Biochemical Engineering Journal 40 (2008) 107115

Modeling of batch fermentation kinetics for succinic

acid production by Mannheimia succiniciproducens
Hyohak Song
a,b
, Seh Hee Jang
a,c
, Jong Myoung Park
a,b
, Sang Yup Lee
a,b,c,d,e,
a
Metabolic and Biomolecular Engineering National Research Laboratory, Korea Advanced Institute of Science and Technology (KAIST),
335 Gwahangno, Yuseong-gu, Daejeon 305-701, Republic of Korea
b
Department of Chemical and Biomolecular Engineering (BK21 Program), Korea Advanced Institute of Science and Technology (KAIST),
335 Gwahangno, Yuseong-gu, Daejeon 305-701, Republic of Korea
c
BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology (KAIST), 335 Gwahangno,
Yuseong-gu, Daejeon 305-701, Republic of Korea
d
Center for Systems and Synthetic Biotechnology, Institute for the BioCentry, Korea Advanced Institute of Science and Technology (KAIST),
335 Gwahangno, Yuseong-gu, Daejeon 305-701, Republic of Korea
e
Department of Bio and Brain Engineering, Bioinformatics Research Center, Korea Advanced Institute of Science and Technology (KAIST),
335 Gwahangno, Yuseong-gu, Daejeon 305-701, Republic of Korea
Received 17 August 2007; received in revised form 15 November 2007; accepted 24 November 2007
Abstract
Kinetic models are proposed for the batch production of succinic acid from glucose by Mannheimia succiniciproducens MBEL55E. The
models include terms accounting for both substrate and product inhibitions. Experimental data collected from a series of batch fermentations
with different initial glucose concentrations were used to estimate parameters and also to validate the models proposed. The optimal values of the
parameters were approximated by minimizing the discrepancy between the model predictions and corresponding experimental data. The growth of
M. succiniciproducens could be expressed by a modied Monod model incorporating inhibitions of glucose and organic acids accumulated in the
culture broth. The LuedekingPiret model was able to describe the formation of organic acids as the fermentation proceeded, in which succinic,
acetic, and formic acids followed a mixed-growth-associated pattern. However, unexpectedly, lactic acid fermentation by M. succiniciproducens
was nearly nongrowth-associated. In all cases, the model simulation matched well with the experimental observations, which made it possible to
elucidate the fermentation characteristics of M. succiniciproducens during efcient succinic acid production from glucose. These models thus can
be employed for the development and optimization of biobased succinic acid production processes.
2007 Elsevier B.V. All rights reserved.
Keywords: Mannheimia succiniciproducens; Succinic acid; Fermentation; Kinetic models; Inhibition
1. Introduction
Succinic acid, an important four-carbon platform chemical,
is mostly being produced by chemical processes using liqueed
petroleum gas or petroleum oil as a starting material. However,
it has been widely accepted that the current petroleum-based
processes will be soon replaced by fermentation of renew-
able resources due to the limited nature of petroleum reserves

Corresponding author at: Department of Bio and Brain Engineering, and

Bioinformatics Research Center, Korea Advanced Institute of Science and Tech-
nology (KAIST), 335 Gwahangno, Yuseong-gu, Daejeon 305-701, Republic of
Korea. Tel.: +82 42 869 3930; fax: +82 42 869 3910.
E-mail address: leesy@kaist.ac.kr (S.Y. Lee).
and growing environmental concerns [1]. Indeed, researchers
have screened several succinic acid-producing microorgan-
isms, including Anaerobiospirillum succiniciproducens [2,3],
Actinobacillus succinogenes [4,5], and Mannheimia suc-
ciniciproducens [6,7]. Among these, M. succiniciproducens
has been studied more extensively based on its complete
genome sequence, and shown to be an efcient succinic acid
producer [8]. In M. succiniciproducens, phosphoenolpyruvate
carboxykinase carboxylates phosphoenolpyruvate to oxaloac-
etate, which is further converted to succinate by the reductive
tricarboxylic acid cycle and menaquinone system. On the
other hand, pyruvate formed from phosphoenolpyruvate is
converted to acetic, formic, and lactic acids via pyruvate
dissimilation pathways. The central carbon metabolism of
1369-703X/$ see front matter 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2007.11.021
108 H. Song et al. / Biochemical Engineering Journal 40 (2008) 107115
Nomenclature
i degree of product inhibition (dimensionless)
j number of experimental data points (dimension-
less)
K
d
specic death rate (h
1
)
K
I
substrate inhibition constant (g L
1
)
K
S
substrate saturation constant (g L
1
)
m
s
specic maintenance coefcient (h
1
)
P product concentration (g L
1
)
P
CRIT
critical product concentration (g L
1
)
r
P
product formation rate (g L
1
h
1
)
r
s
substrate consumption rate (g L
1
h
1
)
r
X
biomass formation rate (g L
1
h
1
)
r
AA
acetic acid formation rate (g L
1
h
1
)
r
FA
formic acid formation rate (g L
1
h
1
)
r
LA
lactic acid formation rate (g L
1
h
1
)
r
SA
succinic acid formation rate (g L
1
h
1
)
S substrate concentration (g L
1
)
X biomass concentration (g L
1
)
Y
X
stoichiometric yield coefcient for biomass on
glucose (g g
1
)
Y
AA
stoichiometric yield coefcient for acetic acid on
glucose (g g
1
)
Y
FA
stoichiometric yield coefcient for formic acid on
glucose (g g
1
)
Y
LA
stoichiometric yield coefcient for lactic acid on
glucose (g g
1
)
Y
SA
stoichiometric yield coefcient for succinic acid
on glucose (g g
1
)
Z minimizing objective function of the discrepancy
between experimental data and simulated results
(dimensionless)
Greek letters

AA
growth-associated acetic acid formation parame-
ter (g g
1
DCW)

FA
growth-associated formic acid formation param-
eter (g g
1
DCW)

LA
growth-associated lactic acid formation parame-
ter (g g
1
DCW)

SA
growth-associated succinic acid formation
parameter (g g
1
DCW)

AA
nongrowth-associated acetic acid formation
parameter (g g
1
DCWh
1
)

FA
nongrowth-associated formic acid formation
parameter (g g
1
DCWh
1
)

LA
nongrowth-associated lactic acid formation
parameter (g g
1
DCWh
1
)

SA
nongrowth-associated succinic acid formation
parameter (g g
1
DCWh
1
)
specic growth rate (h
1
)

m
maximum specic growth rate (h
1
)
M. succiniciproducens is described in detail by Kim et al.
[8].
In order to develop a cost-competitive fermentative suc-
cinic acid production process, strain improvement maximizing
the production of succinic acid but minimizing the formation
of by-products through rational metabolic engineering is of
vital importance. We recently developed an improved succinic
acid producer, M. succiniciproducens LPK7, by genome-based
metabolic engineering, resulting in much increased succinic
acid production while reduced formation of by-products, such
as acetic, formic, and lactic acids [9]. In addition, the pos-
sibilities of cost-effective succinic acid production by M.
succiniciproducens from inexpensive and abundant feedstocks,
including wood hydrolysates and whey were also investigated
[10,11].
Kinetic modeling is regarded as an indispensable step in
developing a fermentation process since the models can be used
to determine an optimal operation condition for the production
of a target metabolite. Both structured and unstructured models
have been used for kinetic modeling. Although the former can
explain complex microbial system at the molecular levels, rela-
tively simpler unstructured kinetic models have frequently been
used for practical applications [12,13]. Several unstructured
kinetic models have been proposed to describe the fermenta-
tion of glucose to organic and amino acids such as citric, lactic,
and glutamic acids [1416]. However, little kinetic study on the
fermentative production of succinic acid has been performed to
date.
In this paper, we present empirical kinetic models to describe
the batch production of succinic acid by M. succiniciproducens
MBEL55E. A series of batch fermentations with different ini-
tial glucose concentrations were conducted in a bioreactor, and
the experimental data were used to estimate parameters and also
to validate the models. The behaviors of M. succiniciproducens
during fermentation were predicted using the models, and the
predictions were compared with the experimental observations.
The models developed were able to successfully explain cell
growth, succinic acid production, by-products (acetic, formic,
and lactic acids) formation, and glucose utilization. The fermen-
tative characteristics of succinic acid production from glucose
by M. succiniciproducens were discussed in detail.
2. Materials and methods
2.1. Organism and growth conditions
Preculture of M. succiniciproducens MBEL55E (KCTC
0769BP; Korean Collection for Type Cultures, Daejeon, Korea)
[6] was performed in a sealed anaerobic ask containing 250 mL
medium composed of yeast extract 5 g L
1
, NaCl 1 g L
1
,
CaCl
2
2H
2
O 0.2 g L
1
, MgCl
2
6H
2
O 0.2 g L
1
, and K
2
HPO
4
8.709 g L
1
. The medium with CO
2
as the gas phase was heat
sterilized at 121

C for 15 min. The pH of the medium was

adjusted to 7.0 by adding 5N NaOH. Separately sterilized glu-
cose was added into the medium to a nal concentration of
50 mM as a carbon source just before inoculation. Inoculation
was done by seeding 2.5 mL glycerol stock culture (15%, w/v,
H. Song et al. / Biochemical Engineering Journal 40 (2008) 107115 109
70

C) into the medium. Anaerobic ask cultures were per-

formed at 39

C for 9 h under a CO
2
atmosphere in an anaerobic
chamber (ThermoForma, Marietta, OH).
Batch fermentations were carried out at 39

Cin a 5-L LiFlus

GX bioreactor (BioTron Inc., Puchon, Korea) at a working vol-
ume of 2.5 L. The initial glucose concentrations in the medium
was varied from 1.0 to 66 g L
1
. The pH was maintained at
6.5 0.1 by the addition of a 28% (v/v) ammonia solution.
The agitation speed and temperature were set to 200 rpm and
39

C, respectively. The bioreactor was continuously sparged

with industrial grade CO
2
(Special gas, Daejeon, Korea) at a
ow rate of 0.25 vvm during the whole period of fermentation.
An oxygen trap (Agilent, Waldbronn, Germany) was tted in the
outlet line of the gas cylinder to completely remove residual oxy-
gen, if any, from CO
2
gas. All chemicals used in this study were
of reagent grade and were obtained from either SigmaAldrich
(St. Louis, MO) or Junsei Chemical (Tokyo, Japan), except for
glucose and yeast extract which were purchased from Samyang
Genex (Inchon, Korea) and Becton Dickinson Company (Le
Pont de Claix, France), respectively.
2.2. Analytical procedures
Quantitative analyses of glucose and organic acids, includ-
ing acetic, formic, lactic, and succinic acids, were performed
by a high-performance liquid chromatography (Varian ProStar
210, Palo Alto, CA) equipped with UV/VIS (Varian Prostar
320) and refractive index (Shodex RI-71, Tokyo, Japan) detec-
tors. A MetaCarb 87H column (300 mm7.8 mm, Varian)
was used to separate the components at 60

C using 0.01N
H
2
SO
4
as a mobile phase at a ow rate of 0.6 mLmin
1
.
Cell growth was monitored by using a spectrophotometer
(Ultrospec3000, Amersham Biosciences, Uppsala, Sweden) at
the optical density of 600 nm (OD
600
), and dry cell weight
(DCW) was determined by the method described previously
[9].
2.3. Development of kinetic models
The kinetics of fermentation can be normally described by a
cell growth model (r
X
), a product formation model (r
P
), and a
substrate utilization model (r
S
). In general, the Monod model
is used to represent bacterial growth under substrate-limited
conditions. Taking into a consideration of the excess substrate
inhibition on the growth of M. succiniciproducens, this well-
known model can be modied as follows [17]:
=

m
S
(S +K
S
+(S
2
/K
I
))
(1)
where is the specic growth rate (h
1
),
m
is the maxi-
mum specic growth rate (h
1
), S is the substrate concentration
(g L
1
), K
S
is the substrate saturation constant (g L
1
), and
K
I
is the substrate inhibition constant (g L
1
). However, Eq.
(1) cannot be directly applicable to many fermentation systems
since cell growth has a tendency to be inhibited gradually by
the fermentation products as cultivation proceeds [1821]. In
addition, the presence of the high concentrations of acids in cul-
ture broth often causes to cell death [2224]. The dependence
of cell growth on products could be described by a generalized
nonlinear equation suggested by Levenspiel [25]. To incorpo-
rate both substrate and product inhibitions on the growth of M.
succiniciproducens, additional terms were introduced into Eq.
(1) as follows:
=

m
S
(S +K
S
+(S
2
/K
I
))

1
P
P
CRIT

i
,
when P < P
CRIT
(2)
and
= K
d
, when P P
CRIT
(3)
where Pis the product concentration(g L
1
), P
CRIT
is the critical
product concentration at which cell growth fully stops (g L
1
),
and K
d
is the specic death rate (h
1
). Exponent i denotes the
degree of product inhibition (dimensionless). M. succinicipro-
ducens produces succinic acid as a major fermentation product
and simultaneously forms acetic, formic, and lactic acids as by-
products during anaerobic glucose fermentation. Accordingly,
P and P
CRIT
in Eq. (2) were given as the sum of the amounts of
succinic and the other acids, rather than considering individual
acids separately. Subsequently, the nal kinetic equations for the
batch growth of M. succiniciproducens could be represented by
the following equations:
r
X
=

m
S
(S +K
S
+(S
2
/K
I
))

1
P
P
CRIT

X,
when P < P
CRIT
(4)
and
r
X
= K
d
X, when P P
CRIT
(5)
where r
X
is the biomass formation rate (g L
1
h
1
) and X is the
biomass concentration (g L
1
).
The formation of organic acids, including succinic, acetic,
formic, and lactic acids, by M. succiniciproducens could be
described by the LuedekingPiret model [26], in which the
product formation rate depends on both r
X
and X:
r
SA
=
SA
r
X
+
SA
X (6)
r
AA
=
AA
r
X
+
AA
X (7)
r
FA
=
FA
r
X
+
FA
X (8)
r
LA
=
LA
r
X
+
LA
X (9)
where r
SA
, r
AA
, r
FA
, and r
LA
represent the formation rates of
succinic, acetic, formic, and lactic acids (g L
1
h
1
), respec-
tively.
SA
,
AA
,
FA
, and
LA
denote the growth-associated
parameters for the formation of succinic, acetic, formic, and lac-
tic acids (dimensionless), respectively.
SA
,
AA
,
FA
, and
LA
denote the non-growth-associated parameters for the formation
of succinic, acetic, formic, and lactic acids (h
1
), respectively.
A carbon substrate, glucose in this study, used by M. suc-
ciniciproducens is converted to form biomass, succinic acid,
110 H. Song et al. / Biochemical Engineering Journal 40 (2008) 107115
and by-products, and also used for the maintenance. Based on
the carbon mass balance, the consumption rate of glucose can
be described as follows:
r
S
=
1
Y
X
r
X
+
1
Y
SA
r
SA
+
1
Y
AA
r
AA
+
1
Y
FA
r
FA
+
1
Y
LA
r
LA
+m
S
X (10)
where r
S
is the consumption rate of glucose (g L
1
h
1
). Y
X
,
Y
SA
, Y
AA
, Y
FA
, and Y
LA
are the stoichiometric yield coefcients
of biomass, and succinic, acetic, formic, and lactic acids on
glucose (g g
1
). m
S
is the specic maintenance coefcient (h
1
).
2.4. Model parameters estimation
Withthe experimental data obtainedfroma series of batchfer-
mentations of M. succiniciproducens, the kinetic parameters of
the models were determinedbyminimizinganobjective function
of Z:
Z
X
=
n

j=1
(X
j,exp
X
j,sim
)
2
, Z
P
=
n

j=1
(P
j,exp
P
j,sim
)
2
,
or Z
S
=
n

j=1
(S
j,exp
S
j,sim
)
2
s (11)
where Z
X
, Z
P
, and Z
S
represent the least-square discrepancies
between experimental data and simulated results of biomass,
product, and substrate, respectively. j denotes the number of
experimental data points. Subscripts exp and sim denote
experimental and simulated, respectively.
3. Results and discussion
3.1. Microbial growth
In order to investigate the behaviors of M. succiniciproducens
on the anaerobic fermentation of glucose, batch fermentations
were carried out using the medium supplemented with varying
concentrations (1, 3, 6, 10, 18, 28, 41, 49, and 66 g L
1
) of glu-
cose. First,
m
and K
S
in Eq. (2) were estimated from the data
collected from the fermentations with low initial glucose con-
centrations (1, 3, and 6 g L
1
). During these fermentations, no
glucose and product inhibitions were observed, implying that
S
2
/K
I
and P/P
CRIT
are close to zero. Consequently, Eq. (2) was
reduced to the classical Monod equation =
m
S/(S +K
S
).
The values of K
S
and
m
were obtained from reciprocal plot-
ting of the Monod equation; 1/ versus 1/S (r
2
=0.991). Next,
K
I
was estimated from the fermentations with high initial glu-
cose concentrations (18, 28, 41, 49, and 66 g L
1
) using the
data collected during the exponential growth phase (until 2.5 h
of inoculation) to avoid the inhibitory effect of products on the
cell growth. In this case, Eq. (2) was reduced to Eq. (1) because
P/P
CRIT
is close to zero. Reciprocal plotting of Eq. (1), 1/ ver-
sus S provided K
I
(r
2
=0.981). Thus, determined values of K
S
,
K
I
, and
m
are listed in Table 1. Using these values, the model
predictions were compared with experimental data as shown
in Fig. 1. As expected, the growth of M. succiniciproducens
Table 1
Values of kinetic parameters

m
(h
1
) 1.324
m
s
(h
1
) 0.061
i (dimensionless) 1.301
Y
AA
(g g
1
) 0.999
Y
FA
(g g
1
) 1.532
Y
LA
(g g
1
) 0.999
Y
SA
(g g
1
) 1.310
Y
X
(g g
1
) 0.765
K
d
(h
1
) 0.010
K
S
(g L
1
) 1.123
K
I
(g L
1
) 88.35
P
CRIT
(g L
1
) 17.23

AA
(g g
1
) 0.626

FA
(g g
1
) 0.665

LA
(g g
1
) 0

SA
(g g
1
) 1.619

AA
(h
1
) 0.124

FA
(h
1
) 0.105

LA
(h
1
) 0.210

SA
(h
1
) 0.355
was negatively inuenced by the high concentration of glucose
in the culture broth. It was found that the maximum specic
growth rate (
m
) of M. succiniciproducens can be achieved is
1.324 h
1
at a glucose concentration of 9.964 g L
1
(given by
S =

K
S
K
I
) in a bioreactor. A higher specic growth rate is
often desirable to grow cells faster to a desirable cell density
as it can reduce the fermentation time and thus improves over-
all productivity. In this sense, M. succiniciproducens showing a
high specic growth rate can be considered as a good succinic
acid producer.
The inhibitory effects of organic acids on microbial growth
have been known to depend on the pH of culture broth, the
dissociation constants of acids (pK
a
), and their molar concen-
trations [27,28]. As mentioned earlier, M. succiniciproducens
produces succinic acid as a major product and acetic, formic,
and lactic acids as minor ones fromvarious carbon sources under
anaerobic conditions. In this study, the pH was maintained at
6.5 during the whole period of fermentation. The dissociation
Fig. 1. Effects of initial glucose concentrations on the specic growth rate of
M. succiniciproducens MBEL55E. Experimental (symbol); simulated (line).
H. Song et al. / Biochemical Engineering Journal 40 (2008) 107115 111
Fig. 2. Time proles of biomass (closed circle, solid line) and glucose (open circle, dashed line) in batch cultures of M. succiniciproducens MBEL55E with different
initial glucose concentrations: (a) 10 g L
1
, (b) 18 g L
1
, (c) 28 g L
1
, (d) 41 g L
1
, (e) 49 g L
1
, and (f) 66 g L
1
. Experimental (symbols); simulated (lines).
constants of organic acids produced by M. succiniciproducens
can be considered not much different for modeling purposes:
acetic acid (pK
a
of 4.75), formic acid (pK
a
of 3.84), lactic acid
(pK
a
of 3.86), and succinic acid (pK
a1
of 4.21). Thus, the prod-
uct term in Eqs. (2)(4) could be assumed to be as the sum
of the amounts of individual acids to simplify the models. As
shown in Figs. 24, the growth of M. succiniciproducens grad-
ually slowed down with increasing amounts of acids, and the
growth completely ceased when it exceeded 17.23 g L
1
(P
CRIT
)
in all experiments. Above this critical value, the concentrations
of biomass declined linearly with K
d
=0.001 h
1
as the fermen-
tation proceeded. The parameter i was estimated to be 1.301.
This is not surprising because the inhibition of microbial growth
by organic acids is common and the acids accumulated to high
concentrations nally cause cell lysis, even though the resistance
levels against acids can be different among microorganisms
[22,27,29,30].
3.2. Succinic acid production and by-products formation
The concentration of succinic acid increased proportionally
to increasing the concentration of biomass in the exponential
growth phase, and its production continued even during the
slow growth and stationary phases (Fig. 3). This means that
the production of succinic acid by M. succiniciproducens fol-
lows the LuedekingPiret model, indicating that the production
rate of a product relies not only on the rate of cell growth (r
X
)
but also on the concentration of biomass (X) [26]. The non-
growth-associated coefcient for succinic acid production by
M. succiniciproducens (
SA
=0.355 h
1
) was rst determined
from the data collected where no cell growth was observed
(PP
CRIT
). Then, the growth-associated coefcient for suc-
cinic acid production by M. succiniciproducens (
SA
=1.619)
was estimated when P<P
CRIT
. During the batch culture with
the initial cell concentration of 0.04 g DCWL
1
, the maxi-
mum volumetric productivity of succinic acid was predicted
to be 1.046 g L
1
h
1
at an initial glucose concentration of
18 g L
1
, which is in excellent agreement with the experimen-
tal result of 1.043 g L
1
h
1
(Fig. 3). The predictions of the
model simulation were compared with the experimental data
and are depicted in Fig. 3, showing a good agreement each
other.
In the same manner, the coefcients for the formation of
acetic, formic, and lactic acids by M. succiniciproducens in
112 H. Song et al. / Biochemical Engineering Journal 40 (2008) 107115
Fig. 3. Time proles of succinic acid concentration in batch cultures of M. succiniciproducens MBEL55E with different initial glucose concentrations: (a) 10 g L
1
,
(b) 18 g L
1
, (c) 28 g L
1
, (d) 41 g L
1
, (e) 49 g L
1
, and (f) 66 g L
1
. Experimental (symbol); simulated (line).
Eqs. (7)(9) were estimated, respectively, and the values are
shown in Table 1. Like succinic acid, the formation of acetic
and formic acids by M. succiniciproducens followed a mixed-
growth-associated product formation pattern (Fig. 4). However,
unexpectedly, lactic acid started to be produced only when
the cell growth rate was nearly zero, which means that the
formation of lactic acid by M. succiniciproducens follows a non-
growth-associated product formation mode under the current
experimental condition. Lactic acid fermentation is presented
as a typical mixed-growth-associated product for most microor-
ganisms includingEscherichiacoli, Lactobacillus, Lactococcus,
and Saccharomyces [15,21,23,27,31]. Lactic acid formation and
succinic acid formation share one common physiological mean-
ing of NADH regeneration during the anaerobic fermentation;
NADH produced during glycolysis needs to be regenerated to
continue fermentation. However, there is a difference in that two
reducing power equivalents are used for succinic acid produc-
tion from phosphoenolpyruvate, while one equivalent is used
for lactic acid production from pyruvate. Production of suc-
cinic acid rather than lactic acid during the cell growth phase
of M. succiniciproducens implies that the pathway involved
in the production of succinic acid is much more favored in
M. succiniciproducens under the given fermentation condition;
anaerobic fermentation of glucose under CO
2
atmosphere. M.
succiniciproducens operates branched TCA cycle; a reductive
branch leading to succinic acid formation fromoxaloacetate and
an oxidative branch producing succinyl-CoAfromcitrate. When
the cell growth is slowed down or stopped, the oxidative branch
operation may be reduced or stopped causing less generation of
reducing powers. This may cause imbalance of reducing power
for the production of succinic acid alone, and cells convert some
pyruvate to lactic acid.
3.3. Substrate utilization
The consumption rate of glucose was calculated based on
the carbon mass balance using the stoichiometric yield coef-
cients and the estimated specic maintenance coefcient
(m
s
=0.061 h
1
). The yield coefcients are shown in Table 1.
In order to calculate the carbon compound incorporated into
H. Song et al. / Biochemical Engineering Journal 40 (2008) 107115 113
Fig. 4. Time proles of the concentrations of acetic (triangle, dotted line), formic (square, solid line), and lactic (diamond, dashed line) acids in batch cultures of
M. succiniciproducens MBEL55E with different initial glucose concentrations: (a) 10 g L
1
, (b) 18 g L
1
, (c) 28 g L
1
, (d) 41 g L
1
, (e) 49 g L
1
, and (f) 66 g L
1
.
Experimental (symbols); simulated (lines).
the cells from glucose, the four main elements of C, H, O,
and N of M. succiniciproducens cells at exponential growth
phase were quantied using an Elemental Analyzer (Flash
EA1112Series, CE instruments, Wigan, UK); it was found to be
CH
1.736
O
0.367
N
0.240
. M. succiniciproducens xes one mole of
CO
2
to yield one mole of succinic acid via three different CO
2
-
xing metabolic reactions catalyzed by phosphoenolpyruvate
carboxykinase, phosphoenolpyruvate carboxylase, and malic
enzyme [9]. All CO
2
used in the production of succinic acid was
assumed to be provided externally in the calculation. Together
with the experimental data, the predicted time proles of glu-
cose are depicted in Fig. 2. Of particular interest from Fig. 2
is that the discrepancies between the experimental data and the
model simulation results were observed at the stationary growth
phase, and were slightly greater when high initial concentra-
tions (41, 49, and 66 g L
1
) of glucose were used (Fig. 2(d)(f)).
For the simplied model development, we did not incorporate
CO
2
production by the cells, which occurs through the decar-
boxylation reactions catalyzed by oxaloacetate decarboxylase,
malic enzyme, pyruvate dehydrogenase, and formate dehydrase
in M. succiniciproducens. The loss of carbon in the formof CO
2
produced by cells thus seems to contribute to this discrepancy.
Also, the released carbon in the form of cell debris as a result
of cell lysis was not included in the calculation. Additionally,
changes in cell composition [3234] during the stationary phase
might have caused this discrepancy as we used one cell com-
position determined at exponential growth phase. Nonetheless,
the simple models developed in this study successfully describe
the cell growth, glucose consumption, and products formation,
which can thus be used for optimizing the process for succinic
acid production. For example, the optimal substrate (glucose)
concentration can be selected for the fed-batch and continuous
fermentation operations.
4. Conclusion
In this study, we developed the simple empirical kinetic mod-
els for the batch production of succinic acid from glucose by M.
114 H. Song et al. / Biochemical Engineering Journal 40 (2008) 107115
succiniciproducens MBEL55E. The results of the model simu-
lations showed very good agreement with the experimental data
obtained at varying initial glucose concentrations. The growth
of M. succiniciproducens could be expressed by an expanded
Monod model, including terms for both substrate and product
inhibitions, which were suggested by Andrews [17] and Leven-
spiel [25], respectively. The models allow identication of the
optimal concentrations of glucose and organic acids in a biore-
actor for the cell growth as well as for the production of succinic
acid. The production of succinic acid by M. succiniciproducens
follows the LuedekingPiret model with the growth- and non-
growth-associated parameter values of 1.619 and 0.355 h
1
,
respectively. The models could elucidate the fermentative char-
acteristics of M. succiniciproducens and also could explain its
superior ability to produce succinic acid under CO
2
atmosphere.
The models proposed here should be useful for developing and
optimizing the succinic acid production processes from renew-
able resources.
Acknowledgement
This work was supported by the Genome-based Integrated
Bioprocess Project of the Ministry of Science and Technol-
ogy through the Korea Science and Engineering Foundation
(KOSEF). Further supports by the LG Chem Chair Professor-
ship and by the Center for Ultramicrochemical Process Systems
(KOSEF) are appreciated.
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