Laboratory 2: Growth Kinetics Study of Microorganism in Bioreactor

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.


Apparatus and Reagent

5L bioreactor
LB medium for E.coli
Antifoam (polypropylene glycol 2000)
Objectives :
To study the growth kinetics of microorganism in bioreactor
To construct a growth curve including lag, log, stationary and
death phases.
To determine the Monod parameters

Nutrient solution
NaOH(Base)
H
3
PO
4
(Acid)
250 ml shake flasks
micropipette, pipette (10 ml)
Syringe
Centrifuge
Spectrophotometer
Eppendorf tubes,
Falcon tubes

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 ensures that the culture is well aerated).

a. Inoculum
Inserted E.coli into the appropriate amount of LB in shake flask and cultivated for 24
hours at 240rpm and 30
o
C. Read the OD600 against water as a blank to verify that
the culture reached stationary phase (OD>1.5).

b. For medium in bioreactor,
Prepared LB broth according to the required volume and inserted into bioreactor
vessel.


ii) Calibration of pH probe with buffer pH 4 and 7
The bench top bioreactor, 2-litre B-Braun fermenter is set to operate at the optimum
growth condition for the microrganism. DCU Tower control unit is switched on.
Connect pH probe with pH connection. Go to control panel, press button calibration
until data for pH calibration appear. Press alter button to set the temperature into
manual mode. Enter to key in the temperature at 25
o
C.Enter again to start calibration.
Cursor will move to the BUFZ 7.00. Soak pH probe into the buffer pH 7. Press enter
to start calibration at pH 7. Cursor will position between pH value and status. Wait
until the cursor automatically moves to BUFS 4.00. Rinse the probe with distilled
water and dried with the paper tissue. Soak the probe again in buffer pH 4. Press
enter button to start calibration. Calibrations start when cursor is in between pH and
status position. Calibration will end when cursor will move to next parameter. Press
enter until first page of pH calibration appear. Complete calibration. Assemble pH
probe into bioreactor

iii) Bioreactor Experimental design
Bioreactor set up is shown in Figure 1. Assembly all of the probe and pH adjuster
bottle at it position. Make sure all off the opening are close and the filters are tightly
attached to the connector. Wrap the opening and filter with cotton wool and aluminum
foil.


a. Dissolve oxygen probe (DO probe).
DO probe must be soak in the medium for at least 6 hour to polarize it before used
(after autoclaved).




























Figure 1: Bioreactor set up

b. Autoclave bioreactor. Make sure no water bubbles are trap in the jacket water.

iv) DO probe calibration and parameter setting
pO
2
is calibrated by using nitrogen gas (N
2
). Connect all of the connectors to the
control panel. Open water inlet and outlet. Press Calibration, the screen below will be
shown.
Press enter until the cursor is located as shown below.




Start to purge nitrogen in the bioreactor and the current DOT will be shown as shown
below.



The cursor will be automatically relocated automatically to next line when the
automatic calibration of DOT 0 % is achieved. The probe will be automatically
calibrated to DOT 0% when the reading of 0% is stable at a steady reading for certain
period. Then change the tubing to aeration from nitrogen supply to the sparger. Start
aeration at certain vvm. Example of vvm setting: 1 vvm

Liquid volume : 2 L
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 (pO
2
), 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).

Table 1 Reference for designed experiment for bioreactor condition optimization
Parameters F1
(pO
2
, %)
F2
(Airflow, vvm)
F3
pH
Run 1
Run 2
Run 3
Run 4
Run 5
Run 6
v) Preparation of inoculum
1 mL of liquid culture is pipetted into a bijou bottle containing 9 mL of media under
aseptic technique. Then, the culture is incubated at their suitable temperature. The 10
mL starter culture in the bijou bottle is then transferred into 250 mL flask containing
90 mL media resulting 100 mL culture and then allowed to incubate in a
thermostatted rotary shaker (for inoculation in bioreactor). The 100 mL inoculum was
transferred aseptically into the inoculum flask.


vi) Stirred Tank Bioreactor
The inoculum flask is taken to the sterilized bioreactor vessel. The connection tube of
the inoculum flask is connected the inoculum pipeline of the bioreactor vessel under
aseptic technique. The connection tube is clamped off, and by gravity action, the
inoculum inside the inoculum flask flowed through the connection tube into the culture
vessel. The inoculum vessels needs to be lifted quite high to ensure stable flow of the
inoculum. Finally, after all the inoculum has been transferred into the bioreactor
vessel, the connection tube was connected aseptically. The stirred tank bioreactor is
running under the parameter desired.
vii) Sampling
a. Required amount of sample is transferred from the sampling port of the bioreactor
into the sampling tube with interval time for every hour.
b. 5 mL of sample is withdrawn every 2 hours into collector during fermentation for
measuring optical density, glucose analysis and total cell number but for enzyme
and product analysis.

viii) Absorbance Analysis (Optical Density)
a. 1 mL of sample is transferred into a cuvette and the optical density measurement
is made using a spectrophotometer with the wavelength set at 600nm.
b. The spectrophotometer is calibrated to zero by blank consisting 1 mL media.
c. This method is used to measure cell growth; higher number of cells means more
absorbance, which is caused by low transmittance and vice versa.

vi) Cell Dry Weight.
a. Aluminum weight of boat are dried in an oven at 80C for 6-8 hours and placed in
a dessicator containing a drying agent for cooling before weighing (for 30min).
b. The cell pellet (after sample is centrifuged at 10,000 rpm) is suspended in 10 mL
centrifuge tube with distilled water.
c. The cell then transferred to aluminum foil boat. The tube was rinsed with water
and placed in an oven at 80C for overnight.
d. The sample is then removed from the oven with tongs and placed in a dessicator
to cool and weighed rapidly on an analytical balance. The weight of the cell pellet
is record.

vii) Glucose Analysis.
a. Sample of 1.5 mL is transferred into the micro centrifuge tube and centrifuged for
10 minutes at 10,000 rpm.
b. Then, the supernatant is taken out into cuvette and put onto turntable of YSI 2700
Select Biochemical Analyzer for direct analysis of glucose (dextrose)
concentration.
c. The glucose analysis is based on Glucose Oxidase that has been immobilized in
the YSI Dextrose Membrane (YSI 2365).


viii) Product analysis
a. The remaining sample is transferred into the falcon tube and centrifuged for 10
minutes at 10,000 rpm.
b. Then, the supernatant is taken out to analyze the product desired by following the
suitable methods.

Report:
1. Abstract/Summary
2. Introduction
3. Aims/Objective
4. Theory
5. Apparatus
6. Methodology/Procedure
7. Results
8. Calculation
9. Discussion
10. Conclusion
11. Recommendation
12. Reference
13. Appendix