chemical structure and sources of the macromolecular ...directory.umm.ac.id/data...

Chemical structure and sources of the macromolecular,resistant, organic fraction isolated from a forest soil

(LacadeÂ e, south-west France)

Natacha Poirier a,b, Sylvie Derenne b, Jean-NoeÈ l Rouzaud c, Claude Largeau b,*,AndreÂ Mariotti a, JeÂ roÃ me Balesdent d, Jocelyne Maquet e

aLaboratoire de BiogeÂochimie Isotopique, INRA-CNRS-UPMC, 4 pl. Jussieu, 75252 Paris cedex 05, FrancebLaboratoire deChimieBioorganique etOrganiquePhysique,UMRCNRS7573,ENSCP, 11 rueP etMCurie, 75231Paris cedex 05, FrancecCentredeRecherche sur laMatieÁreDiviseÂe,UMRCNRS6619,UniversiteÂ d'OrleÂans, 1bis rue de laFeÂrollerie, 45071OrleÂansCedex2,FrancedLaboratoire d'Ecologie Microbienne de la RhizospheÁre, DEVM, CEA Centre de Cadarache, 13108 Saint-Paul les Durance cedex, France

eLaboratoire de Chimie de la MatieÁre CondenseÂe, UPMC, UMR CNRS 7574, 4 pl. Jussieu, 75252 Paris cedex 05, France

Received 26 July 1999; accepted 23 May 2000

(returned to author for revision 13 January 2000)

Abstract

The insoluble, non-hydrolyzable, macromolecular material isolated from a forest soil from LacadeÂ e (south-west

France) was examined via a combination of various methods: FTIR spectroscopy, elemental analysis, ``o�-line'' pyr-olysis and high resolution transmission electron microscopy. Such a resistant material, which accounts for ca. 25% oftotal humin, was shown to be chie¯y composed of melanoidins and black carbon. Two types of black carbon particleswere identi®ed by dark ®eld and lattice fringe electron microscopy. Contrary to previous observations, based on solid

state 13C NMR spectroscopy and Curie point Py/GC/MS, highly aliphatic moieties only a�ord a minor contribution tothe refractory material of the LacadeÂ e soil. Additional studies, using mixtures of model compounds, were carried out toexamine the origin of this conspicuous overestimation of the level of aliphaticity in such heterogeneous material when

the latter two methods are used. # 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Refractory organic matter; Forest soil; Melanoidins; Black carbon; Biased aliphaticity; Solid state 13C NMR; FTIR; Pyr-

olysis; HRTEM

1. Introduction

Three pools, characterized by di�erent turnover rates,are generally distinguished in soil organic matter

(SOM). These include a stable pool with mean residencetimes up to millenia [Balesdent and Mariotti (1996) andreferences therein]. Information on the nature and fate

upon changes in land use of the latter pool is importantsince variations in its abundance would generate large

CO2 ¯uxes between the atmosphere and soils. However,

the mechanism that accounts for the stability of thisrefractory SOM is still far from being completely eluci-dated (e.g. Skjemstad et al., 1996). Protection by miner-

als is often considered but intrinsic resistance todegradation of some SOM constituents, directly relatedto their chemical structure, might also be an important

factor. Nevertheless, as stressed below, the chemicalcomposition of the refractory fraction of SOM is still amatter of debate.Numerous studies point to the occurrence of recalci-

trant aliphatic structures in SOM. Indeed, observationsby solid state 13C NMR on peats, composts and soils(reviewed by Preston, 1996; Baldock et al., 1997) show

0146-6380/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.

PI I : S0146-6380(00 )00067-X

Organic Geochemistry 31 (2000) 813±827

www.elsevier.nl/locate/orggeochem

* Corresponding author. Tel.: +33-1-4427-6761; fax: +33-

1-4325-7975.

E-mail address: [email protected] (C. Largeau).

that the relative contribution of alkyl carbon tends toincrease with increasing degrees of decomposition of theorganic matter. The presence of moieties containing poly-methylenic chains, re¯ected by the formation of

n-alkane/n-alk-1-ene doublets, was also observed via pyr-olysis of (i) soil organic matter (Bracewell andRoberston,1987; Hemp¯ing et al., 1987; van Bergen et al., 1997,

1998; Nierop, 1998), (ii) residues from chemical degra-dation of humic acids and SOM (Saiz-Jimenez and deLeeuw, 1987a,b; Tegelaar et al., 1989a; Almendros et

al., 1991; KoÈ gel-Knabner et al., 1992a,b), (iii) humin(Almendros et al., 1996; Grasset and AmbleÁ s, 1998;Lichtfouse et al., 1998) and (iv) the non-hydrolyzable

fraction of humin isolated via successive, drastic, baseand acid hydrolyses of a forest soil from LacadeÂ e(Augris et al., 1998). Large di�erences in the relativeintensity of the n-alkane/n-alk-1-ene doublets were noted,

in the above studies, when the gas chromatograms of totalpyrolysates were compared, and an especially high con-tribution was observed for the resistant (non-hydrolyzable)

fraction isolated from the humin of the LacadeÂ e soil(Augris et al., 1998). The origin and formation path-ways of aliphatic moieties in the stable fraction of SOM

remain largely unknown (Hedges and Oades, 1997) anddi�erent types of sources have been considered. Thesesources included highly aliphatic, resistant, macro-

molecular components from higher plants, the so-calledcutans and suberans (Saiz-Jimenez and de Leeuw,1987a,b; Tegelaar et al., 1989a; Augris et al., 1998;Nierop, 1998) and from soil microorganisms (Lichtfouse

et al., 1995, 1996, 1998; van Bergen et al., 1998) andmixtures of such higher plant and microbial components(Almendros et al., 1991, 1996; van Bergen et al., 1997).

Moieties with long alkyl chains derived from cross-linking of lipids and/or cutin and suberin polyesterswere also considered (KoÈ gel-Knabner et al., 1992a,b).

In a number of the above-mentioned studies, anabundant contribution of aliphatic moieties to therefractory fraction of SOM was inferred from solid state13C NMR observations. It is well documented, however,

that such spectroscopic methods can markedly over-estimate the aliphatic contribution in SOM fractionsand underestimate the aromaticity (e.g. Hatcher et al.,

1981; Wilson et al., 1987). Indeed, in contrast with theabove ®ndings, it is often considered that aromatic carbontends to accumulate as SOM decomposition proceeds

(Baldock et al., 1997). A part of this refractory aromaticcarbon could correspond to black carbon (e.g. Oades,1995). The complex polyaromatic structures collectively

termed ``black carbon'', a term synonymous in the lit-erature with ``charcoal'', correspond to the residues ofincomplete combustion produced from vegetation ®resand burning of fossil fuels. Black carbon is widely dis-

tributed over the entire surface of the earth (reviewed inGoldberg, 1985) and it was shown to account for asubstantial fraction of total organic carbon in some soils

(Skjemstad et al., 1996; Golchin et al., 1997a; Glaser etal., 1998). Such features re¯ect the origin of black car-bon in widespread burning processes and its refractorynature. The high stability of charcoal, and charred

materials from plants, is illustrated by (i) their greatability to survive severe oxidation treatments whencompared to kerogens (Wolbach and Anders, 1989) and

also to survive severe photo-oxidation (Skjemstad et al.,1996), (ii) the systematic occurrence of charcoal frag-ments in soil pro®les with 14C ages of up to ca. 2000

years in Mediterranean soils (e.g. Thinon, 1978) and (iii)charcoal occurrence in ancient sediments, such as 4 to 8million year old Pliocene samples (Dubar et al., 1995).

In fact, it is considered that only limited degradation ofcharcoal would take place with time through microbialor chemical degradation (Seiler and Crutzen, 1980).Accordingly, charcoal formation is a possible source for

the chemically most stable, aromatic carbon pool insoils (Haumaier and Zech, 1995; Skjemstad et al., 1996;Golchin et al., 1997b). Charcoal formation during

vegetation burning may thus partly convert a potentiallyactive carbon pool into a more inert one and represent away in which long-term protection of OM in soils may

occur. Moreover, recent studies are consistent with animportant role for black carbon as a sink in the globalcarbon cycle via burial in marine sediments (Kuhlbusch

and Crutzen, 1995; Lim and Cachier, 1996; Gustafssonand Gschwend, 1998). Nevertheless, the biological sta-bility of charcoal in soils and sediments, as well as itscontribution to carbon content and distribution remains

largely unknown (Skjemstad et al., 1996; Golchin et al.,1997a,b; Gustafsson and Gschwend, 1998).Melanoidin-type macromolecules might also be a

source for some aliphatic and aromatic moieties in therefractory fraction of SOM. Melanoidins are complex,insoluble macromolecules, highly resistant to chemical

degradation, formed by random condensation ofmonomers and other alteration products of amino acidsand carbohydrates (Maillard, 1917). A large part of thehumic substances in soils is considered to be similar to

such macromolecules by some authors and resultspointing to the presence of melanoidin-type complexesin various soils have been reported (e.g. Benzing-Purdie

and Ripmeester, 1983; van Bergen et al., 1997). Mela-noidins can be easily prepared by the condensation ofsugar and amino acid mixtures in hot alkaline (Hedges,

1978; Ioselis et al., 1981) or acid solutions (Olsson et al.,1978; Allard et al., 1997). The solid state 13C NMRspectra of some synthetic melanoidins exhibit relatively

intense aliphatic peaks (Ikan et al., 1986). Various aro-matic units can also occur in melanoidins, especiallywhen derived from proteins containing tyrosine, pheny-lalanine, tryptophan and proline.

In a previous study of a forest soil from LacadeÂ e(south-west France) we observed that a substantial partof humin corresponds to insoluble, non-hydrolyzable,

814 N. Poirier et al. / Organic Geochemistry 31 (2000) 813±827

macromolecular material (Augris et al., 1998). Thisrefractory resistant organic residue (ROR) accountedfor ca. 25 wt.% of total humin. Solid state 13C NMRand Curie point Py/GC/MS analyses pointed to the

highly aliphatic nature of this ROR. However, as stressedabove, (i) there is much uncertainty about the natureand source(s) of the refractory organic fraction in soils and

(ii) the chemical composition inferred for this fraction(aliphaticity versus aromaticity) might be largely in¯u-enced by the analytical methods used. The major aims

of the present study were therefore to examine the pos-sible contributions of black carbon and melanoidins tothe ROR from the LacadeÂ e soil and to account for the

apparent discrepancy observed between some of theanalytical results for this material. To this end, theLacadeÂ e ROR was examined via Fourier transforminfra-red spectroscopy (FTIR), ``o�-line'' pyrolysis and

high resolution transmission electron microscopy(HRTEM). Elemental analysis was performed so as toderive analytical constraints. Parallel studies by FTIR,

solid state 13C NMR and CuPy/GC/MS on mixtures ofmodel compounds (polyethylene and synthetic melanoidin)were performed.

2. Materials and methods

2.1. Samples

ROR was isolated from the upper layer (0±30 cm) of

LacadeÂ e soil as previously described (Augris et al., 1998).In short, the following treatments were successivelyapplied: disaggregation and sieving (<10 mm), lipid

extraction, extraction of humic and fulvic acids, baseand acid hydrolysis of humin, demineralization usingHF/HCl and densimetric separation to remove the bulk

of the minerals not eliminated by these two acids.Synthetic melanoidin was prepared from glucose and

a mixture of amino acids (aspartic acid/glutamic acid/leucine/glycine, 1/1/1/1 in wt.). Glucose and the above

mixture (9/1, wt./wt.) were condensed by hot acid treat-ment at 100�C for 1 h as previously described (Allard et al.,1997). The insoluble residue thus obtained was ®ltered

on a 0.5 mm PTFE ®lter, washed with water until neutraland then with acetone. Spectrometric grade poly-ethylene powder and amino acids were obtained from

Aldrich. Synthetic melanoidin and polyethylene mixtureswere thoroughly ground together to provide a homo-geneous material and homogeneity was con®rmed from

elemental analysis of 10 di�erent aliquots.

2.2. Spectroscopic studies

FTIR spectra were recorded as KBr pellets with aBruker 45 spectrometer. Solid state 13C NMR spectrawere obtained using the cross-polarization technique

(CP) with magic angle spinning (MAS) on a BrukerMSL 400 spectrometer at 100.62 MHz for carbon, withcontact times ranging from 0.1 to 5 ms and a 5 s pulsedelay. Two di�erent spinning rates were used (3 and 4

kHz) so as to discriminate between signal and spinningside bands.

2.3. Pyrolytic studies

``O�-line'' pyrolysis was performed as previously

described (Largeau et al., 1986). The sample (ca. 2�150mg) into a quartz tube plugged with quartz wool washeated for 1 h at 400�C under a 40 ml minÿ1 He ¯ow

and the products released were trapped in CHCl3 atÿ5�C. The solvent was eliminated under vacuum with arotary evaporator before GC/MS analysis. The pyr-olysis products were separated on a Hewlett-Packard

5890 Serie II gas chromatograph, equipped with a 30 mCPSil5CB capillary column (i.d. 0.25 mm, 0.4 mm ®lmthickness). The temperature of the GC oven was pro-

grammed from 50 to 300�C at a rate of 4�C minÿ1. Thegas chromatograph was coupled with a Hewlett-Pack-ard 5989A mass spectrometer operated at 70 eV.

CuPy/GC/MS was performed using a Fischer 0316Curie-point ¯ash pyrolyser. Samples (0.5 to 3 mg) werepyrolysed for 10 s using a ferromagnetic tube (10 mm

length, 2 mm i.d.) with a Curie temperature of 650�Cunder a 5 ml minÿ1 He ¯ow. The pyrolysis unit wasdirectly coupled to the GC/MS system and GC/MSanalysis was carried out under the same conditions as

above.

2.4. High resolution transmission electron microscopy

Dark ®eld observations were carried out with aPhilips EM 400 electron microscope working at 100 kV

and lattice fringe studies with a Philips CM 20 apparatusworking at 200 kV. Samples for HRTEM observationswere prepared as follows: a few milligrams of ROR wereground in ethanol and sonicated. Drops of the suspension

so-obtained were deposited on TEM grids previouslycovered with a holey carbon ®lm (hole size smaller than1 mm). The thin fragments lying across the holes were

observed after drying.

3. Results and discussion

3.1. ROR bulk features

Elemental analysis showed a relatively low H/Catomic ratio of only 0.9 for the non-hydrolyzable frac-tion isolated from the LacadeÂ e soil. Such a value was at

variance with the highly aliphatic macromolecularstructure previously inferred from solid state 13C NMRand CuPy/GC/MS (Augris et al., 1998). In fact, it

N. Poirier et al. / Organic Geochemistry 31 (2000) 813±827 815

appeared that the ROR is chie¯y composed of highlycondensed moieties and probably consists largely ofaromatic units.The lack of abundant long alkyl chain moieties was

con®rmed by the quantitative results from `ò�-line''pyrolysis of ROR. Under such conditions, the pyrolysisproducts swept away by the helium ¯ow are bubbled

into cold CHCl3 and the crude pyrolysate is weighedand analysed by GC/MS after CHCl3 elimination undervacuum. Accordingly, the volatile pyrolysis products are

lost and, for example, when n-alkanes and n-alkenes areconsidered, only the C11+ compounds are analysed bythis method. The gas chromatogram of the `ò�-line''

pyrolysate of the LacadeÂ e ROR is dominated by n-alkane/n-alk-1-ene doublets (Fig. 1), which accountedfor ca. 60% of the GC-amenable components of thepyrolysate, as previously observed for CuPy/GC/MS

experiments. However, this aliphatic pyrolysate accountedfor only 1.4 wt.% of the mass of the pyrolysed ROR. Incontrast, high yields of trapped products are commonly

observed upon `ò�-line'' pyrolysis of highly aliphaticmacromolecular materials like cutans. Thus, a yield of57 wt.% was obtained from the cutan of Agave amer-

icana (Tegelaar et al., 1989b). In agreement with itsstructure, based on a network of long alkyl chains, thiscutan mostly produced n-alkanes and n-alk-1-enes upon

pyrolysis. Moreover, as shown by parallel CuPy/GC/MS experiments, the corresponding doublets exhibit asmooth distribution so that the loss of the C12- com-pounds during `ò�-line'' pyrolysis remained moderate

and did not strongly lower the yield of the recoveredpyrolysate for A. americana cutan. The extremely lowyield obtained from the LacadeÂ e ROR cannot be

accounted for by the loss of the volatile pyrolysis pro-ducts associated with the `ò�-line'' method since, alsoin that case, CuPy/GC/MS indicated a smooth dis-

tribution for the n-alkane/n-alk-1-ene doublets (Augriset al., 1998). `Ò�-line'' pyrolysis therefore shows, inagreement with the qualitative observations derivedfrom previous CuPy/GC/MS experiments, that the ROR

pyrolysate is dominated by n-alkanes and n-alk-1-enes,

but the quantitative features obtained via `ò�-line''pyrolysis indicate that such compounds are only pro-duced in very low amounts from the LacadeÂ e ROR.These observations are therefore consistent with the

elemental data and they indicate that aliphatic moietiesbased on long alkyl chains are not present in substantialamounts in the non-hydrolyzable residue.

The semi-quantitative information derived from theFTIR spectrum of the ROR (Fig. 2) pointed to (i) arelatively weak contribution of alkyl groups (absorp-

tions in the 2800±3000 cmÿ1 range) and (ii) an abundantcontribution of OH and/or NH groups, of C�O groupsand of C�C groups (probably dominated by aromatic

unsaturations as shown by the maximum around 1600cmÿ1 for C�C stretching vibrations).Taken together the above results showed that alipha-

tic moieties with long alkyl chains are only minor com-

ponents in the LacadeÂ e ROR. Substantial contributionsof cutans and/or suberans, of cross-linked lipids and/orcutin and suberin polyesters can thus be ruled out. In

contrast, these observations could be accounted for bythe presence of a mixture containing black carbon andmelanoidin-type macromolecules. Further studies were

therefore carried out in order to (i) test the presence ofboth types of components and (ii) account for the con-spicuous discrepancy between the indications on ROR

structure previously obtained by a combination of solidstate 13C NMR and CuPy/GC/MS and the presentobservations.

3.2. Black carbon occurrence in ROR

The bulk features of the LacadeÂ e ROR (low H/C

atomic ratio, low pyrolysis yield, intense aromatic IRabsorption) could re¯ect the presence of black carbon inthis resistant residue. The occurrence of black carbon in

soils and sediments can be tested via oxidation reactionswhich are aimed at eliminating the other carbonaceouscomponents. To this end, photo-oxidation (Skjemstad etal., 1996; Golchin et al., 1997a), thermal oxidation

(Gustafsson and Gschwend, 1998) and oxidation with

Fig. 1. Gas chromatogram of the `ò�-line'' pyrolysate of the

LacadeÂ e ROR. Fig. 2. FTIR spectrum of the LacadeÂ e ROR.


HNO3 (Glaser et al., 1998) were previously used. However,some highly refractory, non-black carbon, organic mat-ter such as pollen cell walls might also be retained fol-lowing drastic oxidation (Gustafsson and Gschwend,

1998). In the present study the presence of black carbonwas directly examined via high resolution transmissionelectron microscopy (HRTEM).

HRTEM is a powerful tool for the direct observationof the polyaromatic skeleton of natural or syntheticchars, such as coals, blast-furnace cokes and black carbons

(Boulmier et al., 1982; Rouzaud, 1990; Rouzaud andOberlin, 1990). Such materials consist of polyaromaticbasic structural units (BSU), made of stacks of two or

three aromatic layers, nanometric in size. Depending onthe origin of these chars, BSU are connected by di�erenttypes of bridges, including aliphatic carbons, ethergroups and nitrogen-and sulfur-containing links. More-

over, various numbers of hydrogenated and/or oxygen-ated groups can be grafted on BSU boundaries. SuchBSU can be assimilated to stacks of small graphitic layers

and are periodic enough to scatter the incident electronbeam and to produce di�racted beams. These beamsform the di�raction patterns and the images observed in

the focal plane and in the image plane of the objectivelens, respectively. By selecting the di�erent di�ractedbeams, thanks to the appropriate aperture placed in the

focal plane, the BSU and their mutual arrangement inspace, i.e. char microtexture, can be imaged. The di�erentHRTEM modes are obtained through selection of thedi�racted beams. Details on image formation and the

application of these modes to chars can be found else-where (e.g. Boulmier et al., 1982; Oberlin, 1989; Rou-zaud, 1990; Rouzaud and Oberlin, 1990).

In the present work, two modes were used: the 002dark ®eld (002 DF) and the lattice fringe (002 LF)modes. Both modes use the 002 beams, di�racted by the

stacked polyaromatic planes placed on the Bragg angle,i.e. quasi-parallel to the incident electron beam. In the 002DFmode, a single di�racted beam is used for imaging andthe resolving power is about 1 nm with the aperture

used. BSU then appear as bright zones on a dark ®eld.Depending on the 002 beam selected via the aperture,di�erent images can be obtained permitting one to dis-

criminate between various microtextures resulting fromdi�erent BSU orientations (Fig. 3): (i) BSU at random(object O1), as in low rank coals, give images made of

randomly distributed bright dots (image I1), (ii)domains formed by parallel local orientation of BSU(object O2), as in high rank coals and blast-furnace

cokes, appear as aggregates of bright dots (image I2)and (iii) roughly spherical particles with concentricmicrotextures (object O3), termed ``carbon blacks'' inthe literature on carbonization, are detected as circles

exhibiting characteristic bright sectors (image I3). In thesecond mode (002 LF), with a larger aperture, theunscattered transmitted beam can interfere with the 002

beams. Lattice fringes are thus obtained, correspondingto the pro®le of the aromatic layers forming the BSUand the resolution is 0.14 nm with the HRTEM appa-ratus used. The low magni®cation (ca. 25,000�) of the002 DF mode is useful to describe heterogeneous sam-ples and especially to detect minor phases. The 002 LFmode is used for local observations performed with high

magni®cations (up to 500,000�) in order to accessstructural parameters such as the diameter of the poly-aromatic layers which is imaged by fringe length (Rou-

zaud et al., 1999).

Fig. 3. Carbon materials with various microtextures observed

by 002 dark ®eld imaging. Each pair of fringes represents a

BSU seen edge-on (under the Bragg angle). O1, O2 and O3:

sketches of objects respectively composed of BSU at random

(O1), BSU locally orientated in parallel within domains, them-

selves misorientated (O2), concentric microtexture (O3). D:

simpli®ed di�raction pattern (appearing in the focal plane of

the objective lens): a complete 002 ring is obtained in the three

cases, since the BSU are always globally misorientated ; the

aperture selects a small portion of the ring, formed by the dif-

fraction on the 002 planes in a given orientation. I1, I2 and I3:

sketches of the 002 dark-®eld images corresponding to objects

O1, O2 and O3, respectively; each bright dot corresponds to the

orthogonal projection of a BSU, and in a given 002 image, all

the imaged BSU have the same orientation; I1: randomly dis-

tributed dots coresponding to BSU at random; I2: aggregates

of dots coresponding to locally orientated BSU; I3: two bright

sectors of dots coresponding to concentric microtexture, the

opening of the sectors corresponds to the aperture opening.


In the LacadeÂ e ROR, in the 002 DF mode, roughlyspherical carbon particles with a concentric micro-texture were detected and three size classes wereobserved: ca. 250, 100 and 70 nm in diameter, respec-

tively (Fig. 4). A poorly ordered carbon phase was alsoimaged and the quasi-random distribution of the brightdots indicated the absence of locally organized domains

in the corresponding particles. The relatively low con-centrations of the particles observed on the HRTEMgrids made di�cult the assessment of the relative pro-

portions of these two types of black carbon, howeverthe concentric phase seems to be slightly less abundant.The 002 LF mode a�orded a ®ne imaging of the two

carbon phases. The concentric microtexture of the ®rsttype is clearly revealed (Fig. 5A), whereas the secondtype of particle is seen to be composed of strongly mis-orientated and weakly contrasted BSU (Fig. 5B). The

mean fringe length L, i.e. the average aromatic layer

extent, can be speci®ed thanks to the image analysisprocessing recently developed (Rouzaud et al., 1999). Inthe concentric particles, L is about 1.2 nm, whereas thelargest fringes can reach 3.5 nm. In the amorphous-like

carbon phase, due to the low contrast of the fringes, it isdi�cult to measure L precisely which is smaller than 1 nm.TEM thus provided direct evidence of the presence of

two types of polyaromatic units in the ROR isolatedfrom the LacadeÂ e soil and taken together the two typesof black carbon particles appear to account for a sub-

stantial part of the organic resistant residue. As far asthe ®rst type of carbon phase is concerned, similar con-centric nanoparticles are classically obtained by incom-

plete combustion of hydrocarbons. A realistic sketch oftheir ``onion-like'' organization was given by Heidenreichet al. (1968) (Fig. 6). Such a carbon phase was identi®edin coals and anthracites (Oberlin et al., 1980) and it was

also observed in marine sediments from the North Sea(Oberlin, 1977). These concentric particles are usuallyassumed to be formed during ®res. The second type of

black carbon particle is structurally and microtexturallysimilar to a low rank coal. The latter particles couldoriginate from the aromatization of ligno-cellulosic pre-

cursors due to a partial carbonization process. Both

Fig. 4. (A): HRTEM (002 dark ®eld mode) of the resistant

fraction isolated from the LacadeÂ e soil showing the presence of

various particles of black carbon in a hole of the ®lm (cf) that

covers the TEM grid; (B): schematic drawing of the same area.

Particles with a concentric microtexture (black circles on scheme

B) are easily detected on the dark ®eld image (A) by the presence

of two bright sectors; the poorly organized carbon phase (clear,

egg-shaped zones on scheme B), made of strongly misorientated

BSU, appears as randomly distributed bright dots.

Fig. 5. HRTEM image (002 lattice fringe mode) of the refrac-

tory fraction isolated from the LacadeÂ e soil: (A) two closely

associated particles with a concentric microtexture; (B) amor-

phous-like phase (small and weakly contrasted randomly

orientated BSU).


types might be related to ®re events that resulted in charformation along with the release of gaseous or liquidhydrocarbons. The poorly ordered phase would thencorrespond to the char generated after a solid-state

transformation, whereas the concentric nanoparticleswould result from the incomplete combustion of thesehydrocarbons. In the area of interest, the primary forest

was slashed and burnt to be converted into a grazingarea before the Middle Ages. Thereafter, a pine forestwas planted in the late 18th century. Finally, a part of

the LacadeÂ e forest, including plots adjacent to the onewhere the studied soil was collected, was cleared 40years ago for maize cropping and tree stumps were

burnt on the spot. This may account for the presence ofthese black carbon particles in the LacadeÂ e sample.In both cases, the polyaromatic structures in these

carbon particles, especially in the concentric ones, are

relatively large (about 20 cycles) and connected. Conse-quently, a large number of the carbon atoms in suchstructures could not be detected by solid state 13C

NMR. Indeed, previous studies on model compoundsshowed that carbons in a core position in condensedpolyaromatic structures are not detected (Alemany et

al., 1983). Thus, spin counting techniques using aninternal reference, or comparison with elemental com-position, revealed that at least 40% of the carbons are

not observed in some coals (Dubois Murphy et al.,1982; Hagaman et al., 1986; Botto et al., 1987) andarti®cially matured kerogens (Derenne et al., 1987).Most carbon atoms in polyaromatic materials can be

detected by BlochDecaymeasurements as recently shownfor coals (Maroto-Valer et al., 1996) and carbonized coals(Maroto-Valer et al., 1998). However, such measure-

ments are rarely performed since they are time-consuming.When CuPy/GC/MS experiments are considered, it iswell documented that black carbon is hardly detected

since low weight losses are obtained upon pyrolysis andthe generated products mostly consist of low molecularweight volatiles, including methane and carbon dioxide.

3.3. Melanoidin occurrence in ROR

As shown by HRTEM, a substantial fraction of theLacadeÂ e ROR is comprised of black carbon. However,

the presence of this polyaromatic material alone cannotaccount for the FTIR features of the resistant residue,especially for the abundant contribution of oxygen-

and/or nitrogen-containing groups, whereas the lattergroups may re¯ect the occurrence of melanoidin-typecomponents.

In fact, comparison with previously reported spectraof synthetic melanoidins (Rubinsztain et al., 1984, 1986;Almendros et al., 1989; Allard et al., 1997) indicates that

the FTIR features of the ROR are consistent with thepresence of melanoidin-like moieties. Nevertheless, nei-ther the solid state 13C NMR spectrum nor the CuPy/GC/MS trace clearly indicated the presence of this type

of material. In contrast, conspicuous aliphatic featureswere observed through these two methods although, asdiscussed above, moieties based on alkyl chains only

Fig. 6. Sketch of the ``onion-like'' concentric microtexture of a

``carbon black'' (after Heidenreich et al., 1968).

Fig. 7. CP/MAS 13C NMR spectra recorded at tc=1 ms of the

synthetic melanoidin (a, b) and of mixtures with polyethylene

in various proportions (by weight): 95/5 (c), 80/20 (d) and 50/50

(e). l indicates spinning side bands. Spinning rate was 4 kHz,

except for (a) (3 kHz).


a�ord a minor contribution to the ROR. Accordingly,

mixtures of model compounds (synthetic melanoidinand polyethylene) were prepared and examined via solidstate 13C NMR, FTIR and CuPy/GC/MS so as toaccount for such a discrepancy.

3.3.1. Solid state 13C NMRThe insoluble synthetic melanoidin, obtained in a

yield of 9% from the glucose/amino acid mixture, ischaracterized by a carbon content of 61.3% and anatomic H/C ratio of 1.0. Its solid state 13C NMR spec-

trum, obtained with the classical contact time of 1 ms(Fig. 7a,b) exhibits several intense broad peaks. Thealiphatic signal (0±50 ppm) maximizes at ca. 30 ppm(CH2) with a shoulder at 36 ppm and a substantial peak

at 13 ppm (CH3). The presence of such an aliphatic sig-nal was previously reported in melanoidin spectra(Benzing-Purdie and Ripmeester, 1983; Rubinsztain et

al., 1984; Ikan et al., 1986; Allard et al., 1997). Anintense peak is observed at 150 ppm which can beassigned to aromatic carbons bearing heteroatoms. A

peak can also be observed between 100 and 120 ppm onthe spectrum recorded at 4 kHz (Fig. 7b); it correspondspartly to spinning side bands of the 150 ppm peak but it

also partly comprises a signal as revealed by the spec-trum recorded at 3 kHz (Fig. 7a). This signal, whichmaximizes at 113 ppm is due to carbons in doublebonds; based on the occurrence of the peak at 150 ppm,

these carbons were considered to be located in aromaticstructures, ortho to oxygen or nitrogen (Benzing-Purdieand Ripmeester, 1983; Du� et al., 1988). A peak is also

observed at 89 ppm; such a resonance often occurs in

melanoidins derived from excess of sugars (Ikan et al.,1986) and is assigned to alkyl carbons bearing oxygen ornitrogen. The signal at 173 ppm is due to carboxyl in esterand/or amide groups (Benzing-Purdie and Ripmeester,

1983; Rubinsztain et al., 1984; Ikan et al., 1986; Allardet al., 1997). Peaks at 70 and 190 ppm on Fig. 7b aredue to spinning side bands of the 150 ppm peak.

Three mixtures of melanoidin and polyethylene, con-taining 95, 80 and 50 wt.% of melanoidin, respectively,were prepared and examined via CP/MAS 13CNMR, also

using the classical contact time of 1 ms (Fig. 7c±e). Basedon the elemental analysis of the synthetic melanoidin(61.3% C) and on the carbon content in the poly-ethylene (85.6%), the melanoidin carbon/polyethylene

carbon ratios in the three mixtures are of 13.5, 2.86 and0.71, respectively. In fact, the 13C NMR spectra of allthese melanoidin/polyethylene mixtures are sharply

dominated by a peak at 32 ppm corresponding to CH2

from polyethylene. Moreover, in the 50/50 mixture, themelanoidin signals can hardly be detected. It thus

appears that melanoidin carbons are strongly under-estimated with respect to the polyethylene ones via solidstate 13C NMR.

Such a large underestimation can be a priori relatedeither to intrinsic features of the melanoidin [some car-bons from the melanoidin part could not be observedsince they are too far from protons and they cannot be

reached by spin di�usion as observed, for example, forcondensed polyaromatic materials such as anthracitecoals (Dubois Murphy et al., 1982)] and/or to unsuitable

Fig. 8. CP/MAS 13C NMR spectra of 95/5, wt./wt., melanoidin/polyethylene mixture recorded at various contact times ranging from

0.1 to 5 ms.


conditions for spectrum acquisition, especially anunsuitable contact time, so that the relative numbers ofthe di�erent types of resonant carbons would not beproperly re¯ected by relative signal intensities in the

considered spectrum (Dudley and Fyfe, 1982).The second hypothesis was ruled out by recording

spectra of the 95/5 melanoidin/polyethylene mixture at

contact times ranging from 0.1 to 5 ms (Fig. 8). In thesespectra, two signals were selected to represent the twocomponents of the mixtures: the 150 ppm peak (corre-

sponding to the heteroatom-linked aromatic carbons ofthe melanoidin) and the 32 ppm peak (encompassingboth the signal of all the polyethylene carbons and the

signal of the aliphatic carbons from the melanoidin). Byplotting the intensity of these two signals versus contacttime, we could determine the ratio of the number ofresonant carbons Mo(150 ppm)/Mo(32 ppm)=1.02 for

the 95/5 mixture. Comparison of this ratio with theM(150 ppm)/M(32 ppm) value directly calculated frompeak intensity for each spectrum recorded at a given

contact time (Table 1) revealed that reliable quantitativeinformation on the relative abundance of the di�erenttypes of resonant carbons can be directly deduced from

the spectrum at 2 ms. This contact time was thereforeused to test the ®rst hypothesis, i.e. that melanoidinunderestimation re¯ects the presence of non-resonant

carbons.As alreadymentioned, the 32 ppm peak in the spectra of

the three mixtures, although mostly due to polyethylene,also contains some contribution frommelanoidin carbons.

So as to estimate the fraction of resonant carbons due tomelanoidin in the 32 ppm signal, the pure melanoidinspectrum was subtracted from each mixture spectrum

after having adjusted the intensity so as to ®t the 150 and113 ppm signals, as illustrated in Fig. 9a for the 95/5 mix-ture. The di�erence spectrum thus obtained corresponds

to that of polyethylene (Fig. 9b). The intensity ratio ofthe melanoidin and polyethylene spectra thus calculatedcorresponds to the melanoidin carbons/polyethylene

Table 1

Relative intensity of the 150 and 32 ppm peaks in the solid state13C NMR spectra of the 95/5 melanoidin/polyethylene mix-

tures recorded at di�erent contact times

Contact time (ms) M(150)/M(32)

0.1 2.39

0.25 1.94

0.5 1.41

0.75 1.34

1 1.26

1.5 1.13

2 1.05

3 1.06

5 1.11

Fig. 9. (a) CP/MAS 13C NMR spectrum of 95/5, wt./wt., mel-

anoidin/polyethylene mixture (top) and ®tted spectrum of syn-

thetic melanoidin (bottom); (b) di�erence spectrum between

spectra displayed in (a) showing the polyethylene part in the

95/5 mixture.

Table 2

E�ciency of the detection of the melanoidin carbons by solid state 13C NMR under optimum conditions (contact time of 2 ms) in the

three melanoidin/polyethylene mixturesa

Melanoidin carbon/polyethylene carbon ratios

Melanoidin/polyethylene

mixtures (wt./wt.)

Real

valuesbNMR-derived

values

Melanoidin carbons

undetected by NMR (%)c

95/5 13.5 9.0 33

80/20 2.86 1.44 49

50/50 0.71 0.30 58

a Still lower e�ciencies were obtained with other contact times (e.g. the carbons undetected by NMR correspond to ca. 65% of the

melanoidin carbons, for the 95/5 mixture, with contact times of 1 and 3 ms).b Calculated from the composition (wt./wt.) of the mixture and from carbon content in the synthetic melanoidin and polyethylene

(61.3 and 85.6%, respectively).c As % of total carbons of the melanoidin.


carbons ratio in the mixture, as seen by CP/MAS NMRunder suitable acquisition conditions. Comparison ofthese ratios with the real ones (Table 2) reveals largedi�erences for the three mixtures and the value obtained

from the NMR spectra is always markedly lower. Thisobservation clearly shows that some carbons of themelanoidin cannot be ``seen'' by CP 13C NMR due to

poor magnetization (the cross-polarization time TCH

varies as the sixth power of the carbon±proton distanceand these carbons are too far from protons to be

reached by spin di�usion). From the di�erence betweenthe theoretical and NMR-derived ratios, the percentageof these undetected carbons can be calculated for the

di�erent mixtures (Table 2). Large values are thusobtained for all mixtures; furthermore, the percentage ofundetected carbons tends to increase when the contentof melanoidin decreases. This trend may re¯ect a

decrease in the e�ciency of magnetization transfer tomelanoidin carbons in the presence of increasingamount of polyethylene. The large content of unde-

tected carbons in the synthetic melanoidin probablyre¯ects a highly cross-linked macromolecular structurefor this material and the abundance of oxygen- and/or

nitrogen-containing groups so that a number of mela-noidin carbons are relatively far from protons. In con-trast, the carbons from the aliphatic material are

quantitatively detected. Previous studies, based on spincounting techniques using an internal reference or com-parison with elemental composition, showed that a highpercentage of carbons is not detected, via solid state 13C

NMR, in humic acids (Wilson et al., 1987) and soils(Oades et al., 1987; Preston et al., 1994; Kinshesh et al.,1995a,b). In humic and fulvic acids, the aliphatic con-

tribution was shown from spin countings and analyticalconstraint calculations to be overestimated (Wilson etal., 1987). A similar situation is observed in the present

study from the melanoidin/polyethylene mixtures.

3.3.2. FTIRThe FTIR spectrum of the melanoidin (Fig. 10a) is

similar to previously reported spectra for other syntheticmelanoidins (Rubinsztain et al., 1984, 1986; Almendroset al., 1989; Allard et al., 1997). The main bands are due

to oxygen-containing functions such as those at 3440and 1020 cmÿ1 (OH groups) and at 1706 cmÿ1 (COOHgroups). The relatively high intensity of these bands is

consistent with the fact that the melanoidin was pre-pared with an excess of sugars. Relatively intense bandsare also observed at 1623 and 1510 cmÿ1; they are

usually assigned to aromatic furanic or conjugatedcompounds. A low contribution of CH2 and CH3

groups is re¯ected by the weak absorption in the 2850±2950 cmÿ1 range.The FTIR spectrum of polyethylene (Fig. 10f) shows

the typical bands of polymethylenic chains at 2920 and2850, 1470 and 720±730 cmÿ1.

The spectra of the mixtures (Fig. 10b±e) show a com-bination of the above bands. It can be seen that themelanoidin bands are still clearly visible in the 50/50mixture. When compared to solid state 13C NMR,

FTIR thus appears more suitable for revealing the pres-ence of melanoidin-like structures, but this method onlya�ords semi-quantitative information.

Fig. 10. FTIR spectra of synthetic melanoidin (a), and of mix-

tures of melanoidin with polyethylene in various proportions

(by weight): 98/2 (b), 95/5 (c), 80/20 (d) and 50/50 (e) and of

pure polyethylene (f).


3.3.3. Curie point pyrolysis/gas chromatography/massspectrometry

The pyrolysate of the synthetic melanoidin (Fig. 11a)chie¯y comprises phenolic and furanic compounds andaromatic hydrocarbons and it is dominated by levulinicacid (4-oxo pentanoic acid). Phenolic and furanic products

in such pyrolysates can be derived from sugar moieties(van der Kaaden et al., 1984; Pouwels et al., 1989; Pas-torova et al., 1994) and levulinic acid results from the

dehydration of hexoses (Leonard, 1956). The virtuallack of nitrogen-containing compounds is in agreementwith the very low nitrogen content observed by ele-

mental analysis (0.67 mg of N/g of carbon) and the highsugar to amino acid ratio (9/1) used for the preparationof the synthetic melanoidin. Similar products were

detected in pyrolysates of other synthetic melanoidins(Boon et al., 1984; Allard et al., 1997) and of humicsubstances (Wilson et al., 1983; Gillam and Wilson,1985).

Mixtures of melanoidin and polyethylene containing98, 95, 80 and 50 wt.% of melanoidin were examined byCuPy/GC/MS (Fig. 11). The pyrochromatogram of the

98/2 mixture (Fig. 11b) looks very similar to that of thepure melanoidin (Fig. 11a). Polyethylene pyrolysis is

known to yield a regular series of n-alkane/n-alk-1-enedoublets resulting from the homolytic cleavage of thepolymethylenic chains and extending to high carbonnumbers (Wampler and Levy, 1986). Such doublets

could not be detected in the pyrolysate of the 98/2 mix-ture, even when using selective ion detection at m/z 57.They are hardly visible in the pyrochromatogram of the

95/5 mixture but they become dominant in the 80/20mixture and are overwhelmingly abundant in the 50/50one (Fig. 11c±e). In the latter case, the melanoidin con-

tribution is only revealed by very small peaks at thebeginning of the trace and a hump due to levulinic acidunder the C11 alkane/alkene doublet. When only the gas

chromatogram of the pyrolysis products is considered,the aliphatic moieties are therefore strongly over-estimated when compared to the melanoidin units. Suchan overestimation can easily be explained by the

respective yields of pyrolysis products from melanoidinsand from polymethylenic chains. Melanoidins are char-acterized by low weight losses upon pyrolysis since they

Fig. 11. 610�C Curie point pyrochromatograms of the synthetic melanoidin (a) and of mixtures with polyethylene in various pro-

portions (by weight): 98/2 (b), 95/5 (c), 80/20 (d) and 50/50 (e); f, furanic compounds; p, phenolic compounds; a, levulinic acid; b,

aromatic hydrocarbons.


a�ord large amounts of polyaromatic residue and thereleased products mostly correspond to highly volatilecompounds (Allard et al., 1998) whereas long (CH2)nchains in polyethylene are quantitatively cleaved into

n-alkane/n-alkene doublets that extendover awide carbonnumber range, hence the release of large amounts ofGC-amenable products.

Taken together, these observations account for thepoor detection of the melanoidin fraction in the ROR ofthe LacadeÂ e soil when examined via solid state 13C

NMR and CuPy/GC/MS (Augris et al., 1998).

4. Conclusions

Contrary to previous observations based on solidstate 13C NMR spectroscopy and Curie point Py/GC/

MS, which pointed to a highly aliphatic nature forLacadeÂ e ROR, it appears that aliphatic moieties with longalkyl chains are only minor components. Accordingly, an

important contribution of cutans/suberans, cross-linkedlipids and cutin/suberin polyesters can be ruled out inthis resistant material. In fact, the LacadeÂ e ROR is mostly

composed of melanoidins (complex macromolecularstructures derived from the random condensation ofalteration products of proteins and carbohydrates) and

of black carbon (residues of incomplete combustion).High resolution transmission electron microscopy

observations (dark ®eld mode and lattice fringe mode)provided direct evidence of a substantial contribution of

black carbon corresponding to two types of particles.The slightly more abundant type corresponds to apoorly ordered carbon phase. The second type is com-

posed of roughly spherical particles, ranging, from 70 to250 nm in diameter, with a concentric ``onion-like''microtexture. The formation of both types might be

related to ®re events which resulted in (i) the productionof char via aromatization of ligno-cellulosic precursors(amorphous-like carbon phase) and (ii) the release andthe subsequent incomplete combustion of hydrocarbons

(concentric nanoparticles). The individual polyaromaticunits in these black carbon particles are relatively largeand connected. Accordingly, a large part of the corre-

sponding carbon atoms could not be detected via solidstate 13C NMR. Similarly, the presence of the blackcarbon fraction was not revealed via Curie point pyr-

olysis due to low yields of pyrolysis products.Examination of synthetic melanoidin/polyethylene

mixtures also showed a poor detection of the former

components via solid state 13C NMR, even when usingan optimum contact time. This strong underestimationof the melanoidin carbons is due to poor magnetization,probably related to the highly cross-linked macro-

molecular structure of the melanoidins. Contrary to ali-phatic moieties based on long alkyl chains, the pyrolysisproducts of melanoidins are only generated in low yields,

hence such components are also poorly detected throughpyrolysis experiments. In contrast, FTIR appears moree�cient for the detection of melanoidin-type structures.Observations via solid state 13C NMR spectroscopy

and pyrolysis experiments can therefore lead to a pro-nounced overestimation of highly aliphatic moieties inheterogeneous materials such as the LacadeÂ e ROR, when

applied alone and with no quantitative constraints derivedfrom elemental analysis and/or quantitative pyrolyses.

Acknowledgements

This study was partly supported by the AGRIGESprogram (Institut National de la Recherche Agronomiqueand MinisteÁ re de l'Environnement). We also thank Dr.D. Arrouays (UniteÂ de Science du Sol, INRA, OrleÂ ans)

for providing the soil sample from LacadeÂ e and J. Guil-lemot and Y. Pouet (Py/GC/MS) for technical support.

Associate EditorÐS.J. Rowland

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