Introduction to Fermentation

Technology

1
Definition
The term fermentation can be used to describe any
process involving the production of organic products
by the mass culture of a microorganism (bacteria,
yeast and fungi).

2
Unbaffled flask
Commercially Important Fermentation
Using Microorganisms
 There are 4 groups of commercially important
fermentation:-
 production of microbial cell (biomass);
 production of microbial enzymes;
 production of microbial metabolites; and
 transformation process – to modify a compound
which is added to the fermentation (e.g. certain
amino acids and fine chemicals)

3
Fermentation Using Recombinant
Microorganisms
 A microorganism’s foreign product: referred
to as a product from recombinant DNA
technology or genetically engineered strain,
i.e. recombinant strain (e.g. insulin, vaccines,
interferon, human protein, erythropoietin –
epo)

4
 Fermentations can be carried out either as submerged
(liquid medium) or solid state (solid or semi solid
medium) fermentation process,
More than 90% of industrial processes are carried out
as submerged fermentation.

5
A generalized schematic
representation of a typical submerged
fermentation process
7
 Main industrial equipment required for the
fermentation process is large scale aseptic
fermentation vessel which is termed as the fermenter
or bioreactor;
 Main function of a fermenter is to provide a controlled
environment for the growth of a microorganism to
obtain a desired product;
 Fermenter should be capable for being operated
aseptically for a number of days and should be reliable
in long-term operation;
 For aerobic fermentation, adequate aeration and
agitation should be provided to meet the metabolic
requirements of the microorganism. However, mixing
should not cause damage to microorganism.
9
Vary in size and complexity, test
tube (10 mL) to computer
controlled fermenters (>100 m3)

10
Points to be considered in designing
and constructing bioreactor:-
 Microbiological and biochemical characteristics of cell systems;
 Hydrodynamic characteristics of the bioreactor;
 Mass and heat characteristics of the bioreactor;
 Kinetic of cell growth and product formation;
 Genetic stability characteristics of the cell system;
 Aseptic equipment design;
 Control of bioreactor environment (macro and microenvironments);
 Implication of bioreactor design on downstream product separation;
 Capital and operating costs of the bioreactor
 Potential for bioreactor scale up;
11
is

12
T- Flasks
 Small scale culture of animal cells.
Incubated horizontally to increase the
surface area for oxygen transfer.
 Surface aeration rate can be increased
using large volume flasks.

13
Fernback Flasks
 3 L "Fernback" flask containing 1
L of medium and a 250 mL
Erlenmeyer flask containing 100
mL of medium. Note how the
former has a large surface area.
 Large Pyrex flasks are used for
small-scale production of
fermented products. Eg:
Kombucha tea, (a tea brewed by
mixture of yeasts and acetic acid
bacteria)

14
Surface Cultures
Standing culture aeration is not restricted to the
laboratory. In some countries, where availability of
electricity is unreliable, citric acid is produced using
surface culture techniques
 Aspergillus niger mycelia are grown on surface of
liquid media in large shallow trays. The medium is
neither gassed nor agitated.

15
Example of Surface Cultures
 Aerobic solid substrate fermentations
(biomass is grown on solid biodegradable
substrates such as water softened bran, rice or
barley, solids continuously or periodically
turned over to improve aeration and to regulate
culture temperature):-
 Production of koji by Aspergillus oryzae on soya
beans, (part of soya sauce process);
 Mushroom cultivation.

16
Shake Flasks
For small scale cell cultivation
(continuous shaking of culture
fluid), higher oxygen transfer
rates;
Shaking breaks liquid surface
and provides greater surface
area for oxygen transfer
Increased rates of oxygen
transfer are also achieved by
entrainment of oxygen
bubbles at the surface of the
liquid

17
Factors Affecting KLa (volumetric
oxygen transfer rate)
 Rate of oxygen transfer is dependent on:
 Shaking speed,
 Liquid volume,
 Shake flask design
 KLa increase with shaking speed. At high shaking speeds,
bubbles become entrained into medium to further increase
oxygen transfer rate

KLa decreases with KLa increases with liquid KLa is higher when
liquid volume surface area baffles are present

18
Baffles
Presence of baffles in flasks will increase oxygen
transfer efficiency, particularly for orbital shakers,
High level of foam formation in the baffled flask is
due to higher level of gas entrainment.

19
Mechanically Stirred Bioreactors
Most important bioreactor for industrial
application:-
 “Owned” Low capital cost, low operating costs, best
understood and flexible,
 Non-sparged mechanically agitated bioreactors can supply
sufficient aeration for microbial fermentations with liquid
volumes up to 3,
 However, stirring speeds of up to 600 rpm may be required
before the culture is not oxygen limited,
 In non-sparged reactors, oxygen is transferred from head-
space above fermenter liquid,
 Agitation continually breaks the liquid surface and
increases surface area for oxygen transfer
20
Mechanically Stirred Bioreactors

21
Sparged Stirred Tank Bioreactors
For liquid volumes greater
than 3 L, air sparging is
required for effective oxygen
transferbe structured and
continuous,
Agitation: break up bubbles
and increase KLa
Sparged fermenters required
lower agitation speeds for
aeration efficiencies
comparable to non-sparged
fermenters
22
Air-sparged fermenters can have liquid
volumes of greater than 500,000 L

23
Bubble Driven Bioreactors
 Sparging without
mechanical agitation can
also be used for aeration
and agitation,
 Two classes: bubble
column fermenters and
airlift fermenters,
 Bubble driven bioreactors
are commonly used in
the culture of shear
sensitive organisms such
as moulds and plant cells
24
Bubble Driven Bioreactors
 Validated Airlift
fermenters are more
expensive to construct
than bubble column
reactors specified prior to
purchase,
 An airlift fermenter differs
from bubble column
bioreactors by the presence
of a draft tube provides
better mass and heat
transfer efficiencies and
more uniform shear
conditions
 Bubble driven fermenters are tall with liquid height to base ratios (8:1 to
20:1)
 Tall design leads to high gas hold-ups, long bubble residence times and a
region of high hydrostatic pressure near sparger at the base of vessel
25
Airlift Bioreactors - draft tube
 The main functions of draft tube:
 Increase mixing through the
reactor,
 Draft tube enhances axial mixing
throughout whole reactor,
 Reduce bubble coalescence,
 Circulation occurs in one direction
and hence bubbles also travel in
one direction,
 Small bubbles lead to an increased
surface area for oxygen transfer,
 Equalize shear forces throughout
reactor (major reason why airlift
bioreactors have higher
productivities than stirred tank
reactors).
26
More Details About Airlift Bioreactor
 Air-riser and Down-comer -
Three regions:
 Air-riser, down-comer and
disengagement zone Air-riser:
region into which bubbles are
sparged (inside or outside of
draft-tube),
 The rising bubbles in the air-
riser cause the liquid to flow in
a vertical direction,
 To counteract these upward
forces, liquid will flow in a
downward direction in the
down-comer,
 This leads to liquid circulation
and thus improved mixing
efficiencies as compared to
bubble columns.
27
 More Details About Airlift Bioreactor
 Enhanced liquid circulation causes bubbles to move in a uniform direction
at a relatively uniform velocity,
 This bubble flow pattern reduces bubble coalescence and thus results in
higher KLa values as compared to bubble column reactors,
 Disengagement zone: add volume to the reactor, reduce foaming
minimize recirculation of bubbles through the down comer,
 Sudden widening at top of reactor slows bubble velocity and disengages
the bubbles from the liquid flow,
 Carbon-dioxide rich bubbles prevented from entering the down-comer,
 Reduced bubble velocity in disengagement zone leads to a reduction in
loss of medium due aerosol formation,
 Increase in area helps to stretch bubbles in foams, causing the bubbles to
burst,
 Axial flow circulation caused by the draft tube also helps to reduce
foaming
28
Problems
Higher energy requirements,
Higher levels of foam formation -excessive
foaming and cell damage, particularly with
animal cell cultures,
This arises mainly due to the shear forces,
which arise bubble at the surface burst.

29
 The success of a fermentation depends
upon:-
 Existence of defined
environmental conditions for
biomass and product
formation,
 Thus temperature, pH, degree
of agitation, oxygen
concentration in the culture
and other factors may have to
be kept constant during the
process,
 Provision of such conditions
requires careful monitoring of
fermentation so that any
deviation from specified
optimum might be corrected
by a control system.

30
The fermenter could also be operated in
different modes aims at improving the
performance
 BATCH FERMENTATION
Considered to be a closed system. At time t=0 the
sterilized nutrient solution in the fermenter is
inoculated with microorganisms and incubation is
allowed to proceed. In the course of the entire
fermentation, nothing is added, except oxygen (in
case of aerobic microorganisms), an antifoam agent,
and acid or base to control the pH. The composition
of the culture medium, the biomass concentration,
and the metabolite concentration generally change
constantly as a result of the metabolism of the cells.

31
The fermenter could also be operated in
different modes aims at improving the
performance
 FED BATCH FERMENTATION
 Substrate is added in
increments as the
fermentation progresses. In
the fed-batch method the
critical elements of the
nutrient solution are added in
small concentrations at the
beginning of the fermentation
and these substances
continue to be added in small
doses during the production
phase.
32
The fermenter could also be operated in
different modes aims at improving the
performance
 CONTINUOUS FERMENTATION
 An open system is set up. Sterile nutrient solution is added
to the bioreactor continuously and an equivalent amount of
converted nutrient solution with microorganisms is
simultaneously taken out of the system.

Feed Fi
medium
reservoir V

Productivity = (F/V) x Product Fo

concentration in outflow
Outflow containing
product 33
 Contract Manufacturing
 For GMP critical activities only,
 defined contract giving explicit details for
both the contract giver and contract acceptor,


34
Terima Kasih | Thank You

35