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a W.K. Kellogg Biological Station and Dep. of Crop and Soil Sciences, Michigan State Univ., 3700 E. Gull Lake Drive, Hickory Corners, MI 49060
b Dep. of Geological Sciences, 206 Natural Science Building, Michigan State University, East Lansing, MI 48823
* Corresponding author (tbergsma{at}kbs.msu.edu)
Received for publication April 23, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONCONCLUSIONREFERENCES |
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Abbreviations: BD, bulk density • NOS, nitrous oxide reductase • WFPS, water-filled pore space
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONCONCLUSIONREFERENCES |
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Numerous environmental factors can influence N2O mole fraction, including soil moisture, nitrate and nitrite concentration, pH, aeration, temperature, carbon availability, relative activities of NO-2 and N2O reductases, and moisture history (Colbourn and Dowdell, 1984; Sahrawat and Keeney, 1986; Firestone and Davidson, 1989; Arah and Smith, 1990; Bouwman, 1990; Aulakh et al., 1992; Hutchinson and Davidson, 1993). Moisture history may be particularly important because soil moisture status in most ecosystems can change rapidly; if denitrifying enzymes are induced differentially in response to wetting, then both the overall rate of denitrification (N2O + N2) as well as N2O mole fraction (N2O/[N2O + N2]) will differ substantially among ecosystems.
Many studies have documented ecosystem differences in the rate of denitrification following wetting (e.g., Gilliam et al., 1978; Rice and Smith, 1982; Robertson and Tiedje, 1985, 1988; Sexstone et al., 1986; Groffman and Tiedje, 1989; Ambus and Lowrance, 1991; Groffman et al., 1993), and some have noted denitrification differences between the wet-up and dry-down phases of soil moisture following rainfall events (e.g., Groffman and Tiedje, 1988). Fewer studies have examined the influence of moisture history on the nitrous oxide mole fraction (e.g., Dendooven and Anderson, 1995; Dendooven et al., 1996), and we know of no study that has explained how moisture history effects may differ with ecosystem management.
Moisture history effects on N2O mole fraction may help explain the differences in N2O flux among ecosystems. At our site, N2O flux from a cropped system was three times as high as flux from a successional field on the same soil series (Robertson et al., 2000). Denitrifier taxa—isolated from the cropped system and an adjacent never-tilled successional field—varied considerably in the sensitivity of nitrous oxide reductase (NOS) to oxygen, a soil factor that varies inversely with soil moisture (Cavigelli and Robertson, 2001). In whole soil slurry assays, denitrifying enzymes were more sensitive to oxygen in the cropped soil, and NOS was more active in the successional soil (Cavigelli and Robertson, 2000). We hypothesize that differences in microbial community enzyme activity influence responses of N2O mole fraction to rain events, helping to explain N2O flux differences. Since most N is lost from soils during brief periods following irrigation or rainfall (Smith and Tiedje, 1979; Sexstone et al., 1985; Rolston et al., 1982; Mummey et al., 1994; Davidson, 1991), variation among ecosystems in the response of N2O mole fraction to moisture history could have widespread significance for the global N2O budget.
In this study, we estimated nitrous oxide mole fraction for incubations of soil from two ecosystems (row crop agriculture and early native succession) and for two recent moisture histories in a factorial design. The primary objective was to determine if the effect of recent moisture history on N2O mole fraction depends on ecosystem management. A secondary objective was to compare the use of a 15N-gas evolution method with the acetylene (C2H2) inhibition method for estimating N2 gas flux.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONCONCLUSIONREFERENCES |
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The ongoing LTER experiment is a randomized complete block design with six replicate blocks and seven treatments on the main site, for a total of 42 one-hectare plots. We sampled two treatments: a high-input corn–soybean–wheat rotation and a native succession treatment last plowed in 1988. The annual cropping system is tilled and receives conventional applications of fertilizer and pesticides. The successional treatment is managed by occasional burning and/or felling of woody biomass to maintain an "old field" herbaceous community.
Soil was collected in December 1999 from three replicate blocks of each treatment. For each of six plots (two treatments by three blocks), four soil cores (2 x 16 cm) were collected at each of five sampling stations. Soil was bulked by plot, passed through a 4-mm screen, air-dried for 2 wk to about 1% gravimetric moisture, and stored in plastic bags at room temperature until the start of the experiment.
Experiment and Treatments
We incubated soil treatments for 24 h in 1-L glass mason jars. Twenty-six jars each received 150 g dry soil from one of three replicates of ecosystem. Soil was packed to a volume of approximately 125 mL. Each jar within a replicate set was assigned to one of two moisture histories (long-wet or short-wet, defined below), and one of three gas sampling strategies (15N-labeled soil, unlabeled soil, C2H2–amended soil) or reserved for mineral N analysis. Two additional jars were established without soil to serve as blanks for gas analysis.
Soil from the successional ecosystem was incubated 4 wk later than soil from the cropped system. Soil was sampled for mineral N availability at the start to test for storage effects. Successional soil did not pack as easily as cropped soil, possibly causing bias in repacked bulk density (BD) and water-filled pore space (WFPS). Bulk density and WFPS could not be measured directly. However, soil depth was measured on a subsample of jars (12 per ecosystem) at the conclusion of the experiment as an index of BD.
All soils received 9.75 mg of KNO3 (about 9 mg NO-3–N kg-1 dry soil), 20 mg glucose (about 53 mg C kg-1 dry soil), and 56.6 mL deionized water for a target WFPS of 85% at a BD of 1.2 g cm-3. Gravimetric moisture was 39% on a dry-soil basis. Long-wet soil received 80% of prescribed water 48 h before the start of the incubation; nitrate and glucose were added with the remaining water just before incubation. Short-wet soil received all water, nitrate, and glucose just before incubation. Blank jars received only 56.6 mL water (no soil). Solutions were delivered as a slow trickle down the edge of a tipped jar to minimize soil disturbance and air entrapment. The delivery method produced a wetting front that moved laterally across the soil within 15 min.
The 15N-labeled soil received 9.84 mg K15NO3, the molar equivalent of the 9.75 mg K14NO3 received under the other three strategies. The C2H2 jars received 80 mL C2H2 at the start of the incubation for a headspace concentration of 10 kPa (10% v/v), to inhibit NOS (Yoshinari and Knowles, 1976). All jars were fitted with air-tight lids; rubber septa and Cajon (Macedonia, OH) UltraTorr unions (custom o-ring seal) were added as necessary for syringe sampling and sampling to custom Pyrex vessels (0.5 L, preevacuated, with stopcocks) for 15N analysis (Bergsma et al., 2001).
Sampling and Analysis
Jars for mineral-N analysis of soil were sampled destructively for nitrate and ammonium about 2 h after the start of the incubation (10 g soil, dry weight equivalent, extracted in 100 mL 1 M KCl, followed by analysis using an Alpkem [Wilsonville, OR] autoanalyzer). The N2O concentrations in other jars were measured by gas chromatography at 0, 6, 12, and 24 h after the start of the incubation.
At the close of the incubation (24 h) gas samples were collected from 15N-labeled and unlabeled treatments for analysis by isotope ratio mass spectrometry. The vessel stopcocks were opened for about 10 s and then sealed. Analyses were performed within 2 wk, using the 15N-gas non-equilibrium technique (Bergsma et al., 2001). For N2O we measured m/z ratios 46/44 and 45/44. For N2, we measured ratios 30/28 and 29/28. Equations for estimating the 15N enrichment of the soil mineral N pool and the fraction of headspace gas derived from the soil mineral pool (d) require initial and final measurements of isotopic character (Arah, 1992; Bergsma et al., 1999). Because of the very large sample size, only a final sampling was possible. Therefore, each labeled sample was paired with its corresponding unlabeled sample to represent final and initial conditions, respectively. An advantage of this pairing is that it controls for slight biological and mechanical artifacts that could influence isotopic character under the experimental conditions described above. The N2 flux was calculated as the difference in N2O production between the C2H2–amended and control jars, using controls with unlabeled nitrate for all tests of treatment effects and using controls with labeled nitrate for comparison with N2 flux measured isotopically. Differences among treatment means were examined by analysis of variance (ANOVA), using JMPIN software version 3.1.5 (Sall and Lehman, 1996). The 15N-based estimates of the enrichment of the soil mineral pool were compared with mass-balance estimates calculated from nitrate availability in stock soil and amount of labeled nitrate added.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONCONCLUSIONREFERENCES |
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For the cropped soil, however, the long-wet pretreatment apparently enhanced the activity of NOS relative to the short-wet treatment, but not relative to successional soil. The N2O mole fraction was more than doubled in the absence of prewetting. We suggest that NOS did not persist well in the cropped soil when air-dry, but was restored by 48 h of high soil moisture. Since total denitrification was only slightly less for cropped soil than for successional soil, NOS precursors (e.g., nitrate reductase [NAR], NIR) may have been less affected by drying than was NOS.
Procedural considerations do not account for the patterns of gas production. Actual WFPS, while not measured, could have been greater on average for cropped soils than for successional soil, since achieved BD was apparently greater (mean soil depths and standard deviations were 15.6 ± 0.7 for cropped soil and 17.0 ± 1.1 for the successional soil). Greater WFPS should have favored induction of NOS and total denitrification; however, cropped soil showed NOS limitation and lower total denitrification. High soil moisture in general could have limited the diffusion of C2H2, causing total denitrification and N2 flux to be underestimated and N2O mole fraction to be overestimated. While the cropped soil showed numerically smaller N2 flux and greater N2O mole fraction, its strong response to moisture history remains to be explained.
To the best of our knowledge, no other published study of N2O mole fraction has examined the interaction between ecosystem management and moisture history. However, there are reports of effects of both ecosystem and moisture history on relative proportions of N2 and N2O. Merrill and Zak (1992) reported an N2O mole fraction of 0.7 to 0.9 for well-drained sugar maple (Acer saccharum Marshall subsp. saccharum) forests in northern lower Michigan; in contrast, the N2O mole fraction in a silver maple (Acer saccharinum L.)–red maple (Acer rubrum L.) swamp was 0.25. Dendooven et al. (1996) found an effect of moisture history on relative production of nitrous oxide and dinitrogen (N2O to N2) for pasture soil, but the difference was small: 0.54 for soil cores previously submerged for 96 h, and 0.4 for cores submerged for 6 h. Conversion to nitrous oxide mole fraction yields values of 0.35 and 0.29, similar to the values presented here for successional soil and long-wet cropped soil. Mulvaney and Kurtz (1984) studied N2O and N2 flux for three 15N-amended soils subjected to wetting and drying cycles. We calculate from their Table 1 an average and standard error of 0.33 ± 0.02 (n = 12), similar to the result for our successional soils: 0.33 ± 0.04 (n = 6). In a study of three N-amended soils, Jacinthe et al. (2000) found that N2O mole fraction was initially 0.68, increased to 0.95 with imposition of a water table at a depth of 10 cm, and decreased to 0.35 within 1 wk thereafter.
Our results show that the dependency of nitrous oxide mole fraction on recent moisture history may vary among ecosystems on the same soil series. The observation that N2O mole fraction is higher, shortly after a moisture increase, for the cropped soil than for the successional soil may help explain field data showing threefold greater annual flux of N2O from the cropped system (3.5 ± 0.21 g N2O-N ha-1 d-1) than from the successional system (1.1 ± 0.05 g N2O-N ha-1 d-1; Robertson et al., 2000).
Differences in microbial community enzyme activity may influence responses of N2O mole fraction to rain events. Perhaps lower availability of nitrate in the successional soil relative to the cropped soil (0.63 ± 0.04 and 6.54 ± 0.53 mg NO-3–N kg-1 dry soil, respectively; Robertson et al., 2000) has selected for a community of denitrifiers with the ability to maintain NOS status under dry (aerobic) conditions. Such a community could have a competitive advantage in exploiting the flush of carbon that occurs on soil wet-up (e.g., Groffman and Tiedje, 1988), since it could use N2O as well as NO-3 as a terminal electron acceptor if oxygen were limiting. In cropped soil, the incentive for NOS maintenance would be less, because of the abundance of the more energetically favorable electron acceptor NO-3.
Our work using repacked soil advances the work of others at Kellogg Biological Station. Cavigelli and Robertson (2001) isolated 31 denitrifier taxa from two ecosystems: the cropped ecosystem studied here and a nearby never-tilled successional field. They showed that considerable variability exists among taxa for sensitivity of the NOS enzyme to varying levels of oxygen, a parameter related to soil drying. Furthermore, Cavigelli and Robertson (2000) found differences in denitrifying ability for whole soil microbial communities (slurry assay) for the cropped ecosystem and the never-tilled successional field. Denitrifying enzymes were more sensitive to oxygen levels in the agricultural soil, and NOS was more active in the successional soil. Their results are consistent with our suggestion that the microbial community in the successional soil may have experienced selection for denitrifiers with the ability to maintain NOS status. The convergence of our results with previous results is noteworthy, given the additional structural complexity of repacked soil relative to soil slurries.
Moisture history, as a control on N2O mole fraction, has an important place in the biogeochemistry of nitrogen. Many studies suggest that most N is lost from soils during brief periods following irrigation or rainfall (Smith and Tiedje, 1979; Sexstone et al., 1985; Rolston et al., 1982; Mummey et al., 1994; Davidson, 1991). Dependency of the N2O mole fraction on short-term soil moisture history could have large consequences for the relationship between nitrous oxide production and total denitrification. Given our time scale of about 48 h, our finding is especially relevant for N2O models with a daily time step (e.g., Li et al., 1992a, b), as well as models explicitly invoking the N2O mole fraction (e.g., Parton et al., 1998). Improvement of biogeochemical models helps to constrain the global N2O budget (Davidson, 1991).
Isotopic Analyses
The 15N component of the experiment estimated production of N2 and 15N enrichment of the soil mineral N pool undergoing denitrification using either 15N2 or 15N2O data. Both measures of enrichment agreed well with enrichment estimated by mass balance (Table 2). A well-known weakness of C2H2 inhibition is that C2H2 also inhibits nitrification, potentially leading to underestimation of N gas flux. Agreement of measured and estimated enrichment supports the view that NO-3 was the principal substrate for N2O production in this experiment, implying a negligible contribution from nitrification.
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Methodological bias does not easily account for the differences in the two N2 flux estimates. If the C2H2 had been diffusion-limited by high soil moisture, N2 would have been underestimated rather than overestimated. It seems unlikely that C2H2 stimulated N2O production (e.g., Klemedtsson et al., 1988) in such a short interval, especially since C (glucose) was provided (see Topp and Germon, 1986). The 15N-gas evolution techniques can underestimate N2 flux when the soil mineral N pool undergoing denitrification is not uniform (Boast et al., 1988; Arah, 1992; Bergsma et al., 1999), but for most incubations, N2O derived from soil was in equilibrium or nearly so, implying a well-mixed soil source (above). Since N2O is the direct precursor of N2 (Payne, 1981), one would expect N2 from soil also to be in equilibrium (Focht, 1985) and therefore free of the underestimation ascribed to non-uniform pools. Furthermore, estimates of enrichment (of the soil mineral pool undergoing denitrification) based on N2 in our study agreed well with estimates based on N2O. Others have found similar results (Mulvaney and Kurtz, 1984; Mosier et al., 1986).
The two methods of calculating N2 production may have reflected qualitatively different aspects of the experimental system. Possibly the C2H2 method represented gross N2 production while the 15N-gas evolution technique represented only production from a highly enriched, uniformly labeled pool (i.e., the enriched N2O or its substrate). Apparently a second, unenriched soil mineral N pool was also a source of N2 (indeed the major source), but not a net source of N2O. Under these circumstances, N2 production would have been underestimated without affecting the estimate of enrichment for the labeled pool (see Focht, 1985), explaining the agreement of N2 and N2O data for estimates of pool enrichment (Table 2). The agreement of the mass balance estimates and the 15N estimates suggests further that the putative unlabeled source of N2 is not a static, extractable NO-3 pool. Additional study is needed to characterize potential mineral N sources for the experimental system described.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONCONCLUSIONREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONCONCLUSIONREFERENCES |
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for successional and cropped soil with long-wet and short-wet moisture history.