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Chapter 10
Physiology of Acetobacterand Komagataeibacter spp.: Acetic AcidResistance Mechanism in Acetic Acid
Fermentation
Shigeru Nakano and Hiroaki Ebisuya
Abstract Acetic acid bacteria (AAB) are obligate aerobes that belong to the
α-Proteobacteria and are used for industrial vinegar production because of their
remarkable ability to oxidize ethanol by alcohol dehydrogenase, aldehyde dehy-
drogenase, and terminal oxidase of respiratory chain members on the cell mem-
brane. Acetic acid tolerance is a crucial ability allowing AAB to stably produce
large amounts of acetic acid.
Several molecular machineries responsible for acetic acid tolerance in AAB
have been reported, including (1) prevention of acetic acid influx into the cell,
(2) acetic acid assimilation, (3) acetic acid efflux by transporter or pump, and
(4) protection of cytoplasmic proteins against denaturing by general stress proteins.
(1) AAB optimize the lipid component proportion of the membrane and by further
forming polysaccharide on the surface of the cells to prevent the influx of acetic
acid. (2) AAB acquired the ability to convert the intracellular acetic acid in usable
energy effectively via the alternative TCA cycle. (3) AAB possess two types of
discharging intracellular acetic acid systems, one of which is a putative ABC
transporter, and the other is an efflux pump driven by a proton motive force.
(4) AAB adapt to the environmental changes in cells by inducing chaperones that
stabilize the structure of proteins from acidification of the cell inside, and by
synthesizing the enzymes which decompose reactive oxygen species (ROS) to
maintain the intracellular environment in good condition.
Because acetic acid tolerance in AAB is conferred by several mechanisms, these
mechanisms of acetic acid tolerance are reviewed here.
Keywords Phosphatidylcholine • Tetrahydoxy-bacteriohopane • Polysaccharide •
AarA • AarC • Aconitase • ABC transporter • Efflux pump • Chaperone • Acetic
acid tolerance
S. Nakano (*) • H. Ebisuya
Central Research Institute, Mizkan Holdings Co. Ltd, Handa-shi, Aichi 475-8585, Japan
e-mail: [email protected]
© Springer Japan 2016
K. Matsushita et al. (eds.), Acetic Acid Bacteria,DOI 10.1007/978-4-431-55933-7_10
223
10.1 Introduction
As we discuss the acetic acid tolerance of AAB, we first explain the antimicrobial
activity mechanism of acetic acid. Vinegar is a fermented food that is well known
for its high antimicrobial activity. The antimicrobial activity is the result of acetic
acid, which is the main component of vinegar. The antimicrobial activity of acetic
acid is partly caused by lowering the pH in the environment, and this is similar to
the function of lactic acid, which is produced by lactic acid bacteria in the
manufacturing process of fermented foods, such as alcohol and soy sauce. How-
ever, the most remarkable difference between acetic acid and lactic acid is that the
acid dissociation constant, pKa, of acetic acid and lactic acid is 4.8 and 3.8,
respectively. That is, in a weakly acidic environment, most acetic acid is present
as undissociated acid. Undissociated acetic acid is a hydrophobic small molecule
easily passing through the phospholipid bilayer of the bacterial membrane; it
releases a proton inside the cell, so that the cells are damaged by the decrease in
intracellular pH (Russel 1992; Axe and Bailey 1995; Ishii et al. 2012). For this
reason, acetic acid has higher antimicrobial activity. It has been confirmed that
growth of food poisoning bacteria, such as enterohemorrhagic Escherichia coliO-157 and Staphylococcus aureus, is inhibited by only 0.1% acetic acid (Entani
et al. 1997).
Acetic acid, which is the main component of vinegar, is produced from ethanol
by a respiratory chain consisting of alcohol dehydrogenase (ADH), aldehyde
dehydrogenase (ALDH), and terminal oxidase localized in the cell membrane of
AAB (Fig. 10.1) (Adachi et al. 1978; Ameyama et al. 1981). There are two major
methods of vinegar production. In “surface fermentation,” AAB form a film on the
liquid surface of the tub or pot; in “aerobic submerged fermentation,” the fermen-
tation liquor is aerated and agitated with stirring blades in the fermentation tank
(Fig. 10.2). Acetic acid concentration reached 4–6% acidity at the surface fermen-
tation and reached 5–20% acidity in the aerobic submerged fermentation. AAB is
capable of growth even in the presence of such a high acetic acid concentration. The
acetic acid tolerance mechanism of AAB is very interesting.
Several acetic acid tolerance mechanisms are summarized here.
1. Characteristic membrane structure preventing the inflow of acetic acid into the
cell
2. Efficient consumption of intracellular acetic acid
3. Discharge mechanism of intracellular acetic acid
4. Adaptive mechanism to environmental changes in the cell
These four mechanisms of acetic acid tolerance of AAB are reviewed next.
224 S. Nakano and H. Ebisuya
Fig. 10.1 Schematic representation of ethanol respiration of acetic acid bacteria (AAB). Ethanol
is oxidized in the periplasm by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase
(ALDH), which also donate electrons to an ubiquinol oxidase that in turn generates a proton
motive force. Acetic acid produced outside the cell is easily passed through the phospholipid
bilayer of the bacteria and releases a proton in the cell cytoplasm
Fig. 10.2 Appearance of the two major methods of vinegar production. (A) Surface fermentation
is generally carried out in a tub or pot. The volume of the tab is about 4 Kl. (B, C) Because the
surface fermentation proceeds slowly, it takes 1 week or longer to produce vinegar with a film
forming on the liquid surface. (D) Aerobic submerged fermentation is carried out in a stainless
steel tank, which volume is usually from 20 to 100 Kl. (E) Sparger air is supplied from the bottom
of the tank with stirring blades. One batch of the aerobic fermentation is completed in about 2 days
10 Physiology of Acetobacter and Komagataeibacter spp.: Acetic. . . 225
10.1.1 Characteristic Membrane Structure Preventingthe Inflow of Acetic Acid into the Cell
Acetobacter and Komagataeibacter are used for industrial vinegar production
because of their remarkable ability to oxidize ethanol to acetic acid. Acetobactercan produce acetic acid at 12% concentration, and Komagataeibacter can produce
up to 20% acetic acid acidity. In comparison of their phospholipid chemical
composition, Acetobacter polyoxogenes (presently assigned to genus
Komagataeibacter) showed a significantly higher phosphatidylcholine (PC) contentthan Acetobacter sp. (Goto et al. 2000). Furthermore, in observation of the PC
content of the membrane lipids during acetic acid fermentation of this
Komagataeibacter strain (vinegar-producing strain), it was shown that the ratio of
PC increased and that of phosphatidylglycerol (PG) decreased as the acetic acid
concentration increased (Higashide et al. 1996). The phosphatidylethanolamine N-methytransferase gene (pmt) has been cloned from Acetobacter aceti (Acetobacterpasteurianus) and sequenced (Hanada et al. 2001). pmt catalyzes methylation of
phosphatidylethanolamine (PE) to PC. Although the pmt disruption mutant could
not produce PC and increased PE and PG in the membrane, it showed a reduced
growth rate and lowered maximum cell density as compared to the wild-type strain
in the medium with acetic acid. Therefore, it was suggested that PC is involved in
acetic acid tolerance in Acetobacter and Komagataeibacter.The cell membrane lipids of Komagataeibacter possess a high content of
hopanoids, more than is found in other microorganisms, especially tetrahydoxy-
bacteriohopane (THBH) (Fig. 10.3). THBH is a lipid known as a characteristic
component in the cell membranes of Zymomonas mobilism, that performs alcohol
fermentation of tequila production, and the content of THBH is increased during
alcohol fermentation. It is presumed that THBH is involved in stabilization of the
cell membrane in the presence of a high concentration of ethanol. Because THBH
accounted for 25% of the membrane lipids of Komagataeibacter, it was speculatedthat THBH is involved in the acetic acid tolerance of AAB. The squalene-hopen
cyclase (HpnF) gene, which is involved in the synthesis of THBH precursor, of
A. aceti (A. pasteurianus) was cloned. The A. pasteurianus transformant harboring
the hpnF gene produced THBH of higher ratio than the transformant carrying the
vector in the membranes (Fig. 10.4). Although the significant difference in each
alcohol tolerance was not confirmed, the acetic acid tolerance of the transformant
was higher than that of the vector control.
AAB have the ability to form a pellicle that floats on the liquid medium surface
of the static culture. It is thought to synthesize some polysaccharides on the cell
surface, as a colony has a rough surface on solid agar medium (Matsushita
et al. 1992a). In contrast to the wild-type strain of A. pasteurianus, which makes
extracellular polysaccharide, intracellular acetate/acetic acid content was con-
firmed as significantly higher in the strain unable to produce the polysaccharides.
This result suggests that pellicle polysaccharide surrounding the bacteria suppresses
the influx of acetic acid into the cell (Kanchanarach et al. 2010).
226 S. Nakano and H. Ebisuya
Therefore, it is thought that AAB has specific mechanisms that reduce the influx
of acetic acid by optimizing the proportion of the membrane lipid component and
further forming polysaccharide on the surface of the cells.
10.1.2 Efficient Consumption of Intracellular Acetic Acid
By a genetic approach using an acetic acid-sensitive mutant of A. aceti(A. pasteurianus), aarA and aarC were identified as an acetate-resistant gene
cluster, and they were confirmed to be members of enzymes involved in the
Fig. 10.3 Lipid composition of Gluconacetobacter (Komagataeibacter). Komagataeibactersp. has a characteristic composition with a high content of tetrahydoxy-bacteriohopane (THBH)
and phosphatidylcholine (PC). These lipids are not often found in other microorganisms
Fig. 10.4 Effect of overexpression of hpnF in Acetobacter acti. A. aceti strains were cultured at
400 rpm under 0.2 vvm aeration at 30 �C in YPG medium with constant feeding of ethanol at 1%
(v/v) in a jar-fermenter. The A. aceti transformant harboring pSHC, which was composed of the
hpnF coding sequence and Escherichia coli–Acetobacter shuttle vector pMV24, produced 11.4%
of acetic acid (B), whereas the transformant carrying the vector accumulated 9.5% of acetic acid
(A). Specific growth rate (OD660/h) of the transformant harboring pSHCwas higher than that of the
transformant carrying the vector. OD660 optical density at 660 nm
10 Physiology of Acetobacter and Komagataeibacter spp.: Acetic. . . 227
tricarboxylic acid (TCA) cycle. AarA acts as citrate synthase, and AarC acts as
succinyl CoA:acetate CoA-transferase; thus, they were found to be responsible for
acetate assimilation (Fukaya et al. 1990, 1993b; Mullins et al. 2008). AarB actions
have not yet been defined.
AAB have the alternative TCA cycle that is modified as part of these enzymes.
Although A. pasteurianus does not have any succinyl CoA synthase that involves
interconversion between succinyl-CoA and succinic acid, AarC complements that
function. AarC converts succinyl-CoA to succinate and transfers the CoA to acetic
acid in the cell simultaneously; these are converted to acetyl-CoA and then
converted to citrate by AarA. Thus, AAB consume acetic acid (Fig. 10.5).
In addition, the surface of AarA protein is more basic than that of E. coli, so it isthought that the enzyme functions stably even at lower pH (Francois et al. 2006).
Furthermore, citrate synthase purified from Gluconacetobacter europaeus(Komagataeibacter europaeus) was also shown to be stable in the presence of
acetic acid (Sievers et al. 1997). It appears reasonable that the enzymes of acetic
acid bacteria are intrinsically resistant to inactivation by low pH and functionally
adapt to low pH. It was shown by proteomic analysis that aconitase was induced
with acetic acid in a medium (Nakano et al. 2004). The transformant that highly
expressed aconitase showed higher productivity of acetic acid than the wild-type
strain, which has lower acetic acid tolerance (Fig. 10.6).
In conclusion, AAB acquired the ability to convert the intracellular acetic acid in
usable energy effectively by using the AarC. Also, AAB modified the AarA as
being more robust. Furthermore, aconitase was induced in the presence of acetic
acid. It is thought that AAB can consume intracellular acetic acid efficiently under
the high concentrations of acetic acid.
Fig. 10.5 Overoxidation
pathway of intracellular
acetic acid in A. aceti. It wassuggested that intracellular
acetic acid is consumed by
the actions of the enzymes,
AarC, AarA and aconitase,
which are characteristic of
the AAB described in this
chapter
228 S. Nakano and H. Ebisuya
10.1.3 Discharge Mechanism of Intracellular Acetic Acid
It is confirmed that AAB has two types of systems discharging intracellular acetic
acid. Proteomic analysis of the membrane fraction in A. aceti (A. pasteurianus)revealed that several proteins were induced by acetic acid (Nakano et al. 2006). One
of these proteins has the structure belonging to the family of macrolide antibiotics
transporters, which contain ATP-binding cassette (ABC) sequences and ABC
signature sequences (Mendez and Salas 2001). This protein, named AatA, was a
putative ABC transporter that possibly functioned as an exporter of intracellular
acetic acid. The aatA disruption mutant showed reduced resistance to acetic acid as
compared to the wild-type strain. Although there was no significant difference with
respect to lactic acid and citric acid, formic acid and propionic acid tolerance were
reduced as well as acetic acid tolerance in the aatA mutant (Fig. 10.7). This finding
indicates that AatA is the transporter specific for the linear short-chain fatty acid
that had a dissociation constant (pKa) as high as that of acetic acid. For further
investigation on the possibility that aatA is involved in acetic acid tolerance, an
E. coli transformant harboring the aatA gene was prepared. The transformant
harboring the aatA gene acquired higher levels of tolerance to acetic acid, formic
acid, and propionic acid as compared to the transformant carrying the vector
(Fig. 10.8). These results suggest that the aatA gene product is conferring acetic
acid tolerance to AAB.
Another efflux system different from the ABC transporter was confirmed in
A. aceti (A. pasteurianus). It was shown that acetate accumulation was low in intact
cells in the presence of respiratory substrate and was increased by the addition of
protonophores (Matsushita et al. 2005). Right-side-out (RSO) membrane vesicles
accumulated a high concentration of intracellular acetic acid by passive diffusion,
but accumulation was decreased almost completely by respiration. These phenom-
ena suggest that cells or RSO membrane vesicles actively pump out acetic acid,
which penetrates passively into the cell by using energy produced by respiration.
On the other hand, in-side-out (ISO) membrane vesicles accumulated a high
Fig. 10.6 Acetic acid fermentation by A. aceti transformants. (Figure from Nakano et al. 2004)
Growth (A) and acetic acid concentration (B). The A. aceti transformant was cultured at 400 rpm
under 0.2 vvm aeration at 30 �C in YPG medium with constant feeding of ethanol at 1% (v/v) in a
jar-fermenter. Solid circles wild-type strain, open circles aatA-disrupted strain, OD660 optical
density at 660 nm
10 Physiology of Acetobacter and Komagataeibacter spp.: Acetic. . . 229
concentration of intracellular acetic acid by respiration. Efflux of acetic acid from
RSO membrane vesicles and uptake of acetic acid into ISO membrane vesicles in
the presence of the respiratory substrate were largely decreased by the addition of
protonophores and a respiratory inhibitor such as cyanide. These results suggest
that a proton motive force-dependent acetic acid efflux pump exists in
A. pasteurianus.Ethanol oxidation (acetic acid production) with ADH and ALDH linked to the
terminal oxidase and the following acetic acid assimilation with the cytoplasmic
Fig. 10.7 Effects of various organic acids on growth of A. aceti strains. (Figure from Nakano
et al. 2006) The A. aceti transformants were cultured at 30 �C in the presence of acetic acid (A),
lactic acid, citric acid, formic acid, and propionic acid (B) in YPG medium for 120 h. The growth
of the strains was followed by measuring optical density at 660 nm (OD660). Solid circles wild-type strain carrying the vector, open circles transformant harboring multiple copies of the
aconitase gene
Fig. 10.8 Effect of acetic acids, formic acid, and propionic acid on growth of E. colitransformants cultured at 37 �C for 90 h in LB medium (pH 5.0) in the presence of various
concentrations of acids. Solid symbols transformant harboring pMV24, open symbols transformant
harboring pABC101, which was composed of the aatA coding sequence and E. coli–Acetobactershuttle vector pMV24, OD660 optical density at 660 nm
230 S. Nakano and H. Ebisuya
enzymes, AarA, AarC and aconitase, generate a proton motive force and then ATP,
which in turn is used for the efflux pump and ABC transporter.
Therefore, it is considered that AAB can grow even in an environment of high
acetic acid concentration by acquiring multiple types of discharge mechanisms.
10.1.4 Adaptive Mechanism to Environmental Changesin the Cell
AAB that are applied to vinegar production are exposed to various stressors, such as
ethanol, acetic acid, or heat generated during the acetic acid fermentation. In the
studies of tolerance to various stresses during acetic acid fermentation of AAB, it
was shown that plural types of chaperones were induced, and some chaperones
were identified (Okamoto-Kainuma et al. 2002). All these chaperones, including
GroES and GroEL, have characteristic heat-shock promoter-like sequences for
α-Proteobacteria, and the expressions of these genes are regulated by RpoH
(Okamoto-Kainuma et al. 2011). An rpoH disruption mutant of A. pasteurianusshowed higher sensitivity to ethanol, acetic acid, and temperature as compared to
the wild-type strain. Furthermore, the A. pasteurianus transformant, which highly
expresses GroES, showed higher productivity of acetic acid than the wild-type
strain. These results suggest that RpoH is one of the important factors in acetic acid
fermentation of AAB.
Acetic acid is generated by oxidation of ethanol, and its reaction is catalyzed by
enzymes on the inner membrane in AAB. In this reaction, ADH and ALDH transfer
electrons from ethanol and acetaldehyde to ubiquinones, to generate ubiquiols
(Matsushita et al. 1990, 1992b). Then, ubiquinol oxidase transfers the electrons
from ubiquinol to oxygen to produce water by receiving four electrons (Fukaya
et al. 1993a). When these series of reaction are not going well, reactive oxygen
species (ROS) such as superoxide anion and hydrogen peroxide are generated. In
A. pasteurianus, the mutant strain of catalase-regulating factor showed higher
sensitivity to hydrogen peroxide, and also a more delayed growth, than the wild-
type strain in the culture medium containing ethanol or acetic acid (Okamoto-
Kainuma et al. 2008). These phenomena suggest that efficient and quick degrada-
tion of ROS enables performing stable and effective acetic acid fermentation.
Thus, AAB could adapt to change in the intracellular environment during
fermentation by chaperones that refold proteins denatured by acidification and by
enzymes which decompose generated ROS.
10 Physiology of Acetobacter and Komagataeibacter spp.: Acetic. . . 231
10.2 Summary of Molecular Basis of Acetic Acid Tolerance
in AAB
Previous studies as well as the recent proteomic analysis have revealed several
mechanisms of acetic acid tolerance in AAB (Fig. 10.9).
1. Prevention of acetic acid influx: it was suggested that THBH and PC in the
membrane lipids and polysaccharide on the surface of the cells are related to
acetic acid tolerance.
2. Acetic acid peroxidation: AarA, AarC, and aconitase facilitate the assimilation
of intracellular acetic acid.
Fig. 10.9 Schematic representation of molecular machineries that confer acetic acid resistance in
Acetobacter and Gluconacetobacter. (Schematic diagram quoted from Nakano and Fukaya 2008)
THBH and phosphatidylcholine on the membrane and polysaccharide on the surface of the cells
are suggested to be involved in acetic acid resistance. Acetic acid, which penetrates into the
cytoplasm, is assumed to be metabolized through the TCA cycle by the actions of enzymes typical
for AAB. Furthermore, intracellular acetic acid is possibly pumped out by a putative ABC
transporter and proton motive force-dependent efflux pump using energy produced by ethanol
oxidation or acetic acid overoxidation. Intracellular cytosolic enzymes are intrinsically resistant to
low pH and are protected against denaturation by stress proteins such as molecular chaperones.
ADH membrane-bound alcohol dehydrogenase, ALDH membrane-bound aldehyde dehydroge-
nase, CS citrate synthase, ACN aconitase, PC phosphatidylcholine
232 S. Nakano and H. Ebisuya
3. Acetic acid efflux by transporter or pump: two types of efflux system, a putative
ABC transporter and a proton motive force-dependent efflux pump, function to
pump out intracellular acetic acid.
4. Protection of cytoplasmic proteins against denaturing: several kinds of chaper-
one and ROS scavenger are synthesized to stabilize the cell interior.
These mechanisms will provide clues for breeding a strain having higher toler-
ance to acetic acid for vinegar fermentation, which may be a target for improving
the efficiency of fermentation production.
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