ABSTRACT

Bioreactor can be defined as devise in which a biocatalyst is contacted with nutrients or

substrates to produce industrially useful product. It also known as devises which materials are
treated to promote biochemical transformation of matter by the action of biological agents
such as organism’s in vitro cellular components such as enzymes.1 There are a few type of
reactor of bioreactor which is reactor with internal mechanical agitation, bubble columns,
which rely on gas sparging for agitation and loop reactors in which mixing and liquid circulation
are induced by the motion of an injected gas by mechanical pump or by a combination of the
two.

The example of bioprocess that involve in the bioreactor is fermentation.1 The objective
of this experiment is to measure the volumetric mass transfer coefficient (kLa) of a stirred tank
reactor with bubble aeration, to study the effect of temperature as a parameter towards the
mass transfer coefficient, to study the effect of agitation as a parameter towards the mass
transfer coefficient, to study the effect of aeration as a parameter towards the mass transfer
coefficient and to differentiate the effects of different parameters toward mass transfer
coefficient. There are three parameter that will affect the volumetric mass transfer coefficient
(kLa) which is temperature, rpm and aeration (vvm). Basically we will have three procedures of
conducting this experiment in order to determine the volumetric mass transfer coefficient (kLa)
for temperature, rpm and aeration (vvm) affect.

After several calculation and when graph ln(C*-C) versus time is plotting together and
the gradient from the line graph is the kLa value. For the parameter RPM, the kLa value for RPM
320,440,560,680 and 800 are 0.0051, 0.0075, 0.0092, 0.0362 and 0.0229. Then, for the
parameter aeration (vvm) 0.6, 1.0, 1.2, 1.6, 2.0, 2.4 are 0.0162, 0.0211, 0.0232, 0.0256, 0.0272
and 0.0305. Lastly, for the parameter temperature 35, 40, 45 and 50 are 0.0059, 0.0384, 0.0310
and 0.0429. Finally, all of the objective for this experiment successfully achieved by our group
and the error that occur during the experiment can be handling by our group.
 INTRODUCTION
The determination of kLa of a fermenter is essential in order to establish its aeration
efficiency and to quantify the effects of operating variables on the provision of oxygen. 2 It can
be denied that a lot of biochemical processes need the oxygen as the platform to produce the
desired output or product. For example of biochemical process is fermentation process. The
process that producing the chemical which are from the substrates by using the organisms.
That oxygen exchange rate from a gas will a stock for aerobic fermentation will be a paramount
parameter in the configuration what’s more operation for bioreactors.

Aerobic organism’s necessity oxygen for growth, item formation, and cell maintenance.
Thus, sufficient exchange of oxygen from a gas of the fermentation broth must a chance to be
upheld. All oxygen-consuming variables in the bioreactor which is the rate of oxygen are
consumed for the fermentation process can be seen or observed from the volumetric mass
transfer coefficient, kLa. One of the importance’s of the kLa value is scaling up from laboratory
scale to production scale bioreactors or pilot scale.

The determination of the kLa value for fermentation is important in order to maintain
adequate transfer of oxygen in a bioreactor, for laboratory scale use or when scaling up to a
larger process.3 Exchange of oxygen starting with a gas stage should a fluid stage will be difficult
by vicinity from claiming cells, result formation, ionic species, and antifoaming operators. These
can alter bubble size and liquid film resistance, which affect oxygen solubility.4 Resulting kLa
values are different from those predicted from correlations for oxygen absorption into water.

Therefore, it is important to have a reliable method for measuring kLa in bioreactor

systems.2 In this experiment; we observe the relative effects of agitation rate, aeration rate and
temperature on kLa in distilled water. In addition, the effects of agitation rate on kLa are
examined in a fermentation media. At the end of this experiment, the result obtain for every
different parameter are compared to each other.
 OBJECTIVES
 To determine the mass transfer coefficient of a stirred tank reactor.

 To study the effect of temperature as a parameter towards the mass transfer

coefficient.

 To study the effect of agitation as a parameter towards the mass transfer coefficient.

 To study the effect of aeration as a parameter towards the mass transfer coefficient.

 To differentiate the effects of different parameters toward mass transfer coefficient.

 THEORY
There are several models which can be used to determine kLa. All models used to
evaluate kLa assume ideal mixing of the two phases in the reactor and a negligible resistance of
the gas phase to oxygen transfer across the interface.3 This experiment uses the dynamic
gassing out method, which gives the following oxygen mass transfer model:

𝑑𝐶𝐿
= 𝑘𝐿𝑎(𝐶 ∗ − 𝐶𝐿 )(1)
𝑑𝑡

Where CL is the dissolved oxygen concentration and C* is the saturated dissolved oxygen
concentration in the solution.3, 4, 5

𝑑𝐶𝐿
= 𝑂𝑥𝑦𝑔𝑒𝑛 𝑇𝑟𝑎𝑛𝑠𝑓𝑒𝑟 𝑅𝑎𝑡𝑒 − 𝑂𝑥𝑦𝑔𝑒𝑛 𝑈𝑝𝑡𝑎𝑘𝑒 𝑅𝑎𝑡𝑒 (2)
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 = 𝑘𝐿𝑎(𝐶 ∗ − 𝐶𝐿 )

Where;-

kL = Oxygen transfer coefficient (cm/h)

a = Gas-liquid interfacial area (cm2/cm3)

kLa = Volumetric Oxygen Transfer Coefficient (h-1)

C* = Saturated dissolved oxygen concentration (mg/L)

CL = Actual dissolved oxygen concentration in the broth (mg/L)

OTR = Oxygen transfer rate (mg O2/L.h)

The oxygen uptake rate (OUR) is the 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

After obtain all the equation of OUR and OTR in the above, we substitute into equation (2):-

𝑑𝐶𝐿
= 𝑘𝐿𝑎(𝐶 ∗ − 𝐶𝐿 ) − 𝑞𝑂2 . 𝑋
𝑑𝑡

The saturated oxygen concentration in water (C*) varies with temperature.

 APPARATUS
1. Bioreactor.
2. Distilled water.
3. Stopwatch.
4. Oxygen.
5. Nitrogen gas.
 PROCEDURES
Manipulated variable: Temperature

1. Set up the apparatus by making sure all the apparatus are present and in well condition.
2. PO2 probe is polarized for two hours before the main experiment is started.
3. The bioreactor’s parameter such as aeration and agitation are set fixed which are at
1L/m and 400 RPM respectively.
4. The pump is switched off.
5. Temperature is first set at 30˚C and prepared for 2 point calibration. The setting must be
done before purging the nitrogen.
6. Nitrogen gas is purged into the system until the value of oxygen becomes 0%
7. Make sure that the bioreactor’s monitor stop blinking to indicate the calibration is done
correctly even though the value becomes negative.
8. If the value is negative, the value is adjusted manually until the value is 0
9. The nitrogen valve is closed when reaching 0 and the nitrogen wire is disconnected from
the bioreactor.
10. Pump is switched on.
11. The bioreactor is aerated with air for 100%.
12. Timer is set and reading is taken for every 5 seconds until the increasing DO % reaching
three stable values.
13. The results are recorded and a graph of DO% versus time is plotted.
14. Step is repeated using different temperature which is 35˚C, 40˚C, 45˚C, and 50˚C.

Manipulated variable: aeration

1. Set up the apparatus by making sure all the apparatus are present and in well condition.
2. PO2 probe is polarized for two hours before the main experiment is started.
3. The bioreactor’s parameter such as temperature and agitation are set fixed which are at
30˚C and 400 RPM respectively.
 4. The pump is switched off.
5. Aeration is first set at 1L/m and prepared for 2 point calibration. The setting must be
done before purging the nitrogen.
6. Nitrogen gas is purged into the system until the value of oxygen becomes 0%
7. Make sure that the bioreactor’s monitor stop blinking to indicate the calibration is done
correctly even though the value becomes negative.
8. If the value is negative, the value is adjusted manually until the value is 0
9. The nitrogen valve is closed when reaching 0 and the nitrogen wire is disconnected from
the bioreactor.
10. Pump is switched on.
11. The bioreactor is aerated with air for 100%.
12. Timer is set and reading is taken for every 5 seconds until the increasing DO % reaching
three stable values.
13. The results are recorded and a graph of DO% versus time is plotted.
14. Step is repeated using different aeration which is 1.5, 2.0, 2.5, and 3.0.

Manipulated variable: agitation

1. Set up the apparatus by making sure all the apparatus are present and in well condition.
2. PO2 probe is polarized for two hours before the main experiment is started.
3. The bioreactor’s parameter such as aeration and temperature are set fixed which are at
1L/m and 30˚C respectively.
4. The pump is switched off.
5. Agitation is first set at 200rpm and prepared for 2 point calibration. The setting must be
done before purging the nitrogen.
6. Nitrogen gas is purged into the system until the value of oxygen becomes 0%
7. Make sure that the bioreactor’s monitor stop blinking to indicate the calibration is done
correctly even though the value becomes negative.
8. If the value is negative, the value is adjusted manually until the value is 0
9. The nitrogen valve is closed when reaching 0 and the nitrogen wire is disconnected from
the bioreactor.
10. Pump is switched on.
11. The bioreactor is aerated with air for 100%.
12. Timer is set and reading is taken for every 5 seconds until the increasing DO % reaching
three stable values.
13. The results are recorded and a graph of DO% versus time is plotted.
14. Step is repeated using different agitation which is 400, 600, 800, 100rpm.
RESULTS AND
CALCULATION
 DISCUSSIONS
The purpose of conducting this experiment is to study and to determine the kLa values
of different parameter which are from the manual lab instruction. The parameters that are set
to be manipulated variable for these experiments are agitation rate, aeration rate and also the
temperature. The most important step or procedures before start this experiment is we need
to make sure that the condition of the bioreactor is in the good condition and it must be
calibrated properly before we proceed with the experiments.

In general, values of kLa increased as aeration rates or agitation rates increased. This is
consistent with what is found in the literature.5 It can be prove from our experiment which the
kLa values for aeration rates or agitation rates increase when these two parameters are
increase same for the temperature parameter, the kLa values will increase if the temperature
of the bioreactor is increasing linearly. In our experiment, in order to determine the kLa values
for each different parameter that acts as our manipulated variable, we choose to use the
dynamic gassing out method/model. In this model, it stated that we need to know our C* which
is Saturated dissolved oxygen concentration (mg/L). For our experiment, the value of C * is 100
mg/L. The formula for dynamic gassing out method is dCL/dt=kLa(C*-CL )-qO2.X. The important
of to know the kLa value in order to determine the rate of oxygen flow for the bioreactor.

In our experiment, there are three part of experiment which are for the aeration rate,
agitation rate and lastly for the temperature. For the agitation rate, we used the RPM of
320,440,560,680 and 800. When the we apply the dynamic gassing out method and we plot the
graph of ln(C*-C) versus time, the gradient that we obtain in each different RPM are the kLa
value for each RPM. The value of kLa for each RPM is 0.0051, 0.0075, 0.0092, 0.0362 and
0.0229. From the graph that is plotted in the result and calculation, we can see and observe
that the kLa value increase proportionally when the aeration rates which is the RPM is increase
from 320 to 800. Based on the calculation and result we obtain, the kLa values indicated the
oxygen transferred rate for the bioprocess in the bioreactor. So the higher rate of transfer
oxygen towards the bioreactor is for the RPM 800 because the value of kLa is the highest in our
experiment and the lowest rate of transfer oxygen towards the bioreactor is for RPM 320.

Then the second part of this experiment is the effects of agitation rate towards the kLa
value in the bioreactor. For the agitation rate that we used to determine the kLa value is 0.6,
1.0, 1.2, 1.6, 2.0, and 2.4. After the graph of ln(C*-C) versus time are plotted together and the
gradient of each agitation rate from the line graph that we plotted is the kLa value. The value of
kLa that we obtain from the graph in the result and calculation above is 0.0162, 0.0211, 0.0232,
0.0256, 0.0272 and 0.0305. Same as the parameter of aeration rate, when the agitation rate in
the bioreactor in set up to be increase it will affect the kLa value to be increasing proportionally
by the time. The higher the rates of agitation in the bioreactor, the oxygen will dissolve faster in
the bioreactor and it will cause more oxygen need to be transfer towards the bioreactor. So, in
our experiment the highest kLa value is at the agitation rate of 2.4 which is 0.0305 where the
oxygen transfer rate towards the bioreactor also highest while the lowest kLa value is at the
agitation rate of 0.6 which is 0.0162 where the oxygen transfer rate towards the bioreactor also
lowest.

Lastly, the last part for this experiment is the effects of temperature towards the kLa
value in the bioreactor. The value of different temperature is set up in this bioreactor which is
at 35, 40, 45 and 50. The graph of ln(C*-C) versus time are plotted together and from the line
graph we can obtain the gradient of each temperature where the gradient of the line graph of
the temperature parameter is the kLa value. The kLa value that we obtain for this part of
experiment based on the different temperature is 0.0059, 0.0384, 0.0310 and 0.0429. The
higher the temperature the higher the kLa value of the bioreactor where more of oxygen need
to be transferred into the bioreactor to make the bioprocess more effective. Finally, all the kLa
value that we able to obtain from the dynamic gasses out method is the most accurate value of
kLa because this is the most accurate method that suggested. However, there is other error
that occur during the experiment that might be effect the kLa value that we obtain for our
experiment but the error have been minimized by us in order to get accurate value of kLa.
 CONCLUSIONS
In general for this experiment, when we changes the rates of parameters in this
experiment such as the agitation rates and aeration rates, the kLa values will also increase as
well. It is shown that the kLa a value is affected by the changes of rates parameter in this
experiment such as agitation rates and aeration rates. Values for kLa are also influenced by
factors like fermentation media and the oxygen consumption of aerobic organisms. For aerobic
fermentation, a complete or proper transfer of oxygen is essential for the growth, product
formation and for the cell maintainance of aerobic organisms. The calculation of the kLa values
must consider the oxygen used in this process by the aerobic organisms. In opposite way, which
we did not consider the oxygen used in this process may be cause the values of kLa will appear
or observed lower than the actual values of kLa that we supposed to obtain from the different
parameter in this experiment such as agitation, aeration and temperatures. Lastly, we need to
understand and know which method and model to be used in order to determine the kLa
values for all the parameters in this experiment where the oxygen is provided. When we could
not determine which model and method to be used to determine the kLa values, it is a problem
to us to determine the values of kLa and may cause the values of kLa that we obtain do not
accurate and not valid for all the parameters in our experiment. The most suitable method and
model to be used to determine the kLa values is the dynamic gassing out method/model which
give us the most accurate kLa values for this process.
 RECOMMENDATIONS
Firstly, before doing the 2 point calibration, ensure that the button is set correctly
according to the manual that has been demonstrated. It is need to be done correctly to avoid
from doing the wrong procedure. When calibration process, ensure that the stirrer is on for the
agitation. In the wake of cleansing with nitrogen gas, don't take the reading of the expanding
oxygen straightforwardly or too quick. Besides that, when taking temperature as the
manipulated variable, increase the mixer speed in the bioreactor to increase the agitation. By
doing this, the temperature inside the bioreactor will rise up and reduce the time holding up to
give the temperature a chance to rise up naturally. Ensure that the timer is read for every five
second in accurate and precise value. To increase the accuracy when taking results, divide the
steps to few peoples so that each person can focus on their task. Lastly, if all the
recommendations above are followed properly by student during conduct the experiment we
can ensure that all the result that obtain are accurate and precise. Thus, the errors that occur
during the experiment can be minimized.
 REFERENCES
1. Jump up ^ IUPAC, Compendium of Chemical Terminology, 2nd ed. (the "Gold Book")
(1997). Online corrected version: (2006–) "bioreactor".
2. Lab manual for determination of kLa in different parameters in bioreactor.
3. Gauthier, T., Thibault, J. and LeDuy, A. 1991. Measuring kLa with Randomly Pulsed
Dynamic Method, Biotechnology and Bioengineering,37: 889-893.
4. Schuler, M.L. and Kargi, F. 1992, Bioprocess Engineering: Basic Concepts, Prentice Hall,
Englewood Cliffs, NJ, pp. 277-281.
5. Linek, V., Vacek, V. and Benes, P. 1987. A Critical Review and Experimental Verification
of the Correct Use of the Dynamic Method for the Determination of Oxygen Transfer in
Aerated Agitated Vessels to Water, Electrolyte Solutions and Viscous Liquids, Chemical
Engineering Journal,34:11-34.
APPENDIX