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What is Fermentation?

 Derived from the Latin verb ‘fervere’ meaning ‘to boil’

 Fermentation, according to Pasteur, is directly cause by the life processes of minute

 It is a process by which the living cell is able to obtain energy through the breakdown of
glucose and other simple sugar molecules without requiring oxygen.

 Fermentation results in the production of energy in the form of two ATP molecules, and
produces less energy than the aerobic process of cellular respiration.

 Industrial Fermentation-Any microbial process controlled by humans that produces

useful products.

History of Fermentation

 Louis Pasteur in the 19th century used the term fermentation in a narrow sense to
describe the changes brought about by yeasts and other microorganisms growing in the
absence of air (anaerobically)
 He also recognized that ethyl alcohol and carbon dioxide are not the only products of
 He called the process la vie sans air which means ‘life without air’.
 In 1897, Hans and Eduard Beuchner discovered that fermentation could occur in a cell-
free extract of yeast
 The production of lactic acid in 1880 was the beginning of industrial fermentation to
produce a useful product other than alcohol.
 During World War I, Chaim Weizmann developed a fermentation process to convert
corn to acetone and n-butanol.
 Citric acid and gluconic acid were successfully produced between 1920 and 1940.
 During World War II, the discovery of antibiotics, such as penicillin, set the stage for
great technological processes that are commonly used today.


Category Examples Uses of Remarks

Sour dough, soy sauce, yogurt, Conservation of perishable

Food kefir, cheese, pickles, salami, food by the formation of lactic
anchovy, saucerkraut, vinegar, acid and ethanol
beer, wine, cocoa, coffee, tea
Conservation of green plants by
Feed Silage organic acids

Yeast, lactic acid bacteria, Used as starter cultures, animal

Cell mass single cell protein feed

Ethanol, glycerol, acetone, Cosmetics, pharmaceuticals

Organic solvents butanediol

Lactic, citric, acetic, acrylic, Food, textiles, chemical

Organic acids formic acid intermediates

L-lysine, L-tryptophan, L- Food and feed additives

Amino acids phenylalanine, glutamic acid

Penicillin, streptomycin, Human and veterinary

Antibiotics tetramycin, tetracycline medicines

B12, biotin, riboflavin Food and feed supplements


Amylase, cellulose, protease, Food processing, tanning,

Enzymes lipase, lab detergents additives

Lanthan, dextran, Food additives, medical

Biopolymers polyhydroxybutyrate devices, packaging

Insulin, interferon, Human medicines

Speciality pharmaceuticals erythropoietin (EPO)

Waste and wastewater Public hygiene

Environmental treatment

Ethanol from carbohydrates Fuel additives or heat

Energy and methane from organic generation

Many chemical reactions caused by microorganism are very complex, however, and cannot
easily be classified; so the concept of fermentation itself as a chemical conversion has been
developed. According to Silcox and Lee, the five basic prerequisites of a good fermentation
process are:

1. A microorganism that forms a desired end product. This organism must be readily
propagated and be capable of maintaining biological uniformity, thereby giving
predictable yields.
2. Economical raw materials for the substrate, e.g., starch or one of several sugars.
3. Acceptable yields.
4. Rapid fermentation.
5. A product that is readily recovered and purified.



Industrial alcohol was an outgrowth of alcoholic beverages, but now it has become
important by virtue of its economically useful properties as a solvent and for synthesis of other
chemicals. Alcohol is sold as tax-paid alcohol or, much more widely, as non-taxed denatured
alcohol. There are two classes of the latter:

Completely denatured and Specially denatured alcohol

 The completely denatured formulas comprise mixtures of substances which are difficult
to separate from the alcohol and which smell and taste bad, this all being designed to
render the alcohol non-potable. The public uses it as an anti-freeze and the factories
find it an essential raw material.

A typical completely denatured alcohol formula follows:

To every 100 gal. of ethyl alcohol of not less than 160 proof add:
 0.125 gal. of Pyronate or a compound similar thereto.
 0.5 gal. of acetaldol (β-hydroxybutyraldehyde)
 2.5 gal. of methyl isobutyl ketone.
 1 gal. of kerosene.

Typically specially denatured formulas are:

To every 100 gal of 190-proof ethyl alcohol add:

 Approved wood alcohol.5 gal.

 Benzene, 0.5 gal.
 100 % acetaldehyde, 1 gal, or an alcoholic solution of acetaldehyde(20%), 5 gal.

Uses and Economics

 Tax-paid pure alcohol is used only for medicinal, pharmaceutical, flavoring, or beverage
 Completely denatured alcohol is consumed chiefly as antifreeze.
 The trend has been toward the use of specially denatured alcohols for industrial
 Alcohol is second only to water in solvent value and is employed in nearly all industries.
In addition it is the raw material for making hundreds of chemicals. Most important of
these are acetaldehyde, ethyl acetate, acetic acid, ethylene di-bromide, glycols: and
ethyl chloride.
 During the Second World War when tremendous quantities of butadiene and styrene
were needed for the synthetic-rubber program, alcohol was used as a supplemental raw
material for butadiene although petroleum is the cheaper raw material.
 To produce that much alcohol would require not only using all the surplus grain
available but also radically new technology to convert economically wood, cellulose and
other starchy wastes to alcohol.
 Ethanol is being used as an octane booster for gasoline and small amount is being
consumed as gasohol.


Raw Materials

 Alcohol from cellulosic materials, wood, wood wastes, and sulfite liquors is considered.
 A small amount of glycerin is always found in alcohol fermentation.
 When growing yeast for sale as such or for inoculation, like so many industrial reactions,
is much more involved than these simple reactions.

The principal reactions in alcohol fermentation are:

Equations of Monosaccharide Production


Equations of Fermentation

Energy Requirements, Unit Operations, Unit Processes

 The plant procedures require steam heating for distillation, power for pumping, and
water for condensation and occasionally for cooling during the exothermic
 The heat evolved by this fermentation reaction calculates to 88,940,000 or
353,000,000 B.t.u. per 1,000 gal. of 95 per cent alcohol.

The main steps in the competitive manufacture of alcohol from petroleum cracking are
shown above in parallel comparison.

Figure of industrial alcohol making

Making of Industrial Alcohol.

 Molasses, because of the strong concentration of sugar, does not support direct yeast
fermentation. It must be diluted first to a concentration of about 10 to 14 per cent
sugars. This is called the mash and represents the carbohydrate ready for yeast
 It is pumped to a large steel fermentor (60,000 to 500,000 gal.)
 An ammonium salt and sulfuric acid are added, the one to furnish a nutritive

constituent deficient in molasses. Magnesium sulfate is also added, when deficient.

 Meanwhile a charge of the selected yeast (about 5 per cent of the total volume) has
been growing in the yeast tub.
 Addition of the nutrients, the pH, the temperature (76°F.) and finally the cleaning and
sterilizing of the yeast culture machine in readiness for the next batch.
 Four days are allowed by governmental regulations for a fermentation cycle, though
usually 40 to 72 hr. are used. As alcohol is formed by yeast only from monosaccharides,
it is necessary to split the sucrose, C12H22O11, into d-glucose and d-fructose.
 In alcohol fermentation by yeast, this microorganism furnishes an organic catalyst, or
enzyme, known as invertase which effects this hydrolysis. The yeast also produces
another and more important enzyme, zymase, which changes the monosaccharides into
alcohol and carbon dioxide.



 Anhydrous alcohol was made by absorbing the 4-5% of water present in 95-96%
industrial alcohol, using quicklime, with subsequent distillation.
 This process was expensive and, although it produced a very high quality of anhydrous
alcohol, it has now been superseded largely by improved chemical engineering unit
operations of distillation and extraction involving a third component.
 This has led to lower cost of dehydrating operations, mostly of a continuous nature
using preferably all liquids or solutions, and has resulted in reducing the price of
anhydrous alcohol to a figure only slightly in excess of the usual selling value of the
alcohol contents


The making of fermented beverages was discovered by primitive man and has been
practiced as an art for thousands of years.

Raw Materials

 Grains and fruits supplying carbohydrates are the basic raw materials. The variety of
grains and fruits employed is wide, changing from country to country or from beverage
to beverage.
 Russia ferments potatoes and by distillation obtains vodka; similar treatment of the sap
of the maguey in Mexico yields pulque; but the world's chief raw materials for
fermentations are the cereals, corn, barley, and rice, together with the grape.

Making of Beer

 Beer and allied products are beverages of low alcoholic content (2 to 7 per cent) made
by brewing various cereals with hops , usually added to impart a more or less bitter
taste, followed by fermentation.
 The cereals employed are called brewers' grains and are barley, malted to develop the
necessary enzymes and the desired flavor, as well as malt adjuncts: flaked rice, oats, and

com, with wheat used in Germany and rice and millet in China.
 Brewing sugars and syrups (com sugar or glucose) and yeast complete the raw
materials. For beer the most important cereal is barley, which is converted into malt by
partial germination.

The flow sheet for beer making may be divided into three groups of procedures:

(1) brewing of the mash through to the cooled hopped wort

(2) fermentation

(3) storage, finishing, and packaging for market

 Mashing is the extraction of the valuable constituents of malt, malt adjuncts, and
sugars by macerating the ground materials with water treated to prevent too high pH,
which would tend to make a dark beer.
 The materials so treated, the converter mash, are heated in the pressure cooker in
order to convert insoluble starch into soluble starch, and the soluble malt starch into
dextrin and malt sugars.
 The resulting boiling cooker a spray of decarbonated water at 165°F. is rained through
the grains. This is called sparging. The wort is cooked for approximately 1-1.5 hr.
 At the end of the 1-1.5 hr., the spent hops are, separated from the boiling wort very
quickly through a false bottom in the hop jack or strainer underneath the copper
 The wort is then ready to be cooled. The cooling step is not only to reduce the
temperature but also to allow the wort to absorb enough air to facilitate the start of the
fermentation. In addition, the protein and hop resins are precipitated.
 The cooled wort is mixed with selected yeasts in the line leading to the starting tubs,
between % and 1 lb. of yeast being used per barrel of beer. The initial fermentation
temperature is 40 to 43°F. but, as the fermentation proceeds, the temperature rises to
 The yeast gradually settles to the bottom of the tub, so that at the end of 7 to .10 days
the fermented beer is ready to be vatted. As the beer leaves the fermenting cellar, it
contains in suspension hop resins, insoluble nitrogenous substances, and a fair amount
of yeast.
 The beer is cooled to 32°F. and stored in the cellar for 3 to 6 weeks at this temperature.
During this period, clarification, separation, and precipitation of hard resins and
improvement in palatability (mellowing) occur. At the end of the period the beer is
carbonated and pumped through a pulp filter with or without such non taste-imparting
filter aids as asbestos fiber.

Wine has been made for several thousand years by fermentation of the juice of the
grape. Like other fermentations, many of the primitive procedures have been supplanted by
improved science and engineering, to reduce costs and to make more uniform products..

Wines are classified as natural (alcohol 7 to 14 per cent), fortified (alcohol 14 to 30 per
cent), sweet or dry, still or sparkling. The fortified wines have alcohol or brandy added. In the
sweet wines, some of the sugar remains.

For the manufacture of dry red wine, red or black grapes are necessary

 The grapes are run through a crusher which macerates them but does not crush the
seeds, and also removes part of the stems. The resulting pulp, or must, is pumped into
3,000- to lO,OOO-gal. tanks where sulfurous acid is added to check the growth of wild
 When the fermentation slows up, the juice is pumped out of the bottom of the vat,
back over the top. The wine is finally run into closed tanks in the storage cellar where,
during a period of 2 or 3 weeks, the yeast ferments the remainder of the sugar. The
wine is given a cellar treatment to clear it, improve the taste, and decrease the time of
 The wine is corrected to commercial standards by blending it with other wines and by
the addition of sugar, acids, or tannins.
 By- quick aging methods it is possible to put out a good, sweet wine in 4 months. These
methods include pasteurization, refrigeration, sunlight, ultraviolet light, ozone,
agitation, and aeration. The wine may be held at about freezing for 3 weeks to a month,
and a small amount of oxygen gas bubbled in. Then the wine is racked, clarified, and
further filtered in the usual manner.

Distilled Liquors

Various fermented products, upon distillation and aging, yield the distilled liquors.

Figure above shows the flow sheet for whisky and gin.

 Brandy is distilled from wine or from the marc, which is the pulp left by racking or
straining. By making a beer from a grain mixture containing at least 51 per cent of corn
and distilling and aging it, bourbon whisky is yielded. Similarly rye whisky must have
started with 51 percent of rye in the grain to be mashed and fermented.
 By law, the aging of bourbon or rye whisky of claimed age must take place in charred
new white-oak barrels of approximately 50 gal. These are kept in bonded warehouses at
65 to 85°F. and at a preferred humidity of 65 to 70 per cent for 1 to 5 years usually.
 The distillate from the spirit still is under 160 proof and is subsequently diluted upon
barreling to 100 to 110 proof. It is not pure alcohol but contains small amounts of many
different constituents, generally classed together as congenerics, which by their
reaction with each other or the alcohol, or by their absorption, all catalyzed by the char
of the wood, help greatly in imparting the whisky flavor and bouquet.
 By law, whisky must be fermented from whole grains, so that the germs (containing the
corn oil) and the husks are in suspension in the liquor from the beer still in whisky
manufacture. This discharge liquor is known as slop or stillage.


 Until the First World War, all the acetone produced in the United States was made by
the dry distillation of calcium acetate from pyroligneous acid. Under the stimulus of the
wartime demand for acetone for the manufacture of double-base smokeless powder
the important process became that developed by Chaim Weizmann4 for the
fermentation of starch-containing grains to butyl alcohol and acetone.
 The Commercial Solvents Corporation was organized, and it built and operated two
plants in the corn belt to ferment corn using Clostridium acetobutylicum bacteria.
However, this fermentation gave 2 parts of butyl alcohol to 1 part of acetone and until
the development of the fast-drying nitrocellulose lacquers particularly for the
automotive industry, there was virtually no market for the butyl alcohol produced.
 With the advent of the manufacture of butyl alcohol and especially of acetone by
chemical processes, competition forced the abandonment of corn in favor of the lower-
priced by-product molasses for the monosaccharide raw material for fermentation.

Uses and Economics

 The production of normal butyl alcohol and of acetone amounted to somewhat over
194,000,000 and 478,000,000 lb., respectively, during 1954.
 It is estimated that 40 percent of the butyl alcohol and 4 per cent of the acetone is
produced by fermentation. About 70 per cent of n-butyl alcohol enters directly or
indirectly into lacquer solvents. A percentage breakdown of the end use of acetone is
approximately as follows: cellulose acetate, 50; acetylene (solvent), 5; chemicals, 15;
paint, varnish, and lacquer, 12; drugs, 5; and miscellaneous, 15.



 The aerobic bacterial (Acetobacter aceti or Bacterium aceti) oxidation of alcohol to

dilute acetic l acid (8 per cent) is another ancient procedure, furnishing vinegar, a
flavorea acetic acid solution, fermented from wine, cider, malt, or dilute alcohol. If a
pure dilute alcohol is fermented, pure dilute acetic acid results.
 The yield is from 80 to 90 percent of theory. Air2 must be supplied as these
formulations indicate:

 As these reactions are exothermic, either the alcohol can be slowly trickled through the
apparatus, letting the heat dissipate, or it can be recirculated with special cooling.
 If cider, malt, or wine is fermented, the acetic acid content of the resulting vinegar
rarely exceeds 5 per cent, owing to limitations of the sugar content; if dilute alcohol is
the raw material, the acetic acid may rise to 12 or 14 percent.
 The ancient process consisted in letting alcoholic products like wine or cider stand
around in contact with air until the wine or cider turned to vinegar. This was a slow
procedure, taking many months.
 The modern quick vinegar process, as commercially carried out, trickles the diluted
alcohol, usually mixed with vinegar, down tall wooden tanks with false bottoms (20 ·ft.
high by 10 ft. in diameter) packed with beech wood shavings or coke on which the
bacteria find lodgment commonly as mother of vinegar.
 This takes a week or so, but the production amounts to 10 to 16 gal. of 9 to 10 per cent
spirit vinegar per 24 hr. from a 200-cu. ft. generator.


 Citric acid is one of our widely employed organic acids, its production amounting to
around 40,000,000 or 50,000,000 lb. Its major use is as an acidulant in carbonated
beverages, jams, jellies, and other foodstuffs.
 Another large outlet is in the medicinal field, including the manufacture of citrates and
effervescent salts. Industrial uses, relatively small, include citric acid as a sequestering
agent and acetyl tributyl citrate, a vinyl resin· plasticizer.
 Except for small amounts (less than 7 pecent) produced from citrus fruit wastes, citric
acid is manufactured2 by aerobic fermentation of crude sugar by a special strain of
Aspergillus niger following the classical researches by Currie. The over-all reaction is:

 The fermentation changes sugar, a straight-chain compound, into a branched chain. In

the tray process for citric acid, air is circulated for 9 to 12 days ove hundreds of shallow
pure-aluminum trays (approximately 43 by 43 by 2 in.) filled with a sugar solution. These
trays are placed a few inches above each other in a closed cabinet provided with
facilities for sterilization.



 Lactic acid, 2-hydroxypropionic acid, is one of the oldest known organic acids.
 It is the primary acid constituent of sour milk, from whence it derives its name, having
been formed by the fermentation of milk sugar (lactose) by Streptococcus lactis.
 It has only been since 1930 that lactic acid has been produced commercially from the
milk by-product.
 The small amount of lactic acid remaining is converted into plastics,' solvents, and
certain other chemical products. The U.S.P. grade is an old, well-established standard
pharmaceutical. The general course of the fermentation reactions may be expressed as

 The commercial acid is the inactive or racemic form, consisting of a mixture of the
dextro and levo forms, usually in equal proportions, and it is therefore inactive.
 The procedure consists in fermenting a mash of a carbohydrate substrate together with
suitable nutrients in the presence of an excess of calcium carbonate. The lactic acid as
formed reacts with the CaC03, producing calcium lactate and carbon dioxide.

Miscellaneous Compounds/Product of Fermentation

Monosodium Glutamate

The amino acid glutamic acid may be prepared synthetically, but chemical preparation
produces a racemic mixture. Since only the sodium salt of the naturally occurring L-glutamic
acid is desired for food flavor enhancement, this necessities an expensive resolution step. L-
Glutamic acid can be obtained directly from fermentation of carbohydrates with micrococcus
glutamicas. Many patents have been issued on the variations of the process as this one of the
largest volume compounds produced by fermentation.


L-lysine may be formed by microorganism acting on carbohydrates. The usual organisms are
micrococcus glutamicas, brevibacteriumfalvium, corynebacterium, acetoglutamicum, and
Microbacterium ammoniaphilum. Each of these or organisms requires special conditions and
special additives toproduce in good yields.


Dihydroxyacetone is made by the action of sorbose bacterium fermentation of glycerin. This is

an ingredient of suntan lotion that creates an artificial tan. It is also valuable as chemical
intermediate and as a catalyst in butadiene-styrene polymerization fatty acids esters of the
hydroxyl groups are excellent emulsifying agents.

Pharmaceutical products

The pharmaceutical industry has long employed fermentation to manufacture some of its most
important medicaments, where fermentation is presented for antibiotics, biological, vitamins
and hormones. Controlled microorganisms are the most important chemical processing agent

and assist in performing very complicated chemical recations, in many cases more economically
than purely chemical conversions.

List of certain enzymes, organic catalysts formed in the living cells of plants and animals, which
are essential in bringing about specific biochemical reactions in living cells.

Typical Industrial enzymes sold today: