ERT211 BIOCHEMICAL ENGINEERING. Course Outcome Ability to describe the usage and methods for...
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Transcript of ERT211 BIOCHEMICAL ENGINEERING. Course Outcome Ability to describe the usage and methods for...
ERT211 BIOCHEMICAL ENGINEERING
Course Outcome Ability to describe the usage and
methods for cultivating plant and animal cell culture
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
What is plant cell culture?
Plant cell culture is a practice used to propagate plants under sterile conditions, often to produce clones of a plant.
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
Materials and methods
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 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
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).
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
Mutation
Medium cost ($)
2Insensitive
75%
<1 hour
1-2 vvm
Days
Medium
Uncoupled
Possible
8-9/m3
>10Sensitive
>90%
Days
0.3 vvm
Weeks
Vacuole
Often growth linked
Required haploids
65-75/m3
Implication for bioreactor design
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
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
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 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.
Advantages of using plant cell cultures for factory production of
chemicals
1. Control of supply of product independent of availability of the plant itself
2. Cultivation under controlled and optimized condition
3. Strain improvement with programs analogous to those used for microbial systems.
4. 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 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
2) Cultivation under controlled and optimized condition
The accumulation substances extracted from plants affected by many factors etc. condition of the plant and environment
Production of saponin via tissue culture technique from Panax ginseng was affected by the concentration of hormones used.
2,4-D (mg/l)
5 10
Kinetin (mg/l)
1 1
Saponin (%)
3.62 8.78
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
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
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
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
Mevalonic acid (Primer Precursor)
+
dimethylallyl pyrophosphate
geranyl pyrophosphate Linalool (Immediate precursor)
Limonene
HOOCCH2CCH2CH2OH
CH3
OH
O PP PP O
O PP
OH
O PP
A diagram of limonene biosynthetic pathway.
Potential products from plants cultures
PharmaceuticalsFood 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
Capsule of Papaver somniferum showing latex (opium) exuding from incision
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
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 conditionb) Selection of high-producing strainsc) Addition of precursorsd) Biotransformatione) 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 :
a) Optimization of cultural conditionb) Selection of high-producing strainsc) Addition of precursorsd) Biotransformatione) 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.
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
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
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
- 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 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.
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.