INSTITUTE OF BIO IT SELANGOR

UNIVERSITI INDUSTRI SELANGOR

LABORATORY GUIDE FOR SETTING UP OF A

BIOREACTOR:
1. Installed all components part.
Install all component of the bioreactor, including tubing and filters as instructed.
2. Filled bioreactor with water and test agitation (for height of impeller, airflow, probes). Connect all tubing and filter.
3. Calibrate pH probe (using ph 4.0 and pH 7.0 buffer)
Calibration of pH electrode is carried out before the bioreactor is autoclaved. This
is because calibration of the pH electrode is done externally. To calibrate the pH
electrode, the electrode must be connected to the control unit. The pH electrode is
then taken out from the top plate and mounts on a retort stand in a buffer with pH
7.0. Under the calibration mode on the control panel, the pH of the buffer is read
until it remains constant. The electrode is then immersed in buffer pH 4.0 until it
remain constant.
Notes: Care has to be taken to ensure that the pH electrode is not left submerged in
water/buffer for a long period of time.
4. Prepare media, 1M NaOH, 1 M HCl buffer and antifoam
The media has to be prepared accordingly. The total volume of the media to be
put in the bioreactor has to take into accounts the volume of the inoculum. To
prevent browning effect of glucose, glucose is autoclave separately from the other
component of media such as yeast extract or peptone. The 0.1M NaOH and 0.1M
HCL is to be used to set the desired pH or to maintain pH for a particular period
of time. 4% antifoam either silicon-based or non-silicon based is used to control
foaming.

5. Autoclave bioreactor
Before autoclaving, make sure that all clips are tighten and filters are covered
with aluminium foil. This is to prevent from backflow as well as preventing the
filters from getting wet and blocked. It is very important that the exhaust outlet
remains open and unblocked. This is to prevent pressure build up that may lead to
rupture of the glass bioreactor.
The bioreactor is autoclave for at 121oC for a period of 20 minutes. Let the
bioreactor cool down before taking it out of the autoclave. Use gloves to take out
the bioreactor.
6. Calibrate dissolved oxygen probe.
There are two basic types of dissolved oxygen probe, galvanic and polarographic.
The principles for both types, however, are the same
Before sterilization of the bioreactor, check that the Teflon membrane on the end
of the dissolved oxygen probe is not damaged or kinked, or is leaking electrolyte.
Top up the probe with electrolyte if necessary, or change the membrane.
Autoclave the bioreactor with the dO2 probe in place.
After cooling, set the temperature control module to 30 oC, aeration rate to 0.1-1.0
vvm, and agitation rate ~700 rpm
When the bioreactor has reached 30oC and dO2 probe display has stabilized with
the dO2 adjustment control set the display to 100%, that is, fully saturates with O2.
Switch off the air and sparge reactor with O2-free N2, or simply switch the probe
to zero setting. After the signal has stabilized at a low value, use the zero
adjustment control to set the display to zero. The output of the probe is now
calibrated over the range 0 to 100% dissolved oxygen. The zero adjustment varies
from one bioreactor to another, so consult the manual for the model used before
beginning.
Set the dO2 set-point control to 0 or 100%, depending on the experiment, and set
the minimum and maximum limits for the agitation speed and/or aeration rate
depending on whether the dO2 control is through agitation or aeration
respectively. Consult the bioreactor manuals.

7. Check overnight culture and spore count using haemocytometer and optical
density.
Prepare dilution series if needed and routinely do dilution to get the same number
of spores or cell count for each experiment run. Calculate the number of cells or
spore in four different big squares. One cell for each small square corresponds to
1 x 104 cells/mL
8. Transfer yeast extract and inoculum (spore or overnight culture) of the
appropriate amount to get to the desired number of cells per mL into the inoclum
flask using sterile technique or by using the laminar flow cabinet.
9. Transfer the inoculum and yeast extract into the bioreactor using sterile
technique.
10. Collecting sample and fermentation data collection.
11. Analyzing sample for dry cell weight, optical density, glucose concentration,
specialized method.
12. Processing and presentation of fermentation data.