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Soil Biology & Biochemistry 57 (2013) 1e13

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Soil Biology & Biochemistry

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The role of lignin for the d13C signature in C4 grassland and C3 forest soils

Alexander Dümig a,*, Cornelia Rumpel b, Marie-France Dignac b, Ingrid Kögel-Knabner a,c

a Lehrstuhl für Bodenkunde, Department für Ökologie und Ökosystemmanagement, Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt,Technische Universität München, D-85350 Freising-Weihenstephan, GermanybCNRS-INRA, Laboratoire de Biogéochimie et Ecologie des Milieux Continentaux (UMR 7618, UPMC-CNRS-UPEC-ENS-INRA-AgroParisTech),Centre INRA e Bâtiment EGER de Versailles-Grignon, F-78850 Thiverval-Grignon, Francec Institute for Advanced Study, Technische Universität München, Lichtenbergstrasse 2a, D-85748 Garching, Germany

a r t i c l e i n f o

Article history:Received 5 April 2012Received in revised form26 June 2012Accepted 29 June 2012Available online 20 July 2012

Keywords:13C/12CSoil organic matter13C NMR spectroscopyCuO oxidationGC/C-IRMSCarbohydratesBrazil

* Corresponding author. Tel.: þ49 8161714206; fax:E-mail address: duemig@wzw.tum.de (A. Dümig).

0038-0717/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.soilbio.2012.06.018

a b s t r a c t

13C contents of organic matter are changing during decomposition of plant material and stabilization assoil organic carbon (SOC). In this context, several studies showed 13C enrichment in soil as compared tovegetation for C3 forests, whereas depletion of 13C was frequently reported for C4 grassland soil ascompared to C4 vegetation. These changes were often attributed to selective preservation and/orstabilization of specific organic compounds. This study investigates if changes in the chemical compo-sition of OC and specifically lignin may explain the observed shifts in d 13C values from plant material toSOC. We analyzed aboveground biomass, roots and heavy organo-mineral fractions from topsoils in both,long-term stable C4 grasslands and C3 Araucaria forest situated nearby in the southern Brazilian high-lands on soils with andic properties. The stable carbon isotope (12C/13C) composition was analyzed fortotal organic carbon (OCtot) and lignin-derived phenols. The bulk chemical composition of OC wasassessed by solid-state 13C NMR spectroscopy while neutral sugar monomers were determined after acidhydrolysis. The shifts of the 13C/12C isotope signature during decomposition and stabilization (planttissues versus soil heavy fractions) showed similar trends for VSC phenols and OCtot (

13C depletion in C4

grassland soil and 13C enrichment in C3 forest soil compared to the corresponding vegetation). In thisregard, the isotopic difference between roots and aboveground biomass was not relevant, but maybecome more important at greater soil depths. 13C depletion of VSC lignins relative to OCtot was higher inC3-biomass and C3-derived SOC compared to the C4 counterparts. As lignin contents of heavy fractionswere low, in particular for those with C4 isotopic signature, the influence of lignin on OCtot d

13C values ingrassland topsoils is presumably low. Rather, the presence of charred grass residues and the accumu-lation of alkyl C in heavy fractions as revealed by 13C NMR spectroscopy contribute to decreasing d13Cvalues from grass biomass to C4-derived heavy fractions. In forest topsoils, the accumulation of 13Cdepleted VSC lignin residues in heavy fractions counteracts the prevailing 13C enrichment of OCtot fromplant biomass to heavy fractions. Nonetheless, non-lignin compounds with relatively high 13C contentslike microbial-derived OC have a stronger influence on d13C values of OCtot in forest soils than lignins oraliphatic biopolymers. The mineral-associated SOC is in a late phase of decomposition with largecontributions of microbial-derived carbohydrates, but distinct structural and isotopical alterations oflignin between C4- and C3-derived heavy fractions. This may indicate different processes and/or extent oflignin (and SOM) biodegradation between C4 grassland and C3 forest resulting from other kind ofdecomposer communities in association with distinct types and amounts of plant input as source of SOMand thus, carbon source for microbial transformation. Our results indicate that the importance of ligninfor d13C values of OCtot was overestimated in previous studies, at least in subtropical C4 grassland and C3

forest topsoils.� 2012 Elsevier Ltd. All rights reserved.

þ49 8161714466.

All rights reserved.

1. Introduction

Stable carbon isotopes have been frequently used to describesoil organic carbon (SOC) dynamics (e.g. Balesdent et al., 1987;Bonde et al., 1992; Accoe et al., 2003) or to reconstruct past vege-tation changes (e.g. Deines, 1980; Tieszen and Archer, 1990;

Table 1Properties of heavy fractions in C4 grassland (G) and C3 Araucaria forest (F).

Pedonsa Horizon Depth(cm)

Clay Silt Sand OC(g kg�1)

C/N ratio pH(KCl)

(%)

G1 Ah1 0e9 69 27 4 186.6 19 3.7Ah2 9e20 68 26 5 130.5 22 4.0Ah3 20e31 67 27 6 92.5 26 4.0

G2 Ah1 0e13 65 28 7 136.2 19 3.9F1 Ah1 0e10 48 43 10 80.6 21 3.8

Ah2 10e26 63 29 8 57.7 20 4.12Ah2 26e33 55 23 22 62.7 19 4.2

F2 Ah1 0e4 59 33 8 157.4 13 3.3

a G: grassland; F: Araucaria forest; data from Dümig et al. (2008b).

A. Dümig et al. / Soil Biology & Biochemistry 57 (2013) 1e132

Boutton, 1996) due to climate variations or anthropogenic influ-ences on ecosystems. The extent to which plants discriminateagainst 13CO2 during photosynthesis depends on the photosyn-thetic pathway type (Vogel, 1980; O’Leary, 1988; Farquhar et al.,1989). Accordingly, the natural isotopic difference is especiallygreat between C3 (most trees, shrubs, and cool season grasses) andC4 (most warm season and desert grasses) plants, which showmean d13C values of ca. �27& and �13&, respectively (Martinet al., 1990; Boutton et al., 1998). Both applications use this highisotopic difference between C3 and C4 plants and are based on theassumption that SOC reflects the vegetation origin. However, the13C concentration of organic matter changes from plant material toSOC. This is shown by natural depth gradients of d13C values in soilswithout vegetation change which generally differ between C4grassland (13C depletion) and C3 forest (13C enrichment) ecosys-tems (e.g. Martin et al., 1990; Balesdent et al., 1993; Victoria et al.,1995; Wynn et al., 2006).

Several hypotheses for 13C enrichment or depletion from plantmaterial to SOC andwith increasing soil depth have been discussed.These include environmental conditions (e.g. topography, watersupply and soil redox status) and the Suess effect which is thoughtto produce increasing d13C values with soil depth due to thedecreasing d13C value of atmospheric CO2 since the start of theIndustrial Revolution. However, the influence of these environ-mental factors seems to be smaller than the observed d13C differ-ences in soils (e.g. Balesdent et al., 1993; Boutton, 1996; Ehleringeret al., 2000; Torn et al., 2002; Wynn et al., 2006; Werth andKuzyakov, 2010). Another factor affecting the 13C/12C isotopicsignatures of OC in the plantesoil-system is the so-called “kineticeffect” of most biochemical reactions. This microbial fractionationpreferentially transfers the lighter isotope (12C) into the product(CO2) and leaves the substrate (decomposing plant tissues orindividual organic compounds) enriched in 13C (Hobbie andWerner, 2004). Consequently, microbial fractionation by biodeg-radation of organic matter occurs in C4 grassland and C3 forest, butdifferences in the turnover of organic matter may lead to distinctdegrees of 13C enrichment between vegetation types. The factorscontrolling the magnitude of kinetic isotope 13C fractionation arestill unclear as reviewed by Werth and Kuzyakov (2010).

Moreover, 13C contentsmay differwithin the same plant betweenparts (e.g. Wedin et al., 1995; Schweizer et al., 1999), plant organs(Hobbie and Werner, 2004) or specific organic compounds (e.g.Benner et al.,1987; Collister et al.,1994). Thus, selective consumptionby the decomposing community or preferential stabilization by themineralmatrixof specific organic compoundswould change the d13Cvalue of total organic carbon (OCtot) in soils differing from those ofthe original plantmaterial. In this regard, itwas frequently suggestedthat the isotopic depletion of SOC originates from the relative accu-mulation of 13C depleted lignin owing to a lower decomposition ratecompared to the isotopically heavier cellulose, at least in the earlystages of biodegradation (e.g. Benner et al.,1987; Preston et al., 2005;Osono et al., 2008). Lignin is a high molecular weight, three dimen-sional aromatic polymer with ill-defined size (Crawford, 1981). Itconsists of phenyl propane units and is synthesized only by vascularplants in close associationwithhemicellulose (Kirk andFarrell,1987).The inputs of plant-residue lignin to soils are large as lignin is thesecond most abundant component of vascular plant tissues aftercellulose (Crawford,1981; Kögel-Knabner, 2002). The degradation oflignin is limited to an aerobic environment and only a small numberof microorganisms are involved in lignin mineralization withdifferent efficiency (fungi >> actinomycetes > bacteria), but onlya specific group of fungi (white-rot fungi) is able to mineralize ligninto CO2 (Haider and Trojanowski, 1980; Kirk and Farrell, 1987).However, the extent to which lignin is degraded or stabilized in soilsis still a matter of debate as summarized by Thévenot et al. (2010).

The aim of the present study was to assess if the shift in 13Cconcentration from plant material to SOC is related to changes inthe chemical composition of organic matter with particular refer-ence to lignin. Therefore, we evaluated the contribution of differentSOM components to the d13C signature of the mineral associatedSOM in topsoils under C3 forest and C4 grassland on non-allophanicAndosols in comparison to the corresponding above- and below-ground plant tissues. Solid-state 13C NMR spectroscopy was used toassess the overall SOM composition of alkyl, O-alkyl and aromatic C.Carbohydrate analysis after trifluoroacetic acid hydrolysis providedinformation on the degree of microbial alteration of the carbohy-drate components. Compound-specific gas chromatography-combustion-isotope-ratio mass spectrometry (GC/C-IRMS) incombinationwith alkaline CuO oxidationwas applied to determinethe isotopic composition of lignin-derived phenols. Besides marinesediments (e.g. Goñi and Eglinton, 1996), compound-specific d13Cvalues of individual lignin phenols in soil organic matter have so faronly been reported for agricultural soils by using natural 13Clabeling with maize (e.g. Dignac et al., 2005; Bahri et al., 2006;Heim and Schmidt, 2007b; Hofmann et al., 2009). This is to the bestof our knowledge the first report of 13C values of lignin from forestand grasslands soils.

Our approach allows: (1) to evaluate if the prevailing 13Cdepletion of OCtot in C4 grassland soils could be related to ligninpreservation in heavy soil fractions and (2) to identify if changes inthe chemical composition explain the opposite shifts in OCtot d13Cvalues between vegetation and SOC under C4 grassland and C3

forest.

2. Material and methods

2.1. Study area, sampling and soils

The studyarea belongs to the northern highlands of the southernBrazilian state of Rio Grande do Sul (29� 280e290 S, 50� 100e130 W).The soil profiles are located in the forest reserve CPCN Pró-Mata(Centro de Pesquisas e Conservação da Natureza), which is about35 km east of the city of São Francisco de Paula and 110 km from thecapital Porto Alegre. The subtropical highland climate is character-ized by high precipitation (annual mean 2252 mm) withouta pronounced dry period, moderate temperatures (annual mean14.5 �C) and relative humidity of 80e86% (Bertoletti and Teixeira,1995). Detailed descriptions of field work, sampling, sites and soilcharacteristics are given in Dümig et al. (2008b). The pedons arecarbonate-free and characterized by high clay content, low pH(Table 1) and similar clay mineralogy (quartz, kaolinite, secondarychlorite, gibbsite). Most have humus-rich topsoil and are classifiedas (nonallophanic) Andosols or Umbrisols (Dümig et al., 2008b).

A mosaic of grasslands (C4 plants) and Araucaria forests (C3plants) is established in the highlands. The Araucaria forest iscomposed of deciduous trees and the conifer Araucaria angustifolia.

A. Dümig et al. / Soil Biology & Biochemistry 57 (2013) 1e13 3

In the Pró-Mata forest reserve all forms of land use, for exampletree cutting or grazing and burning of grassland, terminated in1992.

A former study in the southern Brazilian highlands detected 13Cdepletion of total organic carbon (OCtot) in long-term stable C4grasslands (6000e8000 yr BP) from grass tissues to the topsoil andwith increasing soil depth (Dümig et al., 2008a). In C3 Araucariaforest, 13C enrichment was detected from tree tissues to topsoils.Forest topsoils clearly showed C3-derived isotopic signatures as theexpansion of Araucaria forest on grassland occurred a long timeago, and was stopped by grazing and burning of grasslands in morerecent times (Behling et al., 2001; Dümig et al., 2008a).

For this study, the following types of samples were selected inboth, C4 grassland and C3 forest: aboveground biomass (in tripli-cate), roots (in triplicate) and heavy soil fractions (four samples)from two topsoils. The corresponding soil horizons and depths areshown in Table 1. Grass shoots were sampled from Andropogonlateraliswhich is the principal gramineous species of the grasslandsat Pró-Mata (Bertoletti and Teixeira, 1995) as well as from Sor-ghastrum nutans and as mixed sample (cut within a 20 � 20 cmrack) from the grassland soil G2. As aboveground biomass in C3

forest served two organic surface layers (comprising Oi and Oehorizons) and the needles from the conifer A. angustifolia. Rootmaterials were retrieved from the same soil horizons as heavyfractions and alternatively we sampled fine roots fromA. angustifolia.

2.2. Density fractionation and bulk 13C content

The soil material was fractionated by density to separate theheavy organo-mineral fraction from the litter- and/or root-derivedfree and light particulate OM (LF). An aliquot (10 g) from the bulksoil (<2 mm) was saturated with Na polytungstate solution(1.6 g cm�3), gently shaken, allowed to settle (2 h) and centrifugedto separate the LF from the fraction with density >1.6 g cm�3. TheLF fraction was washed with deionized water on a 20 mm meshsieve. The heavy fraction was centrifuged several times withdeionized water until the electrical conductivity had dropped to<2 mS cm�1. All fractions were freeze-dried. Root material waspicked by hand from the LF for further analyses.

The 13C contents were measured using a CHN auto-analyser(CHN NA 1500, Carlo Elba), coupled to an isotope ratio mass spec-trometer (VG Sira 10; Girardin and Mariotti, 1991). The laboratoryreference was calibrated against the Vienna Pee Dee Belemnite (V-PDB) international standard. The carbon isotope ratio values arecalculated in d notation on a per mil basis (&), which is defined asthe 13C/12C ratio of the sample relative to the internationalstandard:

d13Cð&Þ ¼�ð13C=12CÞsample

ð13C=12CÞV�PDB� 1

�� 1000

Analytical precision for isotope measurements was �0.3&.

2.3. Content of lignin-derived phenols (CuO oxidation)

Lignin analysis using CuO oxidation (Hedges and Ertel, 1982)was carried out according to the modified method of Kögel andBochter (1985). Briefly, the samples (50 mg for plant biomass andorganic surface layers, 500 mg for organo-mineral fractions) wereoxidizedwith 250mg CuO and 2MNaOH at 172 �C under N2 for 2 h.Afterward, a standard containing ethylvanillin was added to assessthe recovery of lignin products, which was usually in the range60e70%. The solution was adjusted to pH 1.8e2.2 and left overnight for humic acid precipitation. Thereafter, the lignin-derived

phenols were purified by elution through a C18 column and deriv-atized by adding BSTFA [N, O-bis(trimethylsilyl)trifluoro-acet-amide]. The silylated lignin monomers were separated using an HP6890 gas chromatograph equipped with an SGE BPX-5 column(50 m � 0.25 mm i.d., 0.32 mm coating) and detected with flameionization detection (GC/FID). The GC oven temperature programwas: 100 �C (2 min) to 172 �C at 8� C/min, to 184 �C at 4 �C/min andto 300 �C at 10� C/min. Samples were injected in split mode (1:10).Phenol concentrations were calculated with the internal standardphenylacetic acid. The results are given as mean values obtainedfrom at least two replicates. CuO oxidation products are composedof vanillyl (V)-units (vanillin, acetovanillone, vanillic acid), syringyl(S)-units (syringaldehyde, acetosyringone, syringic acid) and cin-namyl (C)-units (ferulic and p-coumaric acids). The sum of V-, S-and C-type phenols (VSC) expressed as mg VSC g�1 SOC ormg VSC g�1 plant OC is used to estimate the amount of lignin.

2.4. Lignin isotopic analysis

The 13C/12C isotopic signature of the lignin-derived CuO oxida-tion products was determined using compound specific isotopeanalysis (Goñi and Eglinton, 1996). Samples were measured usingan HP5890 gas chromatograph coupled via a combustion interfaceto an Isochrom III isotope ratio mass spectrometer (Micromass-GVIOptima). A volume of 0.3 ml was injected in splitless mode. Thesame column, injection mode and temperature programwere usedas for the quantification of lignin with GC/FID. In order to obtaina concentration of each phenol in the extract greater than 2 nmol C/ml, which represents the detection limit for GC/C-IRMS, the CuOprocedurewas repeated 2e3 times (on 50mg for plant biomass andorganic surface layers, 500 mg for organo-mineral fractions) asdescribed above, and the respective C18 column eluates werecombined. Each composite sample was analyzed three times. The13C content of silylated phenol monomers was corrected for carbonaddition during derivatization using BSTFA. Depending on thephenol, derivatization introduced three to six carbon atoms.Therefore, the d13C values were corrected using the 13C content ofthe derivatizing agent as described by Dignac et al. (2005). Themean d13C values for V-, S-, and C-type lignin were calculated byweighting the 13C contents of each monomer with its relativecontribution to the corresponding lignin type. We used thesevalues and the concentrations of V, S and C to calculate the d13Cvalues of the total analyzable lignin VSC. The precision of theanalytical method is in the range of 0.2e0.8 per mil (Dignac et al.,2005).

2.5. Determination of neutral sugars

Sugar monomers were analyzed according to Eder et al. (2010),which is a modification of the methods of Amelung et al. (1996) aswell as Rumpel and Dignac (2006) for the use in mineral soils.Briefly, the samples were hydrolyzed with 4 M trifluoroacetic acid(TFA) at 105 �C for 4 h. After the samples had cooled down, aninternal standard (methylglucose) was added for calculating therecovery. Thereafter, the hydrolyzed samples were purified byfiltration over glass fiber membrane and dried using a rotaryevaporator (40 �C). Instead of ammonia, ethylenediaminetetra-acetic acid (EDTA) was added to keep iron ions in stable, solublecomplexes and to avoid possible co-precipitation of dissolvedorganic carbon. Derivatization of the sample was carried out inscrew top test tubes. Aldoses were reduced to their correspondingalditols after addition of NaBH4 dissolved in dimethyl sulfoxide(DMSO). Simultaneously, myoinositol was added for calculating theGC response factor. Acetylation was carried out by adding aceticanhydride and glacial acetic acid, and methylimidazole was used as

A. Dümig et al. / Soil Biology & Biochemistry 57 (2013) 1e134

catalyst. After 10 min, the reaction was stopped by adding ice colddeionized water. The derivatized sugar monomers were extractedby liquideliquid extraction with dichloromethane. GC analysis wasperformed on a GC Agilent 6890 (Agilent Technologies, Germany)equipped with a FID. The sugar monomers were separated witha 60m fused silica capillary column (BPX 70, SGE GmbH, Germany).The temperature program started at 180 �C (isothermal for 3 min)and increased to 230 �C at 5 �C min�1, 23 min isothermal at 230 �C.The concentration of individual sugar monomers (arabinose,fucose, galactose, glucose, mannose, rhamnose, ribose, xylose) wascalculated based upon the internal standard myoinositol. Therecovery of the standard methylglucose varied between 65 and93%.

2.6. Solid-state 13C NMR spectroscopy

The samples were analyzed by solid-state 13C NMR spectroscopy(Bruker DSX 200 NMR spectrometer) using the cross-polarizationmagic angle spinning (CPMAS) technique (Schaefer and Stejskal,1976) with a spinning speed of 6.8 kHz and a contact time of1 ms. A ramped 1H-pulse starting at 100% power and decreasinguntil 50% was used during contact time to avoid HartmanneHahnmismatches (Peersen et al., 1993). The pulse delays were 400 ms(heavy fractions) and 2000 ms (plant biomass and organic surfacelayers). In dependence of the signal-to-noise ratio, a line broad-ening between 50 and 100 Hz was used prior to Fourier trans-formation. The chemical shifts of 13C were referred to externaltetramethylsilane (¼ 0 ppm). For quantification, the spectrum wasdivided into four major chemical shift regions according to Knickerand Lüdemann (1995) (0e45 ppm: alkyl-C, 45e110 ppm: O-alkyl-C,110e160 ppm: aromatic C, 160e220 ppm: carboxyl-C) over whichwas integrated. The occurrence of spinning side bands due toinsufficient averaging of the chemical shift anisotropy at the usedspinning speed was considered by adding their intensities to that of

Table 2d13C values (&) of bulk materials and lignin-derived phenols as well as VSC contents (m

d13C values (&) C4 grassland

Shoots Roots

Andropogonlateralis

Sorghastrumnutans

Grassmixture

From soil/horizon

G1 Ah1 G1 Ah2 G2 Ah

OCtot �12.3 �12.9 �12.5 �12.4 �13.3 �12.2Lignin-derived phenolsVanillin �16.3 �12.2 �13.8 �12.4 �19.2 �11.8Acetovanillone �13.1 �15.5 �17.3 �15.6 �17.3 �14.4Syringaldehyde �20.3 �14.3 �19.3 �15.6 �23.2 �14.1Vanillic acid �17.5 �17.2 �15.3 �17.0 �11.4 �17.6Acetosyringone �23.2 �16.1 e �16.8 �22.9 �16.3Syringic acid �20.8 �16.9 �17.8 �17.5 �15.5 �13.0p-Coumaric acid �14.3 �11.3 �11.6 �10.1 �13.0 �10.9Ferulic acid �13.8 �10.4 �11.0 �9.6 �12.1 �6.4

Meansa

V units �16.0 �13.3 �14.5 �13.2 �17.8 �12.7S units �21.1 �15.4 �19.0 �15.9 �22.1 �14.3C units �14.1 �10.8 �11.2 �9.8 �12.4 �7.8VSC �16.7 �12.3 �13.1 �12.3 �15.0 �10.8

Lignin contents (mg VSC g�1 OC)V units 15.1 18.8 12.2 39.5 16.8 39.9S units 20.3 17.8 18.5 32.9 14.4 33.5C units 30.8 47.9 50.4 60.5 56.3 63.1VSC 66.2 84.5 81.0 133.0 87.4 136.5C/V 2.0 2.5 4.1 1.5 3.4 1.6S/V 1.3 0.9 1.5 0.8 0.9 0.8(ac/al)v b 0.14 0.19 0.21 0.13 0.22 0.1(ac/al)s b 0.38 0.37 0.25 0.12 0.17 0.1

V ¼ vanillyl units, S ¼ syringyl units, C ¼ cinnamyl units.a Weighted by the contents of lignin-derived phenols.b (ac/al)v ¼ acid to aldehyde ratio of vanillyl units, (ac/al)s ¼ acid to aldehyde ratio of

the parent signal (Knicker and Lüdemann, 1995; Knicker et al.,2005).

2.7. Statistics

Statistics were conducted with the IBM SPSS Statistic package(release 19.0). We used the ShapiroeWilk’sW-test and the Levene’stest to examine the data for normality and homogeneity of vari-ances with a critical level of p < 0.05, respectively. To identifydifferences between normally distributed data where one-wayANOVA indicated significant differences between C4 grasslandand C3 forest, Fisher’s protected least significant difference (LSD)was used formean separation. For datasets without homogeneity ofvariances, the Dunnett’s-T3 test was applied instead of the LSD.When datasets were not normally distributed, the nonparametricKruskaleWallis test followed by the ManneWhitney-U test wasused to test whether differences between means were significant.Mean values of heavy fractions were calculated for three samplesuntil 20 cm (grassland) or 26 cm (forest) soil depth.

3. Results

3.1. Contents of VSC lignins in C4 grassland and C3 forest

The yield of lignin-derived CuO oxidation products (mg VSC g�1

OC) and the distributions of vanillyl (V), syringyl (S) and cinnamyl(C) units differed between plant species and tissues (Table 2). Thelargest contents of lignin-derived phenols were found in grasstissues, shoots and especially roots (66e137mg VSC g�1 OC), wherethey consist mainly of C units (50e56% of total VSC lignins). Incontrast, tree-derived biomass showed significantly lower contentsof VSC lignins (17e36mg VSC g�1 OC) with generally dominating V-type oxidation products. Thus, grass tissues clearly differed fromthose of trees in the distribution of V- and C-units which is reflected

g VSC g�1 OC) of aboveground biomass and roots in C4 grassland and C3 forest.

C3 Araucaria forest

A. angustifolianeedle

Organic surface layersin Araucaria forest

Roots

A. angustifolia From soil/horizon

1 I II F1 Ah1 F2 Ah1

�26.5 �27.4 �29.0 �23.7 �28.0 �28.5

�31.3 �32.2 �33.0 �31.3 �34.6 �33.4�31.5 �32.5 e �30.8 �33.8 �33.2�34.2 �39.5 �35.5 �32.0 �33.3 �32.0�31.2 �36.2 �35.5 �24.2 �31.8 �32.5�31.9 �30.1 �37.6 �29.1 �33.1 �34.1�24.9 �30.6 �35.0 �26.5 �27.4 �32.2�28.0 �29.6 �29.2 �25.6 �31.8 �26.2�27.1 e �30.5 �21.6 �27.7 �24.3

�31.3 �32.9 �34.2 �30.3 �33.9 �33.1�28.5 �34.8 �35.6 �30.1 �32.6 �32.3�27.7 �29.6 �30.3 �22.0 �28.8 �24.8�30.2 �33.6 �35.1 �28.3 �30.9 �30.8

15.7 16.9 7.7 11.9 9.2 13.15.4 13.1 10.1 1.1 7.4 11.93.3 3.1 3.7 4.1 19.7 8.3

24.3 33.0 21.5 17.1 36.4 33.30.2 0.2 0.5 0.3 2.1 0.60.3 0.8 1.3 0.1 0.8 0.9

3 0.16 0.26 0.85 0.19 0.31 0.323 3.44 0.70 0.27 0.83 0.14 0.18

syringyl units.

A. Dümig et al. / Soil Biology & Biochemistry 57 (2013) 1e13 5

by higher C/V ratios of grasses. When compared to plant tissues,heavy fractions showed opposite results between C3 and C4 origin.On average, C3-derived heavy fractions were characterized byhigher VSC contents (2.7 mg VSC g�1 OC) as well as C/V ratios (0.7)than those with C4 signature (1.3 mg VSC g�1 OC, C/V ratio of 0.3).The VSC yield of the heavy fractions fromAh1 horizons, represented1% (C4 signature) and 12% (C3 signature) of the total VSC yield of theplant biomass (mean of above- and belowground biomass).Comparing the distribution of lignin units in heavy fractions andtheir corresponding plant biomass also evidenced differencesbetween C4 grassland and C3 forest. C4-derived heavy fractionsdiffered from grass tissues in the distribution of lignin phenols, inparticular due to lower contributions of C units, whereas C3-derivedheavy fractions and tree biomass showed similar proportions of V-,S- and C-type phenols. In this context, comparisons betweenC4- andC3-derived heavy fractions are inappropriate as markedly differ-ences already exist between the corresponding plant biomass.

The structural modifications of lignin during oxidative degra-dation (side-chain oxidation, cleavage of CeC bonds, demethox-ylation, degradation of the aromatic nuclei through ring opening)are reflected by changes in the composition of lignin-derivedphenols: relative increases of acid compared to aldehyde forms ofV and S units, and decreasing S/V and C/V ratios due to partialdemethoxylation transforming S-phenols into V-phenols ordifferent resistance (C < S < V units) against biodegradation(Haider et al., 1977; Higuchi, 1982; Ertel and Hedges, 1984; Kirk,1984; Chen and Chang, 1985; Kögel, 1986). With exception of theneedles from Araucaria angustifolia, the ratios of (ac/al)V and (ac/al)S were higher in all heavy fractions compared to plant biomass.

3.2. Stable carbon isotope signatures of plant tissues and heavyfractions in C4 grassland and C3 forest topsoils

The 13C/12C isotopic signatures of total organic carbon (OCtot)and lignin-derived phenols (expressed as VSC lignins) from plant

Table 3d13C values (&) of OCtot and lignin-derived phenols as well as VSC contents (mg VSC g�1

d13C values (&) and VSC lignincontents of heavy fractions

C4 grassland soils

G1 G2

Ah1 Ah2 Ah3

OCtot �14.3 �15.1 �15.1Lignin-derived phenolsVanillin �14.8 �10.5 �12.5Acetovanillone _ �15.6 _Syringaldehyde �16.8 �18.0 �19.8Vanillic acid �11.8 �18.0 �11.4Acetosyringone �20.3 �20.1 �19.7Syringic acid �22.2 �17.1 �13.8p-Coumaric acid �15.8 �16.1 �15.3Ferulic acid �13.6 �12.9 �12.9

Meansa

V units �13.0 �14.1 �12.0S units �20.0 �18.9 �17.9C units �14.3 �16.1 �13.9VSC �16.0 �15.6 �14.8

Lignin contents (mg VSC g�1 OC)V units 0.5 0.7 0.7S units 0.6 0.3 0.5C units 0.5 0.1 0.2VSC 1.6 1.2 1.4C/V 0.9 0.1 0.3S/V 1.0 0.5 0.7(ac/al)v b 1.5 0.6 0.8(ac/al)s b 1.1 1.0 1.5

V ¼ vanillyl units, S ¼ syringyl units, C ¼ cinnamyl units.a Weighted by the contents of lignin-derived phenols.b (ac/al)v ¼ acid to aldehyde ratio of vanillyl units, (ac/al)s ¼ acid to aldehyde ratio of

materials and heavy fractions are presented in Tables 2 and 3,respectively. Mean values and results from statistical analysis canbe found in Table 4. For OCtot and VSC lignins, 13C contents wereclearly higher in C4 materials than in the corresponding C3 coun-terparts. The mean d13C values of OCtot showed smaller differencesbetween C4 grassland and C3 forest (15.1& for abovegroundbiomass, 14.1& for roots and 11.3& for heavy fractions) whencompared to VSC lignins (18.9& for aboveground biomass, 17.3&for roots and 13.3& for heavy fractions). The isotopic differencesbetween C4 and C3 materials were the largest in the abovegroundbiomass and smallest in heavy fractions for OCtot and VSC lignins.Besides the distinct isotopic signatures for C3 forest and C4 grass-land, we found that tree roots were in average 0.9& enriched in 13Crelative to aboveground biomass. In contrast, roots and shoots of C4grasses showed similar OCtot d13C values. Our results are in linewith the findings of Hobbie and Werner (2004) and Werth andKuzyakov (2010). VSC lignins from roots were enriched in 13Cwhen compared to VSC lignins from aboveground biomass (C4grasses: 1.3&, C3 trees: 3.0&). These isotopic differences betweenroots and leaves were not significant.

Generally, the mean 13C depletion of VSC lignin compared toOCtot was larger for aboveground biomass (C4: 1.5&, C3: 5.3&) thanfor roots (C4: 0.1&, C3: 3.2&) and heavy fractions (C4: 1.4&, C3:3.3&). These data also point out that C3-biomass and C3-derivedSOM showed higher degrees of 13C depletion of VSC ligninscompared to OCtot than the C4 counterparts of which d13C values inVSC were similar or even higher compared to OCtot (Fig. 1). Thesetrends were also true at the level of lignin structural units: withexception of two tree root samples, all structural units of ligninwere 13C depleted relative to OCtot in C3-derived biomass and heavyfractions (Fig. 2).

Considerable isotopic differences were detected among ligninmonomers. The highest d13C values were found for cinnamic (C)phenols (p-coumaric and ferulic acids) in all plant tissues and C3-derived heavy fractions (Fig. 2). In contrast, most C4-derived heavy

OC) of heavy fractions in C4 grassland and C3 forest.

C3 Araucaria forest soils

F1 F2

Ah1 Ah1 Ah2 2Ah2 Ah1

�13.8 �24.9 �24.4 �23.6 �27.7

�11.8 �27.0 �28.6 �27.8 �32.4�16.2 �30.1 _ _ _�17.9 _ �27.7 �26.9 �32.1�16.5 �31.8 �25.5 �24.7 �33.4�17.3 �30.6 �26.2 �27.0 �35.7�17.8 �28.3 �26.0 �26.3 �33.7�13.3 �27.5 �24.1 �23.0 �28.5�10.9 �28.0 �26.0 �24.5 �29.2

�14.4 �30.4 �26.5 �25.7 �32.9�17.5 �30.0 �26.6 �26.4 �32.9�12.1 �27.9 �25.3 �23.9 �29.0�15.6 �29.3 �26.1 �25.9 �31.7

0.6 2.5 1.3 1.0 0.30.4 1.1 0.5 0.2 0.50.0 2.1 0.8 0.2 0.31.0 5.8 2.6 1.5 1.00.1 0.9 0.6 0.2 1.00.6 0.5 0.4 0.2 1.70.5 2.7 2.4 1.2 1.10.0 1.4 1.4 2.4 0.2

syringyl units.

Table 4Mean values of properties from aboveground biomass, roots and heavy fractions (three replicates, standard deviation in brackets). Significant differences (p< 0.05) between C4

grassland and C3 forest aremarkedwith asterisk. For d13C values (&) of OCtot and VSClignin, different letters indicate significant differences (p< 0.05) in 13C content between soilheavy fractions and corresponding plant materials (aboveground biomass, roots) in C4 grassland or C3 forest.

Parameters Aboveground biomass Roots Heavy fractionsa

C4 grassland C3 forest C4 grassland C3 forest C4 grassland C3 forest

d13C value (&) of OCtot �12.6 (0.3) b �27.6 (1.3) c* �12.6 (0.6) b �26.7 (2.6) c* �14.4 (0.7) a �25.7 (1.8) c*

d13C value (&) of VSC lignins �14.0 (2.3) c �33.0 (2.5) b* �12.7 (2.1) c �30.0 (1.4) a* �15.8 (0.2) c �29.0 (2.8) a*

d13C value (&) of V units �14.6 (1.4) �32.8 (1.4)* �14.5 (2.8) �32.4 (1.9)* �13.8 (0.7) �29.9 (3.2)*

d13C value (&) of S units �18.5 (2.9) �33.0 (3.9)* �17.4 (4.2) �31.7 (1.4)* �18.8 (1.3) �29.8 (3.2)*

d13C value (&) of C units �12.0 (1.8) �29.2 (1.4)* �10.0 (2.3) �25.2 (3.4)* �14.2 (2.0) �27.4 (1.9)*

Content of OCtot (mg g�1) 453.6 (12.9) 481.4 (23.6) 422.6 (10.5) 448.0 (5.4)* 151.1 (30.9) 98.6 (52.2)VSC content (mg VSC g�1 OC) 77.2 (9.7) 26.3 (6.0)* 119.0 (27.4) 28.9 (10.4)* 1.3 (0.3) 3.1 (2.4)Content of V units (mg V g�1 OC) 15.4 (3.3) 13.4 (5.0) 32.1 (13.2) 11.4 (2.0) 0.6 (0.1) 1.4 (1.1)Content of S units (mg S g�1 OC) 18.8 (1.3) 9.5 (3.9)* 26.9 (10.9) 6.8 (5.4)* 0.4 (0.1) 0.7 (0.3)Content of C units (mg C g�1 OC) 43.0 (10.7) 3.4 (0.3)* 60.0 (3.4) 10.7 (8.1)* 0.2 (0.3) 1.1 (0.9)Content of carbohydrate-C (mg g�1 OC) 374.4 (17.9) 293.8 (13.4)* 486.6 (24.8) 252.2 (54.0)* 271.6 (10.6) 165.6 (56.6)*

GM/AX ratio 0.2 (0.0) 1.0 (0.1) 0.1 (0.0) 0.6 (0.3)* 3.2 (0.5) 5.0 (3.9)Carboxyl C (%) 4 (3) 5 (2) 4 (1) 8 (3) 15 (2) 10 (2)Aromatic C (%) 11 (2) 16 (1)* 16 (1) 22 (3)* 18 (4) 19 (3)O-alkyl C (%) 68 (2) 51 (0)* 64 (1) 48 (11) 35 (3) 31 (1)N-alkyl C (%) 8 (1) 9 (1) 9 (1) 8 (1) 9 (1) 10 (1)Alkyl C (%) 9 (3) 20 (3)* 7 (1) 14 (5) 23 (2) 31 (3)

a From two Ah1 horizons and one Ah2 horizon.

A. Dümig et al. / Soil Biology & Biochemistry 57 (2013) 1e136

fractions showed another pattern of 13C contribution to V, S and Cunits (V> C> S). In almost all samples with C4 signature, C and/or Vunits had higher d13C values relative to OCtot.

The changes in d13C from plant materials to SOC show oppositetrends in C4 grassland and C3 forest (Fig. 1). In the pathway fromplant materials (roots and aboveground biomass) to heavy fractionsof SOC (0e20/26 cm soil depth), we found decreasing d13C values inC4 grassland (1.8 and 2.4& for OCtot and VSC lignins, respectively)and increasing d13C values in C3 forest (1.5 and 2.5& for OCtot andVSC lignins, respectively). Thus, the isotopic signatures of OCtot andVSC lignins in C4 grassland and C3 forest converge during the build-up of soil organic matter. However, the changes in d13C from plantmaterials to SOC were not significant (p < 0.05) for both, OCtot andVSC lignins (Table 4). In C4 grassland, we only found significantdifferences in 13C contents between soil heavy fractions and plantmaterial (roots or aboveground biomass) for OCtot, but not for VSClignins. In C3 forest, significant differences in 13C contents were onlydetected between lignin in soil heavy fractions and lignin inaboveground biomass. The differences between C4- and C3-derivedheavy fractions in 13C abundance of VSC lignin (12.7&) were higherthan those of OCtot (10.6&). In C4 grassland, the decreasing d13Cvalues of lignins mainly result from changes of C- and S-typemonomers. Compared to plant tissues, C-type monomers in heavyfractions showed lower d13C values as well as contributions to totalVSC content, whereas 13C depleted S units were relatively accu-mulated in heavy fractions. In contrast, in C3-derived heavy frac-tions, higher 13C contents in V and S units compared to plant tissueswere detected, and C units showed the average value of roots andaboveground plant tissues. Following the isotopic signature withincreasing soil depth (Ah1 to Ah3), C3-derived heavy fractionsshowed increasing d13C values for VSC lignins (3.4&) and OCtot(1.3&). For C4-derived heavy fractions, VSC lignins were also 13Cenriched (1.2&) with soil depth, whereas OCtot were 13C depleted(0.8&).

3.3. Carbohydrate signatures in C4 grassland and C3 forest

The concentrations of carbohydrate-C are shown inTable 5. Theseconcentrations only account for monosaccharides from hemi-celluloses and microbial products, since TFA does not release crys-talline cellulose. In C4 grassland, the concentration of carbohydrate-Cwas highest in roots (487 � 25 mg g�1 OC) when compared to

aboveground biomass (374 � 18 mg g�1 OC) and heavy fractions(265 � 16 mg g�1 OC). C3-derived materials, in particular tree roots,showed significant lower carbohydrate-C concentrations (above-ground biomass: 294 � 13 mg g�1 OC, roots: 252 � 54 mg g�1 OC,heavy fractions: 149 � 57 mg g�1 OC) than the respective C4 coun-terparts (Table 4). Major differences in the composition ofcarbohydrate-C between grass and tree tissues reside in the higherproportions of xylose and lower ones of galactose and mannose ingrass tissues. As shown in Table 5, this resulted in lower ratios ofgalactose plus mannose over arabinose plus xylose (GM/AX) of grasstissues (ratios of 0.1e0.2) when compared to those of trees (0.3e1.0).Generally, heavy fractions showed higher GM/AX ratios than planttissues, increasing GM/AX ratios with soil depth, and C3-derivedheavy fractionshigherGM/AX ratios thanC4-derivedheavy fractions.Ribose concentrations were very low for all samples.

3.4. Bulk chemical composition of plants and heavy fractions in C4grassland and C3 forest as revealed by solid-state 13C NMRspectroscopy

We found several differences in the relative proportions offunctional groups according to the solid-state 13C NMR spectra of C4grassland and C3 forest plant and soil samples (Table 5). Grasstissues and C4-derived heavy fractions showed higher contribu-tions of O-alkyl C which are mainly attributed to carbohydratecarbons in cellulose and hemicelluloses when compared to treetissues and C3-derived heavy fractions. This is in line withcarbohydrate-C contents (Table 5). On the other hand, the propor-tions of alkyl C were throughout lower in grasses and heavy frac-tions with C4 signature than in the C3 counterparts. The majorsources of alkyl C are aliphatic biopolymers such as lipids, cutin andsuberin, and other long chain carboxylic acids (Kögel-Knabner,1997). Consequently, C3-derived OC is characterized by higheralkyl C/O-alkyl C ratios compared to C4-derived OC. This differencein the chemical composition was significant only for abovegroundbiomass, but not for roots and heavy fractions. The proportions ofaromatic C were significantly higher in all tree tissues compared tograss tissues, whereas C4- and C3-derived heavy fractions showedsimilar proportions of aromatic C. When compared to plant tissues,heavy fractions of Ah1 horizons showed higher proportions ofcarboxyl C and lower ones of O-alkyl C which was particularlypronounced in C4 grassland. In addition, the proportions of alkyl C

Fig. 1. d13C values (&) of total organic carbon (OCtot) and VSC lignins in C4 grassland and C3 forest. Error bars represent the standard deviation of mean values (three replicates).

A. Dümig et al. / Soil Biology & Biochemistry 57 (2013) 1e13 7

and aromatic C similarly increased in C4 grassland and C3 forestfrom plant tissues to heavy fractions.With increasing soil depth, wefound increasing proportions of aromatic C and carboxyl C whichwas associated with decreasing O-alkyl and alkyl C proportions forboth, C4- and C3-derived heavy fractions. No differences at allbetween C4 grassland and C3 forest were detected for N-alkyl Cproportions.

4. Discussion

4.1. Shifts of OCtot d13C values from plant biomass to heavy fractions

of surface horizons in C4 grassland and C3 forest

d13C values of OCtot shifted in opposite directions between C4grassland (13C depletion) and C3 forest (13C enrichment) during thedecay continuum from plant biomass (roots and aboveground plant

materials) to SOC in heavy fractions of surface horizons (Fig. 1).These changes of isotopic signatures were not related to theisotopic difference of OCtot between roots and abovegroundbiomass as (1) we detected similar OCtot d13C values for roots andshoots from C4 grasses, and (2) C3-derived heavy fractions were 13Cenriched compared to both, aboveground biomass (2&) and roots(1&).

Numerous studies investigated the decomposition of organicmatter in litter layers or from plant tissues to surface soil horizonsin C3 forests, and reported increasing, decreasing or almostconstant d13C values of OCtot (Benner et al., 1987; Balesdent et al.,1993; Buchmann et al., 1997; Schweizer et al., 1999; Ehleringeret al., 2000; Osono et al., 2008). Accoe et al. (2003) found 13Cenrichment of SOC compared to fresh grass tissues and with soildepth in C3 grasslands. Direct comparisons between d13C values ofC4 grass tissues and the corresponding SOC or decomposition

Fig. 2. 13C/12C isotopic difference (&) between total organic carbon (OCtot) and VSC lignins as well as vanillyl (V), syringyl (S) and cinnamyl (C) units in C4 grassland and C3 forest.

A. Dümig et al. / Soil Biology & Biochemistry 57 (2013) 1e138

studies with C4 grass materials are rare. Victoria et al. (1995)calculated from the literature data an average d13C value forsurface soils in C4 grasslands of�16.1� 2.2&. The comparisonwiththemean d13C value of�13& for C4 plants (Boutton,1996) indicates13C depletion of C4 grasses OM during the transformation to SOM,similar as observed in the present study. This is in contrast to thegenerally increasing d13C values in the course of OC biodegradationas the decomposing microbial community use preferentially 12C forrespiration (Hobbie and Werner, 2004).

The 13C signatures of the different organic compounds consti-tuting plant litter differ in dependence of their chemical structure(e.g. aliphatic biopolymers, cellulose, lignins) and are usuallydifferent from that of OCtot (e.g. Benner et al., 1987; Collister et al.,1994; Schweizer et al., 1999). Lignin is generally 13C depleted whencompared to OCtot (1e10&) in various C4 and C3 plants analyzed byCuO oxidation (Goñi and Eglinton, 1996; Dignac et al., 2005; Bahriet al., 2006; Hofmann et al., 2009) or other methods for ligninextraction (Benner et al., 1987; Wedin et al., 1995; Schweizer et al.,1999; Dungait et al., 2008). The relative accumulation of lignincompared to isotopically heavier polysaccharides such as celluloseand hemicelluloses during litter decomposition was reported byseveral studies (e.g. Benner et al., 1987; Berg and McClaugherty,2003; Preston et al., 2005). Changes in the relative contents of 13Cenriched or depleted plant biopolymers due to preferentialmicrobial consumption and/or stabilization by the mineral matrixmay therefore control the depletion or enrichment of 13C fromplant biomass to heavy fractions. Ligninmay play a key role in theseprocesses due to its chemical recalcitrance (Benner et al., 1987;Wedin et al., 1995) as well as the preferential sorption to themineral phase (Kaiser and Guggenberger, 2000).

4.2. Is selective microbial preservation and/or consumption oflignins controlling d13C values of OCtot of C4- and C3-derived heavyfractions?

4.2.1. Content and biodegradation of lignin in C4 grassland and C3forest

As shown in Table 2, the yield of lignin-derived CuO oxidationproducts (mg VSC g�1 OC) generally differed between plant parts(roots > aboveground biomass) which was in line with the relativeproportions of aromatic C as measured by 13C NMR spectroscopy(Table 5) as well as with findings from Rasse et al. (2005). Thehigher VSC yields in grass tissues compared to tree tissues mayresult from the different chemical structures and thus, differentextractability of monomer units (Higuchi, 1990). Generally, theyields of VSC lignin were low in heavy fractions when compared totemperate agricultural (Guggenberger et al., 1994; Kiem and Kögel-Knabner, 2003; Dignac et al., 2005) or forest soils (Guggenbergeret al., 1994; Rumpel and Kögel-Knabner, 2002) as well astemperate and semiarid grassland soils (Amelung et al., 1999;Ganjegunte et al., 2005; Otto and Simpson, 2006; Heim andSchmidt, 2007a). Wiesmeier et al. (2009) reported VSC contentsfrom grassland soils in the southern Brazilian highland which weresimilarly low as in this study. In this environment with favorableclimatic conditions for decomposition of organic matter, lignin isapparently biodegraded to a large extent which is in line withmodels indicating rapid lignin turnover in soils similar to or evenfaster than OCtot (Rasse et al., 2006; Heim and Schmidt, 2007a).

Following VSC lignin degradation parameters (Fig. 3), thecomparison with plant tissues showed that the structural trans-formation of lignin differs between C4- and C3-derived heavy

Table 5Contents of organic carbon, carbohydrate-C and VSC lignin as well as relative proportions of functional groups as revealed by 13C NMR spectroscopy.

OC mg g�1 Carbohydrate-C mg g�1 OC

GM/AXa

Functional group/chemical shift (ppm)b Molecular mixing modelc VSCcontentd

Carboxyl C Aromatic C O-alkyl C N-alkyl C Alkyl C Alkyl C/O-alkyl C

Percental proportions of

245e160 160e110 110e60 60e45 45e0 Lignin Char %

C4 grasslandAboveground biomassAndropogon lateralis 468.3 354.6 0.2 4 12 68 8 9 0.1 nd nd 6.6Sorghastrum nutans 447.8 389.6 0.2 7 13 67 7 7 0.1 nd nd 8.4Grass-mix from soil G2 444.6 378.9 0.2 1 9 70 9 12 0.2 nd nd 8.1RootsG1 Ah1 434.3 486.5 0.1 4 15 64 10 7 0.1 nd nd 13.7G1 Ah2 414.0 511.4 0.1 6 17 63 8 6 0.1 nd nd 13.3G2 Ah1 419.5 461.8 0.1 4 16 64 9 7 0.1 nd nd 8.7Heavy fractionsG1 Ah1 186.6 260.0 3.3 14 16 38 9 23 0.6 3 12 0.2G1 Ah2 130.5 280.6 3.6 17 22 32 8 21 0.7 1 20 0.1G1 Ah3 92.5 244.8 4.6 20 27 27 7 19 0.7 0 25 0.1G2 Ah1 136.2 274.3 2.7 15 15 37 9 24 0.6 2 10 0.1

C3 Araucaria forestAboveground biomassOrganic surface layer I 504.4 278.4 1.0 5 16 51 9 18 0.3 nd nd 2.2Organic surface layer II 457.3 299.6 0.8 6 17 51 8 18 0.4 nd nd 3.3A. angustifolia (needles) 482.6 303.3 1.0 3 15 51 8 23 0.5 nd nd 2.4RootsF1 Ah1 449.4 205.3 0.5 9 22 46 8 15 0.3 nd nd 3.3F2 Ah1 442.0 240.2 0.3 11 26 39 7 18 0.5 nd nd 3.6A. angustifolia 452.5 311.3 0.8 4 19 60 8 9 0.1 nd nd 1.7Heavy fractionsF1 Ah1 80.6 164.9 3.5 11 20 31 9 29 0.9 30 2 0.6F1 Ah2 57.7 109.3 9.4 12 21 29 10 29 1.0 28 5 0.3F1 2Ah2 62.7 98.3 9.8 25 24 22 7 22 1.0 36 6 0.2F2 Ah1 157.4 222.5 2.0 8 16 31 11 34 1.1 26 0 0.1

a Ratio of galactose plus mannose over arabinose plus xylose.b Solid-state 13C NMR spectroscopy.c Molecular mixing model (Baldock et al., 2004).d CuO oxidation.

A. Dümig et al. / Soil Biology & Biochemistry 57 (2013) 1e13 9

fractions. d13C values of V, S, and C lignin units also point to distinctlignin alteration in C4 grassland, as C-type phenols (p-coumaric andferulic acids) showed the highest d13C values in all samples apartfrom C4-derived heavy fractions (Fig. 2). The higher 13C content ofC-type units compared to V or S units and sometimes OCtot is in linewith other studies (Goñi and Eglinton, 1996; Dignac et al., 2005;Bahri et al., 2006). This may indicate different processes and/orextent of lignin biodegradation between C4 grassland and C3 forestresulting from other kind of decomposer communities in associa-tionwith distinct compositions of grass and tree tissues (Fig. 4). Ourassumption is supported by (i) the interrelations between the rateof decomposition, litter quality according to the vegetation typeand heavy isotope enrichment (Berg, 2000; Quideau et al., 2003),(ii) the greater 13C enrichment of soil microbial biomass relative toOCtot in C3 than in C4 soils (�Santr�u�cková et al., 2000; Dijkstra et al.,2006), and (iii) the greater bioavailability of C4 biomass whencompared to C3 biomass (Bird and Pousai, 1997) and thus, the fasterdecomposition of C4-derived (grasses) than tree-derived SOC(Wynn and Bird, 2007). With respect to the differences in thestructural transformation of lignin, another explanation could bethe burning of grasslands, as charring of organic materials wasshown to generate a degraded lignin signature (Rumpel et al., 2007;Nocentini et al., 2010) which may differ from microbial ligninalteration.

4.2.2. Evidence from d13C values of VSC ligninsThe shifts of the 13C/12C isotope signature of VSC phenols from

plant tissues to heavy fractions (Ah1 and Ah2 horizons) weresimilar to those of OCtot (13C depletion in C4 grassland and 13C

enrichment in C3 forest), but on a larger scale (Fig. 1). So far, onlyfew data are available for comparison of isotopic values for VSClignin phenols of above- and belowground tissues in C4 grassland.C4 biomass showed smaller isotopic differences (1.5 � 1.6&)between VSC lignin and OCtot (Fig.1) when compared to tree tissues(4.3�1.6&). In addition, we found higher d13C values of VSC ligninsfrom roots relative to aboveground plant tissues (C4: 1.3&, C3:3.0&) which was also reported from Bahri et al. (2006). However,isotopic differences between roots and aboveground biomass arenot sufficient to explain our results as VSC lignins consistentlyshowed 13C enrichment (C3 forest) or depletion (C4 grassland)when roots as well as aboveground plant tissues were compared toheavy fractions.

13C/12C isotopic signatures of lignin-derived phenols in soilshave been reported from agricultural field experiments withnatural 13C labeling by replacement of C3 arable crops with C4maize (Dignac et al., 2005; Bahri et al., 2006; Heim and Schmidt,2007a; Hofmann et al., 2009). These soil lignins were found to be13C enriched (Hofmann et al., 2009) or almost showed no isotopicdifference (Dignac et al., 2005) when compared to VSC lignins inthe corresponding plants. Isotopic fractionation of VSC ligninsduring microbial decomposition associated with the release of 13C-depleted CO2 (Hobbie andWerner, 2004) may explain the observed13C enrichment of lignins from plant biomass to heavy fractions inC3 forest. However, C3-derived VSC lignins were 13C depletedcompared to OCtot and thus, counteract the prevailing 13C enrich-ment of OCtot from plant biomass to heavy fractions in forest soils.This also implies that other organic compounds which induce 13Cenrichment during biodegradation like microbial-derived OC

Fig. 3. Parameters of lignin degradation: ratio of syringyl-to-vanillyl phenols (S/V), ratio of cinnamyl-to-vanillyl phenols (C/V), ratios of acidic to aldehydic forms of the vanillyl-(ac/al)V and syringyl-(ac/al)S phenols.

A. Dümig et al. / Soil Biology & Biochemistry 57 (2013) 1e1310

(�Santr�u�cková et al., 2000; Dijkstra et al., 2006) must be morerelevant than lignin (and 13C depleted aliphatic biopolymers;Hobbie and Werner, 2004) for the isotopic signature of OCtot in C3forest.

In C4-derived heavy fractions, the 13C depletion of lignincompared to OCtot (1.4&) was much smaller than in C3-derivedheavy fractions (3.3&), and both showed 13C depletion from plantbiomass to heavy fractions (VSC lignin: 2.4&, OCtot: 1.8&). Inconsideration of the low lignin content of C4-derived heavy frac-tions, the influence of lignin on OCtot d13C values is presumably low,and thus other compounds may also account for 13C depletion in C4grassland soils. As the charring of C4 grasses could result in 13Cdepletion when compared to unburned grass biomass (Krull et al.,2003), the contribution of charred grass residues to OC in heavyfractions results in lower OCtot d

13C values. In addition, we supposethat the burning of grasslands may also affects the compound-specific d13C values of individual lignin phenols which possiblyexplain the 13C depletion of VSC lignins from plant biomass toheavy fractions. However, the temperature-dependent processes ofstable carbon isotope fractionation by vegetation fires still need tobe clarified (Czimczik et al., 2002).

4.3. Does the bulk chemical composition of SOM explain theopposite changes of OCtot d

13C values from vegetation to SOC underC4 grassland and C3 forest?

Solid-state 13C NMR spectra of C3- and C4-derived heavy frac-tions showed similar relative proportions of aromatic C, whereas

VSC contents were higher for C3-derived heavy soil fractions underC3 vegetation. As the grasslands (C4) were frequently burned in thepast (Behling et al., 2001), charred grass residues may contribute toSOM and thus, produce signals in the aromatic C region in C4-derived heavy fractions. Therefore, we applied the molecularmixing model from Baldock et al. (2004) to differentiate betweensignals in the aromatic C region deriving from chars or lignin. Thismodel uses the distribution of 13C NMR spectral intensity to esti-mate the molecular composition (e.g. carbohydrate, lignin, protein,lipid, charcoal) of organic materials. According to the model(Table 5), char was the major constituent of aromatic C in C4-derived heavy fractions, whereas proportions of lignin were low(0e3%). In contrast, lignin was the predominant compound in thearomatic C region of C3-derived heavy fractions (26e36%). Thus,results from the molecular mixing model are in line with thosefrom CuO oxidation indicating lower lignin contents in C4-derivedheavy fractions when compared to those in C3 forest. The relativeproportion of alkyl C can be used as a proxy for aliphatic biopoly-mers. Lipids of C4 grasses are more depleted in 13C (7e10&) thanthose from C3 woody plants (2.5e6&) relative to OCtot (Collisteret al., 1994; Chikaraishi and Naraoka, 2001; Hobbie and Werner,2004). Thus, the accumulation of char and alkyl C in heavy frac-tionswhich was detected by 13C NMR spectroscopymay also resultsin 13C depletion in C4 grassland soils.

Analysis of non-cellulosic carbohydrates indicated that themineral-associated SOC in C4- and C3-derived heavy fractions is ina late phase of decomposition with large contributions ofmicrobial-derived OC as carbohydrates with high GM/AX ratios of

Fig. 4. Chemical composition of aboveground biomass, roots and heavy fractions in C4 grassland and C3 forest.

A. Dümig et al. / Soil Biology & Biochemistry 57 (2013) 1e13 11

>2 (Table 5). These ratios indicate that carbohydrates may bepredominantly derived from microbial input (Oades, 1984). This isin line with observations from other authors indicating thatmicrobial carbohydrates are preferentially stabilized by interactionwith the mineral phase (Spielvogel et al., 2008; Rumpel et al., 2010)thereby contributing to 13C enrichment of the heavy fractions.These enrichments may be higher under C3 vegetation, in agree-ment with the decreases in the content of carbohydrate-C andproportions of O-alkyl C, which are less pronounced from C3vegetation to C3-derived SOC than from C4 vegetation to C4-derivedSOC (Table 5).

5. Conclusions

Compound-specific 13C/12C isotopic signatures of lignin fromheavy organo-mineral fractions of surface horizons revealeddifferences between long-term stable and frequently burned C4

grassland and C3 Araucaria forest, which were similar to those ofOCtot. Along the decay continuum from plant biomass to heavyfractions in topsoils the d13C values of both, VSC lignins and OCtotshifted in opposite directions between C4 grassland (13C depletion)and C3 forest (13C enrichment). Isotopic differences between rootsand aboveground biomass were not relevant in the topsoilsinvestigated here, but might have an effect on OCtot d13C values atgreater soil depths.

The combined evaluation of lignin-derived CuO oxidationproducts and 13C NMR spectra indicated low lignin contents inheavy fractions, in particular for those in C4 grassland. Thus, the

influence of lignin on OCtot d13C values in grassland topsoils ispresumably low. The presence of charred grass residues and theaccumulation of alkyl C in heavy fractions most likely contribute todecreasing d13C values from grass biomass to C4-derived heavyfractions.

In C3 forest, the accumulation of 13C depleted VSC lignin resi-dues in heavy fractions counteracts the prevailing 13C enrichmentof OCtot from plant biomass to heavy fractions. Nonetheless, organiccompounds which are relatively 13C enriched due to microbialprocessing have a stronger and dominating influence on the d13Csignature in forest topsoils than lignin.

The grassland and forest soils have similar mineral properties,but different chemical compositions of mineral-associated OC asthe vegetation type and land use management (e.g. burning ofgrasslands) determines the type and amount of plant input assource of SOM and thus carbon source formicrobial transformation.These differences may result in distinct structural alteration oflignin between C4 grassland and C3 forest which was shown by VSClignin degradation parameters and d13C values of V, S, and C ligninunits.

Our results indicate that the role of lignin for d13C values of OCtotwas overestimated in previous studies, at least in subtropical C4grassland and C3 forest topsoils.

Acknowledgments

This studywas funded by the Deutsche Forschungsgemeinschaft(DFG) under project Ko 1035/24-1. The research networking

A. Dümig et al. / Soil Biology & Biochemistry 57 (2013) 1e1312

programmeMOLTER from the European Science Foundation (ESF) isacknowledged for supporting this study by a travel grant.Wewouldlike to thank the Pontifícia Universidade Católica do Rio Grande doSul (PUCRS), W. Engels (Eberhard-Karls-Universität Tübingen,Germany) and V. Mosbrugger (Forschungsinstitut und Naturmu-seum Senckenberg) as well as R. Irslinger (University of AppliedForest Sciences Rottenburg, Germany) for realizingour project at theCentro de Pesquisas e Conservação da Natureza Pró-Mata (CPCN).Furthermore, we are grateful to V. De Patta Pillar, E. Klamt and D.Dick of the Universidade Federal do Rio Grande do Sul (UFRGS) aswell as to J. Pfadenhauer (Technische Universität München,Germany) for excellent cooperation.

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