Enzyme and Microbial Technology 39 (2006) 352361

Review

Production of succinic acid by bacterial fermentation

Hyohak Song a , Sang Yup Lee a,b,
a

Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering and
BioProcess Engineering Research Center, Republic of Korea
b Department of BioSystems and Bioinformatics Research Center, Korea Advanced Institute of Science and Technology,
373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea
Received 13 October 2005; received in revised form 18 November 2005; accepted 23 November 2005

Abstract
Succinic acid produced by various microorganisms can be used as a precursor of many industrially important chemicals in food, chemical
and pharmaceutical industries. The assessment of raw material cost and the estimation of the potential market size clearly indicate that the
current petroleum-based succinic acid process will be replaced by the fermentative succinic acid production system in the foreseeable future. This
paper reviews processes for fermentative succinic acid production, especially focusing on the use of several promising succinic acid producers
including Actinobacillus succinogenes, Anaerobiospirillum succiniciproducens, Mannheimia succiniciproducens and recombinant Escherichia
coli. Processes for the recovery of succinic acid from fermentation broth are also reviewed briefly. Finally, we suggest further works required to
improve the strain performance suitable for successful commercialization of fermentative succinic acid production.
2006 Elsevier Inc. All rights reserved.
Keywords: Succinic acid; Fermentation; Recovery; Genome; Metabolic engineering

Contents
1.
2.
3.

4.
5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Succinic acid markets and applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Succinic acid producers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Actinobacillus succinogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Mannheimia succiniciproducens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Anaerobiospirillum succiniciproducens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4. Recombinant E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Recovery of succinic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction
Succinic acid, also known as amber acid or butanedioic
acid, is a dicarboxylic acid having the molecular formula of
C4 H6 O4 . After its first purification of succinic acid from amber
by Georgius Agricola in 1546, it has been produced by microbial

Corresponding author. Tel.: +82 42 869 3930; fax: +82 42 869 8800.
E-mail address: leesy@kaist.ac.kr (S.Y. Lee).

0141-0229/$ see front matter 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2005.11.043

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359

fermentation for the use in agricultural, food and pharmaceutical industries [1]. Currently, most of commercially available
succinic acid is produced by chemical process, in which liquefied petroleum gas (LPG) or petroleum oil is used as a starting
material.
Succinic acid can be used as a precursor of many industrially important chemicals including adipic acid, 1,4-butanediol,
tetrahydrofuran, N-methyl pyrrolidinone, 2-pyrrolidinone, succinate salts and gamma-butyrolactone (Fig. 1). Furthermore, the
increasing demand for succinic acid is expected as its use is

H. Song, S.Y. Lee / Enzyme and Microbial Technology 39 (2006) 352361

353

Fig. 1. Various chemicals and products that can be synthesized from succinic acid.

extended to the synthesis of biodegradable polymers such as

polybutyrate succinate (PBS) and polyamides (Nylon x,4) [2]
and various green solvents [3]. Nonetheless, the global succinic acid market (15,000 t in 1997 with an average annual
growth rate of 610% per year) is much smaller compared with
that of the competing chemical, maleic anhydride (213,000 t
in 1997 with an average annual growth rate of 3.6% per
year) [2,3]. This is partially due to the high conversion cost
of maleic anhydride to succinic acid by the chemical process, which limits the use of succinic acid for the wide range
of applications. On the other hand, recent analysis showed
that fermentative production of succinic acid from renewable
resources can be more cost-effective than the petroleum-based
processes [2]. It is also notable that a greenhouse gas CO2 is
fixed into succinic acid during the fermentation, thus providing an additional advantage of active coping with the Kyoto
protocol.
Many different microorganisms have been screened and studied for succinic acid production from various carbon sources.
Among them, Anaerobiospirillum succiniciproducens [48] and
Actinobacillus succinogenes [912] have been most intensively
studied due to their ability to produce a relatively large amount
of succinic acid. More recently, a new succinic acid producing
bacterium Mannheimia succiniciproducens MBEL55E was isolated from bovine rumen [13]. Also, there has been much effort
in developing recombinant Escherichia coli strains which are
capable of enhanced succinic acid production under aerobic and
anaerobic conditions [1420].

In this paper, we review the metabolic characteristics and

fermentation performance of the most prominent succinic acid
producers. Formation of byproducts such as acetic, formic and
lactic acids is a major problem that has to be solved because it
reduces the succinic acid yield and productivity, while increases
the complexity and cost of succinic acid recovery. Applications
taken to solve this problem are also reviewed. Then, this review
ends with future prospects of fermentative production of succinic
acid from renewable resources.
2. Succinic acid markets and applications
At present, succinic acid is mostly produced by the chemical
process from n-butane through maleic anhydride. It is sold at the
price of $5.9 to 9.0 kg1 depending on its purity. Its manufacturing cost is affected by several factors including succinic acid
productivity and yield, the costs of raw materials, and recovery method. Particularly, the cost of maleic anhydride has been
known to contribute most significantly to the overall cost of
succinic acid production. In 2002, maleic anhydride made from
n-butane was sold at an average selling price of $0.977 kg1 .
Considering that the overall conversion yield of maleic anhydride to succinic acid is 95% (w/w), the major raw material
cost in the chemical process is $1.027 kg1 succinic acid. On
the other hand, more than 30,000,000 t of glucose is produced
annually worldwide, and it is sold at a price of about $0.39 kg1 .
Assuming the succinic acid yield of 91% (w/w) on glucose
[21], the raw material cost in the bioprocess is then $0.428 kg1

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H. Song, S.Y. Lee / Enzyme and Microbial Technology 39 (2006) 352361

succinic acid. It is thus clear that fermentative production of

succinic acid from renewable resources can compete with the
chemical process. More importantly, the limited nature of fossil
reserves and ever increasing environmental concerns are urging us to replace the petroleum-based chemical processes with
bio-based processes. Renewable resources are more than abundant, currently being estimated to be 170 billion tons per year,
of which only 3.5% is being utilized.
The market potential for succinic acid itself and its derivatives is estimated to be more than 270,000 t yr1 in 2004 [2].
The estimated potential market size for the polymers polysuccinate esters and polyamides that can be synthesized from succinic
acid is up to 27 million tons per year in 2001 [22]. Although the
overall economics still limits the bio-based succinic acid production, the assessment of raw material cost and the estimation of
potential market size clearly suggest that the current petroleumbased succinic acid process will be replaced by the fermentative
succinic acid production system in the near future.
3. Succinic acid producers
Since Robert Knock, the Nobel Prize winner, proved that
succinic acid has a positive influence on human metabolism and
there is no risk of its accumulation in the human body, it has
been used in food industries. Succinic acid is an intermediate of
the tricarboxylic acid (TCA) cycle and one of the fermentation
end-products of anaerobic metabolism. Thus, it is synthesized
in almost all microbial, plant and animal cells. Those organisms suitable for the efficient production of succinic acid can be
categorized into fungi and bacteria.
Many researchers have made tremendous efforts to develop
a biological process for the production of succinic acid by
employing fungi such as Aspergillus niger, Aspergillus fumigatus, Byssochlamys nivea, Lentinus degener, Paecilomyces
varioti, Penicillium viniferum and yeast Saccharomyces cerevisiae. These organisms produce succinic acid as a metabolic
byproduct under aerobic and/or anaerobic conditions [23,24].
S. cerevisiae has been best studied among them to achieve
high concentration of succinic acid in the manufacture of wine
[25,26]. A series of its mutant strains were developed by the
inactivation of undesired genes, and some of them showed the
increased levels of succinic acid compared with the wild type
strain [27,28].
A. niger has been recognized as a very important organism
for the production of various organic acids, especially citric acid
and gluconic acid. This organism produces more than 78 g l1
of citric acid with the yield of 65% (w/w) on sucrose [29]. Furthermore, it shows an ability to utilize various carbon sources
with a good yield (115%, w/w) on rapeseed oil [30]. Recently,
the central carbon metabolism of this organism and its metabolic
network were deciphered by combining genomic, biochemical
and physiological information. Based on them, a stoichiometric model composed of 284 metabolites and 335 reactions was
constructed. Simulation of this stoichiometric model suggested
that this organism can produce 1.5 mol succinic acid from 1 mol
glucose under microaerobic condition [31]. However, the use of
fungi has been mostly limited to the manufacture of food and

beverages due to the difficulties in fermentation, separation and

purification as well as low productivities.
Only few Gram-positive bacteria like Corynebacterium glutamicum and Enterococcus faecalis have been studied for succinic acid production. Several engineered C. glutamicum strains
were created by disruption and replacement of genes, and their
optimal culture conditions were developed. It was possible to
increase the succinic acid production rate seven times and the
glucose consumption rate five times under oxygen deprived condition [32]. A two-step succinic acid production process was
developed, in which fumaric acid obtained from the fermentation of glucose and rice bran using Rhizopus sp. is subsequently
converted to succinic acid by E. faecalis RKY1 [33]. The yield of
fumaric acid conversion to succinic acid was 95% (w/w) and the
productivity was 2.2 g l1 h1 . However, it should be noted that
the yield of fumaric acid on glucose and the fumaric acid productivity in the first step were quite low at the levels of 0.5 g g1
and 0.21 g l1 h1 , respectively, limiting its commercialization.
The metabolic pathways leading to the synthesis of succinic
acid are diverse. Some bacteria mainly utilize the phosphoenolpyruvate (PEP) carboxylation reaction, while others use
multiple pathways to form succinic acid [34]. Many different
succinic acid producing Gram-negative bacteria have been isolated in various anaerobic environments such as domestic sludge,
cattle waste, rice paddy, marine shipworm, mouth of dog, rumen
and gastro-intestines. To date, the bacteria isolated from the
rumen, including A. succinogenes and M. succiniciproducens,
are the best candidates for succinic acid production as they produce succinic acid as a major fermentation product. This is most
likely due to that the rumen is a highly efficient organ providing an environment to produce succinic acid. The rumen is a
unique microbial ecosystem found in many species of herbivorous mammals known as ruminants. The primary role of the
rumen is to allow pre-gastric digestion of various polysaccharide materials, which is mediated by a great diversity of rumen
microorganisms, consisting of 109 1010 bacterial, 105 106 protozoan and 103 104 fungal cells ml1 of rumen fluid [35]. The
production of C4 dicarboxylic acids in the rumen reduces energy
loss associated with methanogenesis (3040 mol% of CH4 is
present in the ruminal gas) by increasing the amount of metabolizable energy available to the animal in the form of propionic acid. Although the C4 dicarboxylic compounds, such as
oxaloacetic, malic, fumaric and succinic acids are not detected
in the ruminal fluid, large amounts of these acids are produced
by CO2 fixation reactions, using 6070 mol% of CO2 present in
the ruminal gas. The major C3 compounds in the cell used for
carboxylation reaction are PEP and pyruvate. In particular, succinic acid is converted to propionic acid, which can account for
20% (w/w) of total volatile fatty acids (VFAs) in the rumen, by
succinic acid utilizing bacteria such as Veillonella parvula [36],
Selenomonas ruminantium [37] and Succiniclasticum ruminis
[38]. Propionic acid produced this way is absorbed through
the rumen wall for subsequent oxidation to provide energy and
biosynthetic precursors for the animals. Therefore, it is reasonable to think that some microorganisms present in the rumen will
be a good succinic acid producer. Here, we review the metabolic
characteristics and fermentation performances of two rumen

[17]
[20]
127
89
1.30
0.98
1.10
0.55
99.2
58.3

Data not available.

Cell concentration was represented as optical density by measuring the absorbance.
mg OD1 h1 .
a

Dual-phase fed-batch
Fed-batch

Glucose
Glucose

76
59

10.2
OD600 35b
b

A. succiniciproducens

Recombinant E. coli
AFP111 (pTrc99A-pyc)
HL27659K

544
370

293c
550c
0.88
0.79
1.34
1.87
1.22
2.1
1.35
1.1
33.9
66.4
105.8
14
13.5
24
29.6
33
Repeat-batch
Batch
Batch
Batch
Batch
Continuous
Fed-batch
Batch
A. succinogenes
130Z
FZ53
M. succiniciproducens

Glucose
Glucose
Glucose
Glucose
Whey
Whey
Glucose + glycerol
Glucose

38.5
84
78
7.5
11
D = 0.085 h1
22
30

3.44
3.30

OD660 4.6b
OD660 2.0b

Yield
(g g1 )
Succinic acid
concentration
(g l1 )
Cell concentration
(g l1 )
Time (h)
Substrate
Fermentation strategy
Microorganism

Table 1
Performances of succinic acid production by various microorganisms

A. succinogenes was originally isolated from bovine ruminal contents and belongs to the family Pasteurellaceae based
on its 16S rRNA sequence analysis [9]. The phenotypic analysis
showed that this organism is a facultative anaerobic, non-motile,
pleomorphic, and Gram-negative rod or occasionally filamentous bacterium. A. succinogenes shows a distinctive ability to
produce a relatively large amount of succinic acid from a broad
range of carbon sources such as arabinose, cellobiose, fructose,
galactose, glucose, lactose, maltose, mannitol, mannose, sorbitol, sucrose, xylose or salicin under anaerobic condition [39].
Unlike E. coli or A. succiniciproducens, A. succinogenes is a
moderate osmophile and has good tolerance to a high concentration of glucose, which is beneficial for fermentation.
Extensive physiological and genetic studies relating to succinic acid production in A. succinogenes have been performed.
Five key enzymes responsible for succinic acid production were
identified to be PEP carboxykinase (pck), malate dehydrogenase (mdh), malic enzyme (sfc), fumarase (fum) and fumarate
reductase (frd). Also, enzymatic analysis revealed the presence
of pyruvate kinase (pyk), pyruvate ferredoxin oxidoreductase
(pfo), acetate kinase (ack), alcohol dehydrogenase (adh) and
lactate dehydrogenase (ldh), which affect succinic acid flux in
the central metabolic pathways.
PEP carboxylation, which is the important committed step
for succinic acid production in rumen bacteria, is strongly regulated by CO2 levels. Theoretically, 1 mol of CO2 is required to
form 1 mol of succinic acid. The higher CO2 level resulted in
an increased succinic acid production at the expense of ethanol
and formic acid. This is most likely due to the increased carboxylation of PEP to oxaloacetate rather than PEP conversion
to pyruvate. Also, the addition of extra electron donors including hydrogen and electrically reduced neutral red resulted in the
significant increase of succinic acid production [10,34]. These
observations are consistent with that the use of more reduced
sugars such as arabitol, mannitol and sorbitol resulted in significant increases in the succinic acid and ethanol production
compared with glucose [34].
A. succinogenes 130Z strain and its variant strains (FZ 6, 9,
21, 45 and 53) which are resistant to 18 g l1 of fluoroacetate
were used to evaluate the possibility of commercial succinic acid
production. They were able to produce larger amounts of succinic acid and were more resistant to succinic acid than any other
previously reported succinic acid producers [39,40]. Strain 130Z
produced 66.4 g l1 of succinic acid by consuming 98.3 g l1
of glucose after 84 h fermentation. The batch fermentation was
performed in a 1 l fermenter with 15 g l1 of yeast extract and
corn steep liquor. MgCO3 (80 g) was added to the fermenter
prior to autoclaving in order to moderate the pH drop during
the fermentation. The concentrations of byproducts including
acetic, formic, propionic and pyruvic acids were detected at

Productivity
(g l1 h1 )

3.1. Actinobacillus succinogenes

0.86
0.67
0.8
0.7
0.72
0.72
0.97
0.93

Specific productivity
(mg g DCW1 h1 )

References

bacteria A. succinogenes and M. succiniciproducens along with

two other good candidate bacteria A. succiniciproducens and
recombinant E. coli. Also, their performances are compared in
Table 1.

355

[12]
[39]
[39]
[13]
[41]
[8]
[47]
[5]

H. Song, S.Y. Lee / Enzyme and Microbial Technology 39 (2006) 352361

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H. Song, S.Y. Lee / Enzyme and Microbial Technology 39 (2006) 352361

the values of 12.0, 8.7, 2.5 and 4.3 g l1 , respectively. More

recently, the continuous and repeat-batch biofilm fermentation
of A. succinogenes allowed a significant increase in succinic acid
productivity (8.8 g l1 h1 ), while the yield of succinic acid was
less than 50% (w/w), which is rather low for commercialization [12]. Although the variant strains produced less ethanol,
acetic, formic and lactic acids, formation of these byproducts
could not be completely avoided. Furthermore, the accumulation of propionic and pyruvic acids, which are not generally
detected in the cultivation of other succinic acid producing bacteria, was observed. Considering the costs of separation and
purification of succinic acid from fermentation broth containing
mixed acids, the formation of byproducts should be minimized,
or if possible, completely eliminated by metabolic engineering
and fermentation process optimization. Unfortunately, only a
few studies were conducted on the metabolic engineering of
A. succinogenes due to the limited genetic information and the
lack of appropriate genetic tools [11]. Further improvement
of the performance of A. succinogenes will require genome
analysis and its use in the metabolic engineering in a global
scale.
3.2. Mannheimia succiniciproducens
Another promising succinic acid producing bacterium, M.
succiniciproducens MBEL55E, was recently isolated from the
bovine rumen [13]. Phenotypic and phylogenetic studies suggest
that M. succiniciproducens is a facultative, mesophilic, nonmotile, and capnophilic Gram-negative bacterium. It produces

succinic acid as a major product, acetic and formic acids as the

second major ones from various carbon sources under 100%
CO2 condition at pH of 6.07.5. The succinic acid productivity
of as high as 3.9 g l1 h1 could be achieved, which is the highest
value that has been reported so far. Furthermore, the efficient and
economical production of succinic acid was possible by fermentation of M. succiniciproducens using a whey-based medium
containing corn steep liquor instead of yeast extract [41]. M.
succiniciproducens also efficiently utilizes xylose, which makes
it possible to use untreated wood hydrolysate to reduce the
raw material cost [42]. These observations suggest that M. succiniciproducens can be a good candidate for the cost-effective
succinic acid production from renewable resources.
As mentioned earlier, it is necessary to develop a metabolically engineered strain capable of producing succinic acid
with high yield and productivity without byproducts formation.
Even though rational selection and manipulation of a handful
of genes can lead to the development of a superior strain, it
will be desirable to have a complete genome sequence available for better metabolic engineering based on the genome-wide
analysis. Recently, the complete genome sequence of M. succiniciproducens has been determined [43]. It has a single circular
chromosome of 2,314,078 base pairs without plasmid. Based
on the annotation results, the in silico metabolic network composed of 373 reactions and 352 metabolites was constructed. The
genome-scale metabolic flux analysis was carried out using the
in silico metabolic network to understand the overall metabolic
characteristics under various conditions. The major metabolic
pathways involved in the fermentative production of acids are

Fig. 2. Major metabolic pathways leading to the formation of succinic acid and byproducts in M. succiniciproducens. Intracellular metabolites are shown in single
circles while the excretory metabolic products are shown in double circles. Enzymes catalyzing the reactions are shown in rectangles.

H. Song, S.Y. Lee / Enzyme and Microbial Technology 39 (2006) 352361

shown in Fig. 2. Interestingly, the glycolytic flux under CO2

condition was four times greater than that obtained under N2 .
The PEP carboxylation flux as well as the reductive TCA cycle
flux considerably increased under CO2 atmosphere, which confirms that M. succiniciproducens is a capnophilic bacterium.
When external reducing power was provided in the form of H2 ,
the succinic acid yield on glucose could be further increased
[43]. It was predicted that M. succiniciproducens can produce
as much as 1.71 and 1.86 mol of succinic acid from 1 mol of glucose under CO2 and CO2 H2 environments, respectively. These
results suggest that M. succiniciproducens is a good candidate
for the production of succinic acid with high productivity and
high yield. As mentioned for other bacteria above, formation of
mixed acids is a problem to be solved.
3.3. Anaerobiospirillum succiniciproducens
A. succiniciproducens was isolated from the throat and feces
of beagle dog [44], and produces succinic and acetic acids as
major fermentation products and ethanol and lactic acid as minor
ones under strictly anaerobic condition [21,45]. It belongs to the
family Succinivibrionaceae and is a strictly anaerobic, motile,
and Gram-negative bacterium [46]. Like A. succinogenes and
M. succiniciproducens, it also uses PEP carboxylation pathway
to form succinic acid.
Previous studies showed that A. succiniciproducens can efficiently utilize glucose, glycerol, sucrose, maltose, lactose and
fructose as carbon sources. The use of glycerol as a carbon source
resulted in an increased succinic acid yield (133%, mol/mol) and
much higher ratio of succinic acid to acetic acid (Gram ratio of
25.8:1) than those obtained with glucose [47]. In addition, a possibility of cost-effective succinic acid production was presented
using untreated whey, wood hydrolysate and corn steep liquor,
which are much less expensive than refined carbohydrates and
yeast extract, respectively [6,48].
The levels of CO2 , culture pH, an external electron source
and medium components have been known to be critical factors
affecting both cell growth and succinic acid production. The
increased CO2 availability exerted a positive influence on succinic acid yield in A. succiniciproducens, while it depressed cell
growth resulting in the decreased succinic acid productivity. On
the other hand, the supplementation of hydrogen in the forms
of H2 /CO2 mixture (5:95, v/v) as an external electron donor
significantly enhanced both cell growth, succinic acid yield
(91%, mol/mol) and productivity (1.8 g l1 h1 ) [21]. Also, the
supplementation of biotin improved glucose consumption, cell
growth and succinic acid production rates [5]. The key enzymes
involved in succinic acid production pathways including PEP
carboxykinase, oxaloacetate decarboxylase and pyruvate kinase
have been studied in detail [7,4952]. Especially, the A. succiniciproducens pckA gene encoding PEP carboxykinase has
been cloned, sequenced and expressed in E. coli, which resulted
in the enhanced succinic acid production as well as reduced
byproducts formation [53].
Fluoroacetate resistant variant strains of A. succiniciproducens were isolated and used in the integrated process composed of fermentation, separation and purification [40,5456].

357

Higher succinic acid and lower acetic acid concentrations could

be achieved with the variant strains compared with their parent strain. Production of mixed acids is also a problem to be
solved to prevent high purification cost. Again, the unknown
genomic information is a barrier to the metabolic engineering
of A. succiniciproducens for enhancing succinic acid production
and preventing byproducts formation.
3.4. Recombinant E. coli
A wild type E. coli primarily ferments glucose to ethanol,
formic, acetic and lactic acids with only detectable amounts of
succinic acid under anaerobic condition. The succinic acid yield
on glucose typically obtainable is no more than 0.2 mol mol1 .
It has been known that E. coli utilizes six pathways to form succinic acid, and differently from three bacteria mentioned above,
the PEP carboxykinase plays a minor role [57]. Nevertheless,
metabolic flux analysis showed that the maximum achievable
succinic acid molar yield in E. coli is 1.647 [58]. A number of metabolic engineering strategies have been developed to
enhance succinic acid production by E. coli. The approaches can
be broadly categorized as follows: the inactivation of enzymes
participating in the reactions, which compete with succinic acid
pathways, the amplification of enzymes involved in succinic
acid pathways, and the introduction of heterologous enzymes
catalyzing reactions towards increased succinic acid formation.
The first metabolic engineering approach taken in E. coli
for efficient succinic acid production was the overexpression
of the PEP carboxykinase (pck) and PEP carboxylase (ppc)
genes [14]. Overexpression of the PEP carboxylase resulted in
a 3.5 times increase in the amount of succinic acid, whereas
the PEP carboxykinase overexpression had no effect. The latter phenomenon was due to that PEP carboxykinase functions
as a gluconogenic enzyme in E. coli. With an aim to reduce
byproducts formation, the NZN111 strain was developed by
inactivating the pyruvate:formate lyase (p) and the lactate dehydrogenase (ldh) genes [15,16]. It has been most widely used as
a starting strain for further metabolic engineering of E. coli to
enhance succinic acid production. The inactivation of the p and
ldh genes increased the yield of succinic acid at the expense
of ethanol and acetic, formic and lactic acids under anaerobic condition, but it excreted a significant amount of pyruvic
acid. Furthermore, cell growth was severely inhibited probably
due to the inability to sufficiently regenerating NAD [15,59].
E. coli has a malic enzyme which can interconvert pyruvate
and malate. However, the Km value of E. coli malic enzyme
is 16 and 0.4 mM for pyruvate and malate, respectively, indicating that the reaction proceeds to form pyruvate [45]. After
observing the accumulation of pyruvate in NZN111 strain, it
was reasoned that amplifying the malic enzyme would resulted
in conversion of pyruvate to malate. Indeed, succinic acid production could be enhanced by the overexpression of the sfcA
gene encoding malic enzyme in NZN111 [15,16]. A derivative
of NZN111 which showed restored cell growth was selected by
spontaneous chromosomal mutation. The mutant strain named
AFP111 could grow on glucose and had mutation in the glucosespecific phosphotransferase gene (ptsG). Fermentation of this

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H. Song, S.Y. Lee / Enzyme and Microbial Technology 39 (2006) 352361

strain dramatically increased the succinic acid yield and productivity to 1.0 mol mol1 and 0.87 g l1 h1 , respectively. The
cloning of the heterologous pyruvate carboxylase (pyc) gene
from Rhizobium etli or C. glutamicum into the AFP111 to divert
the accumulated pyruvate to succinic acid was conducted. The
dual-phase fermentation of a recombinant AFP111 strain harboring pTrc99A-pyc, comprised of initial aerobic growth phase
followed by anaerobic succinic acid production phase, resulted
in the succinic acid concentration and productivity of 99.2 g l1
and 1.3 g l1 h1 , respectively [17].
Recently, more effort has been made to develop recombinant E. coli strains capable of producing succinic acid with
high efficiency. The Sorghum vulgare ppc and Lactococcus lactis pyc genes were introduced into the ldhp-inactivated and
ldhptaack-inactivated E. coli mutant strains, respectively, in
order to redirect the accumulated pyruvate to oxaloacetic acid
[18]. In another approach, a recombinant strain was created by
activating the glyoxylate pathway because it requires less NADH
in E. coli [60]. More recently, metabolically engineered E. coli
strain capable of aerobically producing succinic acid through
the glyoxylate pathway and the oxidative branch of the TCA
cycle was developed by inactivating the succinate dehydrogenase (sdh), pyruvate oxidase (poxB), ptaack, aceBAK operon
repressor (iclR) and ptsG genes. The aerobic fed-batch fermentation of this strain resulted in the production of 58.3 g l1 succinic
acid in 59 h with the succinic acid yield of 0.85 mol mol1 glucose [19,20]. However, production of pyruvic (6.1 g l1 ) and
acetic (3.0 g l1 ) acids could not be avoided.
Although metabolically engineered strains of E. coli were
remarkable in their performance compared with the wild type
strain, they still need improvement. In particular, the specific
and volumetric succinic acid productivities are much lower than
those obtained with A. succiniciproducens and rumen bacteria
(Table 1). The low specific succinic acid productivity requires
higher cell density for efficient succinic acid production, which
reduces the succinic acid yield due to the conversion of much
carbon sources to biomass. Co-production of other acids is the
same problem as observed in other bacteria, which needs to be
solved for the efficient purification of succinic acid.
4. Recovery of succinic acid
A typical process for the production of a bioproduct like succinic acid by microbial fermentation consists of seed cultivation,
fermentation, product recovery, concentration and purification.
Considering that the downstream purification cost in the fermentation based process normally accounts for more than 60% of the
total production cost [61], it is crucial to develop an economical
purification process of succinic acid from fermentation broth. In
the case of succinic acid purification, separation of byproducts
including acetic, formic, lactic and pyruvic acids is the most
crucial.
Several methods for the purification of succinic acid, including electrodialysis, acidification and extraction have been developed. Electrodialysis is a widely used separation process, in
which ionized compounds are separated from non-ionized compounds by ion exchange membrane, in wastewater treatment,

pharmaceutical and food processing industries [62,63]. Succinic

acid normally exists in the form of ionized-succinate salt in a
fermentation broth, while others including carbohydrates, proteins and amino acids are mostly non-ionized. Most specialty
and commodity applications of succinic acid require the free
acid form rather than the salt form. Therefore, the succinic acid
purification process composed of conventional electrodialysis
followed by water-splitting electrodialysis membrane stacks,
which removes most of the salt cation and produces highly pure
acid stream, was developed. In order to remove the residual
cationic, anionic and amino acids, cation and anion exchange
resins were integrated into the above process as the final purification step [55]. Although this process increased the concentration
of succinic acid from 51.5% to 79.6% (w/w) and completely
removed proteins and salts, the concentration of acetic acid
increased from 13.2% to 19.9%.
Another purification process employing precipitation of succinate salts was developed [56]. Succinate in the fermentation
broth is precipitated as calcium succinate by adding calcium
dihydroxide, which can neutralize the fermentation broth at the
same time. Calcium succinate is recovered by filtration, and converted to succinic acid by adding sulfuric acid. Succinic acid is
recovered by filtration, and further purified by acidic and basic
ion exchangers. This process dramatically improved the purity
of succinic acid from 44.5% in the fermentation broth to 94.2%
(w/w) after the purification. Unlike the succinic acid recovery
process based on the electrodialysis, it could not completely
remove proteins mainly due to the saturation of the ion exchange
sites with the succinate anion. The above two processes are rather
complex, which results in relatively high purification costs.
The reactive extraction of succinic acid with amine-based
extractant, employing hydrophobic tertiary amines, has been
considered as an effective and economical purification method in
recent years because the process is operated at normal temperature and pressure [64,65]. This process is based on reversible
reaction between the extractant and the extracted carboxylic
acid. The selective separation of specific acid from fermentation broth containing mixed acids can be achieved based on
the pKa values of the acids and operating pH [61,6669]. The
use of tri-n-octylamine as an extractant resulted in the recovery
of succinic acid from the binary mixture of succinic acid and
acetic acid with high selectivity and high extraction efficiency
[70]. More recently, the integrated succinic acid recovery process composed of reactive extraction, vacuum distillation and
crystallization was developed. It allowed purification of succinic
acid with the purity of 99.76% (w/w) and the yield of 73.09%
(w/w) from the actual fermentation broth of M. succiniciproducens. Furthermore, no acetic acid was detected after vacuum
distillation. This process is much simpler and more cost-effective
than those mentioned above.
5. Conclusions and future prospects
In this paper, we reviewed processes for succinic acid production from renewable resources by bacterial fermentation. The
best candidates for succinic acid production including A. succinogenes, M. succiniciproducens and A. succiniciproducens use

H. Song, S.Y. Lee / Enzyme and Microbial Technology 39 (2006) 352361

PEP carboxylation pathway to form succinic acid. The enzymes

that catalyze the reactions competing with succinic acid pathway, such as pyruvate kinase, phosphotransacetylase/acetate
kinase, lactate dehydrogenase, pyruvate:formate lyase and alcohol dehydrogenase, are responsible for the formation of byproducts, which increase the cost of purification and decrease the
yield of succinic acid. Although a few variant strains capable
of producing less byproducts were obtained by mutagenesis,
the formation of byproducts still persisted. This problem can be
solved by rational metabolic engineering based on the systematic
genome-wide analysis. Thanks to the availability of the complete genome sequence of M. succiniciproducens, it is possible
to carry out genome-scale metabolic flux analysis, and genomewide transcriptome and proteome analysis. This will allow us to
develop metabolic engineering strategies for the enhanced production of succinic acid by considering the followings. In silico
metabolic flux analysis will allow us to determine intracellular
flux distribution under various genetic and environmental perturbations. The genetic and environmental conditions that lead to
higher flux toward succinic acid with reduced fluxes to byproducts will be selected and verified experimentally. During this
process, the results of transcriptome and proteome analyses can
be incorporated to fine-tune the constraints. Engineering targets
can be selected directly from transcriptome and proteome analysis as well. Those genes and proteins found to be interesting from
the transcriptome and proteome analyses can be manipulated to
enhance the metabolic performance.
Ultimately, metabolic engineering of a succinic acid producer
should allow production of succinic acid to a high concentration
with high productivity and high yield without byproducts formation. Of course, this strain development process needs to be integrated with the fermentation process development which also
considers the best available carbon source, optimal cell mass,
and other culture conditions. Furthermore, with the advances in
the development of an efficient downstream process, such as the
amine-based reactive extraction for the purification of succinic
acid, the purification cost can be considerably reduced. Taken
together, the future of fermentative production of succinic acid is
bright considering the increasingly acceptable raw material cost,
large potential market size, the additional advantage of carbon
dioxide fixation, and advances in the development of strategies
for strain improvement, fermentation and purification.
Acknowledgements
Our work described in this paper was supported by the
Genome-based Integrated Bioprocess Project of the Ministry
of Science and Technology. Further supports by the LG Chem
Chair Professorship, IBM-SUR program, Microsoft, BK21 program, and by the KOSEF through the Center for Ultramicrochemical Process Systems are appreciated.
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