13
C-contents of sedimentary bacterial lipids in a shallow
sul®dic monomictic lake (Lake CisoÂ, Spain)
Walter A. Hartgers
a,1, Stefan Schouten
b,*, Jordi F. Lopez
a,2,
Jaap S. Sinninghe DamsteÂ
b, Joan O. Grimalt
aaDepartment of Environmental Chemistry (CID-CSIC), Jordi Girona 18-26, 08034 Barcelona, Catalonia, Spain bNetherlands Institute for Sea Research (NIOZ), Department of Marine Biogeochemistry and Toxicology, PO Box 59,
1790 AB Den Burg, The Netherlands
Received 17 February 2000; accepted 20 June 2000 (returned to author for revision 5 April 2000)
Abstract
Stable carbon isotopic analysis was performed on sedimentary biomarkers of a shallow sul®de-rich monomictic lake, Lake Ciso (NE Spain). Speci®c biomarkers derived from phototrophic sulfur bacteria in Lake Ciso were considerably depleted in 13C, most likely due to the depleted 13C-content of the dissolved inorganic carbon that was
photo-synthetically ®xed. Recycling of respired CO2, a well-known phenomenon in monomictic lakes, probably caused this
depletion. The stable carbon isotopic composition of terrestrial markers, such as C25ÿ33 odd-carbon-numbered n
-alkanes and C22ÿ30even-carbon-numberedn-alkan-1-ols and fatty acids, were rather similar to each other and their13C
depleted values (c.ÿ31 toÿ35%) indicated that they were derived from the surrounding vegetation. Phytol was
pre-dominantly derived from the bacteriochlorophylls of phototrophic purple sulfur bacteria as were speci®c fatty acids.
#2000 Elsevier Science Ltd. All rights reserved.
Keywords:Lipids; Stable carbon isotope ratios; Monomictic lake; Purple sulfur bacteria; Green sulfur bacteria; Fatty acids; Car-otenoids; CO2recycling; Lake CisoÂ
1. Introduction
Geochemical studies of strati®ed sul®de-rich lakes have often been performed to investigate the operation of the sulfur cycle in lacustrine environments. Some studies have focused on the competition between iron and organic matter for reaction with inorganic sulfur species (e.g. Hartgers et al., 1997) or on the early incor-poration of sulfur into functionalized lipids (Hartgers et
al., 1996, 1997; Putschew et al., 1996). Reports on the stable carbon isotope composition of dissolved organic carbon (DIC) in the water columns of these lakes have frequently shown that recycling of CO2 plays a major
role in these environments (e.g. Deevey et al., 1963; Rau, 1978; Wachniew and Rozanski, 1997). This is re¯ected in the stable carbon isotopic composition of the organic matter of organisms living at depth on this depleted DIC. For instance, Fry (1986) showed that bulk cell material of zooplankton and bacterioplankton (consisting of Chromatiaceae and Chlorobiaceae) were depleted in13C (
ÿ30 toÿ41%) due to the depleted13
C-content of DIC (up to ÿ21%) in several meromictic
lakes.
Studies of the stable carbon isotopic composition of speci®c bacterial biomarkers deposited in these envir-onments are limited. Such data may be of great interest because strati®ed sul®de-rich lakes usually have speci®c
0146-6380/00/$ - see front matter#2000 Elsevier Science Ltd. All rights reserved. P I I : S 0 1 4 6 - 6 3 8 0 ( 0 0 ) 0 0 0 9 5 - 4
www.elsevier.nl/locate/orggeochem
* Corresponding author. Tel.: 222-369565; fax: +31-222-319674.
E-mail address:schouten@nioz.nl (S. Schouten).
1 Present address: SGS Redwood Nederland B.V., Malledijk
18, P.O. Box 200, 3200 AE Spijkenisse, The Netherlands. 2
microbial communities such as phototrophic sulfur bacteria with speci®c biomarkers having distinct stable carbon isotopic composition. Adequate recognition of these microbial inputs will allow a better understanding of lake systems where CO2-recycling plays a major role,
since the isotopic composition of bulk organic matter may not always re¯ect this process. One example repor-ted in the literature is that of the meromictic Lake Cadagno (Swiss Alps). Putschew et al. (1996) reported some stable carbon isotope data for terrestrially-derived free n-alkanes and bacterially-derived phytol in sedi-ments, whilst Schaeer et al. (1997) determined the13
C-contents of bacterial carotenoids in the same sediments. Both authors found depleted13C-contents for bacterial
lipids, which is due to the low13C-content of the DIC in
Lake Cadagno (Bernasconi and Hanselmann, 1993). The lack of detailed molecular studies prompted us to analyse the stable carbon isotopic composition of bac-terial and terrestrial biomarkers deposited in sediments of a sul®de-rich monomictic lake, Lake Ciso (Catalonia, Spain). This shallow lake is known to contain signi®cant amounts of phototrophic sulfur bacteria that live at the chemocline at depths of 1±2 m (Guerrero et al., 1987; Casamayor et al., 2000). Previous communications have
described the distributions of apolar and polar com-pounds in this sediment, with respect to the formation of organic sulfur compounds (Hartgers et al., 1996, 1997) and the pigment composition (Villanueva et al., 1994). Here we report the13C-composition of lipids that
may allow a more detailed reconstruction of the terres-trial and microbial sources for these compounds.
2. Experimental
2.1. Environmental setting and sampling
Lake Ciso (also commonly referred to as Lake SisoÂ) is located in the Banyoles area of Catalonia (NE Spain; Fig. 1). It is a recent (< 80 years) karstic monomictic lake with a maximum depth of 8 m and a diameter of 25 m. Detailed descriptions of the lake and its biological community are given by Guerrero et al. (1987) and Vil-lanueva et al. (1994). Brie¯y, the lake is characterised by a constant in¯ow of sulfate-rich groundwater, which gives rise to high activity of sulfate reducing bacteria near the sediment surface. The hydrogen sul®de pro-duced is utilised by communities of both purple and
green sulfur bacteria living below the chemocline (1.5±3 m depth). Microscopic analyses suggest that Chroma-tium minus(now namedThiocystis minor; Casamayor et al., 2000),Chlorobium limicolaandC. vibrioforme dom-inate (Guerrero et al., 1987), but recent molecular bio-logical analyses have shown that other, uncultivated purple and green sulfur bacteria are ecologically more signi®cant (Casamayor et al., 2000). The main algal species present are Cryptomonas phaseolus and Ankis-trodesmus spp. (Villanueva et al., 1994) which live just above the chemocline. In addition, Synechococcus-like cyanobacteria are present (Casamayor et al., 2000). During winter, holomixis takes place and the whole water column turns anoxic. A surface sediment (0±20 cm) from was taken near the centre of Lake Ciso at 8.5 m depth in May 1995 when the water column was stra-ti®ed. In addition to the surface sediment, particulate organic matter from the water column was collected by ®ltering c. 1 l of water using a Whatmann GF/F ®lter. Water samples were taken at dierent depths (0.3 and 1.5, above or at the chemocline and 3.0 and 5.9 m, below the chemocline) in July 1996.
2.2. Extraction and fractionation
Extraction of the sediment and subsequent saponi®-cation and fractionation of the extract into fractions containing apolar and polar neutral lipids and fatty acids was performed as previously described (Hartgers et al., 1996). The fatty acids were methylated by heating for 5 min at 60C with BF
3/MeOH. The polar fraction
(c. 10 mg) was reduced using nickel boride (Schouten et al., 1993) for isotopic analysis of carotenoids. Approxi-mately 500 mg of anhydrous NiCl2 and 500 mg of
NaBH4 were used for reduction. These amounts are
approximately 5 times higher than those described in Schouten et al. (1993) and were used in order to improve the yields. After reduction, the fraction was separated into an apolar and polar fraction using column chro-matography (Al2O3 as stationary phase) with hexane/
dichloromethane (9:1. v/v) and dichloromethane/ methanol (1:1, v/v) as eluents, respectively.
2.3. Argentatious thin layer chromatography
Aliquots (c. 10 mg) of the methylated fatty acid frac-tions were further separated by argentatious thin layer chromatography (Ag+-TLC) using toluene as
devel-oper. The AgNO3-impregnated silica plates (Merck;
2020 cm; thickness 0.25 mm) were prepared by dipping them into a solution of 10% AgNO3 in CH3CN for 4
min, drying at 70C for 30 min and subsequent activation
at 110C for 20 min. The silver-loading of the plates is
approx. 23%, as determined by weighing the TLC-plate before and after impregnation. Four fractions (F1,
Rf=0.87±1.00; F2, Rf=0.47±0.87; F3, Rf=0.14±0.47;
F4,Rf=0.00±0.14) were scraped o the TLC plate and
ultrasonically extracted with ethyl acetate (3). Only fractions F1 and F2 contained GC-amenable com-pounds, i.e. saturated and unsaturated fatty acids.
2.4. Derivatisation of unsaturated fatty acids to oxazolines (or 2-alkenyl-4,4-dimethylozaxolines)
To determine the position of the double bond(s) in unsaturated fatty acids, their corresponding 2-alkenyl-4,4-dimethyloxazolines were prepared by a modi®cation of the literature method (Zhang et al., 1988). Typically, 50mg of non-methylated fatty acids were mixed with 250
ml (234 mg) of 2-amino-2-methylpropanol, ¯ushed with N2, in a screw-capped PyrexTMtest tube and heated at
180C for 12 h. After cooling, 1 ml of deionized water
(3) was added to the reaction mixture and the deriva-tised fatty acids were extracted with 1 ml ofn-hexane (3). The location of the double bond(s) is revealed by a mass separation of 12 amu instead of the regular 14 amu in the homologous ion series of the mass spectra (Zhang et al., 1988).
2.5. Fatty acids ofMarichromatium purpuratum
For the investigation of fatty acids of purple sulfur bacteria, a batch culture ofMarichromatium purpuratum
was analysed. Marichromatium purpuratum ( Chroma-tium purpuratum) IO2203 was grown in anoxic seawater medium. Filtered seawater (0.2mm) was amended with KH2PO4(0.1 g), NH4Cl (0.5 g), trace element solution
SL12 (Pfennig and TruÈper, 1992) at 1 ml/l, vitamin solution V7 (Pfennig and TruÈper, 1992) at 1ml/l, Na2S.9H2O (2 mM ®nal concentration) and NaHCO3
(1.5 g). The medium was autoclaved and the pH adjus-ted to 7.2. Cultures were grown in batch cultures at 30
mmols photons mÿ2sÿ1using incandescent light, using a
16 h/8 h light/dark regime. Sul®de was aseptically added several times following its depletion in the culture to obtain a higher biomass (to a ®nal concentration of 2 mM added from sterilised Na2S.9H2O stock solutions).
The cells were harvested and analysed for fatty acids as described above.
2.6. Instrumental analysis
Gas chromatography (GC) was performed using a Carlo Erba 5300 instrument equipped with a splitless injector and a FID detector. A fused silica capillary column (30 m0.25 mm i.d.) coated with DB-5 (J&W Scienti®c; ®lm thickness 0.25mm) was used with hydro-gen as carrier gas (50 cm/s). The samples (in ethyl acet-ate) were injected at 70C and the oven temperature was
subsequently raised to 130C at 10C/min and then at
4C to 320C, at which it was held for 30 min. The
technique), keeping the split valve closed for 35 s. Injector and detector temperatures were 300 and 330C,
respectively. For the analyses of the fatty acids (as their methyl esters) a DB-23 fused silica capillary column (20 m0.18 mm; J&W; ®lm thickness 0.20mm) was used. The carrier gas was helium.
GC±mass spectrometry (MS) was performed with a Fisons MD800 instrument. The gas chromatograph was equipped with a HP-5MS fused silica capillary column (30 m0.25 mm i.d.; Hewlett-Packard; ®lm thickness 0.25 mm). Helium was used as carrier gas. The oven temperature was programmed from 70 to 130C at
10C/min and subsequently at 4C/min to a ®nal
tem-perature of 310C, at which it was held for 30 min.
Injection conditions (300C) were as described above.
Mass spectra were acquired in the electron impact mode (70 eV) scanning from 50 to 700 mass units with a cycle time of 1 s.
The DELTA-C irm-GC-MS-system is similar in principle to the DELTA-S system as described pre-viously (Hayes et al., 1990). The gas chromatograph was equipped with a CP Sil-5 fused silica capillary column (25 m0.32 mm; Chrompack; ®lm thickness 0.20mm) with helium as carrier gas. For the analysis of the fatty acids (as their methyl esters) a DB-23 fused silica capil-lary column (20 m0.18 mm; J&W; ®lm thickness 0.20
mm) with helium as carrier gas was used. The samples (dissolved in n-hexane or ethylacetate) were injected on column at 70C and subsequently the oven was
pro-grammed to 130C at 20C/min, and then at 4C/min to
320 at which it was held for 20 min. The stable carbon isotope compositions are reported in the delta notation against the V-PDB 13C standard. The d13C values of
alcohols were corrected for the isotopic contribution of the trimethylsilyl group which was determined from the stable carbon isotope values measured for 1-hex-adecanol and its silylated counterpart. Likewise, the isotopic contribution of the methyl group in fatty acids was corrected by determining thed13C values (triplicate
injections) of dodecanoic acid and its methyl ester. Bulk stable carbon isotopic compositions of the par-ticulate organic matter samples and the surface sediment were determined by automated on-line combustion (Carlo Erba CN analyser 1502 series) followed by con-ventional isotope ratio±mass spectrometry (Fisons Optima). Prior to analyses of the particulate organic matter, residual carbonate was removed from the ®lter by washing with dilute HCl.
3. Results and discussion
The distributions of compounds in the dierent frac-tions, i.e. the apolar hydrocarbon, polar and reduced polar fractions in the sediment from Lake Ciso have been discussed before (Hartgers et al., 1996). Brie¯y, the
saturated hydrocarbon fraction contains predominantly C20±C35 odd-carbon-numbered n-alkanes, whilst the
polar fraction consists of C20±C32
even-carbon-num-bered n-alkan-1-ols, phytol (I) and 24-ethylcholest-5-ene-3b-ol (II, sitosterol). The apolar fraction isolated from the reduced polar fraction is dominated by phy-tane, derived from the reduction of phytol, small amounts of C27 and C29 n-alkanes and
24-ethylcholes-tane. Furthermore, a range of reduced carotenoids is present, with isorenieratane (III) and ``reduced oke-none'' (IV) in relatively high amounts (Hartgers et al., 1996). In addition, fatty acids were also analysed in the present study and their distribution is shown in Fig. 2. The mixture comprises both free and ester-bound com-ponents released after saponi®cation and is char-acterised by even-numbered alkanoic acids showing a bimodal distribution ranging from C12to C18and C20to
C30.n-Hexadecanoic,n-hexadec-9-enoic (V) andn
-octa-dec-9-enoic acid (VI) dominate the short-chain fatty acids, with minor amounts of n-octadec-11-enoic acid (VII). Signi®cant amounts of 17b,21b (H)-bishomoho-panoic acid (VIII) were also detected. The fractions described above were analysed for their stable carbon isotopic compositions.
3.1. Terrestrial markers
The strong odd-over-even carbon number pre-dominance of the C20±C30 n-alkanes strongly suggests
that they are derived from terrestrial sources. Both the distributions of C20 to C30 n-alkanols and fatty acids
have a strong even-over-odd carbon number pre-dominance suggesting that they are also derived from terrestrial sources (Bianchi, 1995). This is, to some degree, con®rmed by their stable carbon isotopic com-positions (Table 1; Fig. 3). For instance, thed13C-value
of the C25n-alkane (ÿ32.3%), presumably derived from
decarboxylation of C26 fatty acid (Bianchi, 1995), is
similar to that of the C26alkan-1-ol (ÿ32.2%) and the
C26fatty acid (ÿ31.9%). However, there are also some
signi®cant dierences, especially between the fatty acids on the one hand and the n-alkanes/n-alkanols on the other. For instance, the C22fatty acid is 3±4%depleted
compared to the C22n-alkan-1-ol and C21n-alkane. This
may suggest an alternative source for some of the even-carbon-numbered fatty acids. Thed13C values of then
-alkanes (and n-alkanols and fatty acids) point to a contribution of terrestrial C3 plant tissues which are
typically more depleted than ÿ28% (Collister et al.,
usually associated with terrestrial inputs (Huang and Meinschein, 1976) though other non-terrestrial sources are known (Volkman, 1986; Volkman et al., 1998). The
13C-content of 24-ethylcholestane in the reduced polar
fraction is approximately 6%enriched compared to the terrestrially derivedn-alkanes. This is by far larger than the 1% dierence typically observed for the stable
carbon isotopic compositions of sitosterol compared to that n-alkane waxes from tree leaves (Collister et al., 1994; Lockheart et al., 1997). The dierence suggests that this compound is either from a very speci®c13
C-enriched terrestrial source or may partly be derived from an unknown aquatic source, possibly algae. Cryp-tomonas phaseolusandAnkistrodesmusspp. are the main algae in Lake Ciso (Villanueva et al., 1994) but it is, to the best of our knowledge, unknown if they biosynthe-size C29sterols.
3.2. Purple sulfur bacteria
The hydrogenated derivative of okenone present in the reduced polar fraction are derived from the purple sulfur bacteria Thiocystis minor and/or Ameobobacter purpureus (Villanueva et al., 1994). The isotopic com-position of ``reduced'' okenone (IV) of ÿ40% is very depleted in13C with respect to the terrestrial biomarkers
(Table 1; Fig. 3). Culture experiments with dierent species ofChromatiumhave consistently shown a deple-tion ofÿ20 toÿ23%of their biomass compared to their
inorganic carbon source (Wong et al., 1975; Quandt et al., 1977; SirevaÊg et al., 1977; Madigan et al., 1989),
Fig. 2. Gas chromatogram of the fatty acid fraction (methylated) isolated from the extract of Lake CisoÂ. Numbers indicate carbon chain length of the free acid. Key:ai-15=anteiso-pentadecanoic acid;i-15=iso-pentadecanoic acid. Double bond positions of unsa-turated fatty acids are determined by a derivatisation to their corresponding 4,4-dimethyloxazolines (Zhang et al., 1988). Roman numbers refer to numbers of structures in the Appendix.
whilst carotenoids are approximately 3±5% depleted
compared to their biomass (Wong et al., 1975; Madigan et al., 1989). These values are not signi®cantly dierent from those observed for autotrophic algae using the Calvin cycle (Hayes, 1993), suggesting that the max-imum fractionation in 13C during carbon ®xation of
purple sulfur bacteria is also not signi®cantly dierent. Thus, the very depletedd13C value of okenone probably
re¯ects the depleted carbon source used by the purple sulfur bacteria. Indeed, particulate organic matter sam-ples taken at dierent depths (Table 2) also show very
13C-depleted carbon isotopic compositions at 1.5 m
depth where the sul®de-oxidising bacteria are living (visible by their pink color). Thus, the lowd13C values
are likely due to ®xation of 13C-depleted DIC derived
from the microbial decomposition of autochthonous and allochtonous organic matter, as commonly observed in meromictic lake systems (e.g. Rau, 1978; Fry, 1986, Wachniew and Roszanski, 1997). Since these bacteria lived at 1±2 m water depth (Villanueva et al., 1994) and were most abundant during spring and sum-mer strati®cation, the dierence in 13C-content with
respect to DIC must have been very signi®cant. This phenomenon is also commonly observed in meromictic
lakes (e.g Rau, 1978; Fry, 1986; Bernasconi and Han-selmann, 1993; Wachniew and Roszanski, 1997). Indeed, using the data of Schaeer et al. (1997) and Bernasconi and Hasselman (1993) we can estimate an isotopic dierence of 24±26%for okenone of Chroma-tiaceae versus DIC in Lake Cadagno, suggesting that the isotopic composition of DIC in Lake Ciso may have been ca.ÿ14 toÿ16% at a water depth of 1.5 m, the
habitat of the phototrophic purple sulfur bacteria. Interestingly, phytol (I), measured as phytane in the reduced polar fraction, is also depleted in13C, although
it is enriched by 3%compared to reduced okenone (IV)
(Table 1). This tentatively suggests that the bacterio-chlorophylls of purple sulfur bacteria are the main source for phytol. However, it can not be excluded that the algae in Lake Ciso were also depleted in13C and
thus that their chlorophylls were also a source for the sedimentary phytol (I) since no sedimentary algal bio-marker was available. A similar observation has been made for Lake Cadagno (Putschew et al., 1996) where phytol (I) was inferred to be derived from purple sulfur bacteria based on its depleted13C-content.
Some 13C-depleted fatty acids, e.g. n-hexadecanoic
acid (d13C=
ÿ37%) and 11-octadecenoic acid (VII)
Table 1
Carbon isotope data of fractions from the sediment extract of Lake CisoÂ
Fraction/compound d13C (%) Fraction/compound d13C (%)
N-alkanes apolar fraction Fatty acids TLC-F1
C19 ÿ30.70.6 n-C12 ÿ30.31.8
C21 ÿ30.10.2 n-C14 ÿ38.00.4
C23 ÿ31.70.4 i-C15 ÿ34.20.6
C25 ÿ32.30.4 a-C15 ÿ35.41.0
C27 ÿ31.80.2 n-C15 ÿ32.20.9
C29 ÿ32.80.1 i-C16 ÿ30.41.3
C31 ÿ33.20.5 n-C16 ÿ37.40.1
C33 ÿ31.80.1 n-C17 ÿ35.80.5
n-C18 ÿ35.60.3
Polar fraction n-C20 ÿ33.50.1
Phytol ÿ36.80.1 n-C21 ÿ34.20.5
C18alkan-1-ol ÿ33.90.5 n-C22 ÿ34.10.1
C20alkan-1-ol ÿ31.00.2 n-C23 ÿ36.00.8
C22alkan-1-ol ÿ30.70.1 n-C24 ÿ33.20.2
C24alkan-1-ol ÿ32.00.3 n-C25 ÿ34.90.9
C26alkan-1-ol ÿ32.20.1 n-C26 ÿ31.90.1
C28alkan-1-ol ÿ33.10.4 n-C28 ÿ32.10.3
n-C30 ÿ35.21.0
Reduced polar fraction C32hopanoic acid ÿ34.70.4
Phytane ÿ36.00.1
C27n-alkane ÿ32.90.5 Fatty acids TLC- F2
C29n-alkane ÿ33.90.1 n-C18:19 ÿ33.10.4
24-ethyl-5a(H)-cholestane ÿ25.40.1 n-C18:111 ÿ42.60.3
Squalane ÿ27.20.8
Isorenieratane ÿ25.30.2 Bulk organic ÿ26.1
(d13C=
ÿ43%), also occur in Lake Ciso sediments. An
additional clue towards the origin of these compounds was given by a study of the fatty acid distribution of a culture of the purple sulfur bacteriumMarichromatium purpuratum. This bacterium exhibits a simple fatty acid distribution, which consists of 9-hexadecenoic acid (V),
n-hexadecanoic acid and 11-octadecenoic acid (Fig. 4). This distribution and the depleted13C values ofn
-hex-adecanoic acid and 11-octadecenoic acid (VII) in rela-tion to terrestrially-derived fatty acids (Table 1; Fig. 3), suggests that 11-octadecenoic acid (VII) and, in sig-ni®cant part n-hexadecanoic acid, are derived from purple sulfur bacteria. The isotopic depletion of 11-octadecanoic acid (VII) compared to reduced okenone, ca. 2%, is likely due to biosynthetic eects as observed for other organisms using the Calvin cycle (Hayes,
1993). The somewhat enriched isotopic value ofn -hex-adecanoic acid compared to 11-oct-hex-adecanoic acid (VII) shows that biological sources other than purple sulfur bacteria, possibly algae, contribute to this component in the lake as well. The low abundance of 9-hexadecenoic acid (VI) in Lake Ciso is possibly due to a speci®c degradation of this fatty acid or by variations in the fatty acid composition of the purple sulfur bacteria thriving at the anoxic/oxic boundary of Lake Ciso (Guerrero et al., 1987; Casamayor et al., 2000).
3.3. Green sulfur bacteria
Isorenieratane (III), the hydrogenated derivative of isorenieratene, is present in the reduced polar fraction and may derive from the brown-coloured green sulfur bacteriumChlorobium cf. vibrioforme(Villanueva et al., 1994) or related green sulfur bacteria (Casamayor et al., 2000). Its isotopic composition (ÿ25.3%) is enriched in
13C compared to the other sedimentary compounds
(Table 1; Fig. 3), which is due to its speci®c carbon acquisition mechanism, the reversed tricarboxylic acid cycle (Quandt et al., 1977; SirevaÊg et al., 1977). How-ever, thed13C-value of isorenieratane is rather depleted
in13C compared to previously reportedd13C-values of
isorenieratane in other sedimentary settings (e.g. Sin-ninghe Damste et al., 1993; Koopmans et al., 1996; Grice et al., 1996). This phenomenon may have a similar
Table 2
Carbon isotope data of water column particulate organic mat-ter samples from dierent depths in Lake CisoÂ
Depth (m) d13C (%)
0.3 ÿ29.30.2
1.5 ÿ36.10.1
3.0 ÿ34.30.4
5.9 ÿ31.70.3
explanation as that for reduced okenone, i.e. the green sulfur bacteria are assimilating 13C-depleted DIC in
Lake CisoÂ. This is also observed in Lake Cadagno where the isotopic composition of isorenieratene is
ÿ25% (Schaeer et al., 1997) and the isotopic compo-sition of DIC at the chemocline is ÿ13% (Bernasconi
and Hanselmann, 1993). In addition, Fry (1986) observed that the stable carbon isotopic composition of biomass of green sulfur bacteria in Fayetteville Green lake is aroundÿ31%, whilst the isotopic composition of
DIC is betweenÿ11 toÿ15%.
3.4. Sulfate-reducing bacteria
Branched fatty acids, such asiso-andanteiso -penta-decanoic acid and hepta-penta-decanoic acid have been found in cultures of sulfate-reducing bacteria (for a review, see Kaneda, 1991). The stable carbon isotopic compositions of iso- and anteiso-pentadecanoic acid are ca. ÿ35%
(Table 1; Fig. 3). Culture experiments by Mather et al. (1997) showed that these particular lipids in Desulfovi-brio desulfuricans are ca. 15% depleted compared to
their substrate, suggesting that in Late Ciso the isotopic composition of organic substrates on which these bac-teria were living heterotrophically was ca. ÿ20%. This makes it unlikely that they were living on products derived from phototrophic purple sulfur bacterial cell material but were using either isotopically enriched breakdown products (i.e. derived from sugars) of ter-restrial material or possibly algal cell material.
3.5. Bacterial contribution to bulk organic matter
Hartgers et al. (1997) showed that ¯ash pyrolysis of the extracted residue of this sediment yielded primarily signi®cant amounts of lignin-derived phenols. This sug-gested that the bulk organic matter consists of terrestrial organic matter, i.e. lignin and associated poly-saccharides. The stable carbon isotopic composition of the bulk organic matter in the sediment,ÿ26.1%, is in
agreement with this suggestion, since it is far more enri-ched in13C compared to markers of phototrophic
pur-ple sulfur bacteria, even when accounting for the 3±5%
dierence between carotenoids and biomass (Wong et al., 1975; Madigan et al., 1989). Thus, phototrophic purple bacterial biomass does not seem to form a sig-ni®cant part of the total organic matter in the sediments of Lake CisoÂ. Solely based on isotopic composition we cannot exclude a contribution of green sulfur bacterial biomass but the pyrolysis result clearly indicates terres-trial organic matter as the main source. This ®nding is in
agreement with observations for bulk organic matter in other lacustrine and marine sediments (Sinninghe Damste and Schouten, 1997).
4. Conclusions
Stable carbon isotopic analysis showed that bio-markers for phototrophic sulfur bacteria in Lake Ciso are considerably depleted in13C, most likely due to the
depleted 13C-content of the DIC that was
photo-synthetically ®xed. The stable carbon isotopic composi-tion of terrestrial markers (C25ÿ33
odd-carbon-numberedn-alkanes and C22ÿ30even-carbon-numbered n-alkan-1-ols and fatty acids) were similar to each other and indicated that they were derived from the sur-rounding vegetation. Phytol is predominantly derived from the bacteriochlorophylls of phototrophic purple sulfur bacteria as were speci®c fatty acids. The bulk carbon isotopic composition of the organic matter is in agreement with previous suggestions that it is pre-dominantly derived from terrestrial organic matter.
Acknowledgements
Two anonymous reviewers are thanked for their con-structive reviews on an earlier draft. This work was performed under the auspices of the ENOG (Human Capital and Mobility-Network Contract #CHRX-CT94-0474). ENOG (European Network of Organic Geochemistry Laboratories) comprises: Laboratoire de GeÂochimie, IFP, Rueil Malmaison, France; Laboratoire de Chimie Organique des Substances Naturelles, Uni-versite Louis Pasteur, Strasbourg, France; Department of Marine Biogeochemistry and Toxicology, Nether-lands Institute for Sea Research, Den Burg, The Neth-erlands; Organic Geochemistry Unit, Bristol University, Bristol, UK; Department of Environmental Chemistry (CID-CSIC), Barcelona, Spain; Institute of Petroleum and Organic Geochemistry, Research Center JuÈlich (KFA), Germany; and Geological Institute, University of Cologne, Cologne, Germany. Dr. R. de Wit (Uni-versity of Bordeaux, France) is gratefully acknowledged for providing a sample ofMarichromatium purpuratum. The Shell International Petroleum Maatschappij BV is thanked for the ®nancial support for the irm-GC±MS facility at Texel. We thank R. Chaler and M. Dekker for analytical assistance. This is NIOZ contribution no. 3463.
Appendix A
References
Bernasconi, S., Hanselmann, K., 1993. The in¯uence of bacterial activity at the chemocline on particulate organic matter ¯uxes and nutrient cycling in meromictic Lake Cadagno. In: Grimalt, J.O., Dorronsoro, C. (Eds.), Organic Geochemistry: Develop-ments and Applications to Energy, Climate, Environment and Human History. AIGOA, San Sebastian, pp. 1169±1170. Bianchi, G., 1995. Plant waxes. In: Hamilton, R.J. (Ed.),
Waxes: Chemistry, Molecular Biology and Functions. The Oily Press, Dundee, pp. 176±222 (Chapter 5).
Casamayor, E.O., NuÂnÄez-Cardona, M.T., CalderoÂn-Paz, J.I., Mas, J., PedroÂs-Alio, C., 2000. Comparison of pure cultures and natural assemblages of planktonic photosynthetic sulfur bacteria by low molecular mass RNA ®ngerprinting. FEMS Microbiology Ecology 32, 25±34.
Collister, J.W., Rieley, G.R., Stern, B., Eglinton, G., Fry, B., 1994. Compound-speci®cd13C analyses of leaf lipids from plants with diering carbon dioxide metabolisms. Organic Geochemistry 21, 619±627.
Deevey, E.S., Nakai, N., Stuiver, M., 1963. Fractionation of sulfur and carbon isotopes in a meromictic lake. Science 139, 407±408. Fry, B., 1986. Sources of carbon and sulfur nutrition for con-sumers in three meromictic lakes of New York State. Lim-nology and Oceanography 31, 79±88.
Grice, K., Schaeer, P., Schwark, L., Maxwell, J.R., 1996. Molecular indicators of palaeoenvironmental conditions in an immature Permian shale (Kupferschiefer, Lower Rhine Basin, north-west Germany) from free and S-bound lipids. Organic Geochemistry 25, 131±148.
Guerrero, R., PedroÂs-AlioÂ, C., Esteve, I., Mas, J., 1987. Com-munities of phototrophic sulfur bacteria in lakes of the Spanish Mediterranean region. Acta Academiae Aboensis 47, 125±151.
Hartgers, W.A., LoÁpez, J.F., de las Heras, F.X.C., Grimalt, J.O., 1996. Sulphur-binding in recent environments. I. Lipid by-products from Ni2B desulphurization. Organic Geo-chemistry 25, 353±365.
Hartgers, W.A., LoÁpez, J.F., Sinninghe DamsteÂ, J.S., Reiss, C., Maxwell, J.R., Grimalt, J.O., 1997. Sulfur-binding in recent environments. II. Speciation of sulfur and iron and implica-tions for the occurrence of organo-sulfur compounds. Geo-chimica et CosmoGeo-chimica Acta 61, 4767±4788.
Hayes, J.M., Freeman, K.H., Popp, B.N., Hoham, C.H., 1990. Compound-speci®c isotope analysis: a novel tool for recon-struction of ancient biogeochemical processes. In: Durand, B., Behar, F. (Eds.), Advances in Organic Geochemistry 1989.>Pergamon, Oxford, pp. 1115±1128.
Hayes, J.M., 1993. Factors controlling13C contents of sedi-mentary organic compounds: Principles and evidence. Mar-ine Geology 113, 111±125.
Huang, W.-Y., Meinschien, W.G., 1976. Sterols as source indicators of organic material in sediments. Geochimica et Cosmochimica Acta 40, 323±330.
Kaneda, T., 1991. Iso- and anteiso-fatty acids in bacteria: Bio-synthesis, function, and taxonomic signi®cance. Micro-biology Reviews 55, 288±302.
for photic zone anoxia. Geochimica et Cosmochimica Acta 60, 4467±4496.
Lockheart, M.J., van Bergen, P.F., Evershed, R.P., 1997. Variations in the stable carbon isotopic compositions of individual lipids from the leaves of modern angiosperms: implications for the study of higher land plant-derived sedimentary organic matter. Organic Geochemistry 26, 137± 153.
Mather, I.D., Parkes, R.J., Maxwell, J.R., 1997. Unexpected carbon isotopic fractionation eects during growth of sulphate-reducing bacteria. Abstracts of the 18th Interna-tional Meeting on Organic Geochemistry. KFA Julich, pp. 25±26.
Madigan, M.T., Takigiku, R., Lee, R.G., Gest, H., Hayes, J.M., 1989. Carbon isotope fractionation by thermophilic phototrophic sulfur bacteria: Evidence for autotrophic growth in natural populations. Applied and Environmental Microbiology 55, 639±644.
Pfennig, N., TruÈper, H.G., 1992. The family Chromatiaceae. In: Balows, A., TruÈper, H.G., Dworkin, M., Harder, W., Schleifer, K.H. (Eds.), The Prokaryotes. Springer Verlag, New York, pp. 3200±3221.
Putschew, A., Scholz-BoÈttcher, B.M., RullkoÈtter, J., 1996. Early diagenesis of organic matter and related sulphur incorpora-tion in surface sediments of meromictic Lake Cadagno in the Swiss Alps. Organic Geochemistry 25, 379±390.
Quandt, L., Gottschalk, G., Ziegler, H., Stichler, W., 1977. Isotope discrimination by photosynthetic bacteria. FEMS Microbiology Letters 3, 227±230.
Rau, G., 1978. Carbon-13 depletion in a subalpine lake: carbon ¯ow implications. Science 201, 901±902.
Schaeer, P., Adam, P., Wehrung, P., Bernasconi, S., Albrecht, P., 1997. Molecular and isotopic investigation of free and S-bound lipids from an actual meromictic lake (Lake Cadagno, Switzerland). Abstracts of the 18th International Meeting on Organic Geochemistry. KFA Julich, pp. 57±58.
Schouten, S., Pavlovic, D., Sinninghe DamsteÂ, J.S., De Leeuw, J.W., 1993. Nickel boride: an improved desulphurizing agent
for sulphur-rich geomacromolecules in sediments and oils. Organic Geochemistry 20, 901±909.
Sinninghe DamsteÂ, J.S., Wakeham, S.G., Kohnen, M.E.L., Hayes, J.M., de Leeuw, J.W., 1993. A 6000-year sedimentary molecular record of chemocline excursions in the Black Sea. Nature 362, 827±829.
Sinninghe DamsteÂ, J.S., Schouten, S., 1997. Is there evidence for a substantial contribution of prokaryotic biomass to organic carbon in Phanerozoic carbonaceous sediments? Organic Geochemistry 26, 517±531.
SirevaÊg, R., Buchanan, B.B., Berry, J.A., Troughton, J.H., 1977. Mechanisms of CO2®xation in bacterial photosynth-esis studied by the carbon isotope technique. Archives of Microbiology 112, 35±38.
Villanueva, J., Grimalt, J.O., de Wit, R., Keely, B.J., Maxwell, J.R., 1994. Sources and transformations of chlorophylls and carotenoids in a monomictic sulfate-rich karstic lake envir-onment. In: Telnaes, N., van Graas, G., éygard, K. (Eds.), Advances in Organic Geochemistry, 1993. Pergamon, Oxford, pp. 739±757, 22.
Volkman, J.K., 1986. A review of sterol markers for marine and terrigenous organic matter. Organic Geochemistry 9, 83±99. Volkman, J.K., Barrett, S.M., Blackburn, S.I., Mansour, M.P.,
Sikes, E.L., Gelin, F., 1998. Microalgal biomarkers: a review of recent research developments. Organic Geochemistry 29, 1163±1180.
Wachniew, P., Rozanski, K., 1997. Carbon budget of a mid-latitude, groundwater-controlled lake: Isotopic evidence for the importance of dissolved inorganic carbon recycling. Geochimica et Cosmochimica Acta 61, 2453±2465.
Wong, W., Sackett, W.M., Benedict, C.R., 1975. Isotope frac-tionation in photosynthetic bacteria during carbon dioxide assimilation. Plant Physiology 55, 475±479.
