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Soil Biology & Biochemistry 81 (2015) 266e274

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Soil Biology & Biochemistry


journal homepage: www.elsevier.com/locate/soilbio

Carbon quality affects the nitrogen partitioning between plants and


soil microorganisms
hl, Gerd Gleixner*
Angelika Thuille, Judit Laufer 1, Corinna Ho
Max Planck Institute for Biogeochemistry, Hans-Knoell-Str. 10, 07745 Jena, Germany

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 12 May 2014
Received in revised form
21 November 2014
Accepted 24 November 2014
Available online 9 December 2014

We investigated how the carbon quality of soil amendments based upon their carbon (C)-to-nitrogen (N)
-ratio and their degree of aromaticity inuence soil N transformations and affect N partitioning between
soils, plants and microorganisms. A better understanding of these interactions might offer the possibility
to optimize N use efciency in agriculture. We performed a randomized pot experiment with winter
wheat and compared the inuence of naturally 13C labelled soil additives in three increasing condensation degrees, i.e. corn silage, hydrochar and pyrochar, in combination with three levels of 15N labelled
NO
3 on plant growth and N allocation. Corn silage, a lignocellulose material with a wide C-to-N-ratio and
low condensation degree, which was also used as starting material for the two other amendments,
favoured microbial growth and activity while simultaneously leading to N deciency in wheat plants. In
contrast, hydrochar and pyrochar positively inuenced plant growth independent of their C-to-N-ratio
and their degree of aromaticity. After adding hydrochar, plants did not take up the added fertilizer N but
obviously used NH
4 from mineralized hydrochar to meet their N demands. After adding pyrochar, fertilizer NO
3 was used effectively by plants and fertilizer levels were still visible in the soil, while microbial
activity was low. Our results clearly demonstrate that C quality strongly affects the N partitioning in the
plantesoilemicroorganism system. Hydrochars with a low degree of condensation that are slowly
degraded by soil microorganisms might substitute N fertilizers whereas highly condensed pyrochars
decreasing the soil microbial activity might enhance the N use efciency of plants.
2014 Elsevier Ltd. All rights reserved.

Keywords:
Hydrochar
Pyrochar
Nitrogen
15
N
Fertilizer use
Mineralization

1. Introduction
Agricultural use of soils is known for depleting their soil organic
matter (SOM) content (Post and Kwon, 2000; Aka et al., 2005).
This has adverse effects on plant growth and yield since SOM fulls
a plethora of important functions in soils, among others for soil
aeration, water holding capacity and cation exchange capacity.
Various measures have therefore been applied to minimize losses
or to restore C contents by adding amendments like compost,
biochar or other organic materials.
The various amendments added to soils in order to increase
SOM contents vary in their C-to-N ratios and contain C of different
quality: While uncharred organic matter provides mainly lignocellulose material to the soil, hydrochar adds C in the form of

* Corresponding author. Tel.: 49 (0)3641 57 6172.


E-mail addresses: athuille@bgc-jena.mpg.de (A. Thuille), ggleix@bgc-jena.mpg.
de (G. Gleixner).
1
Present address: Leibniz Centre for Agriculture Landscape Research (ZALF) e.V.,
Institute for Landscape Biogeochemistry, Eberswalder Str. 84, 15374 Mncheberg,
Germany.
http://dx.doi.org/10.1016/j.soilbio.2014.11.024
0038-0717/ 2014 Elsevier Ltd. All rights reserved.

aromatic structures arranged as spherical bodies that have a low


degree of condensation and pyrochar as higher condensed layers of
interlinked aromates (Libra et al., 2011). All types of soil amendments do not only increase organic C contents, but they also
interact with N transformations (Libra et al., 2011) according to
their chemical and physical properties, especially, when mineral
fertilizers are applied additionally. Biochars can alter the rate of N
cycling in soils (Clough and Condron, 2010; Nelissen et al., 2012)
and temporarily immobilise N in microbial biomass, especially if
their C-to-N-ratio is wide (Gajic and Koch, 2012). Some studies
found reduced N leaching rates in the presence of biochar (Ventura
et al., 2013; Zheng et al., 2013), which might be caused by an
adsorption of NO
3 from the soil solution to the surface of the char
material (Spokas et al., 2012) or a similar buffering of plantavailable NH
4 (Nelissen et al., 2012). Different char materials may
alter microbial nitrication and denitrication (Spokas et al., 2012).
For example, Nelissen et al. (2012) found increases in gross mineralisation and nitrication rates, leading to an enhanced N turnover and a transfer of N from a stable pool into a more labile pool in
the form of NH
4 . The addition of C to soil via organic amendments

might favour the process of dissimilatory NO


3 reduction to NH4

A. Thuille et al. / Soil Biology & Biochemistry 81 (2015) 266e274

267

15
12.5 and 25 kg N ha1. KNO
3 containing N (10 atom%) was added in
order to be able to track the partitioning of N between plants, microorganisms and soils. The total number of independent replicates
per treatment was three.
All pots were distributed randomly on a table in the greenhouse
and watered regularly three times per week. The appropriate watering amount was dened by previously performed watering tests
with untreated soil to investigate the water holding capacity and
the adsorption quantity of the different soils. Some representative
pots were weighed to estimate the water loss and pots were relled
accordingly. During the course of the experiment, pots with plants
progressively needed more water and, consequently, the watering
amount was adjusted to keep the soil moisture constant. This
included sometimes postponed watering in soils treated with
pyrochar, as their soil moisture persisted at a high level for a longer
period of time after watering.

(DNRA) especially under slightly anaerobic conditions and in the


presence of a wide C to NO
3 ratio (Rtting et al., 2011). On the other
hand, N2O emissions can decrease in the presence of char materials
(Malghani et al., 2013; Zheng et al., 2013) which might favour the
last step in denitrication, thus decreasing the ratio of N2O/N2N2O
in soil N emissions (Cayuela et al., 2013). This effect, however,
seems to be dependent on the nature of the added soil amendment,
since other authors found increases in N2O emissions in the presence of hydrochar char while pyrochar decreased these emissions
(Kammann et al., 2012).
Despite the available information, little is known about the effects that differences in C quality exert on the interaction between
C-containing soil amendments and the N allocation between plants
and soil organisms. A better understanding of the related interactions might offer the possibility to minimize the agricultural
impact on ecosystems by using the gained knowledge to optimize
agricultural management (Spokas et al., 2012). Clough and Condron
(2010) thus recommend research on the effects of various biochars
on N transformations and especially on the fate of N additions to
soils amended with char materials. The comparison of different
materials offers insights into the interaction of C and N cycling in
agricultural ecosystems and help to improve N use efciency of
agricultural crops. We expect that the differences in organic matter
quality cause related variations in the allocation of N. We hypothesize (1) that soil amendments with a high C-to-N-ratio and a low
condensation degree immobilise N thus causing N deciency in
plants if no N fertilizer is added to the system and (2) that soil
microbes are progressively inhibited in the presence of materials
with increasing structural organization of aromatic compounds. To
test these hypotheses, we set up a pot experiment with winter
wheat growing in soils amended with different organic materials
all based on corn silage at various levels of 15N-labelled fertilizer.

2.2. Measurements
2.2.1. Plants
All pots containing seeds were checked for seedlings on days 1,
2, 3, 4, and 7 after sowing. Height measurements of all emerged
plants were performed on days 9, 11, 15, 17, 21, 28, 31 and 38/39
after sowing in order to check for differences in growth rates between the treatments. On day 38 after sowing, chlorophyll uorescence was measured on three representative leaves per pot with
a portable chlorophyll uorometer PAM-2000 (Walz Mess- und
Regeltechnik, Effeltrich, Germany) connected to a HP 200LX
blingen, Germany) in order
Palmtop PC (Hewlett Packard GmbH, Bo
to get an objective measure for plant vitality. Leaves were shaded
with leaf clips 10 min prior to the measurement in order to get a
value for minimal uorescence in the dark (F0). Via the application
of the pulse amplitude modulation technique, maximum emission
of uorescence during a saturating light pulse (Fm) and variable
uorescence (Fv FmF0) were determined. The ratio Fv/Fm is an
indicator for the performance of photosystem II and is lowered
when plants experience stress (Krause and Weis, 1991; Maxwell
and Johnson, 2000; Baker, 2008; Murchie and Lawson, 2013). It
can thus serve as an indicator for plant vitality.
Aboveground plant parts were harvested on day 39 after sowing. Leaf area of three leaves per pot was measured with a leaf
areameter LAI 3000 (Licor inc., Lincoln, Nebraska). Dry weight was
determined separately for the measured leaves and the rest of the
biomass per pot after drying the samples at 70  C for 5 days. Roots
were carefully removed from the soil, washed and dried at 70  C to
determine their dry weight per pot.
The dried plant samples were milled and subsamples were
analysed for organic C and N with a Vario Max and a Vario EL
(Elementar Analysensysteme GmbH, Hanau, Germany), respectively. Further subsamples were analysed for d15N values with a
Delta (Thermo Finnigan MAT, Bremen, Germany) coupled online
to an elemental analyser EA 1100 (CE Instruments, Milano, Italy) via
a ConFlo III (Werner et al., 1999; Steinbeiss et al., 2008a; Gubsch
et al., 2011).

2. Material and methods


2.1. Experimental design
A greenhouse experiment was conducted to compare the effects
of organic amendments differing in their C-to-N ratios or their
physico-chemical structure e corn silage, hydrochar (produced from
corn silage by Fa. CS Carbon Solutions, Kleinmachnow, Germany) and
pyrochar (produced by the authors from corn silage) e on plant
growth and soil properties. Chemical characteristics of the material
are described by Malghani et al. (2013). 54 pots with a diameter of
10 cm were lled with sandy soil, 54 pots were lled with calcareous
soil (Table S1). Half of the pots of both soil types were planted with
nine seeds of winter wheat (Triticum aestivum L., type Bussard,
Bioland Jeebel, Germany) each; the other half was left without
vegetation. The amount of added amendment increased the organic
C content of the original soil by 30% and corresponded to 3.6, 3.5 and
2.0 weight% for silage, hydrochar and pyrochar, respectively. All three
types of additives were produced from corn silage, thus adding a d13C
label to the soils (Table 1). Three different levels of N fertilizer were
applied after germination: no fertilizer, 10 mg KNO
3 eN per pot and
20 mg KNO
3 eN per pot, corresponding to a fertilizer application of 0,
Table 1
Properties of the soils and the soil additives.

Calcareous soil
Sandy soil
Corn silage
Hydrochar
Pyrochar

C [%]

N [%]

C/N

Sand [%]

Silt [%]

Clay [%]

pH 0.01 M CaCl2

d13C []

d15N []

1.8
5.3
43.7
40.6
73.7

0.20
0.29
1.22
2.45
2.20

18
9
36
17
34

9.2
50.4
e
e
e

75.1
43.8
e
e
e

15.7
5.9
e
e
e

7.5
6.8
4.6
5.4
10.6

26.37
27.82
12.33
12.80
12.75

6.81
5.94
7.05
6.88
7.46

(1:2.5)
(1:2.5)
(1:8)
(1:1)
(1:8)

268

A. Thuille et al. / Soil Biology & Biochemistry 81 (2015) 266e274

2.2.2. Soil respiration


Gas samples from the pots without plants were taken on days
24, 31 and 35 of the experiment. For this purpose, a closed but
ventilated chamber was placed on top of each pot and kept there
for three minutes. After that time, a sample was taken with a 50 ml
syringe. 30 ml of this volume were used to ush a 12 ml exetainer in
which the remainder of the air sample was then collected. The gas
sample was analysed for CO2 concentration and d13C with an
isotope ratio mass spectrometer (DeltaPlusXL, Thermo Finnigan,
Bremen, Germany) coupled with an upstream gas chromatographic
system (Thermo Finnigan, Bremen, Germany). The values represent
repeated measurements with an analytical precision of better than
0.2 and were calculated versus V-PDB standard using CO2
reference gas (Werner et al., 2001). CO2 originating from soil
respiration was calculated as the difference between the atmospheric background and the sample. A two component isotopic
mixing model (Gleixner et al., 2002; Miller et al., 2003; Brand and
Coplen, 2012) was applied to calculate the contribution of C originating from the soil additives (C4 signal, cchar) and the soil itself (C3
signal, csoil):

cchar

d13 Ccharsoil  d13 Csoil


d13 Cchar  d13 Csoil

csoil 1  cchar

(1)

(2)

2.2.3. Soil properties


Pots without plants were harvested on day 36, soils with plants
on day 43 after sowing. Between aboveground plant and soil harvest, soil was stored in the climate chamber at 4  C. Fresh weight of
the soil was determined after eventual removal of roots. All soil
samples were taken after removing roots and mixing the soil. A
subsample of around 10 g was dried at 105  C until weight constancy in order to determine the dry weight of the soil. All
elemental and isotope analyses were corrected for the residual
water content (difference between air-dried samples and samples
dried at 105  C).
The pH of the fresh soil material was determined potentiometrically in 0.01 M CaCl2 (VDLUFA, 1991) at a soil solution ratio of
1:2.5 (pH 538 WTW Multical, WTW, Weilheim, Germany). For
mineral N determination (VDLUFA, 1991), 10 g of sieved soil were
extracted with 100 ml 0.5 M KCl solution, shaken for 60 min and

ltered (Whatman 597 lter paper, folded version). NO


3 and NH4
were measured by continuous ow analysis with a SAN plus (Skalar
Analytic GmbH, Erkelenz, Germany) for duplicate samples.
Air-dried soil samples were milled and subsamples were analysed for organic C and N with a Vario Max and a Vario EL (Elementar Analysensysteme GmbH, Hanau, Germany), respectively.
Organic C was determined as the difference between the total C
content and the inorganic C content measured after heating the
sample to 450  C for 16 h in a mufe furnace (Mund and Schulze,
2006; Don et al., 2007; Steinbeiss et al., 2008b). Further subsamples were analysed for d13C and d15N with a Delta (Thermo
Finnigan MAT, Bremen, Germany) coupled online to an EA 1100 (CE,
Milano, Italy) via a ConFlo III (Werner et al., 1999). Soil samples
were pre-treated with 120 ml of 5e6% H2SO3 (Merck, Darmstadt,
Germany) in order to remove carbonates (Steinbeiss et al., 2008b).
2.2.4. Microbial biomass
Chloroform fumigation extraction according to Vance et al.
(1987) was used to analyse microbial biomass. A sample of sieved
soil was divided into two parts: the rst part of the sample (8 g) was
mixed with 40 ml 0.05 M K2SO4 solution, shaken for one hour and

centrifuged for 5 min (Megafuge 3.0R, Heraeus Sepatech, Osterode,


Germany). The ltrate was analysed for organic C content with a
VarioTOC cube (Elementar Analysensysteme GmbH, Hanau, Germany). The second part of the sample was fumigated with ethanolfree chloroform in a desiccator for 24 h and then extracted and
analysed for organic C as described for the rst part. Extractable
microbial biomass C (Cmic) was calculated as the difference between fumigated and non-fumigated extracts (Malik et al., 2013):

Cmic

Cfum  Cnonfum
kEC

(3)

with Cfum amount of carbon measured in soil after fumigation


Cnonfum carbon measured in non-fumigated soil
kEC extractable part of the total soil microbial biomass carbon,
assumed to be 0.45 (Joergensen, 1996)
2.3. Calculation of priming effects
The contribution of soil-derived C in the soil samples taken at
the end of the experiment (csoil) was calculated according to the
equation described for soil respiration (Miller et al., 2003; Brand
and Coplen, 2012). Multiplication with the total mass of organic C
in the samples (Cnal) yielded the remaining mass of soil-derived C
at the end of the experiment (SOCrem):

SOCrem csoil $Cfinal

(4)

The lost percentage of soil organic carbon (SOClost) was calculated in relation to the initial mass of soil organic carbon (SOCinitial)
at the start of the experiment:

SOClost % 100 

SOCrem
100$SOCinitial

2.4. Calculation of the

15

(5)

N recovery rate

Recovery rates for the 15N label (15Nrec) were calculated separately for plants and soil with obs: observed values, bg: background
value and rec: recovered label applying the 4 notation as recommended for enriched samples by Brand and Coplen (2012):

clabel

4N i

15

415 Nobs  415 Nbg


415 Nlabel  415 Nbg

d15 N
Rsa 1

(6)

(6.1)



Rsa d15 N 1 *i Rref

(6.2)

R 15 N=14 N

(6.3)

Nrec clabel $Nrec

(7)

2.5. Data analysis


Statistical analyses were performed with SPSS 16.0 (IBM SPSS
Statistics). Differences between treatments were tested with oneway
and
univariate
analyses
of
variance.
StudenteNewmaneKeuls post hoc tests were performed in order to
group data. In case of data that were not distributed normally or

A. Thuille et al. / Soil Biology & Biochemistry 81 (2015) 266e274

showed non-homogeneous variances, non-parametric tests (KruskaleWallis and Median) were performed to conrm or reject the
results of the respective analyses of variance. Differences were
considered to be signicant if a < 0.05. Graphs were designed with
SigmaPlot10.0 (Systat Software Inc.).
3. Results
We examined the inuence of soil additives in three different
qualities (corn silage, hydrochar and pyrochar), two different soil
types and three levels of KNO
3 fertilizer on the germination success
and growth of winter wheat plants.
Seven days after sowing, germination success on soils treated
with hydrochar was signicantly (p < 0.001) lower (48%) compared
to the two other treatments (75% on corn silage and 77% on pyrochar). This difference was still evident at the end of the experiment
e until day 31 after sowing, germination rate on soils treated with
hydrochar reached 74% and thus was still considerably lower than
on soils treated with corn silage (91%) or pyrochar (93%). A comparable negative effect of hydrochar on plant growth could be
detected during the rst half of the experiment. Nine days after
sowing, plants on soils treated with hydrochar only achieved an
average height of 4 cm, while plants on corn silage reached 6.6 cm
and plants on soils treated with pyrochar 7.5 cm. On days 17 and 21
after sowing, differences in height growth between corn silage and
hydrochar treatments disappeared. Thus, height growth of plants
on hydrochar signicantly surpassed that of the plants on corn
silage at the end of the experiment despite a slower growth in the
initial phase, but remained below that of plants grown on hydrochar (Table 2).
Similarly, shoot dry weight of plants grown on soils with pyrochar reached 64 mg at the time of harvest compared to 45 mg and
24 mg in the hydrochar and the corn silage treatment, respectively
(Table 2). Leaf area was also remarkably higher with 23 cm2 when
plants were amended with pyrochar (hydrochar: 9 cm2, corn silage:
6 cm2). Additionally, specic leaf area was signicantly higher
compared to the corn silage and the hydrochar treatment (Table 2).
The effects of the different soil types and fertilizer levels on these
latter parameters were not signicant. A noteworthy effect caused
by the presence of hydrochar was a larger heterogeneity of the
measured plant parameters compared to the two other treatments.
The root system of winter wheat showed remarkable and
consistent differences between the different treatments (Fig. 1).
Plants on soil amended with hydrochar had a very short root system, which was not reaching the bottom of the pots. Side roots
were short and had a stunted look. In contrast, plants on soils with
corn silage developed very long and ne roots concentrating at the
bottom of the pots. Plants on soils with pyrochar showed an
Table 2
Inuence of amendment types on various plant characteristics. Numbers of days are
given as days after sowing. The parameter Fv/Fm is an indicator of the quantum yield
of photosystem II. Different letters indicate signicant differences at a 0.05.
Plant characteristic

Type of amendment
Corn silage

Germination rate at day 7 [%]


Germination rate at day 31 [%]
Plant height [cm] at day 9
Plant height [cm] at day 17
Plant height [cm] at day 21
Plant height [cm] at day 31
shoot dry weight [mg]
root-to-shoot ratio
chlorophyll uorescence [Fv/Fm]
Leaf area [cm2]
Specic leaf area [cm2 g1]

75
91
6.6
15.3
16.2
18.9
24.4
1.1
0.69
5.7
292

25 b
11 b
2.3 b
4.5 a
4.4 a
5.2 a
7.4 a
0.3 a
0.05 a
1.8 a
28 a

Hydrochar
48
74
4.0
15.0
17.9
24.1
44.5
0.3
0.78
9.2
299

31 a
27 a
2.3 a
4.9 a
4.6 a
4.9 b
27.7 b
0.1 b
0.01 c
3.3 b
32 a

Pyrochar
77
93
7.5
17.8
23.6
18.9
63.6
0.4
0.73
22.5
400

22 b
12 b
3.0 c
4.3 b
5.9 b
6.1 c
5.6 c
0.2 b
0.05 b
5.1 c
43 b

269

intermediate growth with roots that seemed to be only slightly less


developed than those of plants on soils with corn silage.
Consequently, the root-shoot ratio differed signicantly between plants on corn silage-amended soil and plants growing with
either biochar, with plants on corn silage exhibiting the widest
root/shoot ratio. However, no signicant differences were found
between plants on pyrochar and hydrochar (Table 2).
Shoot N contents were measured in order to determine whether
N nutrition was inuenced by the different amendments and thus
resulted in the different growth characteristics. Analyses revealed
that plants growing on hydrochar had highest shoot N contents
despite the fact that the respective plants were smaller than those
growing on pyrochar (Fig. 2). N concentrations were higher in
plants growing on sandy soil. While N concentrations of plants
growing with corn silage or pyrochar amendments increased with
the amount of fertilizer added, N concentrations in plants on
hydrochar-amended soil seemed to be independent of fertilizer
addition.
The good nutritional status of plants growing with hydrochar
was further corroborated by the measurements of chlorophyll
uorescence. The uorescence parameter Fv/Fm, which decreases
when plants experience stress (Krause and Weis, 1991; Maxwell
and Johnson, 2000; Baker, 2008; Murchie and Lawson, 2013) was
highest for plants growing on hydrochar amended soil and lowest
for those on soils with corn silage (Table 2). These results conrmed
the observations made before the harvest of plants: Whereas plants
with biochar treatments had a healthy green leaf colour independent of their actual size, plants grown with corn silage had a more
yellowish look. Soil type and fertilizer level had no signicant effects on chlorophyll uorescence.
Plants growing with hydrochar were vital plants wellnourished with N. Since their N content was independent of fertilizer level, we used the 15N label to examine how much of the
added KNO3 fertilizer was taken up by plants in the different
treatments. While plants growing on soil amended with pyrochar
used large amounts (60e70%) of the added fertilizer N (Fig. 3),
signicantly lower amounts of fertilizer N were taken up by plants
growing with corn silage as well as by plants growing with
hydrochar; the latter using almost no fertilizer N (<10%). Soil type
had no inuence on fertilizer uptake; similarly, there were no
signicant differences between the two fertilizer levels despite a
trend towards higher NO
3 uptake by plants growing with pyrochar
at lower fertilizer levels (Table 3). Independent of soil amendment,
most of the NO
3 eN was detected in shoots. However, on soil
amended with corn silage, this effect was less pronounced: here,
around 30% of the labelled NO
3 eN was found in roots, while with
both biochar treatments, roots only contained around 10% of the
added fertilizer N.
These differences in fertilizer use were also reected by the
amounts of KCl-extractable NO
3 in soil (Fig. 4, left panel). Lowest
values of NO
3 eN were detected in soil amended with corn silage
1
with values lower than 1 mg NO
soil. Soils amended with
3 g

1
hydrochar had NO3 concentrations of around 2e21 mg NO
3 eN g
soil, which were signicantly higher than for silage-amended soils
but considerably lower than values found in pyrochar-amended soil
1
(2e89 mg NO
soil). Fertilizer levels were still visible in
3 eN g
pyrochar-amended soils and the presence of plants caused a
depletion of NO
3 in soils.
Remarkable results were found for NH
4 (Fig. 4, right panel).
Despite the fact that no NH
4 was added via fertilizer, high amounts
1
of more than 150 mg NH
soil were found in the presence of
4 eN g
hydrochar, while NH
concentrations
remained low in the presence
4
1
of the two other amendments (<1.5 mg NH
soil). Soil type
4 N g
and fertilizer level had no inuence on NH
concentrations;
in
4

270

A. Thuille et al. / Soil Biology & Biochemistry 81 (2015) 266e274

Fig. 1. Typical root system of winter wheat plants grown with corn silage (left panel), hydrochar (middle) and pyrochar (right panel).

Fig. 2. Shoot N measured at the day of plant harvest. Results for the sandy soil are shown in the left half of the graph (shaded in grey) and results for the calcareous soil in the right

half. Numbers 0, 1 and 2 indicate the different fertilizer levels: no KNO
3 , 10 and 20 mg KNO3 per pot. A univariate analysis of variance revealed the signicant inuence of soil type,
char type and fertilizer level. Different letters and asterisks denote signicant differences at a 0.05. Data are given with respective standard deviations. HTC: hydrochar, PC:
pyrochar.

contrast, there was a signicant inuence of plant presence for


silage and hydrochar.
In order to gain insight into a potential competition for mineral
N between plants and soil microbes, we also measured soil respiration and microbial biomass C. Both soils amended with corn
silage and with hydrochar showed relatively high soil respiration
compared to pyrochar-treated soil (Fig. 5a). However, the portion of
native soil organic matter respired was considerably higher in the
presence of pyrochar.

Table 3
Inuence of soil type, type of amendment, fertilizer level and plant presence on
selected plant and soil parameters according to univariate ANOVA. A factor is
considered to be of signicant inuence if p < 0.05.
Plant/soil parameter p-values for the inuence of

15
Fig. 3. Percentage of added KNO
N incor3 fertilizer used by plants as revealed by
poration. F1 and F2 denote the fertilizer levels (10 or 20 mg N pot1). Different letters
indicate signicant differences at a 0.05. Soil type had no signicant inuence,
squares give mean values of both fertilizer levels. Data are given with respective
standard deviations. HTC: hydrochar, PC: pyrochar.

Shoot N content
Fertilizer use
NO
3 at harvest
NH
4 at harvest
respired CO2
Microbial biomass C

Soil type Amendment Fertilizer level

Plant presence

0.000
0.073
0.027
0.910
0.293
0.003

e
e
0.000
0.002
e
0.001

0.000
0.000
0.000
0.000
0.000
0.000

0.001
0.200
0.026
0.987
0.103
0.297

A. Thuille et al. / Soil Biology & Biochemistry 81 (2015) 266e274

Microbial biomass C (Fig. 5b) showed a similar pattern with


highest amounts in the presence of silage and low amounts in
pyrochar-treated pots. While fertilizer level neither inuenced the
amount of CO2 respired nor the microbial biomass C, presence of
plants signicantly reduced microbial biomass. In the presence of
hydrochar, microbial biomass showed a rather high variance and
the effect of the different soils was considerably larger than for the
other treatments: There were signicantly higher amounts of microbial C in sandy soil compared to calcareous soil. Nevertheless,
soil respiration did not differ signicantly between the two soils.
However, more native soil organic matter was decomposed when
microbial biomass was higher.
4. Discussion
Both char materials had an overall positive effect on the
aboveground growth of wheat plants in our pot experiment when
compared with the inuence of uncharred corn silage. This is in
agreement with the results from a meta-analysis by Jeffery et al.
(2011) who found an average increase in crop yield of 10% when
soils were amended with biochar produced via pyrolysis.
Taghizadeh-Toosi et al. (2011) found no toxic effects of freshly
produced pyrochar (from pine wood chips) on the growth of Lolium
perenne. In contrast, a reduction in shoot biomass of Medicago
sativa and the occurrence of leaf tip necroses in the presence of
biochar produced via hydrothermal carbonization from spent
brewer's grains was reported by George et al. (2012). Likewise,
Rillig et al. (2010) noticed growth reductions in Taraxacum and
Trifolium plants if hydrochar was applied at rates above 10 vol%. In
our study, the shoot dry weight of plants grown with either type of
char was higher than that of plants grown with corn silage and the
results of the chlorophyll uorescence measurements point to an
active photosystem II, especially in the presence of hydrochar. Toxic
effects as described for hydrochar by Jandl et al. (2013) could thus
be limited to the process of germination, which was reduced in the
presence of hydrochar (compare Bargmann et al., 2013).
While Gajic and Koch (2012) explain initial growth reductions of
sugar beet plants in the presence of hydrochar by the temporary
immobilization of N, we did not detect any N limitation caused by
biochar addition. On the contrary, shoot N contents of plants
growing with hydrochar were even slightly higher compared to

271

those of plants growing with pyrochar. On corn silage amended


soils, however, shoot N concentrations were three times lower and
plants showed signs of N limitation like yellowish leaf tips, a very
extensive root system and reduced values for chlorophyll uorescence which are an indicator for stress (Maxwell and Johnson,
2000; Baker, 2008; Murchie and Lawson, 2013). The low soil NO
3
concentrations found in the presence of corn silage are in agreement with the low shoot N concentrations and point to an immobilisation of NO
3 by soil microbes. While wheat plants growing on
hydrochar took up a large amount of the added fertilizer N as reected by both the their shoot N concentrations and d15N values,
plants on hydrochar obviously used a different N source as their
shoot N contents seem independent of the amount of applied NO
3
fertilizer and only negligible amounts of fertilizer N were
incorporated.
We found unexpectedly high NH
4 concentrations in soils
treated with hydrochar, while NH
4 was only present in minor
amounts in the presence of the two other amendments. It seems
plausible that wheat plants used NH
4 as N source when in contact with hydrochar. Several indicators support this hypothesis:
Plants growing on hydrochar had an underdeveloped root system
compared to the other treatments. It has been shown that contact
of the growing root tip of Arabidopsis plants with NH
4 may lead
to suppressed cell elongation (Li et al., 2010) and thus to stunted
roots. Plants grown with NH
4 as main N source may also show
reduced yield, higher N concentrations in leaves when compared
with plants fed with NO
3 eN (Roosta and Schjoerring, 2008;
n et al., 2013) and a stronger discrimination against 15N
Setie
n et al., 2013). These observations are supported by our
(Setie
data: Compared to plants growing with pyrochar, plants on
hydrochar were less tall and had a signicantly smaller leaf area
and shoot dry weight, while their shoot N concentrations were
highest. We found no discrimination against 15N in our experiment, all d15N values were positive, however, signicantly less
15
N was incorporated in roots and shoots when compared with
plants on pyrochar.
Since we did not add NH
4 , the question arises where the rather

1
large amounts of NH
dry soil) in the
4 (up to 0.15 mg NH4 eN g
soils amended with hydrochar came from and what happened with
the added fertilizer NO
3 . Both microbial biomass and microbial
respiration were relatively high in the presence of hydrochar. This is


Fig. 4. NO
3 and NH4 concentrations in soils after harvest of plants. NO3 concentrations were signicantly inuenced by the different soil types, char amendments and in the case of
pyrochar also by fertilizer level as well as the presence of plants. For hydrochar, values on calcareous soil are shown separately for pots with and without plants because there were
visible differences, however, these differences were not signicant due to a high variance. The square in between shows the mean value for pots with and without plants. NH
4
concentrations were only inuenced by type of amendment and the presence of plants (Silage, hydrochar). Data are given with respective standard deviations. HTC: hydrochar, PC:
pyrochar. Different letters and asterisks denote signicant differences between treatments at a 0.05.

272

A. Thuille et al. / Soil Biology & Biochemistry 81 (2015) 266e274

Fig. 5. Soil respiration measured as CO2 evolution in 3 min (a) and microbial biomass carbon (b) in the different treatments. Fertilizer level had no signicant inuence on CO2
evolution, values were only determined for pots without plants, light brown parts of the bars show fraction arising from the decomposition of native soil organic matter. Microbial
biomass carbon was also independent of fertilizer level but signicantly inuenced by soil type, type of amendment and presence of plants. Here, green bars show results in the
presence of plants, dotted bars symbolize calcareous soil, plain bars sandy soil. Data are given with respective standard deviations. HTC: hydrochar, PC: pyrochar. (For interpretation
of the references to colour in this gure legend, the reader is referred to the web version of this article.)

in agreement with other studies observing an increase in microbial


biomass or activity in the presence of hydrochar (Rillig et al., 2010;
Libra et al., 2011; Kammann et al., 2012; Jandl et al., 2013).
Concurrently, the d13C signature of microbial respiration showed
that microbes mainly respired CO2 from the added hydrochar,
thereby mineralizing about 80% of the hydrochar-C added to the
soil (corresponding to 13.2 mg hydrochar-C g1 dry soil). N losses
from hydrochar amounted to 0.38 mg N g1 dry soil, which represents 40% of the N introduced via hydrochar assuming that all N
losses originated from the mineralization of the soil amendment.
The comparatively narrow C-to-N-ratio of the hydrochar (17) suggests that it was readily accessible for decomposition (Seneviratne,
2000), making the contained N available to plants and microbes.
Another possible process creating NH
4 is the dissimilatory reduc
tion of NO
3 to NH4 (Buresh and Patrick, 1978; Morozkina and
Kurakov, 2007). The addition of easily available organic C via
hydrochar leads to a high C-to- NO
3 -ratio in the soil that is
necessary for this microbial transformation. Since soils amended
with hydrochar were moist, thus enabling the development of
anaerobic microsites, we cannot exclude the possibility that DNRA
occurred in our experiment (compare Rtting et al., 2011). However, plants growing on hydrochar did not incorporate large
amounts of 15N-labelled N into their biomass, which would have
been expected if DNRA played a major role in NH
4 production.
Alternatively, hydrochar might have decreased nitrication rates as
suggested by Ventura et al. (2013) for apple orchards amended with
biochar as well as by Subedi et al. (2013) who found less NO
3 but
more NH
4 in loamy soils amended with pig slurry in the presence
of hydrochar compared to pyrochar. Losses of fertilizer N in the
form of N2O, on the other hand, are considered to be of minor
importance since an experiment with the same chars and soils by
Malghani et al. (2013) revealed reduced emissions in the presence
of hydrochar. Other studies also found decreasing N2O emissions in
the presence of biochar (Cayuela et al., 2013; Zheng et al., 2013).
However, hydrochar might have favoured the last step of denitrication, thus reducing the ration of N2O/(N2N2O) in denitrication end products (Cayuela et al., 2013). As a consequence, it seems
plausible that fertilizer NO
3 was at least partly lost due to volatilization as N2, while NH
4 produced via the mineralisation of the
added hydrochar was conserved in the soileplantesystem due to a
tight coupling of mineralization and NH
4 uptake by plants.

In soils amended with uncharred corn silage, microbes were


even more abundant and respired larger amounts of CO2 than in the
presence of hydrochar. Simultaneously, plants had an extensively
developed root system and low shoot N concentration, suggesting
that microbes were more effective in competing for mineral N, thus
leading to N deciency in wheat plants. Here, the addition of a
material with a wide C-to-N-ratio might lead to processes similar to
those described for the incorporation of crop residues in cereal
cultivation (Jensen, 1997; Bijay-Singh et al., 2001; Montoyalez et al., 2009): the labile C-fraction added via uncharred
Gonza
corn silage might have caused an initial immobilization of N and a
lower rate of fertilizer uptake by plants (compare Kongchum et al.,
2007), which necessitates a higher rate of N fertilization to sustain
plant growth while at the same time promoting microbial growth.
The overall effect of the three soil amendments on the allocation
of N between plants and microbes can be summarized as follows
(Fig. 6):
 Corn silage, consisting mainly of lignocellulose material with a
wide C-to-N-ratio, favours microbes independent of fertilizer
level and probably causes an immobilisation of N, while plants
take up only small amounts of NO
3 fertilizer and remain rather
small showing signs of nutrient deciency. Corn silage is readily
available for microbial decomposition, as shown by d13C values
of microbial respiration.
 Hydrochar, characterized by a narrow C-to-N-ratio and spherically arranged aromatic structures, causes mixed effects: While
plants are well nourished with N mainly originating from the
decomposition of the soil amendment, microbial biomass and
respiration are also high and large amounts of the added material are released as CO2 by microbes, which effectively
mineralize the hydrochar.
 Plants grown with pyrochar (wide C-to-N-ratio, highly aromatic
structure) effectively use the added fertilizer N and compete
successfully against microbes, which only reach a relatively
small biomass and respire signicantly less C compared to the
two other treatments. Additionally, a much smaller percentage
of the respired CO2 originates from pyrochar.
Our results thus conrm the hypothesis that plants suffer from
N immobilisation if lignocellulose material of high C-to-N-ratio is

A. Thuille et al. / Soil Biology & Biochemistry 81 (2015) 266e274

273

Fig. 6. Summary of interactions between the three soil amendments and the nitrogen balance. The dotted line in the right panel symbols interactions between nitrate and the
surface of the pyrochar. Silage structure from: Yinghuai et al. (2013), hydrochar structure from: Kumar et al. (2011), pyrochar structure from: Heidenreich et al. (1968). See text for
further explanation.

added, while pyrochar promotes plant growth despite its equally


wide C-to-N-ratio. Microorganisms, on the other hand are favoured
in the presence of materials which are easily degradable either due
to the absence of aromatic structures (corn silage) or their lower
level of structural organization (hydrochar). In this context,
hydrochar takes an intermediate position e despite containing also
aromatic structures, its narrow C-to-N-ratio enables microbes to
mineralize the material to a similar degree as uncharred corn silage
without immobilising the contained N, thus sustaining a relatively
large amount of microbial biomass while simultaneously guaranteeing a strong plant growth. A study by Bai et al. (2013) conrms
that hydrochar can be similar in its degradability to uncharred
material who produced both types of char from Miscanthus and
found that the half-life of hydrochar was comparable to that of the
educt and much lower than for pyrochar. However, the application
of additional N fertilizer has to be evaluated carefully, since plants
are obviously not able to take up the added N but use the NH
4 set
free via the mineralization of the char material instead. The situation is different with pyrochar: A 15N tracer experiment with
pyrochar produced from corn revealed that pyrochar caused a
transfer of N from a stabile to a more labile pool and thus increased
plant available N (Nelissen et al., 2012). This is in agreement with
Zheng et al. (2013) who argued that biochar might increase the
bioavailability of N and thus lead to reduced N fertilizer requirements in corn plants. Likewise, in our experiment plants
effectively use added NO
3 fertilizer in the presence of pyrochar,
which might help to stabilize NO
3 in soil, as suggested by the fact
that different fertilizer levels are well reected by soil NO
3 concentrations. Both types of char thus have the potential to reduce the
required fertilizer amounts e hydrochar because N might be made
available due to the mineralization of the char material, pyrochar
because it stabilizes N and increases its bioavailability for plants.
Acknowledgements
This study was funded within the framework of the EnerChem
initiative of the Max Planck Society. The authors thank Carbon
Solution Ltd. for providing the hydrochar.

Appendix A. Supplementary data


Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.soilbio.2014.11.024.

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