EXPERIMENT 1
MEDIA DEVELOPMENT AND VESSEL STERILIZATION
1.0 OBJECTIVE
1.1
To study the media development for yeast fermentation in a bioreactor.
1.2
To know the criteria and identify the areas for sterilization before running the
experiment.
2.0 COURSE OUTCOME
CO1: Ability to formulate fermentation media and decide on the types of carbon and
nitrogen source.
CO2: Ability to recognize, compare and draw the schematic diagram for specific
types of bioreactors by applying the knowledge of science and engineering.
3.0 INTRODUCTION
During the fermentation, all atoms of carbon, hydrogen, oxygen, nitrogen and other
elements are consumed by new cells or excreted as products. The growth and
product synthesis are well formulated by metabolic stoichiometry. This stoichiometry
provides a lot of information including mass and energy balance of the fermentation,
comparison on the theoretical and actual product yields, and consistency of the
experimental data and also formulation of the nutrient media. Nutrient media must be
in sufficient to support the cell’s growth and produce the desired products. The
developed media in the bioreactor would then be further autoclave to avoid
contamination of the process by unwanted microorganisms. Knowledge on the
preparation of the bioreactor upon sterilization is important due to safety reason and
to avoid undue release of the culture organism into the environment.
4.0 MATERIALS AND EQUIPMENTS
4.1 Labelled components of a bioreactor
1
2
6
3
5
4
1.
2.
3.
4.
Touch screen control
Start button
Pump
Corrective solution Bottles
5. Culture vessel
6. Stirrer motor
Figure 1: Unit construction for Bioreactor
4.2 Bioreactor set-up
A bioreactor to be used for this experiment consists of the following equipment
and instrumentation:
a. A cylindrical gas of nitrogen.
b. The bioreactor is fitted with ports for the following:
- Electrode sensor: O2 sensor, temperature sensor, pH electrode, antifoam
probe, level probe
- Gassing tube with ring sparger
- Filling port
- Double jacket (heating element)
- Manual sampler
- 3 storage bottles for acid, alkaline, and antifoam agent
- Equipped with baffles
- Stirrer shaft with mechanical seal
- 2 disc impellers
- Culture vessel
4.3 Chemical for media development
a) Distilled water (1.5L)
b) (NH4)2SO4
c) Glucose monohydrate
5.0 PROCEDURES
5.1 Experiment 1: Media Development
The growth of beaker’s yeast (S.cerevisiae) on glucose may be described by the
following equation:
C 6 H12 O 6  3O 2  0.48NH3  0.48C 6 H10 NO3  4.32H 2 O  3.12CO 2
Yeast
By considering the volume for the seed culture is 10% from the working volume of
fermentation media in 2 liter benchtop bioreactor and the desired yeast concentration
of 50 gdw/l,
5.1.1 Determine the concentration and total amount of glucose and (NH4)2SO4
in the nutrient medium
5.1.2 Weigh the required carbon and nitrogen sources
5.1.3 Pour in the prepared media into the bioreactor vessel.
5.1.4 Sterilize the media for 121°C for 15 minutes and let it cool upon
inoculation of yeast
5.2 Experiment 2: Vessel Preparation before Autoclave.
Preparation of Bioreactor before Autoclave.
5.2.1
Switch on the main power of the bioreactor unit and fill the water inside
the jacket heater to the maximum level.
5.2.2
Take out the pH probe and calibrate it appropriately with pH 7 followed by
pH 4. Immerse the probe in KCl solution in a conical flask.
5.2.3
Calibrate each of the dosing pumps to get the correct flow rate
during the experiment.
5.2.4
Remove the lid and detached all the hosing connected from the main
touch panel. All BLACK hosing CANNOT be autoclaved, make sure
to remove them before autoclave. Others colour of hosing CAN be
autoclave.
5.2.5
Remove motor, all hosing and attached probes from bioreactor.
5.2.6
Rinse the vessel with tap water as much time as necessary to make sure
it clean and clear.
5.2.7
Check the electrolyte in the pO2 probe whether it’s still have or not. This is
to ensure membrane inside the pO2 probe is protected during autoclave.
5.2.8
Pour 1L liquid media prepared into the vessel.
5.2.9
Reattach the lid back to the vessel. Mount back pH probe and pO2 probe.
5.2.10 Cover the pH, pO2, antifoam, level, temperature sensor and the hoses
with cotton and aluminum foil.
5.2.11 Make sure relief valve/filter at the condenser and sampling port are not
covered with cotton, just wrap with aluminum foil.
5.2.12 Tubing that is connected to air spurger, sampling pot and from corrective
bottles are clipped.
5.2.13 Wrap the male connector with aluminum foil.
5.2.14 Make sure every procedure is performed successfully before autoclave
session can be done.
5.3 Maintenance And Safety Precautions
5.3.1 All operating instructions supplied with the unit must be carefully read and
understood before attempting to operate the unit.
5.3.2
Turn off the stirrer, pH electrode, pO2 electrode, antifoam, pump at
the bioreactor.
5.3.3
Running the bioreactor MUST BE assisted by a trained PLV at ALL
times.
5.3.4
Please ensure all safety appliances are worn when operating a high
pressure gas line.
5.4 General Shut Down Procedures
5.4.1
Turn the agitator, temperature , dosing pump off.
5.4.2
Close the air sparger valve and turn off the main switch of the touch
panel.
5.4.3
Remove the mixer motor from top of the bioreactor and place on
top of the apparatus.
5.4.4
Carefully remove the pH and pO2 probe and place in the KCl solution for
pH probe, while in the distilled water for pO2 probe.
5.4.5
Remove any hosing attached with the bioreactor and carefully bring the
bioreactor to the sink.
5.4.6
Remove the cover and place the cover on top of the bench so that it will
not roll off.
5.4.7
Pour the reactor media into the sink and wash thoroughly. Rinse the
vessel with distilled water.
5.4.8
Place back the cover to the vessel, and put back the vessel to its position.
6.0
RESULTS AND CALCULATION
6.1
Record the right and wrong parts when preparing the vessel before
autoclaved in the given table.
NO.
1
2
3
4
5
6
7
RIGHT
WRONG
7.0
QUESTIONS
7.1
List down the factors that can cause contamination to your culture.
7.2
Why pH probe needs to be calibrating before autoclave start and pO2 probe
after autoclave?
7.3
What are the differences between impeller design for microbial,
mammalian, and plant bioreactor and why they are designed in
different ways?
7.4
Why microsparger is being used in mammalian bioreactor?
7.5
Give some comment on design for sparger, impeller, baffle and heating
element for these three types of bioreactor.
7.6
How much yeast biomass will be produced theoretically from the
stoichiometric equation above?
7.7
Determine
the
yield
coefficients
YX/S
(biomass/glucose)
and
YX/O2
(biomass/oxygen).
7.8
What will happen if you have mistakenly tighten the lid screw and not clipped
the hose for aeration during sterilization?
8.0
CONCLUSION
8.1
What is your suggestion to improve/optimize the fermentation media?
8.2
In your view, what are the improvements that you would recommend for these
bioreactors?
8.3
Highlight the most important steps in preparing the bioreactor before
sterilization and give your explanation on these steps.