Ethanol fermentation from raw starch of sweet sorghum grains

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

Corresponding Email: najdir@yahoo.com

AbstractEthanol can be readily produced by
fermentation of simple sugars that are converted from
either starchy crops or cellulosic materials. The
hydrolysis and fermentation were used to produce ethanol
from raw starch of sweet sorghum grains by utilizing
commercially available -amylase and glucoamylase.
Ethanol production from hydrolyzed sweet sorghum was
analyzed under different fermentation conditions using
Saccharomyces cerevisiae in batch fermentation. The
concentration of inoculum, urea, NPK (nitrogen,
phosphorus, and potassium), temperature, initial pH, and
agitation were investigated simultaneously through twolevel
factorial
design.
The
maximum
ethanol
concentration (66.70 g/L) were obtained at concentration
of inoculum of 0.3% (w/w), urea of 0.60% (w/w), NPK of
0.05% (w/w), temperature of 30C, initial pH of 6.0, and
agitation of 100 rpm. As shown in the analysis of variance
(ANOVA) result, the concentration of inoculum, urea,
initial pH, and agitation have contributed more
significant effect on fermentation of hydrolyzed sweet
sorghum. The major factors for fermentation were
optimized by the central composite design (CCD) under
the response surface method (RSM). After further
optimization using CCD, the optimum fermentation
conditions for maximum ethanol production of 75.48 g/L
were predicted at 0.43% (w/w) of inoculum concentration,
5.62 of initial pH of fermentation media, and 50 rpm of
agitation speed.
Keywords-ethanol; sweet sorghum; hydrolysis; fermentation
I. INTRODUCTION

Ethanol or ethyl alcohol is the most utilized liquid

biofuel either as a fuel or as a gasoline enhancer [1].
There has been a promotion in new applications and
markets for alcohol as octane enhancer due to
environmental concerns of phasing out of lead from
gasoline [2, 3, 4] and also emission of polluting gases
into the atmosphere, which have caused changes in the
universal climate [1]. Furthermore, the growth of
energy crops contributed to the biofuel production
would boost the agricultural sector [1] since the crops
can be used as a potential source of energy [5].
Ethanol from agricultural crops, which can be referred
as bioethanol, continues to be of interest because of the
renewable nature of the raw materials [6, 7, 8]. Due to the
increasing petroleum shortage, production of fuel ethanol
from renewable materials has the potential to reduce
growing world dependence on petroleum [9, 5].
Fermentation of sugars produces ethanol, and this

process technology has been carried out for well in

practically all regions of the world over 2000 years. Sugars
can also be derived from a variety of sources [10]. The
sources used in the ethanol production via fermentation
can be classified into three main types of materials, which
are sugars, starches, and cellulose materials. Sugars can be
converted into ethanol directly. Meanwhile, starches and
cellulose materials must be hydrolyzed to fermentable
sugars. Once simple sugars are formed, enzymes from
microorganisms can readily ferment the sugars to ethanol
[11].
One type of starchy crops that has been considered as a
particularly important energy plant for the production of
bioethanol is sweet sorghum, which is scientifically known
as Sorghum bicolor (L.) Moench [12, 13]. It is widely
grown in the semiarid tropics of Africa and Asia, and
constitutes a major source of proteins and carbohydrates
for people living in these regions [14]. Also, it has been
considered as an important energy plant for the production
of fuel bioethanol [15, 16, 17].
The starch hydrolysis may be regarded as a first and
important step in starch processing for bioethanol
production. Conventional process for production of
bioethanol from starch basically involved a three-stage
process; liquefaction of starch by -amylase,
saccharification of liquefied starch by glucoamylase and
followed by fermentation of sugar to ethanol using
Saccharomyces cerevisiae [18].
Both ethanol producers and researchers have shown that
grain sorghum is a reasonable raw material (technically
acceptable, fits the infrastructure, and can be economically
viable) for ethanol production and could make a larger
contribution to the nations fuel ethanol requirements [19].
The sweet sorghum can be hydrolyzed and fermented to
produce ethanol for use as a liquid fuel, primarily for
transport purposes. Besides, ethanol can also be used in
ethanol-fuelled lights and cookers [20, 21].
The aim of this study was to investigate the ethanol
fermentation process of hydrolyzed sweet sorghum by
Saccharomyces cerevisiae yeasts. In order to achieve the
optimum conditions, the present study was carried out in
two stages: firstly, the two-level factorial design was
applied to select the most significant conditions of
fermentation such as the concentration of inoculum, urea,
NPK (nitrogen, phosphorus, and potassium), temperature,
initial pH, and agitation were optimized. Secondly, the
central composite design (CCD) was employed to obtain
the optimum level of the significant factors by developing
a model followed by other statistical tests such as analysis
of variance (ANOVA), coefficient of determination, 2D
contour and 3D surface plots for the maximum ethanol.

II. MATERIALS AND METHODS

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

separation were 6.0 x 50 mm SH-1011P and 7.8 x 150 mm

IC-PakTM Ion Exclusion, respectively. 10 l of sample was
injected into HPLC and separation was performed at 75C
with 0.5 mM H2SO4 as the mobile phase at a flow rate of
1.0 mL/min. The temperature of refractive index (RI)
detector was set at 30C with sensitivity at 32, time
constant at 0.2 seconds and sampling rate at 5 pts/sec.
Both pre-column and column were stored using 10%
methanol. Glucose and ethanol were used as standards.
I. Experimental Design
1) Two-Level Factorial Design: The two-level
factorial design was used to identify which factors of
fermentation process have significant effects on the
response, ethanol. The factors selected for the
experiment were the inoculum (A, % (w/w)), urea (B,
% (w/w)), NPK (C, % (w/w)), temperature (D, C),
initial pH (E), and agitation (F, rpm). The factors were
examined at two different levels (low and high) coded
(1 and 2, respectively) as shown in Table I. This design
gave an output of eight experimental runs
(combinations) with six independent variables. All the
experiments were performed in triplicate and the
average of ethanol was used as the response
(dependant variable). The two-level factorial design is
based on the first order model which is as follows:
(1)
where Y is the response (amount of ethanol), b0 is the
model intercept and bi is the linear coefficient, and xi is the
level of the independent variable. This model does not
describe the interaction among the factors and it is used to
evaluate and select the important factors that influence the
response.
TABLE I. INDEPENDENT VARIABLES IN THE TWO-LEVEL
FACTORIAL EXPERIMENTAL DESIGN

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) Central Composite Design (CCD): The central

composite design was used to demonstrate the nature of
the response surface in the experimental region and clarify
the optimal conditions of the most significant independent
variables. According to the CCD for three variables, 15
experimental runs (5 runs at centre point) were executed
and the results were fitted to the following second order
polynomial model:

(2)

agitation (F) is as follows:

where Z is the dependent variable (ethanol); X1, X2 and X3
are the independent variable (inoculum, initial pH, and
agitation); 0 is the regression coefficient at centre point;
1, 2 and 3 are the linear coefficients; 11, 22 and 33 are
the quadratic coefficients; and 12, 13 and 23 are the
second order interaction coefficients.

(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).

TABLE II. INDEPENDENT VARIABLES IN THE CENTRAL

COMPOSITE EXPERIMENTAL DESIGN

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

The developed regression model was evaluated by

analyzing the values of regression coefficients, analysis of
variance (ANOVA), p- and F-values. The quality of fit of
the model equation was expressed by the coefficient of
determination, R2. The statistical software package
Design-Expert v 8.0 (Stat Ease Inc., Minneapolis, USA)
was used to identify the experimental design as well as to
establish a regression model to predict the optimum
combinations considering the effects of linear, quadratic
and interaction on the amount of ethanol produced.
III. RESULTS AND DISCUSSION

A. Screening of Significant Fermentation Parameters

For Ethanol Production Using Two-Level Factorial
Design
Fig. 1 shows the FESEM images of raw sweet
sorghum starch granules. The surfaces of granules were
quite smooth, but there is slight evidence of fissures,
indentations or pores.
Six fermentation parameters were screened by the
two-level factorial design, which showed eight
experimental runs for ethanol production (Table III).
Using the results of the experiments, the regression
model relating the ethanol (Y) with the independent
variables, the inoculum (A), urea (B), initial pH (E), and

Figure 1. FESEM images (1000x) for sweet sorghum (scale bar=10 m).

The main effect of each parameter on ethanol yield

was estimated as the difference between the average of
the measurements made at the low (1) and high level (2)
of the factors. The main effects of each fermentation
parameter are shown in Fig. 2.
Table III shows the achieved ethanol concentrations
after 24 h of fermentation as the maximum ethanol was
obtained at this moment of time. From Table III, out of
eight runs, Run 2 gave the lowest ethanol (61.60 g/L)
while Run 3 gave the highest ethanol (66.70). Also, there
is only small difference between the observed and
predicted ethanol values. The predicted ethanol yield
calculated using (3) was given in Table III along with the
observed value. In this study, the determination
coefficient, R2 = 0.9391 indicates a high correlation
between the experimentally observed and predicted
values.

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

The correlation between the observed and predicted

values is better when the value of R2 is closer to 1. In this
experiment, the value of R2 was 0.9391. This value
indicates a high degree of correlation between the
observed and predicted values. The value of R2 indicates
that 93.91% of the variables: inoculum, urea, initial pH,
and agitation play an important role to the response. The
value of R2 is also a measure of fit of the model and it can
be mentioned that only about 6.09% of the total variations
were not explained by the ethanol production [23].
The results were analyzed using the analysis of
variance (ANOVA) as appropriate to the experimental
design used as shown in Table IV. The computed F-value
(11.57) indicates that the model was highly significant at
high confidence level. The probability p-value was also
relatively low (p-value > F = 0.0362) which indicates the
significance of the model. The Fisher variance ratio, the
F-value is a statistically valid measure of how well the
factors describe the variation in the mean of data. The
greater the F-value indicates that the factors explain
adequately the variation in the data about its mean, and
the estimated factor effects are real. Also, the F-value is
inversely proportional to p-value > F. Higher F-value will
result to lower p-value > F.

followed by amount of inoculum, agitation, urea,

fermentation temperature, and lastly amount of NPK. Positive
linear coefficient means positive effect and vice versa.
Therefore, the most important factors that affect the
hydrolysis process of sweet sorghum are the initial pH the
media (E), amount of inoculums (A), and agitation (F).
In this study, the growth of S. cerevisiae was not monitored
by the method of optical absorbance because the medium
contained precipitate from the sweet sorghum and also the
color of the hydrolysate very dark. However, the fermentation
results, which is the amount of ethanol produced suggested
that S. cerevisiae could grow well in the hydrolysate medium.

TABLE IV. ANALYSIS OF VARIANCE (ANOVA) FOR SCREENING

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

For the independent variables, the p-value for initial pH

is the lowest (0.0254), followed by inoculum (0.0261),
agitation (0.0530), and lastly urea (0.2122). The p-values
were used to check the significance of each coefficient.
The lower the p-value indicates the more significant
correlation of coefficients (p-value < 0.05 indicate the
model terms are significant; p-value < 0.01 indicate the
model terms are highly significant).
When the factor is highly significant, the small change
in the factor (either increase or decrease) will give big
impact on the response. Positive effect means increasing
the factor will result to an increase in the response while
negative effect means reducing the factor will result to an
increase in the response. Linear and quadratic effects of
parameters were significant, meaning that they can act as
limiting factor and little variation in their value would
change either the growth rate or the product formation
rate or both to a considerable extent [24].
From the plot in Fig. 2, three factors, which are NPK,
temperature, and initial pH gave positive effect to the
response. On the other hand, the inoculum, urea, and
agitation gave negative effect to the response. The results
showed that the highest value represents the most
significant factor, by considering the absolute value only
(neglect the positive and negative sign). The initial pH of
the media gave highest impact on ethanol production,

Figure 2. Main effects of the fermentation parameters on ethanol production

(A; Inoculum, B: Urea, C: NPK, D: Temperature, E: Initial pH, F: Agitation).

From Fig. 2, it can be seen that the inoculum has negative

effect on fermentation process, which means lower amount of
inoculum gives higher amount of ethanol. This means the
increase of inoculum concentration did not have pronounced
effect on the final ethanol concentration. As reported by [18],
the duration of fermentation decreased with the increase of
the inoculum concentration. However, conditions for growth
and metabolism at high cell concentration are less favorable
due to difficult access to nutrients, space limitations, and cell
interactions [25].
The medium solution obtained from the hydrolysis process
consisted mainly of glucose, which are an ideal substrate for
the ethanol production. However, the concentration of
nitrogen and phosphorus were too low to support microbial
growth and thus the hydrolyzed sweet sorghum was
supplemented with urea and NPK to cover up for nutrient
shortage. Nitrogen and phosphorus are the main nutritional
requirements for the yeast growth and maximum ethanol
production efficiency [26]. Phosphorus has the major role in
the glycolysis cycle in the yeast cell [27]. During
fermentation, urea was used as nitrogen source due to the
reason that single urea was the best nitrogen source [2].
Meanwhile, NPK was used as both nitrogen and phosphorus
sources.
In this study, the urea gives negative effect because ethanol
yield decreases as the amount of urea increases. Meanwhile,
the amount of NPK provides very low positive effect on the
amount of ethanol. Excess assimilable nitrogen did not lead
to increased rates of ethanol production and to the reduction

of the fermentation time [28]. The reason might be that

urea together with NPK excessively stimulated the growth
of the yeast and decreased its ethanol biosynthesis [2].
S. cerevisiae is reported to grow well within the
temperature range 28-40C [29]. For fermentation
temperature, it has slightly positive effect on the
production of ethanol. Higher ethanol was achieved at
35C. From [30], it is mentioned that the maximum point
for ethanol production from Korean food waste leachate
was at temperature of 38C. Meanwhile, [31] found that
ethanol yield from simultaneous saccharification and
fermentation of citrus peel waste was highest when the
fermentation temperature at 37C. Besides, [32] stated
that higher ethanol productivity from the kitchen waste
was obtained at 37C of fermentation temperature.
It is known that changes in pH of the fermentation
medium significantly affect process parameters [33]. In
this study, the initial pH of fermentation media provides
positive effect on ethanol value. Medium with pH 6.0
provides better condition for ethanol production compared
to pH 5.0. Similar result has been obtained by [31] who
discovered that ethanol production was at maximum when
the initial pH was adjusted to 6.0. Meanwhile, greater
ethanol yield has been reported at the initial pH of 5.85
[32].
Saccharomyces cerevisiae is a facultative anaerobic
microorganism [34]. It can grow in either the presence or
absence of oxygen [35]. In this research, the agitation has
slightly lower negative effect on ethanol production
compared to the effect of inoculum. As reported in [34],
the maximum ethanol concentration obtained using
coculture of Saccharomyces cerevisiae and Candida
tropicalis was higher at 100 rpm compared to 150 rpm,
which are 7.16 and 6.52 g/L respectively. Agitation is
essential for uniform mixing of the medium components
within the fermenter to maintain a homogeneous culture
(dispersion of cells and nutrients). Also, it assists mass
transfer between the different phases present in the
culture. However, agitation speed creates turbulence and
shear force in the cultivation process which will influence
both cell growth and product formation [34].
The maximum achieved ethanol concentration is at 24
h of fermentation. This is because the glucose was
exhausted after 24 h. This indicated that the glucose
consumption was consistent with the time period of
ethanol production. It is clear from the data that lower
agitation time was found to be suitable for ethanol
production. The highest ethanol yield was observed with
24 h of agitation which is primarily due to initial oxygen
requirements of yeast cells [36].
B. Optimization of Fermentation Parameters For
Ethanol Production Using Central Composite Design
The significant hydrolysis parameters such as the
amount of inoculum, initial pH of the media, and
agitation as independent variables were optimized for
the maximum ethanol production from sorghum starch.
Experiments were carried out as designed by using
central composite design (CCD) (Table V), and the
average ethanol production obtained at 24 h of

fermentation was used as the response. The optimal

values of ethanol produced within the experimental
constrains were predicted by fitting a second order
polynomial model to the experimental results for the
dextrose equivalent by the Design-Expert software (v
8.0). The regression model developed relating the
variables are as follows:

(4)
where the ethanol (Z) is a function of inoculum (X1),
initial pH (X 2) and agitation speed (X3).
TABLE V.

THE CENTRAL COMPOSITE DESIGN FOR THE OPTIMIZATION OF

HYDROLYSIS PARAMETERS

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

At the model level, the correlation measures for the

estimation of the regression equation are the
determination coefficient R2 . The correlation between the
observed and predicted values is better when the value of
R2 is closer to 1. In this experiment, the value of R2 was
0.9920. This value indicates a high degree of correlation
between the observed and predicted values. The value of
R2 indicates that 99.20% of the variables: amount of
inoculum, initial pH, and agitation play an important role
to the response.
The optimization results were analyzed using the
analysis of variance (ANOVA) as appropriate to the
experimental design used as shown in Table VI. The
computed F-value (68.50) indicates that the model was
significant at high confidence level. The probability pvalue was also relatively low (p-value > F = 0.0001)
indicates the significance of the model. It was observed
that all of the linear and square terms of inoculum (X 1 ),
initial pH (X 2), and agitation (X 3) were significant (p <
0.05) except for square term of agitation (X 3) (p > 0.05).
Meanwhile, the interactive terms between inoculum and

initial pH (X 1X2), inoculum and agitation (X 1X3), and

initial pH and agitation (X 2X3) shown in the ANOVA
analysis were significant.
TABLE VI.

Source
Model
X1
X2
X3
X12
X22
X32
X 1X 2
X 1X 3
X 2X 3

ANALYSIS OF VARIANCE (ANOVA) FOR OPTIMIZATION

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

The 2D contour plots and 3D response surface are the

graphical representation of the regression model used to
determine the optimum values of the parameters within
the considered ranges [37]. The 2D and 3D plots for the
interaction between two variables among three the
variables are shown in Fig. 3 to Fig. 5. The purpose of
response surface is to determine the optimum values of
the variables, which mean the response is at maximum
value [37]. The contour plot represents an infinitive
number of combinations of the two test variables while
the other variable maintained at zero level (centre). The
maximum predicted value is obtained from the surface
confined in the smallest ellipse in the contour plot.
Elliptical contours are obtained when there is a perfect
interaction between the two independent variables [38].
Meanwhile, the 3D surface plot shows whether the ellipse
in the contour plot is at maximum or minimum.

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)).

Fig. 3 illustrates the response surface of ethanol

production from the interaction of inoculum
concentration and initial pH of the media. The
predicted ethanol yield increases as the amount of
inoculum increases and initial pH decreases. The
maximum ethanol obtained of about 74.48 g/L was
predicted at inoculum concentration and initial pH
around 0.5% (w/w) and 5.5 while agitation speed was
75 rpm.
A response surface in Fig. 4 shows the variation of
ethanol yield as a function of inoculum concentration
and agitation by making the initial pH of the media a
constant. About 74.74 g/L of maximum ethanol yield
was obtained from the response surface at inoculum
concentration, agitation speed, and initial pH were
about 0.5% (w/w), 25 rpm, and 6.5, respectively.
Fig. 5 is the response surface plot for the ethanol
concentration with the interaction of initial pH and
agitation speed. The maximum ethanol concentration
about 71.49 g/L was predicted at two different points,
either at initial pH of 5.5 and agitation of 25 rpm or at
initial pH of 7.5 and agitation of 125 rpm. However,
from Fig. 3 and Fig. 4, the maximum ethanol
production was at both lower initial pH and agitation.
Thus, the maximum ethanol concentration of about
71.49 g/L was obtained when initial pH around 5.5,
agitation speed was 25 rpm, and inoculum
concentration was about 0.3.
The optimum fermentation conditions for maximum
ethanol production of 75.48 g/L were predicted at
0.43% (w/w) of inoculum concentration, 5.62 of initial
pH of fermentation media, and 50 rpm of agitation
speed. According to [27], for both of two commercial
strains of Saccharomyces cerevisiae, Saf-Instant
(Bakers yeast) and Ethanol red (Mutant), the
maximum ethanol content with minimum sugar loss
and minimum undesirable products formation was
with inocula having cell counts of 4108 cells/mL. In
one of the previous study, the ethanol yield increased
with increasing inoculum size and at inoculum size

above 30%, yield of methanol, acetic acid, fusel alcohols,

or aldehydes was the lowest [39]. Meanwhile, as found by
[25] optimized condition of 40 g/L of dry cell mass was
experimentally confirmed and the optimal responses are
80.82.0 g/L of maximal ethanol. From [30], it is
mentioned that the maximum point for ethanol production
from Korean food waste leachate was at pH 5.45. As
reported in [34], when the ethanol fermentation was
performed using coculture of Saccharomyces cerevisiae
and Candida tropicalis, the ethanol productivity increased
when the agitation speed was 50 rpm, but decreased if the
agitation speed was further increased. The highest ethanol
productivity (1.02 g /L.h) was achieved at an agitation
speed of 50 rpm at which the highest maximum ethanol
concentration was also attained
IV. CONCLUSION

From the two-level factorial design, the concentration of

inoculum, urea, initial pH, and agitation have contributed
more significant effect on fermentation of hydrolyzed
sweet sorghum. The maximum ethanol concentration
(66.70 g/L) were obtained at concentration of inoculum of
0.3% (w/w), urea of 0.60% (w/w), NPK of 0.05% (w/w),
temperature of 30C, initial pH of 6.0, and agitation of 100
rpm. Besides, after further optimization using CCD, the
optimum fermentation conditions for maximum ethanol
production of 75.48 g/L were predicted at 0.43% (w/w) of
inoculum concentration, 5.62 of initial pH of fermentation
media, and 50 rpm of agitation speed.
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