TABLE OF CONTENT

PAGE

ABSTRACT 1

INTRODUCTION 2

AIMS 2

THEORY 3-5

APPARATUS 6

PROCEDURE 7-8

RESULTS 9-15

CALCULATIONS 16-19

DISCUSSIONS 20

CONCLUSION 21

RECOMMENDATIONS 22

REFERENCE 22

APPENDIX 23
ABSTRACT

This experiment is conducted to measure the value of volumetric mass transfer

coefficient (kLa) of a stirred tank reactor with bubble aeration. Before the experiment
can begin the reactor must first be calibrated by purging oxygen in the vessel using
nitrogen to obtain 0% dissolved oxygen reading and then allowing air to enter the
vessel to obtain a 100% dissolved oxygen reading. The parameters manipulated in this
experiment are, aeration rate, stirring rate and temperature. The aeration rates used in
this experiment are 0.5 L/min - 2.5 L/min, the stirring rates are 200 rpm - 1000 rpm
and the temperatures are 30C - 50C. The results of the experiment are for 200 rpm,
400 rpm, 600 rpm, 800 rpm and 1000 rpm the value of k La is 0.0205 s-1, 0.0340 s-1,

0.0516 s-1, 0.0707 s-1 and 0.0820 s-1 respectively while for varying temperatures of

30C, 35C, 40C, 45C and 50C the value of k La is 0.0040 s-1, 0.0761 s-1, 0.0409 s-
1
, 0.0387 s-1 and 0.0423 s-1 respectively and for varying aeration rates of 0.5 L/min,

1.0 L/min, 1.5 L/min and 2.0 L/min the value of kLa is 0.0210 s-1, 0.0200 s-1, 0.0364s-
1
and 0.0492 s-1 respectively. In conclusion, an increase in these parameters will affect
the value of volumetric mass transfer coefficient (kLa).

1
INTRODUCTION

Microorganisms are used as 'biological catalysts' to synthesize various, mechanically

Important products in a variety of different bioprocess configurations. For example,
yeasts and molds are essential to the production of many foods, including bread, cheese,
beer, wine, and soy sauce. In the pharmaceutical industries, yeast, bacteria, and
mammalian cells are modified to produce therapeutic proteins and other bioactive
compounds. Finally, bacteria and yeasts are also used to convert lignin, cellulose, and
sugars into alcohol biofuels. A pre-requisite to each of the above functions is the ability to
promote and preserve the health and physiological state of the cells(if you don't keep the
catalyst happy ,it can't/won't do its job effectively). Thus, regardless of the specific
application, the operation any bioprocess first requires an understanding of how to
optimize the growth of cells and enhance their ability to produce the compounds of
interest.

[1]
Bioreactors are vessels or tanks in which whole cells or cell-free enzymes
transform raw materials into biochemical products and/or less undesirable by-products
and is designed to provide the environment for product formation selected by the
scientist, baker, or winemaker which is the heart of many biotechnological systems
that are used for agricultural, environmental, industrial, and medical applications.
[1]
Industrial bioreactors may be operated as batch reactors or continuously, aerobically
or anaerobically, and with pure or mixed cultures whereby in many bioreactors, three
phases (gas, liquid, and solid) are present and mass transfer is an important
consideration.

[2]
The process of mass transfer across an interface, or across a surface in the bulk
of a phase (liquid, gas or solid), is the result of a chemical potential driving force
which is usually expressed in terms of concentrations of a species in liquid phase, or
partial pressures in the case of gas phases. In the case of oxygen transfer in a
[3]
bioreactor, the measurements of the volumetric mass-transfer coefficient, kLa provide
important information about a bioprocess or bioreactor and these determinations
ensure that processing conditions are such it supplies enough oxygen for the growth of
[3]
cells. The kLa value can also be used to optimize control variables (product yield,
power consumption or processing time) over the life cycle of a bioprocess which
would be based on the oxygen demand at various points in the process and growth
phase of the cells.
AIMS
This experiment was conducted to measure the volumetric mass transfer
coefficient (kLa) of a stirred tank reactor with bubble aeration.
To investigate the effect of temperature, agitation and aeration rate on oxygen
mass transfer coefficient in a cell-free system

2
THEORY
[3]
Dissolved oxygen (DO) is often the limiting substrate in fermentation and cell-
culture systems in the case of bacteria and yeast cultures, the critical oxygen
concentration is usually 1050% of air saturation and if the critical level is exceeded,
[3]
the oxygen concentration no longer limits growth. However, for optimum growth, it
is therefore important to maintain DO levels above the critical value by sparging
(bubbling gas through) the bioreactor with air or pure oxygen. Of course, to be
effective, the mass transfer rate of oxygen to the liquid broth must equal or exceed the
rate at which growing cells take up that oxygen.

[4]
Oxygen has comparatively small solubility in aqueous solutions; in distilled
water at standard conditions, the solubility of oxygen is 8ppm. In order for oxygen to
transfer into a cell, it must go through a series of resistances as shown below:

[4]
Figure 1 shows the oxygen transfer through a series of resistances.

[4]
At point (1) the diffusion initially occurs from the bulk gas to the gas-liquid
interface then at point (2) it moves through the gas-liquid interface while at point (3)
[4]
the oxygen diffuses through to the adjacent bulk liquid region. At point (4), the
oxygen travels through the bulk liquid to the cells outer most surface then at point (5)
it diffuses through the cells mucous layer and at point (6) it diffuses into the cellular
mycelia or soil particle and finally at point (7) and (8) the transport occurs across the
cell envelope and into the intracellular reaction site.

3
 In a bioreator it is important to understand the factors affecting oxygen uptake and
[4]
oxygen transfer rates in cell cultures. Oxygen uptake in cell cultures, (OUR) is
affected by the concentration of cells and the rate of oxygen consumption per cell
while the oxygen transfer rates, (OTR) are affected by bubble size, aeration rate,
agitation rate, presence of cells and temperature. The equations representing OUR and
OTR can be represented by the following equations:

OUR:
[4]
QO 2 qO X2

OTR: [4] k L a C*O 2 Cl O 2

[4]
Where,
Q = oxygen uptake rate per volume (gmol/L.s)
O2

q = specific oxygen uptake rate (gmol/g.s)

O2

X = cell concentration (g/L)

kLa = volumetric mass transfer coefficient

C*O 2 = maximum oxygen concentration (g/L)

C lO 2
= critical oxygen concentration (g/L)

[4]
At steady state, there is no accumulation of oxygen anywhere in the bioreactor.
Therefore, the rate of oxygen transfer from the bubbles must be equal to the rate of
oxygen consumption by the cells, OTR = OUR

[4] kLa C*O 2 ClO 2 qO X

2

[4]
In short, an increase in agitation and aeration rates will result in an increase
value of kLa because, an increase in aeration rate causes more bubbles to enter the
vessel which increases the surface area in contact with the contents of the vessel while
increased agitation causes turbulent shear which reduces the thickness of liquid film in
[4]
the vessel. For temperature however, an increase will cause an increase in the value
of kLa but once the temperature goes beyond 40C the solubility of oxygen drops
which results in a lower value of kLa.

4
 The value of volumetric mass transfer coefficient can be determined using a few
methods, namely static gassing out, dynamic gassing out, oxygen balance method and
[4]
sulphite oxidation method. Static gassing out method is used in the absence of
respiring organism (OUR = 0) whereby oxygen concentration in the solution is
lowered by gassing it out with nitrogen. The de-oxygenated liquid is then aerated and
agitated while the increase in dissolved oxygen (DO) is measured using a probe. The
equations represented in this method are as follows:

[4] dC L * l
OTR: dt k L a CO 2 CO 2

By integration, [4] ln CO*2 COl2 kLa t

[4]
Dynamic gassing out on the other hand involves the presence of respiring
organisms. Initially, at time, t = 0 the air supply to the vessel is switched off and the
reduction in DO is measured between t = 0 and t = 1. At time, t = 1 the sir supply is
switched on and the rise in DO is monitored. The equation represented in this method
is as follows:
dC L * l
[4] dt kLa CO 2 CO 2 qO2 X

Figure 2 shows the [4]graph of DO versus time in dynamic gassing out method

5
APPARATUS

Exit Gas Tube

Entering Gas
Tube

Heating Jacket
pO2 Probe

Filter

Flow Regulator

Glass Vessel

Control Panel

Digital Display

Figure 3 shows the bioreator model MINIFOR HT used in this experiment.

6
PROCEDURE

Reactor calibration
1. The reactor display was adjusted to display pO2 (dissolved oxygen reading)
before calibrating the reactor.
2. All oxygen in the reactor was first purged with pure nitrogen by connecting the
air inlet to the nitrogen tube. All the oxygen would have been fully purged when
the display reads 0% then the value was confirmed on the control panel.
3. The nitrogen was then disconnected from the reactor. Next, air was pumped
into the reactor by connecting its inlet to an air pump.
4. Once the display reads 100% the value was confirmed on the control panel and
the air flow into the reactor was stopped and the calibration was completed.

Effects of stirring rate on oxygen transfer

1. All oxygen in the reactor was first purged with pure nitrogen by connecting the
air inlet to the nitrogen tube. All the oxygen would have been fully purged when
the display reads 0%.
2. The nitrogen was then disconnected from the reactor. The stirrer was then set
to 200 rpm, the temperature was set to 30C and the aeration rate was set to 2
L/min Next, air was pumped into the reactor by connecting its inlet to an air
pump.
3. The pO2 displayed on the digital display was taken every 5 seconds until the
display reads 100%. The air flow into the pump was then stopped.
4. Steps 1-3 were repeated for 400 rpm, 600 rpm, 800 rpm and 1000 rpm with
constant temperature and aeration rate.

7
Effects of stirring rate on oxygen transfer
1.All oxygen in the reactor was first purged with pure nitrogen by connecting the
air inlet to the nitrogen tube. All the oxygen would have been fully purged when
the display reads 0%.
2. The nitrogen was then disconnected from the reactor. The stirrer was then set
to 400 rpm, the temperature was set to 30C and the aeration rate was set to 2
L/min Next, air was pumped into the reactor by connecting its inlet to an air
pump.
3. The pO2 displayed on the digital display was taken every 5 seconds until the
display reads 100%. The air flow into the pump was then stopped.
4. Step 1 was repeated before the vessel was heated to 35C.
5. Steps 2-4 were repeated for temperatures of 40C, 45C and 50C with
constant stirring and aeration rate.

Effects of stirring rate on oxygen transfer

1. All oxygen in the reactor was first purged with pure nitrogen by connecting the
air inlet to the nitrogen tube. All the oxygen would have been fully purged when
the display reads 0%.
2. The nitrogen was then disconnected from the reactor. The stirrer was then set
to 200 rpm, the temperature was set to 30C and the aeration rate was set to 0.5
L/min Next, air was pumped into the reactor by connecting its inlet to an air
pump.
3. The pO2 displayed on the digital display was taken every 5 seconds until the
display reads 100%.
4. The air flow into the pump was then stopped. Steps 1-3 were repeated for
aeration rate of 1.0 L/min, 1.5 L/min and 2.5 L/min with constant temperature and
stirring rate.

8
RESULTS
Table 1 shows the dissolved oxygen (DO%) reading for various stirring rate (RPM)

RPM 200 400 600 800 1000

Time DO % DO % DO % DO % DO %
0 0 0 0 0 1.7
5 1.5 3.1 4.9 5.1 2.7
10 4.3 8.0 14.7 16.0 10.7
15 9.9 13.3 23.8 29.8 25.4
20 11.9 19.7 32.0 43.7 41.4
25 15.4 26.4 41.1 53.8 56.9
30 19.3 32.6 50.4 69.8 66.2
35 23.9 38.6 57.9 72.8 77.3
40 27.9 45.8 64.8 79.3 83.5
45 31.8 50.9 70.5 84.1 88.7
50 35.8 55.8 75.7 88.4 92.0
55 39.5 60.6 80.5 91.4 94.8
60 42.7 65.2 84.0 94.0 97.0
65 46.2 69.2 86.8 95.7 98.0
70 49.9 73.0 89.4 97.0 99.0
75 53.0 76.2 91.6 98.3 99.8
80 55.8 79.2 93.5 99.1 100.0
85 58.7 81.6 95.1 99.8
90 61.4 83.9 96.4 100.0
95 63.9 85.9 97.4
100 66.5 87.6 98.3
105 68.7 89.2 98.9
110 70.6 90.5 99.5
115 72.7 91.8 99.9
120 74.5 93.0 100.0
125 76.3 94.1
130 77.9 95.1
135 79.4 95.9
140 80.7 96.6
145 81.9 97.2
150 83.1 97.8
155 84.4 98.3
160 85.5 98.6
165 86.6 99.1
170 87.5 99.4
175 88.4 99.7
180 89.2 100.0
185 90.2

9
190 90.9
195 91.6
200 92.2
205 92.9
210 93.5
215 94.0
220 94.6
225 95.0
230 95.4
235 95.8
240 96.3
245 96.7
250 97.0
255 97.3
260 97.6
265 97.9
270 98.1
275 98.4
280 98.6
285 98.7
290 98.9
295 99.1
300 99.3
305 99.4
310 99.6
315 99.8
320 99.9
325 100.0

10
Table 2 shows the dissolved oxygen (DO%) reading for various temperatures.

pO 2 reading (DO%)
Time T= 30C T= 35C T= 40 C T= 45 C T= 50 C
0 45 11.7 7.80 0.00 -0.10
5 50.5 11.9 15.90 2.77 2.46
10 55.9 13.4 24.20 9.97 8.60
15 61 17.7 31.50 17.70 19.80
20 65.5 24 40.70 28.10 31.80
25 69.6 31.1 47.80 37.50 43.00
30 73.3 39.1 55.50 46.50 52.80
35 77.2 46.3 61.80 55.30 62.50
40 79.5 52.7 68.30 63.30 70.20
45 82.4 58.9 72.90 68.90 77.70
50 84.6 64.5 77.70 75.20 84.50
55 86.3 69.3 81.40 79.90 89.30
60 88.1 73.5 85.40 84.30 93.90
65 89.8 77.5 87.90 88.00 97.50
70 91.2 81 90.90 91.50 100.00
75 92.4 84 93.00 94.20
80 93.5 86.7 95.20 96.90
85 94.6 88.8 96.70 98.60
90 95.3 90.8 98.40 100.00
98 96.1 92.4 99.50
100 96.8 93.9 100.00
105 97.4 95.2
110 97.8 96.3
115 98.3 97.5
120 98.7 98.3
125 99 99
130 99.3 99.7
135 99.5 100
140 99.8
145 100

11