Directory UMM :Data Elmu:jurnal:A:Applied Soil Ecology:Vol13.Issue3.Dec1999:

Applied Soil Ecology 13 (1999) 187±198

Mineralization and immobilization of nitrogen in heath
soil under intact Calluna, after heather beetle
infestation and nitrogen fertilization
H.L. Kristensena,b,*, G.W. McCartyb
a

Department of Terrestrial Ecology, National Environmental Research Institute, Vejlsùvej 25, P.O. Box 314, DK-8600 Silkeborg, Denmark
b
Environmental Chemistry Laboratory, USDA-ARS, Bldg. 007, BARC-West, Beltsville, MD 20705, USA
Received 5 November 1998; received in revised form 25 April 1999; accepted 8 May 1999

Abstract
The maintenance of low availability of mineral N in heath soils is thought to be a key factor for the stability of heathland
ecosystems. We investigated the turnover of NH4‡ and NO3ÿ in the organic surface layer of soils from a Danish heathland
using 15N isotope techniques in laboratory incubations. The soils were sampled under intact and dead Calluna vegetation. The
dead Calluna vegetation had been fertilized at rates of 0, 15 or 35 kg N haÿ1 per year and the death of vegetation had been
caused by a naturally occurring heather beetle infestation. In the soil under intact Calluna, the NH4‡ pool was very low with
no net mineralization, while a substantial mineralization-immobilization turnover of NH4‡ was found with a large capacity for
short term net NH4‡ immobilization (36 mg N gÿ1 during 1 h; 135 mg N gÿ1 during 24 h). The metabolic inhibitor mercury

chloride completely inhibited assimilation of NH4‡ indicating the process was biological. The immobilization of NH4‡ had no
short or long-term (38 days) effect on soil respiration while NH4‡ immobilization stimulated net mineralization of soil N
during long-term incubation. The soils sampled under dead and dead/fertilized Calluna had large pools and high net
mineralization rates of NH4‡ with a decrease of gross NH4‡ immobilization relative to the soil under intact Calluna. Neither
net nor gross nitri®cation activity could be detected in any of the soils. The results indicate that the effects of an increased
atmospheric N deposition to the heathland may be delayed because of the tight cycling of NH4‡ and the storage capacity for N
in the soil and vegetation. The ecosystem may, however, be susceptible to disruption of the tight NH4‡ cycling because of the
limited capacity of the ecosystem to remove excess mineral N from the soil. This may increase the risk of conversion of the
heath into grassland. # 1999 Elsevier Science B.V. All rights reserved.
Keywords: Calluna vulgaris; Gross ammoni®cation; Immobilization; Lochmaea suturalis; Nitri®cation

1. Introduction
Lowland heath ecosystems once covered extensive
areas of Western Europe, but this coverage has been
*Corresponding author. Tel.: +45-8920-1764; fax: +45-8920-1414
E-mail adress: hlk@dmu.dk (H.L. Kristensen)

greatly reduced during the last two centuries as a result
of land use change. During the last two decades much
work has been done to increase our understanding of

heathland ecology and to enable the continued preservation of the remnant heathlands. There has been
growing evidence that the heath areas are threatened
by increased atmospheric deposition of anthropogenic

0929-1393/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 9 - 1 3 9 3 ( 9 9 ) 0 0 0 3 6 - 0

188

H.L. Kristensen, G.W. McCarty / Applied Soil Ecology 13 (1999) 187±198

N that changes heaths into grasslands and by natural
succession of vegetation leading to the development of
forest (Gimingham et al., 1979; Aerts and Heil, 1993).
The dry lowland heaths are dominated by Calluna
vulgaris (L.) Hull. and the litter of this species is
known to be of low palatability for decomposers,
which is thought to be the cause of organic matter
accumulation in the mor layer (O horizon) (Read,
1991). Field and laboratory studies have found little

or no apparent soil N mineralization, resulting in
mineral N pools being maintained at very low content
(Adams, 1986; Rangeley and Knowles, 1988; Kristensen and Henriksen, 1998). In addition, the heath
mor has been found to immobilize substantial amounts
of mineral N (Kristensen and Henriksen, 1998). The
mor layer is, however, considered to be the main
source for plant N uptake and it has been suggested
that the apparent lack of mineral N in the soil is a key
factor for the functioning of heathland ecosystems
(Read, 1991). Studies have indicated the Calluna and
other ericaceous species may be able to grow under
these conditions because they live in symbiosis with
ericoid mycorrhiza which have an ability to degrade
recalcitrant organic N accumulated in the mor layer.
The resulting simple N compounds can then be assimilated by the mycorrhiza and transferred to the host
plant in exchange for photosynthetic products (Smith
and Read, 1997). This may give Calluna and related
species an advantage in the competition with grasses
and herbs which may have arbuscular mycorrhiza and,
therefore, are thought to depend primarily on mineral

N (Michelsen et al., 1996, 1998) or amino acid N for
growth. The latter pathway has recently been indicated
for the grass Deschampsia ¯exuosa (L.) Trin (NaÈsholm
et al., 1998) which is indigenous to lowland heaths
(Gimingham et al., 1979). Free living soil microorganisms may likewise use organic instead of mineral
N during decomposition of organic matter (Jennings,
1995; Barraclough, 1997). With this background, it
can be questioned to what degree mineralization±
immobilization turnover of N is operating in Calluna
mor which has no net mineralization activity. Net
ammoni®cation and nitri®cation have been reported
to occur in several studies of lowland heath soils and
this occurrence has been related to changes in the
species composition of the vegetation as well as to
increased atmospheric N deposition to the ecosystem
(Berendse, 1990; Troelstra et al., 1990; Van Vuuren et

al., 1992; Kristensen and Henriksen, 1998). The atmospheric N deposition has increased in the industrialized countries during the last century as a
consequence of emission of N compounds from agriculture, industry and transportation. In Denmark the
total atmospheric N deposition was, for example, on

average 15 kg N haÿ1 per year in 1996 (Bak et al.,
1999). The deposition is thought to increase the
availability of mineral N in the heathland ecosystem,
which may increase the ability of grasses to compete
with the Calluna vegetation. Studies have found that
grasses like D. ¯exuosa are replacing the Calluna
vegetation in parts of European lowland heath areas
(Aerts and Heil, 1993; Marrs, 1993). This change can
be related to the in¯uence of increased atmospheric N
deposition on decomposition and mineralization in
Calluna mor (French, 1988; Kristensen and Henriksen, 1998), but can also be due to increased N availability in combination with damage to the Calluna
vegetation (Prins et al., 1991). Such damage can result
from naturally occurring epidemic attacks on Calluna
by the heather beetle (Lochmaea suturalis Thoms.)
which may increase in severity and frequency as a
result of increased N input to heathland ecosystems
(Brunsting and Heil, 1985). Little is known, however,
about the in¯uence of beetle infestations on mineralization±immobilization turnover in the mor under
Calluna or the extent to which atmospheric N deposition affects such processes.
The purpose of this study was to investigate the

turnover of NH4‡ and NO3ÿ (i.e., gross mineralization
and immobilization processes) in Calluna mor which
has no net mineralization activity (Kristensen and
Henriksen, 1998). In addition, N and C mineralization
was studied in the mor under Calluna which had been
killed due to infestation by heather beetles (Lochmaea
suturalis) in combination with increased N availability, as these perturbations of the ecosystem are
expected to be key factors in the conversion of Calluna
lowland heaths to grasslands.

2. Materials and methods
2.1. Study sites and sampling
Soil samples were collected from the heathland
Hjelm Hede (088550 E, 568290 N), which is situated

H.L. Kristensen, G.W. McCarty / Applied Soil Ecology 13 (1999) 187±198

in Western Jutland, Denmark, on a ¯uvio-glacial sand
plain from the Weichselian period (10 000 BP). The
soil was a typical Haplorthod; and the total wet and

dry atmospheric N deposition amounted to an average
of 18 kg N haÿ1 per year (Hansen and Nielsen, 1998).
A detailed site description is presented by Kristensen
and Henriksen (1998). Three experimental plots
(5  9.5 m2) were established on the heathland during
the summer of 1993. They were fertilised six times a
year in the periods September±November and March±
May with NH4NO3 in annual doses of 0, 15, and 35 kg
N haÿ1. The fertilizer was applied above the vegetation as 15 l of solution per ®eld plot with a 2.5 m
spraying fan. The western part of the heathland study
area containing the experimental plots was subjected
to a natural epidemic attack by the heather beetle
(Lochmaea suturalis) in July of 1994 which caused the
Calluna to turn reddish and defoliate. The attack did
not spread to the eastern part of the heathland and this
part was therefore left with an intact stand of Calluna
vegetation. The experimental plots were sampled
together with an additional plot which was situated
within a distance of 500 m from the other plots in the
area that was not subjected to beetle infestations. The

experimental plots are distinguished as shown in
Table 1. At the time of soil sampling the I plot was
dominated (>90% coverage) by intact Calluna in the
building phase (last cut in 1991). The D, D ‡ 15, and
D ‡ 35 plots were covered by patches of dead and
decomposing Calluna as well as live Empetrum
nigrum ssp migrum L., D. ¯exuosa, mosses and
lichens. The vegetation in the three plots with dead
Calluna is described in details by Riis-Nielsen (1997)
who found no effect of N fertilization on the phanerogam vegetation in 1995. Soil samples were taken in
November 1995 on a date 21 days after an N fertilizer
application. The samples consisted of three replicate
turfs (15  22 cm; bulk density 0.3 g cmÿ3) of 2±4 cm
thick organic mor layer (the O horizon). The turfs

189

Fig. 1. The gross processes involved in N turnover in soil as
measured by use of isotope dilution techniques.

were taken randomly within each plot, but always
under intact or dead Calluna vegetation. Any aboveground vegetation and fresh litter was removed and the
soils were kept moist and cool until the turfs were
sieved (4 mm mesh size), pooled, ad stored in plastic
bags at 48C.
2.2. Nitrogen kinetics
The gross processes of N turnover presented in
Fig. 1 were estimated by use of 15N labelling and
equations based on the principles of isotope dilution
(Hart et al., 1994):
‡
m ˆ …‰NH‡
4 Š0 ÿ ‰NH4 Št †=t  …log…APE0 =APEt ††=
‡
…log…‰NH‡
4 Š0 =‰NH4 Št ††
‡
i ˆ m ÿ …‰NH‡
4 Št ÿ ‰NH4 Š0 †=t

(1)
(2)

where m ˆ gross N mineralization rate, i ˆ NH4‡
immobilization rate, t ˆ time, APE0 ˆ atom% 15N
excess of the NH4‡ pool at time 0, APEt ˆ atom%
15
N excess of the NH4‡ pool at time t, [NH4‡]0 ˆ total
NH4‡ concentration at time 0, [NH4‡]t ˆ total NH4‡
concentration at time t. Gross nitri®cation and NO3ÿ
immobilization rates were estimated by replacing
NH4‡ with NO3ÿ in the equations. The experiment
involved incubation of soil samples (15 g DW) in
slurries of deionized water (soil : water ratio 1 : 8)
in 250 ml Erlenmeyer ¯asks on an orbital shaker

Table 1
The four experimental plots that were sampled in November 1995
Plot

Treatment

I
D
D ‡ 15
D ‡ 35

Intact Calluna vegetation
Dead Calluna due to heather beetle infestation in July 1994
Dead Calluna due to heather beetle infestation in July 1994, fertilized with 15 kg N haÿ1 per year since Sept. 1993
Dead Calluna due to heather beetle infestation in July 1994, fertilized with 35 kg N haÿ1 per year since Sept. 1993

190

H.L. Kristensen, G.W. McCarty / Applied Soil Ecology 13 (1999) 187±198

(180 rpm) at 228C. At the start of the experiment (time
zero), 5% enriched 15N solution was added to each of
three replicate slurries of each soil. A solution of
15
NH4NO3 was added to the slurries for estimation
of gross ammoni®cation and NH4‡ immobilization
rates, while K15NO3 was added to the slurries for
estimation of gross nitri®cation and NO3 immobilization rates. The ®nal concentration obtained was
200 mg gÿ1 DW soil equivalent to 25 mg mlÿ1 slurry
of NH4‡-N or NO3-N, respectively. After intervals of
1, 24, and 48 h, each slurry was subsampled for 25 g of
slurry and 3 M KCl was added to a ®nal concentration
of 1 M and soil : solution ratio of 1 : 12.5. These
subsamples were extracted for 1 h by shaking; centrifuged at 10 000 rpm for 10 min; and ®ltered through
prerinsed Gelman Acordis glass ®lters. Finally, the
subsamples were stored at 38C until analysis for
amount and isotopic content of N. Additional slurries
of each soil were incubated with KNO3 and C2H2 to
test for occurrence of denitri®cation by analysis of
N2O in gas samples obtained from the sealed incubation ¯asks.
2.3. Inhibition of NH4‡ immobilization and short
term CO2 production
To investigate the chemical/biological nature of the
immobilization process in the soil sampled under
intact Calluna vegetation, the rates of gross NH4‡
immobilization were measured in soil slurries treated
with a metabolic inhibitor by use of isotope dilution
techniques (Eqs. (1) and (2)). The experiment
included a 48 h preincubation at 258C of six replicate
samples of moist soil (10 g DW) in Erlenmeyer ¯asks
(250 ml) with mercury chloride (20 mg HgCl2 gÿ1
DW soil) added to two of the samples to inhibit
biological activity (Wolf and Skipper, 1994). After
preincubation, deionized water was added to all the
samples to make soil slurries (soil : water ratio 1 : 8)
which were then placed on an orbital shaker (180 rpm)
at 228C. The N transformation assay was initiated by
addition of 200 mg 15NH4‡-N gÿ1 DW soil in the form
of 15NH4NO3 (5% 15N enriched) in solution to all
slurries (time zero) except to two replicates which
were incubated without any additions. Subsampling of
slurry for analysis of amount and isotopic content of N
was conducted after 0.25, 6 and 12 h by the same
produce as in the N kinetic experiment. During incu-

bation the ¯asks were sealed and the headspace was
subsampled with a gas syringe for CO2 analysis.
2.4. Long-term N mineralization and CO2 production
To study the long-term effects of increased N
availability on net ammoni®cation, nitri®cation, and
respiration, the soils were incubated with additions of
NH4NO3 during a 38 days incubation period. Two
replicate soil samples (10 g DW) from each plot were
slightly dried and then brought back to the original
moisture content by drop wise addition and mixing of
either deionized water or NH4NO3 solution. The
NH4NO3 addition was equivalent to 200 mg NH4‡N gÿ1 DW soil. The samples were incubated at 258C
in 220 ml bottles which were sealed and subsampled
with a gas syringe for CO2 analysis. The bottles were
opened for aeration every 5±7 days during incubation.
Samples of each soil were extracted at the start and the
end of the experiment by shaking for 1 h in 1 M KCl
(soil : solution ratio 1 : 5) followed by centrifugation,
®ltration, and storage of the ®ltrate for a maximum of
two weeks at 38C until mineral N analysis. The
calculations of the net ammoni®cation rate for the
soil sampled under intact Calluna vegetation were
performed using estimates of the initial NH4‡ pool
as being equal to the sum of added NH4‡ and endogenous NH4‡.
2.5. The microbial biomass and sample analysis
The microbial biomass was estimated by substrate
induced respiration (SIR) as described by Anderson
and Domsch (1978) which included incubation of two
replicate samples (10 g DW) of each soil with additions of 3000 mg glucose gÿ1 DW soil. The soils were
analyzed for soil water content by drying at 1058C for
24 h and all results were calculated on a soil dry
weight basis.The total amount of C and N in the soils
was measured by dry combustion using a C and N
analyzer (Leco CNS-2000). Soil pH was measured in
1 M KCl (soil : solution ratio 1 : 12.5). All soil KCl
extracts were analyzed colorimetrically for NH4‡ and
NO3 content by use of an automated ¯ow-injection
analyzer (Lachat Instruments). The 15 N enriched
extracts were prepared for isotopic analysis of
NH4‡ and NO3 using the diffusion method of Brooks
et al. (1989). Isotope analysis were carried out using

191

H.L. Kristensen, G.W. McCarty / Applied Soil Ecology 13 (1999) 187±198

an isotope mass spectrometer interfaced with an automated N±C analyzer (Europa Scienti®c). The CO2 and
N2O content of the gas samples were measured by gas
chromatography (Tremetrics) (McCarty and BlicherMathiesen, 1996).
2.6. Statistical analysis
The three replicate turfs taken from each ®eld
treatment were combined to form one composite
sample. This procedure eliminated information on
the variation of properties for each treatment and this
limits the ability to perform statistical analyses for
detecting differences between the ®eld treatments
(Hurlbert, 1984). However, the properties found for
each composite soil represented averages for the ®eld
plot. This allows us to use major differences in properties found between the four soil samples as indications
of the differences between the four treatments. Due to
the limitations on statistical analysis, however, conclusions about differences resulting from the ®eld
treatments should be undertaken with some caution.
All laboratory incubations were replicated and standard errors were calculated for data obtained from
each incubation. Differences between means of
laboratory treatments were tested statistically within
each soil by analysis of variance (SAS Institute, Cary,
NC).

3. Results
3.1. Soil N pools and N kinetics
Some chemical and biological properties of the I, D,
D ‡ 15 and D ‡ 35 soils are seen in Table 2. The
NH4‡ pool was very low in the I soil while it was

Fig. 2. The net rates of production and immobilization of NH4‡
and NO3ÿ in the four heath soils (termed according to Table 1)
during 48 h of incubation. Changes in the NH4‡ and NO3ÿ pools
were measured after addition of NH4NO3 and KNO3, respectively.
Error bars indicate standard errors for means obtained during
incubations of a composite sample of each soil.

180 mg N gÿ1 in the D soil with an even greater pool in
the D ‡ 15 and a maximum of 370 mg N gÿ1 in the
D ‡ 15 soil. No NO3ÿ was detected in the I and D
soils and only a small amount (9 mg N gÿ1) was found
in the D ‡ 15 and D ‡ 35 soils. The rates of net
change in inorganic N pools during the N kinetic
experiment are presented as a combination of net
changes of the NH4‡ pool in the NH4NO3 slurries
and net changes in the NO3ÿ pool in the KNO3 slurries
(Fig. 2). A signi®cant part of the NH4‡ that was added
to the slurries of I soil was immobilized at the ®rst
sampling event of the experiment and the added plus
the endogenous NH4‡ pool was therefore used as the
time zero value of the NH4‡ pool during calculation of
rates in this soil. The I soil showed a large net NH4‡
immobilization of almost 3 mg N gÿ1 hÿ1, while the
D, D ‡ 15 and D ‡ 35 soils showed net ammoni®cation rates in the range of 0.4±0.9 mg N gÿ1 hÿ1. In
addition, NO3ÿ was subject to net immobilization in
the I soil at a rate of 0.4 mg N gÿ1 hÿ1 when applied as
KNO3 (Fig. 2) and at a lower rate of 0.1 mg N gÿ1 hÿ1
when applied as NH4NO3 (results not shown)

Table 2
Some chemical and biological properties and N pools of the O horizon of the four heath soils (termed according to Table 1)

I
D
D ‡ 15
D ‡ 35

Soil
water %
H2O

pH(KCl)

C/N

C%

N%

Microbial
biomass
(mg C gÿ1)

NH4‡
pool
(mg N gÿ1)

NO3ÿ
pool
(mg N gÿ1)

150
185
160
200

2.90
3.07
3.22
3.21

26.6
26.3
23.0
24.4

36.3
35.5
30.6
35.8

1.37
1.35
1.33
1.47

5.21
3.96
3.88
4.86

1
180
300
370

0.9
1.1
8.6
8.9

192

H.L. Kristensen, G.W. McCarty / Applied Soil Ecology 13 (1999) 187±198

Table 3
Gross rates of N turnover in the four heath soils (termed according to Table 1) as measured by isotope dilution techniques during 48 h
incubation. Numbers in brackets are standard errors for means obtained during incubations of a composite sample of each soil
Ammonification

NH4‡ immobilization

Nitrification

NO3ÿ immobilization

4.31
0.80
0.56
1.02

0
0
0
0

0.37 (0.05)
0
0
0

mg N gÿ1 hÿ1
I
D
D ‡ 15
D ‡ 35

1.39
1.19
1.43
1.67

(0.04)
(0.05)
(0.04)
(0.06)

(0.02)
(0.10)
(0.15)
(0.20)

(p < 0.01). In either case, the rate of net NO3ÿ immobilization was much lower than that of net NH4‡
immobilization (p < 0.001). Net nitri®cation was not
detectable in any of the soils (Fig. 2).
The gross ammoni®cation rate in the I soil during
the N kinetic experiment (Table 3) was 1.4 mg
N gÿ1 hÿ1 which was in the range of the gross ammoni®cation rates in the soils under dead Calluna vegetation (1.2±1.7 mg N gÿ1 hÿ1). The gross NH4‡
immobilization rate was 4.3 mg N gÿ1 hÿ1 in the I
soil, which was much higher than the rates in the D,
D ‡ 15 and D ‡ 35 soils (0.6±1.0 mg N gÿ1 hÿ1).
None of the soils showed any gross nitri®cation,
and NO3ÿ immobilization was seen in the I soil only.
In the I soil, the NH4‡ process rates were found not to
be constant during the 48 h experimental period of the
N kinetic experiment. This is illustrated in Fig. 3 by
showing the remaining NH4‡ pool in the slurry at each
time of subsampling as well as the calculated gross
process rates between sampling events. The NH4‡

concentration in the slurry decreased very quickly
to an amount around 160 mg N gÿ1 within the ®rst
hour of incubation and then decreased to 66 mg N gÿ1
during the 1±24 h interval. Thereafter, the pool
remained almost constant until the 48 h subsampling
leaving a pool of 60 mg NH4‡-N gÿ1 in the slurry at
the end of the experiment. The gross ammoni®cation
rate decreased from 3.4 to 0.8 mg N gÿ1 hÿ1 during the
experiment while the gross NH4‡ immobilization rate
decreased from 39 to 5.0 and then to 1.1 mg N gÿ1 hÿ1
over the 0±1, 1±24 and 24±48 h interval, respectively.
No denitri®cation was detected from any of the soils
during the incubations with C2H2.

Fig. 3. Net changes (left y axis) in the NH4‡ pool ÐÐÐ; and
gross rates (right y-axis) of ammonification & and NH4‡
immobilization & in NH4NO3 treated soil which was obtained
from under intact Calluna vegetation. Error bars indicate standard
errors.

Fig. 4. Effect of a metabolic inhibitor on the rate of gross NH4‡
immobilization in NH4NO3 treated soil which was obtained from
under intact Calluna vegetation. The treatments were: no inhibitors
(Control); or HgCl2 to inhibit biological acitivity. Error bars
indicate standard errors.

3.2. Inhibition of NH4‡ immobilization and CO2
production
The gross NH4‡ immobilization rates in the slurries
of I soil that were treated with HgCl2 are presented in
Fig. 4. The rate in the soil with NH4NO3 additions was

H.L. Kristensen, G.W. McCarty / Applied Soil Ecology 13 (1999) 187±198

Fig. 5. The CO2 production after no addition or addition of
NH4NO3 to soil obtained from under intact Calluna vegetation.
Error bars indicate standard errors.

7.0 mg N gÿ1 hÿ1 while the rate was close to zero in
the slurries which had been treated with HgCl2 and
NH4NO3 No CO2 was produced in the HgCl2 treated
slurries which indicated that this treatment effectively
stopped soil microbial activity (results not shown).
Comparison of the net change in the NH4‡ pool in the
slurries without additions and the slurries with
NH4NO3 additions showed that NH4‡ was immobilized at a rate of 5.3 mg N gÿ1 hÿ1 in the latter, while
the slurries without additions showed no net change in
the NH4‡ pool of 1.8 mg N gÿ1 during the 12 h
experimental period (results not shown). The respiration rate measured in the two treatments did not,
however, differ either during the ®rst (p < 0.58) or
the last half (p < 0.54) of the experimental period
(Fig. 5).
3.3. Long-term N mineralization and CO2 production
The net ammoni®cation rates in the four soils
during the 38-day incubation period were generally
higher in the samples with N additions as compared to
the soils without additions (Fig. 6). The difference
was signi®cant (p < 0.01) except for the D ‡ 35 soil
(p < 0.59). The largest increase in the net ammoni®cation rate when N was added was seen in the I soil
where the rate increased more than threefold to 0.3 mg
N gÿ1 hÿ1. In the soils without N additions, the rates
increased in the order, I, D, D ‡ 15 and D ‡ 35 while
the order was I, D ‡ 15, D and D ‡ 35 with N
additions. No net nitri®cation or NO3ÿ immobilization
was observed in any soil during the experiment

193

Fig. 6. The net rates of ammonification after no addition or
addition of NH4NO3 to the four heath soils (termed according to
Table 1) during 38-day incubation in the laboratory. Error bars
indicate standard errors and ** indicate differences between
treatments within each soil (F-test, p < 0.01).

(results not shown). The respiration rate was
unchanged with N addition in the I soil (p < 0.20),
while it decreased in the D, D ‡ 15 and D ‡ 35 soils
in the samples where N was added when compared to
the samples where no N was added (Fig. 7), however,
the difference was only statistically signi®cant in the
D ‡ 15 soil while it was close to signi®cant in the
D ‡35 soil and the D soil.

4. Discussion
4.1. Turnover and net immobilization of NH4‡ in the
Calluna mor
The mor under intact Calluna vegetation showed a
substantial capacity for turnover of NH4‡ (Fig. 3)
with gross ammoni®cation rates (0.8±3.4 mg
N gÿ1 hÿ1) comparable to those found by Tietema
(1998) in acid organic forest soils (i.e., 0.4±0.8 mg
N gÿ1 hÿ1) with similar contents of organic matter and
microbial biomass. Thus, the results obtained under
intact Calluna give no support to the theory that the
microbial activity in heath mor is inhibited by the
release of toxic compounds from the Calluna vegetation (Jalal and Read, 1983). This conclusion is in
accordance with the work of Rangeley and Knowles
(1988) who found that microbial activity as measured
by CO2 production in limed Scottish heath mor was
comparable to that of agricultural soil. The present
study found, however, an unusual ability for rapid net
immobilization of added NH4‡ (Fig. 2). In the heath

194

H.L. Kristensen, G.W. McCarty / Applied Soil Ecology 13 (1999) 187±198

Fig. 7. The cumulative CO2 production after no addition or addition of NH4NO3 to the four heath soils (termed according to Table 1) during
38 day incubation. Standard errors and the p-value of F-test of differences between treatments at the end of the experiment are indicated for
each soil.

soil, the NH4‡ pool was maintained at a very low
content (Table 2) and the rate of gross NH4‡ immobilization was much higher than that of gross ammoni®cation after addition of NH4‡ (Fig. 3). This
indicates that the addition of NH4‡ stimulated the
immobilization process. Moreover, this suggests that
the mineralized NH4‡ in general may be re-immobilized in this soil immediately after being released to
the soil NH4‡ pool, and this keeps the pool as well as
the net ammoni®cation rate very low which was also
found in ®eld studies with soil from the same site
(Kristensen and Henriksen, 1998). The cause of this
may be the chemical environment created in the soil
by the Calluna vegetation. For example, Calluna litter
is known to have a high content of soluble phenolic
compounds which enhance the formation of recalcitrant humic complexes through condensation and
microbially mediated immobilization of organic N
(Kuiters, 1990; Read, 1991).
The ability of heathland mor to rapidly immobilize
N could have consequences for the fate of N that is

deposited on the soil from the atmosphere. The conditions for immobilization of inorganic N in short term
slurry experiments differ greatly from ®eld conditions
as regards availability of substrate, temperature, soil
disturbance, etcetera. The results obtained in the present study can, however, indicate the potential capacity of the heath mor under intact Calluna to
immobilize N and was for the 48 h experiment found
to be equivalent to 10±15 kg NH4‡-N haÿ1 and 1±2 kg
NO3ÿ-N haÿ1. Such a capacity for immobilization is
substantial when considering that the total annual N
deposition has been estimated to be 18 kg N haÿ1 per
year for the heathland under study with 65% being
deposited as NHx and the remainder as NOy (Hansen
and Nielsen, 1998). The mor may in this way act as a
short term sink for N from atmospheric deposition,
which may prevent that inorganic N from being
leached to deeper soil layers. This was con®rmed
by studies from the same heathland where intact cores
of the mor layer taken under intact Calluna showed net
immobilization of both NH4‡ and NO3ÿ from rain-

H.L. Kristensen, G.W. McCarty / Applied Soil Ecology 13 (1999) 187±198

water during a 35-day laboratory experiment (Kristensen and Henriksen, 1998). Likewise, studies of
percolating soil water collected under intact Calluna
from this heathland found no NO3ÿ and only traces
of NH4‡ to be leached below the mor layer (Nielsen
et al., 1999). The high capacity for net immobilization
of the mor layer may also maintain low availability of
inorganic N for plant uptake. This probably strengthens the competitive ability of the ericaceous vegetation at the expense of the herbaceous vegetation since
the symbiosis with ericoid mycorrhiza may give the
plant access to recalcitrant organic N (Smith and
Read, 1997).
4.2. Turnover of N in mor under dead Calluna
vegetation
The tight cycling of N in the mineralization±immobilization system in the mor under intact Calluna
was greatly in¯uenced by the heather beetle attack
on the Calluna vegetation. The availability of inorganic N increased from a very low to a high level as
shown by both large pools of NH4‡ and occurrence
of net ammoni®cation (Table 2, Fig. 2). This was
indicated to be a result of a decrease in the gross
immobilization rate while the gross ammoni®cation rates in the three soils sampled under dead
Calluna vegetation were about the same as the rate
in the soil under intact vegetation (Table 3). The
increase of the NH4‡ pool under dead as compared
to intact Calluna was, when calculated on an areal
basis, in the range of the amount of NHx which had
been deposited from the atmosphere since the occurrence of the heather beetle infestation (both around
16 kg N haÿ1). These changes in availability of inorganic N were probably due to a combined effect of the
defoliation and death of the Calluna vegetation with
the heather beetle infestation which resulted in the
cessation of uptake of N by vegetation while litter
production from the dead plants, faeces and dead
beetles was increased. The decrease in gross immobilization after the heather beetle attack could, however, also indicate that the capacity for NH4‡
immobilization under intact Calluna was caused by
a direct in¯uence from the intact Calluna vegetation,
possibly in the form of soluble phenolic compounds
that may be released from leaves and roots (Kuiters,
1990).

195

4.3. Turnover of NO3ÿ
Despite the high availability of NH4‡ as substrate
for autotrophic nitri®cation in the three soils under
dead Calluna vegetation (Table 2), no net or gross
nitri®cation could be detected in any of these soils nor
in the soil under intact Calluna vegetation (Fig. 2,
Table 3). This is one of the few studies that we know
of which has documented a total lack of gross nitri®cation activity in soil. The small amount of NO3ÿ
found in the fertilized plots (Table 2) probably originated from the NH4NO3 fertilizer added 21 days
before the sampling of soil for this study. The lack
of gross nitri®cation can possibly be explained by the
very low pH in the soils (pHKCl around 3 equivalent to
pHH2 O around 4 (Kristensen and Henriksen, 1998)).
However, other studies of for example Dutch heath
soils have found net nitri®cation to occur under similar
acidic conditions (Troelstra et al., 1990). Furthermore,
substantial gross nitri®cation and NO3ÿ immobilization was found in undisturbed forest soils at pH below
4 even though net nitri®cation could not be detected
(Stark and Hart, 1997). It was surprising that there was
a lack of nitri®cation in the soil after the death of the
Calluna vegetation as perturbation of the vegetation in
general is thought to stimulate nitri®cation and this
may lead to N removal from the ecosystem through
NO3ÿ leaching and denitri®cation. The vegetation is
in this way thought to exert biological control over N
losses from some natural ecosystems (Tamm, 1991)
but there was no indication that live Calluna vegetation exerted any direct control over nitri®cation. The
lack of nitri®cation and the subsequent limited capacity for mineral N removal from the heath ecosystem
can help explain the accumulation of NH4‡ in the soil
after perturbation of the vegetation (Table 2) as NH4‡
is adsorbed in the soil and not readily leached with
percolating water. This accumulation of NH4‡ could
be expected to enhance the destabilizing effects of
the beetle attack on the ecosystem by increasing
the availability of inorganic N for competing plant
species.
Nitrate was found to be immobilized in the soil
under intact Calluna vegetation but the process was
found to be inhibited when NH4‡ was also available.
This indicates that the immobilization of NO3ÿ was
probably the result of assimilatory nitrate reductase
(ANR) activity in the microbial biomass which is

196

H.L. Kristensen, G.W. McCarty / Applied Soil Ecology 13 (1999) 187±198

known to be inhibited by microbial assimilation of
NH4‡ (McCarty and Bremner, 1992).

this may further increase the resistance of the immobilized N to decomposition.

4.4. The NH4‡ immobilization process

4.5. Long-term effects of N on mineralization

The use of HgCl2 as an inhibitor of biological
activity showed that the NH4‡ immobilization was
a fully biological process (Fig. 4). The rate of gross
NH4‡ immobilization during the ®rst hour of the
incubation was found to be extraordinarily high at
39 mg N gÿ1 hÿ1 (Fig. 3). This rate may tentatively be
compared with the maximum gross NH4‡ immobilization rate of 23 mg N gÿ1 hÿ1 found by Schimel and
Firestone (1989) in acid coniferous forest ¯oor material. They found that 19% of the immobilization was
due to abiotic processes for gross immobilization rates
in the range of 4±9 mg N gÿ1 hÿ1. However, in the
present study all of the NH4‡ was immobilized by the
microbial population, and the immobilized 15N could
therefore be expected to constitute a signi®cant part of
the biologically active N pool in the soil. An assumption made in Eqs. (1) and (2) is that no immobilized
15
N will be re-mineralized during the experiment
(Hart et al., 1994). With the immobilization of a
large quantity of 15N into the biologically active
pool in the soil, it is likely that this assumption was
violated to some degree. This may have contributed
to decreases in gross ammoni®cation and NH4‡
immobilization rates with the successive periods of
sampling (Fig. 3).
The large microbial assimilation of added NH4‡ in
the soil under intact Calluna suggests that the microbial community was limited by availability of N. But
we found that CO2 production was not stimulated by
addition of NH4NO3 to soil slurries (Fig. 5) and this
contrasts with the expected result if the N assimilation
had stimulated the activity or the growth of the
heterotrophic microorganisms. It is possible, however,
that the assimilated NH4‡ was a type of `luxury'
uptake as described by Fog (1988), where N is
assimilated and stored for later use. For example,
fungi have been found to accumulate amino acids
and it has been suggested that protein inclusions
and other fungal cell structures may act as storage
for N in insoluble form (Jennings, 1995). Moreover,
Kerley and Read (1997) have hypothesized that N may
be incorporated into fungal cell walls through melanisation to enable fungi to persist in the heath soil and

The addition of N during the long-term incubation
experiment was found to increase the net ammoni®cation rate in the soil sampled from under intact Calluna
vegetation while the respiration was largely unaffected. The latter trend was also observed in the
short-term experiment (Figs. 6 and 7). It is possible
that the increase in N availability induced the
decomposition of N-rich organic compounds with a
subsequent release of excess N with no in¯uence
on C mineralization. Studies of acid organic forest
soils have also found either no in¯uence or a negative
effect of N addition on decomposition and respiration,
but the mechanisms behind these ®ndings are not well
understood (Fog, 1988; Martikainen, 1996). Our
results provide evidence, however, that a single addition of NH4‡ of the size of the annual atmospheric
N deposition will induce the release of N from
the large pool of organic N bound in the heath mor.
This suggests that N additions will be detrimental
to the stability of the ecosystem as they may in¯uence
the competition between Calluna and grasses by
increasing nutrient availability. But net ammoni®cation was also observed in the soil under intact
Calluna without N addition (Fig. 6), which otherwise
has been found to be very low in ®eld and laboratory
incubations at temperatures closer to those measured
under ®eld conditions (Kristensen and Henriksen,
1998). Moreover, the amount of N added in our
experiment was well above the amount of N deposited
in a single rain event, and the in¯uence of an intact
Calluna vegetation was missing during the incubation. This indicates that the results of the experiment
may not be directly transferable to the heathland
ecosystem.
Net ammoni®cation was also increased with N
addition during the long-term incubation of the soils
sampled from under dead Calluna (Fig. 6), but,
respiration was found to decrease with the addition
of N (Fig. 7). This may for example have been caused
by a salt effect on the microbial population (Martikainen, 1996) because of the high concentration of
NH4‡ after addition of this compound to the already
high NH4‡ pools in the soils (Table 2). The increases

H.L. Kristensen, G.W. McCarty / Applied Soil Ecology 13 (1999) 187±198

of the NH4‡ pool under dead Calluna when fertilized
with annual doses of 15 and 35 kg N haÿ1 could, when
calculated on an areal basis (11 and 17 kg N haÿ1,
respectively), be accounted for by the amount of
fertilizer NH4‡ applied since the heather beetle attack
(10 and 23 kg N haÿ1, respectively). In addition, the
size of the net ammoni®cation rates during the shortand long-term incubation experiments of the three
soils sampled under dead Calluna were in the same
range and no clear relationship to N fertilization rate
was evident (Figs. 2 and 6). These results indicate that
an increase of N input to the ecosystem will not
in¯uence mineralization of the mor after a heather
beetle attack. This was expected due to the already
large accumulation of NH4‡ in the soil with the
heather beetle attack and the limited capacity of the
ecosystem to remove mineral N in the absence of
nitri®cation.

5. Conclusion
The Calluna mor was found to have a substantial
capacity for mineralization±immobilization turnover
of NH4‡ which was comparable to values reported
in the literature for acid forest soils. The lack of
net mineralization and the high capacity for immobilization of mineral N in the heath soil may point
to a unique in¯uence of the Calluna vegetation on
microbial cycling of N within the ecosystem. The
ability to rapidly immobilize large amounts of N
could play a major role in the competition between
Calluna and grass species because ericaceous
vegetation would be favored by the maintenance
of low N availability. An increased N input to
the heathland ecosystem may increase the frequency
of heather beetle infestations, which in turn was
found to change the balance of N cycling processes
in the ecosystem, resulting in substantial increases
in net ammoni®cation in the soil. These destabilizing in¯uences are likely enhanced by the inability
of the ecosystem to remove excess N because
of the complete lack of nitri®cation in the soil.
Therefore, large accumulations of NH4‡ occur in
soil under Calluna vegetation damaged by heather
beetle attack. Together, these in¯uences may
increase the ability of grasses to gain dominance
on the heathland.

197

Acknowledgements
This work was supported by a fellowship from the
National Environmental Research Institute of Denmark, the Danish Research Academy, and Aalborg
University. We thank the Hjerl Foundation for permission to use the ®eld site and Knud Erik Nielsen for the
establishment and maintenance of the ®eld plots.

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