COMMUNICATION

TO THE

EDITOR

Effects of Dissol ved CO2 Levels on the Growth of

Mannheimia succiniciproducens and
Succinic Acid Production
Hyohak Song,1,2,3 Jeong Wook Lee,1,2,3 Sol Choi,1,2,3 Jong Kyun You,1 Won Hi Hong,1
Sang Yup Lee1,2,3,4
1

Department of Chemical and Biomolecular Engineering (BK21 Program) and

BioProcess Engineering Research Center, Korea Advanced Institute of Science and
Technology (KAIST), 335 Gwahangno, Yuseonggu, Daejeon 305701, Republic of Korea;
telephone: 82 42 869 3930; fax: 82 42 869 3910; e-mail: leesy@kaist.ac.kr
2
Center for Systems and Synthetic Biotechnology, Institute for the BioCentury,
Korea Advanced Institute of Science and Technology (KAIST), Yuseonggu,
Daejeon, Republic of Korea
3
Metabolic and Biomolecular Engineering National Research Laboratory,
Korea Advanced Institute of Science and Technology (KAIST), Yuseonggu,
Daejeon, Republic of Korea
4
Department of BioSystems and Bioinformatics Research Center, Korea Advanced Institute of
Science and Technology (KAIST), Yuseonggu, Daejeon, Republic of Korea
Received 8 March 2007; revision received 13 May 2007; accepted 25 May 2007
Published online 14 June 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.21530

ABSTRACT: A capnophilic rumen bacterium Mannheimia

succiniciproducens produces succinic acid as a major
fermentation end product under CO2-rich anaerobic condition. Since succinic acid is produced by carboxylation
of C3 compounds during the fermentation, intracellular
CO2 availability is important for efficient succinic acid
formation. Here, we investigated the metabolic responses
of M. succiniciproducens to the different dissolved CO2
concentrations (0260 mM). Cell growth was severely
suppressed when the dissolved CO2 concentration was
below 8.74 mM. On the other hand, cell growth and
succinic acid production increased proportionally as the
dissolved CO2 concentration increased from 8.74 to
141 mM. The yields of biomass and succinic acid on glucose
obtained at the dissolved CO2 concentration of 141 mM
were 1.49 and 1.52 times higher, respectively, than those
obtained at the dissolved CO2 concentration of 8.74 mM. It
was also found that the additional CO2 source provided in
the form of NaHCO3, MgCO3, or CaCO3 had positive effects
on cell growth and succinic acid production. However,
growth inhibition was observed when excessive bicarbonate
salts were added. By the comparison of the activities of key
enzymes, it was found that PEP carboxylation by PEP

carboxykinase (PckA) is the most important for succinic

acid production as well as the growth of M. succiniciproducens by providing additional ATP.
Biotechnol. Bioeng. 2007;98: 12961304.
2007 Wiley Periodicals, Inc.
KEYWORDS: Mannheimia succiniciproducens; dissolved CO2
concentration; succinic acid; fermentation

Introduction
Due to the increasing concerns on the limited nature of fossil
resources, there has recently been much interest in the
production of various chemicals and materials by microbial
fermentation from renewable resources. Succinic acid is
a dicarboxylic acid, which can be used as an important
C4 feedstock for industrially important chemicals, biodegradable polymers, and various green solvents (Song and
Lee, 2006; Zeikus et al., 1999). Many organisms have been
known to produce succinic acid as an intermediate of the
tricarboxylic acid (TCA) cycle and also as one of the end

Correspondence to: S.Y. Lee

Contract grant sponsor: Genome-Based Integrated Bioprocess Development
Project, Korean Ministry of Science and Technology
Contract grant number: 2005-01294

1296

Biotechnology and Bioengineering, Vol. 98, No. 6, December 15, 2007

2007 Wiley Periodicals, Inc.

products of anaerobic fermentation. However, only a few

rumen and gastrointestinal bacteria such as Actinobacillus
succinogenes (Guettler et al., 1999), Mannheimia succiniciproducens (Hong et al., 2004), and Anaerobiospirillum
succiniciproducens (Lee et al., 2001) have been reported to
produce large amounts of succinic acid under CO2-rich
anaerobic conditions.
Among these, M. succiniciproducens isolated from the
rumen of a Korean cow (Lee et al., 2002) has been studied in
detail. Its complete genome sequence has been determined,
which allowed construction of a genome-scale metabolic
model for computational studies (Hong et al., 2004).
Genome-based metabolic engineering studies have also been
performed to reduce the by-products formation (Lee et al.,
2006b). Since succinic acid is produced by carboxylation of
C3 compounds, it is obvious that the intracellular CO2
availability is important. However, there have been little
quantitative studies on the effect of CO2 level on succinic
acid production.
Previous studies showed that A. succinogenes (van der
Werf et al., 1997) and A. succiniciproducens (Lee et al., 1999a;
Samuelov et al., 1991) produce succinic acid via PEP
carboxylation pathway using four enzymes, PEP carboxykinase (PckA), malate dehydrogenase, fumarase, and
fumarate reductase, which is different from Escherichia coli
(Clark, 1989). It was reported that PckA is strongly regulated
by CO2 availability and the higher CO2 levels resulted in
higher succinic acid production (Samuelov et al., 1991).
From the complete genome sequence (Hong et al., 2004)
and proteome reference map (Lee et al., 2006a) of M.
succiniciproducens, we were able to identify key enzymes that
catalyze CO2-fixing reactions: PckA, Ppc, and MaeB. Also,
pyruvate kinase (PykA), which competes with PckA and Ppc
for PEP, was identified in M. succiniciproducens. Understanding the relationship between the activities of these
enzymes is thus important for developing an optimal
fermentation condition for the enhanced production of
succinic acid.
In this article, we present the results of quantitative analyses
on the effects of the dissolved CO2 level on the metabolism
of M. succiniciproducens. To calculate the dissolved CO2
concentration in the medium, mathematical models which
consider the pH, temperature, ionic strength, and concentrations of salts and organic substances in the medium, were
developed. M. succiniciproducens was cultivated under varying
dissolved CO2 concentrations, and the fermentation products
were quantitatively analyzed. In addition, the activities of the
key enzymes were measured to elucidate the metabolic
characteristics during succinic acid production in M.
succiniciproducens.

Materials and Methods

Organism and Growth Conditions
Seed culture of M. succiniciproducens MBEL55E (KCTC
0769BP; Korean Collection for Type Cultures, Daejeon,

Korea) was prepared in a sealed anaerobic flask containing

250 mL MH4 medium with CO2 as a gas phase. The MH4
medium contains per liter: 5 g yeast extract, 1 g NaCl, 0.2 g
CaCl2
2H2O, 0.2 g MgCl2
6H2O, and 8.709 g K2HPO4.
The medium was heat sterilized (15 min at 1218C) in an
anaerobic flask with CO2 headspace. Glucose was separately
sterilized and added to the medium at a final concentration
of 50 mM. After adjusting the pH to 7.0 by adding 5 N
NaOH, the medium was inoculated with 2.5 mL of glycerol
stock culture and incubated at 398C for 9 h.
Batch cultures were carried out at 398C in a 6.6 L Bioflo
3000 fermentor (New Brunswick Scientific Co., Edison, NJ)
containing 2.25 L of MH4 medium plus 100 mM glucose
with 10% (v/v) of an inoculum. The pH was controlled at
6.5  0.1 by automatically adding 28% (w/w) ammonia
solution. The agitation speed was kept at 200 rpm. The
fermentor was flushed with a gas mixture of CO2 and N2
(from 0 to 100% CO2, v/v), scrubbed free of oxygen by
passing it through an oxygen trap (Agilent, Waldbronn,
Germany), during the whole period of the fermentation. A
0.2 mm HEPA filter (Millipore, Billerica, CA) was fitted in
the inlet line of the fermentor to maintain sterile conditions.
Gas sparging rate was controlled at 0.25 vvm using a mass
flow controller (MKS Instruments, Wilmington, MA). To
examine the effects of additional CO2 source on fermentation performance, NaHCO3, MgCO3, or CaCO3 was
separately added to the medium at various concentrations
(59.5, 119, and 238 mM) to increase the amount of the
dissolved CO2 in the medium. For these experiments, all
fermentations were carried out at 101.3 kPa CO2 partial
pressure (100% CO2 gas) and the other experimental
conditions were the same with those described previously.
To examine the effects of sodium, magnesium, and calcium
ions derived from the extra CO2 sources on the performance
of fermentation, cells were cultivated in a medium
containing the same molar concentration of NaCl, MgCl2,
or CaCl2, respectively.

Preparation of Cell-Free Extracts

Cells were cultured under 100% CO2 condition. Cells from
the mid-exponential phase were harvested by centrifugation
for 5 min at 10,000g and 48C. After centrifugation, the
supernatant was decanted and the cell pellet was washed
twice with 100 mM Tris-HCl (pH 7.0) containing 20 mM
KCl, 5 mM MnSO4, 2 mM DTT, and 0.1 mM EDTA. The
washed cell pellet was resuspended in the same buffer,
rapidly frozen, and stored at 708C until required. Cell
disruption was performed by using an ultrasonic homogenizer (VCX-600, Sonics and Materials Inc., Newtown,
CT), equipped with a titanium probe 40 T (4-mm diameter
and 127-mm length; Sonics and Materials Inc.) in a conical
tube on ice water. Sonication was carried out at 20 kHz for
six cycles with 30 s of rest time between each cycle; each cycle
was composed of 10 times of 3 s sonication and 3 s rest. The
disrupted cells were centrifuged for 10 min at 16,000g and

Song et al.: Effects of CO2 on Succinic Acid Production

Biotechnology and Bioengineering. DOI 10.1002/bit

1297

48C. The supernatant was immediately used or stored at

708C for future use. Protein concentration in the cellfree extracts was determined by Bradford assay using
bovine serum albumin as a standard (Bradford, 1976). The
average protein concentration in the cell extract prepared
was 17.10 mg mL1.
Enzyme Assays
Enzyme activities were measured spectrophotometrically in
a temperature-controlled spectrophotometer (SpectraMax
M2, Molecular Devices Co., Sunnyvale, CA). All assays were
performed at 308C using the cell extracts prepared from the
cells grown under 100% CO2 condition. Enzyme activities
were determined by averaging three independent measurements using at least two separately prepared cell-free
extracts. All components of the reaction mixture were added
to a quartz cuvette with 1-cm optical path (Sigma, St. Louis,
MO) and reactions were initiated by the addition of the cellfree extracts, which gave a final reaction volume of 1 mL.
The optimal amounts of cell-free extracts for the enzymes to
be assayed were determined after a series of experiments
ranging from 1 to 50 mL mL1. The amounts used in the
final assays of PckA, Ppc, MaeB, and PykA were 1.7, 25.0,
50.0, and 25.0 mL mL1, respectively. Reactions were
monitored by following the NAD(P)H at 340 nm. The
extinction coefficient for NAD(P)H of 6.23 mM1 cm1 at
340 nm was used. One unit (U) of enzyme activity was
defined as the amount of enzyme necessary to catalyze the
conversion of 1 mmol of substrate per minute into specific
products. Specific activity was defined as units per milligram
of protein.
The activity of PckA was measured as described by Kim
et al. (2004) in a reaction mixture containing 100 mM TrisHCl (pH 6.6), 35 mM NaHCO3, 16 mM MgCl2, 0.3 mM
NADH, 5 mM MnCl2, 1 mM DTT, 10 mM ADP, 2 U
phosphoglycerate phosphokinase, 2 U glyceraldehyde
phosphate dehydrogenase, 1 mM DTT, 10 mM ADP,
1.8 mM 3-phosphoglycerate, 5 mM PEP, and the cell-free
extracts. The activity of Ppc was measured in a reaction
mixture containing 66 mM Tris-HCl (pH 9.0), 5 mM PEP,
10 mM MgCl2, 10 mM NaHCO3, 0.15 mM NADH, 2 U
malate dehydrogenase, and the cell-free extracts (Terada
et al., 1991). MaeB was assayed in a reaction mixture
containing 0.1 M Tris-HCl (pH 7.8), 5 mM MgCl2, 2 mM
MnCl2, 0.6 mM NADP, 40 mM L-malate, and the cell-free
extracts (Peng and Shimizu, 2003). PykA was assayed as
described by Sridhar et al. (2000). The assay mixture
contained 0.1 M Tris-HCl (pH 7.5), 5 mM ADP, 1 mM
DTT, 10 mM KCl, 15 mM MgCl2, 0.5 mM PEP, 0.25 mM
NADH, 10 U lactate dehydrogenase, and the cell-free
extracts.
Analytical Procedures
The concentrations of glucose and metabolites, including
acetic, formic, lactic, malic, pyruvic, and succinic acids, and

1298

ethanol were measured by high-performance liquid

chromatography (Varian ProStar 210, Palo Alto, CA)
equipped with UV/VIS (Varian ProStar 320) and RI
(Shodex RI-71, Tokyo, Japan) detectors. A MetaCarb 87H
column (300  7.8 mm, Varian) was eluted isocratically at a
flow rate of 0.6 mL min1 using 0.01 N H2SO4 as a mobile
phase at 608C. Cell growth was monitored by measuring the
absorbance at 600 nm (OD600) using a spectrophotometer
(Ultrospec3000, Amersham Biosciences, Uppsala, Sweden).
Cell concentration defined as gram dry cell weight (DCW)
per liter was calculated from the pre-determined standard
curve relating the OD600 to DCW (1 OD600 0.451 g
DCW L1).

Chemicals and Gases

All chemicals used in this study were of reagent grade and
were purchased from either Sigma or Junsei Chemical
(Tokyo, Japan). Enzymes and coenzymes were obtained from
Sigma and Roche Molecular Biochemicals (Mannheim,
Germany). Gas mixtures were supplied by Special Gas
(Daejeon, Korea).

Modeling of Dissolved CO2 Concentration in a Medium

When a gas mixture is continuously supplied into the
reactor at low agitation and aeration rates, a liquidgas
phase equilibrium can be assumed. The solubility of CO2 in
a pure solvent (water) at an atmospheric pressure can be
described by Henrys law:
pCO2 Ho CCO2

(1)

where pCO2 is the CO2 partial pressure in a gas mixture

(kPa), Ho is the Henrys constant for CO2 in a pure solvent
(kPa L mol1), and CCO2 is the CO2 concentration dissolved
in a liquid (mol L1).
Since a culture medium contains various kinds of salts
and organic substances, the solubility of CO2 in a medium
needs to be described according to an empirical model,
such as the one suggested by Schumpe and Deckwer (1979)
that assumes log-additive of the individual effects of ions
(i) and organic substances (j):

H
log
Ho


aG;o
log
aG
X
X

hi hG ci
Kn;j cn;j
i

(2)

where H is the Henrys constant for CO2 in a medium (kPa L

mol1), ci is the concentration of ion i species (mol L1),
and cn,j is the concentration of organic substance j species
(kg m3). In the above equation, CO2 solubility was
expressed in the form of the Bunsen coefficients (aG and
aG,o) to extend the model to a wide temperature range

Biotechnology and Bioengineering, Vol. 98, No. 6, December 15, 2007

DOI 10.1002/bit

(Weisenberger and Schumpe, 1996). The values of ionspecific parameters, hi (L mol1) are: Na, 0.1143; K,
0.0922; Ca2, 0.1762; Mg2, 0.1694; H, 0; Cl, 0.0318;


2
HPO2
4 , 0.1499; OH , 0.0839; HCO3 , 0.0967; CO3 , 0.1423.
hG was estimated by Equation (3) as suggested by
Weisenberger and Schumpe (1996):
hG hG;o hG;T T  298:15K

(3)

where hG,o and hG,T for CO2 are 0.0172 L mol1 and
0.338  103 L mol1 K1, respectively, at temperatures
between 273 and 313 K (Weisenberger and Schumpe, 1996).
T is the absolute temperature (K). Kn,j in Equation (2) was
estimated by:
Kn;j bn bG

(4)
4

where bn for yeast extract and glucose are 7.90  10 and

6.68  104 m3 kg1, respectively (Rischbieter et al. 1996),
and bG was estimated by Equation (5):
bG bG;o bG;T T  298:15K

medium. The effects of CO2 partial pressure, temperature,

pH, and the concentrations of ions, organic substances, and
carbonate salts in the medium were all incorporated in the
models. The solubility of CO2 in pure water and complex
medium was calculated in wide partial pressure ranges (from
0 to 101.3 kPa) at 398C and an atmospheric pressure by
using Equation (2). Considering the effects of pH, ions, and
organic substances in the medium used in this study on
the CO2 solubility, the calculation was performed using
the values presented in Materials and Methods section. As a
consequence, Equation (2) becomes:


log

H
Ho

log

aG;o
aG


0:0275

(9)

Figure 1a shows a highly correlated linear trend

between the CO2 partial pressure and the dissolved CO2

(5)

where bG,o and bG,T for CO2 are 1.86  104 m3 kg1 and
0.010  104 m3 kg1 K1, respectively, at temperatures
between 288 and 323 K (Rischbieter et al. 1996).
2
When salts in the forms of HCO
3 and CO3 are added to
the medium with a continuous supply of 100% CO2 gas at
2
an atmospheric pressure, CO2, HCO
3 , and CO3 will be in a
chemical equilibrium through the following equations:

CO2 H2 O HCO
3 H

2

HCO
3 CO3 H

K1

K2

HCO
3 H 
CO2 

CO2
3 H 

HCO3 

(6)

(7)

where K1 and K2 are 5.3502  107 and 6.1245  1011 mol

L1, respectively, at 398C in this system (Kent and Eisenberg,
1976). Finally, through the material balance for CO2, the dissolved CO2 concentration in the medium can be expressed
as follows:
CO2  

CT

1 K1 =h K1 K2 =h2

(8)

where CT is the total CO2 concentration (mol L1), h is the

concentration of H ion (mol L1), which can be derived
from the pH value in a medium.

Results and Discussion

Calculation of Dissolved CO2 Concentration
We first developed mathematical models that allow
estimation of the dissolved CO2 concentration in the

Figure 1. The dissolved CO2 concentrations calculated using the models. a: The
dissolved CO2 concentrations in pure water (---) and medium () as a function of CO2
partial pressure. b: The dissolved CO2 concentrations in media supplemented with
different amounts of carbonate salts at the CO2 partial pressure of 101.3 kPa (100% CO2
gas). Symbols are: NaHCO3 (*), MgCO3 (&), and CaCO3 (~).

Song et al.: Effects of CO2 on Succinic Acid Production

Biotechnology and Bioengineering. DOI 10.1002/bit

1299

concentration both in pure water and the complex medium.

Equation (2) suggests that the dissolved substances in the
medium affect the CO2 solubility, referred to as salting-out
effect (Andrzej, 2006); it is evident from Figure 1a that the
amount of dissolved CO2 in the medium was lower than that
obtained in pure water. Under the condition of continuous
100% CO2 supply (101.3 kPa), the Henrys constant of CO2
in the medium at 398C was 4,320 kPa L mol1, which was
6.1% lower than that found in pure water.
The dissolved CO2 concentrations obtained with different
concentrations of three carbonate salts are shown in Table I
and Figure 1b. To determine the amount of dissolved CO2 in
the medium containing NaHCO3, MgCO3, or CaCO3 as an
extra CO2 source, the solubilities of these salts were determined at 398C and the CO2 partial pressure of 101.3 kPa.
Since the maximum solubility of NaHCO3 has been known
to be greater than 1,000 mM in water at 208C, NaHCO3
added into the medium can be assumed to be completely
solubilized. The maximum solubility of MgCO3 in water at
408C has been reported to be 139 mM (Mark et al., 1981).
CaCO3 is a rather insoluble salt and the maximum solubility
in the system exposed to CO2 was calculated from the
equilibrium of CaCO3/CO2/H2O suggested by Mucci
(1983). Based on Equation (8), the dissolved CO2 concentration in the medium was 260 mM when 238 mM of
NaHCO3 was supplemented. This is more than 1.6 and
10 times higher than those obtained by supplementing the
same amounts of MgCO3 and CaCO3, respectively. Thus,
the dissolved CO2 concentration in the medium was found
to be strongly dependent on the type of salt added to the
medium (Fig. 1b and Table I).

101.3 kPa. Under these conditions, the dissolved CO2

concentrations in the medium calculated using Equation (9)
were 0, 5.83, 8.74, 11.6, 17.3, and 23.0 mM, respectively. At
the dissolved CO2 concentrations of 0 and 5.83 mM, only
slight cell growth was observed for the first 4 h, after which
there was no further growth. The maximum cell densities
obtained under these conditions were less than 0.9 g L1. At
higher dissolved CO2 concentrations, cells grew until the
organic acids accumulated in the medium prevented further
growth. Even though more than 5 g L1 glucose was left in
the medium, no further growth was observed at high levels
of organic acids in the medium (Fig. 2). Instead, the residual
glucose was converted into organic acids and glucose was
completely consumed at the end of fermentation.
As shown in Table I and Figure 2, the higher dissolved
CO2 concentration in the medium had positive effects on
cell growth and succinic acid production. At the dissolved
CO2 concentration of 8.74 mM, glucose was completely
consumed at 10 h. Glucose was more rapidly consumed as
the dissolved CO2 concentration increased (Fig. 2). As
the dissolved CO2 concentration increased from 8.74 to
23.0 mM, the concentration and yield of biomass on glucose
obtained at the end of fermentation increased from 2.421
to 2.942 g DCW L1 and 0.126 to 0.162 g g1, respectively.
The maximum specific growth rate obtained at the dissolved
CO2 concentration of 23.0 mM was 1.12 h1, which was
1.43 times higher than that obtained at the dissolved CO2
concentration of 8.74 mM. The yield of succinic acid on
glucose also increased from 0.389 to 0.460 g g1 as the dissolved CO2 concentration increased from 8.74 to 23.0 mM.
The concentrations and yields of organic acids other than
succinic acid were relatively constant regardless of the levels
of the dissolved CO2 in the medium.

Fermentation Under Different CO2 Partial Pressures

The effects of the dissolved CO2 levels on cell growth and
succinic acid production were studied in pH-controlled
batch fermentation at 398C and 1 atm under various
CO2 partial pressures of 0, 25.32, 37.98, 50.65, 75.97, and
Table I.

Effects of Supplementing Bicarbonate Salts

In order to increase the amount of dissolved CO2 in the
medium and possibly to enhance cell growth and succinic

Effects of dissolved CO2 concentration on the growth of M. succiniciproducens and succinic acid production.
Added carbonate salts (mM)
CO2 partial pressure (kPa)a
37.98
b

Dissolved CO2 concentration (mM)

Initial glucose (g L1)
Final biomass (g DCW L1)
Final succinic acid (g L1)
Biomass yield (g DCW g1)
Succinic acid yield (g g1)
Maximum specific growth (h1)
Succinic acid/acetic acid (g g1)
Carbon recovery (%)c

50.65

75.97

101.3

NaHCO3
59.5

119

MgCO3
238

59.5

119

CaCO3
238

59.5

119

238

8.74 11.6
17.3
23.0
81.9
141
260
82.0
141
163
28.3
28.3
28.3
19.00 19.31 19.07 18.05 18.40
17.76
18.03 19.27
18.05
18.43 19.34 18.68 19.07
2.421 2.646 2.675 2.942 3.402
3.348
2.583 3.429
3.348
2.628 1.260 1.224 1.251
7.431 7.792 8.615 8.303 9.280 10.51
10.15
9.616
9.841
9.573 9.887 9.220 9.951
0.126 0.137 0.140 0.162 0.184
0.188
0.143 0.177
0.185
0.142 0.065 0.065 0.065
0.389 0.403 0.451 0.460 0.504
0.591
0.562 0.499
0.545
0.519 0.511 0.493 0.521
0.78
0.74
0.86
1.12
1.13
1.15
1.13
1.13
1.14
1.04
0.76
0.34
0.20
1.98
1.99
2.28
2.26
2.52
3.05
2.64
2.71
2.88
2.57
2.33
2.22
2.36
81.64 85.42 86.17 92.46 96.77 100.4
96.02 92.30
97.82
92.04 85.68 86.32 86.97

Gas mixture was composed of CO2 and N2.

Dissolved CO2 concentrations with the supplementation of carbonate salts were calculated at the CO2 partial pressure of 101.3 kPa (100% CO2).
Carbon recovery was calculated by assuming that all CO2 incorporated came from external sources; thus, 3 mol carbon per mol succinic acid was used.

b
c

1300

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DOI 10.1002/bit

Figure 2. Fermentation profiles of M. succiniciproducens at different CO2 partial pressures: (a) 37.98; (b) 50.65; (c) 75.97; (d) 101.3 kPa. Symbols are: biomass (&), glucose (*),
succinic acid (*), acetic acid (^), formic acid (~), and lactic acid (&).

acid production, batch fermentations were carried out using

the medium supplemented with varying concentrations
(59.5, 119, or 238 mM) of NaHCO3, MgCO3, or CaCO3 as
an extra CO2 source (Fig. 3). The dissolved CO2 concentrations were determined by using Equation (8) and the results
are presented in Table I and Figure 1b. The maximum
dissolved CO2 concentrations of 260, 163, and 28.3 mM
were obtained when NaHCO3, MgCO3, and CaCO3 were
supplemented, respectively (Fig. 1b); different dissolved
CO2 concentrations seem to be due to the different solubilities of NaHCO3, MgCO3, and CaCO3. The precipitation
of unsolubilized MgCO3 and CaCO3 were observed at the
bottom of the fermentor.
When one of the bicarbonate salts was added to the
medium, cell growth and succinic acid production were
slightly enhanced. When 119 mM of NaHCO3 was
supplemented, the yield of succinic acid on glucose was
0.591 g g1, which was 1.28 times higher than that obtained
without supplementation. The highest maximum specific
growth rate and biomass yield on glucose were also obtained
in the medium containing 119 mM of NaHCO3, which
were 1.15 h1, and 0.188 g g1, respectively. These values
were very similar to those obtained in the medium
containing 119 mM MgCO3 (1.14 h1 and 0.185 g g1).
The calculated dissolved CO2 concentrations were 141.65
and 141.39 mM when 119 mM of NaHCO3 and MgCO3
were supplemented, respectively. Interestingly, cell growth
and succinic acid production were suppressed to some
extent in the media containing 238 mM of NaHCO3 and

MgCO3 even though the dissolved CO2 concentrations were

higher. The presence of CaCO3 severely inhibited cell growth
as the maximum specific growth rate and the final cell
density were even lower than those obtained at the CO2
partial pressure of 37.98 kPa (dissolved CO2 concentration
of 8.74 mM).
Although the sodium and magnesium ions have been
known to play important roles in cellular metabolism, the
adverse effect on cell growth caused by their excessive
presence has also been known (Podkovyrov and Zeikus,
1993; Strobel and Russell, 1991). Lee et al. (1999b) reported
that the growth of A. succiniciproducens was suppressed
in the medium containing more than ca. 70 mM NaCl and
4 mM MgCl. Bashir and Matin (2005) also reported that the
optimal concentrations of magnesium and calcium ions
for thermophilic anaerobic bacteria are 13 and 20 mM,
respectively. Thus, it was thought that sodium, magnesium,
and calcium ions might have negatively affected the
performance of fermentation. To examine whether this
was the case, a series of batch fermentations were performed
in the media supplemented with NaCl, MgCl2, and
CaCl2 instead of NaHCO3, MgCO3, and CaCO3, respectively, under the same culture conditions (Table II).
When 59.5 mM of NaCl was supplemented, there were
no apparent changes in cell growth and succinic acid
production. However, supplementing higher concentrations
of NaCl showed negative effects on cell growth and succinic
acid production. Addition of MgCl2 and CaCl2 into the
medium showed more negative effects as can be seen from

Song et al.: Effects of CO2 on Succinic Acid Production

Biotechnology and Bioengineering. DOI 10.1002/bit

1301

Figure 3. Fermentation profiles of M. succiniciproducens with supplementation of various concentrations three different carbonate salts: (a) 59.5 mM NaHCO3; (b) 119 mM
NaHCO3; (c) 238 mM NaHCO3; (d) 59.5 mM MgCO3; (e) 119 mM MgCO3; (f) 238 mM MgCO3; (g) 59.5 mM CaCO3; (h) 119 mM CaCO3; (i) 238 mM CaCO3. Symbols are: biomass (&),
glucose (*), succinic acid (*), acetic acid (^), formic acid (~), and lactic acid (&). Fermentations were performed at the CO2 partial pressure of 101.3 kPa (100% CO2 gas).

Table II; cells could not grow in the medium containing

either 238 mM of MgCl2 or 119 and 238 mM of CaCl2.
Activities of Major Carboxylating Enzymes
In the previous study based on the genome information
of M. succiniciproducens, three CO2-fixing enzymes, including PckA, Ppc, and MaeB, were identified in the central
metabolic pathway (Hong et al., 2004). Furthermore, our
recent genome-based knockout studies (Lee et al., 2006b)
showed that the growth of the pckA-deficient strain was

Table II.

severely suppressed. In order to elucidate the functions of

key enzymes in CO2 fixation necessary for cell growth and
succinic acid formation, the activities of PckA, Ppc, and
MaeB were determined. The activity of PykA, which
catalyzes the conversion of PEP to pyruvate rather than
oxaloacetate, was also measured. In order to avoid potential
effects of the carbonate salts, only the cell-free extracts from
the cells grown at a CO2 partial pressure of 101.3 kPa
without NaHCO3, MgCO3, and CaCO3 were used in this
study. The activity of PckA, 1.27 U mg1 protein, was the
highest among those of enzymes examined in this study.

Effects of sodium, magnesium, and calcium ions on the growth of M. succiniciproducens and succinic acid production.
MgCl2 (mM)

NaCl (mM)
59.5
1

Initial glucose (g L )
Final biomass (g DCW L1)
Final succinic acid (g L1)
Maximum specific growth (h1)
Carbon recovery (%)b

18.94
2.74
9.09
1.02
92.73

119
18.93
2.95
9.03
0.94
93.07

238
18.87
2.08
8.58
0.77
88.91

59.5
18.21
1.01
8.59
0.52
85.60

119
18.78
0.96
8.60
0.27
79.86

CaCl2 (mM)
238
NG
NG
NG
NG
NG

59.5

119

238

18.93
1.06
8.88
0.87
83.94

NG
NG
NG
NG
NG

NG
NG
NG
NG
NG

No growth.
Carbon recovery was calculated by assuming that all CO2 incorporated came from external sources; thus, 3 mol carbon per mol succinic acid was used.

1302

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DOI 10.1002/bit

This value is about 17 and 654 times higher than those of Ppc
(0.0748 U mg1 protein) and MaeB (0.00194 U mg1
protein), respectively, which are also involved in the fixation
of CO2. Although the activity of PykA (0.0463 U mg1
protein) was slightly higher than that of MaeB, it was much
lower than that of PckA. Thus, it was further confirmed
that PckA is the most important carboxylating enzyme for
the growth of M. succiniciproducens and succinic acid
production.
Since the PEP carboxylation reaction catalyzed by PckA
produces ATP in M. succiniciproducens, while Ppc does not,
this high PckA activity suggests that the availability of
enough CO2 is obligate not only for the production of
succinic acid but also for the growth of M. succiniciproducens
by generating ATP. This was supported by the finding
that M. succiniciproducens could not grow well at the
dissolved CO2 concentration of less than 5.83 mM. Pyruvate
is formed from PEP by PykA in M. succiniciproducens (Lee
et al., 2006b) and the level of the activity was quite lower
than that of PckA activity. The level of this enzyme has been
known to be similar under high and low CO2 conditions in
A. succiniciproducens (Samuelov et al., 1991) and did not
have any significant effect on cell growth rate (Pearce et al.
2001; Schaaff et al., 1989). This also seems to be true for M.
succiniciproducens.

Conclusions
In this work, we reported in detail the effects of dissolved CO2
concentration on the growth of M. succiniciproducens and
succinic acid production. As the dissolved CO2 concentration
increased, the maximum specific growth rate, and the yields
of succinic acid and biomass on glucose also increased. The
dissolved CO2 concentration could be increased by supplementing bicarbonate salts, but too excessive addition had a
negative effect on cell growth. CO2 availability was found to
be truly important for the growth of M. succiniciproducens
and succinic acid production, and PckA plays the most
important role in carboxylation reaction generating ATP.

Nomenclature
bG

gas-specific parameter of Rischbieter et al. (1996) model

(m3 kg1)

bn

organic substance-specific parameter of Rischbieter et al. (1996)

model (m3 kg1)

CCO2

CO2 concentration dissolved in a liquid (mol L1)

ci

concentration of ion i (mol L1)

cn,j

concentration of organic substance j (kg m3)

CT
H

total CO2 concentration (mol L1)

Henrys constant for CO2 in a medium (kPa L mol1)

Ho

Henrys constant for CO2 in a pure solvent (water) (kPa L mol1)

H ion concentration (mol L1)

hi

ion-specific parameter of Weisenberger and Schumpe (1996)

model (L mol1)

hG

gas-specific parameter of Weisenberger and Schumpe (1996)

model (L mol1)

Kn,j

organic substance-specific parameter of Weisenberger and

Schumpe (1996) model (m3 kg1)

K1, K2

reaction equilibrium constants (mol L1)

pCO2

CO2 partial pressure in a gas mixture (kPa)

absolute temperature (K)

aG
aG,o

Bunsen coefficient for CO2 in a medium (no dimension)

Bunsen coefficient for CO2 in a pure solvent (water) (no
dimension)

This work was supported by the Genome-Based Integrated Bioprocess

Development Project from Korean Ministry of Science and Technology (No. 2005-01294) through the Korea Science and Engineering
Foundation (KOSEF). Further support by the LG Chem Chair Professorship is appreciated.

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Biotechnology and Bioengineering, Vol. 98, No. 6, December 15, 2007

DOI 10.1002/bit