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; non-
fermentative 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 electrode’s 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 20°C, 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 KLa 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 L 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: L Volume liquid: L
Note: CE = 100% saturation

Agitation speed: rpm

Agitation speed: rpm time CL (% ∆CL/∆t CL(avera Ln(CE –

Air flow rate: L/min (s) saturation) ge) CL)
Volume liquid: L 0
Note: CE = 100% saturation 20
time CL (% CL(averag Ln(CE –
∆CL/∆t 40
(s) saturation) e) CL )
0 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 CBB 20203 Fermentation Technology Page 7

300
 Air flow rate: L/min
Volume liquid: L

time CL (% CL(averag Ln(CE –

∆CL/∆t
(s) saturation) e) CL ) Agitation speed: rpm
0
time CL (% ∆CL/∆t CL(avera Ln(CE –
20
(s) saturation) ge) CL)
40 0

60 20
time CL (% ∆CL/∆t CL(averag Ln(CE – 40
80
(s) saturation) e) CL )
100 0 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 Air flow rate: L/min
Volume liquid: L
290
Note: CE = 100% saturation
300

Agitation speed: rpm

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 Air flow rate: L/min
Volume liquid: L
Agitation speed: rpm
Air flow rate: L/min
Volume liquid: L

time CL (% ∆CL/∆t CL(avera Ln(CE –

Agitation speed: rpm
(s) saturation) ge) CL)
Air flow rate: L/min 0
Volume liquid: L
20
Note: CE = 100% saturation
40
60
80
time CL (% ∆CL/∆t CL(avera Ln(CE –
time CL (% CL(averag Ln(CE – 100
∆CL/∆t (s) saturation) ge) CL)
(s) saturation) e) CL ) 120
0
0
130
20
20
40
140
40
60
150
60
80
160
80
100
170
100
120
180
120
130
190
130
140
200
140
150
210
150
220
160
160
230
170
170
240
180
180
250
190
190
200
260
200
210
270
210
220
280
220
230
290
230
240
300
240
250
250
260
260
270
270
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280
290
290
300
300
 Agitation speed: rpm
Air flow rate: L/min Air flow rate: L/min
Volume liquid: L Volume liquid: L
Note: CE = 100% saturation

time CL (% CL(averag Ln(CE –

∆CL/∆t time CL (% ∆CL/∆t CL(avera Ln(CE –
(s) saturation) e) CL )
0 (s) saturation) ge) CL)
0
20
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: L
Volume liquid: L Note: CE = 100% saturation

time CL (% CL(averag Ln(CE –

∆CL/∆t time CL (% ∆CL/∆t CL(avera Ln(CE –
(s) saturation) e) CL )
0 (s) saturation) ge) CL)
0
20
20
40
40
60
60
80
80
100
100
120
120
130
130
140

150 140

160
150
160
170

180
170

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) KLa (h-1)
0.5
1.0
1.5
2.0
2.5

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

Agitation speed (rpm) KLa(h-1)

200
400
600
800
1000

Effect of salt and antifoam addition on KLa.

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

Substance KLa (h-1)

Salt
Antifoam

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