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RESEARCH ARTICLE
Growth, lipid accumulation, and fatty acid composition in obligate

psychrophilic, facultative psychrophilic, and mesophilic yeasts
Maddalena Rossi1, Pietro Buzzini2, Lisa Cordisco3, Alberto Amaretti1, Maurizio Sala1, Stefano
Raimondi1, Chiara Ponzoni1, Ugo Maria Pagnoni1 & Diego Matteuzzi3
1
Department of Chemistry, University of Modena and Reggio Emilia, Modena, Italy; 2Department of Applied Biology, Industrial Yeasts Collection
DBVPG, University of Perugia, Perugia, Italy; and 3Department of Pharmaceutical Sciences, University of Bologna, Bologna, Italy
Correspondence: Maddalena Rossi,

Department of Chemistry, University of
Modena and Reggio Emilia, via Campi 183,
41100 Modena, Italy. Tel.: 139 059 205
5567; fax: 139 059 373 543; e-mail:
rossi.maddalena@unimore.it
Received 12 November 2008; revised 26 May
2009; accepted 26 May 2009.
Final version published online 14 July 2009.
DOI:10.1111/j.1574-6941.2009.00727.x
MICROBIOLOGY ECOLOGY
Editor: Max Haggblom

Keywords
psychrophilic yeast; acclimation; adaptation;
fatty acids; lipid.
Abstract
Obligate psychrophilic, facultative psychrophilic, and mesophilic yeasts were
cultured in a carbon-rich medium at different temperatures to investigate whether
growth parameters, lipid accumulation, and fatty acid (FA) composition were
adaptive and/or acclimatory responses. Acclimation of facultative psychrophiles
and mesophiles to a lower temperature decreased their specific growth rate, but
did not affect their biomass yield (YX/S). Obligate and facultative psychrophiles
exhibited the highest YX/S. Acclimation to lower temperature decreased the lipid
yield (YL/X) in mesophilic yeasts, but did not affect YL/X in facultative psychrophilic ones. Similar YL/X were found in both groups of psychrophiles, suggesting
that lipid accumulation is not a distinctive characteristic of adaptation to
permanently cold environments. The unsaturation of FAs was one major adaptive
feature of the yeasts colonizing permanently cold ecosystems. Remarkable
amounts of a-linolenic acid were found in obligate psychrophiles at the expense
of linoleic acid, whereas it was scarce or absent in all the other strains. Increased
unsaturation of FAs was also a general acclimatory response of facultative
psychrophiles to a lower temperature. These results improve the knowledge of the
responses enabling psychrophilic yeasts to cope with the cold and may be of
support for potential biotechnological exploitation of these strains.
Introduction
Permanently cold ecosystems make up one of the largest
biospheres on the Earth and represent one of the last
unexplored frontiers of ecology. Psychrophilic microorganisms colonize deep oceans, glaciers, and polar areas and
constitute an important part of cold-adapted biodiversity,
playing an essential role as nutrient cyclers and waste
mineralizers. Conventionally, obligate psychrophiles have a
maximum temperature for growth o 20 1C, an optimum
temperature of c. 15 1C, and a minimum temperature at 0 1C
or lower. On the contrary, facultative psychrophiles can be
thought of as mesophiles that evolved to tolerate cold. They
exhibit optimum temperatures 4 20 1C and are capable of
growth around 0 1C (Cavicchioli & Tortsen, 2000; Price &
Sowers, 2004; Raspor & Zupan, 2006; Margesin et al.,
2007b). Even though psychrophilic microorganisms are still
understudied, they are increasingly being investigated for
FEMS Microbiol Ecol 69 (2009) 363372
valuable biotechnological purposes (Margesin & Schinner,

1999a, b; Russell, 2000; Margesin et al., 2007b, c).
To successfully colonize cold environments, psychrophiles
have evolved a number of genetic-based phenomena,
ascribed to adaptive and acclimatory responses. The timescale exposure is essential to interpret the effects of suboptimal temperature on microbial physiology. An abrupt
temperature shift is likely to generate rapid, high-dynamic
stress-response phenomena on microbial cells, whereas
prolonged exposure to suboptimal temperature leads to
acclimation, which implicates regulatory mechanisms resulting in the full adjustment of the genomic expression and
the physiological state during a lifetime. Over a time scale of
several generations, the evolutionary selection of the gene
alleles, which increase the fitness in a specific environmental
niche, is termed adaptation (Morgan-Kiss et al., 2006).
Yeasts inhabit diverse ecological niches including those
that are considered extreme with respect to low
2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved

c
364
temperatures (Larkin & Stokes, 1968). A few studies reported the isolation of yeasts in meltwater rivers originating
from the glaciers of Patagonia (de Garca et al., 2007), in the
different ice layers of Arctic glaciers (Gostincar et al., 2006),
in sediments, ice, meltwaters, and soils of alpine environments (Margesin et al., 2007a; Turchetti et al., 2008).
Although the ability of yeasts to cope with low environmental temperatures has attracted considerable attention, so
far, the mechanisms underlying this phenomenon have not
been described fully for psychrophilic yeasts, because most
of the studies have been focused on mesophilic ones,
particularly on Saccharomyces cerevisiae (Sahara et al., 2002;
Homma et al., 2003; Schade et al., 2004; Aguilera et al., 2007;
Tai et al., 2007). A temperature decrease from 30 to 12 1C
affects the transcription level of many genes of S. cerevisiae,
such as the ones involved in the regulation of transporters of
growth-limiting nutrients, glycogen metabolism, ribosome
biogenesis, and ribosomal proteins. Trehalose biosynthesis is
involved in cold shock response, but not in steady-state lowtemperature acclimation. A defined group of genes regulating the response of this yeast species to cold is involved in
lipid metabolism. D9 Desaturase, which is the key enzyme in
the synthesis of unsaturated fatty acyl-CoAs and is the only
known desaturase of S. cerevisiae, is overexpressed at low
temperatures (Stukey et al., 1989; Nakagawa et al., 2002;
Martin et al., 2007).
Changes in lipid metabolism are known to constitute the
major adaptation of metabolic functions occurring during
growth at low temperatures. They determine an improved
membrane fluidity causing both the maintenance of the
appropriate physical state of lipid bilayer and the good
functionality of membranes (Los & Murata, 2004; MorganKiss et al., 2006). Differences in fatty acid (FA) composition
have been described since the 1970s in yeasts growing at
diverse temperatures (McMurrough & Rose, 1973; Arthur &
Watson, 1976; Watson & Arthur, 1976; Watson, 1978;
Watson et al., 1978), but no rigorous comparative studies
of the physiological behavior of obligate psychrophilic,
facultative psychrophilic, and mesophilic yeast strains have
been published yet. The present study wanted to fill this
gap by comparing the growth parameters and FA composition of 26 yeasts strains (representative of 12 species)
isolated from different cold and temperate habitats and
clustered as obligate psychrophiles, facultative psychrophiles, and mesophiles.
M. Rossi et al.
Yeast strains
The yeast strains used in this study were obtained from the
Industrial Yeasts Collection DBVPG (University of Perugia,
Italy, http://www.agr.unipg.it/dbvpg), from the American
Type Culture Collection (ATCC), or from our own collection. Twenty psychrophilic yeasts were isolated from sediments, ice, and meltwater of alpine glaciers (Turchetti et al.,
2008). Twelve strains belonged to the species Cryptococcus
gilvescens, Rhodotorula creatinivora, Rhodotorula laryngis,
Rhodotorula glacialis, and the yeast-like species Aureobasidium pullulans. Besides, eight strains were preliminarily
identified as Leucosporidium spp. and Mrakia spp., because
they belong to two so far undescribed new species. Accordingly, their formal taxonomic description is still in progress.
The six mesophilic strains, which were isolated from
temperate habitats and exhibited temperature optima ranging between 25 and 35 1C, belonged to the species
S. cerevisiae, Saccharomyces exiguus, Kluyveromyces marxianus, Pichia farinosa, and Zygosaccharomyces rouxii.
Growth experiments
Materials and methods
All the strains were aerobically cultured in flasks containing

the carbon-rich medium GMY (Buzzini, 2001): 40 g L1
glucose, 8 g L1 KH2PO4, 0.5 g L1 MgSO4 7H2O, and
3 g L1 yeast extract (Difco Laboratories, Sparks, MD), pH
5.5. The yeasts from cold habitats were subcultured at 4 1C
for 14 days, while those from temperate habitats were
subcultured at 30 1C for 5 days.
To determine the growth capabilities at different temperatures, batch cultures were carried out in shake flasks containing 50 mL GMY broth. The flasks were inoculated (10% v/v)
with grown seed cultures and were incubated at 4, 18, or
30 1C. The turbidity (OD600 nm) was monitored during batch
cultivation to calculate the specific growth rate (m) and to
determine the growth kinetic. The residual glucose concentration, the biomass dry weight, the viable count on GMY
agar plates, and lipid composition were determined at the
stationary phase (after 14 days for cultures grown at 4 and
18 1C, and after 5 days for cultures grown at 30 1C).
Biomass dry weight was determined gravimetrically.
Glucose was analyzed by means of HPLC equipped with an
RI detector (HPLC System, 1200 Series, Agilent Technologies, Santa Clara, CA). The analysis was performed using an
Aminex HPX-87H ion exclusion column (300 7.8 mm)
and 5 mM H2SO4 (flow rate 0.6 mL min1) as the isocratic
mobile phase. The biomass/substrate yield (YX/S) was expressed as g dry biomass g1 of consumed glucose.
Chemicals
Lipid analysis
All chemicals were obtained from Sigma-Aldrich (Steinheim, Germany) unless otherwise stated.
Biomass from 50-mL culture samples was harvested (3000 g,

5 min, 0 1C), washed with distilled water, frozen at 80 1C,


c
365
Adaptation and acclimation of psychrophilic yeasts
and lyophilized (Lyolab 3000, Heto-Holten, Allerd, Denmark). Lipids were extracted using the procedure of Folch
et al. (1957), with a few modifications. One gram of
lyophilized biomass was extracted with 50 mL of a chloroform : methanol mixture (2 : 1, v/v) under shaken conditions for 16 h. The extract was filtered through a column of
celite and anhydrous Na2SO4 to remove cell debris and
water. Solvents were removed with a rotavapor and then
lipids were weighed. The lipid/biomass yield (YL/X) was
expressed as g lipid extract g1of dry biomass.
The extract was subjected to methanolysis of total lipids
according to the method of Morrison & Smith (1964).
Lipids were dissolved in 2 mL of a 1 : 1 mixture of hexane
and BF3 solution (14% in methanol, Sigma-Aldrich) and
transferred into a Schlenk tube. Five milligrams of glyceryl
triundecanoate was added to the reaction mixture to generate the internal standard for GC-MS analysis. Transesterification was carried out at 100 1C for 1 h, and then 2 mL of
d.d. water was added to quench the reaction.
The organic phase was collected and the fatty acyl methyl
esters were analyzed by GC. Analysis was performed using a
quadrupole GC-MS system (HP5890 Series II gas chromatograph HP5972 mass selective detector) equipped with an
EI ionization detector (70 eV ionization energy). An HP-5
capillary column was used for the separation (Agilent
Technologies, internal diameter of 0.20 mm, film thickness
of 0.5 mm, and length of 30 m). The injection temperature
was 280 1C and the oven temperature was programmed
from 80 1C (1-min isotherm) to 130 1C at a rate of
50 1C min1, and then to 280 1C at a rate of 5 1C min1 (20min isotherm at 280 1C). High-purity helium was used as
the mobile phase and a constant column head pressure of
9 psi was maintained during the analyses.
FAs were identified by comparison with commercial
standards of fatty acyl methyl esters. Peak areas in the total
ion chromatograms were used to determine their relative
amounts. The quantitative equation of the unsaturation
index (UI) was used to aid the analysis of lipid composition.
The UI was calculated as the number of double bonds of
each FA multiplied by its relative amount (Watson & Arthur,
1976).
Statistical analysis
All values are means of three separate experiments. Differences in means among groups A, B, and C were evaluated
using one-way ANOVA, followed by Tukeys post hoc comparisons. Differences were considered statistically significant at
P 0.05. Differences in means among the growth temperatures were analyzed using two-way ANOVA with repeated
measures with the group as the first factor and temperature
as the second factor, followed by Bonferronis post hoc
comparisons. Differences were considered statistically sigFEMS Microbiol Ecol 69 (2009) 363372
nificant at P 0.05. Statistical analysis was performed using

4.0 (Graphpad Software, San Diego, CA).
GRAPHPAD PRISM
Results
Growth parameters of facultative and obligate
psychrophilic, and mesophilic yeasts
The ability of all the yeasts to grow at 4, 18, and 30 1C was
determined (Table 1). All the strains from cold habitats grew
at 4 1C, but they were divided into obligate and facultative
psychrophiles (groups A and B, respectively) on the basis of
their ability to grow at 18 and 30 1C. The strains belonging
to the species R. creatinivora, R. glacialis, Leucosporidium sp.,
and Mrakia sp. did not grow at 18 and 30 1C and behaved as
obligate psychrophiles (group A). The strains belonging to
the species R. laryngis, C. gilvescens, and A. pullulans grew at
18 and 30 1C and were grouped as facultative psychrophiles
(group B) (Table 1). All the strains from temperate habitats
(group C) were successfully cultured at both 18 and 30 1C,
but only S. cerevisiae L12, S. cerevisiae ATCC 2345, and K.
marxianus L3 grew at 4 1C (Table 1).
The specific growth rate (m) and the biomass/substrate
yield (YX/S) of all the yeasts were determined at the different
growth temperatures (Table 1). Both obligate and facultative
psychrophilic yeasts (groups A and B) grew at 4 1C with a
mean m of 0.039 h1. This value was significantly higher than
that exhibited by the three mesophiles that grew at this
temperature (0.028 h1) (Table 1). When the yeasts of
groups B and C were cultured at 18 and 30 1C, their mean
m significantly increased as the temperature was increased.
At the same time, the strains belonging to group B grown
both at 18 and 30 1C showed a mean m significantly lower
than that exhibited by group C under the same conditions.
The conversion of glucose into biomass (YX/S) was always
significantly higher in psychrophilic (groups A and B) than
in mesophilic (group C) yeasts. In both groups B and C, the
growth temperature did not affect the YX/S (Table 1).
Lipid yield and composition

The lipid/biomass yield (YL/X) was determined at the
stationary phase at the different temperatures (Table 1). At
4 1C, both psychrophilic yeast groups (A and B) exhibited a
mean YL/X significantly higher than that showed by the three
mesophilic strains growing at the same temperature. Yeasts
of group B displayed no significant differences in the mean
YL/X at 4, 18, and 30 1C. In contrast, the YL/X increased in
strains belonging to group C from 4 to 18 1C and 30 1C
(Table 1).
The relative composition of FAs was determined in the
lipid fraction of biomass at the stationary phase (Table 2).
Only linear FAs were found in all the yeasts, independent of
the growth temperatures. Saturated and unsaturated FA

c
366
M. Rossi et al.
Table 1. Specific growth rate (m), biomass/substrate yield (YX/S), and lipid/biomass yield (YL/X) of obligate psychrophilic (group A), facultative
psychrophilic (group B), and mesophilic (group C) yeasts at 4, 18, and 30 1C
m (h1)
Strain
Group A
Leucosporidium sp. DBVPG 4753
Rhodotorula creatinivora DBVPG 4794
Mrakia sp. DBVPG 4775
Rhodotorula glacialis DBVPG 4806
Meanz
Group B
Rhodotorula laryngis DBVPG 4765
Rhodotorula laryngis DBVPG 4772
Aureobasidium pullulans DBVPG 4778
Cryptococcus gilvescens DBVPG 4714
Meanz
Group C
Saccharomyces cerevisiae L 12
Saccharomyces cerevisiae ATCC 2345
Saccharomyces exiguus L 10
Klyveromyces marxianus L 3
Pichia farinosa DBVPG 3626
Zigosaccharomyces rouxii DBVPG 6399
Meanz
YX/Sw
YL/Xw
4 1C
18 1C
30 1C
4 1C
18 1C
30 1C
4 1C
18 1C
30 1C
0.022
0.032
0.035
0.039
0.035
0.033
0.054
0.036
0.038
0.051
0.053
0.046
0.036
0.039b
0.30
0.34
0.33
0.37
0.32
0.33
0.34
0.32
0.33
0.29
0.45
0.38
0.44
0.35b
0.28
0.58
0.46
0.25
0.17
0.34
0.34
0.19
0.13
0.42
0.21
0.17
0.08
0.28b
0.037
0.025
0.045
0.032
0.037
0.046
0.053
0.039b
0.060
0.035
0.064
0.111
0.116
0.087
0.077
0.080az
0.102
0.128
0.109
0.106
0.118
0.087
0.082
0.105ak
0.49
0.47
0.27
0.23
0.29
0.36
0.35
0.35b
0.44
0.43
0.28
0.24
0.29
0.22
0.27
0.31b
0.45
0.49
0.33
0.25
0.30
0.28
0.34
0.35b
0.07
0.28
0.28
0.33
0.21
0.27
0.26
0.25b
0.09
0.34
0.12
0.27
0.27
0.24
0.36
0.24a
0.05
0.04
0.08
0.34
0.30
0.25
0.26
0.19a
0.026
0.027
0.031
0.028a
0.140
0.151
0.147
0.175
0.171
0.099
0.147bz
0.249
0.267
0.323
0.233
0.305
0.192
0.262bk
0.08
0.07
0.25
0.13a
0.13
0.10
0.07
0.20
0.19
0.18
0.15a
0.17
0.11
0.07
0.26
0.18
0.11
0.15a
0.14
0.21
0.05
0.13a
0.44
0.20
0.72
0.21
0.57
0.43
0.43bk
0.21
0.34
0.51
0.12
0.29
0.31
0.30az
Mean values from three experiments; within each strain and condition SD always o 0.005 h1.
w
Mean values from three experiments; within each strain and condition SD always o 0.05.
Means in a column without a common letter differ (P o 0.05). Means in a row without a common symbol differ (P o 0.05).
with chain length ranging from 14 to 18 carbons always

accounted for 4 97%. C14 FA never exceeded 5.3%. The
relative amount of unsaturated FA other than palmitoleic
(C16:1, D9), oleic (C18:1, D9), linoleic (C18:2, D9,12), and
a-linolenic (C18:3, D9,12,15) was negligible.
Significant (P 4 0.05) differences were observed in the
unsaturated FA composition among groups A, B, and C at
4 1C (Table 2). At this temperature, the relative amount of
C18:1 and C18:2 in psychrophilic strains (groups A and B)
was significantly higher than that observed in the yeasts of
group C. Besides, the relative composition of C18:2 significantly differed between groups A and B (14.9% and 24.5%,
respectively). Moreover, obligate psychrophilic strains
(group A) exhibited a significantly (P 4 0.05) higher concentration of C18:3 (16.8%) than those observed in the

c
other two groups (B and C). Among the yeasts belonging to

group C, only K. marxianus L3 produced C18:2 or C18:3 at
4 1C (Table 2). On the other hand, at 4 1C, strains belonging
to group C showed a significantly (P 4 0.05) higher relative
percentage of C16:1 (39.7%) than those observed in yeasts of
groups A and B (2.3% and 0.4%, respectively). No clear
tendency was observed in terms of the relative concentration
of saturated FA (C14, C16, and C18) among the three
groups (Table 2).
Strain-to-strain differences in the FA composition did not
allow distinguishing any common trend as a function of
growth temperature within groups B and C. In group B, the
sole statistically significant difference was the content of
C18:1, which was higher at 18 and 30 1C (58.4% and 57.6%,
respectively) than at 4 1C (41.7%). Likewise, a similar

c
0.0
0.9
0.0
1.0
1.1
0.6
0.0
0.5az
3.1
2.4
3.6
1.1
0.7
1.7
2.1bz
0.8
0.9
0.0
0.9
2.2
0.7
0.0
0.8az
3.3
3.0
0.5
2.3bz
1.6
1.2
4.3
1.8
0.9
1.2
1.8bz
1.6
0.0
0.9
0.2
0.0
0.0
0.3
0.4az
17.5
14.8
23.0
18.4az
26.0
22.6
22.5
26.3
26.2
21.0
17.1
23.1bz
18.5
17.1
25.5
15.7
19.0
20.5
16.9
18.3
23.6
22.1
14.5
13.9
25.5
19.3a
18 1C 30 1C 4 1C
1.6
1.0
2.6
0.3
0.6
0.0
0.2
0.7
0.0
5.3
4.8
2.2
0.3
1.5ab
4 1C
C16
24.1
21.0
28.9
25.2
20.4
16.9
22.8a
23.3
29.1
14.2
19.0
18.5
18.0
20.7
20.4az
18 1C
21.0
21.7
20.3
26.9
23.5
17.5
21.8a
20.4
26.3
26.1
16.4
18.5
16.2
14.9
19.8az
48.1
50.4
20.5
39.7c
1.8
0.7
0.0
0.2
0.0
0.4
0.0
0.4az
3.0
2.2
3.0
3.5
2.2
1.2
2.9
3.2
2.7
2.5
1.4
0.7
1.8
2.3b
30 1C 4 1C
C16:1
0.0
1.9
0.9
0.4
0.0
0.0
0.0
0.5az
31.1
32.2
21.9
11.2
2.6
12.1
18.5bz
11.9
13.4
15.7
8.2
8.3
11.2
11.5bz
C18:1
4.6
5.5
3.9
4.5
4.4
3.4
4.4
4.4az
3.9
16.2
14.1
16.8
8.5
11.5
11.8bz
26.0
27.1
24.8
25.9az
39.4
41.6
57.9
23.9
22.7
37.3
69.0
41.7bz
58.3
60.5
52.5
28.7
23.7
45.4
36.5
27.4
57.9
45.4
39.4
15.5
43.4
41.1b
30 1C 4 1C
18 1C
1.0
1.2
2.1
0.0
7.9
1.4
2.4
4.1
1.7
0.3
15.1
11.4
0.0
19.1
11.2
0.0
8.9
11.5
0.3
2.8
1.8
0.6az
8.4bz 5.9az
0.3
0.6
1.9
4.7
9.0
3.3
1.0
6.8
1.6
1.4
2.6
5.4
10.8
3.8a
4 1C
C18
40.3
5.1
28.6
4.7
35.4
9.0
5.6
2.5
9.9
21.0bz 5.1abz
18 1C 30 1C
Group A
R. creatinivora DBVPG 4794
R. glacialis DBVPG 4806
Meanw
Group B
R. laryngis DBVPG 4765
A. pullulans DBVPG 4778
C. gilvescens DBVPG 4714
Meanw
Group C
S. cerevisiae L 12
S. cerevisiae ATCC 2345
S. exiguus L 10
K. marxianus L 3
P. farinosa DBVPG 3626
Z. rouxii DBVPG 6399
Meanw
Strain
C14
25.2
28.6
23.2
29.6
52.2
38.8
32.9az
45.8
44.8
61.1
64.4
55.9
65.0
72.1
58.4b
18 1C
32.2
31.0
22.1
29.8
53.9
52.3
36.9a
51.1
41.4
51.5
63.3
68.7
61.1
65.9
57.6b
30 1C
16.6
5.5az
0.0
0.0
30.8
23.3
13.0
32.6
29.5
31.4
11.1
24.5cz
12.9
12.1
3.4
17.8
15.7
14.9
17.3
16.7
10.9
11.5
21.2
24.6
14.2
14.9b
4 1C
C18:2
21.3
26.8
15.1
15.2
8.5
19.3
14.1
17.2bz
0.0
2.8
2.4
0.0
0.0
0.0
0.0
0.7az
4.9
6.5
11.0
29.1
29.2
14.7
25.2
26.6
3.4
11.1
15.4
37.3
4.1
16.8b
30 1C 4 1C
0.0
0.0
0.0
0.0
0.0
0.0
4.5
2.3
14.9
8.3
9.1
15.6
10.4
18.7
6.7
9.0a 4.6az 3.0az
25.8
21.8
22.2
3.0
12.9
4.5
5.4
13.7az
18 1C
C18:3
0.0
0.0
0.0
8.7
0.0
0.0
1.5az
3.0
0.0
0.0
0.0
0.0
0.0
0.0
0.4az
0.0
0.0
0.0
6.2
0.0
0.0
1.0az
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0az
18 1C 30 1C
Table 2. Relative composition (%) of fatty acids in the lipid extracts of obligate psychrophilic (group A), facultative psychrophilic (group B), and mesophilic (group C) yeasts at stationary phase at 4, 18,
and 30 1C
367

368
behavior was observed for strains of group C, which

exhibited an increase of both C18 and C18:1 from 4 1C to
18 and 30 1C. These same strains exhibited an increase of
C16 and a simultaneous decrease of C16:1 when the growth
temperature increased from 4 to 18 1C and 30 1C (Table 2).
In order to aid comparison, information about unsaturation was summarized as the UI (Table 3). The different
growth temperatures at 4, 18, and 30 1C did not significantly
affect the UI of strains belonging to groups B and C.
Obligate psychrophilic yeasts (groups A) exhibited a significantly (P 4 0.05) higher UI at 4 1C than those of groups
B and C. By comparing the data reported in Table 3 with
those reported in Table 2, it could be observed that the
different UI between groups A and B was mainly due to the
higher amount of C18:3. On the other hand, the lower UI of
yeasts belonging to group C was mainly due to the general
lower content of unsaturated C18:1, C18:2, and C18:3,
which was only partially balanced by a higher content of
C16:1. On the whole, the major contribution to the UI
provided by strains of group C was due to C16:1 and C18:1,
whereas in psychrophilic yeasts (groups A and B) the main
contribution was due to C18:1, C18:2, and C18:3 (Table 2).
The mean relative compositions of saturated, monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids
(PUFAs), total C16 series (C161C16:1), and C18 series
(C181C18:11C18:21C18:3) for each group grown at the
diverse temperatures are reported in Table 3. At 4 1C, both
psychrophilic yeast groups (A and B) exhibited a significantly higher concentration of PUFAs than those observed
in group C (P 4 0.05). In contrast, under the same conditions, the percentage of MUFAs of group C was significantly
higher than those of both groups A and B. Interestingly,
facultative psychrophilic strains (group B) showed a simultaneous decrease of PUFAs and an increase of MUFAs from
4 to 18 1C and 30 1C. No similar trend of MUFAs and PUFAs
as a function of the growth temperature was observed for
strains belonging to group C, instead (Table 3).
Yeasts belonging to group C always displayed an average
length of the FA chain lower than that found in psychrophilic
ones. At 4 1C, both groups A and B exhibited a significantly
higher concentration of C18 than those observed in group C
(P 4 0.05). On the other hand, under the same conditions,
strains of group C showed a higher percentage of C16 than
those observed in both groups A and B (Table 3). No
differences in the content of C16 and C18 were observed in
group B at 4, 18, and 30 1C. In contrast, the strains of group
C showed a simultaneous decrease of C18 and an increase of
C16 from 4 to 18 and 30 1C.
Discussion
Microbial growth is the net result of a large number of
enzymatic reactions, each affected by temperature because

c
M. Rossi et al.
any decrease in temperature exponentially affects the rate of

biochemical reactions (Price & Sowers, 2004; DAmico et al.,
2006). As expected, the acclimation of facultative psychrophilic and mesophilic yeasts to a lower temperature affected
their specific growth rates. The mean m of both facultative
psychrophiles and mesophiles was positively related to the
temperature, but, unexpectedly, the growth temperature did
not influence the biomass/substrate yield (YX/S) of both the
groups. Obligate and facultative psychrophiles exhibited a
higher efficiency of nutrient conversion into biomass (under
form of YX/S) than mesophiles. Significant differences in
YX/S between cold-adapted yeasts and those from temperate
habitats were observed, likely resulting from adaptive responses. The dramatic gains in ATP at low and subzero
temperatures observed in psycrophilic microorganisms by
Napolitano & Shain (2005) apparently corroborate the
above evidences.
The ability to accumulate high amounts of neutral
storage lipids was reported for a few mesophilic yeasts (Gill
et al., 1977; Evans & Ratledge, 1983a, b; Granger et al., 1993;
Ratledge, 2002). The major neutral lipids are triacyl-glycerol
and steryl-esters, which are uncharged and thus unsuitable
as components of membrane lipid bilayers. Therefore, they
accumulate in hydrophobic lipid particles and serve rather
as storage of the building blocks for membrane lipids
synthesis than as an energy reserve (Wynn et al., 2001;
Wagner & Daum, 2005; Czabany et al., 2007; Daum et al.,
2007). The lipid/protein ratio usually increases markedly at
a low temperature in many eukaryotes and prokaryotes, due
to the relative increase in lipid biosynthesis (Guschina &
Harwood, 2006). Our results indicate that the acclimation of
facultative psychrophilic yeasts at 30, 18, or 4 1C did not
cause any significant increase in the lipid yield (YL/X).
Moreover, similar YL/X were found in facultative and obligate psychrophiles at 4 1C, suggesting that lipid accumulation should not be regarded as a distinctive characteristic of
adaptation to permanently cold environments.
The effects of a low temperature on the stiffening of
membrane bilayers are detrimental for both eukaryotic and
prokaryotic organisms. The need to acclimate and/or adapt
their physiology to cold conditions is generally governed by
the need to maintain membrane functionality (Los &
Murata, 2004; Morgan-Kiss et al., 2006). To regulate membrane fluidity and functionality, facultative and obligate
psychrophilic organisms exploit diverse changes in lipid
composition, consisting in incorporation of unsaturated,
short-chain, branched, or cyclic FA (White et al., 2000;
Chintalapati et al., 2004; Feller, 2007). The role of unsaturation of membrane lipids is one of the most thoroughly
investigated mediators of cold acclimation and/or adaptation (Russell, 1997; Guschina & Harwood, 2006; MorganKiss et al., 2006). Specifically, the presence of unsaturated
and PUFAs is apparently correlated with the ability of

c
1.06
0.90
1.06
0.71
0.82
0.74
0.83
0.87a
0.59
0.62
0.55
0.98
0.86
0.89
0.75a
1.03
0.97
0.91
0.90
0.82
1.01
0.91
0.94a
0.74
0.77
1.06
0.86az
18 1C
1.02
1.06
0.95
1.55
1.46
1.21
1.50
1.44
0.92
1.05
1.30
1.78
0.86
1.24b
4 1C
0.73
0.60
0.63
0.75
0.77
0.76
0.71az
0.95
0.95
0.84
0.94
0.86
1.00
0.94
0.93b
30 1C
25.9
22.5
29.1
25.8ab
28.0
31.4
26.6
42.3
47.5
30.6
19.9
32.3bz
20.4
18.7
30
20.7
28.6
23.8
18.1
25.8
25.2
28.8
21.9
21.5
36.6
24.6a
4 1C
39.1
36.8
48.2
34.5
29.4
29.8
36.3bz
25.4
31.4
15.9
31.4
30.8
30.1
22.5
26.8a
18 1C
Saturatedw
26.5
39.1
38.7
45.5
32.9
30.2
35.5bz
26.5
31.8
30.9
21.2
22.9
19.5
19.6
24.6a
30 1C
74.1
77.5
45.3
65.6bz
41.2
42.3
57.9
24.1
22.7
37.7
69.0
42.1a
61.3
62.7
55.5
32.2
25.9
46.6
39.4
30.6
60.6
47.9
40.8
16.2
45.2
43.5a
4 1C
MUFAsw
56.3
60.8
45.1
40.8
54.8
50.9
51.5a
45.8
46.7
62.0
64.8
55.9
65.0
72.1
58.9az
18 1C
72.5
59.6
57.5
38.8
56.4
62.2
57.8az
52.2
41.4
54.0
63.6
68.7
61.1
66.2
58.2az
30 1C
25.7
8.6b
0.0
0.0
30.8
26.1
15.4
32.6
29.5
31.4
11.1
25.3az
17.8
18.6
14.4
46.9
44.9
29.6
42.5
43.3
14.3
22.6
36.6
61.9
18.3
31.7a
4 1C
PUFAsw
Mean values from three experiments; within each strain and condition SD always o 2.
Group A
R. creatinivora DBVPG 4794
Meanz
Group B
A. pullulans DBVPG 4778
Meanz
Group C
S. cerevisiae L 12
S. cerevisiae ATCC 2345
S. exiguus L 10
K. marxianus L 3
P. farinosa DBVPG 3626
Z. rouxii DBVPG 6399
Meanz
Strain
UI
0.0
0.0
4.5
23.6
15.6
18.7
10.4a
28.8
21.8
22.2
3.0
12.9
4.5
5.4
14.1a
18 1C
0.0
0.0
2.3
14.5
10.4
6.7
5.7a
21.3
26.8
15.1
15.2
8.5
19.3
14.1
17.2b
30 1C
65.7
65.2
43.5
58.1bz
27.8
23.3
22.5
26.5
26.2
21.4
17.1
23.5a
21.5
19.3
28.5
19.2
21.2
21.7
19.8
21.5
26.3
24.6
15.9
14.6
27.3
21.6a
4 1C
55.2
53.2
50.8
36.4
23.0
29.0
41.3b
23.3
31.0
15.1
19.4
18.5
18.0
20.7
20.9a
18 1C
Total C16w
61.3
50.3
55.7
35.9
26.0
27.4
42.8b
21.4
26.3
28.5
16.7
18.5
16.2
15.3
20.4a
30 1C
31.0
31.8
56.1
39.6a
71.4
75.6
77.4
71.6
71.3
77.6
82.9
75.4b
76.4
79.7
68.8
80.3
77.6
78.3
80
77.5
73.8
69.4
78.6
82.8
72.5
76.6b
4 1C
37.1
42.0
43.4
61.4
76.1
68.7
54.8az
76.7
68.0
85.0
78.8
80.0
81.0
79.3
78.4b
18 1C
Total C18w
36.1
47.2
38.5
61.1
72.8
70.5
54.4az
77.0
73.7
70.6
83.1
81.5
83.8
84.4
79.2b
30 1C
Table 3. UI and relative composition (%) of saturated, MUFAs, PUFAs, total C16, and total C18 fatty acids in the lipid extracts of obligate psychrophilic (group A), facultative psychrophilic (group B), and
mesophilic (group C) yeasts at 4, 18, and 30 1C
369

370
growing at a low temperature. Differences in both the FA

composition and the degree of unsaturation between psychrophilic and mesophilic yeasts have been explored
(McMurrough & Rose, 1973; Arthur & Watson, 1976;
Watson & Arthur, 1976; Watson, 1978; Watson et al., 1978),
but an extensive comparison among numerous strains of
mesophilic and psychrophilic species has not been accomplished so far.
Analysis of the lipid composition of yeast strains revealed
that the sum of saturated and unsaturated C14 to C18 FA
always accounted for 4 98%. Psychrophilic yeasts differed
from the mesophilic ones for their response to low temperatures. In the first case, a significant increase of PUFAs was
observed when the temperature decreased to 4 1C, coupled
with a parallel decrease of the MUFAs. In particular, this was
the result of significantly higher amounts of linoleic (C18:2,
D9,12) and a-linolenic (C18:3, D9,12,15) acids. This behavior
was different from that observed in mesophilic strains,
which exhibited both a significant increase of MUFAs and a
decrease of PUFAs at 4 1C. In this case, this was the result of
an increased concentration of palmitoleic acid (C16:1, D9).
This dichotomy may suggest that different desaturases
(specific for different yeast strains and acting on FA characterized by different length chains) could be one of the
acclimation mechanisms acting to restore membrane bilayer
lipid fluidity at low temperatures in either psychrophilic or
mesophilic yeasts.
The highest UI, which was observed in obligate psychrophiles, was mainly due to the highest amounts of a-linolenic
acid (C18:3, D9,12,15), at the expense of linoleic acid (C18:2,
D9,12). At 4 1C, a-linolenic acid was found in all the obligate
psychrophilic strains, accounting for up to 37% of the total
FAs, whereas it was about 3.0% or even absent in the
facultative ones. The remarkable amount of a-linolenic acid
observed in obligate psychrophiles evokes an adaptive
feature, which is probably due to the high activity of a D15
desaturase. a-Linolenic acid was generally absent in mesophilic yeasts, with the sole exception of K. marxianus L3.
Unexpectedly, facultative psychrophilic strains exhibited
no significant increase of the UI at a lower temperature, in
disagreement with previous works (McMurrough & Rose,
1973; Arthur & Watson, 1976; Watson & Arthur, 1976;
Watson, 1978; Watson et al., 1978). This result raises the
question of whether the increased unsaturation is really a
general acclimatory response of yeasts to low temperature.
Based on the results reported herein, the extent of unsaturation of FA could be considered one major adaptive feature
exclusive of obligate psychrophilic yeasts.
A few recent studies reported that the response of diverse
organisms to low temperatures includes the shortening of
the FA chain (Chintalapati et al., 2004; Guschina & Harwood, 2006; Bahrndorff et al., 2007). The results reported
herein confirm this trend only for mesophilic yeasts, which

c
M. Rossi et al.
exhibited an increased concentration of C16:1 at the expense

of C18:1 when cultured at 4 1C. On the contrary, the
production of short-chain FA as a cold adaptation and/or
an acclimation response in psychrophilic yeasts can be
apparently excluded. The production of FA with the C18
chain length (in particular C18:1, C18:2, and C18:3) seems
to be an adaptive feature of the yeasts from cold environments and may occur because elongation beyond C16 is
necessary to introduce additional double bonds by D12 and
D15 desaturases. The acclimation of the obligate psychrophilic strain R. glacialis DBVPG 4785 (selected as a representative strain) to different low temperatures is currently
being investigated in greater detail (data not shown). This
strain was capable of growing in the range between 3 and
15 1C with approximately the same YXS and YLX in the whole
temperature range. As the temperature decreased, a progressive increase of both UI and FA with the C18 chain
length was confirmed, mainly due to the increasing abundance of a-linolenic acid.
The present work improves information about the physiology of both facultative and obligate psychrophilic yeasts
and may be useful for potential biotechnological application
of these strains. Moreover, it sheds light on some physiological and ecological features, which are related to the growth
temperature of psychrophiles, and suggests that obligate and
facultative psychrophilic yeasts presumably use different
metabolic strategies for adapting their life to different
thermal conditions. Even if there is a continuum in temperature adaptation for life with wide or narrow growth
temperature ranges, the results reported herein confirm that
classification based on temperature limits was in good
agreement with differences in the FA composition. The
production of high amounts of PUFAs by obligate psychrophilic yeasts (in particular a-linolenic) correlated with
adaptation to strictly cold environments. Over a time scale
of many generations, the life of obligate psychrophilic yeasts
was likely restricted to permanently cold environments,
whereas facultative psychrophilic yeasts may have evolved
in habitats subjected to wider temperature ranges.
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RESEARCH ARTICLE

Growth, lipid accumulation, and fatty acid composition in obligate

Correspondence: Maddalena Rossi,

Editor: Max Haggblom

valuable biotechnological purposes (Margesin & Schinner,

Materials and methods

All the strains were aerobically cultured in flasks containing

Biomass from 50-mL culture samples was harvested (3000 g,

2009 Federation of European Microbiological Societies

FEMS Microbiol Ecol 69 (2009) 363372

Adaptation and acclimation of psychrophilic yeasts

nificant at P  0.05. Statistical analysis was performed using

Lipid yield and composition

with chain length ranging from 14 to 18 carbons always

other two groups (B and C). Among the yeasts belonging to

FEMS Microbiol Ecol 69 (2009) 363372

Adaptation and acclimation of psychrophilic yeasts

2009 Federation of European Microbiological Societies

behavior was observed for strains of group C, which

any decrease in temperature exponentially affects the rate of

FEMS Microbiol Ecol 69 (2009) 363372

Adaptation and acclimation of psychrophilic yeasts

2009 Federation of European Microbiological Societies

growing at a low temperature. Differences in both the FA

exhibited an increased concentration of C16:1 at the expense

FEMS Microbiol Ecol 69 (2009) 363372

Adaptation and acclimation of psychrophilic yeasts

Megaphorura arctica Tullberg 1876 (Onychiuridae:

FEMS Microbiol Ecol 69 (2009) 363372

Margesin R & Schinner F (eds) (1999a) Biotechnological

2009 Federation of European Microbiological Societies

Stukey JE, McDonough VM & Martin CE (1989) Isolation and

2009 Federation of European Microbiological Societies

psychrophilic and thermotolerant yeasts. Biochem Soc T 6:

FEMS Microbiol Ecol 69 (2009) 363372

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nificant at P 0.05. Statistical analysis was performed using