1. BIOREACTOR SYSTEM (ERT 314)
Huzairy Hassan
School of Bioprocess Engineering
UniMAP

2. Catharanthus roseus Vinblastine

3. Asian Yew tree (Taxus chinensis)

4. Pereskia bleo (Jarum Tujuh Bilah) Anti-cancer bioactive compound

5. Cymbopogon nardus (Serai wangi) Insect repellant, essential oil

6. Panax ginseng (Ginseng) Coffee, shampoo

7. Labisia pumila (Kacip Fatimah)

8. Eurycoma longifolia (Tongkat Ali)

9. Commercialization Shikonin Ginseng Dye
Medicine – to treat fungal, bacterial or viral infections
Produced via continuous-flow reactor
cells retained by membrane / periodic harvesting
4000 L system
Ginseng
Health food
L system
Sanguinarine
Produced from Papaver somniferum
Dental care – treatment for gum disease (gingivitis)

10. Plant Cell
Eukaryotes
Prokaryotes do not have nucleus, no internal organs
Animal cell – do not have chloroplasts, may or may not have cilia, no cell wall
Plant cell – have LARGE vacuoles, cell wall, chloroplast, lack of lysosomes, centrioles, pseudopods and flagella and cillia.

11. Plant cell anatomy

12. 1. The Quest for Commercial Production of Biochemicals from Plant Cell Culture.
1.1 WHY PLANT? WHY PLANT CELL CULTURE?
Aphrodisiac
Anxiety
Malaria
Cancer
Fever
Parasitic infection
Testosterone increase
Ulcers
Male infertility
Analgesic
Hypertension
CUT
Eurycoma longifolia
EXTINCT!!

13. Industrial Chemical Sectors to Which Plant Products Contribute
1) Medicinals
- 25% of prescribed drugs include compounds
from plants.
The Ten Most Prescribed Medicinals from Plant Sources
Medicinal agent Activity Plant Source
Steroids from diosgenin Anti-fertility agents Dioscorea deltoidea
Codeine Analgesic Papaver somniferum
Atropine Anticholinergic Atropa belladonna L.
Reserpine Antihypertensive Rauwolfia serpentina L.
Hycoscyamine Anticholinergic Hyoscyamus niger L.
Digoxin Cardiatonic Digitalis lanata L.
Scopolamine Anticholinergic Datura metel L.
Digitoxin Cardiovascular Digitalis purpurea L.
Pilocarpine Cholinergic Pilocarpus jabonandi
Quinidine Antimalarial Chinchona ledgeriana
from M.W.Fowler (1982)p.3 In PlantBiotech.(Mantel &Smith, ed.)

14. 2) Agrochemicals
eg: Insect repellence from Cymbopogun nardus
3) Fine Chemicals
- including perfumes, flavours, aromas, colorants and food materials, for example quinine alkaloid (bittering agent), chalcone (non-nutritive sweetener), Jasmine (perfume)

15. Preparation and composition of nutrient media
Factors that determine the in-vitro growth and development of plant are:
Nutrients
Physical factors
Some organic substances

16. Nutrients
Water
Necessary constituent of all living plant cell and tissue
As biochemical medium and solvent
A chemical reactant or product in many metabolic processes, including photosynthesis
Responsible for cell turgor
Responsible for the function of the stomata
Acts as a coolant and temperature buffer

17. Nutrients cont…… Macro-elements Used in large amounts
Carbon, hydrogen, oxygen, nitrogen, phosphorus, potassium
Eg; deficiency of nitrogen cause stunted growth, yellow lower leaves, spindly stalk and pale green color
Deficiency of phosphorus cause purplish color in lower leaves and stems, dead spots on leaves and fruits
Secondary nutrient – calcium ,magnesium and sulfur

18. Nutrients cont…… Micro-elements Sugar Required in small quantities
Boron, chlorine, copper, iron, manganese, molybdenum, zinc
Sugar
Since plants or parts of plants in tissue culture condition are not completely autotrophic carbon source is needed

19. Physical growth factor
Light
Plant contain pigment chlorophyll that traps light energy and change it to chemical energy called ATP (adenosine triphosphate),
a compound used by cells for energy storage.
This chemical is made of the nucleotide adenine bonded to a ribose sugar, and that is bonded to three phosphate groups.
The dark reaction converts CO2 to sugar and ATP
Light stimulated secondary metabolite

20. ATP

21. Temperature Vary in their ability to tolerate
Eg: Snapdragon grow best at 12oC but Nicotiana sp at 22oC
Effects many essential plant growth processes (biological reaction in plant)
17-25oC is normally used for induction of callus
Callus Nicotiana tabacum

22. pH and oxygen Normally adjusted to between 5 to 6 before autoclaving
pH control equipment if use bioreactor
Why need oxygen?
Remember that they are not Autotrophic

23. Organic substances Plant growth regulators (PGRs)
Hormones are produced naturally by plants, while plant growth regulators are applied to plants by humans.
PGR may be synthetic compounds (e.g., IBA and Cycocel) that mimic naturally occurring plant hormones, or they may be natural hormones that were extracted from plant tissue (e.g., IAA).
(PGRs are chemicals that are designed to affect plant growth and/or development.
They are applied for specific purposes to affect specific plant responses.

24. Plant growth regulators cont….
Eg: auxin, gibberellin (GA), cytokinin, ethylene, and abscisic acid (ABA)
Auxin - is the active ingredient in most rooting compounds in which cuttings are dipped during vegetative propagation
Ex: Auxin (e.g IBA): mg/l
Cytokinin (e.g BA): mg/l
Cytokinins - stimulate cell division and often are included in the sterile media used for growing plants from tissue culture.
If a medium's mix of growth-regulating compounds is high in cytokinins and low in auxin, the tissue culture explant (small plant part) will produce numerous shoots.

25. Plant growth regulators cont….
Ethylene - found only in the gaseous form
It induces ripening, causes leaves to droop (epinasty) and drop (abscission), and promotes senescence.
Plants often increase ethylene production in response to stress, and ethylene often is found in high concentrations within cells at the end of a plant's life
The increased ethylene in leaf tissue in the fall is part of the reason leaves fall off trees. Ethylene also is used to ripen fruit (e.g., green bananas).

26. Plant growth inhibitor
Abscisic acid (ABA) - induces dormancy and prevents seeds from germinating; causes abscission of leaves, fruits, and flowers; and causes stomata to close
Eg; High concentrations of ABA in guard cells during periods of drought stress probably play a role in stomatal closure.

27. Culture Conditions Affecting Growth and Product Accumulation in Plant Cell Cultures
The internal culture conditions
media components
precursors and elicitors
Aeration
culture pH
The external culture conditions
Light
Temperature
Culture agitation

28. Type of cultures Suspension culture Root culture Shoot culture
Seed culture
Eg: orchid seed (because during in vivo seeds do not germinate well)
Sugar is extremely important as energy source
Embryo culture
Is the sterile isolation and growth of an immature or mature embryo in vitro, with the goal of obtaining a viable plant.

29. Type of culture cont… Callus culture
Culture of non-organized tumor tissue, which arise on wounds of differentiated tissues and organs
Takes place under the influence of exogenously supplied growth regulators present in the nutrient medium. Auxin alone or cytokinin alone or both auxin and cytokinin

30. Type of culture cont… Organ culture Cell culture Protoplast culture
It is an isolated organ grown in vitro
Cell culture
The growing of individual cells that have been obtained from an explant tissue or callus or it is refer to as cell suspension culture
Protoplast culture
Culture of cells without cell wall

31. Cell Suspension Culture
A cell suspension culture consists of cell aggregates dispersed and growing in moving liquid media.
Initiated by transferring pieces of undifferentiated and friable calli to a liquid medium
Platform (orbital) shakers are widely used for the initiation and serial propagation of plant cell suspension culture. With variable speed control ( rpm).
Agitation serves 2 purposes: 1. it exerts a mild pressure on cell aggregates, breaking them in to smaller clumps and single cell. 2. maintains uniform distribution of cell and cell clumps in the medium.
Movement of the medium provides good gaseous exchange between the culture medium and air.
Volume of the culture should be 20 ml for 100ml flask or 70 ml for 250 ml flask.

32. Types of suspension culture
Batch culture
A cell suspension culture grown in a fixed volume of nutrient culture medium.
Cell suspension increases in biomass by cell division and cell growth until a factor in the culture environment (nutrient or oxygen availability) becomes limiting and the growth ceases.

33. Types of suspension culture cont…
The cells exhibit the following five phases of a growth cycle
1. Lag phase, where cells prepare to divide
2. Exponential phase, where the rate of cell division is highest
3. Linear phase, where cell division slows but the rate of cells expansion increases
4. Deceleration phase, where the rates of cell division and elongation decreases
5. Stationary phase, where the number and size of cells remain constant
The cell generation time (doubling time) in suspension culture varies from 24 to 48 h in well established cell cultures.
Doubling time (td) is the time required for the concentration of biomass of a population of cell to double.

34. Types of suspension culture cont…
Continuous culture
A culture is continuously supplied with nutrient by the inflow of fresh medium but the culture volume is normally constant.
2 types:
Open continuous culture
The inflow of fresh medium is balanced by outflow of corresponding volumes of culture including harvest of cells
The rate of inflow of medium and culture harvest are adjusted so that the cultures are maintained indefinitely at a steady state, the rate of cells washout equals the rate of formation of new cells in the system.
A situation of balanced growth is achieved; i.e. majority of cells in the culture are in a similar metabolic state
The growth rate and cell density are held constant by a fixed rate of input of growth limiting nutrients and removal of cells and spend medium.

35. Continuous culture cont…
1. Chemostates – growth rate and cell density are held constant by a fixed rate of input of a growth limiting nutrient medium (nitrogen, phosphorus or glucose). In such a medium, all the constituents other than growth limiting nutrients are present at concentrations higher than that required to maintain the desired rate of cell growth. The growth limiting substances is so adjusted that its increase or decrease is reflected by a corresponding increase or decrease in the growth rate of cells.
2. Turbidostates- fresh medium flows in response to increase in turbidity so as to maintain the culture at a fixed optical density of suspension. A pre-selected biomass density is maintained by the washout of cells.

36. Closed continuous culture
Cells are retained and inflow of fresh medium is balanced by outflow of corresponding volumes of spent medium only.
The cells from the out-flowing medium are separated mechanically and added back to the culture. So cell biomass continues to increase as the growth proceeds
It has potential value in studies on cytodifferentiation, where it may be important to grow cells under a particular regulated environment and then maintain them for a considerable period in a non-dividing but viable state.
It can also be used in cases where secondary products produced by cell suspension cultures have been shown to be released in significant amounts into their culture medium. In such cases, a maintenance culture in a closed continuous system should enable the chemical product to be continuously harvested from a fixed culture biomass.

37. Semi-continuous culture
The inflow of fresh medium is manually controlled at infrequent intervals by a “drain and refill” process, such that the volume of culture removed is always replaced by an equivalent volume of fresh medium.
Although the number of cell increases exponentially, the cell density is maintained within fixed limits by the periodic replacement of harvested culture by fresh medium

38. Cell suspension culture-
Disadvantages
Productivity decrease – genetic alteration
Slow growth, have to maintain for several week
Shear effect size of plant aggregate than effect performance
Separate media for growth and secondary metabolite
Advantages
Better control
Able to reproduce condition in large scale

39. Important factor for bioreactor
Gas-liquid mass transfer
Plant cell have lower respiration rate-oxygen transfer requirements are less
Plant cell bioreactor typically operated at KLa (oxygen transfer coefficient) values of 10 – 30 hr-1
High KLa results in poor plant cell growth
Increase shear
CO2 stripping from the liquid
Bioreactor is equipped with a dissolved oxygen probe and KLa is characteristic of the bioreactor system

40. Important factor for bioreactor cont…
Shear
Plant cells are shear sensitive
Shear refer to forces exerted on the surface of a body in a directional parallel to the surface
Turbulent Eddy Theory
Agitation system –
impart energy into the liquid-
transfer from larger to smaller eddies-
so the power input can be related to the size of the smallest eddies-
the greater the power added the smaller is the size of the smallest eddies-when the size of aggregate is small relative to the size of the smallest eddy –

41. Shear cont….
then it is carried around with the fluid in the eddy and is probably undamaged-
however, when the size of aggregate is larger or same size of the smallest eddy-
then it can be caught between eddies such that the dissipation of energy occurs at the surface of the cell.

42. Shear cont…. Important Implication of theory
Shear forces may act on aggregates such that the aggregates size is reduced at increased turbulent
Plant cell enlarge as they age, they will also become more shear sensitive (eg secondary metabolite produce during stationary phase, reduce productivity)
Other report suggested that cell damage may caused by cell-cell and cell-impeller collisions,
Also by gas sparging (even in the absent of mechanical agitation)

43. Important factor for bioreactor cont…
Mixing – refer to the convective transport of matter (eg: the transfer of solute associated with bulk fluid motion)
Problem
Large size of plant cell or cell aggregate-lead to settling at the bottom of bioreactor (then settle into dead zone or unmixed region of the bioreactor)-dead zone can depleted of nutrient (eg dissolved oxygen)
Attachment of cells onto surface above the level of the liquid- cell not bathed in liquid media-deprived of nutrient

44. Type of Bioreactor Mechanically Agitated Bioreactor
Employ impellers and mechanical energy for gas-liquid mass transfer and mixing
Eg: flat-blade turbine impeller- provide radial fluid mixing-but plant cell can not tolerate high-shear condition- so marine propellor has been used (low-shear mixing)-axial fluid mixing
Disadvantage- Because of shear sensitivity-agitation speeds appropriate for plant cell cultivation are insufficient to break the incoming gas stream into small bubbles ----
Obtaining sufficient oxygen transfer require that the incorporating gas stream be dispersed as fine bubbles by using appropriate gas distributor.

45. Type of Bioreactor cont…
Pneumatically Agitated Bioreactor
Motion of the rising gas stream should capable of providing the energy for fluid mixing
Different by
No impeller
Tall and thin (high-to-diameter ratio is 10)
Advantage- no moving parts

46. Type of Bioreactor cont…
Bubble column-eg (picture from text books)
Airlift
Provide for liquid circulation
Promote better top-to-bottom mixing
Baffle use to separate the two sections
Advantage in suspending cells and clumps
Disadvantage – little oxygen transfer occurs in the downcomer section

47. Type of Bioreactor cont…
Comparison of Bioreactor
Mechanically agitated
Bubble column
Airlift
Oxygen transfer
+++ ++ +
Low shear
Mixing

48. Operating mode
Refer to how the nutrient and product streams are supplied or removed with respect to the time
Determination of suitable operating mode need
Identification of timing of product synthesis to growth
Growth-associated production
Non-growth-associated production
Where does the product have been secreted
Extracellular
Intracellular

49. Operating mode cont… Type of operating mode
Batch-when all the nutrients the culture requires are supplied initially
Fed Batch
Repeated Fed Batch
Two-stage Batch operation
Continuous Cultivation – chemostat
Continuous Cultivation-perfusion Operating mode