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Early diagenesis of organic matter and related sulphur incorporation in surface sediments of meromictic Lake Cadagno in the Swiss Alps
Org. Geoehem. Vol. 25, No. 5-7, pp. 379-390, 1996
Pergamon PII: S0146-6380(96)00143-X
© 1997 ElsevierScienceLtd Printed in Great Britain.All rights reserved 0146-6380/96 $15.00 + 0.00
Early diagenesis of organic matter and related sulphur incorporation in surface sediments of meromictic Lake Cadagno in the Swiss Alps A N K E PUTSCHEW, BARBARA M. SCHOLZ-BOTTCHER and J I ] R G E N RULLKOTTER* Institute of Chemistry and Biology of the Marine Environment (1CBM), Carl von Ossietzky University of Oldenburg, P.O. Box 2503, Oldenburg D-26111, Germany Abstract--Samples of Recent anoxic sediment ( < 100 yr BP) from the meromictic Lake Cadagno (Switzerland) were examined to study sulphur incorporation into organic matter at a very early stage of diagenesis. Trace amounts of two organosulphur compounds were identified in the extractable organic matter. Desulphurization of the heterocompound and asphaltene fractions with nickel boride yielded hydrocarbons with a strong dominance of phytane and minor amounts of n-alkanes, steranes and squalane, but may also have converted ester-bound moieties into hydrocarbons as shown by a control experiment with a chloropyll a standard. The amount of sulphur-bound compounds is always higher than the amount of the free precursors, even in the shallowest sediments. This indicates that the formation of organosulphur compounds starts already in the water column or is very effective in the uppermost millimeters of the sediment layer. Stable carbon isotope data of the sedimentary compounds, and of biological material from the water column and from land biota growing around the lake, show that the sulphur-bound phytane in the youngest samples is dominated by phytane of bacterial origin. With increasing depth the amount of phytane of terrestrial origin slightly increases. ~(7) 1997 Elsevier Science Ltd compounds, Lake Cadagno sediments, anoxic sediment, sulphur incorporation, nickel boride desulphurization Key words--Organosulphur
INTRODUCTION Over more than a decade, organosulphur compounds (OSC) have become a subject of increasing interest in organic geochemistry. About 1500 new structures were identified in this class of compounds (for a review see Sinninghe Damst6 and de Leeuw, 1990). The intense research in the field of OSC has markedly increased our knowledge of the formation of these compounds and their geochemical significance. OSC are products of reactions of reduced inorganic sulphur species with functionalized lipids during early diagenesis, First evidence of such reactions was obtained from the identification of OSC structurally closely related to known biochemical precursors (e.g. Valisolalao et al., 1984; Brassell et al., 1986; Sinninghe Damst6 and de Leeuw, 1987; Sinninghe Damst~ et al., 1987; Kohnen et al., 1990). The products of laboratory simulation of sulphur incorporation into organic substrates are in agreement with the idea of abiotic formation of OSC during early diagenesis (de Graaf et al., 1992; Rowland et al., 1993; Bisseret and Rohmer, 1993; Schouten et al., 1993b, 1994). The reactions involve intra- and/or intermolecular addition of reduced inorganic sulphur species, possibly H S - or polysul*Corresponding author.
phides, into functionalized biogenic lipids (Kohnen et al., 1991a, 1993). Intramolecular sulphur incorporation usually leads to low-molecular-weight components often separable by capillary column gas chromatography (GC) whereas intermolecular reactions produce high-molecular-weight material. The initial structure elucidations, restricted to the GC-amenable OSC fraction, revealed thiophenes, thiolanes, thianes and benzothiophenes as the main compound classes of fossil organosulphur compounds. Advanced analytical techniques like pyrolysis-GC and chemical degradation provided access to the macromolecular OSC by the analysis of their pyrolysis products and the hydrocarbons liberated by desulphurization, respectively (Boudou et al., 1987; Eglinton et al., 1990, 1992; Sinninghe Damst+ et al., 1988a,b, 1989a; Sinninghe Damst~ and de Leeuw, 1990; Schouten et al., 1993a; Kohnen et al., 1991a; Hoffman et al., 1992; Richnow et al., 1992). The biological markers bound via sulphur bridges to the macromolecular sedimentary organic matter provide a particularly well preserved source of information for palaeoenvironmental reconstructions (Sinninghe Damst~ et al., 1989b; de Leeuw and Sinninghe Damst~, 1990), and OSC have to be considered in such assessments in order to avoid biases due to structure- and/or source-specific fractionation between the free and sulphur-bound bio-
379
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Anke Putschew et al. •. ~._~,,Lake Constance
Lake Cadagno Fig. 1. Location map of Lake Cadagno in the Swiss Alps. marker pools during sulphurization (Kohnen et al., 1991b). Until now, virtually all molecular investigations of OSC formation used sediments older than 10,000yr [except those performed on Black Sea (Wakeham et al., 1995) and Peru margin sediments (Kohnen et al., 1991b)] and, with a few exceptions (e.g. Rullk6tter et al., 1990; Barakat and Rullk6tter, 1994, 1995), were restricted to marine deposits. In the present study, recently deposited (< 100 yr) sedimentary organic matter from Lake Cadagno (Switzerland) was analyzed. Lake Cadagno which is located at an altitude of 1923 m above sea level in the Ticino Alps (Fig. 1), is characterized by a chemocline fluctuating over the year between 8 and 12 m water depth. Below the chemocline the water column is permanently anoxic. A constant inflow of sulphate-rich groundwater is responsible for the high activity of sulphate-reducing bacteria in the lower part of the water column and in the surface sediments. Near the chemocline, phototrophic sulphur bacteria (essentially Chromatium spp. based on the intense red colour and microscopic examinations; Ztillig, 1985; Losher, 1989; K. Hanselmann, private communication, 1995) are the main primary producers of organic matter in the water column. The sediment is rich in organic carbon and total sulphur. The organic matter is mainly autochthonous with only a small contribution of allochthonous material of terrestrial higher plant origin (Putschew et al., 1995). In addition to the high organic carbon and sulphur contents, the low concentration of ferrous iron (Losher, 1989) should favour the formation of OSC in this lake system. Relative to an earlier publication of preliminary results (Putschew et al., 1995) which had a strong emphasis on compound identification,
interpretation of sulphurization processes and isotopic data has been significantly modified and extended in this communication.
EXPERIMENTAL Samples
A piston core of 36 cm length and a diameter of 7 cm was provided by A. Losher (ETH Ziirich). It was split into sections of 2 cm thickness at the sampling site, and the sections were immediately frozen. The sediment was homogeneously black and had a strong odour of hydrogen sulphide, which ceased considerably below a depth of 32 cm. In August 1994, grass from the lake shore and green macroalgae floating on the lake surface were sampled. For the recovery of biological material from the water column, water from different water depths (7 m, 11.7 m and 19 m) was pumped and filtered. The particle size of this material ranged from 22 #m down to 0.45 #m, The biological material was immediately frozen at the sampling site. Methods
The freeze-dried sediment samples were ultrasonically extracted with dichloromethane. After addition of internal standards (squalane, thianthrene, 5c¢(H)-androstan-17-one) the extract was separated into fractions soluble in n-hexane and dichloromethane, respectively (asphaltene precipitation). The dichloromethane fraction is operationally termed asphaltenes throughout this paper. The compounds soluble in n-hexane were separated on a 30 cm x 1.6 cm i.d. column filled with 16 g of silica 100 deactivated with 5% water. The nonaromatic hydrocarbon fraction eluted with 40 ml n-hexane,
381
Early diagenesis of organic matter the aromatic hydrocarbon fraction with 60 ml nhexane/dichloromethane (9:1 by volume) and the hetero(NSO)-compounds with 80 ml dichloromethane/methanol (9:1 by volume). Aliquots of the asphaltene and NSO fractions were desulphurized with Ni2B as described by Schouten et al. (1993a). The released hydrocarbons were isolated by column chromatography (dimensions as above) by elution with four times the column volume of n-hexane/dichloromethane (9:1 by volume). The hydrocarbons were analyzed before and after hydrogenation with a PrO2 catalyst. In a control experiment, 1 mg chlorophyll a and 500 #g squalane (internal standard) were dissolved in 2 ml dry tetrahydrofurane and 2 ml methanol and, by addition of 50 mg NiC12 and 50 mg NaBH4 and heating under reflux for 1 h, were treated with nickel boride in the same way as described for the sediment material. Separation of the hydrocarbon fraction was performed as described above, and polar compounds were recovered from the liquid chromatography column with 5 ml methanol/ dichloromethane (1:1). Hydrocarbon products were analyzed without further hydrogenization, the polar fraction was hydrolyzed with KOH in methanol/ water (see below) and, after derivatization of the products with M S T F A (N-methyl-N-trimethylsilyltrifluoroacetamide), analyzed for residual phytol. Recovery of the internal standard in this and a blank experiment (without chlorophyll a) were 39% and 36%, respectively. Selected polar extract fractions and the freezedried biological material were saponified with 20 ml of 5% KOH in methanol/water (8:2 by volume) for 24 h under reflux. The n-hexane-soluble organic matter was separated by liquid chromatography over a silical gel column as described before. Alcohols were derivatized with MSTFA before analysis. GC analysis was carried out on a Hewlett Packard 5890 series II instrument equipped with a temperature-programmable injector (Gerstel KAS 3) and a flame ionization detector (FID) or a sulphur-selective chemoluminescence detector (Sievers SCD). The detection level for sulphur was 17 pg S/ #1. A DB-5 (J&W) fused silica capillary column (30 m x 0.25 mm i.d., 0.25 ttm film thickness) was used with helium as the carrier gas. The temperature of the GC oven was programmed from 60°C (1 min isothermal) to 300°C (50 min isothermal) at 3°C/rain. The injector temperature was programmed from 60°C (5 s hold time) to 300°C (60 s hold time) at 8°C/s. Gas chromatography-mass spectrometry (GC-MS) and gas chromatography-isotope ratio monitoring-mass spectrometry (GC-irm-MS) measurements were performed with the same type of GC system using the conditions described above. For G C - M S analysis, the gas chromatograph was OG 25"5-7 g
coupled to a Finnigan SSQ 710B mass spectrometer operated at 70 eV with a scan range of m/z 50-600 and a scan time of 1 scan/s. For G C - i r m MS analysis the gas chromatograph was attached to a Finnigan MAT 252 isotope ratio mass spectrometer with a Finnigan combustion interface. Carbon isotope ratios were calibrated with a COz standard at the beginning and the end of each analysis and with compounds of known isotopic composition which were co-injected with each sample and served as internal isotopic standards. Isotope ratios are expressed as ~13C values in permil relative to the PDB standard. The 6t3C value of phytol was corrected for the isotopic contribution of the trimethylsilyl group using the following equation: ~13Cphytol_TM S :
20/23~13Cphytol+
(1 - 20/23)313C-rMs with ~I3CTMS = -40.35 :t: 1.38%o.
RESULTS AND DISCUSSION
Bulk and nonaromatic hydrocarbon geochemistry The Lake Cadagno sediments have maximum organic carbon concentrations beween 10% and 14% in the top 10cm and show slightly lower values below this depth (Putschew et al., 1995). Uniform C/N ratios around 10 indicate a more or less constant organic matter type over the entire 36 cm depth interval studied and a dominance of aquatic material. Total sulphur contents vary between 1% and 3.5%, and the Corg/Stot ratios range from 6 to 1.5 with a depth trend virtually paralleling that of the organic carbon content (Putschew et al., 1995). Extract yields are high throughout (41-153mg/g Corg) with the higher values occurring in the upper sediment section. The extracts are dominated by heterocomponents (usually close to, or more than 50%) while nonaromatic hydrocarbons typically amount to 20-30% and asphaltenes to about 10% of total extract. Elemental sulphur is abundant in the shallower sediments. The nonaromatic hydrocarbon fractions are dominated by n-alkanes with a strong odd-overeven carbon number predominance and a maximum at n-C29 or n-C31. In the absence of major tree and bush vegetation at the high altitude, the most likely source is the grass growing around the lake. Hopanoid hydrocarbons of microbial origin are the second-most important group of constituents of the nonaromatic hydrocarbon fractions. Within this group, diploptene (hop-22(29)-ene) is always the compound with the highest relative concentration.
382
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Fig. 2. Partial reconstructed ion chromatogram and mass chromatograms (m/z 98, 111,308) of the aliphatic hydrocarbon fraction of the sample from 0-2cm depth. 21:0 = n-henicosane, 21:1 = n-henicosene, A = 3-methyl-2-(3,7,1l-trimethyldodecyl)thiophene, B = 3-(4,8,12-trimethyltridecyl)thiophene. Phytane and phytenes are detectable in small amounts decreasing with depth. Sterenes are present only in very low concentrations except a compound tentatively identified as 23,24-dimethylcholesta3,5,22-triene (Putschew et al., 1995) which is most likely derived from dinoflagellates known to occur above the chemocline in the lake (K. Hanselmann, private communication 1993). Low-molecular-weight OSC & the nonaromatic hydrocarbon fractions
Despite the anoxic conditions in the lower part of the lake and the high sulphur content of the sediments, only a few GC-amenable OSC were detected which, due to the liquid chromatographic separation scheme applied, occur in the "nonaromatic hydrocarbon" fractions. Mass chromatography of molecular ions and key fragments revealed the presence of 3-methyl-2-(3,7,11-trimethyldodecyl)thiophene and 3-(4,8,12-trimethyltridecyl)thiophene in small amounts (Fig. 2). Both compounds often identified in ancient sediments (Rullk6tter et al., 1982, 1984; ten Haven et al., 1985; Brassell et al., 1986) are thought to be products of early diagenesis. None of the more highly substituted C20 thiophene isomers, e.g. 2,3-dimethyl-5-(3,7,1 ltrimethyldocecyl)thiophene, indicative of higher salinity in the water column at the time of deposition
(de Leeuw and Sinninghe Damst6, 1990), could be detected. Also, no GC-amenable organosulphur compounds were found in the aromatic hydrocarbon fractions (typically only 3% of total extract) within the detection limits of the sulphur-selective detector. Macromolecularly bound O S C
Upon treatment of the NSO fractions and asphaltenes with Ni2B, intended to desulphurize macromotecular entities, major amounts of hydrocarbons were released. Both types of starting materials yielded virtually identical product distributions. Phytane is always the hydrocarbon obtained in highest relative concentration even before hydrogenation. A typical example of a sulphur-bound hydrocarbon distribution (after hydrogenation) from desulphurization of an NSO fraction is shown in Fig. 3. n-Alkanes, squalane and C27, C28 and C29 steranes were minor products. The n-alkanes range from Ci7 to at least C35, and their distributions maximize at n-C26. Other than in the free hydrocarbon fractions, the even-carbon-number homologues dominate over the odd-carbon-numbered n-alkanes, and this preference is only slightly pronounced. The dissimilarity of this n-alkane distribution with the carbon number distributions of both n-fatty acids and n-alkanols suggests a horn-
Early diagenesis of organic matter ologous series of precursors which is not normally observed in the low-molecular-weight bitumen fractions. Strong differences between free n-alkanes and n-alkane distributions obtained after desulphurization of low- and high-molecular-weight OSCs and heterocompound fractions have been noted before, e.g. for the Jurf ed Darawish oil shale (Sinninghe Damst6 et al., 1988a), for sediments from the Monterey Formation (Schouten et al., in press), and for crude oils (Schouten et al., 1995; Rullkrtter and Michaelis, 1990). In most of these cases, the nalkanes from desulphurization showed little resemblance with carbon number distributions of common classes of biogenic compounds with a straight aliphatic carbon chain. A notable exception in this apparent lack of specificity was the occurrence of C37 and C38 n-alkanes in several desulphurized hydrocarbon fractions which were related to longchain aikenones of prymnesiophyte algae (Sinninghe Damst6 et al., 1988a; Rullkrtter and Michaelis, 1990; Schouten et al., 1995). Similar to the n-alkanes, the steranes in the heterocompound (NSO) and asphaltene desulphurization products of the Lake Cadagno sample series show a relatively smooth distribution. Apparently, the C29 steratriene, i.e. 23,24-dimethylcholesta3,5,22-triene, dominating the steroid hydrocarbons
383
in the free nonaromatic hydrocarbon fractions, was not affected by sulphur incorporation to a significant extent. The presence of its sulphur-bound counterpart, in small concentrations in the macromolecular fractions, cannot be readily deduced because in the saturated form 23,24-dimethylcholestane will coelute with 24-ethylcholestane under the analytical conditions applied as has been shown by hydrogenation of a nonaromatic hydrocarbon fraction and coinjection with a 24-ethylcholestane standard during structure elucidation (Putschew et al., 1995). The end member of a pseudohomologous series of diagenetic alteration products of diaromatic carotenoids found in the desulphurization products, although in small concentrations, was either isorenieratane or renieratane (Fig. 4b, compound dA) which are characteristic of phototrophic sulphur bacteria (Liaaen-Jensen, 1978). The isomers cannot be differentiated by their mass spectra which are both dominated by a base peak at m/z 133 (Hartgers et al., 1994a), and standards were not available for identification based on gas chromatographic retention time. The degraded carotenoids, also characterized by a base peak at m/z 133 in their mass spectra, ranged from Cls to C27 with the CI7 and C23 members missing due to the isoprenoid
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384
Anke Putschew et al. tic zone of the lake and shows that degradation of diaromatic carotenoids does not require thermal stress, but may possibly even be induced photochemically. Recently a critical examination of the desulphurization reaction with Raney nickel applied to modern sediments revealed not only that sulphur bonds are broken but also that the chlorophyll a sidechain may be hydrogenolyzed yielding phytane as one of the products (Prahl et al., 1996). The phytane deriving from the carboxylic ester functionality of chlorophyll a certainly was not discernible from phytane produced by desulphurization of macromolecular OSC in the course of the Raney nickel treatment. Nickel boride appeared to be less reactive towards ester bonds because Hartgers et al. (1995) reported that octadecanoic acid methyl ester was not cleaved by Ni2B while other side-reactions with weak bonds comprising heteroatoms other than sulphur did
branching in the chain. They are the dominant compounds in the summed m / z (133 + 134) mass chromatogram in Fig. 4b. The carbon number distribution of the degradation products differs from that observed by Hartgers et al. (1994a) in the highmolecular-weight bitumen fractions of sediments from the Upper Devonian Duvernay Formation (Western Canada Basin). In that case, the distribution had a maximum at Cl4, and the relation to the branching in the chain was less clear due to the presence of small amounts of C~7 and C23 pseudohomologues. Like in the Duvernay Formation sediments, the diaromatic carotenoid degradation products in the Lake Cadagno samples also comprise a series with a base peak at m / z 134 which, in minute concentrations, elutes before the major series in the mass fragmentogram in Fig. 4b. Hartgers et al. (1994a) ascribed this series to a novel diaromatic carotenoid with one benzene ring carrying a 3,4,5-trimethyl-l-alkyl substitution pattern. The identification of the diaromatic carotenoid derivatives in the Lake Cadagno sediments reflects the sulphide-rich anoxic water body within the pho-
o c c u r .
Under the conditions applied during the control experiment in the course of this study, however, the ester bond of a chlorophyll a standard was reduc-
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Early diagenesis of organic matter tively cleaved very efficient. Relative to the internal standard, the yield of phytane and phytenes was 58.5% of the theoretical yield; the remainder was not recoverable as phytol, however, indicating that additional side reactions occurred as well. Furthermore, the overall recovery of the standard - - and, thus, of the ester cleavage products - - was remarkably low, also in a blank experiment. On the other hand, the hydrocarbon distributions in the products of the chlorophyll a reaction with Ni2B (4% phytane, 19% phyt-l-ene, 77% phyt-2-ene) and of the Ni2B reaction with pure phytol (phytene/ phytane ratio of 35:25; Hartgers et al., 1995) are significantly different from the product distribution of the nickel boride treatment of the polar and asphattene fractions extracted from the Lake Cadagno sediments, which typically contained a large abundance of phytane, but only relatively small amounts of phytenes. This altogether indicates, that the discussion of reductive cleavage of sulfur bonds of macromolecular entities in the sediment extracts of Lake Cadagno samples is valid at least in qualitative terms, but that the reaction products may comprise products from ester cleavage to a certain extent which affects the significance of the quantitative data discussed below. Absolute concentrations were determined for the amounts of squalane and phytane released upon desulphurization (after hydrogenation) as well as for their structurally related free analogs, i.e. squalene (aromatic hydrocarbon fraction), phytane, phytenes (nonaromatic hydrocarbon fractions) and phytol (NSO fractions), in the top 36 cm of Lake Cadagno sediments. The yields of sulphur-bound (a)
(b)
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squalane and free squalene are significantly higher in most samples from the shallower half of the sediment section studied than in the deeper samples (Fig. 5). Concentrations of sulphur-bound squalane vary from 100 to 200 pg/g Corg in the top 16 cm of the sedimentary column and from about l0 to 70 #g/g Corg below this depth. Free squalene typically is also more abundant in the shallower sediment section (up to 80gg/g Corg), whereas concentrations in most of the deeper sediments are around 20 #g/g Corg (Fig. 5). The fraction of sulphur-bound squalane, except for the sample at 4 cm depth, is more abundant than the fraction of free squalene, although the difference is less pronounced below a depth of about 16 cm. This can clearly be seen from the percentages of free squalene and sulphur-bound squalane relative to the sum of both fractions (Fig. 5c). At the bottom of Lake Cadagno bacterial sulphate reduction is active down to a depth of about 20 cm into the sediment (Losher. 1989). Should squalene be metabolized by sulphatereducing bacteria, the results of this study would show that macromolecularly bound squalane, as expected, is more resistant to microbial degradation than the corresponding free compound. Figure 6 shows the depth profiles of sulphurbound phytane and the concentrations of the related free compounds phytol, phytane and phytenes. Although the concentrations of sulphur-bound phytane are high in all samples studied (up to more than 2000 #g/g Corg), there are strong fluctuations in the entire sediment core rather then a depth trend. In contrast to this, the concentrations of free phytane, phytene isomers and phytol in the low-
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molecular-weight bitumen fractions decrease with sediment depth even if some scatter is obvious in the upper part of the phytol profile (Fig. 6). It is conceivable that the large scatter in the bound phytane concentrations, particularly in the shallower samples, is affected by contributions of phytane derived from ester-bound phytol as discussed before. While the free phytol concentration in the depth profile decreases, the relative proportion of sulphurbound phytane remains high also at a greater depth. Due to the strong decrease of ester-bound phytol concentrations at a greater depth (Putschew, 1996), it is likely that in these deeper samples nearly all of the phytane released by nickel boride treatment was sulphur-bound. Free phytane and phytenes due to their much lower concentrations do not play a significant role within the C20 isoprenoid pool in the sediment section studied. This may either mean that phytenes are not the main organic reactants in the sulphurization process but that phytol is a more likely reactant or that reaction of phytenes with
Table 1. 6i3c values of sulphur-bound phytane at different depths (mean values of two measurements each, standard deviation = 0.5%0) 6n3C (%o, relative to PDB) Depth ( c m ) 0-2 4-6 16-18 28-30 32-34
reduced sulphur species is extremely fast, and in this case it may even have started in the water column. The co-varying decrease of phytene and phytane concentrations with depth down to about 16 cm and the consistently very low concentrations below may indicate that they are possibly metabolized in the process of bacterial sulphate reduction. Measurements of stable carbon isotope values reveal the origin of the phytane skeleton bound in high concentrations to the high-molecular-weight sedimentary organic matter of Lake Cadagno. In Table 1, the carbon isotope values of sulphurbound phytane are listed for different depth levels. As a reference, Table 2 shows the values for phytol in extant biological material which contributes to the organic matter deposited in the sediment. Phytol in particulate organic matter from the water column at 11.7 m water depth, containing mainly phototrophic sulphur bacteria (Chromatium), is more depleted in ~3C by 14%o than phytol of grass and green algae. The carbon isotope values of the sulphur-bound phytane in the sediments appear to represent a mixture originating from both bacteria
Table 2. 6t3C values of phytol from different biological sources (mean values of two measurements each, standard deviation = 0.5%0)
Phytane released by desulphurization/hydrogenation ---42.0 -42.5 -41.8 -39.0 -38.5
6J3C (%0, relative to PDB) Sample
Phytol
Green algae Grass (surrounding of the lake) (mainly) Chromatium from 11.7 m water depth
-33.9 -33.9 -47.9
Early diagenesis of organic matter
387
Table 3. t~13Cvalues of free n-alkanes at different depths (mean values of two measurements each, standard deviation = 0.5°/**) 613C (%0,relative to PDB) of free n-alkanes n-Alkane C 17H36 CtsH3s CioH40 C2oH42 C21H44 C22H46 C23Has C24H5o C25H52 C26H54 C27H56 C2sHss C29H6o C3oH62 C3tH64 C32H66 C33H6s Mean value
0-2 cm depth
4-6 cm depth
16-18 cm depth
28-30 cm depth
32-34 cm depth
-39.4 n.d. n.d. n.d. -29.5 -30.4 -29.7 -31.8 -35.7 -35.7 -32.7 -31.6 -32.7 -31.9 -33.6 -31.4 -33.0 -32.7
-34.7 -38.5 n.d. n.d. -30.0 -31.3 -30.2 -32.0 -36.2 -35.9 -31.9 -32.3 -32.8 -31.0 -33.1 -33.4 -33.1 -33.7
-27.1 -33.5 -32.2 n.d. -29.6 -30.6 -30.4 -31.1 -31.3 -32.9 -32.0 -31.9 -32.6 -32.5 -33.3 -32.1 -33.6 -31.5
-29.1 -32.5 -35.7 -32.9 -29.6 -32.0 -30.3 -33.1 -33.9 -31.9 -31.7 -33.9 -32.9 -32.9 -33.4 -33.1 -33.9 -32.3
-27.3 -33.6 -36.7 -29.4 -27.9 -32.4 -30.6 -32.8 -32.2 -34.9 -31.6 -32.3 -32.7 -30.3 -32.3 -33.0 -33.8 -32.1
a n d higher organisms. In the u p p e r p a r t o f the sedim e n t a r y c o l u m n the microbial c o n t r i b u t i o n is higher t h a n the c o n t r i b u t i o n o f terrestrial a n d / o r aquatic algal origin. C a r b o n isotopic values o f sulp h u r - b o u n d p h y t a n e become slightly heavier below 20 cm sediment depth, i.e. with increasing d e p t h the c o n t r i b u t i o n o f terrestrial a n d / o r algal origin increases. O n a simple mass balance ground, i.e. neglecting any o t h e r sources t h a n those available for analysis in this study a n d disregarding any possible secondary isotope effects d u r i n g diagenetic reactions, the microbial c o n t r i b u t i o n to the sulphurb o u n d p h y t a n e varies between almost 6 0 % in the u p p e r sediment layers a n d a b o u t 3 5 % in the deeper samples. This is consistent with the o b s e r v a t i o n t h a t organic material o f microbial origin is m o r e readily available for diagenetic reactions and, thus, reacts earlier t h a n t h a t o f higher aquatic or terrestrial plants (Hartgers et aL, 1994b).
It is n o t clear which source organisms contributed to the precursors o f the s u l p h u r - b o u n d nalkanes. C a r b o n isotope values o f free a n d b o u n d n-alkanes (Tables 3 a n d 4) show t h a t the latter are slightly heavier o n average. The isotope values nevertheless indicate t h a t higher aquatic or land C3 plants are the most likely source although, as mentioned before, the distribution patterns o f the b o u n d n-alkanes d o not m a t c h those o f free nalkanes, n-alcohols or n-fatty acids.
CONCLUSIONS Nearly all of the organic sulphur c o m p o u n d s (OSC) identified in the sediments o f Lake C a d a g n o are b o u n d via sulphur bridges to high-molecularweight material in the N S O fraction, the asphaltenes and, as differences between asphaltenes a n d kerogens m a y partly be merely operational based
Table 4. 6J3C values for sulphur-bound n-alkanes at different depths (mean values of two measurements each, standard deviation = 0.5%0) 6J3(%e, relative to PDB) of sulphur-bound n-alkanes
n-Alkane CI7H36 CIsH3s C~9H4o C2oH42 C21H4a C22H46 C23H4s C24H50 C2sH52 C26H54 C27H56 C2sHss C29H6o C30H62 C31H64 C32H66 C33H68 Mean value
0-2 cm depth
4-6 cm depth
16-18 cm depth
28-30 cm depth
32-34 cm depth
-32.7 -38.0 -28.3 -33.1 -34.5 -32.8 -33.8 -30.4 -28.9 -30.9 -29.5 -30.5 -30.6 -30.3 -29.5 -32.1 -32.0 -32.6
-33.1 -32.8 -27.5 -35.7 -29.5 -30.3 -30.5 -30.3 -29.5 -30.1 -29.2 -30.6 -30.8 -30.8 -31.2 -31.9 -32.2 -30.6
n.d. n.d. n.d. n.d. n,d, n,d. -30.1 -29.7 -30.0 -30.8 -29.1 -30.6 -31.4 -31.5 -32.6 -31.8 n.d. -30.7
-34.4 -27.7 -31.7 -33.4 -29.4 -30.2 -29.2 -29.5 -29.6 -30.0 -30.0 -31.4 -30.6 -30.4 -31.9 -33.4 -32.7 -30.9
n.d. n.d. n.d. n.d. n.d. -30.3 -29.8 -29.6 -29.2 -30.2 -30.1 -30.7 -30.3 -29.9 -31.1 -32.2 -29.9 -30.3
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on solvents used, possibly also the kerogen. There are only two GC-amenable OSC in very small concentrations in the hydrocarbon fractions. It is thus possible, that low-molecular-weight OSC are formed to a larger extent only at a later stage of diagenesis. Desulphurization of the N S O and asphaltene fractions with nickel boride released series of hydrocarbons with phytane always being the compound with highest concentration, ranging up to more than 2 mg/g Corg. n-Alkanes and squalane are the next most important constituents in the hydrocarbon fractions obtained after desulphurization, whereas steranes and diaromatic carotenoid derivatives are relatively less important. Control experiments with a chlorophyll a standard showed that nickel boride is able to induce reductive cleavage of ester bonds which may partly perturb the quantitative assessment of macromolecularly bound organosulphur compounds particularly in Recent sediments. A difference in product distribution between reactions applied to standard and Lake Cadagno sediments led us to conclude that in the case of the sediment used, cleavage of sulphur bonds was the dominant reaction, particularly in the deeper sediments. The uncertainties remaining and the low recovery rate in the nickel boride reactions suggest that more systematic studies of the nickel boride reaction may be required. The quantitative comparison between sulphurbound and free components indicates that formation of OSC starts already in the water column or proceeds very rapidly in the upper millimeters of the sediment, because the amounts of sulphurbound squalane and phytane are already very high in the top sediment layer (0-2 cm). The different depth profiles of sulphur-bound phytane and free phytol and the lower abundance of free phytenes by almost two orders of magnitude may, however, suggest that phytol may be the most important organic reactant in sulphurization. Based on molecular carbon isotope analysis, both microbial and higher-plant phytol contribute to the sulphur-bound phytane, but the relative proportion of microbial phytane decreases with depth probably due to the less resistant nature of microbial biomass.
Acknowledgements--We would like to thank Dr A. Losher (formerly ETH Ziirich) for providing the core material and Dr K. Hanselmann (University of Zfirich) and his coworkers for their support in the 1994 sampling campaign. We are indebted to Drs J. S. Sinninghe Damst~ and S. Schouten (NIOZ, Texel) for their help in establishing the desulphurization procedures in our laboratory. S. Schulte and U. Giintner performed the nickel boride control experiments. Careful reviews of the manuscript and many helpful suggestions for improvement by Drs T. Eglinton (Woods Hole Oceanographic Institution) and W. A. Hartgers (CID-CSIC, Barcelona) were highly appreciated. The study was financially supported by the German Science Foundation (DFG, Bonn, Grant No. Ru 458/4).
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Early diagenesis of organic matter and related sulphur incorporation in surface sediments of meromictic Lake Cadagno in the Swiss Alps A N K E PUTSCHEW, BARBARA M. SCHOLZ-BOTTCHER and J I ] R G E N RULLKOTTER* Institute of Chemistry and Biology of the Marine Environment (1CBM), Carl von Ossietzky University of Oldenburg, P.O. Box 2503, Oldenburg D-26111, Germany Abstract--Samples of Recent anoxic sediment ( < 100 yr BP) from the meromictic Lake Cadagno (Switzerland) were examined to study sulphur incorporation into organic matter at a very early stage of diagenesis. Trace amounts of two organosulphur compounds were identified in the extractable organic matter. Desulphurization of the heterocompound and asphaltene fractions with nickel boride yielded hydrocarbons with a strong dominance of phytane and minor amounts of n-alkanes, steranes and squalane, but may also have converted ester-bound moieties into hydrocarbons as shown by a control experiment with a chloropyll a standard. The amount of sulphur-bound compounds is always higher than the amount of the free precursors, even in the shallowest sediments. This indicates that the formation of organosulphur compounds starts already in the water column or is very effective in the uppermost millimeters of the sediment layer. Stable carbon isotope data of the sedimentary compounds, and of biological material from the water column and from land biota growing around the lake, show that the sulphur-bound phytane in the youngest samples is dominated by phytane of bacterial origin. With increasing depth the amount of phytane of terrestrial origin slightly increases. ~(7) 1997 Elsevier Science Ltd compounds, Lake Cadagno sediments, anoxic sediment, sulphur incorporation, nickel boride desulphurization Key words--Organosulphur
INTRODUCTION Over more than a decade, organosulphur compounds (OSC) have become a subject of increasing interest in organic geochemistry. About 1500 new structures were identified in this class of compounds (for a review see Sinninghe Damst6 and de Leeuw, 1990). The intense research in the field of OSC has markedly increased our knowledge of the formation of these compounds and their geochemical significance. OSC are products of reactions of reduced inorganic sulphur species with functionalized lipids during early diagenesis, First evidence of such reactions was obtained from the identification of OSC structurally closely related to known biochemical precursors (e.g. Valisolalao et al., 1984; Brassell et al., 1986; Sinninghe Damst6 and de Leeuw, 1987; Sinninghe Damst~ et al., 1987; Kohnen et al., 1990). The products of laboratory simulation of sulphur incorporation into organic substrates are in agreement with the idea of abiotic formation of OSC during early diagenesis (de Graaf et al., 1992; Rowland et al., 1993; Bisseret and Rohmer, 1993; Schouten et al., 1993b, 1994). The reactions involve intra- and/or intermolecular addition of reduced inorganic sulphur species, possibly H S - or polysul*Corresponding author.
phides, into functionalized biogenic lipids (Kohnen et al., 1991a, 1993). Intramolecular sulphur incorporation usually leads to low-molecular-weight components often separable by capillary column gas chromatography (GC) whereas intermolecular reactions produce high-molecular-weight material. The initial structure elucidations, restricted to the GC-amenable OSC fraction, revealed thiophenes, thiolanes, thianes and benzothiophenes as the main compound classes of fossil organosulphur compounds. Advanced analytical techniques like pyrolysis-GC and chemical degradation provided access to the macromolecular OSC by the analysis of their pyrolysis products and the hydrocarbons liberated by desulphurization, respectively (Boudou et al., 1987; Eglinton et al., 1990, 1992; Sinninghe Damst+ et al., 1988a,b, 1989a; Sinninghe Damst~ and de Leeuw, 1990; Schouten et al., 1993a; Kohnen et al., 1991a; Hoffman et al., 1992; Richnow et al., 1992). The biological markers bound via sulphur bridges to the macromolecular sedimentary organic matter provide a particularly well preserved source of information for palaeoenvironmental reconstructions (Sinninghe Damst~ et al., 1989b; de Leeuw and Sinninghe Damst~, 1990), and OSC have to be considered in such assessments in order to avoid biases due to structure- and/or source-specific fractionation between the free and sulphur-bound bio-
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Anke Putschew et al. •. ~._~,,Lake Constance
Lake Cadagno Fig. 1. Location map of Lake Cadagno in the Swiss Alps. marker pools during sulphurization (Kohnen et al., 1991b). Until now, virtually all molecular investigations of OSC formation used sediments older than 10,000yr [except those performed on Black Sea (Wakeham et al., 1995) and Peru margin sediments (Kohnen et al., 1991b)] and, with a few exceptions (e.g. Rullk6tter et al., 1990; Barakat and Rullk6tter, 1994, 1995), were restricted to marine deposits. In the present study, recently deposited (< 100 yr) sedimentary organic matter from Lake Cadagno (Switzerland) was analyzed. Lake Cadagno which is located at an altitude of 1923 m above sea level in the Ticino Alps (Fig. 1), is characterized by a chemocline fluctuating over the year between 8 and 12 m water depth. Below the chemocline the water column is permanently anoxic. A constant inflow of sulphate-rich groundwater is responsible for the high activity of sulphate-reducing bacteria in the lower part of the water column and in the surface sediments. Near the chemocline, phototrophic sulphur bacteria (essentially Chromatium spp. based on the intense red colour and microscopic examinations; Ztillig, 1985; Losher, 1989; K. Hanselmann, private communication, 1995) are the main primary producers of organic matter in the water column. The sediment is rich in organic carbon and total sulphur. The organic matter is mainly autochthonous with only a small contribution of allochthonous material of terrestrial higher plant origin (Putschew et al., 1995). In addition to the high organic carbon and sulphur contents, the low concentration of ferrous iron (Losher, 1989) should favour the formation of OSC in this lake system. Relative to an earlier publication of preliminary results (Putschew et al., 1995) which had a strong emphasis on compound identification,
interpretation of sulphurization processes and isotopic data has been significantly modified and extended in this communication.
EXPERIMENTAL Samples
A piston core of 36 cm length and a diameter of 7 cm was provided by A. Losher (ETH Ziirich). It was split into sections of 2 cm thickness at the sampling site, and the sections were immediately frozen. The sediment was homogeneously black and had a strong odour of hydrogen sulphide, which ceased considerably below a depth of 32 cm. In August 1994, grass from the lake shore and green macroalgae floating on the lake surface were sampled. For the recovery of biological material from the water column, water from different water depths (7 m, 11.7 m and 19 m) was pumped and filtered. The particle size of this material ranged from 22 #m down to 0.45 #m, The biological material was immediately frozen at the sampling site. Methods
The freeze-dried sediment samples were ultrasonically extracted with dichloromethane. After addition of internal standards (squalane, thianthrene, 5c¢(H)-androstan-17-one) the extract was separated into fractions soluble in n-hexane and dichloromethane, respectively (asphaltene precipitation). The dichloromethane fraction is operationally termed asphaltenes throughout this paper. The compounds soluble in n-hexane were separated on a 30 cm x 1.6 cm i.d. column filled with 16 g of silica 100 deactivated with 5% water. The nonaromatic hydrocarbon fraction eluted with 40 ml n-hexane,
381
Early diagenesis of organic matter the aromatic hydrocarbon fraction with 60 ml nhexane/dichloromethane (9:1 by volume) and the hetero(NSO)-compounds with 80 ml dichloromethane/methanol (9:1 by volume). Aliquots of the asphaltene and NSO fractions were desulphurized with Ni2B as described by Schouten et al. (1993a). The released hydrocarbons were isolated by column chromatography (dimensions as above) by elution with four times the column volume of n-hexane/dichloromethane (9:1 by volume). The hydrocarbons were analyzed before and after hydrogenation with a PrO2 catalyst. In a control experiment, 1 mg chlorophyll a and 500 #g squalane (internal standard) were dissolved in 2 ml dry tetrahydrofurane and 2 ml methanol and, by addition of 50 mg NiC12 and 50 mg NaBH4 and heating under reflux for 1 h, were treated with nickel boride in the same way as described for the sediment material. Separation of the hydrocarbon fraction was performed as described above, and polar compounds were recovered from the liquid chromatography column with 5 ml methanol/ dichloromethane (1:1). Hydrocarbon products were analyzed without further hydrogenization, the polar fraction was hydrolyzed with KOH in methanol/ water (see below) and, after derivatization of the products with M S T F A (N-methyl-N-trimethylsilyltrifluoroacetamide), analyzed for residual phytol. Recovery of the internal standard in this and a blank experiment (without chlorophyll a) were 39% and 36%, respectively. Selected polar extract fractions and the freezedried biological material were saponified with 20 ml of 5% KOH in methanol/water (8:2 by volume) for 24 h under reflux. The n-hexane-soluble organic matter was separated by liquid chromatography over a silical gel column as described before. Alcohols were derivatized with MSTFA before analysis. GC analysis was carried out on a Hewlett Packard 5890 series II instrument equipped with a temperature-programmable injector (Gerstel KAS 3) and a flame ionization detector (FID) or a sulphur-selective chemoluminescence detector (Sievers SCD). The detection level for sulphur was 17 pg S/ #1. A DB-5 (J&W) fused silica capillary column (30 m x 0.25 mm i.d., 0.25 ttm film thickness) was used with helium as the carrier gas. The temperature of the GC oven was programmed from 60°C (1 min isothermal) to 300°C (50 min isothermal) at 3°C/rain. The injector temperature was programmed from 60°C (5 s hold time) to 300°C (60 s hold time) at 8°C/s. Gas chromatography-mass spectrometry (GC-MS) and gas chromatography-isotope ratio monitoring-mass spectrometry (GC-irm-MS) measurements were performed with the same type of GC system using the conditions described above. For G C - M S analysis, the gas chromatograph was OG 25"5-7 g
coupled to a Finnigan SSQ 710B mass spectrometer operated at 70 eV with a scan range of m/z 50-600 and a scan time of 1 scan/s. For G C - i r m MS analysis the gas chromatograph was attached to a Finnigan MAT 252 isotope ratio mass spectrometer with a Finnigan combustion interface. Carbon isotope ratios were calibrated with a COz standard at the beginning and the end of each analysis and with compounds of known isotopic composition which were co-injected with each sample and served as internal isotopic standards. Isotope ratios are expressed as ~13C values in permil relative to the PDB standard. The 6t3C value of phytol was corrected for the isotopic contribution of the trimethylsilyl group using the following equation: ~13Cphytol_TM S :
20/23~13Cphytol+
(1 - 20/23)313C-rMs with ~I3CTMS = -40.35 :t: 1.38%o.
RESULTS AND DISCUSSION
Bulk and nonaromatic hydrocarbon geochemistry The Lake Cadagno sediments have maximum organic carbon concentrations beween 10% and 14% in the top 10cm and show slightly lower values below this depth (Putschew et al., 1995). Uniform C/N ratios around 10 indicate a more or less constant organic matter type over the entire 36 cm depth interval studied and a dominance of aquatic material. Total sulphur contents vary between 1% and 3.5%, and the Corg/Stot ratios range from 6 to 1.5 with a depth trend virtually paralleling that of the organic carbon content (Putschew et al., 1995). Extract yields are high throughout (41-153mg/g Corg) with the higher values occurring in the upper sediment section. The extracts are dominated by heterocomponents (usually close to, or more than 50%) while nonaromatic hydrocarbons typically amount to 20-30% and asphaltenes to about 10% of total extract. Elemental sulphur is abundant in the shallower sediments. The nonaromatic hydrocarbon fractions are dominated by n-alkanes with a strong odd-overeven carbon number predominance and a maximum at n-C29 or n-C31. In the absence of major tree and bush vegetation at the high altitude, the most likely source is the grass growing around the lake. Hopanoid hydrocarbons of microbial origin are the second-most important group of constituents of the nonaromatic hydrocarbon fractions. Within this group, diploptene (hop-22(29)-ene) is always the compound with the highest relative concentration.
382
Anke Putschew et al.
I TIC
[
21:1 21:0
40 , ,~ , ,
/~
f~
?,~_.,,~,
,ool
llllVll,+ll+'~'llll"l!
52
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m/z 98
100 7m/z 308
Retention time (rain)
Fig. 2. Partial reconstructed ion chromatogram and mass chromatograms (m/z 98, 111,308) of the aliphatic hydrocarbon fraction of the sample from 0-2cm depth. 21:0 = n-henicosane, 21:1 = n-henicosene, A = 3-methyl-2-(3,7,1l-trimethyldodecyl)thiophene, B = 3-(4,8,12-trimethyltridecyl)thiophene. Phytane and phytenes are detectable in small amounts decreasing with depth. Sterenes are present only in very low concentrations except a compound tentatively identified as 23,24-dimethylcholesta3,5,22-triene (Putschew et al., 1995) which is most likely derived from dinoflagellates known to occur above the chemocline in the lake (K. Hanselmann, private communication 1993). Low-molecular-weight OSC & the nonaromatic hydrocarbon fractions
Despite the anoxic conditions in the lower part of the lake and the high sulphur content of the sediments, only a few GC-amenable OSC were detected which, due to the liquid chromatographic separation scheme applied, occur in the "nonaromatic hydrocarbon" fractions. Mass chromatography of molecular ions and key fragments revealed the presence of 3-methyl-2-(3,7,11-trimethyldodecyl)thiophene and 3-(4,8,12-trimethyltridecyl)thiophene in small amounts (Fig. 2). Both compounds often identified in ancient sediments (Rullk6tter et al., 1982, 1984; ten Haven et al., 1985; Brassell et al., 1986) are thought to be products of early diagenesis. None of the more highly substituted C20 thiophene isomers, e.g. 2,3-dimethyl-5-(3,7,1 ltrimethyldocecyl)thiophene, indicative of higher salinity in the water column at the time of deposition
(de Leeuw and Sinninghe Damst6, 1990), could be detected. Also, no GC-amenable organosulphur compounds were found in the aromatic hydrocarbon fractions (typically only 3% of total extract) within the detection limits of the sulphur-selective detector. Macromolecularly bound O S C
Upon treatment of the NSO fractions and asphaltenes with Ni2B, intended to desulphurize macromotecular entities, major amounts of hydrocarbons were released. Both types of starting materials yielded virtually identical product distributions. Phytane is always the hydrocarbon obtained in highest relative concentration even before hydrogenation. A typical example of a sulphur-bound hydrocarbon distribution (after hydrogenation) from desulphurization of an NSO fraction is shown in Fig. 3. n-Alkanes, squalane and C27, C28 and C29 steranes were minor products. The n-alkanes range from Ci7 to at least C35, and their distributions maximize at n-C26. Other than in the free hydrocarbon fractions, the even-carbon-number homologues dominate over the odd-carbon-numbered n-alkanes, and this preference is only slightly pronounced. The dissimilarity of this n-alkane distribution with the carbon number distributions of both n-fatty acids and n-alkanols suggests a horn-
Early diagenesis of organic matter ologous series of precursors which is not normally observed in the low-molecular-weight bitumen fractions. Strong differences between free n-alkanes and n-alkane distributions obtained after desulphurization of low- and high-molecular-weight OSCs and heterocompound fractions have been noted before, e.g. for the Jurf ed Darawish oil shale (Sinninghe Damst6 et al., 1988a), for sediments from the Monterey Formation (Schouten et al., in press), and for crude oils (Schouten et al., 1995; Rullkrtter and Michaelis, 1990). In most of these cases, the nalkanes from desulphurization showed little resemblance with carbon number distributions of common classes of biogenic compounds with a straight aliphatic carbon chain. A notable exception in this apparent lack of specificity was the occurrence of C37 and C38 n-alkanes in several desulphurized hydrocarbon fractions which were related to longchain aikenones of prymnesiophyte algae (Sinninghe Damst6 et al., 1988a; Rullkrtter and Michaelis, 1990; Schouten et al., 1995). Similar to the n-alkanes, the steranes in the heterocompound (NSO) and asphaltene desulphurization products of the Lake Cadagno sample series show a relatively smooth distribution. Apparently, the C29 steratriene, i.e. 23,24-dimethylcholesta3,5,22-triene, dominating the steroid hydrocarbons
383
in the free nonaromatic hydrocarbon fractions, was not affected by sulphur incorporation to a significant extent. The presence of its sulphur-bound counterpart, in small concentrations in the macromolecular fractions, cannot be readily deduced because in the saturated form 23,24-dimethylcholestane will coelute with 24-ethylcholestane under the analytical conditions applied as has been shown by hydrogenation of a nonaromatic hydrocarbon fraction and coinjection with a 24-ethylcholestane standard during structure elucidation (Putschew et al., 1995). The end member of a pseudohomologous series of diagenetic alteration products of diaromatic carotenoids found in the desulphurization products, although in small concentrations, was either isorenieratane or renieratane (Fig. 4b, compound dA) which are characteristic of phototrophic sulphur bacteria (Liaaen-Jensen, 1978). The isomers cannot be differentiated by their mass spectra which are both dominated by a base peak at m/z 133 (Hartgers et al., 1994a), and standards were not available for identification based on gas chromatographic retention time. The degraded carotenoids, also characterized by a base peak at m/z 133 in their mass spectra, ranged from Cls to C27 with the CI7 and C23 members missing due to the isoprenoid
I00-Ph~ 'tane
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J
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34
Retention time (min)
Fig. 3. Gas chromatogram of hydrocarbons released by desulphurization of the NSO fraction of the sample from 26-28 cm depth, n-Alkanes are indicated by their carbon numbers. * = internal standards, x = dibutylphthalate.
384
Anke Putschew et al. tic zone of the lake and shows that degradation of diaromatic carotenoids does not require thermal stress, but may possibly even be induced photochemically. Recently a critical examination of the desulphurization reaction with Raney nickel applied to modern sediments revealed not only that sulphur bonds are broken but also that the chlorophyll a sidechain may be hydrogenolyzed yielding phytane as one of the products (Prahl et al., 1996). The phytane deriving from the carboxylic ester functionality of chlorophyll a certainly was not discernible from phytane produced by desulphurization of macromolecular OSC in the course of the Raney nickel treatment. Nickel boride appeared to be less reactive towards ester bonds because Hartgers et al. (1995) reported that octadecanoic acid methyl ester was not cleaved by Ni2B while other side-reactions with weak bonds comprising heteroatoms other than sulphur did
branching in the chain. They are the dominant compounds in the summed m / z (133 + 134) mass chromatogram in Fig. 4b. The carbon number distribution of the degradation products differs from that observed by Hartgers et al. (1994a) in the highmolecular-weight bitumen fractions of sediments from the Upper Devonian Duvernay Formation (Western Canada Basin). In that case, the distribution had a maximum at Cl4, and the relation to the branching in the chain was less clear due to the presence of small amounts of C~7 and C23 pseudohomologues. Like in the Duvernay Formation sediments, the diaromatic carotenoid degradation products in the Lake Cadagno samples also comprise a series with a base peak at m / z 134 which, in minute concentrations, elutes before the major series in the mass fragmentogram in Fig. 4b. Hartgers et al. (1994a) ascribed this series to a novel diaromatic carotenoid with one benzene ring carrying a 3,4,5-trimethyl-l-alkyl substitution pattern. The identification of the diaromatic carotenoid derivatives in the Lake Cadagno sediments reflects the sulphide-rich anoxic water body within the pho-
o c c u r .
Under the conditions applied during the control experiment in the course of this study, however, the ester bond of a chlorophyll a standard was reduc-
~TIC Phi tane
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,
Fig. 4. Partial reconstructed ion chromatogram and mass chromatogram (m/z 133 + 134) of hydrocarbons released by desulphurization of the NSO fraction of the sample from 30-32 cm depth. In the upper trace, n-alkanes are indicated by their carbon numbers. * = internal standards. In the lower trace, numbers indicate carbon numbers of degraded carotenoids derived from diaromatic carotenoid pecursors, dA = renieratene or isorenieratene, * = phthalate contaminants.
Early diagenesis of organic matter tively cleaved very efficient. Relative to the internal standard, the yield of phytane and phytenes was 58.5% of the theoretical yield; the remainder was not recoverable as phytol, however, indicating that additional side reactions occurred as well. Furthermore, the overall recovery of the standard - - and, thus, of the ester cleavage products - - was remarkably low, also in a blank experiment. On the other hand, the hydrocarbon distributions in the products of the chlorophyll a reaction with Ni2B (4% phytane, 19% phyt-l-ene, 77% phyt-2-ene) and of the Ni2B reaction with pure phytol (phytene/ phytane ratio of 35:25; Hartgers et al., 1995) are significantly different from the product distribution of the nickel boride treatment of the polar and asphattene fractions extracted from the Lake Cadagno sediments, which typically contained a large abundance of phytane, but only relatively small amounts of phytenes. This altogether indicates, that the discussion of reductive cleavage of sulfur bonds of macromolecular entities in the sediment extracts of Lake Cadagno samples is valid at least in qualitative terms, but that the reaction products may comprise products from ester cleavage to a certain extent which affects the significance of the quantitative data discussed below. Absolute concentrations were determined for the amounts of squalane and phytane released upon desulphurization (after hydrogenation) as well as for their structurally related free analogs, i.e. squalene (aromatic hydrocarbon fraction), phytane, phytenes (nonaromatic hydrocarbon fractions) and phytol (NSO fractions), in the top 36 cm of Lake Cadagno sediments. The yields of sulphur-bound (a)
(b)
sulphur-bound squnlue
squalane and free squalene are significantly higher in most samples from the shallower half of the sediment section studied than in the deeper samples (Fig. 5). Concentrations of sulphur-bound squalane vary from 100 to 200 pg/g Corg in the top 16 cm of the sedimentary column and from about l0 to 70 #g/g Corg below this depth. Free squalene typically is also more abundant in the shallower sediment section (up to 80gg/g Corg), whereas concentrations in most of the deeper sediments are around 20 #g/g Corg (Fig. 5). The fraction of sulphur-bound squalane, except for the sample at 4 cm depth, is more abundant than the fraction of free squalene, although the difference is less pronounced below a depth of about 16 cm. This can clearly be seen from the percentages of free squalene and sulphur-bound squalane relative to the sum of both fractions (Fig. 5c). At the bottom of Lake Cadagno bacterial sulphate reduction is active down to a depth of about 20 cm into the sediment (Losher. 1989). Should squalene be metabolized by sulphatereducing bacteria, the results of this study would show that macromolecularly bound squalane, as expected, is more resistant to microbial degradation than the corresponding free compound. Figure 6 shows the depth profiles of sulphurbound phytane and the concentrations of the related free compounds phytol, phytane and phytenes. Although the concentrations of sulphur-bound phytane are high in all samples studied (up to more than 2000 #g/g Corg), there are strong fluctuations in the entire sediment core rather then a depth trend. In contrast to this, the concentrations of free phytane, phytene isomers and phytol in the low-
free ~lualeue
(c)
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Fig. 5. Depth profiles of (a) organic carbon-normalized concentrations of sulphur-bound squalane, (b) organic carbon-normalized concentrations of free squalene and (c) percentages of free (I) and sulphurbound (o) squalene relative to the sum of both fractions.
386
Anke Putschew et al. free phytol (Pr/g (?orS)
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Fig. 6. Depth profiles of (a) free phytol, (b) sulphur-bound phytane, (c) free phytenes and (d) free phytane.
molecular-weight bitumen fractions decrease with sediment depth even if some scatter is obvious in the upper part of the phytol profile (Fig. 6). It is conceivable that the large scatter in the bound phytane concentrations, particularly in the shallower samples, is affected by contributions of phytane derived from ester-bound phytol as discussed before. While the free phytol concentration in the depth profile decreases, the relative proportion of sulphurbound phytane remains high also at a greater depth. Due to the strong decrease of ester-bound phytol concentrations at a greater depth (Putschew, 1996), it is likely that in these deeper samples nearly all of the phytane released by nickel boride treatment was sulphur-bound. Free phytane and phytenes due to their much lower concentrations do not play a significant role within the C20 isoprenoid pool in the sediment section studied. This may either mean that phytenes are not the main organic reactants in the sulphurization process but that phytol is a more likely reactant or that reaction of phytenes with
Table 1. 6i3c values of sulphur-bound phytane at different depths (mean values of two measurements each, standard deviation = 0.5%0) 6n3C (%o, relative to PDB) Depth ( c m ) 0-2 4-6 16-18 28-30 32-34
reduced sulphur species is extremely fast, and in this case it may even have started in the water column. The co-varying decrease of phytene and phytane concentrations with depth down to about 16 cm and the consistently very low concentrations below may indicate that they are possibly metabolized in the process of bacterial sulphate reduction. Measurements of stable carbon isotope values reveal the origin of the phytane skeleton bound in high concentrations to the high-molecular-weight sedimentary organic matter of Lake Cadagno. In Table 1, the carbon isotope values of sulphurbound phytane are listed for different depth levels. As a reference, Table 2 shows the values for phytol in extant biological material which contributes to the organic matter deposited in the sediment. Phytol in particulate organic matter from the water column at 11.7 m water depth, containing mainly phototrophic sulphur bacteria (Chromatium), is more depleted in ~3C by 14%o than phytol of grass and green algae. The carbon isotope values of the sulphur-bound phytane in the sediments appear to represent a mixture originating from both bacteria
Table 2. 6t3C values of phytol from different biological sources (mean values of two measurements each, standard deviation = 0.5%0)
Phytane released by desulphurization/hydrogenation ---42.0 -42.5 -41.8 -39.0 -38.5
6J3C (%0, relative to PDB) Sample
Phytol
Green algae Grass (surrounding of the lake) (mainly) Chromatium from 11.7 m water depth
-33.9 -33.9 -47.9
Early diagenesis of organic matter
387
Table 3. t~13Cvalues of free n-alkanes at different depths (mean values of two measurements each, standard deviation = 0.5°/**) 613C (%0,relative to PDB) of free n-alkanes n-Alkane C 17H36 CtsH3s CioH40 C2oH42 C21H44 C22H46 C23Has C24H5o C25H52 C26H54 C27H56 C2sHss C29H6o C3oH62 C3tH64 C32H66 C33H6s Mean value
0-2 cm depth
4-6 cm depth
16-18 cm depth
28-30 cm depth
32-34 cm depth
-39.4 n.d. n.d. n.d. -29.5 -30.4 -29.7 -31.8 -35.7 -35.7 -32.7 -31.6 -32.7 -31.9 -33.6 -31.4 -33.0 -32.7
-34.7 -38.5 n.d. n.d. -30.0 -31.3 -30.2 -32.0 -36.2 -35.9 -31.9 -32.3 -32.8 -31.0 -33.1 -33.4 -33.1 -33.7
-27.1 -33.5 -32.2 n.d. -29.6 -30.6 -30.4 -31.1 -31.3 -32.9 -32.0 -31.9 -32.6 -32.5 -33.3 -32.1 -33.6 -31.5
-29.1 -32.5 -35.7 -32.9 -29.6 -32.0 -30.3 -33.1 -33.9 -31.9 -31.7 -33.9 -32.9 -32.9 -33.4 -33.1 -33.9 -32.3
-27.3 -33.6 -36.7 -29.4 -27.9 -32.4 -30.6 -32.8 -32.2 -34.9 -31.6 -32.3 -32.7 -30.3 -32.3 -33.0 -33.8 -32.1
a n d higher organisms. In the u p p e r p a r t o f the sedim e n t a r y c o l u m n the microbial c o n t r i b u t i o n is higher t h a n the c o n t r i b u t i o n o f terrestrial a n d / o r aquatic algal origin. C a r b o n isotopic values o f sulp h u r - b o u n d p h y t a n e become slightly heavier below 20 cm sediment depth, i.e. with increasing d e p t h the c o n t r i b u t i o n o f terrestrial a n d / o r algal origin increases. O n a simple mass balance ground, i.e. neglecting any o t h e r sources t h a n those available for analysis in this study a n d disregarding any possible secondary isotope effects d u r i n g diagenetic reactions, the microbial c o n t r i b u t i o n to the sulphurb o u n d p h y t a n e varies between almost 6 0 % in the u p p e r sediment layers a n d a b o u t 3 5 % in the deeper samples. This is consistent with the o b s e r v a t i o n t h a t organic material o f microbial origin is m o r e readily available for diagenetic reactions and, thus, reacts earlier t h a n t h a t o f higher aquatic or terrestrial plants (Hartgers et aL, 1994b).
It is n o t clear which source organisms contributed to the precursors o f the s u l p h u r - b o u n d nalkanes. C a r b o n isotope values o f free a n d b o u n d n-alkanes (Tables 3 a n d 4) show t h a t the latter are slightly heavier o n average. The isotope values nevertheless indicate t h a t higher aquatic or land C3 plants are the most likely source although, as mentioned before, the distribution patterns o f the b o u n d n-alkanes d o not m a t c h those o f free nalkanes, n-alcohols or n-fatty acids.
CONCLUSIONS Nearly all of the organic sulphur c o m p o u n d s (OSC) identified in the sediments o f Lake C a d a g n o are b o u n d via sulphur bridges to high-molecularweight material in the N S O fraction, the asphaltenes and, as differences between asphaltenes a n d kerogens m a y partly be merely operational based
Table 4. 6J3C values for sulphur-bound n-alkanes at different depths (mean values of two measurements each, standard deviation = 0.5%0) 6J3(%e, relative to PDB) of sulphur-bound n-alkanes
n-Alkane CI7H36 CIsH3s C~9H4o C2oH42 C21H4a C22H46 C23H4s C24H50 C2sH52 C26H54 C27H56 C2sHss C29H6o C30H62 C31H64 C32H66 C33H68 Mean value
0-2 cm depth
4-6 cm depth
16-18 cm depth
28-30 cm depth
32-34 cm depth
-32.7 -38.0 -28.3 -33.1 -34.5 -32.8 -33.8 -30.4 -28.9 -30.9 -29.5 -30.5 -30.6 -30.3 -29.5 -32.1 -32.0 -32.6
-33.1 -32.8 -27.5 -35.7 -29.5 -30.3 -30.5 -30.3 -29.5 -30.1 -29.2 -30.6 -30.8 -30.8 -31.2 -31.9 -32.2 -30.6
n.d. n.d. n.d. n.d. n,d, n,d. -30.1 -29.7 -30.0 -30.8 -29.1 -30.6 -31.4 -31.5 -32.6 -31.8 n.d. -30.7
-34.4 -27.7 -31.7 -33.4 -29.4 -30.2 -29.2 -29.5 -29.6 -30.0 -30.0 -31.4 -30.6 -30.4 -31.9 -33.4 -32.7 -30.9
n.d. n.d. n.d. n.d. n.d. -30.3 -29.8 -29.6 -29.2 -30.2 -30.1 -30.7 -30.3 -29.9 -31.1 -32.2 -29.9 -30.3
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on solvents used, possibly also the kerogen. There are only two GC-amenable OSC in very small concentrations in the hydrocarbon fractions. It is thus possible, that low-molecular-weight OSC are formed to a larger extent only at a later stage of diagenesis. Desulphurization of the N S O and asphaltene fractions with nickel boride released series of hydrocarbons with phytane always being the compound with highest concentration, ranging up to more than 2 mg/g Corg. n-Alkanes and squalane are the next most important constituents in the hydrocarbon fractions obtained after desulphurization, whereas steranes and diaromatic carotenoid derivatives are relatively less important. Control experiments with a chlorophyll a standard showed that nickel boride is able to induce reductive cleavage of ester bonds which may partly perturb the quantitative assessment of macromolecularly bound organosulphur compounds particularly in Recent sediments. A difference in product distribution between reactions applied to standard and Lake Cadagno sediments led us to conclude that in the case of the sediment used, cleavage of sulphur bonds was the dominant reaction, particularly in the deeper sediments. The uncertainties remaining and the low recovery rate in the nickel boride reactions suggest that more systematic studies of the nickel boride reaction may be required. The quantitative comparison between sulphurbound and free components indicates that formation of OSC starts already in the water column or proceeds very rapidly in the upper millimeters of the sediment, because the amounts of sulphurbound squalane and phytane are already very high in the top sediment layer (0-2 cm). The different depth profiles of sulphur-bound phytane and free phytol and the lower abundance of free phytenes by almost two orders of magnitude may, however, suggest that phytol may be the most important organic reactant in sulphurization. Based on molecular carbon isotope analysis, both microbial and higher-plant phytol contribute to the sulphur-bound phytane, but the relative proportion of microbial phytane decreases with depth probably due to the less resistant nature of microbial biomass.
Acknowledgements--We would like to thank Dr A. Losher (formerly ETH Ziirich) for providing the core material and Dr K. Hanselmann (University of Zfirich) and his coworkers for their support in the 1994 sampling campaign. We are indebted to Drs J. S. Sinninghe Damst~ and S. Schouten (NIOZ, Texel) for their help in establishing the desulphurization procedures in our laboratory. S. Schulte and U. Giintner performed the nickel boride control experiments. Careful reviews of the manuscript and many helpful suggestions for improvement by Drs T. Eglinton (Woods Hole Oceanographic Institution) and W. A. Hartgers (CID-CSIC, Barcelona) were highly appreciated. The study was financially supported by the German Science Foundation (DFG, Bonn, Grant No. Ru 458/4).
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