Directory UMM :Data Elmu:jurnal:O:Organic Geochemistry:Vol31.Issue10.Oct2000:
Organic Geochemistry 31 (2000) 1031±1040
www.elsevier.nl/locate/orggeochem
Sterols of melanized fungi from hypersaline environments
Laurence MeÂjanelle a, Jordi F. LoÁpez a, Nina Gunde-Cimerman b,
Joan O. Grimalt a,*
a
Department of Environmental Chemistry, I.C.E.R.-C.S.I.C., Jordi Girona, 18. 08034-Barcelona, Catalonia, Spain
b
Biology Department, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia
Received 14 December 1999; accepted 20 June 2000
(returned to author for revision 18 February 2000)
Abstract
The lipid compositions of melanized fungi isolated from calcite, gypsum and halite depositional environments of
Mediterranean solar salterns, namely Hortaea werneckii, Alternaria alternata, Cladosporium cladosporioides, Cladosporium sp. and Aureobasidium pullulans, have been examined. Sterols constituted the most distinct lipid fraction.
Ergosterol, 24-methylcholesta-5,7,22-trien-3b-ol, dominated all distributions but major dierences between species
were encountered when considering the subordinate sterols. Thus, 24-methylcholest-7-en-3b-ol, 24-methylcholesta7,24(28)-dien-3b-ol and 4a,24-dimethylcholest-7-en-3b-ol were found in signi®cant proportions in Cladosporium spp
(14±20%), A. alternata (28%) and H. wernekii (29%), respectively. These sterols can be used for discrimination
between these dierent fungal species. 24-Methylcholest-7-en-3b-ol and 24-methylcholesta-7,24(28)-dien-3b-ol were
found in signi®cant proportion in the water column particles and sediments of the gypsum and halite precipitation
ponds (the latter only in the halite domain). In such environments, these sterols may provide a speci®c signature for
these melanized fungi. However, water column particulate matter and sediments from hypersaline depositional settings
show sterol compositions dominated by the constituents typically encountered in phytoplankton and zooplankton, but
not in melanized fungi. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Sterols; Ergosterol; Fungi; Hypersaline environments; Solar salterns
1. Introduction
Historically, the term halophile was only applied to
specialized bacteria until 1975 when it was applied to
foodborne fungi exhibiting superior growth on media
with NaCl as controlling solute (Pitt and Hocking,
1985). Fungi were subsequently described in salt marshes
(Newell, 1996), saline soil (Guiraud et al., 1995) and seawater (Kohlmeyer and Volkmann-Kohlmeyer, 1991) but
were considered unable to grow in highly saline waters.
Very recently, however, high fungal diversity has been
identi®ed in hypersaline waters and surface layers of
microbial mats in environments with salinities ranging
* Corresponding author. Tel.: +34-93-400-6122; fax: +3493-204-5904.
E-mail address: jgoqam@cid.csis.es (J.O. Grimalt).
between 15 and 32%. These fungi were ®rst isolated in
hypersaline waters of marine salterns from Secovlje in
Slovenia (Gunde-Cimerman et al., in press). Subsequent
studies in the solar saltern of La Trinitat (Ebro Delta,
Catalonia, Spain) and BonmatõÂ (Santa Pola, Valencian
Community, Spain) showed the occurrence of the same
dominant species.
The majority of isolates were determined to belong to
melanized meristematic and yeast-like fungi, and a limited number to dierent genera of ®lamentous fungi.
Among the isolated halotolerant/halophilic mycobiota
the following genera were found: Hortaea, Phaeotheca,
Trimmatostroma, Aureobasidium, Alternaria, Cladosporium (Zalar et al., 1999a±c). These dematiaceous fungi
have the common property that they form black, clumplike colonies consisting of isodiametrically dividing cells
in the water, whereas the hyphal growth is mainly exhibited on solid media. This unique in-situ morphology was
0146-6380/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0146-6380(00)00094-2
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L. MeÂjanelle et al. / Organic Geochemistry 31 (2000) 1031±1040
interpreted as a response to multiple stress factors which
helps the fungi to tolerate high temperatures and low
water activity by optimization of the volume-surface
ratio (Zalar et al., 1999a). Among the isolated ®lamentous fungi, better known for their xerophilic nature,
the genera Aspergillus, Penicillium and Wallemia were
prevalent (Gunde-Cimerman et al., 1997).
The recent discovery of these fungal species prompted
us to investigate the geochemical signi®cance of their
organic residues in hypersaline environments. Since
melanized fungi are the most abundant fungal species at
higher salinities, the present study is devoted to this
group of organisms.
Halophilic and halotolerant fungi present in the
diverse depositional environments of La Trinitat solar
saltern were considered for study. As reported previously, the complete sequence of sea water evaporation
including calcite, gypsum and halite precipitation is
represented in this system (Grimalt et al., 1992; de Wit
and Grimalt, 1992; Villanueva et al., 1994; Hartgers et
al., 1997). Water was collected in diverse ponds encompassing the whole salinity range and ®ltered, the ®lters
being incubated on several selective media to stimulate
fungal growth. Agar baits in dialysis tubing were left in
these ponds from June to November. The fungi grown
in them were also cultured on selective media.
The lipid composition of the melanized species isolated from these hypersaline environments was examined. Sterols constituted the most distinct lipid fraction
when compared with the distributions typically encountered in other group organisms, e.g. phytoplankton,
zooplankton and bacteria. Thus, the present study is
focused on this group of compounds, which are discussed as source markers and compared with those present in waters (particulate phase) and sediments of solar
salterns.
2. Experimental
Aliquots of waters (10±100 ml) from the diverse saltern ponds were ®ltered immediately after sampling over
nitrocellulose Millipore membrane ®lters (pore size 0.45
mm) and placed on dierent selective agar media containing either high salt (17±32%) or high sugar concentration (50±70% glucose or fructose). A drop of the
original saline water was applied to the membrane and
dispersed with a Drigalski spatula. For every medium,
four aliquots were ®ltered in parallel and the average
number of colony forming units (CFU) were calculated.
Plates were incubated for 1±10 weeks at 25 C. CFU on
enumeration media were counted every 3, 5, 7, 14, 30
and 60 days of incubation.
Agar baits in dialysis tubing were left in diverse ponds
for 5 months from June to November. After collection,
the agar blocks were pushed out, cut aseptically and
plated out on low water activity media.
An enrichment technique was applied by the addition
of glucose and yeast extract to saline water from the
salterns and incubation on a rotary shaker. Subsequently, the broth was diluted with 17% saline water
several times.
All fungi isolated from selective media were inoculated in parallel on malt extract agar (MEA) and on
MEA+3M NaCl (17%). Only the fungal colonies able
to grow in the presence of 17% salt were further determined taxonomically. Growth rates and morphological
characteristics were determined on both media after 7
and 30 days. The agar medium used for growth yielded
no signi®cant content of sterols. Species identi®cation
was mainly based on descriptions from Ellis (1971;
1976). Identi®cations were con®rmed at the microbiological culture collection CBS (Centraalbureau voor
Schimmelcultures, Baarn, The Netherlands). Culture
purity was ensured by development of spore isolates of
individual fungi and plateing aseptically on fresh sterile
media.
The Saccharomyces cerevisiae strain W303a analyzed
for reference was obtained from the Yeast Stock Center
(Berkeley, CA, USA).
2.1. Site description and cultures of melanized fungi
2.2. Extraction and fractionation
The solar saltern ponds of La Trinitat are located in the
south wing of the Ebro Delta (Villanueva et al., 1994).
This environment is exploited to obtain halite by evaporation of seawater. The salt water circuit consists of a
series of ponds interconnected by sluices. In each pond,
salinity is kept more or less constant. Seawater is pumped
through the system and, because of evaporation, increasing salinity causes the successive precipitation of calcite,
gypsum and halite. A number of studies on the molecular
characteristics of solar saltern microbial mats (Aizenshtat
et al., 1983; Boon et al., 1983; Barbe et al., 1990; Teixidor
et al., 1993; Villanueva et al., 1994) and their microbial
biota (de Wit and Grimalt, 1992; van Gemerden, 1993;
Merino et al., 1995a) have been reported previously.
An aliquot of 1±1.5 g of melanized fungi, gently
scrapped o the MEA surface, was placed in 10150
mm Pyrex tubes. Lipids were extracted ultrasonically in
5 ml of methanol for 20 min, at 30 C. The procedure
was repeated twice with methanol and twice further with
dichloromethane (DCM). Extracts were recovered after
centrifugation. Combined extracts were vacuum concentrated to 1 ml and hydrolyzed by addition of 50 ml
of KOH in methanol (10%). The mixture was sonicated
at room temperature for 10 min, ¯ushed with nitrogen
and kept in the dark for 24 h. Neutral lipids were
extracted with hexane (420 ml). The methanol solution
was then acidi®ed to pH 2 by addition of HCl (aq) and
L. MeÂjanelle et al. / Organic Geochemistry 31 (2000) 1031±1040
further extracted with hexane (420 ml) for isolation of
the fatty acids.
The neutral lipids were fractionated by column chromatography using 1.5 g of 5% water-deactivated silica
gel (40 mesh 70-230 Merck) in a 1806 mm column.
Three fractions were collected: F1 (hydrocarbons), 12
ml of hexane+12 ml hexane/DCM (95:5); F2, 24 ml
DCM; and F3 (alcohols), 24 ml of DCM/Methanol
(90:10). Sterols (in F3) were converted into their trimethylsilylethers by reaction with bis-(trimethylsilyl)tri¯uoroacetamide (70 C, 30 min).
2.3. Instrumental analysis
Samples were dissolved in iso-octane and analyzed by
gas chromatography (GC) using a Carlo Erba 5300
(Carlo Erba, Italy) ®tted with a heated splitless injector
(300 C) and a capillary column (25 m0.25 mm i.d.;
5% phenyl-methyl polysiloxane DB-5; 0.25 mm ®lm
thickness; J&W) using hydrogen as carrier gas. The
oven was kept at 70 C for 1 min, heated to 150 C at
15 C minÿ1, then to 310 C at 4 C minÿ1, and ®nally
held at 310 C for 30 min. The temperature of the ¯ame
ionization detector (FID) was 330 C, the ¯ame was fed
with air (300 ml minÿ1) and hydrogen (30 ml minÿ1).
Nitrogen was used as make up gas (30 ml minÿ1). The
detector response was digitized by a Nelson 900 interface and processed with a Nelson 2600 software package
(Perkin Elmer). Relative concentrations of sterols were
calculated from the GC±FID response areas.
Analyses by GC±mass spectrometry (CG±MS) were
performed using a Fisons 8000 gas chromatograph
coupled to a Fisons MD-800 quadrupole mass analyzer.
Samples were injected in splitless mode at 300 C onto a
capillary column (25 cm0.25 mm i.d.; 5% phenylmethyl polysiloxane HP-5; 0.25 mm ®lm thickness;
Hewlett Packard). Helium was the carrier gas and the
temperature program was the same as for the GC
analyses.
Mass spectra were recorded in electron impact mode
at 70eV by scanning between m/z 50 and 650 every
second. Ion source and transfer line were kept at
300 C. Data were processed with Masslab software
(THERMO Instruments). Sterol identi®cation was
based on mass spectral interpretation and comparison
of mass spectra and retention time data with available
standards.
Lipids of particles and sediments from diverse hypersaline environments were already examined as described
in Barbe et al. (1990) and Grimalt et al. (1992). Sterols
were analyzed as trimethylsilylether derivatives by GC±
MS on a capillary column (25 m0.2 mm i.d.; 5% phenyl-methyl polisiloxane HP-5; 0.11 mm ®lm thickness)
using helium as carrier gas. The injector was kept at
290 C, the oven was programmed from 60 C to 300 C
at 4 C minÿ1 and kept at 300 C for 15 min.
1033
3. Results and discussion
3.1. Melanized fungal species in diverse hypersaline
systems
Hortaea werneckii, Alternaria alternata (Fr.) keissl.,
Cladosporium sphaerospermum, Aureobasidium pullulans
(de Bary) G. Arnaud and Cladosporium spp were identi®ed in La Trinitat solar saltern. Cladosporium spp. are
rather ubiquitous and found in the calcite, gysum and
halite precipitation ponds as well as in the magnesiumcontaining brines. H. werneckii was found in the mixed
calcite/gypsum, gypsum and halite depositional environments. A. alternata and A. pullulans were found in the
more extreme saline conditions. The former was identi®ed in the Mg brines and the halite chrystallizers and
the latter in the halite ponds.
The same major species of halotolerant or halophilic
melanized fungi were identi®ed in the Secovlje saltpans
(Slovenia; Zalar et al., 1999a, b, c; Gunde-Cimerman et
al., in press). This evaporitic system is situated in the
delta of the Dragonja River Delta, on the Adriatic
coast.
The hydrocarbon composition of all fungi except H.
werneckii is largely dominated by squalene, which
accounts for 1.2±7.7% of the neutral lipids (Table 1).
Squalene is also the dominant hydrocarbon in S. cerevisiae which is analyzed here as reference. The fatty acid
compositions of the halotolerant or halophilic species is
similar to the distributions found in algae, with a predominance of saturated and unsaturated C16 and C18
linear homologues.
3.2. Sterol composition
Most sterols identi®ed in the melanized fungal species
have a methyl substituent at C-24, as evidenced by the
molecular weight and fragment ions resulting from the
loss of the side chain (SC) and the trimethylsilylhydroxy
group (Fig. 1, Table 1). Thus, the spectrum of 24methylcholesta-5,7,22-trien-3b-ol shows a m/z 468 molecular ion and the fragment [M-(CH3)3SiOH-SC]+ at m/z
253 which is also indicative of a monounsaturated side
chain. Other dominant ions correspond to fragments [M(CH3)3SiOH-C3H3]+ and [M-(CH3)3SiOH-SC-C2H2]+,
m/z 363 and 211, respectively. The ion [M-SC-C3H5]+,
m/z 337, is typical of sterols with a double bond at position 22 (Rahier and Benveniste, 1989). This spectrum is
in agreement with that of ergosterol reported in Ascomycete yeasts (Parks and Casey, 1995; Weete, 1989).
The other main sterols in these melanized fungi possess
one to four unsaturations at positions 5, 7, 8, 22,
24(28) or 24.
The four tetraunsaturated sterols identi®ed in the
melanized fungi are generally found in minor amounts
although 24-methylcholesta-5,7,22,24(28)-tetraen-3b-ol,
1034
L. MeÂjanelle et al. / Organic Geochemistry 31 (2000) 1031±1040
Table 1
Relative composition of squalene and sterols of cultures of melanized fungi isolated from solar salterns. S. cerevisiae has also been
studied for reference
Compounds
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
31
32
33
Cladosporium
Cladosporium Hortaea Aureobasidium Alternaria Saccharomyces
sphaerospermum sp
werneckii pullulans
alternata cerevisiae
Squalene (% neutrals)
3.0
Cholest-5-en-3b-ol
0.2
24-Methylcholesta-5,7,22,24(28)
3.1
-tetraen-3b-ol
24-Methylcholesta-7,22,24(28)
0.5
-trien-3b-ol
Cholesta-8,24-dien-3b-ol
24-Methylcholesta-8,22-dien-3b-ol
0.5
24-Methylcholestatrien-3b-ol
0.3
24-Methylcholesta-5,7,22-trien-3b-ol
49.1
24-Methylcholesta-7,22-dien-3b-ol
6.2
24-Methylcholestatetraen-3b-ol
0.9
24-Methylcholesta-8,24(28)-dien-3b-ol
0.3
24-Methylcholest-8-en-3b-ol
2.1
24-Methylcholesta-5,7,24(28)-trien-3b-ol
4a-Methylcholestatrien-3b-ol
24-Methylcholestadien-3b-ol
0.5
24-Methylcholesta-5,7-dien-3b-ol
1.3
24-Methylcholesta-7,24(28)-dien-3b-ol
0.7
24-Methylcholest-7-en-3b-ol
19.5
24-Methylcholestatetraen-3b-ol
0.1
24-Ethylcholest-5-en-3b-ol
0.1
4,4,14-Trimethylcholesta-8,24-dien-3b-ol
0.4
4a,24-Dimethylcholest-5-en-3b-ol
tr
24-Methylcholestatetraen-3b-ol
0.8
4a,24-Dimethylcholesta-8,24(28)-dien-3b-ol 1.8
4a,24-Dimethylcholest-8-en-3b-ol
2.4
4a,24-Dimethylcholesta-5,7-dien-3b-ol
0.3
4,4,14,24-Tetramethylcholesta-8,24(28)
2.4
-dien-3b-ol
4a,24-Dimethylcholesta-7,24(28)-dien-3b-ol 0.4
4a,24-Dimethylcholest-7-en-3b-ol
3.8
4,4,14-Trimethylcholestadien-3b-ol
tr
4,4,24-Trimethylcholesta-8,24(28)1.5
dien-3b-ol
4,4,?-Trimethylcholestadien-3b-ol
0.6
the major compound of this group, accounts for 1.9±
4.8% total sterols. This sterol has been identi®ed by
mass spectral interpretation (Fig. 1) and comparison
with the spectra reported in Galli and Maroni (1967),
Rahier and Benveniste (1989) and Gerst et al. (1997).
Sterols with unsaturations at position 7, 24(28) easily
lose the side chain together with two hydrogen atoms
which are extracted from the polycyclic structure (Rahier and Benveniste, 1989). Thus, the base peak of 24methylcholesta-5,7,22,24(28)-tetraen-3b-ol, m/z 251,
corresponds to the fragment [M-(CH3)3SiOH-SC-2H]+.
The other three tetraunsaturated isomers (10, 19, 13)
have not been structurally identi®ed (Table 1, Fig. 2).
Four 24-methylcholestatrien-3b-ol isomers are also
found. The isomer (4; Fig. 2) eluting after 24-methyl-
1.2
0.1
4.8
0.0
0.2
2.2
7.7
0.1
2.1
8.6
0.1
3.1
5.1
0.5
1.9
3.9
0.4
48.3
2.5
0.4
0.4
0.7
1.9
1.1
54.4
4.3
0.3
5.0
7.0
0.9
0.3
57.9
3.3
1.3
0.6
1.3
12.6
1.0
1.9
3.7
0.9
tr
47.0
1.3
0.8
5.6
59.8
4.5
0.8
2.7
2.8
2.1
2.2
0.5
0.8
2.5
14.2
0.3
0.4
0.8
0.9
6.6
0.3
0.4
0.4
5.2
5.8
0.6
0.2
0.1
0.9
0.9
1.1
0.2
1.1
0.3
tr
0.4
0.5
1.3
tr
4.2
0.3
0.3
0.6
1.0
1.4
0.2
1.7
0.3
0.2
0.8
tr
0.6
tr
28.6
0.4
3.4
0.8
1.0
0.7
0.9
28.2
tr
0.4
2.6
6.2
4.3
4.4
tr
4.6
tr
0.3
2.2
0.8
0.1
cholesta-5,7,22,24(28)-tetraen-3b-ol (3; Fig. 2) has been
attributed to 24-methylcholesta-7,22,24(28)-trien-3b-ol
(Gerst et al., 1997). Its retention time is consistent with
the dierence currently observed between 5 sterols and
5a(H) stanols. The intense ion at m/z 337 corresponds to
[M-(CH3)3SiOH-C3H7]+, a fragment generated by
elimination of part of the side chain in the presence of a
double bond at C-22. The assignment of peak No. 13
(Fig. 2) to an isomer with unsaturations at 5, 7, 24(28) is
also based on mass spectral examination and retention
time considerations. Several 24-methylcholestatrien-3bols have also been reported in Ascomycetes (Parks,
1978).
Six 24-methylcholestadien-3b-ols with double bonds
at positions 8, 22, 7,22, 8, 24(28), 5, 7, 7, 24(28) and
L. MeÂjanelle et al. / Organic Geochemistry 31 (2000) 1031±1040
1035
Fig. 1. Selected mass spectra of sterols from melanized fungi. Numbers refer to peak identi®cation in Table 1. No. 4: 24-methylcholesta-5,7,22,24(28)-tetraen-3b-ol, No. 8: 24-methylcholesta-5,7,22-trien-3b-ol; No. 10: 24-methylcholest-8-en-3b-ol; No. 17: 24methylcholesta-7,24(28)-dien-3b-ol; No. 18: 24-methylcholest-7-en-3b-ol; No. 29: 4a,24-dimethylcholest-7-en-3b-ol.
1036
L. MeÂjanelle et al. / Organic Geochemistry 31 (2000) 1031±1040
Fig. 2. Total ion current chromatograms of sterols (as TMSi-ethers) from some melanized fungal cultures. Numbers refer to peak
identi®cation in Table 1.
an unknown compound have been identi®ed in the melanized fungi (Table 1, Fig. 2). 24-Methylcholesta7,24(28)-dien-3b-ol and 24-methylcholesta-8,24(28)dien-3b-ol have similar spectra. They show typical fragments of 8/7 monounsaturated sterols (m/z 211 and
227) and a very intense [M-SC-2H]+ ion at m/z 343. The
GC peaks of these compounds elute before the corresponding monounsaturated 8 or 7 homologues.
24-Methylcholesta-7,24(28)-dien-3b-ol is the most
abundant sterol, constituting up to 28.2% of total sterols in A. alternata. This compound is also the main
diunsaturated sterol in A. pullulans. Other 24-methyl
sterols with unsaturations at C-24(28), e.g. 8, 24(29), are
also very abundant in these two melanized fungi, representing about 5% of total sterols. A diunsaturated sterol
elutes just after 24-methylcholesta-5,7,22-trien-3b-ol. Its
retention time and spectrum are consistent with the
structure of 24-methylcholesta-7,22-dien-3b-ol. This
compound is present in signi®cant amounts (1.3±6.2%)
in all studied melanized fungi. Previous investigations
have shown 24-methylcholesta-5,22-dien-3b-ol to be a
dominant sterol in some fungi (Weete, 1989) but this
compound was not identi®ed in any of the melanized
fungi considered here.
The monounsaturated 24-methylsterols encompass
two main compounds with double bonds at 7 and 8.
Monounsaturated sterols with a double bond at 5
(easily recognizable by characteristic fragments at m/z
129 and [M-129]+), are present at very low levels.
Monounsaturated sterols with double bond at 7 or 8
show strong molecular ions and a strong [M-SC(CH3)3SiOH- 42]+ fragment, m/z 213. They also readily
lose the side chain. The base peak of 7 sterols corresponds to the fragment [M-SC-(CH3)3SiOH]+, m/z 255,
while this ion is also important in 8 sterols. Fragments
at m/z 213 and 229 are speci®cally abundant in these
sterols being produced by cleavage of ring D (Rahier
and Benveniste, 1989). Sterol assignment between these
two isomers has also been performed by taking advantage of GC selectivity, since 8 elute before 7 isomers
(Gerst et al., 1997).
24-Methylcholest-7-en-3b-ol is the second principal
sterol in both studied Cladosporium species (19±14%,
Table 1). These species also contain 24-methylcholesta7,22-dien-3b-ol in signi®cant abundance. In contrast,
24-methylcholest-8-en-3b-ol is the second major sterol
in A. pullulans (7%). This melanized fungus also
contains 24-methylcholesta-8,24(28)-dien-3b-ol in high
abundance.
In addition to these 4-desmethyl sterols, a signi®cant
group comprising 4a,24-dimethylsterols was also found.
The methyl substituent at C-4 gives rise to characteristic
fragments generated by loss of the trimethylsilyl group
and the side chain, m/z 269 and 267, for mono- and
diunsaturated sterols, respectively (Fig. 1). Ions produced by side chain cleavage are also observed, m/z 359
for 8/7 monounsaturated sterols and at m/z 357 for
8, 24(28)/7, 24(28) sterols. The fragments retaining the
C-4 methyl group generated by cleavage of the polycyclic structure have 14 mass units more than those of
the corresponding 4-desmethyl 24-methyl sterols. For
example, cleavage of ring D generates m/z 227 and 243
ions in 8 sterols (Fig. 1) and m/z 227 and 241 ions in
8, 24(28) sterols.
The 4a,24-dimethylsterols encompass the same unsaturated positions as the 4-desmethyl 24-methylsterols:
7, 8, 7,24(28), 8,24(28) and 5,7. In the case of H.
werneckii 4a,24-dimethylcholest-7-en-3b-ol is the second
major sterol constituent after 24-methylcholesta-5,7,22trien-3b-ol. This compound is also abundant in A.
pullulans and C. sphaerospermum. Another abundant
sterol in this group is 4a,24-dimethylcholest-8-en-3b-ol
which is present in concentrations between 1.1 and 2.4%
in all melanized fungi except in A. alternata. This last
L. MeÂjanelle et al. / Organic Geochemistry 31 (2000) 1031±1040
species contains 4a,24-dimethylcholesta-8,24(28)-dien3b-ol (2.6%) which parallels the presence of 24-methylcholesta-8,24(28)-dien-3b-ol in this organism.
Three diunsaturated sterols with 30 carbon atoms,
having methyl substitution at C4a, C4b and C24, are
found in trace amounts. 4,4,24-Trimethylcholesta8,24(28)-dien-3b-ol shows a similar spectrum as that
observed in 24-methylcholesta-8,24(28)-dien-3b-ol. The
base peak at m/z 135 corresponds to the equivalent m/z
107 fragment in the 4-desmethylsterols plus 28 mass
units. Similarly, the fragments [M-(CH3)3SiOH-SC-42]+
and [M-(CH3)3SiOH-SC-42-CH3]+, m/z 241 and 255,
respectively are also relatively abundant. Finally, the
ion m/z 281, [M-(CH3)3SiOH-SC]+, evidences the
substitution of two methyl groups in the steroid nucleus.
4,4,24-Trimethylcholesta-8,24(28)-dien-3b-ol represents
about 1% total sterols in Cladosporium sp. and H.
werneckii.
One C31 sterol, 4,4,14,24-tetramethylcholesta-8,24(28)dien-3b-ol, is also found in signi®cant concentration in all
melanized fungi (0.8±4.2%) considered in this study.
Sterols with a methyl group at C-14 have an intense ion
for fragment [M-(CH3)3SiOH-CH3]+. Cladosporium sp.
and H. werneckii are the species having this compound in
higher abundance, consistent with the higher abundance
of the C30 homologue in these fungi.
3.3. Dierences between species
24-Methylcholesta-5,7,22-trien-3b-ol dominates the
sterol composition of all melanized fungi considered in
this study. It also dominates the composition of S. cerevisiae which was included in this study as reference.
This sterol has been assumed to be ergosterol since this is
the predominant sterol in fungi and S. cerevisiae, although
the C-24 stereochemistry was not speci®cally determined.
All fungi also contain two sterols of similar structure in
lower amounts, 24-methylcholesta-5,7,22,24(28)-tetraen3b-ol and 24-methylcholesta-7,22,-dien-3b-ol, (1.2±4.8%
and 1.3±6.2%, respectively).
Ergosterol is the end product of sterol biosynthesis in
most fungi, whereas cholest-5-en-3b-ol and 24-ethylcholest-5-en-3b-ol are the major products of sterol
synthesis in animals and higher plants, respectively
(Weete, 1989). Ergosterol biosynthesis in fungi proceeds
via various routes (Mercer, 1984; Weete, 1989; Parks
and Casey, 1995) and most of the compounds detected
in melanized fungi are intermediates on these pathways.
Replicates of these species grown under dierent culture
conditions, e.g. dierent salinity concentrations, gave
rise to dierent proportions of ergosterol. Thus, in some
cases this compound could reach up to 75% of total
sterol content. The concentrations of the above reported
sterol intermediates decrease evenly as ergosterol
increases but no qualitative changes are observed. Thus,
the sterols included in Table 1 can be used for source
1037
recognition of these melanized fungal series. A major
dierence between melanized fungi and the reference
yeast is the exclusive presence of cholesta-8,24-dien-3bol in the later, which argues for distinct routes to ergosterol synthesis.
Steroidal compounds with 7 unsaturation are relatively abundant in the Cladosporium and H. werneckii
species studied (Table 1, Fig. 2). The sterols of this series
comprise 24-methyl homologues in the former (with
24-methylcholest-7-en-3b-ol as major compound, 1420% of total sterols) and 4a,24-dimethylcholest-7-en3b-ol in the latter (29% of total sterols). 8 Unsaturated
sterols are also found in these fungi and are represented
by 24-methyl homologues in Cladosporium spp. and
4a,24-dimethyl sterols in H. werneckii, which is consistent with the 7 sterol distributions.
Both 8 and 7 unsaturated sterols constitute the
next major group of sterols (after ergosterol) in A. pullulans, with 24-methylcholest-8-en-3b-ol as the major
homologue (7% of total sterols; Table 1). In this fungus,
7 unsaturated sterols are in minor relative proportion.
A. alternata is constituted by sterols predominated by
x, 24(28) as the major secondary group. The absence of
monounsaturated 7 and 8 sterols is a unique feature
of the composition of this species. In this case, the main
sterols bear two, three or four double bonds, and all
them include a double bond at the C-24(28) site (Table
1, Fig. 2). The main compound of this series is 24methylcholest-7,24(28)-dien-3b-ol (28% total sterols).
The other unsaturated site in the sterol distribution of
this fungus is predominantly situated at 7 or 8.
In S. cerevisiae (Chambon et al., 1991) there is not a
marked predominance of sterols unsaturated at some
speci®c site as a main secondary group (Table 1). The
observed sterols encompass a mixture of compounds
constituted by the same type of steroids as those found
in the previous species, namely 24-methyl homologues
unsaturated at 7, 8, 7, 22, 5, 7, 7, 24(28) and 8, 24(28)
(relative composition in the order of 2±6% total sterols).
Besides 4,4,14-trimethylcholesta-8,24-dien-3b-ol, no other
sterols have more than 28 carbon atoms.
3.4. Sterols of particles and sediments from
Mediterranean solar salterns
The suite of sterols encountered in the halophilic and
halotolerant melanized fungi dier from those commonly reported in marine phytoplankton or zooplankton. Amongst all structures described in Table 1,
only two minor compounds, cholest-5-en-3b-ol and 24ethylcholest-5-en-3b-ol (0.1±0.2%) are commonly
reported in marine environments. Therefore, sterols of
melanized fungi aord a speci®c biomarker imprint.
However, when sterols from particles and sediments of
hypersaline environments are examined (Table 2), the
major fungal sterol, 24-methylcholesta-5,7,22-trien-3b-ol,
L. MeÂjanelle et al. / Organic Geochemistry 31 (2000) 1031±1040
1038
Table 2
Sterol composition of sediments and particles from diverse depositional domains of Mediterranean solar salterns and the melanized
fungi isolated from them
Peak No.
a
2
34
35
36
37
38
39
40
17
18
41
42
43
a
Compounds
Cholest-5-en-3b-ol
5a(H)-cholestan-3b-ol
24-Methylcholesta-5,22-dien-3b-ol
24-Methyl-5b(H)-cholest-22-en-3b-ol
24-Methylcholest-5-en-3b-ol
24-Methyl-5b(H)-cholestan-3b-ol
24-Ethylcholesta-5,22-dien-3b-ol
24-Ethylcholest-22-en-3b-ol
24-Methylcholesta-7,24(28)-dien-3b-ol
24-Methylcholest-7-en-3b-ol
24-Ethylcholest-5-en-3b-ol
24-Ethyl-5a(H)-cholestan-3b-ol
4a,23,24-Trimethyl-5a(H)-cholest-22-en-3b-ol
Melanized fungi
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
+
+
ÿ
ÿ
ÿ
Halite
Gypsum
Gypsum/carbonate
Particles
Particles
Sediments
Sediments
+
ÿ
+
+
+
ÿ
+
+
+
ÿ
+
ÿ
ÿ
+
+
+
ÿ
+
ÿ
+
+
+
+
+
ÿ
ÿ
+
+
+
+
+
+
+
+
+
+
+
+
ÿ
ÿ
+
+
+
+
+
+
+
ÿ
ÿ
+
+
+
Peak numbers are coincident with those in Table 1 when applicable.
cannot be recognized even though abundant melanized
fungi are observed in these ponds. Phyto- and zooplanktonic structures dominating the sterol assemblage,
namely those exhibiting double bonds at positions 5
and/or 22 and methyl or ethyl substitution at C-24,
may hinder the detection of fungal sterols. For instance
the relative retention times of 24-methylcholest-5,22dien-3b-ol and 24-methylcholesta-5,7,22-trien-3b-ol are
close. However, molecules bearing 3 or 4 double bonds
are commonly not preserved in sedimentary systems.
Thus, fungal sterols bearing one or two double bonds
may better meet the stability requirements of biomarkers.
24-Methylcholesta-7,24(28)-dien-3b-ol
and
24methylcholest-7-en-3b-ol (Table 2, Fig. 3), two major
secondary sterols in melanized fungi, were found in signi®cant relative proportion in the particles and sediments of the gypsum and halite precipitation ponds. 24Methylcholesta-7,24(28)-dien-3b-ol and 24-methylcholest-7-en-3b-ol have also been observed to be present in
high relative abundance in the sterol distributions of
decomposed top mats from Gavish Sabkha (de Leeuw et
al., 1985). Besides yeast or fungi, these sterols have also
been identi®ed in some algae, e.g. 24-methylcholest-7en-3b-ol in the dino¯agellate Gymnodinium catenatum
Fig. 3. Gas chromatograms of sterols in particles of halite precipitation ponds (A) and sedimentary gypsum deposits (B) from
Mediterranean solar salterns (Barbe et al., 1990). Numbers refer to peak identi®cation in Tables 1 and 2.
L. MeÂjanelle et al. / Organic Geochemistry 31 (2000) 1031±1040
(Hallegrae et al., 1991) and 24-methylcholesta-7,24(28)dien-3b-ol in some diatom species such as Coscinodiscus
sp. (Barrett et al., 1995) and Thalassiosira pseudana
(Orcutt and Patterson, 1975). However, in the high salinity conditions of the environments under study, these
sterols did not re¯ect inputs from these algae. Thus,
diatoms are found at salinities of seawater or calcite
deposition but not at higher salt concentrations such as
those corresponding to gypsum or halite precipitation
(Clavero et al., 1995; Merino et al., 1995b).
In the context of these hypersaline environments, the
occurrence of this 7, 24(28) sterol but absence of 24methylcholesta-7-en-3b-ol in the halite precipitation
ponds is consistent with the speci®c occurrence of A.
alternata which contains 24-methylcholesta-7,24(28)dien-3b-ol in high relative proportion (28%; Table 1)
but 24-methylcholest-7-en-3b-ol only at trace levels. The
sterol composition of the gypsum ponds is consistent
with contributions from A. pullulans or may re¯ect
mixed inputs from other melanized fungal species.
The abundance of melanized fungal species has been
estimated in some hypersaline systems in terms of cell
numbers (colony forming units). However, the signi®cance of their biomass in relation to that of other
organisms is yet to be assessed. The quantitative examination of their speci®c sterols may be useful for this
purpose and will facilitate the understanding of their
contribution to the organic carbon deposits in hypersaline systems.
4. Conclusions
Until recently, archaea and certain eubacterial taxa
were thought to be the only organisms able to grow in
hypersaline waters. However, it is now clear that melanized fungi can ¯ourish under conditions of halite, gypsum and calcite precipitation. These organisms have
sterol distributions that are consistent with the biosynthesis of 24-methylcholesta-5,7,22-trien-3b-ol (ergosterol). Thus, they are distinct from sterol mixtures in
algal and zooplanktonic species adapted to hypersaline
conditions.
A close study of the sterol composition in the melanized fungi reveals a dominance of 24-methylcholesta5,7,22-trien-3b-ol and signi®cant dierences among the
less abundant sterols. The high degree of unsaturation of
a large proportion of sterols from these melanized fungal
species strongly restricts their preservation and potential
use as biomarkers. However, some of the mono- and
diunsaturated compounds present in higher relative
abundance such as 24-methylcholesta-7,24(28)-dien-3b-ol
and 24-methylcholest-7-en-3b-ol are encountered in signi®cant proportion in sediments and water particulates of
halite and gypsum precipitation systems providing, in this
context, a speci®c signature for melanized fungi.
1039
Acknowledgements
Financial support from the European Union (MAST
Project MAS3-CT98-5057) is acknowledged. This project has also been funded by CICYT (PB93-0190-C0201) from the Spanish Ministry of Education.
Associate EditorÐB.J. Keely
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www.elsevier.nl/locate/orggeochem
Sterols of melanized fungi from hypersaline environments
Laurence MeÂjanelle a, Jordi F. LoÁpez a, Nina Gunde-Cimerman b,
Joan O. Grimalt a,*
a
Department of Environmental Chemistry, I.C.E.R.-C.S.I.C., Jordi Girona, 18. 08034-Barcelona, Catalonia, Spain
b
Biology Department, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia
Received 14 December 1999; accepted 20 June 2000
(returned to author for revision 18 February 2000)
Abstract
The lipid compositions of melanized fungi isolated from calcite, gypsum and halite depositional environments of
Mediterranean solar salterns, namely Hortaea werneckii, Alternaria alternata, Cladosporium cladosporioides, Cladosporium sp. and Aureobasidium pullulans, have been examined. Sterols constituted the most distinct lipid fraction.
Ergosterol, 24-methylcholesta-5,7,22-trien-3b-ol, dominated all distributions but major dierences between species
were encountered when considering the subordinate sterols. Thus, 24-methylcholest-7-en-3b-ol, 24-methylcholesta7,24(28)-dien-3b-ol and 4a,24-dimethylcholest-7-en-3b-ol were found in signi®cant proportions in Cladosporium spp
(14±20%), A. alternata (28%) and H. wernekii (29%), respectively. These sterols can be used for discrimination
between these dierent fungal species. 24-Methylcholest-7-en-3b-ol and 24-methylcholesta-7,24(28)-dien-3b-ol were
found in signi®cant proportion in the water column particles and sediments of the gypsum and halite precipitation
ponds (the latter only in the halite domain). In such environments, these sterols may provide a speci®c signature for
these melanized fungi. However, water column particulate matter and sediments from hypersaline depositional settings
show sterol compositions dominated by the constituents typically encountered in phytoplankton and zooplankton, but
not in melanized fungi. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Sterols; Ergosterol; Fungi; Hypersaline environments; Solar salterns
1. Introduction
Historically, the term halophile was only applied to
specialized bacteria until 1975 when it was applied to
foodborne fungi exhibiting superior growth on media
with NaCl as controlling solute (Pitt and Hocking,
1985). Fungi were subsequently described in salt marshes
(Newell, 1996), saline soil (Guiraud et al., 1995) and seawater (Kohlmeyer and Volkmann-Kohlmeyer, 1991) but
were considered unable to grow in highly saline waters.
Very recently, however, high fungal diversity has been
identi®ed in hypersaline waters and surface layers of
microbial mats in environments with salinities ranging
* Corresponding author. Tel.: +34-93-400-6122; fax: +3493-204-5904.
E-mail address: jgoqam@cid.csis.es (J.O. Grimalt).
between 15 and 32%. These fungi were ®rst isolated in
hypersaline waters of marine salterns from Secovlje in
Slovenia (Gunde-Cimerman et al., in press). Subsequent
studies in the solar saltern of La Trinitat (Ebro Delta,
Catalonia, Spain) and BonmatõÂ (Santa Pola, Valencian
Community, Spain) showed the occurrence of the same
dominant species.
The majority of isolates were determined to belong to
melanized meristematic and yeast-like fungi, and a limited number to dierent genera of ®lamentous fungi.
Among the isolated halotolerant/halophilic mycobiota
the following genera were found: Hortaea, Phaeotheca,
Trimmatostroma, Aureobasidium, Alternaria, Cladosporium (Zalar et al., 1999a±c). These dematiaceous fungi
have the common property that they form black, clumplike colonies consisting of isodiametrically dividing cells
in the water, whereas the hyphal growth is mainly exhibited on solid media. This unique in-situ morphology was
0146-6380/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0146-6380(00)00094-2
1032
L. MeÂjanelle et al. / Organic Geochemistry 31 (2000) 1031±1040
interpreted as a response to multiple stress factors which
helps the fungi to tolerate high temperatures and low
water activity by optimization of the volume-surface
ratio (Zalar et al., 1999a). Among the isolated ®lamentous fungi, better known for their xerophilic nature,
the genera Aspergillus, Penicillium and Wallemia were
prevalent (Gunde-Cimerman et al., 1997).
The recent discovery of these fungal species prompted
us to investigate the geochemical signi®cance of their
organic residues in hypersaline environments. Since
melanized fungi are the most abundant fungal species at
higher salinities, the present study is devoted to this
group of organisms.
Halophilic and halotolerant fungi present in the
diverse depositional environments of La Trinitat solar
saltern were considered for study. As reported previously, the complete sequence of sea water evaporation
including calcite, gypsum and halite precipitation is
represented in this system (Grimalt et al., 1992; de Wit
and Grimalt, 1992; Villanueva et al., 1994; Hartgers et
al., 1997). Water was collected in diverse ponds encompassing the whole salinity range and ®ltered, the ®lters
being incubated on several selective media to stimulate
fungal growth. Agar baits in dialysis tubing were left in
these ponds from June to November. The fungi grown
in them were also cultured on selective media.
The lipid composition of the melanized species isolated from these hypersaline environments was examined. Sterols constituted the most distinct lipid fraction
when compared with the distributions typically encountered in other group organisms, e.g. phytoplankton,
zooplankton and bacteria. Thus, the present study is
focused on this group of compounds, which are discussed as source markers and compared with those present in waters (particulate phase) and sediments of solar
salterns.
2. Experimental
Aliquots of waters (10±100 ml) from the diverse saltern ponds were ®ltered immediately after sampling over
nitrocellulose Millipore membrane ®lters (pore size 0.45
mm) and placed on dierent selective agar media containing either high salt (17±32%) or high sugar concentration (50±70% glucose or fructose). A drop of the
original saline water was applied to the membrane and
dispersed with a Drigalski spatula. For every medium,
four aliquots were ®ltered in parallel and the average
number of colony forming units (CFU) were calculated.
Plates were incubated for 1±10 weeks at 25 C. CFU on
enumeration media were counted every 3, 5, 7, 14, 30
and 60 days of incubation.
Agar baits in dialysis tubing were left in diverse ponds
for 5 months from June to November. After collection,
the agar blocks were pushed out, cut aseptically and
plated out on low water activity media.
An enrichment technique was applied by the addition
of glucose and yeast extract to saline water from the
salterns and incubation on a rotary shaker. Subsequently, the broth was diluted with 17% saline water
several times.
All fungi isolated from selective media were inoculated in parallel on malt extract agar (MEA) and on
MEA+3M NaCl (17%). Only the fungal colonies able
to grow in the presence of 17% salt were further determined taxonomically. Growth rates and morphological
characteristics were determined on both media after 7
and 30 days. The agar medium used for growth yielded
no signi®cant content of sterols. Species identi®cation
was mainly based on descriptions from Ellis (1971;
1976). Identi®cations were con®rmed at the microbiological culture collection CBS (Centraalbureau voor
Schimmelcultures, Baarn, The Netherlands). Culture
purity was ensured by development of spore isolates of
individual fungi and plateing aseptically on fresh sterile
media.
The Saccharomyces cerevisiae strain W303a analyzed
for reference was obtained from the Yeast Stock Center
(Berkeley, CA, USA).
2.1. Site description and cultures of melanized fungi
2.2. Extraction and fractionation
The solar saltern ponds of La Trinitat are located in the
south wing of the Ebro Delta (Villanueva et al., 1994).
This environment is exploited to obtain halite by evaporation of seawater. The salt water circuit consists of a
series of ponds interconnected by sluices. In each pond,
salinity is kept more or less constant. Seawater is pumped
through the system and, because of evaporation, increasing salinity causes the successive precipitation of calcite,
gypsum and halite. A number of studies on the molecular
characteristics of solar saltern microbial mats (Aizenshtat
et al., 1983; Boon et al., 1983; Barbe et al., 1990; Teixidor
et al., 1993; Villanueva et al., 1994) and their microbial
biota (de Wit and Grimalt, 1992; van Gemerden, 1993;
Merino et al., 1995a) have been reported previously.
An aliquot of 1±1.5 g of melanized fungi, gently
scrapped o the MEA surface, was placed in 10150
mm Pyrex tubes. Lipids were extracted ultrasonically in
5 ml of methanol for 20 min, at 30 C. The procedure
was repeated twice with methanol and twice further with
dichloromethane (DCM). Extracts were recovered after
centrifugation. Combined extracts were vacuum concentrated to 1 ml and hydrolyzed by addition of 50 ml
of KOH in methanol (10%). The mixture was sonicated
at room temperature for 10 min, ¯ushed with nitrogen
and kept in the dark for 24 h. Neutral lipids were
extracted with hexane (420 ml). The methanol solution
was then acidi®ed to pH 2 by addition of HCl (aq) and
L. MeÂjanelle et al. / Organic Geochemistry 31 (2000) 1031±1040
further extracted with hexane (420 ml) for isolation of
the fatty acids.
The neutral lipids were fractionated by column chromatography using 1.5 g of 5% water-deactivated silica
gel (40 mesh 70-230 Merck) in a 1806 mm column.
Three fractions were collected: F1 (hydrocarbons), 12
ml of hexane+12 ml hexane/DCM (95:5); F2, 24 ml
DCM; and F3 (alcohols), 24 ml of DCM/Methanol
(90:10). Sterols (in F3) were converted into their trimethylsilylethers by reaction with bis-(trimethylsilyl)tri¯uoroacetamide (70 C, 30 min).
2.3. Instrumental analysis
Samples were dissolved in iso-octane and analyzed by
gas chromatography (GC) using a Carlo Erba 5300
(Carlo Erba, Italy) ®tted with a heated splitless injector
(300 C) and a capillary column (25 m0.25 mm i.d.;
5% phenyl-methyl polysiloxane DB-5; 0.25 mm ®lm
thickness; J&W) using hydrogen as carrier gas. The
oven was kept at 70 C for 1 min, heated to 150 C at
15 C minÿ1, then to 310 C at 4 C minÿ1, and ®nally
held at 310 C for 30 min. The temperature of the ¯ame
ionization detector (FID) was 330 C, the ¯ame was fed
with air (300 ml minÿ1) and hydrogen (30 ml minÿ1).
Nitrogen was used as make up gas (30 ml minÿ1). The
detector response was digitized by a Nelson 900 interface and processed with a Nelson 2600 software package
(Perkin Elmer). Relative concentrations of sterols were
calculated from the GC±FID response areas.
Analyses by GC±mass spectrometry (CG±MS) were
performed using a Fisons 8000 gas chromatograph
coupled to a Fisons MD-800 quadrupole mass analyzer.
Samples were injected in splitless mode at 300 C onto a
capillary column (25 cm0.25 mm i.d.; 5% phenylmethyl polysiloxane HP-5; 0.25 mm ®lm thickness;
Hewlett Packard). Helium was the carrier gas and the
temperature program was the same as for the GC
analyses.
Mass spectra were recorded in electron impact mode
at 70eV by scanning between m/z 50 and 650 every
second. Ion source and transfer line were kept at
300 C. Data were processed with Masslab software
(THERMO Instruments). Sterol identi®cation was
based on mass spectral interpretation and comparison
of mass spectra and retention time data with available
standards.
Lipids of particles and sediments from diverse hypersaline environments were already examined as described
in Barbe et al. (1990) and Grimalt et al. (1992). Sterols
were analyzed as trimethylsilylether derivatives by GC±
MS on a capillary column (25 m0.2 mm i.d.; 5% phenyl-methyl polisiloxane HP-5; 0.11 mm ®lm thickness)
using helium as carrier gas. The injector was kept at
290 C, the oven was programmed from 60 C to 300 C
at 4 C minÿ1 and kept at 300 C for 15 min.
1033
3. Results and discussion
3.1. Melanized fungal species in diverse hypersaline
systems
Hortaea werneckii, Alternaria alternata (Fr.) keissl.,
Cladosporium sphaerospermum, Aureobasidium pullulans
(de Bary) G. Arnaud and Cladosporium spp were identi®ed in La Trinitat solar saltern. Cladosporium spp. are
rather ubiquitous and found in the calcite, gysum and
halite precipitation ponds as well as in the magnesiumcontaining brines. H. werneckii was found in the mixed
calcite/gypsum, gypsum and halite depositional environments. A. alternata and A. pullulans were found in the
more extreme saline conditions. The former was identi®ed in the Mg brines and the halite chrystallizers and
the latter in the halite ponds.
The same major species of halotolerant or halophilic
melanized fungi were identi®ed in the Secovlje saltpans
(Slovenia; Zalar et al., 1999a, b, c; Gunde-Cimerman et
al., in press). This evaporitic system is situated in the
delta of the Dragonja River Delta, on the Adriatic
coast.
The hydrocarbon composition of all fungi except H.
werneckii is largely dominated by squalene, which
accounts for 1.2±7.7% of the neutral lipids (Table 1).
Squalene is also the dominant hydrocarbon in S. cerevisiae which is analyzed here as reference. The fatty acid
compositions of the halotolerant or halophilic species is
similar to the distributions found in algae, with a predominance of saturated and unsaturated C16 and C18
linear homologues.
3.2. Sterol composition
Most sterols identi®ed in the melanized fungal species
have a methyl substituent at C-24, as evidenced by the
molecular weight and fragment ions resulting from the
loss of the side chain (SC) and the trimethylsilylhydroxy
group (Fig. 1, Table 1). Thus, the spectrum of 24methylcholesta-5,7,22-trien-3b-ol shows a m/z 468 molecular ion and the fragment [M-(CH3)3SiOH-SC]+ at m/z
253 which is also indicative of a monounsaturated side
chain. Other dominant ions correspond to fragments [M(CH3)3SiOH-C3H3]+ and [M-(CH3)3SiOH-SC-C2H2]+,
m/z 363 and 211, respectively. The ion [M-SC-C3H5]+,
m/z 337, is typical of sterols with a double bond at position 22 (Rahier and Benveniste, 1989). This spectrum is
in agreement with that of ergosterol reported in Ascomycete yeasts (Parks and Casey, 1995; Weete, 1989).
The other main sterols in these melanized fungi possess
one to four unsaturations at positions 5, 7, 8, 22,
24(28) or 24.
The four tetraunsaturated sterols identi®ed in the
melanized fungi are generally found in minor amounts
although 24-methylcholesta-5,7,22,24(28)-tetraen-3b-ol,
1034
L. MeÂjanelle et al. / Organic Geochemistry 31 (2000) 1031±1040
Table 1
Relative composition of squalene and sterols of cultures of melanized fungi isolated from solar salterns. S. cerevisiae has also been
studied for reference
Compounds
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
31
32
33
Cladosporium
Cladosporium Hortaea Aureobasidium Alternaria Saccharomyces
sphaerospermum sp
werneckii pullulans
alternata cerevisiae
Squalene (% neutrals)
3.0
Cholest-5-en-3b-ol
0.2
24-Methylcholesta-5,7,22,24(28)
3.1
-tetraen-3b-ol
24-Methylcholesta-7,22,24(28)
0.5
-trien-3b-ol
Cholesta-8,24-dien-3b-ol
24-Methylcholesta-8,22-dien-3b-ol
0.5
24-Methylcholestatrien-3b-ol
0.3
24-Methylcholesta-5,7,22-trien-3b-ol
49.1
24-Methylcholesta-7,22-dien-3b-ol
6.2
24-Methylcholestatetraen-3b-ol
0.9
24-Methylcholesta-8,24(28)-dien-3b-ol
0.3
24-Methylcholest-8-en-3b-ol
2.1
24-Methylcholesta-5,7,24(28)-trien-3b-ol
4a-Methylcholestatrien-3b-ol
24-Methylcholestadien-3b-ol
0.5
24-Methylcholesta-5,7-dien-3b-ol
1.3
24-Methylcholesta-7,24(28)-dien-3b-ol
0.7
24-Methylcholest-7-en-3b-ol
19.5
24-Methylcholestatetraen-3b-ol
0.1
24-Ethylcholest-5-en-3b-ol
0.1
4,4,14-Trimethylcholesta-8,24-dien-3b-ol
0.4
4a,24-Dimethylcholest-5-en-3b-ol
tr
24-Methylcholestatetraen-3b-ol
0.8
4a,24-Dimethylcholesta-8,24(28)-dien-3b-ol 1.8
4a,24-Dimethylcholest-8-en-3b-ol
2.4
4a,24-Dimethylcholesta-5,7-dien-3b-ol
0.3
4,4,14,24-Tetramethylcholesta-8,24(28)
2.4
-dien-3b-ol
4a,24-Dimethylcholesta-7,24(28)-dien-3b-ol 0.4
4a,24-Dimethylcholest-7-en-3b-ol
3.8
4,4,14-Trimethylcholestadien-3b-ol
tr
4,4,24-Trimethylcholesta-8,24(28)1.5
dien-3b-ol
4,4,?-Trimethylcholestadien-3b-ol
0.6
the major compound of this group, accounts for 1.9±
4.8% total sterols. This sterol has been identi®ed by
mass spectral interpretation (Fig. 1) and comparison
with the spectra reported in Galli and Maroni (1967),
Rahier and Benveniste (1989) and Gerst et al. (1997).
Sterols with unsaturations at position 7, 24(28) easily
lose the side chain together with two hydrogen atoms
which are extracted from the polycyclic structure (Rahier and Benveniste, 1989). Thus, the base peak of 24methylcholesta-5,7,22,24(28)-tetraen-3b-ol, m/z 251,
corresponds to the fragment [M-(CH3)3SiOH-SC-2H]+.
The other three tetraunsaturated isomers (10, 19, 13)
have not been structurally identi®ed (Table 1, Fig. 2).
Four 24-methylcholestatrien-3b-ol isomers are also
found. The isomer (4; Fig. 2) eluting after 24-methyl-
1.2
0.1
4.8
0.0
0.2
2.2
7.7
0.1
2.1
8.6
0.1
3.1
5.1
0.5
1.9
3.9
0.4
48.3
2.5
0.4
0.4
0.7
1.9
1.1
54.4
4.3
0.3
5.0
7.0
0.9
0.3
57.9
3.3
1.3
0.6
1.3
12.6
1.0
1.9
3.7
0.9
tr
47.0
1.3
0.8
5.6
59.8
4.5
0.8
2.7
2.8
2.1
2.2
0.5
0.8
2.5
14.2
0.3
0.4
0.8
0.9
6.6
0.3
0.4
0.4
5.2
5.8
0.6
0.2
0.1
0.9
0.9
1.1
0.2
1.1
0.3
tr
0.4
0.5
1.3
tr
4.2
0.3
0.3
0.6
1.0
1.4
0.2
1.7
0.3
0.2
0.8
tr
0.6
tr
28.6
0.4
3.4
0.8
1.0
0.7
0.9
28.2
tr
0.4
2.6
6.2
4.3
4.4
tr
4.6
tr
0.3
2.2
0.8
0.1
cholesta-5,7,22,24(28)-tetraen-3b-ol (3; Fig. 2) has been
attributed to 24-methylcholesta-7,22,24(28)-trien-3b-ol
(Gerst et al., 1997). Its retention time is consistent with
the dierence currently observed between 5 sterols and
5a(H) stanols. The intense ion at m/z 337 corresponds to
[M-(CH3)3SiOH-C3H7]+, a fragment generated by
elimination of part of the side chain in the presence of a
double bond at C-22. The assignment of peak No. 13
(Fig. 2) to an isomer with unsaturations at 5, 7, 24(28) is
also based on mass spectral examination and retention
time considerations. Several 24-methylcholestatrien-3bols have also been reported in Ascomycetes (Parks,
1978).
Six 24-methylcholestadien-3b-ols with double bonds
at positions 8, 22, 7,22, 8, 24(28), 5, 7, 7, 24(28) and
L. MeÂjanelle et al. / Organic Geochemistry 31 (2000) 1031±1040
1035
Fig. 1. Selected mass spectra of sterols from melanized fungi. Numbers refer to peak identi®cation in Table 1. No. 4: 24-methylcholesta-5,7,22,24(28)-tetraen-3b-ol, No. 8: 24-methylcholesta-5,7,22-trien-3b-ol; No. 10: 24-methylcholest-8-en-3b-ol; No. 17: 24methylcholesta-7,24(28)-dien-3b-ol; No. 18: 24-methylcholest-7-en-3b-ol; No. 29: 4a,24-dimethylcholest-7-en-3b-ol.
1036
L. MeÂjanelle et al. / Organic Geochemistry 31 (2000) 1031±1040
Fig. 2. Total ion current chromatograms of sterols (as TMSi-ethers) from some melanized fungal cultures. Numbers refer to peak
identi®cation in Table 1.
an unknown compound have been identi®ed in the melanized fungi (Table 1, Fig. 2). 24-Methylcholesta7,24(28)-dien-3b-ol and 24-methylcholesta-8,24(28)dien-3b-ol have similar spectra. They show typical fragments of 8/7 monounsaturated sterols (m/z 211 and
227) and a very intense [M-SC-2H]+ ion at m/z 343. The
GC peaks of these compounds elute before the corresponding monounsaturated 8 or 7 homologues.
24-Methylcholesta-7,24(28)-dien-3b-ol is the most
abundant sterol, constituting up to 28.2% of total sterols in A. alternata. This compound is also the main
diunsaturated sterol in A. pullulans. Other 24-methyl
sterols with unsaturations at C-24(28), e.g. 8, 24(29), are
also very abundant in these two melanized fungi, representing about 5% of total sterols. A diunsaturated sterol
elutes just after 24-methylcholesta-5,7,22-trien-3b-ol. Its
retention time and spectrum are consistent with the
structure of 24-methylcholesta-7,22-dien-3b-ol. This
compound is present in signi®cant amounts (1.3±6.2%)
in all studied melanized fungi. Previous investigations
have shown 24-methylcholesta-5,22-dien-3b-ol to be a
dominant sterol in some fungi (Weete, 1989) but this
compound was not identi®ed in any of the melanized
fungi considered here.
The monounsaturated 24-methylsterols encompass
two main compounds with double bonds at 7 and 8.
Monounsaturated sterols with a double bond at 5
(easily recognizable by characteristic fragments at m/z
129 and [M-129]+), are present at very low levels.
Monounsaturated sterols with double bond at 7 or 8
show strong molecular ions and a strong [M-SC(CH3)3SiOH- 42]+ fragment, m/z 213. They also readily
lose the side chain. The base peak of 7 sterols corresponds to the fragment [M-SC-(CH3)3SiOH]+, m/z 255,
while this ion is also important in 8 sterols. Fragments
at m/z 213 and 229 are speci®cally abundant in these
sterols being produced by cleavage of ring D (Rahier
and Benveniste, 1989). Sterol assignment between these
two isomers has also been performed by taking advantage of GC selectivity, since 8 elute before 7 isomers
(Gerst et al., 1997).
24-Methylcholest-7-en-3b-ol is the second principal
sterol in both studied Cladosporium species (19±14%,
Table 1). These species also contain 24-methylcholesta7,22-dien-3b-ol in signi®cant abundance. In contrast,
24-methylcholest-8-en-3b-ol is the second major sterol
in A. pullulans (7%). This melanized fungus also
contains 24-methylcholesta-8,24(28)-dien-3b-ol in high
abundance.
In addition to these 4-desmethyl sterols, a signi®cant
group comprising 4a,24-dimethylsterols was also found.
The methyl substituent at C-4 gives rise to characteristic
fragments generated by loss of the trimethylsilyl group
and the side chain, m/z 269 and 267, for mono- and
diunsaturated sterols, respectively (Fig. 1). Ions produced by side chain cleavage are also observed, m/z 359
for 8/7 monounsaturated sterols and at m/z 357 for
8, 24(28)/7, 24(28) sterols. The fragments retaining the
C-4 methyl group generated by cleavage of the polycyclic structure have 14 mass units more than those of
the corresponding 4-desmethyl 24-methyl sterols. For
example, cleavage of ring D generates m/z 227 and 243
ions in 8 sterols (Fig. 1) and m/z 227 and 241 ions in
8, 24(28) sterols.
The 4a,24-dimethylsterols encompass the same unsaturated positions as the 4-desmethyl 24-methylsterols:
7, 8, 7,24(28), 8,24(28) and 5,7. In the case of H.
werneckii 4a,24-dimethylcholest-7-en-3b-ol is the second
major sterol constituent after 24-methylcholesta-5,7,22trien-3b-ol. This compound is also abundant in A.
pullulans and C. sphaerospermum. Another abundant
sterol in this group is 4a,24-dimethylcholest-8-en-3b-ol
which is present in concentrations between 1.1 and 2.4%
in all melanized fungi except in A. alternata. This last
L. MeÂjanelle et al. / Organic Geochemistry 31 (2000) 1031±1040
species contains 4a,24-dimethylcholesta-8,24(28)-dien3b-ol (2.6%) which parallels the presence of 24-methylcholesta-8,24(28)-dien-3b-ol in this organism.
Three diunsaturated sterols with 30 carbon atoms,
having methyl substitution at C4a, C4b and C24, are
found in trace amounts. 4,4,24-Trimethylcholesta8,24(28)-dien-3b-ol shows a similar spectrum as that
observed in 24-methylcholesta-8,24(28)-dien-3b-ol. The
base peak at m/z 135 corresponds to the equivalent m/z
107 fragment in the 4-desmethylsterols plus 28 mass
units. Similarly, the fragments [M-(CH3)3SiOH-SC-42]+
and [M-(CH3)3SiOH-SC-42-CH3]+, m/z 241 and 255,
respectively are also relatively abundant. Finally, the
ion m/z 281, [M-(CH3)3SiOH-SC]+, evidences the
substitution of two methyl groups in the steroid nucleus.
4,4,24-Trimethylcholesta-8,24(28)-dien-3b-ol represents
about 1% total sterols in Cladosporium sp. and H.
werneckii.
One C31 sterol, 4,4,14,24-tetramethylcholesta-8,24(28)dien-3b-ol, is also found in signi®cant concentration in all
melanized fungi (0.8±4.2%) considered in this study.
Sterols with a methyl group at C-14 have an intense ion
for fragment [M-(CH3)3SiOH-CH3]+. Cladosporium sp.
and H. werneckii are the species having this compound in
higher abundance, consistent with the higher abundance
of the C30 homologue in these fungi.
3.3. Dierences between species
24-Methylcholesta-5,7,22-trien-3b-ol dominates the
sterol composition of all melanized fungi considered in
this study. It also dominates the composition of S. cerevisiae which was included in this study as reference.
This sterol has been assumed to be ergosterol since this is
the predominant sterol in fungi and S. cerevisiae, although
the C-24 stereochemistry was not speci®cally determined.
All fungi also contain two sterols of similar structure in
lower amounts, 24-methylcholesta-5,7,22,24(28)-tetraen3b-ol and 24-methylcholesta-7,22,-dien-3b-ol, (1.2±4.8%
and 1.3±6.2%, respectively).
Ergosterol is the end product of sterol biosynthesis in
most fungi, whereas cholest-5-en-3b-ol and 24-ethylcholest-5-en-3b-ol are the major products of sterol
synthesis in animals and higher plants, respectively
(Weete, 1989). Ergosterol biosynthesis in fungi proceeds
via various routes (Mercer, 1984; Weete, 1989; Parks
and Casey, 1995) and most of the compounds detected
in melanized fungi are intermediates on these pathways.
Replicates of these species grown under dierent culture
conditions, e.g. dierent salinity concentrations, gave
rise to dierent proportions of ergosterol. Thus, in some
cases this compound could reach up to 75% of total
sterol content. The concentrations of the above reported
sterol intermediates decrease evenly as ergosterol
increases but no qualitative changes are observed. Thus,
the sterols included in Table 1 can be used for source
1037
recognition of these melanized fungal series. A major
dierence between melanized fungi and the reference
yeast is the exclusive presence of cholesta-8,24-dien-3bol in the later, which argues for distinct routes to ergosterol synthesis.
Steroidal compounds with 7 unsaturation are relatively abundant in the Cladosporium and H. werneckii
species studied (Table 1, Fig. 2). The sterols of this series
comprise 24-methyl homologues in the former (with
24-methylcholest-7-en-3b-ol as major compound, 1420% of total sterols) and 4a,24-dimethylcholest-7-en3b-ol in the latter (29% of total sterols). 8 Unsaturated
sterols are also found in these fungi and are represented
by 24-methyl homologues in Cladosporium spp. and
4a,24-dimethyl sterols in H. werneckii, which is consistent with the 7 sterol distributions.
Both 8 and 7 unsaturated sterols constitute the
next major group of sterols (after ergosterol) in A. pullulans, with 24-methylcholest-8-en-3b-ol as the major
homologue (7% of total sterols; Table 1). In this fungus,
7 unsaturated sterols are in minor relative proportion.
A. alternata is constituted by sterols predominated by
x, 24(28) as the major secondary group. The absence of
monounsaturated 7 and 8 sterols is a unique feature
of the composition of this species. In this case, the main
sterols bear two, three or four double bonds, and all
them include a double bond at the C-24(28) site (Table
1, Fig. 2). The main compound of this series is 24methylcholest-7,24(28)-dien-3b-ol (28% total sterols).
The other unsaturated site in the sterol distribution of
this fungus is predominantly situated at 7 or 8.
In S. cerevisiae (Chambon et al., 1991) there is not a
marked predominance of sterols unsaturated at some
speci®c site as a main secondary group (Table 1). The
observed sterols encompass a mixture of compounds
constituted by the same type of steroids as those found
in the previous species, namely 24-methyl homologues
unsaturated at 7, 8, 7, 22, 5, 7, 7, 24(28) and 8, 24(28)
(relative composition in the order of 2±6% total sterols).
Besides 4,4,14-trimethylcholesta-8,24-dien-3b-ol, no other
sterols have more than 28 carbon atoms.
3.4. Sterols of particles and sediments from
Mediterranean solar salterns
The suite of sterols encountered in the halophilic and
halotolerant melanized fungi dier from those commonly reported in marine phytoplankton or zooplankton. Amongst all structures described in Table 1,
only two minor compounds, cholest-5-en-3b-ol and 24ethylcholest-5-en-3b-ol (0.1±0.2%) are commonly
reported in marine environments. Therefore, sterols of
melanized fungi aord a speci®c biomarker imprint.
However, when sterols from particles and sediments of
hypersaline environments are examined (Table 2), the
major fungal sterol, 24-methylcholesta-5,7,22-trien-3b-ol,
L. MeÂjanelle et al. / Organic Geochemistry 31 (2000) 1031±1040
1038
Table 2
Sterol composition of sediments and particles from diverse depositional domains of Mediterranean solar salterns and the melanized
fungi isolated from them
Peak No.
a
2
34
35
36
37
38
39
40
17
18
41
42
43
a
Compounds
Cholest-5-en-3b-ol
5a(H)-cholestan-3b-ol
24-Methylcholesta-5,22-dien-3b-ol
24-Methyl-5b(H)-cholest-22-en-3b-ol
24-Methylcholest-5-en-3b-ol
24-Methyl-5b(H)-cholestan-3b-ol
24-Ethylcholesta-5,22-dien-3b-ol
24-Ethylcholest-22-en-3b-ol
24-Methylcholesta-7,24(28)-dien-3b-ol
24-Methylcholest-7-en-3b-ol
24-Ethylcholest-5-en-3b-ol
24-Ethyl-5a(H)-cholestan-3b-ol
4a,23,24-Trimethyl-5a(H)-cholest-22-en-3b-ol
Melanized fungi
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
+
+
ÿ
ÿ
ÿ
Halite
Gypsum
Gypsum/carbonate
Particles
Particles
Sediments
Sediments
+
ÿ
+
+
+
ÿ
+
+
+
ÿ
+
ÿ
ÿ
+
+
+
ÿ
+
ÿ
+
+
+
+
+
ÿ
ÿ
+
+
+
+
+
+
+
+
+
+
+
+
ÿ
ÿ
+
+
+
+
+
+
+
ÿ
ÿ
+
+
+
Peak numbers are coincident with those in Table 1 when applicable.
cannot be recognized even though abundant melanized
fungi are observed in these ponds. Phyto- and zooplanktonic structures dominating the sterol assemblage,
namely those exhibiting double bonds at positions 5
and/or 22 and methyl or ethyl substitution at C-24,
may hinder the detection of fungal sterols. For instance
the relative retention times of 24-methylcholest-5,22dien-3b-ol and 24-methylcholesta-5,7,22-trien-3b-ol are
close. However, molecules bearing 3 or 4 double bonds
are commonly not preserved in sedimentary systems.
Thus, fungal sterols bearing one or two double bonds
may better meet the stability requirements of biomarkers.
24-Methylcholesta-7,24(28)-dien-3b-ol
and
24methylcholest-7-en-3b-ol (Table 2, Fig. 3), two major
secondary sterols in melanized fungi, were found in signi®cant relative proportion in the particles and sediments of the gypsum and halite precipitation ponds. 24Methylcholesta-7,24(28)-dien-3b-ol and 24-methylcholest-7-en-3b-ol have also been observed to be present in
high relative abundance in the sterol distributions of
decomposed top mats from Gavish Sabkha (de Leeuw et
al., 1985). Besides yeast or fungi, these sterols have also
been identi®ed in some algae, e.g. 24-methylcholest-7en-3b-ol in the dino¯agellate Gymnodinium catenatum
Fig. 3. Gas chromatograms of sterols in particles of halite precipitation ponds (A) and sedimentary gypsum deposits (B) from
Mediterranean solar salterns (Barbe et al., 1990). Numbers refer to peak identi®cation in Tables 1 and 2.
L. MeÂjanelle et al. / Organic Geochemistry 31 (2000) 1031±1040
(Hallegrae et al., 1991) and 24-methylcholesta-7,24(28)dien-3b-ol in some diatom species such as Coscinodiscus
sp. (Barrett et al., 1995) and Thalassiosira pseudana
(Orcutt and Patterson, 1975). However, in the high salinity conditions of the environments under study, these
sterols did not re¯ect inputs from these algae. Thus,
diatoms are found at salinities of seawater or calcite
deposition but not at higher salt concentrations such as
those corresponding to gypsum or halite precipitation
(Clavero et al., 1995; Merino et al., 1995b).
In the context of these hypersaline environments, the
occurrence of this 7, 24(28) sterol but absence of 24methylcholesta-7-en-3b-ol in the halite precipitation
ponds is consistent with the speci®c occurrence of A.
alternata which contains 24-methylcholesta-7,24(28)dien-3b-ol in high relative proportion (28%; Table 1)
but 24-methylcholest-7-en-3b-ol only at trace levels. The
sterol composition of the gypsum ponds is consistent
with contributions from A. pullulans or may re¯ect
mixed inputs from other melanized fungal species.
The abundance of melanized fungal species has been
estimated in some hypersaline systems in terms of cell
numbers (colony forming units). However, the signi®cance of their biomass in relation to that of other
organisms is yet to be assessed. The quantitative examination of their speci®c sterols may be useful for this
purpose and will facilitate the understanding of their
contribution to the organic carbon deposits in hypersaline systems.
4. Conclusions
Until recently, archaea and certain eubacterial taxa
were thought to be the only organisms able to grow in
hypersaline waters. However, it is now clear that melanized fungi can ¯ourish under conditions of halite, gypsum and calcite precipitation. These organisms have
sterol distributions that are consistent with the biosynthesis of 24-methylcholesta-5,7,22-trien-3b-ol (ergosterol). Thus, they are distinct from sterol mixtures in
algal and zooplanktonic species adapted to hypersaline
conditions.
A close study of the sterol composition in the melanized fungi reveals a dominance of 24-methylcholesta5,7,22-trien-3b-ol and signi®cant dierences among the
less abundant sterols. The high degree of unsaturation of
a large proportion of sterols from these melanized fungal
species strongly restricts their preservation and potential
use as biomarkers. However, some of the mono- and
diunsaturated compounds present in higher relative
abundance such as 24-methylcholesta-7,24(28)-dien-3b-ol
and 24-methylcholest-7-en-3b-ol are encountered in signi®cant proportion in sediments and water particulates of
halite and gypsum precipitation systems providing, in this
context, a speci®c signature for melanized fungi.
1039
Acknowledgements
Financial support from the European Union (MAST
Project MAS3-CT98-5057) is acknowledged. This project has also been funded by CICYT (PB93-0190-C0201) from the Spanish Ministry of Education.
Associate EditorÐB.J. Keely
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