l2 Acetic Acid

HEINRICH
EBNER
Linz, Austria

SYLVIASELLMER
HEINRICH
FOLLMANN
Bonn, Germany

1 Introduction 383
2 Bases of Acetic Acid Fermentation 383
3 Raw Materials for Acetic Acid Fermentation 383
3.1 Alcohol 383
3.2 Water for Processing 383
3.3 Nutrients 384
4 Production by Acetic Acid Fermentation 384
4.1 Microorganisms 384
4.1.1 Overview and Basic Problems of Classification 384
4.1.2 Required Properties of Industrially Used Strains 384
4.1.3 Industrially Used Strains 385
4.1.3.1 Plasmid Profile Analysis for Characterization of Acetobacter Strains 385
4.1.4 Genetic Improvement of Acetobacter Strains 386
4.1.5 Bacteriophages 386
4.2 Biochemistry 386
4.2.1 Ethanol 387
4.2.2 Sugar 387
4.2.3 Acetate 387
4.2.4 Carbon Dioxide 387
4.2.5 Nitrogen 387
4.2.6 Growth Factors 388
4.3 Physiology 388
4.3.1 Oxygen Demand and Total Concentration 388
4.3.2 Lack of Ethanol 389
382 12 Acetic Acid

4.3.3 Specific Growth Rate 389

4.3.4 Specific Product Formation 389
4.3.5 Changes in Concentration and Temperature 389
4.3.6 Overoxidation 389
4.4 Industrial Processes 390
4.4.1 Submerged Vinegar Fermentation 390
4.4.1.1 Semicontinuous and Continuous Fermentations 390
4.4.1.2 Two-Stage Processes 390
4.4.1.3 The Frings Acetator@ 391
4.4.1.4 Further Trends in Submerged Processes 392
4.4.1.5 Other Submerged Fermentation Processes 392
4.4.1.6 Continuous Culture and Cell Recycle 392
4.4.1.7 Semicontinuous Culture with Cell Recycle 394
4.4.2 Surface Fermentation 394
4.4.2.1 Old Surface Fermentation Processes 394
4.4.2.2 New Experiments with Surface Fermentation 394
4.4.3 Trickling Processes 394
4.4.4 Experiments with Immobilized Acetic Acid Bacteria 395
4.4.5 Environmental Protection 395
4.5 Production Volumes 396
4.5.1 European Union 396
4.5.2 United States of America 396
4.5.3 Japan 396
4.5.4 World 396
5 Experiments with Anaerobic Fermentation of Glucose 396
6 References 397
 3 Raw Materials for Acetic Acid Fermentation 383

but commonly used calculation see EBNER

1 Introduction and FOLLMANN (1983). The quotient of the
total vinegar concentration produced over the
Worldwide acetic acid is called “vinegar”, if total mash concentration indicates the con-
obtained by oxidative fermentation of etha- centration yield.
nol-containing solutions by acetic acid bacte-
ria. Although “vinegar” does not entirely ex-
clude “diluted chemically produced acetic
acid” in every country of the world, this term
is used here to define acetic acid which is pro- 3 Raw Materials for
duced from ethanol by primary microbial me-
tabolism, the so-called “acetic acid fermenta- Acetic Acid Fermentation
tion” or “vinegar fermentation”.
In the future, acetic acid may possibly also All mashes must contain ethanol and water
be produced by anaerobic fermentation of and provide the acetic acid bacteria with nu-
glucose by Clostridium thermoaceticum. At trients.
least theoretically, this metabolic pathway
should achieve higher yields of acetic acid per
unit of glucose. The basic idea for carrying 3.1 Alcohol
out laboratory research is the potential use of
renewable resources as raw materials for By far the largest percentage of vinegar is
chemical feedstocks, but it is not suitable for alcohol vinegar, which is produced from di-
food production. luted purified ethanol. Other common names
for the same product are “white vinegar” or
“spirit vinegar”. It is customary in almost all
countries to denature the ethanol serving as
raw material for the vinegar industry. In most
2 Bases of Acetic Acid European countries denaturation is carried
out with alcohol vinegar. In the U.S. this is
Fermentation usually done with ethyl acetate, which is split
into ethanol and acetic acid during fermenta-
Acetic acid fermentation (acetous fermen- tion.
tation) is an oxidative fermentation by which Mashes obtained by alcoholic fermentation
solutions of ethanol are oxidized to acetic of natural sugar-containing liquids also serve
acid and water by acetic acid bacteria using as raw materials. The designation of vinegar
atmospheric oxygen. The oxidation proceeds is according to the particular raw material
according to the basic equation used: Wine vinegar, e.g., is produced by acetic
acid fermentation of grape wine; cider vinegar
CZHSOH + 0 2 CH3COOH + H20
+ is produced from fermented apple juice; malt
G o =-455 kJ mol-’ vinegar is the undestilled product of alcoholic
and subsequent acetous fermentation of an
The alcohol-containing solution is called infusion of barley or other cereals, the starch
“mash”. Its alcohol concentration is given in of which was converted by malting: rice vine-
percent per volume. Usually, it also contains gar is made from saccharified rice starch, fol-
some acetic acid, expressed in grams of acetic lowed by alcoholic and acetous fermenta-
acid per 100 mL (% wh). The sum of ethanol tion.
(~01%)and acetic acid (g per 100 mL) is
called “total concentration” because the sum
of these rather incommensurable values gives 3.2 Water for Processing
the maximal concentration of acetic acid that
can be obtained by complete fermentation. The water used for the preparation of
For detailed reasons of this somewhat strange mashes must be clear, colorless, odorless, bac-
384 I2 Acetic Acid

teriologically clean, and without sediments or bacteria. This special primary microbial me-
suspended particles. It must also be free of tabolism at low pH of the surrounding me-
chlorine, ozone, and other chemical com- dium differentiates them from all other bacte-
pounds damaging bacteria. ria. Acetic acid bacteria are polymorphous.
Cells are gram-negative, ellipsoidal to rod
shaped, straight or slightly curved, 0.6-0.8 ym
3.3 Nutrients long, occurring singly, in pairs or in chains.
There are non-motile and motile forms with
Most natural raw materials do not require polar or peritrichous flagella. They are obli-
the addition of extra nutrients. However, ap- gately aerobic, some produce pigments, some
ple cider is usually low in nitrogenous com- cellulose.
pounds, but this can be corrected by the addi- Many attempts to classify acetic acid bacte-
tion of ammonium phosphate. Some grape ria were carried out until 1980. The proposed
wines also require the addition of ammonium classifications were summarized by EBNER
phosphate for an optimal fermentation. In and FOLLMANN(1983). At that time, a major
rare cases all nutrients listed below must be contribution to the exact taxonomy of acetic
added for the production of alcohol vinegar. acid bacteria were the DNA-rRNA hybridi-
For the production of alcohol vinegar, a zation studies of GILLISand DE LEY (1980).
mixture of the required nutrients was devel- A first conclusion from this work was that the
oped. When producing vinegar with up to two groups Gluconobacfer and Acefobacfer,
15% acetic acid, the acetic acid bacteria defi- as classified by DE LEY (1961), were closely
nitely require glucose and potassium, sodium, related groups, justifying their union in the
magnesium, calcium, ammonium as ammo- family of Acetobacteraceae where they are
nium phosphate, sulfate, and chloride. The clearly distinguishable as a separate branch in
following trace minerals are needed: iron, the superfamily IV (the a-subclass; STACKE-
manganese, cobalt, copper, molybdenum, va- BRANDT et al., 1988). Although DE LEYet al.
nadium, zinc. These additions are sufficient (1984) give a set of 32 features to differentiate
for an optimal acetic acid fermentation. the genera Gluconobacfer, Acetobacfer, and
Commercially available mixtures contain Frateuria, not all of them are necessary for a
supplements such as dried yeast extract in or- satisfactory identification.
der to restart a fermentation more quickly if The feature of acetate overoxidation iden-
stopped by a disturbance such as, e.g., a pow- tifies the strains used in “high acid” fermenta-
er failure. Principally, nutrients should be ad- tions as members of the genus Acetobacfer,
ded sparingly in order to exert a selection and thus separates them from the genera Glu-
pressure directing to a low requirement for conobacter, and Frafeuria. SWINGS(1992) di-
nutrients. vided Acetobacrer into 7 species. GtLLts et al.
(1989) described a species capable of fixing
N2 microaerobically, called Acefobacter dia-
zotrophicus.
4 Production by Acetic
4.1.2 Required Properties of
Acid Fermentation Industrially Used Strains
4.1 Microorganisms Considering the interest of many micro-
biologists in acetic acid bacteria it is surpris-
4.1.1 Overview and Basic ing that the available information about these
bacteria has so little effect on vinegar produc-
Problems of Classification tion. “Working” strains were isolated, but lost
again, demonstrating the difficulties to isolate
The microorganisms oxidizing ethanol to and grow Acefobacferstrains from industrial
acetic acid are commonly called acetic acid vinegar fermentations on solid media.
 4 Production by Acetic Acid Fermentation 385

A considerable progress was achieved by Industrial submerged vinegar fermenta-

propagating bacteria from fermenters in Ja- tions are started by inoculation with “inocula-
pan on a special double layer agar. The bacte- tion vinegar”, i.e., microbiologically unde-
ria isolated on this medium have been de- fined remains from previous fermentations.
scribed as Acetobacter polyoxogenes by EN- Acetic acid fermentation can be carried out
TANI et al. (1985). However, A . polyoxogenes for years without interruption or decrease in
was not available from the Japan Collection efficiency or yield, if suitable conditions are
of Microorganisms due to problems of propa- chosen to allow a permanent selection of
gation and preservation. strains tolerating high acidity at a minimum of
A new species in the genus Acetobacter for nutrient concentration.
which SIEVERSet al. (1992) proposed the From industrial practice, it has long been
name Acetobacter europaeus, has been iso- known that the properties of a newly isolated
lated and characterized in pure culture from strain of acetic acid bacteria may change from
vinegar fermentations at high acidity in Ger- the very first moment of cultivation in Petri
many and Switzerland. All investigated dishes and, that this strain, if cultivated over a
strains isolated from submerged fermenters number of generations, may show other prop-
and trickle generators had very low (0-22%) erties, especially as far as the adaptation to
DNA-DNA similarities with the traditional certain concentrations of acetic acid is con-
strains of Acetobacter and Gluconobacter. cerned, but also regarding its phenotypic fea-
The phenotypical differentiation on the spe- tures.
cies level of the genus Acetobacter is rather But, investigations of the last five years us-
difficult, since different strains of a single spe- ing plasmid profile analysis for characteriza-
cies do not necessarily utilize the same carbon tion and identification of acetic acid bacteria
source. A useful and significant criterion for derived from vinegar fermentations could not
the distinction of Acetobacter europaeus from verify this variability.
the other Acetobacter species is the strong to-
lerance to acetic acid of 4 8 % in AE-Agar
and the absolute requirement of acetic acid 4.1.3.1 Plasmid Profile Analysis
for growth. for Characterization of Acetobacter
Independent of all these problems and dif-
ficulties, the first and foremost interest of vi- Strains
negar industry is to use a strain of acetic acid
bacteria that tolerates high concentrations of Since DAVIESet al. (1981) used plasmid
acetic acid and total concentration, requires profile analysis for the identification of lactic
small amounts of nutrients, does not overox- acid bacteria, this method has become a pow-
idize the acetic acid formed, and yields high erful tool to identify and characterize strains
production rates. At present, it also seems to of all genera. Plasmids are circular, extrachro-
be necessary to make this strain phage-resist- mosomal DNA molecules, which can be ex-
ant (SCHOCHER et al., 1979). tracted from bacteria by a sophisticated tech-
nique. The plasmid profile of a strain is very
specific. Plasmids can determine certain prop-
4.1.3 Industrially Used Strains erties of bacteria, i.e., phage resistance or ci-
trate metabolism.
The vinegar industry has always worked The potential of this technique for vinegar
with acetic acid bacteria that, in most cases, fermentations was investigated by INOUEet
were not derived from pure cultures. The al. (1985), FUKAYAet al. (1985a), and TEU-
striking fact that common microorganisms BER et al. (1987b). The majority of the acetic
used on a large scale for industrial vinegar acid bacteria contain 1-8 plasmids from 1 to
production have not been properly described > 17 MDa. SIEVERSet al. (1990) applied plas-
and characterized in taxonomic terms is mid profile analysis to characterize the Aceto-
caused by the difficulty to cultivate them on bacter europaeus strains and the microflora of
semisolid media. industrial vinegar production plants. It was
386 12 Acetic Acid

shown that the microflora in submerged fer- Genetic transformation systems have been
mentations consists of one predominant described for Acetobacter xylinum (VALLA
strain, whereas in trickling generators the mi- et al., 1983), Acetobacter aceti (OKUMURA
croflora is quite heterogenous, containing a et al., 1985), Acetobacter (FUKAYAet al.,
mixture of several Acetobacter europaeus 1985a, b, c), and Gluconobacter suboxydans
strains with quite different plasmid profiles. (FUKAYAet al., 1985d).
This is probably the reason for the suscepti-
bility of submerged fermentations to bacterio-
phage attack. In conclusion, the determina-
tion of plasmid profiles from submerged vine- 4.1.5 Bacteriophages
gar fermenters represents a new and powerful
technique to characterize the microflora of Acetobacter-specific bacteriophages demon-
such fermentations and to answer questions strated by electron microscopy in submerged
that could not have been approached until and trickling fermenters are discussed to be
now, e.g., stability, origin, and identity of the responsible for fermentation problems in in-
prevailing microflora. dustrial vinegar fermenters (TEUBERet al.,
Investigations of the last years have shown 1987a, STAMMet al., 1989; SELLMERet al.,
that the plasmid profiles of these strains are 1992; DEFIVESet al., 1990). In trickling gen-
able to rest stable over a period of at least erators, fermentation is sometimes slowed
five years and are not changing their pheno- down but hardly stops completely. In sub-
typic features during this time. merged fermentations, complete loss of pro-
Fermentations at high total concentrations ductivity has been observed. Only 3 morpho-
are carried out continuously in the laborato- logically different Acetobacter phage types
ries of Heinrich Frings GmbH & Co. KG, were described until 1992. SELLMERet al.
Bonn, Germany, to increase the tolerance of (1992) reported a great variety in phage types
the strains against acetic acid. These cultures regarding the dimensions of the phage heads
are supplied to vinegar factories in all parts of and tails. All phages isolated from vinegar
the world. fermentations in Germany, Austria, and Den-
In 1993 Frings established a Phage and mark showed long contractile tails and iso-
Plasmid Laboratory for further investiga- metric heads belonging to Bradley's group A
tions. (BRADLEY,1967) and the family of Myoviri-
dae (ACKERMANN, 1987), respectively. Al-
though not all phages described have actually
4.1.4 Genetic Improvement of been shown to be infective for defined Aceto-
bacter strains due to a lack of a suitable tech-
Acetobacter Strains nique, their occurrence in high numbers
(lo6- lo9 mL-l) in disturbed fermentations
To improve strains of Acetobacter geneti- suggests that bacteriophages may actually be
cally, recombinant DNA techniques are con- the source of fermentation problems.
sidered to be useful. Host-vector systems and
an efficient transformation method for Aceto-
bacter have been developed. The preparation
of spheroplasts and the development of a 4.2 Biochemistry
spheroplast fusion method for Acetobacter
have been described by FUKAYA et al. During acetic acid fermentation ethanol is
(1989a). A strain growing at 37°C and 4% almost quantitatively oxidized to acetic acid.
acetic acid and a strain unable to grow at Concentration yields between 95% and 98%
35°C and 5% acetic acid were spheroplasted are common, and the remainder is mainly lost
and fused. Fusion products were recieved in the effluent gas. At the same time a suita-
growing at 37°C on agar plates containing 5% ble C6-carbon source (preferably glucose) is
acetic acid, on which both parent strains were oxidized. End products of this oxidation are
unable to grow. C 0 2 and H20.
 4 Production by Acetic Acid Fermentation 387

4.2.1 Ethanol 4.2.2 Sugar

Ethanol oxidation is performed by two se- For the breakdown of sugars, Acetobacter
quential reactions catalyzed by alcohol dehy- is equipped with the hexose monophosphate
drogenase (ADH) and aldehyde dehydrogen- (HMP) pathway and the TCA cycle (ASAI,
ase (ALDH) located at the outer surface of 1968) whereas glycolysis is either absent or
the cytoplasmic membrane. Their function is very weak. The enzymes of the Entner-Dou-
linked to the respiratory chain of the organ- doroff pathway are present in Gluconobacter
ism (AMEYAMA and ADACHI,1982a,b). and in Acetobacter xylinum (KERSTERS and
Alcohol dehydrogenase has been purified DE LEY, 1968). Gluconobacter and Aceto-
from several acetic acid bacteria and is shown bacter are known for their direct oxidative ca-
to contain pyrroloquinoline quinone (PQQ) pacity on sugars, alcohols, and steroids (DE
as well as heme C as prosthetic groups (ADA- LEY and KERSTERS, 1964). For further de-
CHI et al., 1978a, b; MURAOKAet al., 1982). tails, see EBNERand FOLLMANN (1983). The
Extensive purification and characterization of biochemistry of the ketogenic activities of
the enzymes involved in ethanol oxidation of acetic acid bacteria have been reviewed by
Acetobacter aceti, A. rancens, and G. suboxy- KULHANEK(1984).
duns have been reported by ADACHIet al.
(1978a,b, 1980), AMEYAMAet al. (1981),
HOMMEL and KLEBER (1984), and MURAO-
KA et al. (1981, 1982). It has been shown that 4.2.3 Acetate
ADH of acetic acid bacteria donates elec-
trons directly to ubichinone in the cytoplas- Strains of Acetobacter are able to oxidize
mic membranes. Thus, the ethanol oxidase acetate as well as lactate either in the pres-
respiratory chain of acetic acid bacteria is ence or in the absence of ethanol by using en-
constituted of only three membranous respi- zymes of the tricarboxylic cycle.
ratory components - alcohol dehydrogenase,
ubichinone, and terminal oxidase (MATSU-
SHITA et al., 1992).
AMEYAMA et al. (1981) confirmed that al- 4.2.4 Carbon Dioxide
cohol and further aldehyde dehydrogenase of
acetic acid bacteria had the same prosthetic It has been known since the work of RAZU-
group, PQQ, as their glucose dehydrogenase. MOVSKAYA and BELOUSOVA(1952) that
These quinoproteins, glucose, alcohol and al- Acetobacter needs C 0 2 for growth. C02 is in-
dehyde dehydrogenases are involved in sugar corporated into cell substances, where ap-
oxidation that links them to an electron trans- proximately 0.1% of the cell carbon is derived
port system in the membrane of oxidative from C02. A very small but measurable por-
bacteria. FUKAYAet al. (1989b) and TAYA- tion of acetic acid is derived from C02 metab-
MA et al. (1989) purified ADH and ALDH olism (HROMATKA and GSUR,1962).
from A. polyoxogenes sp. nov. This strain ori-
ginated from a vinegar factory showing higher
acid productivity and higher tolerance to ace-
tic acid than strains isolated from natural 4.2.5 Nitrogen
sources. In contrast to the ADH and ALDH
obtained from A. aceti and G. suboxydans, If an organic carbon source is present a
the enzymes of A. polyoxogenes were quite number of acetic acid bacteria can use am-
stable and this high stability seems at least to monium as sole nitrogen source. Some strains
be partially involved in the ability of A. po- need the presence of amino acids, others need
lyoxogenes to produce high concentrations of cofactors such as vitamins or purines
acetic acid. (BROWNand RAINBOW, 1956). No essential
amino acids are known.
388 12 Acetic Acid

4.2.6 Growth Factors aeration. The longer the interruption of aera-

tion and the higher the total concentration of
Growth factor requirement depends on the the mash, the more marked is the effect. At a
carbon source supplied (RAGHAVENDRAtotal concentration of 5% an interruption of
RAO and STOKES,1953). Some strains re- aeration for 2-8 min leads to the same dam-
quire p-aminobenzoic acid, niacin, thiamin, or age as an interruption for 15-60 s at total con-
pantothenic acid as growth factors (GOSSELE centrations of 11-12%. At constant total con-
et al., 1980). AMEYAMA and KONDO(1966) centrations damage increases with increasing
found that some Acetobacter strains did not concentration of acetic acid and with increas-
need vitamins in the presence of glucose. ing fermentation rate. The specific production
But the combined addition of glutathione of cell material is extremely low. For more
with sodium glutamate had a cumulative ef- details see EBNERand FOLLMANN (i983).
fect on the growth of A . aceti, as shown by Experiments conducted by MURAOKAet
ADACHIet al. (1990). al. (1983) confirmed these results. They found
PQQ, the novel prosthetic group in acetic that there was a sharp drop in the oxidation
acid bacteria, also shows an interesting rate of ethanol as well as in the activity of en-
growth-stimulating effect for various microor- zymes involved (ADH and ALDH) after an
ganisms (AMEYAMA et al., 1984, 1985; ADA- interruption of oxygen supply, with an acetic
CHI et al., 1990). One of two PQQ effects was acid concentration of 6%. ADH activity in
the stimulation of both the growth rate and the membrane fraction was highly stable in
the total cell yield with only a trace amount of the culture broth containinp 4% or less acetic
PQQ as an essential growth factor. The sec- acid. But the purified enzyme was very unsta-
ond effect of PQQ observed universally was a ble even in the culture broth of 2% total con-
marked reduction of the lag phase in micro- centration. It was suggested from this fact that
bial growth but no increase of the growth rate the alcohol dehydrogenase has a membrane
at the subsequent exponential phase or in the component contributing to its own stability at
total cell yield at the stationary phase. PQQ acetic acid concentrations of less than 4%,
was produced in culture media by most which becomes gradually liable to damage
strains of microorganisms. with further increasing acidity. But, the rela-
tionship between the alcohol dehydrogenase
system and oxygen has not yet been eluci-
4.3 Physiology dated at all.
Under conditions of industrial acetic acid
4.3.1 Oxygen Demand and Total production the microorganisms undergo con-
siderable stress, caused by a high concentra-
Concentration tion of acetic acid and limited oxygen supply.
A high utilization of oxygen up to 70% can be
During commercial use of many aerobic achieved in industrial production. A sparing
microorganisms it is common practice to let use of air is very important because of the vo-
the inoculum remain in a fermenter for some latility of ethanol and acetic acid. However,
time up to several days without aeration be- the use of pure oxygen or highly oxygen-
fore inoculation into the main fermenter. enriched air may easily damage the bacteria
However, application of this procedure is im- (HROMATKA and EBNER,1951).
possible in the case of acetic acid fermenta- HITSCHMANNand STOCKINGER(1985)
tion. found that at a total concentration of 13%,
Extensive tests have been conducted by the ATP pool and the growth rate show a re-
HROMATKAand EBNER(1951) and HRO- verse behavior. The energy charge (average
MATKA and EXNER(1962) concerning the value of intracellular ATP, ADP, and AMP)
damage of acetic acid bacteria due to an in- showed a rather high value of 0.84 at the be-
terruption of aeration as a function of total ginning of the fermentation. During fermen-
concentration, acetic acid concentration, rate tation, while the acetic acid concentration in-
of fermentation, and length of interruption of creased from 9 to 12%, the energy charge de-
 4 Production by Acetic Acid Fermentation 389

creased to 0.7. After interruption of aeration tion of 10%. This very high value was inde-
for 45 s, the energy charge dropped to 0.58 pendent of the acetic acid concentration of
and after several weeks of storage the charge 4.5-7.2%. MORI and TERUI(1972) found a
of the inoculum was only 0.50. strong dependence between specific product
formation and acetic acid concentration in
continuous fermentations at a total con-
4.3.2 Lack of Ethanol centration of 7%. The maximum specific
product formation was 15 g g - ' h-'. VERA
Acetic acid bacteria are also damaged if a and WANG (1977) found that the specific
vinegar fermentation is carried out to the product formation follows a growth-asso-
point where all of the ethanol has been oxid- ciated mechanism as long as the acetic acid
ized and the addition of fresh ethanol-con- concentration is below 3%. At higher acetic
taining mash is delayed. This is analogous to acid concentrations up to 7% an additional
the damage resulting from an interruption of non-growth associated term must be consid-
oxygen supply and also depends primarily on ered. The highest value obtained with cell re-
the total concentration and the duration of cycle in continuous culture of A . suboxydans
ethanol lack. was 11.5 g g - ' h-I.

4.3.3 Specific Growth Rate 4.3.5 Changes in Concentration

HROMATKA et al. (1953) were the first to and Temperature
calculate the specific growth rate of acetic
acid bacteria. In semicontinuous fermenta- Charging of a fermentation must be done
tions there was no decrease of the specific with mash of the same total concentration
growth rate at increasing acetic acid concen- and under rapid mixing because a locally
trations, but at increasing total concentrations formed strong concentration gradient dam-
the specific growth rate decreased from ages the bacteria. The next cycle starts with-
0.49 h -' at 5% to 0.16 h - ' at 11%. out any lag-phase. At the same time tempera-
In continuous culture at a total concentra- ture has to be kept constant because a re-
tion of 12% the specific growth rate de- peated change in temperature causes a similar
creased from 0.027 h-' at 4.5% ethanol to result (EBNERand FOLLMANN 1983).
0.006 h - ' at 1 % ethanol. It should be men-
tioned that the specific growth rate obtained
in continuous fermentations with constant 4.3.6 Overoxidation
concentrations of acetic acid and ethanol are
less favorable than those obtained under Overoxidation is the undesirable oxidation
semicontinuous conditions. of acetic acid to C 0 2 and H,O that has to be
VERAand WANG(1977) carried out batch prevented by avoiding lack of ethanol and
fermentations with Acetobacter suboxydans keeping the total concentration at a high lev-
and showed that at a total concentration of el. In trickle fermenters this is much more dif-
7.5%, a specific growth rate of 0.30 h - ' is in ficult than in submerged fermenters. For
good agreement with the results found by more details, see EBNER and FOLLMANN
HROMATKA et al. (1953). (1983).

4.3.4 Specific Product Formation

HROMATKAand EBNER(1949) found a
specific product formation of 21 g acetic acid
per gram of bacterial dry substance per hour
in semicontinuous tests at a total concentra-
390 12 Acetic Acid

4.4 Industrial Processes 4.4.1.2 Two-Stage Processes

4.4.1 Submerged Vinegar In the canning industry vinegar with a very
high percentage of acetic acid is in demand.
Fermentation The vinegar industry is interested in produc-
ing vinegar of high acidity in order to save
4.4.1.1 Semicontinuous and storage and transport costs. Therefore, as re-
Continuous Fermentations ported by EBNERand ENENKEL (1978) a
two-stage process for the production of vine-
For the production of vinegar with more gar with more than 15% acetic acid was de-
than 12% and up to 15% acetic acid, as re- veloped. During a submerged fermentation
ported by EBNER(1985), the process has to with a total concentration below 15% in a
be carried out in a semicontinuous manner, first fermenter, alcohol is added slowly to in-
each fermentation cycle taking about the crease the total concentration up to about
same time as the preceding and following cy- 18.5%. After the acetic acid concentration
cles. The starting concentration of each cycle has reached 15%, about 30% of the ferment-
is 7-10% acetic acid and about 5% ethanol. ing liquid are transferred into a second fer-
When an alcohol concentration between menter. The first fermenter is resupplied with
0.05% and 0.3% has been achieved in the fer- new mash of lower total concentration. In the
mentation liquid, a quantity of vinegar is dis- second fermenter the fermentation continues
charged from the fermenter. Refilling with until the alcohol is almost depleted. The
new mash of 0-2% acetic acid and 12-15% al- whole quantity of the finished vinegar is dis-
cohol leads to the starting concentrations for charged. The fermentation liquid in the first
the new cycle mentioned above. Discharging fermenter is supplied with alcohol at the ap-
must be carried out quickly in order to avoid propriate time and later divided again.
complete alcohol depletion. Charging must be Since the end of 1993, vinegar of more than
done slowly under constant fermentation 20% acetic acid can be produced using this
temperature and rapid mixing. process. An automatic control for this process
Since the middle of 1994 it is possible to was described by ENENKEL (1988).
produce vinegar of up to 19% acetic acid in a In 1981, a similar two-stage process for the
modified single-stage process. Starting with production of vinegar with more than 20%
similar concentrations as mentioned above, acetic acid was described by KUNIMATSUet
alcohol is slowly added at a constant alcohol al. (1981, 1982). The second fermentation
level of 2-3% in the fermentation liquid stage is carried out at a reduced temperature
creating a corresponding increase of the total of only 18-24"C, while in the first fermenta-
concentration. Addition of alcohol is stopped tion stage the normal temperature range of
when the desired total concentration has been 27-32°C is used.
reached. When alcohol approaches zero a MASELLIand HORWATH (1984) claimed a
part of the fermentation liquid is discharged similar two-stage process with semicontin-
and replaced by mash with lower total con- uous fermentation in at least two fermenters
centration to bring acetic acid concentration and membrane filtration of the mash from the
and total concentration back to starting con- semicontinuous stage after combining it with
ditions, thereby enabling the bacteria to mul- an oxygen-containing gas. After adding etha-
tiply faster. Later, total concentration is in- nol and nutrients the concentrated mash con-
creased again by adding alcohol. taining the bacteria is subjected to batch fer-
Continuous fermentation is only possible mentation at a substantially lower tempera-
up to a maximum of 9-10% acetic acid be- ture until 18-20% acetic acid are reached.
cause the specific growth rate of the bacteria The membrane-filtered vinegar is used as a
decreases with decreasing ethanol concentra- second end product.
tion. To obtain high yields the fermentation
must be carried out at a low alcohol concen-
tration.
 4 Production by Acetic Acid Fermentation 391

4.4.1.3 The Frings Acetator@ with a shorter operating time of the defoamer
and consequently a lower power uptake and a
Frings Acetator@ is the most common negligibly small volume of the separated li-
equipment for the production of all kinds of quid are the result.
vinegar. At the end of 1993 more than 600 The Frings Alkographs, described by EB-
acetators, continuously improved, enlarged NER and ENENKEL (1966), an automatic in-
and automated, were in operation all over the strument for measuring the amount of etha-
world with a total production of 1354.106 L nol in the fermenting liquid has been in use
a - ' of vinegar with 10% acetic acid. Energy since 1965. Small amounts of liquid flow
consumption is about 400 W L-' of fer- through the analyzer continuously, at first
mented ethanol with decreasing tendency, through a heating vessel and then through
yield is up to 98%. three boiling vessels. The boiling temperature
Commercial sizes of acetators are for the of the incoming liquid is measured in the first
fermentation of up to 3,600 L of pure ethanol boiling vessel. While alcohol is distilled off
in 24 h. continuously from the second and the third
The different processes demand excellent boiling vessel, the higher boiling point of the
performance of the fermenter, especially of liquid from which ethanol has been removed
its aeration system. is measured in the third boiling vessel. The
The Frings aerator, as reported by EBNER difference in temperature corresponds to the
and ENENKEL(1974), is self-aspirating, i.e., ethanol content and is recorded automatical-
no compressed air is needed. The rotor is in- ly. As the flow through the vessels takes some
stalled at the shaft of a motor mounted under time, there is a delay of about 15 min between
the fermenter, connected with an air suction the beginning of the inflow of a sample and
pipe and surrounded by a stator. It sucks air the appearance of the correct value on the re-
and pumps liquid, thereby creating an air- corder. As alcohol concentration is decreas-
liquid emulsion that is ejected radially out- ing slowly during fermentation this delay has
ward through the stator at a given speed. This no disadvantage on fermentation control.
speed must be chosen adequately so that the To carry out the single-stage semicontin-
turbulence of the stream causes a uniform dis- uous process at a defined alcohol content a
tribution of the air over the whole cross sec- contact in the alkograph activates the vinegar
tion of the fermenter. discharge pump. As soon as a preset level has
As foam formation cannot always be been reached the mash pump starts adding
avoided the acetator is equipped with a me- fresh mash. This pump is controlled by the
chanical defoamer. From 1966 until 1991, the fermentation temperature in order to refill
equipment described by EBNER(1966) was under constant temperature. The pump is
used. The rotor, turning in a spiral housing, stopped when the desired level is reached and
separates liquid and gas and returns the liquid an automatic cooling system is activated. A
into the lower part of the fermenter. Only fermentation cycle takes 24-48 h.
during discharging of the finished vinegar a The Frings AlkographB has worked with
part of this returned liquid is pumped out of high reliability for about 30 years in 231 ace-
the fermenter. As this liquid causes foam for- tators all over the world. New biosensors to-
mation, the defoamer must operate most of gether with modern membranes permitted
the time of a fermentation cycle. In an im- the development of a device to measure alco-
proved version described by WITTLER(1991) hol without any time delay: the Frings Alko-
the separated liquid portion is not pumped sen@. HEIDERet al. (1983) described a de-
back into the fermenter, but into a small col- vice for measuring volatile constituents of the
lection vessel to be mixed with the vinegar fermentation medium. A membrane serves to
end product later. Lysis of harmed vinegar filter these volatile constituents from the fer-
bacteria releases foam causing surface-active mentation liquid into a carrier medium, and
substances. By means of the total elimination they are recorded by an electronic sensor. As
of the separated liquid, further foam forma- the measurement relies on diffusion, ambient
tion is avoided. Minimized foam formation temperature has to be kept always at the
392 12 Acetic Acid

same level. Thus the alcohol probe is situated tic acid by electrodialysis, continuous supply
in the fermentation broth. The alcohol value of ethanol and concentrated nutrient solution,
displayed at the analyzer is influenced by the and control of ethanol and pH. The reason
concentration of acetic acid and, therefore, for the continuous removal of acetic acid
new calibrations are needed at different total from the reaction mixture was to alleviate the
concentration levels. This disadvantage has inhibitory effect of the produced acetic acid.
been overcome recently by the new system Ethanol concentration was kept constant at
Frings Alkosens I P , which incorporates a 1%, acetic acid concentration at ca. 2%.
multi-layer membrane. New sensor technolo- Without dialysis and pH control, acetic acid
gies and more sophisticated computer equip- production already stopped after 2 d at ca.
ment permit a fully automated process con- 4%0,while with dialysis it lasted 30 d.
trol of continuous, semicontinuous as well as
single-stage or two-stage high strength fer-
mentations - preparation of mash included. 4.4.1.5 Other Submerged
Fermentation Processes
4.4.1.4 Further Trends in
Other processes of little practical signifi-
Submerged Processes cance and abandoned processes are described
by EBNERand FOLLMANN (1983).
HEKMATand VORTMEYER (1992) applied
a computerized experimental laboratory sys-
tem to submerged acetic acid fermentation 4.4.1.6 Continuous Culture and
with industrial Acetobacter strains at about
10% of final acidity using automated semi- Cell Recycle
continuous cycles over a period of one year.
Based on these data a mathematical model VERAand WANG(1977) and BANSCHand
was developed to predict the time course of a BAUER (1991) invested much work to in-
cycle, even in the case when at the beginning crease the productivity of acetic acid fermen-
of the cycle a lag phase occurred due to a lack tation by continuous culture and cell recycle.
of substrate at the end of the foregoing cycle. It must indeed be admitted that at first
The parameters substrate, product, and bio- sight the productivity in an acetator does not
mass, however, were not sufficient to charac- seem to be very high. The Acetator 3600@,
terize the physiological status of the culture, e.g., produces during 24 h 4,000 kg acetic acid
since the behavior of the microorganisms also with a concentration of up to 15% in a fer-
depends on their past. RNA concentration menting volume of 100 m3, i.e., 1.6 g L-' h - '
per unit of cell mass was found to be an inter- acetic acid in average or about 2.4 g L-' h-'
nal key parameter that showed a reasonable at the maximum fermentation rate. The aera-
agreement of the experimental data and the tion rate is 0.1 vvm only, the nutrient con-
mathematical model. sumption is the same as described in Sect. 4.3.
MORI (1989) summarized the possibilities Kinetic studies carried out by BANSCHand
of on-line measurement of alcohol concentra- BAUER(1991) clarified the dependence of
tion in vinegar production. In addition to the the bacterial growth rate on ethanol concen-
Frings Alkograph" the Alcolyzer", a Japa- tration, on acetic acid concentration, and on
nese instrument based on the same principle, concentration of dissolved oxygen. The opti-
immobilized microbial sensors, immobilized mum ethanol concentration is 23 g L-', the
enzyme sensors, and semiconductor alcohol optimum acetic acid concentration 36 g L-I,
sensors have been described. and the optimum oxygen concentration
NOMURAet al. (1988, 1989) and NOMURA 2.5-3 mg L-'. The productivity of acetic acid
(1992) reported experiments for the contin- formation mainly depends on the growth rate
uous production of acetic acid by submerged but also on the cell concentration. Therefore,
fermentation with continuous removal of ace- the maximum productivity of 6-6.6 g L-' h-I
 4 Production by Acetic Acid Fermentation 393
acetic acid can be obtained at 32 g L - ' etha- tions of acetic acid concentration, oxygen de-
nol and 60-66 g L - ' acetic acid. ficiency, and nutritional limitation.
NANBAet al. (1984) studied the synergistic The effect of dissolved oxygen and acetic
effects of acetic acid and ethanol on the acid concentration on acetic acid production
growth of Acetobacrer sp. at low concentra- in continuous culture has'extensively been in-
tions with similar results. vestigated by PARKet al. (1989b). The opti-
At first sight, the potential productivity is mum concentration of dissolved oxygen was
about 4 times higher than presently achievea- found to be about 2 ppm for acetic acid pro-
ble and could even be further increased by re- duction at low acidity. The specific acetic acid
cycling of the bacteria. However, this high de- production rate, however, was diminished to
gree of productivity can only be achieved at a complete inhibition when the dissolved oxy-
narrow concentration range. A slight change gen concentration was higher than 2 ppm at
of ethanol, acetic acid or oxygen concentra- an acetic acid concentration of more than
tion results in an immediate return to lower 4.5%. This is in good agreement with HRO-
values. MATKA and EBNER(1951), who found that
For technical applications a second fer- the use of pure oxygen during fermentation
menter in series to the fermenter operating at can completely inhibit acetic acid formation.
optimal fermentation conditions would be As viability of the bacteria is important for
necessary to complete the fermentation. To high productivity the factors affecting viabili-
keep a high oxygen concentration in the fer- ty have been investigated by PARKand TODA
mentation liquid at the higher fermentation (1990). To study the effect of cell bleeding
rate, a high aeration rate would be required (withdrawing at constant flow rate) serial ex-
with recovery of ethanol and acetic acid from periments were performed in a bioreactor
the exhaust gas to obtain high yields, and with cell recycle and were supported by math-
probably addition of oxygen would be neces- ematical analysis. Acetic acid concentrations
sary. Higher nutrient consumption as well as were up to 5.6%. The maximum value of pro-
increasing foam formation would result. The ductivity was 123 g L-' h-'. The bleed ratio
cell recycle system would need a continuous and dilution rate at which both concentration
microfiltration avoiding a lack of oxygen and of acetic acid and viable cells are relatively
require an automatic control of cell concen- high can be predicted now.
tration. But all the expenditure in process The authors expect these results to be use-
control only permits the production of the ful for designing a two-stage culture for acetic
same amount of vinegar, containing not more acid production using the active cells in the
than 10% acetic acid, in a smaller fermenter. bleed from the first stage in a second-stage
Apparently, the demand for high productivity vessel beyond a lethal acetic acid concentra-
does not necessarily indicate the optimal de- tion.
velopments for future production. How far these certainly interesting results
PARKet al. (1989a, c) tried to enhance con- will influence the construction of industrial
tinuous acetic acid production in a laboratory equipment in Japan remains to be seen. The
fermenter equipped with a hollow fiber filter costs of such complicated fermenters may
module by increasing the concentration of slow down or even prevent a fast conversion
dissolved oxygen. The maximum acetic acid to industrial scale, although in Japan the de-
concentration achieved was 5%. The highest mand for vinegar with 5% acetic acid is rela-
productivity obtained with yeast extract as tively high. However, the above mentioned
nutrient was 107 g L - ' h-'. The concentra- results will certainly have no influence on the
tion of dissolved oxygen was already lower production of vinegar with more than 10%
than the critical concentration of 1 ppm, al- acetic acid. In a review of the Japanese devel-
though pure oxygen was supplied at an aera- opmental work FUKAYA et al. (1992) point
tion rate of 1 vvm. Furthermore, it was diffi- out that the problem of scale-up remains for
cult to maintain a high concentration of via- further investigation.
ble cells in the cell recycle culture, because
the cells were inactivated by the lethal condi-
394 12 Acetic Acid

4.4.1.7 Semicontinuous Culture authors used a bioreactor with a shallow, ho-

rizontal flow of medium under a bacterial film
with Cell Recycle with a surface of a few hundred cm2. Liquid
depth was up to 10 mm and the acetic acid
PARKet al. (1991a) investigated semiconti- concentration of the effluent was 5.76%. The
nuous cultures with cell recycle and found an oxygen absorption rate through the microbial
increase in productivity compared to semi- film was found to be very high; a kinetic study
continuous culture without cell recycle at an of acetic acid production was published by
acidity of up to 6%. While the concentration PARKet al. (1990).
of total cells increased in every cycle, the via- Kewpie Jozo Kabushiki Kaisa (1970) de-
ble cells reached a much lower quasi steady- scribed a surface fermentation process in a
state value, which was explained by product system of serial intercommunicating fermen-
inhibition. The productivity was 2.9 g L-’ h -’ tation vessels with flow-regulating devices
without and 5.0 g L - ’ h - ’ with cell recycle that enforce the flow to approach plug flow
at 6% final acetic acid concentration, but conditions. The liquid depth under the layer
14 g L - ’ h - ’ at 3% final acetic acid concen- of acetic acid bacteria was about 50 mm. The
tration. inflowing liquid had 2% acidity and con-
In another study PARKet al. (1991b) re- tained 3.5% alcohol. The acidity of the ef-
ported semicontinuous fermentations in fluent stabilized at 5% acetic acid. The resi-
which the ethanol concentration was kept for dence time was about 21 h.
a certain time between 20 and 30 g L - ’ by HIGASHIDE et al. (1992) reported contin-
adding ethanol-rich medium. At the end of uous surface fermentations in a single vessel
the cycle ethanol-poor medium was added. with a working volume of 16L. Under opti-
Cells were recycled by membrane filtration. mum conditions with a liquid depth of 100
The goal was to obtain high concentrations of mm, 2-3% acidity and 20-30 g L - ’ alcohol
acetic acid; the highest amount reached was concentration of the inflowing liquid, vinegar
9%. It was found that the Acetobacrer cells with 4.5% acetic acid was produced during 80
lost their viability at about 6% acetic acid, but d. The flavor of this vinegar was claimed to be
these non-viable cells were still effective for better than that of vinegar produced by other
ethanol oxidation. Already 1976, this interest- processes.
ing feature of Acetobacter cells had been re- For the production of rice vinegar with
ported by EBNER. good quality surface fermentation develops to
an automatic fermentation process at lower
costs in Japan.
4.4.2 Surface Fermentation
4.4.2.1 Old Surface Fermentation 4.4.3 Trickling Processes
Processes
The old semicontinuous generator process
CONNERand ALLGEIER(1976) published used beech wood shavings, birch twigs, or
detailed descriptions of the history of vinegar corn cobs as carrier material for the vinegar
fermentation. Today, the equipment used in bacteria. Large wooden tanks with up to
the famous “Orleans Process” for the produc- 100 m3 shavings are still in use. The ferment-
tion of wine vinegar can only be seen in mu- ing liquid is collected in the lower part of the
seums. tank and continuously pumped under cooling
over the carrier column. Air is aspirated or
blown through the column by ventilators. A
4.4.2.2 New Experiments with number of generators older than 40 years are
Surface Fermentation still in operation.
The inhomogeneity of the carrier column in
A new approach for surface fermentation general makes a sufficiently homogenous dis-
was made by TODAet al. (1989, 1990). The tribution of air and trickling liquid impossi-
 4 Production by Acetic Acid Fermentation 395

ble. Variations in temperature within the col- tages of all known carriers SAEKIet al. (1991)
umn can hardly be avoided. Mashes contain- tried a new ceramic carrier called “Aphro-
ing a high amount of nutrients that are low in cell” with a continuous pore structure, but the
total concentration form slimy deposits in the oxygen supply to this structure was difficult.
carrier material which may clog the column. None of the tested carriers proved to be ide-
Nevertheless, in 1993, Carl Kuhne KG pro- al.
posed the use of a silicate carrier material SUNet al. (1990) carried out experimental
with a large specific inner surface and an and theoretical studies on the continuous ace-
open pore structure. tic acid production by immobilized cells in a
three-phase fluidized-bed bioreactor at very
low acetic acid concentrations. The influence
4.4.4 Experiments with of gel-entrapped and suspended cells, gel size,
Immobilized Acetic Acid Bacteria k L a , solid holdup on productivity was studied
by theoretical calculations and experiments.
Many Japanese scientists investigated the MORI(1985, 1992) and MORI et al. (1992)
fermentation of rice or fruit vinegar with the stated that immobilized acetic acid bacteria
usual concentration of about 5% acetic acid are highly oxygen-dependent. Therefore, it is
in continuous fermentation of high productiv- advantageous to supply oxygen to a closed
ity with immobilized acetic acid bacteria. and pressurized culture. Feeding medium at a
MORIet al. (1989) reported a 460 days run higher dilution rate gives a higher production
in an air lift bioreactor with acetic acid bacte- rate. As acetic acid inhibits the bacteria, it is
ria immobilized on K-carrageenan gel beads, considered that multiple reactors in series
where living cells were newly released into would be the best solution to obtain an in-
the reactor. These newly released bacteria creasing acidity, thereby keeping the dilution
showed extremely high growth rate and respi- rate higher and creating the prolonged resi-
ratory activity which they retained for a few dence time necessary. An output acidity of
generations after leakage. The fact that the 5.5% was obtained in two fluidized-bed type
bacteria entrapped in K-carrageenan gel and tabletop reactors in series. In scaling-up to a
treated in a fluidized-bed reactor were grad- pilot reactor carrageenan gel beads were not
ually released from the gel beads had already used any longer because many of them had
been published before by OSUGA et al. broken. Porous chitosan beads were selected
(1984). with a diameter of 0.3-3 mm, that gave a
OKUHARA (1985) and NANBAet al. (1985) higher productivity up to 9 g L - ’ h-I. The
proposed polypropylene fibers as carrier ma- microbial layer, however, was easily peeled
terial for Acetohacter producing rice vinegar off from the chitopearls. The authors state
of 3% acetic acid. KONDOet al. (1988) used that more operational know-how is needed
ceramic honeycomb-monolith as carrier in a for the practical application of this technolo-
fixed-bed reactor and achieved a maximum of gy.
4% acetic acid. SAEKI(1990a) investigated
calcium alginate beads in a fluidized-bed
reactor and obtained 3,5% acetic acid. The 4.4.5 Environmental Protection
same author (SAEKI,1990b, 1991) used this
reactor in combination with an ethanol fer- Today, it is possible to produce vinegar
menter to produce rice vinegar of up to 6,5% without any environmental pollution.
acetic acid from a saccharified rice medium. The exhaust gas contains alcohol, acetic
TAKADA and HIRAMITSU (1991) tested a acid, and ethyl acetate according to their va-
two column bioreactor of porous ceramics to por tension at a fermentation temperature of
produce vinegar of an orange wine with up to about 30°C. To obtain high yields the aera-
8,4% acetic acid. YAMASHITA et al. (1991) tion rate is chosen as low as possible. It is fur-
fixed the cells on woven cotton fabric to pro- thermore common practice today to cool the
duce vinegar of kiwi fruit and persimmon of exhaust gas down with cooling water as far as
4,5% acetic acid. Because of the disadvan- possible, and in order to absorb the rest of
396 12 Acetic Acid

the volatiles it can be scrubbed with water

that is recycled and used for mash prepara-
5 Experiments with
tion later.
The raw vinegar contains bacteria that have
Anaerobic Fermentation
to be filtered off. Today most modern facto- of Glucose
ries use cross-flow filtration without adding
any filter aid. The very small quantity of re-
maining solids of about 100 g per 1,000 L of A novel route to the production of acetic
vinegar formed by acetic acid bacteria are de- acid by direct anaerobic fermentation of glu-
graded during waste water treatment. cose has been under investigation since 1978
using an anaerobic and thermophilic microor-
ganism, Clostridium thermoaceticum. While
4.5 Production Volumes during anaerobic fermentation of glucose to
ethanol and further aerobic fermentation to
acetic acid, a maximum yield of 0.67 g acetic
4.5.1 European Union acid per g glucose is only achieveable. An - at
least theoretical - maximum yield is 1 g g - ' if
In 1992, the European Union produced the acid is produced directly by Clostridium
325.10' L of alcohol vinegar, 143.10' L of thermoaceticum.
wine vinegar, and 45-10' L of other kinds of WANGet al. (1978) carried out batch fer-
vinegar (all with 10% acetic acid). mentations in a 3 L fermenter at 58°C with
anaerobiosis maintained by an overlay of
C02. Maximum final acetic acid concentra-
4.5.2 United States of America tion was 30 g L-'. This yield could only be
maintained at a constant pH of 7.0 by adding
Years ago, the U.S. vinegar industry ceased NaOH. Total inhibition of growth resulted at
to control and publish vinegar production fig- a sodium acetate concentration of 40 g L-'.
ures. Therefore, older figures must be quoted The yield was 0.85 g acetic acid per g glu-
here. In 1987, production volumes were cose.
581.106 L of distilled vinegar, 54.10' L of SCHWARTZand KELLER(1982) compared
cider vinegar, and 126.10' L of other types of different strains of Clostridium thermoaceti-
vinegar - all of different acetic acid concen- cum with regard to growth rate, tolerance to
trations. acetate, efficiency of converting glucose to
acetic acid, and to cell mass. An interesting
discovery was the effect of the redox potential
4.5.3 Japan on the growth rate. At pH 7 a redox potential
of about -360 mV was needed before growth
In 1992, Japan produced 56. 10' L of rice occurred. At pH 6 the potential had to be
vinegar, 146.10' L of other grain vinegars, about -300 mV and at pH 5 -240 mV. The
and 161* 10' L of other brewed vinegar, i.e., a cells are able to reduce the redox potential,
total of 394.106 L at concentrations of 4 5 % but the nature of the reducing agent is un-
acetic acid. known. At pH 7 the maximum acetic acid
concentration obtained was 20 g L-'.
WANGand WANG(1983) reported contin-
4.5.4 World uous culture experiments for the production
of acetic acid by immobilized whole cells of
From statistics and personal knowledge of Clostridium therrnoaceticurn in a fermenter
the situation in many countries, the world with 500 mL working volume. Carrageenan
production (excluding the USSR and China) gels were used for immobilization. High cell
of vinegar of 10% acetic acid is assumed to be loading (60 g L - ' of gel) can be achieved in-
about 1,900-106 L a - ' or 190,000 t of pure side the gel. Cell concentration is significantly
acetic acid. higher than in free-cell fermentations. Conse-
 6 References 397

quently, dilution rates that are much higher

than the maximum growth rate could be at-
6 References
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The highest volumetric acetic acid productivi- omy, Microbiol. Sci. 4, 214-218.
ty achieved was 6.9 g L - ' h-' at a dilution ADACHI, O., TAYAMA, K., SHINAGAWA, E., MAT-
rate of 0.40 h-', an acetic acid concentration SUSHITA, K., AMAYAMA, M. (1978a), Purifica-
of 19 g L-', and a pH adjusted to 6.9. tion and characterization of particulate alcohol
WANG and WANG (1984) carried out pH dehydrogenase from Gluconobacter suboxydans,
controlled batch fermentations in a 5 L fer- Agric. Biol. Chem. 42, 2045-2056.
menter. The initial glucose concentration was ADACHI,O., MIYAGAWA, E., SHINAGAWA, E.,
18 g L -' and a concentrated glucose solution MATSUSHITA, K., AMAYAMA, M. (1978b), Pu-
rification and properties of particulate alcohol
was fed periodically maintaining the dissolved dehydrogenase from Acetobacter aceti, Agric.
glucose concentration between 5 and 15 g L -'. Biol. Chem. 42, 2331-2340.
At pH 6.9, 56g L - ' acetic acid were ob- ADACHI, O., TAYAMA, K., SHINAGAWA, E., MAT-
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of Clostridium thermoaceticum was inhibited. and characterization of membrane-bound alde-
At pH values below 6.0 undissociated acetic hyde dehydrogenase from Gluconobacter subox-
acid is responsible for growth inhibition, and ydans, Agric. Biol. Chem. 44, 503-515.
at pH values above 6.0 ionized acetate is re- ADACHI,O., OKAMOTO, K., MATSUSHITA,K.,
SHINAGAWA, E., AMEYAMA, M. (1990), An ide-
sponsible for the same effect. al basal medium for assaying stimulating activity
SUGAYA et al. (1986) carried out batch fer- of pyrroloquinoline quinone with Acetobacter
mentations in 16 L fermenters, and contin- aceti, Agric. Biol. Chem. 54, 2751-2752.
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growth associated and non-growth associated. brane bound, in: Methods in Enzymology,
The obtained yield was 0.67 g g - ' glucose (WOOD,W. A., Ed.) Vol. 89, pp. 45W57. New
only. Reactions with phosphate may be a ma- York: Academic Press.
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novel prosthetic group, PQQ, in membrane-
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of 2 t d - ' acetic acid and vinegar from mo- Lett. 130 (2), 179-183.
lasses via ethanol was established in Turkey AMEYAMA, M., SHINAGAWA, K., MATSUSHITA,
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398 12 Acetic Acid

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