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Studies of Antarctic Yeast Isolates for
Exopolysaccharide Synthesis
S. Rusinova-Videva
a
, K. Pavlova
a
& R. Metcheva
b
a
Department of Microbial Biosynthesis and Biotechnology, Institute of Microbiology,
Bulgarian Academy of Sciences, Plovdiv, Bulgaria
b
Institute of Zoology, Bulgarian Academy of Sciences, Sofia, Bulgaria
Published online: 15 Apr 2014.
To cite this article: S. Rusinova-Videva, K. Pavlova & R. Metcheva (2009) Studies of Antarctic Yeast Isolates
for Exopolysaccharide Synthesis, Biotechnology & Biotechnological Equipment, 23:sup1, 888-891, DOI:
10.1080/13102818.2009.10818565
To link to this article: http://dx.doi.org/10.1080/13102818.2009.10818565
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STUDIES OF ANTARCTIC YEAST ISOLATES FOR EXOPOLYSACCHARIDE
SYNTHESIS
S. Rusinova-Videva
1
, K. Pavlova
1
, R. Metcheva
2
1
Department of Microbial Biosynthesis and Biotechnology, Institute of Microbiology, Bulgarian Academy of Sciences,
Plovdiv, Bulgaria
2
Institute of Zoology, Bulgarian Academy of Sciences, Sofia, Bulgaria
Correspondence to: Snezhana Rusinova-Videva
E-mail: jrusinova@abv.bg
ABSTRACT
From Antarctic samples were obtained yeast isolates that were investigated for exopolysaccharide synthesis. The screening
process revelated that significant number of psychrophilic yeasts had the ability to produce biopolymers.Perspective strains
were selected. A physico-chemical analysis of Antarctic samples was made.

Keywords: Antarctica, polysaccharides, psychrophylic yeast
Introduction
The Antarctic continent is characterized with unique
combination of extreme environment conditions low
temperature and humidity, high winds, extended light and
dark periods, nutrient and water deficiency (11). The land
areas of the Antarctic are or constantly frozen or they thaw
only for several weeks during the summer (2,6). These
natural habitats include cold soils, plants (mosses, lichens,
grasses), lakes, sediments, cold-blooded animals (fishes). A
great number of microorganisms (fungi, bacteria, yeasts,
algae) are capable of growing in extreme environments (4).
Commonly, the eukaryotes are psychrophiles, acidophiles,
alkaliphiles, piezophiles, xerophiles, and halophiles, which
respectively thrive at low temperatures, low pH, high pH,
under extreme pressure, desiccation, and salinity (12).
Extremophilic microorganisms have recently attracted
considerable attention not only for their metabolite adaptation
at extreme conditions, but also for their biotechnological
potential (1,5). A progress in the research of Antarctic
microorganisms is made by the creation of culture collections
and screenings for perspective strains, producing biologically
active compounds enzymes

(7,8,14) polysaccharides (9,10),
lipids (3,16), etc. The interest for biotechnological production
of polysaccharides is determined by the possibility of
different microorganisms to synthesize exopolysaccharides,
applied in the food, pharmaceutical, cosmetic, and other
industries as emulsifiers, stabilizers, binding and gelling
agents, coagulants, lubricants, film forms, thickening and
suspending agents. The Antarctic yeasts are insufficiently
studied as producers of exopolisaccharides with new
functional properties and applications.
In this paper we present the results from physical and
chemical characterization of Antarctic soil and moss samples,
from which yeast isolates were obtained and perspective
strains, producing new exopolisaccharides were selected.
Materials and methods
Antarctic samples from Livingston, Antarctica and
microorganisms. Livingston Island is the second island
among the South Shetlands located between 62 27 - 62 48
S and 59 45 - 6115 W of the northern sea coast of the
Antarctic Peninsula. Samples (soil and moss) were taken
from different sites of Livingston Island by the Bulgarian
Antarctic Expedition in the summer of 2006-2007 and 2007-
2008. The samples were suspended in sterile water and after
suitable dilution they were plated on malt agar. The
cultivation was carried out at 4C for 3 to 14 days. The
isolated colonies were reinoculated several times for purity,
maintained on malt slant agar and stored at 4C. The yeasts
studied belonged to the psychrophilic yeast collection
isolated from Antarctic samples.
Exopolysaccharide (EPS) production. The fermentation
medium contained (g l
-1
): sucrose, 40; (NH
4
)
2
SO
4
, 2.5;
KH
2
PO
4
, 1.0; MgSO
4
.7H
2
O, 0.5; NaCl, 0.1; CaCl
2
.2H
2
O, 0.1;
yeast extract, 1.0. The initial pH was adjusted to pH 5.3. The
XI ANNIVERSARY SCIENTIFIC CONFERENCE BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/SE
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inoculum from psychrophilic isolates were obtained in 100
ml Erlenmeyer flasks containing 50 ml of Sabouraud
medium (Merck, Germany), on a rotary shaker at 220 rpm at
22C for 48 h.The fermentation medium was inoculated with
10.0% w/v inoculum. The cultivation was performed at 22C
for 120 h with shaking at 220 rpm. Cells were collected by
centrifugation. The exopolysaccharides in the supernatant
were precipitated with two volumes of cold absolute ethanol,
held at 4C overnight and then centrifuged at 6000 g for 30
min, washed with ethanol and dried.
Analyses. The yield of exopolysaccharides and the dry
biomass were determined by the weight method after drying
to constant mass at 65C and 105C. The viscosity was
determined by the method of Stoks, 20C (13).
The IR spectra of exopolysaccharide were implemented using
KBr tablets on a spectrometer Nicolet Avatar 330 FT-IR,
Thermor Electron Corporation, Madison, USA.
Results and Discussion
The Bulgarian scientific research expeditions on Livingston
Island provided us with Antarctic soil and moss samples. The
structural and functional characteristics of the samples were
determined, since these features were preconditions for the
existence of microorganisms and uncovered the potentiality
for their isolation. The results from the physical and chemical
analysis of the Antarctic soil and moss samples are shown in
Table 1.
The structure of soil sample 1 was coarse-grained and the
sample contained plant tissues such as small roots and leaves.
Sample 2 was sandy structured with the presence of small
roots. Sample 3 was definitely fine-grained and sandy. The
soil samples were characterized with neutral pH values, at
which microorganisms display the highest life activity. The
humidity of the samples was determined to be low, which
limited the presence and growth of microorganisms. The
number of the isolates selected from the three samples was as
follows: eight isolates from sample 1, five isolates from
sample 2, and three isolates from sample 3. The chemical
analysis of the soil samples, regarding carbon and nitrogen
content, showed that these basic nutrient sources are available
in small quantities. This fact is explained by the extreme
environment conditions on the continent, which limit flora
and fauna diversity. The ratios of carbon and nitrogen source
in the soil samples had close values: 10:1, 12:1, and 11.9:1
respectively. The high phosphorus content of sample 3 is
probably the reason for the small number of isolates obtained.
Sixteen isolates were selected from the Antarctic moss
samples. The moss samples were characterized with up to ten
folds higher carbon content and two folds higher nitrogen
content in comparison to the soil samples the ratio C:N is
61:1 in sample 4, and 41:1 in sample 5. The higher
concentration of carbon and nitrogen and the higher humidity
of the plant samples explain the higher percentage of yeasts
isolated. Additionally, nine isolates were selected from
Antarctic samples (soil and penguin feather) taken from the
Polish base, situated on King Georgia Island. Totally, 41
psycrophilic yeast isolates were collected.
The metabolite potential of the isolated yeasts for
biosynthesis of exopolysaccharides was studied. Eleven
strains, producing exopolysaccharides were selected by
submerged cultivation on a synthetic nutrient medium,
containing 4 % sucrose. The potential for exopolysaccharides
biosynthesis was evaluated by the yield of polysaccharides,
by the measured viscosity, and by the quantity of
accumulated biomass (Fig. 1).
The concentration of produced biopolymers varied in the
range of 4.00 gL
-1
to 5.15 gL
-1
. The content of biomass was
in the range of 2.40 % to 4.22 %, and the viscosity was from
1.97 Pa.s to 2.77 Pa.s. According to the results, the strains,
producing over 4.70 gL
-1
exopolysaccharides were
considered to be of interest. The first six strains synthesized
an almost equal quantity of biopolymers. For this reason they
will be used for further selection of a perspective strain after
determination of some physical and chemical parameters.
The biomass concentrations were significantly lower than
exopolysaccharides concentrations. Probably, this was due to
biotransformation of carbon source to biopolymers in a
higher degree in comparison to its utilization as energy
supplement for cell growth. The measurement of the
supernatant viscosity by the method of Stocks provided us
with preliminary information for the reologic properties of
the exopolysaccharides, produced by the tested isolates. The
results showed no correlation between the three parameters
determined, probably the isolates taxonomically belong to
different genera and species. During the cultivation of the
psychrophilic yeast isolates on a nutrient medium with an
initial pH value of 5.3, a decrease of pH was detected at hour
24 of the process pH values varied in the range of 1.92 to
2.60. The process was under its own control, which ensured
sterility of the fermentations.

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SPECIAL EDITION/ONLINE 120 YEARS OF ACADEMIC EDUCATION IN BIOLOGY
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ABLE 1
Characterization of Antarctic siol and moss by Livingston Island
Livingston Island Humidity,
%
arbon,
g kg
-1

Nitrogen,
g kg
-1

Phosphorus,
g kg
-1

1. 623814.6 S 17. . .
602136.0 W
7.36 0.6 15.1 1.0 1.45 0.13 1093 170
2. 623849.1 S 36. . .
602218.2 W
7,06 1,3 43.0 3,2 3,52 0.31 1576 244
3. 623835,8 S 43. . .
602214.4 W
6.12 0.8 43.7 3.2 3.67 0.33 2272 352
4. 624009 S .
602408 W
7.15 2.1 472 34 7.6 0.7 1232 191
5. 624009,8 S
602408,1 W
7.21 3.2 259 19 6.3 0.6 2436 223

E
P
S

(
g
/
L
)
,

B
i
o
m
a
s
s

(
g
/
L
)

,

V
i
s
c
o
s
i
t
y

(
P
a
.
s
)
0
1
2
3
4
5
6
7
E P S
B io m a s s
V is c o s ity
1
0
0
1
0
2
3
4

6
5
6
6
1
0
1

6
2
1
0
5

9
5
8
6
9
9
I s o l a t e s

Fig. 1. Biosyntesis of exopolysaccharide, biomass and Viscosity from different psychrophilic yeast Isolates.

100

95
981.65

1432.50
90

85

80
616.99

2341.99
75


Fig. 2. IR spectra of polysaccharide from psyhrophilic yeast Isolate 100

656.54

668.63

1096.94

1138.35
2359.88
70

%
T
r
a
n
s
m
i
t
t
a
n
c
e

65

3303.42

60

1195.71
55

50

30

35

40

45

500 1000 1500 2000 2500 3000 3500 4000
Wavenumbers (cm-1)
XI ANNIVERSARY SCIENTIFIC CONFERENCE BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/SE
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We observed in the IR spectra of exopolysaccharide of yeast
isolate
100
characteristic absorption bands typical of natural
polysaccharides. The band at 2900-3300 m
-1
was distinctive
of the -
2
group, and at 3300-3600 m
-1
of the presence of
groups, involved in the formation of inter-molecular
hydrogen bonds. The band we observed at 1648-1654 m
-1

according Zhang et al., 2001 (15) is due to the in plane
deformation of the water molecule. This water is the strongly
bound water of crystallization.
In the neutral Antarctic samples from soil and moss with
low humidity and different quantities of the basic carbonic
and nitric nutrient sources, there exist conditions for growth
of microbic populations from which we have isolated and
selected psychrophilic yeasts with high biological potential
for synthesis of exopolysaccharides.
Acknowledgment
This investigation was supported by the Grant B-1615
from the National Fund Scientific Investigation.
BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/SE XI ANNIVERSARY SCIENTIFIC CONFERENCE
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