BIOCHEMICAL
ENGINEERING
Cell Cultivations
CHAPTER-5
cell cultivations
The purpose of this chapter is to give
an introduction to the basics of
microbial, animal, and plant cells
and their cultivation techniques and
applications.
MICROBIAL CELL
CULTIVATIONS
Microorganisms are not only used
for the traditional microbial
processes
but also for new processes such
as the
production of pharmaceuticals,
industrial
chemicals, enzymes, agricultural
chemicals, waste water
MICROBIAL CELL
CULTIVATIONS
MICROBIAL CE
CULTIVATIONS
The protists refer relatively simple
biological organisms compared to
plants and animals and include
algae,
protozoa, fungi, and bacteria.
MICROBIAL CELL
CULTIVATIONS
MICROBIAL CE
CULTIVATIONS
MICROBIAL CE
CULTIVATIONS
MICROBIAL CE
CULTIVATIONS
The more complex eukaryotic
cell
is the unit structure in plants,
animals,
protozoa, fungi, and algae.
The eukaryotic cell has internal
unit
MICROBIAL CE
CULTIVATIONS
The nucleus controls hereditary
properties and all vital activities of the
cell.
The chromosomes are long and
threadlike bodies and are found in the
nuclei of cells, which contain the
genes
arranged in linear sequence in
nucleoproteins
MICROBIAL CE
CULTIVATIONS
The cytoplasm contains
numbers
of granules, called ribosom
are
involved in continuous rea
synthesize cell mater
MICROBIAL CE
CULTIVATIONS
The ribosome is especially concentrate
the
MICROBIAL
NOMENCLATUR
MICROBIAL
NOMENCLATURE
The second word in the name of a
microorganism is the species name and
is not capitalized. There may be
several species with the same genus
name, for example, Bacillus subtilis,
B. albus, and B. coagulans. Note that
when the same genus name is repeated
several times, it is abbreviated
INDUSTRIAL
APPLICATIONS
SHAPES OF BACTERIA: OF
MICROORGANISMS
Bacteria occur in a variety of shapes such as:
cocci: spherical or ovoid
bacilli: cylindrical or rod shaped
spirilla: helically coiled.
INDUSTRIAL
APPLICATIONS OF
MICROORGANISMS
Bacteria reproduces predominantly by
a process known as binary fission.
This is an asexual reproductive process.
The steps involved are:
1. Cell elongation,
2. Invagination of the cell wall,
3. Distribution of nuclear material
INDUSTRIAL
APPLICATIONS OF
MICROORGANISMS
Growth pattern of bacteria
NUTRITIONAL REQUIREM
BACTERIA
NUTRITIONAL REQUIREM
BACTERIA
NUTRITIONAL REQUIREM
BACTERIA
PHYSICAL CONDITIO
NECESSARY
FOR BACTERIAL GRO
PHYSICAL CONDITIO
NECESSARY
FOR BACTERIAL GRO
For most of the bacteria the optimum pH i
7.5,
Some bacteria can survive under extreme
ranging
PHYSICAL CONDITIO
NECESSARY
FOR BACTERIAL GRO
PHYSICAL CONDITIO
NECESSARY
FOR BACTERIAL GRO
PHYSICAL CONDITIO
NECESSARY
FOR BACTERIAL GRO
PHYSICAL CONDITIO
NECESSARY
• Fungi are plants devoid of chlorophyll, therefore
FOR GROWTH
incapable of synthesising its own food like other OF FU
plants.
• There are two important fungi: YEAST, and MOLDS.
• Fungi range in size and shape from single-celled
yeasts multicellular mushrooms.
PHYSICAL CONDITIO
NECESSARY
FOR GROWTH OF YE
Yeasts are widely distributed in nature.
They are found in fruits, grains and
other
food containing sugar. They are also in
the soil, in the air, on the skin and in the
intestines of animals. Since yeasts do
not have chlorophyll, they depend on
PHYSICAL CONDITIO
NECESSARY
FOR GROWTH OF YE
Yeasts are generally unicellular
organisms
and their shape is spherical to ovoid.
Their size is 1 to 5 μm in width and
from 5 to 30 μm in length. The cell wall
is
quite thin in young cells but thickens
with age.
GROWTH PATTERN OF
PHYSICAL CONDITIO
NECESSARY
FOR GROWTH
Molds are filamentous fungi. A single reproductive cell or
OF MO
spore is
germinated to form long thread, hyphae which branches
repeatedly as it
elongates to form a vegetative structure called a mycelium.
PHYSICAL CONDITIO
NECESSARY
FOR GROWTH OF MO
mycelium consists of a multinucleate
mass of
cytoplasm with in a rigid, much
branched system of tubes.
Since mycelium is capable of growing
indefinitely, it can attain macroscopic
dimensions.
PHYSICAL CONDITIO
NECESSARY
FOR GROWTH OF MO
The most important classes of molds
industrially are Aspergillus and
Penicillium.
Molds are used in the production of
antibiotics,
industrial chemicals, enzymes, and
food
additives.
CULTURE MEDI
The growth of microbial population
in
artificial environments is called
cultivation.
A culture that contains only one
kind of
microorganism is a pure culture. A
mixed
CULTURE MEDI
2. Sterilizing in order to eliminate all living organisms in the vessel.
3. Inoculating the microorganism in the prepared medium.
There are two main types of culture
media: natural (or empirical,
or complex) and synthetic (or
NATURAL CULTURE
NATURAL CULTURE
Examples of a relatively simple liquid and a
solid medium
that supports the growth of many common
heterotrophs
are nutrient broth and nutrient agar. Their
composition
Nutrient broth: 3 g of beef extract,
is as follows:
5 g of
peptone, 5 g of yeast extract,
and water to make 1 L.
Nutrient agar: the same
ingredient as
Nutrient broth, 15 g of agar and
SYNTHETIC CULTU
MEDIA
They are often required for research
purposes.
The medium may be as simple as
inorganic
ammonium salt plus minerals and a
sugar,
STERILIZATION
or as complex as purified casein with
added
INOCULATION
vitamins, minerals, and a sugar.
ANIMAL CELL CULTIV
Cells removed from animal tissue
can
be cultivated in nutritional
medium
outside the donor's body.
Cultured cells
grow by increasing in number and
size.
ANIMAL CELL CULTIV
Tissue culture methodology has
given researchers the opportunity
to
study cancer cells; to classify
malignant tumors, to determine
tissue compatibility in
transplantation,
ANIMAL CELL CULTIV
The mammalian cell culture
technique can be
employed to produce clinically
important
biochemicals such as human growth
hormones, interferon, plasminogen
activator,
viral vaccines, and monoclonal
ANIMAL CELL CULTIV
Some mammalian gene products can
be also produced by bacterial systems
using recombinant DNA technology.
Fast growth
rate and inexpensive medium
requirements
of bacterial cells make them an
economical
ANIMAL CELL CULTIV
However, it is difficult to cultivate large
quantities
of mammalian cells because of the following
reasons
(Feder and Tolbert, 1983):
1. They are larger and more complex than
most microorganisms.
2. Their growth rate is very slow compared
to the microorganisms.Therefore, the
ANIMAL CELL CULTIV
3. They are enclosed with a delicate plasma membrane
without the tough
cell wall normally found in microorganisms or plant cells.
As a result,
they are fragile.
4. Their nutritional requirements are not fully defined yet,
requiring
expensive blood serum for medium.
5. They are part of an organized tissue rather than an
individual cellular
organism.
6. Most animal cells only grow when attached to a surface.
ANIMAL CELL CULTIV
ANIMAL CELL CULTIVA
CELL TYPES: SUSPE
CELLS
Blood and lymph are rather a typical
connective
tissues with liquid matrices. Cells from
blood or
lymph fluids are suspension cells, or
nonanchorage
dependent when grown in culture.
Nonanchorage-
dependent cells do not require a surface to
ANIMAL CELL CULTIVA
CELL TYPES; ANCHO
DEPENDENT CEL
Most mammalian cells are anchorage dependent
cells,
they require a surface for attachment and growth.
The most
widely used anchorage-dependent cell types are
epithelial or
fibroblast (broadly classified as connective) cells
(Fig. 5.6 a, c.)
Anchorage dependent cell require wettable surface
to grow on
ANIMAL CELL CULTIV
GROWTH MED
CELL GROWTH MEASU
Balanced growth is defined as
growth
during which a doubling of the
biomass
is accompanied by a doubling
of all other measurable properties of
the population such as protein, DNA,
RNA, and intracellular water.
CELL GROWTH MEASU
In other words, cultures
undergoing
balanced growth maintain a
constant
chemical composition. In an
adequate
medium to which they have
1. Measurement of cell number: microscopic
counts, viable plate count, coulter counter.
2. Measurement of cell mass: cell dry weight,
turbidity.
3. Indirect methods: nutrient consumption,
product formation, cell components, heat
evolution and viscosity.
CELL GROWTH MEASU
MICROSCOPIC CO
The number of cells in a population
can be measured under a microscope
by counting cells placed in special
counting chambers.
There are two types of chambers used
for counting cell number in liquid samples:
1. hemocytometer: a blood cell counting
CELL GROWTH MEASU
MICROSCOPIC CO
The desirable features of direct-counting
methods are:
1. Minimal equipment is required.
2. Results are obtained rapidly.
3. The morphological characteristics
of the organisms can be observed.
CELL GROWTH MEASU
MICROSCOPIC CO
The disadvantages are:
1. Dead cells cannot usually be
distinguished from live cells.
2. The method is not suitable for
cell suspensions of low density.
3. Small cells are difficult to see
under the microscope and can be missed
when counting.
CELL GROWTH MEASU
VIABLE PLATE CO
A viable cell is defined as one that is
able
to divide and form a colony. There are
two ways of performing a plate count the
spread
plate method and pour plate method.
With the spread plate method, a volume
of no larger than 0.1 mL is spread over
the agar surface. With the pour plate
CELL GROWTH MEASU
COULTER COUNT
CELL GROWTH MEASU
MEASUREMENT OF CE
Cell dry weight can be measured
directly
by taking an aliquot of cell suspension
and
centrifuging it. After the supernatant is
discarded, the cells are thoroughly
washed with distilled water to
eliminate
all soluble matter. The suspension
CELL GROWTH MEASU
MEASUREMENT OF CE
TURBIDITY
When a beam of light is passed through
a suspension of organisms, the
reduction
in the amount of light transmitted as a
consequence of scattering is thus a
measure of the cell density.
CELL GROWTH MEASU
MEASUREMENT OF CE
TURBIDITY
Such measurements are usually made in
a
spectrophotometer, which reads in
absorbency (A) units. The absorbency
is defined as the logarithm of the ratio
of the intensity of light striking the
suspension (I0) to that transmitted by the
suspension (I):
CELL GROWTH MEASU
INDIRECT METHO
CELL GROWTH MEASU
INDIRECT METHO
1. Nutrient Consump
2. Product Formation,
3. Cell Components,
4. Heat Evolution and,
5. Viscosity.
CELL GROWTH MEASU
INDIRECT METHO
CELL GROWTH MEASU
INDIRECT METHO
CELL GROWTH MEASU
INDIRECT METHO
CELL GROWTH MEASU
INDIRECT METHO
CELL GROWTH MEASU
INDIRECT METHO
CELL GROWTH MEASU
INDIRECT METHO
CELL
IMMOBILISATION
By immobilizing the cells, process
design
can be simplified since cells
attached to
large particles or on surfaces are
easily
separated from product stream.
This
CELL
IMMOBILISATION
Immobilization can also provide
conditions
conducive to cell differentiation and
cell-to-cell
communication, thereby encouraging
production of high yields of secondary
metabolites. Immobilization can
protect
cells and thereby decrease problems
CELL
IMMOBILISATION
Cell immobilization methods can be
divided into four major categories:
attachment to a surface, entrapment
within a porous matrix, containment
behind a barrier, and self-aggregation
as summarized in Table 5.8 (Karel et
al., 1985).
CELL
IMMOBILISATION
•
CELL
IMMOBILISATION
CELL
IMMOBILISATION:
ENTRAPMENT WITH
IN POROUS MATRIX
Cells are allowed to diffuse into
preformed porous matrices such
as bricks, cordierite, and pore glass,
in
which they will grow and be trapped.
The main advantages of this method
are that
CELL
IMMOBILISATION:
ENTRAPMENT WITH
IN POROUS MATRIX
However, it is difficult to reach a
high cell
concentration due to the limited pore
volume usually available for
entrapment
within typical support materials.
Another way is to entrap cells within
CELL
IMMOBILISATION:
CONTAINMENT
BEHIND A BARRIER
CELL
IMMOBILISATION:
SELF
SEGGREGATION
