European Journal of Soil Biology 65 (2014) 7e14
Contents lists available at ScienceDirect
European Journal of Soil Biology
journal homepage: http://www.elsevier.com/locate/ejsobi
Original article
Inhibitory and side effects of acetylene (C2H2) and sodium chlorate
(NaClO3) on gross nitrification, gross ammonification and soilatmosphere exchange of N2O and CH4 in acidic to neutral montane
grassland soil
Changhui Wang a, b, *, Michael Dannenmann a, c, Rudi Meier a, Klaus Butterbach-Bahl a
a
Institute for Meteorology and Climate Research, Atmospheric Environmental Research (IMK-IFU), Karlsruhe Institute of Technology (KIT), GarmischPartenkirchen 82467, Germany
b
State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, The Chinese Academy of Sciences (IBCAS), Beijing 100093, China
c
University of Freiburg, Institute of Forest Botany and Tree Physiology, Chair of Tree Physiology, 79110 Freiburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 February 2014
Received in revised form
14 August 2014
Accepted 22 August 2014
Available online 23 August 2014
Nitrification is a central component of the terrestrial nitrogen (N) cycle, but the contribution of autotrophic and heterotrophic nitrification to total gross nitrification remains poorly understood. To clarify
their relative importance in neutral and moderate acid soils, an incubation experiment was conducted
with 15N-ammonium isotopic pool dilution techniques and combined with acetylene (C2H2, 10 Pa) as a
specific inhibitor of autotrophic nitrification and sodium chlorate (NaClO3) as a potential inhibitor of
heterotrophic nitrification. Additionally, CO2, N2O and CH4 fluxes were measured to identify potential
side-effects of inhibitors on soil respiration and CH4 fluxes.
The presence of C2H2 completely eliminated gross nitrification in all investigated soil samples. The
addition of NaClO3 affected neither gross nitrification nor gross ammonification in soils of both investigated grassland sites. This provided strong evidence that heterotrophic nitrification was not an
important process in the investigated grassland soils. Acetylene but not NaClO3 decreased net CH4 uptake, likely due to homology of the enzymes ammonia monooxygenase. Overall, the present study shows
a dominant role of autotrophic nitrification in gross nitrate production for both neutral and slightly acid
soils and illustrates the potential of acetylene as an inhibitor of gross autotrophic nitrification.
© 2014 Published by Elsevier Masson SAS.
Keywords:
Autotrophic/heterotrophic nitrification
Nitrification inhibitor
Microbial activity
Gross ammonification/nitrification rate
Grassland
1. Introduction
Nitrification, the microbial oxidation of ammonia (NH3) to ni
trite (NO
2 ) or further to nitrate (NO3 ), plays a key role for nitrogen
(N) cycling in terrestrial ecosystems [1] by affecting major ecological processes such as net primary productivity [2e4] and net
ecosystem exchange [5,6] as well as ecosystem N losses via leaching
[7,8] and gaseous pathways, e.g. via the potent greenhouse gas
nitrous oxide (N2O) [9,10]. Nitrous oxide is either directly produced
by nitrification or by subsequent denitrification [11]. Nitrification is
performed by both autotrophic and heterotrophic microorganisms
with pH playing an important role in the regulation of the
* Corresponding author. Institute of Meteorology and Climate Research, Atmospheric Environmental Research (IMK-IFU), Karlsruhe Institute of Technology (KIT),
Kreuzeckbahnstr. 19, 82467 Garmisch-Partenkirchen, Germany.
E-mail address: wangch@ibcas.ac.cn (C. Wang).
http://dx.doi.org/10.1016/j.ejsobi.2014.08.006
1164-5563/© 2014 Published by Elsevier Masson SAS.
importance of autotrophic vs. heterotrophic nitrification. Heterotrophic nitrification has been assumed to be of importance only in
acidic soils [12e15]. In contrast, for soils with neutral pH values, it
has been assumed for decades that autotrophic nitrification is the
sole process with no contribution of heterotrophic nitrification
[16e18]. However, these studies were usually based on methodologies with constraints such as the determination of net nitrification rate [19] or culture studies [12,20], i.e., methods not
providing actual gross nitrification rates.
Improved understanding of nitrification has been achieved by
the application of the 15N pool dilution technique which provides
gross rather than net rates of nitrification [21e23]. However, due to
methodological difficulties, the separation of total gross NO
3 production into process-specific pathways, i.e. gross autotrophic
nitrification (oxidation of free soil ammonium) and gross heterotrophic nitrification (also involving direct oxidation of organic N
compounds to NO
3 ) [4,24] has seldom been studied [12,13,25].
8
C. Wang et al. / European Journal of Soil Biology 65 (2014) 7e14
Specific inhibitors such as C2H2 at low concentrations (10 Pa) have
been used to inhibit the NH3 oxidation by the activity of autotrophic nitrifiers [1,26]. To current knowledge heterotrophic nitrification does not rely on the enzyme NH3 monooxygenase and thus,
is not inhibited by common nitrification inhibitors such as C2H2 or
nitrapyrin [26]. However, chlorate was found to inhibit heterotrophic nitrification in acid forest humus [16], which was confirmed by
Lang (1986), who found that chlorate blocks nitrification in acid
forest soil from the Solling site, i.e. a site at which autotrophic nitrifiers could not be detected. On the other hand, several studies
reported that NaClO3 also inhibits ammonia monooxygenase
[17,19]. Such inhibitors have often been used in combination with
indirect parameters of soil N turnover such as microbial biomass,
net nitrification, enzyme activity and C and N gas fluxes, culture
studies [12,17,20,24,27,28], but have rarely been linked with studies
determining gross rates of N turnover and simultaneous determination of soil-atmosphere exchange of N2O, CH4 and CO2 [29].
Indeed very few studies investigated the role of autotrophic vs.
heterotrophic gross nitrification for soils with slightly acidic or
neutral pH values [30]. Therefore, inhibitor effects on total gross
nitrification as well as the quantitative contribution of autotrophic
versus heterotrophic nitrification to gross NO
3 production and N2O
formation are actually much more uncertain than is suggested by
abundant studies using traditional indirect methods for the characterization of nitrification. Furthermore, inhibitor side effects on
CH4 and CO2 are uncertain [31].
Recently the application of numerical 15N tracing models [4,32]
15
and studies using analytical 15NHþ
NO
4 and
3 pool dilution approaches [32] indicated a significant contribution of the direct
conversion of organic N to nitrate, ascertained to heterotrophic
nitrification. This challenged the previous views of the absolute
dominance of autotrophic nitrification in slightly acidic or neutral
soils. In order to contribute to the clarification of this issue, we
combined 15N pool dilution approaches to quantify gross nitrification and the selective inhibitor C2H2 to investigate the role of
heterotrophic versus autotrophic nitrification in slightly acidic and
neutral grassland soils of Southern Germany. We also included a
NaClO3 treatment to compare C2H2 vs. NaClO3 effects on gross
nitrification with the aim to contribute to understanding of the
contradictory results of NaClO3 effects on either autotrophic or
heterotrophic nitrification in earlier studies.
Inhibitor treatments and measurements of gross N turnover
were accompanied by measurements of soil-headspace exchange
of CO2, CH4 and N2O to evaluate the importance of heterotrophic vs.
autotrophic nitrification in the production of N2O at different pH
levels and to identify potential side-effects of inhibitors on soil
respiration and CH4 fluxes.
2. Material and methods
2.1. Soil sampling and measurement of soil properties
Soil samples were collected at two typical pre-alpine grassland
ecosystems of Southern Germany (Graswang site: 11.03 E; 47.57 N;
and Wielenbach site: 11.15 E, 47.89 N) with calcareous soil and pH
values of 7.33 and 5.94 (Table 1). Both sites are located in the flood
plain of the Ammer river catchment. The Graswang site is located in
the upper part of the catchment at 865 m a.s.l., surrounded by
calcareous alpine mountain ranges, while the Wielenbach site is
located downstream in the lower pre-alpine part of the catchment
at 545 m a.s.l. This altitudinal gradient induces a climatic gradient:
the mean annual temperature at the Graswang site is 6.0 C and the
mean annual precipitation is 1437.6 mm, while at the Wielenbach
site, the mean annual temperature is 7.8 C and the mean annual
precipitation is 1020.5 mm during 50 years (1955e2005).
Table 1
Topsoil (0e10 cm soil depth) characteristics of grasslands in Graswang and Wielenbach (n ¼ 3).
N3
Graswang
Soil
Soil
C:N
Soil
Soil
Soil
Soil
13.6
0.81
16.9
7.33
0.98
19.1
0.29
total organic carbon (%)
total nitrogen (%)
pH value
bulk density (mg cm3)
nitrate concentration (mg kg1 SDW)
ammonium concentration (mg kg1 SDW)
±
±
±
±
±
±
±
0.3
0.04
0.5
0.08
0.03
3.9
0.01
Wielenbach
6.8
0.68
10.0
5.94
1.08
41.4
0.25
±
±
±
±
±
±
±
1.1
0.10
0.2
0.01
0.04
1.9
0.04
Soil samples of the Ah horizon (0e10 cm) were collected in
March 2010. Soil was sampled randomly at three locations at each
site as intact surface soil of approximately 3 kg each (Fig. 1).
Following immediate transfer to the laboratory of IMK-IFU, soil was
pooled within sites, air dried and sieved for removal of stones
>5 mm, roots >1 cm and soil macrofauna such as earthworms.
Subsequently soil was stored at 4 C until further processing. All
experiments were carried out in the laboratory.
2.2. Laboratory incubation
Three different incubation treatments were performed: a) control, i.e. no addition of inhibitors; b) addition of C2H2 (10 Pa) to the
headspace to inhibit autotrophic nitrification and, c) addition of
NaClO3 (100 mg per kg soil dry weight). This general experimental
set-up was repeated three times (Fig. 1).
Three days prior to experiments all soil samples were re-wetted
to 65% maximum water holding capacity by adding the respective
amount of standard rain solution [33]. Following the preincubation period of three days a subsample was taken for the
determination of soil NHþ
4 and NO3 concentrations as well as microbial biomass. Thereafter, the soil was split into three subsamples
(treatments: control, C2H2, NaClO3), and these subsamples were
further divided into two subsamples for determination of gross
ammonification and gross nitrification rates. Subsamples used to
quantify gross N turnover were further subdivided into six subsamples of 30 g each, with three subsamples each being used for
the first and second extraction after isotope labeling to quantify
gross N turnover (see below) (Fig. 1).
2.3. Determination of gross rates of ammonification and
nitrification
Gross rates of ammonification and nitrification were determined by the 15N pool dilution method [34]. For this purpose, soil
was labeled with either 15Ne(NH4)2SO4 or 15NeKNO
3 solution at
50 atom% 15N enrichment and an application rate of 3 ml 100 g1
dry soil equivalent. The 15N solution was sprayed on soil in five
steps with subsequent mixing to ensure homogenous labeling. The
amount of added N corresponded to 2 mg N kg1 soil. Hence, we
increased soil NHþ
4 availability by a factor of approximately five in
soil samples used for the determination of gross ammonification
rates (see Table 1 for background NHþ
4 and NO3 concentrations).
However, adding 2 mg of NO
3 eN to the soil for determination of
gross nitrification did alter soil NO
3 concentrations only marginally
(factor 1:10 for Graswang and factor 1:20 for Wielenbach, respectively). Thirty grams of labeled soil was filled into glass flasks
(338 ml volume), which were immediately closed gas-tight with
rubber stoppers. Inhibitors were added either with the labeling
solution (in case of NaClO3) or by injecting C2H2 to the headspace of
the gas-tightly closed incubation vessels so that an end concentration of 10 Pa was achieved.
C. Wang et al. / European Journal of Soil Biology 65 (2014) 7e14
Graswang/Wielenbach site plot 1
Graswang/Wielenbach site plot 2
9
Graswang/Wielenbach site plot 3
Air drying, sieving, pooling of soil (one composite sample per site); rewetting to 65% of WHC prior to experiment
Control
10 Pa C2H2
100 mg NaClO3 kg-1 SDW
(no inhibitor treatment)
to inhibit autotrophic nitrification
to inhibit heterotrophic
3 different experiments
3 different experiments
3 different experiments
2 mg NH4+-N kg-1 SDW at 50 atom % 15N
2 mg NO3--N kg-1 SDW 50 atom% 15N
for determination of gross ammonification
for determination of gross nitrification
Time 1 extraction 3 h
After 15N application
(30 g each, triplicated)
N2O, CH4, CO2 flux
Measurements (0, 3,
6, 9, 24 h after 15N
application, 30 g
each, triplicated)
Time 1 extraction 3 h
After 15N application
(30 g each, triplicated)
Time 2 extraction 27 h
after 15N application
(30 g each, triplicated)
N2O, CH4, CO2 flux
measurements (0, 3,
6, 9, 24 h after 15N
application, 30 g
each, triplicated)
Time 2 extraction 27 h
after 15N application
(30 g each, triplicated)
Fig. 1. Scheme showing the applied soil processing procedure used in this study to identify effects of two inhibitors (10 Pa C2H2 for autotrophic nitrification and 100 mg NaClO3 per
kg SDW for heterotrophic nitrification) on soil microbial N turnover rates and soil fluxes of N2O, CH4 and CO2.
At T0 (3 h after label addition) soils from three bottles of each
treatment were extracted with 60 ml of 0.5 M K2SO4 by rigorously
shaking the soil solution for 1 h in a reciprocal shaker and filtering
through Whatman No. 1 filter paper (10.5 cm in diameter). The
same process was repeated for the remaining three bottles per
treatment 24 h after 15N labeling (T1). Soil solutions were immediately frozen until colorimetrical determination of NHþ
4 eN and
NO
3 eN concentrations (Landwirtschaftliches Labor Dr. Janssen,
Gillersheim, Germany). Subsamples of soil extract were analyzed
for 15N enrichment in NHþ
4 and NO3 following the diffusion method
by Dannenmann et al. (2006) [35]. Briefly, NaOH was added to the
soil solution to increase pH and convert NHþ
4 to NH3, which is
trapped on an acidified filter disk (Whatman ashless paper filters)
in the headspace of the gastight incubation flask. Following complete removal of NHþ
4 /NH3 from the solution, Devarda's alloy (50%
þ
Cu, 45% Al, 5% Zn) was added in order to convert NO
3 to NH4 . This
step was followed by a second diffusion step to trap the NHþ
4 eN
originating from NO
3 on filter disks. Finally, filter disks were dried
and analyzed for 15N enrichment on an ion ratio mass spectrometer
(Flash EA coupled to Thermo Delta VPlus, Thermo Fisher Scientific,
Bremen, Germany) at the Center of Stable Isotopes of IMK-IFU in
Garmisch-Partenkirchen [36].
synthetic air containing 380 ppmv CO2 was re-injected. The headspace gas samples were analyzed immediately using a gas chromatograph equipped with a thermal conductivity detector for CO2
detection, a flame ionization detector for CH4 detection, and a 63Ni
electron capture detector (ECD) for N2O detection. A standard gas
mixture of 397 ppmv CO2, 4.09 ppmv CH4, and 408 ppbv N2O (Air
Liquide, Düsseldorf, Germany) was used as a reference. Since N2
was used as carrier gas, we used an ascarite pre-column prior of the
analytical column (Hayesep N, 3 m, 1/800 for N2O; for CH4 Hayesep
Q) to remove CO2 and thus to avoid cross interference of N2O
detection with CO2 in sample air [37]. CO2 and N2O fluxes were
calculated from the linear change of gas concentrations with time.
For calculation of CH4 fluxes a non linear change approach was
used. Fluxes were corrected for air pressure and temperature.
While CH4 and N2O fluxes were calculated over the full incubation
period of 24 h, CO2 flux calculation was based only on the first three
sampling points, as linearity was given only in this period of 9 h.
Further details on measurements, gas chromatographic conditions
and flux calculation are provided by Wu et al. (2010) [38] for CO2
and CH4 and Yao et al. (2010) [39] for N2O.
2.4. Gas measurement
The concentration of microbial biomass C and N were determined in triplicate prior to inhibitor additions using the
fumigation-extraction technique as described by Vance et al. (1987)
[40]. Briefly, the moist samples were fumigated for 24 h with
ethanol-free chloroform. Soil extracts from the fumigated and
control samples were obtained by shaking soil samples with 60 ml
of 0.5 M K2SO4 for 60 min. Extracts were filtered through 0.45-mm
In order to assess the influence of inhibitors on microbial production and consumption of CO2, N2O and CH4 we measured
changes in headspace gas concentration in the T1 incubation bottles at 0, 3, 6, 9 and 24 h. For this purpose, 10 ml of headspace air
was sampled at each time interval. To avoid underpressure, 10 ml of
2.5. Further soil parameters
C. Wang et al. / European Journal of Soil Biology 65 (2014) 7e14
2000
-1
(mg kg SDW)
filters and frozen at 20 C before analysis of extractable dissolved
organic carbon (DOC) and total N [38]. Microbial biomass C and N
were calculated from the difference between extractable C and N
contents in the fumigated and control samples using conversion
factors (kEC ¼ 0.45 and kEN ¼ 0.54) [40,41]. All results were
expressed on an oven-dried soil basis (105 C, 24 h). Soil pH was
measured with triplicated soil samples in distilled water (soil:water
ratio of 1:5) using a combined electrode.
Microbial biomass C
10
a
1500
A
b
1000
500
2.6. Statistical analyses
3. Results
3.1. Physicochemical soil characteristics
Mean values for soil pH at the Wielenbach grassland site were
significantly lower (5.94 ± 0.01) as compared to soil samples taken
from Graswang grassland site (7.33 ± 0.08; P < 0.05). Concentrations of microbial biomass C (MBC) and N (MBN) were on average
by 26% and 128% larger in soil of the Graswang site than in soil of
the Wielenbach site, respectively (Fig. 2, P < 0.05). The C:N ratio of
microbial biomass was significantly higher in soil of the Wielenbach site (15.7 ± 0.75) compared to soil of the Graswang site
(8.5 ± 0.11) (P < 0.01).
3.2. Inhibitors and gross N turnover
(mg kg SDW)
200
B
a
-1
Microbial biomass N
0
Ratio of microbial biomass
C to N
Results were expressed as mean values from three replicates per
site and experimental treatment and evaluated statistically by use
of analysis of variance (ANOVA). Differences in CO2, N2O, CH4 fluxes
and gross N transformation rates between the control, C2H2 and
NaClO3 treatments were determined by multiple comparisons
(Duncan test). Linear correlation analysis was used to investigate
the relationship between gas fluxes and gross ammonification/
nitrification rate. Weighted 95% confidence intervals calculated
were transformed into standard error of the mean (SAS Institute
Inc. 1995).
b
100
0
20
a
C
15
10
b
5
0
Graswang
Wielenbach
Fig. 2. Microbial biomass carbon (MBC) and nitrogen (MBN) in higher altitude grassland site (Graswang) and lower altitude grassland site (Wielenbach) (mean ± 1SD).
Statistical differences (P < 0.05) across different treatment are depicted by different
letters in each panel.
Mean gross rates of ammonification in soil of the Wielenbach
site exceeded the respective rates of the Graswang soil by more
than a factor of two (P < 0.01, Fig. 3 upper panel). Across sites
addition of 10 Pa C2H2 to the headspace significantly increased rates
of gross ammonification rate by 63%. In contrast, addition of
100 mg NaClO3 kg1 fresh soil weight did not significantly affect
gross rates of ammonification (P > 0.05).
No differences in gross rates of nitrification were found between
samples taken from the Wielenbach and Graswang grassland sites
(P > 0.05, Fig. 3 lower panel). However, addition of 10 Pa C2H2 to the
headspace significantly decreased gross nitrification (P < 0.01) for
soil sampled at the Wielenbach site with rates under C2H2 addition
not significantly different from zero. For gross nitrification in soil of
the Graswang site, a similar but non-significant trend was
observed. The addition of NaClO3 did not affect gross nitrification.
this was not statistically significant. Addition of NaClO3 increased
N2O fluxes of soil samples at Wielenbach site by a factor of 5e6.
CH4 uptake was significantly larger for soils of the Graswang site
(0.67 ± 0.16 mg CH4eC kg sdw1 h1) than for soils taken from the
Wielenbach site (0.16 ± 0.10 mg CH4eC kg sdw1 h1) when no
inhibitors were present (Fig. 4). Addition of 10 Pa C2H2 to the
headspace significantly decreased or even completely eliminated
net CH4 uptake. However, no significant effect of NaClO3 on CH4
uptake was found.
In contrast to N2O production and CH4 consumption, no significant positive or negative effect of C2H2 and NaClO3 on microbial
soil respiration was observed, with rates of soil respiration being
comparable across sites (Wielenbach: 7.1 ± 0.8 mg CO2eC kg1 sdw
h1; Graswang: 6.6 ± 0.7 mg CO2eC kg1 sdw h1).
3.3. Inhibitors and CO2, N2O and CH4 fluxes
3.4. N transformation rates and C and N gas exchange
N2O production by the mineral topsoil of both sites varied
between 0.02 and 0.85 mg N2OeN kg sdw1 h1 for all treatments, with rates tending to be higher for soil of the Wielenbach
site (mean value: 0.19 mg N2OeN kg sdw1 h1) than soil of the
Graswang site (mean value: 0.06 mg N2OeN kg sdw1 h1). An ef15
fect of 15NHþ
NO
4 or
3 labeling on N2O fluxes was not observed,
even though soil NHþ
4 concentrations were increased approximately by a factor of five (Fig. 4). Addition of 10 Pa C2H2 to the
headspace tended to decrease soil N2O fluxes at both sites, however
Nitrous oxide fluxes decreased linearly with increasing gross
ammonification for soil samples taken from the Graswang site
(R2 ¼ 0.51, P ¼ 0.03; Fig. 5), whereas such a relationship was not
found for soils of the Wielenbach site (Fig. 5). However, only for soil
of the latter site a significantly positive correlation between N2O
fluxes and gross nitrification was observed (Fig. 5).
Only for soils of the Graswang site, our data indicated that CH4
uptake increased with increasing rates of gross ammonification
(R2 ¼ 0.39, P ¼ 0.07; Fig. 5) but significantly decreased with
a
ab
a
a
a
a
10 Pa C
0.6
H
100 mg NaClO
/kg S DW
0.3
b
a
a
b
a
a
a
1
b
-1
a
5
-1
a
(μg CH4 -C kg SDW h )
0.0
a
CH4 fluxes
(mg N kg SDW day )
-1
-1
Gross nitrification rate
0
3
Control
-1
/kg S DW
b
-1
2
A
-1
B
11
0.9
(μg N2 O-N kg SDWh )
-1
4
Control
10 Pa C H
100 mg NaClO
N2 O Production
6
(mg N kg SDW day )
Gross ammonification rate
C. Wang et al. / European Journal of Soil Biology 65 (2014) 7e14
a
-0.2
ab
b
a
-0.5
-1
b
b
-3
(mg CO2 -C kg SDW h )
-0.8
Wielenbach
a
increasing gross nitrification rates (R2 ¼ 0.43, P ¼ 0.05; Fig. 5).
Microbial respiration rates were neither correlated with other gas
fluxes nor with gross N turnover.
a
a
4.1. Importance of gross autotrophic vs heterotrophic nitrification
We expected that heterotrophic nitrification may account for
part of total gross nitrate production specifically in the slightly
acidic soil of the Wielenbach site (pH ¼ 5.9) rather than in the
neutral soil of the Graswang site (pH ¼ 7.3). This expectation was
based on a series of field studies showing that heterotrophic
nitrification gains in importance or even dominates if soils are more
acidic [14,17,18,42] due to reduced availability of gaseous ammonia
[29,43] However, all of our experiments did not provide any evidence that heterotrophic nitrification is indeed of importance in the
investigated grassland soils. Low concentrations of C2H2 fully
inhibited gross nitrification, so that measured rates were not
significantly different from zero. Furthermore, an inhibitory effect
of NaClO3, probably an inhibitor of heterotrophic nitrification, could
not be demonstrated. Only for the pH-neutral Graswang soils, a
tendency to lower rates of gross nitrification was found under
presence of NaClO3, while such an effect was not detectable at all
for the slightly acidic soils of the Wielenbach site.
For distinguishing gross rates of autotrophic from gross rates of
heterotrophic nitrification, we used low concentrations (10 Pa) of
C2H2 as a well established inhibitor of autotrophic nitrification,
which is thought to not affect heterotrophic nitrification [13,14,29].
In contrast to C2H2, representing an effective and unambiguous
competitive inhibitor of the autotrophic ammonia monooxynase
enzyme, the inhibiting mechanism of NaClO3 is less clear. The hypothesis that chlorate indeed may inhibit heterotrophic nitrification is further supported by the study of Lang (1986) [44], showing
that chlorate blocked nitrification in acid forest soil, which was
characterized by absence of autotrophic nitrifiers. In contrast,
a
a
6.0
3.0
0.0
Graswang
4. Discussion
a
-1
Fig. 3. Acetylene injection and sodium chlorate addition effect on gross ammonification rate (GA) and gross nitrification rate (GN) in soil incubated at Graswang (higher
altitude) and Wielenbach (lower altitude) sites (mean ± 1SD). Statistical differences
(P < 0.05) across different treatment and two sites are depicted by different letters in
each panel.
CO2 production
-1
Graswang
Wielenbach
Fig. 4. Acetylene injection and sodium chlorate addition effect on CO2, N2O, CH4
production of soil incubated at two grassland sites of different altitude to simulate
climate change (mean ± 1SE). Graswang site: high elevation, control; Wielenbach site:
low elevation, climate change treatment. Statistical differences (P < 0.05) across
different treatment are depicted by different letters in each panel.
Schimel et al. (2004) [45] found no effect of chlorate on net rates of
heterotrophic nitrification. However, there were still some studies
which found that chlorate can inhibit autotrophic nitrification
[17,19].
The observed absence of heterotrophic nitrification in our
slightly acidic to neutral grassland soil is contradictory to recent
findings of Müller et al. (2011) [30], who repeatedly analyzed 15N
dynamics in a temperate grassland soil of Central Germany
(pH ¼ 6.2) without application of inhibitors but by use of a 15N
tracing model optimizing kinetic rate parameters of heterotrophic
and autotrophic nitrification. In these studies it was concluded that
heterotrophic nitrification was of similar importance as autotrophic
nitrification, i.e. the oxidation of free soil NHþ
4 to NO3 [31], despite
heterotrophic nitrification could have been promoted by organic
material in applied slurry. Furthermore, there is increasing evidence that heterotrophic nitrification may play a dominant role in
grasslands during freeze-thaw events, also under conditions of high
pH values of around 6e7 [32]. These conflicting results indicate that
pH may be a less powerful predictor of the importance of heterotrophic nitrification in grassland soils than previously thought.
4.2. Implications of observed inhibitor effects for the determination
of gross N turnover
In our study, gross nitrification rates occasionally tended to be
higher than gross ammonification rates, though this was
12
C. Wang et al. / European Journal of Soil Biology 65 (2014) 7e14
-1
-0.1
2
R = 0.07
P > 0.05
2
R = 0.13
P > 0.05
2
R = 0.51
P = 0.03
-1
0.2
-0.4
2
R = 0.39
P = 0.07
0.1
-0.7
0.0
-1.0
0
1
2
3
Gross ammonification
-1
0
1
2
3
Gross ammonification
-1
-1
-1
(mg NH4 -N kg SDW day )
(mg NH4 -N kg SDW day )
0.2
0.3
-0.1
2
R = 0.11
P > 0.05
2
R = 0.37
P = 0.08
-1
(μg N2 O-N kg SDW h )
-1
-1
Soil N2 O production
-1
0.4
CH4 fluxes (μg CH4 -C kg SDW h )
-1
CH4 fluxes (μg CH4 -C kg SDW h )
0.2
-1
(μg N2 O-N kg SDW h )
Soil N2 O production
0.3
0.2
-0.4
2
R = 0.12
P > 0.05
0.1
-0.7
2
R = 0.43
P = 0.05
0.0
-1.0
-4
-1
2
Gross nitrification
-1
5
-1
(mg NO3 -N kg SDW day )
-7
-4
-1
2
5
Gross nitrification
-1
8
-1
(mg NO3 -N kg SDW day )
Graswang
Wielenbach
Graswang
Wielenbach
Graswang
Wielenbach
Graswang
Wielenbach
Fig. 5. The relationship between N2O and CH4 effluxes and gross ammonification and nitrification rates for soil incubated at high altitude (Graswang) and low altitude (Wielenbach)
sites.
statistically not significant. This is often taken as an indication that
the direct oxidation of monomeric organic N compounds via heterotrophic nitrification [46] is a significant source for soil NO
3 [32].
This finding e contradictory to the full inhibition of gross nitrification by C2H2 e left us puzzled since a microbial NO
3 producing
process is needed to explain that nitrification is equal or tended to
be higher than microbial production of NHþ
4 . Though, there was an
explanation with an over exploration of the native soil NHþ
4 pool
[30], this explanation is unlikely for soils in our study, since soil
NHþ
4 concentrations are very low (Table 1). Other reasons which
may explain the conundrum of nitrification: ammonification ratios
>1, while C2H2 is completely inhibiting nitrification, are:
a) C2H2 may not only inhibit autotrophic but also heterotrophic
nitrification. In consequence rates of autotrophic nitrification
are overestimated,
b) NaClO3 is not a suitable inhibitor for heterotrophic (and autotrophic) nitrification and C2H2 partly also blocks heterotrophic
nitrification. This would result in an overestimation of autotrophic and an underestimation of heterotrophic nitrification
c) rates of gross ammonification were underestimated.
An underestimation of gross ammonification may have occurred
in view of the very low soil NHþ
4 concentrations, indicating high
competition for NHþ
4 as substrate for autotrophic nitrifiers, as a
metabolic N source for microbial biomass as well as for enzyme
þ
15
production. Under NHþ
4 limiting conditions added N NH4 may be
immobilized by microbes for microbial growth and formation of
exo-enzymes. A high turnover of immobilized N due to die back and
remineralization of microbial formed biomass, with part of the
remineralization occurring already within 24 h, would violate
principal assumptions of the pool dilution technique. The 15N pool
dilution model of Kirkham and Bartholomew (1954) applied here
does not account for rapid remineralization. Consequently a rapid
immobilization-remineralization of added 15NHþ
4 will result in an
underestimation of gross ammonification [22]. However, the
comparably short incubation period of this study of one day only is
regarded as sufficient to avoid such errors [22], despite such
experimental results for temperate grassland soils are rare [47].
Given the generally short life cycles of microbial biomass and recent
hypotheses and experimental findings about rapid microbial succession in soil directly fed by microbial residues [32], a potential
underestimation of gross ammonification due to rapid remineralization cannot be excluded.
Another potential reason for underestimation of gross ammonification may be incomplete mixing of added 15NH4 with ambient
þ
NHþ
4 , in combination with preferential nitrification of native NH4
over added 15N enriched NHþ
[47].
This
type
of
bias
is
typically
4
occurring in soils with low NHþ
4 concentrations and high nitrification potentials such as the soils investigated here and can be
C. Wang et al. / European Journal of Soil Biology 65 (2014) 7e14
13
avoided by use of C2H2 as an inhibitor of autotrophic nitrification
[47]. In our study rates of gross ammonification were significantly
larger in presence of C2H2 than in absence of C2H2 for the Wielenbach soils, and also tended to be larger in C2H2 than in control
treatments for soil of the Graswang site. Therefore, an underestimation of gross ammonification due to preferential nitrification of
native NHþ
4 indeed may have occurred. Consequently, we recommend determining gross ammonification in this type of soil using a
15
NH4 pool dilution technique in presence of C2H2.
Inhibitors provide an approach to determine gross nitrification
without isotopes. This method is based on the assumption that
under complete inhibition of gross nitrification, the difference between net nitrification with and without the presence of this inhibitor is providing an estimate of gross nitrification [30]. Here, we
provide evidence, that C2H2 is a powerful inhibitor for such approaches in pre-alpine grassland soils, but NaClO3 is not.
acidic pre-alpine montane grassland soil. As a side effect of its
inhibitory effect on autotrophic ammonia oxidation, 10 Pa C2H2
appeared to have minimized the methodological artifact of prefþ
15
15
erential use of native soil NHþ
4 over added NH4 in the applied N
pool dilution technique for determination of gross ammonification.
Consequently, we recommend the use of C2H2 in 15NHþ
4 pool dilution experiments for the determination of gross ammonification
rates mainly under conditions of low ambient NHþ
4 concentrations.
Also, low concentrations of C2H2 effectively inhibited net CH4 uptake in soil either due to the homology between the enzymes
ammonia monooxygenase and CH4 monooxygenase. Therefore, low
concentrations of acetylene may be used in field studies to effectively disentangle CH4 fluxes into production and consumption
pathways.
4.3. Effects of inhibitors in soil-atmosphere exchange of C and N
trace gases
This work was financially supported by the Helmholtz funded
joined Sino-German laboratory ENTRANCE of the Institute of Atmospheric Physics, Chinese Academy of Sciences (IAP-CAS) and the
IMK-IFU. Further funding was received by the German Science
Foundation under contract number BU1173/12-1, by the Helmholtz
TERENO Initiative and by the FORKAST project funded by the
Bavarian Government. We thank Allison Kolar for language
correction.
The addition of C2H2 at 10 Pa as well as the addition of NaClO3 at
a rate of 100 mg kg1 soil did not alter microbial respiration in the
investigated soils (Fig. 4), which is consistent with earlier investigations using grassland and forest soils [20,47,48].
In contrast to microbial respiration, net N2O losses were affected
by the inhibitors. Generally, the contribution of heterotrophic
nitrification to N2O formation may be as negligible as rates of
heterotrophic nitrification are. Consequently, it was plausible that
the C2H2-induced inhibition of ammonia monooxygenase of autotrophic nitrifiers tended to decrease N2O emissions (Fig. 4). Since
N2O fluxes were not fully inhibited by our inhibitor treatments,
denitrification pathways uncoupled from ammonia oxidation [10]
must have significantly contributed to soil N2O losses. This
conclusion is also supported by the comparably weak explanatory
power of the relationships between gross nitrification rates and soil
N2O loss (R2 ¼ 0.12 and 0.37 for the Wielenbach and Graswang sites,
respectively, Fig. 5). Consequently, the increase in N2O emissions in
the NaClO3 treatment observed for soil of the Wielenbach site is
either caused by an unknown inhibitor effect on denitrification (e.g.
on the N2O reductase enzyme activity) or the result of a minor
reduction of nitrite oxidation and associated increased nitrite
reduction to N gases, which was not visible in our 15NO
3 pool
dilution approaches. In this context, it needs to be noted that even
the increased N2O emissions in the Wielenbach soil are more than
two orders of magnitude smaller than corresponding gross nitrate
production rates, so that a tiny reduction in nitrite oxidation due to
NaClO3 would have been more visible as a change in N2O emission
than as reduced gross nitrification rates.
The strongest inhibitor effect found in this study was the C2H2induced reduction of net CH4 uptake (Fig. 4). This observation
confirms earlier work on the inhibitory effect of C2H2 on the
enzyme methane monooxygenase [49]. Similar inhibition effects of
C2H2 on gross nitrification and net CH4 oxidation as observed in this
study may be related to the homology between the enzymes
ammonia monooxygenase and CH4 monooxygenase [50]. Furthermore, CH4 oxidation can be conducted also by some chemoautotrophic ammonia oxidizers and vice versa [51].
5. Conclusions
Combined application of the 15NO
3 pool dilution technique with
the inhibitors C2H2 or NaClO3 proved to be a powerful tool to
distinguish process-specific pathways of nitrification. However, the
inhibitor of NaClO3 did not provide evidence of a significant presence of heterotrophic nitrification neither in neutral nor in slightly
Acknowledgments
References
[1] C. Gurbry-Rangin, G.W. Nicol, J.I. Prosser, Archaea rather than bacteria control
nitrification in two agricultural acidic soils, FEMS Microbiol Ecol. 74 (2010)
566e574.
[2] P.M. Vitousek, R.W. Howarth, Nitrogen limitation on land and in the seas: how
can it occur? Biogeochemistry 13 (1991) 87e115.
[3] P.B. Reich, D.F. Grigal, J.D. Aber, S.T. Gower, Nitrogen mineralization and
productivity in 50 hardwood and conifer stands on diverse soils, Ecology 78
(1997) 335e347.
[4] C. Müller, R.J. Stevens, R.J. Laughlin, A 15N tracing model to analyse N transformations in old grassland soil, Soil Biol. Biochem. 36 (2004) 619e632.
[5] B.A. Hungate, J.S. Dukes, M.R. Shaw, Y.Q. Luo, C.B. Field, Nitrogen and climate
change, Science 302 (2003) 1512e1513.
[6] K.S. Pregitzer, A.J. Burton, D.R. Zak, A.F. Talhelm, Simulated chronic nitrogen
deposition increases carbon storage in Northern Temperate forests, Glob.
Change Biol. 14 (2008) 142e153.
[7] S. Korsaeth, T.M. Henriksen, L.R. Bakken, Temperal changes in mineralization
and immobilization of N during degradation of plant material: implications for
the plant N supply and nitrogen losses, Soil Biol. Biochem. 34 (2002) 789e799.
[8] S. Zechmester-Boltenstern, M. Hahn, S. Meger, R. Jandl, Nitrous oxide emissions and nitrate leaching in relation to microbial biomass dynamics in a
beech forest soil, Soil Biol. Biochem. 34 (2002) 823e832.
[9] J.E.T. McLain, D.A. Martens, N2O production by heterotrophic N transformations in a semiarid soil, Appl. Soil Ecol. 32 (2006) 253e263.
[10] K. Butterbach-Bahl, P. Gundersen, P. Ambus, J. Augustin, C. Beier, P. Boeckx,
M. Dannenmann, B.S. Gimeno, R.M. Rees, K.A. Smith, C. Stevens, T. Vesala,
S. Zechmeister-Boltenstern, Nitrogen processes in terrestrial ecosystems, in:
M.A. Sutton, C.M. Howard, J.W. Erisman, G. Billen, A. Bleeker, P. Grennfeldt,
H. Van Grinsen, B. Grozetti (Eds.), The European Nitrogen Assessment: Sources
Effects, and Policy Perspectives, Cambridge University Press, 2011, pp.
99e125.
[11] a) K. Butterbach-Bahl, E.M. Baggs, M. Dannenmann, R. Kiese, S. ZechmeisterBoltenstern, Nitrous oxide emissions from soils, how well do we understand
the processes and their controls, Philos. Transac. Royal Soc. B e Biol. Sci. 368
(2013) 1621, http://dx.doi.org/10.1098/rstb.2013.0122;
b) J. Bauhus, A.C. Meyer, R. Brumme, Effects of the inhibitors nitrapyrin and
sodium chlorate on nitrification and N2O formation in an acid forest soil, Biol.
Fert. Soils 22 (1996) 318e325.
[12] H. Barraclough, G. Puri, The use N-15 pool dilution and enrichment to separate
the heterotrophic and autrotrophic pathways of nitrification, Soil Biol. Biochem. 27 (1995) 17e22.
[13] H. Pedersen, K.A. Dunkin, M.K. Firestone, The relative importance of autotrophic and heterotrophic nitrification in a conifer forest soil as measured by
15
N tracer and pool dilution techniques, Biogeochemistry 44 (1999) 135e150.
[14] W.R. Cookson, C. Müller, P.A. O'Brien, D.V. Murphy, P.F. Grierson, Nitrogen
dynamics in an Australian semiarid grassland soil, Ecology 87 (2004)
2047e2057.
[15] J.A. Adams, Identification of heterotrophic nitrification in strongly acid larch
humus, Soil Biol. Biochem. 18 (1986) 339e341.
14
C. Wang et al. / European Journal of Soil Biology 65 (2014) 7e14
[16] P.I. Pennington, R.C. Ellis, Autotrophic and heterotrophic nitrification in acidic
forest and native grassland soils, Soil Biol. Biochem. 25 (1993) 1399e1408.
[17] D.L. Tong, R.K. Xu, Effects of urea and (NH4)2SO4 on nitrification and acidification of ultisols from Southern China, J. Environ. Sci. 24 (2012) 682e689.
[18] L.W. Belser, E.L. Mays, Specific inhibition of nitrite oxidation by chlorate and
its use an assessing nitrification in soils and sediments, Appl. Environ.
Microbiol. 39 (1980) 505e510.
[19] T.Y. Zhang, X.K. Xu, X.B. Luo, L. Han, Y.H. Wang, G.X. Pan, Effects of acetylene at
low concentrations on nitrification, mineralization and microbial biomass
nitrogen concentration in forest soils, Chin. Sci. Bull. 54 (2009) 296e303.
[20] E.A. Davidson, S.C. Hart, M.K. Firestone, Internal cycling of nitrate in soils of a
mature coniferous forest, Ecology 73 (1992) 1148e1156.
[21] D.V. Murphy, S. Recous, E.A. Stockdale, I.R.P. Fillery, L.S. Jensen, D.J. Hatch,
K.W.T. Goulding, Gross nitrogen fluxes in soil: theory, measurement and
application of 15N pool dilution techniques, Adv. Agron. 79 (2003) 69e118.
[22] M.S. Booth, J.M. Stark, E. Rastetter, Controls on nitrogen cycling in terrestrial
ecosystems: a synthetic analysis of literature data, Ecol. Monogr. 75 (2005)
139e157.
[23] R.K. Hynes, R. Knowles, Inhibition of chemoautotrophic nitrification by sodium chlorate and sodium chlorite: a reexamination, Appl. Environ. Microbiol.
45 (1983) 1178e1182.
[24] K. Killham, Heterotrophic nitrification, in: J.I. Prosser (Ed.), Nitrification, Special Publications for Society for General Microbiology, vol. 20, IRL Press, Oxford, 1987, pp. 117e126.
[25] R. Conrad, Microbiological and biochemical background of production and
consumption of NO and N2O in soil, in: R. Gasche, H. Papen, H. Rennenberg
(Eds.), Trace Gas Exchange in Forest Ecosystems, Kluwer Academic Publishers,
Dordrecht, 2002, pp. 3e33.
[26] A. Bollmann, R. Conrad, Acetylene blockage technique leads to underestimation of denitrification rates in oxic soils due to scavenging of intermediate
nitric oxide, Soil Biol. Biochem. 29 (1997) 1067e1077.
[27] M. Matsushima, W.K. Choi, K. Inubushi, Nitrification inhibitor reduces nitrous
oxide production from different soil profiles of an Andosol soil, Commun. Soil
Sci. Plan. 40 (2009) 3181e3193.
[28] M. Kuroiwa, K. Koba, K. Isobe, R. Tateno, A. Nakanishi, Y. Inagaki, H. Toda,
S. Otsuka, K. Senoo, Y. Suwa, M. Yoh, R. Urakawa, H. Shibata, Gross nitrification
rates in four Japanese forest soils: heterotrophic versus autotrophic and the
regulation factors for the nitrification, J. For. Res. 16 (2011) 363e373.
[29] J.M. Norton, J.M. Stark, Regulation and measurement of nitrification in
terrestrial systems, in: M.G. Glotz (Ed.), Methods in Enzymology, vol. 486,
Academic Press, Burlington, 2011, pp. 343e368.
[30] C. Müller, R.J. Laughlin, P. Christie, P. Christie, C.J. Watson, Effects of repeated
fertilizer and cattle slurry application over 38 years on N dynamics in a
temperate grassland soil, Soil Biol. Biochem. 43 (2011) 1362e1371.
[31] A.K. Herrmann, E. Witter, T. K€
atterer, Use of acetylene as a nitrification inhibitor to reduce biases in gross N transformation rates in a soil showing rapid
disappearance of added ammonium, Soil Biol. Biochem. 39 (2007)
2390e2400.
[32] H.H. Wu, M. Dannenmann, B. Wolf, X.G. Han, X.H. Zheng, K. Butterbach-Bahl,
Seasonality of soil microbial nitrogen turnover in continental steppe of Inner
Mongolia-insights from a full year dataset of gross and net nitrogen turnover,
Ecosphere 3 (2012). Article 34.
[33] L. Breuer, R. Kiese, K. Butterbach-Bahl, Temperature and moisture effects on
nitrification rates in tropical rain forest soils, Soil Sci. Soc. Am. J. 66 (2002)
834e844.
[34] D. Kirkham, W.V. Bartholomew, Equations for following nutrient transformations in soil utilizing tracer data, Soil Sci. Soc. Am. Pro 18 (1954) 33e34.
[35] M. Dannenmann, R. Gasche, A. Ledebuhr, H. Papen, Effects of forest management on soil N cycling in beech forests stocking on calcareous soils, Plant
Soil 287 (2006) 279e300.
€gel[36] M. Dannenmann, J. Simon, R. Gasche, J. Holst, R. Pena, P.S. Naumann, I. Ko
Knabner, H. Knicker, H. Mayer, M. Schloter, A. Polle, H. Rennenberg, H. Papen,
Tree girdling provides insight on the role of labile carbon in nitrogen partitioning between soil microorganisms and adult European beech, Soil Biol.
Biochem. 41 (2009) 1622e1631.
[37] X.H. Zheng, B.L. Mei, Y.H. Wang, B.H. Xie, Y.S. Wang, H.B. Dong, et al., Quantification of N2O fluxes from soil plant systems may be biased by the applied
gas chromatograph methodology, Plant Soil 311 (2008) 211e234.
[38] X. Wu, Z.S. Yao, N. Brüggemann, Z.Y. Shen, B. Wolf, M. Dannenmann, X. Zheng,
K. Butterbach-Bahl, Effects of soil moisture and temperature on CO2 and CH4
soil-atmosphere exchange of various land use/cover types in a semi-arid
grassland in Inner Mongolia, China, Soil Biol. Biochem. 42 (2010) 773e787.
[39] Z.S. Yao, X. Wu, B. Wolf, M. Dannenmann, K. Butterbach-Bahl, N. Brüggemann,
W. Chen, X. Zheng, Soil-atmosphere exchange potential of NO and N2O in
different land use types of Inner Mongolia, as affected by soil temperature, soil
moisture, freeze-thaw and dryingerewetting events, J. Geophys. Res. 115
(2010) D17116, http://dx.doi.org/10.1029/2009JD013528.
[40] E.D. Vance, P.C. Brookes, D.S. Jenkinson, An extraction method for measuring
soil microbial biomass C, Soil Biol. Biochem. 19 (1987) 703e707.
[41] P.C. Brookes, A. Landman, G. Pruden, D.S. Jenkinson, Chloroform fumigation
and the release of soil nitrogen: a rapid direct extraction method to measure
microbial biomass nitrogen in soil, Soil Biol. Biochem. 17 (1985) 837e842.
[42] H. Papen, R. von Berg, A Most Probable Number method (MPN) for the estimation of cell numbers of heterotrophic nitrifying bacteria in soil, Plant Soil
199 (1998) 123e130.
[43] E.P. Serjeant, B. Dempsey, Ionization Constants of Organic Acids in Solution,
IUPAC Chemical Data Series No. 23, Pergamon Press, Oxford, UK, 1979.
[44] E. Lang, Heterotrophe und autotrophe Nitrifikation untersucht and Bodenproben von drei Buchenstandorten, Gottinger Bodenkd. Ber. 89 (1986)
199e210.
[45] J.P. Schimel, J. Bennett, Nitrogen mineralization: challenges of changing
paradigm, Ecology 85 (2004) 591e602.
[46] A. Islam, D. Chen, R.E. White, Heterotrophic and autotrophic nitrification in
two acid pasture soils, Soil Biol. Biochem. 39 (2007) 972e975.
[47] J.P. Malone, R.J. Stevenes, R.J. Laughlin, Combine the 15N and acetylene inhibition techniques to examine the effect of acetylene on denitrification, Soil
Biol. Biochem. 30 (1998) 31e37.
[48] K.F. Bronson, A.R. Mosier, Suppression of methane oxidation in aerobic soil by
nitrogen fertilizers, nitrification inhibitors, and urease inhibitors, Biol. Fert.
Soils 17 (1994) 263e268.
[49] K. Urmann, M.H. Schroth, J. Zeyer, Recovery of in-situ methanotrophic activity
following acetylene inhibition, Biogeochemistry 89 (2008) 347e355.
[50] P.L.E. Bodelier, P. Frenzel, Contribution of methanotrophic and nitrifying
bacteria to CH4 and NHþ
4 oxidation in the rhizosphere of rice plants as
determined by new methods of discrimination, Appl. Environ. Microbiol. 65
(1999) 1826e1833.
[51] R.D. Jones, R.Y. Morita, Methane oxidation by nitrosococcus-oceanus and
nitrosomonas-europaea, Appl. Environ. Microbiol. 45 (1983) 401e410.