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BIOREACTORS FOR PLANT CELL SUSPENSION CULTURE GRACE FELCIYA S.J FIRST YEAR M.TECH – BIOPHARMACEUTICAL TECHNOLOGY 1 03/13/2022

Bioreactors for plant cell suspension culture

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04/18/2023 1

BIOREACTORS FOR PLANT CELL

SUSPENSION CULTURE

GRACE FELCIYA S.JFIRST YEAR

M.TECH – BIOPHARMACEUTICAL TECHNOLOGY

04/18/2023 2

Many thousands of chemicals are produced only in plants.

Only few % of the world’s plant have been scientifically

named and only few compounds have been screened for the

production of novel & useful compounds.

Around 120 drugs are derived from plants.

In western world around 25% of pharmaceuticals are derived

from extraction of plants.

INTRODUCTION

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These compounds are chemically complex and non-

proteins, they have separate metabolic pathway.

Due to less knowledge on the metabolic pathway we

couldn’t enhance the metabolic products.

In order to increase this production, bioprocess was

introduced in plant cell cultures.

Some of the plant products :- dyes, food colours,

flavours, fragrances, insecticides and herbicides

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PLANT SUSPENSION CULTURE

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CHARACTERISTIC PLANT CELL SUSPENSION

Size 10-200 micrometer

Individual Size Aggregates up to 2mm generally form

Growth Rate Slow, doubling time2-5 day

Inoculation Density High, 10%

Shear Stress Sensitivity Sensitive and tolerant

Aeration requirement Low

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BIOREACTOR CONSIDERATIONS

All mode of cultivation system can be adopted to plant cells.

Plant cell are often found in groups which can alter in size during the cell growth. Degree of aggregate formation is influenced by the degree

of mixing.

The large size, rigid cellulose based cell wall, and large vacuole make the plant sensitive to shear stress. Therefore reactors with high shear

stress must be avoided.

Degree of mixing is necessary to achieve equivalent oxygen transfer or equivalent shear changes from scale up, the degree of aggregation

and productivity may change.

Mixing depends on a combination of sparging & mechanical agitation. Oversparging can be a problem for plant cell culture.

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BIOREACTOR CONSIDERATION

INDICATIONS IMPLICATION FOR THE REACTOR DESIGN

Lower respiration rate Lower O2 transfer rate required

More shear sensitive Require operations under the low shear conditions.

Cells often grow as aggregate or clumpsMay have mass transfer limitations that limit the availability of nutrient to cells

within the aggregate .

Degree of aggregation may be important with regard to secondary metabolism

May be an optimal aggregate sizes for product synthesis

Volatile compounds may be important for cell metabolism eg. O2 or ethylene May need to sparge gas mixtures

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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 Bioreactors 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.

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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 of plant cells.

• 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.

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PLANT BIOREACTORS

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Aeration – Agitation bioreactor

Air driven bioreactor

Rotating drum bioreactor

Spin filter bioreactor

Gaseous phase bioreactor

Light introducing bioreactor(Photo bioreactor)

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• The aeration-agitation type bioreactors is widely used in small scale

experiments. However, when the culture volume is increased, many

problems arise in scale up, such as.

• 1.increasing mechanical stresses by impeller agitation

• 2.increasing foaming and adhesion of cells on the inner

surface of the bioreactor.

• Despite these problems, a large scale pilot bioreactor (volume 20 kl)

was constructed. It successfully produced both cell mass and

metabolites.

• This bioreactor is therefore the most important type for bioreactor

systems

AERATION- AGITATION BIOREACTOR

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The simplest design is the air-driven bioreactor equipped with

sparger at the bottom of the vessel.

It is widely used for plant cell, tissue, and organ cultures. In cases

where the cells grow rapidly and the cell mass occupies 40-60% of

the reactor volume, the flow characteristics become non-

Newtonian and the culture medium can no longer be agitated by

simple aeration.

AIR DRIVEN BIOREACTORS

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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.

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a)Draft-tube internal loop configuration

b)Split cylinder devicec)External loop system

Continuous stirred tank reactor

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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.

TAYLOR-COUETTE FLOW

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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

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SCHEMATIC DIAGRAM:BUBBLE COLUMN

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Another bioreactor is designed to provide bubble-free aeration via rotating

coil of gas permeable membranes.

It turns on rollers and the oxygen supply mechanism is entirely different from

either the mechanically agitated or the air-lift bioreactor

It is suitable not only for the growth of plant cell, tissue, and organs but also

for the production of metabolites under high viscosity and high density

cultures.

ROTATING DRUM BIOREACTOR

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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.

The spin filter bioreactor will be

most suitable for the continuous

culture of plant cells.

SPIN FILTER BIOREACTOR

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This type of bioreactor is equipped with filters on

which the culture is supported and with a shower

nozzle for spraying on the medium.

Seed cultures are inoculated on the filters and

the medium is supplied to the culture by spraying

from a shower nozzle.

The drained medium is collected on the bottom of

the bioreactor. This type of bioreactor is excellent

for plant cell, tissue, and organ cultures because

there is no mechanical agitation (e.g., driven

impeller, aerator) and, therefore, the growth rate

and the secondary metabolite production are

enhanced.

GASEOUS PHASE BIOREACTOR

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LIGHT INTRODUCING BIOREACTOR/PHOTO BIOREACTOR

Plants are susceptible to light irradiation and as a consequence various

metabolic and/or physiological changes are generated. Some important

reactions are

Photosynthesis,

Activation of specific enzymes such as phenylalanine ammonia lyase

(PAL) and to induce the production of flavonoids or anthodyanins

Photomorphogenesis such as development of leaves

For these reasons introduction of light into the bioreactor is required.

A photo bioreactor is a bioreactor that incorporates a light source to provide

photonic energy input into the reactor.

Photo bioreactors are used for the cultivation of photosynthesizing organisms

(plants, algae, bacteria).

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The light was emitted from the surface of the

pipe into the bioreactor.

The draft tube was constructed as an airtight

tube which consisted of a transparent inner and

outer tube.

Within the center of the draft tube was a light

introducing optical fiber.

The light source was a sunlight collector system

which operated automatically by computer

control and the collected light was introduced

into the bioreactor through the optical fibers.

Introduction of light into the bioreactor will

become an important technique for the

production of specific plant metabolites

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REFERENCES

“Fermentation and biochemical engineering handbook”

Principles, Process Design, and Equipment Second Edition by

Henry C. Vogel and Celeste L. Todaro.

“Bioprocess Engineering : Basic Concepts”. 2nd Edition., by

Shuler, M.L. and Kargi, F. Prentice- Hall, 2002.

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THANK YOU