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Factors regulating denitrification in a soil under pasture
PERGAMON
Soil Biology and Biochemistry 31 (1999) 913±927
Factors regulating denitri®cation in a soil under pasture J. Luo *, R.W. Tillman, P.R. Ball Institute of Natural Resources, Massey University, Palmerston North, New Zealand Accepted 9 December 1998
Abstract Experiments were conducted to obtain insights into factors regulating denitri®cation rate in a silt loam soil under permanent pasture in New Zealand, by removing possible limitations to denitri®cation during the incubation for the denitri®cation measurement. Soil temperature in the ®eld was found to limit denitri®cation rate in all seasons relative to the denitri®cation rate measured at 258C in the laboratory. This temperature eect was greatest in the cool±wet season. Additions of nitrate solution to soil cores stimulated denitri®cation rates in all seasons. This increase in denitri®cation rate suggests the availability of NO3ÿ may have limited denitri®cation in this pasture soil. Denitri®cation rates also increased when soluble-carbon was added to the soil cores, but the magnitude of the eect depended on other edaphic factors. A large increase in denitri®cation rate was obtained by saturating the soil cores collected in most seasons, but particularly during the warm±dry period. However, little enhanced eect on denitri®cation rate by anaerobic incubation of soil cores was observed. These results suggest that the observed eect of water addition on denitri®cation rate may have been due to the easier diusive movement of NO3ÿ , or possibly soluble-C, to the microsites where denitri®cation was occurring in this pasture, and the creation of anaerobic sites in the soil may not have been as important to the increase of denitri®cation rate. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction Environmental factors which aect microbiological denitri®cation have been identi®ed (Firestone, 1982; Tiedje, 1988), and the relative importance of these factors on both denitri®cation rate and its variability has been investigated in a number of ®eld studies (e.g. Ryden, 1983; Myrold et al., 1989; Aulakh et al., 1992; Schipper et al., 1993; Avalakki et al., 1995). In general, denitri®cation is promoted by high soil moisture conditions, high soil temperature, a low rate of oxygen diusion as well as the presence of soluble organic matter and nitrate. It has been often suggested that the spatial and temporal variations observed in soil denitri®cation rate in the ®eld could be attributed
* Corresponding author. Environmental Management Team, MIRINZ Food Technology and Research, P.O. Box 617, Hamilton, New Zealand. Tel.: +64-7-8299844; fax: +64-7-8299842; e-mail: [email protected].
to spatial and temporal changes in soil water content, O2 content, availability of C and NO3ÿ ±N, and to complex interactions between these variables (Smith, 1980; Ryden, 1986; Parkin, 1987; Estavillo et al., 1994). Large variations in soil denitri®cation rates in the grazed pasture in New Zealand have been observed in a few studies (e.g. Luo et al., 1994a, 1998; Ruz-Jerez et al., 1994). However, little is known of the factors controlling in situ denitri®cation and its variations, and few studies have been conducted to obtain insights into factors regulating the denitri®cation process in pastures. In our study individual soil cores were assessed for denitri®cation rate in the ®eld and then amended with water, NO3ÿ ±N or soluble-C and the denitri®cation rate was remeasured in the laboratory. It was hoped by this process to examine more closely the factors most limiting denitri®cation rate in dierent contrasting seasons and to understand the causes of the variation in denitri®cation rate in pastures.
0038-0717/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 0 1 3 - 9
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air was withdrawn from the tube and the same amount of puri®ed C2H2 (by passing industrial C2H2 gas through a high concentration of H2SO4) was injected into each tube using a syringe. The syringe was pumped several times to mix the C2H2 within the tube. The tubes were incubated for 24 h on the ground in a shaded place close to the paddock. At 1 and 24 h after the addition of C2H2, samples of the headspace gases were collected in 5 ml venoject evacuated test tubes (Becton Dickinson Vacutainer Systems).
2. Materials and methods 2.1. Site description The research was carried out using soil samples collected from a paddock (No. 17) of the Massey University No. 4 Dairy-farm, Palmerston North, New Zealand. The paddock was under ryegrass±white clover pasture and was periodically grazed by cows. During the period of study, the paddock received no N fertilisers. The soil at this site, the Tokomaru silt loam (Cowie, 1974), is classi®ed as a Yellow Grey Earth (Taylor and Pohlen, 1968) or a Pallic Soil (Hewitt, 1992). It is a poorly-drained soil with wet conditions in winter, and relatively dry conditions in summer. The paddock was predominantly ¯at with a small gully running through it.
2.3. Measurement of denitri®cation rate in the laboratory After the last gas sampling for ®eld denitri®cation measurement in individual cores, soil cores were removed to the laboratory and treated as follows. Of these 112 cores, 20 received no additional treatment, 12 were saturated with distilled water, 20 were amended with approximately 50 mg NO3ÿ ±N g ÿ 1 soil and were saturated, 20 were amended with approximately 300 mg glucose±C g ÿ 1 soil and were saturated, 20 were amended with both approximately 50 mg NO3ÿ ±N g ÿ 1 soil, and approximately 300 mg glucose± C g ÿ 1 soil and were saturated, and 20 received no N or C amendments but were incubated under unsaturated and anaerobic conditions. To achieve the desired N and C concentrations in soil cores, tests were made to decide how much water was needed to saturate a single soil core. The appropriate concentrations of KNO3 or glucose solutions were then calculated to provide the required quantities of N or C. Soil cores were carefully dipped into the solutions to absorb the required water and to obtain the enhanced NO3ÿ ±N or C concentrations. Table 2 presents information on the variation in soil properties between amended cores sampled on 25 January 1993. To obtain the anaerobic incubation conditions in the ®nal treatment, PVC
2.2. Measurement of denitri®cation rate in the ®eld Samples were collected on nine occasions in three contrasting seasons throughout a year from the ¯at land site in the paddock. On two occasions samples were taken from grazed and ungrazed areas in order to investigate the direct eect of grazing. The grazing management on these two occasions in winter of 1993 and summer of 1994 were described by Luo et al. (1999). Some soil properties on each of the sampling dates are given in Table 1. On each of the sampling occasions 112 soil cores were collected randomly from the site. The rate of denitri®cation was measured using the acetylene-inhibition technique (Yoshinari et al., 1977), using the individual soil core incubation system under ®eld conditions as described by Ryden et al. (1987). A core was approximately 2 cm dia. and 7.5 cm long. Individual cores were transferred from corers into PVC tubes (2.5 cm dia., 15 cm long). The tubes were closed at both ends with rubber septa. Six ml of
Table 1 Soil properties on sampling dates Climatic conditions
Date
Moisture (% ww ÿ 1)
Nitrate (mg NO3ÿ ±N kg ÿ 1)
Respiration (mg CO2±C kg ÿ 1 d ÿ 1)
Temperature (8C)
Warm±moist
17 08 07 25 30 30 09 20 05 05
35.0 38.1 40.0 26.6 15.9 16.0 51.3 52.6 47.0 48.5
0.6 0.6 0.8 10.1 8.1 12.9 1.9 0.6 0.8 3.1
19.0 18.6 N.D. 15.0 16.4 17.4 16.2 17.1 19.3 20.9
18 20 14 19 18 18 13 10 7 7
Warm±dry Cool±wet
a
Nov 1992 Dec 1992 Oct 1993 Jan 1993 Jan 1994a Jan 1994b June 1993 July 1993 Aug 1993a Aug 1993b
Ungrazed control site,bGrazed site.
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Table 2 Variation in soil properties in individual soil cores after application of treatments to samples collected on 25 January 1993
Moisture Nitrate concentration Respiration rate Denitri®cation rate
Treatment
Control
Saturation
NO3ÿ ±N amendment plus saturation
Glucose±C amendment plus saturation
NO3ÿ ±N and glucose±C amendment plus saturation
Unsaturated but anaerobic
mean (% ww ÿ 1) standard deviation mean (mg NO3ÿ ±N kg ÿ 1) standard deviation mean (mg CO2±C kg ÿ 1 d ÿ 1) standard deviation mean (mg N2O±N kg ÿ 1 d ÿ 1) standard deviation
26.3 2.4 11.0 7.8 30.3 4.0 0.02 0.01
54.2 3.3 12.8 4.8 30.1 3.5 0.68 0.19
55.1 2.7 48.2 5.5 35.8 2.1 0.40 0.024
54.0 3.9 15.8 10.9 60.0 2.2 0.37 0.19
54.8 4.0 47.9 5.5 59.5 2.6 3.22 0.19
25.1 3.8 7.1 5.0 5.9 1.2 0.021 0.022
tubes were evacuated and ¯ushed with pure N2 3 times and vented to atmospheric pressure. All cores were then incubated in the PVC tubes for 5 h with 6 ml of C2H2 at 258C, and at 1 and 5 h gas samples for N2O and CO2 analyses were collected.
2.4. Analytical methods A g.c. equipped with a 63 Ni e.c.d was used to measure the concentrations of N2O in the gas samples. The details of the measurements and the calculations were described by Luo et al. (1999). Carbon dioxide concentration was determined from the same gas samples as those used for N2O analysis. No signi®cant dierences in CO2 concentration had been observed between systems with or without added C2H2 during previous incubation tests, which indicates that the respiration rate in the soil was not signi®cantly aected by the addition of C2H2. The same ®ndings were made by Ryden (1982). Carbon dioxide was measured in a g.c. equipped with a t.c.d. Soil NO3ÿ ±N concentrations in each individual core, immediately following the incubation procedure, were analyzed using the method described by Luo et al. (1996). Original NO3ÿ ±N in each soil core was estimated by adding the amounts of the measured NO3ÿ ± N and denitri®ed N2O±N formed during the incubation. Moist soil were dried at 1058C for 24 h to determine the gravimetric soil moisture content. Due to the large spatial variation in denitri®cation rates among replicates in the ®eld (Luo et al., 1994b), the mean soil denitri®cation rates were calculated using Uniform Variance Unbiased Estimators. The arithmetic means of replicate denitri®cation rates were calculated for the laboratory data. Statistical analyses were performed using the Statistical Analysis System (SAS) (SAS Institute, 1985).
3. Results 3.1. Responses of denitri®cation to treatments The eects of soil amendment and subsequent incubation in the laboratory on denitri®cation rate are presented in Fig. 1 for samples collected when soils were warm and moist (October±December); in Fig. 2 for samples collected when soils were warm and dry (January); and in Fig. 3 for samples collected when soils were cool and moist (July±August). In each ®gure the ®lled circles and squares represent the mean rates of denitri®cation in the 12±20 (depending on treatment) individual cores at ®eld temperature prior to amendment and incubation in the laboratory at 258C, respectively. The initial mean rates were generally similar across treatments although there was some variation re¯ecting the large variability between individual cores that was identi®ed (Luo et al., 1994b). On each sampling occasion incubation of the unamended control cores at 258C in the laboratory increased the rate of denitri®cation above that measured at the cooler ®eld temperature (Figs. 1±3). As would be expected this dierence was greatest in the cool, wet season. In the amended treatments denitri®cation rates were also higher than in the original samples incubated at ®eld temperature (Figs. 1±3). In some treatments and at some sampling times these increases in denitri®cation rate were very large Ð sometimes greater than three orders of magnitude. The response of denitri®cation rates to saturation depended on sampling time (Figs. 1±3). During the warm±moist season, the increases in denitri®cation rate obtained by saturating the soil cores and incubating at 258C were higher than in the control cores but were consistently lower than the increases in denitri®cation rate in NO3ÿ ±amended soils (Fig. 1). In contrast, during the warm±dry period saturation alone
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Fig. 1. Denitri®cation rates in untreated soil cores collected in warm moist seasons and incubated at ®eld temperature (circle) and in the same cores after application of treatments and incubated at 258C (square) (Number adjacent to each data point indicates S.D.).
was sucient to induce a very large increase in denitri®cation rate (Fig. 2). However, there was no signi®cant dierence in the increase in denitri®cation rates between saturated and control cores collected during the cool±wet season (Fig. 3). Regardless of the season, denitri®cation rates were always strongly enhanced by NO3ÿ additions, although
the responses to added N in the warm±dry season were not as large as those in the other seasons (Figs. 1±3). The very large increases in denitri®cation rate after amendment with NO3ÿ ±N in all seasons suggest that availability of NO3ÿ ±N is likely to be one factor limiting denitri®cation in this pasture soil. The response of denitri®cation rate to glucose±C
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Fig. 2. Denitri®cation rates in untreated soil cores collected in warm dry seasons and incubated at ®eld temperature (circle) and in the same cores after application of treatments and incubated at 258C (square) (Number adjacent to each data point indicates S.D.).
solution addition also diered with season, with the largest increase being found in the warm±dry season (Figs. 1±3). In most cases, the eects of glucose±C on denitri®cation were much less than those of NO3ÿ ±N. The maximum rates of denitri®cation were observed when both NO3ÿ ±N and glucose±C
solutions were added (Figs. 1±3). These maximum denitri®cation rates were similar irrespective of when the soils were sampled. Anaerobic incubation of soil cores had little eect on the increase in denitri®cation rate compared with the control in all seasons (Figs. 1±3).
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Fig. 3. Denitri®cation rates in untreated soil cores collected in cool wet seasons and incubated at ®eld temperature (circle) and in the same cores after application of treatments and incubated at 258C (square) (Number adjacent to each data point indicates S.D.).
3.2. Relationships between NO3ÿ concentration, C availability and denitri®cation rate Examples of the relationship between soil NO3ÿ ±N concentration and denitri®cation rate for each set of treated cores on three sampling dates (representing the three contrasting seasons) are presented in Figs. 4±6.
Good linear relationships between denitri®cation rate (log-transformed data) and soil NO3ÿ ±N concentration were observed after saturation of soil cores collected during both the warm±moist and warm±dry seasons, or after saturation and addition of glucose to soil cores collected in all three seasons (Figs. 4±6). Under anaerobic incubation conditions, denitri®cation rates
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Fig. 4. Relationship between denitri®cation rates and soil nitrate concentrations in cores receiving the indicated treatments (17 Nov 1992) (Warm±moist season).
were proportional to soil NO3ÿ ±N concentrations in cores collected during the warm±dry season (Fig. 5). A positive relationship between denitri®cation rates and NO3ÿ ±N concentrations was also observed in the control soils sampled in the cool±wet season (Fig. 6).
Similar plots of denitri®cation rates (log± transformed data) against soil respiration rates gave good linear relationships when soils were incubated after NO3ÿ ±N amendment and saturation in all three seasons (Figs. 7±9). A good relationship between denitri®cation rate and soil respiration rate was also
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Fig. 5. Relationship between denitri®cation rates and soil nitrate concentrations in cores receiving the indicated treatments (25 Jan 1993) (Warm±dry season).
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Fig. 6. Relationship between denitri®cation rates and soil nitrate concentrations in cores receiving the indicated treatments (9 June 1993) (Cool± moist season).
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Fig. 7. Relationship between denitri®cation rates and soil respiration rates in cores receiving the indicated treatments (17 Nov 1992) (Warm±moist season).
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Fig. 8. Relationship between denitri®cation rates and soil respiration rates in cores receiving the indicated treatments (25 Jan. 1993) (Warm±dry season).
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Fig. 9. Relationship between denitri®cation rates and soil respiration rates in cores receiving the indicated treatments (9 June 1993) (Cool±moist season.)
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found when ®eld moist soils taken during January (warm±dry season) were incubated in the laboratory with no amendment (Fig. 8). 4. Discussion 4.1. In¯uence of soil temperature and soil water content on denitri®cation Biological denitri®cation is an anaerobic microbial process that depends on temperature (Knowles, 1982). Our data con®rm that denitri®cation in this pasture soil was limited by temperature in all seasons relative to the rates observed after incubation at 258C in the laboratory (Figs. 1±3). The majority of the soil cores that were saturated had higher rates of denitri®cation than those in the control (Figs. 1±3). This agrees with the results of Bremner and Shaw (1958), Grundmann and Rolston (1987), Myrold (1988) and Ruz-Jerez et al. (1994) who demonstrated that soil water content is a major factor determining the rate of denitri®cation. Water in soil pores controls denitri®cation through aecting both soil aeration and substrate movement. Denitri®cation requires anaerobic conditions, hence, many studies have demonstrated that the rate of denitri®cation can increase when the O2 concentration in soil decreases (e.g. Parkin and Tiedje, 1984; Tiedje, 1988; Arah et al., 1991). Surprisingly, our results revealed a general lack of denitri®cation rate response to removal of O2 (Figs. 1±3). This may suggest that factors other than O2 status in this pasture were more important in controlling denitri®cation. With little apparent eect of O2 concentration on denitri®cation, we suggest that the observed eect of soil water content on denitri®cation may have been due to the easier diusive movement of NO3ÿ or soluble-C to the microsites where denitri®cation was occurring in this pasture. This eect was most noticeable in samples collected in the warm±dry summer season (Fig. 2) when initial soil moisture was at its lowest. Other studies have also suggested that diusion can limit NO3ÿ , or even C, availability to denitri®cation even when these materials are present at relatively high concentrations in well-aggregated soils (Ryden, 1983; Myrold and Tiedje, 1985). 4.2. Availability of nitrate in soil associated with denitri®cation Considerable increases in denitri®cation rate after addition of NO3ÿ solutions were observed in our study in all seasons. This suggests that low availability of NO3ÿ may be a principal factor keeping the denitri®cation rate low in this pasture (Luo et al., 1994a). This
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result was consistent with other studies in dierent soils (Keller et al., 1988; Elliott et al., 1991). It is interesting to note therefore that in the studies on the spatial and temporal variation of denitri®cation in the study area (Luo et al., 1994a,b) denitri®cation rate was not strongly related to soil NO3ÿ ±N concentrations. The smallest eect of NO3ÿ addition was found in summer (Fig. 5) probably because there were relatively high concentrations of soil NO3ÿ ±N at that time. But even then denitri®cation was still substrate limited after water addition (Fig. 2). This may be because NO3ÿ was rapidly depleted in the denitri®cation sites, and diusion of NO3ÿ could then limit denitri®cation (Murray et al., 1989; Ambus and Christensen, 1993). Although it has been suggested that NO3ÿ does not limit denitri®cation in most agricultural soils (Parkin and Robinson, 1989), in those cases soil NO3ÿ ±N concentrations were generally higher than in the pasture soil studied here. 4.3. Availability of C in soil associated with denitri®cation Carbon availability has been recognised as one of the most important factors controlling the denitri®cation rate and the spatial variability in denitri®cation rate (Burford and Bremner, 1975; Parkin, 1987; Christensen et al., 1990; Weier et al., 1993). We found that the response of denitri®cation to C addition was not much greater than that obtained by the addition of water alone (Figs. 1±3). A separate study has also indicated that the distribution of available-C in this pasture was not the main reason for high spatial variation in denitri®cation rate (Luo, unpublished data). It seems likely that soil C was not an important regulatory factor for denitri®cation in this pasture. This is consistent with some other ®eld studies in grasslands (Elliott et al., 1991; Estavillo et al., 1994; Schnabel et al., 1996) and contrasts with the results in some cropped soils (e.g. Christensen et al., 1990). These results could be explained by the relatively high organic C concentrations in our study pasture compared with many cropped soils. The small eect of added glucose±C may also be due to immobilisation of available N in this N-limited soil in the presence of relatively high amounts of available-C. This is supported by the observation that denitri®cation rate appeared to be correlated with soil respiration when N was made non-limiting by amendment with NO3ÿ and saturation (Figs. 7±9). Therefore, the eect of C on denitri®cation may be in¯uenced by other soil factors in this pasture. The smaller response of denitri®cation to water plus glucose additions in winter and spring compared to summer re¯ects the lower available NO3ÿ concentrations in those seasons (Figs. 1±3). The greater quantities of denitri®cation in
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soil cores receiving both NO3ÿ and glucose further suggest that at high NO3ÿ concentration available-C may limit denitri®cation. This stimulation of denitri®cation may be due to either provision of C directly to denitrifying organisms or stimulation of the other heterotrophic organisms activity leading to reduced O2 concentrations in the soil (Beauchamp et al., 1989). This in turn may stimulate the denitri®cation rate if the denitri®ers have access to NO3ÿ (Sexstone et al., 1985). 4.4. In¯uence of grazing on amendment eect Our results indicate that grazing events can in¯uence denitri®cation from this pasture. Since NO3ÿ and denitri®cation enzyme activity were increased by grazing events (Luo et al., 1999), the eects of added substrates or water were slightly greater than that in the control sites (Figs. 2 and 3). However, the eect of grazing on observed denitri®cation response to the various amendments was less than the seasonal eects observed. 4.5. Conclusions Data from our experiments suggest that denitri®cation rates in all seasons were limited by availability of NO3ÿ ; in particular, the accessibility of NO3ÿ to the microsites of denitri®cation in the soil. Low availability and accessibility of NO3ÿ , in¯uenced by low water contents, was considered to be the most important factor limiting denitri®cation in the warm, dry summer, whereas low absolute concentrations of NO3ÿ were important in the other seasons. The interactive eects of NO3ÿ , C and water on denitri®cation were clearly demonstrated in our study. The use of NO3ÿ concentration or CO2 production as predictor variables for denitri®cation is reasonable in light of observations of good associations between denitri®cation rates and NO3ÿ ±N concentrations or CO2 emission rates under no C and N limiting, respectively. However, it is dicult to establish good correlations between denitri®cation rate and NO3ÿ ±N concentrations or CO2 emission rates on a single soil core in the study paddock for the whole year (Luo et al., 1994a). This may be due to the various limitations of C or N in dierent seasons, as we found in our investigation. References Ambus, P., Christensen, S., 1993. Denitri®cation variability and control in a riparian fen irrigated with agricultural drainage water. Soil Biology & Biochemistry 25, 915±923. Arah, J.R.M., Smith, K.A., Crichton, I.J., Li, H.S., 1991. Nitrous oxide production and denitri®cation in Scottish arable soils. Journal of Soil Science 42, 351±367.
Aulakh, M.S., Doran, J.W., Mosier, A.R., 1992. Soil denitri®cation ± signi®cance, measurement, and eects of management. Advances in Soil Science 18, 1±57. Avalakki, U.K., Strong, W.M., Sagna, P.G., 1995. Measurement of gaseous emissions from denitri®cation of applied nitrogen-15. III. Field measurement. Australian Journal of Soil Research 33, 101±111. Beauchamp, E.G., Trevors, J.T., Paul, J.W., 1989. Carbon sources for bacterial denitri®cation. Advances in Soil Science 10, 113±142. Bremner, J.M., Shaw, K., 1958. Denitri®cation in soil. II. Factors aecting denitri®cation. Journal of Agricultural Science 51, 39±52. Burford, J.R., Bremner, J.M., 1975. Relationships between the denitri®cation capacities of soils and total, water-soluble and readily decomposable soil organic matter. Soil Biology & Biochemistry 7, 389±394. Christensen, S., Simkins, S., Tiedje, J.M., 1990. Spatial variation in denitri®cation: dependency of activity centres on the soil environment. Soil Science Society of America Journal 54, 1608±1613. Cowie, J.D., 1974. Soils of Palmerston North City and Environs. New Zealand Soil Survey Report No. 24. New Zealand Soil Bureau, D.S.I.R. Wellington. Elliott, P.W., Knight, D., Anderson, J.M., 1991. Variables controlling denitri®cation from earthworm casts and soil in permanent pastures. Biology and Fertility of Soils 11, 24±29. Estavillo, J.M., Rodriguez, M., Domingo, M., Munoz-Rueda, A., Gonzalez-Murua, C., 1994. Denitri®cation from a natural grassland in the Basque Country under organic and inorganic fertilization. Plant and Soil 162, 19±29. Firestone, M.K., 1982. Biological denitri®cation. In: Stevenson, F.J. (Ed.), Nitrogen in Agricultural Soils. American Society of Agronomy, Madison, pp. 289±326. Grundmann, G.L., Rolston, D.E., 1987. A water function approximation to degree of anaerobiosis associated with denitri®cation. Soil Science 144, 437±441. Hewitt, A.E., 1992. New Zealand Soil Classi®cation. DSIR Land Resources Scienti®c Report No. 19. Keller, M., Kaplan, W.A., Wofsy, S.C., daCosta, J.M., 1988. Emissions of N2O from tropical forest soils: response to fertilization with NH4+ , NO3ÿ , and PO3ÿ 4 . Journal of Geophysical Research 93, 1600±1604. Knowles, R., 1982. Denitri®cation. Microbiological Reviews 46, 43± 70. Luo, J., Tillman, R.W., Ball, P.R., 1994a. Nitrogen loss by denitri®cation from a pasture. In: Currie, L.D., Loganathan, P. (Eds.), The Ecient Use of Fertilizers in a Changing Environment: Reconciling Productivity and Sustainability. Massey University, Palmerston North, pp. 139±151. Luo, J., Tillman, R.W., Ball, P.R., 1994b. Spatial variability of denitri®cation in a pasture. Transactions of International Congress of Soil Science 5b, 44±45. Luo, J., White, R.E., Ball, P.R., Tillman, R.W., 1996. Measuring denitri®cation activity in soils under pasture: optimizing conditions for the short-term denitri®cation enzyme assay and the eects of soil storage on denitri®cation activity. Soil Biology & Biochemistry 28, 409±417. Luo, J., Tillman, R.W., White, R.E., Ball, P.R., 1998. Variation in denitri®cation activity with soil depth under pasture. Soil Biology & Biochemistry 30, 897±903. Luo, J., Tillman, R.W., Ball, P.R., 1999. Grazing eect on denitri®cation in a soil under pasture during two contrasting seasons. Soil Biology & Biochemistry 903±912. Murray, R.E., Parsons, L.L., Smith, M.S., 1989. Kinetics of nitrate utilization by mixed populations of denitrifying bacteria. Applied and Environmental Microbiology 55, 717±721. Myrold, D.D., 1988. Denitri®cation in ryegrass and winter wheat cropping systems of Western Oregon. Soil Science Society of America Journal 52, 412±416.
J. Luo et al. / Soil Biology and Biochemistry 31 (1999) 913±927 Myrold, D.D., Tiedje, J.M., 1985. Diusional constraints on denitri®cation in soil. Soil Science Society of America Journal 49, 651± 657. Myrold, D.D., Matson, P.A., Peterson, D.L., 1989. Relationship between soil and microbial properties and above ground stand characteristics of conifer forests in Oregon. Biogeochemistry 8, 265±281. Parkin, T.B., 1987. Soil microsites as a source of denitri®cation variability. Soil Science Society of America Journal 51, 1194±1199. Parkin, T.B., Robinson, J.A., 1989. Stochastic models of soil denitri®cation. Applied and Environmental Microbiology 55, 72±77. Parkin, T.B., Tiedje, J.M., 1984. Application of a soil core method to investigate the eect of oxygen concentration on denitri®cation. Soil Biology & Biochemistry 16, 331±334. Ruz-Jerez, B.E., White, R.E., Ball, P.R., 1994. Long-term measurement of denitri®cation in three contrasting pastures grazed by sheep. Soil Biology & Biochemistry 26, 29±39. Ryden, J.C., 1982. Eects of acetylene on nitri®cation and denitri®cation in two soils during incubation with ammonium nitrate. Journal of Soil Science 33, 263±270. Ryden, J.C., 1983. Denitri®cation loss from a grassland soil in the ®eld receiving dierent rates on nitrogen as ammonium nitrate. Journal of Soil Science 34, 355±365. Ryden, J.C., 1986. Gaseous losses of nitrogen from grassland. In: van der Meer, H.G., Ryden, J.C., Ennik, G.C. (Eds.), Nitrogen Fluxes in Intensive Grassland Systems. Nijho, Dordrecht, pp. 59±73. Ryden, J.C., Skinner, J.H., Nixon, D.J., 1987. Soil core incubation system for the ®eld measurement of denitri®cation using acetyleneinhibition. Soil Biology & Biochemistry 19, 753±757.
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SAS Institute, 1985. Users guide: Statistics Version 5 Edition. Statistical Analysis System Institute Inc., Cary, NC. Schipper, L.A., Cooper, A.B., Harfoot, C.G., Dyck, W.J., 1993. Regulators of denitri®cation in an organic riparian soil. Soil Biology & Biochemistry 25, 925±933. Schnabel, R.R., Cornish, L.F., Stout, W.L., Shaer, J.A., 1996. Denitri®cation in a grassed and a wooded, valley and ridge, riparian ecotone. Journal of Environmental Quality 25, 1230±1235. Sexstone, A.J., Parkin, T.B., Tiedje, J.M., 1985. Temporal response of soil denitri®cation rates to rainfall and irrigation. Soil Science Society of America Journal 49, 99±103. Smith, K.A., 1980. A model of the extent of anaerobic zones in aggregated soils, and its potential application to estimates of denitri®cation. Journal of Soil Science 31, 263±277. Taylor, N.H., Pohlen, I.J., 1968. Classi®cation of New Zealand soils. In: Soils of New Zealand. N.Z. Soil Bureau Bulletin No. 26, D.S.I.R. Wellington. Tiedje, J.M., 1988. Ecology of denitri®cation and dissimilatory nitrate reduction to ammonium. In: Zehnder, A.J.B. (Ed.), Biology of Anaerobic Microorganisms. John Wiley, New York, pp. 179± 244. Weier, K.L., Doran, J.W., Power, J.F., Walters, D.T., 1993. Denitri®cation and the dinitrogen/nitrous oxide ratio as aected by soil water, available carbon, and nitrate. Soil Science Society of America Journal 57, 66±72. Yoshinari, T., Hynes, R., Knowles, R., 1977. Acetylene inhibition of nitrous oxide reduction and measurement of denitri®cation and nitrogen ®xation in soil. Soil Biology & Biochemistry 9, 177±183.
Soil Biology and Biochemistry 31 (1999) 913±927
Factors regulating denitri®cation in a soil under pasture J. Luo *, R.W. Tillman, P.R. Ball Institute of Natural Resources, Massey University, Palmerston North, New Zealand Accepted 9 December 1998
Abstract Experiments were conducted to obtain insights into factors regulating denitri®cation rate in a silt loam soil under permanent pasture in New Zealand, by removing possible limitations to denitri®cation during the incubation for the denitri®cation measurement. Soil temperature in the ®eld was found to limit denitri®cation rate in all seasons relative to the denitri®cation rate measured at 258C in the laboratory. This temperature eect was greatest in the cool±wet season. Additions of nitrate solution to soil cores stimulated denitri®cation rates in all seasons. This increase in denitri®cation rate suggests the availability of NO3ÿ may have limited denitri®cation in this pasture soil. Denitri®cation rates also increased when soluble-carbon was added to the soil cores, but the magnitude of the eect depended on other edaphic factors. A large increase in denitri®cation rate was obtained by saturating the soil cores collected in most seasons, but particularly during the warm±dry period. However, little enhanced eect on denitri®cation rate by anaerobic incubation of soil cores was observed. These results suggest that the observed eect of water addition on denitri®cation rate may have been due to the easier diusive movement of NO3ÿ , or possibly soluble-C, to the microsites where denitri®cation was occurring in this pasture, and the creation of anaerobic sites in the soil may not have been as important to the increase of denitri®cation rate. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction Environmental factors which aect microbiological denitri®cation have been identi®ed (Firestone, 1982; Tiedje, 1988), and the relative importance of these factors on both denitri®cation rate and its variability has been investigated in a number of ®eld studies (e.g. Ryden, 1983; Myrold et al., 1989; Aulakh et al., 1992; Schipper et al., 1993; Avalakki et al., 1995). In general, denitri®cation is promoted by high soil moisture conditions, high soil temperature, a low rate of oxygen diusion as well as the presence of soluble organic matter and nitrate. It has been often suggested that the spatial and temporal variations observed in soil denitri®cation rate in the ®eld could be attributed
* Corresponding author. Environmental Management Team, MIRINZ Food Technology and Research, P.O. Box 617, Hamilton, New Zealand. Tel.: +64-7-8299844; fax: +64-7-8299842; e-mail: [email protected].
to spatial and temporal changes in soil water content, O2 content, availability of C and NO3ÿ ±N, and to complex interactions between these variables (Smith, 1980; Ryden, 1986; Parkin, 1987; Estavillo et al., 1994). Large variations in soil denitri®cation rates in the grazed pasture in New Zealand have been observed in a few studies (e.g. Luo et al., 1994a, 1998; Ruz-Jerez et al., 1994). However, little is known of the factors controlling in situ denitri®cation and its variations, and few studies have been conducted to obtain insights into factors regulating the denitri®cation process in pastures. In our study individual soil cores were assessed for denitri®cation rate in the ®eld and then amended with water, NO3ÿ ±N or soluble-C and the denitri®cation rate was remeasured in the laboratory. It was hoped by this process to examine more closely the factors most limiting denitri®cation rate in dierent contrasting seasons and to understand the causes of the variation in denitri®cation rate in pastures.
0038-0717/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 0 1 3 - 9
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air was withdrawn from the tube and the same amount of puri®ed C2H2 (by passing industrial C2H2 gas through a high concentration of H2SO4) was injected into each tube using a syringe. The syringe was pumped several times to mix the C2H2 within the tube. The tubes were incubated for 24 h on the ground in a shaded place close to the paddock. At 1 and 24 h after the addition of C2H2, samples of the headspace gases were collected in 5 ml venoject evacuated test tubes (Becton Dickinson Vacutainer Systems).
2. Materials and methods 2.1. Site description The research was carried out using soil samples collected from a paddock (No. 17) of the Massey University No. 4 Dairy-farm, Palmerston North, New Zealand. The paddock was under ryegrass±white clover pasture and was periodically grazed by cows. During the period of study, the paddock received no N fertilisers. The soil at this site, the Tokomaru silt loam (Cowie, 1974), is classi®ed as a Yellow Grey Earth (Taylor and Pohlen, 1968) or a Pallic Soil (Hewitt, 1992). It is a poorly-drained soil with wet conditions in winter, and relatively dry conditions in summer. The paddock was predominantly ¯at with a small gully running through it.
2.3. Measurement of denitri®cation rate in the laboratory After the last gas sampling for ®eld denitri®cation measurement in individual cores, soil cores were removed to the laboratory and treated as follows. Of these 112 cores, 20 received no additional treatment, 12 were saturated with distilled water, 20 were amended with approximately 50 mg NO3ÿ ±N g ÿ 1 soil and were saturated, 20 were amended with approximately 300 mg glucose±C g ÿ 1 soil and were saturated, 20 were amended with both approximately 50 mg NO3ÿ ±N g ÿ 1 soil, and approximately 300 mg glucose± C g ÿ 1 soil and were saturated, and 20 received no N or C amendments but were incubated under unsaturated and anaerobic conditions. To achieve the desired N and C concentrations in soil cores, tests were made to decide how much water was needed to saturate a single soil core. The appropriate concentrations of KNO3 or glucose solutions were then calculated to provide the required quantities of N or C. Soil cores were carefully dipped into the solutions to absorb the required water and to obtain the enhanced NO3ÿ ±N or C concentrations. Table 2 presents information on the variation in soil properties between amended cores sampled on 25 January 1993. To obtain the anaerobic incubation conditions in the ®nal treatment, PVC
2.2. Measurement of denitri®cation rate in the ®eld Samples were collected on nine occasions in three contrasting seasons throughout a year from the ¯at land site in the paddock. On two occasions samples were taken from grazed and ungrazed areas in order to investigate the direct eect of grazing. The grazing management on these two occasions in winter of 1993 and summer of 1994 were described by Luo et al. (1999). Some soil properties on each of the sampling dates are given in Table 1. On each of the sampling occasions 112 soil cores were collected randomly from the site. The rate of denitri®cation was measured using the acetylene-inhibition technique (Yoshinari et al., 1977), using the individual soil core incubation system under ®eld conditions as described by Ryden et al. (1987). A core was approximately 2 cm dia. and 7.5 cm long. Individual cores were transferred from corers into PVC tubes (2.5 cm dia., 15 cm long). The tubes were closed at both ends with rubber septa. Six ml of
Table 1 Soil properties on sampling dates Climatic conditions
Date
Moisture (% ww ÿ 1)
Nitrate (mg NO3ÿ ±N kg ÿ 1)
Respiration (mg CO2±C kg ÿ 1 d ÿ 1)
Temperature (8C)
Warm±moist
17 08 07 25 30 30 09 20 05 05
35.0 38.1 40.0 26.6 15.9 16.0 51.3 52.6 47.0 48.5
0.6 0.6 0.8 10.1 8.1 12.9 1.9 0.6 0.8 3.1
19.0 18.6 N.D. 15.0 16.4 17.4 16.2 17.1 19.3 20.9
18 20 14 19 18 18 13 10 7 7
Warm±dry Cool±wet
a
Nov 1992 Dec 1992 Oct 1993 Jan 1993 Jan 1994a Jan 1994b June 1993 July 1993 Aug 1993a Aug 1993b
Ungrazed control site,bGrazed site.
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Table 2 Variation in soil properties in individual soil cores after application of treatments to samples collected on 25 January 1993
Moisture Nitrate concentration Respiration rate Denitri®cation rate
Treatment
Control
Saturation
NO3ÿ ±N amendment plus saturation
Glucose±C amendment plus saturation
NO3ÿ ±N and glucose±C amendment plus saturation
Unsaturated but anaerobic
mean (% ww ÿ 1) standard deviation mean (mg NO3ÿ ±N kg ÿ 1) standard deviation mean (mg CO2±C kg ÿ 1 d ÿ 1) standard deviation mean (mg N2O±N kg ÿ 1 d ÿ 1) standard deviation
26.3 2.4 11.0 7.8 30.3 4.0 0.02 0.01
54.2 3.3 12.8 4.8 30.1 3.5 0.68 0.19
55.1 2.7 48.2 5.5 35.8 2.1 0.40 0.024
54.0 3.9 15.8 10.9 60.0 2.2 0.37 0.19
54.8 4.0 47.9 5.5 59.5 2.6 3.22 0.19
25.1 3.8 7.1 5.0 5.9 1.2 0.021 0.022
tubes were evacuated and ¯ushed with pure N2 3 times and vented to atmospheric pressure. All cores were then incubated in the PVC tubes for 5 h with 6 ml of C2H2 at 258C, and at 1 and 5 h gas samples for N2O and CO2 analyses were collected.
2.4. Analytical methods A g.c. equipped with a 63 Ni e.c.d was used to measure the concentrations of N2O in the gas samples. The details of the measurements and the calculations were described by Luo et al. (1999). Carbon dioxide concentration was determined from the same gas samples as those used for N2O analysis. No signi®cant dierences in CO2 concentration had been observed between systems with or without added C2H2 during previous incubation tests, which indicates that the respiration rate in the soil was not signi®cantly aected by the addition of C2H2. The same ®ndings were made by Ryden (1982). Carbon dioxide was measured in a g.c. equipped with a t.c.d. Soil NO3ÿ ±N concentrations in each individual core, immediately following the incubation procedure, were analyzed using the method described by Luo et al. (1996). Original NO3ÿ ±N in each soil core was estimated by adding the amounts of the measured NO3ÿ ± N and denitri®ed N2O±N formed during the incubation. Moist soil were dried at 1058C for 24 h to determine the gravimetric soil moisture content. Due to the large spatial variation in denitri®cation rates among replicates in the ®eld (Luo et al., 1994b), the mean soil denitri®cation rates were calculated using Uniform Variance Unbiased Estimators. The arithmetic means of replicate denitri®cation rates were calculated for the laboratory data. Statistical analyses were performed using the Statistical Analysis System (SAS) (SAS Institute, 1985).
3. Results 3.1. Responses of denitri®cation to treatments The eects of soil amendment and subsequent incubation in the laboratory on denitri®cation rate are presented in Fig. 1 for samples collected when soils were warm and moist (October±December); in Fig. 2 for samples collected when soils were warm and dry (January); and in Fig. 3 for samples collected when soils were cool and moist (July±August). In each ®gure the ®lled circles and squares represent the mean rates of denitri®cation in the 12±20 (depending on treatment) individual cores at ®eld temperature prior to amendment and incubation in the laboratory at 258C, respectively. The initial mean rates were generally similar across treatments although there was some variation re¯ecting the large variability between individual cores that was identi®ed (Luo et al., 1994b). On each sampling occasion incubation of the unamended control cores at 258C in the laboratory increased the rate of denitri®cation above that measured at the cooler ®eld temperature (Figs. 1±3). As would be expected this dierence was greatest in the cool, wet season. In the amended treatments denitri®cation rates were also higher than in the original samples incubated at ®eld temperature (Figs. 1±3). In some treatments and at some sampling times these increases in denitri®cation rate were very large Ð sometimes greater than three orders of magnitude. The response of denitri®cation rates to saturation depended on sampling time (Figs. 1±3). During the warm±moist season, the increases in denitri®cation rate obtained by saturating the soil cores and incubating at 258C were higher than in the control cores but were consistently lower than the increases in denitri®cation rate in NO3ÿ ±amended soils (Fig. 1). In contrast, during the warm±dry period saturation alone
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Fig. 1. Denitri®cation rates in untreated soil cores collected in warm moist seasons and incubated at ®eld temperature (circle) and in the same cores after application of treatments and incubated at 258C (square) (Number adjacent to each data point indicates S.D.).
was sucient to induce a very large increase in denitri®cation rate (Fig. 2). However, there was no signi®cant dierence in the increase in denitri®cation rates between saturated and control cores collected during the cool±wet season (Fig. 3). Regardless of the season, denitri®cation rates were always strongly enhanced by NO3ÿ additions, although
the responses to added N in the warm±dry season were not as large as those in the other seasons (Figs. 1±3). The very large increases in denitri®cation rate after amendment with NO3ÿ ±N in all seasons suggest that availability of NO3ÿ ±N is likely to be one factor limiting denitri®cation in this pasture soil. The response of denitri®cation rate to glucose±C
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Fig. 2. Denitri®cation rates in untreated soil cores collected in warm dry seasons and incubated at ®eld temperature (circle) and in the same cores after application of treatments and incubated at 258C (square) (Number adjacent to each data point indicates S.D.).
solution addition also diered with season, with the largest increase being found in the warm±dry season (Figs. 1±3). In most cases, the eects of glucose±C on denitri®cation were much less than those of NO3ÿ ±N. The maximum rates of denitri®cation were observed when both NO3ÿ ±N and glucose±C
solutions were added (Figs. 1±3). These maximum denitri®cation rates were similar irrespective of when the soils were sampled. Anaerobic incubation of soil cores had little eect on the increase in denitri®cation rate compared with the control in all seasons (Figs. 1±3).
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Fig. 3. Denitri®cation rates in untreated soil cores collected in cool wet seasons and incubated at ®eld temperature (circle) and in the same cores after application of treatments and incubated at 258C (square) (Number adjacent to each data point indicates S.D.).
3.2. Relationships between NO3ÿ concentration, C availability and denitri®cation rate Examples of the relationship between soil NO3ÿ ±N concentration and denitri®cation rate for each set of treated cores on three sampling dates (representing the three contrasting seasons) are presented in Figs. 4±6.
Good linear relationships between denitri®cation rate (log-transformed data) and soil NO3ÿ ±N concentration were observed after saturation of soil cores collected during both the warm±moist and warm±dry seasons, or after saturation and addition of glucose to soil cores collected in all three seasons (Figs. 4±6). Under anaerobic incubation conditions, denitri®cation rates
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Fig. 4. Relationship between denitri®cation rates and soil nitrate concentrations in cores receiving the indicated treatments (17 Nov 1992) (Warm±moist season).
were proportional to soil NO3ÿ ±N concentrations in cores collected during the warm±dry season (Fig. 5). A positive relationship between denitri®cation rates and NO3ÿ ±N concentrations was also observed in the control soils sampled in the cool±wet season (Fig. 6).
Similar plots of denitri®cation rates (log± transformed data) against soil respiration rates gave good linear relationships when soils were incubated after NO3ÿ ±N amendment and saturation in all three seasons (Figs. 7±9). A good relationship between denitri®cation rate and soil respiration rate was also
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Fig. 5. Relationship between denitri®cation rates and soil nitrate concentrations in cores receiving the indicated treatments (25 Jan 1993) (Warm±dry season).
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Fig. 6. Relationship between denitri®cation rates and soil nitrate concentrations in cores receiving the indicated treatments (9 June 1993) (Cool± moist season).
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Fig. 7. Relationship between denitri®cation rates and soil respiration rates in cores receiving the indicated treatments (17 Nov 1992) (Warm±moist season).
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Fig. 8. Relationship between denitri®cation rates and soil respiration rates in cores receiving the indicated treatments (25 Jan. 1993) (Warm±dry season).
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Fig. 9. Relationship between denitri®cation rates and soil respiration rates in cores receiving the indicated treatments (9 June 1993) (Cool±moist season.)
J. Luo et al. / Soil Biology and Biochemistry 31 (1999) 913±927
found when ®eld moist soils taken during January (warm±dry season) were incubated in the laboratory with no amendment (Fig. 8). 4. Discussion 4.1. In¯uence of soil temperature and soil water content on denitri®cation Biological denitri®cation is an anaerobic microbial process that depends on temperature (Knowles, 1982). Our data con®rm that denitri®cation in this pasture soil was limited by temperature in all seasons relative to the rates observed after incubation at 258C in the laboratory (Figs. 1±3). The majority of the soil cores that were saturated had higher rates of denitri®cation than those in the control (Figs. 1±3). This agrees with the results of Bremner and Shaw (1958), Grundmann and Rolston (1987), Myrold (1988) and Ruz-Jerez et al. (1994) who demonstrated that soil water content is a major factor determining the rate of denitri®cation. Water in soil pores controls denitri®cation through aecting both soil aeration and substrate movement. Denitri®cation requires anaerobic conditions, hence, many studies have demonstrated that the rate of denitri®cation can increase when the O2 concentration in soil decreases (e.g. Parkin and Tiedje, 1984; Tiedje, 1988; Arah et al., 1991). Surprisingly, our results revealed a general lack of denitri®cation rate response to removal of O2 (Figs. 1±3). This may suggest that factors other than O2 status in this pasture were more important in controlling denitri®cation. With little apparent eect of O2 concentration on denitri®cation, we suggest that the observed eect of soil water content on denitri®cation may have been due to the easier diusive movement of NO3ÿ or soluble-C to the microsites where denitri®cation was occurring in this pasture. This eect was most noticeable in samples collected in the warm±dry summer season (Fig. 2) when initial soil moisture was at its lowest. Other studies have also suggested that diusion can limit NO3ÿ , or even C, availability to denitri®cation even when these materials are present at relatively high concentrations in well-aggregated soils (Ryden, 1983; Myrold and Tiedje, 1985). 4.2. Availability of nitrate in soil associated with denitri®cation Considerable increases in denitri®cation rate after addition of NO3ÿ solutions were observed in our study in all seasons. This suggests that low availability of NO3ÿ may be a principal factor keeping the denitri®cation rate low in this pasture (Luo et al., 1994a). This
925
result was consistent with other studies in dierent soils (Keller et al., 1988; Elliott et al., 1991). It is interesting to note therefore that in the studies on the spatial and temporal variation of denitri®cation in the study area (Luo et al., 1994a,b) denitri®cation rate was not strongly related to soil NO3ÿ ±N concentrations. The smallest eect of NO3ÿ addition was found in summer (Fig. 5) probably because there were relatively high concentrations of soil NO3ÿ ±N at that time. But even then denitri®cation was still substrate limited after water addition (Fig. 2). This may be because NO3ÿ was rapidly depleted in the denitri®cation sites, and diusion of NO3ÿ could then limit denitri®cation (Murray et al., 1989; Ambus and Christensen, 1993). Although it has been suggested that NO3ÿ does not limit denitri®cation in most agricultural soils (Parkin and Robinson, 1989), in those cases soil NO3ÿ ±N concentrations were generally higher than in the pasture soil studied here. 4.3. Availability of C in soil associated with denitri®cation Carbon availability has been recognised as one of the most important factors controlling the denitri®cation rate and the spatial variability in denitri®cation rate (Burford and Bremner, 1975; Parkin, 1987; Christensen et al., 1990; Weier et al., 1993). We found that the response of denitri®cation to C addition was not much greater than that obtained by the addition of water alone (Figs. 1±3). A separate study has also indicated that the distribution of available-C in this pasture was not the main reason for high spatial variation in denitri®cation rate (Luo, unpublished data). It seems likely that soil C was not an important regulatory factor for denitri®cation in this pasture. This is consistent with some other ®eld studies in grasslands (Elliott et al., 1991; Estavillo et al., 1994; Schnabel et al., 1996) and contrasts with the results in some cropped soils (e.g. Christensen et al., 1990). These results could be explained by the relatively high organic C concentrations in our study pasture compared with many cropped soils. The small eect of added glucose±C may also be due to immobilisation of available N in this N-limited soil in the presence of relatively high amounts of available-C. This is supported by the observation that denitri®cation rate appeared to be correlated with soil respiration when N was made non-limiting by amendment with NO3ÿ and saturation (Figs. 7±9). Therefore, the eect of C on denitri®cation may be in¯uenced by other soil factors in this pasture. The smaller response of denitri®cation to water plus glucose additions in winter and spring compared to summer re¯ects the lower available NO3ÿ concentrations in those seasons (Figs. 1±3). The greater quantities of denitri®cation in
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soil cores receiving both NO3ÿ and glucose further suggest that at high NO3ÿ concentration available-C may limit denitri®cation. This stimulation of denitri®cation may be due to either provision of C directly to denitrifying organisms or stimulation of the other heterotrophic organisms activity leading to reduced O2 concentrations in the soil (Beauchamp et al., 1989). This in turn may stimulate the denitri®cation rate if the denitri®ers have access to NO3ÿ (Sexstone et al., 1985). 4.4. In¯uence of grazing on amendment eect Our results indicate that grazing events can in¯uence denitri®cation from this pasture. Since NO3ÿ and denitri®cation enzyme activity were increased by grazing events (Luo et al., 1999), the eects of added substrates or water were slightly greater than that in the control sites (Figs. 2 and 3). However, the eect of grazing on observed denitri®cation response to the various amendments was less than the seasonal eects observed. 4.5. Conclusions Data from our experiments suggest that denitri®cation rates in all seasons were limited by availability of NO3ÿ ; in particular, the accessibility of NO3ÿ to the microsites of denitri®cation in the soil. Low availability and accessibility of NO3ÿ , in¯uenced by low water contents, was considered to be the most important factor limiting denitri®cation in the warm, dry summer, whereas low absolute concentrations of NO3ÿ were important in the other seasons. The interactive eects of NO3ÿ , C and water on denitri®cation were clearly demonstrated in our study. The use of NO3ÿ concentration or CO2 production as predictor variables for denitri®cation is reasonable in light of observations of good associations between denitri®cation rates and NO3ÿ ±N concentrations or CO2 emission rates under no C and N limiting, respectively. However, it is dicult to establish good correlations between denitri®cation rate and NO3ÿ ±N concentrations or CO2 emission rates on a single soil core in the study paddock for the whole year (Luo et al., 1994a). This may be due to the various limitations of C or N in dierent seasons, as we found in our investigation. References Ambus, P., Christensen, S., 1993. Denitri®cation variability and control in a riparian fen irrigated with agricultural drainage water. Soil Biology & Biochemistry 25, 915±923. Arah, J.R.M., Smith, K.A., Crichton, I.J., Li, H.S., 1991. Nitrous oxide production and denitri®cation in Scottish arable soils. Journal of Soil Science 42, 351±367.
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