Institute of Plant Nutrition, Justus-Liebig-University,. Südanlage 6, D-35390 Giessen, Germany. Fax: c49-641-99-39169. Abstract The effects of inorganic N and ...
Biol Fertil Soils (1998) 28 : 27–35
Q Springer-Verlag 1998
ORIGINAL PAPER
B. W. Hütsch
Methane oxidation in arable soil as inhibited by ammonium, nitrite, and organic manure with respect to soil pH
Received: 31 October 1997
Abstract The effects of inorganic N and organic manure, applied to a loamy arable soil, on CH4 oxidation were investigated in laboratory incubation experiments. Applications (40 mg N kg P1) of NH4Cl, (NH4)2SO4, and urea caused strong instantaneous inhibition of CH4 oxidation by 96%, 80%, and 84%, respectively. After nitrification of the added N the inhibitory effect was not fully reversible, resulting in an residual inhibition of 21%, 16%, and 25% in the NH4Cl, (NH4)2SO4, and urea treatments, respectively. With large NHc 4 applications [240 mg N kg P1 as (NH4)2SO4] the residual inhibition was as high as 64%. Exogenous NOP 2 (40 mg P1 -N kg ) initially inhibited CH oxidation by 84%, NOP 2 4 decreasing to 41% after its oxidation. Therefore, applied NOP 2 was a more effective inhibitor of CH4 conP sumption than NHc 4 . Temporary accumulation of NO2 during nitrification of added N was small (maximum: P1 ) and thus of minor importance 1.9 mg NOP 2 -N kg with respect to the persistent inhibition after NHc 4 or urea application. CH4 oxidation after NaNO3 (40 mg N kg P1) and NaCl addition did not differ to that of the untreated soil. The effect of organic manures on CH4 oxidation depended on their C/N ratio: fresh sugar beet leaves enhanced mineralization, which caused an instantaneous 20% inhibition, whereas after wheat straw application available soil N was rapidly immobilized and no effect on CH4 oxidation was found. The 28% increase in CH4 oxidation after biowaste compost application was not related to its C/N ratio and was probably the result of an inoculation with methanotrophic bacteria. Only with high NHc 4 application rates (240 mg N kg P1) could the persistent inhibitory effect partly be attributed to a pH decrease during nitrification. The exact reason for the observed persistent inhibition after a single, moderate NHc 4 or urea application is still unknown and merits further study. B. W. Hütsch (Y) Institute of Plant Nutrition, Justus-Liebig-University, Südanlage 6, D-35390 Giessen, Germany Fax: c49-641-99-39169
Key words Methane oxidation 7 Ammonium 7 Soil pH 7 Nitrite 7 Organic manure
Introduction Although the current concentration of CH4 is much lower than the concentration of CO2 in the atmosphere (1.7 ml l P1 and 350 ml l P1, respectively), CH4 absorbs infrared irradiation more effectively than CO2, and reemission of the absorbed radiant energy causes global warming. Therefore, the current radiative forcing by CH4 is 26 times that of CO2 (calculated on a mole CO2/mole CH4 basis; Lelieveld et al. 1993), making CH4 the second most important greenhouse gas. The estimated contribution to the anthropogenic greenhouse effect is 15% for CH4 and 60% for CO2 (Rodhe 1990). The atmospheric CH4 concentration has been increasing by 0.8–1.0% per year until recently, when smaller increases have been reported (Blake and Rowland 1988; Steele et al. 1992). Obviously, the increase in atmospheric CH4 concentrations results from an imbalance between CH4 production and consumption. Sources of the increasing CH4 concentrations include the expansion of paddy rice production, the burning of biomass and an increasing number of ruminants. This trend is driven by population growth, particularly in the developing world, and greater demand for meat and dairy products in industrial nations. However, the average increase in the total CH4 emission flux accounts only partly for the annual CH4 increase in the atmosphere. Another contributing factor along with the increase in CH4 emissions is the decrease in CH4 sinks. An amount equal to approximately 90% of the annual emissions is oxidized through photochemical reactions initiated by OH radicals in the troposphere, and 5% are considered to be lost by microbial uptake at the Earth’s surface (Crutzen 1995). Although the soil sink strength is relatively small, its absence would cause the atmospheric concentration of CH4 to increase at about 1.5 times the current rate
28
(Duxbury 1994). Thus a change in this sink has important implications for overall atmospheric CH4 concentrations. Methanotrophic bacteria are responsible for CH4 oxidation in aerobic soils. They are unique in their ability to utilize CH4 as a sole C and energy source. The key enzymes, methane-monooxygenases (MMOs), present in aerobic methanotrophic bacteria, exhibit a striking lack of substrate specificity, resulting in the fortuitous metabolism of a very large number of compounds. Methanotrophs and chemoautotrophic ammonia-oxidizing bacteria are very similar in the ways they oxidize CH4 and NH3 (Hanson and Hanson 1996). All CH4-oxidizing bacteria examined oxidized NH3 to NOP 2 , although the specific rates of NH3 oxidation by methanotrophs were 2 orders of magnitude lower than those of the chemoautotrophic nitrifiers (Bedard and Knowles 1989). The inhibition of CH4 oxidation by NH3 has profound effects on the ecology of methanotrophs in agricultural soils, forests, and rice paddies. Several studies have been conducted to test the immediate effect of NHc 4 application on the CH4-oxidizing ability of soils. In laboratory incubations Adamsen and King (1993), Bronson and Mosier (1994), Crill et al. (1994), and Boeckx and Van Cleemput (1996) found strong inhibitory effects of NHc 4 on CH4 oxidation in forest, grassland, peatland, and landfill cover soils, respectively. In addition, in some investigations on regularly fertilized arable soils, NHc 4 was also inhibitory (Bender and Conrad 1994; Flessa et al. 1996; Hütsch et al. 1996). However, in field measurements on arable soils no immediate effect on CH4 uptake was observed after the addition of NHc 4 -containing fertilizers or urea (Dobbie and Smith 1996; Delgado and Mosier 1996). From the investigations of King and Schnell (1994a), NOP 2 was identified as an additional, non-competitive inhibitor of methanotrophs. In pure cultures of the methanotrophs Methylobacter albus BG8 and Methylosinus trichosporium OB3b, CH4 oxidation was inhibited by NH4Cl and NaNO2, and both species produced NOP 2 from NHc 4 (King and Schnell 1994b). Schnell and King (1994) found in investigations of an acid forest soil that exogenous NOP 2 was a more effective inhibitor of CH4 consumption than NHc 4 (59% and 42% inhibition, respectively), and that a single treatment with NHc 4 had a long-term inhibitory effect on soil CH4 consumption (unchanged within 39 days). Conflicting results were obtained by Dunfield and Knowles (1995), who investiP gated the effect of NHc 4 and NO2 addition on CH4 oxidation of a humisol with a pH near neutral. In their study the inhibition of CH4 oxidation by both NHc 4 and NOP 2 was fully reversible. In the work I report here, incubation experiments were conducted with sieved soil from an arable site, annually cultivated by plowing. The soil is a Luvisol derived from loess, belonging to the most fertile soils in Germany. The effects of commonly applied rates of P inorganic N fertilizers (including NHc 4 , NO3 , and urea on CH4 oxidation were tested. It was of particular inter-
est whether an inhibitory effect of NHc 4 is only a transient one or if there is also a long-term influence, persisting after nitrification of the added N. In addition, the soil was amended with biowaste compost and crop residues, and the effect of NOP 2 on CH4 oxidation was also investigated. It is well known that NOP 2 is toxic to all living organisms, but it remains to be shown whether in representative agricultural soils toxic levels of NOP 2 are attained after NHc 4 fertilizer application. During the incubation period the actual inorganic N concentrations in soil, as well as the pH, were determined at the time of CH4 measurement. To evaluate how important pH changes are for the methanotrophs, the soil was gradually acidified and its CH4-oxidizing ability measP ured. As the oxidation of NHc 4 to NO3 causes soil acidification, a possible persistent inhibition of CH4 oxidation by NHc 4 may be partly the result of a concurrent drop in soil pH.
Materials and methods Soil sampling and preparation Random samples were collected from the surface layer (0–12 cm) of a Luvic Phaeozem derived from loess at Ossenheim near Friedberg (Germany) on 27 February 1995, before the first fertilizer was applied in spring. The soil is a silty loam with 21% clay and 67% silt in the topsoil. At the time of sampling the chemical properties of the soil were as follows: total N 1.47 g kg P1, organic C 15.9 g kg P1, pH (in H2O) 8.0, 201 mg P kg P1 and 187 mg K kg P1 (calcium-acetate-lactate extractable P and K). The site is used for arable farming and plowed annually (plowing depth: 25 cm). Winter wheat was growing after sugar beet, and fertilizer and pesticides were applied as usual for this area. At the time of sampling the soil moisture content was near field capacity and the soil samples were slightly air-dried at ambient temperatures (5–10 7C; H2O content: 12% w/w) prior to sieving (^5 mm mesh width). No difference in CH4 oxidation ability occurred between 11.4% and 29% H2O (w/w), equivalent to 28–70% water-holding capacity. The soil was stored at 5 7C before usage.
Incubation experiments The sieved, slightly air-dried soil samples were weighed in 100-g units into 250-ml Erlenmeyer flasks, sealed, and pre-incubated at 25 7C to adapt to this temperature. Organic material in solid form was added to the soil just before the pre-incubation stage. After 24 h the flasks were gently flushed with compressed air, and either 2 ml of fertilizer solution or, in the case of organic manure and control treatment, 2 ml of deionized H2O were applied to the soil. The effects of the following treatments on CH4 oxidation were tested: 1. Control. 2. (NH4)2SO4 (a) 40 mg N kg P1 soil and (b) 240 mg N kg P1 soil. 3. NH4Cl, 40 mg N kg P1 soil. 4. NaNO3, 40 mg N kg P1 soil. 5. urea, 40 mg N kg P1 soil. 6. NaNO2, 40 mg N kg P1 soil. 7. Sugar beet leaves, 97 mg N kg P1 soil (C : N ratio 8). 8. Biowaste compost, 87 mg N kg P1 soil (C : N ratio 16). 9. Wheat straw, 2.3 g straw kg P1 soil (C : N ratio 109). 10. NaCl.
29 The set of samples with the NaCl application was prepared to test the effect of the accompanying ions, Na c and Cl P. The experiment with the large N application (240 mg N kg P1) was extended by a second incubation via flushing the flasks with compressed air after the first 168 h and setting up the next CH4 measurement as described below. The sugar beet leaves had not been treated with pesticides for at least 6 weeks prior to usage, and immediately after collection they were cut into small pieces and mixed with the soil (1.5 g fresh weight per flask). The biowaste compost was collected after a maturation period of approximately 4 months, homogenized and chopped to pass a 2-mm sieve; 1.0 g fresh weight per flask were applied. The wheat straw was dried (105 7C) and ground prior to usage (0.2 g dry weight per flask). The application rates of inorganic N (apart from 240 mg N kg P1) and of organic substances employed are commonly used in agriculture. After thoroughly but gently mixing the soil with a spatula, the flasks were capped with glass stoppers, which were given a thin film of vacuum grease to prevent leakage. At the top of the glass stoppers a silicon septum was inserted, which allowed gas sampling with a syringe and sideport needle. Incubation took place at 25 7C in the dark. In total, six separate incubation experiments were conducted. Each experiment comprised 54 flasks at the start of the incubation, always consisting of unamended soil samples (18 flasks) and either two inorganic N treatments (18 flasks each) or one organic manure treatment (36 flasks). To each flask, 8 ml CH4 l P1 was added (2.1 ml of 0.1% CH4) to give a final concentration of 10 ml CH4 l P1. In addition, two flasks were prepared by adding CH4 (2.5 ml of 0.1% CH4), but no soil. The headspace CH4 and CO2 concentrations either in three flasks of the inorganic N treatments, or in six flasks of the organic manure treatments, in three flasks of the unamended samples, and in the two vessels without soil were measured after 0, 24, 48, 72, 120, and 168 h. The gas analyses were performed with a gas chromatograph as described previously (Hütsch et al. 1996). The first CH4 measurement took place approximately 1–1.5 h after fertilizer application. Immediately after the gas measurements, the soil was removed from the flasks, and 50 g was weighed into 250-ml extraction bottles and frozen until required for further investigation. The remaining soil was dried in a drying cabinet (40 7C, 24 h), milled, and sieved (^1 mm mesh width). A separate set of samples was prepared to evaluate the effect of pH changes on CH4 oxidation. Firstly, a buffer curve was set up to determine the amounts of HCl necessary to lower the soil pH in small steps of 0.2–0.3 units. Additions of 0.0, 0.5, 1.25, 2.5, and 5.0 mmol H c per 100 g moist soil resulted in pH values of 8.0, 7.8, 7.6, 7.4, and 7.1, respectively. The appropriate amount was applied in solution (2 ml) with six replicates per pH level. The amended soil was preincubated in sealed 250-ml Erlenmeyer flasks at 25 7C for 72 h. Thereafter, for each pH level, three repli-
c cate samples were used for soil analyses (NOP 3 , NH4 and pH). With the remaining samples, CH4 measurements were performed as described above. The CO2 concentrations in the headspace were determined after 24 h and 168 h, and the soil was analyzed again at the end of the entire incubation.
Soil analyses The moisture content of the soil, as well as of sugar beet leaves and compost, was measured gravimetrically by drying at 105 7C. To determine soil pH, 40 7C-dried samples were ground (^1 mm), mixed with freshly degassed, deionized water (soil : water ratio 1 : 2.5), and the pH measured in the supernatant liquid with a glass electrode. The soils (50 g fresh weight, ^5 mm, deep frozen before use) were extracted by shaking them with 200 ml 10 mM CaCl2 for 1 h and were filtered through Macherey and Nagel filter paper (MN 261 1/4). The NOP 2 concentrations were determined immediately P after extraction, and for NHc 4 and NO3 the filtrates were stored in P a refrigerator until measurement within 1–2 days. The NOP 2 , NO3 and NHc concentrations were determined colorimetrically with a 4 continuous-flow three-channel autoanalyser (Autoanalyser II, Bran and Luebbe, Germany). Concentrations are expressed as mg N kg P1 dry soil.
Results and discussion In the following tables and figures the average of three or six replicates, measured in one incubation, and the corresponding SD or SE are given. Data from the control soil were averaged for the three replicates of all six separate incubations (np18, Tables 1, 2).
Effect of NHc 4 and urea on CH4 oxidation The CH4-oxidizing capacity of a loamy arable soil was measured at a water content of 14% w/w and an incubation temperature of 25 7C. In Fig. 1a the CH4 concentration in the headspace of the incubation flasks is plotted against time; the curves for the treatments NH4Cl, NaNO3 and for the control are given. The unamended soil oxidized CH4 rapidly with a final concentration of 0.5 ml CH4 l P1 after 168 h. Addition of 40 mg N kg P1
Table 1 CH4 concentrations (ml CH4 l P1) in headspace of control and treatments (40 mg N kg P1 as mineral fertilizer). MeansBSD are given Time of CH4 measurement (h)
Control (np18) (NH4)2SO4 (np3) NH4Cl (np3) NaNO3 (np3) urea (np3) NaNO2 (np3) Sugar beet leaves (np6) a Biowaste compost (np6) Wheat straw (np6) NaCl (np3) a
0
24
48
72
120
168
10.0B0.3 10.6B0.0 10.0B0.4 9.9B0.0 10.4B0.1 10.4B0.3 9.7B0.1 9.7B0.2 10.0B0.2 10.2B0.1
6.8B0.4 9.9B0.3 9.9B0.2 6.6B0.4 9.9B0.2 9.9B0.1 6.7B0.2 6.8B0.1 7.0B0.2 6.8B0.1
4.8B0.4 8.7B0.1 8.9B0.2 5.0B0.3 8.5B0.2 9.7B0.3 5.5B0.1 4.9B0.1 4.9B0.2 4.8B0.0
3.4B0.5 5.8B0.2 6.4B0.1 3.3B0.2 6.2B0.2 7.6B0.1 4.2B0.2 3.4B0.1 3.1B0.1 3.5B0.1
1.6B0.3 2.8B0.0 3.0B0.1 1.5B0.1 2.8B0.0 3.9B0.1 2.4B0.1 1.6B0.1 1.5B0.1 1.6B0.1
0.7B0.2 1.3B0.1 1.3B0.0 0.6B0.0 1.4B0.1 1.9B0.0 2.8B0.1 0.7B0.1 0.7B0.1 0.7B0.0
Anaerobic conditions at the end of the incubation
30 Table 2 Inorganic N concentrations (mg N kg P1 soil) of control and treatments (40 mg N kg P1 as mineral fertilizer) at time of CH4 P1 measurements. MeansBSD are given. b.d. Below detection limit (~0.03 mg NHc ) 4 -N7l Time of CH4 measurement (h) 0
24
48
72
120
168
Control (np18) a
NOP 2 -N NOP 3 -N
0.3B0.0 11.3B0.5
0.3B0.0 11.7B0.5
0.3B0.0 12.0B0.4
0.3B0.0 12.2B0.4
0.3B0.0 12.9B0.7
0.4B0.0 13.6B0.7
(NH4)2SO4 (np3)
NHc 4 -N NOP 2 -N P NO3 -N
13.9B0.6 1.3B0.1 13.5B0.2
2.3B0.2 1.9B0.1 38.7B0.4
b.d. 0.3B0.0 49.5B1.1
b.d. 0.3B0.0 48.7B0.5
b.d. 0.3B0.0 50.4B0.3
b.d. 0.4B0.0 49.9B1.4
NH4Cl (np3)
NHc 4 -N NOP 2 -N P NO3 -N
14.0B0.2 1.1B0.1 14.0B0.2
3.4B0.1 1.6B0.1 40.1B0.3
b.d. 0.3B0.0 51.9B1.0
b.d. 0.3B0.0 51.5B0.2
b.d. 0.3B0.0 52.8B0.4
b.d. 0.4B0.0 53.2B0.8
NaNO3 (np3) a, b
NOP 3 -N
53.8B1.1
54.8B1.7
54.5B1.2
54.9B0.3
55.5B0.9
56.7B2.0
urea (np3)
NHc 4 -N NOP 2 -N NOP 3 -N
8.1B0.3 1.0B0.0 14.2B0.1
3.1B0.3 1.9B0.1 40.5B1.2
b.d. 0.3B0.0 51.5B0.3
b.d. 0.3B0.0 52.7B1.2
b.d. 0.3B0.0 53.4B0.2
b.d. 0.4B0.0 53.1B0.5
NaNO2 (np3) a
NOP 2 -N NOP 3 -N
36.8B0.5 12.1B0.6
22.3B0.1 26.1B0.3
4.6B0.3 45.0B0.5
0.6B0.0 50.1B0.5
0.5B0.0 50.9B0.5
0.6B0.0 51.9B0.4
Sugar beet leaves (np6) b, c
NHc 4 -N NOP 3 -N
1.0B0.3 17.0B0.5
1.8B0.2 22.0B0.9
1.3B0.2 27.1B1.0
1.3B0.2 32.1B1.2
0.8B0.2 39.5B2.0
3.0B0.2 17.1B1.5
Biowaste compost (np6) a, b
NOP 3 -N
12.0B0.4
12.2B0.1
12.0B0.3
12.4B0.6
13.4B0.5
14.2B0.4
NO -N
11.3B0.1
10.3B0.1
7.6B0.3
3.6B0.3
1.5B0.2
1.3B0.2
NOP 3 -N
12.0B0.3
12.7B0.0
12.8B0.1
12.9B0.3
12.9B0.2
13.3B0.1
Wheat straw (np6) NaCl (np3) a, b a b
a, b
P 3
NHc 4 -N concentrations always b.d. NOP 2 -N concentrations have not been determined
soil as NaNO3 did not change the CH4-oxidizing ability of this soil, whereas the same amount of N applied as NH4Cl caused a strong inhibition for the first 48 h of incubation. Thereafter, the added NHc 4 had been entirely nitrified (Fig. 1b), and CH4 was oxidized at a high rate (Fig. 1a). During the incubation, soil NOP 3 increased only slightly in the NaNO3 treatment and in the control, whereas nitrification of the added NHc 4 resulted in a steep increase in NOP 3 concentrations during the first 48 h with almost constant values thereafter P1 (maximum nitrification rate: 26 mg NOP 3 -N kg P1 day ). During this nitrification process a transient accumulation of soil NOP 2 occurred with a maximal conP1 at time (t)p24 h (Tacentration of 1.6 mg NOP 2 -N kg ble 2). At the same time the concentration in the conP1 with almost no change trol was 0.3 mg NOP 2 -N kg during the incubation. Regarding the effects of NH4Cl on CH4 oxidation, P soil NOP 3 and NO2 concentrations, identical results were obtained after application of (NH4)2SO4 and urea in similar amounts (Tables 1, 2). The accompanying ions, Na c and Cl P did not affect the CH4-oxidizing ability, showing the same pattern as NaNO3 and the control (Table 1). There was also no difference in NOP 3 concentrations between the NaCl treatment and the control (Table 2). At the start of incubation only 35% of the added NHc 4 was extractable with 10 mM CaCl2 (Table 2, (NH4)2SO4 and NH4Cl treatments). Loss through NH3 volatilization can be excluded, as the final NOP 3 concen-
c
Anaerobic conditions at the end of the incubation
trations match almost exactly the amount of added N plus the amount in the control (53 mg N kg P1). In this Luvisol derived from loess with a high concentration of exchangeable K (187 mg K kg P1), cation substitution is most likely with subsequent adsorption of NHc 4 by clay minerals, as postulated earlier (Hütsch et al. 1996). Dunfield and Knowles (1995) also observed that 60– 70% of the NHc 4 added to a humisol was not recoverable shortly after addition; it was assumed to be held on exchange sites. Nevertheless, the adsorbed NHc 4 was rapidly nitrified and not protected from microbial attack (Fig. 1b, Table 2). The results clearly show that NH3 oxidation and CH4 oxidation were mutually exclusive, and only when nitrification was almost completed did CH4 metabolism commence. Methanotrophic bacteria are able to oxidize both CH4 and NH3 (Bedard and Knowles 1989). As MMOs exhibit a striking lack of substrate specificity (Hanson and Hanson 1996), the methanotrophs fortuitously metabolize NH3 as soon as it is added to the soil, and can oxidize CH4 only after NH3 is almost entirely decomposed. This explanation is strengthened by the fact that the relation of added N to CH4 available in the headspace of the jars was 2400 to 1 at the start of the incubation (NH3 to CH4). The inhibition of CH4 oxidation by NH4Cl, (NH4)2SO4 and urea was 96%, 80%, and 84%, respectively, immediately after application (tp0–24 h), and still 21%, 16%, and 25%, respectively, between tp48– 72 h, when the NHc 4 concentration in the soil was near
31
Fig. 1a, b CH4 oxidation of a loamy arable soil (a), and NOP 3 concentration in the soil (b), after NH4Cl and NaNO3 application (40 mg N kg P1) in comparison to the untreated soil. Each data point is the averageBSD of three replicates. CH4 oxidation in the control and NaNO3 treatment was virtually the same resulting in only one curve (a)
Table 3 Mean CH4 oxidation rates (mg C kg P1 day P1), calculated for time (t)p0–24 h and tp48–72 h, of treatments (40 mg N kg P1 as mineral fertilizer) and control, and the percentage inhibiCH4 oxidation rate tp0–24 h
(NH4)2SO4 NH4Cl NaNO3 urea NaNO2 Sugar beet leaves Biowaste compost Wheat straw NaCl
Treatments
Control
1.1B0.2 0.2B0.3 4.9B0.4 0.8B0.2 0.8B0.2 4.5B0.2 4.4B0.1 4.6B0.2 5.0B0.2
5.2B0.1 4.7B0.1 4.7B0.1 4.8B0.2 5.2B0.1 5.6B0.1 3.5B0.1 4.8B0.1 4.8B0.2
a Maximum rate for the entire incubation, when the added NHc 4 was completely nitrified; the other treatments had their maximum rate at tp0–24 h
zero and the highest CH4 oxidation occurred in the treated soils (Table 3). Although the strong inhibitory effects of NHc 4 and urea were only temporary, the treated soils were unable to reach the CH4-oxidizing ability of the unamended soil at tp0. The transient, strong inhibition of CH4 oxidation by NHc 4 , lasting only for 48 h, seems to be contradictory to previously reported results, where NHc 4 addition to the same soil suppressed CH4 oxidation completely during a 168-h incubation (Hütsch et al. 1996). In these previous experiments 240 mg N kg P1 were applied, and P1 there were still 41 mg NHc extractable at the 4 -N kg P end of incubation; from the NO3 concentration (147 mg P1 ) it was concluded that, in addition, apNOP 3 -N kg P1 were adsorbed by clay proximately 50 mg NHc 4 -N kg minerals. This experiment was repeated by adding 240 mg N kg P1 as (NH4)2SO4 to the soil, and extending the measurements by a second 168-h incubation. CH4 oxidation was completely suppressed for 216 h (Table 4). Thereafter, when the added NHc 4 was almost entirely nitrified (at tp72 h of the second incubation), CH4 oxidation commenced at a rate of 1.83 mg CH4-C kg P1 day P1 (mean rate for tp72–120 h). However, this is still a 64% inhibition in comparison to the control (5.07 mg CH4-C kg P1 day P1 at tp0–24 h of the second incubation). Therefore, the higher the NHc 4 application rate the stronger the inhibitory residual effect on CH4 oxidation. In laboratory experiments Nesbit and Breitenbeck (1992) investigated the effect of NH4Cl on CH4 oxidation of a hardwood swamp soil. The inhibitory effects P1 were clearof amending soils with 100 mg NHc 4 -N kg ly evident at the end of a 5-day study despite the fact that more than 75% of N had been nitrified by that time. Unlike the results found here, CH4 oxidation was not completely suppressed immediately after NHc 4 application. Because of this observation it is suggested that the methanotrophs in this swamp soil were less
tion [(rate of controlPrate of treatment/rate of control)!100]. MeansBSE are given. n.c. Not calculated Inhibition (%)
CH4 oxidation rate tp48–72 h a
Inhibition (%) b
Treatments 80B5 96B6 P 6B10 84B4 84B4 20B3 P28B5 4B6 P 6B7 b
4.4B0.3 3.7B0.1 n.c. 3.5B0.3 3.1B0.2 n.c. n.c. n.c. n.c.
16B5 21B2 n.c. 25B8 41B4 n.c. n.c. n.c. n.c.
Calculated with the rate of the control at tp0–24 h
32 sphere was renewed before start of the second incubation. MeansBSD (np3) are given
Table 4 CH4 concentrations in headspace, inorganic N concentrations, and pH after application of 240 mg N kg P1 as (NH4)2SO4 in comparison to untreated samples; the gas atmoFirst incubation Time of CH4 measurement (h)
Second incubation Time of CH4 measurement (h) 0a
0
168
CH4 (ml CH4 l P1) P1 NHc ) 4 (mg N kg P1 NOP ) 3 (mg N kg pH
10.9B0.2 110.3B1.0 17.6B0.8 7.6B0.2
(NH4)2SO4 treatment (240 mg N kg P1) 10.6B0.0 10.2B0.1 10.2B0.0 10.0B0.2 26.0B1.7 13.8B0.7 5.6B1.5 179.1B2.5 207.6B0.8 230.0B1.9 7.4B0.0 7.4B0.0 7.3B0.1
CH4 (ml CH4 l P1) P1 NOP ) 3 (mg N kg pH
10.7B0.1 16.3B0.2 8.1B0.1
a b
0.8B0.0 17.5B0.2 8.0B0.0
10.0B0.1
24
48
Control b 6.7B0.1 17.9B0.4 8.0B0.0
4.2B0.1 18.0B0.3 8.0B0.0
72
120
168
9.1B0.2 1.2B0.1 243.3B2.8 7.4B0.0
6.7B0.1 0.3B0.0 244.2B6.5 7.2B0.1
4.9B0.5 0.2B0.0 240.2B3.0 7.3B0.0
2.7B0.0 19.2B0.5 8.0B0.0
1.1B0.0 19.1B0.4 8.0B0.0
0.5B0.1 19.5B0.5 8.0B0.0
Inorganic nitrogen concentrations and pH are identical with values of first incubation at tp168 h P1 c NHc ) 4 -N concentrations always below detection limit (~0.03 mg NH4 -N l
sensitive to the actual NHc 4 concentration than in the arable soil investigated in the present study. However, the inhibitory effects of N fertilization persisted after c nitrification of NHc 4 or NH4 -producing fertilizers, which is in line with my results, in particular after addition of P1 ). Therefore, a large amount of N (240 mg NHc 4 -N kg c both investigations suggest that NH4 acts as a competitive inhibitor for methanotrophic microorganisms, and that this effect is not fully reversible. These findings confirm previously published results, where long-term N fertilization (NH4NO3) inhibited CH4 oxidation strongly, although the actual NHc 4 concentrations in soil were below 1 mg N kg P1 (Hütsch et al. 1993). In addition, the larger the amount of fertilizer applied the lower the rate of CH4 oxidation (in the range of 48–144 kg N ha P1 year P1). This could be confirmed in the present study with a much higher degree of residual inhibition after NHc 4 application of 240 mg N kg P1 compared to 40 mg N kg P1 (64% compared to 16%, respectively). Contradictory results were obtained by Dunfield and Knowles (1995) in incubation studies of a humisol. NHc 4 acted as a simple competitive inhibitor for CH4 oxidation and the effect was fully reversible. The authors suggested that the absolute concentration and time of exposure affect the ability of methanotrophs to recover from NHc 4 inhibition, and that the extremely high natural nitrification rate of the humisol shields mec thanotrophs from NHc 4 . The maximum NH4 concentration in their incubation studies was approximately 13 mg N kg P1, and indeed much lower than those used in the experiments of Nesbit and Breitenbeck (1992) and in my own incubations. However, the nitrification rate P1 day P1 in my studies was as high as 26 mg NOP 3 -N kg c P1 after application of 40 mg N kg as NH4 or urea. Under field conditions one would expect lower nitrification rates, as suboptimal temperature and moisture conditions prevail at the time of fertilizer application in spring. Therefore, an even stronger residual inhibitory
effect on CH4 oxidation is most likely under natural nitrification conditions in the field than under laboratory conditions (25 7C, 14% H2O w/w in the present study). This results in long-term inhibition of CH4 oxidation in c regularly NHc 4 fertilized soils, although the actual NH4 concentrations were very low at the time of measurement (Hütsch et al. 1993; Hütsch 1996). In the urea-amended soil, CH4 oxidation was also strongly inhibited, and a 25% inhibition was still present after nitrification of the added N (Table 3). This result is consistent with results from measurements of CH4 fluxes from control and urea-N fertilized soils of a slash pine plantation (Castro et al. 1994). The daily average uptake of atmospheric CH4 by the fertilized soils (180 kg urea-N ha P1 year P1) was 5–20 times lower than that by control soils. The uppermost soil horizon of 0–2 cm was mainly responsible for this difference. At the three dates of flux measurements there were only small differences in NHc 4 concentrations between control and fertilized plots, being in the range of 1–10 mg P1 in the 0–2 cm depth (Castro et al. 1994). NHc 4 -N kg Thus, also in these experiments the inhibitory effect on CH4 oxidation persisted after nitrification of the major part of the added N. In contrast, flux measurements in corn and spring barley fields showed no effect of urea fertilization on CH4 consumption (Bronson and Mosier 1993; Delgado and Mosier 1996). The fertilizer was applied at a depth of 15 cm and 8 cm to corn and barley, respectively. From the inorganic N content in the 0–15 cm layer, 29 days after fertilization of the barley plot, when almost P no NHc 4 and only a small amount of NO3 was left (0.9 c P P1 P1 kg NH4 -N ha , 8.1 kg NO3 -N ha ), it can be assumed that the applied N was either taken up by the plants very quickly or was leached into deeper horizons favored by regular irrigation. Both factors, that in the case of the barley field the top 0–8 cm were almost not contaminated with N fertilizer, and that this horizon is very important for atmospheric CH4 uptake, result in
33
distinct locations of CH4 oxidation and nitrification. Therefore, under the experimental conditions reported by Bronson and Mosier (1993) and Delgado and Mosier (1996) an inhibitory effect of freshly applied urea on atmospheric CH4 consumption could not be expected. Effect of NOP 2 on CH4 oxidation P1 ) initially inhibited Addition of NOP 2 (40 mg N kg CH4 oxidation by 84%, decreasing to 41% after 48 h (Table 3). Exogenous NOP 2 was rapidly oxidized in this loamy arable soil, reaching levels slightly above backP1 in treatground at tp72 h (0.6 vs. 0.3 mg NOP 2 -N kg ment and control, respectively; Table 2). Gaseous N losses could be excluded, as the soil NOP 3 concentration at the end of incubation was in the range of that in the control plus the amount of applied N (52 mg NOP 3 -N kg P1). Thus a strong inhibition of CH4 oxidation ocP curred until the NOP 2 added was oxidized to NO3 . This non-competitive inhibition of CH4 oxidation via NOP 2 seems to be more persistent than the competitive inhibition via NHc 4 (41% vs. 16% and 21%, Table 3). Schnell and King (1994) also found in their incubation studies with a forest soil that exogenous NOP 2 was a more effective inhibitor of CH4 consumption than NHc 4. The fact that NHc 4 is partly adsorbed to soil particles immediately after application, whereas NOP 2 is fully present in the soil solution, could account for the observed differences. According to King and Schnell (1994a), the extent of NHc 4 inhibition increases with increasing CH4 concentration, which cannot be explained by competitive inhibition at the enzyme level. The authors propose that c NOP 2 formation from methanotrophic NH4 oxidation accounts for much of the observed inhibition. This might be true for the acid forest soil (pH 4) investigated, although NOP 2 was never detected in any soil sample and NOP 3 was the only product from NH3 oxidation (Schnell and King 1994). However, in my studies the NOP 2 oxidizers showed a high activity resulting in only a small accumulation of NOP 2 during nitrification P1 P ; of added NHc 4 or urea (maximum: 1.9 mg NO2 -N kg P P1 Table 2). Even the applied NO2 (40 mg N kg ) was decomposed after 72 h of incubation. Although NOP 2 is the final product of NH3 oxidation by methanotrophs, it can only accumulate and become detrimental under certain conditions. Either there are no NOP 2 oxidizers present (e.g. in pure cultures of methanotrophs; King and Schnell 1994b), or their activity is strongly restricted (e.g. in acid soils; Schnell and King 1994). In soils with optimal conditions for nitrification the inhibiP P tory effect of NHc 4 via NO2 is unlikely as NO2 is immediately oxidized as soon as it is produced. In incubation studies with a neutral landfill cover soil, Boeckx et al. (1996) observed the accumulation of P1 after NHc 31–59 ng NOP 2 -N g 4 application. The inP crease in NO2 was only detectable at a moisture con-
tent of 5%, and the authors concluded that under these dry conditions the accumulation of small amounts of NOP 2 could considerably reduce CH4 uptake. It is doubtful that these low NOP 2 concentrations caused an inhibition of the CH4 oxidizers, because in my experiments the NOP 2 -N concentration in the control soil was already 5–10 times higher. Probably the physiological stress of the microorganisms, caused by low water availability, resulted in the very low CH4 oxidation rates reported for the landfill soil. Effect of organic manure on CH4 oxidation Application of sugar beet leaves caused an instantaneous 20% inhibition of CH4 oxidation (Table 3). Steadily increasing concentrations of NOP 3 in the soil (Table 2) and of CO2 in the headspace of the incubation flasks (data not shown) indicated that mineralization of this fresh plant material proceeded well. During this process small amounts of NHc 4 accumulated (maxiP1 ; Table 2). Towards the end mum: 1.8 mg NHc 4 -N kg of incubation (tp120–168 h) anaerobic conditions occurred under limited availability of O2 for the microorganisms. Wheat straw application had no effect on CH4 oxidation (Tables 1, 3). Immobilization of inorganic N ocP P1 curred with a decrease in NOP 3 by 10 mg NO3 -N kg during the incubation (Table 2). The effect of organic residue amendments obviously depends on their C/N ratios, as postulated by Boeckx and Van Cleemput (1996). In their investigations, crop residues with a high C/N ratio (e.g. wheat straw) also stimulated N immobilization and did not affect CH4 oxidation, whereas crop residues with a low C/N ratio (e.g. sugar beet leaves) stimulated N mineralization resulting in a strong inhibition of CH4 oxidation. The almost 100% inhibition of CH4 oxidation after application of potato or sugar beet leaves is not in agreement with my results, where only a 20% inhibition occurred. In the experiments of Boeckx and Van Cleemput (1996) the amended soils had a 7-day pre-incubation before the first CH4 measurement was carried out. The P proceeding mineralization resulted in NHc 4 and NO2 P1 concentrations of 31 and 13 mg N kg , respectively, in the treatment with sugar beet leaves. Although these P amounts of accumulated NHc 4 and NO2 are unusually high and point to conditions unfavorable for nitrification, they were certainly sufficient for a 100% inhibition of CH4 oxidation. In my experiments the NHc 4 concentrations were rather small during the entire incubation and did not exceed 2.0 mg N kg P1 when aerobic conditions prevailed. CH4 oxidation increased by 28% (i.e. negative inhibition) after addition of biowaste compost to the soil (Table 3). During the incubation only small amounts of NOP 3 accumulated, which were comparable to levels in the control, and NHc 4 was not detectable (Table 2). Thus, compost affected neither mineralization nor im-
34
mobilization of N, and the support of CH4 oxidation is not related to the C/N ratio, pointing to another reason. The biowaste compost was collected after a maturation period of approximately 4 months. During this phase part of the organic material was anaerobically degraded in microsites of the compost heap, resulting in CH4 production. According to Bender and Conrad (1995), CH4 mixing ratios exceeding about 100–1000 ml CH4 l P1 are suitable to increase the numbers of methanotrophic bacteria. Although CH4 concentrations in compost heaps have not been measured so far, the evolving CH4 could be favorable for growth of methanotrophs in aerobic sites of the heap, e.g. at the surface, where both CH4 and O2 are available. Compost application to the soil is therefore comparable to an inoculation with CH4 oxidizers, which temporarily results in an increased CH4 oxidation rate. However, this effect lasts only for a short period, as these “additional” methanotrophs are not able to survive under natural conditions. In field experiments there was no difference in CH4 oxidation detectable between amended and unamended plots, measured 2 years after compost addition (application rate equivalent to that used in the laboratory incubations; Hütsch et al. 1997). Effect of pH on CH4 oxidation and its relationship with N turnover Methanotrophic bacteria in the loamy arable soil showed a strong sensitivity to decreases in soil pH. No difference in CH4 oxidation occurred between pH 8.0 (control) and pH 7.8; pH 7.6 caused a slight inhibition, whereas a pH of 7.4 and lower inhibited CH4 oxidation strongly (Fig. 2). Thus, in the range of pH 7.6–7.1, small changes of 0.2–0.3 units already had a strong impact on the activity of methanotrophs. The differences in soil pH between moderate N treatments and controls lay between 0.0 units and 0.3 units (individual results are not given). The largest
Fig. 2 CH4 oxidation of a loamy arable soil, adjusted to different pH values by HCl additions (native soil pH: 8.0). Each data point is the averageBSD of three replicates
changes in pH occurred after application of (NH4)2SO4 and NH4Cl with a decrease of 0.3 units. This small decrease had presumably no effect on CH4 oxidation (see Fig. 2). The inhibitory residual effect, which occurred after complete nitrification of the added NHc 4 and urea (16–25% inhibition, Table 3), was thus not a result of a decrease in soil pH. However, this is not the case after application of a higher amount of ammonium (6 times P1 ). When CH4 oxidation higher, i.e. 240 mg NHc 4 -N kg commenced after an entire incubation period of 216 h the pH was 7.3 (Table 4). Therefore, the observed 64% inhibition of CH4 oxidation after nitrification of the added N could have partly resulted from the concurrent drop in soil pH. Similar results could probably be obtained after frequent additions of N at moderate rates, usually applied in agriculture. The NHc 4 is, depending on the environmental conditions, more or less rapidly nitrified, whereas a pH shift is persistent until the next liming. Also in investigations of Arif et al. (1996) a sandy arable soil had a rather narrow pH range (5.9–7.7) which allowed CH4 oxidation. Both a decrease and an increase in soil pH by 1 unit from the native soil pH (6.8) retarded CH4 oxidation, and was regained after a delay of 2 days. After application of 100 mg NHc 4 -N kg P1 the soil pH dropped by more than 1 unit as a result of nitrification. In comparison to my results, this decrease is rather large, and reflects the differences in buffer capacity of the two soils investigated (sandy vs. loamy soil). Therefore, long-term inhibitory effects of N fertilization can partly be attributed to decreases in soil pH, provided that they exceed the limits of the optimum pH range of the prevailing methanotrophic population. Regular liming is a prerequisite for maintaining the soils ability to oxidize CH4. However, if the impact of NHc 4 lasted too long, an increase in pH could not restimulate CH4-oxidizing activity, as could be clearly demonstrated with measurements on the Park Grass Experiment at Rothamsted (Hütsch et al. 1994). In conclusion, with moderate, generally used agricultural application rates of NHc 4 or of substrates producduring decomposition, the strong inhibitory efing NHc 4 fect on CH4 oxidation depends on the competitive inhibition of the methanotrophs via NH3, which is, for still unknown reasons, only partly reversible after nitrification of the added N. The non-competitive inhibition of P NHc 4 via NO2 , which is the end product of NH3 oxidation by methanotrophic bacteria, is unlikely in well-aerated arable soils with a neutral pH, where NOP 2 is very rapidly oxidized to NOP 3 as soon as it is produced. A decrease in pH during nitrification could be partly responsible for the persistent inhibitory effect if either an unusually high amount of NHc 4 is applied or if pH decreases caused by frequent, moderate N additions are not counteracted by regular liming treatments. The application of NHc 4 fertilizers undoubtedly restrict CH4 oxidation in agricultural areas, which has significant ecological implications. The inhibition of CH4 oxida-
35
tion persists for long periods, and because of the large areas involved, the reduction in the atmospheric CH4 oxidation potential is significant. Acknowledgements Thanks are due to T. Appel and K. Mengel, Giessen, for helpful discussions. These investigations were financially supported by the Deutsche Forschungsgemeinschaft.
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