Temporal and spatial variation of nitrogen transformations in a
coniferous forest soil
A.M. Laverman
a,*, H.R. Zoomer
a, H.W. van Verseveld
b, H.A. Verhoef
aa
Department of Ecology and Ecotoxicology, Institute of Ecological Science, Vrije Universiteit, De Boelelaan 1087, 1081 HV, Amsterdam, The Netherlands
b
Department MCF, section Molecular Microbial Ecology, Vrije Universiteit, De Boelelaan 1087, 1081 HV, Amsterdam, The Netherlands
Accepted 23 March 2000
Abstract
Forest soils show a great degree of temporal and spatial variation of nitrogen mineralization. The aim of the present study was to explain temporal variation in nitrate leaching from a nitrogen-saturated coniferous forest soil by potential nitri®cation, mineralization rates and nitrate uptake by roots. Variation in nitrate production in time and space, between the dierent organic horizons, has been related to temperature, moisture content, substrate availability and pH. Temporal variation in concentrations of nitrate and ammonium in the forest ¯oor was signi®cant during a one-year cycle, when randomly taken samples were pooled. Nitrogen concentrations diered between the dierent organic horizons with highest concentrations found in the litter layer, decreasing with increasing depth. Ammonium concentrations always exceeded nitrate concentrations by a factor ten, indicating
that ammonium was not limiting nitri®cation. Nitri®cation potential, the nitrate production at ®eld moisture at 258C, was
highest in the litter layer, lower in the fragmentation layer and hardly measurable in the mineral soil. Uptake of nitrate by roots and changes in mineralization rates turned out to be unimportant to explain variation in time, as seasonal ¯uctuations seem to be less important than spatial variation. We found that horizontal spatial variation in potential nitrate production, leaching of nitrate and nitrogen concentrations from non-pooled ®eld samples was higher than variation in time. All this re¯ects the actual spatial variation in the ®eld, which is not explained by dierences in moisture content or temperature. Overall neither pH nor substrate availability could explain this observed variation, however, local variation in microsites may be responsible for small-scale spatial variation. Allelopathic compounds and/or the composition of the microbial community are suggested as factors
possibly aecting nitrate production.72000 Elsevier Science Ltd. All rights reserved.
Keywords:Nitrogen; Mineralization; Nitri®cation; Spatio-temporal variation; Abiotic factors
1. Introduction
During the last few decades the development of ani-mal husbandry and the excessive use of manure as fer-tilizer has led to increased atmospheric nitrogen deposition in Western Europe and parts of the USA (Berg et al., 1997; Erisman and Bleeker, 1995; Jeries and Maron, 1997). This high nitrogen input has
chan-ged nitrogen-limited ecosystems, characterized by intensively cycled nitrogen by microorganisms and soil fauna, to nitrogen-saturated systems (Aber et al., 1989). In the latter systems, availability of nitrogen exceeds plant and microbial nutritional demand, which can lead to nitrogen loss from the system. This loss of nitrogen is mainly due to nitrate leaching and N2O
and N2-production, whereas cation leaching, soil
acidi-®cation and, eventually, forest decline are additional environmental consequences of nitrogen saturation (Van Breemen et al., 1982, 1987). Nitri®cation is of special interest in acidi®ed soils because autotrophic nitri®ers are known to be sensitive to relatively low pH
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* Corresponding author. Tel.: 4447073; fax: +31-20-4447123.
values and are inactive below pH 4.5 in pure cultures (Focht and Verstraete, 1977). Nevertheless, nitrate pro-duction has been observed in acid forest and heathland soils at pH values lower than 4.5 in environments with high N deposition (Berg et al., 1997; De Boer et al., 1988; Weber and Gainey, 1962). Using speci®c inhibi-tors, such as acetylene and nitrapyrin, it has been shown that in these acid soils, nitrate production is due to the activity of autotrophic nitri®ers, located in microsites with relatively high pH values (De Boer et al., 1990; Hankinson and Schmidt, 1984).
Besides high pH microsites with a random distri-bution, there is an overall vertical variation in pH in acid forest soils and nitrifying potentials vary between these dierent organic layers (Persson and Wiren, 1995; Tietema et al., 1992). Vertical distribution of nitri®cation depends on soil type (Clays-Josserand et al., 1988; Persson and Wiren, 1995; Tietema et al., 1992). In Norway spruce soils, complete nitri®cation occurred only in soil layers with a relatively high pH (>4.1). Coniferous soils (Douglas ®r and Scots pine) showed dominant nitrate production in the organic horizons, whereas in deciduous forests (oak and beech) the top and the ®rst 5 cm of mineral soil contributed equally or even more to nitrate production than the mineral soil (Tietema et al., 1992).
Apart from vertical variation in net nitrate pro-duction, temporal variation has been found in conifer-ous and deciduconifer-ous forest soils (Berg et al., 1997; Tietema and Verstraten, 1992; Vitousek et al., 1982). Seasonal ¯uctuations in nitri®cation and nitrogen min-eralization rates in forest soil were positively correlated with temperature (Tietema and Verstraten, 1992). Nitrate leaching showed seasonal ¯uctuations from trenched forest soils, where nitrate leaching peaked in the growing season (Vitousek et al., 1982). In nitrogen saturated forest soils nitrate leaching, measured in rootless lysimeter systems, was highest in autumn, probably related to high mineralization rates in this period (Berg et al., 1997). In these systems without root uptake, nitrate leaching mainly results from nitrate production. However, the presence of roots will aect nitrate dynamics in soils (Willison et al., 1990). Nitrate leaching in the presence of roots possibly peaks in autumn, when root uptake is low, together with high mineralization and nitri®cation rates. In order to resolve how the balance between root uptake and mineralization aect nitrate leaching in autumn, we determined both nitrate leaching in the presence of roots and the actual nitrate and ammonium concen-trations in time. In order to relate nitrate leaching to the presence and activity of nitri®ers, nitrifying tials were determined as well as mineralization poten-tials throughout the year in dierent organic horizons. Using acetylene blockage techniques, discrimination was possible between autotrophic and heterotrophic
nitri®cation (Hynes and Knowles, 1982; Sahrawat et al., 1987). Temperature, substrate, pH and moisture content were investigated as factors in¯uencing nitrate production and possibly causing variation in time or between the dierent organic horizons.
2. Materials and methods
2.1. Site description
Experiments were carried out in Wekerom forest, a ®rst generation Scots pine forest (Pinus sylvestris L.) and in the laboratory. The ®eld site is situated near Wekerom, the Netherlands (latitude 528 06' N; longi-tude 5841'W; elevation 23 m). The trees were planted in 1955 on an inland dune in a former sand-drift area. The undergrowth consisted mainly of wavy hair grass, Deschampsia ¯exuosa(L.). The soil type was a non-cal-careous, acid pHKCl2:6±3:4young developing
pod-zol (Albic Arenosol). The organic layer was characterized as a mor pro®le; a litter (L), fragmenta-tion (F) and a thin humus (H) horizon were distin-guished. A more extensive description of the Wekerom forest is given by Berg and Verhoef (1998).
2.2. Experimental set-up and sampling
Nine tension ceramic plate lysimeters with a dia-meter of 13 cm and thickness of 0.5 cm and a pore size of 0.2 mm were placed just under the organic hor-izon in the forest soil. The distance between the plates was at a minimum of 1 m and a maximum of 30 m. The experimental plot size was 4050 m. The under-pressure in the plates was maintained at 0.2 bar. Plots (0.25 m2) containing a ceramic plate were covered by a perspex roof at a height of 0.5 m. The plots were watered every two weeks with 4.5 l of arti®cial rain, containing 0.56 mMNH4, 0.13 mMNO3ÿ and 0.6 mM Clÿ, representing the local nitrogen load of ca. 37 kg
of the upper Flayer, whereas the H layer included the lowerFand the thinH layer.
In a plot of 3535 m, within the experimental plot, soil samples were collected for laboratory determi-nation of soil organic nitrogen concentrations and po-tential N mineralization and nitri®cation. The sampling took place monthly from January to Decem-ber 1997. Within the 3535 m plot, eight random lo-cations of 0:350:35 m were chosen (using two
random tables). In these locations 5 core samples were collected. To reduce spatial variation, eight core samples (one per random location) were bulked to obtain ®ve composite samples. The cores were directly divided, into a L, F layer and mineral soil (including the humus layer). The samples were transported to the laboratory immediately, stored at 48C and processed the following day.
2.3. Soil incubation experiments to measure nitri®cation and nitrogen mineralization
The subsamples of soil were thoroughly mixed by hand. The litter layer was cut with scissors to achieve a well-homogenised sample. Approximately 15 g of the homogenised soil with original water content was incu-bated in a 250 ml screw-cap serum bottle. Monthly, ®ve replicates (as described above) ofL, Fand mineral soil were incubated at 258C for three weeks. Controls were incubated with acetylene (10 Pa) to inhibit auto-trophic nitri®cation (Hynes and Knowles, 1982). Acetylene was added through the septum to the con-trols and was present throughout the incubation time as acetylene was still detectable in the headspace at the end of the incubation (data not shown). The presence of acetylene was necessary to keep nitri®cation inhib-ited; previous reports have shown recovery of nitri®ca-tion after removal of acetylene (e.g., Bollmann and Conrad, 1997). After the incubation period, 10 g was used to determine extractable nitrate and ammonium. These concentrations were used to compare with the initial values in order to determine nitrogen transform-ation rates. The nitrogen transformtransform-ation rates were expressed as mg N kgÿ1 dry weight per week. The net mineralization rate was calculated as the dierence between inorganic nitrogen at the end and start, whereas the net nitri®cation rate was determined as the dierence between nitrate at the end and start of the incubation period. As nitrate concentrations in samples incubated with inhibitor (acetylene) decreased compared to start values, the dierence in nitrate con-centrations between start and soil incubated with in-hibitor was indicated as nitrate uptake (immobilisation and/or denitri®cation). This nitrate uptake was assumed to occur in samples incubated without inhibi-tor; therefore the dierence in nitrate concentrations at
the end of incubation with and without inhibitor was indicated as total nitri®cation rate.
2.4. Chemical analyses
Nitrate, ammonium and chloride in the percolate were measured on an autoanalyzer (Skalar SA 400). Exchangeable nitrate and ammonium were determined by extraction of the samples in 1 M KCl in a 1:12.5 ratio (w/v). After ®ltration, (Schleicher & Schuell, 595 1/2) the extract was analyzed for nitrate, ammonium (Skalar autoanalyzer SA 400), and pHKCl (Consort
P907). Total nitrogen and carbon contents were deter-mined using a Carlo Erba Strumentazione elemental analyzer (model 1106). The moisture content was determined gravimetrically by drying the samples at 608C for 72 h. The nitrogen concentrations were expressed asmg N per gram dry weight.
2.5. Statistics
One way analysis of variance (ANOVA) test was used to determine eects of time. An ANOVA with repeated measures was applied to data obtained for or-ganic layers originating from the same plot. Parametric tests were only performed when homogeneity of var-iance was determined by the Bartlett test. When necessary log transformations were applied to data to establish homogeneity of variance. Pearson's lation coecients were calculated to check for corre-lation between nitrate production and pH, moisture content, CN ratio and nitrate and ammonium concen-trations. All statistical analyses were done using SYSTAT15.2.1 software package (SYSTAT, 1992).
3. Results
3.1. Characteristics of soil horizons
Table 1 shows the thickness, the dierences in nitro-gen and carbon content and pH of the dierent or-ganic horizons. The total carbon content decreased with depth and the total nitrogen content reached highest values in the litter layer, then decreasing with depth like the carbon content. The pH decreased from freshly fallen needles to the L and F and H layer. In the mineral soil pH is slightly higher.
3.2. Nitrogen leaching
production in the top layers. Three plates (Fig. 1(A); plates 2, 8, 9) showed increased nitrate leaching in autumn. Plates 4, 6 and 7 showed little variation throughout the year. Leachable ammonium showed less temporal ¯uctuations than nitrate leaching (Fig. 1). During the incubation period plate 4 collected low amounts of ammonium, whereas plates 7 and 9 started high but decreased to levels comparable to plate 4 during the measured seven months. The highest am-monium values were from plate 2, reaching values up to 0.5 mM, which was almost the ammonium concen-tration present in the rainwater. The two remaining plates (6 and 8) collected intermediate concentrations of ammonium and ¯uctuated only slightly. There was no consistent correlation between nitrate and am-monium leaching, meaning that plots with low nitrate leaching did not necessarily leach large amounts of ammonium or vice versa.
3.3. Nitrogen concentrations
The nitrogen concentrations (determined in 10 fold) in the dierent layers in time are presented in Fig. 2. Ammonium concentrations always exceeded nitrate concentrations by a factor of ten. In general, nitrate concentrations in theL andFlayer were quite similar, in the H and mineral layer these concentrations were lower. Ammonium concentrations were highest in the L layer reaching values up to 600 mg per gram dry weight and decreased gradually with depth to less than 10 mg per gram dry weight in the mineral soil. The variations in nitrogen concentrations were highest in the top layers (L, F and H) and were relatively con-stant in the mineral soil (see Fig. 2). Besides these gen-eral dierences between layers, the time course of both nitrate and ammonium concentrations diered between layers (ANOVA [repeated measures] timelayer inter-action: F3:6 for nitrate, F5:9 for ammonium,
bothp<0:001). Thus, the pattern in time was not the
same for the dierent layers, the nitrate course in the Llayer being signi®cantly dierent from that in the F, H and mineral layer. Table 2 shows the overall mean in nitrate and ammonium concentrations in the
dier-ent horizons, including the spatial and temporal stan-dard deviations. Nitrate and ammonium in the L and Flayer showed more variation in space than in time.
3.4. Nitrogen mineralization and nitri®cation potential
The nitrate and ammonium concentrations of the pooled samples (5 soil samples each composed of 8 subsamples) are presented together with the moisture contents (Fig. 3). Moisture content showed its highest variation in the top layer and decreased with depth. The highest and most ¯uctuating moisture contents were found in the L layer (60±80%), with June being an exception due to a dry period (25%, Fig. 3(A)). Moisture levels in the F layer ¯uctuated between 50 and 60%, again with the exception for June (Fig. 3(B)). In the mineral layer moisture content was fairly con-stant at 10±20% (Fig. 3(C)).
A signi®cant variation in time was found for nitrate in the L F9:9, p<0:001), F F22:8,
p<0:001 and the mineral soil F30:8,
p<0:001). The same was found for ammonium in
L F21:1, p<0:001), F F12:1, p<0:001 and
M F14:3, p<0:001). Nitrate values were highest
in March and August in L and F layers; in the mineral soil highest values were reached in January and December. Nitrate concentrations ¯uctuated between 10 and 30 mg per gram, whereas am-monium varied between 100 and 600 mg per gram Table 1
Some properties of the dierent organic horizons from Wekerom foresta
Thickness (cm) TotalN(%) TotalC(%) pHKCl
Freshly fallen needles ± 0.96 (0.11)b 50.37 (0.31)b 4.53 (0.09)b
Litter 0.5±2 1.78 (0.09) 47.01 (1.72) 3.79 (0.24)c
Fragmentation 3±8 1.36 (0.13) 36.61 (3.99) 2.76 (0.10)c
Humus 5±10 0.19 (0.02) 4.18 (0.39) 2.90 (0.05)
Mineral soil > 20 ± ± 3.40 (0.30)c
aValues shown are means and standard deviations (in parentheses)n60. bSamples were only collected in October (needle fall),n5.
cn35.
Table 2
Mean values, spatial variation and temporal variation for nitrate and ammonium in the dierent layers
dry weight (Fig. 3(A)). Nitrogen concentrations decreased with depth as was shown in Fig. 2. In the F layer nitrate varied between 5 and 15 mg and ammonium between 20 and 100 mg per gram dry weight. The lowest concentrations were found in the
mineral soil, only 1±6 mg gÿ1 of nitrogen (nitrate or ammonium) was detected in this layer.
The mineralization and nitri®cation rates of pooled samples (5 samples each consisting of 8 subsamples) in the dierent layers throughout the year are shown in
Table 3. All nitri®cation was inhibited by low concen-trations of acetylene in the control experiments (Table 3) indicating autotrophic nitri®cation. At the end of the incubation period low concentrations of acetylene were still detectable (data not shown). Both nitrogen mineralization and nitri®cation rates decreased with increasing depth. Mineralization rates were always highest in the litter layer, varying between 296 mg N in June till up to 1314 mg N kgÿ1 wkÿ1 in October. In the other months rates between 600 and 800 mg kgÿ1 wkÿ1were found, the variation in miner-alization rates in the litter being signi®cant in time F7:4,p<0:001). The main part of the N
mineraliz-ation is due to ammoni®cmineraliz-ation, with nitrate production accounting only for a small part: in the L layer not more than 1%, in the F layer between 5 and 10%. In the F layer less variation was observed in mineraliz-ation rates, varying between 70 and 150 mg N kgÿ1 wkÿ1and variation in these rates was not signi®cant in time. In the mineral layer the lowest amount was pro-duced, between 5 and 15 mg N kgÿ1wkÿ1, which var-ied signi®cantly in time F15:1,p<0:001).
Net nitri®cation rates varied more than net nitrogen mineralization rates in all layers, especially in the L layer, with no signi®cant variation in time for nitri®ca-tion rates. The L layer showed the highest variation
and the highest nitri®cation rates, reaching values up to 60 mg kgÿ1 weekÿ1 for some samples in January. Nitri®cation in the F layer varied less, between zero and approximately 8 mg kgÿ1 weekÿ1 in February. It seems however, that in almost all cases observed, nitrate uptake (denitri®cation and/or immobilization) took place, meaning that total nitrate production was higher than net nitrate production. The total nitrate
production in theF layer ranged between 0 and 10 mg kgÿ1 weekÿ1. In the mineral soil hardly any nitrate was produced, whereas mineralization rates were low but measurable, exceeding the nitrate concentrations at the end of the incubation. Nitrate uptake was low and followed the same pattern as most concentrations and activities, showing signi®cant variation in time in theL and F layer F9:2 and F15:4 respectively, both
p<0:001). No signi®cant correlation was found for
nitrate production in the L layer with moisture, CN ratio, pH or ammonium concentrations. In the Flayer as well as in the mineral soil signi®cant correlations were observed between nitrate start values and nitrate production R2 0:53,p<0:05; R20:79, p<0:001,
respectively). Other factors showed no signi®cant cor-relations with nitrate production.
4. Discussion
The horizontally spatial variation in nitrate leaching and nitrogen concentrations in the nitrogen-saturated coniferous forest soil in Wekerom dominated temporal ¯uctuation. Therefore, immobilisation of nitrate by roots or increased mineralization turned out to be
unimportant in terms of seasonal variation in nitrate leaching. Uptake of nitrate by plant and/or tree roots is most likely responsible for the lower nitrate leaching in this study compared to the values found by Berg et al. (1997) who used isolated cores in the same ®eld without interference of roots. Signi®cant temporal variation in nitrate and ammonium concentrations from the ®eld was only found when randomly collected samples were pooled, reducing spatial variation. No signi®cant temporal variation in nitri®cation potential rates was observed, indicating the presence and poten-tial activity of nitri®ers throughout the year.
Vertical variation in nitrogen concentrations and transformations was more obvious, both being highest in the top layers and low in the mineral soil, as has also been observed in other studies (De Boer et al., 1992; Tietema et al., 1992). Especially in the L layer,
Table 3
Net mineralization and nitri®cation rates expressed in mg kgÿ1dry soil wkÿ1for the dierent layers throughout 1997a
Net mineralization (mg N kgÿ1wkÿ1) Nitri®cation (mg N kgÿ1wkÿ1)
Net Uptake Total
January L 878 (144) 32.2 (34.5) ÿ4.5 (1.9) 36.7 (36.0)
F 95 (32) 2.7 (1.6) ÿ2.3 (0.8) 5.0 (1.3)
M 5 (1) ÿ0.2 (0.2) ÿ0.3 (0.2) 0.2 (0.4)
February L 342 (58) ÿ2.7 (0.3) ÿ2.6 (0.4) ÿ0.2 (0.3)
F 87 (14) 7.7 (2.3) ÿ0.7 (0.3) 8.3 (2.0)
M 10 (2) 0.0 (0.2) 0.21 (0.2) ÿ0.2 (0.2)
March L 606 (184) ÿ2.4 (7.8) ÿ9.8 (1.5) 7.4 (7.3)
F 124 (61) 3.9 (1.2) ÿ4.5 (0.7) 8.4 (1.8)
M 4 (1) ÿ0.3 (0.1) ÿ0.5 (0.1) 0.2 (0.1)
April L 732 (197) 9.2 (17.0) ÿ3.7 (1.0) 12.9 (16.2)
F 120 (45) 0.9 (3.2) ÿ1.8 (0.7) 2.7 (3.0)
M 13 (3) ÿ0.1 (0.1) ÿ0.2 (0.1) 0.1 (0.1)
May L 699 (728) ÿ4.9 (1.3) ÿ5.7 (1.3) 0.8 (1.3)
F 75 (22) 0.2 (3.2) ÿ2.5 (0.6) 2.7 (3.2)
M 6 (1) ÿ0.1 (0.1) ÿ0.4 (0.1) 0.4 (0.1)
June L 296 (142) ÿ0.9 (1.0) ÿ1.3 (0.5) 0.4 (0.7)
F 79 (8) ÿ0.2 (0.3) ÿ0.4 (0.5) 0.1 (0.5)
M 5 (1) 0.0 (0.1) ÿ0.0 (0.1) 0.1 (0.0)
July L 842 (110) 9.0 (32.5) ÿ5.0 (3.1) 14 (30.9)
F 102 (7) 3.0 (4.5) ÿ1.4 (0.7) 4.5 (4.1)
M 8 (1) 0.2 (0.2) ÿ0.2 (0.2) 0.4 (0.2)
August L 812 (184) 7.0 (21.3) ÿ7.7 (1.6) 14.7 (22.7)
F 100 (20) 3.9 (3.8) ÿ4.7 (1.3) 8.6 (4.8)
M 8 (1) ÿ0.1 (0.1) ÿ0.4 (0.2) 0.4 (0.2)
September L 851 (227) 12.1 (16.2) ÿ4.3 (1.4) 16.4 (15.1)
F 115 (26) 0.9 (2.1) ÿ3.4 (0.9) 4.3 (3.0)
M 8 (0.81) ÿ0.0 (0.2) ÿ0.3 (0.0) 0.3 (0.2)
October L 1314 (458) ÿ5.7 (1.8) ÿ6.9 (1.1) 1.2 (1.1)
F 116 (11) ÿ1.4 (1.7) ÿ3.9 (1.0) 2.5 (1.7)
M 9 (2) ÿ0.1 (0.1) ÿ0.2 (0.0) 0.1 (0.1)
November L 878 (295) ÿ2.8 (2.3) ÿ4.4 (2.7) 1.7 (2.1)
F 125 (18) ÿ0.2 (0.4) ÿ2.8 (0.9) 2.6 (1.0)
M 6 (2) ÿ0.1 (0.1) ÿ0.2 (0.1) 0.1 (0.2)
December L 934 (190) 8.2 (13.0) ÿ5.92 (1.4) 14.2 (12.1)
F 145 (32) ÿ0.9 (1.1) ÿ4.67 (1.4) 3.8 (2.4)
M 9 (2) ÿ0.3 (0.1) ÿ0.51 (0.1) 0.2 (0.1)
a
potential nitrate production was highest and also most variable. In this layer high nitrate uptake was observed. Whether this was due to nitrate immobilis-ation or denitri®cimmobilis-ation remains unclear. Nitrate immo-bilisation seems unlikely as ammonium is present in high levels, although, preference for nitrate uptake over ammonium has been suggested in soils (Drury et al., 1991). Denitri®cation is thought to be of minor im-portance in coniferous forest soils (Martikainen et al., 1993) but cannot be excluded. The vertical variation in nitrifying potential strongly indicates a layer speci®c nitrifying activity. Explanations for the dierent nitri®-cation rates between the organic horizons could be re-lated to dierences in nitrogen levels, pH, structure and moisture content. As ammonium was always pre-sent in concentrations exceeding the nitrate values, substrate de®ciency does not seem to be a factor explaining dierences between the dierent layers. Although Persson and Wiren (1995) concluded that the pH in the mineral layer is responsible for the high nitri®cation potential in this layer, we found that nitri-®cation potential was not related to vertical variation in pH. The nitri®cation potentials in the F layer were higher than those in the mineral layer, while pH was higher in the latter. Nitri®cation potentials seem to vary in their vertical distribution in dierent soil types with dierent properties; bulk pH does not seem to be the controlling factor for these variations in nitrate production, although small scale pH could be.
The observed horizontally spatial variation in nitro-gen leaching and concentrations were in agreement with overall nitri®cation rates in other deciduous and pine forest soils in the Netherlands (De Boer and Kester, 1996; Tietema et al., 1992), however, the fac-tors causing this variation remain uncertain. Overall no correlations were found between pH, CN ratio, moisture and ammonium concentration, only in the F layer and mineral soil signi®cant correlation was observed between nitrate start concentrations and nitrate production. One of the factors that can be ruled out as a factor determining the dierences in nitrate leaching, nitrate concentrations and nitrate pro-duction is temperature, as samples taken at a certain time were all exposed to the same ®eld temperature and incubation temperatures were kept constant. How-ever, some factors can be suggested to cause variation in nitrogen transformation, for example the substrate concentration could play a role. In the ®eld it was shown that not only nitrate but also ammonium is lea-ched from the soil, indicating that the soil is am-monium-saturated. However, a low availability of ammonium at the microsite level due to the compo-sition of the soil matrix could limit nitri®cation (Davidson and Hackler, 1994; Drury et al., 1991). In addition, competition for ammonium between hetero-trophic bacteria and nitri®ers (Verhagen et al., 1992)
and plant roots (Verhagen et al., 1995), is always in favor of the heterotrophs. The spatial dierences in ammonium concentrations showed locations with high and very low substrate levels, thus indicating spatial variation in substrate availability at this scale. Another potential factor to in¯uence nitrate production, pH did not show any relation with nitri®cation rates, however, dierences in microsite pH could play a role (Strong et al., 1998). Moisture content too, could not explain the spatial variation, as within one month, moisture con-tents were comparable.
The question remains which factors are in¯uencing nitri®cation rates, a possibility being allelopathic com-pounds, such as monoterpenoids, which have been shown to in¯uence nitri®cation (Olson and Reiners, 1983; Paavolainen et al., 1998; White, 1988). Other factors causing spatial variation in nitri®cation rates can be the composition of the microbial population (De Boer and Kester, 1996) or the composition and ac-tivity of the soil fauna community (Verhoef and Brus-saard, 1990). Despite the numerous studies addressing measured variation in the ®eld, more detailed studies regarding the possible factors responsible for the high spatial variation in nitrogen concentrations and trans-formations are still necessary for a detailed under-standing of the mechanisms involved.
Acknowledgements
The authors thank Michel Peereboom, Elisabete Alves and Peter Overweg for assistance in the ®eld; Dr. A. Tietema for providing ceramic plates; Dr. J.J.M. Bedaux for statistical advice and Prof. N.M. van Straalen, Prof. H.J. Laanbroek, Dr. M.P. Berg and two anonymous reviewers for critically reading the manuscript.
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