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BIOETHANOL PRODUCTION FROM SAGO STARCH

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 BIOETHANOL PRODUCTION
FROM SAGO STARCH
Maizirwan Mel, Husna Bt Muhammad Nadzri, Mohd Hider Kamarudin, Mohd Ismail Abd Karim
Bioprocess and Molecular Engineering Research Unit,
Faculty of Engineering,
International Islamic University Malaysia, P.O. Box 10, 50728, Kuala Lumpur, Malaysia
Corresponding Email: maizirwan@iium.edu.my
Abstract— Two stage processes prior to ethanol
production has been proposed which is hydrolysis and
fermentation. Commercial enzymes were used in this two
steps hydrolysis; α-amylase (liquefaction step) and
glucoamylase (saccharification step). Optimization was
carried out in both stages; hydrolysis and fermentation.
Three parameters are involved in optimization of %
dextrose equivalent (DE); sago starch concentration (20%
(w/v), 30% (w/v), 40% (w/v)), glucoamylase enzyme (52
U/g, 78 U/g, 104 U/g) and time during saccharification (1,
2, 3 hours). Three parameters are involved in optimization
of ethanol; agitation (100 rpm, 150 rpm, 200 rpm),
inoculums (1% (v/v), 3% (v/v), 5% (v/v)) from constant
initial stock of 2.5x106 cells/mL and pH (4, 5, 6). Both
optimization studies were carried out using Box-Behnken
design. The experiment showed that the optimum
parameters for hydrolysis study was identified to be
glucoamylase (75.87 U/g), substrate concentration
(28.49% (w/v)), and time (2 hours) which produced 62.15
g/L glucose as the fermentation substrate. For ethanol
fermentation study, it was identified that the optimum
parameters that produced 29.25 g/L were 167 rpm
agitation, 3.43% (v/v) inoculums and pH 5.
Sago; fermentation; hydrolysis; ethanol; optimization
I. INTRODUCTION
Due to the scarce resources and increasing
environmental concern, replacing the non renewable
energy with alternative resources is one of the most
studied opportunities nowadays. One way is by utilizing
the solar energy in form of biomass to produce ethanol
also called as bioethanol [1]. Balat et. al [2] pointed out
that an appropriate blending of ethanol with
conventional fuels has proven not only in promoting a
cleaner environment but also helps balancing the
economical value of fuel price.
Bioethanol is mainly produced from sugar base
crops, starchy and cellulosic raw materials via
fermentation process. Ethanol from sugar base crops
such as sugar cane can be converted directly since the
sugar is already in fermentable form. For starchy raw
materials, hydrolysis must be conducted first before
proceeding with fermentation process [3]. Hydrolysis
process can be divided into two parts; liquefaction and
saccharification. Liquefaction process utilizes α-amylase
enzyme, whereas saccharification process utilizes
glucoamylase enzyme [3].This is an early process to
produce sugar in a readily fermentable form.
One type of starchy crops that has promising future
in bioethanol is sago palm or scientifically known as
Metroxylon sagu [4]. As cited in [4], sago palm is also
known as ‘the starch crops of the 21st century’ by most
scientists. This proves that sago palm has brighter
prospect as the source for carbohydrate as well as for
bioethanol production. Starch from sago palm is the only
commercial starch source that is derived from stem and
contains bulky amount of starch in its trunk [5].
Karim et al [5] also stated that, sago palm plantation
mainly concentrated in most tropical countries. In
Malaysia, approximately 12% of Sarawak total area is
mainly covered with sago plantation.
Generally, good production of bioethanol depends on
the availability to produce large ethanol concentrations
and maintaining high quality yield at the end of the
process [6]. Ratnam et al [6] pointed out that, substrate
selection is the main cost factor for ethanol industry.
Traditionally, ethanol fermentation greatly depends on
sugar rich substrates such as sugar cane since they are
already in form of readily fermented carbohydrate [7].
Nevertheless, sugar cane is costly and difficult to obtain
since it is categorized under seasonal crops [7].
Therefore, it is a great economic advantage to expand
the substrate choice to either starchy crops and cellulosic
materials which are cheaper compared to sugar rich
substrate [7].
Seeing it as the potential for carbohydrate sources,
the study on sago as the source for ethanol industry
should be done intensively. Starchy crops primarily
involved for ethanol production includes cereal grains
(corn, wheat, barley, sorghum), tuber roots (potato),
roots (cassava) and legumes (peas, beans) [8]. Due to the
availability and lack of involvement of sago for ethanol
production, it is a challenging opportunity to study and
optimize the parameters involved in producing high
level of ethanol product with excellent yield.
This project involved two stages of bioconversion
namely, hydrolysis and fermentation using sago starch as
the raw material. In general, hydrolysis process is carried
out to convert the long polymer like chain of
carbohydrate into monomers of glucose. In this study,
two steps of enzymatic hydrolysis process which
involves liquefaction and saccharification was carried
out.
High concentration of ethanol depends on the
availability substrate [3]. Therefore, optimization of
glucose yield is necessary to improve the overall ethanol
production. Here, the optimization process of glucose
yield focuses at saccharification step of hydrolysis.
There are three parameters involved which include
substrate, glucoamylase enzyme and time. After that,
fermentation process is carried out by optimizing the
parameter involved in the process. Three parameters
were analyzed at fermentation stage which includes pH,
agitation and inoculums size. All experiments are
conducted in shake flask in Bioprocess Engineering
Laboratory of IIUM.
The initial studies from laboratory level can be further used in
upscale level of bioethanol industry.
Mojovic et al [9] stated that US and Brazil are the major
contributors toward ethanol industry. Perhaps, studying sago
as an ethanol fermentable substrate will be able to break the
monopoly of ethanol and provides better opportunity for
Malaysia in improving their economy. In addition, the
abundance amount of sago palm provides cheap selection of
subtract that will subsequently reduce the overall cost
production of ethanol. Moreover, this will also solve the over
supply of agricultural resources and offers a great opportunity
in helping farmers in contributing towards agricultural
industry. Hence, diverging part of sago crop to ethanol
production does not only help in reducing the fuel price but
also maintaining the price of sago derived products [10].
Furthermore, it will also provide agricultural sustainability for
sago palm since it is fully utilized not only for food, but also
energy consumption [11].
The aims of this study were to optimize the process
condition for hydrolysis of sago starch to glucose with α-
amylase and glucoamylase enzyme and to optimize
fermentation process of saccharified sago starch to ethanol
using Saccharomyces cerevisiae in shake flask experiment.
II. MATERIALS AND METHODS
A. Substrate
Sago starch (powdered form containing 39% dry weight
starch content) was obtained from Sarawak Craun Research
Institute. Initially, 100 mL beaker was filled with 20, 30 and
40 g sago starch (powdered form) and added with 100 mL
distilled water (to make a total concentration of 20% (w/v),
30% (w/v) and 40% (w/v)).The resulting slurry was heated to
80ºC for 15 minutes for starch gelatinization [7].
B. Enzymes
α-Amylase for liquefaction was used and produced from
Bacillus subtilis with an activity of 25,000 U/mL. One unit of
α-amylase equals to the amount of enzyme which liquefies
soluble starch to get 1 mg dextrins at 70ºC and pH 6.0 in one
minute. Glucoamylase for saccharification was also used and
produced from Aspergillus niger with an activity of 130,000
U/mL. One unit of glucoamylase equals to the amount of
enzyme which hydrolyzes soluble starch to 1 mg glucose at
40ºC and pH4.5 in one hour.
C. Hydrolysis
Initially, 100mL beaker was filled with 20, 30 and 40 g
sago starch (powdered form) according and added with 100
mL distil water (to make a total concentration of 20% (w/v),
30% (w/v) and 40% (w/v)).The resulting slurry was heated to
80ºC for 15 minutes for starch gelatinization [7]. After
gelatinization, 25 U/g of α-amylase was added to the slurry
and heated at 80ºC using hot plate with mixing for 1 hour
using magnetic stirrer [12]. Then, the temperature was
lowered down to 50ºC for 3 minutes to make sure that the
overall temperature has already drop in preparation for
saccharification to take place. Then, the desired concentration
of glucoamylase (52 U/g, 78 U/g, and 104 U/g) was added and
left to react within the specified hours (1, 2, 3 hours). Mixing
was carried out throughout the whole reaction using a
magnetic stirrer.
D. Analyses
After hydrolysis, all samples were collected and cooled
down to 35ºC. Then, centrifugations of samples were carried
out at 5000 rpm for 30 minutes. The supernatant was collected
and reducing sugar was analyzed using dinitrosalicylic acid
(DNS) method [13]. Then % dextrose equivalent (DE) is
calculated based on equation (1) below according to [14].
% DE=g reducing sugar expressed as glucose x 100% (1)
g dry solid weight sago starch
% DE is calculated to determine the effective conversion
process to glucose based on the amount of sago starch.
After fermentation, the samples of 10 mL were collected
every 8 hours of fermentation and centrifuged at 5000 rpm for
15 minutes at 4ºC to remove the cell debris. The supernatants
left were then used for ethanol and reducing sugar analysis.
The reducing sugar analysis was carried out based on [13]
during hydrolysis. For ethanol determination, two milliliters
of the supernatant was pipette to a test tube, followed by the
addition of 2-3 drops of potassium dichromate solution
(dissolve 2 g of potassium dichromate in 80 cm³ of distilled
water and carefully add 10 cm³ of concentrated sulphuric
acid). The tube was vigorously swirled in order to ensure
homogenous suspension. The tube then was placed in a water
bath at 60ºC for about 20 minutes. Color development was
observed and the color was left to stable for at least 30
minutes at room temperature.
The color density was measured at 600 µm wavelength by
using the spectrophotometer with the blank being distilled
water. The ethanol content of the samples will be estimated by
reference to the ethanol standard curve
E. Inoculums Preparations
One colony of S. cerevisiae was isolated and inoculated
into 100 mL shake flask containing 50 mL of yeast malt under
sterilized condition. Then, it was incubated for 24 hours at
30ºC and 150 rpm. The inoculums size was prepared to have
an initial concentration of 2.5x106 cells per mL. Dilutions are
made if the concentration is too high.
F. Fermentation
For medium preparation, 0.05% (w/v) of urea and 0.05%
(w/v) of NPK (nitrogen, phosphorus and potassium) were
added to 250 mL shake flask containing 100 mL of
saccharified sago starch [12]. The glucose concentration for
the fermentation media was 62.15 g/L which was obtained
from the hydrolysis process using the optimum condition. The
pH was adjusted to pH 4, 5 and 6 by the addition of required
volume of 1M NaOH and 1M H2SO4. Then, the inoculated
yeast (1 %v/v, 3 %v/v, 5 %v/v) was added to the medium
from the same inoculums stock (2.5x10 6 cells per mL). Then,
the sample was transferred into the shaker and agitation speed
was set to 100, 150 and 200 rpm with fixed temperature of 30º
C for 72 hours [12]. Fermentation process was carried out
under sterilized condition. Readings were taken every 12
hours of fermentation for 72 hours and recorded.
G. Statistical Analysis The average triplicate value of % dextrose equivalent (DE)
Analysis of the results was done statistically with the aid was referred as dependent variable or response of Y. Table 1
of Design Expert software v 8.01. The analysis was done shows the predicted levels of response Y using equation (4)
based on Ratnam’s method [6]: were given along with actual values.
Analysis of Variance (ANOVA) is used to investigate and
TABLE 1. Actual and predicted value of % dextrose equivalent (DE)
model the relationship between a response variable with one
or more independent variable. Response Surface Methodology Factors
(RSM) uses quantitative data from appropriate experiments to Glucoamylase Substrate Time Actual Predicted
simultaneously determine and solve multi variant equations. Std. (A) (B) (C) value value
The factors that should be taken into consideration: i) system Order U/g starch (%w/v) (h) %DE %DE
is well understood where all the critical factors are known ii) 1 52 20 2 17.40 16.55
region of interest where the factor levels influencing product is 2 104 20 2 10.65 10.43
known iii) factors vary continuously throughout experimental
3 52 40 2 10.33 10.56
range has been tested iv) a mathematical function relates the
factors to the measured response and v) the response defined 4 104 40 2 12.59 13.44
by the function is a smooth curve. 5 52 30 1 11.09 11.92
6 104 30 1 10.07 10.27
From the designed experiment, 15 runs have been done for the
optimization of the parameters during hydrolysis and 7 52 30 3 11.18 10.97
fermentation. The model parameters during both optimization 8 104 30 3 10.22 9.38
processes will be: 9 78 20 1 14.43 14.45
10 78 40 1 16.85 15.79
Y= f (A,B,C) (2)
11 78 20 3 15.30 16.36
Y=b0+b1A+b2B+b3C+b11A2+b22B2+b33C2+b12AB 12 78 40 3 12.07 12.05
+b23BC+b13AC (3) 13 78 30 2 21.90 22.67
14 78 30 2 23.40 22.67
Where Y is the output (glucose (% glucose conversion),
ethanol (g/L)), predicted response; A, B and C the independent 15 78 30 2 22.70 22.67
variables, b0 the offset term, b1, b2, b3 the linear effects, b11, b22,
b33 the squared variables and b12, b23, b13 are the interaction predicted vs actual
effects.
(%DE)

25.00
III. RESULTS AND DISCUSSION R2 = 0.9799

A. Design of experiment and Statistical Analysis for 20.00

predicted value

Hydrolysis
15.00
For the optimization process of hydrolysis, three factors
namely; sago starch concentration (A), glucoamylase (B) and 10.00
time (C) were observed to study the most effective conversion
of sago starch to glucose. The overall optimization study result 5.00

of hydrolysis is shown in Table 2. In order to search for the

0.00
optimum parameter for % dextrose equivalent yield, a total of 0.00 5.00 10.00 15.00 20.00 25.00
15 treatments for each optimization were established using
actual value (%DE)
Design Expert Software with applying Box-Behnken Design
with different range of the said parameters. The quadratic
relating to the % dextrose equivalent (DE) with independent Figure 1. Parity plot showing the distribution of predicted versus actual values
variables A, B and C are as follows: of % DE

Y (%dextrose equivalent) = -69.40933 + 1.31737A
+ 1.30216B + 23.95647C
- 0.010317A2 - 0.029489B2
- 5.05582C2 + 0.00865894AB
+ 0.000580769AC - 0.14128BC (4)
Figure 1 proves that a satisfactory correlation coexists
between the actual and predicted value, wherein, the points
cluster around the linear line which indicates a good fit model.
From the ANOVA analysis, the determination coefficient R2
was evaluated as to test fit of the design experiment. The
mathematical adjust of those values generated a R2 = 0.9799,
revealing that the model could not explain only 2.01% of the
overall effects, showing that it is a robust statistical model
[15]. Table 2 presented the corresponding analysis of variance
(ANOVA).
 DESIGN-EXPERT Plot dextros e equivalent (%)
TABLE 2. ANOVA for Response Surface Quadratic Model during hydrolysis dextrose eq uivalent
40. 00

Desig n Points
14.5355 20.6994
Sum of Mean Prob > X = A: g lucoamylase

B: substrate (%w/v)
16.5901
Y = B: substrate
Source Squares DF Square F Value F Actual Factor
35. 00

C: time = 2.00
18.6448
Model 311.38 9 34.60 27.03 0.0010 significant 18.6448

30. 00 3 16.5901

A 5.25 1 5.25 4.10 0.0988

B 4.42 1 4.42 3.45 0.1222 25. 00

14.5355

C 1.69 1 1.69 1.32 0.3022

20. 00
52. 00 65. 00 78. 00 91. 00 104. 00

A2 179.60 1 179.60 140.31 0.0001 significant

A: gluc oam y las e (U/g)
B2 32.11 1 32.11 25.08 0.0041 significant DESIGN-EXPERT Plot

dextrose eq uivalent
X = A: g lucoamylase
C2 94.38 1 94.38 73.74 0.0004 significant Y = B: substrate

Actual Factor 22.7541

C: time = 2.00
AB 20.27 1 20.27 15.84 0.0105 significant

(%)
19.6721

16.5901

AC 0.000912 1 0.000912 0.0007125 0.9797

dextrose equivalent
13.5082

10.4262

BC 7.98 1 7.98 6.24 0.0547

Residual 6.40 5 1.28

40.00
Lack of not 35.00
104.00

Fit 5.28 3 1.76 3.13 0.2513 significant 30.00

91.00

78.00
Pure B: s ubs trate 25.00 65.00
A: gl uc oamyl as e
Error 1.12 2 0.56 (%w/v) 20.00 52.00
(U/g)
Cor
Total 317.78 14
Figure 2. Response surface described by the model equation to estimate %
dextrose equivalent value over independent variables substrate concentration
To determine the significance of the overall model and % (w/v) and glucoamylase enzyme U/g. (i) Upper: Contour plot surface
individual model term, it depends on the value of Prob. > F. response (ii) Lower: 3D plot surface response.
Prob. > F less than 0.0500 indicates that the model is
significant. From the result, the overall ANOVA for response DESIGN-EXPERT Plot dextros e equivalent (%)
surface for quadratic model demonstrates a significant model dextrose equivalent
3. 00
12.4808

with value of Prob > F= 0.0010. Based on Table 4.2, A2 Design Points 14.5355
14.5355
X = A: glucoamylase
(0.0001), B2 (0.0041), C2 (0.0004) and AB (0.0105) are Y = C: time
2. 50
16.5901
C: time (hours)

significant model terms. Other than Prob. > F, F-test also can Actual Factor
B: substrate = 30.00

be used to determine the significance of the individual model 16.5901

2. 00 3
terms. As the F value >10, indicates the model terms are
significant. From Table 4.2, A2, B2, C2 and AB show
significant model terms with F value 140.31, 25.08, 73.74 and 1. 50
20.6994

15.84 respectively. 14.5355

The “Lack of Fit-F value” implies the Lack of Fit is not 1. 00

14.5355 18.6448

significant relative to the pure error. There is a 25.13% 52. 00 65. 00 78. 00 91. 00 104. 00

chances that the “Lack of Fit-F value” this large could occur A: gluc oam y las e (U/g)
due to noise. Based on the ANOVA analysis, it can be DESIGN-EXPERT Plot

concluded that the significant variables with largest effect on dextrose equivalent
X = A: glucoamylase
% dextrose equivalent are squared term of glucoamylase Y = C: time

Actual Factor
enzyme (A2), squared term of sago starch concentration (B 2) B: substrate = 30.00
22.6783
(%)

19.3543
and squared term of time (C2) and interaction between 16.0303

glucoamylase and sago starch (AB). Other variables which

dextrose equivalent

12.7063

excluded from the said variables do not have great interaction 9.38228

towards % dextrose equivalent (DE).

To investigate the effects of each independent variable,
3.00
the 3D response surface and contour plots response surface are 2.50
104.00

used. These two techniques are the graphical representation of 2.00

78.00
91.00

regression equation in order to determine the optimum values C: time 1.50 65.00
A: gluc oamylas e
(hours)
of variables [16]. The maximum predicted value is referred by 1.00 52.00
(U/g)

the surface defined in the smallest ellipse in the contour

diagram. The perfect interaction between the independent Figure 3. Response surface described by the model equation to estimate %
variables can be shown when elliptical contours are obtained dextrose equivalent value over independent variables time (hrs) and
[17]. The graph representations are shown in Figure 2 to glucoamylase enzyme (U/g). (i)Upper:Contour plot surface response(ii)
Figure 4. Lower: 3D plot surface response
DESIGN-EXPERT Plot
3. 00
dextros e equivalent (%) TABLE 3. Actual and predicted value of ethanol (g/L) after 72 hours
14.5355
dextrose eq uivalent
Desig n Points 18.6448 16.5901

X = B: substrate Actual Predicted

Y = C: time
2. 50
Factors value value
Actual Factor Agitation Inoculums pH ethanol ethanol
A: g lucoamylase = 78.00
(A) (B) (C) g/L g/L
(hours) Std. Order RPM (%v/v)
C: time
2. 00 3

1 100 1 5 9.7 10.58

1. 50
2 200 1 5 16.02 16.23
20.6994

16.5901
3 100 5 5 14.36 14.15
18.6448
1. 00
20. 00 25. 00 30. 00 35. 00 40. 00 4 200 5 5 21.54 20.66

DESIGN-EXPERT Plot B: s ubs t rat e (%w/v) 5 100 3 4 20.04 19.06

dextrose eq uivalent
X = B: substrate
Y = C: time 6 200 3 4 17.67 17.37
Actual Factor 22.7119
A: g lucoamylase = 78.00 7 100 3 6 9.07 9.37
(%)

20.0455

17.379
8 200 3 6 22.25 23.23
dextrose equivalent

14.7126

12.0461
9 150 1 4 16.73 16.83

10 150 5 4 18.3 19.48

3.00

2.50
40.00 11 150 1 6 14.75 13.57
35.00
2.00
30.00
C: ti me 1.50
12 150 5 6 19.01 18.91
25.00
(hours) B: s ubs trate
1.00 20.00 (%w/v)
13 150 3 5 28.09 25.30
Figure 4. Response surface described by the model equation to estimate
% dextrose equivalent value over independent variables time (hrs) and 14 150 3 5 22.59 25.30
substrate (%w/v). (i) Upper: Contour plot surface response (ii) Lower: 3D
plot surface response 15 150 3 5 25.22 25.30
Based on the three figures of surface responds and
contour plots, it is observed that maximum dextrose equivalent
was found to be between 2 to 2.5 hours, glucoamylase enzyme predicted vs actual value
between 65 U/g to 78 U/g and substrate concentration 25% to
30% (w/v) in order to obtain an optimum of 22.76% dextrose 30
equivalent value for this experiment. R2 = 0.944
predicted value (g/L)

25
From the optimization study of hydrolysis, it is
suggested by Design Expert software that the combination 20
process between glucoamylase (75.87 U/g), substrate
concentration (28.49% w/v), and time (2 hours) will produce 15
an optimum degree of hydrolysis of 22.76% dextrose
10
equivalent.
5

B. Design of experiment and statistical analysis for 0

fermentation 0 5 10 15 20 25 30
In optimization of ethanol fermentation study there are actual value (g/L)
three parameters involved; agitation (A), inoculums (B) and
pH (C) in determination of optimum ethanol yield. In order to Figure 5. Parity plot showing the distribution of predicted versus actual values
search for the optimum combination parameters, a total of 15 of ethanol yield after 72 hours
treatments for each optimization were established using
Design Expert Software with applying Box-Behnken Design The parity plot which is in figure 5, shows a satisfactory
at different range of the said parameters. The quadratic correlation coexists between the actual and predicted values,
relating to the production of ethanol with independent wherein, the point’s lines within the linear line which indicates
variables A, B and C are as follows: a good fit model. From the ANOVA analysis, the
determination coefficient R2 was evaluated as to test fit of the
Y (ethanol g/L) = -51.2887+0.255675 A+6.46315 B design experiment. For this study, the model shows a good
+17.62125C-0.00197A2-1.24438B23.125C2 determination coefficient R2 =0.944 which implies 94.4% of
+0.00215AB+0.07775AC-0.33625BC (5) the sample variation in the ethanol production is attributed to
the independent variables; agitation, inoculums and pH. The
The average triplicate value of ethanol production after 72 R2 value also indicates that about 5.6% of the variation is not
hours sampling was referred as dependent variable or response explained by the model [15]. Table 4 shows the corresponding
of Y. Table 3 shows the predicted levels of response Y using analysis of variance (ANOVA) is presented in.
equation (5) were given along with actual values.
 TABLE 4. ANOVA for Response Surface Quadratic Model during
fermentation DESIGN-EXPERT Plot
5.00
ethanol
ethanol (g/L)
Sum of Mean F Prob > Design Points
Source Squares DF Square Value F X = A: agitation
Y = B: inoculum 4.00
Model 365.38 9 40.60 9.362 0.0120 significant

B: inoculum (%v/v)
Actual Factor 23. 4159
C: pH = 5.00
A 73.87 1 73.87 17.036 0.0091 significant
3.00 3
B 32.04 1 32.04 7.389 0.0419 significant
18. 2816

C 7.33 1 7.33 1.691 0.2501 20. 8487

2.00

A2 89.29 1 89.29 20.591 0.0062 significant 15. 7144

13. 1472
B2 91.48 1 91.48 21.096 0.0059 significant 1.00
18. 2816

100.00 125.00 150.00 175.00 200.00

C2 36.06 1 36.06 8.315 0.0344 significant

A: agitation
AB 0.18 1 0.18 0.043 0.8445 DESIGN-EXPERT Plot
(rpm)
ethanol
AC 60.45 1 60.45 13.941 0.0135 significant X = A: agitation
Y = B: inoculum
BC 1.81 1 1.81 0.417 0.5468
Actual Factor 25.9831
C: pH = 5.00
Residual 21.68 5 4.34 22.1323

Lack of Not 18.2816

Fit 6.55 3 2.18 0.288 0.8341 significant

ethanol(g/L)
14.4308
Pure 10.58
Error 15.13 2 7.57
Cor
Total 387.06 14
5.00
200.00
The probability P-value was also relatively low (Pmodel> F 4.00
175.00
3.00
=0.012) indicates the significance of the model where B: i nocul um
150.00
2.00 125.00
significance is judge on Prob > F less than 0.0500. Meanwhile, 1.00 100.00
A: agi tati on

model terms with values of Prob > F greater than 0.1000 (%v/v)
(rpm)
indicate the model terms are not significant. Based on Table Figure 6. Response surface described by the model equation to ethanol yield
value over independent variables agitation (rpm) and inoculums % (v/v). (i)
4.4, A (0.0091), B (0.0419), A2 (0.0062), B2 (0.0059), C2
Upper: Contour plot surface response (ii) Lower: 3D plot surface response.
(0.0344) and AC (0.0135) are significant model terms.
The “Lack of Fit-F value” implies the Lack of Fit is not
significant relative to the pure error. There is 83.41% chances DESIGN-EXPERT Plot
6.00
ethanol (g/L)
that the “Lack of Fit-F value” this large could occur due to ethanol
Design Points 13.1472

noise. Based on the ANOVA analysis, it can be concluded that X = A: agitation

15.7144

Y = C: pH
the significant variables with largest effect on % dextrose 5.50 18.2816

Actual Factor
equivalent are agitation (A), inoculums (B), squared term of B: inoculum = 3.00 20.8487

agitation (A2), squared term of inoculums (B2), squared term

C: pH

5.00 3
of pH (C2) and interaction of agitation and pH (AC).Other
variables which excluded from the said variables do not have
great interaction ethanol production. The 3D response surface 4.50

and contour plots response surface are used to further 23.4159

20.8487

investigate the effects of each independent variable. The

4.00
maximum predicted value is referred by the surface defined in 100.00 125.00 150.00 175.00 200.00

the smallest ellipse in the contour diagram. The graph

A: agitation (rpm)
representations are shown in Figure 6 to Figure 8. DESIGN-EXPERT Plot

Based on the three plots, an optimum ethanol production ethanol

X = A: agitation
which was about 26 g/L was found to achieved at an Y = C: pH

Actual Factor
approximate parameter of pH 5, 167 rpm and 3% (v/v) B: inoculum = 3.00
25.7691

inoculums from 62.15 g/L of saccarified sago starch by two 21.6702

(g/L)

17.5714
stages bioconversion processes (hydrolysis and fermentation). 13.4726

Where, approximately 41.8% of substrate was converted into

ethanol

9.37375

ethanol. The ethanol yield was slightly lower compared to the

theoretical maximum yield of ethanol stated by Chiaramonti
[3] where from 1 kg of mash can yield approximately 0.5111 6.00

kg of ethanol. In Ratnam’s study [6], an optimum ethanol 5.50

175.00
200.00

production of 70.68 g/L was produced from 140 g/L sago 5.00
150.00

starch with agitation speed 200 rpm, pH5 and 5% (v/v) C: pH 4.50 125.00
A: agi tati on
4.00 100.00
inoculums which is about 50.48% of the sago starch was (rpm)

converted into ethanol using simultaneous saccharification and

Figure 7. Response surface described by the model equation to ethanol yield
fermentation with glucoamylase (AMG) and Zymomonas value over independent variables agitation (rpm) and pH. (i) Upper: Contour
mobilis. plot surface response (ii) Lower: 3D plot surface response.
 DESIGN-EXPERT Plot
6.00
ethanol (g/L)
15.7144 20.8487
ethanol
Design Points IV. CONCLUSION
X = B: inoculum
Y = C: pH 5.50
18.2816
The present study has been performed to produce ethanol
Actual Factor by two stage conversion process namely; hydrolysis and
A: agitation = 150.00 20.8487
fermentation using sago starch as a substrate. Sago starch was
supplied from Sarawak Craun Research Institute. The
C: pH
5.00 3

production of bioethanol from sago starch in this project has

proven to be feasible in somewhat its large scale production
4.50
promising high economic advantages as well as fulfill the
23.4159
demand for ethanol. Likewise, this laboratory scale study also
4.00 offers the basis in scaling up the potential process for the
1.00 2.00 3.00 4.00 5.00
production of bioethanol in industrial scale. The optimum %
DESIGN-EXPERT Plot B: inoculum (%v/v) dextrose equivalent (DE) has been obtained at was 22.76% (g
ethanol
X = B: inoculum
glucose/g starch) with optimal parameters at 75.87 U/g
Y = C: pH glucoamylase, 28.49% substrate concentration and 2 hours
Actual Factor 25.5522
A: agitation = 150.00
incubation.
22.5557

The results obtained from validation study proved that

(g/L)

19.5592

16.5627 the optimize parameters can be used for the improvement of

ethanol

13.5663
the process. However, this value is comparatively low with
other studies and further improvement throughout the
hydrolysis stage can be carried out to overall improve the
6.00
5.00
ethanol production.
5.50
4.00
5.00
3.00 ACKNOWLEDGMENT
C: pH 4.50 2.00

4.00 1.00
B: i nocul um
(%v/v)
We would like to acknowledge the Faculty of Engineering
IIUM Malaysia for the support. Also, we thank Najiah Nadir
Figure 8. Response surface described by the model equation to ethanol yield for her useful guidance, Mohd Hafizul Shaibon and Sukiman
value over independent variables pH and inoculums % (v/v). (i) Upper:
Sengat for their technical assistance.
Contour plot surface response (ii) Lower: 3D plot surface response.
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