Sterols of melanized fungi from hypersaline environments
Laurence MeÂjanelle
a, Jordi F. LoÁpez
a, Nina Gunde-Cimerman
b,
Joan O. Grimalt
a,*
aDepartment of Environmental Chemistry, I.C.E.R.-C.S.I.C., Jordi Girona, 18. 08034-Barcelona, Catalonia, Spain bBiology 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, Clados-porium 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-methylcholesta-7,24(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 sea-water (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
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 lim-ited number to dierent genera of ®lamentous fungi. Among the isolated halotolerant/halophilic mycobiota the following genera were found:Hortaea,Phaeotheca,
Trimmatostroma,Aureobasidium,Alternaria, Cladospor-ium (Zalar et al., 1999a±c). These dematiaceous fungi have the common property that they form black, clump-like colonies consisting of isodiametrically dividing cells in the water, whereas the hyphal growth is mainly exhib-ited on solid media. This unique in-situ morphology was
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* Corresponding author. Tel.: 93-400-6122; fax: +34-93-204-5904.
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 ®la-mentous fungi, better known for their xerophilic nature, the genera Aspergillus, Penicillium andWallemia 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 pre-viously, 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 encom-passing 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 iso-lated from these hypersaline environments was exam-ined. Sterols constituted the most distinct lipid fraction when compared with the distributions typically encoun-tered in other group organisms, e.g. phytoplankton, zooplankton and bacteria. Thus, the present study is focused on this group of compounds, which are dis-cussed as source markers and compared with those pre-sent in waters (particulate phase) and sediments of solar salterns.
2. Experimental
2.1. Site description and cultures of melanized fungi
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 eva-poration 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, increas-ing 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.
Aliquots of waters (10±100 ml) from the diverse salt-ern ponds were ®ltered immediately after sampling over nitrocellulose Millipore membrane ®lters (pore size 0.45
mm) and placed on dierent selective agar media taining either high salt (17±32%) or high sugar con-centration (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 25C. 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. Subse-quently, the broth was diluted with 17% saline water several times.
All fungi isolated from selective media were inocu-lated 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 deter-mined 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 micro-biological 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.
TheSaccharomyces cerevisiaestrain W303a analyzed for reference was obtained from the Yeast Stock Center (Berkeley, CA, USA).
2.2. Extraction and fractionation
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 30C. The procedure
was repeated twice with methanol and twice further with dichloromethane (DCM). Extracts were recovered after centrifugation. Combined extracts were vacuum con-centrated 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
further extracted with hexane (420 ml) for isolation of
the fatty acids.
The neutral lipids were fractionated by column chro-matography 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 tri-methylsilylethers by reaction with bis-(trimethylsilyl)tri-¯uoroacetamide (70C, 30 min).
2.3. Instrumental analysis
Samples were dissolved iniso-octane and analyzed by gas chromatography (GC) using a Carlo Erba 5300 (Carlo Erba, Italy) ®tted with a heated splitless injector (300C) 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 70C for 1 min, heated to 150C at
15C minÿ1, then to 310C at 4C minÿ1, and ®nally
held at 310C for 30 min. The temperature of the ¯ame
ionization detector (FID) was 330C, 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 inter-face 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 300C onto a
capillary column (25 cm0.25 mm i.d.; 5%
phenyl-methyl 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 300C. 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 hyper-saline 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%
phe-nyl-methyl polisiloxane HP-5; 0.11 mm ®lm thickness) using helium as carrier gas. The injector was kept at 290C, the oven was programmed from 60C to 300C
at 4C minÿ1and kept at 300C for 15 min.
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 andCladosporiumspp were iden-ti®ed in La Trinitat solar saltern.Cladosporiumspp. are rather ubiquitous and found in the calcite, gysum and halite precipitation ponds as well as in the magnesium-containing brines.H. werneckiiwas found in the mixed calcite/gypsum, gypsum and halite depositional envir-onments.A. alternataandA. pullulanswere 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 exceptH. 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 inS. cere-visiaewhich 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 pre-dominance 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 24-methylcholesta-5,7,22-trien-3b-ol shows am/z468 mole-cular ion and the fragment [M-(CH3)3SiOH-SC]+atm/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/z363 and 211, respectively. The ion [M-SC-C3H5]+, m/z337, is typical of sterols with a double bond at posi-tion22(Rahier and Benveniste, 1989). This spectrum is
in agreement with that of ergosterol reported in Asco-mycete yeasts (Parks and Casey, 1995; Weete, 1989). The other main sterols in these melanized fungi possess one to four unsaturations at positions5,
7, 8,
22, 24(28)or
24.
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 (Rah-ier and Benveniste, 1989). Thus, the base peak of 24-methylcholesta-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-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 between5sterols and
5a(H) stanols. The intense ion atm/z337 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 at5, 7, 24(28)is
also based on mass spectral examination and retention time considerations. Several 24-methylcholestatrien-3b -ols have also been reported in Ascomycetes (Parks, 1978).
Six 24-methylcholestadien-3b-ols with double bonds at positions8, 22,7,22,8, 24(28),5, 7,7, 24(28)and
Table 1
Relative composition of squalene and sterols of cultures of melanized fungi isolated from solar salterns.S. cerevisiaehas also been studied for reference
Compounds Cladosporium
sphaerospermum
Cladosporium
sp
Hortaea werneckii
Aureobasidium pullulans
Alternaria alternata
Saccharomyces cerevisiae
1 Squalene (% neutrals) 3.0 1.2 0.0 7.7 8.6 12.6
2 Cholest-5-en-3b-ol 0.2 0.1 0.2 0.1 0.1 1.0
3 24-Methylcholesta-5,7,22,24(28) -tetraen-3b-ol
3.1 4.8 2.2 2.1 3.1 1.9
4 24-Methylcholesta-7,22,24(28) -trien-3b-ol
0.5 5.1 0.5 1.9 3.9
5 Cholesta-8,24-dien-3b-ol 3.7
6 24-Methylcholesta-8,22-dien-3b-ol 0.5 0.9 1.9 0.9
7 24-Methylcholestatrien-3b-ol 0.3 0.3 0.4 1.1 tr
8 24-Methylcholesta-5,7,22-trien-3b-ol 49.1 57.9 48.3 54.4 47.0 59.8
9 24-Methylcholesta-7,22-dien-3b-ol 6.2 3.3 2.5 4.3 1.3 4.5
10 24-Methylcholestatetraen-3b-ol 0.9 1.3 0.4 0.3 0.8 0.8
11 24-Methylcholesta-8,24(28)-dien-3b-ol 0.3 0.6 0.4 5.0 5.6 2.7
12 24-Methylcholest-8-en-3b-ol 2.1 1.3 0.7 7.0 2.8
13 24-Methylcholesta-5,7,24(28)-trien-3b-ol 2.1
14 4a-Methylcholestatrien-3b-ol 2.2
15 24-Methylcholestadien-3b-ol 0.5 0.5 0.4 0.4
16 24-Methylcholesta-5,7-dien-3b-ol 1.3 0.8 0.8 0.4 6.2
17 24-Methylcholesta-7,24(28)-dien-3b-ol 0.7 2.5 0.9 5.2 28.2 4.3
18 24-Methylcholest-7-en-3b-ol 19.5 14.2 6.6 5.8 4.4
19 24-Methylcholestatetraen-3b-ol 0.1 0.3 0.3 0.6 tr tr
20 24-Ethylcholest-5-en-3b-ol 0.1
21 4,4,14-Trimethylcholesta-8,24-dien-3b-ol 0.4 0.2 0.3 0.3 0.3 4.6
22 4a,24-Dimethylcholest-5-en-3b-ol tr 0.1 tr 0.3 tr
23 24-Methylcholestatetraen-3b-ol 0.8 0.9 0.4 0.6 0.4 0.3
24 4a,24-Dimethylcholesta-8,24(28)-dien-3b-ol 1.8 0.9 0.5 1.0 2.6 2.2 25 4a,24-Dimethylcholest-8-en-3b-ol 2.4 1.1 1.3 1.4
26 4a,24-Dimethylcholesta-5,7-dien-3b-ol 0.3 0.2 tr 0.2 27 4,4,14,24-Tetramethylcholesta-8,24(28)
-dien-3b-ol
2.4 1.1 4.2 1.7 0.8
28 4a,24-Dimethylcholesta-7,24(28)-dien-3b-ol 0.4 0.2 tr 0.4 0.8 29 4a,24-Dimethylcholest-7-en-3b-ol 3.8 0.8 28.6 3.4
31 4,4,14-Trimethylcholestadien-3b-ol tr tr 32
4,4,24-Trimethylcholesta-8,24(28)-dien-3b-ol
1.5 0.6 1.0 0.7 0.9
an unknown compound have been identi®ed in the mel-anized fungi (Table 1, Fig. 2). 24-Methylcholesta-7,24(28)-dien-3b-ol and 24-methylcholesta-8,24(28)-dien-3b-ol have similar spectra. They show typical frag-ments of8/7monounsaturated sterols (m/z211 and 227) and a very intense [M-SC-2H]+ion atm/z343. The
GC peaks of these compounds elute before the corre-sponding monounsaturated8or
7homologues.
24-Methylcholesta-7,24(28)-dien-3b-ol is the most abundant sterol, constituting up to 28.2% of total ster-ols 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, repre-senting 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 at7and
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 at7or8
show strong molecular ions and a strong [M-SC-(CH3)3SiOH- 42]+fragment,m/z213. They also readily
lose the side chain. The base peak of 7sterols
corre-sponds to the fragment [M-SC-(CH3)3SiOH]+,m/z255,
while this ion is also important in8sterols. 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 advan-tage of GC selectivity, since8elute before
7isomers
(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-methylcholesta-7,22-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 pro-duced by side chain cleavage are also observed,m/z359 for8/
7monounsaturated sterols and atm/z357 for 8, 24(28)/7, 24(28) sterols. The fragments retaining the C-4 methyl group generated by cleavage of the poly-cyclic structure have 14 mass units more than those of the corresponding 4-desmethyl 24-methyl sterols. For example, cleavage of ring D generatesm/z227 and 243 ions in8sterols (Fig. 1) andm/z227 and 241 ions in 8, 24(28)sterols.
The 4a,24-dimethylsterols encompass the same unsa-turated positions as the 4-desmethyl 24-methylsterols:
7,8, 7,24(28), 8,24(28)and 5,7. In the case of H. werneckii4a,24-dimethylcholest-7-en-3b-ol is the second major sterol constituent after 24-methylcholesta-5,7,22-trien-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 inA. alternata. This last
species contains 4a ,24-dimethylcholesta-8,24(28)-dien-3b-ol (2.6%) which parallels the presence of 24-methyl-cholesta-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-Trimethylcholesta-8,24(28)-dien-3b-ol shows a similar spectrum as that observed in 24-methylcholesta-8,24(28)-dien-3b-ol. The base peak atm/z135 corresponds to the equivalentm/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 C31sterol,
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.
andH. werneckiiare the species having this compound in higher abundance, consistent with the higher abundance of the C30homologue 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 ofS. cer-evisiae which was included in this study as reference. This sterol has been assumed to be ergosterol since this is the predominant sterol in fungi andS. 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)-tetraen-3b-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-ethyl-cholest-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
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-3b -ol in the later, which argues for distinct routes to ergos-terol synthesis.
Steroidal compounds with7 unsaturation are rela-tively abundant in the Cladosporium andH. 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, 14-20% of total sterols) and 4a ,24-dimethylcholest-7-en-3b-ol in the latter (29% of total sterols).8Unsaturated
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 con-sistent with the7sterol distributions.
Both 8 and 7 unsaturated sterols constitute the next major group of sterols (after ergosterol) inA. pull-ulans, with 24-methylcholest-8-en-3b-ol as the major homologue (7% of total sterols; Table 1). In this fungus,
7unsaturated sterols are in minor relative proportion. A. alternatais constituted by sterols predominated by
x, 24(28)as the major secondary group. The absence of
monounsaturated7and8sterols 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 24-methylcholest-7,24(28)-dien-3b-ol (28% total sterols). The other unsaturated site in the sterol distribution of this fungus is predominantly situated at7or8.
InS. 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 at7,
(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
cannot be recognized even though abundant melanized fungi are observed in these ponds. Phyto- and zoo-planktonic 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-5,22-dien-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 24-methylcholest-7-en-3b-ol (Table 2, Fig. 3), two major secondary sterols in melanized fungi, were found in sig-ni®cant relative proportion in the particles and sedi-ments of the gypsum and halite precipitation ponds. 24-Methylcholesta-7,24(28)-dien-3b-ol and 24-methylchol-est-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-7-en-3b-ol in the dino¯agellate Gymnodinium catenatum 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. Compounds Melanized fungi Halite Gypsum Gypsum/carbonate
Particles Particles Sediments Sediments
2a Cholest-5-en-3b-ol
ÿ + + + ÿ
34 5a(H)-cholestan-3b-ol ÿ ÿ + + +
35 24-Methylcholesta-5,22-dien-3b-ol ÿ + + + +
36 24-Methyl-5b(H)-cholest-22-en-3b-ol ÿ + ÿ + +
37 24-Methylcholest-5-en-3b-ol ÿ + + + +
38 24-Methyl-5b(H)-cholestan-3b-ol ÿ ÿ ÿ + +
39 24-Ethylcholesta-5,22-dien-3b-ol ÿ + + + +
40 24-Ethylcholest-22-en-3b-ol ÿ + + + +
17 24-Methylcholesta-7,24(28)-dien-3b-ol + + + + ÿ
18 24-Methylcholest-7-en-3b-ol + ÿ + + ÿ
41 24-Ethylcholest-5-en-3b-ol ÿ + + + +
42 24-Ethyl-5a(H)-cholestan-3b-ol ÿ ÿ ÿ + +
43 4a,23,24-Trimethyl-5a(H)-cholest-22-en-3b-ol ÿ ÿ ÿ ÿ +
a Peak numbers are coincident with those in Table 1 when applicable.
(Hallegrae et al., 1991) and 24-methylcholesta-7,24(28)-dien-3b-ol in some diatom species such asCoscinodiscus
sp. (Barrett et al., 1995) and Thalassiosira pseudana
(Orcutt and Patterson, 1975). However, in the high sali-nity 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
24-methylcholesta-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 sig-ni®cance of their biomass in relation to that of other organisms is yet to be assessed. The quantitative exam-ination 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 hypersa-line 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 mela-nized fungi can ¯ourish under conditions of halite, gyp-sum and calcite precipitation. These organisms have sterol distributions that are consistent with the bio-synthesis of 24-methylcholesta-5,7,22-trien-3b-ol (ergos-terol). 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 mela-nized fungi reveals a dominance of 24-methylcholesta-5,7,22-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 sig-ni®cant proportion in sediments and water particulates of halite and gypsum precipitation systems providing, in this context, a speci®c signature for melanized fungi.
Acknowledgements
Financial support from the European Union (MAST Project MAS3-CT98-5057) is acknowledged. This pro-ject has also been funded by CICYT (PB93-0190-C02-01) from the Spanish Ministry of Education.
Associate EditorÐB.J. Keely
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