agronomy

Article
Management Intensity Controls Nitrogen-Use-Efficiency
and Flows in Grasslands—A 15N Tracing Experiment
Marcus Zistl-Schlingmann † , Steve Kwatcho Kengdo † , Ralf Kiese and Michael Dannenmann *
Karlsruhe Institute of Technology (KIT), Institute of Meteorology and Climate Research-Atmospheric
Environmental Research (IMK-IFU), Kreuzeckbahnstrasse 19, 82467 Garmisch-Partenkirchen, Germany;
[email protected] (M.Z.-S.); [email protected] (S.K.K.);
[email protected] (R.K.)
* Correspondence: [email protected]
† These authors contributed equally to this study.




Received: 6 March 2020; Accepted: 19 April 2020; Published: 23 April 2020


Abstract: The consequences of land use intensification and climate warming on productivity, fates
of fertilizer nitrogen (N) and the overall soil N balance of montane grasslands remain poorly
understood. Here, we report findings of a 15 N slurry-tracing experiment on large grassland plant–soil
lysimeters exposed to different management intensities (extensive vs. intensive) and climates (control;
translocation: +2 ◦ C, reduced precipitation). Surface-applied cattle slurry was enriched with both
15 NH + and 15 N-urea in order to trace its fate in the plant–soil system. Recovery of 15 N tracer in
4
plants was low (7–17%), while it was considerably higher in the soil N pool (32–42%), indicating
N stabilization in soil organic nitrogen (SON). Total 15 N recovery was only 49% ± 7% indicating
substantial fertilizer N losses to the environment. With harvest N exports exceeding N fertilization
rates, the N balance was negative for all climate and management treatments. Intensive management
had an increased deficit relative to extensive management. In contrast, simulated climate change had
no significant effects on the grassland N balance. These results suggest a risk of soil N mining in
montane grasslands under land use intensification based on broadcast liquid slurry application.

Keywords: 15 N tracing; nitrogen-use-efficiency; nitrogen balance; montane grasslands; climate change;

management intensity

1. Introduction
Grasslands in Germany cover about 30% of the total national agricultural area. One fifth of
these grasslands are located in the alpine and pre-alpine belt in southern Germany [1]. Due to a
predominantly cool-moist climate and traditional organic fertilization over the last centuries, permanent
grassland soils in the pre-alpine region represent a large storage pool of soil organic carbon (SOC) and
total nitrogen (TN) [2]. Furthermore, the traditional extensive management [3] remains common and
is vital for the maintenance of ecosystem services such as biodiversity and socioeconomic services [4,5].
High SOC and TN contents are a crucial factor in the provisioning of ecological soil functions such as
nutrient retention and groundwater protection, while also facilitating high plant productivity [6,7].
Increasing demand for meat and dairy products [8] in turn increases demand for both forage
production and adequate disposal areas for animal waste. With climate warming, biomass production
of grasslands in the alpine and pre-alpine region may increase [9,10], thus enabling farmers to achieve
higher yields and to expand the intensive management from the lowlands into montane regions.
This is of special interest in the European alpine and pre-alpine regions, where climate warming
is occurring at twice the rate compared to global average, a trend, projected to continue during
the next decades [11–15]. Changes in seasonal precipitation and in precipitation extremes are also

Agronomy 2020, 10, 606; doi:10.3390/agronomy10040606 www.mdpi.com/journal/agronomy

Agronomy 2020, 10, 606 2 of 15

expected, but the direction and magnitude of effects in the Alps may vary strongly across regions and
elevation [12,14–17].
Another driver of intensification may originate from recent changes in the legislation. The new
German fertilizer ordinance, which became operative in June 2017, limits use of organic fertilizer to
170 kg N ha−1 year−1 in order to reduce nitrate leaching rates to groundwater bodies. Beforehand,
slurry application rates in the pre-alpine region were up to 305 kg N ha−1 year−1 [18]. The new legal
constraints for organic fertilizer could cause farmers to take the now excess slurry from intensively
managed grasslands (five fertilization-harvest events per season) and apply it to extensively used
grasslands that previously received only 1–2 slurry applications, equaling ca. 30–80 kg N ha−1 year−1 .
Earlier studies have indicated that climate change in the alpine and pre-alpine grassland region
could lead to SOC loss [2] and nitrogen (N) mining (when N export exceeds input causing a depletion
of soil N) from extensively managed grassland soils [19]. This is mainly activated by increased
mineralization of soil organic matter (SOM) and associated liberation of nutrients, thereby stimulating
both N losses and grassland productivity [2,15,20]. This can be relevant for the overall N balance
because harvest export is the dominant N loss pathway in montane grasslands [21].
Various studies have investigated the impact of warming and management on grassland biomass
production [10,22,23], N leaching losses [20,24] and nitrous oxide (N2 O) emissions [25–28]. However,
little is known on the fates of slurry N in the grassland plant–soil system and how it is partitioned
to plant nutrition, storage in soil, total leaching and gaseous N losses. Hence, consequences of
intensification for the N balance under a warming climate are largely unclear. Understanding the fate
of fertilizer N while considering plant N-use-efficiency and total N losses is therefore important for the
development of sustainable and climate adapted management strategies.
Most available literature on the fate of N fertilization examined arable systems [29–31] and focused
on the use of mineral fertilizers or urea rather than cattle slurry [30,32–35]. Furthermore, most of the
available literature only reported short term N-use-efficiency rather than tracing the fate of fertilizer N
during a complete growing season. This knowledge gap prevents adequate adaptation of grassland
management to future climates.
In order to provide a comprehensive insight into fates of slurry N in a typical montane grassland
system, we applied 15 N labeled slurry to large lysimeters over a full growing season. This was done
for two management intensities (extensive vs. intensive) and two climate treatments (ambient climate
as control, experimentally simulated climate change via translocation to a lower elevation) in a full
factorial design. Our climate change treatment represented a realistic future scenario of increased
temperature with reduced precipitation but was unable to parse these effects. We traced fertilizer N
fates into plant biomass and soil (down to 60 cm) and derived N balances for the four treatments.
We hypothesized that climate change would increase productivity, which will result in increased
N exports and thus, negative ecosystem N balances and risk of N mining from SOM. Furthermore, we
expected that intensive management would counteract the risk of N mining by increased N addition
and stabilization in soil.

2. Materials and Methods

2.1. Study Sites and Experimental Design

The study was conducted in the TERENO pre-alpine observatory in the Ammer river catchment
in southern Germany at the sites of Graswang and Fendt [36]. The Graswang site (860 m ASL, 47◦ 570 N,
11◦ 030 E, the control site herein referred to as high elevation (HE) is characterized by a mean annual
temperature (MAT) of 6.9 ◦ C and mean annual precipitation (MAP) of 1347 mm (2014–2017). The soil
at HE is characterized as C and N rich Haplic Cambisol according to World Reference Base for Soil
Resources [37] with neutral pH. The Fendt site (600 m ASL, 47◦ 830 N, 11◦ 070 E, herein referred to as
low elevation “LE”), is characterized by a MAT of 8.9 ◦ C and 956 mm MAP and was used to simulate
climate change conditions for intact lysimeters translocated within a space-for-time approach from
Agronomy 2020, 10, 606 3 of 15

Graswang to the Fendt site. This climate change treatment represents projected temperature increases
of +2 ◦ C and summer precipitation reduction as projected by regional climate modeling of the mid-21st
century [12–16]. However, it does not include free air CO2 enrichment.
In 2011, a total of 12 large intact soil cores (112 cm diameter, 1.5 m height) from three replicated
sites within a 5 km distance from the HE site were excavated without disturbance of plants and soil [38].
Half remained at HE and half were transferred to LE. Lysimeters operated since the end of 2011 and
organized in sets of six surrounding a service unit that hosted all steering and data recording devices.
For further details of Lysimeter sampling, setup and instrumentation see [20,38]. Immediately after
translocation, half of the lysimeters were exposed to extensive management (two to three cuts and one
to two fertilization events with liquid cattle slurry surface application). This followed the previous
management regime at HE. The other half underwent an intensive management regime with four to
five fertilization-cutting cycles per year. These management regimes correspond to regional farmers
practice. The present study was conducted in 2017 after an operational period of almost six years and
used an application of 15 N labeled slurry for all fertilization events and all treatments.

2.2. Production and Use of 15 N Labeled Cattle Slurry

The 15 N labeling of the lysimeters was conducted via the application of 15 N enriched liquid cattle
slurry. Slurry was supplied by the local farmer at the Fendt field site and analyzed by a commercial
laboratory (Raiffeisen-Laborservice, Ormont, Germany). The intended 15 N enrichment of the slurry
was ca. 5%, executed via the addition of 99% 15 N enriched Ammonium sulfate and Urea in equal
N-amounts. The cattle slurry contained on average 4.2% total N, which was 53% NH4 + -N and 47%
organic-N (including urea). Addition of the 15 N label slightly increased the N content of slurry by an
average of 3.5%, which was accounted for in the N balance calculations.
Immediately prior to fertilization of the lysimeters, the 15 N tracer was added to the slurry in a
polyethylene barrel and vigorously shaken to ensure a homogenous 15 N labeling of the slurry. Each
lysimeter was fertilized with 1.8 L of liquid slurry per fertilization event, representing the regional
farmers’ practice of an addition of 18 m3 ha−1 . This resulted in an addition of 253 mg 15 N in excess
of natural abundance per lysimeter and fertilization event. For application of 15 N labeled slurry, we
followed actual farmers’ practices, which resulted in four fertilization/labeling events and five cuts for
intensive management. Extensive management involved one fertilization/labeling event in spring and
three harvests. Both management treatments received another unlabeled fertilization event after the
final November sampling that was considered in the N balance calculations.

2.3. Sampling and Sample Preparation

Sampling of aboveground biomass (AGB) was conducted at each cutting event by cutting the
vegetation of each lysimeter at ground level. Samples were transported to the facilities of KIT/IMK-IFU
in Garmisch-Partenkirchen, Germany for further processing and analysis. Plant biomass yield and N
export was measured during 2012–2017, while analysis of 15 N recovery was only conducted during
labeling with 15 N enriched slurry in 2017.
Soil sampling of all lysimeters was conducted during October 2017, i.e., the end of the growing
season. For this purpose, three soil cores per lysimeter were retrieved using an auger filled with clear
PVC soil liner in order to preserve the integrity of the soil cores. Soil cores were sampled at a distance
of at least 30 cm to the edge of the lysimeter, to avoid edge effects. For each lysimeter, sampled soil
columns were cut into four depths (0–5; 5–15; 15–30 and 30–60 cm), cleaned of displaced material and
homogenized. Three replicated samples of each depth were pooled to one representative sample that
was subsequently processed and analyzed. Soil samples and aboveground biomass were dried at
55 ◦ C until a constant weight and subsequently analyzed for TN concentrations and 15 N enrichment as
described in detail by [21].
The ratio of N isotopes in soil and plant samples were analyzed using an isotope ratio mass
spectrometer (Delta PlusXP, Thermo Scientific, Waltham, MA, USA) coupled to an elemental analyzer
Agronomy 2020, 10, 606 4 of 15

(Flash EA, Thermo Scientific, Waltham, MA, USA) (EA-IRMS) at the Center for Stable Isotope Analysis
(CSI) at KIT/IMK-IFU in Garmisch-Partenkirchen, Germany [39].

2.4. Calculation of 15 N Recovery

Excess 15 N amount (mg) in all investigated pools was calculated using the following Equation (1).

APE
 
Npool ∗ (1)
100

where Npool is the amount of N in mg in the plant or depth-specific soil N pool and (atomic percent
excess) APE is the 15 N excess enrichment (atom% 15 N measured minus the natural abundance atom%
15 N) of the respective N pool. Natural 15 N abundance in plants were determined for plant samples of

the lysimeters used in this study but from 2016, the year prior to labeling. Natural abundance of soil N
was determined in 2011 using soil from the sampling locations of the lysimeters.
Excess 15 N recovery at the sampling time, expressed as a percentage, was calculated by dividing
the excess amount of 15 N in the analyzed pools by the cumulative 15 N excess addition following slurry
fertilization at the sampling time (253 mg 15 N excess per fertilization event).
All results were scaled to the lysimeters by multiplying the calculated 15 N excess (mg kg−1 sdw) by
the respective sdw (kg) of the lysimeter layer sampled. Tracer recovery in AGB was always calculated
with cumulated 15 N excess, thereby accounting for the previously exported 15 N excess across multiple
seasonal biomass harvests.

2.5. Nitrogen Balance

Slurry-N-input, plant-N-harvest exports (N withdrawn via plant harvest for forage production)
and total slurry-N-loss (the percentage of unrecovered 15 N multiplied by N input via slurry application)
of the N balance were calculated with the data gathered during the experiment. N deposition rates
of 33 ± 7 kg N ha−1 year−1 were taken from measurements of dry and wet deposition at three
comparable, fertilized grassland sites in southern Bavaria, located within a distance of 100 km from our
study sites [40]. Biologic nitrogen fixation (BNF) was estimated based on the measured contribution
of legumes to total plant biomass yield and previously published relationships between legume
abundance and BNF rates [41] from comparable grassland sites in Switzerland. In the latter study,
a contribution of 5% to 15% of legume abundance lead to a contribution of fixed N to plant biomass N
ranging from 9–16%, equaling 1–26 kg N ha−1 . Applying these relationships, the observed proportion
of legumes to total dry matter yield (2% and 3% for intensive and extensive management, respectively)
translated into BNF estimates of 10 (extensive) and 15 kg N ha−1 year−1 (intensive).
The origin of plant N was calculated based on the recovery rate of excess 15 N in plants multiplied
by the total amount of applied slurry N. This amount was divided by total plant N, giving the
share of slurry N in plant N. The rest of plant N was considered to originate predominantly from
SOM mineralization.

2.6. Statistical Analysis

Each lysimeters (N = 3) was used as a statistical replicate in this study. Results were tested for
the ANOVA requirements of normality and homoscedasticity. Since these requirements were not met,
the non-parametric Kruskal–Wallis test was carried out to test for significant effects of climate and
management treatments on measured parameters. All statistical analyses were carried out with IBM
SPSS 25.0 (IBM, Armonk, NY, USA).
Agronomy 2020, 10, 606 5 of 15

3. Results

3.1. Meteorological Conditions

During the experimental year 2017, annual temperature for HE and LE were 7.1 ◦ C and 8.7 ◦ C,
respectively. Precipitation was higher at HE with 1571 mm, compared to 1249 mm at LE. The translocated
lysimeters were therefore exposed to a warming of 1.6 ◦ C and a precipitation decrease of 322 mm.
Summer precipitation was higher than winter precipitation (Figure 1a,b). The vegetation period
(average daily air Temperature > 5 ◦ C) was 212 and 235 days long with an average air temperature of
12.3 and 13.3 ◦ C, at HE and LE, respectively.

Figure 1. Meteorological conditions during the experimental period in the year 2017 for both high
elevation (HE) (a) and low elevation (LE) (b) with daily precipitation and air temperature values and
volumetric water content (VWC) in 10 cm depth at both sites (c).

Soil moisture showed generally similar temporal dynamics at HE and LE, albeit with several key
seasonal treatment effects (Figure 1c). Spring snow cover and a later snow melt led to reduced soil
moisture at HE compared to LE (March-April), while the opposite pattern was observed in the summer
months (June, July and August), which generally showed more pronounced variations in soil moisture
with several distinct drying and wetting cycles at LE.
1

3.2. Plant Biomass Yields, N Content and Associated N Export

In 2017, cumulative plant biomass yields for the complete growing season ranged from 7.5 to
11.6 t ha−1 across treatments (Table 1). Intensive management increased biomass production in both
climate treatments by 30% at the HE site and 39% at the LE site. Simulated climate change increased
dry matter yield under intensive management by 20% (Table 1).
Agronomy 2020, 10, 606 6 of 15

Table 1. Yield (t/ha) of the aboveground biomass for each harvest (±SE). Lower case letters depict
statistical differences (p < 0.05) between climate treatments within one land use. Capital letters depict
differences between management treatments within one sampling date and climate treatment (p < 0.05).
HE: high elevation; LE: low elevation.

Intensive Extensive
Date HE LE HE LE
23.05./08.06.17 2.2 ± 0.2 a 3.8 ± 0.4 b 3.7 ± 0.4 a 3.7 ± 0.4 a
05.07.17 3.1 ± 0.4 a 2.4 ± 0.2 b
07.08.17 1.8 ± 0.1 a 1.9 ± 0 a 2.8 ± 0.2 a 3.0 ± 0.6 a
21.09.17 2.1 ± 0.1 a 1.9 ± 0.3 a
08.11.17 0.6 ± 0.3 a 1.5 ± 0.9 a 0.9 ± 0.2 a 1.6 ± 0.5 a
Sum 9.8 ± 0.6 Aa 11.6 ± 0.7 Ab 7.5 ± 0.5 Ba 8.3 ± 1.2 Ba

Intensive management significantly increased plant-N concentrations (Table 2) and N export

rates between 2012–2017 (Figure 2, Table 3) compared to extensive management. However, N export
rates for single harvest events in 2017 did not show major differences between treatments. Rather,
the increased number of harvests accounted for increased cumulative N export under intensive
management (Figure 2). Exports persistently exceeded fertilizer N addition for single dates in 2017
(Figure 2). Climate change did not affect cumulative annual N export (Figure 2, Table 3).

Table 2. Mean nitrogen (N) concentration (%) of the aboveground biomass for each harvest (±SE).
Lower case letters depict statistical differences (p < 0.05) between climate treatments within one land
use. Capital letters depict differences between management treatments within one sampling date and
climate treatment (p < 0.05). HE: high elevation; LE: low elevation.

Intensive Extensive
HE LE HE LE
N concentration 2.3 ± 0.3 Aa 2.1 ± 0.1 Aa 1.7 ± 0.1 Ba 1.6 ± 0.1 Ba

Table 3. Plant harvest nitrogen (N) export (kg N ha-1 ) during the years 2012–2017 on the lysimeters
investigated in this study (±SE). Lower case letters depict statistical differences (p < 0.05) between climate
treatments within one land use. Capital letters depict differences between management treatments
within one sampling date and climate treatment (p < 0.05). HE: high elevation; LE: low elevation.

Intensive Extensive
Year HE LE HE LE
2012 291 ± 44 Aa 316 ± 46 Aa 165 ± 31 Ba 167 ± 37 Ba
2013 365 ± 13 Aa 416 ± 26 Aa 210 ± 40 Ba 244 ± 28 Ba
2014 310 ± 10 Aa 283 ± 14 Aa 157 ± 22 Ba 156 ± 16 Ba
2015 189 ± 7 Aa 219 ± 4 Ab 169 ± 7 B 130 ± n.a. B
2016 n.a. n.a. n.a. n.a
2017 213 ± 25 Aa 230 ± 26 Aa 128 ± 16 Ba 127 ± 28 Ba
Agronomy 2020, 10, 606 7 of 15
Agronomy 2020, 10, x FOR PEER REVIEW 7 of 15

Addition of
Figure 2. Addition of slurry
slurry nitrogen
nitrogen(N)
(N) and
and export
export of
of plant
plant N
N for
for each
each fertilization
fertilization and harvest
event under intensive (a) and extensive (b) management. Dates are the respective
respective dates of slurry/label
slurry/label
application and plant harvest. Numbers represent the sum of each bar. Error bars represent standard
deviation of each single date. HE: high elevation; LE: low elevation.

3.3. Slurry N
3.3. Slurry N Input
Input
Slurry N input showed a constant decline during the year 2017, with the highest N addition
Slurry N input showed a constant decline during the year 2017, with the highest N addition
occurring
occurring during
during the
the first
first application
applicationininspring
springand
andsubsequently
subsequentlydecreasing
decreasingthroughout
throughoutthetheyear
yearvia a
via
decrease
a decrease inin
slurry NN
slurry content. Analyses
content. Analyses ofof
slurry samples
slurry revealed
samples a constant
revealed decline
a constant in in
decline drydry
matter in
matter
the slurry, from 8% during the first sampling period down to 2% in the slurry applied
in the slurry, from 8% during the first sampling period down to 2% in the slurry applied in in November.
November.
3.4. 15 N Recovery from Labeled Fertilizer
3.4. 15N Recovery from Labeled Fertilizer
3.4.1. Plant 15 N Recovery
3.4.1.Recovery
Plant 15N of excess 15 N in plant biomass was significantly higher under intensive management
Recovery
throughout the entire growing season, except for the first sampling date (Figure 3). In the intensive
Recovery of excess 15N in plant biomass was significantly higher under intensive management
treatment, even considering the continuous addition of 15 N labeled slurry, percent recovery of excess
throughout the entire growing season, except for the first sampling date (Figure 3). In the intensive
15 N increased during the growing season with a maximum recovery in November of 15.7% and 16.5%
treatment, even considering the continuous addition of 15N labeled slurry, percent recovery of excess
at HE and LE, respectively. Recovery under extensive management only marginally increased during
15N increased during the growing season with a maximum recovery in November of 15.7% and 16.5%
the year with a maximum of 7.2% and 8.0% at LE and HE, respectively. Climate change did not show
at HE and LE, respectively. Recovery under extensive management only marginally increased during
any significant effects on 15 N recovery in plant biomass.
the year with a maximum of 7.2% and 8.0% at LE and HE, respectively. Climate change did not show
The contribution of slurry N to harvested plant biomass N was 3.0% ± 0.3% under extensive
any significant effects on 15N recovery in plant biomass.
management. This contribution increased more than threefold to 10.7% ± 0.8% for plant biomass of the
The contribution of slurry N to harvested plant biomass N was 3.0% ± 0.3% under extensive
intensively managed lysimeters (Table 4).
management. This contribution increased more than threefold to 10.7% ± 0.8% for plant biomass of
the intensively managed lysimeters (Table 4).
Table 4. Origin of plant nitrogen (N) from fertilizer and other N sources (mainly soil organic matter
(SOM) mineralization). Error depicts standard deviation. Capital letters depict significant (p < 0.05)
Table 4. Origin of plant nitrogen (N) from fertilizer and other N sources (mainly soil organic matter
differences between management treatments and lower-case letters between climate treatments. HE:
(SOM) mineralization). Error depicts standard deviation. Capital letters depict significant (p < 0.05)
high elevation; LE: low elevation.
differences between management treatments and lower-case letters between climate treatments. HE:
high elevation; LE: low elevation.
Management Site N from Fertilizer (%) N from Other Sources (%)

Management Site 10.9 ± 0.8 (%)

HE N from Fertilizer Aa 89.1
N from Other Sources (%)
Intensive
LE 10.6 ± 0.6 Aa 89.4
Intensive HEHE
10.9 ±3.1
0.8±Aa
0.3 Ba
89.1
96.9
Extensive LE 10.6 ± 0.6±Aa 89.4
LE 2.8 0.3 Ba 97.2
Extensive HE 3.1 ± 0.3 Ba 96.9
LE 2.8 ± 0.3 Ba 97.2
Agronomy 2020, 10, x FOR PEER REVIEW
606 88 of 15
Agronomy 2020, 10, x FOR PEER REVIEW 8 of 15

Figure 3. Recovery of excess 15N in plant biomass across one growing season. Error bars represent
Figure
Figure 3. deviation.
standard Recovery
Recovery ofof excess 15N
excess 15
Asterisks Nin in plant
represent biomass
significant
plant across
across one
biomassdifferences growing
onebetween season.
season. Error
growingmanagement bars
bars represent
treatments
Error (blue
represent
standard
standard
for HE site deviation.
and red Asterisks
deviation. represent
represent
for LE site; significant
significant
p < 0.05). differences
differences
No significant effects between
between management
management
of the climate treatments
treatments
change (blue
(blue for
were
for
HEHE site site
observed. and
andHE: red
red for for
LE
high site; < 0.05).
LEpsite;
elevation; p <LE:
0.05).
Nolow Noelevation;
significant
significant effects effects
of
INT: of the change
theintensive
climate climate change treatments
treatments
management; were
wereextensive
EXT: observed.
observed. HE: highLE:
HE: high elevation;
management. elevation; LE: low
low elevation; INT:elevation; INT: intensive
intensive management; EXT:management; EXT: extensive
extensive management.
management.
15
3.4.2. 15NNRecovery
3.4.2. Recoveryin inTotal
TotalSoil
SoilNitrogen
Nitrogen
3.4.2.Recovery
15 N Recoveryratesinof
ofTotal Soil15
applied Nitrogen
15 Nfertilizer
fertilizerin inthe
the soil
soil were
were around
around twice
twice as
as high
high as
as plant
plant recovery
recovery
Recovery rates applied N
under intensive management, but four to five times higher under extensive management. The 15 N
under Recovery
intensive rates of applied but
management, 15 N fertilizer
four to five in the soilhigher
times were around twice as high
under extensive as plant recovery
management. The 15N
excess intensive
under
excess recovery in
recovery in the
the soil
soilcolumn
management, but down
column four
down tototo6060cm
five cmdepth
times was
higher
depth was40–42%
under in the
inextensive
extensive
40%–42% the treatment,
management.
extensive Thewith
15N
treatment,
no significant
excess
with no recovery difference to intensive
in thedifference
significant soil column management
to down
intensive (32–34%).
to 60management
cm depth was Soil exposed
40%–42% in
(32%–34%). to intensive
theexposed
Soil extensivemanagement
totreatment,
intensive
showed 15 N recovery between topsoil and deeper soil compared to soil from the
with no larger
management differences
significant
showed in differences
difference
larger to intensive
in 15N management
recovery between (32%–34%).
topsoil andSoildeeper
exposed to intensive
soil compared to
extensive treatment (Figure 4). Climate change simulation had no effect on slurry 15 N recovery in the
management showed larger differences in 15N recovery between topsoil and
soil from the extensive treatment (Figure 4). Climate change simulation had no effect on slurry N deeper soil compared 15to
TN pool.
soil from in
recovery thethe
extensive
TN pool.treatment (Figure 4). Climate change simulation had no effect on slurry 15N
recovery in the TN pool.

Figure
Figure 4.
4. Recovery
Recovery of
of excess
excess 15N
15
N in
in the
the soil
soil as
as aa function
function of
of depth.
depth. Asterisks
Asterisks represent
represent significant
significant
Figure 4.
differencesRecovery
between of excess
management15 N in the soil
treatments as a
(blue function
for HE; of
red depth.
for LE;Asterisks
p
differences between management treatments (blue for HE; red for LE; p < 0.05). < 0.05). represent significant
differences between management treatments (blue for HE; red for LE; p < 0.05).
3.4.3. Total 15N Recovery
3.4.3. Total 15N Recovery
Total recovery of applied 15N fertilizer in the soil up to 60 cm depth and plant biomass ranged
Total48%–50%
between recoveryandof applied 15N fertilizer in the soil up to 60 cm depth and plant biomass ranged
was affected by neither management nor climate change treatments. Higher
between
recovery 48%–50% and wasofaffected
in plant biomass by neither
the intensive management
treatment nor climate
was counteracted bychange treatments.
lower recovery Higher
in the soil
recovery
and vice in plant
versa inbiomass of the treatment.
the extensive intensive treatment was counteracted
Consequently, the estimatedbyloss
lower
of recovery
slurry N in the soil
based on
and vice versa in the extensive treatment. Consequently, the estimated loss of slurry N based on
Agronomy 2020, 10, 606 9 of 15

3.4.3. Total 15 N Recovery

Total recovery of applied 15 N fertilizer in the soil up to 60 cm depth and plant biomass ranged
between 48–50% and was affected by neither management nor climate change treatments. Higher
recovery in plant biomass of the intensive treatment was counteracted by lower recovery in the soil
Agronomy 2020, 10, x FOR PEER REVIEW 9 of 15
and vice versa in the extensive treatment. Consequently, the estimated loss of slurry N based on
unrecovered
unrecovered tracer
tracerwaswassimilar in both
similar management
in both treatments
management (50–52%),
treatments despite different
(50%–52%), despite recovery
different
patterns of slurry N in the plant–soil system.
recovery patterns of slurry N in the plant–soil system.
3.5. Nitrogen Balance
3.5. Nitrogen Balance
The N balance was negative for all treatments (Figure 5). Due to high fertilizer N loss and
The N balance was negative for all treatments (Figure 5). Due to high fertilizer N loss and
particularly high N exports through biomass harvest, the N deficit increased over the growing season
particularly high N exports through biomass harvest, the N deficit increased over the growing season
with every fertilization/cutting cycle. Consequently, the more frequent fertilization and cutting events
with every fertilization/cutting cycle. Consequently, the more frequent fertilization and cutting
in the intensive treatment resulted in a larger N deficit compared to the extensive treatment. The deficit
events in the intensive treatment resulted in a larger N deficit compared to the extensive
−1 in thetreatment.
in the grassland N balance amounted to an average of 86 ± 29 kg N ha−1 year intensive
The deficit in the grassland N balance amounted to an average of 86 ± 29 kg N ha yr−1 in the intensive
−1
treatment, significantly larger than values (47 ± 18 kg N−1 ha−1−1 year−1 ) obtained for the extensive
treatment, significantly larger than values (47 ± 18 kg N ha yr ) obtained for the extensive treatment
treatment (n = 6, p < 0.05). In contrast to management, the experimentally simulated climate change
(n = 6, p < 0.05). In contrast to management, the experimentally simulated climate change had no
had no effects on the N balance (Figure 5).
effects on the N balance (Figure 5).

Figure 5. N balance for both climate and management scenarios. Because of the low values, leaching
rates
rates were
wereexcluded
excludedfrom thethe
from graph, but included
graph, in thein
but included calculation of the balance.
the calculation Error barsError
of the balance. represent
bars
standard
representdeviation.
standard deviation.

4. Discussion
4. Discussion
4.1. N Cycling in Montane Grassland Soil
4.1.N Cycling in Montane Grassland Soil
High plant productivity and associated plant N exports that persistently exceeded total N inputs
High plant productivity and associated plant N exports that persistently exceeded total N inputs
by fertilization were pervasive across the experiment. Surprisingly, plants used slurry N at very low
by fertilization were pervasive across the experiment. Surprisingly, plants used slurry N at very low
efficiency, so that the high plant N demand was largely met by mineralization of SON rather than
efficiency, so that the high plant N demand was largely met by mineralization of SON rather than
recently applied fertilizer N. Since refueling of SON stocks by fertilizer addition was also inefficient
recently applied fertilizer N. Since refueling of SON stocks by fertilizer addition was also inefficient
due to high slurry N losses, N balances were negative, thereby indicating soil N mining in all climate
due to high slurry N losses, N balances were negative, thereby indicating soil N mining in all climate
and management treatments. The high productivity observed in this study (7.5–11.6 t ha−1 year−1 ) is
and management treatments. The high productivity observed in this study (7.5–11.6 t ha−1 yr−1) is in
in line with studies conducted in other temperate grasslands, which reported yields ranging from 5.5
line with studies conducted in other temperate grasslands, which reported yields ranging from 5.5 to
to 14.5 t ha−1 year−1 [42–44].
14.5 t ha−1 yr−1 [42–44].
N balances were calculated from tracing fertilizer N and quantifying plant and soil N pools in a
N balances were calculated from tracing fertilizer N and quantifying plant and soil N pools in a
lysimeter study. This approach has inherent methodological limitations. Since organic N in slurry was
lysimeter study. This approach has inherent methodological limitations. Since organic N in slurry
not labeled, except urea, it remained untraced in this study. However, the organic N components in
was not labeled, except urea, it remained untraced in this study. However, the organic N components
slurry require depolymerization to become available for plant-microbe competition pathways, thus the
in slurry require depolymerization to become available for plant-microbe competition pathways,
partitioning of organic N to plant and microbial uptake or loss may follow comparable patterns as
thus the partitioning of organic N to plant and microbial uptake or loss may follow comparable
revealed by 15 N tracing of ammonium and urea from slurry. Further uncertainty is associated with
patterns as revealed by 15N tracing of ammonium and urea from slurry. Further uncertainty is
associated with BNF, which was estimated from literature. However, with an estimate of 10–15 kg N
ha−1 yr−1 this is likely to be a comparably low contributor in the N balance in any case.
The 15N recovery from slurry (49% ± 7%) is in general agreement with other 15N tracing studies,
including a 24%–62% loss of 15N labeled slurry in a permanent grass sward in Ireland [43] and a 32%
recovery in a 15NH4+ tracing experiment in grassland mesocosms [45]. Overall 15N recovery may be
slightly underestimated since recovery in belowground biomass remains unmeasured within this
Agronomy 2020, 10, 606 10 of 15

BNF, which was estimated from literature. However, with an estimate of 10–15 kg N ha−1 year−1 this
is likely to be a comparably low contributor in the N balance in any case.
The 15 N recovery from slurry (49% ± 7%) is in general agreement with other 15 N tracing studies,
including a 24–62% loss of 15 N labeled slurry in a permanent grass sward in Ireland [43] and a 32%
recovery in a 15 NH4 + tracing experiment in grassland mesocosms [45]. Overall 15 N recovery may
be slightly underestimated since recovery in belowground biomass remains unmeasured within this
study. In a similar slurry tracing experiment in the study region, 15 N excess recovery in roots was only
3.4% [21], therefore of minor importance for the overall recovery.

4.2. Effects of Climate Change Simulation

Diverging effects of warming on grassland productivity were found in earlier studies, ranging
from productivity increase [9] in N-rich montane soils, to no warming effect in North American
pastures [46] and a Mediterranean grassland in California [47] or to a decrease of biomass production
in alpine/subalpine grasslands of Switzerland [48]. Ref [21] reported an increase in productivity and
plant N export by translocation of extremely organic matter-rich grassland plant–soil-mesocosms from
1260 m to the HE site of this study, but with no further productivity increase or even a decreasing
productivity upon translocation to LE due to soil moisture limitations. Other studies have similarly
suggested a high dependency of grassland productivity on soil moisture conditions [23]. In our study,
we only found minor effects of simulated climate change on plant biomass production, with a slight
increase of dry matter yield under intensive management at LE (Table 1). However, plants were not
able to increase N acquisition alongside increased biomass production, resulting in N export rates that
were not elevated under experimental climate change. Similarly, recovery of excess 15 N from labeled
slurry remained unchanged by climate change. It appears that under the climatic gradient used in this
study, any potential warming-induced changes are counterbalanced by soil moisture limitations in the
summer season (Figure 1c). Hence, in contrast to our hypothesis, our results do not show increased
soil N mining under climate change simulation. However, a design that incorporated elevated CO2
mixing ratios could yield the expected changes. It remains to be explored whether higher plant water
use efficiency under elevated CO2 can partly relieve water limitations.

4.3. Effects of Intensification

In contrast to largely absent climate change effects, we found a significant effect of management
intensity. Yields increased under intensive management by 31–40% compared to extensive management
and were accompanied by an increased N content in the plants, as predicted by [49]. Intensification is
commonly associated with plant biodiversity decrease due to shifts in competitive hierarchies [50].
While unmeasured in this study, our treatment potentially promoted the abundance of species with
higher plant tissue N concentrations [51]. Furthermore, under intensive management, plants showed
higher fertilizer N-use-efficiency than under extensive management. We assume that this is closely
related to the timing and frequency of fertilization: While the single 15 N-labeled fertilization under
extensive treatment was conducted in spring (i.e., at lower temperatures and less plant growth),
intensively managed plants receive continuous fertilization including during peak growing season,
possibly enhancing plant N uptake during periods of high productivity.
Generally, it is assumed that low fertilization rates increase the plant-microbe competition under
extensive management, leading to lower direct plant uptake and increased immobilization by microbial
biomass [52]. Higher recovery of excess 15 N in the TN pool in extensive management, indicates efficient
microbial immobilization of N; fixation in SON further supports this theory. However, irrespective of
management, higher recovery in total soil N than in plants generally demonstrates that immobilization
by free living heterotrophic soil microbes at the expense of plant uptake [52,53] is the dominant fate of
fertilizer N. This microbial driven nutrient retention may be enabled by high C availability in the organic
matter rich soil [27], leading to bioavailable N being limiting rather than C under both management
types. This finding is further strengthened by the low N leaching rates (1–5 kg ha−1 year−1 ) reported
Agronomy 2020, 10, 606 11 of 15

by [20] for the same lysimeters. Instead, the high proportion of unrecovered 15 N indicates high direct
losses from slurry along gaseous pathways as indicated by the direct measurements of [54].
Low recovery of excess 15 N in plants (7.6% ± 1.6% and 16.1% ± 1.7% for extensive and intensive,
respectively) was slightly higher, but generally in the same range (5–6%) as found by [55] in a 15 NH4
tracing study in an annual grassland in California. In contrast, recovery found in maize crops ranged
from 57% to 70% of applied 15 N slurry fertilizer [30], a considerably higher range compared to
this study. This highlights that research findings from arable systems are inadequate for informing
grassland management given the vastly different fates and functions of fertilizer N. Our observation
that mineralized SON is the main source of plant N means that the additional fertilization alone does
not explain increasing yields and N exports in the intensive treatment. In contrast, increased N exports
through mowing in the intensive treatments may be supported by priming effects of slurry-N on SOM
mineralization, thus increasing N and possibly P supply for plant nutrition [56,57]. Furthermore,
the combination of fertilization, priming and the frequency of mowing may have increased biomass
production. However, the effect of mowing frequency is controversial, as biomass production was
observed to decrease at higher cutting frequencies [22,58], suggesting threshold dynamics.
These observations challenge the paradigm that slurry N application in grasslands directly fuels
plant N demand. Rather, it appears that N supplied by slurry fertilization serves to refuel SON
stocks that are mineralized to meet direct plant N demand. Depending on recalcitrance of organic N
compounds [59] formed from slurry N (e.g., through stabilization of microbial residues), this slurry N
will be mineralized and eventually taken up by plants on the mid to long term. The time span of this
flow of slurry-N through SON requires further investigation.
Compared to surface-applied slurry-N, mineralized soil N may be more available for rhizosphere
uptake given sufficiently high mineralization rates due to spatial synchrony. In this context, the extremely
high plant N uptake from older SON observed in this study (88 to 236 kg N ha−1 season−1 ) requires sufficient
depolymerization and gross N mineralization rates. For the Haplic Cambisol soil of this study, very
high ammonification gross rates of 200 (HE) to 500 kg N ha−1 year−1 (LE) under extensive management
for only 10 cm depth were reported. In addition, heterotrophic nitrification, i.e., a direct oxidation of
organic N to nitrate, strongly contributed to soil mineral N availability on top of mineralization [19].
These findings confirm that there is sufficient N mineralization to meet the high plant N demand. For
slurry-N to refuel SON stocks, low N losses are essential and thus unfulfilled by the slurry surface
application technique in this study, clearly shown by the high proportion of unrecovered slurry N
(35–63%). The combination of high harvest N exports and high loss of slurry N led to a deficit in the N
balance at all sites and treatments (Figure 5), indicating insufficient supply of fertilizer N to maintain
SON stocks. Nitrogen mining increased from 53 ± 17 (HE) and 52 ± 19 kg N ha−1 year−1 (LE) under
extensive management to 88 ± 21 (HE) and 103 ± 34 kg N ha−1 year−1 (LE) in intensive treatments.
The high plant N export rates for the years 2012–2017 show that such N mining was likely not unique
to 2017 when 15 N tracers were used. These results conflict with [60], who reported a net C and N
sequestration under intensive management in a temperate grassland, while extensive management
led to a net loss of C and N. In contrast, our findings suggests that current management techniques
with broadcast liquid slurry application are not suitable to sustain SON and—given the intimately
linked N and C cycles—potentially also SOC stocks in our study system. This, however, remains
unresolved since C and N cycles may respond differently to changes in climate and management as
well with changes in soil C:N ratios. With plant productivity and associated N export representing the
key component for the N balance, it is speculative as to whether SOC is affected in a similar magnitude
as SON. For example, increased productivity under intensive management could result in increased
plant C transfer to soil and further C gain for soil could arise from slurry C.
Agronomy 2020, 10, 606 12 of 15

5. Conclusions
The investigated pre-alpine grassland with C- and N-rich soil was characterized by high plant
biomass production in both extensive and intensive treatments. Management was identified as the
dominant controlling factor of N fates in this grassland, while combined warming and precipitation
reduction had little effect on fates of slurry N in the plant–soil system. Disentangling effects of single
versus combined climate change factors in multi-factorial experiments in future studies may help to
better predict grassland N cycling in a changing climate.
High plant N demand was largely fed by mineralization of SOM but not by fertilizer-N, resulting
in very low N-use-efficiency and high gaseous environmental N losses. In sum, this resulted in soil
N mining that increased with management intensity but not with climate change. A potential soil N
mining of~50–100 kg N ha−1 year−1 as indicated by our results is able to significantly reduce the SON
stocks of ca. 18 t N ha−1 at decadal timescales.
On the long term, associated soil functions such as productivity and eventually SOC stocks may
decline if no grassland management with improved N-use-efficiency is implemented. Therefore,
we recommend testing of alternative slurry application such as slurry acidification, slurry injection
into soil, as well as traditional fertilization with solid manure, in combination with reduced cutting
frequency for their potential towards climate-smart sustainable management of grasslands.

Author Contributions: Conceptualization, M.D., R.K. and M.Z.-S.; methodology, M.Z.-S., S.K.K. and M.D.; data
curation, S.K.K., M.Z.-S. and M.D.; writing—original draft preparation, S.K.K. and M.Z.-S.; writing—review and
editing, M.D. and R.K.; visualization, S.K.K. and M.Z.-S.; supervision, M.D. and R.K.; project administration, R.K.,
M.D.; funding acquisition, M.D. and R.K., M.Z.-S. and S.K.K. equally contributed to this study. All authors have
read and agreed to the published version of the manuscript.
Funding: This research was funded by the German Federal Ministry of Education and Research (BMBF) in in
the SUSALPS project (Sustainable use of alpine and pre-alpine grassland soils in a changing climate), embedded
within the BonaRes initiative, Grant Number FKZ 031B0516A. Further funding was obtained from the Helmholtz
TERENO initiative.
Acknowledgments: We would like to thank Rainer Gasche for technical support during lysimeter sampling;
Sascha Klefenz for support during sampling and sample preparation; Katrin Schneider for analysis and quality
check of soil moisture data; Paul Töchterle for the analysis of plant and soil samples in the Center for stable Isotopes
(CSI) at the KIT/IMK-IFU. We furthermore want to express our gratitude to Peter Wilfahrt for proofreading and
language editing.
Conflicts of Interest: The authors declare no conflict of interest.

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