ABSTRACK

This experiment is to study the growth kinetics of microorganism in shake flask. Erlenmeyer
flask is used in this experiment to grow microorganisms. Escherichia coli (E.Coli) is grown in Terrific
Broth (TB) medium at 350 rpm and 37C. They were fermented for 24 hours. Throughout every 1hour
for 4 hour then 2 hours for rest 20 hours, cell is taken out to obtain the value of absorbance by using
spectrophotometer. Cell dry is then obtained after the mass concentration inside the flask is dried
overnight. As for the optical density analysis, the absorbance reading from the spectrophotometer is
taken. The cell dry weight, in the other hand, is taken after the mass concentration is being dried
overnight in the oven. The weight of the tube which contains the biomass before and after the drying
process is recorded to get the cell dry weight. Then, the obtained value can be turned into graph to
observed the changing in growth kinetics of the cell . Graph of growth curve including lag, log,
stationary and death phases. The others parameters that we studied includes cell concentration,
absorbance reading, and cell dry weight. This experiment get a a few of human error when taking the
reading for absorbance and mass of cell growth. But after we can observe the growth of microorganisms
and construct the growth curve and determine the Monod parameters, the value of Monod parameter

of maximum growth gate max which is equal to the slope of the graph is 0.2443h-1, maximum
net growth rate,net is 0.2747 h-1, specific growth rate, net is 0.7985 h-1 and mass doubling time,
is 0.8681 h. This experiment was consider succeed .

INTRODUCTION

When microbial cells are inoculated into a batch reactor containing fresh culture
medium and their increase in concentration is monitored, several distinct phases of growth can
be observed. There is an initial lag phase, which is of variable duration. This is then followed
by the exponential growth phase, where cell number (and dry weight) increases exponentially.
This is also referred to as the logarithmic phase, the name arising from the common method of
plotting the logarithm of cell number against time. Following this is a short phase of declining
growth, and then the stationary phase. Here the cell numbers are highest. Finally the cell
numbers decline during the death phase.
Fermentation process such as batch, continuous and fed-batch processes. In this
experiment, the shake flask fermentation was used. The shake flask fermentation is an example
of batch fermentation. In shake flask, the culture flask usually Erlenmeyer flask is being used
to place and growing the microorganisms. It is a small scale equipment which equivalent to
stirred tank bioreactor. It is the cheapest and easiest way to culture microorganism aerobically,
in small volumes of nutrient broth.
The cultures are incubated at certain temperature 37C and shaking frequency 350 rpm
for 4hours in an incubator shaker to achieve a required growth rate. The shaking agitates the
medium and the culture to keep the mixture relatively homogeneous and also to ensure aeration,
creating an aerobic condition. In batch culture, it is closed environment that means there is
neither input supplied nor output generated throughout the fermentation. The medium culture
is initially inoculated with the microorganism. The growth keeps increasing until at certain
extent, the grow this inhibited because of the decreasing substrate concentration and the
presence of toxic metabolites.
In order to prevent any contamination to the culture, shake flask must be plugged. The
plug has to prevent airbone microorganism from getting into the medium while at same time
allow free flow of air into the flask. Different plug can be made of cotton-wool, glass wool,
polyurethane foam, gauze or synthethic fibrous material.
The microorganism that we used to study in this experiment is E .coli . There are many
specific media for growth of E. coli but in this experiment we used Terrific Broth (TB) media
because it is the most commonly used medium in molecular biology for E. coli cell culture .The
relationship between the specific growth rate () of a microbial population and the substrate
concentration (s), is an indispensable tool in all fields of microbiology, be it physiology,
2

genetics, ecology, or biotechnology, and therefore it is an important part of the basic teaching
of microbiology (Kovrov-Kovar. K, et. al, 1998) .

OBJECTIVE

1. To study/observe the growth kinetics of microorganism in shake flask experiment.

2. To construct a growth curve including lag, log, stationary and death phases.
3. To determine the Monod parameters.

THEORY

Rate of microbial growth is known as

net

1 1

[ ]

Yield coefficients

/ =

[g cells/g substrate]

Mass doubling time (d)

ln 2

[h]

Monodequation
=

= net when =0
= Endogenous metablosim
= Saturation constant
= =
When =

, S<<

Figure 1:Growth curve of microorganism based on cell number analysis

1. During lag phase, bacteria adapt themselves to growth conditions. It is the period where
the individual bacteria are maturing and not yet able to divide. During the lag phase of
the bacterial growth cycle, synthesis of RNA, enzymes and other molecules occurs.
2.

The log phase is a period characterized by cell doubling. The number of new bacteria
appearing per unit time is proportional to the present population. If growth is not limited,
doubling will continue at a constant rate so both the number of cells and the rate of
population increase doubles with each consecutive time period.

3. The stationary phase is the point where the growth rates has declined to zero. Stationary
phase results from a situation in which growth rate and death rate are equal. The number
of new cells created is limited by the growth factor and as a result the rate of cell growth
matches the rate of cell death. The result is a smooth, horizontal linear part of the
curve during the stationary phase.
4. At death phase (decline phase), number of bacteria became decline because death of
bacteria. This could be due to lack of nutrients, a temperature which is too high or low,
or the wrong living conditions.

MATERIAL AND APPARATUS

1. Microbe:Escherichia Coli .
2. Shake flask (250mL flasks and 1000 mL flasks)
3. Eppendorf tubes / falcon tube (1.5 mL)
4. Cuvettes (spectrophotometer)
5.

Incubator Shaker

6. Refrigerated Centrifuge
7. Media (for specific microbe)
8. Ethanol (70% ethanol for swabbing for sterility)
9. Spectrophotometer (wavelength: 600nm)
10. Bunsen burner for sterility
11. Graduated Flask for measuring media (1000mL, 100mL, 10mL)
12. Laminar Flow hood for sterility
13. Biochemical Analyzer
14. HPLC for product measurement like ethanol
15. Cotton plugged

PROCEDURE

(i) Preparation of media

a) Terrific Broth (TB) preparation
1. The recipe as stated at the bottle is followed.
2. The media is autoclaved at 121C for 20 minutes
3. Glycerol and media has been autoclaved together.4. pH reading should be near 7 as
the media is a readied phosphate buffer solution

(ii) Preparation of cell culture

a) Seed culture preparation (inoculums)
1. 5 loops of grown E coli is taken on agar plates and added to the sterilized media of 150mL
in 1000mL shake flask. (you may need 2 of 1000mL shake flask to ensure enough inoculums
needed)
2. Sterility must be sustained during transfer.
3. The media is grown at 350 rpm for 4 hours and it is assumed as exponential growth of E
coli.
4. At this stage, the seed cultures are assumed to be at its most active condition.
5. OD reading is taken for seed culture using spectrophotometer during this time.

b) Main experiment
1. 10% of inoculum to the main experiment media is transferred using aseptic technique.(For
instance, if the working volume is 150ml, therefore, 10% of inoculum would be15mL of seed
culture needed).
2.The shake flask is then capped (cotton plugged) and swabbed with 70% ethanol before
incubation in a thermostated rotary shaker at required rotational speed and temperature for 24
hours.
(iii) sampling
1. Required amount of sample is transferred into the sampling tube with interval time for
every hour or every 2 or 3 hours.
2. 5 mL of sample is withdrawn every time sampling is done during fermentation for
measuring optical density (OD) and total cell number (biomass concentration: g/L).

3. Table below is referred for planned usage of sample volume:

No.

Sample

Volume (L)

Use for

Name
1

OD

1000

Optical measurement using spectrophotometer

CDW

1500

For Cell Dry Weight measurement

iv) Absorbance Analysis (Optical Density) (OD)

1. 1 mL of sample is transferred into a cuvette and the optical density measurement ismade
using a spectrophotometer with the wavelength set at 600nm.
2. The spectrophotometer is calibrated to zero by blank consisting 1 mL chosen media.
3. This method is used to measure cell growth; higher number of cells means more
absorbance, which is caused by low transmittance and vice versa.

v) Cell Dry Weight. (Biomass Concentration) (X) (g/L)

1. Dried centrifuge tubes is weighted and noted as initial mass.(empty container)
2. 1.5 mL sample is added to weighted centrifuge tube.
3. The sample is centrifuged at 10,000 rpm and at T of 4C. for 20 minutes.
4. The supernatant is taken out and washing with distilled water and centrifuging may be
repeated.
5. The centrifuge tube is dried(left with biomass only) in oven at 80C for overnight.
6. The dried centrifuged tubes is left in desiccators for half an hour.
7. The centrifuge tube weighted and note this as final mass (with biomass = Cell Dry Weight).
Cell Dry Weight = Final mass Initial mass

RESULT AND CALCULATION

Time , t (h)

Absorbance (real OD)

Diluted

Not Diluted

Empty

Dried

Cell mass

centrifuge

centrifuge

concentration ,

m1 , (g)

+sample , m2

X=(m2-m1)/0.002L

(g/L)

(g)
0

-0.156

0.066

1.0847

1.0865

0.9

-0.109

0.416

1.0680

1.0720

2.0

0.188

1.739

1.0877

1.0933

2.8

0.433

2.014

1.0272

1.0336

3.2

0.484

2.225

1.0818

1.0902

4.2

0.583

2.269

0.9493

0.9611

5.9

1.542

2.397

1.0193

1.0355

8.1

10

1.607

2.494

1.0731

1.0855

6.2

12

1.863

2.571

1.0771

1.0862

4.6

14

2.063

2.644

1.0724

1.0812

4.4

16

1.135

2.681

1.0741

1.0826

3.6

18

0.996

2.603

1.0677

1.0760

4.2

20

1.334

2.753

1.0699

1.0793

4.7

22

1.210

2.606

1.0631

1.0721

4.5

24

2.377

2.688

1.0820

1.0912

4.6

Growth Curve
time
0
1
2
3
4
6
8
10
12
14
16
18
20
22
24

Absorbance
-0.156
-0.109
0.188
0.433
0.484
0.583
1.542
1.607
1.863
2.063
1.135
0.996
1.334
1.410
2.377

Absorbance nm vs time

absorbance (nm)

2.5
2
1.5
1
0.5
0
0

10

15

20

25

30

-0.5

time
Graph 1: Absorbance (nm) for optical density against time

Cell mass concentration,X against time

time

X
0
1
2
3
4
6
8
10
12
14
16
18
20
22
24

0.9
2.0
2.8
3.2
4.2
5.9
8.1
6.2
4.6
4.4
3.6
4.2
4.7
4.5
4.6

cell mass concentration,X

Cell mass concentration,X against time

9
8
7
6
5
4
3
2
1
0
0

10

time,h

15

20

25

30

Graph 2:Cell mass concentration,X against time,h

Cell dry weight

= m2 m1
= 1. 0865g 1.0847g
= 0.0018g
Cell mass concentration, X

=
=

volume of sample = 0.002L

0.0018
0.002

= 0.9g/L
10

Exponential growth phase against time

time

Ln(X/X0)
0
0.7985
1.1350
1.2685
1.5404
1.8803
2.1972

0
1
2
3
4
6
8

Ln(X/X0) against time

2.5
y = 0.2443x + 0.4225
R = 0.9015

ln(X/Xo)

1.5
1
0.5
0
0

time
Graph 3 : ln(X/Xo) against time

maximum growth rate, max

the value of max which is equal to the slope of the graph is 0.2443h-1

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Growth Rate, net

time

ln
-0.1054
0.6931
1.0296
1.1632
1.4351
1.7750
2.0919
1.8245
1.5261
1.4816
1.2809
1.4351
1.5476
1.5041
1.5261

0
1
2
3
4
6
8
10
12
14
16
18
20
22
24

lnX vs time
2.5
2

y = 0.0311x + 1.0154
R = 0.2298

lnX

1.5
1
0.5
0
0

10

15

20

25

30

-0.5

time
Graph 4 : LnX against tim(h)

Maximum net growth rate, net =

=

=8=0
=8 =0
ln 8.10.9
80

= 0.2747 h-1

12

Growth Rate, net

=
=

1
1
20.9
10

= 0.7985 h-1

Mass doubling time,

ln 2

ln 2

= 0.7985
= 0.8681 h

DISCUSSION

In this experiment , we selected E.Coli as the cell and it is cultivated in shake flask . The
flask is shaken during cultivation ensuring it is in homogenous. The duration for this experiment
is 24 hours. The cell is taken out every 1 hour for first 4 hours, then every 2 hours for the rest
20 hours. We took the cell out to analyze the concentration of the cell (g/L) and cell dry weight.
In order to analyze the concentration of the cell inside the flask, absorbance reading for
the optical density is taken from the spectrophotometer. The higher the absorbance reading
means higher number of cell presence inside the flask at a particular time. As for this
experiment, the diluted absorbance reading is increase from the beginning of the experiment
until the 14st hour and decrease slightly at the 16th hour until 18th hour and increase again until
24thhour. For non-diluted absorbance reading is increasing from begining of the experiment to
24th hour. It can be explained that the number of cell increase throughout the cultivation
indicating that the cell is growing. In the other hand, the decrease in cell number in 16th hour
for diluted absorbance is indicating that due to error when take the reading of absorbance.
During growth stage there are feed/back mechanisms that regulate the bacterial enzymes
involved in key metabolic steps to enable the bacteria to withstand starvation. There is much
turnover of protein for the culture to cope with this period of low substrate availability.for nondiluted absorbance, the higher the absorbance reading means greater number of cells inside the
13

flask at a particular time. Although it did not increase the absorbance reading steadily, the
pattern roughly shows an increase from the beginning of the experiment until the end of the
experiment. From this observation , we can explain that the number cell increasing throughout
the cultivation indicating that the cell is growing. In cell growth, the cell will go through several
phases like lag, exponential, deceleration, stationary and death phase.
The cell sample is analyze by taking the dry weight of the cell. In this method, the cell
is being taken out from cultivation flask and transferred into viral tube. The tube is the being
centrifuged to separate the supernatant with the cell. Then , the supernatant was removed and
the remaining cell ware dried inside oven for 12 hours and then take the reading of cell dry
weight. The cell dry weight is increased from 0th hour until 10th hour and gradually decreased
from the 12th hour to 18th hour and 20th hour until 24th hour, the values are inconsistent in
increasing and decreasing. This error occurs due to the ways students in separating the
supernatant from the samples . The students might have extracted and removed the cells along
with the supernatant. This human error can cause inaccurate in weight reading. The value of
Monod parameter of maximum growth gate max which is equal to the slope of the graph is
0.2443h-1, maximum net growth rate,net is 0.2747 h-1, specific growth rate, net is 0.7985 h-1
and mass doubling time, is 0.8681 h.

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CONCLUSION

This

experiment

was

carried

out

to

investigate

the

growth

kinetics

of

microorganism in shake flask . Microorganisms will go through several phases during their
growth, analysts has been made to obtain the kinetics of the cell and duration for each phase.
we also want to construct a growth curve including lag, log, stationary and death phases. The
others parameters that we studied includes cell concentration, absorbance reading, and cell dry
weight. From the graph plotted and equations that have been given, we succeeded in calculating
the Monod parameter of maximum growth rate, specific growth rate and mass doubling time
but the calculating is not well accurate due to error in reading. Some parameters that cannot be
calculated because of technical problem that we cannot avoid . This experiment succeed.
In conclusion, the microbial culture in batch culture system (shake flask system) goes through
a lag phase, exponential growth phase, decelerating growth phase, stationary phase and
sometimes the death phase depends on the end product desired. The substrate concentration in
the culture medium and growth parameters, such as glucose concentration changes
correspondingly throughout the growth phases. Thus, the physiology of the microorganisms is
always in a transient stage, subjected to a continually changing culture conditions.
Consequently, product formation is confined to a certain period of cultivation, for example
antibiotics would only be produced in the decelerating and stationary growth phases.

RECOMMENDATION

Aseptic technique must be practised when handling biomass concentration to avoid any
contamination.

Cuvette must be wiped cleanly to prevent any scratch that would affect the
spectrophotometer reading during protein test.

This experiment must be carried out under the laminar flow to prevent any
contamination to the culture.

Dispose of all contaminated materials in appropriate containers.

Make a dilution if the reading of spectrophotometer is more than 1 and take the Optical
Density reading again.

15

The supernatant of cell concentration should be taken out carefully without any taking
out of the biomass.

REFERENCES

1. Kovrov-Kovar, K., & Egli, T. (1998). Growth Kinetics of Suspended Microbial Cells:
From Single-Substrate-Controlled Growth to Mixed-Substrate Kinetics. Retrieved on
October 4, 2015 from http://mmbr.asm.org/content/62/3/646.full .
2. Suresh, S. et al. (2009) Techniques for oxygen transfer measurement in bioreactors: a
review. J. Chem. Technol. Biotechnol. 84, 10911103
3. Crueger W and Crueger A., (1990). Biotechnology: A Textbook of Industrial
Microbiology. Sinauer Associates, Sunderland Massachusetts.
4. Bernstein, A.M., et al., Dietary protein sources and the risk of stroke in men and
women. Stroke, 2012. 43(3): p. 637-44.
5. Manual laboratory of Chemical Reaction Engineering. UiTM Shah Alam.

16

APPENDICES

Figure 1: Spectrometer

Figure 2 : Taking sample

Figure 3 : Remove supernatant

17

Figure 4: Incubator Shaker YSI

18