Production of Vinegar: Steps, Raw Materials, Process and Treatment | Fermentation | Industrial Microbiology

In this article we will discuss about the production of vinegar:- 1. Basics of Vinegar Fermentation 2. Raw Materials Required for Vinegar Production 3. Trickling Process of Vinegar Fermentation 4. Treatment of Raw Vinegar.

Basics of Vinegar Fermentation:

The vinegar fermentation is an oxidative fermentation in which diluted solutions of ethanol are oxidized by Acetobacter with the oxygen of air to acetic acid and water.

The oxidation proceeds according to the basic equa­tion-

The alcohol-containing solution is called a mash. Its alcohol concentra­tion is given in volume percent. Usually it also contains some acetic acid which is determined by titration with 1 N NaOH and expressed as grams acetic acid per 100 ml. The sum of the vol. % ethanol and the weight percent (g/100 ml) of acetic acid is called “total concentration.”

The sum of these rather incommensurable values gives the maximal concentration of acetic acid which can be obtained by complete fermentation. The quotient of the “total concentration” of the produced vinegar over the “total concentration” of the mash gives the concentration yield. The quotient of the acetic acid concentration of the produced vinegar over the “total concentration” of the mash gives the acid yield.

Raw Materials Required for Vinegar Production:

All mashes must contain ethanol, water, and nutrients for the acetic acid bacteria. By far the largest percentage of vinegar is distilled vinegar which is produced from diluted, purified ethanol or from fuel oil containing crude spirit. Other common names for the same product are white vinegar, spirit vinegar, alcohol vinegar, or grain vinegar.

It is customary in almost all countries to denature the ethanol which serves as raw material for the vinegar industry. In the United States this is mostly done with ethyl acetate, which is split during the fermentation to ethanol and acetic acid. In most European countries denaturation is carried out with distilled vinegar.

Mashes obtained by the alcoholic fermentation of natural sugar-containing liquids also serve as a raw material. The vinegar is designated according to the particular raw material used. For instance, wine vinegar is produced by vinegar fermentation of grape wine. In some countries with large wine production the use of ethanol as raw material for vinegar production is not permitted.

Cider vinegar is produced from fermented apple juice. It is particularly well known in the United States, Switzerland, and Austria because of its desirable aroma.

Malt vinegar is the product made by the alcoholic and subsequent acetous fermentation, without distillation, of an infusion of barley malt or cereals whose starch has been converted by the malt. It is well known in the United States, in England, and in South Africa.

Whey vinegar is produced by the alcoholic and subsequent acetous fer­mentation of concentrated whey. Fruit vinegar is made from fruits which are available in surplus in some countries. Its production from dates, citrus fruit, or bananas, for instance, is gaining in importance.

Sugar vinegar is made by the alcoholic and subsequent acetous fermen­tation of sugar syrup, molasses, or refiners’ syrup.

Glucose vinegar is made by the alcoholic and subsequent acetous fermen­tation of glucose solutions.

Rice vinegar is made by the saccharification of rice starch, followed by alcoholic and acetous fermentation.

Water for Processing:

The water used for the preparation of mashes must be clear, colorless, odorless, and without the presence of sediment or suspended particles. It may be hard or soft but frequent changes in hardness as they occur in municipal water supplies may interfere with the fermentation. In extreme cases the water must be demineralized followed by the addition of minerals. The water must be bacteriologically clean. Therefore, care has to be used when well water or water previously used for cooling purposes is used. If municipal water is used, it must be free from chlorine.

Nutrients:

Most natural raw materials do not require the addition of extra nutrients. Apple cider is usually low in nitrogenous materials which can be corrected by the addition of 100 g ammonium phosphate to 1000 liters of mash. Some grape wines require the addition of up to 400 g ammonium phosphate for a satisfactory fermentation. In rare cases the same nutrients must be added as those for the production of distilled vinegar.

For the production of distilled vinegar a mixture of the required nutrients had to be developed. Acetobacter definitely require between 500 and 1000 g of glucose per 1000 liters mash. Further, a total of 300 g of the following substances are required per 1000 liters mash- potassium, magnesium, cal­cium, ammonium as phosphate, sulfate, and chloride salts. The following trace minerals are required- iron, manganese, cobalt, copper, molybdenum, and vanadium. Demineralized water requires the addition of 100 g of calcium carbonate and 100 g of sodium chloride per 1000 liters.

These additions are sufficient for a satisfactory acetous fermentation. Commercial mixtures containing supplements such as malt extract or dried yeast are available. These are used to restart fermentation more quickly if it has been stopped by a disturbance, for instance, by an interruption of the power supply. The amount added is not more than 200 g per 1000 liters mash.

The amounts of nutrients mentioned are required for the submerged fermentation process. For the trickle process about one-third of the listed amounts are needed. In principle, nutrients should be added sparingly in order to exert a selection pressure in the direction of a low requirement for nutrients.

Acetobacter:

Since the time of Pasteur’s work, it has been known that acetic acid bacteria cause the souring of stored wine. In 1900 Beijerinck suggested the designation Acetobacter for the genus, a designation which is still current today. The Acetobacter belong to the family Pseudomona- daceae, but this classification is in dispute.

The first attempt to classify acetic acid bacteria was made by Hansen in 1894, followed by classifications of other investigators. The number of isolated strains became larger and larger and their classification more complicated. But already Visser’t Hooft (1925) had indicated briefly that acetic acid bacteria of prior collections did not retain their properties.

For a while it appeared as if Frateur (1950) had succeeded in the final classification of the group by the designation of 4 main groups- peroxydans, oxydans, mesoxydans, and suboxydans. But subsequently new strains with different properties were discovered, and these had to be accommodated in ever more complex schemes of classification.

As the result of an extensive study, Shimwell (1959) found that a strain of Acetobacter changes its properties right at the time of its isolation. The identification of a strain of Acetobacter reflects therefore only its properties at the time of isolation and gives no information on its properties at an earlier or later date. The mutants which arise immediately give rise to new mutants.

However, it is not certain that the word “mutation” can properly be applied to this change. Other concepts such as cytoplasmic variation, segregation, recombination, or other more complex types of reproduction are under consideration. The variability of Acetobacter goes so far that the property of oxidizing ethanol to acetic acid may be lost. Hence, Acetobacter really defy classification.

The vinegar industry is only interested in using a strain of Acetobacter which tolerates high concentrations of acetic acid and of “total concentra­tion,” which requires small amounts of nutrients, and which does not over-oxidize the formed acetic acid. By 1950 it had already been observed that with each cultivation of a strain in Petri dishes there was a danger of a change in properties.

Therefore, vinegar fermentations are carried out continuously in the laboratories of the firm Heinrich Frings in Bonn in order to supply the strain for fermentations in all parts of the world. A newly installed fermentor is directly inoculated by means of a transportable fermentor.

If this is not possible, vinegar from the mentioned laboratory fermentation is poured into bottles (partially filled) which are transported in the pressurized cabin of a plane. At the destination the vinegar is aerated until the fermentation begins again; and this is used to inoculate the new commercial fermentor.

In this manner variations in the Acetobacter strain can be avoided. In spite of the propensity of Acetobacter for variation, it is possible to perform vinegar fermentations year-in year-out without inter­ruption. This can be done if one does not give mutants which require more nutrients or which are more sensitive to acetic acid a chance to survive. This is accomplished by keeping the total concentration high and the concen­tration of nutrients low.

Biochemistry of Vinegar Fermentation (Ethanol):

In satisfactory vinegar fermentation, ethanol is almost quantitatively oxidized to acetic acid. Yields between 95 and 98% are normal, and the remainder is mainly lost in the effluent gas. Acetic acid should not be oxidized further to water and CO₂ (over-oxidation).

From Wieland and Bertho (1929) it is known that the oxidation of ethanol takes place in two steps with acetaldehyde as the intermediate product. Other investigators have studied this reaction in detail. However, this work has been carried out with varying strain of Acetobacter. This means that the properties of the Acetobacter at the time of the investigation varied, and that they often did not correspond to the properties of the strains during the vinegar fermentation.

King and Cheldelin (1954) successfully purified an alcohol dehydroge­nase from Acetobacter suboxydans which is NAD-dependent and which does not act on acetaldehyde. The same authors also purified an acetaldehyde dehydrogenase which required NADP as coenzyme. Prieur (1968) found two enzyme systems in the oxidation of ethanol by Acetobacter xylinum. One of these with maximum activity at pH 5.7 requires no NAD; the other one with maximum activity at pH 8.1 is NAD-dependent.

The most exhaustive investigations have been carried out by Nakayama (1959, 1960, 1961A,B). He isolated a highly purified ethanol oxidizing enzyme from an Acetobacter sp. with some similarity to A. suboxydans. It contains a heme protein and has an absorption spectrum similar to that of cytochrome C and an absorption maximum at 553 nm.

In the presence of ethanol the enzyme reduces several oxidation-reduction dyes, but not TPN and DPN. Its optimum pH is at 3.8. Its temperature inactivation curve parallels that of the denaturation of the heme protein. With ferricyanide as electron acceptor, the enzyme shows broad substrate specificity by oxida­tion of many saturated and unsaturated straight chain mono-alcohols.

The heme protein is also reduced by acetaldehyde in presence of an aldehyde dehydrogenase which can also be isolated from Acetobacter and which is not coenzyme-dependent. Further, an aldehyde dehydrogenase has been iso­lated which is TPN-dependent and has broad substrate specificity.

The following scheme of oxidation is derived:

Ethanol is oxidized by the alcohol-cytochrome-553-reductase (E₁) to acet­aldehyde. The electrons are transferred to the iron of the heme protein of E₁ Acetaldehyde is further oxidized by the coenzyme-independent aldehyde dehydrogenase (E₂) or by the TPN-dependent aldehyde dehydrogenase (E₃). For oxidation by E₂ the freed electrons are also transferred to the heme iron of E₁.

The reduced cytochrome 553 is oxidized by a cytochrome oxidase present in the cell. Electrons liberated by the oxidation of acetaldehyde by E₃ reduce TPN to TPNH₂. It is assumed that the presence of TPNH₂ inter­feres with the further oxidation of acetic acid by the tricarboxylic acid cycle. The acid pH optima of E₁ and E₂ favor the accumulation of acetic acid by the microorganism. TPN must be available for these reactions through the metabolism of the cell.

Loitsyanskaya (1955) was the first to show that Acetobacter schutzenbachii, A. curvum, or A. aceti does not oxidize glucose to gluconic acid in a medium containing both glucose and ethanol (which is not true in the absence of ethanol). Apparently the glucose is used for cell metabolism. It is not absolutely necessary to use a microorganism classified as A. suboxydans according to Frateur in order to avoid over-oxidation.

However, it must be assumed that the Acetobacter used commercially in concentrated alcohol vinegar fermentations approaches the properties of this group. For in­stance, it was possible to adapt a lyophilized strain of A. aceti (ATCC 15937) to the production of 13.5% alcohol vinegar.

This strain could not then be distinguished in its fermentation properties from a previously used A. suboxydans strain which had been slowly adapted to such high concentra­tions. Therefore, it seems appropriate to consider primarily investigations with A. suboxydans if one wishes to gather information on the carbohydrate metabolism of acetic acid bacteria during the vinegar fermentation.

King and Cheldelin (1952) were the first to point out that glucose may be oxidized directly or after phosphorylation. The metabolism of phosphorylated glucose may occur along 3 pathways- the glycolytic path of Embden-Meyerhof-Parnas, the pentose cycle, or the Entner-Doudoroff pathway.

The presence of enzymes of the EMP pathway does not mean that this glycolytic path actually operates since each of the enzymes of this scheme also par­ticipates in the pentose cycle or the Entner-Doudoroff pathway. The active participation of the pentose cycle and the presence of the corresponding enzymes in A, suboxydans have been demonstrated by Hauge et al. (1955).

A quantitative study of the pentose cycle as respiration mechanism in A. suboxydans has been done by Kitos et al. (1958) with aerated resting cells. In pure oxygen, 100 molecules of glucose were metabolized as follows- 28 molecules were oxidized to 2-ketogluconic acid, and of the remaining 72 molecules 63 entered the pentose cycle.

All of the CO₂ produced from glucose was derived from the pentose cycle. During oxidation with air the pentose cycle delivered the major portion of the CO₂; a minor portion of the oxidation of trioses followed a different path. The tricarboxylic acid cycle as well as the Entner-Doudoroff pathway seems to be of minor importance.

According to Kitos etal. (1958), acetate is not oxidized by A. suboxydans. If CH₃¹⁴COOH is added to respiring cells, only a negligible part of the ¹⁴C appears in respiratory CO₂, while 25% of it can be recovered in the lipid fraction of the cell wall. Kitos et al. (1957) showed that the latter occurs only in the presence of glucose.

Raghavendra Rao and Stokes (1953) found that A. suboxydans and A. melanogenum require sugar to initiate growth; but then ethanol may be used as an additional carbon source and incorporated into cell material although most of the ethanol is oxidized to acetic acid.

Razumovskaya and Belousova (1952) were the first to show that A. schutzenbachii is inhibited in its growth if CO₂ is removed from the air, particularly in mineral media in the presence of ethanol. Loitsyanskaya (1958) showed that A. schutzenbachii and A. aceti fix CO₂, particularly during growth, and that the uptake rate depends on the nitrogen source.

Inorganic nitrogen causes a higher CO₂ uptake than peptone or yeast extract. An increase in the CO₂ concentration in the air to 1% stimulates growth, while higher concentrations are inhibitory. Hromatka et al. (1962) confirmed that the growth of A. suboxydans is also inhibited if CO₂ is removed from the air.

A. suboxydans uses some CO₂ carbon for incorpora­tion into cell substance as shown by experiments with ¹⁴CO₂ so that approx­imately 0.1% of the cell carbon is derived from CO₂. A very small but measurable portion of acetic acid is derived from CO₂ metabolism. Claus et al. (1969) showed that dialyzed extracts of A. suboxydans catalyze the assimilation of ¹⁴CO₂ in the presence of phosphor enolpyruvate and divalent cations.

In contrast to A. suboxydans, strains of Acetobacter which belong to other groups (as classified by Frateur) prefer different paths of glucose metab­olism. King et al. (1956) found that A. pasteurianum which belongs to the oxydans group showed strong activity of the tricarboxylic acid cycle. In strains of the mesoxydans group, use of the EMP pathway has been shown, while for A. peroxydans the func­tion of the TCA cycle is still in doubt.

It is likely that the enzymes for the different pathways are always present in the Acetobacter cells and that repression of one or the other pathway for instance that of the TCA cycle is caused by particular conditions of growth, such as, for instance, a high acid concentration.

The oxidation of ethanol to acetic acid is not entirely dependent on cell multiplication. Even after cell growth stops, for instance, when a high concentration of acetic acid has been reached, the cells are capable of oxidizing ethanol to acetic acid for a certain period of time. After this period the cells die quickly and oxidation ceases.

In confirmation Vera and Wang (1977) found that the formation of acetic acid follows growth kinetics up to an acetic acid concentration of 3 g/100 ml, while a mixed kinetic model is required for concentrations between 4 and 7 g per 100 ml in which the formation of acetic acid depends both on growth rate and on actual cell concentration.

The exact connection of ethanol oxidation with the energy balance of cell metabolism, the mechanism which enables cells to withstand high concen­trations of acetic acid, and the reasons for the unusual properties of Acetobacter during vinegar fermentations have not yet been clarified.

Trickling Process of Vinegar Fermentation:

Older Trickling Processes:

Detailed descriptions of the history of the vinegar fermentation have been published by Haeseler (1955), Allgeier et al. (1974) and Connor and Allgeier (1976). The equipment used for the surface fermentations of the New Orleans method can only be seen in museums of factories.

The next step in the development of equipment for the fermentation of vinegar was the so-called trickling process in which the bacteria adhere to the large surface areas of a carrier material which is surrounded by air. The carrier material up to this date is still beechwood shavings, birch twigs, or corn cobs. Installations which operate on the Schutzenbach principle are today very rare. In such factories a number of smaller fermentors with approximately 2 m³ of carrier material are joined in a battery. Periodically mash is poured over the carrier material.

The mash has a relatively high concentration of acetic acid and such a low concentration of ethanol that the fermentation is completed after the liquid has passed 1, 2, or 3 times through the column of carrier material. The fermentors have openings in the lower portion through which air is sucked in because of the evolved heat.

This equipment can be enlarged by the addition of a collection tank from which the liquid is pumped continuously over the carrier column, with cooling of the liquid and with openings for the aspiration of air, or by blowing air in by means of ventilators. This scheme leads to the generator process. The process has some basic disadvantages in comparison with equipment using the submerged fermentation process.

It is impossible to distribute the liquid trickling over the carrier material so uniformly that the ethanol content is everywhere the same. There is, therefore, always the danger that the ethanol content will drop to zero at some points of the fermentor and lead to losses of fermentation capacity and to over-oxidation.

Mashes high in nutrient content and low in ethanol concentration aggra­vate this problem by formation of slimy deposits on the carrier material which may plug up the column. Therefore, generators filled with birch twigs are still in use today for the production of malt vinegar.

The non-homogeneity of the carrier column makes it impossible to distribute the air, which has been sucked in or blown in, evenly. This results in variations in temperature within the column which cannot be corrected. The interrup­tion of aeration affects the fermentation less rapidly than with the sub­merged system because of the reserves of air within the carrier material.

However, once the fermentation rate has been reduced, it cannot be as quickly reestablished as with the submerged process, since the recolonization of those parts of the column where the Acetobacter have died is a slow process. It is difficult to stop the fermentation completely; and switching from one raw material to another produces mixed types of vinegar for some time.

Yields are also lower than with the submerged process. Today quite a number of generators are still in operation which were designed and built by vinegar factories.

The Frings Generator:

The problem of automatic temperature control within the carrier column has been solved, as far as that is possible, by the Frings generator process. Mash is pumped from the collecting chamber of the fermentor (B) by pump (D) continuously through the heat exchanger (H) into the feed tank (I).

From there it returns to the collection chamber either indirectly through the distributing wheel (K) and the beechwood shavings (A) or directly through the overflow pipe (C). The column of shavings contains 3 contact thermometers (M) which activate valves in the feed tank (E) for automatic control of the circulating liquid. A contact thermometer (G) in the mash pipe controls the flow of cooling water to the heat exchanger (H) by means of a magnetic valve (F).

The mash is cooled in this fashion to 28°C and at very high fermentation rates to 26°C. The contact points of the thermometers which protrude into the column of shavings are set between 28° and 33°C. Air is blown into the carrier column from the bottom and escapes through the exit gas pipe (N).

When a residual ethanol content of 0.3 vol.% has been reached, the collection tank is largely emptied. It is subsequently refilled in 2 to 3 steps over a period of several days. There is a decided drop in rate of fermentation at the beginning of each fermentation period which is caused by the dying off of bacteria in the upper part of the carrier column due to the rapid change in alcohol and acetic acid concentration.

Once the newly added mash has been mixed with the vinegar which was absorbed by the shavings, favorable conditions (e.g., 8% acetic acid and 4% ethanol) are established for the production of vinegar with 11% acetic acid. The yield depends on the age of the shavings column. It is between 85 and 90%.

Oxygen utilization during the peak fermentation period is about 50%. The length of the fermentation period depends on the volume ratio of the column of shavings to the collection chamber. It is generally between 4 and 10 days. With beechwood shavings as carrier material, a productivity of 5 liters of acetic acid per m³ of shavings per day can be obtained.

The durability of such installations is such that at the end of 1980 Frings generators with capacities of 20, 40, and 60 m³ for the shavings column were still in operation. These generators were first delivered more than 25 years before. Total output of such installations is estimated at 400 million liters per year.

Treatment of Raw Vinegar:

The pH of the mash drops during the vinegar fermentation. With mashes from natural raw materials there is certain liability after completion of the fermentation with regard to the solubility of previously dissolved com­pounds. This liability is greater the smaller the drop in the pH during the fermentation.

For instance, insoluble materials will precipitate over a period of several months if a cider vinegar of 5% is produced from fresh apple juice soon after its alcoholic fermentation. But the process of precipitation is completed much faster if apple juice concentrate is fermented to 10% cider vinegar. A rest period of several months is, therefore, recommended for all kinds of vinegar produced from natural raw materials. This does not apply to distilled vinegar.

It is a well-recognized fact that the quality of vinegar improves on storage. To a lesser extent this is also true for distilled vinegar. Residual ethanol which forms esters plays a part in this improvement of quality. In general there is no difference in quality between vinegar produced by submerged fermentation and by trickling processes.

Poor quality of vinegar produced in generators usually indicates that the column of shavings has been used too long or that slime has built up on the shavings. Poor quality of vinegar from the submerged process indicates problems with the fermentation, such as a low concentration of acetic acid, a high concentration of nutrients, poor selection of bacteria, or poor aeration. Vinegar should have a clean aroma which is related to the raw material that has been used.

During storage of vinegar, precipitated materials settle. This facilitates the subsequent operations.

Refining:

Raw vinegar contains acetic acid bacteria which make it opaque. This is also true, although to a lesser extent, for vinegar produced in generators. A portion of the acetic acid bacteria settles during storage. Subsequent filtration is facilitated if the vinegar is fined with bentonite, but this is not absolutely essential. For this purpose an aqueous suspension of bentonite is added to the vinegar and mixed with it intimately. The suspended particles are permitted to settle for several hours, so the supernate is usually clear and easy to filter.

Filtration:

Filtration is carried out with suspensions of diatomaceous earth whether the vinegar has been stored for some time or not, and whether it has been fined or not. Filtration must remove all suspended material such as vinegar bacteria or the occasionally appearing “vinegar eels.” The latter occur often during production by the trickling process, but rarely during submerged fermentations. For the filtration of distilled vine­gar produced by the trickling process, simple plate and frame filters are also satisfactory.

A membrane ultrafiltration process has been developed by Ebner and Enenkel (1976) which eliminates the fining process and simplifies the filtration. This process permits the continuous and automatic production of a bacteria-free filtrate from unrefined vinegar from the submerged process.

At low hydrostatic pressure, the flux through the filter modules is so rapid that there is no concentration polarization at the surface of the membranes, which means that the pores of the filter always remain open. The vinegar, which contains only about 0.04% of filter aid, is re-circulated through the system and the amount of filtrate is replaced with raw vinegar. The filtrate is automatically checked for cloudiness and pumped off. The addition of raw vinegar is stopped after 4 weeks of continuous operation.

Filtration is continued until a concentrate is obtained which constitutes only about 0.5% of the filtered volume. The ultra-filter is now emptied, cleaned, and the process started again. This ecologically sound process requires only about 10 g of filter aid per 1000 liters of filtered vinegar.

It is assumed that this continuous method of ultrafiltration will replace other methods of filtration because of its considerable advantages. The labor-saving process may also eliminate the use of sterilizing filters directly before bottling and avoid the undesirable presence of bentonite and diatomaceous earth in the plant effluents.

Pasteurization and Addition of SO₂:

Vinegar is often pasteurized by short heating just before bottling, since entirely reliable methods of sterile filtration did not exist until very recent­ly. This is particularly true for fruit vinegars which are low in total concentration and high in nutrient content. In some countries, sulfiting of vinegar up to 50 mg SO₂ per liter is permitted. In this case, sulfiting replaces pasteurization. However, it is necessary to add sulfite immediately before bottling, since SO₂ oxidizes rapidly in vinegar with high extract values and thereby loses its effectiveness.

Bottling and Keeping Quality:

Vinegar for domestic use is sold exclusively in bottles of glass, poly vinyl- chloride, or polyethylene with plastic screw top closures. The closure must be air tight. For institutional consumers, such as restaurants, vinegar is sold in 5 to 25 liter plastic containers. Industrial users of vinegar use tank trucks of stainless steel.

The keeping quality of bottled vinegar must be guaranteed. This presents no problems with distilled vinegar. However, vinegar of low “total concen­tration” and high extract value is more readily subject to spoilage. The best guarantees for a long shelf life are a sufficient storage period to complete the precipitation of cloud-forming materials, ultrafiltration, or fining followed by filtration, absence of bacteria, and tight closures of the bottles. Pasteur­ization provides additional protection but can cause new problems with after-precipitation due to the heating. Heavy metals can also cause prob­lems and their presence should be eliminated.

Concentration:

Vinegar is used in large quantities by the canning and other food industries. For some purposes, vinegar with an acetic acid con­centration higher than can be obtained by fermentation is required. The producer of vinegar also prefers a product with high acetic acid content because of the savings in freight and in storage capacity.

Vinegar of 20 to 30% acetic acid is produced by freezing of vinegar with 10 to 13% acetic acid. The ice which forms during this process contains very little acetic acid. It is removed by centrifuging, leaving vinegar with the desired high concentration of acetic acid. The ice can be thawed and reused for the preparation of mashes. Unfortunately, the process of concentration is rather costly.

It is advantageous to reduce the cost of the freezing process by producing vinegar with the highest possible concentration during the fermentation. It is possible to produce vinegar with up to 15 g/100 ml of acid in the Acetator. This limit is set by the ability of the Acetobacter to multiply. Recently a 2-step production sequence has been developed which permits the manufacture of vinegar at higher percentages of acetic acid.

At a certain stage of the fermentation the “total concentra­tion” of a fermentor is increased by the addition of ethanol. A portion of the contents is pumped into a second fermentor where the process of acidifica­tion is completed in a second fermentation stage. The remaining portion of the fermentor contents of the first stage is replenished with fresh mash until its “total concentration” has dropped to the original value.

This per­mits the acetic acid bacteria to multiply again so that a sufficiently large number of bacteria are again available for the second stage. The first fer­mentor is thus used for semi-continuous fermentations at varying “total concentrations.” The second fermentor completes the fermentation as a batch process.

It is then emptied and refilled from the first fermentor. At the end of 1979 the first unit (Acetator 1200) was in commercial operation and produced vinegar with 18.5% acetic acid per 100 ml with this procedure. It is anticipated that the acetic acid concentration can be further increased.