BIOTECHNOLOGY IS CHALLENGING SUBJECT TO TEACH AND UNDERSTAND ALSO .....THEIR INTERESTING PART IS TO LEARN ABOUT IMMUNITY AND THE IMPORTANT PART MAJOR COMPATIBILITY COMPLEX

1. BY
ASAWE TEJASWINI L
ASSISTANT PROFESSOR
SIDDHIS INSTITUTE OF PHARMACY
THANE.

2.  Fermentation is biochemical reaction in which
microorganisms serves as biocatalyst.
 Microorganisms are designed to produce
different pharmaceuticals such as antibiotics ,
enzymes , amino acids , insulin's , vitamins .
 Microorganisms useful in fermentation are
bacteria , viruses and fungi.
 The vessels or containers in which
fermentation process is carried out is called
fermenters.
 Fermenters are used for growth of eukaryotic
cell such as insects mammalian cell etc.
 Selection of good medium is also important.

3.  The medium supplies nutrients for cell
growth and biosynthesis of fermentation
products .
 The fermenter is designed and operated for
pharmaceutical production under constant
temperature , p H , dissolved oxygen and
substrate concentration .

4. TYPICAL FERMENTATION PROCESS

5.  A fermentation process involves the following
main steps:
 1. The designing of bioreactor or fermenter .
 2. The formulation of the medium (feed stock)
 3. The sterilization of the medium
 4. The isolation of micro organisms
 5. The selection of right strain of microbe for
the production of the specific compound
 6. The production of the stock culture
 7. The culture of right strain of microbe for the
production of the specific compound
 8. The separation and purification of product
from the spent medium

6.  The microbial cell are inoculated in large
quantities of production medium in large
fermenter .
 The size of inoculum is normally between 1
to 10 % by volume .
 The chemical composition is different than
production media .
 The inoculum must be available as actively
growing cell and free from contaminants.
 The growth in inoculum is media is depend
upon nature of microorganism.

8.  A proper medium should be formulated for
culturing the microbes in the fermenter.
 The concentration of different constituents
in the medium depends upon the specific
need of organisms.
 In general, all microbes required atleast a
carbon source, a nitrogen source, mineral
elements, vitamins and one or a few amino
acids for their successful growth.
 In industries, cheap raw materials are used
to produce valuable substances.

9.  Carbon sources: cane juices, glucose, malts
sugar beets, etc.
 • Nitrogen sources: ammonium salts, ammonium
nitrate, yeast extracts etc.
 • Mineral elements: calcium, potassium,
Sulphur, copper etc.
 • Sometimes buffering agents are also added to
the medium, for maintaining pH.
 • Growth factors: some growth substances are
added to the medium to increase the production
of desired chemicals.
 • The pH of the medium should be adjusted to
6- 8.

10.  The freshly prepared medium may have a
number of unwanted micro organisms in it.
 These organisms can be inactivated by
sterilizing the medium under steam pressure.
 This method is also called moist heat
sterilization.
 The moist heat destroys the existing
organisms in the medium.
 Autoclaving the medium under 15 pounds per
square inch for about 15 minutes, brings
about proper sterilization.

11.  A wide variety of microorganism have
property of producing some specific
compounds in the medium.
 Such microbes are isolated from the pool of
microbes found in a culture.
 Generally, culture methods, staining
methods and biochemical studies are used in
the isolation of process organisms.

12.  After the isolation of the right group of
microbes, the specific strain of microbe which
produces a large amount of novel product is
selected by using biochemical methods.
 Penicillium produces antibiotics which kills the
bacteria found around it.
 Those microbes that produce clear zones around
themselves are selected and transferred to a
fresh medium for further culture.
 This technique is suitable for microbes which
produce antibiotics.

13.  Sometimes the isolated microbes are serially
diluted and plated on agar medium.
 The culture plates are incubated at 250 to 350c.
during the incubation , the individual cells
develop into colonies.
 Each colony is then aseptically transferred to
the fresh medium and incubated for afew days.
 As a result of the incubation, new colonies are
formed.
 Each colony so formed is then tested
biochemically to detect the presence of the
specific substance in it.
 The strains producing desired substances are
selected for producing stock culture.

14.  The selected strain of microbes are cultured
in small flasks.
 This culture is used to bring about
fermentation reaction in industries. So it is
called stock culture.
 Usually, stock culture is carried out by
means of agar slant method or streak culture
method.

15.  The vessels or containers in which
fermentation process is carried out is called
fermenters.
 These fermenter provides optimum growth
and metabolism to microbial strains for
production of the desired product.
 The term bioreactor often used for
fermenter.
 Fermenter are used for growth of prokaryotic
cells while bioreactors are used for growth of
eukaryotic cells.

16.  It should provide best possible growth to
industrial strains.
 Microorganism during fermentation should free
from contamination.
 The temperature , agitation(mechanical force
applied ) aeration , p H etc should be under
control.
 The fermenter vessels must have sterile air and
stirring facility .
 Fermenter should have additional inoculum ,
seed or media tank .

17.  1.Submerged fermenters
I. Mechanically stirred fermenters
a. Batch operated fermenters
b. Continuous stirred tank fermenters
II. Forced convection fermenters
III. Pneumatic fermenters
 2.Surface fermenters
 a. Tray fermenters
 b. Packed bed column fermenters

18.  In submerged fermenters suspended growth
system the microorganisms are dispersed in
nutrient medium [ liquid] at maintain
environmental conditions.
 on the basis of mechanism of agitation
fermenters are further classified as follows

19.  it is commonly used to carryout fermentation
reaction.
 It is an upright cylindrical vessel.
 Both the ends of the vessels are closed with
hemi spherical basins.
 A stirred is fitted inside the vessel in a
horizontal position.
 The stirrer properly mixes the air and the
nutrients during fermentation.
 At the lateral side of the fermenter there is an
opening through which culture medium is being
pumped into the fermenter.
 This opening is the outlet for harvesting the
products.

20.  Sterile air is pumped into the fermenter through
a pipeline found just below the stirrer.
 A heating coil is used for raising the
temperature inside the vessel.
 The vessel is covered with a water jacket which
has 2 openings.
 Through one opening water is pumped into the
jacket and the other opening is used to release
the water from the jacket.
 This upper portion of this vessel has two
openings, one of them is used to add inoculum
into the fermenter and the other opening is used
to release gases produced during fermentation

22.  a continuous stirred tank fermenter is basically the
same as batch fermenters
 In continuous fermentation fresh medium is added
continuously in the fermentation vessel and the other
in medium means withdrawn for recovery of
fermentation products
 A continuous fermenters can be conducted in various
techniques
 It can be perform as a single stage in which single
fermentor is inoculated and then kept in continuous
operation by balancing the input and output culture
medium
 In a recycle continues fermentation a portion of
withdrawn culture or residual unused substrate plus
the withdrawn culture is recycled to the
fermentation vessel
 The multistage continues Continuous fermenters
involves two or more stages with the fermenters
being operated in a sequence.

24.  Less risk of contamination or sale mutation
because of short growth.
 More flexibility with different products and
biological system process is more economical
and simple raw material conversion level is more
 disadvantages of batch fermentation
 low productivity due to time required faster
sterilizing , filing , cooling, emptying ,cleaning
etc
 more expenses are required for subculture for
inoculation labor and process control
 more focus on instrumentation due to frequency
larger industrial hygiene risks due to potential
contact with pathogenic microorganisms

25.  Less labour expenses due to automation of
fermentation process
 Less toxicity risk to operator by any toxins
producing by microorganisms
 High yield and good quality products due to
invariable operating parameters and automation
of the process
 Less stress on fermentor as a sterilization is not
very frequent
 Disadvantages of continuous fermentation
 Higher investment cost in control and
automation equipment
 More risk of contamination and sale mutation
 Slight variation is possible in continuous process

26.  In forced convection fermenter the agitation is
affecting by using a pump instead of mechanical
stirrer
 Loop fermenter and deep jet fermenter are
available involving liquid movement and gas
entertainer
 In the loop fermenter gas distribution device is
subsidiary vessel where liquid saturated with gas is
circulated by force convection into a fermenter
vessels .
 In deep jet fermenter gas is entertained into high
power jet of liquid into the liquid of the fermenter
 Two different types of force convection fermenter
are available i.e gas lift or air lift fermenter and
bubble column or sparged tank fermenter

28.  Air lift fermenters are classified as a draught tube
or internal loop fermenters and external loop
fermenters
 In the gas slip fermenters internal liquid
circulation individual is achieved by sparger ring
the vessel with gas
 Fluid volume of the vessel is divided into 2
interconnected zones by draught tube
 Air is typically feed through a sparger ring into
the bottom of the central draught tube that
controls the circulation of air and the medium
 The air lift external loop reactor system is used
for circulation of direct air and liquid throughout
the vessel
 This system consists of riser and external
downcomer which are connected at the bottom
and the top respectively

29.  Bubble column reactor is cylindrical column in which the
gas is sparged at the bottom through nozzles on a
perforated or porous distributor plates
 The gas bubbles rise through the liquid in the vessel and
may be redispersed by succession of horizontal perforated
baffle plates
 Temperature controls are maintained by the temperature
jacket or internal coils.
 Advantages of Airlift fermenter –
 High flexibility, control flow and efficient mixing
 Simple design without any moving parts like agitator
 Easy maintenance less risk of defect and easy sterilization
 Large volume tanks , possible specific interfacial contact
area with low energy input
 Higher mass transfer due to enhance oxygen solubility
achieved in a larger tank with greater pressure

30.  Fluidized bed bioreactor is an example of
pneumatic bioreactor used in fermentation
involving fluid with suspended particulate
biocatalyst i.e enzyme or cell particles.
 The cell particles are Fluidized with
upcoming stream of liquids
 Top part of the fluidized Fermenter is
expanded to reduce the superficial velocity
of fluidized bed
 Solid set easily in setting zone and then drop
into the fluidized zone .

32.  Microbial cells are cultured on the surface
layer of the nutrient medium i.e solid or
liquid held in a dish or a tray and is
generally called the surface culture system
or supported growth system
 The technique is commonly used for
production of Citric acid from aspergillus
niger and nicotine acid from Aspargillus
Terrus.
 Packed bed column fermenter and tray
fermenter are example of surface
fermenters.

33.  This is type of surface culture bioreactor in
which solid particles form the packed bed on
which enzymes are immobilized
 A liquid nutrients is allowed to flow
continuously through the packed bed
 Metabolic products are released in the fluid
and removed in the outflow
 When the film of microorganisms is formed
on the surface of packing material then this is
called as film bioreactor.

34. PACKED BED FERMENTER

35.  In the tray for Mantar Solid as well as liquid media
are used for the fermentation .
 If liquid medium is used cells are allowed to float
easily and to make process continuous
 If solid medium is used for tray fermentation then
the process is called solid state fermentation.
 Solid state fermentation is defined as the growth of
microorganism mainly fungi on moist solid materials
in the absence of free flowing water
 This process have been used for the production of
antibiotics , enzymes , organic acids and for bio
remediation of hazardous compounds , nutrition
enrichments, biopharmaceutical products etc
 Solid fermentation has become more attractive
alternative to liquid fermentation for many
productions.

36. TRAY FERMENTER

37.  Solid state fermentation produce higher yield
than submerged liquid fermentation
 All natural habitats from fungi are easily
maintained in solid state fermentation
 The possibility of contamination by bacteria
and Yeast are very less because of low
availability of water
 Simple design of fermentor and low energy
requirements
 Small reactors are used due to concentrated
nature of the substrate
 Low moisture availability

38.  The solid nature of substrate caused problem
in the monitoring of the process parameters
i.e. (PH, moisture content , substrate and
oxygen concentration)
 Agitation and biomass determination is very
difficult
 Solid state fermentation is only suitable for
microorganism that can grow at low moisture
level.
 Aeration may be difficult due to high level of
solid contents
 Possibility of contamination by different
types of undesirable fungi.

39.  Fermenter is a system consisting of different
types of equipments to provide microbial
growth by controlling environmental
condition.
 A typical large scale fermenter consists of
three parts such as the culture vessels ,
associated supply and environmental systems
and measurement and control system .
 The various designing aspects required to be
considered in constructing a ideal fermenter
for production of pharmaceuticals are :

40.  Provide operation free from contamination
 Adequate mixing and aeration
 Maintain specific temperature and p H
 Access point for inoculation and sampling
 Non – toxic to microorganisms and safety
 Minimize liquid loss from fermenter
 Monitoring and control of dissolved oxygen
 Allow feeding of nutrient solution and
reagent
 Suitability for wide range of microbial
cultures
 Cheapest material and smooth internal
surface for vessel.

41.  Measurement and control of environment
condition and biological variable is called
fermentation
 Process parameter are the optimum
conditions which are to be maintained for
the production organism
 Following are monitoring parameter such as
temperature , p H , dissolved oxygen ,
pressure, nutrient concentration and foam
formation .

42.  Temperature control –
 The Temperature control in the fermenter is
important parameter for good fermentation
process.
 Lower Temperature causes reduced product
formation , while higher Temperature adversely
affects the growth of microorganism
 The temperature can be measure by mercury in
glass thermometer or thermistors.
 p H monitoring and control –
 Many microbial cells growth optimally between p
H 5.5 and 8.5
 Microbial cell grow rapidly in fermentation
medium and release metabolites are responsible
for p H .
 Combined glass electrode is used for p H
measurement

43.  Dissolved oxygen –
 In most aerobic fermentation it is essential to
ensure that the dissolved oxygen concentration
does not fall below a specified minimal level.
 Oxygen monitoring is important for optimal
product formation
 Pressure measurement –
 Positive pressure is maintained in fermentation
vessels and it may differ from process to
process.
 The pressure maintained is 12 to 15 lb per sq.
inch .

44.  Foam formation is dependent on composition of
media , type of microorganism , aeration and
agitation .
 Antifoaming agents are used in fermentation.
 Mineral oils based on silicone or vegetable oils
are commonly used as antifoaming agents.