Bioprocess Biosyst Eng (2011) 34:779–787
DOI 10.1007/s00449-011-0526-y
ORIGINAL PAPER
New reactive extraction systems for separation of bio-succinic acid
Tanja Kurzrock • Dirk Weuster-Botz
Received: 17 December 2010 / Accepted: 10 February 2011 / Published online: 25 February 2011
Ó Springer-Verlag 2011
Abstract Biotechnologically produced succinic acid has
the potential to displace maleic acid and its uses and to
become an important feedstock for the chemical industry. In
addition to optimized production strains and fermentation
processes, an efficient separation of succinic acid from the
aqueous fermentation broth is indispensable to compete
with the current petrochemical production processes. In this
context, high molecular weight amines are known to be
effective extractants for organic acids. For this reason, as a
first step of isolation and purification, the reactive extraction
of succinic acid was studied by mixing aqueous succinic
acid solutions with 448 different amine–solvent mixtures as
extraction agents (mixer-settler studies). The extraction
agents consist either of one amine and one solvent (208
reactive extraction systems) or two amines and two solvents
(240 reactive extraction systems). Maximum extraction
yields of succinic acid from an aqueous solution with
423 mM succinic acid at pH 2.0 were obtained with more
than 95% yield with trihexylamine solved in 1-octanol or
with dihexylamine and diisooctylamine solved in 1-octanol
and 1-hexanol. Applying these optimized reactive extrac-
tion systems with Escherichia coli fermentation broth
resulted in extraction yields of 78–85% due to the increased
ionic strength of the fermentation supernatant and the co-
extraction of other organic acids (e.g., lactic acid and acetic
acid), which represent typical fermentation byproducts.
Keywords Succinic acid
Reactive extraction

Fermentation broth
Co-extraction
Ionic strength
T. Kurzrock
D. Weuster-Botz (&)
Institute of Biochemical Engineering,
Technische Universität München,
Boltzmannstr. 15, 85748 Garching, Germany
e-mail: d.weuster-botz@lrz.tum.de
Introduction
Succinic acid is applied in the food and pharmaceutical
industry and other chemical industry sectors and is used as
feedstock for the syntheses of biodegradable polyester
resins. Nowadays, succinic acid is mainly produced pet-
rochemically by hydrogenation of maleic anhydride and
subsequent hydration or direct hydrogenation of maleic
acid [1]. The current annual market size is about
20,000–30,000 tonnes (*400 million US $) [2].
The petrochemical production uses heavy metal cata-
lysts in organic solvents at high temperature and pressure.
Alternatively succinic acid can be produced by microor-
ganisms from renewable resources at ambient temperature
and pressure. If bio-succinic acid could be produced
competitively, it will have the potential to displace maleic
acid and its uses. The estimated market potential is up to
270,000 tonnes and 15 billion US $ per year [2–4].
In the recent years, bio-succinic acid yields of up to
1.78 mol per mol glucose and final succinic acid concen-
trations of up to 146 g L-1 have been reported with opti-
mized fermentation processes [5, 6]. The cost-effective
separation and isolation of succinic acid from the aqueous
fermentation broth remains a so far not finally solved prob-
lem for industrial production of bio-succinic acid. A variety
of different downstream processing methods for bio-succinic
acid, like precipitation with calcium hydroxide or ammo-
nium, electrodialysis, ion exchange and adsorption, crys-
tallisation and liquid–liquid extraction have been reviewed
recently [7]. Most of these separation technologies showed
low selectivities and/or low yields with the exception of
reactive extraction with high molecular weight amines
solved in non-water-miscible organic solvents [8–14].
The objective of this study is to broaden the number of
reactive extraction systems which may be applicable for
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the extraction of succinic acid from aqueous solutions and
to identify new improved reactive extraction systems. The
application of primary, secondary and tertiary amines as
well as alkylamines and arylamines will be studied with
simple mixer-settler configurations. All of the amines will
be applied as solutions in organic solvents due to their
physical properties such as high viscosity and corrosivity.
It is known that the solvents will have a high impact on the
stoichiometry of the formed amine–acid complexes. In this
context, solvents can be classified into active solvents
(polar, proton-donating), which can stabilize the amine–
acid complexes, and inert solvents, which are poorly sol-
vating media [15–17]. To identify the reactive extraction
systems with the highest yields and selectivities for suc-
cinic acid, simple reactive extraction systems (one amine
and one solvent) and complex reactive extraction systems
(two amines and two solvents) will be designed and stud-
ied. Reactive extraction systems already described in the
literature will be used as reference. The most efficient
reactive extraction systems will be applied for succinic acid
extraction from fermentation broth to identify the effects of
ions, ionic strength and other organic acids formed in the
fermentation as byproducts on the succinic acid extraction
yield.
Materials and methods
Materials
Octylamine was used as primary amine extractant, dibu-
tylamine, dihexylamine, dioctylamine, diisooctylamine
and diphenylamine as secondary amine extractants and
tripropylamin, tributylamin, tripentylamin, trihexylamin,
trioctylamin, triisooctylamin, tridodecylamin, methyldioc-
tylamine, methyldidecylamine and methyldodecylamine as
tertiary amine extractants. All amines were obtained from
Merck (Darmstadt, Germany), apart from tripentylamine
and trihexylamine which were purchased from Sigma
(Hamburg, Germany). The amines were always dissolved
in different active and inert solvents. As polar and proton-
donating solvents 1-butanol, 1-hexanol, 1-octanol, 2-octa-
nol, 1-nonanol, 1-decanol and 1-dodecanol were used.
Applied polar aprotic solvents were methylisobtylketone
(MIBK) and diisobutylketone (DIBK). As non-polar sol-
vents n-pentane, n-hexane, n-heptane, n-octane, n-nonane,
n-decane, n-dodecane, kerosene and toluene were applied.
Most of them were purchased from Merck. 1-decanol,
1-dodecanol and kerosene were obtained from Sigma and
MIBK and n-heptane from Roth (Karlsruhe, Germany).
The amines were used without further purification with an
initial amine concentration of 1.0 or 1.5 mol kg-1 based on
the solvent (first screening). Amine concentrations between
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Bioprocess Biosyst Eng (2011) 34:779–787
0.033 and 1.0 mol kg-1 based on the solvent mixture were
applied in the second screening.
Succinic acid and lactic acid were obtained from Roth,
whereas acetic acid was purchased from Merck. NaCl
(Roth) was used for adjustment of ionic strength of the acid
solutions. The inorganic salts NH4Cl (Roth), NaCl (Roth),
MgCl2 (Roth), MgSO4 (Merck), CaCl2 (Merck) and
KH2PO4 (Roth) were dissolved in aqueous succinic acid
solutions in different concentrations to achieve a constant
ionic strength of 1.0 mol L-1 in the particular solution.
Adjustment of pH was always carried out with NaOH
(Roth) or HCl (Roth).
Methods
Succinic acid solutions
For the extraction experiments from fermentation broth,
succinic acid was dissolved in fermentation supernatant
(without cells and proteins) of a cultivation of E. coli K12
DSM 428. The cultivation medium was a defined salt
medium [18] with NH4Cl 3.7 mM, (NH4)2SO4 15.1 mM,
KH2PO4 23.9 mM, K2HPO4 8.6 mM, NaH2PO4 12.5 mM,
MgSO4 4.2 mM, CaCl2
2H2O 0.07 mM, ZnSO4
7H2O
0.002 mM, CuCl2
2H2O 0.0015 mM, MnSO4
1H2O 0.015
mM, CoCl2
6H2O 0.007 mM, H3BO3 0.002 mM, AlCl3

6H2O 0.01 mM, Na2MoO4
2H2O 0.002 mM and FeSO4
0.04 mM. Glucose was applied as carbon source in dif-
ferent concentrations according to the particular process
phase. After batch cultivation for 4 h with an initial glu-
cose concentration of 2 g L-1, the substrate limited expo-
nential fed-batch cultivation phase was started [19]. This
phase was divided into two feeding phases: Feeding phase
1 with a glucose concentration of 100 g L-1 to control the
growth rate to 0.5 h-1 (4 h) and feeding phase 2 with
300 g L-1 glucose to assure a growth rate of 0.1 h-1
(16 h). Fermentation was performed at 37 °C and pH was
controlled to 7.0 with the addition of 25% NH4OH.
The final byproduct concentrations after the fermenta-
tion were 0.3 g L-1 pyruvate, 0.114 g L-1 acetate and
0.5 g L-1 fumarate. No glucose remained in the fermen-
tation broth, because of the substrate limited conditions.
The cells were separated by a centrifugation step (30 min,
4,500 rpm, 3,270g). After addition of succinic acid, pre-
cipitated proteins were removed by a second centrifugation
(30 min, 4,500 rpm, 3,270g).
For extraction of succinic acid from an aqueous solu-
tion, succinic acid was dissolved in distilled water.
The pH of the aqueous succinic acid solution and the
fermentation broth was adjusted with NaOH or HCl. A low
pH of about 2–3 was preferred for succinic acid extrac-
tions, because only the undissociated acid molecules are
extractable. Anyhow, some experiments had to be carried
Bioprocess Biosyst Eng (2011) 34:779–787
out with higher pH up to 4.5 to assure the dissolution of all
additives such as NaCl or other salts.
Extraction
For extraction studies, 500 lL of the succinic acid solution
(aqueous succinic acid solution or fermentation broth) were
mixed with 500 lL of the respective simple or complex
reactive extraction system. The mixing was carried out in
1.5 mL glass vials with a mixer mill MM200 from Retsch
(Haan, Germany) at 30 Hz for 10 min to assure thermo-
dynamic equilibrium conditions. Afterwards, the phases
were separated by centrifugation at 4,500 rpm (3,270g) for
5 min.
Analytics
The acid concentrations in the aqueous phase were mea-
sured by a HPLC 1100 from Agilent (Waldbronn, Ger-
many) with an Aminex HPX-87H column from Biorad
(München, Germany) using 0.5 mM H2SO4 (from Merck)
as mobile phase and a flow rate of 0.6 mL min-1. The
concentrations of succinic acid in the organic phase were
calculated by applying mass balances.
Screening
The first screening of simple reactive extraction systems
was carried out with an aqueous 0.5 M succinic acid
solution with pH 2.3. The amines were applied with a
concentration of 1.0 mol kg-1 based on the particular
solvent. Altogether, 208 simple reactive extraction systems
were compared.
16 amines and 16 solvents were applied in varying
amounts to form the complex reactive extraction systems
under study (combination of two solvents and two amines).
This would have resulted in a high number of
3,317,760,000 different reactive extraction systems in total.
Therefore a stochastic search strategy was chosen to reduce
the experimental effort: Genetic Algorithm for Multiob-
jective Experimental Design (GAME.opt) [20]. GAME.opt
generates binary bitstrings of the scalar quantities. These
bitstrings encode the composition of the particular reactive
extraction system. The first set of parallel experiments
(generation) is designed randomly. All reactive extraction
systems of one generation are experimentally validated in
parallel and the results are valuated on the basis of the
objectives (maximization of extraction yield, minimization
of amine concentration). The best reactive extraction sys-
tems are selected with highest probability; two bitstrings
are randomly coupled and varied by recombinant cross-
overs. Premature convergence to local optima is prevented
by applying randomly generated point mutations with
781
reduced probability. That way, a new generation of reactive
extraction systems is generated, which has to be evaluated
with further parallel experiments. This procedure is repe-
ated as long as optima are identified or the experimental
effort becomes too high.
To tune into different objectives, a strict Pareto principle
is used. The reactive extraction systems with the highest
yields of one generation are clustered and copied in a so-
called external population, if they show improved results
with respect to the objectives compared to the already
copied reactive extraction systems from the previous
generations.
The parameter settings of the GAME.opt software
applied for the second screening are listed in Table 1.
Effects of ions
To study the effects of salts and ions on reactive extraction
from fermentation broth, succinic acid was dissolved in
distilled water (0.423 mol L-1, pH 4.5). The ionic strength
of the solution was varied between 0.5 and 3.0 mol L-1 by
addition of NaCl.
Experiments to study the effect of different salts were
carried out with 0.423 M succinic acid solutions (pH of
2.5) containing NH4Cl, NaCl, MgCl2, MgSO4, CaCl2 or
KH2PO4. The salt concentrations varied whilst the ionic
strength of the solutions was constant 1.0 mol L-1.
Co-extraction experiments
Aqueous mixtures of 0.423 mol L-1 succinic acid,
0.083 mol L-1 acetic acid and 0.022 mol L-1 lactic acid
were used for the co-extraction experiments. The pH was
varied between 2.0 and 8.0 by adding NaOH or HCl.
Results and discussion
Screening of simple reactive extraction systems
The nature of the amine and of the solvent has a high
impact on the extraction efficiency (yields), as well as the
stability and the stoichiometry of the generated amine–acid
complexes. In Fig. 1, the final yields of all the tested
simple reactive extraction systems are compared. The yield
was not determined if aggregate formation was observed
(marked with grey), because back-extraction of succinic
acid from aggregates will not be possible. 94 from 208
simple reactive extraction systems resulted in aggregate
formation.
Regarding the applied solvents, two different factors are
influencing the extraction yield of succinic acid from
aqueous solutions: the polarity of the solvent and the
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Table 1 Parameter settings of

Seven variables Amine 1 (16 possible amines, 4 bits)
the GAME.opt software (second
screening) Concentration of amine 1 (0–500 mM, 4 bits)
Amine 2 (16 possible amines, 4 bits)
Concentration of amine 2 (0–500 mM, 4 bits)
Solvent 1 (16 possible solvents, 4 bits)
Faction of solvent 1 (0–100%, 4 bits)
Solvent 2 (16 possible solvents, 4 bits)
Fraction of solvent 2 (was calculated)
Possible 3,317,760,000
combinations
Two objectives Maximization of the extraction yield
Minimization of the amine concentration
Number of 30 individuals per generation (three parallel experiments per individual)
experiments
Crossover points 2
Mutation rate 1%
N extern 15
N cluster 15
Applied amines Octylamine, dibutylamine, dihexylamine, dioctylamine, diisooctylamine,
tripropylamine, tributylamine, tripentylamine, trihexylamine, trioctylamine,
triisooctylamine, tridodecylamine, methyldioctylamine, dimethyldodecylamine,
methyldidecylamine and diphenylamine
Applied solvents 1-Octanol, 2-octanol, 1-hexanol, 1-nonanol, 1-decanol, 1-dodecanol, kerosin, toluol,
n-pentane, n-hexane, n-heptane, n-nonane, n-decane, n-dodecane, MIBK and DIBK
Extraction 0.847 mol L-1 succinic acid in H2O; pH 4.0
conditions

presence of additional functional groups. Polar solvents

Fig. 1 Extraction yields and aggregate formation (grey fields) of
simple reactive extraction systems. Extraction of succinic acid from a
0.5 M aqueous solution (pH 2.3) was carried out with an amine
concentration of 1.0 mol kg-1 solvent in a mixer mill. Bold Reactive
extraction systems described in literature. Framed Most promising
simple reactive extraction system (T1) in this screening
like alcohols or ketones show higher extraction yields of
succinic acid from aqueous solutions than non-polar sol-
vents like alkanes. The higher the polarity, the higher is the
ability of the solvent to solvate the generated amine–acid
complexes and the higher are the achievable yields. This
effect is also observable with respect to the tested alcohols.
The polarity decreases with an increasing chain length of
the alcohols. Therefore, higher extraction yields are pos-
sible with short-chain alcohols. Furthermore, solvents with
additional functional groups (alcohols) show much higher
yields than expected from their polarity [15, 16, 21–23].
These solvents are able to release a proton. Thereby, the
amine–acid complexes are stabilized and high yields can be
achieved.
Comparing the different groups of applied amines, two
effects were observed. A combination of amines with
alcohols shows the following order of extraction yields,
which was already described for amines in polar solvents
[24, 25]:
Primary\Secondary [ Tertiary:
On the one hand, increasing extraction yields of succinic
acid from aqueous solutions are observed with increasing
alkylation and therewith increasing basicity of the amines.

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On the other hand, lower extraction yields occur with long-
chain tertiary amines because of steric hindrance due to the
three long side chains. Therefore, the highest extraction
yields of 95–96% are measured with secondary long-chain
amines, such as diisooctylamine (C8), or shorter tertiary
amines, such as trihexylamine (C6).
The order of extraction yields of succinic acid from
aqueous solutions differs in non-polar solvents. Secondary
amines are also more efficient than primary ones, but ter-
tiary amines show very low extraction yields for succinic
acid. According to this, the order of extraction yields of
reactive extraction systems with non-polar solvents (like
alkanes) follows:
Primary  Tertiary\Secondary:
Primary amines are less alkaline than secondary and
tertiary amines and achieve only low extraction yields. The
steric hindrance of tertiary amines and the low solvating
ability of alkanes lead also to strongly reduced extraction
yields. Therefore, only dibutylamine or short-chain tertiary
amines (BC5) are able to extract succinic acid, even though
with low yields.
As described generally in the literature [26], arylamines
achieve lower extraction yields than alkylamines, due to
their low basicity. Comparing the extraction yields of
diphenylamine (arylamine) with dihexylamine (alkyla-
mine), diphenylamine is less adequate for succinic acid
extraction, although both amines are secondary amines
with two C6 side chains or two C6 rings.
About 45% of the 208 reviewed simple reactive
extraction systems showed aggregate formation. First of
all, combinations of non-polar alkanes with long-chain
secondary and tertiary amines tend to aggregation. This
correlates to the data by Tamada et al. [16]. Reason for this
is the low solvation ability of alkanes and the low solubility
of amine–acid complexes containing long-chain secondary
or tertiary amines, due to steric hindrance. Aggregation has
also occurred in combinations with long-chain alcohols
(CC8). Independent of the applied solvent, secondary and
tertiary amines with more than eight carbon atoms tend to
aggregation.
Moreover, the reactive extraction systems, which show
the highest yields, contain partially water-miscible short-
chain alcohols (1-butanol or 1-hexanol). First of all,
1-butanol is water miscible up to a concentration of
1.04 mol L-1. The water miscibility of 1-hexanol is about
0.06 mol L-1. However, they are much more miscible in
comparison to the other applied solvents with a maximum
miscibility in water of 8.6 mmol L-1.
Ranking of reactive extraction systems with respect to
yields, no aggregate formation and low water solubility of
maximal 2.7–8.6 mM results in the following list of
783
systems which are the most applicable for the succinic acid
extraction:
1. diisooctylamine ? 1-octanol (yield: 92.3%)
2. trihexylamine ? 1-octanol (yield: 91.5%)
3. dioctylamine ? 1-octanol (yield: 90.3%)
4. trihexylamine ? 2-octanol (yield: 89.9%)
5. trioctylamine ? 1-octanol (yield: 86.5%).
The reactive extraction system with trioctylamine and
1-octanol is the most efficient reactive extraction system
for succinic acid described in the literature so far [10, 12].
According to this, four reactive extraction systems with
higher yields were identified. Altogether, the reactive
extraction system with trihexylamine and 1-octanol (in the
following called ‘T1’) seems to be more adequate than the
reactive extraction system with diisooctylamine, because
no aggregate formation has occurred in all tested systems,
independent on the polarity of the applied solvent.
Screening of complex reactive extraction systems
To minimize the aggregate formation and the water mis-
cibility of some reactive extraction systems, another
screening was carried out with the software GAME.opt. At
this, a combination of two amines in two organic solvents
should minimize aggregation by an improved solvation of
the amine–acid complexes with the application of a polar
solvent. At the same time, a reduction of the polar water-
miscible solvent (short-chain alcohol) is achieved by a
combination with a less water-miscible solvent. To assure
enough open space for the optimization, the extraction
conditions were modified. The initial succinic acid con-
centration was increased to 0.847 mol L-1 and the pH to
4.0. As a consequence, the maximum batch extraction
yields are lower, due to the strong influence of the pH on
the extraction.
180 batch extractions with varying combination of
amines and solvents were performed within six sets of
parallel experiments (generations). In Fig. 2, the Pareto-
fronts of the first and the sixth generation of reactive
extraction systems are compared. The highest batch
extraction yields were achieved at the highest initial amine
concentrations. The average yields of the sixth generation
are about 5% higher than the yields of the first generation.
In the following, the complex reactive extraction system
29 of the sixth generation (in the following called ‘29/6’) is
used for further experiments in comparison to the simple
reactive extraction system T1 of the first screening, because
it has shown an effective extraction of succinic acid under
these (and many other conditions, data not shown) without
aggregate formation. To assure comparable conditions to
reactive system T1, the overall amine concentration of
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Fig. 3 Batch extraction yields of succinic acid from an aqueous

solution (grey bar) or an E. coli fermentation supernatant (white bar)
Fig. 2 Best results of the first (grey triangles) and the sixth (black with the reactive extraction systems T1 and 29/6 with amine
rhombus) generation of the screening of complex reactive extraction concentrations of 1.0 or 1.5 mol kg-1 solvent. Succinic acid concen-
systems with a stochastic search strategy (GAME.opt). Extraction of a tration in the aqueous model solution and the fermentation superna-
0.847 M aqueous succinic acid solution (pH 4.0) was carried out with tant was set to 0.423 mol L-1 at pH 2.0
an amine concentration of 1.0 mol kg-1 solvent in a mixer mill.
White circle is the complex reactive extraction system 29/6 (system amine concentrations of the reactive extraction systems T1
29 of the sixth generation) which is used for all following experiments and 29/6.
in comparison to reactive extraction system T1
The succinic acid extraction yields with fermentation
Table 2 Composition of reactive extraction system T1 and 29/6
supernatant are reduced to 84.4–85.1% compared to a pure
(1.5 mol amine kg-1 solvent) aqueous solution (91.4–95.5%) with reactive system 29/6.
With the simple reactive extraction system T1, yields are
Reactive extraction Components Volume Concentration
system (mL L-1) (mol L-1) solvent still lower. Instead of 89.2–94.7% (aqueous solution) only
78.3–82.9% of the succinic acid present in the fermentation
29/6 Diisooctylamine 101.66 0.34 supernatant was extracted. Reasons for the decreased yields
Dihexylamine 79.04 0.34 may be other ions and salts as well as other organic acids
1-Octanol 162.25 1.03 present in the fermentation supernatant.
1-Hexanol 657.06 5.27
T1 Trihexylamine 217.19 0.64 Effects of ionic strength
1-Octanol 783.54 4.99
It was shown that the extraction yields with reactive
extraction system T1 as well as with reactive extraction
system 29/6 was also set to 1.0 or 1.5 mol kg-1 based on system 29/6 decrease with increasing ionic strength of the
the solvent mixture. succinic acid solution (Fig. 4).
The detailed composition of the best simple reactive An ionic strength of above 2.0 mol L-1 makes the
extraction system T1 and of the chosen complex reactive extraction of succinic acid completely impossible. An ionic
extraction system 29/6 is specified in Table 2 for an overall strength of 0.6 mol L-1 represents the ionic strength of the
amine concentration of 1.5 mol kg-1 based on the solvent applied E. coli fermentation broth. The corresponding
or solvent mixture. reduction in extraction yield of about 7% is the same as
compared to the data of the extraction of succinic acid from
Reactive extraction of succinic acid from fermentation real fermentation broth, shown in Fig. 3. The maximum
broth batch extraction yields are lower than those shown in
Fig. 3, because of the higher pH of 4.5, which was nec-
Batch extraction of succinic acid from E. coli fermentation essary to assure the dissolution of the added NaCl.
supernatant was studied with the reactive extraction sys-
tems T1 and 29/6. The succinic acid concentration of the Effects of ions
fermentation supernatant was adjusted to 0.423 mol L-1
and a pH of 2.0. In Fig. 3, the extraction yields from water Batch extraction yields of succinic acid were measured in
and fermentation supernatant are shown for two different the presence of different salts like NH4Cl, NaCl, MgCl2,

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Fig. 4 Extraction yield as function of ionic strength (varied between
0.5 and 3.0 mol L-1) of an aqueous succinic acid solution
(0.423 mol L-1, pH 4.5). The extractions were carried out with the
reactive extraction systems T1 (white circles) and 29/6 (black
rhombus) with amine concentrations of 1.0 mol kg-1 solvent. The
extraction yield marked with (star) corresponds to the ionic strength
of the applied E. coli medium (*0.6 mol L-1)
MgSO4, CaCl2 or KH2PO4. The ionic strength of each
solution was kept constant at 1.0 mol L-1 with exception
of the reference conditions with \0.5 mol L-1. The pH
was set to pH 2.5 to assure the dissolution of succinic acid
and the particular salt. The type of added salt changes the
maximum extraction yields dramatically (Fig. 5).
NH4Cl, NaCl and MgCl2 show a high impact on suc-
cinic acid extraction, whereas the other salts are less
inhibiting. A clear correlation between the ion concentra-
tion and the observed yields was measured. Increasing ion
concentrations result in the decrease of the extraction
yields of succinic acid with both reactive extraction sys-
tems. This can be explained by the salt anions, which
compete with the carboxylic groups of succinic acid for the
protons [27]. The corresponding acids (HCl, H2SO3 or
H3PO4) were co-extracted with succinic acid, which leads
to a decrease in the extraction yields. Therefore, a
Fig. 5 Effects of NH4Cl, NaCl, MgCl2, MgSO4, CaCl2 and KH2PO4
on the extraction yields of succinic acid (0.423 mol L-1, pH 2.5) with
the reactive extraction systems T1 (white bar) and 29/6 (grey bar)
with amine concentrations of 1.0 mol kg-1 solvent. In addition, the
extraction yields under reference conditions (-) and the ion concen-
tration of the particular solution are indicted (black square)
785
minimized concentration of these components should be
one objective of fermentation process optimization if
reactive extraction is applied for downstream processing.
Reactive extraction of other organic acids
Main byproducts of succinic acid fermentations with pro-
duction strains of Corynebacterium glutamicum, E. coli,
Anaerobiospirillum succiniciproducens, Actinobacillus
succinogenes, Mannheimia succiniciproducens or Basfia
succiniciproducens are ethanol, pyruvate, acetate, formate,
propionate, malate and lactate [5, 6, 28–31]. Alcohols, such
as ethanol, show no influence on the reactive extraction of
succinic acid, whilst other organic acids can be co-
extracted according to their pKa and the pH in the aqueous
phase. The dissociation constants pKa of these organic
acids are listed in Table 3.
All organic acids, with the exception of pyruvic acid,
have a pKa in the same range as succinic acid. Pyruvic acid
has a lower pKa. Therefore, pyruvic acid formed as a
byproduct will not be extracted in significant amounts at
pH [ 3, whereas the byproduct formation of all other
organic acids discussed above has to be avoided. Due to the
fact that acetic and lactic acid are the predominantly
formed byproducts, mixtures of them with succinic acid
were studied in more detail making use of reactive
extraction systems T1 and 29/6 (Fig. 6).
As expected, the extraction yield of all organic acids
increases with decreasing pH because solely undissociated
acids can be extracted by secondary and tertiary amines.
All organic acids show dissociation constants around
pKa = 4–5. This explains why high extraction yields are
observed at pH below pKa.
At pH 5 or 6 acetic acid can be extracted with 10–15%
higher yields compared to the other two organic acids
under study, because acetic acid has a higher pKa compared
to the other organic acids. At pH 4 the extraction yields
correlate with the pKa of the acids. Lactic acid has a pKa of
3.86 and shows the lowest yield, acetic acid has a pKa of
4.76 and shows the highest yield and the yield of succinic
acid is in between the two other organic acids (pKa1 of
Table 3 Dissociation constants pKa of some organic acid
Acid pKa1 pKa2
Succinic acid 4.17 5.64
Acetic acid 4.76 –
Lactic acid 3.86 –
Formic acid 3.77 –
Malic acid 3.46 5.10
Propionic acid 4.87 –
Pyruvic acid 2.49 –
123
786
Fig. 6 Reactive extraction yields of succinic acid (black bar), acetic
acid (striped bar) and lactic acid (grey bar) from an aqueous solution
as function of pH. The applied acid concentrations were 0.423
mol L-1 succinic acid, 0.083 mol L-1 acetic acid and 0.022 mol L-1
lactic acid. Reactive extraction systems T1 and 29/6 were applied
with an amine concentration of 1.0 mol kg-1 solvent
succinic acid = 4.17). Low pH of 2–3 allows for a selec-
tive extraction of succinic acid due to its two carboxylic
groups. Nevertheless, these results show that organic acids
produced as byproducts in the fermentation will be co-
extracted in significant amounts. The suppression of
organic acids formation as byproducts during bio-succinic
acid fermentation should be another objective of strain and
fermentation process optimization if reactive extraction is
applied for downstream processing.
Conclusions
The new identified reactive extraction systems with trihe-
xylamine solved in 1-octanol or with dihexylamine and
diisooctylamine solved in 1-octanol and 1-hexanol result in
the best yields of succinic acid extraction from aqueous
solutions so far reported (up to 95.5% with a simple mixer-
settler). These high batch extraction yields can only be
achieved at a pH of the aqueous fermentation broth far
below the pKa1 of succinic acid (pH  4.17). The appli-
cation of reactive extraction in the bypass to a fermentation
process (process integrated product separation) will thus
demand for a microbial succinic acid production process at
very low pH or titrations of the fermentation broth. The
succinic acid producers presently under study (C. glutam-
icum, E. coli, Saccharomyces cerevisiae, A. succinogenes,
A. succiniciproducens, M. succiniciproducens, B. succi-
niciproducens) operate best at 6.0 \ pH \ 7.8 [5, 6, 28–34].
123
Bioprocess Biosyst Eng (2011) 34:779–787
Consequently, one focus in strain and fermentation process
optimization for succinic acid production should be the
development of low pH fermentation processes if fermen-
tation process integrated reactive extraction of succinic
acid is envisioned.
Another important issue is the high impact of ions and
other organic acids on succinic acid extraction yields,
which was shown in this study. Fermentation process
development for succinic acid production should thus focus
on minimization of ionic strength (salt additions) as well as
strain optimization to minimize byproduct formation,
which is a general strategy to achieve higher fermentation
yields.
Reactive extraction processes will need efficient back-
extraction to isolate the extracted succinic acid from the
organic phase. The development of an efficient back-
extraction process in combination with crystallization of
the isolated succinic acid is presently under study in our
labs. One important issue of these studies will be the
achievement of total recyclability of the organic phase with
the solved amines as often as possible to minimize the
downstream processing costs.
Acknowledgments This study was generously supported by the
Deutsche Bundesstiftung Umwelt (DBU), particularly by a DBU
Ph.D. grant for Tanja Kurzrock. The authors gratefully acknowledge
for the support of the TUM Graduate School.
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