how to increase production of secondary metabolite by using cell immobilization technique.

1. PLANT CELL IMMOBILIZATION
PRESENTED BY
MR. SURAJ B. MANATKAR
B-PHARM SEMESTER VIITH
UNDER GUIDANCE OF
PROF. SHANKUL KUMAR
M-PHARM
DEPT. OF PHARMACOGNOSY
S. G. S. P. S. Institute of Pharmacy, Akola
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2. CONTENT:
 Introduction.
 Need of Immobilization.
 Immobilization Technique.
 Viability testing.
 Bioreactor.
 Advantages.
 Disadvantages.
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3. Definition :
Immobilization is technique, which confines to a
catalytically active enzyme or to cell within reactor
system and prevents its entry into the mobile phase,
which carries substrate and product.
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4. Need of Immobilization
 Plant cell culture has been use for production of secondary
metabolite.
 Characteristics of plant cell culture such as slow growth,
large size, sensitive to shear and oxygen and need of cell to
cell contact for metabolite production, the compound
produced should be of high value and low volume.
The use of high biomass level for extended period
would be one method of increasing productivity and hence
reducing the costs this can be achieved by the
immobilization of plant cell.
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5. Different types of immobilization technique.
1. Physical retention within the framework of different pore size and
permeability.
a. Entrapment
b. Micro-encapsulation
2. Direct intracellular binding due to natural affinity.
a. Adsorption
b. Adhesion
c. Agglutination
3. Intracellular connection via bi or poly functional reagent.
a. Cross – linking
4. Mixing with suitable material changing their consistency with
temperature.
a. Embedding.
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6. Selection of immobilization system.
 The polymer material used for immobilization must be
available in large quantities, inert, non-toxic, cheap.
 Able to carry large quantities of biomass and its fixing
potential must be high
 The immobilization process must not diminish
enzymatic activity of biological catalyst.
 Manipulation of biological catalyst must be as simple as
possible.
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7. METHOD
ENTRAPMENT :
1. Entrapment by polymerization.
2. Entrapment by ionic network formation.
3. Entrapment by precipitation.
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9. 1. Gel entrapment by polymerization
 A monomer or mixture of monomers is polymerized in the
presence of a cell suspension, which is entrapped cell inside the
lattice of the polymer.
 The method is based on the free radical polymerization of
acrylamide in an aqueous solution.
 The free radical polymerization of acrylamide is conducted in an
aqueous solution containing the cell and the cross linking agent.
 Initiator – N, N, N’,N’- tetramethy/ethylene
 An initiator & cross –linking agent are toxic to the cells &
therefore viability can be lost.
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10. 2.Gel entrapment by ionic network formation.
 In this method the polymerization of poly electrolyte is
achieved by addition of multivalent ions the most common
method is the entrapment in calcium alginate.
 This is non-toxic process in which sodium alginate solution
is dropped into mixture or counter ion solution such as
calcium chloride.
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11. Immobilization by Embedding :
 The temperature dependent solubility of macromolecules like
agarose, agar, carrageenam.
 Insoluble are formed under cold condition (agar) or in aqueous
solution of CaCl2 .
 Their structure in non-uniform, with different pore size at the
surface and in deeper layer.
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12. Encapsulation:
Encapsulation is the process of forming a continuous membrane
around cells to be immobilized that denote the core of the system in
which the inner matrix is protected by means of the outer membrane.
Liquid form of active substance is the core material and polymeric
wall is the outer membrane.
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13. Example :
Cells mixed in aqueous polymeric solution
Dispersed
Oil phase
stirred
Droplet formation
cool
Solidification of polymer
centrifugation
Microencapsulated beads collect.
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14. Adsorption/Surface Immobilization:
 The adhesion of cells on the surface of support matrix is initiated by the
attraction of cells on the matrix followed by adsorption.
 The interaction between the cells and matrix is provided by vander
Waals, electrostatic, hydration, and hydrophobic forces.
 For the immobilization of viable cells adsorption process is well suited
when compared with entrapment technique. This type of
immobilization is considered as one of the easiest technique.
 Fiberglass mats, unwoven short fibrepolyster.
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15. Viability testing of immobilized cells.
1. Viability staining
a. Fluroscein diacetate ( FDA) – green
b. Phenosafranin - Red
2. Plasmolysis:
Determine Integrity of Plasma membrane by adding
plasmolysing agent
e.g – glycerol & sorbitol.
3. Cell Growth.
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16. Bioreactor for Cell Immobilization.
1. Packed bed reactors :-
In this reactor, cells can be immobilizes
either on surface or throughout the support.
When the cell are immobilized through the
support the placed bed can accommodate
large no. of cells per reactor volume.
Disadvantage
 Law degree of mixing
 Large incompressible support particle are
needed.
 The packed bed reactor are having filter,
when the support particle fragment during
operation they will block the pathways for
fluid flow.
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17. 2. Fluidized Bed Reactors
It utilize energy of the flowing fluid to
suspend the particle.
Small immobilized particle are employed
Fluid flow rate should be sufficient to
suspend particle.
Large gas volume can be used to suspend
the immobilized cell while maintaining low
fluid flow rates.
These condition leads to large degree of
fluid mixing.
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18. Membrane Reactors
In these, the cells are separated from growth medium by
membrane are suitable for fragile cells which can be entrapped
more readily on membrane.
The environment in membrane reactor is more homogeneous.
a. Flat plate membrane reactor.
1. One side flow.
2. Two side flow.
b. Multimembrane reactor.
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19. a. Flat plate membrane reactor
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20. b. Multimembrane reactor
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21. Advantages of Cell Immobilization
1. It may enable prolonged use of biomass.
2.The entrapped cells are protected against shear, reduce problem of
aggregate.
3. High biomass level (compared to cell suspension culture).
4. Separates cell from medium and therefore if the product is
extracellular. It can simplify downstream processing .
5. It allows a continuous process, which increase volumetric
productivity and allow removal of metabolic inhibitors.
6.It uncouples growth and product formation which allows product
optimization without affecting growth.
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22. Disadvantages of Cell Immobilization
1. The efficiency of the production process depends on the rate
of release of product rather than actual rate of biosynthesis.
2. The immobilization process may reduce biosynthesis
capacity.
3. Product must be released from cell into medium
4. Secretion of secondary metabolite requires cellular transport
or artificial altered membrane permeability.
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23. Reference:
 Medicinal Plant Biotechnology by Ciddi Veeresham, first edition
2008 published by CBS Publisher and Distributors.
 Plant Tissue Culture by S. S. Bhojawani and M. K. Razdan,
First edition 2013 , published by Elsevier Pvt. Ltd.
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