Historical development

Fermentation products – wine,

bread etc.,
Invention of microbes responsible
for these conversion – science of
microbiology
Penicillin fermentation – sterility
concepts, pilot plant concepts
Single cell production by ICI – air
lift fermenters, continuous
cultivation, process control
Biotechnology Products

 Microbial biomass
 Enzymes
 Primary and secondary

metabolites
 Recombinant products
 Biotransformation
 Operations in
Fermentation
 Inoculum preparation
 Medium preparation
 Sterilization
 Cultivation
 Batch, Fed Batch, Continuous etc.,
 Downstream processing
 Effluent treatment
 Fermentation Industry
Overview
Microbiology & Quality Control

Downstream Effl
Utility Fermentation bay Processing uen
t
trea
tme
nt

STORES
 Utilities
 Steam – 4 BAR & 1.5 BAR
 Air – 1.5 BAR
 Chilled water
 Cooling water
 Deionised water
 Softwater
 MCC
 Fermentation bay
 Seed Fermenter
 Main fermenter
 Acid, Alkali & Antifoam tanks
 Nutrient feeding
 Harvest tank
Microbiology Laboratory
 Media preparation
 Autoclaving
 Inoculation chamber
 Microscopic observation
 Quality control
 Culture stock maintenance
 Culture Stock
Maintenance
 Plates – 15 days
 Stab/slant – 6 months
 Glycerol stock – 6 months
 Lyophilisation – 15 years
 Sand cultures – 15 years
 Liquid nitrogen storage – 15
years

Culture Validation
 Bioreactor
How a bioreactor differs from a chemical reactor ?

Both are agitated tanks

Bioreactor should be capable of being operated

aseptically for number of days
Adequate aeration and agitation should be
provided to meet the metabolic requirements
which will vary from time to time
Material of Construction
 Lab fermenters are made up of
glass
 Pilot and industrial scale
fermenters are made up of
Stainless steel
 Steel having chromium more
than 4% are called us stainless
steel.
 The corrosion resistance of
stainless steel depend on the
The pillar has attracted the attention
of archaeologists and materials scientists because of
its high resistance to corrosion and has been called a
"testimony to the high level of skill achieved by the
ancient Indian iron smiths in the extraction and
processing of iron. The corrosion resistance results
from an even layer of crystalline iron hydrogen
phosphate hydrate forming on the high-phosphorus-
content iron, which serves to protect it from the effects
of the Delhi climate.
 The composition of this film
varies with different steel alloys
and manufacturing process
 This oxide film is stabilized by
chromium
 If the film is damaged it will
repair itself when exposed to air
or oxidizing agent.
 Minimum amount of chromium
needed to resist corrosion will
depend on the corroding agent in
environment (acid, alkali, salt
etc)
 10 – 13% chromium will develop
effective oxide film
 Also addition of nickel to this
high percent chromium steel
enhances the resistance to
corrosion and give strength.
 Addition of molybdenum
improves the resistance of
stainless steel to solution of salts
or halogens such as chlorides.
 Corrosion resistance can also be
increased using tungstan silicone
 Most commonly used stainless
steel for fermentation equipment
is
 SS316 Cr Ni Mo : 17 12 2
 SS316L Cr Ni Mo : 18 14 3
 SS317 Cr Ni Mo : 17 13 4
 SS304 Cr Ni : 18.5 10
 Normally the main tank is made
up of 316 grades and jackets are
made up of 304 to reduce the
cost.
 Height to Diameter ratio :
 Height of the fermenter is one of the critical

parameters in the fermenter design.

 If the height of the fermenter is more the

bubble residence time is more and better

oxygen efficiency.
 H/D ratio of the fermenter 2 to 3 is preferred.

Shape of the vessel

For Design
Purpose the
effective working
volume of the
reactor is
considered to be
80% of Total
volume of the 80%
reactor.

Fermenter will be
running 330 days
 Mixing
 One of the major task of bioreactor is mixing

 Mixing is a physical operation which reduces

non uniformities in fluid by eliminating
gradients of concentration, temperature and
other properties

 Mixing in the Bioreactor involves

 Blending the soluble medium components eg. Sugars
 Dispersing air in the form of small bubbles
 Suspension of solid particles such as cells
 Promoting heat transfer and dispersion of immiscible
liquids such as antifoam
Circular flow
 Baffle
 Normally four Baffles are installed in the
Bioreactor
 Purpose of Baffle is to avoid the vortex formation
and increasing the aeration efficiency
 Baffles are normally 1/10th of the dia of the vessel.
 Increasing the size of the baffle increase the
agitation effect marginally.
 Decreasing that will have substantial drop in
efficiency.
 Baffles should be installed such that gap exist
between the vessel and baffle which minimizes
dead space and avoid microbial growth
Baffles can be
effectively used
to increase the
heat transfer
efficiency
Baffles can be
used as level
indicators
Baffles can be
used as inlet and
exit point in big
fermenters
Propeller Turbine Pitched Blade

Types of
agitator
Radial flow Axial flow
with baffle with baffle
 Stagnant
Multiple
zones in big
impellers
reactors
Geometrical ratios of
Fermenter

 H/D : 2 – 3
 Imp dia P : 0.3 – 0.5 D
 Baffle : 0.1 D
 W/P : 1.5 – 2.0
 P/Z : 0.5-1.0
Problem:

In a lab scale bioreactor

production of gluconic acid in
optimized medium is 180 g/l. The
time duration of cultivation is 36
hours and downtime for cleaning,
sterilization, medium preparation
etc. is 12 hours. You have been
asked to design a fermentor for
the production of 2000 Tonnes per
annum. State your assumptions
and geometrical ratios for your
Air Filter
Air Inlet
Line in
Bioreactor
Air outlet
line in
Bioreactor
Rupture
disk
Drain
Sampling
valve
 Ports
 Small reactor -12 mm ports
 Pilot scale – 19 and 25 mm ports
 Big reactors - any size depending
on the probes.
 INSTRUMENTATION
Measurement type

Inline measurement : The sensor is an integrated

part of the fermentation equipment and
measure the process variable real time
continuously.
Online measurement: Continuous/discontinuous
measurement but the sensor is not an
integrated part of the bioreactor but connected
to the system
Offline sensor: Discontinuous measurement and
the sensor is not an integrated part of the
system. Samples have to be connected and
When evaluating sensors to use in
measurement and control it is
important to consider the following:
 Response time
 Gain and sensitivity

 Accuracy

 Ease and speed of calibration

 Reliability

 Material of construction

 Sterilization
 Bioreactor variables
 Physical variables
 Temperature, Pressure, Flow, Agitation
speed, Mass, Volume, Foam

 Chemical variables
 pH, Dissolved oxygen, Dissolved carbon
dioxide, ion concentration, substrate
concentration

 Biological variables
 Cell concentration, Protein, DNA,RNA,
Enzyme activities etc
 Temperature
Measurement
 Mercury in glass thermometers,
thermocouples, bimetallic thermometers,
thermistors, Metal resistance
thermometers etc.,

 Metal resistance thermometers are most

commonly used.

 Mercury in glass thermometers are used

for precise calibration of other
measurement systems
 Metal resistance
thermometers
Platinum resistance thermometers are
commonly used in bioreactors. (Pt 100)
Electrical resistance of platinum changes
with change in temperature.
Electrical resistance wire of 100 ohms is used
This is connected to one side of Wheatstone
bridge and other three sides are having
known resistance.
The known electrical voltage is applied across
the bridge and change in the voltage occurs
when the resistance changes according to
the temperature
Change in resistance 0.385 to 0.392 ohms/oC
 Pressure Measurement
 Pressure is one of the critical
measurements mainly for the safety
of the bioreactor.
 Always bioreactors are operated
under positive pressure i.e pressure
above atmospheric pressure to
prevent contamination and better
oxygen transfer.
 Pressure will buidup in the
bioreactor during sterilization
 Devices
 C tube bourdon gauge - manual
 If a wire is subject to strain its electrical
resistance changes.
 Flow Measurement
 Gas flow measurement – area rotameter
 Mass flow meter
 Liquid flow – weighing the mass or
metering pump
 Rotameter
Advantages:
 A rotameter requires no external power or fuel, it uses only
the inherent properties of the fluid, along with gravity, to
measure flow rate.
 A rotameter is also a relatively simple device that can be
mass manufactured out of cheap materials, allowing for
widespread use in places such as third-world countries.
Disavantages:
 Due to its use of gravity, a rotameter must always be
vertically oriented and right way up, with the fluid flowing
upward.
 Due to its reliance on the ability of the fluid or gas to
displace the float, graduations on a given rotameter will
only be accurate for a given substance. The main property
of importance is the density of the fluid; however, viscosity
may also be significant. Floats are ideally designed to be
insensitive to viscosity; however, this is seldom verifiable
from manufacturers specs. Either separate rotameters for
different densities and viscosities may be used, or multiple
scales on the same rotameter can be used.
 Rotameters normally require the use of glass (or other
THERMAL MASS FLOWMETER

Temperature at the sensors varies

depending upon the mass flow
 Speed
 Measurement by Tachometer fixed in the
motor – electromagnetic voltage
generation
Weight
 Measurement by load cell
 Foam
 Capacitance probes
 Conductivity probes
 Thermal conductivity
 Ultrasonic
 Rotating disk
 Biomass Measurement
 Dry weight
 OD 600
 PCV
 Cell count
 Fluorescence probe
pH Probe
 The pH probe measures pH as the
activity of hydrogen ions
surrounding a thin-walled glass bulb
at its tip. The probe produces a
small voltage (about 0.06 volt per
pH unit) that is measured and
displayed as pH units by the meter
Oxygen sensors have a thin organic membrane covering
a layer of electrolyte and two metal electrodes. Oxygen
diffuses through the membrane at a rate proportional to
its partial pressure—the greater the oxygen partial
pressure, the more oxygen diffuses through the
membrane. Oxygen meters measure the current as
oxygen is reduced at the cathode and more oxygen
diffuses through the membrane. Since the diffusion
current is directly proportional to the concentration of
dissolved oxygen, the calibrated meter simply converts
measured current into concentration units.
 The reactions are as follows:
At cathode: O2 + 4H+ + 4 e- →
2H2O
 ASSIGNMENT
 Temperature measurement
 Air flow measurement
 Pressure measurement
 Foam measurement
 pH PROBE
 DO Probe
 O2 Analyser
 CO2 analyser