Unit : 3

Fermentation Industries
 2.1 Alcohol production
• Almost all major antibiotic are obtained by fermentation

•Important factors involved in fermentation

• Temperature, PH, aeration-agitation, Pure culture fermentation

and uniformity of the yield.

Industrial alcohol
•It is the outgrowth of alcoholic beverage

•Ethanol has been recognized as an important renewable and

sustainable fuel source for modern industries

• It is used as solvent and for synthesis of other chemicals

• Alcoholic Beverage
• Beer and wine
The Alcohol Production Process
• Ethanol can be derived from any substance yielding fermentable
sugars.

• The nature of the feedstock affects how sugars are obtained.

• Sugars can be obtained directly from crops such as sugar cane, sugar
beet and fruits simply by crushing the material and extracting the
juice.

• Feedstock containing starch such as wheat and maize must first be

treated with the enzymes α-amylase and amyloglucosidase to break
down the starch to glucose.

• Lignocellulosic materials such as wood, paper and straw require

extensive pre-treatment using chemicals and/or high pressure and
high temperature treatments;
• The exact production process may vary depending upon individual
circumstances.

• The process for potable alcohol is broadly similar to that for fuel
alcohol but differs in additives that can be used.

• Fuel alcohol can use commercial enzymes and chemicals,

• Neutral alcohol can use commercial enzymes for saccharification

but no chemicals

• Potable alcohol production is constrained to using only grains,

yeast and water so no chemicals and commercial enzymes are
used.
• Principal reaction in alcohol fermentation
I. Milling
• The milling process increases the grain surface area, allowing
more effective slurring, cooking and liquefaction and more
effective enzymatic breakdown of starch

• Milling of grains for bioethanol production may be carried out by

either a “dry grind” or a “wet grind” process.

• With a “dry grind”, the whole grain is milled without any

separation of grain components.

• This is the cheapest and most common process found in existing

bioethanol production facilities, and is also common in potable
alcohol distilleries.
• Wet grind” process separates the grain into its constituent components,
starch, fibre, protein and germ after a period of soaking (or steeping) in
dilute sulphuric acid prior to milling.

II. Liquefaction/ Gelatinisation

• A high temperature “cooking” step is commonly used to gelatinise the starch
and make it more accessible to enzymes for degradation.

• The high temperatures also help to reduce microbial contamination.

• The milled grain is mixed with water to form a mash and heated to 120-
150ºC.

• High temperature and high pressure cause mechanical shearing forces on the
starch molecule.

• Release of pressure (blowdown) further disrupts the remaining endosperm

• If the starch is cooked for too long or at too high temperature,
browning (or Malliard) reactions may occur, resulting in reduced
alcohol yields.

• Novel enzyme mixtures of α amylases and gluco-amylases are

now commercially available which are able to break down starch

• Cold cooking’ methods whereby ground grain is either not

cooked before enzymatic saccharification or cooked at a reduced
temperature (e.g. 80°C).

• These gave higher alcohol yields but the energy saved by cold
cooking may be offset or even increased by the need to mill the
grain more finely.
III. Saccharification

• In fuel alcohol production, after cooling to 90-100°C, a heat stable

α-amylase is added to breakdown starch to smaller subunits.

• This step significantly reduces the viscosity of the mash

• The mash is then cooled further to 80-90˚C and amyloglucosidase

is added.

• Amyloglucosidase removes successive glucose residues at the

ends of the starch molecules.

• Breakdown of starch leaves a sugar solution called ‘wort’.

IV. Fermentation
• Under anaerobic (oxygen limiting) environments, yeasts produce
ethanol and carbon dioxide from sugars in a process called
fermentation.

• The mash from saccharification is cooled and yeast added.

• Fermentation typically occurs for 48-72 hours at approximately

30°C-35°C and results in wort with a typical final alcohol content of
8-12%. depending upon

 The initial substrate level,

 Amount of yeast added (pitching rate) and

 The degree of bacterial contamination.

V. Distillation and Dehydration

• Distillation allows the concentration of alcohol to be increased by

separating ethanol from water.

• At sea level, ethanol vaporizes at 78°C the ethanol and water can be

separated to leave a 95% ethanol and 5% water azeotrope.

• Distillation for potable alcohol stops at this stage but for transport

alcohol further dehydration is necessary.

VI. Stillage Separation

• After fermentation and distillation, the residual mash, termed

‘whole stillage’ is separated by centrifugation or pressing and
extrusion into wet grain (containing heavy particulate matter) and
thin stillage (containing water and small particulate matter).

• The thin stillage fraction is dried to a syrup, then mixed with the
wet grain fraction and dried further to form Dried Distillers
Grains with Soluble (DDGS).
Co-Products
• The remaining part (non fermented part) 1/3rd of the grain consists
of non-starch polysaccharides, non-degraded starch, proteins and
lipids.

• There is still considerable scope to find uses for co-products other

than in animal feeds, such as in pharmaceutical, nutraceutical and
cosmetic products.

• Wheat has the potential to provide gluten (used in the baking

industry and as an emulsifier or thickener; bran (used in cereal
foods), germ (used in bakery products and for some high value
cosmetic uses) and flour, in addition to DDGS, the standard co-
product of bioethanol production.
VII. Purification of Bio-ethanol
• As fermentation is completed, the next stage of ethanol production
is purification.

• Purification separated the ethanol from the other components of

fermentation; since water is the largest to remove.

• This step is necessary because fuel purposes required very pure

ethanol to proper blend with gasoline.

• The current separation technology used in the ethanol industry is

comprised of two steps:
1) Distillation

2) Dehydration
VIII. Distillation Process

• Distillation operates under the principles that different materials

boil at different temperatures.

• The material with the higher boiling point remains a liquid and is
found at the bottom of the pot as stillage.

• Using this principle, a two-component mixture of ideal liquids can

be heated in a “pot” under controlled conditions, with the boiling
point material completely vaporized and collected at the top of the
pot.
• When the amount of water reduced to Azeotropic ethanol-water
mixture, fractional distillation not able to separate the mixture.

IX. Dehydration Process

• After fermentation and distillation are completed, the next stage
of ethanol purification is dehydration.

• Dehydration is necessary because traditional distillation leaves

approximately 4.37% water (by weight) water in the finished
ethanol.

• If the ethanol is required to blend with gasoline, and the presence

of water significantly enhances the natural molecular polarity of
ethanol.
• it will act more like water than ethanol when mixed with
hydrocarbon solutions, such as gasoline.

• When “wet” ethanol is mixed with gasoline, the ethanol separates

out with all the ethanol settling to the bottom of the container and
the gasoline to the top.

• There are different dehydration techniques in the industry,

Azeotropic distillation

Molecular sieve dehydration

Chemical action separation

Pressure swing distillation.

Absolute alcohol
• An azeotrope is a mixture of two or more liquids whose
proportions cannot be altered by simple distillation.

• The boiling point of an azeotrope is either less than the boiling

point temperatures of any of its constituents (a positive
azeotrope), or greater than the boiling point of any of its
constituents (a negative azeotrope).

• A well-known example of a positive azeotrope is 95.63% ethanol

and 4.37% water (by weight).
• Ethanol boils at 78.4 °C, water boils at 100 °C, but the azeotrope
boils at 78.2 °C, which is lower than either of its constituents.

• Indeed 78.2 °C is the minimum temperature at which any

ethanol/water solution can boil at atmospheric pressure.

• An example of a negative azeotrope is hydrochloric acid at a

concentration of 20.2% and 79.8% water (by weight).

• Hydrogen chloride boils at 84 °C and water at 100 °C, but the

azeotrope boils at 110 °C, which is higher than either of its
constituents.

• The maximum temperature at which any hydrochloric acid

solution can boil is 110 °C.
Homogeneous and heterogeneous azeotropes

• If the constituents of a mixture are not completely miscible, the

azeotrope is called heterogeneous azeotrope.

• If the constituents of the mixture are completely miscible, the type

of azeotrope is called a homogeneous azeotrope.

• Azeotropes consisting of two constituents are

called binary azeotropes.

• If it consisting of three constituents it is called ternary azeotropes.

A. Azeotropic Distillation
• It is used for the separation of ethanol and water by adding a third
chemical such as benzene or cyclohexane as entrainer chemical
during the distillation process.

• When benzene is fed as the entrainer, three distinct regions are

present with different composition in the distillation column.

• The bottom of the column contains the first fraction which is

composed of almost pure water.

• The middle of the column contains a near-ternary azeotrope

mixture of water, benzene, and alcohol.

• the top of the column contains anhydrous ethanol.

Mechanism
• Azeotropes can only form when a mixture deviates from Raoult's law.

• Raoult's law applies when the molecules of the constituents stick to

each other to the same degree as they do to themselves.

– For example, if the constituents are X and Y, then X sticks to Y with roughly
equal energy as X does with X and Y does with Y.

• A positive deviation from Raoult's law results when the constituents

have a disaffinity for each other – that is X sticks to X and Y to Y
better than X sticks to Y.

• Because this results in the mixture having less total sticking together
of the molecules than the pure constituents, they more readily escape
from the stuck-together phase, which is to say the liquid phase, and
into the vapor phase.
• When X sticks to Y more aggressively than X does to X
and Y does to Y, the result is a negative deviation from
Raoult's law.

• In this case because there is more sticking together of the

molecules in the mixture than in the pure constituents,
they are more reluctant to escape the stuck-together
liquid phase.

• When the deviation is great enough to cause a maximum

or minimum in the vapor pressure versus composition
function, it is a mathematical consequence that at that
point, the vapor will have the same composition as the
liquid, and so an azeotrope is the result.
B. Pressure swing distillation
• Some binary azeotrope mixtures lose azeotropic behaviour when
the system pressure is changed.
• Pressure-swing distillation is a method for separating a pressure-
sensitive azeotrope that utiltises 2 columns operated in sequence at
2 different pressures.

• However ethanol/water is not affected by pressure very much to be

easily separated by pressure swings.

C. Molecular sieves
• For the distillation of ethanol for gasoline addition, the most
common means of breaking the azeotrope is the use of molecular
sieves .
• Ethanol is distilled to 96%, then run over a molecular sieve which
adsorbs water from the mixture.
D. Chemical Action separation
• Another type of entrainer is one that has a strong chemical
affinity for one of the constituents.

• In water/ethanol azeotrope, the liquid can be shaken with

calcium oxide, which reacts strongly with water to form the
nonvolatile compound, calcium hydroxide.

• Nearly all of the calcium hydroxide can be separated by filtration

and the filtrate redistilled to obtain 100% pure ethanol.

• Anhydrous calcium chloride is used as a desiccant for drying a

wide variety of solvents since it is inexpensive and does not react
with most nonaqueous solvents.
2.2 Beer Production
Main ingredients
• Beer is usually made with four primary ingredients:
(1) water
(2) malted barley and adjunct
(3) hops
(4) yeast
Malt
• Malts are made from selected cereal grain usually barley, (but
sometimes wheat, oats, sorghum or millet).

• Malt is germinated barley or other cereals like wheat and

sorghum:

• First the grains are "steeped" bringing the water content from
about 12% to 45%, then they are allowed to germinate for 4-6
days and finally the germination is stopped by heating (kilning).

• Some enzymes are already present in the barley, e.g. β-amylases,

but the majority of enzymes are produced during the germination,
e.g. α-amylases and proteases, and in the final malt all the
enzymes needed for the conversion of "grains" into a fermentable
liquid (wort).
• The crucial role of malting is enriching the malt with enzymes
(amylolytic, proteolytic, etc.), modification of kernel endosperm,
and formation of flavor and aroma compounds.

• Starch-degrading enzymes (such as α-amylase, β-amylase, α-

glucosidase, and limit dextrinase) produced during germination are
better characterized than the proteolytic counterparts.

• At this point, germination is arrested by kilning (drying). A

varieties of malt can be derive from this point by kilning or
roasting at different temperatures.

• However, the more the malt is heat treated, the greater is the
damage to the enzymes. So, while Pilsener malts are the richest in
enzymes, chocolate malt (thoroughly roasted) have no enzymatic
activity at all.
• Mainly, two species of barley are used in brewing: the two-
row barley (with one grain per node) and the six-row barley
(with three grains per node).

• The fewer are the kernels per node, the bigger and richer in
starch they are.
• Conversely, the six-row barley has less starch but higher protein
content. Therefore, if the brewer wants to increase the extract
content, the two-row barley is the best option, whereas if
enzymatic strength is the aim, the six-row will be the best choice

Adjuncts
• Adjuncts are unmalted grains that are added.

• Worldwide, most breweries use alternative starch sources

(adjuncts) in addition to malted barley.

• Adjuncts are used to reduce the final cost of the recipe and/or
improve beer’s color and flavor/aroma.

• The most common adjuncts are unmalted barley, wheat, rice, or

corn, but other sugar sources such as starch, sucrose, glucose, and
corresponding sirup are also used.
• The use of adjuncts is only feasible because light malts (i.e., Pilsener
malt) have enough enzymes to breakdown up to twice their weight
of starch granules.

Water
• Water is the primary raw material used not only as a component of
beer, but also in the brewing process for cleaning, rinsing, and other
purposes.

• Thus, the quality of the “liquor,” which is how brewers call the
water as an ingredient, will also determine the quality of the beer.

• It has to be potable, free of pathogens. In addition, different beer

styles require different compositions of brewing liquor.

• Water has to be often adjusted previously to be ready as brewing

liquor.
• Adjustments involve removal of suspended solids, reduction of
unwanted mineral content, and removal of microbial
contamination.

• Calcium is perhaps the most important ion in the brewing liquor.

• It protects α-amylase from the early inactivation by lowering the

pH toward the optimum for enzymatic activity.

• Throughout boiling, it not only supports the precipitation of the

excess of nitrogen compounds, but also acts in the prevention in
over-extraction of hops components

• Furthermore, calcium also plays a crucial role through

fermentation, since it is mandatory for yeast flocculation
Minerals and chemicals needs to adjusted:
• Calcium
• Bicarbonate (Alkalinity)
• Magnesium
• Sodium
• Potassium
• Sulphate and Chloride
• Nitrate
Hops
• Compared to water and malts, hops are lesser of the ingredients
used in brewing, but no lesser is the contribution it makes to the
final beer.

• Hops influence to a large extent the final character of beer.

• Thus, some hops are known as “aroma/flavor hops” while others

as “bitter hops.”

• The α-acids are responsible for the bitterness of a given hop,

whereas aroma is tied to essential oils from hope cones.

• aroma hops are usually weaker in α-acids but rich in essential oils.
Conversely, bitter hops have higher contents of α-acid but may
lack on essential oils
• In addition to the bittering, flavoring and aromatic qualities that
hops bring to beer, they also serve as a stability agent, preventing
spoilage, contribute to head retention and act as a natural
clarifier.

• Nowadays, breweries rarely use cones, but pellets and hop extracts
instead.
• Pellets are made from raw hops by drying, grinding, screening,
mixing, and pelletizing.

• Extracts result from extraction with ethanol or carbon dioxide.

• The resulting product is a concentrated, resin-like sticky substance.

• The extracts and pallets are easier to be stored and have higher
shelf life but also different chemical compositions than hop cones.
Yeast
• Enzymes are responsible convert the starch to sugar so that the
yeast can use the energy stored in the starch.

• Yeast metabolizes the sugars from the grains and produces alcohol
and CO2.

• It is said that brewers make wort and yeast makes beer.

• Yeast also produces other fermentation by-products such as

phenols and esters that add significant flavor and aroma character
to beer ranging from delectable fruitiness to peppery spice
• Higher fermentation temperatures encourage the production of
greater amounts of esters and phenols, resulting in beers with
more yeast derived flavor and aroma.

• Lager yeasts ferment colder, between 45° F and 55° F.

• Colder temperatures inhibit the production of the various

fermentation by-products resulting in beers with a “clean”
yeast profile.
• Based on the type of fermenting yeast, beer can be classified in to
top fermenting yeast and bottom fermenting yeast.

Ale (top-fermenting yeasts)

• Ale yeasts ferment at warmer temperatures between 15°C and
20°C, and occasionally as high as 24°C.

• Pure ale yeasts form a foam on the surface of the fermenting beer,
because of this they are often referred to as "top-fermenting" yeast
- though there are some ale yeast strains that settle at the bottom.

• Ales are generally ready to drink within three weeks after the
beginning of fermentation, however, some styles benefit from
additional aging for several months or years. Ales range in color
from very pale to black opaque.
• Most of the time Saccharomyces cerevisiae strains are used as top
fermenting yeast.

• Fermentation systems developed to allow for the property of

separating the yeast from the top of the fermenter at the end of
fermentation.

• Removal of the yeast at the end of fermentation is traditionally by

manual skimming or suction of the froth or `head' directly from
the bulk of the fermented wort in the vessel.
Lager (bottom-fermenting yeasts)
• Lager yeast tends to collect at the bottom of the fermenter
and is often referred to as "bottom-fermenting" yeast.
• Saccharomyces ovarum is bottom fermented yeast.
• This allows most of the yeast to be separated, leaving the
beer comparatively free of yeast.
• Lager is fermented at much lower temperatures, around
10°C, which is lower as we compare it with top fermented
beer .
• It is stored for about 30 days or longer close. During the
storing or "lagering" process, the beer mellows and
flavors become smoother.
• Sulfur components developed during fermentation
dissipate.
• The fermenter must be equipped with a cooling system
to remove heat generated during fermentation and to
allow the control of temperature to the required profile.
Brewing process
Process consists of the following steps:
• Milling
• Mashing
• Boiling
• Cooling
• Fermenting
• Conditioning
• Packaging
Milling
• The malt is crushed in a malt mill to break apart the grain kernels,
increase their surface area, and separate the smaller pieces from the
husks, which allows it to contact enzymes in the mashing
processing.

• The more the kernel is broken up, the more sugars can be extracted
from the grains; but if it is broken up too much, the husk that
surrounds the kernel may get broken down, which can cause a stuck
mash.
Mashing
• The resulting grist from the milling unit operation is mixed with
heated water and heating continued in a vat called a "mash tun“.
Natural enzymes within the malt break down much of the starch
into sugars which play a vital part in the fermentation process.
• Mashing usually takes 1 to 2 hours, and during this time various
temperature rests (waiting periods) activate different enzymes
depending upon the type of malt being used, its modification
level, and the desires of the brew master.

• The activity of these enzymes convert the starches of the grains to

dextrines and then to fermentable sugars such as maltose.

• A mash rest at 40 °C activates beta-glucanase, which breaks

down gummy beta-glucans in the mash, making the sugars flow
out more freely later in the process.
• A mash rest from 49 °C to 55 °C activates various proteinases,
which break down proteins that might otherwise cause the beer to
be hazy.

• But care is of the essence since the head on beer is also composed
primarily of proteins, so too aggressive a protein rest can result in
a beer that cannot hold a head.

• A mash rest temperature of 65 to 71 °C is used to convert the

starches in the malt to sugar, which is then usable by the yeast
later in the industrial brewing process.

• Finally the mash temperature may be raised to (about 75°C

(known as a mashout) to deactivate enzymes.
• After the mashing, the mash is pumped to a lauter
tun where the resulting liquid is strained from the grains
in a process known as lautering.
• The lauter tun generally contains a slotted "false bottom"
or other form of manifold which acts as a strainer
allowing for the separation of the liquid from the grain.
Lautering
• Lautering is the separation of the extracts from the spent
grain.
• It is achieved in either a lauter tun, a wide vessel with a
false bottom, or a mash filter, a plate-and-frame filter
designed for this kind of separation.
• Lautering has two stages: first wort run-off, during
which the extract is separated in an undiluted state
from the spent grains, and sparging, in which extract
which remains with the grains is rinsed off with hot
water.
• At this point the liquid is known as wort.
• Before the mash starts, the grains don't taste at all
sweet, but the liquid that is drained off from the grains
at the end of the mash is very sweet and sticky. This
liquid, which now contains mostly fermentable sugars,
goes on to the boil.
Boiling
• The wort is moved into a large tank known as a "cooking tun" or
kettle where it is boiled with hops and sometimes other
ingredients such as herbs or sugars.

• The boiling process serves to terminate enzymatic processes,

precipitate proteins, isomerize hop resins, concentrate and sterilize
the wort.

• During the boil hops are added, which contribute bitterness,

flavor, and aroma compounds to the beer, and, along with the heat
of the boil, causes proteins in the wort to coagulate and the pH of
the wort to fall.

• Finally, the vapors produced during the boil volatilize off flavors,
including dimethyl sulfide precursors.
• In this process, it consumes about half of the energy in the
brewing process.
Whirlpool
• At the end of the boil, the wort is set into a whirlpool.
• The so-called teacup effect forces the more dense solids
(coagulated proteins, vegetable matter from hops) into a cone in
the center of the whirlpool tank.
Wort cooling
• After the brewing, the wort is filtered and quickly cooled to a
point that yeast can be safely added.
• This is the preparation of fermentation. The yeast does not grow in
a high temperature condition.
• After the hop filtration, the wort must be brought down to
fermentation temperatures before yeast is added.
Fermenting
• Fermentation, as a step in the brewing process, starts as soon as
yeast is added to the cooled wort. This is also the point at which
the product is first called beer.

• It is during this stage that extracted sugars from the malt are
metabolized into alcohol and carbon dioxide.

• Most breweries today use cylindroconical tanks, have a conical

bottom and a cylindrical top, in the case of lager beer.

• The cone's aperture is typically 60°, an angle that will allow the
yeast to flow toward the cones apex, but is not so steep as to take
up too much vertical space.
• At the end of fermentation, the yeast and other solids
which have fallen to the cones apex can be simply
flushed out a port at the apex.
Conditioning processes
• Primary stage is over, the majority of the wort sugars have been
converted to alcohol, and a lot of the yeast are going dormant;
but there is still yeast activity.
• During the earlier phases, many different compounds were
produced by the yeast in addition to ethanol and CO 2, e.g.,
acetaldehyde, esters, amino acids, ketones- diacetyl,
pentanedione, dimethyl sulfide, etc.
• Once the easy food is gone, the yeast start re-processing these
by-products. Diacetyl and pentanedione are two ketones that
have buttery and honey-like flavors.
• These compounds are considered flaws when present in large
amounts and can cause flavor stability problems during
storage.
• Acetaldehyde is an aldehyde that has a pronounced green apple
smell and taste.
• It is an intermediate compound in the production of ethanol.
The yeast reduce these compounds during the later stages of
fermentation
Filtering
• Filtering the beer stabilizes the flavor, and gives beer its
polished shine and brilliance. Not all beer is filtered.
• Filters come in many types. Many use pre-made filtration
media such as sheets or candles, while others use a fine
powder made of, for example, diatomaceous earth, also
called kieselguhr, which is introduced into the beer and
recirculated past screens to form a filtration bed.
• Filters range from rough filters that remove much of the
yeast and any solids (e.g. hops, grain particles) left in the
beer, to filters tight enough to strain color and body from the
beer.
• Normally used filtration ratings are divided into rough,
fine and sterile.
• Rough filtration leaves some cloudiness in the beer, but it
is noticeably clearer than unfiltered beer.
• Fine filtration gives a glass of beer that you could read a
newspaper through, with no noticeable cloudiness.
• Finally, as its name implies, sterile filtration is fine
enough that almost all microorganisms in the beer are
removed during the filtration process.
• A beer which is filtered is stable, so all conditioning has
stopped - as such it is termed "brewery-conditioned".
Beers which are in contact with the yeast are known as
bottle-conditioned or cask-conditioned
• The filtered beer is checked for CO2 levels, then it will
be adjusted prior to packaging.
• Then the beer either goes keg filler Or to bottling line for
packaging.
Packaging
• Packaging is putting the beer into the containers
in which it will leave the brewery.
• Typically this means in bottles and kegs, but it
might include cans or bulk tanks for high-volume
customers.
• What is the difference between drought and beer?
• Why brown glasses are used for bottling?
• What is non alcoholic beer and how it is produced?