Critical Reviews in Biotechnology

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Biotechnological Applications of Acetic Acid

Bacteria

Peter Raspor & Dušan Goranovič

To cite this article: Peter Raspor & Dušan Goranovič (2008) Biotechnological Applications
of Acetic Acid Bacteria, Critical Reviews in Biotechnology, 28:2, 101-124, DOI:
10.1080/07388550802046749

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Critical Reviews in Biotechnology, 28:101–124, 2008
Copyright
c Informa Healthcare USA, Inc.
ISSN: 0738-8551 print / 1549-7801 online
DOI: 10.1080/07388550802046749

Biotechnological Applications of Acetic Acid Bacteria

Peter Raspor and Dušan Goranovič
Department of Food Science and Technology, University of Ljubljana, Ljubljana, Slovenia

The acetic acid bacteria (AAB) have important roles in food and beverage production, as well
as in the bioproduction of industrial chemicals. In recent years, there have been major ad-
vances in understanding their taxonomy, molecular biology, and physiology, and in methods
for their isolation and identification. AAB are obligate aerobes that oxidize sugars, sugar al-
cohols, and ethanol with the production of acetic acid as the major end product. This special
type of metabolism differentiates them from all other bacteria. Recently, the AAB taxonomy
has been strongly rearranged as new techniques using 16S rRNA sequence analysis have been
introduced. Currently, the AAB are classified in ten genera in the family Acetobacteriaceae.
AAB can not only play a positive role in the production of selected foods and beverages, but
they can also spoil other foods and beverages. AAB occur in sugar- and alcohol-enriched envi-
ronments. The difficulty of cultivation of AAB on semisolid media in the past resulted in poor
knowledge of the species present in industrial processes. The first step of acetic acid production
is the conversion of ethanol from a carbohydrate carried out by yeasts, and the second step is
the oxidation of ethanol to acetic acid carried out by AAB. Vinegar is traditionally the product
of acetous fermentation of natural alcoholic substrates. Depending on the substrate, vinegars
can be classified as fruit, starch, or spirit substrate vinegars. Although a variety of bacteria can
produce acetic acid, mostly members of Acetobacter, Gluconacetobacter, and Gluconobacter are
used commercially. Industrial vinegar manufacturing processes fall into three main categories:
slow processes, quick processes, and submerged processes. AAB also play an important role in
cocoa production, which represents a significant means of income for some countries. Microbial
cellulose, produced by AAB, possesses some excellent physical properties and has potential for
many applications. Other products of biotransformations by AAB or their enzymes include 2-
keto-L-gulonic acid, which is used for the production of vitamin C; D-tagatose, which is used as
a bulking agent in food and a noncalorific sweetener; and shikimate, which is a key intermediate
for a large number of antibiotics. Recently, for the first time, a pathogenic acetic acid bacterium
was described, representing the newest and tenth genus of AAB.

Keywords acetic acid bacteria (AAB), acetic acid, acetous fermentation, Acetobacter, biosensors,
biotransformation, cellulose, cocoa, D-tagatose, Gluconobacter, Gluconacetobacter,
kombucha, shikimate, spoilers, vinegar

INTRODUCTION
The microorganisms oxidizing ethanol to acetic acid are com-
monly called acetic acid bacteria (AAB). This special primary
metabolism at low pH differentiates them from all other bacte-
ria. AAB can play not only a positive role in the production of
selected foods and beverages, such as vinegar, kombucha bev-
erage, and cocoa, but they can also spoil other foods and bever-
ages, such as wine, beer, soft drinks, and fruits. Physiologically,
AAB convert alcohols to acids by oxidation, and even though
Address correspondence to Dr. Peter Raspor, Department of Food
Science and Technology, Biotechnical Faculty, University of Ljubljana,
Jamnikarjeva 101, 1000 Ljubljana, Slovenia. E-mail: peter.raspor@
bf.uni-lj.si
101
AAB have been extensively studied, researchers are still unrav-
eling the mechanisms of their unique metabolism. People have
benefited from the actions of AAB long before they were recog-
nized as the causative agent in acetous fermentation, and today,
many new fields in biotechnology are exploiting the benefits
that AAB offer. The AAB taxonomy has recently been strongly
rearranged as new techniques using 16S rRNA sequence anal-
ysis have been introduced. Identification and differentiation of
AAB on the species level is still difficult, despite the occurrence
of new molecular methods, which are continuously being im-
proved. We face new improvements in technology of AAB due
to technical developments. As our knowledge and technolog-
ical possibilities increase, so do the benefits that microorgan-
isms, such as AAB, offer. In recent years, there have been major
advances in understanding AAB taxonomy, molecular biology,
102 P. RASPOR AND D. GORANOVIČ
and physiology, and in methods for their isolation and identifica-
tion, but the knowledge obtained remains somewhat scattered.
Despite the fact, that some applications involving AAB (e.g.,
production of vinegar and cocoa) have been practiced for a long
time, on an industrial scale, and represent important economic
niches, it is surprising that much of the knowledge needed for
a complete understanding of these processes remains absent.
There is much new fundamental and applied information about
these bacteria that would be of interest to many microbiolo-
gists and biotechnologists, and this review aims to fill the gap in
knowledge concerning AAB.
ECOLOGY
Natural Environment
In the environment, AAB occur in fruits, flowers, palm sap,
garden soil, and honey bees. Gluconobacter strains prefer sugar-
enriched environments in contrast to Acetobacter strains, which
prefer alcohol-enriched environments. Some AAB species cause
pink disease of pineapple fruit and rot of apples and pears. A
few species are nitrogen fixers, inhabiting the roots and stems
of plants (Cavalcante and Döbereiner, 1988; Fuentes-Ramı́rez
et al., 2001; Gillis et al., 1989; Loganathan and Nair, 2004).
Artificial Environment
Artificial and manmade environments that support AAB
growth include soft drinks, sake, tequila, palm wine, grape wine,
cider, beer, kefir, sugar cane juice, tea fungus, vegetable tanning
liquors, and canal water (De Ley, Gills, and Swings, 1984).
Acetic Acid Bacteria as Spoilers
AAB are particularly known in beverage production for their
ability to produce acetic acid, and therefore vinegary off-flavors,
turbidity, and ropiness, when basic hygienic and technical pro-
cedures are not correctly performed. Due to the presence of
oxygen, even in microconcentrations, if the temperature is in a
proper range, the spoiling process can start with ethanol oxida-
tion. Ropiness is characterized by an increase in the viscosity
of the beverage, which pours as an “oily stream” (Guizani and
Mothershaw, 2006). AAB of the genera Acetobacter and Glu-
conobacter are known as a spoiling agent of beer and wine. The
surface contamination they cause is often apparent as an oily or
moldy film (velum). AAB can be transferred with the pitching
yeast to the following batch, and flies, particularly the fruit fly,
can also spread the infection. Gluconobacter spp. range from
aerobic to microaerophilic and can tolerate conditions of low
pH and the ethanol concentrations normally found in beer, and
they grow well at 18◦ C, using proline as a nitrogen source. Ace-
tobacter spp. can further oxidize the generated acetic acid to
carbon dioxide and water. Consequently, the alcoholic beverage
infected with AAB has a vinegary flavor, with a lowered pH
and ethanol content (Odhav, 2004). The main species of AAB
observed on unspoiled grapes is Gluconobacter oxydans, which
was also found to predominate in fresh must with Acetobacter
pasterianus occurring at lower numbers. G. oxydans numbers
decrease after alcoholic fermentation due to its low alcohol tol-
erance and Acetobacter aceti, A. pasterianus, and Gluconace-
tobacter hansenii normally start to take over. The number of
AAB increases drastically on rotten or Botrytis-infected grapes,
and in those conditions, Acetobacter species can start to domi-
nate. Although AAB are obligate aerobes, they can survive and
even grow in semianaerobic conditions occurring in wine bar-
rels. During the aging of wine in wooden barrels, about 30 mg
oxygen L−1 penetrates through the wood and into the wine in
a year. This could sustain a viable population of AAB, and the
exposure of wine to air, even for a very short period of time,
drastically enhances the risk of spoilage. Prevention of wine
spoilage by AAB can be achieved by using healthy grapes, a
high inoculum of yeasts, the addition of SO2 to the must, mini-
mum pick-up of air, clarification of the must, and the lowering of
the pH by addition of acid. Wine in barrels should also be filled
regularly, due to the process of evaporation. Failure to do this
could present the AAB with a surface to grow on (Du Toit and
Pretorius, 2002). Gluconobacter spp. are the most frequently
encountered cause of bacterial spoilage of soft drinks at low
pH. Spoilage changes flavor properties and may lead to pack
swelling and haze. Gluconobacter spp. are resistant to preserva-
tives such as sorbic acid, benzoic acid, and dimethyldicarbonate,
but are heat sensitive and absolutely dependent on the presence
of free oxygen. Spoilage is a problem only in gas-permeable
packaging, such as still soft drinks in plastic beakers (Stratford
and Capell, 2003).
Pathogenesis
Chronic granulomatous disease (CGD) is a rare inherited dis-
ease of the phagocyte NADPH oxidase system, which leads
to defective production of superoxide and hydrogen peroxide
(Segal et al., 2000). Patients suffer from recurrent life-
threatening infections with catalase-producing organisms and
also develop tissue granulomas (Winkelstein et al., 2000). In
2003, a novel gram-negative rod was isolated from cervical and
supraclavicular lymph nodes of a CGD patient. This organism
was subsequently isolated from the same patient three more
times throughout 2005. A similar gram-negative rod was later
isolated from two other CGD patients. Phenotypic and geno-
typic analyses were consistent with this being a previously un-
described bacterium in the group of AAB. Like Acidomonas
methanolica, the novel bacterium is a facultative methylotroph
that is able to use methanol as a sole carbon source. However, it
is distinguished from A. methanolica in that it generates acetic
acid poorly on ethanol-CaCO3 agar (with 2% CaCO3 ), oxidizes
lactate and grows on glutamate and mannitol agar, and forms yel-
low colonies. Whereas the optimum temperature for growth of
most AAB is 25◦ C to 30◦ C, Granulibacter bethesdensis prefers
higher temperatures (35◦ C–37◦ C) (Greenberg et al., 2006).
The analyses of the 16S, ITS, and RecA sequences broadly
supported previously published findings for members of the
 BIOTECHNOLOGICAL APPLICATIONS OF ACETIC ACID BACTERIA
Acetobacteriaceae (Cleenwerck et al., 2002; Tanasupawat et al.,
2004; Yamada et al., 2000), while establishing a separate lin-
eage for this organism. The results confirmed that the isolate
represented a member of the Acetobacteriaceae family and that
it was distinct enough to warrant separate genus-level desig-
nation. The name G. bethesdensis was proposed. The cells of
G. bethesdensis are nonmotile and coccobacillus to rod-shaped,
strictly aerobic, catalase positive, and oxidase negative, and the
optimum pH for growth is 5.0 to 6.5. (Greenberg et al., 2006).
TAXONOMY
The microorganisms oxidizing ethanol to acetic acid are com-
monly called acetic acid bacteria (AAB). This special primary
microbial metabolism at low pH of the surrounding medium
differentiates them from all other bacteria. Recently, the AAB
taxonomy has been strongly rearranged, and some species pre-
viously ascribed to the genera Acetobacter and Gluconobacter
are now located in different genera: Acetobacter liquefaciens,
Acetobacter xylinus, Acetobacter europaeus, Acetobacter in-
termedius, Acetobacter oboediens, and A. hansenii were trans-
ferred in the genus Gluconacetobacter (Yamada, Hoshino, and
Ishikawa, 1997). Nowadays, AAB are classified in the follow-
ing genera: Acetobacter (A), Gluconobacter (G), Gluconaceto-
bacter (Ga), Granulibacter (Gr), Acidomonas (Ac), Asaia (As),
Kozakia (K ), Neoasaia (N ), Swaminathania (Sw), and Saccha-
ribacter (S). These genera are phylogenetically joined into a
broad rRNA cluster in the family Acetobacteriaceae, within the
α-subclass of the Proteobacteria, in the AAB lineage (Cleen-
werck et al., 2002; Gullo et al., 2006). The genus Asaia was
introduced as the fifth genus in the family Acetobacteriaceae
with a single species, Asaia bogorensis (Yamada et al., 2000).
In addition, two species, Asaia siamensis (Katsura et al., 2001)
and Asaia krungthepensis (Yukphan et al., 2004), were de-
scribed. The genus Kozakia was subsequently proposed as the
sixth genus with a single species, Kozakia baliensis (Lisdiyanti
et al., 2002). In 2004, the seventh and the eighth genera were de-
scribed: Saccharibacter (Jojima et al., 2004) and Swaminatha-
nia (Loganathan and Nair, 2004), with a single species each,
Saccharibacter floricola and Swaminathania salitolerans, re-
spectively. The genus Neoasaia was proposed in 2005 with a
single species Neoasaia chiangmaiensis (Yukphan et al., 2005).
The most recent addition in the family Acetobacteriaceae was
the genus Granulibacter with the first known pathogenic species
of AAB?G. bethesdensis. The novel organism showed unique
phenotypic characteristics compared with isolates belonging to
the nine described genera of AAB, and therefore clearly repre-
sented a new taxon (Greenberg et al., 2006). Among the ten gen-
era, the AAB recovered from vinegar production are mainly dis-
tributed in the genera Acetobacter and Gluconacetobacter and
the most frequently isolated species are A. pasterianus, Aceto-
bacter polyoxogenes, Gluconacetobacter xylinus, Ga. hansenii,
Gluconacetobacter europaeus, Gluconacetobacter oboediens,
Gluconacetobacter intermedius, and Gluconacetobacter entanii
(Yamada, 2000). Identification below the genus level is difficult.
103
AAB are polymorphous, cells are gram negative, ellipsoidal to
rod shaped, straight or slightly curved, 0.6 to 0.8 µm long, and
occurring singly, in pairs, or in chains. There are nonmotile
and motile forms with polar or peritrichous flagella. They are
obligately aerobic, and some produce pigments, some cellulose.
The striking fact is that common microorganisms used on a large
scale for industrial vinegar production have not been properly
described and characterized in taxonomic terms (Ebner, Sellmer,
and Follmann, 1996) caused by the difficulty to cultivate them
on semisolid media.
Methods for Cultivation and Identification
Cultivation
One of the greatest hurdles when managing AAB is their cul-
tivation and maintenance in pure culture, especially for strains
isolated from a high acetic acid level source (Entani et al., 1985;
Gullo et al., 2006). Considerable progress in the isolation and
cultivation of the AAB from submerged bioreactors was made by
using a double-layer medium and the more efficient AE medium
(acetic acid-ethanol), which provides colonies growing on the
surface with a constant supply of ethanol and moisture from a
lower, semisolid layer. The RAE (reinforced acetic acid-ethanol)
medium contains glucose, yeast extract, peptone, Na2 HPO4 , and
citric acid, supplemented with acetic acid and ethanol after au-
toclaving (Entani et al., 1985; Sokollek and Hammes, 1997;
Sokollek, Hertel, and Hammes, 1998a). These methods permit
the cultivation of AAB that are capable of producing acetic
acid concentrations of up to 10% to 15% (Sokollek, Hertel,
and Hammes, 1998a). Other media used for growth and enu-
meration of AAB include GYC (glucose, yeast extract, calcium
carbonate), YPM (yeast extract, peptone, mannitol), YPE (yeast
extract, peptone, ethanol), etc. Antibiotics are often added to
prevent the growth of other microorganisms such as yeasts (pi-
maricin, cycloheximide) and lactic acid bacteria (nisin, peni-
cillin). Reported incubation times vary from two to eight days at
temperatures from 25◦ C to 30◦ C. The strains of AAB isolated
from vinegar form round, beige-yellowish colonies of 1 to 2 mm
within two days of incubation on RAE agar. AAB can be stored
on GYC agar slants at 4◦ C and freeze-dried, but the survival rate
is also good during storage with glycerol at −80◦ C (Du Toit and
Pretorius, 2002). The use of 20% malt extract as a cryoprotectant
was also effective for the preservation of AAB strains (Du Toit
and Pretorius, 2002; Sokollek, Hertel, and Hammes, 1998a). Be-
cause a large proportion of the microbial population can be in a
viable but nonculturable state, conventional culture-dependent
methods have the limit to isolate only easily cultivable species
or strains, even if less abundant (De Vero et al., 2006; Millet and
Lonvaud-Funel, 2000). From industrial practice, it has long been
known that the properties of a newly isolated strain of AAB may
change from the very first moment of cultivation in Petri dishes.
Strains cultivated over a number of generations, may show dif-
ferent phenotypic features properties, especially as far as the
adaptation to certain concentrations of acetic acid is concerned
(Ebner, Follmann, and Sellmer, 1995).
104 P. RASPOR AND D. GORANOVIČ
Identification
Overoxidation of acetic acid has been used for the differenti-
ation between Acetobacter and Gluconobacter strains (Swings,
1992). Acetobacter spp. can overoxidize acetic acid, but this
ability depends on the composition of the media. It takes place
in the absence of ethanol (Adams and Moss, 2000; De Ley,
Gills, and Swings, 1984; Drysdale and Fleet, 1989; Guizani
and Mothershaw, 2006; O’Toole and Lee, 2003). The genus
Acidomonas is characterized by its ability to grow on methanol
(Swings, 1992), and the genus Asaia by its inability to grow on
a 0.35% acetic acid–containing medium (Yamada et al., 2000).
The two other genera, Acetobacter and Gluconacetobacter, can
be differentiated from each other on the basis of ubiquinone Q-9
and ubiquinone Q-10 contents (Yamada, Hoshino, and Ishikawa,
1997). The phenotypic identification of the AAB, especially on
the species level, is difficult (Swings, 1992). One of the rea-
sons for this difficulty is the high frequency of spontaneous
mutations, attributed to the presence of insertion elements in
the AAB (Takemura, Horinouchi, and Beppu, 1991). The iden-
tification methods based on phenotypic characteristics are not
only inaccurate, but also time consuming. Therefore, the ap-
plication of molecular methods that do not require microor-
ganism isolation based on the identification and characteriza-
tion of specific DNA segments could offer the solution for
quick and accurate identification of AAB (Trček and Teuber,
2002). Molecular phylogeny based on small subunit rRNA se-
quences reflects natural relationships, and therefore, 16S rDNA
sequences are useful for identification and differentiation be-
tween most AAB species (Escalante et al., 2004; Franke et al.,
2000; Prieto et al., 2007; Sievers, Ludwig, and Teuber, 1994;
Sievers et al., 1995; Sievers et al., 1996; Sokollek, Hertel, and
Hammes, 1998a). However, the 16S rDNA sequences of AAB
are very similar to each other, which causes problems in de-
lineating the species on the basis of restriction fragment length
polymorphism (RFLP) of the 16S rDNA alone (Poblet et al.,
2000; Yamada et al., 2000). The polymerase chain reaction
(PCR)-RFLP method of the 16S rRNA using different com-
binations of restriction enzymes proved to be fast and reliable,
but it was also unable to differentiate some species (González
et al., 2006a). Fast and sensitive identification methods such as
real-time PCR and nested PCR have been developed to iden-
tify AAB species. These methods are ideal for detection of food
spoilage organisms because they allow a rapid, high-throughput,
and automated procedure (Gammon et al., 2007; González et al.,
2006b). Much higher variability in sequence composition of the
spacer regions, which separate the rDNA sequences, provides
a better starting point for accurate species differentiation of the
AAB. 16S-23S rDNA internal transcribed spacer region analy-
sis has been proven successful for identification of AAB from
vinegar and cocoa fermentations (Barry et al., 1991; González
et al., 2006a; Nielsen et al., 2007; Prieto et al., 2007; Trček and
Teuber, 2002). The use of microsatellites for identification at the
level of strain was applied for assessing the population dynam-
ics during the microbial process of cocoa production. Analyses
of the repetitive sequence−based PCR, combined with other
culture-independent and culture-dependent methods, proved to
yield objective and specific data regarding biodiversity, pop-
ulation dynamics, and microbial succession during microbial
processing (Camu et al., 2007). Denaturing gradient gel elec-
trophoresis (DGGE) is a well-documented culture-independent
method for analysis of microbial communities in environmental
and food samples. The PCR-DGGE method was successfully
used for rapid and cost-effective screening of AAB in vinegar,
but it was not possible to differentiate all species using this
method alone (De Vero et al., 2006). Plasmid profile analysis
is also a powerful tool for identification and characterization of
bacteria, and the plasmid profile is strain specific. The major-
ity of AAB contain one to eight plasmids from 1 to >17 MDa.
Determination of plasmid profiles represents a useful technique
to characterize the main AAB of fermentations and to answer
questions that concern stability, origin, and identity of the pre-
vailing strains (Ebner, Sellmer, and Follmann, 1996; Mariette
et al., 1991; Teuber, Sievers, and Andersen, 1987; Trček, Ras-
por, and Teuber, 2000). Besides other characteristics, each genus
of the AAB typically possesses a distinctive feature, which can
be easily used for identification on the genus level.
PHYSIOLOGY
The acetic acid bacteria are mesophilic obligate aerobes that
oxidize sugars, sugar alcohols, and ethanol, with the production
of acetic acid as the major end product. During acetic acid pro-
duction, ethanol is almost quantitatively oxidized to acetic acid.
AAB exhibit resistance to high acetic acid concentrations and
low pH.
Oxidation of Ethanol to Acetic Acid
Ethanol oxidation is performed by two sequential reactions:
CH3 CH2 OH → CH3 CHO + 2H → CH3 COOH + 2H
The first reaction is catalyzed by alcohol dehydrogenase (ADH)
and the second by aldehyde dehydrogenase (ALDH), which
is located at the outer surface of the cytoplasmic membrane
(Adachi et al., 1978; Ebner, Sellmer, and Follmann, 1996; Moat,
Foster, and Spector, 2002; Saeki et al., 1997). These dehydro-
genase enzymes consist of quinoproteins and flavoproteins,
which have pyrroloquinoline quinone and covalently linked
flavin adenine dinucleotide as prosthetic groups, respectively
(Matsushita, Toyama, and Adachi, 1994; Saeki et al., 1997).
The ADH consists of two or three subunits, which include the
dehydrogenase and cytochrome c subunits that are essential for
the activity of the enzyme. The three-component ADH, consist-
ing of a 72- to 78-kDa dehydrogenase, a 48-kDa cytochrome c,
and a 20-kDa subunit, were found in A. aceti and A. pasterianus.
The two-component ADH was found in A. polyoxogenes. The
two larger subunits play a role in the intramolecular transport
of electrons from the ADH to ubiquinone and further to the
terminal oxidase during the oxidation of ethanol. The smaller
 BIOTECHNOLOGICAL APPLICATIONS OF ACETIC ACID BACTERIA
subunit helps the two functional subunits with their association
with the membrane (Kondo and Horinouchi, 1997; Saeki et al.,
1997). This membrane-bound ADH has pyrroloquinoline as a
cofactor and is independent of NAD(P)+, although a cytoplas-
mic NAD(P)+-dependent alcohol dehydrogenase has also been
identified. The latter, however, has a much lower specific activ-
ity than the membrane-bound ADH and a higher optimal pH of
6 to 8, which limits its contribution to the oxidation process of
ethanol (Adachi et al., 1978; Matsushita, Toyama, and Adachi,
1994; Takemura et al., 1993). The ADH activity of Acetobacter
spp. is more stable under acetic conditions than that of Glu-
conobacter spp., explaining the higher production of acetic acid
by Acetobacter spp. (Matsushita, Toyama, and Adachi, 1994).
The ALDH is also located in the cytoplasmic membrane, it is
independent of NAD(P)+, and it has an optimum pH of between
4 and 5. It is, in addition, also able to catalyze the oxidation of
acetaldehyde to acetate at lower pH values (Adachi et al., 1980).
Acetobacter strains can further oxidize acetic acid to CO2 and
water through the tricarboxylic acid cycle. This occurs in case
of ethanol depletion, and it seems to be an irreversible change in
their metabolism, after which they are not able to oxidize ethanol
anymore. In the presence of ethanol, this metabolic pathway is
repressed (Adams and Moss, 2000; De Ley, Gills, and Swings,
1984; Drysdale and Fleet, 1989; Guizani and Mothershaw, 2006;
O’Toole and Lee, 2003). Strains of Gluconobacter are unable
to do this as a result of a nonfunctional tricarboxylic acid cycle.
This is due to two enzymes of this cycle, α-ketoglutarate dehy-
drogenase and succinate dehydrogenase, being nonfunctional
(Adams and Moss, 2000; Greenfield and Claus, 1972; Guizani
and Mothershaw, 2006; Moat, Foster, and Spector, 2002).
Metabolism of Various Compounds
Sugar is normally preferred as a carbon source more by Glu-
conobacter than by Acetobacter (De Ley, Gills, and Swings,
1984). AAB also possess an NAD(P)+-dependent glucose dehy-
drogenase in the cytoplasm and a membrane-bound NAD(P)+-
independent glucose dehydrogenase, with the latter being
responsible for most of the glucose conversion. Glucose is oxi-
dized to glucono-δ-lactone and then to gluconic, 2-ketogluconic
and 2,5-diketogluconic acid, respectively. The route through
which AAB oxidize glucose is dependent on pH and glu-
cose concentration (Qazi et al., 1991). G. oxydans can pro-
duce up to 120 g of gluconic acid L−1 . Acetobacter strains are
also able to produce high levels of gluconic acid, but not as
high as G. oxydans (Attwood, Van Dijken, and Pronk, 1991;
Seiskari and Linko, 1985). AAB are also able to metabolize
different organic acids. This is achieved through the tricar-
boxylic acid cycle through which these acids are oxidized to
CO2 and water. It is not surprising then that Gluconobacter
spp., which lack a functional tricarboxylic acid cycle, are un-
able to oxidize most organic acids. These organic acids include
acetic, citric, fumaric, lactic, malic, pyruvic, and succinic acids
(Holt et al., 1994). G. oxydans is unsurpassed by other or-
ganisms in its ability to incompletely oxidize a great variety
105
of carbohydrates, alcohols, and related compounds. The cor-
responding products are secreted almost completely into the
medium. The entire genome of G. oxydans has been sequenced
(Prust et al., 2005), and it revealed a large and diverse set of
genes encoding membrane-bound dehydrogenases, including
four novel PQQ-dependent dehydrogenases that channel elec-
trons into the respiratory chain and many intracellular oxidore-
ductases. Seventy-five open reading frames (ORFs) were iden-
tified that encode putative dehydrogenases/oxidoreductases of
unknown functions. These findings may lead to the development
of new strategies for the employment of G. oxydans in a much
greater variety of incomplete oxidations. The genome sequence
of G. oxydans also helped resolve a long-standing controversy
over the components of the respiratory chain in Gluconobac-
ter species. The sequence analysis revealed a simple core sys-
tem consisting of a non−proton-pumping NADH:ubiquinone
oxidoreductase and two quinol oxidases. The organism lacks a
proton-translocating NADH:ubiquinone oxidoreductase (com-
plex I) and a cytochrome c oxidase (complex IV). Therefore,
the ability to translocate protons in the course of redox reactions
is rather limited. The electrochemical proton gradient is used to
generate ATP via an F1 F0 -type ATP synthase (Prust et al., 2005).
It has been shown that membrane-bound dehydrogenases trans-
fer electrons to ubiquinone, which functions as an electron donor
for the quinol oxidases (Matsushita, Toyama, and Adachi, 1994).
The active sites of the dehydrogenases are oriented toward the
periplasm. Thus, substances that are used as energy sources can
be oxidized in the periplasmic space without the need to enter
the cytoplasm (Deppenmeier, Hoffmeister, and Prust, 2002). The
products of the oxidations are easily released into the medium
via porins in the outer membrane. In addition to the membrane-
bound dehydrogenases, there is an alternative route for the oxi-
dation of sugars, alcohols, and polyols in G. oxydans. The second
set of enzymes is located in the cytoplasm, indicating that the
uptake of their substrates into the cell is required. These enzymes
catalyze reversible reactions and are NAD(P)+ dependent. The
substrates are oxidized and the resulting intermediates are phos-
phorylated and further metabolized via the pentose phosphate
pathway. The soluble, NAD(P)+-dependent enzymes are be-
lieved to participate in the synthesis of biosynthetic precursors
and are obviously involved in the maintenance of cells in the sta-
tionary growth phase (Matsushita, Toyama, and Adachi, 1994).
Within the genome, no ORF could be assigned that potentially
encodes a phosphoenolpyruvate synthase or other similar en-
zymes, indicating that G. oxydans cannot produce C6 sugars
via gluconeogenesis. Hence, the formation of glucose, for ex-
ample, from pentoses or glycerol must depend on the oxidative
pentose phosphate pathway. Further genome analysis showed
that G. oxydans contains metabolic pathways for the de novo
synthesis of all nucleotides, amino acids, phospholipids, and
most vitamins. A gene encoding a pyruvate decarboxylase was
also identified, indicating G. oxydans can produce acetaldehyde
from pyruvate (Prust et al., 2005). For the breakdown of sugars,
Acetobacter spp. are equipped with the hexose monophosphate
106 P. RASPOR AND D. GORANOVIČ
pathway and the tricarboxylic acid (TCA) cycle, whereas gly-
colysis is either absent or very weak. In A. aceti, the composi-
tion of the respiratory chain varies according to the degree of
aeration during growth. The enzymes of the Entner-Doudoroff
pathway are present in Gluconobacter spp. and in Ga. xylinus
(De Ley, Gills, and Swings, 1984; Ebner, Sellmer, and Follmann,
1996). The respiratory chain of Gluconobacter suboxydans con-
sists of cytochrome c, ubiquinone, and a terminal cytochrome-o-
ubiquinol oxidase. G. suboxydans lacks a functional TCA cycle,
although all enzymes of this cycle except succinate dehydroge-
nase are present. This organism cannot ferment glucose or other
carbohydrates since the Embden-Meyerhoff-Parnas and Entner-
Doudoroff pathways are not present. A modified pentose cycle
is used for the metabolism of glucose under aerobic conditions.
The acetyl phosphate resulting from C2 -C3 cleavage of pentose
is oxidized to CO2 , and energy is generated via oxidative phos-
phorylation (Moat, Foster, and Spector, 2002). AAB can also
use compounds, such as quinones and reducible dyes, which
are present in wine, as electron acceptors. G. oxydans exhibits
a four-fold higher oxidation reaction rate of glycerol with ρ-
benzoquinone as electron acceptor than it does with oxygen.
The by-product formed from this, hydroquinone, can be oxi-
dized back to ρ-benzoquinone for reuse (Du Toit and Pretorius,
2002). Strains of Acetobacter are able to oxidize acetate, as well
as lactate, either in the presence or absence of ethanol by us-
ing enzymes of the TCA cycle (Ebner, Sellmer, and Follmann,
1996). AAB are very susceptible to interruptions to the air sup-
ply, indicating that in order to survive suspended in a medium
with a pH of 2.5 and 10% to 14% acidity, the bacteria need a
constant supply of energy from respiration (Adams and Moss,
2000). Among the AAB, Gluconacetobacter diazotrophicus is
rather unique because it carries out nitrogen fixation (Cavalcante
and Döbereiner, 1988), is a true endophyte, and is often found in
plants grown in soils where nitrogen fertilizer input is low (Lee
et al., 2004). Optimal nitrogen fixation by G. diazotrophicus in
culture demands high aerobic conditions (Flores-Encarnación
et al., 1999). This unusual lifestyle requires an efficient mech-
anism for protection of its nitrogenase activity from the dele-
terious action of oxygen. Therefore, it was relevant to find that
G. diazotrophicus has significantly high respiratory activity in
N2 -dependent grown cells, suggesting that it uses a respiratory
protection mechanism to preserve nitrogenase activity in highly
aerobic environments (González et al., 2006c).
Acetic Acid Resistance
For the majority of microorganisms, acetic acid is detrimen-
tal at 0.5% (Conner and Kontrola, 1995). The resistance of
AAB to acetic acid is strain dependent, with ethanol functioning
synergistically with acetic acid to inhibit the bacteria (Nanba,
Tamura, and Nagai, 1984). In acidic conditions, A. aceti cy-
toplasm also becomes acidic, yet the cells continue to grow
and oxidize ethanol even as the cytoplasmic pH drops as low
as 3.7 (Menzel and Gottschalk, 1985). In this respect, A. aceti
and other AAB differ from other acidophiles, most of which
tolerate only membrane-impermeant mineral acids (e.g., HCl)
and avoid cytoplasmic acidification (Booth, 1985). The enzyme
citrate synthase, a hexameric type II citrate synthase, plays a
key role in this resistance, which detoxicates acetic acid by in-
corporation into the tricarboxylic or glyoxylate cycles. Citrate
synthase could also supply the large amounts of ATP necessary
to overcome the toxic effect of the acid (Francois et al., 2006;
Fukaya et al., 1990; Sievers, Stöckli, and Teuber, 1997). In the
search for other mechanisms of resistance to acetic acid, the most
plausible was an efflux pump in the cytoplasmic membrane that
pumps acetic acid out of the cell, and indeed, it was found. A.
aceti IFO 3283 possesses a proton motive force-dependent ef-
flux pump for acetic acid. The efflux pump of A. aceti IFO 3283
was examined using intact cells and membrane vesicles. The
accumulation of acetic acid/acetate in intact cells was increased
by the addition of a proton uncoupler and/or cyanide, suggesting
the presence of an energy-dependent efflux system. To confirm
this, right-side-out and inside-out membrane vesicles were pre-
pared from A. aceti IFO 3283, and the accumulation of acetic
acid/acetate in the vesicles was examined. On the addition of
a respiratory substrate, the accumulation of acetic acid/acetate
in the right-side-out vesicles was largely decreased, while its
accumulation was very much increased in the inside-out vesi-
cles. These respiration-dependent phenomena observed in both
types of membrane vesicles were all sensitive to a proton un-
coupler. Acetic acid/acetate uptake in the inside-out membrane
vesicles was dependent not on ATP but on the proton motive
force. Furthermore, uptake was shown to be rather specific for
acetic acid and to be pH dependent because higher uptake was
observed at lower pH (Matsushita et al., 2005). Another enzyme
contributing to acetic acid resistance was identified using two-
dimensional gel electrophoresis of a soluble fraction of A. aceti.
It revealed the presence of several proteins whose production
was enhanced, to various extents, in response to acetic acid in
the medium. A protein with an apparent molecular mass of 100
kDa was significantly enhanced in amount by acetic acid and
identified to be aconitase by NH2 -terminal amino acid sequenc-
ing and subsequent gene cloning. Amplification of the aconitase
gene by use of a multicopy plasmid in A. aceti enhanced the en-
zymatic activity and acetic acid resistance. These results showed
that aconitase is concerned with acetic acid resistance. Enhance-
ment of the aconitase activity turned out to be useful for acetic
acid fermentation because the A. aceti transformant harboring
multiple copies of the aconitase gene produced a higher concen-
tration of acetic acid with a reduced growth lag time (Nakano,
Fukaya, and Horinouchi, 2004). Other AAB cytoplasmic pro-
teins also show enhanced stability in acidic conditions, which
could be a result of altered enzyme chemistry due to semicontin-
uous exposure to acid. Bacterial alanine racemase is an enzyme
that catalyzes the interconversion of the D- and L-isomers of
alanine and normally has a basic pH optimum of >8 (Esaki
and Walsh, 1986; Seow et al., 2000), including one from an
acidophile that, unlike A. aceti, maintains a near-neutral cyto-
plasmic pH (Seow et al., 1998). Bacteria use alanine racemase to
 BIOTECHNOLOGICAL APPLICATIONS OF ACETIC ACID BACTERIA
make D-alanine bound for peptidoglycan. Acetobacter spp. con-
tain alanine that is up to 8% D-alanine, indicating the presence
of an active alanine racemase (Bruckner, Becker, and Lupke,
1993). A. aceti alanine racemase was shown to be intrinsically
resistant to acid-mediated inactivation and have the catabolic
properties of an ordinary alanine racemase (Francois and Kap-
pock, 2007). Two-dimensional gel electrophoresis was also used
to detect changes in the membrane fraction protein profile in
response to acetic acid. A 60-kDa protein named AatA was pro-
duced in response to acetic acid, and it proved to be a putative
ATP-binding cassette transporter, which possibly functions as
an exporter of acetic acid. Higher sensitivity to acetic acid of a
mutant with a disrupted aatA gene coding for AatA and enhance-
ment in acetic acid resistance that occurred by overexpression
of aatA in A. aceti and E. coli clearly indicated that AatA is
involved in acetic acid resistance (Nakano, Fukaya, and Hori-
nouchi, 2006). The mechanisms of acetic acid resistance have
mostly been studied in A. aceti, and because vinegar-producing
species of the genus Gluconacetobacter generally exhibit much
higher acetic acid resistance than A. aceti, Gluconacetobacter
species were studied regarding their acetic acid resistance (Trček
et al., 2006). The highest resistance against acetic acid was de-
scribed for the following species: Ga. europaeus, Gluconace-
tobacter intermedius, Gluconacetobacter oboediens, and Glu-
conacetobacter entanii (Boesch et al., 1998; Schüller, Hertel,
and Hammes, 2000; Sievers and Teuber, 1995; Sokollek, Her-
tel, and Hammes, 1998b). Ga. europaeus, Ga. intermedius, and
A. pasterianus were compared for their growth characteristics,
acetic acid production rate, acetic acid tolerance, and molecular
characteristics of pyrroloquinoline quinone (PQQ)-dependent
ADH. Ga. europaeus exhibited the highest resistance to acetic
acid (10%), whereas Ga. intermedius and A. pasterianus re-
sisted up to 6% of acetic acid. In media with different concen-
trations of acetic acid, the maximal acetic acid production rate
of Ga. europaeus slowly increased, but specific growth rates
decreased concomitant with increased concentration of acetic
acid in medium. The lag phase of A. pasterianus was twice
and four times longer in comparison to the lag phases of Ga.
europaeus and Ga. intermedius, respectively. PQQ-dependent
ADH activity was twice as high in Ga. europaeus and Ga. in-
termedius as in A. pasterianus. The purified enzymes showed
almost the same specific activity to each other, but in the pres-
ence of acetic acid, the enzyme activity decreased faster in A.
pasterianus and Ga. intermedius than in Ga. europaeus. Higher
ADH activity may produce a bigger energy pool available for
other membrane-associated processes, such as an acetate/acetic
acid export system (Matsushita et al., 2005; Steiner and Sauer,
2003), which might also explain a capability of Ga. europaeus
to resist higher concentrations of acetic acid. These results sug-
gest that high ADH activity in the Ga. europaeus cells and high
acetic acid stability of the purified enzyme represent two of the
unique features that enable this species to grow and stay metabol-
ically active at extremely high concentrations of acetic acid
(Trček et al., 2006).
107
pH
The optimum pH for the growth of AAB is 5.5 to 6.3 (Holt
et al., 1994). However, these bacteria can survive at the low
pH values of between 3.0 and 4.0, and some strains have been
isolated from an aerated media containing acetate that could
grow at pH values as low as 2.0 to 2.2 (Du Toit and Pretorius,
2002). It was postulated that there are three groups of strains that
might exist in vinegar production, namely, acetophilic strains
that grow only at a pH value of about 3.5, acetophobic strains
that only grow at pH levels higher than 6.5, and acetotolerant
strains that can grow at both these values. There may be a gradual
development from acetophobic to acetotolerant strains and, with
prolonged exposure to low pH and high acetic acid concentra-
tions, to acetophilic strains. This suggests the development of a
gradual acid resistance in these bacteria (Kittelman et al., 1989;
Kösebalan and Özilgen, 1992).
Temperature
The optimum growth temperature for most AAB is 25◦ C
to 30◦ C (Holt et al., 1994). The maximum temperature for the
growth of A. aceti was found to be about 35◦ C (De Ory, Romero,
and Cantero, 1998). Thermotolerant AAB that are able to grow at
37◦ C to 40◦ C have also been isolated. These bacteria were able to
oxidize ethanol at 38◦ C to 40◦ C at the same rate that mesophilic
strains do at 30◦ C, as well as being able to oxidize ethanol more
rapidly than the mesophilic strains at the higher temperatures
(Ndoye et al., 2006; Saeki et al., 1997). AAB can also be active
at lower temperatures, and weak growth was observed even at
10◦ C (Joyeux, Lafon-Lafourcade, and Ribérau-Gayon, 1984).
BIOTECHNOLOGICAL APPLICATIONS IN
BIOTECHNOLOGY USING ACETIC ACID BACTERIA
Vinegar and Acetic Acid Production
Vinegar is a transparent liquid, colorless or the color of the
raw material, or colored by caramel with a prescribed content
of acetic acid between 40 and 150 g acetic acid L−1 (Ebner,
Follmann, and Sellmer, 1995). Worldwide, acetic acid is called
vinegar if it is obtained biologically by oxidative conversion of
ethanol-containing solutions by AAB. Although vinegar does
not entirely exclude diluted chemically produced acetic acid in
every country of the world, this term is used here to describe
acetic acid that is produced by primary microbial metabolism,
traditionally called “acetic acid fermentation” or “vinegar fer-
mentation” (Ebner, Sellmer, and Follmann, 1996). In 1916, the
first dedicated plant for the production of acetic acid, by chemical
rather than by biological means, became commercial. However,
traditional bioprocessing is still the main source of vinegar for
the food market. The first vinegar was probably a result of a
spoiled alcoholic beverage, considering that the Latin word ace-
tum means sour or sharp wine. Thus, it has been produced as
long as wine making has been practiced and therefore dates back
to at least 10,000 BC. Vinegar is traditionally a product of acetic
acid fermentation from natural alcoholic solutions (10%−15%
108 P. RASPOR AND D. GORANOVIČ
by volume of ethyl alcohol). Raw materials used for vinegar
production can be wine, cider, beer, and other liquors deriving
from the alcoholic fermentation of cereals, fruit and potatoes,
and sugar solutions, such as molasses, honey, and whey, and also
diluted pure ethanol with the addition of nutrients. These are the
so-called brewed vinegars, derived from the oxidation activity
of aerobic microorganisms. The common types of vinegar in a
region often reflect the local alcoholic beverage (e.g., rice vine-
gar is popular in Japan, wine vinegar in France, and malt vine-
gar in the UK) (Guizani and Mothershaw, 2006; Plessi, 2003).
Most natural raw materials do not require the addition of extra
nutrients, but for the production of spirit vinegar, a mixture of re-
quired nutrients was developed for optimal bioprocessing. Com-
mercially available mixtures contain supplements such as dried
yeast extract in order to restart the bioprocess more quickly if
stopped by a disturbance. Principally, nutrients should be added
sparingly to exert a selection pressure directing to a low require-
ment for nutrients. The water used for the preparation of mashes
must be clear, colorless, odorless, bacteriologically clean, and
without sediments or suspended particles. It must also be free
of chlorine, ozone, and other chemical compounds damaging to
bacteria (Ebner, Sellmer, and Follmann, 1996). The main pro-
ducing organisms for aerobic processes are AAB, mostly of the
genus Acetobacter (some species now belonging to the genus
Gluconacetobacter), and for anaerobic processes, bacteria of
the genus Clostridia. Aerobically, food-grade acetic acid is pro-
duced by the two-step vinegar process. The first step is the fer-
mentation to produce ethanol from a carbohydrate source such as
glucose. This is carried out at 30◦ C to 32◦ C using the yeast Sac-
charomyces cerevisiae. The second step is the biooxidation of
ethanol to acetic acid (Cheryan, 2000; Ebner, Sellmer, and Foll-
mann, 1996). The alcohol-containing liquor is called “mash.”
Usually, it also contains some acetic acid, expressed in grams of
acetic acid per 100 mL (% w/v). The sum of ethanol (vol %) and
acetic acid (g/100 mL) is called “total concentration” because the
sum of these rather incommensurable values gives the maximal
concentration of acetic acid that can be obtained by complete
bioprocessing. The quotient of the total mash concentration in-
dicates yield (Ebner, Sellmer, and Follmann, 1996). AAB are
able to produce very high concentrations of acetic acid. Some
strains can easily produce more than 50 and up to 150 g acetic
acid L−1 (Lu, Lee, and Chen, 1999; Sievers, Stöckli, and Teuber,
1997). Primary metabolic conversion of ethanol to acetic acid
is accompanied by secondary metabolism, which combines pro-
ducing flavor and typical aroma. Small quantities of volatile sub-
stances are formed during secondary metabolism, which include
ethane, acetaldehyde, ethyl formate, ethyl acetate, isopentyl ac-
etate, butanol, methylbutanol, and 3-hydroxy-2-butanone, which
vary from vinegar to vinegar, depending on the starting mate-
rial, and because of their individual properties produce vinegars
with a variety of odor, taste, color, and other properties. The bio-
process is usually stopped at a minimum residual ethanol level
to avoid overoxidation. If ethanol concentration falls under this
level, acetic acid is oxidized to water and CO2 (Plessi, 2003).
The most important properties of a production strain in the vine-
gar industry are tolerance to high concentrations of acetic acid
and total concentration, low nutrient requirements, inability to
overoxidize the formed acetic acid, high production rate, and
resistance to phage infections (Ebner, Sellmer, and Follmann,
1996). Although a variety of bacteria can produce acetic acid,
mostly members of Acetobacter (Gluconacetobacter) are used
commercially, typically the aerobic bacterium A. aceti at 27◦ C to
37◦ C (Adams and Moss, 2000; Guizani and Mothershaw, 2006;
Josephsen and Jaspersen, 2006; O’Toole and Lee, 2003; Plessi,
2003). A. europaeus, now named Ga. europaeus, was found to
be the main producing species of industrial vinegar bioreactors
in central Europe (Sievers, Sellmer, and Teuber, 1992). Other
species frequently isolated from vinegar fermentations include
A. pasterianus, A. polyoxogenes, Ga. xylinus, Ga. hansenii, Ga.
oboediens, and Ga. intermedius (Gullo et al., 2006; Yamada,
2000). Recently, thermoresistant strains of Acetobacter tropi-
calis and A. pasterianus have been isolated and selected for
their ability to grow and produce acetic acid at high tempera-
tures. The thermoresistant property of these strains is important
in tropical regions, where temperature is regularly higher than
30◦ C and where cooling expenses can readily become limit-
ing for industrial application (Ndoye et al., 2006; Ndoye et al.,
2007). In the production of traditional Chinese vinegars, cultures
of Acetobacter lovaniensis are used (Zheng, Wang, and Zheng,
2006). Pure cultures are not widely used in the acetic acid indus-
try (Josephsen and Jaspersen, 2006). Acetobacter spp. are better
acid producers and are more commonly used in commercial
vinegar production, but they suffer from the need to overoxi-
dize acetic acid, which is not a problem with Gluconobacter
spp. (Adams and Moss, 2000; Guizani and Mothershaw, 2006).
Ethanol is dehydrogenated to acetic acid and the reduced co-
substrates are oxidized via the respiratory chain (Plessi, 2003).
This bioprocess is an incomplete oxidation because the reduc-
ing equivalents generated are transferred to oxygen and not to
carbon dioxide. In any industrial process, the identification and
quantification of different species and strains involved in the
bioprocess is relevant. It is also important to monitor the likely
presence or absence of microorganisms in the final product after
the manufacturing process. Thus, a quick and accurate proce-
dure for detection and enumeration of these bacteria is important
from the industrial point of view. AAB have traditionally been
enumerated by quantifying viable colonies by plating on solid
culture media. Some work has been done using selective media,
but the limitations of cultural methods are the time required and
the inability to detect viable but nonculturable bacteria. To over-
come the disadvantages of traditional enumeration of AAB, a
new method based on epifluorescence and direct counting has
been developed, which yields more accurate estimates of to-
tal and viable cell number as an alternative to plate counting
(Baena-Ruano et al., 2006). The submerged bioprocess has al-
most completely replaced surface methods. Industrial processes
have evolved from the simple “let-alone” method involving a
partially filled open container of wine exposed to air to the “field”
 BIOTECHNOLOGICAL APPLICATIONS OF ACETIC ACID BACTERIA
method in which a series of casks are filled with wine and inoc-
ulated in series by the vinegar produced in the previous casks.
A certain amount of vinegar is still manufactured following the
centuries-old empirical methods of the small producer; however,
during the past century, a flourishing vinegar manufacturing in-
dustry has developed (Cheryan, 2000).
Technological Solutions in Vinegar Production
There are various techniques for acetification that differ in
the means by which the three interacting components, ethanol,
bacteria and oxygen, are brought together. The industrial bio-
process involves a series of casks filled with wine and inoculated
in series by the vinegar produced in the previous casks. Vine-
gar production usually requires lower capital investment, has
shorter start-up times, and can generate different types of vine-
gar when different carbohydrate sources are used (Guizani and
Mothershaw, 2006; Plessi, 2003). The overall theoretical yield
of acetic acid produced from glucose is 0.67 g acetic acid per
gram glucose. Complete aeration and strict control of the oxygen
concentration during bioprocessing are important to maximize
yields and keep the bacteria viable (Cheryan, 2000). An inter-
ruption in the oxygen supply, even for a short period of time,
will result in death of the bacteria and failure of the process
(Guizani and Mothershaw, 2006). The theoretical amount of air
required for 1 L of vinegar containing 6% of acetic acid is about
120 L, whereas, in practice, given the slow rate of liquid-gas
exchange, the amount required is much greater. Although the
industrial submerged processes are more efficient, the product
is less aromatic than vinegars obtained by slower processes. This
is due to the fact that, given the brief period of contact, the es-
terases do not have time to perform their function adequately
and the characteristic volatile substance content is low as a re-
sult. The product is therefore filtered and, in some cases, put into
wooden casks to age. The result is vinegar of superior quality,
which is rich in biological principles, generated by bacterial cell
autolysis, and perfectly clear. Industrial vinegar manufacturing
processes fall into three main categories: slow processes, quick
processes, and submerged processes (Plessi, 2003).
Traditional Slow Orleans Process
Although the slow processes, which are the oldest commer-
cial procedures and resemble home-brewing techniques, are
mostly no longer used, the “Orleans” process is still in use for
the production of high-quality vinegar (Adams and Moss, 2000;
Guizani and Mothershaw, 2006; Plessi, 2003). The procedure
has remained unchanged throughout the years but is very slow
and requires a lot of space. In the Orleans method, holes are
bored into the casks, and a glass tube is inserted to allow the ad-
dition and removal of vinegar. The starting liquor is placed in a
large cask, containing wood shavings or grape stalks, where the
acetification process begins (Guizani and Mothershaw, 2006). If
the liquor is left undisturbed, a surface culture of AAB forms at
the interface between the acetifying medium and the air, form-
109
ing a bacterial “mat” or pellicle (velum). As oxygen enters the
liquor through the mat, the bacteria use it and convert the ethanol
to acetic acid. Therefore, vinegar is produced at the surface and
diffuses throughout the body of the liquor. In a similar man-
ner, the ethanol diffuses to the pellicle, where it is used by the
bacteria (O’Toole and Lee, 2003). The cask is left undisturbed
until the acidity reaches the required level, after about eight days,
then the liquid is withdrawn and transferred into barrels, which
are left only one-half or two-thirds full, and where the liquid
remains until acidity reaches its peak (about three months). Af-
ter the acidity has reached the peak, two-thirds or three-fourths
of the vinegar is withdrawn from the bottom of the barrel every
week, and an equal volume of liquid from the generator cask is
added from the top (Plessi, 2003).
The Generator Process (German Process)
Generator processes, also called the “trickling” or “German”
processes because they were first introduced in Germany, have
been in general use for almost 200 years. The first generator pro-
cess for the production of vinegar was introduced by Schutzen-
bach in 1823 (Cheryan, 2000). Commercially, the quick vinegar
process is the most common biofilm process in current operation
(Pometto and Demirci, 2006). The bioreactors (generator tanks)
are usually made from wood or sometimes steel, are equipped
with cooling coils, and are vented to allow the air to circulate
(Cheryan, 2000; Plessi, 2003). The volume of a typical com-
mercial vinegar bioreactor is 50 to 60 m3 (O’Toole and Lee,
2003). This process is principally a surface process in which the
microbial population is immobilized on wood shavings (prefer-
ably beech) or grape stalks, with which the tanks are filled. The
alcoholic liquid is distributed over the surface of the packed bed
using a spray mechanism, and it then trickles over the wood
shavings or grape stalks covered with AAB, while a large vol-
ume of air is forced up from the bottom of the bioreactor. The
liquid is collected at the bottom and continuously pumped back
to the sprinkler on the top of the bioreactor, until acetification
is complete, which generally takes about 1 week, providing the
temperature is kept in the optimal range of 27◦ C to 30◦ C. A
measured volume of vinegar is then withdrawn from the bot-
tom of the bioreactor and is replaced with an equal volume of
fresh liquid (Cheryan, 2000; Plessi, 2003). Ethanol conversion
to acetic acid is 88% to 90%, and the rest of the substrate is used
in biomass production or lost by volatilization. Advantages of
this bioprocess include low costs, ease of control, high acetic
acid concentrations, and lower space requirements. The costs
of the wood shavings, which have to be replaced at least once
a year, long start-up time, loss of ethanol by volatilization, and
production of slime-like material by the Ga. xylinus, are prob-
lems. Furthermore, there are zones of overoxidation, uneven
aeration, and heat development (Cheryan, 2000; Guizani and
Mothershaw, 2006; Plessi, 2003). Nevertheless, this mechanism
for vinegar production is still in practice due to high sensorial
quality of the final product.
110 P. RASPOR AND D. GORANOVIČ
Submerged Process
The third category involves the submerged processes, which
were introduced around 1952. The Frings acetator was the first
submerged culture process, but others were subsequently de-
veloped, including Yeoman’s cavitator used in the United States
and Japan, the Bourgeois process used in Spain and Italy, and the
Fardon process used in Africa (O’Toole and Lee, 2003). These
are the most up to date, benefiting as they do from industrial
experience with submerged culture techniques employed in the
field of antibiotics (Plessi, 2003). Only a few studies have been
done on preparing and preserving starter cultures of AAB for
an optimum acetification process. Such studies have widely de-
scribed the microbiological conditions to obtain a high level of
biomass and to revitalize the starter culture as quickly as possi-
ble (Ndoye et al., 2007; Sokollek and Hammes, 1997; Sokollek,
Hertel, and Hammes, 1998a). The bioreactor is made of stain-
less steel or polypropylene reinforced with fiberglass, and it is
equipped with devices that ensure adequate and continuous air
flow and thermometers and cooling coils for monitoring and
maintaining liquor temperature of about 30◦ C. Sometimes, an
automatic gage for measuring the alcohol content of the fer-
menting liquid is applied (Plessi, 2003). Due to the sensitivity
of the bacteria, submerged cultures require continuous monitor-
ing. The air is forced into the liquid, and the level of gas-phase
oxygen is crucial to the process; thus, efficiency is based on
broth aeration with oxygen (Adams and Moss, 2000). Interrup-
tion of aeration is damaging to AAB and a break of only 1 min
can completely arrest acetification, which cannot be recovered
on its own when aeration is resumed. The longer the interruption
of aeration and the higher the total concentration of the mash,
the greater is the damage to the bacteria. At constant total con-
centration, damage increases with an increasing concentration
of acetic acid and with increasing bioconversion rate. The ox-
idation rate of ethanol drops rapidly, as well as the activity of
enzymes involved (ADH, ALDH) after an interruption of the
oxygen supply and high acetic acid concentration. Under con-
ditions of industrial acetic acid production, the microorganisms
undergo considerable stress, caused by a high concentration of
acetic acid and limited oxygen supply. A high utilization of
oxygen, up to 70%, can be achieved in industrial production.
A sparing use of air is very important because of the volatility
of ethanol and acetic acid. However, the use of pure oxygen
or highly oxygen-enriched air may easily damage the bacteria
(Ebner, Sellmer, and Follmann, 1996). For industrial processes,
10% to 18% ethanol and five times the nutrients used for sur-
face bioprocesses are the starting conditions for fermentation.
Oxidation of the alcohol starts slowly by means of aeration,
and bioconversion of ethanol is apparent after about twenty-four
hours. Air is then introduced at regular hourly intervals, and it
uniformly permeates the body of the liquor and enables a rapid
bioprocess. The productivity is high, and the process is operated
in a semicontinuous manner that helps minimize variation in the
product. When the concentration of ethanol reaches minimum
residual level (depending on the type of vinegar), a quantity of
the solution is removed and replaced with fresh substrate con-
taining 10% to 18% ethanol (Cheryan, 2000). Monitoring of
the ethanol concentration is important because in the case of
ethanol depletion, the Acetobacter strains undergo a change in
their metabolism and begin overoxidizing acetic acid to CO2
and water. Ethanol represses overoxidation and, consequently,
reduces the risk of overoxidation during acidification; a resid-
ual level of ethanol is always maintained (Ebner, Sellmer, and
Follmann, 1996; Guizani and Mothershaw, 2006). Exhaustion of
ethanol is also damaging to the bacteria, which is analogous to
the damage resulting from an interruption of the oxygen supply
and also depends primarily on the total concentration and the
duration of ethanol lack (Ebner, Sellmer, and Follmann, 1996).
Monitoring of ethanol concentration during an acetification cy-
cle also provides data for estimating the mean acetification rate,
which is an important factor when optimizing the process. Op-
timizing the process entails examining the influence of diverse
factors on the reactor performance, which involves much exper-
imental work, owing to the large number of variables potentially
affecting the process and the typical difficulty encountered in ex-
periments with microbial reactions (Garcı́a-Garcı́a et al., 2007;
Garrido-Vidal, Pizarro, and González-Sáiz, 2003). Specifically,
when optimizing the acetification process, the overall acetifica-
tion rate in each semicontinuous work cycle must be determined
in order to compare it with values obtained under different exper-
imental conditions. The overall acetification rate is usually deter-
mined from the final acidity of the culture medium immediately
prior to unloading the reactor, the unloaded volume, and the total
duration of the production cycle. However, the rate can also be
determined from the variation of the ethanol concentration dur-
ing a cycle. A number of devices for continuously monitoring the
ethanol concentration in the culture medium (e.g., Alkosens and
Frings probes) are currently available. Effectively optimizing
vinegar production entails determining the acetification rate in
both ways. Its determination from the ethanol concentration al-
lows one to establish the variation of the biological oxidation rate
throughout the cycle, which is especially interesting with a view
to assessing the influence of operational variables on the differ-
ent steps of the process and alter them as required to improve
the outcome. However, determining the acetification rate from
final acidity data provides an additional measure of the amount
of substrate evaporated through aeration in each cycle as well as
other forms of ethanol consumptions. In any case, it is known that
different ethanol consumptions to that used for acetic acid for-
mation are very small and can be neglected (Garcı́a-Garcı́a et al.,
2007; Garrido-Vidal, Pizarro, and González-Sáiz, 2003; Gómez
and Cantero, 1998; González-Sáiz, Pizarro, and Garrido-Vidal,
2003). Charging and refilling of the bioreactor must be done with
mash of the same total concentration and under rapid mixing be-
cause a locally formed strong concentration gradient damages
the bacteria. At the same time, temperature has to be kept con-
stant because a repeated change in temperature causes a similar
result (Ebner, Sellmer, and Follmann, 1996). It is important to
prevent damage to the bacteria because dead cells cause foaming.
 BIOTECHNOLOGICAL APPLICATIONS OF ACETIC ACID BACTERIA
Mechanical defoaming techniques are used to eliminate this
problem. Comparing submerged bioprocesses to surface bio-
processes results in higher productivity, faster conversion of
ethanol, smaller reaction volumes, fewer interruptions due to
clogging by shavings, and lower capital investment per product
amount. Much of the work done has been performed with batch
mode, in which all carbohydrates and nutrients are added at the
beginning. For bioprocesses suffering from substrate inhibition,
the fed-batch mode should be practiced (Cheryan, 2000). Vine-
gars with a high acid concentration of 170 g acetic acid L−1
were obtained by fed-batch fermentation (Berraud, 2000). Pro-
duction of highly concentrated vinegar would be advantageous
to the vinegar production industry because of decreased storage
capacity and filtration volumes. However, reaching these high
acetic acid concentrations would require control of parameters
such as alcohol and acetic acid concentrations in the medium
because the growth of AAB is directly associated with these
variables. Batch mode of operation is not the most suitable and
does not allow for the optimization of these parameters. Usually,
the number of viable AAB stops increasing above 40 g acetic
acid L−1 , and moreover, this number is inhibited by ethanol (the
substrate) at about 40 g ethanol L−1 (Berraud, 2000; Park et al.,
1989). Cell-recycle bioreactors using a membrane module as the
separation device have been shown to increase the productivity
and may have some advantages over immobilized cells, such as
higher concentration of free cells, no diffusion limitation, ex-
cellent mixing in the bioreactor, and a cell-free product stream.
The greatest advantage is that cell concentrations far in excess
of normal levels can be used with no danger of cell washout.
A “draw-and-fill” bioreactor in combination with a membrane
appears to be the optimum design. In this design, the reaction
vessel is operated as a batch fermenter. At the end of the fermen-
tation, broth is withdrawn through the membrane module. The
cells are recycled, and the reaction vessel is charged with fresh
substrate. For all types of bioreactors studied, increasing dilu-
tion rate increases volumetric productivity but decreases specific
productivity (grams acetate produced per gram cells). Thus, in
cell-recycle bioreactors, the nutrient supply should be increased
in proportion to cell concentration to realize the full potential of
the microorganisms (Cheryan, 2000). During vinegar produc-
tion using the Frings acetator, bacteriophages can be a problem
(Adams and Moss, 2000; Ebner, Sellmer, and Follmann, 1996;
Guizani and Mothershaw, 2006; O’Toole and Lee, 2003). As
in the dairy industry, these bacterial viruses attack and dam-
age bacteria. Acetobacter-specific bacteriophages demonstrated
by electron microscopy in submerged and trickling fermenters
are discussed to be responsible for bioprocess interruption in
industrial vinegar bioreactors. In trickling generators, the pro-
cess is sometimes slowed down but hardly stops completely. In
submerged processes, complete loss of productivity has been
observed (Ebner, Sellmer, and Follmann, 1996). Bacteriophage
infections are a lesser problem if a variety of strains are used be-
cause some strains are less susceptible to infection and can take
over the fermentation in case of phage infection. The solution
111
is to use filtered, sterilized air and pasteurized substrate (Adams
and Moss, 2000). Raw vinegar is more or less turbid because it
contains AAB and deposits that originate from the raw material,
and it therefore needs further treatment (Ebner, Follmann, and
Sellmer, 1995). In downstream processing, the first operation is
cell separation, which can be done by cross-flow microfiltration.
When a microfilter or ultrafilter is combined in a semi–closed-
loop configuration to the bioreactor, it improves productivity,
while also providing a cell-free broth for subsequent downstream
processing. Depending on the physical and chemical nature of
the bioproducts, the cell-free broth is subjected to chromatog-
raphy, electrophoresis, crystallization, precipitation, extraction,
distillation, and/or membranes. Freeze concentration is the pre-
ferred method for vinegar (Cheryan, 2000).
Several methods and different types of bioreactors for sub-
merged acetification have been described and patented, such as
Frings acetator, cavitator, bubble column bioreactor, or biore-
actors with different aeration systems, such as the jet or effigas
turbine (vinegator). Each system offers very different values for
oxygen mass transfer coefficients. The Frings acetator and the
bubble column fermenter are the most widely used in vinegar-
producing industries. The bubble column fermenter consists of
a column with a diameter height ratio of 1:5 (or more). The up-
per part of the column has the major diameter, which permits
the reduction of the bubble volume at the surface and facilitates
the sedimentation of the bacterial culture in order to reduce the
removal of these along the finished product. The air is supplied
from a compressor and passes through a diffuser (perforated
plate) at the base of the column by which aeration and mechan-
ical agitation are attained (Tesfaye et al., 2002).
The most commercially successful bioreactor is the Frings
acetator (Ebner, Follmann, and Sellmer, 1995; Guizani and
Mothershaw, 2006). The Frings acetator is the most common
equipment for the production of all kinds of vinegar. Energy
consumption is about 400 W/L of ethanol, and the yield is as
high as 98%. Commercial sizes of acetators are sufficient for
the conversion of up to 3,600 L of pure ethanol in twenty-
four hours. The different processes demand excellent perfor-
mance of the bioreactor, especially of its aeration system. The
Frings aerator is self-aspirating, and no compressed air is needed.
The rotor is installed at the shaft of a motor mounted under
the bioreactor, connected with an air suction pipe, and sur-
rounded by a stator. It sucks air and pumps liquid, thereby
creating an air-liquid emulsion that is ejected radially outward
through the stator at a given speed. This speed must be cho-
sen adequately so that the turbulence of the stream causes a
uniform distribution of the air over the whole cross-section of
the bioreactor. Because bacterial cell lysis cannot be avoided
completely, the surface-active substances are released from the
cells and cause foaming. The bioreactor is therefore equipped
with a mechanical defoamer. The Frings alkograph is an au-
tomatic instrument for measuring the amount of ethanol in
the fermenting liquid, and it is installed in the fermenter. To
carry out the single-stage semicontinuous process at defined
112 P. RASPOR AND D. GORANOVIČ
alcohol content, a contact in the alkograph activates the vine-
gar discharge pump. As soon as a preset level has been reached,
the mash pump starts adding fresh mash. This pump is controlled
by the temperature in order to refill under constant temperature.
The pump is stopped when the desired level is reached, and an
automatic cooling system is activated. A bioprocess cycle takes
twenty-four to forty-eight hours. The Frings Alkosens is also a
device for measuring the amount of ethanol, but it is newer and
has some advantages over the older Frings alkograph because it
gives faster measurements. New sensor technologies and more
sophisticated computer equipment permit a fully automated pro-
cess control of continuous, semicontinuous, and single-stage
or two-stage high-strength bioprocesses. Comparing the single-
and dual-stage high-strength bioprocesses, vinegars can be pro-
duced with acidity up to 17.5% and 20.5%, respectively (Ebner,
Sellmer, and Follmann, 1996).
Fruit Substrate Vinegars
Wine Vinegar
Wine vinegar, which is obtained from acetous conversion of
wine, is largely produced in continental Europe. The wines used
for acetification are those with low ethanol content (7%−9%
v/v) or those in which volatile acidity is too high. Spoilt wines
cannot be used. If wines with high alcohol content are to be
used, they need to be appropriately diluted with water because
a high concentration of alcohol will inhibit the development
of AAB. For the same reason, the wine must be free of sulfur
dioxide. Both white and red or rose wines can be used to pro-
duce white or red vinegar, respectively. In the home or small-
scale production of wine vinegar, the wine is poured into small
wooden barrels, together with the vinegar starter, which consists
of colonies of Acetobacter taken from barrels in which vinegar
has already been produced. The barrel must contain air, so for
this reason it is not filled completely. Acetification is slow and
stops spontaneously when acidity reaches 7% to 8%. The slow
transformation of wine into vinegar leads to the formation of
many substances that impart excellent sensorial qualities to the
end product. The most important of these are acetaldehyde and
ethyl acetate. The vinegar is then partly drawn off for use and re-
placed with fresh wine. Vinegar produced in this way is bound to
vary in composition and character; it may be more or less cloudy,
and its acidity may vary according to the degree of alcohol in
the wine and the bioprocess nature (Plessi, 2003). Wine vine-
gars contain the same spectrum of amino acids as spirit vinegar,
but in larger amounts. Polyphenol compounds have been shown
to be of great interest regarding the stability of wine vinegars.
The phenolic compounds are generally contributed by the solid
parts of the grapes. Therefore, in the case of wines kept in longer
contact with the grapes, a larger amount of polyphenols will be
extracted. To avoid instability due to enzymes and microorgan-
isms, wine vinegar is pasteurized prior to bottling or afterward.
At lower temperatures, only the enzymes are inactivated, and at
higher temperatures, the microorganisms as well (Ebner, Foll-
mann, and Sellmer, 1995).
Cider Vinegar
Cider vinegar is produced from cider that has undergone ace-
tous bioconversion and is widely used as table vinegar. It is
yellowish in color and may be darkened with caramel. Its acid-
ity is not very high and its acidic, astringent flavor recalls the
fruit of origin (Plessi, 2003). Many of the compounds found in
cider vinegar were not found in the cider from which the vinegar
was produced. The compounds in cider vinegar correspond ap-
proximately to those found in wine vinegar (Ebner, Follmann,
and Sellmer, 1995).
Honey Vinegar
This vinegar is obtained from honey, to which the right quan-
tity of water is added. After alcoholic fermentation is finished,
the acetic acid bacteria take over. At the right temperature condi-
tions and oxygen levels, AAB produce acetic acid. The vinegar
is then clarified by bland filtration or by decanting, so that all
the qualities of honey remain unaltered (Plessi, 2003).
Fruit Vinegars
Fermented juices from a variety of other fruits, such as
peaches and berries, are also used to produce vinegar. Because
these alcoholic liquids are not distilled, they maintain the subtle
flavors and aromas of the raw ingredients (Plessi, 2003).
Starch Substrate Vinegars
Malt Vinegar
Malt vinegar is produced from malted barley with or with-
out the addition of other cereals. Malt vinegar manufacture in-
volves mashing, fermentation, and acetification. During mash-
ing, the malted barley, sometimes mixed with other cereals such
as maize and rice, is milled and mixed with hot water in mash
casks, where the starch is converted by α-amylase into maltose,
dextrose, and dextrins. The sweet liquor drains off the mash
through the perforated false bottom of the cask and is collected
in vessels, where it is fermented by the addition of yeasts that
convert the fermentable sugars to ethanol and carbon dioxide.
When fermentation is complete, the alcoholic liquor is separated
from the yeasts and acetified by inoculation with Acetobacter
cultures. The resultant alcohol is thus oxidized to acetic acid in
the presence of atmospheric oxygen. The process lends itself to
the different systems in current use. The vinegars are more or
less aromatic, and the superior varieties are those produced by
the old and slow Orleans process. Malt vinegar is straw colored
and must contain 4% w/v of acetic acid (Plessi, 2003).
Rice Vinegar
In the Far East, where rice is the staple cereal, vinegar is
prepared from rice, sake, or the by-products of sake manufac-
ture. Traditional methods, similar to the Orleans process, are
still in use but have been largely superseded by modern sub-
mersion techniques. This vinegar has a fairly low acidity and
 BIOTECHNOLOGICAL APPLICATIONS OF ACETIC ACID BACTERIA
high amino acid content. It is light in color and has a clean,
delicate flavor. It is therefore highly prized in oriental cooking
because it does not significantly alter the taste of the food (Plessi,
2003). The Chinese vinegars are rich in amino acids, and, be-
sides acetic acid, pyroglutamic acid and lactic acid predominate
(Ebner, Follmann, and Sellmer, 1995).
Molasses Vinegar
This vinegar is prepared from sugar syrup or molasses. It
serves to make use of the by-products of the sugar industry, but
is not widely used (Plessi, 2003).
Spirit Vinegar
By far the largest percentage of vinegar is spirit vinegar,
sometimes referred to as white, distilled, or alcohol vinegar,
which is produced from diluted purified ethanol. In countries
where it is permitted by law, wide use is made of synthetic
ethanol, diluted to 10% to 14% v/v. Mashes obtained by alco-
holic fermentation of natural sugar–containing liquids may also
serve as raw materials. Spirit vinegar is strongly acidic but not
aromatic. It contains a small amount of amino acids, which are
mainly products of autolysis of the AAB. This vinegar is less
expensive and is the most widespread in the world (Ebner, Sell-
mer, and Follmann, 1996; Plessi, 2003). In a number of coun-
tries, spirit vinegar is sold completely colorless, which requires
a treatment with activated carbon. In other countries, it is col-
ored yellow with caramel or other colorants admitted for food
(Ebner, Follmann, and Sellmer, 1995).
Special Vinegars
Balsamic Vinegar
A particular type of highly prized vinegar has been pro-
duced for centuries in Northern Italy. The raw material is grape
must, preferably Trebbiano. When alcoholic fermentation be-
gins, about twenty-four hours after pressing, the must is boiled
gently until it has reduced to about one-half or one-third of its
starting volume. The result is a liquor with a high sugar concen-
tration (about 30%) in which alcoholic and acetous fermentation
take place together very slowly in special barrels, with decreas-
ing volumes from 60 to 20 L arranged in succession, also known
as a barrel battery. The vinegar barrel battery consists of a vari-
able number of barrels (between five and twelve or even more) of
different woods, which permit different transpiration and, con-
sequently, different biochemical reactions. Some of the more
commonly used woods to make the barrels are ash, cherry, oak,
juniper, mulberry, and chestnut. Each wood will influence the
balsamic vinegar in a unique way. The chestnut is rich in tannin
and gives good color. The cherry is sweet. The oak, because of
its density, helps concentrate the vinegar and is often used in the
first and last barrels. The barrel battery is set up in well-ventilated
areas that are hot and dry in summer and cold in winter. Accord-
ing to the traditional method, part of the contents of the smallest
barrel is drawn off annually for consumption and replaced with
113
an equal volume from the next largest barrel in the battery. This
is in turn replenished from its neighbor, and so on up the line.
Finally, the largest barrel is topped up with the season’s boiled
must. The process takes at least twelve years, although it is not
uncommon to find vinegars that are fifty or more years old. On
average, the process for traditional balsamic vinegar production
takes about twenty years. The yield is very low (no more than 1
L of vinegar from 100 kg of fresh must); however, the end prod-
uct is of exceptionally high quality, dark brown in color, of a
syrupy consistency, sweet and sour to the taste, and with a char-
acteristically pleasant aromatic smell. During the bioprocess,
maturing, and aging, the product becomes highly concentrated
and the sugars, alcohols, aldehydes, and organic acids undergo
gradual biochemical transformation. The sugar tolerance is an
important trait of AAB for the production of traditional balsamic
vinegar because it is made from cooked must with a high sugar
concentration. Yeasts (Saccharomyces and Zygosaccharomyces)
and AAB (Acetobacter and Gluconobacter) are needed for the
formation of this vinegar. In particular, few species are able to
grow at elevated sugar concentration (e.g., Gluconobacter dia-
zotrophicus species are able to grow at 30% of D-glucose) (Gullo
et al., 2006). In traditional balsamic vinegar, the total solids are
very high (20%−70%), acidity varies between 6% and 18% w/v
acetic acid, and there are large amounts of sugars, essentially
glucose and fructose, as well as numerous aromatic substances
that have gradually been formed over the years (Plessi, 2003).
Chinese Vinegar
A very detailed description of vinegar making was recorded
in an ancient Chinese book, Qimin Yaoshu. Just like soy sauce,
vinegar is another important condiment in Chinese cuisines. In
China, Shanxi aged vinegar and Zhenjiang scented vinegar are
considered the best among the traditional Chinese vinegars. Both
are produced using a mixed solid culture. Shanxi aged vinegar
is a typical example of a Big-Qu process. Big-Qu is a brick-
shaped starter culture and is made of wheat, barley, or green
peas. During the time course of fermentation, saccharification
and alcoholic fermentation occur at low temperature. The freshly
obtained vinegar is then placed outdoors, heated in summer by
the sun and frozen in the winter. The Shanxi aged vinegar is
viscous in texture, dark purple in color, sweet in taste, and has a
long shelf-life. The Zhenjiang scented vinegar presents a typical
example of Small-Qu production. Regular rice or waxy rice is
used, and the whole process normally takes about sixty days. The
finished product is famous for its delicate combination of color,
fragrance, sourness, mellowness, and richness (Zheng, Wang,
and Zheng, 2006).
Kombucha Beverage Production
Kombucha is a traditional fermented beverage obtained by
the mixed fermentation of sugared tea with a symbiotic culture
of AAB and yeasts, which together form the so-called tea fungus.
Kombucha has a history of several thousand years in the East and
114 P. RASPOR AND D. GORANOVIČ
is also quite popular today in the West because of its beneficial
effects on human health. Analysis of the fermented liquid has
revealed the presence of acetic, lactic, and gluconic acids as ma-
jor chemical compounds. A diverse range of flavor compounds,
including a range of alcohols, aldehydes, ketones, esters, and
amino acids have also been identified (Teoh, Heard, and Cox,
2004). Bacteria and yeasts present in kombucha form a powerful
symbiosis able to inhibit the growth of potential contaminating
bacteria. The main AAB found in the tea fungus are Ga. xyli-
nus, Acetobacter xylinoides, A. aceti, A. pastorianus, and some
Gluconobacter species. Ga. xylinus has the ability to synthesize
a floating cellulose network, which enhances the association
formed between bacteria and yeasts. Caffeine and related xan-
thines of the tea infusion stimulate the cellulose formation by Ga.
xylinus. The yeasts convert sucrose into fructose and glucose,
and produce ethanol. AAB convert glucose to gluconic acid and
ethanol to acetic acid. The resultant low pH and presence of an-
timicrobial metabolites reduce the competition of other bacteria,
yeasts, and filamentous fungi. Black tea and white sugar are the
best substrate for the preparation of kombucha, although green
tea can also be used. Tea leaves are added to boiling water and
allowed to infuse for about 10 min, after which the leaves are
removed. Sucrose (50 g/L) is dissolved in the hot tea, and the
preparation is left to cool. Tea is poured into a wide-mouthed
clean vessel and is acidified by the addition of vinegar or already
prepared kombucha. The tea fungus is laid on the tea surface, and
the jar is carefully covered with a clean cloth. The preparation is
left to incubate at room temperature for 1 to 8 weeks. When the
bioprocess is completed, the beverage is filtered and stored in
capped bottles at 4◦ C. The taste of the kombucha changes during
mixed fermentation from a pleasant fruit sour-like flavor after a
few days, to a mild vinegar-like taste with prolonged incubation.
Although kombucha is becoming more and more popular, it is
still being produced traditionally on a small scale (Dufresne and
Farnworth, 2000).
Cocoa Production
Cacao beans, the starting material for cocoa and chocolate
manufacture, are the seeds from fruits (pods) of the Theobroma
cacao tree. Each fruit pod contains thirty to forty beans embed-
ded in a mucilaginous pulp (Lopez and Dimick, 1995; Thomp-
son, Miller, and Lopez, 2001). The popularity of cocoa is not its
nutritional value but rather its unique flavor, color, and aroma.
To obtain these sensory properties, the beans must be fermented
because roasting unfermented dried cacao does not result in
the desired flavor, aroma, or color. Cocoa is therefore classi-
fied into that category of foods whose characteristic flavor is
developed through a bioprocess. The mixed fermentation and
drying processes are also referred to as “curing.” The principal
objectives are the removal of mucilage to provoke aeration of
the fermenting seeds, facilitate later drying, and provide heat
and acetic acid necessary for killing (preventing germination)
and curing the seeds. The biochemical changes that affect cur-
ing may be divided into those that occur in the external pulp,
owing to microbial action and those initiated within the seed as
a consequence of the former (Lopez and Dimick, 1995). The
pulp is predominantly water with 10% to 15% sugars and has
a low pH, between pH 3.6 to 4.0, primarily due to a high con-
centration of citric acid (1%−3%) (Ardhana and Fleet, 2003;
Guizani and Mothershaw, 2006; Thompson, Miller, and Lopez,
2001). The bioprocess is spontaneous, and therefore, the micro-
bial species present also differ between batches and different
geographic locations. Occasionally, a more controlled situation
is required, and starter cultures are used; however, these gen-
erally do not compare favorably with the spontaneous biopro-
cesses. The beans are fermented for two to twelve days. During
this time, the sticky pulp becomes a turbid broth from which
the beans absorb flavors (Guizani and Mothershaw, 2006). The
events taking place during mixed microbial pulp fermentation
correspond to a succession of microorganisms metabolizing the
pulp. When starting mixed fermentation, the low pH value and
the high sugar content of the pulp allow fermentation by yeasts
and lactic acid bacteria (Biehl and Ziegleder, 2003; Thompson,
Miller, and Lopez, 2001). As the microorganisms ferment the
sugars, they produce heat, and high temperatures up to 45◦ C
to 50◦ C develop (Gotsch, 1997; Lagunes-Gálvez et al., 2007;
Thompson, Miller, and Lopez, 2001). As the temperature rises
and alcohol accumulates, the proportion of yeasts falls rapidly,
and AAB predominate. Lactic acid bacteria start to grow, al-
though their numbers and activity are believed to be subordinate
to that of the AAB. The pulp is stirred and drained, which in-
creases the level of aeration. The presence of oxygen and the low
pH favor the growth of AAB, mostly Acetobacter spp. (Guizani
and Mothershaw, 2006). Study of the microflora of cocoa pro-
duction in the Dominican Republic determined A. lovaniensis
as the predominating AAB (Lagunes-Gálvez et al., 2007), and
in the case of Ghanaian cocoa production, Acetobacter syzy-
gii, A. pasterianus and A. tropicalis were found to predominate
(Nielsen et al., 2007). Acetic acid and lactic acid are the most
significant metabolites penetrating the beans and are responsible
for the acidic taste. In addition, Gluconobacter spp. were found
(Biehl and Ziegleder, 2003). Bioprocesses vary between dif-
ferent countries and even between different processors. Efforts
have been made toward standardizing practices, and today, many
of the primitive methods, such as fermentation in holes in the
ground, canoes, and banana and bamboo frames, are the excep-
tion rather than the rule. In general, the seeds are placed in some
kind of receptacle and covered and weighed down. Here, natu-
rally occurring microorganisms that contaminated the external
pulp during handling ferment the sugars (Lopez and Dimick,
1995). Most of the world’s cocoa is produced on drying plat-
forms, in baskets, in banana leaf–covered heaps, or in boxes. In
the case of the bioprocess on drying platforms, wet cocoa seeds
are spread on platforms where they undergo microaerophilic
fermentation while drying and fermentation when the seeds are
heaped into piles each night. Fermentation in heaps covered with
plantain leaves is popular and can produce good quality cocoa.
 BIOTECHNOLOGICAL APPLICATIONS OF ACETIC ACID BACTERIA
Cocoa seeds varying in quantities from 25 to 1,000 kg are heaped
on a floor of plantain leaves (Lopez and Dimick, 1995; Thomp-
son, Miller, and Lopez, 2001). The seeds are mixed on the spot
or by making another heap. The duration of the bioprocess is
from four to seven days. The box process, which requires a
large, fixed volume of cocoa, is the method of choice on large
estates. Variations occur not only in the size of boxes, but also in
methods of drainage, aeration, and duration of the bioprocess.
The box contents are mixed every forty-eight hours by turn-
ing the seed into an adjacent empty box to ensure uniformity.
Processing generally requires six to seven days, depending on
the season and the state of the harvested pods. The progress of
the bioprocess is assessed by the odor, as well as the external
and internal color changes of the seeds. When the bioprocess
is complete, the beans have a moisture content of about 50%,
which must be reduced to 6% to 8% to keep them safe during
storage. Sun drying is the preferred method because it allows
the biological and oxidative chemical changes to progressively
decrease with moisture loss. Drying temperatures must not ex-
ceed 60◦ C, and times should not be less than forty-eight hours.
Rapid drying at elevated temperatures tends to produce acidic,
bitter cocoa with brittle shells. The beans are then roasted at
121◦ C to obtain the characteristic smell and flavor of chocolate
(Guizani and Mothershaw, 2006; Lopez and Dimick, 1995). In
2002, the world production of cocoa exceeded the 3 million ton
mark, and even with the increase in production, it is still carried
out traditionally (Lagunes-Gálvez et al., 2007). Cocoa produc-
tion and its export present a significant part of the income for
some countries; therefore, improvements of the bioprocess and
increases of yield are of great importance and interest for future
research (Nielsen et al., 2007).
Cellulose Production
Cellulose, a polymer of β-1,4-linked glucose units, is syn-
thesized by a number of Acetobacter species (some of which
have been transferred in the genus Gluconacetobacter), and
Ga. xylinus is the most recognized cellulose producer. Cellu-
lose produced by Ga. xylinus has excellent properties such as
transparency, tensile strength, fiber-binding ability, adaptabil-
ity to the living body, and biodegradability (Takai and Erata,
1998). During the process of actual biosynthesis, various car-
bon compounds of the nutrition medium are used by the bac-
teria; polymerized into single, linear β-1,4-glucan chains; and,
finally, secreted outside the cells through a linear row of pores
located on their outer membrane. The subsequent assembly of
the β-1,4-glucan chains outside the cell is a precise, hierarchi-
cal process. Initially, they form subfibrils (consisting of 10–15
nascent β-1,4-glucan chains) and then later microfibrils, and,
finally, bundles of microfibrils consisting of a loosely wound
ribbon, which is comprised of about 1,000 individual glucan
chains (Ross, Mayer, and Benziman, 1991). The thick, gelati-
nous membrane formed in static culture conditions as a result
of these processes is characterized by a 3-D structure consisting
115
of an ultrafine network of cellulose nanofibers (3–8 nm), which
are highly uniaxially oriented (Czaja, Romanovicz, and Brown,
2004). Such a 3-D structure, not found in vascular plant cellu-
lose, results in high cellulose crystallinity (60%–80%) and enor-
mous mechanical strength. Particularly impressive is the fact that
the size of microbial cellulose fibrils is about 100 times smaller
than that of plant cellulose. This unique nanomorphology results
in a vast surface area that can hold a large amount of water (up
to 200 times its dry mass) and that, at the same time, displays
great elasticity, high wet strength, and conformability. The cel-
lulose produced in the form of a gelatinous membrane can be
molded into any shape and size during its synthesis, depending
on the production technique and conditions used (Bielecki et al.,
2002; Czaja et al., 2006). Although cellulose synthesis has been
studied extensively in both higher plants and bacteria, the inter-
mediary steps in the pathway have not been entirely elucidated.
Labeling studies and demonstration of enzyme activities in cell-
free extracts indicate that the sequence of reactions leading to
cellulose synthesis is
Glucose → G-6-P → G-1-P → UDPG → cellulose
Lipid- and protein-linked cellodextrins (partially polymerized
glucose units) may function as intermediates between UDPG
(uridine glucose-5 -phosphate) and cellulose. A multienzyme
complex orchestrates intracellular cellulose synthesis. Extrusion
through pores in the lipopolysaccharide layer results in the ag-
gregation of bundles of cellulose that undergo crystallization
by self-assembly at the cell surface (Moat, Foster, and Spector,
2002). Alternatively to static cultures, the bacteria can be grown
in submerged culture, although the design of the fermenter and
the degree of aeration are important factors in optimizing cellu-
lose yield. Medium costs limit commercial use of bacterial cel-
lulose, so low-cost substrates are being used, such as molasses
from the sugar cane process with a content of 55% to 60% su-
crose (Yoshinaga, Tonouchi, and Watanabe, 1997), or corn steep
liquor. These are preferable to glucose because it is converted
to gluconic acid with a consequent rapid fall in pH (Sutherland,
1996). Food process effluents such as potato effluents, cheese
whey permeate, and sugar beet raffinate have also been stud-
ied as substrates for bacterial cellulose production (Thomson
and Hamilton, 2001). D-xylose has been demonstrated as a car-
bon source for bacterial cellulose production and lignocellulosic
materials will be an attractive feedstock for bacterial cellulose
production because of their availability at low costs and large
quantities (Ishihara et al., 2002). Product yields of up to 28
g dry polysaccharide L−1 have been reported. Some cellulose
from bacteria is produced commercially as a source of highly
pure polymer in the so-called cellulose I form, free from lignin
and other related noncellulose material. Production may either
be in the form of pellicle from static cultures or from submerged,
fed-batch culture in stirred tank reactors. Low shear conditions
are usually preferred. For such purposes, high yielding strains
must be selected that are capable of at least 20% conversion of
116 P. RASPOR AND D. GORANOVIČ
substrate to polysaccharide (Sutherland, 1996). For commercial
production, it is needed to establish a mass production system,
which allows high rates of cellulose production by monitoring
the consumption of sugars, controlling the pH of medium and the
aeration and agitation rate, and adding some cell growth stimu-
lators such as methionine and lactate in the early stages (Ishihara
et al., 2002; Matsuoka et al., 1996). The general failure of a large-
scale commercialization effort for microbial cellulose seems to
have been mainly caused by the lack of an efficient production
system. Sheer stresses in agitated bioreactors always damage the
cellulose during synthesis. Another factor that contributes to the
reduced cellulose yield in submerged cultures is the spontaneous
selection of cellulose-negative variants. A scale-up approach has
been based on a combination of stationary and agitated culture
and takes place in horizontal bioreactors, where optimal con-
ditions for media supply and cell attachment to the surface of
rotating discs or roller are created (Krystynowicz et al., 2002).
In static cultures, cellulose formation at the air–cellulose pellicle
interface rather than at the liquid−cellulose pellicle interface has
been reported (Borzani and de Sourza, 1995). In addition, the
cellulose production and the pellicle performance are affected
by oxygen tension in the gaseous phase. Thus, oxygen plays
a very important role in microbial cellulose production (Chien
et al., 2006; Watanabe and Yamanaka, 1995). The presence of
water-soluble polysaccharides such as acetan, which increases
the viscosity of the medium, have been reported to be indispens-
able for quantity production of bacterial cellulose in the highly
agitated tank cultures (Ishida et al., 2002; Ishida et al., 2003).
For the preparation of material for use in wound dressings, the
Gluconacetobacter spp. was grown at 28◦ C in an unshaken,
complex medium supplemented with yeast extract. The prod-
uct resembles hyaluronic acid in its very high water retention
capacity. The disadvantage of this technique is the presence of
the bacterial cell material within the pellicle. Growth in shaken,
aerated cultures with glucose as carbon substrate provides an
alternative means of obtaining the polymer (Sutherland, 1996).
Bacterial cellulose has a number of potential uses, including the
manufacture of wound dressings for patients with burns, chronic
skin ulcers, or other extensive loss of tissue (Alvarez et al., 2004;
Czaja et al., 2006; Fontana et al., 1990). The cellulose acts as
a temporary skin substitute, and the high water capacity of the
oxygen-permeable film appears to stimulate regrowth of the skin
tissue while assisting in the prevention of infection. Bacterial cel-
lulose also forms the basis for high-quality acoustic diaphragm
membranes in which the distribution of the fibrils containing par-
allel orientation of the glucan chains results in fibers possessing
high tensile strength (Yamanaka et al., 1989). Other applica-
tions use bacterial cellulose as binders for ceramic powders and
minerals and thickeners for adhesives. Less pure material can be
used for binding and coating purposes in the mining industry,
but cost remains relatively high in comparison with other, com-
peting polymers. A further area for potential development lies in
the applications for cellulose-synthetic copolymers (Sutherland,
1996).
Biotransformations/Bioconversions
Biotransformation deals with the use of biological catalysts
to convert a substrate into a product in a limited number of en-
zymatic steps. The chemical industry currently takes advantage
of biocatalysts in various sectors, namely, in the food, phar-
maceutical, and detergent sectors. An ongoing trend toward the
implementation of commercial processes based on the use of bio-
catalysts in other areas (e.g., polymers, fine chemicals, miscel-
laneous chemicals) is noticeable (Fernandes and Cabral, 2006).
The ability of AAB to oxidize several substrates has long been
known and is still attracting attention. The enzymatic oxidation
of primary alcohols for the production of aldehydes is attractive
because it can be carried out under mild conditions that are also
suited for labile products. The use of isolated enzymes is often
complicated by the requirements for cofactors and systems for
their regeneration, and whole microbial cells have been used.
The oxidation of alcohols by AAB is an old and established mi-
crobial method for obtaining production of vinegar and various
carboxylic acids, sometimes with remarkable chemo- and enan-
tioselectivity (Romano et al., 2002; Villa et al., 2002). One of the
earliest bioconversions is the production of vinegar from ethanol.
In AAB, Gluconobacter strains are generally more ketogenic
than Acetobacter strains. This characteristic is brought about by
many different primary dehydrogenases, most of which are lo-
calized on the outer surface of the cytoplasmic membranes of the
organisms. Thus, Gluconobacter strains oxidize a broad range of
substrates, such as alcohols, sugars, sugar acids, and sugar alco-
hols, and the corresponding oxidation products are accumulated
in the culture medium. Based on this characteristic, several use-
ful oxidation products are produced industrially, and such pro-
cesses are called oxidative conversions. These reactions are done
in the periplasmic space; no cytosolic NAD(P)-dependent dehy-
drogenase is involved in oxidation. Several quinoproteins have
been found in AAB, all of which can be applied to microbial or
enzymatic production of useful materials by means of oxidative
bioprocessing (Adachi et al., 2003a; Matsushita, Toyama, and
Adachi, 1994).
Other biotransformations involving AAB include the pro-
duction of L-sorbose from D-sorbitol (Gonzales, 2006; Han-
cock and Viola, 2002; Kim, Kim, and Shin, 1999; Ku-
bicek and Karaffa, 2006; Stefanova et al., 1987), L-ribulose
from ribitol (Adachi et al., 2001; Adachi et al., 2003a), L-
erythrulose from meso-erythrol (Adachi et al., 2003a; Moon-
mangmee et al., 2002), D-tagatose from D-galactitol (Rollini
and Manzoni, 2005; Suresh, Ritu, and Ravishankar, 2006),
phenylacetaldehyde and phenylacetate from 2-phenylethanol
(Çelik, Bayraktar, and Mehmetoǧlu, 2004; Manzoni et al.,
1993; Villa et al., 2002), dihydroxyacetone from glycerol
(Hekmat, Bauer, and Neff, 2007; Nabe et al., 1979), (R)-3-
hydroxy-2-methyl propionic acid from 2-methyl-1,3-propandiol
(Molinari et al., 2003), 3-dehydroshikimate from quinate
(Adachi et al., 2003c; Adachi et al., 2006), and acetoin
and diacetyl from 2,3-butanediol (De Faveri et al., 2003;
Romano et al., 2002).
 BIOTECHNOLOGICAL APPLICATIONS OF ACETIC ACID BACTERIA
L-Ascorbic Acid (Vitamin C) Production
L-ascorbic acid also known as vitamin C is a water-soluble
vitamin that is indispensable for several physiological functions.
Approximately 50% of the synthetic L-ascorbic acid is used in
vitamin supplements and pharmaceutical preparations, 25% in
food processing, 15% in beverage manufacturing, and 10% in
animal feeds (Bremus et al., 2006; Hancock and Viola, 2002).
Commercially, ascorbic acid is mainly produced by a combina-
tion of five synthetic organic chemical steps and one biotrans-
formation step, known collectively as Reichstein synthesis. Its
basic principle is reduction of C-1 of D-glucose and oxidation
at C-5 and C-6, while preserving the chirality at C-2 and C-3.
The microbially catalyzed step is the oxidation of D-sorbitol to
L-sorbose, which is carried out by G. oxydans. Modern bacte-
rial strains of G. oxydans have been developed that give excep-
tionally high yields of almost 100% for this biotransformation
(De Wulf, Soetaert, and Vandamme, 2000; Kim, Kim, and Shin,
1999; Kulhanek, 1970; Stefanova et al., 1987). Large-scale pro-
cessing occurs at 30◦ C to 35◦ C and at a pH of 4 to 6. All six
steps of Reichstein’s synthesis generally have a yield of >90%,
and thus, the final yield of ascorbic acid is about 60% (Hancock
and Viola, 2002; Kubicek and Karaffa, 2006). Over the past two
decades, several innovative bioconversion systems have been
proposed in order to simplify the long-time market-dominating
Reichstein method (Bremus et al., 2006). The two most com-
mercially advanced methods are the oxidation of D-glucose to 2-
keto-L-gulonate (2-KLG) via D-gluconate, 2-keto-D-gluconate,
and 2,5-diketo-D-gluconate (2,5-DKG pathway), and the oxida-
tion of D-sorbitol or L-sorbose to 2-KLG via the intermediate
L-sorbosone (sorbitol pathway) (De Wulf, Soetaert, and Van-
damme, 2000). 2-KLG is later converted into ascorbic acid by
conventional chemical processing technology (i.e., esterification
and lactonization). Therefore, efforts have been focused on en-
gineering strains capable of efficiently producing 2-KLG from
different sugars. Two strains of G. oxydans were engineered to
convert sorbitol into 2-KLG. Genes encoding the enzymes sor-
bitol dehydrogenase and sorbose dehydrogenase were cloned
from G. oxydans T-100 into G. oxydans G624, allowing the pro-
duction of 2-KLG in three steps (Bremus et al., 2006; Gonzales,
2006; Saito et al., 1998).
D-Tagatose Production
D-tagatose is a rare ketohexose and a C-4 fructose epimer.
D-tagatose is generally recognized as safe substance under U.S.
Food and Drug Administration regulation, is used as a bulking
agent in food and a noncalorific sweetener (sweetness 0.92),
and has a taste very similar to sucrose, with no aftertaste or
cooling effect. In addition, D-tagatose does not show a laxative
effect, unlike other polyols. Although it occurs naturally, the
amounts are too small for economic recovery, a major impedi-
ment to its use in the food industry (Levin et al., 1995). Being
a nondigestible food ingredient, D-tagatose exhibits a probiotic
and prebiotic effect and is able to benefit humans by selectively
117
stimulating growth and/or activity of a limited number of bacte-
ria, thus improving host health (Levin et al., 1995). By increas-
ing the growth of lactic acid bacteria, D-tagatose promotes the
production of butyrate, which plays an important role in colon
epithelium protection (Johnson, 1995). Several methods have
been studied for the manufacture of D-tagatose, and biologi-
cal production has been studied intensely in recent years. D-
tagatose production from galactitol using galactitol dehydroge-
nase is well known. Galactitol, however, is more expensive than
galactose and seems to have only little potential for commercial
production. D-tagatose is obtained by the biotransformation of
D-galactitol and bioconversion of D-galactose (Suresh, Ritu,
and Ravishankar, 2006). AAB can carry out the oxidative bio-
transformation of D-galactitol into D-tagatose, catalyzed by the
enzyme galactitol dehydrogenase. AAB strains were screened
for metabolic characteristics appropriate for processes involving
dehydrogenation reactions. The best biotransformation results
were achieved using G. oxydans, where galactitol was added
to 24-h old cultures, when the bacteria were in the stationary
growth phase. Gluconobacter spp. are better tagatose produc-
ers than Acetobacter spp., which could be attributable to the
fact that the species G. oxydans, originally named A. suboxy-
dans, is characterized by its inability to catabolize synthesized
and accumulated oxidized metabolites (Manzoni, Rollini, and
Bergomi, 2001; Rollini and Manzoni, 2005). Recently, a new
approach to the production of D-tagatose by biotransformation
of D-galactitol was reported using AAB adapted in the pres-
ence of high glycerol levels and D-galactitol as an enzymatic
activity inducer (Rollini and Manzoni, 2005). At the end of the
biotransformation, the reaction mixture containing D-tagatose is
centrifuged to remove the bacterial cells. The supernatant fluid
is deionized and then concentrated at 40◦ C under vacuum. Af-
ter two days of standing in absolute ethyl alcohol at 5◦ C, white
crystals of D-tagatose are obtained. In the future, the use of D-
tagatose as a limited substitute for sucrose may prove feasible.
Chocolates and chewing gum, both of which were successfully
formulated by substituting D-tagatose for sucrose in each, are
planned as initial products, along with the use of D-tagatose as
a tabletop sweetener for coffee and tea and in single-use packets
(Suresh, Ritu, and Ravishankar, 2006).
Shikimate Production
Shikimate is a key intermediate for aromatic amino acids and
for large numbers of antibiotics, alkaloids, and herbicides. Re-
cently, another immediate impact has been given to shikimate as
a precursor for Oseltamivir (an antiviral drug) synthesis, protect-
ing people from pandemic flu infection. Despite global World
Health Organization warnings about pandemic flu, including
avian influenza, there are insufficient measures for Oseltamivir
around the world. One reason is the technical difficulty in prepar-
ing shikimate, because two different metabolic pathways, the
glycolysis and the pentose phosphate pathway, must be com-
bined in forming 3-deoxy-7-phospho-D-arabinoheptulosonate
118 P. RASPOR AND D. GORANOVIČ
before reaching shikimate. Furthermore, the metabolic location
of shikimate is far from D-glucose, and it is difficult to lead
the metabolic flow to shikimate production by classic fermenta-
tion technology or by modern molecular biotechnology (Draths,
Knop, and Frost, 1999; Kramer et al., 2003). Total synthesis of
shikimate by organic chemistry has not been practical. Neverthe-
less, it is necessary to develop a novel method for more effective
and convenient shikimate production contributing to Oseltamivir
synthesis. Quinoprotein quinate dehydrogenase (QDH) and 3-
dehydroquinate dehydratase are localized predominately on the
outer surface of the cytoplasmic membranes of some species of
Gluconobacter and quinate is oxidized to 3-dehydroshikimate in
a sequential manner (Adachi et al., 2003c; Vangnai et al., 2004).
In the cytoplasm, NADP-dependent shikimate dehydrogenase
(SKDH) catalyzes a reversible reaction of 3-dehydroshikimate
to shikimate. At first, shikimate production was proposed us-
ing a singular cellular system of AAB, but such systems have
given insufficient production of shikimate (Adachi et al., 2003a;
Adachi et al., 2003b; Adachi et al., 2003c; Adachi et al., 2003d).
A better strategy has been developed for high shikimate produc-
tion using the two separately localized enzymatic systems to
work sequentially. Dried cells or the membrane fraction involv-
ing QDH and 3-dehydroquinate dehydratase can be used for
3-dehydroshikimate production in the first reaction. The sec-
ond reaction is a coupling reaction composed of two cytosolic
enzymes, SKDH and D-glucose dehydrogenase as an NADPH
regenerating enzyme. The coupling reaction results in the pro-
duction of shikimate from 3-dehydroshikimate in the presence
of excess D-glucose. Because SKDH showed strong reactivity
with 3-dehydroshikimate, even in highly diluted solutions, the
reaction system for shikimate production can be scaled up suc-
cessfully, and quinate can be supplied readily from industrial
processing of coffee wastes (Adachi et al., 2006).
ACETIC ACID BACTERIA IN BIOSENSORS
A biosensor is an analytical device that combines a biological
sensing element with a transducer to produce a signal propor-
tional to the analyte concentration. This signal can result from a
change in the proton concentration, release or uptake of gases,
light emission, absorption, and so forth, brought about by the
metabolism of the target compound by the biological recogni-
tion element. The transducer converts this biological signal into
a measurable response such as current, potential, or absorption
of light through electrochemical or optical means, which can
be further amplified, processed, and stored for later analysis
(Lei, Chen, and Mulchandani, 2006; Turner, Karube, and Wil-
son, 1992). Bacteria belonging to the genus Acetobacter and
Gluconobacter, and enzymes isolated from them have been ex-
tensively used for biosensor construction in the past decade. Ace-
tobacter and Gluconobacter and/or enzymes isolated from them
can be effectively combined with amperometric transducers to
construct amperometric biosensors. Future developments in am-
perometric biosensors depend mainly on the availability of oxi-
doreductive enzymes. The potential range of analyzable analytes
is practically limited by the number of commercially available
enzymes. There is currently only one commercially available
Gluconobacter enzyme, fructose dehydrogenase. The main ad-
vantage of cell-based biosensors is that a wide variety of cells are
easy and inexpensive to prepare; the enzymes do not need to be
isolated because they are present in their natural environment
and therefore usually more stable; and cofactors, if required, are
being regenerated in situ in the cell. The disadvantage, however,
is that there are usually more enzymatic activities present in
cells, and their ratio is difficult to control. Both approaches to
Gluconobacter biosensors, enzymes, and cells are currently used
and being reported, and both have their distinctive advantages
in practical applications. The range of compounds analyzable
by Gluconobacter biosensors includes mono- and polyalcohols,
multiple aldoses and ketoses, several disaccharides, triacylglyc-
erols, and complex parameters such as utilizable saccharides or
biological O2 demand. In addition to multiple enzyme activities,
the enzyme nonspecificity also widens the range of potential ana-
lytes (Švitel et al., 2006). Studies of newly isolated enzymes and
their specificity characterizations, usually followed by biosensor
applications, continue to appear (Lapenaite et al., 2005; Malin-
auskas et al., 2004). A Clark-type O2 probe biosensor (with
glucose oxidase) was historically the first type of biosensor.
This concept was much later adapted also to Gluconobacter
biosensors and used to detect xylose (Reshetilov et al., 1997).
The function of O2 probe-based biosensors relies on dissolved
O2 depletion in the bacteria-containing layer covering the O2
probe. This construction corresponds to the general definition of
a biosensor: a biological sensing element (bacteria) being closely
integrated with a physicochemical transducer (O2 probe). More
recent works use a true amperometric biosensor configuration: a
working-reference electrode pair, where the working electrode
is covered with a biocatalyst. The analyte is oxidized on the
working electrode, and electrons resulting from the oxidation
are transferred to the electrode. There are multiple strategies of
how to use catalytic activities present in microbial cells, ranging
from using viable cells, nonviable cells, permeabilized cells, or
membrane fractions. These cell-derived biocatalysts serve as an
economical substitute for enzymes and the additional benefit for
the biosensor performance is that the enzymes are still linked
to the respiratory chain. The features that make Gluconobacter
spp. particularly suitable for biosensor construction include that
they grow fast in simple cultivation media, contain high enzy-
matic activities, and are relatively stable during immobilization,
and that their PQQ dehydrogenases do not require an external
cofactor to be added. A typical experimental setup, including an
amperometric biosensor based on Gluconobacter spp. cells, is
comprised of a measurement cell with controlled temperature
and O2 concentration. The dehydrogenase activities present in
Gluconobacter cells are dependent on the carbon sources used
to grow the cells (Lee et al., 2002). The ability of Gluconobacter
oxydans to oxidize a wide range of compounds was employed
to prepare a 1,3-propanediol biosensor (Katrlı́k et al., 2006).
Gluconobacter spp. cannot oxidize disaccharides; however,
 BIOTECHNOLOGICAL APPLICATIONS OF ACETIC ACID BACTERIA
detection of sucrose or lactose was achieved by coimmobiliza-
tion of Gluconobacter cells with permeabilized cells contain-
ing invertase or β-galactosidase activity, respectively (Švitel,
Čurilla, and Tkáč, 1998). Coupling Gluconobacter cells with a
triglyceride-hydrolyzing catalyst was used do develop a triglyc-
eride biosensor assay (Tkáč et al., 2000a; Tkáč et al., 2000b).
A potentiometric biosensor is another concept that can be ap-
plied to Gluconobacter-based biosensors; extracellular changes
of pH resulting from xylose dehydrogenation were monitored
by a field effect transistor (Reshetilov et al., 1996). The ability
of quinohemoprotein alcohol dehydrogenase isolated from Glu-
conobacter spp., as well as other direct electron transfer enzymes
to directly generate electric potential, opens new possibilities for
designing new potentiometric sensors (Ramanavicius, Kausaite,
and Ramanaviciene, 2006). There are still many possibilities for
using Gluconobacter spp. catalytic properties that remain to be
explored. The knowledge of the genome sequence (Prust et al.,
2005) will likely lead to novel applications of Gluconobacter in
biotransformations and biosensors. There are numerous possible
implications and great potential of biosensor applications, and
there are obviously plenty of opportunities for creative biosensor
scientists (Švitel et al., 2006).
FUTURE PROSPECTS
Recent research has been focused on increasing yields and
improving the quality of the product. To accomplish these pur-
poses, different strategies can be faced that include designing
a better acetification system, setting the optimum conditions
for the process, and selecting more productive strains. To im-
prove producing bacterial strains genetically, thus enhancing tol-
erance and acquiring higher yields, new recombinant techniques
are being used. Succeeding to improve the tolerance to ethanol
and acid would not only bring benefits to vinegar production,
but also other bioprocesses, using different production organ-
isms that face similar limitations. New substrates, measurement
techniques, materials, and optimization processes will also con-
tribute to greater yields and product quality. Some research in
the field has also been focused on setting up validated analyti-
cal methods that can ensure the authenticity of the products and
differentiate defective or adulterated vinegars from authentic
ones. Apart from novel experimental techniques, a more com-
prehensive statistical data analysis has recently emerged. Future
trends in vinegar production will be directed toward a reduction
of the aging period, while still maintaining similar quality and
yield. The possibility of sensorial analysis as a tool to classify
vinegars according to its quality must also be explored (Tesfaye
et al., 2002). Molecular methods for successful identification
and differentiation of AAB will continue to develop, improve,
and simplify the research being done in the field of AAB. New
knowledge of genetics and the discovery of novel enzymes will
further expand the potential use of AAB in biotransformations
and other applications such as biosensors and alternative energy
sources.
119
SUMMARY
Although the presence of AAB is undesirable in some cases,
regarding spoilage of certain foods and beverages and the recent
discovery of pathogenic strains, they generally play an impor-
tant positive role in the production of certain foods and in many
bioprocesses. Applications of AAB and their enzymatic poten-
tial have been widely expanded since their sole initial role as
vinegar producers. As our knowledge and technological possi-
bilities increase, so do the benefits that microorganisms such as
AAB offer. The unique metabolism of the AAB, governed by
their specialized enzymes, enables their use in various fields of
biotechnology, such as food production, pharmaceutics, medici-
nal biotechnology, biosensorics, biotransformations, fine chemi-
cals production, alternative energy source production, etc. Vine-
gar production will probably continue to be the main field of use
of AAB, but their role will definitely continue to expand in the
future.
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