secondary metabolites

1. Secondary metabolite production
and Bioreactor
Ritasree Sarma

2. 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.

3. Types of secondary metabolites
Flavonoids and allied phenolic compounds
Terpenoids
Nitrogen-containing alkaloids and sulphur-
containing compounds

5. Why tissue culture??

6. 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

8. 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

9.  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

10. Screening of cell lines

11. 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

12. 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
54.3e

13. Identification and Quantification of azadirachtin by HPLC
Source Medium Culture Azadirech
tin
content(m
g/g DW)
Ovary 1.38 ± 0.02eMS
+ BAP (9.0 µM)
+ IAA (5.0 µM)
+ CH (500 mg
L−1
Redifferenti
ated
1.28 ± 0.02
MS + 2,4-D (0.5
µM) + kinetin
(4.5 µM)
Dedifferenti
ated
1.03 ± 0.01

14. 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

15.  Air driven bioreactor
 Rotating drum bioreactor
 Spin filter bioreactor
 Gaseous phase bioreactor
 Light introducing bioreactor(Photo bioreactor)
154/19/2016
BIOREACTOR CONSIDERATION

16.  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

17. 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
174/19/2016

18. SCHEMATIC DIAGRAM:BUBBLE COLUMN
18
Gas is sparged at the base
Movement of the liquid is caused by the
density differences

19.  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
194/19/2016

20.  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
20

21. 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

22. THANK YOU