Experiment 5

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.

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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)

Mass balance for the system;

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Rate of change in O2 conc. = Rate of O2 in - Rate of O2 out - Rate of usage Qd

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)

Can be rewritten as,

dCL/dt = -KLa.CL + KLa.CE

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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

at a 500 rpm and air flow rate of 1 L/min. Then, add 5 mL of

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.

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Results
Non fermentative method
Agitation speed:

rpm

Agitation speed:

rpm

Air flow rate:

L/min

Air flow rate:

L/min

Volume liquid:

Volume liquid:

Agitation speed:

rpm

Note: CE = 100% saturation

Agitation speed:

rpm

Air flow rate:

L/min

Volume liquid:

Note: CE = 100% saturation

time

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

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Air flow rate:

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

Air flow rate:

L/min

Volume liquid:

Note: CE = 100% saturation

300

Agitation speed:

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Air flow rate:

L/min

Volume liquid:

Agitation speed:

rpm

Air flow rate:

L/min

Volume liquid:

Agitation speed:

rpm

Air flow rate:

L/min

Volume liquid:

time

CL (%

(s)
0

saturation)

CL/t

CL(avera

Ln(CE

ge)

CL)

CL(avera

Ln(CE

ge)

CL)

20

Note: CE = 100% saturation

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

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290
300

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Agitation speed:

rpm

Air flow rate:

L/min

Air flow rate:

L/min

Volume liquid:

Volume liquid:

Note: CE = 100% saturation

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

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Effect of salt and antifoam addition on KLa

Agitation speed:

rpm

Agitation speed : _____________ rpm

Air flow rate:

L/min

Air flow rate:

L/min

Volume liquid:

Volume liquid:

Note: CE = 100% saturation

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
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

Effect of airflow rate on KLa (500 rpm)

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Airflow rate (L/min)

0.5
1.0
1.5
2.0
2.5

KLa (h-1)

Effect of agitation speed on KLa (air flow rate = 1 L/min)

Agitation speed (rpm)
200
400
600
800
1000

KLa(h-1)

Effect of salt and antifoam addition on KLa.

(Agitation = 500 rpm; Airflow rate = 1 L/min)
Substance
Salt
Antifoam

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KLa (h-1)

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