Directory UMM :Data Elmu:jurnal:O:Organic Geochemistry:Vol31.Issue9.2000:

Organic Geochemistry 31 (2000) 813±827
www.elsevier.nl/locate/orggeochem

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 e
a

Laboratoire de BiogeÂochimie Isotopique, INRA-CNRS-UPMC, 4 pl. Jussieu, 75252 Paris cedex 05, France
Laboratoire de Chimie Bioorganique et Organique Physique, UMR CNRS 7573, ENSCP, 11 rue P et M Curie, 75231 Paris cedex 05, France
c
Centre de Recherche sur la MatieÁre DiviseÂe, UMR CNRS 6619, Universite d'OrleÂans, 1bis rue de la FeÂrollerie, 45071 OrleÂans Cedex 2, France
d
Laboratoire d'Ecologie Microbienne de la RhizospheÁre, DEVM, CEA Centre de Cadarache, 13108 Saint-Paul les Durance cedex, France
e
Laboratoire de Chimie de la MatieÁre CondenseÂe, UPMC, UMR CNRS 7574, 4 pl. Jussieu, 75252 Paris cedex 05, France
b

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'' pyrolysis 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
state 13C NMR spectroscopy and Curie point Py/GC/MS, highly aliphatic moieties only a€ord 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 state
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 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

* Corresponding author. Tel.: +33-1-4427-6761; fax: +331-4325-7975.
E-mail address: clargeau@ext.jussieu.fr (C. Largeau).

13

C NMR; FTIR; Pyr-

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 elucidated (e.g. Skjemstad et al., 1996). Protection by minerals 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 recalcitrant aliphatic structures in SOM. Indeed, observations
by 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.
PII: S0146-6380(00)00067-X

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

that the relative contribution of alkyl carbon tends to
increase with increasing degrees of decomposition of the
organic matter. The presence of moieties containing polymethylenic chains, re¯ected by the formation of
n-alkane/n-alk-1-ene doublets, was also observed via pyrolysis 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 degradation 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 di€erences in the relative
intensity of the n-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 contribution was observed for the resistant (non-hydrolyzable)
fraction isolated from the humin of the LacadeÂe soil
(Augris et al., 1998). The origin and formation pathways of aliphatic moieties in the stable fraction of SOM
remain largely unknown (Hedges and Oades, 1997) and
di€erent types of sources have been considered. These
sources included highly aliphatic, resistant, macromolecular 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 crosslinking 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
13
C NMR observations. It is well documented, however,
that such spectroscopic methods can markedly overestimate 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 literature with ``charcoal'', correspond to the residues of
incomplete combustion produced from vegetation ®res
and burning of fossil fuels. Black carbon is widely distributed 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 carbon 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 fragments 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 stability 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). Melanoidins 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 aromatic units can also occur in melanoidins, especially
when derived from proteins containing tyrosine, phenylalanine, tryptophan and proline.
In a previous study of a forest soil from LacadeÂe
(south-west France) we observed that a substantial part
of humin corresponds to insoluble, non-hydrolyzable,

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

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¯uenced by the analytical methods used. The major aims
of the present study were therefore to examine the possible 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 state 13C 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 (