Seasonality of soil microbial nitrogen turnover
in continental steppe soils of Inner Mongolia
H. WU,1,2,3 M. DANNENMANN,1,4, B. WOLF,1 X. G. HAN,2,5 X. ZHENG,6
AND
K. BUTTERBACH-BAHL1
1
Karlsruhe Institute of Technology (KIT), Institute for Meteorology and Climate Research (IMK-IFU),
Kreuzeckbahnstrasse 19, 82467 Garmisch-Partenkirchen, Germany
2
State Key Laboratory of Forest and Soil Ecology, Institute of Applied Ecology, Chinese Academy of Sciences,
Shenyang 110016 China
3
State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences,
Xiangshan, Beijing 100093 China
4
Institute of Forest Botany and Tree Physiology, University of Freiburg, Freiburg im Breisgau 79110 Germany
5
Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016 China
6
State Key Laboratory of Atmospheric Boundary Layer Physics and Atmospheric Chemistry (LAPC),
Institute of Atmospheric Physics, Chinese Academy of Sciences (CAS), Beijing 100029 China
Citation: Wu, H., M. Dannenmann, B. Wolf, X. G. Han, X. Zheng, and K. Butterbach-Bahl. 2012. Seasonality of soil
microbial nitrogen turnover in continental steppe soils of Inner Mongolia. Ecosphere 3(4):34. http://dx.doi.org/10.1890/
ES11-00188.1
Abstract. Annual estimates and seasonal patterns of gross nitrogen turnover in terrestrial soils are poorly
understood due to the lack of experimental evidence. Based on year round sampling in wintergrazed and
ungrazed steppe soils of Inner Mongolia, we show that measurements of net rates of ammonification (9 to
6 kg N ha1 year1) or nitrification (19 to 31 kg N ha1 year1) do not at all reflect the pronounced dynamics
of gross rates of ammonification (215–240 kg N ha1 year1) or nitrification (362–417 kg N ha1 year1).
Four different seasons with characteristic functional patterns of N turnover were identified: (1) Growing
season dynamics as characterized by drying/rewetting cycles and negatively correlated temporal courses of
net microbial growth and periods with intensive gross ammonification, contributing 40–52% and 29–32%
to cumulative annual gross ammonification and nitrification, respectively. Net N mineralization was
almost exclusively observed during the growing season. (2) Microbial N dynamics during the autumn
freeze-thaw period was characterized by a sharp decline in microbial biomass in conjunction with a peak of
gross nitrification contributing 19–36% to cumulative annual fluxes. (3) During winter at constantly frozen
soil, a net build-up of microbial biomass was observed, whereas gross N turnover rates were low,
contributing 7–10% and 6–11% to cumulative annual gross ammonification or gross nitrification,
respectively. (4) The spring freeze-thaw period showed extremely dynamic changes in gross N turnover
and soil nitrate concentrations. This period contributed 34–44% and 21–46% to cumulative annual gross
ammonification and nitrification, respectively. This study highlights that freeze-thaw cycles are key periods
for understanding patterns and magnitudes of gross N turnover in semi-arid continental steppe
ecosystems. The results further imply that the observed patterns of microbial biomass and gross N
turnover dynamics are likely the consequence of a seasonal succession of microbial communities and
turnover of microbial biomass. Our findings emphasize the necessity for high resolution studies on gross N
turnover as a prerequisite to infer functioning and annual budgets of ecosystem N cycling.
Key words: annual nitrogen budget; drying-rewetting; freeze-thaw; microbial biomass; nitrification; N mineralization;
steppe.
Received 30 June 2011; revised 17 January 2012; accepted 24 January 2012; final version received 21 March 2012;
published 27 April 2012. Corresponding Editor: S. Frey.
Copyright: Ó 2012 Wu et al. This is an open-access article distributed under the terms of the Creative Commons
Attribution License, which permits restricted use, distribution, and reproduction in any medium, provided the original
author and sources are credited.
E-mail: [email protected]
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WU ET AL.
temporal resolution of these studies is strongly
limited. The few available studies examining the
temporal variability of gross N turnover in soils
(e.g., Dannenmann et al. 2006, Rosenkranz et al.
2010) show that rates may markedly fluctuate
across seasons. Hence, the available studies on
gross N turnover in general neither allow for
understanding seasonal dynamics or for calculating annual rates of gross N turnover. Actual
gross rates of N turnover in soil subjected to
winter conditions have only been determined a
few times in laboratory studies (Müller et al.
2002, Ludwig et al. 2004, Freppaz et al. 2007), but
not in situ. The latter studies determined
significant rates of gross ammonification and/or
nitrification in soil subjected to freeze-thaw
events, though still being restricted to single
point in time measurements. So far, no study has
investigated gross N turnover in permanently
frozen soil. However, based on the investigation
of more indirect parameters of N turnover such
as soil mineral N concentrations, several studies
showed that significant biogeochemical C and N
turnover can occur in frozen soils and during
freeze/thaw periods (Vogt et al. 1986, Clein and
Schimel 1995, Brooks et al. 1999). Soil microbial
N turnover may occur in winter under snowpack
in the soil, as indicated e.g., by measurements of
significant net N turnover rates in continental
steppe of China (Zhou et al. 2009, Zhao et al.
2010), boreal forests (Kielland et al. 2006), arctic
tundra (Schimel et al. 2004), and temperate
hardwood forests (Groffman et al. 2001b). Furthermore, microbial biomass and the activities of
several soil enzymes were even found to peak in
late winter in alpine soils (Lipson et al. 1999,
2002). Furthermore, significant plant litter decomposition was found in winter in seasonally
snow-covered ecosystems (Taylor and Jones
1990, Hobbie and Chapin 1996, Schmidt and
Lipson 2004).
Soil freeze-thaw cycles have been reported to
kill a significant portion of the soil microbial
biomass (Clein and Schimel 1995, Schimel and
Clein 1996, DeLuca et al. 2002). The resulting
increase of easily degradable N and C substrates
in soil during spring snowmelt has been shown
to prime fine root turnover, increase net N
mineralization (Groffman et al. 2001a, Schmidt
et al. 2007, Matzner and Borken 2008), nutrient
leaching (Brooks et al. 1999), and N2O emissions
INTRODUCTION
Biogeochemical nitrogen (N) cycling in terrestrial ecosystems is complex, since it comprises
many players ranging from microorganisms to
higher plants performing and involving a wide
range of processes with very different magnitudes of N turnover (Schimel and Bennett 2004,
Booth et al. 2005, Kreutzer et al. 2009, Rennenberg et al. 2009, Butterbach-Bahl et al. 2011). The
terrestrial N cycle is dominated by the soil
microbial N turnover processes of ammonification (the conversion of organic N compounds to
ammonium), nitrification (conversion of organic
N or ammonium to nitrate), and the subsequent
allocation of bioavailable N such as ammonium
and nitrate to plants and microorganisms as well
as to N loss pathways. Soil nitrogen cycling
regulates plant productivity, and ecosystem N
retention and loss. Hence, N cycling affects soil
acidification, stream water quality, and eutrophication, as well as atmospheric chemistry and
radiative forcing (Galloway et al. 2008, Butterbach-Bahl et al. 2011). Consequently, the ecological significance of N turnover processes has been
well acknowledged. However, due to its complexity and methodological difficulties in the
quantification of actual soil N turnover processes,
our understanding of soil N cycling is still
fragmentary. The current state of knowledge on
N cycling may correspond to the state of
knowledge on the carbon (C) cycle several
decades ago (Schlesinger 2009).
In the past most studies focused on measurements of net rates of ammonification and
nitrification across a wide variety of terrestrial
ecosystems. However, since net rates comprise
both production and consumption of inorganic
N, such studies do not necessarily provide
insight into actual gross rates of N turnover.
Therefore, they are a poor approximation of the
real N status of ecosystems (Davidson et al. 1991,
1992, Schimel and Bennett 2004). Since the
determination of gross rates of ammonification
and nitrification is based on time- and resourceintensive techniques mostly involving the use of
stable 15N isotopes, actual gross rates of N
turnover have been determined less often than
net rates. Nevertheless, studies on gross N
turnover are available for a range of terrestrial
ecosystems (Booth et al. 2005), though the
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WU ET AL.
from soil (Wolf et al. 2010).
The knowledge gap on gross N turnover in
snow covered/and or frozen soils still hampers a
functional and quantitative understanding of soil
N biogeochemistry at the annual scale. In
particular, it remains uncertain whether our
understanding of the contribution of the winter
season to annual N flux is valid, as estimated
from dynamics and magnitude of measurements
of net rates of N turnover, enzyme activities and
microbial community parameters.
The Inner Mongolia grassland, part of the
Eurasian Steppe, is the largest contiguous grassland area in the world (Bai et al. 2004) covering
approx. 8% of the Earth’s land surface. Rapid
degradation and desertification have taken place
in these grasslands, primarily caused by overgrazing that reduced vascular plant cover,
accelerated soil loss, and decreased soil nutrient
level (Graetz 1994, Kang et al. 2007). Few studies
on gross N turnover are available for such
ecosystems (Holst et al. 2007, Wu et al. 2011),
and data that are available were based on a few
measurements made during the growing seasons
only and thus cannot provide insight into the
temporal dynamics and environmental controls
of N turnover at the annual scale. Hence, a
functional understanding of the importance of
the winter and freeze-thaw periods for annual
soil N turnover has not yet been achieved for
semi-arid continental steppe.
Sheep grazing could either decrease or increase
gross ammonification, nitrification and nitrate
concentrations in such soils (Holst et al. 2007, Wu
et al. 2011). However, also these grazing studies
were based on only one or few measurements
during the growing season, and were additionally not supported by plot replication. In the light
of the observation, that spring thaw is triggering
N2O pulse emissions in steppe soils, which
decrease with increasing grazing intensity (Wolf
et al. 2010), it is essential to also understand the
full annual course of N turnover and the
dynamics of soil mineral N and microbial
biomass concentrations as influenced by grazing.
Therefore, we measured gross and net N
turnover as well as dynamics of soil inorganic N
and microbial biomass concentrations in monthly
or bi-weekly temporal resolution over an entire
year at replicated plots of either winter grazed or
ungrazed steppe. The aim of this study was to
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characterize and quantify the temporal dynamics,
and season-specific patterns, as well as obtaining
annual estimates of microbial N turnover over a
full year. In particular, we tested the following
hypotheses: (1) There is significant gross N
turnover in frozen steppe soils, (2) both N
turnover during the growing season as well as
during freeze-thaw periods are of major importance for the annual budget of gross N fluxes; (3)
grazing decreases microbial N turnover due to
reduced plant cover and altered soil microclimate.
Furthermore, we were interested in elucidating
the significance of potential environmental controls (temperature, soil moisture, microbial biomass) on gross N turnover and to investigate
whether net rates of N turnover provide insight
into the magnitude and dynamics of gross N
turnover over an annual cycle.
MATERIAL
AND
METHODS
Study site description
Our study was carried out in the Xilin River
Basin, Inner Mongolia Autonomous Region,
China (43838 0 N, 116842 0 E). The mean annual
temperature is 0.38C with mean monthly temperatures ranging from 21.68C in January to
19.08C in July. Mean annual precipitation is 346
mm (1984–2005) with 60–80% falling during the
growing season from May to August. Only 10%
of annual precipitation occurs during the winter
months as snow. The soil is classified as
alkalescent Phaeozem with a loamy sand texture
(Steffens et al. 2008). We investigated three
ungrazed and three grazed plots (1 ha each) of
Leymus chinensis grassland. The ungrazed plots
(UG) were fenced in 1999 to prevent grazing by
large animals. The winter grazed plots (WG)
were fenced and grazed in the winter period
from November to April by 3–4 sheep units per
hectare, representing a heavy grazing intensity.
Further site information is given by Wolf et al.
(2010).
Experimental and sampling design
Soil sampling was conducted at the three
ungrazed and grazed plots by use of soil cores
of 5 cm diameter and 10 cm depth. At each of the
six investigated plots, ten sampling spots were
randomly selected over the entire plot every
sampling date. For the determination of gross
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WU ET AL.
were determined using an in situ 15N pool
dilution technique described previously by Dannenmann et al. (2006) and Wolf et al. (2010). The
experimental procedure was adapted to the
logistical and climatic conditions at the remote
experimental site. Samples bulked from the 10
cores were sieved (5 mm mesh width) and
homogeneously sprayed with (NH4 )2SO4 or
KNO3 solution at 30 atom% 15N enrichment
(Dannenmann et al. 2009) on the day of
sampling. Label application increased soil water
content by 3% and the total NH4þ-N- or NO3-N
content by approximately 1 mg N kg1 sdw,
which is generally well below the measured
mean pool sizes in unlabelled soil (compare Fig.
1F, G). Six subsamples of 30 g each for every
labelling treatment and plot were placed in
parafilm-sealed plastic bottles (250 ml volume)
and buried in situ close to the sampling location
after labelling. Eighteen hours (time 1) and 42
hours after labelling (time 2), half of the bottles,
i.e., three bottles per labelling treatment and plot,
were excavated and immediately extracted with
60 ml 1 M KCl as described by Dannenmann et
al. (2006). Diffusion steps for trapping on acid
filter traps and subsequent GC-IRMS analyses for
15
N-enrichment were performed as described
earlier (Dannenmann et al. 2009). Total NH4þ
and NO3 concentrations in the extracts were
determined at Inner Mongolia Grassland Ecosystem Research Station (IMGERS) as described
below. For frozen soil conditions, a different 15N
labelling technique was applied (Wolf et al.
2010). Here, the labelling solution was amended
with triple hot washed and autoclaved quartz
sand at a ratio of 1:2.5, frozen, and subsequently
crushed to a fine powder. This 15N-labelled
quartz-sand/ice mixture was homogenously
mixed with the still frozen soil for labelling. All
experimental steps until the amendment of the
KCl solution for soil extraction were performed
outdoors at air temperatures below 108C in
order to ensure that the investigated soil and the
labelling solution/quartz sand mixture remained
constantly frozen. Incubations took place in situ
in the plots by burying the incubation bottles in
winter and by maintaining a representative snow
cover, if applicable.
Calculation of rates of gross ammonification
and nitrification were based on the equations
given by Kirkham and Bartholomew (1954). We
rates of ammonification and nitrification, one soil
core was taken per sampling spot, and subsequently the soil of the 10 cores of every plot was
bulked for the 15N experiments, which immediately started after sampling. In order to avoid the
necessity for soil storage, separate soil samples
were used for determination of net rates of N
turnover, soil mineral N concentrations and
microbial biomass C and N. Samples for these
analyses were taken at the same sampling spots
after 15N experiments were finished, i.e., with a
time shift of approximately five days. For this
purpose, two paired soil cores were inserted at
every sampling spot (i.e., 20 cores at each plot).
One of the paired cores was immediately
sampled and analyzed for soil mineral N
concentrations as well as microbial biomass C
and N. The second core was covered with plastic
film that allowed gas exchange but prevented
water penetration and was analyzed for soil
mineral N concentrations after an in situ incubation period of two weeks in order to facilitate the
determination of net rates of N turnover. Inorganic N concentrations, net rates of N turnover
and soil microbial biomass were analyzed separately in the spatial replicates, i.e., samples were
not bulked. All soil samples were immediately
processed after sampling in order to avoid
storage artifacts. Both paired intact soil cores
for net rate assays and homogenized soil samples
for the determination of gross N turnover were
incubated in situ at the sampling spots. Soil
inorganic N concentrations, net rates of N
turnover, microbial biomass C and N and gross
ammonification were measured 20–21 times at 8–
38 day intervals from August 16, 2007 to October
13, 2008. On 15 occasions, gross nitrification was
also determined simultaneously to gross ammonification.
Based on analysis of variance of the results of
the first four sampling dates, the spatial replicates were reduced from ten to eight (net rates of
N turnover, soil mineral N concentrations) and to
five (microbial biomass C and N). Overall, net
rates of N turnover and soil mineral N concentrations were determined for 952 samples and
microbial biomass C and N were determined for
618 samples.
Gross rates of microbial N turnover
Gross rates of ammonification and nitrification
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did not calculate microbial NH4þ immobilization
rates from 15N consumption rates (Davidson et
al. 1991), since our data indicated the presence of
heterotrophic nitrification (direct oxidation of
organic N to NO3 without dilution of 15NH4þ).
Similarly, NO3 immobilization was not considered to equal 15NO3 consumption, since the
contribution of denitrification to NO3 consumption remained unclear. In this context, the
observations of dry soil and low N2O emissions
may be insufficient criteria to conclude that
dinitrogen emissions and total denitrification
are negligible for the NO3 mass balance
(Dannenmann et al. 2011).
area, 10 cm depth) were obtained by use of a
spate. One half was immediately extracted and
analyzed as described above. The second half
was packed into polyethylene bags with pinholes
(Dannenmann et al. 2006) and reburied for the
second extraction two weeks later. If applicable,
the incubated soil was covered with snow of a
representative height. In winter, soil samples
were kept frozen during transport from the field
sites to a nearby provisional laboratory until
extraction.
Microbial biomass C and N
Microbial biomass C and N was estimated
using the chloroform fumigation-extraction (FE)
method (Brookes et al. 1985, Vance et al. 1987,
Tate et al. 1988) as described in detail by
Dannenmann et al. (2006) and Wu et al. (2011).
After removal of coarse organic materials and
stones, samples were divided into paired subsamples of 30 g each. One subsample was
immediately extracted with 60 ml 0.5 M K2SO4
while the second subsample was fumigated
under chloroform vapour for 24 h in a desiccator.
Subsequently, ten vacuum/release purge cycles
ensured the complete removal of chloroform, and
fumigated subsamples were extracted as described above. Extracts were filtered using a
0.45 lm syringe filter (Schleicher and Schuell,
Dassel, Germany) and immediately frozen until
analysis for total organic carbon (TOC) and total
chemically bound nitrogen (TNb) using a TOC
analyzer with a coupled TNb module (Dimatec
Analysentechnik GmbH, Essen, Germany). Total
carbon (TC) and total inorganic carbon (TIC)
were determined based on non-dispersive infrared photometrical detection of evolving CO2 after
thermic and chemical oxidation of the samples.
TOC was calculated as TC TIC. TNb was
analyzed by use of a chemoluminescence detector. Correction factors (0.54 for microbial biomass
N and 0.379 for microbial biomass C; Brookes et
al. 1985, Vance et al. 1987) were applied to the
difference in total extractable N and TOC
between paired untreated and fumigated subsamples to estimate microbial biomass C and N.
Net rates of N turnover and
mineral N concentrations
Net rates of N turnover were determined
according to the paired soil core method described by Wang et al. (2006). One of the paired
soil cores was immediately harvested, soil was
removed from the core and organic debris and
rocks were separated from the soil. Representative subsamples were analysed for inorganic N
concentrations and microbial biomass C and N
(see below). Soil extraction for mineral N
concentrations was conducted with 30 g of soil
by use of 1 M KCl (soil to solution ratio 1:2) as
described in detail by Dannenmann et al. (2006).
Concentrations of inorganic N (NH4þ-N and
NO3-N) in the filtered extracts were conducted
at IMGERS using a flow injection autoanalyzer
(Zhou et al. 2009; FIAstar 5000 Analyzer, Foss
Tecator, Denmark). Soil water content was
determined gravimetrically at 1058C for 24 h.
After two weeks, the remaining soil cores were
harvested, extracted and analyzed for mineral N.
The NH4þ-N and NO3-N concentrations from
the first harvesting date are referred to in situ soil
ammonium and nitrate concentrations (Fig.
1F, G). Net rates of ammonification and nitrification (Fig. 1H, I) were calculated from the
difference between the two sampling times
(Wang et al. 2006).
Under frozen soil conditions, the use of the
polyvinyl chloride plastic (PVC) soil cores was
not possible. Here, an in situ incubation buried
bag technique (Dannenmann et al. 2006) was
used for the determination of net N turnover
instead of the soil core technique. For this
purpose, paired intact portions of soil (50 cm2
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Meteorological data
Soil temperature at 0.05 m depth was recorded
continuously in the plots with PT100 thermometers (Th2-h, UMS GmbH, Muenchen, Germany)
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WU ET AL.
at one minute intervals. Soil moisture was
recorded with the same frequency using three
FD probes (ECH2O-5, Decagon Devices, Pullman, WA, USA) per site. The FD sensors were
installed in a way that they integrated soil
moisture over a depth of 0–0.05 m. During
wintertime when soil temperatures dropped
below 08C, soil samples from 0–0.05 m soil depth
were taken by means of 100 ml core cutters at
least twice a week. The samples were dried in the
oven at a temperature of 1058C for 24 hours in
order to determine volumetric water content.
Precipitation data was provided by the IMGERS
station in daily resolution.
values, precipitation events during three consecutive days in mid September increased soil
moisture again to almost 30 vol%, followed again
by a rapid drying out of soil in the last week of
September. Following these two drying/rewetting cycles, soil temperatures declined slowly
towards 08C. The first freeze-thaw event in
topsoil took place in the first week of October
(Fig. 1A). It needs to be noted, that the first night
time freeze events in the uppermost cm of the
topsoil occurred at temperatures well above 08C
as given in Fig. 1, since the presented temperature is daily mean temperature at 5 cm soil depth.
From mid November 2007 to the beginning of
March 2008 the soil down to at least 15 cm was
permanently frozen.
The spring-thaw period with frequent freezethaw events lasted from the beginning of March
until the beginning of May. The beginning of this
period is clearly marked by a sharp increase in
soil moisture, which was more pronounced in
UG as compared to WG plots. Following these
spring freeze-thaw cycles, the growing season of
the year 2008 was characterized by pronounced
soil drying-rewetting cycles as driven by episodic
strong convective rainfall events (Fig. 1A).
Statistics and calculations
In general, the plot mean values were used as
the statistical unit in this study (n ¼ 3 for grazed
and n ¼ 3 for ungrazed). The Wilcoxon test was
applied to test for significant differences between
WG and UG at a given sampling date. For the
calculation of cumulative annual N turnover on
an area basis, gross and net N turnover was
calculated on a daily basis by linear interpolation
considering the bulk density of the uppermost 10
cm of soil. Thus, the given cumulative curves of
N turnover are representative for the uppermost
10 cm of the soil.
Pearson’s correlation coefficients were calculated to illustrate dependency between N fluxes and
potential controls. This was done (1) using data
from specific seasons only (growing season,
freeze-thaw periods, winter), and (2) using the
whole annual dataset. When several potential
environmental controls correlated significantly
with N turnover rates at the annual scale,
multiple linear regression models with a forward
stepwise procedure were used to identify dominant environmental controls of N turnover
processes. The threshold value for significant
correlations or differences was set at P , 0.05. All
statistical analyses were performed with SPSS
10.0 (SPSS, Chicago, USA).
Temporal dynamics of gross and N turnover
and microbial biomass
Soil microbial biomass N as well as N turnover
were characterized by pronounced temporal
dynamics between and within seasons over the
investigated time span of 14 months (Fig. 1).
During the growing season, microbial biomass
oscillated along with drying-rewetting cycles
(range approx. 20 to 100 mg N kg1 sdw; Fig.
1D). After the first topsoil freeze events in
autumn, soil microbial biomass declined dramatically by more than 80% (Fig. 1D), while at the
same time the microbial C:N ratio increased by
approximately a factor of two (Fig. 1E). After an
initial strong increase in winter, microbial biomass N declined in the beginning of January 2008
in conjunction with an intensification of soil frost,
i.e., a drop in soil temperatures to approx. 108C
at the ungrazed plots and to approx. 158C at the
wintergrazed plots (Fig. 1A, D). Subsequently,
the sudden increase in soil moisture due to
spring thaw events coincided with increases in
soil microbial biomass N.
Gross ammonification (Fig. 1B) during the
RESULTS
Meteorological data
The start of our study in August 2007 was
characterized by a sharp decrease in soil moisture
from 25 vol% to values well below 10% (Fig. 1A).
After a few weeks of such low soil moisture
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Fig. 1. Dynamics of gross rates of N turnover, net rates of N turnover, extractable soil inorganic N
concentrations, soil microbial biomass N, microbial C:N ratio and meteorological data over the investigation
period (August 2007 to October 2008).
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growing season was characterized by huge
variability following drying-rewetting cycles.
However, it oscillated in opposite directions
compared to soil moisture and microbial biomass, i.e., soil drying and microbial biomass
decline were accompanied by the highest rates of
gross ammonification (approximately 3 mg N
kg1 sdw d1). In the autumn freeze-thaw period,
a moderate increase of gross ammonification at
low levels occurred (turnover rates of less than
0.5 mg N kg1 sdw day1; Fig. 1B). In winter,
gross ammonification was either not significantly
different from zero or slightly but significantly
positive throughout the soil frost period. A
pronounced temporal dynamic was not evident
(Fig. 1B). In contrast, high rates of ammonification were observed in the spring freeze-thaw
period (Fig. 1B).
Compared to gross ammonification, smaller
temporal variation was observed for gross
nitrification (rates between 0 and 1.5 mg N kg1
sdw d1) during the growing season (Fig. 1C).
Due to the lower temporal resolution of measurements of gross nitrification, clear evidence
for effects of summer drying-rewetting events on
gross nitrification could not be established (Fig.
1B). However, gross nitrification dramatically
increased to values of approximately 2.2–4 mg
N kg1 sdw day1 after the first topsoil freeze
events in autumn (Fig. 1C). Similar to gross
ammonification, very small or non-significant
rates of gross nitrification were observed in
winter, followed by a strong increase of gross
nitrification in the spring freeze-thaw period.
Net ammonification was close to zero over the
whole year, but became significantly negative in
summer 2008 (Fig. 1H). This did not affect
extractable soil ammonium concentrations which
showed little temporal variation over the whole
year (Fig. 1F). However, peaks of soil nitrate
concentrations were observed in the spring
freeze-thaw period, whereas smaller soil nitrate
levels were observed during the growing season
(Fig. 1G).
Several peaks of net nitrification were observed during the growing season (Fig. 1I).
However, despite pulses of gross nitrification
and soil nitrate concentrations in the spring
freeze-thaw period, a corresponding dynamic of
net nitrification in that period was not observed
(Fig. 1I).
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Cumulative annual N turnover and
contribution of seasons
To obtain cumulative rates of N turnover,
linear interpolation between measuring points
was used. These calculations illustrated the
importance of both autumn and spring freezethaw periods for annual gross N turnover (Fig.
2). Neither magnitude nor dynamics of cumulative net N turnover were related to cumulative
gross N turnover. For the period from October 1,
2007 to September 30, 2008, annual gross
ammonification for the uppermost 10 cm of soil
was 240 and 215 kg N ha1 year1, while annual
gross nitrification was 417 and 362 kg N ha1
year1 for UG and WG, respectively (Table 1). In
contrast, annual net ammonification was 9 and
6 kg N ha1 year1, while annual net nitrification was 31 and 19 kg N ha1 year1 at UG and
WG, respectively (Table 1). The autumn freezethaw period (46 days) contributed 6% and 7% to
annual sums of gross ammonification, but 19%
and 36% to annual sums of gross nitrification at
UG and WG, respectively (Table 1). Approximately one third (WG: 34%) to one half (UG:
44%) of annual gross ammonification was observed during the spring freeze-thaw period (79
days). Also with regard to gross nitrification the
spring thaw period was of key importance: 46%
and 21% of annual gross nitrification at UG and
WG, respectively, was observed during this
period of time. Both freeze-thaw periods contributed 50% and 41% to annual cumulative gross
ammonification and 65% and 57% to annual
cumulative gross nitrification. In contrast, the
growing season contributed 40% and 52% to
annual cumulative gross ammonification, 29%
and 32% to annual cumulative gross nitrification,
while the winter period was of minor importance
for both gross rates of N turnover (Table 1). In
contrast, different patterns were observed for
seasonal contribution to annual net N turnover.
Almost all net nitrification occurred during the
growing season (Table 1). Net ammonification
was negative at the annual scale and the
calculated annual value was mainly influenced
by net ammonium consumption during the
growing season (Table 1).
Season-specific environmental controls
on gross N turnover
The role of soil drying-rewetting cycles during
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April 2012 v Volume 3(4) v Article 34
WU ET AL.
Fig. 2. Cumulative gross N turnover and cumulative net N turnover during the investigation period (August
2007 to October 2008).
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April 2012 v Volume 3(4) v Article 34
WU ET AL.
Table 1. Contributions of seasons to annual gross and net N turnover (in kg N ha1y1) in winter-grazed (WG)
and ungrazed (UG) sites.
Gross ammonification
Season
Date
WG
Autumn freeze-thaw 01.10.2007–
15.11.2007
Spring freeze-thaw
18.02.2007–
07.05.2008
Growing season
08.05.2008–
30.09.2008
Winter
16.11.2007–
17.02.2008
Whole year
01.10.2007–
30.09.2008
UG
Autumn freeze-thaw 01.10.2007–
15.11.2007
Spring freeze-thaw
18.02.2007–
07.05.2008
Growing season
08.05.2008–
30.09.2008
Winter
16.11.2007–
17.02.2008
Whole year
01.10.2007–
30.09.2008
Gross nitrification
Net ammonification
Net nitrification
Mean 6 SE
%
Mean 6 SE
%
Mean 6 SE
%
Mean 6 SE
%
14 6 4
7
130 6 11
36
0.20 6 0.34
3
1.53 6 0.45
8
74 6 7
34
77 6 20
21
0.02 6 0.59
0
1.05 6 0.78 6
112 6 17
52
117 6 18
32
6.92 6 2.26
117
19.18 6 5.18 102
16 6 6
7
40 6 109
11
0.77 6 2.15
13
0.81 6 2.65 4
215 6 35
5.94 6 5.35
362 6 158
18.84 6 9.05
15 6 3
6
79 6 36
19
0.92 6 0.19
10
0.67 6 0.31
2
107 6 5
44
191 6 63
46
0.56 6 0.34
6
4.36 6 0.54
14
95 6 29
40
122 6 31
29
7.48 6 3.2
84
27.72 6 3.51
89
23 6 9
10
25 6 47
6
0.10 6 1.9
1
1.53 6 3.11 5
240 6 45
417 6 177
8.86 6 5.63
31.22 6 7.47
Notes: Duration of seasons: autumn freeze-thaw period: 46 days; spring freeze-thaw period: 79 days; growing season: 146
days; winter: 94 days. Percentages are the contributions to annual flux.
period only, a positive relationship between soil
moisture and gross ammonification, was observed (R ¼ 0.778, P ¼ 0.003).
the growing season as a driver of microbial
biomass dynamics was confirmed by highly
significant positive correlations between soil
moisture and soil microbial biomass N (R ¼
0.93, P , 0.001). Remarkably the oscillations of
soil microbial biomass N along drying-rewetting
cycles (range approx. 20 to 100 mg N kg1 sdw;
Fig. 1D) coincided with simultaneous counter
rotating cycles of gross rates of ammonification
(range from close to zero to .3 mg N kg1 sdw
d1; Fig. 1B). That is, gross rates of ammonification during the vegetation period were highest
following declines in microbial biomass. This
inverse relationship was confirmed by a significant negative correlation between gross ammonification and soil microbial biomass N during
the growing seasons (Fig. 3).
A similar relationship between microbial biomass dynamics and gross N turnover was
evident during the first topsoil freeze events in
fall, when microbial biomass declined dramatically, while at the same time the microbial C:N
ratio increased by approx. a factor of two (Fig.
1E), followed by a minor increase in gross
ammonification but a dramatic increase in gross
nitrification (Fig. 1C). For the spring freeze-thaw
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Environmental controls on soil N dynamics
over the full annual course
While within-season dynamics of gross ammonification was partly correlated with other
parameters (see above), gross rates of ammonification rates were not correlated with any of the
determined potential controls (i.e., soil temperature, soil moisture, microbial biomass C and N as
well as microbial C:N ratio) when the full annual
dataset was analyzed. In contrast, correlations at
the annual scale were found for rates of gross
nitrification and net N turnover. Multiple regression analysis indicated a positive relationship
between microbial C:N ratio and gross nitrification, while other parameters, though in part
positively correlated, were removed from the
regression model (Table 2). Both for net nitrification and for net ammonification, soil moisture
remained as a single parameter in the multiple
regression models (Table 2). While the relationship between soil moisture and net nitrification
was positive, it was negative for net ammonifi10
April 2012 v Volume 3(4) v Article 34
WU ET AL.
Fig. 3. Linear correlation between soil microbial biomass N and the gross rates of ammonification during the
growing seasons. Points are means of treatments (wintergrazed, ungrazed) as calculated from three replicate
plots. Error bars represent standard errors of the mean.
than in UG plots (Fig. 1B). Gross nitrification was
almost twice as large at UG than at WG in the
spring freeze-thaw period (Fig. 1C), however,
due to large variation between plots, this
difference was not significant. As a consequence
of different turnover rates in the freeze-thaw
period, grazing reduced gross ammonification by
11% and gross nitrification by 13% on an annual
basis (Fig. 3).
cation, or, in other words, there was a positive
relationship between net ammonium consumption and soil moisture (Table 2).
Grazing effects on N turnover
Wintergrazing significantly reduced microbial
biomass N, soil nitrate concentrations and net
nitrification at only a few single sampling dates
during the growing season (Fig. 1D). A pronounced grazing-induced reduction in microbial
biomass N was found in winter, when soil
temperatures in the WG but not the UG plots
dropped below 108C (Fig. 1A, D). However, the
most obvious effect was observed in the spring
freeze-thaw period, with persistently reduced
soil microbial biomass N in WG (Fig. 1D),
accompanied by increased microbial C:N ratios
(Fig. 1E). At the same time, gross rates of
ammonification were significantly larger in WG
DISCUSSION
Temporal dynamics of N turnover
With a hitherto not realized temporal resolution of mostly sub-monthly measurements (8–38
day intervals) over a period of 14 months we
followed the temporal dynamics of soil gross and
net microbial N turnover in typical steppe soils of
Inner Mongolia. Our measurements show an
Table 2. Results of multiple stepwise regression analysis using data from the whole investigation period in order
to identify major environmental controls of gross and net N turnover. Gross ammonification was not correlated
with any of the potential controls at the annual scale. The unit for gravimetric soil moisture was g H2O g1 sdw.
Metric
Stepwise multiple regression model
R2
corrected R 2
P
Gross nitrification
Net nitrification
Net ammonification
0.106 3 microbial C:N ratio 0.480
1.652 3 gravimetric soil moisture 0.100
0.453 3 gravimetric soil moisture þ 0.003
0.202
0.351
0.356
0.17
0.333
0.338
0.019
,0.001
,0.001
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April 2012 v Volume 3(4) v Article 34
WU ET AL.
Fig. 4. Linear correlation between gravimetric soil moisture and soil microbial biomass N. Data are at the level
of single soil samples for both treatments (grazed, ungrazed, N ¼ 618). The outliers at highest soil moisture were
determined using samples from ungrazed plots collected at the onset of the first spring freeze-thaw event.
Retarded microbial growth after suddenly optimal conditions for microbial succession is hypothesized to have
caused these outliers (see text). These outliers were included in the correlation analysis presented here.
often assumed but rarely demonstrated high
temporal variability of gross inorganic N turnover (Grenon et al. 2004, Dannenmann et al.
2006, Rennenberg et al. 2009) and microbial
biomass, driven by periods of environmental
stress or transition, such as drying-rewetting
cycles or freeze-thaw events.
So far, most studies on gross N turnover in
various ecosystems (see summary by Booth et al.
2005) were limited to growing seasons. For
example, Holst et al. (2007) determined gross N
turnover at adjacent unreplicated steppe sites
during the growing seasons of the years 2004–
2005 and determined mean gross ammonification
rates of 0.6–4.1 mg N kg1 sdw day1 and gross
nitrification rates of 0.5–3.1 mg N kg1 sdw
day1, thus being of similar magnitude as
determined in this study. Though measurements
of Rosenkranz et al. (2010) also showed a high
variability of gross inorganic N production
between and within all sampling periods from
February to November for an N-saturated
temperate spruce forest in Southern-Germany,
winter measurements of gross N turnover for
frozen soil were not reported.
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Contribution of seasons to annual N turnover
In our study four distinct seasons with
characteristic patterns and magnitudes of N
turnover could be identified: growing season,
autumn freeze-thaw period, winter with permanently frozen soil, and spring freeze-thaw period.
Here we demonstrate that not only the growing
season, but also autumn and spring freeze-thaw
periods were of major importance for gross N
turnover. Though gross N turnover in permanently frozen soil was of little importance for the
annual budget, significant and persistent activity
and even microbial immobilization of inorganic
N in frozen soil was found. Microbial immobilization was indicated by frequently significantly
positive 15NO3 and 15NH4þ-consumption rates
(data not shown). Microbial activity in frozen soil
is further confirmed by the net growth of
microbial biomass over winter, and by net nitrate
immobilization (Fig. 1I). To our knowledge, there
is no other study investigating gross N turnover
in frozen soil, but Zhang et al. (2011) reported
significant net nitrification or net nitrate immobilization under frozen soil conditions for steppe
sites of Duolun County, Inner Mongolia, in a
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April 2012 v Volume 3(4) v Article 34
WU ET AL.
range as observed in this study.
Since the very minor net immobilization of
inorganic N during winter (,0.03 mg N kg1
sdw d1) cannot explain the net increase in
microbial biomass (up to 40 mg N kg1 sdw)
(Fig. 1) and—if at all—only little gross inorganic
N production was observed, we hypothesize that
during conditions of frozen soils microbial
growth was fuelled by the metabolism of
monomeric organic N compounds. This view is
supported by several studies detecting significant
turnover of monomeric organic N compounds
such as amino acids in seasonally frozen continental soils (Lipson and Monson 1998, Kielland
et al. 2007, Näsholm et al. 2009).
A few studies have investigated gross N
turnover rates in thawing soils, e.g., Müller et
al. (2002) for grassland soil, Ludwig et al. (2004)
for agricultural Luvisol, and Freppaz et al. (2007)
for alpine forest soils. These studies—though
restricted to single point in time measurements
only—showed that significant gross N turnover
may occur in freeze-thaw periods. Comparable
results were also obtained in this study. With the
on-set of soil thawing a vigorous revival of gross
N turnover was observed driving pulse emissions of N2O, with N2O emissions during the
spring thaw period even dominating the annual
N2O budget (Wolf et al. 2010).
The observed variability of gross N turnover as
well as the importance of the freeze-thaw periods
for annual N turnover questions the low-temporal resolution approach of most previous studies
on gross N turnover. It can be doubted if lowtemporal resolution studies may provide a
sufficient insight into functioning and magnitude
of ecosystem N cycling. Though determination of
gross N turnover remains time- and resourceintensive, we suggest that a concentration of
scientific resources to determine gross N turnover with a reasonable temporal resolution of
monthly values at a reduced number of sites may
be more beneficial for a better understanding of
magnitudes and controls of ecosystem soil N
cycling than conducting abundant low temporal
resolution studies.
seasonality of rainfall, which peaks in the
vegetation period from May-August, we also
observed high values for microbial biomass.
Similarly, Liu et al. (2010) have shown for
another semi-arid steppe in Duolun County,
Inner Mongolia, that water availability and plant
productivity regulate the interannual variability
of soil microbial N and C pools. The few outliers
observed for our relationship between soil
moisture and microbial biomass at high soil
moisture contents (see Fig. 4) were determined in
soil samples taken during the first soil thaw
event after the winter in the ungrazed plots.
Hence, these outliers may arise from a retarded
microbial growth at suddenly extremely high soil
water content. Pronounced changes in environmental conditions such as freeze-thaw in autumn
and spring, as well as drying events during the
growing season triggered a rapid decline in
microbial biomass, but stimulated gross N
turnover. Hence, gross rates of N turnover were
not related to soil moisture or temperature, but
gross N turnover seems to depend on the
availability of C and N substrates, most of which
may originate from microbial residues. Significant changes in microbial C:N ratios in such
periods of environmental stress or transition (Fig.
1E) indicate microbial succession of functional
microbial groups well adapted to the new
environmental conditions (Schmidt et al. 2007).
For the autumn freeze-thaw period we calculated
mean residence times of microbial N (estimated
by microbial biomass N divided by the dominating process of gross N turnover) of 3–6 days
only while it was 47–63 days on an annual scale.
This confirms that such transition periods are
characterized by a very active microbial community and that successions of microbial communities may be fuelled by feeding on the residues of
the preceding microbial community. High microbial immobilization in periods of environmental
transition was also indicated by the observation
that the accompanying pulses of gross N
turnover were usually not associated with
increased net inorganic N production, also
during absence of competing vegetation in
freeze-thaw periods. The dynamic of gross N
turnover and microbial biomass observed here
supports a recent hypothesis about biogeochemical consequences of rapid microbial succession
in soils, i.e., that turnover and succession of
Environmental controls of gross N turnover
It was very obvious that soil moisture exerted a
dominant influence on soil microbial biomass N
dynamics (Fig. 4). In close relationship with the
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April 2012 v Volume 3(4) v Article 34
WU ET AL.
microbial biomass are the major source of
bioavailable N in soil (Schmidt et al. 2007).
Similar to recent observations in a temperate
beech forest in Austria (Kaiser et al. 2011), the
observed net build-up of microbial biomass in
winter steppe soils may represent a large pool of
bio-available nitrogen for the consecutive growing season, thus improving ecosystem productivity.
Both the observed absence of effects of soil
moisture on gross N turnover and the negative
relationships between microbial biomass and
gross N turnover over most periods of the year
are contradictory to findings in other ecosystems.
Gross nitrification in a temperate beech forest
was found to be positively related to soil
microbial biomass and to soil moisture content
up to an optimum soil water content of approximately 60% of the water holding capacity
(Dannenmann et al. 2006). Furthermore, a
laboratory parameterization of gross ammonification in temperate hardwood forest soil revealed a significantly positive effect of soil
moisture (Rennenberg et al. 2009). Positive
relationships between soil moisture and gross
ammonification as well as gross nitrification were
also reported for the uppermost mineral soil of a
nitrogen saturated temperate spruce forest of
Southern Germany (Rosenkranz et al. 2010).
These contrasting findings may imply that the
temporally explicit mechanistic understanding of
gross N turnover as developed in this study for
semi-arid continental steppe with their extremely
pronounced dynamics in environmental conditions is not directly transferable to temperate
forests or other ecosystems.
ammonium (autotrophic nitrification). This interpretation explains why the 15N excess was only
diluted in the nitrate pool but not in the
ammonium pool (Huygens et al. 2008). Heterotrophic nitrification is likely to be performed by
fungi characterized by higher microbial C:N
ratios than bacteria (Landi et al. 1993, Paul and
Clark 1996), which coincides with our finding of
high microbial C:N ratios during key periods of
gross nitrification (Table 2). Heterotrophic nitrification has been reported to dominate over
autotrophic nitrification in several soils ranging
from various forest ecosystems (Burton et al.
2007, Grenon et al. 2004, Pedersen et al. 1999,
Koyama et al. 2010) to grassland (Cookson et al.
2006, Müller et al. 2002, 2009, Huygens et al.
2008, Rütting et al. 2008). Nonetheless, the actual
contribution of heterotrophic nitrification to total
gross nitrification remains unclear in our as well
as in most other studies, unless 15NO3 pool
dilution studies combined with selective inhibitors of autotrophic or heterotrophic nitrification
(Barraclough and Puri 1995) are performed.
Grazing effects on N turnover
Negative effects of winter grazing on N
turnover and microbial biomass were only
significant in the spring freeze-thaw period.
Comparable results were also found by Wolf et
al. (2010), who showed that at grazed sites
spring-thaw N2O pulse emissions were significantly lower or even missing. These authors
showed that grazing-induced reductions in vegetation height, surface roughness length and
snow capture resulted in a reduction of the
insulation of the soil and thus lower winter soil
temperatures (Fig. 1A, D). Furthermore, reduced
snow cover reduces wintertime water retention,
and thus strongly decreases soil moisture in the
spring freeze-thaw period. The effects of grazing
on both soil temperatures and soil moisture
explain grazing-induced reduction of microbial
biomass N, gross ammonification as observed in
this study (Fig. 1B, D), and the strongly reduced
N2O emissions in the spring freeze-thaw period
as described by Wolf et al. (2010). Here, we also
found that gross nitrification was smaller at
grazed plots in the spring freeze-thaw period
by a factor of two, but these differences remained
insignificant due to large variation between the
three investigated plots (Fig. 1C). Since winter
Gross nitrification exceeded
gross ammonification during freeze-thaw periods
and at the annual scale
During freeze-thaw periods gross nitrification
was considerably higher than gross ammonification (Fig. 1B, C). For several adjacent sites, Holst
et al. (2007) also reported that monthly mean
gross nitrification in August was several fold
larger than the corresponding gross ammonification. However, since a depletion of the soil
ammonium pool was not observed, nitrification
must have largely been based on the direct
oxidation of organic N (heterotrophic nitrification) and not on the oxidation of free soil
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April 2012 v Volume 3(4) v Article 34
WU ET AL.
41 kg N ha1 year1 determined for the years
2004–2006 (Giese et al. Institute of Plant Production and Agroecology in the Tropics and Subtropics, University of Hohenheim, Germany,
unpublished). The same authors reported belowground net primary production (BNPP) of 24–39
kg N ha1 year1. Thus, total net N uptake by
plants may amount to 52–80 kg N ha1 year1,
which is several times higher than the growing
season net N mineralization at ungrazed plots of
20.2 kg N ha1 year1 determined in this study
for the uppermost 10 cm of the soil. Further net
inorganic N production may have occurred in
deeper soil layers, however is expected to
strongly decline with deeper soil horizons
(Matejek et al. 2010). This imbalance between
net N mineralization and net plant N uptake may
be related to plant acquisition of organic N
(Näsholm et al. 2009) as well as to successful
competition for bioavailable N of plants over
microorganisms in the rhizosphere (Schimel and
Bennett 2004, Chapman et al. 2006).
In sum, our study shows that the determination of net N turnover is of little use in steppe
systems to understand magnitudes, patterns,
mechanisms and dynamics of soil N turnover,
as well as the magnitude of plant N availability
and N loss. We hypothesize that this may also
apply to other ecosystems.
grazing completely eliminated spring thaw N2O
pulse emissions (Wolf et al. 2010), but insignificantly reduced gross nitrification (Fig. 1C), this
points to the fact that denitrification was the
major source of freeze-thaw pulse emissions of
N2O in the investigated steppe soils.
Do net rates provide insight into ecosystem N
dynamics and N status of semi-arid steppe?
The parallel full annual courses of gross and
net N turnover represent a unique prerequisite to
judge to what extent net rates of N turnover,
which are much easier to determine, allow for
insight into actual status and dynamics of N
turnover. Growing season net N mineralization
(calculated as the sum of net ammonification and
net nitrification) of this study (year 2008) was
20.2 and 12.2 kg N ha1 year1 for UG and WG,
respectively. These values are in the range of the
values reported by Zhou et al. (2009) for
comparable ungrazed steppe sites. These authors
reported net N mineralization in the uppermost
10 cm of 13.2 kg N ha1 year1 for the growing
season of 2007 and 30.7 kg N ha1 year1 for the
growing season of 2006. In our study, net N
mineralization was approximately one order of
magnitude smaller than gross N turnover (Table
2). More surprisingly, the dynamics of net N
turnover were not related to gross N turnover
(Figs. 1–3, Table 2) either. In particular, pulses of
gross N turnover during the freeze-thaw periods
were not accompanied by a concomitant increase
in net N turnover (Fig. 1). Thus, N2O peak
emissions during spring freeze-thaw (Wolf et al.
2010) were not indicated by net rates of N
turnover. This is contradictory to the suggestion
that net rates of N turnover may be predictors of
N loss (e.g., Holmes and Zak 1999, Venterea et al.
2003, Matejek et al. 2009) at least for steppe
systems.
Net rates of N turnover have been frequently
used to estimate plant available N (Schimel et al.
2004). Indeed, highest net N mineralization
occurred during the growing season in this
study. Nitrogen uptake into aboveground biomass was 27–46 kg N ha1 year1 in the year
2008 at adjacent ungrazed plots (Schönbach,
University of Kiel, Institute of Agronomy and
Plant Breeding, Germany, personal communication). This corresponds well to a mean aboveground net primary productivity (ANPP) of 36–
v www.esajournals.org
CONCLUSIONS
Four different seasons with characteristic
patterns of gross N dynamics could be distinguished in the investigated semi-arid continental
Asian grassland. Both freeze-thaw cycles and the
growing season were key periods for understanding patterns and magnitudes of gross N
turnover. Various patterns of biogeochemical N
turnover between seasons appeared to be closely
related to microbial succession in periods of
environmental stress or transition. In this context,
turnover and succession of soil microbial biomass appeared to be the major driver of gross N
fluxes, and hence, are of outstanding importance
for nutrient retention and availability and may
mitigate nutrient shortage in drought periods of
the growing season. Net rates of N turnover may
be of limited use in steppe ecosystem studies,
since they provided only a very poor approximation to the magnitude, dynamics and status of
15
April 2012 v Volume 3(4) v Article 34
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actual N turnover in soil. The observed high
temporal variation of N turnover within and
between seasons emphasizes the necessity for
high resolution sampling of gross N turnover as
a prerequisite to infer functioning and annual
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ACKNOWLEDGMENTS
Funding of this work by the German Research
Foundation/Deutsche Forschungsgemeinschaft (DFG)
within the framework of the MAGIM research group is
greatly acknowledged. Co-Funding was obtained by
the Helmholtz/CAS ENTRANCE joint laboratory. We
thank Shenghui Han, Nicole Fanselow, Yuandi Zhu
and Weiwei Chen for logistic support. We also
acknowledge Nicolas Brüggemann and Rudi Meier
for providing the technical support of Centre of Stable
Isotopes at IMK-IFU. Furthermore we are grateful to
Qiang Yu for providing comments on the manuscript.
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