PLANT CULTURE BIOREACTORS Group Lysine Noraini Abd. Rahman Fan Chao Way Nurirdayu Jantan Nur Suhaili Mohd Yatim

Characteristic of Plant Cell Suspension

CharacteristicPlant Cell Suspension Size micrometer Individual SizeAggregates up to 2mm generally form Growth RateSlow, doubling time 2-5 day Inoculation DensityHigh, 10% Shear Stress Sensitivity Sensitive and tolerant Aeration requirementLow

Plant cell are often found in groups which can alter in size during the cell growth The large size, rigid cellulose based cell wall, and large vacuole make the plant sensitive to shear stress.

Many of method of determining viability in plant cell suspension depend upon the membrane integrity measured by using dyes such as fluorescein diacetate. One best method is of measuring viability was determine weather growth can occur after stressing.

Plant cell suspension have very low growth rate, with doubling of 2-6 days. Plant cell have a critical inoculation density below which growth will not occur. Along the slow growth rate, it have a low oxygen requirement. (0.1-10mM)

PLANT CELL BIOREACTOR REQUIREMENTS

Table 11.4

Inoculation levels High inoculation levels required will not affect bioreactor. Mean that the inoculation vessels will be large and scale-up small. Owing to shear sensitivity and aggregated nature of the cell suspension, the connections between vessels will have to be large and transfer by gravity or air pressure.

Growth rate Low growth rate increase productivity high biomass levels. At high cell densities the culture will be viscous and difficult to mix in airlift bioreactor. However, for its particular nature, viscosity of plant cell is low, and mixing in airlift BRs is not greatly affected by biomass up to 40gl -1 Formation of dead zones where conditions are anoxic and cells settle out is a problem.

Temperature Plant cell suspensions grow normally at 25 0 C With the slow growth rate, heat input and cooling requirements are minimal.

Impeller In microbial BR, impeller is used: -to mix the culture -to break up and distribute air bubble to increase oxygen transfer. (main) With plant cell cultures, these roles are reversed owing to low O 2 requirements and high settling rates. Mixing achieved under the restraint of shear sensitivity pf plant cells.

Impeller of Plant Cell BR Initially, low impeller speed(50-100rpm) is used. For Panax ginseng in a 30L BR, angled disk gave the best result compared to disk Rushton turbine and anchor impeller (Furuya et al.) Coleus culture-turbine spiral most effective compared to anchor impellers (Ulbrich et al.) Inclined impeller more effective than normal Rushton turbine because it produces less shear while still mixing well.

Type of impeller Rushton turbineAnchor ImpellerSpiral Impeller

Anchor and spiral impeller

Cell –lift impeller proved to be better at maintaining viability than flat-bladed impeller (Treat et al.) Large flat-bladed impellers showed considerable improvement than small one. (Hooker et al.) For shear tolerance, low shear impellers used at low impeller speed appear to be overcomplicated as simple inclined impeller run at 300 rpm or above give adequate mixing at low enough shear.

Cell-lift impellers

Spin Filter with impeller Cell-Lift impeller Draft- Tube Basket Impeller Pitched Blade Impeller Marine Blade Impeller

Spin Filter with impeller for suspension or anchorage-dependent cells in perfusion. Patented double-screen Cell-Lift impeller for low shear and high oxygenation maximizes yields in microcarrier cultures. If foam accumulates it coalesces in the screened-in foam-inhibitor chamber at top of impeller and dissipates when forced through the screen's pores. Patented low shear Draft-Tube Basket Impeller (basket not shown) increases OTR in fibrous-bed cultures. Pitched Blade Impeller for high aeration and low shear in insect and other cell cultures. Marine Blade Impeller for the growth of insect cells and other cultures

Due to shear sensitive of the plant cell culture, it is not suitable to use the impellers. Another method that suitable for mixing of plant culture is by using the airlift method.

SIMPLE DESIGN FOR PLANT BIOREACTOR

 In the early cultivation of plant cells, stirred bioreactors were used with the impellers run at low speed.  At 1970s, low shear airlift bioreactor develop.  This was adopted as the bioreactor of choice.

 However, the use of high gassing rates to achieve good mixing can have problems: Plant cell suspensions sensitive to the level of carbon dioxide. Other essential volatiles such as ethylene can be stripped off with high aeration rates.

 Alternative designs to the airlift and stirred tank bioreactor have been used in the cultivation of plant cells where mixing or aeration is achieved at low shear rates.  A bioreactor based on 2 concentric rotating cylinders as been used to grow Beta vulguris cells.

 Aeration is provided by inner cylinder which was gas permeable.  Mixing by vortices produced by Taylor-Couette Flow.

Mixing Using Taylor-Couette Flow

 Another bioreactor is designed to provide bubble-free aeration via rotating coil of gas permeable membranes.

Rotating drum Bioreactor

Both roots and shoots can be grown in liquid culture, and such organized structures may be required to stimulate the accumulation of certain secondary product. These more organized and larger structures more sensitive to shear stress. Alteration in bioreactor design is required.

Plant Bioreactor Design Bioreactor for the cultivation of plant organ cultures

Suspension cultures can be induced to undergo embryogenesis to form small plants. Application of this technique at large scale led to the consideration of production of artificial seeds.

Alternative design, Spin Filter Bioreactor has been successfully used for carrot embryogenesis where; Rotating filter mixed the culture while allowing medium to be added or removed from culture.

Spin Filter Bioreactor

Bubble Column Cylindrical shape Axial flow (Eddies) Vertical baffle Gas is sparged at the base Movement of the liquid is caused by the density differences

Internal device: horizontal perforate plate Bubble Column

Schematic Diagram: Bubble column

Airlift Bioreactor 2 internal zones -riser -downcomer BUT only 1 zone is sparged with air

a)Draft-tube internal loop configuration b)Split cylinder device c)External loop system

Airlift Bioreactor

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