Directory UMM :Data Elmu:jurnal:O:Organic Geochemistry:Vol31.Issue5.2000:
Organic Geochemistry 31 (2000) 409±419
www.elsevier.nl/locate/orggeochem
Structure elucidation of soil macromolecular lipids by
preparative pyrolysis and thermochemolysis
V. GobeÂ, L. LemeÂe, A. AmbleÁs *
Laboratoire de SyntheÁse et ReÂactivite des Substances Naturelles, UMR 6514, Faculte des Sciences,
40 Avenue du Recteur-Pineau, 86022 Poitiers, France
Abstract
The structure of macromolecular lipids from an acid anmoor soil was investigated using preparative pyrolysis and
thermochemolysis. The matrix is formed by cross-linked aliphatic chains. Alkanols, triterpenoid alcohols e.g. a-amyrin,
sterols, stanols can be bound to the matrix via ether or ester groups. Fatty acids can be incorporated through esteri®cation. Dicarboxylic acids and other compounds such as hydroxyacids represent alkyl bridges between the polymethylene chains. Various components are trapped in the macromolecular network. The two techniques give
complementary results. Despite higher yields, thermochemolysis does not permit one to distinguish between methyl
ester groups present in the structure and methyl ester groups formed on degradation. # 2000 Elsevier Science Ltd. All
rights reserved.
Keywords: Macromolecular lipids; Pyrolysis; Thermochemolysis; Hydrocarbons; Fatty acids; Alcohols; Aldehydes; Ketones
1. Introduction
Lipids play an important role in soil processes (Stevenson, 1982) because they aect plants or soil micro¯ora
and in¯uence soil physical properties (Jambu et al., 1983).
Previous studies concerning lipids present in soils have
shown that this soluble fraction includes simple lipids
with a plant or a microbial origin but also complex,
macromolecular lipids, corresponding to the polar lipid
fraction when using the McCarthy and Duthie (1962)
procedure for lipid fractionation. This latter fraction has
been studied using chemical degradation methods such
as alkaline hydrolysis under phase catalysis conditions,
ether bond cleavage with boron tribromide or alkaline
potassium permanganate oxidation (AmbleÁs et al.
1991, 1993a). It was shown that soil macromolecular
lipids contains preserved biomacromolecules of plant
or microbial origin (AmbleÁs et al., 1991). The (reversible) incorporation of exogenic organic molecules in
* Corresponding author. Tel.: +33-5-4945-3866; fax: +335-4945-3501.
E-mail address: andre.ambles@campus.univ-poitiers.fr
(A. AmbleÁs).
macromolecular lipid fraction, for instance by ester
groups, has then been clearly demonstrated (AmbleÁs et
al., 1994).
In hydromorphous environments, these macromolecular lipids can partly escape biodegradation
(AmbleÁs et al., 1991) and become part of the sedimentary organic matter. As a consequence, some analogy
between these soil macromolecular lipids and the
organic matter present in ancient sediments may exist
and macromolecular lipids can be compared with a
proto-kerogen in some soils (Shioya and Ishiwatari,
1983; AmbleÁs et al., 1991).
In this paper, the structure of macromolecular lipids
from an acidic anmoor soil was investigated using preparative o-line pyrolysis (Behar and Pelet, 1985; Largeau et al., 1986; Metzger and Largeau, 1994) and a new
preparative thermochemolysis technique which permits
the treatment of more than one gram of product
(Grasset and AmbleÁs, 1998b). In the latter technique, an
alkylating agent is used for the thermally assisted
hydrolysis and alkylation, as developed for analytical
¯ash on-line pyrolysis (del Rio et al., 1996). The soil
samples originated from a peaty soil (Plateau de Millevaches, France) where organic matter is accumulating
and as such the structural study of macromolecular
0146-6380/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0146-6380(00)00009-7
410
V. Gobe et al. / Organic Geochemistry 31 (2000) 409±419
lipids from these soils may give information about early
diagenetic processes.
2. Experimental
2.1. Samples
Samples were collected from a peaty soil (Sphagnum
peat), an acidic anmoor located on the Plateau de Millevaches (North CorreÁze). The macromolecular lipids
(ML) from the surface 0±20 cm horizon were previously
described by AmbleÁs et al. (1991; 1993a). The samples
used for the present work originated from the 30±50 cm
horizon which is waterlogged, organic-rich, with a
higher degree of decomposition of plant debris (saprist)
than the surface horizon (®brist).
2.2. Separation of the macromolecular lipid fraction
The procedure is shown in Fig. 1. Representative soil
samples were dried and sieved to 2 mm. Total lipids
were extracted with chloroform in a Soxhlet (324 h).
The original soil sample contained 56% of total organic
matter (TOM) (determined by combustion). Lipids contributed 5% to the TOM. The total lipid extract was
fractionated on a silica-potassium hydroxide column. In
order to avoid problems of separation between the acid
and the polar fractions in the classical McCarthy±Duthie
procedure (AmbleÁs et al., 1991) on the one hand, and to
release lipids possibly trapped by the macromolecular
network (such as HCT, Fig. 1) (AmbleÁs et al., 1991) on
the other hand, the procedure of separation was slightly
modi®ed: the ``classical'' acid and the polar fractions
were mixed and then methylated using trimethylsilyldiazomethane (Hashimoto et al., 1981). The most
polar fraction (corresponding to the polar fraction of
the McCarthy±Duthie procedure) obtained after liquid
chromatography (MLA) contains macromolecular
lipids. Other macromolecular lipids (MLN) were
obtained by liquid chromatography of the neutral fraction (Fig. 1). The total macromolecular lipid fraction
(ML) was obtained after addition of MLA and MLN. It
gave no elution in thin layer chromatography using
polar eluents. The other fractions (in italics in Fig. 1)
were not studied in this work.
Macromolecular lipids contributed 15% to the total
lipid extract (0.7% of TOM). The elemental composition of the ML is : C 72.39%, H 10.16%, O 16.96% (by
dierence), N 0.27%. The value of the atomic H/C ratio
is 1.68.
2.3. Preparative o-line pyrolysis
The same device as that described by Behar and Pelet
(1985) was used. A quartz tube containing the complex
lipids (60±70 mg) was placed in a stainless steel tube and
put in an oven, at 425 C for 1 h under helium (¯ow rate
30 ml minÿ1). Pyrolysis products were collected in two
successive traps containing solvent cooled to ÿ30 C. After
evaporation of the solvent, the pyrolysate from seven
experiments was separated on a SiO2/KOH column
(McCarthy and Duthie, 1962) in a neutral fraction and an
acid + polar fraction. The later was methylated. Each
fraction was chromatographed on a silica column, the
various components were eluted with diethyl ether/petroleum ether mixtures of increasing polarity (Fig. 1).
Three chromatographic fractions were obtained for
hydrocarbons: HC I:12 mg, HC II:1 mg; HC III:1.5 mg.
2.4. Preparative o-line thermochemolysis
Fig. 1. Procedure of fractionation of total lipid extract and of
pyrolysis product.
ML (349 mg) was placed in a ceramic boat and
moistened with 3 ml of a methanol 50% w/w solution of
tetramethylammonium hydroxide (TMAH) (Aldrich).
The ceramic boat was then transferred into a 603 cm
i.d. Pyrex1 tube. The tube was maintained at 425 C
(oven) for 1 h under helium (¯ow rate 100 ml minÿ1).
The pyrolysate was collected in two successive traps, as
described above. The pyrolysis products were directly
V. Gobe et al. / Organic Geochemistry 31 (2000) 409±419
411
separated by liquid chromatography on SiO2 (diethyl
ether/petroleum ether elution).
2.5. Analyses
The products were analyzed by capillary GC and
GC±MS. GC separations were carried out with a Shimadzu GC-14A gas chromatograph in splitless injection
mode, using a CPSil 8-CB (Chrompack) capillary column
(50 m0.25 mm i.d., 0.12 mm ®lm thickness) and a FID.
The temperature of the column was programed from 50 C
(2 min isothermally) to 200 C (2 min isothermally) at 5 C
minÿ1 then to 300 C (30 min isothermally) at 3 C minÿ1.
The temperature of the injector and the detector was
300 C.
The quantities of each kind of products are given as
apparent concentrations; they were determined on the
basis of liquid chromatography for the various families and on the basis of GC response (peak area) for
a determined family. No correction was made for
derivatization.
GC±MS were performed on a FINNIGAN MATINCOS 500 mass spectrometer coupled with a VARIAN 3400 gas chromatograph. The GC conditions were
the same as for GC analysis. The mass spectrometer
was operated in the electron impact mode (70 eV).
Transfer line and ion source were 290 and 180 C,
respectively. Mass spectra were acquired by scanning
the region m/z 50±600 at a rate of 1 s per decade and
recorded by a Data General 20 computer. The various
products were identi®ed on the basis of their GC retention times and their mass spectra (comparison with
standards). Alcohols were identi®ed as acetates (acetylation with acetic anhydride/pyridine). Acids were
methylated with trimethylsilyldiazomethane (Hashimoto et al., 1981). Fourier transform infra-red (FT-IR)
spectra were obtained in CCl4 using a Nicolet 750 spectrometer. NMR spectra were recorded in CDCl3 solution on a BRUKER AVANCE DPX 300 (300 MHz for
1
H and 75 MHz for 13C). Tetramethylsilane was used as
internal reference.
3. Results and discussion
3.1. Bulk properties
The results of the elemental analysis and the value of
the atomic H/C ratio (see experimental section) indicated a rather aliphatic nature for the macromolecular
lipids of the studied soil. The point was corroborated by
the FT-IR spectrum (Fig. 2) which exhibited strong
bands centered at 2926, 2853, 1464 and 1377 cmÿ1 which
can be attributed to CH3 and/or CH2 groups (GobeÂ,
1998). NMR measurements gave con®rmation of this
predominantly aliphatic nature: 1H NMR spectrum
Fig. 2. FT-IR spectrum of macromolecular lipid fraction (CCl4
solution).
presented bands in the 0.5±1.7 ppm region (Fig. 3a) and
the 13C NMR spectrum (Fig. 3b) showed signals in the
aliphatic 9±35 ppm region. The FT-IR spectrum
revealed also the presence of hydroxyl groups (broad
band centered at 3413 cmÿ1) and broad bands at 1737
cmÿ1 (carbonyl), 1252 (nCsp3-O), 1174, 1132 and 1032
cmÿ1 (nCsp3-O) which can be attributed to ester and
ether groups. In the 1H NMR spectrum (Fig. 3a), signals at 3.6 ppm (H±C±O), 2.3 ppm (H±C±CO) and 1.5
ppm (very broad) (H-C-C-O) can con®rm the presence
of ester groups, as well as the signal at 169.5 ppm
(CO) on the 13C NMR spectrum.
3.2. Pyrolysis and thermochemolysis
ML were investigated using o-line preparative pyrolysis (Behar and Pelet, 1985 ; Largeau et al., 1986) and
o-line thermochemolysis as described by Grasset and
AmbleÁs (1998b). The results are given in Table 1. Seven
elemental o-line pyrolysis on 415 mg of material yielded 98 mg of pyrolysis products corresponding to
23.6% of the original macromolecular lipids. The
remaining residue represented 64.8% of initial ML. The
loss of weight was probably due to volatile compounds.
Preliminary experiments (GobeÂ, 1998) clearly showed
that the pre-pyrolysis used for the study of sedimentary
organic matter (Largeau et al., 1986) is not useful for
the study of soil organic matter, pre-pyrolysis and pyrolysis yielding the same products.
Preparative thermochemolysis aorded degradation
products with a much higher yield (234 mg; 67% of
ML) but TMAH was present among the pyrolysis products. The loss of products was 23 mg (6.6% of the
initial ML).
412
V. Gobe et al. / Organic Geochemistry 31 (2000) 409±419
Fig. 3. 1H NMR (a) and 13C NMR (b) spectra of macromolecular lipids (CDCl3 solution).
The composition of the pyrolysates from both
experiments was determined by liquid chromatography.
The main series of compounds are listed in Table 2.
Polar products corresponded to the remaining material
and/or to more or less transformed macromolecular
lipids. The yield of GC amenable components was much
higher for thermochemolysis (Table 2).
The hydrocarbon fraction formed in pyrolysis (HC I,
Fig. 1) showed the classical series of n-alkane/n-alk-1ene doublets, ranging from C16 to C36 (Fig. 4a). The
same distribution was obtained upon thermochemolysis.
Alkenes can be produced by b-scission of radicals (Larter
and Hors®eld, 1993). The origin of n-alkane/n-alkene
doublets on pyrolysis is diverse. One origin can be the
thermal degradation of esters which is known to produce
chie¯y n-alkanes from an acid moiety and n-alk-1-enes
from the alcohol moiety (Van de Meent et al., 1980).
These esters correspond to preserved protective layers of
higher plants, as suberin, cutin (de Leeuw and Largeau,
1993 ; Macko et al., 1993) from above-ground parts as
well as from roots (Nierop, 1998). The presence of
alkene/alkane doublets in pyrolysates is indicative of the
V. Gobe et al. / Organic Geochemistry 31 (2000) 409±419
413
Table 1
Results (mg) of pyrolysis and thermochemolysis of macromolecular lipids
Pyrolysis
Thermochemolysis
a
b
Mass of
sample
Pyrolysate
Residue
Loss of
weight
415
349
98a
234b
270
922
47
23
Seven elemental pyrolysis.
Presence of TMAH.
Table 2
Components produced by thermal degradation of macromolecular lipids from Plateau de Millevaches (% of pyrolysate)
Pyrolysis Thermochemolysis Range
Hydrocarbons
14.5
Fatty acids
4.1
(methl esters)a
Fatty acid methylesters 6.1
26
C16±C36
C14±C34
19
Aliphatic diacids
(dimethylesters)a
Alcohols (acetates)a
Ketones
Aldehydes
a-Hydroxymethylesters
o-Hydroxymethylesters
(o-1)-Ketomethylesters
(o-1)-Ketoalcohols
Fatty acid octylesters
Polar products
7.2
15
C14±C19;
C20±C32
C11±C28
2.6
1.7
0.7
1
2.1
2.1
1
1.9
55
5
5
0.1
2.1
2.8
ND
ND
ND
25
a
C14±C32
C17±C33
C19±C31
C22±C27
C16±C28
C22±C29
C17±C29
C14±C22
After derivatization.
presence of resistant aliphatic macromolecules (cutan,
suberan...) in soils (Tegelaar et al., 1989b; van Bergen et
al., 1997a; Augris et al., 1998). Various terpenoids, norhopanes, norhopenes, hopadienes, hopenes and hopanes
(derived from bacteriohopanetetrol or related compounds) and a sterene, 24-ethyl 5a(H) cholest-2-ene were
also identi®ed, eluting between nC29 and nC36 alkanes
(Fig. 4a; Table 3). These triterpenoid compounds were
not identi®ed on hydrolysis (AmbleÁs et al., 1991; 1993a)
which indicates a possible ether linkage to the macromolecular matrix on carbon 3 of the A ring of the terpenoid/steroid. Despite a higher quantity of released
hydrocarbons (Table 2), the number of polycyclic structures were less numerous on thermochemolysis. Only 5
components were present 22,29,30-trisnorhop-17 (21)
ene, 17a(H),22,29,30-trisnorhopane, 30-norhop-17(21)
ene, 17b(H), 21 b(H)-30-norhopane, 17a(H),21 a(H)hop-22(29)-ene. The presence of the two isoprenoids
prist-1-ene and prist-2-ene, indicated the occurrence of
chemically-bound phytol (Larter et al., 1979) and/or
tocopherols (Goossens et al., 1984). Among the pyrolysis
Fig. 4. Chromatograms of hydrocarbons obtained by pyrolysis
(a) n-alkane/n-alkene doublets with triterpenoids (HC I fraction).(The vertical numbers correspond to triterpenoids listed
in Table 3) (b) n-alkane distribution of the HC III fraction of
hydrocarbons.
products, two minor chromatographic fractions were
also obtained (Fig. 1). The HC II fraction (1 mg) contained only n-alkanes with abundant C27 and C29 members, superimposed on the Gaussian C21±C37
distribution. The HC III fraction (1.5 mg) contained
dominant even C18, C20, C22 and C24 saturated members
(Fig. 4b). The origin of these even hydrocarbons is very
probably fatty acid or alcohol reduction. These hydrocarbons were obviously trapped in the macromolecular
network and released after alteration of the matrix.
OkomeÂ-Mintsa (1991) reported the presence of such nalkanes in the hydrolysis products of macromolecular
lipids from the same soil. The same phenomenon was
observed after chemical degradation of kerogens from
ancient sediments (Baudet et al., 1991; Halim et al.,
1997).
The alcohol fraction from both experiments presented
the same composition. n-Alkanols were present in the range
C14±C32 with dominant even members and a clear maximum at C22, accompanied with neolupenol [identi®ed in
some roots (Ageta et al., 1981)], a-amyrin, sterols also
V. Gobe et al. / Organic Geochemistry 31 (2000) 409±419
414
Table 3
Triterpenoid hydrocarbons detected in Fig. 4
Number Name
Structure
Number Name
1
17a(H)-22,29,30-trisnorhopane
8
17b(H),21a(H)-hop-22(29)-ene
2
24-ethyl-5a(H)-cholest-2-ene
9
17a(H),21b(H)-hopane
3
C29 17b(H),21a(H)-hopadiene
10
C32 17b(H),21a(H)-hopane
4
C30 17b(H),21a(H)-hopadiene
11
17b(H),21b(H)-homohopane
5
17a(H),21b(H)-30-nor-neohop-13(18)-ene
12
C32 17b(H),21b(H)-hopane
6
17b(H),21b(H)-norhopane
13
C32 17b(H),21b(H)-hopene
7
30-norhop-17(21)-ene
14
C33 17b(H),21b(H)-hopene
mainly of plant origin (i.e. stigmasterol, 24-methyl- and 24ethylcholesterol) and their corresponding stanols. The
sterol/stanol ratio ranged from 0.5 (cholesterol/cholestanol) to 0.9 (24-ethylcholesterol/24-ethylcholestanol). The
sterol distribution with a dominant 24-ethylcholesterol is
the same as that from the simple lipids from the same
soil providing evidence that simple lipids can become
incorporated into a macromolecular lipid fraction
(AmbleÁs et al., 1991). As the same structures (including
stanols) were found after alkaline hydrolysis (AmbleÁs et
al., 1991; 1993a), incorporation was most probably via
ester groups. The presence of stanols is a consequence of
anaerobic conditions in this hydromorphic soil, as in
marine or lacustrine sediments (Gaskell and Eglinton,
1975). Microbial reduction of sterols occurs when the OH
at position 3 is not bound, the attachment to the matrix
occurring later probably via a transesteri®cation process
(de Leeuw and van Bergen, personal communication).
The distribution of n-alkanols formed by thermal
degradation of macromolecular lipids does not resemble
that of alcohols present either in the above-ground parts
of plant in the studied sample or in the soil simple lipid
fraction [highly dominant C28 member (AmbleÁs et al.
1991)]. They can partly be inherited from plant wax
esters (Jambu et al., 1993; AmbleÁs et al., 1993a), or from
highly resistant biopolymer in root material as suberin
(Nierop, 1998), or result from the enzymatic reduction
Structure
of fatty acids (Kolattukudy, 1976). No close correlation
with the distribution of fatty acids can be expected
inasmuch as the occurring processes are biological processes (with discrimination with chain length) and
maybe competitive processes.
The distribution of aliphatic monocarboxylic acids
formed on pyrolysis of macromolecular lipids (Fig. 5a)
showed a strong even over odd carbon number predominance. These acids could originate from plant protective
polyesters (Tegelaar et al., 1989a,b). Unsaturated C15:1,
C16:1, C18:1, C18:2 components, and branched iso- and
anteiso C15, C16 and C17 acids of bacterial origin (Boon
et al., 1977 ; Perry et al., 1979), C31 and C32 1721b(H),
21b(H) hopanoic acids were also present. Fatty acid
methyl esters, corresponding to 6.1% of the pyrolysate
(Table 2) which were also identi®ed amongst the products (Fig. 5b), showed a similar distribution to that of
fatty acids present in simple lipids. Thermochemolysis
aorded only methyl esters arising mainly from the
transesteri®cation of esters (Martin et al., 1995; Gonzalez-Vila et al., 1996). The relative yield of products was
much higher than in pyrolysis. Conversely, thermochemolysis does not permit to distinguish fatty acids
bound by ester linkage from fatty acid methyl esters, if
any, trapped in the macromolecular matrix. The distribution of the fatty acid methyl esters corresponded to
the combined distributions of Fig. 5.
V. Gobe et al. / Organic Geochemistry 31 (2000) 409±419
415
Dicarboxylic acids in pyrolysis or dimethylesters in
thermochemolysis were identi®ed in the C11±C28 range
with an even predominance and a dominance of the C22
member (and an important C16 component). They were
bound as diesters in the macromolecular matrix. Both
experiments exhibited the same distributions.
a- and o-hydroxyacid methyl esters were observed in
both experiments, respectively in the C20±C29 range
(max. C24) (Fig. 6) and C16 to C28 with a maximum at
C22. a- hydroxyacid methyl esters give a prominent m/z
90 ion on mass spectrometry (McLaerty rearrangement) while the o- isomers led to the classical m/z 74.
Although they were produced (as methyl esters, without
derivatization) in pyrolysis with a lower yield, this
method indicated that they were bound to the matrix via
the hydroxyl groups only, the methyl ester group being
pre-existant in the macromolecular structure. Otherwise,
if the COOH groups were esteri®ed to the matrix,
hydroxyacids would have been produced upon pyrolysis. o-Hydroxyacids are present in cuticular waxes,
cutins and suberin from plants (Eglinton et al., 1968;
Ketola et al., 1987) while a-hydroxyacids have a microbial origin, resulting from the oxidation of fatty acids
(Eglinton et al.,1968; Cranwell, 1984). The presence of
a- and o-hydroxyacids in the degradation products of
macromolecular lipids con®rms that this fraction contains partly preserved biopolymers of plant or microbial
origin (AmbleÁs et al., 1991).
Ketones (Fig. 7) were essentially methylketones in the
C13-C29 range accompanied with the isoprenoidal
6,10,14-trimethyl-pentadecan-2-one and steroidal 24ethylcholest-4-en-3-one, 24-methyl- and 24-ethylcholesta-3,5-dien-7-one (or stigmastadienone). The isoprenoid ketone originates probably from the microbial
degradation of phytol, as reported by Brooks and
Maxwell (1974). The origin of methylketones in thermal
degradation products is not well established. Among
soil (simple) lipids, they arise exclusively from oxidation
of plant hydrocarbons or b-oxydation-decarboxylation
of fatty acids (AmbleÁs et al., 1993b) and are predominantly odd-carbon numbered components. The
distribution of Fig. 7 could indicate that these ketones
were trapped in the macromolecular matrix. Methylketones could also have been produced from the scission
of an ether linkage on carbon 2 or the scission of an
alkyl chain on the a position of a hydroxyl group. An
other mechanism involving a radical scission at the b
position of a free hydroxyl group was postulated by
Hartgers et al. (1995). Several mechanisms contribute
probably to the formation of methylketones on thermal
degradation of macromolecular material. At present,
this point remains unclear. Steroidal ketones could also
have been trapped compounds or bound to the network
by an ether bond at the carbon 3 position. The occurrence of ester bond can also be postulated insofar as the
presence of esteri®ed cholesterol, cholestanol, 24-ethylcholesterol and 24-ethylcholestanol in the macromolecular lipids of the surface 0±20 cm horizon was
previously reported (AmbleÁs et al., 1991; 1993a).
Linear C19±C31 aldehydes with maxima at C23 and
C29 were present in minor amount, (Table 2). They
Fig. 6. Distribution of a-hydroxyacids obtained by pyrolysis
(m/z 90 fragmentogram).
Fig. 7. Distribution of methylketones obtained by thermochemolysis (m/z 58 fragmentogram).
Fig. 5. Fatty acids produced upon pyrolysis [m/z 74 fragmentogram of the methyl ester derivatives (i : iso ; a : anteiso)].(a)
Aliphatic monocarboxylic acids (as methyl esters); (b) aliphatic
monocarboxylic acid methyl esters produced on pyrolysis.
416
V. Gobe et al. / Organic Geochemistry 31 (2000) 409±419
could have been formed from chains bearing an OH
group (Gelin et al., 1994; Hartgers et al., 1995) or could
arise from the cleavage of an ether group (Grasset, 1997;
GobeÂ, 1998). Series of (o-1)-ketoacid methyl esters in
the C22±C29 range with a Gaussian distribution centered
on C25, and (o-1)-ketoalcohols in the range C17±C29 with
a regular distribution also centered on C25 were also
produced upon pyrolysis. Several tentative hypotheses
could explain the presence of these products. For
example, (o-1)-ketoalcohols could have been trapped in
the macromolecular matrix, or were covalently bound to
the matrix with one linkage (acting as a monosubstituent of the matrix) or covalently bound with two
linkages (acting as bridges in the matrix). Keto groups
could also partly originate from dehydrogenation of
hydroxyl groups.
Fig. 8. Schematic modes of occurence of soil macromolecular lipids. The rectangle represents the macromolecular matrix. Molecules
in the rectangle represent trapped molecules. Aliphatic bridges are placed outside the rectangle for enhanced legibility.
V. Gobe et al. / Organic Geochemistry 31 (2000) 409±419
Octyl esters of C14, C16 (abundant), C18 (dominant)
and C20 fatty acids were only detected upon pyrolysis.
They were also found in the pyrolysates of other soil
samples and of kerogen from phosphate rocks (unpublished results). They are never found in the products
from thermochemolysis whatever material is degraded.
Their origin is maybe related to a transesteri®cation
process occurring only under the pyrolysis conditions.
4. Conclusions
In this work, a detailed study of the structure of
complex, macromolecular lipids present in soil was
conducted using preparative o-line pyrolysis and preparative thermochemolysis. The two techniques provided evidence that the studied soil organic fraction is
aliphatic in nature (AmbleÁs et al., 1991; 1993a). The
identi®cation of degradation products permits one to
advance a schematic representation of the modes of
occurrence of structural building blocks (Fig. 8). The
matrix of soil macromolecular lipids is formed by crosslinked aliphatic chains, a part of which are partly preserved biopolyesters. Ether and ester groups are prominently involved in the cross-linking of the chains. Some
aliphatic moieties are acting as monosubstituents of the
matrix, re¯ected as aliphatic monocarboxylic acids (or
methyl esters), alcohols, methylketones and aldehydes
upon pyrolysis or thermochemolysis. The components
with two functional groups (aliphatic dicarboxylic acids,
(o-1)-ketoalcohols, etc.) provide evidence of the occurrence of alkyl bridges in the network. Nevertheless,
some of these components are bound to the matrix by
only one linkage releasing a- and o-hydroxyacid methyl
esters and (o-1)-ketoacid methyl esters on thermal
degradation. As in kerogens from ancient sediments
after chemical degradation (Halim et al., 1997), aliphatic
compounds such as some alkanes, fatty acid methyl
esters, possibly methylketones etc. are trapped in the
macromolecular structure and are released when the
structure of the matrix is altered. Direct ``thermoevaporation'' of the trapped compounds could also
occur on pyrolysis and thermochemolysis. The use of
more ecient extraction methods, such as supercritical
¯uid extraction could free the trapped compounds as
evidenced from a study of the released triterpenoids.
Taking into account hydrolysis products (AmbleÁs et al.,
1991; 1993a), it can be established that sterols, stanols,
neolupenol, a-amyrin are bound to the matrix by ester
(and maybe ether) linkages through the 3 carbon atom,
hopanoic acids are bound by ester groups. The loss of
the alcoholic group resulted in the formation of the
corresponding unsaturated compounds. Contrary to
other reported results using ¯ash Curie point pyrolysis
(van Bergen et al., 1997b), the loss of the OH group is
not total, probably as a consequence of a lower tem-
417
perature. Hopanes are present as trapped molecules.
Stigmastadienone, and related compounds could also be
present as trapped molecules. Alternatively, they are
linked to the matrix as etheri®ed 7-ketosterols (hydrolysis did not aord any 7-ketosterol), the stable conjugated dienone being formed on thermal degradation.
It is interesting to note that, as reported for ¯ash online pyrolysis, preparative thermochemolysis in the presence of tetramethylammonium hydroxide is a very useful and powerful technique for the study of natural
macromolecules. Preparative thermochemolysis aords
higher yields of products than conventional preparative
pyrolysis, which is useful for representative structure
determination. Heating in the presence of an alkylating
agent causes additional chemolysis of the resistant aliphatic parts of the matrix, allowing, as an example,
transalkylation of polyesters resistant under classical
pyrolysis conditions. As a consequence, the importance
of structural parts can be largely underestimated when
using the classical method (del Rio et al., 1996). Nevertheless, pyrolysis only permits one to distinguish the
groups present as methyl esters from the acid groups
linked as esters to the matrix. Conversely, the separation
procedure is much more time consuming than for preparative thermochemolysis.
From the results presented in this work, it can be
concluded that there is, on the one hand an analogy
between soil macromolecular lipids and the aliphatic
part of humin from the same soil (Grasset and AmbleÁs,
1998a) and, on the other hand similarities with the
structure of kerogen present in immature ancient sediments (Tegelaar et al., 1989a,b; AmbleÁs et al., 1991,
1993a; AmbleÁs et al., 1996; Kribii et al., 1996).
Acknowledgements
The authors gratefully acknowledge the authorities of
the French ReÂgion Poitou-Charentes for a doctoral grant
(V.GobeÂ.) and C.N.R.S. for ®nancial support. The
authors thank Dr. P.F. van Bergen and an anonymous
reviewer for very helpful comments and suggestions.
Appendix
418
V. Gobe et al. / Organic Geochemistry 31 (2000) 409±419
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www.elsevier.nl/locate/orggeochem
Structure elucidation of soil macromolecular lipids by
preparative pyrolysis and thermochemolysis
V. GobeÂ, L. LemeÂe, A. AmbleÁs *
Laboratoire de SyntheÁse et ReÂactivite des Substances Naturelles, UMR 6514, Faculte des Sciences,
40 Avenue du Recteur-Pineau, 86022 Poitiers, France
Abstract
The structure of macromolecular lipids from an acid anmoor soil was investigated using preparative pyrolysis and
thermochemolysis. The matrix is formed by cross-linked aliphatic chains. Alkanols, triterpenoid alcohols e.g. a-amyrin,
sterols, stanols can be bound to the matrix via ether or ester groups. Fatty acids can be incorporated through esteri®cation. Dicarboxylic acids and other compounds such as hydroxyacids represent alkyl bridges between the polymethylene chains. Various components are trapped in the macromolecular network. The two techniques give
complementary results. Despite higher yields, thermochemolysis does not permit one to distinguish between methyl
ester groups present in the structure and methyl ester groups formed on degradation. # 2000 Elsevier Science Ltd. All
rights reserved.
Keywords: Macromolecular lipids; Pyrolysis; Thermochemolysis; Hydrocarbons; Fatty acids; Alcohols; Aldehydes; Ketones
1. Introduction
Lipids play an important role in soil processes (Stevenson, 1982) because they aect plants or soil micro¯ora
and in¯uence soil physical properties (Jambu et al., 1983).
Previous studies concerning lipids present in soils have
shown that this soluble fraction includes simple lipids
with a plant or a microbial origin but also complex,
macromolecular lipids, corresponding to the polar lipid
fraction when using the McCarthy and Duthie (1962)
procedure for lipid fractionation. This latter fraction has
been studied using chemical degradation methods such
as alkaline hydrolysis under phase catalysis conditions,
ether bond cleavage with boron tribromide or alkaline
potassium permanganate oxidation (AmbleÁs et al.
1991, 1993a). It was shown that soil macromolecular
lipids contains preserved biomacromolecules of plant
or microbial origin (AmbleÁs et al., 1991). The (reversible) incorporation of exogenic organic molecules in
* Corresponding author. Tel.: +33-5-4945-3866; fax: +335-4945-3501.
E-mail address: andre.ambles@campus.univ-poitiers.fr
(A. AmbleÁs).
macromolecular lipid fraction, for instance by ester
groups, has then been clearly demonstrated (AmbleÁs et
al., 1994).
In hydromorphous environments, these macromolecular lipids can partly escape biodegradation
(AmbleÁs et al., 1991) and become part of the sedimentary organic matter. As a consequence, some analogy
between these soil macromolecular lipids and the
organic matter present in ancient sediments may exist
and macromolecular lipids can be compared with a
proto-kerogen in some soils (Shioya and Ishiwatari,
1983; AmbleÁs et al., 1991).
In this paper, the structure of macromolecular lipids
from an acidic anmoor soil was investigated using preparative o-line pyrolysis (Behar and Pelet, 1985; Largeau et al., 1986; Metzger and Largeau, 1994) and a new
preparative thermochemolysis technique which permits
the treatment of more than one gram of product
(Grasset and AmbleÁs, 1998b). In the latter technique, an
alkylating agent is used for the thermally assisted
hydrolysis and alkylation, as developed for analytical
¯ash on-line pyrolysis (del Rio et al., 1996). The soil
samples originated from a peaty soil (Plateau de Millevaches, France) where organic matter is accumulating
and as such the structural study of macromolecular
0146-6380/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0146-6380(00)00009-7
410
V. Gobe et al. / Organic Geochemistry 31 (2000) 409±419
lipids from these soils may give information about early
diagenetic processes.
2. Experimental
2.1. Samples
Samples were collected from a peaty soil (Sphagnum
peat), an acidic anmoor located on the Plateau de Millevaches (North CorreÁze). The macromolecular lipids
(ML) from the surface 0±20 cm horizon were previously
described by AmbleÁs et al. (1991; 1993a). The samples
used for the present work originated from the 30±50 cm
horizon which is waterlogged, organic-rich, with a
higher degree of decomposition of plant debris (saprist)
than the surface horizon (®brist).
2.2. Separation of the macromolecular lipid fraction
The procedure is shown in Fig. 1. Representative soil
samples were dried and sieved to 2 mm. Total lipids
were extracted with chloroform in a Soxhlet (324 h).
The original soil sample contained 56% of total organic
matter (TOM) (determined by combustion). Lipids contributed 5% to the TOM. The total lipid extract was
fractionated on a silica-potassium hydroxide column. In
order to avoid problems of separation between the acid
and the polar fractions in the classical McCarthy±Duthie
procedure (AmbleÁs et al., 1991) on the one hand, and to
release lipids possibly trapped by the macromolecular
network (such as HCT, Fig. 1) (AmbleÁs et al., 1991) on
the other hand, the procedure of separation was slightly
modi®ed: the ``classical'' acid and the polar fractions
were mixed and then methylated using trimethylsilyldiazomethane (Hashimoto et al., 1981). The most
polar fraction (corresponding to the polar fraction of
the McCarthy±Duthie procedure) obtained after liquid
chromatography (MLA) contains macromolecular
lipids. Other macromolecular lipids (MLN) were
obtained by liquid chromatography of the neutral fraction (Fig. 1). The total macromolecular lipid fraction
(ML) was obtained after addition of MLA and MLN. It
gave no elution in thin layer chromatography using
polar eluents. The other fractions (in italics in Fig. 1)
were not studied in this work.
Macromolecular lipids contributed 15% to the total
lipid extract (0.7% of TOM). The elemental composition of the ML is : C 72.39%, H 10.16%, O 16.96% (by
dierence), N 0.27%. The value of the atomic H/C ratio
is 1.68.
2.3. Preparative o-line pyrolysis
The same device as that described by Behar and Pelet
(1985) was used. A quartz tube containing the complex
lipids (60±70 mg) was placed in a stainless steel tube and
put in an oven, at 425 C for 1 h under helium (¯ow rate
30 ml minÿ1). Pyrolysis products were collected in two
successive traps containing solvent cooled to ÿ30 C. After
evaporation of the solvent, the pyrolysate from seven
experiments was separated on a SiO2/KOH column
(McCarthy and Duthie, 1962) in a neutral fraction and an
acid + polar fraction. The later was methylated. Each
fraction was chromatographed on a silica column, the
various components were eluted with diethyl ether/petroleum ether mixtures of increasing polarity (Fig. 1).
Three chromatographic fractions were obtained for
hydrocarbons: HC I:12 mg, HC II:1 mg; HC III:1.5 mg.
2.4. Preparative o-line thermochemolysis
Fig. 1. Procedure of fractionation of total lipid extract and of
pyrolysis product.
ML (349 mg) was placed in a ceramic boat and
moistened with 3 ml of a methanol 50% w/w solution of
tetramethylammonium hydroxide (TMAH) (Aldrich).
The ceramic boat was then transferred into a 603 cm
i.d. Pyrex1 tube. The tube was maintained at 425 C
(oven) for 1 h under helium (¯ow rate 100 ml minÿ1).
The pyrolysate was collected in two successive traps, as
described above. The pyrolysis products were directly
V. Gobe et al. / Organic Geochemistry 31 (2000) 409±419
411
separated by liquid chromatography on SiO2 (diethyl
ether/petroleum ether elution).
2.5. Analyses
The products were analyzed by capillary GC and
GC±MS. GC separations were carried out with a Shimadzu GC-14A gas chromatograph in splitless injection
mode, using a CPSil 8-CB (Chrompack) capillary column
(50 m0.25 mm i.d., 0.12 mm ®lm thickness) and a FID.
The temperature of the column was programed from 50 C
(2 min isothermally) to 200 C (2 min isothermally) at 5 C
minÿ1 then to 300 C (30 min isothermally) at 3 C minÿ1.
The temperature of the injector and the detector was
300 C.
The quantities of each kind of products are given as
apparent concentrations; they were determined on the
basis of liquid chromatography for the various families and on the basis of GC response (peak area) for
a determined family. No correction was made for
derivatization.
GC±MS were performed on a FINNIGAN MATINCOS 500 mass spectrometer coupled with a VARIAN 3400 gas chromatograph. The GC conditions were
the same as for GC analysis. The mass spectrometer
was operated in the electron impact mode (70 eV).
Transfer line and ion source were 290 and 180 C,
respectively. Mass spectra were acquired by scanning
the region m/z 50±600 at a rate of 1 s per decade and
recorded by a Data General 20 computer. The various
products were identi®ed on the basis of their GC retention times and their mass spectra (comparison with
standards). Alcohols were identi®ed as acetates (acetylation with acetic anhydride/pyridine). Acids were
methylated with trimethylsilyldiazomethane (Hashimoto et al., 1981). Fourier transform infra-red (FT-IR)
spectra were obtained in CCl4 using a Nicolet 750 spectrometer. NMR spectra were recorded in CDCl3 solution on a BRUKER AVANCE DPX 300 (300 MHz for
1
H and 75 MHz for 13C). Tetramethylsilane was used as
internal reference.
3. Results and discussion
3.1. Bulk properties
The results of the elemental analysis and the value of
the atomic H/C ratio (see experimental section) indicated a rather aliphatic nature for the macromolecular
lipids of the studied soil. The point was corroborated by
the FT-IR spectrum (Fig. 2) which exhibited strong
bands centered at 2926, 2853, 1464 and 1377 cmÿ1 which
can be attributed to CH3 and/or CH2 groups (GobeÂ,
1998). NMR measurements gave con®rmation of this
predominantly aliphatic nature: 1H NMR spectrum
Fig. 2. FT-IR spectrum of macromolecular lipid fraction (CCl4
solution).
presented bands in the 0.5±1.7 ppm region (Fig. 3a) and
the 13C NMR spectrum (Fig. 3b) showed signals in the
aliphatic 9±35 ppm region. The FT-IR spectrum
revealed also the presence of hydroxyl groups (broad
band centered at 3413 cmÿ1) and broad bands at 1737
cmÿ1 (carbonyl), 1252 (nCsp3-O), 1174, 1132 and 1032
cmÿ1 (nCsp3-O) which can be attributed to ester and
ether groups. In the 1H NMR spectrum (Fig. 3a), signals at 3.6 ppm (H±C±O), 2.3 ppm (H±C±CO) and 1.5
ppm (very broad) (H-C-C-O) can con®rm the presence
of ester groups, as well as the signal at 169.5 ppm
(CO) on the 13C NMR spectrum.
3.2. Pyrolysis and thermochemolysis
ML were investigated using o-line preparative pyrolysis (Behar and Pelet, 1985 ; Largeau et al., 1986) and
o-line thermochemolysis as described by Grasset and
AmbleÁs (1998b). The results are given in Table 1. Seven
elemental o-line pyrolysis on 415 mg of material yielded 98 mg of pyrolysis products corresponding to
23.6% of the original macromolecular lipids. The
remaining residue represented 64.8% of initial ML. The
loss of weight was probably due to volatile compounds.
Preliminary experiments (GobeÂ, 1998) clearly showed
that the pre-pyrolysis used for the study of sedimentary
organic matter (Largeau et al., 1986) is not useful for
the study of soil organic matter, pre-pyrolysis and pyrolysis yielding the same products.
Preparative thermochemolysis aorded degradation
products with a much higher yield (234 mg; 67% of
ML) but TMAH was present among the pyrolysis products. The loss of products was 23 mg (6.6% of the
initial ML).
412
V. Gobe et al. / Organic Geochemistry 31 (2000) 409±419
Fig. 3. 1H NMR (a) and 13C NMR (b) spectra of macromolecular lipids (CDCl3 solution).
The composition of the pyrolysates from both
experiments was determined by liquid chromatography.
The main series of compounds are listed in Table 2.
Polar products corresponded to the remaining material
and/or to more or less transformed macromolecular
lipids. The yield of GC amenable components was much
higher for thermochemolysis (Table 2).
The hydrocarbon fraction formed in pyrolysis (HC I,
Fig. 1) showed the classical series of n-alkane/n-alk-1ene doublets, ranging from C16 to C36 (Fig. 4a). The
same distribution was obtained upon thermochemolysis.
Alkenes can be produced by b-scission of radicals (Larter
and Hors®eld, 1993). The origin of n-alkane/n-alkene
doublets on pyrolysis is diverse. One origin can be the
thermal degradation of esters which is known to produce
chie¯y n-alkanes from an acid moiety and n-alk-1-enes
from the alcohol moiety (Van de Meent et al., 1980).
These esters correspond to preserved protective layers of
higher plants, as suberin, cutin (de Leeuw and Largeau,
1993 ; Macko et al., 1993) from above-ground parts as
well as from roots (Nierop, 1998). The presence of
alkene/alkane doublets in pyrolysates is indicative of the
V. Gobe et al. / Organic Geochemistry 31 (2000) 409±419
413
Table 1
Results (mg) of pyrolysis and thermochemolysis of macromolecular lipids
Pyrolysis
Thermochemolysis
a
b
Mass of
sample
Pyrolysate
Residue
Loss of
weight
415
349
98a
234b
270
922
47
23
Seven elemental pyrolysis.
Presence of TMAH.
Table 2
Components produced by thermal degradation of macromolecular lipids from Plateau de Millevaches (% of pyrolysate)
Pyrolysis Thermochemolysis Range
Hydrocarbons
14.5
Fatty acids
4.1
(methl esters)a
Fatty acid methylesters 6.1
26
C16±C36
C14±C34
19
Aliphatic diacids
(dimethylesters)a
Alcohols (acetates)a
Ketones
Aldehydes
a-Hydroxymethylesters
o-Hydroxymethylesters
(o-1)-Ketomethylesters
(o-1)-Ketoalcohols
Fatty acid octylesters
Polar products
7.2
15
C14±C19;
C20±C32
C11±C28
2.6
1.7
0.7
1
2.1
2.1
1
1.9
55
5
5
0.1
2.1
2.8
ND
ND
ND
25
a
C14±C32
C17±C33
C19±C31
C22±C27
C16±C28
C22±C29
C17±C29
C14±C22
After derivatization.
presence of resistant aliphatic macromolecules (cutan,
suberan...) in soils (Tegelaar et al., 1989b; van Bergen et
al., 1997a; Augris et al., 1998). Various terpenoids, norhopanes, norhopenes, hopadienes, hopenes and hopanes
(derived from bacteriohopanetetrol or related compounds) and a sterene, 24-ethyl 5a(H) cholest-2-ene were
also identi®ed, eluting between nC29 and nC36 alkanes
(Fig. 4a; Table 3). These triterpenoid compounds were
not identi®ed on hydrolysis (AmbleÁs et al., 1991; 1993a)
which indicates a possible ether linkage to the macromolecular matrix on carbon 3 of the A ring of the terpenoid/steroid. Despite a higher quantity of released
hydrocarbons (Table 2), the number of polycyclic structures were less numerous on thermochemolysis. Only 5
components were present 22,29,30-trisnorhop-17 (21)
ene, 17a(H),22,29,30-trisnorhopane, 30-norhop-17(21)
ene, 17b(H), 21 b(H)-30-norhopane, 17a(H),21 a(H)hop-22(29)-ene. The presence of the two isoprenoids
prist-1-ene and prist-2-ene, indicated the occurrence of
chemically-bound phytol (Larter et al., 1979) and/or
tocopherols (Goossens et al., 1984). Among the pyrolysis
Fig. 4. Chromatograms of hydrocarbons obtained by pyrolysis
(a) n-alkane/n-alkene doublets with triterpenoids (HC I fraction).(The vertical numbers correspond to triterpenoids listed
in Table 3) (b) n-alkane distribution of the HC III fraction of
hydrocarbons.
products, two minor chromatographic fractions were
also obtained (Fig. 1). The HC II fraction (1 mg) contained only n-alkanes with abundant C27 and C29 members, superimposed on the Gaussian C21±C37
distribution. The HC III fraction (1.5 mg) contained
dominant even C18, C20, C22 and C24 saturated members
(Fig. 4b). The origin of these even hydrocarbons is very
probably fatty acid or alcohol reduction. These hydrocarbons were obviously trapped in the macromolecular
network and released after alteration of the matrix.
OkomeÂ-Mintsa (1991) reported the presence of such nalkanes in the hydrolysis products of macromolecular
lipids from the same soil. The same phenomenon was
observed after chemical degradation of kerogens from
ancient sediments (Baudet et al., 1991; Halim et al.,
1997).
The alcohol fraction from both experiments presented
the same composition. n-Alkanols were present in the range
C14±C32 with dominant even members and a clear maximum at C22, accompanied with neolupenol [identi®ed in
some roots (Ageta et al., 1981)], a-amyrin, sterols also
V. Gobe et al. / Organic Geochemistry 31 (2000) 409±419
414
Table 3
Triterpenoid hydrocarbons detected in Fig. 4
Number Name
Structure
Number Name
1
17a(H)-22,29,30-trisnorhopane
8
17b(H),21a(H)-hop-22(29)-ene
2
24-ethyl-5a(H)-cholest-2-ene
9
17a(H),21b(H)-hopane
3
C29 17b(H),21a(H)-hopadiene
10
C32 17b(H),21a(H)-hopane
4
C30 17b(H),21a(H)-hopadiene
11
17b(H),21b(H)-homohopane
5
17a(H),21b(H)-30-nor-neohop-13(18)-ene
12
C32 17b(H),21b(H)-hopane
6
17b(H),21b(H)-norhopane
13
C32 17b(H),21b(H)-hopene
7
30-norhop-17(21)-ene
14
C33 17b(H),21b(H)-hopene
mainly of plant origin (i.e. stigmasterol, 24-methyl- and 24ethylcholesterol) and their corresponding stanols. The
sterol/stanol ratio ranged from 0.5 (cholesterol/cholestanol) to 0.9 (24-ethylcholesterol/24-ethylcholestanol). The
sterol distribution with a dominant 24-ethylcholesterol is
the same as that from the simple lipids from the same
soil providing evidence that simple lipids can become
incorporated into a macromolecular lipid fraction
(AmbleÁs et al., 1991). As the same structures (including
stanols) were found after alkaline hydrolysis (AmbleÁs et
al., 1991; 1993a), incorporation was most probably via
ester groups. The presence of stanols is a consequence of
anaerobic conditions in this hydromorphic soil, as in
marine or lacustrine sediments (Gaskell and Eglinton,
1975). Microbial reduction of sterols occurs when the OH
at position 3 is not bound, the attachment to the matrix
occurring later probably via a transesteri®cation process
(de Leeuw and van Bergen, personal communication).
The distribution of n-alkanols formed by thermal
degradation of macromolecular lipids does not resemble
that of alcohols present either in the above-ground parts
of plant in the studied sample or in the soil simple lipid
fraction [highly dominant C28 member (AmbleÁs et al.
1991)]. They can partly be inherited from plant wax
esters (Jambu et al., 1993; AmbleÁs et al., 1993a), or from
highly resistant biopolymer in root material as suberin
(Nierop, 1998), or result from the enzymatic reduction
Structure
of fatty acids (Kolattukudy, 1976). No close correlation
with the distribution of fatty acids can be expected
inasmuch as the occurring processes are biological processes (with discrimination with chain length) and
maybe competitive processes.
The distribution of aliphatic monocarboxylic acids
formed on pyrolysis of macromolecular lipids (Fig. 5a)
showed a strong even over odd carbon number predominance. These acids could originate from plant protective
polyesters (Tegelaar et al., 1989a,b). Unsaturated C15:1,
C16:1, C18:1, C18:2 components, and branched iso- and
anteiso C15, C16 and C17 acids of bacterial origin (Boon
et al., 1977 ; Perry et al., 1979), C31 and C32 1721b(H),
21b(H) hopanoic acids were also present. Fatty acid
methyl esters, corresponding to 6.1% of the pyrolysate
(Table 2) which were also identi®ed amongst the products (Fig. 5b), showed a similar distribution to that of
fatty acids present in simple lipids. Thermochemolysis
aorded only methyl esters arising mainly from the
transesteri®cation of esters (Martin et al., 1995; Gonzalez-Vila et al., 1996). The relative yield of products was
much higher than in pyrolysis. Conversely, thermochemolysis does not permit to distinguish fatty acids
bound by ester linkage from fatty acid methyl esters, if
any, trapped in the macromolecular matrix. The distribution of the fatty acid methyl esters corresponded to
the combined distributions of Fig. 5.
V. Gobe et al. / Organic Geochemistry 31 (2000) 409±419
415
Dicarboxylic acids in pyrolysis or dimethylesters in
thermochemolysis were identi®ed in the C11±C28 range
with an even predominance and a dominance of the C22
member (and an important C16 component). They were
bound as diesters in the macromolecular matrix. Both
experiments exhibited the same distributions.
a- and o-hydroxyacid methyl esters were observed in
both experiments, respectively in the C20±C29 range
(max. C24) (Fig. 6) and C16 to C28 with a maximum at
C22. a- hydroxyacid methyl esters give a prominent m/z
90 ion on mass spectrometry (McLaerty rearrangement) while the o- isomers led to the classical m/z 74.
Although they were produced (as methyl esters, without
derivatization) in pyrolysis with a lower yield, this
method indicated that they were bound to the matrix via
the hydroxyl groups only, the methyl ester group being
pre-existant in the macromolecular structure. Otherwise,
if the COOH groups were esteri®ed to the matrix,
hydroxyacids would have been produced upon pyrolysis. o-Hydroxyacids are present in cuticular waxes,
cutins and suberin from plants (Eglinton et al., 1968;
Ketola et al., 1987) while a-hydroxyacids have a microbial origin, resulting from the oxidation of fatty acids
(Eglinton et al.,1968; Cranwell, 1984). The presence of
a- and o-hydroxyacids in the degradation products of
macromolecular lipids con®rms that this fraction contains partly preserved biopolymers of plant or microbial
origin (AmbleÁs et al., 1991).
Ketones (Fig. 7) were essentially methylketones in the
C13-C29 range accompanied with the isoprenoidal
6,10,14-trimethyl-pentadecan-2-one and steroidal 24ethylcholest-4-en-3-one, 24-methyl- and 24-ethylcholesta-3,5-dien-7-one (or stigmastadienone). The isoprenoid ketone originates probably from the microbial
degradation of phytol, as reported by Brooks and
Maxwell (1974). The origin of methylketones in thermal
degradation products is not well established. Among
soil (simple) lipids, they arise exclusively from oxidation
of plant hydrocarbons or b-oxydation-decarboxylation
of fatty acids (AmbleÁs et al., 1993b) and are predominantly odd-carbon numbered components. The
distribution of Fig. 7 could indicate that these ketones
were trapped in the macromolecular matrix. Methylketones could also have been produced from the scission
of an ether linkage on carbon 2 or the scission of an
alkyl chain on the a position of a hydroxyl group. An
other mechanism involving a radical scission at the b
position of a free hydroxyl group was postulated by
Hartgers et al. (1995). Several mechanisms contribute
probably to the formation of methylketones on thermal
degradation of macromolecular material. At present,
this point remains unclear. Steroidal ketones could also
have been trapped compounds or bound to the network
by an ether bond at the carbon 3 position. The occurrence of ester bond can also be postulated insofar as the
presence of esteri®ed cholesterol, cholestanol, 24-ethylcholesterol and 24-ethylcholestanol in the macromolecular lipids of the surface 0±20 cm horizon was
previously reported (AmbleÁs et al., 1991; 1993a).
Linear C19±C31 aldehydes with maxima at C23 and
C29 were present in minor amount, (Table 2). They
Fig. 6. Distribution of a-hydroxyacids obtained by pyrolysis
(m/z 90 fragmentogram).
Fig. 7. Distribution of methylketones obtained by thermochemolysis (m/z 58 fragmentogram).
Fig. 5. Fatty acids produced upon pyrolysis [m/z 74 fragmentogram of the methyl ester derivatives (i : iso ; a : anteiso)].(a)
Aliphatic monocarboxylic acids (as methyl esters); (b) aliphatic
monocarboxylic acid methyl esters produced on pyrolysis.
416
V. Gobe et al. / Organic Geochemistry 31 (2000) 409±419
could have been formed from chains bearing an OH
group (Gelin et al., 1994; Hartgers et al., 1995) or could
arise from the cleavage of an ether group (Grasset, 1997;
GobeÂ, 1998). Series of (o-1)-ketoacid methyl esters in
the C22±C29 range with a Gaussian distribution centered
on C25, and (o-1)-ketoalcohols in the range C17±C29 with
a regular distribution also centered on C25 were also
produced upon pyrolysis. Several tentative hypotheses
could explain the presence of these products. For
example, (o-1)-ketoalcohols could have been trapped in
the macromolecular matrix, or were covalently bound to
the matrix with one linkage (acting as a monosubstituent of the matrix) or covalently bound with two
linkages (acting as bridges in the matrix). Keto groups
could also partly originate from dehydrogenation of
hydroxyl groups.
Fig. 8. Schematic modes of occurence of soil macromolecular lipids. The rectangle represents the macromolecular matrix. Molecules
in the rectangle represent trapped molecules. Aliphatic bridges are placed outside the rectangle for enhanced legibility.
V. Gobe et al. / Organic Geochemistry 31 (2000) 409±419
Octyl esters of C14, C16 (abundant), C18 (dominant)
and C20 fatty acids were only detected upon pyrolysis.
They were also found in the pyrolysates of other soil
samples and of kerogen from phosphate rocks (unpublished results). They are never found in the products
from thermochemolysis whatever material is degraded.
Their origin is maybe related to a transesteri®cation
process occurring only under the pyrolysis conditions.
4. Conclusions
In this work, a detailed study of the structure of
complex, macromolecular lipids present in soil was
conducted using preparative o-line pyrolysis and preparative thermochemolysis. The two techniques provided evidence that the studied soil organic fraction is
aliphatic in nature (AmbleÁs et al., 1991; 1993a). The
identi®cation of degradation products permits one to
advance a schematic representation of the modes of
occurrence of structural building blocks (Fig. 8). The
matrix of soil macromolecular lipids is formed by crosslinked aliphatic chains, a part of which are partly preserved biopolyesters. Ether and ester groups are prominently involved in the cross-linking of the chains. Some
aliphatic moieties are acting as monosubstituents of the
matrix, re¯ected as aliphatic monocarboxylic acids (or
methyl esters), alcohols, methylketones and aldehydes
upon pyrolysis or thermochemolysis. The components
with two functional groups (aliphatic dicarboxylic acids,
(o-1)-ketoalcohols, etc.) provide evidence of the occurrence of alkyl bridges in the network. Nevertheless,
some of these components are bound to the matrix by
only one linkage releasing a- and o-hydroxyacid methyl
esters and (o-1)-ketoacid methyl esters on thermal
degradation. As in kerogens from ancient sediments
after chemical degradation (Halim et al., 1997), aliphatic
compounds such as some alkanes, fatty acid methyl
esters, possibly methylketones etc. are trapped in the
macromolecular structure and are released when the
structure of the matrix is altered. Direct ``thermoevaporation'' of the trapped compounds could also
occur on pyrolysis and thermochemolysis. The use of
more ecient extraction methods, such as supercritical
¯uid extraction could free the trapped compounds as
evidenced from a study of the released triterpenoids.
Taking into account hydrolysis products (AmbleÁs et al.,
1991; 1993a), it can be established that sterols, stanols,
neolupenol, a-amyrin are bound to the matrix by ester
(and maybe ether) linkages through the 3 carbon atom,
hopanoic acids are bound by ester groups. The loss of
the alcoholic group resulted in the formation of the
corresponding unsaturated compounds. Contrary to
other reported results using ¯ash Curie point pyrolysis
(van Bergen et al., 1997b), the loss of the OH group is
not total, probably as a consequence of a lower tem-
417
perature. Hopanes are present as trapped molecules.
Stigmastadienone, and related compounds could also be
present as trapped molecules. Alternatively, they are
linked to the matrix as etheri®ed 7-ketosterols (hydrolysis did not aord any 7-ketosterol), the stable conjugated dienone being formed on thermal degradation.
It is interesting to note that, as reported for ¯ash online pyrolysis, preparative thermochemolysis in the presence of tetramethylammonium hydroxide is a very useful and powerful technique for the study of natural
macromolecules. Preparative thermochemolysis aords
higher yields of products than conventional preparative
pyrolysis, which is useful for representative structure
determination. Heating in the presence of an alkylating
agent causes additional chemolysis of the resistant aliphatic parts of the matrix, allowing, as an example,
transalkylation of polyesters resistant under classical
pyrolysis conditions. As a consequence, the importance
of structural parts can be largely underestimated when
using the classical method (del Rio et al., 1996). Nevertheless, pyrolysis only permits one to distinguish the
groups present as methyl esters from the acid groups
linked as esters to the matrix. Conversely, the separation
procedure is much more time consuming than for preparative thermochemolysis.
From the results presented in this work, it can be
concluded that there is, on the one hand an analogy
between soil macromolecular lipids and the aliphatic
part of humin from the same soil (Grasset and AmbleÁs,
1998a) and, on the other hand similarities with the
structure of kerogen present in immature ancient sediments (Tegelaar et al., 1989a,b; AmbleÁs et al., 1991,
1993a; AmbleÁs et al., 1996; Kribii et al., 1996).
Acknowledgements
The authors gratefully acknowledge the authorities of
the French ReÂgion Poitou-Charentes for a doctoral grant
(V.GobeÂ.) and C.N.R.S. for ®nancial support. The
authors thank Dr. P.F. van Bergen and an anonymous
reviewer for very helpful comments and suggestions.
Appendix
418
V. Gobe et al. / Organic Geochemistry 31 (2000) 409±419
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