294

Chiang Mai J. Sci. 2014; 41(X)

Chiang Mai J. Sci. 2015; 42(2) : 294-303
http://epg.science.cmu.ac.th/ejournal/
Contributed Paper

Effects of Initial Glucose Concentration on

Fermentation Performance of Kluyveromyces
marxianus Cells Immobilized on Banana Leaf Sheath
Pieces
Hoang Du Le and Van Viet Man Le*
Department of Food Technology, Ho Chi Minh City University of Technology 268 Ly Thuong Kiet Street,
District 10, Ho Chi Minh City, Viet Nam.
*Author for correspondence; e-mail: lvvman@hcmut.edu.vn
Received: 4 June 2013
Accepted: 23 December 2013

ABSTRACT
Ethanol fermentation was carried out by Kluyveromyces marxianus cells immobilized
on banana leaf sheath pieces at different initial glucose concentrations (120, 160, 200, 240 and
280 g.L-1). Fermentation performance of the free yeast was also evaluated and compared.
Increase in glucose concentration from 120 to 200 g.L-1 did not affect the growth and ethanol
formation of both immobilized and free yeast. In the glucose concentration ranging from
200 to 280 g.L-1, the growth and ethanol formation of both immobilized and free yeast
decreased. The immobilized yeast demonstrated faster sugar assimilation and higher final ethanol
concentration in comparison to the free yeast at all glucose concentrations. The maximum
ethanol concentration produced by the immobilized yeast was 1.12 to 1.67 times higher than
that produced by the free yeast as the glucose concentration increased from 120 to 280 g.L-1.
Keywords: banana leaf sheath pieces, ethanol fermentation, Kluyveromyces marxianus,
immobilized yeast
1. INTRODUCTION
From the nineties of the last century,
the thermotolerant yeast strain Kluyveromyces
marxianus has emerged as a realistic candidate
for commercial ethanol production at high
temperature [1]. Ethanol fermentation at high
temperature is a key requirement for effective
ethanol production in tropical countries
where average day-time temperatures are
usually high throughout the year [2].
Other advantages associated with ethanol
fermentation at high temperature are rapid

bioconversion rates, low contamination [1]

and low cooling costs [2].
The free cells of K. marxianus were
reported to produce ethanol at an elevated
temperature up to 45C [1]. In addition,
K. marxianus cells demonstrated better
fermentation performance than S.cerevisiae
in terms of ethanol productivity [3]. The
application of the immobilized cell system was
reported to improve ethanol performance of
K. marxianus under environmental stresses such

Chiang Mai J. Sci. 2014; 41(X)

as high temperature [4, 5], low pH [4, 5] and

high osmotic pressure [6]. In addition, increase
in substrate concentration was reported to
increase ethanol yield, ethanol volumetric
productivity and ethanol concentration in
the fermented medium [7]. Until now,
the effects of initial substrate concentration
on fermentation performance of K. marxianus
have been investigated with lactose [7],
fructose from Jerusalem Artichoke extract
[4, 6, 8] and mixture of glucose and fructose
[9]. In this study, fermentation performance
of K.marxianus immobilized on banana leaf
sheath pieces was carried out at different
initial glucose concentrations.
Banana is one of the most common,
widely distributed, and useful fruits in
tropical countries. The banana fruit is
consumed by local population in Asia,
America and Africa [10] while the banana
leaf sheath is usually discarded as waste [11].
The banana leaf sheath was used as adsorbents
for removal of metal ions from waste water
due to their highly porous structure [12, 13].
In this study, banana leaf sheath pieces were
used as support for yeast immobilization.
The aim of this work was to clarify the
effects of initial glucose concentration on the
growth, glucose assimilation and ethanol
formation of K. marxianus cells immobilized
on banana leaf sheath pieces. The fatty acid
composition of the immobilized and free cells
was examined to give a clearer understanding
about the difference in ethanol performance
between the immobilized and free yeast.
2. MATERIALS AND METHODS

2.1. Yeast and Media

A strain of K. marxianus was used for
ethanol fermentation. For the inoculum
preparation, the yeast strain was cultivated
in 10 mL of growth medium containing
30 g.L-1 of glucose, 5 g.L-1 of yeast extract,
1 g.L-1 of NH4Cl, 1 g.L-1 of KH2PO4, and

295

0.3 g.L-1 of MgSO4.7H2O in a test-tube

(150 16 mm). The test-tube was shaken at
30 C, 150 rpm for 24 h. Ten mL of the
preculture was then inoculated into a 250 mL
Erlenmeyer flask containing 90 mL of
growth medium. The flask was also shaken
at 30 C, 150 rpm for 24 h. The preculture
was subsequently centrifuged at 5 C,
5000 rpm for 20 min. Yeast cells were
collected and washed with sterile water.
The medium composition for yeast
immobilization and ethanol fermentation
was similar to that for the preculture
preparation except glucose level. The glucose
concentration in the medium for yeast
immobilization was adjusted to 120 g.L-1.
The glucose concentration in the media for
ethanol fermentation was changed from
120 to 280 g.L-1. The initial pH of the media
was 5.5. All media were sterilized at 121 C
for 20 min before use.
2.2. Carrier
Leaf sheath of banana (Musa acuminata)
was used as carrier for yeast immobilization.
Firstly, the leaf sheath was washed by water
and then cut into tubular shape with 20 mm
in diameter and 5 mm in height. Secondly,
the leaf sheath pieces (LSP) were treated
with 0.01 N NaOH solution at 120 rpm for
30 min for tannin removal and subsequently
washed with distilled water three times.
Finally, the LSP were sterilized at 121 C for
20 min before use.
2.3. Yeast Immobilization on Banana Leaf
Sheath Pieces
Twenty grams of sterilized LSP and
100 mL of medium for yeast immobilization
were added into a 250 mL shaking flask.
The yeast biomass was then introduced into
the flask in order to reach a cell density of
3.0 107 cfu.mL-1. The flasks were shaken at
30 C, 120 rpm for 20 h. The liquid fraction

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was decanted and the LSP with immobilized

cells were washed with the fermentation
medium twice. The immobilized biocatalyst
was sampled for cell quantification.
2.4. Ethanol Fermentation
Ethanol fermentations were carried out
in 500 mL flasks containing 250 mL medium
under stationary conditions. The immobilized
yeasts were introduced into the medium with
the inoculum size of 1.0 107 cfu.mL-1.
The initial glucose concentration was
varied: 120, 160, 200, 240 and 280 g.L-1.
The fermentation temperature was 35C.
The fermentations lasted for 72 h. During
the fermentation, samples were taken at
12 h intervals for analysis. The fermentations
with the free yeast were also performed under
the same conditions.
2.5. Analytical Methods
2.5.1. Cell density
Total viable cell number in the
immobilized yeast culture included the cells
adsorbed on the LSP and the cells released
into the fermented medium. The cells
adsorbed on the LSP were quantified by the
procedure as described previously [14] with
slight modification. One hundred millimeters
of sterile water and 10 g of the LSP with the
immobilized cells were ground by a grinder
at 3,500 rpm for 5 minutes. Afterwards, the
cell number was determined by plate count
agar with the incubation at 30 C for 48 h.
For counting yeast cells released into the
fermented medium, the liquid fraction of the
medium was sampled and the cell number
was determined by plate count agar with the
incubation at 30 C for 48 h [15]. Total cell
number in the immobilized yeast culture
was calculated and expressed as cfu per
milliliter of the fermented medium.
Total viable cell number in the free
yeast culture was quantified by plate count

Chiang Mai J. Sci. 2014; 41(X)

agar with the incubation at 30 C for 48 h

[15]
The average growth rate (cfu.mL-1h-1) of
both immobilized and free yeast was
calculated by the formula: (X2-X1)/t; where
X1: Total cell number in the immobilized
or free yeast culture at the start of the
fermentation (cfu.mL-1); X2: Total cell number
in the immobilized or free yeast culture at
the end of the fermentation (cfu.mL -1);
t: Fermentation time (h).
2.5.2. Glucose
Glucose content was quantified
by spectrophotometric method with
dinitrosalicylic acid reagent [16].
2.5.3. Ethanol
Ethanol concentration was determined
by enzymatic method using ethanol kit with a
reflectometer model 116970 (MercK KgaA,
Germany).
2.5.4. Fatty acid composition of yeast cell
membrane
Prior to determine fatty acid
composition, the lipid in yeast cell membrane
was extracted by the method [17] previously
described with slight modification. Mixture
of yeast biomass and methanol was treated
with ultrasound by a ultrasonic probe model
VC 750 (Sonics & Materials Inc., The United
States) at an ultrasonic power of 5 W.g-1 for
1 min to break down the cell wall. The lipid
extraction was then carried out by adding
chloroform and methanol (2:1 v:v) to the
sonicated mixture. The weight ratio of
material and solvent was 5:2. The extraction
was performed under agitation (200 rpm) for
2 h. The organic phase was then transferred
into a glass screw tube containing 0.88%
KCl solution. The mixture was centrifuged
at 25 C and 3,000 rpm for 5 min. The
organic phase was then collected and used

Chiang Mai J. Sci. 2014; 41(X)

for determination of fatty acid composition.

Fatty acid composition was determined
by gas chromatography using a HewlettPackard model 5890A (Hewlett - Packard,
The United States). The extract was injected
into an FFAP-HP column of 25 m 0.2 mm
with an HP automatic injector. Helium was
used as carrier gas at 1.0 mL.min -1 and
heptadecanoic acid methyl ester (1 g.L-1)
was added as an internal standard.
Column inlet pressure was 150 kPa.
The injector temperature was 250 C.
Detector temperature was 250 C. The
temperature program was 25 C.min -1
from 70 C to 200 C. Peak areas were
measured using a Hewlett-Packard model
3396A integrator.
2.6. Statistical Analysis
All experiments were performed in
triplicate. Mean values were considered
significantly different when P < 0.05.
One-way and multi-way analyses of variance
were performed using the software
Statgraphics Centurion XV.
3. RESULTS AND DISCUSSION

3.1. Effects of Initial Glucose

Concentration on Yeast Growth
Yeast growth was evaluated by
maximum cell density in the cultures and
average growth rate of the immobilized
and free yeast. The results in Table 1 show
that the maximum cell density in the cultures
and average growth rate of both immobilized
and free yeast remained stably when the
glucose concentration increased from 120
to 200 g.L-1. In this glucose concentration

297

range, the free yeast demonstrated higher

maximum cell density and faster average
growth rate than the immobilized yeast.
Differences in growth between the cells
adsorbed on solid supports and the free cells
have not been clearly explained. In case of
S. cerevisiae yeast entrapped in gelatin gel,
the multiplication of the immobilized cells
could not proceed as readily as that of the
free cells; bud emergence in the immobilized
cells was limited due to the attachment of
the cells to gel matrix [18]. Hence, the
replication of the immobilized yeast was
restricted. Figure 1 shows SEM picture of
K. marxianus cells adsorbed on banana LSP.
Cell aggregation was clearly observed and that
could restrict the cell replication. As a result,
the growth of the immobilized yeast was
reduced.
As the glucose concentration increased
from 200 to 280 g.L-1, the growth rate of the
immobilized and the free yeast decreased by
32 and 54%, respectively. Similar results were
reported in a previous study [7] when the
authors used lactose as substrate for ethanol
fermentation with the free K. marxianus cells.
In the glucose concentration ranging from
240 to 280 g.L-1, the immobilized cells always
demonstrated higher maximum cell density
and faster average growth rate than the free
cells. It was reported that carrier would
protect the immobilized yeast from
environmental stresses, including high
osmotic stress [14]. Therefore, the growth of
the immobilized yeast was better than that
of the free yeast when the fermentations
were carried out at high initial glucose
concentration.

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Chiang Mai J. Sci. 2014; 41(X)

Table 1. Effects of initial glucose concentration on maximum cell density and average growth
rate of the immobilized and free Kluyveromyces marxianus yeast.
Initial glucose
concentration
(g.L-1)
120
160
200
240
280

Log (maximum cell density)

Immobilized cells
7.930.02k
7.920.01k
7.930.01k
7.820.01j
7.710.01h

Free cells
7.970.02l
7.970.01l
7.980.01l
7.750.01i
7.660.03g

Average growth rate

(106 cfu.mL-1.h-1)
Immobilized cells
Free cells
3.160.05e
3.980.05f
3.180.03e
4.000.04f
e
3.180.03
3.940.03f
2.510.03d
2.240.03c
b
2.150.03
1.830.07a

Various superscripts in table indicate significant differences (p < 0.05)

Figure 1. Inside structure of banana leaf sheath before (a) and after immobilization of
Kluyveromyces marxianus cells (b).
3.2. Effects of Initial Glucose
Concentration on Glucose Assimilation
of K. marxianus Yeast
Table 2 presents that the increase in
glucose concentration from 140 to 200 g.L-1
augmented the glucose uptake rate of both
immobilized and free yeast. No study has
been done to investigate the effect of initial
substrate concentration on sugar assimilation
rate of the K. marxianus cells adsorbed on
solid carriers. In case of K. marxianus cells
entrapped in calcium alginate gel, the sugar
uptake rate of the immobilized yeast was
unchanged when the initial substrate
concentration increased from 150 to
250 g.L-1 [6]. For free cells of K. marxianus,
it was reported that the lactose uptake rate
of the yeast increased as the initial lactose

concentration augmented from 85 to

170 g.L -1 [7]. Glucose assimilation of
K. marxianus cells depended on the molecular
transport across cellular membrane [19].
This transport was performed by symporter
that located inside the yeast cell membrane
[20]. When the osmotic pressure gradually
increased, more substrate molecules were
transported into the cells and that led to an
increase in substrate assimilation rate.
In this study, the residual glucose level
in both immobilized and free yeast cultures
was similar (4 g.L-1) as the initial glucose
concentration varied from 140 to 200 g.L-1.
Similar results were reported in a previous
study [8] when the authors used free cells of
K. marxianus for ethanol fermentation at
different initial fructose concentrations.

Chiang Mai J. Sci. 2014; 41(X)

299

Table 2. Effects of initial glucose concentration on glucose assimilation of the immobilized

and free Kluyveromyces marxianus yeast.
Initial glucose
concentration
(g.L-1)
120
160
200
240
280

Glucose uptake rate (g.L-1.h-1)

Immobilized cells
4.230.12c
5.580.17e
7.180.18h
6.760.13g
4.910.16d

Free cells
3.090.12a
4.260.09c
6.470.11f
3.560.08b
3.230.16a

Residual sugar (g.L-1)

Immobilized cells
4.10.1c
4.10.2c
4.00.1c
42.01.0i
75.31.5j

Free cells
4.10.2c
4.20.1c
4.10.1c
73.01.0j
121.31.3k

Various superscripts in table indicate significant differences (p < 0.05)

Increase in glucose concentration from

200 to 280 g.L-1 decreased the glucose uptake
rate of both immobilized and free yeast.
Too high substrate concentration was
reported to change the yeast cell
permeability [8] and to alter glucose-specific
proton symport systems of K. marxianus
cells [20]. Hence, glucose assimilation of K.
marxianus cells slowed down.
In this study, the immobilized yeast
exhibited faster glucose uptake rate than the
free yeast at all initial glucose concentrations.
The glucose uptake rate of the immobilized
yeast was from 1.1 to 1.5 times higher than
that of the free yeast as the glucose
concentration increase from 200 to 280 g.L-1.
Generally, the improvement in substrate
assimilation of the immobilized yeast was
explained through the protection of the
carrier [14].
The response of the free cells of S.
cerevisiae to environmental stresses through
changes in fatty acid composition in cellular
membrane was reported [17, 21-22].
However, no study compared fatty acid
composition in cellular membrane of the
immobilized and free cells of K. marxianus.
Our investigation showed that the level of
long chain fatty acids (C16 and C18) was

dominant in comparison with that of other

fatty acids in cellular membrane of the
immobilized and free cells of K. marxianus.
The relative percentage of palmitic (C16:0),
stearic (C18:0), oleic (C18:1) and linoleic acid
(C18:2) in cellular membrane of the
immobilized and free yeast is shown in
Figure 2. The immobilized yeast exhibited
higher level of unsaturated fatty acid and
lower level of saturated fatty acid than the
free yeast. Consequently, the immobilization
process reduced the level of saturated fatty
acid in the cellular membrane of K. marxianus
cells. The changes in immobilized yeast cell
components was due to the disturbance of
the yeast cell cycle which caused by cell
attachment to the support [18]. In case of
S. cerevisae, it was reported that the
immobilized cells of S. cerevisae on sintered
glass contained significantly higher level of
saturated fatty acids compared to free cells
[22]. The difference between our results and
those of Hilge-Rotmann and Rehm [22] was
possibly due to the difference in yeast genus.
In our study, adsorption of K. marxianus cells
on banana LSP increased unsaturated fatty acid
level in cellular membrane and these
changes might improve osmotic tolerance
of the immobilized yeast.

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Chiang Mai J. Sci. 2014; 41(X)

Figure 2. Relative percentage of long chain fatty acids in cellular membrane of the
immobilized ( ) and free ( ) K.marxianus at the beginning of the fermentation.
3.3. Effects of Glucose Concentration on
Ethanol Formation of K.Marxianus
Yeast
The impacts of initial glucose
concentration on ethanol formation of the
immobilized and free cells of K. marxianus
are shown in Table 3. The final ethanol
concentration and ethanol formation rate
increased as the glucose concentration

increased from 120 to 200 g.L-1. In this

range of glucose concentration, the ethanol
yield of both immobilized and free
yeast remained stably. Similar results
were reported in some previous studies
[7, 9] when the lactose or mixture of
glucose and fructose were used for
ethanol fermentation with the free cells of
K. marxianus.

Table 3. Effects of initial glucose concentration on final ethanol concentration, average ethanol
formation rate and ethanol yield of the immobilized and free Kluyveromyces marxianus yeast.
Final ethanol
Initial glucose
concentration concentration (%v/v)
Immobilized Free cells
(g/L)
cells
6.850.16o 5.830.16n
120
160
9.000.08r 8.040.06q
200
11.080.11u 9.860.18t
240
9.430.12s 7.480.05p
280
9.160.04r 5.480.05m

Average ethanol
Ethanol yield
formation rate (g/L.h) (g ethanol/g glucose)
Immobilized Free cells Immobilized Free cells
cells
cells
h
g
2.100.01 1.800.01 0.450.01e 0.390.01d
2.390.02j 2.120.03h 0.450.01e 0.410.01d
2.770.04l 2.450.02k 0.450.01e 0.390.01d
2.470.02k 1.800.02g 0.360.01c 0.340.01b
2.210.01i 1.060.01f 0.350.01b 0.270.00a

Various superscripts in table indicate significant differences (p < 0.05)

Chiang Mai J. Sci. 2014; 41(X)

Increase in glucose concentration from

240 to 280 g.L-1 decreased final ethanol
concentration, average ethanol formation
rate and ethanol yield of both immobilized
and free yeast. Similar results were reported
in a previous study [7] in which the free cells
of K. marxianus and medium with different
lactose levels were used for ethanol
fermentation. However, it was reported
that when the initial lactose concentration
increased from 70 to 100 g.L-1, the ethanol
formation rate of K. marxianus cells
immobilized on delignified cellulosic
material slowed down [5].
At all investigated glucose concentrations,
the immobilized yeast always exhibited
higher final ethanol concentration, faster
average ethanol formation rate and better
ethanol yield than the free yeast. When the
glucose concentration increased from 200
to 280 g.L-1, the ethanol formation rates of
the immobilized and free yeast decreased
by 20% and 57%, respectively while the
final ethanol concentration produced by the
immobilized yeast was 1.12 to 1.67 times
higher than that produced by the free yeast.
In case of S. cerevisiae cells entrapped in agar,
carrageenan, alginate and polyacrylamide gels,
the ethanol formation rate of the immobilized
cells was about 20-25% higher than that of
the free cells as the initial glucose concentration
increased from 100 to 300 g/L [24]. The
change in membrane fatty acids of the
immobilized yeast cells was one of the
explanations for the improvement in ethanol
formation of the immobilized yeast.
4. CONCLUSION

The final ethanol concentration in the

culture with both immobilized and free cells
of K. marxianus increased as initial glucose
concentration augmented from 120 to
200 g.L -1 . Increase in initial glucose
concentration from 200 to 280 g.L-1 decreased

301

the growth, substrate assimilation and

ethanol formation of both immobilized and
free yeast. The adsorption of K. marxianus
cells on banana LSP increased membrane
unsaturated fatty acid level and these
changes might improve ethanol formation of
the immobilized yeast.
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