See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/45603629

Acetic Acid Fermentation of Acetobacter pasteurianus : Relationship between

Acetic Acid Resistance and Pellicle Polysaccharide Formation

Article in Bioscience Biotechnology and Biochemistry · August 2010

DOI: 10.1271/bbb.100183 · Source: PubMed

CITATIONS READS

64 454

6 authors, including:

Watchara Kanchanarach Gunjana Theeragool

12 PUBLICATIONS 140 CITATIONS

Kasetsart University
93 PUBLICATIONS 1,196 CITATIONS
SEE PROFILE
SEE PROFILE

Kazunobu Matsushita
Yamaguchi University
434 PUBLICATIONS 11,124 CITATIONS

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Genome View project

All content following this page was uploaded by Watchara Kanchanarach on 20 April 2020.

The user has requested enhancement of the downloaded file.

 Bioscience, Biotechnology, and Biochemistry

ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: https://www.tandfonline.com/loi/tbbb20

Acetic Acid Fermentation of Acetobacter

pasteurianus: Relationship between Acetic Acid
Resistance and Pellicle Polysaccharide Formation

Watchara KANCHANARACH, Gunjana THEERAGOOL, Taketo INOUE,

Toshiharu YAKUSHI, Osao ADACHI & Kazunobu MATSUSHITA

To cite this article: Watchara KANCHANARACH, Gunjana THEERAGOOL, Taketo INOUE,

Toshiharu YAKUSHI, Osao ADACHI & Kazunobu MATSUSHITA (2010) Acetic Acid Fermentation
of Acetobacter�pasteurianus: Relationship between Acetic Acid Resistance and Pellicle
Polysaccharide Formation, Bioscience, Biotechnology, and Biochemistry, 74:8, 1591-1597, DOI:
10.1271/bbb.100183

To link to this article: https://doi.org/10.1271/bbb.100183

Published online: 22 May 2014.

Submit your article to this journal

Article views: 1846

Citing articles: 39 View citing articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=tbbb20
Biosci. Biotechnol. Biochem., 74 (8), 1591–1597, 2010

Acetic Acid Fermentation of Acetobacter pasteurianus: Relationship

between Acetic Acid Resistance and Pellicle Polysaccharide Formation
Watchara K ANCHANARACH,1; * Gunjana T HEERAGOOL,2 Taketo INOUE,1 Toshiharu Y AKUSHI,1
Osao ADACHI,1 and Kazunobu M ATSUSHITA1; y
1
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan
2
Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand

Received March 15, 2010; Accepted April 22, 2010; Online Publication, August 7, 2010
[doi:10.1271/bbb.100183]

Acetobacter pasteurianus strains IFO3283, SKU1108,
and MSU10 were grown under acetic acid fermentation
conditions, and their growth behavior was examined
together with their capacity for acetic acid resistance
and pellicle formation. In the fermentation process, the
cells became aggregated and covered by amorphous
materials in the late-log and stationary phases, but
dispersed again in the second growth phase (due to
overoxidation). The morphological change in the cells
was accompanied by changes in sugar contents, which
might be related to pellicle polysaccharide formation.
To determine the relationship between pellicle forma-
tion and acetic acid resistance, a pellicle-forming R
strain and a non-forming S strain were isolated, and
their fermentation ability and acetic acid diffusion
activity were compared. The results suggest that pellicle
formation is directly related to acetic acid resistance
ability, and thus is important to acetic acid fermentation
in these A. pasteurianus strains.
Key words: acetic acid bacteria; Acetobacter pasteuria-
nus; acetic acid resistance; pellicle polysac-
charide; acetic acid diffusion
Vinegar is produced industrially with acetic acid
bacteria (AAB) and is widely used in many food
products. AAB being obligate aerobes are unique
microorganisms characterized by their strong ability to
oxidize alcohols such as ethanol. In addition, AAB,
especially Acetobacter and Gluconacetobacter species,
tolerate high concentrations of acetic acid, and thus both
genera are used industrially as vinegar producers.
However, in addition to their acetic acid accumulation
ability, Acetobacter and Gluconacetobacter species are
also able to oxidize acetic acid. This phenomenon is
called acetate overoxidation, and it is a nuisance during
vinegar fermentation. The ethanol oxidation (or acetic
acid production), tolerance to acetic acid, and over-
oxidation of acetic acid are important characteristics of
AAB not only for vinegar fermentation, but also for their
microbial physiology.1)
Acetic acid fermentation is catalyzed by two mem-
brane-bound enzymes, alcohol dehydrogenase and alde-
hyde dehydrogenase,2) which produce large amounts of
y
To whom correspondence should be addressed. Tel: +81-83-933-5857; Fax: +81-83-933-5859; E-mail: kazunobu@yamaguchi-u.ac.jp
*
Present address: Department of Biology, Faculty of Science, Mahasarakham University, Mahasarakham 44150, Thailand
Abbreviations: AAB, acetic acid bacteria; AO-phase, acetate over-oxidation phase; AR-phase, acetate resistance phase; CCCP, carbonylcyanide
m-chlorophenylhydrazone; CFU, colony-forming unit; EO-phase, ethanol oxidation phase
acetic acid outside the cells. Thus, as well as ethanol
oxidation capacity, acetic acid resistance is a crucial
factor for AAB to perform fermentation stably. Many
studies of the acetic acid resistance of AAB have been
reported. The molecular mechanisms conferring acetic
acid resistance on Acetobacter aceti have been inves-
tigated using acetic acid-sensitive mutants, and the aarA
gene encoding citrate synthase and the aarC gene were
found to be responsible for acetate assimilation.3,4)
Later, enhanced expression of aconitase was found to
lead the acetic acid resistance,5) and the acetic acid
resistance protein AarC was identified as succinyl-
CoA:acetyl-CoA transferase, converting succinyl-CoA
and acetate to succinate and acetyl-CoA, which process
allows acetic acid to be removed efficiently without
substrate-level phosphorylation.6) The enzymes working
in the TCA cycle are related to acetate overoxidation,
together with acetyl-CoA synthase and phosphoenolpyr-
uvate carboxylase,7,8) but the mechanism responsible for
acetic acid resistance cannot be explained only by
acetate assimilation, because Acetobacter and Glucona-
cetobacter species survive without assimilation of
acetate under high concentrations of acetic acid under
fermentation conditions.1,9) Acetobacter species have
been found to have proton motive force-dependent and
ABC-transporter-like efflux pump systems for acetic
acid in Acetobacter pasteurianus IFO328310) and Ace-
tobacter aceti11) respectively. An additional mechanism
has also been shown that changes in membrane lipid
composition such as phosphatidylcholine and/or a cis-
vaccenic acid are related to acetic acid resistance.9,12)
Hence it has been suggested that acetic acid resistance in
AAB is conferred by several different mechanisms.
Bacteria capable of growing under environmental
stress can survive by developing a variety of mecha-
nisms of resistance. Biofilm formation is one of such the
defense mechanisms. Thus Pseudomonas aeruginosa
able to produce biofilm are more resistant to heavy
metals than planktonic bacteria,13) and biofilmed cells of
Lactobacillus plantarum subsp. plantarum JCM1149 are
more resistant to acetic acid and ethanol than planktonic
cells.14) Acetobacter species, Acetobacter tropicalis
SKU1100 and Acetobacter aceti IFO3284, have been
shown to produce capsular polysaccharides as a pellicle
1592 W. KANCHANARACH et al.
15,16)
or biofilm, and have two different types of cells, the
R (rough colony) and S (smooth colony) strains, where
the R strain but not the S strain produces the
pellicle.17,18) Thus, pellicle formation migh also be a
defense mechanism for acetic acid resistance, though
this has never been confirmed.
In the present study, we investigated acetic acid
fermentation capacity with A. pasteurianus IFO3283,
and also with thermotolerant A. pasteurianus SKU1108
and MSU10, from which the S and R strains were
isolated and examined as to acetic acid fermentation and
polysaccharide production capacity. The R strains of
all three A. pasteurianus, which are able to produce
pellicles, were found to perform fermentation better than
any S strain. Furthermore, the R strains but not the S
strains were found to resist the accumulation of acetic
acid. Thus the results obtained suggest that the pellicle
polysaccharide produced on the cell surface disturbs the
diffusion of acetic acid so as to boost acetic acid
resistance in these A. pasteurianus strains.
Materials and Methods
Materials. [1-14 C] Sodium acetate (9.25 MBq, 60 mCi/mmol,
200 mCi/ml) was purchased from Amersham Biosciences (renamed
GE Healthcare Bio-Science, Tokyo). Hoechst solution and Mounting
Fluorescent Solution were from Dojindo (Kumamoto) and Dako
(Tokyo) respectively. Yeast extract was kindly provided by Oriental
Yeast (Osaka). All other chemicals were commercial products of
guaranteed grade.
Bacterial strains, culture media, and culture conditions. A.
pasteurianus SKU1108 (NBRC101655; National Institute of Technol-
ogy and Evaluation (NITE) Biological Resource Center, Japan), A.
pasteurianus MSU10 (NBRC105870), and A. pasteurianus IFO3283
(Institute for Fermentation, Osaka, IFO, Japan) were used in this study.
Potato medium consisted of 5 g of D-glucose, 20 g of glycerol, 10 g of
yeast extract, 10 g of peptone, and 100 ml of potato extract in 1 liter of
tap water. YPG medium consisted of 2 g each of glycerol, yeast extract,
and peptone in 1 liter of tap water. YPGD medium consisted of 5 g
each of D-glucose, yeast extract, glycerol, and peptone, filled to 1 liter.
Each strain of AAB was first cultivated in 5 ml of potato medium at
30
C with rotary shaking at 200 rpm until a klett unit (Klett-
Summerson photoelectric colorimeter, Klett New York, NY) of 250
(mid-exponential phase) was reached. The seed culture was then
transferred to 100 ml of YPG or YPGD medium supplemented with 2%
or 4% ethanol in a 500-ml shaking flask. The flasks were incubated at
30
C or 37
C with rotary shaking at 200 rpm for 12 d.
Isolation of the S and R strains from A. pasteurianus strains.
A. pasteurianus IFO3283, SKU1108, and MSU10 original strains were
cultured 30 times by transferring the culture to 5 ml of potato medium
every 24 h under shaking or static conditions at 30
C. Finally, the
culture was diluted and spread onto potato agar plates, from which
S-type and R-type colonies were isolated as the S or the R strain.
Transport assay. The A. pasteurianus S or R strains were grown to
late exponential phase in YPGD medium without ethanol, washed
twice in 50 mM potassium phosphate buffer (pH 6.5), and kept on ice.
Before transport assay, cells were suspended in McIlvaine buffer
(pH 6.5) or YPGD medium for acetate uptake assay and acetic acid
diffusion assay respectively, at a final dry weight of about 0.2 g/ml.
The reaction was started by adding acetic acid containing [1-14 C]
sodium acetate (8 mCi, 6.25 mCi/mmol) to final concentrations of
0.4 mM and 80 mM for uptake or diffusion assay respectively into the
cell suspension. Samples of cells, 50 ml in volume, were incubated at
25
C for 10 min for acetate uptake assay and 15 min for acetic acid
diffusion assay, and then withdrawn at intervals and filtered through
0.45-mm pore cellulose acetate filters. The filters were quickly washed
twice with 3 ml of cold 50 mM potassium phosphate buffer (pH 6.5).
After 5 ml of scintillator solution (ACS II Scintillation Cocktail,
Amersham) was added to vials having each filter membrane, radio-
activity was measured with a scintillation counter.
Microscopic observation. The original A. pasteurianus SKU1108
and A. pasteurianus IFO3283 strains were grown on YPGD medium
containing 4% ethanol at each growth phase. The samples (1 ml) were
harvested in an Eppendorf tube and centrifuged at 9;000  g for 5 min
at 4
C, and then resuspended and washed twice under the same
conditions with 50 mM potassium phosphate buffer (pH 6.5). The pellet
was carefully and completely resuspended in 100 to 200 ml of the same
buffer, to which Hoechst, Calcein-AM, and PI solutions were added
quickly at a final concentration of 1 ml/ml. After they were left in the
dark for 15 min, the samples were observed by optical or fluorescent
microscope. They were putting on a slide glass with one drop of
Mounting Fluorescent Solution.
Other analytical methods. The sugar content was measured by the
phenol-sulfuric acid method19) using glucose as standard. The protein
content was measured by a modified Lowry method.20) The acetate
concentration in the culture media was determined by titration with
0.8 N sodium hydroxide in the presence of 5 mM phenolphthalein as pH
indicator.
Results
Growth behavior of A. pasteurianus IFO3283 in
ethanol culture
In ethanol culture (acetic acid fermentation), as shown
in Fig. 1, A. pasteurianus IFO 3283 exhibited three clear
growth phases, in which acetic acid was produced and
remained in the culture medium for a long time before
assimilation. In both 4% ethanol culture and 3% ethanol
culture in the presence of 1% acetic acid, the cell growth
of the IFO3283 strain increased concomitantly with the
increase in acetic acid, and then reached a plateau after
converting all ethanol to acetic acid. We call this
stationary phase the acetic acid resistance phase (the AR
phase). After remaining on the plateau for about 120 h,
cell turbidity increased again, concomitantly with the
consumption of accumulated acetic acid, and hence was
called the acetate overoxidation phase (AO phase),
whereas viable cell numbers, measured as colony-
forming units (CFU), started to decrease during the late
ethanol oxidation phase (EO phase) and decreased
continuously in accordance with acetic acid accumula-
tion. After reaching a threshold (around 104 CFU in this
case), it increased again before the onset of the AO
phase, whereas in the culture containing 1% acetic acid
and not any ethanol, the IFO3283 strain grew after a
relatively long lag phase, which appeared to be
equivalent to the AR phase. In this case, viable cell
numbers also decreased, to nearly 104 CFU, before
commencing utilization of acetic acid.
When we examined the cell situation and morphol-
ogy during ethanol culture directly by light microscope,
the cells appeared to aggregate as ethanol oxidation
proceeded. That is, acetate accumulation proceeded in
the late EO phase and especially in the AR phase,
while such aggregates appeared to vanish during the
AO phase (data not shown). When the same cell
suspension was observed by fluorescent microscope
after staining with fluorescent dyes, the aggregated cell
mass appeared to be covered by amorphous materials
(Fig. 1B). Although the staining principle is not clear,
we think that the amorphous materials might be related
to pellicles that have been found to be produced at the
 Acetic Acid Fermentation of A. pasteurianus 1593

4 10
A 300
a b c

Growth (Klett units)

3
8

Acidity (%)
200

log CFU
2
6
100
1
4

0 0
0 2 4 6 8 0 2 4 6 8 0 2 4 6 8
Time (d)

B a b c d

Fig. 1. Growth Behavior of A. pasteurianus IFO3283 in YPG Medium Containing Ethanol and/or Acetic Acid, and Morphological Changes
Observed under Fluorescent Microscopy in 4% Ethanol Culture.
A, A. pasteurianus IFO3283 was cultivated in 100 ml of YPG medium containing 4% ethanol (a), 3% ethanol and 1% acetic acid (b), and 1%
acetic acid (c), and cultivated at 30
C, as described in ‘‘Materials and Methods.’’ Growth (open circle) and acidity (open triangle) were measured
as described in ‘‘Materials and Methods.’’ CFU (closed circle) was counted after diluting and spreading onto potato-agar plates at each time
point. B, Microscopic analysis of cells during ethanol culture. A. pasteurianus IFO3283 was cultured in YPG medium containing 4% ethanol,
and 1 ml of culture was harvested in the mid EO phase (a, 36 h), in the late EO phase (b, 72 h), in the late AR phase (c, 144 h), and in the mid AO
phase (d, 288 h). The cells were collected by centrifugation, resuspended with 200 ml of 50 mM KPB, pH 6.5, stained for 15 min with fluorescent
dyes, and then observed under a fluorescent microscope.

3.0
A B
Sugar content (µmol/mg of protein)

800 5
Growth (Klett units)

4 2.0
600
Acidity (%)

3
400
2
200
1 1.0

0 0
0 2 4 6 8 10 12
Time (d)

0
1 2 4 12
Days

Fig. 2. Growth Behavior and Changes in Sugar Contents of the Cells in 4% Ethanol Culture of A. pasteurianus SKU1108.
A, A. pasteurianus SKU1108 was cultivated in 100 ml of YPGD medium containing 4% ethanol at 37
C. Growth (closed diamond) and
acidity (open circle) were measured as described in ‘‘Materials and Methods.’’ B, Sugar contents of cells collected at the various growth phases
shown by arrows in (A) at the mid EO phase (1 d), at the late EO phase (2 d), in the AR phase (4 d) and in the late AO phase (12 d). The cells were
harvested, washed, and resuspended in distilled water, and the sugar and protein contents were measured directly using a cell suspension, as
described in ‘‘Materials and Methods.’’

cell surface as capsular polysaccharides in Acetobacter ined relationship between acetic acid fermentation and
species.15,16) pellicle polysaccharide production in SKU1108 strain. In
this study, we assessed acetic acid fermentation with
Acetic acid fermentation and pellicle polysaccharide SKU1108 strain, together with morphological changes
production of thermotolerant A. pasteurianus SKU1108 and with the polysaccharide production, in YPGD
in ethanol culture medium supplemented with 4% ethanol at 37
C
Since growth behavior similar to that of IFO3283 (Fig. 2A). We found that SKU1108 strain converted
strain, as described above, has been observed in ethanol almost completely to acetic acid for 2 d under
thermotolerant A. pasteurianus SKU1108,21) we exam- these culture conditions and started overoxidation after
1594 W. KANCHANARACH et al.

A a b c

S R S R S R
SKU1108 IFO3283 MSU10

B a b c

S R S R S R

SKU1108 IFO3283 MSU10

Fig. 3. Colony Morphology (A) and Growth Behavior in Static Culture (B) of the S and R Strains Isolated from A. pasteurianus SKU1108,
IFO3283, and MSU10.
A, The colony shapes of the S and R strains from A. pasteurianus SKU1108, IFO3283, and MSU10 was compared. B, These strains were
cultivated statically in 5 ml of YPGD medium at 30
C for 5 d.

5–6 d, earlier than the case of IFO3283 grown at 30
C. Table 1. Sugar Contents of Cells of A. pasteurianus SKU1108,
IFO3283, and MSU10 S and R Strains Grown on 4% Ethanol in YPGD
As in the case of IFO3283 strain, the cells appeared to be Medium for 3 d
aggregated and covered by amorphous components, Strains were cultivated in YPGD medium supplemented with 4%
especially in the AR phase (data not shown). Moreover, ethanol by shaking for 3 d, or in potato medium for 1 d at 37
C. The
we collected cells at each phase, the early and late EO cells were then harvested, washed, and resuspended in distilled water.
phases, the AR phase, and the AO phase, and examined The sugar and protein contents of the intact cells were determined
directly using the cell suspension by the phenol-sulfuric acid method
the sugar contents of the cells to estimate polysaccharide and a modified Lowry method respectively.
production. As shown in Fig. 2B, the sugar contents of
the cells increased from the early EO phase to the AR Sugar contenta (mmol/mg of protein)
phase, and finally decreased again in the AO phase. Since A. pasteurianus strains YPGD-4%
the sugar contents of cells have been found to be related Potato medium
ethanol medium
to the polysaccharide attached to the cells, which could SKU1108 S 0:86  0:04 1:33  0:18
be detected only in the R strain, as described,14,15) the SKU1108 R 2:03  0:12 3:61  0:49
culture appeared to be rich in cells having pellicle IFO3283 S 0:74  0:06 0:82  0:01
polysaccharides in the late EO phase to the AR phase. IFO3283 R 1:34  0:004 2:88  0:15
This might be related to the acetic acid resistance of the MSU10 S 0:66  0:02 0:86  0:13
cells. This observation is contrast with the notion that the MSU10 R 1:50  0:02 2:55  0:30
cells of the R strain, producing pellicle polysaccharides, a
The sugar content is shown as a mean  SD of three assays.
do not predominate in shaking culture at least in YPG
medium.22)
these A. pasteurianus strains grown on YPGD medium
Isolation of the S and R strains from A. pasteurianus containing 4% ethanol or potato medium were also
and their acetic acid fermentation and acetic acid examined, and were found to be approximately 2-fold
resistance capacities and 3-fold respectively higher in the R strain cells than
Acetobacter species were found to be separated into in the S strain cells (Table 1). The difference in sugar
a pellicle-forming R strain and a non-producing S contents between the R and S strains appeared to
strain. Therefore, to determine the relationship between reflect the content of pellicle polysaccharides, since the
the pellicle polysaccharide and acetic acid fermentation sugar content detected in the S strain must have been
capacities, the S and R strains were isolated from the due to other components such as lipopolysaccharides,
original cultures of A. pasteurianus IFO3283 and not to the pellicle.15) Thus, all the R strains of these
SKU1108, together with another thermotolerant A. Acetobacter species appeared to produce pellicle
pasteurianus, MSU10,23) as described in ‘‘Materials polysaccharides.
and Methods.’’ As shown in Fig. 3, S colonies obtained
from IFO 3283, SKU1108, and MSU10 strains all Acetic acid fermentation with the S and R strains of
showed shiny smooth-surfaced colonies (the S type) on A. pasteurianus strains
agar plates, while the R colonies were rough-surfaced Acetic acid fermentation of the S and R strains of
colonies (the R type). All the R strains grew on static IFO3283, SKU1108, and MSU10 strains, were exam-
culture to form a pellicle on the medium surface, while ined in YPGD medium containing 4% ethanol at 37
C,
the S strains grew poorly and did not produce any as shown in Fig. 4. It was found that the SKU1108 and
pellicle. The sugar contents of the S and R strains of IFO3283 R strains accumulated 3.4% acetic acid and
 Acetic Acid Fermentation of A. pasteurianus 1595

S strains R strains
A 800 5
SKU1108 4
600
3
400
2
200 1

0 0
B IFO3283 4
600
3
400
Growth (Klett units)

2
200
1

Acidity (%)
0 0

C MSU10
4
600
3
400
2
200
1

0 0
D MSU10
600 4

3
400
2
200
1

0 0
0 2 4 6 8 10 0 2 4 6 8 10 12

Time (d)

Fig. 4. Acetic Acid Fermentation with the S and R Strains from A. pasteurianus SKU1108, IFO3283, and MSU10.
A to C, the A. pasteurianus SKU1108 S and R strains (A), the A. pasteurianus IFO3283 S and R strains (B), and the A. pasteurianus MSU10 S
and R strains (C) were cultivated in YPGD medium containing 4% ethanol at 37
C. D, the A. pasteurianus MSU10 S and R strains were
cultivated in YPGD medium containing 2% ethanol at 37
C. Growth (closed diamond) and acidity (open circle) were measured as described in
‘‘Materials and Methods.’’

exhibited the next overoxidation of acetic acid 5 d and
10 d later respectively, although the MSU10 R strain
accumulated acetic acid to only 2.4% without over-
oxidation, which may have been due to the low acetic
acid resistance capacity of the strain. Hence acetic acid
fermentation was repeated with the MSU10 R and S
strains in YPGD containing 2% ethanol. The MSU10 R
strain accumulated 1.9% acetic acid, and then exhibited
overoxidation after 5 d. On the other hand, the S strains
of the three strains produced only 1.2–1.8% acetic acid,
including MSU10 S strain with 2% ethanol, and did not
exhibit overoxidation. Thus the S strains appeared not to
tolerate the acetic acid produced by themselves.
Based on these results, it is tentatively concluded that
the R strains exhibit higher resistance to acetic acid than
the S strains, suggesting that the pellicle polysaccharide
in the cells gives them high acetic acid resistance capacity.
Acetic acid and acetate transport of the S and R
strains of A. pasteurianus strains
Since the pellicle polysaccharides were expected to
disturb acetic acid diffusion into the cells, acetic acid
uptake and diffusion into the cells were examined with
both the R and the S strain. First, using cells of the
A. pasteurianus S and R strains, acetic acid (apparently
as acetate) uptake was measured with 0.4 mM acetic acid
at pH 6.5 (Fig. 5A). All three S strains, IFO 3283,
SKU1108, and MSU10, exhibited acetate uptake 3–4
fold higher than the R strains. Uptake was decreased
by the addition of an ionophore, carbonylcyanide m-
chlorophenylhydrazone (CCCP), and of a respiratory
inhibitor, NaN3 , the effects of which were different
depending on the strain, although the reasons are not
clear. In order to show more clearly the effects of the
polysaccharides on the diffusion of acetic acid, we
measured acetic acid diffusion using a higher concen-
tration of acetic acid (80 mM instead of 0.4 mM for the
uptake experiment) under more acidic conditions
(pH 3.9 after the addition of 80 mM acetic acid into
the YPGD medium). However, since it was very hard to
measure the uptake of acetic acid under such a high
concentration of cold acetic acid, which disturbs
measurements by competing with the uptake of the
labeled acetic acid, we carefully repeated the uptake
experiments with the high concentration of acetic acid in
the presence of CCCP, which can disturb the efflux of
acetic acid. As shown in Fig. 5B, acetic acid uptake
under these diffusion conditions also showed a higher
accumulation of acetic acid in the S strains than in the R
strains of at least SKU1108 strain, although no signifi-
cant difference was seen clearly in IFO3283 or MSU10
strain. These results indicate that acetic acid enters into
the S strain more easily than the R strain.
Discussion
AAB generally have a strong capacity to oxidize
ethanol to acetic acid and to accumulate it outside the
cell, resulting a decrease in the culture pH to less than
4.1) In such a low pH environment, acetate is protonated
1596 W. KANCHANARACH et al.

A S strains R strains B 60
a b a
SKU1108
15
40
S
10
**
20
5
R
0 0
nmol acetate / mg DW

nmol acetate / mg DW
c d IFO3283 b
15
40

10 S

20 R
5

0 0
e f MSU10 c
15
40
10 S
20
5 R

0 0
0 2 4 6 8 10 0 2 4 6 8 10
Time (min)

Fig. 5. Acetic Acid and Acetate Uptake and Diffusion Assays in the Cells of the A. pasteurianus S and R Strains.
A, Acetate uptake was measured by the addition of 0.4 mM (final) acetic acid including [1-14 C] acetate, as described in ‘‘Materials and
Methods.’’ The reaction mixtures (cell suspensions) were prepared with McIlvaine buffer (pH 6.5) in the absence (open circle) and the presence
of 10 mM CCCP (closed diamond) or 20 mM NaN3 (closed triangle). The assay was done with the A. pasteurianus SKU1108 S and R strains
(a, b), the A. pasteurianus IFO3283 S and R strains (c, d), and the A. pasteurianus MSU10 S and R strains (e, f). B, Acetic acid diffusion was
measured by the addition of 80 mM (final) acetic acid including [1-14 C] acetate, as described in ‘‘Materials and Methods.’’ The cell suspensions
(reaction mixture) were prepared in YPGD medium supplemented with 10 mM CCCP, and incubated for 15 min after the addition of acetic acid
(the pH of the mixture became 3.9 after the addition of 80 mM acetic acid). The white and gray columns represent the S and R strains respectively
of A. pasteurianus SKU1108 (a), A. pasteurianus IFO3283 (b), and A. pasteurianus MSU10 (c). Assays were performed in triplicate, and bars
indicate averages and standard deviation.  Significantly different as between the R and S strains at p < 0:01.

to acetic acid (pKa ¼ 4:76), which can penetrate into the
cells through a membrane by passive diffusion and
decrease the intracellular pH by releasing the proton to
kill the cells. Thus, AAB must have some acetic acid
resistance mechanisms to grow in a medium with a high
concentration of acetic acid. As described in the
introduction, AAB have several mechanisms responsible
for acetic acid resistance, including acetic acid assim-
ilation (detoxification), acetic acid efflux, and protection
against acetic acid diffusion by modification of the lipid
compositions of the cytoplasmic membranes.
Acetobacter species have been found to produce
pellicle polysaccharides on their cell surface, which
might be a kind of biofilm useful for drug resistance,
including acetic acid resistance.17) To examine this idea,
we cultured several A. pasteurianus strains on YPGD
medium containing 4% ethanol. These strains exhibited
three growth phases: they first grow by completely
oxidizing ethanol to acetic acid (EO phase), then they
stop growing and persist for a while in a culture medium
filled with a high concentration of acetic acid (AR
phase), and finally they start growing again by utilizing
the accumulated acetic acid during the AO phase
(Figs. 1, 2). During the late EO and AR phases,
A. pasteurianus were found to have an amorphous layer
surrounding the cells, and also to exhibit higher sugar
contents in the cells, which might be due to the
production of pellicle polysaccharides, because it has
been found that a pellicle-forming R strain produces
non-cellulose-hetero-polysaccharides which yield cells
exhibiting such high sugar contents.15) Hence the R
strains are expected to become predominant in the late
EO and AR phases in ethanol culture. This change in
cell type in proportion to the accumulation of acetic acid
in the culture medium must be due to an adaptive
response for survival under acetic acid stress. A similar
adaptive response for acetic acid was seen in Glucona-
cetobacter europaeus V3, in which the cells changed
from short cells to long rods covered with a spongy
layer.9) On the other hand, in the AO phase, the cells
became dispersed again and perhaps were reduced in the
sugar content, suggesting increases in the S strains. The
change from the S to the R strain and vice versa have
been shown to be due to the changes in the cell
populations, not to conditional expression for the
pellicle, because such a S-R exchange has been found
to occur by adaptive and spontaneous mutation.17,18) The
increase in the pellicle polysaccharide surrounding the
cells may confer the resistance against acetic acid
accumulated during the late EO and AR phases.
In order to determine the relation between acetic acid
resistance and the pellicle polysaccharide, we isolated S
and R cells from A. pasteurianus IFO3283, SKU1108,
and MSU10 and compared acetic acid fermentation
 Acetic Acid Fermentation of A. pasteurianus
capacities related to acetic acid resistance between the S
and R strains. In all three A. pasteurianus strains, the R
strains showed clearly high capacity for acetic acid
fermentation. The cells produced high acetic acid
production of nearly 3.5%, with a typical diauxic growth
during the EO, AR, and AO phases, whereas the S
strains did not complete fermentation, so as to produce
only 1.5% acetic acid, and thus had no AO phase.
Thus the R strain, with the pellicle polysaccharide, had
higher acetic acid resistance ability than the S strain,
which did not produce a pellicle. This suggests that the
polysaccharide surrounding the cells is related to acetic
acid diffusion into the cells.
In order to determine the function of the polysaccha-
ride related to acetic acid resistance, we examined the
difference in acetic acid diffusion between the S and the
R strains. Acetate uptake (influx) measured at neutral pH
was found to be lower in the R strain than in the S strain,
which suggests that pellicle polysaccharides are a barrier
to acetic acid or acetate reaching the cytoplasmic
membranes, but real acetic acid diffusion should be
measured at lower pH, less than the pKa value of acetic
acids, and also with higher concentrations of acetic
acid. Although this experiment was very difficult, as
described in ‘‘Results,’’ the R strain had a lower acetic
acid accumulation than the S strain in the acetic acid
diffusion experiment.
The results obtained in this study suggest that pellicle
polysaccharides are involved in the acetic acid resist-
ance of the A. pasteurianus strains in that it functions as
a biofilm-like barrier to passive diffusion of acetic acid
into the cells.
Acknowledgments
W. K. is grateful to Mahasarakham University for
financial support to pursue her Ph.D. program. This
work was supported by the Program for the Promotion
of Basic Research Activities for Innovative Biosciences.
Part of this work was carried out through a collaboration
in Asian Core Program between Yamaguchi University
and Khon Kaen University, supported by the Japan
Society for the Promotion of Science (JSPS) and the
National Research Council of Thailand (NRCT). We are
grateful to Mr. Nobuhiko Kawabe for technical support,
and to Dr. Hirohide Toyama of the University of the
Ryukyus and Dr. Mamoru Yamada of Yamaguchi
University, for helpful discussion during the course of
this work.
View publication stats
1597
References
1) Matsushita K, Inoue T, Theeragool G, Trček J, Toyama H, and
Adachi O, ‘‘Survival and Death in Bacteria,’’ ed. Yamada M,
Research Signpost, Kerala, pp. 169–181 (2005).
2) Matsushita K, Toyama H, and Adachi O, ‘‘Advances in
Microbial Physiology’’ Vol. 36, eds. Rose AH and Tempest
DW, Academic Press, London, pp. 247–301 (1994).
3) Fukaya M, Takemura H, Okumura H, Kawamura Y, Horinouchi
S, and Beppu T, J. Bacteriol., 172, 2096–2104 (1990).
4) Fukaya M, Takemura H, Tayama K, Okumura H, Kawamura Y,
Horinouchi S, and Beppu T, J. Ferment. Bioeng., 76, 270–275
(1993).
5) Nakano S, Fukaya M, and Horinouchi S, FEMS Microbiol. Lett.,
235, 315–322 (2004).
6) Mullins EA, Francois JA, and Kappock TJ, J. Bacteriol., 190,
4933–4940 (2008).
7) Saeki A, Taniguchi M, Matsushita K, Toyama H, Theeragool G,
Lotong N, and Adachi O, Biosci. Biotechnol. Biochem., 61,
317–323 (1997).
8) Saeki A, Matsushita K, Takeno S, Taniguchi M, Toyama H,
Theeragool G, Lotong N, and Adachi O, Biosci. Biotechnol.
Biochem., 63, 2102–2109 (1999).
9) Trček J, Jernejc K, and Matsushita K, Extremophiles, 11, 627–
635 (2007).
10) Matsushita K, Inoue T, Adachi O, and Toyama H, J. Bacteriol.,
187, 4346–4352 (2005).
11) Nakano S, Fukaya M, and Horinouchi S, Appl. Environ.
Microbiol., 72, 497–505 (2006).
12) Hanada T, Kashima Y, Kosugi A, Koizumi Y, Yanagida F, and
Udaka S, Biosci. Biotechnol. Biochem., 65, 2741–2748 (2001).
13) Teitzel GM and Parsek MR, Appl. Environ. Microbiol., 69,
2313–2320 (2003).
14) Kubota H, Senda S, Nomura N, Tokuda H, and Uchiyama H,
J. Biosci. Bioeng., 106, 381–386 (2008).
15) Moonmangmee S, Kawabata K, Tanaka S, Toyama H, Adachi
O, and Matsushita K, J. Biosci. Bioeng., 93, 192–200 (2002).
16) Moonmangmee S, Toyama H, Adachi O, Theeragool G, Lotong
N, and Matsushita K, Biosci. Biotechnol. Biochem., 66, 777–783
(2002).
17) Deeraksa A, Moonmangmee S, Toyama H, Yamada M, Adachi
O, and Matsushita K, Microbiology, 151, 4111–4120 (2005).
18) Azuma Y, Hosoyama A, Matsutani M, Furuya N, Horikawa H,
Harada T, Hirakawa H, Kuhara S, Matsushita K, Fujita N, and
Shirai M, Nucleic Acids Res., 37, 5768–5783 (2009).
19) Dubois M, Gilles KA, Hamilton JK, Robers PA, and Smith F,
Anal. Chem., 28, 350–356 (1956).
20) Dulley JR and Grieve PA, Anal. Biochem., 64, 136–141 (1975).
21) Chinnawirotpisan P, Theeragool G, Limtong S, Toyama H,
Adachi O, and Matsushita K, J. Biosci. Bioeng., 96, 564–571
(2003).
22) Matsushita K, Ebisuya H, Ameyama M, and Adachi O,
J. Bacteriol., 174, 122–129 (1992).
23) Kanchanarach W, Theeragool G, Yakushi T, Toyama H, Adachi
O, and Matsushita K, Appl. Microbiol. Biotechnol., 85, 741–751
(2010).