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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

mailto:[email protected]

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).


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


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


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


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


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.


10.2 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


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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