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Soil Biology & Biochemistry 32 (2000) 2019±2030
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Overestimation of gross N transformation rates in grassland soils due to
non-uniform exploitation of applied and native pools
C.J. Watson a,*, G. Travers a,b, D.J. Kilpatrick c, A.S. Laidlaw d, E. O'Riordan b
a

Agricultural and Environmental Science Division, Department of Agriculture for Northern Ireland, Newforge Lane, Belfast BT9 5PX
and School of Agriculture and Food Science, The Queen's University of Belfast, Newforge Lane, Belfast, Ireland
b
Teagasc, National Beef Research Centre, Grange, Dunsany, County Meath, Ireland
c
Biometrics Division, Department of Agriculture for Northern Ireland, Newforge Lane, Belfast BT9 5PX and School of Agriculture and Food Science,
The Queen's University of Belfast, Newforge Lane, Belfast, Ireland
d
Applied Plant Science Division, Department of Agriculture for Northern Ireland, Newforge Lane, Belfast BT9 5PX
and School of Agriculture and Food Science, The Queen's University of Belfast, Newforge Lane, Belfast, Ireland
Accepted 3 May 2000

Abstract

The study tested the validity of some of the assumptions in the 15N pool dilution technique in short-term soil incubations. Microbial N
transformation rates were calculated using 15N pool dilution during 24 h in four grassland soils in April 1998. The change in concentration
and enrichment of the NH41-N and NO32-N pools was determined at 0, 1.5, 4, 10, 16 and 24 h following application of differentially 15N
labelled NH4NO3 in solution at a rate of either 2 or 15 mg N kg 21 oven-dry soil and at an enrichment of 99.8 atom% excess. Rapid 15N pool
dilution occurred in all soils. Rates of gross mineralisation and NH41 consumption were not constant during the 24 h incubation in contrast to
nitri®cation rates. An application of 15 mg N kg 21 decreased gross mineralisation and NO32 consumption and increased nitri®cation rates
compared to an application of 2 mg N kg 21. Applied 15NH41 was rapidly nitri®ed with up to 55% of the added label recovered as 15NO32 after
24 h. This rapid conversion of 15NH41 to 15NO32 occurred without a proportional and concurrent increase in the size of the unlabelled NO32
pool. Gross and net nitri®cation rates were signi®cantly different due to 15NO32 consumption. The results suggest that there was non-uniform
exploitation of the 14N and 15N pools by soil microorganisms, invalidating one of the key assumptions in the 15N pool dilution technique.
Preferential consumption of applied NH41 and NO32 led to an overestimate of gross mineralisation and nitri®cation rates due to the greater rate
of decline of the 15N enrichment of the added N pool. In future studies care should be taken to ensure that gross N transformation rates are not
altered by the method used to quantify them. q 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Ammonium consumption; 15N pool dilution; Grassland soils; Gross nitrogen mineralisation; Nitrate consumption; Nitri®cation

1. Introduction
Net N mineralisation studies provide information on
changes in overall nitrogen cycling but, do not give any
indication of gross mineralisation±immobilisation turnover
(MIT) which can only be studied using isotope techniques.

There are several mathematical equations available to
calculate gross N transformation rates using the data from
experiments with 15N (Barraclough, 1991; Bjarnason, 1988;
Kirkham and Bartholomew, 1954). These calculations rely
on certain key assumptions (Hart et al., 1994) namely: (1)
all rate processes can be described by zero-order kinetics
over the experimental period; (2) microorganisms do not
* Corresponding author. Tel: 144-28-90-255359; fax: 144-28-90662007.
E-mail address: c.watson@qub.ac.uk (C.J. Watson).

discriminate between 14N and 15N; (3) there is uniform
mixing of added label with the soil inorganic N pool; and
(4) labelled N immobilised over the experimental period is
not remineralised.
Few 15N pool dilution experiments have been undertaken
in grassland soils over short (,3 d) time periods. Indigenous process rates can be studied by adding small concentrations of highly-enriched 15NH41 or 15NO32 to soil. Gross rates
of N mineralisation (NH41 production) and NH41 consumption (immobilisation and nitri®cation) can be calculated
from the rate of dilution in 15N enrichment of the NH41
pool as organic 14N is mineralised to 14NH41 and from the
change in the size of the total NH41 pool. Gross nitri®cation

and NO32 consumption are determined in a similar manner
with 15NO32 being applied to soil. As one of the assumptions
in the pool dilution technique is that microorganisms do not
discriminate between 14N and 15N, consumption of NH41-N

0038-0717/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0038-071 7(00)00103-6

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C.J. Watson et al. / Soil Biology & Biochemistry 32 (2000) 2019±2030

Table 1
Average physical and chemical characteristics of the soils (5 cm depth)
Grass sward

Location

N (%)

C (%)

pH

Silt (%)

Clay (%)

Sand (%)

Grazed grass/clover
Cut 0 kg N ha 21 y 21
Cut 300 kg N ha 21 y 21
Grazed 300 kg N ha 21 y 21

Grange (Grange GC)
Grange (Grange 0)
Grange (Grange 300)
Hillsborough (CENIT)

0.39
0.37
0.40
0.40

3.91
3.76
4.11
4.95

6.1
6.2
5.9
5.6

42
42
44
31

23
24
25
21

35
34
31
48

and NO32-N will change the pool sizes but will not affect the
15
N enrichment allowing gross rates of production of NH41
and NO32 to be separated from concurrent consumption
rates. Gross immobilisation is the difference between NH41
consumption and nitri®cation.
A further key assumption in calculating gross N transformation rates is that there is uniform mixing of added 15N
label with the soil inorganic N pool. However, this is dif®cult to achieve because ambient inorganic 14N is not
uniformly distributed in soil (Hart et al., 1994). Any preferential use of applied N by soil microorganisms would result
in erroneously high gross MIT rates, due to the greater rate

of decline of the 15N enrichment of the added N pool. High
gross mineralisation and immobilisation rates have been
reported in a range of soils (Bjarnason, 1988; Davidson et
al., 1991; Schimel, 1986; Watson and Mills, 1998). The
current study was undertaken to establish if there was any
preferential use of applied N in short-term incubations by
measuring 15N pool dilution at six time intervals during a
24 h period having applied highly enriched 15N to four
grassland soils with different management histories. In addition, the rates of microbial N transformations were calculated to determine whether they were constant during the
incubation period.

2. Materials and methods
2.1. Site characteristics
Samples from four grassland soils were collected in April
1998 from the Central Nitrogen Experimental Site (CENIT)
at the Agricultural Research Institute for Northern Ireland
(ARINI), Hillsborough, Co. Down and the Teagasc, Grange
Research Centre, Co. Meath. The CENIT grassland site at
ARINI was established in 1987 on a relatively free draining
clay-loam soil. The grass sward received an annual input of

300 kg N ha 21, applied in six equal dressings between April
and August and was continuously grazed by beef steers from
April to October to maintain a constant sward height of
7 cm. The three grassland swards at Grange Research
Centre were established in 1994, on a moderately well
drained Brown Earth soil and consisted of two grass swards
which were cut at 4-week intervals between April and
September. Nitrogen fertiliser was applied to one sward
after each cut giving a total N application of
300 kg ha 21 y 21, while the other cut sward did not receive

any N fertiliser. The remaining grass sward at Grange
Research Centre was a rotationally grazed grass/clover
sward (21 d cycle; beef steers) which received no N fertiliser. All swards received a single annual application of P and
K, according to soil analysis and standard recommendations. Selected soil properties (average of three replicates)
are given in Table 1.
2.2. Sampling and incubation procedure
Prior to N fertiliser application, soil cores (2.5 cm
diameter £ 5 cm deep) were collected randomly and bulked
for each of 3 replicate swards of the 4 soils. The freshly

collected soil was coarsely sieved through a 6.7 mm sieve to
remove large root and shoot material. Fresh soil (equivalent
to 72 g on an oven-dry weight basis) was weighed into
500 cm 3 Kilner jars and acclimatised in a controlled environment cabinet at 13.58C for 24 h. The surface area of
exposed soil was 81 cm 2.
Differentially 15N labelled NH4NO3 was applied at a rate
of either 2 or 15 mg 15N kg 21 oven-dry soil to allow paired
soil incubations. Half of the Kilner jars received 15NH4NO3
and the other half received NH4 15NO3, each of the labelled
moieties being at an enrichment of 99.8 at% excess. The 15N
labelled substrates were applied uniformly over the soil
surface in a solution (10 ml) using a ®ne tipped pipette.
The average moisture content of the soils was 30% (g g 21)
initially and increased to 40% (g g 21) after substrate addition.
The jars were sealed with glass lids and incubated in a
temperature controlled cabinet at 13.58C in the dark. This
temperature was selected as it was the mean soil temperature at the Grange Research Centre at a depth of 5 cm
during the period April to September 1997. The soil in the
jars was destructively sampled at 0, 1.5, 4, 10, 16 and
24 h. There were 288 jars in total (4 soils £ 2 labels £ 2

concentrations £ 6 times £ 3 replicates).
2.3. Chemical analysis
At each sample time 3 replicates per treatment were
destructively harvested. The soil in the jars was shaken
with 200 ml of 2 M KCl for 1 h and ®ltered (Whatman
GF/C). The NH41-N and NO32-N concentration in the ®ltrate
was determined using a Technicon Random Access Automated Chemistry System (TRAACS 800 1 ) (Bran and
Luebbe, 1995) and expressed as mg N kg 21 oven-dry soil.
The KCl extracts were stored at 48C and analysed for

C.J. Watson et al. / Soil Biology & Biochemistry 32 (2000) 2019±2030

mineral N within 24 h of extraction and for 15N within one
week. The extraction at time zero occurred instantaneously
after application of the 15N label.
Determination of the 15N enrichment of the NO32-N in
the KCl soil extracts was based on the production of
N2O from nitrite and hydroxylamine intermediates
during reduction with Cd/Cu (Stevens and Laughlin,
1994). The 15N enrichment of NH41-N in the KCl

extracts was determined by ®rstly generating NH3 by
addition of MgO. The NH3 was absorbed by a CuSO4/
H2SO4 solution, which was later dried to a residue. The
N2O produced as a side reaction on the addition of
sodium hypobromite was analysed by isotope ratio
mass spectrometry (Laughlin et al., 1997).
2.4. Calculation of gross mineralisation, consumption and
nitri®cation rates
Rates of gross mineralisation, NH41 consumption, nitri®cation and NO32 consumption were calculated for each of
the ®ve possible time periods (0±1.5, 1.5±4, 4±10, 10±16,
16±24 h) using Eqs. (1) and (2) (Kirkham and Bartholomew, 1954), separately for each of the three replicates.
m ˆ ‰…M0 2 M1 †=tŠ log…H0 M1 =H1 M0 †=log…M0 =M1 †

…1†

c ˆ ‰…M0 2 M1 †=tŠ log…H0 =H1 †=log…M0 =M1 †

…2†

where
M0
M1
H0
H1
m
c
t

initial 14115N pool (mg N kg 21)
post-incubation 14115N pool (mg N kg 21) at time t
initial 15N pool (mg N kg 21)
post-incubation 15N pool (mg N kg 21) at time t
mineralisation rate (mg N kg 21 h 21)
consumption rate (mg N kg 21 h 21)
time (h)

and where m ± c: Kirkham and Bartholomew (1954)
provided another equation for the condition when m ˆ c
(i.e. when the mineral N pool size stays constant with
time), which did not occur in this study.
For samples that received 15NH41 the NH41 pool was used
for M and H. For samples that received 15NO32 the NO32 pool
was used for M and H in Eqs. (1) and (2), to give the rate of
nitri®cation (mg kg 21 h 21) and NO32 consumption, respectively. Gross immobilisation was the difference between
NH41 consumption and nitri®cation.
The average mineral N concentrations of the NH41-N and
NO32-N labelled moieties were calculated for each of the
three replicates for each soil and concentration at each time.
There were three replicates for all determinations of 15N
enrichment and calculation of gross N transformation
rates. The data were analysed as a split-plot design with
treatments as the main plot factor and concentration and
time as sub-plot factors. However, two problems were identi®ed with this approach when calculating and analysing

2021

gross N transformation rates:
1. There was considerable variation between the replicates
leading to large standard errors for the mean rates. This is
a common statistical problem due to the calculation
being based on the means of ratios, which tends to
produce highly variable results, rather than the more
stable ratio of means.
2. The rates ¯uctuated erratically between the various time
periods. This was particularly true for the calculation of
gross mineralisation. Examination of the data showed
that this was due to erratic variability in the M and H
values.
Accordingly another approach was investigated. The ®rst
problem was addressed by basing the calculation on the
means for M and H over the three replicates. A bootstrap
technique was then used to estimate standard errors for the
mean rates. As described by Manly (1997), the bootstrap
technique allows the distribution of values in a population to
be investigated in the absence of any prior knowledge. The
method is to repeatedly resample the sampled values and
calculate the parameter of interest for each resample. This
resampling is done ªwith replacementº i.e. some values may
appear two or more times in the resample while others may
not appear at all. If a large number of independent resamples
are taken, then the overall mean and standard deviation of
the parameter provides unbiased estimates of the parameter
and its standard error. In relation to the current dataset, the
bootstrap resampling procedure involves randomly selecting a sample of size 18 with replacement from the 18 actual
values (6 times £ 3 replicates) for both M and H.
The second problem was addressed by ®tting smoothing
curves to the mean M and H values over time (t). A random
bootstrap resample of size 18 was selected as described in
the previous paragraph. Three types of curve were ®tted to
these values Ð (1) linear y ˆ a 1 bt; (2) exponential y ˆ
a 1 br t ; and (3) spline which does not have a functional
form but corresponds to an iterative mathematical procedure
to ®t cubic functions to segments of the curve between
adjacent time points constrained to be ªsmoothº at the junctions between segments. The Kirkham and Bartholomew
(1954) equations were applied to both the original and the
®tted M and H values from each of these three types of
curve. This provided estimates of the rates of gross mineralisation, consumption, immobilisation and nitri®cation
both for each time period and for the overall 24 h period.
Net nitri®cation was estimated from direct linear regression
of the NO32 pool size against time. The difference between
the gross nitri®cation rate, calculated from the Kirkham and
Bartholomew (1954) equations, and the net nitri®cation rate
was also calculated. Net mineralisation was estimated from
direct linear regression of the total mineral N pool size
against time. The resampling procedure was repeated
1000 times. The means and standard errors of the various
rates over these 1000 resamples were calculated. These

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C.J. Watson et al. / Soil Biology & Biochemistry 32 (2000) 2019±2030

were used to compare the rates in successive time periods
and also to test whether the difference between gross and net
nitri®cation was equal to zero. The difference between daily
gross mineralisation and immobilisation should indicate the
net production of N. This calculated value was compared
with the measured change in total mineral N (net m) over the
24 h incubation.
All random re-sampling and calculations were carried out
using the Genstat (1993) statistical package.

3. Results
3.1. Soil mineral N concentrations

Fig. 1. Change in NH41-N during a 24 h incubation with (a) 2 mg N kg 21
and (b) 15 mg N kg 21. (soil £ time £ concentration sem ˆ 0.66). V CENIT;
B Grange GC; K Grange 0; £ Grange 300.

Figs. 1 and 2 show the changes in NH41-N and NO32-N,
respectively, during the 24 h incubation for soils that
received (a) 2 mg 15N kg 21 or (b) 15 mg 15N kg 21. When
data for all soils were analysed together there was a signi®cant decrease …P , 0:001† in NH41-N and a signi®cant
increase …P , 0:001† in NO32-N with time at both N applications. The NH41-N and NO32-N content of the CENIT soil
was signi®cantly …P , 0:05† greater than the Grange soils at
the start of the incubation. The decrease in NH41-N and
increase in NO32-N content was greater when 15 mg
N kg 21 was applied than when 2 mg N kg 21 was applied.
Net NO32-N production after 24 h was signi®cantly …P ,
0:001† greater with the CENIT soil than with the Grange
soils.

Fig. 2. Change in NO32-N during a 24 h incubation with (a) 2 mg N kg 21 and (b) 15 mg N kg 21. (soil £ time £ concentration sem ˆ 0.35). V CENIT; B Grange
GC; K Grange 0; £ Grange 300.

C.J. Watson et al. / Soil Biology & Biochemistry 32 (2000) 2019±2030

Fig. 3. Change in atom% excess of (a) 15NH41 (soil £ time £ concentration sem ˆ 2.11) and (b)
application of 2 mg N kg 21. V CENIT; B Grange GC; K Grange 0; £ Grange 300.

15

2023

NO32 (soil £ time £ concentration sem ˆ 1.08) with an

3.2. Atom% excess 15N in NH41-N and NO32-N

Fig. 4. Change in atom% excess of (a) 15NH41 (soil £ time £ concentration
sem ˆ 2.11) and (b) 15NO32 (soil £ time £ concentration sem ˆ 1.08) with
an application of 15 mg N kg 21. V CENIT; B Grange GC; K Grange 0; £
Grange 300.

Fig. 3 shows the change in atom% excess of (a) 15NH41
and (b) 15NO32 during 24 h when 2 mg 15N kg 21 was
applied. There was a highly signi®cant …P , 0:001† decline
in both labelled moieties with time; however, the 15NH41
decreased exponentially from an average of 24.0 at% excess
at time zero to 1.9 at% excess after 24 h, whereas 15NO32
decreased linearly over the time period from an average of
28.8 at% excess to 14.3 at% excess. When 15 mg 15N kg 21
was applied the atom% excess of both 15N moieties
decreased in a linear manner (Fig. 4). The rate of decline
in atom% excess 15NO3 was greater …P , 0:001† at 15 mg
15
N kg 21 than at 2 mg 15N kg 21, being 0.94 and 0.60at%
excess h 21, respectively, averaged for all soils. At both
application rates there was a highly signi®cant …P ,
0:001† difference between soils and a signi®cant soil £ time
interaction for both labelled moieties. This was because the
CENIT soil had a higher initial NH41-N and NO32-N content
than the other soils, which resulted in a signi®cantly lower
atom% excess at time zero.
Fig. 5 shows the signi®cant …P , 0:001† appearance of
15
NH41 during the experimental period in soils that had
received 15NO3 labelled NH4NO3 at (a) 2 mg N kg 21 and
(b) 15 mg N kg 21. With 15 mg 15N kg 21 the atom% excess
15
NH41 continued to increase over the duration of the study,
however, with 2 mg 15N kg 21 the 15NH41 peaked at 4 h in the
Grange GC soil and at 1.5 h in the CENIT soil. There was a
signi®cantly …P , 0:001† higher atom% excess 15NH41-N
with 15 mg N kg 21 than with 2 mg N kg 21 which, averaged
for the duration of the incubation and soils, was 0.63 and

2024

C.J. Watson et al. / Soil Biology & Biochemistry 32 (2000) 2019±2030

the current study the % recovery of 15NH41 and 15NO32 at
time zero was 98.1 and 99.9%, respectively with the CENIT
soil when 2 mg N kg 21 was applied and 102.4 and 95.4%
when 15 mg N kg 21 (Table 2) was applied. Abiotic NH41
®xation did not occur in the CENIT soil, in contrast to the
Grange soils where the recovery of 15NH41 at time zero
averaged 88.1 and 89.4% with an application of 2 and
15 mg N kg 21, respectively.
There was a high recovery of 15NO3-N in soils that
received 15NH41-N (Fig. 6). The % recovery was highest
with the CENIT soil and after 24 h was equivalent to 55.2
and 45.1% of the 15NH41-N applied with 2 and 15 mg
N kg 21, respectively. Table 2 shows the size of the labelled
and unlabelled NH41 and NO32 moieties at the start and end
of the incubation, when 15NH41 was applied at the rate of
15 mg N kg 21. The rapid conversion of 15NH41 into 15NO32
occurred in the Grange soils without a concurrent increase
in the size of the unlabelled NO32 pool. For example, in the
Grange GC soil 69.6% of the NH41 at the start (0 h) was 15N
labelled. If the 14NH41 and 15NH41 pools were exploited in
proportion to their size the expected increase in 14NO32 and
15
NO32 pools after 24 h would be 1.34 and 3.06 mg N kg 21,
respectively. The observed increase in the 14NO32 pool
(0.1 mg N kg 21) was considerably lower than expected
whereas, the increase in the 15NO32 pool of 4.30 mg
N kg 21 was greater than expected. A similar ®nding was
observed with the lower rate of N application (results not
shown). Although there was an increase in the unlabelled
NO32 pool in the CENIT soil after 24 h, the increase in
labelled NO32 was proportionately greater.
When 2 mg 15NO32-N kg 21 was applied to the soils there
was a signi®cant …P , 0:001† decrease in the recovery of
15
NO32 (expressed as a % of the time zero value) after 24 h
(Table 3). The % recovery of 15NO32 was higher …P , 0:01†
in the CENIT soil compared to the Grange soils and was
signi®cantly greater …P , 0:001† at the higher application
rate.

Fig. 5. Appearance of 15NH41 in soils that received 15NO32 at (a) 2 mg
N kg 21 and (b) 15 mg N kg 21 (soil £ time £ concentration sem ˆ 0.12).
V CENIT; B Grange GC; K Grange 0; £ Grange 300.

0.36 at% excess, respectively. There was a signi®cant …P ,
0:05† difference between soils and a signi®cant …P , 0:001†
soil £ time interaction. There was an increase in 15NH41
throughout the incubation in the Grange 0 soil which after
24 h was 0.76 and 1.63 at% excess with 2 and 15 mg
N kg 21, respectively. The other soils were more variable.
When the 15NH41-N content was expressed as a % of 15NO32N applied the recovery was small and did not exceed 3.3%
when 2 mg 15NO32-N was applied. When 15 mg 15NO3-N
was applied no more than 1.7% of the 15N was recovered
as 15NH41-N.
The ®xation of NH41 to clay minerals can occur in some
soils. Davidson et al. (1991) suggest that abiological reactions occur rapidly and that initial 14N and 15N pool sizes
should be adjusted by undertaking a time zero extraction. In

3.3. Hourly gross N transformation rates
Gross N transformation rates were calculated at each time
using the equations of Kirkham and Bartholomew (1954),
having smoothed the data using a curve ®tting procedure
and the results were expressed as mg kg 21 h 21. For gross
mineralisation and NH41 consumption an exponential ®t was

Table 2
Labelled and unlabelled NH41 and NO32 pool sizes (mg N kg 21) at the start (0 h) and end of the incubation (24 h) after linear smoothing when
applied at the rate of 15 mg N kg 21. Figures in brackets are the standard errors of the means; n ˆ 3
Pool size (mg N kg 21)

15

NH41
14
NH41
15
NO32
14
NO32

CENIT

Grange GC

Grange 0

15

NH41 was

Grange 300

0h

24 h

0h

24 h

0h

24 h

0h

24 h

15.36 (0.615)
8.04 (0.837)
0.00
23.85 (2.652)

3.46 (0.329)
10.44 (1.668)
6.92 (0.762)
26.95 (2.156)

13.05 (0.585)
5.71 (1.272)
0.00
18.23 (0.369)

5.79 (0.741)
6.65 (1.932)
4.30 (0.556)
18.33 (0.939)

13.48 (0.105)
5.52 (0.323)
0.00
18.53 (0.205)

4.02 (0.724)
11.92 (2.006)
4.54 (0.341)
17.78 (0.904)

13.71 (0.017)
8.00 (0.746)
0.00
18.25 (0.711)

6.23 (0.906)
8.29 (0.619)
3.09 (0.219)
16.70 (0.874)

C.J. Watson et al. / Soil Biology & Biochemistry 32 (2000) 2019±2030

Fig. 6. Recovery of 15NO32 in soils that received
Grange GC; K Grange 0; £ Grange 300.

15

2025

NH41 at (a) 2 mg N kg 21 and (b) 15 mg N kg 21 (soil £ time £ concentration sem ˆ 1.80). V CENIT; B

best. Table 4 shows the hourly gross mineralisation rates for
the different soils receiving either 2 or 15 mg 15N kg 21.
Rates varied signi®cantly with time and were generally
higher with the low N application than with the high N
application. Gross NH41 consumption rates also varied
signi®cantly with time, generally decreasing (Table 5).
However, the rate of N application had little or no effect.
Gross mineralisation and NH41 consumption rates were
highest in the CENIT soil. The estimate of gross mineralisation and NH41 consumption during the incubation obtained
using the zero and 24 h smoothed data from the exponential
curve ®t, agreed reasonably well with the values calculated
using the raw data at time zero and time 24 h (Tables 4 and
5).
In contrast, gross nitri®cation rates were generally
constant with time and were higher when 15 mg N kg 21

was applied than when 2 mg N kg 21 was applied (Table
6). The gross nitri®cation rate was higher in the CENIT
soil than in the other soils. The estimate of hourly gross
nitri®cation rate from the linear, exponential and spline
smoothing procedures agreed well with each other and
with the calculation using the 0 and 24 h raw data. As
neither the exponential nor spline smoothing gave a signi®cantly better ®t than the linear smoothing, only the linear
results are shown in Table 6. Nitrate consumption was
generally constant with time so only the hourly rates from
the linear smoothed data (0±24 h) are shown in Table 7.
Nitrate consumption was generally higher when 2 mg
N kg 21 was applied than when 15 mg N kg 21 was applied
and was higher in the Grange soils than in the CENIT soil.
The rate of consumption in the CENIT soil receiving 15 mg
N kg 21 was not signi®cantly different from zero (Table 7).

Table 3
Percentage recovery of applied 15NO32 in each soil after 24 h (sem ˆ 2.31;
n ˆ 3)

3.4. Daily N transformation rates

N applied (mg N kg 21)

CENIT Grange GC

Grange 0

Grange 300

2
15

83.1
103.2

56.9
91.8

66.9
90.0

71.5
95.5

Daily gross mineralisation and immobilisation rates are
shown in Table 8. Gross immobilisation was calculated as
the difference between NH41 consumption and gross nitri®cation. Daily net mineralisation was determined from linear
regression of the change in the total mineral N pool with

2026

C.J. Watson et al. / Soil Biology & Biochemistry 32 (2000) 2019±2030

Table 4
Hourly gross mineralisation rates (mg N kg 21 h 21) calculated after exponential smoothing at different times during a 24 h incubation. Figures in brackets are
the standard errors of the means estimated from the bootstrap technique …n ˆ 3†
Time (h)

1.5
4
10
16
24
0±24 h (smoothed)
0±24 h (raw)

Application rate of 2 mg N kg 21

Application rate of 15 mg N kg 21

CENIT

Grange GC

Grange 0

Grange 300

CENIT

Grange GC

Grange 0

Grange 300

0.89 (0.433)
1.24 (0.151)
1.22 (0.076)
0.89 (0.085)
0.42 (0.075)
0.94 (0.061)
0.99 (0.042)

1.18 (0.262)
1.06 (0.126)
0.82 (0.075)
0.48 (0.145)
0.30 (0.193)
0.61 (0.133)
0.73 (0.100)

0.84 (0.237)
1.01 (0.112)
0.89 (0.043)
0.45 (0.059)
0.11 (0.038)
0.57 (0.048)
0.65 (0.069)

0.97 (0.148)
0.91 (0.118)
0.76 (0.062)
0.49 (0.072)
0.12 (0.079)
0.44 (0.050)
0.50 (0.034)

0.64 (0.244)
0.53 (0.236)
0.75 (0.089)
0.73 (0.112)
0.63 (0.175)
0.70 (0.093)
0.74 (0.100)

0.61 (0.307)
0.50 (0.211)
0.36 (0.077)
0.16 (0.074)
0.07 (0.052)
0.19 (0.057)
0.26 (0.076)

0.14 (0.148)
0.24 (0.136)
0.53 (0.104)
0.67 (0.048)
0.80 (0.150)
0.65 (0.045)
0.75 (0.053)

0.50 (0.159)
0.47 (0.128)
0.40 (0.075)
0.29 (0.062)
0.13 (0.089)
0.25 (0.020)
0.29 (0.029)

time. Net mineralisation was greater in the CENIT soil than
in the Grange soils where there was little or no net increase
in total mineral N during the 24 h incubation (Table 8). The
difference between daily gross mineralisation and immobilisation should indicate the net production of N. However,
this calculated net production did not always agree with the
measured change in total mineral N over the 24 h incubation
(Table 8), with signi®cant differences found for Grange 0 at
both N concentrations and for Grange 300 at the lower N
concentration.
Daily net nitri®cation was determined from linear regression of the change in the NO32 pool with time. Table 9 shows
that daily gross nitri®cation rates were signi®cantly higher
(at least P , 0:01† than net nitri®cation rates, except in the
CENIT soil receiving 15 mg N kg 21, where there was no
signi®cant difference.
4. Discussion
The calculation of gross N transformation rates using the
N pool dilution technique relies on certain key assumptions (Hart et al., 1994)
15

1. All rate processes can be described by zero-order kinetics
over the experimental period.
2. Microorganisms do not discriminate between 14N and
15
N.
3. There is uniform mixing of added label with the soil

inorganic N pool.
4. Labelled N immobilised over the experimental period is
not remineralised.
These assumptions will be appraised in turn for this study.
4.1. Zero-order kinetics
The current study has shown that gross mineralisation and
gross NH41 consumption rates cannot be described by zeroorder kinetics during a 24 h incubation. Calculated hourly
rates varied with time, although the best estimate of the
hourly rate from the smoothed data agreed reasonably
well with the hourly rate calculated using the raw data at
time zero and 24 h. As the rates of gross mineralisation and
NH41 consumption generally decreased with time, rates
calculated over the ®rst 24 h would likely be higher than
if a longer incubation interval had been used. The daily rates
calculated in this study were considerably higher than other
reported studies with grassland soils (Jamieson et al., 1998;
Ledgard et al., 1998; Murphy et al., 1999), where 15N pool
dilution was measured several days after 15N application.
Nitrogen transformation rates were also affected by the
amount of N applied. The current study has shown that
generally an application of 15 mg N kg 21 decreased gross
mineralisation and NO32 consumption and increased nitri®cation rates compared to an application of 2 mg N kg 21.
Nitri®cation is known to be stimulated by the addition of
an NH41-N substrate (Recous et al., 1999; Willison et al.,

Table 5
Hourly gross NH41 consumption rates (mg N kg 21 h 21) calculated after exponential smoothing at different times during a 24 h incubation. Figures in brackets
are the standard errors of the means estimated from the bootstrap technique …n ˆ 3†
Time (h)

1.5
4
10
16
24
0±24 h (smoothed)
0±24 h (raw)

Application rate of 2 mg N kg 21

Application rate of 15 mg N kg 21

CENIT

Grange GC

Grange 0

Grange 300

CENIT

Grange GC

Grange 0

Grange 300

1.74 (0.137)
1.49 (0.052)
1.27 (0.053)
0.90 (0.085)
0.43 (0.076)
1.06 (0.082)
1.12 (0.073)

1.14 (0.174)
1.07 (0.120)
0.84 (0.085)
0.49 (0.171)
0.22 (0.162)
0.60 (0.087)
0.69 (0.029)

1.43 (0.093)
1.20 (0.058)
0.92 (0.037)
0.46 (0.056)
0.11 (0.033)
0.64 (0.048)
0.73 (0.048)

0.98 (0.138)
0.93 (0.114)
0.79 (0.060)
0.55 (0.080)
0.27 (0.094)
0.53 (0.038)
0.57 (0.021)

1.33 (0.224)
1.20 (0.135)
1.05 (0.063)
0.91 (0.066)
0.78 (0.149)
1.02 (0.076)
1.13 (0.074)

1.22 (0.404)
0.95 (0.210)
0.59 (0.063)
0.31 (0.117)
0.16 (0.113)
0.45 (0.040)
0.52 (0.047)

0.77 (0.127)
0.73 (0.102)
0.71 (0.064)
0.73 (0.053)
0.82 (0.180)
0.83 (0.068)
0.88 (0.047)

0.65 (0.162)
0.63 (0.124)
0.59 (0.056)
0.54 (0.085)
0.48 (0.187)
0.52 (0.060)
0.59 (0.066)

2027

C.J. Watson et al. / Soil Biology & Biochemistry 32 (2000) 2019±2030

Table 6
Hourly gross nitri®cation rates (mg N kg 21 h 21) calculated after linear smoothing at different times during a 24 h incubation. Figures in brackets are the
standard errors of the means estimated from the bootstrap technique …n ˆ 3†
Time (h)

1.5
4
10
16
24
0±24 h (smoothed)
0±24 h (raw)

Application rate of 2 mg N kg 21

Application rate of 15 mg N kg 21

CENIT

Grange GC

Grange 0

Grange 300

CENIT

Grange GC

Grange 0

Grange 300

0.29 (0.053)
0.29 (0.054)
0.30 (0.054)
0.31 (0.055)
0.32 (0.056)
0.30 (0.055)
0.33 (0.068)

0.17 (0.017)
0.17 (0.017)
0.18 (0.017)
0.20 (0.018)
0.22 (0.018)
0.19 (0.017)
0.19 (0.022)

0.18 (0.016)
0.18 (0.016)
0.20 (0.018)
0.22 (0.020)
0.27 (0.023)
0.22 (0.019)
0.21 (0.025)

0.18 (0.023)
0.18 (0.023)
0.20 (0.025)
0.22 (0.028)
0.24 (0.032)
0.21 (0.027)
0.21 (0.033)

0.55 (0.084)
0.55 (0.084)
0.55 (0.083)
0.55 (0.083)
0.55 (0.082)
0.55 (0.084)
0.53 (0.096)

0.28 (0.023)
0.29 (0.023)
0.29 (0.023)
0.29 (0.023)
0.30 (0.022)
0.29 (0.023)
0.29 (0.026)

0.30 (0.013)
0.30 (0.013)
0.31 (0.014)
0.32 (0.016)
0.34 (0.018)
0.32 (0.016)
0.31 (0.016)

0.27 (0.046)
0.28 (0.047)
0.29 (0.048)
0.30 (0.052)
0.32 (0.056)
0.30 (0.051)
0.27 (0.059)

1998). Although nitri®cation rates may have been overestimated in the current study, due to the addition of NH41, they
may represent the potential nitrifying activity of the soil.
The CENIT soil would appear to have a higher nitrifying
potential than the Grange soils, which may re¯ect its
previous grazing management. In the case of gross mineralisation, because the product pool is labelled with 15N
rather than the substrate pool, rates of NH41 production
should not be affected by the amount of N applied (Hart
et al., 1994). However, this was not the case.
4.2. Isotopic fractionation and uniform mixing
The assumption that microorganisms do not discriminate
between 14N and 15N is not strictly true. Delwiche and Steyn
(1970) showed some discrimination in favour of 14N in the
®xation of N, the oxidation of NH41 to NO22 and in the
assimilation of NH41. However, the error due to fractionation during an incubation of a few days is small relative to
the large decreases in 15N enrichment of the product pool
that occur from production (Hart et al., 1994). The current
study suggests that microorganisms exploit the indigenous
and applied N pools at different rates. For example 15NH41
was rapidly nitri®ed with 24.5±55% of the added label
recovered as 15NO32 after 24 h. This rapid conversion of
15
NH41 to 15NO32 occurred without a concurrent increase
in the size of the unlabelled NO32 pool. This suggests that
there was non-uniform mixing of the 14N and 15N pools. The
newly applied 15NH41 in solution would appear to be more
accessible to nitri®ers compared to indigenous soil NH41
located or produced at microsites. Preferential consumption
of applied NH41-N leads to an overestimate of gross N
mineralisation rates due to the greater rate of decline in

the enrichment of the added 15NH41-N pool. The magnitude
of this overestimation is dependent on the soil and the
concentration of N applied.
The net ¯ux of 14NH41 and 15NH41 between the native soil
solution and the added 15N labelled solution will depend on
the concentration difference between the two solutions.
Prior to N application the NH41 pool size in the CENIT
soil averaged 8.9 mg N kg 21 and the moisture content was
32.2% on an oven-dry weight basis. The concentration in
the soil solution was 28 mg NH41-N l 21. In comparison the
concentration of NH41-N applied was 14 and 108 mg NH41N l 21 at the low and high application rates, respectively.
Homogeneous mixing of the indigenous and applied N
pools would take longer at the low N than at the high N
application rate due to a less pronounced concentration
gradient. One way of ensuring uniform mixing of the
added label with the soil inorganic N pool would be to
use soil suspensions. This could be useful for comparative
purposes but the MIT rates obtained could not be extrapolated to the ®eld. Application of 15N label in solution has
been found to stimulate N transformation processes
compared to dry application techniques (Murphy et al.,
1999; Willison et al., 1998).
The signi®cant difference between gross and net nitri®cation rates observed in the current study was due to 15NO32
consumption. Gross nitri®cation rates would be overestimated if 15NO32 consumption takes place. For example
substantial NO32 consumption occurred in the Grange 0
soil receiving 2 mg N kg 21, which resulted in daily gross
nitri®cation rates being 3.6 times higher than net nitri®cation rates. There was no evidence that the addition of NO32
stimulated NO32 consumption (Stark and Hart, 1997), as the
rate was lower when 15 mg N kg 21 was applied than when

Table 7
Rate of NO32 consumption (mg N kg 21 h 21). Figures in brackets are the standard errors of the means estimated from the bootstrap technique …n ˆ 3†
Time interval 0±24 h

Linear (smoothed)
Raw

Application rate of 2 mg N kg 21

Application rate of 15 mg N kg 21

CENIT

Grange GC

Grange 0

Grange 300

CENIT

Grange GC

Grange 0

Grange 300

0.07 (0.024)
0.10 (0.014)

0.09 (0.010)
0.10 (0.011)

0.16 (0.009)
0.16 (0.010)

0.11 (0.010)
0.11 (0.012)

0.01 (0.008)
-0.04 (0.015)

0.07 (0.016)
0.04 (0.019)

0.14 (0.016)
0.07 (0.012)

0.16 (0.019)
0.08 (0.022)

2028
Table 8
Daily gross N mineralisation and immobilisation rates (mg N kg 21 d 21) calculated using 0±24 h data after exponential smoothing. Figures in brackets are the standard errors of the means estimated from the
bootstrap technique …n ˆ 3†. (Daily net mineralisation (mg N kg 21 d 21) was determined from linear regression of the change in the total mineral N pool with time; ns, not signi®cant; *P , 0:05; **P , 0:01 and
***P , 0:001; any small discrepancy in scaling up from hourly to daily rates is due to rounding of the means to 2 decimal places)
Application rate of 2 mg N kg 21
CENIT

Grange GC

Grange 0

Grange 300

CENIT

Grange GC

Grange 0

Grange 300

22.60 (1.459)
18.06 (2.372)
4.54 (1.815)
4.96 (1.975)
ns

14.57 (3.197)
9.76 (2.120)
4.80 (2.323)
1.97 (1.015)
ns

13.72 (1.147)
10.18 (1.238)
3.54 (0.610)
2 0.168 (0.480)
***

10.49 (1.188)
7.53 (1.110)
2.97 (1.173)
0.52 (1.061)
*

16.69 (2.234)
11.14 (2.716)
5.55 (2.391)
5.52 (2.258)
ns

4.62 (1.358)
3.67 (1.092)
0.94 (1.813)
1.28 (1.711)
ns

15.50 (1.075)
12.16 (1.678)
3.34 (1.289)
2 1.24 (0.593)
***

6.08 (0.478)
5.28 (1.878)
0.81 (1.734)
0.64 (1.025)
ns

Table 9
Daily gross nitri®cation rates (mg N kg 21 d 21) calculated using 0±24 h data after linear smoothing. Figures in brackets are the standard errors of the means estimated from the bootstrap technique …n ˆ 3†. (Daily
net nitri®cation (mg N kg 21 d 21) was determined from linear regression of the change in the NO32-N pool with time; ns, not signi®cant; **P , 0:01; ***P , 0:001; any small discrepancy in scaling up from
hourly to daily rates is due to rounding of the means to 2 decimal places)
Application rate of 2 mg N kg 21

Gross nitri®cation (n)
Net nitri®cation (net n)
n 2 net n
Signi®cance of
difference between n
and net n

Application rate of 15 mg N kg 21

CENIT

Grange GC

Grange 0

Grange 300

CENIT

Grange GC

Grange 0

Grange 300

7.31 (1.310)
5.70 (1.529)
1.61 (0.554)
**

4.64 (0.408)
2.45 (0.550)
2.19 (0.245)
***

5.28 (0.466)
1.46 (0.463)
3.81 (0.218)
***

5.08 (0.646)
2.36 (0.576)
2.71 (0.238)
***

13.21 (2.006)
13.36 (2.143)
2 0.15 (0.362)
ns

7.05 (0.542)
5.44 (0.749)
1.61 (0.382)
***

7.63 (0.382)
4.37 (0.262)
3.26 (0.391)
***

7.14 (1.217)
3.25 (1.099)
3.90 (0.485)
***

C.J. Watson et al. / Soil Biology & Biochemistry 32 (2000) 2019±2030

Gross mineralisation (m)
Gross immobilisation (i)
m2i
Net mineralisation (net m)
Signi®cance of difference
between gross m 2 i and net m

Application rate of 15 mg N kg 21

C.J. Watson et al. / Soil Biology & Biochemistry 32 (2000) 2019±2030

2 mg 15NO32-N kg 21 was applied. This would explain the
higher % recovery of 15NO32 after 24 h when 15 mg N kg 21
was applied. The CENIT soil had a lower rate of NO32
consumption compared to the Grange soils, which resulted
in a higher % recovery of 15NO32 at the end of the incubation. Nitrate consumption was negligible in the CENIT soil
receiving 15 mg N kg 21 and with this treatment there was
no signi®cant difference between gross and net rates of
nitri®cation. Consumption of NO32 would include denitri®cation, dissimilatory NO32 reduction and microbial assimilation. Although gaseous losses were not measured in the
current study, it is unlikely that denitri®cation alone would
have resulted in the observed loss of NO32 as the soils were
aerated and their moisture content was well below ®eld
capacity. Dissimilatory NO32 reduction is also unlikely as
this pathway occurs in strictly anaerobic environments such
as sediments (Cole, 1988). Evidence that rapid microbial
assimilation of 15NO32 occurred in the current study comes
from the appearance of 15NH41 within 1.5 h in soil that
received 15NO32. There are a number of recent reports that
indicate that rapid microbial assimilation of NO32 is an
important process in undisturbed forest soils (Stark and
Hart, 1997) and in aquatic (Caraco et al., 1998) and marine
(Kirchman and Wheeler, 1998) ecosystems.
Recent workers have taken the initial extraction time as
24 h after 15N application and have calculated daily gross N
transformation rates using the time interval from 24 h (time
zero) to 72 h (time 1) (Murphy et al., 1999). However,
unless it can be established that preferential use of applied
N is not occurring after 24 h, calculated gross N transformation rates will still be overestimated. The rapid decrease in
enrichment of the 15NH41 pool observed in the current study
after applying 2 mg N kg 21 meant that after 24 h there was
no further 15N pool dilution. Signi®cantly increasing the
15
NH41-N pool size, by applying 15 mg N kg 21, ensured
continued pool dilution after 24 h but stimulated the rate
of nitri®cation. Nitri®cation inhibitors could be used to
prevent the conversion of NH41 to NO32. However, their
use would maintain an elevated NH41 pool size that might
stimulate immobilisation or decrease mineralisation by
feedback inhibition. There was evidence that the rate of
gross mineralisation was lower with an application of
15 mg N kg 21 compared to 2 mg N kg 21. Due to rapid 15N
pool dilution in some soils it may not be possible to determine indigenous N transformation rates at time intervals
greater than 24 h. However, information on gross N transformations could be obtained in response to a simulated
fertiliser application. In this case differentially labelled
NH4NO3 would be the preferred N source.
4.3. Remineralisation
Remineralisation of immobilised 15N can lead to substantial error in estimating mineralisation±immobilisation rates,
but it is not believed to be a major process if incubations are
less than one week (Bjarnason, 1988). The ®xation of NH41

2029

to clay minerals can be allowed for by undertaking a time
zero extraction (Davidson et al., 1991). The % recovery of
15
NH41 at time zero was close to 100% in the CENIT soil.
However, abiotic NH41 ®xation occurred in the Grange soils.
Trehan (1996) noted that where ®xation of NH41 occurred at
time zero the loss of 14NH41 from the soil solution via nitri®cation remobilised the ®xed 15NH41 from the clay minerals
into the soil solution. This could alter calculated mineralisation rates if nitri®cation was rapid (Scherer and Werner,
1996).
The current study has shown that preferential consumption of applied 15NH41 and 15NO32 by soil microorganisms
invalidated some of the assumptions used in the 15N pool
dilution technique. This led to an overestimate of gross
mineralisation and nitri®cation rates, due to the greater
rate of decline of the 15N enrichment of the added N pool.
In future studies it will be important to establish that preferential use of applied N is not occurring during the experimental period and that steady-state conditions have been
reached following 15N application. Care should be taken
to ensure that process rates are not altered by the methods
used to quantify them.
Acknowledgements
Gerard Travers would like to thank Teagasc for receipt of
a Walsh Fellowship. The authors would also like to thank
Mr P. Poland and Mr R.J. Laughlin for analysis of samples
and Dr R.J. Stevens for helpful discussions.
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