Transcript ert 211 biochemical engineering

ERT 211 BIOCHEMICAL
ENGINEERING
Course Outcome
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Ability to describe the usage and methods for
cultivating plant and animal cell culture.
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Ability to discuss the technologies available in
bioconversion.
Chapter 5
Considerations in Using
Plant and Animal Cell
Cultures
Chapter 5(i) Outline
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Usage of plant cell cultures
Potential products from plants cultures
Approaches to increase productivity
Difference of plant cells from microbes
- implications of bioreactor design
Usage of plant cell cultures
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Control of supply of product independent of
availability of the plant itself.
Cultivation under controlled and optimized
conditions.
Strain improvement with programs analogous to
those used for microbial systems.
With the feeding of compounds analogous to
natural substances, novel compounds not present
in nature can be synthesized.
Control of supply of product independent of availability of
the plant itself
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Many drugs contained active ingredients source
from plant parts.
For example, paclitaxel (taxol) an anticancer agent
extracted from bark of Pacific yew tree (Taxus
brevifolia).
Three 100-year-old trees is needed to supply
enough paclitaxel for one patient.
The semi-synthetic productions concerns with
environment contaminants from chemical
processing.
Cultivation under controlled and optimized
conditions
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The accumulation substances extracted from plants
affected by many factors etc. condition of the plants
and environment.
Production of saponin via tissue culture technique
from Panax ginseng was effected by the
concentration of hormones used.
3.62% of total saponins was detected in callus
cultivated in MS medium containing 5 mg/L 2,4-D
and 1 mg/L kinetin, while 8.78% was produced in 10
mg/L and 1 mg/L kinetin medium.
Callus cultures of Oxalis reclinata are yellow in the light, but when
they are transferred to the dark, they start producing red anthocyanin pigments.
Strain improvement with programs analogous to those
used for microbial systems.
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The physiological characteristics of individual plant
cells are not always uniform.
In tissue culture, selection of cell lines for specific
compounds production will increased in yield.
A strain of Euphorbia milli was recognized to
accumulate about 7 times higher amounts of
anthocyanins than that of the parent strain after 24
selections.
With the feeding of compounds analogous to natural substances,
novel compounds not present in nature can be synthesized.
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Addition to the culture media of appropriate
precusors or related compounds sometimes
stimulates secondary metabolite productions.
Precursor can be synthetic or natural substances.
This approach is advantageous if the precursors are
inexpensive.
The production of limonene is improved with the
addition of mevalonic acid (primer precursor).
Acetyl CoA
3-hydroxyl-3-methylglutaryl CoA
CH3
HOOCCH2CCH2CH2OH
Mevalonic acid (Primer Precursor)
OH
O PP
O PP
+
dimethylallyl pyrophosphate
OH
O PP
geranyl pyrophosphate
Linalool (Immediate precursor)
O PP
A diagram of limonene
biosynthetic pathway.
Limonene
Potential products from
plants cultures
1. Shikonin production
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Shikonin and its derivatives such as acetyl shikonin
and isobutyl shikonin accumulated in roots of
Lithospermum erythrorhizon.
They are reddish purple pigments and have been
used in traditional dyeing and as a herbal medicine.
Because of shortage of this plant, Mitsui
Petrochemical company in Japan investigated mass
cultivation of L. erythrorhizon cells to produce
shikonin compounds.
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1. Lithospermum erythorrhizon plant
2. L. erythorrhizon dry root
3. Suspension cultures of L. erythorrhizon in bioreactor
4. Cell agregates of L. erythorrhizon
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First step was to established a cell line for shikonin
production.
Second step is to promote the cell growth in
Linsmair-Skoog’s medium.
Third step is to accumulate shikonin compound in
White’s medium.
This type of culture is called two-stage culture.
Later they found that selecting the cell lines from the
protoplasts is more efficient than from aggregated
cells.
They also feeding nutrients into the medium in order
to increase the cell mass and therefore will increase
the compounds productions.
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Another researchers established a hairy root
culture of L erythrorhizon with Agrobacterium
rhizogenes.
Assignment 1
With labeled diagram, explain in details twostage culture process for shikonin production
2. Morphinan alkaloids
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Codeine is an analgesic and cough-suppressing
drug.
Morphine from Papaver somniferum L. (opium
poppy) is a traditional commercial source.
Morphine can be converted to codiene.
Thebaine from capsules of P. bracteatum also can
be converted to codeine.
Little success has been achieved from culture of
undifferentiated cells of these plants for codiene
production.
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After many attempts, researchers still failed
to get direct codiene and morphine from the
cell cultures.
Later, Japanese researchers (Furuya et al),
studied the biotransformation of codeinone to
codiene using immobilized cells of P.
somniferum.
The conversion yield was 70.4% of codiene
produced and out of that, 88% was converted
was excreted into the medium.
Assignment 2
Explain techniques that can be used to
increase production of codein
3. Berberine production
4. Rosmarinic acid production
5. Antocyanins production
Reference
Suri, S.S., Sharma, R., Ramawat, K.G. 1999. Production of secondary
metabolites in bioreactor. In: Role of biotechnology in medicinal and
aromatic plants. Volume II. Ukaaz Publications.
Approaches to increase productivity
1.
Optimization of Cultural Conditions
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explain on the use of two-stage culture under medium
Selection of High-producing Strains
Addition of precursors
Biotransformation
Elicitor Treatment
1.
Optimization of Cultural Conditions
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The cultural conditions includes medium,
surrounding environment (temperature, pH light &
O2) and culture density.
The most important is the medium that influence
both the growth of cells and yield of desirable
products.
Various basal medium have been used and
Murashige & Skoog (MS) is among the most
widely applicable.
Sucrose and glucose are carbon source for plant
tissue cultures and affects cell growth and yield of
products.
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Phytohormones such as auxins and kinetins have
shown the most remarkable effects on growth and
productivity of plant metabolites.
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Generally, a temperature of 17-25°C is used for
induction of callus tissues and growth of cultured
cells.
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The pH is usually adjusted between 5-6 before
autoclave.
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The use of light depends on the type of culture and
the desire products.
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Oxygen is not critical for plant cultures but still has
an effect on the growth and production.
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The use of high cell density cultures in a suitable
bioreactors found to increase yield in some cultures.
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Some plant cells needs different media for the cell
growth and secondary metabolite production.
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This cultural condition is called two-stage culture.
It means that 2 types of culture medium are used.
both culture medium may differ in the concentration or
types of nutrient, hormone or vitamins used.
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two types of medium were used because the
medium used for promoting growth is not suitable for
promoting metabolites production.
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In such system, the first medium is used to promote
the growth of cells in the culture system.
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the second culture is to promote the production of
metabolites in the culture.
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Cells of plant will be first cultivate in the first medium
and after certain period, they will be transferred into
the second growth until harvesting period.
2. Selection of High-producing Strains
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The physiological characteristics of individual plant
cells are not always uniform.
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Therefore a rapid assay method is crucial in the
selection of a high yielding cell line.
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The specific cell line is obtained from the selection a
number of strains producing high level of desirable
product .
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The strains then were subjected to further cell cloning
to increase the level of secondary metabolites.
3. Addition of precursors
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Precursors are related compounds sometimes
stimulates secondary metabolites.
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This approach is advantageous if the precursors are
inexpensive.
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For exp. Phenylalanine is one of the biosynthetic
precursors of rosmarinic acid. Addition of this amino
acid to Salvia officialis suspension cultures
stimulated the production of rosmarinic acid.
4. Biotransformation
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Almost similar to precursor addition where, a suitable
substrate compound may be biotransformed to a
desired product using plant cell.
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Biotransformation has been extensively applied in the
fermentation using microorganisms and their
enzymes.
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For example, L-aspartic acid and L-malic acid can be
biotransformed from fumaric acid using
microorganisms.
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Using plant cells, for exp. Digitalis lanata, the
biotransformation of -mrthyldigitoxin to methyldigoxin has been investigated.
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Digoxin has a large market as a cardiac glycoside.
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This approach beside precursors feeding are the
most commercially realistic approaches because of
economic reasons.
5. Elicitor Treatment
- Elicitor is an agent of microbial infections on intact
plants that cause the synthesis of specific secondary
metabolite.
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Some studies reviewed possible correlations between
stress and secondary metabolism in cultured cells.
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Some suggested that upon infection, plants shows
their defense mechanism by secreting secondary
metabolite.
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Elicitors that have been used in plant cell cultures are
yeast extract, chitosan, inorganic and organic
molecules and many more.
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Plants grow under stress condition also show
elicitation effects. Phosphate limitation in hairy root
cultures of Hyoscyamus muticus had increased
production of the sesquiterpene solavetivone.
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Examples of inorganic compounds used are sodium
chloride, potassium chloride, sorbitol and abscisic
acid.
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For economical use of the elicitors, they should be
cheap and easy to obtain.
Implications of bioreactor
design
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
>10
Sensitive
>90%
Days
0.3 vvm
Weeks
Vacuole
Uncoupled
Possible
8-9/m3
Often growth linked
Required haploids
65-75/m3
Factors for growth in bioreactor
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Gas-liquid mass transfer
Shear
Mixing
Assignment 3
Compare the plant cells with microbes and
discuss the implications for bioreactor design.
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Shear Stress
Microorganisms cells are less sensitive to plant cells. Plant
cells may require operation under low-shear conditions.
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Aeration and Agitation
In aerobic fermentation processes, microorganisms require
high aeration compare to plant cells.
Require to change the bioreactor system that provide high
aeration rate.
This system can supply necessary oxygen to the
microorganisms because they have high metabolic activities.
system can be equipped with mechanical devices used to
agitate the liquid broth or by air compressor.
3.
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Size
Plant cells are large compared to
microorganisms. Cell often grow as
aggregates or clumps.
The large size, rigid cellulose-based cell
wall, and large vacuole making plant cells
sensitive to shear stress. (fan and balloon in
a room).
Cell often grow as aggregates or clumps
and may have mass transfer limitations that
limit the availability of nutrients to cells
within the aggregate.
4.
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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.