Vinegar (Acetic Acid) Production

2.1. Vinegar (Acetic Acid) Production

Vinegar fermentation enjoys the reputation of being one of the oldest known fermentative pro- cedures to the mankind; and historically, it was obtained as an unwanted spoilage of wine as a natural event. In true sense, vinegar (acetic acid) is nothing but a fermentation-derived food product essen- tially comprising of not less than 4 g of acetic acid per 100 ml (i.e., 4% w/v) ; and besides, it inherently possesses an unique special flavour characteristic features, which evidently protects and provides a clear-cut edge over the artificial direct products obtained via pure synthetic routes in flavour, mellow- taste, and above all the competition.

The production of vinegar on commercial scale may be accomplished by several time-tested procedures as stated below, namely :

(i) Traditional method, (ii) Aerobic fermentation process, (iii) Orleans process, (iv) Packed-generator process,

(v) Trickling generator, and (vi) Submerged fermentor. The aforementioned methods shall now be described individually in the sections that follows :

2.1.1. Traditional Method

In this particular instance the production of vinegar essentially requires two different modes of fermentations, namely :

(a) Utilizing a specific yeast — i.e., to produce ethanol from cane-sugar (sucrose — obtained from fruits, malt etc.), and

(b) Utilizing Acetobacter species — i.e., to carry out the oxidation of ethanol via acetaldehyde to ultimately acetic acid.

In usual practice, the substrate for the first-stage of fermentation may be provided by almost a host of naturally occurring starting materials, such as : ripe and sweet fruits e.g., apples, pears, plums, grapes, berries etc.; honey ; wine, malt and the like. It is, however, pertinent to mention here that mostly the modern trends in the production of vinegar extensively make use of apple-cider and wine, in the presence of two predominantly employed fermentation organisms, namely :

(i) Saccharomyces cerevisiae ; and (ii) Saccharomyces cerevisiae var. ellipsoideus. The introduction of pressurized sulphur dioxide (SO 2 ) gas into the fermentative broth is an

absolute necessity in order to control as well as monitor the bacterial growth effectively.

CAUTION : It is quite important to remove the inducted SO 2 -the gas from the fermentative broth either by aeration or by other suitable means just prior to the oxidation of generated alcohol further to the desired vinegar (i.e., acetic acid).

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Once the alcohol-fermentation is fully accomplished, the resulting mass of yeast cells along with various other sedimented solid residues are permitted to settle down as completely as possible, and the supernatant fermented alcoholic broth is withdrawn carefully.

Finally, the vinegar production is initiated from the above ‘alcoholic broth’ by following the various steps detailed below sequentially :

(i) alcohol content adjusted between 10-13%, (ii) vinegar in small quantity added to enhance the acidity of the broth, and (iii) alternatively, Acetobacter cells may also be incorporated in such processes that would

require inoculation.

2.1.2. Aerobic Fermentation Process

In general, the microbial oxidation of ethanol to acetic acid is nothing but an aerobic fermenta- tion process which essentially demands an excessive oxygen (O 2 ) requirement.

Salient Features : The various salient features of an aerobic fermentation process are as given below :

(1) Acetobacter cells are usually of a highly aerobic nature; and, therefore, any circumstantial development of an ‘oxygen deficiency’ in the fermentation medium would directly affect their normal activities.

(2) Oxidative conversion of ‘ethanol’ to ‘acetic acid’ per se needs an enormous quantum of oxygen which is evidenced by the following example :

1 L of Ethanol when converted to Acetic Acid requires 552 g of oxygen. (3) Evolution of Heat Energy—Oxidation of ethanol to acetic acid evolves a considerable amount

of heat energy that should ordinarily be dissipated from the fermentative broth in due course, example :

4.5 L ( ≡ 1 Gallon) of Ethanol when converted to Acetic Acid liberates approximately

30, 250 BTUs (i.e., British Thermal Units). (4) Interestingly, the more latest developments in process design have adequately provided enough

manipulative measures and controls with regard to the supply of sufficient oxygen to the fermentation broth, and also sufficient arrangement for dissipation of heat either through circulation of chilled air or chilled-water coils in the bioreactors.

In the age-old vinegar fermentation processes, besides those wherein a fortuitous souring of wine took place, were duly obtained by the introduction of an ‘alcoholic liquid’, either in the form of fermented fruit juice or wine, in shallow open vessel. The exposed air adequately caused due inoculation of the

alcoholic solution by Acetobacter organism, whereas the ensuing large surface area catered for the much needed aeration. It has been duly observed that during an extended incubation period, the apparent growth of a ‘bacterial scum’ essentially comprising of the alcohol-oxidizing bacteria got eventually developed on the surface of the liquid. In addition to the above, certain nematodes* very specific to vinegar usually termed as vinegar eels, also found to have multiplied in these vessels both progressively and aggressively.

* A class of the phylum. Nemathelmenthes that includes the true roundworms or threadworm, many species of which are parasitic in nature.

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2.1.3. Orleans Process

Orleans, in France, first and foremost worked out gradually the ‘production of vinegar’ from the original vat fermentation to the rather more sophisticated ‘Orleans Process’ particularly for wine vinegars. Importantly, the said methodoloy is still being adopted in certain units in France to produce

fine and top quality table vinegars.

In fact, the Orleans process, essentially makes use of large fermentation vessels or barrels or casks placed either in vertical or in horizontal positions. A number of holes made in the walls of these fermentative vessels, strategically located above the ‘level of liquid’, allow the cross-sectional move-

ment of air in them adequately. Consequently, the Acetobacter organism (bacteria) generate a substan- tial quantum of ‘slime’ which eventually grow into a layer or filu or vinegar mother, right upon the surface of the alcoholic broth, carefully supported at the surface of the floating raft made up of wooden grating. It has been duly established that the culture cells in the slime layer are further placed together in position by the corresponding cellulosic strands caused by one of the Acetobacter species known as Acetobacter xylinum. However, in actual practice, a natural inoculum of Acetobacter cells is allowed to pile up either in the casks, or a portion of the ‘vinegar mother’, which is essentially transferred from the previous casks to boost and promote the process of ‘acetification’.

It is pertinent to state here that the phenomenon of oxidation responsible for the conversion of ethanol to acetic acid via this route is rather slow and sluggish. It usually demands an incubation span varying between 1 to 3 months at a stretch, and during this fermentative duration a plethora of other non- Acetobacter organisms are also rendered active, thereby giving rise to organic acids viz., lactic acid, propionic acid that eventually as their respective ‘esters’ do impart/induce an exceptionally unique and pleasant fruity flavour and aroma to the ultimate vinegar thus produced.

Note : Nevertheless, long incubation period normally gives rise to a relatively higher loss of ethanol via evaporation and over oxidation.

2.1.4. Packed-Generator Process

Packed-generator process was developed initially by Schuzenbach, a German scientist, whereby the microbial oxidation of ethanol to acetic acid could be accomplished efficiently and adequately by a rather more rapid methodology. However, it is also known as the ‘quick method’ and the ‘trickle method’.

In the packed-generator process the fermentation vessel i.e., the ‘vinegar generator’ usually consists of a large vertical tank, that could be either open or closed at the top, and loosely packed with beechwood* shavings, small twigs, evenly cut corn cobs, bamboo-stick bundles, or similar other pack- ing substances. Consequently, a bacterial film made up of mixed Acetobacter species, other than those for the Orleans Process (section 4.2.1.3), eventually grows upon the exposed surface of the supporting agent. At this stage, the alcoholic broth (obtained separately by many known methods) is introduced in small lots at intervals from the top of the packed generator, which trickles down gradually via the packing medium (support) in order to make available sufficient contact of the alcohol with the bacterial cells ; and this ultimately leads to the oxidation of alcohol into acetic acid. In usual practice, the packed

generators (vinegar generators) are strategically provided with air-inlet devices positioned very much near the bottom to permit air to move-up through the generator, because the ‘rise-of-air’** gets acceler-

* A tree with smooth bark and glossy leaves. ** Whenever fermentation takes place, CO 2 is evolved, which generates heat ; and the hot-air being lighter

than cold-air has a tendency to move up and escape through the vents at the top of the generator.

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ated progressively by virtue of the ‘heat’ being generated during the process of fermentation. Precisely, it is absolutely important and necessary to control and monitor the evolution of heat so as to allow the fermentative process to continue without any adverse/harmful effect ; and it may be accomplished by any one of the following procedures, namely :

• rate of addition of added ‘alcoholic broths’, • temperature of the added ‘alcoholic broths’,

• cooling-coils provided in sophisticated generators, and • chilling of incoming or recycled ‘alcoholic broths’ Salient Features : The salient features of packed-generator process are enumerated below : (1) Alcoholic broth is either adequately recycled through the same fermentor until the entire

ethanol content gets ‘oxidized’ completely, or it is passed through the several generators kept in series (i.e., connected to one another) in order to oxidize the alcohol successively at each of the connected generators.

(2) These generators (connected in series) invariably yield an appreciable concentration of acetic acid even upto 15% (v/v) ; and, therefore, are being employed extensively and specifically for the genuine production of ‘white table vinegar’ across the globe.

(3) Modern day recirculating packed-generators are duly stuffed with specially sized beechwood shavings (air-dried), 2 × 1.25 inches, and not-so-tightly rolled, that may take up approximately 2000 cubic feet of the said material.

(4) Satisfactory accumulation of ‘bacterial film’ upon the supporting agent in the ‘packed gen- erator’ would render it operational effectively from several to many months at a stretch or until an unavoidable serious contamination caused due to the slime-forming bacterium, Acetobacter xylinum becomes prevalent.

2.A. Fermentor Designs

The broad spectrum of fermentor designs involving the sustained performance of the submerged fermentation approach for ‘vinegar fermentation’ has duly acclaimed a reasonably widely recognized

development in process technology. In fact, the present day submerged fementations being utilized, extensively in the large-scale production of ‘table vinegars’ and are entirely based upon the well-elabo- rated intensive aeration studies performed on antibiotic fermentations both during and after World War II.

Importantly, these submerged fermentations make use of two entirely different ‘fermentor de-

signs’ invariably termed as : (a) acetator ; and (b) cavitator.

Acetator : Fig. 4.1 illustrates the schematic diagram of the Fring’s submerged culture acetator, wherein the following essential components are as given below :

1 = Aerator motor ;

2 and 3 = Aerator assembly ;

4 = Heat-exchange coils ;

5 = Baffles ;

6 = Cooling water valve ;

7 = Mechanical defoamer ; and

8 = Waste-air stack.

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Fig. 4.1. Schematic Diagram of Fring’s Submerged Culture Acetator. [Adapted from : Casida LE, Industrial Microbiology, 2004]

Acetator normally operates as a semi-batch-mode ; and, however, with certain modifications may now be utilized as a continuous-flow-mode. An effective ‘aeration’ in the Acetator may be adequately accomplished with the aid of a fast-rotating ceramic disc strategically placed over an air

nozzle to produce extremely fine dispersed air-bubbles, having consequent solution of O 2 in liquid-phase (i.e., culture medium).

At the initial stage, the Acetator is fed with a mixture of fresh stock of alcoholic solution together with actively oxidizing vinegar obtained from a simultaneously operating fermentor. It is a common practice to determine the alcoholic and acid contents of the mixed alcohol stock with vinegar ; and, if required, necessary adjustments are duly made to achieve adequate starting concentrations. Ideally, the acid content should vary between 1-1.5%, and an alcohol content between 4.5-10.8%. It is pertinent to mention here that start up alcohol concentration seems to be too high for sustaining the continuous operation ; and, therefore, the fermentation broth is neither withdrawn for harvest, nor is fresh alcohol stock added, till such time when the fermentation has adequately gone through to accomplish an alcohol content nearly 0.5%. Fresh alcohol stock solution is further added automatically and the completed fermentation broth is withdrawn from the Acetator accordingly.

It is absolutely important to maintain the actual alcohol content of the ‘harvested broths’ to stand nearly at 0.3% because of the following two reasons, namely :

(a) strength of alcohol content less than 0.3% shall cause harm to the organism, and (b) alcohol content < 0.3% also gives rise to excessive foaming (which may be controlled by the

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Cavitator : Just like the Acetator, the Cavitator is also an ‘automated device’ that essentially caters for both charging with alcoholic solution and discharging of completed fermentation broth. Because this fermentator i.e., Cavitator is rated as highly efficient and capable of producing acetic acid at a much faster speed and pace, it is appreciably smaller in size and dimension in comparison to the correspond- ing packed vinegar generators (see section 2.1.4).

Advantages : The various distinct advantages of the Cavitator are as enumerated under : (1) It neither forms ‘slime’ nor the ‘vinegar eel’.

(2) Since, the rate of O 2 -removal from the culture medium by the Acetobactor cells is so large and predominant that neither the ‘oxidative browning’ of the ‘apple cider’ nor of ‘wine mash’ ever occurs.

(3) A small quantum of ‘residual alcohol’ is invariably left behind in the vinegar as it is recov- ered from the fermentor ; and, therefore, in this particular process the vinegar is usually stored to permit broth ‘aging’ and subsequent ‘microbial depletion’ of this residual alcohol.

(4) Superior Vinegar. In actual practice, the vinegar removed from the Cavitator prominently contains a host of ‘suspended microorganisms’, quite unlike the ‘packed vinegar genera- tors’ (wherein the microorganisms are intimately attached to the beechwood shavings), and during storage of the vinegar these microorganisms specifically oxidize the residual alcohol and simultaneously attribute splendid additional ‘pleasing alterations’ both in the overall aroma and taste to yield a ‘superior vinegar’.

Methodology : The various steps involved are as follows : (1) It is a complete continuous fermentation process. (2) It essentially comprises of five vital stages, namely :

Stage-1 : The cavitation force developed by the rotor at the time air-bubbles are formed. Stage-2 : The air-diffusion takes place during the passage of the bubbles to the surface. Stage-3 : An advanced stage whereby the air-diffusion at the point at which the bubbles

usually burst at the surface. Stage-4 : At this stage, the surface-aeration caused by the atmospheric pressure on account

of the constant change of surface exposed to atmosphere both by the agitation and circulation of the liquid very much within the tank.

Stage-5 : This is the final stage whereby the air induced into the liquid by a vortex action

as the liquid flows over the cone and through the draft tube. All the aforesaid five distinct and critical stages have been duly shown in the

following Fig. 4.2.

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Air under atmospheric pressure introduced to

hollow shaft and rotor

S tage4 S urfac e aeration caus ed by atm ospheric pres sure bec aus e

Stage3 of the constant change of

Air diffusion at the point surface exposed to atm osphere by the agitation and circ ulation

at which the bubbles of teh liquid w ithin the tank

burst at the surface Mash in

Vinegar out Cooling coil

Baffile Stage 5

Stage 2 Air induced into the liquid by

Air diffusion during the a vortex action as the liquid

passage of the bubbles flows over the cone and

to the surface through the draft tube

Stage 1 Draft tube The cavitation force developed

by the rotor at the time air bubbles are formed

Fig. 4.2. Schematic Diagram of the Cavitator. [Adopted from : Casida LE, Industrial Microbiology, 2004]

(3) Diammonium hydrogen phosphate [(NH 4 ) 2 HPO 4 ], as an additional nutrient supplement is judiciously incorporated both at start-up and in the course of continuous operation. Eventu- ally, the progress acquired in, fermentation is estimated periodically by finding the ‘acid’ and ‘alcohol’ contents of the broths. However, it is quite important to induct fresh-air into the fermentors, during this period, along with the recirculated air of the fermentor so as to

afford just sufficient O 2 to fulfil the combined demands of the prevailing microbial respira- tion as well as alcohol oxidation. Besides, adequate care needs to be taken with respect to the feed-rate of the fresh alcohol stock to maintain the required and stipulated low-alcohol- content in the finished vinegar i.e., the fermented broth.

2.1.5. Trickling Generator

The trickling generator still holds a coveted position and extensively employed in vinegar pro- duction, as depicted in Fig. 4.3. In reality, the ‘wooden bioreactor’ has an overall capacity approxi- mately 60 m 3 and is usually packed with beechwood shavings.

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Fig. 4.3. Diagram of a Trickling Generator for Acetic Acid.

In actual practice, the starting material is adequately sprayed over the surface and trickles gradu- ally through the shavings containing organisms into a basin located in the bottom, where the partially converted solution (i.e., alcohol converted to acetic acid), as shown below, is duly chilled and pumped again to the top of the generator.

2 × 3 AT P + H O 2

+ T P = A d en o sin e

trip h o sp ha te ;

N A D (P )H 2 NADP

3 C H C H (O H ) 2 CH COOH 3

E th an o l

A c etald eh y d e

A c etald eh y d e

A c etic A c id

H y d rate

Oxidation of Ethanol to Acetic Acid.

It has been duly observed that the ‘trickling generator’ process is capable of converting be- tween 88-90% of total ethanol added into acetic acid ; whereas, the rest of ethanol (10-12%) gets either used up for the primary metabolism or released along with the waste air. Interestingly, the prevailing temperature in the upper portion of the trickling generator stands at about 29°C ; whereas, in the lower segment it remains at 35°C (hence, it is partially cooled and recirculated from the top).

In nutshell, it takes almost 72 hours to yield 12% acetic acid by this methodology.

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2.1.6. Submerged Fermentor

The submerged fermentor first and foremost makes use of either fruit wines or special mashes having reasonably rather low concentrations of ethanol. Indeed with such a low-yielding methodology/ technique the aeration was not absolutely critical and important. However, as on date the emergence and recognition of high-yielding methodologies that essentially yield 13% acetic acid in amounts ranging up

to 50 m 3 , predominantly demands highly controlled and regulated means of aeration. Interestingly, the fermentors very much look alike other bioreactors (see chapter 3). The fementation

tanks are usually made up of stainless steel that have an arrangement of stirring at the bottom. The aeration assembly comprises of a suction rotor having the incoming air flowing down via a SS pipe located at the top of the fermentor. In order to dissipate the elevated temperature during fermentation process efficient heat-exchangers are engaged (through which cold water is passed constantly) to con- trol the temperature around 25 ± 2°C ; besides, mechanical foam eliminators should be pressed into service to arrest the nuisance caused due to foaming.

Methodology : The various steps involved in the production of acetic acid (household vinegar)

by the submerged fermentor process are as stated under : (1) Household vinegar (13% acetic acid) is usually produced in a semicontinuous, absolutely

automatic process, in an environment (atmosphere) of adequate aeration and constant me- chanical stirring together with a start-up material which essentially composed of approxi- mately 7-10% acetic acid plus 5% ethanol.

(2) The concentration of ethanol is determined continuously by suitable method during the fermentative process ; and when the concentration dips down to a level varying between 0.05 and 0.3% achievable within a span of 36 hours. At this particular juncture, about 50-60% of the fermented solution is removed and duly replaced with a new lot of ‘mash’ containing 0 to 2% acetic acid and 10-15% ethanol so as to recharge the fermentor accordingly.

It has been duly established by researchers that one may obtain upto 98% yields at 40°C via fully continuous processes, as shown in Fig. 4.4.

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Mechanical foam separator FUNDAFOM*

Nozzle for

Electro motor

effluent gas Separator cone nest.

Air-suction pipe Substrate feed-line

Reactor Vessel Heat exchanger plates

Central suction tube

EFFIGAS* turbine

Bearing Harvest-valve

Electro motor

Fig. 4.4. Diagrammatic Sketch of Submerged Fermentor for Acetic-Acid Production. [Adopted from : Crueger and Crueger : Biotechnology, 2004]

Advantages Over Trickling Generator : The advantages score of the submerged process

upon the trickling generator are as described below : (1) The production rate with the submerged process per m 3 is almost 5 folds greater than the corresponding trickling generator process and 10 folds higher than the surface fermenta-

tion process.

(2) Submerged process require much lesser capital investment per production amount, merely 1/5th of the total plant area is required for its due installation, vulnerability of faster conver- sion to different mash variants in a much shorter duration, and above all reasonably lower manpower cost involved on account of highly automatic controls and measures.

Recovery : The end-product acetic acid (household vinegar) obtained by the sub-merged process is invariably turbid in apperance by virtue of the presence of microorganisms (mostly in suspended form) ; and, therefore, the product must be clarified by adequate filtration. One may make use of plate-type filters along with appropriate filter-aids are mostly recommended and used gainfully. The coloured filtrate (acetic

acid) may be decolourized by the help of potassium ferrocyanide [K 4 Fe(CN) 6 ], if necessary.

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