This action might not be possible to undo. Are you sure you want to continue?
1
8
7
4
Research Article
Received: 18 September 2012 Revised: 7 January 2013 Accepted article published: 31 January 2013 Published online in Wiley Online Library: 13 March 2013
(wileyonlinelibrary.com) DOI 10.1002/jctb.4042
Kinetic studies of ethanol fermentation using
Kluyveromyces sp. IIPE453
Sachin Kumar,
a,c∗,†
Pratibha Dheeran,
a,‡
Surendra P. Singh,
b
Indra
M. Mishra
c
and Dilip K. Adhikari
c
Abstract
BACKGROUND: To operate the fermentation process effectively and efﬁciently, the kinetic modeling of cell growth and ethanol
fermentation is necessary to predict the results of industrial fermentations under the optimized conditions.
RESULTS: A kinetic study was conducted for glucose utilization for growth of the yeast strain Kluyveromyces sp. IIPE453 and
ethanol formation. Effect of temperature, pH and initial glucose concentration on cell growth and ethanol formation was
studied. The data obtained experimentally were validated with existing kinetic models for product and/or substrate inhibition.
CONCLUSION: Of all the models, the Aiba model for substrate inhibition was found to be the best ﬁt to the experimental data
for the growthof Kluyveromyces sp. IIPE453, andthe Luong model for product inhibitionwas foundto be the best ﬁt for ethanol
formation using Kluyveromyces sp. IIPE453.
c 2013 Society of Chemical Industry
Keywords: ethanol fermentation; thermotolerant yeast; Kluyveromyces sp.; growth kinetics; fermentation kinetics
NOTATION
K
CM
Maintenance coefﬁcient (h
−1
)
K
d
Speciﬁc death rate (h
−1
)
K
I
Substrate inhibition constant for cell growth
(g L
−1
)
K
IP
Substrate inhibition constant for ethanol
production (g L
−1
)
K
P
Ethanol inhibition constant for cell growth (g L
−1
)
K’
P
Ethanol inhibition constant for ethanol
production (g L
−1
)
K
S
Saturation constant for cell growth (g L
−1
)
K
SP
Saturation constant for ethanol production
(g L
−1
)
m,n, j Empirical numbers
P Product concentration (g L
−1
)
q
p
Volumetric ethanol productivity (g L
−1
h
−1
)
q
s
Speciﬁc sugar consumption rate (g g
−1
h
−1
)
q
sp
Speciﬁc productivity (g g
−1
h
−1
)
r
P
Rate of ethanol formation (g L
−1
h
−1
)
r
S
Rate of sugar consumption (g L
−1
h
−1
)
r
X
Rate of cell formation (g L
−1
h
−1
)
S Rate limiting substrate concentration (g L
−1
)
S
j
Variance of error of residues
S
o
Initial substrate concentration (g L
−1
)
w
j
Weight factor
X Cell concentration (g L
−1
)
Y
P/S
Yield coefﬁcient for ethanol formation per unit
substrate consumed (g g
−1
)
Y
X/S
Yield coefﬁcient for cells formation per unit
substrate consumed (g g
−1
)
Y
P/S
Experimental ethanol yield (g g
−1
)
Greek symbols
µ Speciﬁc growth rate (h
−1
)
µ
m
Maximum speciﬁc growth rate (h
−1
)
ν Speciﬁc ethanol production rate (h
−1
)
ν
m
Maximum speciﬁc ethanol production rate (h
−1
)
α,β Empirical numbers
j
Mean standard deviation
λ Error statistic
ij
Difference between model and experimental
value of the jth variable in the ith experimental
point
INTRODUCTION
Overexploitation of fossil fuels has been a matter of concern for
energy security and climate change. Green energy sources such as
bioethanol offer numerous advantages, especially as a transport
fuel to improve the quality of urban air accompanied with the
∗
Correspondence to: Dr. Sachin Kumar, Biotechnology Area, Indian Institute of
Petroleum, Dehradun 248 005, India. Email: sachin.biotech@gmail.com
† Present address: Sardar Swaran Singh National Institute of Renewable Energy,
Kapurthala144 601, India
‡ Present address: SRM Research Institute, SRM University, Kattankulathur,
Tamilnadu, India
a Biotechnology Area, Indian Institute of Petroleum, Dehradun 248 005, India
b Department of Chemical Engineering, Indian Institute of Technology Roorkee,
Roorkee 247 667, India
c Department of Paper Technology, Indian Institute of Technology Roorkee,
Saharanpur Campus 247 001, India
J ChemTechnol Biotechnol 2013; 88: 1874–1884 www.soci.org c 2013 Society of Chemical Industry
1
8
7
5
Kinetic studies of ethanol fermentation www.soci.org
reduction in the emission of greenhouse gases, nitrogen oxides
and hydrocarbons. Ethanol has been traditionally manufactured
through fermentation route, using sugary raw materials such as
molasses from sugarcane and sugar beet. Lignocellulosic biomass
is a favorable feedstock for ethanol production based on the
life cycle analysis of the carbon neutral process.
1
However,
fermentation of sugars produced from the sacchariﬁcation
of lignocellulosic biomass such as glucose, xylose, mannose,
galactose, arabinose and cellobiose, to ethanol has limitations due
to the wellknown ethanologens such as Saccharomyces cerevisae
or Zymomonas mobilis because of their metabolic inefﬁciency.
2,3
A
thermotolerant yeast strainKluyveromyces sp. IIPE453(MTCC5314)
showed growth and fermentation efﬁciency on a wide range of
substrates such as glucose, xylose, mannose, galactose, arabinose,
sucrose and cellobiose for growth as well as fermentation to
ethanol at a moderately high temperature of 50
◦
C.
4
The yeast
strainwas alsoabletoconvert sugars present insugarcanebagasse
hydrolysate, sugarcane juice, molasses, mahua ﬂower extract and
their mixtures, to ethanol efﬁciently.
5
Thermophilic/thermotolerant microorganisms and ther
mostable enzymes are of great scientiﬁc interest, principally
with regard to their potential industrial applications due to their
stability at high temperatures.
6,7
Thermophiles have distinct
advantages over mesophiles for ethanol production in terms
of increased solubility of substrates, improved mass transfer
due to decreased viscosity, increased diffusion rates, high
bioconversion rates, ability to use a variety of inexpensive
biomass feedstocks, low risk of contamination, and facilitated
product recovery.
8–12
To operate the fermentation process effectively and efﬁciently,
kinetic modeling of cell growth and ethanol fermentation is
necessary to predict the results of industrial fermentations
under optimized conditions.
6,13
Kinetic modeling of ethanol
fermentation is important to understand metabolic processes, to
estimateprocess parameters andtheir inﬂuenceoncell growthand
product utilization, in the scaleup of the process and the control
of bioreactors.
14–17
In biological processes, such parameters
as temperature, pH, osmotic pressure, substrate and product
concentrations play key and important roles.
18
Monod’s equation
is the most commonmodel, but is applicable for fermentationwith
noinhibition.
17
This model is alsonot validfor theinitial adaptation
phrase just after inoculation of the substrate. An appropriate
ethanol fermentation model should account for the four kinetic
factors, namely
14
substratelimitation, substrateinhibition, ethanol
inhibition and cell death. Phisalaphong et al.
13
considered all four
kinetics parameters for ethanol fermentation by the ﬂocculating
yeast, Saccharomyces cerevisiae M30 and investigated the effect of
temperature on these parameters.
In the present work, the growth of thermotolerant yeast
Kluyveromyces sp. IIPE453 (MTCC 5314) and fermentation of
glucose to ethanol at 50
◦
C are reported. The experimental data
are used for the determination of the kinetic parameters and the
existing mathematical models were tested with the experimental
data and the best ﬁt models were reported.
MATERIALS ANDMETHODS
Growth Conditions
The growth of Kluyveromyces sp. IIPE453 was carried out in a
Bioﬂow110bioreactor (NewBrunswick, USA) (5Lworkingvolume)
under aerobic conditions at a temperature of 50
◦
C and pH 5.0. 1
mol L
−1
phosphoric acid and 1 mol L
−1
NaOH were used as acid
and base, respectively, to maintain the pH. The dissolved oxygen
(DO) was controlled at 40% by agitation and an aeration rate of
1 vvm. The growth medium, salt medium (SM), contained (in g
L
−1
) disodium hydrogen ortho phosphate, 0.15; potassium di
hydrogen ortho phosphate, 0.15; ammonium sulphate, 2.0; yeast
extract, 1.0. Themediumwas sterilizedfor 20minat 121
◦
Cusingan
autoclave. To determine the effect of temperature and pH on the
growth of Kluyveromyces sp. IIPE453, the temperature was varied
from 40 to 60
◦
C at constant pH 5.0 and pH was varied from 4.0 to
6.0 at a constant temperature of 50
◦
C, respectively. The effect of
glucose concentration on the growth of Kluyveromyces sp. IIPE453
was observed by varying its concentration from 5 to 40 g L
−1
at a
temperature of 50
◦
C and pH 5.0.
Fermentation conditions
Ethanol fermentation was carried out in a Bioﬂow110 bioreactor
(New Brunswick, USA) (2 L working volume) under anaerobic
conditions at a temperature of 50
◦
C and pH 5.0. The fermentation
mediumwas the growth medium, except an ammoniumsulphate
concentration of 1.0 g L
−1
, was prepared. The medium was
sterilized for 20 min at 121
◦
C using an autoclave. To determine
the effect of temperature and pH on ethanol fermentation, the
temperature was varied from 40 to 60
◦
C at constant pH 5.0 and
pH was varied from 4.0 to 6.0 at a constant temperature of 50
◦
C.
The effect of glucose concentration on ethanol fermentation was
observed by varying its concentration from 200 to 300 g L
−1
at a temperature of 50
◦
C and pH 5.0. All the experiments were
conducted in duplicate and average values are reported.
Analytical methods
Glucose was analyzed by HPLC using a high performance
carbohydrate column (Waters) at 30
◦
C with acetonitrile and water
mixture (75:25) as a mobile carrier at a ﬂow rate of 1.4 mL min
−1
and detected by a Waters 2414 refractive index detector. Ethanol
was analyzed by gas chromatography using an Ashco Neon II gas
analyzer with a 2 m long and 1/8
diameter porapakQS column
with a mesh range of 80/100. The sample was injected at an
inlet temperature of 220
◦
C, oven temperature of 150
◦
C and ﬂame
ionization detector temperature of 250
◦
C using nitrogen gas as a
carrier.
KINETICS
Ethanol fermentation is governed by the speciﬁcity of the
microorganisms and the metabolic regulations, which are
dependent on the process parameters. Hence, the growth of
microbial cells and fermentation, the cell behavior towards the
substrate and the product, particularly on their concentration and
the role of cells in the overall productivity of the process, need
careful monitoring. A functional relationship between speciﬁc
growth rate (µ) and the rate limiting substrate concentration (S) is
generally expressed by the Monodtype equation:
µ =
µ
m
S
K
S
+S
(1)
where µ
m
is the maximumspeciﬁc growth rate, and K
S
is the value
of the rate limiting substrate concentration at which the speciﬁc
growth rate is half of its maximum value, generally referred to as
the saturation constant. In most processes, high concentrations
of substrate and/or product often lead to inhibitory effects.
J ChemTechnol Biotechnol 2013; 88: 1874–1884 c 2013 Society of Chemical Industry wileyonlinelibrary.com/jctb
1
8
7
6
www.soci.org S Kumar et al.
Equation (1) does not incorporate inhibition due to substrate
or product or both. Therefore, the proposed kinetics can be
modiﬁed in terms of both substrate and product inhibition and
combined with the death rate and cell maintenance. The rate of
cell mass formation, ethanol formationandsubstrateconsumption
are related to the cell concentration (X), ethanol concentration (P)
and substrate concentration (S) as follows:
19
Cells : r
X
=
dX
dt
= µX −K
d
X (2)
Ethanol : r
P
=
dP
dt
= νX (3)
Substrate : −r
S
= −
dS
dt
=
1
Y
X/S
_
dX
dt
_
+
1
Y
P/S
_
dP
dt
_
+K
CM
X (4)
where K
d
is the speciﬁc death rate (h
−1
); K
CM
is the maintenance
coefﬁcient (h
−1
); Y
X/S
and Y
P/S
are the yield coefﬁcients for cell
mass and ethanol formation per unit substrate consumed (g
g
−1
), respectively. The Monod equation is not valid during initial
adaptation phase following inoculation. This is basically because
of the time needed for the establishment of glycolysis and other
pathways by the enzyme pool.
The rate of cell growth, substrate consumption and ethanol
formation for fermentation without any growth can then be
related as follows:
Cells : r
X
=
dX
dt
= −K
d
X (5)
Substrate : −r
S
= −
dS
dt
=
1
Y
P/S
_
dP
dt
_
+K
CM
X (6)
Ethanol : r
P
=
dP
dt
= νX (7)
ν =
ν
m
S
K
S
+S
(8)
where
ν =
1
X
dP
dt
= speciﬁc product formation rate
_
g g
−1
h
−1
_
Ethanol fermentation with sugary substrates generally
experiences inhibition of cell growth by the substrate (feed)
and ethanol (product). Several inhibition models have been
proposed by various researchers for either substrate or product
or both. These are summarized in Table 1 Equations (T1) to
(T28). Most of the models have been developed for substrate
inhibition. The models are categorized into three types: (1)
substrate inhibition; (2) product inhibition; (3) both substrate and
product inhibition.
Various researchers have validated their models for substrate
and/or product inhibition with experimental data. The initial
parameters for growth µ and Y’
X/S
can be calculated from the
experimental data. Further, the parameters µ
m
, K
d
, K
CM
and Y’
X/S
can be calculated from Equations (1) to (4) using Newton’s
method (Solver, Microsoft Excel 2003, Microsoft Corporation,
USA) by minimizing the sum of the squared deviations between
experimental and calculated data. The parameters µ
m
, K
S
, K
I
and
K
P
can be calculated by using the best ﬁt model from those given
in Table 1 odd numbered Equations (T1) to (T28) using Newton’s
method.
The initial parameters for fermentation ν and Y’
P/S
can be
calculated from the experimental data. Further, the parameters
ν
m
, K’
d
, K’
CM
and Y’
P/S
can be calculated from Equations (5) to (8)
using Newton’s method. The parameters ν
m
, K
SP
, K
IP
and K
P
can
be calculated by using the best ﬁt model from the models given
in Table 1 even numbered Equations (T1) to (T28) using Newton’s
method.
The ethanol yield (Y
P/S
), (g g
−1
), speciﬁc sugar consumption rate
(q
s
), (g g
−1
h
−1
), volumetric productivity (q
p
), (g L
−1
h
−1
), and
speciﬁc productivity (q
sp
), (g g
−1
h
−1
), can be calculated as:
Y
P/S
=
P
(S
o
−S)
(9)
q
s
=
S
o
−S
Total fermentation time ×Average dry cell weight
(10)
q
p
=
P
Total fermentation time
(11)
q
sp
=
q
p
Average dry cell weight
(12)
where P is the ethanol concentration; S
o
is the initial sugar
concentration and S is the residual sugar concentration.
Estimation of model parameters
Error minimization between the model predicted values and the
corresponding experimental data was carried out by using various
error estimates including minimization of the weighted sum of
squares of residuals (SSWR), the mean standard deviation (
j
), the
variance of error of residues (S
j
), an error statistic (λ), and the root
mean square error (RMSE) as given by Khan et al.
20
The weighted
sum of squares of residuals is deﬁned as:
SSWR =
n
i=1
m
j=1
2
ij
w
2
j
(13)
where n and m are the number of experimental data points and
the number of process variables, respectively; w
j
is the ‘weight
factor’, which is the maximum value of the variables; and
ij
is
the difference between model and experimental value of the jth
variable in the ith experimental point.
The mean standard deviation of the variable (
j
) is calculated
as follows:
j
=
1
n
n
i=1
ij
j = 1, m (14)
The variance of error of residues (S
j
) is estimated as:
S
j
=
1
n −1
n
i=1
(
ij
−
j
)
2
j = 1, m (15)
The error statistic (λ) is deﬁned as:
λ =
(n −m) n
(n −1) m
m
j=1
2
j
S
j
(16)
wileyonlinelibrary.com/jctb c 2013 Society of Chemical Industry J ChemTechnol Biotechnol 2013; 88: 1874–1884
1
8
7
7
Kinetic studies of ethanol fermentation www.soci.org
Table 1. Kinetic models for microbial growth and fermentation with inhibition effect
S.No. Model Equation for cell growth Equation for fermentation
A. Substrate inhibition
1 Haldane
30
(1965) µ = µ
m
S
K
S
+S+
S
2
K
I
(T1) ν = ν
m
S
K
SP
+S+
S
2
K
IP
(T2)
2 Andrews
31
(1968) µ = µ
m
S
(S+K
S
)
_
1+
S
K
I
_
(T3) ν = ν
m
S
(S+K
SP
)
_
1+
S
K
IP
_
(T4)
3 Aiba et al.
24
(1968) µ = µ
m
S
(S+K
S
)
exp
_
−
S
K
I
_
(T5) ν = ν
m
S
(S+K
SP
)
exp
_
−
S
K
IP
_
(T6)
4 Yano and Koga
32
(1969) µ =
µmax
1+
K
S/S+
n
J=1
_
S
/K
j
_
J
(T7) ν =
νmax
1+
K
SP/S+
n
J=1
_
S
/K
j
_
J
(T8)
5 Orhon and T¨ unay
33
(1979) µ = µ
m
S
K
S
+S+
S.S
I
K
I
(T9) ν = ν
m
S
K
SP
+S+
S.S
IP
K
IP
(T10)
6 Luong
34
(1987) µ = µ
m
S
(S+K
S
)
_
1 −
S
K
I
_
α
(T11) ν = ν
m
S
(S+K
SP
)
_
1 −
S
K
IP
_
α
(T12)
7 Han and Levenspiel
35
(1988) µ = µ
m
_
1 −
S
K
I
_
n
S
S+K
S
_
1−
S
K
I
_
m
(T13) µ = µ
m
_
1 −
S
K
IP
_
n
S
S+K
SP
_
1−
S
K
IP
_
m
(T14)
8 Sivakumar et al.
36
(1994) µ = µ
m
_
1 −
S
K
I
_
n
S
S+K
S
_
1−
S
So
_
m
(T15) ν = ν
m
_
1 −
S
K
IP
_
n
S
S+K
SP
_
1−
S
So
_
m
(T16)
B. Product inhibition
9 Aiba et al.
24
(1968) µ = µ
m
S
(S+K
S
)
exp(−P.K
P
) (T17) ν = ν
m
S
(S+K
SP
)
exp
_
−P.K
P
_
(T18)
10 Levenspiel
37
(1980) µ = µ
m
_
1 −
P
K
P
_
β
(T19) ν = ν
m
_
1 −
P
K
P
_
β
(T20)
11 Luong
27
(1985) µ = µ
m
_
1 −
_
P
K
P
_
β
_
(T21) ν = ν
m
_
1 −
_
P
K
P
_
α
_
(T22)
C. Substrate and product inhibition
12 Heuvel and Beeftink
38
(1988) µ = µ
m
S
K
S
+S+
S
2
K
I
.
K
P
K
P
+P
(T23) ν = ν
m
S
K
SP
+S+
S
2
K
IP
.
K
P
K
P
+P
(T24)
13 Gonc¸alves et al.
39
(1991) µ = µ
m
_
1 −
S
S
∗
_
n
_
1 −
P
P
∗
_
m
(T25) ν = ν
m
_
1 −
S
S
∗
_
n
_
1 −
P
P
∗
_
m
(T26)
14 Phisalaphong et al.
13
(2006) µ = µ
m
S
K
S
+S+
S
2
K
I
_
1 −
P
K
P
_
(T27) ν = ν
m
S
K
SP
+S+
S
2
K
IP
_
1 −
P
K
P
_
(T28)
S, S
o
and P are substrate, initial substrate and product concentrations, respectively; µ
m
and ν
m
are the maximum speciﬁc growth rate (h
−1
) and
maximum speciﬁc ethanol production rate (h
−1
), respectively; K
S
, K
I
and K
P
are the saturation, substrate inhibition and ethanol inhibition constants,
for cell growth (g L
−1
), respectively; K
SP
, K
IP
and K’
P
are the saturation, substrate inhibition and ethanol inhibition constants, for ethanol production
(g L
−1
), respectively; α, β, m, n and j are empirical numbers.
The error statistic (λ) can be calculated using Equations (13) and
(16). The error statistic has the F
m,nm
distribution and is used to
ﬁnd the statistical adequacy for acceptance of the model. If λ is
less than F
m,nm
value obtained from the Ftable, the method is
acceptable for representing the data for a particular percentage
conﬁdence level.
The root mean square error (RMSE), commonly used to
check the validity of the model for a single variable, can be
determined as:
RMSE =
¸
¸
¸
_
1
n
n
i=1
(Observedvalues −Predictedvalues)
2
(17)
Values of SSWR, S
j
and RMSE close to zero provide better
estimates of the model parameters. The variance of error of
residues (S
j
), and error statistic (λ) take into account the mean
standard deviation (
j
) in their estimates.
RESULTS ANDDISCUSSION
Growth of Kluyveromyces sp. IIPE453
The growth of the yeast Kluyveromyces sp. IIPE453 was studied
on glucose. Figure 1 shows the residual glucose concentration
(S) during the growth of the yeast at 50
◦
C with different initial
concentrations of glucose (10 g L
−1
≤S
o
≤40 g L
−1
). The glucose
could be utilized for both growth of the cells as well as ethanol
formation under aerobic conditions (Fig. 1). The cell mass yield,
Y
X/S
, on glucose was obtained as 0.2 g cells g
−1
glucose, whereas
the ethanol yield was found to be 0.35 g g
−1
glucose. The ethanol
yield, Y
P/S
, was obtained as 0.35 g g
−1
. The speciﬁc growth rate
of Kluyveromyces sp. IIPE453 was found to be 0.23 h
−1
on glucose
at a temperature of 50
◦
C and pH 5.0. Banat et al.
21
reported a
speciﬁc growth rate of Kluyveromyces marxianus IMB3 as 0.63 h
−1
on glucose in batch fermentation at 50
◦
C.
Figure 2 shows the variation of residual glucose concentration
with its initial value S
o
at different times during the growth
J ChemTechnol Biotechnol 2013; 88: 1874–1884 c 2013 Society of Chemical Industry wileyonlinelibrary.com/jctb
1
8
7
8
www.soci.org S Kumar et al.
Figure 1. Timecourse of glucose concentration (S) ; dry cell weight
(X) ; and ethanol concentration (P) during the growth of
Kluyveromyces sp. IIPE453 at different initial glucose concentrations (S
o
) at
T =50
o
C; pH =5.0 [S
o
(g L
−1
): 10; 20; 40].
Figure 2. Variation of residual glucose concentration (S) ; dry cell
weight (X) and ethanol formation (P) [ ] with initial glucose
concentrations (S
o
) with time (t) as a parameter during the growth of
Kluyveromyces sp. IIPE453 at T = 50
o
C; pH = 5.0 [t (h): 0; 4; 8; 12;
16].
of Kluyveromyces sp. IIPE453. Although an increase in S
o
hastens glucose consumption at any time. The residual glucose
concentration was higher at higher S
o
at fermentation time 12 or
16 h. Figure 2 shows the variation of cell mass concentration (X)
with S
o
and time as the parameters. It is found that X increases
from S
o
= 5 g L
−1
to S
o
= 10 g L
−1
and then decreases as S
o
is
further increased. The peak of X at S
o
= 10 g L
−1
shows that cell
growth is maximum at S
o
= 10 g L
−1
for time up to 16 h (Fig. 2).
The inﬂuence of S
o
on ethanol concentration is shown in Fig. 2. It
is seen that the increase in ethanol concentration with S
o
is low
initially. As time progresses, the increase in ethanol concentration
becomes more and more pronounced. At t = 16 h, the ethanol
concentration increases from P = 0.6 g L
−1
at S
o
= 5 g L
−1
to P
= 6.5 g L
−1
at S
o
= 40 g L
−1
. This trend is in contrast with that
for X versus S
o
plots in Fig. 2. It can be hypothesized that with an
increase in S
o
(S
o
> 10 g L
−1
), ethanol formation is at the expense
of cell growth.
The timecourse of rate of glucose consumption (−r
s
) and cell
mass formation (r
x
) is shown in Fig. 3. It is found that the rate of
glucose consumption is faster up to t = 12 h and, thereafter, the
increase in rate is slower. The rate of cell mass formation increases
faster up to ˜8 h and, thereafter, the rate of increase slows. For S
o
=
10gL
−1
, r
x
shows a decreasingtrendbeyondt =24h. For S
o
=40g
L
−1
, r
x
becomes almost constant after t =16 h. Figure 3 shows the
Figure 3. Timecourse of rate of glucose consumption (r
s
) ; cell mass
formation (r
x
) and ethanol formation (r
p
) with initial glucose
concentration (S
o
) as a parameter at T = 50
o
C; pH = 5.0 [S
o
(g L
−1
): 10;
20; 40].
Table 2. Effect of temperature on kinetic parameters for cell growth
at S
o
=20 g L
−1
; pH =5.0
Temperature (
◦
C)
Parameters 40 45 50 55 60
µ
m
(h
−1
) 0.25 0.32 0.34 0.23 0.12
K
S
(g L
−1
) 14.2 15.0 16.8 18.5 21.2
K
d
(h
−1
) 0.02 0.028 0.005 0.039 0.045
K
CM
(h
−1
) 0.011 0.011 0.011 0.013 0.012
Y’
X/S
0.45 0.56 0.52 0.38 0.29
Y’
P/S
0.4 0.5 0.54 0.36 0.28
timecourse of rate of ethanol formation (r
p
) at 50
◦
C at different
initial glucose concentrations. It is seen that the rate of ethanol
formation increases at a faster rate with an increase in S
o
. For S
o
=
40 g L
−1
, the increase in r
p
becomes much slower at t >8 h.
Evaluation of kinetic parameters for cell growth
Using the experimental growth data under aerobic conditions, the
Monod model was ﬁtted to determine kinetic parameters and the
effect of temperature, pH and initial glucose concentration using
Equations (1) –(4) as shown in Tables 2, 3 and 4, respectively. As
shown in Table 2, the maximumspeciﬁc growth rate (µ
m
) for S
o
=
20 g L
−1
and pH= 5.0 increases with an increase in temperature
up to 50
◦
C and, thereafter, shows a sharp decline. Table 3 shows
the maximum growth rate at pH 5.0. Table 4 shows the maximum
growth rate at the glucose concentration of 10 g L
−1
. The values
of µ
m
at S
o
= 10 g L
−1
and S
o
= 20 g L
−1
are almost the same.
However, S
o
> 20 g L
−1
, µ
m
decreased considerably. Similarly,
µ
m
is maximum at pH = 5.0 and decreases considerably when
pH is decreased or increased. Wang et al.
22
estimated the kinetic
parameters using the Monod model on glucose using S. cerevisiae
strain CCTCC M201022 as µ
m
=0.094 h
−1
, Y’
X/S
=0.22 g g
−1
, Y’
P/S
=1.22 g g
−1
and K
CM
=0.114 h
−1
.
Simulation of the growth of Kluyveromyces sp. IIPE453 cells
The Monod Equation (1) is valid for noninhibitory growth of
microbial cells but is not valid for the initial adaption period,
as stated earlier. Figure 2 shows that the cell concentration (X)
decreases with an increase in S
o
beyond 10 g L
−1
. However,
wileyonlinelibrary.com/jctb c 2013 Society of Chemical Industry J ChemTechnol Biotechnol 2013; 88: 1874–1884
1
8
7
9
Kinetic studies of ethanol fermentation www.soci.org
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0 5 10 15 20 25 30 35 40
S (g l
1
)
0 5 10 15 20 25 30 35 40
S (g l
1
)
µ
(
h

1
)
m
m
= 0.177 h
1
m
m
= 0.13 h
1
m
m
= 0.15 h
1
m
m
= 0.19 h
1
m
m
= 0.21 h
1
(a)
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
µ
(
h

1
)
(b)
K
S
= 30g l
1
K
S
= 25.2 g l
1
K
S
= 35 g l
1
K
S
= 20 g l
1
K
S
= 15 g l
1
Figure 4. Simulation of growth of Kluyveromyces sp. IIPE453 at S
o
=40 g L
−1
; pH =5.0; T =50
◦
C using Monod equation for (a) different values of µ
m
at
K
S
=25.2 g L
−1
; (b) different values of K
S
at µ
m
=0.177 h
−1
( model curve; experimental data).
Table 3. Effect of pH on kinetic parameters for cell growth at S
o
=
20 g L
−1
; T =50
◦
C
pH
Parameters 4.0 4.5 5.0 5.5 6.0
µ
m
(h
−1
) 0.18 0.26 0.34 0.31 0.24
K
S
(g L
−1
) 2.5 2.2 16.8 2.9 6.2
K
d
(h
−1
) 0.035 0.038 0.036 0.042 0.46
K
CM
(h
−1
) 0.012 0.012 0.011 0.015 0.014
Y’
X/S
0.42 0.5 0.52 0.35 0.23
Y’
P/S
0.48 0.5 0.54 0.39 0.31
Table 4. Effect of initial glucose concentration (S
o
) on kinetic
parameters for cell growth at T =50
◦
C; pH =5.0
S
o
(g L
−1
)
Parameters 5 10 20 40
µ
m
(h
−1
) 0.27 0.35 0.34 0.18
K
S
(g L
−1
) 1.7 5.5 16.8 25.2
K
d
(h
−1
) 0.036 0.038 0.036 0.036
K
CM
(h
−1
) 0.011 0.011 0.011 0.017
Y’
X/S
0.52 0.54 0.52 0.48
Y’
P/S
0.44 0.56 0.54 0.47
the experimental cell growth data are found to be correlated
satisfactorily by the Monod equation for 10 g L
−1
< S
o
< 40 g
L
−1
. The Monod model ﬁts with the experimental data varying µ
m
and K
S
are shown in Fig. 4. Table 5 shows the sensitivity of the
model parameters µ
m
and K
S
and error estimates for the model
ﬁt with the experimental data. RMSE is found to be lowest at
µ
m
= 0.177 h
−1
and K
s
= 25.2 g L
−1
. The Monod model does
not represent the transient data during the initial adaptation
phase, therefore, as S
o
increases, higher values of K
S
are used
to ﬁt the data. Although, Ren et al.
23
used a pathway metabolic
approachtodeal withsucha situation, whichmakes K
S
maintainits
value within a reasonable and meaningful range, this approach is
not used here.
Various models are available for representing the experimental
cell growth data under substrate inhibition conditions (Table 1).
Of all the models, the model of Aiba et al.,
24
Equation (T5) was
Table 5. Sensitivity analysis of Monod model parameters and error
estimates for the experimental Kluyveromyces sp. IIPE453 growth data
Model
equation with
parameters
Parametric
value S
j
λ RMSE Figure
Equation (1) µ
m
=0.177 h
−1
K
s
=15 g L
−1
0.00011 0.0020 0.0616 4(a)
K
s
=20 g L
−1
4.0x10
5
0.0007 0.0287
K
s
=25.2 g L
−1
9.0x10
6
0.0002 0.0092
K
s
=30 g L
−1
4.4x10
6
7.6x10
5
0.0214
K
s
=35 g L
−1
1.4x10
5
0.0002 0.0374
Equation (1) K
s
=25.2 g L
−1
µ
m
=0.13 h
−1
6.5x10
5
0.0011 0.0567 4(b)
µ
m
=0.15 h
−1
1.93x10
5
0.0003 0.0331
µ
m
=0.177 h
−1
9.0x10
6
0.0002 0.0092
µ
m
=0.19 h
−1
2.6x10
5
0.0004 0.0188
µ
m
=0.21 h
−1
7.8x10
5
0.0014 0.0416
found to ﬁt the experimental data satisfactorily and adequately.
Figure 5 shows the ﬁt of the experimental data of µ versus S for
various values of µ
m
, K
s
and K
I
. Table 6 shows the error estimates
for ﬁtting the model Equation (T5) to the experimental growth
data with variation in parametric values. It is found that the model
of Aiba et al.
24
has the best ﬁt with the experimental µ versus S
data for parametric values µ
m
= 0.44 h
−1
, K
s
= 71.7 g L
−1
and
K
I
= 99.3 g L
−1
. Comparison of Table 5 with Table 6 shows that
the Monod equation gives a better ﬁt to the experimental speciﬁc
growthdatawiththeerror estimates lower thanthecorresponding
values for the model of Aiba et al.
24
Figure 6 shows the simulation
of residual sugar concentration (S), cell concentration (X) and
ethanol concentration (P) with time for S
o
= 40 g L
−1
, pH = 5.0
at 50
◦
C temperature. It is seen that all the data ﬁt well with the
model predicted curves. Thus, the growth rate data can be best
represented by either the Monod model equation:
µ =
0.177 ×S
25.2 +S
(18)
or the model of Aiba et al.:
24
µ = 0.44 ×
S
(S +71.7)
exp
_
−
S
99.3
_
(19)
J ChemTechnol Biotechnol 2013; 88: 1874–1884 c 2013 Society of Chemical Industry wileyonlinelibrary.com/jctb
1
8
8
0
www.soci.org S Kumar et al.
m
m
= 0.44 h
1
m
m
= 0.36 h
1
m
m
= 0.40 h
1
m
m
= 0.48 h
1
m
m
= 0.52 h
1
K
S
= 80 g l
1
K
S
= 71.7 g l
1
K
S
= 90 g l
1
K
S
= 60 g l
1
K
S
= 50 g l
1
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
µ
(
h

1
)
(a)
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
µ
(
h

1
)
(b)
0 5 10 15 20 25 30 35 40
S (g l
1
)
K
l
= 80 g l
1
K
l
= 99.3g l
1
K
l
= 120 g l
1
K
l
=110 g l
1
K
l
= 90 g l
1
0
0.02
0.04
0.06
0.08
0.1
0.12
µ
(
h

1
)
(c)
0 5 10 15 20 25 30 35 40
S (g l
1
)
0 5 10 15 20 25 30 35 40
S (g l
1
)
Figure 5. Simulation of growth of Kluyveromyces sp. IIPE453 at S
o
= 40 g L
−1
; pH = 5.0; T = 50
◦
C using the model of Aiba et al. for different values of
(a) µ
m
at K
S
= 71.7 g L
−1
; K
I
= 99.3 g L
−1
; (b) K
S
at µ
m
= 0.44 h
−1
; K
I
= 99.3 g L
−1
; (c) K
I
at µ
m
= 0.44 h
−1
; K
S
= 71.7 g L
−1
( model curve;
experimental data).
0
5
10
15
20
25
30
35
40
45
0 4 8 12 16 20 24 28 32 36
Time (h)
S
(
g
l

1
)
0
2
4
6
8
10
12
14
16
X
,
P
(
g
l

1
)
Figure 6. Simulation of residual glucose concentration (S), dry cell mass
(X) and ethanol concentration (P) using the model of Aiba et al. at S
o
=40
g L
−1
; pH = 5.0; T = 50
◦
C; µ
m
= 0.44 h
−1
; K
S
= 71.7 g L
−1
( model
curves; Sexp; Xexp; Pexp).
Krishnan et al.
25
determined the kinetic parameters for growth
of recombinant Saccharomyces 1400(pLNH33) as µ
m
=0.662 h
−1
,
K’
S
=0.565 g L
−1
, K’
I
=283.7 g L
−1
, K’
CM
=0.1 h
−1
and Y’
X/S
= 0.115
on glucose, and µ
m
= 0.19 h
−1
, K’
S
= 3.4 g L
−1
, K’
I
= 18.1 g L
−1
,
K’
CM
=0.07 h
−1
and Y’
X/S
= 0.162 on xylose.
Ethanol fermentation
Batch fermentation studies were conducted for S
o
= 200 g L
−1
,
250 g L
−1
and 300 g L
−1
. The concentration of glucose to ethanol
at a S
o
=300 gL
−1
and temperature of 50
◦
C was found to be 57%.
The initial glucose concentrations (200≤ S
o
≤300 g L
−1
) did not
have any effect onthe maximumethanol concentrationwhichwas
found to be about 86.5 g L
−1
(Fig. 7). The ethanol yield was 90%
of its theoretical yield at S
o
= 300 gL
−1
. The volumetric ethanol
Table 6. Sensitivity analysis of kinetic parameters for the model of
Aiba et al.
24
and error estimates for the experimental Kluyveromyces
sp. IIPE453 growth data
Model
equation with
parameters
Parametric
value S
j
λ RMSE Figure
Equation (T5) µ
m
=0.36 h
−1
0.00002 0.0002 0.0404 5(a)
K
S
=71.7 g L
−1
µ
m
=0.40 h
−1
0.00001 0.0001 0.0222
K
I
=99.3 g L
−1
µ
m
=0.44 h
−1
0.00001 0.0001 0.0101
µ
m
=0.48 h
−1
0.00003 0.0003 0.0214
µ
m
=0.52 h
−1
0.00008 0.0008 0.0395
Equation (T5) K
S
=50 g L
−1
0.00015 0.0014 0.0643 5(b)
µ
m
=0.44 h
−1
K
S
=60 g L
−1
0.00006 0.0005 0.0316
K
I
=99.3 g L
−1
K
S
=71.7 g L
−1
0.00001 0.0001 0.0101
K
S
=80 g L
−1
0.000004 0.00004 0.0199
K
S
=90 g L
−1
0.00001 0.00013 0.0359
Equation (T5) K
I
=80 g L
−1
0.00001 0.0001 0.0160 5(c)
µ
m
=0.44 h
−1
K
I
=90 g L
−1
0.00001 0.0001 0.0115
K
S
=71.7 g L
−1
K
I
=99.3 g L
−1
0.00001 0.0001 0.0101
K
I
=110 g L
−1
0.00022 0.0001 0.0113
K
I
=120 g L
−1
0.00002 0.0002 0.0137
productivity for S
o
= 200 g L
−1
, 250 g L
−1
and 300 g L
−1
were
found to be 2.28 g L
−1
h
−1
, 2.17 g L
−1
h
−1
and 2.16 g L
−1
h
−1
,
respectively, whereas the speciﬁc ethanol productivity for S
o
=
200 g L
−1
, 250 g L
−1
and 300 g L
−1
were found to be 0.63 g
g
−1
h
−1
, 0.57 g g
−1
h
−1
and 0.48 g g
−1
h
−1
, respectively. The
volumetric ethanol productivity for S
o
= 200 g L
−1
was found
to be marginally higher than that for S
o
= 250 g L
−1
or 300 g
wileyonlinelibrary.com/jctb c 2013 Society of Chemical Industry J ChemTechnol Biotechnol 2013; 88: 1874–1884
1
8
8
1
Kinetic studies of ethanol fermentation www.soci.org
Figure 7. Timecourse of glucose concentration (S) and ethanol
concentration (P) during batch fermentation with initial glucose
concentration (S
o
) as a parameter at T =50
◦
C; pH =5.0 [S
o
(g L
−1
): 200;
250; 300].
L
−1
whereas speciﬁc productivity for S
o
= 200 g L
−1
was found
to be much higher than that for S
o
= 250 g L
−1
or 300 g L
−1
.
After 40 h of fermentation, with 200 g L
−1
≤ S
o
≤ 300 g L
−1
, the
ethanol concentration (P) was found to be the same, 86.8 g L
−1
.
This shows high tolerance of Kluyveromyces sp. IIPE453 for the
substrate glucose and the product ethanol at 50
◦
C temperature
and pH 5.0. After 32 h of fermentation, the ethanol formation
diminisheddue to product inhibitionas the ethanol concentration
in the broth was found to be more than 86 g L
−1
. Banat et al.
26
reported a maximum ethanol concentration of 69.46 g L
−1
with
an ethanol yield of 97.8%of its theoretical yield and 1.67 g L
−1
h
−1
ethanol productivity when glucose was used at a concentration of
S
o
=140 g L
−1
by Kluyveromyces marxianus at 45
◦
C.
Figure 8 shows the variation of residual glucose concentration
with its initial value S
o
at different times during the ethanol
fermentation. It was foundthat glucoseconsumptionwas constant
for S
o
= 50 to S
o
= 200 g L
−1
,
4
whereas it decreased for S
o
= 250
and 300 g L
−1
after 8 h. At t =12 h, the glucose was exhausted for
S
o
=50 g L
−1
, whereas, the glucose was exhausted at t =16 h for
S
o
=100 g L
−1
. The glucose remained unutilized for S
o
=250 g L
−1
toS
o
=300gL
−1
duetoincreaseintheethanol concentrationinthe
broth. Figure8shows thevariationof ethanol concentration, Pwith
S
o
withtime as the parameter. It is seenthat the increase inethanol
concentration with S
o
is initially slow. P increases from S
o
= 50 g
L
−1
to S
o
= 100 g L
−1
and then decreases slightly as S
o
increases.
As time progresses, P becomes constant and at t =32 to 40 h, the
ethanol concentrationincreases very slightly. This trendshows the
inhibition due to increase in ethanol concentration after t =32 h.
Figure 9 shows the timecourse of rate of glucose consumption
(−r
s
) and the rate of ethanol formation (r
p
), respectively, during
batch fermentation at 50
◦
C temperature and pH5.0. It is seen that
the rate of glucose consumption and ethanol formation increase
with time, form a plateau and then decrease. Initially, the rates
decrease withincrease inS
o
, but at the endof fermentationat 40 h,
the rates become the same. As shown in Fig. 9, the rate of ethanol
formation starts decreasing after 32 h, showing the inhibition due
to high ethanol concentration in the broth.
Kinetic parameters for ethanol fermentation
Using the experimental data for ethanol fermentation, the kinetics
of fermentation was studied. The kinetic parameters for ethanol
fermentation were determined by ﬁtting Equations (5) –(8) to the
experimental data at different temperatures, pH and S
o
as shown
in Tables 7, 8 and 9, respectively.
Figure 8. Variation of residual glucose concentration (S) and ethanol
formation (P) with initial glucose concentrations (S
o
) with time (t) as
a parameter during batch fermentation of glucose using Kluyveromyces sp.
IIPE453 at T =50
o
C; pH =5.0 [t (h): 0; 4; 8; 12; 16; 20; 24;
28; ×32; −36; +40].
Figure 9. Timecourse of rate of glucose consumption (r
s
) and
ethanol formation (r
p
) during batch fermentation of glucose with
initial glucose concentration (S
o
) as a parameter at T =50
◦
C; pH =5.0 [S
o
(g L
−1
): 200; 250; 300].
Table 7. Effect of temperature on kinetic parameters for ethanol
fermentation at S
o
=200 g L
−1
; pH =5.0
Temperature (
◦
C)
Parameters 40 45 50 55 60
ν
m
(h
−1
) 0.8 0.98 1.08 0.69 0.42
K
SP
(g L
−1
) 77 83 85 56 43
K’
d
(h
−1
) 0.01 0.01 0.012 0.025 0.038
K’
CM
(g L
−1
) 0.024 0.025 0.027 0.029 0.034
Y’
P/S
0.48 0.5 0.5 0.39 0.34
As shown in Table 7, the maximum speciﬁc ethanol formation
rate (ν
m
) was observed at a temperature of 50
◦
C when S
o
= 200
g L
−1
and pH was kept at 5.0. The maximum speciﬁc ethanol
formation rate was also observed at pH 5.0 for S
o
= 200 g L
−1
and the temperature was maintained at 50
◦
C (Table 8). The effect
of S
o
on ethanol fermentation can be observed from Table 9. It is
seen that S
o
has very little effect on ν
m
for 200 g L
−1
≤S
o
≤300 g
L
−1
. Apart from ν
m
, Y’
P/S
was also found to be highest (Y
P/S
= 0.5)
during fermentation at a temperature of 50
◦
C and pH = 5.0. Y’
P/S
was found to be invariant with S
o
over 200 g L
−1
≤S
o
≤300 g L
−1
.
Krishnan et al.
25
determined the kinetic parameters as ν
m
= 2.0
h
−1
, K’
S
=1.34 g l
−1
, K’
CM
=0.1 h
−1
and Y’
P/S
= 0.47.
J ChemTechnol Biotechnol 2013; 88: 1874–1884 c 2013 Society of Chemical Industry wileyonlinelibrary.com/jctb
1
8
8
2
www.soci.org S Kumar et al.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1 (a)
0 50 100 150 200 250
ν
m
= 0.85 h
1
ν
m
= 0.89 h
1
ν
m
= 0.93 h
1
K'
P
= 88.3 g l
1
K'
P
=86 g l
1
K'
P
= 90 g l
1 ν
(
h

1
)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1 (b)
ν
(
h

1
)
S (g l
1
)
α = 7.1
α = 8.5
α = 6.5
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1 (c)
ν
(
h

1
)
0 50 100 150 200 250
S (g l
1
)
0 50 100 150 200 250
S (g l
1
)
Figure 10. Simulation of speciﬁc rate of ethanol formation at S
o
=250 g L
−1
; T =50
◦
C; pH =5.0 for different values of (a) ν
m
at K
p
=88.3 g L
−1
; α =7.1;
(b) K
P
at ν
m
=0.89 h
−1
; α =7.1; (c) α at ν
m
=0.89 h
−1
; K
P
=88.3 g L
−1
[ model curve; experimental data].
0
50
100
150
200
250
300
0 4 8 12 16 20 24 28 32 36 40
Time (h)
S
(
g
l

1
)
0
10
20
30
40
50
60
70
80
90
100
P
(
g
l

1
)
Figure 11. Simulation of residual glucose concentration (S) and ethanol
concentration (P) during ethanol fermentation using Luong model at S
o
=
250 g L
−1
; T = 50
◦
C; pH = 5.0; ν
m
= 0.89 h
−1
; K
p
= 88.3 g L
−1
; α = 7.1
[ model curves; Sexp; Pexp].
Simulation of ethanol fermentation using Luong model
27
The experimental data for the fermentation of glucose to ethanol
usingKluyveromyces sp. IIPE453at theinitial glucoseconcentration
(S
o
) = 250 g L
−1
were tested for validity of the models listed in
Table1. Of thesemodels, theLuongmodel for product inhibition,
27
Equation (T22) was found to be the best ﬁt by Kluyveromyces
sp. IIPE453. Luong
27
correlated his experimental data of ethanol
fermentation using glucose as a substrate by S. cerevisiae ATCC
4126satisfactorily. Themaximumallowableethanol concentration
was predicted to be 112 g L
−1
. The calculations are shown in
Table 8. Effect of pHon kinetic parameters for ethanol fermentation
using glucose at S
o
=200 g L
−1
; T =50
◦
C
pH
Parameters 4.0 4.5 5.0 5.5 6.0
ν
m
(h
−1
) 0.48 0.82 1.08 0.92 0.7
K
SP
(g L
−1
) 60.2 46 85 76 85
K’
d
(h
−1
) 0.025 0.02 0.012 0.018 0.03
K’
CM
(g L
−1
) 0.029 0.034 0.027 0.023 0.026
Y’
P/S
0.35 0.39 0.5 0.488 0.5
Table 10 for various values of the model parameters along with
the error estimates for ﬁtting the experimental speciﬁc ethanol
formation rate (ν). The simulated results and the experimental
data are presented in Fig. 10. It is seen that the Luong model ﬁts
well with the experimental data for the fermentation of glucose to
ethanol using Kluyveromyces sp. IIPE453, which shows the product
inhibition. As shown in Table 10, all the error parameters, namely
S
j
, λ and RMSE are lowest at ν
m
= 0.89 h
−1
; K
P
= 88.3 g L
−1
and
α = 7.1. The values of simulated S and P are calculated using
Equations (6) and (7) and compared with the experimental data
as shown in Fig. 11. The simulated values of S and P are in good
agreement with the experimental data. Thus, the fermentation
wileyonlinelibrary.com/jctb c 2013 Society of Chemical Industry J ChemTechnol Biotechnol 2013; 88: 1874–1884
1
8
8
3
Kinetic studies of ethanol fermentation www.soci.org
Table 9. Effect of initial glucose concentration (S
o
) on kinetic
parameters for ethanol fermentation using glucose at T = 50
◦
C;
pH =5.0
S
o
(g L
−1
)
Parameters 200 250 300
ν
m
(h
−1
) 1.08 1.09 1.1
K
SP
(g L
−1
) 85 88 84.9
K’
d
(h
−1
) 0.008 0.012 0.02
K’
CM
(g L
−1
) 0.027 0.027 0.0275
Y’
P/S
0.5 0.5 0.5
Table10. Effect of kinetic parameter of Luong
27
model andthe error
estimates
Model
equation with
parameters
Parametric
value S
j
λ RMSE Figure
Equation (T22) ν
m
=0.85 h
−1
0.00495 0.0891 0.0756
10(a) K
P
=88.3 g L
−1
ν
m
=0.89 h
−1
0.00475 0.0856 0.0671
α =7.1 ν
m
=0.93 h
−1
0.00492 0.0886 0.0745
Equation (T22) K
P
=86 g L
−1
0.00857 0.1545 0.1015
10(b) ν
m
=0.89 h
−1
K
P
=88.3 g L
−1
0.00475 0.0856 0.0671
α =7.1 K
P
=90 g L
−1
0.00617 0.1110 0.0815
Equation (T22) α =6.5 0.00485 0.0872 0.0685
10(c) ν
m
=0.89 h
−1
α =7.1 0.00475 0.0856 0.0671
K
P
=88.3 g L
−1
α =8.5 0.00518 0.0933 0.0734
data can be best represented by the Luong model:
ν = 0.89
_
1 −
_
P
88.3
_
7.1
_
(20)
ArellanoPlaza et al.
28
evaluated the Luong model to predict
product inhibition of the tequila fermentation process and the
calculated kinetic parameters for the Luong model as reported by
them were K
P
= 130 g L
−1
and α = 9. Krishnan et al.
25
evaluated
fermentation kinetics of ethanol production from glucose by
recombinant Saccharomyces 1400(pLNH33) to predict product
inhibition and calculated the kinetic parameters of the Luong
model as ν
m
= 2.0 h
−1
, K
P
= 103 g L
−1
and α = 1.42. Ge and
Bai
29
evaluated the kinetics of continuous ethanol production of a
ﬂocculatingfusant yeast strainSPSC01 usingthe Luongmodel and
reported the values as ν
m
=1.99 h
−1
, K
P
=125 g L
−1
and α = 1.72.
CONCLUSIONS
Optimumtemperature, pH and initial glucose concentration were
found to be 50
◦
C, 5.0 and 10 g L
−1
, respectively, for the growth
of Kluyveromyces sp. IIPE453. Optimum conditions for ethanol
fermentation using the yeast strain with glucose as the substrate
were found to be 50
◦
C temperature, pH 5.0 and initial glucose
concentration of 200 g L
−1
. On validating experimental data with
existingkinetic models for product and/or substrate inhibition, the
Aiba model was found to best ﬁt the experimental growth data of
Kluyveromyces sp. IIPE453. The Luong model for product inhibition
was found to best represent the ethanol fermentation data with
Kluyveromyces sp. IIPE453.
ACKNOWLEDGEMENTS
We thank Dr MO Garg, Director IIP, Dehradun for valuable
suggestions and encouragement to carry out this research work.
One of the authors (Sachin Kumar) gratefully acknowledges a
Senior Research Fellowship awarded by the Council of Scientiﬁc
and Industrial Research (CSIR), India.
REFERENCES
1 KemppainenAJ andShonnardDR, Comparative lifecycle assessments
for biomasstoethanol production from different regional
feedstocks. Biotechnol Prog 21:1075–1084 (2005).
2 Mart´ın C, Galbe M, Wahlbom CF, HahnH¨ agerdal B and J ¨ onsson
LJ, Ethanol production from enzymatic hydrolysates of sugarcane
bagasse using recombinant xyloseutilising Saccharomyces cere
visiae. Enzyme Microbial Technol 31:274–282 (2002).
3 DienBS, Cotta MAandJeffries TW, Bacteria engineeredfor fuel ethanol
production: current status. Appl Microbiol Biotechnol 63:258–266
(2003).
4 Kumar S, Singh SP, Mishra IM and Adhikari DK, Ethanol and xylitol
production from glucose and xylose at high temperature by
Kluyveromyces sp. IIPE453. J Ind Microbiol Biotechnol 36:1483–1489
(2009).
5 Kumar S, Singh SP, Mishra IM and Adhikari DK, Feasibility of
ethanol production with enhanced sugar concentration in bagasse
hydrolysate at high temperature using Kluyveromyces sp. IIPE453.
Biofuels 1:697–704 (2010).
6 HeitmannT, WenzigE andMersmannA, Akinetic model of growthand
product formation of the anaerobic microorganism Thermoanaer
obacter thermohydrosulfuricus. J Biotechnol 50:213–223 (1996).
7 Kumar S, Singh SP, Mishra IM and Adhikari DK, Recent advances in
production of bioethanol from lignocellulosic biomass. Chem Eng
Technol 32:517–526 (2009).
8 Anderson PJ, McNeil K and Watson K, Highefﬁciency carbohydrate
fermentation to ethanol at temperatures above 40
◦
C by
Kluyveromyces marxianus var. marxianus isolated from sugar mills.
Appl Environ Microbiol 51:1314–1320 (1986).
9 Lynd LR, Production of ethanol from lignocellulosic materials using
thermophilic bacteria. Critical evaluation of potential and review,
in Advances in Biochemical Engineering/Biotechnology, Vol. 38,
Lignocellulosic Materials, ed by Fiechter A. Springer, New York,
1–52 (1989).
10 Holst O, Manelius A, Krahe M, M¨ arkl H, Rawen N and Sharp R,
Thermophiles and fermentation technology. Comparative Biochem
Physiol 118A:415–422 (1997).
11 Knutson BL, Strobel HJ, Nokes SE, Dawson KA, Berberich JA and Jones
CR, Effect of pressurized solvents on ethanol production by the
thermophilic bacterium Clostridium thermocellum. J Supercrit Fluids
16:149–156 (1999).
12 Avci A and D¨ onmez S, Effect of zinc on ethanol production by two
Thermoanaerobacter strains. Process Biochem41:984–989 (2006).
13 Phisalaphong M, Srirattana N and Tanthapanichakoon W, Mathe
matical modeling to investigate temperature effect on kinetic
parameters of ethanol fermentation. BiochemEngJ 28:36–43(2006).
14 Ghaly AE and ElTaweel AA, Kinetic modelling of continuous
production of ethanol from cheese whey. Biomass Bioenergy
16:461–472 (1997).
15 Lee WC and Huang CT, Modeling of ethanol fermentation using
Zymomonas mobilis ATCC 10988 grown on the media containing
glucose and fructose. BiochemEng J 4:217–227 (2000).
16 Altıntas MM, Kırdar B,
¨
Onsan Z
˙
I and
¨
Ulgen K
¨
O, Cybernetic modelling
of growth and ethanol production in a recombinant Saccharomyces
cerevisiae strain secreting a bifunctional fusion protein. Process
Biochem37:1439–1445 (2002).
17 Georgieva TI, Skiadas IV and Ahring BK, Effect of temperature on
ethanol tolerance of a thermophilic anaerobic ethanol producer
Thermoanaerobacter A10: modeling and simulation. Biotechnol
Bioeng 98:1161–1170 (2007).
18 Ge XM, Zhang L and Bai FW, Impacts of temperature, pH, divalent
cations, sugars and ethanol on the ﬂocculating of SPSC01. Enzyme
Microbial Technol 39:783–787 (2006).
19 Shuler ML and Kargi F, Bioprocess Engineering, 2nd edn. PrenticeHall
of India Private Limited, New Delhi (2002).
J ChemTechnol Biotechnol 2013; 88: 1874–1884 c 2013 Society of Chemical Industry wileyonlinelibrary.com/jctb
1
8
8
4
www.soci.org S Kumar et al.
20 Khan NS, Mishra IM, Singh RP and Prasad B, Modeling the growth of
Corynebacteriumglutamicumunder product inhibitioninLglutamic
acid fermentation. BiochemEng J 25:173–178 (2005).
21 Banat IM, Singh D and Marchant R, The use of thermotolerant
fermentative Kluyveromycesmarxianus IMB3 yeast strain for ethanol
production. Acta Biotechnol 16:215–223 (1996).
22 Wang D, Xu Y, Hu J and Zhao G, Fermentation kinetics of different
sugars by apple wine yeast Saccharomyces cerevisiae. J Inst Brew
110:340–346 (2004).
23 Ren HT, Yuan JQ and Bellgardt KH, Macrokinetic model for
methylotrophic Pichia pastoris based on stoichiometric balance.
J Biotechnol 106:53–68 (2003).
24 Aiba S, Shoda M and Nagatani M, Kinetics of product inhibition in
alcohol fermentation. Biotechnol Bioeng 10:845–864 (1968).
25 Krishnan MS, Ho NWY and Tsao GT, Fermentation kinetics of ethanol
productionfromglucoseandxylosebyrecombinant Saccharomyces
1400(pLNH33). Appl BiochemBiotechnol 77–79:373–388 (1999).
26 Banat IM, Nigam P and Marchant R, The isolation of thermotolerant
fermentative yeasts capable of growth at 52
◦
C and ethanol
production at 45
◦
C and 50
◦
C. W J Microbiol Biotechnol 8:259–263
(1992).
27 Luong JHT, Kinetics of ethanol inhibition in alcohol fermentation.
Biotechnol Bioeng 27:280–285 (1985).
28 ArellanoPlaza M, HerreraL´ opez EJ, D´ıazMonta˜ no DM, Moran A and
Ram´ırezC´ ordova JJ, Unstructured kinetic model for tequila batch
fermentation. Int J Math Comput Sim1 (2007).
29 Ge XMand Bai FW, Intrinsic kinetics of continuous growth and ethanol
production of a ﬂocculating fusant yeast strain SPSC01. J Biotechnol
124:363–372 (2006).
30 Haldane JBS, Enzymes. MIT Press, Cambridge, MA (1965).
31 Andrews JF, A mathematical model for the continuous culture of
microorganisms utilizing inhibitory substrate. Biotechnol Bioeng
10:707–723 (1968).
32 Yano T and Koga S, Dynamic behaviour of the chemostat subject to
substrate inhibition. Biotechnol Bioeng 11:139–153 (1969).
33 Orhon D and T¨ unay O, Mathematical models of biological waste
treatment process for thedesignof aerationtanksdiscussion. Water
Res 13:553–556 (1979).
34 Luong JHT, Generalization of Monod kinetics for analysis of growth
datawithsubstrateinhibition. Biotechnol Bioeng29:242–248(1987).
35 Han K and Levenspiel O, Extended Monod kinetics for substrate,
product and cell inhibition. Biotechnol Bioeng 32:430–437 (1988).
36 Sivakumar A, Srinivasaraghavan T, Swaminathan T and Baradarajan A,
Extendedmonodkinetics for substrateinhibitedsystems. Bioprocess
Eng 11:185–188 (1994).
37 Levenspiel O, The monod equations: a revisit and a generalization
to product inhibition situations. Biotechnol Bioeng 22:1671–1687
(1980).
38 Heuvel JCVDandBeeftink HH, Kinetic effect of simultaneous inhibition
of substrate and product. Biotechnol Bioeng 31:718–724 (1988).
39 Gonc¸alves LMD, Xavier AMRB, Almedia JS and Carrondo MJT,
Concomitant substrate and product inhibition kinetics in lactic
acid production. Enzyme Microbial Technol 13:314–319 (1991).
wileyonlinelibrary.com/jctb c 2013 Society of Chemical Industry J ChemTechnol Biotechnol 2013; 88: 1874–1884