lab 1 bioreactor

Fermentation Technology Lab Bioreactor-Basics Ms. Phoeurk Chanrith

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LABORATORY 1: BIOREACTOR-BASICS

OBJECTIVE

- To understand the basic design and operation features of the bioreactor.

THEORY

Key parts of the bioreactor are described below:

Bioreactor – can be described as a vessel which has provision of cell cultivation under sterile

condition & control of environmental conditions e.g., pH, Temperature, Dissolved oxygen

etc. It can be used for the cultivation of microbial plant or animal cells. A typical bioreactor

consists of following parts.

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 inlet /exit gases etc. However mechanical driven bottom impellers need high

quality mechanical seals to prevent leakage of the broth.

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.

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 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.

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.

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


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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.

Identification of pH controller control settings for Bio-Engineering AG (Switzerland)

bioreactor – For this specific pH controller one has to suitably identify the right control

action setting for the addition of certain concentration of acid / alkali in the desired

fermentation broth which can give quick control action with-out any oscillations/offset of

measured value around the set point. The controller panel and the different knobs are

adequately described in the following figure –

Before the start of autoclaving of the broth for any cultivation experiment, it is

essential to calibrate the pH probe (as described above). Thereafter the set point (say 5) and p-

band (1.0) is entered on the controller. This essentially means that now the pH controller will

control the pH value in the range 4.0 to 6.0 For example if the measured pH value in the


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bioreactor is 4.5 then the controller will trigger alkali addition to the reactor to bring the

measured value to 5.0 and similarly if the pH value is 5.5 it activates the acid pump to bring

down the pH value to the set point. It is ensured that the p-band value is not kept too small or

else it may lead to oscillation of the measured value around the set point similarly if the p-

band is too large then it may give rise to offset between the measured value and set point of

the controller. It may also be noted that the activation of acid/alkali pumps is done in a phased

manner. For example if the controller is adding alkali to bring down the pH, the addition of

the alkali is not done in one shot. In fact the alkali addition pump is kept on for some time and

then it is off for some time. This ensures adequate mixing of first installment of acid/alkali in

the broth before next installment is added. This strategy avoids over addition of acid/alkali for

the pH control. The On/Off time of the controller has to be adjusted by separate experiments

and will depend on the buffering capacity of the broth, concentration of acid/alkali etc.

However it is absolutely essential to identify & maintain these setting before the start of the

experiment in order to have efficient control action of the control which features stable quick

control action with-out oscillations &/or off set around set point. There is another knob td in

the control panel which provides the setting of the safety time during which if the control

action is not achieved it raises the alarm for the operator.

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 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.

PROCEDURE AND SIMULATOR

1. Speed control system

The purpose of Speed Control System is to provide mixing of reactor contents

(Microorganism and Medium).

To start the operation of the reactor enter “Set Lower Limit” as some value let us say 100.

Then enter “Set Upper Limit” as some value Let us say 400.

Then enter “Set Actual Speed” as some value Let us say 200.

Then press RUN.

The agitator will start rotating at 200 RPM.

To stop the agitator press STOP button (Do not do it now!!).


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2. Speed control system

The purpose of Speed Control System is to provide mixing of reactor contents

(Microorganism and Medium).

To start the operation of the reactor enter “Set Lower Limit” as some value let us say 100.

Then enter “Set Upper Limit” as some value Let us say 400.

Then enter “Set Actual Speed” as some value Let us say 200.

Then press RUN.

The agitator will start rotating at 200 RPM.

To stop the agitator press STOP button (Do not do it now!!).

Press enter NEXT button to go to Gas supply system


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3. Temperature Control System

The purpose of Temperature control system is to maintain the right cultivation temperature in

the bioreactor. The temperature is measured by the temperature sensor (Blue). This value is

then compared with the set point value and depending on the error the temperature

manipulation is done by passing either cooling (blue dots) or hot water (red dots) in the jacket

of the bioreactor.

4. pH measurement and Control System

Enter any pH value say 5.0 in the set point and press RUN.

The pH in-side the bioreactor is measured by the sensor (orange) and is compared with the set

point and depending on the error either acid (green) or base (orange) peristaltic pump is

triggered from the controller.


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5. Dissolved Oxygen measurement and Control Enter value of Dissolved oxygen level in the bioreactor say (30% Saturation) and press

RUN.

The Dissolved oxygen inside the bioreactor is measured by magenta sensor, the value

is compared with the set point and depending on error the controller action is activated. For

example if the Dissolved oxygen in the bioreactor is registered low the controller speeds up

the air supply and/or the agitation speed to increase the level of dissolved oxygen in the

bioreactor. Similarly if the level of the dissolved oxygen is measured low in the bioreactor it

will reduce the rate of supply of oxygen &/or RPM in the bioreactor. This way the set point

value of 30 % saturation level of dissolved oxygen is maintained in the bioreactor.

6. Foam Control system

Foam is defined as dispersion of liquid in gas and it is invariably produced in any cell

cultivation primarily due to presence of nitrogen and protein material in the medium. Though

it is essential to keep the nitrogenous material in the medium as high as possible because it is

essentially required to give good growth however the foam is on the other hand highly

undesirable as it can lead to heavy loss of medium from the bioreactor. The foam is detected

by Red sensor and antifoam addition is triggered through peristaltic pump to kill the foam.

Peristaltic pump adding the antifoam solution (blue bottle) is made to work at a very slow

speed primarily to add as minimum amount as possible. Over addition of antiform may create

serious problem in isolation of the product from the fermentation broth.


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REFFERENCE

1. iitd.vlab.co.in,. (2011). Bioreactor- Basics. Retrieved 20 September 2015, from

iitd.vlab.co.in/?sub=63&brch=177&sim=647&cnt=1

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University of Tokyo Press, Japan

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Hill Book Company, New Delhi

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Englewood Cliffs, New Jersey

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& K.L. Nelsen John Wiley and Sons Inc. New York

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Publishers, Weinheim.


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8. Process Biotechnology Fundamentals, S.N. Mukhopadhyay, Viva Books Pvt. Limited,

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Dunnill, A.E. Humphrey & M.D. Lilly John Wiley and sons, New York

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Sinauer Associates Inc, Sunderland, MA & Science Tech, Inc. Madison, WI

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Green, K Jayaraman. University Press, Hyderabad

14. Manual of Industrial Microbiology and Biotechnology, Arnold L. Demain, Nadine A.

Solomon, American Society for Microbiology, D.C.

15. Introduction to Industrial Sterilization, Richards

lab 1 bioreactor

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