FEMS Microbiology Letters 204 (2001) 375^379
www.fems-microbiology.org
Physiological di¡erence during ethanol fermentation
between calcium alginate-immobilized
Candida tropicalis and Saccharomyces cerevisiae
Latifa Jamai a , Khalid Sendide a , Khalil Ettayebi a , Faouzi Errachidi a ,
Omar Hamdouni-Alami a , Mohamed Ali Tahri-Jouti a , Timothy McDermott b ,
Mohamed Ettayebi a;b; *
b
a
Biotechnology Units, University Sidi Mohamed Ben Abdallah, P.O. Box 1796 Atlas, Fes, Morocco
Thermal Biology Institute, Montana State University, P.O. Box 173142, Bozeman, MT 59717-3142 USA
Received 2 July 2001; received in revised form 4 September 2001 ; accepted 7 September 2001
First published online 23 October 2001
Abstract
Calcium alginate-immobilized Candida tropicalis and Saccharomyces cerevisiae are compared for glucose fermentation. Immobilized
C. tropicalis cells showed a slight morphological alteration during ethanol production at 40³C, but their fermentation capacity was reduced
by 25%. Under immobilization conditions, the two species demonstrated two different mathematical patterns when the relationship between
growth rate, respiration rate, and ethanol tolerance was assessed. The interspecific difference in behavior of immobilized yeast cells is mainly
due to their natural metabolic preference. The production of CO2 by calcium alginate-immobilized C. tropicalis, as well as the lower supply
of oxygen to the cells, are the major factors that reduce ethanol production. ß 2001 Federation of European Microbiological Societies.
Published by Elsevier Science B.V. All rights reserved.
Keywords : Calcium alginate immobilization ; Ethanol production ; Thermotolerance ; Candida tropicalis; Saccharomyces cerevisiae
1. Introduction
Candida tropicalis, an asporogenous diploid yeast, has
long been neglected for its valuable potential in energyrelated biotechnology. Unlike most other yeast species, a
very large variety of carbon sources can be utilized by
C. tropicalis including many sugars, disaccharides, phenols,
alkanes, alkane derivatives, and fatty acids [1,2]. C. tropicalis grows on each compound as a sole carbon and energy source, generating high biomass yields since its metabolism becomes purely oxidative when enough oxygen is
supplied in the culture medium. While a special interest
was given to the induction of peroxisomal enzymes of
C. tropicalis grown on fatty acids and alkanes [2,3], a considerable e¡ort is still to be made to optimize the production of ethanol by C. tropicalis from soluble starch, cellu-
* Corresponding author. Tel. : +1 (406) 994 5549;
Fax: +1 (406) 994 1848.
E-mail address : [email protected] (M. Ettayebi).
lose and wood-derived hemicellulose hydrolysates [4].
Candida species are considered strong candidates for thermotolerance and ethanol tolerance needed to produce
ethanol from lignocellulosic biomass [5].
The immobilization of living cells facilitates the separation of cells from media as well as their recirculation.
Immobilized yeast-cell technology has found various applications, particularly in ethanol production [6^8]. Immobilized Saccharomyces cerevisiae cells displayed an increase
in ethanol productivity compared to free cells [9]. However, Senac and Hahn-Hagerdal found no improvement of
the productivity after cell immobilization [10]. On the other hand, immobilization of C. tropicalis in agarose lowered
ethanol yield and productivity, indicating that the physiology of immobilized cells di¡er from that of free cells
[11]. The di¡erence in behavior between immobilized and
free cells is related to several factors. Nutrient limitations
and microenvironment surrounding the cells are widely
used to explain physiological and morphological changes
of cells after immobilization [12].
In the present paper, we compare ethanol production,
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 4 3 0 - X
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by free and immobilized C. tropicalis and S. cerevisiae
cells, by integrating aerobic respiration, immobilization,
growth rate, and ethanol tolerance during glucose fermentation. Thermotolerance of C. tropicalis is also discussed.
for observation with scanning electron microscope according to a standard procedure described by Van Neerven et
al. [15].
2. Materials and methods
Glucose concentrations were assayed using Miller's
method [16]. Ethanol was determined by gas chromatography (PYE UMICAM PU 4500). Oxygen was measured
polarographically with a biological oxygen monitor YSI
model 5300 (Varian model 9176). CO2 was measured following a previously described method [17].
2.1. Yeast strains
C. tropicalis YMEC14 and S. cerevisiae YMES2 used in
this study were isolated from Moroccan olive-mill wastewater and traditional bread dough respectively. The species were identi¢ed following classical and molecular techniques [13,14]. C. tropicalis Y1552 and S. cerevisiae 41278
were provided by C.P. Kurtzman (US Department of
Agriculture, Peoria, IL, USA) and S. Vissers (ULB, Belgium) respectively. Yeast strains were routinely grown on
in YPG medium containing, in g l31 , yeast extract 5, bacterial peptone 10 and glucose 5 (Difco, Detroit, MI, USA).
2.2. Cell growth and immobilization
Yeast strains were cultured at the appropriate temperature in Bio£o 2000 fermentor (New Brunswick Scienti¢c,
NJ, USA) with a 1.3-l working volume of oxidative YPG
broth containing 2% glucose. Agitation and oxygen £ow
were maintained at 450 rpm and 0.6 l min31 respectively.
pH was maintained at 4.5 by automatic addition of 4 N
NaOH. At the end of the exponential growth phase, yeast
cells were harvested by centrifugation at 2000Ug for 10
min and washed twice with 0.9% NaCl (w/v). 8 g fresh
wet-weight cells were mixed with 1.6% calcium alginate
and added dropwise with a syringe into a sterile solution
of 0.1 M CaCl2 . The droplets gelled in contact with Ca2‡
cations forming beads which were left to harden for 15
min at 37³C, then washed three times with 0.9% NaCl
before storing them in saline solution at 4³C. The ethanol
fermentation, by free and immobilized cells, was conducted under the same conditions in 250 ml of oxidative
YPG medium (15% glucose) except for the temperature,
which was 30³C for S. cerevisiae and 40³C for C. tropicalis. Free cells were prepared under the same conditions.
2.3. Scanning electron microscopy
Immobilized and free yeast cells were ¢xed by 1% glutaraldehyde in cocadylic bu¡er, dehydrated and prepared
2.4. Analytical methods
3. Results
The choice of C. tropicalis YMEC14 and S. cerevisiae
YMES2 used in this study were achieved according to the
performances they displayed in growth rate and ethanol
production compared to yeast strains from our collection.
Their taxonomic identity was demonstrated by classical
methods and con¢rmed by tRNA pro¢le analysis [13,14].
Ethanol production rate is identical in free and immobilized S. cerevisiae YMES2. However, immobilized S. cerevisiae 41278 demonstrated a small increase in ethanol
productivity compared to suspended cells. On the other
hand, ethanol production of immobilized C. tropicalis cells
was 8^20% lower than that of S. cerevisiae (Table 1). The
immobilization of C. tropicalis YMEC14 and Y1552
caused a decrease in ethanol production of 25 and 27%
respectively.
Under aerobic conditions, growth rates of free and immobilized C. tropicalis strains in YPG broth, supplemented with di¡erent concentrations of ethanol, were stable between 0 and 0.85 M ethanol. Higher ethanol
concentrations decreased their growth rate in a linear
manner (Fig. 1). Both strains of S. cerevisiae are inhibited
at lower ethanol concentrations; the inhibition in this case
followed an exponential pattern. Furthermore, S. cerevisiae YMES2 and 41278 demonstrated intraspecies di¡erences. In fact, in both conditions of free and immobilized
cells, S. cerevisiae YMES2 showed more resistance to
ethanol than S. cerevisiae 41278. In addition, the growth
rate of suspended cells is lower than immobilized cells in
both strains when ethanol concentrations are higher than
0.42 M. The phenomenon is reversed in C. tropicalis.
To check whether these di¡erences were due to yeast
respiration tendency, respiration rates were measured.
Table 1
Bioconversion of glucose to ethanol by free (F) and immobilized (I) C. tropicalis (1) YMEC14, (2) Y1552, and S. cerevisiae (3) YMES2, (4) 41278 yeast
cells
Ethanol yield (% theoretical)
Ethanol productivity (g l31 h31 )
1I
1F
2I
2F
3I
3F
4I
4F
70
3.58
70
4.47
64.7
3.31
66
4.21
78.3
5.06
78.3
5.06
86
5.5
80
5.16
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377
Similar to the growth rate, the respiration rate was inhibited by exogenous ethanol, following two di¡erent mathematical models in the two yeast species (Fig. 2). Furthermore, the release of CO2 showed more important variation
in C. tropicalis than in S. cerevisiae strains. The release of
CO2 decreased by a factor of 0.5, but that of S. cerevisiae
was slightly a¡ected (Fig. 3).
Finally, scanning electron microscopy showed limited
alterations (less than 5%) of C. tropicalis cells due to immobilization, while S. cerevisiae remained intact (Fig. 4).
4. Discussion
The ethanol concentration at the end of the fermentation cycle is similar for both free and immobilized C. tropicalis cells, but the time required to reach these values is
longer under immobilized conditions. While free and immobilized S. cerevisiae achieve complete fermentation at
the same time, immobilized C. tropicalis required 2^3 h
more than free C. tropicalis to complete the fermentation
process. This decrease in ethanol production of C. tropicalis-immobilized cells could be related to the fact that
these cells have a less rigid cell wall. It has been reported
that cell immobilization causes biochemical changes of
wall polysaccharides and glycoproteins [7]. It is possible
that these changes a¡ect C. tropicalis more than S. cerevisiae. The fact that negligible morphological changes were
detected by electron microscopy lets us suppose that metabolic alterations have more signi¢cant e¡ect on ethanol
fermentation.
The inhibitory e¡ect of exogenous ethanol on respiration and growth rates of free or immobilized C. tropicalis
and S. cerevisiae di¡ers according to whether the respira-
Fig. 1. E¡ect of ethanol concentration on the growth rate of free (F)
and immobilized (I) C. tropicalis and S. cerevisiae cells. (1) YMEC14;
(2) Y1552; (3) YMES2; (4) 41278.
Fig. 2. E¡ect of ethanol concentration on the respiration rate of free
(F) and immobilized (I) C. tropicalis and S. cerevisiae cells. (1)
YMEC14; (2) Y1552; (3) YMES2; (4) 41278.
tion is occurring or not (Fig. 1). The relationship between
growth rate and ethanol concentration follows an exponential pattern in both strains of S. cerevisiae. The relationship is linear in immobilized and free C. tropicalis
strains. We conclude that under the same culture conditions, S. cerevisiae utilizes fermentation, and thus is not
a¡ected by ethanol as much as C. tropicalis, which utilizes
respiration. In S. cerevisiae, the glucose causes a catabolic
repression inhibiting respiratory enzymes and inducing fermentation. In the strain 41278, the Crabtree e¡ect seems
more important, which explains the slight increase in ethanol productivity after immobilization.
The situation is di¡erent in C. tropicalis, which has a
lower glycolytic £ux on one hand and higher oxygen consumption in media containing glucose on the other hand.
Under immobilization conditions, calcium alginate matrix
creates a barrier to the oxygen needed by C. tropicalis,
reducing its ethanol productivity [18]. Moreover, oxygen
a¡ects the permeability and composition of the cell mem-
Fig. 3. CO2 production by free (F) and immobilized (I) C. tropicalis
and S. cerevisiae cells. (1) YMEC14 ; (2) Y1552; (3) YMES2; (4) 41278.
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in a continuous fermentation system. We found that immobilized C. tropicalis cells remained physiologically stable after 20 cycles of fermentation (data not shown).
Because of its thermotolerance, C. tropicalis is well
suited for simultaneous sacchari¢cation and fermentation
of hemicellulose that can be achieved at a temperature
between 42 and 45³C. This solves the problems encountered by S. cerevisiae fermentation at temperatures higher
than 30³C. Moreover, C. tropicalis has the ability to tolerate concentrations of lignin-like polyphenols that are
highly toxic to other yeast species (our unpublished results). S. cerevisiae requires exogenous phenol oxidase
genes to enhance its resistance to fermentation phenolic
inhibitors [20]. C. tropicalis is known by its vigorous
growth on various carbon sources, which makes it a
good candidate for industrial production of ethanol from
renewable energy sources [21]. In fact, S. cerevisiae is unable to convert the C5 -sugars present in hemicellulose, but
C. tropicalis has the enzymatic machinery to ferment them.
All the above properties demonstrate that C. tropicalis is a
very attractive system for biofuel production.
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Fig. 4. Scanning electron microscopy of immobilized yeast cells
(U3000). (A) C. tropicalis ; (B) S. cerevisiae.
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