Bioenerg. Res.
(2008) 1:239–247
DOI 10.1007/s12155-008-9018-6
Optimization of Process Conditions Using Response Surface
Methodology (RSM) for Ethanol Production from Pretreated
Sugarcane Bagasse: Kinetics and Modeling
Ezhumalai Sasikumar & Thangavelu Viruthagiri
Published online: 19 September 2008
# Springer Science + Business Media, LLC. 2008
Abstract Based on a five level central composite design
(CCD) involving the variables substrate concentration (C),
pH (P), incubation temperature (T) and fermentation time
(H), a response surface methodology (RSM) for the
production of ethanol from pretreated sugarcane bagasse
by cellulase and yeast Kluyveromyces fragilis was stan-
dardized. The design contains a total of 31 experimental
trials in which the first 24 organized in a factorial design
and from 25 to 31 involving the replications of the central
points. Data obtained from RSM on ethanol production
were subjected to the analysis of variance (ANOVA) and
analyzed using a second order polynomial equation.
Maximum ethanol concentration (32.6 g/l) was obtained
from 180 g/l pretreated sugarcane bagasse at the optimized
process conditions (temperature 35°C, pH 5.5) in 72 h
aerobic batch fermentation. Various kinetic models such as
logistic model, logistic incorporated leudeking piret model
and logistic incorporated modified leudeking piret model
have been evaluated and the constants were predicted.
Keywords Ethanol . Central composite design (CCD) .
Response surface methodology (RSM) . Pretreated sugarcane
bagasse . Kluyveromyces fragilis
Introduction
Over the last century, energy consumption has increased
progressively as the result of growing world population and
E. Sasikumar (*) : T. Viruthagiri
Bioprocess Engineering Research Laboratory,
Department of Technology, Annamalai University,
Annamalai Nagar 608 002, India
e-mail: sashikumar_ess@yahoo.co.in
industrialization. Ethanol is a renewable energy source
produced through fermentation of sugars unlike the fossil
fuels. Interest in the bioconversion of abundant and
renewable cellulosic biomass into fuel ethanol as an
alternative to petroleum is rising around the world owing
to the realization of diminishing natural oil and gas
resources [20]. Lignocellulosic biomass, such as agricul-
tural residues (corn stover, wheat straw, sugarcane bagasse),
wood and energy crops, is an attractive material for
bioethanol (biomass ethanol) fuel production since it is
the most abundant renewable resource on the earth [38].
Lignocellulosic biomass could produce up to 442 billion
liters per year of bioethanol [5]. Lignocellulosic materials
constitute an abundant and cheap feedstock, but the
processing technique required for ethanol production are
presently extensive and costly. The cost of ethanol
produced from lignocellulosic materials with currently
available technology and under the present economic
conditions is not competitive with the cost of gasoline
[11]. Comprehensive process development and optimiza-
tion are still required to make the process economically
viable. In reality, environmental considerations and energy
and tax policies will determine the extent of fuel ethanol
utilization in the future. Approximately 80% of the ethanol
produced in the world is still obtained from fermentations;
the remainder comes largely by synthesis from the
petroleum product, ethylene [23]. Lignocellulosic materials
generally contain 75–80% of cellulose and hemicellulose,
which cannot be easily converted to simple monomeric
sugars due to their recalcitrant nature [29].
Furthermore, lignocellulose is a more complex substrate
than starch. It is composed of a mixture of carbohydrate
polymers (cellulose and hemicellulose) and lignin. One of
the largest cellulosic agro-industrial by-products is sugar-
cane bagasse (or, bagasse as it is generally called); a fibrous
240
residue of cane stalks left over after the crushing and
extraction of the juice from the sugar cane. Bagasse
consists of approximately 450–500 mg/g cellulose and
250–300 mg/g hemicellulose and 150–200 mg/g lignin.
Chemically, bagasse contains about 500 mg/g cellulose,
300 mg/g pentosans, and 24 mg/g ash. Because of its low
ash content, bagasse offers numerous advantages in
comparison to other crop residues such as rice straw and
wheat straw, which have 175 mg/g and 110 mg/g,
respectively, ash contents, for usage in bioconversion
processes using microbial cultures. Sugarcane bagasse is a
suitable cellulosic substrate for a bioconversion process
because of its relatively low lignin content and its
availability at central collection sites [12, 30].
The biological process for converting the lignocellulosic
materials into fuel ethanol requires the following: deligni-
fication to liberate cellulose and hemicellulose from their
complex with lignin, depolymerization of the carbohydrate
polymers to produce free sugars, and fermentation of mixed
hexose and pentose sugars to produce ethanol. Lynd et al.
[24] has summarized the desirable properties for an ideal
lignocellulose material after chemical pretreatment, i.e. it
should (a) produce reactive fibers, (b) yield pentoses in
non-degraded form, (c) not release the compounds that
significantly inhibit fermentation, (d) work in reactors of
reasonable size with moderate cost and (e) be effective at
low moisture contents. A number of pretreatment options
are available; acid pretreatment, alkaline treatment, steam
explosion, wet oxidation, organic solvent pretreatment, and
hot water [27]. Among all these methods, steam explosion
and acid pretreatment are still the methods of choice in
several model processes [18, 21].
Enzymatic hydrolysis is the most promising approach to
get high product yields vital to economic success [31, 39].
The high cost of cellulase enzyme production hinders the
application of these enzymes to bioethanol production [1,
17]. The common end products of enzymatic cellulose
hydrolysis is cellobiose, a dimer of two glucose units joined
by a β-1,4 glycosidic linkage and glucose which were the
major inhibitors for the hydrolysis process [4]. One way to
overcome the inhibition problem is to ferment the glucose
into ethanol as soon as it appears in solution. Simultaneous
saccharification and fermentation (SSF) is the ideal method
of producing ethanol from sugarcane bagasse. In this
process, a cellulose hydrolyzing enzyme (cellulase) is
combined with an ethanol producing organism (yeast) to
carry out simultaneous hydrolysis of cellulose to glucose
and the conversion of glucose to ethanol in the same
reactor. The result is improved hydrolysis rates and yields
of ethanol when compared to those involving separate
hydrolysis and fermentation steps [32]. This method keeps
the concentration of glucose low and the accumulation of
ethanol in the fermentor does not inhibit cellulase as much
Bioenerg. Res. (2008) 1:239–247
as high concentrations of glucose, so SSF is a good strategy
for increasing the overall rate of cellulose to ethanol
conversion [6]. Other advantages of this approach are a
shorter fermentation time and reduced risk of contamination
with external microflora, due to the high temperature of the
process, the presence of ethanol in the reaction medium and
the anaerobic conditions. One problem associated with SSF
is the different optimum temperatures of saccharification
and fermentation. The use of thermotolerant yeasts capable
of fermenting glucose to ethanol at temperatures above
45°C, which are closer to the optima for the activity of the
cellulolytic complex in the range of 35–45°C, is therefore
advisable when employing coupled SSF processes [3, 7,
35].
In the present study, optimization of process conditions
(substrate concentration, temperature, pH and fermentation
time) for the production of ethanol from pretreated
sugarcane bagasse by using cellulase and yeast Kluyver-
omyces fragilis NCIM 0557 has been carried out by
response surface methodology and also various kinetic
models such as logistic model (growth kinetics), logistic
incorporated leudeking piret model (product formation) and
logistic incorporated modified leudeking piret model
(substrate utilization) has been evaluated.
Materials and Methods
Materials
Sugarcane bagasse sample was obtained from M.R.K.
Sugar Mills Ltd. Sethiyathope, Tamilnadu. The bagasse
sample was made into 100 mesh (0.15 mm) fine powder by
use of laboratory blender at 3,000 rpm. Sample was
preserved in a sealed plastic bag at 4°C to prevent any
possible degradation or spoilage. Pure cellulose powder
was used in reference of cellulose estimation and fermen-
tation tests. The control and pretreated bagasse samples
were analyzed for cellulose content using anthrone reagent
at 630 nm in Bio spectrophotometer, Elico BL 198 [37].
The estimated cellulose content of control and pretreated
sample was 420 mg/g and 420 mg/g respectively.
Micro Organism and Culture Conditions
Commercially available cellulase enzyme was obtained
from SISCO Laboratories, Mumbai. The activity of
cellulase was found to be 15 FPU/ml and it was used
throughout the experimentation. The cellulase activity was
measured by standard Mandel’s method [25]. Yeast
Kluyveromyces fragilis-NCIM 0557 was obtained from
National Collection of Industrial Microorganism (NCIM),
National Chemical Laboratory, Pune, India. Culture was
Bioenerg. Res. (2008) 1:239–247
maintained on yeast extract–malt extract agar (YMA)
medium. After 3 days incubation at 40°C the agar slants
were stored at 4°C.The liquid medium for the growth of
inoculum for yeast was yeast extract–malt extract–peptone–
glucose medium (YMP) composed of 3 g/l of yeast extract,
3 g/l of malt extract, 5 g/l of peptone and 10 g/l of glucose.
Inocula were grown aerobically in 250 ml Erlenmeyer
flasks containing the above mentioned medium at 40°C in
an Environmental Shaker (Remi Scientific) at 200 rpm for
24 h. Active cells were centrifuged in a clinical centrifuge
(1,200 rpm), washed with sterile water, and were used as
inoculum. Fermentations for ethanol production were
conducted on a shaker at 200 rpm in 250 ml flasks with
100 ml medium.
Pretreatment of Sugarcane Bagasse
The pretreatment of bagasse was generally done using
different quantities of sodium chlorite and acetic acid at
100°C. Samples of 5 g of dry bagasse (with known
moisture) were transferred to 250 ml Erlenmeyer flasks
with 160 ml of distilled water, 0.5 ml of acetic acid and
1.5 g of sodium chlorite. The samples were heated in a
water bath at 70–80°C with agitation at each 10 min. After
1 h reaction, 0.5 ml of acetic acid and 1.5 g of sodium
chlorite were added. The addition was repeated at each
hour, until the final time of 4 h of reaction. The samples
were filtered in crucibles and washed with 1.6 l of hot
distilled water under suction. Again the samples were
washed with acetone and dried at room temperature.
Simultaneous Saccharification and Fermentation (SSF)
The samples of pretreated sugarcane bagasse (PSB) were
weighed (10 g/flask) and distributed into 250 ml Erlen-
meyer flasks with the addition of nutrient medium (YMP
without glucose) and 0.05 M Sodium phosphate buffer
(pH-5.0) followed by sterilization for 15 min, at 15 psi
(121°C) in an autoclave. Cellulase dosage of 15 FPU/g of
substrate was used for hydrolysis. Yeast cells at the amount
of 2 g/l based on the dry weight were inoculated into the
production medium. Flasks were then covered with cotton
to allow CO2 produced during fermentation to escape. The
flasks were incubated in a rotary shaker (200 rpm) at 40°C
for 96 h. Samples were withdrawn periodically, centrifuged
in a laboratory desktop centrifuge at 1,200 rpm, and the
supernatants were analyzed for glucose and ethanol
concentrations.
Cell Growth and Chemical Analysis
Cell growth (except during SSF) was monitored directly by
reading the optical density (OD) using systronics colorim-
241
eter (420–820). The sugarcane bagasse sample was ana-
lyzed for sugar and Klason lignin content following the
procedures described in NREL Standard Procedure (No.
002). Total reducing sugar concentration was determined by
the dinitrosalicylic acid (DNS) method [26]. Then it was
confirmed by bio spectrophotometer (Elico BL 198) at
510 nm. Ethanol was estimated using NUCON 5765 gas
chromatography (GC) with a flame ionization detector and
CHROMATO-PAK (10%C–20 M) column (3.0 m ×
0.32 cm) using nitrogen as the carrier gas at 40 µl/min.
The oven temperature was held at 80°C. The injector and
detector temperature was maintained at 200°C.
Experimental Design and Statistical Analysis
Optimization of process conditions is one of the most
critical stages in the development of an efficient and
economic bioprocess [34]. Classical and statistical method-
ologies are available for optimizing process conditions such
as response surface methodology (RSM), Taguchi, SX, etc.
RSM is a powerful mathematical model with a collection of
statistical techniques wherein, interactions between multiple
process variables can be identified with fewer experimental
trials [8, 9].
The RSM used in the present study is a central
composite design (CCD) involving four different factors.
Experiments were conducted in a randomized fashion. The
CCD contains a total of 31 experimental trials with the first
16 organized in a factorial design and from 25 to 31
involving the replications of the central points (Table 1).
The dependent variables selected for this study were
ethanol yield (g/l). The independent variables chosen were
substrate composition, C (g/l); pH of the fermentation
medium, P; incubation temperature, T (°C); fermentation
time, H (h). Once the experiments are performed, a second
order polynomial Eq. (1) shown below was used to describe
the effect of variables in terms of linear, quadratic and cross
product terms [15].
X
k X
k k X
X k
Y ¼ b0 þ bi Xi þ bij Xi2 þ bij Xi Xj þ e ð1Þ
i¼1 i¼1 ii <j j
Where, i, j are linear, quadratic coefficients, respectively,
while ‘b’ is regression coefficient, Y is the ethanol yield, k
the number of factors studied and optimized in the
experiment and ‘e’ is random error. The quality of fit of
the second order equation was expressed by the coefficient
of determination R2, and its statistical significance was
determined by F-test. The significance of each coefficient
was determined using Student’s t-test. The coefficients of
the equation were determined by employing MINITAB 15
software. Analysis of variance (ANOVA) for the final
242
predictive equation was done using the same software
package. The response surface equation was optimized for
maximum yield in the range of process variables using
MATLAB 7.0.1 version. Three dimensional plots and their
respective contour plots were obtained based on the effect
of the levels of two parameters (at five different levels each)
and their interactions on the yield of ethanol by keeping the
other three parameters at their optimal concentrations. From
these contour plots, the interaction of one parameter with
another parameter was studied and also the model is
developed to explain the quadratic interaction effects by
conducting the pairwise regression analysis of experimental
data which is not fully described in the multiple regression
analysis due to limited degrees of freedom. The optimum
concentration of each parameter was identified based on the
hump in the three-dimensional plots.
Results and Discussion
Optimization of Ethanol Production from Pretreated
Sugarcane Bagasse
Table 1 shows the four independent variables (substrate
composition, pH, temperature, fermentation time) and their
concentrations at different coded and actual levels of the
variables employed in the design matrix. The data obtained
from the five level central composite design matrix were
used to develop models in which each dependent variable
(ethanol yield) was obtained as the sum of the contributions
of the independent variable through second order polyno-
mial equation and interaction terms. The actual yields of
ethanol obtained in the experiments and the yields predicted
by the model Eq. (1) are given in Table 2.
Based on the experimental response the quantity of
ethanol produced by K. fragilis ranged from 1.50 to 4.11
g/l. Runs # 1 and # 25 had the minimum and maximum
ethanol production respectively. The ANOVA result of
quadratic regression model for ethanol yield is described in
Table 3. ANOVA of the regression model for ethanol yield
demonstrated that the model was significant due to an
F-value of 14.96 and a very low probability value (P model
Table 1 Codes and actual levels of the independent variables for
design of experiment
Independent variables Symbols Coded levels
−2 −1 0 1 2 axes of the contour plots between the variables pH and
Substrate conc. (g/l) C 10 20 30 40 50
pH P 4 5 6 7 8
Temperature (°C) T 25 30 35 40 45
Fermentation time (h) H 24 48 72 96 120
Bioenerg. Res. (2008) 1:239–247
>F—0.005). ANOVA (F-test) for the model explained the
response of the dependent variable Y. Table 3 also showed
that the experimental yields fitted the second order
polynomial equation well as indicated by high R2 values
(0.929). F-value several times greater than the tabulated
F-value showed that the model predicted the experimental
results well and the estimated factors effects were real.
The regression coefficients, along with the corresponding
P-values, for the model of ethanol production by K. fragilis,
are described in Table 3 and Table 4. It showed that the
regression coefficients of all the linear term and all quadratic
coefficients of X1, X2, X3 and X4 were significant at <1%
level. ANOVA suggested the model to be significant at P<
0.01. The P-values are used as a tool to check the
significance of each of the coefficients, which in turn
indicate the pattern of the interactions between the variables.
Smaller value of P then it was more significant to the
corresponding coefficient [19]. From the Figs. 1, 2, 3 and 4,
it was evident that the interaction effects are significant but
the ANOVA and multiple regression analysis suggests that
the interaction effects are insignificant hence it is essential to
analyze the experimental data for quadratic interactions by
pairwise regression analysis in an attempt to show the
significance of interactions. Neglecting the unimportant
terms with the P-value greater than 0.01 the model is
reduced and the stepwise regression analysis is conducted on
the experimental data to show the significance of quadratic
interactions. From Table 5, it is clear that the models
developed by pairwise regression analysis of quadratic
interactions P×T, C×T, P×H and T×H are significant with
an R2 value of 0.581, 0.549, 0.395 and 0.439 respectively.
The contour plots described by the regression model
were drawn to illustrate the effects of the independent
variables, and effects of interactions of each independent
variable, on the response variables. The shape of the
corresponding contour plots indicates whether the mutual
interactions between the independent variables are signifi-
cant or not. Interactions of variables can be better
determined by the orientation of the principal axes of the
contour plots. From the contour plots, the optimal values of
the independent variables could be observed, and the
interaction between each independent variable pair can de
described.
The contour plots based on independent variable was
obtained using the same software package (Figs. 1, 2, 3,
and 4) indicated that a local optimum exists in the area
experimentally investigated. The orientation of the principal
temperature, substrate concentration and temperature, pH
and fermentation time and temperature and fermentation
time indicated that the mutual interactions between these set
of variables had a significant effect on the ethanol yield.
The isoresponse contour plots of RSM as a function of two
Bioenerg. Res. (2008) 1:239–247 243

Table 2 Four level central composite design and the experimental responses of dependent variable (ethanol yield)

Run order Substrate conc. (g/l) pH Temp. (°C) Fermentation time (h) Ethanol conc. (g/l)

Experimental Predicted

1 −1(20) −1(5) −1(30) −1(48) 1.5 1.7

2 1(40) −1(5) −1(30) −1(48) 2.3 2.4
3 −1(20) 1(7) −1(30) −1(48) 2.1 2.0
4 1(40) 1(7) −1(30) −1(48) 1.9 2.1
5 −1(20) −1(5) 1(40) −1(48) 1.9 2.1
6 1(40) −1(5) 1(40) −1(48) 2.6 2.7
7 −1(20) 1(7) 1(40) −1(48) 3.3 3.5
8 1(40) 1(7) 1(40) −1(48) 2.2 2.3
9 −1(20) −1(5) −1(30) 1(96) 2.2 2.3
10 1(40) −1(5) −1(30) 1(96) 2.6 2.8
11 −1(20) 1(7) −1(30) 1(96) 1.9 2.1
12 1(40) 1(7) −1(30) 1(96) 2.9 3.1
13 −1(20) −1(5) 1(40) 1(96) 2.3 2.4
14 1(40) −1(5) 1(40) 1(96) 2.4 2.5
15 −1(20) 1(7) 1(40) 1(96) 2.5 2.7
16 1(40) 1(7) 1(40) 1(96) 2.8 2.9
17 −2(10) 0(6) 0(35) 0(72) 2.0 1.8
18 2(50) 0(6) 0(35) 0(72) 2.8 2.7
19 0(30) −2(4) 0(35) 0(72) 1.8 1.7
20 0(30) 2(8) 0(35) 0(72) 2.9 3.1
21 0(30) 0(6) −2(25) 0(72) 2.1 2.0
22 0(30) 0(6) 2(45) 0(72) 2.2 2.1
23 0(30) 0(6) 0(35) −2(24) 2.6 2.8
24 0(30) 0(6) 0(35) 2(120) 3.8 3.2
25 0(30) 0(6) 0(35) 0(72) 4.1 4.2
26 0(30) 0(6) 0(35) 0(72) 4.1 4.2
27 0(30) 0(6) 0(35) 0(72) 4.1 4.2
28 0(30) 0(6) 0(35) 0(72) 4.1 4.2
29 0(30) 0(6) 0(35) 0(72) 4.1 4.2
30 0(30) 0(6) 0(35) 0(72) 4.1 4.2
31 0(30) 0(6) 0(35) 0(72) 4.1 4.2

factors at a time, holding all other factors at fixed level, are
helpful for understanding both the main and the interaction
effects of these two factors. The effect of varying levels of
pH and temperature on the ethanol production, while other
two variables (substrate concentration at 30 g/l and fer-
mentation time at 72 h) were fixed at central level, is shown
in Fig. 1. When the other two variables kept constant, the
interaction between the two variables (pH and temperature)
showed that the ethanol yield was sensitive even when pH
and temperature were subject to small alterations (Fig. 1).
Under certain conditions a maximal contour (ethanol
concentration of 4.10 g/l) could be determined, meaning
that further change in temperature and pH would not
increase the ethanol yield any further. The contour plot for
substrate concentration and temperature on the yield of
ethanol, where the two variables fermentation time and pH
were kept constant at 72 h and 6 h respectively, showed that
the ethanol yields were obtained in the middle level of the
process variables (Fig. 2).
The other pair of the independent variables pH and
fermentation time showed similar effects while keeping the
other independent variables, temperature and substrate
concentration as constant at 35°C and 30 g/l respectively
(Fig. 3). Also the effect of temperature and fermentation
time at a constant level of substrate concentration, (30 g/l)
and pH (6) was studied and was observed that the ethanol
yields were affected when the two independent variables
(temperature and fermentation time) subject to small
alterations (Fig. 4). The results showed that as the values
of process variables increased, the yield also increased but
only up to the midpoint of range of variables and thereafter
the yield decreased even though the values of variables
increased. The ethanol yield was significantly affected by
substrate concentration, pH, temperature and fermentation
time, where pH produces greater effect.
Based on the model, the optimal working conditions
were obtained to attain high ethanol yield. Response
analysis revealed the maximum ethanol yield by K. fragilis
244 Bioenerg. Res. (2008) 1:239–247

Table 3 Results of regression analysis and corresponding t and p- Table 5 Stepwise regression analysis for quadratic interactions
value of second order polynomial model for optimization of ethanol affecting ethanol yield of sugarcane bagasse
production
Model and independent Estimate Standard t-value P-
Term Regression Std t- P- variablesa error value
constant coefficient deviation statistic value
pH×temperature (P×T)
Intercept 4.1000 0.1179 34.834 <0.001 Intercept 3.627 0.180 20.151 < 0.001
Ca 0.1500 0.0637 2.354 0.032 P2 −0.319 0.115 −2.790 0.009
Pb 0.1667 0.0637 2.616 0.019 T2 −0.369 0.115 −3.231 0.003
Tc 0.1167 0.0637 1.831 0.086 P2 ×T2 −0.601 0.229 −2.630 0.014
Hd 0.1750 0.0637 2.746 0.014 Substrate conc.×temperature (C×T)
C×C −0.4588 0.0583 −7.859 <0.001 Intercept 3.625 0.183 19.850 < 0.001
P×P −0.4713 0.0583 −8.073 <0.001 C2 −0.306 0.116 −2.630 0.014
T×T −0.5213 0.0583 −8.929 <0.001 T2 −0.369 0.116 −3.170 0.004
H×H −0.2588 0.0583 −4.432 <0.001 C2 ×T2 −0.612 0.233 −2.630 0.014
C×P −0.1250 0.0780 −1.602 0.129 pH×fermentation time (P×H)
C×T −0.1250 0.0780 −1.602 0.129 Intercept 3.443 0.212 16.280 <0.001
C×H 0.1000 0.0780 −1.281 0.218 P2 −0.273 0.135 −2.030 0.053
P×T 0.0875 0.0780 1.121 0.279 H2 −0.061 0.135 −0.450 0.656
P×H −0.0375 0.0780 −0.480 0.637 P2 ×H2 −0.771 0.270 −2.860 0.008
T×H −0.1125 0.0780 −1.441 0.169 Temperature×fermentation time (T×H)
Intercept 0.3479 0.204 17.090 <0.001
R2 =0.929
a T2 −0.332 0.130 −2.560 0.016
Substrate concentration (g/l)
b
pH H2 −0.070 0.130 −0.540 0.595
c
Temperature (°C) T2 ×H2 −0.740 0.260 −2.850 0.008
d
Fermentation time (h) a
R2 =0.581 (P×T), 0.549 (C×T), 0.395 (P×H), 0.439 (T×H)

could be achieved at the conditions when substrate Kinetics and Modeling

concentration is 31 g/l, pH of growth media is 5.5;
temperature is 35°C and fermentation time is 72 h. The In general, formulating a useful kinetic model of a cell
matching quality, of the data obtained by the model population is an art which requires consideration of the
proposed in Eq. (1), was evaluated considering the intended end use of the kinetic model, careful thought and
correlation coefficient, R2, between the experimental and experiment to identify the key variables and parameters
modeled data. The mathematical adjust of those values which influence the processes of primary interest, and some
generated a R2 =0.962, revealing that the model could not conceptual and mathematical agility in translating this
explain only 3.74% of the overall effects, showing that it is qualitative view of the system into a workable mathematical
a robust statistical model. representation [2, 10]. Model predictions about the dynamic
behavior of the system offer a more stringent test of validity
than does comparison to steady state experiments [4, 16].
The process is simply that experimental evidence suggests
models which lead to testable predictions and to further
Table 4 Analysis of variance (ANOVA) for the fitted quadratic
experiments which lead to refinements in the models
polynomial model for ethanol production
resulting in new hypothesis and experiments etc. [13, 33].
Source Sum of Degrees of Mean F-value P-value The following desirable aspects are kept in mind for
squares freedom (df) square (MS) selecting the models (1) it should be capable of representing
the entire course of fermentation, (2) the equation has to be
Regression 20.4076 14 1.45768 14.960 <0.001
Linear 2.2683 4 0.56708 5.820 0.004 flexible to fit different types of data, (3) model parameters
Square 17.1317 4 4.28293 43.950 <0.001 should be such that they could be easily estimated and (4)
Interaction 1.0075 6 0.16792 1.720 0.180 the model should be easily usable once the parameters are
Residual 1.5592 16 0.09745 – – determined [9, 28]. Monod and logistic are the models for
error growth kinetics, modified leudeking piret and Logistic
Lack-of-fit 1.5592 10 0.15592 – – incorporated modified leudeking piret are the models for
Pure error 0.0000 6 0.00000 – –
product formation and substrate utilization kinetic models
Total 21.9667 30 – – –
respectively [14, 22].
Bioenerg. Res. (2008) 1:239–247 245

45 120

Fermentation time (h)

Temperature (°C)

40 96

35 4 72 4
3

2 3
30 48
2
1
25 24
4 5 6 7 8 4 5 6 7 8
pH pH
Hold values: Subst. conc.: 30.0 Time: 72.0 Hold values: Subst. conc.: 30.0 Temp: 35.0
Fig. 1 Contour plot for the effect of pH and temperature on ethanol Fig. 3 Contour plot for the effect of pH and fermentation time on
production ethanol production

the process simulation from the various models using the

The objective of this modeling is to develop a
comprehensive mechanistic model based on the experimen-
tal observations. A comprehensive mechanistic kinetic
model has been derived based on the mechanism of ethanol
production from lignocellulosic materials by using yeast
strains. The validity of the proposed model under different
experimental conditions has been tested. The cellmass,
product formation and substrate utilization kinetics using
K. fragilis with different parameters are studied. Monod,
Logistic and Modified logistic models are attempted for
representing the batch growth kinetics. Leudeking piret and
Modified leudeking piret model, which are modified by
incorporating the logistic models, are tried for product
formation and substrate utilization kinetics. The results of
experimental data are compared.
(a) Logistic model (growth)
Under optimal growth conditions and when the inhibi-
tory effects of substrates and product play no role, the rate
of cell growth is given by Eq. (2)
dX
¼ m0 X ð2Þ
dt
where μ0 is a constant defined as the initial specific
growth rate. The logistic model equation implies that
the growth rate increases with increase in cellmass
concentration and is independent of the substrate
concentration. In reality the growth of cell is governed

45 2
Fermentation time (h)
Temperature (°C)

40 1

4
35 4 0 3
3

30 2 -1 2

1 1
25 -2
10 20 30 40 50 -2 -1 0 1 2
Substrate conc. (g/l) Temperature (°C)
Hold values: pH: 6.0 Time: 72.0 Hold values: Subst. conc.: 30.0 pH: 6.0

Fig. 2 Contour plot for the effect of substrate concentration and Fig. 4 Contour plot for the effect of temperature and fermentation
temperature on ethanol production time on ethanol production
246 Bioenerg. Res. (2008) 1:239–247

Table 6 Model parameters for ethanol production from pretreated The model parameter values are evaluated using MATLAB
sugarcane bagasse
program and are presented in Table 6. The simulation result
Model Models of the logistic incorporated leudeking piret model is in good
parameters agreement with the experimental data obtained from the
Logistic Logistic Logistic
pretreated sugarcane bagasse and the minimum average
incorporated incorporated
leudeking piret modified
error of 4.46%.
leudeking piret (c) Logistic incorporated modified leudeking piret model
(substrate utilization)
X0a 1.2 g/l – –
Xmb 5.5 g/l – – The substrate utilization kinetics is the modified form of
μ0c – 0.20 h−1 0.20 h−1 the leudeking piret model which can be used for substrate
μmd 0.086 h−1 –
utilization kinetics. Substrate consumption depends on the
–
Kse 0.52 g/l – –
magnitude of three sink terms, the instantaneous cellmass
αf 0.48 0.38 0.42 growth rate, the instantaneous product formation rate and a
βg 0.018 0.012 0.014 cellmass maintenance function. The assumed kinetic form
γh 5.80 8.80 8.20 is a linear combination of these terms.
ηi  
0.0542 0.016 0.008 Xo emt
Avg. error 6.47% 4.46% 3.02% St ¼ So
g
Xo
R2 0.942 0.966 0.972
1
ðXo =Xm Þð1
emt Þ
   
Xm Xo
a
Initial biomass concentration (g/l)
h ln 1
ð1
emt Þ ð5Þ
b
Maximum biomass concentration (g/l) m Xm
c
Initial specific growth (h−1 )
d
Specific growth rate (h−1 ) The model parameter values shown in Table 6 are then used
e
Growth associated constant for substrate consumption (g substrate/g to simulate the experimental data of substrate concentration
biomass)
f
Non growth associated constant for substrate at any time during the entire fermentation. Better substrate
g
Consumption (g substrate/g biomass h) utilization kinetics is obtained using the logistic incorpo-
h
Growth associated constant (g product/g biomass) rated modified leudeking piret model Eq. (5) and is well
i
Non growth associated constant (g product/g biomass h) suited for ethanol production from pretreated sugarcane
by a hyperbolic relationship and the logistic Eq. (3) is bagasse with a minimum average error of 3.02%.
given by [36]


X ðt Þ ¼ X0 emo t =1
X0=Xm ð1
emo t Þ ð3Þ Conclusion

The logistic equation is utilized to describe the kinetics of Due to dwindling of fossil fuel microbial production of bio
several polysaccharides fermentation systems. The model fuel from biomass (mass of living matter) by products has
parameter values are evaluated using MATLAB 7.0.1 acquired significant fuel for the future. Bagasse, the solid
program and are shown in Table 6. A better prediction of residue after extraction of the sugarcane juice, is mostly
cellmass concentrations is obtained using the logistic utilized for producing steam and electricity required for the
model and it is most suited for ethanol production with cane processing plant. The price of bagasse ranges from Rs.
pretreated sugarcane bagasse as substrate. 100 to 400 (~$2 to 9) per tonne with an average of about Rs.
(b) Logistic incorporated leudeking piret model (product 100 ($2) per tonne. Because of its high carbohydrate content,
formation) relatively low lignin content and its availability as an
industrial waste product, bagasse is a particularly appropriate
substrate for bioconversion to ethanol. This study examines
Logistic incorporated leudeking piret model is developed the possibility of ethanol production from sugarcane bagasse
by rearranging the leudeking piret model as shown below using cellulase and K. fragilis. Conventional optimization
by equation (4). studies are time consuming and expensive. To overcome
2 3 these problems, a central composite design (CCD) was used
m0 t
Pt ¼ P0 þ aX0 4

e
 5 þ bXm for the optimization of process conditions. From the
X
1
0=Xm ð1
e 0 Þm t m0 experimentation, it is evident that the use of statistical
  process condition optimization approach, response surface
X0 methodology has helped to locate the most significant
 ln 1
ð1
em0 t Þ ð4Þ
Xm conditions with minimum effort and time. In addition, it
Bioenerg. Res. (2008) 1:239–247
has also proved to be useful in increasing ethanol concentra-
tion. A maximum ethanol concentration of 32.6 g/l was
obtained from 180 g/l in of sugarcane bagasse at the optimized
process conditions of temperature 35°C, pH 5.5 and fermen-
tation time 72 h in aerobic batch fermentation. Kinetic models
were evaluated and the model constants were predicted on
ethanol production from pretreated sugarcane bagasse.
Acknowledgements The authors express their sincere thanks to
UGC for the financial support through the Rajiv Gandhi National
Fellowship Scheme (RGNFS) for the fellowship to one of the authors
(E. Sasikumar) and to the Department of Technology, Annamalai
University for providing the necessary facilities for the successful
completion of this research work.
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