ARTICLE

Production of Polymalic Acid and Malic

Acid by Aureobasidium pullulans
Fermentation and Acid Hydrolysis
Xiang Zou,1,2 Yipin Zhou,2,3 Shang-Tian Yang2
1

College of Pharmaceutical Sciences, Southwest University, Chongqing, P.R. China

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State
University, 140 West 19th Ave., Columbus, Ohio 43210; telephone: 1-614-292-6611;
fax: 614-293-3769; e-mail: yang.15@osu.edu
3
Boprocessing Innovative Company, 4734 Bridle Path Ct., Dublin, Ohio
2

Introduction
ABSTRACT: Malic acid is a dicarboxylic acid widely used in
the food industry and also a potential C4 platform chemical
that can be produced from biomass. However, microbial
fermentation for direct malic acid production is limited by
low product yield, titer, and productivity due to end-product inhibition. In this work, a novel process for malic acid
production from polymalic acid (PMA) fermentation followed by acid hydrolysis was developed. First, a PMAproducing Aureobasidium pullulans strain ZX-10 was
screened and isolated. This microbe produced PMA as
the major fermentation product at a high-titer equivalent
to 87.6 g/L of malic acid and high-productivity of 0.61 g/L h
in free-cell fermentation in a stirred-tank bioreactor. Fedbatch fermentations with cells immobilized in a fibrous-bed
bioreactor (FBB) achieved the highest product titer of
144.2 g/L and productivity of 0.74 g/L h. The fermentation
produced PMA was purified by adsorption with IRA-900
anion-exchange resins, achieving a
100% purity and a high
recovery rate of 84%. Pure malic acid was then produced
from PMA by hydrolysis with 2 M sulfuric acid at 858C,
which followed the first-order reaction kinetics. This process
provides an efficient and economical way for PMA and malic
acid production, and is promising for industrial application.
Biotechnol. Bioeng. 2013;110: 21052113.
2013 Wiley Periodicals, Inc.
KEYWORDS: Aureobasidium pullulans; polymalic acid;
malic acid; fermentation; acid hydrolysis

Correspondence to: S.-T. Yang

Contract grant sponsor: This work was supported in part by grants from the National
Key New Drug Creation and Development Program
Contract grant number: 2010ZX09401-306-4-4
Contract grant sponsor: Chongqing Science and Technology Innovation Ability
Construction Program
Contract grant number: CSTC 2009CB1010
Received 22 November 2012; Revision received 6 February 2013;
Accepted 11 February 2013
Accepted manuscript online 22 February 2013;
Article first published online 16 March 2013 in Wiley Online Library
(http://onlinelibrary.wiley.com/doi/10.1002/bit.24876/abstract)
DOI 10.1002/bit.24876

2013 Wiley Periodicals, Inc.

Malic acid (2-hydroxybutanedioic acid) is a C4 dicarboxylic

acid and an intermediate in the tricarboxylic acid (TCA)
cycle. As an acidulant and flavor enhancer, malic acid is
widely used in the food industry (Bressler et al., 2002;
Goldberg et al., 2006; Takata and Tosa, 1993; Takata et al.,
1980). Malic acid is also used in metal cleaning, textile
finishing, pharmaceuticals (Goldberg et al., 2006; Vrsalovic
Presecki et al., 2009) and the synthesis of biodegradable
polymer, polymalic acid (PMA) (Gross and Kalra, 2002;
Tsao et al., 1999). In addition, the US Department of Energy
has also identified malic acid as a potential building-block
chemical that can be mass produced from renewable
biomass and converted to various chemical products,
including polymeric resins (Werpy et al., 2004). The future
use of malic acid is predicted to be over 200,000 tons per year
(Sauer et al., 2008).
Currently, malic acid is mainly produced through either
the hydration of fumaric or maleic acid under hightemperature and pressure, yielding a racemic mixture, or the
biotransformation of fumarate using immobilized cells (e.g.,
Brevibacterium flavum) (Bressler et al., 2002; Chibata et al.,
1983; Takata et al., 1980) or fumarase (Stojkovic et al., 2011;
Vrsalovic Presecki et al., 2009; Zheng et al., 2009), producing
L-malic acid. However, these processes require petroleumderived maleic anhydride or fumaric acid, and relatively
expensive catalysts for the chemical or biotransformation
reaction, which limit their economical competitiveness.
Therefore, recent research efforts have focused on the
development of microbial fermentation using renewable
feedstocks for malic acid production. Many microorganisms
including various Aspergillus species (Battat et al., 1991;
West, 2011), Zygosaccharomyces rouxii (Taing and Taing,
2007), Schizophyllum commune (Kawagoe et al., 1997), and
engineered Saccharomyces cerevisiae and Escherichia coli
(Moon et al., 2008; Zelle et al., 2008; Zhang et al., 2011b) can
produce malic acid from glucose and other carbohydrates.

Biotechnology and Bioengineering, Vol. 110, No. 8, August, 2013

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Among them, A. flavus can produce malic acid at the highest

titer of 113 g/L with a high-yield of 0.94 g/g and good
productivity of 0.59 g/L h (Battat et al., 1991). However,
the intrinsic pathogenicity of this microbe has prevented
its industrial application (Hedayati et al., 2007). The
co-production of other organic acids, such as succinic
and acetic acids (Goldberg et al., 2006), also render the
downstream process of separating and purifying malic acid
relatively complex and expensive (Zhang et al., 2011b). On
the other hand, malic acid fermentation by genetically
engineered E. coli and S. cerevisiae is severely limited by
end-product inhibition, resulting in a low product titer of
<60 g/L (see Table I). This is because malic acid with a low
pKa of 3.4 can inhibit cell growth and hamper the
fermentation. Metabolic and evolutionary engineering offer
promising strategies to improve industrial organic acids
fermentation, but increasing product titer, yield, and productivity to economical levels requires dramatic improvements in acid tolerance of the microorganisms, which
remains a daunting challenge for malic acid fermentation
(Cao et al., 2011). To date, no fermentation process has been
developed for industrial production of malic acid.
In this study, Aureobasidium pullulans capable of
producing large amounts of PMA from glucose was isolated
and used for malic acid production. A. pullulans, a
cosmopolitan yeast-like fungus, can produce various
bioproducts including pullulan, PMA, xylanase, heavy oil,
and lipase (Liu et al., 2008; Manitchotpisit et al., 2011, 2012;
Wu et al., 2012). Some strains of A. pullulans synthesized
PMA from the only monomer L-malic acid and secreted into
the fermentation broth (Cao et al., 2012a, 2012b; Leathers
and Manitchotpisit, 2012; Liu and Steinbuchel, 1997;
Manitchotpisit et al., 2012; Zhang et al., 2011a). Unlike
malic acid, PMA is not toxic to cells and can be easily

Table I.

separated from the fermentation broth by simple ethanol

precipitation (Holler, 2010). However, the co-production of
pullulan, organic acids, and color compounds by this
organism not only would lower the PMA yield but also
render down-stream processing more complicated and
challenging (Singh and Saini, 2008). In this work, we
successfully isolated a strain A. pullulans ZX-10 that
produced PMA at a high yield and purity without any
pigment, and developed a fed-batch fermentation process
for PMA production with a high product titer and
productivity. Separation and purification of the fermentation-produced PMA by adsorption with IRA-900 anionexchange resins, and acid hydrolysis of PMA to malic acid
were also investigated and are discussed in this article.

Materials and Methods

Cultures and Media
A. pullulans NRRL 12974, 12996, Y2311, 58012, and Y23111 obtained from the NRRL Culture Collection (Peoria, IL)
were used as the starting strains to screen for the highproducer of PMA. The strain NRRL Y2311-1 with high
PMA production and forming light yellow or creamy
colonies on potato dextrose agar (PDA) plates were
selected and further purified by serial subculturing and
isolation on PDA plates to obtain the strain ZX-10, which
formed white or colorless colonies. This strain was
maintained on PDA slants and stored in the refrigerator.
Unless otherwise stated, the fermentation medium contained glucose (60150 g/L), NH4NO3 (2 g/L), KH2PO4
(0.1 g/L), MgSO4  7H2O (0.1 g/L), KCl (0.5 g/L), ZnSO4
(0.1 g/L), and CaCO3 (30 g/L), which was used to maintain

Comparison of PMA and malic acid production by different strains in various fermentation processes.

Microorganism

Bioreactor

Natural malic acid producers

Aspergillus flavus ATCC13697
Aspergillus niger ATCC9142
Zygosaccharomyces rouxii
Monascus araneosus ST91
Schizophyllum commune

16 L fermentor
Shake flask
Test tube
Shake flask
Bubble column
fermentor
Aureobasidium pullulans ZD-3d 10 L fermentor
A. pullulans CBS591.75
1 L fermentor
A. pullulans ipe-1
7.5 L fermentor
A. pullulans ZX-10
5 L fermentor

Engineered strains
Escherichia coli WGS-10
Escherichia coli XZ658
Saccharomyces cerevisiae

Operating
mode

Time PMAa Malic acid Productivitya

(h) (g/L)
(g/L)
(g/L h)
Yielda (g/g)

Batch
Batch
Batch
Batch
Batch

190
192
140
120
112

162
216

FBB

Batch
Batch
Repeated-batch
Batch
Fed-batch
Fed-batch

96
140
192

57.2
13.9
63.2
41.2
76.2
123.7

5 L fermentor
3 L fermentor
Shake flask

Batch
Batch
Batch

12
72
310

113
17
75
28
18

0.59
0.1
0.52
0.23
0.16

0.94
0.80
0.40
0.37
0.36

Battat et al. (1991)

West (2011)
Taing and Taing (2007)
Lumyong and Tomita (1993)
Kawagoe et al. (1997)

65.7
16.0
72.6
47.3
87.6
142.2

0.35
0.06
1.15
0.49
0.61
0.74

0.47
0.15
0.25a
0.47
0.49
0.55

Zhang et al. (2011a)

Liu and Steinbuchel (1997)
Cao et al. (2012a)
This study

0.74
0.47
0.19

0.42
1.06
0.31

Moon et al. (2008)

Zhang et al. (2011b)
Zelle et al. (2008)

9.25
34
59

, None or not reported; a, calculated from data in this literature.

PMA yield and productivity are based on the malic acid that can be released from PMA after hydrolysis.

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Refs.

the medium pH around 6 during fermentation. The seed

culture was grown in a 250 mL shake-flask containing 50 mL
of liquid medium and incubated at 258C on a rotary shaker
(220 rpm) for 2 days.

Fermentation
Batch fermentation kinetics was first studied in 250 mL
shake flasks each containing 50 mL of the fermentation
medium with 60150 g/L of glucose to study the effect of
glucose concentration on PMA production. Batch fermentation kinetics was then studied in a 5 L stirred tank
fermentor (BioFlo II, NBS) containing 3 L of the medium
with glucose as the substrate. Unless otherwise noted, the
fermentor was inoculated with 300 mL of a culture grown in
a shake flask for 48 h, and operated at 258C with agitation at
500800 rpm and aeration at 1 vvm. For fed-batch
fermentation, the fermentation medium containing concentrated glucose of
600 g/L was fed into the fermentor
when the residual glucose was below
50 g/L. Throughout
the fermentation, broth samples were taken periodically for
analysis of residual glucose and metabolic products (PMA,
acetic acid, and succinic acid). Fed-batch fermentation was
also studied with cells immobilized in a 0.5 L fibrous-bed
bioreactor (FBB) connected to the 5-liter stirred-tank
fermentor with liquid recirculation at
100 mL/min.
Detailed description of the FBB construction can be found
elsewhere (Suwannakham and Yang, 2005; Yang and Shu,
1996). Other conditions were the same as those in the freecell fermentation.

378C, and redissolved in distilled water. The PMA was then

hydrolyzed to malic acid and analyzed with HPLC for purity
and yield.
The purified PMA was hydrolyzed with H2SO4 at various
concentrations (1, 2, and 3 M) at 858C in sealed centrifuge
tubes to study the PMA hydrolysis kinetics. The concentration of released malic acid was analyzed with HPLC.

Analytical Methods
The concentration of glucose was measured by using a
glucose analyzer (YSI2700 analyzer, Yellow Spring, OH). For
PMA, the fermentation broth was centrifuged, and the
supernatant was mixed with an equal volume of 2 M H2SO4
and incubated at 858C overnight. The hydrolyzed PMA
sample was analyzed for malic acid by HPLC (LC-20AT,
Shimadzu, Japan) using an organic acid column
(Phenomenex Rezex ROA) at 408C eluted with
5 mM H2SO4 at 0.6 mL/min. Succinic acid and acetic acid
in the fermentation broth were also analyzed by HPLC using
the same column at 408C eluted with 0.01 M H3PO4 at
0.6 mL/min and detected with a UV detector at 210 nm. The
amount of PMA was estimated from the amount of malic
acid obtained after hydrolysis based on the assumption that
1 M water was added to each mol malic acid released or
[PMA] (g/L) 0.87  [malic acid] (g/L).

Results and Discussion

Separation and Hydrolysis of PMA

Screening for High-Level PMA Production Strain

PMA was separated from fermentation broth by either

adsorption with Amberlite IRA-900 anion-exchange resins
(Rohm and Haas, Philadelphia, PA) or by ethanol
precipitation. For adsorption, fermentation broth was first
diluted 10, adjusted to pH 8.0 with 5 N NaOH solution
and clarified by centrifugation at 8,000 rpm for 45 min
followed by microfiltration (0.45 mm membrane pore size).
Then, 300 mL of pretreated PMA solution (
14 g/L) were
loaded to an XK 16/40 column packed with 50 mL of IRA900 ion-exchange resins at 2.8 mL/min, washed with 3 bed
volumes of distilled water at the same flow rate, and then
eluted with five bed volumes of 1.2 M NaCl solution at
5.6 mL/min. The eluent was collected in a fractional
collector at 1 min intervals. The collected PMA was then
hydrolyzed to malic acid and analyzed with high performance liquid chromatography (HPLC) described below to
determine its purity and recovery yield.
For ethanol precipitation, two volumes of anhydrous
ethanol were mixed with PMA solutions of various
concentrations (final concentrations: 377 g/L) and incubated at 48C overnight. The resulting precipitates were
collected by filtration with Whatman1 No. 41 filter paper
(Whatman Ltd, Oakland, CA), washed with ethanol, dried at

Various strains of A. pullulans were cultured on PDA plates

and tested in shake flasks for PMA production from glucose.
The results showed that the strain NRRL Y2311-1 formed
light-yellowish and creamy colonies and had a higher PMA
production level, reaching 22.05  3.83 g/L in 120 h, than
other strains (Fig. 1). Other strains either produced much
less PMA or formed dark colonies. A. pullulans usually form
yellowish to blackish colonies due to the formation of
chlamydospore at a later stage of its life cycle (Chi et al.,
2009). Strains producing dark pigments (melanin) usually
produced less PMA. Therefore, screening for colonies with
light or no color provides a simple and efficient method for
PMA-producing strain screening. A white single colony was
isolated from the strain NRRL Y2311-1 after serial
subculturing and isolation on PDA plates. The isolated
colony remained white on the plate, indicating no dark
pigment accumulation, and this purified strain was named
ZX-10 and used for PMA production. It should be noted
that the strain NRRL 58012 also gave good PMA production
but formed dark reddish colonies, indicating the production
of pigments that could complicate downstream processing
for PMA purification (Zhang et al., 2011a) and thus was not
selected.

Zou et al.: Production of Polymalic Acid and Malic Acid

Biotechnology and Bioengineering

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Figure 1. Screening of A. pullulans strains for PMA production. A: Morphology and appearance of A. pullulans on agar plates; B: Comparison of PMA (reported as malic acid)
production from glucose by various A. pullulans strains in shake-flasks.

Effect of Glucose Concentration on PMA Production

Batch and Fed-Batch Fermentations

The effect of the initial glucose concentration in the

fermentation medium on PMA production was studied in
shake flasks and the results are shown in Table II. Malic acid
is an intermediate in the TCA cycle and its overproduction,
which is the prerequisite for PMA biosynthesis, requires an
over-supply of glucose. Therefore, a high-glucose concentration should favor PMA production with higher yield and
productivity. PMA production increased
78% when the
initial glucose concentration increased from 60 to 90 g/L
in the fermentation media, with both PMA yield and
productivity also increased significantly. However, further
increasing the glucose concentration did not seem to
increase PMA production because the fermentation stopped
with significant amounts of residual glucose when the PMA
concentration reached
48 g/L (or
55 g/L malic acid after
hydrolysis) in the shake flasks. Based on the consumed
glucose, the highest product (malic acid) yield of 0.72 g/g
was obtained when the initial glucose concentration was
150 g/L.

Figure 2 shows free-cell batch and fed-batch fermentations

for PMA production in a 5 L stirred-tank fermentor. About
41.2 g/L PMA (47.3 g/L malic acid after hydrolysis) was
produced from
100 g/L glucose in 96 h in batch
fermentation, with a malic acid productivity of 0.49 g/L h.
In the fed-batch fermentation, a high final PMA titer of
76.2 g/L (87.6 g/L malic acid after hydrolysis) was reached in
144 h with a corresponding malic acid productivity of 0.61 g/
L h. It is noted that the production of PMA stayed at a
relatively constant rate throughout the fermentation,
indicating that PMA had no significant inhibition effect
on the fermentation or cell growth, which continued until
glucose was completely consumed and PMA reached
41.2 and 76.2 g/L in batch and fed-batch fermentations,
respectively. The fermentation pH dropped slightly from the
initial
6.8 to
6.0 due to the production of succinic and
acetic acids, which however, were relatively low, reaching 0.6
and 1.2 g/L, respectively, in batch fermentation and 0.38 and
0.67 g/L, respectively, in the fed-batch fermentation. These

Table II.

Effects of initial glucose concentration on PMA production in shake-flasks.

Initial glucose
(g/L)
60
90
120
150

Residual glucose
(g/L)

Cell biomass
(g/L)

Malic acida
(g/L)

Productivity
(g/L h)

Yield
(g/g)

0.04  0.02
0.10  0.01
26.82  0.00
77.40  1.44

19.09  0.86
21.09  0.26
21.16  1.36
21.38  0.08

30.77  1.16
54.90  2.33
52.48  0.52
52.39  1.85

0.26  0.01
0.46  0.02
0.44  0.01
0.44  0.02

0.51  0.02
0.61  0.02
0.56  0.01
0.72  0.02

PMA production was reported as malic acid obtained after hydrolysis of PMA with H2SO4.

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Biotechnology and Bioengineering, Vol. 110, No. 8, August, 2013

Figure 3. Kinetics of fed-batch fermentation for PMA production by A. pullulans

immobilized in a fibrous-bed bioreactor. PMA production is reported as malic acid
after acid hydrolysis of PMA.

Figure 2. Fermentation kinetics of PMA production by A. pullulans in a stirredtank fermentor. A: Batch fermentation; B: Fed-batch fermentation. PMA production is
reported as malic acid after acid hydrolysis of PMA.

results indicated that PMA could be produced at a relatively

high-productivity with a high-product titer in the free-cell
fermentation with good aeration and agitation. However,
the product yield was relatively low, less than 0.5 g/g, due to
a large amount of the glucose was used to grow cell biomass
and lost in CO2 in the free-cell fermentation.
It has been reported that cell immobilization in the FBB
increased cell density and thus the reactor productivity, and
directed more substrate toward the metabolic product
instead of cell biomass (Jiang et al., 2010; Suwannakham and
Yang, 2005; Wu and Yang, 2003). Therefore, to improve
PMA production, fed-batch fermentation was studied with
cells immobilized in the FBB. As shown in Figure 3, a high
final product titer (123.7 g/L of PMA or 142.2 g/L of malic
acid after hydrolysis) was produced in 192 h from the
immobilized-cell fermentation. The productivity and yield
of malic acid in the fermentation were 0.74 g/L h and 0.55 g/
g glucose, respectively, which were 21.3% and 12.2% higher
than those in the free-cell fed-batch fermentation. Although

a significant amount of acetic acid was produced initially,

resulting in a rapid drop in the pH due to a lack of pH
control in the FBB, the final acetic and succinic acid
concentrations in the fermentation broth was only 2.81 and
0.47 g/L, respectively. The higher PMA yield and lowbyproducts formation in the FBB fermentation, which
might be attributed to lowered demands for glucose and
ATP because of reduced cell growth, suggested that the cells
immobilized in the FBB were effective for PMA production
from glucose with a high product titer, productivity and
yield.
It should be noted that malic acid, the precursor for PMA
biosynthesis, is mainly derived from CO2 and pyruvate via
the pyruvate carboxylationoxaloacetate reduction pathway
in the presence of carbonate (Lee et al., 1999). Adding
CaCO3 in the fermentation medium thus could greatly
increase PMA production in a dose dependent manner as
previously reported (Zhang et al., 2011a). Therefore, a
relatively high concentration of CaCO3 (
30 g/L) was used
in this study to maintain the fermentation pH around 6 as
well as to stimulate PMA production. A near-neutral pH of
66.5 is important for the PMA biosynthesis because a
lower pH may cause the hydrolysis of PMA (at pH < 5)
(Holler et al., 1992) and induce the production of
extracellular polysaccharides (at pH 4.5) and chlamydospore (at pH 3) (Li et al., 2009), which may compete for
limited carbon source and result in lower PMA yield.
As can be seen in Table I, the PMA fermentation by the
strain ZX-10 in the FBB produced the highest product titer
(123.7 g/L of PMA or 142.2 g/L of malic acid), which could
greatly reduce downstream processing costs. In contrast,
most of the fermentations with either native or engineered
malic acid producing strains gave a low product titer of less
than 75 g/L. In addition, the FBB fermentation with ZX-10

Zou et al.: Production of Polymalic Acid and Malic Acid

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2109

gave a high productivity (0.64 g/L h for PMA or 0.74 g/L h

for malic acid), which can be attributed to the high viable
cell density achieved with immobilized cell fermentation
(Jiang et al., 2010; Suwannakham and Yang, 2005).
Nevertheless, the productivity could be further improved
to greater than 1 g/L h as obtained with A. pullulans ipe-1
cells maintained in the exponential phase in repeated batch
fermentation (Cao et al., 2012a). The FBB fermentation gave
an overall product yield of 0.55 g/g, which was significantly
lower than the 0.72 g/g achieved in shake-flask fermentation
with a higher initial glucose concentration of 150 g/L and
those with A. flavus (0.94 g/g) and an engineered E. coli
strain (1.06 g/g) (Zhang et al., 2011b). The maximum
theoretical yield of malic acid production from CO2-fixing
pyruvate carboxylationoxaloacetate reduction pathway is
2 mol malic acid per mol glucose (1.48 g/g) (Kenealy et al.,
1986; Zelle et al., 2008). The product yield could be greatly
increased in fermentation using non-growing or resting but
metabolically active cells (Nakajima-Kambe et al., 1996) to
direct the carbon flux toward PMA biosynthesis instead of
cell biomass and other metabolites.

recovered PMA had a
100% purity based on the HPLC

analysis of the acid hydrolysate, which contained only malic
acid (see insert in Fig. 4). The overall recovery yield of this
adsorptiondesorption process was
84%, which could
be improved by optimizing process conditions such as the
feed flow rate, pH and temperature. It is also noted that a
lower concentration of NaCl could be used in desorption
although its effect on the recovery yield would need further
studies.
Ethanol precipitation for PMA recovery from fermentation broth was also studied and the results are shown in
Figure 5. In general, the recovery yield of PMA increased
with increasing the PMA concentration in the solution and
reached 80% when the initial PMA concentration was 77 g/
L. However, the recovery yield was lower than that of
adsorption and the ethanol precipitates contained significant amounts of glucose and acetic acid (Fig. 5B), requiring
further purification to remove the impurities. In addition, it
is necessary to recover and recycle the ethanol present in the
solution, which increases energy consumption. Overall, the
adsorptiondesorption process would be more energyefficient for PMA recovery and purification, although the
process needs further optimization and its economics
remains to be investigated.

Separation and Purification of PMA from Fermentation

Broth
PMA separations by ethanol precipitation and by adsorption
with IRA-900 followed with desorption or elution with
1.2 M NaCl solution were studied. As shown in Figure 4,
nearly 100% of the PMA adsorbed on the IRA-900 could be
recovered by desorption with 1.2 M NaCl solution, and the

Hydrolysis of PMA for Malic Acid Production

The kinetics of PMA hydrolysis by sulfuric acid was studied
at various acid concentrations and the results are shown in
Figure 6. A strong acid or H can break the ester bonds in

Figure 4. Elution of PMA with NaCl solution after adsorption on Amberlite IRA-900 ion-exchange resins. Insert: HPLC analysis of the eluted product. PMA is reported as malic
acid after acid hydrolysis of PMA.

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Biotechnology and Bioengineering, Vol. 110, No. 8, August, 2013

Figure 6. Kinetics of acid hydrolysis of PMA to malic acid with various concentrations of H2SO4. Symbols show experimental data and curves show model
predictions.

Figure 5. PMA recoveries from fermentation broth by ethanol precipitation. A:

Effects of the initial PMA concentration on the residual PMA concentration in the
solution containing 66.7% (v/v) ethanol and PMA recovery yield; B: HPLC analysis of
the redissolved ethanol precipitates.

PMA and release malic acid, which follows the first-order

reaction and can be represented by the following equations.
k

PMA ! Malic acid

rMA 

dCPMA
kCPMA
dt

CMA CPMA;o  CPMA CPMA;o 1  ekttlag

(1)
(2)
(3)

Under this condition, all PMA was hydrolyzed to malic

acid with no detectable degradation product, such as 3oxopropanoic acid (Whitford, 1925). The PMA hydrolysis
rate or malic acid releasing rate (rMA) is proportional to
the concentration of PMA (CPMA) or the total molar
concentration of malic acid in the solution. In Equation (3),
k is the reaction rate constant (h1) and tlag accounts for the
delay in the hydrolysis (h1) possibly contributed by the
time required for heating and diffusion of H to ester bonds.
CPMA,o is the initial PMA concentration, which should be

equal to the final malic acid concentration when all PMA has
been hydrolyzed. In general, the model fits the data well,
with R2 > 0.95. Table III lists the best values for k, tlag and
CPMA,o. As indicated by the k and tlag values, increasing the
sulfuric acid concentration from 1 to 3 M also increased
the rate and reduced the lag time of PMA hydrolysis. The
hydrolysis of PMA with dilute H2SO4 (0.10.5 M) was also
investigated at a higher temperature of 1218C for 30 min,
which however, gave a lower product yield of 7493%
possibly due to the formation of coumalic acid from malic
acid via a self-condensation reaction (Wiley and Smith,
1963). Further studies with cost analysis would be necessary
in order to find the optimal conditions for the acid
hydrolysis process.
The simple downstream process for high-purity PMA
purification from the fermentation broth with IRA-900
followed with acid hydrolysis of PMA to malic acid offers an
attractive method to produce high-purity malic acid. In
conventional malic acid fermentation processes, other
organic acids were simultaneously produced as byproducts.
Although some metabolic engineering strategies and newengineered host strains have been explored, the acid

Table III.
data.

CPMA,o
(g/L)

k
(h1)

tlag
(h)

R2

79.1
84.4
79.9

0.44
0.83
2.30

1.46
0.83
0.89

0.985
0.984
0.956

Zou et al.: Production of Polymalic Acid and Malic Acid

2111

Sulfuric
acid (M)
1
2
3

Kinetic coefficients in Eq. (3) and R2 for the model fitting of the

Biotechnology and Bioengineering

byproducts were difficult to be completely removed (Zhang

et al., 2011b). Similar phenomena were also detected in
other dicarboxylic acids (e.g., succinic acid and fumaric
acid) fermentations (Engel et al., 2008; Jantama et al., 2008).
In this regard, the strain ZX-10 has remarkable advantages
over malic acid producing strains in that it produced mainly
PMA with little acid byproducts. As demonstrated in this
study, PMA can be readily separated from other fermentation byproducts by adsorption onto IRA-900 and eluted
with NaCl solution. A high-purity malic acid can then be
obtained from PMA after a simple acid hydrolysis treatment
with no detectable impurity (see Fig. 4). Compared to
ethanol precipitation and the more complicated separation
process for malic acid production (Uslu and Kirbaslar,
2009), the simpler and more efficient downstream process
with a high recovery yield of 84% and high purity of
100%
developed in this work should be more economical, and can
provide a novel approach for malic acid production.

Conclusions
Microbial production of malic acid from glucose is severely
limited by end-product inhibition of the accumulated malic
acid and high costs for separating malic acid from other acid
byproducts. In this work, a novel process based on high-level
PMA production and hydrolysis was developed to circumvent the acid inhibition problem. A high-yield PMA
producing strain A. pullulans ZX-10 was isolated and
used for PMA production in fed-batch fermentation,
achieving a high product (malic acid) titer of 142.2 g/L,
productivity of 0.74 g/L h, and yield of 0.55 g/g glucose. No
product inhibition was observed in the fermentation,
confirming that malic acid in the polymerized form does
not exert any negative effect on cell metabolism and growth.
Considering that PMA can be easily hydrolyzed to release
malic acid, PMA fermentation followed with acid hydrolysis
offers a promising process for industrial malic acid
production from glucose and other carbohydrates.

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