Abstract The polar fraction, isolated from the o-line pyrolysate at 400 C of a Late Tithonian, sulphur-and oxygen-rich, kerogen was examined via Raney Nickel desulphurization and TMAH thermochemolysis. Important information on this kerogen, not accessible via conventional pyrolysis, was thus obtained: (i) its structure is not simply based on alkyl skeletons cross-linked by ether and (poly)sulphide bridges, (ii) TMAH thermochemolysis aorded direct evidence of the survival of proteinaceous moieties in this 140 million years old kerogen and (iii) encapsulation within an aliphatic organic matrix was probably the main pathway reponsible for such a conspicuous preservation, also possibly favoured by the presence of numerous sulphur links. 

A Garcette-Lepecq

A Riboulleau

C Largeau

F Baudin

S Derenne

Th Mongenot

Y Pouet

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Note
Occurrence of proteinaceous moieties in S- and O-rich Late
Tithonian kerogen (Kashpir oil Shales, Russia)
Th. Mongenot a,b,*, A. Riboulleau a,b, A. Garcette-Lepecq a, S. Derenne a,
Y. Pouet a, F. Baudin b, C. Largeau a
aLCBOP, CNRS UMR 7573, ENSCP, 11 rue P. & M. Curie 75005 Paris, France
bStratigraphie PaleÂontologie, CNRS ESA 7073, UPMC, 4 pl. Jussieu 75005 Paris, France
Received 25 May 2000; accepted 18 October 2000
(returned to author for revision 11 July 2000)
Abstract
The polar fraction, isolated from the o-line pyrolysate at 400C of a Late Tithonian, sulphur- and oxygen-rich,
kerogen was examined via Raney Nickel desulphurization and TMAH thermochemolysis. Important information on
this kerogen, not accessible via conventional pyrolysis, was thus obtained: (i) its structure is not simply based on alkyl
skeletons cross-linked by ether and (poly)sulphide bridges, (ii) TMAH thermochemolysis aorded direct evidence of
the survival of proteinaceous moieties in this 140 million years old kerogen and (iii) encapsulation within an aliphatic
organic matrix was probably the main pathway reponsible for such a conspicuous preservation, also possibly favoured
by the presence of numerous sulphur links. # 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Sulphur- and oxygen-rich kerogen; TMAH thermochemolysis; Amino acids; Encapsulation
1. Introduction
The 140 m.y. sulphur- and organic-rich Kashpir oil
shales form a widespread deposit on the Russian plat-
form (total area ca. 90,000 km2). Recent studies
(Riboulleau et al., 2000) on the kerogen of the organic-
richest level (45% TOC) of the Gorodische section,
termed ``ftop'', showed that intermolecular sulphur
incorporation was a major process in its formation.
These studies also pointed to a substantial role for oxi-
dative cross-linking via ether bridges, re¯ected by (i) the
high O/C atomic ratio (ca. 0.1) of ftop kerogen and (ii) a
relatively abundant production of oxygen-containing
products, especially n-alkanones, upon o-line pyrolysis
at 400C. However, as expected for such a heteroatom-
rich kerogen, large amounts of dicult to identify polar
products occur in the o-line pyrolysate. Indeed, the
most polar components (methanol-eluate) account for
ca. 40% of the total pyrolysate, and the bulk, especially
the predominant non-acid components, cannot be iden-
ti®ed by gas chromatography/mass spectrometry (GC±
MS). Polar pyrolysis products contain important infor-
mation which cannot be retrieved through analysis of
less polar ones (e.g. RullkoÈ tter and Michaelis, 1990).
The present study was therefore focused on the metha-
nol-eluted non-acid (MeNA) fraction from the 400C
o-line pyrolysate of ftop. It was submitted to Raney
Nickel desulphurization, additional pyrolysis via con-
ventional Curie Point pyrolysis at 650C and thermo-
chemolysis with tetramethylammonium hydroxide
(TMAH) at 358 and 650C.
2. Experimental
Isolation of ftop kerogen, o-line pyrolysis at 400C for
1 h, pyrolysate separation by column chromatography
into fractions of increasing polarity using n-heptane,
toluene and methanol, separation of the latter into an
0146-6380/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PI I : S0146-6380(00 )00154-6
Organic Geochemistry 32 (2001) 199±203
www.elsevier.nl/locate/orggeochem
* Corresponding author. Tel.: +33-1-4427-6716; fax: +33-
1-4325-7975.
E-mail address: mongenot@ccr.jussieu.fr (Th. Mongenot).
acid and a non-acid fraction by extraction, elemental
analysis, GC±MS analyses and conventional Curie point
pyrolysis-GC±MS (CuPy-GC±MS) were as previously
described (Riboulleau et al., 2000).
Raney Nickel desulphurization, and hydrogenation of
a part of the MeNA, fraction, was carried out according
to Sinninghe DamsteÂ et al. (1988). The desulphurized
mixture was separated by thin layer chromatography
(TLC) on silica gel plates, developed with cyclohexane,
into two subfractions (Rf: 0±0.5 and 0.75±0.9), recov-
ered by CH2Cl2 extraction and analysed by GC±MS.
CuPy-GC-MS and TMAH thermochemolysis (TMAH/
CuPy-GC±MS) of the MeNA fraction were performed
on ca 1.5 mg aliquots to which a solution of 200 ml of
internal standard (n-icosane, 1.7 mg/1600 ml of CH2Cl2)
was added. Thermochemolysis was performed according
to Challinor (1989) by adding 300 ml of a 25% solution
of TMAH in methanol toMeNA aliquots. Identi®cations
were based on elution orders and mass spectra previously
published (e.g. Knicker and Hatcher, 1997; Zang et al.,
2000). An analytical ¯ow chart is given in Fig. 1.
3. Results and discussion
The MeNA fraction accounts for ca. 25% of the total
400C o-line pyrolysate of ftop kerogen. Elemental ana-
lysis of this fraction showed high contents of oxygen and
sulphur (9.7 and 7.2 wt.%, respectively) and a substantial
level of nitrogen (1.5 wt.%). The atomic ratios (O/C:
0.1, S/C: 0.038 and N/C: 0.018) are similar to those of
ftop kerogen (Riboulleau et al., 2000), except for S/C
which substantially decreased (0.068 in ftop). As observed
for other S-rich kerogens (e.g. Mongenot et al., 1999) a
part of the (poly)sul®de bridges are cleaved upon pyrolysis
and the MeNA fraction mostly corresponds to building
blocks of ftop kerogen freed through the partial clea-
vage of such thermally labile links. Accordingly, these
blocks are rapidly released upon pyrolysis and can
retain some potentially labile moieties. The GC trace of
the fraction shows a large hump, re¯ecting a complex
composition and extensive co-elutions, with only two
well resolved peaks (Fig. 2a). The latter were previously
identi®ed as C16 and C18 n-alkanols (Riboulleau et al.,
2000) and only C9-C16 n-alkylphenols and n-alkoxy-
phenols could be further identi®ed in this complex
mixture, via selective detection of characteristic ions.
3.1. Desulfurization with Raney Nickel and hydrogenation
This treatment is commonly used to release and iden-
tify hydrocarbon skeletons in S-bound moieties (e.g.
Sinninghe DamsteÂ et al., 1988). Elemental analysis
showed an almost complete removal of sulphur (>95%)
from the MeNA fraction. C14±C28 n-alkanes, derived
from moieties with linear skeletons were formed
through the cleavage of sulphur links. Nevertheless, (i)
the GC trace still showed a prominent hump (Fig. 2b)
and (ii) hydrocarbons separated by TLC only accounted
for ca. 6% of the desulphurized fraction which is still
dominated (> 90%) by a complex, oxygen-rich and
nitrogen-containing, mixture of chie¯y unidenti®ed
compounds. Indeed, GC/MS only showed the presence,
in addition to the above n-alkanes, of the few alkanols
and phenols already detected in the untreated MeNA
fraction. In contrast, Raney Nickel desulphurization of
the complex MeNA fraction isolated from the o-line
pyrolysate at 400C of a S-rich kerogen of Orbagnoux
(Mongenot et al., 1999) resulted in a sharp decrease in
hump intensity and in a large production of alkanes. In
Fig. 1. Analytical ¯ow chart for structural analysis of the methanol-eluted, non-acid pyrolysis products of ftop kerogen. (*) Previously
analysed (Riboulleau et al., 2000).
200 Th. Mongenot et al. / Organic Geochemistry 32 (2001) 199±203
both cases, the MeNA fraction corresponds to sulphur
bridge-comprising building blocks. Nevertheless, unlike
the Orbagnoux sample, the building blocks of ftop kero-
gen do not simply correspond to alkyl skeletons cross-
linked by such S-bridges since a simple mixture of hydro-
carbons is not obtained following desulphurization.
3.2. Conventional pyrolysis
The trace of the pyrolysate obtained via pyrolysis of
the MeNA fraction (CuPy-GC±MS at 650C) still exhi-
bits a large hump. Moreover, all the pyrolysis products
identi®ed had already been detected via direct CuPy-
GC±MS at 650C of ftop kerogen and/or GC±MS
analysis of the less polar fractions of its o-line pyr-
olysate (Riboulleau et al., 2000). Pyrolysis at 650C
therefore resulted, as expected, in further cracking of
the MeNA fraction but this was not associated with the
release of new series aording further information on its
composition.
3.3. TMAH thermochemolyses
TMAH thermochemolysis is a powerful tool used
to examine the structure of oxygen-containing
macromolecules: (i) ecient cleavage is achieved for
esters and ethers (Challinor, 1989) and peptide bonds
(Knicker and Hatcher, 1997) and (ii) methylation makes
easier GC±MS identi®cation of polar pyrolysis pro-
ducts. TMAH thermochemolysis was used for the study
of various samples, including kerogens (Kralert et al.,
1995), aliphatic biopolymers (McKinney et al., 1996),
algal sapropels (Knicker and Hatcher, 1997), peat
humic acids (Zang et al., 2000) and Recent sediments
(Garcette-Lepecq et al., 2000).
The nature and relative abundances of TMAH ther-
mochemolysis products can be in¯uenced by tempera-
ture (Hatcher and Cliord, 1994). TMAH/CuPy-GC±
MS of the MeNA fraction was performed at 358 and
650C. The traces of the two pyrolysates show a simple
pro®le without any signi®cant hump. The same pyr-
olysis products, methylated fatty acids and amino acids,
are identi®ed in both cases (Figs. 2c and d). However,
comparison with the internal standard indicated impor-
tant quantitative dierences for the amino acids. Thus,
358C is too low a temperature for obtaining an ecient
release and much higher yields are observed at 650C.
Furthermore, no signi®cant amounts of secondary pro-
ducts, from the thermal degradation of amino acids, were
detected even at 650C. Thus, the latter temperature is
Fig. 2. GC-traces of (a) untreatedMeNA and (b)MeNA after Raney Nickel desulphurization and hydrogenation; TIC trace of TMAH/
CuPy/GC±MS of MeNA at (c) 650C and (d) 358C.^, n-Alkanols;&, n-alkanes;&, amino acids and derivatives; *, saturated fatty
acids;*, unsaturated fatty acids; I.S., internal standard, n-icosane; (Cn), carbon number; Gly, glycine; Ala, alanine; Asp, aspartic acid;
Phe, phenylalanine; AA-d98, amino acid derivative with base peak atm/z 98; AA-d116, amino acid derivative with base peak atm/z 116.
Th. Mongenot et al. / Organic Geochemistry 32 (2001) 199±203 201
suitable for the release of amino acid moieties from the
MeNA fraction. Almost no dierence was noted in fatty
acid abundance.
3.3.1. Fatty acids
The fatty acids (C6±C18, maximum at C16) show a
strong even-over-odd predominance and comprise unsa-
turated compounds. Re-examination of the pyrolysate
from conventional CuPy at 650C, via selective detection
of characteristic fragments, con®rmed that fatty acids
are not generated under these conditions from the
MeNA fraction. This production of acids via thermo-
chemolysis at 650C, and even at 358C, illustrates the
high eciency of TMAH for cleaving the ester bonds
that linked such moieties to non-GC-identi®able struc-
tures in this fraction. The distribution and nature of
these acids is consistent with the early incorporation,
and very ecient protection against subsequent degra-
dation, of weakly altered acids of microalgal and/or
cyanobacterial origin in the macromolecular structure
of ftop kerogen
3.3.2. Amino acids
Several amino acids were identi®ed in the TMAH
pyrolysates of the MeNA fraction (Fig. 2c and d). An
amino acid derivative (base peak: m/z 98) possibly rela-
ted to lysine or phenylalanine (Knicker and Hatcher,
1997; Zang et al., 2000) and an a-amino acid (base peak:
m/z 116) were also observed. Parallel control experiments
did not show any signi®cant amount of such compounds.
Diketopiperazines have been observed previously in
conventional pyrolysates of various proteinaceous
materials. However, it seems that such a production is
restricted to proline-containing materials and do not
occur in the presence of TMAH (e.g. Zang et al., 2000).
Amide-N in proteinaceous structures is generally
considered as highly sensitive to diagenetic degradation.
Nevertheless, recent studies using solid state 15N NMR
showed that the bulk of the refractory nitrogen in algal
compost (Knicker et al., 1996), algal sapropels of Man-
grove Lake sediments (Knicker and Hatcher, 1997),
degraded biomass of Botryococcus braunii deposited
under highly oxic conditions (Derenne et al., 1998),
Everglades peat (Zang et al., 2000) and Black Sea sur-
face sediments (Garcette-Lepecq et al., 2000) corresponds
to amide groups. Moreover, TMAH thermochemolysis
demonstrated that this amide-N is mostly proteinaceous
in the 4000 years old sapropel of the Mangrove Lake
(Knicker and Hatcher, 1997), in the Everglades peat
(Zang et al., 2000) and in the Black Sea surface sedi-
ments (Garcette-Lepecq et al., 2000). Phytoplankton
decay in laboratory incubations (Nguyen and Harvey,
1998) also showed that proteinaceous material can sur-
vive early diagenesis. All these observations were related
to recent materials. In contrast, the present study deals
with a ca. 140 million years old kerogen and indicates
that protein-derived moieties also survived in this
ancient material. To the best of our knowledge, this is
the ®rst time that TMAH thermochemolysis has pro-
vided evidence of the survival of proteinaceous material
over such a time scale.
The cause of the recalcitrance of proteins, or derived
moieties, in organic-rich materials is still a matter of
debate. Three processes have been considered: Firstly,
studies on marine organic matter suggest that some
speci®c proteins exhibit a high resistance to degradation
and are selectively preserved (Tanoue et al., 1996). Sec-
ondly, refractory, amide-containing materials could be
formed via the degradation±recondensation pathway of
Tissot and Welte (1984): that is formation of highly
resistant geomacromolecules via abiotic random con-
densation of partly degraded proteins, polysaccharides
and lipids. Thirdly, encapsulation of proteins, and
derived moieties, into intrinsically refractory organic
substances was recently shown to be an important
pathway (Knicker and Hatcher, 1997; Zang et al., 2000)
which can provide eective physical protection, within
three-dimensional structures, to potentially labile com-
pounds. The protective matrix would correspond to ali-
phatic biopolymers for the Mangrove Lake sapropel
(Knicker and Hatcher, 1997) and humic substances for
the Everglades peat (Zang et al., 2000). In the latter case,
direct evidence supporting this pathway was obtained via
simulated encapsulation of proteins in humic acids.
The MeNA fraction corresponds to building blocks
of ftop kerogen freed via partial cleavage of sulphide
bridges. The nature of its thermochemolysis products
points to an important role for the encapsulation path-
way in the survival of proteinaceous moieties in this
ancient material. Indeed, the presence of aliphatic
chains in the organic matrix is probably a major feature
for the ecient protection of the encapsulated molecules
(Knicker and Hatcher, 1997; Zang et al., 2000). Thus,
the production of both fatty acids and amino acids from
the MeNA fraction strongly supports the encapsulation
process. In addition, Nguyen and Harvey (1998) con-
sidered that protection may also be aorded by encap-
sulation into S-rich macromolecules. Accordingly, for
ftop, the eciency of the encapsulation might be
increased by the presence of sulphur links, hence the
conspicuous survival, on a geological time scale,
observed for proteinaceous moieties in this ancient
material.
Acknowledgements
We thank Dr. P. Hatcher for review and constructive
comments and ``SocieÂ teÂ de Secours des Amis des Sci-
ences'' for ®nancial support to T.M.
Associate EditorÐA.G. Douglas
202 Th. Mongenot et al. / Organic Geochemistry 32 (2001) 199±203
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Occurrence of proteinaceous moieties in S-and O-rich Late Tithonian kerogen (Kashpir oil Shales, Russia)

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Occurrence of proteinaceous moieties in S-and O-rich Late Tithonian kerogen (Kashpir oil Shales, Russia)

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