Fuel 88 (2009) 1602–1607

Contents lists available at ScienceDirect

Fuel
journal homepage: www.elsevier.com/locate/fuel

Bioethanol production from corn meal by simultaneous enzymatic

saccharification and fermentation with immobilized cells of Saccharomyces
cerevisiae var. ellipsoideus
Svetlana Nikolić a,*, Ljiljana Mojović a, Marica Rakin a, Dušanka Pejin b
a
Faculty of Technology and Metallurgy, Department of Biochemical Engineering and Biotechnology, University of Belgrade, Karnegijeva 4, 11000 Belgrade, Serbia
b
Faculty of Technology, University of Novi Sad, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia

a r t i c l e i n f o a b s t r a c t

Article history: The simultaneous enzymatic saccharification and fermentation (SSF) of corn meal using immobilized
Received 23 September 2008 cells of Saccharomyces cerevisiae var. ellipsoideus yeast in a batch system was studied. The yeast cells were
Received in revised form 4 November 2008 immobilized in Ca-alginate by electrostatic droplet generation method. The process kinetics was assessed
Accepted 17 December 2008
and determined and the effect of addition of various yeast activators (mineral salts: ZnSO4
7H2O and
Available online 12 January 2009
MgSO4
7H2O, and vitamins: Ca-pantothenate, biotin and myo-inositol) separately or mixed, was inves-
tigated. Taking into account high values of process parameters (such as ethanol concentration, ethanol
Keywords:
yield, percentage of the theoretical ethanol yield, volumetric productivity and utilized glucose) and sig-
Ethanol
SSF
nificant energy savings the SSF process was found to be superior compared to the SHF process. Further
Saccharomyces cerevisiae var. ellipsoideus improvement in ethanol production was accomplished with the addition of mineral salts as yeast activa-
Immobilized cells tors which contributed to the highest increase in ethanol production. In this case, the ethanol concentra-
tion of 10.23% (w/w), percentage of the theoretical ethanol yield of 98.08%, the ethanol yield of 0.55 g/g
and the volumetric productivity of 2.13 g/l
h were obtained.
Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction
As a consequence of industrial development and population
growth there is an increase of energy consumption in the world.
The world-wide energy consumption has increased 17-fold in the
last century [1]. However, conventional energy resources, like fos-
sil fuels, cannot meet the increasing energy demand. The quantities
of non-renewable (conventional) energy resources are limited and
they have a considerable negative environment impact e.g. in-
creased greenhouse gas emissions. Therefore, the use of biofuels
as alternative energy sources has many advantages, such as contri-
bution to the reduction of CO2 emission, lower dependency on the
import of oil for non-oil producing countries, new employment
opportunities and development of rural communities; they are
easily available from common biomass sources, biodegradable
and contribute to the sustainability [2]. Bioethanol is one of the
most promising biofuels from renewable resources. Fermentation
derived ethanol can be produced from sugar, starch or lignocellu-
losic biomass. Sugar and starch based feedstocks are currently pre-
dominant at the industrial level and they are so far economically
favorable. In Serbia, one of the most suitable and available agricul-
tural raw material for industrial bioethanol production is corn. It is
* Corresponding author. Tel.: +381 113370423; fax: +381 113370387.
E-mail address: tehnikol@yahoo.com (S. Nikolić).
reported that in this year the average corn yield in Serbia is 6–7
million ton (calculated domestic needs for corn are only 4–4.5 mil-
lion ton) [3]. This means that there is enough corn for other pur-
poses besides the food; therefore significant amounts can be
used for bioethanol production. These are the reasons why we used
corn meal as a substrate in this work.
The current world bioethanol research is driven by the need to
reduce the costs of production. Until now the cost of bioethanol
has significantly dropped due to continuous improvement in feed-
stock pretreatment, enzyme application and fermentation [4]. One
of the possibilities to reduce production costs and to make the pro-
duction more efficient is to use simultaneous enzymatic sacchari-
fication and fermentation (SSF) process. This process is favorable
compared with conventional separate hydrolysis and fermentation
(SHF) process in terms of higher ethanol yield, lower energy con-
sumption and shorter processing time. In SSF process the end-
product inhibition of the enzymes is avoided because fermenting
organism immediately consumes the released sugars. Also, capital
investments are lower in this process as the total reactor volume is
decreased due to higher productivity. On the other hand, the crit-
ical problem with SSF is that it operates at non-optimal hydrolysis
temperature since optimal temperatures for the yeast and the en-
zymes differ [5].
Saccharomyces strains are well known ethanol producing micro-
organisms. In this work we used immobilized cells of S. cerevisiae

0016-2361/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fuel.2008.12.019
 S. Nikolić et al. / Fuel 88 (2009) 1602–1607
var. ellipsoideus. Previously, this microorganism was found to be
the most superior for the fermentation of corn meal hydrolyzates
among four tested yeasts: S. cerevisiae, S. cerevisiae var. ellipsoideus,
S. carlsbergensis and Schizosaccharomyces pombe [6]. In addition,
Okunowo and Osuntoki achieved greater ethanol production by
using this yeast for wine fermentation compared to the production
obtained with S. cerevisiae and S. carlsbergensis [7]. One of the most
critical components of management of the yeast fermentation is to
make sure that the yeasts have all the requisite nutrients to main-
tain fermentation rates and optimal ethanol and temperature tol-
erance. The doses of the mineral compounds and vitamins, as
micronutrients, depend on the raw material used, which can be
sometimes characterized by too poor chemical composition as a
substrate for yeasts. The addition of mineral salts and vitamins
has a stimulatory effect on yeasts and contributes to an increase
in efficiency of ethanol fermentation. These compounds have a
protective effect either on growth, fermentation, or viability, which
overall stimulates the rate of ethanol production [8]. The use of
immobilized yeast has attracted considerable attention during
the past few years due to the advantages over the processes with
free yeast. Immobilization of the cells can eliminate inhibition
caused by high concentration of substrate and product, and also
enhance ethanol yield and productivity [9]. Other advantages in-
clude increased yeast stability and simpler bioreactor design. Many
inorganic compounds, such as calcium alginate, c-alumina, kissiris
(volcanic material found in Greece), k-carrageenan gel, polyacryl-
amide, and porous ceramics have been used as a support material
for cell immobilization [10].
The aim of this study was to investigate the possibilities of
improving productivity of bioethanol production from corn meal
by applying simultaneous enzymatic saccharification and fermen-
tation with immobilized yeast S. cerevisiae var. ellipsoideus in a
batch system. The kinetics of SSF process and the improvement
of the ethanol production by adding different yeast activators
(mineral salts: ZnSO4
7H2O and MgSO4
7H2O, and vitamins: Ca-
pantothenate, biotin, myo-inositol) separately or mixed was
assessed.
2. Materials and method
2.1. Starch
Corn meal obtained by dry milling process was a product of
corn processing factory (‘‘RJ Corn Product”, Sremska Mitrovica, Ser-
bia). The corn meal consisted of particles with diameter 0.2–
1.7 mm (95% or more particles pass through a 1.70 mm sieve).
The content of the main components in the corn meal, determined
by chemical analysis, was the following: starch, 73.75% (w/w); pro-
teins, 7.98% (w/w); lipids, 5.86% (w/w); fibers, 1.37%; ash, 0.70%
(w/w) and water, 10.34% (w/w).
2.2. Enzymes and microorganisms
Termamyl SC, a heat-stable a-amylase from Bacillus lichenifor-
mis was used for corn meal liquefaction. The enzyme activity was
133 KNU/g (KNU, kilo novo units a-amylases – the amount of en-
zyme which breaks down 5.26 g of starch per hour according to
Novozyme’s standard method for the determination of a-amylase).
SAN Extra L, Aspergillus niger glucoamylase, activity 437 AGU/g
(AGU is the amount of enzyme which hydrolyses 1 lmol of malt-
ose per minute under specified conditions) was used for corn meal
saccharification. The enzymes were gift from Novozymes,
Denmark.
S. cerevisiae var. ellipsoideus was used for the fermentation of
hydrolyzed corn meal. The culture originated from the collection
1603
of Department of Biochemical Engineering and Biotechnology, Fac-
ulty of Technology and Metallurgy, Belgrade (BIB-TMFB), and was
maintained on a malt agar slant. The agar slant consisted of malt
extract (3 g/l), yeast extract (3 g/l), peptone (5 g/l), agar (20 g/l)
and distilled water (up to 1 l). Before use as an inoculum for the
fermentation, the culture was aerobically propagated in 500 ml
flasks in a shaking bath at 30 °C for 48 h and then separated by cen-
trifugation. The liquid media consisted of yeast extract (3 g/l), pep-
tone (3.5 g/l), KH2PO4 (2.0 g/l), MgSO4
7H2O (1.0 g/l), (NH4)2SO4
(1.0 g/l), glucose (10 g/l) and distilled water.
2.3. Immobilization and cultivation procedure
The yeast cells were immobilized in Ca-alginate using an elec-
trostatic droplet generation method. The 2% (w/w) Na-alginate
solution was prepared by dissolving 4.8 g of Na-alginate powder
(Sigma medium viscosity) into 240 ml of distilled water. Poly-
mer/cell suspension was formed by mixing of 240 ml of Na-algi-
nate solution with 60 ml of thick yeast suspension at room
temperature. Spherical microbeads were formed by extrusion of
Na-alginate/yeast cell suspension through a blunt stainless steel
needle using a syringe pump (Harvrad Apparatures, Pump 11) with
a 20 ml plastic syringe and an electrostatic droplet generator
(Nisco Encapsulator, Switzerland). The cell suspension was forced
out of the tip of the needle at constant flow rate (0.25 ml/min),
and droplets were formed by the action of electrostatic and gravi-
tational forces. Electrostatic potential was formed by connecting
the positive electrode of a high voltage dc unit to the gelling bath
which was 2.65% (w/v) CaCl2 solution while the needle was
grounded. In this way, the yeast cells were entrapped in the gel
matrix of Ca-alginate. After gelling the microbeads were placed
in double distilled water to remove unreacted material. Micro-
beads with cells were stored in physiological solution at 8 °C.
2.4. Liquefaction and SSF experiments
A 100 g of corn meal was mixed with water at the weight ratio
(hidromodul) 1:3, and 60 ppm of Ca2+ (as CaCl2) ions was added.
The liquefaction was carried out at 85 °C and pH of 6.0 for 1 h by
adding 0.026% (v/w of starch) enzyme Termamyl SC. The liquefac-
tion and SSF process were performed in flasks in thermostated
water bath with shaking (100 rpm), as described by Mojović
et al. [11].
The liquefied mash was cooled, pH was adjusted to 5.0 using
2 M HCl, and KH2PO4 (4.0 g/l), MgSO4
7H2O (0.4 g/l) and
(NH4)2SO4 (2.0 g/l) were added. The SSF process was performed
by adding 0.156% (v/w of starch) enzyme SAN Extra L and 2% (w/
v) of Ca-alginate beads with entrapped cells (5.0  107 CFU/g of
beads) to liquefied mash for a period up to 48 h at 30 °C. Improve-
ment of the ethanol production was investigated by adding differ-
ent yeast activators: additional mineral salts, ZnSO4
7H2O (0.3 g/l)
and MgSO4
7H2O (2.0 g/l); and vitamins, Ca-pantothenate
(30.0 mg/l); biotin (64.0 lg/l) and myo-inositol (350.0 mg/l). It
was considered that the pasteurization of the substrate achieved
during the enzymatic liquefaction (85 °C for 1 h) was sufficient
thermal treatment, and thus no additional sterilization prior to
SSF process was performed.
2.5. Analytical methods
The starch content was determined by Ewers polarimetric
method [12]. The water content in the corn meal was determined
by the standard drying method in an oven at 105 °C to constant
mass [13]. Lipid concentration was determined according to the
Soxhlet method [13]. The protein content was estimated as the to-
tal nitrogen by the Kjeldahl method multiplied by 6.25 [13], the
1604 S. Nikolić et al. / Fuel 88 (2009) 1602–1607
ash content was determined by slow combustion of the sample at
650 °C for 2 h [13], and the fiber content was determined by the
Scharrer–Kürschner method [13]. During the SSF process, the con-
tent of reducing sugars, calculated as glucose, was determined by
3.5-dinitrosalicylic acid [14]. A standard curve was drawn by mea-
suring the absorbance of known concentrations of glucose solu-
tions at 570 nm. The ethanol concentration was determined
based on the density of the alcohol distillate obtained from the fer-
mentation broth. The density of the sample was calculated as fol-
lowing: q (g/l) = (Wsample W)/(Wwater W), where Wsample is the
mass of picnometer containing ethanol distilled from the sample
i.e. filtrated mash (50 ml) and then filled up with distilled water
up to the marked level; W is the mass of clean and dry pycnometer
(50 ml); Wwater is the mass of the picnometer filled up with dis-
tilled water. The ethanol concentration expressed in weight%
(w/w) was calculated from the density of the sample by using stan-
dard tables [13]. Indirect counting method i.e. pour plate technique
was used to determine the number of viable cells. Serial dilutions
of the samples were performed, and after the incubation time at
30 °C, colonies grown in Petri dishes were used to count the num-
ber of viable cells. The first dilution was performed in 2% sodium
citrate solution (instead of physiological solution) in order to dis-
solve alginate gel. At least three measurements were made for each
condition and the data given were averages.
3. Results and discussion
The Ca-alginate microbeads with immobilized cells had rather
uniform average diameter of 0.8 mm, and are presented in Fig. 1.
Small diameter beads are generally preferred because of the
more favorable mass transfer. The significance of this immobiliza-
tion method is that the matrix is porous enough for substrate and
products to traverse where a level of cell retention is maintained
within the immobilization matrix [15]. Alginate as a suitable cell
entrapment matrix is non-toxic, less expensive, reversible and
has good mechanical properties [16]. However, a disadvantage of
the use of calcium alginate as an immobilization matrix is that
phosphates, EDTA, cations Mg2+ and K+ can disrupt the gel by sol-
ubilising the Ca2+. This leads to the breaking of the bonds between
Ca and alginate, resulting in a loss of the mechanical stability of the
gel and to its complete disruption [15]. This phenomenon was not
noticed in this study.
In order to reduce the time of the complete process and make
beneficial energy savings, we performed a simultaneous process
Fig. 1. Yeast S. cerevisiae var. ellipsoideus cells entrapped in the gel matrix of Ca-
alginate.
14 10
13 ethanol
12 glucose
11 cells
number
10 9
Ethanol (% w/w)
Glucose (% w/v)
9
Log (CFU/g)
8
7
6 8
5
4
3
2 7
1
0
0 10 20 30 40 50
Time (h)
Fig. 2. Time course of glucose consumption, number of viable cells and ethanol
production from corn meal hydrolyzates by immobilized cells of S. cerevisiae var.
ellipsoideus. Process conditions: pH 5.0, 30 °C, 100 rpm, enzyme SAN Extra L was
added in concentration of 0.156% (v/w of starch), inoculum concentration 2% (w/v).
of the second hydrolysis step (saccharification) and fermentation
of corn meal at 30 °C. Fig. 2 presents the time course of ethanol
production, glucose consumption and the number of cells in the
SSF process.
As shown in Fig. 2, during the SSF process the ethanol concen-
tration gradually increased without any declining phase, probably
due to the fact that the immobilization increased the ethanol toler-
ance of the yeast cells, as reported earlier [17]. This behavior sug-
gests protective effect of immobilization against substrate and
product inhibition. Also, these results show that applied immobili-
zation method by electrostatic droplet generation was appropriate
and thus no substrate diffusion restrictions were noticed.
The maximum ethanol concentration of 9.42% w/w, the ethanol
yield of 0.51 g/g, percentage of the theoretical ethanol yield of
90.32%, and the volumetric productivity of 1.96 g/l
h were
achieved after 48 h of the SSF process (Fig. 2, Table 1).
As shown in Fig. 2, the glucose consumption was in accordance
with the results of ethanol concentration since the glucose was
consumed as a carbon source by the yeast. An increase in glucose
concentration was observed at the beginning of the SSF process.
This indicates that the hydrolysis was much faster than the ethanol
fermentation. During this time there was not a significant ethanol
production, because the yeast cells were still in the adaptation
phase. This phenomenon was also observed by other authors
[18,19]. In contrast, Öhgren et al. [20] explained an increase in glu-
cose concentration during the first 10 h of SSF of corn stover by S.
cerevisiae by high viscosity and uneven slurry distribution caused
by ineffective stirring.
At the end of the SSF process the total amount of utilized glu-
cose was 97.72% (Table 1) indicating the end of fermentation since
the glucose was almost completely consumed.
As shown in Fig 2, the lag phase of yeast growth wasn’t ob-
served or it was too short, and the number of cells gradually in-
creased during the SSF process. Maximum number of viable cells
of the immobilized yeast was 1.05  109 CFU/g of beads. These re-
sults correspond well to the fact that there was no decline in eth-
anol production during the SSF process.
Previous studies showed that the yeast viability significantly
decreased in free cell system due to the great accumulation of
intracellular ethanol which altered the structure of the membrane
decreasing its functionality [21,22]. On the other hand, Ciesarová
et al. [23] and Verbelen et al. [24] reported that immobilized cells
are considered to be more tolerant against ethanol because the ma-
trix provides a protective environment against ethanol toxicity.
 S. Nikolić et al. / Fuel 88 (2009) 1602–1607 1605

Table 1
Values of the significant parameters obtained during the SSF and SHF process of corn meal by immobilized cells of S. cerevisiae var. ellipsoideus.

Type of the process Ethanol concentration Ethanol yield YP/S (g ethanol/ Percentage of the theoretical Volumetric productivity Utilized
(% w/w) g starch) ethanol yield (%) P (g/l
h) glucose (%)b
SHF without media 8.01 ± 0.16 0.43 ± 0.009 76.79 ± 1.54 1.67 ± 0.03 86.25 ± 0.06
supplementationa
SHF with addition of 8.41 ± 0.17 0.45 ± 0.008 80.64 ± 1.47 1.75 ± 0.05 88.64 ± 0.07
mineralsa
SSF without media 9.42 ± 0.12 0.51 ± 0.007 90.32 ± 1.52 1.96 ± 0.03 97.72 ± 0.09
supplementation
SFF with addition of minerals 10.23 ± 0.11 0.55 ± 0.004 98.08 ± 1.50 2.13 ± 0.04 99.08 ± 0.05

Process conditions are the same as in Fig. 2.

a
Ref. [25].
b
Utilized glucose (%) was calculated as the ratio of the consumed mass of glucose to initial mass of glucose.

This was in agreement with our results since there was no declin-
ing in number of cells and ethanol production during the SSF
process.
In our previous study we investigated SHF process of corn meal
by the immobilized S. cerevisiae var. ellipsoideus and reported sig-
nificant process parameters [25]. In Table 1, we compared the re-
sults obtained in SHF [25] and SSF (this study) under the same
process conditions. The SSF has been found to be economically
favorable in terms of all presented process parameters and also
considering the lower energy consumption. This is also shown by
other authors [5,11,26,27]. Additionally, it is important to point
out that in this study (SSF process) a superior process was accom-
plished with the same concentration of glucoamylase as in SHF
process [25].
Based on the obtained results the time of the overall process of
ethanol production may be reduced for 4 h (the time required for
the saccharification). In addition, the energy savings could be at-
tained since the overall process was effectively performed at
30 °C, which is a lower temperature than the optimal temperature
for the action of enzyme glucoamylase itself (55 °C).
3.1. Effect of the yeast activators (mineral salts and vitamins)
Vitamins and minerals, as yeast micronutrients, are needed to
facilitate the biochemical reactions. The mineral compounds take
part in metabolism of yeasts as the activators of enzymes or ele-
ments of cell components [28]. The yeast cells also require vita-
mins (such as myo-inositol, pantothenic acid, biotin and
thiamine) for their growth and acceleration of fermentation [8].
It is known that the pantothenic acid is a part of acetyl coen-
zyme A which is involved in many steps of intermediate metab-
olism of carbohydrates, fats and proteins. It increases the yeast
Further experiments were conducted in order to investigate
improvement of the ethanol production and yeast growth by addi-
tion of different yeast activators: mineral salts, ZnSO4
7H2O (0.3 g/
l corresponding to 1 mM Zn2+) and MgSO4
7H2O (2.0 g/l corre-
sponding to 10 mM Mg2+); and vitamins, Ca-pantothenate
(30.0 mg/l); biotin (64.0 lg/l) and myo-inositol (350.0 mg/l). The
concentration of Mg2+ before the mineral salts addition was
1.5 mM. The ethanol concentration and the number of viable cells
of immobilized S. cerevisiae var. ellipsoideus achieved after 48 h of
the process with addition of only mineral salts, only vitamins
and a mixture of mineral salts and vitamins were compared with
a control (samples without any activators), and presented in Figs.
3 and 4.
As shown in Fig. 3, a maximum increase in ethanol concentra-
tion (8.60% compared to the control sample) was achieved when
mineral salts were added. In this case, the ethanol concentration
of 10.23% (w/w), percentage of the theoretical ethanol yield of
98.08%, the ethanol yield of 0.55 g/g and the volumetric productiv-
ity of 2.13 g/l h were obtained. High values of all these parameters
were also achieved in the sample with addition of a mixture of
mineral salts and vitamins (final ethanol concentration was
9.97% and the increase in ethanol concentration was 5.80% com-
pared to the control sample). Very small improvement of the eth-
anol production was observed in the sample with only vitamins
added (increase in ethanol concentration was 1.40% compared to
the control sample).
12
ethanol
11 8.60% 1.40% 5.80%
10
Ethanol concentration (% w/w)

tolerance to alcohol because it stimulates the synthesis of lipids 9

[28]. Biotin is a cofactor of many enzymes involved in carboxyl- 8
ation reactions, such as gluconeogenesis, amino acid metabolism,
7
fatty acid biosynthesis and energy metabolism. Its assimilation
and storage conditions the growth rate of yeasts and ethanol 6
production [8,29]. Myo-inositol is also essential growth factor 5
for yeasts and it contributes to the high ethanol tolerance and 4
increased cell viability [30]. Metal ions (such as K+, Mg2+, Ca2+
3
and Zn2+) can change the rate of glycolysis and the conversion
of pyruvate to ethanol and therefore significantly impact on 2
the progress and efficiency of the fermentation. Magnesium is 1
involved in many physiological functions – yeast growth, cell
0
division and enzyme activity, and it has an important role in
Control Mineral salts Vitamins Min.+ vit.
the cellular protection of toxic levels of ethanol. If the media is
magnesium-limited the conversion of sugar to ethanol may lead Fig. 3. Effect of mineral salts, vitamins and a mixture of mineral salts and vitamins
to slow or incomplete fermentation process [31]. Zinc is a trace on ethanol concentration after 48 h of SSF process of corn meal by immobilized cells
element that is necessary for several enzymes activities such as of S. cerevisiae var. ellipsoideus. Process conditions are the same as in Fig. 2.
Activators: ZnSO4
7H2O, 0.3 g/l; MgSO4
7H2O, 2.0 g/l; Ca-pantothenate, 30.0 mg/l;
alcohol dehydrogenase, aldolase, alkaline phosphatase, DNA and
biotin, 64.0 lg/l; myo-inositol, 350.0 mg/l. The numbers above the bars represent
RNA polymerase [32]. the percentage of the increase in ethanol concentration.
1606 S. Nikolić et al. / Fuel 88 (2009) 1602–1607
12
number of viable cells
11
10 0.50% 2.77% 1.10%
9 can be toxic to yeast even at lower concentrations. The toxicity
8
Log (CFU/g)
7 bilization [36,37]. In this study applied concentrations of zinc were
6 not found toxic to the yeast.
5
4 of vitamin mixture (biotin, pantothenic acid, nicotinic acid, myo-
3
2 increase the ethanol productivity. They also pointed out the exis-
1 tence of an uncoupling between the growth rate and the ethanol
0
Control Mineral salts Vitamins Min.+vit.
Fig. 4. Effect of mineral salts, vitamins and a mixture of mineral salts and vitamins
on number of viable cells of immobilized S. cerevisiae var. ellipsoideus after 48 h of
SSF process of corn meal. Other process conditions are the same as in Fig. 2. The
numbers above the bars represent the percentage of the increase in logarithmic
value of number of cells.
Investigating the effect of added activators on yeast growth
(Fig. 4) an increase in the number of yeast cells in the samples with
the addition of mineral salts and a mixture of mineral salts and
vitamins was rather small, 0.50% and 1.10% (compared to the con-
trol sample), respectively. The addition of vitamins only, increased
the number of viable cells the most i.e. for 2.77% compared to the
control sample. In this case, 1.86  109 CFU/g was obtained.
According to the results presented in Figs. 3 and 4, the addition
of only mineral salts contributed to the achievement of the maxi-
mum increase in ethanol concentration and the lowest increase
in yeast growth, because the consumption of substrate was more
directed to the production of ethanol than to the production of bio-
mass. In contrast, vitamins caused the highest increase in cell num-
ber and the lowest increase in ethanol concentration. Thus, our
preposition is to use only mineral salts as the most appropriate
activators for the SSF process by immobilized yeast, since the max-
imum increase in ethanol concentration was obtained. In addition,
it is also economically more favorable than the addition of the mix-
ture of mineral salts and vitamins.
Metal ions in fermentation media are important factors that
have an effect on yeast cell physiology and alcohol production.
Magnesium ions directly influence the rate of yeast growth, sugar
consumption and ethanol production [33]. It is generally required
by the yeast in millimolar concentration range. In this study addi-
tion of 10 mM of Mg2+ successfully contributed to the increase in
ethanol production. The impact of magnesium on ethanol produc-
tion by S. cerevisiae has been reported by several authors. Dombek
and Ingram [34] have shown that supplementing the ethanol fer-
mentation with magnesium at a level of 0.5 mM prolonged the
exponential growth, increased the yeast cell mass accumulation
and reduced the decrease in fermentation rate during the comple-
tion of batch fermentation. Birch and Walker [31] reported that
elevated concentrations of magnesium in medium (up to 50 mM)
resulted in the improvement in survival of the cells under condi-
tions with high ethanol concentrations. Also, magnesium exerted
protective effects on stressed cells resulting in reduction in cell
mortality, prevention of cell-surface damage and repression of
stress protein biosynthesis. Zinc as a trace element is very impor-
tant in yeast fermentative metabolism not only because it is essen-
tial for alcohol dehydrogenase (terminal alcohologenic Zn-
metalloenzyme), but also because it can stimulate uptake of malt-
ose and maltotriose into yeast cells, thereby increasing fermenta-
tion rates [35]. However, zinc and other necessary trace elements
for yeast growth (such as calcium, manganese, iron and copper)
could be diminished due to interactions with other trace elements
or nutrients of the substrate as well as due to protection by immo-
Alfenore et al. [8] carried out fed-batch fermentation experi-
ments on synthetic medium by S. cerevisiae with various amounts
inositol, thiamine, pyridoxine and para-aminobenzoic acid) using
an exponential feeding strategy, and they managed to significantly
production rate, and the ability of non-growing cells to sustain eth-
anol formation (this phenomenon occured for higher ethanol con-
centration). This agrees well with our results. Kotarska et al. [28]
investigated the effect of various activators on alcoholic fermenta-
tion of rye mash. They reported that application of magnesium sul-
phate (2 g/l), biotin (0.001 g/l) and biotin with thiamine (0.1 g/l)
had no significant effect on the improvement of ethanol fermenta-
tion. However, the best results were obtained with following acti-
vators: mineral mix ((NH4)2SO4, 1 g/l; KH2PO4, 1 g/l; MgSO4
7H2O,
2 g/l), soybean flour (2 g/l) and soybean oil (3.4 ml/l).
Additionally, Ca-alginate gel could express instability in the
presence of certain concentrations of substances which have a high
affinity for Ca2+ ions such as phosphate, citrate and lactate, and
ions such as K+ and Mg2+. On the other hand, avoidance of magne-
sium, phosphate and other necessary nutrients in the medium is
not possible as they are essential for the yeast metabolism, main-
tenance of cell viability and cell wall integrity. In this study we
managed to attain a high productivity of batch SSF process of corn
meal and to preserve physical and chemical stability of gel beads
by addition of mineral salts in appropriate amounts. Our current
goal is to investigate the effect of these minerals in repeated
batches during a longer period, before establishing the continuous
fermentation mode.
The values of significant process parameters, such as ethanol
concentration, ethanol yield, percentage of the theoretical ethanol
yield, volumetric productivity and utilized glucose obtained after
48 h of the SSF with and without addition of yeast activators were
assessed and presented in Table 1. These parameters are compared
to the process parameters obtained in SHF [25].
As shown in Table 1, in both the free and immobilized yeast sys-
tems addition of mineral salts contributed to the maximum in-
crease in ethanol concentration and other process parameters,
compared to the control samples. As mentioned above, the mineral
salts caused the lowest increase in yeast growth in these systems.
Comparing the values of all process parameters obtained in these
four systems, the SSF process with media supplementation by min-
eral salts has been found to be the most superior system.
4. Conclusion
Taking into consideration parameters such as ethanol concen-
tration, ethanol yield, percentage of the theoretical ethanol yield,
volumetric productivity and utilized glucose, the SSF process has
been found to be economically favorable in terms of all process
parameters and also considering lower energy consumption com-
pared to the SHF process. The maximum ethanol concentration of
9.42% w/w, the ethanol yield of 0.51 g/g, percentage of the theoret-
ical ethanol yield of 90.32%, the volumetric productivity of 1.96 g/
l h and utilized glucose of 97.72% were achieved after 48 h of the
 S. Nikolić et al. / Fuel 88 (2009) 1602–1607
SSF process. By immobilization of the yeast into Ca-alginate using a
method of electrostatic droplet generation a system exhibited low
substrate inhibition and high tolerance to ethanol since there was
not declining phase in ethanol production and the number of via-
ble cells during the SSF process. The yeast S. cerevisiae var. ellipsoi-
deus cells entrapped in Ca-alginate was appropriate for SSF of corn
meal since no substrate and product diffusion restrictions were
noticed.
The most appropriate yeast activators for immobilized cell sys-
tem were mineral salts (10 mM of Mg2+ and 1 mM of Zn2+) which
caused an increase in ethanol concentration of 8.60% compared
to the control samples. In this case, ethanol concentration of
10.23% (w/w) was achieved after 48 h of the process. Addition of
the applied amounts of magnesium and zinc contributed to the
achievement of high productivity of the batch SSF of corn meal,
while still preserving a physical and chemical stability of Ca-algi-
nate gel beads.
We are expecting additional benefits of the utilization of S. cere-
visiae var. ellipsoideus immobilized system in a continuous fermen-
tation mode, which is a part of our further research.
Acknowledgements
This work was funded by the Serbian Ministry of Science and
Technological Development (TR 18002).
References
[1] Demirbas A. Progress and recent trends in biofuels. Prog Energ Combust
2007;33:1–18.
[2] Baras J, Gaćeša S, Pejin D. Ethanol is a strategic raw material. Chem Ind
2002;56:89–105.
[3] http://www.b92.net/biz/vesti/srbija.php.
[4] Mielenz J. Ethanol production from biomass: technology and
commercialization status. Curr Opin Microbiol 2001;4:324–9.
[5] Öhgren K, Bura R, Lesnicki G, Saddler J, Zacchi G. A comparison between
simultaneous saccharification and fermentation and separate hydrolysis and
fermentation using steam-pretreatment corn stover. Process Biochem
2007;42:834–9.
[6] Rakin M, Mojovic L, Nikolić S, Vukašinović M, Nedović V. Comparative study of
bioethanol production from corn hydrolyzates using different yeast
preparations. In: Proceedings of the 15th European biomass conference and
exhibition: from research to market deployment, CD ed. Berlin,
Germany;2007:2058–63.
[7] Okunowo WO, Osuntoki AA. Quantitation of alcohols in orange wine
fermented by four strains of yeast. African J Biochem Res 2007;1:95–100.
[8] Alfenore S, Molina-Jouve C, Guillouet SE, Uribelarrea JL, Goma G, Benbadis L.
Improving ethanol production and viability of Saccharomyces cerevisiae by a
vitamin feeding strategy during fed-batch process. Appl Microbiol Biotechnol
2002;60:67–72.
[9] Najafpour G, Younesi H, Syahidah K, Ismail K. Ethanol fermentation in an
immobilized cell reactor using Saccharomyces cerevisiae. Biores Technol
2004;92:251–60.
[10] Banat IM, Nigam P, Singh D, Marchant R, McHale AP. Review: Ethanol
production at elevated temperatures and alcohol concentrations: Part I – Yeast
in general. World J Microb Biotech 1998;14:809–21.
[11] Mojović L, Nikolić S, Rakin M, Vukašinović M. Production of bioethanol from
corn meal hydrolyzates. Fuel 2006;85:1750–5.
[12] International Standards: ISO 10520. Determination of starch content – Ewers
polarimetric method; 1997.
[13] Official Methods 923.03, 925.09, 930.15, 955.04, 960.39. In: Official methods of
analysis of AOAC international, 17th ed. Gaithersburg, MD, USA: AOAC
International; 2000.
1607
[14] Miller GL. Use of dinitrosalycilic acid for determining reducing sugars. Anal
Chem 1959;31:426–8.
[15] Margaritis A, Kilonzo PM. Production of ethanol using immobilised cell
bioreactor systems. In: Nedović V, Willaert R, editors. Applications of cell
immobilisation biotechnology. Netherlands: Springer; 2005. p. 375–405.
[16] Vogelsang C, Wijffels RH, Østgaard K. Rheological properties and mechanical
stability of new gel-entrapment system applied in bioreactors. Biotechnol
Bioeng 2000;70:247–53.
[17] Nikolić S, Mojović L, Rakin M, Pejin D, Nedović V. Effect of different
fermentation parameters on bioethanol production from corn meal
hydrolyzates by free and immobilized cells of Saccharomyces cerevisiae var.
ellipsoideus. J Chem Technol Biotech 2008. doi:10.1002/jctb.2068.
[18] Montesinos T, Navarro JM. Production of alcohol from raw wheat flour by
amyloglucosidase and Saccharomyces cerevisiae. Enzyme Microb Technol
2000;27:362–70.
[19] Zhu S, Wu Y, Yu Z, Zhang X, Wang C, Yu F, et al. Simultaneous saccharification
and fermentation of microwave/alkali pre-treated rice straw to ethanol.
Biosyst Eng 2005;92:229–35.
[20] Öhgren K, Rudolf A, Galbe M, Zacchi G. Fuel ethanol production from steam-
pretreated corn stover using SSF at higher dry matter content. Biomass
Bioenerg 2006;30:863–9.
[21] Torija MJ, Rozès N, Poblet M, Guillamón JM, Mas A. Effects of fermentation
temperature on the strain population of Saccharomyces cerevisiae. Int J Food
Microbiol 2003;80:47–53.
[22] Lucero P, Peňalver E, Moreno E, Lagunas R. Internal trehalose protects
endocytosis from inhibition by ethanol in Saccharomyces cerevisiae. Appl
Environ Microb 2000;66:4456–61.
[23] Ciesarová Z, Dömény Z, Šmogrovičová D, Pátková J, Šturdík E. Comparison of
ethanol tolerance of free and immobilized Saccharomyces uvarum yeasts. Folia
Microbiol 1998;43:55–8.
[24] Verbelen PJ, De Schutter DP, Delvaux F, Verstrepen KJ, Delvaux FR.
Immobilized yeast cell systems for continuous fermentation applications.
Biotechnol Lett 2006;28:1515–25.
[25] Nikolic S, Mojovic L, Pejin D, Rakin M, Vučurović V. Improvement of ethanol
fermentation of hydrolyzates of corn semolina by immobilized Saccharomyces
cerevisiae var. elipsoideus by media supplementation. Food Technol Biotech
2009;47:84–90.
[26] Marques S, Alves L, Roseiro JC, Gírio FM. Conversion of recycled paper
sludge to ethanol by SHF and SSF using Pichia stipitis. Biomass Bioenerg
2008;32:400–6.
[27] Yamashita Y, Kurosumi A, Sasaki C, Nakamura Y. Ethanol production from
paper sludge by immobilized Zymomonas mobilis. Biochem Eng J 2008;42:
314–9.
[28] Kotarska K, Czupryński B, Kłosowski G. Effect of various activators on the
course of alcoholic fermentation. J Food Eng 2006;77:965–71.
[29] Wu H, Ito K, Shimoi H. Identification and characterization of a novel biotin
biosynthesis gene in Saccharomyces cerevisiae. Appl Environ Microb
2005;71:6845–55.
[30] Furukawa K, Kitano H, Mizoguchi H, Hara S. Effect of cellular inositol content
on ethanol tolerance of Saccharomyces cerevisiae in sake brewing. J Biosci
Bioeng 2004;38:107–13.
[31] Birch RM, Walker GM. Influence of magnesium ions on heat shock and ethanol
stress responses of Saccharomyces cerevisiae. Enzyme Microb Tech
2000;26:678–87.
[32] Mayalagu S, Patturajan M, Chatterji D. The presence of two tightly bound Zn2+
ions is essential for the structural and functional integrity of yeast RNA
polymerase II. Gene 1997;190:77–85.
[33] Rees EMR, Stewart GG. The effects of increased magnesium and calcium
concentrations on yeast fermentation performance in high gravity worts. J I
Brewing 1999;103:287–91.
[34] Dombek KM, Ingram LO. Magnesium limitation and its role in apparent
toxicity of ethanol during yeast fermentation. Appl Eviron Microb
1986;52:975–81.
[35] Magonet E, Hayen P, Delforge D, Delaive E, Remacle J. Importance of the
structural zinc atom for the stability of yeast alcohol dehydrogenase. J
Biochem 1992;287:361–5.
[36] Filipović-Kovačević Ž, Mikšaj M, Berčuk N, Jukić M. Amperometric biosensor
for monitoring respiration activity of Saccharomyces cerevisiae in the presence
of cobalt and zinc. Food Technol Biotech 2002;40:111–7.
[37] Stehlik-Thomas V, Grba S, Runjic-Peric V. Zinc uptake by Saccharomyces
cerevisiae and its impact on alcoholic fermentation. Chem Biochem Eng Q
1997;11:147–51.