CBE 661 Lab 2

Edited 2 Feb 2015

Laboratory 2:

Lab Manual

Growth Kinetics Study of Microorganism in Bioreactor

Objectives

:
To study/observe the growth kinetics of microorganism in shake flask
experiment

To construct a growth curve including lag, log, stationary and death

phases.

To determine the Monod parameters of maximum growth rate (max),

yield of substrate (Yx/s), mass doubling time (td), saturation constant
(Ks), specific growth rate (net).

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.

CBE 661 Lab 2

Edited 2 Feb 2015

Lab Manual

Apparatus and Reagent

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)

Preparation of media and inoculum

Prepared the media according to the needs of microorganism used. (LB broth are
used for inoculation of E.coli strain).The size of inoculums can be varied between 1%
to 10% of the intended fermentation. For a 5 L fermenter, the required inoculums of
50 to 500 ml can be conveniently prepared in 250 ml to 2500 ml shakes flasks (a
maximum ratio of liquid/ flask volume of 0.3 in order to ensure well aeration during
fermentation
a. Inoculum
Ferment the bacteria as used in Lab 1 in the shaken culture experiment in shake
flask. (sterility condtion as needed for the experiment must be abided as taught in Lab
1).
b. For medium in bioreactor,
Specific volume of main media in bioreactor is prepared and sterilized.
Refer to specific volume of culture to volume of flask ratio from standard fermentation
process.

ii)

Setup of bioreactor (before sterilization)

Setup the bioreactor accordingly based on the manual for the specific bioreactor
(refer to bioreactor manual from lab assistant).

iii)

Calibration of pH probe with buffer pH 4 and 7

There are few steps involved.
Calibration of the pH probe must be done prior to sterilization by using buffer
solutions of pH 4 and pH 7.

CBE 661 Lab 2

Edited 2 Feb 2015

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.

Figure 1: Bioreactor set up

*** please insert bioreactor

v)

DO probe calibration and parameter setting

pO2 is calibrated by using nitrogen gas (N2) in alternate to air for 0% of air and 100%
of air.

CBE 661 Lab 2

Edited 2 Feb 2015

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)

Absorbance Analysis (Optical Density) (OD)

Please refer to Lab 1 for the method for OD measurement.
Cell Dry Weight. (X)
Please refer to Lab 1 for the method for X measurement.

x)

Glucose Analysis. (S)

Please refer to Lab 1 for the method for S measurement.

CBE 661 Lab 2

Edited 2 Feb 2015

xi)

Lab Manual

Product analysis (P) optional

Please refer to Lab 1 for the method for P measurement.

Optional method / precautions

During fermentation, cascading can be adopted to ensure specific Dissolved Oxygen reading
which does not go below the critical value of DO reading.
Ensure antifoam liquid volume in the specific antifoam bottle is adequate during fermentation.
Monitor the foaming throughout the fermentation.
Monitor the pH readings for the experiment during fermentation.

Collect data from the measurement for instance:

Time

Table 1 Data Collection

DO
X
S
pH
(g/L)

(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