Origin and formation pathways of kerogen-like
organic matter in recent sediments o the Danube delta
(northwestern Black Sea)
Anouk Garcette-Lepecq
a,b,*, Sylvie Derenne
a, Claude Largeau
a,
Ioanna Bouloubassi
b, Alain Saliot
baLaboratoire de Chimie Bioorganique et Organique Physique, UMR CNRS 7573, E.N.S.C.P., 11, rue P. et M. Curie,
75231 Paris Cedex 05, France
bLaboratoire de Physique et Chimie Marines, ESA CNRS 7077, Universite P. et M. Curie, 4 place Jussieu, 75252
Paris cedex 05, France
Abstract
The chemical structure, source(s), and formation pathway(s) of kerogen-like organic matter (KL) were investigated in recent sediments from the northwestern Black Sea, o the Danube delta. Three sections from a sediment core col-lected at the mouth of the Sulina branch of the delta, under an oxic water column, were examined: S0(0±0.5 cm bsf),
S10(10±13 cm bsf), and S20(20±25 cm bsf). The bulk geochemical features of these sediments (total organic carbon,
organic C/N atomic ratio,d13C
org) were determined. Thereafter, KL was isolated from the samples, as the insoluble
residue obtained after HF/HCl treatment. KL chemical composition was investigated via spectroscopic (FTIR, solid state13C and15N NMR) and pyrolytic (Curie point pyrolysis±gas chromatography±mass spectrometry) methods, and
the morphological features were examined by scanning and transmission electron microscopy. Similar morphological features and chemical composition were observed for the three KLs and they suggested that the selective preservation of land-plant derived material as well as of resistant aliphatic biomacromolecules (probably derived from cell walls of freshwater microalgae) was the main process involved in KL formation. Besides, some melanoidin-type macro-molecules (formed via the degradation-recondensation of products mainly derived from proteinaceous material) and/or some encapsulated proteins also contributed to the KL chemical structure. #2000 Elsevier Science Ltd. All rights reserved.
Keywords:Kerogen-like organic matter; Recent sediments; Danube delta; Northwestern Black Sea; Preservation mechanisms; Selec-tive preservation pathway; Degradation-recondensation
1. Introduction
A wide variety of organic and inorganic substances are transported from the continent to coastal marine environments via river discharge. The fate of riverine organic material in coastal areas largely depends on the physico±chemical partition between particulate and dis-solved phases, and how this distribution is modi®ed as freshwater mixes with saline water (Stumm and
Mor-gan, 1970; Olsen et al., 1982). The rapid ¯occulation of terrestrially derived humates in seawater has been demonstrated (Narkis and Rebhun, 1975; Sholkovitz, 1976; Sholkovitz et al., 1978), and a large part of both riverborne particulate organic matter (POM) and ¯oc-culated colloids is expected to be exported from the water column to the sediment underlying estuarine sys-tems.
The Danube delta is, after the Volga, the second lar-gest delta in Europe, and 23rd in the world. It lies at the mouth of a river extending over a distance of 2860 km (exclusive of the delta itself) and drains an area of 805,300 km2. Within the delta, two major bifurcations
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* Corresponding author at ®rst address. Fax: +33-1-43-25-79-75.
from rivers, and from sources such as fertilisers from agriculture, and industrial and municipal wastewaters along the shoreline (especially phosphate), is the main cause for the increased eutrophication observed in the semi-enclosed Black Sea basin during the last decades (Mee, 1992). Indeed, due to a strong thermohaline stra-ti®cation and the long residence time of water masses, the Black Sea ecosystem is highly sensitive to increased production of OM (Cociasu et al., 1996). In addition, the mainly wind-driven water circulation along the Romanian coast and the Coriolis force lead to a long residence time of the riverine waters on the Romanian shelf and the resulting negative eects on the coastal ecosystem have been observed (Mee, 1992). Distinct increases in phytoplankton cell densities, as well as the frequency of algal blooms, were reported. There was a more than double decrease of benthic biomass in the Romanian shelf area due to suboxia in the bottom water after the sedimentation of algal blooms (Cociasu et al., 1996).
The present study was designed to investigate the insoluble, HF/HCl resistant, OM isolated from three sections of a recent sediment core collected o the Sulina branch of the Danube delta. This organic frac-tion corresponds to the ``kerogen'' de®ned by Durand and Nicaise (1980) as the fraction of sedimentary OM isolated via organic solvent extraction and HF/HCl treatment, and is therefore referenced as ``kerogen-like'' material (KL) in this paper. KL is characterised by a macromolecular structure with a high resistance to bac-terial, chemical, and diagenetic alterations. Only a few studies about KL have been reported so far, whilst such a fraction can account for a substantial part of total OM in marine sediments (e.g. Eglinton et al., 1994; Zegouagh et al., 1999). The aim of the present work is to derive information on the mechanism(s) and sour-ce(s) involved in KL formation and preservation in these Black Sea sediments. To this end, the morpho-logical and chemical features of KLs were examined via a combination of electron microscopy observations and spectroscopic and pyrolytic methods, so as to deconvoluate autochthonous versus allochthonous sources contributing to KLs in such a deltaic environ-ment.
frozen until analysis. The sections correspond to inter-vals 0±0.5 cm bsf (S0sample, oxic level), 10±13 cm bsf
(S10sample), and 20±25 cm bsf (S20sample). The
sedi-ments were freeze dried and ground before further treatment.
2.2. Total organic carbon (TOC)
TOC contents were determined after decarbonatation by high temperature catalytic oxidation (HTCO) using a Shimadzu Solid Sample Module SSM 5000A coupled to a Shimadzu TOC 5000 analyser.
2.3. KL isolation
About 10 g of dry sediment were treated to isolate the ``kerogen-like'' fraction. Lipids were ®rst removed by extraction with CH2Cl2/CH3OH, 2/1, v/v (stirring
over-night at room temperature). The bulk of the mineral constituents of the lipid-free samples was then elimi-nated via a classical HF/HCl treatment (Durand and Nicaise, 1980). The organic residue was submitted to extraction with CH2Cl2/CH3OH, 2/1, v/v (stirring
overnight at room temperature), and ®nally the inso-luble organic material was recovered by centrifugation and dried under a nitrogen ¯ow. It is important to note
that HF/HCl treatment, when applied to recent materi-als, results not only in the elimination of most minerals but also in the removal of some insoluble, macro-molecular, organic constituents (e.g. labile protein- and polysaccharide-derived moieties) due to acid hydrolysis (Durand and Nicaise, 1980). However, some acid-labile organic constituents could be retained due to incorpora-tion within the macromolecular network of the KLs.
2.4. Elemental analysis
Elemental composition of the three KLs obtained after the above treatments was determined by the ``Ser-vice Central de Microanalyse'', CNRS, Vernaison.
2.5. Scanning (SEM) and transmission (TEM) electron microscopy
KL samples were ®xed by 1% glutaraldehyde in cacodylate buer (pH 7.4), post-®xed in 1% OsO4and
embedded in Araldite. Ultrathin sections were stained with uranyl acetate and lead citrate, and TEM observa-tions were performed on a Philips 400 microscope.
For SEM, the ®xed samples were dehydrated using the CO2 critical point technique and coated with gold,
as previously described by Largeau et al. (1990), prior to observations on a Jeol 840 microscope.
2.6. Fourier transform infra red spectroscopy (FTIR)
FTIR spectra were recorded on a Bruker IFS 48 spectrometer using pellets prepared by grinding 1 wt.% of KL with KBr.
2.7. Solid-state13C and15N nuclear magnetic resonance spectroscopy (13C and15N NMR)
Solid-state13C NMR spectra were obtained at 100.62
MHz for13C on a Bruker MSL400 spectrometer with a
recycle time of 5 s. High power decoupling, cross polar-isation (contact time 1 ms) and magic angle spinning at 4.5 kHz, were used. The spectra were the results of ca. 8000 scans. Since all spectra recorded at 4.5 kHz are similar, only one sample (S20) was analysed at a higher
spin frequency (15 kHz), on a Bruker MSL400 with the same parameters, to remove spinning side bands which make interpretation of some resonances dicult in the spectra at 4.5 kHz.
A Bruker DMX 400 operating at 40.56 MHz was used for acquiring the solid-state CP/MAS 15N NMR
spectrum of one of the three samples (S20), applying a
contact time of 1 ms and a recycle time of 150 ms. Ca. 2 million scans were accumulated at a magic angle spin-ning speed of 5.5 kHz. The chemical shift is referenced to the nitromethane scale (= 0 ppm) and was adjusted with15N labeled glycine (
ÿ347.6 ppm).
2.8. Curie point pyrolysis±gas chromatography±mass spectrometry (CuPy±GC/MS)
KL aliquots (ca. 3 mg) were loaded in tubular ferro-magnetic wires with a Curie temperature of 650C and pyrolysis was performed by inductive heating of the sample-bearing wires to their Curie temperature in 0.15 s (10 s hold time). A Curie point high frequency gen-erator was used to produce the magnetic ®eld and the pyrolysis unit (Fisher 0316M) was directly coupled to GC±MS (a Hewlett Packard HP-5890 gas chromato-graph and a Hewlett Packard HP-5889A mass spectro-meter, electron energy 70 eV, ion source temperature 250C, scanning from 40 to 650 a.m.u., 0.7 scan/s). Separation of the products was achieved by a 30 m fused silica capillary column coated with chemically-bound Restek RTX-5MS (0.25 mm i.d., ®lm thickness 0.50mm). Helium was used as carrier gas. The tempera-ture of the GC oven was programmed from 50 to 300C at a rate of 4C/min, after a ®rst stage at 50C for 10 min to allow a better separation of the most volatile pyrolysis products. The wires were weighed after pyr-olysis, and the weight loss calculated. Compounds were identi®ed based on their mass spectra, GC retention times, comparisons with library mass spectra, and with published mass spectra (Danielson and Rogers, 1978; van de Meent et al., 1980a,b; van der Kaaden et al., 1983; Saiz-Jimenez and de Leeuw, 1984; Tsuge and Matsubara, 1985; Ralph and Hat®eld, 1991).
3. Results and discussion
3.1. Bulk geochemical features of the untreated sediments
TOC contents, C/N atomic ratios andd13C
orgvalues
of the three sediment samples are reported in Table 1. TOC content varies between 1.34 and 1.61%. Pre-vious studies on marine surface sediments revealed a wide range of OM contents. Most surface sediments from deltaic or estuarine systems exhibit TOC values around 0.1±1% in temperate, subtropical and equatorial climates (e.g. van Vleet et al., 1984; Kennicutt II et al., 1987; Bigot et al., 1989; Bouloubassi and Saliot, 1993), although higher values (1±5% range) were recorded for the Rhone delta (Bouloubassi et al., 1997), the Ebro delta (Grimalt and AlbaigeÂs, 1990), the Mackenzie River delta (Yunker et al., 1993) and the Lena River delta (Zegouagh et al., 1996). Intermediate TOC con-tents are, therefore, observed in the present study.
The stable carbon isotopic composition (d13C org) of
OM produced by photosynthetic organisms principally re¯ects the dynamics of carbon assimilation and the isotopic composition of the inorganic carbon source (e.g. Hayes, 1993). The dierence in d13C
cessfully been used to trace the sources and distribution of OM in coastal ocean sediments (e.g. Prahl et al., 1994). Average values of ÿ24 to ÿ23% have been reported for Black Sea suspended particles dominated by marine phytoplankton (Calvert and Fontugne, 1987; Fry et al., 1991; Arthur et al., 1994), whereas the ter-restrial end-member has ad13C
org value ofÿ27 %, on
average (Arthur and Dean, 1998). In contrast to marine algae, freshwater algae have d13C
org values typically
indistinguishable from those of OM from vascular land plants, i.e. betweenÿ30 andÿ25%(e.g. Benson et al., 1991; Tenzer et al., 1997). The stable carbon isotopic ratios of bulk OM in the three studied sediments, from ÿ26.3 toÿ25.9%, point to a strong contribution from land plants and/or freshwater algae. Indeed, surface particles collected near the rivermouth during the same period exhibitedd13C
orgvalues identical to those found
in the present study (Saliot et al., in press).
Organic carbon to nitrogen atomic ratios (C/N) also have been widely used to distinguish between algal and land-plant sources of sedimentary OM (Premuzic et al., 1982; Ishiwatari and Uzaki, 1987; Meyers, 1994; Prahl et al., 1994; Silliman et al., 1996). Algae typically have atomic C/N ratios between 4 and 10, whereas vascular land plants have C/N ratios greater than 20 (Meyers, 1997). This dierence arises from the protein richness of algal OM and is largely preserved in sedimentary OM (Jasper and Gagosian, 1990; Meyers, 1994). Riverine and estuarine sediments typically contain a large con-tribution of OM derived from land plants and thus show elevated atomic C/N ratios, in the 13±35 range (Kennicutt II et al., 1987). Interestingly, our samples show relatively low C/N ratios with values in the 9±10 range, that characterise OM of mainly algal origin. Diagenetic modi®cations cannot explain these low values since early diagenesis rather results in an increase of C/N ratios due to the preferential loss of labile N-bearing components. Lowering of C/N ratios also has been observed in soils (e.g. Sollins et al., 1984) and in ocean sediments (e.g. MuÈller, 1977), where it re¯ects the absorption of ammonia derived from OM decomposition, accompanied by remineralisation and release of carbon dioxide (Meyers, 1997; Bouloubassi et al., 1998). In addition, the C/N ratios of OM in ®ne-sized sediments
nents derived from freshwater algae in the studied area (Ragueneau et al., in press; Saliot et al., in press), as well as the good preservation of protein-derived material, which could explain the unusually low C/N ratio asso-ciated to a lowd13C
orgvalue.
Consequently atomic C/N ratios and d13C
org values
found in the sediment core indicate an admixture of terrigenous and freshwater materials as the main sour-ces (Meyers, 1997).
3.2. Geochemical features and relative abundance of the three KLs
The amounts of organic carbon remaining after the HF/HCl treatment (as wt.% of the total organic carbon in the raw sediments), along with the elemental compo-sitions of KLs in the three samples are reported in Tables 2 and 3, respectively.
As expected, the HF/HCl treatment resulted in the release of large amounts of organic carbon. However,
Table 2
KL abundance (as wt.% of TOC in the raw sediments)
Sample S0 S10 S20
KL abundance 50.4 54.3 55.8
Table 3
Elemental compositiona (wt.%) and atomic ratios of the iso-lated KLs
Sample C H N Stotb Fe Ash H/C N/C Sorg/Cc
S0 40.2 3.4 2.5 3.9 2.6 29.4 1.01 0.05 0.01 S10 36.7 3.3 2.7 6.2 3.5 24.3 1.08 0.06 0.02 S20 32.2 2.8 2.1 4.8 3.0 36.3 1.04 0.06 0.02
a Oxygen analysis was not performed because the ash con-tent was too high.
b Stot=Sorg+Spyrite.
the resistant carbon retained after this treatment accounts for ca. 50 wt.% of the total initial carbon for the three samples (Table 2). The slightly more pro-nounced decrease observed for the surface sample S0is
in agreement with the presence of intact (or slightly modi®ed) biomacromolecules such as proteins and polysaccharides, which are acid labile. Indeed, the latter compounds are known to be partly retained in surface samples and to be rapidly degraded with depth in the upper layers of sediments (Hedges and Oades, 1997).
The organic sulphur to carbon atomic ratio is rela-tively low (Sorg/C0.02), indicating a low contribution
of organic sulphur compounds (OSC) in the three KLs. The hydrogen to carbon atomic ratio (H/C 1.04) indicates a substantial contribution of aromatic and/or unsaturated structures. Elemental analysis also revealed relatively high levels of nitrogen (ca. 2.5%) and iron (ca. 3%) in the three KLs. In a H/C ratio vs. N/C ratio dia-gram, the KL representative point falls close to OM in soils and terrestrial humic acids, thus pointing to a mainly continental origin.
X-ray analyses revealed that the residual inorganic fraction chie¯y corresponds to ¯uorides, neo-formed during the HF/HCl treatment and not eliminated after-wards, which account for the substantial ash content of the KLs (ca. 30%).
3.3. KL morphological features
Observations of the three KLs by SEM (Fig. 2) showed that they are mainly composed of shapeless aggregates of various sizes ranging from ca. 810 up to ca. 3040mm (Fig. 2a and b). Most of these aggregates have a granular appearance (Fig. 2b and d), while a few of them exhibit a ``gel-like'' appearance (Fig. 2c). A number of pollen grains and/or land-plant spores (Fig. 2e and f), and of ligneous debris (Fig. 2g) were recog-nised, pointing to some contribution of land plants. A few framboids of pyrite, a mineral known to resist to HF/HCl attack, were also observed (Fig. 2h).
TEM observations (Fig. 3) revealed that the three KLs are largely composed of various types of lamellar structures with dierent thicknesses. The thinner ones (ca. 20±40 nm thick; Fig. 3a) represent a relatively minor fraction and are tightly associated into bundles. Similar lamellar structures, termed ultralaminae, have been recently observed by TEM in a number of kero-gens from source rocks and oil shales (Largeau et al., 1990), so far considered as amorphous based on light microscopy observations. Ultralaminae have been shown to derive from the selective preservation of the thin outer walls of some microalgae (Largeau et al., 1990; Derenne et al., 1991, 1992a,b; Gillaizeau et al., 1996), which are composed of highly resistant bioma-cromolecules termed algaenans (Tegelaar et al., 1989). Thicker structures (ca. 50±500 nm thick) resembling
preserved cell walls of dierent shapes and thicknesses predominate in the three KLs (Fig. 3b±f). Based on their abundance these various lamellar structures likely cor-respond to the shapeless aggregates observed by SEM. Ligneous debris (Fig. 3g), pollen grains and/or land-plant spores (Fig. 3h), as well as a nanoscopically amorphous fraction (Fig. 3e) were also observed by TEM.
The combination of SEM and TEM observations thus allowed to conclude that the selective preservation of resistant cell walls (probably of microalgal origin) and of terrestrial material played an important role in the formation of the kerogen-like material of the three samples.
3.4. Spectroscopic studies
Because the three samples exhibit similar spectro-scopic features, only the spectra of S20 are discussed
here in detail.
The FTIR spectrum of S20 (Fig. 4) is characterised
by an intense absorption centred at 3400 cmÿ1 (wide
band in the 3600±3000 cmÿ1 range) corresponding to
OH and/or NH groups. Carbonyl and/or carboxyl functions are responsible for the relatively weaker absorption at 1710 cmÿ1. An intense band in the 1685±
1585 cmÿ1 range comprises two contributions: the
stronger one, centred at 1655 cmÿ1, corresponds to
car-bonyl groups in primary amide functions, while ole®nic and aromatic unsaturations lead to a wide absorption occurring as a shoulder of the former band. The occurrence of aromatic groups is also suggested by absorptions at 790 and 750 cmÿ1, although the bands
in the aromatic C±H out-of-plane deformation region (900-700 cmÿ1) are overlapped by relatively strong
absorptions caused by residual minerals at wavelengths below 800 cmÿ1. A weak absorption centred at 1540
cmÿ1is assigned to amide N±H bonds. Several
absorp-tions are observed between 1000 and 1200 cmÿ1which
can be due to C±O bonds (ethers and alcohols) and/or minerals. A wide band in the 1300±1200 cmÿ1 range
(maximum at 1230 cmÿ1) is also noted. The latter may
correspond to C±N bonds in aromatic amine functions. Relatively weak absorptions in the 3000±2800 cmÿ1
range re¯ect the contribution of alkyl groups. The intensity ratio of the 1450 cmÿ1 (CH
3and CH2
asym-metric bending) and 1380 cmÿ1(CH
3symmetric
bend-ing) absorptions shows that CH3 groups are relatively
abundant, pointing to a high degree of branching. A very weak absorption at 420 cmÿ1re¯ects the presence
of a low amount of pyrite, in agreement with SEM observations.
The solid-state 13C NMR spectrum of S
20 KL (15
wide indicating a substantial level of branching. The latter feature, which is in agreement with FTIR data, is also indicated by the occurrence of a shoulder at 15 ppm due to CH3groups. Several peaks re¯ect the occurrence
of heteroelements in the KLs. The peak at 74 ppm cor-responds to carbons bearing O or N atoms, including carbons from carbohydrates. The presence of the latter compounds is con®rmed by a weak signal at 105 ppm due to anomeric carbons (acetal groups). Carbonsato those occurring at 74 ppm are responsible for the peak at 55 ppm. However, the latter peak can also include carbons in methoxyl groups and/or carbons bearing the amino group in amino acids. The broad resonance centred at 130 ppm with a high relative intensity
corre-sponds to aromatic and ole®nic carbons. A weak signal at 150 ppm is assigned to phenols. A narrow peak centred at 175 ppm corresponds to ester and/or amide functions.
The solid-state15N NMR spectrum of S
20KL (Fig. 6)
is dominated by a peak atÿ258 ppm assigned to amides. The shoulder betweenÿ200 andÿ230 ppm indicates the occurrence of both N-alkyl-substituted pyrroles: indoles or carbazoles (ÿ200 ppm), and unsubstituted pyrroles (ÿ230 ppm) (Derenne et al., 1993). A weak signal at ÿ344 ppm may be due to amine functions.
The combination of FTIR and solid-state 13C and 15N NMR data showed that oxygenated and
unsaturations, are predominant features for the three KLs. These bulk chemical observations were completed by pyrolytic studies to derive further information about the molecular structure and origin(s) of KLs.
3.5. Pyrolytic studies
CuPy-GC/MS of the KLs isolated from samples S0,
S10, and S20resulted in a substantial weight loss (ca. 20
wt.%). The ¯ash pyrolysates generated from the three KLs are highly complex and show similar compositions. Their total ion current (TIC) traces (Fig. 7) are char-acterised by (i) an abundant presence of phenols and methoxyphenols, (ii) the presence of a complex mixture of alkylbenzenes, naphthalenes, indenes, indoles, pyr-roles, pyridines and aromatic nitriles, with a low degree of alkylation (44 carbons) for the alkylated homo-logues of each family, (iii) a substantial contribution of normal alkanes and alk-1-enes, (iv) a virtual lack of sulphur compounds, (v) a low relative abundance of C6±
C18saturated fatty acids. The distributions of these
dif-ferent types of pyrolysis products are described in the following paragraphs and their origin and signi®cance are discussed.
3.5.1. n-Alkanes/n-alk-1-enes
The mass chromatogram ofm/z57 (Fig. 8) shows an homologous series ofn-alkanes ranging from C9to C33
(maximum C13), with no signi®cant odd- or
even-car-bon-number dominance. A series of n-alk-1-enes with
similar features was also observed.n-Alk-1-ene/n-alkane doublets are abundant in the pyrolysates of the highly aliphatic, resistant, biopolymers present in some higher plant tissues such as cutans (in cuticles), or suberans (in periderm tissues), as well as in tegmens (inner seed coats) (Nip et al., 1986a, b; van Bergen et al., 1993, 1994). Owing to the presence of such highly resistant components, these tissues and inner seed coats can retain their morphological features upon fossilisation. TEM observations on the three KLs revealed the abun-dant presence of relatively thick ``lamellar'' structures with dierent types of organisation (Fig. 3). A part of these structures might be related to some of the above resistant materials and correspond to selectively pre-served structures derived from higher plants. In addi-tion, SEM observations showed the presence of some plant spores and/or pollen grains (Fig. 2e and f). It is well documented that pollen and spore walls are com-posed of highly resistant materials, the so-called spor-opollenins, which comprise aliphatic moieties and are also selectively preserved in the fossil record. Accord-ingly, a signi®cant part of then-alkanes andn-alk-1-enes observed in KL pyrolysates can be derived from land-plant materials.
However, straight-chain hydrocarbons have also been described in the pyrolysates of lacustrine and marine materials (Fukushima and Ishiwatari, 1988; Fukushima et al., 1989). These hydrocarbons are likely derived from the algaenans which occur in the outer walls of numer-ous freshwater and marine microalgae (Largeau et al., 1984, 1986; Goth et al., 1988; Derenne et al., 1992b, 1995; de Leeuw et al., 1995; Gelin et al., 1999). The chemical structure of a number of algaenans was shown to be based on a network of long polymethylenic chains (Largeau et al., 1986; Derenne et al., 1991, 1992 a,b; Fig. 5. Solid-state13C NMR spectrum of S20KL (at 15 kHz).
Fig. 4. Fourier transform infrared spectrum of S20KL (similar spectroscopic features were observed for the two other KLs).
Gelin et al., 1996, 1997), which generate large amounts of n-alkanes and n-alk-1-enes (up to C35) upon
pyr-olysis. Moreover, owing to their extremely high resis-tance to chemical and microbial degradation (Tegelaar et al., 1989) such macromolecular constituents can sur-vive almost unaected during diagenesis. However, depending upon the thickness of the wall the initial morphology of the cell may be retained upon fossilisa-tion, which leads to microfossils as in Torbanite (Lar-geau et al., 1984, 1986) or in Messel Oil Shale (Goth et al., 1988), or may be partly altered as in ultralaminae (Derenne et al., 1991, 1992a). Such lamellar structures were observed by TEM in the KLs (Fig. 3) and they are probably derived from algaenan-composed thin outer walls of microalgae. However, the lack of predominance of the n-alk-1-ene/n-alkane doublets in the KL pyr-olysates points to a moderate contribution of selectively preserved, resistant, aliphatic macromolecules. The lat-ter are probably derived both from lat-terrestrial plants and from freshwater microalgae (in agreement with the
d13C
organd C/N values).
3.5.2. Isoprenoid hydrocarbons
Prist-1-ene and trace amounts of prist-2-ene were identi®ed in KL pyrolysates (Fig. 7 and 8). Several ori-gins were previously put forward for these ubiquitous
pyrolysis products. The phytyl side chain of chlorophyll a was proposed as a precursor of pristene upon pyr-olysis (Ishiwatari et al., 1991, 1993). However, Goossens et al. (1984) demonstrated that the pyrolysis of toco-pheryl moieties produces pristene through an intramo-lecular rearrangement, in much higher yields than chlorophylla. Tocopherols are known to be relatively abundant in the chloroplasts of most photosynthetic microorganisms. Prist-1-ene and prist-2-ene in pyr-olysates of fossil leaf material (Nip et al., 1986a; Logan et al., 1993) and tegmens of fossil water plants (van Bergen et al., 1994) were considered as being derived from tocopherol. The absence of phytadienes, which have been shown also to originate from chlorophyll-bound phytol (van de Meent et al., 1980a,b; Ishiwatari et al., 1991), suggests that chlorophyllawas probably not the precursor of pristenes in KL pyrolysates. Indeed the phytyl chain is esteri®ed to the cyclic tetrapyrrole moiety of chlorophylla, which renders it less stable than tocopheryl units upon hydrolysis during HF/HCl treatment.
3.5.3. Organic sulphur compounds (OSC)
The three KLs are characterised by a very low Sorg/C
Damste et al., 1986, 1989; Schmid et al., 1987; Adam et al., 1993) only played a negligible role in their forma-tion. The presence of pyrite (Fig. 2h) shows that some bacterial sulphate-reducing activity took place in the sediment. Nevertheless, this activity was probably low, and there was enough active iron to scavenge the reduced sulphur species.
3.5.4. Phenols
The KL pyrolysates are characterised by large amounts of phenolic compounds, including a large number of methoxyphenols (Fig. 7 and Table 4). Phenol and its alkylated homologues were identi®ed by selective ion detection (m/z=94+107+108+121+122; Fig. 9). This series is dominated by phenol, 3- and 4-methyl-phenols, and 4-ethylphenol, which also correspond to relatively important peaks in the TIC traces (Fig. 7). The abundance of the alkylated isomers decreases with the number of alkyl carbons, the amount of C3+
homologues being quasi negligible.
Alkylphenols, associated to short-chain (C1±C3)
methoxy-substituted alkylphenols are typical pyrolysis products of unaltered or weakly altered lignin (e.g. Martin et al., 1979; Obst, 1983; Saiz-Jimenez and de Leeuw, 1986a; Hatcher and Spiker, 1988), thus re¯ect-ing a terrigenous input. Catechols (1,2-benzenediols) are often assigned to demethylated units formed from
microbial degradation of lignin (Hatcher et al., 1988; Stout et al., 1988). Therefore, the recognition of a wide range of lignin monomer units (Table 4) with intact methoxyl groups and the minor amount of catechols indicate that weakly altered lignins were signi®cant pre-cursors of the phenols in KL pyrolysates. This terrige-nous contribution as evidenced by the pyrolytic study is consistent with SEM observations showing selectively preserved ligneous debris (Fig. 2g), as well as with the relatively 13C
org-depleted isotopic composition (ca.ÿ26
%0).
Lignin pyrolysis products are characterised by the occurrence (or predominance) of monomer units with very speci®c substitution patterns (e.g. Martin et al., 1979; Obst, 1983). Gymnosperms (non-¯owering plants) and angiosperms (¯owering plants) synthesise dierent types of lignin, which allows for the identi®cation of the general vascular plant sources of sedimentary lignin-derived material (Hedges and Mann, 1979). The pre-dominance of guaiacyl (vanillyl) over syringyl phenols (G vs. S) among KL pyrolysis products is consistent with the predominance of gymnosperms in the water-shed vegetation of the Danube delta.
Sicre et al., 1994; Peulve et al., 1996a,b; van Heemst et al., 1993, 1997; Zegouagh et al., 1999). In some of these previous studies (Sicre et al., 1994; Peulve et al., 1996a,b) the distribution is relatively invariant with
depth, thus indicating that this series of alkylphenols is probably derived from complex resistant macro-molecules, corresponding to melanoidin-type compo-nents. Melanoidins originate from random Table 4
Phenolic compounds identi®ed in KL pyrolysates
Peak numbera Main mass fragments Lignin parameter
1 94,66 Phenol ±
2 122, 121, 77 (4-Methoxytoluene) ± 3 108, 107, 79 2-Methylphenol (o-cresol) ± 4 107, 108, 77 4-Methylphenol (p-cresol) ± 5 107, 108, 79 3-Methylphenol (m-cresol) ±
6 109, 124, 81 Guaiacol G
7 107, 122, 77 2,6-Dimethylphenol ±
8 2-Ethylphenol ±
9 107, 122, 121 2,4-Dimethylphenol+2,5-dimethylphenol ±
10 107, 122 4-Ethylphenol ±
11 110, 64 1,2-Benzenediol (catechol) ? ± 12 123, 138, 95 2-Methoxy-4-methylphenol (4-methylguaiacol) G 13 3-Ethylphenol+3,5-dimethylphenol ±
14 2,3-Dimethylphenol ±
15 3,4-Dimethylphenol ±
16 Trimethylphenol ? ±
17 120, 91 4-Vinylphenol ±
18 121, 136 4-Ethyl-2-methylphenol ? ± 19 121, 136 Ethyl-methylphenol ? ±
20 Propylphenol ? ±
21 137, 152 2-Methoxy-4-ethylphenol (4-ethylguaiacol) ± 22 150, 135, 77, 107 2-Methoxy-4-vinylphenol (4-vinylguaiacol) G 23 154, 139 2,6-Dimethoxyphenol (syringol) S 24 164, 77, 103, 149 2-Methoxy-4-(2-propenyl)-phenol (eugenol) G 25 137, 166 2-Methoxy-4-propylphenol (4-propylguaiacol) G 26 164, 77, 103, 149 2-Methoxy-4-(1-propenyl)-phenol
(cissoeugenol)
G
27 168, 153, 125 2,6-Dimethoxy-4-methylphenol (4-methylsyringol)
S
28 164, 77, 103, 149 2-Methoxy-4-(1-propenyl)-phenol (transisoeugenol)
G
29 147, 162, 91 1-(4-Hydroxy-3-methoxyphenyl)-propyne ? G 30 151, 166 1-(4-Hydroxy-3-methoxyphenyl)-ethanone
(acetovanillone)
G
31 167, 182 4-Ethyl-2,6-dimethoxyphenol (4-ethylsyringol) S 32 137, 180 1-(4-Hydroxy-3-methoxyphenyl)-2-propanone
(guaiacylacetone)
G
33 180, 165, 137 2,6-Dimethoxy-4-vinylphenol (4-vinylsyringol) S 34 151, 180 1-(4-Hydroxy-3-methoxyphenyl)-1-propanone
(propiovanillone)
G
35 194, 119 4-Allyl-2,6-dimethoxyphenol (4-allyl-syringol) S 36 194, 119 cis2,6-Dimethoxy-4-propenylphenol S 37 167, 210 trans2,6-Dimethoxy-4-propenylphenol S 38 181, 196 1-(4-Hydroxy-3,5-dimethoxyphenyl)-ethanone
(acetosyringone)
S
39 167, 210 1-(4-Hydroxy-3,5-dimethoxyphenyl)-2-propanone (syringylacetone)
S
40 181, 210 1-(4-Hydroxy-3,5-dimethoxyphenyl)-1-propanone (propiosyringone)
S
condensations of altered moieties derived from proteins and polysaccharides, and provide OM preservation via the so-called degradation-recondensation pathway. Melanoidins are characterised by relatively high oxygen contents, and their infra red spectra exhibit intense absorptions due to OH and CO groups (Rubinsztain et al., 1984, 1986a,b). Their general IR features, as well as the solid-state 13C and 15N NMR spectra obtained
for some synthetic, amino acid-rich, melanoidins (Ikan et al., 1986; Benzing-Purdie et al., 1983), show impor-tant similarities with those of the KLs. Van Heemst et al. (1997) also prepared synthetic melanoidins from mixtures of a tyrosine-containing protein and carbohy-drate components, leading to similar distributions of alkylphenols upon pyrolysis as observed in the present study. These observations therefore point to melanoidin contribution to the three KLs. In addition, some con-tribution of proteinaceous material which would have survived diagenesis and HF/HCl hydrolysis cannot be excluded. Indeed, alkylphenols are also generated upon pyrolysis of tyrosine-containing proteins or derived materials (Tsuge and Matsubara, 1985), and as recently shown (Knicker and Hatcher, 1997; Zang et al., in press), proteins can be protected from diagenetic and chemical alterations via encapsulation in macro-molecular organic substances. The presence of melanoi-dins and/or of such protected proteins is supported by the predominance of amides in the solid-state15N NMR
spectra of the three KLs (Fig. 6), as well as by their relatively low C/N atomic ratios.
The above observations suggest the signi®cant occur-rence of another type of precursor, in addition to lignins and lignin-derived components, for the alkylphenols of KL pyrolysates: tyrosine-derived moieties preserved from degradation by incorporation in melanoidin-type macromolecules and/or by encapsulation within the macromolecular network of the KLs.
3.5.5. Alkylbenzenes
Toluene, an ubiquitous pyrolysis product of sedi-mentary OM, represents a relatively important con-tribution to the TIC traces of the three KL pyrolysates (Fig. 7). Several isomers of alkylbenzenes with C2- to
C16-chains were identi®ed in mass chromatogram
m/z=91+92+105+106+119+120 (Fig. 10), but the relative abundance of the latter series is low and strongly decreases with the number of alkyl carbons.
Some of these compounds (e.g.n-alkylbenzenes with a C5- to C16-chain, orn-alkyl-methylbenzenes with a C6
-to C11-chain) are probably formed during pyrolysis by
secondary reaction via cyclisation/aromatisation pro-cesses (Hartgers et al., 1994a). This is con®rmed by the ortho-position observed for the latter series. Never-theless, some isomers with a low alkyl carbon number (i.e. in the C1±C4 range) might be primary pyrolysis
products formed by-cleavage of substituted aromatic rings linked to the macromolecular structure via an alkyl chain (Hartgers et al., 1994b).
Toluene and C2-alkylbenzenes can be produced upon
protein derivatives (Tsuge and Matsubara, 1985; Stan-kiewicz et al., 1998). The former source cannot be ruled out for the three KLs since, as already stressed, some proteins may have survived diagenesis and HF/HCl hydrolysis owing to encapsulation in the macro-molecular structure of the KLs. Similar distributions of alkylbenzenes with C1- to C3-chains were recently
observed in pyrolysates of particulate OM from the northwestern Mediterranean Sea (Peulve et al., 1996a) and of refractory OM isolated from sediments of the northwestern African upwelling system (Zegouagh et al., 1999). In the above studies, these alkylbenzenes have been considered as originating from phenylalanine-con-taining moieties derived from altered proteins, and incorporated within melanoidin-type structures. This suggests, as in the case of alkylphenols, that the degra-dation-recondensation preservation mechanism also has played a role in the formation of the three KLs.
Alkylbenzenes, sometimes including long chain com-pounds, and probably derived from algaenans, were detected as major pyrolysis products in a few previous studies on kerogens (e.g. Hors®eld et al., 1992; Sin-ninghe Damste et al., 1993). Such compounds were also observed in the pyrolysis products of resistant bioma-cromolecular material isolated from marine microalgae (Derenne et al., 1996; Kokinos et al., 1998). TEM observations of the three KLs showed the presence of numerous cell walls, probably containing resistant algaenans or algaenan-like, biopolymers, based on their
excellent morphological preservation. Consequently, resistant macromolecular material from microalgal cell walls might be an additional source of some of the alkylbenzenes detected in the KL pyrolysates.
The presence of 1,2,3,4-tetra-methylbenzene (TMB) in the pyrolysates of various kerogens was ascribed to speci®c aromatic carotenoids of Chlorobiaceae (Hart-gers et al., 1994b,c; Guthrie, 1996; Clegg et al., 1997). Chlorobiaceae are anaerobic, photosynthetic, green sul-phur bacteria, and their presence is an indicator of anoxia in the photic zone. Since the water column is oxic in the zone where the studied sediment core was collected, such a bacterial origin can be ruled out. The occurrence of 1,2,3,4-TMB in pyrolysates of the resis-tant material isolated from several marine algae (Hoefs et al., 1995) also suggests an algal origin, which would be consistent with TEM observations in the present case.
3.5.6. Other aromatic hydrocarbons
Naphthalenes and indenes were identi®ed by selective ion detection at m/z=128+142+156+170 (Fig. 11) and m/z=115+116+129+130 (Fig. 12), respectively. Naphthalene and indene are the dominant compounds of both series, but their contribution to the KL pyr-olysates is minor. The distribution pattern of C1- to C3
observed in previous studies of marine, particulate and sedimentary, OM (Peulve et al., 1996a; Zegouagh et al., 1999). However, the sources and precursors of both families are unknown at present.
3.5.7. Nitrogen-containing compounds
Pyrroles, indoles, pyridines, and aromatic nitriles were identi®ed in KL pyrolysates. These series were identi®ed in mass chromatograms m/z=103+117+ Fig. 11. Mass chromatogram (m/z128+142+156+170) of the 650C Curie-point pyrolysate of S20KL showing alkylnaphthalene distribution.
131+144 (alkylindoles and aromatic nitriles; Fig. 13), m/z=67+81+95 (pyrroles), and m/z=79+93+107 (pyridines). In each family the unsubstituted compound predominates, whereas C1alkylated isomers are minor
compounds and C2 homologues occur in negligible
amounts.
Alkylated pyrroles can be derived from chlorophyll pigments. But, in the present case, such an origin cannot be considered since their distribution pattern diers from that reported for alkylpyrroles present in pyr-olysates of porphyrin derivatives (Sinninghe Damste et al., 1992).
All the above mentioned classes of N-containing compounds can be ascribed to pyrolysis products of proteins or protein-derived units. For instance, indoles and pyrroles are often associated with tryptophan and proline moieties, respectively (Danielson and Rogers, 1978; Tsuge and Matsubara, 1985). As already dis-cussed, some proteins may have been protected via encapsulation in the three KLs. Similar distributions of alkylindoles and alkylpyrroles were also observed in previous studies of dissolved OM from Paci®c Ocean, suspended OM from the Rhone delta, sediment trap material from the northwestern Mediterranean Sea, and marine sediments from the northwestern African upwelling system (van Heemst et al., 1993; Sicre et al., 1994; Peulve et al., 1996a; Zegouagh et al., 1999) and considered to be related to melanoidin-type structures. Accordingly, as already concluded for alkylphenols and
alkylbenzenes, these N-containing pyrolysis products may be related to melanoidins comprising degraded proteinaceous material, and/or to proteins encapsulated in the most refractory part of the KL structure.
3.5.8. Polysaccharide pyrolysis products
Polysaccharides are ubiquitous compounds in the marine environment, occurring as constituents of cell walls, storage material, constituents of marine snow, and mucus (de Leeuw and Largeau, 1993).
Pyrolysis of polysaccharides and polysaccharide-derived components produces furans and furan-deriva-tives (Helleur et al., 1985a,b). Dimethylfuran, 2-fur-aldehyde, 5-methyl-2-furaldehyde and 2-furanmethanol were identi®ed in the KL pyrolysates in very low amounts, 2-furaldehyde being the major furanic frag-ment. The presence of polysaccharidic moieties in the three KLs is con®rmed by the signals at 74 and 105 ppm in their solid-state 13C NMR spectra. Furfurals and
methylfurfurals have also been observed by Boon et al. (1986) in the pyrolysates of sedimentary OM from the estuary of the Rhine river, and cellulose was considered as a potential source for these pyrolysis products. Ther-mal degradation of cellulose results in a wide variety of products including furans and pyrans (van der Kaaden et al., 1983; Pouwels et al., 1989). These compounds are generally detected in minor amounts, whereas high yields of levoglucosan were obtained from dierent cel-luloses (20±60 wt.%) (Sha®zadeh et al., 1979). Since (i)
most of these compounds, and in particular anhy-drosugars like levoglucosan, were not detected in KL pyrolysates, and (ii) the bulk of the polysaccharides that occurred in the sediments was most likely eliminated by the HF/HCl attack applied for eliminating the mineral matrix, the presence of furanic components in KL pyr-olysates can be attributed to degraded ligno-cellulosic complexes of higher plants and polysaccharide-derived moieties in melanoidin-type components.
3.5.9. Fatty acids
A minor series of straight-chain saturated fatty acids (C6-C18) was detected in KL pyrolysates (chromatogram
of m/z=60+73; Fig. 14). The C16 homologue is
pre-dominant and is observed in a signi®cant relative abun-dance in the TIC traces of KL pyrolysates. This series is dominated by even-carbon-numbered homologues. Considering the moderate level of alteration of the acid moieties (indicated by this even predominance), it is likely that weakly altered saturated fatty acids were incorporated early within melanoidin-type structures, as previously observed for sediments from the north-western African upwelling system (Zegouagh et al., 1999). Indeed, Larter and Douglas (1980) reported on the participation of some lipids in the condensation reactions occurring during melanoidin formation. The absence of branched acids among KL pyrolysis pro-ducts points to a negligible contribution from bacterial lipids in the condensation steps.
4. Conclusions
The main conclusions of this study, carried outviaa combination of microscopic, spectroscopic, isotopic, and pyrolytic methods, on the KL isolated from three sections of a recent sediment core o the Danube delta, are summarised below:
(i) The kerogen-like material accounts for a sub-stantial fraction of total OM in the three samples (ca. 50%).
(ii) Similar morphological features and chemical composition are observed for the KL isolated from these sediments.
(iii) The main source of the KLs is an admixture of land-plant and freshwater microalgal material, whereas the contribution of bacterial material was low.
(iv) The selective preservation of terrigenous material (including ligneous debris and pollen and plant spores) as well as of resistant aliphatic bioma-cromolecules (probably derived from cell walls of microalgae) was the main process for KL for-mation.
proteinaceous and polysaccharidic materials, and/or encapsulation of proteins within the macromolecular network of the three KLs. (vi) The role of natural sulphurisation was negligible
in KL formation.
Acknowledgements
The authors wish to thank Dr. H. Knicker (T.U. MuÈnchen) for the solid-state 15N NMR spectrum, and
P. Florian (CRPHT, CNRS, OrleÂans) for the solid-state
13C NMR spectrum. SEM observations were performed
in the CIME (Universite Pierre et Marie Curie). Dr. J. M. Martin, Co-ordinator of the EROS 21 Project, C. Lancelot, Chief Scientist during the EROS 21 cruises, and the crew of the Research Vessel ``Professor Vodya-nitsky'' are gratefully acknowledged. This work was carried out in the framework of the EROS 21 (European River-Ocean System) project. We kindly acknowledge the support of the European Commission under the 4th Environment Programme (ENV-4CT 96-0286). Anonymous referees are sincerely thanked for their constructive reviews of the manuscript.
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