Detailed Notes on Immobilization Techniques

Detailed Notes on Immobilization Techniques

1. Introduction
• In a continuous culture higher dilution rates can lead to
- higher biomass productivity
But
- higher substrate concentrations in the effluent
and lower biomass concentrations in the reactor
• When the dilution rate exceeds the critical dilution rate
then washout occurs.

2. Introduction
•In chemostat processes similar consequences can occur. If the
substrates are expensive, e.g animal cell culture, high dilution
rates can dramatically affect process profitablility.
•Immobilizing cells in the fermenter ensures that cells do not
washout when the critical dilution rate is exceeded.
•By immobilizing the cells in the fermenter, high cell numbers
can be maintained at dilution rates which exceed µ
m
.
•Therefore in an immobilised continuous fermenter system high
cell counts can be maintained leading to higher biomass
productivity as compared to a normal chemostat.

3. Advantages over
suspension cultures
(1). Immobilisation provides high cell concentration
(2). Immobilisation provides cell reuse and eliminates the
costly processes of cell recovery and cell recycle
(3). Immobilisation eliminates cell washout problems at
high dilution rates
(4). Combination of high cell concentrations and high flow
rates allows high volumetric productivities
(5). Favourable microenvironmental conditions
(6). Improves genetic stability
(7). Protects against shear damage

4. Advantages of immobilised
cell reactors
•Being able to maintain high cell concentrations in the reactor at
high dilution rates provides immobilised cell bioreactors with
advantages over chemostats.
•More biomass means that the fermenter contains more
biocatalysts, thereby high bioconversion rates can be achieved.
•Immobilised cell bioreactors are also more stable than
chemostats.
•The higher productivity and greater stability of immobilized
fermenters thus leads to smaller fermenter requirements and
considerable savings in capital and energy costs.

5. Support selection
The support selection is one of the crucial decisions to be made in the course of
preparation of the immobilization process. Selection criteria differ among one
another, depending on the biocatalyst of interest, but there are still few basic
features that must be considered.
•Material used as a carrier should have chemical, physical and biological stability
during processing, as well as in the reaction conditions; sufficient mechanical
strength, especially for its utilization in reactors and industry; should be nontoxic
both for the immobilized cell/bioparticle, as well for the product; also should have
adequate function groups for binding biocatalyst and high loading capacity.
•Profitability of the material application and its processing costs always have to be
taken upon consideration.
•Other criteria, such as physical characteristics (porosity, swelling, compression,
material and mean particle behaviour), as well as possibility for microbial growth,
biodegradability, solubility, are more application specific.

6. Types of immobilisation
•Active immobilisation
•Passive immobilisation

7. Active immobilisation
•Is entrapment or binding of cells by physical or
chemical forces
•Physical entrapment within porous matrices is the
most widely used method of cell immobilisation
•Immobilised beads should be porous enough to allow
transport of substrates and products in and out of the
beads

8. Active immobilisation
Beads can be prepared by

1) Gelation of polymers
2) Precipitation of polymers
3) Ion exchange gelation
4) Polycondensation
5) Polymerisation
6) Encapsulation

9. Matrix Entrapment
The solution is mixed with a polymeric fluid that solidifies
into various forms, depending on application (usually small
beads). The polymeric material is semi-permeable. Large
molecular weight enzymes can not diffuse out, but smaller
substrate and product molecules can.
Matrices for Entrapment
• Ca-alginate
• Agar
• Polyacrylamide
• Collagen

10. Membrane Entrapment
Enzymes solution may be confined between thin semi-
permeable membranes.
Membrane materials include;
• Nylon • Cellulose
• Polysulfone • Polyacrylate
Membrane Configurations
Hollow fiber configuration is a popular arrangement for
separating enzyme from substrate and product solution.

11. •One major problem encountered in immobilizing cells with physical
entrap
­ment is the lack of expansibility of the conventional cell carriers;
cell growth is restricted in the carrier, resulting in limited catalyst life as
well as limited cata
­lyst stability (Joung and Royer, 1990). Conventional
carriers such as polyacry- lamide gels or carrageenan gels, which have
frequently been used for cell im
­mobilization, have limited expansibility.

12. Passive immobilisation
•Biological films
•The multilayered growth of cells on solid support
surfaces
•The support material can be inert or biologically active
•Biofilm formation is common in natural and industrial
fermentation systems, i.e biological wastewater
treatment and mold fermentations

13. •Polymer producing organism facilitate bio film
formation and stability
• Micro environmental condition always vary
• Nutrient and product profile affects cellular
physiology & metabolism
• Thickness of the film directly relates to performance
of the system:
–Thin - low rate of bioconversion
–Thick – Diffusionally limited growth, nutrient depleted region
• Rate limiting nutrient – D.O

14. Types of immobilized cell
reactors
•There are many types of immobilized cell reactors
either in use or under development.
•In this section we will look at four major classes of
immobilized cell reactors:
Cell recycle systems
Fixed bed reactors
Fluidized bed reactors
Flocculated cell systems

15. Cell recycle systems
•In a fermenter with cell recycle the cells are
separated from the effluent and then recycled
back to the fermenter; thus minimising cell
removal from the fermenter:

16. Effluent
Biomass recycle
Fermenter
Fresh feed
Biomass
separation
system
Cell recycle system

17. Cell recycle systems
•Cell recycle is used in activated sludge systems. A portion of
the cells are separated in a settling tank and returned to the
activated sludge fermenter.
•Biomass recycling for product or biomass production is more
difficult due to the need for maintaining sterility during cell
separation. Centrifugation which is a faster process than
settling would be used to separate the cells.
•Biomass recycle systems can be easily modelled.

18. Fixed bed reactors
•In fixed bed fermenters, the cells are immobilized by
absorption on or entrapment in solid, non-moving solid
surfaces.

19. Fixed bed reactors
•In one type of fixed bed fermenter, the cells are immobilized
on the surfaces of immobile solid particles such as
plastic blocks
concrete blocks
wood shavings or
fibrous material such as plastic or glass wool.
•The liquid feed is either pumped through or allowed to trickle
over the surface of the solids where the immobilized cells
convert the substrates into products.

20. Fixed bed reactors
•Once steady state has been reached there will be a
continuous cell loss from the solid surfaces. These
types of fermenters are widely used in waste treatment
•In other types of fixed-bed fermenters, the cells are
immobilized in solidified gels such as agar or
carrageenin

21. Fixed bed reactors
•In these fermenters, the cells are physically trapped inside
the pores of the gels and thus giving better cell retention
and a large effective surface area for cell entrapment.
•In order to increase the surface area for cell immobilization,
some researchers have investigated the use of hollow
fibres and pleated membranes as immobilization surfaces.
•Industrial applications of fixed bed reactors include
waste water treatment
production of enzymes and amino acids
steroid transformations

22. Fluidised bed reactors
•In fluidized-bed fermenters the cells are immobilized on or
in small particles.
•The use of small particles increases the surface area for
cell immobilization and mass transfer.
•Because the particles are small and light, they can be
easily made to flow with the liquid (ie. fluidised).

23. Small
moving
particles
Fluidised bed reactors

24. Fluidized bed reactors
•The fluidisation of the particles in the reactor leads to
the surface of the particles being continuously turned
over. This also increases the mass transfer rate.
•Fluidised beds are typically categorized as either
being a
2 phase system which are not aerated and
3 phase system which is aerated by sparging
•Fluidized bed bioreactors are used widely in
wastewater treatment.

25. Fluidized bed reactors
•Fluidized bed bioreactors are also used for animal cell
culture.
•Animal cells are trapped in gels or on the surface of
special particles known as "microcarriers".
•Fluidized bed reactors are one example of perfusion
culture technology used for animal cell culture.

26. Comparing fluidised bed
and fixed reactors
• Fluidised bed reactors are considerably more efficient than
fixed bed reactors for the following reasons:
1) A high concentration of cells can be immobilized in the
reactor due to the larger surface area for cell immobilization is
available
2) Mass transfer rates are higher due to the larger surface
area and the higher levels of mixing in the reactor.
3) Fluidised bed reactors do not clog as easily as fixed bed
reactors.

27. Comparing fluidised bed
and fixed reactors
•Fluidised bed reactors are however more difficult to
design than fixed bed reactors.
•Design considerations include:
Setting the flow rate to achieve fluidisation
Ensuring that bubble size remains small
during the fermentation.
Prevention of the cells from falling or
"sloughing" off the particles.
Minimising particle damage.

28. Flocculated cell reactors
•In flocculated cell reactors, the cells are trapped in the
reactor due to an induced or natural flocculation process.
In flocculation cells tend to group together causing them to
come out of solution and to sink towards the base of the
reactor.
•Flocculated cell reactors are used widely in anaerobic
waste treatment processes.
•In these reactors, the methanogenic and other bacteria
form natural flocs. The flocs move due to the release of
methane and carbon dioxide by the cells.

29. Flocculated cell reactors
Cells form flocs which
gently fall and rise with
gases they produce

30. Limitations
(1). Often the product of interest has to be excreted
from the cell
(2). Complications with diffusional limitations
(3). Control of microenvironment conditions is difficult
due to heterogeneity in the system
(4). Growth and gas evolution can lead to mechanical
disruption of the immobilised matrix

31. Conclusions
1. Increased reaction rates e.g. higher flow rates
2. Higher cell densities
3. Repeated use of biocatalyst
4. Minimal cost for cell separation