Initial differentiation of vertical soil organic matter distribution and composition under juvenile beech (Fagus sylvatica L.) trees

REGULAR ARTICLE

Initial differentiation of vertical soil organic matterdistribution and composition under juvenile beech(Fagus sylvatica L.) trees

Carsten W. Mueller & Nicolas Brüggemann & Karin Pritsch & Gunda Stoelken &

Sebastian Gayler & J. Barbro Winkler & Ingrid Kögel-Knabner

Received: 3 November 2008 /Accepted: 9 February 2009 /Published online: 28 February 2009# Springer Science + Business Media B.V. 2009

Abstract In a lysimeter experiment with juvenilebeech trees (Fagus sylvatica L.) we studied thedevelopment of depth gradients of soil organic matter(SOM) composition and distribution after soil distur-bance. The sampling scheme applied to the given soillayers (0–2 cm, 2–5 cm, 5–10 cm and 10–20 cm) wascrucial to study the subtle reformation of SOMproperties with depth in the artificially filled lysim-eters. Due to the combination of physical SOMfractionation with the application of 15N-labelledbeech litter and 13C-CPMAS NMR spectroscopy wewere able to obtain a detailed view on verticaldifferentiation of SOM properties. Four years aftersoil disturbance a significant decrease of the mass of

particulate OM (POM) with depth could be found. Aclear depth distribution was also shown for carbon (C)and nitrogen (N) within the SOM fractions related tobulk soil. The mineral fractions <63 µm clearly dom-inated C storage (between 47 to 60% of bulk soil C) andN storage (between 68 to 86% of bulk soil N). A drasticincrease in aliphatic C structures concomitant todecreasing O/N-alkyl C was detected with depth,increasing from free POM to occluded POM. Only aslight depth gradient was observed for 13C but a clearvertical incorporation of 15N from the applied labelledbeech litter was demonstrated probably resulting fromfaunal and fungal incorporation. We clearly demon-strated a significant reformation of a SOM depthprofile within a very short time of soil evolution. Oneimportant finding of this study is that especially in soils

Plant Soil (2009) 323:111–123DOI 10.1007/s11104-009-9932-1

Responsible Editor: Philippe Hinsinger.

C. W. Mueller (*) : I. Kögel-KnabnerLehrstuhl für Bodenkunde,Technische Universitaet Muenchen,D-85350 Freising, Germanye-mail: [email protected]

N. BrüggemannForschungszentrum Karlsruhe, Institute for Meteorologyand Climate Research,Atmospheric Environmental Research (IMK-IFU),D-82467 Garmisch-Partenkirchen, Germany

K. Pritsch : S. GaylerChair of Soil Ecology, Helmholtz Zentrum Muenchen,German Research Center for Environmental Health,D-85764 Neuherberg, Germany

G. StoelkenChair of Tree Physiology,Institute of Forest Botany and Tree Physiology,University of Freiburg,D-79110 Freiburg, Germany

J. B. WinklerDepartment of Environmental Engineering,Helmholtz Zentrum Muenchen,German Research Center for Environmental Health,D-85764 Neuherberg, Germany

with reforming SOM depth gradients after land-usechanges selective sampling of whole soil horizons canbias predictions of C and N dynamics as it overlooks apotential development of gradients of SOM propertieson smaller scales.

Keywords Lysimeter . Particulate organic matter .

Mineral bound organic matter . Isotopictracer . 13C . 15N . 13C-CPMAS NMR . Fungal hyphae

AbbreviationsC carbonfPOM free particulate organic matterN nitrogenV-PDB Vienna-Pee Dee BelemniteoPOM occluded particulate organic materialoPOMsmall occluded particulate organic material

< 20 µmSOM soil organic matter

Introduction

The equilibrium between carbon (C) input andmineralization is altered by land use change until anew potential equilibrium is reached (Guo andGifford 2002). For example after afforestation ofcroplands, a fast accumulation of C takes place (Walland Hytonen 2005). But also the distribution andcomposition of different soil organic matter (SOM)fractions was demonstrated to be influenced bymanagement alterations (Mueller and Kögel-Knabner2008). For a site which was ploughed and subse-quently planted with Norway spruce 76 years ago, theauthors showed still lower contents of free particulateOM (fPOM) compared to an adjacent continuouslyforested site. Nevertheless, C accumulation is closelyrelated to the decay rates within the C cycle andtherefore to the quantity and quality of the C input(Tate et al. 2000). Thus, the fPOM is mostly related toprimary litter input, whereas in soil aggregatesoccluded POM (oPOM) is mainly affected by theaggregate turnover of the particular soils (Six et al.1999). Both fractions represent light OM which is notfirmly associated to soil minerals. By density frac-tionation it is possible to separate light particulate OMfrom heavy OM bound on mineral surfaces orassociated to oxides. Therefore, the study of differentSOM fractions representing different functional pools

can help to evaluate anthropogenic influences onSOM dynamics. This separation allows for studyingdifferent C stabilization processes, like chemicalrecalcitrance, spatial inaccessibility and the mineralassociation of SOM (von Lützow et al. 2006).Especially the labile C pool of fresh SOM is knownto be influenced by soil homogenisation, e.g. plough-ing (Grandy and Robertson 2007; Oorts et al. 2007).

A common tool to track the incorporation of freshorganic material into soils and differently stabilizedSOM fractions is the application of plant materialsenriched in 13C and/or 15N (Aita et al. 1997; Haynes1997; Kölbl et al. 2006). By an experiment with 15Nlabelled mustard litter (Sinapis alba) Kölbl et al.(2006) showed a rapid transfer of the labelled materialfrom the fPOM into fine mineral fractions within thefirst 5 months after litter application. In an experimentabout the N movement between different leavesSchimel and Hättenschwiler (2007) showed via 15Nlabelled leaves that at the absence of fungi the grossmovement of N between leaves of different N statuswas controlled by the N availability of the sourceleave. Thus, the use of a 15N tracer led to a betterunderstanding of the N limitation of microbial litterdecomposition. In a tracer study with the applicationof (15NH4)2SO4 and 13C labelled wheat straw to soilmaterial from an Alfisol, Frey et al. (2003) demon-strated the reciprocal fungal transfer of litter-C intosoil and soil-N into the litter layer. The authorsdemonstrated the transfer of litter derived C intomacroaggregates by fungal hyphae. Therefore, theapplication of labelled plant material enables the directstudy of the vertical fate of C and N from the litter layerto the mineral soil and variably stabilized SOMfractions.

Especially in the course of land-use change fromcropland to forest the new establishment of SOMdepth gradients is of major concern for C and Ncycles, but also for the nutrient supply of the growingtrees. Therefore, studying lysimeters stocked withtrees offers a unique chance to analyse these SOMprocesses at an early stage after the disturbance of thesoil system. In the given work we studied how depthgradients of SOM content and composition developedwithin 4 years after soil homogenisation. Using eightlysimeters filled with the same soil material allowedus to gain a unique data set to demonstrate SOMchanges on a statistically significant level. Weassumed that the soil material, which was taken at a

112 Plant Soil (2009) 323:111–123

beech forest site and homogenised afterwards, had nodepth gradient at the beginning of the experiment. Totrack the differentiation of certain SOM fractions withdepth we combined a set of different techniques. By acoupled density and particle-size fractionation protocoltogether with the application of 15N labelled beech litterwe aimed to study the incorporation of litter derived Nin different SOM fractions. The spectroscopic analyses(13C-CPMAS NMR) of the SOM fractions helped tounravel the development of depth gradients of SOMcomposition after soil homogenisation.

Materials and methods

Experimental design

The experiment was conducted at the outdoorlysimeter station of the Helmholtz Centre Munich(48°13’ N 11°36’ E, 490 m), Germany. Mean annualprecipitation at the lysimeter field ranged from546 mm (dry year in 2003) to 876 mm (in 2005)(Winkler et al. 2009). Mean annual temperaturesranged from 8.2°C (2005) to 8.9°C (2003, 2006).The experimental setup of the forest soil lysimeters isdescribed in detail by Winkler et al. (2009). In brief,eight lysimeters, each with a surface area of 1 m2 anda depth of 2 m, were filled in 1999 with soil material(Haplic Cambisol dystric) from the forest site “Högl-wald” (48°18’ N 11°05’ E, 540 m) near Augsburg(Bavaria, Germany). The upper 30 cm of the lysimeterswere filled in summer 2002 with freshly homogenisedsoil material. Therefore, a homogeneous soil materialwas assumed for the upper 30 cm at the start of theexperiment similar to a topsoil after ploughing. Thesoil comprised of 24% sand, 56% silt and 21% claywith pH values (in CaCl2) of about 3.8 (Table 1). Eachlysimeter was planted with four 3-year-old saplings of

Fagus sylvatica L. in autumn 2002. The space aroundthe lysimeter cylinders was filled with the same soiland also planted with beech trees in the density as inthe lysimeters to provide a homogeneous standclimate and to avoid edge effects.

In spring 2006, the original leaf litter layer of eachlysimeter was removed and weighed. Average leaf drymass per lysimeter was 134 g. In May 2006, thecontrol lysimeters #5–8 were again covered with134 g of their original leaf litter. Lysimeters #1–4were covered with 134 g of 15N-labelled beech(Fagus sylvatica L.) leaves (approx. 1 atom% 15Nenrichment), corresponding to a total N mass of18.6 mg and a 15N content of 0.176 mg g−1. Afterdistributing the leaves on the surface of the lysim-eters, they were covered with a fine mesh to avoid arelocation of litter to the neighbouring lysimeters anda contamination with unlabelled litter.

Soil sampling

In September 2006 all eight lysimeters were finallyharvested. The topmost 100 cm of each lysimeterwere cut into slices of 20 cm thickness (Reth et al.2007). Bulk soil samples were taken at differentdepths, i.e. 0–2 cm, 2–5 cm, 5–10 cm, 10–20 cm. Inorder to maintain the natural aggregation, soil materialwas gently sieved under field moist condition with asieve of 6.3 mm mesh size. The sieved bulk soilmaterial was air-dried for all further analyses.

Physical fractionation

A combined density and particle-size fractionationwas applied. Briefly, 20 g of air-dried soil material(<6.3 mm), were capillary-saturated with sodiumpolytungstate solution (1.8 g cm−3) and allowed tosettle over night. The floating free particulate organic

Table 1 Soil characteristics of the upper four soil layers (0–20 cm) of the analysed beech lysimeters at the Helmholtz CentreMünchen given as mean values with standard deviation (n=8)

Soil layer Sand Siltmg g−1

Clay pH (CaCl2) Cmg g−1

Nmg g−1

Ckg m−2

Ng m−2

C to N

0–2 cm 235±10 559±10 206±17 3.9±0.0 44.1±6.3 2.8±0.4 0.97±0.14 61.3±7.6 15.8±0.3

2–5 cm 3.8±0.1 32.8±5.4 2.1±0.3 1.08±0.18 69.6±10.0 15.5±0.4

5–10 cm 3.8±0.1 25.4±5.2 1.7±0.3 1.56±0.32 104.4±15.0 14.9±1.5

10–20 cm 3.7±0.0 15.8±1.9 1.1±0.1 2.05±0.25 141.4±12.9 14.5±0.7

Plant Soil (2009) 323:111–123 113

matter (fPOM) was extracted by aspiration via a waterjet pump. To remove the Na-polytungstate from thePOM fractions, the samples were washed severaltimes with deionised water on a sieve of 20 µm meshsize. The remaining slurry was dispersed ultrasonical-ly (Bandelin, Sonopuls HD 2200) with an energyinput of 440 J ml−1 in order to break down soilaggregates. The energy input had been tested beforeto avoid disruption of coarse POM along withaggregate disruption. Centrifugation (30 min at3,000 rpm) was used to separate the occluded POM(oPOM) from the mineral residue. The fraction<20 µm (oPOMsmall) obtained from the washing ofthe oPOM was washed via pressure filtration until theelectric conductivity dropped below 5 µS cm−1. Sand(>63 µm) and coarse silt (>20 µm to 63 µm) wereseparated by wet sieving. Medium silt (6.3 µm to20 µm), fine silt (2 µm to 6.3 µm) and clay (<2 µm)were obtained by sedimentation. The mass recoveryof the SOM fractionation was in the range of 97.5±2.1% from the initial mass. All fractions were freeze-dried, weighed and subjected to C and N elementalanalysis, and analysis of 13C and 15N.

Chemical analyses

The C and N contents were measured in duplicate bydry combustion (Elementar, vario MAX CNS Ana-lyzer for bulk soils; vario EL CN Analyzer for SOMfractions). Since all samples were free of carbonates(cf. Table 1), the measured C concentrations equalledthe organic C concentrations.

For isotopic measurements all three POM fractionsand the fine silt and clay fractions were taken, as thesefractions store most of the C and N within the studiedsoils and represent the major SOM pools. All samplesof SOM fractions were analyzed in tin capsules(elementar Analysensysteme GmbH, Hanau, Ger-many) for δ13C against Vienna–Pee Dee Belemnite(V-PDB) and δ15N against air-N2 with an isotope ratiomass spectrometer (Delta V Plus, ThermoFisher,Bremen, Germany) coupled to an elemental analyzer(Vario EL, elementar Analysensysteme GmbH,Hanau, Germany) at the Centre of Stable Isotopes ofForschungszentrum Karlsruhe, Institute for Meteorol-ogy and Climate Research (IMK-IFU, Garmisch-Partenkirchen, Germany). Working standard (mineralsoil), calibrated against the primary standards USGS40 (glutamic acid, δ13CV-PDB=−26.389) and USGS

41 (glutamic acid, δ13CV-PDB=+37.626) for δ13C and

USGS 25 (ammonium sulphate, δ15NAir=−30.4) andUSGS 41 (δ15NAir=+47.6) for δ15N, was analyzedafter every twelfth sample to detect a potentialinstrument drift over time and to determine theanalytical precision of the instrument which wassmaller than ±0.15 for δ13C and ±0.25 for δ15N (SDfor n=6–8). For δ13C analyses a blank correction wasperformed to account for the background signal of thetin capsules. For δ15N analyses no blank correction wasnecessary, as the background signal of the tin capsuleswas negligible. Due to the large range of δ15N values acorrection of δ15N of the samples was performed,using a two-point calibration with USGS 25 and USGS41 as anchor points.

13C-CPMAS NMR spectroscopy was accom-plished with a Bruker DSX 200 spectrometer (BrukerBioSpin GmbH, Karlsruhe, Germany). Samples werefilled into zirconium dioxide rotors and spun in amagic angle spinning probe at a rotation speed of6.8 kHz to minimize chemical anisotropy. A ramped1 H pulse was used during a contact time of 1 ms toprevent Hartmann–Hahn mismatches. The delay timesranged from 300 ms for bulk soil samples to 1,000 msfor litter and POM samples. Chemical shifts arereferenced to tetramethylsilane (TMS=0 ppm). Forintegration, chemical shift regions were used as given:alkyl C ((−10) to 45 ppm), O/N-alkyl C (45 to110 ppm), aromatic C (110 to 160 ppm) and carbonyl/carboxyl/amide C (160 to 220 ppm). In contrast to theisotopic measurements we did not obtain 13C-CPMASNMR spectra of the fine silt as the acquisition timeand the signal-to-noise ratio was not sufficient due tothe low C contents.

Mathematical analysis

The atom% 15N excess of the SOM fractions (Table 5)was calculated as difference between the atom% 15Nof the labelled SOM fractions and the atom% 15N ofthe SOM fractions from the control lysimeters.

For statistical analysis of the datasets, SPSS 16.0 forWindows (SPSS Inc., Chicago) was used. Significantdifferences of parameters of the SOM fractionsbetween the studied soil layers were tested by thenonparametric Mann-Whitney U test. We tested sig-nificant differences of the datasets with eight repli-cates, only for the 15N values we took the unlabelledlysimeters as a control leading to n=4.

114 Plant Soil (2009) 323:111–123

Results

Masses, OC and N in soil fractions

The mass of the particle size fractions (sand, silt andclay) was evenly distributed within the top 20 cm ofthe lysimeters (Fig. 1), with a clear dominance of thesilt fraction. A clear trend of decreasing mass wasobserved for all fPOM (−79±8%), oPOM (−74.6±13.3%) and oPOMsmall (−57.9±27.7%) fractions fromthe 0–2 cm to the 10–20 cm layer. Although theobserved mass range of POM fractions was large, thedecrease in mass with depth was in most casessignificant at p<0.05.

The highest C and N contents were found for theoPOM, followed by the fPOM and oPOMsmall

fractions (Table 2). A significant decrease of Ccontents was only observed with depth for oPOMsmall,whereas the N decreased significantly with depth inall three POM fractions.

The C/N ratio of the fPOM at 0–2 cm (26.3±1.6)was not significantly lower than the initial C/N ratioof the litter of the Oi layer (28.7±2.4). A clearincrease (p<0.05) of C/N ratios with depth wasdetected for all POM fractions with a maximum ofthe oPOMsmall (62.5±7.1) in the 10–20 cm layer(Fig. 1). For all replicates within a single soil layer aclear decrease could be demonstrated in C/N ratiosfrom the POM to the mineral-bound SOM fractions.

Large proportions of C (Fig. 2) were stored withinthe fraction <63 µm (silt and clay), from 47±3.7%(10–20 cm) to 60.3±5.9% (5–10 cm). Within the

0

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Fig. 1 Mass distributionand C/N ratios of the SOMand particle size fractionsfor the four studied soillayers. Data points are themean of 8 replicates witherror bars representing stan-dard deviations. Significantdifferences of the massesand C/N ratios between dif-ferent soil layers of a singleSOM fraction are indicatedby letters and * (p<0.05),** (p<0.01)

Plant Soil (2009) 323:111–123 115

Table 2 Carbon and nitrogen contents of the density (POM) and particle size fractions (sand, silt and clay)

Depth Fraction

fPOM oPOM oPOMsmall Sand Silt Clay

OC mg (g bulk soil)−1 0 to 2 cm 312.4±16.8 a 447.3±28.8 305.0±29.5 a 3.6±1.0 a 19.4±2.7 a 73.1±8.5 a

2 to 5 cm 324.3±33.5 a 452.2±27.6 297.0±45.7 ab 2.6±0.6 a 12.7±2.0 b** 61.4±8.3 b**

5 to 10 cm 332.4±30.1 a 449.3±54.7 267.7±30.6 b* 1.6±0.3 b** 8.6±1.7 c** 51.8±7.0 c*

10 to 20 cm 337.8±22.5 b* 408.7±92.9 353.6±83.1 ac* 1.2±0.2 c* 4.7±1.0 d** 28.1±4.3 d**

N mg (g bulk soil)−1 0 to 2 cm 11.9±0.9 a 14.5±1.4 a 13.7±1.5 a 0.2±0.0 1.3±0.2 a 6.4±0.7 a

2 to 5 cm 11.1±1.2 ab 12.9±1.4 b* 11.2±1.0 b* 0.1±0.0 0.9±0.1 b** 5.3±0.6 b**

5 to 10 cm 9.9±1.4 b* 11.2±2.1 b* 9.9±1.6 b* 0.1±0.0 0.6±0.1 c** 4.4±0.4 c**

10 to 20 cm 8.5±1.0 b* 9.5±2.2 b** 5.8±0.9 c** 0.1±0.0 0.4±0.1 d** 2.8±0.4 d**

Data is given as mean values with standard deviation (n=8)

Significant differences related to the soil layer of C and N contents of a single SOM fraction is indicated by letters and *(p<0.05),**(p<0.01)

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fPOM oPOM oPOM small sand silt clay

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fPOM oPOM oPOM small sand silt clay

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a b*

a

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aa

b*

b**

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a

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c**

Fig. 2 Carbon and nitrogendistribution within SOMand particle size fractionsrelated to bulk soil(< 6.3 mm) for the four soillayers studied. Data pointsare the mean of 8 replicateswith error bars representingstandard deviations. Signifi-cant differences of theC— and N-amounts be-tween different soil layers ofa single SOM fraction areindicated by letters and* (p<0.05), ** (p<0.01)

116 Plant Soil (2009) 323:111–123

<63 µm fraction the C storage was clearly dominatedby the clay separates in all soil layers, culminating at46.4±5.2% clay-bound C in the 5–10 cm layer. Fromthe 0–2 cm layer to the 5–10 cm layer the C contentof the POM fractions clearly decreased (p<0.05). Forevery POM fraction a slight increase in C content inrelation to the bulk soil was observed in the 10–20 cmlayer. Thus, the C content of the oPOMsmall had itsmaximum within the 10–20 cm layer. The N storagealso was clearly dominated by the mineral fractionsaccounting for about 82.1±7.7% on average over allsoil layers (Fig. 2). A clear trend of decreasing Nstorage with depth was found for both fPOM andoPOM fractions (p<0.05). Nitrogen amounts related tothe clay size separates increased significantly (p<0.01)with depth.

Chemical composition of soil fractions

In Fig. 3 the 13C-CPMAS NMR spectra of the SOMfractions of Lysimeter #1 are given exemplarily forthe set of the spectra obtained, relative intensitiesof the integrated shift regions are given in Table 3.From the initial litter to the fPOM of the 0–2 cm layerthe O/N-alkyl C intensities decreased significantly(p<0.01), while the content of alkyl C increased(p<0.01, Table 3). The trend of increasing alkyl / O/N-alkyl C ratios of the fPOM fraction continued through-out the topmost 10 cm with significant (p<0.01) higherratios in the 5–10 cm layer. Increasing alkyl / O/N-alkyl C ratios with depth were also detected for the

oPOM for the upper 10 cm. A remarkable increasewas observed for the alkyl C from the fPOM to theoPOM and oPOMsmall. With a relative intensity ofalkyl C of 52.7±1.6% the oPOMsmall showed a veryhigh content of aliphatic structures in the 5–10 cmlayer. For the aromatic and carbonyl C contents noclear trend was observed in the soil layers andfractions. Also no depth-dependent trend was observedfor the relative intensities of the chemical shift regionsof the mineral-bound SOM of the clay fractions. Bycontrast, the aromatic C intensities of the clay boundSOM showed a slight increase with depth.

13C and 15N in soil fractions

The δ13C values for the three POM, fine silt and clayfractions are displayed in Table 4. The δ13C valuesof the oPOMsmall were only significantly different(p<0.01) from the other two POM fractions withinthe 0–2 cm soil layer. Although it was not significant,a trend of decreasing δ13C values in the order ofoPOMsmall to oPOM and fPOM was observed in allsoil layers. The δ13C values of the fine silt weresignificantly lower (p<0.01) than the δ13C values ofthe clay fractions in all studied soil layers. Only asubtle depth gradient was observed for the δ13Cvalues for particulate and mineral-bound SOM,leading to lower values for the POM and highervalues for the mineral bound SOM.

The δ15N values of the SOM fractions of thestudied soil layers are shown in Fig. 4. The natural

350 250 150 50 0 -100

chemical shift (ppm)

fPOM

oPOM

oPOMsmall

clay

0 - 2 cm

350 250 150 50 0 -100

chemical shift (ppm)

2 - 5 cm

350 250 150 50 0 -100

chemical shift (ppm)

5 - 10 cm Fig. 3 13C-CPMAS NMRspectra of SOM fractions ofthe soil layers 0–2 cm,2–5 cm and 5–10 cm fromLysimeter 1

Plant Soil (2009) 323:111–123 117

abundance δ15N values of the control lysimetersincreased with depth and from POM to mineral boundfractions. A high variability of the δ15N values wasfound for the SOM fractions of the labelled lysimeterscompared to the control. Four months after labelapplication, significantly higher 15N concentrations(p<0.05) of the POM fractions could be found in thetopmost layer of 0–2 cm of the labelled lysimeterscompared to the control. A significant enrichment(p<0.05) was detected for the fPOM and oPOMsmall

fractions of the soil layer of 2–5 cm. Within the5–10 cm layer a clear trend of enrichment was foundfor fPOM and oPOM. A variable enrichment in 15N(0.1‰ to 4.4‰ δ15N vs. air) was observed for theoPOMsmall within the 10–20 cm layer. Besides the

oPOM, no SOM fractions showed a significantenrichment in the 10–20 cm layer. Besides the smallincrease in 15N values in the fine silt and clay of the0–2 cm layer, no enrichment was found in the mineralfractions. A similar pattern as for the 15N concen-trations was observed for the atom% 15N excess of thelabelled SOM fractions (Table 5). Here a clear trendof increased 15N excess due to the labelling could beshown for fPOM and oPOM fractions. The largest15N excess was shown for the oPOM fractions. TheoPOMsmall and the mineral fractions only showed apronounced atom% 15N excess in the 0–2 cm layer.For the mineral bound N of the fine silt and clay, theatom% 15N excess was at reasonable detection limitbetween 5 to 20 cm depth.

Table 3 Relative contents of alkyl C, O/N-alkyl C, aryl C, carbonyl C and alkyl / O/N-alkyl C ratios obtained by 13C-CPMAS NMRspectroscopy of the litter and the fPOM, oPOM, oPOMsmall and clay fractions of the three uppermost soil layers

Layer Fraction Alkyl C % O/N-alkyl C % Aromatic C % Carbonyl C % Alkyl / O/N-alkyl C

Litter (n=8) 17.9±1.9 ** 56.7±4.0 ** 18.4±2.1 7.0±1.8 * 0.32±0.05 **

0–2 cm fPOM (n=7) 25.7±1.8 a 45.7±2.3 a 19.7±1.2 8.9±1.4 0.56±0.05 a

oPOM (n=6) 39.9±3.2 a 34.7±1.0 a 16.8±1.4 8.6±1.8 1.15±0.11a

oPOMsmall (n=4) 47.9±4.5 a 26.7±1.6 a 15.7±1.7 9.7±1.4 1.80±0.29 a

clay (n=3) 37.9±3.3 37.4±1.2 13.0±2.5 11.7±1.6 1.01±0.08

2–5 cm fPOM (n=6) 28.7±2.8 a 43.5±3.5 a 19.7±1.7 8.1±0.8 0.67±0.11 a

oPOM (n=3) 45.8±2.0 b* 30.6±1.8 b* 15.9±0.2 7.6±0.5 1.50±0.15 b*

oPOMsmall (n=3) 48.4±2.6 b* 25.4±0.3 b* 15.8±0.7 10.4±1.8 1.90±0.12 b*

clay (n=3) 38.1±6.3 37.0±3.4 14.6±3.2 10.3±2.3 1.05±0.28

5–10 cm fPOM (n=6) 33.5±2.2 b** 35.8±5.1 b** 22.0±3.2 8.8±1.3 0.96±0.21 b**

oPOM (n=4) 46.6±2.9 b* 29.3±2.4 b* 16.8±2.5 7.3±1.5 1.60±0.17 b*

oPOMsmall (n=3) 52.7±1.6 b** 22.9±0.8 b** 15.5±0.8 8.9±1.2 2.08±0.15 b**

clay (n=3) 33.1±3.5 35.7±0.1 18.9±2.8 12.3±0.5 0.93±0.10

Data is given as mean value with standard deviation. Significant differences between single fractions according to depth are indicatedby letters and *(p<0.05), **(p<0.01), the litter was tested versus the fPOM of the 0–2 cm layer

Table 4 δ13C values (vs. V-PDB) of the three POM fractions (fPOM, oPOM and oPOMsmall) and the fine silt and clay fractions

Depth fraction; δ13C vs. V-PDB (‰)

fPOM oPOM oPOMsmall Fine silt Clay

0 to 2 cm −28.7±0.1 a −28.9±0.2 −27.9±0.3 a −27.7±0.2 a −26.7±0.2 a

2 to 5 cm −28.6±0.3 a −29.0±0.1 −28.1±0.3 a −27.5±0.1 a −26.4±0.1 b*

5 to 10 cm −29.0±0.2 b* −29.0±0.1 −28.1±0.7 a −27.4±0.2 a −26.4±0.1 b*

10 to 20 cm −29.0±0.3 b* −28.8±0.9 −29.0±0.2 b** −26.3±0.4 b** −25.6±0.2 c**

Data is given as mean values with standard deviations (n=8), significant differences between different soil layers of a single SOM orparticle size fraction are indicated by letters and *(p<0.05) and **(p<0.01). The oPOM showed no significant depth gradient

118 Plant Soil (2009) 323:111–123

Discussion

Vertical differentiation of SOM after 4 years

Due to the equal mass distribution of the particle sizefractions (sand, silt and clay) among the lysimeters(Table 1) it can be assumed that the eight studiedpedons were still homogeneous 4 years after the startof the experiment. This assumed homogeneity of the

eight lysimeters is supported by Gayler et al. (2009)who could show a high conformity of soil propertiesamong the studied eight soil systems. The pH valueswithin the topmost 20 cm also showed no develop-ment of a depth gradient (Table 1). Nevertheless, thedepth gradient of the amount of the POM fractionsclearly implied an ongoing soil evolution over theexperiment duration. The clear decrease in fPOMmass indicated the beginning development of a depth

-5

0

5

10

15

20

25

30

15N

vs.

air

(‰

)

fPOM oPOM oPOMsmall fSi C

fraction

15N labelledcontrol

0 to 2 cm

*

*

*

*

*

-10

-5

0

5

10

15

20

25

30

35

40

15N

vs.

air

(‰

)

fPOM oPOM oPOMsmall fSi C

fraction

15N labelledcontrol

2 to 5 cm

**

-6

-4

-2

0

2

4

6

8

15N

vs.

air

(‰

)

fPOM oPOM oPOMsmall fSi C

fraction

15N labelledcontrol

5 to 10 cm

-5

-4

-3

-2

-1

0

1

2

3

4

5

6

15N

vs.

air

(‰

)fPOM oPOM oPOMsmall fSi C

fraction

15N labelledcontrol

10 to 20 cm

*

Fig. 4 15N abundancesexpressed as δ values vs.air of SOM fractions incontrol lysimeters andlysimeters treated with 15N-labelled beech litter. Datapoints are the mean of 4replicates with error barsrepresenting standard devia-tions. Significant differencesbetween 15N labelled andcontrol lysimeters areshown by *(p<0.05). fSi,fine silt; C, clay

Table 5 Atom% 15N excess of the SOM fractions, calculated as difference between atom% 15N of SOM fractions from labelled andcontrol lysimeters

Depth Fraction; atom% 15N excess

fPOM oPOM oPOMsmall Fine silt Clay

0 to 2 cm 0.0065 (0.0013) 0.0058 (0.0025) 0.0023 (0.0007) 0.0013 (0.0004) 0.0016 (0.0008)

2 to 5 cm 0.0026 (0.0057) 0.0067 (0.0035) 0.0005 (0.0005) 0.0007 (0.0003) 0.0002 (0.0024)

5 to 10 cm −0.0027 (0.0044) 0.0029 (0.0021) 0.0008 (0.0005) 0.0000 (0.0003) −0.0001 (0.0023)

10 to 20 cm 0.0004 (0.0001) 0.0005 (0.0004) −0.0037 (0.0013) −0.0002 (0.0003) 0.0001 (0.0002)

Standard error is given in parentheses

Plant Soil (2009) 323:111–123 119

gradient of free SOM particles. Nevertheless, from theinitial litter to the fPOM within the 0–2 cm layer nosignificant decrease in C/N ratios was observed.

The amount and the composition of occluded SOMdepend on the extent and the turnover of soil aggregates(Six et al. 2002). From the significant decrease of themass of the two oPOM fractions (oPOM, oPOMsmall)with depth, a fast aggregate turnover can be assumed.This is supported by the high decomposition degreesrevealed by 13C-NMR (alkyl / O/N-alkyl C in Table 3)of the occluded SOM particles. Therefore, we assumethe clear dominance of highly aliphatic C structureswithin the oPOM fractions resulted from favourabledecomposition conditions accompanied by a rapidaggregate turnover leading to a pronounced decompo-sition of compounds rich in O/N-alkyl C. Due to thespatial inaccessibility of the oPOM for microbialdecay, soils with slower aggregate turnover showlower alkyl C contents (Golchin et al. 1994; Muellerand Kögel-Knabner 2008). The given assumption isalso supported by the significant decrease in C whichis attributed to the oPOM fractions in relation to bulksoil (Fig. 2). The increase of the decomposition degreewith depth for the three POM fractions shown by theincreasing alkyl / O/N-alkyl C ratios (Table 3) iscorroborated by the increasing C/N ratios. From theinitial litter to both oPOM fractions a distinctiveincrease in the C/N ratios took place. The relativeincrease in long-chain aliphatic structures (alkyl C) andthe decomposition of O/N-alkyl-dominated SOM isaccompanied by a relative decrease in N (Table 2).Therefore the increase of the C/N ratios of the singlePOM fractions with depth was explained by theincreased contents of aliphatic C with concomitantdecreasing N contents. The assessment of the decom-position degree for the studied SOM fractions exclu-sively by C/N ratios would have led to wrongassumptions.

Although the decrease in aromatic C representsonly a trend (Table 3), an obvious decrease of thepeaks at 115, 128 and 152 ppm (Fig. 3 and Table 3)was detected. The decrease in aromatic C was in therange reported by Parfitt and Newman (2000) for thedecomposition of pine needles. The mineral-boundSOM dominated both the C and N storage. Theincreasing dominance in C storage of the clay-boundSOM (Fig. 2) also demonstrated the starting develop-ment of the natural shift from particulate C tomineral-bound C with depth. For undisturbed soils

this shift towards a dominance of mineral-bound SOMwith depth was already documented (Eusterhues et al.2003; Mueller and Kögel-Knabner 2008). For thestudied soils an accelerated SOM decomposition canbe assumed due to the drastic disturbance byhomogenisation at the start of the experiment. Alsoin field studies it could be shown that ploughing andtherefore soil disruption and homogenisation led tosimilar effects on labile C pools (Grandy andRobertson 2007; Oorts et al. 2007). Therefore, thenew C input of the young beech trees and theaccelerated decomposition of old SOM may havecounteracted in the studied soils. Nevertheless, thehigh standard deviation of the POM-related propertiesdemonstrated the wide range of differentiation ofSOM after topsoil homogenisation, e.g. after plough-ing. This led to the assumption that we observedthe beginning development of heterogeneous soilprofiles comparable to the heterogeneity of naturalsoil systems.

Isotopic composition of C and N with depth

The δ13C values of the POM fractions indicated onlysubtle but significant depth gradients for the pedonsof the lysimeter. We observed an increase of δ13Cvalues of the mineral-bound SOM in the 10–20 cmlayer in relation to the upper 10 cm. This increase isin accordance to other reported depth profiles of 13C(Wynn et al. 2006). By isotopic studies on permanentgrasslands Accoe et al. (2003) could show a reason-able correlation between C decomposition rates andδ13C values for the upper 20 cm. On the contraryKramer et al. (2003) found no clear trend for δ13Cvalues in relation to the decomposition degreemeasured as aliphaticity of the SOM. In the studiedsoil the POM fractions mainly influenced by litterinput and aggregate turnover showed no clear verticalisotopic fractionation. We assume the mineral-boundSOM of deeper layers as more influenced by anaccelerated decomposition due to the initial soildisruption. Therefore a lower influence of freshSOM inputs on mineral bound SOM can be statedfor deeper soil layers, as indicated by vertical 13Cenrichment.

For all studied soil layers, an enrichment in 15Nwas observed from fPOM to oPOM to mineral boundSOM. An natural enrichment in 15N takes place bythe accumulation of N as mineral bound N and

120 Plant Soil (2009) 323:111–123

recalcitrant N within aggregates (Högberg 1997). Inaddition to the found differences between fractions wealso found a subtle depth gradient of the δ15N valuesfor every studied SOM fraction of control (naturalabundance 15N) and labelled lysimeters. More posi-tive 15N values for the whole soil in relation to plantand decaying litter was reported before (Poertl et al.2007). Koba et al. (1998) also found increasing δ15Nvalues for total and inorganic N with depth. Due tothe dilution of the upper soil layers with fresh litterwith low 15N contents a depth gradient developed asthe residual N becomes enriched in 15N with ongoingdecomposition (Högberg 1997). Kramer et al. (2003)demonstrated a positive relationship between naturalabundance δ15N and the degree of aliphaticity andtherefore the degree of decomposition. Furthermorethe preservation and illuviation of 15N-enrichedmaterial may also have contributed to the enrichmentof 15N with depth (Koba et al. 1998).

The POM fractions of the topmost soil layer of0–2 cm from the labelled lysimeters showed apredicted decrease in excess 15N concentrations fromfree to occluded particles. Swanston and Myrold(1997) also observed the highest 15N recovery in thetopmost soil layers <5 cm and the light fraction, whilebelow 5 cm depth the 15N recoveries were low andvariable. In a decomposition study with 15N labelledbeech litter Zeller et al. (2000) found after 3 years62% of the released N in the surface soil but only12% below 2 cm. From the given 15N concentrationswe assume a natural incorporation cascade for thisfirst layer (0–2 cm). The pathway of OM into the soilmatrix of the lysimeters may have been initialised byfaunal fragmentation (Setälä et al. 1996) of partlydecomposed litter which we separated as fPOM, and asubsequent occlusion of POM in the form of oPOMand oPOMsmall. This assumption is also supported bythe atom% 15N excess shown in Table 5. However,for soil from 2–20 cm depth no such clear pattern wasobserved. Here we could show an enhanced 15Nenrichment of occluded particles, whereas in the 10–20 cm layer no enhanced 15N enrichment of thefPOM was found. Kölbl et al. (2006) explained thehigh recovery of 15N labelled material in the oPOMafter 161 days by a relatively fast aggregate formationdue to the fresh partly decomposed litter additionswhich promote aggregation. Nevertheless, the 15Nenrichment especially of the POM fractions at ourlysimeters showed a very large variability. As C

contents and the amounts of SOM fractions were verysimilar between the lysimeters (Figs. 1 and 2)differing aggregate dynamics could not explain thisvariability. Buchmann et al. (1996) could demonstratethe high ability of fungal hyphae for N transport byhighly 15N-enriched fruit bodies of fungi growing onplots with no 15N addition of a labelling study inPicea abies plantations. In the labelled lysimeters ofthis study highly enriched ectomycorrhizae with δ15Nvalues of up to 24‰ vs. air for Tomentella sp. werefound at 0–20 cm depth. Since even more interest-ingly rhizomorphs of Tomentella sp. showed δ15Nvalues of up to 32‰ vs. air (Pritsch, unpublishedresults), we assume the high 15N concentrations of theoccluded POM fractions as influenced by fungalhyphae which may contribute to POM during frac-tionation. But also an active contribution of fungalhyphae to the aggregate build-up seems to bereasonable for the studied soil, as fungal hyphae playan important role as binding agents in the develop-ment of macroaggregates >250 µm (Six et al. 2004).From these findings we hypothesise an active trans-port for litter-derived N, but presumably also C byfungal hyphae into inner-aggregate spheres duringaggregate build-up. Some indication comes from asample of Xerocomus sp. representing a long-distanceexploration type (Agerer 2001) with extensive rhizo-morphs. The fact that we found δ15N values of 4.01‰vs air in mycorrhizae of Xerocomus even at a depth of60 cm may indicate mobilisation by hyphae andtransport from the litter through rhizomorphs. Theextensive mycelium of such species as Xerocomusand of course also saprotrophic fungi may provide thestructure to distribute C and N in soil. The reciprocaltransfer of litter C into soil and soil N into the litterlayer was found by Frey et al. (2003), whereas theauthors assumed a preferential fungal transfer of litterderived C into macroaggregates (> 250 µm).

Nevertheless, we found a very high variability ofδ15N values especially in the enriched POM fractions.The application of whole leaves on top of the soilwithout artificial incorporation into the soil material,i.e. ploughing resulted in spatially heterogeneousincorporation degrees of the material. But also fungalmycelia with their locally heterogeneous distributionmay influence the transfer of N compounds into thegiven SOM fractions.

We only found clear excessive δ15N values of themineral bound SOM fractions in the 0–2 cm layer. We

Plant Soil (2009) 323:111–123 121

did not find a rapid translocation of 15N labelledmaterial from fPOM to fine mineral fractions as it wasshown by Kölbl et al. (2006) who found up to 25% ofthe applied 15N in clay-sized fractions after 161 dayson a cropland. Swanston and Myrold (1997) showedthat after 21 months of incubation only one third ofthe 15N recovered from labelled red alder leaves weresituated in the heavy fraction. Microbial immobiliza-tion as one of the key factors leading to stabilisationof N (Herrmann and Witter 2008) but also C in themineral associated fractions did therefore only lead tosignificant 15N enrichments in the uppermost soillayer. Thus, on the short timescale of 15N-litterexposure within the presented study faunal and fungalmechanisms may have dominated the incorporationmechanisms for the labelled compartments.

Conclusions

In the present lysimeter study, 4 years after homogeni-sation of soil material were sufficient for the develop-ment of depth gradients of SOM properties. However,differences between the soil layers could only bedetected due to the dense soil sampling scheme. Besidesthe mass distribution of SOM fractions also the chemicalcomposition of the soil showed a clear depth-dependentdifferentiation. Although the SOM pools were far fromequilibrium, a fast development of depth profiles wasshown after severe soil disturbance. Fungal hyphae wereassumed to play an important role in the transport of Ncompounds into soil aggregates. In the present study wecould clearly show that selective sampling of soilhorizons can severely bias predictions of the develop-ment of vertical differentiation. Especially after land-usechanges and soil disturbances the knowledge about thereforming processes and dynamics of SOM fractions iscrucial for the prediction of future C and N sequestrationin managed soils.

Acknowledgements We like to thank Livia Wissing andMaria Greiner at TU Muenchen for their energetic help in thelaboratory. Rudolf Meier at the IMK-IFU in Garmisch-Partenkirchen is gratefully acknowledged for the isotopicmeasurements. Financial support of this study was granted bythe Helmholtz Gemeinschaft within the frame of the virtualinstitute “Center for Stable Isotope Analysis in EcosytemResearch” under contract no. VH-VI-129 and by the DeutscheForschungsgemeinschaft (DFG) within the frame of theCollaborative Research Centre “SFB 607”.

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