Chemical structure and sources of the macromolecular,
resistant, organic fraction isolated from a forest soil
(LacadeÂe, south-west France)
Natacha Poirier
a,b, Sylvie Derenne
b, Jean-NoeÈl Rouzaud
c, Claude Largeau
b,*,
Andre Mariotti
a, JeÂroÃme Balesdent
d, Jocelyne Maquet
eaLaboratoire de BiogeÂochimie Isotopique, INRA-CNRS-UPMC, 4 pl. Jussieu, 75252 Paris cedex 05, France
bLaboratoire de Chimie Bioorganique et Organique Physique, UMR CNRS 7573, ENSCP, 11 rue P et M Curie, 75231 Paris cedex 05, France cCentre de Recherche sur la MatieÁre DiviseÂe, UMR CNRS 6619, Universite d'OrleÂans, 1bis rue de la FeÂrollerie, 45071 OrleÂans Cedex 2, France dLaboratoire 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 LacadeÂe (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% of total humin, was shown to be chie¯y composed of melanoidins and black carbon. Two types of black carbon particles were identi®ed by dark ®eld and lattice fringe electron microscopy. Contrary to previous observations, based on solid state13C NMR spectroscopy and Curie point Py/GC/MS, highly aliphatic moieties only aord a minor contribution to the refractory material of the LacadeÂe soil. Additional studies, using mixtures of model compounds, were carried out to examine 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 state13C NMR; FTIR;
Pyr-olysis; HRTEM
1. Introduction
Three pools, characterized by dierent turnover rates, are generally distinguished in soil organic matter (SOM). These include a stable pool with mean residence times up to millenia [Balesdent and Mariotti (1996) and references therein]. Information on the nature and fate upon changes in land use of the latter pool is important since variations in its abundance would generate large
CO2¯uxes between the atmosphere and soils. However, the mechanism that accounts for the stability of this refractory 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 to degradation of some SOM constituents, directly related to their chemical structure, might also be an important factor. Nevertheless, as stressed below, the chemical composition of the refractory fraction of SOM is still a matter of debate.
Numerous studies point to the occurrence of recalci-trant aliphatic structures in SOM. Indeed, observations by solid state13C NMR on peats, composts and soils (reviewed by Preston, 1996; Baldock et al., 1997) show
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* Corresponding author. Tel.: 1-4427-6761; fax: +33-1-4325-7975.
that the relative contribution of alkyl carbon tends to increase with increasing degrees of decomposition of the organic 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 and Roberston, 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 de Leeuw, 1987a,b; Tegelaar et al., 1989a; Almendros et al., 1991; KoÈgel-Knabner et al., 1992a,b), (iii) humin (Almendros et al., 1996; Grasset and AmbleÁs, 1998; Lichtfouse et al., 1998) and (iv) the non-hydrolyzable fraction of humin isolated via successive, drastic, base and acid hydrolyses of a forest soil from LacadeÂe (Augris et al., 1998). Large dierences in the relative intensity of then-alkane/n-alk-1-ene doublets were noted, in the above studies, when the gas chromatograms of total pyrolysates were compared, and an especially high con-tribution was observed for the resistant (non-hydrolyzable) fraction isolated from the humin of the LacadeÂe 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) and dierent types of sources have been considered. These sources included highly aliphatic, resistant, macro-molecular components from higher plants, the so-called cutans 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) and mixtures 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 polyesters were also considered (KoÈgel-Knabner et al., 1992a,b).
In a number of the above-mentioned studies, an abundant contribution of aliphatic moieties to the refractory fraction of SOM was inferred from solid state 13C NMR observations. It is well documented, however,
that such spectroscopic methods can markedly over-estimate the aliphatic contribution in SOM fractions and underestimate the aromaticity (e.g. Hatcher et al., 1981; Wilson et al., 1987). Indeed, in contrast with the above ®ndings, it is often considered that aromatic carbon tends to accumulate as SOM decomposition proceeds (Baldock et al., 1997). A part of this refractory aromatic carbon 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 of incomplete combustion produced from vegetation ®res and burning of fossil fuels. Black carbon is widely dis-tributed over the entire surface of the earth (reviewed in Goldberg, 1985) and it was shown to account for a substantial fraction of total organic carbon in some soils
(Skjemstad et al., 1996; Golchin et al., 1997a; Glaser et al., 1998). Such features re¯ect the origin of black car-bon in widespread burning processes and its refractory nature. The high stability of charcoal, and charred materials from plants, is illustrated by (i) their great ability to survive severe oxidation treatments when compared 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 8 million year old Pliocene samples (Dubar et al., 1995). In fact, it is considered that only limited degradation of charcoal would take place with time through microbial or chemical degradation (Seiler and Crutzen, 1980). Accordingly, charcoal formation is a possible source for the chemically most stable, aromatic carbon pool in soils (Haumaier and Zech, 1995; Skjemstad et al., 1996; Golchin et al., 1997b). Charcoal formation during vegetation burning may thus partly convert a potentially active carbon pool into a more inert one and represent a way in which long-term protection of OM in soils may occur. Moreover, recent studies are consistent with an important role for black carbon as a sink in the global carbon cycle via burial in marine sediments (Kuhlbusch and Crutzen, 1995; Lim and Cachier, 1996; Gustafsson and Gschwend, 1998). Nevertheless, the biological sta-bility of charcoal in soils and sediments, as well as its contribution 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 the refractory fraction of SOM. Melanoidins are complex, insoluble macromolecules, highly resistant to chemical degradation, formed by random condensation of monomers and other alteration products of amino acids and carbohydrates (Maillard, 1917). A large part of the humic substances in soils is considered to be similar to such macromolecules by some authors and results pointing to the presence of melanoidin-type complexes in 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 of sugar 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 NMR spectra of some synthetic melanoidins exhibit relatively intense aliphatic peaks (Ikan et al., 1986). Various aro-matic units can also occur in melanoidins, especially when derived from proteins containing tyrosine, pheny-lalanine, tryptophan and proline.
macromolecular material (Augris et al., 1998). This refractory resistant organic residue (ROR) accounted for ca. 25 wt.% of total humin. Solid state 13C NMR and Curie point Py/GC/MS analyses pointed to the highly aliphatic nature of this ROR. However, as stressed above, (i) there is much uncertainty about the nature and 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 to the ROR from the LacadeÂe soil and to account for the apparent discrepancy observed between some of the analytical results for this material. To this end, the LacadeÂe ROR was examined via Fourier transform infra-red spectroscopy (FTIR), ``o-line'' pyrolysis and high resolution transmission electron microscopy (HRTEM). Elemental analysis was performed so as to derive analytical constraints. Parallel studies by FTIR, solid state13C NMR and CuPy/GC/MS on mixtures of model 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 LacadeÂe soil as previously described (Augris et al., 1998). In short, the following treatments were successively applied: disaggregation and sieving (<10 mm), lipid
extraction, extraction of humic and fulvic acids, base and acid hydrolysis of humin, demineralization using HF/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 100C for 1 h as previously described (Allard et al.,
1997). The insoluble residue thus obtained was ®ltered on a 0.5mm PTFE ®lter, washed with water until neutral
and then with acetone. Spectrometric grade poly-ethylene powder and amino acids were obtained from Aldrich. Synthetic melanoidin and polyethylene mixtures were thoroughly ground together to provide a homo-geneous material and homogeneity was con®rmed from elemental analysis of 10 dierent aliquots.
2.2. Spectroscopic studies
FTIR spectra were recorded as KBr pellets with a Bruker 45 spectrometer. Solid state 13C NMR spectra were obtained using the cross-polarization technique
(CP) with magic angle spinning (MAS) on a Bruker MSL 400 spectrometer at 100.62 MHz for carbon, with contact times ranging from 0.1 to 5 ms and a 5 s pulse delay. Two dierent spinning rates were used (3 and 4 kHz) so as to discriminate between signal and spinning side bands.
2.3. Pyrolytic studies
``O-line'' pyrolysis was performed as previously described (Largeau et al., 1986). The sample (ca. 2150 mg) into a quartz tube plugged with quartz wool was heated for 1 h at 400C under a 40 ml minÿ1He ¯ow
and the products released were trapped in CHCl3 at ÿ5C. The solvent was eliminated under vacuum with a
rotary evaporator before GC/MS analysis. The pyr-olysis products were separated on a Hewlett-Packard 5890 Serie II gas chromatograph, equipped with a 30 m CPSil5CB capillary column (i.d. 0.25 mm, 0.4mm ®lm
thickness). The temperature of the GC oven was pro-grammed from 50 to 300C at a rate of 4C minÿ1. The gas chromatograph was coupled with a Hewlett-Pack-ard 5989A mass spectrometer operated at 70 eV.
CuPy/GC/MS was performed using a Fischer 0316 Curie-point ¯ash pyrolyser. Samples (0.5 to 3 mg) were pyrolysed for 10 s using a ferromagnetic tube (10 mm length, 2 mm i.d.) with a Curie temperature of 650C
under a 5 ml minÿ1 He ¯ow. The pyrolysis unit was directly coupled to the GC/MS system and GC/MS analysis was carried out under the same conditions as above.
2.4. High resolution transmission electron microscopy
Dark ®eld observations were carried out with a Philips EM 400 electron microscope working at 100 kV and lattice fringe studies with a Philips CM 20 apparatus working at 200 kV. Samples for HRTEM observations were prepared as follows: a few milligrams of ROR were ground in ethanol and sonicated. Drops of the suspension so-obtained were deposited on TEM grids previously covered with a holey carbon ®lm (hole size smaller than 1mm). The thin fragments lying across the holes were
observed after drying.
3. Results and discussion
3.1. ROR bulk features
appeared that the ROR is chie¯y composed of highly condensed moieties and probably consists largely of aromatic units.
The lack of abundant long alkyl chain moieties was con®rmed by the quantitative results from ``o-line'' pyrolysis of ROR. Under such conditions, the pyrolysis products swept away by the helium ¯ow are bubbled into cold CHCl3 and the crude pyrolysate is weighed and analysed by GC/MS after CHCl3elimination under vacuum. Accordingly, the volatile pyrolysis products are lost and, for example, whenn-alkanes andn-alkenes are considered, only the C11+ compounds are analysed by this method. The gas chromatogram of the ``o-line'' pyrolysate of the LacadeÂe ROR is dominated by n -alkane/n-alk-1-ene doublets (Fig. 1), which accounted for ca. 60% of the GC-amenable components of the pyrolysate, as previously observed for CuPy/GC/MS experiments. However, this aliphatic pyrolysate accounted for only 1.4 wt.% of the mass of the pyrolysed ROR. In contrast, high yields of trapped products are commonly observed upon ``o-line'' pyrolysis of highly aliphatic macromolecular materials like cutans. Thus, a yield of 57 wt.% was obtained from the cutan of Agave amer-icana (Tegelaar et al., 1989b). In agreement with its structure, based on a network of long alkyl chains, this cutan mostly producedn-alkanes andn-alk-1-enes upon pyrolysis. Moreover, as shown by parallel CuPy/GC/ MS experiments, the corresponding doublets exhibit a smooth distribution so that the loss of the C12- com-pounds during ``o-line'' pyrolysis remained moderate and did not strongly lower the yield of the recovered pyrolysate for A. americana cutan. The extremely low yield obtained from the LacadeÂe ROR cannot be accounted for by the loss of the volatile pyrolysis pro-ducts associated with the ``o-line'' method since, also in that case, CuPy/GC/MS indicated a smooth dis-tribution for the n-alkane/n-alk-1-ene doublets (Augris et al., 1998). ``O-line'' pyrolysis therefore shows, in agreement with the qualitative observations derived from previous CuPy/GC/MS experiments, that the ROR pyrolysate is dominated byn-alkanes andn-alk-1-enes,
but the quantitative features obtained via ``o-line'' pyrolysis indicate that such compounds are only pro-duced in very low amounts from the LacadeÂe ROR. These observations are therefore consistent with the elemental data and they indicate that aliphatic moieties based on long alkyl chains are not present in substantial amounts in the non-hydrolyzable residue.
The semi-quantitative information derived from the FTIR spectrum of the ROR (Fig. 2) pointed to (i) a relatively weak contribution of alkyl groups (absorp-tions in the 2800±3000 cmÿ1range) and (ii) an abundant contribution of OH and/or NH groups, of CO groups and of CC groups (probably dominated by aromatic unsaturations as shown by the maximum around 1600 cmÿ1for CC stretching vibrations).
Taken together the above results showed that alipha-tic moieties with long alkyl chains are only minor com-ponents in the LacadeÂe ROR. Substantial contributions of cutans and/or suberans, of cross-linked lipids and/or cutin and suberin polyesters can thus be ruled out. In contrast, these observations could be accounted for by the presence of a mixture containing black carbon and melanoidin-type macromolecules. Further studies were therefore carried out in order to (i) test the presence of both types of components and (ii) account for the con-spicuous discrepancy between the indications on ROR structure previously obtained by a combination of solid state 13C NMR and CuPy/GC/MS and the present observations.
3.2. Black carbon occurrence in ROR
The bulk features of the LacadeÂe ROR (low H/C atomic ratio, low pyrolysis yield, intense aromatic IR absorption) could re¯ect the presence of black carbon in this resistant residue. The occurrence of black carbon in soils and sediments can be tested via oxidation reactions which are aimed at eliminating the other carbonaceous components. To this end, photo-oxidation (Skjemstad et al., 1996; Golchin et al., 1997a), thermal oxidation (Gustafsson and Gschwend, 1998) and oxidation with
Fig. 1. Gas chromatogram of the ``o-line'' pyrolysate of the
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 carbon was directly examined via high resolution transmission electron microscopy (HRTEM).
HRTEM is a powerful tool for the direct observation of the polyaromatic skeleton of natural or synthetic chars, such as coals, blast-furnace cokes and black carbons (Boulmier et al., 1982; Rouzaud, 1990; Rouzaud and Oberlin, 1990). Such materials consist of polyaromatic basic structural units (BSU), made of stacks of two or three aromatic layers, nanometric in size. Depending on the origin of these chars, BSU are connected by dierent types of bridges, including aliphatic carbons, ether groups and nitrogen-and sulfur-containing links. More-over, various numbers of hydrogenated and/or oxygen-ated groups can be grafted on BSU boundaries. Such BSU can be assimilated to stacks of small graphitic layers and are periodic enough to scatter the incident electron beam and to produce diracted beams. These beams form the diraction patterns and the images observed in the focal plane and in the image plane of the objective lens, respectively. By selecting the dierent diracted beams, thanks to the appropriate aperture placed in the focal plane, the BSU and their mutual arrangement in space, i.e. char microtexture, can be imaged. The dierent HRTEM modes are obtained through selection of the diracted 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 002 dark ®eld (002 DF) and the lattice fringe (002 LF) modes. Both modes use the 002 beams, diracted by the stacked polyaromatic planes placed on the Bragg angle, i.e. quasi-parallel to the incident electron beam. In the 002 DF mode, a single diracted beam is used for imaging and the 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, dierent images can be obtained permitting one to dis-criminate between various microtextures resulting from dierent 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 concentric microtextures (object O3), termed ``carbon blacks'' in the literature on carbonization, are detected as circles exhibiting characteristic bright sectors (image I3). In the second mode (002 LF), with a larger aperture, the unscattered transmitted beam can interfere with the 002
beams. Lattice fringes are thus obtained, corresponding to the pro®le of the aromatic layers forming the BSU and the resolution is 0.14 nm with the HRTEM appa-ratus used. The low magni®cation (ca. 25,000) of the 002 DF mode is useful to describe heterogeneous sam-ples and especially to detect minor phases. The 002 LF mode is used for local observations performed with high magni®cations (up to 500,000) in order to access structural parameters such as the diameter of the poly-aromatic layers which is imaged by fringe length (Rou-zaud et al., 1999).
In the LacadeÂe ROR, in the 002 DF mode, roughly spherical carbon particles with a concentric micro-texture were detected and three size classes were observed: ca. 250, 100 and 70 nm in diameter, respec-tively (Fig. 4). A poorly ordered carbon phase was also imaged and the quasi-random distribution of the bright dots indicated the absence of locally organized domains in the corresponding particles. The relatively low con-centrations of the particles observed on the HRTEM grids made dicult the assessment of the relative pro-portions of these two types of black carbon, however the concentric phase seems to be slightly less abundant.
The 002 LF mode aorded a ®ne imaging of the two carbon phases. The concentric microtexture of the ®rst type is clearly revealed (Fig. 5A), whereas the second type 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 analysis processing recently developed (Rouzaud et al., 1999). In the concentric particles,Lis about 1.2 nm, whereas the largest fringes can reach 3.5 nm. In the amorphous-like carbon phase, due to the low contrast of the fringes, it is dicult to measureLprecisely which is smaller than 1 nm. TEM thus provided direct evidence of the presence of two types of polyaromatic units in the ROR isolated from the LacadeÂe soil and taken together the two types of black carbon particles appear to account for a sub-stantial part of the organic resistant residue. As far as the ®rst type of carbon phase is concerned, similar con-centric nanoparticles are classically obtained by incom-plete combustion of hydrocarbons. A realistic sketch of their ``onion-like'' organization was given by Heidenreich et al. (1968) (Fig. 6). Such a carbon phase was identi®ed in 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 usually assumed to be formed during ®res. The second type of black carbon particle is structurally and microtexturally similar to a low rank coal. The latter particles could originate 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 LacadeÂe 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.
types might be related to ®re events that resulted in char formation along with the release of gaseous or liquid hydrocarbons. The poorly ordered phase would then correspond to the char generated after a solid-state transformation, whereas the concentric nanoparticles would result from the incomplete combustion of these hydrocarbons. In the area of interest, the primary forest was slashed and burnt to be converted into a grazing area before the Middle Ages. Thereafter, a pine forest was planted in the late 18th century. Finally, a part of the LacadeÂe forest, including plots adjacent to the one where the studied soil was collected, was cleared 40 years ago for maize cropping and tree stumps were burnt on the spot. This may account for the presence of these black carbon particles in the LacadeÂe 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 such structures could not be detected by solid state 13C NMR. Indeed, previous studies on model compounds showed that carbons in a core position in condensed polyaromatic structures are not detected (Alemany et al., 1983). Thus, spin counting techniques using an internal 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) and arti®cially matured kerogens (Derenne et al., 1987). Most carbon atoms in polyaromatic materials can be detected by Bloch Decay measurements as recently shown for 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 is well documented that black carbon is hardly detected since low weight losses are obtained upon pyrolysis and the generated products mostly consist of low molecular weight volatiles, including methane and carbon dioxide.
3.3. Melanoidin occurrence in ROR
As shown by HRTEM, a substantial fraction of the LacadeÂe ROR is comprised of black carbon. However, the presence of this polyaromatic material alone cannot account for the FTIR features of the resistant residue, especially for the abundant contribution of oxygen-and/or nitrogen-containing groups, whereas the latter groups may re¯ect the occurrence of melanoidin-type components.
In fact, comparison with previously reported spectra of 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 the presence of melanoidin-like moieties. Nevertheless, nei-ther the solid state13C NMR spectrum nor the CuPy/ GC/MS trace clearly indicated the presence of this type of material. In contrast, conspicuous aliphatic features were observed through these two methods although, as discussed 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/MAS13C NMR spectra recorded att
c=1 ms of the
aord a minor contribution to the ROR. Accordingly, mixtures of model compounds (synthetic melanoidin and polyethylene) were prepared and examined via solid state 13C NMR, FTIR and CuPy/GC/MS so as to account for such a discrepancy.
3.3.1. Solid state13C NMR
The insoluble synthetic melanoidin, obtained in a yield of 9% from the glucose/amino acid mixture, is characterized by a carbon content of 61.3% and an atomic H/C ratio of 1.0. Its solid state13C NMR spec-trum, obtained with the classical contact time of 1 ms (Fig. 7a,b) exhibits several intense broad peaks. The aliphatic 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). An intense peak is observed at 150 ppm which can be assigned to aromatic carbons bearing heteroatoms. A peak can also be observed between 100 and 120 ppm on the spectrum recorded at 4 kHz (Fig. 7b); it corresponds partly 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, which maximizes at 113 ppm is due to carbons in double bonds; based on the occurrence of the peak at 150 ppm, these carbons were considered to be located in aromatic structures, ortho to oxygen or nitrogen (Benzing-Purdie and 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 or nitrogen. The signal at 173 ppm is due to carboxyl in ester and/or amide groups (Benzing-Purdie and Ripmeester, 1983; Rubinsztain et al., 1984; Ikan et al., 1986; Allard et al., 1997). Peaks at 70 and 190 ppm on Fig. 7b are due 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/MAS13C NMR, also using the classical contact time of 1 ms (Fig. 7c±e). Based on the elemental analysis of the synthetic melanoidin (61.3% C) and on the carbon content in the poly-ethylene (85.6%), the melanoidin carbon/polypoly-ethylene carbon ratios in the three mixtures are of 13.5, 2.86 and 0.71, respectively. In fact, the13C NMR spectra of all these melanoidin/polyethylene mixtures are sharply dominated by a peak at 32 ppm corresponding to CH2 from polyethylene. Moreover, in the 50/50 mixture, the melanoidin signals can hardly be detected. It thus appears that melanoidin carbons are strongly under-estimated with respect to the polyethylene ones via solid state13C NMR.
Such a large underestimation can be a priori related either to intrinsic features of the melanoidin [some car-bons from the melanoidin part could not be observed since they are too far from protons and they cannot be reached by spin diusion as observed, for example, for condensed polyaromatic materials such as anthracite coals (Dubois Murphy et al., 1982)] and/or to unsuitable
Fig. 8. CP/MAS13C NMR spectra of 95/5, wt./wt., melanoidin/polyethylene mixture recorded at various contact times ranging from
conditions for spectrum acquisition, especially an unsuitable contact time, so that the relative numbers of the dierent types of resonant carbons would not be properly 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 these spectra, two signals were selected to represent the two components of the mixtures: the 150 ppm peak (corre-sponding to the heteroatom-linked aromatic carbons of the melanoidin) and the 32 ppm peak (encompassing both the signal of all the polyethylene carbons and the signal of the aliphatic carbons from the melanoidin). By plotting the intensity of these two signals versus contact time, we could determine the ratio of the number of resonant carbons Mo(150 ppm)/Mo(32 ppm)=1.02 for the 95/5 mixture. Comparison of this ratio with the M(150 ppm)/M(32 ppm) value directly calculated from peak intensity for each spectrum recorded at a given contact time (Table 1) revealed that reliable quantitative information on the relative abundance of the dierent types of resonant carbons can be directly deduced from the spectrum at 2 ms. This contact time was therefore used to test the ®rst hypothesis, i.e. that melanoidin underestimation re¯ects the presence of non-resonant carbons.
As already mentioned, the 32 ppm peak in the spectra of the three mixtures, although mostly due to polyethylene, also contains some contribution from melanoidin carbons. So as to estimate the fraction of resonant carbons due to melanoidin in the 32 ppm signal, the pure melanoidin spectrum was subtracted from each mixture spectrum after having adjusted the intensity so as to ®t the 150 and 113 ppm signals, as illustrated in Fig. 9a for the 95/5 mix-ture. The dierence spectrum thus obtained corresponds to that of polyethylene (Fig. 9b). The intensity ratio of the melanoidin and polyethylene spectra thus calculated corresponds to the melanoidin carbons/polyethylene
Table 1
Relative intensity of the 150 and 32 ppm peaks in the solid state
13C NMR spectra of the 95/5 melanoidin/polyethylene
mix-tures recorded at dierent 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/MAS13C NMR spectrum of 95/5, wt./wt.,
mel-anoidin/polyethylene mixture (top) and ®tted spectrum of syn-thetic melanoidin (bottom); (b) dierence spectrum between spectra displayed in (a) showing the polyethylene part in the 95/5 mixture.
Table 2
Eciency of the detection of the melanoidin carbons by solid state13C 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 valuesb
NMR-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 eciencies 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).
carbons ratio in the mixture, as seen by CP/MAS NMR under suitable acquisition conditions. Comparison of these ratios with the real ones (Table 2) reveals large dierences for the three mixtures and the value obtained from the NMR spectra is always markedly lower. This observation clearly shows that some carbons of the melanoidin 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 distance and these carbons are too far from protons to be reached by spin diusion). From the dierence between the theoretical and NMR-derived ratios, the percentage of these undetected carbons can be calculated for the dierent mixtures (Table 2). Large values are thus obtained for all mixtures; furthermore, the percentage of undetected carbons tends to increase when the content of melanoidin decreases. This trend may re¯ect a decrease in the eciency of magnetization transfer to melanoidin carbons in the presence of increasing amount of polyethylene. The large content of unde-tected carbons in the synthetic melanoidin probably re¯ects a highly cross-linked macromolecular structure for 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 spin counting techniques using an internal reference or com-parison with elemental composition, showed that a high percentage of carbons is not detected, via solid state13C 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 analytical constraint calculations to be overestimated (Wilson et al., 1987). A similar situation is observed in the present study from the melanoidin/polyethylene mixtures.
3.3.2. FTIR
The FTIR spectrum of the melanoidin (Fig. 10a) is similar to previously reported spectra for other synthetic melanoidins (Rubinsztain et al., 1984, 1986; Almendros et al., 1989; Allard et al., 1997). The main bands are due to oxygen-containing functions such as those at 3440 and 1020 cmÿ1(OH groups) and at 1706 cmÿ1(COOH groups). 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 bands are also observed at 1623 and 1510 cmÿ1; they are
usually assigned to aromatic furanic or conjugated compounds. A low contribution of CH2 and CH3 groups is re¯ected by the weak absorption in the 2850± 2950 cmÿ1range.
The FTIR spectrum of polyethylene (Fig. 10f ) shows the typical bands of polymethylenic chains at 2920 and 2850, 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 the melanoidin bands are still clearly visible in the 50/50 mixture. When compared to solid state 13C NMR, FTIR thus appears more suitable for revealing the pres-ence of melanoidin-like structures, but this method only aords semi-quantitative information.
3.3.3. Curie point pyrolysis/gas chromatography/mass spectrometry
The pyrolysate of the synthetic melanoidin (Fig. 11a) chie¯y comprises phenolic and furanic compounds and aromatic hydrocarbons and it is dominated by levulinic acid (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 virtual lack of nitrogen-containing compounds is in agreement with the very low nitrogen content observed by ele-mental analysis (0.67 mg of N/g of carbon) and the high sugar to amino acid ratio (9/1) used for the preparation of the synthetic melanoidin. Similar products were detected in pyrolysates of other synthetic melanoidins (Boon et al., 1984; Allard et al., 1997) and of humic substances (Wilson et al., 1983; Gillam and Wilson, 1985).
Mixtures of melanoidin and polyethylene containing 98, 95, 80 and 50 wt.% of melanoidin were examined by CuPy/GC/MS (Fig. 11). The pyrochromatogram of the
98/2 mixture (Fig. 11b) looks very similar to that of the pure melanoidin (Fig. 11a). Polyethylene pyrolysis is known to yield a regular series ofn-alkane/n-alk-1-ene doublets resulting from the homolytic cleavage of the polymethylenic chains and extending to high carbon numbers (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 atm/z57. They are hardly visible in the pyrochromatogram of the 95/5 mixture but they become dominant in the 80/20 mixture and are overwhelmingly abundant in the 50/50 one (Fig. 11c±e). In the latter case, the melanoidin con-tribution is only revealed by very small peaks at the beginning of the trace and a hump due to levulinic acid under the C11alkane/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. Such an overestimation can easily be explained by the respective yields of pyrolysis products from melanoidins and from polymethylenic chains. Melanoidins are char-acterized by low weight losses upon pyrolysis since they
Fig. 11. 610C Curie point pyrochromatograms of the synthetic melanoidin (a) and of mixtures with polyethylene in various
aord large amounts of polyaromatic residue and the released products mostly correspond to highly volatile compounds (Allard et al., 1998) whereas long (CH2)n
chains in polyethylene are quantitatively cleaved into
n-alkane/n-alkene doublets that extend over a wide carbon number range, hence the release of large amounts of GC-amenable products.
Taken together, these observations account for the poor detection of the melanoidin fraction in the ROR of the LacadeÂe soil when examined via solid state 13C NMR and CuPy/GC/MS (Augris et al., 1998).
4. Conclusions
Contrary to previous observations based on solid state 13C NMR spectroscopy and Curie point Py/GC/ MS, which pointed to a highly aliphatic nature for LacadeÂe ROR, it appears that aliphatic moieties with long alkyl chains are only minor components. Accordingly, an important contribution of cutans/suberans, cross-linked lipids and cutin/suberin polyesters can be ruled out in this resistant material. In fact, the LacadeÂe ROR is mostly composed of melanoidins (complex macromolecular structures derived from the random condensation of alteration 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 a poorly ordered carbon phase. The second type is com-posed of roughly spherical particles, ranging, from 70 to 250 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 production of char via aromatization of ligno-cellulosic precursors (amorphous-like carbon phase) and (ii) the release and the subsequent incomplete combustion of hydrocarbons (concentric nanoparticles). The individual polyaromatic units in these black carbon particles are relatively large and connected. Accordingly, a large part of the corre-sponding carbon atoms could not be detected via solid state 13C NMR. Similarly, the presence of the black carbon fraction was not revealed via Curie point pyr-olysis due to low yields of pyrpyr-olysis products.
Examination of synthetic melanoidin/polyethylene mixtures also showed a poor detection of the former components via solid state13C NMR, even when using an optimum contact time. This strong underestimation of 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 pyrolysis products of melanoidins are only generated in low yields,
hence such components are also poorly detected through pyrolysis experiments. In contrast, FTIR appears more ecient 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 in heterogeneous materials such as the LacadeÂe ROR, when applied alone and with no quantitative constraints derived from elemental analysis and/or quantitative pyrolyses.
Acknowledgements
This study was partly supported by the AGRIGES program (Institut National de la Recherche Agronomique and MinisteÁre de l'Environnement). We also thank Dr. D. Arrouays (Unite de Science du Sol, INRA, OrleÂans) for providing the soil sample from LacadeÂe and J. Guil-lemot and Y. Pouet (Py/GC/MS) for technical support.
Associate EditorÐS.J. Rowland
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