Eect of NH
4
Cl addition on methane oxidation by paddy soils
Zucong C. Cai
a,*, Arvin R. Mosier
ba
Institute of Soil Science, Chinese Academy of Sciences, P.O. Box 821, Nanjing, People's Republic of China
b
USDA/ARS, P.O. Box E, Ft Collins, CO 80522, USA
Accepted 23 February 2000
Abstract
Methane emission from ¯ooded rice ®elds is a net product of CH4 production and oxidation. The ability of paddy soils to
oxidize CH4 produced endogenously is an important natural feature for mitigating CH4 emission to the atmosphere. We
conducted a series of studies on CH4oxidation by two fresh paddy soils (Wuxi soil and Yingtan soil). The soils were amended
with NH4Cl at rates equivalent to 0, 10 and 50 mg N gÿ1 soil, respectively, and incubated over ®ve consecutive periods at
elevated CH4 concentrations of0500,01000,01500 or02000 ml lÿ1 in the headspace, respectively, at 258C. NH4Cl addition
inhibited the rates of CH4 oxidation by the paddy soils at all initial CH4 concentrations during the ®rst incubation. The
inhibitory eect was strengthened with increasing NH4Cl addition and alleviated with increasing initial CH4 concentration, i.e.
there was an interaction of NH4Cl and initial CH4concentration on the inhibitory eect. If the initial CH4 concentration was
suciently high (above02000ml lÿ1
for Wuxi soil and01000ml lÿ1
for Yingtan soil), the initial inhibitory eect was alleviated and then eliminated during subsequent incubations. Eventually, NH4Cl addition stimulated the CH4oxidation rate. If the initial
CH4 concentration was not suciently high (less than 500 ml lÿ1 in the headspace for both soils), the inhibitory eect was
maintained during subsequent incubations. Considering the fact that the CH4 concentration in paddy soils when they are
¯ooded is generally higher than02000ml lÿ1, we conclude that the initial eect of NH
4Cl addition on CH4oxidation in paddy
soils is temporary.72000 Elsevier Science Ltd. All rights reserved.
1. Introduction
Methane emission from ¯ooded rice ®elds contrib-utes up to 12% of global CH4 emission to the atmos-phere (IPCC, 1992). The increase in ¯ooded rice harvested area is considered to be one of the factors responsible for the continuous increase in the atmos-pheric CH4concentration. It is well-known that only a portion of the CH4 produced in ¯ooded rice ®elds escapes into the atmosphere. The remainder is oxidized in oxic sites, such as interfaces between the ¯oodwater and the soil surface, and rhizospheres. The proportion of CH4 oxidized in micro-oxic sites in ¯ooded rice ®elds to that produced varies from 39 to 92% (Hol-zapfel-Pschorn et al., 1985; Galchenko et al., 1989; Sass et al., 1991; Frenzel et al., 1992). The oxidation
of endogenously produced CH4 is also important in the CH4 emissions from swamps, lakes and seas (Gal-chenko et al., 1989).
It has been demonstrated that CH4emissions from ¯ooded rice ®elds are aected by N fertilization (SchuÈtz et al., 1989; Lindau et al., 1991; Cai et al., 1997), however, the results are in con¯ict with each other. Lindau et al. (1991) observed that application of urea stimulated CH4 emission from ¯ooded rice ®elds in LA, USA, but SchuÈtz et al. (1989) and Cai et al. (1997) found that urea depressed CH4 emission. Cicer-one and Shetter (1981) reported that the CH4 ¯ux from rice paddies supplied with ammonium sulfate was ®ve times higher than that from plots without N appli-cation. Since then, most results show that application of ammonium sulfate decreases CH4 emission (SchuÈtz et al., 1989; Kimura et al., 1992; Cai et al., 1997). N fertilization should aect both CH4 production and oxidation in ¯ooded rice ®elds through NH4+ and counter-ions, such as SO42ÿand CO32ÿ. The response of
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* Corresponding author.
rice plant growth to N fertilization would also aect CH4production and oxidation through the changes in the releases of exudates as carbon sources of CH4 pro-duction and oxygen required for CH4oxidation. Com-pared to the con¯icting results of changes in CH4 ¯uxes from rice ®elds by N fertilization, we know even less about how N fertilizers in¯uence CH4production and oxidation individually in rice ®eld soils.
Laboratory and ®eld studies have demonstrated that NH4+ inhibits CH4oxidation in forest, grassland, arable and paddy soils (Steudler et al., 1989; Mosier et al., 1991, Nesbit and Breitenbeck 1992; HuÈtsch et al., 1993; Flessa et al., 1995; Cai and Yan, 1999). Though at least one soil slurry incubation at high CH4 concen-trations showed that the inhibition of NH4+ addition on CH4oxidation was completely reversible (Dun®eld and Knowles, 1995), most ®eld measurements and lab-oratory incubations showed that the inhibition of NH4+ addition on the oxidation of CH4at the atmos-pheric concentration or at low concentration was maintained even after the disappearance of added NH4
+
(Mosier et al., 1991; Nesbit and Breitenbeck, 1992). Mosier et al. (1996) found that sandy loam soils in shortgrass steppe that was fertilized with nitrogen, 5±13 years earlier, consumed 30±40% less CH4 than unfertilized soils. Aerobic soils might completely lose their ability to consume the atmospheric CH4 after long-term application of ammonium N fertilizers inde-pendent of the mineral N content of the soil at the time of measurement (HuÈtsch et al., 1993; Powlson et al., 1997). Similar to upland soils, laboratory exper-iments demonstrated that NH4+ addition depressed CH4 oxidation by paddy soils and the eect increased with increasing N addition concentration (Conrad and Rothfuss, 1991; Cai and Yan, 1999). However, no report has shown that paddy soils lose their ability to oxidize endogenously produced CH4, even though the soils are usually supplied with ammonium based N fer-tilizers continuously for rice production.
This study was conducted to determine if rice ®eld soils that have been N-fertilized for many years con-tinue to oxidize methane or if concon-tinued cropping with N-fertilization decreased CH4oxidation as observed in upland soils. A series of incubations were conducted with a variety of NH4Cl and CH4 concentrations to represent the water management practices (¯ooding and drainage) that are used in the rice ®elds from which soils were collected for this study.
2. Materials and methods
2.1. Soils
Two typical paddy soils, collected from rice ®elds on 1 September 1998 while rice was growing, were used
for this experiment. One paddy soil was collected from a rice ®eld in Wuxi (31836'N, 120824'E), Jiangsu, China. The ®eld is rotated between rice and winter wheat and fertilized with nitrogen at a rate of ca. 300 kg N haÿ1 per crop season. Urea, ammonium
bicar-bonate and complex fertilizers with NH4 +
±N, NH4 +
±K and NH4+±P are the main N fertilizers. Phosphorus and potassium were applied to rice ®elds at rates of about 46 kg P haÿ1 and 22 kg K haÿ1, respectively.
The other paddy soil was collected from a rice ®eld located at the bottom of a slope in Yingtan (28812'N, 11786'E), Jiangxi, China. This ®eld is planted with double rice crops and usually fallowed in winter sea-son. Chemical fertilizers are typically applied for each rice crop at the rate of about 150 kg N haÿ1
as urea, 31 kg P haÿ1
as superphosphate and 71 kg K haÿ1 as potassium chloride, of which 70% is applied as a basal and 30% as single topdressing. This soil was used to evaluate salt eects on CH4oxidation by Cai and Yan (1999). Soil samples were collected from ®ve points distributed randomly in the ®eld and then mixed. The soil samples were collected from the plough layer of each ®eld, and the distinction between plow layer and plow pan was identical visually. The depth of the plough layer was about 15 cm. In the laboratory, fresh soils were passed through an 8-mm sieve and mixed well for use. The major properties of the soils are listed in Table 1.
2.2. Consecutive incubation
The rice ®elds in China are usually intermittently irrigated and multiple mid-season aeration is com-monly employed. With such water management, elev-ated CH4 supplies for methanotrophic bacteria are also intermittent. For simulating the pattern of CH4 supplies in ¯ooded rice ®elds, the headspace gas was renewed after a given period of incubation in this ex-periment. Twenty-®ve gram samples of soil (oven-dry basis) were incubated in closed jars (470 ml). The treatments imposed were CH4 concentration (4 rates)
NH4Cl addition (3 rates). Designed initial CH4 con-centrations in jar headspace were 500 (M1), 1000 (M2), 1500 (M3) and 2000 (M4) ml lÿ1
; and NH4Cl ad-dition rates were equivalent to 0 (N0), 10 (N1) and 50 (N2) mg N gÿ1
Incu-bations were carried out at a constant room tempera-ture of 258C. Five milliliters of headspace gas was sampled by a 5-ml syringe with a nylon stopcock for analyses of CO2, N2O and CH4concentrations. Before each sampling, 5 ml standard gas having CO2, N2O and CH4 concentrations of 406, 0.447 and 1.97ml lÿ
1 , respectively, was injected into the headspace to keep pressure constant inside jars after sampling. When headspace gases were renewed, incubation jars were opened and ¯ushed with compressed air for 30 s, and then covered, and headspace CH4 concentration was re-adjusted by pure CH4 gas to the same initial con-centration as in the ®rst period. Exact initial CH4 con-centration of each period was determined immediately after injection of pure CH4 (taken as zero time for each incubation period). The average of the initial CH4concentrations of ®ve incubation periods (average 2standard deviation) were 48428, 1030219, 15462
47 and 2084275 ml lÿ1
for Wuxi soil and 490213, 1028232, 1572240 and 2063256ml lÿ1 for Yingtan
soil, respectively. The mean rates of CH4 oxidation, CO2and N2O evolutions in the ®rst 19 h incubation of each consecutive incubation period were calculated by the dierences between their concentrations at the onset and at 19 h after each incubation. The reasons for using the mean rates of the ®rst 19 h incubation were for the comparisons of the rate changes in the consecutive incubation periods and avoiding the underestimation of CO2evolution rates because a feed-back of CO2 evolution was observed when CO2 con-centration buildup was very high in the headspace. Table 2 gave the mean rates in the ®rst and ®fth incu-bation periods.
2.3. CH4, N2O, CO2and ancillary measurements
Gas samples were analyzed using a gas chromato-graph equipped with ¯ame ionization detector for CH4 and with electron capture detector for N2O and CO2 (Mosier et al., 1991).
After the consecutive incubation at elevated CH4 concentration, the soil was incubated under atmos-pheric CH4concentration for 146 h (the results are not discussed in the present paper). Then, the soils were extracted with 2 mol lÿ1KCl for analyses of
extracta-ble NH4+ and NO3ÿ. The extracts were analyzed by using a ¯ow injection analyzer.
2.4. Simulation of CH4oxidation by a kinetic model
The plots of logarithm of CH4 concentration in the headspace versus incubation hours were not straight lines and the deviations from the straight lines became larger and larger with increasing initial CH4 concen-tration during the ®rst incubation period (data not shown). The increase in the mean CH4 oxidation rate of the ®rst 19 h incubation with the increase in the number of the consecutive incubation period (Table 2) also indicated that the CH4 oxidation activity was stimulated by the elevated CH4 concentration. This suggested that the CH4 oxidation by paddy soils did not follow ®rst-order reaction kinetics, which assumes that the activity is a constant. It has been well docu-mented that the community of CH4oxidizers increases under high CH4concentration (Segers, 1998). The pat-tern of CH4oxidation with changes in the community of CH4 oxidizers is successfully described by the kin-etic model introduced by Cai and Yan (1999). This model, which de®nes the CH4oxidation activity as the product of the CH4 oxidation activity of individual bacteria (k) and the number of bacteria (m), ®ts the time course of CH4 oxidation during the ®rst period very well. The minimum correlation coecients between measured and simulated values were 0.9995 (P< 0.001) for Wuxi soil and of 0.9997 (P< 0.001) for Yingtan soil. Due to the limitation of sampling times, the time courses of CH4 oxidation were not simulated by the model in the subsequent incubation periods.
For testing the signi®cance of the initial CH4 con-centration and NH4Cl addition on CH4 oxidation by the paddy soils, the two-factor analysis of variance with the initial CH4concentration and NH4Cl addition was employed in this study.
3. Results
3.1. Eect of CH4concentration on CH4oxidation
Before elevated CH4 incubation, an incubation was
Table 1
Major properties of paddy soils studied
Soil Soil moisture (%WHC) Organic C (mg gÿ1DW) Total N (mg gÿ1DW) KCl extractable N (mg gÿ1)
NH4 +
±N NO3ÿ±N
Wuxi 71 27.9 2.7 2.1 6.1
carried out, which showed that both soils could not oxidize CH4 under ambient atmospheric concen-trations (data not shown). However, they were able to consume CH4 at elevated CH4 concentrations. The CH4 concentration in the headspace declined rapidly with incubation time. Fig. 1 gives examples of the time courses of CH4 oxidation by the soils amended with NH4Cl with the initial CH4 concentration of0500 ml lÿ1(M1). At the end of the ®rst period (139 h), the
CH4 concentration remaining in the headspace fell below the atmospheric CH4 concentration in the treat-ments M3N0, M4N0 and M4N1 for Wuxi soil. But for Yingtan soil, there was no treatment in which CH4 concentration in the headspace fell below the atmos-pheric concentration (data not shown). Simulating the experimental data by the kinetic model introduced by Cai and Yan (1999) showed that the initial CH4 oxi-dation activities did not dier signi®cantly among treatments in the same soil and between two soils (0.01520.004 hÿ1for Wuxi soil and 0.01520.004 hÿ1
for Yingtan soil, P > 0.05). However, the activities increased quickly with incubation time and were in¯u-enced by the initial CH4concentration. At the end of the ®rst incubation, the activity increased with the increase in the initial CH4concentration (Fig. 2). Due to the increases in the CH4 oxidation activities of the soils, the mean CH4 oxidation rates increased in the consecutive incubation at the same initial CH4 concen-trations (Table 2). The CH4oxidation rates became so high that the CH4 concentration remaining in the headspace went down below the atmospheric
concen-tration just 43 h after the ®fth period in the treatments M3 and M4 with all added NH4Cl concentrations in the Wuxi soil. The CH4 concentrations remaining in
Table 2
Mean CH4oxidation and CO2and N2O release rates in the ®rst 19 h of the ®rst and ®fth incubation periods a
Treatmentb CH4oxidation rate (ng C gÿ 1
hÿ1
) CO2evolution rate (mg C gÿ 1
hÿ1
) N2O evolution rate (ng N gÿ 1
hÿ1
)
Wuxi soil Yingtan soil Wuxi soil Yingtan soil Wuxi soil Yingtan soil
1st 5th 1st 5th 1st 5th 1st 5th 1st 5th 1st 5th
M1N0 84 163 111 101 1.18 0.45 1.21 0.51 0.33 0.03 2.67 ÿ0.02
M1N1 81 165 77 91 1.39 0.51 1.29 0.43 1.82 0.02 2.96 0.03
M1N2 54 152 46 75 1.57 0.49 1.23 0.43 10.2 0.03 2.66 1.24
M2N0 189 411 230 344 1.08 0.64 1.09 0.62 0.32 0.01 2.12 0.02
M2N1 189 413 166 340 1.19 0.55 1.08 0.54 1.06 0.02 3.05 0.03
M2N2 147 408 101 353 1.39 0.62 1.01 0.51 11.0 0.01 2.83 1.45
M3N0 279 651 338 601 0.56 0.57 0.71 0.67 0.27 0.05 2.37 0.04
M3N1 273 644 251 610 0.83 0.61 0.74 0.63 1.29 ÿ0.03 2.21 0.05
M3N2 215 650 166 642 1.00 0.68 0.83 0.62 9.82 0.26 3.16 0.19
M4N0 399 890 435 846 0.74 0.45 0.77 0.76 0.43 0.03 2.50 0.08
M4N1 398 886 325 856 0.89 0.58 0.87 0.73 1.47 0.03 2.04 0.07
M4N2 320 892 228 876 0.99 0.58 0.83 0.68 8.58 0.03 2.93 0.13
LSD.05 20 17 14 21 0.27 0.19 0.17 0.05 2.37 0.25 1.20 0.83
LSD.01 27 22 19 29 0.37 0.26 0.24 0.06 3.20 0.33 1.63 1.12
a
Twenty-®ve gram of paddy soil was incubated in a 470-ml jar under various elevated initial CH4concentrations and NH4Cl addition rates at
258C.
b
M1, M2, M3 and M4 refer to the initial CH4concentration in the headspace of0500,01000,01500 and02000ml lÿ1and N0, N1 and N2
to NH4Cl addition rate equivalent to 0, 10 and 50mg N gÿ1soil, respectively.
Fig. 1. Eect of NH4Cl addition on CH4 oxidation during the ®rst
incubation period of a consecutive incubation at the initial CH4
con-centration of0500ml lÿ1(M1) with NH
4Cl addition rate equivalent
to 0 (N0), 10 (N1) and 50 (N2)mg N gÿ1soil, respectively, at 258C.
the headspace also decreased below the atmospheric concentration 63 h after the ®fth period in the treat-ment M4 with all added NH4Cl concentrations in the Yingtan soil and in the treatment M2 with all added NH4Cl concentrations in the Wuxi soil (data not shown). For M1 in Yingtan soil, CH4 oxidation ac-tivity reached a maximum value at 19 h after the incu-bation of the ®rst period and decreased with incubation time afterwards (Fig. 2b). Consistent with this phenomenon, the mean CH4 oxidation rate in the interval of 0±19 h did not increase signi®cantly in the subsequent incubations in the treatment without N ad-dition (Table 2).
3.2. Eect of NH4Cl addition on CH4oxidation
Addition of NH4Cl signi®cantly depressed CH4 oxi-dation rate during the ®rst period (P< 0.01 for both soils). The inhibition increased with the increase in added NH4Cl during the ®rst period (Fig. 1). The simulation of the experimental data with the kinetic
model showed that NH4Cl addition signi®cantly aected the increase in soil CH4oxidation activity. As a result, the CH4 oxidation activities decreased with increasing NH4Cl addition rate at the end of the ®rst incubation period, especially for Yingtan soil (Fig. 3. There was, however, a signi®cant interaction between added NH4Cl and initial CH4 concentration on the CH4oxidation activity at the end of the ®rst period (P < 0.05 for Wuxi soil; and P< 0.01 for Yingtan soil). The interaction suggests that the inhibitory eect of NH4Cl addition is alleviated with the increase in the initial CH4 concentration. Under the initial CH4 con-centration of0500 ml lÿ1
(M1), the CH4oxidation ac-tivity was reduced by 3 and 18% for Wuxi soil and 37 and 55% for Yingtan soil in the treatments N1 and N2, respectively. In contrast, under the initial CH4 concentration of02000ml lÿ1
(M4), the CH4oxidation activity was not aected signi®cantly for Wuxi soil (Fig. 3a) and less reduced (22 and 37%) for Yingtan soil in the treatments N1 and N2, respectively (Fig. 3b). In the subsequent incubations, the CH4 oxidation rates increased in NH4Cl treated samples to be the
Fig. 3. CH4 oxidation activities of the paddy soils (a, Wuxi soil; b,
Yingtan soil) at the end of the ®rst incubation period of a consecu-tive incubation at elevated CH4 concentrations and NH4Cl addition
rate equivalent to 0 (N0), 10 (N1), and 50 (N2)mg N gÿ1 soil,
re-spectively, at 258C. Bars refer to one standard deviation. Fig. 2. Change of CH4 oxidation activity of paddy soils (a, Wuxi
soil; b, Yingtan soil) during the ®rst incubation period of a consecu-tive incubation at the initial CH4concentration of0500 (M1),01000
(M2),01500 (M3) and02000 (M4)ml lÿ1, respectively, at 258C (the
same as the samples without NH4Cl addition. For Yingtan soil, the accelerating rates of CH4 oxidation were even higher in the treatments amended with NH4Cl than in those without NH4Cl addition, except for the treatments of the initial CH4 concentration of
0500 ml lÿ1. Therefore, the dierences of mean CH
4 oxidation rates between the treatments with or without NH4Cl addition became smaller with increasing num-ber of the consecutive incubation periods. Eventually, the CH4 oxidation rates were even higher in the treat-ments with NH4Cl addition than without NH4Cl ad-dition (Table 2). The higher the initial CH4 concentration and the more NH4Cl addition, the shorter the time required for this to occur. The same trend was observed in Wuxi soil, but the change was slower and the eect was smaller. However, the CH4 oxidation rates were always smaller in the treatment with NH4Cl addition than without NH4Cl addition at the initial CH4 concentration of 0500 ml lÿ1 and decreased with the increase in N addition rate for both soils (Table 2).
3.3. Relationships between CH4oxidation and CO2and N2O evolution
For both the soils, the mean CO2 evolution rates were signi®cantly aected by the initial CH4 concen-tration. In the ®rst incubation period, the CO2 evol-utions were depressed by the elevation of CH4 concentration (P< 0.01 for both soils, Table 2). There were negative relationships between the mean CH4 oxi-dation rate and CO2 evolution rate (r = 0.73, P< 0.01 for Wuxi soil andr= 0.73,P< 0.01 for Yingtan soil). However, the relationships between the rates of the CO2 evolution and CH4 oxidation were signi®-cantly positive in most subsequent incubations. In the ®rst incubation period, the mean CO2 evolution rates were higher than the mean CH4 oxidation rates at all the CH4 initial concentrations. In contrast, in the sub-sequent incubations, the CO2 evolution rates were smaller than the CH4 oxidation rates at high CH4 in-itial concentrations (Table 2).
Apparently, after CH4 oxidizers established their dominant positions, the CO2 that evolved from soil was mainly from the activities of CH4oxidizers, and a portion of the consumed CH4 were immobilized by microorganisms. Analysis of variance showed that not only did the initial CH4 concentration in¯uence CO2 evolution, but NH4Cl addition also aected CO2 evol-ution. However, the eect was dierent in both soils. For Wuxi soil, NH4Cl addition increased the mean CO2evolution rate in the ®rst incubation period (P< 0.01) and the eect disappeared in the subsequent in-cubations. For Yingtan soil, the addition of NH4Cl did not signi®cantly aect the CO2 evolution rate
during the ®rst incubation and afterwards decreased CO2evolution rate signi®cantly (P< 0.01, Table 2).
The pattern of N2O evolution was also dierent between the tested soils. Addition of NH4Cl to the Yingtan soil did not increase N2O evolution rate in the ®rst incubation (P> 0.05, Table 2). In the following incubation periods, however, N2O evolution rates increased signi®cantly as NH4Cl addition increased until the fourth period (P< 0.01), although the rates were decreased as compared to the ®rst period. N2O evolution from the second to ®fth periods accounted for 19 to 51% of the total in the treatments N2 with all initial CH4 concentrations. In contrast, N2O evol-ution rates increased signi®cantly with NH4Cl addition in the ®rst period in Wuxi soil (P< 0.01, Table 2). But the eect of NH4Cl was not signi®cant in the sub-sequent incubations (P > 0.05). The amount of N2O evolved from the second to ®fth periods made a minor contribution to the total N2O emission during the in-cubations. For Wuxi soil, added ammonium was almost completely oxidized into NO3ÿ±N and the amount of extractable NH4
+
±N in KCl extract was around the detection limit of the instrument. In con-trast, for Yingtan soil, an appreciable amount of NH4+±N was maintained at the end of all incubations in the treatment N2 with all initial CH4concentrations (Table 3). Both the KCl extractable N and the pattern of N2O emission suggest that the transformation of added ammonium was nearly completed in Wuxi soil during the ®rst incubation, but in Yingtan soil, not by the end of the experiment.
Apparent ratios of evolved N2O±N to added N were calculated by subtracting N2O evolution in the treat-ment without N addition from those in the treattreat-ments with NH4Cl addition. For Wuxi soil, the ratio ranged from ÿ0.02 to 0.37% and increased with the increase in N addition rate. On an average, the apparent ratio was 0.09% for the treatment N1 and 0.29% for the treatment N2. In the treatment N1, the ratio was higher in the initial CH4 concentration of0500 ml lÿ1 (M1) than in higher initial CH4 concentration (M2, M3 and M4). But the CH4 eect was small in the treatments N2. For Yingtan soil, the apparent ratio ranged fromÿ0.16 to 0.30% and was in¯uenced by in-itial CH4 concentration pronouncedly, decreasing with the increase in initial CH4 concentration, while the eect of NH4Cl addition rate was not signi®cant.
4. Discussion
CH4 oxidation, i.e. the inhibitory eect was strength-ened with increasing NH4Cl addition and alleviated with increasing initial CH4 concentration in the head-space. The inhibitory eect was more signi®cant for Yingtan soil than for Wuxi soil. The results suggest that if the CH4 concentration is suciently high (r2000ml lÿ1for Wuxi soil andr1000ml lÿ1for
Ying-tan soil) and continuously supplied for methanotrophic bacteria growth, NH4Cl inhibition on CH4 oxidation is only temporary and the inhibitory eect is reduced with time. With successive incubations, NH4Cl ad-dition stimulated CH4oxidation rather than remaining inhibitory. However, if the CH4concentration was not suciently high (<500 ml lÿ1for both soils in this
ex-periment), the inhibitory eect of NH4Cl addition was maintained and not alleviated during the incubations. Since the CH4concentration in paddy soils is typically higher than this critical concentration while soils are ¯ooded (Frenzel et al., 1992; Nouchi et al., 1994; Gil-bert and Frenzel, 1995), the inhibitory eect of NH4Cl addition on CH4 oxidation maintaining after NH4+±N disappearance in upland soils (Mosier et al., 1996; Powlson et al., 1997) should not occur in paddy soils.
Because of the similarity in shape and size of CH4 and NH4
+
and the relatively low speci®city of mono-oxygenase enzymes responsible, both methanotrophs and ammonia oxidizers can oxidize CH4 and NH4+ (BeÂdard and Knowles, 1989). So it is hypothesized that the competition between NH4+ and CH4 is one of the mechanisms for the inhibitory eect of NH4+ on CH4 oxidation (Schimel et al., 1993). Competitive inhibition has been demonstrated in upland soils oxidizing at-mospheric CH4 (Dun®eld and Knowles, 1995;
Gul-ledge and Schimel, 1998). However, the toxicity to methanotropic bacteria of NO2ÿ produced from nitri®-cation was also hypothesized to be another mechanism for inhibitory eect of NH4+ addition on CH4 oxi-dation (Schnell and King, 1994; Dun®eld and Knowles, 1995). Moreover, the inhibition of N fertili-zation on CH4 oxidation would result not only from the NH4+ speci®c eect, but also from the salt eect (Gulledge and Schimel, 1998). No signi®cant dier-ences were observed in the initial inhibitory eect between NH4Cl and KCl on the CH4 oxidation; but the eect of NH4Cl became stronger than KCl with in-cubation time in Yingtan soil in the study of Cai and Yan (1999). Because there was no salt control, the competitive and salt eects could not be distinguished in this experiment. However, the interaction of NH4Cl addition and the initial CH4concentration on the CH4 oxidation in the ®rst period is in accordance with the mechanism of the enzymatic substrate competition. The shift of NH4Cl eect from inhibition to stimu-lation could also be explained by the same mechanism. Soil CH4 oxidation activity increased with increasing initial CH4 concentration (Fig. 2). The result suggests the growth of methanotrophic bacteria community during the consecutive incubations (Cai and Yan, 1999). The growth of the community of methano-trophic bacteria under high CH4 concentration has also been reported (Bender and Conrad, 1992, 1995; Kightley et al., 1995). But as shown in Fig. 3, the growth was inhibited by the NH4Cl addition in the ®rst period. It might be reasonable to assume that the activities of ammonium oxidation in the paddy soils were stimulated by the NH4+ addition, but depressed,
Table 3
KCl extractable NO3ÿ±N and NH4 +
±N contents in paddy Wuxi soil and Yingtan soil 146 h extra incubation under the atmospheric CH4
concen-tration after consecutive incubations under elevated CH4concentration
Treatmenta Wuxi soil [mg N gÿ1
soil (DW)] Yingtan soil [mg N gÿ1
soil (DW)]
NO3ÿ±N NH4+±N Total NO3ÿ±N NH4+±N Total
M1N0 16.3 0.77 17.1 33.8 0.96 34.7
M1N1 23.3 0.61 23.9 44.8 0.94 45.8
M1N2 49.1 0.29 49.4 55.1 13.9 69.0
M2N0 15.2 0.50 15.7 34.2 0.96 35.1
M2N1 22.5 0.33 22.8 43.4 1.22 44.6
M2N2 48.1 0.48 48.6 55.8 12.4 68.2
M3N0 14.6 0.42 15.0 35.1 1.42 36.5
M3N1 21.1 0.65 21.7 43.0 1.08 44.1
M3N2 47.9 0.44 48.3 55.0 11.4 66.4
M4N0 13.5 0.46 14.0 31.7 0.92 32.6
M4N1 21.0 0.33 21.3 42.5 1.22 43.7
M4N2 46.3 0.65 46.9 53.6 13.8 67.3
LSD0.05 1.89 0.64 1.79 1.92 1.64 2.16
LSD0.01 2.57 0.86 2.43 2.61 2.22 2.93
a
M1, M2, M3 and M4 refer to the initial CH4concentration in headspace of0500,01000,01500 and02000ml lÿ 1
at least to a certain extent, by the injection of CH4, as indicated by the relationship between the initial CH4 concentration and the apparent ratio of evolved N2O± N to added N. With the decrease in NH4+±N concen-tration in the subsequent incubation (Table 3), the contribution of ammonium oxidizers to CH4oxidation would increase. Therefore, the rapid increase in the CH4 oxidation rate later in the experiment might be partially contributed by the ammonium oxidizers. Thereby, the inhibitory eect of NH4+ on CH4 oxi-dation would be compensated by the induced growth of ammonium oxidizers. When the enhancement of nitri®ers is greater than the inhibition on the popu-lation growth of methanotrophs, the net eect of NH4+±N addition would be to increase CH4oxidation. This would be the case under the condition that CH4 concentration was suciently high in this experiment. Otherwise, the net eect would be negative, such as in the case when the initial CH4was less than 500 ml lÿ1. The shift in the NH4
+
eect might occur in ¯ooded rice ®elds. It was reported that the CH4 ¯uxes from ¯ooded rice plots applied with urea were higher in 2±3 weeks after the fertilization but lower afterwards, com-pared to the control without application of urea (Cai et al., 1997).
5. Conclusions
We found that addition of NH4Cl inhibited CH4 oxidation by paddy soils signi®cantly during the ®rst incubation period. However, by employing a set of sequential incubations, we further found that there was a signi®cant interaction between NH4Cl addition and initial CH4concentration. The inhibitory eect of NH4Cl was strengthened with the increase in NH4Cl addition but was alleviated by raising initial CH4 con-centration. If CH4 concentration was suciently high (r2000ml lÿ1for Wuxi soil andr1000ml lÿ1for
Ying-tan soil) and was supplied continuously, the inhibitory eect of NH4Cl addition was decreased, and eventually stimulated CH4 oxidation in paddy soils. The higher the initial CH4 concentration, the shorter the time required for the shift from inhibition to stimulation. If the CH4concentration was not higher than 500 ml lÿ1, the inhibitory eect of NH4Cl addition on the CH4 oxidation was not alleviated during ®ve consecutive periods of incubation. It would probably become the maintaining eect after disappearance of added NH4+± N, similar to that observed in upland soils. The threshold of CH4 concentration varied with soils, but was less than the typical CH4 concentration in paddy soils when they are ¯ooded. Therefore, shifting the eect of NH4Cl addition on CH4oxidation from inhi-bition to stimulation is likely under ®eld conditions
and a maintaining eect of NH4Cl on the oxidation of CH4 produced endogenously would not exist in paddy soils.
Acknowledgements
We thank the Chinese Academy of Sciences for pro-viding travel and living support for Dr Zucong Cai's research work at USDA/ARS in Ft Collins. This pro-ject was also supported by a grant (49771073) from the National Natural Science Foundation of China, NKBRDF (G1999011805) and USDA/ARS. We also thank Mary Smith and Susan Crookall for their tech-nical assistance.
References
BeÂdard, C., Knowles, R., 1989. Physiology, biochemistry, and speci®c inhibitors of CH4, NH4
+
and CO oxidation by methano-trophs and nitri®ers. Microbiological Reviews 53, 68±84. 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., 1995. Eect of CH4concentrations and soil
conditions on the induction of CH4 oxidation activity. Soil
Biology & Biochemistry 27, 1517±1527.
Cai, Z.C., Xing, G.X., Yan, X.Y., Xu, H., Tsuruta, H., Yagi, K., Minami, K., 1997. Methane and nitrous oxide emissions from rice paddy ®elds as aected by nitrogen fertilizers and water man-agement. Plant and Soil 196, 7±14.
Cai, Z.C., Yan, X.Y., 1999. A kinetic model for methane oxidation by paddy soil as aected by temperature, moisture and N ad-dition. Soil Biology & Biochemistry 31, 715±725.
Cicerone, R.J., Shetter, J.D., 1981. Sources of atmospheric methane: measurements in rice paddies and a discussion. Journal of Geophysical Research 86, 7203±7209.
Conrad, R., Rothfuss, F., 1991. Methane oxidation in the soil sur-face layer of a ¯ooded rice ®eld and the eect of ammonium. Biology and Fertility of Soils 12, 28±32.
Dun®eld, P., Knowles, R., 1995. Kinetics of inhibition of methane oxidation by nitrate, nitrite, and ammonium in a Humisol. Applied and Environmental Microbiology 61, 3129±3135. Flessa, H., DoÈrsch, P., Beese, F., 1995. Seasonal variation of N2O
and CH4 ¯uxes in dierently managed arable soils in southern
Germany. Journal of Geophysical Research 100, 23115±23124. Frenzel, P., Rothfuss, F., Conrad, R., 1992. Oxygen pro®les and
methane turnover in a ¯ooded rice microcosm. Soil Biology & Biochemistry 14, 84±89.
Galchenko, V.A., Lein, A., Ivanov, M., 1989. Biological sinks of methane. In: Andreae, M.O., Schimel, D.S. (Eds.), Exchange of Trace Gases between Terrestrial Ecosystems and the Atmosphere. Wiley, Chichester, pp. 59±71.
Gilbert, B., Frenzel, P., 1995. Methanotrophic bacteria in the rhizo-sphere of rice microcosms and their eect on porewater methane concentration and methane emission. Biology and Fertility of Soils 20, 93±100.
Gulledge, J., Schimel, J.P., 1998. Low-concentration kinetics of at-mospheric CH4 oxidation in soil and mechanism of NH4
Holzapfel-Pschorn, A., Conrad, R., Seiler, W., 1985. Production, oxidation, and emission of methane in rice paddies. FEMS Microbiology Ecology 31, 343±351.
HuÈtsch, B.W., Webster, C.P., Powlson, D.S., 1993. Long-term eects of nitrogen fertilization on methane oxidation in soil of the Broadbalk Wheat Experiment. Soil Biology & Biochemistry 25, 1307±1315.
IPCC Ð Intergovernmental Panel on Climate Change, 1992. In: Houghton, J.T., Callender, B.A., Varney, S.K., (Eds.) Climate Change. The Supplementary Report to the IPCC Scienti®c Assessment, Cambridge University Press, Cambridge.
Knightley, D., Nedwell, D.B., Cooper, M., 1995. Capacity for methane oxidation in land®ll cover soils measured in laboratory-scale soil microcosms. Applied & Environmental Microbiology 61, 592±601.
Kimura, M., Asai, K., Watanabe, A., Murase, J., Kuwatsuka, S., 1992. Suppression of methane ¯uxes from ¯ooded paddy soil with rice plants by foliar spray of nitrogen fertilizers. Soil Science and Plant Nutrition 38, 735±740.
Lindau, C.W., Bollich, P.K., deLaune, R.D., Patrick Jr, W.H., Law, V.J., 1991. Eect of urea fertilizer and environmental factors on CH4 emissions from a Louisiana, USA rice ®eld. Plant and Soil
136, 195±203.
Mosier, A.R., Parton, W.J., Valentine, D.W., Ojima, D.S., Schimel, D.S., Delgado, J.A., 1996. CH4and N2O ¯uxes in the Colorado
shortgrass steppe. Part I: Impact of landscape and nitrogen ad-dition. Global Biochemical Cycles 10, 387±399.
Mosier, A.R., Schimel, D., Valentine, D., Bronson, K., Parton, W., 1991. Methane and nitrous oxide ¯ux in native, fertilized and cul-tivated grasslands. Nature 350, 330±332.
Nesbit, S.P., Breitenbeck, G.A., 1992. A laboratory study of factors
in¯uencing methane uptake by soils. Agriculture Ecosystems & Environment 41, 39±54.
Nouchi, I., Hosono, T., Aoki, K., Minami, K., 1994. Seasonal vari-ation in methane ¯ux from rice paddies associated with methane concentration in soil water, rice biomass and temperature, and its modeling. Plant and Soil 161, 195±208.
Powlson, D.S., Goulding, K.W.T., Willison, T.W., Webster, C.P., HuÈtsch, B.W., 1997. The eect of agriculture on methane oxi-dation in soil. Nutrient Cycling in Agroecosystems 49, 59±70. Sass, R.L., Fisher, F.M., Harcobe, P.A., Turner, F.T., 1991.
Methane production emission in a Texas agricultural wetland. Global Biogeochemical Cycles 4, 47±68.
Schimel, J.P., Holland, E.A., Valentine, D., 1993. Controls on methane ¯ux from terrestrial ecosystem. In: Harper, L.A., Mosier, A.R., Duxbury, J.M., Rolston, D.E. (Eds.), Agricultural Ecosystem Eects on Trace Gases and Global Climate Change. American Society of Agronomy, Madison, pp. 167±182.
Schnell, S., King, G., 1994. Mechanistic analysis of ammonium inhi-bition of atmospheric methane consumption in forest soils. Applied and Environmental Microbiology 60, 3514±3521. SchuÈtz, H., Holzapfel-Pschorn, A., Conrad, R., Rennenberg, H.,
Seiler, W., 1989. A 3-year continuous record on the in¯uence of day-time, season and fertilizer treatment on methane emissions rates from an Italian rice paddy. Journal of Geophysical Research 94, 16405±16416.
Segers, R., 1998. Methane production and methane consumption: a review of processes underlying wetland methane ¯uxes. Biogeochemistry 41, 23±51.
