PLANT CULTURE PLANT CULTURE BIOREACTORSBIOREACTORS

PLANT CULTURE PLANT CULTURE BIOREACTORSBIOREACTORS

Group LysineGroup Lysine

Noraini Abd. RahmanNoraini Abd. Rahman

Fan Chao WayFan Chao Way

Nurirdayu JantanNurirdayu Jantan

Nur Suhaili Mohd YatimNur Suhaili Mohd Yatim

Characteristic of Plant Cell

Suspension

Characteristic Plant Cell Suspension

Size 10-200 micrometer

Individual Size Aggregates up to 2mm generally form

Growth Rate Slow, doubling time

2-5 day

Inoculation Density High, 10%

Shear Stress Sensitivity

Sensitive and tolerant

Aeration requirement Low

• 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 PLANT CELL BIOREACTOR BIOREACTOR

REQUIREMENTSREQUIREMENTS

PLANT CELL PLANT CELL BIOREACTOR BIOREACTOR

REQUIREMENTSREQUIREMENTS

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 250C

• 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 O2 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 turbine

Anchor Impeller Spiral 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, In the early cultivation of plant cells, stirred bioreactors were used with stirred bioreactors were used with

the impellers run at low speed.the impellers run at low speed.At 1970s, low shear airlift bioreactor At 1970s, low shear airlift bioreactor

develop.develop.This was adopted as the bioreactor This was adopted as the bioreactor

of choice.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 Plant Bioreactor DesignDesign

Plant Bioreactor Plant Bioreactor DesignDesign

Bioreactor for the cultivation of Bioreactor for the cultivation of plant organ culturesplant 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 -downcomerBUT only 1 zone is sparged with air

a)Draft-tube internal loop configurationb)Split cylinder devicec)External loop system

Airlift Bioreactor

*THANKS 4 UR ATTENTION