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Laboratory 2:
Lab Manual
Objectives
:
To study/observe the growth kinetics of microorganism in shake flask
experiment
Introduction:
A bioreactor is a vessel in which is carried out a chemical process which involves
organisms or biochemically active substances derived from such organisms. Bioreactors
are commonly cylindrical, ranging in size from some liter to cube meters, and are often made
of stainless steel.
Bioreactor design is quite a complex engineering task. Under optimum conditions the
microorganisms or cells will reproduce at an astounding rate. The vessel's environmental
conditions like gas (i.e., air, oxygen, nitrogen, carbon dioxide) flowrates, temperature, pH and
dissolved oxygen levels, and agitation speed need to be closely monitored and controlled.
Most industrial bioreactor manufacturers use vessels, sensors and a control system
networked together. Fouling can harm the overall sterility and efficiency of the bioreactor,
especially the heat exchangers. To avoid it, the bioreactor must be easily cleaned and as
smooth as possible (therefore the round shape). A heat exchanger is needed to maintain the
bioprocess at a constant temperature. Biological fermentation is a major source of heat,
therefore in most cases bioreactors need refrigeration. They can be refrigerated with an
external jacket or, for very large vessels, with internal coils.
In an aerobic process, optimal oxygen transfer is perhaps the most difficult task to
accomplish. Oxygen is poorly soluble in watereven less in fermentation brothsand is
relatively scarce in air (20.95%). Oxygen transfer is usually helped by agitation, which is also
needed to mix nutrients and to keep the fermentation homogeneous. There are, however,
limits to the speed of agitation, due both to high power consumption (which is proportional to
the cube of the speed of the electric motor) and to the damage to organisms caused by
excessive tip speed. In practice, bioreactors are often pressurized; this increases the
solubility of oxygen in water.
Lab Manual
5L bioreactor
Nutrient Media for bacteria (same bacteria used on Lab 1)
Antifoam liquid
Buffer solution for acid and base for pH meter calibration
Inoculum for main media in bioreactor (seed culture)
Micropipette, pipette (10 ml)
Syringe
Centrifuge
Spectrophotometer
Eppendorf tubes
Falcon tubes
Bunsen burner
Safe sampling method
Flask, graduated cylinders etc
Others where appropriate to ensure sterility method
Experimental Procedures:
i)
ii)
iii)
Lab Manual
Refer to bioreactor manual for calibration steps (Obtain the manual from lab
assistant)
Once the calibration of the pH probe is complete, prepare the setup of the bioreactor
for sterilization.
iv)
Do not switch off the main base (control panel and computer) of bioreactor in order to
ensure the calibrated date of the pH probe is kept.
Setup of bioreactor (after sterilization)
Reconnect the bioreactor after sterilization to the main base. (control panel and
computer)
Dissolve oxygen probe (DO probe).
Polarization of the DO probe must be conducted of at least 6 hours after sterilization.
v)
Lab Manual
Connect all of the connectors to the control panel. Open water inlet and outlet. Press
Calibration, the screen below will be shown.
Please refer to the bioreactor manual for further explanation and details of specific
steps.
Start the aeration at a certain vvm. For example of vvm setting: 1 vvm
Liquid volume
:2L
Gas volume
: 2NL
Specific aeration rate : Volume of gas/volume of liquid/min
: 2NL/2L/min
Let the aeration until steady readings which will be automatically detected by the
probe under DOT 100%. Once detected ad steady reading indicating saturated
oxygen concentration in the bioreactor liquid media, the calibration is finished.
Then, the medium parameter such as pH, partial oxygen pressure (pO2), agitation,
aeration and temperature are set according to the predetermined optimum values.
The dissolved oxygen level, temperature and pH, are monitored for 24 hours (during
the fermentation process).
vi)
Preparation of inoculum
Inoculum is prepared as in Lab 1. 10% of inoculum (seed culture) is added to the
main media in bioreactor.
Transfer the inoculum to the main media in bioreactor during its exponential phase
(active condition). (only after the main bioreactor is ready for fermentation: after
sterilization)
vii)
Sampling
Required amount of sample is transferred from the sampling port of the bioreactor
into the sampling tube with interval time for every hour or certain selected period.
Samples taken are measured for optical density (OD), glucose analysis (substrate
concentration S in g/L) and cell dry weight (biomass concentration) as denoted as X
in g/L.
viii)
ix)
x)
xi)
Lab Manual
Time
(g/L)
Time
Report: (100M)
1. Abstract/Summary (5M)
2. Introduction
(10M)
3. Aims/Objective
(5M)
4. Theory
(10M)
5. Apparatus
(5M)
6. Methodology/Procedure (10M)
7. Results
(10M)
8. Calculations
(10M)
9. Discussion
(20M)
10. Conclusion
(5M)
11. Recommendation
(5M)
12. Appendix
(5M)
vvm