This document discusses different methods for immobilizing whole cells, including perfusion bioreactors and biofilm formation. Perfusion bioreactors culture cells continuously over long periods by feeding fresh media and removing waste, while various separation methods like hollow fiber membranes or centrifuges keep cells in the bioreactor. Perfusion offers advantages like improved product quality, smaller reactor size, and lower costs compared to traditional fed-batch systems. The document also covers immobilizing cells through entrapment in polymers, attachment to surfaces, or passive biofilm formation on supports.

1. IMMOBILIZED WHOLE CELLS
AND CHARACTERISTICS
G.Vijay chithra – 117011101311
H.Vishalachi – 117011101313
R.Yokesh - 117012101314

2. Fed-batch bioreactors
• Traditional fed-batch bioreactor systems consist of tanks
that are usually between 10,000-25,000 liters. Cells are
cultured in batches that typically run between 7-21 days by
which time media nutrients have been consumed and toxic
waste has begun to accumulate. During the run, cells
secrete the protein of interest into the media and at the
end of the run the protein is separated from cell mass as a
batch. Typical product yields are in the range of 1 to 4
grams per liter depending on the clone and antibody. While
regular improvements have moved product yield from
under 1 gram per liter to where they are now, it has not
improved other issues in fed batch manufacturing including
large manufacturing footprints and challenges with
scalability.

3. Perfusion systems
• In contrast, perfusion bioreactors culture cells over
much longer periods, even months, by continuously
feeding the cells with fresh media and removing spent
media while keeping cells in culture. In perfusion
there are different ways to keep the cells in culture
while removing spent media. One way is to keep the
cells in the bioreactor by using capillary fibers or
membranes, which the cells bind to. Another does not
bind the cells, but rather relies on filtration systems
that keep the cells in the bioreactor while allowing
the media to be removed.

4. • Another method is the use of a centrifuge to separate cells and
return them to the bioreactor.
• EXAMPLES OF CELL SEPARATION METHODS:
• GE Healthcare’s Hollow Fiber Microfiltration Cartridges – “In this
system, the retentate consists of the cells, which flow past the
membrane and are sent back to the bioreactor. The spent
medium is the permeate that passes through the membrane.”
• ATMI’s iCELLis Single Use Fixed Bed – In this system, cells are
bound to custom microcarriers, which allows cells to stay in place
while media flows around them.
• Centrifuge method – In this system, a centrifuge is used to
separate cells from culture media and then cells are returned to
the bioreactor.
•

6. Advantages of Perfusion systems
• PRODUCT QUALITY AND STABILITY :
By continuously removing spent media
and replacing it with new media, nutrient levels are
maintained for optimal growing conditions and cell
waste product is removed to avoid toxicity. In addition,
the product is regularly removed before being
exposed to excessive waste that causes protein
degradation. Product is also harvested and purified
much more quickly, which is particularly helpful when
producing a product that is unstable.

7. • SCALABILITY
perfusion bioreactors are smaller in size and can produce
the same product yield in less space. Typically perfusion
bioreactors operate at 10-30x concentrations compared to
fed-batch bioreactors. For example, it has been shown that
a 50-liter perfusion bioreactor can produce the same yield
as a 1,000-liter fed-batch bioreactor. Therefore, the use of
perfusion should enable the replacement of typical 10,000
L bioreactors with 1,000L bioreactors without negatively
impacting the yearly yield of manufactured product.
•

8. • COST SAVINGS
perfusion bioreactors require less on
utilities cost and they are less labor intensive
to operate. Thus requiring significantly less
capital investment on the front end and less
on operating costs to manufacture the same
yield as fed-batch bioreactors.

9. • REMOVAL OF COMPONENTS:
potential removal of cell debris and
inhibitory by-products, removal of enzymes
released by dead cells that may destroy
product.

10. Disadvantages of Perfusion systems
• Large amount of medium used.
• Nutrients in the medium are less completely
usedd than in batch 0r fed-batch systems.
• High cost of rawmaterials.
• High cost for waste treatment

11. Immobilization of cells
INTRODUCTION :
common method : “ immobilization of cells
as biocatalysts”
• Cell immobilization can be defined as
entrapment or localization of living cells to a
certain region of space with preservation of their
metabolic and/or catabolic activity.
• Cell immobilization improves the efficiency of
the cultures by mimicking cell natural
environment.

12. Advantages of immobilised cell
cultures over suspension culture
• Provides high cell concentration
• Allows cell reuse and reduces cost of cell recovery and
cell recycle
• Eliminates cell wash out problems at high dilutions.
• High volumetric productivity = high cell concentration
+ high flow rates
• Provides favourable microenvironental conditions for
cells which leads to better performance of
biocatalysts.
• Improves genetic stability in some cases.
• Protection against shear damage for some cells.

13. Limitations of immobilization
• Product of interest should be excreated by
the cell
• It often leads to system for which diffusional
limitations are important.
• Control of microenvironmental culture is
difficult
• They can lead to mechanical disruption of
immobilizing matrix

14. Active immobilisation of cells
• Entrapment of cells in gel or behind semi-permeable membranes
is the most popular method for immobilization of plant cells.
• Some polymers used to entrap plant cells are alginate, agar,
agarose and carrageenan. Of these, alginate has been most
widely used because it can be polymerized at room temperature
using Ca 2+.
• Polyurethane foam has also been used to immobilize a range of
plant cells. Alternatively, plant cells can be entrapped by inclusion
within membrane reactors.
• A semi-permeable membrane is introduced between the cells
and the recirculating medium so that the cells can be packed at a
very high density under very mild conditions. Some designs of
membrane reactors are shown in Figure.

15. Fig A: Flat plate membrane reactor with
one side flow of nutrients
Fig B: Flat plate membrane reactor
with two side flow of nutrients
Fig C:Multi membrane reactor system

16. • Immobilization of cells on the surface of an inert
support, such as fibreglass mats and unwoven short
fibre polyester, has also been examined for in vitro
production of secondary metabolites.
• For surface immobilization of cells, a bioreactor (air
lift or mechanically agitated design), provided with
the support matrix, is inoculated with a plant cell
suspension of suitable density and operated for an
initial period as a suspension bioreactor. During this
period virtually all cells spontaneously adhere to the
surface of the support.

19. Advantages
Special advantage of this method over the other
methods of immobilization of cells is the absence of
any physical restriction to mass transfer between the
culture medium and the biomass surface.
Since the surface immobilized cells grow on the
surface of the support matrix, it should facilitate
visual monitoring of the conditions, distribution and
extent of the biomass and to routinely sample the
biomass, if desired.

20. PASSIVE IMMOBILIZATION:
BIOLOGICAL FILMS
• Biological films are the multilayer growth of cells
on solid support surfaces.
• The support material can be insert or biologically
active.
• Biofilm formation is common in natural and
industrial fermentation systems, such as
biological waste-water treatment and mold
fermentation.
• The interaction among cells and the binding
forces between the cell and support material may
be very complicated.

21. In mixed-culture microbial films;
• The presence of some polymer-producing organisms
facilitates biofilm formation and enhances the stability of
the biofilms.
• Micro environmental conditions inside a thick biofilm vary
with position and affect the physiology of the cells.
In a stagnant biological film;
• Nutrients diffuse into the biofilm and products diffuse out
into liquid nutrient medium.
• Nutrient and product profiles within the biofilm are
important factors affecting cellular physiology and
metabolism.
• Biofilm cultures have almost the same advantages as those
of the immobilized cell systems over suspension cultures.

23. Thickness of a biofilm:-
The thickness of the biofilm is an important
factor affecting the performance of the biotic
phase.
Thin biofilms – will have low rates of
conversion due to low biomass concentration
Thick biofilms – May experience diffusionally
limited growth, which may or may not be
beneficial depending on the cellular system and
objectives. Nutrient-depleted regions may also
develop within the biofilm for thick biofilms.

24. • In many cases, an optimal biofilm thickness resulting
in the maximum rate of bioconversion exists and can
be determined.
• In some cases,growth under diffusion limitations
may result in higher yields of products as a result of
changes in cell physiology and cell-cell interactions.
• In this case, improvement in reaction stoichiometey
(e.g., high yield) may overcome the reduction in
reaction rate, and it may be more beneficial to
operate the system under diffusion limitations.
• Usually, the most sparingly soluble nutrient, such as
dissolved oxygen, is the rate-limiting nutrient within
the biofilm.