 and control treatment during the entire incubation. Nitri®cation started after a lag phase of 4 d (Fig. 7C and D). The NH41 content started to decrease on d 5, while a parallel, but opposite, trend was observed for the NO32 1 NO22 content. In soil B, the NO22 concentrations were always much lower than the NO32 concentrations (Table 2).
4. Discussion 4.1. Effect of elevated CH4 concentrations The CH4 oxidation rates for soils conditioned at ambient CH4 concentrations were 0.95 ^ 0.13 ng CH4 g 21 h 21 and 0.90 ^ 0.07 ng CH4 g 21 h 21 for soil A and B, respectively. The measured oxidation rates were higher than those
reported by other authors using fresh samples of agricultural soils. Methane oxidation rates of 0.013 ng CH4 g 21 h 21 (Tlustos et al., 1998) and 0.19±0.31 ng CH4 g 21 h 21 (HuÈtsch, 1998) were determined in arable soil in the UK and Germany, respectively. In soils conditioned at elevated CH4 concentrations, the oxidation rates increased approx. fourfold and were 3.64 ^ 0.24 ng CH4 g 21 h 21 and 3.58 ^ 0.40 ng CH4 g 21 h 21 in soil A and B, respectively. Exposure to high CH4 concentrations was found to raise the atmospheric CH4 uptake in agricultural soils (Bender and Conrad, 1992; Jensen and Olsen, 1998), however, not in undisturbed natural forest soils (Jensen and Olsen, 1998). Although soil texture was found to be an important factor for CH4 oxidation (DoÈrr et al., 1993), the soils in this study showed a similar CH4 uptake rate despite differences in texture (Table 1, Fig. 1). This could be explained by the fact that, in our experimental design the soils were loosely packed and that gas diffusion is not expected to be a limiting factor (Boeckx and Van Cleemput, 1996).
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Fig. 3. In¯uence of different NH41 concentrations on the CH4 oxidation rate constants (k1) in soil A and soil B. Arrows indicate the NH41 concentrations that decreased the k1 value to 50% (ki-values) in soil A and B.
4.2. Short-term effect of NH41 on CH4 oxidation and N2O emission In Fig. 2 a non-linear relation between the CH4 oxidation rates and the NH41 content of the soil are shown. Studies using different types of soil have reported a negative linear (Boeckx and Van Cleemput, 1996) or sigmoõÈd (Kravchenko, 1999) correlation between CH4 oxidation rates and the NH41 content. Rapid inhibition of CH4 oxidation upon NH41 application has been well documented. This has been attributed to the combined effect of substrate competition at the level
of the MMO (Bosse et al., 1993; HuÈtsch et al., 1993; Castro et al., 1995) and non-competitive inhibition (King and Schnell, 1994; Gulledge and Schimel, 1998). Moreover, it has been suggested that CEC could be an important factor controlling CH4 oxidation (De Visscher et al., 1998). The degree of extractable NH41 is lower in soil A than in soil B, especially when low NH41 doses were applied (Fig. 4). This effect might partly explain the higher ki value calculated for soil A as compared to soil B (Fig. 3). The ki value corresponds to the NH41 addition that is needed to reduce the CH4 oxidation (k1 values) by 50%. Since, the
Fig. 4. Fraction of NH41 extracted from soil A and soil B as a function of added NH41 concentrations. NH41 salts (5±100 mg NH41 ±N kg 21) were added to the soils and extracted after 2 h of equilibration.
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Fig. 5. Relationship between CH4 uptake and N2O emission in soil A and soil B based on 24 h incubations with different NH41 applications (0±100 mg NH41 ± N kg 21).
amount of extractable NH41 is partly affected by the CEC of the soil, this might be re¯ected in the extent of the inhibitory effect of NH41 on the CH4-oxidising capacity. However, the CEC or the degree of extractable NH41 could not explain the observed N2O emission trends in soil A and B. From the experimental design (aerobic conditions plus NH41 addition) and from the nitri®cation results in
the medium-term experiment (see Figs. 6C,D and 7C,D) it was evident that N2O must originate from nitri®cation and not denitri®cation. From the medium-term experiment (see Fig. 7C and D) is was also clear that in soil B the nitri®cation activity showed a lag-phase of 4 d. Therefore, the nitri®cation might not be at the optimum rate in the short-term experiment of soil B, which lasted only 24 h. These results
Fig. 6. Changes in CH4 oxidation rates (A) N2O emission rates, (B) NH41 concentrations, (C) NO32 1 NO22 concentrations, (D) during an incubation of 30 d of soil A. Error bars represent the standard deviation n 3:
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Table 2 NO22 ±N concentrations (mg N kg 21) in soil A and soil B in the mediumterm CH4 oxidation experiment (ND not determined)
NH41 results in more N2O emission and more inhibition of the CH4 oxidation.
Day
Soil A
Soil B
1 3 5 8 11 16
1.06 0.36 0.36 0.41 ND ND
ND ND 0.31 0.43 0.38 0.48
4.3. Medium-term of NH41 on CH4 oxidation, N2O emission and nitri®cation
were con®rmed in a short-term NO emission experiment. The NO emission of soil A was sevenfold higher than that in soil B (data not shown). It is suggested that this lag-phase effect might be related to a difference in the structure or response of the microbial community in soil A and B. Moreover, in soil A (medium texture), N2O could additionally be produced from denitri®cation or via nitri®er denitri®cation in anoxic micro-sites. Correlation analysis between the CH4 oxidation rates and the N2O ¯uxes for soil A and B showed a clear negative relation (R2 0:73 for soil A and R2 0:68 for soil B) (Fig. 5). This clearly demonstrates that a higher availability of
Methane oxidation, N2O emission and nitri®cation dynamics were also investigated in the medium-term incubation experiment. HuÈtsch (1998) presented comparable results for a silt loam soil. During the ®rst day of the incubation of soil A, the CH4 oxidation capacity was considerably decreased (Fig. 6A). On d 1, also the highest NO22 ±N concentrations were found in soil A (Table 2). Nitrite is strongly toxic with respect to CH4 oxidation. As such, NO22 might at least partly be responsible for the observed inhibitory effect. However, the inhibition effect of residual NH41 concentrations during the medium-term experiment was lower, compared to the same initial NH41 concentrations in the short-term experiment. For example, it was only 9.6% after 3 d (54.4 mg NH41 ±N kg 21 remaining) as compared to 56.8% for the 50 mg NH41 ±N kg 21 dose in the shortterm experiment. Moreover, soil A showed a varying CH4oxidising activity, which after reaching a maximum, decreased signi®cantly between d 8 and 20 (Fig. 6A). Similar
Fig. 7. Changes in CH4 oxidation rates (A) N2O emission rates, (B) NH41 concentrations, (C) NO32 1 NO22 concentrations, (D) during an incubation of 30 d of soil B. Error bars represent the standard deviation n 3:
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losses of the CH4 oxidising activity have been observed in laboratory experiments using forest soils (Amaral et al., 1998). Both phenomena may re¯ect a changing methanotrophic community. We propose that the methantrophic community might have been increased during the conditioning at high CH4 concentrations, leading initially to a recovery of the CH4 oxidation. The loss of the CH4oxidising activity during incubations at lower CH4 concentrations and high NH41 concentrations could be the result of a decreasing methanotrophic community size or activity. The N2O emission showed a peak on d 1 and decreased thereafter (Fig. 6B). Thus, a relationship between the CH4 oxidation rate and N2O emission could also be seen. In soil A the net nitri®cation rate was calculated by linear regression analysis of the increase of NO32 1 NO22 concentrations from d 0 to 7 and was 10.5 mg N kg 21 d 21 (Fig. 6D). These results suggest that soil A contained an active nitrifying community and substrate addition caused an immediate response. It has been shown that NH41 application to arable soil caused a rise in the nitrifying activity but did not change the size of NH41-oxidising community (Mendum et al., 1999). In soil B a persistent inhibition of the CH4 oxidation could be observed upon NH41 addition. The reason for this persistent inhibition is not clear. It could not be explained by the NO22 accumulation during the medium-term incubation (Table 2). Whalen (2000) has also observed the existence of a persistent reduction in CH4 oxidation in similar laboratory studies. It has been proposed by MacDonald et al. (1997) and Whalen (2000) that such a medium-term effect could be explained by a non-speci®c salt effect of NH4(SO4)2 addition, rather than by the competition between NH41 and CH4. Also, in this experiment soil B showed low N2O emission rates compared to soil A. The net nitri®cation rate in soil B (between d 4 and 11) was 8.3 mg N kg 21 d 21. The rise in nitri®cation rates after 4 d (lag-phase) may suggest a phenotypic or size change of the communities of nitrifying or methanotrophic microorganisms upon NH41 addition. The NH41 was nitri®ed almost completely and the NO32 1 NO22 concentration (about 100 mg N kg 21) remained virtually constant thereafter. The latter could eventually explain the absence of a recovery of CH4 oxidation, because in some cases, persistent inhibition of CH4 oxidation has been observed upon NO32 addition (Prieme and Christensen, 1997; Whalen, 2000), in contradiction to experiments where no effect of NO32 could be observed (Boeckx and Van Cleemput, 1996). 4.4. Conclusions From the short-term (24 h) experiment it was clear that NH41 addition was a key factor controlling the interaction between CH4 oxidation and N2O emission in both arable soils. Ammonium was found to act as a competitive inhibitor for CH4 oxidation in both arable soils. It is suggested that the CEC and the NH41 extractability might affect the CH4
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oxidation kinetics. However, the microbial community structure or response might be even more important for the observed CH4 and N2O emissions. From the medium-term experiment it became clear that the effect of NH41 on CH4 oxidation was different when a certain concentration was added directly (short-term effect) or reached after a certain period upon addition of an initially higher NH41 concentration (medium-term effect). The nitrifying activity of soil A and B responded differently to NH41 additions. In soil B, nitri®cation showed a lag phase. Moreover, soil A and B showed a different course for CH4 oxidation and N2O emission. All these observations cannot be explained entirely by the physical-chemical properties of both soils. Therefore, it is suggested that the microbial communities present in soil A and B, responded or developed differently to the observed NH41, NO32, NO22 or CH4 contents. Thus, an important future goal in explaining the mechanisms of CH4 oxidation and N2O emission in soils could be the identi®cation of the methanotrophic and nitrifying communities or diversity in soils. Acknowledgements The ®nancial support of the Belgian Of®ce for Scienti®c, Technical and Cultural Affairs for the research fellowship of Irina Kravchenko is highly appreciated. References Amaral, J.A., Ren, T., Knowles, R., 1998. Atmospheric methane consumption by forest soils and extracted bacteria at different pH values. Applied and Environmental Microbiology 64, 2397±2402. Bender, M., Conrad, R., 1992. Kinetics of CH4 oxidation in oxic soils exposed to ambient air or high CH4 mixing ratios. FEMS Microbiology Ecology 101, 261±270. Bender, M., Conrad, R., 1994. Microbial oxidation of methane, ammonium and carbon monoxide, and turnover of nitrous oxide and nitric oxide in soils. Biogeochemistry 27, 97±112. Boeckx, P., Van Cleemput, O., 1996. Methane oxidation in a neutral land®ll cover soil: in¯uence of moisture content, temperature and nitrogenturnover. Journal of Environmental Quality 25, 178±183. Boeckx, P., Van Cleemput, O., Meyer, T., 1998. The in¯uence of land use and pesticides on methane oxidation in some Belgian soils. Biology and Fertility of Soils 27, 293±298. Bosse, U., Frenzel, O., Conrad, R., 1993. Inhibition of methane oxidation by ammonium in the surface layer of littoral sediment. FEMS Microbiology Ecology 13, 123±134. Castro, M.S., Steudler, P.A., Melillo, J.M., Aber, J.D., 1995. Factors controlling atmospheric methane consumption by temperate soils. Global Biogeochemical Cycles 9, 1±10. De Visscher, A., Boeckx, P., Van Cleemput, O., 1998. Interaction between nitrous oxide formation and methane oxidation in soils: in¯uence of cation exchange phenomena. Journal of Environmental Quality 27, 679±687. De Visscher, A., Thomas, D., Boeckx, P., Van Cleemput, O., 1999. Methane oxidation in simulated land®ll cover soil environments. Environmental Science and Technology 33, 1854±1859. Delgado, J.A., Mosier, A.R., 1996. Mitigation alternatives to decrease
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nitrous oxides emissions and urea-nitrogen loss and their effect on methane ¯ux. Journal of Environmental Quality 25, 1105±1111. Dobbie, K.E., Smith, K.A., 1996. Comparison of CH4 oxidation rates in woodland, arable and set aside soils. Soil Biology and Biochemistry 28, 1357±1365. DoÈrr, H., Katruff, L., Levin, I., 1993. Soil texture parameterization of the methane uptake in aerated soils. Chemosphere 26, 697±713. Gulledge, J., Schimel, J.P., 1998. Low-concentration kinetics of atmospheric CH4 oxidation in soil and mechanisms of NH41 inhibition. Applied and Environmental Microbiology 64, 4291±4298. Gulledge, J., Doyle, A.P., Schimel, J.P., 1997. Different NH41 inhibition patterns of soil CH4 consumption: a result of distinct CH4-oxidizer populations across sites? Soil Biology and Biochemistry 29, 13±21. HACH, 2000. DR/2010 Spectrophotometer Procedures Manual. HACH, Loveland, CO. HuÈtsch, B.W., 1998. Methane oxidation in arable soil as inhibited by ammonium, nitrite, and organic manure with respect to soil pH. Biology and Fertility of Soils 28, 27±35. HuÈtsch, B.W., Webster, C.P., Powlson, D.S., 1993. Long-term effects of nitrogen fertilization on methane oxidation in soil of the Broadbalk wheat experiment. Soil Biology and Biochemistry 25, 1307±1315. HuÈtsch, B.W., Russell, P., Mengel, K., 1996. CH4 oxidation in two temperate arable soils as affected by nitrate and ammonium application. Biology and Fertility of Soils 23, 86±92. IPCC (Intergovernmental Panel on Climate Change), 2001. Climate Change 2001. The Scienti®c Basis. Cambridge University Press, Cambridge. Jensen, S., Olsen, R.A., 1998. Atmospheric methane consumption in adjacent arable and forest systems. Soil Biology and Biochemistry 30, 1187±1193. Keeney, D.R., Nelson, D.W., 1982. NitrogenÐinorganic forms. In: Page, A.L., Miller, R.H., Keeney, D.R. (Eds.). Methods of Soil Analysis. Soil Science Society of America, Madison, pp. 643±698. King, G.M., Schnell, S., 1994. Ammonium and nitrite inhibition of methane oxidation by Methylobacter albus BG8 and Methylosinus trichosporium OB3b at low methane concentrations. Applied and Environmental Microbiology 60, 3508±3513. Kravchenko, I.K., 1999. The inhibiting effect of ammonium on the activity of the methanotrophic microbial community of a raised Spagnum bog in
West Siberia. Microbiology (Translation of Mikrobiologiya) 68, 241± 246. MacDonald, J.A., Skiba, U., Sheppard, L.J., Ball, B., Roberts, J.D., Smith, K.A., Fowler, D., 1997. The effect of nitrogen deposition and seasonal variability on methane oxidation and nitrous oxide emission rates in an upland spruce plantation and moorland. Atmospheric Environment 31, 3693±3706. Mendum, T.A., Sockett, R.E., Hirsch, P.R., 1999. Use of molecular and isotopic techniques to monitor response of autotrophic ammoniaoxidizing populations of the subdivision of the class Proteobacteria in arable soils to nitrogen fertilizer. Applied and Environmental Microbiology 65, 4155±4162. Mosier, A., Schimel, D., Valentine, D., Bronson, K., Parton, W., 1991. Methane and nitrous oxide ¯uxes in native, fertilized and cultivated grasslands. Nature (London) 350, 330±332. PriemeÂ, A., Christensen, S., 1997. Mechanistic analysis of ammonium inhibition of atmospheric methane oxidation in a Danish spruce forest. Soil Biology and Biochemistry 29, 1165±1172. Smith, K.A., Dobbie, K.E., Ball, B.C., Bakken, L.R., Sitaula, B.K., Hansen, S., Brumme, R., Borken, W., Christensen, S., PriemeÂ, A., Fowler, D., Macdonald, J.A., Skiba, U., Klemedtsson, L., Kasimir-Klemedtsson, A., Degorska, A., Orlanski, P., 2000. Oxidation of atmospheric methane in northern European soils, comparison with other ecosystems, and uncertainties in the global terrestrial sink. Global Change Biology 6, 791±803. Steudler, P.A., Jones, R.D., Castro, M.S., Melillo, J.M., Lewis, D., 1996. Microbial controls of methane oxidation in temperate forest and agricultural soils. In: Murrell, J.C., Kelly, D.P. (Eds.). The Microbiology of Atmospheric Trace Gases. Springer, Berlin, pp. 69±84. Tlustos, P., Willison, T.W., Baker, J.C., Murphy, D.V., Pavlikova, D., Goulding, K.W.T., Powlson, D.S., 1998. Short-term effects of nitrogen on methane oxidation in soils. Biology and Fertility of Soils 28, 64±70. Whalen, S.C., 2000. In¯uence of N and non-N-salts on atmospheric methane oxidation by upland boreal forest and tundra soils. Biology and Fertility of Soils 31, 279±287. WMO (World Meteorological Organization), 1995. Scienti®c assessment of ozone depletion: 1994. Global Ozone Research and Monitoring Project, Report No. 37, Geneva, Switzerland.
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Short- and medium-term effects of NH41 on CH4 and N2O ¯uxes in arable soils with a different texture I. Kravchenko a,1, P. Boeckx b,*, V. Galchenko a,1, O. Van Cleemput b a
Laboratory of Unique Micro-organisms Classi®cation and Storage, Institute of Microbiology, Russian Academy of Sciences, Prospect 60-let Octyabrya 7/2, 117811 Moscow, Russian Federation b Laboratory of Applied Physical Chemistry, Faculty of Agricultural and Applied Biological Sciences, Gent University, Coupure 653, B-9000 Gent, Belgium Received 13 October 2001; received in revised form 30 October 2001; accepted 9 November 2001
Abstract The short- (24 h) and medium-term (30 d) effect of NH41 on the CH4 and N2O ¯uxes from two arable soils with different textures have been tested. Soil A had a medium (loam) texture and a cation exchange capacity (CEC) of 8.4 cmol(1) kg 21. Soil B had a coarse (loamy sand) texture and a CEC of 4.7 cmol(1) kg 21. During the short-term experiment, both soils showed a decreasing CH4-oxidising capacity upon increasing NH41 application rates. These CH4 oxidation rates were inversely related to the N2O emissions. The two soils showed slightly different CH4 oxidation kinetics. The ki value has been determined as the NH41 concentration of the soil resulting in a reduction by 50% of the initial (no NH41 added) CH4 oxidation rate constant (k1). The ki value in soil A (22.2 mg NH41 ±N kg 21) was twice as high as that of soil B (12.4 mg NH41 ±N kg 21). Similarly, the CEC of soil A was about twice as high as the CEC of soil B. This could, however, not explain the different N2O ¯uxes in soil A and B. Therefore, it was suggested that differences in the microbial community in both soils might be even more important in controlling the CH4 and N2O ¯uxes. This suggestion could also be made from the results obtained in the medium-term experiment. Herein, soil A showed a transient recovery of the CH4 oxidation rate upon NH41 addition and a peak emission of N2O when the CH4 oxidation was inhibited. Soil B, however, gave a persistent inhibition of the CH4 oxidation upon NH41 addition, a low N2O ¯ux and a lag phase of 4 d of the nitri®cation activity. It is hypothesised that the microbial communities of each soil responded or developed differently to the CH4, NH41, NO32 or NO22 concentrations. Therefore, it is suggested that identi®cation of the microbial structure, response or diversity might be as important as soil physical-chemical parameters in explaining CH4 and N2O ¯uxes from aerobic soils. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Methane oxidation; Nitrous oxide emission; Ammonium addition; Soil texture
1. Introduction Methane (CH4) and nitrous oxide (N2O) are important atmospheric trace gases in¯uencing radiative forcing and atmospheric chemistry (WMO, 1995). The atmospheric concentration of CH4 and N2O has been increasing considerably since pre-industrial times (IPCC, 2001). Exchange of CH4 and N2O between terrestrial ecosystems and the atmosphere plays a signi®cant role in the global budgets of these trace gases. The sink strength of aerobic soils for atmospheric CH4 has been estimated at 29 (uncertainty range is 7± . 100) Tg CH4 yr 21 (Smith et al., 2000). On the other hand, natural and agricultural soils are the most * Corresponding author. Tel.: 132-9-264-60-00; fax: 132-9-264-62-30. E-mail addresses: [email protected] (I. Kravchenko), [email protected] (P. Boeckx). 1 Tel.: 17-095-135-01-80; fax 17-095-135-65-30.
important global source of N2O (6.0 and 4.2 Tg N2O± N yr 21, respectively) (IPCC, 2001). It has been demonstrated that the sink and source functions of aerobic soils for CH4 and N2O depend largely on land use, management and climate. In general, conversion of natural soils to agricultural use reduces their sink strength for CH4 and enhances their N2O emission (Boeckx et al., 1998; Smith et al., 2000). The processes of CH4 oxidation and N2O production are strongly interconnected via soil N transformations. Ammonium is an important inhibitor of CH4 oxidation (e.g. Bender and Conrad, 1994; King and Schnell, 1994; Boeckx and Van Cleemput, 1996; HuÈtsch et al., 1996). However, in some cases, no immediate effect was observed upon addition of NH41 (Dobbie and Smith, 1996; Delgado and Mosier, 1996; Gulledge et al., 1997). The mechanism of NH41 inhibition is complex and thought to include both a competitive inhibition of the CH4 mono-oxygenase (MMO) enzyme, as well as a non-competitive (toxic) inhibition by hydroxylamine
0038-0717/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0038-071 7(01)00232-2
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Table 1). Soil A had a medium (loam) texture and a CEC of 8.4 cmol(1) kg 21 and soil B had a coarse (loamy sand) texture and a CEC of 4.7 cmol(1) kg 21.
Table 1 Characteristics of the investigated soils
General Location Land use Soil Texture Clay (%) Sand (%) Loam (%) pH-H2O NH41 (mg N kg 21) NO32 (mg N kg 21) FC (%) CEC (cmol(1) kg 21) CaCO3 (%) Organic matter (%) a b
Soil A
Soil B
51810 0 N, 2855 0 E Arable
5185 0 N, 3831 0 E Arable
a
Loam , medium 20 47 33 7.6 0.31 0.23 26.4 8.4 1.03 2.54
b
a
Loamy sand , coarse 4 87 9 6.7 0.44 1.49 23.4 4.7 0.26 2.92
2.2. Soil conditioning
b
US Department of Agriculture textural classes. FAO textural classes.
(NH2OH) or nitrite (NO22) produced via NH41 oxidation (King and Schnell, 1994). Moreover, additional noncompetitive inhibition resulting from a general salt effect has also been reported (MacDonald et al., 1997; Gulledge and Schimel, 1998). De Visscher et al. (1998) demonstrated that the amount of exchangeable NH41 could be another important factor controlling the inhibitory effect of NH41. The effect of NO32, another product of NH41 oxidation, on CH4 oxidation is ambiguous. Some authors did not observe an effect of NO32 (Boeckx and Van Cleemput, 1996), while others observed the contrary (Whalen, 2000). It has also been suggested that N-turnover rates (mineralisation and gross nitri®cation) (Mosier et al., 1991; Steudler et al., 1996) in¯uence CH4 uptake rather than the actual soil mineral N (NH41, NO32 or NO22) content. We studied the relationship between, and controlling factors of, CH4 oxidation and N2O emission by aerobic soils receiving NH41. Firstly, the short-term (24 h) effect of NH41 applications, ranging from 5 to 100 mg NH41 ± N kg 21, on CH4 oxidation and N2O emission by two different arable soils was investigated. Secondly, the mediumterm (30 d) effect of addition of 100 mg NH41 ±N kg 21 on CH4 oxidation, N2O emission and nitri®cation was tested in the same arable soils.
2. Materials and methods 2.1. Sites and soils The CH4-oxidising capacity and N2O emission were investigated for two arable soils (soil A: 51810 0 N, 2855 0 E and soil B: 5185 0 N, 3831 0 E). The sites were comparable in terms of climatic conditions (temperate), land use (arable soil), pH, organic matter and inorganic N content, but had a different texture and cation exchange capacity (CEC) (see
The soil samples were taken from the surface (0±15 cm) and were air-dried, ground and sieved (2 mm) before use. The effect of NH41 on CH4 and N2O ¯uxes was investigated using batch incubation experiments. To stimulate the methanotrophic community, the soils were re-wetted up to 75% ®eld capacity (FC), being optimal for CH4 oxidation (Boeckx and Van Cleemput, 1996). Thereafter, the soils were placed in Plexiglas cylinders and were continuously fed with pure CH4 from the base. Methane was introduced at a rate of 5 ml min 21 to obtain a CH4 input of 10 mol CH4 m 22 d 21. The headspace was continuously ¯ushed with moist air (11 min 21) to prevent drying of the soil. More details concerning the set-up can be found in De Visscher et al. (1999). At regular intervals gas samples (100 ml) were taken from the outlet of the cylinders during the 10 d of conditioning and analysed for CH4 in order to estimate the CH4-oxidising activity of the soils. A similar conditioning experiment was conducted using ambient CH4 concentrations. 2.3. NH41 ®xation The short-term NH41 ®xation capacity of both soils was estimated. Ammonium sulphate ((NH4)2SO4) solutions (0.5 ml) were added to the soil samples (equal to 30 g dry weight) using the same N concentration range as used in the short-term incubation experiment (5±100 mg NH41 ± N kg 21, see later). The samples were mixed carefully and equilibrated for 2 h at room temperature. Then, the soil samples were extracted with 2 M KCl and analysed for NH41 ±N. 2.4. Experimental design The following experimental design was used to determine the short-term effect of NH41 on CH4 and N2O ¯uxes. Soil samples (equal to 30 g dry weight) were transferred from the conditioning cylinders into 200 ml bottles. Ammonium sulphate was dissolved in distilled water and added to the soil in 0.5 ml and carefully mixed to ensure homogeneous distribution of NH41. The following concentrations were used: 5, 10, 25, 50 and 100 mg NH41 ±N kg 21 soil. Addition of 0.5 ml of distilled water served as a control treatment. If necessary, additional water was added to maintain 75% FC. All experiments were carried out in triplicate and at room temperature (22±23 8C). The bottles were sealed with rubber stoppers and CH4 was added up to an initial concentration of 10 nl CH4 ml 21 in the headspace. Methane uptake and N2O emission was determined simultaneously by analysing the headspace of the bottles 0, 2, 4, 6, 8 and 24 h after CH4 addition. Zero time samples were taken
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5 min after CH4 addition to allow uniform diffusion of the added CH4. A separate set of samples was prepared to evaluate the medium-term (30 d) effect of NH41 (100 mg NH41 ±N kg 21) on CH4 oxidation, N2O emission and nitri®cation. Gas analysis (0, 2, 4 and 6 h after CH4 addition) and analysis of the mineral N content (NH41, NO32 1 NO22 and NO22) of the soil were performed daily during the ®rst week of the incubation and once or twice a week later on. Triplicate treatments were used for soil A and B. Changes in the water content were determined gravimetrically during the incubation and losses were adjusted when necessary. During the short-term experiment, CH4 oxidation followed ®rst order kinetics: dCH4 =dt k1 £ CH4 : From the slope of the log-transformed CH4 concentrations as a function of time, the CH4 oxidation rate constants (k1, h 21) could be calculated via linear regression. The CH4 oxidation rates were calculated by multiplying k1 with the CH4 concentration at time zero. A t-test a 0:01 was used to identify signi®cantly different oxidation rates between the different treatments. The NH41 inhibition constant (ki) was de®ned as the NH41 concentration, that reduced the initial oxidation rate constant (k1) (no NH41 added) by 50%. The ki values were calculated using an optimisation procedure in Solver in Microsoft Excel, using the measured CH4 oxidation rate constants as a function of the applied NH41 concentrations. During the medium-term experiment the decrease of the CH4 concentration was linear during each gas sampling period of 6 h. Thus, zero order kinetics dCH4 =dt k0 were applied to calculate the CH4 oxidation rates. A t-test a 0:01 was used to identify signi®cantly different oxidation rates between the different treatments. The increase of N2O concentration was linear (R 2 $ 0.95) during the ®rst 6±8 h of the incubation in the short- and medium-term experiment. Nitrous oxide ¯uxes were expressed using zero order kinetics: dN2 O=dt k0 and N2O ¯uxes were estimated via linear regression analysis. A t-test a 0:01 was used to identify signi®cantly different oxidation rates between the different treatments. 2.5. Measurements The CH4 concentrations were measured using a Chrompack CP9000 gas chromatograph (Chrompack, The Netherlands) equipped with a ¯ame ionisation detector and a NaI aluminium column. Helium was the carrier gas (37 ml min 21) and the settings were as follows: injector temperature 65 8C, oven temperature 55 8C and detector temperature 200 8C. Nitrous oxide was determined using a Chrompack 437A gas chromatograph (Chrompack, The Netherlands) with a stainless steel packed column (Chromosorb 102) and a 63Ni electron capture detector. The following conditions were used: injector temperature 90 8C, oven temperature 90 8C and detector temperature 300 8C. Dinitrogen was the carrier gas (32 ml min 21). The concen-
671
trations of CH4 and N2O were calculated from the peak area in the chromatograms, which were registered and analysed using the WOW software package (Thermo Separation Products, USA). Soil analyses (see Table 1) were carried out after the soil conditioning procedure. The soil moisture content was determined gravimetrically (drying at 105 8C). Soil pH was determined in soil/water extractions (1/5 w/w) using a combined pH electrode connected to a C832 multichannel analyser (Consort, Belgium). Inorganic N compounds were extracted from the soil samples by adding 2 M KCl (soil/KCl ratio 1:2). The extracts were analysed for NH41 and (NO32 1 NO22) via steam distillation of the extract and subsequent titration (Keeney and Nelson, 1982). Nitrite was determined separately using the diazotisation method (HACH, 2000). 3. Results 3.1. Effect of elevated CH4 concentrations During the conditioning incubation at elevated CH4 concentrations, soils A and B showed a substantial increase of the CH4 uptake. Methane uptake in the soil cylinders stabilised after 5±7 d at about 3±4 mol CH4 m 22 d 21 for both soils. The CH4 uptake rates were signi®cantly higher compared to soils conditioned under ambient CH4 concentrations (P , 0.01). In a short-term batch experiment (identical set-up as in the short-term experiments assessing the effect of NH41, except that no NH41 was applied) the headspace CH4 concentrations decreased from about 10 nl CH4 ml 21 to 2.1±2.3 nl CH4 ml 21 and to 7 nl CH4 ml 21 in activated and non-activated soils, respectively (Fig. 1). 3.2. Short-term effect of NH41 on CH4 oxidation and N2O emission From Fig. 2 it is clear that addition of NH41 caused a reduction of the CH4 oxidation rates of soil A and B for all tested NH41 concentrations (5±100 mg NH41 ±N kg 21). The extent of the inhibitory effect of NH41 depended on its concentration. In soil A, treatments with 10±100 mg NH41 ± N kg 21 showed signi®cantly (P , 0.01) lower CH4 oxidation rates than the control (no NH41 added). This effect occurred for all amounts of NH41 added to soil B. Soil A and B showed clear differences in N2O emission rates as a function of the NH41 application (Fig. 2). The N2O ¯uxes increased upon increasing NH41 addition. Soil A showed the highest N2O ¯uxes. Addition of 100 mg NH41 ±N kg 21 gave a N2O ¯ux of 17.6 ^ 2.4 ng N2O g 21 h 21. In soil B, addition of the same amount of NH41 resulted in a much lower N2O emission, 2.8 ^ 0.3 ng N2O g 21 h 21. Moreover, soil A showed slightly different CH4 oxidation kinetics as a function of the NH41 content compared to soil B. In Fig. 3 the k1 and ki values of soil A and B are shown. The ki value was higher in soil A (22.2 mg NH41 ±N kg 21) than in soil B (12.4 mg NH41 ±N kg 21). Similarly, the CEC
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Fig. 1. Change in headspace CH4 concentrations during the incubation of soil A and B. Samples were conditioned at elevated or at ambient CH4 concentrations. Error bars represent standard deviations n 3:
of soil A is a factor twofold than the CEC of soil B (Table 1). Fig. 4 shows the degree of extractable NH41 of soil A and B. In soil A, only 54±99%, depending on the initial NH41 concentration, of the added NH41 could be extracted 2 h after NH41 addition. In contrast, in soil B the added NH41 was extracted completely for all concentrations. In Fig. 5 the relation between the CH4 oxidation rates and the N2O emission rates in soil A and B is shown. A signi®cant (P , 0.01) linear correlation (R2 0:73 and 0.68 for soil A and B, respectively) between CH4 and N2O ¯uxes could be observed. In both cases the slope was signi®cantly different from zero (P , 0.01). 3.3. Medium-term of NH41 on CH4 oxidation, N2O emission and nitri®cation The effect of NH41 supply on CH4 oxidation, N2O emission and nitri®cation dynamics were investigated in a medium-term incubation experiment (30 d). In soil A, NH41 addition caused a strong inhibition of the CH4
oxidation until d 1. Thereafter, the CH4 oxidation increased again and was practically restored between d 3 and 8, reaching comparable CH4 oxidation values as in the control soil (Fig. 6A). Both in the control and the NH41 treatment, the CH4-oxidising capacity started to decrease after d 8. In soil A, the N2O ¯ux was maximum at d 1 (42.8 ng N2O g 21 h 21) and declined sharply thereafter (Fig. 6B). In the control soil, the N2O emission remained low throughout the entire 30 d of the incubation (Fig. 6B). Nitri®cation of the added NH41 resulted in a signi®cant increase in NO32 1 NO22 concentrations during the ®rst 10 d (P , 0.01) of the incubation (Fig. 6C and D). In soil A, the NO22 concentrations were always much lower than the NO32 concentrations (Table 2). The highest NO22 content was observed on d 1. Soil B behaved completely different from soil A. A strong inhibition of the CH4 oxidation (about 90%) was observed during the entire medium-term incubation, despite the fact that more than 92% of the added NH41 was nitri®ed after 14 d (Fig. 7A and C). In the control treatment, the
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673
Fig. 2. The in¯uence of NH41 addition on the CH4 oxidation rates and the N2O emission in soil A and B. Each data point represents the average ^ standard deviations n 3:
CH4-oxidising capacity started to decrease after d 8, as was the case for soil A. The N2O emission remained low in the NH41 and control treatment during the entire incubation. Nitri®cation started after a lag phase of 4 d (Fig. 7C and D). The NH41 content started to decrease on d 5, while a parallel, but opposite, trend was observed for the NO32 1 NO22 content. In soil B, the NO22 concentrations were always much lower than the NO32 concentrations (Table 2).
4. Discussion 4.1. Effect of elevated CH4 concentrations The CH4 oxidation rates for soils conditioned at ambient CH4 concentrations were 0.95 ^ 0.13 ng CH4 g 21 h 21 and 0.90 ^ 0.07 ng CH4 g 21 h 21 for soil A and B, respectively. The measured oxidation rates were higher than those
reported by other authors using fresh samples of agricultural soils. Methane oxidation rates of 0.013 ng CH4 g 21 h 21 (Tlustos et al., 1998) and 0.19±0.31 ng CH4 g 21 h 21 (HuÈtsch, 1998) were determined in arable soil in the UK and Germany, respectively. In soils conditioned at elevated CH4 concentrations, the oxidation rates increased approx. fourfold and were 3.64 ^ 0.24 ng CH4 g 21 h 21 and 3.58 ^ 0.40 ng CH4 g 21 h 21 in soil A and B, respectively. Exposure to high CH4 concentrations was found to raise the atmospheric CH4 uptake in agricultural soils (Bender and Conrad, 1992; Jensen and Olsen, 1998), however, not in undisturbed natural forest soils (Jensen and Olsen, 1998). Although soil texture was found to be an important factor for CH4 oxidation (DoÈrr et al., 1993), the soils in this study showed a similar CH4 uptake rate despite differences in texture (Table 1, Fig. 1). This could be explained by the fact that, in our experimental design the soils were loosely packed and that gas diffusion is not expected to be a limiting factor (Boeckx and Van Cleemput, 1996).
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Fig. 3. In¯uence of different NH41 concentrations on the CH4 oxidation rate constants (k1) in soil A and soil B. Arrows indicate the NH41 concentrations that decreased the k1 value to 50% (ki-values) in soil A and B.
4.2. Short-term effect of NH41 on CH4 oxidation and N2O emission In Fig. 2 a non-linear relation between the CH4 oxidation rates and the NH41 content of the soil are shown. Studies using different types of soil have reported a negative linear (Boeckx and Van Cleemput, 1996) or sigmoõÈd (Kravchenko, 1999) correlation between CH4 oxidation rates and the NH41 content. Rapid inhibition of CH4 oxidation upon NH41 application has been well documented. This has been attributed to the combined effect of substrate competition at the level
of the MMO (Bosse et al., 1993; HuÈtsch et al., 1993; Castro et al., 1995) and non-competitive inhibition (King and Schnell, 1994; Gulledge and Schimel, 1998). Moreover, it has been suggested that CEC could be an important factor controlling CH4 oxidation (De Visscher et al., 1998). The degree of extractable NH41 is lower in soil A than in soil B, especially when low NH41 doses were applied (Fig. 4). This effect might partly explain the higher ki value calculated for soil A as compared to soil B (Fig. 3). The ki value corresponds to the NH41 addition that is needed to reduce the CH4 oxidation (k1 values) by 50%. Since, the
Fig. 4. Fraction of NH41 extracted from soil A and soil B as a function of added NH41 concentrations. NH41 salts (5±100 mg NH41 ±N kg 21) were added to the soils and extracted after 2 h of equilibration.
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675
Fig. 5. Relationship between CH4 uptake and N2O emission in soil A and soil B based on 24 h incubations with different NH41 applications (0±100 mg NH41 ± N kg 21).
amount of extractable NH41 is partly affected by the CEC of the soil, this might be re¯ected in the extent of the inhibitory effect of NH41 on the CH4-oxidising capacity. However, the CEC or the degree of extractable NH41 could not explain the observed N2O emission trends in soil A and B. From the experimental design (aerobic conditions plus NH41 addition) and from the nitri®cation results in
the medium-term experiment (see Figs. 6C,D and 7C,D) it was evident that N2O must originate from nitri®cation and not denitri®cation. From the medium-term experiment (see Fig. 7C and D) is was also clear that in soil B the nitri®cation activity showed a lag-phase of 4 d. Therefore, the nitri®cation might not be at the optimum rate in the short-term experiment of soil B, which lasted only 24 h. These results
Fig. 6. Changes in CH4 oxidation rates (A) N2O emission rates, (B) NH41 concentrations, (C) NO32 1 NO22 concentrations, (D) during an incubation of 30 d of soil A. Error bars represent the standard deviation n 3:
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Table 2 NO22 ±N concentrations (mg N kg 21) in soil A and soil B in the mediumterm CH4 oxidation experiment (ND not determined)
NH41 results in more N2O emission and more inhibition of the CH4 oxidation.
Day
Soil A
Soil B
1 3 5 8 11 16
1.06 0.36 0.36 0.41 ND ND
ND ND 0.31 0.43 0.38 0.48
4.3. Medium-term of NH41 on CH4 oxidation, N2O emission and nitri®cation
were con®rmed in a short-term NO emission experiment. The NO emission of soil A was sevenfold higher than that in soil B (data not shown). It is suggested that this lag-phase effect might be related to a difference in the structure or response of the microbial community in soil A and B. Moreover, in soil A (medium texture), N2O could additionally be produced from denitri®cation or via nitri®er denitri®cation in anoxic micro-sites. Correlation analysis between the CH4 oxidation rates and the N2O ¯uxes for soil A and B showed a clear negative relation (R2 0:73 for soil A and R2 0:68 for soil B) (Fig. 5). This clearly demonstrates that a higher availability of
Methane oxidation, N2O emission and nitri®cation dynamics were also investigated in the medium-term incubation experiment. HuÈtsch (1998) presented comparable results for a silt loam soil. During the ®rst day of the incubation of soil A, the CH4 oxidation capacity was considerably decreased (Fig. 6A). On d 1, also the highest NO22 ±N concentrations were found in soil A (Table 2). Nitrite is strongly toxic with respect to CH4 oxidation. As such, NO22 might at least partly be responsible for the observed inhibitory effect. However, the inhibition effect of residual NH41 concentrations during the medium-term experiment was lower, compared to the same initial NH41 concentrations in the short-term experiment. For example, it was only 9.6% after 3 d (54.4 mg NH41 ±N kg 21 remaining) as compared to 56.8% for the 50 mg NH41 ±N kg 21 dose in the shortterm experiment. Moreover, soil A showed a varying CH4oxidising activity, which after reaching a maximum, decreased signi®cantly between d 8 and 20 (Fig. 6A). Similar
Fig. 7. Changes in CH4 oxidation rates (A) N2O emission rates, (B) NH41 concentrations, (C) NO32 1 NO22 concentrations, (D) during an incubation of 30 d of soil B. Error bars represent the standard deviation n 3:
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losses of the CH4 oxidising activity have been observed in laboratory experiments using forest soils (Amaral et al., 1998). Both phenomena may re¯ect a changing methanotrophic community. We propose that the methantrophic community might have been increased during the conditioning at high CH4 concentrations, leading initially to a recovery of the CH4 oxidation. The loss of the CH4oxidising activity during incubations at lower CH4 concentrations and high NH41 concentrations could be the result of a decreasing methanotrophic community size or activity. The N2O emission showed a peak on d 1 and decreased thereafter (Fig. 6B). Thus, a relationship between the CH4 oxidation rate and N2O emission could also be seen. In soil A the net nitri®cation rate was calculated by linear regression analysis of the increase of NO32 1 NO22 concentrations from d 0 to 7 and was 10.5 mg N kg 21 d 21 (Fig. 6D). These results suggest that soil A contained an active nitrifying community and substrate addition caused an immediate response. It has been shown that NH41 application to arable soil caused a rise in the nitrifying activity but did not change the size of NH41-oxidising community (Mendum et al., 1999). In soil B a persistent inhibition of the CH4 oxidation could be observed upon NH41 addition. The reason for this persistent inhibition is not clear. It could not be explained by the NO22 accumulation during the medium-term incubation (Table 2). Whalen (2000) has also observed the existence of a persistent reduction in CH4 oxidation in similar laboratory studies. It has been proposed by MacDonald et al. (1997) and Whalen (2000) that such a medium-term effect could be explained by a non-speci®c salt effect of NH4(SO4)2 addition, rather than by the competition between NH41 and CH4. Also, in this experiment soil B showed low N2O emission rates compared to soil A. The net nitri®cation rate in soil B (between d 4 and 11) was 8.3 mg N kg 21 d 21. The rise in nitri®cation rates after 4 d (lag-phase) may suggest a phenotypic or size change of the communities of nitrifying or methanotrophic microorganisms upon NH41 addition. The NH41 was nitri®ed almost completely and the NO32 1 NO22 concentration (about 100 mg N kg 21) remained virtually constant thereafter. The latter could eventually explain the absence of a recovery of CH4 oxidation, because in some cases, persistent inhibition of CH4 oxidation has been observed upon NO32 addition (Prieme and Christensen, 1997; Whalen, 2000), in contradiction to experiments where no effect of NO32 could be observed (Boeckx and Van Cleemput, 1996). 4.4. Conclusions From the short-term (24 h) experiment it was clear that NH41 addition was a key factor controlling the interaction between CH4 oxidation and N2O emission in both arable soils. Ammonium was found to act as a competitive inhibitor for CH4 oxidation in both arable soils. It is suggested that the CEC and the NH41 extractability might affect the CH4
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oxidation kinetics. However, the microbial community structure or response might be even more important for the observed CH4 and N2O emissions. From the medium-term experiment it became clear that the effect of NH41 on CH4 oxidation was different when a certain concentration was added directly (short-term effect) or reached after a certain period upon addition of an initially higher NH41 concentration (medium-term effect). The nitrifying activity of soil A and B responded differently to NH41 additions. In soil B, nitri®cation showed a lag phase. Moreover, soil A and B showed a different course for CH4 oxidation and N2O emission. All these observations cannot be explained entirely by the physical-chemical properties of both soils. Therefore, it is suggested that the microbial communities present in soil A and B, responded or developed differently to the observed NH41, NO32, NO22 or CH4 contents. Thus, an important future goal in explaining the mechanisms of CH4 oxidation and N2O emission in soils could be the identi®cation of the methanotrophic and nitrifying communities or diversity in soils. Acknowledgements The ®nancial support of the Belgian Of®ce for Scienti®c, Technical and Cultural Affairs for the research fellowship of Irina Kravchenko is highly appreciated. References Amaral, J.A., Ren, T., Knowles, R., 1998. Atmospheric methane consumption by forest soils and extracted bacteria at different pH values. Applied and Environmental Microbiology 64, 2397±2402. Bender, M., Conrad, R., 1992. Kinetics of CH4 oxidation in oxic soils exposed to ambient air or high CH4 mixing ratios. FEMS Microbiology Ecology 101, 261±270. Bender, M., Conrad, R., 1994. Microbial oxidation of methane, ammonium and carbon monoxide, and turnover of nitrous oxide and nitric oxide in soils. Biogeochemistry 27, 97±112. Boeckx, P., Van Cleemput, O., 1996. Methane oxidation in a neutral land®ll cover soil: in¯uence of moisture content, temperature and nitrogenturnover. Journal of Environmental Quality 25, 178±183. Boeckx, P., Van Cleemput, O., Meyer, T., 1998. The in¯uence of land use and pesticides on methane oxidation in some Belgian soils. Biology and Fertility of Soils 27, 293±298. Bosse, U., Frenzel, O., Conrad, R., 1993. Inhibition of methane oxidation by ammonium in the surface layer of littoral sediment. FEMS Microbiology Ecology 13, 123±134. Castro, M.S., Steudler, P.A., Melillo, J.M., Aber, J.D., 1995. Factors controlling atmospheric methane consumption by temperate soils. Global Biogeochemical Cycles 9, 1±10. De Visscher, A., Boeckx, P., Van Cleemput, O., 1998. Interaction between nitrous oxide formation and methane oxidation in soils: in¯uence of cation exchange phenomena. Journal of Environmental Quality 27, 679±687. De Visscher, A., Thomas, D., Boeckx, P., Van Cleemput, O., 1999. Methane oxidation in simulated land®ll cover soil environments. Environmental Science and Technology 33, 1854±1859. Delgado, J.A., Mosier, A.R., 1996. Mitigation alternatives to decrease
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