Bioresource Technology 304 (2020) 122990

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Case Study

Poly(malic acid) production from liquefied corn starch by simultaneous T

saccharification and fermentation with a novel isolated Aureobasidium
pullulans GXL-1 strain and its techno-economic analysis
⁎
Wei Zeng, Bin Zhang, Li Jiang, Yao Liu, Su Ding, Guiguang Chen, Zhiqun Liang
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi Microorganism and Enzyme Research Center of Engineering Technology,
College of Life Science and Technology, Guangxi University, 100 Daxue Road, Nanning 530004, Guangxi, China

A R T I C LE I N FO A B S T R A C T

Keywords: In this study, a novel Aureobasidium pullulans GXL-1 strain without melanin secretion was isolated for efficient
Poly(β-L-malic acid) polymalic acid (PMA) production. The PMA produced by GXL-1 was characterized, and its molecular mass was
Aureobasidium pullulans determined to be 1.621 kDa by gel permeation chromatography. Liquefied corn starch was shown to replace
Liquefied corn starch glucose for PMA production by GXL-1 through simultaneous saccharification and fermentation. The PMA titer
Fermentation
obtained from batch fermentation was up to 49.0 ± 1.6 g/L in a 10 L fermentor, and the PMA yield and
Techno-economic analysis
productivity obtained from repeated-batch fermentation were up to 0.50 g/g and 0.34 g/L·h, respectively.
Furthermore, process design and techno-economic analysis were performed at an annual output level of 5000
metric tons by SuperPro Designer. Results showed that the production cost of $2.046/kg and payback period of
6.9 years were achieved by repeated-batch fermentation; this provides an economically feasible strategy for
industrial-scale production of PMA.

1. Introduction et al., 1969) have been shown to exhibit PMA synthesis ability. The
PMA titer of P. polycephalum was only 2.7 g/L, but the PMA molecular
Poly(β-L-malate) (PMA) is a linear anionic homopolymer of L-malic mass was as high as 50 ~ 300 kDa (Lee and Holler, 1999). In com-
acid monomers linked via ester bonds between their α-hydroxyl and β- parison, PMA with titers between 9.8 and 62.6 g/L and molecular
carboxyl groups (Chi et al., 2016). As a result of their excellent water masses between 2.8 kDa and 19 kDa were obtained from A. pullulans by
solubility, biocompatibility, and biodegradability, PMA and its deri- batch fermentation (Zou et al., 2019). Furthermore, low-molecular-
vates have prospective applications in the food industry, medicine, and mass PMA has superior water solubility, biocompatibility, and biode-
other industrial fields (Portilla-Arias et al., 2008). An increasing gradability than high-molecular-mass PMA (Cao et al., 2016; Li et al.,
number of researches have focused on the industrial-scale production of 2015). Thus, no matter in terms of PMA titer or molecular mass, A.
PMA, which is a prerequisite for its commercial applications. Both pullulans is more valuable for PMA production. To date, several of A.
chemical synthesis and microbial fermentation can be used for PMA pullulans strains, dubbed GXZ-6 (Zeng et al., 2019a), YJ6-11 (Zou et al.,
production. However, chemical synthesis is not conducive to industrial 2016), ZX-10 (Zou et al., 2013), ipe-1 (Cao et al., 2013), ZD-3d (Zhang
PMA production because of the complex process route, harsh reaction et al., 2011), CBS591.75 (Liu and Steinbuchel, 1996), and A-91 (Nagata
conditions and high degree of pollution involved (Vert, 1998). By et al., 1993), have been isolated and studied extensively. Furthermore,
contrast, microbial fermentation may be more conducive to industrial PMA titers of 124.1 g/L and 75.7 g/L were obtained by fed-batch fer-
PMA production owing to the simpler operation and mild conditions mentation and repeated-batch fermentation, respectively, in A. pull-
involved (Zou et al., 2019). However, the isolation of excellent strains ulans GXZ-6 (Zeng et al., 2019b). However, most of these A. pullulans
and reduction of production costs remain two major challenges to be strains secreted melanin, a black pigment with polyphenol-like struc-
resolved in relation to the application of microbial fermentation for ture, during PMA production (Jiang et al., 2017). This melanin secre-
PMA production. tion makes it difficult to recover and purify PMA, resulting in decreased
Aureobasidium pullulans (Nagata et al., 1993), Physarum poly- PMA recovery rates and increased costs. Therefore, for PMA produc-
cephalum (Fischer et al., 1989), and Penicillium cyclopium (Shimada tion, it is necessary to isolate strains that do not secrete melanin and

⁎
Correspondence author.
E-mail address: zqliang@gxu.edu.cn (Z. Liang).

https://doi.org/10.1016/j.biortech.2020.122990
Received 21 December 2019; Received in revised form 6 February 2020; Accepted 7 February 2020
Available online 11 February 2020
0960-8524/ © 2020 Elsevier Ltd. All rights reserved.
W. Zeng, et al. Bioresource Technology 304 (2020) 122990

produce low-molecular-mass PMA. 2.3. Characterization of PMA

In the fermentation industry, the use of low-cost feedstock as sub-
strate is an important strategy to reduce production costs. To date, PMA was recovered and purified using an ethanol precipitation
various lignocellulosic feedstocks, such as bagasse (Cao et al., 2020), method reported previously (Liu and Steinbuchel, 1996; Zeng et al.,
barley straw (Yegin et al., 2019), soybean hull (Cheng et al., 2017), 2019a), and identified by analyzing its monomers as follows: the pur-
corncob (Zou et al., 2015, 2016), corn fiber and wheat straw (Leathers ified PMA was dissolved in 2 M H2SO4 and hydrolyzed overnight at
and Manitchotpisit, 2013), have been reportedly used for PMA pro- 90 °C. The hydrolysate was neutralized with 2.5 M NaOH and filtered
duction. However, these feedstocks need to be hydrolyzed first, by acid using 0.45 μm microporous membrane filter (Nagata et al., 1993).
treatment and heating, to release fermentable carbohydrate. Further- Then, the filtrate was analyzed by a C18 column (JADE-PAK ODS-AQ,
more, various toxic compounds, such as furfural, and 5-hydroxymethyl 4.6 mm × 250 mm; Techway, China) in a HPLC system (LC-20A, Shi-
furfural, etc., are produced during the hydrolysis process, which would madzu Corp., Japan) with a UV detector. The detection conditions are
affect adversely cell growth and PMA synthesis (Jonsson and Martin, as follows: injection volume of 5 μL, detection temperature of 25 °C,
2016). Therefore, starchy feedstocks and molasses, such as Jerusalem detection wavelength of 210 nm, eluent of KH2PO4 (50 mM, pH 2.5),
artichoke tuber (Cao et al., 2019), soy molasses (Cheng et al., 2017), and flow rate of 0.7 mL/min (Zeng et al., 2019b). L-malic acid was used
cane molasses (Xia et al., 2016), and sweet potato (Zan and Zou, 2013) as standard reference. The PMA was characterized by Fourier transform
were used to replace lignocellulose for PMA production. However, infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopy. A
these feedstocks still require some pretreatment. For instance, Jer- total of 100 mg purified PMA was mixed and ground with 1 g of dried
usalem artichoke tuber, soy molasses, and sweet potato must be hy- KBr powder, and then analyzed with a FTIR spectrometer (Nicolet iS10,
drolyzed at high temperature by sulfuric acid, while cane molasses Thermo Fisher Scientific Inc., USA) from 4000 to 400 cm−1. A total of
require treatment with a combination of sulfuric acid, potassium fer- 50 mg purified PMA was dissolved in 0.5 mL of D2O solution for 1H
rocyanide, and tricalcium phosphate. It should be noted that A. pull- NMR and 13C NMR spectral analysis with a NMR spectrometer
ulans is able to produce PMA using the above-mentioned feedstocks (AVANCE 600, Bruker Corp., Switzerland) at 600 MHz. PMA molecular
because its genome carries a large number of genes encoding enzymes mass was determined with a previously reported gel permeation chro-
involved in sugar hydrolysis and metabolism (Gostincar et al., 2014). matography (GPC) method (Zeng et al., 2019a).
This raises the question of whether starchy feedstocks can be used for
PMA production only after simple liquefaction treatment, thereby not 2.4. Optimization of PMA production in a shake-flask
only reducing the cost of pretreatment, but also eliminating the effect of
toxic compounds generated by acid treatment during PMA production. The effects of using glucose, fructose, sucrose, and maltose as
In order to answer these questions, here, a strain of A. pullulans GXL- carbon sources, with concentration of 140 g/L, for PMA production
1 that did not secrete melanin, and which produced low-molecular- were first investigated by single-factor analysis. Then, the effects of
mass PMA, was isolated from fresh plant leaves. Liquefied corn starch nitrogen sources, including yeast extract, beef extract, peptone, am-
was used as a substrate for PMA production by GXL-1 through si- monium chloride, ammonium nitrate, and sodium nitrate, on PMA
multaneous saccharification and fermentation (SSF). Then, process production were studied in detail. The concentration of the nitrogen
design and techno-economic analysis were performed at an annual sources was set at 3 g/L. To further improve PMA production, the ef-
output level of 5000 metric tons (MT) by SuperPro Designer. fects of metabolic intermediates (fumaric acid, succinic acid, and citric
acid), activator (biotin) and inhibitor (sodium malonate) on PMA pro-
2. Materials and methods duction were investigated. Except for that of biotin (5 mg/L), the
concentrations of other metabolic intermediates and inhibitor were set
2.1. Medium at 5 g/L. Furthermore, the concentration of glucose, ammonium
chloride, and succinic acid were also optimized. Wheat starch, corn
A mannitol-enriched medium composed of mannitol 10 g/L, starch, and cassava starch were liquefied and substituted for glucose as
NH4NO3 1 g/L, KH2PO4 0.5 g/L, MgSO4·7H2O 0.2 g/L, and citric acid carbon sources for PMA production. The liquefied starch solution was
2 g/L, and a glucose-screening medium composed of glucose 120 g/L, prepared as follows: 15% (w/v) starch aqueous solution (pH 5.5) was
NH4NO3 1 g/L, KCl 0.5 g/L, KH2PO4 0.1 g/L, MgSO4·7H2O 0.2 g/L, and heated and stirred to slurry; then, 0.25% (w/v) α-amylase (AOBOX,
CaCO3 20 g/L were used for isolation of PMA-producing strains. The China) was added as the temperature was lowered to 60 °C, and the
seed medium consisted of glucose 80 g/L, NH4Cl 2 g/L, KCl 0.5 g/L, mixture was hydrolyzed at 60 °C for 60 min.
KH2PO4 0.1 g/L, MgSO4·7H2O 0.1 g/L, ZnSO4·7H2O 0.05 g/L, and
CaCO3 20 g/L. The fermentation medium was the same as the seed 2.5. Culture conditions
medium, except for the differences in concentration of glucose, NH4Cl
and ZnSO4·7H2O: the concentration of glucose and NH4Cl were changed The strain was inoculated into 100 mL of seed medium in a 500 mL
to 140 and 3 g/L, respectively, based on carbon and nitrogen source shake-flask, and then oscillation-cultured at 180 rpm and 30 °C for 36 h;
optimization experiments. Additionally, the concentration of this culture was defined as the primary seed culture. Then, 100 mL of
ZnSO4·7H2O was changed to 0.2 g/L because of the PMA titer obtained primary seed culture was transferred to 550 mL of seed medium in a 3-L
from 0.2 g/L ZnSO4·7H2O was increased by 4.5% relative to that from shake-flask, and then oscillation-cultured at 180 rpm and 30 °C for 36 h;
0.1 g/L ZnSO4·7H2O (data not shown in Results section). this culture was defined as the secondary seed culture. To carry out
batch fermentation in shake-flask, 4 mL of the primary seed culture was
2.2. Isolation and identification of the GXL-1 strain transferred to 36 mL of fermentation medium in a 250 mL shake-flask,
and then oscillation-fermented at 180 rpm and 30 °C for 8 days. To
The strain of interest was isolated from fresh plant leaves using carry out batch fermentation in the fermentor, 0.6 L of the secondary
mannitol-enriched medium and glucose-screening medium, according seed culture was transferred to 6.9 L of fermentation medium in a 10-L
to a method reported previously (Nagata et al., 1993). Strain identifi- fermentor and fermented at 30 °C. The aeration rate was set at 0.6 vvm,
cation was performed by lawn morphological observation on potato- and the agitation speed was varied between 200 rpm and 400 rpm to
dextrose agar plate, cell morphological observation under scanning maintain the dissolved oxygen level over 10% (v/v) during fermenta-
electron microscope (SEM), and phylogenetic affiliation analysis with tion. Repeated-batch fermentation with three batches was performed
deoxyribonucleic acid internal transcribed spacer (ITS) sequence (Zeng using the same conditions as the batch fermentation, except for the
et al., 2019a). differences in fermentation medium: the initial medium was the same

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as that used for batch fermentation. After fermentation for 144 h and 1556 cm−1 indicated the presence of arboxylic acid salt (–COO−).
252 h, 5.5 L of fermentation broth was discharged and another 5.5 L of Furthermore, an O–H stretching vibration band at 3390 cm−1 and a
fresh fermentation medium was added into the fermentor. –CH2 bending vibration band at 1427 cm−1 were also observed. The
chemical shifts of 175.71 (–COOH), 171.68 (–CO–), 71.69 (–CH), and
2.6. Analytical methods 36.05 (–CH2) corresponding to four carbon atoms of L-malic acid were
showed in the 13C NMR spectrum. The chemical shifts of 5.10 (–CH
The biomass was determined using a dry cell weight (DCW) method proton) and 2.88 (–CH2 proton) were observed in the 1H NMR spec-
reported previously (Zou et al., 2016). Since one mol H2O was added to trum. The above described characterization of the polymer indicated
each mol L-malate released in PMA hydrolysis, the PMA concentration that PMA was the fermentation product of A. pullulans GXL-1. The
was determined with a correction factor by measuring the difference molecular mass of PMA produced by different strains is variable (Lee
between L-malate levels before and after hydrolysis of fermentation and Holler, 1999; Zeng et al., 2019a). More importantly, PMA with
broth (Wei et al., 2017; Zou et al., 2013). The specific calculation for- different molecular masses is needed for various application fields (Cao
mula is as follows: [PMA] (g/L) = 0.87 × [L-malate] (g/L). The con- et al., 2016). In this study, the molecular mass of PMA from GXL-1 was
centration of L-malate in hydrolysate and fermentation broth was determined to be 1.621 kDa by GPC, which indicated that it consisted of
measured using a HPLC method as described in section 2.3. Maltotriose, 14 malic acid monomers. Such low-molecular-mass PMA represents an
maltose and glucose concentrations in the supernatant were measured excellent choice for application in biomedical fields.
by a NH2 column (4.6 mm × 250 mm; Chiral, China) in a HPLC system
(LC-20A, Shimadzu Corp., Japan) with a refractive index detector. The 3.3. Fermentation optimization of PMA production in shake-flask
detection conditions are as follows: injection volume of 5 μL, detection
temperature of 40 °C, eluent of acetonitrile : water (70 : 30, v/v), and 3.3.1. Effect of carbon sources
flow rate of 1.0 mL/min. The solid content in the liquefied starch so- Monosaccharides and disaccharides have been commonly used as
lution was measured by the refractive index method. The process design carbon sources for PMA production in previous reports (Cao et al.,
and techno-economic analysis of PMA production were performed by 2013; Wei et al., 2017; Zeng et al., 2019b; Zhang et al., 2011). Thus, the
SuperPro Designer (Intelligen, Inc.), which is a useful software program effects of glucose, fructose, sucrose, and maltose on PMA titer and DCW
for integrated industrial process modeling, evaluation, and optimiza- were investigated (Fig. 2a). Glucose was not only the optimal carbon
tion. source for PMA production, but also the most beneficial carbon source
for cell growth by GXL-1. The maximum PMA titer and DCW were
3. Results and discussion 40.7 ± 0.8 g/L and 18.2 ± 0.5 g/L, respectively. However, the PMA
titer was less than 30 g/L when other monosaccharides and dis-
3.1. Isolation and identification of the GXL-1 strain accharides were used as the carbon source. In particular, GXL-1 could
use maltose, a disaccharide formed of two units of glucose, as a carbon
A strain, dubbed GXL-1, with the ability to produce PMA was iso- source for PMA production; however, the PMA titer and DCW were
lated from fresh plant leaves. The lawn of GXL-1 on potato-dextrose much lower than when glucose was used as the carbon source. The
agar plate showed a moist yeast-like appearance in the middle and was reason might be that glucose availability at the early stage of culturing
surrounded by small claw shapes. Furthermore, GXL-1 cells showed in maltose medium was not sufficient to support rapid cell growth and
yeast-like morphology under SEM observation. It was interesting that synthesis of maltose hydrolase in large quantities. Furthermore, glucose
there was no significant change in lawn morphology even though the concentration was further optimized (Fig. 2b). In the glucose con-
culture time was extended to 18 days. Unlike A. pullulans var. melano- centration range of 80 g/L to 160 g/L, both PMA titer and DCW ap-
genum lawns, which turn black at the late stage of culture (Zeng et al., peared to first increase and then decrease. It was interesting that the
2019a), the color of the present GXL-1 lawns was light orange at the maximum DCW was achieved at 100 g/L glucose, but the maximum
early stage, and then light pink at the late stage. This indicated that PMA titer was achieved at 140 g/L glucose. Thus, glucose with a con-
GXL-1 did not secrete melanin, a common but undesirable by-product, centration of 140 g/L was selected for further study.
during PMA production.
For phylogenetic affiliation of GXL-1, the ITS fragment of the GXL-1 3.3.2. Effect of nitrogen sources
genome was amplified and sequenced. Then, the sequence of this ITS A. pullulans is able to use a variety of nitrogen sources for PMA
fragment with 540 nucleotides was submitted to GenBank, and ob- production (Zou et al., 2019). The effects of yeast extract, beef extract,
tained an accession number of MK0412909. Comparison of the ob- peptone, ammonium chloride, ammonium nitrate, and sodium nitrate,
tained sequence with other sequences available in the NCBI database as nitrogen sources, on PMA titer and DCW were investigated (Fig. 2c).
using the Nucleotide BLAST program revealed highest identity to the Results showed that the PMA titer of GXL-1 was lower than 20 g/L
corresponding sequence of A. pullulans. Furthermore, phylogenetic tree when yeast extract, beef extract, or peptone was used as nitrogen
analysis between GXL-1 with other Aureobasidium strains using MEGA 7 source. Although many studies have reported that trace elements and
software also revealed that GXL-1 was more related to A. pullulans biotin in organic nitrogen source promote PMA production (Cheng
(Fig. 1). Based on the above lawn morphological observation, cell et al., 2017; Wang et al., 2015), inorganic nitrogen sources were more
morphological observation, and ITS sequence analysis, GXL-1 was conducive to cell growth and PMA production. The PMA titer was as
identified as an A. pullulans strain. It was deposited at the China Center high as 43.8 ± 1.1 g/L when ammonium chloride was used as the
for Type Culture Collection with the preservation number CCTCC M nitrogen source; this finding is different from other reports showing that
2018519. ammonium nitrate or sodium nitrate is the most suitable nitrogen
source for PMA production by other A. pullulans strains (Zeng et al.,
3.2. Characterization of polymer 2019a; Zou et al., 2016). Moreover, it should be noted that ammonium
nitrate was beneficial to cell growth but not suitable for PMA produc-
The purified polymer was hydrolyzed by sulfuric acid and analyzed tion in GXL-1, while the opposite result was observed for sodium nitrate
by HPLC, and results showed that the hydrolyzed product was L-malic as nitrogen source. These results suggest that there is no positive cor-
acid. Furthermore, FTIR, 13C NMR, and 1H NMR spectra were used to relation between PMA titer and DCW. Furthermore, the effect of the
characterize this polymer. The FTIR spectral data showed that a char- concentration of ammonium chloride on PMA production was also
acteristic peak at 1731 cm−1 (–C=O) and 1170 cm−1 (–C–O–C) for studied (Fig. 2d). PMA titer and DCW showed similar trends with in-
ester bonds were observed. Then, a characteristic band observed at creasing ammonium chloride concentration. The maximum PMA titer

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Fig. 1. Neighbor-joining phylogenetic tree of the GXL-1 strain was constructed using MEGA 7 software based on ITS sequences. The number in parentheses is the
GenBank ID of ITS sequence. The number at the node represents the bootstrap value. The scale bar represents 0.005 nucleotide substitutions per position.

was obtained at 3 g/L ammonium chloride (defined as mode I), which to replace glucose for PMA production, results showed that they sup-
was chosen for further study. ported PMA synthesis and cell growth well (Fig. 3b); DCW exceeded
20 g/L regardless of which form of liquefied starch was used, which is
comparable to the corresponding value for glucose. Furthermore, a
3.3.3. Effect of metabolic intermediates, activator, and inhibitor
PMA titer of 57.2 ± 1.2 g/L was obtained from liquefied corn starch,
L-malic acid was the only monomer used in the synthesis of PMA;
which was 17.6% and 13.3% higher than that from liquefied cassava
therefore, the amount of intracellular L-malic acid directly affects PMA
starch and wheat starch, respectively. These results suggest that li-
titer. Furthermore, malate synthesis is related to the TCA cycle,
quefied starch could replace glucose for PMA production by GXL-1
glyoxylate cycle, and non-oxidative pathway in A. pullulans (Zeng et al.,
through SSF.
2019c). Thus, the effects of metabolic intermediates, activator, and
inhibitor related to these cycles on PMA titer and DCW were in-
vestigated (Fig. 2e). Compared with the control group, cell growth was
3.5. Scaled-up production of PMA in fermentor
not inhibited regardless of which metabolites were added. In particular,
DCW was increased by 16.3% and 20.3% with the addition of citric acid
3.5.1. Batch fermentation with glucose and liquefied corn starch
and succinic acid, respectively. Moreover, succinic acid could effec-
To further verify the effect of fermentation optimization on PMA
tively promote PMA production, and PMA titer was increased by 11.2%
production, batch fermentation for PMA production with glucose as
to 51.3 ± 1.3 g/L. However, citric acid and fumaric acid, metabolic
substrate was performed in a 10 L fermentor (Fig. 4a, mode III). Glucose
intermediates of the TCA cycle, could not improve PMA titer. Sodium
was continuously consumed as fermentation proceeded, with a corre-
malonate, as an inhibitor of succinate dehydrogenase in the TCA cycle,
sponding increase in PMA titer and DCW. The PMA titer of
did not significantly inhibit or promote PMA synthesis and cell growth.
41.2 ± 1.5 g/L and DCW of 18.9 ± 0.4 g/L were obtained at 192 h,
Biotin, which is an activator of pyruvate carboxylase in the non-oxi-
which were lower than values for shake-flask fermentation (Table 1).
dative pathway, promoted PMA synthesis, and PMA titer was increased
When succinic acid was added to fermentor at the beginning of fer-
by 9.1%. Based on the above results, the effect of succinic acid con-
mentation (Fig. 4b, mode IV), the PMA titer and DCW were significantly
centration on PMA production was further studied (Fig. 2f). The max-
increased as observed for shake-flask fermentation. The maximum PMA
imum PMA titer was obtained at 3 g/L succinic acid (mode II), but the
titer reached 51.8 ± 1.4 g/L with a productivity of 0.27 g/L·h; these
maximum DCW was obtained at 5 g/L succinic acid. Thus, succinic acid
values were 25.8% and 28.6% higher, respectively, than those in mode
with a concentration of 3 g/L was selected.
III. Moreover, DCW reached 29.2 ± 0.7 g/L, which was 1.5 times that
in mode III. It was interesting that the residual glucose concentration in
3.4. Liquefied starch as a replacement for glucose for PMA production mode IV was also higher than that in mode III. The PMA yield reached
0.45 g/g, which indicated that cell growth and PMA synthesis were
A. pullulans has been reported to produce several glycoside hydro- improved by consuming less glucose under regulation by succinic acid.
lases such as amylase, cellulase, xylanase, and mannanase (Chi et al., Furthermore, batch fermentation for PMA production from liquefied
2009). These enzymes enable A. pullulans to utilize various biomass corn starch was also performed in a 10 L fermentor (Fig. 4c, mode V).
substrates for cell growth and metabolites synthesis. Thus, the effects of Compared with shake-flask fermentation, the PMA titer was decreased
liquefied wheat starch, corn starch, and cassava starch on PMA pro- but DCW was significantly increased in mode V, possibly because dis-
duction were investigated. The product composition of liquefied starch solved oxygen and mass transfer in fermentor were superior to that in
was analyzed first (Fig. 3a). Results showed that liquefied starch mainly the shake-flask, which led to rapid growth of GXL-1 cells and therefore
contained glucose, maltose, maltotriose, and other oligosaccharides. limited production of PMA. Compared with that in mode III, PMA titer,
The concentration of glucose and maltose in liquefied corn starch was yield, and productivity were all increased in mode V; in particular, PMA
higher than in liquefied cassava starch and wheat starch. However, the yield was increased by 26.5% because of high residual glucose con-
concentration of maltotriose and other oligosaccharides was lower in centration (Table 1). Furthermore, the time course for sugar in mode V
liquefied corn starch. When these three liquefied starch types were used was more complex. Maltotriose and maltose were consumed

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Fig. 2. Effects of fermentation medium components and concentrations on DCW and PMA titer produced by A. pullulans GXL-1 in shake-flasks. (a) carbon sources; (b)
glucose concentration; (c) nitrogen sources; (d) ammonium chloride concentration; (e) metabolic intermediates, activator and inhibitor; (f) succinic acid con-
centration.

continuously as fermentation proceeded, as a result of mal- of fermentation, PMA titer and DCW continued to show an obvious
tosyltransferase hydrolysis, and then depleted at 96 h and 144 h, re- increase. The reason for this might be that glucose hydrolyzed from by-
spectively. Glucose was decreased in the first 48 h of fermentation, but product polysaccharide at the late stage of fermentation was used for
increased to 34.1 ± 1.5 g/L at the end of fermentation. It was inter- cell growth and PMA synthesis. When succinic acid was added into the
esting that although total sugars were no longer consumed after 132 h fermentor at the beginning of fermentation (Fig. 4d, mode VI),

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Fig. 3. (a) The product composition of liquefied starch solution. (b) Effects of liquefied cassava starch, corn starch and wheat starch on DCW and PMA titer produced
by A. pullulans GXL-1in shake-flasks.

maltotriose and maltose were also depleted at 96 h and 144 h, re- than that in mode V. Thus, the maximum PMA titer and DCW reached
spectively, but the consumption rate in the first 24 h of fermentation 49.0 ± 1.6 g/L and 41.3 ± 0.9 g/L in mode VI, respectively. Through
was higher than that in mode V. As in mode V, glucose concentration batch fermentation experiments in a 10 L fermentor, it was confirmed
also decreased first and then increased over the entire course of fer- that liquefied corn starch can be used to replace glucose for PMA pro-
mentation. However, the residual sugar concentration was only duction, and succinic acid as metabolic intermediates could improve
24.0 ± 1.4 g/L at the end of fermentation, which was 29.8% lower PMA titer.

Fig. 4. PMA production was performed in a 10 L fermentor by batch fermentation: (a) glucose as substrate; (b) glucose as substrate with addition of succinic acid; (c)
liquefied corn starch as substrate; (d) liquefied corn starch as substrate with addition of succinic acid. Other components of the fermentation medium are the same in
the above experiments. Each fermentation was repeated three times, and the error bars represent the standard deviation.

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Table 1
Results of PMA production in shake-flask and fermentor by batch fermentation for 192 h.
Mode Initial sugar (g/L) Residual sugar (g/L) DCW (g/L) PMA (g/L) Yield (g/g) Productivity (g/L·h)

Mode Ia
140 – 19.8 ± 0.6 47.8 ± 1.2 – 0.25
Mode II b 140 – 23.4 ± 0.8 51.6 ± 1.2 – 0.27
Mode III c 140 18.5 ± 1.3 18.9 ± 0.4 41.2 ± 1.5 0.34 0.21
Mode IV d 140 25.5 ± 1.6 29.2 ± 0.7 51.8 ± 1.4 0.45 0.27
Mode Ve 140 34.1 ± 1.5 37.8 ± 0. 8 45.7 ± 1.4 0.43 0.24
Mode VI f 140 24.0 ± 1.4 41.3 ± 0.9 49.0 ± 1.6 0.42 0.26

a
Mode I represents PMA production from glucose in shake-flask.
b
Mode II represents PMA production from glucose with addition of succinic acid in shake-flask.
c
Mode III represents PMA production from glucose in fermentor.
d
Mode IV represents PMA production from glucose with addition of succinic acid in fermentor.
e
Mode V represents PMA production from liquefied corn starch in fermentor.
f
Mode VI represents PMA production from liquefied corn starch with addition of succinic acid in fermentor.

3.5.2. Repeated-batch fermentation with liquefied corn starch an annual output level of 5000 MT by SuperPro Designer. The fer-
In batch fermentation, PMA can be produced by GXL-1 from li- mentation parameters such as substrate concentration, PMA titer, yield,
quefied corn starch; however, this is hindered by the prolonged fer- productivity, and fermentation time were collected from mode III,
mentation time and low productivity. To further obtain higher PMA mode V and mode VII, respectively. The process parameters such as
titer, yield, and productivity, repeated-batch fermentation was per- PMA recovery rate were as previously described (Zou, et al., 2013; Zeng
formed in a 10 L fermentor (Fig. 5, mode VII). In the first batch, al- et al., 2019b). The costs of raw material and energy were calculated
though the fermentation time was only 144 h, PMA titer and DCW also with reference to local market prices. The process includes medium
reached 37.3 ± 1.0 g/L and 30.8 ± 0.7 g/L, respectively. In the preparation, fermentation, cell separation by filtration, PMA solution
second and third batch, although the initial sugar concentration was concentration and purification by ultrafiltration, PMA recovery by
reduced by ~30 g/L and the fermentation time was further shortened to ethanol precipitation, and PMA product formation by spray drying. In
108 h, the PMA titer was still comparable to that of the first batch. The addition, ethanol was recycled by distillation to reduce the costs. The
yield and productivity were also increased by ~48.4% and ~26.9%, economic data for this process were automatically calculated by the
respectively (Table 2). Thus, the PMA titer, yield, and productivity economic evaluation module of SuperPro Designer.
reached 36.7 g/L, 0.42 g/g, and 0.31 g/L·h, respectively, on average in Table 3 shows the results of techno-economic analysis for PMA
three batches. Furthermore, maltotriose and maltose were depleted in production from glucose and liquefied corn starch by batch or repeated-
36 h and 72 h, respectively, in the second and third batches, which batch fermentation. When glucose was used as the substrate in batch
much faster than that in the first batch. The reason for this might be fermentation, the unit production cost and return on investment were
that cells in the second and third batch do not need to go through a lag $2.488/kg and 8.0%, respectively. Compared with that when glucose
phase, but go directly to the logarithmic growth phase. Unlike the first was used as substrate, the total capital investment using liquefied corn
batch, glucose concentrations in the second and third batch increased starch as substrate in batch fermentation was higher because of the
first and then decreased; therefore, the residual glucose concentration addition of the corn starch liquefaction operation unit, but the annual
was ~30 g/L. Based on the above results, repeated-batch fermentation operating cost was lower because of the higher PMA titer and yield.
may be more suitable for efficient PMA production with liquefied corn Thus, the unit production cost was reduced to $2.320/kg, and the re-
starch as substrate. turn on investment was increased to 9.8%. However, regardless of
whether glucose or liquefied corn starch was used as the substrate in
3.6. Process design and techno-economic analysis batch fermentation, the payback period was more than 10 years.
Compared with batch fermentation, repeated-batch fermentation with
To evaluate the feasibility of industrial-scale production of PMA by liquefied corn starch not only reduced the total capital investment
GXL-1, process design and techno-economic analysis were performed at owing to the reduction of seed fermentation operation unit, but also

Fig. 5. PMA production was performed in a 10 L fermentor by repeated-batch fermentation with three batches from liquefied corn starch: (a) time courses of sugar
concentration; (b) time courses of DCW and PMA titer. This repeated-batch fermentation was repeated three times; only one typical run is shown here.

7
W. Zeng, et al. Bioresource Technology 304 (2020) 122990

Table 2
Results of PMA production from liquefied corn starch by repeated-batch fermentation.
Mode Batch Initial sugar (g/L) Residual sugar (g/L) Fermentation time (h) DCW (g/L) PMA (g/L) Yield (g/g) Productivity (g/L·h)

Mode VII Batch 1 140 18.3 ± 1.4 144 30.8 ± 0.7 37.3 ± 1.0 0.31 0.26
Batch 2 107.6 ± 1.9 30.5 ± 1.6 108 33.2 ± 0.8 35.7 ± 0.8 0.46 0.33
Batch 3 110.8 ± 2.4 36.9 ± 1.8 108 29.7 ± 0.7 37.1 ± 1.1 0.50 0.34

reduced annual operating costs because of the shorter fermentation typically require a temperature of 25 °C during fermentation. It is well
duration and low initial sugar concentration required. As a result of the known that fermentation is an exothermic process; therefore, in order
above advantages, the unit production cost of $2.046/kg and the return to maintain the fermentation temperature at 25 °C, additional energy is
on investment of 14.5% were obtained. Thus, repeated-batch fermen- needed for cooling. Therefore, fermentation at 30 °C enables reduction
tation with liquefied corn starch, whose payback period was 6.9 years, of energy consumption. Based on the above comparison and analysis,
has better application value for industrial-scale production of PMA. the production of PMA by A. pullulans GXL-1 from liquefied corn starch
has great potential in industrial application.

3.7. Comparison with other studies

4. Conclusions
PMA production by different wild strains of A. pullulans from var-
ious biomass substrates, via batch fermentation and repeated-batch A novel A. pullulans GXL-1 strain without melanin secretion was
fermentation, has been reported (Leathers and Manitchotpisit, 2013; isolated and identified, and the low-molecular-mass PMA produced by
Zan and Zou, 2013; Zou et al., 2015, 2016; Xia et al., 2016; Wei et al., this strain was characterized. PMA could be produced efficiently by
2017; Cheng et al., 2017; Yegin et al., 2019; Zeng et al., 2019b; Cao GXL-1 from liquefied corn starch through SSF. A high PMA titer of 49 g/
et al., 2019, 2020). PMA titers ranging from 10 g/L to 55 g/L, with a L was achieved in batch fermentation, and PMA yield of 0.50 g/g and
yield of 0.19 ~ 0.54 g/g and productivity of 0.06 ~ 0.52 g/L·h were productivity of 0.34 g/L·h were achieved in repeated-batch fermenta-
obtained from these strains by batch fermentation with multiple low- tion. Furthermore, the production cost of $2.046/kg and payback
cost feedstocks as substrates. Furthermore, the maximum PMA titer, period of 6.9 years were achieved by repeated-batch fermentation
yield, and productivity could reach 75 g/L, 0.56 g/g and 0.78 g/L·h, based on techno-economic analysis of a 5000 MT plant, which provided
respectively, by repeated-batch fermentation. However, most of these an economically feasible strategy for industrial-scale PMA production.
low-cost feedstocks require pretreatment by acid hydrolysis, and toxic
compounds such as 5-hydroxymethylfurfural, furfural, formic acid, and
acetic acid in the hydrolysate inhibit cell growth and PMA synthesis. CRediT authorship contribution statement
Further, although strain GXZ-6 identified in our previous research is
able to utilize malt syrup without any pretreatment to produce PMA Wei Zeng: Conceptualization, Methodology, Writing - original
efficiently, melanin secretion by this strain during PMA production draft, Writing - review & editing, Visualization, Funding acquisition.
makes it difficult to recover and purify the PMA produced (Zeng et al., Bin Zhang: Methodology, Investigation, Visualization. Li Jiang:
2019a, 2019b). Compared with the above-mentioned strains, the pre- Investigation, Validation. Yao Liu: Investigation. Su Ding: Data cura-
sent A. pullulans GXL-1 strain exhibited several excellent properties for tion. Guiguang Chen: Project administration. Zhiqun Liang: Writing -
industrial PMA production. First, GXL-1 was able to produce PMA review & editing, Supervision, Funding acquisition.
through SSF using only liquefied starch as substrate. The PMA titer of
49 g/L was achieved in batch fermentation using liquefied corn starch
as substrate, and the yield of 0.50 g/g and productivity of 0.34 g/L·h Declaration of Competing Interest
were achieved in repeated-batch fermentation. Second, GXL-1 did not
secrete melanin during PMA production, which makes the recovery and The authors declare that they have no known competing financial
purification of PMA easier. Third, GXL-1 produced PMA at a higher interests or personal relationships that could have appeared to influ-
temperature of 30 °C; in comparison, other PMA-producing strains ence the work reported in this paper.

Table 3
Comparison of costs of PMA production from glucose and liquefied corn starch at an annual output of 5,000 MT with different fermentation strategies.
Item Glucose Liquefied corn starch

Batch Batch Repeated-batch

Total Capital Investment $20,750,000 $22,545,000 $21,438,000

Annual Operating Cost $12,438,000 $11,600,000 $10,323,000
Raw Materials 7,658,000 (61.6%) 6,378,000 (55.0%) 5,840,000 (57.1%)
Labor 1,944,000 (15.6%) 2,180,000 (18.8%) 1,770,000 (17.3%)
Depreciation 1,154,000 (9.3%) 1,212,000 (10.5%) 1,038,000 (10.1%)
Utilities 1,180,000 (9.5%) 1,252,000 (10.8%) 1,060,000 (10.4%)
Other Misc 502,000 (4.0%) 578,000 (5.0%) 524,000 (5.1%)
Unit Product Cost $2.488/kg $2.320/kg $2.046/kg
Gross Revenues ($3.0/kg) a $15,000,000 $15,000,000 $15,000,000
Gross Profits $2,562,000 $3,400,000 $4,768,000
Net Profit after Tax (35%) $1,665,300 $2,210,000 $3,099,200
Net Profit Margin 13.4% 19.1% 30.3%
Return on Investment 8.0% 9.8% 14.5%
Payback Period 12.5 years 10.2 years 6.9 years

a
The selling price of PMA in reference to that reported by Wei et al. (2017).

8
W. Zeng, et al. Bioresource Technology 304 (2020) 122990

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