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Soil Biology & Biochemistry 81 (2015) 266e274

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Soil Biology & Biochemistry

journal homepage: www.elsevier .com/locate/soi lbio

Carbon quality affects the nitrogen partitioning between plants andsoil microorganisms

Angelika Thuille, Judit Laufer 1, Corinna H€ohl, Gerd Gleixner*

Max Planck Institute for Biogeochemistry, Hans-Knoell-Str. 10, 07745 Jena, Germany

a r t i c l e i n f o

Article history:Received 12 May 2014Received in revised form21 November 2014Accepted 24 November 2014Available online 9 December 2014

Keywords:HydrocharPyrocharNitrogen15NFertilizer useMineralization

* Corresponding author. Tel.: þ49 (0)3641 57 6172.E-mail addresses: athuille@bgc-jena.mpg.de (A. T

de (G. Gleixner).1 Present address: Leibniz Centre for Agriculture La

Institute for Landscape Biogeochemistry, EberswaldeGermany.

http://dx.doi.org/10.1016/j.soilbio.2014.11.0240038-0717/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

We investigated how the carbon quality of soil amendments based upon their carbon (C)-to-nitrogen (N)-ratio and their degree of aromaticity influence soil N transformations and affect N partitioning betweensoils, plants and microorganisms. A better understanding of these interactions might offer the possibilityto optimize N use efficiency in agriculture. We performed a randomized pot experiment with winterwheat and compared the influence of naturally 13C labelled soil additives in three increasing conden-sation degrees, i.e. corn silage, hydrochar and pyrochar, in combination with three levels of 15N labelledNO3

� on plant growth and N allocation. Corn silage, a lignocellulose material with a wide C-to-N-ratio andlow condensation degree, which was also used as starting material for the two other amendments,favoured microbial growth and activity while simultaneously leading to N deficiency in wheat plants. Incontrast, hydrochar and pyrochar positively influenced plant growth independent of their C-to-N-ratioand their degree of aromaticity. After adding hydrochar, plants did not take up the added fertilizer N butobviously used NH4

þ from mineralized hydrochar to meet their N demands. After adding pyrochar, fer-tilizer NO3

� was used effectively by plants and fertilizer levels were still visible in the soil, while microbialactivity was low. Our results clearly demonstrate that C quality strongly affects the N partitioning in theplantesoilemicroorganism system. Hydrochars with a low degree of condensation that are slowlydegraded by soil microorganisms might substitute N fertilizers whereas highly condensed pyrocharsdecreasing the soil microbial activity might enhance the N use efficiency of plants.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Agricultural use of soils is known for depleting their soil organicmatter (SOM) content (Post and Kwon, 2000; Akça et al., 2005).This has adverse effects on plant growth and yield since SOM fulfilsa plethora of important functions in soils, among others for soilaeration, water holding capacity and cation exchange capacity.Various measures have therefore been applied to minimize lossesor to restore C contents by adding amendments like compost,biochar or other organic materials.

The various amendments added to soils in order to increaseSOM contents vary in their C-to-N ratios and contain C of differentquality: While uncharred organic matter provides mainly ligno-cellulose material to the soil, hydrochar adds C in the form of

huille), ggleix@bgc-jena.mpg.

ndscape Research (ZALF) e.V.,r Str. 84, 15374 Müncheberg,

aromatic structures arranged as spherical bodies that have a lowdegree of condensation and pyrochar as higher condensed layers ofinterlinked aromates (Libra et al., 2011). All types of soil amend-ments do not only increase organic C contents, but they alsointeract with N transformations (Libra et al., 2011) according totheir chemical and physical properties, especially, when mineralfertilizers are applied additionally. Biochars can alter the rate of Ncycling in soils (Clough and Condron, 2010; Nelissen et al., 2012)and temporarily immobilise N in microbial biomass, especially iftheir C-to-N-ratio is wide (Gajic and Koch, 2012). Some studiesfound reduced N leaching rates in the presence of biochar (Venturaet al., 2013; Zheng et al., 2013), which might be caused by anadsorption of NO3

� from the soil solution to the surface of the charmaterial (Spokas et al., 2012) or a similar buffering of plant-available NH4

þ (Nelissen et al., 2012). Different char materials mayalter microbial nitrification and denitrification (Spokas et al., 2012).For example, Nelissen et al. (2012) found increases in gross min-eralisation and nitrification rates, leading to an enhanced N turn-over and a transfer of N from a stable pool into a more labile pool inthe form of NH4

þ. The addition of C to soil via organic amendmentsmight favour the process of dissimilatory NO3

� reduction to NH4þ

A. Thuille et al. / Soil Biology & Biochemistry 81 (2015) 266e274 267

(DNRA) especially under slightly anaerobic conditions and in thepresence of a wide C to NO3

� ratio (Rütting et al., 2011). On the otherhand, N2O emissions can decrease in the presence of char materials(Malghani et al., 2013; Zheng et al., 2013) which might favour thelast step in denitrification, thus decreasing the ratio of N2O/N2þN2Oin 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 pres-ence of hydrochar char while pyrochar decreased these emissions(Kammann et al., 2012).

Despite the available information, little is known about the ef-fects that differences in C quality exert on the interaction betweenC-containing soil amendments and the N allocation between plantsand soil organisms. A better understanding of the related in-teractions might offer the possibility to minimize the agriculturalimpact on ecosystems by using the gained knowledge to optimizeagricultural management (Spokas et al., 2012). Clough and Condron(2010) thus recommend research on the effects of various biocharson N transformations and especially on the fate of N additions tosoils amended with char materials. The comparison of differentmaterials offers insights into the interaction of C and N cycling inagricultural ecosystems and help to improve N use efficiency ofagricultural crops. We expect that the differences in organic matterquality cause related variations in the allocation of N. We hypoth-esize (1) that soil amendments with a high C-to-N-ratio and a lowcondensation degree immobilise N thus causing N deficiency inplants if no N fertilizer is added to the system and (2) that soilmicrobes are progressively inhibited in the presence of materialswith increasing structural organization of aromatic compounds. Totest these hypotheses, we set up a pot experiment with winterwheat growing in soils amended with different organic materialsall based on corn silage at various levels of 15N-labelled fertilizer.

2. Material and methods

2.1. Experimental design

A greenhouse experiment was conducted to compare the effectsof organic amendments differing in their C-to-N ratios or theirphysico-chemical structure e corn silage, hydrochar (produced fromcorn silage by Fa. CS Carbon Solutions, Kleinmachnow, Germany) andpyrochar (produced by the authors from corn silage) e on plantgrowth and soil properties. Chemical characteristics of the materialare described by Malghani et al. (2013). 54 pots with a diameter of10 cmwere filled with sandy soil, 54 pots were filled with calcareoussoil (Table S1). Half of the pots of both soil types were planted withnine seeds of winter wheat (Triticum aestivum L., type “Bussard”,Bioland Jeebel, Germany) each; the other half was left withoutvegetation. The amount of added amendment increased the organicC content of the original soil by 30% and corresponded to 3.6, 3.5 and2.0weight% for silage, hydrochar and pyrochar, respectively. All threetypes of additives were produced from corn silage, thus adding a d13Clabel to the soils (Table 1). Three different levels of N fertilizer wereapplied after germination: no fertilizer, 10 mg KNO3

�eN per pot and20mgKNO3

�eNper pot, corresponding to a fertilizer application of 0,

Table 1Properties of the soils and the soil additives.

C [%] N [%] C/N Sand [%] Silt

Calcareous soil 1.8 0.20 18 9.2 75.1Sandy soil 5.3 0.29 9 50.4 43.8Corn silage 43.7 1.22 36 e e

Hydrochar 40.6 2.45 17 e e

Pyrochar 73.7 2.20 34 e e

12.5 and 25 kgN ha�1. KNO3� containing 15N (10 atom%)was added in

order to be able to track the partitioning of N between plants, mi-croorganisms and soils. The total number of independent replicatesper treatment was three.

All pots were distributed randomly on a table in the greenhouseand watered regularly three times per week. The appropriate wa-tering amount was defined by previously performed watering testswith untreated soil to investigate the water holding capacity andthe adsorption quantity of the different soils. Some representativepots wereweighed to estimate thewater loss and pots were refilledaccordingly. During the course of the experiment, pots with plantsprogressively needed more water and, consequently, the wateringamount was adjusted to keep the soil moisture constant. Thisincluded sometimes postponed watering in soils treated withpyrochar, as their soil moisture persisted at a high level for a longerperiod of time after watering.

2.2. Measurements

2.2.1. PlantsAll pots containing seeds were checked for seedlings on days 1,

2, 3, 4, and 7 after sowing. Height measurements of all emergedplants were performed on days 9, 11, 15, 17, 21, 28, 31 and 38/39after sowing in order to check for differences in growth rates be-tween the treatments. On day 38 after sowing, chlorophyll fluo-rescence was measured on three representative leaves per pot witha portable chlorophyll fluorometer PAM-2000 (Walz Mess- undRegeltechnik, Effeltrich, Germany) connected to a HP 200LXPalmtop PC (Hewlett Packard GmbH, B€oblingen, Germany) in orderto get an objective measure for plant vitality. Leaves were shadedwith leaf clips 10 min prior to the measurement in order to get avalue for minimal fluorescence in the dark (F0). Via the applicationof the pulse amplitude modulation technique, maximum emissionof fluorescence during a saturating light pulse (Fm) and variablefluorescence (Fv ¼ Fm�F0) were determined. The ratio Fv/Fm is anindicator for the performance of photosystem II and is loweredwhen plants experience stress (Krause and Weis, 1991; Maxwelland Johnson, 2000; Baker, 2008; Murchie and Lawson, 2013). Itcan thus serve as an indicator for plant vitality.

Aboveground plant parts were harvested on day 39 after sow-ing. Leaf area of three leaves per pot was measured with a leafareameter LAI 3000 (Licor inc., Lincoln, Nebraska). Dry weight wasdetermined separately for the measured leaves and the rest of thebiomass per pot after drying the samples at 70 �C for 5 days. Rootswere carefully removed from the soil, washed and dried at 70 �C todetermine their dry weight per pot.

The dried plant samples were milled and subsamples wereanalysed for organic C and N with a Vario Max and a Vario EL(Elementar Analysensysteme GmbH, Hanau, Germany), respec-tively. Further subsamples were analysed for d15N values with aDeltaþ (Thermo Finnigan MAT, Bremen, Germany) coupled onlineto an elemental analyser EA 1100 (CE Instruments, Milano, Italy) viaa ConFlo III (Werner et al., 1999; Steinbeiss et al., 2008a; Gubschet al., 2011).

[%] Clay [%] pH 0.01 M CaCl2 d13C [‰] d15N [‰]

15.7 7.5 (1:2.5) �26.37 6.815.9 6.8 (1:2.5) �27.82 5.94

e 4.6 (1:8) �12.33 7.05e 5.4 (1:1) �12.80 6.88e 10.6 (1:8) �12.75 7.46

A. Thuille et al. / Soil Biology & Biochemistry 81 (2015) 266e274268

2.2.2. Soil respirationGas samples from the pots without plants were taken on days

24, 31 and 35 of the experiment. For this purpose, a closed butventilated chamber was placed on top of each pot and kept therefor three minutes. After that time, a sample was taken with a 50 mlsyringe. 30ml of this volumewere used to flush a 12ml exetainer inwhich the remainder of the air sample was then collected. The gassample was analysed for CO2 concentration and d13C with anisotope ratio mass spectrometer (DeltaPlusXL, Thermo Finnigan,Bremen, Germany) coupled with an upstream gas chromatographicsystem (Thermo Finnigan, Bremen, Germany). The values representrepeated measurements with an analytical precision of better than±0.2‰ and were calculated versus V-PDB standard using CO2reference gas (Werner et al., 2001). CO2 originating from soilrespiration was calculated as the difference between the atmo-spheric background and the sample. A two component isotopicmixing model (Gleixner et al., 2002; Miller et al., 2003; Brand andCoplen, 2012) was applied to calculate the contribution of C origi-nating from the soil additives (C4 signal, cchar) and the soil itself (C3signal, csoil):

cchar ¼d13CðcharþsoilÞ � d13CðsoilÞd13CðcharÞ � d13CðsoilÞ

(1)

csoil ¼ 1� cchar (2)

2.2.3. Soil propertiesPots without plants were harvested on day 36, soils with plants

on day 43 after sowing. Between aboveground plant and soil har-vest, soil was stored in the climate chamber at 4 �C. Fresh weight ofthe soil was determined after eventual removal of roots. All soilsamples were taken after removing roots and mixing the soil. Asubsample of around 10 g was dried at 105 �C until weight con-stancy in order to determine the dry weight of the soil. Allelemental and isotope analyses were corrected for the residualwater content (difference between air-dried samples and samplesdried at 105 �C).

The pH of the fresh soil material was determined potentio-metrically in 0.01 M CaCl2 (VDLUFA, 1991) at a soil solution ratio of1:2.5 (pH 538 WTW Multical, WTW, Weilheim, Germany). Formineral N determination (VDLUFA, 1991), 10 g of sieved soil wereextracted with 100 ml 0.5 M KCl solution, shaken for 60 min andfiltered (Whatman 597½ filter paper, folded version). NO3

� and NH4þ

weremeasured by continuous flow analysis with a SAN plus (SkalarAnalytic GmbH, Erkelenz, Germany) for duplicate samples.

Air-dried soil samples were milled and subsamples were ana-lysed for organic C and N with a Vario Max and a Vario EL (Ele-mentar Analysensysteme GmbH, Hanau, Germany), respectively.Organic C was determined as the difference between the total Ccontent and the inorganic C content measured after heating thesample to 450 �C for 16 h in a muffle furnace (Mund and Schulze,2006; Don et al., 2007; Steinbeiss et al., 2008b). Further sub-samples were analysed for d13C and d15N with a Deltaþ (ThermoFinniganMAT, Bremen, Germany) coupled online to an EA 1100 (CE,Milano, Italy) via a ConFlo III (Werner et al., 1999). Soil sampleswere pre-treated with 120 ml of 5e6% H2SO3 (Merck, Darmstadt,Germany) in order to remove carbonates (Steinbeiss et al., 2008b).

2.2.4. Microbial biomassChloroform fumigation extraction according to Vance et al.

(1987) was used to analyse microbial biomass. A sample of sievedsoil was divided into two parts: the first part of the sample (8 g) wasmixed 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 filtrate was analysed for organic C content with aVarioTOC cube (Elementar Analysensysteme GmbH, Hanau, Ger-many). The second part of the sample was fumigated with ethanol-free chloroform in a desiccator for 24 h and then extracted andanalysed for organic C as described for the first part. Extractablemicrobial biomass C (Cmic) was calculated as the difference be-tween fumigated and non-fumigated extracts (Malik et al., 2013):

Cmic ¼Cfum � Cnonfum

kEC(3)

with Cfum ¼ amount of carbon measured in soil after fumigationCnonfum ¼ carbon measured in non-fumigated soilkEC ¼ 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 atthe end of the experiment (csoil) was calculated according to theequation described for soil respiration (Miller et al., 2003; Brandand Coplen, 2012). Multiplication with the total mass of organic Cin the samples (Cfinal) yielded the remaining mass of soil-derived Cat the end of the experiment (SOCrem):

SOCrem ¼ csoil$Cfinal (4)

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

SOClost½%� ¼ 100� SOCrem100$SOCinitial

(5)

2.4. Calculation of the 15N recovery rate

Recovery rates for the 15N label (15Nrec) were calculated sepa-rately for plants and soil with obs: observed values, bg: backgroundvalue and rec: recovered label applying the 4 notation as recom-mended for enriched samples by Brand and Coplen (2012):

clabel ¼415Nobs � 415Nbg

415Nlabel � 415Nbg(6)

4N ¼ d15NiRsa þ 1

(6.1)

iRsa ¼�d15Nþ 1

�*iRref (6.2)

iR ¼ ½15N�=½14N� (6.3)

15Nrec ¼ clabel$Nrec (7)

2.5. Data analysis

Statistical analyses were performed with SPSS 16.0 (IBM SPSSStatistics). Differences between treatments were tested with one-way and univariate analyses of variance. Stu-denteNewmaneKeuls post hoc tests were performed in order togroup data. In case of data that were not distributed normally or

A. Thuille et al. / Soil Biology & Biochemistry 81 (2015) 266e274 269

showed non-homogeneous variances, non-parametric tests (Krus-kaleWallis and Median) were performed to confirm or reject theresults of the respective analyses of variance. Differences wereconsidered to be significant if a < 0.05. Graphs were designed withSigmaPlot™10.0 (Systat Software Inc.).

3. Results

We examined the influence of soil additives in three differentqualities (corn silage, hydrochar and pyrochar), two different soiltypes and three levels of KNO3

� fertilizer on the germination successand growth of winter wheat plants.

Seven days after sowing, germination success on soils treatedwith hydrochar was significantly (p < 0.001) lower (48%) comparedto the two other treatments (75% on corn silage and 77% on pyro-char). This difference was still evident at the end of the experimente until day 31 after sowing, germination rate on soils treated withhydrochar reached 74% and thus was still considerably lower thanon soils treated with corn silage (91%) or pyrochar (93%). A com-parable negative effect of hydrochar on plant growth could bedetected during the first half of the experiment. Nine days aftersowing, plants on soils treated with hydrochar only achieved anaverage height of 4 cm, while plants on corn silage reached 6.6 cmand plants on soils treated with pyrochar 7.5 cm. On days 17 and 21after sowing, differences in height growth between corn silage andhydrochar treatments disappeared. Thus, height growth of plantson hydrochar significantly surpassed that of the plants on cornsilage at the end of the experiment despite a slower growth in theinitial phase, but remained below that of plants grown on hydro-char (Table 2).

Similarly, shoot dry weight of plants grown on soils with pyro-char reached 64 mg at the time of harvest compared to 45 mg and24 mg in the hydrochar and the corn silage treatment, respectively(Table 2). Leaf area was also remarkably higher with 23 cm2 whenplants were amendedwith pyrochar (hydrochar: 9 cm2, corn silage:6 cm2). Additionally, specific leaf area was significantly highercompared to the corn silage and the hydrochar treatment (Table 2).The effects of the different soil types and fertilizer levels on theselatter parameters were not significant. A noteworthy effect causedby the presence of hydrochar was a larger heterogeneity of themeasured plant parameters compared to the two other treatments.

The root system of winter wheat showed remarkable andconsistent differences between the different treatments (Fig. 1).Plants on soil amended with hydrochar had a very short root sys-tem, which was not reaching the bottom of the pots. Side rootswere short and had a stunted look. In contrast, plants on soils withcorn silage developed very long and fine roots concentrating at thebottom of the pots. Plants on soils with pyrochar showed an

Table 2Influence of amendment types on various plant characteristics. Numbers of days aregiven as days after sowing. The parameter Fv/Fm is an indicator of the quantumyieldof photosystem II. Different letters indicate significant differences at a ¼ 0.05.

Plant characteristic Type of amendment

Corn silage Hydrochar Pyrochar

Germination rate at day 7 [%] 75 ± 25 b 48 ± 31 a 77 ± 22 bGermination rate at day 31 [%] 91 ± 11 b 74 ± 27 a 93 ± 12 bPlant height [cm] at day 9 6.6 ± 2.3 b 4.0 ± 2.3 a 7.5 ± 3.0 cPlant height [cm] at day 17 15.3 ± 4.5 a 15.0 ± 4.9 a 17.8 ± 4.3 bPlant height [cm] at day 21 16.2 ± 4.4 a 17.9 ± 4.6 a 23.6 ± 5.9 bPlant height [cm] at day 31 18.9 ± 5.2 a 24.1 ± 4.9 b 18.9 ± 6.1 cshoot dry weight [mg] 24.4 ± 7.4 a 44.5 ± 27.7 b 63.6 ± 5.6 croot-to-shoot ratio 1.1 ± 0.3 a 0.3 ± 0.1 b 0.4 ± 0.2 bchlorophyll fluorescence [Fv/Fm] 0.69 ± 0.05 a 0.78 ± 0.01 c 0.73 ± 0.05 bLeaf area [cm2] 5.7 ± 1.8 a 9.2 ± 3.3 b 22.5 ± 5.1 cSpecific leaf area [cm2 g�1] 292 ± 28 a 299 ± 32 a 400 ± 43 b

intermediate growth with roots that seemed to be only slightly lessdeveloped than those of plants on soils with corn silage.

Consequently, the root-shoot ratio differed significantly be-tween plants on corn silage-amended soil and plants growing witheither biochar, with plants on corn silage exhibiting the widestroot/shoot ratio. However, no significant differences were foundbetween plants on pyrochar and hydrochar (Table 2).

Shoot N contents were measured in order to determine whetherN nutrition was influenced by the different amendments and thusresulted in the different growth characteristics. Analyses revealedthat plants growing on hydrochar had highest shoot N contentsdespite the fact that the respective plants were smaller than thosegrowing on pyrochar (Fig. 2). N concentrations were higher inplants growing on sandy soil. While N concentrations of plantsgrowing with corn silage or pyrochar amendments increased withthe amount of fertilizer added, N concentrations in plants onhydrochar-amended soil seemed to be independent of fertilizeraddition.

The good nutritional status of plants growing with hydrocharwas further corroborated by the measurements of chlorophyllfluorescence. The fluorescence parameter Fv/Fm, which decreaseswhen plants experience stress (Krause and Weis, 1991; Maxwelland Johnson, 2000; Baker, 2008; Murchie and Lawson, 2013) washighest for plants growing on hydrochar amended soil and lowestfor those on soils with corn silage (Table 2). These results confirmedthe observationsmade before the harvest of plants:Whereas plantswith biochar treatments had a healthy green leaf colour indepen-dent of their actual size, plants grown with corn silage had a moreyellowish look. Soil type and fertilizer level had no significant ef-fects on chlorophyll fluorescence.

Plants growing with hydrochar were vital plants well-nourished with N. Since their N content was independent of fer-tilizer level, we used the 15N label to examine how much of theadded KNO3 fertilizer was taken up by plants in the differenttreatments. While plants growing on soil amended with pyrocharused large amounts (60e70%) of the added fertilizer N (Fig. 3),significantly lower amounts of fertilizer N were taken up by plantsgrowing with corn silage as well as by plants growing withhydrochar; the latter using almost no fertilizer N (<10%). Soil typehad no influence on fertilizer uptake; similarly, there were nosignificant differences between the two fertilizer levels despite atrend towards higher NO3

� uptake by plants growing with pyrocharat lower fertilizer levels (Table 3). Independent of soil amendment,most of the NO3

�eN was detected in shoots. However, on soilamended with corn silage, this effect was less pronounced: here,around 30% of the labelled NO3

�eN was found in roots, while withboth biochar treatments, roots only contained around 10% of theadded fertilizer N.

These differences in fertilizer use were also reflected by theamounts of KCl-extractable NO3

� in soil (Fig. 4, left panel). Lowestvalues of NO3

�eN were detected in soil amended with corn silagewith values lower than 1 mg NO3

� g�1 soil. Soils amended withhydrochar had NO3

� concentrations of around 2e21 mg NO3�eN g�1

soil, which were significantly higher than for silage-amended soilsbut considerably lower than values found in pyrochar-amended soil(2e89 mg NO3

�eN g�1 soil). Fertilizer levels were still visible inpyrochar-amended soils and the presence of plants caused adepletion of NO3

� in soils.Remarkable results were found for NH4

þ (Fig. 4, right panel).Despite the fact that no NH4

þ was added via fertilizer, high amountsof more than 150 mg NH4

þeN g�1 soil were found in the presence ofhydrochar, while NH4

þ concentrations remained low in the presenceof the two other amendments (<1.5 mg NH4

þ�N g�1 soil). Soil typeand fertilizer level had no influence on NH4

þ concentrations; in

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 righthalf. Numbers 0, 1 and 2 indicate the different fertilizer levels: no KNO3

�, 10 and 20 mg KNO3� per pot. A univariate analysis of variance revealed the significant influence of soil type,

char type and fertilizer level. Different letters and asterisks denote significant differences at a ¼ 0.05. Data are given with respective standard deviations. HTC: hydrochar, PC:pyrochar.

Fig. 3. Percentage of added KNO3� fertilizer used by plants as revealed by 15N incor-

poration. F1 and F2 denote the fertilizer levels (10 or 20 mg N pot�1). Different lettersindicate significant differences at a ¼ 0.05. Soil type had no significant influence,squares give mean values of both fertilizer levels. Data are given with respectivestandard deviations. HTC: hydrochar, PC: pyrochar.

A. Thuille et al. / Soil Biology & Biochemistry 81 (2015) 266e274270

contrast, there was a significant influence of plant presence forsilage and hydrochar.

In order to gain insight into a potential competition for mineralN between plants and soil microbes, we also measured soil respi-ration and microbial biomass C. Both soils amended with cornsilage and with hydrochar showed relatively high soil respirationcompared to pyrochar-treated soil (Fig. 5a). However, the portion ofnative soil organic matter respired was considerably higher in thepresence of pyrochar.

Table 3Influence of soil type, type of amendment, fertilizer level and plant presence onselected plant and soil parameters according to univariate ANOVA. A factor isconsidered to be of significant influence if p < 0.05.

Plant/soil parameter p-values for the influence of

Soil type Amendment Fertilizer level Plant presence

Shoot N content 0.000 0.000 0.001 e

Fertilizer use 0.073 0.000 0.200 e

NO3� at harvest 0.027 0.000 0.026 0.000

NH4þ at harvest 0.910 0.000 0.987 0.002

respired CO2 0.293 0.000 0.103 e

Microbial biomass C 0.003 0.000 0.297 0.001

A. Thuille et al. / Soil Biology & Biochemistry 81 (2015) 266e274 271

Microbial biomass C (Fig. 5b) showed a similar pattern withhighest amounts in the presence of silage and low amounts inpyrochar-treated pots. While fertilizer level neither influenced theamount of CO2 respired nor the microbial biomass C, presence ofplants significantly reduced microbial biomass. In the presence ofhydrochar, microbial biomass showed a rather high variance andthe effect of the different soils was considerably larger than for theother treatments: There were significantly higher amounts of mi-crobial C in sandy soil compared to calcareous soil. Nevertheless,soil respiration did not differ significantly between the two soils.However, more native soil organic matter was decomposed whenmicrobial biomass was higher.

4. Discussion

Both char materials had an overall positive effect on theaboveground growth of wheat plants in our pot experiment whencompared with the influence of uncharred corn silage. This is inagreement with the results from a meta-analysis by Jeffery et al.(2011) who found an average increase in crop yield of 10% whensoils were amended with biochar produced via pyrolysis.Taghizadeh-Toosi et al. (2011) found no toxic effects of freshlyproduced pyrochar (from pine wood chips) on the growth of Loliumperenne. In contrast, a reduction in shoot biomass of Medicagosativa and the occurrence of leaf tip necroses in the presence ofbiochar produced via hydrothermal carbonization from spentbrewer's grains was reported by George et al. (2012). Likewise,Rillig et al. (2010) noticed growth reductions in Taraxacum andTrifolium plants if hydrochar was applied at rates above 10 vol%. Inour study, the shoot dry weight of plants grownwith either type ofchar was higher than that of plants grown with corn silage and theresults of the chlorophyll fluorescence measurements point to anactive photosystem II, especially in the presence of hydrochar. Toxiceffects as described for hydrochar by Jandl et al. (2013) could thusbe limited to the process of germination, which was reduced in thepresence of hydrochar (compare Bargmann et al., 2013).

While Gajic and Koch (2012) explain initial growth reductions ofsugar beet plants in the presence of hydrochar by the temporaryimmobilization of N, we did not detect any N limitation caused bybiochar addition. On the contrary, shoot N contents of plantsgrowing with hydrochar were even slightly higher compared to

Fig. 4. NO3� and NH4

þ concentrations in soils after harvest of plants. NO3� concentrations were

pyrochar also by fertilizer level as well as the presence of plants. For hydrochar, values on cavisible differences, however, these differences were not significant due to a high variance.concentrations were only influenced by type of amendment and the presence of plants (Silapyrochar. Different letters and asterisks denote significant differences between treatments

those of plants growing with pyrochar. On corn silage amendedsoils, however, shoot N concentrations were three times lower andplants showed signs of N limitation like yellowish leaf tips, a veryextensive root system and reduced values for chlorophyll fluores-cence which are an indicator for stress (Maxwell and Johnson,2000; Baker, 2008; Murchie and Lawson, 2013). The low soil NO3

�

concentrations found in the presence of corn silage are in agree-ment with the low shoot N concentrations and point to an immo-bilisation of NO3

� by soil microbes. While wheat plants growing onhydrochar took up a large amount of the added fertilizer N as re-flected by both the their shoot N concentrations and d15N values,plants on hydrochar obviously used a different N source as theirshoot N contents seem independent of the amount of applied NO3

�

fertilizer and only negligible amounts of fertilizer N wereincorporated.

We found unexpectedly high NH4þ concentrations in soils

treated with hydrochar, while NH4þ was only present in minor

amounts in the presence of the two other amendments. It seemsplausible that wheat plants used NH4

þ as N source when in con-tact with hydrochar. Several indicators support this hypothesis:Plants growing on hydrochar had an underdeveloped root systemcompared to the other treatments. It has been shown that contactof the growing root tip of Arabidopsis plants with NH4

þ may leadto suppressed cell elongation (Li et al., 2010) and thus to stuntedroots. Plants grown with NH4

þ as main N source may also showreduced yield, higher N concentrations in leaves when comparedwith plants fed with NO3

�eN (Roosta and Schjoerring, 2008;Seti�en et al., 2013) and a stronger discrimination against 15N(Seti�en et al., 2013). These observations are supported by ourdata: Compared to plants growing with pyrochar, plants onhydrochar were less tall and had a significantly smaller leaf areaand shoot dry weight, while their shoot N concentrations werehighest. We found no discrimination against 15N in our experi-ment, all d15N values were positive, however, significantly less15N was incorporated in roots and shoots when compared withplants on pyrochar.

Since we did not add NH4þ, the question arises where the rather

large amounts of NH4þ (up to 0.15 mg NH4

þeN g�1 dry soil) in thesoils amended with hydrochar came from andwhat happened withthe added fertilizer NO3

�. Both microbial biomass and microbialrespirationwere relatively high in the presence of hydrochar. This is

significantly influenced by the different soil types, char amendments and in the case oflcareous soil are shown separately for pots with and without plants because there wereThe square in between shows the mean value for pots with and without plants. NH4

þ

ge, hydrochar). Data are given with respective standard deviations. HTC: hydrochar, PC:at a ¼ 0.05.

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 significant influence 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. Microbialbiomass carbon was also independent of fertilizer level but significantly influenced by soil type, type of amendment and presence of plants. Here, green bars show results in thepresence of plants, dotted bars symbolize calcareous soil, plain bars sandy soil. Data are given with respective standard deviations. HTC: hydrochar, PC: pyrochar. (For interpretationof the references to colour in this figure legend, the reader is referred to the web version of this article.)

A. Thuille et al. / Soil Biology & Biochemistry 81 (2015) 266e274272

in agreement with other studies observing an increase in microbialbiomass 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 showedthat microbes mainly respired CO2 from the added hydrochar,thereby mineralizing about 80% of the hydrochar-C added to thesoil (corresponding to 13.2 mg hydrochar-C g�1 dry soil). N lossesfrom hydrochar amounted to 0.38 mg N g�1 dry soil, which rep-resents 40% of the N introduced via hydrochar assuming that all Nlosses originated from the mineralization of the soil amendment.The comparatively narrow C-to-N-ratio of the hydrochar (17) sug-gests that it was readily accessible for decomposition (Seneviratne,2000), making the contained N available to plants and microbes.Another possible process creating NH4

þ is the dissimilatory reduc-tion of NO3

� to NH4þ (Buresh and Patrick, 1978; Morozkina and

Kurakov, 2007). The addition of easily available organic C viahydrochar leads to a high C-to- NO3

�-ratio in the soil that isnecessary for this microbial transformation. Since soils amendedwith hydrochar were moist, thus enabling the development ofanaerobic microsites, we cannot exclude the possibility that DNRAoccurred in our experiment (compare Rütting et al., 2011). How-ever, plants growing on hydrochar did not incorporate largeamounts of 15N-labelled N into their biomass, which would havebeen expected if DNRA played a major role in NH4

þ production.Alternatively, hydrochar might have decreased nitrification rates assuggested by Ventura et al. (2013) for apple orchards amendedwithbiochar as well as by Subedi et al. (2013) who found less NO3

� butmore NH4

þ in loamy soils amended with pig slurry in the presenceof hydrochar compared to pyrochar. Losses of fertilizer N in theform of N2O, on the other hand, are considered to be of minorimportance since an experiment with the same chars and soils byMalghani et al. (2013) revealed reduced emissions in the presenceof hydrochar. Other studies also found decreasing N2O emissions inthe presence of biochar (Cayuela et al., 2013; Zheng et al., 2013).However, hydrochar might have favoured the last step of denitri-fication, thus reducing the ration of N2O/(N2þN2O) in denitrifica-tion end products (Cayuela et al., 2013). As a consequence, it seemsplausible that fertilizer NO3

� was at least partly lost due to volatil-ization as N2, while NH4

þ produced via the mineralisation of theadded hydrochar was conserved in the soileplantesystem due to atight coupling of mineralization and NH4

þ uptake by plants.

In soils amended with uncharred corn silage, microbes wereevenmore abundant and respired larger amounts of CO2 than in thepresence of hydrochar. Simultaneously, plants had an extensivelydeveloped root system and low shoot N concentration, suggestingthat microbes were more effective in competing for mineral N, thusleading to N deficiency in wheat plants. Here, the addition of amaterial with awide C-to-N-ratio might lead to processes similar tothose described for the incorporation of crop residues in cerealcultivation (Jensen, 1997; Bijay-Singh et al., 2001; Montoya-Gonz�alez et al., 2009): the labile C-fraction added via uncharredcorn silage might have caused an initial immobilization of N and alower rate of fertilizer uptake by plants (compare Kongchum et al.,2007), which necessitates a higher rate of N fertilization to sustainplant growth while at the same time promoting microbial growth.

The overall effect of the three soil amendments on the allocationof N between plants and microbes can be summarized as follows(Fig. 6):

� Corn silage, consisting mainly of lignocellulose material with awide C-to-N-ratio, favours microbes independent of fertilizerlevel and probably causes an immobilisation of N, while plantstake up only small amounts of NO3

� fertilizer and remain rathersmall showing signs of nutrient deficiency. Corn silage is readilyavailable for microbial decomposition, as shown by d13C valuesof microbial respiration.

� Hydrochar, characterized by a narrow C-to-N-ratio and spheri-cally arranged aromatic structures, causes mixed effects: Whileplants are well nourished with N mainly originating from thedecomposition of the soil amendment, microbial biomass andrespiration are also high and large amounts of the added ma-terial are released as CO2 by microbes, which effectivelymineralize the hydrochar.

� Plants grownwith pyrochar (wide C-to-N-ratio, highly aromaticstructure) effectively use the added fertilizer N and competesuccessfully against microbes, which only reach a relativelysmall biomass and respire significantly less C compared to thetwo other treatments. Additionally, a much smaller percentageof the respired CO2 originates from pyrochar.

Our results thus confirm the hypothesis that plants suffer fromN immobilisation if lignocellulose material of high C-to-N-ratio is

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 thesurface 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 forfurther explanation.

A. Thuille et al. / Soil Biology & Biochemistry 81 (2015) 266e274 273

added, while pyrochar promotes plant growth despite its equallywide C-to-N-ratio. Microorganisms, on the other hand are favouredin the presence of materials which are easily degradable either dueto the absence of aromatic structures (corn silage) or their lowerlevel of structural organization (hydrochar). In this context,hydrochar takes an intermediate position e despite containing alsoaromatic structures, its narrow C-to-N-ratio enables microbes tomineralize the material to a similar degree as uncharred corn silagewithout immobilising the contained N, thus sustaining a relativelylarge amount of microbial biomass while simultaneously guaran-teeing a strong plant growth. A study by Bai et al. (2013) confirmsthat hydrochar can be similar in its degradability to uncharredmaterial who produced both types of char from Miscanthus andfound that the half-life of hydrochar was comparable to that of theeduct and much lower than for pyrochar. However, the applicationof additional N fertilizer has to be evaluated carefully, since plantsare obviously not able to take up the added N but use the NH4

þ setfree via the mineralization of the char material instead. The situa-tion is different with pyrochar: A 15N tracer experiment withpyrochar produced from corn revealed that pyrochar caused atransfer of N from a stabile to a more labile pool and thus increasedplant available N (Nelissen et al., 2012). This is in agreement withZheng et al. (2013) who argued that biochar might increase thebioavailability of N and thus lead to reduced N fertilizer re-quirements in corn plants. Likewise, in our experiment plantseffectively use added NO3

� fertilizer in the presence of pyrochar,which might help to stabilize NO3

� in soil, as suggested by the factthat different fertilizer levels are well reflected by soil NO3

� con-centrations. Both types of char thus have the potential to reduce therequired fertilizer amounts e hydrochar because N might be madeavailable due to the mineralization of the char material, pyrocharbecause it stabilizes N and increases its bioavailability for plants.

Acknowledgements

This study was funded within the framework of the EnerCheminitiative of the Max Planck Society. The authors thank CarbonSolution 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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