Directory UMM :Data Elmu:jurnal:S:Soil Biology And Chemistry:Vol32.Issue11-12.oct2000:
Soil Biology & Biochemistry 32 (2000) 1661±1670
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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 a
a
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 smallscale spatial variation. Allelopathic compounds and/or the composition of the microbial community are suggested as factors
possibly aecting nitrate production. 7 2000 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 animal husbandry and the excessive use of manure as fertilizer 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-
* Corresponding author. Tel.: +31-20-4447073; fax: +31-204447123.
E-mail address: laverman@bio.vu.nl (A.M. Laverman).
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
0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 8 2 - 1
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A.M. Laverman et al. / Soil Biology & Biochemistry 32 (2000) 1661±1670
values and are inactive below pH 4.5 in pure cultures
(Focht and Verstraete, 1977). Nevertheless, nitrate production 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 inhibitors, 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 distribution, 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 production, temporal variation has been found in coniferous and deciduous forest soils (Berg et al., 1997;
Tietema and Verstraten, 1992; Vitousek et al., 1982).
Seasonal ¯uctuations in nitri®cation and nitrogen mineralization 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 concentrations in time. In order to relate nitrate leaching to
the presence and activity of nitri®ers, nitrifying potentials were determined as well as mineralization potentials 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; longitude 58 41 ' 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-calcareous, acid pHKCl 2:6±3:4 young developing podzol (Albic Arenosol). The organic layer was
characterized as a mor pro®le; a litter (L ), fragmentation (F ) and a thin humus (H ) horizon were distinguished. 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 diameter of 13 cm and thickness of 0.5 cm and a pore
size of 0.2 mm were placed just under the organic horizon 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 40 50 m. The underpressure 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,
ÿ
containing 0.56 mM NH
4 , 0.13 mM NO3 and 0.6 mM
ÿ
Cl , representing the local nitrogen load of ca. 37 kg
ÿ1
ÿ
yearÿ1 (Berg and
NH
4 ±N and 8.5 kg NO3 ±N ha
Verhoef, 1998). Percolate was collected from September 1995 to June 1996. The samples were analyzed in
the laboratory for nitrate, ammonium and chloride.
Nitrate and ammonium concentrations were corrected
using chloride as the tracer anion. The concentrations
of extractable nitrate and ammonium in the soil were
determined in the same period of sampling of the lysimeters, ten soil-cores (diameter 9.5 cm) sampled
monthly from ten plots (0.25 m2) covered with roofs
and rained similar to the plots containing the tension
plates. These soil-cores were immediately divided,
using morphological criteria, into a L, F, H layer and
mineral soil and used for the determination of ammonium and nitrate. In this case, the F layer consisted
A.M. Laverman et al. / Soil Biology & Biochemistry 32 (2000) 1661±1670
of the upper F layer, whereas the H layer included the
lower F and the thin H layer.
In a plot of 35 35 m, within the experimental plot,
soil samples were collected for laboratory determination of soil organic nitrogen concentrations and potential N mineralization and nitri®cation. The
sampling took place monthly from January to December 1997. Within the 35 35 m plot, eight random locations of 0:35 0:35 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 incubated in a 250 ml screw-cap serum bottle. Monthly,
®ve replicates (as described above) of L, F and mineral
soil were incubated at 258C for three weeks. Controls
were incubated with acetylene (10 Pa) to inhibit autotrophic nitri®cation (Hynes and Knowles, 1982).
Acetylene was added through the septum to the controls 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 inhibited; previous reports have shown recovery of nitri®cation 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 transformation rates. The nitrogen transformation 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 concentrations between start and soil incubated with inhibitor was indicated as nitrate uptake (immobilisation
and/or denitri®cation). This nitrate uptake was
assumed to occur in samples incubated without inhibitor; therefore the dierence in nitrate concentrations at
1663
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 determined 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 as mg 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 organic layers originating from the same plot. Parametric
tests were only performed when homogeneity of variance was determined by the Bartlett test. When
necessary log transformations were applied to data to
establish homogeneity of variance. Pearson's correlation coecients were calculated to check for correlation between nitrate production and pH, moisture
content, CN ratio and nitrate and ammonium concentrations. All statistical analyses were done using
SYSTAT1 5.2.1 software package (SYSTAT, 1992).
3. Results
3.1. Characteristics of soil horizons
Table 1 shows the thickness, the dierences in nitrogen and carbon content and pH of the dierent organic 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
Average nitrate leaching ¯uctuated in time, with a
large variation in concentrations between individual
lysimeters (as shown in Fig. 1(A)). Nitrate leaching
was consistently higher than nitrate input by rainwater
and varied between 0.3 and 1.5 mM, indicating nitrate
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A.M. Laverman et al. / Soil Biology & Biochemistry 32 (2000) 1661±1670
Table 1
Some properties of the dierent organic horizons from Wekerom foresta
Freshly fallen needles
Litter
Fragmentation
Humus
Mineral soil
Thickness (cm)
Total N (%)
±
0.5±2
3±8
5±10
> 20
0.96
1.78
1.36
0.19
±
(0.11)b
(0.09)
(0.13)
(0.02)
Total C (%)
pHKCl
50.37 (0.31)b
47.01 (1.72)
36.61 (3.99)
4.18 (0.39)
±
4.53
3.79
2.76
2.90
3.40
(0.09)b
(0.24)c
(0.10)c
(0.05)
(0.30)c
a
Values shown are means and standard deviations (in parentheses) n 60.
Samples were only collected in October (needle fall), n 5.
c
n 35.
b
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 ammonium values were from plate 2, reaching values up
to 0.5 mM, which was almost the ammonium concentration 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 ammonium 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 the L and F layer 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 constant in the mineral soil (see Fig. 2). Besides these general dierences between layers, the time course of both
nitrate and ammonium concentrations diered between
layers (ANOVA [repeated measures] time layer interaction: F 3:6 for nitrate, F 5:9 for ammonium,
both p < 0:001). Thus, the pattern in time was not the
same for the dierent layers, the nitrate course in the
L layer 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 standard deviations. Nitrate and ammonium in the L and
F layer 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 constant at 10±20% (Fig. 3(C)).
A signi®cant variation in time was found for
nitrate in the L F 9:9, p < 0:001), F F 22:8,
p < 0:001
and
the
mineral
soil
F 30:8,
p < 0:001). The same was found for ammonium in
L F 21:1, p < 0:001), F F 12:1, p < 0:001 and
M F 14:3, 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 ammonium varied between 100 and 600 mg per gram
Table 2
Mean values, spatial variation and temporal variation for nitrate and
ammonium in the dierent layers
NO3±N (mg gÿ1)
L
F
H
M
a
b
NH4±N (mg gÿ1)
Mean
SDa
SDb
Mean
SDa
SDb
23.1
19.5
5.8
2.4
6.2
4.7
2.1
0.4
7.1
5.6
1.6
0.2
384.0
174.3
60.3
9.1
64.9
32.3
40.1
7.1
76.4
69.4
31.6
6.5
Temporal standard deviation.
Spatial standard deviation.
A.M. Laverman et al. / Soil Biology & Biochemistry 32 (2000) 1661±1670
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
1665
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
Fig. 1. Nitrate (A) and ammonium (B) concentrations in leachate collected with ceramic plates in the ®eld. The arrows on the left side indicate
the amount of nitrate or ammonium in the rainwater. Average of all plates (*), plate 2 (w), 4 (q), 6 (r), 7 (r), 8 (t) and 9 (w).
No data are
shown from plates 1, 3 and 5 as the amount of collected leachate was too low at most time points; missing data for the other plates are also due
to low leaching.
1666
A.M. Laverman et al. / Soil Biology & Biochemistry 32 (2000) 1661±1670
Table 3. All nitri®cation was inhibited by low concentrations 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 wkÿ1 were found, the variation in mineralization rates in the litter being signi®cant in time
F 7:4, p < 0:001). The main part of the N mineraliz-
ation is due to ammoni®cation, 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 mineralization rates, varying between 70 and 150 mg N kgÿ1
wkÿ1 and variation in these rates was not signi®cant in
time. In the mineral layer the lowest amount was produced, between 5 and 15 mg N kgÿ1 wkÿ1, which varied signi®cantly in time F 15:1, 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®cation rates. The L layer showed the highest variation
Fig. 2. Nitrate (A) and ammonium (B) concentrations as measured in the L (dark gray), F (light gray), H (black) and mineral (white) soil in
time. Data represent the mean of 10 replicates. Vertical bars indicate standard deviation.
A.M. Laverman et al. / Soil Biology & Biochemistry 32 (2000) 1661±1670
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
1667
production in the F 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 the L
and F layer F 9:2 and F 15:4 respectively, both
Fig. 3. Ammonium (q) and nitrate (*) concentrations and moisture content (R) throughout the year in the litter (A), fragmentation (B) and
mineral (C) layers. Data represent the mean of ®ve replicates. Vertical bars indicate standard deviation.
1668
A.M. Laverman et al. / Soil Biology & Biochemistry 32 (2000) 1661±1670
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 F layer
as well as in the mineral soil signi®cant correlations
were observed between nitrate start values and nitrate
production R 2 0:53, p < 0:05; R 2 0:79, p < 0:001,
respectively). Other factors showed no signi®cant correlations 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 potential 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 kgÿ1 dry soil wkÿ1 for the dierent layers throughout 1997a
Net mineralization (mg N kgÿ1 wkÿ1)
January
February
March
April
May
June
July
August
September
October
November
December
a
L
F
M
L
F
M
L
F
M
L
F
M
L
F
M
L
F
M
L
F
M
L
F
M
L
F
M
L
F
M
L
F
M
L
F
M
878 (144)
95 (32)
5 (1)
342 (58)
87 (14)
10 (2)
606 (184)
124 (61)
4 (1)
732 (197)
120 (45)
13 (3)
699 (728)
75 (22)
6 (1)
296 (142)
79 (8)
5 (1)
842 (110)
102 (7)
8 (1)
812 (184)
100 (20)
8 (1)
851 (227)
115 (26)
8 (0.81)
1314 (458)
116 (11)
9 (2)
878 (295)
125 (18)
6 (2)
934 (190)
145 (32)
9 (2)
Values shown are means n 5 and standard deviations (in parentheses).
Nitri®cation (mg N kgÿ1 wkÿ1)
Net
Uptake
Total
32.2 (34.5)
2.7 (1.6)
ÿ0.2 (0.2)
ÿ2.7 (0.3)
7.7 (2.3)
0.0 (0.2)
ÿ2.4 (7.8)
3.9 (1.2)
ÿ0.3 (0.1)
9.2 (17.0)
0.9 (3.2)
ÿ0.1 (0.1)
ÿ4.9 (1.3)
0.2 (3.2)
ÿ0.1 (0.1)
ÿ0.9 (1.0)
ÿ0.2 (0.3)
0.0 (0.1)
9.0 (32.5)
3.0 (4.5)
0.2 (0.2)
7.0 (21.3)
3.9 (3.8)
ÿ0.1 (0.1)
12.1 (16.2)
0.9 (2.1)
ÿ0.0 (0.2)
ÿ5.7 (1.8)
ÿ1.4 (1.7)
ÿ0.1 (0.1)
ÿ2.8 (2.3)
ÿ0.2 (0.4)
ÿ0.1 (0.1)
8.2 (13.0)
ÿ0.9 (1.1)
ÿ0.3 (0.1)
ÿ4.5 (1.9)
ÿ2.3 (0.8)
ÿ0.3 (0.2)
ÿ2.6 (0.4)
ÿ0.7 (0.3)
0.21 (0.2)
ÿ9.8 (1.5)
ÿ4.5 (0.7)
ÿ0.5 (0.1)
ÿ3.7 (1.0)
ÿ1.8 (0.7)
ÿ0.2 (0.1)
ÿ5.7 (1.3)
ÿ2.5 (0.6)
ÿ0.4 (0.1)
ÿ1.3 (0.5)
ÿ0.4 (0.5)
ÿ0.0 (0.1)
ÿ5.0 (3.1)
ÿ1.4 (0.7)
ÿ0.2 (0.2)
ÿ7.7 (1.6)
ÿ4.7 (1.3)
ÿ0.4 (0.2)
ÿ4.3 (1.4)
ÿ3.4 (0.9)
ÿ0.3 (0.0)
ÿ6.9 (1.1)
ÿ3.9 (1.0)
ÿ0.2 (0.0)
ÿ4.4 (2.7)
ÿ2.8 (0.9)
ÿ0.2 (0.1)
ÿ5.92 (1.4)
ÿ4.67 (1.4)
ÿ0.51 (0.1)
36.7 (36.0)
5.0 (1.3)
0.2 (0.4)
ÿ0.2 (0.3)
8.3 (2.0)
ÿ0.2 (0.2)
7.4 (7.3)
8.4 (1.8)
0.2 (0.1)
12.9 (16.2)
2.7 (3.0)
0.1 (0.1)
0.8 (1.3)
2.7 (3.2)
0.4 (0.1)
0.4 (0.7)
0.1 (0.5)
0.1 (0.0)
14 (30.9)
4.5 (4.1)
0.4 (0.2)
14.7 (22.7)
8.6 (4.8)
0.4 (0.2)
16.4 (15.1)
4.3 (3.0)
0.3 (0.2)
1.2 (1.1)
2.5 (1.7)
0.1 (0.1)
1.7 (2.1)
2.6 (1.0)
0.1 (0.2)
14.2 (12.1)
3.8 (2.4)
0.2 (0.1)
A.M. Laverman et al. / Soil Biology & Biochemistry 32 (2000) 1661±1670
potential nitrate production was highest and also most
variable. In this layer high nitrate uptake was
observed. Whether this was due to nitrate immobilisation or denitri®cation remains unclear. Nitrate immobilisation 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 importance 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 related to dierences in nitrogen levels, pH, structure
and moisture content. As ammonium was always present 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 nitrogen 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 factors 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 production is temperature, as samples taken at a certain
time were all exposed to the same ®eld temperature
and incubation temperatures were kept constant. However, 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 leached from the soil, indicating that the soil is ammonium-saturated. However, a low availability of
ammonium at the microsite level due to the composition of the soil matrix could limit nitri®cation
(Davidson and Hackler, 1994; Drury et al., 1991). In
addition, competition for ammonium between heterotrophic bacteria and nitri®ers (Verhagen et al., 1992)
1669
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 contents were comparable.
The question remains which factors are in¯uencing
nitri®cation rates, a possibility being allelopathic compounds, 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 activity of the soil fauna community (Verhoef and Brussaard, 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 transformations are still necessary for a detailed understanding 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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www.elsevier.com/locate/soilbio
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 a
a
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 smallscale spatial variation. Allelopathic compounds and/or the composition of the microbial community are suggested as factors
possibly aecting nitrate production. 7 2000 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 animal husbandry and the excessive use of manure as fertilizer 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-
* Corresponding author. Tel.: +31-20-4447073; fax: +31-204447123.
E-mail address: laverman@bio.vu.nl (A.M. Laverman).
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
0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 8 2 - 1
1662
A.M. Laverman et al. / Soil Biology & Biochemistry 32 (2000) 1661±1670
values and are inactive below pH 4.5 in pure cultures
(Focht and Verstraete, 1977). Nevertheless, nitrate production 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 inhibitors, 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 distribution, 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 production, temporal variation has been found in coniferous and deciduous forest soils (Berg et al., 1997;
Tietema and Verstraten, 1992; Vitousek et al., 1982).
Seasonal ¯uctuations in nitri®cation and nitrogen mineralization 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 concentrations in time. In order to relate nitrate leaching to
the presence and activity of nitri®ers, nitrifying potentials were determined as well as mineralization potentials 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; longitude 58 41 ' 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-calcareous, acid pHKCl 2:6±3:4 young developing podzol (Albic Arenosol). The organic layer was
characterized as a mor pro®le; a litter (L ), fragmentation (F ) and a thin humus (H ) horizon were distinguished. 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 diameter of 13 cm and thickness of 0.5 cm and a pore
size of 0.2 mm were placed just under the organic horizon 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 40 50 m. The underpressure 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,
ÿ
containing 0.56 mM NH
4 , 0.13 mM NO3 and 0.6 mM
ÿ
Cl , representing the local nitrogen load of ca. 37 kg
ÿ1
ÿ
yearÿ1 (Berg and
NH
4 ±N and 8.5 kg NO3 ±N ha
Verhoef, 1998). Percolate was collected from September 1995 to June 1996. The samples were analyzed in
the laboratory for nitrate, ammonium and chloride.
Nitrate and ammonium concentrations were corrected
using chloride as the tracer anion. The concentrations
of extractable nitrate and ammonium in the soil were
determined in the same period of sampling of the lysimeters, ten soil-cores (diameter 9.5 cm) sampled
monthly from ten plots (0.25 m2) covered with roofs
and rained similar to the plots containing the tension
plates. These soil-cores were immediately divided,
using morphological criteria, into a L, F, H layer and
mineral soil and used for the determination of ammonium and nitrate. In this case, the F layer consisted
A.M. Laverman et al. / Soil Biology & Biochemistry 32 (2000) 1661±1670
of the upper F layer, whereas the H layer included the
lower F and the thin H layer.
In a plot of 35 35 m, within the experimental plot,
soil samples were collected for laboratory determination of soil organic nitrogen concentrations and potential N mineralization and nitri®cation. The
sampling took place monthly from January to December 1997. Within the 35 35 m plot, eight random locations of 0:35 0:35 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 incubated in a 250 ml screw-cap serum bottle. Monthly,
®ve replicates (as described above) of L, F and mineral
soil were incubated at 258C for three weeks. Controls
were incubated with acetylene (10 Pa) to inhibit autotrophic nitri®cation (Hynes and Knowles, 1982).
Acetylene was added through the septum to the controls 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 inhibited; previous reports have shown recovery of nitri®cation 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 transformation rates. The nitrogen transformation 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 concentrations between start and soil incubated with inhibitor was indicated as nitrate uptake (immobilisation
and/or denitri®cation). This nitrate uptake was
assumed to occur in samples incubated without inhibitor; therefore the dierence in nitrate concentrations at
1663
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 determined 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 as mg 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 organic layers originating from the same plot. Parametric
tests were only performed when homogeneity of variance was determined by the Bartlett test. When
necessary log transformations were applied to data to
establish homogeneity of variance. Pearson's correlation coecients were calculated to check for correlation between nitrate production and pH, moisture
content, CN ratio and nitrate and ammonium concentrations. All statistical analyses were done using
SYSTAT1 5.2.1 software package (SYSTAT, 1992).
3. Results
3.1. Characteristics of soil horizons
Table 1 shows the thickness, the dierences in nitrogen and carbon content and pH of the dierent organic 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
Average nitrate leaching ¯uctuated in time, with a
large variation in concentrations between individual
lysimeters (as shown in Fig. 1(A)). Nitrate leaching
was consistently higher than nitrate input by rainwater
and varied between 0.3 and 1.5 mM, indicating nitrate
1664
A.M. Laverman et al. / Soil Biology & Biochemistry 32 (2000) 1661±1670
Table 1
Some properties of the dierent organic horizons from Wekerom foresta
Freshly fallen needles
Litter
Fragmentation
Humus
Mineral soil
Thickness (cm)
Total N (%)
±
0.5±2
3±8
5±10
> 20
0.96
1.78
1.36
0.19
±
(0.11)b
(0.09)
(0.13)
(0.02)
Total C (%)
pHKCl
50.37 (0.31)b
47.01 (1.72)
36.61 (3.99)
4.18 (0.39)
±
4.53
3.79
2.76
2.90
3.40
(0.09)b
(0.24)c
(0.10)c
(0.05)
(0.30)c
a
Values shown are means and standard deviations (in parentheses) n 60.
Samples were only collected in October (needle fall), n 5.
c
n 35.
b
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 ammonium values were from plate 2, reaching values up
to 0.5 mM, which was almost the ammonium concentration 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 ammonium 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 the L and F layer 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 constant in the mineral soil (see Fig. 2). Besides these general dierences between layers, the time course of both
nitrate and ammonium concentrations diered between
layers (ANOVA [repeated measures] time layer interaction: F 3:6 for nitrate, F 5:9 for ammonium,
both p < 0:001). Thus, the pattern in time was not the
same for the dierent layers, the nitrate course in the
L layer 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 standard deviations. Nitrate and ammonium in the L and
F layer 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 constant at 10±20% (Fig. 3(C)).
A signi®cant variation in time was found for
nitrate in the L F 9:9, p < 0:001), F F 22:8,
p < 0:001
and
the
mineral
soil
F 30:8,
p < 0:001). The same was found for ammonium in
L F 21:1, p < 0:001), F F 12:1, p < 0:001 and
M F 14:3, 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 ammonium varied between 100 and 600 mg per gram
Table 2
Mean values, spatial variation and temporal variation for nitrate and
ammonium in the dierent layers
NO3±N (mg gÿ1)
L
F
H
M
a
b
NH4±N (mg gÿ1)
Mean
SDa
SDb
Mean
SDa
SDb
23.1
19.5
5.8
2.4
6.2
4.7
2.1
0.4
7.1
5.6
1.6
0.2
384.0
174.3
60.3
9.1
64.9
32.3
40.1
7.1
76.4
69.4
31.6
6.5
Temporal standard deviation.
Spatial standard deviation.
A.M. Laverman et al. / Soil Biology & Biochemistry 32 (2000) 1661±1670
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
1665
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
Fig. 1. Nitrate (A) and ammonium (B) concentrations in leachate collected with ceramic plates in the ®eld. The arrows on the left side indicate
the amount of nitrate or ammonium in the rainwater. Average of all plates (*), plate 2 (w), 4 (q), 6 (r), 7 (r), 8 (t) and 9 (w).
No data are
shown from plates 1, 3 and 5 as the amount of collected leachate was too low at most time points; missing data for the other plates are also due
to low leaching.
1666
A.M. Laverman et al. / Soil Biology & Biochemistry 32 (2000) 1661±1670
Table 3. All nitri®cation was inhibited by low concentrations 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 wkÿ1 were found, the variation in mineralization rates in the litter being signi®cant in time
F 7:4, p < 0:001). The main part of the N mineraliz-
ation is due to ammoni®cation, 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 mineralization rates, varying between 70 and 150 mg N kgÿ1
wkÿ1 and variation in these rates was not signi®cant in
time. In the mineral layer the lowest amount was produced, between 5 and 15 mg N kgÿ1 wkÿ1, which varied signi®cantly in time F 15:1, 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®cation rates. The L layer showed the highest variation
Fig. 2. Nitrate (A) and ammonium (B) concentrations as measured in the L (dark gray), F (light gray), H (black) and mineral (white) soil in
time. Data represent the mean of 10 replicates. Vertical bars indicate standard deviation.
A.M. Laverman et al. / Soil Biology & Biochemistry 32 (2000) 1661±1670
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
1667
production in the F 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 the L
and F layer F 9:2 and F 15:4 respectively, both
Fig. 3. Ammonium (q) and nitrate (*) concentrations and moisture content (R) throughout the year in the litter (A), fragmentation (B) and
mineral (C) layers. Data represent the mean of ®ve replicates. Vertical bars indicate standard deviation.
1668
A.M. Laverman et al. / Soil Biology & Biochemistry 32 (2000) 1661±1670
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 F layer
as well as in the mineral soil signi®cant correlations
were observed between nitrate start values and nitrate
production R 2 0:53, p < 0:05; R 2 0:79, p < 0:001,
respectively). Other factors showed no signi®cant correlations 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 potential 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 kgÿ1 dry soil wkÿ1 for the dierent layers throughout 1997a
Net mineralization (mg N kgÿ1 wkÿ1)
January
February
March
April
May
June
July
August
September
October
November
December
a
L
F
M
L
F
M
L
F
M
L
F
M
L
F
M
L
F
M
L
F
M
L
F
M
L
F
M
L
F
M
L
F
M
L
F
M
878 (144)
95 (32)
5 (1)
342 (58)
87 (14)
10 (2)
606 (184)
124 (61)
4 (1)
732 (197)
120 (45)
13 (3)
699 (728)
75 (22)
6 (1)
296 (142)
79 (8)
5 (1)
842 (110)
102 (7)
8 (1)
812 (184)
100 (20)
8 (1)
851 (227)
115 (26)
8 (0.81)
1314 (458)
116 (11)
9 (2)
878 (295)
125 (18)
6 (2)
934 (190)
145 (32)
9 (2)
Values shown are means n 5 and standard deviations (in parentheses).
Nitri®cation (mg N kgÿ1 wkÿ1)
Net
Uptake
Total
32.2 (34.5)
2.7 (1.6)
ÿ0.2 (0.2)
ÿ2.7 (0.3)
7.7 (2.3)
0.0 (0.2)
ÿ2.4 (7.8)
3.9 (1.2)
ÿ0.3 (0.1)
9.2 (17.0)
0.9 (3.2)
ÿ0.1 (0.1)
ÿ4.9 (1.3)
0.2 (3.2)
ÿ0.1 (0.1)
ÿ0.9 (1.0)
ÿ0.2 (0.3)
0.0 (0.1)
9.0 (32.5)
3.0 (4.5)
0.2 (0.2)
7.0 (21.3)
3.9 (3.8)
ÿ0.1 (0.1)
12.1 (16.2)
0.9 (2.1)
ÿ0.0 (0.2)
ÿ5.7 (1.8)
ÿ1.4 (1.7)
ÿ0.1 (0.1)
ÿ2.8 (2.3)
ÿ0.2 (0.4)
ÿ0.1 (0.1)
8.2 (13.0)
ÿ0.9 (1.1)
ÿ0.3 (0.1)
ÿ4.5 (1.9)
ÿ2.3 (0.8)
ÿ0.3 (0.2)
ÿ2.6 (0.4)
ÿ0.7 (0.3)
0.21 (0.2)
ÿ9.8 (1.5)
ÿ4.5 (0.7)
ÿ0.5 (0.1)
ÿ3.7 (1.0)
ÿ1.8 (0.7)
ÿ0.2 (0.1)
ÿ5.7 (1.3)
ÿ2.5 (0.6)
ÿ0.4 (0.1)
ÿ1.3 (0.5)
ÿ0.4 (0.5)
ÿ0.0 (0.1)
ÿ5.0 (3.1)
ÿ1.4 (0.7)
ÿ0.2 (0.2)
ÿ7.7 (1.6)
ÿ4.7 (1.3)
ÿ0.4 (0.2)
ÿ4.3 (1.4)
ÿ3.4 (0.9)
ÿ0.3 (0.0)
ÿ6.9 (1.1)
ÿ3.9 (1.0)
ÿ0.2 (0.0)
ÿ4.4 (2.7)
ÿ2.8 (0.9)
ÿ0.2 (0.1)
ÿ5.92 (1.4)
ÿ4.67 (1.4)
ÿ0.51 (0.1)
36.7 (36.0)
5.0 (1.3)
0.2 (0.4)
ÿ0.2 (0.3)
8.3 (2.0)
ÿ0.2 (0.2)
7.4 (7.3)
8.4 (1.8)
0.2 (0.1)
12.9 (16.2)
2.7 (3.0)
0.1 (0.1)
0.8 (1.3)
2.7 (3.2)
0.4 (0.1)
0.4 (0.7)
0.1 (0.5)
0.1 (0.0)
14 (30.9)
4.5 (4.1)
0.4 (0.2)
14.7 (22.7)
8.6 (4.8)
0.4 (0.2)
16.4 (15.1)
4.3 (3.0)
0.3 (0.2)
1.2 (1.1)
2.5 (1.7)
0.1 (0.1)
1.7 (2.1)
2.6 (1.0)
0.1 (0.2)
14.2 (12.1)
3.8 (2.4)
0.2 (0.1)
A.M. Laverman et al. / Soil Biology & Biochemistry 32 (2000) 1661±1670
potential nitrate production was highest and also most
variable. In this layer high nitrate uptake was
observed. Whether this was due to nitrate immobilisation or denitri®cation remains unclear. Nitrate immobilisation 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 importance 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 related to dierences in nitrogen levels, pH, structure
and moisture content. As ammonium was always present 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 nitrogen 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 factors 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 production is temperature, as samples taken at a certain
time were all exposed to the same ®eld temperature
and incubation temperatures were kept constant. However, 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 leached from the soil, indicating that the soil is ammonium-saturated. However, a low availability of
ammonium at the microsite level due to the composition of the soil matrix could limit nitri®cation
(Davidson and Hackler, 1994; Drury et al., 1991). In
addition, competition for ammonium between heterotrophic bacteria and nitri®ers (Verhagen et al., 1992)
1669
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 contents were comparable.
The question remains which factors are in¯uencing
nitri®cation rates, a possibility being allelopathic compounds, 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 activity of the soil fauna community (Verhoef and Brussaard, 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 transformations are still necessary for a detailed understanding 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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