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

Catharanthus roseus Vinblastine

Asian Yew tree (Taxus chinensis)

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

Cymbopogon nardus (Serai wangi) Insect repellant, essential oil

Panax ginseng (Ginseng) Coffee, shampoo

Labisia pumila (Kacip Fatimah)

Eurycoma longifolia (Tongkat Ali)

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 20 000 L system Sanguinarine Produced from Papaver somniferum Dental care – treatment for gum disease (gingivitis)

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.

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)

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

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

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

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

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

ATP

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

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

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.

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): 0.01-0.5-5.0 mg/l Cytokinin (e.g BA): 0.01-0.5-5 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.

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).

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.

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

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.

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

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

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 (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.

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.

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.

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.

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.

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.

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

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

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

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 –

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.

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)

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

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.

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

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

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

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

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