World Journal of Microbiology & Biotechnology (2005) 21:399404

DOI 10.1007/s11274-004-2461-4

Springer 2005

Optimization of medium constituents and fermentation conditions for the production

of ethanol from palmyra jaggery using response surface methodology
B.V.V. Ratnam1,*, S. Subba Rao3, M. Damodar Rao2, M. Narasimha Rao3 and C. Ayyanna3
1
Department of Neurology, Johns Hopkins University School of Medicine, Pathology 233/Meyer 222, 600 North
Wolfe Street, Baltimore, MD 21287, USA
2
Department of Pediatrics, Division of Infectious Diseases, Johns Hopkins University School of Medicine, 720 Rutland
Avenue, Ross 1135B, Baltimore, MD-21205, USA. Tel: 410 614 0058, Fax: 410 614 1315, e-mail: dmendu1@jhmi.edu
3
Center for Biotechnology, Department of Chemical Engineering, College of Engineering, Andhra University,
Visakhapatnam 530 003, India
*Author for correspondence: E-mails: vbandar2@jhmi.edu/ratnam72@redimail.com
Keywords: Central composite design (CCD), ethanol, palmyra jaggery, response surface methodology (RSM),
Saccharomyces cerevisiae

Summary
The quantitative effects of sugar concentration, nitrogen concentration, EDTA, temperature, pH and time of
fermentation on ethanol production were optimized using a Box-Wilson central composite design (CCD)
experiment. It was found that palmyra jaggery (sugar syrup from the palmyra palm) is a suitable substrate for the
production of high concentrations of ethanol using Saccharomyces cerevisiae NCIM 3090 by submerged
fermentation. A maximum ethanol concentration of 129.4 g/l was obtained after optimizing media components
and conditions of fermentation. The optimum values were a temperature of 26.2 C, pH of 8.4, time of fermentation
of 4.2 days with 398.5 g of substrate/l, 3.1 g of urea/l and 0.51 g of EDTA/l. Thus by using the CCD, it is possible
to determine the accurate values of the fermentation parameters where maximum production of ethanol occurs.

Introduction
Ethanol is one of the largest volume organic chemicals
that are industrially produced. The study of ethanol
fermentation has gained importance because of increasing demand for it in recent years as a motor fuel
supplement to gasoline. Rapid fermentation and high
ethanol levels are desirable to minimize capital costs and
distillation energy, while good yields are necessary for
process economics. The substrate is the main cost
component for industrial ethanol production and it is
essential that ethanol production should be carried out
with cheap substrates (Lee & Woodward 1983; Elisson
et al. 2001). Palmyra jaggery, sugar syrup from the
palmyra palm (Borassus abellifer) is an agricultural
product abundantly available in the India, Peninsula
and the Northern of Sri Lanka and is an alternative
substrate for producing ethanol.
To develop a process for the maximum production of
ethanol, standardization of media and fermentation
conditions is crucial. Medium optimization by the
classical method: a single dimensional search involving changing one variable while xing the others at a
certain level is laborious and time consuming, especially when the number of variables is large. An
alternative and more efcient approach in microbial
systems is the use of statistical methods (Duff et al.

1973). Statistical inference techniques can be used to

assess the importance of individual factors, the appropriateness of this functional form and sensitivity of the
response to each factor (Mason et al. 1989). Recently
many statistical experimental design methods have been
employed in bioprocess optimization. Among them,
response surface methodology (RSM) is the one suitable
method for identifying the eect of individual variables
and for seeking the optimum conditions for a multivariable system eciently. This method has been successfully applied to optimize alcoholic fermentation and
other fermentation media (Maddox & Reichert 1977;
Chen 1981, 1996; Zertuche & Zall 1985; Coteron et al.
1993; Sunitha et al. 1998; Ambati & Ayyanna 2001;
Ratnam 2001; Ratnam et al. 2003). A detailed account
of this technique has been outlined (Cochran & Cox
1968). Basically, this optimization process involves three
major steps: performing the statistically designed experiments, estimating the coecient in a mathematical
model and predicting the response and checking the
adequacy of the model.
In this study, the RSM approach was adopted to
locate optimum levels of substrate concentration, urea
concentration, EDTA concentration, temperature, pH
and time of fermentation using palmyra jaggery as a
substrate, since these parameters play a key role in the
enhancement of ethanol yield.

400

B.V.V. Ratnam et al.

Materials and methods

Substrate
Palmyra jaggery is the dark solid obtained from the
sweet today, which is collected from the palmyra tree
(Borassus abellifer) grown in West Godavari District,
Andhra Pradesh, India.

80 mesh, manufactured by Nucon Engineers, India)

were used. The column oven was operated isothermally at 150 C and the detector and injection ports
were kept at 170 C. Nitrogen was used as carrier gas
at a ow rate of 30 cm3/min and the combustion gas
was a mixture of hydrogen and air. Sugars were
determined using Millers method (1959).

Microorganism

Experimental design and optimization

Saccharomyces cerevisiae NCIM 3090 obtained from

National Chemical Laboratory, Pune, India, was used
throughout the study.

Central composite experimental design (CCD, Box and

Wilson 1951) was used in the optimization of ethanol
production. Substrate (X1, g/l), urea (X2, g/l), EDTA
(X3, g/l), temperature (X1, C), pH (X2) and time of
fermentation (X3, days) were chosen as the independent
variables shown in Tables 1 and 2. Ethanol concentration (Yi, g/l) was used as the dependent output variable.
For statistical calculations the variables Xi were coded
as xi according to Equation (1)

Growth medium and growth conditions

The culture was maintained on agar slants having the
composition (%): malt extract 0.3, glucose 1.0, yeast
extract 0.3, peptone 0.5 and agar agar 2.0. The pH of the
medium was adjusted to 6.46.8 and cultures were
incubated at 30 C for 48 h.
Production media and fermentation conditions
Palmyra jaggery with 70% sugars was used as the sole
carbon source for the fermentation and the syrup contains
a sugar concentration of 280 g/l. A weighed amount of
palmyra jaggery was dissolved in water and sterilized at
121 C for 15 min. The fermentation was carried out in a
Biostat M fermentor supplied by B. Braun Co., Germany
with all necessary controls. The reactor was of 2 l capacity
and working volume was 1 l. The operating conditions
were maintained at a temperature of 30 C, pH 5.0, stirrer
speed 200 rev/min and inoculum size 5% (v/v). Inoculum
was prepared in 500-ml ask containing 100-ml fermentation medium by incubating it at 30 C for 48 h on a
rotary shaker. The reactor was maintained under anaerobic conditions without aeration.


Xi Xi
X
i = DXj i 1; 2; 3; . . . ; k

where, is the dimensionless value of an independent

variable, Xi the is real value of an independent variable,

xi ; is the real value of the independent variable at the
center point and DXj is step change.
p
A 23-factorial CCD, with six axial points a 3
and six replications at the center points (n0 6)
leading to a total number of 20 experiments was
employed (Table 2) for the optimization of the constituents of fermentation. The second degree polynomials (Equation (2)) were calculated with the
statistical package (Stat-Ease Inc, Minneapolis, MN,
USA) to estimate the response of the dependent
variable.
Yi b0 b1 X1 b2 X2 b3 X3 b11 X12 b22 X22
b33 X32 b12 X1 X2 b23 X2 X3 b13 X1 X3

Analytical methods
Ethanol was estimated by GC in which a ame
ionization detector and stainless steel column (2.0 m
length, 3.0 mm i.d.) packed with Porapak-Q (50

where Yi is the predicted response, X1, X2, X3 are

independent variables, b0, is the oset term, b1, b2, b3 are
linear eects, b11, b22, b33 are squared eects and b12,
b23, b13 are interaction terms.

Table 1. Independent variables in the experimental plan.

Variables

Equation 3
Substrate (g/l), X1
Urea (g/l), X2
EDTA (g/l) , X3
Equation 4
Temperature (C), X1
pH, X2
Time (days), X3

Coded levels
)1.682

)1

1.682

316.9
1.318
0.3318

350
2
0.4

400
3
0.5

450
4
0.6

484.1
4.682
0.6682

16.58
6.318
2.318

20
7
3

25
8
4

30
9
5

33.4
9.682
5.682

401

Ethanol production from palmyra jaggery

Table 2. The CCD matrix employed for three independent variables
(actual values are given in Table 1).
Run no.

X1

X2

X3

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

)1
1
)1
1
0
0
)1
1
)1
1
0
0
)1.682
1.682
0
0
0
0
0
0

)1
)1
1
1
0
0
)1
)1
1
1
0
0
0
0
)1.682
1.682
0
0
0
0

)1
1
1
)1
0
0
1
)1
)1
1
0
0
0
0
0
0
)1.682
1.682
0
0

urea concentration and EDTA concentration of ethanol.

Hence these three factors are considered as major
constituents of the medium to optimize by keeping the
mineral constituents of the medium constant.
Using CCD, a total number of 20 experiments with
different combinations of substrate, urea, EDTA were
performed (Tables 1 and 2). The response was taken at
the maximum ethanol production which was observed at
4 days. The results were analysed using the analysis of
variance (ANOVA) and v2 test as appropriate to the
experimental design being used. The calculated regression equation for the optimization of medium constituents showed that the ethanol production (Yi, g/l) is a
function of the concentration of substrate (X1, g/l), urea
(X2, g/l) and EDTA (X3, g/l). By applying multiple
regression analysis on the experimental data, the following second order polynomial equation was found to
represent the ethanol production adequately.
Yi
889:849 5:0625X1
17:642X2 114:1887X3

0:0068X12
3:1970X22
188:886X32
0:0885X1 X2 4:3687X2 X3
0:1619X1 X3 :

Results and discussion

Optimization of medium constituents

RSM is a sequential procedure with an initial objective
of leading the experimenter rapidly and efciently to the
general vicinity of the optimum. Since the location of the
optimum is unknown prior to running RSM experiments, it makes sense to have a design that provides
equal precision of estimation in all directions is
employed. The three factors which inuence highly the
fermentative production are substrate concentration,

The predicted levels of ethanol production from

palmyra jaggery medium using the above equation
are given in Table 3 along with experimental data. The
goodness of the model can be checked by different
criteria. The coefcient of determination, R2 is 0.9788,
implies that 97.88% of the sample variation in the
ethanol production is attributed to the independent
variables. The R2 value also indicates that the only 1%
of the variation is not explained by the model. The
value of R is 0.9893. The corresponding analysis of

Table 3. Experimental and predicted yields for ethanol.

X1

350
450
350
450
400
400
350
450
350
450
400
400
315.9
484.1
400
400
400
400
400
400

X2

2
2
4
4
3
3
2
2
4
4
3
3
3
3
1.318
4.682
3
3
3
3

X3

0.4
0.6
0.6
0.4
0.5
0.5
0.6
0.4
0.4
0.6
0.5
0.5
0.5
0.5
0.5
0.5
0.3318
0.6682
0.5
0.5

Ethanol yield (g/l)

Experimental

Predicted

105.16
96.68
94.19
100.67
121.14
120.59
103.66
94.19
93.19
104.16
122.50
121.77
79.26
69.23
107.96
118.54
117.29
116.61
120.72
122.44

105.37
93.74
97.05
101.84
121.57
121.57
103.51
92.36
97.16
104.97
121.57
121.57
75.66
71.38
111.25
113.79
115.69
116.76
121.57
121.57

402

B.V.V. Ratnam et al.

Table 4. ANOVA for full quadratic model.

Source of
variation

Sum of
squares
(SS)

Regression 4385.5
Residual
95.13
Total
4480.6

Degrees of Mean
freedom
squares
(DF)
(MS)
9
10
19

487.3
9.5

F value

Probe > F

51.2

variance (ANOVA) is presented in Table 4. test was

also carried out to check the best t of the model. The
model was a good t. Since v2cal < v2tab , where v2cat is 0.98
and v2tab is 30.14. The predicted optimum levels of
substrate, urea and EDTA were obtained by applying
the regression analysis to the Equation (3). The
predicted and experimental ethanol production at the
optimum levels of medium constituents was also
determined by using Equation (3). Figures 13 represent the response surface and contour plots for the
optimization of medium constituents of ethanol production. The optimum medium constituents for higher
metabolic production can be attained at the concentration of 398.5 g of substrate/l, 3.1 g of urea/l and
0.51 g of EDTA/l. At these optimum medium consentrations maximum ethanol production of 125.4 g/l
was obtained. Experimental and predicted ethanol
production at the optimum levels of media constituents
were also determined (Table 7).
Optimization of fermentation conditions
The most important physical factors which affect the
fermentative production of ethanol are the temperature,
initial pH and time of fermentation. The suitable levels
for these parameters were also determined using statis-

Figure 1. Response surface and contour plot of substrate concentration vs. urea concentration on ethanol production (EDTA was kept
constant at 0.5 g/l).

Figure 2. Response surface and contour plot of substrate concentration vs. EDTA concentration on ethanol production (urea was kept
constant at 3 g/l).

tical CCD. The experimental design matrix is given in

Tables 1 and 2. Twenty experiments were performed
using different combinations of the variables as per the
CCD. Using the results of the experiments, the following
second order polynomial equation giving the ethanol as
a function of temperature (X1, C), pH (X2) and time of
fermentation (X3, days) was obtained.
Yi
1394:95 69:9918X1 117:3672X2
53:7724X3
1:0407X12
4:2425X22

6:8737X32
1:8253X1 X2
0:4824X2 X3

0:0216X1 X3

Figure 3. Response surface and contour plot of urea concentration vs.

EDTA concentration on ethanol production (substrate concentration
was kept constant at 400 g/l).

403

Ethanol production from palmyra jaggery

Table 5. Experimental and predicted yields for ethanol.
X1

X2

X3

Ethanol yield (g/l)

Experimental Predicted

20
30
20
30
25
25
20
30
20
30
25
25
16.58
33.4
25
25
25
25
25
25

7
7
9
9
8
8
7
7
9
9
8
8
8
8
6.318
9.682
8
8
8
8

3
5
5
3
4
4
5
3
3
5
4
4
4
4
4
4
2.318
5.682
4
4

45.44
99.27
80.4
101.75
124.72
125.00
47.42
107.22
85.98
105.23
125.12
124.98
36.34
77.62
117.15
120.13
100.26
122.13
125.10
123.99

55.02
109.17
89.26
98.51
125.13
125.13
58.48
106.16
83.88
103.46
125.13
125.13
23.84
79.13
103.39
122.85
102.15
109.21
125.13
125.13

Figure 4. Response surface and contour plot of temperature vs. pH on

ethanol production (time was kept constant at 4 days).

Table 6. ANOVA for full quadratic model.

Source of
variation

Sum of
squares
(SS)

Degrees of Mean
freedom
squares
(DF)
(MS)

Regression 14904.6
9
Residual
936.99 10
Total
15841.6 19

1656.1
93.7

F value

Probe > F

17.7

Table 7. Optimum values of media constituents, fermentation conditions and the experimental and predicted yields for ethanol.
Variables

Equation 3
Substrate (g/l), X1
Urea (g/l), X2
EDTA (g/l), X3
Equation 4
Temperature (C), X1
pH, X2
Time (days), X3

Optimum
values

398.5
3.1
0.51
26.2
8.4
4.2

Optimum ethanol yield (g/l)

Experimental

Predicted

125.4

125.6
Figure 5. Response surface and contour plot of temperature vs. time
on ethanol production (pH was kept constant at 8.0).

129.4

129.8

The predicted production of ethanol using the above

equation is given in Table 5 along with the experimental values. The coefcient of determination, R2 is
0.9408, implies that the sample variation of 94.08%
for ethanol production is attributed to the independent variables, viz., temperature, pH and fermentation
time. The R2 value also indicates that only 1% of the
variation is not explained by the model. The value of
R is 0.97. The corresponding analysis of variance
(ANOVA) was presented in Table 6. v2 test shows

that the model is a good t since v2cal < v2tab , where v2cal
is 15.77 and v2tab is 30.14. The predicted optimum
levels of temperature, initial pH and time of fermentation were obtained by applying the regression
analysis to the Equation (4). The predicted and
experimental ethanol productions at the optimum
levels of fermentation conditions were also determined. Figures 46 represent the isoresponse contour
and surface plots for the optimization of fermentation
conditions of ethanol production. The maximum
ethanol concentration of 129.4 g/l appeared at temperature, pH and time of fermentation of 26.2 C, 8.4
and 4.2 days respectively. The experimental and predicted ethanol production at optimum conditions of
fermentation were also determined (Table 7).

404

Figure 6. Response surface and contour plot of pH vs. time on ethanol

production (temperature was kept constant at 25 C).

Thus the present study using the technique of central

composite design enables to nd the accurate values of
the medium constituents and fermentation conditions
for the maximum product concentration of ethanol
using Saccharomyces cerevisiae.

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