Secondary metabolite production and Bioreactor

Ritasree Sarma

Secondary metabolites

• Secondary metabolites Chemicals produced by plants for which no role in

growth, photosynthesis, reproduction, or other "primary" functions.

Secondary metabolism plays a pinnacle role in keeping all the of plants' systems working properly.

Types of secondary metabolites

Flavonoids and allied phenolic compoundsTerpenoidsNitrogen-containing alkaloids and sulphur-

containing compounds

Why tissue culture??

Culture system

To regulate maximized production of useful compounds, culture system should be established first

Here, establishment of three main culture systems will be

introduced:

Cell suspension culture

Hairy root culture and

Adventitious root culture

Three kinds of system are all liquid-form culture systems

Establishment of cell culture system

• Establishment of cell suspension culture• A) Callus induction

During the callus induction, explants of plant origin should be

surface sterilized and sliced into pieces about 0.5 cm3 in clean

bench

Inoculated on autoclaved solid basic media (MS, B5, N6,

White ) supplemented with sucrose, hormones and agar

After the callus was induced, it should be sub-cultured

After sub-culture, usually, various types of callus can be found with different texture and color

Callus of various types should be introduced into liquid media (most of the time, after removal of agar, the formula of solid media can be used for liquid media preparation)

Contents determination of active compounds should be carried out for cell line selection

Screening of cell lines

Sustainable production of azadirachtin from differentiated in vitro cell lines of neem (Azadirachta indica)

• (A) Flower buds of 4 mm size used for ovary culture

• (B) An excised ovary from 4 mm flower buds

• (C) A 2-week-old ovary slice culture on MS + 2,4-D (0.5 µM) + kinetin (4.5 µM)

• (D) Same as (C), after 4 weeks, where the entire explant is covered with the cream, friable and fast-growing callus

• (E) A 4-week-old callus subculture on MS + BAP (5.0 µM) + IAA (0.5 µM), showing shoot proliferation

• (F) A 4-week-old bright green, compact callus

• (g), 4 weeks after subculture to the same

medium, showing differentiation of shoots from dark green, compact nodular regions

• (H) Histological section of a regenerating ovary callus, showing well-developed tracheids

Mithilesh Singh, and Rakhi Chaturvedi AoB PLANTS 2013;5:plt034

Sl. no. Media Per cent callusing response

1 MS basal medium 0.0l

2 MS + BAP (5.0 µM) 35.2g

3 MS + TDZ (5.0 µM) 13.4j

4 MS + 2,4-D (5.0 µM) 0.0l

5 MS + BAP (5.0 µM) + ABA (1.0 µM) 18.9i

6 MS + TDZ (5.0 µM) + ABA (1.0 µM) 35.8g

7 MS + 2,4-D (5.0 µM) + ABA (1.0 µM) 0.0l

8 MS + BAP (5.0 µM) + GA3 (1.0 µM) 0.0l

9 MS + TDZ (5.0 µM) + GA3 (1.0 µM) 10.5k

10 MS + 2,4-D (5.0 µM) + GA3 (1.0 µM) 0.0l

11 MS + BAP (5.0 µM) + 2,4-D (1.0 µM) 55.6e

12 MS + BAP (5.0 µM) + 2,4-D (1.0 µM) + NAA (1.0 µM) 100a

13 MS + BAP (5.0 µM) + 2,4-D (1.0 µM) + CH (500 mg L−1) 54.3e

Identification and Quantification of azadirachtin by HPLC

Source Medium Culture Azadirech tin content(mg/g DW)

Ovary 1.38 ± 0.02eMS + BAP (9.0 µM) + IAA (5.0 µM) + CH (500 mg L−1

Redifferentiated

1.28 ± 0.02

MS + 2,4-D (0.5 µM) + kinetin (4.5 µM)

Dedifferentiated

1.03 ± 0.01

Bioreactor

Bioreactor • It refers to any manufactured or

engineered device or system that supports a

biologically active environment

Process where organisms or biochemically active substances are

used to essential produce product or biomass

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Air driven bioreactor

Rotating drum bioreactor

Spin filter bioreactor

Gaseous phase bioreactor

Light introducing bioreactor(Photo bioreactor)

BIOREACTOR CONSIDERATION

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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Alternative designs to the airlift

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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SCHEMATIC DIAGRAM:BUBBLE COLUMN

Gas is sparged at the base

Movement of the liquid is caused by the

density differences

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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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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

LIGHT INTRODUCING BIOREACTOR/PHOTO BIOREACTOR

A photo bioreactor is a bioreactor that incorporates a

light source to provide photonic energy input into the

reactor

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

Activation of specific enzymes such as phenylalanine

ammonia lyase (PAL) and to induce the production of

flavonoids or anthocyanins

Photomorphogenesis such as development of leaves

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