Appl Biochem Biotechnol (2018) 186:217–232

https://doi.org/10.1007/s12010-018-2732-4

Producing Acetic Acid of Acetobacter pasteurianus

by Fermentation Characteristics and Metabolic
Flux Analysis

Xuefeng Wu 1,2 & Hongli Yao 1 & Qing Liu 1 &

Zhi Zheng 1,2 & Lili Cao 1,2 & Dongdong Mu 1 &
Hualin Wang 1,2 & Shaotong Jiang 1,2 & Xingjiang Li 1,2

Received: 3 January 2018 / Accepted: 28 February 2018 /

Published online: 19 March 2018
# Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract The acetic acid bacterium Acetobacter pasteurianus plays an important role in
acetic acid fermentation, which involves oxidation of ethanol to acetic acid through the ethanol
respiratory chain under specific conditions. In order to obtain more suitable bacteria for the
acetic acid industry, A. pasteurianus JST-S screened in this laboratory was compared with
A. pasteurianus CICC 20001, a current industrial strain in China, to determine optimal
fermentation parameters under different environmental stresses. The maximum total acid
content of A. pasteurianus JST-S was 57.14 ± 1.09 g/L, whereas that of A. pasteurianus CICC
20001 reached 48.24 ± 1.15 g/L in a 15-L stir stank. Metabolic flux analysis was also
performed to compare the reaction byproducts. Our findings revealed the potential value of
the strain in improvement of industrial vinegar fermentation.

Keywords Acetic acid fermentation . Ethanol respiratory chain .

Acetobacter pasteurianus JST-S . Environmental stress . Metabolic flux analysis

Introduction

Acetic acid bacteria (AAB) include three genera, i.e., Acetobacter, Gluconobacter, and
Gluconoacetobacter, and are involved in vinegar fermentation [1–4]. A. pasteurianus, partic-
ularly A. pasteurianus CICC 20001 (also known as A. pasteurianus Huniang 1.01), is the main

* Xingjiang Li
lixingjiang1978@hfut.edu.cn

1
School of Food Science and Engineering, Hefei University of Technology, No.193 Tunxi Road, Hefei
City 230009 Anhui Province, People’s Republic of China
2
Key Laboratory for Agricultural Products Processing of Anhui Province, Hefei 230009 Anhui
Province, People’s Republic of China
218 Appl Biochem Biotechnol (2018) 186:217–232

species used in industrial acetic acid production in China and can rapidly oxidize ethanol into
acetic acid through the ethanol respiratory chain [5–7]. In order to adapt to varying conditions,
AAB possess resistance against stress induced by high ethanol concentrations, high acetic acid
concentrations, and high temperatures [4, 8, 9]. This tolerance to environmental stress (i.e.,
ethanol oxidation, temperature, and acetic acid resistances) has been studied using Acetobacter
species by analyzing various parameters, including cell growth, biomass, and acetic acid
production [8, 10–13].
The response of oxidized ethanol to acetic acid with AAB mainly involves two key
enzymes in the ethanol respiratory chain, i.e., alcohol dehydrogenase (ADH), which catalyzes
the oxidation of ethanol to aldehyde, and aldehyde dehydrogenase (ALDH), which catalyzes
the oxidation of id [4, 14, 15]. In particular, ADH plays an important role in the fermentation
characteristics of acetic acid bacteria under high alcohol, temperature, and acetic acid stresses
[8, 15, 16]. Previous studies have shown that A. pasteurianus produces large amounts of acetic
acid by oxidizing ethanol and shows high tolerance to acetic acid, even at higher temperatures;
these features are thought to be related to the activity of ADH [8, 15.
The metabolism of acetic acid in A. pasteurianus mainly involves the Embden-Meyerhof-
Parnas (EMP), pentose phosphate (PPP), pyruvate metabolism, ethanol oxidation respiratory
chain, and tricarboxylic acid (TCA) cycle pathways [17–20]. The establishment of effective
metabolic pathways is the basis of quantitative analysis of metabolic logistics and its regula-
tion, guaranteeing the successful implementation of metabolic flux analysis (MFA) [21, 22].
MFA has been applied to improve the yield and productivity of native products synthesized by
microorganisms [20, 23–25].
In this study, the characteristic parameters of A. pasteurianus JST-S, including cell growth
and ADH and ALDH activities, were compared with those of A. pasteurianus CICC 20001 to
identify the most suitable strain for application in industrial vinegar fermentation. The
fermentation cultivation of A. pasteurianus JST-S and A. pasteurianus CICC 20001 was
processed in a 15-L stir tank. The metabolic network was constructed, and a metabolic
equation was given based on genomic information and the metabolic mechanism. Metabolic
flux analysis of the pathway for metabolism from ethanol and glucose was also investigated by
comparison of the two strains.

Materials and Methods

Bacterial Strains and Media

In this study, A. pasteurianus JST-S was isolated from the solid fermentation substrate of
vinegar (Yantai Di Boshi brewing machine Co., Ltd., Yantai, China). It was identified as one of
the A. pasteurianus subspecies by physicochemical characteristics and 16S rDNA gene
homology assay. The 16S rDNA nucleotide sequences of isolated strain was submitted to
NCBI database (the accession number, MF457917). A. pasteurianus CICC 20001 was sup-
ported by China Center of Industrial Culture Collection (CICC). All bacterial strains were
stored in the lab of the College of Food Science and Engineering, the Key Laboratory for
Agricultural Products Processing of Anhui Province, Hefei University of Technology.
Seed medium (YG1) contained 11 g/L glucose, 11 g/L yeast extract, 1.1 g/L MgSO4·7H2O,
3.3 g/L K2HPO4, and 2% (v/v) ethanol. Fermentation medium (YG2) contained 5 g/L glucose,
5 g/L yeast extract, 1.1 g/L MgSO4·7H2O, 3.3 g/L K2HPO4, and different concentrations of
Appl Biochem Biotechnol (2018) 186:217–232 219

ethanol. YPG medium contained 5 g/L yeast extract, 5 g/L peptone, 5 g/L glucose, 17 g/L agar,
and different concentrations of ethanol and acetic acid. Media were divided into 50-mL
aliquots in 250-mL Erlenmeyer flasks and were then sterilized at 121 °C for 20 min prior to
the addition of ethanol and acetic acid. Cells were seeded in YG1 and cultured on an intelligent
thermostatic shaking incubator (ZHP-Y2102L; Shanghai Sanfa Scientific Instruments Co.,
Ltd., Shanghai, China) at 30 °C for 36 h at 150 rpm. Fermentation cultivation was performed
in YG2 medium at 32 °C at 150 rpm. The chemicals used in this study were provided by
Songong Biotech Co., Ltd., Shanghai, China.

Growth Status of the Strains Under Different Ethanol Concentrations

The seed fluid of both strains from YG1 was inoculated into YPG medium containing 4, 6, 8,
10, and 12% (v/v) ethanol. The incubation was performed in a biochemical incubator (LRH-
250; Shanghai Lei Yun Testing Instrument Manufacturing Co., Ltd., Shanghai, China) at 30 °C
for 3 days in order to observe and compare the growth of the strains.

Growth Status of the Strains Under Different Acetic Acid Concentrations

The seed fluid of both strains from YG1 was inoculated into YPG medium with 3% (v/v) ethanol
and 0.9, 1.2, 1.5, 1.8, 2.1, or 2.4% (v/v) acetic acid to determine the acetic acid tolerance of the
two strains. The other conditions of the incubation were the same as described above.

Growth Status of the Strains Under Different Temperature Conditions

The seed fluid of both strains from YG1 was inoculated into YPG medium containing 3% (v/v)
ethanol and 0.9% (v/v) acetic acid and cultivated at 28, 32, 36, 40, or 42 °C for 3 days. The
growth statuses of A. pasteurianus JST-S and A. pasteurianus CICC 20001 were examined
and compared at different temperatures.

Determination of Total Acid Content

The fermentation cultivation was performed in a stirred acetator (Pilot-Acetator 15 L; Sartorius

Stedim Biotech GmbH Shanghai Representative Office, Shanghai, China) with 10 L YG2
medium with 46 g/L ethanol and 12 g/L acetic acid at 32 °C for 72 h at 150 rpm. The aeration
rate was 0.15 L/min, and the inoculation was 10% of the seed liquid cultured for 36 h. The
results are expressed as means ± standard deviations (n = 3). The acidity of the fermentation
medium was measured using 0.1 M NaOH with phenolphthalein as a pH indicator [15, 26, 27].
Biomass was measured by reference to the literature [28].

Determination of Ethanol Content

Ethanol contents were measured using an Agilent 7890A GC System (Agilent Technologies
Investment Co., Ltd., Shanghai, China) with a 30 m × 1.25 mm × 0.4 μm capillary column GC
(DB-624) [29, 30]. N-butyl alcohol (0.1 mL n-butyl alcohol per 1 mL fermentation liquid) was
added as an internal standard. The flow rate of the carrier gas (nitrogen) was 30 mL/min. The injector
and detector temperatures were 200 and 250 °C, respectively. The temperature program of the
capillary column GC was set to 100 °C for 1 min, ramped to 190 °C at 15 °C/min, and then held for
220 Appl Biochem Biotechnol (2018) 186:217–232

3 min. According to the internal standard curve and the peak area ratio of ethanol and n-butanol
alcohol in the sample, the residual ethanol content of the fermentation liquid was calculated.

Determination of Activity of ADH/ALDH Enzymes

AAB were inoculated and cultivated in YG2 medium containing 2, 4, 6, or 8% (v/v) ethanol or 0.9,
1.2, 1.5, or 1.8% (v/v) acetic acid at 32 °C for 36 h at 150 rpm. The crude enzyme was obtained
with reference to former studies [28, 31]. Cultures were centrifuged at 8000×g for 10 min at 4 °C
to collect cells. The samples were washed twice with 0.1 M phosphate buffer saline (PBS pH 7.0,
3 ml buffer per 1 g of wet bacteria) with the same centrifugal condition and resuspended in the
same buffer. Subsequently, the suspension was broken into some active fragments by JY92-II
ultrasonic disintegration (220 W, 3 s 3 s−1) (Ningbo Scientz biotechnology Co., Ltd., Zhejiang,
China) for 10 min in ice-water bath. Fractured fluid was further centrifuged at 10000×g for 30 min
at 4 °C to get supernatant fraction. The ADH and ALDH were measured by reaction with
potassium ferricyanide as an electron acceptor coupled with dehydrogenation of substrate, which
was catalyzed by the enzyme [32]. One unit of enzyme activity was defined as the amount of
enzyme catalyzing the oxidation of 1 μmol of substrate per minute. Specific enzyme activity was
introduced to evaluate enzyme activity level. The supernatant fraction was used instead of pure
enzyme for ADH and ALDH activity assay. All determinations of enzymes activities were
performed at 25 °C. The protein concentration was measured by the modified Lowry method,
with bovine serum albumin as a standard protein [33].

Analysis of Glucose and Acetic Acid Production

High-performance liquid chromatography (HPLC) was performed to determine the concentrations

of glucose and acetic acid in the fermentation liquid [34–36]. The fermentation liquid that had been
cultivated was centrifuged at 25 °C and 10,000×g for 10 min, and 1 mL of the supernatant was
diluted with distilled water and filtered through a 0.22-μm membrane. An Atlantis dC18 150 mm ×
4.6 mm (3.0 μm) chromatographic column (Waters, USA) was used as an analytical column with a
flow rate of 0.6 mL/min, and the column temperature was 30 °C [25, 37]. We used 0.05 M
NH4H2PO4 buffer adjusted to pH 2.5 with phosphoric acid as the mobile phase. The concentrations
of glucose and acetic acid were analyzed by HPLC on a Waters 600 instrument (American Waters
Co., Milford, USA) equipped with a UV detector (Waters 2487) at 210 nm and a refractive index
detector (Waters 2414) at 40 °C. The injection volume of the samples was 10 μL.

MFA

The suggested metabolic network was constructed by substrate utilization and combining some
references and genomic information from the Kyoto Encyclopedia of Genes and Genomes
(www.kegg.jp) [19, 20, 38, 39]. MFA, which was based on the pseudo-steady-state assumption
that there was no accumulation of any intermediates for a certain period of time, was carried out
for the calculation of volumetric rates of intracellular metabolite formation [17, 23, 40]. The
total stoichiometric matrix of all reactants and reaction products (A) was calculated as follows:
AT B ¼ R ð1Þ

In Eq.1, A represents the total stoichiometric matrix for all reactants and products of
reactions, B represents the internal reaction rate (mmol/g DW h), and R represents the net
Appl Biochem Biotechnol (2018) 186:217–232 221

formation rate of metabolites (mmol/g DW h). MF was computed using MINVERSE and
MMULT in Excel 2010 (Microsoft Co., Redmond, WA, USA) [25, 41, 42].

Results

Growth Status of A. pasteurianus JST-S and CICC 20001 Under Different

Environmental Conditions

In order to compare the alcohol tolerance of A. pasteurianus JST-S and A. pasteurianus CICC
20001, the bacteria were cultivated on YPG medium containing different concentrations of
ethanol at 30 °C for 3 days. The results are shown in Fig. 1a. A. pasteurianus JST-S and
A. pasteurianus CICC 20001 showed good growth characteristics, and growth was not
inhibited by ethanol when the ethanol concentration ranged from 4 to 6%. Notably, after
addition of 8% ethanol, A. pasteurianus CICC 20001 showed relatively controlled growth,
with an observable reduction in growth rate, whereas A. pasteurianus JST-S showed no
changes in growth. A. pasteurianus CICC 20001 growth was abolished after addition of
10% ethanol, whereas A. pasteurianus JST-S grew poorly under these conditions. However,
both of strains did not grow well in medium containing 12% ethanol.
Acetic acid was added to YPG medium containing 3% ethanol prior to inoculation of both
strains to study the acetic acid tolerance of the strains (Fig. 1b). When acetic acid was added at
concentrations of 0.9–1.5%, both of the stains could grow well in YPG medium. Increasing the

(A) In turn, a, b, c, d, e

H B

H B

(B) In turn, a, b, c, d, e, f

(C) In turn, a, b, c, d, e
Fig. 1 Growth status of A. pasteurianus CICC 20001 and JST-S under different conditions. H represents
A. pasteurianus CICC 20001 and B represents A. pasteurianus JST-S. In a, a, b, c, d, and e, respectively,
response to 4, 6, 8, 10, and 12% (v/v) of ethanol contents. In b, a, b, c, d, e, and f, respectively, response to 0.9,
1.2, 1.5, 1.8, 2.1, and 2.4% (v/v) of acetic acid contents. In c, a, b, c, d, and e, respectively, response to 28, 32, 36,
40, and 42 °C
222 Appl Biochem Biotechnol (2018) 186:217–232

acetic acid concentration to 1.8% slightly inhibited the growth of both strains. However,
A. pasteurianus CICC 20001 could not grow well, and the growth area of A. pasteurianus
JST-S decreased following the addition of 2.1% acetic acid. Both of strains were not able to
grow when the acetic acid concentration was 2.4%.
Based on studies of ethanol and acetic acid tolerance, the temperature tolerance of
A. pasteurianus JST-S and A. pasteurianus CICC 20001 was also compared by incubation
of the strains at 28, 32, 36, 40, and 42 °C. As described in Fig. 1c, the growth of
A. pasteurianus JST-S and A. pasteurianus CICC 20001 was optimal at 32 °C. If the
temperature was increased or decreased, it was not conducive to the growth of the strains.
When the temperature was 36 °C, the growth area of the strains was decreased, although the
growth area of A. pasteurianus JST-S was slightly larger than that of A. pasteurianus CICC
20001. Both strains showed decreased growth rates at a temperature of 40 °C, and at 42 °C,
A. pasteurianus JST-S showed only very slight growth, and A. pasteurianus CICC 20001 did
not grow.

Assays of Activity of ADH/ALDH Enzymes

To clarify the effects of ethanol assimilation on A. pasteurianus JST-S and A. pasteurianus

CICC 20001, the activities of these key enzymes were measured. As shown in Fig. 2a, the
ADH and ALDH activities of the two strains showed similar trends. The two strains showed
the highest enzyme activities when the ethanol concentration was 4%. ADH and ALDH of
A. pasteurianus JST-S were 5.96 ± 0.26 and 4.82 ± 0.22 U/mg, respectively, whereas ADH and
ALDH of A. pasteurianus CICC 20001 reached 4.78 ± 0.28 and 4.12 ± 0.24 U/mg, respec-
tively. When EC was added to 8%, the ADH and ALDH activities of the two strains were the
lowest, i.e., those of A. pasteurianus JST-S were 2.01 ± 0.10 and 1.74 ± 0.08 U/mg, respec-
tively, and those of A. pasteurianus CICC 20001 were 0.96 ± 0.12 and 1.26 ± 0.12 U/mg,
respectively. The ADH and ALDH activities of A. pasteurianus JST-S were higher than those
of A. pasteurianus CICC 20001 by comparison of different ethanol contents with the whole
fermentation process.
Previous studies have shown that the more stable ADH and ALDH enzymes are at high
acidity, the higher the yield of vinegar [15]. Thus, the activities of ADH and ALDH for the two
strains were compared during vinegar fermentation according to different acetic acid contents.

Ap. JST-S ADH

Ap. CICC 20001 ADH
7 Ap. JST-S ADH
Ap. JST-S ALDH
(A) Ap. CICC 20001 ADH (B) 6
Ap. CICC 20001 ALDH
6 Ap. JST-S ALDH
ADH/ALDH activities (U/mg)
ADH/ALDH activities (U/mg)

Ap. CICC 20001 ALDH 5

5
4
4
3
3

2 2

1 1

0 0
2% 4% 6% 8% 0.9 1.2 1.5 1.8
EC(%) AAC(%)

Fig. 2 The comparison of ADH and ALDH activities with A. pasteurianus CICC 20001 and JST-S. a Different
ethanol contents (2, 4, 6 and 8% (v/v)) and b different acetic acid contents (0.9, 1.2, 1.5 and 1.8% (v/v))
Appl Biochem Biotechnol (2018) 186:217–232 223

As shown in Fig. 2b, the two strains showed the highest enzyme activities, ADH and ALDH of
A. pasteurianus JST-S were 6.08 ± 0.18 and 5.47 ± 0.16 U/mg, respectively, and those of
A. pasteurianus CICC 20001 were 5.66 ± 0.16 and 4.87 ± 0.12 U/mg, respectively, in the
presence of 1.2% acetic acid. Moreover, A. pasteurianus JST-S showed higher ADH and
ALDH activities than A. pasteurianus CICC 20001 for all acetic acid concentrations. ADH
and ALDH from A. pasteurianus JST-S also showed more stability to different acid contents
than those of A. pasteurianus CICC 20001, as shown in Fig. 2b.

Comparison of the Metabolism of A. pasteurianus JST-S and CICC 20001

Vinegar fermentation with A. pasteurianus JST-S and A. pasteurianus CICC 20001 was
carried out in a stirred acetator (15 L) under the same culture conditions in order to compare
their metabolic diversity. As shown in Fig. 3, the maximum total acid yield of A. pasteurianus
JST-S was 57.14 ± 1.09 g/L when the fermentation time was 72 h, whereas that of
A. pasteurianus CICC 20001 was only 48.24 ± 1.15 g/L at the same time. Finally, the residual
ethanol content of A. pasteurianus JST-S was 5.02 ± 1.19 g/L, and that of A. pasteurianus
CICC 20001 was 11.12 ± 1.02 g/L at an initial ethanol concentration of 46.0 g/L. Dry cell
weight was used as an index to compare the cell growth of the two strains; the lag phase of
A. pasteurianus JST-S lasted 12 h, whereas that of A. pasteurianus CICC 20001 lasted for
18 h. The cell growth rates of the two strains slowed down after 48 h of fermentation. When
the fermentation time was 72 h, the dry cell weight of A. pasteurianus CICC 20001 was 0.262
± 0.007 g/L, whereas that of A. pasteurianus JST-S was 0.339 ± 0.008 g/L.

Comparison Between Metabolic Fluxes of A. pasteurianus JST-S and CICC 20001

The main pathways of glucose and ethanol metabolism by A. pasteurianus are shown in Fig. 4.
The ethanol oxidation respiratory chain, pyruvate metabolism, EMP, PPP, and TCA pathways

Fig. 3 The change of substance content during acetic acid fermentation in a 15-L stir tank. Open squares
represent ethanol content of A. pasteurianus JST-S, open circles represent ethanol content of A. pasteurianus
CICC 20001, closed squares represent total acid content of A. pasteurianus JST-S, closed circles represent total
acid content of A. pasteurianus CICC 20001, closed triangles represent dry cell weight of A. pasteurianus JST-S,
and open triangles represent dry cell weight of A. pasteurianus CICC 20001. Error bars show standard deviations
of three replicated measurements
224 Appl Biochem Biotechnol (2018) 186:217–232

Glucose

G6P
PPP

F6P Ru5P
EMP
G3P

PEP
Lactate
Pyruvate Ac.H EtOH

AC-CoA PM
OAA Ace
Malate Citrate
TCA
Fumarate Isocitrate
Succinate

Fig. 4 The main pathways of glucose and ethanol metabolism by Acetobacer pasteurianus. EMP Embden-
Meyerhof-Parnas pathway, PPP pentose phosphate pathway, PM pyruvate metabolism, TCA tricarboxylic acid
cycle, G6P glucose-6-phosphate, F6P fructose-6-phosphate, Ru5P ribulose 5-phosphate, G3P glyceraldehydes-3-
phosphate, PEP phosphoenolpyruvate, AC.H acetaldehyde, OAA oxaloacetate, Acetyl-CoA acetyl coenzyme A,
Ace acetate, EtOH ethanol

were the primary pathways of A. pasteurianus metabolism. A. pasteurianus JST-S and

A. pasteurianus CICC 20001 could use glucose and ethanol to produce acetic acid as well
as many other byproducts, e.g., lactic acid, malic acid, citric acid, and succinic acid [19, 43].
The production of these byproducts by the two strains was compared to evaluate the metabolic
fluxes of the strains (Fig. 5). Reactions and enzymes in the metabolic pathway of acetic acid in
A. pasteurianus are shown in Table 1. According to the basal mass balance and the biochem-
ical reactions shown in Table 1, 33 equations were obtained, as described in Table 2. The
coefficients of 10 equations were obtained from previous studies (Table 2) [37, 44, 45],
whereas those of the others were calculated by dividing the net change in the concentration
of metabolites during the short period of the nearly stationary phase by the duration of this
phase [17, 23, 25].
A. pasteurianus JST-S and A. pasteurianus CICC 20001 were inoculated in YG2 at 32 °C
with 46 g/L ethanol. Ethanol, acetic acid, and glucose concentrations in fermentation liquid
were measured when the fermentation time reached 72 h. The fermentation time was selected
Appl Biochem Biotechnol (2018) 186:217–232 225

Glucose

P1
CO2 P38 BM
ATP

Glucose-6P P3 Ribulose-5P
P37
P4 P5
P2 NAD(P)H
BM
P16 P15 Xylulose-5P
Fructose-6P
P40 P14
P18 P13
BM ATP Eyr-4P P39 BM
Fructose-1,6-2P
P12 P6
NADH P17
P19 P10 P9 P7 Ribose-5P
Sep-7P
P11
G3P BM
P8
P41 P25 Lactate
NADH
P20 NADH
BM BM
P43 AC.H
PEP P22 Pyruvate P23 EtOH
Acety-CoA AC.H P27
P42
P21 ATP P28
P36 P26
BM NADH P24 NADH
ATP
P35 OAA Acety-CoA P29 Ace
P46
BM P44 BM
NADH P29

Malate
Citrate

P34 P30

Fumarate BM Isocitrate
FADH
P45 NADH

P33 P32 a-Ket P31 CO2

Succinate

Fig. 5 Metabolic network of Acetobacer pasteurianus. Glucose-6P glucose-6-phosphate, Fructose-6P fructose-

6-phosphate, Fructose-1,6-2P fructose-1,6-bisphosphate, G3P glyceraldehydes-3-phosphate, PEP phosphoenol-
pyruvate, Ribulose-5P ribulose5-phosphate, Ribose-5P ribose 5-phosphate, Xylulose-5P xylulose-5-phosphate,
Sep-7P sedoheptulose-7-phosphate, Eyr-4P erythrose-4-phosphate, OAA oxaloacetate, Acetyl-CoA acetyl coen-
zyme A, a-K a-ketoglutaric acid, Ac.H acetaldehyde, Ace acetate, EtOH ethanol, BM biomass

at 72 h because cell growth and the increase in total acid contents were nearly abolished at this
time. At this time point, the acetic acid content of the reaction with A. pasteurianus JST-S was
53.43 g/L, and the amount of residual ethanol was 5.96 g/L; for A. pasteurianus CICC 20001,
226 Appl Biochem Biotechnol (2018) 186:217–232

Table 1 Reactions and enzymes in the metabolic pathway of acetic acid in A. pasteurianus JST-S

No. Enzymes Reactions

1 Glucokinase ATP + Glucose = ADP + Glucose-6P

2 Glucose-phosphate-isomerase Glucose-6P = Fructose-6P
3 Phosphofructokinase ATP + Fructose-6P = ADP + Fructose-1,6-2P
4 Fructose-bisphosphate-aldolase Fructose-1,6-2P = 2 × G3P
5 Phosphoglycerate-kinase G3P + NAD+ + Pi + ADP = PEP + NADH + ATP + H2O + H+
6 Glucose-6-phosphate dehydrogenase Glucose-6P + 2NADP+ = 2NADPH + Ribulose-5P + CO2
7 Ribose 5-phosphate isomerase Ribulose-5P = Ribose-5P
8 Ribulose-phosphate-3-epimerase Ribulose-5P = Xylulose-5P
9 Transketolase Ribose-5P + Xylulose-5P = Sep-7P + G3P
10 Transaldolase Sep-7P + G3P = Eyr-4P + Fructose-6P
11 Transketolase Eyr-4P + Xylulose-5P = G3P + Fructose-6P
12 Pyruvate-kinase ADP + PEP = ATP + Pyruvate
13 Alcohol dehydrogenase EtOH + NAD+ = AC.H + NADH + H+
14 Acetaldehyde dehydrogenase AC.H + NAD+ = Ace + NADH + H+
15 Pyruvate dehydrogenase Pyruvate + CoA + NAD+ = Acetyl-coA + CO2 + NADH
16 Pyruvate decarboxylase Pyruvate = AC.H. + CO2
17 Lactate dehydrogenase Pyruvate + NADH + H+ = Lactate + NAD+
18 Pyruvate carboxylase ATP + Pyruvate + HCO3- = ADP + Pi + OAA
19 Phosphotransacetylase/acetate kinase Acety-CoA + Pi + ADP = CoA + Ace + ATP
20 Citrate synthase Acety-CoA + H2O + OAA = Citrate + CoA
21 Aconitase Citrate = Isocitrate
22 Isocitrate dehydrogenase Isocitrate + NADP+ = α-Ket + NADH + CO2
23 Succinate dehydrogenase FAD+ + Succinate =Fumarate + FADH
24 Fumarase Fumarate + H2O = Malate
25 Malate dehydrogenase Malate + NAD+=OAA + NADH + H+

Glucose-6P glucose-6-phosphate, Fructose-6P fructose-6-phosphate, Pi phosphate, G3P glyceraldehydes-3-

phosphate, PEP phosphoenolpyruvate, Ribulose-5P ribulose5-phosphate, Fructose-1,6-2P fructose-1,6-
bisphosphate, Ribose-5P ribose5-phosphate, Xylulose-5P xylulose-5-phosphate, Sep-7P sedoheptulose-7-phos-
phate, Eyr-4P erythrose-4-phosphate, EtOH ethanol, AC.H acetaldehyde, Ace. acetic acid, OAA oxaloacetate,
Acetyl-CoA acetyl coenzyme A, a-K a-ketoglutaric acid

the acetic acid content was 44.68 g/L, corresponding to 12.9 g/L residual ethanol content.
Original flux data for A. pasteurianus CICC 20001 and A. pasteurianus JST-S were obtained
(Table 3) by calculating the 47 equations in Table 2. As described in Table 3, the total flux of
acetic acid in A. pasteurianus JST-S was 6.18186 mmol/g DW h, whereas that of
A. pasteurianus CICC 20001 was 5.16947 mmol/g DW h.
The fluxes of glucose uptake and ethanol uptake in A. pasteurianus JST-S were 0.11994
and 6.04125 mmol/g DW h, respectively, whereas those of A. pasteurianus CICC 20001 were
0.12958 and 5.13596 mmol/g DW h, respectively. In the TCA cycle, the fluxes of citric,
succinic, fumaric, and malic acids in A. pasteurianus JST-S were 0.04293, 0.04139, 0.04139,
and 0.04139 mmol/g DW h, respectively, whereas those of A. pasteurianus CICC 20001 were
0.15216, 0.15063, 0.15063, and 0.15063 mmol/g DW h, respectively.

Discussion

In order to obtain the optimal bacterium for brewing with acetic acid, we compared
A. pasteurianus JST-S screened by our experimental staff with A. pasteurianus CICC
20001, the primary strain used for acetic acid industrial brewing in China. The tolerabilities
of A. pasteurianus JST-S and A. pasteurianus CICC 20001 were studied by comparing the
Appl Biochem Biotechnol (2018) 186:217–232 227

Table 2 Flux equations of A. pasteurianus JST-S

Metabolic Metabolic
Flux equations Flux equations
notes notes
Glucose-6P P1-P2-P3-P37=0 Acety-CoA P26-P29-P44=0
P3-P4-P5-P38=0 Citrate P29-P30=0
Ribulose-5P
P4-P5=0 Isocitrate P30-P31=0
Ribose-5P P5-P7=0 a-Ket P31-P32-P45=0
P4-P6-P15=0 Succinate P32-P33=0
Xylulose-5P
P6-P15=0 Fumarate P33-P34=0
P2+P13+P16-P18-P40=0 Malate P34-P35=0
Fructose-6P
P18-0.5×P19=0 OAA P21-P29+P35-P36-P46=0
G3P P8-P11+P17+P19-P20-P41=0 NADPH 2×P3-P35=0
P20-P21-P22-P42=0 Ace P24+P28=0
PEP
6×P21-P22=0 Glucose P1=0
P22-P23-P25-P26+P36-P43=0 EtOH P27=0
Pyruvate
4×P25-P36=0 BM P47=0
Pyruvate Ace P23-P24=0 P37-0.00535P47=0
AC.H P27-P28=0 P38-0.00895P47=0
P6+P7-P8-P9=0 P39-0.00605P47=0
P8-P9=0 P40-0.00164P47=0
P9-P10=0 BM P41-0.00129P47=0
Sep-7P and P10-P11=0 synthesis P42-0.00689P47=0
Eyr-4P P10-P12=0 P43-0.0086P47=0
P11-P13=0 P44-0.00414P47=0
P12-P14-P39=0 P45-0.00274P47=0
P14+P15-P16-P17=0 P46-0.01959P47=0
P16-P17=0
Glucose-6P glucose-6-phosphate, Fructose-6P fructose-6-phosphate, Ribulose-5P ribulose5-phosphate, Ribose-
5P ribose 5-phosphate, Xylulose-5P xylulose-5-phosphate, G3P glyceraldehydes-3-phosphate, PEP phospho-
enolpyruvate, Sep-7P sedoheptulose-7-phosphate, Eyr-4P erythrose-4-phosphate, OAA oxaloacetate, Acetyl-CoA
acetyl coenzyme A, a-K a-ketoglutaric acid, EtOH ethanol, AC.H acetaldehyde, Ace. acetic acid, BM biomass

growth status under different conditions, based on previous studies on environment tolerance
of bacteria, including alcohol tolerance, acetic acid resistance, and temperature resistance [4, 8,
12]. The results of this study confirmed that there were some differences in the tolerabilities of
A. pasteurianus JST-S and A. pasteurianus CICC 20001 under different environmental
conditions.
Finally, under different alcohol conditions, a comparative study on the growth status of the
two strains showed that A. pasteurianus JST-S and A. pasteurianus CICC 20001 were only
slightly inhibited by alcohol concentrations below 6%, and the ethanol tolerance of
A. pasteurianus JST-S was better than that of A. pasteurianus CICC 20001 when ethanol
was added at a concentration of more than 8% in the medium. Furthermore, the results of
acetic acid tolerance showed that both strains had good tolerance to acetic acid corresponding
to good growth when the acetic acid concentration was 0.9–1.5%. The acid tolerance of
228 Appl Biochem Biotechnol (2018) 186:217–232

Table 3 Original flux data of

A. pasteurianus CICC 20001 and Flux data A B
JST-S
P1 0.12958 0.11994
P2 0.04472 0.09625
P3 0.08187 0.02070
P4 0.03843 0.00784
P5 0.03843 0.00784
P6 0.01921 0.00392
P7 0.03843 0.00784
P8 0.02882 0.00588
P9 0.02882 0.00588
P10 0.02882 0.00588
P11 0.02882 0.00588
P12 0.02882 0.00588
P13 0.02882 0.00588
P14 0.02543 0.00249
P15 0.01921 0.00392
P16 0.02232 0.00321
P17 0.02232 0.00321
P18 0.09494 0.10442
P19 0.18989 0.20884
P20 0.21149 0.21132
P21 0.02966 0.02964
P22 0.17797 0.17783
P23 0.01843 0.14061
P24 0.01843 0.14061
P25 0.00429 0.00428
P26 0.16759 0.04525
P27 5.15105 6.04125
P28 5.15105 6.04125
P29 0.16527 0.04293
P30 0.16527 0.04293
P31 0.16527 0.04293
P32 0.16374 0.04139
P33 0.16374 0.04139
P34 0.16374 0.04139
P35 0.16374 0.04139
P36 0.01715 0.01713
P37 0.00300 0.00300
P38 0.00501 0.00501
P39 0.00339 0.00339
P40 0.00092 0.00092
P41 0.00072 0.00072
P42 0.00386 0.00386
P43 0.00482 0.00482
P44 0.00232 0.00232
P45 0.00153 0.00153
A, A. pasteurianus CICC 20001; P46 0.01097 0.01097
P47 0.56004 0.56004
B, A. pasteurianus JST-S

A. pasteurianus JST-S was superior to that of A. pasteurianus CICC 20001 when comparing
the growth statuses of both strains at acetic acid concentrations of more than 1.8%. Finally, a
comparison of the temperature tolerance of the strains indicated that the resistance of
A. pasteurianus JST-S to temperature was higher than that of A. pasteurianus CICC 20001.
We also studied the ADH and ALDH activities of both strains in the presence of different
concentrations of alcohol and acetic acid in order to further compare the tolerance of
Appl Biochem Biotechnol (2018) 186:217–232 229

A. pasteurianus JST-S with that of A. pasteurianus CICC 20001 [14, 18]. Notably, for alcohol
concentrations of 2 to 8% (v/v), ADH and ALDH enzyme activities initially increased and then
decreased. ADH and ALDH enzyme activities were highest at 4% alcohol content and lowest
at 8% alcohol content, which indicated that ADH and ALDH enzyme activities were improved
by adding a certain amount of ethanol. ADH and ALDH enzyme activities decreased due to
inhibition of cell growth when the ethanol content was higher. A comparison of ADH and
ALDH enzyme activities between A. pasteurianus JST-S and A. pasteurianus CICC 20001
under the same alcohol concentrations showed that the alcohol tolerance of A. pasteurianus
JST-S was higher than that of A. pasteurianus CICC 20001. Similarly, the effects of different
acetic acid concentrations on the enzyme activities also confirmed that the acetic acid tolerance
of A. pasteurianus JST-S was superior to that of A. pasteurianus CICC 20001 [28].
Fermentation with A. pasteurianus JST-S and A. pasteurianus CICC 20001 was carried out
in a 15-L stir stank for 72 h to obtain the strain with highest acid production and good growth
under high alcohol concentrations. Importantly, we found that the maximum total acid content
of A. pasteurianus JST-S was higher than that of A. pasteurianus CICC 20001, whereas the
residual alcohol content of A. pasteurianus JST-S was lower than that of A. pasteurianus CICC
20001, indicating that the average rate of acetic acid production by ethanol in A. pasteurianus
JST-S was greater than that of Ap. CICC 20001. The dry cell weight of A. pasteurianus JST-S
was higher than that of A. pasteurianus CICC 20001 because A. pasteurianus JST-S was more
resistant to alcohol than A. pasteurianus CICC 20001. The growth rate of A. pasteurianus JST-
S was higher than that of A. pasteurianus CICC 20001 under the same fermentation condi-
tions. Hence, A. pasteurianus JST-S could reduce the production cost of acetic acid by
shortening the fermentation time, making it attractive for industrial applications.
In order to obtain strains with higher acetic acid yields, MFA was applied to compare
metabolites of A. pasteurianus JST-S and A. pasteurianus CICC 20001 during the acetic acid
fermentation process [23, 46]. Our results showed that the optimal fermentation time was 72 h,
and the total flux of acetic acid in A. pasteurianus JST-S was higher than that in
A. pasteurianus. CICC 20001, whereas the glucose uptake in the former was lower than that
in the latter. These results indicated the metabolic flux of A. pasteurianus JST-S flowing into
the PPP pathway was lower than that of A. pasteurianus CICC 20001, whereas the ability of
A. pasteurianus JST-S to oxidize ethanol to acetic acid was superior to that of A. pasteurianus
CICC 20001. The fluxes of citric, succinic, fumaric, and malic acids in A. pasteurianus JST-S,
as byproducts during acetic acid fermentation, were lower than those in A. pasteurianus CICC
20001, which may be because the increase in acetic acid production in bacteria bodies
inhibited the metabolism of pyruvate to various organic acids in the TCA cycle [43].
In this study, we found that tolerance of A. pasteurianus JST-S to ethanol, acetic acid, and
temperature was superior to that of A. pasteurianus CICC 20001 by a comparison of their
growth statuses and ADH and ALDH activities. Moreover, MFA showed that the internal
reaction rates of the byproducts in A. pasteurianus JST-S were lower than those in
A. pasteurianus CICC 20001. Thus, our findings demonstrated that these strains may have
applications in the improvement of industrial acetic acid productivity and as a useful reference
for similar fermentation of organic acids by other bacteria. In the future, the tolerance
mechanism of A. pasteurianus JST-S must be studied in more depth by combining molecular
biology and proteomics. To improve acetic acid production of A. pasteurianus JST-S, the
modifications of genetic and proteome will be used. MFA, as an essential tool for metabolic
engineering [47, 48], was not only widely applied to calculate the maximum theoretical yield
of a desired metabolite to be produced, but also to reduce the time, money, and effort required
230 Appl Biochem Biotechnol (2018) 186:217–232

to develop metabolically engineered strains for the enhanced production of acetic acid. Along
these lines, engineered strains of A. pasteurianus JST-S will be constructed in our laboratory.

Funding Information This work was supported by National Natural Science Foundation of China
(31601465), Project of Hefei University of Technology (JZ2017YYPY0247/JZ2016YYPY0041), and
Anhui Science and Technology Project (15CZZ03100).

Compliance with Ethical Standards

Conflict of Interest The authors declare that they have no conflict of interest.

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