Experiment 2: Kinetics Study Batch Fermentation of Baker’s Yeast

Objective

 To study the growth kinetic of Baker’s yeast in batch production.

 To familiarize the students with culture media which used to maintain pure culture
and develop the fermentation system.

Introduction

Baker’s yeast is the type of yeast used in home and commercial bread baking. It is
widely available in a number of forms, including Cake Yeast (also known as Wet, Fresh, or
Compressed Yeast), Active Dry Yeast and Instant (or fast-rising) Yeast. Bakers’ yeast
performs its leavening function by fermenting such sugars as glucose, fructose, maltose, and
sucrose. It cannot use lactose, the predominant sugar of milk, or certain
other carbohydrates. The principal products of fermentation are carbon dioxide,
the leavening agent, and ethanol, an important component of the aroma of freshly baked
bread. Other yeast activity products also flavour the baked product and change the dough’s
physical properties.

The rate at which gas is evolved by yeast during the various stages of dough
preparation is important to the success of bread manufacture. Gas production is partially
governed by the rate at which fermentable carbohydrates become available to the yeast.
The sugars naturally present in the flour and the initial stock of added sugar are rapidly
exhausted. A relatively quiescent period follows, during which the yeast cells become
adapted to the use of maltose, a sugar constantly being produced in the dough by the action
of diastatic enzymes on starch. The rate of yeast activity is also governed by temperature
and osmotic pressure, the latter primarily a function of the water content and salt
concentration.

Baker's yeast is of the species Saccharomyces cerevisiae, which is the same species,
commonly used in alcoholic fermentation, and so is also called brewer's yeast. Batch
fermentation mode is used in the process of Baker’s yeast, also known as a closed system.
This fermentation process is carried out at optimum conditions without any addition of
nutrient medium during the fermentation process. As the fermentation process continues,
the pH, cell concentration and substrate concentration as well as product concentration will
change.

Methodology
Microorganism
The Baker’s yeast, Saccharomyces cerevisiae, was used in all experiment. This is hybrid yeast
produced for commercial baker.

Media
Most practical industrial fermentation processes are based on complex media because
of the cost and the choice of the nutrients and the ease of nutrient preparation.
The well formulated defined medium was used in this experiment.

Table 1: Stock culture medium

Compounds Concentration (g/L)
Glucose 50
Yeast extract 5
KH2PO4 2
MgCl2 1
NH4Cl2. H2O 1
Technical agar 15
pH 5.5

Table 2: Batch fermentation medium

Compound Concentration (g/L)

Batch fermentataion
Glucose 50
Yeast extract 5
KH2PO4 2
MgCl2 1
NH4Cl2. H2O 1
pH 5.5

Preparation of inoculum (Stock Culture)

A single colony of pure culture from a slant agar (containing solid gel) (Table 1) is inoculated
into 150 mL of medium in 300 mL shake flask (Table 2) and incubated in an incubator shaker
at 37 °C for 12-14 hours to reach exponential growth phase or log phase.

Batch Fermentation
Batch fermentation was carried out in fermenter with 1L culture volume. The inoculum was
prepared as instructed. A single colony of pure culture from a petri dish, or a slant
tube (containing solid gel) was inoculated into the medium flask (250mL) containing
100mL pre-seed medium. Medium was sterilized by autoclaving at 121°C, 15psi for 20 min
prior to inoculation. The inoculated flask was constantly agitated in a temperature
control flask shaker for 12-14 hours to reach exponential growth phase, or log phase.
The S.cerevisiae culture was then inoculated into sterile fermenter containing 900mL batch
culture medium to start the fermentation. During the fermentation, samples were
withdrawn at time intervals. In normal practice for preparation of inoculums,
subculturing was done repeatedly for several times to ensure that the culture is
acclimated before employed in fermentation.

Sample Analysis
15mL of samples from every shake flask is taken out to measured pH of culture and
absorbance at 610 nm. 1.5 mL sample is allocated for dry weight determination and the
sample was put into eppendoft tube after weight it and centrifuged at 8000 rpm for 10 min.
the steps repeated to the duplicate samples. The supernatant is discarded and the cell pellet
dried at 80 °C overnight and reweighed. The dry cell weigh (X) calculated.

Dry cell weight is also determined using whatman filter paper no.1. The filter paper
dried overnight at 80 °C and placed in a desiccator containing drying agent for cooling
before weight. The pellet resuspended in 20 mL distilled water and filtered through the filter
paper. The cake dried at 80 °C overnight and reweight. The dry cell (X) is calculated.

Result
Value of pH culture and optical density:
Time (hour) pH value Optical Density (A)
0 4.77 0.088
2 3.96 1.610
4 3.70 1.772
6 3.59 1.820
8 3.50 1.837
 10 3.38 1.851

X, cell biomass concentration:

Wt . of dry filter paper +dry cell ( g )−wt . of dry filter paper ( g)
x 1000
sample volume

hour Filter paper Dry cell + filter X

s weight (g) paper (g)

0 0.896 0.896−0.896
× 1000=0
1.5
2 0.896 0.904 0.904−0.896
× 1000=5.333
1.5
4 0.911 0.911−0.896
× 1000=10.000
1.5
6 0.918 0.918−0.896
×1000=14.667
1.5
8 0.920 0.920−0.896
×1000=16.000
1.5
10 0.917 0.917−0.896
× 1000=14.000
1.5

Specific growth rate, µ:

Dx/dt = µX

µ = 1/X (dX/dt)

Hours 1/X dX dt µ
0 1 0 0 0
=0
0
2 1 5.333 2 5.333
=0.1875 0.1875 x = 0.5000
5.333 2
4 1 10.000 4 10.000
=0.1 0.1 x = 0.2500
10.000 4
6 1 14.667 6 14.667
=0.0682 0.0682 x = 0.1667
14.667 6
8 1 16.000 8 16.000
=0.0625 0.0625 x = 0.1250
16.000 8
10 1 14.000 10 14.000
=0.0714 0.0714 x = 0.9996
14.000 10
Specific growth rate:

Hours X (g/L) 1/X dX dt µ

0 0 0 0 0 0
2 5.333 0.1875 5.333 2 0.5000
4 10.000 0.1000 10.000 4 0.2500
6 14.667 0.0682 14.667 6 0.1667
8 16.000 0.0625 16.000 8 0.1250
10 14.000 0.0714 14.000 10 0.9996

Graft:

OD, pH & cell vs time

18

16

14

12

10

0
0 2 4 6 8 10

OD pH cell (g)

Discussion

From this experiment, there is 0 concentration of biomass at 0 hour and the

concentration is began to increase as the hours passes. Then the concentration of biomass
reduces when reach 10 hours of fermentation. It indicates that, the growth of yeast
undergoes lag phase at the 0 hours because it takes time to adaption and the growth
increase as it reached log phase which is started to breed and fed simultaneously, keep on
multiplying in number. The concentration of glucose influences the growth of yeast through
aerobic function which oxygen is required. As the time passes, the medium became limited
due to consumption by yeast and it reduced the growth of yeast. The yeast achieved
stationary or death phase from 8 to 10 hours because the value of concentration of biomass
decreases.

Besides that, the pH value becomes more acidic as growth proceeds as a result of
metabolic by-product which was accumulate in the medium.

Besides that, spectrophotometer was used for monitoring the growth of cell.
Spectrophotometer is an instrument which measures the amount of light of a specified
wavelength which passes through a medium. According to Beer's law, the
amount of l i g h t absorbed by a medium is proportional to the
c o n c e n t r a t i o n o f t h e a b s o r b i n g material or solute present. However, this method
does not directly indicate the cell concentration in the culture (especially when
different medium type, condition, or cell strain was analysed). In this experiment, error of
spectrophotometer reading can result from cell shape and medium colour.
Spectrophotometer does not able to distinguish between the cell and large
particles of debris and viable cells or not.

Conclusion
The growth kinetic of Baker’s yeast in batch production is studied. Culture medium used to
maintain pure culture and developing the fermentation system is familiarized.

References
 Bread processing. Retrieved from https://www.slideshare.net/smrutikudalkar/bread-
37165351 on 1 March 2017.
 Panikov, N.S. 1995.Microbial Growth Kinetics Chapman & Hall, London. 378p
 T r u n , N . J . a n d T r e m p y , J . E . 2 0 0 3 . Fundamental Bacterial Genetics
Blackwell Publishing,Oxford. 287p
Questions

1. Did the pH change during fermentation? If so, what was the cause of the change in
pH? Did the change coincide with different batch growth phases?
The pH generally seemed to decrease. This could be due to release of metabolic
waste by the Baker’s yeast, creating a more acidic environment. Yes, almost all the
growth phases showed a reduction in pH except samples from 4 hours.

2. Comment on ways to improve the experiment.

Reading of spectrophotometer should be more improvised.

3. Explain in detail process involved in bread making using Baker’s yeast.

Bread making process using Baker’s yeast involves mixing, fermenting, kneading,
moulding, baking and cooling the bread. During mixing stage, all the necessary
ingredients to make bread, including the Baker’s yeast are mixed together to evenly
distribute various ingredients and allow development of gluten to give the best
bread possible. Once mixed, the bread is fermented and allowed to rise. This is due
to action of Baker’s yeast which maximizes carbon dioxide production, which raises
the bread. Any large gas holes formed during rising are released by kneading. The
dough is then moulded into desired loaf shape. During the final rising, the loaf fills up
with more bubbles of gas and once this has proceeded far enough, they are
transferred to the oven for baking. The loaf is then placed into a preheated oven to
bake. The high temperature kills the yeast which stops its growth and prevents the
bread from rising. The whole loaf is finally cooled to 35 ℃ before it is cut and
wrapped to avoid any damage to the loaf.
Experiment 3: Calibration of Peristaltic Pump
Objective

To introduce to the calibration technique of peristaltic pump in order to obtain the value of
flow rate and know the important of the flow rate value before running fed-batch and
continuous culture.

Introduction

A peristaltic pump is a type of positive displacement pump used for transferring a wide
variety of fluids and also known as roller pump. This creates a vacuum which allows the fluid
to be drawn through the tubing. The fluid is contained within a flexible tube fitted inside a
circular pump casing (though linear peristaltic pumps have been made). A rotor with a
number of "rollers", "shoes" or "wipers" attached to the external circumference compresses
the flexible tube. As the rotor turns, the part of the tube under compression closes (or
"occludes") thus forcing the fluid to move through the tube. Additionally, as the tube opens
to its natural state after the passing of the cam ("restitution") fluid flow is induced to the
pump. This process is called peristalsis and is used in many biological systems such as the
gastrointestinal tract.

In simpler terms, the peristaltic pump works by squeezing a flexible tube that is filled
with liquid so that the liquid squirts out of the open end of the tube. In water treatment,
peristaltic pumps are used mainly to inject chemical treatment agents into pressurized
water lines. They are commonly used to inject chlorine, soda ash, polyphosphates, and
hydrogen peroxide because the flexible tubing is the only wet part maintenance and clean-
up is simple and convenient. Peristaltic pumps are typically used to pump clean/sterile or
aggressive fluids, because cross contamination cannot occur.
 The peristaltic pump can be regulated to provide high and/or low fluid flows, making
this instrument very lucrative in any field of study. The pump has been designed to be
customized depending on the relevance of the research or the definitive industry for which
it is utilized. Compatibility is one key factor the pump has become so prominent. The tubing
is manufactured such that the elasticity allows it to maintain the same circular cross-
sectional area after extensive application.

There are several reasons the calibration process is a valued asset in any industry.
The process guarantees that instruments are constructed to specifications in accordance
with the national and international measuring standards. This provides the public with a
product of high caliber. It also provides the public with the knowledge that manufacturers
are providing its customers with high quality, systematically tested, and calibrated
instrumentation they can trust. Moreover, the value of the product increases. In order to
maximize the effectiveness of a peristaltic pump the materials selected must be of optimal
quality. Many formulations of these materials are available to industries worldwide. The
wide range of usage allows for this instrument to be one of the most lucrative in the
transferring of viscous and or liquid fluids.

Methodology
Material that have used was beakers, volumetric flask, stop watch, tubing, peristaltic
pump and distilled water.
Peristaltic pump was set up and it was tested with different time by recording time
from 1-minute intervals until 6 minutes by using distilled water using stopwatch. T volume
of solution and the flow rates were calculated the speed was fixed.
Then the pump speed was increased every 1 minute from 2, 4, 6, 8 and 10. The
volume of distilled water and the flow rate were calculated.
volume (ml)
Flow rate=
time( min)
Result
Table 1: Every 1 minute the volume of distilled water recorded at fixed speed, 6
Pump speed Volume of solution(ml) Flow rate (ml/min)
 1 7.0 7.0
=¿7.0
1
2 15 15
=¿7.5
2
3 23 23
=¿7.7
3
4 31 31
=¿7.8
4
5 39 39
=¿7.8
5
6 47 47
=¿ 7.8
6

Flow Rate Against Time

8.0

7.8

7.6
Flow rate (ml/min)

7.4

7.2

7.0

6.8

6.6

6.4
0 1 2 3 4 5 6 7

Pump speed

The slope value of graph,

7.8−7.38
gradient , m=
5−2
= 0.14

Table 2: every 1 minute, volume of distilled water recorded

Pump speed Volume of solution (ml) Flow rate (ml/min)

2 1.6 1.6
4 4.9 4.9
 6 7.9 7.9
8 10 10
10 11 11

Flow rate vs speed

12

10
Flow rate (ml/min)

0
1 2 3 4 5 6 7 8 9 10 11

Pump speed

The slope value of graph,

12.0−7.0
gradient , m=
10−6
= 1.25

Discussion:

The results from graph show the flow rate of distilled water were directly
proportional to the speed of pump even though the volumes were different. This is because
the tubing size, specifically the inside diameter or ID, is directly proportional to the flow rate
in all pump heads. Larger tubing sizes and larger diameter rotors have a greater "pillow"
volume (the fluid space in the tubing between adjacent rollers in the pump head). This
volume determines the flow per revolution of the pump head. Although the speed of pump
was same, small tubing did not give a consistent flow rate compared to that of large tubing.
This could be due to systematic error in the peristaltic pump itself or random error in
recording the time and amount of fluid. It can be said that the flow rate may be more
consistent when large tubing is used.

Peristaltic pump which plays important role to pump various fluid, need to be
maintained so that the pump produces effective function and achieve higher life time
application. There are few ways in maintaining the pump in appropriate condition which
include the tubing maintenance. When using new tube sets, it is advisable to run the pump
at full speed for 20 min to 30 min to allow the pump tube to stretch into its final shape.
Besides that, covered container or one with a small surface area must be use for calibration
of pump at very low flow rates as this will prevent evaporation that can take place easily. It
is also recommendable to use the same liquid that typically used under actual operating
conditions. Both the inlet and outlet pressures of the pump should be considered as
pressures acting on the tubing will directly affect the peristaltic pump life.

Conclusion

The flow rate is directly proportional to the speed of pump, either in constant speed or
various speed. The gradient for fixed speed graph is 0.14 and 1.25 for various speed graph.

Reference

 Lindsey, J. (2011). Pump Troubleshooting: Cleaning and Maintenance. Retrieved

November 23, 2016, from http://www.scinomix.com/sci-pump-maintenance.
 Marlow, W. (2016). Peristaltic and Sinusoidal Pumps. Retrieved November 23, 2016,
from http://www.watson-marlow.com/gb-en/support/how-it-works.
 Thomas, Walker. (1966). Fluid Motions in Peristaltic Pump. Retrieved November 23,
2016, from http://dspace.mit.edu/handle/1721.1/17282.
Experiment 4: Harvesting Technique In Downstream

Objectives

 To learn the harvesting techniques such as sedimentation, centrifugation and

filtration in downstream processing
 To study the effectiveness of the different methods on solid-liquid separation of
yeast Saccharomyces cerevisiae

Introduction

Downstream processing is the part of a bioprocess where the cell mass from the
upstream is processed to meet purity and quality requirements. The isolation and
purification of a biotechnological product to a form suitable for its intended use, is termed
DSP. Typically, this means recovery of a product from an aqueous solution. The products of
biotechnology include whole cells, organic acids, amino acids, solvents, antibiotics, industrial
enzymes, therapeutic proteins, vaccines etc. As these products vary greatly in size and
nature, different separation principles are required for their recovery and purification.
 There are few stages in order to bring a product from its natural state as a
component of a tissue, cell or fermentation broth through progressive improvements in
purity and concentration. The first stage in the downstream processing are solid-liquid
separation phase. This step involves the separation of whole cells (cell biomass) and other
insoluble ingredients from the culture broth. Several methods are in use for solid-liquid
separation which are flotation, flocculation, filtration and centrifugation. While, the
technique of centrifugation is based on the principle of density differences between the
particles to be separated and the medium which is why this process is mostly used for
separating solid particles from liquid phase.

Besides that, filtration also the most commonly used technique for separating the
biomass and culture filtrate. The efficiency of filtration depends on many factors such as the
size of the organism, presence of other organisms, viscosity of the medium, and
temperature. Several filters such as depth filters, absolute filters, rotary drum vacuum filters
and membrane filters are in use. Depth Filters are composed of a filamentous matrix such as
glass wool, asbestos or filter paper where the particles are trapped within the matrix and
the fluid passes out. In Membrane Filters, membranes with specific pore sizes can be used.
However, clogging of filters is a major limitation. There are two types of membrane
filtrations which are static filtration and cross-flow filtration where cross flow filtration
reduces the clogging process and hence better than the static filtration.

Other than that, sedimentation method is the process of allowing particles in

suspension in water to settle out of the suspension under the effect of gravity. The particles
that settle out from the suspension become sediment, and in water treatment is known as
sludge. When a thick layer of sediment continues
to settle, this is known as consolidation. When a
thick layer of sediment continues to settle, this is
known as consolidation. When consolidation of
sediment, or sludge, is assisted by mechanical
means then this is known as thickening. There is
a variety of methods for applying sedimentation
and include: horizontal flow, radial flow, inclined plate, ballasted floc and floc blanket
sedimentation.

Downstream processing is usually considered a specialized field in biochemical

engineering, itself a specialization within chemical engineering, though many of the key
technologies were developed by chemists and biologists for laboratory-scale separation of
biological products. This process also applies to large-scale production of biological products
too, which are nowadays extensively used for varied applications like medical diagnostics,
research and development, biotransformation and also in food, pharmaceutical and
cosmetic industries.

Material and Method

Microorganism

The Baker’s yeast is saccharomyces cerevisae is used in all experiments. This is hybrid yeast
produced for commercial Baker.
Media

Compounds Concentration (g/L)

Glucose 50
Yeast extract 5
KH2PO4 2
MgCl2 1
NH4Cl2. H2O 1
Technical agar 15
pH 5.5
Table 1: Stock Culture Medium

Table 2: Batch fermentation medium

Compound Concentration (g/L)

Batch fermentataion
Glucose 50
Yeast extract 5
KH2PO4 2
MgCl2 1
NH4Cl2. H2O 1
pH 5.5

Preparation for inoculum

A single colony of pure culture from a slant agar (containing solid gel) (table 1) is
inoculated into the 150 mL of medium in 300 mL shake flask (table 2) and incubated in an
incubator shaker at 37 °C for 12-14 hour to reach exponential growth phase or log phase.

Batch fermentation

Batch fermentation carried out in 300 mL shake flask containing 150 mL

fermentation medium. Batch fermentation is started by inoculate a 10% of inoculum into
the medium flask (table 2). Medium is sterilized by autoclaving at 121 °C, 15 psi for 20
minutes before inoculation. The inoculated flasks are constantly agitated in an incubator
shaker for 24h.

Sample analysis
  Sedimentation

15 mL of sample was taken out from a shake flask and put into a test tube. Then the
sample is leaved to stand in normal condition and wait till the cells sediment at the bottom
of the test tube.

 Centrifugation

15 mL of sample was taken out from a shake flask and put into a centrifuge bottle.
The sample centrifuged at 4000 rpm for 15 minutes. Then the separation of cells and liquid
after centrifugation is observed.

 Filtration

15 mL of sample was taken out from a shake flask and filtered through 0.2 µm nylon
membrane filter using vacuum pump. Then the separation of cells and liquid after
centrifugation is observed.

Result

filtration pump
Centrifugation

Filtration conventional

Discussion

From the experiment, the sedimentation method took about a day for the
Saccharomyces cerevisiae to settle down in the bottom of the tube. The medium was still
turbid even after the harvesting. This indicates that the medium was not harvest completely
and there is possibility for some of the cells to be suspended in the medium.

Centrifugation done using a centrifuge machine for 15 minutes. The separation of

cells and liquid can see clearly, and the medium was less turbid compared to medium of
sedimentation after harvesting. Most of the cells were pelleted to the bottom of tube by

gravitational force. Centrifugation can be viewed as an extension of the conventional

filtration and gravitational sedimentation. Centrifugal force replaces the pressure force in
filtration and gravitational force in sedimentation.

Filtration on the other hand had the clearest medium after harvesting but it took
about 18 minutes when done in conventional method using filter paper. Filtration was
quicker when done with a vacuum pump which took only around 2 minutes. Therefore, the
most efficient harvesting method of S. cerevisae is filtration as it has the clearest medium
indicating close to complete removal of cells from medium and it can be done quick when
using vacuum pump.

Conclusion

The filtration method is more effective than centrifugation and sedimentation of solid-liquid
separation of Saccharomyces cerevisiae.
Reference

 Bracke, M. (2013). Large-Scale Algal Cultivation, Harvesting and Downstream

Processing. Journal of Extracellular Vesicles, 3(1), 35-49. Retrieved 4 November,
2016, from http://www.journalofextracellularvesicles.net/index.php/jev/article/view/
24858.
 Lim, J. (2013). Methods in Harvesting of Downstream Processing. Journal of Science
and Biotechnology, 31(6), 862-876. Retrieved 4 November 2016, from
https://www.ncbi.nlm.nih.gov/pubmed.
 Mayo, J. (2015). Centrifuge and Filter/Dryer Product Comparison. Retrieved 4 November,
2016, from http://www.ddpsinc.com/blog-0/centrifuge-and-filter/dryer-product-
comparison.

Experiment 5: Detrmination of KLa

Objective
To understand the method to determine KLa value (non-fermentative method).

Introduction

KLa is known as the volumetric mass-transfer coefficient that describes the efficiency
with which oxygen can be delivered to a bioreactor for a given set of operating conditions.
In cell culture, oxygen is a key substrate for growth, production, and maintenance activities.
Cells obtain their oxygen in free and non-compound forms, called dissolved oxygen (DO).
One of the most important functions of bioreactors is providing dissolved oxygen to cells
continuously through a process called aeration. Aeration in the bioreactor typically occurs in
two conditions whereas, when oxygen diffuses through overlay to the cell culture medium
interface and oxygen from the spargers dissolves in the cell culture through convection with
the help of agitation.

Agitation disperses the oxygen bubbles and promotes mass transfer of the gas
bubbles through the gas-liquid (cell culture medium) interface. The rate of oxygen transfer
(OTR) from gas to liquid interface is a function of physicochemical properties of the cell
culture medium, the geometrical parameters of the bioreactor, and presence of cells.

Diagram of a gas bubble in liquid, showing how the bubble is released, solubilized, and transferred to
a cell.

However, dissolved oxygen (DO) is often the limiting substrate in fermentation and
cell-culture systems. 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. Therefore,
measurements of KLa provide
important information about a bioprocess or bioreactor. These determinations ensure that
processing conditions are such that an adequate supply of oxygen is available for
proliferation of cells. The KLa value can also be used to optimize control variables over the
lifecycle of a bioprocess. Such optimization would be based on the oxygen demand at
various points in the process and growth phase of the biological. Criteria for optimization
may be product yield, power consumption, or processing time. There are two methods can
be employed to achieve this lowering of the dissolved oxygen concentration; non-
fermentative and fermentative.

In non-fermentative method, the KLa value can be determine by lowering the

concentration of oxygen so that the solution is free of oxygen gas. Then, aeration initiated at
a constant air flow rate and the dissolved oxygen gas tension (DOT) is monitor. While, in
batch fermentation, the air supply is shut off and the agitation speed set at 50 rpm to
decrease the DO about 20% saturation. Then the air supply open and increase agitation
speed to the set level (100, 300, 600 rpm). The rate of oxygen transfer from the buble air to
the liquid phase may be described by the equation: dC L/dt = KLa(C* - CL). The KLa value is
equal to the slope of graph ln (C*- CL) against t which produce straight line.

CL = concentration of dissolved O2 in fermentation broth, moles dm3

t = time, h
dCL/dt = changes in O2 concentration over a time period
C* = saturated dissolved O2 concentration, moles dm3

Material and method

The fermentation process held in 2.0 litre bioreactor. Distilled water, oxygen and
nitrogen gas were used. Time was recorded using stopwatch.

Results
Table 1: Agitation speed for 100rpm
Time (min) DO (%) C* - CL ln (C*- CL)
1 18.8 100 – 18.8 = 81.2 4.397
2 30.7 100 – 30.7 = 69.3 4.237
3 45.3 100 – 45.3 = 54.7 4.000
4 57.2 100 – 57.2 = 42.8 3.756
5 68.4 100 – 68.4 = 31.6 3.453
6 74.7 100 – 74.7 = 25.3 3.230
7 79.2 100 – 79.2 = 20.8 3.035
8 80.5 100 – 80.5 = 19.5 2.970
 9 84.6 100 – 84.6 = 15.4 2.734
10 88.7 100 – 88.7 = 11.3 2.425
11 91.6 100 – 91.6 = 8.4 2.128
12 94.7 100 – 94.7 = 5.3 1.668
13 95.8 100 – 95.8 = 4.2 1.435
14 96.3 100 – 96.3 = 3.7 1.308
15 96.3 100 – 96.3 = 3.7 1.308
Graph

ln (C*- CL) against ti me (min)

5
4.5
4
3.5
ln(C*- CL)

3
2.5
2
1.5
1
0.5
0
0 2 4 6 8 10 12 14 16

Time (min)

The slope value of graph,

3.035−3.756
gradient , m=
7−4
= - 0.240

KLa = 0.240 x 60 = 14.4 h-1

Time (min) DO (%) C* - CL ln (C*- CL)

1 48.2 100 – 48.2 = 51.8 3.947
2 80.5 100 – 80.5= 19.5 2.970
3 91.8 100 – 91.8 = 8.2 2.104
 4 93.5 100 – 93.5 = 6.5 1.872
5 95.9 100 – 95.9 = 4.1 1.411
6 96.0 100 – 96.0 = 4.0 1.386
7 96.2 100 – 96.2 = 3.8 1.335
8 96.2 100 – 96.2 = 3.8 1.335

Table 2: Agitation speed for 300rpm

Graph

Y-Values
4.5
4
3.5
3
ln (C*- CL)

2.5
2
1.5
1
0.5
0
0 1 2 3 4 5 6 7 8 9

Time (min)

The slope value of graph,

1.500−3.250
gradient , m=
6−1
= - 0.350

KLa = 0.350 x 60 = 21 h-1

Table 3: Agitation speed for 600rpm

Time (min) DO (%) C* - CL ln (C*- CL)

 1 66.8 100 – 66.8 = 33.2 3.503
2 90.4 100 – 90.4 = 9.6 2.262
3 93.8 100 – 93.8 = 6.2 1.825
4 95.1 100 – 95.1 = 4.9 1.589
5 95.7 100 – 95.7 = 4.3 1.459
6 95.7 100 – 95.7 = 4.3 1.459

Graph

ln (C*- CL) against ti me (min)

4

3.5

2.5
ln (C*- CL)

1.5

0.5

0
0 1 2 3 4 5 6 7

Time (min)

The slope value of graph,

1 . 45 9−2.250
gradient , m=
5−3
= - 0.3955

KLa = 0.3955 x 60 = 23.73 h-1

Table 4: KLa value for agitation speed of 100, 300 and 600(rpm)
 Agitation speed (rpm) KLa (h-1)
100 14.4
300 21
600 23.73

Graph

KLa (h-1) against Agitation Speed (rpm)

25
23.73
21
20

15 14.4
KLa (h-1)

10

0
100 300 600

Agitation speed (rpm)

Discussion

In non-fermentative method, the KLa value can be determine by lowering the

concentration of oxygen so that the solution is free of oxygen gas. Then, aeration initiated at
a constant air flow rate and the dissolved oxygen gas tension (DOT) is monitor. While, in
batch fermentation, the air supply is shut off and the agitation speed set at 50 rpm to
decrease the DO about 20% saturation. Then the air supply open and increase agitation
speed to the set level (100, 300, 600 rpm). The rate of oxygen transfer from the buble air to
the liquid phase may be described by the equation: dCL/dt = KLa(C* - CL).

Where CL is the dissolved oxygen concentration and C* is the saturated dissolved oxygen
concentration in the solution. Aeration to an active culture is briefly turned off and the unsteady-state mass
balance of oxygen was tracked. 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.

There are two possible cause of error in this method. First, when the air supply is
turned off, the dissolved oxygen concentration at point B has to be above the critical
dissolved oxygen concentration (Critical). If the dissolved oxygen concentration is below C ritical,
anaerobic metabolism will occur rather than the aerobic metabolism. This method also
requires an oxygen probe with fast response time which will result in less accurate outcome
if neglecting these facts.

Transfer of oxygen from a gas phase to a liquid phase is complicated by presence of

cells, product formation, ionic species, and antifoaming agents. These can alter bubble size
and liquid film resistance, which affect oxygen absorption into water. Therefore, it is
important to have a reliable method for measuring KLa in fermentation system.

Relationship between agitation speed and dissolved oxygen concentration is the

time to reach saturated level of dissolved oxygen concentration shorter if the agitation
speed (rpm) is faster.

Relationship between dissolved

oxygen concentration and time in
dynamic gassing out method

Point A shows level of DO before it

was consumed (air supply off). Point B represent air is pumped into the culture and the
dissolved oxygen concentration increases as a function of time. Point C represent steady-
state value.
Conclusion

The KLa value for 100 rpm is 14.4 KLa h-1, 300 rpm is 21 KLa h-1 and 600 rpm is 23.73 KLa h-1.
The KLa value increased as the agitation speed increased.

Reference

 Jane K., Measuring kLa for better bioreactor performance, Article of Vendor Voice.
Retrieved
fromURL:http://www.bioprocessintl.com/wpcontent/uploads/2014/07/BPI_A_1210
03AR10_O_176218a.pdf
 7 factors that affect oxygen transfer to cells in bioreactors, Retrieved from URL:
https://www.gelifesciences.com/en/us/solutions/bioprocessing/knowledge-center/7-
factors-that-affect-oxygen-transfer-to-cells-in-bioreactors

 Alvin C., (2016), Measurement of KLa It is extremely difficult to measure both ’KL‘
and 'a' in a fermentation and, therefore, the two terms are generally combined in
the. Retrieved from URL: https://slideplayer.com/slide/5894959/