LECTURE FIVE

INDUSTRIAL PRODUCTION OF SOME PRODUCTS USING MICROORGANISMS

COMMERCIAL PRODUCTION OF BEER

Beer is a product of the fermentation of barley grains by yeasts. Barley seed are allowed to
germinate are allowed to germinate, then dried or roasted. During germination the naturally
occurring amylases convert the grain starch to sugars, most of which being maltose. The process
is called malting. Germinating the grains cause the production of a number of enzymes, most
notably amylases. The dried or roasted mailed grains are ground into a grist and mixed with
heated water in a process called mashing.

After mashing, the spent grain is separated from the liquid. The spent grain is usually sold
for livestock feed. The liquid which is called wort is transferred to a large kettle where it is
boiled with hops and times other herbs or flavours. Dried petals of a vine Humulus lupulus are
called hops .Hops gives wort flavor, colour, clarity and stability. Hops also prevent
contamination of the wort, due to the presence of two antimicrobial substances in petals. At this
stage the fluid is filtered and yeast is added in large quantities. The a-acids (humulone,
cohumulone and adhumulone) in hops provide the bitter flavour and large proteins settle out with
tamins to clarify the beer. Trans-isohumulone in hops are responsible for the antibacterial
properties.

After boiling and addition of hops, the wort is clarified by spinning it and transferred to
fermentation tanks. In the fermentation tank, yeast is pitched (added). Top fermenting yeast
produce ales bottom -fermenting yeasts produce lagers. Generally, a week is needed for normal
fermentation to take place. After a week, the young beer is transferred to vats for primary and
secondary ageing. It may take 6 months. For canning or bottling the beer is to be pasteurized at
1400cF to kill the yeast or may be filtered to remove the years. Some yeasts are used to seed new
wort, the rest as animal feed or pressed to tablets for human consumption (SCP). Alcohol content
of beer is roughly 4%.

SPOILAGE OF BEER

Beer can be spoiled either by wild yeasts or bacteria. The bacteria generally associated with beer
spoilage are lactic acid bacteria,acetic acid acid bacteria and Zymomonas anaerobia. However
the lactic acid bacteria are most prevalent both in beer during processing and in packaged
beer.The two most prominent species are Lactobacillus pastorianus and L.diastaticus.. The
acetic acid bacteria are strict aerobes hence they are able to exclude air. Gluconobacter and
Acetobacter are able to oxidize ethanol to acetic acid in the presence of air. Wild yeasts acan
spoil beer at almost any point in the process, including the packaged product
COMMERCIAL PRODUCTION OF WINE

Wine production, or the science of enology(Greek oinos, wine, and olology,

the science of), starts with the collection of grapes, continues with their crushing
and the separation of the liquid (must) before fermentation, and concludes with a
variety of storage and aging steps. All grapes have white juices. To make a red
wine from a red grape, the grape skins are allowed to remain in contact with the
must before fermentation to release their skin colouring components. Wines can be
produced by using the natural grape skin microorganisms but this natural mixture
of bacteria and yeast gives unpredictable fermentation results. To avoid such
problems, fresh must be treated with a sulphurdioxide fumigant and a desired strain
of yeast Saccharomyces cerevisae or S.ellipsoideus is added. After inoculation the
juice is fermented for 3 to 5 days at temperatures between 20 and 28 0c. depending
on the alcohol tolerance of the yeast strain (the alcohol eventually kills the yeast
that produce it), the final product may contain 10 to 14% alcohol.

Clearing and development of flavor occur during the aging process. The
malolactic fermentation is an important part of wine production. Grape juice
contains high levels of organic acids, including the malic and tartaric acids. If the
levels of these acids are not decreased during the fermentation process, the wine
will be too acidic, and have poor stability and “Mouth feel”. This essential is
carried out by the bacteria Leuconostoc oenos, L. plantarum, L. hilgardii, L. brevis,
and L. casei. The activities of these microbes transform malic acid (a four-carbon
tricarboxylic acid) to lactic acid (a three-carbon monocaboxylic acid) and carbon
dioxide.

This results in deacidification (pH increase), improvement of flavor stability,

and in some cases the possible accumulation of bacteriocins in the wines.

A critical part of wine making involves the choice of whether to produce a

dry (no remaining free sugar) or a sweeter (varying amounts of free sugar) wine.
This can be controlled by regulating the initial must sugar concentration. With
higher level of sugar, alcohol will accumulate and inhibit the fermentation before
the sugar can be completely used, thus producing a sweeter wine.

During final fermentation in aging process, flavoring compounds accumulate and

influence the bouquet of the wire.
Microbial growth during the fermentation process produces sediments, which are
removed during racking. Racking can be carried out at the time the fermented wine
is transferred to bottles or casks for aging or even after the wine is placed in bottles

VINEGAR (ACETICACID) PRODUCTION

This production involves two types of biochemical changes: (1) an alcoholic fermentation of a
carbohydrate and (2) oxidation of the alcohol to acetic acid. There are several kinds of vinegars,
and the differences among them are primarily associated with the kind of material used in the
alcoholic fermentation e.g. fruit juices, e.g. saphite sugar-containing syrups, and hydrolysed
starchy materials.

As listed above the fermentation involved in vinegar manufacture can be separated principally on
the basis of the fermenting organisms to oxygen. The first step involving conversion of the sugar
of engr apple juice to alcohol may begin under aerobic conditions to stimulate yeast growth and
increased cell mass. But conditions are soon made anaerobic to favor the actual yeast
fermentation of the sugar to alcohol. The second step involving the conversion of alcohol. The
second step involving the conversion of alcohol to acitic acid is promoted but highly aerobic
conditions since this transformation is really oxidative fermentation. This conversion of alcohol
to acetic acid is commonly carried out in a vinegar generator. Vinegar generators differ in assign
but generally consist of large tanks or vats packed with wood shavings to provide a large aerobic
surface area. The alcoholic cider, after heavy inoculation with vinegar bacteria (Acetobacter
aceti), is trickled through the wood shavings while air is blown up through the shavings. The
vinegar is removed from the generator when its acetic acid concentration reaches four percent (or
somewhat higher). Operation of a vinegar generator demands close control. Aerobic conditions
can encourage mould development, and this breakdown the acid. In addition, excessive aeration
can itself oxidize acetic acid further to carbon dioxide and water.

Frings vinegar generator. A dilute solution of alcohol percolates through wood shavings that are
covered with a growth of acetobacter. The bacteria oxidize the alcohol to acetic acid.

2nd C2H50H + O2 + Acebacter accti = CH2COOH + H2O

Alcohol + Oxygen + Acetic acid bacteria = Acetic + Water

The rate of conversion of alcohol to acetic acid depends on the activity of the organism, the
amount of alcohol present, the temperature, and the amount of surface expanded per unit of
volume. The time required for the slow process, barred type, is about three months or more. In
large-scale generator processes where the surface exposed to air is large, the time for the
fermentation may be shortened to a matter of hours.
COMMERCIAL PRODUCTION OF VITAMINS

All phototrophic microorganisms arc capable of synthesizing vitamins and other growth
stimulating compounds for their vegetative growth by using the chemical constituents from the
culture medium. When synthesis of these compounds exceed beyond their requirement, it
accumulates in cultures; from where it is recovered. Nowadays, commercial exploitation of such
microorganisms is being done which synthesize vitamins on large scale under different cultural
conditions. Microbiologically produced vitamins are: carotene, precursor of Vitamin A

COMMERCIAL PRODUCTION OF BREAD

Bread is made by using yeasts that carry out an alcoholic fermentation. The substrate for
this fermentation are the sugars from the carbohydrates in wheat flour which are broken down by
the enzyme amylase from added malt or adding more sugars. i.e. the yeasts ferments the sugar to
produce carbon dioxide and ethanol.

The principal yeast used in bread making in saccharomyces cerevisiae known as Baker’s
yeast. Carbon dioxide formed by baker’s yeasty leaves the dough and causes it to rise.

After the leavening, the bread is baked. Carbon dioxide bubbles are trapped in the dough
and give rise to the honey comb texture and increased volume of the baked bread.

Although, the interior of the bread does not reach 100 0c. The heating performs these
functions:

Kill the yeasts

Inactivate the enzymes

Expand the gas

Evaporate the ethanol produced during the fermentation and

Establish the structure of the bread of loaf.

Gluten, a glycoprotein in wheat that solidifies when heated (1) gives the structural support
and elasticity to the dough.

In addition to leavening bread, microorganisms produce the characteristics flavours of

some bread. e.g. the production of San Francisco Sourdough bread uses yeast (Torulopsis holmii)
and lacto and bacterium (Lacto bacillus San francisco) to sour the dough and gives this bread its
characteristic sour flavor.
VITAMIN B12 (CYANOCOBALAMINE)

In nature, Vitamin B12 is synthesized by microorganisms. For industrial production of

vitamin B12 a number of bacteria and streptomycetes are used. The amount of Vitamin B12
produced by them has been estimated to be about 20mg/litre. It is used in medicine and feed
supplements, and is most essential for human growth.

Commercially Vitamin B12 is produced in a continuous culture where two fermentors are
used in a series. In each fermentor culture is kept for about 60 hours. Precaution taken is that the
first fermenter should be anaerobic, while the second aerobic. In the second fermenter 5,6 -
dimethyl benziminazol is added continuously, sterilized culture medium, containing glucose,
corn steep, betain (5%), cobalt (5ppm) and pH 7.5 in fermenter, is inoculated with
Propionibacterium freudenreichii and allowed undergo anaerobic fermentation for about 70
hours. During this period, cobinamide is produced which accumulates in the broth. There after
5,6-dimethyl benziminazole (0.1%) is added to it. The fermentor is then kept further for 50 hours
for aerobic fermentation - During this period nucleotide is synthesized and linked with cobamide
molecule to yield about 20ppm cobalamin. Culture is acidified to pH 2.0 to 3.0 gently heated to
100°C and filtered to remove cell debris. Finally potassium cyanide (5ppm) is added to filtrate to
give cyanocobalamin. Generally sodium sulphite is mixed with the solution so that
cyanocobalamin could not be oxidized. P.denitrificans is able to produce 50,000 times more
Vitamin B12 than its parental strain.

COMMERCIAL PRODUCTION OF ORGANIC ACIDS

There are 3 methods for commercial production of citric acid:

i. Koji fermentation process: This method is used in Japan which accounts forabout one
fifth of citric acid produced per annum. Strains of A.niger are used in this method.

ii. Liquid surface culture fermentation process: In this method, liquid cultures are
inoculated with the spores of A.niger which germinate within 24hours. Mycelin
cover and float on whole surface of the solution.

iii. Submerged culture fermentation process: In this case the mycelia of A. japonicum
grown in solution of about 1.5cm depth by liquid surface culture fermentation.

For commercial production strains of A.niger are selected from the mutants developed by
certain procedure. For large scale production, continuous culture is not suitable. Therefore, a
multi tank system is any process in which cell growth and met and metabolic products occur at
different times.

Culture medium contains carbohydrate, KH2PO4 (0.01-0.3%), MgSO4, 7H2O (0.25%) and
a few trace elements. Solution sucrose, pH of the culture medium is maintained to about 3.5 by
adding ammonium nitrate (0.25%). It avoids the formation of oxalic acid gluconic acids

After inoculation, the culture medium must be aerated by bubbling the air to allow
maximum growth for the fungus. Fermentation 27-33°C. Lime (Ca(OH) 2 is added to allow the
precipitation of citric acid in the form of calcium citrate. Again the precipitate is treated with
sulphuric acid to precipitate insoluble calcium sulphate. H is the filtered. The filtered containing
citric acid is purified by passing through column of carbon granules. Passing through ion
exchange beds solution is demineralised, which is then concentrated under vaccum to form
crystals. Citric acid is used in food industry (e.g. fruit, drinks, confectionary, jams, jellies,
preserved fruits, candies, wines), pharmacy, cosmetics and industries (electroplating, etc.)

COMMERCIAL PRODUCTION OF CITRIC ACID

Citric acid is an important chemical used in medicines, flavouring extracts, food and
candies, the manufacture of ink, dyeing and engraving. Several different species of moulds have
the ability to convert sugar to citric acid, but Aspergillus nigcr is most widely used for its
commercial production. The development of this industry in the United States illustrates the
value of applying new ideas is an old industry. Many sugars may serve as the substrate for the
production of citric acid; however, molasses is generally used. The carbohydrate is incorporated
into a medium containing an inorganic nitrogen compound as well as inorganic salts. The sterile
medium containing an inorganic nitrogen compound as well as inorganic salts. The sterile
medium is dispensed into shallow pans and inoculated with the mould spores. This is an aerobic
process; consequently a large surface area provides an adequate supply of oxygen. An alternative
to this method of production is the sub merged-culture technique, in which the inoculated
medium is contained in large tanks through which a supply of sterile air is forced. The strain of
mould employed, the composition of the medium, the degree of aeration, and the temperature of
incubation all have an effect on the yield of citric acid.

LACTIC ACID PRODUCTION

Carbohydrate substances such as corn and potato starch, molasses, and whey can be used for the
production of lactic acid. Starch must be hydrolysed to glucose by acid or enzymatic treatment.
The choice of carbohydrate material depends upon its availability, treatment required prior to
fermentation and cost.

PRODUCTION OF LACTIC ACID FROM WHEY

Large quantities of whey constitute a waste predict in the manufacture of certain dairy products
such as cheese. From the stand point of pollution problems created by the disposal of intreated
whey, as well as economic reasons, it is desirable to use it to make some useful product. Whey
satisfactory medium for the growth of certain bacteria, since it contains carbohydrate (lactose),
nitrogenous substances including vitamins and salts. The first requirement for the development
of a method of producing lactic acid is an organism capable of growing in whey and fermenting
most if not all the lactose to lactic acid. Lactobacilli are suitable for this purpose, particularly
Lactobaciiius bulgaricus. This organism grows rapidly and is homofermentative and thus is
capable of converting the lactose to single product i.e. lactic acid. Stock cultures of the organism
used are maintained in a skim-milk medium. To prepare a sufficient amount of inculum for
addition to the main fermentation tank, the culture is successively transferred and incubated in
increasing amount of sterile, skim milk, paseurised skim mild and finally whey. Milk is used in
“building up” the inoculums, since it is a superior medium. Inoculum from the whey-incubation-
tank is added to the fermentation tank in an amount equivalent to 5 to 10% of starter culture (in
milk) the volume to be fermented. An incubation temperature of 43°C is used and has the
desirable effect of inhibiting growth of many extraneous microorganisms. During the
fermentation, a slurry of lime, Ca(OH)2, is added infermittently to neutralize the acid, and
calcium lactate is formed; otherwise the accumulation of acid would retard fermentation. Upon
completion of fermentation (approx. 2days) the material in the tank boiled to coagulate the
protein, which is filtered and processed for use as an animal-feed supplement. The filtrate
containing the calcium Iactate is then concentrated by removal of water under a vacuum,
followed by additional treatments to purify the compound. The process is illustrated below. The
biochemical reactions performed by microorganism in producing the lactic acid can be
summarized:

C12H220H + H2O 2C6H12O6 2CH3CHOHCOOH

Derivatives of lactic are used in the treatment of calcium deficiency (calcium lactate) and of
anaemia (iron lactate), as a solvent in lacquers (n-butyl lactate), and as a plasticizer and
humechant (sodium lactate).

Illustration on Lactic acid production from whey by Lactobacillus bulgaricus

Starter culture

Agar plate or slope culture

Starter vessel or shake flask

Seed fermentation

Secondary fermenter

Production fermenter

Scaling-up of inoculums

(Building up).

PENICILLIN PRODUCTION

The commercial production of penicillin and other antibiotics represents on of the most
dramatic case histories in the development antibiotic industry microbiology. The antibiotics
industry did not exist in 1941, but 10 years later net sales of these products had reached $344
million per year.

Penicillin was the first antibiotic to be learned in transforming Fleming's laboratory large-
scale operation paved the way for success antibiotic as they were discovered.

The mould related by Nonder Fleming (Penicilum notadum) and as grown in his
laboratory, yielded only a few units of penicillin per milliter, an exceedingly small amount when
one considers that a patient may require treatment with millions of units. However, within a
relatively short time after discover, the yield of penicilum was increased about a thousand times.
The developments contributing to this enormous increase in yield were as follows:

1. Improvement in composition of medium

2. Isolation of a better penicillin-producing mould species, penicillum crysogenum

 3. Development of the submerged-culture technique: cultivation of mould in large volumes
of liquid medum through which sterile air is forced.

4. The production of mutant strains of P.crysogenum which were capable of producing large
amounts of penicillin. A series of mutants, produced by x-ray and ultraviolet radiation,
resulted in strains with a remarkable capacity for synthesis of penicillin.

5. The addition of chemicals to the medium which served as precursors for synthesis of
penicillin.

6. Refinements in methods of recovering penicillin from the fermentation mixture.

The major steps in the commercial production of penicillin are:

1. Preparation of inoculums

2. Preparation and sterilization of medium

3. Inoculation of the medium in the fermenter

4. Forced aeration with sterile air during incubation

5. Removal of the mould mycelium after fermentation

6. Extraction and purification of penicillin.

This process is illustrated in the diagram below.

The production of most other antibiotics follows a similar plan. The major differences related to
the organism, composition of medium, and method of extraction. Some manufacturers employ
the same fermentation equipment for the production of several different antibiotics.

COMMERCIAL PRODUCTION OF CHEESE

Various cheeses are produced by microbial fermentation.

Cheeses consist of milk curds that have been separated from the liquid portion of the milk
(whey). The curdling of enzyme rennin (casein co agulase or chymosin) andlactic acid
bacterialfromcalfstomachs or by microbial productionfromthemuccorpussilus.

The production of cheese involves of streptoccus and lacto bacillus species fermentation.

After the formation of the curds, they are separated from the whey, washed, pressed,
often cooked and salted. The cheese istheripeneda process involving additional transformation
performed by proteolytic enzymes.

Different types of cheese are ripened at various temperatures to encourage and promote
the growth of specific organisms. Sometimes a cheese is soaked in a brine to encourage the
development of selected bacterial and fungal population during ripening.

Cheese are categorized based on texture as:

1) Soft cheese as cottage cheese, Green cheese.

2) Semi soft cheese e.g. blue cheese

3) Hard cheese e.g. cheddar or swiss

4) Very hard cheese e.g. romano

The hardness of cheese partially depends on the length of the ripening period and the
amount of the water removed from the curd. Soft cheese are ripened for 1 to 5 months, hand
cheese, 3 to 12 months and very hard cheese may take up to 16 months to ripen.

During ripening the curds are softened by enzymes and the cheese acquires its
characteristics aroma and flavor from microbial by products.

Swiss cheese formation involves a late propionic acid fermentation, with ripening
accomplished by propioni bacterium shermani and propionibacterium freudenriechii. The
propionic acid yields the characteristics aroma and flavor, and the carbondioxide produced
during this late fermentation forms the holes or eyes in the swiss cheese.

Various fungi may also be used in the ripening of different cheese. The unripened cheese is
normally inoculated with fungal spores and incubated in a warm, most room to promote the
growth of filamentous fungi e.g. blue cheese are produced by using penicillin species. Roquefort
cheese is produced using P. roqueforti and camembert and brie by using P. camemberti and P.
candidum.