Methods of Cell Immobilization

Introduction
intact or disintegrated dead cells that contain
active enzymes
resting or growing cells
(this technique is used especially with eukaryotic
cells where the whole metabolic machinery is often
required for their specific application)
 Methods of Cell Immobilization
The reactions catalyzed by immobilized whole-cell
biocatalysts can be classified as follows:
1. Reactions involving single enzymes
(bioconversions)
2. Reactions involving multienzyme systems with
or without cofactors
3. Reactions involving a complete metabolic
pathway yielding primary or secondary metabolites
 Methods of Cell Immobilization
The use of immobilized microbial cells is advantageous in the following
areas

1.when the desired enzymes are intracellular and the extracted, purified
enzymes become unstable after immobilization

2. when the microorganism does not contain interfering enzymes or

when such enzymes can be inactivated without loss of desired catalytic
activity

3. when substrates and products do not have a high molecular mass

and can diffuse through the cell membrane
 Methods of Cell Immobilization
1.The material should be available in sufficient quantities and at low
price.
2. The material should have a large surface area accessible to cells
and reactants
3. The material must be mechanically, chemically, and thermally stable
under process and storage conditions.
4. The matrix should contain a sufficient number of functional groups to
bind the cells.
5. The material should not reduce cell activity or initiate cell lysis.
6. The material should be easy to handle in the immobilization
procedure.
7. The material should be capable of recycling or safe disposal.
8. In the case of viable growing cells, the matrix should have a
sufficiently large void volume or be elastic enough to accommodate
new cells.
 Methods of Cell Immobilization
Immobilization Techniques
1. Carrier-free immobilization
2. Immobilization of a given biomass onto a
preformed carrier surface
3. Immobilization of a given biomass during
the course of carrier formation (e.g., by
polymerization)
4. Immobilization by controlled growth of an
inoculum or by germination of
immobilized spores
Classification of cell immobilization
methods
 Cell Immobilization without a
Support
intrinsic tendency to aggregate or flocculate at high
cell densities.
Yeast, mold, and plant cells aggregand are
relatively stable to shear fields in fluidized-bed
reactors
Flocculation can also be induced by
polyelectrolytes such as chitosan
Cell aggregation can be induced by low molecular
mass bi- or multifunctional reagents such as
glutaraldehyde, diazotized diamines, or toluene
diisocyanate
 Binding of Cells to a Carrier
Physical Adsorption.
• Additional chemicals are usually unnecessary, and fixation is carried
out under growth conditions; viable cell preparations can, therefore,
be obtained.
mammalian cells bound to preformed surfaces to produce
therapeutic biochemicals. The cells are immobilized on microcarriers
(small-diameter beads, 100 – 200 µm) manufactured from different
synthetic polymers (e.g., polystyrene, gelatin, dextran,
polyacrylamide, or glass) that offer a large specific surface area for
cell growth
monolithic ceramic matrix has been developed for large-scale cell
cultures. For adherent cell growth, the scalability is almost linear and
depends on the available surface area.

• Ionic Binding. Ionic binding is a special case of physical adsorption

where charged microbial cells can electrostatically interact with the
ions on a carrier surface to form stable complexes. Synthetic ion-
exchange resins, modified cellulose derivatives, or inorganic
materials can be used as carriers. Cell adsorption is mainly affected
by factors such as pH, ionic strength, surface charge, cell age, or
composition of the carrier surface.
 Covalent Binding
Cells can also bind to the functional groups
of the carrier surface by covalent bonds.
This technique is frequently used for
enzyme immobilization but has only limited
use in whole-cell immobilization because the
toxicity of the coupling agents often results
in loss of cell viability or enzyme activity
 Immobilization of Cells by
Entrapment
Lattice Entrapment.
retention of cells within the network of a polymer
matrix.

forms pellets
high viable cell density
(e.g., polyacrylamide gel, alginate gel, k-
carrageenan, and photo-cross-linkable resins)
 Polyacrylamide Gel.

free-radical polymerization in an aqueous acrylamide

monomer solution containing the cells at low temperature.
The degree of cross-linking determines the porosity and
mechanical properties of the pellets. The major
disadvantage of the method is the toxicity of the acrylamide
monomer, the cross-linking agent (e.g.,
methylenebisacrylamide [110-26-9]), and the
polymerization initiator (e.g.,tetramethylethylenediamine
[110-18-9]) which can decrease cell viability and enzyme
activity], Hydroxyethyl methacrylate [868-77-9] .....less toxic
alternative to acrylamide
With acrylamide monomers, optimal results are achieved in
isotonic buffered solution at low temperature (4 – 12 °C) by
keeping exposure time to a minimum (3 – 4 min).
 Alginate [9005-38-3]
is extracted from seaweed and is a linear
copolymer of b-D-mannuronic acid and a-L-
guluronic acid linked by 1,4-glycosidic
bonds. It forms a gel in the presence of
multivalent ions, usually calcium or
aluminum. The controlled entrapment of
cells is simple and generally nontoxic.
Various cell types can be immobilized with
negligible loss of viability.
 κ-Carrageenan [11114-20-8]
sulfonated polysaccharide extracted from
seaweed. It consists of b-D-galactose 4-sulfate
and 3,6-anhydro-D-galactose units. Only the
sodium salt of k-carrageenan is soluble in cold
water. Induction of gelation with potassium ions
can be performed under very mild conditions
without the use of chemicals that decrease
enzyme activity. Another advantage of this
technique is that the immobilized cell biocatalysts
can be tailor-made into pellets, sheets, beads, etc.
one-step procedure
 Photo-cross-linkable resins

Active groups (e.g., vinyl groups) are

coupled to polyethylene or poly-(propylene
glycol) oligomers of controlled chain length
to prepare prepolymers. The prepolymers
are then mixed with the cell culture, and
cross--linking is initiated with UV radiation
 Immobilization of Cells by
Entrapment
The advantages of this method are:
1. entrapment is simple and proceeds under very
mild conditions
2. prepolymers do not contain toxic monomers
3. the network structure of the gels can be adapted
as required
4. optimal physicochemical gel properties can be
achieved by selecting suitable prepolymers
 Immobilization of Cells by
Entrapment
Membrane Entrapment

The supply of oxygen and nutrients to the

cells and the removal of carbon dioxide from
the cells occur by diffusion and may be
insufficient.
 Microencapsulation
entrapped in hollow spheres where they can
be cultured.
Different capsule diameters (from 20 nm to
2 mm) and controlled membrane porosity
can be achieved
In larger microcapsules, however, radial
concentration gradients of nutrients or
oxygen can result in a necrotic core
 Methods of Organelle
Immobilization
mitochondria, chloroplasts, peroxisomes
(microbodies), vacuoles, and nuclei.
chloroplasts as potential solar energy
photoconverters
Immobilization of rat liver mitochondria on a solid
support consisting of alkysilanized glass beads
Rat liver mitochondria have also been entrapped in
polyacrylamide gel
yeast mitochondria have been entrapped in a
polyurethane matrix
Coimmobilization of Biocatalysts

• galactosidase and Saccharomyces cerevisiae

for the conversion of cellobiose to ethanol
• Yeast cells or dead mycelia have been
employed for direct coupling of enzymes to cell
walls in food-processing applications
• bioproduction of hydrogen gas with hydrogenase
and chloroplasts Coimmobilization results in a
fivefold increase in hydrogen production
compared with the system employing two
separately immobilized catalytic species.
 Economic Aspects
25 % of the world pharmaceutical market is
already covered by biotechnologically
manufactured products. In the chemical industry,
this fraction is approaching 10 %. According to
market growth projections, U.S. biotechnology
industry sales will increase from about $ 400×106
in 1987 to $ 2.5×109 by 1990, and more than
$ 25×109 by 2000 . The world biotechnology
market in the year 2004 is predicted to be
$ 75×109
 Immobilized Enzymes.
The total market for industrial enzymes is about $ 400×106 and is
growing by about 15 % each year. Enzyme immobilization has not
initiated the expected revolution in the enzyme industry
Soluble use-and-discard enzymes have, by far, the most dominant
market share. Only one immobilized enzyme product, immobilized
glucose isomerase, is used in amounts > 50 t a year; 1500 – 1750 t are
used worldwide annually. The three other major immobilized enzymes
are aminoacylase (about 5 t/a), lactase (< 5 t/a), and penicillin G
acylase (3 – 4 t/a) .Other immobilized enzymes are also available but
are used in even smaller amounts: glucoamylase, hydantoinase,
invertase, nitrilase, RNAse, and penicillin V acylase.
 Immobilized microorganisms
multiple enzyme reactions especially where cofactor regeneration is
essential.

Recombinant Escherichia coli can be used for the production of

relatively simple peptide hormones with pharmaceutical applications
such as human insulin, human growth factor, or human interferons An
immobilized biocatalyst is reportedly used for the conversion . A
hepatitis B surface antigen vaccine prepared in recombinant yeast was
approved for marketing, and many other products from genetically
engineered microorganisms and yeast are in the clinical test stage
(interferons, lymphokines, hormones, enzymes, and vaccines.
 Immobilized Mammalian Cells.
Many complex proteins of pharmacological interest have precise folding and
glycosylation requirements that cannot be fulfilled in simple microorganisms. By using
hybridoma or recombinant DNA techniques, mammalian cells can often be persuaded to
produce and secrete useful quantities of these compounds
Tissue plasminogen activator (TPA) recombinant E. coli or yeast or in glycosylated form
in mammalian cells. The first three months' sales in the United States were almost
$ 60×106. Worldwide sales are projected to reach about $ 500×106 in the next few years.
.
Monoclonal antibodies 100 monoclonal antibodies are offered, with annual sales of nearly
$ 500×106.
Generally, the economics of microbial systems are more favorable than those of
mammalian cell culture systems for recombinant proteins. When the use of recombinant
microbial systems is impossible or impractical, cell culture systems will gain a market
position if the products are high-value biologicals (> $ 100/g).
 Immobilized Plant Cells.
Plant cell cultures can also be used for the production of metabolites
such as pharmaceuticals, chemicals, flavors, and fragrances. The first
product obtained from mass plant cell cultures was shikonin [517-89-5],
a red pigment composed of eight naphthoquinone molecules. Shikonin
is produced by a two-stage fermentation process and is a high-value
chemical ($ 4000/kg) with a limited annual market capacity of ca. 15 kg.
Immobilized plant cell systems will be used mainly for products of cells
in the stationary growth phase. The release of intracellularly stored
products by intermittent permeabilization of immobilized cells can be a
great economic advantage, allowing reutilization of the biomass. The
continuous immobilized plant cell process in combination with strain
selection and improved product leakage allows production of plant-
derived chemicals in the range of $ 20 – 25/kg. However, in the present
industrial state of technology for plant cell cultures, a relatively small
number of products have both high value per weight and sufficient
market size.
Safety, Environmental, and Legal
Aspects
• Immobilized Enzymes
1. catalytic activity of the pure enzyme,
2. allergenic reactions produced by the
enzyme itself or by other proteins in the
preparation,
3. presence of toxic metabolites such as
mycotoxins or antibiotics.
Safety, Environmental, and Legal
Aspects
Joint FAO/WHO Expert Committee on Food Additives (JECFA)

The committee laid down criteria for evaluating enzymes according to

the source material . Enzymes obtained from animal, plant, or microbial
sources commonly used as food, or used in the preparation of food are
accepted as foods if satisfactory chemical and microbiological
specifications can be established. No toxicological studies are required
for such enzymes. For enzymes obtained from nonpathogenic
microorganisms commonly found as contaminants in food, JECFA
recommended short-term toxicity experiments. More extensive
toxicological studies, as well as chemical and microbiological
specifications, are required for enzymes obtained from less well-known
microorganisms.
Safety, Environmental, and Legal
Aspects