1.

0 ABSTRACT

The objectives of this experiment are to study the effect of three bioreactor parameters which are
temperature, aeration and agitation speed on the dissolved oxygen percentage and to measure the
volumetric mass transfer coefficient (KLa) of a stirred tank reactor with bubble aeration. Gassing
out method are used in this experiment. This method performed in the absence of cells. Graph
ln(C*-Cl) against time was plotted. Based on the graph value of KLa can be obtained based on the
slope of the graph. Graph volumetric mass transfer coefficient (KLa) against the three parameter
which are temperature, aeration and agitation speed was plotted. For temperature 30oC, 40 oC and
50oC the KLa value obtained are 77.4 h-1, 107.28 h-1 and 135.36 h-1 respectively. For aeration 1vvm,
2vvm and 3vvm, the value of KLa obtained are 77.4 h-1, 73.8 h-1 and 72 h-1 respectively. For
agitation speed 200 rpm, 300 rpm and 400 rpm, the value KLa obtained are 43.92h-1, 67.68h-1and
95.76 h-1. The result show if temperature and agitation speed increasing, the value of KLa will
increasing. Meanwhile if aeration increasing, the value of KLa will decreasing. There are some
error during conducting experiment as the result of aeration parameter are not satisfying. The
experiment are successful as it achieve all objective accept when the aeration parameter was set as
manipulated variable.

1
2.0 INTRODUCTION

Biochemistry, is the study of chemical processes within and relating to living organisms.
Biochemistry deals with structures, functions and interactions of biological macromolecules, like
proteins, nucleic acids, carbohydrates and lipids. Most of biochemical process require oxygen as
the source to yield output. One of example of bioprocess is aerobic fermentation. Aerobic
fermentation is a metabolic process by which cells digest sugar via fermentation in the presence
of oxygen. This phenomenon can be observed in yeast.

In aerobic bioprocesses, oxygen is required due to its solubility in aqueous solution and it
has to be supplied continuously. The oxygen transfer rate (OTR) must be known, to achieve an
optimum design operation and scale-up bioreactors. The dissolved oxygen concentration in a
suspension of aerobic microorganisms depends on the rate of oxygen transfer from the gas phase
to the liquid, on the rate at which oxygen is transported into the cells (where it is consumed), and
on the oxygen uptake rate (OUR) by the microorganism for growth, maintenance and production
(Felix G et al, 2009). The magnitude of the OTR is influenced by volumetric mass-transfer
coefficient (KLa), the overall mass transfer coefficient, which is a characteristic of the diffuser
which is depends on the bubble size and the gas being transferred.

Aeration and agitation are important variables to provide effective oxygen transfer rate
during aerobic bioprocesses (D. Moutafchieva et al, 2013). Hence, the knowledge of the
volumetric mass transfer coefficient (kLA) is required. KLA, describes the efficiency with which
oxygen can be delivered to a bioreactor for a given set of operating condition (James K, 2012).
There are many method to determine value of kLA and two of it are: the static or gassing out
method and the dynamic method with growing culture (Lab manual). The values of kLA are
affected by many factors, such as geometrical and operating characteristics of the reactor (the
agitation speed, air flow rate, type of impeller and the geometry of the bioreactor), media
composition and properties, concentration and microorganism’s morphology and biocatalyst’s
properties (D. Moutafchieva et al, 2013).

2
3.0 OBJECTIVES

The objective of this experiment are:

 To identify the effect of bioreactor parameters which are air flow rate, agitation speed tand
temperature on the dissolved oxygen percentage.
 To measure the volumetric mass transfer coefficient (kLA) of a stirred tank reactor with bubble
aeration.

4.0 THEORY

The volumetric mass transfer coefficient (kLA), is the parameter that characterizes the gas-liquid
mass transfer in bioreactors (M.Tobojas et al, 2000). The determination of kLA of a fermentation
is essential in order to establish its aeration efficiency and to quantify the effects of operating
variables on the provision of oxygen (D. Moutafchieva et al, 2013).

Among numerous method of determining value of, kLA, it is often determined by gassing-
out techniques (static and dynamic methods) (M.Tobojas et al, 2000). In order, to monitor the
increase in dissolved oxygen over an adequate range, it is necessary to decrease the oxygen level
to a low value. Two methods can be used to s lower the dissolved oxygen concentration; non-
fermentative and fermentative.

In non-fermentative method, the O2 concentration of the solution is lowered by gassing

the liquid out with the nitrogen gas, so the solution is ‘scrubbed’ free of O2. Aeration is then
initiated at a constant air flow rate and the increase in dissolved O2 tension (DOT) is monitored
using dissolved O2 electrode.

Figure 4.1: Relationship between dissolved oxygen concentration and time dynamic gassing out method.

3
 Based on figure 1, point A1 shows the level of dissolved oxygen before it was consumed
which the air is supply off. Point B illustrate the air is pumped into the culture and the dissolved
oxygen concentration increases as the function of time. While point C represent steady-state
value.

The mass balance for the dissolved oxygen in the well-mixed liquid phase can be written as:

𝑑𝐶𝐿
= 𝑂𝑥𝑦𝑔𝑒𝑛 𝑇𝑟𝑎𝑛𝑠𝑓𝑒𝑟 𝑅𝑎𝑡𝑒 − 𝑂𝑥𝑦𝑔𝑒𝑛 𝑈𝑝𝑡𝑎𝑘𝑒 𝑅𝑎𝑡𝑒
𝑑𝑡

Where:
CL = dissolved oxygen concentration (mmol O2/L or mgO2/L)

The oxygen transfer rate is the rate at which oxygen is transferred into solution and can
be expressed in term of kLA:

𝑂𝑇𝑅 = 𝑘𝐿 𝑎 ( 𝐶 ∗ − 𝐶𝐿 )

Where:
kL = Oxygen transfer coefficient. (cm /h)
a = Gas-liquid interfacial area (cm2/cm3)
kLa = Volumetric oxygen transfer rate coefficient (h-1)
C* = Saturated dissolved oxygen concentration (mg/L)
CL = Actual Dissolved Oxygen Concentration in the broth (mg/L)
OTR = Oxygen transfer rate (mgO2/L.h)

4
 The oxygen uptake rate at which bacteria or other microorganisms consume oxygen
(typical units of mmol O2/L. h)

𝑂𝑈𝑅 = 𝑞𝑂2 . 𝑋

Where:
qO2 = Specific Rate of Oxygen Consumption (mmol O2/gdw.h)
X = Bacteria Concentration (gdw/L)
gdw = gram dry weight of cells

Substitute equation OUR and OTR into the first equation:

𝑑𝐶𝐿
= 𝑘𝐿 𝑎 (𝐶 ∗ − 𝐶𝐿 ) − 𝑞𝑂2 . 𝑋
𝑑𝑡

The oxygen uptake rate (OTR) can be considered as zero when there are no presence of
bacteria or microorganisms. Hence, the equation used is for the static or gassing out method which
is:

𝑑𝐶𝐿
= 𝑘𝐿 𝑎 (𝐶 ∗ − 𝐶𝐿 )
𝑑𝑡

5
5.0 APPARATUS AND MATERIALS

Figure 5.1: Bioreactor.

Apparatus:

a) Bioreactor
b) Stopwatch
c) HI-BLOW (HP 80) linear air pump aerator.

Materials:

a) Distilled water
b) Oxygen
c) Nitrogen gas.

6
6.0 PROCEDURES

1. The bioreactor was turned on and left for a while in order to stabilize the oxygen probe before
the experiment starts.
2. The airline of the air compressor was connected to the reactor.
3. The ‘F1’ button on the control panel was selected to choose the desired parameter. The value
of selected parameter was the displayed on the window. The other ‘F’ button was then selected
to the desired parameter.
4. After the parameters were set with temperature of 30℃, 200 rpm and the “ON” button on the
far right was selected on the control panel.
5. The oxygen probe (PO2) was ensure to turn on during the calibration.
6. The ‘cAL’ was displayed on the window. For calibration, 2 points was needed which are 0 and
100% of air. The ‘+’ and ‘-‘button were pressed to obtain the number 2 displayed on the settling
display window.
7. The gas valve was then opened. The flow rate was set to 1.0 vvm by using the rotameter.

8. The calibration started when the air was purged out from the vessel with nitrogen.
9. The airline was replaced with nitrogen at the back of the reactor. The air compressor was then
switched off when the airline has been pulled out.
10. During purging, the value of “cAL” was displayed on the window. The value was kept blinking
until it was reached to the set value which is 0.
11. When the reading was 0, this indicate that the air was being purged out.
12. The “c2” was displayed and the reactor was then used for the second calibration point.
13. For the second calibration point which is 100% air, the airline was then changed. The nitrogen
line was pulled out and was replaced with the airline.
14. After the “c2” sign has stopped blinking, the “ON/OFF” button was pressed at the end of the
calibration. The value of “cAL End” was then displayed.
15. When the air was in, the stopwatch is started until it reached 100.
16. The readings were recorded for every 5 seconds. After the value has reached to 100, the flow
rate was changed to 2.0 vvm.
17. The zeroing procedure was repeated. The air was purged out until the reading is back to ‘0’.
The gas line was changed and supplied to the reactor.

7
18. The timer was started and the reading of PO2 was recorded for every 5 seconds.
19. The same procedure was repeated for the next flow rate which is 3.0 vvm.
20. The air flow rate/ aeration was vary to determine the effect of aeration in bioreactor. Agitation
and temperature value are constant.
21. The parameters were switched off after the experiment has done.
22. This experiment was repeated using other temperature witch are 40 oC and 50 oC to determine
the effect of temperature. Agitation and aeration value are constant.
23. This experiment was repeated using other agitation which are 300 rpm and 400 rpm to
determine the effect of agitation. Aeration and temperature value are constant.

8
7.0 RESULTS

EFFECT OF TEMPERATURE ON BIOREACTOR:

The agitation and aeration value are constant.
Aeration = 1vvm
Agitation=400rpm

Table 7.1: Dissolved oxygen concentration in the bioreactor at 30 oC

Time(s) PO2 %Air Cl C*-Cl ln(C*-Cl)

10 0.27 0.0027 0.004 1.403 0.339

20 0.4 0.004 0.006 1.401 0.337
30 0.53 0.0053 0.007 1.400 0.336
40 5.32 0.0532 0.075 1.332 0.287
50 15.7 0.157 0.222 1.185 0.170
60 28.1 0.281 0.397 1.010 0.010
70 39.7 0.397 0.561 0.846 -0.167
80 49.8 0.498 0.703 0.704 -0.352
90 59.2 0.592 0.836 0.571 -0.561
100 66.3 0.663 0.937 0.470 -0.754
110 72.1 0.721 1.018 0.389 -0.945
120 77.2 0.772 1.091 0.316 -1.150
130 81.2 0.812 1.147 0.260 -1.347
140 84.5 0.845 1.194 0.213 -1.545
150 87.5 0.875 1.236 0.171 -1.766
160 89.3 0.893 1.261 0.146 -1.927
170 91.2 0.912 1.288 0.119 -2.131
180 92.8 0.928 1.311 0.096 -2.342
190 93.8 0.938 1.325 0.082 -2.501
200 94.8 0.948 1.339 0.068 -2.690
210 95.7 0.957 1.352 0.055 -2.898

9
220 96.4 0.964 1.362 0.045 -3.095
230 96.9 0.969 1.369 0.038 -3.265
240 97.4 0.974 1.376 0.031 -3.469
250 97.8 0.978 1.382 0.025 -3.670
260 98 0.98 1.384 0.023 -3.787
270 98.3 0.983 1.389 0.018 -3.994
280 98.5 0.985 1.391 0.016 -4.161
290 98.7 0.987 1.394 0.013 -4.361
300 98.8 0.988 1.396 0.011 -4.478
310 99 0.99 1.398 0.009 -4.764
320 99.1 0.991 1.400 0.007 -4.945
330 99.2 0.992 1.401 0.006 -5.166
340 99.3 0.993 1.403 0.004 -5.450
350 99.3 0.993 1.403 0.004 -5.450
360 99.4 0.994 1.404 0.003 -5.849
370 99.5 0.995 1.406 0.001 -6.522
380 99.5 0.995 1.406 0.001 -6.522
390 99.5 0.995 1.406 0.001 -6.522
400 99.6 0.996 1.407 0.000 -9.747
410 99.5 0.995 1.406 0.001 -6.522
420 99.6 0.996 1.407 0.000 -9.747
430 99.6 0.996 1.407 0.000 -9.747

10
 2.000

0.000
0 50 100 150 200 250 300 350 400 450 500

-2.000
ln (C* - CL)

-4.000

-6.000

y = -0.0215x + 1.4144
R² = 0.9377
-8.000

-10.000

-12.000
Time (s)

Figure 7.1: In(C*-CL) with temperature of 30°C against time.

y = mx + c

y = -0.0215X + 1.4144

m = -0.0215

KLa = 0.0215s-1

⸫ KLa = 77.4h-1

11
Aeration = 1vvm
Agitation =400rpm
Table 7.2: Dissolved oxygen concentration in the bioreactor at 40 oC

Time(s) PO2 %Air Cl C*-Cl lnC*-Cl

5 4.17 0.0417 0.069 1.584 0.460

10 4.60 0.046 0.076 1.577 0.455
15 8.32 0.0832 0.138 1.515 0.416
20 13.40 0.134 0.222 1.431 0.359
25 25.10 0.251 0.415 1.238 0.213
30 30.30 0.303 0.501 1.152 0.141
35 37.70 0.377 0.624 1.029 0.029
40 42.20 0.422 0.698 0.955 -0.046
45 45.60 0.456 0.755 0.898 -0.107
50 51.40 0.514 0.850 0.803 -0.220
55 55.50 0.555 0.918 0.735 -0.308
60 58.70 0.587 0.971 0.682 -0.383
65 62.50 0.625 1.034 0.619 -0.480
70 65.60 0.656 1.085 0.568 -0.566
75 69.20 0.692 1.145 0.508 -0.677
80 71.70 0.717 1.186 0.467 -0.762
85 74.40 0.744 1.231 0.422 -0.863
90 76.60 0.766 1.267 0.386 -0.953
95 78.80 0.788 1.304 0.349 -1.052
100 80.70 0.807 1.335 0.318 -1.147
105 82.50 0.825 1.365 0.288 -1.245
110 84.20 0.842 1.393 0.260 -1.348
115 87.00 0.87 1.440 0.213 -1.544
120 88.30 0.883 1.461 0.192 -1.651
125 89.60 0.896 1.483 0.170 -1.769
130 90.60 0.906 1.499 0.154 -1.872

12
135 91.60 0.916 1.516 0.137 -1.985
140 92.70 0.927 1.534 0.119 -2.127
145 93.70 0.937 1.550 0.103 -2.277
150 94.20 0.942 1.559 0.094 -2.361
155 95.00 0.95 1.572 0.081 -2.512
160 95.70 0.957 1.583 0.070 -2.666
165 96.20 0.962 1.592 0.061 -2.793
170 96.90 0.969 1.603 0.050 -3.003
175 97.30 0.973 1.610 0.043 -3.146
180 97.70 0.977 1.617 0.036 -3.313
185 98.10 0.981 1.623 0.030 -3.514
190 98.60 0.986 1.631 0.022 -3.839
195 98.90 0.989 1.636 0.017 -4.101
200 99.20 0.992 1.641 0.012 -4.458
205 99.30 0.993 1.643 0.010 -4.612
210 99.60 0.996 1.648 0.005 -5.304
215 99.90 0.999 1.653 0.000 -12.126

13
 2.000

0.000
0 50 100 150 200 250

-2.000

-4.000
ln (C* - CL)

y = -0.0298x + 1.4354
-6.000 R² = 0.7082

-8.000

-10.000

-12.000

-14.000
Time (s)

Figure 7.2: In(C*-CL) with temperature of 40°C against time.

y = mx + c
y = -0.0298X + 1.4354
m = -0.0298

KLa = 0.0298s-1

⸫ KLa = 107.28h-1

14
Aeration = 1vvm
Agitation =400rpm
Table 7.3: Dissolved oxygen concentration in the bioreactor at 50 oC

Time(s) PO2 %Air Cl C*-Cl ln(C*-Cl)

0 0 0 0.000 1.919 0.652

10 2.99 0.0299 0.057 1.862 0.621
20 17.3 0.173 0.332 1.587 0.462
30 36.9 0.369 0.708 1.211 0.191
40 53.5 0.535 1.027 0.892 -0.114
50 67.1 0.671 1.288 0.631 -0.460
60 77.1 0.771 1.480 0.439 -0.823
70 84.1 0.841 1.614 0.305 -1.188
80 89.2 0.892 1.712 0.207 -1.575
90 93 0.93 1.785 0.134 -2.010
100 96.5 0.965 1.852 0.067 -2.705
110 98.7 0.987 1.894 0.025 -3.703
120 100 1 1.919 0.000 #NUM!

15
 2.000

1.000

0.000
0 20 40 60 80 100 120
ln(C*-Cl)

-1.000
y = -0.0376x + 1.1807
R² = 0.9421

-2.000

-3.000

-4.000
Time (s)

Figure 7.3: In(C*-CL) with temperature of 50°C against time.

y = mx + c
y = -0.0376X + 1.1807
m = -0.0376

KLa = 0.0376s-1

⸫ KLa = 135.36h-1

16
Table 7.4: Mass transfer coefficient in bioreactor with the changes of temperature.

Temperature (°C ) KLa(h-1 )

30 77.4
40 107.28
50 135.36

160

140

120

100
KLa(h-1 )

80

60

40

20

0
0 10 20 30 40 50 60
Temperature (oC )

Figure 7.4: Mass transfer coefficient against temperature.

17
EFFECT OF AERATION RATE ON BIOREACTOR:
The temperature and agitation value are constant.

Temperature = 30°C
Agitation = 400 rpm

Table 7.5: The result for aeration at 1vvm

Time(s) PO2 %Air Cl C*-Cl ln(C*-Cl)

10 0.27 0.0027 0.004 1.403 0.339

20 0.4 0.004 0.006 1.401 0.337
30 0.53 0.0053 0.007 1.400 0.336
40 5.32 0.0532 0.075 1.332 0.287
50 15.7 0.157 0.222 1.185 0.170
60 28.1 0.281 0.397 1.010 0.010
70 39.7 0.397 0.561 0.846 -0.167
80 49.8 0.498 0.703 0.704 -0.352
90 59.2 0.592 0.836 0.571 -0.561
100 66.3 0.663 0.937 0.470 -0.754
110 72.1 0.721 1.018 0.389 -0.945
120 77.2 0.772 1.091 0.316 -1.150
130 81.2 0.812 1.147 0.260 -1.347
140 84.5 0.845 1.194 0.213 -1.545
150 87.5 0.875 1.236 0.171 -1.766
160 89.3 0.893 1.261 0.146 -1.927
170 91.2 0.912 1.288 0.119 -2.131
180 92.8 0.928 1.311 0.096 -2.342
190 93.8 0.938 1.325 0.082 -2.501
200 94.8 0.948 1.339 0.068 -2.690
210 95.7 0.957 1.352 0.055 -2.898
220 96.4 0.964 1.362 0.045 -3.095

18
230 96.9 0.969 1.369 0.038 -3.265
240 97.4 0.974 1.376 0.031 -3.469
250 97.8 0.978 1.382 0.025 -3.670
260 98 0.98 1.384 0.023 -3.787
270 98.3 0.983 1.389 0.018 -3.994
280 98.5 0.985 1.391 0.016 -4.161
290 98.7 0.987 1.394 0.013 -4.361
300 98.8 0.988 1.396 0.011 -4.478
310 99 0.99 1.398 0.009 -4.764
320 99.1 0.991 1.400 0.007 -4.945
330 99.2 0.992 1.401 0.006 -5.166
340 99.3 0.993 1.403 0.004 -5.450
350 99.3 0.993 1.403 0.004 -5.450
360 99.4 0.994 1.404 0.003 -5.849
370 99.5 0.995 1.406 0.001 -6.522
380 99.5 0.995 1.406 0.001 -6.522
390 99.5 0.995 1.406 0.001 -6.522
400 99.6 0.996 1.407 0.000 -9.747
410 99.5 0.995 1.406 0.001 -6.522
420 99.6 0.996 1.407 0.000 -9.747
430 99.6 0.996 1.407 0.000 -9.747

19
 2.000

0.000
0 50 100 150 200 250 300 350 400 450 500

-2.000
ln (C*-Cl)

-4.000
y = -0.0215x + 1.4144
R² = 0.9377
-6.000

-8.000

-10.000

-12.000
Time (s)

Figure 7.5: ln(C*- CL) against time for aeration rate at 1.0 vvm.

y = mx + c
y = -0.0215X + 1.4144
m = -0.0215

KLa = 0.0215s-1

⸫KLa = 77.4h-1

20
Temperature = 30°C
Agitation = 400 rpm
Table 7.6: The result for aeration at 2 vvm
Time(s) PO2 %Air Cl C*-Cl ln(C*-Cl)

0 0 0 0.000 1.410 0.34

10 0 0 0.000 1.410 0.34
20 4.5 0.045 0.064 1.346 0.30
30 18.6 0.186 0.263 1.147 0.14
40 33.3 0.333 0.470 0.940 -0.06
50 47.5 0.475 0.671 0.739 -0.30
60 60.6 0.606 0.856 0.554 -0.59
70 70.1 0.701 0.990 0.420 -0.87
80 77.6 0.776 1.096 0.314 -1.16
90 83 0.83 1.172 0.238 -1.44
100 87 0.87 1.229 0.181 -1.71
110 90.4 0.904 1.277 0.133 -2.02
120 92.8 0.928 1.311 0.099 -2.31
130 94.6 0.946 1.336 0.074 -2.61
140 95.9 0.959 1.355 0.055 -2.89
150 96.8 0.968 1.367 0.043 -3.16
160 97.5 0.975 1.377 0.033 -3.42
170 98 0.98 1.384 0.026 -3.66
180 98.4 0.984 1.390 0.020 -3.91
190 98.8 0.988 1.396 0.014 -4.24
200 98.9 0.989 1.397 0.013 -4.35
210 99.1 0.991 1.400 0.010 -4.59
220 99.3 0.993 1.403 0.007 -4.92
230 99.4 0.994 1.404 0.006 -5.14
240 99.4 0.994 1.404 0.006 -5.14
250 99.4 0.994 1.404 0.006 -5.14

21
260 99.5 0.995 1.406 0.004 -5.41
270 99.5 0.995 1.406 0.004 -5.41
280 99.6 0.996 1.407 0.003 -5.79
290 99.6 0.996 1.407 0.003 -5.79
300 99.6 0.996 1.407 0.003 -5.79
310 99.7 0.997 1.408 0.002 -6.41
320 99.7 0.997 1.408 0.002 -6.41
330 99.7 0.997 1.408 0.002 -6.41
340 99.7 0.997 1.408 0.002 -6.41
350 99.8 0.998 1.410 0.000 -8.36
360 99.7 0.997 1.408 0.002 -6.41
370 99.8 0.998 1.410 0.000 -8.36
380 99.7 0.997 1.408 0.002 -6.41
390 99.7 0.997 1.408 0.002 -6.41
400 99.8 0.998 1.410 0.000 -8.36
410 99.8 0.998 1.410 0.000 -8.36
420 99.8 0.998 1.410 0.000 -8.36
430 99.8 0.998 1.410 0.000 -8.36
440 99.8 0.998 1.410 0.000 -8.36
450 99.8 0.998 1.410 0.000 -8.36
460 99.8 0.998 1.410 0.000 -8.36

22
 2.00

0.00
0 50 100 150 200 250 300 350 400 450 500

-2.00
ln (C*-Cl)

-4.00

y = -0.0205x + 0.2191
R² = 0.9658
-6.00

-8.00

-10.00
Time (s)

Figure 7.6: ln(C*- CL) against time for aeration rate at 2.0 vvm.

y = mx + c
y = -0.0205X + 0.2191
m = -0.0205

KLa = 0.0205s-1

⸫KLa = 73.8h-1

23
Temperature = 30°C
Agitation = 400 rpm
Table 7.7: The result for aeration rate at 3.0 vvm
Time(s) PO2 %Air Cl C*-Cl ln(C*-Cl)

10 7.19 0.0719 0.102 1.306 0.267

20 23.3 0.233 0.329 1.079 0.076
30 39.7 0.397 0.561 0.847 -0.166
40 54.8 0.548 0.774 0.634 -0.456
50 67 0.67 0.946 0.462 -0.773
60 75.9 0.759 1.072 0.336 -1.091
70 82.7 0.827 1.168 0.240 -1.428
80 87.5 0.875 1.236 0.172 -1.760
90 90.9 0.909 1.284 0.124 -2.088
100 93.4 0.934 1.319 0.089 -2.423
110 95.3 0.953 1.346 0.062 -2.784
120 96.6 0.966 1.365 0.043 -3.136
130 97.4 0.974 1.376 0.032 -3.438
140 98.1 0.981 1.386 0.022 -3.806
150 98.5 0.985 1.391 0.017 -4.099
160 98.8 0.988 1.396 0.012 -4.393
170 99 0.99 1.398 0.010 -4.653
180 99.2 0.992 1.401 0.007 -5.004
190 99.3 0.993 1.403 0.005 -5.241
200 99.4 0.994 1.404 0.004 -5.551
210 99.4 0.994 1.404 0.004 -5.551
220 99.4 0.994 1.404 0.004 -5.551
230 99.5 0.995 1.406 0.002 -6.003
240 99.5 0.995 1.406 0.002 -6.003
250 99.6 0.996 1.407 0.001 -6.851
260 99.6 0.996 1.407 0.001 -6.851

24
270 99.6 0.996 1.407 0.001 -6.851
280 99.6 0.996 1.407 0.001 -6.851
290 99.6 0.996 1.407 0.001 -6.851
300 99.6 0.996 1.407 0.001 -6.851
310 99.6 0.996 1.407 0.001 -6.851
320 99.6 0.996 1.407 0.001 -6.851
330 99.6 0.996 1.407 0.001 -6.851
340 99.6 0.996 1.407 0.001 -6.851
350 99.7 0.997 1.408 0.000 #NUM!

25
 1.000

0.000
0 50 100 150 200 250 300 350 400

-1.000

-2.000

-3.000
Axis Title

-4.000
y = -0.02x - 0.4973
R² = 0.6609
-5.000

-6.000

-7.000

-8.000
Axis Title

Figure 7.7: ln(C*- CL) against time for aeration rate at 3.0vvm.

y = mx + c
y = -0.02X + 0.4973
m = -0.02

KLa = 0.02s-1

⸫KLa = 72h-1

26
Table 7.8: Mass transfer coefficient in bioreactor at changes of aeration rate.
Aeration rate(vvm) KLa(h-1 )

1 77.4
2 73.8
3 72

78

77

76

75
KLa(h-1 )

74

73

72

71
0 0.5 1 1.5 2 2.5 3 3.5
Aeration (vvm)

Figure 7.8: Mass transfer coefficient against aeration rate.

27
EFFECT OF AGITATION SPEED ON BIOREACTOR:
The temperature and aeration value are constant.

Temperature = 50℃
Aeration = 1vvm

Table 7.9: Results when agitation speed at 200rpm.

Time(s) PO2 %Air Cl C*-Cl ln(C*-Cl)

0 0 0 0.000 1.919 0.652

10 0.31 0.0031 0.006 1.913 0.649
20 3.37 0.0337 0.065 1.854 0.618
30 9.13 0.0913 0.175 1.744 0.556
40 15.7 0.157 0.301 1.618 0.481
50 22 0.22 0.422 1.497 0.403
60 28.5 0.285 0.547 1.372 0.316
70 34.7 0.347 0.666 1.253 0.226
80 40.9 0.409 0.785 1.134 0.126
90 46.4 0.464 0.891 1.028 0.028
100 52.2 0.522 1.002 0.917 -0.087
110 57.6 0.576 1.106 0.813 -0.206
120 62.1 0.621 1.192 0.727 -0.319
130 65.1 0.651 1.249 0.670 -0.401
140 70 0.7 1.344 0.575 -0.553
150 73.4 0.734 1.409 0.510 -0.673
160 76.4 0.764 1.466 0.453 -0.793
170 79.2 0.792 1.520 0.399 -0.919
180 82.3 0.823 1.580 0.339 -1.081
190 84.6 0.846 1.624 0.295 -1.220
200 86.7 0.867 1.664 0.255 -1.367
210 88.8 0.888 1.704 0.215 -1.539

28
220 90.6 0.906 1.739 0.180 -1.714
230 92.2 0.922 1.770 0.149 -1.901
240 93.9 0.939 1.802 0.117 -2.147
250 95.3 0.953 1.829 0.090 -2.409
260 96.7 0.967 1.856 0.063 -2.764
270 97.9 0.979 1.879 0.040 -3.219
280 99 0.99 1.900 0.019 -3.969
290 100 1 1.919 0.000 #NUM!

29
 2.000

1.000

0.000
0 50 100 150 200 250 300 350
ln (C*-Cl)

-1.000

-2.000

y = -0.0122x + 0.998
-3.000 R² = 0.7584

-4.000

-5.000
Time (s)

Figure 7.9: ln(C*- CL) against time at agitation speed of 200 rpm.

y = mx + c
y = -0.0122X + 0.998
m = -0.0122

KLa = 0.0122s-1

⸫KLa =43.92h-1

30
Temperature = 50℃
Aeration = 1vvm
Table 7.10: Results when agitation speed at 300rpm.

Time(s) PO2 %aAir Cl C*-Cl ln(C*-Cl)

0 0 0 0.000 1.919 0.652

10 0.87 0.0087 0.017 1.902 0.643
20 4.43 0.0443 0.085 1.834 0.606
30 10.7 0.107 0.205 1.714 0.539
40 18.8 0.188 0.361 1.558 0.444
50 26.8 0.268 0.514 1.405 0.340
60 35.6 0.356 0.683 1.236 0.212
70 44.7 0.447 0.858 1.061 0.059
80 53.2 0.532 1.021 0.898 -0.108
90 63 0.63 1.209 0.710 -0.343
100 69.2 0.692 1.328 0.591 -0.526
110 74.5 0.745 1.430 0.489 -0.715
120 79.2 0.792 1.520 0.399 -0.919
130 85.9 0.859 1.649 0.270 -1.308
140 90.8 0.908 1.743 0.176 -1.736
150 93.8 0.938 1.800 0.119 -2.131
160 97.1 0.971 1.864 0.055 -2.894
170 99.1 0.991 1.902 0.017 -4.076
180 100 1 1.919 0.000 #NUM!

31
 2.000

1.000

0.000
0 20 40 60 80 100 120 140 160 180 200

-1.000
ln(Cl*-Cl)

-2.000 y = -0.0188x + 1.0966

R² = 0.6437

-3.000

-4.000

-5.000
Time,s

Figure 7.10: ln(C*- CL) against time at agitation speed of 300 rpm.

y = mx + c
y = -0.0188X + 1.0966
m = -0.0188

KLa = 0.0188s-1

⸫KLa =67.68h-1

32
Temperature = 50℃
Aeration = 1vvm
Table 7.11: Results when agitation speed at 400rpm.

Time(s) PO2 %Air Cl C*-Cl ln(C*-Cl)

0 0 0 0.000 1.919 0.652

10 2.99 0.0299 0.057 1.862 0.621
20 17.3 0.173 0.332 1.587 0.462
30 36.9 0.369 0.708 1.211 0.191
40 53.5 0.535 1.027 0.892 -0.114
50 67.1 0.671 1.288 0.631 -0.460
60 77.1 0.771 1.480 0.439 -0.823
70 84.1 0.841 1.614 0.305 -1.188
80 89.2 0.892 1.712 0.207 -1.575
90 93 0.93 1.785 0.134 -2.010
100 96.5 0.965 1.852 0.067 -2.705
110 98.7 0.987 1.894 0.025 -3.703
120 100 1 1.919 0.000 #NUM!

33
 1.500

1.000

0.500

0.000
0 20 40 60 80 100 120 140
-0.500
ln(C*-Cl)

-1.000 y = -0.0266x + 0.778

R² = 0.5812
-1.500

-2.000

-2.500

-3.000

-3.500

-4.000
Time (s)

Figure 7.11: ln(C*- CL) against time at agitation speed of 400 rpm.

y = mx + c
y = -0.0266X + 0.778
m = -0.0266

KLa = 0.0266s-1

⸫KLa =95.76h-1

34
Table 7.12: Mass transfer coefficient in bioreactor at changes of agitation speed.
Agitation speed (rpm) KLa (h-1 )

200 43.92
300 67.68
400 95.76

120

100

80
KLa (h-1)

60

40

20

0
0 50 100 150 200 250 300 350 400 450
Agitation (rpm)

Figure 7.12: Mass transfer coefficient against agitation speed.

35
8.0 SAMPLE CALCULATION

The sample data below is the percentage of dissolve oxygen, DO (%) at temperature of 30oC
for every 10 seconds.

From Table 7.1

Time(s) PO2 %Air Cl C*-Cl ln(C*-Cl)

10 0.27 0.0027 0.004 1.403 0.339

20 0.4 0.004 0.006 1.401 0.337

30 0.53 0.0053 0.007 1.400 0.336

To calculate the percentage difference,

C* - CL

Where,
C* = 100% saturation
CL = the percentage of dissolve oxygen, DO (%)

From the data at 10 seconds,

C* = 1.407 mmol/L (the highest value of Cl)

Cl = 0.004 mmol/L

C* - Cl = 1.403
ln(C*- C) = 0.339

The graph of ln(C*- CL) vs time was plotted to obtain the gradient of the graph. Based on figure
7.1 for details.

From the static gassing-out method, the equation to determine the KLa:

𝐥𝐧(𝑪*−𝑪𝑳) = −𝑲𝑳𝒂(𝒕)

36
 2.000

0.000
0 50 100 150 200 250 300 350 400 450 500
-2.000
ln (C* - CL)

-4.000

-6.000
y = -0.0215x + 1.4144
R² = 0.9377
-8.000

-10.000

-12.000

Time (s)

Figure 7.1: In(C*-CL) with temperature of 30°C against time.

So,

y = mx + c

y = -0.0215X + 1.4144

m = -0.0215

KLa = 0.0215s-1

m= - KLa = 0.0215s-1

Convert units from second to hour

0.0215 60𝑠 60 𝑚𝑖𝑛

× × = 77.4 h-1
𝑠 1 𝑚𝑖𝑛 1ℎ

⸫ KLa = 77.4h-1

37
9.0 DISCUSSION

The purpose of this experiment are to identify the effect of bioreactor parameters which are
temperature, air flow rate or aeration and agitation speed on the dissolved oxygen percentage and
to measure the volumetric mass transfer coefficient (KLa) of a stirred tank reactor with bubble
aeration. During the experiment was conducted the value of temperature, air low rate and agitation
speed are being manipulate. For the first parameter, temperature was changing with an increment
of 10oC for each temperature which are 30oC, 40oC and 50oC. For air flow rate, the values was
vary from 1vvm, 2vvm and 3vvm. Whereas, the agitation speed was manipulated with an
increment of 100 rpm for each speed which are 200 rpm, 300 rpm and 400 rpm. The experiment
stop when dissolved oxygen value in bioreactor remain constant. When the dissolve oxygen
become constant it indicates that the dissolve oxygen has achieved its concentration value.

The value of KLa can be determine based on the slope of the graph ln(C*-Cl) against time.
For first parameter which is temperature, the data has been recorded and calculated. Based on
Figure 7.4 the graph of mass transfer coefficient (KLa) against temperature (oC) was plotted .For
the temperature 30oC, 40 oC and 50 oC, the value of KLa obtained are 77.4 h-1, 107.28 h-1 and 135.36
h-1 respectively. Throughout an experiment that manipulates temperature value, the aeration and
agitation value are remain constant which are 1vvm and 400 rpm respectively. The graph that has
been plotted shows that the value of KLa are increasing as the temperature increasing. When
temperature increase will lead to the decreasing of solubility of oxygen. Higher temperature will
lead to the faster rate of reaction. However, when temperature exceed optimum temperature will
destroy or denature the enzyme.

For aeration or air flow rate parameter, graph on (Figure 7.8) mass transfer coefficient
(KLa) against aeration (vvm) was plotted. For the aeration rate 1 vvm, 2 vvm and 3 vvm, the value
of KLa obtained are 77.4 h-1, 73.8 h-1 and 72 h-1 respectively. Throughout an experiment that
manipulates aeration value, the temperature and agitation value are remain constant which are
30oC and 400 rpm respectively. The graph has been plotted shows the value of KLa decreasing
with increasing of aeration. There are several error during conducting this experiment because the
value of KLa should increase with increasing of aeration. Higher aeration or air flow rate, will

38
dissolve the molecule faster in liquid. However, if aeration exceed optimum aeration will lead to
the reduction in the volume of fermentation broth.

For agitation speed parameter, graph on (Figure 7.12) mass transfer coefficient (KLa)
against agitation speed (rpm) was plotted. For the agitation speed 200 rpm, 300 rpm and 400 rpm,
the value of KLa obtained are 43.92h-1, 67.68h-1and 95.76 h-1 respectively. Throughout the
experiment that manipulates agitation speed value, the aeration and temperature value are remain
constant which are 1vvm and 50oC respectively The graph has been plotted shows the value of
KLa increasing with the increasing of agitation speed. The higher the agitation speed, the faster the
reaction take place and mix between oxygen and the solution.

There are error occur during an experiment when aeration parameter was set as
manipulated variable as the result obtained are not satisfying. Error that may be occur during
experiment is when handling air supply, when turn it off it lead to the dissolve oxygen has been
above the critical dissolved oxygen concentration. If the oxygen is below critical oxygen,
anaerobic metabolism will occur rather than anaerobic metabolism. This matter causing the KLa
values cannot be achieved.

Overall experiment can be conclude as successful as it achieve the objective of this

experiment except for experiment that set aeration parameter as manipulated variable as the result
obtain are not satisfying.

39
10.0 CONCLUSION

The aim of this experiment are to study the effect of three bioreactor parameters which are
temperature, aeration and agitation speed on the dissolved oxygen percentage and to measure the
volumetric mass transfer coefficient (KLa) of a stirred tank reactor with bubble aeration. The KLa
can be obtained based on the slope of the graph ln(C*-Cl) against time. For temperature
parameter, the value of KLa calculated are 77.4 h-1, 107.28 h-1 and 135.36 h-1 respectively with the
30oC, 40 oC and 50oC. For aeration parameter, the value of KLa calculated are 77.4 h-1, 73.8 h-1
and 72 h-1 respectively with the 1vvm, 2vvm and 3vvm. For agitation speed, the value of K La
calculated are 43.92h-1, 67.68h-1and 95.76 h-1 respectively with the 200 rpm, 300 rpm and 400
rpm. For temperature, the KLa value increase as the temperature increase. For aeration, the K La
value decrease as the aeration increase. Lastly, for the agitation speed the value of KLa increase as
the agitation speed increase. It can be concluded the overall experiment are success as all the
objective are achieved except experiment when aeration parameter set as manipulated variable as
the desired KLa values cannot be obtain.

11.0 RECCOMENDATION

There are several factor that can be fix and take into consideration for the experiment run smoothly
and get accurate result. First recommendation is to ensure all the equipment is properly calibrated,
functioning, clean and ready to use. Equipment that are malfunction can affected the result.
Secondly, student must follow the procedure given, general start up need to be done correctly so
the bioreactor run in good condition during main experiment. Thirdly, during an experiment make
sure all parameter such as temperature, agitation and aeration was set properly before start in order
to get all the results needed. Other than that, to obtain desired value of dissolve oxygen make sure
the oxygen probe (PO2) must be turn on during the calibration. Lastly, during this experiment the
result need to take every 5 seconds. So, to ensure no data left behind record the reading of the
bioreactor using camera or phone along the experiment, then jot down in the piece of paper.

40
12.0 REFERENCES

1. Felix G, O., Emilio G. (2009). Bioreactor scale-up and oxygen transfer rate in microbial
processes. Biotechnology Advances, 27(2), pp 153-176.
2. D.Moutafchieva,D.Popova, M.Dimitrova, S.Tchaoushev. (2013). Experimental Determination
Of The Volumetric Mass Transfer Coefficient. Chemical Technology and Metallurgy, 48(4),
pp 351-356.
3. James K. (2012). Measuring kLa for Better Bioreactor Performance
4. M.Tobojas., E. Garcia Calvo. (2000). Comparison of experimental methods for determination
of the volumetric mass transfer coefficient in fermentation processes. Heat and Mass Transfer,
36(3), pp 201-207.
5. CHE 506 – Lab Manual 7

41
13.0 APPENDIX

42
BIOREACTOR

Nitrogen tank

43