FEMS Microbiology Letters 244 (2005) 229–234
www.fems-microbiology.org
Yeast diversity in hypersaline habitats
L. Butinar a, S. Santos b, I. Spencer-Martins b, A. Oren c, N. Gunde-Cimerman
a,d,*
a
Laboratory of Biotechnology, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia
Centro de Recursos Microbiológicos (CREM), Biotechnology Unit, Faculty of Sciences and Technology,
New University of Lisbon, 2829-516 Caparica, Portugal
The Institute of Life Sciences, and The Moshe Shilo Minerva Center for Marine Biogeochemistry, The Hebrew University of Jerusalem,
Jerusalem 91904, Israel
d
Biology Department, Biotechnical Faculty, University of Ljubljana, Večna pot 111, SI-1000 Ljubljana, Slovenia
b
c
Received 29 November 2004; received in revised form 21 January 2005; accepted 25 January 2005
First published online 3 February 2005
Edited by C. Edwards
Abstract
Thus far it has been considered that hypersaline natural brines which are subjected to extreme solar heating, do not contain nonmelanized yeast populations. Nevertheless we have isolated yeasts in eight different salterns worldwide, as well as from the Dead Sea,
Enriquillo Lake (Dominican Republic) and the Great Salt Lake (Utah). Among the isolates obtained from hypersaline waters,
Pichia guilliermondii, Debaryomyces hansenii, Yarrowia lipolytica and Candida parapsilosis are known contaminants of low water
activity food, whereas Rhodosporidium sphaerocarpum, R. babjevae, Rhodotorula laryngis, Trichosporon mucoides, and a new species
resembling C. glabrata were not known for their halotolerance and were identified for the first time in hypersaline habitats. Moreover, the ascomycetous yeast Metschnikowia bicuspidata, known to be a parasite of the brine shrimp, was isolated as a free-living
form from the Great Salt Lake brine. In water rich in magnesium chloride (bitterns) from the La Trinitat salterns (Spain), two new
species provisionally named C. atmosphaerica – like and P. philogaea – like were discovered.
Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Yeast; Hypersaline water; Biodiversity; Halotolerance; 26S rDNA sequence
1. Introduction
Xerophilic and xerotolerant fungi are known contaminants of low water activity (aw) foods. Yeasts predominate in liquid products such as syrups, honey and
brines. Xerotolerant food borne yeasts include the
genera Candida (C. lambica), Debaryomyces (D. hansenii), Pichia (P. anomala, P. guilliermondii, P. ohmeri),
*
Corresponding author. Tel.: +386 (0)1 423 33 88; fax: +386 (0)1 257
33 90.
E-mail address: [email protected] (N. GundeCimerman).
Rhodotorula (R. glutinis) and Zygosaccharomyces (Z.
rouxii, Z. bisporus) [1]. Some yeasts are recognized
for their tolerance to high concentrations of sugars
(aw as low as 0.62), while others tolerate high salt concentrations. Torulopsis famata, Rhodotorula rubra, Pichia etchelsii, Candida parapsilosis and Debaryomyces
hansenii are able to grow above 10–15% NaCl [1,2].
Some are more tolerant at higher pH values, while
others show better tolerance under more acidic conditions [3].
Osmotolerant yeasts frequently occur on the phylloplane of plants, especially in the Mediterranean area
[4]. They were occasionally isolated from moderately
0378-1097/$22.00 Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsle.2005.01.043
230
L. Butinar et al. / FEMS Microbiology Letters 244 (2005) 229–234
saline environments such as saline soil [5–8], estuarine
water and sediments [9], salt marshes [10] and from
deep-sea environments [11,12]. Yeast populations are
more sparse in marine water than in fresh water, and decrease with depth and distance from land [13]. Few reports exist on the occurrence and biodiversity of yeasts
in natural hypersaline waters. Most strains of the variety
Metschnikowia bicuspidata var. bicuspidata originate in
salt lakes and ponds (ca. 10% NaCl), often associated
with diseased brine shrimp (Artemia salina). Some initially did not grow in the usual media unless they were
supplemented with 2% NaCl [14]. However, this feature
did not hold when the cultures were maintained in artificial laboratory media. Kritzman in 1973 [15] isolated
an osmophilic yeast from the Dead Sea, although no
cultures have been preserved. Lahav et al. [16] discovered two yeast species in hypersaline chemical wastewater evaporation ponds. It is generally considered that
hypersaline brines that experience extreme solar heating
do not harbor any yeast populations [17]. To our knowledge, no further studies on the occurrence of yeast species in this natural extreme habitat were reported. In this
study we present the biodiversity and occurrence of nonmelanized yeasts in hypersaline waters of different salterns and salt lakes worldwide.
2. Materials and methods
2.1. Sampling sites and determination of environmental
parameters
Water samples were taken in 1997 and 1999 from
the active Slovenian solar salterns Sečovlje in the
northern Adriatic coast, at the border between Slovenia and Croatia. Because of the sub-Mediterranean climate, salt is collected only during the salt production
season [18]. Outside the season of salt production the
salinity of the water mainly remains below 5% (w/v)
NaCl. Four successive evaporitic ponds were selected
for fungal isolations, covering salinities from 3% to
30% NaCl.
Sampling within the same salinity range was performed as well in Eilat salterns on the Red Sea coast
in Israel, while in the salterns on the Mediterranean
coast in Spain (Santa Pola and Ebre delta) and in
France (Camargue salterns), on the Atlantic coast in
Namibia (Skeleton coast), in the Dominican Republic
and in Portugal (Samouco salterns, south of Lisbon)
only hypersaline water from the crystallisation ponds
was sampled. Hypersaline water samples were also taken
from the Dead Sea, the Great Salt Lake (Utah) and the
Enriquillo Lake (Dominican Republic).
Salinity (areometer) and water activity (aw) (CX-1
system, Campbell Scientific Ltd., Loughborough, UK)
were determined in all water samples.
2.2. Isolation methods and preservation of isolates
To enable discrimination between xerophilic/xerotolerant and halophilic/halotolerant fungi present in the
salterns, selective media were used in which the water
activity was lowered by addition of high concentrations
of glycerol (18%), NaCl (from 10% to 32%) or sugar
(50–70%) [19]. The fungal population dynamics were
followed as described by Gunde-Cimerman et al. [19].
In 1997, agar baits in dialysis tubing were incubated in
different ponds in the Adriatic salterns, and biofilms
covering the brine were spread on low water activity
selective media [19].
The isolated and identified strains from all sampling
sites are maintained in the Culture Collection of the National Institute of Chemistry (MZKI), Slovenia and in
the Portuguese Yeast Culture Collection (PYCC),
Portugal.
2.3. Taxonomy
For morphological characterization cultures were
grown on cornmeal (Difco), malt [20] and acetate agar
[20] at 25 °C and studied with phase-contrast optics.
A selection of physiological tests (urease, Diazonium
Blue B colour reaction, fermentation of glucose, assimilation of inositol and D-glucuronate as sole carbon
sources; assimilation of nitrate as sole nitrogen source,
and production of starch-like compounds) was performed as described by Yarrow [20].
For DNA extraction, two loopfuls of MYP agar
grown cultures were suspended in 500 ll lysing buffer
(50 mmol l1 Tris; 250 mmol l1 NaCl; 50 mmol l1
EDTA; 0.3% w/v SDS; pH 8) and the equivalent to a
volume of 200 ll of 425–600 lm glass beads (Sigma)
was added. After vortexing for 3 min, the tubes were
incubated for 1 h at 65 °C. The suspensions were then
centrifuged for 30 min at 4 °C. Finally, the collected
supernatant was diluted 1:750, and 5 ll were directly
used in the PCR.
For rDNA sequence analysis DNA was amplified
using primers NS7 (5 0 GAG GCA ATA ACA GGT
CTG TGA TGC) or ITS5 (5 0 GGA AGT AAA AGT
CGT AAC AAG G) and LR6 (5 0 CGC CAG TTC
TGC TTA CC). Cycle sequencing of the 600–650 base
pair region at the 5 0 end of the 26S rDNA D1/D2 domain employed the forward primer NL1 (5 0 GCA TAT
CAA TAA GCG GAG GAA AAG) and reverse primer
NL4 (5 0 TCC TCC GTC TAT TGA TAT GC). Sequences were obtained using either Amersham Pharmacia ALF Express II or ABI 310 (capillary) automated
DNA sequencer, following the manufacturerÕs instructions. For identification, the obtained sequences were
compared with those of all known yeast species, available at the GenBank database.
L. Butinar et al. / FEMS Microbiology Letters 244 (2005) 229–234
231
3. Results
3.2. Characterization of the isolates
3.1. Physicochemical conditions at the isolation sites
A total of 43 yeast isolates from eight different salterns worldwide, as well as from the Dead Sea, Enriquillo Lake (Dominican Republic) and the Great Salt Lake
(Utah) were studied. The yeasts strains isolated from the
hypersaline water were identified as Pichia guilliermondii, Debaryomyces hansenii, Yarrowia lipolytica, Metschnikowia bicuspidata, C. parapsilosis, Rhodosporidium
sphaerocarpum, R. babjevae, Rhodotorula laryngis and
Trichosporon mucoides (Table 1).
Sequence analysis of the D1/D2 domains of the 26S
rDNA revealed three novel yeast species. Sequences of
three strains (MZKI K-173, K-174 and K-175) differed
significantly (>2%) from the type strain of Candida
atmosphaerica, one strain (MZKI K-203) from the type
The salinity in the Adriatic saltern ponds ranged from
3% to 30% and the aw was between 0.72 and 0.96. Water
samples from other sampled salterns were in the salinity
range from 8% to 32% NaCl (aw 0.67–0.95). The aw in
the Dead Sea was 0.67, in the Eilat saltern it ranged from
0.73 to 0.95, in the Ebro River Delta from 0.73 to 0.83
and in Santa Pola from 0.73 to 0.94, in the Camargue salterns and Samouco salterns it was 0.73, in Namibian salterns it was 0.78 and in the Dominican Republic salterns
it ranged from 0.73 to 0.95. The Great Salt Lake (Utah)
water activity was 0.88, while at the Enriquillo Lake
(Dominican Republic) the water activity was 0.94.
Table 1
Spatiotemporal occurrence of yeast species from different hypersaline environments and ecological data reported in the literature
Species
Hypersaline water
Candida
atmosphaerica-like
Candida
glabrata-like
Candida parapsilosis
Bitterns, salterns La Trinitat; media with
10–17% NaCl; 30 CFU l1
Dead Sea; media without NaCl and with
18% glycerol; 1000 CFU l1
21% salinity in crystallization period;
Adriatic and Samouco salterns, Dead
Sea; media with 10–17% NaCl;
10 CFU l1
Salterns on the Atlantic coast in Namibia
and in Great Salt Lake; media with 25%
NaCl and 18% glycerol; 50 CFU l1
Great Salt Lake; media with 18%
glycerol; 10 CFU l1
Debaryomyces
hansenii
Metschnikowia
bicuspidata
Pichia guilliermondii
Pichia philogaea-like
Rhodosporidium
babjevae
Rhodosporidium
sphaerocarpum
Rhodotorula laryngis
Trichosporon
mucoides
Yarrowia lipolytica
8–25% salinity in crystallization period; 4%
salinity in precrystallization period;
Adriatic salterns; media with 10–17%
NaCl; 10–270 CFU l1
Bitterns, salterns La Trinitat; media
without NaCl; 10 CFU l1
5% salinity in precrystallization period;
Adriatic salterns; media with 10% NaCl;
20 CFU l1
5% salinity in precrystallization period;
Adriatic salterns; agar baits; media with
10–17% NaCl and 18% glycerol; 10–
80 CFU l1 and up to 1000 CFU l1 in
crystallisation period
Dead Sea; media without NaCl and with
18% glycerol; 1000 CFU l1
14-33% salinity; salterns Eilat, Dead Sea;
media without NaCl and with 18%
glycerol; 100–1000 CFU l1
25% salinity; Eilat salterns; media with
18% glycerol; 23 CFU l1
Habitats
Association with humans and
representative infections
Soil, water, spruce, olives, pickled
cucumbers, rotten tree trunk [13]
Onchomycosis, endocarditis,
endophthalmitis; skin, sputa,
nail, toe, bladder [13]
Cheese, sausages, sake moto, rennet,
tobacco, salmon [13]
Psoriasis, patient with angina,
infected nails, infected hand,
wound [13]
Salt lakes, often associated with diseased
brine shrimp, and sometimes in fresh
water lakes, possibly associated with
Daphnia magnaor with trematode
sporocysts, sea water [13]
Soil, water [13]
Otitis (rare), endocarditis,
joint infection; sputa, feces [13]
Herbaceus plants, soil, silage, peat, birch
leaves [13]
A commonly occuring species in marine
habitats, from the Antarctic Peninsula to
Palmer Archipelago and the Caribbean
Sea, salt farm [13]
Wine grapes [29]
Laryngeal swab [30]
Soil, water [13]
Meningitis, spinal fluid of human
patient, pubic white piedra (AIDS),
skin lesion, disseminated; sputum,
skin, hair [13]
Cornea of a human eye [13]
Refrigerated meat products, petroleum
products, agricultural (corn) processing
plant compost soil [13]
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L. Butinar et al. / FEMS Microbiology Letters 244 (2005) 229–234
strain of C. glabrata, and one strain (MZKI K-176)
from the type strain of Pichia philogaea.
3.3. Yeast diversity and spatiotemporal occurrence
The frequency of occurrence of yeasts isolated from
all sampled solar salterns was low, on average between
0 and 300 CFU l1, rarely exceeding 1000 cells l1, primarily outside the season of salt production (Adriatic
salterns) or in waters with NaCl concentrations below
20%. In all cases yeasts were mainly isolated on medium
with 10% NaCl, and to a lesser extent on media with 17–
25% NaCl, but never on 32% NaCl medium. No yeasts
were isolated from biofilms covering the brine.
The most frequently occurring species in the Adriatic
salterns and sporadically isolated as well in other salterns were identified as Pichia guilliermondii (up to
270 CFU l1) and C. parapsilosis, the latter being present in lower numbers. In the Adriatic salterns it was followed by Rhodosporidium sphaerocarpum and R.
babjevae, which were isolated at up to 20 CFU l1. C.
parapsilosis, R. sphaerocarpum and P. guilliermondii
were recovered in both years, while R. babjevae was
found only in 1999. R. sphaerocarpum was isolated as
well from agar baits incubated in the saltern ponds. In
the salterns on the Atlantic coast in Namibia and in
the Great Salt Lake brine Debaryomyces hansenii was
isolated (up to 50 CFU l1). In the Great Salt
Lake Metschnikowia bicuspidata has also been found
(Table 1).
Different yeast species were isolated in Israel,
although they occurred on media within the same salinity range (10–25% NaCl). The species that appeared
most frequently, T. mucoides (up to 1000 CFU l1),
was the only species isolated both from Eilat as well as
from the Dead Sea. Yarrowia lipolityca was isolated only
from the Eilat saltern (23 CFU l1), while Rhodotorula
laryngis, C. parapsilosis and a new species resembling
Candida glabrata was only recovered from the Dead
Sea (Table 1).
In the water rich in magnesium chloride (bitterns)
from the La Trinitat salterns (Spain), two new species
were isolated on different media, including the medium
with 17% NaCl. They are provisionally called C. atmosphaerica – like (MZKI K-173, K-174 and K-175) and P.
philogaea – like (MZKI K-176) (Table 1).
4. Discussion
Yeasts able to grow under reduced aw conditions, described as osmotolerant, were known primarily as contaminants in the food industry, but they remained
largely unknown in natural low water activity habitats.
Our study, conducted in diverse hypersaline habitats
around the world, therefore represents the first attempt
towards a survey of yeast biodiversity in such environments. Low yeast counts, about 10 cells l1, are typical
for open ocean water [13]. In hypersaline waters of the
salterns these counts occasionally increased to several
hundred cells per liter, although they seldom exceeded
this range.
Yeast isolates from hypersaline waters, of ascomycetous affinity, included members of the genera Candida,
Debaryomyces, Metschnikowia and Pichia. Different species of the genusPichia have been found in similar habitats, with P. membranefaciens being the most common
species isolated [9]. The opportunistic pathogen Pichia
guilliermondii was the only species of this genus isolated
from the salterns sampled in our work. It has been recognized by Lahav et al. [16] as one of the two dominant
microorganisms (together with Rhodotorula mucilaginosa) in hypersaline waste waters, growing on a wide
range of salinities and pH. Neither the halotolerant
food-borne P. etchelsii, nor the highly osmotolerant P.
anomala were detected.
Debaryomyces hansenii, known also as an opportunistic pathogen, a halotolerant food-borne yeast and a
model organism to understand fungal halophilic adaptive mechanisms [21] has been found mainly in cold
sea water [22]. To our knowledge, this was the first time
that it has been discovered in natural hypersaline habitats. Even so, the only hypersaline habitats in which
D. hansenii was obtained were salterns at the Namibian
Skeleton coast and the Great Salt Lake, both exposed to
seasonal low temperatures.
Belonging to a group of species in the genus Metschnikowia that are predominantly associated with aquatic
environments and often parasitic in invertebrates [23],
M. bicuspidata was isolated.
So far, only one unidentified yeast species was discovered in the Dead Sea [15]. In our study, a new yeast species was discovered (C. glabrata-like), as well as diverse
opportunistic pathogenic species of the genus Candida.
These included C. glabrata, C. parapsilosis and C.
atmosphaerica, which were also isolated from the more
polluted intertidal estuarine water and sediments [9]
and hypersaline water of the Dead Sea. Among these,
only C. parapsilosis was previously known as a foodborne halotolerant yeast, whereas others were not
known for any particular halotolerance [2].
A special ecological niche within the salterns are the
nutrient-rich bitterns that remain after the crystallization of halite. It was supposed that these are devoid of
life, as no organisms appeared to exist that would tolerate the extremely high Mg2+ concentrations [24]. In our
study, two novel yeasts species were recovered from the
bitterns.
Ascomycetous yeasts, recognized for their tolerance
to high concentrations of sugars, such as Zygosaccharomyces rouxii and Z. bisporus, were not isolated from
the salterns.
L. Butinar et al. / FEMS Microbiology Letters 244 (2005) 229–234
The predominant basidiomycetous yeasts in many
aquatic environments are primarily members of the genera Rhodotorula, Rhodosporidium, Trichosporon, Cryptococcus and Sporobolomyces [11]. In our investigations,
R. sphaerocarpum, R. babjevae, Rh. laryngis and T.
mucoides have been isolated from the salterns.
Saltern biotopes worldwide differ significantly with
respect to the nutrient status of the brines and the retention time of the water in the system, which mainly depends on climatic conditions. As a result of such local
differences, the diversity and quantity of microorganisms that occur in the ponds may vary [25,26]. In this
study, yeasts were isolated irregularly on space-time
scales or were isolated only once, while black yeasts,
the dominant fungi present in the salterns, have been
so far isolated from all sampled salterns and salt lakes,
apart from Eilat salterns and the Dead Sea in Israel
[19,27,28]. The fact that the isolation of most yeast species found did not follow a consistent spatial or temporal pattern indicates that many of the species are not
native to the hypersaline environments. No significant
correlation was observed between species richness and
samples recovered at different localities, which suggests
that most of the mycobiota diversity may represent contaminants from the incoming sea water or from the
areas surrounding the sampling sites. Noteworthy, the
species that showed the largest geographical distribution
and higher temporal frequency of occurrence were P.
guilliermondii, C. parapsilosis and T. mucoides. These
species could be indigenous and they probably represent
a stable core of the microbial communities in the hypersaline environments analysed.
Acknowledgement
This study was supported in part by the Slovenian
Ministry of Education, Science and Sport, by the Centro
de Recursos Microbiológicos (CREM), New University
of Lisbon, Portugal, and by the Moshe Shilo Minerva
Center for Marine Biogeochemistry, The Hebrew University of Jerusalem, Israel.
References
[1] Samson, R.A., Hoekstra, E.S., Frisvad, J.C. and Filtenborg, O.
(2000) Introduction to Food- and Airborne Fungi. Centraalbureau voor Schimmelcultures, Utrecht, p. 389.
[2] Lages, F., Silva-Graça, M. and Lucas, A. (1999) Active glycerol
uptake is a mechanism underlying halotolerance in yeasts: a study
of 42 species. Microbiology 145, 2577–2585.
[3] Praphailong, W. and Fleet, G.H. (1997) The effect of pH, sodium
chloride, sucrose, sorbate and benzoate on the growth of food
spoilage yeasts. Food Microbiol. 14, 459–468.
[4] Inacio, J., Pereira, P., de Carvalho, M., Fonseca, A., AmaralCollaco, M.T. and Spencer-Martins, I. (2002) Estimation and
diversity of phylloplane mycobiota on selected plants in a
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
233
Mediterrenean-type ecosystem in Portugal. Microb. Ecol. 44,
344–353.
Elliott, J.S.B. (1929) The soil fungi of the Dovey salt marshes.
Ann. Biol. XVII, 284–305.
Bergen, L. and Wagner-Merner, D.T. (1977) Comparative survey
of fungi and potential pathogenic fungi from selected beaches in
the Tampa bay area. Mycologia 69, 299–308.
Abdel-Hafez, S.I.I., Moubasher, A.H. and Abdel-Fattah, H.M.
(1978) Cellulose-decomposing fungi of salt marshes in Egypt.
Folia Microbiol. 23, 37–44.
Steiman, R., Guiraud, P., Sage, L. and Seigle-Murandi, F.
(1997) Soil mycoflora from the Dead Sea oases of Ein Gedi
and Einot Zuqim (Israel). Antonie van Leeuwenhoek 72, 261–
270.
Soares, C.A.G., Maury, M., Pagnocca, F.C., Araujo, F.V. and
Mendoca-Hagler, L.C. (1997) Ascomycetous yeast from tropical
intertidal dark mud of southeast Brazilian estuaries. J. Gen. Appl.
Microbiol. 43, 265–272.
Pitt, J.I. and Hocking, A.D. (1985) The ecology of fungal food
spoilage In: Fungi and Food Spoilage (Schweigert, B.S. and
Stewart, G.F., Eds.), pp. 5–18. Academic Press, Sydney.
Nagahama, T., Hamamoto, M., Nakase, T., Takami, H. and
Horikoshi, K. (2001) Distribution and identification of red yeasts
in deep-sea environments around the northwest Pacific Ocean.
Antonie van Leeuwenhoek 80, 101–110.
Nagahama, T., Hamamoto, M., Nakase, T. and Horikoshi, K.
(2001) Rhodotorula lamellibrachii sp. nov., a new yeast species
from a tubeworm collected at the deep-sea floor in Sagami bay
and its phylogenetic analysis. Antonie van Leeuwenhoek 80, 317–
323.
van Uden, N. and Fell, J.W. (1968) Marine yeasts. Adv.
Microbiol. Sea 1, 167–201.
Kurtzman, C.P. and Fell, J.W. (1998). The Yeasts, a Taxonomic
Study. Elsevier, Amsterdam, p. 1055.
Kritzman G. (1973) Observations on the microorganisms in the
Dead Sea. M.Sc. thesis, The Hebrew University of Jerusalem (in
Hebrew).
Lahav, R., Fareleira, P., Nejidat, A. and Abeliovich, A. (2002)
The identification and characterisation of osmotolerant yeast
isolates from chemical wastewater evaporation ponds. Microb.
Ecol. 43, 338–396.
Hernandez-Saavedra, N.Y., Ochoa, J.L. and Vazquez-Dulhalt, R.
(1995) Osmotic adjustment in marine yeast. J. Plankton Res. 17,
59–69.
Schneider, J. (1995) Eindunstung von Meerwasser und Salzbildung in Salinen. Kali Steinsalz 11, 325–330.
Gunde-Cimerman, N., Zalar, P., de Hoog, G.S. and Plemenitaš,
A. (2000) Hypersaline water in salterns – natural ecological niches
for halophilic black yeasts. FEMS Microbiol. Ecol. 32, 235–240.
Yarrow, D. (1998) Methods for the isolation, maintenance and
identification of yeasts. In: The Yeasts (Kurtzman, C.P. and Fell,
J.W., Eds.), A Taxonomic Study, pp. 77–100. Elsevier,
Amsterdam.
Prista, C., Almagro, A., Loureiro-Dias, M.C. and Ramos, J.
(1997) Physiological basis for the high salt tolerance of Debaryomyces hansenii. J. Gen. Appl. Microbiol. 63, 4005–4009.
Norkrans, B. (1966) Studies on marine occurring yeasts: growth
related to pH, NaCl concentration and temperature. Arch.
Mikrobiol. 54, 374–392.
Mendonça-Hagler, L.C., Hagler, A.N. and Kurtzman, C.P.
(1993) Phylogeny of Metschnikowia species estimated from partial
rRNA sequences. Int. J. Syst. Bacteriol. 43, 368–373.
Javor, B.J. (1983) Planktonic standing crop and nutrients in a
saltern ecosystem. Limnol. Oceanogr. 28, 153–159.
Davis, J.S. (1974) Importance of microorganisms in solar salt
production. In: 4th Symposium on Salt (Coogan, A.L., Ed.), pp.
369–372. Northern Ohio Geological Society, Cleveland.
234
L. Butinar et al. / FEMS Microbiology Letters 244 (2005) 229–234
[26] Litchfield, C.D. and Oren, A. (2001) Polar lipids and
pigments as biomarkers for the study of the microbial
community structure of solar salterns. Hydrobiologia 466,
81–89.
[27] Buchalo, A.S., Nevo, E., Wasser, S.P., Oren, A. and Molitoris,
H.P. (1998) Fungal life in the extremely hypersaline water of
the Dead Sea: first records. Proc. R. Soc. London B 265, 1461–
1465.
[28] Butinar L., Sonjak S., Zalar P., Plemenitaš A. and GundeCimerman N. (2005) Melanized halophilic fungi are eukaryotic
members of microbial communities in hypersaline waters of solar
salterns. Botanica Marina, (in press).
[29] Prakitchaiwattana, C.J., Fleet, G.H. and Heard, G.M. (2004)
Application and evaluation of denaturing gradient gel electrophoresis to analyse the yeast ecology of wine grapes. FEMS Yeast
Res. 4, 865–877.
[30] Scorzetti, G., Fell, J.W., Fonseca, A. and Statzell-Tallman, A.
(2002) Systematics of basidiomycetous yeasts: a comparison of
large subunit D1/D2 and internal transcribed spacer rDNA
regions. FEMS Yeast Res. 2, 495–517.