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Formation of insoluble, nonhydrolyzable, sulfur-rich macromolecules via incorporation of inorganic sulfur species into algal carbohydrates
Geochimica et Cosmochimica Acta, Vol. 64, No. 15, pp. 2689 –2699, 2000 Copyright © 2000 Elsevier Science Ltd Printed in the USA. All rights reserved 0016-7037/00 $20.00 ⫹ .00
Pergamon
PII S0016-7037(00)00382-3
Formation of insoluble, nonhydrolyzable, sulfur-rich macromolecules via incorporation of inorganic sulfur species into algal carbohydrates MARIKA D. KOK, STEFAN SCHOUTEN,* and JAAP S. SINNINGHE DAMSTE´ Department of Marine Biogeochemistry and Toxicology, Netherlands Institute for Sea Research (NIOZ), PO Box 59, 1790 AB Den Burg, Texel, the Netherlands (Received September 1, 1999; accepted in revised form March 8, 2000)
Abstract—The process of sulfur incorporation into organic matter was simulated in the laboratory by sulfurization of cell material of the prymnesiophyte alga Phaeocystis in sea water with inorganic polysulfides at 50°C. Flash pyrolysis of the residue, obtained after extraction and several hydrolysis steps, yielded mainly C1–C4 alkylbenzenes and C1–C4 alkylphenols and, in contrast to control and blank experiments, relatively high amounts of C0–C4 alkylthiophenes. The distribution of the thiophenes is very similar to that in pyrolysates of type II-S kerogens. The formation of high-molecular-weight sulfur-rich macromolecules co-occurs with a marked drop in the content of hydrolyzable carbohydrates. This indicates that sulfurization results in the preservation of algal carbohydrate carbon in a macromolecular structure composed of (poly) sulfidic cross-linked carbohydrate skeletons, which upon pyrolysis yields alkylthiophenes. Sulfurization of glucose under similar conditions resulted in the formation of a nonhydrolyzable, solid material, which yielded high amounts of organic sulfur compounds upon pyrolysis, mainly short-chain alkylthiophenes, although with a different distribution than that in the pyrolysate of the sulfurized algal material. The carbon numbers of these organic sulfur compounds extend beyond six, indicating that the length of the carbon skeleton of the pyrolysis products is not limited by the length of the carbon skeleton of the substrate. These results suggest that the sulfurization of carbohydrates may be an important pathway in the preservation of organic matter in euxinic depositional environments. Copyright © 2000 Elsevier Science Ltd weight (LMW) OSC, whereas studies on high-molecularweight (HMW) OSC are scant, probably because HMW OSC are often not gas chromatography (GC)-amenable. In some studies such HMW OSC have been analyzed by pyrolysis gas chromatography (py-GC-MS). For instance, Moers et al. (1988) reacted glucose with hydrogen sulfide and pyrolyzed the extracted and dried reaction mixtures, which yielded small amounts of thiophenic pyrolysis products. Philp et al. (1992) pyrolyzed the extracted residues of cyanobacterial mats, which were reacted with almost pure H2S, yielding trace amounts of thiophenes. Laboratory sulfurization experiments described in the literature have shown that sulfur incorporation can occur under relatively mild conditions, at temperatures of 50°C or less, without applying either extreme acidic or basic conditions or ultraviolet radiation. However, the reaction conditions applied and reagents used vary widely, e.g., elemental sulfur, hydrogen sulfide, and polysulfides have been used as inorganic sulfur source (Lalonde et al., 1987; Lalonde, 1990; Moers et al., 1988; Rowland et al., 1993). Other differences are associated with the reaction medium employed, e.g., (sea) water, a two-phase system of ethyl acetate and water or pure dimethyl formamide (Fukushima et al., 1992; de Graaf et al., 1992, 1995; Schouten et al., 1993, 1994). Sometimes, a base or a phase transfer catalyst was added to the reaction mixture (Krein and Aizenshtat, 1993, 1994; Rowland et al., 1993). All these different reactions were used to indicate the sulfurization potential of geochemically relevant organic compounds. Despite the use of the same substrate, differences in reaction conditions can result in the formation of different sulfurized reaction products. For example, sulfurization of phytenal produced (i) isoprenoid thiophenes when elemental sulfur was used in a dichloromethane
1. INTRODUCTION
In many sediments and oils, organic sulfur compounds (OSC) have been identified (Sinninghe Damste´ and de Leeuw, 1990). It is generally accepted that these OSC are formed during early diagenesis via the incorporation of inorganic sulfur species into functionalized lipids (Valisolalao et al., 1984; Brassell et al., 1986; Sinninghe Damste´ et al., 1989b; ten Haven et al., 1990; Kohnen et al., 1990; Wakeham et al., 1995). The location of sulfur incorporation corresponds with the position of the functional group in the precursor lipid molecule. Sulfurization of lipids is thus thought to represent a sink for relatively labile organic components. In this way, information on the precursor molecule, that otherwise would have been completely mineralized during diagenesis, will be preserved, which makes OSC carriers of paleoenvironmental information. The processes and timing of sulfur incorporation have been examined in several ways. Firstly, detailed molecular studies were performed of organic matter in recent sediments where these reactions actually take place (Wakeham et al., 1995; Hartgers et al., 1997; Kok et al., 2000; Werne et al., 2000). Secondly, structural studies have been done on sulfur bonding in organic matter in older sediments (Schouten et al., 1995; van KaamPeters and Sinninghe Damste´, 1997). Finally, the sulfurization process has been simulated in the laboratory (Lalonde et al., 1987; Vairavamurthy and Mopper, 1987; de Graaf et al., 1992, 1995; Krein and Aizenshtat, 1993, 1994; Kok et al., 1995; Adam et al., 1998). Most of these latter studies have concentrated on the formation and characterization of low-molecular-
*Author to whom ([email protected]).
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(DCM)/methanol (MeOH)/water mixture (48:48:4) with the aid of trimethylamine (Rowland et al., 1993); (ii) isoprenoid dithiolanes and trithianes in a two-phase system of ethyl acetate and water (1:1) using polysulfides and a phase transfer catalyst (Schouten et al., 1993); and (iii) oligomers consisting of polysulfidic cross-linked phytane skeletons when using polysulfides and a phase transfer catalyst in a two-phase system of toluene/ water (1:1) (Krein and Aizenshtat, 1994). Thus, reaction conditions in simulated sulfurization experiments should be kept as close to the natural situation as possible, as small differences can produce different products. In this paper we present the results of laboratory sulfurization experiments with a complex mixture of components of biologic origin, i.e., a phytoplankton concentrate collected during the bloom period of the alga Phaeocystis (Prymnesiophyceae). To mimic the natural situation as closely as possible, the reaction was performed in sea water, without the aid of any catalyst or organic solvent and polysulfides were used as the reduced inorganic sulfur source. In addition, we performed a laboratory sulfurization of glucose with polysulfides in water and studied the fate of Phaeocystis carbohydrates under oxic conditions in sediments from Balsfjord (northern Norway). The focus of these experiments was on the formation of highly resistant HMW sulfur-rich material and on comparisons of artificially produced sulfur-rich HMW organic matter with that in kerogens, to better understand the processes of HMW OSC formation. 2. EXPERIMENTAL 2.1. Sulfurization of Algal Material The algal material was collected during a spring bloom period and is predominantly composed of the prymnesiophyte Phaeocystis spp., a widespread algal species, mainly living in polar and temperate seas, but also in equatorial waters (Baumann et al., 1994). It forms massive spring blooms and has a high carbohydrate content. Phaeocystis colonies are composed of a ca. 7 m-thick layer of mucus surrounding an aqueous lumen, with cells present at the inside of the mucus layer (van Rijssel et al., 1997), instead of a colony filled with mucus. Although total carbohydrate concentrations of up to 90% have been reported previously (Rousseau et al., 1990), van Rijssel et al. (1997) showed that Phaeocystis species can have a carbohydrate content of up to ca. 35– 40% of total carbon during the stationary growth phase and is mainly present as the storage carbohydrate -glucan (Janse et al., 1996b). Algal material was collected during spring blooms of Phaeocystis spp. in the western Dutch Wadden Sea and the southern North Sea with plankton nets (50 m mesh) and stored frozen until use. Three different experiments were performed, i.e., a sulfurization, a control, and a blank experiment (Fig. 1). In the sulfurization experiment wet algal material (ca. 70 g dry weight), elemental sulfur (3 g), and NaHS (110 g) were gently stirred in sea water (1.5 L) for 4 weeks at a temperature of 50°C under a nitrogen atmosphere. As a control experiment, algal material was stirred for 8 weeks in sea water under a nitrogen atmosphere at 50°C, without the addition of any inorganic sulfur compounds. As a blank experiment, untreated fresh algal material was analyzed. The reaction mixtures of the sulfurization, control, and blank experiments were centrifuged and washed to remove most of the sea water and subsequently freeze dried, ultrasonically extracted (step 1; Fig. 1) with MeOH and DCM, followed by KOH- (step 2), HCl- (step 3), and H2SO4- (step 4) hydrolysis as described by Gelin et al. (1997). After each step, reaction mixtures were extracted with MeOH and DCM and the collected extracts were washed with distilled water. Elemental sulfur was removed from the extracts by reaction with activated Cu-curls and subsequent filtration. Before analysis by gas chromatography–mass spectrometry (GC-MS) the extracts were deri-
Fig. 1. Analytical scheme.
vatized with diazomethane and bis(trimethylsilyl)trisfluoro acetamide (BSTFA) in pyridine. The residues left after extraction were analyzed by flash pyrolysis– gas chromatography–mass spectrometry (py-GCMS). Carbohydrates in the final residues of the sulfurized algal material, the control experiment, and the fresh algal material were analyzed as described by Janse et al. (1996a) according to the slightly modified method of Kamerling and Vliegenthart (1989). Monosaccharide compositions were analyzed gas chromatographically with parallel analysis of derivatized standards of monosaccharides and mannitol for identification and quantification. 2.2. Sulfurization of Glucose Glucose (200 mg) dissolved in 6 ml of distilled water was heated at 50°C under a nitrogen atmosphere for 12 weeks after addition of 560 mg NaHS and 16 mg of elemental sulfur. The solid material formed was collected, washed with distilled water, and extracted with hexane to remove elemental S. The residual material was analyzed by py-GCMS. 2.3. Black Sea Sediments Sediment from Unit II of the Black Sea was Soxhlet extracted with MeOH/DCM (1:7.5; vol/vol) for 16 h. The residue was hydrolyzed with KOH (1N; 2 h reflux), brought to pH 4 with HCl and extracted with MeOH/H2O (1:1, 1⫻), MeOH (1⫻), and DCM (3⫻). Subsequently, the residue was hydrolyzed with HCl (4N; 6 h, 100°C), brought to pH 8 with KOH and, after freeze drying, another KOH hydrolysis and additional extraction were performed. The remaining residue was analyzed by py-GC-MS. 2.4. Balsfjord (Northern Norway) Samples Before, during, and after a bloom (15th April–28th May 1996) of the prymnesiophyte Phaeocystis sp., sediments were collected in Balsfjord, northern Norway (sediment depths ranging from 0 –20 cm). During the peak of the bloom period (29th April) a concentrate of the phytoplank-
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Fig. 2. (a) Total ion chromatogram (TIC) of the pyrolysate of the control algal material, (b) TIC of the pyrolysate of the sulfurized algal material, and (c) distribution of alkylthiophenes in the pyrolysate of the sulfurized algal material, as revealed by the partial accurate mass chromatogram of m/z 84 ⫹ 97 ⫹ 98 ⫹ 111 ⫹ 112 ⫹ 125 ⫹ 126 ⫹ 139 ⫹ 140 ⫹ 153 ⫹ 154. Compound assignments are listed in Table 1.
ton was collected with a plankton net. After extraction of the sediments with MeOH and DCM, the residues were analyzed by py-GC-MS and
compared with the pyrolysate of MeOH and DCM extracted phytoplankton material (mainly Phaeocystis sp.) collected during the bloom.
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M. D. Kok, S. Schouten, and J. S. Sinninghe Damste´ Table 1. Compounds identified in the pyrolysates.a
2.5. GC GC was performed using a Carlo Erba 5300 or a Hewlett-Packard 5890 instrument, both equipped with an on-column injector. A fused silica capillary column (25 m ⫻ 0.32 mm) coated with CP-Sil 5 (film thickness 0.12 m) was used with helium as carrier gas. The effluent of the Carlo Erba 5300 was monitored with a flame ionization detector (FID). The effluent of the Hewlett-Packard 5890 was monitored using both a FID and a sulfur-selective flame photometric detector (FPD), applying a stream-splitter with a split ratio of FID:FPD of ca. 1:2. The samples were injected at 70°C and the oven was programmed to 130°C at 20°C/min and then at 4°C/min to 320°C, at which it was held for 15 min. 2.6. GC-MS GC-MS analyses were performed with a Hewlett-Packard 5890 gas chromatograph interfaced with a VG Autospec Ultima mass spectrometer operated at 70 eV with a mass range of m/z 40 – 800 and a cycle time of 1.7 s (resolution 1000). The gas chromatograph was equipped with a fused silica capillary column (25 m ⫻ 0.32 mm) coated with CP Sil-5 (film thickness 0.12 m). The carrier gas was helium. The samples were injected on column at 60°C and subsequently the oven was programmed to 130°C at 20°C/min and then at 4°C/min to 320°C at which it was held for 10 min. 2.7. Curie-point py-GC-MS Py-GC-MS analyses were carried out on a Hewlett-Packard 5890 gas chromatograph equipped with a FOM-3LX unit for pyrolysis and interfaced to a VG Autospec Ultima mass spectrometer operated at 70eV with a mass range of m/z 35–700 and a cycle time of 1.7 s (resolution 1000). The samples were applied to a ferromagnetic wire with a Curie temperature of 610°C. The gas chromatograph, equipped with a cryogenic unit, was programmed from 0°C (5 min) to 300°C (10 min) at a rate of 3°C/min. Separation was achieved using a fused silica capillary column (30 m ⫻ 0.25 mm) coated with DB1701 (film thickness 0.25 m). Helium was used as a carrier gas. 3. RESULTS AND DISCUSSION
To study the sedimentary fate of carbohydrate-rich algal material under euxinic conditions, collected Phaeocystis cell material was reacted, for 4 weeks, with (poly)sulfides in sea water under a nitrogen atmosphere. As a control experiment, algal material was subjected to the same reaction conditions except for the absence of (poly)sulfides. The duration of this experiment was twice as long as the sulfurization experiment, i.e., 8 weeks. Fresh algal material was used as a blank. The reaction mixtures of the sulfurized and control experiment and of the blank algal material were subjected to extraction and several hydrolysis steps (Fig. 1) and the obtained extracts and residues were analyzed for the presence of OSC. 3.1. Extracts Major compounds present in the extracts obtained after steps 1, 2, 3, and 4, respectively, (Fig. 1) of the fresh, control, and sulfurized algal material are very similar and consist predominantly of fatty acids (mainly C14:0, C16:0, and C18:0), phytol, and C27 sterols. GC analysis using a sulfur-selective FPD revealed no OSC in the extracts of the sulfurized algal material. Even traces of OSC, e.g., isoprenoid thiophenes, could not be detected by GC-MS, indicating that under the conditions used no sulfur incorporation into lipids, resulting in GC-amenable and extractable OSC, had occurred. A similar laboratory sulfurization experiment with the marine microalga Nannochloropsis salina did result in the formation of minor amounts of
1 Toluene 2 Ethylbenzene 3 1,3-Dimethylbenzene ⫹ 1,4-Dimethylbenzene 4 Styrene 5 2-Methylpyrrole 6 3-Methylpyrrole 7 1-Ethyl-3-methylbenzene 8 1,2,4-Trimethylbenzene 9 1,2,3-Trimethylbenzene 10 Tetramethylbenzene 11 Methoxyphenol 12 Phenol 13 Methylphenol a
a b c d e f g h i j k l
Thiophene 2-Methylthiophene 3-Methylthiophene 2-Ethylthiophene 2,5-Dimethylthiophene 2,4-Dimethylthiophene 2,3-Dimethylthiophene 2-Propylthiophene 2-Ethyl-5-methylthiophene 2,3,5-Trimethylthiophene 2-Methyl-5-propylthiophene 2,3-Dimethyl-5-(2,6,10-trimethylundecyl)thiophene
Peak numbers refer to Fig. 2.
isoprenoid thiophenes (Gelin et al., 1998). It is unclear what the reason is for this difference.
3.2. Residues The pyrolysates of the final residue (residue 4, Fig. 1) of the control experiment and the fresh algal material are very similar and consist mainly of aromatic compounds (e.g., Fig. 2a, Table 1). The major compounds present are C1–C4 alkylated benzenes and C1–C4 alkylated phenols. However, the pyrolysate of the sulfurized algal cell material contains, apart from the aromatic compounds also encountered in the control and the blank experiment, large amounts of alkylated thiophenes (Fig. 2b). The thiophene distribution is dominated by C0–C4 alkylated thiophenes with a linear carbon skeleton (Fig. 2c). Other OSC present in the pyrolysate of the sulfurized algal material are C20 isoprenoid thiophenes, thiolanes, dithiophenes, and (methyl) benzothiophenes. All these thiophenes are almost absent from the pyrolysates of the final residues of the blank algal material and the control experiment. A co-pyrolysis experiment was performed to exclude the possibility that thiophenes were formed during pyrolysis by reaction of remnant inorganic sulfur with the algal material. Fresh algal material was mixed with an excess amount of elemental sulfur and the mixture was subsequently pyrolyzed. This resulted in the formation of traces of thiophenes in the co-pyrolysate, comparable to the relative amounts of thiophenes present in the pyrolysates of the fresh and the control experiment material. Hence, the presence of elemental sulfur during pyrolysis has no major influence on the formation of thiophenes. Allard et al. (1997) showed that during the process used to isolate resistant algal and bacterial material artifactual formation of melanoidin-like polymers can occur. Hence, it may be that the OSC present in the pyrolysate of the final residue were derived from artifacts formed during the isolation procedure. However, the difference in the relative abundance of the thiophenes in the pyrolysates of sulfurized experimental material versus control experimental material and blank algal biomass is already observed in residue 1. Furthermore, the thiophene distributions in the pyrolysates of residues 1 and 4 of the sulfurized algal material are very similar. Thus, melanoidin
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Fig. 3. Distribution of alkylthiophenes in the pyrolysates of (a) sulfurized Phaeocystis and (b) Black Sea sediment (0.006 Ma), a type II-S kerogen, as revealed by the partial accurate mass chromatograms of m/z 84 ⫹ 97 ⫹ 98 ⫹ 111 ⫹ 112 ⫹ 125 ⫹ 126 ⫹ 139 ⫹ 140 ⫹ 153 ⫹ 154.
formation is of no importance to the formation of the Sorg-rich moieties which yield alkylthiophenes upon pyrolysis. 3.3. Comparison with Type II-S Kerogens In flash pyrolysates of many type II-S kerogens, thiophenes are major compounds and, even in cases where thiophenes do not dominate the FID-traces, they are the most abundant OSC (Sinninghe Damste´ et al., 1989a; Eglinton et al., 1990a,b). Figure 3 shows the thiophene distribution in pyrolysates of the sulfurized algal cell material and of a typical type II-S kerogen of a Holocene Black Sea sediment. The thiophene distributions are very similar, suggesting that the formation of OSC in type II-S kerogens could proceed in a similar way to the formation of sulfurized algal cell material, as observed in the laboratory simulation experiment. Eglinton et al. (1992) showed that the 2-methylthiophene over toluene (2MT/Tol) ratio can be used to estimate the atomic Sorg/C ratio in pyrolysates of kerogens and that this ratio is
comparable to the 2,3-dimethylthiophene over 1,2-dimethylbenzene ⫹ n-non-1-ene ratio (Eglinton et al., 1990a). Type II-S kerogens have a Sorg/C ratio of ⬎0.04 (Orr, 1986), which corresponds to a 2MT/Tol ratio of ⬎0.38 (Eglinton et al., 1992). When the 2MT/Tol ratio is used to obtain an indication of the Sorg-richness of the algal material, an increase in this ratio is observed for the sulfurized material after each extraction and hydrolysis step (Table 2). The 2MT/Tol ratio of the pyrolysate of the sulfurized algal material after extraction with DCM and MeOH (residue 1) is an order of magnitude higher than the 2MT/Tol ratio of the corresponding pyrolysate of the control experiment or blank algal material (Table 2). After the various hydrolysis steps, which are applied to remove protein and carbohydrates, the 2MT/Tol ratio of the pyrolysate of the final residue of the sulfurized algal material has increased to 0.60, indicating that the Sorg is relatively more resistant to hydrolysis compared to the Corg material. Assuming that the composition of the nonhydrolyzable, sulfurized algal material
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Table 2. 2-Methylthiophene/toluene ratios in pyrolysates of laboratory simulation experiments of Phaeocystis, type II-S kerogen and Black Sea kerogen. Pyrolysates
2MT/Tol
Laboratory simulation experiments of Phaeocystis Blank experiment extracted residue (1) Blank experiment nonhydrolyzable residue (4) Control experiment extracted residue (1) Control experiment nonhydrolyzable residue (4) Sulfurized experiment extracted residue (1) Sulfurized experiment nonhydrolyzable residue (4) Type II-S kerogen (Sorg/C ⬎ 0.04) Black Sea kerogen
⬍0.01 0.02 0.01 0.06 0.13 0.60 ⬎0.38 0.22
may be compared with kerogen, (i.e., the several hydrolysis and extraction steps give results comparable to natural degradation), the 2MT/Tol ratio of 0.60 indicates that the Sorg content is similar to that of a type II-S kerogen. 3.4. Origin of Thiophenes Previously it has been suggested that alkylthiophenes, which are common in pyrolysates of type II-S kerogens, are generated from thiophene units with longer carbon skeletons already present in the macromolecular matrix by cleavage of C–C bonds, predominantly the -bond relative to the thiophene unit (Sinninghe Damste´ et al., 1990). However, laboratory sulfurization experiments with phytadienes, phytol, or phytenal as substrates resulted in the formation of cyclic isoprenoid sulfides or polysulfide cross-linked polymers, which only after heating (250 –300°C) resulted in the formation of isoprenoid thiophenes (de Graaf et al., 1992; Schouten et al., 1993, 1994; Krein and Aizenshtat, 1994). When Fukushima et al. (1992) and Rowland et al. (1993) used the same lipid substrates, this resulted directly in the formation of isoprenoid thiophenes. In a recent study, Sinninghe Damste´ et al. (1998) proposed a new pathway to explain the presence of alkylthiophenes in kerogen pyrolysates. Microscale sealed vessel (MSSV) pyrolysis of sulfur-rich kerogen shows that even at low pyrolysis temperatures (150°C) linear LMW alkylthiophenes are produced as the major S-containing pyrolysis products (apart from hydrogen sulfide). MSSV pyrolysis of a long-chain alkylthiophene and an alkylbenzene indicated that at 300°C for 72 h no -cleavage, leading to the generation of LMW alkylated thiophenes and benzenes, occurred. Several recent studies (Krein and Aizenshtat, 1994; Schouten et al., 1994; Koopmans et al., 1995; Tomic et al., 1995) have indicated that long-chain alkylthiophenes are stable thermal products formed at temperatures ranging from 250 –500°C, from carbon skeletons linked intermolecularly via several sulfide-bridges. This would indicate that linear LMW alkylthiophenes are predominantly formed by thermal degradation (i.e., during pyrolysis) of multiple (poly) sulfide-bound linear C5–C7 skeletons. This rearrangement model, proposed by Sinninghe Damste´ et al. (1998), would explain the early loss during maturation of the precursor moieties of the LMW alkylthiophenes as observed by Eglinton et al. (1990a,b), due to the relative weakness of the (poly)sulfide bonds. This model is also supported by a sulfur speciation study with X-ray absorption near edge structure (XANES) spectros-
copy of artificially matured kerogens (Nelson et al., 1995), which indicated that the relative amounts of organic (poly) sulfides show a significant decrease with increasing maturation temperature, whereas at the same time the relative amount of thiophenic sulfur remained rather constant. The thiophenes present in our pyrolysates may thus originate mainly from sulfurization of a precursor characterized by a small carbon skeleton and with multiple functionalities available for sulfur incorporation and sulfur cross-linking. Furthermore, it should be omnipresent in relatively high amounts in the environment. Carbohydrate substrates seem to be the perfect candidates, as they meet these requirements. Circumstantial evidence for the sulfurization of carbohydrates in the natural environment stems from investigations on the carbon isotopic composition of organic matter from the Kimmeridge Clay Formation (van Kaam-Peters et al., 1998). During these studies a positive relationship between the ␦13CTOC and the degree of sulfurization of the organic matter was observed. The ␦13CTOC was also positively correlated with the relative abundance of ␦13C-enriched C1–C3 alkylated thiophenes, generated during pyrolysis. It was suggested that these thiophenes are isotopically heavy because they might originate from carbohydrates that are relatively enriched in ␦13C (Deines, 1980). Thus, in the Kimmeridge Clay samples the relationship between an enrichment in ␦13CTOC with increasing TOC was explained by an increasing proportion of sulfurized carbohydrates in the kerogen (van Kaam-Peters et al., 1998). This is also in agreement with the presence of relatively isotopically heavy short-chain alkylthiophenes in a Monterey kerogen pyrolysate (Eglinton, 1994). Thus, it is suggested that carbohydrates are the precursor compounds for the sulfur-rich organic matter formed during the sulfurization experiments in this study. It is the same reason why carbohydrates, but not lipids, are likely to be the precursor compounds. As only HMW OSC are formed (no LMW OSC were observed in the extracts) the precursor compounds require a high number of functionalities for sulfur incorporation and cross-linking, resulting in the formation of a sulfidic crosslinked polymer. The precursor compounds should be abundant and have short (linear) carbon skeletons, as during pyrolysis linear C0–C4 alkylthiophenes are generated upon cleavage of the relatively weak C–S and S–S bonds (compared to C–C) present in the HMW OSC. To further support the idea that carbohydrates are the precursor compounds for the HMW OSC formed, the amounts of carbohydrates present in the fresh algal material, the control experiment, and the sulfurized algal cell material were determined. They amount to 35, 27, and 14 wt%, respectively, of the cell material (Fig. 4; Table 3). Thus, sulfurization results in a decrease of ca. 60% in carbohydrate content compared to the fresh material. In contrast, stirring alone at 50°C without added inorganic sulfur species for twice as long as the sulfurization experiment gives a decrease of only 23%, possibly due to melanoidin formation. This suggests that under the sulfurization conditions a carbohydrate-consuming reaction took place, i.e., sulfurization, in addition to removal of carbohydrates by simple heating. These results demonstrate that S-incorporation of carbohydrates is very likely under the conditions used and may also occur in nature. However, it does not exclude the formation of
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3.5. Sulfurized Glucose
Fig. 4. Absolute amounts (wt%) of hydrolyzable carbohydrate monomers present in the blank, control, and sulfurized algal material.
HMW OSC through the sulfurization of lipids. For instance, Gelin et al. (1998) showed that lipids of Nannochloropsis salina can form HMW OSC using the same sulfurization procedure. Probably the formation of HMW OSC depends on the composition of the reactive organic matter pool, i.e., the type and number of functionalized lipids and the amount of sugars available for sulfurization.
To further test the idea that sulfurization of carbohydrates leads to the formation of precursor moieties of the alkylthiophenes, a laboratory sulfurization with glucose as substrate was performed, under reaction conditions similar to those applied during the sulfurization of algal material. After 12 weeks of reaction a substantial amount of a blackish solid material had formed. After washing with water and extraction with hexane, this solid material yielded upon pyrolysis relatively high amounts of C0–C2 alkylthiophenes, with both branched and linear carbon skeletons, as well as some other OSC (Fig. 5a). Small amounts of trimethyl- and tetramethylthiophenes are also present, indicating that sulfurization of a hexose carbohydrate and subsequent pyrolysis can yield products with more than six C-atoms. The composition of the co-pyrolysate of glucose and an excess of elemental sulfur is rather different and contains mainly levoglucosan and other glucose-related pyrolysis products, with only relatively small amounts of 2-methylthiophene (Fig. 5b). This indicates that a solid, polymeric material composed of glucose carbon skeletons with multiple (poly-) S-links was formed during sulfurization. Upon pyrolysis of this material, C–S and S–S bonds are cleaved and thiophenes and other short-chain OSC are formed. S-incorporation is likely to occur at the hydroxylated C-atoms, similar to the sulfurization of the side chain of bacteriohopanetetrol, which contains four hydroxyl groups. Sulfurization of this compound results in the formation of up to four (poly)sulfide bonds, binding it tightly to the kerogen matrix (Richnow et al., 1992, 1993). When Moers et al. (1988) sulfurized glucose in the presence of either hydrogen sulfide or polysulfides, py-GC-MS of the reaction mixtures revealed low amounts of linear (acetyl)thiophenes formed as pyrolysis products. This difference may be due to the different sulfurization procedures used. Both the sulfurized algal material and the sulfurized glucose yield short-chain (linear) C0–C4 alkylthiophenes upon pyrolysis, though with different distribution patterns and relative abundances. The branched alkylthiophenes and other OSC present in the pyrolysate of the sulfurized glucose are absent or only minor compounds in the pyrolysate of the sulfurized algal material. These differences are likely due to the difference in composition of the substrates, i.e., the monomer glucose versus
Table 3. Amounts of hydrolyzable carbohydrate monomers present in the blank, control, and sulfurized algal material. Fresh algal material (blank)
Glucose Rhamnose O-Methylated hexoses Ribose O-Methylated pentoses Mannose Xylose Galactose Arabinose Total
Control algal material
Sulfurized algal material
abs. (wt%)
rel. (mol%)
abs. (wt%)
rel. (mol%)
abs. (wt%)
rel. (mol%)
0.5 0.8 0.9 1.3 2.3 2.3 6.3 6.7 13.5 34.6
1 2 2 4 6 6 19 17 43 100
0.3 0.4 0.7 0.3 1.1 1.1 5.2 5.9 12.2 27.2
1 2 2 1 4 4 20 19 47 100
0.2 0.4 0.1 0.4 0.8 0.8 2.1 4.0 5.0 13.9
2 3 1 3 6 5 16 26 38 100
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Fig. 5. TIC of the pyrolysates of (a) the sulfurized glucose and (b) the co-pyrolysis of glucose and elemental sulfur.
the complex mixture of polymeric algal carbohydrates, comprised of many monomers (Table 3). 3.6. Environmental Controls on Sulfurization of Carbohydrates The Balsfjord (northern Norway) is a permanently oxic fjord, with Phaeocystis spp. present throughout all seasons and dominating the phytoplankton during the bloom period in April/ May (Eilertsen et al., 1981a,b; Lutter et al., 1989; Schuman, personal communication). Before, during, and after this bloom, surface sediments and some deeper sediments (up to 20 cm depth) were collected to investigate whether some of the algal carbohydrates are sulfurized. Although the exact depth of oxygen penetration into the sediment is unknown, at least the deeper sediments are expected to be anoxic and, therefore, a possible site for sulfurization. However, the compositions of the pyrolysates of these sediments are all very similar and only
the pyrolysate of a surface sediment collected shortly after the peak of the Phaeocystis bloom is shown here (Fig. 6b). No thiophenes or carbohydrate pyrolysis products are present in these pyrolysates. The pyrolysate of a phytoplankton concentrate collected during the peak of the bloom period however, does contain a large amount of levoglucosan (Fig. 6a). Hence, it can be concluded that under oxic conditions in the water column and surface sediment, carbohydrates are degraded or consumed before a sedimentary carbohydrate reservoir can be formed, which would be available for sulfurization once the sediments become anoxic. Previous studies with sediment traps in Balsfjord (Northern, Norway) by Lutter et al. (1989) are in agreement with this conclusion. These authors showed that direct sedimentation of Phaeocystis is unlikely despite the massive spring bloom, due to grazing by herbivores and degradation and remineralization in the photic zone, resulting in transport of only highly resistant
Formation of sulfur-rich macromolecules via sulfurization of algal carbohydrates
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Fig. 6. TIC of the pyrolysates of the Balsfjord (Northern Norway) samples (a) phytoplankton concentrate and (b) sediment surface collected shortly after the Phaeocystis bloom.
phytoplankton material to the sediment via fecal pellets. In contrast, if sinking velocity is high and degradation low, particulate material from a sinking bloom may reach deeper layers and the sediment, generating sticky mats of Phaeocystis material (e.g., Ross Sea; Wassmann, 1994). Under these conditions a carbohydrate sink may be formed which then can become sulfurized. Similarly, a partially euxinic water column may also serve as a site suitable for sulfurization of algal carbohydrates assuming that the carbohydrates enter the euxinic zone before being completely degraded in the oxic part of the water column (e.g., the present day Black Sea). 4. CONCLUSIONS
Laboratory experiments aimed to simulate natural sulfurization of Phaeocystis spp. algal material resulted in the formation of a nonhydrolyzable, sulfur-rich macromolecular material, which was not present in the blank and the control experiment. No traces of extractable OSC were detected. A remarkable 60%
decrease in the carbohydrate content after sulfurization occurred. Most likely these carbohydrates, present in large amounts in the Phaeocystis algal material, are the precursors for the sulfur-rich macromolecular material. Sulfur incorporation, presumably at the position of the hydroxyl groups, yields a macromolecular matrix of polysulfidic cross-linked carbohydrate skeletons. Upon pyrolysis the relatively weaker S–S and C–S bonds (as compared to C–C) are cleaved and via rearrangement reactions mainly linear C0–C4 alkylthiophenes are generated. Upon laboratory sulfurization of glucose a blackish solid material is formed, which upon pyrolysis yields almost solely OSC, though with a different composition than those present in the pyrolysate of the sulfurized algal material. Both linear and branched alkylthiophenes and other organic sulfur compounds are formed, with their carbon numbers extending beyond six. The differences in the pyrolysis products are likely due to the differences in substrate, i.e., the monomer glucose compared to the complex mixture of polymeric algal carbohy-
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drates. The thiophenes in the pyrolysate of the sulfurized Phaeocystis material show a distribution similar to that of thiophenes in pyrolysates of type II-S kerogens, suggesting that a ubiquitous process in the formation of sulfur-rich kerogen could be the result of sulfur incorporation into (otherwise labile) carbohydrates. Therefore, sulfurization of carbohydrates is probably an important pathway in the preservation of organic matter in euxinic environments. This implies that if S-bound short-chain carbon skeletons are indeed important components of sulfur-rich kerogens then these moieties may have a major impact on oil and gas generation profiles and the timing of petroleum generation from source rocks. Acknowledgments—Dr. R. Osinga is thanked for help with initiating this project and for supplying the Phaeocystis spp. phytoplankton material. We thank Dr. I. Janse from the University of Groningen for performing the carbohydrate analyses. Dr. V. Schuman is gratefully acknowledged for collecting the Balsfjord sediments and phytoplankton sample. Ms. M. Dekker and Dr. W. Pool are thanked for analytical assistance. This work was supported by a PIONIER grant to JSSD by the Netherlands Organization for Scientific Research (NWO). This is NIOZ contribution no. 3460. REFERENCES Adam P., Philippe E., and Albrecht P. (1998) Photochemical sulfurization of sedimentary organic matter: A widespread process occurring at early diagenesis in natural environments? Geochim. Cosmochim. Acta 62, 265–271. Allard B., Templier J., and Largeau C. (1997) Artifactual origin of mycobacterial bacteran: Formation of melanoidin-like artifact macromolecular material during the usual isolation process. Org. Geochem. 26, 691–703. Baumann M. E. M., Lancelot C., Brandini F. P., Sakshaug E., and John D. M. (1994) The taxonomic identity of the cosmopolitan prymnesiophyte Phaeocystis: A morphological and ecophysiological approach. J. Mar. Syst. 5, 5–22. Brassell S. C., Lewis C. A., de Leeuw J. W., de Lange F., and Sinninghe Damste´ J. S. (1986) Isoprenoid thiophenes: Novel diagenetic products in sediments? Nature 320, 160 –162. de Graaf W., Sinninghe Damste´ J. S., and de Leeuw J. W. (1992) Laboratory simulation of natural sulphurization I. Formation of monomeric and oligomeric isoprenoid polysulphides by low-temperature reactions of inorganic polysulphides with phytol and phytadienes. Geochim. Cosmochim. Acta 56, 4321– 4328. de Graaf W., Sinninghe Damste´ J. S., and de Leeuw J. W. (1995) The low temperature additions of hydrogenpolysulphides to olefins: Formation of secondary polysulphides from 1-alkenes and heterocyclic and polymeric organic sulfur compounds from ␣,-dienes. J. Chem. Soc. Perkins Trans. I. 6, 635– 640. Deines P. (1980) The isotopic composition of reduced organic carbon. In Handbook of Environmental Isotope Geochemistry, Vol. 1, The Terrestrial Environment (eds. P. Fitz and J. Ch. Fontes), pp. 329 – 406, Elsevier, Amsterdam. Eglinton T. I. (1994) Carbon isotopic evidence for the origin of macromolecular aliphatic structures in kerogen. Org. Geochem. 21, 721–735. Eglinton T. I., Sinninghe Damste´ J. S., Kohnen M. E. L., and de Leeuw J. W. (1990a) Rapid estimation of the organic sulphur content of kerogens, coals and asphaltenes by pyrolysis– gas chromatography. Fuel 69, 1394 –1404. Eglinton T. I., Sinninghe Damste´ J. S., Kohnen M. E. L., de Leeuw J. W., Larter S. R., and Patience R. L. (1990b) Analysis of maturityrelated changes in organic sulfur composition of kerogens by flash pyrolysis– gas chromatography. In Geochemistry of Sulphur in Fossil Fuels (eds. W. L. Orr, and C. M. White), ACS Symp. Ser. 429, 529 –565. Eglinton T. I., Sinninghe Damste´ J. S., Pool W., de Leeuw J. W., Eijkel G., and Boon J. J. (1992) Organic sulphur in macromolecular sedimentary organic matter. II. Analysis of distributions of sulphur-
containing pyrolysis products using multivariate techniques. Geochim. Cosmochim. Acta 56, 1545–1560. Eilertsen H. C., Falk-Petersen S., Hopkins C. C. E., and Tande K. (1981a) Ecological investigations on the plankton community of Balsfjorden, northern Norway: Program for the project, study area, topography, and physical environment. Sarsia 66, 25–34. Eilertsen H. C., Schei B., and Taasen J. P. (1981b) Investigations on the plankton community of Balsfjorden, northern Norway: The phytoplankton 1976 –1978. Abundance, species composition, and succession. Sarsia 66, 129 –141. Fukushima K., Yasukawa M., Muto N., Uemara H., and Ishiwatari R. (1992) Formation of C20 isoprenoid thiophenes in modern sediments. Org. Geochem. 18, 83–92. Gelin F., Boogers I., Noordeloos A. A. M., Sinninghe Damste´ J. S., Riegman R., and de Leeuw J. W. (1997) Resistant biomacromolecules in marine microalgae of the classes Eustigmatophyceae and Chlorophyceae: Geochemical implications. Org. Geochem. 26, 659 – 675. Gelin F., Kok M. D., de Leeuw J. W., and Sinninghe Damste´ J. S. (1998) Laboratory sulfurisation of the marine microalga Nannochloropsis salina. Org. Geochem. 29, 1837–1848. Hartgers W. A., Lopez J. F., Sinninghe Damste´ J. S., Reiss C., Maxwell J. R., and Grimalt J. O. (1997) Sulphur-binding in recent environments. II. Speciation of sulphur and iron and implications for the occurrence of organo-sulphur compounds. Geochim. Cosmochim. Acta 61, 4769 – 4788. Janse I., van Rijssel M., Gottschal J. C., Lancelot C., and Gieskes W. W. C. (1996a) Carbohydrates in the North Sea during spring blooms of Phaeocystis: A specific fingerprint. Aquat. Microb. Ecol. 10, 97–103. Janse I., van Rijssel M., van Hall P. J., Gerwig G. J., Gottschal J. C., and Prins R. A. (1996b) The storage glucan of Phaeocystis globosa (Prymnesiophyceae) cells. J. Phycol. 32, 382–387. Kamerling J. P. and Vliegenthart J. F. G. (1989) In Clinical Biochemistry Principles, Methods, Applications (ed. A. M. Lawson), Vol. I, Mass Spectrometry, pp. 175–263, de Gruyter, Berlin. Kohnen M. E. L., Sinninghe Damste´ J. S., Kock-van Dalen A. C., ten Haven H. L., Rullko¨tter J., and de Leeuw J. W. (1990) Origin and diagenetic transformations of C25 and C30 highly branched isoprenoid sulphur compounds: Further evidence for the formation of organically bound sulphur during early diagenesis. Geochim. Cosmochim. Acta 54, 3053–3063. Kok M. D., Schouten S., and Sinninghe Damste´ J. S. (1995) Laboratory simulation of natural sulphurization: The reactivity of some functionalized model compounds and the catalytic influence of sediment. In Organic Geochemistry: Developments and Applications to Energy, Climate, Environment and Human History (eds. J. O. Grimalt et al.), A.I.G.O.A., Donostia-San Sebastia´n, pp. 1047–1050. Kok M. D., Rijpstra W. I. C., Robertson L., Volkman J. K., and Sinninghe Damste´ J. S. (2000) Steroid sulfurisation in surface sediments from Ace Lake, Antarctica. Geochim. Cosmochim. Acta 64, 1425–1436. Koopmans M. P., Sinninghe Damste´ J. S., Lewan M. D., and de Leeuw J. W. (1995) Thermal stability of thiophene biomarkers as studied by hydrous pyrolysis. Org. Geochem. 23, 583–596. Krein E. B. and Aizenshtat Z. (1993) Investigations of polysulphide phase transfer catalyzed (PTC) sulphur incorporation; mechanism and geochemical significance. In Organic Geochemistry (ed. K. Øygard), pp. 596 – 602, Falch Hurtigtrykk, Oslo. Krein E. B. and Aizenshtat Z. (1994) The formation of isoprenoid sulfur compounds during diagenesis: Simulated sulfur incorporation and thermal transformation. Org. Geochem. 21, 1015–1025. Lalonde R. T. (1990) Polysulphide reactions in the formation of organosulphur and other organic compounds in the geosphere. In Geochemistry of Sulfur Compounds in Fossil Fuels (eds. W. L. Orr and C. M. White), ACS Symp. Ser. 249, 68 – 82. Lalonde R. T., Ferrara L. M., and Hayes M. P. (1987) Low-temperature, polysulfide reactions of conjugated ene carbonyls: A reaction model for the geological origin of S-heterocycles. Org. Geochem. 6, 563–571. Lutter S., Taasen J. P., Hopkins C. C. E., and Smetacek V. (1989) Phytoplankton dynamics and sedimentation processes during spring and summer in Balsfjord, northern Norway. Polar Biol. 10, 113–124.
Formation of sulfur-rich macromolecules via sulfurization of algal carbohydrates Moers M. E. C., de Leeuw J. W., Cox H. C., and Schenck P. A. (1988) Interaction of glucose and cellulose with hydrogensulphide and polysulphides. In: Advances in Organic Geochemistry 1987 (eds. L. Mattavelli and L. Novelli), Org. Geochem. 13, 1087–1091. Nelson B. C., Eglinton T. I., Seewald J. S., Vairavamurthy M. A., and Miknis F. P. (1995) Transformations in organic sulfur speciation during maturation of Monterey Shale: Constraints from laboratory experiments. In Geochemical Transformation of Sedimentary Sulfur (eds. M. A. Vairavamurthy and M. A. A. Schoonen), ACS Symp. Ser. 612, 138 –166. Orr W. L. (1986) Kerogen/asphaltene/sulfur relationships in sulfur-rich Monterey oils. In Advances in Organic Geochemistry 1985 (eds. D. Leythaeuser and J. Rullko¨tter); Org. Geochem. 10, 499 –516. Philp R. P., Suzuki N., and Galvez-Sinibaldi A. (1992) Early-stage incorporation of sulfur into protokerogens and possible kerogen precursors. In Organic Matter: Productivity, Accumulation and Preservation in Recent and Ancient Sediments (eds. J. K. Welan and J. W. Farrington), pp. 264 –282, Columbia, New York. Richnow H. H., Jenisch A., and Michaelis W. (1992) Structural investigations of sulphur-rich macromolecular oil fractions and a kerogen by sequential chemical degradation. In: Advances in Organic Geochemistry 1991 (eds. C. B. Eckard, J. R. Maxwell, S. R. Larter, and D. A. C. Manning), Org. Geochem. 19, 351–370. Richnow H. H., Jenisch A., and Michaelis W. (1993) The chemical structure of macromolecular fractions of a sulfur-rich oil. Geochim. Cosmochim. Acta 57, 2767–2780. Rousseau V., Mathot S., and Lancelot C. (1990) Calculating carbon cell material of Phaeocystis sp. from microscopic observations. Marine Biology 107, 305–314. Rowland S., Rockey C., Al-Lihaibi S. S., and Wolff G. A. (1993) Incorporation of sulphur into phytol derivatives during simulated early diagenesis. Org. Geochem. 20, 1–5. Schouten S., van Driel G. B., Sinninghe Damste´ J. S., and de Leeuw J. W. (1993) Natural sulphurization of ketones and aldehydes: A key reaction in the formation of organic sulfur compounds. Geochim. Cosmochim. Acta 57, 5111–5116. Schouten S., de Graaf W., Sinninghe Damste´ J. S., van Driel G. B., and de Leeuw J. W. (1994) Laboratory simulation of natural sulphurization. II. Reaction of multi-functionalized lipids with inorganic polysulphides at low temperatures. In Advances in Organic Geochemistry 1993 (eds. N. Telnaes, G. van Graas, and K. Øygard), Org. Geochem. 22, 825– 834. Schouten S., Eglinton T. E., Sinninghe Damste´ J. S., and de Leeuw J. W. (1995) Influence of sulphur-crosslinking on the molecular size distribution of sulphur-rich macromolecules in bitumen. In Geochemical Transformations of Sedimentary Sulfur (eds. M. A. Vairavamurthy and M. A. A. Schoonen), ACS Symp. Ser. 612, 80 –92. Sinninghe Damste´ J. S. and de Leeuw J. W. (1990) Analysis, structure and geochemical significance of organically-bound sulphur in the geosphere: State of the art and future research. In Advances in Organic Geochemistry 1989 (eds. B. Durand and F. Behar), Org. Geochem. 16, 1077–1101. Sinninghe Damste´ J. S., Eglinton T. I., de Leeuw J. W., and Schenck P. A. (1989a) Organic sulphur in macromolecular sedimentary organic matter. I. Structure and origin of sulphur-containing moieties
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in kerogen, asphaltene and coal as revealed by flash pyrolysis. Geochim. Cosmochim. Acta 53, 873– 889. Sinninghe Damste´ J. S., Rijpstra W. I. C., Kock-van Dalen A. C., de Leeuw J. W., and Schenck P. A. (1989b) Quenching of labile functionalised lipids by inorganic sulphur species: Evidence for the formation of sedimentary organic sulphur compounds at the early stage of diagenesis. Geochim. Cosmochim. Acta 53, 1343–1355. Sinninghe Damste´ J. S., Eglinton T. I., Rijpstra W. I. C., and de Leeuw J. W. (1990) Characterisation of organically-bound sulfur, in highmolecular-weight sedimentary organic matter using flash pyrolysis and Raney Ni desulfurisation. In Geochemistry of Sulfur in Fossil Fuels (eds. W. L. Orr and C. M. White), ACS Symp. Ser. 429, 486 –528, American Chemical Society, Washington, DC. Sinninghe Damste´ J. S., Kohnen M. E. L., and Horsfield B. (1998) Origin of low-molecular-weight alkylthiophenes in pyrolysates of sulphur-rich kerogens as revealed by micro-scale sealed vessel pyrolysis. Org. Geochem. 29, 1891–1998. ten Haven H. L., Rullko¨tter J., Sinninghe Damste´ J. S., and de Leeuw J. W. (1990) Distribution of organic sulphur compounds in Mesozoic and Cenozoic sediments from the Atlantic and Pacific Oceans and the Gulf of California. In Geochemistry of Sulphur in Fossil Fuels (eds. W. L. Orr and C. M. White), ACS Symp. Ser. 429, 613– 632. Tomic J., Behar F., Vandenbroucke M., and Tang Y. (1995) Artificial maturation of Monterey kerogen (Type II-S) in a closed system and comparison with Type II kerogen: Implications on the fate of sulphur. Org. Geochem. 23, 647– 660. Vairavamurthy A. and Mopper K. (1987) Geochemical formation of organosulphur compounds (thiols) by additions of H2S to sedimentary organic matter. Nature 329, 623– 625. Valisolalao J., Perakis N., Chappe´ B., and Albrecht P. (1984) A novel sulphur-containing C35 hopanoid in sediments. Tetrahedron Lett. 25, 1183–1186. van Kaam-Peters H. M. E. and Sinninghe Damste´ J. S. (1997) Characterisation of an extremely organic sulfur-rich, 150 Ma old carbonaceous rock: Palaeoenvironmental implications. Org. Geochem. 23, 607– 615. van Kaam-Peters H. M. E., Schouten S., Ko¨ster J., and Sinninghe Damste´ J. S. (1998) Controls on the molecular and carbon isotopic composition of organic matter deposited in a Kimmeridgian euxinic shelf sea: Evidence for preservation of carbohydrates through sulfurisation. Geochim. Cosmochim. Acta 62, 3259 –3283. van Rijssel M., Hamm C. E., and Gieskes W. W. C. (1997) Phaeocystis globasa (Prymnesiophyceae) colonies: Hollow structures built with small amounts of polysaccharides. Eur. J. Phycol. 32, 185–192. Wakeham S., Sinninghe Damste´ J. S., Kohnen M. E. L., and de Leeuw J. W. (1995) Organic sulfur compounds formed during early diagenesis in the Black Sea. Geochim. Cosmochim. Acta 59, 521–533. Wassmann P. (1994) Significance of sedimentation for the termination of Phaeocystis blooms. J. Mar. Syst. 5, 81–100. Werne J. F., Hollander D. J., Behrens A., Schaeffer P., Albrecht P., and Sinninghe Damste´ J. S. (2000) Timing of early diagenetic sulfurization of organic matter: A precursor-product relationship in Holocene sediments of the anoxic Cariaco Basin, Venezuela. Geochim. Cosmochim. Acta 64, 1741–1751.
Pergamon
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Formation of insoluble, nonhydrolyzable, sulfur-rich macromolecules via incorporation of inorganic sulfur species into algal carbohydrates MARIKA D. KOK, STEFAN SCHOUTEN,* and JAAP S. SINNINGHE DAMSTE´ Department of Marine Biogeochemistry and Toxicology, Netherlands Institute for Sea Research (NIOZ), PO Box 59, 1790 AB Den Burg, Texel, the Netherlands (Received September 1, 1999; accepted in revised form March 8, 2000)
Abstract—The process of sulfur incorporation into organic matter was simulated in the laboratory by sulfurization of cell material of the prymnesiophyte alga Phaeocystis in sea water with inorganic polysulfides at 50°C. Flash pyrolysis of the residue, obtained after extraction and several hydrolysis steps, yielded mainly C1–C4 alkylbenzenes and C1–C4 alkylphenols and, in contrast to control and blank experiments, relatively high amounts of C0–C4 alkylthiophenes. The distribution of the thiophenes is very similar to that in pyrolysates of type II-S kerogens. The formation of high-molecular-weight sulfur-rich macromolecules co-occurs with a marked drop in the content of hydrolyzable carbohydrates. This indicates that sulfurization results in the preservation of algal carbohydrate carbon in a macromolecular structure composed of (poly) sulfidic cross-linked carbohydrate skeletons, which upon pyrolysis yields alkylthiophenes. Sulfurization of glucose under similar conditions resulted in the formation of a nonhydrolyzable, solid material, which yielded high amounts of organic sulfur compounds upon pyrolysis, mainly short-chain alkylthiophenes, although with a different distribution than that in the pyrolysate of the sulfurized algal material. The carbon numbers of these organic sulfur compounds extend beyond six, indicating that the length of the carbon skeleton of the pyrolysis products is not limited by the length of the carbon skeleton of the substrate. These results suggest that the sulfurization of carbohydrates may be an important pathway in the preservation of organic matter in euxinic depositional environments. Copyright © 2000 Elsevier Science Ltd weight (LMW) OSC, whereas studies on high-molecularweight (HMW) OSC are scant, probably because HMW OSC are often not gas chromatography (GC)-amenable. In some studies such HMW OSC have been analyzed by pyrolysis gas chromatography (py-GC-MS). For instance, Moers et al. (1988) reacted glucose with hydrogen sulfide and pyrolyzed the extracted and dried reaction mixtures, which yielded small amounts of thiophenic pyrolysis products. Philp et al. (1992) pyrolyzed the extracted residues of cyanobacterial mats, which were reacted with almost pure H2S, yielding trace amounts of thiophenes. Laboratory sulfurization experiments described in the literature have shown that sulfur incorporation can occur under relatively mild conditions, at temperatures of 50°C or less, without applying either extreme acidic or basic conditions or ultraviolet radiation. However, the reaction conditions applied and reagents used vary widely, e.g., elemental sulfur, hydrogen sulfide, and polysulfides have been used as inorganic sulfur source (Lalonde et al., 1987; Lalonde, 1990; Moers et al., 1988; Rowland et al., 1993). Other differences are associated with the reaction medium employed, e.g., (sea) water, a two-phase system of ethyl acetate and water or pure dimethyl formamide (Fukushima et al., 1992; de Graaf et al., 1992, 1995; Schouten et al., 1993, 1994). Sometimes, a base or a phase transfer catalyst was added to the reaction mixture (Krein and Aizenshtat, 1993, 1994; Rowland et al., 1993). All these different reactions were used to indicate the sulfurization potential of geochemically relevant organic compounds. Despite the use of the same substrate, differences in reaction conditions can result in the formation of different sulfurized reaction products. For example, sulfurization of phytenal produced (i) isoprenoid thiophenes when elemental sulfur was used in a dichloromethane
1. INTRODUCTION
In many sediments and oils, organic sulfur compounds (OSC) have been identified (Sinninghe Damste´ and de Leeuw, 1990). It is generally accepted that these OSC are formed during early diagenesis via the incorporation of inorganic sulfur species into functionalized lipids (Valisolalao et al., 1984; Brassell et al., 1986; Sinninghe Damste´ et al., 1989b; ten Haven et al., 1990; Kohnen et al., 1990; Wakeham et al., 1995). The location of sulfur incorporation corresponds with the position of the functional group in the precursor lipid molecule. Sulfurization of lipids is thus thought to represent a sink for relatively labile organic components. In this way, information on the precursor molecule, that otherwise would have been completely mineralized during diagenesis, will be preserved, which makes OSC carriers of paleoenvironmental information. The processes and timing of sulfur incorporation have been examined in several ways. Firstly, detailed molecular studies were performed of organic matter in recent sediments where these reactions actually take place (Wakeham et al., 1995; Hartgers et al., 1997; Kok et al., 2000; Werne et al., 2000). Secondly, structural studies have been done on sulfur bonding in organic matter in older sediments (Schouten et al., 1995; van KaamPeters and Sinninghe Damste´, 1997). Finally, the sulfurization process has been simulated in the laboratory (Lalonde et al., 1987; Vairavamurthy and Mopper, 1987; de Graaf et al., 1992, 1995; Krein and Aizenshtat, 1993, 1994; Kok et al., 1995; Adam et al., 1998). Most of these latter studies have concentrated on the formation and characterization of low-molecular-
*Author to whom ([email protected]).
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(DCM)/methanol (MeOH)/water mixture (48:48:4) with the aid of trimethylamine (Rowland et al., 1993); (ii) isoprenoid dithiolanes and trithianes in a two-phase system of ethyl acetate and water (1:1) using polysulfides and a phase transfer catalyst (Schouten et al., 1993); and (iii) oligomers consisting of polysulfidic cross-linked phytane skeletons when using polysulfides and a phase transfer catalyst in a two-phase system of toluene/ water (1:1) (Krein and Aizenshtat, 1994). Thus, reaction conditions in simulated sulfurization experiments should be kept as close to the natural situation as possible, as small differences can produce different products. In this paper we present the results of laboratory sulfurization experiments with a complex mixture of components of biologic origin, i.e., a phytoplankton concentrate collected during the bloom period of the alga Phaeocystis (Prymnesiophyceae). To mimic the natural situation as closely as possible, the reaction was performed in sea water, without the aid of any catalyst or organic solvent and polysulfides were used as the reduced inorganic sulfur source. In addition, we performed a laboratory sulfurization of glucose with polysulfides in water and studied the fate of Phaeocystis carbohydrates under oxic conditions in sediments from Balsfjord (northern Norway). The focus of these experiments was on the formation of highly resistant HMW sulfur-rich material and on comparisons of artificially produced sulfur-rich HMW organic matter with that in kerogens, to better understand the processes of HMW OSC formation. 2. EXPERIMENTAL 2.1. Sulfurization of Algal Material The algal material was collected during a spring bloom period and is predominantly composed of the prymnesiophyte Phaeocystis spp., a widespread algal species, mainly living in polar and temperate seas, but also in equatorial waters (Baumann et al., 1994). It forms massive spring blooms and has a high carbohydrate content. Phaeocystis colonies are composed of a ca. 7 m-thick layer of mucus surrounding an aqueous lumen, with cells present at the inside of the mucus layer (van Rijssel et al., 1997), instead of a colony filled with mucus. Although total carbohydrate concentrations of up to 90% have been reported previously (Rousseau et al., 1990), van Rijssel et al. (1997) showed that Phaeocystis species can have a carbohydrate content of up to ca. 35– 40% of total carbon during the stationary growth phase and is mainly present as the storage carbohydrate -glucan (Janse et al., 1996b). Algal material was collected during spring blooms of Phaeocystis spp. in the western Dutch Wadden Sea and the southern North Sea with plankton nets (50 m mesh) and stored frozen until use. Three different experiments were performed, i.e., a sulfurization, a control, and a blank experiment (Fig. 1). In the sulfurization experiment wet algal material (ca. 70 g dry weight), elemental sulfur (3 g), and NaHS (110 g) were gently stirred in sea water (1.5 L) for 4 weeks at a temperature of 50°C under a nitrogen atmosphere. As a control experiment, algal material was stirred for 8 weeks in sea water under a nitrogen atmosphere at 50°C, without the addition of any inorganic sulfur compounds. As a blank experiment, untreated fresh algal material was analyzed. The reaction mixtures of the sulfurization, control, and blank experiments were centrifuged and washed to remove most of the sea water and subsequently freeze dried, ultrasonically extracted (step 1; Fig. 1) with MeOH and DCM, followed by KOH- (step 2), HCl- (step 3), and H2SO4- (step 4) hydrolysis as described by Gelin et al. (1997). After each step, reaction mixtures were extracted with MeOH and DCM and the collected extracts were washed with distilled water. Elemental sulfur was removed from the extracts by reaction with activated Cu-curls and subsequent filtration. Before analysis by gas chromatography–mass spectrometry (GC-MS) the extracts were deri-
Fig. 1. Analytical scheme.
vatized with diazomethane and bis(trimethylsilyl)trisfluoro acetamide (BSTFA) in pyridine. The residues left after extraction were analyzed by flash pyrolysis– gas chromatography–mass spectrometry (py-GCMS). Carbohydrates in the final residues of the sulfurized algal material, the control experiment, and the fresh algal material were analyzed as described by Janse et al. (1996a) according to the slightly modified method of Kamerling and Vliegenthart (1989). Monosaccharide compositions were analyzed gas chromatographically with parallel analysis of derivatized standards of monosaccharides and mannitol for identification and quantification. 2.2. Sulfurization of Glucose Glucose (200 mg) dissolved in 6 ml of distilled water was heated at 50°C under a nitrogen atmosphere for 12 weeks after addition of 560 mg NaHS and 16 mg of elemental sulfur. The solid material formed was collected, washed with distilled water, and extracted with hexane to remove elemental S. The residual material was analyzed by py-GCMS. 2.3. Black Sea Sediments Sediment from Unit II of the Black Sea was Soxhlet extracted with MeOH/DCM (1:7.5; vol/vol) for 16 h. The residue was hydrolyzed with KOH (1N; 2 h reflux), brought to pH 4 with HCl and extracted with MeOH/H2O (1:1, 1⫻), MeOH (1⫻), and DCM (3⫻). Subsequently, the residue was hydrolyzed with HCl (4N; 6 h, 100°C), brought to pH 8 with KOH and, after freeze drying, another KOH hydrolysis and additional extraction were performed. The remaining residue was analyzed by py-GC-MS. 2.4. Balsfjord (Northern Norway) Samples Before, during, and after a bloom (15th April–28th May 1996) of the prymnesiophyte Phaeocystis sp., sediments were collected in Balsfjord, northern Norway (sediment depths ranging from 0 –20 cm). During the peak of the bloom period (29th April) a concentrate of the phytoplank-
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Fig. 2. (a) Total ion chromatogram (TIC) of the pyrolysate of the control algal material, (b) TIC of the pyrolysate of the sulfurized algal material, and (c) distribution of alkylthiophenes in the pyrolysate of the sulfurized algal material, as revealed by the partial accurate mass chromatogram of m/z 84 ⫹ 97 ⫹ 98 ⫹ 111 ⫹ 112 ⫹ 125 ⫹ 126 ⫹ 139 ⫹ 140 ⫹ 153 ⫹ 154. Compound assignments are listed in Table 1.
ton was collected with a plankton net. After extraction of the sediments with MeOH and DCM, the residues were analyzed by py-GC-MS and
compared with the pyrolysate of MeOH and DCM extracted phytoplankton material (mainly Phaeocystis sp.) collected during the bloom.
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M. D. Kok, S. Schouten, and J. S. Sinninghe Damste´ Table 1. Compounds identified in the pyrolysates.a
2.5. GC GC was performed using a Carlo Erba 5300 or a Hewlett-Packard 5890 instrument, both equipped with an on-column injector. A fused silica capillary column (25 m ⫻ 0.32 mm) coated with CP-Sil 5 (film thickness 0.12 m) was used with helium as carrier gas. The effluent of the Carlo Erba 5300 was monitored with a flame ionization detector (FID). The effluent of the Hewlett-Packard 5890 was monitored using both a FID and a sulfur-selective flame photometric detector (FPD), applying a stream-splitter with a split ratio of FID:FPD of ca. 1:2. The samples were injected at 70°C and the oven was programmed to 130°C at 20°C/min and then at 4°C/min to 320°C, at which it was held for 15 min. 2.6. GC-MS GC-MS analyses were performed with a Hewlett-Packard 5890 gas chromatograph interfaced with a VG Autospec Ultima mass spectrometer operated at 70 eV with a mass range of m/z 40 – 800 and a cycle time of 1.7 s (resolution 1000). The gas chromatograph was equipped with a fused silica capillary column (25 m ⫻ 0.32 mm) coated with CP Sil-5 (film thickness 0.12 m). The carrier gas was helium. The samples were injected on column at 60°C and subsequently the oven was programmed to 130°C at 20°C/min and then at 4°C/min to 320°C at which it was held for 10 min. 2.7. Curie-point py-GC-MS Py-GC-MS analyses were carried out on a Hewlett-Packard 5890 gas chromatograph equipped with a FOM-3LX unit for pyrolysis and interfaced to a VG Autospec Ultima mass spectrometer operated at 70eV with a mass range of m/z 35–700 and a cycle time of 1.7 s (resolution 1000). The samples were applied to a ferromagnetic wire with a Curie temperature of 610°C. The gas chromatograph, equipped with a cryogenic unit, was programmed from 0°C (5 min) to 300°C (10 min) at a rate of 3°C/min. Separation was achieved using a fused silica capillary column (30 m ⫻ 0.25 mm) coated with DB1701 (film thickness 0.25 m). Helium was used as a carrier gas. 3. RESULTS AND DISCUSSION
To study the sedimentary fate of carbohydrate-rich algal material under euxinic conditions, collected Phaeocystis cell material was reacted, for 4 weeks, with (poly)sulfides in sea water under a nitrogen atmosphere. As a control experiment, algal material was subjected to the same reaction conditions except for the absence of (poly)sulfides. The duration of this experiment was twice as long as the sulfurization experiment, i.e., 8 weeks. Fresh algal material was used as a blank. The reaction mixtures of the sulfurized and control experiment and of the blank algal material were subjected to extraction and several hydrolysis steps (Fig. 1) and the obtained extracts and residues were analyzed for the presence of OSC. 3.1. Extracts Major compounds present in the extracts obtained after steps 1, 2, 3, and 4, respectively, (Fig. 1) of the fresh, control, and sulfurized algal material are very similar and consist predominantly of fatty acids (mainly C14:0, C16:0, and C18:0), phytol, and C27 sterols. GC analysis using a sulfur-selective FPD revealed no OSC in the extracts of the sulfurized algal material. Even traces of OSC, e.g., isoprenoid thiophenes, could not be detected by GC-MS, indicating that under the conditions used no sulfur incorporation into lipids, resulting in GC-amenable and extractable OSC, had occurred. A similar laboratory sulfurization experiment with the marine microalga Nannochloropsis salina did result in the formation of minor amounts of
1 Toluene 2 Ethylbenzene 3 1,3-Dimethylbenzene ⫹ 1,4-Dimethylbenzene 4 Styrene 5 2-Methylpyrrole 6 3-Methylpyrrole 7 1-Ethyl-3-methylbenzene 8 1,2,4-Trimethylbenzene 9 1,2,3-Trimethylbenzene 10 Tetramethylbenzene 11 Methoxyphenol 12 Phenol 13 Methylphenol a
a b c d e f g h i j k l
Thiophene 2-Methylthiophene 3-Methylthiophene 2-Ethylthiophene 2,5-Dimethylthiophene 2,4-Dimethylthiophene 2,3-Dimethylthiophene 2-Propylthiophene 2-Ethyl-5-methylthiophene 2,3,5-Trimethylthiophene 2-Methyl-5-propylthiophene 2,3-Dimethyl-5-(2,6,10-trimethylundecyl)thiophene
Peak numbers refer to Fig. 2.
isoprenoid thiophenes (Gelin et al., 1998). It is unclear what the reason is for this difference.
3.2. Residues The pyrolysates of the final residue (residue 4, Fig. 1) of the control experiment and the fresh algal material are very similar and consist mainly of aromatic compounds (e.g., Fig. 2a, Table 1). The major compounds present are C1–C4 alkylated benzenes and C1–C4 alkylated phenols. However, the pyrolysate of the sulfurized algal cell material contains, apart from the aromatic compounds also encountered in the control and the blank experiment, large amounts of alkylated thiophenes (Fig. 2b). The thiophene distribution is dominated by C0–C4 alkylated thiophenes with a linear carbon skeleton (Fig. 2c). Other OSC present in the pyrolysate of the sulfurized algal material are C20 isoprenoid thiophenes, thiolanes, dithiophenes, and (methyl) benzothiophenes. All these thiophenes are almost absent from the pyrolysates of the final residues of the blank algal material and the control experiment. A co-pyrolysis experiment was performed to exclude the possibility that thiophenes were formed during pyrolysis by reaction of remnant inorganic sulfur with the algal material. Fresh algal material was mixed with an excess amount of elemental sulfur and the mixture was subsequently pyrolyzed. This resulted in the formation of traces of thiophenes in the co-pyrolysate, comparable to the relative amounts of thiophenes present in the pyrolysates of the fresh and the control experiment material. Hence, the presence of elemental sulfur during pyrolysis has no major influence on the formation of thiophenes. Allard et al. (1997) showed that during the process used to isolate resistant algal and bacterial material artifactual formation of melanoidin-like polymers can occur. Hence, it may be that the OSC present in the pyrolysate of the final residue were derived from artifacts formed during the isolation procedure. However, the difference in the relative abundance of the thiophenes in the pyrolysates of sulfurized experimental material versus control experimental material and blank algal biomass is already observed in residue 1. Furthermore, the thiophene distributions in the pyrolysates of residues 1 and 4 of the sulfurized algal material are very similar. Thus, melanoidin
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Fig. 3. Distribution of alkylthiophenes in the pyrolysates of (a) sulfurized Phaeocystis and (b) Black Sea sediment (0.006 Ma), a type II-S kerogen, as revealed by the partial accurate mass chromatograms of m/z 84 ⫹ 97 ⫹ 98 ⫹ 111 ⫹ 112 ⫹ 125 ⫹ 126 ⫹ 139 ⫹ 140 ⫹ 153 ⫹ 154.
formation is of no importance to the formation of the Sorg-rich moieties which yield alkylthiophenes upon pyrolysis. 3.3. Comparison with Type II-S Kerogens In flash pyrolysates of many type II-S kerogens, thiophenes are major compounds and, even in cases where thiophenes do not dominate the FID-traces, they are the most abundant OSC (Sinninghe Damste´ et al., 1989a; Eglinton et al., 1990a,b). Figure 3 shows the thiophene distribution in pyrolysates of the sulfurized algal cell material and of a typical type II-S kerogen of a Holocene Black Sea sediment. The thiophene distributions are very similar, suggesting that the formation of OSC in type II-S kerogens could proceed in a similar way to the formation of sulfurized algal cell material, as observed in the laboratory simulation experiment. Eglinton et al. (1992) showed that the 2-methylthiophene over toluene (2MT/Tol) ratio can be used to estimate the atomic Sorg/C ratio in pyrolysates of kerogens and that this ratio is
comparable to the 2,3-dimethylthiophene over 1,2-dimethylbenzene ⫹ n-non-1-ene ratio (Eglinton et al., 1990a). Type II-S kerogens have a Sorg/C ratio of ⬎0.04 (Orr, 1986), which corresponds to a 2MT/Tol ratio of ⬎0.38 (Eglinton et al., 1992). When the 2MT/Tol ratio is used to obtain an indication of the Sorg-richness of the algal material, an increase in this ratio is observed for the sulfurized material after each extraction and hydrolysis step (Table 2). The 2MT/Tol ratio of the pyrolysate of the sulfurized algal material after extraction with DCM and MeOH (residue 1) is an order of magnitude higher than the 2MT/Tol ratio of the corresponding pyrolysate of the control experiment or blank algal material (Table 2). After the various hydrolysis steps, which are applied to remove protein and carbohydrates, the 2MT/Tol ratio of the pyrolysate of the final residue of the sulfurized algal material has increased to 0.60, indicating that the Sorg is relatively more resistant to hydrolysis compared to the Corg material. Assuming that the composition of the nonhydrolyzable, sulfurized algal material
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Table 2. 2-Methylthiophene/toluene ratios in pyrolysates of laboratory simulation experiments of Phaeocystis, type II-S kerogen and Black Sea kerogen. Pyrolysates
2MT/Tol
Laboratory simulation experiments of Phaeocystis Blank experiment extracted residue (1) Blank experiment nonhydrolyzable residue (4) Control experiment extracted residue (1) Control experiment nonhydrolyzable residue (4) Sulfurized experiment extracted residue (1) Sulfurized experiment nonhydrolyzable residue (4) Type II-S kerogen (Sorg/C ⬎ 0.04) Black Sea kerogen
⬍0.01 0.02 0.01 0.06 0.13 0.60 ⬎0.38 0.22
may be compared with kerogen, (i.e., the several hydrolysis and extraction steps give results comparable to natural degradation), the 2MT/Tol ratio of 0.60 indicates that the Sorg content is similar to that of a type II-S kerogen. 3.4. Origin of Thiophenes Previously it has been suggested that alkylthiophenes, which are common in pyrolysates of type II-S kerogens, are generated from thiophene units with longer carbon skeletons already present in the macromolecular matrix by cleavage of C–C bonds, predominantly the -bond relative to the thiophene unit (Sinninghe Damste´ et al., 1990). However, laboratory sulfurization experiments with phytadienes, phytol, or phytenal as substrates resulted in the formation of cyclic isoprenoid sulfides or polysulfide cross-linked polymers, which only after heating (250 –300°C) resulted in the formation of isoprenoid thiophenes (de Graaf et al., 1992; Schouten et al., 1993, 1994; Krein and Aizenshtat, 1994). When Fukushima et al. (1992) and Rowland et al. (1993) used the same lipid substrates, this resulted directly in the formation of isoprenoid thiophenes. In a recent study, Sinninghe Damste´ et al. (1998) proposed a new pathway to explain the presence of alkylthiophenes in kerogen pyrolysates. Microscale sealed vessel (MSSV) pyrolysis of sulfur-rich kerogen shows that even at low pyrolysis temperatures (150°C) linear LMW alkylthiophenes are produced as the major S-containing pyrolysis products (apart from hydrogen sulfide). MSSV pyrolysis of a long-chain alkylthiophene and an alkylbenzene indicated that at 300°C for 72 h no -cleavage, leading to the generation of LMW alkylated thiophenes and benzenes, occurred. Several recent studies (Krein and Aizenshtat, 1994; Schouten et al., 1994; Koopmans et al., 1995; Tomic et al., 1995) have indicated that long-chain alkylthiophenes are stable thermal products formed at temperatures ranging from 250 –500°C, from carbon skeletons linked intermolecularly via several sulfide-bridges. This would indicate that linear LMW alkylthiophenes are predominantly formed by thermal degradation (i.e., during pyrolysis) of multiple (poly) sulfide-bound linear C5–C7 skeletons. This rearrangement model, proposed by Sinninghe Damste´ et al. (1998), would explain the early loss during maturation of the precursor moieties of the LMW alkylthiophenes as observed by Eglinton et al. (1990a,b), due to the relative weakness of the (poly)sulfide bonds. This model is also supported by a sulfur speciation study with X-ray absorption near edge structure (XANES) spectros-
copy of artificially matured kerogens (Nelson et al., 1995), which indicated that the relative amounts of organic (poly) sulfides show a significant decrease with increasing maturation temperature, whereas at the same time the relative amount of thiophenic sulfur remained rather constant. The thiophenes present in our pyrolysates may thus originate mainly from sulfurization of a precursor characterized by a small carbon skeleton and with multiple functionalities available for sulfur incorporation and sulfur cross-linking. Furthermore, it should be omnipresent in relatively high amounts in the environment. Carbohydrate substrates seem to be the perfect candidates, as they meet these requirements. Circumstantial evidence for the sulfurization of carbohydrates in the natural environment stems from investigations on the carbon isotopic composition of organic matter from the Kimmeridge Clay Formation (van Kaam-Peters et al., 1998). During these studies a positive relationship between the ␦13CTOC and the degree of sulfurization of the organic matter was observed. The ␦13CTOC was also positively correlated with the relative abundance of ␦13C-enriched C1–C3 alkylated thiophenes, generated during pyrolysis. It was suggested that these thiophenes are isotopically heavy because they might originate from carbohydrates that are relatively enriched in ␦13C (Deines, 1980). Thus, in the Kimmeridge Clay samples the relationship between an enrichment in ␦13CTOC with increasing TOC was explained by an increasing proportion of sulfurized carbohydrates in the kerogen (van Kaam-Peters et al., 1998). This is also in agreement with the presence of relatively isotopically heavy short-chain alkylthiophenes in a Monterey kerogen pyrolysate (Eglinton, 1994). Thus, it is suggested that carbohydrates are the precursor compounds for the sulfur-rich organic matter formed during the sulfurization experiments in this study. It is the same reason why carbohydrates, but not lipids, are likely to be the precursor compounds. As only HMW OSC are formed (no LMW OSC were observed in the extracts) the precursor compounds require a high number of functionalities for sulfur incorporation and cross-linking, resulting in the formation of a sulfidic crosslinked polymer. The precursor compounds should be abundant and have short (linear) carbon skeletons, as during pyrolysis linear C0–C4 alkylthiophenes are generated upon cleavage of the relatively weak C–S and S–S bonds (compared to C–C) present in the HMW OSC. To further support the idea that carbohydrates are the precursor compounds for the HMW OSC formed, the amounts of carbohydrates present in the fresh algal material, the control experiment, and the sulfurized algal cell material were determined. They amount to 35, 27, and 14 wt%, respectively, of the cell material (Fig. 4; Table 3). Thus, sulfurization results in a decrease of ca. 60% in carbohydrate content compared to the fresh material. In contrast, stirring alone at 50°C without added inorganic sulfur species for twice as long as the sulfurization experiment gives a decrease of only 23%, possibly due to melanoidin formation. This suggests that under the sulfurization conditions a carbohydrate-consuming reaction took place, i.e., sulfurization, in addition to removal of carbohydrates by simple heating. These results demonstrate that S-incorporation of carbohydrates is very likely under the conditions used and may also occur in nature. However, it does not exclude the formation of
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3.5. Sulfurized Glucose
Fig. 4. Absolute amounts (wt%) of hydrolyzable carbohydrate monomers present in the blank, control, and sulfurized algal material.
HMW OSC through the sulfurization of lipids. For instance, Gelin et al. (1998) showed that lipids of Nannochloropsis salina can form HMW OSC using the same sulfurization procedure. Probably the formation of HMW OSC depends on the composition of the reactive organic matter pool, i.e., the type and number of functionalized lipids and the amount of sugars available for sulfurization.
To further test the idea that sulfurization of carbohydrates leads to the formation of precursor moieties of the alkylthiophenes, a laboratory sulfurization with glucose as substrate was performed, under reaction conditions similar to those applied during the sulfurization of algal material. After 12 weeks of reaction a substantial amount of a blackish solid material had formed. After washing with water and extraction with hexane, this solid material yielded upon pyrolysis relatively high amounts of C0–C2 alkylthiophenes, with both branched and linear carbon skeletons, as well as some other OSC (Fig. 5a). Small amounts of trimethyl- and tetramethylthiophenes are also present, indicating that sulfurization of a hexose carbohydrate and subsequent pyrolysis can yield products with more than six C-atoms. The composition of the co-pyrolysate of glucose and an excess of elemental sulfur is rather different and contains mainly levoglucosan and other glucose-related pyrolysis products, with only relatively small amounts of 2-methylthiophene (Fig. 5b). This indicates that a solid, polymeric material composed of glucose carbon skeletons with multiple (poly-) S-links was formed during sulfurization. Upon pyrolysis of this material, C–S and S–S bonds are cleaved and thiophenes and other short-chain OSC are formed. S-incorporation is likely to occur at the hydroxylated C-atoms, similar to the sulfurization of the side chain of bacteriohopanetetrol, which contains four hydroxyl groups. Sulfurization of this compound results in the formation of up to four (poly)sulfide bonds, binding it tightly to the kerogen matrix (Richnow et al., 1992, 1993). When Moers et al. (1988) sulfurized glucose in the presence of either hydrogen sulfide or polysulfides, py-GC-MS of the reaction mixtures revealed low amounts of linear (acetyl)thiophenes formed as pyrolysis products. This difference may be due to the different sulfurization procedures used. Both the sulfurized algal material and the sulfurized glucose yield short-chain (linear) C0–C4 alkylthiophenes upon pyrolysis, though with different distribution patterns and relative abundances. The branched alkylthiophenes and other OSC present in the pyrolysate of the sulfurized glucose are absent or only minor compounds in the pyrolysate of the sulfurized algal material. These differences are likely due to the difference in composition of the substrates, i.e., the monomer glucose versus
Table 3. Amounts of hydrolyzable carbohydrate monomers present in the blank, control, and sulfurized algal material. Fresh algal material (blank)
Glucose Rhamnose O-Methylated hexoses Ribose O-Methylated pentoses Mannose Xylose Galactose Arabinose Total
Control algal material
Sulfurized algal material
abs. (wt%)
rel. (mol%)
abs. (wt%)
rel. (mol%)
abs. (wt%)
rel. (mol%)
0.5 0.8 0.9 1.3 2.3 2.3 6.3 6.7 13.5 34.6
1 2 2 4 6 6 19 17 43 100
0.3 0.4 0.7 0.3 1.1 1.1 5.2 5.9 12.2 27.2
1 2 2 1 4 4 20 19 47 100
0.2 0.4 0.1 0.4 0.8 0.8 2.1 4.0 5.0 13.9
2 3 1 3 6 5 16 26 38 100
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Fig. 5. TIC of the pyrolysates of (a) the sulfurized glucose and (b) the co-pyrolysis of glucose and elemental sulfur.
the complex mixture of polymeric algal carbohydrates, comprised of many monomers (Table 3). 3.6. Environmental Controls on Sulfurization of Carbohydrates The Balsfjord (northern Norway) is a permanently oxic fjord, with Phaeocystis spp. present throughout all seasons and dominating the phytoplankton during the bloom period in April/ May (Eilertsen et al., 1981a,b; Lutter et al., 1989; Schuman, personal communication). Before, during, and after this bloom, surface sediments and some deeper sediments (up to 20 cm depth) were collected to investigate whether some of the algal carbohydrates are sulfurized. Although the exact depth of oxygen penetration into the sediment is unknown, at least the deeper sediments are expected to be anoxic and, therefore, a possible site for sulfurization. However, the compositions of the pyrolysates of these sediments are all very similar and only
the pyrolysate of a surface sediment collected shortly after the peak of the Phaeocystis bloom is shown here (Fig. 6b). No thiophenes or carbohydrate pyrolysis products are present in these pyrolysates. The pyrolysate of a phytoplankton concentrate collected during the peak of the bloom period however, does contain a large amount of levoglucosan (Fig. 6a). Hence, it can be concluded that under oxic conditions in the water column and surface sediment, carbohydrates are degraded or consumed before a sedimentary carbohydrate reservoir can be formed, which would be available for sulfurization once the sediments become anoxic. Previous studies with sediment traps in Balsfjord (Northern, Norway) by Lutter et al. (1989) are in agreement with this conclusion. These authors showed that direct sedimentation of Phaeocystis is unlikely despite the massive spring bloom, due to grazing by herbivores and degradation and remineralization in the photic zone, resulting in transport of only highly resistant
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Fig. 6. TIC of the pyrolysates of the Balsfjord (Northern Norway) samples (a) phytoplankton concentrate and (b) sediment surface collected shortly after the Phaeocystis bloom.
phytoplankton material to the sediment via fecal pellets. In contrast, if sinking velocity is high and degradation low, particulate material from a sinking bloom may reach deeper layers and the sediment, generating sticky mats of Phaeocystis material (e.g., Ross Sea; Wassmann, 1994). Under these conditions a carbohydrate sink may be formed which then can become sulfurized. Similarly, a partially euxinic water column may also serve as a site suitable for sulfurization of algal carbohydrates assuming that the carbohydrates enter the euxinic zone before being completely degraded in the oxic part of the water column (e.g., the present day Black Sea). 4. CONCLUSIONS
Laboratory experiments aimed to simulate natural sulfurization of Phaeocystis spp. algal material resulted in the formation of a nonhydrolyzable, sulfur-rich macromolecular material, which was not present in the blank and the control experiment. No traces of extractable OSC were detected. A remarkable 60%
decrease in the carbohydrate content after sulfurization occurred. Most likely these carbohydrates, present in large amounts in the Phaeocystis algal material, are the precursors for the sulfur-rich macromolecular material. Sulfur incorporation, presumably at the position of the hydroxyl groups, yields a macromolecular matrix of polysulfidic cross-linked carbohydrate skeletons. Upon pyrolysis the relatively weaker S–S and C–S bonds (as compared to C–C) are cleaved and via rearrangement reactions mainly linear C0–C4 alkylthiophenes are generated. Upon laboratory sulfurization of glucose a blackish solid material is formed, which upon pyrolysis yields almost solely OSC, though with a different composition than those present in the pyrolysate of the sulfurized algal material. Both linear and branched alkylthiophenes and other organic sulfur compounds are formed, with their carbon numbers extending beyond six. The differences in the pyrolysis products are likely due to the differences in substrate, i.e., the monomer glucose compared to the complex mixture of polymeric algal carbohy-
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drates. The thiophenes in the pyrolysate of the sulfurized Phaeocystis material show a distribution similar to that of thiophenes in pyrolysates of type II-S kerogens, suggesting that a ubiquitous process in the formation of sulfur-rich kerogen could be the result of sulfur incorporation into (otherwise labile) carbohydrates. Therefore, sulfurization of carbohydrates is probably an important pathway in the preservation of organic matter in euxinic environments. This implies that if S-bound short-chain carbon skeletons are indeed important components of sulfur-rich kerogens then these moieties may have a major impact on oil and gas generation profiles and the timing of petroleum generation from source rocks. Acknowledgments—Dr. R. Osinga is thanked for help with initiating this project and for supplying the Phaeocystis spp. phytoplankton material. We thank Dr. I. Janse from the University of Groningen for performing the carbohydrate analyses. Dr. V. Schuman is gratefully acknowledged for collecting the Balsfjord sediments and phytoplankton sample. Ms. M. Dekker and Dr. W. Pool are thanked for analytical assistance. This work was supported by a PIONIER grant to JSSD by the Netherlands Organization for Scientific Research (NWO). This is NIOZ contribution no. 3460. REFERENCES Adam P., Philippe E., and Albrecht P. (1998) Photochemical sulfurization of sedimentary organic matter: A widespread process occurring at early diagenesis in natural environments? Geochim. Cosmochim. Acta 62, 265–271. Allard B., Templier J., and Largeau C. (1997) Artifactual origin of mycobacterial bacteran: Formation of melanoidin-like artifact macromolecular material during the usual isolation process. Org. Geochem. 26, 691–703. Baumann M. E. M., Lancelot C., Brandini F. P., Sakshaug E., and John D. M. (1994) The taxonomic identity of the cosmopolitan prymnesiophyte Phaeocystis: A morphological and ecophysiological approach. J. Mar. Syst. 5, 5–22. Brassell S. C., Lewis C. A., de Leeuw J. W., de Lange F., and Sinninghe Damste´ J. S. (1986) Isoprenoid thiophenes: Novel diagenetic products in sediments? Nature 320, 160 –162. de Graaf W., Sinninghe Damste´ J. S., and de Leeuw J. W. (1992) Laboratory simulation of natural sulphurization I. Formation of monomeric and oligomeric isoprenoid polysulphides by low-temperature reactions of inorganic polysulphides with phytol and phytadienes. Geochim. Cosmochim. Acta 56, 4321– 4328. de Graaf W., Sinninghe Damste´ J. S., and de Leeuw J. W. (1995) The low temperature additions of hydrogenpolysulphides to olefins: Formation of secondary polysulphides from 1-alkenes and heterocyclic and polymeric organic sulfur compounds from ␣,-dienes. J. Chem. Soc. Perkins Trans. I. 6, 635– 640. Deines P. (1980) The isotopic composition of reduced organic carbon. In Handbook of Environmental Isotope Geochemistry, Vol. 1, The Terrestrial Environment (eds. P. Fitz and J. Ch. Fontes), pp. 329 – 406, Elsevier, Amsterdam. Eglinton T. I. (1994) Carbon isotopic evidence for the origin of macromolecular aliphatic structures in kerogen. Org. Geochem. 21, 721–735. Eglinton T. I., Sinninghe Damste´ J. S., Kohnen M. E. L., and de Leeuw J. W. (1990a) Rapid estimation of the organic sulphur content of kerogens, coals and asphaltenes by pyrolysis– gas chromatography. Fuel 69, 1394 –1404. Eglinton T. I., Sinninghe Damste´ J. S., Kohnen M. E. L., de Leeuw J. W., Larter S. R., and Patience R. L. (1990b) Analysis of maturityrelated changes in organic sulfur composition of kerogens by flash pyrolysis– gas chromatography. In Geochemistry of Sulphur in Fossil Fuels (eds. W. L. Orr, and C. M. White), ACS Symp. Ser. 429, 529 –565. Eglinton T. I., Sinninghe Damste´ J. S., Pool W., de Leeuw J. W., Eijkel G., and Boon J. J. (1992) Organic sulphur in macromolecular sedimentary organic matter. II. Analysis of distributions of sulphur-
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