Directory UMM :Data Elmu:jurnal:S:Soil Biology And Chemistry:Vol32.Issue2.2000:
Soil Biology & Biochemistry 32 (2000) 229±239
www.elsevier.com/locate/soilbio
Dierent pathways of formation of N2O, N2 and NO in black
earth soil
I. Wolf a, b,*, R. Russow a
a
Department of Soil Science, UFZ Centre for Environmental Research Leipzig-Halle, Theodor-Lieser-Strasse 4, D-06120 Halle, Germany
b
Institute of Soil Science and Forest Nutrition, University of GoÈttingen, BuÈsgenweg 2, D-37077 GoÈttingen, Germany
Accepted 17 August 1999
Abstract
The use of 15N tracer provides a suitable technique to investigate the processes of N transformation in soils and the origin of
the environmentally relevant gaseous N compounds N2O and NO from nitri®cation and denitri®cation. The results of
incubation experiments with black earth soil under two dierent water contents are presented here. Nitri®cation and
denitri®cation proceeded simultaneously, but the importance of these two microbial processes shifted depending on the water
ÿ
content of the soil. Under water-unsaturated conditions the microbial oxidation of NH+
4 to NO3 predominated, but a reduction
ÿ
of NO3 also occurred. The emission of NO exceeded the emission of N2O by a factor of up to 20 at the beginning of the
experiments. Under water-saturated conditions denitri®cation was the dominant process of N transformation in the soil.
However, nitri®cation also occurred to a considerable extent. The emission of N2O was greater than under unsaturated
conditions. The formation of NO could hardly be observed. N loss by molecular nitrogen from denitri®cation could be detected
under saturated conditions. The N loss amounted to 60% of NOÿ
3 and thereby the cumulative N ratio of N2 to N2O was 3.
+
ÿ
Under either unsaturated or saturated conditions NO arose from NOÿ
2 or during the microbial oxidation of NH4 to NO2 .
However, N2O mainly formed from denitri®cation under both conditions. Furthermore, NO could not be observed as a
precursor of N2O and the free NOÿ
2 could not be detected as a common N pool for the formation of N2O and NO. High
emissions of NO could be a problem for the black earth soil in the semi-arid climate in central Germany, if there are large
amounts of NH+
4 in the soil after fertilisation. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Denitri®cation; Di-nitrogen; Nitri®cation;
15
N; Nitric oxide; Nitrous oxide
1. Introduction
Nitrous oxide and nitric oxide are directly or indirectly involved in global warming, the destruction of
the stratospheric ozone, the production and consumption of atmospheric oxidants, and the photochemical
formation of nitric acid (Bouwman, 1990; Williams et
al., 1992). Additionally, the emission of N2O, NO and
N2 from agricultural soils is a loss of N fertiliser
(Granli and Bùckman, 1994).
During nitri®cation and denitri®cation N2O and NO
* Corresponding author. Tel.: +49-551-393518; fax: +49-551393310.
E-mail address: [email protected] (I. Wolf).
as well as N2 are emitted (Firestone and Davidson,
1989). Nitrous oxide and NO can also be consumed by
microorganisms in the soil (Hutchinson and Davidson,
1993). Although the mechanism of N2O and NO formation in soils is generally well-known, it is still not
clear how nitri®cation and denitri®cation contribute to
the formation of N2O under dierent amounts of
water saturation in the soil. Further uncertainties
include the role of NO in these processes. Nitric oxide
could be a by-product or an obligate and free precursor of N2O in the denitri®cation path. Not much is
known about the ratios between N2 and N2O-N or
N2O-N and NO-N under dierent conditions.
Recent 15N studies with agricultural soil have shown
that under unsaturated conditions nitri®cation was the
0038-0717/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 1 5 1 - 0
230
I. Wolf, R. Russow / Soil Biology & Biochemistry 32 (2000) 229±239
Fig. 1. Incubation vessel and the container holding incubation vessels to determine the N emission rates.
dominant process for N2O production (Stevens et al.,
1997). The formation of N2O and NO via nitri®cation
could be signi®cant for the black earth soils in central
Germany because of the absence of typical denitri®cation conditions (water saturation) under the prevailing
semi-arid climate conditions. However, N2O can be
formed in soils by denitri®cation due to anoxic microsites within soil aggregates (Renault and Stengel, 1994)
or by aerobic denitri®cation (Robertson and Kuenen,
1984). Studies on the formation of NO by these microbial processes using 15N have not been carried out
yet.
The eect of soil moisture on the formation of N2O,
NO and N2 was studied by the kinetic 15N isotope
15
NOÿ
method (Neiman and Gal, 1971) using 15NH+
4 ,
3
ÿ
15
15
and NO2 as tracer. Comparisons of the N abunÿ
ÿ
dance of NH+
4 , NO3 , NO2 , N2O, NO and N2 allowed
us to identify the N processes in the soil and the formation processes of the gaseous N compounds.
2. Materials and methods
2.1. Soil
Soil was taken from the surface of a plot at the
research ®eld belonging to the Centre for
Environmental
Research
Leipzig-Halle
(Bad
LauchstaÈdt). The soil type is Haplic Phaeozem and the
soil form is a well-textured loess±black earth (21%
clay, 68% silt, 11% sand, Nt=0.31%, Ct=3.45%).
The soil was air-dried and then sieved (2 mm).
QP 2000) with column switching of a PORAPAK Q
packed column and a PLOT column containing molecular sieve 5 AÊ (Russow et al., 1995). The gas was
injected via a gas injection and preconcentration device
(Sich and Russow, 1998).
ÿ
ÿ
2.3. Analysis of NH+
4 , NO3 and NO2
ÿ
For the quantitative analysis of NH+
4 and NO3 in
the soil samples, steam distillation of the KCl extract
was used (Faust et al., 1981; Bremner and Mulvaney,
1982) followed by the determination of 15N abundance
using emission spectrometry (Fischer and Meier, 1992).
15
N enrichment was
The content of NOÿ
2 and its
determined by producing nitric oxide followed by an
analysis with a continuous ¯ow quadrupole mass spectrometer (CF±QMS; Russow et al., 1996a).
2.4.
15
N tracer methods
The experiments were carried out on the basis of the
kinetic isotope method explained in detail by Neiman
and Gal (1971) using the stable isotope 15N (15NH+
4 ,
15
15
ÿ
NOÿ
and
NO
).
It
included
the
comparison
of
the
3
2
15
N abundance and concentration of the participating
N compounds during the incubation. With the analytical equipment used and developed by us, it was possible to record and quantify the concentration and 15N
ÿ
ÿ
abundance of NH+
4 , NO3 and NO2 as well as N2O,
NO and N2 during incubation.
N2 losses due to denitri®cation were determined by
the 15N gas ¯ux method (Hauck and Melsted, 1958;
Boast et al., 1988; Arah, 1992; Russow et al., 1996b).
2.2. Gas analysis
The concentrations of N2O, NO and N2, as well as
their 15N abundance in the incubation atmosphere
were recorded by a gas chromatograph linked to a
quadrupole mass spectrometer (GC±QMS, Shimadzu
2.5. Conditions for the series of experiments and
experimental design
Experiments were conducted under either unsaturated (50±55% water-holding capacity (WHC)) or
I. Wolf, R. Russow / Soil Biology & Biochemistry 32 (2000) 229±239
Fig. 2. Procedure for sampling the soil and the N gases evolved for one
saturated conditions (about 95% WHC). The uniform
application of the N fertiliser at the start of the experiments was important (154 mg N kgÿ1 air-dried soil;
about 280 kg N haÿ1). Application was carried out
ÿ
ÿ
using NH+
4 , NO2 and NO3 salts dissolved in distilled
water. To promote nitri®cation under unsaturated conÿ
ditions the N ratio of NH+
4 to NO3 was 2, and to
promote denitri®cation under saturated conditions the
ÿ
N ratio of NH+
4 to NO3 was 0.5. The initial concenÿ
tration of NO2 was 5 mg N kgÿ1 air-dried soil in all
experiments. Three separate experiments were done at
each moisture content with the ®rst experiment using
15
15
NH+
NOÿ
4 , the second experiment using
2 , and the
ÿ
15
third experiment using NO3 as tracer. An incubation
system was used which permitted a high frequency
sampling of the soil and the N gases evolved (Figs. 1
and 2). After a conditioning phase (air-dried soil incuÿ1
air-dried soil and 40%
bated with 8 mg NH+
4 -N kg
WHC over 3 d) 15 g of the soil were placed in an incubation vessel and compressed (soil bulk density 1.3 g
15
231
N tracer experiment with three replications.
cmÿ3). The application of the fertiliser was carried out
by pipetting the solutions of the salts over the soil.
After the treatment the resulting moisture contents of
the soil were 50±55% WHC or about 95% WHC. The
vessels were kept at 308C in the dark. With three replications a total of 39 incubation vessels were used for
one 15N tracer experiment (Fig. 2). For each GC±
QMS measurement of the soil atmosphere seven
vessels (soil mass 105 g air-dried soil) were placed in
one of three containers. The other vessels were incubated in parallel outside of the containers. Each container with the incubation vessels was connected to the
analytical system (GC±QMS) via a gas circulation
pump leading to the GC's injection and preconcentration device (Fig. 3). After measurement of the emitted
N gases the soil of one vessel from each container was
extracted with 75 ml of 1 M KCl (Fig. 2). The extract
was analysed for concentrations and 15N contents of
ÿ
ÿ
NH+
4 , NO3 and NO2 . For each vessel removed a new
one from the stock of vessels was placed into the con-
232
I. Wolf, R. Russow / Soil Biology & Biochemistry 32 (2000) 229±239
Fig. 3. Container connected to the analytical system (injection device, GC±QMS).
tainer to ensure equal number of vessels in the container for the next GC±QMS measurement of the soil
atmosphere.
3. Results
3.1. Water-unsaturated conditions
The inorganic N compounds exhibited a typical
nitri®cation time course. The concentration of NH+
4
decreased and the concentration of NOÿ
3 increased
during the course of incubation (Fig. 4a), and N2O
and NO were formed (Fig. 4b). The emission of NO
was high at the beginning of the experiments but
decreased thereafter. The emissions of N2O followed
an opposite temporal course. It increased constantly
from 1±2 mg N kgÿ1 air-dried soil hÿ1 during the ®rst
4 d to 3±6 mg N kgÿ1 air-dried soil hÿ1 after d 4.
Mineralisation was slow as indicated by the constant
15
15
N abundance of the NH+
NH+
4 at the start of the
4
experiment (Fig. 5a). Due to the decrease of the
amounts of NH+
4 during the experiments (Fig. 4a),
small amounts of NH+
4 from the mineralisation contributed to the remarkable 15N dilution of the labelled
15
NH+
4 pool after d 8. Nitri®cation was the dominant
N transformation process, 15N from the NH+
4 pool
ÿ
being transformed via NOÿ
to
NO
to
increase
the
2
3
ÿ
15
N enrichment of the NOÿ
and
NO
(Fig.
5a).
2
3
From the pro®les of the 15N abundance of the NOÿ
2,
which is between the 15N abundance of NH+
4 and
ÿ
NOÿ
3 (Fig. 5a,c,e), it is apparent that the NO2 pool
Table 1
15
N balances for the experiments under unsaturated conditions
Nitrogen label
NH+
4
NOÿ
2
15
NOÿ
3
15
15
a
Amount of
15
N (mg
15
N kgÿ1 soil) (15N recovery (%))
start
end
Ntotal
NH+
4
NOÿ
2
NOÿ
3
Norg
N2O
NO
Ntotal
45.59
5.63
16.53a
0
0
0
0.01 (0.02)
0
0
40.37 (88.6)
4.21 (74.8)
16.48 (99.7)
3.82 (8.4)
0.91 (16.2)
0.89 (5.4)
0.30 (0.7)
0.02 (0.4)
0.09 (0.5)
0.20 (0.4)
0.10 (1.8)
0.04 (0.2)
44.70 (98.1)
5.24 (93.2)
17.50 (105.8)
Impurity with nitrite 410 mg
15
N kgÿ1 soil.
I. Wolf, R. Russow / Soil Biology & Biochemistry 32 (2000) 229±239
233
Fig. 4. Changes in concentrations and emission rates of the N components with time under water-unsaturated (a, b) and water-saturated conditions (c, d) (mean values for the three experiments, error bars=standard deviation).
ÿ
was fed by both NH+
4 via nitri®cation and NO3 via
denitri®cation. This is in agreement with the results of
Burns et al. (1996). However, there is also the possibility that two or more separate pools of NOÿ
2 exist in
dierent soil aggregates. As we analysed a mixture of
NOÿ
2 in the soil KCl extract, we were unable to distinguish two or more pools of the very reactive NOÿ
2
in the soil.
In the 15NOÿ
3 experiments we have to take into consideration the high 15N abundance of NOÿ
2 at the
beginning of the experiments. These high 15N abun-
dance was due to impurities of the used salt (K15NO3)
with 15NOÿ
2.
In the 15N tracer experiment with 15NOÿ
3 , the enrichment of the NOÿ
pool
decreased
due
to
an input of
3
ÿ
and
NO
pools
by nitri®unlabelled N from the NH+
4
2
cation (Fig. 5e). After 8 to 9 d the 15N abundance of
the NOÿ
3 reached a constant value in all cases (Fig.
5a,c,e). In all experiments NOÿ
3 was the ®nal product.
At the end of the experiments between 75 and 100%
of the added 15N was found in the NOÿ
3 pool (Table
1).
234
I. Wolf, R. Russow / Soil Biology & Biochemistry 32 (2000) 229±239
Fig. 5. Changes in the 15N abundance of the N components with time under water-unsaturated conditions with
15
NOÿ
3 (e, f) as tracer (error bars=standard deviation).
15
NH+
4 (a, b),
15
NOÿ
2 (c, d) and
235
I. Wolf, R. Russow / Soil Biology & Biochemistry 32 (2000) 229±239
Fig. 6. Changes in the 15N abundance of the N components with time under water-saturated conditions with
15
NOÿ
3 (e, f) as tracer (error bars=standard deviation).
15
NH+
4 (a, b),
15
NOÿ
2 (c, d) and
236
I. Wolf, R. Russow / Soil Biology & Biochemistry 32 (2000) 229±239
Table 2
15
N balances for the experiments under saturated conditions
Nitrogen label
NH+
4
NOÿ
2
15
NOÿ
3
15
15
a
b
Amount of
15
N (mg
15
N kgÿ1 soil) (15N recovery (%))
start
end
Ntotal
NH+
4
NOÿ
2
NOÿ
3
Norg
N2O
NO
Ntotal
N2
30.18
1.60
52.21b
0.63 (2.1)
0.01 (0.6)
0.07 (0.1)
0.02 (0.1)
0.01 (0.6)
0.05 (0.1)
12.07 (40.0)
0.02 (1.2)
8.64 (16.6)
5.58 (18.5)
0.12 (7.5)
1.29 (2.5)
0.54 (1.8)
0.27 (16.8)
10.57 (20.2)
0.03 (0.1)
0.01 (0.4)
0.02 (0.04)
18.86 (62.5)
0.44 (27.1)
20.64 (39.5)
ND (37.5)a
ND (72.9)a
31.57a (60.5)a
Calculated from the 15N balance.
Impurity with nitrite 1.24 mg 15N kgÿ1 soil.
From the time-course of the 15N abundance of N2O
and NO during the incubation we inferred dierent
pathways for their formation. On d 1 of the incubation
the 15N abundance of N2O and NO was the same, and
were similar to that for NOÿ
2 (Fig. 5). Thus, the N2O
and NO may have originated directly from NOÿ
2.
After d 2, the 15N abundance of N2O approached the
15
N abundance of NOÿ
3 . From d 8 onwards N2O and
15
N abundance. Therefore, N2O
NOÿ
3 had the same
was formed only from NOÿ
3 via denitri®cation. The
15
N abundance of NO followed more or less the value
of NOÿ
2 after d 2. Therefore, NO must have had a
quite dierent formation path to N2O. It could have
ÿ
originated during the oxidation of NH+
4 to NO2 or
ÿ
directly from NO2 . Due to the dierent pro®les of the
15
N abundance of N2O and NO after d 2 of incubation
(Fig. 5b,d,f) extracellular NO was not an intermediate
product or a precursor for the formation of N2O
under unsaturated conditions.
3.2. Water-saturated conditions
Incubations under saturated conditions produced
data which were more variable and more disparate
than under unsaturated incubations. The concenÿ
trations of NH+
4 and NO3 decreased during the incubation (Fig. 4c). Denitri®cation was the most common
microbial process, but nitri®cation also occurred to
some extent. In comparison with the experiments
under unsaturated conditions large amounts of N2O
were formed at the beginning of the incubation, but
little emission of NO was ascertained (Fig. 4d).
The 15N abundance of the 15N labelled NH+
4 pool
decreased from the beginning of the experiment (Fig.
6a). Therefore, a rapid N mineralisation process took
place during the incubation. It exceeded the N mineralisation rate under unsaturated conditions. The
recovery of 15N in the organic N pool indicated that
immobilisation of NH+
4 was also faster than under
unsaturated conditions (Tables 1 and 2). Similar to
unsaturated conditions the NOÿ
3 pool supplied from
pool
by
nitri®cation
was
enriched during the
the NH+
4
incubation (Fig. 6a). A decrease in 15N abundance of
15
NOÿ
the NOÿ
3 in the experiment with
3 as tracer conÿ
®rmed the N input to the NO3 pool by nitri®cation
(Fig. 6e).
15
N
The 15N abundance of NOÿ
2 was between the
+
ÿ
abundance of NH4 and NO3 (Fig. 6a,e). Therefore
similar to unsaturated conditions the NOÿ
2 was formed
from both N pools, by reduction of the NOÿ
3 and by
oxidation of the NH+
4 .
On d 1 of the experiments the 15N abundance of
N2O and NO were similar and followed the value of
NOÿ
2 (Fig. 6). N2O and NO must have originated
from the same NOÿ
2 pool and is similar to the experiment under unsaturated conditions. After d 1 of incubation, the 15N abundance of N2O was the same as
the 15N abundance of NOÿ
3 . This indicated that the
NOÿ
3 was the mean N pool for the formation of N2O.
The 15N abundance of NO followed either those of the
15
N abundance of NH+
NOÿ
2 or was between the
4 and
ÿ
NO2 . Again, the NO was coming directly from the
+
ÿ
NOÿ
2 pool or from the oxidation of NH4 to NO2 .
Therefore, under these typical denitri®cation conditions (water saturation) the role of NO as a free intermediate of N2O in the denitri®cation path could not
be con®rmed.
From the 15N balance it was obvious that there is a
high loss of N gases (Table 2). This could not be
explained by the emission of N2O and NO. However,
the emission of N2 from denitri®cation could lead to
these N losses. 15N in N2 could be detected only in the
15
N
experiment with 15NOÿ
3 as tracer due to the high
ÿ
enrichment in the NO3 pool. The N2 rate of denitri®cation was calculated according to the 15N gas ¯ux
method. About 60% of the soil NOÿ
3 was reduced to
N2. At the end of the experiment the cumulative N
ratio of N2 to N2O was 3.
4. Discussion
Based on the results a ¯ow chart was developed
with respect to the N transformation processes in the
237
I. Wolf, R. Russow / Soil Biology & Biochemistry 32 (2000) 229±239
Fig. 7. Flow chart of the paths of N transformation in black earth soil under water-unsaturated (a) and water-saturated (b) conditions related to
N2O, NO and N2 formation (thickness of the arrows refer to the importance of processes of N transformation in the soil and of formation of
ÿ
the N gases; SOM=soil organic matter; [NOÿ
2 ]e=enzyme-bound NO2 ).
soil under unsaturated and saturated conditions, and
the resulting emission of N2O, NO and N2 (Fig. 7).
In the soil, nitri®cation and denitri®cation proceeded
under both unsaturated (nearly aerobic) and saturated
(nearly anaerobic) conditions.
The total gaseous N loss and the portions of N2,
N2O and NO in this N loss during the saturated and
unsaturated incubations were clearly dierent. Under
saturated conditions the total gaseous N loss was 90.4
mg N kgÿ1 air-dried soil for the experiment with
15
NOÿ
3 as tracer, apportioned between N2, N2O-N and
NO-N in the ratio 1:0.3:0.002. However, the cumulative emission of N2O and NO under unsaturated conditions averaged only 2.2 mg N kgÿ1 air-dried soil.
The proportion of N2O-N and NO-N in the total N
gases evolved were equal. The N ratios of N2O to NO
were considerably larger under saturated conditions
than under unsaturated conditions during incubation
(Table 3). This is in contrast to the results provided by
VanCleemput and Baert (1984) and BaumgaÈrtner and
Conrad (1992), who observed high emissions of NO
under saturated conditions.
Under unsaturated as well as saturated conditions
N2O was mainly formed by denitri®cation of NOÿ
3
(Fig. 7). Only at the beginning of soil incubation did a
large part originate from nitri®cation in the aerobic
zones. As incubation under unsaturated conditions
progressed, denitri®cation became the dominant process for N2O formation, due to the development of anaerobic microsites (Renault and Stengel, 1994).
Increasing water saturation promoted N2O emission
via denitri®cation caused by an increasing number of
anaerobic sites in the soil. It was possible to calculate
the contribution of NOÿ
3 reduction to N2O formation
by using the isotope dilution equation for the experiment with 15NOÿ
3 as tracer (Table 4; Sich, 1997).
Under unsaturated conditions the percentage of NOÿ
3
reduction to N2O formation was between 69±98%
from d 3 to 17. This is in contrast to other investigations with agricultural soil, where under unsaturated
conditions the nitri®cation was responsible for 70% of
the N2O ¯ux (Stevens et al., 1997). Under saturated
conditions from d 2 onwards 64±100% of the N2O
reduction as expected.
was formed from NOÿ
3
However, N2O was probably not directly formed from
the NOÿ
3 pool. Nitrous oxide may have arose from a
stage between NOÿ
and free NOÿ
3
2 , possibly an
ÿ
enzyme-bound NO2 ([NOÿ
2 ]e, Fig. 7) (Ye et al., 1994).
The formation of N2O from the free NOÿ
2 pool in the
soil as suggested by Firestone and Davidson (1989)
and Kroneck and Zumft (1990) could not be con®rmed by our results.
The free NO desorbed into the soil atmosphere was
mainly produced by nitri®cation as a by-product of
ÿ
ÿ
the oxidation of NH+
4 to NO2 or directly by NO2 decomposition under oxygen limitation through nitri®er
denitri®cation (Poth and Focht, 1985) (Fig. 7). No evidence was found for NO being a free obligatory intermediate of N2O in the consecutive elementary
denitri®cation steps NOÿ
2 4 NO 4 N2O 4 N2 as we
Table 3
N ratio of N2O to NO during the experiments under unsaturated and saturated conditions
Duration (d)
0±3
4±11
12±17
Unsaturated conditions N2O-N to NO-N
Saturated conditions N2O-N to NO-N
minimum
maximum
minimum
maximum
0.05
1.0
16.3
0.5
16.0
38.8
60.3
7.9
10.9
293.0
53.8
52.8
238
I. Wolf, R. Russow / Soil Biology & Biochemistry 32 (2000) 229±239
Table 4
ÿ
Contribution of NOÿ
3 reduction to N2O formation (NO3 R) for the
experiment with 15NOÿ
under
unsaturated
and
saturated
conditions
3
with high amounts of NH+
4 after fertilisation, high
emissions of NO could be observed for the black earth
soils in central Germany exceeding N2O emissions by
a factor of up to 20.
Unsaturated conditions
Saturated conditions
Duration (d)
NOÿ
3 R (%)
Duration (d)
0.1±0.2
0.2±0.24
0.24±1
1±3
3±6
6±10
10±15
15±17
17
17.8
41.5
22.2
46.8
69.3
80.8
88.5
97.8
85.6
0.1±0.17
21.9
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0.17±2
25.0
2±6
6±9
9±14
86.5
64.0
80.5
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Health Studies 28, 96±100.
Granli, T., Bùckman, O.C., 1994. Nitrous oxide from agriculture.
Norwegian Journal of Agricultural Sciences 12.
Hauck, R.D., Melsted, S.W., 1958. Use of N-isotope distribution in
nitrogen gas in the study of denitri®cation. Soil Science 86, 287±
291.
Hutchinson, G.L., Davidson, E.A., 1993. Processes for production
and consumption of gaseous nitrogen oxides in soil. In:
Agricultural Ecosystem Eects on Trace Gases and Global
Climate Change. American Society of Agronomy, Madison, pp.
79±93.
Kroneck, P.M.H., Zumft, W.G., 1990. Bio-Inorganic aspects of denitri®cation: structures and reactions of NxOy compounds and their
interaction with iron and copper proteins: denitri®cation in soil
and sediment. Federation of European Microbiological Societies,
Symposium 56, 1±20.
Neiman, M.B., Gal, D., 1971. The Kinetic Isotope Method and its
Application. AkadeÂmi Kiado, Budapest.
Poth, M., Focht, D.D., 1985. 15N-Kinetic analysis of N2O production by Nitrosomonas europaea: an examination of nitri®er
denitri®cation. Applied and Environmental Microbiology 49,
1134±1141.
Renault, P., Stengel, P., 1994. Modelling oxygen diusion in aggregated soils, 1. Anaerobiosis inside the aggregates. Soil Science
Society of America Journal 58, 1017±1023.
Robertson, L.A., Kuenen, J.G., 1984. Aerobic denitri®cation: a controversy revived. Archives of Microbiology 139, 351±354.
17
NOÿ
3 R (%)
100
assumed from the literature (Firestone and Davidson,
1989; Ye et al., 1994). This ®nding could be caused by
diusion limitation of NO in the soil water of the anaerobic microsites. Nitric oxide arising from NOÿ
3 can
not desorb from the liquid phase into the gaseous
phase before its denitri®cation continued to N2O.
Further investigations without diusion limitation and
15
N labelled NO are needed to elucidate this problem.
The emission of N2 from denitri®cation was only
detected under saturated conditions (Fig. 7b). Thereby
the N loss via N2 was about 60% of the NOÿ
3 and
exceeded the emission of N2O by a factor of 3.
5. Conclusion
The results obtained and the ¯ow chart developed
show that in the black earth soil the N transformation
processes nitri®cation and denitri®cation took place
simultaneously under aerobic as well as anaerobic conditions and were closely coupled. The relationship
between nitri®cation and denitri®cation depended on
the degree of water saturation and thus the development of aerobic and anaerobic microsites within the
same soil aggregate.
The processes of formation of N2O and NO in the
black earth soil were dierent. The N2O was mainly
formed via denitri®cation and NO via nitri®cation or
nitrite decomposition. These formation pathways for
N2O and NO were the same under unsaturated as well
as saturated conditions. During the pathway of denitri®cation NO was not a free precursor of N2O.
Black earth soils in the semi-arid climate of central
Germany (unsaturated conditions) are a permanent,
low source for N2O. Under saturated conditions the
emission of N2O could increase to 280 mg N kgÿ1 airdried soil hÿ1. However, N2O mainly originated from
the anaerobic microsites via denitri®cation.
These experiments highlighted the formation of NO
from black earth soil. Under water unsaturation and
I. Wolf, R. Russow / Soil Biology & Biochemistry 32 (2000) 229±239
Russow, R., Sich, I., FoÈrstel, H., 1995. A GC±QMS aided incubation system for trace gas studies in soils using stable isotopes.
In: International Atomic Energy Agency, International
Symposium, pp. 63±72.
Russow, R., Sich, I., Stevens, R.J., 1996a. Fast and highly selective
15
N analysis of 15N enriched nitrite in water samples and soil
extracts by reaction continuous ¯ow mass spectrometry. Isotopes
in Environmental and Health Studies 32, 323±328.
Russow, R., Stevens, R.J., Laughlin, R.J., 1996b. Accuracy and precision for measurements of the mass ratio 30/28 in dinitrogen
from air samples, and its application to the investigation of N
losses from soil by denitri®cation. Isotopes in Environmental and
Health Studies 32, 289±297.
Sich, I., 1997. 15N-Traceruntersuchungen zur Nitri®kation/
Denitri®kation, insbesondere zur Bildung von Stickstooxiden in
BoÈden und waÈssrigen Medien, UFZ-Bericht, no. 17.
239
Sich, I., Russow, R., 1998. Cryotrap enrichment of nitric oxide and
nitrous oxide in their natural air concentration for 15N analysis
by GC±QMS. Isotopes in Environmental and Health Studies 34,
279±283.
Stevens, R.J., Laughlin, R.J., Burns, L.C., Arah, J.R.M., Hood,
R.C., 1997. Measuring the contribution of nitri®cation and denitri®cation to the ¯ux of nitrous oxide from soil. Soil Biology &
Biochemistry 29, 139±151.
VanCleemput, O., Baert, L., 1984. Nitrite: a key compound in N
loss processes under acid conditions. Plant and Soil 76, 233±241.
Williams, E.J., Hutchinson, G.L., Fehsenfeld, F.C., 1992. NOx and
N2O emissions from soil. Global Biogeochemical Cycles 6, 351±
388.
Ye, R.W., Averill, B.A., Tiedje, J.M., 1994. Denitri®cation: production and consumption of nitric oxide. Applied and
Environmental Microbiology 60, 1053±1058.
www.elsevier.com/locate/soilbio
Dierent pathways of formation of N2O, N2 and NO in black
earth soil
I. Wolf a, b,*, R. Russow a
a
Department of Soil Science, UFZ Centre for Environmental Research Leipzig-Halle, Theodor-Lieser-Strasse 4, D-06120 Halle, Germany
b
Institute of Soil Science and Forest Nutrition, University of GoÈttingen, BuÈsgenweg 2, D-37077 GoÈttingen, Germany
Accepted 17 August 1999
Abstract
The use of 15N tracer provides a suitable technique to investigate the processes of N transformation in soils and the origin of
the environmentally relevant gaseous N compounds N2O and NO from nitri®cation and denitri®cation. The results of
incubation experiments with black earth soil under two dierent water contents are presented here. Nitri®cation and
denitri®cation proceeded simultaneously, but the importance of these two microbial processes shifted depending on the water
ÿ
content of the soil. Under water-unsaturated conditions the microbial oxidation of NH+
4 to NO3 predominated, but a reduction
ÿ
of NO3 also occurred. The emission of NO exceeded the emission of N2O by a factor of up to 20 at the beginning of the
experiments. Under water-saturated conditions denitri®cation was the dominant process of N transformation in the soil.
However, nitri®cation also occurred to a considerable extent. The emission of N2O was greater than under unsaturated
conditions. The formation of NO could hardly be observed. N loss by molecular nitrogen from denitri®cation could be detected
under saturated conditions. The N loss amounted to 60% of NOÿ
3 and thereby the cumulative N ratio of N2 to N2O was 3.
+
ÿ
Under either unsaturated or saturated conditions NO arose from NOÿ
2 or during the microbial oxidation of NH4 to NO2 .
However, N2O mainly formed from denitri®cation under both conditions. Furthermore, NO could not be observed as a
precursor of N2O and the free NOÿ
2 could not be detected as a common N pool for the formation of N2O and NO. High
emissions of NO could be a problem for the black earth soil in the semi-arid climate in central Germany, if there are large
amounts of NH+
4 in the soil after fertilisation. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Denitri®cation; Di-nitrogen; Nitri®cation;
15
N; Nitric oxide; Nitrous oxide
1. Introduction
Nitrous oxide and nitric oxide are directly or indirectly involved in global warming, the destruction of
the stratospheric ozone, the production and consumption of atmospheric oxidants, and the photochemical
formation of nitric acid (Bouwman, 1990; Williams et
al., 1992). Additionally, the emission of N2O, NO and
N2 from agricultural soils is a loss of N fertiliser
(Granli and Bùckman, 1994).
During nitri®cation and denitri®cation N2O and NO
* Corresponding author. Tel.: +49-551-393518; fax: +49-551393310.
E-mail address: [email protected] (I. Wolf).
as well as N2 are emitted (Firestone and Davidson,
1989). Nitrous oxide and NO can also be consumed by
microorganisms in the soil (Hutchinson and Davidson,
1993). Although the mechanism of N2O and NO formation in soils is generally well-known, it is still not
clear how nitri®cation and denitri®cation contribute to
the formation of N2O under dierent amounts of
water saturation in the soil. Further uncertainties
include the role of NO in these processes. Nitric oxide
could be a by-product or an obligate and free precursor of N2O in the denitri®cation path. Not much is
known about the ratios between N2 and N2O-N or
N2O-N and NO-N under dierent conditions.
Recent 15N studies with agricultural soil have shown
that under unsaturated conditions nitri®cation was the
0038-0717/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 1 5 1 - 0
230
I. Wolf, R. Russow / Soil Biology & Biochemistry 32 (2000) 229±239
Fig. 1. Incubation vessel and the container holding incubation vessels to determine the N emission rates.
dominant process for N2O production (Stevens et al.,
1997). The formation of N2O and NO via nitri®cation
could be signi®cant for the black earth soils in central
Germany because of the absence of typical denitri®cation conditions (water saturation) under the prevailing
semi-arid climate conditions. However, N2O can be
formed in soils by denitri®cation due to anoxic microsites within soil aggregates (Renault and Stengel, 1994)
or by aerobic denitri®cation (Robertson and Kuenen,
1984). Studies on the formation of NO by these microbial processes using 15N have not been carried out
yet.
The eect of soil moisture on the formation of N2O,
NO and N2 was studied by the kinetic 15N isotope
15
NOÿ
method (Neiman and Gal, 1971) using 15NH+
4 ,
3
ÿ
15
15
and NO2 as tracer. Comparisons of the N abunÿ
ÿ
dance of NH+
4 , NO3 , NO2 , N2O, NO and N2 allowed
us to identify the N processes in the soil and the formation processes of the gaseous N compounds.
2. Materials and methods
2.1. Soil
Soil was taken from the surface of a plot at the
research ®eld belonging to the Centre for
Environmental
Research
Leipzig-Halle
(Bad
LauchstaÈdt). The soil type is Haplic Phaeozem and the
soil form is a well-textured loess±black earth (21%
clay, 68% silt, 11% sand, Nt=0.31%, Ct=3.45%).
The soil was air-dried and then sieved (2 mm).
QP 2000) with column switching of a PORAPAK Q
packed column and a PLOT column containing molecular sieve 5 AÊ (Russow et al., 1995). The gas was
injected via a gas injection and preconcentration device
(Sich and Russow, 1998).
ÿ
ÿ
2.3. Analysis of NH+
4 , NO3 and NO2
ÿ
For the quantitative analysis of NH+
4 and NO3 in
the soil samples, steam distillation of the KCl extract
was used (Faust et al., 1981; Bremner and Mulvaney,
1982) followed by the determination of 15N abundance
using emission spectrometry (Fischer and Meier, 1992).
15
N enrichment was
The content of NOÿ
2 and its
determined by producing nitric oxide followed by an
analysis with a continuous ¯ow quadrupole mass spectrometer (CF±QMS; Russow et al., 1996a).
2.4.
15
N tracer methods
The experiments were carried out on the basis of the
kinetic isotope method explained in detail by Neiman
and Gal (1971) using the stable isotope 15N (15NH+
4 ,
15
15
ÿ
NOÿ
and
NO
).
It
included
the
comparison
of
the
3
2
15
N abundance and concentration of the participating
N compounds during the incubation. With the analytical equipment used and developed by us, it was possible to record and quantify the concentration and 15N
ÿ
ÿ
abundance of NH+
4 , NO3 and NO2 as well as N2O,
NO and N2 during incubation.
N2 losses due to denitri®cation were determined by
the 15N gas ¯ux method (Hauck and Melsted, 1958;
Boast et al., 1988; Arah, 1992; Russow et al., 1996b).
2.2. Gas analysis
The concentrations of N2O, NO and N2, as well as
their 15N abundance in the incubation atmosphere
were recorded by a gas chromatograph linked to a
quadrupole mass spectrometer (GC±QMS, Shimadzu
2.5. Conditions for the series of experiments and
experimental design
Experiments were conducted under either unsaturated (50±55% water-holding capacity (WHC)) or
I. Wolf, R. Russow / Soil Biology & Biochemistry 32 (2000) 229±239
Fig. 2. Procedure for sampling the soil and the N gases evolved for one
saturated conditions (about 95% WHC). The uniform
application of the N fertiliser at the start of the experiments was important (154 mg N kgÿ1 air-dried soil;
about 280 kg N haÿ1). Application was carried out
ÿ
ÿ
using NH+
4 , NO2 and NO3 salts dissolved in distilled
water. To promote nitri®cation under unsaturated conÿ
ditions the N ratio of NH+
4 to NO3 was 2, and to
promote denitri®cation under saturated conditions the
ÿ
N ratio of NH+
4 to NO3 was 0.5. The initial concenÿ
tration of NO2 was 5 mg N kgÿ1 air-dried soil in all
experiments. Three separate experiments were done at
each moisture content with the ®rst experiment using
15
15
NH+
NOÿ
4 , the second experiment using
2 , and the
ÿ
15
third experiment using NO3 as tracer. An incubation
system was used which permitted a high frequency
sampling of the soil and the N gases evolved (Figs. 1
and 2). After a conditioning phase (air-dried soil incuÿ1
air-dried soil and 40%
bated with 8 mg NH+
4 -N kg
WHC over 3 d) 15 g of the soil were placed in an incubation vessel and compressed (soil bulk density 1.3 g
15
231
N tracer experiment with three replications.
cmÿ3). The application of the fertiliser was carried out
by pipetting the solutions of the salts over the soil.
After the treatment the resulting moisture contents of
the soil were 50±55% WHC or about 95% WHC. The
vessels were kept at 308C in the dark. With three replications a total of 39 incubation vessels were used for
one 15N tracer experiment (Fig. 2). For each GC±
QMS measurement of the soil atmosphere seven
vessels (soil mass 105 g air-dried soil) were placed in
one of three containers. The other vessels were incubated in parallel outside of the containers. Each container with the incubation vessels was connected to the
analytical system (GC±QMS) via a gas circulation
pump leading to the GC's injection and preconcentration device (Fig. 3). After measurement of the emitted
N gases the soil of one vessel from each container was
extracted with 75 ml of 1 M KCl (Fig. 2). The extract
was analysed for concentrations and 15N contents of
ÿ
ÿ
NH+
4 , NO3 and NO2 . For each vessel removed a new
one from the stock of vessels was placed into the con-
232
I. Wolf, R. Russow / Soil Biology & Biochemistry 32 (2000) 229±239
Fig. 3. Container connected to the analytical system (injection device, GC±QMS).
tainer to ensure equal number of vessels in the container for the next GC±QMS measurement of the soil
atmosphere.
3. Results
3.1. Water-unsaturated conditions
The inorganic N compounds exhibited a typical
nitri®cation time course. The concentration of NH+
4
decreased and the concentration of NOÿ
3 increased
during the course of incubation (Fig. 4a), and N2O
and NO were formed (Fig. 4b). The emission of NO
was high at the beginning of the experiments but
decreased thereafter. The emissions of N2O followed
an opposite temporal course. It increased constantly
from 1±2 mg N kgÿ1 air-dried soil hÿ1 during the ®rst
4 d to 3±6 mg N kgÿ1 air-dried soil hÿ1 after d 4.
Mineralisation was slow as indicated by the constant
15
15
N abundance of the NH+
NH+
4 at the start of the
4
experiment (Fig. 5a). Due to the decrease of the
amounts of NH+
4 during the experiments (Fig. 4a),
small amounts of NH+
4 from the mineralisation contributed to the remarkable 15N dilution of the labelled
15
NH+
4 pool after d 8. Nitri®cation was the dominant
N transformation process, 15N from the NH+
4 pool
ÿ
being transformed via NOÿ
to
NO
to
increase
the
2
3
ÿ
15
N enrichment of the NOÿ
and
NO
(Fig.
5a).
2
3
From the pro®les of the 15N abundance of the NOÿ
2,
which is between the 15N abundance of NH+
4 and
ÿ
NOÿ
3 (Fig. 5a,c,e), it is apparent that the NO2 pool
Table 1
15
N balances for the experiments under unsaturated conditions
Nitrogen label
NH+
4
NOÿ
2
15
NOÿ
3
15
15
a
Amount of
15
N (mg
15
N kgÿ1 soil) (15N recovery (%))
start
end
Ntotal
NH+
4
NOÿ
2
NOÿ
3
Norg
N2O
NO
Ntotal
45.59
5.63
16.53a
0
0
0
0.01 (0.02)
0
0
40.37 (88.6)
4.21 (74.8)
16.48 (99.7)
3.82 (8.4)
0.91 (16.2)
0.89 (5.4)
0.30 (0.7)
0.02 (0.4)
0.09 (0.5)
0.20 (0.4)
0.10 (1.8)
0.04 (0.2)
44.70 (98.1)
5.24 (93.2)
17.50 (105.8)
Impurity with nitrite 410 mg
15
N kgÿ1 soil.
I. Wolf, R. Russow / Soil Biology & Biochemistry 32 (2000) 229±239
233
Fig. 4. Changes in concentrations and emission rates of the N components with time under water-unsaturated (a, b) and water-saturated conditions (c, d) (mean values for the three experiments, error bars=standard deviation).
ÿ
was fed by both NH+
4 via nitri®cation and NO3 via
denitri®cation. This is in agreement with the results of
Burns et al. (1996). However, there is also the possibility that two or more separate pools of NOÿ
2 exist in
dierent soil aggregates. As we analysed a mixture of
NOÿ
2 in the soil KCl extract, we were unable to distinguish two or more pools of the very reactive NOÿ
2
in the soil.
In the 15NOÿ
3 experiments we have to take into consideration the high 15N abundance of NOÿ
2 at the
beginning of the experiments. These high 15N abun-
dance was due to impurities of the used salt (K15NO3)
with 15NOÿ
2.
In the 15N tracer experiment with 15NOÿ
3 , the enrichment of the NOÿ
pool
decreased
due
to
an input of
3
ÿ
and
NO
pools
by nitri®unlabelled N from the NH+
4
2
cation (Fig. 5e). After 8 to 9 d the 15N abundance of
the NOÿ
3 reached a constant value in all cases (Fig.
5a,c,e). In all experiments NOÿ
3 was the ®nal product.
At the end of the experiments between 75 and 100%
of the added 15N was found in the NOÿ
3 pool (Table
1).
234
I. Wolf, R. Russow / Soil Biology & Biochemistry 32 (2000) 229±239
Fig. 5. Changes in the 15N abundance of the N components with time under water-unsaturated conditions with
15
NOÿ
3 (e, f) as tracer (error bars=standard deviation).
15
NH+
4 (a, b),
15
NOÿ
2 (c, d) and
235
I. Wolf, R. Russow / Soil Biology & Biochemistry 32 (2000) 229±239
Fig. 6. Changes in the 15N abundance of the N components with time under water-saturated conditions with
15
NOÿ
3 (e, f) as tracer (error bars=standard deviation).
15
NH+
4 (a, b),
15
NOÿ
2 (c, d) and
236
I. Wolf, R. Russow / Soil Biology & Biochemistry 32 (2000) 229±239
Table 2
15
N balances for the experiments under saturated conditions
Nitrogen label
NH+
4
NOÿ
2
15
NOÿ
3
15
15
a
b
Amount of
15
N (mg
15
N kgÿ1 soil) (15N recovery (%))
start
end
Ntotal
NH+
4
NOÿ
2
NOÿ
3
Norg
N2O
NO
Ntotal
N2
30.18
1.60
52.21b
0.63 (2.1)
0.01 (0.6)
0.07 (0.1)
0.02 (0.1)
0.01 (0.6)
0.05 (0.1)
12.07 (40.0)
0.02 (1.2)
8.64 (16.6)
5.58 (18.5)
0.12 (7.5)
1.29 (2.5)
0.54 (1.8)
0.27 (16.8)
10.57 (20.2)
0.03 (0.1)
0.01 (0.4)
0.02 (0.04)
18.86 (62.5)
0.44 (27.1)
20.64 (39.5)
ND (37.5)a
ND (72.9)a
31.57a (60.5)a
Calculated from the 15N balance.
Impurity with nitrite 1.24 mg 15N kgÿ1 soil.
From the time-course of the 15N abundance of N2O
and NO during the incubation we inferred dierent
pathways for their formation. On d 1 of the incubation
the 15N abundance of N2O and NO was the same, and
were similar to that for NOÿ
2 (Fig. 5). Thus, the N2O
and NO may have originated directly from NOÿ
2.
After d 2, the 15N abundance of N2O approached the
15
N abundance of NOÿ
3 . From d 8 onwards N2O and
15
N abundance. Therefore, N2O
NOÿ
3 had the same
was formed only from NOÿ
3 via denitri®cation. The
15
N abundance of NO followed more or less the value
of NOÿ
2 after d 2. Therefore, NO must have had a
quite dierent formation path to N2O. It could have
ÿ
originated during the oxidation of NH+
4 to NO2 or
ÿ
directly from NO2 . Due to the dierent pro®les of the
15
N abundance of N2O and NO after d 2 of incubation
(Fig. 5b,d,f) extracellular NO was not an intermediate
product or a precursor for the formation of N2O
under unsaturated conditions.
3.2. Water-saturated conditions
Incubations under saturated conditions produced
data which were more variable and more disparate
than under unsaturated incubations. The concenÿ
trations of NH+
4 and NO3 decreased during the incubation (Fig. 4c). Denitri®cation was the most common
microbial process, but nitri®cation also occurred to
some extent. In comparison with the experiments
under unsaturated conditions large amounts of N2O
were formed at the beginning of the incubation, but
little emission of NO was ascertained (Fig. 4d).
The 15N abundance of the 15N labelled NH+
4 pool
decreased from the beginning of the experiment (Fig.
6a). Therefore, a rapid N mineralisation process took
place during the incubation. It exceeded the N mineralisation rate under unsaturated conditions. The
recovery of 15N in the organic N pool indicated that
immobilisation of NH+
4 was also faster than under
unsaturated conditions (Tables 1 and 2). Similar to
unsaturated conditions the NOÿ
3 pool supplied from
pool
by
nitri®cation
was
enriched during the
the NH+
4
incubation (Fig. 6a). A decrease in 15N abundance of
15
NOÿ
the NOÿ
3 in the experiment with
3 as tracer conÿ
®rmed the N input to the NO3 pool by nitri®cation
(Fig. 6e).
15
N
The 15N abundance of NOÿ
2 was between the
+
ÿ
abundance of NH4 and NO3 (Fig. 6a,e). Therefore
similar to unsaturated conditions the NOÿ
2 was formed
from both N pools, by reduction of the NOÿ
3 and by
oxidation of the NH+
4 .
On d 1 of the experiments the 15N abundance of
N2O and NO were similar and followed the value of
NOÿ
2 (Fig. 6). N2O and NO must have originated
from the same NOÿ
2 pool and is similar to the experiment under unsaturated conditions. After d 1 of incubation, the 15N abundance of N2O was the same as
the 15N abundance of NOÿ
3 . This indicated that the
NOÿ
3 was the mean N pool for the formation of N2O.
The 15N abundance of NO followed either those of the
15
N abundance of NH+
NOÿ
2 or was between the
4 and
ÿ
NO2 . Again, the NO was coming directly from the
+
ÿ
NOÿ
2 pool or from the oxidation of NH4 to NO2 .
Therefore, under these typical denitri®cation conditions (water saturation) the role of NO as a free intermediate of N2O in the denitri®cation path could not
be con®rmed.
From the 15N balance it was obvious that there is a
high loss of N gases (Table 2). This could not be
explained by the emission of N2O and NO. However,
the emission of N2 from denitri®cation could lead to
these N losses. 15N in N2 could be detected only in the
15
N
experiment with 15NOÿ
3 as tracer due to the high
ÿ
enrichment in the NO3 pool. The N2 rate of denitri®cation was calculated according to the 15N gas ¯ux
method. About 60% of the soil NOÿ
3 was reduced to
N2. At the end of the experiment the cumulative N
ratio of N2 to N2O was 3.
4. Discussion
Based on the results a ¯ow chart was developed
with respect to the N transformation processes in the
237
I. Wolf, R. Russow / Soil Biology & Biochemistry 32 (2000) 229±239
Fig. 7. Flow chart of the paths of N transformation in black earth soil under water-unsaturated (a) and water-saturated (b) conditions related to
N2O, NO and N2 formation (thickness of the arrows refer to the importance of processes of N transformation in the soil and of formation of
ÿ
the N gases; SOM=soil organic matter; [NOÿ
2 ]e=enzyme-bound NO2 ).
soil under unsaturated and saturated conditions, and
the resulting emission of N2O, NO and N2 (Fig. 7).
In the soil, nitri®cation and denitri®cation proceeded
under both unsaturated (nearly aerobic) and saturated
(nearly anaerobic) conditions.
The total gaseous N loss and the portions of N2,
N2O and NO in this N loss during the saturated and
unsaturated incubations were clearly dierent. Under
saturated conditions the total gaseous N loss was 90.4
mg N kgÿ1 air-dried soil for the experiment with
15
NOÿ
3 as tracer, apportioned between N2, N2O-N and
NO-N in the ratio 1:0.3:0.002. However, the cumulative emission of N2O and NO under unsaturated conditions averaged only 2.2 mg N kgÿ1 air-dried soil.
The proportion of N2O-N and NO-N in the total N
gases evolved were equal. The N ratios of N2O to NO
were considerably larger under saturated conditions
than under unsaturated conditions during incubation
(Table 3). This is in contrast to the results provided by
VanCleemput and Baert (1984) and BaumgaÈrtner and
Conrad (1992), who observed high emissions of NO
under saturated conditions.
Under unsaturated as well as saturated conditions
N2O was mainly formed by denitri®cation of NOÿ
3
(Fig. 7). Only at the beginning of soil incubation did a
large part originate from nitri®cation in the aerobic
zones. As incubation under unsaturated conditions
progressed, denitri®cation became the dominant process for N2O formation, due to the development of anaerobic microsites (Renault and Stengel, 1994).
Increasing water saturation promoted N2O emission
via denitri®cation caused by an increasing number of
anaerobic sites in the soil. It was possible to calculate
the contribution of NOÿ
3 reduction to N2O formation
by using the isotope dilution equation for the experiment with 15NOÿ
3 as tracer (Table 4; Sich, 1997).
Under unsaturated conditions the percentage of NOÿ
3
reduction to N2O formation was between 69±98%
from d 3 to 17. This is in contrast to other investigations with agricultural soil, where under unsaturated
conditions the nitri®cation was responsible for 70% of
the N2O ¯ux (Stevens et al., 1997). Under saturated
conditions from d 2 onwards 64±100% of the N2O
reduction as expected.
was formed from NOÿ
3
However, N2O was probably not directly formed from
the NOÿ
3 pool. Nitrous oxide may have arose from a
stage between NOÿ
and free NOÿ
3
2 , possibly an
ÿ
enzyme-bound NO2 ([NOÿ
2 ]e, Fig. 7) (Ye et al., 1994).
The formation of N2O from the free NOÿ
2 pool in the
soil as suggested by Firestone and Davidson (1989)
and Kroneck and Zumft (1990) could not be con®rmed by our results.
The free NO desorbed into the soil atmosphere was
mainly produced by nitri®cation as a by-product of
ÿ
ÿ
the oxidation of NH+
4 to NO2 or directly by NO2 decomposition under oxygen limitation through nitri®er
denitri®cation (Poth and Focht, 1985) (Fig. 7). No evidence was found for NO being a free obligatory intermediate of N2O in the consecutive elementary
denitri®cation steps NOÿ
2 4 NO 4 N2O 4 N2 as we
Table 3
N ratio of N2O to NO during the experiments under unsaturated and saturated conditions
Duration (d)
0±3
4±11
12±17
Unsaturated conditions N2O-N to NO-N
Saturated conditions N2O-N to NO-N
minimum
maximum
minimum
maximum
0.05
1.0
16.3
0.5
16.0
38.8
60.3
7.9
10.9
293.0
53.8
52.8
238
I. Wolf, R. Russow / Soil Biology & Biochemistry 32 (2000) 229±239
Table 4
ÿ
Contribution of NOÿ
3 reduction to N2O formation (NO3 R) for the
experiment with 15NOÿ
under
unsaturated
and
saturated
conditions
3
with high amounts of NH+
4 after fertilisation, high
emissions of NO could be observed for the black earth
soils in central Germany exceeding N2O emissions by
a factor of up to 20.
Unsaturated conditions
Saturated conditions
Duration (d)
NOÿ
3 R (%)
Duration (d)
0.1±0.2
0.2±0.24
0.24±1
1±3
3±6
6±10
10±15
15±17
17
17.8
41.5
22.2
46.8
69.3
80.8
88.5
97.8
85.6
0.1±0.17
21.9
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0.17±2
25.0
2±6
6±9
9±14
86.5
64.0
80.5
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NOÿ
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100
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5. Conclusion
The results obtained and the ¯ow chart developed
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The processes of formation of N2O and NO in the
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formed via denitri®cation and NO via nitri®cation or
nitrite decomposition. These formation pathways for
N2O and NO were the same under unsaturated as well
as saturated conditions. During the pathway of denitri®cation NO was not a free precursor of N2O.
Black earth soils in the semi-arid climate of central
Germany (unsaturated conditions) are a permanent,
low source for N2O. Under saturated conditions the
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the anaerobic microsites via denitri®cation.
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