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ERT 211 BIOCHEMICAL ENGINEERING
TUTORIAL
(Consideration in using plant and animal cell culture)
Answer all questions
1) In a cell culture system of Papaver somniferum for codeine production, it was
found that the end yield was only 15%. The company aims to produce codeine up
to 30% in their next operation.As a bioprocess engineer in the company, suggest
how to improve the production of codeine to meet the company’s demand.
1) Optimization of culture condition
-Find medium that influence both growth and yields
-Optimize the pH, temp of medium, cell density and oxygen transfer
2) Selection of high producing strain
-The specific cell line is obtained from the selection a number of strains
producing high level of desirable product.
-the strains then were subjected to further cell cloning to increase the level of
secondary metabolites
3) Addition of precursors
-Precursors are compound that participate in the chemical reaction that
produce another compound
-Addition of precursor to the culture media sometimes stimulates secondary
metabolites production
-This approach is advantageous if the precursors are inexpensive
4) Biotransformation
-A suitable substrate compound may be biotransformed to a desired product
using plant cell
-Biotransformation has been extensively applied in the fermentation using
microorganisms and their enzymes
-For example, L-aspartic acid and L-malic acid can be biotransformed from
fumaric acid using microorganisms
5) Elicitor treatment
-Elicitor is an agent of microbial infections on intact plants that cause the
synthesis of specific secondary metabolite.
-upon infection, plants shows their defense mechanism by secreting secondary
metabolite.
-Elicitors that have been used in plant cell cultures are yeast extract, chitosan,
inorganic and organic molecules and many more
2) Shikonin are reddish purple pigment, accumulated in roots of Lithospermum
erythrorhizon. Shikonin had been produced commercially in a two-stage culture
system.
(i)
Explain what is two-stage culture system and why such condition is
needed for the production of shikonin.
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Two stage culture system mean two type of culture mediums are used.
Both culture medium may differ in the concentration or type of
nutrients, hormones or vitamins used.
Such condition is needed because cells of Lithospermum erythrorhizon
need different media for growth and shikonin production.
3) Define the term below :
(i) Plant cell culture - is a practice used to propagate plants under sterile
conditions, often to produce clones of a plant.
(ii) Callus - tissues arising by proliferation from explants on agar medium/Mass
of undifferentiated cells
(iii) suspension culture - cells or small cells aggregates growing dispersed in
liquid medium
4) Briefly explain basic protocols for callus suspension culture
1) Excised plant material is placed on solidified medium
2)Callus forms can be quite large (> 1 cm)
3)Callus transfer to liquid medium (for propagation)
4)A piece of callus is placed in liquid medium in shake flask.
5)With moderate agitation, cells or small aggregates of cells will slough off
6)These suspended cell then replicate
7)After 2/3 weeks, suspended cells are transferred to fresh medium
5) State three (3) products from plant cell cultures.
1) shikonin
2) codein
3) ginseng
6) Compare the plant cells with microbes and discuss the implications for bioreactor
design.
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Characteristics
Microorganism
Plant Cell
Size
Shear stress
Water content
Duplication time
Aeration
Fermentation time
Product accumulation
Product phase
Mutation
Medium cost ($)
2
Insensitive
75%
<1 hour
1-2 vvm
Days
Medium
Uncoupled
Possible
8-9/m3
>10
Sensitive
>90%
Days
0.3 vvm
Weeks
Vacuole
Often growth linked
Required haploids
65-75/m3
1.Shear Stress
Plant cells are large compared to microorganism
When they are expose to high shear stress, the cell will damage
Reactors with high shear must be avoided
Plant cells may require operation under low-shear conditions.
2. Aeration
In aerobic fermentation processes, microorganisms require high aeration (have
high metabolic actv) compare to plant cells
Require to change the bioreactor system that provide high aeration rate.
system can be equipped with mechanical devices used to agitate the liquid broth
or by air compressor.
Eg airlift reactor
3. Size
 Plant cells are large compared to microorganisms. Cell often grow as aggregates
or clumps.
 Cell often grow as aggregates or clumps and may have mass transfer limitations
that limit the availability of nutrients to cells within the aggregate.
 In some case, aggregates are necessary to achieve a mix of cell types essential for
good secondary-metabolites formation
4.Duplication times
 Duplication time in microorganisms is within days compare to plants within
weeks.
 To contain the rapid multiplication of cells, larger bioreactor volumes is needed
and usually in the range of 10,000 – 100,000 L.
 Products are produce within shorter period.
 Shorter period in the culture systems means less contamination and less cost for
the maintenance.
7) Define the terms below :
i)
Animal Cell culture - is the process of culture animal cells outside the
tissue (in vitro) from which they were obtained. It will continue grow if
supplied with appropriate conditions and nutrients.
ii)
Primary culture - The cells directly taken from animal tissue (kidney,
lung) and placed into a suitable culture environment. Grow in a form
of monolayer on support surface. Also refer as anchorage-dependent
iii)
Secondary culture - A cell line obtained from the primary culture is
known as the secondary culture. Also refer as nonanchorage
dependent
iv)
Anchorage-dependent culture - requirement of cells for a solid
substratum for attachment before growth can occur.
v)
vi)
Nonanchorage-dependent culture - cells can grow in suspension
culture.
8) Briefly outline basic technique for cultivating animal cell
 Excise tissues from specific organ of animals (lung, kidney) under aseptic
conditions.
 Transfer tissues into a growth medium containing serum and antibiotics in small
T-flasks.
 These cells form a primary culture that usually attach onto the glass surface of
flask in monolayer form.
 The cells growing on support surfaces are known as anchorage-dependent cells.
 Remove cell from the surface of flasks using trypsin and add serum to the culture
bottle.
 The serum containing suspension is then use to inoculate secondary cultures.
 Many secondary lines can be adapted to grow in suspension and are
nonanchorage dependent.
9) Describe briefly basic features of animal cell bioreactor
1) Reactor should be gently agitated and aerated. Agitation speed ≈20rpm.
Bubble-column & airlift reactor operating at high aeration may cause damage of
cells
2) Supply of CO2-enriched air
3) Removal of toxic products from metabolism eg lactic acid, ammonium
10) XYZ Sdn Bhd is attempting to use animal cells culture in their operation.
(i)
Suggest the best culture condition to help the company to growth these
types of cells.

Best culture condition provide the cells with appropriate temperature and
pH and good substrate for attachment.
Temperature
 Usually set at the same point as the body temp of the host from which the
cell obtained
 Cold-blooded vertebrates – 18-25°C
 Mammalian cells – 36-37°C
 Temp maintained by use of carefully calibrated and frequently checked
incubators
pH
 Most cells in culture grow best at pH 7.4
 Common used buffer bicarbonate-CO2 or HEPES
 Keep the pH medium in a range 7-7.4
 When using bicarbonate-CO2 buffer, need to regulate the amount of CO2
dissolved in the medium
 Done by using an incubator with CO2 control set to provide an
atmosphere with between 2% and 10% CO2.
Good substrate for attachment
 Anchorage-dependent cells require good substrate for attachment
 Commonly used substrate – glass and specially treated plastics
 Attachment factors – collagen, gelatin, fibronectin, laminin (used as
substrate coating to improve growth & function of cells derived from
brain, blood vessels)
(ii)
What are the components of a typical culture medium.
a) Carbohydrates (glucose, fructose)
* provide an energy sources as well as a precursor for
biosynthesis
b) amino acids (Glutamine)
* as a sources of precursors for protein synthesis
Glutamine is normally included at higher concentrations in order
to act as a precursor for the TCA cycle intermediates. However,
ammonia is formed from the metabolic breakdown of glutamine
and can be inhibitory to growth in some cultures.
c) Vitamins & hormones
*are present at relatively low concentrations and are utilized as
metabolic cofactors.
Helps regulate and control the cell’s growth rate and functional
characteristics
d) Salts
*are included so that the solution is isotonic and has no
imbalances with the intracellular content
Helps regulate the flow of substances in and out of the cell
5) Phenol red
*usually added as a pH indicator of the medium and accounts for
the color of culture media
6) Additional media supplements
 Serum – is a cell free-free liquid recovered from blood. Eg fetal bovine
serum, calf serum, horse serum
normally added to culture media to promote cell growth.
 Antibiotic – are often included in media for short-term cultures in
order to reduce the risk of contamination