See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/358266686

industrial microbiology book 11

Book · February 2022

CITATIONS READS
0 1,965

1 author:

Constance Chinyere Ezemba

ChyChy Gilgal Laboratories and Consultancy @Chukwuemeka Odumegwu Ojukwu University
124 PUBLICATIONS   191 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Production and Comparative Physicochemical Analysis of Vinegar from Locally Grown Fruits in Nigeria and Industrial Produced Vinegar View project

Fungal Quality of Fresh Shrimp View project

All content following this page was uploaded by Constance Chinyere Ezemba on 01 February 2022.

The user has requested enhancement of the downloaded file.

CHUKWUEMEKA ODUMEGWU OJUKWU UNIVERSITY ULI, ANAMBRA STATE.
DEPARTMENT OF MICROBIOLOGY
FACULTY OF NATURAL AND APPLIED SCIENCES SECOND SEMESTER
MCB 426 INDUSTRIAL MICROBIOLOGY II (2 CREDIT LOAD)
LECTURER: DR. CHY. EZEMBA YEAR 4

COURSE OUTLINE

COURSE UNITS/CHAPTERS

Chapter 1
1.0 STERILITY AND STERILIZATION: STEAM, FILTRATION.

2.0 EXTRACTION OF INUDUSTRIAL MATERIALS: CENTRIFUGATION,

FILTRATION, SEDIMENTATION e.t.c. i.e.downstream processing which include:
-Cell separation (separation of cells or extracellular products from the culture broth).
-Cell disruption(for intracellular products)
-Clarification (separation of cell debris after disruption)
-Concentration of products.
-Purification of products
-Extraction
-Purification of product
-Product polishing.

3.0 PRODUCTION OF ALCOHOL-BASED GOODS: INDUSTRIAL ALCOHOL BEER,

STOUT, SPIRIT. NON-ALCOHOL; MALT, YOUGURT, VINEGAR.

Dr. Chy. Ezemba

CHAPTER 1: STERILITY AND STERILIZATION
1.0 Media Sterilization & It Importance
A. In a laboratory, culture media are contained with glass or silicon, petri dishes & test
tubes, bottles with screw caps and flasks, magnetic stirrrer bars, silicon tubing.
Where in industries, media are contained in a kegs, drums, tanks,
B. Sterilization of nutrition media, Prior to be used the culture media are all being labelled,
properlly dispensed sealed with plastic closure or pluged with cotton wool and sterilized.
Culture media is sterilized in an autoclave (steam sterilzation) at15psi (pounds of
pressure) at 121oC for 30min.
C. It is also important to make sure that the containers are not filled to capacity, otherwise
they may boil over in the autoclave or the container may crack or break. Prior to being
used, all containers should be labeled with :
-the type of culture media that they contain.
-Date, when the culture media was prepared
-Initials of the person who prepared them.
Most importantly the pettri dish must be labled on the bottom plate, never on the lid.
D. Sterilization of glass wares: glass wares (petri plates, vials, culture tube, flasks, pipettes
& metallic instruments are sterilized in a hot air-oven at 160 – 180 0C for 2 – 4 hours.
E. Sterilization of instruments: The metallic instruments e.g. forceps, scalpels, needles,
spatulas etc are flame sterilized. i.e. dipping them in 75% ethanol followed by flaming
and cooling (incineration).
F. Sterilization of culture room & transfer area: The floor and table are 1 st washed with
detergent then 2% sodium hypochlomte or 95% ethanol. Lager surface area is sterilized
by exposure to UV light.
The cabinet of laminar airflow is also sterilized by exposing UV light for 30min & 95% ethanol
15 minutes before beginning to work. The UV radiation is harmful to eyes & skin.
Major difference between sterilization of media in laboratory & industry is the much greater
scale of the former. In the laboratory a liter of medium would probably require ten minutes to
attain the sterilizing temperature 15 min at 1210C then another 10 – 15 min, making a total of 40
– 45 min. while a 10,000 liter medium the equivalent period may well take several hours for each
of the three periods.

1.1 Industrial Sterilization

In the fermentation industry contaminates by unwanted MOS could pose serious problems
because of the vastly increased scale of the operation in comparison with laboratory work.
Contamination in industrial microbiology could lead to huge financial losses to
fermentation firm. Imagine in the beer industry when lactic acid bacteria Pediococus
streptococcus damnosus contaminate the fermentating wort, they utilize sugars present therein to
produce unwanted lactic acid which renders the beer sour.

 Losses by lytic MOS such as bacteriophages or Bdellovibrio may lead to the death of
producing organism.
  Contaminant may utilize the components of the fermentation both to produce unwanted
end-products, alter the environmental condition such as pH or oxidation-reduction
potential and therefore reducing yield in the final product. Removal of these by-products
through extraction process may be expensive & time consuming since it not already
established in the factory.
Sterilization is the process by which all living cells, viable spore viruses and viroids are either
destroyed or removed from an object or habitat. A sterile object is totally free of viable mos,
spores and other infectious agent.

Control and Sterilization Methods

They are categorized into physical & chemical methods.
A. Physical Method
1. Asepsis is a procedure routinely observed in many microbiological, pharmaceutical and
food industries. It involves general cleanliness hand wash, washing of pipes, utensils,
fermentation vats, wearing protective clothing such as gloves, masks & laboratory coats.
This controls and reduces the load of microorganisms, also hence lessens the stringency
of the sterility measures employed.
Heat and other physical affects are normally used to control microbial growth and
sterilize objects, as seem from the continual operation of the autoclave in every
microbiology laboratory.
The four most frequently employed physical agents are heat, low temperature filtration &
radiation.
2. Heat – may be applied dry or moist
Dry heat: Many objects are best sterilized in the absence of H 2O by heat serilization. It is used to
sterilize glassware, but in large scale used to sterilize some types of air filters.
Principally, the air is sterilized by compression.
When air is compression the temperature rises in accordance with the gas law.
PV = RT
P = Pressure R = Gas constant
V = Volume T= Temperature

e.g incineration(reduced to ash), sterilization oven 170 0C for 2hrs.

Advantages of dry heat
It does not corrode glassware and a metal instrument as moist heat does.
It can be use to sterilized powders, oils and similar items.
Disadvantages of dry heat
It is slow and not suitable for heat sensitive materials like many plastics and rubber items.
Most laboratories sterilize glass petri dishes and pipette.
it is expensive.
Moist heat:
Sterilization must be carried out at temperature above 1000C, in order to destroy bacterial
endospores and this requires the use of saturated steam under pressure.
 1. Boiling: at 1000C to kill vegetative cells and most viruses.
2. Autoclaving: Steam sterilization where by the air initially present with chamber is forced
out until the chamber is filled with saturated steam and the outlays are closed at
temperature of 121oC & pressure of is 15 psi developed by chamberland in 1884.
3. Tyndallization: consists of boiling the material for one half hour on three consecutive
days. 1st days, vegetative cells are killed, 2nd day spores germinated will be killed then 3rd
day those vegetative spores resulting from the 2nd day spores are killed. This method is
used for sterilizing heat - labeled media where medium is too viscous for filtration.

4. Pasteurization: It consists of exposing the food or material to a temperature well below

boiling for a sufficiently long period to destroy pathogenic or spoilage organisms
developed by Louis Pasteur in 1880. It can either be:
A. Batch: Using low temperature long time (LTLT) technique heating at about (63 0C for
30mins). When used for large scale it is attained by gradual increase of temperature,
holding at the pasteurization temperature and cooling all the equally divided by 90
minutes.
B. Continuous: Involves the use of flash method or high temperature short time (HTST)
pasteurization, heating at about 720C for 15 seconds, then rapid cooling.
C. Ultra high temperature (UHT) sterilization, heatmilk and milk products at 140 – 150 0C
for 1 – 3 sec in the dairy industry . It used for treating beer and wine (food industry).
Advantages
It kills pathogens (vegetable cells) and food spoilage MOS without destroy the food.
Disadvantage
The prolong time at high temperature may give rise to a “burnt” odor is the major
deficiency of batch in comprision with continuous pasteurization.
3. Steam under pressure: is used for sterilization of equipment in the laboratory, as well as in the
industry (pipes, fermentors & media). This steam is under pressure, because higher the pressure
higher the temperature. It kills vegetable spore, viruses & endospores. Although dry air heat is
less effective than most heat. Clostridium botulium spores are killed in 5min at 1210C by moist
heat but only after 2 hours at 1600C with dry heat. It is also used for heat.
4. Low temperatures:
Although, our emphasis has been on the destruction of MOS, often the most convenient control
technique is to inhibit the growth and reproduction by the use of either;
FREEZING: At 200Cor lower stops microbial growth & kill but does not destroy contaminating
microbes.
REFRIGERATION: Greatly slows microbial growth reproduction but does not halt it
completely.
5. Filtration – Is an excellent way to reduce the microbial population in solutions of heat-
sensitive material rather directly destroying contaminating MOS, the filter simply removes them.
Large volumes of sterile air and other gases are required especially in pharmaceuticals industries
where injection and vaccines are handled. They are used for aeration in most fermentation. They
are two (2) types of filter: Depth filters; consist of fibrous or granular materials that have been
bonded into a thick layer filled with twist channels of small diameter. The solution containing
MOS is sucked through this layer under vaccum and microbial cells are removed by physical
screening or entrapment. And also by absorption to the surface of ..
6.Radiations: The radiations used for sterilizing are Ultra Violet light, X – rays and Gamma
rays. X – rays and Gamma rays are extremely high frequency electromagnetic waves, which
have enough photon energy to produce ionization (create positive and negative electrically
changed atoms or parts of molecules by knocking off the electrons on the outer orbits of atoms of
the materials through which they pass. The atoms losing the electrons and those accepting them
become ionized on account of the electron changes.
Gamma ray are generated from X-ray machine & produced by the spontaneous decay of
radioactive metal such as cobalt 60 (C0). They are used to sterilize plastic syringes, rubber
gloves and other materials which are liable to damage by heat or chemicals.
Ultraviolet light: Visible light falls between wavelength of 400 and 700nm. Ultraviolet light
(UV) ranges from 100 – 400nm with germicidal range is 254nm peak between 200 – 300nm. The
process of the killing of microbes by UV involves absorption of a UV photon by DNA chains,
which causes a distribution in the DNA chain by causes adjacent thyamine bases to dimerism or
become linked. The organism’s metabolism is disrupted and it may eventually die.
Disadvantage
UV light does not penetrate, so act mainly on the surface. Therefore, it used for sterilizing the air
in fermentation halls & other such large open spaces and for creating mutations in culture
improvement.

B. Chemical Method: These can be divided into 2 groups

i. Chemosterilants: when sterilization is achieved by the use of chemical agent, the
chemical is called sterilant. which kill both vegetative cells as well as spores of bacteria,
fungi, viruses & protozoa.
ii. Disinfectants: Which can at least kill, inhibit & remove unwanted pathogenic(MOS
causing diseases) or spoilage organisms on an inanimate object. But does not kill spores
or even some vegetative cells.
iii. Sanitization: is reduction of microbial population which is reduced to levels that are
considered safe by public health standards.
iv. Antisepsis: is the prevention of infection or sepsis by the use of antiseptics.

Revision Question
1. Discuss the effects of contaminants on microbial cell cultures.
2. Discuss any four methods used for sterilization of culture media, pointing out their advantages
and disadvantages.
3. Discuss the adverse effects of heat sterilization on media components and methods of reducing
such effects.
4. What is autoclave? Discuss the factor that affect the efficacy of sterilization by high-pressure
steam
CHAPTER 2

2.0 EXTRACTION OF INDUSTRIAL MATERIALS

Downstream processing
Downstream processing refers to the isolation and purification of a biotechnologically formed
product to a state suitable for the intended use. The term is also used for recovery and
purification of natural products from plants, animals, microorganisms and purification of natural
products from cell culture broth.
In most, but not all, biotechnology processes the desired product(s) will be in dilute aqueous
solution and the ultimate level of downstream processing will mirror the type of product and
required degree of purity. The range of products is considerable and varied in form and can
include whole cells, amino acids, vitamins, organic acids, solvents, enzymes, vaccines,
therapeutic proteins and monoclonal antibodies, which some are cell biomass, while some cases
the products are either stored within the cell (intra-cellular products or secreted into the culture
broth (extra-cellular products). Within these products there will be considerable variation in
molecular size and chemical complexity, and a wide range of separation methods will be
required for recovery and purification. While many of the products are relatively stable in
structure, others can be highly labile and require careful application of the methodology. In any
case, the product must be separated and purified to degree of purity.
The primary objective of most cell cultures is to produce some products, although microbial,
plant and animal cells can synthesize many classes of useful metabolites, such metabolites can
only be commercially produced if they are synthesized in the form and quantity that can be
economically isolated and purified to desired degree of purity. Downstream processing will
primarily be concerned with initial separation of the bioreactor broth into a liquid phase and a
solids phase and subsequent concentration and purification of the product. Downstream
processing is a multistage operation (Figure 1).
It is the last step in manufacture of purified products fit for a specific use, which must meet a st
of standard for the product. the amount of contaminates (biological, chemical and physical
contaminants) must be low enough to pass the set standards.
The design and efficient operation of downstream processing operations are vital elements in
getting the required products into commercial use and should reflect the need not to lose more of
the desired product than is absolutely necessary. An example of the effort expended in
downstream processing is provided by the plant Eli Lilly built to produce human insulin
(Humulin). Over 90% of the 200 staff are involved in recovery processes. Thus, downstream
processing of biotechnological processes represents a major part of the overall costs of most
processes but is also the least-heralded aspect of biotechnology.
Improvements in downstream processing will benefit the overall efficiency and costs of the
processes.
The major unit operations in downstream processing include:
1. Cell separation (separation of cells or extracellular products from the culture broth).
2. Cell disruption(for intracellular products)
3. Clarification (separation of cell debris after disruption)
4. Concentration of products.
5. Purification of products
6. Extraction
7. Purification of product
8. Product polishing. Then Final product!!!
 Crude products

Extracellular Intracellular
Biomass

Cell disruption Mechanical and

non mechanical
Cell separation Sedimentation methods.
Clarification Filtration
Centrifugation

Washing

Concentration Evaporation
Adsorption
Precipitation
Ultra filtration
Disruption

Purification
Chromatography
Electrophoresis
Crystallization

Product polishing Freeze drying

Spray drying
Sterile filtration

Final products

Fig. 1: Schematic Flow Diagram of Unit Operations in Downstream

Processing (Ogbonna, J.C. 2012)
2.1 Cell Separation Techniques of Industrial Materials

Cell separation:
Cell separation, also commonly referred to as cell isolation or cell sorting, is a process to isolate
one or more specific cell populations from a heterogeneous mixture of cells. There are a number
of cell separation methods available, each with its own advantages and disadvantages.

Types of Cell Separation Techniques:

There are many different ways to isolate cells from complex biological samples. Common
characteristics used to isolate cells include; cell size, cell density, cell shape, and surface
protein expression. The most common cell separation techniques include:
 Immunomagnetic cell separation
 Fluorescence-activated cell sorting (FACS)
 Density gradient centrifugation
 Immunodensity cell separation
 Microfluidic cell sorting
 Sedimentation
 Adhesion
There are also less commonly used cell separation methods, including buoyancy-activated cell
sorting, aptamer-based cell isolation, complement depletion, and more.

Note: The cell separation method you choose typically depends on what you intend to use the
isolated cells for, and the choice may involve a trade-off. For example, if you need very pure
cells, you will likely choose a method with high purity but that may result in lower yield.

1.Immunomagnetic Cell Separation Technique:

Immunomagnetic cell separation is a technique whereby magnetic particles are used to isolate
target cells from heterogeneous mixtures. To accomplish this, the magnetic particles are bound to
specific cell surface proteins on the target cells via antibodies, enzymes, lectins, or streptavidin.
The sample is then placed in an electromagnetic field that pulls on the magnetic particles,
bringing the labeled cells with them. The unlabeled cells remain in the supernatant, thus creating
a physical separation between target and non-target cells within the sample.
Due to its speed and simplicity, immunomagnetic cell separation is one of the most commonly
used methods by which scientists isolate highly purified populations of specific cell subsets.
Immunomagnetic cell separation has several advantages, including:
 High purity
 Fast protocols
 Ease of use
 Low equipment cost
 Many cells can be isolated at once
 Potential for automation
 High cell viability

Both positive and negative selection can be performed using magnetic cell isolation methods.
When a positive selection is performed, the supernatant can be discarded and the magnetically-
labeled cells of interest remain immobilized until removed from the electromagnetic field. When
a negative selection is performed, the desired cells are located in the supernatant.

2.Fluorescence-Activated Cell Sorting (Facs):

Fluorescence-activated cell sorting (FACS) is a method that uses flow cytometry and fluorescent
probes to sort heterogeneous mixtures of cells. Fluorophore-tagged antibodies bind to epitopes
on specific antigens on the target cells within a single-cell suspension. After tagging, the flow
cytometer focuses the cell suspension into a uniform stream of single cells. This stream is then
passed through a set of lasers that excites the cell-bound fluorophores, causing light scattering
and fluorescent emissions. Based on the wavelengths produced by the laser excitation, the
resulting photon signals are converted into a proportional number of electronic pulses that assign
a charge to the droplet that is formed around the cell. As each droplet falls between the deflection
plates, its charge causes the droplet to either be deflected into collection tubes or fall into the
waste chamber.
Immunomagnetic cell separation is a much faster and simpler procedure than FACS, and is often
the preferred cell isolation method for common cell types. FACS has several advantages over
immunomagnetic cell separation including the ability to:
 Sort single cells
 Isolate cells based on intracellular markers (e.g. GFP)
 Isolate cells based on surface marker expression levels
 Sort complex cell types with multiple markers at higher purity

3.Density Gradient Centrifugation

Density gradient centrifugation relies on the varying densities of cells within a heterogeneous
sample. The sample is layered on top of a density gradient medium before being centrifuged.
During centrifugation, each cell type will sediment to its isopycnic point, which is the place in
the medium gradient where the density of the cells and medium are equal.
Common applications include;
 Fractionation of peripheral blood mononuclear cells
 Exclusion of dead cells from a cell culture
 Separation of plasma from blood cells.

There are several types of density gradient media, each with unique properties that render them
ideal for different purposes. The following are examples of the most well-known types:

 Lymphoprep™, Lympholyte®, and Ficoll-Paque® are similar media that consist of

saccharides and sodium diatrizoate; they have a density of 1.077 g/mL. These media are
commonly used to isolate mononuclear cells from peripheral blood, cord blood, and bone
marrow.
 Percoll® (density: 1.131 g/mL) consists of colloidal silica particles coated with
polyvinylpyrrolidone (PVP) and is widely used to separate cells, organelles, viruses, and
other subcellular particles.
 OptiPrep™ is a medium consisting of iodixanol in water that is used to isolate viruses,
organelles, macromolecules, and cells.
Density gradient centrifugation is an inexpensive cell separation technique but has limited
specificity, low purity, and low throughput. In addition, even though it is a common laboratory
technique, density gradient centrifugation can be a slow and laborious process that is difficult to
master. Scientists typically need to carefully layer their sample over the density gradient
medium, centrifuge for 30 minutes without brakes, then carefully harvest and wash the
appropriate layer of cells. Technologies like SepMate™ make this method easier and faster.
SepMate ™ is a specialized tube that allows users to quickly layer blood over the density
gradient medium, prevents the layers from mixing and facilitates fast and easy harvesting of the
target cells. With SepMate™, cells can be obtained in as little as 15 minutes.

4.Immunodensity Cell Separation:

Immunodensity cell separation, also referred to as erythrocyte rosetting, is a negative selection
method that uses a combination of antibody-based labeling and density gradient centrifugation.
With this method, antibodies are added to a whole blood sample, labeling the unwanted cells and
cross-linking them to red blood cells. These results in the formation of complexes called
immunorosettes that are much denser than the mononuclear cells being isolated. During
centrifugation, the unwanted cells pellet with the red blood cells, leaving the target cells in a
layer above the density medium.
Immunodensity cell separation doesn’t require any specialized equipment beyond a centrifuge,
can be easily incorporated into established density gradient centrifugation protocols, and can be
used to isolate specific cell subsets. However, the technique is limited to negative selection.

5. Microfluidic Cell Separation:

Microfluidics is an umbrella category of cell separation methods. Designed to manipulate fluids
on a microscopic level in order to facilitate single-cell isolation, microfluidic technologies are
frequently built onto microchips and are also commonly known as “lab-on-a-chip" devices.
These devices have several advantages, including the smaller volumes of samples and reagents
required for use. Lab-on-a-chip devices are also portable, allowing them be used virtually
anywhere, making them particularly useful as field-based diagnostic tools.
Microfluidic methods can be divided into active and passive systems. Active microfluidic
systems involve external forces, whereas passive microfluidics make use of the cell’s density and
mass in combination with gravity. These methods can also be classified by the presence or
absence of cell labeling; although some methods involve labeling cells with antibodies, most
methods are known for being label-free. There are several different microfluidic methods used
for cell isolation, including:
Acoustophoresis
Aqueous two phase systems
Biomimetic microfluidics
Cell affinity chromatography
Deterministic lateral displacement
Electrophoretic sorting
Field flow fractionation
Gravity and sedimentation
Magnetophoresis
Microfiltration
Optical sorting.
6.Sedimentation:
Sedimentation works on the basis that gravity will cause larger and denser components to
sediment faster than materials that are smaller and less dense. The largest and densest
components in a sample can be pelleted through an initial low-force centrifugation due to their
high rate of sedimentation. The supernatant can then be spun again. Through successive
centrifugations, components with an increasingly lower rate of sedimentation can be isolated.
NB: sedimentation is inexpensive but generally results in lower purity than other methods.

7.Adhesion:
The unique adhesion profiles of different cell types can be used to separate target cells from
heterogeneous populations. By choosing suitable growth factors and culture plates to selectively
favor or inhibit adhesion, adherent cells can be separated from cells in suspension.

2.2 Cell Disruption

Cell disruption: is the process of obtaining intracellular fluid via methods that open the cell
wall. The overall goal in cell disruption is obtain the intracellular fluid without disrupting any of
its components. The method used may vary depending on the type of cell and its
composition.Irrespective of the method used, the main aim is that the disruption must be
effective and the method should not be too harsh so that the product recovered remains in its
active form.
Cell disruption can be categorized in to mechanical and non- mechanical
Mechanical methods are divided in to solid shear methods and liquid shear methods.
Non- mechanical methods can be divided in to physical methods and chemical methods and
enzymatic methods.

a.Mechanical methods:are those methods which required some sort of force to separate out
intracellular protein without adding chemical or enzyme.
1. Mortar and pastel/grinding
2. Blender
3. Bead beating
4. Ultra sonication
5. Homogenization
Mortar &pestle
 It involves the grinding of the cells such that they are disrupted.
 This does not have to be in suspension and is often done with plant samples frozen in
liquid nitrogen.
 When the material has been disrupted, metabolities can be extracted by adding solvents.
Blenders
 The use of blenders which employ high speed can be used to disrupt cell walls.
 It is the same process used by centrifugation, which separates or concentrates materials
suspended in a liquid medium.
Bead beating
 Glass or ceramic beads are used to crack open cells
 The kind of mechanical shear is gentle enough to keep organelles intact.
  It can be used with all kinds of cells, just add beads to an equal amount of cell suspension
and vortex.

Ultra sonication
 Ultra sonic homogenizers work by inducing vibration in a titanium probe that is
immersed in the cell solution.
 A process called cavitation occurs, in which tiny bubbles are formed and explode,
producing a local shockwave and disrupting cell walls by pressure change.
 This method is very popular for disruption of plant and fungal cells.
Homogenization
 Liquid-based homogenization is the most widely used cell disruption technique for small
volumes and cultured cells.
 Cells are lysed by forcing the cell or tissue suspension through a narrow space
 Homogenizers use shearing forces on the cell similar to the bead method.
 Homogenization can be performed by squeezing cells through a tube that is slightly
smaller than beads beating.

b.Non –mechanical methods are further divided in to three classes, Fig 2 which are following:
Physical methods
Freeze thaw
 It is suitable when working with soft plant material and algae.
 Disruption is achieved via a series of freezing and thawing cycles.
 Freezing forms ice crystals which expand upon thawing, and this ultimately causes the
wall to rupture.

Microwave/thermolysis
 Microwave (along with autoclave and other high temperature methods) are used to disrupt
the bonds within cell walls, and also to denature proteins.
 However, uncontrolled amount of heat can easily denature or damage target proteins and
substances.

Osmotic shock
 Through the process of osmosis, water can be moved in to the cell causing its volume to
increase to the point that it bursts.
 The method however, can only work with animal cells and protozoa, since they do not
have cell walls.
Electric discharge
 It is also possible to achieve cell disruption via electrical discharges in mammalian and
other cells that are bounded by plasma membranes only.
Chemical methods
 They are often used with plants cells(and sometimes in combination with shearing)
 Organic solvents such as toluene, ether, benzene, methanol, surfactants, and phenyl ethyl
alcohol DMSO can be used to permeate cell walls.
 EDTA can be used specifically to disrupt the cells walls of gram negative bacteria, whose
cell walls contain lipopolysaccharides that are stabilized by cations like mg2+ and ca2+.
 EDTA will chelate the cations leaving holes in the cells.
Fig. 2: Non –mechanical methods
2.3 INDUSTRIAL CLARIFICATION

This the process of making clear or transparent by freeing visible impurities, particularly, the
clearing or fining of liquid substances from feculent matter by the separation of the insoluble
particles which prevent the liquid from being transparent.

The term, “clarification,” is usually applied to the removal of small concentration of solid
particles from fluids. The amount of particles present and to be removed is typically less than 1%
and is often as low as or lower than 100 parts per million (ppm). The particle size, however, may
vary significantly, ranging from the larger particles, which can be easily removed by
sedimentation, to those of colloidal size. For solid–liquid systems, Purchas (1967) classifies
these processes according to the type of driving force applied: (1) gravity, (2) vacuum, (3)
pressure, and (4) centrifugal force. According to this classification, granular filtration may be
operated with either gravity or pressure. Alternatively, one may characterize a clarification
process by the size of particles it is capable of removing. The limit of clarifying power of various
filter media given by Purchase.

Most clarification processes operate on the principle of exclusion; in other words, the
dimensions of the pore spaces of the filter media are such that particles present in the liquid are
excluded. Such a particle-deposition mechanism is known as straining or sieving

A.Clarification of Wine
Natural clarification takes place as wine ages in barrel, its suspended particles gradually falling
to the bottom.
In wine tasting, a wine is considered "clear" when there are no visible particles suspended in the
liquid and, especially in the case of white wines, when there is some degree of transparency. A
wine with too much suspended matter will appear cloudy and dull, even if its aroma and flavor
are unaffected; wines therefore generally undergo some kind of clarification.

[1] Pectins are structural molecules in the cell walls of fruits which have the important function
of 'gumming' plant cells together. The pectin content of grapes increases steadily throughout
ripening, reaching levels of about 1 g/l, although it varies by varietal and pre-fermentation
handling processes. Large pectin molecules can affect the amount of juice yielded at pressing,
ease of filtration and clarification, and extraction of tannins. Grapes contain natural pectolytic
enzymes responsible for softening the grape berries during ripening, but these are not active
under wine-making conditions (due to pH level, SO2, and alcohol.) Therefore, fungal pectolytic
enzymes are often added to white must to break up pectins, decrease the viscosity of the juice,
and speed up settling. In red musts, this increases color and tannin extraction.

After fermentation, the force of gravity may eventually cause the wine to "fall bright" or clarify
naturally, as the larger suspended particles gradually settle to the bottom of the storage vessel.
The wine can then be siphoned or "racked" off the compact solids into a new container. But this
process may take many months, or even years, as well as several rackings, in order to produce a
perfectly clear wine.

Producers can accelerate the process by using fining agents, filtration and/or flotation.[1]

Clarification and stabilization may involve fining, filtration, centrifugation, flotation,

refrigeration, pasteurization, and/or barrel maturation and racking.

Fining
In winemaking, fining is the process where a substance (fining agent) is added to the wine to
create an adsorbent, enzymatic or ionic bond with the suspended particles, producing larger
molecules and larger particles that will precipitate out of the wine more readily and rapidly.
Unlike filtration, which can only remove particulates (such as dead yeast cells and grape
fragments), fining can remove soluble substances such as polymerized tannins, coloring phenols
and proteins; some of these proteins can cause haziness in wines exposed to high temperatures
after bottling. The reduction of tannin can reduce astringency in red wines intended for early
drinking. Many substances have historically been used as fining agents, including dried blood
powder.
Today, there are two general types of fining agents — organic compounds and solid/mineral
materials.

Organic compounds used as fining agents are generally animal based, a possible cause of
concern to vegans.

The most common organic compounds used include egg whites, casein derived from milk,
gelatin and isinglass obtained from the bladders of fish. Pulverized minerals and solid materials
can also be used, with bentonite clay being one of the most common, thanks to its effectiveness
in absorbing proteins and some bacteria. Activated carbon from charcoal is used to remove some
phenols that contribute to browning as well as some particles that produce "off-odors" in the
wine.

In a process known as blue fining, potassium ferrocyanide is sometimes used to remove any
copper and iron particles that have entered the wine from bentonite, metal winery and vineyard
equipment, or vineyard sprays such as Bordeaux mixture. Because potassium ferrocyanide may
form hydrogen cyanide its use is highly regulated and, in many wine producing
countries,[which?] illegal. Silica and kaolin are also sometimes used.

Some countries, such as Australia and New Zealand, have wine labeling laws that require the use
of fining agents that may be an allergenic substance to appear on the wine label. A study
conducted by the University of California, Davis Department of Viticulture and Enology,
however, found that no detectable amount of inorganic fining agents, and only trace quantities of
proteinaceous agents, are left in the wine.

There is the risk of valuable aromatic molecules being precipitated out along with the less
desirable matter. Some producers of premium wine avoid fining, or delay it in order to leach
more flavor and aroma from the phenols before they are removed.

Filtration
Diatomaceous earth, often used in depth filtration.
While fining clarifies wine by binding to suspended particles and precipitating out as larger
particles, filtration works by passing the wine through a filter medium that captures particles
larger than the medium's holes. Complete filtration may require a series of filtering through
progressively finer filters. Many white wines require the removal of all potentially active yeast
and/or lactic acid bacteria if they are to remain reliably stable in bottle, and this is usually now
achieved by fine filtration.
Most filtration in a winery can be classified as either the coarser depth filtration or the finer
surface filtration.

In depth filtration, often done after fermentation, the wine is pushed through a thick layer of pads
made from cellulose fibers, diatomaceous earth, or perlite.

In surface filtration, the wine passes through a thin membrane. Running the wine parallel to the
filter surface, known as cross-flow filtration, will minimize the filter clogging. The finest surface
filtration, microfiltration, can sterilize the wine by trapping all yeast and, optionally, bacteria,
and so is often done immediately prior to bottling. An absolute rated filter of 0.45 µm is
generally considered to result in a microbially stable wine and is accomplished by the use of
membrane cartridges, most commonly polyvinylidene fluoride (PVDF). Certain red wines may
be filtered to 0.65 µm, to remove yeast, or to 1.0 µm to remove viable brettanomyces only.

Flotation
The winemaking technique of flotation was adapted from the froth flotation process used in the
mining industry for ore refining. In this process, small bubbles of air (or compressed nitrogen)
are injected into the bottom of a tank. As the bubbles rise through the must, grape solids,
including phenolic compounds prone to oxidation and browning, will tend to cling to the
bubbles, creating a froth that can be removed from the wine. This must be done prior to
fermentation, since yeast will inhibit the flocculation involved.

B.Water Clarification

Water clarification involves removing solids and other suspended particles from water to make it
clear. Water clarification is the typical first step for treatment of surface water and the next step
prior the biological treatment of waste water. There are a variety of different clarification
systems in different configurations and sizes that can meet your individual needs.

Circular Clarifier
Circular clarifiers are more commonly used in water and waste water treatment particularly in
domestic or municipal waste water treatment plants where footprint is not a major concern.
Solids and floating+ scum are removed by gravity separation. In the process, the sludge settles
and accumulates at the bottom of the tank. The circular clarifier is configured with sludge
removal mechanisms to efficiently collect and evacuate the sludge. Circular clarifiers have the
option of a full or a half bridge design. There is also the option of a steel or concrete tank.

Vertical Parallel Plate or Lamella Plate Clarifier

Vertical parallel plate or lamella plate clarifier are usually used to remove suspended solids and
coagulated-flocculated particles from process waters and industrial waste waters. Lamella
clarifier are designed so that the settling surface area is several times more than the actual cross-
sectional area or floor surface area of the clarifier. This is done through its inclined lamella plate
design. The resulting design doesn’t take up as much space as other similar sized circular
clarifier. This design is cost effective to install and is low maintenance and uses no moving parts
in the settling area.

Parallel Plate Horizontal Clarifier

Parallel plate horizontal clarifier separate solids and aid in oil recovery. The parallel plate
horizontal clarifier can do practically all the applications that a circular clarifier can do using less
space. The size depends on the use of the tank so that it is most effective. This means that each
clarifier is custom built.

C.Clarification of Sugarcane
The clarification of sugar cane juice occurs by coagulation, flocculation, and precipitation of the
colloids and pigmented substances, which are later eliminated by decanting and filtration, i.e., an
insoluble precipitate which absorbs and drags such compounds from the juice is formed.
Flocculation can be carried out by changing the pH, using chemical reagents, or through heating

D.Pharmaceutical Clarification

Key points
• Clarification processes are widely employed in the industry in the preparation of drug
substances and drug products.
• Clarification can be achieved using either filtration or centrifugation techniques.
• The main pharmaceutical uses of clarification are to remove unwanted solid particles from
fluids or to separate a required solid from a fluid.
 • Straining/sieving and impingement are the main mechanisms by which filtration occurs.
• The rate at which a filtration process occurs depends on the properties of the product being
filtered and the design and operation of the filtration equipment.
• Darcy’s equation combines the factors responsible for determining the filtration rate and
shows how these factors may be manipulated to control the rate of filtration.
• Filters used for liquid products may be classified as either gravity filters (e.g. simple
laboratory filters), vacuum filters (e.g. the rotary vacuum filter) or pressure filters (e.g.
cartridge filters).
• Centrifugal force can be utilized to enhance the separate of solids from liquids (e.g.
perforated-basket centrifuges).

Introduction
Clarification is a term used to describe processes that involve the removal or separation of a solid
from a fluid, or a fluid from another fluid. The term ‘fluid’ encompasses both liquids and gases.
Clarification can be achieved using either filtration or centrifugation techniques, both of which
are described in this chapter.
In pharmaceutical processing there are two main reasons for such processes:
• to remove unwanted solid particles from either a liquid product or from air
• to collect the solid as the product itself (e.g. following crystallization).

Filtration
Types of Filtration
Solid/fluid filtration
Solid/fluid filtration can be defined as the separation of an insoluble solid from a fluid by means
of a porous medium that retains the solid but allows the fluid to pass. It is the most common type
of filtration encountered during the manufacture of pharmaceutical products. Solid/fluid filtration
may be further subdivided into two types, namely solid/liquid filtration and solid/gas filtration.
Solid/liquid filtration.
There are numerous applications of solid/liquid filtration in pharmaceutical processing, some of
which are listed below:
• improvement of the appearance of solutions, mouthwashes, etc., to give them a ‘sparkle’ or
‘brightness’; this is often referred to as ‘clarifying’ a product
• removal of potential irritants, e.g. from eye drop preparations or solutions applied to mucous
membranes
• production of water of appropriate quality for pharmaceutical production
• recovery of desired solid material from a suspension or slurry, e.g. to obtain a drug or
excipient after a crystallization process
• certain operations, such as the extraction of vegetable drugs with a solvent, may yield a turbid
product with a small quantity of fine suspended colloidal matter. This can be removed by
filtration
 • sterilization of liquid or semi-solid products where processes involving heat (such as
autoclaving) are not appropriate
• detection of microorganisms present in liquids. This can be achieved by analysing a suitable
filter on which the bacteria are retained. This method can also be used to assess the efficiency
of preservatives.
Solid/gas filtration.
There are two main applications of solid/gas filtration in pharmaceutical processing. One of
particular importance in manufacturing is the removal of suspended solid material from air in
order to supply air of the required standard for either processing equipment or manufacturing
areas. This includes the provision of air for equipment such as fluidized-bed processors. film-
coating machinery and bottle-cleaning equipment, so that product appearance and quality are
maintained. The use of suitable filters also enables the particulate contamination of air in
manufacturing areas to be at an appropriate level for the product being manufactured; for example,
air free from microorganisms can be supplied to areas where sterile products are being
manufactured.
It is also often necessary to remove particulate matter generated during a manufacturing
operation from the process air in order to prevent the material being vented to the atmosphere.
Examples of this include filtering of exhaust air from fluidized-bed and coating processes.

Fluid/fluid filtration
Flavouring oils are sometimes added to liquid preparations in the form of a spirit, i.e. dissolved
in alcohol. When these spirits are added to aqueous-based formulations some of the oil may
come out of solution, giving the product a degree of turbidity. Removal of the oil droplets by
passing them through an appropriate filter (a liquid/liquid filtration process) is used to produce
the desired product appearance.
Compressed air is used in a number of pharmaceutical processes, e.g. film-coating spray guns
bottle-cleaning equipment and fluid energy mills. Before use, the compressed air needs to be
filtered to ensure that any entrained oil or water droplets are removed. This is an example of a
fluid/gas filtration process.

Mechanisms of Filtration
The mechanisms by which material may be retained by a filter medium (i.e. the surface on or in
which material is deposited) are discussed below.

Straining/sieving
If the pores in the filter medium through which the fluid is flowing are smaller than the material
that is required to be removed, the material will be retained. Filtration occurs on the surface of
the filter in this case, and therefore the filter can be very thin (typically around 100 µm). Filter
media of this type are referred to as membrane filters. Because filtration occurs on the surface
there is a tendency for them to become blocked unless the filter is carefully designed (see later).
Filters employing the straining mechanism are used where the contaminant level is low or small
volumes need to be filtered. Examples of the use of membrane filters include the removal of
bacteria and fibres from parenteral preparations.

Impingement
As a flowing fluid approaches and passes an object, for example a filter fibre, the fluid flow
pattern is disturbed, as shown in Figure 3. Suspended solids may, however, have sufficient
momentum so that they do not follow the fluid path but impinge on the filter fibre and are
retained, owing to attractive forces between the particle and the fibre. Where the pores between
filter fibres are larger than the material being removed, some particles may follow the fluid
streamlines and miss the fibre, this being more likely if the particles are small (owing to their
lower momentum) and as the distance from the centre of the fibre towards which they approach
increases. To ensure the removal of all unwanted material, filter media using the impingement
mechanism must be sufficiently thick so that material not trapped by the first fibre in its path is
removed by a subsequent one. These types of filter are therefore referred to as depth filters. The
fluid should flow through the filter medium in a streamlined manner to ensure the filter works
effectively, as turbulent flow may carry the particles past the fibres. Depth filters are the main
type of filter used for removing material from gases.

Attractive forces
Electrostatic and other surface forces may exert sufficient hold on the particles to attract and
retain them on the filter medium (as occurs during the impingement mechanism).
Air can be freed from dust particles in an electrostatic precipitator by passing the air between
highly charged surfaces, which attract the dust particles.

Autofiltration
Autofiltration is the term used to describe the situation when filtered material (termed the filter
cake) acts as its own filter medium. This mechanism is used by the metafilter which is covered
later in this chapter.

Factors Affecting the Rate Of Filtration

The filtration process chosen must remove the required ‘contaminants’ or product but must also
do so at an acceptably fast rate to ensure that the manufacturing process can be carried out
economically. The laboratory Buchner funnel and flask (Fig. 4) is a convenient filter that can be
used to illustrate the factors that influence the rate at which a product can be filtered. This filter
is used for solid/liquid filtration processes but the same basic principles are valid whatever
filtration process is being evaluated.
FIG. 3 Filtration by impingement.

FIG. 4 Buchner funnel and vacuum flask.

 The rate of filtration (volume of filtered material ((V, m3) obtained in unit time (t,( s) ) depends
on the following factors:
• the area available for filtration ((A, m2), which in this case is the cross-sectional
sectional area of the
funnel
• the pressure difference (ΔP,, Pa) across the filter bed (filter medium and any cake formed).
With the Buchner funnel apparatus, this difference is due to the ‘head’ of unfiltered
suspension and therefore it decreases as filtration proceeds and the level drops. Note that it is
the pressure difference across the filtration medium that is im important,
portant, and this can be
increased by drawing a vacuum in the collection flask. The difference between atmospheric
pressure and the lower pressure in the flask is added to the pressure due to the unfiltered
product to give the total pressure difference
• the
he viscosity of the fluid passing through the filter, i.e. the filtrate ((µ,, Pa s). A viscous fluid
will filter more slowly than a mobile one owing to the greater resistance to movement offered
by more viscous fluids (see Chapter 6)
• the thickness of the filter
ilter medium and any deposited cake ((L,, m). The cake will increase in
thickness as filtration proceeds so if this is not removed, the rate will fall.

Darcy’s equation
The above factors are combined in the Darcy equation:

(25.1)
In this equation the driving force for this particular ‘rate process’ is the pressure difference
across the filter, and the resistance to the process is a function of the properties of the filter bed,
its thickness and the viscosity of the filtrate. The contribution to resista
resistance
nce to filtration from the
filter medium is usually small compared to that of the filter cake, and can often be neglected in
calculations.

2.4.CONCENTRATION: For a product concentration application, one would select a

membrane with a rating to ensure re retention
tention of the target material. The liquid passing to the
permeate side results in a proportionate increase in the product concentration on the feed side of
the membrane. But, there are noteworthy limitations to this type of process. First, the material
must
st remain with a low enough viscosity so that a recirculation flow can be maintained without
excessive pressure drop. Secondly, there is a need to anticipate a minimum working volume
within the system that allows the desired degree of concentration without causing air induction in
the feed vessel. For some applications seeking a 100 × concentration, either a special feed tank
may be required or the process may require a two
two-stage
stage system to reduce the minimum working
volume associated with the 100 × final co
concentration.
 2.5 Purification of Industrial Materials
Purification
It aims at recovery of the product in a highly purified state.

Purification is achieved by the following procedures:

Sublimation: Some solids can directly pass to the vapor state without going through the liquid
phase. The purification technique which exploits this property is called sublimation. It is helpful
in separating sublimable compounds from non-sublimable ones.
Crystallization: Crystallization is defined as a process by which a chemical is converted from a
liquid solution into a solid crystalline state. This is used for the low molecular mass compound
like antibiotics.

Types of Crystallization
 Evaporative crystallization.
 Cooling crystallization from solution or the melt.
 Reactive crystallization or precipitation

 Distillation: is the process of separating the components or substances from a liquid mixture by
using selective boiling and condensation. Distillation may result in essentially complete
separation (nearly pure components), or it may be a partial separation that increases the
concentration of selected components in the mixture. In either case, the process exploits
differences in the relative volatility of the mixture's components. Distillation is used mostly in
separation of fermented products.
 Chromatographic methods: Chromatography is based on the principle where molecules in
mixture applied onto the surface or into the solid, and fluid stationary phase (stable phase) is
separating from each other while moving with the aid of a mobile phase. it is generally, used for
the purification of low molecular mass compound from mixture of similar molecules, e.g.,
antibiotics(homologous) and macromolecules (enzymes).

The different chromatographic procedures are:

1. Ion exchange chromatography: Ion- exchange chromatography is based on electrostatic
interactions between charged protein groups, and solid support material (matrix). Matrix has
an ion load opposite to that of the protein to be separated, and the affinity of the protein to the
column is achieved with ionic ties. Proteins are separated from the column either by
changing pH, concentration of ion salts or ionic strength of the buffer solution. Positively
charged ion- exchange matrices are called anion-exchange matrices, and adsorb negatively
charged proteins. While matrices bound with negatively charged groups are known as cation-
exchange matrices, and adsorb positively charged proteins
2. Affinity chromatography: This chromatography technique is used for the purification of
enzymes, hormones, antibodies, nucleic acids, and specific proteins. A ligand which can
make a complex with specific protein (dextran, polyacrylamide, cellulose etc) binds the
 filling material of the column. The specific protein which makes a complex with the ligand is
attached to the solid support (matrix), and retained in the column, while free proteins leave
the column. Then the bound protein leaves the column by means of changing its ionic
strength through alteration of pH or addition of a salt solution
3. Gas chromatographyIn this method stationary phase is a column which is placed in the
device, and contains a liquid stationary phase which is adsorbed onto the surface of an inert
solid. Gas chromatography is a “gas-liquid” chromatography. Its carrier phase consists of
gases as He or N2. Mobile phase which is an inert gas is passed through a column under high
pressure. The sample to be analyzed is vaporized, and enters into a gaseous mobile phase
phase. The components contained in the sample are dispersed between mobile phase, and
stationary phase on the solid support. Gas chromatography is a simple, multifaceted, highly
sensitive, and rapidly applied technique for the extremely excellent separation of very minute
molecules. It is used in the separation of very little amounts of analytes.
4. Gel filtration
5. Adsorption
6. Partition chromatography.
7. Thin layer chromatography

2.6. Extraction of Industrial Product.

Extraction is the first step to separate the desired natural products from the raw materials.
The three most common types of extractions are:
• Liquid–liquid extraction
• Solid-phase extraction (Solid/Liquid extraction)
• Acid-base extraction (also known as a chemically active extraction).

Other types are:

• Supercritical fluid extraction
• Ultrasound-assisted extraction
• Heat reflux extraction
• Mechanochemical-assisted extraction
• Microwave-assisted extraction
• Instant controlled pressure drop extraction (DIC, from the French, Détente instantanée
contrôlée)
• Perstraction

Liquid–liquid extraction (LLE): (Solvent extraction and partitioning) is a method to separate

compounds or metal complexes, based on their relative solubility in two different immiscible
liquids, usually water (polar) and an organic solvent (non-polar). There is a net transfer of one or
more species from one liquid into another liquid phase, generally from aqueous to organic. The
transfer is driven by chemical potential, i.e. once the transfer is complete, the overall system of
chemical components that make up the solutes and the solvents are in a more stable
configuration (lower free energy). The solvent that is enriched in solute(s) is called extract. The
feed solution that is depleted in solute(s) is called the caffeinate.

LLE is a basic technique in chemical laboratories, where it is performed using a variety of

apparatus, from separator funnels to countercurrent distribution equipment called as mixer
settlers. This type of process is commonly performed after a chemical reaction as part of the
work-up, often including an acidic work-up. The term partitioning is commonly used to refer to
the underlying chemical and physical processes involved in liquid–liquid extraction, but on
another reading may be fully synonymous with it. The term solvent extraction can also refer to
the separation of a substance from a mixture by preferentially dissolving that substance in a
suitable solvent. In that case, a soluble compound is separated from an insoluble compound or a
complex matrix.

Solid-phase extraction (SPE) is an extractive technique by which compounds that are dissolved
or suspended in a liquid mixture are separated from other compounds in the mixture according to
their physical and chemical properties. The coffee and tea examples are both of the liquid/solid
type in which a compound (caffeine) is isolated from a solid mixture by using a liquid extraction
solvent (water).

Analytical laboratories use solid phase extraction to concentrate and purify samples for analysis.
Solid phase extraction can be used to isolate analytes of interest from a wide variety of matrices,
including urine, blood, water, beverages, soil, and animal tissue.

SPE uses the affinity of solutes dissolved or suspended in a liquid (known as the mobile phase)
for a solid through which the sample is passed (known as the stationary phase) to separate a
mixture into desired and undesired components. The result is that either the desired analytes of
interest or undesired impurities in the sample are retained on the stationary phase. The portion
that passes through the stationary phase is collected or discarded, depending on whether it
contains the desired analytes or undesired impurities. If the portion retained on the stationary
phase includes the desired analytes, they can then be removed from the stationary phase for
collection in an additional step, in which the stationary phase is rinsed with an appropriate
eluent.

Acid-base extraction is a procedure using sequential liquid–liquid extractions to purify acids

and bases from mixtures based on their chemical properties.

Acid-base extraction is routinely performed during the work-up after chemical syntheses and for
the isolation of compounds and natural products like alkaloids from crude extracts. The product
is largely free of neutral and acidic or basic impurities. It is not possible to separate chemically
similar acids or bases using this simple method.
The fundamental theory behind this technique is that salts, which are ionic, tend to be water-
soluble while neutral molecules tend not to be.

The addition of an acid to a mixture of an organic base and acid will result in the acid remaining
unchanged, while the base will be protonated to form a salt. If the organic acid, such as a
carboxylic acid, is sufficiently weak, its self-ionization can be suppressed by the added acid.

Conversely, the addition of a base to a mixture of an organic acid and base will result in the base
remaining unchanged, while the acid is deprotonated to give the corresponding salt. Once again,
the self-ionization of a strong base is suppressed by the added base.

The acid-base extraction procedure can also be used to separate very weak acids from stronger
acids and very weak bases from stronger bases, as long as the difference of their pKa (or pKb)
constants is large enough.

Extraction methods include solvent extraction, distillation method, pressing and sublimation
according to the extraction principle. Solvent extraction is the most widely used method. The
extraction of natural products progresses through the following stages:

1. the solvent penetrates into the solid matrix;

2. the solute dissolves in the solvents;
3. the solute is diffused out of the solid matrix;
4. the extracted solutes are collected.

Any factor enhancing the diffusivity and solubility in the above steps will facilitate the
extraction. The properties of the extraction solvent, the particle size of the raw materials, the
solvent-to-solid ration, the extraction temperature and the extraction duration will affect the
extraction efficiency.

The selection of the solvent is crucial for solvent extraction. Selectivity, solubility, cost and
safety should be considered in selection of solvents. Based on the law of similarity and
intermiscibility (like dissolves like), solvents with a polarity value near to the polarity of the
solute are likely to perform better and vice versa. Alcohols (EtOH and MeOH) are universal
solvents in solvent extraction for phytochemical investigation.

Generally, the finer the particle size is, the better result the extraction achieves. The extraction
efficiency will be enhanced by the small particle size due to the enhanced penetration of solvents
and diffusion of solutes. Too fine particle size, however, will cost the excessive absorption of
solute in solid and difficulty in subsequent filtration.

High temperatures increase the solubility and diffusion. Temperatures that too high, however,
may cause solvents to be lost, leading to extracts of undesirable impurities and the
decomposition of thermolabile components.
The extraction efficiency increases with the increase in extraction duration in a certain time
range. Increasing time will not affect the extraction after the equilibrium of the solute is reached
inside and outside the solid material.

The greater the solvent-to-solid ratio is, the higher the extraction yield is; however, a solvent-to-
solid ratio that is too high will cause excessive extraction solvent and requires a long time for
concentration.

The conventional extraction methods, including maceration, percolation and reflux extraction,
usually use organic solvents and require a large volume of solvents and long extraction time.
Some modern or greener extraction methods such as super critical fluid extraction (SFC),
pressurized liquid extraction (PLE) and microwave assisted extraction (MAE), have also been
applied in natural products extraction, and they offer some advantages such as lower organic
solvent consumption, shorter extraction time and higher selectivity. Some extraction methods,
however, such as sublimation, expeller pressing and effleurage are rarely used in current
phytochemical investigation.

Chromatography. It is in this area that several potential industrial applications of modern

biotechnology have come to grief either because the extraction has defeated the ingenuity of the
designers or, more probably, because the extraction process has required so much energy input as
to render it uneconomic.
Final products of the downstream purification stages should have some degree of stability for
commercial distribution. Stability is best achieved for most products by using some form of
drying. In practice, this is achieved by spray drying, fluidised-bed drying or by freeze drying.
The method of choice is product- and cost-dependent. Products sold in the dry form include
organic acids, amino acids, antibiotics, polysaccharides, enzymes, SCP and many others. Many
products cannot be supplied easily in a dried form and must be sold in liquid preparations. Care
must be taken to avoid microbial contamination and deterioration and, when the product is
proteinaceous, to avoid denaturation.

The role of downstream processing will continue to be one of the most challenging and
demanding parts of many biotechnological processes. Purity and stability are the hallmarks of
most high-value biotechnological products.
It can be said that biotechnological processes will, in most part, need to be contained within a
defined area or bioreactor and, to a large extent, the ultimate success of most of the processes
will depend on the correct choice and operation of these systems. For most high-value products,
cultivation of the producer organism will normally be by monoculture, requiring complete
asepsis to maximise product formation. On the industrial side, the scale of operation will, for
economic reasons, mainly be very large, and in almost all cases the final success will require the
closest cooperation between the bioscientist, the chemist and the process or biochemical engineer
– in this way demonstrating the truly interdisciplinary nature of biotechnological processes.
2.7. Product polishing:
This is the last stage in downstream processing. It involves removal of minor impurities and it is
applied only when very high degree of purity is required. Non-functional isoforms, denature
proteins and other minor impurities are removed at this stage. The unit operations include
desiccation, sterile filtration, lyophilzation (drying in a frozen state under vacuum). And spray
drying. Depending on the product, this phase also includes operations to sterilize the product and
remove or de-activate trace contaminants. It provide the desired product in a form suitable for
final formulation and blending.

Revision Question
1. What do you understand by downstream processing?
2. Explain the different methods used to disrupt cells for extraction of intracellular products.
3. How are precipitation and crystallization used to concentrate products in culture broth.
4. Using a schematic flow diagram, enumerate the various unit operations in downstream
processing.
CHAPTER 3
PRODUCTION OF AALCOHOL-BASED GOODS: BEER, STOUT, SPIRIT,
INDUSTRIAL ALCOHOL, VINEGAR.
Learning Objectives
- Define industrial fermentation and bioreactor.
- Differentiate primary from secondary metabolites.
-Describe the role of microorganisms in the production of industrial chemicals and
pharmaceuticals.
- List biofuels that can be made by microorganisms.

3.1 FERMENTATION: AN ANCIENT TRADITION

Fermentation has been known and practiced by humankind since prehistoric times, long before
the underlying scientific principles were understood. That such a useful technology should arise
by accident will come as no surprise to those people who live in tropical and subtropical regions,
where, as Marjory Stephenson put it, “every sandstorm is followed by a spate of fermentation in
the cooking pot” (Stephenson 1949). For example, the productions of bread, beer, vinegar,
yogurt, cheese, and wine were well-established technologies in ancient Egypt. It is an interesting
fact that archaeological studies have revealed that bread and beers, in that order, were the two
most abundant components in the diet of ancient Egyptians. Everyone, from the pharaoh to the
peasant, drank beer for social as well as ritual reasons. Archaeological evidence has also revealed
that ancient Egyptians were fully aware not only of the need to malt the barley or the emmer
wheat but also of the need for starter cultures, which at the time may have contained lactic acid
bacteria in addition to yeast.
3.2 THE RISE OF FERMENTATION MICROBIOLOGY
With the advent of the science of microbiology, and in particular fermentation microbiology, we
can now shed light on these ancient and traditional activities. Consider, for example, the age-old
technology of wine making, which relies upon crushing grapes and letting nature take its course
(i.e., fermentation). Many microorganisms can grow on grape sugars more readily and efficiently
than yeasts, but few can withstand the osmotic pressure arising from the high sugar
concentrations. Also, as sugar is fermented, the alcohol concentration rises to a level at which
only osmotolerant, alcohol-tolerant cells can survive. Hence inhabitants of ancient civilizations
did not need to be skilled microbiologists in order to enjoy the fruits of this popular branch of
fermentation microbiology.
In fact, the scientific understanding of fermentation microbiology and, in turn, biotechnology
only began in the 1850s, after Louis Pasteur had succeeded in isolating two different forms of
amyl alcohol, of which one was optically active (L, or laevorotatory) while the other was not.
Rather unexpectedly, the optically inactive form resisted all of Pasteur’s attempts to resolve it
into its two main isomers, the laevorotatory (L) and the dextrorotatory (D) forms. It was this
observation that led Pasteur into the study of fermentation, in the hope of unraveling the
underlying reasons behind his observation, which was contrary to stereochemistry and
crystallography understandings at the time.
In 1857, Pasteur published the results of his studies and concluded that fermentation is associated
with the life and structural integrity of the yeast cells rather than with their death and decay. He
reiterated the view that the yeast cell is a living organism and that the fermentation process is
essential for the reproduction and survival of the cell. In his paper, the words cell and ferment are
used interchangeably (i.e., the yeast cell is the ferment). The publication of this classic paper
marks the birth of fermentation microbiology and biotechnology as a new scientific discipline.
Guided by his critical and unbiased approach to experimental design, Pasteur was able to
confidently challenge and reject Liebig’s perception that fermentation occurs as a result of
contact with decaying matter. He also ignored the well-documented view that fermentation
occurs as a result of “contact catalysis,” although it is possible that this concept was not suspect
in his view. The term “contact catalysis” probably implied that fermentation is brought about by
a chain of enzyme-catalyzed reactions. In 1878, Wilhelm Kühne (1837–1900) was the first to use
the term enzyme, which is derived from the Greek word ενζυμον (“in leaven”) to describe this
process. The word enzyme was used later to refer to nonliving substances such as pepsin, and the
word ferment used to refer to chemical activity produced by living organisms.
Although Pasteur’s interpretations were essentially physiological rather than biochemical, they
were pragmatically correct. During the course of his further studies, Pasteur was also able to
establish not only that alcohol was produced by yeast through fermentation but also that souring
was a consequence of contamination with bacteria that were capable of converting alcohol to
acetic acid. Souring could be avoided by heat treatment at a certain temperature for a given
length of time. This eliminated the bacteria without adversely affecting the organoleptic qualities
of beer or wine, a process we now know as pasteurization.
A second stage in the development of fermentation microbiology and biotechnology began in
1877, when Moritz Traube proposed the theory that fermentation and other chemical reactions
are catalyzed by protein-like substances and that, in his view, these substances remain unchanged
at the end of the reactions. Furthermore, he described fermentation as a sequence of events in
which oxygen is transferred from one part of the sugar molecule to another, culminating in the
formation of a highly oxidized product (i.e., CO2) and a highly reduced product (i.e., alcohol).
Considering the limited knowledge of biochemistry in general and enzymology in particular at
the time, Traube’s remarkable vision was to prove 50 years ahead of its time. In 1897 Eduard
Buchner, two years after Pasteur died, discovered that sucrose could be fermented to alcohol by
yeast cell-free extracts and coined the term “zymase” to describe the enzyme that catalyses this
conversion. The term “zymase” Is derived from the Greek word “zymosis”, which means
fermentation. In 1907, he received the Nobel Prize in Chemistry for his biochemical research and
his discovery of cell-free fermentation. In the early 1900s, the views of Pasteur were modified
and extended to stress the idea that fermentation is a function of a living, but not necessarily
multiplying, cell and that fermentation is not a single step but rather a chain of events, each of
which is probably catalyzed by a different enzyme.
3.3 DEVELOPMENTS IN METABOLIC AND BIOCHEMICAL ENGINEERING
The outbreak of the First World War provided an impetus and a challenge to produce certain
chemicals that, for one reason or another, could not be manufactured by conventional means. For
example, there was a need for glycerol, an essential component in the manufacture of
ammunition, because no vegetable oils could be imported due to the naval blockade. German
biochemists and engineers were able to adapt yeast fermentation, turning sugars into glycerol
rather than alcohol. Although this process enabled the Germans to produce in excess of 100 tons
of glycerol per month, it was abandoned as soon as the war was over because glycerol could be
made very cheaply as a by-product of the soap industry. There was also, of course, a dramatic
drop in the level of manufacture of explosives and, in turn, the need for glycerol.
The diversion of carbon flow from alcohol production to glycerol formation was achieved by
adding sodium bisulfite, which reacts with acetaldehyde to give an adduct that cannot be
converted to alcohol Consequently, NADH accumulates intracellularly, thus perturbing the
steadystate redox balance (NAD+:NADH ratio) of the cell. The drop in the intracellular level of
NAD+ is accompanied by a sharp drop in the flux through glyceraldehyde-3-phosphate
dehydrogenase, which in turn allows the accumulation of the two isomeric forms of triose
phosphate (i.e., glyceraldehyde- 3-phosphate and dihydroxyacetone-3-phosphate).
Accumulations of the latter together with high intracellular levels of NADH trigger the
expression of glycerol-3-phosphate dehydrogenase, which in turn leads to the diversion of
carbon flux from ethanol production to glycerol formation, thus restoring the redox balance
within the cells by regenerating NAD+ (Figure 1.3).
Although this explanation is with the hindsight of modern biochemistry, the process can be
viewed as an early example of metabolic engineering.
Following the First World War, research into yeast fermentation was largely influenced by the
work of Carl Neuberg and his proposed scheme (biochemical pathway) for the conversion of
sugars to alcohol (alcohol fermentation). Although Neuberg’s scheme was far from perfect and
proved erroneous in many ways, it provided the impetus and framework for many scientists at
the Delft Institute, who vigorously pursued research into oxidation/reduction mechanisms and
the kinetics of product formation in a wide range of enzyme-catalyzed reactions. Such studies
were to prove important in the development of modern biochemistry as well as fermentation
biotechnology.
Wine producers who allowed wine to be exposed to air found that it soured from the growth of
aerobic bacteria that converted the ethanol in the wine to acetic acid. The result was vinegar (vin
= wine; aigre = sour). The process is now used deliberately to make vinegar. Ethanol is first
produced by anaerobic fermentation of carbohydrates by yeasts. The ethanol is then aerobically
oxidized to acetic acid by acetic acid–producing bacteria of the genera Acetobacter and
Gluconobacter.

The industrial uses of microbiology had their beginnings in large-scale food fermentations that
produced lactic acid from dairy products and ethanol from brewing. These two chemicals also
proved to have many industrial uses unrelated to foods. During World Wars I and II, microbial
fermentation and similar technologies were used in the production of armament-related chemical
compounds, such as glycerol and acetone. Present industrial microbiology dates largely from the
technology developed to produce antibiotics following World War II. There is renewed interest
in some of these classic microbial fermentations, especially if they can be used as feedstocks,
products that are renewable, or, ideally, use products that would otherwise be wasted.
In recent years, industrial microbiology has been revolutionized by biotechnology, the
application of genetically modified organisms.

3.4 FERMENTATION TECHNOLOGY

The industrial production of microbial products usually involves fermentation. Industrial
fermentation is the large-scale cultivation of microbes or other single cells to produce a
commercially valuable substance. We will discussed the most familiar examples: the anaerobic
food fermentations used in the dairy, brewing, and winemaking industries. Much of the same
technology, with the frequent addition of aeration, has been adapted to make other industrial
products, such as insulin and human growth hormone, from genetically modified
microorganisms.

Industrial fermentation is also used in biotechnology to obtain useful products from genetically
modified plant and animal cells.

Vessels for industrial fermentation are called bioreactors; they are designed with close attention
to aeration, pH control, and temperature control. There are many different designs, but the most
widely used bioreactors are of the continuously stirred type. The air is introduced through a
diffuser at the bottom (which breaks up the incoming airstream to maximize aeration), and a
series of impeller paddles and stationary wall baffles keep the microbial suspension agitated.
Oxygen is not very soluble in water, and keeping the heavy microbial suspension well aerated is
difficult. Highly sophisticated designs have been developed to achieve maximum efficiency in
aeration and other growth requirements, including medium formulation.

The high value of the products of genetically modified microorganisms and eukaryotic cells has
stimulated the development of newer types of bioreactors and computerized controls for them.

Bioreactors are sometimes very large, holding as much as 500,000 liters. When the product is
harvested at the completion of the fermentation, this is known as batch production. There are
other designs of fermentors. For continuous flow production, in which the substrates (usually a
carbon source) are fed continuously past immobilized enzymes or into a culture of growing cells,
spent medium and desired product are continuously removed.

Generally speaking, the microbes in industrial fermentation produce either primary metabolites,
such as ethanol, or secondary metabolites, such as penicillin. A primary metabolite is formed
essentially at the same time as the new cells, and the production curve follows the cell population
curve almost in parallel, with only minimal lag. Secondary metabolites are not produced until the
microbe has largely completed its logarithmic growth phase, known as the trophophase, and has
entered the stationary phase of the growth cycle. The following period, during which most of the
secondary metabolite is produced, is known as the idiophase.

The secondary metabolite may be a microbial conversion of a primary metabolite. Alternatively,

it may be a metabolic product of the original growth medium that the microbe makes only after
considerable numbers of cells and a primary metabolite have accumulated. Cellular metabolism
leaves behind small-molecule chemical fingerprints of the cellular processes: a metabolic profile.

3.5 PRODUCTION OF ETHANOL AND RELATED COMPOUNDS

3.5.1 Production of Ethanol
Ethanol is a primary metabolite produced by fermentation of sugar, or of a polysaccharide that
can be depolymerized to a fermentable sugar. S. cerevisiae is used for the fermentation of
hexoses, whereas the Kluyveromyces fragilis or Candida species can be used if lactose or a
pentose, respectively, is the substrate. Under optimum conditions, approximately 10–12%
ethanol by volume is obtained within five days. At present, all beverage alcohol is made by
fermentation. Industrial ethanol is mainly manufactured by fermentation, but some is still
produced from ethylene by the petrochemical industry. Bacteria such as Clostridia and
Zymomonas are being reexamined for ethanol production after years of neglect. Clostridium
thermocellum, an anaerobic thermophile, can convert waste cellulose and crystalline cellulose
directly to ethanol (see Chapter 9 on renewable resources conversion to fine chemicals). The
available cellulosic feedstock in the United States could supply 20 billion gallons of ethanol in
comparison to the 3 billion gallons currently made from corn. This would be enough to add 10%
ethanol to all gasoline used in the United States.
Other Clostridia produce acetate, lactate, acetone, and butanol and will be used to produce these
chemicals when the global petroleum supplies begin to become depleted.
Ethyl alcohol is produced in Brazil from cane sugar at over 4 billion gallons per year and is used
either as a 25% blend or as a pure fuel. Most new cars in Brazil use pure ethanol, whereas the
remainder utilizes a blend of 20–25% ethanol in gasoline. In the United States, over 3.4 billion
gallons of ethanol were made from starchy crops (mainly corn) in 2004. It is chiefly added to
gasoline to reduce CO2 emissions by improving the overall oxidation and performance of
gasoline.
Fuel ethanol produced from biomass would provide relief from air pollution caused by the use of
gasoline and would not contribute to the greenhouse effect. E. coli has been converted into an
excellent ethanol producer (43% yield, v/v) by cloning and expressing the alcohol
dehydrogenase and pyruvate decarboxylase genes from Zymomonas mobilis and Klebsiella
oxytoca. The recombinant strain was able to convert crystalline cellulose to ethanol in high yield
when fungal cellulase was added. Other genetically engineered strains of E. coli can produce as
much as 60 g l−1 of ethanol.
An ethanol-resistant mutant of S. cerevisiae makes 96 g g l−1.
3.5.2 PRODUCTION OF BEER AND STOUT
ALCOHOLIC DRINKS (BEER AND STOUT)
Alcohols: These are both chemical and a psychoactive drug which exist when a hydroxyl group,
a pair of oxygen and hydrogen atoms replaces the hydrogen atom in a hydrocarbon. usually
alcohols bind with other atoms to create three types of alcohols that humans use everyday.
Methanol, isopropanol and ethanol. Ethanol is consumable by humans and produced by the
fermentation of yeast, sugars and starch.
Alcoholic Drinks are categories as:Distilled andUndistilled
3.5.2.1 Beer production: brewing is the process of production of malt beverages. Beers, ale and
lagers are the main malt beverages produced by sa method called brewing. Brewing is a complex
fermentation process. It differs from other industrial fermentation because flavor, aroma, clarity,
color, foam production , foam stability and percentage of alcohol are the factors associated with
finished product.
Steps involved in beer production:
A. Malting: beer is produced from barley grains.
Barley grains are first cleaned and then soaked in water for about 2days. Then excess water is
drained away and barley are incubated for 4-5days to allow germination.
Malt adjuncts;
Barley contains considerable amount of protein. So, if only barley are used for beer production,
the final beer will be dark and unstable. Therefore, protein present in malt should be diluted by
adding additional starch or sugary materials. Such sugary or starchy materials are called adjuncts
and includes dextrose sugar syrup.

Stages of malting
.Steeping: here the seed is soaked in water to order to encourage growing
.Germination: here the bailey grows under controlled conditions however the changes are just
inside the seed.
B. Kilning: the germinated seed are then killed by slow heating at 80c. This process is
called kilning. The kilning temperature must not harm amylase enzyme. Furthermore, if kilning
temeperature is higher, darker will be the beer produce.

.Mailing; the dried barley grains are then crushed between rollers to produced coarse powder
called grist.

.Mashing: grist is mixed with warm water and the resulting materials is maintained at 65c for
about an 1hour. In doing so, starch is hydrolyzed by amylase enzyme to produce single sugar,
maltose, dextrose etc. similarily, protein is hydrolyzed by proteolytic enzymes in to small
fragments and amino acids.
The degree of enzymatic hydrolysis is strongly depends on ph and temperature. B- amylase has
optimum activity at temperature 57-65c whereas a-amylase has optimum activity at temperature
70-75.
The liquid obtained by mashing is called wort. The husks and other grains residue as well as
precipitated proteins are removed filtration.
Stages of mashing
Milling: these stage the malt is cruched
Mashing: here the crushed malt is mixed with water to start the conversion process
Conversion: here the starch is converted in to sugars by the malt enzymes

.Boiling of the wort;

The filtrate is then boiled with stirring for 2-3 hours and hop flowers are added at various
interval during boiling. Reason for the wort:
-For extraction of hop flavor from hop flower.
-Boiling coagulase remaining protein and partially hydrolyze protein and help in removal of
protein.
-Boiling inactivates enzymes that were active during mashing, otherwise causes caramelization
of sugar
-Boiling also sterilize and concentrate the wort

.Hops
Hops are dried female flower of hop plant humuluslupulus. Approximately one quarter pound of
hop flower is added per barrel of beer and up to 2 pound barrel of ale.
Advantages of hop addition in beer;
-Provide beer with its pungent and aromatic character.
-Provide tannin which helps in coagulation of remaining protein
-Contains a-resin and B-resin which gives bitter flavor as well as preservative action against
gram positive bacteria.
-Contains pectin which is responsible for foam characteristic of beer

Fermentation
Beer production utilize strain of saccharomyces carlsbergens and S. varum which are bottom
yeast and S.cerevisiae which is a top yeast.
Yeast cells for inoculation are usually recover from previous fermentation tank by treatment with
phosphoric acid, tartaric acid or ammonium persulphate to reduce the P H and removed
considerable bacterial contamination.
Fermentation is usually carried out 3-4oc but it may range from 3-14ofermentation usually
completes in 14days.
During fermentation yeast converts sugar mainly into ethanol and co2 plus some amount of
glycerol and acetic acid.
For fermentation open tank fermenter can be used however closed fermenter tank is preferred,
so that CO2 liberated during fermentation can be collected for later carbonation step.
C02 evolution is maximum by fifth day of fermentation, there is no evolution of co2 by 7-9
days because yeast cells become inactivate and flocculate.
Most beer contains 3.5% alcohol.

.Finishing, Ageing, Maturation and Carbonation

The young and green beer is stored in vat at 0oc for several weeks to several months. During
this period, precipitation of protein, yeast, resin and other undesired substances take place and
beer become clear.
Ester and other compounds are also produced during ageing which gives taste and aroma.
After ageing, the beer is carbonated by carbondioxide of 0.45-0.52%.
The beer is then cooled, clarified, filtered and packed in bottles, barrels and cans.

3.5.2.2 Production of stout

Stouts are some of the world most recognizable and iconic beers due to their dark color. Stouts
originated as a strong type of porter in fact, they were originally called stout portersand are
made with a portion of heavily roasted barley. Malted barley, or barley that has been germinated
and then dried, is what gives any beer its flavor. However, the color of the resulting brew
depends on how much is roasted, unmalted barley is later added, as well as how long and at
what temperature the barley has been roasted. The higher the heat while roasting, the darker the
kernels come out. There are two things that influence a beers color during that time. During the
roasting process, the heat helps convert starch in green barley kernels to sugar, and as things
heat up, the liquid sugar decomposes in a process known as caramelization. The process is
responsible for that delicious golden brown color you see when you hold a marshmallow over a
campfire, and creates rich notes of toffee in beer. Dried grain kernels are also roasted to create
coffee and chocolate notes. Once these are roasted barley can darken the color of a beer quite a
bit.

Types of stout
Dry or irish stout: is very dark in color, and it often has a roasted or coffee-like taste e.g
Guinness, murphys and beamish. It alcoholic content is characterized as light depending on the
country or brewery.
Imperial stout: imperial stout is a strong, dark beer in the style that was brewed in 28th century
by thrales brewery in London.
Milk stout: milk stout contains lactose, a sugar that is derived from milk because lactose is
unfermentable by beer yeast, it can adds sweetness body and colouries to the finished beer
example mackeson.
Oatmeal stout: this is a stout with a proportion of oats normally a maximum of 30%, added
during the brewing process. During the medieval period in Europe, oats were a common
ingredient in ale, and proportions of up to 35% were standard.
Chocolate stout: chocolate stout is a name brewers sometimes give to certain stout having a
noticeable dark chocolate flavor through the use of darker, more romantic malt, particularly
chocolate malt, kilned or roasted malt that has acquired a chocolate color after roasting.
Examples of chocolate stout. Young double chocolate stout, muskoka brewing is double
chocolate cranberry stout, rogue brewing chocolate stout.

3.5.3. PRODUCTION OF SPIRITS/ LIQUOR

Spirit/Liquor (also hard liquor, hard alcohol, distilled alcohol, fire water, or spirit water) is an
alcoholic drink produced by distillation of grains, fruits, or vegetables that have already gone
through alcoholic fermentation. The distillation process purifies the liquid and removes diluting
components like water, for the purpose of increasing its proportion of alcohol content
(commonly expressed as alcohol by volume, ABV). As liquors contain significantly more
alcohol than other alcoholic drinks, they are considered "harder" – in North America, the term
hard liquor is used to distinguish distilled alcoholic drinks from non-distilled ones, whereas the
term spirits is used in the UK. There is no evidence for a health benefit for liquor at any level of
consumption. Compared to other types of alcohol, excess consumption of liquor is more strongly
associated with harmful health effects. The term "spirit" refers to liquor that contains no added
sugar and has at least 20% alcohol by volume (ABV).

Types of spirits
Gin
Liquer
Vermouth
Borbourn
Scotch
Whisky
Vodka
Rum
Cognac

3.5.3.1 Production of Gin

Definition:
The legal EU definition of gin means that a drink must be a juniper-dominated spirit, with an
agricultural origin and at least 37.5% alcohol by volume (ABV). You will learn about how the
strength of an alcoholic drink is measured by volume in Week 8.
So, for a substance to be classed as gin, the main natural flavouring (known as a botanical) must
be juniper, the base alcohol must be made from something natural such as wheat, barley, rye,
molasses, potatoes or grapes, and there must be at least 37.5% of pure alcohol in the total volume
of liquid.

There are three fundamental types of gin:

 Distilled gin
 London Dry Gin
 Flavored gin.

Distilled gin
Distilled gin is the first of our three types of gin and is produced exclusively by redistilling
ethanol of agricultural origin with an initial strength of 96% ABV in the presence of juniper
berries and of other natural botanicals, provided that the juniper taste is predominant.

London Dry Gin

London Dry Gin is the second of our three types of gin. This type of gin is not exclusive to
London geographically but simply refers to a gin which is deemed to be the highest-quality gin
you can produce. It has to comprise only natural ingredients, be made with high-quality alcohol
and only contain 0.01 g of sugar per litre of alcohol.

Flavoured gins
A visit to the supermarket shelves in the gin aisle will also likely reveal a variety of so-called
‘flavoured’ gins. Strictly speaking, these do not represent a separate type of gin as such, since
some flavoured gins simply add additional flavours to the gin post-distillation. Examples of
readily available flavoured gins include rhubarb, raspberry, lemon and even chocolate flavoured
gins.

At this point, it is also worth mentioning sloe gin. Despite the name, sloe gin isn’t actually a gin
but a liqueur. This is because the ABV is in the range 20–25%, well below the minimum
requirement of a spirit. Sloe gin also tends to have a much higher sugar content than is permitted
in a London Dry Gin.

The stages of gin production

The process used to produce gin is relatively simple (in comparison to the brewing process) and
is based mostly on the fundamentals of distillation.
Commercial distillation:
Once the addition of the botanicals is complete, the actual distillation begins within the copper
still (Figure 4). The still is heated to a sufficient temperature to start boiling. If you recall from
our laboratory still earlier, once a sufficient temperature is reached to enable a single component
of the mixture to boil, this component alone boils, evaporates and condenses. In doing so, it is
separated from the remainder of the mixture which has not yet reached boiling temperature.

The same principle is operating here in the production of gin, but on a much larger scale. Initially
when the still is heated, the first distillate to appear is called the ‘heads’ of the run – it is quite
harsh and astringent and often contains impurities so it is discarded.

The middle or ‘hearts’ of the run is the only distillate that is used by the distillery to produce
their gin and it is carefully collected. A drop in the % ABV is usually an indication of the end of
the hearts component of the distillation.

The final section of the run is the ‘tails’, and this is also discarded. By being selective in this
way, only the finest part of the run makes it into the final gin. This does, however, mean that
only small quantities of gin can be produced from a single distillation run, but the quality of this
gin will be superior.

Producing the final gin – the final taste

The hearts, which are at 83% ABV, are then rested for five days to allow the various flavours to
come together and settle. The very last stage in the gin production process is to reduce the
alcohol content. To do this, filtered water is added to reduce the strength to the Cotswold
Distillery-desired ABV of 46%. The gin is then bottled in-house at the distillery.

Commercially, this is known as ‘single-shot distillation’ and results in the best possible flavour
and quality of the final product. Some larger-scale distilleries produce gin for the mass market by
adding more neutral grain spirit to the distilled batch to increase the number of bottles made from
each run.

Some gin distilleries chill-filter their gin in order to remove oils and esters to make sure the gin
stays crystal clear.

3.5.3.2 Production of Liqueurs

Definition of Liqueurs:
Liqueurs are mixtures of spirits, sweeteners, and flavorings like herbs, fruits, nuts and flowers.
They are sometimes served as after-dinner drinks, but they’re more often poured on desserts or
mixed into cocktails, milk or coffee.
Production of Liqueurs
The production of liqueurs involves the following steps:
 Extraction
 Distillation
 Compounding
 Maturing
 Fining
 Bottling
-Extraction:
In this process, the flavors are extracted from the main ingredient. The methods of extraction are:
 Pressure: mechanical presses are used to extract the oil from citrus peel.
 Maceration: the flavoring agents are soaked in cold spirit then meshed and squeezed to
extract maximum flavor.
 Infusion: maceration in warm spirit which is maintained at a constant temperature for
several days.
 Percolation: the spirit is continuously bubbled through the flavoring or the spirit is boiled
and the vapours pass up through the flavoring agents and condensed.
-Distillation:
The natural products are steeped in the alcohol until it is well impregnated with flavor, then it is
distilled. This liquid is further purified by re-distillation to remove impurities, which would
change the flavor.

-Compounding:
The ingredients are then blended in strict sequence to produce the desired flavor. Most liqueurs
are made to secrete recipes, many of which are century old.

-Maturing:
Liqueurs must be given time to allow the ingredients to develop flavor and character. The finest
liqueurs are matured in oak casks, which aid in mellowing the liquid.

-Fining:
Impurities are still suspended in the liquid and must be removed. For that a substance is added to
the liqueur and to which attracts all the impurities. Then a final filtration is carried out.

-Bottling:
Spirit is added to the liqueur to bring it to the correct alcoholic strength. Sugar syrup may also be
added to adjust the sweetness. Harmless organic colors may be added at this point.
In conclusion, liqueurs;
 contain at least 2.5% sugar by weight
 are generally preferred as digestives
  are generally served frappe (on crushed ice) in a cocktail glass or served straight in a
liqueur glass/sherry glass.
 Are most commonly used in preparations of various cocktails/mocktails and flavored
coffees.

3.5.3. 3 Production of Vermouth

Vermouth is an aromatized, fortified wine, flavored with various botanicals (roots, barks,
flowers, seeds, herbs, and spices) and sometimes colored.
Vermouth is produced by starting with a base of a neutral grape wine or unfermented wine must.
Each manufacturer adds additional alcohol and a proprietary mixture of dry ingredients,
consisting of aromatic herbs, roots, and barks, to the base wine, base wine plus spirit, or spirit
only – which may be redistilled before adding to the wine or unfermented wine must. After the
wine is aromatized and fortified, the vermouth is sweetened with either cane sugar or
caramelized sugar, depending on the style.

Production of Vermouth
Several wine grapes, including Clairette blanche, Piquepoul, Bianchetta Trevigiana,
Catarratto and Trebbiano, are generally used as the base ingredients for vermouths. From these
grapes, a low-alcohol white wine is produced by vermouth manufacturers. The wine may be aged
for a short while before the addition of other ingredients. For sweet vermouths, sugar syrup is
added before the wine is fortified with extra alcohol. The added alcohol is usually a neutral grape
spirit, but may also come from vegetable sources such as sugar beets. The wine is then placed in
large barrels or tanks to which the dry ingredients have already been added. The mixture is
stirred at intervals until the dry ingredients have been absorbed and the drink is ready for
bottling.
Spice ingredients often used in vermouths include cloves, cinnamon, quinine, citrus peel,
cardamom, marjoram, chamomile, coriander, juniper, hyssop, ginger, and labdanum.
Sweet vermouths usually contain 10–15% sugar. The sugar content in dry vermouths generally
does not exceed 4%. Dry vermouths usually are lighter in body than sweet vermouths.
In addition to pale and red vermouths, there exist golden and rosé versions, but these are not as
internationally popular.

According to Stuart Walton and Brian Glover, vermouth "is as far removed from the natural
produce of the vine as it is possible for a fortified wine to get."

3.5.3.4 Production of Bourbon

Bourbon is a type of whiskey where the mashbill which is the recipe of grains used to produce
the whiskey. Consists of 51% - 80% corn. Typically, distillers use approximately 70% corn
content and free to choose other grains for the remainder of the mashbill. The type of grain used
will affect the style and flavors of the whiskey.
The process: most bourbon starts with sour mash. It is taken from a previous batch of mash
(crushed grain going through the fermentation process) set out to sour overnight, and then added
to new batch. The process is much like that of starting sourdough bread.

The recipe: Bourbon is distilled from a fermented mash of grain, yeast and water. The “mash
bill” must have a minimum of 51% corn. For most bourbons, the average is about 70% other
grains such as rye, malted barley and wheat are considered the considered the “flavor” grain.

Length of aging: Bourbon must be aged for a minimum of the years. Many premium bourbons
on the market are aged between 5-12, with some as long as 27 years.

The barrel: Bourbon must be aged in brand new barrels which are made of white oak and have
charred on the inside. Brands determine the varying levels of char for their barrels from 1 to 4.

Flavor: By law nothing can be added at bottling except water. Nothing is added that might
enhance flavor, add sweetness or alter color.

Proof: Bourbon is bottled at between 80 and 125 proof. Only water may be used to lower the
alcohol.

3.5.3. 6 Production of Scotch Whisky

Whisky is a widely consumed global distilled spirit and is one of the highest revenue alcoholic
beverages. Whisky (or whiskey, when referring to Irish or American whiskies) derives from the
ancient term uisge beatha, which is Gaelic for the Latin aqua vitae or 'water of life', was
corrupted in the 18th century to usky, and then to whisky. It is a distilled spirit made from
cereals. Scotland produces the most widely exported and most recognized whisky brands and
Scotch whiskies represent large sources of revenue and employment in Scotland. The Scotch
Whisky Regulations 2009 defined five categories of Scotch Whiskey, they include:

 Single Malt
 Single Grain
 Blended
 Blended Malt
 Blended Grain

What are the main kinds of Scotch Whisky?

There are two kinds of Scotch Whisky - Malt Whisky and Grain Whisky.
The Malt Whiskies are divided into four groups according to the geographical location of the
distilleries in which they are made, as follows:
  Lowland Malt Whiskies: made south of an imaginary line drawn from Dundee in the
east to Greenock in the west.
 Highland Malt Whiskies: made north of that line.
 Speyside Malt Whiskies: from the valley of the River Spey. Although these whiskies
come from within the area designated as Highland Malt Whiskies, the concentration of
distilleries and the specific climatic conditions produce a whisky of an identifiable
character and require a separate classification.
 Islay Malt Whiskies: from the island of Islay.
 Campbeltown Whiskies: from the Campbeltown peninsula on the west coast.

Each group has its own clearly defined characteristics, ranging from the lighter Lowland Malt
Whiskies to those distilled on Islay which are generally regarded as the heaviest Malt Whiskies.

Malt Whiskies, which differ considerably in flavor according to the distillery from which they
come, have a more pronounced bouquet and flavor than the Grain Whiskies. The production of
Grain Whisky is not so influenced by geographical factors and it may be distilled anywhere in
Scotland.

-The production of malt whisky

The origins of malt whisky distilling in Scotland are lost in the mists of antiquity. They date back
at least to the monks of the 15th century and probably long before.
Although the distillers' art has been understood since earliest times, the subtle aromas and
flavours of whisky have never been fully explained, even today. The following description is a
generalisation of the process.
It should be remembered that each distillery has its own unique specifications.

1.Malting
Best quality barley is first steeped in water and then spread out on malting floors to germinate. It
is turned regularly to prevent the build-up of heat. Traditionally, this was done by tossing the
barley into the air with wooden shovels in a malt barn adjacent to the kiln.
During this process enzymes are activated which convert the starch into sugar when mashing
takes place. After 6 to 7 days of germination the barley, now called green malt, goes to the kiln
for drying. This halts the germination. The heat is kept below 70°C so that the enzymes are not
destroyed. Peat may be added to the fire to impart flavor from the smoke.

2. Mashing
The dried malt is ground into a coarse flour or grist, which is mixed with hot water in the mash
tun. The water is added in 3 stages and gets hotter at each stage, starting around 67°C and rising
to almost boiling point.
The quality of the pure Scottish water is important. The mash is stirred, helping to convert the
starches to sugar. After mashing, the sweet sugary liquid is known as wort. The spent grains -
the draff - are processed into cattle feed.

3.Fermentation
The wort is cooled to 20°C and pumped into washbacks, where yeast is added and fermentation
begins. The living yeast feeds on the sugars, producing alcohol and small quantities of other
compounds known as congeners, which contribute to the flavor of the whisky. Carbon dioxide is
also produced and the wash froths violently. Revolving switchers cut the head to prevent it
overflowing. After about 2 days the fermentation dies down and the wash contains 6-8% alcohol
by volume.

4.Pot Stills
In some mysterious way the shape of the pot still affects the character of the individual malt
whisky, and each distillery keeps its stills exactly the same over the years.
In distillation, the still is heated to just below the boiling point of water and the alcohol and
other compounds vaporise and pass over the neck of the still into either a condenser or a worm -
a large copper coil immersed in cold running water where the vapour is condensed into a liquid.

5.Distillation
The wash is distilled twice - first in the wash still, to separate the alcohol from the water, yeast
and residue called pot ale - the solids of which are also saved for use in animal feeds.
The distillate from the wash still, known as low wines, and containing about 20% alcohol by
volume, then goes to the spirit still for the second distillation. The more volatile compounds
which distil off first - the foreshots, and the final runnings called feints where more oily
compounds are vaporised, are both channelled off to be redistilled when mixed with the low
wines in the next batch. Only the pure centre cut, or heart of the run, which is about 68% alcohol
by volume is collected in the spirit receiver.

6. Spirit Safe
All the distillates pass through the spirit safe - whose locks were traditionally controlled by the
Customs & Excise. The stillman uses all his years of experience to test and judge the various
distillates without being able to come into physical contact with the spirit.
The newly distilled, colourless, fiery spirit reduced to maturing strength, 63% alcohol by
volume, is filled into oak casks which may have previously contained Scotch whisky, bourbon or
sherry, and the maturation process begins.

-The production of grain whisky

Scotch grain whisky is usually made from 10-20% malted barley and then other unmalted cereals
such as maize or wheat. The starch in the non-malted cereals is released by pre-cooking and
converted into fermentable sugars. The mashing and fermentation processes are similar to those
used for malt whisky.
The wash is distilled in a continuous or Coffey still, named after its inventor Aeneas Coffey. It
has two tall columns - a rectifier and an analyzer. Cold wash is pumped in at the top of the
rectifier and meets steam. The columns in fact act like a heat exchanger. The alcohol is cooled,
condenses and flows away as Scotch grain spirit at about 94% alcohol by volume.
The distilled grain spirit is lighter in character and aroma than most malt whiskies and therefore
requires rather less time to mature. The bulk of matured grain whisky is used for blending.
The maturation process
While maturing, the whisky becomes smoother, gains flavour, and draws its golden colour from
the cask. A proportion of the higher alcohols turn into esters and other complex compounds
which subtly enhance each whisky's distinctive characteristics.
By law all Scotch whisky must be matured for at least 3 years, but most single malts lie in the
wood for 8, 10, 12, 15 years or longer. Customs & Excise allow for a maximum of 2% of the
whisky to evaporate from the cask each year - the Angels' Share. Unlike wine, whisky does not
mature further once it is in the bottle.

The art of blending

While the distinctive single malts produced by individual distilleries are becoming increasingly
popular, blending creates over 90% of the Scotch whisky enjoyed throughout the world.

By nosing samples in tulip-shaped glasses the blender selects from a wide palate - from the
numerous Highland and Speyside malts to the strongly flavoured and peaty Island malts, and the
softer and lighter Lowland malts. These malts are combined with grain whiskies - usually 60-
80% grain whiskies to 20-40% malt whiskies, and are then left to 'marry' in casks before being
bottled as one of the world-renowned blended whiskies.

A blend of a range of malt whiskies, with no grain whisky included is known as blended malt.
The way we make Scotch whisky has evolved over several centuries, but the history of Scotch
whisky embraces a much wider heritage; that of Scotland and its people.

3.5.3. 7 Production of Vodka

Vodka is a neutral spirit made from the fermentation and distillation. It is a clear
distilled alcoholic beverage with different varieties originating in Poland, Russia
and Sweden Fig 5. It is composed primarily of water and ethanol, but sometimes with traces of
impurities and flavorings. Traditionally it is made by distilling the liquid from cereal grains that
have been fermented, with potatoes arising as a substitute in more recent times, and some
modern brands using fruits as the base. Since the 1890s, the standard alcohol volume has been
40%.
Fig 5: Vodka
Vodka can be manufactured from almost anything, which contains starch including potatoes,
grains, sugar cane, and grapes but generally produced by rye or corn. All these ingredients have
been commercially used although grain is the source of most vodka produced in the world today.

Production
The grain is first pressure-cooked and then grounded. For this, a very special type of pressure
cooker is used. The mash is then cooled and mixed with water. Special types of enzymes are
added to convert starch into sugar.

Fermentation
Yeast is added and fermentation takes place as much as in whiskey making.

Distillation
Then mash goes into a continuous still for distillation where it is distilled to very high proof
around 190 U.S proof which eliminates all possible flavoring & congeners from the spirit.

Rectification
Continuous still generally have two columns, one for distillation and another for rectification.
But in stills where the neutral spirit is produced, there is an additional column for purification.
Generally, very high-quality vodkas are passed through a bed of vegetable charcoal. When vodka
comes out from the tank of charcoal it is absolutely clear and free of all sorts of characters.
Bottling
The purified neutral spirit is diluted with natural river water (not distilled water) to reduce its
alcoholic strength to 40% v/v. Aging is not required as vodka can be bottled the same day it is
made. Vodka certainly does not have the bold flavor but neither it is as tasteless.
The vodkas are perfectly fit for use in cocktails like Bloody Mary, Screw Driver, Vodka Martini,
Bullshot, Black and White Russian, etc. Russian vodkas are enjoyed either on the rocks or
straight preferably the latter. The Polish vodka is also enjoyed in the same way as
their Russian counterparts.

3.5.3.8 Production of Rum

It takes about 4-10 days to complete. Rum has been in production since the 17 th century in the
Caribbean, where the majority of rum production still place. It was traditionally made from
sugarcane juice, but now is usually made from sugarcane juice, but now is usually made from
molasses or brown sugar.
Procedure for preparing rum
A, making of mash
1. Begin by placing 20l of water in a clean boiler. The operative word is clean. The slightest
bit of contamination can spoil the rum. Before you start, make sure you are dealing with
the cleanest ingredients and a sanitary area. Clean and then submerge any utensils you
will be using in boiling water and soak your pot or barrel in the near- boiling water. Then
dump the water. This will help kill off any potentially harmful germs.
2. Dissolve both the sugar and molasses in 20 liters of water over medium heat. The sugar
will dissolve easily, but the molasses will be tougher dissolve, as it is very sticky. Try not
to boil the water. Continue to let it heat up until bubbles just start to form, and then turn
the burner off.
3. Cool the solution to 280C (820F) and add the hydrated yeast.

B, Fermentation
1. Allow solution to ferment at 250C (770F) until the airlock on your pot stops bubbling.
2. Once airlock has finished bubbling allow the mash to sit for 3-7 days.
3. Knock your yeast down by lowering the temperature.
C, Distillation
1. Place a collection vessel under the distillative valve to catch your alcohol solution.
2. Connect a water source to content input.
3. Now siphon the solution into still
4. Begin slowly bringing the solution to a boil
5. Discard the first 100ml (3.38 fl. Ounces) the clear liquid.
6. Collect the next 2-3 l of distillate exiting the spout. Stop collecting once temperature
reached 960C ( 204. 8F)
 7. Turn off the heat source, the turn off the cold water.
8. Open the lid to still so as to avoid creating a vacuum inside your still.
D, Finishing of the rum
1. Age your rum in oak barrels or toasted oak (optional).
2. Use water to dilute alcohol to expected proof.
3. Add flavors of other additives to improve the taste.

3.5.3.9 Preparation of Cognac

The manufacturing process

Pressing the grapes: The grapes are pressed and the juice is allowed to ferment naturally. No
sugar or sulfur dioxide is added.

First Distillation- as soon as the wine has fermented, it is poured into pot stills enclosed in brick
kilns. Each still holds approximately 660 gallons, or the equivalent of 3000 bottles. The kilns are
heated to a temperature range between 1730F (78.30C) and 2120F (1000C) until the alcohol
vaporizes and separates from the rest of the liquid.

The vapors are collected in the cowl and the swan’s neck of the still. They then pass into the
serpentine-like condenser coil. The condensed liquid, called “broullis”, is reduced one third from
the original amount and measures about 30% alcohol by volume.

Second distillation: The broullis is heated a second time in a process known as “Bonnechauffe.”
This is a exacting process because the distiller has to decide at what moment to isolate what is
known as the “heart” of the liquid, to separate it from the “head” and “tails.” The head portion is
too high in alcohol content while the tail is lacking in substance. These portions are redistilled
several times and used in blending.

Casking the distilled brandy: Generally speaking, the brandy is first stored in newer casks for
periods between one and two years. The amount of time is dependent on the level of tannin that
is desired. Tannin is strongest in the new oak, so the brandy must possess enough character to
absorb large amounts of tannin.

Aging and blending the Cognac

Bottling the Cognac: The Cognac is bottled and packaged. Many of the bottles are handcrafted
of crystal. They are often sealed with wax and draped with satin ornaments. After bottling, the
cognac is either packed for shipping or stored for future shipments.
3.5.3. 10 Production of Tequila
Tequila is a distilled beverage made from the blue agave plant, primarily in the area surrounding
the city of tequila 65 km northwest of Guadalajara and in the Jaliscan highlands of the central
western Mexican state of Jalisco. The production of tequila is divided into eight steps:
harvesting, extraction, cooking, fermentation, distillation, filtration, aging and bottling.
1. Harvesting: the agave is harvested from the plantations. Experts come in to take samples
of agave lots, and determine the total sugars within the plants using a titration method. The agave
pina (fruit/ succulent core) is shredded through 3 shredding processes, the agave fibers are
prepared for sugar extraction.
2. Extraction: A diffuser extracts the sugar within the shredded agave fibers using a gentle
extraction process, producing a solution of the water and agave sugar. Agave juice can then be
obtained from this solution, as can bagasse, which is later used as compost for the agave
plantations.
3. Cooking: Agave contains complex sugars, like inulin, that must be transformed into
fermentable sugars through a hydrolysis process. The hydrosis process. The hydrolysis process
takes about 6 hours to convert inulin into fructose and glucose.
4. Fermentation: Anaerobic fermentation is carried out in stainless steel tanks using a
proprietary blend of yeasts and nutrients. Each round ferments for 24-48 hours with varying
temperatures, from 320C (98.6 0F). Fermentation process is the fast and clean, because we use
closed tanks and an automated cleaning process that guarantees a safe tequilla product.
5. Distillation: The first distillation process takes place in steel distillation columns, during
which water is removed and alcohol is concentrated from fermented agave juice. The second
distillation takes place in stills to obtain tequila with a range of 55-65% alcohol, depending on
the brand. All of the distillation by-products are treated in a wastewater treatment plant, in full
compliance with environment regulations.
6. Filtration: Every tequila is filtered using slightly different methods. We use carbon filters,
through which tequila is filtered through in cold temperatures to remove the excess fatty
compounds that give tequila a cloudy appearance. Other filtration processes assure that there are
no particulates present in the liquid, and ultimately work to give our tequila a shiny finish.
7. Aging: Tequila is aged in the white oak wood containers for at least 2 months. Tequila is
kept in the white oak barrels for at least 12 months, and extra-aged tequilas are kept in white
oak barrels for at least 36 months.
8. Bottling: the tequila is then bottled, labeled and ready for distribution.
3.5.4 PRODUCTION OF ETHANOL AND INDUSTRIAL ALCOHOLS
Ethanol (also called ethyl alcohol, grain alcohol, drinking alcohol, spirits, or simply alcohol) is
an organic chemical compound. It is a simple alcohol with the chemical formula C 2H6O or
C2H5OH. Ethanol is a volatile, flammable, colorless liquid with a slight characteristic odor. It is a
psychoactive substance, recreational drug, and the active ingredient in alcoholic drinks. Ethanol
is used in medical wipes and most commonly in antibacterial hand sanitizer gels as an antiseptic
for its bactericidal and anti-fungal effects. Ethanol kills microorganisms by dissolving their
membrane lipid bilayer and denaturing their proteins, and is effective against most bacteria and
fungi and viruses. However, it is ineffective against bacterial spores, but that can be alleviated by
using hydrogen peroxide.[16] A solution of 70% ethanol is more effective than pure ethanol
because ethanol relies on water molecules for optimal antimicrobial activity.

Production
Ethanol is produced both as a petrochemical, through the hydration of ethylene and, via
biological processes, by fermenting sugars with yeast. Which process is more economical
depends on prevailing prices of petroleum and grain feed stocks. In the 1970s most industrial
ethanol in the United States was made as a petrochemical, but in the 1980s the United States
introduced subsidies for corn-based ethanol and today it is almost all made from that source.the
processes of production include;
Ethylene hydration

Ethanol for use as an industrial feedstock or solvent (sometimes referred to as synthetic ethanol)
is made from petrochemical feed stocks, primarily by the acid-catalyzed hydration of ethylene:
C2H4 + H2O → CH3CH2OH

The catalyst is most commonly phosphoric acid, adsorbed onto a porous support such as silica
gel or diatomaceous earth. This catalyst was first used for large-scale ethanol production by the
Shell Oil Company in 1947. The reaction is carried out in the presence of high pressure steam at
300 °C (572 °F) where a 5:3 ethylene to steam ratio is maintained. This process was used on an
industrial scale by Union Carbide Corporation and others in the U.S., but now only
LyondellBasell uses it commercially.

In an older process, first practiced on the industrial scale in 1930 by Union Carbide, [88] but now
almost entirely obsolete, ethylene was hydrated indirectly by reacting it with concentrated
sulfuric acid to produce ethyl sulfate, which was hydrolyzed to yield ethanol and regenerate the
sulfuric acid:[89]
C2H4 + H2SO4 → CH3CH2SO4H
CH3CH2SO4H + H2O → CH3CH2OH + H2SO4
From CO2

CO2 can also be used as the raw material.

CO2 can be reduced by hydrogen to produce ethanol, acetic acid, and smaller amounts of 2,3-
butanediol and lactic acid using Clostridium ljungdahlii, Clostridium autoethanogenum or
Moorella sp.

CO2 can be converted using electrochemical reactions at room temperature and pressure. In a
system developed at Delft University of Technology, a copper nanowire array used as a cathode
adsorbs molecules of carbon dioxide and reduced intermediate species such as CO and COH.
However, even in the best results about half the current went into producing hydrogen and only a
small amount of ethanol was produced. Other products, produced in larger quantities, were (in
decreasing order) formic acid, ethylene, CO, and n-propanol.
From lipids

Lipids can also be used to make ethanol and can be found in such raw materials such as algae.

Fermentation
Ethanol in alcoholic beverages and fuel is produced by fermentation. Certain species of yeast
(e.g., Saccharomyces cerevisiae) metabolize sugar, producing ethanol and carbon dioxide. The
chemical equations below summarize the conversion:
C6H12O6 → 2 CH3CH2OH + 2 CO2
C12H22O11 + H2O → 4 CH3CH2OH + 4 CO2

Fermentation is the process of culturing yeast under favorable thermal conditions to produce
alcohol. This process is carried out at around 35–40 °C (95–104 °F). Toxicity of ethanol to yeast
limits the ethanol concentration obtainable by brewing; higher concentrations, therefore, are
obtained by fortification or distillation. The most ethanol-tolerant yeast strains can survive up to
approximately 18% ethanol by volume.

To produce ethanol from starchy materials such as cereals, the starch must first be converted into
sugars. In brewing beer, this has traditionally been accomplished by allowing the grain to
germinate, or malt, which produces the enzyme amylase. When the malted grain is mashed, the
amylase converts the remaining starches into sugars.

Celtic Renewables produces ethanol using waste that results from the production of whisky, and
low-grade potatoes, via ABE fermentation.
Cellulose

Sugars for ethanol fermentation can be obtained from cellulose. Deployment of this technology
could turn a number of cellulose-containing agricultural by-products, such as corncobs, straw,
and sawdust, into renewable energy resources. Other agricultural residues such as sugar cane
bagasse and energy crops such as switchgrass may also be fermentable sugar sources.

Ethanol purification
Distillation

Ethylene hydration or brewing produces an ethanol–water mixture. For most industrial and fuel
uses, the ethanol must be purified. Fractional distillation at atmospheric pressure can concentrate
ethanol to 95.6% by weight (89.5 mole%). This mixture is an azeotrope with a boiling point of
78.1 °C (172.6 °F), and cannot be further purified by distillation. Addition of an entraining
agent, such as benzene, cyclohexane, or heptane, allows a new ternary azeotrope comprising the
ethanol, water, and the entraining agent to be formed. This lower-boiling ternary azeotrope is
removed preferentially, leading to water-free ethanol

At pressures less than atmospheric pressure, the composition of the ethanol-water azeotrope
shifts to more ethanol-rich mixtures, and at pressures less than 70 torr (9.333 kPa), there is no
azeotrope, and it is possible to distill absolute ethanol from an ethanol-water mixture. While
vacuum distillation of ethanol is not presently economical, pressure-swing distillation is a topic
of current research. In this technique, a reduced-pressure distillation first yields an ethanol-water
mixture of more than 95.6% ethanol. Then, fractional distillation of this mixture at atmospheric
pressure distills off the 95.6% azeotrope, leaving anhydrous ethanol at the bottom.
Molecular sieves and desiccants

Apart from distillation, ethanol may be dried by addition of a desiccant, such as molecular
sieves, cellulose, and cornmeal. The desiccants can be dried and reused. [84] Molecular sieves can
be used to selectively absorb the water from the 95.6% ethanol solution. [97] Synthetic zeolite in
pellet form can be used, as well as a variety of plant-derived absorbents, including cornmeal,
straw, and sawdust. The zeolite bed can be regenerated essentially an unlimited number of times
by drying it with a blast of hot carbon dioxide. Cornmeal and other plant-derived absorbents
cannot readily be regenerated, but where ethanol is made from grain, they are often available at
low cost. Absolute ethanol produced this way has no residual benzene, and can be used to fortify
port and sherry in traditional winery operations.
Membranes and reverse osmosis

Membranes can also be used to separate ethanol and water. Membrane-based separations are not
subject to the limitations of the water-ethanol azeotrope because the separations are not based on
vapor-liquid equilibria. Membranes are often used in the so-called hybrid membrane distillation
process. This process uses a pre-concentration distillation column as first separating step. The
further separation is then accomplished with a membrane operated either in vapor permeation or
pervaporation mode. Vapor permeation uses a vapor membrane feed and pervaporation uses a
liquid membrane feed.
Other techniques

A variety of other techniques have been discussed, including the following:

 Salting using potassium carbonate to exploit its insolubility will cause a phase separation
with ethanol and water. This offers a very small potassium carbonate impurity to the
alcohol that can be removed by distillation. This method is very useful in purification of
ethanol by distillation, as ethanol forms an azeotrope with water.
 Direct electrochemical reduction of carbon dioxide to ethanol under ambient conditions
using copper nanoparticles on a carbon nanospike film as the catalyst.
 Extraction of ethanol from grain mash by supercritical carbon dioxide;
 Pervaporation;
 Fractional freezing is also used to concentrate fermented alcoholic solutions, such as
traditionally made Applejack (beverage);
 Pressure swing adsorption.

3.5.5 WINE PRODUCTION

a detailed team work on the basic knowledge, production, advantages and side effects of
alcoholic and non alcoholic wine
INTRODUCTION
Wine is a typical alcoholic drink made from fermented grape juice. Yeast consumes the sugar in
the grapes, converting it to ethanol, carbon (IV) oxide and heat. It is used to prevent heart
disease, stroke, and decline in thinking skills later in life, diabetes and digestive tract infections
leading to ulcers (no scientific backing). It contains alcohol that exerts a depressant action,
blocking various pathways in the brain and preventing blood platelets from forming clots.
It also contains
 Calcium concentrates; to reduce the acidity of the finished wine and sometimes added if
the grapes have had trouble ripening. It is usually added before or at the start of
fermentation, so it doesn’t impact on the aroma of the wine.
 Flavors; oak barrels, oak chips, powders or staves can be added to help evenly distribute
the flavors.
 Grape juice concentrates; usually derived from Teinturer grapes. It is sometimes included
to boost the color of red wine and add a bit of sugar to smooth out the mouth feel.
  Non vegan material; used as a fining agent and clarifier. This includes egg whites,
bentonite clay and mammal proteins.
 Powdered tannins; naturally occur in the skins of the grapes and are good for adding
complexity to wines. Powdered forms are preferable.
 Potassium sorbate and potassium metabisulphate; they are both used to guard against
bacteria and protect the yeast from spoiling. It is commonly used together.
 Sulphur dioxide; this is usually known as sulfites. It is used to preserve the grapes and
prevent oxidation during the wine making process.
 Sugar; added to help boost the alcohol content in a process called chaptalization. It also
assist the yeast during the fermentation process
 Water; added at the beginning of the wine making process to help bring down high
alcohol levels and add balance tom the wine.
 Yeast; it is the key ingredient in wine making. It help convert sugars into alcohol. Some
producers use cultured yeast to enhance certain flavor profiles or use yeast in second
fermentation called malolactic fermentation, where naturally present malic acids are
converted into softer lactic acids.

Wine Production
It includes five major processes;
 Harvesting
 Pressing
 Fermentation
 Clarification
 Aging/bottling

.Harvesting
The grapes are cautiously watched during its growth and plucked in bunches when ripened,
preferably by hand to minimize spoils, carefully selected to remove the under ripe and rotten
fruits from the whole.

.Pressing
The grapes are made to undergo crushing through a diastemer mechanically which is important
for sanitation quality and longetivity of the wine. The grapes are quickly crushed and pressed to
separate the juice from the seed, skin and solids when producing white wine, while those add
flavors and color to red wine.

.Fermentation
This is the crucial part in wine making, where the yeast helps to convert the sugars to alcohol. It
involves two stages. The primary stage that lasts for 5-14 days and the secondary stage that
takes a maximum of 5-10 days. The requirements for fermentation includes
  Considerations
o Temperature, speed of fermentation, oxygen levels
 Equipments
o Tank(stainless steel for white wine, open wooden vat, wine barrels, wine bottles)
Wild yeast fermentation includes the use of naturally occurring ambient yeasts, which can
produce high quality, unique flavored wine but are often unpredictable and can introduce
undesirable traits to it. Examples include Klockera/Hansenia spora, Pichia, Metschnikoviaceae,
and Zygosaccharomyces.
Cultured yeast fermentation involves the introduction of Saccharomyces cerevisiae, a sugar
yeast with a continuous supply of carbon, nitrogen, sulphur, phosphorus, vitamins and minerals
to achieve an estimable result.

.Clarification
This involves the use of non-vegan materials such as egg whites, bentonite clay and mammalian
proteins to sieve through the wine to get rid of solids and impurities. This is a rather cautious
stage.

.Aging/Bottling
This stage involves the intensifying of flavors in the wine by using bottles, stainless steels tanks,
oaks barrels etc. type of aging and how long it takes influences the quality and taste of the wine
produced.
At this stage, either an alcoholic or non alcoholic wine could be produced. If the manufacturer
desires an alcoholic wine, he continues to the bottling stage, but if he desires to produce a non
alcoholic wine, he removes the alcohol from the wine using either of these two major processes

Non-Alcoholic Wine Production Processes

Reverse osmosis/filtration; this is an ingenious process that filters out the aroma
compounds and phenolics before the alcohol is removed by distillation. This is done with
extreme pressure and the water is added back afterwards. This is a rather expensive
method with up to 2-4 passes to completely remove the alcohol in the wine.
Vacuum distillation; this method evaporates the alcohol more like boiling at a much
lower temperature of about 70 degrees Fahrenheit with the use of a vacuum chamber.
Unfortunately this process causes most of the pleasant aroma compounds to fly
off(volatilize) with the evaporating alcohol.
Once alcohol is removes, it is ready for bottling and consumption.
Some examples of non-alcoholic wine includes Ariel Cabernet. Sauvignon dealcoholized,
martinelli’s sparkling apple cider amongst others.
Conclusion
Alcohol is sometimes seen as a stimulant because it is rapidly absorbed in the
gastrointestinal tract, no digestive process involved, it moves to the blood and also to the brain
exerting its depressant action.
Side effects include flushing confusion, rapid mood changes, and if taken in excess could
bring about blackouts, walking troubles, vomiting, seizures, diarrhea etc. long term abuse could
cause heart, liver pancreas and cancer problems.

3.5.6 PRODUCTION OF NON-ALCOHOLIC BEVERAGES

MALT DRINKS

INTRODUCTION
Malt drink is a non-alcoholic, wholesome, nourishing and satisfying food drink with zero or
negligible level of alcohol. A malt drink is a fermented drink in which the primary ingredient is
the grain, or seed, of the barley plant, which has been allowed to sprout slightly in a traditional
way called "malting" before it is processed. Non-alcoholic drinks are additional products to beer
which are produced and marketed by several breweries in Nigeria. There are more potential
customers for the malt drinks than beer in view of its non-alcoholic nature. Malt drink production
involves the use of similar raw materials, machinery and procedure as in beer brewing.
Traditionally, barley malt has been used in the production of extract for making malt drinks. In
recent times, there has been an increased utilization of locally grown cereals such as sorghum
and maize as adjunct of barley malt in Nigerian breweries for the production of brewing extract.
Malting is the process of converting insoluble sugar in cereal grain to soluble sugars, modifying
protein, generating nutrient for yeast and development of enzymes. In other words, it is the
process of modifying (force germinating) the grain in other to make the sugar trapped inside the
kernel available for mashing. There are three main processes involved in malting of cereals for
production of malt drinks. Breweries typically purchase malted grain (malt) from malting
operations. In the malting process, grain is first soaked in water-filled steeping tanks for
softening. After softening, the grain is transferred to germination tanks, in which the grain
germinates, typically over a 1-week period. From the germination tanks, the grain enters a kiln,
which halts germination by drying the grain. To begin the brewing process, malt (usually barley
malt) is transported by truck or rail to a brewery and is conveyed to storage silos. The malt is
then ground into malt flour by malt mills and transferred to milled malt hoppers. Many small
breweries purchase malt flour (malted and milled grain) from facilities with malt mills. Malt
provides the starch-splitting and protein-splitting enzymes that are necessary to convert grain
starches into fermentable sugars.
 3.5.7 PRODUCTION OF YOGHURT
The industrial manufacture of yogurts is organized along three main steps:
1. The preparation of the mix and all corresponding physical treatments such as homogenization, heat
treatment, cooling, and deaeration
2. The fermentation process starting after inoculation of the mix
3. The yogurt harvesting, post-treatment, and packaging. Depending on the steps performed, at least four
types of yogurt can be considered. One has to notice that each step of the manufacture affects the final
quality of the yogurts and that, except for set-type yogurts, the product flavoring and the cup filling are
performed after fermentation.

PREPARATION OF THE MIX

Milk standardization
In order to obtain the mix to be fermented, milk preparation involves mainly fat and protein
content standardization and optional addition of sweeteners and stabilizers. Fat standardization consists
of fat removal by centrifugation (at about 55 C), followed by cream reincorporation to reach the targeted
fat content, ranging from nonfat (0.01%), to low- or light-fat (1–2%), to whole-fat yogurts (>3.2%).
Protein standardization aims at increasing the protein content of the mix (from 3% to 5–15%) in order to
improve the yogurt firmness (texture) and reduce its syneresis. It is mostly done by addition of milk
powder, which is the easier and traditional way. The use of milk proteins or milk replacers as caseinates
or whey powders is also common. A complete mixing of the dry ingredients without air incorporation is
recommended. Concentration of milk by membrane processes (ultrafiltration and reverse osmosis) is an
alternative method to increase the protein content of the mix. For some yogurt recipes, sugars or other
sweetening agents are added to the mix, generally after the physical treatments described in the
succeeding text. In some countries, the use of thickeners and stabilizers (gelatin, pectin, xanthan gum,
carrageenan, starch, etc.) at concentrations varying from 5% to 10% is allowed by FAO/WHO to
improve the yogurt texture.

Physical treatments of the mix

Heat treatment is an essential step of the mix preparation. It allows removing spoilage
microorganisms, inactivating lacto peroxidases and producing stimulatory compounds in milk. In
parallel, heat treatment contributes to improved yogurt texture by allowing whey protein denaturation
and interaction with casein, resulting in a decrease of gel syneresis and an increase of gel firmness.
During industrial yogurt manufacture, the mixes are generally heated at 90 or 95 C for 3–7 min before
cooling down to fermentation temperature. Plate heat exchangers, with a tubular holding zone, are
generally used and are designed in order to cool the mix accurately at the fermentation temperature
(between 37 and 43 C). Two other physical treatments of the mix, deaeration and homogenization, are
closely associated with the heat treatment, and the design of the heat exchangers takes into account the
temperature favoring their effect. Homogenization is compulsory for yogurt quality, as it increases the
gel texture and reduces syneresis. It provokes a reduction of the size of the fat globules (near 2 mm) and
a better link between fat and hydrophilic proteins. Homogenization of the mix is done at high pressure
(20 or 25 MPa) and at a temperature close to 70 C. Associated with the heat treatment of the mix, it
takes place just after the holding section of the heat exchanger. Double-stage high-pressure
homogenizers are recommended for high-fat yogurts. Vacuum deaeration of the mix is performed at
large industrial scale to reduce its oxygen content and consequently shorten the fermentation time, as to
improve the yogurt texture and to remove off-flavors. This step is generally performed at 70 C, before
homogenization

The Fermentation Process

Inoculation of the mix
At industrial scale, yogurts are prepared through inoculation of the mix with concentrated starter
cultures of the two yogurt bacteria (Streptococcus Thermophilus and Lactobacillus delbrueckii subsp.
bulgaricus). The commercial starter cultures are composed of specific blends of selected and well-
defined strains, at a concentration higher than 1010 colony-forming units (CFU) g1 , and are preserved
as frozen or freeze-dried formulations. The inoculated mix contains generally 106 –107 CFU ml1 of
bacteria. After mixing, it is transferred to the fermentation tanks (for stirred, drinking, or concentrated
yogurt manufacture) or directly to the packaging machine for fermentation in cups (for set-type yogurt
manufacture).

Fermentation step
During the lactic acid fermentation of milk, numerous parameters vary as a function of time,
The consumption of lactose and nitrogenous compounds permits the growth of both strains and leads to
the accumulation of many relevant metabolites. Lactic acid, galactose, acetaldehyde, and
exopolysaccharides are the most important ones, contributing to flavor and texture of the yogurt. The
synthesis of extracellular lactic acid provokes an acidification of the mix characterized by a decrease of
the pH , the coagulation of proteins, and the subsequent gel formation. Acetaldehyde confers to yogurt
its particular aroma, and exopolysaccharides contribute to its texture.
The acidification process is controlled by the final pH of the yogurt and the acidification rate,
which are key factors to master quality. The fermentation is stopped (by a fast cooling of the product)
when the final pH of the yogurt is reached. The targeted final pH varies from 4.8 to 4.5, as a function of
the type of yogurt. A significant postacidification during the yogurt’s cooling, harvesting, and storage
has to be considered in defining this target. Generally, online measurement of pH of the mix is avoided,
because the glass pH probes may break inside the mix and need to be submitted to cumbersome
protocols of cleaning and calibration. Consequently, only manual sampling is done during the
acidification process to allow offline pH measurements, and the decision to stop the fermentation by
cooling requires a good expertise of the process. The acidification rate acts directly on the fermentation
time, so that its knowledge and control are very important to properly schedule industrial production. It
is influenced by various factors, such as starter composition and activity, mix composition and physical
treatments, and fermentation temperature. However, accurate temperature control is quite impossible
during yogurt fermentation because of the coagulation phenomenon that occurs at about pH 5.2. As a
consequence, the fermentation time can vary in important ranges. For probiotic yogurt, fermentation
 time can reach 6–8 h, whereas for stirred yogurt, a 3–4 h process is the common target. However,
industrial manufacturers have difficulties to master perfectly all the parameters of the process, and
longer fermentation times (5 or 8 h) are frequently attained. The fermentation of set-type yogurts is
generally performed in cabinets, incubation rooms, or large tunnels in which the pallets move forward
gradually with forced ventilation of warm air. The fermentation of stirred yogurts is performed in large
tanks (15–20 m3 for the largest ones) equipped with mixing devices for mix homogenization, starter
mixing, and gel breaking after fermentation Fig. 6.

Yogurt Harvesting and Packaging

Cooling and harvesting of yogurt
The first step in yogurt harvesting corresponds to a fast cooling of the product in order to stop its
acidification. It takes place when the required final pH of yogurt is obtained. Set yogurts are cooled
within 1 or 2 h to 4 or 5 C using cold air in ventilated cabinets, cooling rooms, or tunnels, as a function
of the size of the manufacturing unit. For stirred yogurt, the cooling is performed in an external heat
exchanger reaching an intermediate temperature (between 18 and 25 C) in less than 1 h (20–60 min for
industrial tanks). At this temperature, some additives as aroma compounds, sweeteners, and fruits (jam,
pulp, and pieces) can be added to stirred yogurts. In modern large plants, these additions are generally
performed online at the level of the packaging machine, using metering pumps and mixers. The final
texture of yogurts, especially stirred ones, is a critical factor for consumer acceptance. As the texture is
influenced by many factors (mix composition, strains used, and processing conditions), it is a real
challenge to obtain the targeted texture. The mechanical constraints exerted on stirred yogurt by all the
harvesting devices (pumps, heat exchangers, pipes, mixers, filling machine, etc.) tend to reduce its
texture but can give them some smoothness.

Packaging of yogurt
Yogurt packaging ensures its hygiene and protection during distribution. If plastic and glass
cups are always used for set type yogurts, large up-to-date packaging units use the ‘form–fill–seal’
technology. The same packaging machine realizes the three main following operations
1. The thermoformation of the containers at 150–200 C, using multilayer thermoplastic material
2. the filling of the preformed containers under a closed environment and sterile air overpressure
3. The thermosealing of the filled containers with an aluminum lid labeled to deliver product information.
These high-tech packaging machines allow reaching high security and high capacity (up to 70 000 cups
per hour) standards. Consequently, they correspond to the most expensive investment in an industrial
manufacturing unit of yogurt. Overpackaging is then carried out, in the form of multipacks of 2, 4, 8, or
16 cups, with the help of an automatic tray packer. After packaging and overpackaging, the yogurts are
stored at low temperature (4 or 5 C), which is maintained during transportation and commercialization.
This low temperature maintenance permits limiting the postacidification in the products and preserving
their safety
 Reception and storage of milk (4 C)

Fat standardization (not-fat, low-fat, full-fat)

Protein standardization (5–15%)

Heat treatment (90–95 C, 3–7 min

Homogenization (20–25 Mpa at 70 C)

Deaeration (70 C)

Cooling to incubation temperature (42 C)

Inoculation (106–107 CFU ml–1)

Flavoring Fermentation in tank (42 C)

Filling (10 C) Mixing of the coagulum

Fermentation in cup (42 C) Cooling (18–25 C) Homogenization Concentration

Flavoring Flavoring Cooling at 10–12 C

Filling (10 C, aseptic conditions)

Cooling (5 C), cold storage, transportation, delivery

Set Type Stirred Drinking Concentrated

Fig 6: SCHEMATIC DIAGRAM OF THE PRODUCTION PROCESSES OF SET-TYPE,

STIRRED, DRINKING, AND CONCENTRATED YOGURT
3.5.8 VINEGAR PRODUCTION
Introduction
Vinegar is a non-alcoholic acid liquid produced from the fermentation of ethanol. it contains
about 4-8% acetic acid in water, varying amounts of fixed fruits acids, coloring matter,salt and a
few other fermentation products of which impart characteristic flavors and aroma to the products.
Vinegar is primarily used to flavor and preserve foods and as aningredient in salad dressings and
marinades. Vinegar is also usedas a cleaning agent. Natural vinegar is a superior food additive
oversynthetic vinegar as it carries essential amino acids from its fruitsource and is reported to act
as a medicine for aches and gastrictroubles. However, it is generally ignored by both the
consumer (dueto the higher price) and the producer (due to the long fermentationtime of 5–6
weeks).
There are different types of vinegar,produced in different parts of the worldand each type
depends on the raw material or source and ranges from wine vinegar, cider vinegar, honey
vinegar, fruit vinegar, balsamic vinegar, malt vinegar,coconut vinegar, palm vinegar, raisin
vinegar, to sugar cane vinegar.

-Raw Materials
The major raw materials for the production of vinegar are alcoholcontaining liquid, Acetobacter,
a genus of aerobic bacteria, oxygen, and sometimes herbs and fruits as a flavoring agent.

Alcohol Containing Liquid

Vinegar can be made from a variety of diluted alcohol products, themost common being wine
and beer. Alternatively, an alcohol productcan be prepared through fermenting carbohydrate in
rice, sugar cane,or malt anaerobically by yeast. The resulting alcohol product ispasteurized,
filtered and then diluted to adjust the alcohol contentand then used for vinegar production.

Bacterial Cultures
Acetobacter acetii cultures are used for vinegar production. Theseperfectly work at a temperature
of 28 °C (82 °F) with full air injection. The lowest temperature that the bacteria can tolerate is 20
°C(8 °F) and the maximum temperature is 33 °C (91 °F). Below andabove these temperatures,
there is no conversion from alcohol intoacetic acid. The starting alcohol should be lower than 7.5
% (v/v)and there should be no free Sulfites. In the natural processes, Acetobacters are allowed to
grow overtime. However, mother of vinegar is added as a source of Acetobacter for commercial
production. Mother of vinegar is the gooey film thatappears on the surface of the alcohol product
as it is converted tovinegar. Mother of vinegar is skimmed off the top and added tosubsequent
batches of alcohol to speed the formation of vinegar. Itconsists natural carbohydrate called
cellulose and this film holds thehighest concentration of Acetobacters. Sometimes in the
vinegarfactory acetozym nutrients are added in to the alcohol liquid as abacterial culture.
Acetozym nutrients are manmade powdered formof mother of vinegar.
Production Methods
Vinegar is the product obtained as a result of impartial oxidation ofalcohol in a fermenting sugar
containing fruit or cane juice, molasses,fermented mash of malted grain, honey, syrups, etc. It is
made fromthe fermentation of ethanol by acetic acid bacteria. The ethanol maybe derived from
many different sources including wine, cider, beeror fermented fruit juice. For wine vinegar and
beer vinegar theproduction process only includes fermentation for the conversion ofthe alcohol
present in the raw materials into acetic acid. However,in other types of vinegar such as fruit
vinegars or cane vinegars twomajor processing steps are carried out; one for the production
ofethanol alcohol from raw materials and the other for the conversionof the ethanol produced
into acetic acid. Vinegar contains, mainly, acetic acid by weight and small quantities of alcohol,
glycerol, esters,sugars, and salts. To find pure acetic acid the vinegar is subjectedto purification
by distillation.The transformation of wine or fruit juice to vinegar is a chemicalprocess in which
ethyl alcohol undergoes partial oxidation that results in the formation of acetaldehyde. Then, the
acetaldehyde is convertedinto acetic acid. Thus, it can be said that the production of
vinegarinvolves two types of biochemical reactions: alcoholic fermentationand oxidation of
alcohol into acid.
Alcoholic fermentation of carbohydrate is the first critical step in the production of vinegar and
takes place under anaerobic condition.
In this step sugar is fermented to alcohol by the action of yeast species as follows;
C6H12O6 (Glucose) 2C2H5OH (Ethyl Alcohol) + 2CO2 Yeast + Energy
Oxidation of alcohol to acid is the second major step in theproduction of vinegar and is an
aerobic process. In this step alcohol isoxidized to acetic acid by the action of acetic bacteria; the
species ofAcetobacter.
Commercial vinegar is produced either by fast or slow fermentationprocesses. In the slow, or
natural, process, vats of cider are allowedto sit open at room temperature. During a period of
several months,the fruit juices ferment into alcohol and then oxidize into acetic acid.Slow
methods generally are used with traditional vinegars;fermentation proceeds slowly over the
course of weeks or months.The longer fermentation period allows for the accumulation of
anontoxic slime composed of acetic acid bacteria and soluble cellulose,known as the mother of
vinegar.Fast methods add mother of vinegar (i.e., bacterial culture) to thesource liquid before
adding air using a Venturi pump system or aturbine to promote oxygenation to obtain the fastest
fermentation. Infast production processes, vinegar may be produced in a periodranging from 20
hours to three days. In the modern commercialproduction of vinegar, the generator method and
the submergedfermentation method are employed. These methods are based onthe goal of
infusing as much oxygen as possible into the alcoholproduct.
The three common methods used for vinegar production are; thegenerator or trickling method,
the submerged fermentation or theAcetator method and the Orleans traditional method.
Traditionallynatural spontaneous fermentation is also used. The Generatormethod is the quicker
one and is generally used in commercial vinegarproduction.
The Orleans Method
The Orleans process is one of the oldest and well known methodsfor the production of vinegar. It
is a slow, continuous process, whichoriginated in France. High grade vinegar is used as a starter
culture,to which wine is added at weekly intervals. The vinegar is fermentedin large (200 liter)
capacity barrels. Approximately 65 to 70 liters ofhigh grade vinegar is added to the barrel along
with 15 liters of wine.After one week, a further 10 to 15 liters of wine are added and this
isrepeated at weekly intervals. After about four weeks, vinegar can bewithdrawn from the barrel
(10 to 15 liters per week) as more wine isadded to replace the vinegar. One of the problems
encountered withthis method is that of how to add more liquid to the barrel withoutdisturbing the
floating bacterial mat. This can be overcome by usinga glass tube which reaches to the bottom of
the barrel. Additionalliquid is poured in through the tube and therefore does not disturbthe
bacteria. Wood shavings are sometimes added to the fermentingbarrel to help support the
bacterial mat.
Procedures
1) Wooden barrels are laid on their sides. Bungholes are drilledinto the top side and plugged with
stoppers. Holes are also drilledinto the ends of the barrels.
2) The alcohol is poured into the barrel via long-necked funnelsinserted into the bungholes.
Mother of vinegar is added at thispoint. The barrel is filled to a level just below the holes on
theends. Netting or screens are placed over the holes to preventinsects from getting into the
barrels.
3) The filled barrels are allowed to sit for several months. The roomtemperature is kept at
approximately 85°F (29°C). Samples aretaken periodically by inserting a spigot into the side
holes and drawing liquid off. When the alcohol has converted to vinegar, itis drawn off through
the spigot. About 15% of the liquid is left inthe barrel to blend with the next batch.

The Generator Method

Because the Orleans process is slow, other methods have beenadapted to try and speed up the
process. This method uses agenerator (figure 1), which is an upright tank filled with beech
woodshavings and fitted with devices which allow the alcoholic solution totrickle down through
the shavings in which the acetic acid bacteriaare living.
Generators of various sizes (15 feet in diameter and 20 feet inlength) are used. The generator
consists of a cylindrical tank with a perforated false bottom supporting beech-wood shavings or
similarmaterial that will help increase the flow of air from this bottom thathas an exit at the top.
A mix is prepared which consist of an adjusted solution of alcohol acidified with acetic acid and
special nutrients forthe growth of acetic acid bacteria. The latter, spies of the genusAcetobacter,
are inoculated into the beech-wood shavings. The mixis applied in a trough at the top of the
chamber and allowed to trickledown over the shavings. The mix is collected at the bottom of
thegenerator and is re-circulated over the shavings resulting in moreoxidation of alcohol until
vinegar of the desired strength is obtained.Oxidation of alcohol by bacteria may result in the
development oftemperatures high enough to kill them. In order to keep thetemperature down to
25 to 300C, cooling coils need to be provided.The generator method is quicker in comparison to
other methodsand the vinegar may be produced within a period of 10 days. Thismethod is
usually used to manufacture distilled vinegar. Aftercollection of the vinegar distillation process
takes place to concentratethe product.

Fig. 7: Generator for Vinegar Production

Source: www.studentsguide.in

The Submerged Fermentation Method

In the submerged fermentation method, a tank filled with alcohol ispumped with oxygen and
maintained at warm temperature. Primarilyused to produce wine vinegars, this process was
developed in the1950s, using tanks called acetators. The wine is kept at a temperaturebetween 26
and 38ºC while nutrients and air arepumped in to the mixture. The submerged fermentation
method iscommonly used in the production of wine vinegars.Submerged vinegar systems are
most commonly used bycorporations who produce high quantities of vinegar with a highcontent
of acetic acid. Submerged vinegar processing systems workwith the continuous aeration of the
liquid. The vinegar bacteria arefloating in the liquid and do not produce a vinegar mother. Using
thissystem, there is no slime in the machine and the ready vinegar isexceptionally clean and
typical. Two systems are used:

Turbine Systems
work with a turbine at the bottom of the tank and bring the air into theliquid. Those turbine
machines can be controlled automatically withan electronic system: The vinegar is pumped out
of the machinewhen it is ready and is then refilled with wine. These systems aresuitable for
producers who plan to make more than 50,000 liters ofvinegar per year.

Venturi Air Systems

are smaller and cheaper than turbine systems. The processing isdone with a pumping system in a
closed stainless steel tank. The airis brought into the liquid with a Venturi air nozzle, which
brings air into the pumped liquid. The air bubbles have the same size like theturbine bubbles.
The processing time of venturi systems is around30 % longer than turbine systems, but this is
better for quality,because of the lower aeration there is not so much flavor loss. Astastings
showed, vinegars from these machines are always betterand fruitier.
The changing is done after manual controlling of acetic acidcontent with a pump or with the
pump inside the machine. The refillingis done with an external pump. These machines are very
competitiveand are available from 20 liters filling up to 600 liters filling. At themoment the
venturi air system is the most common processing systemfor small and medium vinegar makers
around the world.

Procedures
1) Production plants are filled with large stainless steel tanks calledacetators. The acetators are
fitted with centrifugal pumps in thebottom that pump air bubbles into the tank in much the
sameway that an aquarium pump does.
2) As the pump stirs the alcohol, acetozym nutrients are piped intothe tank. The nutrients spur
the growth of Acetobacter on theoxygen bubbles. A heater in the tank keeps the
temperaturebetween 80 and 100°F (26-38°C).
3) Within a matter of hours, the alcohol product has been convertedinto vinegar. The vinegar is
piped from the acetators to a plate and-frame filtering machine. The stainless steel plates pressthe
alcohol through paper filters to remove any sediment, usuallyabout 3% of the total product. The
sediment is flushed into rain while the altered vinegar moves to the dilution station.

Conclusion
Vinegar is the product obtained as a result of impartial oxidation ofalcohol in a fermenting sugar
containing fruit or cane juice, molasses,fermented mash of malted grain, honey, syrups, etc.
Vinegar isprimarily used to flavor and preserve foods and as an ingredient insalad dressings.The
three common methods used for vinegar production are thegenerator or trickling method, the
submerged fermentation or theAcetator method and the Orleans traditional method. The
generatormethod is quicker in comparison with the others. Submerged vinegarsystems are most
commonly used by corporations who produce highquantities of vinegar with a high content of
acetic acid.Generally, vinegar production allows utilization of over-ripped fruits,sugarcane
rejects, ethyl alcohol rejects and cane by-products suchas molasses and bagasse.
SUMMARY: Industrial Microbiology
The microbial production of primary metabolites, through fermentation, contributes significantly
to the quality of life that we enjoy today. Microorganisms are capable of converting inexpensive
carbon and nitrogen sources into voluble metabolites such as amino acids, nucleotides, organic
acids, and vitamins, which can be added to food as a flavor enhancer and/or to increase its
nutritional value. Also, many primary metabolites are proving invaluable as biosynthetic
precursors for the manufacture of therapeutics.
Overproduction of microbial metabolites is related to developmental phases of microorganisms.
Inducers, effectors, inhibitors, and various signal molecules play roles in different types of
overproduction.
Biosynthesis of enzymes catalyzing metabolic reactions in microbial cells is controlled by well-
known positive and negative mechanisms (e.g., induction, nutritional regulation, and feedback
regulation).
. Microorganisms produce alcohols and acetone that are used in industrial processes.
. Industrial microbiology has been revolutionized by the ability of genetically modified cells to
make many new products.
. Biotechnology is a way of making commercial products by using living organisms.
. The growth of cells on a large scale is called industrial fermentation.
. Industrial fermentation is carried on in bioreactors, which control aeration, pH, and
temperature.
. Primary metabolites such as ethanol are formed as the cells grow (during the trophophase).
. Secondary metabolites such as penicillin are produced during the stationary phase (idiophase).
. Mutant strains that produce a desired product can be selected.
. Most amino acids used in foods and medicine are produced by bacteria.
. Microbial production of amino acids can be used to produce l-isomers; chemical production
results in both d- and l-isomers.
Citric acid, used in foods, is produced by Aspergillus niger.
Enzymes used in manufacturing foods, medicines, and other goods are produced by microbes.
. Some vitamins used as food supplements are made by microorganisms.
. Vaccines, antibiotics, and steroids are products of microbial growth.
. Yeasts are grown for wine- and breadmaking; other microbes (Rhizobium, Bradyrhizobium,
and Bacillus thuringiensis) are grown for agricultural use.
. Organic waste, called biomass, can be converted by microorganisms into the alternative fuel
methane, a process called bioconversion.
. Fuels produced by microbial fermentation are methane, ethanol, and hydrogen.
. Biofuels include alcohols and hydrogen (from microbial fermentation) and oils (from algae).
Review
1. What is industrial microbiology/fermentation? Why is it important?
2. How does commercial sterilization differ from sterilization procedures used in a hospital or
laboratory?
3. Why is a can of blackberries preserved by commercial sterilization typically heated to 100°C
instead of at least 116°C?
4. Beer is made with water, malt, and yeast; hops are added for flavor.
What is the purpose of the water, malt, and yeast? What is malt?
5 Why is a bioreactor better than a large flask for industrial production of an antibiotic?
6. Describe an example of bioconversion. What metabolic processes can result in fuels?
7. Which one of the following is not a fuel produced by microorganisms?
a. algal oil
b. ethanol
c. hydrogen
d. methane
e. uranium
8. Which type of radiation is used to preserve foods?
a. ionizing
b. nonionizing
c. radiowaves
d. microwaves
e. all of the above
9. Which of the following reactions is undesirable in winemaking?
a. Sucrose ➝ ethanol
b. Ethanol ➝ acetic acid
c. Malic acid ➝ lactic acid
d. Glucose ➝ pyruvic acid
10. Which of the following reactions is an oxidation carried out by Acidithiobacillus
ferrooxidans?
a. Fe2+ ➝ Fe3+
b. Fe3+ ➝ Fe2+
c. CuS ➝ CuSO4
d. Fe0➝ Cu0
e. none of the above
Application Question
a. Researchers at the CDC inoculated apple cider with 105 E. coli O157:H7 cells/ml to determine
the fate of the bacteria in apple cider (pH 3.7). They obtained the following results: Number of E.
coli O157:H7 cells/ml after 25 days
Apple cider at 25°C 104 (mold growth evident by 10 days)
Apple cider with potassium sorbate at 25°C 103
Apple cider at 8°C 102
What conclusions can you reach from these data? What disease is caused by E. coli O157:H7?
View publication stats

ANSWER
1. Industrial microbiology is the science of using microorganisms to produce products or
accomplish a process. Industrial microbiology provides (1) chemicals such as antibiotics that
would not otherwise be available, (2) processes to remove or detoxify pollutants, (3) fermented
foods that have desirable flavors or enhanced shelf life, and (4) enzymes for manufacturing a
variety of goods.
2. The goal of commercial sterilization is to eliminate spoilage and disease-causing organisms.
The goal of hospital sterilization is complete sterilization.
3. The acid in the berries will prevent the growth of some microbes.
4. Nutrients must be dissolved in water; water is also needed for hydrolysis. Malt is the carbon
and energy source that the yeast will ferment to make alcohol. Malt contains glucose and maltose
from the action of amylases on starch in seeds (e.g., barley).
5. A bioreactor provides the following advantages over simple flask containers:
■ Larger culture volumes can be grown.
■ Process instrumentation for monitoring and controlling critical environmental conditions such
as pH, temperature, dissolved oxygen, and aeration can be used.
■ Sterilization and cleaning systems are designed in place.
■ It offers aseptic sampling and harvest systems for in-process sampling.
■ Improved aeration and mixing characteristics result in improved cell growth and high final cell
densities.
■ A high degree of automation is possible.
■ Process reproducibility is improved.
6. The production of ethyl alcohol from corn; or methane from sewage. Alcohols and hydrogen
are produced by fermentation; methane is produced by anaerobic respiration.
Multiple Choice
7. e
8. a
9. B
10. a