INTRODUCTION

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Atmospheric CH₄ contributes substantially to the greenhouse effect, and the concentration of atmospheric CH₄ has increased dramatically in the past century because of human activity associated with agriculture, land use changes, and industry (34, 35). Bacterial oxidation of atmospheric CH₄ in well-drained soils is an important regulator of atmospheric CH₄ concentration, yet the organisms responsible remain unidentified and the physiology of the process is poorly understood (9, 35, 36). Although soil CH₄ consumption is inhibited by a wide variety of anthropogenic disturbances, such as agriculture, N deposition, and forestry (12, 17, 22, 23, 32, 43, 44), predictable inhibition patterns have failed to emerge, which has made it difficult to predict the effects of disturbance on soil CH₄ flux in various ecosystems. The most commonly reported disturbance effect is that of NH₄⁺ fertilizers, which can suppress soil CH₄ consumption by up to 70% (1, 8, 10, 17, 22, 32, 33, 37, 38, 43). In the field, inhibition may occur immediately following fertilization, may be delayed for months to years, or may never occur despite years of chronic fertilization (9, 17). This variety of responses may stem at least in part from the distribution of physiologically diverse methane oxidizer populations across sites (17, 18, 20).

Of the various NH₄⁺ inhibition patterns, immediate inhibition is the best documented. As in field studies, however, physiological laboratory studies have produced variable results, suggesting that there may be multiple inhibition mechanisms (15, 17, 26-28, 36, 39). Physicochemical similarities between CH₄ and NH₃ may permit these two compounds to compete for enzyme active sites so that fortuitous NH₃ oxidation competitively inhibits CH₄ oxidation (38). Although this mechanism has been demonstrated to occur in pure cultures of methanotrophic bacteria (6) and in a CH₄-producing agricultural soil (15), it has not been demonstrated to occur in well-drained, nonagricultural mineral soils, which comprise the dominant terrestrial sink for atmospheric CH₄ (14, 38, 45), nor has it been demonstrated to occur at near-atmospheric CH₄ concentrations. In many cases, the kinetics of immediate NH₄⁺ inhibition in soil cannot be reconciled easily with substrate competition (15, 16, 26-28, 39). An alternative mechanism has been proposed, whereby the toxicity of NO₂ or NH₂OH produced by fortuitous NH₄⁺ oxidation suppresses methanotrophic activity (26, 27, 39). Hence, multiple inhibition mechanisms may be involved, and these mechanisms may vary with the physiology of different CH₄ oxidizer populations (17).

Two physiologically distinct communities of CH₄ oxidizers apparently exist in soil. One group, generally associated with atmospheric CH₄ consumption, exhibits an extremely high affinity for CH₄. Representatives of this group have yet to be cultivated or otherwise identified (9). The second group exhibits a much lower affinity for CH₄ and is generally associated with common methanotrophs, such as those that have been studied in pure culture for many years (2, 9). In upland mineral soils, only high-affinity activity is usually detectable without artificial enrichment with high CH₄ concentrations in the laboratory. However, the only prior study in which kinetic constants for NH₄⁺ inhibition of soil CH₄ oxidation were reported was conducted in a periodically moist, organic matter-rich agricultural soil with demonstrable methanogenesis (15, 16). Such a soil potentially harbors a rich community of CH₄ oxidizers representing a continuum from low-affinity organisms to high-affinity organisms. Although this important investigation demonstrated that NH₄⁺ inhibits CH₄ oxidation via enzymatic substrate competition in an agricultural humisol, it is unclear to what extent its results apply to well-drained mineral soils lacking endogenous CH₄ sources. Physiological studies of soil CH₄ oxidation typically derive kinetic constants from oxidation rates at CH₄ concentrations ranging from atmospheric levels (~1.7 µl liter¹) to K_m for high-affinity CH₄ oxidizers. Even in soil in which only high-affinity organisms are active, the CH₄-oxidizing enzyme(s) could respond differently to NH₄⁺ at high CH₄ concentrations than at near-atmospheric concentrations (15, 39). Thus, to study NH₄⁺ inhibition of high-affinity CH₄ oxidizers per se, it would be preferable to examine inhibition kinetics at near-atmospheric CH₄ concentrations in a soil with no apparent endogenous CH₄ source.

A common shortcoming of NH₄⁺ inhibition studies, regardless of the organisms involved, has been a lack of attention to nonammoniacal salt effects despite numerous reports of substantial inhibition by such salts (1, 10, 15, 17, 24). King and Schnell (28) examined the effects of several Cl and SO₄² salts and concluded that nonammoniacal salts indirectly inhibit CH₄ oxidation by desorbing endogenous NH₄⁺ from cation exchange sites in the soil, which then directly inhibit CH₄ oxidation. Many N-limited soils, however, have extremely low concentrations of exchangeable NH₄⁺, yet are substantially inhibited by nonammoniacal salts (17), suggesting that these salts have NH₄⁺-independent effects on atmospheric CH₄ oxidizers. Additional mechanisms may alter inhibition kinetics, thus hindering the diagnosis of NH₄⁺-specific inhibition.

With the limitations described above in mind, we used a simple steady-state kinetics approach to assess the mechanism of NH₄⁺ inhibition of CH₄ oxidation at near-atmospheric concentrations (1.8 to 15 µl liter¹) in three well-drained, N-limited forest soils that lack known endogenous CH₄ sources. In addition, we examined the effects of nonammoniacal salts in parallel samples to judge the utility of these salts as experimental controls for neutralizing NH₄⁺-independent salt effects.

	MATERIALS AND METHODS

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Field sites. We studied soils from two temperate forests and one taiga forest, the major characteristics of which are listed in Table 1. The two temperate soils were from the Harvard Forest Long-Term Ecological Research site in western Massachusetts (29), where fertilizer inhibition of atmospheric CH₄ consumption was first observed (43). The sites and their biogeochemical cycles have been described in detail previously (7, 8, 30). The taiga site is approximately 120 years postburn, and the understory is dominated by Rosa acicularis and Equisetum spp. The mineral soil consists of a uniform layer of silty glacial loess. The pedology, ecology, and biogeochemistry of this site are similar to the pedology, ecology, and biogeochemistry of nearby sites that have been described previously (17). All three sites are well drained and have never been observed to produce CH₄ (7, 8, 19).

Soil processing and bioassays. All experiments were performed at the University of Alaska, Fairbanks. At each site, soil was collected in bulk from the upper 10 cm of the mineral soil, which included the zone of maximum CH₄ oxidation, and stored in perforated plastic bags for transport to the laboratory. The soil was homogenized by sieving it through a 4-mm-mesh screen. The water-holding capacity of each soil type was determined as described previously (18), and the moisture was adjusted so that the final water content was 30 to 35% of the water-holding capacity (Table 1) after the final treatment with deionized water or salt solutions. This moisture level was determined previously to be optimal for atmospheric CH₄ consumption in a wide variety of soils (18). Samples were treated with deionized water, K₂SO₄, (NH₄)₂SO₄, Na₂SO₄ (taiga soil only), KCl, or NH₄Cl. Each salt solution was added to a single bulk sample (0.1 ml g of dry soil¹), which was then mixed thoroughly and subdivided into individual samples. The salts were added at a rate of 5.6 µmol of cations per g of dry soil, so that all of the salts were equinormal with respect to cations. The resulting NH₄⁺ additions were equivalent to 75 mg of N per kg of dry soil, which matched the treatments used in previous experiments (17). This amount of NH₄⁺ was intended to overwhelm the endogenous soil N (Table 1) yet remain within the range of soil NH₄⁺ concentrations reported for forest soils with various land use histories (13, 31, 42).

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View larger version (30K): [in this window] [in a new window]   FIG. 1.   CH4 oxidation kinetics in three upland forest soils, a birch taiga soil (a), a temperate hardwood soil (b), and a temperate pine soil (c). The data for the birch taiga soil are shown with linear regression lines, whereas the data for the temperate soils are shown with nonlinear regression curves fit to the Michaelis-Menten equation. Each point represents a single rate measurement.

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Statistical analyses and calculations. The effects of the salt treatments and CH₄ concentration on oxidation rates in each soil were analyzed by analysis of covariance by using treatment as the independent factor and the initial CH₄ concentration as a covariate; Bonferroni contrasts were used in multiple comparisons. Because the incubation times were the same for all treatments in a given soil, the treatments with higher oxidation rates consumed more substrate than the treatments with lower oxidation rates. For regression analyses, therefore, the oxidation rate from an individual assay was paired with the corresponding midpoint CH₄ concentration (Fig. 1) rather than the initial concentration. This standard technique normalizes consumption rates for unequal substrate concentrations among treatments and also minimizes the deviation from standard Michaelis kinetics that can result from substrate depletion (41). First-order kinetics were modeled by linear regression, and the rate constants were estimated from the slope of the regression line, with both variables expressed in picomoles. Michaelis constants were obtained from a least-squares nonlinear regression fit of the data to the Michaelis-Menten equation. For treatments exhibiting Michaelis kinetics, pseudo-first-order rate constants were calculated as V_max/K_m, with both constants expressed in picomoles. Relative inhibition was calculated for each treatment as follows: relative inhibition = (1  k₂/k₁) × 100, where k₁ is the first-order (or pseudo-first-order) rate constant for the control sample and k₂ is the first-order (or pseudo-first-order) rate constant for the treated sample.

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View larger version (27K): [in this window] [in a new window]   FIG. 2.   Effect of CH4 concentration on inhibition of CH4 oxidation. General salt inhibition compared to water controls in the birch taiga soil (a) and in the temperate hardwood soil (b). (c) Specific NH4+ inhibition compared to K+ controls in the temperate hardwood soil.

	RESULTS

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Birch taiga soil. In the birch taiga soil, the CH₄ oxidation kinetics at concentrations of 15 µl liter¹ were approximately first order (R² > 0.99 except for Cl salts) for all treatments, but the rate constants varied among treatments (Fig. 1a; Table 2). Analyses at higher CH₄ concentrations (10 to 800 µl liter¹) (data not shown) yielded a K_m(app) for oxidation in this soil of 39 µl liter¹ (57 nM in solution), which is typical for upland soils (2, 3, 36, 46, 47). Neither K₂SO₄ nor Na₂SO₄ significantly inhibited CH₄ oxidation compared to deionized water (P = 0.78), and the curves for K₂SO₄ and Na₂SO₄ were indistinguishable (P = 0.91) (Na₂SO₄ data not shown). Specific NH₄⁺ inhibition, calculated using K₂SO₄ as the control, was relatively weak (19%) but was statistically significant (P = 0.04). Both KCl and NH₄Cl inhibited CH₄ oxidation severely (~90%; slopes were significantly different from zero at P < 0.01) (Fig. 1a). Unlike the comparison of K₂SO₄ and (NH₄)₂SO₄, the effects of KCl and NH₄Cl were indistinguishable (P = 0.84) (Fig. 1a). All four salts caused relative inhibition to increase as CH₄ concentration increased (Fig. 2a). The slopes of the increases were similar for all salts regardless of which cation was added and regardless of the final soil NH₄⁺ concentration. All salts inhibited total microbial respiration, but like CH₄ oxidation, CO₂ production was more sensitive to Cl salts than to SO₄² salts; the relative inhibition was ~18% for both K₂SO₄ and (NH₄)₂SO₄, whereas it was 22 to 25% for KCl and NH₄Cl.

Temperate forest soils. The relative inhibition patterns for the various salts in the temperate hardwood and pine forest soils were similar to the patterns in the birch taiga soil, except that CH₄ oxidation conformed well to Michaelis-Menten kinetics (R² > 0.98 in most cases) (Fig. 1b and c; Table 2). With minor differences in magnitude, the pine soil exhibited the same patterns as the hardwood soil. As in the birch taiga soil, the Cl salts were the most inhibitory salts, followed by (NH₄)₂SO₄ and then K₂SO₄. K₂SO₄ inhibition and specific NH₄⁺ inhibition (relative to K⁺) were stronger in the temperate soils than in the taiga soil; the levels of specific NH₄⁺ inhibition in the temperate hardwood and pine soils were 54 and 34%, respectively (Table 2). As in the birch taiga soil, inhibition of CH₄ oxidation increased with the CH₄ concentration when K⁺ salts were added. Unlike the taiga soil, however, inhibition in the temperate soils decreased as CH₄ concentration increased when NH₄⁺ salts were added (Fig. 2b) (pine forest results not shown). When specific NH₄⁺ inhibition was calculated using K⁺ salts as controls, inhibition decreased sharply from ~50% in the presence of 1.8 µl of CH₄ liter¹ to ~20 to 30% in the presence of 15 µl of CH₄ liter¹ in the temperate hardwood soil (Fig. 2c).

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	DISCUSSION

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Often, soil CH₄ oxidation at near-atmospheric CH₄ concentrations follows first-order reaction kinetics (3, 39, 46), as was the case in the birch taiga soil in this study (Fig. 1a). In fine-textured soils, first-order kinetics at lower CH₄ concentrations may result from restricted gas diffusion from the atmosphere into the soil, a purely first-order process (14, 37, 45). The birch taiga soil studied is a fine silt soil and therefore strongly limits gas diffusion from the atmosphere to the CH₄ oxidizers (14, 37), thus possibly increasing the K_m(app) and creating a problem for studying low-concentration CH₄ oxidation kinetics in this soil (Fig. 3). By contrast, the two temperate forest soils studied have a coarse sandy texture, which enhances CH₄ diffusion, allowing uptake kinetics to reflect enzyme activity more closely and permitting standard kinetic analyses of NH₄⁺ and salt inhibition of CH₄ oxidation at near-atmospheric CH₄ concentrations. The maximum CH₄ concentration used in our experiments (15 µl liter¹) was similar to the K_m(app) values in the temperate forest soils. The regression curves resulting from the kinetic analyses provided very good fits to the actual data (generally, R² > 0.98) (Fig. 1; Table 2), indicating that the kinetic models which we used accurately described the process as measured in this study.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   Potential effect of soil texture on CH4 oxidation kinetics in soil. Because gas transport by diffusion is purely first order, a fine-textured soil may exhibit first-order kinetics at lower CH4 concentrations, whereas a coarse-textured soil containing the same CH4-oxidizing enzyme or a similar enzyme may exhibit Michaelis-Menten kinetics at the same concentrations.

Although there have been numerous reports of steady-state kinetic constants for soil CH₄ oxidation (2-4, 15, 36, 46, 47), only one previous study reported kinetic parameters for NH₄⁺ inhibition (15). The soil studied previously was an agricultural humisol with an organic matter content of ~70% and demonstrable methanogenic activity (15, 16). These conditions probably supported a much different CH₄ oxidizer community than the community expected in upland mineral soils that lack an endogenous CH₄ source. Indeed, the K_m(app) values for our temperate forest soils (Table 2) were substantially lower than the K_m(app) values reported in the previous study (15), suggesting that there were physiological differences in the CH₄ oxidizer communities in the upland mineral soils and the agricultural humisol. In the present study we focused specifically on high-affinity CH₄ oxidation in three non-CH₄-producing upland soils from two North American biomes, subarctic taiga forest and northeastern temperate forest.

Salt effects. Interpreting inhibition mechanisms based on kinetic parameters in a system that is as biologically and chemically complex as soil requires careful consideration of how ions added to the system may affect the process of interest, both directly and indirectly (15, 28). All of the ions used in this study potentially could affect CH₄ oxidation in three basic ways. First, they could change the soil osmotic potential and impose water stress on the microbial community (18, 40); second, they could affect ion exchange, thereby altering NH₄⁺ availability (28); and third, they could affect the CH₄ oxidizers directly in a number of ways (11, 15, 21). Any of these factors could alter CH₄ oxidation rates and kinetics and thus affect the interpretation of the specific NH₄⁺ inhibition mechanism.

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Specific NH₄⁺ inhibition. Determining the physiological mechanism of specific, immediate NH₄⁺ inhibition has proven to be difficult (6, 15, 17, 26-28). Dunfield and Knowles (15) demonstrated that NH₄⁺ inhibited CH₄ oxidation by enzymatic substrate competition in an agricultural humisol assayed at high CH₄ concentrations. The kinetics varied between samples, however, indicating that an additional mechanism may have been involved. King and Schnell (27, 39) examined NH₄Cl inhibition at low CH₄ concentrations and found that relative inhibition increased with CH₄ concentration. They concluded that this phenomenon resulted from the fortuitous oxidation of NH₄⁺ to toxic NO₂ or NH₂OH, which in turn reduced the activity of the methanotroph population (39). They did not examine Michaelis constants or comparable kinetic parameters. We observed similar increases in inhibition with increasing CH₄ concentrations in all three of the soils we examined. In the taiga soil, this phenomenon occurred with nonammoniacal salts as well as NH₄⁺ salts, and the slope of the increase was not affected by the NH₄⁺ concentration (Fig. 2a). In the temperate soils, K⁺ salts caused inhibition to increase, whereas NH₄⁺ salts caused inhibition to decrease as the CH₄ concentration increased (Fig. 2b and c). The same pattern occurred whether Cl or SO₄² salts were added, indicating that it was not specific to a particular counterion (Fig. 2a and b). These results indicate that the increase in inhibition did not result from NH₄⁺ or its by-products. Thus, although NO₂ undoubtedly inhibits atmospheric CH₄ oxidation when it is added directly to soil (19, 26), an increase in NH₄⁺ salt inhibition when the CH₄ concentration increases more likely results from a general salt effect than from by-products of fortuitous NH₄⁺ oxidation.

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Summary and conclusions. Our approach using K⁺ salts as controls, and the resulting interpretation, provided a plausible NH₄⁺ inhibition mechanism that is consistent with the data presented here and can also account for the contrasting results of previous studies (15, 27, 39). Whereas Dunfield and Knowles (15) observed competitive inhibition kinetics in their agricultural humisol, Schnell and King observed increasing inhibition with increasing CH₄ concentrations in a temperate forest soil, a result that, by itself, is inconsistent with competitive inhibition. Both phenomena occurred simultaneously in our temperate forest soils and could be explained by a mixed- type inhibition resulting from at least two independent mechanisms, enzymatic substrate competition by NH₄⁺ and one or more noncompetitive or uncompetitive mechanisms common to salts in general. Although the inhibition mechanism in the birch taiga soil could not be determined directly because it displayed first-order kinetics (Fig. 1a), the relative inhibition pattern for the various treatments was consistent with the patterns obtained with the two temperate soils, so that all three soils may have shared the same mechanisms. Moreover, it is notable that despite very different soil characteristics, we found essentially the same inhibition mechanism that Dunfield and Knowles (15) found in an agricultural humisol. This convergence of physiological responses in ecologically diverse environments suggests that enzymatic substrate competition is an important NH₄⁺ inhibition mechanism in a wide variety of soils.