DiscussionApproximately 70% of the

Discussion
Approximately 70% of the isolated yeasts could grow at
temperatures above 20°C, and 16% of them were able to
grow at ≥30°C. The predominance of psychrotolerant
fungi in cold environments has been previously noted,
and is attributable to seasonal and local increases in soil
temperature due to solar radiation [2]. In our study, the
temperature measured in situ at the different sampling
sites ranged from 0 to 11.9°C, but temperatures up to
20°C have been reported in this region [15-17]. The
main obstacle to assessing the yeast communities in Antarctic regions is the scant knowledge regarding their
environmental and nutritional requirements. Because
the yeast populations/species inhabiting terrestrial and
aquatic environments can colonize specific niches, no
appropriate method exists for efficiently isolating all species
[18]. In this work the yeasts were isolated using rich
media supplemented with glucose, because almost all
known yeasts can assimilate this sugar [19]. However,
this culture condition could favor the proliferation of
yeasts with high metabolic rates, to the detriment of
slow-growing yeasts. Nevertheless, large numbers and
high species diversity were attained in this study (22 species
from 12 genera). Cold-loving yeasts have been isolated
mainly from the Antarctic and the Arctic, and
from European and South American glaciers [10]. In all
of these environments, the most ubiquitous species are
Rhodotorula laryngis and Cr. victoriae. On the other
hand, C. sake, D. fristingensis, G. antarctica and Sp. salmonicolor
have been isolated only in the Southern Cone
(South American glaciers and Antarctica). This work
reports for the first time the isolation of Cryptococcus
gastricus, Cryptococcus gilvescens, D. fristingensis and
Leucosporidiella creatinivora from an Antarctic region.
Also isolated was W. anomalus, which is not generally
found in cold regions

During molecular analysis of the yeasts, most isolates
assigned to the same species possessed identical D1/D2
and ITS sequences. Thus, combining these rDNA
regions is a useful technique for rapid identification and
typing of yeasts, as others have suggested [14,20,21].
However, the isolates identified as Leuconeurospora sp.
were 0.7% and 0.9% different in their D1/D2 (578 bp)
and ITS (534 bp) sequences, respectively. Similarly, the
isolates identified as D. fristingensis exhibited identical
D1/D2 (456 bp) sequences, but their ITS (479 bp)
sequences were markedly different (4.4%), and their
overlap was punctuated with several gaps. Furthermore,
given the physiological differences between isolates that
are identical or similar at molecular level, strongly support
that the definitions of yeast species must be supplemented
by classical characterizations.


Most yeast isolates showed lipase activity, consistent
with a previous report in which all of the filamentous
fungi from Antarctica displayed this activity [22]. Among
the “cold loving” yeasts, lipase activity has been
described in Pseudozyma antarctica [23], Leucosporidium
antarcticum [24] and in species of Cryptococcus
and Rhodotorula [25]. Unlike this last-mentioned study,
we detected lipase activity in R. laryngis also. Lipase activity
has also been described in W. anomalus from tropical
environments [26]. The least common extracellular
activity was xylanase, observed only in the D. fristingensis
isolate. Although this activity has been previously
described in Cryptococcus species [27,28], no xylanase
activity was observed in the Cryptococcus isolates identified
here. Consistent with our results, protease, amylase
and esterase extracellular activities have been
reported in several yeast species isolated from cold
and tropical environments [24-26,29-33]. However, we
present the first report of extracellular amylase activity
in Le. creatinivora, H. watticus, Leuconeurospora sp.
and D. fristingensis. In addition to Mrakia and Rhodotorula
species, for which extracellular pectinase activity
has been described [33], we detected pectinase activity
in species of Wickerhamomyces, Metschnikowia, Dioszegia,
Leucosporidiella and Candida. All Mrakia species
isolated in this work showed cellulase activity,
which has been previously described in Mrakia frigida
isolated from King George Island [34]; furthermore,
this activity was observed in Cryptococcus and Dioszegia
species, contrary to a previous report [25]. Extracellular
chitinase activity has been reported in
Cryptococcus species [26], but here we observed this
activity in M. psychrophila, Sp. salmonicolor, Metschnikowia
sp., Leuconeurospora sp. and D. fristingensis. We
detected cellulase and chitinase activities in yeasts species
that have not been described from cold regions,
probably because our sampling sites included areas
with vegetation and animal contact and/or were
located close to the sea. Cellulose is one of the most
abundant carbohydrates produced by plants [35] and
chitin is the most abundant renewable polymer in the
ocean, where it constitutes an important source of
carbon and nitrogen [36]. Furthermore, significant
quantities of lipids exist in phytoplankton [37] and in
sediments of this region [38], which can explain the
high incidence of lipase activity found in the yeasts.
All of the extracellular enzyme activities analyzed in
this work are potentially useful to industry: amylases
in food processing, fermentation and pharmaceutical
industries; cellulases and pectinases in textiles, biofuel
processing and clarification of fruit juice; esterase in
the agro-food industries; lipases and proteases in food
and beverage processing, detergent formulation and
environmental bioremediations; chitinases in biocontrol
and treatment of chitinous waste; xylanase as a
hydrolysis agent in biofuel and solvent industries