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Batch Fementation For Succinic Acid Production
Batch Fementation For Succinic Acid Production
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 modified 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 efficient 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 liquefied
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 renewable resources due to the limited nature of petroleum reserves
108
Nomenclature
i
j
Kd
KI
KS
ms
P
PCRIT
rP
rs
rX
rAA
rFA
rLA
rSA
S
X
YX
YAA
YFA
YLA
YSA
Z
Greek letters
AA
growth-associated acetic acid formation parameter (g g1 DCW)
growth-associated formic acid formation paramFA
eter (g g1 DCW)
LA
growth-associated lactic acid formation parameter (g g1 DCW)
SA
growth-associated succinic acid formation
parameter (g g1 DCW)
AA
nongrowth-associated acetic acid formation
parameter (g g1 DCW h1 )
nongrowth-associated formic acid formation
FA
parameter (g g1 DCW h1 )
nongrowth-associated lactic acid formation
LA
parameter (g g1 DCW h1 )
SA
nongrowth-associated succinic acid formation
parameter (g g1 DCW h1 )
70 C) into the medium. Anaerobic flask cultures were performed at 39 C for 9 h under a CO2 atmosphere in an anaerobic
chamber (ThermoForma, Marietta, OH).
Batch fermentations were carried out at 39 C in a 5-L LiFlus
GX bioreactor (BioTron Inc., Puchon, Korea) at a working volume of 2.5 L. The initial glucose concentrations in the medium
was varied from 1.0 to 66 g L1 . 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 CO2 (Special gas, Daejeon, Korea) at a
flow rate of 0.25 vvm during the whole period of fermentation.
An oxygen trap (Agilent, Waldbronn, Germany) was fitted in the
outlet line of the gas cylinder to completely remove residual oxygen, if any, from CO2 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, including 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) detectors. A MetaCarb 87H column (300 mm 7.8 mm, Varian)
was used to separate the components at 60 C using 0.01N
H2 SO4 as a mobile phase at a flow rate of 0.6 mL min1 .
Cell growth was monitored by using a spectrophotometer
(Ultrospec3000, Amersham Biosciences, Uppsala, Sweden) at
the optical density of 600 nm (OD600 ), 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 (rX ), a product formation model (rP ), and a
substrate utilization model (rS ). 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 wellknown model can be modified as follows [17]:
=
m S
(S + KS + (S 2 /KI ))
(1)
where is the specific growth rate (h1 ), m is the maximum specific growth rate (h1 ), S is the substrate concentration
(g L1 ), KS is the substrate saturation constant (g L1 ), and
KI is the substrate inhibition constant (g L1 ). 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
109
addition, the presence of the high concentrations of acids in culture 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 incorporate both substrate and product inhibitions on the growth of M.
succiniciproducens, additional terms were introduced into Eq.
(1) as follows:
i
P
m S
,
=
(S + KS + (S 2 /KI ))
PCRIT
when
P < PCRIT
(2)
and
= Kd ,
when
P PCRIT
(3)
when
P < PCRIT
(4)
and
rX = Kd X,
when
P PCRIT
(5)
(6)
rAA = AA rX + AA X
(7)
rFA = FA rX + FA X
(8)
rLA = LA rX + LA X
(9)
where rSA , rAA , rFA , and rLA represent the formation rates of
succinic, acetic, formic, and lactic acids (g L1 h1 ), respectively. SA , AA , FA , and LA denote the growth-associated
parameters for the formation of succinic, acetic, formic, and lactic acids (dimensionless), respectively. SA , AA , FA , and LA
denote the non-growth-associated parameters for the formation
of succinic, acetic, formic, and lactic acids (h1 ), respectively.
A carbon substrate, glucose in this study, used by M. succiniciproducens is converted to form biomass, succinic acid,
110
(10)
n
(Xj,exp Xj,sim )2 ,
ZP =
j=1
or ZS =
n
(Sj,exp Sj,sim )2 s
m (h1 )
ms (h1 )
i (dimensionless)
YAA (g g1 )
YFA (g g1 )
YLA (g g1 )
YSA (g g1 )
YX (g g1 )
Kd (h1 )
KS (g L1 )
KI (g L1 )
PCRIT (g L1 )
AA (g g1 )
FA (g g1 )
LA (g g1 )
SA (g g1 )
AA (h1 )
FA (h1 )
LA (h1 )
SA (h1 )
1.324
0.061
1.301
0.999
1.532
0.999
1.310
0.765
0.010
1.123
88.35
17.23
0.626
0.665
0
1.619
0.124
0.105
0.210
0.355
(Pj,exp Pj,sim )2 ,
j=1
n
Table 1
Values of kinetic parameters
(11)
j=1
111
Fig. 2. Time profiles 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 L1 , (b) 18 g L1 , (c) 28 g L1 , (d) 41 g L1 , (e) 49 g L1 , and (f) 66 g L1 . Experimental (symbols); simulated (lines).
112
Fig. 3. Time profiles of succinic acid concentration in batch cultures of M. succiniciproducens MBEL55E with different initial glucose concentrations: (a) 10 g L1 ,
(b) 18 g L1 , (c) 28 g L1 , (d) 41 g L1 , (e) 49 g L1 , and (f) 66 g L1 . Experimental (symbol); simulated (line).
113
Fig. 4. Time profiles 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 L1 , (b) 18 g L1 , (c) 28 g L1 , (d) 41 g L1 , (e) 49 g L1 , and (f) 66 g L1 .
Experimental (symbols); simulated (lines).
114
succiniciproducens MBEL55E. The results of the model simulations 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 Levenspiel [25], respectively. The models allow identification of the
optimal concentrations of glucose and organic acids in a bioreactor 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 nongrowth-associated parameter values of 1.619 and 0.355 h1 ,
respectively. The models could elucidate the fermentative characteristics of M. succiniciproducens and also could explain its
superior ability to produce succinic acid under CO2 atmosphere.
The models proposed here should be useful for developing and
optimizing the succinic acid production processes from renewable resources.
[11]
[12]
[13]
[14]
[15]
[16]
[17]
Acknowledgement
This work was supported by the Genome-based Integrated
Bioprocess Project of the Ministry of Science and Technology through the Korea Science and Engineering Foundation
(KOSEF). Further supports by the LG Chem Chair Professorship and by the Center for Ultramicrochemical Process Systems
(KOSEF) are appreciated.
[18]
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[20]
[21]
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