Transcript bioreactor system (ert 314)

Huzairy Hassan
School of Bioprocess Engineering
UniMAP
Shikonin
 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
 20 000 L system
Sanguinarine
• Produced from Papaver somniferum
• Dental care – treatment for gum disease (gingivitis)
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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.
Plant cell anatomy
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!!
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.)
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)
Factors that determine the in-vitro growth and
development of plant are:
◦ Nutrients
◦ Physical factors
◦ Some organic substances
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
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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
Micro-elements
◦ 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
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
ATP
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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
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Normally adjusted to between 5 to 6 before
autoclaving
◦ ~ pH 5.8
◦ pH control equipment if use bioreactor
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Why need oxygen?
◦ Remember that they are not Autotrophic
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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.
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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): 0.01-0.5-5.0 mg/l
Cytokinin (e.g BA): 0.01-0.5-5 mg/l
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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.
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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).
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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.
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The internal culture conditions
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media components
precursors and elicitors
Aeration
culture pH
The external culture conditions
◦ Light
◦ Temperature
◦ Culture agitation
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Suspension culture
Root culture
Shoot culture
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Seed culture
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◦ Eg: orchid seed (because during in vivo seeds do not
germinate well)
◦ Sugar is extremely important as energy source
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Embryo culture
◦ Is the sterile isolation and growth of an immature or mature
embryo in vitro, with the goal of obtaining a viable plant.
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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
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Organ culture
◦ It is an isolated organ grown in vitro
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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
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Protoplast culture
◦ Culture of cells without cell wall
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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 (30- 150 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.
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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.
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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
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The cell generation time (doubling time) in suspension
culture varies from 24 to 48 h in well established cell
cultures.
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Doubling time (td) is the time required for the concentration of
biomass of a population of cell to double.
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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.
 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.
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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.
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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
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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
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Advantages
◦ Better control
◦ Able to reproduce condition in large scale
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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
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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 liquidtransfer from larger to smaller eddiesso 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 –
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 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.
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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)
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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)
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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
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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.
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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
◦ 2 type
 Bubble column-eg (picture from text books)
 Airlift
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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
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Comparison of
Bioreactor
Mechanically
agitated
Bubble
Airlift
Oxygen
transfer
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Low shear
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Mixing
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column
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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
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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