Special Behavior of Acetobacter during Vinegar Fermentation | Industrial Microbiology

In this article we will discuss about the special behavior of acetobactor during vinegar fermentation. The special behavior are:- 1. Sen­sitivity toward Lack of Oxygen 2. Sensitivity to Lack of Ethanol 3. Sensitivity to Changes in Temperature 4. Specific Growth Rate 5. Sensitivity to Changes in Concentration and 6. Over-Oxidation.

Sen­sitivity toward Lack of Oxygen:

During commercial use of many aerobic microorganisms, it is common practice to permit the inoculum to remain in a fermentor for several days without aeration before inoculation into the main fermentor. This is also true for the oxidation of sorbitol to sorbose by A. suboxydans. However, this procedure is unthinkable with vinegar fermentation.

Even today the work of Hromatka and Ebner (1949) forms the basis of all commercial submerged vinegar fermentations. According to these authors it is customary to express productivity by the Eta value, that is, the increase in acetic acid in g per 100 ml per 24 hr.

If the log of the Eta value is plotted against time, a straight line is obtained during exponential growth of the bacteria. Its slope is a function of the growth rate. A change of the slope or any curvature of the line indicates a change in the conditions of the fer­mentation.

The use of Acetobacter strains which are adapted to the highest “total concentration” and the highest acetic acid concentration occurring in an experiment is essential if one wishes to draw valid conclusions from experimental work. One cannot generalize from the results if these con­ditions are not fulfilled.

Extensive tests have been conducted by Hromatka et al. (1951) and Hromatka and Exner (1962) on the damage to Acetobacter due to an inter­ruption of aeration as a function of several variables. The percentage of killed bacteria was calculated from the change of the log Eta curve. Table 18.1 shows the effect of “total concentration,” acetic acid concentration, ethanol concentration, rate of fermentation, and length of interruption of aeration.

First one notices a dependence of damage on “total concentration.” At a “total concentration” of 5% an interruption of aeration from 2 to 8 min leads to the same damage as an interruption from 15 to 60 sec at a “total concentration” of 11 -12%. The degree of damage is between 10 and 100%.

It increases sharply with increasing “total concentration” for equal periods of interruption of aeration. At constant total concentration, damage in­creases with increasing concentrations of acetic acid and with an increasing fermentation rate which is proportional to the number of bacteria.

At Eta = 1 there are about 50 x 10⁹ cells per liter, corresponding to about 20 mg/liter of dry substance. Commercial vinegar fermentations have an Eta = 4, that is, they have about 80 mg/liter of dry bacterial solids. Today up to 150 g of acetic acid can be produced per liter, which means that only 0.6 mg of bacterial solids is needed for each g of final product.

The productivity of commercial vinegar fermentation is kept at about 1.7 g acetic acid per hr per liter. Higher values can be obtained but pre­suppose higher additions of nutrients which are costly and which may interfere with the maintenance of the strain. Vera and Wang (1977) found in laboratory experiments that the rate of a continuous fermentation at a “total concentration” of 7% could be increased by recycling bacterial cells to the fermentor.

The organisms were recovered by ultrafiltration, but the death rate of the cells was so high that 50% of the cells in the fermentor were dead in spite of the aeration with oxygen during separation and reinoculation of the cells. Therefore, it seems that the high rate of productivity, 11.5 g acetic acid per liter per hr, cannot yet be attained commercially.

Experiments with ¹⁴C-ethanol and A. rancens showed that the uptake of oxygen corresponded to the theoretical value of the oxidation of ethanol. The cell material in grams produced per gram atom of oxygen which was taken up is only about one-tenth that of other microorganisms. It was concluded that the cell yield based on ATP was very small.

Meyrath (1973) considered the reason for the extreme sensitivity of Acetobacter during the fermentation to be a lack of oxygen. He assumes that Acetobacter has high apyrase activity so that ATP which accumulates during the oxidation of ethanol is rapidly hydrolyzed and therefore only poorly available for other metabolic activities of the cell. When aeration is interrupted, the ATP pool disappears so quickly that the cell does not have the ability to adjust to the changed conditions.

Experiments to substantiate this theory have not been published. Also, it does not account for the dependence of the degree of cell damage on acetic acid concentration and “total concentration.” It may be assumed that the ATP pool is required to prevent the entry of ethanol or acetic acid into the cell interior.

The effect of air bubble size on the achievement of maximal Eta values has been studied by Hromatka and Ebner (1951). At a “total concentration” of 9% in submerged vinegar fermentation, decreasing bubble size leads to higher Eta values. The utilization of oxygen in the airstream can be raised to the point where only 4 vol.% residual oxygen remains in the effluent gas without adverse effects on the fermentation. Such a high utilization of oxygen, up to 80%, can also be achieved commercially. A sparing use of air is quite important because of the volatility of ethanol and acetic acid. The use of pure oxygen or highly oxygen-enriched air damages the acetic acid bacteria.

Sensitivity to Lack of Ethanol:

Acetic acid bacteria are damaged if vinegar fermentation is carried on to the point where all of the ethanol has been oxidized and if the addition of fresh ethanol-containing mash is de­layed beyond that point. This is analogous to the damage resulting from an interruption of the oxygen supply and depends also primarily on “total concentration” and the duration of the interruption. The interruption of the ethanol supply can, therefore, cause severe damage to the bacteria.

Sensitivity to Changes in Temperature:

The fermentation is not af­fected if the temperature is cycled every 2 hr between 32° and 26°C. Howev­er, the maximal Eta value decreases markedly if the change in temperature takes place every 30 min. If cooling is stopped during vinegar fermentation, the temperature rises higher and higher. Damage to the cells of Acetobacter increases with the duration of the interruption of cooling, with higher temperatures, and with higher concentrations of acetic acid. Ultimately the fermentation ceases.

Specific Growth Rate:

The specific growth rates were calculated from the slopes of the log-Eta curves for fermentations which were carried out with varying “total concentrations” and at varying temperatures. The values are shown in Table 18.2. For such semi-continuous fermentations the specific growth rate did not depend on the acetic acid concentration but it decreased rapidly at higher “total concentrations.” At the same time the optimal fermentation temperature decreased with increasing “total concentration.”

There is a trend toward higher fermentation temperatures in commer­cial fermentations in order to permit the reuse of water from evaporative coolers in warmer climates. The trend toward higher “total concentrations,” which is also present in order to save nutrients, storage, and transportation costs, makes the solution of this problem more difficult. But fermentation temperatures between 31° and 33°C at a total concentration of 12% are possible.

In a continuous culture with a “total concentration” of 12% in the feed, the dilution rate was kept constant until a constant concentration of ethanol arid acetic acid was reached in the effluent. This showed that the specific growth rate of 0.027 hr^-1 at 7.5 g/100 ml acetic acid and 4.5 vol.% ethanol decreased in linear fashion to a growth rate of 0.006 hr^-1 at 11.0 g acetic acid per 100 ml and 1.0 vol.% ethanol.

It should be mentioned that the values obtained with continuous fermentations, that is, with con­stant acetic acid and ethanol concentrations, are less favorable than those obtained under semi-continuous conditions with variable acetic acid and ethanol concentrations.

Additionally, there is a decrease in the specific growth rate with decreasing ethanol concentrations. Therefore, it makes sense to run commercial continuous fermentations, which have to be car­ried out with low alcohol concentrations, only up to a “total concentration” between 9 and 10%.

Vera and Wang (1977) carried out batch fermentations with A. suboxydans at a “total concentration” of 7.5%. Yeast extract was used as nutrient. The value for the specific growth rate of 0.30 hr¹ agrees with that of Hromatka et al. (1953).

The specific growth rate of A. aceti rises sharply with the rate of aeration at an acetic acid concentration of 2%. At an increased acetic acid concentration of 5%, the dependence of the growth rate on aeration becomes rather slight. The same dependence could be observed for the oxidation capacity of the Acetobacter cells. Mori et al. (1970) did batch experiments with A. rancens at a “total concentration” of 7%, starting with an acetic acid concentration of 2 g/100 ml.

Acetic acid formation paralleled cell growth. In confirmation of Alian et al. (1963), the specific growth rate depended on oxygen concentration at low acetic acid concentrations but not at higher concentrations. Increasing concentrations of acetic acid inhibited the uptake of oxygen by the bacterial cells, which was explained by inhibi­tion of the cytochrome system. Most likely this is really due to insufficient adaptation of the Acetobacter strain to acetic acid concentrations between 5 and 7%.

Mori and Terui (1972A) worked with A. rancens in continuous fermenta­tions. The specific growth rate was strongly dependent on the acetic acid and ethanol concentrations. It rose from 0.05 hr^-1 at 6 g/100 ml acetic acid to 0.4 hr^-1 at 2.5 g/100 ml acetic acid. They also found a strong dependence of the specific product formation on the acetic acid concentra­tion at a “total concentration” of 7%. Maximum specific product formation was 15 g/g-hr at 3.0 g/100 ml acetic acid, and dropped in approximately linear fashion to 2 g/g-hr at 7.0 g/100 ml acetic acid.

In contrast to these results Hromatka and Ebner (1949) found a specific product formation of 21 g/g-hr in semi-continuous tests and at a “total concentration” of 10%. Specific product formation was independent of the concentration of acetic acid between 4.5 and 7.2 g per 100 ml.

The explana­tion may be in part that their strain was better adapted to industrial conditions. This is supported by the fact that the respiratory activity was high, namely 7750 ml O₂ per g of bacterial dry substance, and independent of acetic acid concentration between 4.5 and 7.2 g per 100 ml.

The strain used by Mori and Terui (1972A) had a maximal respiratory activity of only 3160 ml O₂/g-hr with strong dependence on the concentration of acetic acid. Again, it appears that a change in process conditions (continuous vs. semi-continuous) has influenced the results.

Vera and Wang (1977) found a maximal value for product formation of only 5.5 g/g-hr in a continuous fermentation and at a “total concentration” of 7%. This shows the effect of the particular strain and of the process technique.

Mori et al. (1972) reported an inhibition of the specific growth rate as a function of the starting concentration of acetic acid for A. rancens. However, successive inoculations of these bacteria during the logarithmic growth phase permitted adaptation and the disappearance of this dependence.

According to Hromatka and Ebner (1959), ethanol concentrations above 6 vol.% should be avoided in commercial fermentations since such higher ethanol concentrations reduce the specific growth rate.

Mori and Terui (1972C) attempted to determine the effect of the ethanol concentration at the beginning of the fermentation on the specific growth rate of A. rancens. This included experimeats with starting concentrations of up to 10 vol.% ethanol, but none of the experiments gave a higher yield of acetic acid than 5.5 g 100 ml since the strain of Acetobacter was only adapted to a “total concentration” of 6%. That means that fermentations started at higher “total concentrations” became stuck.

This was primarily due to lack of adaptation to higher “total concentrations” and not —as the authors concluded—to the starting concentration of ethanol. It is clear that ethanol as well as acetic acid and the “total concentration” influence cell metabolism. But we are far from a detailed understanding since these concentrations cannot be varied independently of each other.

Sensitivity to Changes in Concentration:

Commercial vinegar fermen­tations are generally conducted as semi-continuous fermentations. Shortly before the concentration of ethanol reaches zero, about 40% of the fermentor contents are pumped out of the fermentor which may contain at this point 13.3 g/100 ml acetic acid and 0.2 vol.% ethanol.

Without interruption of the aeration and at a constant temperature of the fermen­tation, the fermentor is refilled with new mash containing, for instance, 1.0 g/100 ml acetic acid and 13.0 vol.% ethanol. Strong concentration gradients of acetic acid and ethanol are formed locally if the fresh mash is merely pumped through a pipe ending near the wall of the fermentor.

The cells of Acetobacter are unavoidably exposed to this concentration gradient and damaged to the point where the cells die. The new fermentation starts then, for instance, with only 10% of the number of bacteria of the preceding fermentation period instead of the 60% which would have been otherwise available. The dead cells cause considerable foaming. In practice, damage to the cells is avoided by adding fresh mash in such a way that it is rapidly mixed with the fermentor contents by the aerator.

There is no lag phase at all if the time for pumping out of the fermentor and the method of adding fresh mash is chosen correctly not even with very high “total concentrations.” This presupposes however, that the “total concentration” of the new mash does not differ greatly from the “total concentration” in the fermentor. If this is not the case, then the changes in concentration will have a detrimental effect on the fermentation.

Over-Oxidation:

Over-oxidation is the undesirable oxidation of acetic acid to carbon dioxide and water. Continued aeration in the absence of ethanol may trigger a change in cell metabolism which occurs more rapidly at lower “total concentrations” than at higher ones. Further, over-oxidation is always correlated with bacterial growth; and the reaction is faster at lower concentrations.

It is well known in industrial practice that over-oxi­dation once started can proceed simultaneously with the oxidation of eth­anol to acetic acid. It can be recognized by lower yields which are caused, by the drop in “total concentration.” An abundance of nutrients in the mash favors over-oxidation. According to Hitschmann and Meyrath (1972), over-oxidation is stimulated by the addition of 10^-6 molar DPN or 10^-3 molar pyruvate.

Divies et al. (1969) showed that addition of succinic acid causes over-oxidation. On the basis of extensive experiments, Hitschmann and Meyrath stated that over-oxidation takes place only at acetic acid concen­trations below 6%, and that it does not proceed at all in the presence of ethanol. This is not in accord with the experience gained in industrial practice.

It is most important to make sure that the fermentation does not proceed until ethanol is used up if one wishes to avoid over-oxidation. In the sub­merged process this can be achieved by appropriate automation. Natural mashes which are low in total concentration and high in nutrients, such as apple cider, are best fermented continuously but with automatic control of the ethanol concentration. Distilled vinegar should be produced semi-continuously and preferably at high “total concentrations” and at low concen­trations of nutrients.

It is more difficult to avoid over-oxidation in trickle fermentations. In such fermentations the bacterial cells adhere to a carrier, usually beechwood shavings which are aerated and through which the fermentor liquid trick­les. The unavoidable formation of clumps (due in part to the formation of slime), uneven aeration, and uneven development of heat often lead to zones in which ethanol has been completely used up, while other parts still contain a sufficient concentration. This alone can cause over-oxidation. Once over-oxidation has been recognized in a commercial fermentor, it becomes necessary to stop the fermentation and to clean and sterilize the fermentor and all tanks, lines, and pumps ahead of the fermentor.