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Najiah Nadir1, Maizirwan Mel2, Mohd Ismail Abd Karim3, Rosli Mohd Yunus4
123
Bioprocess and Molecular Engineering Research Unit, Faculty of Engineering, International Islamic University
Malaysia, P.O. Box 10, 50728, Kuala Lumpur, Malaysia
4
Department of Chemical Engineering, Faculty of Chemical and Natural Resources Engineering, Universiti Malaysia
Pahang, MEC City, 26300 Gambang, Kuantan, Pahang
A. Substrates
Sweet sorghum grains were obtained from Indonesian
Bioenergy Foundation.
B. Microorganisms
Dried-form industrial Saccharomyces cerevisiae
yeast was used in this research.
C. Enzymes
Both -amylase from Bacillus subtilis and
glucoamylase from Aspergillus niger were obtained from
enzyme industry in Riau, Indonesia. The activities of the
two enzymes were identified to be 25,000 U/mL and
130,000 U/mL, respectively.
D. Substrate Preparation
Sweet sorghum grains were blended into small size
of approximately 20 m to enhance the hydrolysis
process. The microstructure of the starch granules were
viewed with a field emission scanning electron
microscope (FESEM).
E. Hydrolysis
Hydrolysis was performed based on previous
optimization study [22]. The shake flask was filled with
100 mL of distilled water and heated to 90C. Then, 25 g
of sweet sorghum was added to the flask (to make 25%
(w/v) of substrate). After that, 25 U/g of -amylase (from
the amount of sorghum) was added and the mixture was
cooked at 90C for 1 hour. After 1 hour, the mixture was
cooled down to 47C and 313 U/g of glucoamylase was
added and the mixture was left for 2 hours. Mixing was
carried out throughout the whole reaction using a
magnetic stirrer. After 2 hours, the mixture was cooled
down to 35C for fermentation purpose.
F. Inoculum Preparation
For inoculum preparation by using incubator shaker,
10 mL of distilled water was heated to 40C in a shake
flask. After that, 0.3% (w/w) of S. cerevisiae yeast was
added into the warmed water to activate the yeast. The
mixture was left for 10 minutes at 150 rpm.
G. Fermentation
For fermentation, 0.3% (w/w) of urea and 0.05%
(w/w) of NPK (nitrogen, phosphorus, and potassium)
were added to the flask containing 90 mL of
hydrolyzed sorghum and the mixture was mixed well.
The pH was then adjusted to pH 6.0. After 10 minutes,
the activated yeast solution was added to the media.
The fermentation was performed at 35C and 150 rpm
for 72 hours. The procedure was repeated according to
the experimental design. During the fermentation, data
was collected for every 12 hours for the measurement
of glucose and ethanol concentrations. For screening
part, the fermentation was done by using incubator
shaker. Meanwhile, the optimization part was
performed by using 2L bioreactor.
H. Glucose and Ethanol Determinations
Samples for glucose and ethanol determination were
centrifuged at 5000 rpm for 30 minutes to remove the
substrates and cells. The supernatant was filtered through
a 0.45 m membrane and analyzed by high performance
liquid chromatography (HPLC) equipped with a refractive
index detector. The pre-column and column used for
Variables
Symbol
Inoculum, % (w/w)
Urea, % (w/w)
NPK, % (w/w)
Temperature, C
Initial pH
Agitation, rpm
A
B
C
D
E
F
Coded levels
Low
High
(1)
(2)
0.3
0.6
0.3
0.6
0.05
0.1
30
35
5.0
6.0
100
150
(2)
(3)
where Y is the dependent variable (ethanol yield); A, B,
E, and F are the independent variable (amount of
inoculum, urea, pH, and agitation, respectively).
Variables
-2
Coded levels
-1
0
+1
+2
X1
0.1
0.2
0.3
0.4
0.5
X2
X3
5.5
25
6.0
50
6.5
75
7.0
100
7.5
125
Symbol
Inoculum, %
(w/w)
Initial pH
Agitation, rpm
Figure 1. FESEM images (1000x) for sweet sorghum (scale bar=10 m).
TABLE III. THE OBSERVED AND PREDICTED RESULTS FOR ETHANOL YIELD
Run
1
2
3
4
5
6
7
8
A
(% (w/w))
0.3
0.6
0.3
0.6
0.3
0.6
0.3
0.6
B
(% (w/w))
0.3
0.3
0.6
0.6
0.3
0.3
0.6
0.6
Factors
C
(% (w/w))
0.05
0.05
0.05
0.05
0.1
0.1
0.1
0.1
D
(C)
35
30
30
35
35
30
30
35
E
6.0
5.0
6.0
5.0
5.0
6.0
5.0
6.0
F
(rpm)
150
150
100
100
100
100
150
150
Response
Y (g/L)
Observed
Predicted
65.66
65.90
61.60
61.59
66.70
66.69
62.15
62.39
66.08
65.35
64.89
65.38
62.43
62.91
63.66
62.93
Source
Model
A
B
E
F
Sum of Squares
25.12
9.17
1.36
9.35
5.24
F-value
11.57
16.90
2.50
17.23
9.65
p-value > F
0.0362
0.0261
0.2122
0.0254
0.0530
(4)
where the ethanol (Z) is a function of inoculum (X1),
initial pH (X 2) and agitation speed (X3).
TABLE V.
Factors
Run
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
X1
(%
(v/w))
0.4
0.4
0.2
0.2
0.1
0.5
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
X2
7.0
6.0
7.0
6.0
6.5
6.5
5.5
7.5
6.5
6.5
6.5
6.5
6.5
6.5
6.5
X3
(rpm
)
50
100
100
50
75
75
75
75
25
125
75
75
75
75
75
Response
Z (g/L)
Observed
Predicted
70.26
70.94
70.08
66.11
64.14
71.10
70.29
69.15
69.58
70.77
70.80
70.27
70.03
70.09
70.40
70.34
71.02
70.16
66.18
64.10
71.06
70.25
69.11
69.54
70.73
70.30
70.30
70.30
70.30
70.30
Source
Model
X1
X2
X3
X12
X22
X32
X 1X 2
X 1X 3
X 2X 3
Sum of Squares
51.31
24.22
0.65
0.71
10.82
0.56
0.04
2.01
3.28
0.63
F-value
68.50
290.97
7.78
8.47
130.03
6.75
0.49
24.13
39.38
7.63
p-value > F
0.0001
< 0.0001
0.0385
0.0334
< 0.0001
0.0484
0.5143
0.0044
0.0015
0.0398
Figure 3. 2D contour plot and 3D response surface show the effect of amount of inoculum (% (w/w)) and initial pH on the ethanol production (g/L)
(agitation was 75 rpm).
Figure 4. 2D contour plot and 3D response surface show the effect of amount of inoculum (% (w/w)) and agitation (rpm) on the ethanol production
(g/L) (pH was 6.50).
Figure 5. 2D contour plot and 3D response surface show the effect of initial pH and agitation (rpm) on the ethanol production (g/L) (amount of
inoculum was 0.30% (w/w)).
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