Process Improvement of Pharmaceutical Grade Ethanol Production Using

Sweet Sorghum
1

Najiah Nadir, 1Maizirwan Mel, 1Mohd Ismail Abdul Karim, 2Rosli Mohd Yunus

Bioprocess Engineering Research Group

Department of Biotechnology Engineering,
Faculty of Engineering,
International Islamic University Malaysia, P. O Box 10, 50728 Kuala Lumpur, Malaysia
2

Department of Chemical Engineering

Faculty of Chemical & Natural Resources Engineering,
Universiti Malaysia Pahang, MEC City, 26300 Gambang, Kuantan, Pahang, Malaysia

______________________________________________________________________________
Abstract
The conversion of starch to sugar can be achieved by hydrolysis process. The two-step enzymatic hydrolysis of
sweet sorghum was performed by commercially available -amylase and glucoamylase and further ethanol
fermentation of the obtained hydrolyzates by Saccharomyces cerevisiae was studied. In order to attain a higher
ethanol yield, optimization study was carried out in the 2 litres stirred tank bioreactor, B-Braun fermenter to
investigate the effect of main factors of the hydrolysis process, namely, amount of substrate, temperature, time, and
amount of enzyme for the maximum production of bioethanol. As shown in the analysis of variance (ANOVA)
result, the amount of substrate, liquefaction and saccharification temperature have contributed more significant
effect on hydrolysis process of sweet sorghum.
KEYWORDS: sweet sorghum; Saccharomyces cerevisiae; hydrolysis; fermentation; ethanol

1. Introduction
The worlds leading manufacturers and
industries are seeking to substitute petrochemicalbased feedstock with agricultural-based materials as
petroleum supplies continue to decline [1]. Great
attention has been given to the ethanol production
using various substrates which can be classified into
three main types of materials, which are sugars (from
sugarcane, sugar beet, sweet sorghum, molasses, and
fruits), starches (from sweet sorghum grain, cassava,
corn, potato, and root crops), and cellulose materials
(from agricultural residue, wood, and paper mills)
[2], because of the increase in demand for ethanol
which is considered as an alternative energy source
[3].
Sweet sorghum (Sorghum biocolor (L.) Moench)
is one of the most favourable crops for industrial
applications [1]. Sorghum is a C4 plant characterized
by a high biomass- and sugar-yielding crop [4]. It
contains approximately equal quantities of soluble
(glucose and sucrose) and insoluble carbohydrates
(cellulose and hemicellulose) [5]. Sweet sorghum has
the ability of remaining dormant during the driest
periods and is often judged to be one of the most

drought resistant agricultural plants [6, 7]. Thus, it

can be planted primarily in semiarid and dried parts
of the world, especially in areas too dry for maize [1].
Also, it has been considered as an important energy
plant for the production of fuel bioethanol [8].
Sweet sorghum grain is a starch-rich grain [1].
Starch consists of two types of polysaccharides, the
linear molecule, amylose and a highly branched
molecule, amylopectin [9]. Amylose is a linear
molecule of (14) linked -D-glucopyranosyl units
(-D-(14)-glucan), but it is well established that
some molecules are slightly branched by (16)-linkages. Meanwhile, amylopectin is a highly
branched component of starch formed through chains
of -D-glucopyranosyl residues linked together
mainly by (14) linkages but with 5-6% of (16)
bonds at the branch points. It is a branched
polysaccharide composed of hundreds of short
(14)--glucan chains, which are interlinked by
(16)--linkages [10, 11]. In most common types of
cereal endosperm starches, the relative weight
percentages of amylose range between 18-33% and
amylopectin range between 72-82% amylopectin
[10].

Starchy grains and effluent generated from starch

processing units are the cheap feedstocks and could
be used as potential raw materials for ethanol
fermentation [12]. The sweet sorghum starch
hydrolysis may be regarded as a first and important
step in sorghum processing for bioethanol production
[11]. Enzymatic hydrolysis is essential for the
production of glucose syrups from starch because of
the specificity of enzymes allows the sugar syrups
production with well-defined physical and chemical
properties and the milder enzymatic hydrolysis
results in few side reactions and less browning
[2]. Conventional process for production of
bioethanol from starch basically involved a threestage process liquefaction of starch by -amylase,
saccharification of liquefied starch by glucoamylase
and followed by fermentation of sugar to ethanol
using Saccharomyces cerevisiae [11, 12].
The aim of this study was to investigate the
liquefaction and saccharification processes of sweet
sorghum by commercially available -amylase and
glucoamylase and ethanol fermentation of the
obtained hydrolyzates by Saccharomyces cerevisiae
yeasts. The conditions of starch hydrolysis such as
the substrate and enzyme concentration and the
temperature and time required for the enzymatic
action were optimized taking into account both the
effects of hydrolysis and the ethanol fermentation.
Then, the most significant factors that affect the
hydrolysis and fermentation processes have been
quantified from the STATISTICA 8 of Taguchi
Orthogonal Array Design for the maximum ethanol
yield.
2. Materials and Methods
2.1. Substrates
Sweet sorghum grains were obtained from
Indonesian supplier and blended into small size of
approximately 100 m to enhance the hydrolysis
process.
2.2. Microorganisms
Saccharomyces cerevisiae yeast was obtained from
local market in dry form. For inoculum, 100 ml of
distilled water was heated to 40 C in a shake flask.
After that, 0.5% (w/w) of Saccharomyces cerevisiae
yeast was added into the warmed water to activate the
yeast. The mixture was left for 5-10 min at 150 rpm.

2.3. Enzymes

Both -amylase from Bacillus subtilis and

glucoamylase from Aspergillus niger were obtained
from local market. The activities of the two enzymes
were 90% each.
2.4. Hydrolysis
The two litres B-Braun bioreactor was filled with
200 g of sweet sorghum and 900 ml of distilled water.
Then, 0.05% (v/w) of -amylase (from the amount of
sorghum) was added and the mixture was cooked at
80 C and 500 rpm for 1 h. After 1 h, the mixture was
cooled down to 50 C and 0.05% (v/w) of
glucoamylase was added and the mixture was left for
2 h with 250 rpm agitation. Next, the solution was
cooled down to 35 C and the pH was adjusted to pH
5.
2.5. Fermentation
In the meantime, 0.5% (w/w) of urea and 0.05%
(w/w) of NPK (nitrogen, phosphorus, and potassium)
were added to the bioreactor. After 10 min, the
activated yeast solution was added to the two liters
bioreactor. The mixture was mixed well and left for 8
hours without agitation. Then, the agitation was
changed to 50 rpm until 72 h of incubation.
2.6. Analysis
Data was collected for every 8 hours. The
concentration of glucose and ethanol were
determined by centrifuging at 5000 rpm for 30
minutes to remove cells. The supernatant was
analyzed by HPLC using an SUPELCOGEL C-610H
column equipped with a refractive index detector.
Separations were performed at 30 C, eluted at 0.5
mL/min using 0.1% H3PO4.
2.7. Experimental design and optimization
Taguchi Orthogonal Array Design was used to
improve the hydrolysis process for maximum
bioethanol production. Amount of substrate (A,
gram), liquefaction temperature (B, C), liquefaction
time (C, hour), amount of -amylase (D, % (v/w)),
saccharification temperature (E, C), saccharification
time (F, hours), and amount of glucoamylase (G, %
(v/w)) were chosen for the independent variables as
shown in Table 1. Ethanol concentration (Y, % (v/v))
was used as the dependent output variable.

Table 1.
Independent variables in the experimental design
Variables

Symbol

Amount of substrate, g
Liquefaction temperature, C
Liquefaction time, h
Amount of -amylase, % (v/w)
Saccharification temperature, C
Saccharification time, h
Amount of glucoamylase, % (v/w)

A
B
C
D
E
F
G

3. Results and Discussion

regression model relating the ethanol (Y) with the

independent variables, amount of substrate (A),
liquefaction temperature (B), saccharification
temperature (E) and saccharification time (F) is as
follows:
Y = -5.41975 + 0.030825A + 0.11170B 0.048800E - 0.18325F
(1)

3.1. Optimization of hydrolysis process

Eight experiments were performed using different
combinations of the variables according to Taguchi
OA Design. Using the results of the experiments, the
Table 2.
The observed and predicted results for ethanol yield
Run
Factors
A
B
C
D
E
(g)
(C)
(h)
(% (v/w))
(C)
1
2
3
4
5
6
7
8

200
200
200
200
300
300
300
300

80
80
90
90
80
80
90
90

1
1
2
2
2
2
1
1

0.05
0.1
0.05
0.1
0.05
0.1
0.05
0.1

50
60
50
60
60
50
60
50

The predicted ethanol concentration using

Equation (1) was given in Table 2 along with the
observed value. The results were analyzed using the
analysis of variance (ANOVA) as appropriate to the
experimental design used as shown in Table 3. The
Table 3.
ANOVA for the entire model
Effect
Sums of Squares
Regression
22.24392
Residual
0.08453
Total
22.32846
In this study, the determination coefficient, R 2 =
0.9962 indicates a high correlation between the
experimentally observed and predicted values and
indicates the degree of precision with which the
ethanol yield is attributed to the independent

Coded levels
Low (1)
High (2)
200
300
80
90
1
2
0.05
0.1
50
60
2
4
0.05
0.1

F
(h)

G
(% (v/w))

2
4
4
2
2
4
4
2

0.05
0.1
0.1
0.05
0.1
0.05
0.05
0.1

Response
Y
(% (v/v))
Observed
6.899
6.161
7.607
7.357
9.451
9.444
10.244
11.215

Predicted
6.875
6.020
7.625
7.504
9.469
9.591
10.220
11.074

computed F-value (197.3507) indicates that the

model was highly significant at high confidence
level. The probability p-value was also relatively low
(pmodel > F = 0.000581) indicates the significance of
the model.

df
4
3

Mean Squares
5.560981
0.028178

F-value
197.3507

p-level
0.000581

variables. The matching quality of the data obtained

by the model proposed in Equation (1) was evaluated
considering the correlation coefficient, R2, between
the experimental and modeled data.

Table 4.
Regression Summary
Intercept
Substrate
Liq Temp
Sacch Temp
Sacch Time

Beta

Standard Error
of Beta

0.922547
0.334302
-0.146051
-0.109688

0.035524
0.035524
0.035524
0.035524

Table 4 shows the regression summary and the

corresponding values of the variable estimation. The
p-values were used to check the significance of each
coefficient. The lower the p-value (less than 0.05)
indicates the more significant correlation of
coefficients. Thus, from the statistics, it can be
evaluated that only the amount of substrate,
liquefaction and saccharification temperature are the
significant factors which affect the production of
ethanol.

B
3.529750
3.082500
1.117000
-0.488000
-0.366500

Standard
Error of B
0.361004
0.118697
0.118697
0.118697
0.118697

t(3)

p-level

9.77759
25.96939
9.41048
-4.11129
-3.08768

0.002273
0.000125
0.002542
0.026061
0.053811

3.2. Effect of substrate concentration

Fig. 2. Effect of amount of substrate on ethanol

production

Fig. 1. Means plot for main factors of the hydrolysis

process
The mean plot analysis (Fig. 1) indicates the
amount of substrate is the most significant factor
which affects the ethanol production from sweet
sorghum, followed by liquefaction temperature,
saccharification temperature, saccharification time,
liquefaction time, glucoamylase and -amylase.

In this study, the substrate concentration has

positive effect on both hydrolysis and fermentation
processes was assessed, which means higher amount
of substrate gives higher amount of ethanol. The
maximum concentration of ethanol was obtained for
the highest sweet sorghum starch concentration in the
mixture, which is 300 g as shown in Fig. 1. However,
according to Mojovi et al. [11], the substrate
concentration had a pronounced effect on the starch
hydrolysis and the ethanol fermentation. Regarding
the yields, lower amount of substrate is more
appropriate as substrate inhibition could be avoided.
On the other hand, Aggarwal et al. [13] mentioned
that 25% of sorghum slurry was more appropriate for
liquefaction process. Meanwhile, from Fig. 2, the
interaction between substrate concentration and
liquefaction temperature provides higher effect
compared to the interaction of substrate with other
factors as the p-value is the lowest. But the factors
interaction was not significant to the ethanol
production as the p-value is greater than 0.05.

Fig. 4. Effect of liquefaction time on ethanol

concentration
3.3. Effect of liquefaction temperature
As shown in Fig. 1, liquefaction time has slightly
negative effect on ethanol production. As the
liquefaction time increases, the amount of ethanol
produced decreases. This is because the longer
exposure of the enzyme to high temperatures, which
are needed for gelatinization of the starch granules
and for achieving a good susceptibility to enzyme
action, could lead to slight enzyme deactivation [11].
Furthermore, Fig. 4 shows that the interaction
between liquefaction time and substrate concentration
provides higher effect compared to other factors.
Also, this interaction is significant to the production
of ethanol since the p-value is around 0.03.
Fig. 3. Effect of liquefaction temperature on ethanol
concentration
From Fig. 1, it can be seen that the liquefaction
temperature gives positive effect because ethanol
concentration increases as the temperature increases.
As stated by Aggarwal et al. [13], liquefaction under
pressurized steam at 104 C was more effective
compared to using a water bath at 95 C. On the other
hand, Apar and zbek [14] found that the optimum
temperature of starch hydrolysis was found as 65 C
from the studies at various temperature values
because the relative enzyme activity (in both
presence and absence of the starch) decreases as the
temperature increases and reaches a value of zero at
70 C due to the inhibitory effects of temperature.
However, Snchez and Cardona [15] mentioned that
for amylases to attack starch, these suspensions
should be brought to high temperatures (90-110 C)
for the breakdown of starch kernels. Moreover, as
shown in Fig. 3, the interaction between liquefaction
temperature and substrate concentration provides
higher effect, similar to Fig. 2.
3.4. Effect of liquefaction time

3.5. Effect of concentration of -amylase

Fig. 5. Effect of the amount of -amylase enzyme on

ethanol concentration
For -amylase enzyme, it provides almost no
effect or very low positive effect on the ethanol
concentration as shown in Fig. 1. However, Aggarwal
et al. [13] mentioned that the optimum -amylase
enzyme concentration was 0.15% (v/w) for 60 min of
liquefaction time, which is higher than the
concentration used in this study. Meanwhile, from
Fig. 5, the interaction between -amylase enzyme
and amount of substrate provides higher effect
compared to the interaction with others. All
interactions provide significant effect as all p-values
are smaller than 0.05.

3.6. Effect of saccharification temperature

compared to the effect of saccharification

temperature. As the saccharification time increases,
the amount of ethanol produced decreases.
Furthermore, Fig. 7 shows that the interaction
between saccharification time and substrate
concentration provides higher effect compared to
other factors. However, this interaction is not
significant to the production of ethanol since the pvalue is around 0.07.
3.8. Effect of concentration of glucoamylase

Fig. 6. Effect of saccharification temperature on

ethanol concentration
From Fig. 1, it can be seen that the
saccharification temperature gives negative effect
because ethanol concentration decreases as the
temperature increases. As stated by Aggarwal et al.
[13], the maximum saccharification occurred at 45 C
because the rate of saccharification reduced
substantially at higher temperature. However, the
glucoamylase
enzyme
activity
increased
progressively with an increase in temperature from
20 C and reaching maximum at 60 C [16].
Meanwhile, from Fig. 6, the interaction between
saccharification
temperature
and
substrate
concentration provides highest effect compared to the
interaction of other factors as the p-value is the
lowest. But the interaction was not significant to the
ethanol production as the p-value is greater than 0.05.
3.7. Effect of saccharification time

Fig. 8. Effect of the amount of glucoamylase enzyme

on ethanol concentration
For glucoamylase enzyme, it provides slightly
positive effect on the ethanol concentration as shown
in Fig. 1. According to Mojovi et al. [11], the lower
amount of enzyme is sufficient for the effective
conversion of the substrate. Also, the two-step
enzymatic process was shown to be more efficient
than one-step enzymatic action. Moreover, as shown
in Fig. 8, the interaction between glucoamylase
enzyme and substrate concentration provides highest
effect, which is significant. As indicated by all
ANOVA result, the mean for ethanol yield has been
achieved at 8.5 % (v/v) at Sigma value of 1.7860.
4. Conclusion
From the Taguchi OA Design, the most important
factors that affect the hydrolysis and fermentation
processes of sweet sorghum are the amount of
substrate,
liquefaction
and
saccharification
temperature.
References

Fig. 7. Effect of saccharification time on ethanol

concentration
As shown in Fig. 1, saccharification time has
slightly lower negative effect on ethanol production

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