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Organic Geochemistry 31 (2000) 777±786
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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 Damste b, Joan O. Grimalt a

a
Department of Environmental Chemistry (CID-CSIC), Jordi Girona 18-26, 08034 Barcelona, Catalonia, Spain
Netherlands Institute for Sea Research (NIOZ), Department of Marine Biogeochemistry and Toxicology, PO Box 59,
1790 AB Den Burg, The Netherlands

b

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 photosynthetically ®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 nalkanes and C22ÿ30 even-carbon-numbered n-alkan-1-ols and fatty acids, were rather similar to each other and their 13C
depleted values (c. ÿ31 to ÿ35%) indicated that they were derived from the surrounding vegetation. Phytol was predominantly 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; Carotenoids; CO2 recycling; 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 incorporation of sulfur into functionalized lipids (Hartgers et
* Corresponding author. Tel.: +31-222-369565; fax: +31222-319674.
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
Present address: InstituÈt fur Chemie und Dynamik der
GeosphaÈre, Forschungszentrum JuÈlich GmbH, D-52425 JuÈlich,
Germany.

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 in 13C (ÿ30 to ÿ41%) due to the depleted 13Ccontent of DIC (up to ÿ21%) in several meromictic
lakes.
Studies of the stable carbon isotopic composition of
speci®c bacterial biomarkers deposited in these environments are limited. Such data may be of great interest

because strati®ed sul®de-rich lakes usually have speci®c

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778

W.A. Hartgers et al. / Organic Geochemistry 31 (2000) 777±786

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 reported 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 sediments, whilst Schae€er et al. (1997) determined the 13Ccontents of bacterial carotenoids in the same sediments.
Both authors found depleted 13C-contents for bacterial

lipids, which is due to the low 13C-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 bacterial 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 compounds 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 the 13C-composition of lipids that
may allow a more detailed reconstruction of the terrestrial 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 Villanueva 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 produced is utilised by communities of both purple and

Fig. 1. Maps showing the location of Lake CisoÂ.

W.A. Hartgers et al. / Organic Geochemistry 31 (2000) 777±786

779

green sulfur bacteria living below the chemocline (1.5±3
m depth). Microscopic analyses suggest that Chromatium minus (now named Thiocystis minor; Casamayor et
al., 2000), Chlorobium limicola and C. vibrioforme dominate (Guerrero et al., 1987), but recent molecular biological 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 Ankistrodesmus 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 strati®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 di€erent depths (0.3 and
1.5, above or at the chemocline and 3.0 and 5.9 m,
below the chemocline) in July 1996.

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 compounds, i.e. saturated and unsaturated fatty acids.

2.2. Extraction and fractionation

2.5. Fatty acids of Marichromatium purpuratum

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 60 C with BF3/MeOH. The polar fraction
(c. 10 mg) was reduced using nickel boride (Schouten et
al., 1993) for isotopic analysis of carotenoids. Approximately 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 chromatography (Al2O3 as stationary phase) with hexane/
dichloromethane (9:1. v/v) and dichloromethane/
methanol (1:1, v/v) as eluents, respectively.

For the investigation of fatty acids of purple sulfur
bacteria, a batch culture of Marichromatium purpuratum
was analysed. Marichromatium purpuratum (Chromatium purpuratum) IO2203 was grown in anoxic seawater
medium. Filtered seawater (0.2 mm) 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 adjusted to 7.2. Cultures were grown in batch cultures at 30
mmols photons mÿ2 sÿ1 using 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.

2.4. 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-alkenyl4,4-dimethyloxazolines were prepared by a modi®cation
of the literature method (Zhang et al., 1988). Typically,
50 mg of non-methylated fatty acids were mixed with 250
ml (234 mg) of 2-amino-2-methylpropanol, ¯ushed with
N2, in a screw-capped PyrexTM test tube and heated at

180 C for 12 h. After cooling, 1 ml of deionized water
(3) was added to the reaction mixture and the derivatised fatty acids were extracted with 1 ml of n-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).

2.3. Argentatious thin layer chromatography
2.6. Instrumental analysis
Aliquots (c. 10 mg) of the methylated fatty acid fractions were further separated by argentatious thin layer
chromatography (Ag+-TLC) using toluene as developer. 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 70 C for 30 min and subsequent activation
at 110 C 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;

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 0.25 mm) was used with hydrogen as carrier gas (50 cm/s). The samples (in ethyl acetate) were injected at 70 C and the oven temperature was
subsequently raised to 130 C at 10 C/min and then at
4 C to 320 C, at which it was held for 30 min. The
injection was in the splitless mode (hot needle

780

W.A. Hartgers et al. / Organic Geochemistry 31 (2000) 777±786

technique), keeping the split valve closed for 35 s.
Injector and detector temperatures were 300 and 330 C,
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 0.20 mm) 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 130 C at
10 C/min and subsequently at 4 C/min to a ®nal temperature of 310 C, at which it was held for 30 min.
Injection conditions (300 C) 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 previously (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 0.20 mm)
with helium as carrier gas. For the analysis of the fatty
acids (as their methyl esters) a DB-23 fused silica capillary 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 70 C and subsequently the oven was programmed to 130 C at 20 C/min, and then at 4 C/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-hexadecanol and its silylated counterpart. Likewise, the
isotopic contribution of the methyl group in fatty acids
was corrected by determining the d13C values (triplicate
injections) of dodecanoic acid and its methyl ester.
Bulk stable carbon isotopic compositions of the particulate organic matter samples and the surface sediment
were determined by automated on-line combustion
(Carlo Erba CN analyser 1502 series) followed by conventional 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 di€erent fractions, 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-numbered n-alkan-1-ols, phytol (I) and 24-ethylcholest-5ene-3b-ol (II, sitosterol). The apolar fraction isolated
from the reduced polar fraction is dominated by phytane, derived from the reduction of phytol, small
amounts of C27 and C29 n-alkanes and 24-ethylcholestane. Furthermore, a range of reduced carotenoids is
present, with isorenieratane (III) and ``reduced okenone'' (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 components released after saponi®cation and is characterised by even-numbered alkanoic acids showing a
bimodal distribution ranging from C12 to C18 and C20 to
C30. n-Hexadecanoic, n-hexadec-9-enoic (V) and n-octadec-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)-bishomohopanoic 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 predominance 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 predominance suggesting that they are also derived from
terrestrial sources (Bianchi, 1995). This is, to some
degree, con®rmed by their stable carbon isotopic compositions (Table 1; Fig. 3). For instance, the d13C-value
of the C25 n-alkane (ÿ32.3%), presumably derived from
decarboxylation of C26 fatty acid (Bianchi, 1995), is
similar to that of the C26 alkan-1-ol (ÿ32.2%) and the
C26 fatty acid (ÿ31.9%). However, there are also some
signi®cant di€erences, especially between the fatty acids
on the one hand and the n-alkanes/n-alkanols on the
other. For instance, the C22 fatty acid is 3±4% depleted
compared to the C22 n-alkan-1-ol and C21 n-alkane. This
may suggest an alternative source for some of the evencarbon-numbered fatty acids. The d13C values of the nalkanes (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.,
1994; Lockheart et al., 1997). Indeed, the terrestrial
vegetation around Lake Ciso is dominated by populations of Populus alba, Fraxinus angustifolia and Ulmus
minor (Garcia-Gil, pers. comm.). Interestingly, 24ethylcholestane dominates the steranes in the reduced
polar fraction, whilst sitosterol dominates the sterols.
Such a dominance of C29 steroids over other steroids is

W.A. Hartgers et al. / Organic Geochemistry 31 (2000) 777±786

781

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 unsaturated 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.

usually associated with terrestrial inputs (Huang and
Meinschein, 1976) though other non-terrestrial sources
are known (Volkman, 1986; Volkman et al., 1998). The
13
C-content of 24-ethylcholestane in the reduced polar
fraction is approximately 6% enriched compared to the
terrestrially derived n-alkanes. This is by far larger than
the 1% di€erence 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 di€erence suggests
that this compound is either from a very speci®c 13Cenriched terrestrial source or may partly be derived
from an unknown aquatic source, possibly algae. Cryptomonas phaseolus and Ankistrodesmus spp. are the main
algae in Lake Ciso (Villanueva et al., 1994) but it is, to
the best of our knowledge, unknown if they biosynthesize C29 sterols.
3.2. Purple sulfur bacteria

Fig. 3. Graph showing the distribution of stable carbon isotopic composition of compounds (shaded areas) derived from
the di€erent source organisms present in Lake CisoÂ.

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 composition of ``reduced'' okenone (IV) of ÿ40% is very
depleted in 13C with respect to the terrestrial biomarkers
(Table 1; Fig. 3). Culture experiments with di€erent
species of Chromatium have consistently shown a depletion of ÿ20 to ÿ23% 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),

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W.A. Hartgers et al. / Organic Geochemistry 31 (2000) 777±786

Table 1
Carbon isotope data of fractions from the sediment extract of Lake CisoÂ
Fraction/compound

d 13C (%)

Fraction/compound

d 13C (%)

N-alkanes apolar fraction
C19
C21
C23
C25
C27
C29
C31
C33

ÿ30.70.6
ÿ30.10.2
ÿ31.70.4
ÿ32.30.4
ÿ31.80.2
ÿ32.80.1
ÿ33.20.5
ÿ31.80.1

Fatty acids TLC-F1
n-C12
n-C14
i-C15
a-C15
n-C15
i-C16
n-C16
n-C17
n-C18

ÿ30.31.8
ÿ38.00.4
ÿ34.20.6
ÿ35.41.0
ÿ32.20.9
ÿ30.41.3
ÿ37.40.1
ÿ35.80.5
ÿ35.60.3

Polar fraction
Phytol
C18 alkan-1-ol
C20 alkan-1-ol
C22 alkan-1-ol
C24 alkan-1-ol
C26 alkan-1-ol
C28 alkan-1-ol

ÿ36.80.1
ÿ33.90.5
ÿ31.00.2
ÿ30.70.1
ÿ32.00.3
ÿ32.20.1
ÿ33.10.4

n-C20
n-C21
n-C22
n-C23
n-C24
n-C25
n-C26
n-C28
n-C30

ÿ33.50.1
ÿ34.20.5
ÿ34.10.1
ÿ36.00.8
ÿ33.20.2
ÿ34.90.9
ÿ31.90.1
ÿ32.10.3
ÿ35.21.0

Reduced polar fraction
Phytane
C27 n-alkane
C29 n-alkane
24-ethyl-5a(H)-cholestane
Squalane
Isorenieratane
Reduced okenone

C32 hopanoic acid

ÿ34.70.4

ÿ36.00.1
ÿ32.90.5
ÿ33.90.1
ÿ25.40.1
ÿ27.20.8
ÿ25.30.2
ÿ40.30.6

Fatty acids TLC- F2
n-C18:1
9
n-C18:1
11

ÿ33.10.4
ÿ42.60.3

Bulk organic

ÿ26.1

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 di€erent
from those observed for autotrophic algae using the
Calvin cycle (Hayes, 1993), suggesting that the maximum fractionation in 13C during carbon ®xation of
purple sulfur bacteria is also not signi®cantly di€erent.
Thus, the very depleted d13C value of okenone probably
re¯ects the depleted carbon source used by the purple
sulfur bacteria. Indeed, particulate organic matter samples taken at di€erent depths (Table 2) also show very
13
C-depleted carbon isotopic compositions at 1.5 m
depth where the sul®de-oxidising bacteria are living
(visible by their pink color). Thus, the low d13C 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 summer strati®cation, the di€erence 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 Hanselmann, 1993; Wachniew and Roszanski, 1997).
Indeed, using the data of Schae€er et al. (1997) and
Bernasconi and Hasselman (1993) we can estimate an
isotopic di€erence of 24±26% for okenone of Chromatiaceae versus DIC in Lake Cadagno, suggesting that
the isotopic composition of DIC in Lake Ciso may have
been ca. ÿ14 to ÿ16% 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 in 13C, although
it is enriched by 3% compared to reduced okenone (IV)
(Table 1). This tentatively suggests that the bacteriochlorophylls 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 in 13C and
thus that their chlorophylls were also a source for the
sedimentary phytol (I) since no sedimentary algal biomarker 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 depleted 13C-content.
Some 13C-depleted fatty acids, e.g. n-hexadecanoic
acid (d13C=ÿ37%) and 11-octadecenoic acid (VII)

W.A. Hartgers et al. / Organic Geochemistry 31 (2000) 777±786
Table 2
Carbon isotope data of water column particulate organic matter samples from di€erent depths in Lake CisoÂ
Depth (m)

d 13C (%)

0.3
1.5
3.0
5.9

ÿ29.30.2
ÿ36.10.1
ÿ34.30.4
ÿ31.70.3

783

1993). The somewhat enriched isotopic value of n-hexadecanoic acid compared to 11-octadecanoic 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 CisoÂ
(Guerrero et al., 1987; Casamayor et al., 2000).
3.3. Green sulfur bacteria

(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 bacterium Marichromatium
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 depleted 13C values of n-hexadecanoic acid and 11-octadecenoic acid (VII) in relation to terrestrially-derived fatty acids (Table 1; Fig. 3),
suggests that 11-octadecenoic acid (VII) and, in signi®cant part n-hexadecanoic acid, are derived from
purple sulfur bacteria. The isotopic depletion of 11octadecanoic acid (VII) compared to reduced okenone,
ca. 2%, is likely due to biosynthetic e€ects as observed
for other organisms using the Calvin cycle (Hayes,

Isorenieratane (III), the hydrogenated derivative of
isorenieratene, is present in the reduced polar fraction
and may derive from the brown-coloured green sulfur
bacterium Chlorobium cf. vibrioforme (Villanueva et al.,
1994) or related green sulfur bacteria (Casamayor et al.,
2000). Its isotopic composition (ÿ25.3%) is enriched in
13
C 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). However, the d13C-value of isorenieratane is rather depleted
in 13C compared to previously reported d13C-values of
isorenieratane in other sedimentary settings (e.g. Sinninghe Damste et al., 1993; Koopmans et al., 1996;
Grice et al., 1996). This phenomenon may have a similar

Fig. 4. Gas chromatogram of the fatty acid fraction (methylated) of the extract of Marichromatium purpuratum. Numbers indicate
carbon chain length of the free acid. Key: *=contamination. Double bond positions of unsaturated fatty acids were determined by a
derivatisation to their corresponding 4,4-dimethyloxazolines (Zhang et al., 1988).

784

W.A. Hartgers et al. / Organic Geochemistry 31 (2000) 777±786

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% (Schae€er et al., 1997) and the isotopic composition 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 around ÿ31%, whilst the isotopic composition of
DIC is between ÿ11 to ÿ15%.
3.4. Sulfate-reducing bacteria
Branched fatty acids, such as iso- and anteiso-pentadecanoic acid and heptadecanoic 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 Desulfovibrio desulfuricans are ca. 15% depleted compared to
their substrate, suggesting that in Late Ciso the isotopic
composition of organic substrates on which these bacteria 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 terrestrial 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 suggested that the bulk organic matter consists of terrestrial
organic matter, i.e. lignin and associated polysaccharides. 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 enriched in 13C compared to markers of phototrophic purple sulfur bacteria, even when accounting for the 3±5%
di€erence between carotenoids and biomass (Wong et
al., 1975; Madigan et al., 1989). Thus, phototrophic
purple bacterial biomass does not seem to form a signi®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 terrestrial 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 biomarkers for phototrophic sulfur bacteria in Lake CisoÂ
are considerably depleted in 13C, most likely due to the
depleted 13C-content of the DIC that was photosynthetically ®xed. The stable carbon isotopic composition of terrestrial markers (C25ÿ33 odd-carbonnumbered n-alkanes and C22ÿ30 even-carbon-numbered
n-alkan-1-ols and fatty acids) were similar to each other
and indicated that they were derived from the surrounding 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 predominantly derived from terrestrial organic matter.

Acknowledgements
Two anonymous reviewers are thanked for their constructive reviews on an earlier draft. This work was
performed under the auspices of the ENOG (Human
Capital and Mobility-Network Contract #CHRXCT94-0474). ENOG (European Network of Organic
Geochemistry Laboratories) comprises: Laboratoire de
GeÂochimie, IFP, Rueil Malmaison, France; Laboratoire
de Chimie Organique des Substances Naturelles, Universite Louis Pasteur, Strasbourg, France; Department
of Marine Biogeochemistry and Toxicology, Netherlands Institute for Sea Research, Den Burg, The Netherlands; 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 (University of Bordeaux, France) is gratefully acknowledged
for providing a sample of Marichromatium 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.
Associate EditorÐG.A. Wol€

W.A. Hartgers et al. / Organic Geochemistry 31 (2000) 777±786

785

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