Transcript ert211 biochemical engineering

ERT211
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
Topic Outline
Usage of plant cell cultures
 Potential product from plants cell cultures
 Approaches to increase productivity
 Difference of plant cells from microbes
- implications for bioreactor design
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What is plant cell culture?
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Plant cell culture is a practice used to
propagate plants under sterile conditions,
often to produce clones of a plant.
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The purpose of practicing plant and cell culture
includes for regeneration/propagation,
embryogenesis and secondary metabolites
production.
In this technique, culture medium components,
explant source and plant growth regulator is the
important factors.
Procedures should be follows includes laboratory
set up, medium preparation, sterilization, initiation
and maintenance of cultures.
Plant cell culture techniques
Laboratory requirements for plant
cell culture
Laminar air flow cabinets
 Autoclave
 Oven for dry sterilization
 Water distillation apparatus
 Incubator
 Shakers
 Fermenters or bioreactors
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Materials and methods
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Plants – any part obtained from any plant
species can be employed to induce callus tissue
Media - inorganic salt
- Carbon sources
- Vitamins
- phytohormones
- organic supplements
Or ready made medium – Murashige and
Skoog, Gamborg, B5, Nitsch etc
Establishment of plant cell culture
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Callus and suspension cultures have been
established from hundreds of different plants
A callus can be formed from any portion of the
whole plant containing dividing cells
Callus – tissues arising by proliferation from
explants on agar medium/Mass of
undifferentiated cells produced in tissue culture is
called callus. The callus is highly vacuolated and
unorganised cells.
Suspension cultures – cells or small cells
aggregates growing dispersed in liquid medium
Basic protocols for suspension culture
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Excised plant material is placed on solidified
medium
Callus forms can be quite large (> 1 cm)
Callus transfer to liquid medium (for propagation)
Establishment of suspension culture from callus
is straightforward if callus is friable
A piece of callus is placed in liquid medium in
shake flask.
With moderate agitation, cells or small
aggregates of cells will slough off
A platform shaker is used to give a circular/orbital
motion in a variable speed control (a range form
30-150 rpm).
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Cond. 25°C, pH5.5 in the dark
These suspended cell then replicate
After 2/3 weeks, suspended cells are transferred
to fresh medium
For large scale suspension cultures, bioreactors
system is being used for the purpose of
producing high value plant products.
Implication for bioreactor
design
Plant cell vs microbes
Characteristics
Microorganism
Plant Cell
Size
Shear stress
Water content
Duplication time
Aeration
Fermentation time
Product
accumulation
Product phase
2
Insensitive
75%
<1 hour
1-2 vvm
Days
Medium
>10
Sensitive
>90%
Days
0.3 vvm
Weeks
Vacuole
Uncoupled
Mutation
Medium cost ($)
Possible
8-9/m3
Often growth
linked
Required haploids
65-75/m3
Implication for bioreactor design
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Many of the diff between plant cell and
microbes have direct implication for the
design and scale up of suspension culture
Differences
Implication for bioreactor design
More shear sensitive
May require operation under
low-shear condition
Lower growth rate
May need large bioreactor
volume
Lower respiration rate
Lower O2 transfer rates required
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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
Airlift reactor
•Mixing in airlift bioreactors
usually accomplished
without any mechanical
agitation
•The bioreactors are used
for tissue culture because
the tissues are shear
sensitive and normal
mixing (aeration plus
mechanical agitation) is
not possible
3. Size
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Plant cells are large compared to
microorganisms. Cell often grow as
aggregates or clumps.
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Cell often grow as aggregates or clumps and
may have mass transfer limitations that limit
the availability of nutrients to cells within the
aggregate.
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In some case, aggregates are necessary to
achieve a mix of cell types essential for good
secondary-metabolites formation
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.
Advantages of using plant cell
cultures for factory production of
chemicals
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4.
Control of supply of product independent of
availability of the plant itself
Cultivation under controlled and optimized
condition
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
1) 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.
Paclitaxel (taxol) - anticancer agent
- isolated from bark of Pacific
yew tree (Taxus brevifolia)
- Three 100-year-old trees is needed
to supply enough paclitaxel for one
patient
Supply problems greatly impeded the clinical
development of this drug
Production of paclitaxel in large-scale
bioreactors provides a product with fewer
contaminants, highly controllable and
reproducible.
 Recent, commercial production of
paclitaxel in stirred tank vessel 30000 l
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2) Cultivation under controlled and
optimized condition
The accumulation
2,4-D
5
10
substances extracted
(mg/l)
from plants affected by
Kinetin 1
1
many factors etc.
(mg/l)
condition of the plant
Saponin 3.62 8.78
and environment
Production of saponin via (%)
tissue culture technique
from Panax ginseng was
affected by the
concentration of
hormones used.
Callus cultures of Oxalis reclinata are yellow in the light, but when
they are transferred to the dark, they start producing red anthocyanin
pigments.
3) 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
compound production will increase in yield.
Selection using radioimmunoassay. Similar method to
monocolony isolation of bacteria
radioimmunoassay (RIA), highly sensitive laboratory
technique used to measure minute amounts of
substances including antigens, hormones, and chemicals
present in plant cells
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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.
4) With the feeding of compounds
analogous to natural substances, novel
compounds not present in the nature can be
synthesized
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Addition to the culture media of appropriate
precursors or related compounds sometimes
stimulates secondary metabolite production
Precursors can be synthetic or natural
substances
This approach is advantages 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)
A diagram of limonene
biosynthetic pathway.
O PP
Limonene
Potential products from plants
cultures
Pharmaceuticals
Food additives
Morphinan alkaloids
Codeine is an analgesic & cough-suppressing
drug
 Morphine from Papaver somniferum L.
(opium poppy) is a traditional commercial
source of codein. Morphine can be converted
to codeine
 Thebaine from P.bracteatum also can be
converted to codeine
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Capsule of Papaver somniferum
showing latex (opium) exuding
from incision
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Little success has been achieved from culture of
undifferentiated cells of these plants for codiene
production
After many attempts, researchers still failed to get
direct codeine and morphine from cell culture
Later, Japanese researchers (Furuya et al), studied
the biotransformation of codeinone to codeine
using immobilized cells of P.somniferum
The conversion yield was 70.4% and about 88% of
codeine converted was excreted into the medium
Shikonin compounds
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Shikonin and its derivatives (e.g. acetyl shikonin
& isobutyl shikonin) are reddish purple pigments
accumulated in roots of Lithospermum
erythrorhizon
They have been used in traditional dying 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.
Two-stage culture for shikonin production
Approaches to increase
productivity
a) Optimization of cultural condition
b) Selection of high-producing strains
c) Addition of precursors
d) Biotransformation
e) Elicitor treatment
In order to obtain products in high
concentration, many effort have been made
to stimulate or restore biosynthetic activities
of cultured cells using various methods.
 several typical approaches that may
increase productivity of cultured plant cells :
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a) Optimization of cultural condition
b) Selection of high-producing strains
c) Addition of precursors
d) Biotransformation
e) Elicitor treatment
1) Optimization of cultural conditions
Medium
-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
-Phytohormones such as auxins and cytokinin have
shown the most remarkable effects on growth and
productivity of plant metabolites.
Surrounding environment (temp,pH,light &
O2 )
- Generally, a temperature of 17-25°C is normally
used for induction of callus tissues and growth of
cultured cells
- The pH is usually adjusted between 5-6 before
autoclave
- The use of light depends on the type of culture
and the desire products
- Oxygen is not critical for plant cultures but still
has an effect on the growth and production
Cell density
- The use of high cell density cultures in a suitable
bioreactors found to increase yield in some cultures
TWO-STAGE CULTURE
- Some plant cells needs different media for the cell
growth and secondary metabolite production.
- 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.
- In such system, the first medium is used to
promote the growth of cells in the culture system.
- the second culture is to promote the production
of metabolites in the culture.
- 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.
Two-stage culture for shikonin production
2) Selection of high-producing
strains
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The physiological characteristics of individual
plant cells are not always uniform
Therefore a rapid assay method is crucial in the
selection of a high yielding cell line
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
Precursor – a compound that participate in
the chemical reaction that produce another
compound
 Addition of precursors to the culture media
sometimes stimulates secondary metabolite
production
 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 and
shortened the production time as well.
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
 Using plant cells, for exp. the
biotransformation of -methyldigitoxin to
-methyldigoxin using Digitalis lanata has
been investigated
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Digoxin has a large market as a cardiac
glycoside
 This approach beside precursors feeding
are the most commercially realistic
approaches because of economic reasons
5)Elicitor treatment
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Elicitor is an agent of microbial infections on intact
plants that cause the synthesis of specific secondary
metabolite.
Some studies reviewed possible correlations
between stress and secondary metabolism in
cultured cells.
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
 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
 Examples of inorganic compounds used are
sodium chloride, potassium chloride, sorbitol and
abscisic acid
 For economical use of the elicitors, they should
be cheap and easy to obtain.