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Kinetics is a study that deals with rate of the reaction. It is based on the mechanisms of
any processes-physical, chemical or biological. The chemical kinetics deals with how the
rate of reaction is dependent upon the concentration of the reactants. In a biochemical
reaction, kinetics is a little complicated. A living cell, during fermentation or any
biological processes, require essential nutrients and suitable environment to survive.
Hence a biochemical reaction proceeds with the intervention of microbial cells in a liquid
medium containing nutritive sources under conditions like pH, temperature. The cells
multiply and grow, consuming a part of substrate and other necessary components from
the fermentation broth. Cell growth is associated with two processes namely, uptake of
some material from the cells environment and consuming it into metabolic end
products.
A kinetic model describes the behavior of the cellular processes through possible
mathematical equations and it serves to be a very effective tool to test and eliminate the
extremities. Different kinetic models have been proposed in several research works for
microbial growth, substrate utilization and product formation in various fermentation
modes.
In order to construct a simple kinetic model, different approximation and
representations of cellular population are taken into consideration. An individual cell
in a liquid broth is a complicated multi component system, which is not spatially
homogenous even at a single cell level. Nutrient solution used for the inoculum growth
and production is also a multi component system containing all the complex nutrients for
cell growth and accumulates various products as the result of metabolic activities.
According to number of components used in microbial representations and considering
cells as heterogenous mass or wholly as average cell clusters, different perspective are
put forward for cell population kinetic representations.
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Fig 1: Certain important parameters, phenomena, and interactions which determine cell
population kinetics.
Structured model is one perspective in which the microbial cells are considered as
multicomponent systems. When cell population is treated as one component system, it
is referred to as unstructured model. The rate of reaction depends only on the
parameters inside the fermenter. These models only contain growth, substrate
consumption and product formation kinetics. Cell to cell heterogeneity and discrete cells
compose a multi component entity, constituting ultimately a segregated perspective. An
unsegregated view considers average cellular properties. In actual cellular cases, the
situation is a structured and segregated one. In balanced growth of cells, the metabolic
activities are coordinated in such a way that the average cell composition is not
influenced by increase in cell mass. In such cases, models that do not consider the multi
component nature of cells are necessary. Hence unsegregated, unstructured model is
the most idealized situation when analyzing the growth of cells.
Models Unstructured Structured
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Examples of Kinetic Models:
1. Structured Model: Williams model
2. Unstructured Model: Monods model, Malthus model
Advantages:
1. The general strength of the kinetic modelling approach is that it quantitatively
takes into account the factors that determine the rate of reactions.
2. The principles of kinetic modelling are applicable for all parts of the cell as well as
the extracellular environment. The many different levels of control, regulation and
coordination of biochemistry are essential features of living cells and a modelling
framework with a broad applicability is clearly an advantage if one desires to
study the integration of different cellular processes.
3. Kinetic models can assist in understanding the complex behaviours of biological
systems.
4. Kinetic modelling can turn understanding of how cell factories work into
predictions about how to improve them. When models have been set up linking
relevant aspects on the system-level with the properties of the system components,
they become valuable for predicting and optimizing the performance of cell
factories.
Challenges:
1. The overall challenge lies in producing predictive models of high quality that
really can make a difference for improving cell factory performance.
2. The difficulty of producing high quality predictive models is that it requires a lot
of detailed information about the system that one wishes to model.
3. Producing the right kind of data is critical for parameter estimation in kinetic
models. Ideally, methods from identifiability analysis and experimental design
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should assist in laying down the directions for what data to collect, rather than
uncritically basing these decisions common practice or on intuition.
4. The time it takes to set up kinetic models must be reduced.
Traditionally, enzymes in free solutions (i.e. in soluble or free form) react with substrates
to result in products. Such use of enzymes is wasteful, particularly for industrial
purposes, since enzymes are not stable, and they cannot be recovered for reuse.
Immobilization of enzymes (or cells) refers to the technique of confining/anchoring the
enzymes (or cells) in or on an inert support for their stability and functional reuse. By
employing this technique, enzymes are made more efficient and cost-effective for their
industrial use. Some workers regard immobilization as a goose with a golden egg in
enzyme technology. Immobilized enzymes retain their structural conformation necessary
for catalysis.
Advantages of immobilized enzymes:
Stable and more efficient in function.
Can be reused again and again.
Products are enzyme-free.
Ideal for multi-enzyme reaction systems.
Control of enzyme function is easy.
Suitable for industrial and medical use.
Minimize effluent disposal problems.
Immobilized enzymes are generally preferred over immobilized cells due to specificity to
yield the products in pure form. However, there are several advantages of using
immobilized multi-enzyme systems such as organelles and whole cells over immobilized
enzymes. The immobilized cells possess the natural environment with cofactor
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availability (and also its regeneration capability) and are particularly suitable for multiple
enzymatic reactions.
Methods of Immobilization:
The commonly employed techniques for immobilization of enzymes areadsorption,
entrapment, covalent binding and cross-linking.
1. Adsorption:
Adsorption involves the physical binding of enzymes (or cells) on the surface of an inert
support. The support materials may be inorganic (e.g. alumina, silica gel, calcium
phosphate gel, glass) or organic (starch, carboxymethyl cellulose, DEAE-cellulose,
DEAE-sephadex).
Adsorption of enzyme molecules (on the inert support) involves weak forces such as van
der Waals forces and hydrogen bonds (Fig. 21.3). Therefore, the adsorbed enzymes can
be easily removed by minor changes in pH, ionic strength or temperature. This is a
disadvantage for industrial use of enzymes.
2. Entrapment:
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3. Microencapsulation:
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microencapsulation. Hybridoma cells have also been immobilized successfully by this
technique.
4. Covalent Binding:
5. Cross-Linking:
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Glutaraldehyde is the most extensively used cross-linking reagent. It reacts with lysyl
residues of the enzymes and forms a Schiffs base. The cross links formed between the
enzyme and glutaraldehyde are irreversible and can withstand extreme pH and
temperature. Glutaraldehyde cross- linking has been successfully used to immobilize
several industrial enzymes e.g. glucose isomerase, penicillin amidase. The technique of
cross-linking is quite simple and cost-effective. But the disadvantage is that it involves
the risk of denaturation of the enzyme by the poly-functional reagent.
Immobilization of Cells:
Immobilized individual enzymes can be successfully used for single-step reactions. They
are, however, not suitable for multi-enzyme reactions and for the reactions requiring
cofactors. The whole cells or cellular organelles can be immobilized to serve as multi-
enzyme systems. In addition, immobilized cells rather than enzymes are sometimes
preferred even for single reactions, due to cost factor in isolating enzymes. For the
enzymes which depend on the special arrangement of the membrane, cell immobilization
is preferred.
Immobilized cells have been traditionally used for the treatment of sewage. The
techniques employed for immobilization of cells are almost the same as that used for
immobilization of enzymes with appropriate modifications. Entrapment and surface
attachment techniques are commonly used. Gels, and to some extent membranes, are also
employed.
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Advantages of whole cell immobilization:
Multiple enzymes can be introduced to a single step
Extraction and purification of enzymes are not required
Enzymes are stable for long time
Native conformation of enzyme is best maintained
Cost effective method
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Applications of Immobilized Cells:
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5. Require gentler culture condition and control systems that are optimized for lower
metabolic rates.
6. Therefore, the design, mode of operation and control systems of Stirred Tank
Reactor used for animal cells are distinctly different from those that would be
applicable to bac The fragility of animal cells culture has been a subject of
considerable for fermenter design.
7. Although cells in suspension can be damaged by various forces acting in a stirred
culture, the major damaging force is from bubble bursting on the culture surface
resulting from culture aeration.
8. The simplest stirring operation involves the rotation of suspended bar by magnetic
stirrer.
9. This is the system used in glass spinner bottles and is suitable for stirring cultures
up to 1 liter bacterial or fungal cells.
10. At larger volumes, magnetic stirrer are not suitable because of the increased
energy required for rotation.
11. In order to ensure adequate mixing at low strirring speeds, the culture vessels are
designed with a round bottom, which distinguishes them from the flat-bottomed
bacterial fermenter.
12. Impeller blades which are fitted at the end of mechanical drive shafts are designed
to allow vertical as well as horizontal liquid flow.
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Roller bottle
2. SPINNER FLASKS:
Spinner Flask
3. STIRRED TANK REACTORS:
1) Sail type and Axial flow hydrofoil agitators reduce shear stress
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2) Agitation rate: 10-40rpm
3) Preferred for suspension cells.
4) Shear protecting agents Pluronic F-68 reduce shear at gas-liquid interface
due to bursting of bubbles.
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8) Solution: Macroporous Microcarriers: cells enter the porous microcarrier
and are shielded from shear forces BUT mass transfer (diffusional) limitations
and heterogeneous conditions.
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Monoclonal Antibodies
Immuno-biological Regulators
Vaccines
Hormones
Enzymes
Whole cells and tissue cultures
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