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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
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Table I.
Comparison of PMA and malic acid production by different strains in various fermentation processes.
Microorganism
Bioreactor
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
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
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
0.74
0.47
0.19
0.42
1.06
0.31
9.25
34
59
2106
Refs.
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.
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).
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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.
Table II.
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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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.
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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.
2110
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.
dCPMA
kCPMA
dt
(1)
(2)
(3)
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
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Sulfuric
acid (M)
1
2
3
Kinetic coefficients in Eq. (3) and R2 for the model fitting of the
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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