(6th Sem B.Pharma Pharmaceutical Biotechnology) Microbial genetics: • Transformation, • Transduction, • Conjugation, • Plasmids and transposons, • Study of the production of - Penicillins, Citric acid, Vitamin B12, Glutamic acid, Griseofulvin, • Blood Products: Collection, Processing, and Storage of whole human blood,Dried human plasma, Plasma substitutes BY- SHYAM BASS

1. BIOTECHNOLOGY
UNIT IV
Microbial genetics:
• Transformation,
• Transduction,
• Conjugation,
• Plasmids and transposons,
• Study of the production of - Penicillins, Citric acid, Vitamin B12, Glutamic acid,
Griseofulvin,
• Blood Products: Collection, Processing and Storage of whole human blood,Dried
human plasma, Plasma substitutes
PRESENTED BY : SHYAM BASS
B.Sc BIOTECHNOLOGY (HONS), B.PHARMA
LOVELY SCHOOL OF PHARMACEUTICAL SCIENCES
e-mail: shyambass0925@gmail.com

2. MICROBIAL GENETICS
• Microbial genetics is a joint narrative of microbiology and genetic engineering.
• Microbial Genetics is the study of the mechanism of heritable information in micro-organisms including bacteria,
archaea, viruses and some protozoa and fungi.
• Genetic Engineering is a field of using the study of DNA recombinant technology within microbial genetics.(fig.1)
• The process involves creating recombinant DNA molecules through manipulating a DNA sequence.That
manipulated DNA is allowed to come in contact with a host organism. Cloning is also an example of genetic
engineering.
Fig.1.1 The use of microbial genetics in gene transfer systems that have been mostly studied in bacteria include genetic
transformation, conjugation and transduction.
TRANFORMATION
TRANSDUCTION
CONJUGATION

3. GENE TRANSFER AND ITS METHODS
• Gene transfer is defined as transfer of gene from one DNA molecule to another DNA molecule or introduction
of foreign gene into the genome of target cell. (Fig 1.2)
• The stable integration of foreign gene, expression of foreign gene in the genome of target cell and which is
inherited into the successive generations is referred as the genetic transformation.
• The foreign gene which is transferred is called as transgene and the acceptor of the transgene is called as
transgenic.(fig 1.2)
Bacteriophage DNA
transferring
TRANSGENE to the host
bacterial cell
The Bacteria accepted the
transgene In its genome
called as TRANSGENIC
bacterial host cell
Fig 1.2 Gene transfer from one DNA molecule to another. Transfer
of transgene to transgenic bacterial cell.

4. GENE TRANSFER AND ITS METHODS
GENE TRANSFER METHODS
TRANSFORMATION TRANSDUCTION CONJUGATION
Agrobacterium
Mediated gene transfer
Direct
gene transfer
1)Chemical Method
2)Lipofection(Liposome transfection)
3)Particle Gun delivery
4)Micro-injection
5)Macro-injection
6)Pollen grain transformation
7)Fibre mediated gene transfer
8)Gene transfer by Ultra-sonication
9)Electroporation
10)Lazer induced gene transfer
Generalized
Transduction
Specialized
Transduction
General bacterial
conjugation
Conjugation
in F’ bacterial
strain
Conjugation in
Hfr strain
Flowchart 1.1 Gene transfer methods

5. GENE TRANSFER METHOD: TRANSFORMATION
TRANSFORMATION is the process of uptake of transgene (foreign DNA molecule/ desired DNA molecule) by the host
cell from the extracellular environment.
• The uptake and incorporation of external DNA into the cell thereby resulting in the alteration of the genome.
• Transformation is broadly classified into 2 major classes:
A. Agrobacterium mediated gene transfer
B. Direct gene transfer
A. Agrobacterium mediated gene transfer: (fig1.3)
The Carrier Plasmid DNA
is of Agrobacterium
Tumefaciens I.e Ti plasmid
Fig 1.3 Agrobacterium mediated gene transfer: 1) Removal of Ti plasmid and cut, 2)Foreign DNA or Gene of interest is cut,
3)Desired DNA is inserted in Ti Plasmid, 4)The Recombinant Plasmid is reinserted into the bacterium, 5)The recombinant
Plasmid is inserted into the chromosome of plant cell, 6)The culture of new plant cells, 7) Plants are generated with new trait.

6. GENE TRANSFER METHOD: TRANSFORMATION
B. Direct gene transfer :
It is the method in which transgene is directly inserted into the host cell by any physical means like injection, ultra-
sonication etc.
S.no. Method Explanation
1
Chemical
method
• Some chemical substances like Polyvinyl Alcohol, Polyethylene Glycol, Dextran,
Calcium Phosphate increases the uptake of transgene by the host cell.
• Chemical substance +Host cell + transgene are cultured for particular time period
during which transgene is transferred into host cell.
2 Lipofection
• Liposomes(which act as lipid carriers) are used to transfer the transgene to the host
cell.
3
Particle gun
delivery
• Particles of Tungsten/Gold of diameter 1-2μm are used in which these particles are
coated with transgene which are to be delivered into the host cell, with a particular
velocity, by a gun like apparatus.
• The velocity can be regulated by using pressurised helium gas or electrostatic energy.
4
Micro-
injetion
• DNA solution is injected into the host cell by using capillary glass micropipette.
5
Macro-
injection
• DNA solution as well as plasmids are injected into the host cell by using Hypodermic
needle.
• The DNA solution can be directly inserted into the lumen of developing flower head
using this needle.

7. GENE TRANSFER METHOD: TRANSFORMATION
S.no. Method Explanation
6
Pollen grain
Transformation
• Pollen grains are soaked with DNA solution which results in adsorbance of DNA
over the surface of pollen grains , which are than used as carrier for gene transfer.
7
Fibre mediated
gene transfer
• Transgene is transferred to host cell by means of Silicon Carbide fibres as carrier
(of Diameter 0.6μm and length of 10μm).
8
Gene transfer by
ultra-sonication
• Transgene+Host cell+Culture media are mixed by Ultra sonication (using sonicator)
to transfer the gene.
• Vigorous vibration in the medium and high hydrostatic pressure may result in
localized rupture in the membrane which can facilitate uptake of exogenous DNA.
9 Electroporation
• Due to application of high- voltage electricity, pores are developed in the plasma
membrane of host cell, thus the DNA molecule easily transfers in the cell.
• Due to presence of electric field , permeability of the membrane also increases.
10
Lazer induced
gene transfer
• Pores are developed in the plasma membrane when exposed to laser rays, which
allows the direct transfer of gene into host cell.
Table 1.1 Methods of transformation

8. GENE TRANSFER METHOD: TRANSDUCTION
TRANSDUCTION is the process of transfer of genetic material from one bacterium to other by Bacteriophage.
• Bacteriophage is a Virus , used to replicate within bacteria or Archaea. Discovered by- Frederick twort.
• Bacteriophage can infect some specific bacteria like E.coli , Salmonella spp., Staphylococcus species.
• Bacteriophage can be of 2 types:
Virulence Bacteriophage Temperate Bacteriophage
This particular virus can able to cause infection to
bacterial cell and also cause Immediate lysis of bacterial
cell .
Example: T4 - bacteriophage
This particular virus uses bacterial cell for its replication
and after some cycle(life cycle) covered , it becomes
virulent and causes death of bacteria.
Example: α-bacteriophage
&
• Transduction can be of major 2 types: (Fig 1.6)
Generalised Transduction Specialised Transduction
Generalized transduction is the process by which any
bacterial DNA may be transferred to another bacterium
via a bacteriophage
Specialized transduction is the process by which
a restricted set of bacterial genes is transferred to another
bacterium.
&

9. GENE TRANSFER METHOD: TRANSDUCTION
Life cycle of bacteriophage: LYTIC CYCLE
LYSOGENIC CYCLE
S.no. Lytic Cycle Lysogenic Cycle
1
Destruction of cell membrane and rest of
cellular structure of the cell
Integration of viral DNA with bacterial DNA occurs
2 Viral DNA replication occurs independently
Viral DNA replication occurs with the bactrial DNA
replicateon machinery
3 Occurs in short period of time Takes time
4
Lysis occurs with the formation of many
viruses within cell
Lysis doesn't occur and no virus is formed within cell
5
Doesn't allow genetic recombination in the
host bacterium
Allows genetic recombination in the host bacterium
6 Cannot follow Lysogenic cycle Can follow Lytic cycle
7
Host’s cellular mechanism is completely taken
by viral genome
Host’s cellular mechanism is slightly disturbed by viral
genome
8
Host cell is lysed during release of viral
particles
Host cell is not lysed
9 Doesn't have prophage stage Has a prophage stage
Table1.2 Difference between Lytic and Lysogenic cycle

10. LYTIC CYCLE: Simply means bursting or rupturing cycle. It is one of the cycles of bacteriophage in which there is
master-slave relationship with host cell or bacterial cell. (Fig 1.4)
GENE TRANSFER METHOD: TRANSDUCTION
Fig 1.4 Lytic cycle of Bacteriophage
1) Attachment- Bacteriophage attach to the bacterium cell.
2) Penetration- Bacteriophage injects its DNA into host cell.
3) Biosynthesis- Bacterial cell allows the multiplication of phage DNA into number of copies and phage DNA is
produced.
4) Maturation- all components of Phage assembles and maturation of Bacteriophage takes place
5) Lysis- Bacterial cell undergoes lysis and release the newly matured phages.

11. LYSOGENIC CYCLE: The lysogenic cycle is a viral mode of reproduction where it inserts its genome in the host's DNA,
then remains at rest as time goes by. However, it replicates itself by letting the host cell copy the virus' genetic material in
cell division. If the host cell divides, the viral genome is also copied, therefore replicating itself without exerting much effort .
The lysogenic cycle can also shift to the lytic cycle. This is noticeable in viruses that can be at rest for years and then attacks
whenever the host’s immune system is suppressed or compromised. (Fig.1.5)
GENE TRANSFER METHOD: TRANSDUCTION
Fig 1.5 Lysogenic cycle of Bacteriophage
1-Attachment- the virus attaches to the host cell
2-Penetration- penetration of Viral DNA into the host cell
3B-Integration- the Viral DNA integrates into the host DNA.
4B-Replication-Chromosomes with integrated Prophage replicates and allows mitosis (cell division).
5- the Recombinant and infected cell formed- In very stressful conditions the prophage DNA separates from Host DNA and enters Lytic cycle.

12. GENE TRANSFER METHOD: TRANSDUCTION
• Transduction can be of major 2 types:
Generalised transduction is the process by which any bacterial DNA may be
transferred to another bacterium via a bacteriophage(occurs during lytic cycle+
bacteriophage is carrier)
Specialised transduction is the process by which a restricted set of bacterial genes is
transferred to another bacterium.(occurs during lysogenic cycle+ Temperate
bacteriophage is carrier)
&
Generalised Transduction Specialised Transduction
Fig 1.6 Generalised Transduction and Specialised Transduction
1)The bacterial donor cell got infected by phage .
2)The DNA of host cell and DNA of phage is broken
down Into pieces + Protein and enzymes
Of phage is synthesised(for body of Bacteriophage/
viral capsids) with in host cell.
3)The DNA of bacterial host cell
Is packed into Viral capsids(The body of
Bacteriophage contains The DNA of bacterial host
cell) which is released on lysis of host cell.
4)Now the Phage containing the host DNA infect the
new recipient cell- here Recombination also takes
place.
5)The Recombinant new cell formed is mixture of
Donor DNA+ its own DNA.
1)The bacterial donor cell got infected by temperate
phage DNA.
2)A part of bacterial donor DNA and temperate
phage DNA is cut in the host cell and separates out +
Protein and enzymes of phage is synthesised (for body
of Bacteriophage/viral capsids).
3)Phage capsid encapsulate-part of bacterial donor
DNA and temperate phage DNA.
4)Now the Phage containing the host DNA+
temperate phage DNA infect the new recipient cell-
here Recombination also takes place.
5)The Recombinant new cell formed is a mixture of
Donor DNA+ temperate phage DNA+ its own DNA.

13. GENE TRANSFER METHOD: CONJUGATION
• CONJUGATION is one of the methods of horizontal gene transfer, mainly in bacteria.
• Generally, conjugation occurs through the direct contact of two bacterial cells through a pilus.
• Here, genes for the antibiotic resistance, xenobiotic tolerance, etc. are transferred from one to the second bacterium.
Therefore, this transformation of genes is beneficial to the recipient bacterium.
• However, conjugation does not form a generation of new organisms. It also occurs through the exchange of genetic
material but, not the exchange of gametes.
• Therefore, conjugation is not considered as a method of sexual reproduction. Moreover, the transferring of genetic
materials can be either plasmids or transposons.
• Bacterial conjugation is the method in which the genetic material or trans gene is transferred from one bacterial to
another cell followed by recombination.
• The bacterial conjugation can be classified into 3 types.
General bacterial Conjugation
This type of conjugation generally
occurs between F+ (have F factor)and
F- (devoid of F factor) cells.
Conjugation in Hfr strain
Here conjugation takes place in
HIGH FREQUENCY
RECOMBINANT STRAIN
Conjugation in F’ bacterial strain
Here conjugation occurs between F’ cells
and F- cells

14. GENE TRANSFER METHOD: CONJUGATION
General bacterial Conjugation Conjugation in Hfr strain Conjugation in F’ bacterial strain
1. F+ cell (consist extra genetic material called
as F factor or F plasmid) attaches to F- cell
(devoid of F factor) with the help of sex pilli.
2. Development of conjugation tube takes
place between donor and recipient cell.
3. F factor of donor cell is cut and transfers into
the recipient cell through tube.
4. The recipient cell synthesize the
complimentary stand of F factor and
becomes F+ cell.
5. As a result the formation of 2 F+ cell takes
place. (Fig 1.7)
Fig 1.7 General Bacterial conjugation
1. When F factor of
F+ cell gets
integrated with its
own chromosome
it develops a Hfr
strain sell (High
frequency
recombinant
strain). (Fig 1.8)
Fig 1.8 Hfr strain is the integrated
form of F+ plasmid
and its chromosomes
Fig 1.9 Conjugation in Hfr strain
2. Hfr attaches to the F- cell via sex pilli-
conjugation tube.
3. The integrated F factor and chromosomal
DNA fragment transfers into the F- cell.
4. The recombination of donor and recipient
DNA takes place followed by synthesis of
complete DNA
5. As a result formation of a recombinant cell
containing a part of Hfr +its own chromosome
is formed and Hfr cell regains its identity and
remains as it is.
1. It occurs between F’ cell and F- cell
2. The F’ cell is when from Hfr the F factor
de-integrates and remake in free state in
the cell such cell is called as F’ cell.
3. The attachment causes the formation of
conjugation tube.
4. The F’ factor cuts and transferred into the
F- cell via tube.
5. The F- cell will synthesise the complete
complementary F’ factor and becomes F’
cell. Simultaneously F’ cell repairs itself
and remain as it is.
6. As a result the formation of two F’ cells
takes place. (Fig 1.10)
Fig 1.10 Conjugation in F’bacterial strain

15. PLASMIDS AND TRANSPOSONS
• Plasmids and Transposons are two types of mobile genetic elements, which are involved in the transfer of genetic
material .
• Insertion sequences (IS) and episomes are the other types of mobile genetic elements.
• A plasmid is an extra-chromosomal, self-replicative DNA molecule that naturally occurs in bacteria. (Fig. 1.11)
• Transposon is a DNA sequence that moves around different positions within a genome due to which it is also called
jumping genes. (Fig 1.12)
• The main difference between plasmid and transposon is that plasmid transfer genetic material between genomes
whereas transposon transfer genetic material between chromosomes within the same genome.
Fig 1.11 The figure showing the Plasmid in the
bacterial cell other than chromosomal DNA- these
plasmids are extracted out and allowed to form a
recombinant DNA molecule to transfer genetic
material between genomes. Fig 1.12 The green coloured DNA sequence are the
Transposons which are moving from DNA A to DNA B -
“Jumping genes”. These jumping genes or transposons
transfer genetic material between chromosomes.

16. PLASMIDS AND TRANSPOSONS
• A plasmid refers to a genetic element that replicates independently of the chromosomes.
• Plasmids are double-stranded, circular DNA molecules, occurring in the cytoplasm of the bacteria, archaea, yeast,
and protozoans.
• The size of a plasmid may vary from 1 – 1,000 kbp. One to thousands of different plasmid types can be identified in
different types of cells.
• The main function of plasmids in nature is to participate in conjugation, which is a mechanism of horizontal gene
transfer (HGT). HGT refers to the movement of genetic material between organisms.
• Features of Plasmids:
•Can self-replicate inside a cell
•Can be readily isolated from cells
•Have a unique restriction site for one or more restriction enzymes
•The insertion of a foreign DNA piece may not alter its replication properties
•Do not occur freely in nature
Five classes of plasmids can also be identified based on their function:
1. F-plasmids – F plasmids contain tra genes. Which are capable of expressing sex pilli during conjugation.
2. Resistance plasmids – Resistance plasmids contain genes that provide resistance to antibiotics or poisons. It was
historically known as R-factors before the nature of plasmids was understood.
3. Col plasmids – Col plasmids contain genes that code for bacteriocins, proteins that can kill other bacteria.
4. Degradative plasmids – Degradative plasmids enable the digestion of unusual substances such as toluene and
salicylic acid.
5. Virulence plasmids – Virulence plasmids turn the bacterium into a pathogen.
PLASMID

17. PLASMIDS AND TRANSPOSONS
• A transposon refers to a chromosomal segment that can be translocated between chromosomal, plasmid or phage DNA.
• Transposons are also known as transposable elements (TE).
• Transposons cause mutations in the genome.
• During transposition, the size of the genome can be either increased or decreased. As transposons may contain genes,
they are known as jumping genes.
• Transposon, class of genetic elements that can “jump” to different locations within a genome. Although these elements
are frequently called “jumping genes,” they are always maintained in an integrated site in the genome.
• Three basic types of transposons have been identified. These include class
TRANSPOSONS
Retrotransposons
(class I transposons).
II transposons
Miniature inverted-repeat transposable elements
(MITEs, or class III transposons)
• Class II Transposons are segments of DNA that
move from one place to another via a “cut and
paste” mechanism.
• Most of these elements encode an enzyme called
transposase, which acts to cleave the ends of the
transposon, to free it from its initial location in the
genome.
• Transposase also cleaves target sites where the
element is to be inserted.
• Once the transposon is ligated (bound) into its new
position, gaps that are left in the DNA sequence
are filled in through the synthesis of nucleotides.
• Class II transposons range in length from 1,000 to
as many as 40,000 base pairs.
• MITEs are characterized by their short lengths,
generally about 400 to 600 bp, and of about 15
base pairs that occurs at each end of each
element in an inverted fashion (as mirror
sequences).
• Thousands of MITEs have been identified in the
genomes of Oryza
sativa (cultivated rice), Caenorhabditis
elegans (a type of nematode), and other
organisms.
• Unlike some types of transposons, MITEs do
not appear to encode proteins.
• Retrotransposons represent a highly unique group
of transposable elements and form large portions of
the genomes of many eukaryotes.
• Retrotransposons function by a “copy and paste”
mechanism. Thus, they leave behind the original
copy and generate a second copy that is inserted
elsewhere in the genome. This process results in
the insertion of repetitive sequences of DNA
throughout the genome.
• The first step in retrotransposition occurs when the
transposable DNA is copied into RNA. The RNA
segment then jumps to another location in the
genome.

18. PLASMIDS AND TRANSPOSONS
PLASMIDS TRANSPOSONS
• A plasmid refers to a genetic element that replicates
independently.
• A transposes refers to a chromosomal segment that can
be translocated between chromosomal, plasmid or
phage DNA.
• An extra chromosomal, self replicative DNA molecule,
naturally occurring in bacteria.
• A DNA sequence that moves around different positions
within a genome.
• Consist of an origin of replication, promoter, antibiotic
resistance genes, and multiple cloning sites.
• Consist of coding region of transposase, transposable
genes and terminal repeats.
• F plasmids, resistance plasmids ,col
plasmids ,degradative plasmid and virulence plasmids
are the five classes.
• Class II transposons, Miniature inverted repeats and
Retrotransposons are the three classes.
• Used to insert new genes into the genome of another
organism.
• Act as mutagens that sometimes cause genetic diseases.
• Plasmids can be used to insert new genes into the
genome of another organism.
• Transposons are mutagens that sometimes cause
genetic diseases.
• Plasmids are used as vectors to produce recombinant
DNA.
• Transposons are used as vectors to insert several bases
in insertional mutagenesis.
• The difference between PLASMIDS and TRANSPOSONS are as follows:
Table 1.3 The difference between PLASMIDS and TRANSPOSONS

19. STUDY OF PRODUCTION OF SEVERAL COMPONENTS
PENICILLIN
• Penicillin (PCN) is a group of antibiotics, derived originally from common moulds known as Penicillium moulds.
• It is a β- lactam antibiotic, discovered by Alexander Fleming.
• Originally, Penicillin was discovered from isolated strain of Penicillium notated , but this species gave low yield of
penicillin.
• The Fleming discovered Penicillium chrysogenum, that gave high yield of penicillin.
• The uses of Penicillin to treat infection like Respiratory tract infection, Pneumonia, Throat infection, Skin infection etc.
• Production of Penicillin: (Fig 1.13)
Inoculum:
1. Early strains of P.chrysogenum was isolated from mouldy spoiled fruits.
2. The isolated strains are allowed to get subjected to treatment with Mutagenic Agents like UV-radiation, X-ray and
mechlorethamine (Nitrogen Mustard). [for the purpose development of Highly productive mutant i.e Q-176 strain
of P.chrysogenum - which has the ability to produce maximum amount of Penicillin] .
3. Q-176 strain can produce more than 100units of penicillin/mL.
Cuture Media: (pH maintained at 7-7.5 and temperature -28 ℃)
1. Carbon source- lactose, Glucose, Nitrogen source- Yeast extract
2. N2 source- corn steep liquor solids.
3. Salt-calcium carbonate
4. Buffering agent- Potassium Hydrogen Phosphate.
5. Penicillin precursor - Phenyl acetic acid.
6. Oil- edible oil (cooling oil)

20. Performed the filtration , collected the filtrate and checked it pH.
The pH of filtrate was maintained between 2-2.5 by adding Phosphoric acid/Sulphuric acid
( to convert the resulting Penicillin into its anionic form).
The resulting fermented broth is immediately extracted with solvent like Amyl acetate/Butyl
acetate or Methyl isobutyl ketone by using Podbielniak-Counter Current Solvent Extractor.
The organic solvent extract is added to KOH/NaOH to get aqueous form of Potassium/Sodium salts of Penicillin.
Resulting aqueous solution was again acidified and re-extracted with Methyl isobutyl ketone
(The Shifting of Aqueous to Organic solvent or vice-versa helps in ultimate purification). The resulting Solvent extract is
finally subjected o back-extracted with KOH/NaOH to get aqueous medium.
The Penicillin was crystallised , washed ,dried and preserved.
STUDY OF PRODUCTION OF SEVERAL COMPONENTS
PENICILLIN
Process: (Optimum pH 7-7.5 and temperature 28 ℃)
1. The culture is kept for 20-30 hours for rapid growth.
2. The actual yield of penicillin is carried out within 48-96 hours.
3. The complete in fermentation is indicated by pH rise to 8.
Purification and Recovery: After completion of fermentation
Discard the mycelium
and other solid residue
Flowchart 1.2 Production of penicillin

21. STUDY OF PRODUCTION OF SEVERAL COMPONENTS
PENICILLIN
Fig 1.13 Flowchart representing the commercial production of Penicillin

22. STUDY OF PRODUCTION OF SEVERAL COMPONENTS
CITRIC ACID
• Citric is one of the most common acid found in primarily in several Fruits and Vegetables.
• It is used as food additive, Preservative, in cosmetics etc.
• It is a weak organic acid used as natural preservative , food and pharmaceutical industry.
• Citric avid was first isolated in 1783 by Swedish chemist CARL WILHELM SCHEELE, who crystallised it from
lemon juice.
• Large number of micro-organisms including bacteria (Bacillus licheniformis, Corynebacterium paraffins etc), fungi
(Aspergillus nagger, Penicillium janthinelum etc.) and yeasts(Saccahromicopsis lipolytica, Hansenula anamosa etc)
have been employed to produce citric acid.
• Fermentation is the most economical and widely used way of citric acid synthesis.
• The industrial citric acid production can be carried in following Three ways:
SURFACE FERMENTATION
• Surface fermentation uses
Aspergillus niger , on rice bran, or
in liquid solution in flat
aluminium or stainless steel pans.
• Special strains of Aspergillus
niger which can produce citric
acid despite of rice bran due the
high content of trace metals, are
used.
SUBMERGED FERMENTATION
• In this case , the strains are
inoculated of about 15cm depth
in fermentation tank.
• The culture is enhanced by giving
aeration using air bubbles and
kept it for 5 to 14 days.
• The citric acid is produced in the
fermentation tank and is purified.
SOLID STATE FERMENTATION
• It is simplest method for citric acid
production.(Koji process).
• Citric acid production reached a
maximum(88g/kg dry
matter)when fermentation is
carried out with cassava having
initial moisture of 62% at 26℃ for
120 hours.

23. STUDY OF PRODUCTION OF SEVERAL COMPONENTS
CITRIC ACID
• Separation:
-The biomass is separated by filtration
-The Liquid id transferred to recovery process.
-Separation of citric acid from the liquid precipitation.
-Calcium hydroxide is added to obtain Calcium citrate.
Tetra
hydrate
Wash the
precipitate
Dissolved it with dilute sulphuric
acid, yield citric acid and calcium
sulphate precipitate.
Bleach and
crystallisation
Anhydrous
mono hydrate
citric acid
• Purification:
-Purification is a simple process of getting pure product i.e. Citric acid.
- Here purification occurs by => Precipitation & =>Filtration.
The reaction
between Citric acid
and calcium
carbonate was
allowed
Filter and
precipitate
Sulphuric acid is allowed
to react with precipitate
Filter and
precipitate
Purified citric
acid

24. STUDY OF PRODUCTION OF SEVERAL COMPONENTS
VITAMIN B12 (Cyanocobalamin)
• Vitamin B12 (Cyanocobalamin) is produces commercially by Streptomyces species i.e. from Streptomyces oliveceus.
• The fermentation is done in the medium composed of Glucose, Corn steep liquor,Cobalt chloride.
• Best Method of production: the latest method adopted these days for production is carried out by using the strains of
Propionibacterium species- Propionibacterium shermanii. Or from pseudomonas species.
• The uses of it to treat neuronal disorders, to treat Megaloblastic anaemia (Vitamin B12 deficiency) etc.
• Three different types of medium are used in the production of Vitamin B12 from the species of Propionibacterium
shermanii.
MAINTAINANCE MEDIUM SEED CULTURE MEDIUM MAIN CULTURE MEDIUM
• Microbes are added to
maintenance medium to develop
culture.
• Composition -
- Thytome
- Yeast extract
- Filtered tomato juice
- Agar
• pH maintained at 7.2
• Incubation period 96hours
• Temperature 30℃
First stage
medium
• Composition-
- Thytone
- Yeast extract
- Filtered tomato
juice
• Incubation
period 48hours
• Temperature
30℃ without
agitation.
Second stage
medium
• Composition-
- Glucose
- Corn Steep
Liquor
• pH maintained at
6.5
• Incubation
period 24 hours
• Temperature
30℃
• Composition -
- Glucose
- Corn steep liquor
- Cobalt chloride
• pH maintained at 7.0
• Incubation period first 80hours
without aeration but with slight
introduction of N2 with agitation.
• Temperature 30℃

25. STUDY OF PRODUCTION OF SEVERAL COMPONENTS
VITAMIN B12 (Cyanocobalamin)
• General steps of production process :
S.olivaceus is allowed to grow at constant aeration at 27℃ in culture medium(Glucose as carbon source and cobalt
chloride as cobalt source as precursor).
Total fermentation process last upto 3-4 days or until such time when mycelium lysis takes place(before autolysis
occur because a major portion of Vitamin produced ,remains within microbial cell until autolysis occur .
After completing incubation, mycelium is collected before autolysis and destruction of vitamin occurs.
Mycelium and the fermented broth are separated either by filtration or by centrifugation(dried form of mycelium can
be used as vitamin B12 -enriched animal/poultry feed supplement)
Now the filtrate (mycelium culture) and fermentation broth is acidified / treated with alcohol.
Treated with sodium sulphite (to stabilise the vitamin)
Heated the content by steam heated coils along with proper agitation in order to obtain the vitamin.
The mixture is again filtered (to eliminate mycelial growth)
The filtrate is concentrated and evaporated to dryness under vacuum ( in certain cases it is further filtered and treated
with acetone and ion-exchange resin to obtain ultra purified crystalline form of Vitamin B12.
Flowchart 1.3 Production of Vitamin B12

26. STUDY OF PRODUCTION OF SEVERAL COMPONENTS
GLUTAMIC ACID
• Glutamic acid is an α- amino acid that is used in biosynthesis of proteins.
• It is non-essential in humans i.e. the body can synthesise on its own.
• Amino acids are important as Nutrients(food), flavouring and starting material for pharmaceutical cosmetics and other
chemicals.
• Amino acids always play an important role in biology of life , biochemistry and industrial applications.
• Large scale chemical and microbial production processes have been commercialised for the production of number of
essential and non-essential amino acids.
• L-glutamic acid is one of the major amino acids that is present in a wide variety of foods. It is mainly used as a food
additive and flavour enhancer in the form of sodium salt. Corynebacterium glutamicum is one of the major organisms
widely used for glutamic acid production.
• In biotechnological processes, Corynebacterium species are used for economic production of glutamic acid by
submerged fermentation .
• Glucose is one of the major carbon sources for production of glutamic acid.
• Glutamic acid was produced with various kinds of raw materials using sub-merged fermentation of palm waste
hydrolysate , cassava starch , sugar cane bagasse (dry pulpy fibrous residue) , date waste.
• The manufacturing of Glutamic acid by fermentation comprises - Fermentation, isolation and purification are as
follows:

27. STUDY OF PRODUCTION OF SEVERAL COMPONENTS
GLUTAMIC ACID
The glutamic acid crystal is added to the sodium hydroxide solution and converted into MonoSodium Glutamate
(MSG)
MonoSodium Glutamate (MSG) is more soluble In water , and is cleaned by using active carbon.
The cleaned MonoSodium Glutamate (MSG) is concentrated by heating and the monosodium glutamate crystal is
formed.
The monosodium glutamate crystal produced are dried in a closed system under hot air
The crystals are packed and ready for use.
Sugar cane is taken and squeezed to make syrup and sterilised.
The heated sterilised raw material and other requirements are put it into the Fermentor.
The micro-organism Corynebacterium glutamicum which produces glutamic acid is added to the fermentation broth.
The micro-organism reacts with sugar to produce glutamic acid
The Fermentation broth is acidified and glutamic acid is crystallised.
After the Fermentation process, specific method is require to separate and purify the amino acid product, which
include- centrifugation, filtration, crystallisation , ion exchange, evaporation etc.
Flowchart 1.4 Production of Glutamic acid

28. STUDY OF PRODUCTION OF SEVERAL COMPONENTS
GRISEOFULVIN
• Griseofulvin is an Anti fungal drug .
• Its was first isolated from Penicillin griseofulvin as secondary metabolite in 1939.
• It is used for the treatment of Ringworm infection on body.
• The process includes several process are as follows:
• Preparation of fermentation medium: Czapek dox medium
-Composition Glucose, Sodium Nitrate, Potassium Hydrogen Phosphate, Magnesium sulphate.
• Steps included in manufacturing process are:

29. STUDY OF PRODUCTION OF SEVERAL COMPONENTS
GRISEOFULVIN
Fermentation: Adjusted the pH of medium between 6-7.2 and added into the fermenter.
Added the suspension of fungus which is obtained from Rapper steep agar (Czapek dox medium+Corn steep+Agar)
The mixture is allowed to ferment for 14days at 24℃
Pre-treatment of fermentation broth: After the fermentation , the broth is heated at 60℃ for 20-30min or at 80℃
for 5-10min.(heating offers suitable flow rate for filtration process or improvement in separation features of broth)
Filtration: the Broth is filtered big using rotatory vacuum filter
Solid residue form cake Filterate
Washed with water
Immediately dewatered by blowing air over it
Used as animal feed supplements
Solid cake can immersed into the medium and filtrate can be gain collected
Extraction: Filtrate is extracted with cold Acetone which is highly efficient
solvent to extract Griseofulvin - 75-96%
De-coloration of extract: by Calcium hydroxide at more the pH 10
Isolation and separation : Impurities are removed by washing with Hexane
Precipitation and purification: the separated product is added with alkaline water(ammonia+water/
alkali metal carbonates/alkali metalhydoxideso at pH 8.5) which allows precipitation of Griseofulvin .
Precipitate are purified by menthol followed by drying and collection of final product
Flowchart 1.5 Production of Griseofulvin

30. BLOOD PRODUCTS: COLLECTION, PROCESSING AND STORAGE
WHOLE HUMAN BLOOD
PROTHROMBIN
(INACTIVE ENZYME)
THROMBIN
(ACTIVE ENZYME)
FIBRINOGIN FIBRIN
(BLOOD CLOT)
THROMBOPLASTIN AND CALCIUM IONS
THROMBIN
• Blood is a body fluid in humans and animals that carries Essential constituents like nutritions and oxygen to the cells
and allows the metabolic waste aways from cells.
• Any person with good health can be a donor.
• Donor shouldn't suffer from any transmitted disease.
• Haemoglobin content shouldn't less than 12.5g/dl of blood for female and 13.5g/dl of blood for male. Blood is
around 7% of whole body weight with total volume of 5-7 L of which comprises of plasma and different kinds of
cells.
• Whole blood is combination of cellular components, colloids and crystalloids.
Collection-
• Blood is collected aseptically from median cubital vein, in front of the elbow into a sterile container containing anti
coagulant.
• During collection the container is gently shaken, to ensure that blood and anti coagulate are mixed well which
prevents the formation of Fibrin clots.
• At a time not more than 420mL blood is taken.
• The collected blood is sealed and freezes at 4-6℃
• Blod clotting factors are:

31. BLOOD PRODUCTS: COLLECTION, PROCESSING AND STORAGE
WHOLE HUMAN BLOOD
• Heparin- naturally occurring anti-coagulant, expensive and looses its anticoagulant activity in in-vitro condition.
• EDTA Di sodium salt- chelating agent and prevents blood coagulation but less stable.
• Citrates-Acid citrate dextrose(ACD) solution is the most commonly used Anti coagulant.
Processing or testing after collection:
• Rh system- it consist of 50 different types of blood group antigens also determine that whether donor’s blood is
having Rhesus factor or not i.e. Rh+/Rh-
• Rh factor- group of antigen in RBCs. This antigen will cause agglutination when it reacts with antibodies from
Individuals without this antigen.
• Determination of ABO grouping and determination of transmitted disease .
Storage:
• Sterile container/ disposable bag containing anticoagulants.
• At temperature 4-6℃ until required for use. Can be stored for 42days.
• If stored ant room temperature (for 1 day), it may reduce post transfusion survival of Erythrocytes.
Use:
• To be given to treat every haemorrhage, uncontrolled Diarrhoea and vomiting.
• Major surgery etc.
Groups Can donate to Can receive from Comments
A A and AB A and O
B B and AB B and O
AB AB A, B,AB and O Universal acceptor
O A, B,AB and O O Universal donor
Table 1.4 Blood grouping

32. BLOOD PRODUCTS: COLLECTION, PROCESSING AND STORAGE
DRIED HUMAN PLASMA
• In some circumstances , dried human plasma is used as substitute for whole blood.
• The major advantage is , if it is stored properly can be used upto 5 years, if protected from light can be kept at 20℃
from refrigerator, can be given to patients of any blood group.
Collection:
• The whole human citrated blood is used as source and supernatant fluid Is separated by centrifugation.
• Batches of not more than 10 bottles are collected , mixed in correct ratio to neutralise Agglutinins (plasma antibody).
• The mixture is stored at 4-6 ℃ (sterility testing is also done periodically).
Processing:
• Preliminary freezing- 400 mL mixture is taken in the bottles and sealed with bacteriologically efficient pads and
centrifuged at -18℃. The liquid snap-freezes and become distressed inside the bottle.
• Primary drying- the bottles of frozen material are mounted horizontally in the drying chamber under high vacuum for
2 days after which the moisture content of residual is about 2%.
• Secondary drying- then the bottle are transferred to another chamber by vacuum desiccation over phosphorus
peroxide.
• For one day product is left with only 0.5% moisture content.
Storage:
• Dried plasma kept below 20 ℃, protected away from light, moisture and oxygen.

33. BLOOD PRODUCTS: COLLECTION, PROCESSING AND STORAGE
DRIED HUMAN PLASMA
• Direction of use: Dried plasma should be reconstituted in
- Water for injection
- Sodium chloride injection
- Solution containing 2.5% dextrose and 0.45% sodium chloride.
Must be used immediately after reconstitution.
• Dried plasma must be soluble in these solvents and form solution within 10min.
• If gel formation took place / incomplete solution is obtained . It indicates dried plasma is deteriorated.
Uses :
• Mostly preferred in the treatment of severe burns where excessive loss of fluid and protein occurs.
• May be given as substitutes of whole human blood (condition- in emergency whole Human blood is not available/
blood group matching tests are not known)

34. BLOOD PRODUCTS: COLLECTION, PROCESSING AND STORAGE
PLASMA SUBTITUTES
• These are of non human origin , which could be use to restore the blood volume temporary.
• Properties of such substitutes are:
- Should have same osmotic pressure as whole blood.
- Should have same viscosity as plasma.
- Should have low rate of excretion but must completely eliminate form the body.
- Free from antigen, pyrogen and must be sterile.
- Stable in liquid form at normal and sterilising temperature.
- Economic and easy to prepare.
• Examples of Plasma substitutes:
1. Gum saline- (injection of sodium chloride and Acacia) British Pharmacopeia in 1932 prepared Gum saline by
mixing 6% Acacia in 0.9% NaCl solution.
2. PVP- (Polyvinylpyrrolidone) in 1950’s century it was used a plasma substitute but has major problem was its
Carcinogenic nature.
3. Dextran- It is the most satisfactory plasma substitute.it was discovered by LOUIS PASTEUR as microbial product in
wine.

35. The dextran produced may have high molecular weight which cannot be used as plasma
substitutes as it may yield very viscous solution which may not be administered parenterally.
BLOOD PRODUCTS: COLLECTION, PROCESSING AND STORAGE
PLASMA SUBTITUTES
DEXTRAN
• Inoculum - Leuconostoc mesenteroides. This organism secrets an enzyme Dextran sucrase which converts sucrose
into dextran.
• Medium composition - Sucrose, peptones, yeast extract, Dipotassium hydrogen phosphate, manganese chloride
and calcium chloride.
• Prepration- Fresh culture of L.mesenteroides is added to fermentation medium.
Fixed the fermentation at 80 ℃ and incubate for 20 hours at pH 6-8
fermentation broth is added with equal volume of chilled ethanol for precipitation of dextran
Centrifuged at 10,000rpm for 15min
Discarded Supernatant Precipitates are dried under in vacuum u in presence of Calcium chloride
It may cause renal damage and allergic reaction. So molecular weight of Dextran can be
reduced by following three methods I.e.Acid hydrolysis, Thermal Degradation and Ultra
sonication.
Flowchart 1.6 Production of Dextran

36. BLOOD PRODUCTS: COLLECTION, PROCESSING AND STORAGE
PLASMA SUBTITUTES
• Quality control tests performed- Pyrogen testing , Sterility testing and Serological testing for determination of
antigen. Example: injections like Dextran 110 and Dextran 40.
• Uses- in treatment of severe burns and in treatment of acute Peritonitis.
Three methods of molecular weight reduction of dextran
Dextran hydrolysis
• pH of Dextran solution is
adjusted to 2 at 90℃. This
reduces viscosity of the
preparation due to decrease
in molecular weight.
Thermal Degradation
• Solution of dextran is heated
under pressure at 160℃ in
presence of Sodium Sulphate
and calcium carbonate.
Ultra Sonication
• Dextran Solution is ultra-
sonicated to split the large
molecules into smaller one
• Acceptable range of
molecules weight of dextran
is 10,0000(1 lakh) to
2,50,000 (2lakh
50thousand) daltons.

37. REFERENCES
1. https://www.nature.com/subjects/microbial-genetics
2. https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-cloning-tutorial/a/bacterial-
transformation-selection
3. https://www.thermofisher.com/in/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-
of-molecular-biology/molecular-cloning/transformation/bacterial-transformation-workflow.html
4. https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Boundless)/
7%3A_Microbial_Genetics/7.11%3A_Genetic_Transfer_in_Prokaryotes/7.11C%3A_Bacterial_Transduction
5. https://www.merriam-webster.com/dictionary/transduction
6. https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/bacterial-conjugation
7. https://www.asmscience.org/content/book/10.1128/9781555817923.chap6
8. https://www.acs.org/content/acs/en/education/whatischemistry/landmarks/penicillin.html
9. http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822010000400005

38. THANK YOU
PRESENTED BY : SHYAM BASS
B.Sc BIOTECHNOLOGY (HONS), B.PHARMA
LOVELY SCHOOL OF PHARMACEUTICAL SCIENCES
e-mail: shyambass0925@gmail.com