Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
KLa measurement
Objectives
1. To determine KLa of a fermentation system by dynamic gassing out techniques
depends upon the monitoring of the increase in dissolve oxygen in agitation and
aeration range.
2. To monitor the increase in dissolved oxygen over an adequate range, it is
necessary to fast decrease the O2 level to a low value. Two methods can be
employed to achieve this lowering of the dissolved oxygen concentration; nonfermentative and fermentative.
3. To study the effect of medium viscosity on KLa value.
Apparatus
Bioreactor including pO2 probe.
Stopwatch
Chemicals
NaCl
Antifoam
Distilled water
Calibration of dissolved oxygen electrode
Before calibration the pO2 must be polarized. The polarization must be repeated any
time the electrode is disconnected from the amplifier for more than 10 min, but may
require less time then. The calibration of pO 2-electrode includes zero and slope
calibration. The zero is the electrodes current, when no oxygen is present in the
culture medium meanwhile the slope is usually the pO 2 after saturation of the medium
with air at the maximum air supply intended for the process.
The calibration of the pO2 electrode involved several steps;
1. Temperature in the culture vessel is adjusted at the operating temperature.
Page 1
2. For "zero" calibration, it could be measured the pO 2 of the culture medium before
starting the air supply. The medium will be degassed almost completely due to the heat
impact during sterilization and thus should not contain dissolved oxygen. Alternatively,
we can supply an oxygen-free gas (such as nitrogen of 99.98 purity) to the culture
medium to displace the dissolved oxygen until a constant pO2 near 0" can be read at the
measurement and control system.
3. For slope adjustment, the air supply is activated and the stirring speed is adjusted at
the operating value. The medium should be optimally gassed (max. flow rate intended
for the process) and mixed. At a stable display of the measured value we can calibrate
this as 100 pO2".
4. After calibration, the gas supply rate required for the start up of the intended
fermentation process can be adjusted on the rotameter of the control unit. Note that the
rotameter is calibrated according to standard conditions (temperature 20C, with air at 2
barabs). If it is important to maintain precise operating air flow-rates for further
calculations, this makes it necessary to recalculate the indicated flowrate with a
correction factor.
The calibration of the p02 -electrode is made in the culture vessel after autoclaving and
under the conditions of fermentation.
Non-fermentative method
In this technique, the oxygen concentration of the solution is lowered by gassing the
liquid out with nitrogen gas, so that the solution is "scrubbed" free of oxygen. Aeration is
then initiated at a constant sir flow rate and the increase in dissolved oxygen tension
(DOT) is monitored using dissolved oxygen electrode. The profile of DOT during
deaeration and aeration is shown in Figure 1. Increase in DOT during aeration can be
expressed by Eq. 1;
dCL/dt = KLa (CE-CL)-Qd (1)
(1)
Page 2
Figure 1: Dynamic gassing out for the determination of K La values. Aeration was
terminated at point A and recommenced at point B.
Since microorganism is not present in the solution, Qd = 0. Eq. 1 becomes
dCL/dt=KLa (CE-CL)
(2)
Page 3
Page 4
Page 5
Experimental procedures
1. Set the agitation speed of 500 rpm and 1.0L/min. Purge the nitrogen gas until reach
0% DO. Determine K L a of stirred tank reactor at different air flow rate (0.5, 1.0, 1.5,
2.0 and 2.5 L/min). For this experiment, set the agitation speed at 500 rpm.
2. Determine the effect of increasing agitation speed (200, 400, 600, 800 and 1000
rpm) on K L of a 2 L stirred tank fermenter. For this experiment, set the air flow rate at
1 L/min.
3. In experiment 1 and 2, the fermenter is filled with 1.5 L of distilled water.
4. Investigate the effect of salt (NaCI) and antifoam addition to distilled water on K L a.
In this experiment, add 1.5 g of NaCI to 1.5 L distilled water in a fermenter.
Determined the K
antifoam in a salt solution and determine KLa at the same agitation speed and air
flow rate.
Presentation of Results and Discussion
1. Plot a graph to show the effect of air flow rate and agitation speed on K La. Also
discuss the effect of the addition of salt and antifoam on KLa.
2. Compare the KLa value determined using different rpm and air flow rate.
3. Discuss the possible cause of error in determination of KLa by using this dynamic
gassing out technique.
Page 6
Results
Non fermentative method
Agitation speed:
rpm
Agitation speed:
rpm
L/min
L/min
Volume liquid:
Volume liquid:
Agitation speed:
rpm
Agitation speed:
rpm
L/min
Volume liquid:
CL (%
(s)
0
saturation)
CL/t
time
CL (%
(s)
0
saturation)
CL/t
CL(avera
Ln(CE
ge)
CL)
20
CL(averag
Ln(CE
e)
CL )
40
60
20
80
40
100
60
120
80
130
100
140
120
150
130
160
140
170
150
180
160
190
170
200
180
210
190
220
200
230
210
240
220
250
230
260
240
270
250
280
260
290
270
300
280
290
300
Page 7
L/min
Volume liquid:
time
CL (%
(s)
0
saturation)
CL/t
CL(averag
Ln(CE
e)
CL )
20
40
60
time
80
(s)
100 0
Agitation speed:
time
CL (%
(s)
0
saturation)
rpm
CL/t
CL(avera
Ln(CE
ge)
CL)
20
CL (%
saturation)
CL/t
CL(averag
Ln(CE
e)
CL )
40
60
12020
80
13040
100
14060
120
15080
130
160100
140
170120
150
180130
160
190140
170
200150
180
210160
190
220170
200
230180
210
240190
220
250200
230
260210
240
270220
250
280230
260
290240
270
300250
280
260
290
270
300
280
290
L/min
Volume liquid:
300
Agitation speed:
rpm
Page 8
L/min
Volume liquid:
Agitation speed:
rpm
L/min
Volume liquid:
Agitation speed:
rpm
L/min
Volume liquid:
time
CL (%
(s)
0
saturation)
CL/t
CL(avera
Ln(CE
ge)
CL)
CL(avera
Ln(CE
ge)
CL)
20
40
60
time
CL (%
(s)
0
saturation)
20
40
60
80
100
120
130
140
150
160
170
180
190
200
210
220
230
240
250
260
270
280
290
300
CL/t
CL(averag
Ln(CE
e)
CL )
80
time
100
(s)
120
0
CL (%
saturation)
CL/t
130
20
40
140
60
150
80
160
100
170
120
180
130
190
140
200
150
210
220
160
230
170
240
180
250
190
200
260
210
270
220
280
230
290
240
300
250
260
270
Page 9
Agitation speed:
rpm
L/min
L/min
Volume liquid:
Volume liquid:
time
CL (%
(s)
0
saturation)
CL/t
CL(averag
Ln(CE
e)
CL )
20
time
CL (%
(s)
0
saturation)
CL/t
CL(avera
Ln(CE
ge)
CL)
20
40
40
60
60
80
80
100
100
120
120
130
130
140
140
150
150
160
160
170
170
180
180
190
190
200
200
210
210
220
220
230
230
240
240
250
250
260
260
270
270
280
280
290
290
300
300
Page 10
rpm
L/min
L/min
Volume liquid:
Volume liquid:
CL (%
(s)
0
saturation)
CL/t
CL(averag
Ln(CE
e)
CL )
20
time
CL (%
(s)
0
saturation)
CL/t
CL(avera
Ln(CE
ge)
CL)
20
40
40
60
60
80
80
100
100
120
120
130
130
140
140
150
150
160
160
170
170
180
190
180
200
190
210
200
220
210
230
220
240
230
250
240
260
250
270
260
280
270
290
280
300
290
300
Page 11
KLa (h-1)
KLa(h-1)
KLa (h-1)
Page 12