BTH 401 Bioprocessing Engineering

PROF.
BALASUBRAMANIAN SATHYAMURTHY
2016 EDITION
BTH 401: BIOPROCESSING ENGINEERING
FOR MSC BIOTECHNOLOGY STUDENTS

2014 ONWARDS
Biochemistry scanner
THE IMPRINT
BTH-401: BIOPROCESS ENGINEERING
As per Bangalore University (CBCS) Syllabus

2016 Edition
BY: Prof. Balasubramanian Sathyamurthy
Supported By:
Ayesha Siddiqui
Kiran K.S.
THE MATERIALS FROM THE IMPRINT (BIOCHEMISTRY SCANNER) ARE NOT

FOR COMMERCIAL OR BRAND BUILDING. HENCE ONLY ACADEMIC CONTENT
WILL BE PRESENT INSIDE. WE THANK ALL THE CONTRIBUTORS FOR
ENCOURAGING THIS.
BE GOOD DO GOOD & HELP OTHERS
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PROF. BALASUBRAMANIAN SATHYAMURTHY
2016 EDITION
DEDICATION
I dedicate this material to my spiritual guru Shri Raghavendra swamigal,
parents, teachers, well wishers and students who always increase my morale
and confidence to share my knowledge to reach all beneficiaries.
beneficiaries.
PREFACE
Biochemistry scanner THE IMPRINT consists of last ten years solved question
paper of Bangalore University keeping in mind the syllabus and examination
pattern of the University. The content taken from the reference books has been
presented in a simple language for better understanding.
The Author Prof. Balasubramanian Sathyamurthy has 15 years of teaching

experience and has taught in 5 Indian Universities including Bangalore
University and more than 20 students has got university ranking under his
guidance.
THE IMPRINT is a genuine effort by the students to help their peers with their
examinations with the strategy that has been successfully utilized by them.
These final year M.Sc students have proven their mettle in university
examinations and are College / University rank holders.
This is truly for the students, by the students. We thank all the contributors for
their valuable suggestion in bringing out this book. We hope this will be
appreciated by the students and teachers alike. Suggestions are welcomed.
For any comments, queries, and suggestions and to get your free copy write us
at theimprintbiochemistry@gmail.com or call
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CONTRIBUTORS:
CHETAN ABBUR
ANJALI TIWARI
AASHITA SINHA
ASHWINI BELLATTI
BHARATH K
CHAITHRA
GADIPARTHI VAMSEEKRISHNA
KALYAN BANERJEE
KAMALA
KISHORE
KIRAN
KIRAN H.R
KRUTHI PRABAKAR
KRUPA S
LATHA M
MAMATA
MADHU PRAKASHHA G D
MANJUNATH .B.P
NAYAB RASOOL S
NAVYA KUCHARLAPATI
NEHA SHARIFF
DIVYA DUBEY
NOOR AYESHA M
PAYAL BANERJEE
POONAM PANCHAL
PRAVEEN
PRAKASH K J M
PRADEEP.R
PURSHOTHAM
PUPPALA DEEPTHI
RAGHUNATH REDDY V
RAMYA S
RAVI
RESHMA
RUBY SHA
SALMA H.
SHWETHA B S
SHILPI CHOUBEY
SOUMOUNDA DAS
SURENDRA N
THUMMALA MANOJ
UDAYASHRE. B
DEEPIKA SHARMA
EDITION
: 2016
PRINT
: Bangalore
CONTACT : theimprintbiochemistry@gmail.com or 9980494461

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2016 EDITION
M. SC. BIOTECHNOLOGY FOURTH SEMESTER

BTH 401: BIOPROCESS ENGINEERING
4 units (52 hrs)
Unit 1 - Introduction:
Scope and importance of bioprocess engineering technology, development and strain
improvement of industrially important microorganisms.
Unit 2 - Bioreactors:
Typical structure of advanced Bioreactor and their working mechanism;Design features;
Heat transfer and Mass transfer; Specialised bioreactors-design and their functions;
Airlift bioreactor, Tubular bioreactors, Membrane bioreactors, Tower bioreactors,
Fluidized bed bioreactor, Packed bed reactors and photo bioreactors.
Unit 3 - Fermentation media and Fermentation process:
Natual and synthetic media; Strategies for media formulation, sources of carbon,
nitrogen, vitamins and mineral.Role of buffers, precursors, inhibitors, inducers and
antifoam agents.
Types of fermentation process-submerged fermentation, surface or solid state
fermentation, batch fermentation, continuous fermentation, kinetics of fermentation
process, bioprocess control, monitoring of variables-temperature, agitation, pH and
pressure.
Unit 4 - Downstream processing:
Cell distruption, precipitation methods, solid liquid separation, liquid-liquid extraction,
filtration, centrifugation, chromatography, drying devices (Lyophiliztion and spray dry
technology),
crystallization,
biosensors-construction
and
applications,
Food
processsiong:
Food preservation, and spoilage. Sterilization and pasteurization , canning and packing
of foods.
Unit 5 - Immobilization and Biotransformation:
Methods of immobilization, adsorption, cross-linking, ionic bonding, entrapment,
encapsulation, Advantages and industrial applications of Immobilization of enzymes
and whole cells. Biotransformation of antibiotics, steroids and their applications.
Unit 6 Production of Industrially important products:
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Alcohol: Ethanol, glycerol, butanol, Acetone, Organic streptomycin, tetracycline,

Vitamins:
riboflavin,
Enzymes:
amylase,
protease,
Biodegradable
plastic:
polyhydroxyalkanoates (butyarate, propionate), Recombinant protein-Insulin, hepatitisB vaccine,Fermented foods-sausages,olives,bread,idly and acidophilus milk.
Unit7 Intellectual Property Rights (IPRs) and Entrepreneurship:
IPRs- implications of India, WTO, WIPO, GATT, TRIPS. Patenting and the procedures
involved in the application ofr patents and granting biotechnology, legal implications,
traditional knowledge commercial exploitation, protection.
Entrepreneurship- Potential entrepreneurship activities in biotechnology, product
development, marketing, research and training units. Industrial licensing, venture
capital, Biotechology Industries in India and the potential job opportunites.
References
1.
Jackson AT., Bioprocess Engineering in Biotechnology, Prentice Hall, Engelwood Cliffs,

1991.
2.
Shuler ML and Kargi F., Bioprocess Engineering: Basic concepts, 2nd Edition, Prentice
Hall, Engelwood Cliffs, 2002.
3.
Stanbury RF and Whitaker A., Principles of Fermentation Technology, Pergamon press,

Oxford, 1997.
4.
Mansi EMTEL, Bryle CFA. Fermentation Microbiology and Biotechnology, (2nd Ed).
Taylor & Francis Ltd, UK, 2007.
5.
Colin Ratledge and Bjorn Kristiansen, Basic Biotechnology (2nd Ed.).Cambridge

University Press. 2002.
6.
Prescott, Sc and Dunn, C. Industrial Microbiology, McGraw Hill, New York. 1984
7.
Michael, L. Shulers and Fikret Kargi. Bioprocess Engineering: Basic concepts (2nd Ed.)
Prientice Hall Publishers. 2001
8.
Paulins, M. D. Bioprocess Engineering Principles. John Wiley Publishers.2003
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UNIT 1 - INTRODUCTION:
Scope and importance of bioprocess engineering technology, development and
strain improvement of industrially important microorganisms.
SCOPE AND IMPORTANCE OF BIOPROCESS ENGINEERING TECHNOLOGY

Fermentation:
Fermentation Latin- fervere, meaning to boil. Fermentation in a strict sense is a
biological process that occurs in the absence of oxygen. It is no longer used bcoz the
word Industrial Fermentation is used for large-scale cultivation of microorganisms
(Many are aerobic).Bioprocess technology is a recent replacement to fermentation
technology
Bioprocess Engineering:
Bioprocessing makes use of microorganisms, cells in culture or enzymes to
manufacture products. Improving the strain using molecular biology and rDNA
technology for commercial use Bioprocess Engineering
Applications
Application areas commonly associated with bioprocess engineering include the
production of biofuels
Design and operation of fermentation systems
Development of food processing systems
Application and testing of product separation technologies
Design of instrumentation to monitor and control biological processes.
Production of biological and many more.
Career Options
Process Development
Manufacturing Operations
Quality
Assurance/Compliance
Regulatory
Affairs
(cGMP)Project
Management
Environmental Remediation
Food Technology
Therapeutic Stem Cells
Development and Manufacture of Gene Therapy Vectors and Vaccines
Production of Renewable Biofuels
New Enterprise Development
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Recombinant Protein Production Protein Recovery, Purification and Analysis

Beer and Wine Fermentation
DEVELOPMENT AND STRAIN IMPROVEMENT OF INDUSTRIALLY IMPORTANT
MICROORGANISMS.
Converting a wild type strain into a commercial or Industrial strain
Need for strain improvement:
For making a normal metabolic product in an amount required at commercial level the
wild type organism isolated from natural environment, requires genetic programming
and after genetic improvement it is called the Production Strain.
Removing or shunting of undesired genes
Addition of genes which improves expression of the product.
Production strains to confirm Generally Recognized as Safe (GRAS) norms for
manufacture of food products and ingredients.
Characteristics of a Production Strain
Ideal characteristics of a Production Strain
Should be a high yielding strain
Should have stable biochemical characteristics
Should not produce undesirable substances
Should be easily cultivated on a large scale
Limited or no need for vitamins and additional growth factors
Utilization of a wide range of low cost and readily available carbon sources
Ready breakage, if the product is intracellular
Production of limited byproducts to ease subsequent purification problems.
Development of Production Strain
Primary Screening
To identify and isolate orgs with potentiality
Crowded Plate technique
Auxanography
Enrichment Culture technique
Indicator dye method
Secondary Screening
To isolate industrially useful orgs from Primary Screening
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Primary Screening merely allows the detection and isolation whereas secondary
screening helps in detecting really useful orgs in fermentation process.
Optimization of the orgs to fermentable parameters like pH, temp, pressure,
substrate concentration, air, etc.
Strain Improvement
Preservation
Reason for preferring microbial sources:
Over 2000 different enzymes are available and only few are used commercially.
Commercial enzymes include enzymes from plant, animal and microbial sources. From
1960s microbial source is preferred for several reasons, *Economical produced on
large scale within limited space and time. *Can be easily extracted and purified. *Can
grow in a wide range of environmental conditions *Capable of producing a wide variety of
enzymes. *They can be genetically manipulated to increase the yield of enzymes
Genetic Improvement
This can improve the product yield by more than 100 times or more. Success of the
product depends on the success of strain improvement.
Methods of Strain Improvement
Mutation:
Site directed Mutation
Major Mutation
Minor Mutation
Recombination
Recombinant DNA Technology
Mutations:
One of the most successful approaches for strain improvement. A mutation is any
change in the base sequence of DNA - deletion, insertion, inversion, substitution.
The types include:
Spontaneous mutation
Induced mutation
Site directed mutation
Spontaneous mutation:
Occur spontaneously at the rate of 10-10 and 10-15 per generation and per gene. Occur
at low frequency and hence not used much in industrial strain improvement.
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Induced mutation:
The rate of mutation can be increased by various factors and agents called mutagens.
Ionizing radiations (e.g. X-rays, gamma rays)
Non-ionizing radiations (e.g. ultraviolet radiations)
Various chemicals (e.g. mustard gas, benzene, ethidium bromide, NitrosoguanidineNTG)
Site directed mutations (SDM) (site-specific mutagenesis):
Change in the base sequence of DNA changing the codon in the gene coding for that
amino acid. Can be done by protein engineering method. Desired improvements might
be *increased thermostability, *altered substrate range, *reduction in negative feedback
inhibition, *altered pH range, etc.
Steps involved in site directed mutagenesis:
1.
Isolate required enzyme gene, e.g. via mRNA and its conversion into cDNA,
2.
Sequence the DNA of the gene (in order to decide on change required for primer in stage
5).
3.
Splice gene into M13 vector dsDNA and transduce E. coli host cells.
4.
Isolate ssDNA in phage particles released from host cells.
5.
Synthesize an oligonucleotide primer with the same sequence as part of the gene but
with altered codon (mismatch/mispair) at desired point(s). For example, one of the
codons in DNA coding for the amino acid Alanine is CGG. If the middle base is changed
by SDM from G to C the codon sequence becomes CCG which codes for a different
amino acid (Glycine).
6.
Mix oligonucleotide with recombinant vector ssDNA.
7.
Carried out at low temperature (0-10oC) and in high salt concentration to allow
hybridization between oligonucleotide and part of gene.
8.
Use DNA polymerase to synthesize remainder of strand. (Oligonucleotide acts as a

primer for the DNA synthesis).
9.
Then add ligase to join primer and new strand
10. Double strand DNA molecule.

11. Transform E. coli cells and allow them to replicate recombinant vector molecule.
12. DNA replication is semi-conservative, therefore two types of clone are produced each of
which excretes phage particles containing ssDNA: Type 1: contain the wild-type gene
(i.e. unaltered) Type 2: contain the mutated gene!!!
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Minor Mutation
Affects only the amount of product synthesized. Variants remain phenotypically same
like parent with faster growth. More suitable for fungi. Population tested must be large
and assay of desired product should also be accurate.Eg. P.chrysogenum Q-176 showed
significantly improved antibiotic titres after series of minor mutation
Major Mutation
Mutant with a pronounced change in a biochemical character of practical interest. They
are Surviving population after prolonged exposure to mutagen .Eg. Non pigmenting
P.chrysogenum.Str.aureofacies S-604 synthesized 6-dimethyl tetracycline, a new
antibiotic, not produced by parental strains
Recombination:
Protoplast fusion:
Crossing of two high yielding strains or high yielding with wild type
Gradual decline in the product yield after each mutation, can be prevented by using
recombination.
Heat Transfer in Fermentation
Introduction:
Several important chemical engineering concepts in Bioprocess Engineering are
transport phenomena (fluid flow, mixing, heat and mass transfer), unit operations,
reaction engineering, and bioreactor engineering.
Two types of common heat transfer application in bioreactor operation:
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In situ batch sterilization of liquid medium: In this process, the fermenter vessel
containing medium is heated using steam and held at the sterilization temperature for a
period of time; cooling water is then used to bring the temperature back to normal
operating conditions
Temperature control during reactor operation: Metabolic activity of cells generates heat.
Some
microorganisms
need
extreme
sensitive
temperature
conditions.
(e.g.
psycrophilic, thermophilic microorganisms)

Heat transfer configurations for bioreactors: jacketed vessel, external coil, internal
helical coil, internal baffle-type coil, and external heat exchanger.
Pros and cons of the heat exchanger configurations
External jacket and coil give low heat transfer area. Thus, they are rarely used for
industrial scale.
Internal coils are frequently used in production vessel; the coils can be operated with
liquid velocity and give relatively large heat transfer area. But the coil interfere with the
mixing in the vessel and make cleaning of the reactor difficult. Another problem is film
growth of cells on the heat transfer surface.
External heat exchanger unit is independent of the reactor, easy to scale up, and
provide best heat transfer capability. However, conditions of sterility must be met, the
cells must be able to withstand the shear forces imposed during pumping, and in
aerobic fermentation, the residence time in the heat exchanger must be small enough to
ensure the medium does not become depleted of oxygen.
Heat exchangers in fermentation processes
Double-pipe heat exchanger
Shell and tube heat exchanger
Plate heat exchanger
Spiral heat exchanger
In bioprocess, the temperature difference is relatively small. Thus, plate heat exchanger
is almost never being used
Heat exchangers:-
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Double-pipe heat exchanger
Shell and tube heat exchanger
Plate heat exchanger
Spiral heat exchanger
Mass Transfer in Fermentation

Introduction:
The Henrys law of Solubility, C * =
Po
H
C* is the oxygen saturation concentration of the nutrient solution,Po is the partial

pressure of the gas in the gas phase, H is Henrys constant,which is specific for the gas
and the liquid phase
Oxygen Transfer:
Oxygen to be transferred from a gas bubble to an individual cell has to overcome several
individual partial resistances
Resistance within the gas film to the phase boundary
Penetration of the phase boundary b/n gas bubble and liquid
Transfer from the phase boundary to the liquid
Movement within the nutrient solution
Transfer to the surface of the cell
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Role of diffusion in Bioprocess

Scale of mixing:
Mixing on a molecular scale relies on diffusion as the final step in mixing process
because of the smallest eddy size
Solid-phase reaction:
The only mechanism for intra particle mass transfer is molecular diffusion
Mass transfer across a phase boundary
Oxygen transfer to gas bubble to fermentation broth, penicillin recovery from aqueous
to organic liquid, glucose transfer liquid medium into mould pellets are typical example.
Film theory:
The two film theory is a useful model for mass transfer between phase. Mass transfer of
solute from one phase to another involves transport from bulk of one phase to the
interface, and then from the interface to the bulk of the second phase. This theory is
based on idea that a fluid film or mass transfer boundary layer forms whenever there is
contact between two phases. According to film theory, mass transfer through the film is
solely by molecular diffusion and is the major resistance:-
Convective mass transfer

It refers to mass transfer occurring in the presence of bulk fluid motion.
N A = ka(C Ao C Ai )
NA: Volume dependent Mass transfer[mM O2/I.h], k: mass transfer coefficient [m/s],a:
area available for mass transfer [m2/m3],CAo: concentration of A or dissolved gas in
Phase boundary ,CAi: concentration of A at interface, For gas-liquid system, A from gas
to liquid:
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Overall mass transfer coefficient

Oxygen transport to fermentation broth can be modeled as diffusion of A through
stagnant or non-diffusing B.
If A is poorly soluble in the liquid B, e.g. oxygen in aqueous solution, the liquid-phase
mass transfer resistance dominates and kGa is much larger than kLa. Hence, KLa kLa.
Gas Absorption Rate (GAR)
GAR is not constant during fermentations
There is a characteristic difference in the OAR during the fermentation of single celled
and mycelium producing organisms.
During Log phase, OAR increases and the O2 content in the broth decreases until it
becomes limiting.
In unicellular fermentations, OAR is constant until another substrate becomes limiting.
In mycelial fermentations,OAR decreases when O2 becomes limiting,due to increase in
mycelial volume
X . Qm = kLa . (C* - CL ) (mM O2/l.h)
GAR is not constant during fermentations
There is a characteristic difference in the OAR during the fermentation of single celled
and mycelium producing organisms.
During Log phase, OAR increases and the O2 content in the broth decreases until it
becomes limiting.
In unicellular fermentations,OAR is constant until another substrate becomes limiting.
In mycelial fermentations,OAR decreases when O2 becomes limiting,due to increase in
mycelial volume
X . Qm = kLa . (C* - CL ) (mM O2/l.h)
Critical Oxygen Concentration
It is value of the specific oxygen absortion rate which permits respiration without
hindrance.
They are 5-25% of the oxygen saturation value in cultures
There is no correlative dependence b/n respiration rate and dissolved oxygen beyond
this value.
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Oxygen transfer from gas bubble to cell

Eight steps involved:
Transfer from the interior of the bubble to the gas-liquid interface
Movement across the gas-liquid interface
Diffusion through the relatively stagnant liquid film surrounding the bubble
Transport through the bulk liquid
Diffusion through the relatively stagnant liquid film surrounding the cells
Movement across the liquid-cell interface
If the cells are in floc, clump or solid particle, diffusion through the solid of the
individual cell
Transport through the cytoplasm to the site of reaction.
Analysis for most bioreactors in each step involved
Transfer through the bulk phase in the bubble is relatively fast
The gas-liquid interface itself contributes negligible resistance
The liquid film around the bubble is a major resistance to oxygen transfer
In a well mixed fermenter, concentration gradients in the bulk liquid are minimized and
mass transfer resistance in this region is small, except for viscous liquid.
The size of single cell <<< gas bubble, thus the liquid film around cell is thinner than
that around the bubble. The mass transfer resistance is negligible, except the cells form
large clumps.
Resistance at the cell-liquid interface is generally neglected
The mass transfer resistance is small, except the cells form large clumps or flocs.
Intracellular oxygen transfer resistance is negligible because of the small distance
involved.
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UNIT 2 - BIOREACTORS:
Typical structure of advanced Bioreactor and their working mechanism;Design
features; Heat transfer and Mass transfer; Specialised bioreactors-design and their
functions; Airlift bioreactor, Tubular bioreactors, Membrane bioreactors, Tower
bioreactors, Fluidized bed bioreactor, Packed bed reactors and photo bioreactors.
TYPICAL STRUCTURE OF ADVANCED BIOREACTOR AND THEIR WORKING

MECHANISM
Bioreactor:
An apparatus for growing organisms (yeast, bacteria, or animal cells) under controlled
conditions or any manufactured or engineered device or system that supports a
biologically active environment
Types of Bioreactors
Batch:
Media and cells are added to the reactor and it is run until a predetermined set point
(i.e. time, concentration). The bioreactor has a constant volume (the initial volume).
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Fed-Batch:
The bioreactor is a batch process in the beginning and after a certain point a feed input
is introduced and the volume in the vessel increases.
Continuous:
The bioreactor starts with an initial volume and media is constantly introduced and
product is constantly taken out. The inputs and outputs are at the same rate, so the
volume always remains the same.
DESIGN FEATURES
Bioreactor Components
Bioreactors consist of: Vessel , Agitator, Sparger, Baffles Condenser ,Back Pressure
Valve, Inlet Filters and Exhaust Filters, Valves, Load Cell , Cooling Jacket, Ports for
input and output of material , Probes:- Temperature, Dissolved Oxygen, pH, Pressure
Gauge
Bioreactor - Vessel
The bioreactor vessel is a container which holds the media and the cells.
Vessels can be made of glass, stainless steel, or a durable plastic.
The durable plastic vessels are disposable (single-use).
All the additional parts of a bioreactor connect to the vessel.
The top of the vessel is called a head plate; most additional parts are located on the
head plate.
Bioreactor - Agitator
An agitator is required to mix the contents in the vessel to ensure a homogeneous
environment.
Agitators consist of a shaft and impellers.
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Mixing of the bioreactor is crucial in order to supply nutrients and oxygen to the culture
and to maintain a constant pH and temperature.
Impellers come in many different shapes and sizes depending on what type of mixing
are needed.
Bioreactor - Sparger
The sparger is an apparatus used to introduce gasses into the vessel.
Spargers are located at the bottom of the vessel and consist of a tube with tiny holes for
the gas to escape through into the culture.
The gas coming out of the sparger helps to aerate and mix the contents in the vessel, as
well as supply oxygen to the cells.
Types of Spargers:
Bioreactor - Baffles
Baffles are obstructions on the side of the vessel that generate turbulence in the flow of
the culture.
Baffles are made out of stainless steel and are welded to the inside of the vessel.
Baffles help to mix the culture by creating a more turbulent flow.
Bioreactor - Probes
Bioreactors require probes to monitor the culture in the vessel.
The probes are found at different locations on the vessel: head plate, top probe belt,
bottom probe belt.
Useful probes include temperature, pH, DO (dissolved oxygen), and CO2
Bioreactor Cooling Jacket
Cells give off heat when growing and dividing.
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To maintain a constant temperature in the reactor, the vessel is covered by a cooling

jacket.
Coolant (cold water or glycol) flows through the cooling jacket to regulate the
temperature.
The temperature is controlled by the flow rate of the coolant.
Bioreactor - Ports
Bioreactors require addition ports, where material is either introduced or removed from
the vessel.
Ports are needed to add the media (media port), cells (inoculation port), and nutrients
(feed ports).
Ports are also used for the addition of acid and base for pH control.
A sample port is also located on each vessel to remove culture for off-line investigation.
Bioreactor - Condenser
A condenser is an apparatus that captures saturated air leaving the vessel.
The condenser is cooler than the saturated air allowing it to condense on the surface
and return to the vessel as a liquid.
Condensers help to minimize the loss of material out of the reactor due to evaporation.
Bioreactor - Filters
Bioreactors need inlet filters to ensure the gasses entering the vessel are sterile.
They require exhaust filters to keep the reactor sterile and allow gas to escape to
regulate pressure.
Filters require filter housing a stainless steel cabinet to hold and sterilize the filter.
Bioreactor - Valves
Valves are used to control the flow of either gas or liquid
Bioreactors use many different types of valves.
Manual Valves open and close the valves with your hand.
Pneumatic Valves automatic valve which opens and closes with the use of high
pressured air (instrument air).
Steam Lock Valves a two valve assembly so you can allow a fluid to flow through a
portion of the valve and then close and use the other portion of the valve to sterilize the
valve assembly after use to reduce contamination.
These valves can be manual or
pneumatic.
Valves are used at many different locations on the bioreactor
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Sample valve where a sample from the bioreactor can be obtained.

Harvest valve location where the tank can be drained for harvest.
Ports contain an automatic steam lock valve, so liquid can be added to the vessel
Used in:
Industrial processes to produce pharmaceuticals, vaccines, or antibodies.
To convert raw materials into useful byproducts such as in the bioconversion of corn
into ethanol.
To treat sewage and wastewater.
To grow cells or tissues in the context of cell culture.
HEAT TRANSFER AND MASS TRANSFER
Heat transfer
Heat transfer is necessary during both sterilization and for temperature maintenance
during operation.
Transfer into liquid from the gaseous phase is enhanced by agitation: It prolongs
retention of air bubbles in suspension, reduces bubble size to increase the surface area
for oxygen transfer, prevents bubble coalescence and decreases the film thickness at
the gas-liquid interface.
Maintenance of suitable shear conditions during the fermentation is very important:
Certain agitation systems develop high shear that may damage shear-sensitive cells.
Low shear systems can lead to cell flocculation or unwanted growth on surfaces, such
as on the vessel walls, stirrer and electrodes.
The mixing of nutrients and gaseous exchange within any fermenter is influenced by:
Automatic temperature control during the fermentation is accomplished by injecting
either cold or hot water into the outer jacket and/or internal coils.
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In some circumstances alternative cooling media may be used, e.g. glycol.

Mass transfer
Transfer of nutrients from the aqueous phase into the microbial cells during
fermentation is relatively straightforward as the nutrients are normally provided in
excess.
Design of a typical bioreactor and the parts

Body construction:
Construction materials differ with small scale, pilot and large scale. In small scale for
vessel construction glass or stainless steel may be used. For pilot and large scale
process, stainless steel (>4% chromium), mild steel (coated with glass or epoxy
material), wood, plastic or concrete may be used as vessel construction material. Any
vessel used should not have any corners and smooth surface is essential. The
construction material must be non toxic and corrosion proof. Glass vessel (borosilicate
glass)
Type I glass vessel round or flat bottom with top plate. It can be sterilized by
autoclaving and the largest diameter is 60cm.
Type II glass vessel flat bottom with top and bottom stainless steel plate. This type is
used in insitu sterilization process and the largest diameter 30cm.
Sealing:
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Sealing between top plate and vessel is an important criteria to maintain airtight
condition, aseptic and containment. Sealing have to be done between three types of
surfaces viz. between glass-glass, glass- metal and metal-metal. There are three types of
sealing. They are gasket, lipseal and O ring. This sealing ensures tight joint in spite of
expansion of vessel material during fermentation. The materials used for sealing may be
fabric-nitryl or butyl rubbers. The seals should be changed after finite time. There are
two way of sealing in O ring type simple sealing and double sealing with steam between
two seals.
Baffles
Baffles are metal strips that prevent vortex formation around the walls of the vessel.
These metal strips attached radially to the wall for every 1/10th of vessel diameter.
Usually 4 baffles are present but when the vessel diameter is over 3dm3 around 6-8
baffles are used. There should be enough gaps between wall and baffle so that scouring
action around vessel is facilitated. This movement minimizes microbial growth on
baffles and fermentation walls. If needed cooling coils may be attached to baffles.
Aeration system (sparger)
Sparger is a device for introducing air into fermenter. Aeration provides sufficient
oxygen for organism in the fermenter. Fine bubble aerators must be used. Large
bubbles will have less surface area than smaller bubbles which will facilitate oxygen
transfer to a greater extent. Agitation is not required when aeration provides enough
agitation which is the case Air lift fermenter. But this is possible with only for medium
with low viscosity and low total solids. For aeration to provide agitation the vessel
height/diameter ratio (aspect ration) should be 5:1. Air supply to sparger should be
supplied through filter. There are three types of sparger viz. porous sparger, orifice
sparger and nozzle sparger.
Exit gas cooler
Similar to liebig condenser, condenses the moisture from the exhaust gas in the
fermenter. This removes as much moisture as possible from the gas leaving the
fermenter and prevent excess fluid loss.
Agitation provides uniform suspension of cells in homogenous nutrient medium. This
agitation provides bulk fluid and gas phase mixing, air dispersion, facilitates oxygen
transfer and heat transfer and uniform environment through out the vessel. There are
four classes, namely Disc turbine, Vaned disc, Open turbine of variable pitch and
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Marine impeller. Disc turbine prevents flooding by air bubbles. Flooding occurs when
the air bubble is not properly dispersed the air pocket is formed one area. Flooded only
at 120min/hour of air discharge when disc turbine is used. When open turbine and
propeller are used the medium is flooded at 21min per hour of air discharge.
Stirrer glands and bearings
The entry point of stirrer into fermenter may be from top to bottom or sides. Mostly
used from bottom so that that leaves more space for entry ports on top. There are four
types of stirrer glands and bearings.
Steam traps
This steam trap is important to remove any steam condensate. There are two
components viz. valve and seat assembly and open / close device.
SPECIALISED BIOREACTORS DESIGN AND THEIR FUNCTIONS
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Factors to be considered for designing a fermentor:

The function of the fermenter or bioreactor is to provide a suitable environment in
which an organism can efficiently produce a target productthe target product might
be
Cell biomass
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Metabolite
Bioconversion Product
The sizes of the bioreactor can vary over several orders of magnitudes.
The microbial cell culture (few mm3), shake flask ( 100 -1000 ml), laboratory fermenter
( 1 50 L), pilot scale (0.3 10 m3) to plant scale ( 2 500 m3) are all examples of
bioreactors.
The performance of any fermenter depends on the following key factors:
Agitation rate
Oxygen transfer
pH
Temperature
Foam production
The design and mode of operation of a fermenter mainly depends on the production
organism, the optimal operating condition required for target product formation,
product value and scale of production.
The design also takes into consideration the capital investment and running cost.
Large volume and low value products like alcoholic beverages need simple fermenters
and do not need aseptic condition.
High value and low volume products require more elaborate system of operation and
aseptic condition.
The Designing of a Bioreactor also has to take into considerations the Unique Aspects
of Biological Processes:
The concentrations of starting materials (substrates) and products in the reaction
mixture are frequently low; both the substrates and the products may inhibit the
process.
Cell growth, the structure of intracellular enzymes, and product formation depend on
the nutritional needs of the cell (salts, oxygen) and on the maintenance of optimum
biological conditions (temperature, concentration of reactants, and pH) within narrow
limits.
Certain substances, inhibitors, effectors, precursors, metabolic products influence the
rate and the mechanism of the reactions and intracellular regulation.
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Microorganisms can metabolize unconventional or even contaminated raw materials

(cellulose, molasses, mineral oil, starch, ores, wastewater, exhaust air, biogenic waste),
a process which is frequently carried out in highly viscous media.
In contrast to isolated enzymes or chemical catalysts, mos adapt the structure and
activity of their enzymes to the process conditions, whereby selectivity and productivity
can change.
Mutations of the microorganisms can occur under sub optimal biological conditions.
Microorganisms are frequently sensitive to strong shear stress and to thermal and
chemical influences.
Reactions generally occur in gas-liquid -solid systems, the liquid phase usually being
aqueous.
Continuous bioreactors often exhibit complicated dynamic behavior H) The microbial
mass can increase as biochemical conversion progresses.
Effects such as growth on the walls, flocculation, or autolysis of microorganisms can
occur during the reaction.
There is no universal bioreactor.
The general requirements of the bioreactor are as follows:
The design and construction of bioreactors must keep sterility from the start point to
end of the process.
Optimal mixing with low, uniform shear.
Adequate mass transfer, oxygen.
Clearly defined flow conditions.
Feeding substrate with prevention of under or overdosing.
Suspension of solids.
Gentle heat transfer.
Compliance with design requirements such as: ability to be sterilized; simple
construction; simple measuring, control, regulating techniques; scale-up; flexibility;
long term stability; compatibility with up- downstream processes; antifoaming
measures.
To achieve this fermenter should have:
Heat and oxygen transfer configuration
Sterilization procedures
Foam control
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Fast and thorough cleaning system

Proper monitoring and control system
Traditional design is open cylindrical or rectangular vessels made from wood or stone.
Most fermentation is now performed in close system to avoid contamination.
It should be constructed from non-toxic, corrosion-resistant materials.
Small fermentation vessels of a few liters capacity are constructed from glass and/or
stainless steel.
Pilot scale and many production vessels are normally made of stainless steel with
polished internal surfaces
Very large fermenters are often constructed from mild steel lined with glass or plastic,
in order to reduce the cost.
If aseptic operation is required, all associated pipelines transporting air, inoculum and
nutrients for the fermentation need to be sterilizable, usually by steam.
Most vessel cleaning operations are now automated using spray jets, and called
cleaning in place CIP. And located within the vessel.
Associated pipe work must also be designed to reduce the risk of microbial
contamination. There should be no horizontal pipes or unnecessary joints and dead
stagnant spaces where material can accumulate; otherwise this may lead to ineffective
sterilization.
Normally, fermenters up to 1000 L capacity have an external jacket, and larger vessels
have internal coils.
Pressure gauges and safety pressure valves must be incorporated, (required during
sterilization and operation).
For transfer of media pumps are used. Centrifugal pumps (generate high shear forces
and path for easy contaminations), magnetically coupled, jet and peristaltic pumps.
Alternate methods of liquid transfer are gravity feeding or vessel pressurization.
In fermentations operating at high temperatures or containing volatile compounds, a
sterilizable condenser may be required to prevent evaporation loss.
Fermenters
are
often
operated
under
positive
pressure
to
prevent
entry
contaminants.
AIRLIFT BIOREACTOR
They are impeller free system,
Air-lift fermenter uses air injected from the bottom of its draft-tube,
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The air rises up through the riser column it reaches the upper part and comes down the
down comer column (space between the outer wall of draft tube and inner wall of the
fermentor)
Entire reactor is divided into 2 Halves
By a draft tube
Inner gassed region riser/outer ungassed region
Air is pumped in to the riser region
Down comer has degassed media and cells
Airlift fermentor
Uses
Mammalian cell culturers
Waste water treatment
To produce biopharma proteins
Advantages
They are more energy efficient as mixing of oxygen is not required
Types of airlift fermentor
Split cylinder type
Draft tube internal loop
Draft tube external
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It is ideally suited for aerobic cultures since oxygen mass transfer coefficient are quite
high in comparison to stirred tank reactors.
This is ideal for SCP production from methanol as carbon substrate.
This is used mainly to avoid excess heat produced during mechanical agitation
Disadvantage
Low local mixing intensity hence can be use for low viscosity
Bubble cap fermentor:
It consists of horizontal plates, which contain medium, and inoculated with the
respective microorganism
Each horizontal Plate has vertical pipes on its upper surface which is projecting just
above the surface medium
The vertical pipe has a hole at the bottom, which helps in contact with the atmosphere
above fermentation medium
Inverted cap covers the top of vertical pipe such that its lower rim lies inside
fermentation medium
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Hydrogen gas (gaseous carbon nutrients i.e., methane gas) is introduced from the
bottom of horizontal plate
Gas rises through the vertical pipe sent through the tube, and escapes, because the
inverted cap loosely covers the pipe
Gas which is not oxidized at each level is exposed to next plate level
Remaining un oxidized hydrocarbon gas rises up and is recycled to the bottom of the
fermentor for subsequent use
Use of bubble cap fermentor

For production of SCP
Citric acid production.
Disadvantages
Low local mixing intensity hence can be use for low viscosity
TUBULAR BIOREACTORS
The concept of multiple series of C.S.T.R.s has been applied in the multistage perforated
plate column bioreactor which is shown in Fig. 21. Even though an increase in
productivity and reduction in overall volume is obtained, the mechanical complex ity of
the perforated plate column reactor is a major factor against its industrial application.
Bioreactors operated with cell recycle are another group of reactors. Cell recycle has
been employed in many cases in order to increase reactor productivity and
concentration of biomass. The use of continuous cell recycle reactors has been
thoroughly investigated and applied at large scale. A cell recycle system is shown in Fig.
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The system is identical to the simple C.S.TR.s except that a centrifuge is used to
separate yeast from the product overflow and this yeast is returned to the reactor. With
continued cell growth, and cell escape prevented, the cell concentration in the reactor
becomes extremely high and total productivity is greatly increased.
Complexities arise due to the requirement for some separation device in this process.
Mechanical centrifuges increase capital costs and require considerable maintenance.
Electrical energy costs are increased, and added supervision is required to monitor the
centrifuge. These disadvantages have been shown to be more than offset by the great
increase in productivity and reduction in equipment size.
MEMBRANE BIOREACTORS
The membrane bioreactor enables reduction of the product inhibitory effect in
consequence of its partial and continuous removal from the fermentation zone in
addition to the effective immobilization of cells in the bioreactor and maintenance of
their metabolic activity. In the simple continuous membrane system which is shown in
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Fig, extremely high cell densities can be achieved since cells cannot escape the reaction
zone. For this system, reaction rate is limited by the rate at which substrate can diffuse
across the limited membrane surface area. This practical diffusion limitation prevents
very rapid reaction in a simple system.
The limitations of low substrate diffusion rate, which is another problem of a membrane
bioreactor, can be prevented by using the pressure membrane which is shown in Fig.
However, this system has a disadvantage since free proteins (the product of cell lysis)
rapidly pile up on the membrane pores and prevent further flow.
TOWER BIOREACTORS
They consist of a long cylindrical vessel with an inlet at the bottom, an exhaust at the
top, and a jacket to control temperature.
They
do
not
require
agitation
hence
there
are
no
shafts,
impellers
or
blades.
The fermentor has height to diameter ratio of 6:1
Disadvantages:
Despite of being simple and agitation free (to keep yeast cells in suspension) the tower
fermentors have following drawbacks:
Long start up
Technical complexity
Skilled personnel required
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No product consistency
Use
Tower fermenters are used for continuous fermentation of beer, yeast and SCP
Types of tower fermentor
Bubble column tower fermentor-used for citric acid and tetracycline production
capacity 3L to 950L
Vertical tower beer fermentor-capacity 20,000L for beer production
Multi stage tower fermentor-where the tower is divided in to compartments by
perforated plates
Media is fed from the top and air is sent from the bottom
Used for culturing of E.coli and S.cerevisiae
TRICKLE-BED FERMENTER
These consist of a cylindrical vessel packed with support material (e.g. wood chips,
rocks, plastic structure).
The support has large open spaces, for the flow of liquid and gas and the growth of
micro organisms on the solid support.
A liquid nutrient broth is sprayed onto the top of support material, and trickles down
the bed.
Air may flow up the bed, countercurrent to the liquid flow.
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These fermenters are used in vinegar production, these are suitable for liquids with low
viscosity and few suspended solids.
FLUIDIZED BED BIOREACTOR
These are similar to bubble columns with an expanded cross section near the top.
Fresh or recirculated liquid is continuously pumped into the bottom of the vessel, at a
velocity that is sufficient to fluidize the solids or maintain them in a suspension.
These fermenters need an external pump.
The expanded section slows down the local velocity of the upward flow of medium, such
that the solids are not washed out of the bioreactors.
In this type of fermenter, a fluid (gas or liquid) is passed through a granular solid
material at high enough velocities to suspend the solid and cause it to behave as
though it were a fluid.
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This process, known as fluidization, imparts many important advantages to the FBR.
As a result, the fluidized bed reactor is now used in many industrial applications
PACKED BED REACTORS
The design and operation of fluidized-bed bioreactors is by no means an easy task,
especially when the recirculation of solids is involved. There is much more technological
operation knowledge involved than with packed beds. The use of packed-bed
bioreactors for bioconversions using immobilized cells or enzymes has the advantage of
simplicity of operation, high mass transfer rates, and high volumetric reaction rates (for
processes which are not inhib ited by the substrate concentration). High biomass
loadings are achieved by immobilizing the cells; hence washout, which is a problem of
several systems, can also be prevented.
However, the immobilization materials, gels, are somewhat compressible and the
production of CO2 causes the beads to be compressed and the void fraction to decrease.
The result is a high pressure drop and phase separation together with a decrease in
ethanol productivity.
Packed-bed cylindrical bioreactors have some disadvantages in use such as an increase
in bed surface area in the direction of flow resulting in a drop in fluid velocity and a
reduction in the pressure drop. One approach to successfully circumvent these
disadvantages has been the development of two novel packed-bed bioreactor
geometries, which are known as a radial- flow packed-bed, and a tapered packed-bed
reactor
PHOTO BIOREACTORS.
A photo bioreactor is a bioreactor that incorporates some type of light source to
provide photonic energy input into the reactor.
It is an illuminated culture vessel designed for controlled biomass production
of phototrophic liquid cell suspension cultures.
They are used for the phototrophic cultivation of algae and cyanobacteria.
Algal culturing can be achieved in two ways: open ponds and photobioreactors (PBR).
A photobioreactor is closed equipment which provides a controlled environment and
enables high productivity of algae.
As it is a closed system, all growth requirements of algae are introduced into the system
and controlled according to the requirements.
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PBRs facilitate better control of culture environment such as carbon dioxide supply,
water supply, optimal temperature, efficient exposure to light, culture density, pH
levels, gas supply rate, mixing regime, etc.,
PBR
From the feeding vessel, the flow progresses to the diaphragm pump which moderates
the flow of the algae into the actual tube. Built into the pump is the CO2 inlet valve.
The photobioreactor itself is used to promote biological growth by controlling
environmental parameters including light.
The tubes are made of acrylic and are designed to have light and dark intervals to
enhance the growth rate.
The photobioreactor has a built-in cleaning system that internally cleans the tubes
without stopping the production.
After the algae have completed the flow through the photobioreactor, the optical Cell
Density sensor determines the harvesting rate.
When the algae are ready for harvesting, they pass through the connected filtering
system.
This filter collects the algae that are ready for processing, while the remaining algae
passes back to the feeding vessel.
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Advantages of PBR over open systems.

They can prevent or minimize contamination,
They offer better control over bicultural conditions (pH, light intensity, carbon dioxide,
and temperature).
Prevent water evaporation, lower carbon dioxide losses due to our gassing,
Permit higher cell concentrations.
Space saving - Can be mounted vertically, horizontally or at an angle, indoors or
outdoors.
Reduced Fouling - Recently available tube self cleaning mechanisms can dramatically
reduce fouling.
Disadvantages of Photobioreactors
Capital cost of construction is very high. This is hindering the progress of
algae fuel industry.
The difficulty in sterilizing the photobioreactors has hindered their application for algae
culture for specific end-products such as high value pharmaceutical products.
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UNIT 3 - FERMENTATION MEDIA AND FERMENTATION PROCESS:

Natual and synthetic media; Strategies for media formulation, sources of carbon,
nitrogen, vitamins and mineral.Role of buffers, precursors, inhibitors, inducers
and antifoam agents.
Types of fermentation process-submerged fermentation, surface or solid state
fermentation,
batch
fermentation,
continuous
fermentation,
kinetics
of
fermentation process, bioprocess control, monitoring of variables-temperature,

agitation, pH and pressure.
NATUAL AND SYNTHETIC MEDIA

Medium improvement
Medium designed for the initial production of antibiotic usually does not have to be
developed very skillfully since the potential for antibiotic production is quite low with
wild-type strains.
Media for ultra-high antibiotic-producing strains, which have been developed through
repeated genetic manipulations, must be formulated with utmost care.
In the past, strain improvement and media development were the responsibilities of
different research groups. Today we know that each higher-producing clone, after
mutation and screening, requires a medium optimized for its performance.
The importance of medium improvement
Only small to moderate increases in the level of production can result in an actual
reduction in production cost so that it can economically be sold in competition with
others.
There is little published literature on the complex substrates that have been developed
for the production of various products. Most fermentation processes, which include the
production of fermentation media, are closely guarded trade secrets.
Different technical objectives of media formulation
Inoculum (starter culture) propagation steps / pilot-scale fermentations / main
production fermentation
Biomass or primary metabolites production / secondary metabolite production
Considerations in seed culture media formulation
The seed stages are designed to give rapid and reproducible growth without nutrient
depletion, autolysis, or an adverse change in pH.
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There is less concern with the cost of ingredients in the seed stages since the volume is
usually only 5% of the fermentation volume and excellent uniformity of medium
ingredients is highly desirable.
Some specially prepared dairy products have been used quite extensively in primary
and secondary seeds.
Industrial Fermentation Medium
The basic ingredients of media include carbon source nitrogen source inorganic salt
growth factor and distilled water etc.
Medium Development
To maintain economic competitiveness, low-cost crude materials are frequently used
Levels of minerals and growth factors may be critical
Kinds of culture media
The source of media constituent
Complex media
Synthetic or defined media
Semi-defined medium
Physics states
Liquid medium
Solid medium
Semi-solid medium
For use
Basic medium
Enriched medium
Differential medium
Selective medium
Manufacture intention
Seed medium
Ferment medium
Nutrient sources for industrial fermentation
Carbon & energy source + nitrogen source + O2 + other requirements
Biomass + Product + byproducts + CO2 + H2O + heat
Fermentation media
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Nutrient
Raw material
Carbon
molasses, starch
Nitrogen
corn steep liquor, soybean meal, pure ammonia or

ammonium salts, urea, nitrate salts, phosphate salts
Vitamin and
biotin, yeast extract, beef extract, corn steep liquor, wheat
growth factors
germ meal
Constituents of medium
Water
Carbon source / Nitrogen source / Sources of phosphorous and sulfur / Minor and
trace elements / Vitamins such as biotin and riboflavin
Oxygen:
even
some
anaerobic
fermentation
require
initial
aeration,
e.g.
beer
fermentations
Buffers or controlled by acid and alkali additions
Antifoam agents
Precursor, inducer or inhibitor compounds
STRATEGIES FOR MEDIA FORMULATION
Nutritional Requirements
Environmental Requirements
Techno-economic Factors:
Nutritional requirements
Nutritional requirements include elemental, specific nutrient, and energy requirements
Elemental requirements:
The stoichiometry for growth and product formation
C-source + N-source + O2 +
minerals + specific nutrients cell mass + product + CO2 + H2O + heat

Specific nutrient requirements:
Auxotroph: To use a complex medium or to identify the specific nutrient
Elemental requirements
Main elemental formula of microbial cells C4H7O2N (dry weight basis 48% C, 7% H,
32% O, 14% N), e.g. Bakers yeast C3.72H6.71O1.95N0.61S0.017P0.035K0.056
Average:
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Sources
Carbon
45 - 55
Nitrogen
6 14
Potassium
0.5 2
Phosphorous
13
Manganese
0.1 - 1
Sulphur
0.02-1
Minor minerals (mg /100g cell)

Copper
0.1-1
Iron
1-10
Zinc
~1
Magnesium
0-5
Chemical composition of fermentation product

Typical concentration of fermentation products in the broth (dry wt / vol, %):
Lactic acid (13), citric acid (12), glutamic acid (10), ethanol (8), bakers yeast (5), benzyl
penicillin (3), riboflavin (1), vitamin B12 (0.002)
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Environmental requirements
Effect of growth temperature on cell yield / below optimal temperature for growth
Effect of water activity (Aw = Ps/Pw) on growth rate, vapor pressure of water in solution
(Ps) or in pure water(Pw)
Combined effect of temperature and pH on growth / opt pH for growth and production
is not always the same
Environmental effect of substrate

Substrate concentration: Monod equation,
= m S / (Ks + S)
Ks for C-source 1 ~ 10 mg/L, when S = 10 ~ 100 mg/L, m; Ks for amino acid 0.003
~ 0.2 mg/L; Ks for ammonia 0.1 ~ 1.0 mg/L
Substrate inhibition: carbohydrate 50 to 100 ~ 150 g/L (osmotic pressure); phenol,
toluene, butanol a few g/L (damage cell membrane); ammonia 3 ~ 5 g/L
Catabolite repression
NO3- NO2- toxic effect
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Phosphate repression and sulfate repression

Techno-economic factors that affect the choice of individual raw materials:
Cost: transport and storage, e.g. temperature control
Availability: consistent quality and year round availability
Ease of handling: solid or liquid forms
Sterilization: thermal damage and inhibitory byproduct
Operational characteristics: formulation, mixing, complexing and viscosity
characteristics that may influence agitation, aeration, foaming and recovery
Supply: the concentration of target product attained, its rate of formation and yield per
gram of substrate utilized
Purification: levels and range of impurities, potential for generating undesired products
Pollution control
Health and safety implications
Cost analysis
Raw materials (consumed in production or recovery) constitute a major part of the
manufacturing cost
30 to 80% of the production cost for biologically based production system
10 to 50% of the production cost for conventional chemical production plants
Nutrients: up to 60% of the production cost
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Examples;
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SOURCES OF CARBON
Molasses
Byproduct of cane or beet sugar production / residues remaining after most of the
sucrose has been crystallized from the plant extract
Dark colored viscous syrup containing 50-60% (w/v) carbohydrate, primarily sucrose,
with 2% (w/v) nitrogenous substances, along with some vitamins and minerals.
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Overall composition varies depending upon the plant source, the location of the crop,
the climatic conditions under which it was grown, and the factory where it was
processed
The carbohydrate concentration may be reduced during storage by contaminating
microorganisms
Hydrol molasses, containing primarily glucose, is a byproduct of maize starch
processing
Malt extract
Concentrated aqueous extracts of malted barley to form syrups / particularly useful for
the cultivation of filamentous fungi, yeasts and actinomycetes
App. 90% carbohydrate (w/w) and some vitamins and app. 5% nitrogenous substances,
proteins, peptides and amino acids / carbohydrate comprising 20% hexoses (glucose
and small amounts of fructose), 55% disaccharides (maltose and traces of sucrose),
10% maltotriose, and additionally contain 15-20% branched and unbranched dextrins,
which may or may not be metabolized, depending upon the microorganisms
Careful sterilization to prevent over-heating /Maillard reaction products (brown
condensation products resulting from the reaction of amino groups and carbonyl
groups) when heated at low pH / color change, loss of fermentable materials, some toxic
products
Starch and dextrins
Can be directly metabolized by amylase-producing microorganisms, particularly
filamentous fungi
Maize starch is most widely used
To allow use in a wide range of fermentations, the starch is usually converted into sugar
syrup, containing mostly glucose. It is first gelatinized and then hydrolyzed by dilute
acids or amylolytic enzymes, often microbial glucoamylases that operate at elevated
temperatures
Sulfite waste liquor
Sugar containing wastes derived from the paper pulping industry are primarily used for
the cultivation of yeasts
Waste liquors from coniferous trees contain 2-3% (w/v) sugar, 80% hexoses (glucose,
mannose and galactose) and 20% pentoses (mostly xylose and arabinose) / Liquors
derived from deciduous trees contain mainly pentoses
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Usually the liquor requires processing before use as it contains sulfur dioxide / The low
pH is adjusted with calcium hydroxide or calcium carbonate, and these liquors are
supplemented with sources of nitrogen and phosphorus
Cellulose
Predominantly as lignocellulose (composed of cellulose, hemicellulose and lignin)
Available from agricultural, forestry, industrial and domestic wastes
Relatively few microorganisms can utilize it directly / The cellulose component is in part
crystalline, encrusted with lignin, and provides little surface area for enzyme attack
At present, mainly used in solid-substrate fermentations (e.g. mushrooms)
Potentially a very valuable renewable source of fermentable sugars once hydrolyzed,
particularly in the bioconversion to ethanol for fuel use
Whey
An aqueous byproduct of the dairy industry / Annual worldwide production is over 80
million tons,
containing over 1 million tons of lactose and
0.2 million tons
of milk proteins
Expensive to store and transport / Lactose concentrates are often prepared for later
fermentation by evaporation of the whey, following removal of milk proteins for use as
food supplements
Lactose is less useful than sucrose / e.g. S. cerevisiae does not ferment lactose
Formerly used extensively in penicillin fermentation / Still employed for producing
ethanol, single cell protein, lactic acid, xanthan gum, vitamin B12 and gibberellic acid
Alkanes and alcohols
n-Alkanes (C10-C20): readily metabolized by certain microorganisms / industrial use is
dependent upon the prevailing price of petroleum
Methane: utilized by a few microorganism, but its conversion product methanol is often
preferred for
industrial fermentations
High purity methanol is readily obtained / completely miscible with water / has a high
per cent carbon content and is relatively cheap / only limited organisms will metabolize
methanol / only low conc., 0.1-1% (v/v) are tolerated by microorganisms / oxygen
demand and heat of fermentation are high, but this is even more problematic when
growing on alkanes
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Ethanol is less toxic than methanol / used as a sole or cosubstrate / too expensive for
general use as a carbon source / its biotransformation to acetic acid remains a
major
fermentation process
Fats and oils
Hard animal fats (composed mainly of glycerides of palmitic and stearic acids) are rarely
used in fermentation
Plant oils (primarily from cotton seed, linseed, maize, olive, palm, rape seed and soy)
and occasionally fish
oil, may be used as the primary or supplementary
source, especially in antibiotic production /
carbon
Plant oils are mostly composed of oleic
and linoleic acids, but linseed and soy oil also have a substantial amount of linolenic
acid
Oils contain more energy per unit weight than carbohydrates / Oils can be particularly
useful in fed-batch operations than carbohydrates (aqueous solutions less than 50%,
w/v; occupy a greater volume)
SOURCES OF NITROGEN, VITAMINS AND MINERAL

Corn steep liquor
Byproduct of starch extraction from maize / first use in fermentations for penicillin
production in the 1940s
Exact composition varies depending on the quality of maize and the processing
conditions / Concentrated extracts generally contain about 4% (w/v) nitrogen,
including a wide range of amino acids, along with vitamins and minerals / Any residual
sugars are usually converted to lactic acid (9-20%, w/v) by contaminating bacteria Can
sometimes be replaced by liquor derived from potato starch production
Yeast extract - 1
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Produced from waste bakers and brewers yeast, or other strains of S. cerevisiae / Or
Kluyveromyces marxianus (formerly K. fragilis) grown on whey and Candida utilis
cultivated using ethanol, or wastes from wood and paper processing
Extracts used in the formulation of fermentation media are normally salt-free
concentrates of soluble components of hydrolyzed yeast cells / Extracts with sodium
chloride concentrations greater than 0.05%
(w/v) cannot be used in fermentation
processes due to potential corrosion problems

Yeast cell hydrolysis is often achieved by autolysis, which can be initiated by
temperature or osmotic shock, causing cells to die but without inactivating their
endogenous enzymes
Yeast extract - 2
Temperature and pH are controlled throughout an optimal and standardized autolysis
process / Temperature control is particularly important to prevent loss of vitamins
Autolysis (50-55oC for several hours before the temperature is raised to 75oC to
inactivate enzymes), plasmolysis or mechanical disruption of cells / filtration or
centrifugation to remove cell wall materials and other debris / rapid concentration
Extracts are available as liquids containing 50-65% solids, viscous pastes or dry
powders
They contain amino acids (35-40%, w/v), peptides (30-45%, w/v), water-soluble
vitamins and some glucose derived from the yeast storage carbohydrates (trehalose and
glycogen)
Peptones
Peptones are usually too expensive for large-scale industrial fermentations
Prepared by acid or enzyme hydrolysis of high protein materials: meat, casein, gelatin,
keratin, peanuts, soy meal, cotton seed, etc.
Amino acids compositions vary depending upon the original protein source / Gelatinderived peptones are rich in proline and hydroxyproline, but almost devoid
of sulfur-
containing amino acids / Keratin peptone is rich in both proline and cystine, but lacks
lysine
Plant peptones invariably contain relatively large quantities of carbohydrates
Soya bean meal
Residuals after extraction of soy oil
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Composed of 50% protein, 7% non-protein nitrogenous compounds, 30% carbohydrates

and 1% oil
Often used in antibiotic fermentation because the components are only slowly
metabolized, thereby eliminating the possibility of repression of product formation
ROLE OF BUFFERS, PRECURSORS, INHIBITORS, INDUCERS AND ANTIFOAM

AGENTS.
Water
Use for media, cleaning, cooling
A reliable source of large quantities of clean water, of consistent composition, is
essential
Before use, removal of suspended solids, colloids and microorganisms is usually
required
Hard water is treated to remove salts such as calcium carbonate
Iron and chlorine may also require removal
Water is becoming increasingly expensive / recycle / reuse wherever possible /
minimizes water costs and reduces the volume requiring waste-water treatment
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Antifoams -1
Foaming is largely due to media proteins that become attached to the air-broth interface
where they denature to form a stable foam
If foaming is minimized, then throughputs can be increased
Three approaches to controlling foam production: modification of medium composition,
use of mechanical foam breakers and addition of chemical antifoams
Chemical antifoams are surface-active agents which reduce the surface tension that
binds the foam together
Antifoams -2
Ideal antifoam:
Readily and rapidly dispersed with rapid action
High activity at low concentration
Prolonged action
Non-toxic to fermentation microorganisms, humans or animals
Low cost
Thermostable
Compatibility with other media components and the process , i.e. having no effect on
oxygen transfer rates or downstream processing operations (e.g. some may adversely
affect membrane filtration)
Natural antifoams include plant oils (e.g. from soy, sunflower and rapeseed), deodorized
fish oil, mineral oils and tallow.
Synthetic antifoams are mostly silicon oils, poly alcohols and alkylated glycols
Special compounds -1
Precursors: phenylacetic acid or phenylacetamide as side-chain precursors in penicillin
production
phenethylamine into benzyl penicillin
D-threonine in L-isoleucine production by Serratia marsescens
Anthranillic acid for L-tryptophan production by yeast Hansenula anomala
Inducers and elicitors: Inducers are often necessary
for genetically modified
microorganisms (GMMs) / Production of secondary metabolites, such as flavonoids and

terpenoids, in plant cell culture can be triggered by adding elicitors, which may be
isolated from various microorganisms, particularly plant pathogens
Special compounds -2
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Inhibitors: 1. Used to redirect metabolism towards the target product and reduce
formation of other metabolic intermediates (e. g. sodium bisulfite in production of
glycerol by S. cerevisiae) 2. Antibiotics for some GMMS containing plasmids bearing an
antibiotic resistance gene
Cell permeability modifiers: e.g. penicillins and surfactants added to amino acid
fermentations, including processes for producing L-glutamic acid by members of the
genera Corynebacterium and Brevibacterium
TYPES OF FERMENTATION PROCESS
Small and large scale production of commercial products, and also to carry out complex
chemical transformations. Microbes become biocatalysts
An industrial microorganism must:
Produce the product of interest in high yield
Grow rapidly on inexpensive culture media available in bulk quantity (corn steep liquor,
whey [NONanimal (Mad Cow Disease BSE] a major proportion of the production cost
of commodity chemicals is the substrate.
Commodity chemicals are inexpensive chemicals produced in bulk, including ethanol,
citric acid, and many others - DEE antibiotics).
Be amenable to genetic manipulation mutation, genetically engineered - yet stable
Be non-pathogenic
Primary metabolites *
Ethanol Antibiotics
Amino-acids Statins cholesterol lowering agents
Secondary metabolites *
Not essential for growth
Dependent on growth conditions (repression)
Over-production often achievable (not growth related)
Often produced as a series of closely related
Compounds
Characteristics of Large-Scale Fermentations
Fermentation (generic term) is carrier out in a Fermentor (a vat) by a Fermenter
(microbe). Scale laboratory 5 10 l -- Production up to 500,000 l
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Large-scale industrial fermentation presents several engineering problems while the

microbial
process
must
be
continuously
monitored
to
ensure
yield
and
no
contamination.
Fermentation scale-up is an art in itself optimization of laboratory scale to production
scale.
Industrial fermentations will be anaerobic and aerobic processes, aeration in the latter
being critical, and requires special attention to stirring (impellor)/ bubbling (sparger).
Both require heat removal systems.
SUBMERGED FERMENTATION
Submerged fermentation refers to those fermentations wherein microorganisms
employed grows in submerged state within the fermentation media.many fermentations
fall in this category like penicillin submerged fermentation technique.
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SURFACE OR SOLID STATE FERMENTATION

Surface fermentation refers to those fermentations in which the microorganisms utilised
grows on the surface of the fermentation media.Examples being citric acid surface
fermentation process wherein the microorganism aspergillus Niger grows as a thick
floating mycelial mat over the surface of the media used.
The growth of microorganisms on moist solid substrate particles in the absence or mere
absence of visible liquid water between the particles.
The moisture content of solid substrate ranges between 12-80%.
The water content of a typical submentation is more than 95%.
SSFs are usually used for the fermentation of agricultural products or foods, such as
rice, wheat, barley, corn and soybeans.
Some food fermentations involving Solid state fermentation:
Wheat by Aspergillus
Soybean by Rhizopus
Soybean by Aspergillus
Phases of solid state fermentation
They are 3 phases:
Solid phase
Liquid phase
Gaseous phase
Solid phase of there are two types:
Natural solid materials
An inert solid support
Characteristics of solid state fermentation
The substrate may require preparation or pretreatment like,
Chopping or grinding-reduce particle size
Cooking or chemical hydrolysis
Pasteurization or sterilization-reduce contaminants
Microorganism is usually a filamentous fungus requiring aerobic condition.
The Inoculum is mixed into substrate to fermentation.
Advantage of SSF
A lower chance of contamination due to low moisture levels
Ease of product separation
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Energy efficiency
Development of fully differentiated structures
Disadvantages
Heterogeneous nature of the media, due to poor mixing characteristics.
At high agitation speeds, mycelial cells may be damage.
Rotary-tray or rotating-drum fermenters are often used.
Flow chart
BATCH FERMENTATION
Today, most ethanol is produced by the same processes developed in the beverage
industry more than a hundred years ago. These methods are based on the simple batch
fermentation of carbohydrate feedstocks. In batch fermentation, substrate and
separately grown cell slurry are charged into the bioreactor together with nutrients and
enzymes required (Keim, 1983).
The general characteristics of batch systems are well known. Usually the time required
to completely utilize the substrate is 36-48 h. The temperature is held at 10-30 C and
initial pH is adjusted to 4.5. Depending upon the nature of the carbohydrate material,
conversion efficiency lies in the range of 90-95% of the theoretical value with a final
ethanol concentration of 10-16% (w/v).
There is a specific reaction period for the cultivation. During this time, cell, substrates
(carbon source, nutrient salts, vitamins, etc.) and product concentrations alter. This
rapid increase slows down towards the end of the cultivation period with the rate
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approaching zero asymptotically. After as long as 50-60 h, the fermented material is

pumped to distillation supply tank. Then, the bioreactor is washed, sterilized, and
recharged with the new batch. The time lost in emptying, cleaning, and filling reduces
the effective reactor volume by about 20%.
Advantages:
Low investment costs are required with not much control.
Low requirement for complete sterilization.
Use of unskilled labor.
Low risk of financial loss.
Easy management of feedstocks.
Greater flexibility is achieved by using a bioreactor for various products specifications.
Well-defined cultivation periods are possible, so higher conversion levels are obtained.
Less risk of infection and cell mutation, since relatively short cultivation periods are
used.
Disadvantages
Non-productive idle time for emptying, cleaning, sterilizing, cooling, heating, and
recharging the bioreactor, so only 80% of the reactor is effective.
Frequent sterilization results in drifts of measuring instruments.
Preparing several subcultures for inoculum and the control of this non-stationary
process requires more expenditure.
Greater risk to service personnel from possible contact with some pathogenic
microorganisms or toxic products.
Initial growth lag decrease the reactor productivity while microbes are locked in the
exponential phase of their growth cycle in continuous processes.
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Fed-batch Process:
Fed-batch operation which can be regarded as a combination of batch and continuous
operations is a very popular type of process in ethanol industry. In this operation, feed
solution which contains substrate, yeast culture, required minerals and vitamins are
fed at constant intervals while effluent is removed discontinuously.
Advantages of fed-batch operation:
High yield is achieved since well-defined cultivation time (no cells are added or taken
away during the cultivation period) is possible in fed-batch operation. Hence, the
reactor operates batchwise as far as the microorganisms are concerned.
High level of flexibility.
Semi-stationary
method
of
operation
even
in
the
case
of
slightly
mutating
microorganisms and those involving an infection risk.

Optimization of the environmental conditions such as the growth or production phase
and the age of the culture of the microorganisms is possible.
Disadvantages of fed-batch operation:
Non-productive idle time is required for filling, heating, sterilizing, cooling, emptying,
and cleaning the reactor.
Higher manpower requirements or expensive instruments such as process computers
are required (e.g., to keep the substrate concentration requires expensive instruments).
Greater possible risk to service personnel of contact with pathogenic microorganisms or
toxic products.
More wear and tear on instruments from frequent sterilization.
Semi-continuous processes:
The semi-continuous processes (Yarovenko, 1978) comprise the so-called outflowinflow and overflow processes and also a wide range of battery and cyclic fermentation
variants.
In semi-continuous processes a portion of the culture is withdrawn at intervals and
fresh medium is added to the system. Repeated fed-batch culture, which can be
maintained indefinitely, is another name of the semi-continuous process.
The advantages of continuous and batch operations:
There is no need for a separate inoculum vessel, except at the initial start-up.
Time is not wasted in non-productive idle time for cleaning and re-sterilization.
High flexibility of operation.
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Less wear and tear on instruments from sterilization.

Not much control is required.
It also has some disadvantages:
Slightly higher investment costs are required due to larger reactor volumes.
High risk of contamination and mutation due to long cultivation periods and periodic
handling.
Complications exist resulting from the combined biomass and product formation stages.
KINETICS OF FERMENTATION PROCESS and BIOPROCESS CONTROL
Batch Fermentation Process
Dynamic processes that are never in a steady state.
Often, the critical parameter is gas exchange or balance between respiration rate and
oxygen transfer.
Sterilized media components are supplied at the beginning of the fermentation with no
additional feed after inoculation.
Cells are grown in a batch reactor; they go through a series of stages:
Lag phase
Log or Exponential phase
Stationary phase
Death phase
Lag Phase
Microbial population remains constant as there is no growth. However it is the period of
intense metabolic activity.
Factors Influencing the Lag Phase
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Chemical composition of the fermentation media influences the length of the lag phase.
Longer lag phase is observed if the inoculums are transferred into a fresh medium of
different carbon source.
Age of the inoculums. If the inoculums are in exponential growth phase, it will exhibit
shorter lag in the fresh medium.
Concentration of the inoculums.
Viability and morphology of the inoculums.
Exponential Phase
Cell divides with increasing frequency till it reaches the maximum growth rate (max)
At this point logarithmic growth begins and cell numbers or cell biomass increase at a
constant rate.
Mathematical Expression of Growth

It can be based on cell mass (x) or cell number (N).
Rate of Cell Growth based on Cell Biomass
Rate of change of biomass at a given time is
dx/dt = x 1
=1/x.dx/dt 2
Where, x= concentration of biomass (g/l), = specific growth rate (h-1), t= time (h)
On integrating equation 1.1
xt = x0et 3
Where, xt= biomass concentration after time t, x0= biomass concentration at the start
of exponential growth, e= Log constant (Natural log)
Taking natural log, log (ln) of eqn 3
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lnxt = lnx0+t 4
= (lnxt - lnx0)/t 5
This equation is of the form y = c (intercept on y axis) + mx, = gradient ( in
equation4)
For cells in exponential phase, a plot of natural log of biomass concentration against
time, a semi log plot should yield a straight line with the slope (gradient) equal to .
Growth Rate Constant based on Cell Number
If the number of cells at the start of exponential growth is N0.
If the cell population at the time of starting of exponential growth is 1 i.e. N0 =1, then
undergoing binary fission the number of cells after time t becomes Nt.
Nt is given by the equation:
Nt = 2n N0
Where, N0= initial population size, n= Number of generations or divisions

Taking natural logarithms gives
ln Nt = ln N0 + In n2
Number of divisions (n) is given by

n = (ln Nt lnN0) / ln2 3
Growth rate or division rate constant is the average number of generations per hour, K
= n/t= (ln Nt lnN0 )/tln2
In industrial processes we need to know the generation time or doubling time rather
than division rate constant.
Generation time or doubling time is the average time for population to double or time
taken for one division
td = t/n=1/k = t ln2/ (ln Nt lnN0)
Calculation of Doubling Time on the Basis of Cell Biomass
If we consider the initial cell biomass to be xo, and after time t, the microbial biomass
doubles i.e. xt =2xo then (t) will be the doubling time td.
Substituting these parameters into equation. 1
2xo = xoetd 1
Taking log of eqn. 1
ln2xo = lnxo + td 2
td = ln2 3
td = ln2/ 4
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Monod showed that growth rate is an approximate hyperbolic function of the

concentration of the growth limiting nutrient(s).
This impact of essential nutrient depletion on growth can be described mathematically
by the Monod equation, in a form similar to that used in biochemistry, where MichaelisMenten kinetics define the rate of an enzyme catalyzed reaction in relation to its
substrate concentration:
The relationship between substrate concentration and specific growth rate:
where max = maximum specific growth (per hour) of the cells, i.e. when substrate
concentration is not limiting, S = concentration of limiting substrate (g/L)
Ks = saturation constant i.e. concentration (g/L) of limiting nutrient enabling growth at

half the maximum specific growth rate, i.e. = 1/2 max and is a measure of the
affinity of the cells for this nutrient.
When a microorganism is provided with the limiting substrate at a concentration

greater than the Ks, and with all other nutrients in excess, the microorganism will grow
exponentially at its maximum rate i.e. when SKs, then = max.
However, as the level of this substrate decreases, it eventually becomes limiting and can
no longer sustain max.
This is the beginning of the deceleration phase.
As the residual concentration of the limiting substrate approaches Ks and then falls
below this concentration, there is an accompanying gradual decrease in growth rate ().
The growth rate of a microorganism with a very high affinity for a rate-limiting substrate
(i.e. a low Ks) will not be affected until the substrate concentration becomes very low.
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However, where there is a low affinity for the limiting substrate (i.e. a high Ks), the
growth rate will begin to fall even at relatively high substrate concentrations and the
organism exhibits a longer deceleration phase.
Stationary Phase
The specific growth rate of the microorganism continues decelerating until the substrate
is completely depleted.
Overall growth rate has declined to zero and there is no net change in cell numbers/
biomass i.e. rate of cell division equals rate of cell death.
Microorganisms are still metabolically active, metabolizing intracellular storage
compounds, utilizing nutrients released from lysed cells and in certain cases produce
secondary metabolites.
Death Phase
Cells die at constant rate and often undergo lysis.
Growth Parameters for Process Optimization
Yield Coefficient
Yield coefficient (Y), is determined on the basis of the quantity of rate- limiting nutrient,
normally the substrate converted into the microbial product.
In case of biomass production, the yield coefficient relates to the quantity of biomass
produced per gram of substrate utilized and is depicted by the equation
x = Yx/s(S Sr) 1
where x = biomass concentration (g/L), Yx/S= yield coefficient (g biomass/g substrate
utilized), S =initial substrate concentration (g/L), and Sr = residual substrate
concentration (g/L)
Therefore, the higher is the yield coefficient, the greater the percentage of the original
substrate converted into microbial biomass.
CONTINUOUS FERMENTATION
Continuous ethanol production eliminates much of the unproductive downtime
associated with batch culture. This includes stripping and cleaning the apparatus,
recharging with media, and time required for the lag phase. Feed, which contains
substrate, culture medium and the other required nutrients, is pumped continuously
into an agitated vessel where the microorganisms are active. The sugar is largely
consumed and ethanol and new cell mass are produced during the process. The
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product, which is taken from the top of the bioreactor, contains ethanol, cells, and
residual sugar.
The advantages of continuous operation are:
Mechanization and automation are made for large scales.
Continuous process requires lower manpower.
Lower reactor volumes, as there are no unproductive down-time taken up with
emptying, filling, and sterilization of the reactor.
Since operating conditions are invariant, product quality is constant.
Less possible danger to service personnel from pathogenic microorganisms or toxic
materials, due to improved mechanization.
Less wear and tear on instruments from sterilization.
There are disadvantages in continuous systems, such as:
Raw material quality must be uniform as operating conditions cannot be adapted so
easily.
Problems in maintaining a high fermentation rate.
Low flexibility is reached since only slight changes on throughput; medium
composition, temperature, and oxygen concentration are possible.
Continuous sterilization of the medium, using expensive control and automation
equipment cause mainly high investment costs.
High risk of microorganism mutation due to long cultivation periods (either internal
through mutation or external by an invading microbe).
Continuous removal of non-soluble, solid substrates can be very expensive.
MONITORING OF VARIABLES -TEMPERATURE, AGITATION, PH AND PRESSURE
Agitation:
Agitation of suspended cell fermentations is performed in order to mix the three phases
within a fermenter
Liquid phase contains dissolved nutrients and metabolites
Gaseous phase is predominantly oxygen and carbon dioxide
Solid phase is made up of the cells and any solid substrates that may be present.
Mixing should produce homogeneous conditions and promote
a) Nutrient transfer
b) Gas transfer
c) Heat transfer
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Heat transfer is necessary during both sterilization and for temperature maintenance
during operation.
Transfer into liquid from the gaseous phase is enhanced by agitation: It prolongs
retention of air bubbles in suspension, reduces bubble size to increase the surface area
for oxygen transfer, prevents bubble coalescence and decreases the film thickness at
the gas-liquid interface.
Maintenance of suitable shear conditions during the fermentation is very important:
Certain agitation systems develop high shear that may damage shear-sensitive cells.
Low shear systems can lead to cell flocculation or unwanted growth on surfaces, such
as on the vessel walls, stirrer and electrodes.
The mixing of nutrients and gaseous exchange within any fermenter is influenced by:
Automatic temperature control during the fermentation is accomplished by injecting
either cold or hot water into the outer jacket and/or internal coils.
In some circumstances alternative cooling media may be used, e.g. glycol.
Mass transfer
Transfer of nutrients from the aqueous phase into the microbial cells during
fermentation is relatively straightforward as the nutrients are normally provided in
excess.
Transport of Nutrients
The performance of the reactor is affected if the rate of the transport of the limiting
nutrients is slower than the rate of utilization by the cells.
Efficiency of the bioprocess could be increased by increasing the rate of transport of a
limiting nutrient.
Transport of Oxygen
Compressed air entering a fermenter is usually stripped of moisture and any oil vapors
that may originate from the compressor.
To prevent the risk of contamination, gases introduced into the fermenter should be
passed through a sterile filter.
A similar filter on the air exhaust system avoids environ-mental contamination.
Sterile filtered air or oxygen normally enters the fermenter through a sparger system,
and airflow rates for large fermenters rarely exceed 0.5-1.0 volumes of air per volume of
medium per minute (v/v/m).
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To promote aeration in stirred tanks, the sparger is usually located directly below the
agitator.
Sparger structures can affect the overall transfer of oxygen into the medium, as it
influences the size of the gas bubbles produced.
Small bubbles are desirable because the smaller the bubble, the larger the surface area
to volume ratio, which provides greater oxygen transfer.
However, spargers with small pores that are effective in producing small air bubbles are
more prone to blockage and require a higher energy input.
The availability of the oxygen depends on:
Solubility
Mass transfer rate of oxygen in the fermentation broth
Rate of utilization of DO by microbial biomass.
Sparger structures can affect the overall transfer of oxygen into the medium, as it
influences the size of the gas bubbles produced.
Small bubbles are desirable because the smaller the bubble, the larger the surface area
to volume ratio, which provides greater oxygen transfer.
However, spargers with small pores that are effective in producing small air bubbles are
more prone to blockage and require a higher energy input.
The availability of the oxygen depends on:
Solubility
Mass transfer rate of oxygen in the fermentation broth
Rate of utilization of DO by microbial biomass.
It is essential with any fermentation to ensure that only the desired bacteria, yeasts or
moulds start to multiply and grow on the substrate. This has the effect of suppressing
other micro-organisms which may be either pathogenic and cause food poisoning or will
generally spoil the fermentation process, resulting in an end-product which is neither
expected nor desired. An everyday example used to illustrate this point is the
differences in spoilage between pasteurised and unpasteurised milk. Unpasteurised
milk will spoil naturally to produce a sour tasting product which can be used in baking
to improve the texture of certain breads. Pasteurised milk, however, spoils (nondesirable fermentation) to produce an unpleasant product which has to be disposed of.
The reason for this difference is that pasteurisation (despite being a very important
process
to
destroy
pathogenic micro-organisms) changes the micro-organism

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environment and if pasteurised milk is kept unrefrigerated for some time, undesirable
micro-organisms start to grow and multiply before the desirable ones. In the case of
unpasteurised milk, the non-pathogenic lactic acid bacteria start to grow and multiply
at a greater rate that any pathogenic bacteria. Not only do the larger numbers of lactic
acid bacteria compete more successfully for the available nutrients, but as they grow
they produce lactic acid which increases the acidity of the substrate and further
suppresses the bacteria which cannot tolerate an acid environment.
Most food spoilage organisms cannot survive in either alcoholic or acidic environments.
Therefore, the production of both these end products can prevent a food from spoilage
and extend the shelf life. Alcoholic and acidic fermentations generally offer cost effective
methods of preserving food for people in developing countries, where more sophisticated
means of preservation are unaffordable and therefore not an option.
The principles of microbial action are identical both in the use of micro-organisms in
food preservation, such as through desirable fermentations, and also as agents of
destruction via food spoilage. The type of organisms present and the environmental
conditions will determine the nature of the reaction and the ultimate products. By
manipulating the external reaction conditions, microbial reactions can be controlled to
produce desirable results. There are several means of altering the reaction environment
to encourage the growth of desirable organisms. These are discussed below.
Manipulation of microbial growth and activity
There are six major factors that influence the growth and activity of micro-organisms in
foods. These are moisture, oxygen concentration, temperature, nutrients, pH and
inhibitors (Mountney and Gould, 1988). The food supply available to the microorganisms depends on the composition of the food on which they grow. All microorganisms differ in their ability to metabolise proteins, carbohydrates and fats.
Obviously, by manipulating any of these six factors, the activity of micro-organisms
within foods can be controlled.
Moisture
Water is essential for the growth and metabolism of all cells. If it is reduced or removed,
cellular activity is decreased. For example, the removal of water from cells by drying or
the change in state of water (from liquid to solid) affected by freezing, reduces the
availability of water to cells (including microbial cells) for metabolic activity. The form in
which water exists within the food is important as far as microbial activity is concerned.
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There are two types of water - free and bound. Bound water is present within the tissue
and is vital to all the physiological processes within the cell. Free water exists in and
around the tissues and can be removed from cells without seriously interfering with the
vital processes. Free water is essential for the survival and activity of micro-organisms.
Therefore, by removing free water, the level of microbial activity can be controlled. The
amount of water available for micro-organisms is referred to as the water activity (aw).
Pure water has a water activity of 1.0. Bacteria require more water than yeasts, which
require more water than moulds to carry out their metabolic activities. Almost all
microbial activity is inhibited below aw of 0.6. Most fungi are inhibited below aw of 0.7,
most yeasts are inhibited below aw of 0.8 and most bacteria below aw 0.9. Naturally,
there are exceptions to these guidelines and several species of micro-organism can exist
outside the stated range. See table for further information on water activity and
microbial action. The water activity of foods can be changed by altering the amount of
free water available. There are several ways to achieve this drying to remove water;
freezing to change the state of water from liquid to solid; increasing or decreasing the
concentration of solutes by adding salt or sugar or other hydrophylic compounds (salt
and sugar are the two common additives used for food preservation). Addition of salt or
sugar to a food will bind free water and so decrease the aw. Alternatively, decreasing the
concentration will increase the amount of free water and in turn the aw. Manipulation of
the aw in this manner can be used to encourage the growth of favourable microorganisms and discourage the growth of spoilage ones.
Oxidation-Reduction potential
Oxygen is essential to carry out metabolic activities that support all forms of life. Free
atmospheric oxygen is utilised by some groups of micro-organisms, while others are
able to metabolise the oxygen which is bound to other compounds such as
carbohydrates. This bound oxygen is in a reduced form.
Micro-organisms can be broadly classified into two groups - aerobic and anaerobic.
Aerobes grow in the presence of atmospheric oxygen while anaerobes grow in the
absence of atmospheric oxygen. In the middle of these two extremes are the facultative
anaerobes which can adapt to the prevailing conditions and grow in either the absence
or presence of atmospheric oxygen. Microaerophilic organisms grow in the presence of
reduced amounts of atmospheric oxygen. Thus, controlling the availability of free
oxygen is one means of controlling microbial activity within a food. In aerobic
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fermentations, the amount of oxygen present is one of the limiting factors. It determines
the type and amount of biological product obtained, the amount of substrate consumed
and the energy released from the reaction.
Moulds do not grow well in anaerobic conditions, therefore they are not important in
terms of food spoilage or beneficial fermentation, in conditions of low oxygen
availability.
Temperature
Temperature affects the growth and activity of all living cells. At high temperatures,
organisms are destroyed, while at low temperatures, their rate of activity is decreased or
suspended. Micro-organisms can be classified into three distinct categories according to
their temperature preference.
Classification of bacteria according to temperature requirements.

Temperature required for growth 0C
Type of
Minimum
optimum
maximum
bacteria
Psychrophilic
General sources of
bacteria
0 to 5
15 to 20
30
Water and frozen

foods
Mesophilic
10 to 25
30 to 40
35 to 50
Pathogenic and nonpathogenic bacteria
Thermophilic
25 to 45
50 to 55
70 to 90
Spore forming
bacteria from soil
and water
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Nutritional requirements
The majority of organisms are dependent on nutrients for both energy and growth.
Organisms vary in their specificity towards different substrates and usually only
colonise foods which contain the substrates they require. Sources of energy vary from
simple sugars to complex carbohydrates and proteins. The energy requirements of
micro-organisms are very high. Limiting the amount of substrate available can check
their growth.
Hydrogen ion concentration (pH)
The pH of a substrate is a measure of the hydrogen ion concentration. A food with a pH
of 4.6 or less is termed a high acid or acid food and will not permit the growth of
bacterial spores. Foods with a pH above 4.6. are termed low acid and will not inhibit the
growth of bacterial spores. By acidifying foods and achieving a final pH of less than 4.6,
most foods are resistant to bacterial spoilage.
The optimum pH for most micro-organisms is near the neutral point (pH 7.0). Certain
bacteria are acid tolerant and will survive at reduced pH levels. Notable acid-tolerant
bacteria include the Lactobacillus andStreptococcus species, which play a role in the
fermentation of dairy and vegetable products. Moulds and yeasts are usually acid
tolerant and are therefore associated with spoilage of acidic foods.
Micro-organisms vary in their optimal pH requirements for growth. Most bacteria favour
conditions with a near neutral pH (7). Yeasts can grow in a pH range of 4 to 4.5 and
moulds can grow from pH 2 to 8.5, but favour an acid pH. The varied pH requirements
of different groups of micro-organisms is used to good effect in fermented foods where
successions of micro-organisms take over from each other as the pH of the environment
changes. For instance, some groups of micro-organisms ferment sugars so that the pH
becomes too low for the survival of those microbes. The acidophilic micro-organisms
then take over and continue the reaction. The affinity for different pH can also be used
to good effect to occlude spoilage organisms.
Inhibitors
Many chemical compounds can inhibit the growth and activity of micro-organisms.
They do so by preventing metabolism, denaturation of the protein or by causing
physical damage to the cell. The production of substrates as part of the metabolic
reaction also acts to inhibit microbial action.
Controlled fermentation
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Controlled fermentations are used to produce a range of fermented foods, including

sauerkraut, pickles, olives, vinegar, dairy and other products. Controlled fermentation
is a form of food preservation since it generally results in a reduction of acidity of the
food, thus preventing the growth of spoilage micro-organisms. The two most common
acids produced are lactic acid and acetic acid, although small amounts of other acids
such as propionic, fumaric and malic acid are also formed during fermentation.
It is highly probable that the first controlled food fermentations came into existence
through trial and error and a need to preserve foods for consumption later in the
season. It is possible that the initial attempts at preservation involved the addition of
salt or seawater. During the removal of the salt prior to consumption, the foods would
pass through stages favourable to acid fermentation. Although the process worked, it is
likely that the causative agents were unknown. Today, there are numerous examples of
controlled fermentation for the preservation and processing of foods. However, only a
few of these have been studied in any detail - these include sauerkraut, pickles, kimchi,
beer, wine and vinegar production. Although the general principles and processes for
many of the fermented fruit and vegetable products are the same -relying mainly on
lactic acid and acetic acid forming bacteria, yeasts and moulds, the reactions have not
been quantified for each product. The reactions are usually very complex and involve a
series of micro-organisms, either working together or in succession to achieve the final
product.
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UNIT 4 - DOWNSTREAM PROCESSING

Cell distruption, precipitation methods, solid liquid separation, liquid-liquid
extraction,
filtration,
centrifugation,
chromatography,
drying
devices
(Lyophiliztion and spray dry technology0, crystallization, biosensors-construction

and applications
Food processsiong:
Food preservation and spoilage. Sterilization and pasteurization , canning and
packing of foods.
DOWNSTREAM PROCESSING
Downstream processing is an essential part of bioprocess technology in that the desired

product needs to be isolated, purified and for different end uses. A variety of
microorganisms including genetically engineered species are used for the production of
desired products. The products formed may be secreted into the broth or may be
retained within the cell introducing complexity in the recovery of the product. In view of
the complexity, downstream processing involves various techniques and methodologies.
Bioproducts differ greatly in their nature hence different separation principles and
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mechanisms depending on molecular mass, charge distribution, hydrophobicity,

distribution coefficient, structure and immunogenic structure and specific affinity
towards other biomolecules becomes necessary for their isolation and purification. The
choice of the separation methodology depends to a large extent on the nature of the
product, its quantity and the extent of purity required.
The desired for isolation by Down Stream Processing are most frequently metabolized
which may be present as follows;
Intracellular metablize: e.g. vitamins and enzymes.
Extracellular metabolize: e.g. most antibiotic,amino acids,alcohol,citric acid.
Both intracellular and extracellular metabolize: e.g. vitamin B12 flavomycin.
Downstream processing is any treatment of culture broth after fermentation to
concentrate and purify products. It follows a general sequence of steps:
Cell removal (filtration, centrifugation)
Primary isolation to remove components with properties significantly different from
those of the products (adsorption, liquid extraction, precipitation). Large volume,
relatively non selective
Purification. Highly selective (chromatography, ultra filtration, fractional precipitation)
Final isolation (crystallization, followed by centrifugation or filtration and drying).
Typical for high-quality products such as pharmaceuticals.
Downstream processing mostly contributes 40-90 % of total cost.
CELL DISTRUPTION
Mechanical cell disruption methods
French press (pressure cell) and high-pressure homogenizers. In these devices, the cell
suspension is drawn through a check valve into a pump cylinder. At this point, it is
forced under pressure (up to 1500 bar) through a very narrow annulus or discharge
valve, over which the pressure drops to atmospheric. Cell disruption is primary
achieved by high liquid shear in the orifice and the sudden pressure drop upon
discharge causes explosion of the cells.
Ultrasonic disruption
It is performed by ultrasonic vibrators that produce a high-frequency sound with a wave
density of approximately 20 kHz/s. A transducer convert the waves into mechanical
oscillations via a titanium probe immersed in the concentrated cell suspension. For
small scale
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The equation for Manton-Gaulin homogenizer
Rm
= kNp
ln
R
R
m
Rm: maximum amount protein available for release

R: amount of protein release after N passes through the homogenizer
k: temperature-dependent rate constant
p: operating pressure drop
: resistance parameter of the cells, for S. cerevisiae is 2.9
Cell Disruption
Physical method of cell disruption

Liquid shear
Solid shear
Aggitation with abrassive
Freeze thawing
Ultra sonication
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Liquid shear
The method is mainly used in enzyme purification
Liquid-based homogenization is the most widely used cell disruption technique for
small volumes and cultured cells.
Cells are lysed by forcing the cell suspension through a narrow space, thereby shearing
the cell membranes or cell wall.
Three different types of liquid shear homogenizers are
Dounce homogenizer
Dounce homogenizer -consists of a round glass pestle that is manually driven into a
glass tube.
French Press
A French press consists of a piston that is used to apply high pressure to a sample
volume of 40 to 250mL, forcing it through a tiny hole in the press. Only two passes are
required for efficient lysis due to the high pressures used with this process.
The equipment is expensive, but the French press is often the method of choice for
breaking bacterial cells mechanically.
Solid shear
In this method the cells are disrupted by a shearing force generated between the cells
and solid abrasive
The cells are cooled at -25 oC
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This method damage the subcellular organells such as mitochondria ,nuclei and
chloroplast
The cells are placed in a motor and pestle and is ground in the presence of small
amount of abrasive like coarse sand or fine silica
The pestle and motor is precooled before use to maintain low temperature
Or the prechilled cells are passed through a small orifice
X-press
The X-press has been developed for the disintegration of cells in the frozen state (mostly
at 25C).
By forcing the frozen cells contained in a cylinder through a hole much smaller than the
diameter of the cylinder but much larger than the size of the cells
By forcing the cells through the hole repeatedly rather than once, a higher degree of
disintegration can be achieved.
The disintegrated material is very easily recoveredit is simply taken out of the press in
the form of a cylinder containing the frozen, disintegrated material which is then easily
homogenized.
The method is used for the disintegration of a large number of cells, viz. bacteria,
yeasts, moulds,
Solid shear-X press
Aggitation with abrasives
Solid shear-X press figure:
In this method cells are disrupted with the help of mechanically resistant beads
The beads are made of glass, alumina or titanium compounds.
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Inside a hollow chamber, cells are agitated with beads by a system of agitator shaft.
The shear forces so generated cause cell disintegration.
Theprocess is exothermic and an efficient cooling system must be associated to prevent
thermaldenaturation of the enzyme.
Freeze-Thaw
The freeze-thaw method is commonly used to lyse bacterial and mammalian cells.
The technique involves freezing a cell suspension in a dry ice/ethanol bath or freezer
and then thawing the material at room temperature or 37C.
This method of lysis causes cells to swell and ultimately break as ice crystals form
during the freezing process and then contract during thawing.
Multiple cycles are necessary for efficient lysis, and the process can is quite lengthy.
The method is used to obtain beta glucosidase from s.cerevisiae
Ultrasonication
Ultrasonic waves are used to break the cells at small scale.
The process relies on the cavitation generated due to the sonic waves and the shear
force generated thereby.
A high electrical power is converted in to mechanical energy in terms of sonic wave
This causes cavitation.
Draw bracks
High power consumption, heat generation, small operable volume, etc.
Chemical methods of cell disruption
Detergent
Alkali
Osmotic shock
Detergents
Detergent-based cell lysis is an alternative to physical disruption of cell membranes,
The method is sometimes used in conjunction with homogenization and mechanical
grinding.
Detergents
disrupt
the lipid barrier
surrounding
cells
by
disrupting
lipid:lipid,
lipid:protein and protein:protein interactions.

The ideal detergent for cell lysis depends on cell type and source and on the
downstream applications following cell lysis.
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Animal cells, bacteria and yeast all have differing requirements for optimal lysis due to
the presence or absence of a cell wall.
nonionic(ex
triton),
resulting
in
less
protein
denaturation
upon
cell
lysis,
than ionic detergents

In contrast, ionic detergents are strong solubilizing agents and tend to denature
proteins, thereby destroying protein activity and function..
Alkali treatment
Treatment of cells with alkali is used in small scale and large scale extraction of
proteins
For example the therapeuttic enzyme
L asparginase can be released from Erwinia Chrysanthemi by exposing the cells to pH
values between 11 to 12.5 for 20mins
Osmotic Shock
Osmotic shock is applied for the mild release of the enzymes from the cells.
A sudden change in the salt concentration changes the osmotic balance within the cells
and the cell is disrupted.
The method is not very efficient for the microbial cells having tough cell wall.
The method has proved to be very efficient and unique for
the release of luciferase
enzyme from Photobacterium fischeri.

Enzymatic method
Various enzymes can break different bonds present in the cell wall and facilitate the
release of enzymes.
The process is the mildest one used for the release of intracellular enzymes.
The enzymes used are lysozyme, enzyme extracts from Streptomyces sp., Penicillum
sp.,Trichoderma sp., snail etc.
The method is very costly but highly effective for the release of enzymes with lesser
impurities.
During the downstream processing, the used enzyme must be removed.
Sometimes this process is also used in combination with other cell disintegration
methods.
Extraction
The process of recovering the compound from a mixture or from cells into a solvent
phase is called extraction
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PRECIPITATION METHODS
Precipitation is widely used in downstream processing of biological products, such as
proteins. This unit operation serves to concentrate and fractionate the target product
from various contaminants. For example, in the biotechnology industry protein
precipitation is used to eliminate contaminants commonly contained in blood.
Protein solubility
The solubility of proteins in aqueous buffers depends on the distribution of hydrophilic
and hydrophobic amino acid residues on the proteins surface. Hydrophobic residues
predominantly occur in the globular protein core, but some exist in patches on the
surface. Proteins that have high hydrophobic amino acid content on the surface have
low solubility in an aqueous solvent. Charged and polar surface residues interact with
ionic groups in the solvent and increase solubility. Knowledge of amino acid
composition of a protein will aid in determining an ideal precipitation solvent and
method.
Precipitate formation
Protein precipitate formation occurs in a stepwise process. The addition of a
precipitating agent and steady mixing destabilizes the protein solution. Mixing causes
the precipitant and the target product to collide. Enough mixing time is required for
molecules to diffuse across the fluid eddies. During the following nucleation phase,
submicroscopic sized particles are generated. Growth of these particles is under
Brownian diffusion control. Once the growing particles reach a critical size (0.1 m to
10 m for high and low shear fields, respectively), by diffusive addition of individual
protein molecules, they continue to grow by colliding into each other and sticking or
flocculating. This phase occurs at a slower rate. During the final step, aging in a shear
filed, the precipitate particles repeatedly collide and stick, then break apart, until a
stable mean particle size is reached, which is dependent upon individual proteins. The
mechanical strength of the protein particles correlates with the product of the mean
shear rate and the aging time, which is known as the Camp number. Aging helps
particles withstand the fluid shear forces encountered in pumps and centrifuge feed
zones without reducing in size.
Salting out
Salting out is the most common method used to precipitate a target protein. Addition of
a neutral salt, such as ammonium sulfate, compresses the solvation layer and
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increases protein-protein interactions. As the salt concentration of a solution is

increased, more of the bulk water becomes associated with the ions. As a result, less
water is available to partake in the solvation layer around the protein, which exposes
hydrophobic patches on the protein surface. Proteins may then exhibit hydrophobic
interactions, aggregate and precipitate from solution.
Isoelectric point precipitation
The isoelectric point (pI) is the pH of a solution at which the net primary charge of a
protein becomes zero. At a solution pH that is above the pI the surface of the protein is
predominantly negatively charged and therefore like-charged molecules will exhibit
repulsive forces. Likewise, at a solution pH that is below the pI, the surface of the
protein is predominantly positively charged and repulsion between proteins occurs.
However, at the pI the negative and positive charges cancel, repulsive electrostatic
forces are reduced and the dispersive forces predominate. The dispersive forces will
cause aggregation and precipitation. The pI of most proteins is in the pH range of 4-6.
Mineral acids, such as hydrochloric and sulfuric acid are used as precipitants. The
greatest disadvantage to isoelectric point precipitation is the irreversible denaturation
caused by the mineral acids. For this reason isoelectric point precipitation is most often
used to precipitate contaminant proteins, rather than the target protein. The
precipitation of casein during cheesemaking, or during production of sodium caseinate,
is an isoelectric precipitation. tkr
Precipitation with organic solvents
Addition of miscible solvents such as ethanol or methanol to a solution may cause
proteins in the solution to precipitate. The solvation layer around the protein will
decrease as the organic solvent progressively displaces water from the protein surface
and binds it in hydration layers around the organic solvent molecules. With smaller
hydration layers, the proteins can aggregate by attractive electrostatic and dipole forces.
Important parameters to consider are temperature, which should be less than 0 C to
avoid denaturation, pH and protein concentration in solution. Miscible organic solvents
decrease the dielectric constant of water, which in effect allows two proteins to come
close together. At the isoelectric point the relationship between the dielectric constant
and protein solubility is given by:
S0 is an extrapolated value of S, e is the dielectric constant of the mixture and k is a
constant that relates to the dielectric constant of water. The Cohn process for plasma
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protein fractionation relies on solvent precipitation with ethanol to isolate individual

plasma proteins
Precipitation reactors
There are numerous industrial scaled reactors than can be used to precipitate large
amounts of proteins, such as recombinant DNA polymerases from a solution.
Batch reactors
Batch reactors are the simplest type of precipitation reactor. The precipitating agent is
slowly added to the protein solution under mixing. The aggregating protein particles
tend to be compact and regular in shape. Since the particles are exposed to a wide
range of shear stresses for a long period of time, they tend to be compact, dense and
mechanically stable.
Tubular reactors
In tubular reactors, feed protein solution and the precipitating reagent are contacted in
a zone of efficient mixing then fed into long tubes where precipitation takes place. The
fluid in volume elements approach plug flow as they move though the tubes of the
reactor. Turbulent flow is promoted through wire mesh inserts in the tube. The tubular
reactor does not require moving mechanical parts and is inexpensive to build. However,
the reactor can become impractically long if the particles aggregate slowly.
Continuous stirred tank reactors (CSTR)
CSTR reactors run at steady state with a continuous flow of reactants and products in a
well-mixed tank. Fresh protein feed contacts slurry that already contains precipitate
particles and the precipitation reagents.
SOLID LIQUID SEPARATION
The Miller Vacuband filter (Miller, 1980; Kehse, 1979) which is a stationary bed vacuum
filter was used in semi-commercial scale production of milk fat fractions. This system,
offered by CJC, shows important advantages over the open vacuum systems.
Crystallisation of milk fat was carried out in a jacketed stainless steel vessel of virtually
identical design to that of a batch stirred tank reactor (BSTR). Nominal working
capacity of the vessel was approximately 400 kg charge of anhydrous milk fat (AMF)
feedstock. To achieve good heat transfer characteristics, the vessel was fitted with a
variable speed, full sweep, anchor-type agitator arranged to prohibit mass rotation.
Agitator speeds were possible within the range 4-30 r.p.m. although the optimum range
was found to be 7-10 r.p.m., i.e. 0.36-0.52 m s-1. The vessel was additionally rated at
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3.3 bar, for positive pressure nitrogen blanketing of product. During crystallisation, the
head space was purged to establish a nitrogen blanket. The temperature difference
between the oil and water jacket was maintained at a maximum of 5C. Separation of
milk fat crystals was carried out on the novel, stationary-bed, vacuum band filter, the
Vacuband, surface area 1 m2, Fig.
The novelty lies in being able to filter and separate the liquid from the solid phase,
under vacuum, within an enclosed upper chamber. This solid-liquid separation system
is being used in a variety of liquid processing industries and in the edible oil industry
during bleaching earth filtration, winterisation, and hydrogenation catalyst filtration.
The unit comprised an indexing, horizontal rolled stored filter medium (paper), arranged
over a static lower vacuum chamber and with a second upper movable (vertically)
vacuum/feed chamber in opposition. The standard design utilised the upper chamber
to recreate a self-feeding system using upper chamber vacuum level, and on completion
of each filtration cycle the vacuum in each chamber was released, and the upper
chamber opened by lifting up, allowing the band to be indexed forward to its discharge.
A stainless steel wire, fixed along the width of the band, ensured that the cake was
dislodged from the filter paper and dropped into the heated trough in front of the filter.
When the cake liquefied it was transferred via a butterfly valve at the base for packaging
or texturisation for food use. The filtrate (olein) drawn under vacuum during filtration,
was transferred via an intermediate vacuum tank, filtrate receiver, into the filtrate
storage tank before being drummed. The most suitable filtration medium was found to
be Paper/Binzer Type 67/N, 80 g, roll, 0.108 m in width and approximately 200 m in
length, of bleached crepe quality.
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The yields for milk fat were typically 76-80%, Table 8.1, compared to 67-72% for the
Florentine filter. This is attributed to the improved efficiency achieved by using the
integral, vacuum sealed, upper chamber. The fastest crystallization rate was
established as 6C h-', cool down to 28C for satisfactory filtration.
LIQUID-LIQUID EXTRACTION
During the liquid-liquid extraction a suitable solvent, is used .
The desired products are extracted out of the feed solution into the solvent phase.
After the extraction is completed, the solvent-rich phase is called the extract and the
residual liquid from which solute has been removed is called the raffinate.
Single-stage Extraction
Extraction can be carried out as a single-stage operation either in batch or continuous
mode.
The most widely used extractor is the mixer-settler that is a cylindrical vessel with one
or several agitators.
The vessel is usually equipped with four equally spaced baffles to prevent the vortex
formation
The mixer and settler can be combined in one vessel or separated.
Multistage Extraction co- current extraction
There is n mixers/seperator vessel in line and the raffinate goes from vessel 1 to vessel
to n
Fresh solvent is added to each stage
The feed and the extracting solvent pass through the cascade in the same direction
stage
Extract is recovered from each vessel
Multistage Counter current Extraction
In this system a number of mixer/seperator vessel are in line
The raffinate goes from vessel 1 to n
While the product enriched solvent is flowing from vessel n to 1
The feed and the extracting solvent pass through the cascade in opposite direction
Aqueous two-phase extractions
Aqueous (or water-based) solutions, being polar, are immiscible with non-polar organic
solvents (chloroform, toluene, hexane etc.) and form a two-phase system.
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The system is used to separate compounds based on their relative solubilities in two
different immiscible liquids,
Usually water and an organic solvent are used as the two phases
It is an extraction of a substance from one liquid phase into another liquid phase.
Types of aqueous two-phase extractions
Polymerpolymer systems
In a Polymerpolymer system, The heavy phase will generally be Polyethylene
glycol (PEG), and the light phase is generally a polysaccharide
If the target compound being separated is a protein then a ligand is incorporated to one
of the polymer phases.
This improves the target's affinity to that phase, and improves its ability to partition
from one phase into the other
Polymersalt systems
Aqueous two-phase systems can also be generated by introducing a high concentration
of salt to a polymer solution.
The polymer phase used is generally PEG. A kosmotropic salt, such as Na3PO4 is used
Since polymersalt systems demix readily they are easier to use.
At high salt concentrations, proteins generally either denature, or precipitate from
solution. Thus, polymersalt systems are not useful for purifying proteins.
Solvent recovery
After the extraction process it is necessary to remove the solvent
Because the cost of solvent is very high
Solvent may also cause contamination of the product
Distillation process is used for solvent recovery and recycling of solvent
Batch distillation and continuous distillation
Batch distillation
In batch distillation the vapour from the boiler passes up the column through the plate
containing the liquid
The liquid with the vapour is condensed in the condenser and it is collected depending
on their boiling point
Continuous distillation
Plate or tray column is used
It consists of distinct chambers which are seperated by perforated plates or trays
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The rising vapour passes through the liquid present in the tray
The liquid present in the tray is converted vapours
This is then passed in to the condenser
The remaining liquid in the tray reaches the reboiler through a series of down pipes.
FILTRATION
Cell aggregation and Flocculation
Microorganisms in solution are usualy held as discrete units in three ways
Surfaces are negatively charged repul each other
Hydrophillic cellwall act as thermodynamic barrier to aggregation
Irregular shapes of cell walls steric hindrance
Still flocculation is induced by
Neutralization of anionic charges,primarily carboxyl and phosphate groups,on the
surface of microbial cells
Reduction in surface hydrophilicity
Use of high molecular weight polymer bridges anionic, non-ionic and cationic
polymers
Flocculation compounds can also be added like alum,calcium salts,ferric salts,tannic
acid,titaniun tetra chloride
Majority zof the agents currently in use are polyelectrolytes- act by cherge
nuetralisation and hydrophobic interactions
Foam Separation
Foam separation of solid materials like microbial cells or proteins and colloidal
materials is mainly based on the surface properties of the materials.
Cells or molecules are absorbed on surfaces of the gas bubbles are separated by
skimming.
Surfactants used generally are called collector and the material that is made surface
active and collected are termed as colligends.
Separation process
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Schematic representation of the foam separation process

Filtration
Type of filtration unit:
Plate and frame filter. For small fermentation batches
Rotary-drum vacuum filter. Continuous filtration that is widely used in the
fermentation industry. A horizontal drum 0.5-3 m in diameter is covered with filter
cloth and rotated slowly at 0.1-2 rpm.
CENTRIFUGATION
Centrifugation is used to separate materials of different density when a force greater
than gravity is desired
The type of industrial centrifugation unit:
Tubular bowl centrifuge (Narrow tubular bowl centrifuge or ultracentrifuge, decanter
centrifuge, etc). Simple and widely applied in food and pharmaceutical industry.
Operates at 13000-16000 G, 105-106 G for ultracentrifuge
Disc-stack bowl centrifuge. This type is common in bioprocess. The developed forces are
5000-15000 G with minimal density difference between solid and liquid is 0.01-0.03
kg/m3. The minimum particle diameter is 5 m
Types Centrifugation
Perforated bowl basket centrifuge
Tubular bowl
Solid bowl scroll(decanter)
Multichamber
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Centrifugation (dry solid decanter centrifuge)
CHROMATOGRAPHY
Purification
In fermentation process chromatographic techniques are used to isolate and purify
relatively low concentration of metabolic products
Types of chromatography used
Adsorption chromatography
Ion exchange chromatography
Gel permeation chromatography
Affinity chromatography
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Reverse phase chromatography

High performance liquid chromatography
Adsorption chromatography
The stationary phase of this particular technique is a solid material on which the
sample compounds are adsorbed. Mobile phase is either a liquid (solid-liquid
chromatography) or a gas (gas-solid chromatography).
Adsorption chromatography is based on the interaction between the solute molecules
and active sites on the stationary phase.
Stationary phase can be inorganic absorbants like (active carbon, aluminium oxide,
aluminium hydroxide, magnesium oxide, silica gel) and macroporous resins
Streptomycin is extracted using activated charcoal column
Macroporous resins are used in purification of cephalosporin
The individual components are retained by the stationary phase differently
They then separate from each other while they are running at different speeds through
the column with the eluent.
At the end of the column they elute one at a time.
During the entire chromatography process the eluent is collected in a series of fractions.
The composition of the eluent flow can be monitored and each fraction is analyzed for
dissolved compounds
Ion exchange chromatography

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It is used in the purification of biological materials.

There are two types of exchange columnCation exchange column in which the stationary phase resins carries a negative charge,
The resins in cationic exchange column may contain sulphonic acid,carboxylic acid or
phosphonic acid active groups
The commonly used cation exchange resin is carboxy methyl cellulose resin
Positively charged solutes ex proteins will bind to the resins
They are then eluted by passing buffer of increasing pH or ionic strength anion
exchange column- in which the stationary phase carries a positive charge.
Charged molecules in the liquid phase pass through the column until a binding site in
the stationary phase appearance resin is
Anionic exchange resins include secondary amine, quaternary amonium active groups
A common anion exchange resin is DEAE-diethylaminoethyl
The molecule is eluted from the bypassing buffer of increasing ph or ionic strength
In recovery of streptomycin, the harvested filterate is fed in to a column of weak acid
cationic resin such as amberlite IRC 50 Which is in the sodium form
The streptomycin are adsorbed on the column and the sodium ions are displaced
Gel permeation chromatography
Gel permeation chromatography (GPC) is a type of size exclusion chromatography (SEC)
that separates analytes on the basis of size.
GPC separates based on the size of the analytes.
Separation occurs via the use of porous beads packed in a column (stationary phase)
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The smaller analytes can enter the pores more easily and therefore spend more time in
these pores, increasing their retention time.
On the other hand larger analytes spend little if any time in the pores and are eluted
quickly.
Example it is used in the purification of tetanus and diphtheria broth
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GPC instrument
Sample holder, Column, Pump, Refractive Index, Detector, UV-vis Detector
Affinity Chromatography
Affinity chromatography separates the protein of interest on the basis of a reversible
interaction between it and its antibody coupled to a chromatography bead (here labeled
antigen).
To elute the protein of interest from the affinity beads, the interaction can be reversed
by changing the pH or ionic strength.
Making antibodies to the protein of interest is expensive, so affinity chromatography is
the least economical choice for production chromatography.
Example-Used in protein and enzyme purification from cell extracts
Streptavidin is a terameric protein purified from the bacterium streptomyces avidin by
affinity chromatography
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Reversed-phase chromatography
Reversed-phase chromatography (also called RPC or hydrophobic chromatography)
Chromatography, results from the adsorption of hydrophobic molecules onto a
hydrophobic solid support (silica) in a mobile phase.
Decreasing the mobile phase polarity by adding more organic solvent reduces the
hydrophobic interaction between the solute and the solid support resulting in
desorption.
The more hydrophobic the molecule the more time it will spend on the solid support
and the higher the concentration of organic solvent that is required to promote desorption.
RPC is not typically used for protein purification due to the presence of the organic
solvent which can cause denaturation of proteins and destroys their biological activity.
Elution is usually performed by an increase in organic solvent concentration.
Acetonitrile, methanol, ethanol, and propanol are most commonly used.
Uses-- RPC is used for the final purification step of proteins that are stable in the
organic solvents used.
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High-performance liquid chromatography

Pressurized liquid and a sample mixture are made to pass through a column filled with
a sorbent, leading to the separation of the sample components.
The sorbent, is a granular material made of solid particles (e.g. silica, polymers) 2-50
micrometers in size.this is the stationary phase
The components of the sample mixture are separated from each other due to their
different degrees of interaction with the sorbent particles.
Instead of a solvent being allowed to drip through a column under gravity, it is forced
through under high pressures of up to 400 atmospheres. That makes the process
faster.
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The pressurized liquid is typically a mixture of solvents (e.g. water, acetonitrile and/or
methanol) and is referred to as "mobile phase".
Its composition and temperature plays a major role in the separation process by
influencing the interactions taking place between sample components and sorbent
DRYING DEVICES (LYOPHILIZTION AND SPRAY DRY TECHNOLOGY)
It is the last stage of manufacturing process
It involves the final removal of water from a heat sensitive material
Ensuring that there is minimum loss in viability ,activity or nutritional value.
Drying is undertaken becauseCost of transport can be reduced
The material is easier to handle and packaged
The material can be stored more conveniently in the dry state
The products are dried using driers
The driers can be classified by the method of heat transfer to the product and the
degree of agitation of the product
Drum drier
In this method slurry of the product is run on to a slowly rotating steam heated drum,
evaporation takes place
The dry product is removed by a scraper blade
The solid is in contact with the heating surface for 6-15 seconds
Spray drier
This type of drier is used when the starting material is in the form of liquid or paste
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The material to be dried is atomized in to small droplets through a nozzle or by contact

with a rotating disc
The droplets is then made to fall in to a spiral stream of hot gas at 150C to 250C
The high surface area: volume ratio of the droplets results in rapid evaporation
The drying takes place in few seconds
The evaporative cooling effect prevents the material from becoming over heated and
damaged
The gas flow rate must be carefully regulated so that the gas has the capacity to contain
the required moisture content at the cool air exhaust temperature
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Freeze drying
In this method the material is first frozen and then dried by sublimation in a high
vacuum
This method does not harm heat sensitive items
Fluidised bed drier

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These driers are used in pharmaceutical industry

Heated air is fed in to a chamber of fluidized solids, to which material is continuously
added and dry material is continuously removed.
CRYSTALLIZATION
It is a method of purification of the product
It is used in the purification of low molecular weight compounds
Crystallisation is used in the purification of antibiotics,citric acid,glutamic acid
For example in citric acid production the filtered broth is treated with Ca(OH)2
Durring this process insoluble calcium citrate crystals will be precipitated from solution
BIOSENSORS - CONSTRUCTION AND APPLICATIONS
A biosensor is a self-contained integrated device i.e. capable of providing specific
quantitative or semi- quantitative analytical information using a biological recognition
element which is in direct spatial contact with a transduction elemet (IUPAC, 1996)
Biosensor- 1. Bioanalytical system,2. An enzyme electrode is a biosensor
Biosensor Any device that uses specific biochemical reactions to detect chemical
compounds in biological samples.
Current Definition
A sensor that integrates a biological element with a physiochemical transducer to
produce an electronic signal proportional to a single analyte which is then conveyed to a
detector
Father of the Biosensor: Professor Leland C Clark Jnr 19182005
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Basic Characteristics of a Biosensor

Linearity:
Linearity of the sensor should be high for the detection of high substrate concentration.
Sensitivity:
Value of the electrode response per substrate concentration.
Selectivity:
Chemicals Interference must be minimised for obtaining the correct result.
Response time:
Time necessary for having 95% of the response.
Working of biosensor
a- Bio-element,
b- Transducer,
c- Amplifier, d- Processor, e- Display
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Biosensor
The Analyte (What do you want to detect)
Molecule - Protein, toxin, peptide, vitamin, sugar, metal ion
Sample
handling
(How
to
deliver
the
analyte
to
the
sensitive
region?)
(Micro) fluidics - Concentration increase/decrease), Filtration/selection

Detection/Recognition (How do you specifically recognize the analyte?)
Signal (How do you know there was a detection)
Example of biosensors
Glucose monitoring device (for diabetes patients) Monitors the glucose level in the
blood.
Infectous disease biosensor from RBS
Research Biosensors
Typical Sensing Techniques for Biosensors
Fluorescence,
DNA
Microarray,
SPR
Surface
plasmon
spectroscopy,SPM (Scanning probe microscopy, AFM,
resonance,Impedance
STM) ,QCM (Quartz crystal
microbalance),SERS (Surface Enhanced Raman Spectroscopy),Electrochemical

Types of Biosensors
Electrochemical Biosensor
Potentiometric Biosensor
Amperometric Biosensor
Conductometric Biosensors
Optical Biosensor
Piezo-electric Biosensor
Acoustic Biosensor
Thermometric Biosensors
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Piezo-Electric Biosensors
Piezo-electric devices use gold to detect the specific angle at which electron waves are
emitted when the substance is exposed to laser light or crystals, such as quartz, which
vibrate under the influence of an electric field.
The change in frequency is proportional to the mass of absorbed material.
Electrochemical Biosensors
For applied current: Movement of e- in redox reactions detected when a potential is
applied between two electrodes.
Potentiometric Biosensor
For voltage: Change in distribution of charge is detected using ion-selective electrodes,
such as pH-meters.
Optical Biosensors
Bioluminescence and Chemiluminescence
Colorimetric for color
Measure change in light adsorption
Photometric for light intensity
Photon output for a luminescent or fluorescent process can be detected with
photomultiplier tubes or photodiode systems.
Calorimetric Biosensors
If the enzyme catalyzed reaction is exothermic, two thermistors may be used to measure
the difference in resistance between reactant and product and, hence, the analyte
concentration.
Electrochemical DNA Biosensor
Steps involved in electrochemical DNA hybridization biosensors:
Formation of the DNA recognition layer
Actual hybridization event
Transformation of the hybridization event into an electrical signal
DNA biosensor
Motivated by the application to clinical diagnosis and genome mutation detection
Types DNA Biosensors
Electrodes, Chips, Crystals,
Glucose biosensor
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Glucose reacts with glucose oxidase (GOD) to form gluconic acid. Two electrons & two
protons are also produced.
Glucose mediator reacts with surrounding oxygen to form H2O2 and GOD.
Now this GOD can react with more glucose.
Higher the glucose content, higher the oxygen consumption.
Glucose content can be detected by Pt-electrode.
Wearable Biosensors: eg Ring Sensor, Smart Shirt
Biosensors on the Nanoscale
Molecular sheaths around the nanotube are developed that respond to a particular
chemical and modulate the nanotubes optical properties.
A layer of olfactory proteins on a nanoelectrode react with low-concentration odorants
(SPOT-NOSED Project). Doctors can use to diagnose diseases at earlier stages.
Nanosphere lithography (NSL) derived triangular Ag
streptavidin down to
nanoparticles are used to detect
one picomolar concentrations.
The School of Biomedical Engineering has developed an antibody
based
piezoelectric
nanobiosensor to be used for anthrax, HIV hepatitis detection.

Application of Biosensor
Food Analysis
Study of biomolecules and their interaction
Drug Development
Crime detection
Medical diagnosis (both clinical and laboratory use)
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Environmental field monitoring

Quality control
Industrial Process Control
Detection systems for biological warfare agents
Manufacturing of pharmaceuticals and replacement organs
FOOD PRESERVATION
Foods can be preserved by a variety of methods (table).
It is vital to eliminate or reduce the populations of spoilage and disease-causing

microorganisms and to maintain the microbiological quality of a food with proper
storage and packaging. Contamination often occurs after a package or can is opened
and just before the food is served. This can provide an ideal opportunity for growth and
transmission of pathogens, if care is not taken.
Removal of Microorganisms
Microorganisms can be removed from water, wine, beer, juices, soft drinks, and other
liquids by filtration. This can keep bacterial populations low or eliminate them entirely.
Prefilters and centrifugation often are used to maximize filter life and effectiveness.
Several major brands of beer are filtered rather than pasteurized to better preserve the
flavor and aroma of the original product. Low Temperature
Refrigeration at 5C retards microbial growth, although with extended storage,
psychrophiles and psychrotrophs will eventually grow and produce spoilage. Slow
microbial growth at temperatures below 10C has been described, particularly with fruit
juice concentrates, ice cream, and some fruits. Some microorganisms are very sensitive
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to cold and their numbers will be reduced, but cold does not lead to significant
decreases in overall microbial populations.
High Temperature
Controlling microbial populations in foods by means of high temperatures can
significantly limit disease transmission and spoilage. Heating processes, first used by
Nicholas Appert in 1809, provide a safe means of preserving foods, particularly when
carried out in commercial canning operations.
Canned food is heated in special containers called retorts at about 115C for intervals
ranging from 25 to over 100 minutes. The precise time and temperature depend on the
nature of the food. Sometimes canning does not kill all the microorganisms, but only
those that will spoil the food (remaining bacteria are unable to grow due to acidity of the
food, as an example). After heat treatment the cans are cooled as rapidly as possible,
usually with cold water. Quality control and processing effectiveness are sometimes
compromised, however, in home processing of foods, especially with less acidic (pH
values greater than 4.6) products such as green beans or meats.
Pasteurization involves heating food to a temperature that kills disease-causing
microorganisms and substantially reduces the levels of spoilage organisms. In the
processing of milk, beers, and fruit juices by conventional low-temperature holding
(LTH) pasteurization, the liquid is maintained at 62.8C for 30 minutes. Products can
also be held at 71C for 15 seconds, a high-temperature, short-time (HTST) process;
milk can be treated at 141C for 2 seconds for ultra-high-temperature (UHT) processing.
Shorter-term processing results in improved flavor and extended product shelf life.
Such heat treatment is based on a statistical probability that the number of remaining
viable microorganisms will be below a certain level after a particular heating time at a
specific temperature.
Despite efforts to eliminate spoilage microorganisms during canning, sometimes canned
foods are spoiled. This may be due to spoilage before canning, underprocessing during
canning, and leakage of contaminated water through can seams during cooling. Spoiled
food can be altered in such characteristics as color, texture, odor, and taste. Organic
acids, sulfides, and gases (particularly CO2 and H2S) may be produced. In flat sour
spoilage no gas is generated and the can does not swell, but its contents are rendered
sour by the presence of fermentation acids. If spoilage microorganisms produce gas,
both ends of the can will bulge outward to give a swell. Sometimes the swollen ends can
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be moved by thumb pressure (soft swells); in other cases the gas pressure is so great
that the ends cannot be dented by hand (hard swells). It should be noted that swelling
is not always due to microbial spoilage. Acid in high-acid foods may react with the iron
of the can to release hydrogen and generate a hydrogen swell. Hydrogen sulfide
production by Desulfotomaculum can cause sulfur stinkers.
Water Availability
Dehydration, such as lyophilization to produce freeze-dried foods, is now a common
means of eliminating microbial growth. The modern process is simply an update of
older procedures in which grains, meats, fish, and fruits were dried. The combination of
free-water loss with an increase in solute concentration in the remaining water makes
this type of preservation possible.
Chemical-Based Preservation
Various chemical agents can be used to preserve foods, and these substances are
closely regulated by the U.S. Food and Drug Administration and are listed as being
generally recognized as safe or GRAS (table). They include simple organic acids,
sulfite, ethylene oxide as a gas sterilant, sodium nitrite, and ethyl formate. These
chemical agents affect microorganisms by disrupting a critical cell factor. For example,
they may damage the plasma membrane or denature various cell proteins. Still other
compounds interfere with the functioning of nucleic acids, thus inhibiting cell
reproduction. The effectiveness of many of these chemical preservatives depends on the
food pH.
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As an example, sodium propionate is most effective at lower pH values, where it is

primarily undissociated and able to be taken up by lipids of microorganisms. Breads,
with their low pH values, often contain sodium propionate as a preservative. Chemical
preservatives are used with grain, dairy, vegetable, and fruit products. Sodium nitrite is
an important chemical used to help preserve ham, sausage, bacon, and other cured
meats by inhibiting the growth of Clostridium botulinum and the germination of its
spores. This protects against botulism and reduces the rate of spoilage. Besides
increasing meat safety, nitrite decomposes to nitric acid, which reacts with heme
pigments to keep the meat red in color. Current concern about nitrite arises from the
observation that it can react with amines to form carcinogenic nitrosamines. Nitrite is
added in very small amounts, and eventually it may be possible to eliminate its use
entirely.
Radiation
Radiation, both ionizing and nonionizing, has an interesting history in relation to food
preservation. Ultraviolet radiation is used to control populations of microorganisms on
the surfaces of laboratory and food-handling equipment, but it does not penetrate food.
The major method used for radiation sterilization of food is gamma irradiation from a
cobalt-60 source. Such electromagnetic radiation has excellent penetrating power and
must be used with moist foods because the radiation produces peroxides from water in
the microbial cells, resulting in oxidation of sensitive cellular constituents. This process
of radappertization, named after Nicholas Appert, can extend the shelf life of seafoods,
fruits, and vegetables. To sterilize meat products, commonly 4.5 to 5.6 megarads are
used. Among the more interesting radiation-resistant bacteria that have been studied is
Deinococcus radiodurans.
This bacterium has a complex cell wall structure and tetrad-forming growth patterns. It
also has an extraordinary capacity to withstand high doses of radiation, although the
mechanism for its resistance is not understood. Microbial ProductBased Inhibition
There is increasing interest in the use of bacteriocins for the preservation of foods.
Bacteriocins are bactericidal proteins active against closely related bacteria, which bind
to specific sites on the cell, and affect cell membrane integrity and function.
The only currently approved product is nisin. Nisin, produced by some strains of
Streptococcus lactis, is a small hydrophobic protein. It is nontoxic to humans and affects
mainly gram-positive bacteria, especially Enterococcus faecalis. Nisin can be used
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particularly in low-acid foods to improve inactivation of Clostridium botulinum during

the canning process or to inhibit germination of any surviving spores.
Bacteriocins function by dissipating the proton motive force (PMF) of a susceptible
bacterium. These compounds have a wide variety of names, depending on the
organisms that produce them.
Bacteriocins function by the formation of hydrophilic pores in a susceptible bacterium
and the release of low-molecular-weight molecules; this may occur as a result of
peptidoglycan synthesis inhibition and detergent-like effects on the cytoplasmic
membrane. Bacteriocin addition to foods such as cheddar cheese can lead to a two- to
threefold
reduction
in
Listeria
monocytogenes
in
180-dayold
cheeses.
Similar
compounds also occur in the eucaryotes.

FOOD SPOILAGE
Foods, because they provide nutrients for us, also are excellent environments for the
growth of microorganisms. Microbial growth is controlled by factors related to the food
itself, or intrinsic factors, and also to the environment where the food is being stored,
or what are described as extrinsic factors, as shown in figure.
The intrinsic or food-related factors include pH, moisture content, water activity or
availability, oxidation-reduction potential, physical structure of the food, available
nutrients, and the possible presence of natural antimicrobial agents. Extrinsic or
environmental factors include temperature, relative humidity, gases (CO2, O2) present,
and the types and numbers of microorganisms present in the food.
Intrinsic Factors
Food composition is a critical intrinsic factor that influences microbial growth. If a food
consists primarily of carbohydrates, spoilage does not result in major odors. Thus foods
such as breads, jams, and some fruits first show spoilage by fungal growth. In contrast,
when foods contain large amounts of proteins and/or fats (for example, meat and
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butter), spoilage can produce a variety of foul odors. One only need think of rotting
eggs. This proteolysis and anaerobic breakdown of proteins that yields foul- smelling
amine compounds is called putrefaction.
One major source of odor is the organic amine cadaverine (imagine the origin of that
name). Degradation of fats ruins food as well. For example, the production of shortchained fatty acids from fats renders butter rancid and unpleasant.
The pH of a food also is critical because a low pH favors the growth of yeasts and molds.
In neutral or alkaline pH foods, such as meats, bacteria are more dominant in spoilage
and putrefaction. Depending on the major substrate present in a food, different types of
spoilage may occur (table).
The presence and availability of water also affect the ability of microorganisms to
colonize foods. Simply by drying a food, one can control or eliminate spoilage processes.
Water, even if present, can be made less available by adding solutes such as sugar and
salt. Water availability is measured in terms of water activity (aw). This represents the
ratio of relative humidity of the air over a test solution compared with that of distilled
water. When large quantities of salt or sugar are added to food, most microorganisms
are dehydrated by the hypertonic conditions and cannot grow (table). Even under these
adverse conditions, osmophilic and xerophilic microorganisms may spoil food.
Osmophilic [Greek osmus, impulse, and philein, to love] microorganisms grow best in
or on media with a high osmotic concentration, whereas xerophilic [Greek xerosis, dry,
and philein, to love] microorganisms prefer a low aw environment and may not grow
under high aw conditions.
Water activity and microbial growth.
The oxidation-reduction potential of a food also influences spoilage. When meat
products, especially broths, are cooked, they often have lower oxidation-reduction
potentials. These products with their readily available amino acids, peptides, and
growth factors are ideal media for the growth of anaerobes, including Clostridium.
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The physical structure of a food also can affect the course and extent of spoilage. The
grinding and mixing of foods such as sausage and hamburger not only increase the food
surface
area
and
alter
cellular
structure,
but
also
distribute
contaminating
microorganisms throughout the food. This can result in rapid spoilage if such foods are
stored improperly. Vegetables and fruits have outer skins (peels and rinds) that protect
them from spoilage. Often spoilage microorganisms have specialized enzymes that help
them weaken and penetrate protective peels and rinds, especially after the fruits and
vegetables have been bruised. Many foods contain natural antimicrobial substances,
including complex chemical inhibitors and enzymes. Coumarins found in fruits and
vegetables exhibit antimicrobial activity. Cows milk and eggs also contain antimicrobial
substances. Eggs are rich in the enzyme lysozyme that can lyse the cell walls of
contaminating gram-positive bacteria. Other interesting foods with antimicrobial
activities include the hot sauces used on raw oysters and other seafoods. Tabasco and
other hot red pepper sauces apparently have particularly desirable antimicrobial
characteristics.
Herbs and spices often possess significant antimicrobial substances; generally fungi are
more sensitive than most bacteria. Sage and rosemary are two of the most antimicrobial
spices. Aldehydic and phenolic compounds are found in cinnamon, mustard, and
oregano. These compounds inhibit microbial growth. Other important inhibitors are
garlic, which contains allicin, and cloves, which have eugenol. However, spices also can
contain pathogenic and spoilage organisms. Coliforms, B. cereus, C. perfringens, and
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Salmonella species have been detected in most spices. Microorganisms can be

eliminated or reduced by ethylene oxide sterilization. This treatment can result in
Salmonella-free spices and herbs and a 90% reduction in the levels of general spoilage
organisms.
Unfermented green and black teas also have welldocumented antimicrobial properties
because of their polyphenol contents, which apparently are diminished when the teas
are fermented. Such teas are active against bacteria, viruses, and fungi and may have
anticancer properties.
Extrinsic Factors
Temperature and relative humidity are important extrinsic factors in determining
whether a food will spoil. At higher relative humidities microbial growth is initiated
more rapidly, even at lower temperatures (especially when refrigerators are not
maintained in a defrosted state). When drier foods are placed in moist environments,
moisture absorption can occur on the food surface, eventually allowing microbial
growth. The atmosphere in which the food is stored also is important. This is especially
true with shrink-packed foods because many plastic films allow oxygen diffusion, which
results in increased growth of surface-associated microorganisms. Excess CO2 can
decrease the solution pH, inhibiting microbial growth. Storing meat in a high CO2
atmosphere inhibits gram-negative bacteria, resulting in a population dominated by the
lactobacilli. The observation that food storage atmosphere is important has led to the
development modified atmosphere packaging (MAP). By the use of modern shrinkwrap materials and vacuum technology, it is possible to package foods with controlled
atmospheres. With a carbon dioxide content of 60% or greater in the atmosphere
surrounding a food, spoilage fungi will not grow, even if low levels of oxygen are present.
Some oxygen is kept because if all the oxygen is removed, the psychrophile Clostridium
gasigenes can grow. This organism can produce gases in 14 days at 2C, which leads to
swollen food packages. Such MAP procedures, while assisting in controlling spoilage,
also cause a shift in the general structure of the microbial community, from gram
negative to gram-positive organisms.
STERILIZATION
Sterilization can be defined as a means of complete of destruction of bacteria or fungal
spores effectively in microbiology. The glass wares and media used should be free from
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micro organisms and hence subjected to sterilization. Sterilization is achieved by

physical, chemical methods.
Physical methods of sterilization:
1.
Heat:
For general sterilisation a time and temperature that kill all organisms including heatresistant spores is used. The methods generally adopted are as follows:
Dry heat: Dry heat kills microorganisms by oxidative destruction of cell components.
This is done in a hot air oven, where a temperature of 160 C for two hours is usually
required.
Moist heat: This is more effective than dry heat because moist heat condition in more
rapid in killing all the bacteria and spores.
This is done in autoclave in which the
media is exposed to steam under pressure at approximately 15lb/in2 that will give a
temperature of 121C maintained for 30min. This is the best method, if practicable.
Tyndallization: This is a course of three periods of boiling at 100C for 30 min. at daily
intervals.
2.
Filtration:
The liquid or gas to be sterilised is passed through a filter with a porosity sufficient to
remove any microorganism in suspension. The cotton wool is used for gases. For
liquids, a variety of (filters are available, made of materials such as cellulose nitrate
(millipore filters). This method is very useful for sterilisation of liquids containing heatlabile components.
3.
Radiation:
Ultraviolet light is very effective in sterilisation of air. For solids, we generally use
gamma rays or x-rays from a source such as radioactive cobalt. Ionizing radiation is
often used to sterilize plastics and other heat labile materials.
Chemical methods of sterilization: This can be achieved by using disinfectants;
a.
Ethyl alcohol:
Alcohols are effective antiseptics when used as 50%-70% aqueous solutions alcohols
precipitate proteins and solubilise lipids present in the cell membrane. As solvents, they
also effectively clean human tissues. Alcohol kills the vegetative cells of many bacteria
but they dont effect microbial spores fungi and most viruses.
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Alcohols cannot produce a sterile condition but they can be relied upon the
substantially reduce the concentration of the microbial flora. Alcohols can also damage
viral proteins by mechanically removing them by detergent action.
b.
Aldehydes:
Formaldehyde-Active against bacteria, viruses, and spores.
Used for specimen preservation, but is highly irritant to skin and eyes.
Gluteraldehyde-Less toxic than formaldehyde.
Used for the sterilization of bronchoscope,face masks,rubber tubes.
c.
Phenol:
Lysol and cresol used as floor disinfectant, denature the proteins.
Chlorophenol and chloroxyphenol acts as disinfectant and are less toxic.
d.
Halogens
Iodine Sporucidal, fungicidal,virsucidal and amoebicidal. Eg:Providone
Chlorine-Chlorine compounds used for disinfection are hypochlorites and chloramines.
Eg:NaClO and Ca(OCl)2
e.
Metallic salts
Heavy metals like Hg, Ag, Cu, Zn have antimicrobial activity. Eg:HgCl2 inactivate
enzymes that contains SH groups.
AgNO2 used for treating ophthalmia neonatarum.
f.
Gases:
Ethylene dioxide inactivate micro organisms by alkylation of SH groups, OH groups,
NH2 group, carboxyl group of enzymes.
Formaldehyde gases inactivates cellular proteins and nucleic acids by reacting with
the amino group
PASTEURIZATION
Pasteutization is a process of heating every particle of milk or milk product at a least
62.8 degree C for 30 mins followed by sudden cooling.
Methods of Pasteurization:
Based on the temperature and time relationship, there are mainly 3 methods of
pasteurization,
LTH (Low temperature holding method):
It is also known as LTLT or Low temperature long time. In LTH method the milk is
heated at 62.8oC for 30 mins and then suddenly cooled to 10-15 oC.
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HTST (High temperature short time method):

It is also known as Flash treatment method. Most widely used method in the industries.
In this process raw milk is heated at 71.2 oC for 15 mins with sudden cooling.
UHT method (Ultra high temperature method):
The ultra high temperature is also a pasteurization process in which the milk is heat at
130oC for 1-2 seconds.UHT process divided into 2 types
Direct heating systems
Indirect heating system
In direct heating system, the heated steam is directly injected into milk and in indirect
method; the heating agent and the milk are separated by a heat conducting barrier.
Temperature at 100 oC (Boiling):
This method was performed in boiling bath. Boiling at 100o C for 5-10 mins is sufficient
to kill as non -sporing and many but not all sporing organisms.
This method can be used for sterilization of scalpels, forceps, scissors and syringes of
metal and glass that do not stand high temperature.
Temperature above 100 oC:
Moist heat is the form of pressurized steam is regarded as the most dependable method
for the destruction of all forms of life, indeed bacterial spores. This method is
incorporated in a device known as Autoclave. In Autoclave media is exposed to 121 oC
temperature for 20-30 mins at 15 psi.
Tyndallization or Fractional sterilization:
This method was developed by John Tyndall. In the years before develop of autoclave
liquids and other objects were sterilized by exposure to free flowing steam at 100 degree
C for 30 mins on each of 3 successive days. The method was called fractional
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sterilization because a fraction was accomplished on each day. This is known as

tyndalization or fraction sterilization.
Importance:
Pasteurization is one of the physical methods used for the sterilization.
Since the temperature is below 1000c the quality of milk wont be affected.
Other than milk and milk products other fermented foods like beer, wine are also
sterilized.
During the process of Low Temperature Holding (LTH) method and a High Temperature
Short Time (HTST) method, the equipment is designed and operated so that every
particle of milk is heated to the required temperature and held for the specified time.
Example: Mycobacterium tuberculosis was regarded as one of the most heat resistant
pathogen likely to occur in milk.
This organism is destroyed when exposed to a temperature of 60.60c for 10 minutes

.The Pasteurization temperature was set at 61.90c for 30 minutes.
Later it was discovered that Coxiella burnetti, the causative agent of Q fever which can
be transmitted by 62.80c milk, can survive in milk heated to 61.90c for 30 minutes.
Thus the temperature of 62.80c for 30 minutes.
CANNING OF FOODS
It is defined as the preservation of foods in sealed containers and usually implies that
heat treatment as the principle factor in the prevention of food spoilage.
The method was invented and introduced by a Franchmen Nicolas Appert in 1810,
called the father of canning.
Method:
The basic steps in canning are
1.
Cleaning:
The food materials are washed to remove dirt and as many microorganisms as possible.
This reduces microbial load.
2.
Blanching:
The raw food materials are immersed in hot water. This removes adhering material s
which cannot be removed with cold water.
3.
Exhausting:
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The food is then filled in cans while still hot. The open filled containers are passed
through an exhaust box in which hot water or steam is used. Exhausting expands food,
drives out air or gas bubbles.
4.
Sealing:
Each container is immediately sealed, after exhausting, before it is subjected to the
heating process. This prevents recontamination of the contents.
5.
Heat processing:
Microorganisms are more easily killed in acid environment than in a non acid one. Acid
foods, such as fruits therefore require very little heat processing as compared to non
acid foods such as meat, fish, poultry and certain vegetables. A longer heating period is
required for larger containers than for smaller ones.
6.
Cooling:
The processed containers are immediately cooled in air or in changes in texture and
flavour. It also prevents multiplication of thermophilic organisms which might survive
an in adequate heat treatment.
Advantages:
Canning process is designed to produce foods that are bacterially inactive, commercially
sterile or practically sterile.
Disadvantages:
Some thermophilic aerobic bacteria may survive in the process of canning but are
unable to grow in the absence of oxygen.
Common defects associated with canned food:
a)
Chemical defect:
The main important kind of chemical defect of canned food is hydrogen swell. Hydrogen
swell resulting from a presence of hydrogen gas released by the can hydrogen swells are
favoured by:
Increasing acidities of foods.
Increasing temperature of storage.
A poor exhaust.
Presence of soluble sulphur and phosphorous compounds.
b)
Biological defects:
Microorganisms may result from either or both of the two causes:
1.
Survival of organisms after the administration of the heat treatment.

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Leakage of the container after the heat process, permitting entrance of organisms.
Spoilage in acid products is usually caused by non spore forming lactobacilli and
yeasts.
Stages of defected can:Flat can: Normally ends of the canned foods are turned flat.
Flipper can: A can that normally appears flat, but one of the end become convex.
Springer can: A can with one end or both the ends permanently bulged.
Breather: It is a can with minute leak that permits air to move in or out.
PACKING OF FOODS
Packaging has become a modern socio-scientific discipline having the following roles:
Containing and safety of product that is of paramount importance.
Facilitating the handling, storage and distribution.
Protecting against biological, chemical and distribution damages.
Providing convenience.
Informing through the medium of labeling.
Security through a tamper evident design.
Contribution to the product image through structural and graphic design.
Increasing the shelf-life and ensuring longer availability.
As a marketing and advertising tool.
Environment protection by taking responsibility of empty packaging material after its
use.
The packaging industry in India is a heterogeneous mix of both organized and
unorganized sectors. The industry comprises of manufacturers of basic materials,
converted package forms, ancillary materials and packaging machinery. The packaging
conversion machinery and ancillary materials production units are primarily in the
small-scale sector and being gradually updated to reach international standards. The
packaging lines generally occupy 50% of the floor space, and the packaging and related
activities engage about 60% of the 5 million labor force concerned with the Indian food.
NEED FOR PACKAGING OF FOODS:
Packaging and package labelling have several objectives:
Physical protection
The objects enclosed in the package may require protection from, among other things,
shock, vibration, compression, temperature, etc.
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Barrier protection
A barrier from oxygen, water vapor, dust, etc., is often required. Package permeability is
a critical factor in design. Some packages contain desiccants or Oxygen absorbers to
help extend shelf life. Modified atmospheres or controlled atmospheres are also
maintained in some food packages. Keeping the contents clean, fresh and safe for the
intended shelf life is a primary function.
Containment or agglomeration
Small objects are typically grouped together in one package for reasons of efficiency. For
example, a single box of 1000 pencils requires less physical handling than 1000 single
pencils. Liquids, powders, and flowables need containment.
Information transmission
Information on how to use, transport, recycle, or dispose of the package or product is
often given on the package or label. With pharmaceutical, food, medical, and chemical
products, some types of information are required by legislation.
Marketing
The packaging and labels can be used by marketers to encourage potential buyers to
purchase the product. Package design has been an important and constantly evolving
phenomenon for dozens of years. Marketing communications and graphic design are
applied to the surface of the package and (in many cases) the point of sale display.
Security
Packaging can play an important role in reducing the security risks of shipment.
Packages can be made with improved tamper resistance to deter tampering and also
can have tamper-evident features to help indicate tampering. Packages can be
engineered to help reduce the risks of package pilferage: Some package constructions
are more resistant to pilferage and some have pilfer indicating seals. Packages may
include authentication seals to help indicate that the package and contents are not
counterfeit. Packages also can include anti-theft devices, such as dyepacks, Radio
Frequency Identification (RFID) tags, or electronic article surveillance tags, that can be
activated or detected by devices at exit points and require specialized tools to deactivate.
Using packaging in this way is a means of loss prevention.
Convenience
Packages can have features which add convenience in distribution, handling, display,
sale, opening, reclosing, use and reuse.
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Portion control
Single serving or single dosage packaging has a precise amount of contents to control
usage. Bulk commodities (such as salt) can be divided into packages that are a more
suitable size for individual households. It is also aids the control of inventory: selling
sealed one-literbottles of milk, rather than having people bring their own bottles to fill
themselves.
TYPES OF PACKAGING:
Individual packaging:
This means the packaging of individual items of goods and includes the technique of
application of appropriate materials and containers, etc. to protect each individual item
of goods, or to increase the merchandise value as well as the conditions of the goods to
which those techniques are applied. This could also be called as Primary Packaging.
Inner packaging:
This means the inner packaging of packaged goods, the techniques of application of the
appropriate materials or container, etc., with consideration of the protection of goods
against water vapour, light, heat, impact, etc. as well as the condition of the goods to
which these techniques have been applied. This could also be called as Secondary
Packaging.
External packaging:
This indicates the outer packaging of packed goods, in other words, the techniques of
placing the goods in a box, bag or other container such as a barrel or can, etc., or
bundling without the use of a container, and adding markings to identify the goods as
cargo; as well as the conditions of application of these procedures. This could also be
called as Tertiary Packaging.
In case of food packaging, the word goods can be substituted by food.
Another classification of food packaging is into `Rigid Containers` like cans, glass
bottles etc.; semi-rigid containers like standi-packs, Tetra-bricks and plastic bottles,
etc. and Flexible packages like LDPE milk pouches, laminates containing spices and
Tetrafino pillow pouches etc.
FORMS OF PACKAGING
Metal cans:
These can be classified into round, square, oval or pill-shaped, flat, etc. Cans are often
classified into 2-piece or 3-piece cans. The latter uses tin-plate as its basic material,
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and the can is joined by soldering or welding. In the case of tin-free-steel (TFS) cans,
body making is done by using an organic adhesive agent. 2-piece cans include cans
punched out by a press machine, aluminium can is made by impact extraction, D1
cans ironed after contraction process and DR cans are manufactured by carrying out
contraction process two or more times. D1 cans are used where high internal pressure
resistance is required. Bonded and welded cans may be used as alternative to
conventional soldered cans.
Glass bottles:
Glass bottles and containers are available in many different shapes such as large freesize bottles, small one-shot styles, light-weight bottles for soft drinks, heavy-weight
hand-crafted type liquor bottles returnable bottles, etc.
Stretch-wrap packaging:
In this method, food is placed in a tray and film is stretched over the food to cover it.
Stretchable PVC films, PE films, etc. are used as packaging materials. Shrink packaging
is a form of packaging in which one or more items are covered with film, which shrinks
when heated. The film is shrunk using either dry or moist heat. Films that are used for
this application include PVC, PP and PE.
Flexible pouches:
A pouch is a container made of a flexible packaging material, such as plastic film,
aluminium foil, paper, etc. which is used either singly or in continuation.
Bag-in-box packages:
Bag-in-carton or bag-in-box containers have double construction, with both an inner
and an outer package. The former type is used for several food items including liquids
while the latter is used for institutional use and for bulk shipment. The external
package provides mechanical strength, while the inner bag protects the contents
against water vapour, gases and volatiles. This can be made of single substance or
multi-layer structure.
Cups/trays:
The types of cups used as containers include thermo-formed, air- Food Packaging
pressure formed and expanded plastic sheets. Recently, a cup with a barrier layer
manufactured using pressurized air with laminated sheet and a composite cup with an
inner layer of aluminium foil has been introduced. Paper cups, with PE, PP or PET
inserts, thermo-formed are also being used.
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Paper-board containers:
The pure-pak/`tetra-pack` type of containers, with its distinctive gable-top and the
brick-type smaller containers are the predominant packages used for milk products.
Paper laminated to PE is used for ordinary milk while for long-life milk, fruit drinks, etc.
laminated aluminium foils are used.
Choosing an appropriate packaging material
In modern food business which is heavily dependent on the retailing sector, it is
important to choose the best packaging for the food being marketed in the most cost
effective manner. Choice of an appropriate packaging material is governed by several
factors such as:
The specific sensitivities of the contents, e.g. moisture, oxygen, etc.
Factors changing the contents viz. temperature, RH, pH, and the reaction mechanism
involved.
Weight and shape of container.
Effect on filling and sealing speeds.
Contamination of food by constituents of the packing material.
Storage conditions- How long the product needs to be protected.
Bio-degradability and recycling potential.
Most of the food production has been in the rural pockets of the country, while the
major markets are in the urban areas. So the need for its transportation over long
distances has become a necessity.
Dairy and fruit products being highly perishable products, utmost care is needed in its
preservation during storage, handling and transportation.
Food products spoil fast at high temperatures, in the presence of oxygen and other
contaminating agents present in the atmosphere.
There are many more peculiarities, which could be identified under the following
headings for determining the packaging of processed food products.
-> Product range
-> Market
-> Consumer needs
-> Operating margins
In India, there are 12 different types of thermoplastics used as raw materials for
manufacturing of plastic products along with separate standard on positive list of
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constituents that have been formulated. These standards prescribe requirements,

methods of test and sampling for respective materials, vis--vis positive list of
constituents of respective material such as homopolymer, polymer, etc. The standards
are used for food contact application and to be used in combination to provide a system
of control to the plastic manufactures as well as fabricators of thermoplastic packaging
material to derive maximum benefits.
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UNIT 5 - IMMOBILIZATION AND BIOTRANSFORMATION:

Methods of immobilization, adsorption, cross-linking, ionic bonding, entrapment,
encapsulation, Advantages and industrial applications of Immobilization of
enzymes and whole cells. Biotransformation of antibiotics, steroids and their
applications.
METHODS OF IMMOBILIZATION
Immobilization techniques can be classified by basically two methods, the chemical and
the physical method. The former is covalent bond formation dependent and the latter is
noncovalent bond formation dependent.
ADSORPTION
This physical method is the simplest way to immobilize enzymes. Enzymes can be
adsorbed physically on a surface-active adsorbent by contacting an aqueolls solution of
enzyme with an adsorbent. Commonly employed adsorbents are (Zaborsky, 1973):
alumina, anion-exchange resins, calcium carbonate, carbon, cation exchange resins,
celluloses, clays, collagen, colloid-ion, conditioned metal, glass plates, diatomaceous
earth, and hydroxyapatite.
The advantages of adsorption techniques are as follows:
The procedure of immobilization is simple.
Is possible to separate and pllrify the enzymes while being immobilized.
The enzymes are not usually deactivated by adsorption.
The adsorption is a reversible process.
However, adsorption techniques also have several disadvantages:
The bonding strength is weak.
The state of immobilization is very sensitive to solution pH, ionic strength, and
temperature.
The amount of enzymes loaded on a unit amount of support is usually low.
CROSS-LINKING
Covalent Attachment: The covalent attachment of enzyme molecules via nonessential
arnino
acid residues (that
is,
amino
acids
minus
water)
to
water-insoluble,
functionalized supports are the most widely used method for immobilizing enzymes.
Functional (<sroups of the nonessential amino acid residues that are suitable for the
immobilization process are free , or -carboxyl groups, a- or
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-amino groups, and phenyl, hydroxyl, sulfhydryl, or imidazole groups.

Another variation of immobilization by covalent attachment is the copolymerization of
the enzyme with a reactive monomer (M) such as
where M E may have the following structure:
Commonly employed water-insoluble supports for the covalent attachment of enzymes

include: synthetic supports such as acrylamide-based polymers, maleic anhydride-based
polymers, methacrylic acid-based polymers, styrene-based polymers, and polypeptides,
and natural supports such as agarose (Sepharose), cellulose, dextran (Sephadex), glass,
and starch (Zaborsky, 1973).
Already active polymers such as maleic anhydride copolymers will be simply mixed with
enzymes to produce immobilized enzymes. Normally, natural or synthetic polymers
need to be activated by treating them with reagents before adding the enzyme.
The activation involves the chemical conversion of a functional group of the polymer.
The enzyme's active site should not be involved in the attachment, in which case the
enzyme would lose its activity upon immobilization.
The following experiment illustrates the immobilization of glucose oxidase in agarose
(Gutcho, 1974). oxidase in agarose (Gutcho, 1974).
Activation of agarose:
Eliminate
excess
water
from
the
water-swollen,
ball-shaped
agarose
particles
(Sepharose 2B) by subjecting them to suction on a glass filter. Add 3.9 grams of the
particles (corresponding to 100 mg of shrunken and dried agarose) to 4 mL of a
cyanogen bromide solution containing 25 mg cyanogen bromide per mL of water. Allow
the particles to be activated for 6 minutes at pH 11 by adding 2 M sodium hydroxide
solution with an automatic titrator at 23C. Wash the activated product on a glass filter
with 1 L of ice water. Finally wash the particles rapidly with 0.1 Mphosphate buffer (pH
of 7.4).
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Binding with glucose oxidase:

React the activated polymer for 10 hours (with mild stirring) with 24 mg of glucose
oxidase dissolved in 1 mL of 0.1 M phosphate buffer at pH 7.4. Cross-linking Using
Multifunctional Reagents: Water-insoluble enzymes can be prepared by using
multifunctional agents that are all bifunctional in nature and have low molecular
weight, such as glutaraldehyde.
There are several different methods for producing immobilized enzymes with
multifunctional reagents, as illustrated in Figure.
Enzymes can be reacted with multifunctional reagent alone so that they are crosslinked intermolecularly by the reagent to form a water insoluble derivative. Another
method is to adsorb enzymes on a water-insoluble, surface-active support followed by
intermolecular
cross-linking
with
multifunctional
reagents
to
strengthen
the
attachment. Multifunctional reagents can be also used to introduce functional groups

into water-insoluble polymers, which then react covalently with water-soluble enzymes.
IONIC BONDING
An obvious approach to the reversible immobilization of enzymes is to base the protein
ligand interactions on principles used in chromatography. For example, one of the first
applications of chromatographic principles in the reversible immobilization of enzymes
was the use of ion-exchangers.
The method is simple and reversible but, in general, it is difficult to find conditions
under which the enzyme remains both strongly bound and fully active. More recently,
the use of immobilized polymeric-ionic ligands has allowed for modulation of protein
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matrix interactions and has thus optimized the properties of the derivative. A number of
patents have been filed on the use of polyethyleneimine to bind a rich variety of
enzymes and whole cells.
However, problems may arise from the use of a highly charged support when the
substrates or products themselves are charged; the kinetics are distorted as a result of
partition or diffusion phenomena. Therefore, enzyme properties, such as pH optimum or
pH stability, may change.
Although this could pose a problem it could also be useful to shift the optimal
conditions of a certain enzyme towards more alkaline or acidic conditions, depending on
the application.
ENTRAPMENT
Entrapment: Enzymes can be entrapped within cross-linked polymers by forming a

highly cross-linked network of polymer in the presence of an enzyme. This method has
a major advantage in the fact that there is no chemical modification of the enzyme;
therefore, the intrinsic properties of an enzyme are not altered. However, the enzyme
may be deactivated during the gel formation. Enzyme leakage is also a problem. The
most commonly employed crosslinked polymer is the polyacrylamide gel system. This
has been used to immobilize alcohol dehydrogenase, glucose oxidase, amino acid
oxidase, hexokmase, glucose isomerase, urease, and many other enzymes.
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ENCAPSULATION
Microencapsulation: Enzymes can be immobilized within semipermeable membrane
microcapsules. This can be done by the interfacial polymerization technique. Organic
solvent containing one component of copolymer with surfactant is agitated in a vessel
and aqueous enzyme solution is introduced. The polymer membrane is formed at the
liquid-liquid interface while the aqueous phase is dispersed as small droplets. One
example of this process is the polyamide nylon system, in which 1, 6-diaminohexane is
the watersoluble diamme and 1,10-decanoyl chloride is the organic-soluble diacid
halide. The organic solvent for this system is a chloroformcyclohexane mixture (1:4 v Iv)
containing usually 1 percent (v Iv) Span85 surfactant (Zaborsky, 1973). The
immobilized enzyme produced by this technique provides an extremely large surface
area.
ADVANTAGES AND INDUSTRIAL APPLICATIONS OF IMMOBILIZATION OF
ENZYMES AND WHOLE CELLS
The use of immobilized cells has found applications in a wide range of biological
processes, ranging from the production of ethanol to the degradation of phenol.
Industrial methods for cell immobilization are classified under two broad categories:
immobilized-free-cell method and modified-cell methods.
In the free-cell method, cells are immobilized by confining them behind dialysis/
filtration membranes. An example of this is the hollow fiber system.
However, the cost of hollow fibers and a steady decline in the filtration rate are the two
major limitations.
In the modified-cell methods, cells are either bound to a support or entrapped within a
matrix such as polyacrylamide, polyurethane, alginate, collagen, and k-carragenan.
Entrapment inside a matrix has limitations of a high degree of mass transfer resistance
between the cell and the surroundings.
Additionally, the use of entrapment sometimes requires harsh conditions and may
result in damage or loss of viability.
Cells could be attached to the surface by taking advantage of their property to naturally
adhere to surfaces. Although easy to perform, mild on the cells, and potentially free of
diffusion, cell immobilization by simple adsorption has serious drawbacks. Changes in
pH, temperature, or ionic strength can easily release the cells from the support matrix.
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These limitations can be alleviated by covalently coupling the cells to the support
matrix.
This technique involves linkage of any reactive component, generally the amine groups,
on the cell to an activated support. This technique produces a system free of diffusion
limitation and provides very strong binding.
However, covalent immobilization involves the use of cross-linking agents such as
glutaraldehyde for the attachment of cells to the support, which results in a loss of
activity and cell viability.
Additionally, since covalent bonds are very strong and for most purposes irreversible,
the immobilization matrix cannot be regenerated when the biological activity decreases
to an undesirable level.
A significant improvement, in terms of both economics and technology, could be
achieved with reversible and specific adhesion to the support.
Many biological molecules can be attached non-covalently to a support by receptormediated specific adhesion.
Advantages of immobilization through an affinity tag are specificity and the ability to
reverse the binding for regeneration of the support matrix when the wholecell activity
drops to an undesirable level. Several noncovalent adhesions such as biotin-avidin and
antibody- antigen have been reported for immobilization of proteins.
However, to date, use of such tags has not been demonstrated for whole-cell
immobilization. Additionally, because these supports are relatively expensive, their
commercial application for cell immobilization may be economically infeasible.
BIOTRANSFORMATIONS.
Introduction:
Biotransformation, the enzyme-catalyzed process in which toxic, hydrophobic molecules
are converted to less toxic, water-soluble molecules, consists of two types of biochemical
reaction class: phase I and phase II. Phase I reactions introduce or unmask polar
functional groups in hydrophobic molecules. In phase II the water solubility of
substrate molecules is substantially improved by the conjugation of functional groups
with substances such as glucuronic acid.
Principles:
To perform (large-scale) synthesis of drugs, materials, or chemicals, one can principally
follow three different approaches with various degrees of complexity:
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Use a purely chemical strategy.

Use a chemoenzymatic route combining chemical and biocatalytic steps. In this case,
the biocatalyst is preferentially used to perform the key reaction(s) requiring high
selectivity or specificity or to replace environmentally intolerable reaction steps.
Use a biological total synthesis by fermentation or multistep biotransformation.
Enzymes and whole-cell biocatalysts have several attractive properties, which make
them privileged catalysts for organic synthesis; these are as follows:
They have high chemo-, regio-, and stereo-selectivities.
They require mild reaction conditions. Therefore, biocatalysis offers great chances and
advantages for successful applications (also in cases where either the substrates or the
products of the reaction are chemically labile).
Biocatalysis is normally performed in an aqueous environment but can, in many cases,
also be conducted in solvent mixtures, liquidliquid two-phase systems, and even in
pure organic solvents. A relevant practical example is the use of esterases and lipases to
catalyze esterifications in organic solvents such as vinyl acetate.
There is no, or only limited use of protecting groups, for example, for the
chemoenzymatic synthesis of complex carbohydrates and glycoconjugates.
Screening for Biocatalysts
Three main screening strategies have been followed so far:
Testing and evaluating inexpensive commercially available (bulk) biocatalysts or
suitable microorganisms. Enzymes that do not require coenzymes, such as hydrolases
(lipases, esterases, proteases) are still the preferred biocatalysts for the preparation of
optically active compounds.
Searching and screening for suitable novel microbial biocatalysts from natural sources,
for example, by selective enrichment techniques from environmental samples, starting
from soil or sewage samples, or alternatively and in rare cases, by testing extracts of
plant or animal tissues. This laborious and more demanding approach is essential
when no suitable enzyme or microbe is commercially accessible for the conversion of a
defined substrate to a defined product using a particular reaction type. Both
approaches 1 and 2 are complementary.
A third approach, related to the second, is the search for completely novel enzymes
catalyzing difficult reaction types, where no indications or prior knowledge on their
existence has been available. Typical examples for this approach from the last decades
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are the discovery and investigation of enzymes catalyzing BaeyerVilliger oxidations or

DielsAlder-type reactions.
Technical Aspects of Biocatalysis
To set up a practical bioconversion process screening and optimization, work is
required at the following different levels:
Screening for suitable microorganisms or plant cells, and so on, and/or enzymeswith
the required catalytic properties and selectivities.
Screening for the optimum conditions for culture growth and production of the desired
enzyme(s).
This can be very demanding work in cases where enzyme expression is not constitutive.
In this case, the best and most economical process parameters, such as the conditions
for enzyme induction and the right harvesting time must be identified.
Screening for the optimum reaction conditions with whole cells, crude, or purified
enzymes including engineering and technological aspects such as cofactor regeneration,
immobilization, type of reactor, and so on.
Optimization of the biocatalyst by directed evolution or other enzyme engineering
technologies in order to obtain the ideal biocatalyst under the given process conditions.
In order to perform bioreactions, the biocatalyst can be operated under various process
conditions.
Batch or fed-batch application of whole cells in free or immobilized form in aqueous
environment;
Continuous application of whole cells in immobilized form in aqueous environment;
Application of whole cells in two-liquid phase, multiphase systems, or in micelles;
Use of acetone-dried or permeabilized cells;
Batch or fed-batch application of free crude or purified enzymes in aqueous or organic
environment;
Use of polyethyleneglycol (PEG)-modified enzymes in organic solvents;
Continuous application of immobilized enzymes;
Use of enzyme membrane reactors; the method of choice for systems with cofactor
recycling or for reactions with expensive enzymes.
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BIOTRANSFORMATION OF ANTIBIOTICS
Tetracyclines
Synonyms: Oxytetracycline, chlortetracycline, tetracycline, and doxycycline are several
types of this antibiotic.
Mechanism of Action
The tetracyclines exert their antimicrobial action by inhibiting the 30s ribosomal unit of
bacteria resulting in reduced protein synthesis. The mechanisms of toxicosis do not
appear to be related to the antimicrobial action of the drug. One proposed mechanism
for the cardiovascular effects is due to a propylene glycol vehicle. It is thought that the
propylene glycol induces histamine release leading to cardiovascular collapse. Another
proposed mechanism for cardiovascular collapse relates to the ability of tetracycline to
chelate calcium in serum acute renal failure. Hepatotoxicity may be noted with
tetracycline administration that is due to triglyceride accumulation in the mitochondria
of hepatocytes.
Toxicity and Risk Factors
Animals that are dehydrated are at greater risk of toxicosis. Intravenous administration
is a risk factor for cardiovascular toxicosis. Cattle given tetracycline dissolved in saline
at a dose of 10 mg/kg intravenously over a period of 1 minute collapsed.
Dogs receiving 25 mg/kg intravenously for 2 days exhibited renal toxicosis. The risk of
enamel hypoplasia and tooth discoloration is greater if the dam is given tetracycline in
the later term of pregnancy or early in the neonatal period.
Treatment
Animals that exhibit cardiovascular collapse may be treated symptomatically with fluids
and calcium gluconate. Concurrent intravenous administration of fluids and diuretics
(mannitol and furosemide) may prevent renal toxicosis by maintaining normal urine
production. Hemodialysis may be necessary if the renal function is severely
compromised
Prognosis. The prognosis is guarded with cardiovascular collapse.
Prevention and Control
Tetracycline that is given intravenously should be diluted in fluids and administered
slowly to prevent possible cardiovascular effects. The hydration status must be
addressed in animals with decreased renal function.
Chloramphenicol
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Synonyms:
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Chloramphenicol is also known as Tevcocin, Chlorasol, Chloromycetin,
Chlora-Tabs, Chloricol, and Anacetin.

Toxicokinetics:
The drug is metabolized by the liver and eliminated as a glucuronide conjugate. This
may partially explain the increased sensitivity of the cat to potential toxic effects.
Mechanism of Action
Antibacterial activity of chloramphenicol is related to the actions of the drug on peptidyl
transferase that is associated with the 50s ribosomal unit. The mammalian and
bacterial mitochondria are similar, and chloramphenicol may inhibit the replication of
cells in the bone marrow.
Toxicity and Risk Factors

Cats appear to be more sensitive to the hematologic effects of chloramphenicol. Cats
that received 100 to 120/mg/kg/day for more than 2 weeks developed bone marrow
suppression. Hepatic disease or dysfunction can predispose the animal to intoxication.
Chronic chloramphenicol therapy is also a risk factor for developing toxicosis. Some
humans develop a lifethreatening aplastic anemia in response to chloramphenicol
exposure. This is the reason that this drug is not approved for use in food-producing
animals.
Treatment
The initial step in treatment is to discontinue the use of the drug. No specific antidote
exists, and the animal must receive supportive and symptomatic therapy.
BIOTRANSFORMATION OF STEROIDS
Heart attacks and strokes are responsible for over a quarter of a million deaths in
Britain every year. Raised cholesterol levels are believed to be a future indication of the
possibility of such attacks. Cholesterol is a water-insoluble steroid that is an essential
component of cell membranes and the precursor of steroidal hormones and bile acids. It
enters the body through the small intestine and is transported through the circulatory
system by lipoproteins. These lipoproteins are macromolecules that consist of a
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spherical shell of a monolayer of lipoproteins and carbohydrates in which are embedded

a few cholesterol molecules. This shell surrounds a non-polar core containing
cholesterol, triglycerides and other lipid substances. The polar groups in the shell are
orientated into the plasma, which ensures that the structure remains soluble, and the
complete structure is held together by noncovalent bonds.
Humans either obtain cholesterol from their diet or by biosynthesis from ethanoate by a
long and complicated pathway first outlined by Konrad Bloch. The rate limiting step in
this pathway is the reduction of b-hydroxy-b-methylglutaryl-coenzyme A (HMG-CoA) to
mevalonate by HMG-CoA reductase.
This point in the biosynthesis was selected for the development of an inhibitor for
HMG-CoA in an attempt to control the level of cholesterol in the plasma and hence its
arterial deposition. This approach led to the discovery of lovastatin and mevastatin.
Lovastatin was originally isolated from a number of different fungi by several different
companies. For example, Merck obtained it from Aspergillus terreus. Mevastatin was
isolated from Penicillium cillium citrum, but although it inhibited HMG-CoA reductase
it failed an initial toxicity test and was withdrawn from clinical trials. Further SAR
investigations yielded a number of other compounds, collectively referred to as statins
that are now in clinical use. They are reasonably effective in reducing the levels of LDLcholesterol circulating in the plasma.
Lovastatin and Simvastatin are prodrugs. They are administered orally and pass
unchanged into the liver where the lactone ring is hydrolysed to the active hydroxyheptanoic acid form of the drug.
Pravastatin, fluvastatin, atorvastatin and cerivastatin have hydroxy-heptanoic acid side
chains that correspond to the lactone containing side chains of lovastatin and
simvastatin. However, cerivastatin was withdrawn in 2001 as it was associated with a
higher incidence of rhabomyolysis than other statins.
Rhabomyolysis is a condition where muscle fibres degenerate. This together with
muscular pains is one of the unwanted side effects of the use of statins. These
muscular problems are believed to arise because all statins inhibit coenzyme Q10
(CoQ10) as well as HMG-CoA reductase.
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The hydroxy acid side chains of pravastatin, fluvastatin and atorvastatin are more
hydrophilic than the lactone rings of lovastatin and simvastatin and consequently are
more polar and less able to cross the bloodbrain barrier than lovastatin and
simvastatin and so have fewer CNS side effects than these drugs. It is emphasised that
statins reduce the level of LDL-cholesterol in the plasma. Many but
not all of the other types of lipoproteins may be reduced by changes in diet and the use
of other suitable drugs.
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APPLICATIONS
The ability of microorganisms, e.g., bacteria, to produce large amounts of biomass and
a great variety of different enzymes in a short time.
The chemo-, regio-, and enantioselectivity of enzymes, because of their small size
bacteria have by far the largest surface- to-volume ratio in the living world, which
allows them to maximize their metabolic rates because of a high exchange of molecules
and metabolites through their surface.
Microorganisms have great potential for inducing new or novel enzyme systems capable
of converting foreign substrates.
Microorganisms are capable of producing unique enzymes which are stable toward
heat, alkali and acid.
Umplung type reactions can be carried out.
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UNIT 6 PRODUCTION OF INDUSTRIALLY IMPORTANT PRODUCTS:

Alcohol: Ethanol, glycerol, butanol, Acetone, Organic streptomycin, tetracycline
Vitamins: riboflavin, Enzymes: amylase, protease, Biodegradable plastic:
polyhydroxyalkanoates (butyarate, propionate), Recombinant protein-Insulin,
hepatitis-B vaccine, Fermented foods-sausages, olives, bread, idly and acidophilus
milk.
PRODUCTION OF ALCOHOL ETHANOL
A flow diagram for a conventional fermentation plant producing 76.0 x 103 m3 a"1
anhydrous ethanol from 816.5 x 103 kg d"1 corn is shown in Fig.
Corn from storage is fed to a grinder where the kernel size is reduced to expose the
interior portion of the grain. Water is added, pH adjusted, and the ground grain is then
cooked to solubilize and gelatinize the starch. After cooking and partial solubilization,
fungal amylase is added. Yeast, which has also been propagated in the plant, is added
and the fermentation is allowed to continue for approximately 48 h at a temperature of
32 C.
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During this time about 90% of the original starch in the grain is converted to alcohol.
The processing proceeds on a cyclic or batch basis, with some vessels containing
fermenting mash while other vessels are being filled, emptied, or sterilized.
Once fermentation is completed, the mixture is fed to the beer still where essentially all
of the alcohol is distilled overhead to about 50 vol.%.The diluted alcohol is purified by
further distillation, which removes fermentation byproducts (aldehydes, ketones, fusel
oils), yielding 95 vol.% alcohol. If it is desired to produce anhydrous ethanol, the 95 vol.
% ethanol is fed to a dehydration section consisting of an extractive distillation with
benzene. In the beer still, the remaining water with dissolved and undissolved solids is
drawn from the bottom of the still and fed to a centrifuge.
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The liquid phase from the centrifuge is concentrated to 50% dissolved solids in a
multiple-effect evaporator and mixed with the solids from the centrifuge. This mixture is
then dried in a fluidized transport-type dryer to 10% moisture and is used as cattle
feed. The cattle feed contains all the proteins that were originally present in the grain,
plus the additional proteins from the yeast, resulting in a product containing 28-36%
protein by weight.
In addition to alcohol and cattle feed, the original 816.5 x 103 kg of corn yield 175.0 x
103 kg of carbon dioxide and 95 kg of by-product aldehydes, ketones, and fusel oils.
Ethanol production can be incorporated into a wet milling corn processing plant. In this
case, the substrate is the isolated corn starch and the byproduct is gluten feed.
PRODUCTION OF GLYCEROL
Glycerol, a 1,2,3-propanetriol, is a simple alcohol with many uses in the cosmetic,
paint, automotive, food, tobacco, pharmaceutical, pulp and paper, leather and textile
industries (Table) or as a feedstock for the production of various chemicals. Glycerol is
also known as glycerin or glycerine. Glycerol has also been considered as a feedstock for
new industrial fermentations in the future. For example, glycerol can be fermented to
1,3 propanediol, which is used for the chemical synthesis of
poly(trimethylene
terephthalate), a new polyester with novel fiber and textile applications that combines
excellent properties (good resilience, inherent stain resistance, low static generation)
with an environmentally benign manufacturing process.
The transformation of glycerol to dihydroxyacetone by the bacterium Acetobacter
suboxidans is another example of a potential process. In a submerged fermentation, the
bacteria produce dihydroxyacetone in yields of 7590% from a 515% solution of
glycerol. The dihydroxyacetone can be transformed further by a dihydroxyacetone
kinase to dihydroxyacetone phosphate, which serves as an essential substrate for some
aldolases to produce various optically active sugar derivatives (Itoh et al., 1999).
Glycerol can be produced either by microbial fermentation or by chemical synthesis
from petrochemical feedstocks or can be recovered as a by-product of soap manufacture
from fats.
Traditionally, glycerol is produced as a by-product of the hydrolysis of fats in soap and
other related materials and contributes significantly to the present glycerol production
volume of about 600 000 tons annually. This process is now of lesser importance in
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industrial nations and many developing countries, because of the replacement of soap
with detergents.
Currently, approximately 25% of world glycerol production occurs by the oxidation or
chlorination of propylene to glycerol, but this route has declined in relative importance
since the early 1970s (Hester, 2000) partially because of environmental concerns.
Furthermore, as the cost of propylene has increased and its availability has decreased
especially in developing countries, glycerol production by fermentation has become
more attractive as an alternative route.
A significant amount of glycerol is also synthesized from allyl alcohol. Currently, the
price of glycerol is between US$1.10/kg and US$1.25/kg and is expected to increase in
line with inflation over the next 10 years. Glycerol production costs by microbial
fermentation are difficult to estimate. Recently, High Plains Corporation (Wichita, KS)
reported that glycerol production costs between US$0.40/kg and US$0.53/kg would
result in a profitable operation.
Glycerol metabolism in yeast:
Glycerol is synthesized in the cytosol of the yeast S. cerevisiae from the glycolytic
intermediate dihydroxyacetone phosphate in two steps that are catalyzed by glycerol-3phosphate dehydrogenase (Gpd) and glycerol-3-phosphatase (Gpp), respectively (Fig.).
Each enzyme has two isoenzymes, the osmotically induced Gpd1p, the constitutive
Gpd2p , the osmotic-induced Gpp2p and the constitutive Gpp1p. The
GPD1 and/or GPD2 genes encoding glycerol-3-phosphate dehydrogenase have been
cloned
and
sequenced
from
S.
cerevisiae,
Saccharomyces
diastaticus,
Schizosaccharomyces pombe, Candida glycerinogenes and Zygosaccharomyces rouxii.

This, together with the report of enzyme glycerol-3-phosphate dehydrogenase activity in
Debaryomyces hansenii, suggests that synthesis of glycerol via glycerol-3-phosphate is
fairly common in yeasts. Of the two enzymes, Gpd1p is the key enzyme in glycerol
formation in S. cerevisiae. Glycerol-3-phosphate and dihydroxyacetone
phosphate are also important metabolic intermediates for synthesis of other substances
besides glycerol. For example, glycerol-3-phosphate and dihydroxyacetone phosphate
can serve as precursors to synthesize phospholipids and glycerolipids
and
dihydroxyacetone phosphate is also a precursor for amino acid synthesis.
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Fig. Biochemical pathways of glycerol metabolism in yeast

ACS:
acetyl
CoA
synthetase;
ADH:
alcohol
dehydrogenase;
ALD:
aldehyde
dehydrogenase; DAK: dihydroxyacetone kinase; FBA: fructose bisphosphate aldolase;

FBP: fructose bisphosphatase; FPS: glycerol facilitator; GCY: glycerol dehydrogenase;
GLK:
glucokinase; GND: 6-phosphogluconate dehydrogenase; GPD: cytoplasmic glycerol-3phosphate dehydrogenase; GPP: glycerol-3-phosphatase; GUP: glycerol uptake protein;
GUT1: glycerol kinase; GUT2: mitochonchial glycerol-3-phosphate dehydrogenase; HXK:
hexokinase; HXT: hexose transferase; PDC: pyruvate decarboxylase; PDH: pyruvate
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dehydrogenase;
PFK:
2016 EDITION
fructose-6-phosphate
kinase;
PGI:
glucose-6-phosphate
isomerase; PGL: 6-phosphogluconolactonase; PYK: pyruvate kinase; TAL: transketolase;

TKT: transaldolase; TDH: glyceraldehyde3-phosphate dehydrogenase; TPI: triose phosphate isomerase; ZWF: glucose-6phosphate dehydrogenase. Ac.carn. = Acetylcarnitine; Ac.CoA= Acetyl-coenzymeA;
DHA= Dihydroxyacetone; DHAP= Dihydroxyacetone phospate; E-4-P = Erythose-4phospate; F-6-P = Fructose-6-phospate; F-1,6-BP= Fructose-1,6-biphospate;
GAP = Glyceraldehyde-3-phospate; Gly-3-P =Glycerol-3-phospate; G-6-P =Glucose-6phospate; PEP = Phosphoenolpyruvate; R-5-P= Ribulose-5-phospate; RI-5-P= Ribose-5phospate; SH-7-P= Sedoheptulose-7-phospate; TCA= Tricarboxylic acid cycle; XI-5-P =
Xylulose-5-phospate. Blocks indicate the proteins encoded by the genes regulated by
osmotic stress.
PRODUCTION OF BUTANOL / ACETONE

The
Conventional
Butanol
Fermentation
Process
Fermentation
prodtrcts
are
conventionally produced in batch fermentation. A plant using batch fer.mentation to

produce butanol was designed to act as a benchmark for comparison with the extractive
fennentation process. The batch process design relies heavily on descriptions of
commercial butanol fermentation facilities and on a prior economic analysis of the
conventional butanol fermentation of molasses.
Figure shows a schematic of the batch fermentation process. Molasses, containin g 55
wtVo fermentable sugars and 30 wtVo nonfermentable solids, is diluted to 60 g/L sugar
and mixed with nutrients in the feed mix tank. Butanol inhibition prevents the use of
higher sugar con centrations in the fermentor. The diluted feed is continuously
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sterilized by direct steam injection and charged into batch fermentors. Fermentors are
inoculated with actively growing cells of a strain of Clostridiunt acetobugtlicum
produced in smaller seed fermentors. After 30 hours of fermentation, the broth,
containing (in SIL): 13.7 butanol, 5.4 acetone, 1.5 ethanol, 0.2 butyric acid, 0.3 acetic
acid, and 3.0 cells, is discharged to the broth surge tank. The batch fermentors are
operated on a staggered schedule so that downstream processing is continuous.
Butanol, acetone, and ethanbl are stripped frorn the broth with 50 psig steam in the
beer stripper after being heated to 100'C by heat exchange with the stripper bottoms
product. The stripped broth, containing acetic and butyric acids, cells, proteins and
nonfermentable molasses solids, is evaporated to 5O wt% solids in the multiple-effect
stillage evaporators and then dried to 85 wtTo solids in a rotary dryer to give a dried
stillage product that can be used as an animal feed supplement.
The overhead vapor from the beer stripper, containing approximately 7O wtVo water
and 30 wt%o acetone, butanol, and ethanol, is separated in a series of four distillation
coltrmns. 99.5 wt% acetone is taken overhead from the first column. This column is
operated at 0.7 atm so that low pressure steam from the last effect of the stillage
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evaporators can be used in the reboiler. The bottoms product from the acetone column
is fed to the ethanol column, which operates at 0.3 atm pressure.
Vacuum operation reduces the reflux needed to produce the 95 wtTo ethanol overhead
product and allows the total reboiler duty to be met by condensing the overhead vapors
from the beer stripper in the ethanol column reboiler. The bottoms product from the
ethanol still and the overhead streams from the water and butanol strippers are fed to a
decanter where an aqueous-rich phase is allowed to separate from a butanol rich
phase. The water rich phase, containing approximately 9.5 wtVo butanol, is refluxed to
the water stripper, which produces water containing less than O.Ol wt%o butanol. The
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butanol-rich phase, containing about 23 wt% water, is refluxed to the butanol stripper,
which produces a 99.7 wtTo butanol product. The operating pressures of the stripping
colurnns are set such that about half of the heat duty in the water stripper reboiler is
met by condensing the overhead vapors from the butanol stripper.
PRODUCTION OF ORGANIC STREPTOMYCIN
Streptomycin and Rifampicin are potent antibiotic drugs. Streptomycin is broad
spectrum drug effective against both gram positive and gram negative bacteria.
However, its major use is as an anti-TB drug. Rifampicin also is used extensively as an
anti-TB drug but it is also used in treatment of leprosy. As both the diseases are
rampant in India, their importance can not be over emphasized.
Streptomycin is available in its sulphate form. It is poorly absorbed from the gut and
hence is normally available in injection form. Normal dose is 0.5 to 1.0 gm of the base,
given daily. Streptomycin is normally given along with other anti-TB drugs.
Streptomycin is especially useful during initial phase of a therapy as it is very effective
against rapidly multiplying bacilli.
Manufacture
Streptomycin is manufactured by fermentation process. The process comprises three
major steps:
Inoculum preparation.
Fermentation.
Extraction, recovery and purification
Streptomycin griseus is the micro-organism whose strain is used for the purpose of
manufacturing streptomycin. First step is the preparation of inoculum from the stock
culture of the strain/The inoculum is transferred to a germinator, where total quantity
of biomass is increased. This biomass is sent to the first of a series of fermentors in
which medium has been introduced. Fermentation processis aerobic submerged type
fermentation. Sterile air is introduced through spargers. Fermentation takes place in
controlled environment with suitable media ingredients consisting of carbohydrates,
soybean flour, corn steep liquor, sodium sulphate etc. Other chemicals like antifoaming
agents are also added. Fermentation usually takes about 200 hours.
Broth, after harvest, is filtered, diluted and passed through Ion Exchange Resin
columns where streptomycin is adsorbed. Streptomycin is eluted from resin column as
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streptomycin sulphate. It is further treated with sodium hypochlorite, EDTA,

activatedcarbon and de-ashed in resin column to remove impurities.
Purified streptomycin sulphate solutiorf ii concentrated under vacuum and dried under
aseptic conditions.
DEVELOPMENTS
The research efforts directed exclusively towards streptomycin are meager since 1970.
Since research requires sustained commitment of money and manpower, major
developments for any antibiotic have come from advanced countries. With negligible
requirement, production has stopped in these countries and so have the research
efforts However, major development did take place in first two decades of its discovery.
Strain Selection
At the beginning in 1940s, strains were of very poor quality and yields were about 50
units per ml of broth. These strains were steadily improved to give yields of as high as
25,000 units per ml. Yields of 16-18000 units per ml became common. Further
research could have yielded higher potency as in the case of penicillin.
Second major area was development of strains where no streptomycin- B is produced.
Streptomycin-B is an undesirable product. If present, it could lower overall efficiency by
10-15% and increase the cost of production.
Fermentation
Fermentor size has increased substantially making larger batches possible. Material of
construction has improved. Close loop process control and complete automation have
resulted in finer control and reduction in number of batches lost. Process time has
reduced by more than 50 % and media quality has improved. All these have helped in
improving the yields and in reducing the manufacturing costs.
Extraction & Recovery
Major changes have taken place, Ion exchange resin columns have replaced traditional
solvent extraction. Number of steps have been reduced and savings made in expensive
solvents. Extraction efficiency of upto 10% have been achieved.
World Production Status
All the above developments took place before 1970. World production has considerably
reduced since then. Presently, People's Republic of China end India are the leading
manufacturers in the world.
Status of production and technology in India:
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India, today, is a major producer of streptomycin. (Production has almost stopped in all
the major developed countries). Indigenous demand is fully met from indigenous
supply.
PRODUCTION OF TETRACYCLINE
The production of tetracycline1 by fermentation was disclosed by Minieri et al. (1953).
Details of this fermentation process in synthetic media of low chloride content are
presented, and the culture isolation program which was carried out in conjunction with
this study is discussed.
In this fermentation the composition of the medium and the strain of streptomycete are
both important factors, since Streptomyces aureofaciens is capable of producing at least
two antibiotic substances as pointed out by Backus et al. (1954). In the presence of
chloride, which is incorporated within the chlortetracycline2 molecule (Broschard et al.,
1949) and which is therefore essential for its production as disclosed by Petty and
Matrishin (1950), the antibiotic formed was predominately chlortetracycline.
In media low in chloride tetracycline predominates and the chlortetracycline fraction
diminishes since 1 ppm of available chloride ion can produce at most 14, ug/ml
chlortetracycline. The simultaneous production of two or more antibiotics in
fermentation is well known, and the substances formed may be either closely related on
a chemical or a biological basis or widely separated.
Typical examples of closely related compounds produced simultaneously by the same
organism
are
the
penicillins
(Clarke,
1949),
streptomycins
(Waksman,
1949),
polymyxins (Brownlee, 1949), bacitracins (Newton and Abraham, 1950), cephalosporins

(Crawford et al., 1952), nisins (Berridge et al., 1952), neomycins
(Waksman, 1953), rhodomycins (Brockmann et al., 1951), and candicidins (Lechevalier
et al., 1953).
Compounds which differ in their structure and in their biological activity and are
produced simultaneously by the same organism are illustrated by spinulosin,
fumigatin, and gliotoxin (Menzel et al., 1944); actidione, grisein, and streptomycin
(Waksman et al., 1948; Whiffen, 1948); rimocidin and oxytetracycline (Davisson et al.,
1951); fradicin and neomycin (Waksman, 1953); chlortetracycline and an antifungal
compound (Duggar et al., 1954); and fungicidin and an actidione-like antibiotic (Hazen
and Brown, 1951).
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The effect of medium and strain upon the concurrent production of these antibacterial
and antifungal agents is well established. Calam and Levi (1944) found different types of
penicillin produced in synthetic and natural media, and Smith and Bide (1944)
established the phenylacetyl grouping as a necessary component of the medium for the
production of penicillin G in contrast to penicillin F, which was formed in its absence.
That different isolates derived from the same parent culture were capable of producing
different types of penicillin was demonstrated by Calam and Levi (1944).
Changes in medium were found by Whiffen (1948) to alter the ratio between
streptomycin and actidione, and strain selection led to the sole production of either
component. Perlman (1949) showed that substrains could be chosen which produced
more of the desired streptomycin and less of mannosidostreptomycin than the parent.
Mayer et al. (1951) reported on two antibacterial substances produced by Actinomyces
vinaceous which were markedly influenced by the medium. Waksman (1953) pointed
out that Streptomyces fradiae produced fradicin, neomycin A, and a subtilis factor
along with neomycin, and that medium and strain influenced the proportion of the
individual components of the neomycin complex. Backus et al. (1954) disclosed that
different strains of S. aureofaciens possess the capacity to produce the antibiotics
chlortetracycline and tetracycline under controlled conditions of fermentation, and
Martin et al. (1955) demonstrated the effect of medium upon the production by S.
aureofaciens of an antifungal agent and chlortetracycline as well as other antibacterial
agents.
VITAMINS
PRODUCTION OF RIBOFLAVIN
In the fermentation production of riboflavin by Ashhya gossypii, the culture medium,
comprising glucose (corn sugar), corn steep liquor (byproduct of corn wet milling), and
animal stick liquor (a packing-house byproduct of wet rendering), is prepared in a
mixing tank. The medium is pumped at a controlled rate through
a steam jet heater, where by injection of high-pressure steam the solution is almost
instantaneously heated to 275 F. (135 C.). The hot solution circulates through
insulated pipes to retain the high temperature for 5 minutes, then through additional
pipes or coils surrounded by cold water to reduce the temperature to 82 to 86 F. (28
to 30 C.).
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Through steam-sterilized pipe lines, the cooled solution is pumped to a sterile

fermentation vessel. This is a closed tank equipped with a jacket or coils hy which the
tank contents may be maintained at a uniform temperature of 82 F. (28 G.). In the
bottom of the tank are fine-porosity stone; or perforated coils through which sterile air
is supplied. A mechanical agitator assists in providing adequate air distribution.
After the sterile culture medium is transferred to the tank^ a small volume of a day-old
culture of Ashhya gossypii is added, and sterile air is introduced through the air
distribution system at a rate of one-fourth to one-half volume per volume of medium per
minute.
By the fourth day, the maximum yield of riboflavin has been obtained, and the culture
medium has acquired a beautiful, intense yellow color.
Two types of products can be produced. A potent riboflavin concentrate, ideally suited
to enriching poultry and livestock feeds, can be had by evaporating the water from the
fermented m.edium to prepare a sirup of about 30 percent solids. The sirup is converted
to a dry powder by such conventional equipment as a drum or spray drier.
The drum drier has a pair of cylindrical rolls, mounted horizontally, which are steam
heated. The sirup is continuously fed to the valley between the rolls, and as the rolls
rotate in opposite directions a thin film of sirup adheres to each. The water rapidly
evaporates before a revolution is completed, and the dry material, scraped off by knives,
is conveyed to bagging equipment.
In the spray-drier method, the sirup is sprayed into a chamber through which heated
air is passed; the air absorbs the water; and the dry riboflavin concentrate is
mechanically removed to packaging equipment. Concentrates containing 25,000
micrograms of riboflavin per gram (11,350 milligrams per pound, or 2.5 percent
riboflavin) are thereby produced.
Pure crystalline riboflavin may be recovered from the fermented solution. Synthetic
riboflavin was marketed in 1938.
ENZYMES
PRODUCTION OF AMYLASE
Production of amylase was carried out by SSF using the substrates of zero cost namely
Wheat Bran, Rice Husk, Vegetable Waste (Potato, Tomato, Brinjal) and Banana Peel. For
SSF 20gm of powdered wheat bran and rice husk were taken in 250ml flasks and
moistened with nearly 50ml of MSM containing the following in gm/l (0.8 g NaCl , 0.8 g
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KCl , 0.1 g CaCl2 , 2.0 g Na2HPO4 , 0.2g MgSO4 , 0.1 g FeSO4, 8.0 g Glucose, 2.0 g
NH4Cl pH 6.2). Flasks were autoclaved, cooled to room temperature, inoculated with
1ml of 48 hour old grown broth culture of showing maximum hydrolysis during
screening and incubated at 28C for 5 days. Vegetable waste and peels of banana to be
used as substrate were washed several times with distilled water, dried under shed,
rinsed with 0.1% H2SO4, cut into small pieces and ground by the help of sterile mortar
and pestle. 20 gm of the resultant pastes of vegetable waste and banana peels were
transferred into 250ml flask, and moistened with MSM. Both the flasks were
autoclaved, cooled to room temperature, inoculated with 1ml of 48 hour old grown
broth culture of showing maximum hydrolysis during screening and incubated at 28C
for 5 days.
Extraction of Crude Enzyme
Crude enzyme was extracted from fermented media by adding 100ml of 100mM Tris
buffer pH 6.2, agitating the flask in shaker at 180rpm for 1hour, the mixture was
filtered through cheese cloth and centrifuged at 8000 rpm at 4oC for 5 min. The
supernatant was collected and treated as crude enzyme.
Protein Estimation in Crude Enzyme
Concentration of protein in crude enzyme (extracted from four flasks containing
different substrates) was determined by Lowrys method of protein estimation in which
enzyme was reacted with the Lowrys reagents and the absorbance obtained was
compared with a standard graph plotted by reacting a standard protein with known
concentrations with the Lowrys reagents and plotting a graph between concentration of
standard protein (BSA) on X axis and absorbance at 660nm on Y axis.
Enzyme assay in Crude Enzyme
Enzyme assay was carried out by DNS method of [11] in which 0.5ml enzyme was
reacted with 0.5ml of substrate (1% starch in 100mM Tris buffer) under standard
reaction conditions and the reaction was stopped by adding DNS reagent, amount of
maltose released was determined by comparing the absorbance reading of the test
enzyme at 540 nm with the standard graph plotted by reacting the known
concentration of maltose ranging from 0.05mg/ml to 0.5mg/ml. One unit amylase
activity was defined as amount of enzyme that releases 1 micromoles of maltose per
minute under standard reaction conditions.
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PRODUCTION OF PROTEASE
Microorganism and inoculum preparation
A culture of Bacillus subtilis previously isolated from water and identified by standard
method for bacterial identification. Stock cultures were maintained in nutrient broth
medium (Difco) with 70% glycerol, cultures were preserved at -20 C. a Loopful of
bacterial strain (Bacillus subtilis) were transferred to a tube of sterile nutrient broth
and allowed to grow overnight at 37 C before being used to inoculation. A stock
suspension was prepared and adjusted to 7 X103 cell/ml-1
Fermentation procedure:
Protease crude enzyme was produced by fermentation of the (50 ml-1/flask). The
nutrient broth {production medium (PM)} was supplemented with gelatin (10 g) and then
autoclaved at 120 C for 20 min before inoculation. The contents of the flasks were
mixed thoroughly and then incubated for 24 h at 37 C) before enzyme assay.
Extraction of Protease:
The whole contents of fermented containing protease were filtered through Whitman No.
1 filter paper to obtain the extracted volume then preserved in the refrigerator at 4 C as
a crude protease filtrate.
BIODEGRADABLE PLASTIC
PRODUCTION OF POLYHYDROXYALKANOATES (BUTYARATE, PROPIONATE)
There has been considerable interest in the development and production of
biodegradable polymer to solve the current problem of pollution caused by the
continuous use of synthetic polymer of petroleum origin. Polyhydroxyalkanoates (PHAs)
are known to be accumulated as intracellular inclusion in some bacteria. The materials
properties exhibited by PHAs, ranging from stiff, brittle to rubber-like makes it a close
substitute for the synthetic plastic. The high cost of PHAs production has restricted its
applications. The possibility of producing this polymer commercially and at comparable
cost has been the main focus in this area.
Biosynthetic pathways of PHA
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Polyhydroxyalkanoates are polyesters of hydroxyalkanoates (HAs) with the general

structural formula as shown in Figure. (Steinbchel, 1991; Lee, Ojumu et al. 19
1996). It was first discovered in 1926 as constituent of the bacterium Bacillus
megaterium (Lemoigne, 1926).
Over 250 different bacteria, including gram-negative and gram-positive species, have
been reported to accumulate various PHAs (Steinbchel, 1991; Steinbchel, 1992; Lenz
et al., 1992). PHAs can be divided into two broad groups based on the number of
carbon atoms in the monomer units; the short chain length polyhydroxyalkanoates
PHAs
(SCL),
which
consist
of
C3-C5
atoms,
and
medium
chain
length
polyhydroxyalkanoates PHAs (MCL) consisting of C6-C14 atoms. This grouping is due to

the substrate specificity of the PHA synthesis that only accepts 3-hydroxyalkanoates
(3HAs) of a certain range of carbon length (Anderson and Dawes, 1990). The PHA
synthetases of A. eutrophus can only polymerize 3HAs(SCL) while that of seudomonas
oleovorans only polymerize 3HAs (MCL). For PHAs (SCL), the monomer units are
oxidized at positions other than the third carbons while for PHAs(MCL), all the
monomers units are oxidized at the third position except in few cases (Valentin et al.,
1994). A lot of PHAs (MCL) containing various functional groups such as olefins,
branched alkyls, halogens, aromatic and cyano has been reported (Fritzsche el al.,
1990; Huijberts et al., 1992; Kim et al., 1992; Hazer et al., 1994). This flexibility of PHA
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biosynthesis makes it possible to design and produce related biopolymers having useful
physical properties ranging from stiff and brittle plastic to rubbery polymers
Production of polymer
PHA are synthesized and intracellularly accumulated in most bacteria a under
unfavourable growth condition such as limitation of nitrogen, phosphorus, oxygen or
magnesium in the presence of excess supply of carbon source (Du et al., 2001a, Du and
Yu, 2002a; Lee, 1996). Strategies are still being developed to simulate conditions for
efficient production of PHAs. (Yu, 2001, Du et al., 2001b; Du and Yu, 2002b). Some
bacteria such as A. eutrophus, A. Latus and mutant strain of Azotobacter vinelandii are
known to accumulate PHA during growth in the absence of nutrient limitation. Several
factors need to be considered in the selection of microorganism for the industrial
production of PHA such as the ability of the cell to utilize an inexpensive carbon source,
growth rate, polymer synthesis rate and the maximum extent of polymer accumulation
of a particular cell based on the substrate. Some workers have derived equation that
predicts the PHA yield on several carbon source (Yamane, 1992;Yamane, 1993; Yu and
Wang, 2001) which could be used for the preliminary calculation of PHA yields.
In order to reduce the overall cost, it is important to produce PHA with high productivity
and high yield. Several methods such as Fed-batch and continuous cultivations have
been carried out to improve productivity (Lee, 1996; Du and Yu, 2002a; Du and Yu,
2002b; Du et al 2001b; Yu and Wang, 2001). Only three prominent PHAs [PHB, poly (3hydroxybutyrate-co-3-
hydroxyvalerate)
and
poly
(3-hydroxyhexanoate-co-3-
hydroxyoctanoate)] have been produced to a relatively high concentration with high

productivity. Recently, workers have been exploring cultivation strategies involving
inexpensive, renewable carbon substrates in order to reduce production cost and obtain
high productivity (Lee, 1996; Byrom, 1992; Kim et al., 1994, Park and Damodarau,
1994; Ishizaki and Tanaka, 1991; Poirier et al., 1995; Doi et al., 1988).
Recovery of PHA should also be considered because it significantly affects the overall
process economics. The last stage of PHB production involves separating the polymer
from the cells. To do this a solvent of aqueous extraction can be used. In the aqueous
process, the cell walls are broken and the polymer is then extracted and purified. The
aqueous process is less expensive, but the process reduces the polymer molecular
weight. For example, solvent extraction can produce copolymer weights of 1 million,
whereas typical molecular weights of aqueously extracted copolymer are in the range
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600,000 (Luzier, 1992). But solvent extraction, according to Luzier(1992) present some
safety concerns.
In
the
solvent
extraction
process,
the
solvent
employed
include
chloroform,
methylenechloride, propylene carbonate, and dichloroethane (Baptist, 1962; Lafferty et

al., 1988; Ramsay et al., 1994). Lee (1996) reported that large amount of solvent is
required due to high viscosity of PHA, this makes the method economically unattractive.
Sodium hypochlorite is used for the aqueous process. Although the use of sodium
hypochlorite significantly increased PHA degradation, polymer purity greater than 95%
is achieved. Lee (1996) also reported aqueous enzymatic digestion method as an
alternative to solvent extraction and washing with an anionic surfactant to solubilise
non-PHA cellular materials. Since recovery of PHA contributes significantly to the
overall economics, development of a process that allows the simple and efficient
extraction of polymers will be well rewarded.
Due to the relative high cost of producing PHA, several researches have been
considering the possibility of producing PHA as cheap as starch. Starch and lipids are
two of the most industrially useful and versatile product harvested from crop plants.
There has being some investigations on the possibility of producing PHB in transgenic
plant (Poirier et al., 1995; Lee, 1996; Nawrath et al., 1994). It has been reported that
the first enzymes of PHA synthesis, -Ketothiolase, is found in the cytoplasm of some
higher plants (Poirier et al., 1995), this means that only the reductase and the PHA
synthase are required by the plant to synthesize PHA.
A small oil seed plant (transgenic plant) was engineered to harbour the A. eutrophus
PHA biosynthesis genes, this was found to accumulate PHB granules of 0.2-0.5 m
diameter in the nucleus, vacuole and cytoplasm. The accumulation was 100 g/g fresh
weight (Lee, 1996; Poirier et al., 1995) but the transgenic plant growth was impaired,
the reason attributable to the severe depletion of substrate from the mevalonate
pathway (Nawrath et al., 1994; Lee, 1996). This problem was solved and the polymer
accumulation was improved upon by further genetic manipulations in order to divert
reduced carbon away from endogenous metabolic pathways and to regulate the tissue
specificity and the timing of gene expression. Thus genetically engineered genes of A.
eutrophus were successfully transferred to the plastids of Arabidopsis thaliana. The
hybrid plant expressing the A. eutrophus PHA synthesis enzymes accumulated PHB up
to 10 mg/g fresh weight, about 14% of dry weight (Lee,1996 and Poirier et al.,1995).
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These show that production of PHA by transgenic plant may be economically viable if
efforts are concentrated at improving this process. It may be possible in the near future
to see farmers growing plastics in their field and new agricultural product plastics
fruit may appear in our market.
RECOMBINANT PROTEIN - PRODUCTION OF INSULIN
The first step in creating synthetic human insulin is to extract human DNA. The
method to extracting human DNA is as follows:
First charge a sample of blood, normally between 300 and 2500 microlitres, and allow it
to
undergo
treatment
of
lysis
by
cationic
detergent,
for
example
tetradecyltrimethilammoniumbromide (TTAB) or dodecyltrimethilammoniumbromide

(DTAB) both with sodium chloride at a concentration higher than 0.5 M. Then mix up
the solution and heat it up to 68C and incubate it for five minutes. Add 1 volume of
chloroform or another organic solvent. Allow the mixture to undergo centrifuging with a
normal bench centrifuge for a few minutes to eliminate the protein portion which forms
a clog with the organic base. After centrifuging, to the aqueous phase add a quantity of
water to decrease the ionic strength below 0.5 M NaCl, and a cationic detergent (for
instance a solution of 5% of Cetyl-trimetil-ammonium bromide), so that precipitation of
the cationic detergent micellar complex-DNA takes place after a short mixing operation.
The solution now containing the micellar-DNA complex then undergoes filtration. The
ultrafiltration takes place with a filter (for instance, sintered borosilicate glass or an
organic matrix like polypropilene or polyethylene) of known and tested porosity (pore
size between 5 and 15 micron), which retains the DNA-cationic detergent micellar
complex in a satisfactory way. The hydrophilic surface enables DNA to be recovered
easily and speedily after the washing operations. The organic matrix filter allows a
slower recovery of DNA so it is not commonly used at the moment. As genomic DNA is
immobilized on the filter, it is then eluted.
Once the human DNA has been extracted, it is necessary to isolate the exact gene for
insulin. It is located in the top of the short arm of the eleventh chromosome. The DNA
sequence for the A chain is compromised of sixty three nucleotides and the sequence for
the B chain is compromised of ninety nucleotides. An extra codon must be placed at the
end of both sequences to signal the termination of protein synthesis. Also, an anticodon, consisting of the amino acid methionine, must be placed at the beginning of
each chain in order to make the removal of the insulin possible. In order to cut the
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genes for insulin production from the DNA you must mix the human DNA with the
enzymes HincII and BamHI.
Once the DNA has been cleaved and the insulin gene and been isolated, the mixture
must be run through an agarose gel electrophoresis in order to be able to remove the
insulin gene. First you must cast the agarose gel. To do this place the well-forming
comb into the gel-casting tray and pour enough agarose solution to fill the tray to 6mm.
After the gel has set (15-20 minutes), place it into the gel box, fill the box with enough
TBE buffer to cover the gel, and remove the comb.
After these steps have been completed, the DNA must be loaded into the gel by inserting
it into the wells with a pipet. After all of the DNA has been loaded, close the
electrophoresis chamber and connect the electrical leads to a power supply and turn it
on. Wait until the bands are almost to the end and then remove the gel and stain it.
Once the gel has been stained, elute the DNA fragments by shaking the gel in a solution
of 0.2 N NaCl, 1 mm EDTA, and 10 mm Trk. Now that the insulin gene has been
isolated and removed, it must be inserted into the plasmid of the vector cell, which is E.
coli. The genes for the A and B chains of insulin are inserted into the gene for the
bacterial enzyme, B-galactosidase. Once the genes have been inserted into the plasmid,
the plasmid is reinserted into the E. coli cell. When the cell replicates, the Bgalactosidase is formed with either the A or B chain of insulin attached to it. The two
chains are then extracted and purified. Then they are mixed together thus connecting to
each other by forming disulfide cross bridges. The end result is Humulin, or synthetic
human insulin.
PRODUCTION OF HEPATITIS-B VACCINE
Hepatitis B virus (HBV) is wide spread in man and produces several chronic liver
disorders such as Fulminant chronic hepatitis, cirrhosis and primary liver cancer. HBV
DNA is a double stranded circular molecule of about 3Kb size and has a large single
stranded gap which must be required with an endogenous polymerase before digestion
with restriction enzyme for DNA cloning (Glover, 1984). After infection in human being,
HBV fails to multiply and infect a large number of cells and even does not grow in
cultured cells. This property has been explained to be due to hindrance of its molecular
characterization and development of vaccines. Plasma of human has been detected to
have varying amount of antigens. Three types of viral proteins are recognized to be
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antigenic: (i) viral surface antigen (HBsAg), (ii) viral core antigen (HBcAg), and (iii) the eantigen (HBeAg).
Although the whole viral genome has been cloned and sequenced, yet there is limited
information about amino acid sequence of surface and core antigens. Recently, HBV
DNA has been successfully cloned in E. coli and mammalian cells, and synthesis of
HBsAg and HBcAg particles has been done in the cells. Burrell et al. (1979) inserted
HBcAg genes inPBR322 near b-glactosidase gene (Edman et al., 1981). Production of
these genes is needed in order to get production of vaccines on a large scale. In yeast or
mammalian systems, these antigens are synthesized more efficiently than in
prokaryotes.
Recombinant vaccine for Hepatitis B virus. After infection, HBV fails to grow and even
in cultured cells it does not grow. This property has been explained to be due to
inhibition of its molecular expression and development of vaccines. Recombinant
vaccine for HBV was produced by cloning HBsAg gene of the virus in yeast cells. The
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yeast system has its complex membrence and ability of secreting glycosylate protein.
This have made it possible to build an autonomously replicating plasmid containing
HBsAg gene near the yeast alcohol dehydrogenase (ADH) Ipromoter.
The HBsAg gene contains 6 bp long sequence preceding the AUG that synthesizes Nterminal methionine. This is joined to ADH promoter cloned in the yeast vector PMA-56.
The recombinant plasmid is inserted into yeast cells. The transformed yeast cells arc
multiplied in trytophan-free medium. The transformed cells are selected. The cloned
yeast cells are culture for expression of HBsAg gene. This inserted gene sequence
expresses and produces particles similar to the 22 mm particle of HBV as these
particles are produced in serum of HBV patients. The expressed HBsAg particles have
similarity in structure and immunogenicity with those isolated from HBV-infected cells
of patients. Its high immunogenicity has made it possible to market the recombinant
product as vaccine against HBV infection.
Indigenous Hepatitis-B vaccine
India's
first
genetically
engineered
vaccine (Guni) against
HBV
developed
by
Hyderabad based laboratory (Shantha Biotechnics Pvt. Ltd.) was launched on August
18, 1997. India is the fourth country (after the U.S.A., France and Beligum) to develop
this highly advanced vaccine. The indigenous yeast-desired HBV vaccine is one third
the cost of the imported vaccine. This new vaccine had undergone human clinical trials
at Nizam's Institute of Medical Sciences, Hyderabad and K.E.M. Hospital, Mumbai. The
clinical trials clearly proved that the seroprotection is about 98%. It was found more
effective than the imported vaccine. The Drug Controller General of India has permitted
it for commercial manufacture.
FERMENTED FOODS
PRODUCTION OF SAUSAGES
Fermented sausages can be divided into two groups:
Sliceable raw sausages (Salami, Summer Sausage, Pepperoni))
Spreadable raw sausages (Teewurst, Mettwurst)
Salamis can be divided into:
Fast-fermented
Medium-fast-fermented
Slow-fermented:
With mold
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Without mold (often smoked) or as:

Moist - 10% weight loss
Semi-dry - 20% weight loss
Dry - 30% weight loss
Meat selection
It is the first step to a successful production of a fermented sausage.
Going into details on selecting meats according to their pH or using terms like PSE,
GFD or MDM meats is beyond the scope of these pages and will make them confusing
to read. What we want to stress is that meat must be perfectly fresh with the lowest
count of bacteria possible. Commercial producers try to keep this number between
100 and 1000 per gram of meat but a home based sausage maker has to make sure
that:
Meat is very fresh and kept cold
Facilities and tools are very clean
Working temperatures are as low as possible
Typical values of meats selected for commercial production are: pork: pH-<5.9-6.0, beef:
pH-<5.8 and Aw- 0.98 - 0.99
Curing
The application of salt and nitrite is actually our first line of defense against the growth
of spoilage bacteria as in many cases (home production) there is very little we can do
about a selected meat's bacteria count except making sure that the meat is fresh. As
the sausage slowly dries out it loses moisture but not the original amount of salt which
remains inside. As a result, in time the sausage becomes much saltier to bacteria. In
about 3-6 days the Aw drops to about 0.95 and the sausage is microbiologically more
stable as some pathogenic bacteria (for example Salmonella) stop multiplying now.
pH
Foods
with
low
pH
value
(high
acidity)
develop
resistance
against
microbiological spoilage. Pickles, sauerkraut, eggs, pig feet, anything submerged in

vinegar will have a long shelf life. Even ordinary meat jelly (headcheese) will last longer
if some vinegar is added and this type of headcheese is known as "souse". Bacteria hate
acidic foods and this fact plays an important role in the production and stabilization of
fermented sausages.
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Sugar is mainly added to provide food for starter cultures. The pH drop in sausage
depends on the type and amount of sugar utilized. Introduction of more sugar generally
leads to lower pH and stronger acidification.
Water activity (Aw)
It is an indication of how tightly water is "bound" inside of a product. It does not say
how much water is there, but how much is available to support the growth of bacteria,
yeasts or molds (fungi). Adding salt or sugar "binds" some of this free water inside of the
product and lowers the amount of available water to bacteria which compete very poorly
with salt. Molds are very good competitors for free water. We could make Aw lower by
lowering the temperature of the product but that is not practical as the temperatures
for making fermented sausages are well defined. A much better solution is to lower
water activity by drying.
Smoking
It may or may not be utilized in a production of fermented sausages. It has been used in
countries in Northern Europe where due to colder climate and shorter seasons, the
drying conditions were less favorable than in Spain or Italy. Smoking imparts a different
flavor, has some effect of fighting bacteria, especially on the surface of the product and
thus prevents growth of molds on fermented sausages. Mold is desired on some
traditionally made Italian salamis and obviously smoking is not deployed. Smoking
temperature must correspond with temperatures of fermenting and drying and in
traditionally made sausages, cold smoke of less than 22 C (70 F) is applied after
fermentation stage. Cold smoking is after all "drying with smoke".
Manufacturing Technology
The first manufacturing steps such as meat selection, grinding, mixing and stuffing are
common to all sausages whether fresh, smoked or fermented types. After being stuffed
with meat the fermented sausages are submitted to different steps:
Conditioning (optional)
Fermenting
Drying
Storing
Conditioning is an optional step for a home sausage maker and he has to exercise his
own judgement. In commercial plants the process of grinding, mixing and stuffing
salami is undertaken at a low temperature (0 C, 32 F) and as the cold sausage is
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placed in a warmer (fermenting/drying) room, not needed condensation will appear on

the surface of the sausage. The sausage must remain there for 1-6 hours (depending on
its diameter) at low humidity (no air draft) until the moisture evaporates. Then we can
start the fermentation process. If the sausage casing is dry there is no need for
conditioning. It should also be very carefully monitored (or even eliminated) in small
diameter casings which can dry out too quickly on the surface. This will eliminate
moisture (food) for lactic bacteria and they will not reduce pH within the outer layers.
As a result the sliced sausage will have a different color in its outer layer (see effects of
too fast drying above).
Fermentation refers to the production of lactic acid and to produce consistent quality
product parameters such as temperature, humidity and air flow should be carefully
monitored. The humidity in a drying room is increased to about 92-95 % and the
temperature is increased to 18 -26 C, (66 -78 F). The temperature range depends
on the type of the sausage produced (fast, medium or slowfermented) and the type
of the starter culture used. The air flow is kept about 0.8 m/sec. Commercial plants
monitor Aw (water activity) of the sausage and readjust the correspondingly humidity
level of the drying chamber. There is normally a difference of less than 5% between
moisture level of the sausage and relative humidity of the room, the latter figure being
lower. This means if the Aw of the sausage is 0.95, the humidity is set at 90%. Then
when Aw drops to 0.90, the humidity drops to 85% and so on.
When the fermentation starts the main hurdles against microbiological spoilage of the
sausage are the low bacteria count of the meat, the presence of nitrite and salt. Keep in
mind that in time the sausage will be losing more and more moisture but the salt
remains inside and the percentage of salt in a finished sausage will be higher. In about
48 hours lactic bacteria metabolize enough sugar to produce a sufficient amount of
lactic acid to drop pH (increase acidity) of the sausage and this stabilizes the sausage
making it more resistant to spoilage.
Drying
It is a very important process especially in the initial stages of production. One may say
why not dry a sausage very fast which will remove moisture and be done with all this
pH stuff and bacteria. Well, there are basically two reasons:
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The outside layer of the sausage must not be hardened as it may prevent removal of the
remaining moisture. It may effect the curing of the outside layer which will become
visible when slicing the sausage (see the drawings below).
Naturally existing in meat, bacteria and/or introduced starter cultures need moisture
and some time before they can metabolize sugar and produce lactic acid which lowers
pH.
PRODUCTION OF OLIVES
Olive oil production is divided into three fields:
Oil mills, which process the olives into oil and oil cake Refineries, where the nonconsumable oil is refined, Plants where the oil cake is processed and residual oil is
extracted from the waste resulting from olive oil extraction.
To get the highest possible yield of valuable virgin oil, the olives have to be processed at
low temperature (< 40 C). For olive oil extraction three processes are applied:
Pressing, traditional system.
Continuous process with three-phase decanters.
Continuous process with two-phase decanters. It generates alpeorujo, the waste treated
in the project IMPROLIVE.
Smaller plants work in batch operation. Here the washed olives are grinded together
with the stones, the mixed pulp is then pressed several times by means of hydraulic
open cage presses. The off-running oil is collected and clarified by settling in containers.
The preliminary separation of the stones from the pulp can be advantageous. With this
production variant the stones are processed separately into oil. In larger plants,
however, high-mechanised methods are largely used, which allow a quasi-continuous
material flow. After grinding the pulp is led into malaxeurs. With the help of this mixing
and kneading device the coagulation process and, with this, the development of larger
oil droplets is favoured.
Oil extraction can be divided into seven steps:
Delivery
The olives are delivered and stored in the yard in collection boxes. This offers the
possibility to determine the quality and rate of yield, and serves as basis for the
settlement of accounts between the oil mill and olive farmer. The production plant is fed
in charges. The olives are filled in baskets, transported by donkey carts to the soil
funnel, and from there by a belt conveyor to the first processing stage.
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Washing of the olives

The olives are filled in charges into a soil funnel and transported by a belt conveyor into
a sucking device, where leaves, wood particles and other disturbing solids are removed.
Subsequently they are cleaned in a washing plant. In some plants the washing water is
recycled into the process after sedimentation of the solids or filtration. In other plants
the olives are directly processed without being washed. For washing of the olives 100 120 l of water per 1000 kg of olives are required. With the help of a perforated vibrating
screen they are transported into a funnel. By the funnel tip they get into the loading
opening of an eccentric worm pump which conveys them into a grinding device.
Grinding
The olives are ground up, together with their stones, and mixed into a homogeneous
pulp. This is carried out in a stone-mill consisting of a horizontally lying granite block
with a granite millstone resting perpendicular to it. Metal mills and hammer mills are
also used for this purpose. If frozen or very dry olives are processed, a small quantity of
water is added (100 - 150 l per 1000 kg of olives.
Mixing/beating of the olive pulp
In downstream-arranged malaxeurs the pulp is mixed after adding of warm water. For
further breakdown of the olive cells and to create large oil droplets, the pulp is beaten.
For this purpose salt is often added which aids the osmotic breakdown of cells in the
olives and so the separation of the oil and water from each other is eased. Beating of the
olives is repeated several times. For oil extraction by centrifuges the pulp is heated to
improve the separation process. After beating the pulp is further ground up. In a
malaxeur up to 100 % water are added before conveying the pulp by an eccentric worm
pump into the two-phase- or three-phase decanter.
Oil extraction
In small oil mills the olive oil is extracted in batch operation using the traditional press
method. The oil extracted is collected in containers and clarified by sedimentation.
About 200 kg of oil result on an average from one ton of processed olives. To improve
the separation of oil and pulp, biological or chemical aids can be added that attack the
cell walls. With traditional presses the energy demand for olive oil extraction processes
is 40 - 63 kWh per ton of processed olives, and 90 - 117 kWh with three-phase
decanters.
Purification of the oil
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By means of a vibrating screen fine solids that might still be contained are removed
from the oil. To enable the separation of small waste water quantities and suspended
solids that accompany the oil, 100 - 150 l of water per 1000 kg of centrifuged oil are
normally added to the purification process, which is achieved with the help of
centrifugation.
Extraction of residual oil
The high-loaded waste water from the three-phase decanter is treated by means of a
vibrating screen and a centrifuge, the residual oil is extracted. The oil centrifuged twice
flows into a collecting tank and is pumped by eccentric worm pumps in surface or
underground storage tanks. The solid waste from oil extraction by pressing still
contains about 6 % oil, using the continuous three-phase decanter, still 4 % olive oil.
The oil content in the solid-liquid mixture from the dual-phase decanting process is
2.5% to 3.5 %. In specialised plants the solvent extraction process is used for this
purpose. First the waste is completely dried and then extracted using hexane as
solvent. The dry residues can be used as concentrated fodder. In some extraction plants
the stones are separated from the pulp after extraction and used as fuel for heating of
the driers. The pulp is sold as fertiliser or fodder. In some oil mills the solid waste from
the press is directly used as fuel for the heating of water.
PRODUCTION OF BREAD
The 14 steps of bread baking is the best way to improve your bread-baking. They are
simple fundamentals that should be applied to every recipe and will turn your bread
from average to extraordinary.
Step 1 Select Ingredients
Selecting the right ingredients is the most overlooked step in bread baking, especially
by beginners. Ingredients change flavor, texture and other attributes of the dough.
Many types of bread only have 4 ingredients flour, salt, water and yeast but are
combined with different techniques to create hundreds of variations.
What type of flour, how large the salt is, water vs milk vs other liquid, what type of
yeast are all things that need to be considered before baking.
Step 2 Weigh Ingredients
Weighing ingredients, instead of volume measurement, is the best way to prepare for
baking. Weighing helps keep results consistent and gives a benchmark for comparing
results.
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Step 3 Mix
Mixing is the most technical aspect of bread baking. There are different mixing
methods that produce different results. Mixing methods affect crumb size, open or
closed, color of the crust and crumb and how long the dough needs to ferment.
Hand Mixing
Autolyse is sciences happy gift to bread-baking. Simply mix the dough without salt,
in most recipes it is simply flour and water. Let the dough sit at room temperature,
covered, for between 15-30 minutes. During this time the flour hydrates and gliadin
and glutenin begin to form gluten.
After the autolyse period the dough will have developed a lot of strength and most of the
gluten needed for baking. It is a small amount of effort for a great result. it is also what
makes no-knead bread possible and provides several benefits to the crumb.
Slap and Fold This technique was supposedly developed by Richard Bertinet. It isnt
effective on lower hydration dough, but it works very well for dough with around 70
percent or more hydration. I find it is one of the only ways to knead 80 percent or
higher dough by hand.
Hand Mixing - This is what most people think of when making dough. Mix your
ingredients together until they resemble a shaggy mess and turn it onto a table. Fold
the outside edge (the one that is furthest away from you) towards you with your
fingertips and use your palms to press that edge into the center of the dough. Rotate
the dough 45 degrees and repeat. Continue this motion until the flour is completely
hydrated and the gluten is fully developed.
Machine Mixing
Short Mix - This method is exactly what it sounds like. Mix the dough ingredients on
low-speed for 5 minutes. Friction from mixing by machine heats up the dough, which
shortens the fermentation period. Using the short mix doesnt heat up the dough as
much. The fermentation period is longer letting the gluten develop naturally as the
dough sits. Dough mixed by this method usually requires a few folds during
fermentation to develop the right gluten strength for baking. This is a great method for
creating open crumb artisan breads while still using a machine to do most of the work.
Intensive Mix - This method requires 5 minutes of mixing on low-speed and then 10 or
so minutes on high-speed. The idea is to develop the gluten very quickly to produce
massive amounts of bread; I.E. Wonderbread. There are two big problems with this
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method for most home-bakers. Mixing at high speeds with most counter-top mixers
tends to burn out the motors, especially on lower hydration dough. The second problem
is the oxidation that occurs. The extra air that is literally whipped into the dough
destroys flavors and aromas that would have been present with other mixing types. This
is why 90 percent of all dough on our store shelves takes like nothing. There are ways
to improve flavors using preferments, but most industrial bakers dont have the space
or time to develop preferments properly.
Improved Mix - Because the intensive mix created such a poor product that ended up
on store shelves, bakers started to figure out a way to decrease mixing times without
losing too many flavors. This led to the improved mixing method. For the improved
method, mix for 5 minutes on low-speed. Follow this with a 3-5 minute rest and then
mix on medium for 2 minutes. If the gluten doesnt completely develop at this point fold
the dough during fermentation.
Step 4 Bulk Fermentation
After
mixing,
the
dough
starts
its
first
fermentation
period,
called
the bulk
fermentation. This is where most of the flavor in your bread will come from. The dough
sits, covered, usually until it doubles in size. At this point yeast and enzymes are
developing flavors and aromas. The gluten in the dough also becomes stronger, as
mentioned in step 3.
The most important aspects of bulk fermentation are time and temperature.
That is where professionals make their bread and butter; pun intended. Recipes are
designed around time and temperature regardless if they say it. According to Peter
Reinhart Yeast will double its rate of fermentation for every 17F increase in heat, up to
the killing point. What this means is that if a recipe calls for the dough to double in
two hours at 72F, but your apartment is at 89F, than it will ferment and double twice
as fast than the recipe states 1 hour instead of 2. Flavor comes from long regimented
bulk ferments. If you speed things along, you lose flavor and texture. If you slow things
down you can let enzymes do too much work on the dough and lose flavor and texture.
Step 5 Fold
Folding is a way of strengthening the dough, redistributing food for the yeast and
evening the temperature throughout the day. It is one of the easiest ways to take your
bread to the next level and is always overlooked because of the silly name punching
that most recipes use.
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Folding may be done inside or outside the bulk fermentation container. Lightly wet your
hands and grab one edge of your dough. Fold it towards the center and gently press the
fold into the body of the dough. Grab the opposite side and repeat. Repeat again for the
top and the bottom.
Step 6 -Divide
After the bulk fermentation we must divide the dough into the right size for baking. If
you are making two loaves, simply divide the dough evenly and continue on. For three
or more, use a scale to accurately way the dough to help the loaves cook evenly and
uniformly. Its not necessary, but it helps to divide the dough in even shapes. It will
help during pre-shaping and with final shaping.
Step 7 Pre-Shape
The pre-shape is the beginning of the hardest technical parts in bread baking.
Step 8 Bench Rest
After pre-shaping, the dough must go through an intermediate resting period. This
lets the gluten in the dough relax so it can be properly shaped for baking. It also adds
slightly more flavor to the dough as it is continually fermenting during this entire
process.
Step 9 Final Shape
If your pre-shaping is successful, final shaping wont be difficult. The main objective in
the final shape is to develop a tight skin on the outside of the loaf. This helps control
the rise of the dough while baking and makes it easier to score loaves, if need be.
Step 10 Proof
After shaping and working the dough, most of the CO2 from the yeast has been
expelled from the dough. Proofing lets the dough come back to life, develop more flavors
and gain the texture we recognize in professional bread. Some recipes call for a 10
minute proof, others, like bagels, may take all night. If the air is too try a skin develops
on the outside of the loaf that prevents the dough from expanding in the oven. I usually
proof my dough on a floured linen towel resting on a wooden board. I cover the board
with a plastic bag to keep the moisture inside. It is very effective in preventing the skin.
Step 11 Score
Scoring does more than just look nice, although it really does look nice. It creates weak
spots in the gluten structure which encourage the dough to spring in certain directions
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when in the oven. Oven spring is the term used to described how the dough jumps in
volume when put into a warm environment.
Step 12 Bake
Baking bread has two general temperature ranges. 300F-400F and 425F-500F plus.
Enriched dough or dough with with sugars, eggs, milks etc are usually baked in the
lower range. Straight dough, or dough with only flour, water, salt and yeast, bake at the
higher temperature in order to gain a deep brown crust and increase oven spring. If a
recipe with at least 50g of sugar calls for being baked at 450F or higher, I recommend
you pay attention to the crust to see how dark it gets. A lot of times the crust will
darken before the interior is cooked.
Another rule of them is that the interior temperature of the bread should end up
between 200-210F when the bread has finished cooking. Many resources describe how
only a few degree differences at the end of baking can change texture, flavor and shelflife.
Steam is a very important part of bread-baking. Professional ovens inject steam into the
baking deck. The steam acts similarly to what happens when you baste a turkey. The
extra moisture causes something called evaporative cooling. Evaporative cooling slows
down the browning and stops the crust from forming too quickly. This is how hearth
breads from straight dough rise and expand so drastically while baking.
Its very difficult to replicate the steam injection from professional ovens, but a few
simple techniques can still give great results. The goal is to create steam for the first 1015 minutes of baking. If you have a cast iron or heavy duty pan, place it on the bottom
shelf of the oven as it preheats. When you place the bread in the oven pour about 1 cup
of water into the pan. It should instantly spatter and boil and begin to release steam.
Close the door and wait 1 minute before checking to see if the pan has gone dry. Try to
keep water in the pan until the ten minute mark. At that point you can either remove
the pan or, if the pan is dry, open the door and let any remaining moisture dissipate.
The bread should rise drastically and then begin developing a beautiful brown crispy
crust.
Step 13 Cool
The easiest step in the 14 steps of bread baking to accomplish, but the hardest one to
obey. When bread comes out of the oven it isnt close to being done. The center of the
bread is most likely very gummy and moist and will continue to cook as the steam on
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the inside of the loaf cools. Breads need to fully cool before being cut into. If you would
like to serve warm bread, bake the bread and cool it properly, then reheat the bread to
serve.
Step 14 Store
Storage is another important part of enjoying bread properly. There are three great ways
to store bread, plastic, paper bags and a bread-box. Enriched breads should be stored
in plastic. It helps retain moisture and keeps breads soft. Paper bags work best for
artisan breads, straight dough breads that are meant to be enjoyed with a crisp crust.
PRODUCTION OF IDLY
Idli, a very popular fermented breakfast food consumed in the Indian subcontinent,
consists mainly of rice and black gram. Idli fermentation was carried out in the
conventional way in a batter having rice to black gram in the ratios of 2:1, 3:1 and 4:1
at room temperature. It makes an important contribution to the diet as a source of
protein, calories and vitamins, especially B-complex vitamins, compared to the raw
unfermented ingredients. It can be produced locally and used as a dietary supplement
in developing countries to treat people suffering from protein calorie malnutrition and
kwashiorkor.
Adding Saccharomyces cerevisiae, along with natural bacterial flora of the ingredients,
was the best method for standardizing idli fermentation in terms of improved
organoleptic characteristics, leavening and nutritional constituents. Traditional idli
fermentation involves several bacteria and yeasts, contributed by the ingredients rice
(Oryza sativa), black gram (Phaseolus mungo) and the environment, with overall idli
batter was prepared from soaking polished parboiled rice and decorticated black gram.
The blend a ratio of 2:1, 3:1 and 4:1 (v/v) and the batter was allowed for fermentation
(0, 6, 12, 18 and 24 h) adding two percent of salt. Other legumes such as soybeans and
Great Northern beans could be substituted for black gram in preparation of idli.
Fermentation time of the batter varies from 14 to 24 h with overnight fermentation
being the most frequent time interval. The ingredients for idli are carefully washed,
soaked in water separately, grounded, mixed, and finally allowed to ferment overnight.
When the batter has been raised sufficiently, it is cooked by steaming and served hot.
The product has a very soft and spongy texture and a desirably sour flavour and taste.
The black gram was washed several times, first with tap water and finally with distilled
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water to remove the surface microorganisms. These were found to produce off flavour in
the idli unless they were washed out.
The microorganisms responsible for the characteristic changes in the batter were
isolated and identified. Although there is a sequential change in the bacterial flora, the
predominant microorganism responsible for souring, as well as for gas production, was
found to be Leuconostoc mesenteroides. In the later stages of fermentation, growth of
Streptococcus faecalis and, followed by Pediococcus cerevisiae became significant. The
fermentation of idli demonstrates a leavening action caused by the activity of the hetero
fermentative lactic acid bacterium, L. mesenteroides. As far as is known, this is the first
record of a leavening action produced exclusively by the activity of a lactic acid
bacterium.
Idli is traditional fermented rice and black gram based breakfast food of South India.
Idli batter was prepared from soaking polished parboiled rice and decorticated black
gram for 4 h at 30 1oC in water. The soaked mass was ground to 0.5 to 0.7 mm
particle size batter using wet grinder with adequate amount of water. The idli batter
parameters such as bulk density, pH, total acidity, flow behaviour index and
consistency coefficients were studied for different fermentation times and blend ratios.
The bulk density, pH and percentage total acidity of batter during different fermentation
times and blend ratios ranged between 0.94 and 0.59 g/cm3, 5.9 and 4.1 and 0.443
and 0.910%, respectively. The consistency coefficient at any fermentation time shows
increasing trend as the rice to black gram ratio increased.
The
flow
behaviour
index
indicated
strong
non-Newtonian
fluid
behaviour
(pseudoplastic) of idli batter at different fermentation times and blend ratios.

The rheology of the idli batter was assessed using a Brookfield viscometer having disc
spindles. Power law model with yield stress adequately fitted the data. Yield stress
values were in the range of 13-43 Pa and reached a maximum value at 7 h of
fermentation. Flow behaviour indices were in the range of 0.287-0.605. Flow behaviour
indices at 23 h were significantly lower than those at 0 h. Consistency index values, at
any fermentation time, increased as the rice to black gram ratio increased. Mean
particle size ranged from 500 to 600 micro meters and there was no definite trend
noticed with respect to time of fermentation and rice to black gram ratio. There was a
steep change in volume increase after 4-h fermentation.
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The idli batter comprises lactic acid bacteria and yeasts and causes an improvement in
the nutritional, textural and flavour characteristics of the final product. The desirable
flavor compounds such as ketones, diols and acids were found to be present up to eight
days of storage, whereas undesirable flavours like sulphurous and oxazolidone
compounds, ethanone and thiazole appeared in the batter subsequent to six days of
storage. The sensory attributes of idli (final product) prepared from the stored batter
related well to the determined flavour profile.
The work done by Nisha et al., (2005) stabilized the idli batter at room temperature (2830C) and refrigerated storage (4-8C) by using various hydrocolloids and some
surfaceactive agents. The batter was evaluated in terms of decrease in volume, and
whey separation. While hydrocolloids gave good stabilization, surface-active agents
failed to stabilize the batter and they reduced whey separation. Among the various
hydrocolloids, 0.1percent guar gave best batter stabilization, and idli made after ten
days of room temperature and 30 days of refrigerated storage of batter were found to be
of acceptable quality.
Reduction in the fermentation time of the idli batter is of great commercial significance
for large-scale idli production and can be potentially achieved by addition of enzymes.
The idli batter fermentation process by adding an exogenous source of -amylase
enzyme. 5, 15 and 25 U per 100 g batter of amylase added to the idli batter was allowed
to ferment. Different parameters were monitored and sensory attributes were also
studied and compared with that of the control set. The fermentation time was reduced
from a conventional 14 h to 8 h and the sensory attributes of the final product were
also successfully maintained.
PRODUCTION OF ACIDOPHILUS MILK.
The use of acidophilus milk as a treatment for scours in calves originated at the Oregon
Experiment Station in 1935. The results which followed experimental trial appeared
better than those obtained by any other known treatment. Since that time this
treatment has been quite generally used and in most instances reports on its use
indicate the saving of the lives of a large number of calves.
The production of acidophilus milk requires special equipment and training in
bacteriology.
Fresh skimmed cow's milk is sterilized by heating in a steam sterilizer at 15 pounds for
30 minutes. It is then cooled and inoculated with cultures of Lactobacillus acidophilus.
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After inoculation it is incubated at 100F. until a curd has formed and the lactic acid
content has reached about 1 per cent. At this time, the milk will contain in the
neighborhood of 400,000,000 organisms per cubic centimeter. The number of
organisms starts diminishing a few days after preparation, but remains high for about
two weeks if the milk is not subjected to extremes of heat or cold. It should be kept at
room temperature (about 70F.) and should never be kept in a refrigerator.
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UNIT 7 INTELLECTUAL PROPERTY RIGHTS (IPRS) AND ENTREPRENEURSHIP:

IPRs- implications for India, WTO, WIPO, GATT, TRIPS. Patenting and the
procedures involved in the application for patents and granting biotechnology,
legal implications, traditional knowledge commercial exploitation, protection.
Entrepreneurship
Potential
entrepreneurship
activities
in
biotechnology,
product development, marketing, research and training units. Industrial licensing,

venture
capital,
Biotechology
Industries
in
India
and
the
potential
job
opportunites.
IPRS - IMPLICATIONS FOR INDIA, WTO, WIPO, GATT, TRIPS.
Intellectual property (IP) rights are the rights awarded by society to individuals or
organizations principally over creative works: inventions, literary and artistic works,
and symbols, names, images, and designs used in commerce. They give the creator the
right to prevent others from making unauthorised use of their property for a limited
period. IP is categorised as Industrial Property (functional commercial innovations), and
Artistic and Literary Property (cultural creations). Current technological developments
are blurring, to some extent, this distinction, and some hybrid sui generis systems are
emerging.
Industrial Property
Patents: A patent is an exclusive right awarded to an inventor to prevent others from
making, selling, distributing, importing or using their invention, without license or
authorisation, for a fixed period of time (TRIPS stipulates 20 years minimum from filing
date). In return, society requires that the patent applicant disclose the invention in a
manner that enables others to put it into practice. This increases the body of knowledge
available for further research. As well as sufficient disclosure of the invention, there are
three requirements (although details differ from country to country) that determine the
patentability of an invention: novelty (new characteristics which are not prior art),
non-obviousness (an inventive step not obvious to one skilled in the field), and utility
(as used in the US) or industrial applicability (as used in the UK).
Utility models are similar to patents, but in some countries confer rights of shorter
duration to certain kinds of small or incremental innovations.
Industrial Designs: Industrial designs protect the aesthetic aspects (shape, texture,
pattern, and colour) of an object, rather than the technical features. TRIPS requires that
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an original design be eligible for protection from unauthorised use by others for a
minimum of 10 years.
Trademarks: Trademarks provide exclusive rights to use distinctive signs, such as
symbols, colours, letters, shapes or names to identify the producer of a product, and
protect its associated reputation. In order to be eligible for protection a mark must be
distinctive of the proprietor so as to identify the proprietors goods or services. The main
purpose of a trademark is to prevent customers from being misled or deceived. The
period of protection varies, but a trademark can be renewed indefinitely. In addition
many countries provide protection against unfair competition, sometimes by way of
preventing misrepresentations as to trade origin regardless of registration of the
trademark.
Geographical
Indications:
Geographical
Indications
(GIs)
identify
the
specific
geographical origin of a product, and the associated qualities, reputation or other

characteristics. They usually consist of the name of the place of origin. For example,
food products sometimes have qualities that derive from their place of production and
local environmental factors. The geographical indication prevents unauthorized parties
from using a protected GI for products not from that region or from misleading the
public as to the true origin of the product.
Trade Secrets: Trade secrets consist of commercially valuable information about
production methods, business plans, clientele, etc. They are protected as long as they
remain secret by laws which prevent acquisition by commercially unfair means and
unauthorised disclosure.
Artistic and Literary Property
Copyright: Copyright grants exclusive rights to the creators of original literary,
scientific and artistic works. Copyright only prevents copying, not independent
derivation. Copyright protection begins, without formalities, with the creation of the
work, and lasts (as a general rule) for the life of the creator plus 50 years (70 years in
the US and EU). It prevents unauthorized reproduction, public performance, recording,
broadcasting, translation, or adaptation, and allows the collection of royalties for
authorised use. Computer programs are protected by copyrights, as software source
and code have been defined as a literary expression.
Sui Generis systems
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Integrated Computer Circuits: A specific sui generis form of protection for design of
integrated computer circuits. As the inventive step is often minimal and originality is
the only requirement, the minimum period of protection under TRIPS is 10 years.
Plant Breeders Rights: Plant breeders rights (PBRs) are granted to breeders of new,
distinct, uniform and stable plant varieties. They normally offer protection for at least
fifteen years (counted from granting). Most countries have exceptions for farmers to
save and replant seeds, and for the use of protected materials for further breeding.
Database Protection: The EU has adopted legislation to provide sui generis protection
in respect of databases, preventing unauthorised use of data compilations, even if nonoriginal. Exclusive rights to extract or utilize all or a substantial part of the contents of
the protected database are granted.
International Organizations & Treaties
A UN agency, namely, World Intellectual Property Organization (WIPO) based in Geneva
administers treaties in the field of intellectual property. India is a member of WIPO.
Department of Industrial Policy & Promotion is the nodal Department in the
Government of India for all matters concerning WIPO.
India is also a member of the World Trade Organization (WTO).
The WTO agreement on Trade Related Aspects of Intellectual Property (TRIPS).
This Agreement made protection of intellectual property an enforceable obligation of the
Member States.
TRIPS Agreement sets out minimum standards of intellectual property protection for
Member States.
The Patents (Amendment) Act, 1999 passed by the Indian Parliament on March 10,
1999 to amend the Patents Act of 1970 that provides for establishment of a mail box
system to file patents and accords exclusive marketing rights for 5 years.
The Trade Marks Bill, 1999 which repeals and replaces the Trade and Merchandise
Marks Act, 1958 passed by the Indian Parliament in the Winter Session that concluded
on December 23, 1999.
The Copyright (Amendment) Act, 1999 passed by both houses of the Indian Parliament,
and signed by the President of India on December 30, 1999.
Geographical Indications of Goods (Registration & Protection) Bill, 1999 approved by
both houses of the Indian Parliament on December 23, 1999.
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The Industrial Designs Bill, 1999 which replaces the Designs Act, 1911 on December
23, 1999 and is presently before the Lower House for its consideration.
The Patents (Second Amendment) Bill, 1999 to further amend the Patents Act, 1970
and make it TRIPS compliant was introduced in the Upper House of Indian Parliament
on December 20, 1999.
Projects relating to the modernization of patent information services and trademarks
registry have been implemented with help from WIPO/UNDP.
The Patents Law provides for compulsory license to avoid misuse of an Exclusive
Marketing Right by the right holder.
In terms of the TRIPS Agreement, India has time till January 1, 2005 to extend patent
protection to this area. Intellectual Property Appellate Board (IPAB)
An Intellectual Property Appellate Board (IPAB) has been set up at Chennai to hear
appeals against the decisions of Registrar of Trademarks, Geographical Indications and
the Controller of Patents.
Both foreign and domestic IPR holders are treated equally under Indian law.
The Government also brought out A Handbook of Copyright Law to create awareness
about copyright amongst the stakeholders, enforcement agencies, professional users
like the scientific and academic communities and members of the public.
At present there are three registered copyright societies. These are The Society for Copyright Regulations of Indian Producers of Films & Television
(SCRIPT) for cinematography films,
Indian Performing Rights Society Limited (IPRS) for musical works and
Phonographic Performance Limited (PPL) for sound recordings.
These societies, particularly the PPL and the IPRS, have been quite active in anti-piracy
work.
The PPL has even set up a special anti-piracy cell under a retired Director General of
Police, and this cell has been working in tandem with the police.
WIPO Guide to Intellectual Property Worldwide
This Guide is the first of its kind published by World Intellectual Property Organization
(WIPO).
It gives essential information on intellectual property by means of individual country
profiles on WIPO Member States.
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The
profiles
include
Basic
2016 EDITION
legislation,
membership
of
international
treaties,
administrative structures, governmental and non-governmental bodies for information

and enforcement, educational institutions and industrial property statistics; useful
contact addresses are provided for readers needing further information.
It is intended as a tool for all kinds of readers, not only for officials working in this field,
but for legal practitioners, teachers, students, researchers, creators or owners of
intellectual property, as well as for interested members of the general public.
Certain Internet links to some country offices are provided for information purposes
only.
The
World
Intellectual
Property
Organization
(WIPO)
is
an
international
intergovernmental organization dedicated to ensuring that the rights of creators and

owners of intellectual property are protected worldwide and that inventors and authors
are thus recognized and rewarded for their ingenuity.
This international protection acts as a spur to human creativity, pushing forward the
boundaries of science and technology and enriching the world of literature and the arts.
By providing a stable environment for the marketing of intellectual property products,
such protection also facilitates international trade.
The information in this Guide is drawn from data supplied by the governmental
administrations of the countries concerned, as well as, in some cases, from WIPO
sources, supplemented in parts by information from the World Trade Organization
(WTO).
The titles of the laws and of the government administrations responsible for intellectual
property in English, French and Spanish speaking countries are given, whenever
available, in their original language.
Research and private study Fair dealing with a literary, dramatic, musical or artistic
work for the purposes of research for a non-commercial purpose
Does not infringe any copyright in the work provided that it is accompanied by a
sufficient acknowledgement.
Copying for purpose of research or private study
Copying for purpose of criticism or review.
Copying for non-profit educational purposes
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PATENTING AND THE PROCEDURES INVOLVED IN THE APPLICATION FOR

PATENTS AND GRANTING BIOTECHNOLOGY
Types of patent applications
1.
Ordinary Application
2.
Application for Patent of Addition (granted for Improvement or Modification of the

already patented invention, for an unexpired term of the main patent).
3.
Divisional Application (in case of plurality of inventions disclosed in the main

application).
4.
Convention application, claiming priority date on the basis of filing in Convention

Countries.
5.
National Phase Application under PCT.

Who may apply?
The inventor may make an application, either alone or jointly with another, or his/their
assignee or legal representative of any deceased inventor or his assignee.
General precautions for applicant
The first to file system is employed, in which, among persons having filed the same
invention, first one is granted a patent, therefore,a patent application should be filed
promptly after conceiving the invention. It is common experience that through
ignorance of patent law, inventors act unknowingly and jeopardize the chance of
obtaining patents for their inventions. The most common of these indiscretions is to
publish their inventions in newspapers or scientific and technical journals, before
applying for patents.
Publication of an invention, even by the inventor himself, would (except under certain
rare circumstances) constitute a bar for the subsequent patenting of it. Similarly, the
use of the invention in Public, or the commercial use of the invention, prior to the date
of filing patent application would be a fatal objection to the grant of a patent for such
invention, thereafter. There is, however, no objection to the secret working of the
invention by way of reasonable trial or experiment, or to the disclosure of the invention
to others, confidentially.
Another mistake, which is frequently made by the inventors, is to wait until their
inventions are fully developed for commercial working, before applying for patents. It is,
therefore, advisable to apply for a patent as soon as the inventor's idea of the nature of
the invention has taken a definite shape.
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It is permissible to file an application for a patent accompanied by a "Provisional

Specification" describing the invention. The application may, therefore, be made even
before the full details of working of the invention are developed. The filing of an
application for a patent disclosing the invention would secure priority date of the
invention, and thereby, enable the inventor to work out the practical details of the
invention and to file complete specification within 12 months from the date of filing of
provisional specification.
What is patentable invention?
A new product or process, involving an inventive step and capable of being made or
used in an industry. It means the invention to be patentable should be technical in
nature and should meet the following criteria
Before the date of filing of the patent application in India.
Inventive Step: The invention is not obvious to a person skilled in the art in the light of
the prior publication/knowledge/ document.
Industrially applicable: Invention should possess utility, so that it can be made or used
in an industry.
What is not patentable?
The following are Non-Patentable inventions within the meaning of the Act: -
An invention which is frivolous or which claims anything obviously contrary to well

established natural laws;
An invention the primary or intended use or commercial exploitation of which could be

contrary to public order or morality or which causes serious prejudice to human,
animal or plant life or health or to the environment;
The mere discovery of a scientific principle or the formulation of an abstract theory (or
discovery of any living thing or non-living substances occurring in nature);
The mere discovery of a new form of a known substance which does not result in the
enhancement of the known efficacy of that substance or the mere discovery of any new
property or mere new use for a known substance or of the mere use of a known process,
machine or apparatus unless such known process results in a new product or employs
at least one new reactant ;
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Metabolites, pure form, particle size, isomers, mixtures of isomers, complexes, combinations
and other derivatives of known substance shall be considered to be the same substance,
unless they differ significantly in properties with regard to efficacy.
A substance obtained by a mere admixture resulting only in the aggregation of the

properties of the components thereof or a process for producing such substance;
The mere arrangement or re-arrangement or duplication of known devices each

functioning independently of one another in a known way;
A method of agriculture or horticulture;
Any process for the medicinal, surgical, curative, prophylactic,diagnostic, therapeutic or

other treatment of human beings or any process for a similar treatment of animals to
render them free of disease or to increase their economic value or that of their products.
Plants and animals in whole or any part thereof other than micro-organisms but
including seeds, varieties and species and essentially biological processes for
production or propagation of plants and animals;
A mathematical or business method or a computer programme per se or algorithms;
A literary, dramatic, musical or artistic work or any other aesthetic creation whatsoever
including cinematographic works and television productions;
Amere scheme or rule or method of performing mental act or method of playing game;
Apresentation of information; (n) topography of integrated circuits;
An invention which in effect, is traditional knowledge or which is an aggregation or

duplication of known properties of traditionally known component or components.
Inventions relating to atomic energy and the inventions prejudicial to the interest of
security of India.
Appropriate office for filing an application & for other proceedings
Application is required to be filed according to the territorial limits where the applicant
or the first mentioned applicant in case of joint applicants, for a patent normally resides
or has domicile or has a place of business or the place from where the invention
actually originated. If the applicant for the patent or party in a proceeding having no
business place or domicile in India, the appropriate office will be according to the
address for service in India given by the applicant or party in a proceeding . The
appropriate office once decided in respect of any proceedings under the Act shall not
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ordinarily be changed. The four patent offices are located at Kolkatta, Mumbai, Delhi &
Chennai (Annexure 1).
Publication & examination of patent applications
Publication:
All the applications for patent, except the applications prejudicial to the defence of India
or abandoned due to non-filing of complete specification within12 months after filing
the provisional or withdrawn within 15 months of filing the application, are published
in the Patent Office Journal just after 18 months from the date of filing of the
application or the date of priority whichever is earlier. The publication includes the
particulars of the date of the application, application number, name and address of the
applicant along with the abstract. The applications for patent are not open for public
inspection before publication. After the date of publication of the application, as stated
above, the complete specification along with provisional and drawing, if any, abstract,
application on any form or on plain paper and any correspondence between the office
and applicant may be inspected at the appropriate office by making a written request to
the Controller in the prescribed manner and on the payment of prescribed fee..
Early Request for Publication:
The applicant may also file a request for early publication in Form-9 with a prescribed
fee of Rs 2500/- or Rs 10,000/- for natural person and other than natural person
respectively.The above application is published ordinarily within one month from the
date of the request on Form-9. The applicant shall have provisional Rights from the date
of publication.
Request for examination
No application for patent will be examined if no request is made by the applicant or by
any other interested person in Form-18 with prescribed fee of Rs.2,500/- or
Rs.10,000/- for natural person and other than natural person respectively, within a
period of 48 months from the date of priority of the application or from the date of filing
of the application ,whichever is earlier. Where no request for examination of the
application for patent has been filed within the prescribed period, the aforesaid
application will be treated as withdrawn and, thereafter, application cannot be revived.
Examination
Application for patent, where request has been made by the applicant or by any other
interested person, will be taken up for examination, according to the serial number of
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the requests received on Form 18. A First Examination Report (FER) stating the
objections/requirements is communicated to the applicant or his agent according to the
address for service ordinarily within six (06) months from the date of request for
examination or date of publication whichever is later. Application or complete
specification should be amended in order to meet the objections/requirements within a
period of 12 months from the date of First Examination Report (FER). No further
extension of time is available in this regard. If all the objections are not complied with
within the period of 12 months, the application shall be deemed to have been
abandoned. When all the requirements are met the patent is granted, after 6 months
from the date of publication, the letter patent is issued, entry is made in the register of
patents and it is notified in the Patent Office, Journal ..
Withdrawal of patent application
The application for patent can be withdrawn at least 3(Three) months before the first
publication which will be 18(Eighteen) months from the date of filing or date of priority
whichever is earlier.
The application can also be withdrawn at any time before the grant of the patent.
The application withdrawn after the date of publication cannot be refiled as it is already
laid open for public inspection. However, application withdrawn before the publication
can be refiled provided it is not opened to public otherwise.
Opposition proceedings to grant of patents
Pregrant opposition
Where an application for a patent has been published but a patent has not been
granted, any person may, in writing represent by way of opposition to the Controller
against the grant of any Patent. The representation shall be filed at the appropriate
office and shall include a statement and evidence, if any, in support of the
representation and a request for hearing if so desired.
The above representation may be made on the following grounds
That the applicant for the patent or the person under or through whom he claims,
wrongfully obtained the invention or any part thereof from him or from a person under
or through whom he claims;
That the invention so far as claimed in any claim of the complete specification has been
published before the priority date of the claim
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(i) In any specification filed in pursuance of an application for a patent made in India on
or after the 1st day of January, 1912; or (ii) in India or elsewhere, in any other
document: Provided that the ground specified in sub-clause (ii) shall not be available
where such publication does not constitute an anticipation of the invention by virtue of
sub-section (2) or sub-section (3) of section 29;
(c) that the invention so far as claimed in any claim of the complete specification is
claimed in a claim of a complete specification published on or after the priority date of
the applicant's claim and filed in pursuance of an application for a patent in India,
being a claim of which the priority date is earlier than that of the applicant's claim;
(d) that the invention so far as claimed in any claim of the complete specification was
publicly known or publicly used in India before the priority date of that claim.
Explanation For the purposes of this clause, an invention relating to a process for
which a patent is claimed shall be deemed to have been publicly known or publicly used
in India before the priority date of the claim if a product made by that process had
already been imported into India before that date except where such importation has
been for the purpose of reasonable trial or experiment only;
(e) that the invention so far as claimed in any claim of the complete specification is
obvious and clearly does not involve any inventive step, having regard to the matter
published as mentioned in clause (b) or having regard to what was used in India before
the priority date of the applicant's claim;
8 or has furnished the information which in any material particular was false to his
knowledge; (i) that in the case of convention application, the application was not made
within twelve months from the date of the first application for protection for the
invention made in a convention country by the applicant or a person from whom he
derives title;
(j) That the complete specification does not disclose or wrongly mentions the source or
geographical origin of biological material used for the invention;
(k) that the invention so far as claimed in any claim of the complete specification is
anticipated having regard to the knowledge, oral or otherwise, available within any local
or indigenous community in India or elsewhere, but on no other ground.
The Controller shall, if requested by such person for being heard, hear him and dispose
of such representation. If the opposition is decided in favour of the applicant, the patent
is granted and the grant of Patent is published in the Patent Office Journal thereby
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opening the application, specification and other related documents for public inspection
on payment of prescribed fee.
GRANT OF PATENT
When all the requirements of the FER are met or in case of opposition under section
25(1), if the opposition is decided in favour of the applicant, the patent is granted, after
6 months from the date of publication under section 11 A, the letter patent is issued,
entry is made in the register of patents and it is notified in the Patent Office, Journal,
thereafter opening the application ,specification and other related documents for public
inspection on payment of prescribed fee.
Term and date of patent
Term of every patent will be 20 years from the date of filing of patent application,
irrespective of whether it is filed with provisional or complete specification. Date of
patent is the date on which the application for patent is filed.
Post grant opposition:
Any interested person can file notice of opposition (along with written statement and
evidence, if any) anytime after the grant of Patent but before the expiry of a period of
one year from the date of publication of grant of a Patent in the Patent Office Journal.
The above notice under Section 25(2) shall be filed on Form-7 along with a fee of Rs.
1500/ or Rs. 6000/- for natural person and other than natural person respectively, in
duplicate at the appropriate office. The grounds of opposition under section 25 (2) are
the same as given before in case of pre grant opposition. The post grant opposition is
decided by an Opposition Board followed by a hearing and the reasoned decision by the
Controller.
Rights of the patentee
Where a patent covers a product, the grant of patent gives the patentee the exclusive
right to prevent others from performing, without authorisation, the act of making,
using, offering for sale, selling or importing that product for the above purpose.
Where a patent covers a process, the patentee has the exclusive right to exclude others
from performing, without his authorisation, the act of using that process, using and
offering for sale, selling or importing for those purposes, the product obtained directly
by that process in India. These rights created by statute are circumscribed by various
conditions and limitations as provided in the Patents Act, 1970 as amended by The
Patents (amendment) Act, 2002.
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Register of patent
The Register of Patents are kept in the Patent offices and can be inspected or extract
from it can be obtained on payment of prescribed fee. Register of Patent contains the
name and address of the patentee, notification of assignment etc., particulars in respect
of validity or proprietorship of patent and payment of renewal fee.
Renewal fee
To keep the patent in force, Renewal fee is to be paid every year. The first renewal fee is
payable for the third year and must be paid before the expiration of the second year
from the date of patent If the patent has not been granted within two years the renewal
fees may be accumulated and paid immediately after the patent is granted, or within
three months of its recordal in Register of Patents or within extended period of 9
months, by paying extension fees of six month on Form 4, from the date of recordal. If
the renewal fees are not paid within the prescribed time, the patent will cease to have
effect. However, provision to restore the patent is possible provided application is made
within eighteen months from the date of cessation.
Renewal fee is counted from the date of filing of the Patent application. Six month's
grace time is available with extension fee for payment of renewal fee. No renewal fees is
payable on Patents of Addition, unless the original patent is revoked and if the Patent of
Addition is converted into an independent patent; renewal fee, then, becomes payable
for the remainder of the term of the main patent.
Restoration
Application for restoration of a patent that lapses due to non-payment of renewal fees
must be made within 18 months of lapse. The application is to be filed in the
appropriate office according to the jurisdiction.
Documents required for filing an application
Application form in duplicate (Form 1).
1.
Provisional or complete specification in duplicate. If the provisional specification is filed,

it must be followed by the complete specification within 12 months.(Form 2).
2.
Drawing in duplicate (if necessary).
3.
Abstract of the invention in duplicate.
4.
Information & undertaking listing the number, filing date & current status of each
foreign patent application in duplicate (Form 3).
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Priority document (if priority date is claimed) in convention application,when directed

by the Controller.
6.
Declaration of inventor-ship where provisional specification is followed by complete

specification or in case of convention/PCT national phase application (Form 5).
7.
Power of attorney (if filed through Patent Agent).
8.
Fee (to be paid in cash/by cheque/by demand draft) (See Schedule I). (Note: The cheque
or demand draft should be payable to the "Controller of Patents" drawn on any schedule
bank at a place where the appropriate office is situated).
Request for permission to file abroad:
If any application is to be filed abroad ,without filing in India ,it should be made only
after taking a written permission from the Controller .The request for permission for
making patent application outside India shall be made in Form-25 along with a fee of
Rs 1000/- or Rs 4000/- for natural person and other than natural person respectively.
A gist of invention should also be filed along with the Form-25.
Provisional specification
Application for patent may be accompanied by the provisional specification. It should
contain the description of invention with drawing, if required. It is not necessary to
include Claim. However, the complete specification should be fairly based on the matter
disclosed in the provisional specification and should be filed within 12 months. If the
complete specification is not filed within 12 months the application is deemed to have
been abandoned.
Complete specification*
The complete specification is an essential document in the filing of patent application
along with the drawing to be attached according to the necessity. Complete specification
shall fully describe the invention with reference to drawing, if required, disclosing the
best method known to the applicant and end with Claim/Claims defining the scope of
protection sought.. The specification must be written in such a manner that person of
ordinary skill in the relevant field, to which the invention pertains, can understand the
invention. Normally, it should contain the following matter-
1.
Title of invention
2.
Field of invention
3.
Background of invention with regard to the drawback associated with known art
4.
Object of invention
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5.
Statement of invention
6.
A summary of invention
7.
A brief description of the accompanying drawing
8.
Detailed description of the invention with reference to drawing/examples
9.
Claim(s)
10. Abstract.
The specification must start with a short title, which describes the general nature of
invention. The title should not contain anyone's name, a fancy name and trade name or
personal name or any abbreviation etc.
Description:
The specification must be written in good and clear English or Hindi. The specification
should indicate those features which are essential for the operation of the invention as
well as those features for which a choice can be made. The description must be
sufficiently detailed for someone who works in the same area of technology to be able to
perform the invention from the information given in the description. The best method of
putting the invention into effect is required to be described.
In case of biological invention, it is required to mention the source or geographical
origin of biological material used for the invention
Claim:
A set of properly drafted claims is an important part of complete specification. The
complete specification must have at least one Claim. . The first claim is the main claim.
The subsidiary claims refer to the main claim and include qualifying or explanatory
clauses on the various integers of the main claim or optional features. They may also
contain independent claims. Although the claim clauses consist of a number of claims,
the totality of the claims must relate to one invention only. It should be noted that a
claim is a statement of technical facts expressed in legal terms defining the scope of the
invention sought to be protected.
Abstract
The abstract is the concise summary of the invention preferably within 150 words and
shall commence with the title of the invention. It should be prepared in such a way that
one can understand the technical problem and solution with its usefulness. If
necessary, most relevant drawing should also be included in the abstract, particularly,
in mechanical type inventions. Each main feature mentioned in the abstract and
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illustrated by a drawing should be indicated by reference numerals. In case of Chemical

invention, it should contain the Chemical Formula for understanding the invention.
However, it cannot be used for the purpose of interpreting the scope of protection in
legal proceeding.
DRAWING*
Drawing should be filed on standard A4 size sheet in duplicate. Drawing should be
drawn on the sheet with margin of 4 cm on top and left hand and 3 cm at the bottom
and right hand side. Figure should be shown clearly on sufficient scale in upright
position with respect to top and bottom position of the sheet. At left-hand top corner of
the sheet, the name of applicant should be mentioned, with the application No.
therebelow. No. of sheets and sheet no. should be mentioned at the right hand top
corner. At the right-hand bottom, signature of the applicant/agent should be made
mentioning the name there under. Reference letter/numerals as used in the description
should also be used in denoting the corresponding component/part in the figure(s). No
descriptive matter should appear on drawing except under certain cases such as flow
sheet, chemical and other reactions etc. No drawing or sketch should appear in the
specification.
LEGAL IMPLICATIONS
Intellectual property laws and enforcement vary widely from jurisdiction to jurisdiction.
Intellectual property laws confer a bundle of exclusive rights in relation to the particular
form or manner in which ideas or information are expressed or manifested, and not in
relation to the ideas or concepts themselves
TYPES OF IPR
The term Intellectual Property" denotes the specific legal rights
Copyrights (e.g. book)
Patents (e.g. new drug)
Trademark (e.g. symbol)
Trade Secrets (e.g. new method)
Copyright:
It may subsist in creative and artistic works (e.g. books, movies, music, paintings,
photographs, and software) and give a copyright holder the exclusive right to control
eproduction or adaptation of such works for a certain period of time (historically a
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period of between 10 and 30 years depending on jurisdiction, more recently the life of
the author plus several decades).
Patent
A patent is a right to gives the patent holder to prevent others from practicing the
invention without a license from the inventor for a certain period of time (typically 20
years from the filing date of a patent application).
Trade Mark
A trademark is a distinctive sign which is used to distinguish the products or services
of different businesses.
An industrial design right protects the form of appearance, style or design of an
industrial object (e.g. spare parts, furniture, or textiles).
A trade secret ("confidential information") is secret, non-public information concerning
the commercial practices or proprietary knowledge of a business, public disclosure of
which may sometimes be illegal
Patents, trademarks, and designs rights are sometimes collectively known as industrial
property, as they are typically created and used for industrial or commercial purposes.
Copyright (e.g. books, paintings, films)
Protects the work of expressions, not the ideas (ie. The form a creator/publisher gives to
ideas). Limited protection against substitute.
Protection focuses on copying
Long but limited protection (life + 50years in Canada)
Covers original work of authorship in a tangible medium of expression (e.g. book)
Registration is inexpensive. Copyrights are easy to Obtain
Patents (i.e. inventions)
Protect the ideas, not just expressions
Registration is necessary (patent office)
Requirements: utility, novelty, non-obviousness.
Difficult to obtain
Short protection periods but greater protection against infringers
Trademarks (e.g. brand names, product logos)
Protect symbols and phrases
Registration is not necessary but easy
Protection periods vary
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Distinctiveness is required
Trade Secrets (e.g. formula, program, technique, method, process)
Derives independent economic value from not being known
Registration is not required. Perpetual protection is possible
Intellectual Property Rights in India
There is a well-established statutory, administrative and judicial framework to
safeguard intellectual property rights in India, whether they relate to patents,
trademarks, copyright or industrial designs etc.
Well-known international trademarks have been protected in India even when they were
not registered in India.
The Indian Trademarks Law has been extended through court decisions to service
marks in addition to trade marks for goods.
Features
IPR are property rights over information, knowledge and ideas
IPR are exclusive, trade able and temporary
IPR differ in the information being protected (copyright, patent, trade secrets,
trademarks)
IPR protect information that has public good characteristics (non-excludability and nonrivalry consumption)
IPR : Digital Era
Computer software companies have successfully curtailed piracy through court orders.
Computer databases have been protected.
The courts, under the doctrine of breach of confidentiality, accorded an extensive
protection of trade secrets.
Right to privacy, which is not protected even in some developed countries, has been
recognized in India.
IP, protected through law, like any other form of property can be a matter of trade, that
is, it can be owned, bequeathed, sold or bought. The major features that distinguish it
from other forms are their intangibility and non-exhaustion by consumption.
IP is the foundation of knowledge-based economy.
It pervades all sectors of economy and is increasingly becoming important for ensuring
competitiveness of the enterprises.
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TRADITIONAL KNOWLEDGE - COMMERCIAL EXPLOITATION PROTECTION

Human communities have always generated, refined and passed on knowledge from
generation to generation. Such traditional knowledge1 is often an important part of
their cultural identities. Traditional knowledge has played, and still plays, a vital role in
the daily lives of the vast majority of people. Traditional knowledge is essential to the
food security and health of millions of people in the developing world. In many
countries, traditional medicines provide the only affordable treatment available to poor
people. In developing countries, up to 80% of the population depends on traditional
medicines to help meet their healthcare needs.
In addition, knowledge of the healing properties of plants has been the source of many
modern medicines. The use and continuous development by local farmers of plant
varieties and the sharing and diffusion of these varieties and the knowledge associated
with them play an essential role in agricultural systems in developing countries.
Only recently, however, has the international community sought to recognise and
protect traditional knowledge. In 1981, WIPO and UNESCO adopted a model law on
folklore. In 1989 the concept of Farmers Rights was introduced by the FAO into its
International Undertaking on Plant Genetic Resources and in 1992 the Convention on
Biological Diversity (CBD) highlighted the need to promote and preserve traditional
knowledge.3 In spite of these efforts which have spanned two decades, final and
universally acceptable solutions for the protection and promotion of traditional
knowledge have not yet emerged.
The CBD also set out principles governing access to genetic resources and the
knowledge associated with them, and the sharing of benefits arising from such access.
We therefore consider the relationship between the IP system and the access and
benefit sharing principles of the CBD in the context of both knowledge, traditional or
otherwise, and genetic resources.
The Nature of Traditional Knowledge and the Purpose of Protection
How can traditional knowledge be defined? Whilst the vast majority of the knowledge is
old in the sense that it has been handed down through the generations, it is continually
refined and new knowledge developed, rather as the modern scientific process proceeds
by continual incremental improvement rather than by major leaps forward. One of the
speakers at our conference suggested that the term folklore be replaced by the more
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appropriate expressions of culture which represents living, functional traditions,

rather than souvenirs of the past.
Whilst most traditional knowledge and folklore is passed on orally, some of it, such as
textile designs and Ayurveda medicinal knowledge is codified. The groups that hold
traditional knowledge are very diverse: individuals, groups or groups of communities
may all be custodians. Such communities might be indigenous to the land or
descendents of later settlers. The nature of the knowledge is also diverse:
it covers, for example, literary, artistic or scientific works, song, dance, medical
treatments and practices and agricultural technologies and techniques.
Whilst a number of definitions for traditional knowledge and folklore have been put
forward, there is no widely acceptable definition for either of them. It is not only the
broad scope of traditional knowledge that has confounded the debate so far. There is
also some confusion about exactly what is meant by protection and its purpose. It
should certainly not be equated directly with the use of the word protection in its IP
sense. In its report on a series of fact-finding missions,
WIPO sought to summarise the concerns of traditional knowledge holders as follows:
Concern about the loss of traditional life styles and of traditional knowledge, and the
reluctance of the younger members of the communities to carry forward traditional
practices
Concern about the lack of respect for traditional knowledge and holders of traditional
knowledge
Concern about the misappropriation of traditional knowledge including use of
traditional knowledge without any benefit sharing, or use in a derogatory manner
Lack of recognition of the need to preserve and promote the further use of traditional
knowledge.
Making Use of the Existing IP System to Protect and Promote Traditional
Knowledge
Examples are emerging which illustrate how the current intellectual property system
can be utilized to commercialise traditional knowledge or prevent its misuse. For
example, Aboriginal and Torres Strait Islander artists in Australia have obtained a
national certification trademark.
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Like any other trademark, this certification mark or Label of Authenticity is intended to
help promote the marketing of their art and cultural products and deter the sale of
products falsely claiming to be of Aboriginal origin.
In recent surveys of the existing protection of traditional knowledge and folklore, a
number of countries have provided further examples of how IP tools have been utilised
to promote and protect traditional knowledge and folklore.12 These include the use of
copyright protection in Canada to protect tradition-based creations including masks,
totem poles and sound recordings of Aboriginal artists; the use of industrial designs to
protect the external appearance of articles such as head dresses and carpets in
Kazakhstan and the use of geographical indications to protect traditional products such
as liquors, sauces and teas in Venezuela and Vietnam.
The ability to extend the life of trademarks indefinitely and the possibility of collective
ownership of such rights suggest that they may be especially suitable for protecting
traditional knowledge.
This is also the case with geographical indications, which may be used to protect
traditional products or crafts if particular characteristics of such products can be
attributed to a particular geographical origin. However, trademarks and geographical
indications can only prevent the use of the protected marks or indications; they do not
protect the knowledge, or the technologies embracing that knowledge, as such.
Other IP rights, especially those requiring some form of novelty or those with fairly
limited periods of protection seem less appropriate for protecting traditional knowledge.
Nevertheless it is clear from these surveys, and indeed other research, that existing
IPRs do have a role to play in protecting traditional knowledge. Whether that role is a
significant one remains to be seen. Experience elsewhere would suggest that the impact
may not be great, not least because of the high cost of obtaining and enforcing rights. If
the majority of small companies in developed countries have found the intellectual
property system, particularly the patent system, to be unattractive, then it seems
unlikely that local communities in developing countries, or individuals within such
communities, will derive much benefit.
Sui Generis Protection of Traditional Knowledge
Some countries have already decided that the existing intellectual property system is
not, on its own, adequate to protect traditional knowledge. A number of these have
enacted or are in the process of enacting sui generis systems of protection.The
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Philippines has enacted legislation, and is considering further provisions, giving

indigenous communities rights over their traditional knowledge. These rights extend to
controlling access to ancestral lands, access to biological and genetic resources and to
indigenous knowledge related to these resources. Access by other parties will be based
on the prior informed consent (PIC) of the community obtained in accordance with
customary laws. Any benefits arising from genetic resources or associated knowledge
will be equitably shared. The legislation however seeks to maintain the free exchange of
biodiversity among local communities. The law also seeks to ensure that indigenous
communities are able to participate at all levels of decision-making.
Whilst the primary objectives of these pieces of legislation is to recognise, protect and
promote the rights of communities and indigenous people, including those relating to
biological resources and associated traditional knowledge, they also recognise the
potential for exploiting these resources. However, Guatemalan law also seeks to
preserve and promote the wider use of its traditional knowledge by placing expressions
of national culture, including for example medicinal knowledge and music, under the
protection of the state. Such expressions cannot under the law be sold or be subject of
any remuneration. Thus, different types of models are being developed at the national
level, seeking to adapt legislation and practice to local needs.
A particularly important question is the extent to which any form of protection
recognises the customary laws under which the knowledge evolved. Countries such as
Bangladesh and organisations such as the AU are considering sui generis legislation
that provides communitybased rights over biological resources and associated
traditional knowledge and are seeking to give increased recognition to the cultural and
customary practices of communities. The sui generis system of protection in the
Philippines also takes account of customary laws.
WIPO is also examining the extent to which information on traditional knowledge is
already available on the Internet. Initial findings from WIPO indicate that the amount of
traditional knowledge-related information available is substantial and growing.
However, much of it is not in a form that would make it either searchable or useable by
patent examiners.
The greater documentation of traditional knowledge may not only be of value in
preventing the granting of unwarranted patents but also, more importantly, it may
contribute to the preservation, promotion and possible exploitation of traditional
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knowledge. In this respect it is crucial that the documentation process does not
prejudice possible IPRs in the material being documented. Indias National Innovation
Foundation provides an example of an attempt to address these issues. One of the
concerns raised by both WIPO, and a number of developing countries, about many of
the databases unearthed by WIPO was whether the information had been recorded with
the prior informed consent of the holders of the knowledge. During discussions in WIPO
on the documentation of traditional knowledge, differences were also evident among
developing countries as to the type of data that could or should be included in any
databases. Some countries, for example, argued that such databases are appropriate
only for information that was already publicly available in a codified form. Others
indicated that traditional knowledge that had not yet been codified could also be
included.
Digital libraries of traditional knowledge should, as soon as it is practical, be
incorporated into the minimum search documentation lists of patent offices therefore
ensuring that the data contained within them will be considered during the processing
of patent applications. Holders of the traditional knowledge should play a crucial role in
deciding whether such knowledge is included in any databases and should also benefit
from any commercial exploitation of the information.
Traditional medicine is an area that has the potential to be quite well documented. In
Lao Peoples Democratic Republic, for example, the Government established the
Traditional Medicines Resource Centre (TRMC) which is working with local healers to
document details of all traditional medicines with a view to promoting a sharing of
practices within Laos. The TRMC is also collaborating with the International Cooperative Biodiversity Group (ICBG) in efforts to discover prospective medicinal
products. Any benefits, profits or royalties realised from plants and knowledge
recovered during the collaboration will be shared with all the communities involved.
IPRs clearly may have a role in exploiting products based on traditional medicine. But
the primary objective must be to promote the application of this knowledge to improve
human health, rather than to generate income. Indeed it would be unfortunate if the
objective of benefit sharing based on commercialisation resulted only in a few people
getting richer at the price of restricting access to medicines needed particularly by the
poor. The WHO Traditional Medicine Strategy for 2002Contact for your free pdf & job opportunities theimprintbiochemistry@gmail.com
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2005 clearly bring outs the public health objective.26 Lessons learnt from this exercise
and other similar initiatives should be freely shared and technical assistance provided
to assist other countries managing initiatives relating to documentation.
It must however be recognised that much traditional knowledge will continue to be
undocumented. The concept of absolute novelty whereby any disclosure including
through use, anywhere in the world, is sufficient to destroy the novelty of an invention
therefore remains a necessary safeguard. Without this safeguard, patents could
continue to be granted for traditional knowledge that is already in the public domain,
albeit not through written disclosure. Some countries do not include use outside their
country as prior art. Those countries that only include domestic use in their definition
of prior art, should give equal treatment to users of knowledge in other countries. In
addition, account should be taken of the unwritten nature of much traditional
knowledge in any attempts to develop further the patent system internationally.
For some communities the granting of IPRs such as patents over their knowledge can
cause great offence. Although provisions exist in most countries to prevent the granting
of IPRs on moral grounds, it is questionable whether intellectual property offices will be
able to apply them in respect of small indigenous communities. For example, moral
grounds for rejecting trademark applications have existed for some time in New Zealand
but it has now been considered necessary to define more clearly the scope of this
provision. The amendment under consideration would prevent the registration of a
trademark where, on reasonable grounds, the use or registration of the mark is likely to
offend a significant section of the community, including the Maori. Such measures as
this, together with the greater use of searchable databases of traditional knowledge
already in the public domain should go some way to preventing the issuing of IP rights
on material that is not novel, obvious or likely to cause offence.
However, as we have noted, there is a second group of patents and indeed other IPRs
that cause concern. These are rights which essentially meet the usual criteria for
patentability or protection but which nevertheless:
Are based on, or consist of, material obtained illegally or without the consent of the
holder of the material
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Do not fully recognise the contribution made by others to the invention either in terms
of ownership of the rights, or in the sharing of any benefits accruing from the
commercialisation of the patented invention.
These concerns do not apply just to patents relating to traditional knowledge although,
in light of the CBD, the most contentious patents in this area are likely to be those
relating to biological resources and/or traditional knowledge associated with such
resources. In the Hoodia case, the concern was essentially not about whether the
patents should have been granted, but about whether the San would receive a fair
share of the benefits of commercialisation.
ENTREPRENEURSHIP
Entrepreneur (Oxford Dictionary) Person who undertakes an enterprise with chances
of profit or loss. (As I have understood, Entrepreneur is a person who undertakes a
business activity of which he has no background and faces considerable risks in the
process. If either of the two elements, i.e., no background or considerable risk is
missing in the venture, it is no entrepreneurship).
Enterprise (Oxford Dictionary) Bold Undertaking
Entrepreneur (New Encyclopaedia Britannica) An individual who bears the risk of
operating business in the face of uncertainty about the future conditions.
Common Meaning one who starts his own, new and small business.
Entrepreneurship It is a philosophy or process through which an entrepreneur seeks
innovation and employment.
Entrepreneur - Person
Entrepreneurship - Process or Philosophy
Enterprise Object
Entre preneurship Theories
1600 French verb Entreprendre to undertake.
1700 Person bearing Risk or Profit in a fixed price contract (Risk)
1725 Richard Cantillon Person bearing risks is different from Capital Supplier (Risk)
1803 J. B. Say Shifts economic resources out from an area of lower to higher
productivity & greater yields (Value Addition)
1934 Joseph Schumpeter Innovator and develops untried technology (Productivity &
Innovation)
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1961 David McClelland Highly motivated, energetic, moderate risk taker (Need for
achievement)
1964 Peter Drucker Searches for change, responds to it & exploits as opportunity
(Opportunity Focused)
1980 Karl Vesper Behaviour Perceptions Economists, Psychologists, Businessmen,
Politicians (Environment)
1983 Gifford Pinchot Intrapreneur
1985 Robert Hisrich Creating something different with value, devoting time & effort,
assuming risks (FPS); results rewards and satisfaction (Leadership & Vision)
Please note that key word in Entrepreneurship is RISK. Any venture where risk is
mitigated due to any reason does not qualify to be called entrepreneurship.
Entrepreneurs are people who create new business activity in the economy and bear
considerable business risk in the process. This is often done by starting new
companies. But they can also create new business activity by introducing a new
product or creating a new market
Standard (New) Definit ion
Entrepreneurship is the process of creating something different with value by
devoting the necessary time and effort, assuming the accompanying financial, psychic,
social risks and receiving the resulting rewards of monetary and personal satisfaction
and independence.
Word Entrepreneur stems from French Verb Entreprendre means between; taker or
go between
New Definition involves four aspects
The creation process
Devotion of time and efforts
Assumption of risks
Rewards of independence, satisfaction, money.
Advantages of Entrepreneur ship
To an Individual
Provides Self Employment for the entrepreneur
Entrepreneur can provide employment for near & dear one as well
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Entrepreneurship often provides an employment and livelihood for next generations as

well.
Freedom to use own ideas Innovation and creativity
Unlimited income / higher retained income Bill Gates has risen to become richest in
the world in a single life time through entrepreneurship
Independence
Satisfaction
To the nation
Provides larger employment Entrepreneurs provide employment for self as well as
other people and is source of employment creation.
Results in wider distribution of wealth This is a logical sequel of above issue. Higher
the employment, greater the distribution of wealth
Mobilizes local resources, skills and savings
Accelerates the pace of economic development Entrepreneurship is the govts one of
the most trusted vehicles for economic development
Stimulates innovation & efficiency
Factor s Favour ing Ent reprene ur ship
Developed Infrastructure Facilities Availability of infrastructure reduces the cost
& efforts and improves viability of projects through higher profit margins.
Financial Assistance Easy availability of cheap funds is vital for promoting
entrepreneurship.
Protective and Promotional Policies Most of the entrepreneurship projects start
very small and have no resilience. They are extremely vulnerable to competitors,
market, money markets, etc, for considerable time. Favourable Govt policies shelter
them from such vagaries.
Growth of Education Science, Technology & Management Growth of education is
believed to be promoting entrepreneurship. However, there are enough examples to
suggest otherwise. A very large proportion of first generation entrepreneurs are low
educated.
Risk Taking Abilities Risk taking ability is one of the pillars of entrepreneurial
spirits.
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Hunger for Success (Capitalistic View) Fire in the belly and dreams of riches is
what drives most entrepreneurs on this risky path. Any person content with what he
has would take the easier route of salaries job.
Environment/Culture Impact Entrepreneurship is contagious. Communities like
Punjabies and Marwaries are historically entrepreneurial. They are known for seeking
and exploiting business opportunities in most remote areas. It is a culture that propels
them.
Social Security Social security acts as a safety net against failure of enterprise.
Social security guarantees basic roti, kapada aur makan in case of failure.
Entrepreneurial spirit of United States is born partly out of this security.
Technical/Industrial Training Facilities Industrial Training facilities on one hand
generate skilled manpower so vitally required for setting up enterprises while on the
other hand they are also nursery for future entrepreneurs. Among the educated
entrepreneurs, a majority is product of technical institutes from IIT to ITI
(Tier I to Tier III institutes).
Globalization Globalization has provided another avenue for business. Many dare
devils have taken a head along plunge into this uncharted water and have written new
success stories.
Wha t makes a Successful Entrepreneur?
The urge for achievement (most often monetary ambitions) Most Important
Willingness to take moderate risks (High risk takers are not entrepreneurs but
gamblers).
Determination to win
Win Win Personality
Ability to identify & explore opportunities
Analytical ability to take strategic decisions
Perseverance
Flexibility
Capacity to plan and organize
Preparedness to undergo physical and emotional stress
Positive self concept/Self Belief
Future orientation Vision
Ethics and Values Mission
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Who can be an Entrepr eneur?

Who feels the need for achievement?
Who can take moderate risks?
Who possess skills in organizing?
Who can capitalize on opportunities?
Who has some financial strength On his own or borrowed
Who has ability to work hard?
Who has desire for responsibility?
Who has a clear perception of probability of success?
Who gets stimulation by feedback?
Anyone He can be male, female or even a Eunuch
Who does not have previous experience?
Charact eristics of an Entrepreneur
Mental ability
Clear objectives
Business secrecy
H.R. ability
Communication ability
Technical knowledge
Achievement oriented
Perseverance
Ethical
Motivator
Self confident
Long term involvement
High energy level
Problem solver
Initiator
Goal setter
Risk taker
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POTENTIAL ENTREPRENEURSHIP ACTIVITIES IN BIOTECHNOLOGY - PRODUCT

DEVELOPMENT , MARKETING, RESEARCH AND TRAINING UNITS
Biotech parks are promoted in our country to help the entrepreneurs and
technopreneurs for starting their own ventures in biotechnology. These are essentially
built-up structures where common facilities are available for smooth initiation and
conduct of business. It is specially suited for technopreneurs who have a technology
with them but have no idea on the commercial viability of that technology. The presence
of the core-shared facility would help in reducing capital costs for the clients in the
Park as they would be able to use state of the art equipment in the facility without
having to invest on their own in various fields. The biotech parks will also have an
incubation centre and business facilitation centre to help create economic value.
Concept of Biotechnology Park:
Biotechnology parks are science and technology parks with a special emphasis on
biotechnology development. The broad concept is that of a facility, where the interface of
research with commerce and industry is facilitated for better utilization of the
technology. The UK Science Parks Association (UKSPA) defines a science park as a
business support and technology transfer initiative, which:
Encourages and supports the startup and integration of innovation led high-growth,
knowledge-based business.
Provides an environment where larger and international businesses can develop specific
and close interactions with a particular center of knowledge creation for their mutual
benefit.
Has formal and operational links with centers of knowledge creation, such as
universities, higher education institutes and research organizations.
The science park movement has been playing an important role in the development of
knowledge-based economy through technology innovation, networking, sound business
management and entrepreneurial partnering and nurturing.
There has been an increased activity towards applied research and technology
development by both the academia and the industry leading to increase in potential
filing for protection of the innovation. There is also an increasing trend to set up start
up companies by technocrats based on technology leads. Moreover a number of first
generation entrepreneurs and established companies operating in other areas are
showing interest in investment in biotechnologies developed indigenously or sourced
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from other countries. This has led to increase in corporate research activities. The
major constraints faced by such entrepreneurs, which has retarded the pace of
investments, include the following:
Large requirement of funds and long gestation period for developing the inventions into
commercial projects.
Difficulty in obtaining financial support for commercialization, particularly venture
capital for innovative inventions,
High-risk perception and lack of adequate incentives necessary to cover such risk and
encourage such investment.
Poor public private interaction and partnership due to lack of information about
technologies, technical capabilities, infrastructure availability in institutes and
mechanisms and incentives to promote such interactions.
High rate of technology obsolescence coupled with difficulty in accessing proprietary
technologies and intellectual property which are concentrated in the private sector of
industrialized nations.
Cumbersome regulatory protocols in different countries based on diverse biosafety
regulations are often difficult and costly to implement.
Scarcity of appropriately skilled manpower.
Inadequate information sources resulting in poor regional and inter regional
interactions for technology collaborations and market access.
Weakness in specialized areas such as IPR and biosafety resulting in delays and
inability to gear up to rapid global technological advancements.
To promote biotech investments, biotech parks in India should be modeled to promote
technology incubation and scale up as well as manufacturing activities by providing
infrastructure support, facilities and incentives so as to minimize the above constraints
and make it a preferred location for investments.
Functions
Biotechnology parks could perform the functions as encouraging and supporting
incubation and development of biotech innovations. The facilities in the incubator could
include physical facilities in the form of office space, dry and wet laboratories,
specialized equipment and instrumentation facilities, pilot plant and scale up units and
good communication network. In addition, the park should provide developed land at
cost effective rate for carrying out manufacturing activity.
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The park should establish close formal linkages with a local universities and research
institutions, specializing in biotechnology. These centers could become a primary
source for the basic technologies for incubation and development into commercial
ventures, particularly catering to the needs of small and startup enterprises. They can
also be assisted through consultancy assistance in technology up gradation,
troubleshooting, contract research etc. Linkage with a research institution of repute will
also attract research and development activities of major industries to the park.
Consultancy and networking for advice on IPR protection, regulatory issues, project
management and sourcing of funds from venture capitalists etc. is also critical for
success of park. FACILITIES/SERVICES & INCENTIVES AT BIOTECH PARKS
Biotech parks are equipped with state-of-the-art facilities, along with which it comprise
the following:
Technology incubation center (TIC)
Specialized facilities
Common facilities
Business support facilities (BSF)
Business enterprise zone (BEZ)
Technology Incubation Center TIC would consist of laboratory modules, specialized
facilities and a well-equipped instrumentation facility. The laboratory space could be
leased out to the tenant for specified periods. TIC would enable the tenants to translate
their research ideas into commercialisable technologies as well as upgradation of
existing technologies without making huge investments on buildings, equipment etc.,
initially. This facility would be useful for technically competent small and medium scale
enterprises (SME) and entrepreneurial scientist with limited financial resources
particularly because of non-availability of financial assistance for R & D projects under
conventional funding schemes. This facility could also be used for small scale
manufacturing of the biotech products by SMEs.
Specialized Facilities
These would consist of fully equipped laboratories in the areas of commercial
importance and of relevance to the state. The new quality control norms and
certification system demands proper testing and validation of bio products
(biofertilizers, biopesticides, tissue cultured plants etc) and so the facilities proposed in
the park can help the tenants in the initial phase to meet the quality standards. These
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facilities could be leased out to companies, contract research organizations or contract

manufacturing organizations for facilitating cost effective development of
technologies/products and manufacturing. In the first phase of the park the following
for facilities are proposed to be set up. It consists of facilities for Plant Tissue Culture,
animal Testing, Medicinal plant extraction facility, biopesticides & biofertilizer
production etc.
Common Facilities
The common facilities in the park are cold storages and warehouses to facilitate smallscale companies to store their finished goods and raw materials. Other facility includes
the common effluent treatment plant to ensure elimination of harmful live
microorganisms, as bacteria, will be carried out by the units in the parks themselves.
Some parks specializing in medical biotechnology will have a common secondary
effluent treatment plant that will collect all the bacteria killed in effluent from the
primary effluent plants of individual units for further treatment and disposal.
Business Support Facilities

This facility consists of Administrative office consisting of management, and other
linked departments responsible for parks administration. Biotechnology Information
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Centre (BIC), a bioinformatics facility for accessing all the on line journals and other IT
tools a facility, Meeting rooms for making presentations etc., would be equipped with
state of the art equipment such as LCD/digital projector, slide projector, overhead
projector, public address system etc. Apart from these amenities the park is also
equipped with a Consultancy cell to encourage biotech consultants specialized in
providing the following services by offering space initially at concessional rates such as.
Technology transfer assistance.
Fund syndication.
Project assistance.
Marketing assistance.
Single window clearance.
Facilitation of regulatory approvals
Besides these facilities the park also consists of commercial shops, cafeteria, parking
space for tenants and PFUs would be dealing in items such as stationery, raw
materials, laboratory consumables and lab ware etc. as required by the tenants. These
shops could be leased/ purchased by stationery sellers and other service providers
including biotech consultants. Business Enterprise Zone
The Business Enterprise Zone would consist of developed plots, roads, water supply &
sewerages, effluent treatment facility, telecom network and power supply. The developed
plots would be required by medium and large-scale enterprises. These plots would be
leased out to the tenants.
INDUSTRIAL LICENSING
The Biotechnology industry in India is governed by the following enactments,
regulations and guidelines:
Environment Protection Act, 1986;
EXIM Policy;
Foreign Exchange Management Act, 1999;
Laws pertaining to Intellectual Property Rights;
Rules for the Manufacture, Use/Import/Export and Storage of Hazardous;
Micro Organisms/Genetically Engineered Organisms or Cells, 1989; notified by Ministry

of Environment & Forests on December 5, 1989 under Environment and Protection Act,
1986;
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Revised Recombinant DNA Safety Guidelines;
Guidelines for Research in Transgenic Plants & Guidelines for Toxity and
Allergenicity Evaluation of Transgenic Seeds, Plants and Plant Parts,
1998;
National Seed Policy, 2002;
Seeds Act, 1966;
The Plants, Fruits and Seeds [Regulation of import in India] Order 1989 issued under
the Destructive Insects and Pests Act, 1914;
Guidelines for Generating Preclinical and Clinical Data for Rdna Therapeutics, 1999;
Drugs & Cosmetic Act 1940 along with Drugs and Cosmetic Rules;
Drug Policy, 2002;
Biological Diversity Act.

VENTURE CAPITAL
India is ranked among the top 12 biotech destinations in the world and the Indian
biotech sector is the third largest in the Asian region after Australia and China.
The Indian biotechnology sector has excellent potential and is expected to touch five
billion-dollar mark by 2010.
The biotechnology sector in India is growing rapidly at a rate of over 40 percent
annually. India is the largest producer of recombinant Hepatitis B vaccine in the world.
In addition, Indian vaccine market, which is over $100 million, is growing at over 20
percent annually. The Indian market of Contract Research Organizations is growing at
the rate of 30% to 40%.
India occupies a significant position in the world pharma market, 8% by volume (fourth
largest in the world) and 1% by value. The pharma industry ranks 17th in terms of
export value. India accounts for 22% of the global generics market
Exports:
During 2007-08, the biotech exports grew to Rs 5,733.7 crore in revenues. Exports
account for 56% share of the total biotech sector. Exports from biopharma alone
accounted for over 70 percent of the total industry (Rs 3,999 crore), while the
bioservices sector had 26 percent share in exports (Rs 1,502 crore). The bioservices
sector registered 53 percent growth. The bioagri sector grew by 30 percent to Rs 1,202
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crore, the bioindustrial sector by 4 percent to Rs 410, and the bioinformatics sector by
31 percent to clock Rs 190 crore in revenues.
Government Initiatives:
The Central government as well as the State governments have taken various initiatives
to boost the biotechnology sector in India. Some of the key steps taken include the
following:
Commercial cultivation of BT cotton was approved by the government in early 2002
which today accounts for about 70 per cent of the total area under cotton cultivation
and thus makes agri-biotech the fastest growing biotech industry.
National Biotechnology Policy:
The government has announced a National Biotechnology Policy as recognition of the
importance of the sector as a key growth area. The key elements of strategy include:
A National Biotechnology Regulatory Authority would be set up as an independent,
autonomous and professionally led body to provide a single window mechanism for
biosafety clearance of genetically modified products and processes.
A high-powered Inter-ministerial Committee is to be set up under the chairmanship of
secretary, DBT, to effectively coordinate the development of the sector by addressing
cross cutting issues.
30 percent of DBT's Budget to be spent on public-private partnership programs.
A Biotechnology Industry Partnership Programme (BIPP) for Advanced Technology
would be launched.
The existing Small Business Innovation Research Industry (SBIRI) scheme to promote
innovation in SMEs has been a success.
Approval has been accorded for the expansion of SBIRI during the 11th Plan.
A Biotechnology Industry Research Assistance Council (BIRAC) is to be launched to act
as an interface between academic and private sector, particularly SMEs and startups;
nurture and catalyze R&D and innovation in biotechnology in the private sector and
promotes public-private partnerships.
State Biotechnology Policies: Karnataka was the first state in the country to announce a
millennium biotechnology policy as early as 2001 to promote the nascent biotech
sector. Maharashtra, Tamil Nadu, Himachal Pradesh and Andhra Pradesh followed it
with their own biotechnology policies during the year. Later other states like Haryana
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(2002), Punjab, Kerala, Madhya Pradesh (2003), Uttar Pradesh, Rajasthan (2004) and
Gujarat (2005) joined the ranks by announcing their respective state policies.
Biotech Parks:
The government has set up exclusive Biotech Parks such as Shapoorji Pallonji Biotech
Park, Hyderabad; ICICI Knowledge Park, Hyderabad; International Biotech Park, Pune.
These biotechnology parks are acting as bioclusters - where companies, universities
and R&D institutes are all located in one place. These parks have emerged as a focal
point of some of the leading biotech clusters such as Genome Valley in Hyderabad and
Hinjewadi in Pune. States such as Andhra Pradesh, Kerala, Maharashtra, Punjab,
Tamil Nadu and Uttar Pradesh have made substantial progress in establishing biotech
parks, whereas Himachal Pradesh, Karnataka, Madhya Pradesh, Rajasthan and
Uttaranchal are in development stage. The state governments too are supporting the
industry players who are looking at setting up their units at the parks by offering
incubation facilities, tax holidays and incentive packages, venture funding initiatives
and so on.
The government has approved the National Policy on Biofuels along with setting up of
an empowered National Biofuel Coordination Committee and a Biofuel Steering
Committee.
The government has initiated a project to conduct genome-wide research on a range of
agronomically important crops.
Nanoelectronics Centres have been launched by the government.
The Patent Act (Third Amendment), 2005: India amended its Patent Act in 2005 to
usher in a new product patent regime. Provisions in the amended Act which relate
specifically to biotechnology include the following:
Plants and animals, seeds, including essentially biological processes used for
propagating plants and animals are not patentable. The area of patentability in relation
to microorganisms is not clear. Going by the US and European precedents, it would
appear that only such microorganisms that are the result of human intervention would
be patentable.
Synthetic genes (as distinct from naturally occurring gene segments) too would now be
the subject matter of patentability.
Genetic inventions will include SNP (single nucleotide polymorphism), vectors,
recombinant products such as vaccines, enzymes, hormones, etc.
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In order to get a patent, the Act requires the deposit of biological material with the
International Depository Authority (IDA). IMT, Chandigarh, is the IDA in India for some
of biological materials such as bacteria and plasmids.
Tax Incentives (Budget 2008-09):
Excise duty on all goods produced in the pharmaceutical sector reduced from 16 per
cent to 8 per cent.
A reduction in Customs duty from 10 per cent to 5 per cent on certain specified life
saving drugs and on the bulk drugs used for the manufacture of such drugs.
Reduction in Central Sales Tax from 3 percent to 2 percent.
A total exemption of excise duty on certain specified life-saving drugs and bulk-drugs.
Amounts spent in R&D eligible for a 125 per cent weighted deduction.
Increase in healthcare allocation by 15 percent to Rs 16,354 crore.
Increase in healthcare allocation by 15 percent to Rs 16,354 crore.
Biotech Parks:
Concessions to biotech companies located in biotech parks
Duty free import of equipment, instruments and consumables
Tax holiday granted under the Income Tax Act which provides such a special provision
in respect of newly established industrial undertakings in free trade zones and newly
established hundred per cent export-oriented undertakings.
Biotech companies located in biotech parks to be allowed a five -year time frame to meet
the export obligation norms under the Special Economic Zone scheme.
Foreign Direct Investment Policy:
FDI up to 100% is permitted through the automatic route for the manufacture of drugs
and pharmaceuticals provided the activity does not attract compulsory licensing or
involve the use of recombinant DNA technology and specific cell/tissue targeted
formulations
FDI proposals for the manufacture of licensable drugs and pharmaceuticals and bulk
drugs produced by recombinant DNA technology, and specific cell / tissue targeted
formulations will require prior Government approval
BIOTECHOLOGY INDUSTRIES IN INDIA
The biotech companies have developed in three major clusters across the country. The
largest in terms of revenue generated is the Western cluster (Ahmedabad, Aurangabad,
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Mumbai and Pune), followed by the Southern cluster (Bangalore, Chennai and
Hyderabad) and the Northern cluster (Delhi, Gurgaon and Noida).
The biotech cluster in Bangalore makes the city the leading biotech destination in
the country. The Knowledge Park, the Biotech Park, Genome Valley and other projects
in Hyderabad make it another very preferred destination having operations of over 53
international biotech companies in the Genome Valley over the last one year.
Additionally, two biotech special economic zones (SEZs) and three biotech parks will be
developed in the city in the next couple of years. A genomics centre is being set up at
Tidel Park in Chennai and three more biotech parks and a biotech SEZ will be
developed in the coming years.
Madurai, Kolkata, Gurgaon, Thrissur, Nagpur and Tiruchirapalli have immense
potential to attract investments and emerge as biotech destinations.
Location of Biotech Parks in India:
Andhra Pradesh - Development and scale-up of bioprocesses and technologies
Karnataka - Drugs and Pharma
Punjab - Agribusiness and Certification of Export Goods
Kerala - Traditional Medicines
Himachal Pradesh - Medicinal and Aromatic Plants, Horticulture
Key Indian Biotech Companies:
Biocon
Shantha Biotechnics
Bharat Biotech
Wockhardt
Dr. Reddy's Laboratories
Serum Institute of India
Zydus Cadila
Aventis Pharma
Reliance Life Sciences
Panacea Biotec
Ranbaxy Laboratories Ltd.
Sun Pharmaceuticals Industries Ltd.
GVK Biosciences Pvt. Ltd.
Nuziveedu Seeds
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Rasi Seeds
Novo Nordisk
Novozymes South Asia
Indian Immunologicals
Mahyco
Jubilant
Themis Medicare
Haffkine Biopharma
Rossari Biotech
GlaxoSmithKline
Biological E
International Biotech Companies in India:
Monsanto
Pfizer
Astra Zeneca
Unilever
Dupont
Bayer
Eli Lilly
Hoechst Roussel Vet
Millipore
Novozymes
THE POTENTIAL JOB OPPORTUNITES.
Biotechnology is expected to offer investment opportunities of Rs. 9-10 billion in the
next 5 years and the growth is expected in the following areas:
Vaccines:
Vaccines and recombinant therapeutics are the leading sectors driving the growth of the
biotechnology industry in India. It is estimated to reach $20 billion in 2012. The next
few years will also witness launch of newer therapies, prominent among these would be
the monoclonal antibodies products, stem cell therapies, growth factors and others.
Indias huge population makes it among the worlds largest markets for vaccines.
Bioactive Therapeutic Proteins:
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Production of proteins and antibodies and fabrication of diagnostic protein chips would
be a promising area for investment. Stem cell research, cell engineering and cell based
therapeutics could be another area where India could cash in its expertise. Up to 25%
growth is expected in this field.
Agriculture sector:
India has the potential to become a major producer of transgenic rice and several
genetically modified (GM) or engineered vegetables by 2010.There is an increasing use
of molecular markers in crop breeding and a growing realization that some of these new
technologies could lead to future (brinjal), tomato and ladys finger. Therefore hybrid
seeds, including genetically modified seeds represent new business opportunities in
India based on yield improvement, and development of a production base in
biopesticides and biofertilisers would facilitate Indias entry into the growing organic or
natural foods market.
Contract Research:
The R&D Sector has got huge potential and with the number of foreign companies
stepping in India it has opened doors of opportunities to this sector. The cutting edge of
the biotech sector is development of new products. Indian pharma companies possess
competitive skills in chemical synthesis and process engineering, which they can
leverage to develop new chemical entities, and with the application of bioinformatics
tools, tap into the high-potential biogenerics segment.
Clinical Trials and outsourcing:
India offers a suitable population for clinical trials because of its diverse gene pools
covering a large number of diseases. Due to the rising costs of R&D abroad, many
global companies are looking for contract research in India especially US and European
companies. Cost-effectiveness, competition and the increased confidence on capabilities
and skill sets have propelled many global pharmaceutical players to expand their own
clinical research investment in India. Contract research supported by IT skills has led
to promising outsourcing business in various other segments including Clinical trial
data management, statistical analysis, and electronic data capture.
Bioinformatics:
Indian bioinformatics companies can play a significant role in critical areas such as
data mining, mapping and DNA sequencing, besides functional genomics, proteonics
and molecule design simulation in the US$ 2 billion world market for bilinformatics
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services. The IT skill of Indian manpower also offers services in bioinformatics and data
mining. Besides the above, the following thrust areas offer tremendous scope for
potential investment in the Biotechnology sector:
Medicinal and Aromatic plants
Animal Biotechnology
Aquaculture and Marine Biotechnology
Seri biotechnology
Stem Cell Biology
Human Genetics and Genome Analysis
Environmental Biotechnology
Microbial and Industrial Biotechnology
Agriculture and Plant Biotechnology
Healthcare
Bio-Fuels
Bio Pesticides
Bio-Informatics
Software Support
ALL THE BEST
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PROF.

BTH 401: BIOPROCESSING ENGINEERING

FOR MSC BIOTECHNOLOGY STUDENTS

As per Bangalore University (CBCS) Syllabus

BY: Prof. Balasubramanian Sathyamurthy

THE MATERIALS FROM THE IMPRINT (BIOCHEMISTRY SCANNER) ARE NOT

PROF. BALASUBRAMANIAN SATHYAMURTHY

BTH 401: BIOPROCESSING ENGINEERING

The Author Prof. Balasubramanian Sathyamurthy has 15 years of teaching

Contact for your free pdf & job opportunities theimprintbiochemistry@gmail.com

PROF. BALASUBRAMANIAN SATHYAMURTHY

BTH 401: BIOPROCESSING ENGINEERING

CONTACT : theimprintbiochemistry@gmail.com or 9980494461

PROF. BALASUBRAMANIAN SATHYAMURTHY

BTH 401: BIOPROCESSING ENGINEERING

M. SC. BIOTECHNOLOGY FOURTH SEMESTER

Contact for your free pdf & job opportunities theimprintbiochemistry@gmail.com

PROF. BALASUBRAMANIAN SATHYAMURTHY

BTH 401: BIOPROCESSING ENGINEERING

Alcohol: Ethanol, glycerol, butanol, Acetone, Organic streptomycin, tetracycline,

Jackson AT., Bioprocess Engineering in Biotechnology, Prentice Hall, Engelwood Cliffs,

Stanbury RF and Whitaker A., Principles of Fermentation Technology, Pergamon press,

Colin Ratledge and Bjorn Kristiansen, Basic Biotechnology (2nd Ed.).Cambridge

Paulins, M. D. Bioprocess Engineering Principles. John Wiley Publishers.2003

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PROF. BALASUBRAMANIAN SATHYAMURTHY

BTH 401: BIOPROCESSING ENGINEERING

SCOPE AND IMPORTANCE OF BIOPROCESS ENGINEERING TECHNOLOGY

PROF. BALASUBRAMANIAN SATHYAMURTHY

BTH 401: BIOPROCESSING ENGINEERING

Recombinant Protein Production Protein Recovery, Purification and Analysis

PROF. BALASUBRAMANIAN SATHYAMURTHY

BTH 401: BIOPROCESSING ENGINEERING

PROF. BALASUBRAMANIAN SATHYAMURTHY

BTH 401: BIOPROCESSING ENGINEERING

Isolate ssDNA in phage particles released from host cells.

Mix oligonucleotide with recombinant vector ssDNA.

Use DNA polymerase to synthesize remainder of strand. (Oligonucleotide acts as a

Then add ligase to join primer and new strand

10. Double strand DNA molecule.

PROF. BALASUBRAMANIAN SATHYAMURTHY

BTH 401: BIOPROCESSING ENGINEERING

PROF. BALASUBRAMANIAN SATHYAMURTHY

BTH 401: BIOPROCESSING ENGINEERING

psycrophilic, thermophilic microorganisms)

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PROF. BALASUBRAMANIAN SATHYAMURTHY

Double-pipe heat exchanger

BTH 401: BIOPROCESSING ENGINEERING

Shell and tube heat exchanger

Plate heat exchanger

Spiral heat exchanger

Mass Transfer in Fermentation

C* is the oxygen saturation concentration of the nutrient solution,Po is the partial

PROF. BALASUBRAMANIAN SATHYAMURTHY

BTH 401: BIOPROCESSING ENGINEERING

Role of diffusion in Bioprocess

Convective mass transfer

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PROF. BALASUBRAMANIAN SATHYAMURTHY

BTH 401: BIOPROCESSING ENGINEERING

Overall mass transfer coefficient

PROF. BALASUBRAMANIAN SATHYAMURTHY