Journal of Applied Microbiology 1999, 86, 55–62

A thermotolerant and high acetic acid-producing bacterium

Acetobacter sp. I14–2
S.-F. Lu, F.-L. Lee and H.-K. Chen
Food Industry Research and Development Institute, Hsinchu, Taiwan, Republic of China
6714/04/98: received 28 April 1998, revised 13 July 1998 and accepted 16 July 1998

A thermotolerant bacterium with high production

S .- F. L U, F. - L. LE E AN D H . -K . C H EN . 1999.
of acetic acid was isolated from spoiled banana in Taiwan. The isolate, I14–2 ,was considered
to be an Acetobacter sp. according to phenotypic and chemotaxonomic characteristics.
Optimal cultural conditions for Acetobacter sp. I14–2 to produce acetic acid were
studied under cultivation in a medium containing 2 mg l−1 acetic acid and 5% ethanol
at 30 °C. Acetic acid productivity by Acetobacter sp. I14–2 was almost two and
three times the amount produced by Acet. aceti IFO3283 and Acetobacter sp. CCRC
12326, respectively. The isolate retained 22% residual acetic acid-producing
activity after 3 d incubation in a medium containing 8% ethanol, and produced acetic
acid in a medium containing 10 g l−1 acetic acid. This bacterium is thermotolerant
and retained 97% and 68% of acetic acid-producing activity after 3 d incubation at
35 °C and 37 °C, respectively, compared with that when incubated at 30 °C.

INTRODUCTION controlled at 30 °C. In recent years, the temperature in sum-

mer has gradually increased in many countries. In Taiwan,
Production of acetic acid can result from biological oxidation
the average temperature indoors at night during the summer
of ethanol by acetic acid bacteria. It involves a two-step
is over 30 °C. Therefore, almost all domestic vinegar manu-
reaction catalysed by two membrane-bound enzymes, alcohol
facturers can produce vinegar for only half the year. Today,
dehydrogenase and aldehyde dehydrogenase. Besides these
several technologies, including recombinant DNA and
two enzymes, cytochrome c and terminal oxidase are two
spheroplast fusion, and immobilized cells have been
important components for ethanol oxidation (Tamaki et al.
developed to improve these industrial bacterial properties
1991). Acetic acid bacteria were classified into two genera,
such as an increase in acetic acid production rate or enhance-
Acetobacter and Gluconobacter, based on their abilities to over-
ment of the tolerance to high acetic acid, ethanol and fer-
oxidize acetate or lactate and the positions of their flagella
mentation temperature (Lotong et al. 1989; Fukaya 1994).
(De Ley et al. 1984). Since Acetobacter prefers to oxidize
Ohmori et al. (1980) isolated three Acetobacter strains with
ethanol more strongly than glucose and Gluconobacter prefers
the ability to produce acetic acid at 37 °C from vinegar mash.
glucose more than ethanol, most strains useful in vinegar
Saeki et al. (1997) screened some thermophilic acetic acid
manufacture belong to Acetobacter, whereas Gluconobacter
bacteria showing the same fermentation efficiency at 38–40 °C
is used for industrial applications such as fermentation of
as that of mesophilic strains at 30 °C. In this report, the
ketogluconic acid, sorbose and dihydroxyacetone (Swings
isolation of a thermotolerant Acetobacter sp. I14–2 strain cap-
1991). Among the Acetobacter species, Acet. aceti, Acet. past-
able of producing high acetic acid is described and its proper-
eurianus, Acet. polyoxogenes and Acet. europaneus are the most
ties characterized.
popular strains for making acetic acid in vinegar factories as
their oxidization for ethanol is better and they do not attack
acetic acid later (Entani et al. 1985; Sievers et al. 1992).
As these bacteria useful for acetic acid production are MATERIALS AND METHODS
mesophilic strains with optimum temperature for growth at
about 30 °C, most industrial vinegar production is strictly Bacterial strains

Correspondence to: F.-L. Lee, Food Industry Research and Development Some acetic acid-producing bacteria, including Acet. aceti
Institute, P.O.Box 246, Hsinchu, Taiwan, Republic of China. IFO 3283, Acet. aceti DSM 2002, Acet. xylinum ATCC 11142,
© 1999 The Society for Applied Microbiology
56 S .- F. L U E T A L .
Acet. pasteurianus ATCC 9432, Acet. pasteurianus ATCC 6438
and Acetobacter sp. CCRC 12326, were isolated from vinegar
and used in this study as reference strains.
Culture media and conditions
The acetic acid bacteria in the samples tested were first
enriched in an enrichment medium at 30 °C with shaking in
a rotary shaker at 150 rev min−1 for 3–5 d. The medium
contained (g l−1): potato dextrose broth (Difico), 110;
peptone, 3; yeast extract, 5; ethanol, 5; acetic acid, 0·3; pH 4·2.
A screening medium (AC medium) modified from Yamada
(1986) was used to isolate acetic acid-producing bacteria from
the enrichment medium culture. The AC medium consisted
of all the components of enrichment medium except acetic
acid and was amended with (g l−1): CaCO3, 5; agar, 20. For
evaluation of acetic acid production, the isolates were grown
in 10 ml seed medium for 2 d and 1·5 ml of this liquid culture
was transferred to 50 ml fresh seed medium and incubated at
30 °C with shaking at 150 rev min−1 for 2 d. A medium
modified from Saeki (1993) was used as seed medium and
contained (g l−1): glycerol, 5; dextrose, 5; polypeptone
(Wako), 5; yeast extract, 5; ethanol, 20; acetic acid, 2; pH 4·3.
The main culture was prepared by inoculating 1 ml of the
second seed culture into a medium containing (g l−1):
glycerol, 2; dextrose, 30; polypeptone (Wako), 2; yeast extract,
2; ethanol, 50; acetic acid, 2; pH 4·1 and shaking at 50
rev min−1 for 3 d. The culture was centrifuged at 4 °C and
13 000 g for 15 min, and the supernatant fluid collected to
determine the amounts of acetic acid, ethanol and gluconic
acid. Growth of the micro-organism was determined by the
absorbance of the culture broth at 600 nm using a spec-
trophotometer (DU 640, Beckman, Palo Alto, CA, USA).
Characterization and identification of micro-
organisms
Phenotypic characteristics. The morphological charac-
teristics of the isolate were determined by the conventional
method (De Ley et al. 1984). Ninety-five carbon source util-
ization patterns of the isolate were determined using a rapid
identification system, Biolog MicroStationTM (Biolog, Inc.,
CA, USA) (Fredrickson et al. 1991). To examine the oxi-
dation of acetate and lactate, the isolate was cultivated in a
medium containing 2 g l−1 sodium acetate or sodium lactate
and 0·02 g l−1 bromothymol blue at 30 °C for 5–7 d (Asai
et al. 1964).
Cellular fatty acid composition. Cellular fatty acid com-
position of the isolate was analysed using the MIDI Microbial
Identification System (Microbial ID, Inc., DE, USA). The
cells were grown on trypticase soy broth (BBL) containing
© 1999 The Society for Applied Microbiology, Journal of Applied Microbiology 86, 55–62
20 g l−1 agar at 30 °C for 24 h, harvested, and lysed according
to the manufacturer’s instructions to prepare methyl esters
of cellular fatty acids. The esters were analysed by gas chro-
matography according to the method of Steele et al. (1992).
Isoprenoid quinones. Isoprenoid quinones of the isolate were
extracted with chloroform–methanol (2:1, v/v) and purified
by thin layer chromatrography (TLC) on silica gel 60F254
(Merck, 20 × 20 cm) using benzene as developing solvent.
Quinones recovered from the TLC plates were dissolved in
acetone and analysed by high performance liquid chro-
matrography (HPLC) as described by Akagawa-Matsushita
et al. (1992) with minor modification. The HPLC system
was equipped with a reversed-phase column (Nova-Pak C18,
3·9 × 150 mm, Waters, Milford, MA, USA), and a mixture
of methanol and isopropanol (2:1, v/v) was used as the mobile
phase at 1 ml min−1 flow rate. Types of quinone were ident-
ified by absorption at 275 nm and comparison with standards.
DNA base composition and DNA homology. Fresh cells were
harvested from AC broth after incubation for 2 d and digested
with lysozyme and protease K at 37 °C for 1 h. Bacterial DNA
was isolated and purified using the Qiagen Genomic DNA
Kit (Qiagen Inc., Germany). The molar percentage of guan-
osine plus cytosine of chromosomal DNA was determined by
HPLC (Tamaoka and Komagata 1984). DNA homologies
between the isolate and Acetobacter type strains were deter-
mined according to the method of Ezaki et al. (1989).
Acetic and gluconic acids assay
Acetic acid production of the isolate was assayed according
to the method of Pecina et al. (1984). A BioRad (Hercules,
CA, USA) Aminex HPX-87H column was used in the HPLC
system and maintained at 40 °C, and 5 mmol l−1 H2SO4 was
used as the mobile phase with a flow rate of 0·6 ml min−1.
Acetic acid and ethanol in the eluate were detected and quant-
ified by monitoring with an RI detector. Gluconic acid pro-
duced by the isolate was evaluated by HPLC with two
connecting Shodex (Showa Denko Co., Tokyo, Japan) KC
811 columns maintained at 40 °C; 5 mmol l−1 HClO4 was
used as the mobile phase with a flow rate of 1·0 ml min−1. A
reagent containing 0·2 mmol l−1 bromothymol blue in
15 mmol l−1 Na2HPO4 was used to separate organic acid
components, and gluconic acid was detected by the absorb-
ance at 445 nm. Samples were mixed with 100 ml 200 g l−1
sulphosalicylic acid and centrifuged at 9000 g for 10 min
before analysis.

Effects of initial acetic acid, and ethanol
concentrations and temperature, on acetic acid
production
The effect of acetic acid on acetic acid production was studied
by inoculating the isolate in media amended with different
amounts of acetic acid ranging from 0 to 40 g l−1. The effect
of ethanol concentration on acetic acid production was stud-
ied by incorporating 1–10% (v/v) ethanol in the media.
Optimum temperature of acetic acid production was deter-
mined by incubating the isolate at various temperatures for 3
d, and the residual activity of acetic acid production in each
sample was compared with the control sample which was
cultured at 30 °C.
RESULTS
Isolation of acetic acid-producing bacteria
Eighty-six samples including 35 from fresh fruits, 24 from
flowers, 14 from rotten fruits, six from soils, five from wine
with dregs and two from the wood of fruit trees were used to
isolate acetic acid-producing bacteria. The colonies with acid
production, which was indicated by the formation of a clear
zone around the cells on AC screening medium, were isolated.
All isolated strains were preliminarily characterized by mor-
phology and cellular fatty acid composition. Sixty-nine
strains of acetic acid-producing bacteria were obtained, 21
from fresh fruits, 11 from flowers, 28 from rotten fruits, one
from soil, three from wine with dregs and five from the wood
of fruit trees. According to Swings (1991), acetic acid bacteria
occur in many natural niches such as flowers, fruits, beehives
and tea-fungus. In the present study, it was found that the
frequency of isolating acetic acid bacteria from rotten fruits
was higher than that from other samples; two strains on
average were isolated from each rotten fruit sample and fewest
strains were isolated from soils. The abilities of the tested
isolates to oxidize ethanol to acetic acid in a medium con-
taining 5% ethanol are listed in Table 1. Acetobacter aceti IFO
3283, an excellent bacterium for vinegar production (Saeki
1993), produced 23 g l−1 acetic acid after incubation for 3 d
in this study. Six of the isolated strains produced more acetic
acid than all the reference strains, and seven strains had
abilities to oxidize ethanol similar to Acet. aceti IFO 3283.
One strain, isolate I14–2, with the highest acetic acid pro-
ductivity, was chosen for further studies. Acetic acid pro-
ductivity of this isolate was twice that of Acet. aceti IFO 3283.
Isolate I14–2 was isolated from spoiled banana collected at
Taichung, Taiwan.
Taxonomic studies on isolate I14–2
Selected phenotypic and chemotaxonomic characteristics of
this isolate are shown in Table 2. The results of studies on
© 1999 The Society for Applied Microbiology, Journal of Applied Microbiology 86, 55–62
T HE RM O TO LE R AN T A C ET OB A CT ER S P. I1 4 -2 57
its ability to oxidize acetate and lactate, two important charac-
teristics for differentiating the genus Acetobacter from Glu-
conobacter, showed that isolate I14–2 belongs to the genus
Acetobacter. This isolate contains a straight-chain unsaturated
fatty acid, C18:1, as the major component and a small amount
of a straight-chain saturated fatty acid C14:0, which is an
important characteristic of the genus Acetobacter (Yamada
et al. 1981). This result indicates that isolate I14–2 is an
Acetobacter strain. In order to identify the isolate, quinone
type, G ¦ C content of DNA, and DNA homology were
determined. Isolate I14–2 contains Q9 as a major quinone and
Q8 as a minor quinone. This distinguished the isolate from
Acet. liquefaciens, Acet. xylinum, Acet. diazotrophicus and Acet.
methanolicum (Gillis et al. 1989). The G ¦ C content of the
DNA of isolate I14–2 was 51·7 mol l−1 %. The quinone type
and the G ¦ C content of the DNA of isolate I14–2 were
close to those of Acet. aceti and Acet. pasteurianus. Using these
two Acetobacter type strains, Acet. aceti NCIMB 8621 and
Acet. pasteurianus NCIMB 12228, as probes, the isolate I14–
2 exhibited low levels (3·0 and 55·7%) of DNA homology
with strain NCIMB 8621 and strain NCIMB 12228, respec-
tively. Based on the above results, the isolate I14–2 should
be considered as a species that is genetically distinct from
previously described Acetobacter species. Therefore, we pro-
pose that this isolate should be a new Acetobacter species.
Time course of acetic acid production from ethanol
by Acetobacter sp. I14–2
In the time course study of acetic acid production from
ethanol by isolate I14–2, it was found that acetic acid accumu-
lation paralleled cell growth (Fig. 1). The lag period for acetic
acid production by isolate I14–2 was not obvious (within
12 h), while the lag period for cell growth was not observed.
Ethanol was consumed steadily until cell growth reached
stationary phase after 3·5 d of cultivation, whereupon acetic
acid accumulation reached a maximum level of about 50
g l−1. It was found that gluconic acid formation occurred during
the log phase along with acetic acid accumulation, and no
further production was observed during the stationary phase.
Effect of initial acetic acid concentration on acetic
acid production
Acetic acid was added to the culture medium at con-
centrations of 0, 2, 5, 10, 15, 20, 30 and 40 g l−1, giving initial
medium pH values of 5·44, 3·71, 3·44, 3·22, 3·11, 3·01, 2·93
and 2·83, respectively. Acetic acid production by isolate I14–
2 reached the highest acetic acid accumulation level of 38
g l−1 at an initial acetic acid concentration of 2 g l−1, which
is similar in trend to results with Acet. aceti IFO 3283 and
Acetobacter sp. CCRC 12326 but with different levels of
accumulation (Fig. 2). This isolate produced the same amount
58 S .- F. L U E T A L .

Table 1 Acetic acid production from ethanol by isolated strains and reference strains
–—–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Strains Acetic acid (g l−1) Ethanol (%) Gluconic acid (g l−1)
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
—
Acetobacter aceti IFO 3283 23·0 1·68 1·14
Acet. aceti DSM 2002 14·0 2·80 1·76
Acet. pasteurianus ATCC 9432 6·1 3·17 0·35
Acet. pasteurianus ATCC 6438 13·3 2·83 0·91
Acetobacter sp. CCRC 12326 15·2 2·80 0·81
I4-4 24·8 1·93 0·38
I8-1 23·1 2·06 0·43
I10-3 30·3 1·35 0·72
I14-2 43·6 0·39 1·26
I16-2 25·8 2·01 0·59
I21-2 24·4 1·87 1·74
I34-2 25·4 1·88 1·04
I40-1 26·0 1·59 0·52
I41-1 31·1 1·48 0·42
I42-1 33·6 1·24 0·69
I48-1 28·7 1·66 0·52
I48-3 32·5 1·38 2·41
I56-2 24·5 2·08 0·47
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
—
All strains were tested in a medium containing 5% (v/v) ethanol and incubated at 30 °C with shaking at 150 rev min−1 for 3 d before
analysis.

of acetic acid as it did in a medium without the addition of
acetic acid. At an initial acetic acid concentration of 10 g l−1,
isolate I14–2 retained about 20% of its maximal activity of
producing acetic acid at 2 g l−1 (Fig. 2a), while Acet. aceti
IFO 3283 retained about 68% (Fig. 2b) and Acetobacter sp.
CCRC 12326 retained none (Fig. 2c). At an initial acetic acid
concentration of 15 g l−1, however, only Acet. aceti IFO 3283
retained about 50% of its maximal activity of acetic acid
production. Gluconic acid production by these three strains
was inhibited steadily by increasing initial acetic acid con-
centration. This indicates that gluconic acid formation is
repressed either by acetic acid added initially and produced
during fermentation, or by low pH caused by acetic acid.
Effect of initial ethanol concentration on acetic acid
production
The acetic acid produced by isolate I14–2 showed an almost
symmetrical profile over a range of initial ethanol con-
centration of 0–10% with a peak at 5% (Fig. 3a). At initial
ethanol concentrations of 1–5%, the amount of acetic acid
produced by the isolate increased in proportion to the initial
ethanol concentration, while the amount of gluconic acid
produced decreased with the initial ethanol concentration. At
initial ethanol concentrations of 5–10%, the amount of acetic
acid produced by the isolate decreased in proportion to the
initial ethanol concentration, while the amount of gluconic
© 1999 The Society for Applied Microbiology, Journal of Applied Microbiology 86, 55–62
acid produced remained low. At the peak at 5% ethanol,
Acetobacter sp. I14–2 produced about 44 g l−1 of acetic acid,
which is twice that produced by Acet. aceti IFO 3283 at the
same ethanol concentration (Fig. 3b), but the amounts of
gluconic acid produced by both strains were almost the same.
The optimum ethanol concentration for acetic acid pro-
duction was 5% for both isolate I14–2 and Acet. aceti IFO
3283, and 3% for Acetobacter sp. CCRC 12326 (Fig. 3c). The
production of gluconic acid, a product oxidized from glucose,
by the three strains reached the highest level in a medium
without ethanol addition. It is reasonable to suppose that
Acetobacter would oxidize glucose to gluconic acid when etha-
nol is absent, but would prefer to use ethanol rather than
glucose when ethanol is present. In addition to higher acetic
acid productivity, isolate I14–2 revealed a higher ethanol
tolerance than the two reference strains. This isolate was still
active in the presence of 9% ethanol (Fig. 3a). The tolerance
to ethanol of mesophilic strains such as the two reference
strains is lower than 8% ethanol. The relative activities for
producing acetic acid at 8 and 9% ethanol were about 22 and
7·3%, respectively, that at 5% of ethanol.
Effect of temperature on acetic acid production
Acetic acid was produced by Acetobacter sp. I14–2 over a
temperature range from 20 to 37 °C with an optimum at 30 °C
(Fig. 4a). The 3 day acetic acid productivities were 97% and
 T HE RM O TO LE R AN T A C ET OB A CT ER S P. I1 4 -2 59

Table 2 Selected phenotypic and chemotaxonomic 5 60 1·5 4

characteristics of the isolate I14-2

Residual ethanol (%)

50

Gluconic acid (g l )
4

–1
—
–––––––––––––––––––––––––––––––––––––––––––––––––––––

Acetic acid (g l–1)

3
Phenotypic characteristics 40 1·0

O.D.600
1. Rod-shaped 2. Gram-negative 3
30 2
3. Motility: (−) 4. Anaerobic growth: (−)
2
5. Catalase: (¦) 6. Oxidase: (−) 20 0·5
7. Lactate oxidation: (¦) 8. Acetate oxidation: (¦) 1
1 10
9. Assimilation of carbon sources
(¦): a-D-glucose, D-arabitol, D-fructose, D-galactose, methyl
0 0 0 0
pyruvate, acetic acid, formic acid, r-hydroxy butyric 0 1 2 3 4 5 6 7 8 9 10
acid, D,L-lactic acid, bromo succinic acid, L alanine, Cultivation time (d)
and glycerol
Fig. 1 Time courses of acetic acid and gluconic acid production
(−): L-arabinose, D-mannose, maltose, D-mannitol, N-
in relation to cell growth of Acetobacter sp. I14–2. Seed culture (1%)
acetyl-D-glucosamine, L-fucose, D-trehalose,
was inoculated into a main culture containing 5% (v/v) ethanol
dextrin, glycogen, adonitol, phenylacetate, cellobiose, a-
and 2 g l−1 acetic acid, and cultivation was conducted with shaking
lactose, L-rhamnose, D-raffinose, turanose, D-
gluconic acid, malate, caprate, citrate, adipate, succinic at 50 rev min−1 and 30 °C. The amounts of ethanol, acetic acid
acid, psicose, and sucrose and gluconic acid of the fermentation broth were determined as
described in the text. Ethanol (Ž); acetic acid (ž); gluconic
Cellular fatty acid composition acid (); O.D.600 (t)
1. C18:1 51·71% 6. C18:0 2·69%
2. C19:0 cyclo 16·30% 7. C14:0 2·43%
3. C16:0 12·47% 8. C16:0 3OH 1·87%
4. C14:0 2OH 5·74% 9. C16:1 iso 1·26% DISCUSSION
5. C16:0 2OH 4·37% 10. C20:3 1·15%
Acetobacter sp. I14–2 showed a higher acetic acid productivity
Quinone Q9 (Q8) than either Acet. aceti IFO 3283 or Acetobacter sp. CCRC
G ¦ C content of DNA 51·7 mol % 12326, and could oxidize ethanol to acetic acid at tem-
DNA homology 3% with Acetobacter aceti peratures above 30 °C. When incubated as a static culture,
NCIMB 8621 this isolate did not form a pellicle on the liquid surface, but
56% with Acetobacter generated aggregates suspended in the culture. By taxonomic
pasteurianus NCIMB 12228 studies, it was classified into the genus Acetobacter based on
—––––––––––––––––––––––––––––––––––––––––––––––––––––– the results of Table 2. Ohmori et al. (1980) reported that
(−), Negative; (¦), Positive. thermophilic strains able to ferment acetic acid at higher
temperatures might belong to the genus Acetobacter because
they express a wide fluctuation in their sensitivity to tem-
perature. Based on the results of quinone, G ¦ C content of
68% at 35 °C and 37 °C, respectively, compared with that at DNA, and DNA homology studies, this isolate does not seem
30 °C. The effects of temperature on gluconic acid production to belong to any known Acetobacter species and is therefore a
and cell growth were coincident in trend with acetic acid novel and very interesting strain. Further research required
formation. In this study, Acet. aceti IFO 3283 and Acetobacter to identify this isolate is comparison of its DNA sequence
sp. CCRC 12326 did not grow or produce acetic acid when with other Acetobacter species.
they were incubated at 35 °C. They had an optimum tem- Industrial vinegar production by acetic acid bacteria is
perature for acetic acid production at 30 and 25 °C, respec- carried out at about 30 °C, either by traditional processes or by
tively (Fig. 4b, c). When the temperature was raised, a delay continuous submerged culture. Most strains used in vinegar
in ethanol oxidation was observed. The lag period for acetic production are mesophilic. These strains could not grow at
acid production from ethanol by Acetobacter sp. I14–2 was temperatures above 30 °C or produce acetic acid when they
prolonged to about 24 h at 35 and 37 °C (Fig. 5). However, were cultured at temperatures higher than 32 °C (Nakayama
ethanol was completely exhausted in 3–4 d and acetic acid 1961). Saeki et al. (1997) found that the lag phase for ethanol
production reached almost the same maximum level when oxidation was prolonged when the fermentation temperature
incubated at both 30 and 35 °C. Although the consumption was elevated for thermophilic strains and the two mesophilic
of ethanol at 37 °C was obviously delayed, isolate I14–2 could strains, Acet. rancens IFO 3298 and Acet. aceti IFO 3283. The
still produce 41 g l−1 of acetic acid. The decrease in amount lag time for producing acetic acid in Acet. aceti IFO 3283 was
of acetic acid produced might be caused by ethanol vol- very long (about 3 d) when it was cultured at 37 °C. We
atilization at a higher temperature. found that the very low productivity of acetic acid by Acet.
© 1999 The Society for Applied Microbiology, Journal of Applied Microbiology 86, 55–62
60 S .- F. L U E T A L .

5 50 1·5 2·0 10 50 1·5 20

(a) (a)
4 40 8 40
1·5 15
1·0 1·0
3 30 6 30
1·0 10
2 20 4 20
0·5 0·5
0·5 5
1 10 2 10

0 0 0·0 0·0 0 0 0 0
5 50 1·5 2·0 10 50 1·5 20
Acetic acid produced (g 1–1)

(b)

Acetic acid produced (g l–1)

(b)
Residual ethanol (%)

Gluconic acid (g l )

Residual ethanol (%)

–1
4 40

Gluconic acid (g l )
8 40

–1
1·5 15
1·0 1·0
3 30 O.D.600
6 30

O.D.600
1·0 10
2 20 4 20
0·5 0.5
0·5 5
1 10 2 10

0 0 0·0 0 0 0 0 0
5 50 1·5 2·0 10 50 1·5 20
(c) (c)
4 40 8 40
1·5 15
1·0 1·0
3 30 6 30
1·0 10
2 20 4 20
0·5
0·5
0·5
1 10 5
2 10

0 0 0
0 2 4 6 8 10 0 0 0 0
0 2 4 6 8 10
Initial acetic acid (g l–1)
Ethanol concentration (%)
Fig. 2 Effect of initial acetic acid concentration on acetic acid Fig. 3 Effect of initial ethanol concentration on acetic acid
production. Various concentrations of acetic acid and 5% (v/v) production. Main culture media supplemented with 2 g l−1 acetic
ethanol were added to the main culture medium before the test acid and various amounts of ethanol were inoculated with
strain was inoculated. Ethanol, acetic acid, gluconic acid and Acetobacter sp. I14–2 (a), Acet. aceti IFO 3283 (b), or Acetobacter sp.
cell growth were determined after Acetobacter sp. I14–2 (a), Acet. CCRC 12326 (c) and incubated at 30 °C with shaking at 50
aceti IFO 3283 (b), or Acetobacter sp. CCRC 12326 (c) was rev min−1 for 3 d. Residual ethanol (Ž); acetic acid produced
inoculated into the main culture medium and incubated at 30 °C (ž); gluconic acid (); O.D.600 (t)
with shaking at 50 rev min−1 for 3 d. Residual ethanol (Ž); acetic
acid produced (ž); gluconic acid (); O.D.600 (t)

of acetic acid by Acet. altoacetigenes MH-24 reached 92%

aceti IFO 3283 might be attributed to the same cause. On the after 5 d of fermentation (Entani et al. 1987). Acetobacter aceti
other hand, Acetobacter sp. I14–2 still actively produced acetic ATCC 23746 and Gluconobacter oxydans subsp.
acid when it was cultured at 35 and 37 °C for 3d, and retained sphaericus IFO 12467 exhibited 80 and 92% yields, respec-
about 97 and 68%, respectively, of the activity compared tively (Bar et al. 1987; Saeki 1993). Acetobacter sp. 249–1,
with that at 30 °C. The same result was observed for ethanol which could grow well at a wide temperature range of 30–
consumption by Acetobacter sp. I14–2; the lag time was pro- 37 °C, showed a yield of 95% (Lotong et al. 1989). The yields
longed when the temperature was elevated to 35 and 37 °C of three thermophilic strains, no. 550, 554 and S-13, were
(Fig. 5). The yield of acetic acid from ethanol by Acetobacter about 87·5% at 30 °C (Ohmori et al. 1980). However, the
sp. I14–2 was 95% after incubation at 30 °C for 3·5 d. In yield of strain no. 554 was very low at 37 °C, and strain S-30
comparison, the usual yield of acetic acid by acetic acid had lost its ability to produce acetic acid at 35 °C. Acetobacter
bacteria ranges from 60 to 98% (Bar et al. 1987). The yield sp. I14–2 expressed an outstanding thermotolerance with a
© 1999 The Society for Applied Microbiology, Journal of Applied Microbiology 86, 55–62
 T HE RM O TO LE R AN T A C ET OB A CT ER S P. I1 4 -2 61

5 50 1·5 2·0 50
(a)

4 40
1·5
1·0 40
3 30
1·0

Acetic acid (g l )
–1
2 20
0·5 30
0·5
1 10

0 0 0 20
5 50 1·5 2·0
Acetic acid produced (g l–1)

(b)
Residual ethanol (%)

Gluconic acid (g l )
4 40

3 30
2 20
1 10
0 0
5 50
(c)
4 40
3 30
2 20
1 10
0 0
15 20 25 30 35 40
Temperature (°C)
Fig. 4 Effect of temperature on acetic acid production. Main
culture media supplemented with 2 g l−1 acetic acid and 5%
(v/v) ethanol were inoculated with Acetobacter sp. I14–2 (a),
Acet. aceti IFO 3283 (b), or Acetobacter sp. CCRC 12326 (c) and
incubated at various temperatures with shaking at 50 rev min−1
for 3d. Residual ethanol (Ž); acetic acid produced (ž); gluconic
acid (); O.D.600 (t)
yield of 85 and 82% when it was cultured at 35 and 37 °C,
respectively, for 6 d. The yield at 37 °C by Acetobacter sp.
I14–2 is comparable with that of Acet. lovaniensis SKU 1108, a
thermophilic bacteria (Saeki et al. 1997). Acetobacter sp. I14–2
generated acetic acid at a rate of 0·61 and 0·59 g l−1 h−1 at
30 and 35 °C, respectively. Acetic acid produced by Frings
generator method had a production rate of 0·16 g l−1 h−1.
The acetic acid production rates of Acet. rancens strains S3
and F11 were 0·23 and 0·26 g l−1 h−1, respectively (Harada
and Mori 1971), while the rate for Acetobacter sp. 249–1 was
0·23 g l−1 h−1 (Lotong et al. 1989). Therefore, the acetic
acid production rate of Acetobacter sp. I14–2 at 30 °C was
© 1999 The Society for Applied Microbiology, Journal of Applied Microbiology 86, 55–62
–1
1·5 10
1·0 O.D.600
1·0
0·5 0 1 2 3 4 5 6 7 8 9
0·5 Cultivation time (d)
Fig. 5 Time course of acetic acid production from ethanol by
0 Acetobacter sp. I14–2 at various temperatures: 30 °C (ž);
1·5 2·0
35 °C (); 37 °C (R)
1·5
1·0
1·0 comparable with those of some thermoplilic strains such as
0·5 Acetobacter sp. no.550, 554 and S-23 (Ohmori et al. 1980). In
0·5 addition, the production rate at 35 or 37 °C was higher than
that of a protoplast fusant strain, no. 116, which possessed a
0 high resistance to acetic acid and ability to grow at high
45 temperatures (Fukaya et al. 1989). The reasons for thermo-
stability of thermophilic strains are still uncertain. Ohmori
et al. (1980) suggested that the increase in tolerance to acetic
acid or ethanol might account for their thermophilic proper-
ties. Saeki et al. (1997) studied the stability of aldehyde
dehydrogenase and alcohol dehydrogenase, but they did not
find any significant differences between thermophilic and
mesophilic strains. We considered that tolerance to acetic
acid or ethanol did not contribute to the thermotolerance of
Acetobacter sp. I14–2 as there was no remarkable increase in
tolerance to these compounds. Further studies are needed to
elucidate the increase in stability of enzymes, or the change
in membrane structure in Acetobacter sp. I14–2, which might
account for its thermostability.
Some authors have reported that several thermophilic
strains lose their acetic acid resistance and ethanol oxidation
capability in the stationary phase (Ohmori et al. 1982; Tak-
emura et al. 1991), but it was not observed in Acetobacter sp.
I14–2. With the advantages of thermotolerance, resistance to
ethanol, high acetic acid productivity, and easy preservation
by lyophilization, isolate I14–2 is suitable for vinegar making.
Future research will be to evaluate which metabolites can
stimulate acetic acid production by Acetobacter sp. I14–2 and
how to apply this strain in large-scale fermentation.
62 S .- F. L U E T A L .
ACKNOWLEDGEMENTS
The authors thank Dr L. L. Chern for her critical reading of
the manuscript. This work was supported by the Ministry of
Economic Affairs of the Republic of China.
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