15BT206L Bioprocess and Enzyme Technology Laboratory

EXP.No. 1. BATCH BIOREACTOR OPERATION

Aim:

To study the design, construction and control systems of a bioreactor.

Principle:

A bioreactor is a device in which a substrate of low value is utilized by living cells or enzymes
to generate a product of higher value. Bioreactors are extensively used for food processing,
fermentation, waste treatment, etc. On the basis of the agent used, bioreactors are grouped into the
following two broad classes: (i) those based on living cells and, (ii) those employing enzymes. But in
terms of process requirements, they are of the following types: (i) aerobic, (ii) anaerobic, (iii) solid
state, and (iv) immobilized cell bioreactors.

A bioreactor should provide for the following: (i) agitation (for mixing of cells and medium), (ii)
aeration (aerobic fermenters; for O2 supply), (iii) regulation of factors like temperature, pH, pressure,
aeration, nutrient feeding, liquid level, etc., (iv) sterilization and maintenance of sterility, and (v)
withdrawal of cells/medium (for continuous fermenters). Modern fermenters are usually integrated with
computers for efficient process monitoring, data acquisition, etc.

Basic Functions of a Fermenter:

1. It should provide a controlled environment for optimum biomass/product yields.

2. It should permit aseptic fermentation for a number of days reliably and dependably, and meet the
requirements of containment regulations. Containment involves prevention of escape of viable cells
from a fermenter or downstream processing equipment into the environment.

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3. It should provide adequate mixing and aeration for optimum growth and production, without
damaging the microorganisms/cells. The above two points (items 2 and 3) are perhaps the most
important of all.

4. The power consumption should be minimum.

5. It should provide easy and dependable temperature control.

6. Facility for sampling should be provided.

7. It should have a system for monitoring and regulating pH of the fermentation broth.

8. Evaporation losses should be as low as possible.

9. It should require a minimum of labour in maintenance, cleaning, operating and harvesting operations.

10. It should be suitable for a range of fermentation processes. But this range may often be restricted by
the containment regulations.

11. It should have smooth internal surfaces, and joints should be welded wherever possible.

12. The pilot scale and production stage fermenters should have similar geometry to facilitate scale-up.

13. It should be contrasted using the cheapest materials that afford satisfactory results.

Key parts of the bioreactor:

Agitator – This facilitates the mixing of the contents of the reactor which eventually keeps the “cells”
in the perfect homogenous condition for better transport of nutrients and oxygen for adequate
metabolism of cell to the desired product(s).

The agitator can be top driven or bottom which could be basically magnetic / mechanically
driven. The bottom driven magnetic /mechanical agitators are preferred as opposed to top driven
agitators as it saves adequate space on the top of the vessel for insertion of essential probes
(Temperature, pH, dissolved oxygen foam, CO2 etc) or inlet ports for acid, alkali, foam, fresh media

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inlet /exit gases etc. However mechanical driven bottom impellers need high quality mechanical seals
to prevent leakage of the broth.

* Types of agitators:

 Disc turbine
 Open turbines of variable patch.
 Propellers

Baffle – The purpose of the baffle in the reactor is to break the vortex formation in the vessel, which is
usually highly undesirable as it changes the centre of gravity of the system and consumes additional
power.
 Baffles are metal stripes roughly 1/10th of the vessel diameter and are attached radially to the
wall.
 Normally 4 baffles are used, but in vessel over 3 dm3 diameter 6-8 baffles may be used.

Sparger – In aerobic cultivation process the purpose of the sparger is to supply oxygen to the growing
cells. Bubbling of air through the sparger not only provide the adequate oxygen to the growing cells but

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also helps in the mixing of the reactor contents thereby reducing the power consumed to achieve a
particular level of (mixing) homogeneity in the culture.

Three basic types of sparger are used:

 Porous sparger.
 Orifice sparger.
 Nozzle sparger

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Bioreactor (Bottom Driven)

Jacket – The jacket provides the annular area for circulation of constant temperature water which keeps
the temperature of the bioreactor at a constant value. The desired temperature of the circulating water is
maintained in a separate Chilled Water Circulator which has the provision for the maintenance of
low/high temperature in a reservoir. The contact area of jacket provides adequate heat transfer area
wherein desired temperature water is constantly circulated to maintain a particular temperature in the
bioreactor.

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Body construction:
For a small scale (1 to 30 dm3) glass and/ or stainless steel is used because it can withstand
repeated steam sterilization cycles.
Two basic types are used:
 A glass vessel with a round or flat bottom and top flanged carrying plates.
 A glass cylinder with stainless steel top and bottom plates. This bioreactor may be sterilized in
situ. (AISI graded steel are now commonly used in bioreactor construction).

Peripheral parts:
*Reagent pumps

 Pumps are normally part of the instrumentation system for pH and antifoam control.
 Peristaltic pumps are used and flow rate is usually fixed with a timed shot and delay feed
system of control.

*Medium feed pumps and reservoir bottles

Medium feed pumps are often variable speed to give the maximum possible range of feed rates.
The reservoir bottles are usually larger, but are prepared in the same as normal reagent bottles.

*Rotameter

A variable area flow meter indicates the rate of gas flow into a bioreactor. A pressure regulator
valve before the rotameter ensures safe operation.

*Stirrer glands and bearings:

These are used for the sealing of the stirrer shaft assembly and can be operated aseptically for a
long duration. Four basic types of seal assembly have been used,

 The stuffing box (packed gland seal)

 The simple bush seal

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 The mechanical seal
 Magnetic drive.

Basic control systems for the operation of the bioreactor are described below:

 Temperature Measurement and control – The measurement of the temperature of the

bioreactor is done by a thermocouple or Pt -100 sensor which essentially sends the signal to the
Temperature controller. The set point is entered in the controller which then compares the set
point with the measured value and depending on the error, either the heating or cooling finger of
the bioreactor is activated to slowly decrease the error and essentially bring the measured
temperature value close to the set point.

 pH measurement and control – The measurement of pH in the bioreactor is done by the

autoclavable pH probe. The measured signal is compared with the set point in the controller unit
which then activates the acid or alkali to bring the measured value close to the set point.
However before the pH probe is used, it needs to be calibrated with two buffers usually in the
pH range which is to be used in the bioreactor cultivation experiment. The probe is first inserted
in (let us say) pH 4 buffer and the measured value is corrected by the zero knob of the
controller. Thereafter the probe is put in pH 7 buffer and if needed the measured value is
corrected by the asymmetry knob of the controller. The pH probe is now ready for use in the
range 0-7 pH range.

 Dissolved oxygen controller – The dissolved oxygen in the bioreactor broth is measured by a
dissolved oxygen probe which basically generates some potential corresponding to the
dissolved oxygen diffused in the probe. Before the measurement can be done by the probe it is
to be calibrated for its zero and hundred percent values. The zero of the probe is set by (zero
knob) the measured value of the dissolved oxygen when the broth is saturated with nitrogen
purging. Similarly the hundred percent of the instrument is calibrated by the measured value of
dissolved oxygen when broth is saturated with purging air in it. After calibration the instrument
is ready for the measurement of the dissolved oxygen in the broth. In the event of low oxygen in
the fermentation broth, more oxygen can be purged in the bioreactor &/or stirrer speed can be

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increased to enhance the beating of the bubbles which essentially enhances the oxygen transfer
area and net availability of oxygen in the fermentation broth.

 Foam control – The fermentation broth contains a number of organic compounds and the broth
is vigorously agitated to keep the cells in suspension and ensure efficient nutrient transfer from
the dissolved nutrients and oxygen. This invariably gives rise to lot of foam. It is essential that
control of the foam is done as soon as possible.
 Speed control- Speed control relies on the feedback from tachometer located with drive motor
determining the power delivered by the speed controller to maintain the speed set point valve
set by the user. A digital display shows the actual speed in rpm, as determined by the
tachometer signals.

Table 1: Measurements of various parameters in a bioreactor

Remarks
Measurements Methods
Agitator speed Frequency counter tacho generator More precise less reliable.

Agitator power Torque sensor. Difficult.

Electrical power. Recommended.
Temperature Resistance Probably best
Thermometer Fragile
Thermistor Satisfactory
Thermocouple Not recommended
Flow rate Rota meter Satisfactory
Orifice meter Less accurate
Thermal mass flow meter Set-point control
Dissolved oxygen Galvanic probe Widely used
Polarographic probe Widely used
pH pH electrode Widely used
Foam Conductivity probe Widely used
Redox Redox electrode Empirical valve
Turbidity
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SRM sensor
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Liquid feed rate Peristaltic pump Widely used

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Significance of the experiment:

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