Fermentor
Krishna Priya.K
Lecturer
Dept. of Microbiology
What is fermentation?
Pasteur’s definition: “life without air”, anaerobe red ox
reactions in organisms
New definition: a form of metabolism in which the end
products could be further oxidized
For example: a yeast cell obtains 2 molecules of ATP per
molecule of glucose when it ferments it to ethanol
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What is fermentation technique?
Techniques for large-scale production of microbial products.
It must both provide an optimum environment for the
microbial synthesis of the desired product and be
economically feasible on a large scale. They can be divided
into surface (emersion) and submersion techniques. The latter
may be run in batch, fed batch, continuous reactors
In the surface techniques, the microorganisms are cultivated
on the surface of a liquid or solid substrate. These techniques
are very complicated and rarely used in industry
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What is fermentation technique?
In the submersion processes, the microorganisms grow in a
liquid medium. Except in traditional beer and wine
fermentation, the medium is held in fermenters and stirred to
obtain a homogeneous distribution of cells and medium. Most
processes are aerobic, and for these the medium must be
vigorously aerated. All important industrial processes
(production of biomass and protein, antibiotics, enzymes and
sewage treatment) are carried out by submersion processes.
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Typical Bioprocessing
Stock Culture Raw Materials
Shake Flasks Medium Formulation
Seed Sterilization
Fermenter
Air Agitator
Recovery
Purification Products
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View looking down into a stainless steel
fermentor
Agitator (impeller)
Achieve mixing objectives – bulk
fluid and gas-phase mixing, air
dispersion, oxygen transfer, heat
transfer, suspension of solid particles
and maintaining uniform environment
throughout vessel contents.
Introduction
The function of the fermenter or bioreactor is to provide a suitable
environment in which an organism can efficiently produce a target product—
the target product might be
· Cell biomass
· Metabolite
· Bioconversion Product
The sizes of the bioreactor can vary over several orders of
magnitudes.
The microbial cell culture (few mm3), shake flask ( 100 -1000 ml),
laboratory fermenter ( 1 – 50 L), pilot scale (0.3 – 10 m3) to plant
scale ( 2 – 500 m3) are all examples of bioreactors.
Cell culture
fermenter Shake flask fermenter laboratory fermenter
Pilot fermenter Plant fermenter
Fermentation Process
Upstream Processing
Fermentation Raw Materials Production Microorganism
Fermentation
Downstream
Product Purification Processing
Effluent Wastes Product
Upstream Processing
• Three main areas:
• A) Producer microorganism
• This include processes for
• obtaining a suitable microorganism
• strain improvement to increase the productivity and yield
• maintenance of strain purity
• preparation of suitable inocullum
• B ) Fermentation media
• C) Fermentation Process
Downstream Processing
The processes that follows fermentation:
A) Cell harvesting
B) Cell disruption
C) Product purification from cell extracts or the growth
medium
Key Factor of Fermenter design
The performance of any fermenter depends on the following key
factors:
· Agitation rate
· Oxygen transfer
· pH
· Temperature
· Foam production
The design and mode of operation of a fermenter mainly depends
on the production organism, the optimal operating condition
required for target product formation, product value and scale of
production.
The design also takes into consideration the capital investment and
running cost.
Requirements of Bioreactors
There is no universal bioreactor.
The general requirements of the bioreactor are as follows:
A) The design and construction of bioreactors must keep sterility from the
start point to end of the process.
B) Optimal mixing with low, uniform shear.
C) Adequate mass transfer, oxygen.
D) Clearly defined flow conditions.
E) Feeding substrate with prevention of under or overdosing.
F) Suspension of solids.
G) Gentle heat transfer.
H) Compliance with design requirements such as: ability to be sterilized;
simple construction; simple measuring, control, regulating techniques; scale-
up; flexibility; long term stability; compatibility with up- downstream
processes; antifoaming measures.
Some important fermentation products
Product Organism Use
Ethanol Saccharomyces Industrial solvents,
cerevisiae beverages
Glycerol Saccharomyces Production of
cerevisiae explosives
Lactic acid Lactobacillus Food and
bulgaricus pharmaceutical
Acetone and Clostridium Solvents
butanol acetobutylicum
α-amylase Bacillus subtilis Starch hydrolysis
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Some important fermentation products
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Some important fermentation products
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Some important fermentation products
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Winemaking fermenter
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Flow sheet of a multipurpose fermenter and
its auxiliary equipment
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Component parts of a fermention
1. Formulation of media to be used in culturing the organism during
development of inoculum and in the production fermenter
2. Sterilization of the medium, fermenter and ancillary equipment
3. Production of an active, pure culture in sufficient quantity to inoculate the
production vessel
4. The growth of the organism in the production fermenter under optimum
conditions for product formation
5. The extraction of the product and its purification
6. Disposal of effluents produced by the process
Typical fermentation process involves :-
Upstream processing(USP)
Downstream processing(DSP)
USP is associated with all factors and processes leading to and
including the fermentation
Consists of three main areas.
1. The producer micro organism
2. The fermentation medium
3. The fermentation
DSP includes all processes that follow fermentation
They involve
1. cell harvesting
2. cell disruption
3. product purification from cell extracts or the growth
medium
4. Disposal of effluent wastes
APPLICATIONS OF FERMENTATION
Microbial fermentations may be classified into the following major groups:-
(i) Those that produce microbial cells (biomass) as the product.
(ii) Those that produce microbial metabolites.
(iii) Those that produce microbial enzymes.
(iv) Those that modify a compound which is added to the
fermentation- the transformation processes.
(v) Those that produce recombinant products.
Microbial Growth Kinetics
• Microbial Growth Kinetics describe
how the microbe grows in the
fermenter. This information is
important to determine optimal batch
times. The growth of microbes in a
fermenter can be broken down into
four stages:
Lag Phase
Exponential Phase
Stationary Phase
Death Phase
Microbial Growth Kinetics
• Lag Phase
This is the first phase in the fermentation process
The cells have just been injected into a new environment and
they need time to adjust accordingly
Cell growth is minimal in this phase.
Microbial Growth Kinetics
• Exponential Phase
The second phase in the fermentation process
The cells have adjusted to their environment and rapid growth
takes place
Cell growth rate is highest in this phase
Microbial Growth Kinetics
• Exponential Phase (Continued)
At some point the cell growth rate will level off and become
constant
The most likely cause of this leveling off is substrate limited
inhibition
• Substrate limited inhibition means that the microbes do not have enough
nutrients in the medium to continue multiplying.
Microbial Growth Kinetics
• Stationary phase
This is the third phase in the fermentation process
The cell growth rate has leveled off and become constant
The number of cells multiplying equals the number of cells
dying
Microbial Growth Kinetics
• Death phase
The fourth phase in the fermentation process
The number of cells dying is greater than the number of cells
multiplying
• The cause of the death phase is usually that the cells have consumed most
of the nutrients in the medium and there is not enough left for
sustainability
Most fermentations require liquid media, often referred to as
broth
Fermentation media must satisfy all the nutritional requirements
of the microorganism
Most fermentations, except those involving solid substrates,
require large quantities of water in which the medium is
formulated.
General media requirements include
a carbon source- provides both energy and carbon units for
biosynthesis
sources of nitrogen, phosphorus and sulphur.
Other minor and trace elements
some microorganisms -vitamins, such as biotin and riboflavin.
Aerobic fermentations are dependent on a continuous input of
molecular oxygen, and even some anaerobic fermentations require
initial aeration of media, e.g. beer fermentations
buffers, or the pH is controlled by acid and alkali additions
antifoam agents
For some processes, precursor, inducer or inhibitor compounds
must be introduced at certain stages of the fermentation.
The main factors that affect the final choice of individual raw materials
are as follows.
1 Cost and availability
2 Ease of handling in solid or liquid forms, along with associated transport and storage
costs
3 Sterilization requirements and any potential denaturation
problems.
4 Formulation, mixing, and viscosity -characteristics that may influence agitation, aeration
and foaming during fermentation and downstream processing stages.
5 The concentration of target product attained, its rate of formation and yield of
product per gram of substrate utilized.
6 The levels and range of impurities, and the potential for generating further undesired
products during the process.
7 Overall health and safety implications.
Control of Physicochemical Parameters
A) Agitation:
Agitation of suspended cell fermentations is performed in order to
mix the three phases within a fermenter
liquid phase contains dissolved nutrients and metabolites
gaseous phase is predominantly oxygen and carbon dioxide
solid phase is made up of the cells and any solid substrates that may
be present.
Mixing should produce homogeneous conditions and promote
a) Nutrient transfer b) Gas transfer c) Heat transfer
Heat transfer is necessary during both sterilization and for
temperature maintenance during operation.
AGITATION
Helps to mix 3 phases within a fermentor
liquid phase-nutrients and metabolites
gaseous phase-O2 and CO2
solid phase-cells any solid substrates present
Mixing –homogenous conditions and promote nutrient, gas and
heat transfer
Heat transfer- sterilization and temp maintenance during
fermentation process
Aerobic fermentations- mixing for transfer of O2 from gaseous to
liquid phase.
Stirred tank reactors-agitators or impellors are used for agitation
Impellor-connected to a strong and straight shaft
Shaft passes through the lid of the fermentation tank
Shaft rotated with electric motor mounted externally on top of the
fermentor
Appropriate seals
Effectiveness of agitation depends on
design of impeller blades
speed of agitation
depth of liquid
Vortex formation-reduced agitation and reduced aeration as
only a small amount of the medium surface is exposed to the
atmosphere in the head space of the tank.
To avoid this problem, flat vertical plates-width of 1/10 th of
the vessel called baffles
Baffles are present on the wall of the fermentor
Disc type
Marine type
Inclined type
Open turbine
They increase turbulence, prevent vortex formation and
eliminates dead spaces .
Usually 4-6 baffles per fermentor
Agitation should be controlled to suite a particular fermentation
High shear- damage shear sensitive cells
Low shear agitation systems –flocculation of cells or unwanted
growth on the surfaces such as vessel walls, stirrers and
electrodes
Disadvantages of mechanical agitation systems:-
Damage to shear sensitive cells
high initial costs
maintenance costs
higher power consumption
excessive foam formation
Advantages of mechanical agitation systems:-
efficient mixing of contents
proper distribution of dissolved
O2, temp, and pH
Other systems of agitation-Airlift fermentor
No moving parts
Uses expansion of compressed gas to bring about the mixing
Liquid movement initiated by injection of compressed air at the
bottom of the column
Air bubbles expand in column causing the upward movement of
liquid –initiate cycling within the fermentor.
Agitator (impeller)
Achieve mixing objectives – bulk
fluid and gas-phase mixing, air
dispersion, oxygen transfer, heat
transfer, suspension of solid particles
and maintaining uniform environment
throughout vessel contents.
Baffles
Four baffles incorporated
into agitated vessels of all
sizes to prevent vortex
and to improve aeration
efficiency
Metal strips roughly one-
tenth of vessel diameter and
attached radially to the wall
Minimizes microbial growth
on baffles and fermenter
walls.
Control of Physicochemical Parameters
B. Transport of Oxygen
To prevent the risk of contamination, gases introduced into the
fermenter should be passed through a sterile filter.
A similar filter on the air exhaust system avoids environ-mental
contamination.
Sterile filtered air or oxygen normally enters the fermenter through a
sparger system,
To promote aeration in stirred tanks, the sparger is usually located
directly below the agitator.
Aeration system (sparger)
Introduces air into liquid of
fermenter
Three basic types – porous
sparger
1. Orifice sparger – a perforated
pipe
2. Nozzle sparger – an open or
partially closed pipe
3. Combined sparger-agitator
Aeration
Majority of fermentations are aerobic
Requires large quantities of sterile air or O2
Also removes unwanted volatile metabolic products from the
medium
Air or gases- passed through filter???
Filter at the exhaust ???
Sterile air enters the fermentor through a sparger system
Air flow rarely exceeds 0.5 to 1.0 volume of air per volume
of medium
Stirred tank-sparger is placed directly below the agitator
Different kinds of sparger used
Smaller the size of air bubbles produced more efficient is the
aeration
OXYGEN SUPPLY
Oxygen is normally supplied to microbial cultures in the form of air,
this being the cheapest available source of the gas. The method for
provision of a culture with a supply of air varies with the scale of the
process:
i. Laboratory-scale cultures may be aerated by means of the shake-
flask technique where the culture (50 to 100 cm3) is grown in a
conical flask (250 to 500 cm3) shaken on a platform contained
in a controlled environment chamber.
ii. Pilot and industrial-scale fermentations are normally carried
out in stirred, aerated vessels, termed fermenters.
Ring type
Grid type
Ring sparger and grid sparger-used when unicellular
organisms are used for fermentation
Multicellular organisms clog the holes
Single long pipe used to pump air to avoid clogging
Or ring sparger with orifice facing downward can also avoid
clogging
Antifoams
Antifoams are necessary to reduce foam formation during
fermentation.
Foaming is largely due to media proteins that become attached to the
air–broth interface where they denature to form a stable foam.
Proteolytic bacteria
If uncontrolled the foam may
1)block air filters, resulting in the loss of aseptic conditions; the
fermenter becomes contaminated and microorganisms are
released into the environment
2)Considerable loss of medium
3)Reduces aeration by hindering gas exchange b/w medium
and atmosphere in head space
The ideal antifoam should have the
following properties:
1 readily and rapidly dispersed with rapid action;
2 high activity at low concentrations;
3 prolonged action;
4 non-toxic to fermentation microorganisms, humans
or animals;
5 low cost;
6 thermostability; and
7 compatibility with other media components and the
process, i.e. having no effect on oxygen transfer rates or
downstream processing operations.
There are three possible approaches to controlling foam
production:
modification of medium composition,
use of mechanical foam breakers
addition of chemical antifoams
Mechanical foam control- impeller blade
ultrasonic waves
Chemical antifoams- added manually or can be automated
Natural antifoams include plant oils (e.g. from soya,sunflower
and rapeseed), deodorized fish oil, mineral oils.
The synthetic antifoams are mostly
silicon oils, poly alcohols and alkylated glycols
Transfer of Heat in Bioreactors
To maintain a constant temperature in the
fermenter, heat is either supplied or removed from
the fermentation broth during the course of
fermentation.
In fixed bed microbial reactors heat transfer takes
place by natural convection or phase change
(evaporation-condensation).
Heat Transfer Configurations:
The primary heat transfer configurations in fermentation vessels are:
i. External jackets
ii. Internal coils
iii. External surface heat exchanger
The internal coils though provide better heat transfer capabilities, but they
cause problems of microbial film growth on coil surfaces, alteration of mixing
patterns and fluid velocities.
VALVES
To control the flow of liquids and gases
Valves may be:-
Simple on /off-fully open or fully closed
Coarse control of flow rates
Valves that can be adjusted precisely-flow rates can be
controlled accurately
Safety valves
Gate valves
Sliding disc is moved in or out of the path by turning the stem of
the valve
Suitable for general purposes on a steam or water line
Fully open or fully closed
Should not be used for regulating flow
Not suitable for aseptic conditions
----mash solids can pack in the groove where the gate slides
----Leakage round the stem of the valve
Globe valves
Horizontal disc or plug is raised or lowered in its seating
Regulates the flow of water or steam
Not suitable for aseptic operation-leakage round the valve
stem(similar to gate valve)
Flexible metallic membrane around the stem of gate and
globe valves—---aseptic operations--- but expensive
Piston valves
Similar to globe valve
Except ---flow is controlled by a piston passing between two
packing rings
Very efficient for aseptic operation
Needle valves
Similar to globe valve
Except-disc is replaced by a tapered plug or needle fitting
into a tapered valve seat
Used to give fine control of steam or liquid flow
Not suitable for aseptic operations
Plug valves
Parallel or tapered plug sitting in a housing through which an
orifice has been machined
When plug is turned through 90 degree the valve is fully
open
Flow path determined by the cross-sectional area of the
orifice
Orifice is not as large as that of the pipeline
Ball valves
Developed from the plug valve
Valve is a stainless steel ball through which an orifice is
machined
Ball is sealed between two wiping surfaces
Wipe the surface and prevent deposition of matter at this
point
Orifice –same diameter as that of the pipe line, giving an
excellent flow path
Suitable for aseptic operations
Can handle mycelial broths
Can be operated under high temperatures and pressures
Butterfly valves
Consists of a shaft which rotates about a shaft in a housing
Disc closes against a seal to stop the flow of liquid
Valves usually used in large diameter pipes operating under
low pressure where absolute closure is not essential
Not suitable for aseptic operations
Pinch Valves
Flexible sleeve closed by a pair of pinch bars
Suitable for aseptic operations
Diaphragm valves
Flexible closure
Suitable for aseptic operations
Diaphragm must be made of a material that can withstand
repeated sterilization
Check valves
Prevent accidental reversal of flow of liquid or gas in a pipe due
to breakdown in some part of the eqpt
Pressure control valves
Pressure reduction valves
Pressure retaining valves
Safety valves
Safety valve
Types of fermenter
Simple fermenters (batch and continuous)
Fed batch fermenter
Air-lift or bubble fermenter
Cyclone column fermenter
Tower fermenter
Other more advanced systems, etc
The size is few liters (laboratory use) - >500 m3
(industrial applications)
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Tower fermentor
Elongated non mechanically stirred fermentor
10:1 diameter ratio
Unidirectional flow of gases
Tube with sparger at the base
Initially used in the batch fermentor mode-citric acid
production
Later in 1965- continous mode for brewing industry
Settling zone or separator- at the top of the fermentor- induces
the gas bubbles produced during the reaction to coalesce and
escape from the liquid phase
Quiescent zone within the separator –free of rising gas – so
that yeast cells could settle and return to the main body of
the tower and clear beer can be removed
Attemporator jacket- encloses the tower
temperature regulation of the contents
Little yeast lost due to the flocculant nature of the yeast
Wort is introduced into the base of the fermentor and it
passes through a porous plug of yeast
Yeast concentration is30-35A% by weight at the bottom of
the tower and as low as 5-10% at the top due to the
flocculent nature of the yeast
Progressive and continuous fall in specific gravity of the nutrient
medium between the bottom and the top of the tower???
Steam sterilized b4 fermentation
Vessel filled partially with sterile wort and inoculated with
flocculant yeat
Initial stages- designed for high biomass production----- by
periodic addition of wort for about 9 days
Porous plug of yeast develops at base of the tower
Flow rate of the wort is gradually increased over the next 9-
12 days and a steady state is achieved in this time
Beer produced in the initial 3 weeks does not contain
sufficient alcohol concentration
Thus more than 3 months operations necessary to
compensate for the initial losses of the process
Air lift fermenter
Advantage:- low energy requirement
create less shear than stirred
tank reactors
Deep jet fermentor:- pump is used externally for circulation
and reinjection
Smaller the bubble, larger the surface area to volume ratio and
better O2 transfer o the liquid phase
Spargers with small pores-prone to blockage, energy
requirement is high
Size of the holes ranges from 1/64 to 1/32 of an inch or larger
Tall fermentor aeration is better as bubbles remain longer in the
medium
