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INTRODUCTION

Fermentation can be carried out as batch, continuous and fed-batch processes. In this
experiment, the shake flask fermentation is being used. Shake flask fermentation is the
example of batch fermentation. In shake flask, the culture flask usually Erlenmeyer flask is
being used to place and growing the microorganisms. It is the cheapest and easiest way to
culture microorganism aerobically, in small volumes of nutrient broth. It is a small scale
equipment which equivalent to stirred tank bioreactor.I n this experiment the microorganism
used is E.coli.The growth curve of E coli in a batch culture is been studies. The graph below
shows the phases of a typical growth curve of E coli in a batch culture.

Figure 1 Phases of a typical growth curve of E.coli in a batch culture

Lag phase is the time between inoculation and reaching the maximum growth rate.
There are two sub phases in the lag phase. In the first phase, there is no growth identified
whereas in the second sub phase which is also known as acceleration phase, there is a
constant growth begins.
The second phase is exponential phase. The cells begin to proliferate with their
maximum growth rate. The doubling time of E.coli is 20 minutes. Exponential phase is
important for determining the maximum growth rate, and doubling time, d since the growth
at this time is the most constant and ideal.
Retardation phase is the period between exponential and stationary phase, or in other
words, the phase before the growth becomes stationary. Among the factors that inhibit the

growth are reduced dissolved oxygen tension (DOT), substrate concentration, pH changes
and presence of inhibiting metabolites.
After retardation phase, the growth phase enters stationary phase where the growth
becomes constant for a period of time before it declines. Finally, the growth declines from its
stationary phase due to the cells lysation. This is indicated by the decrease of the viable cell
number

OBJECTIVES

To study and observe the growth kinetics of microorganism in shake flask

experiment.
To construct a growth curve including lag, log, stationary and death phases.
To determine the Monod parameters of maximum growth rate (max), yield of
substrate (Yx/s), mass doubling time (td), saturation constant (KS) and specific
growth rate ( net).

THEORIES

Rate of microbial growth net is characterized by specific growth rate:

net =

1 dX
X dT

(1/h)

Yield Coefficients (Yx/s) are defined based on the amount of consumption of another material
X
Yx/s = - S

(g cells/g substrate )

Mass doubling time (d) is calculated based on cell numbers and the net specific rate of
replication
2
d = net

(h)

For substrate limited growth Monod equation is applicable in cellular system. Monod
equation is as the following:

S) /(KS + S)

(1/h)

where :

m:

maximum specific growth rate when S >>Ks

g ;

net

when endogeneous metabolism is unimportant

KS : saturation constant or half-velocity constant


KS = S when
S >>Ks ,

S << Ks,

S) /(KS + S)

APPARATUS AND MATERIALS

1.

Microbe : Escherichia Coli

2.

Shake flask (250mL flasks and 1000mL flasks)

3.

Eppendorf tubes/falcon tube (1.5mL)

4.

Cuvettes (spectrophotometer)

5.

Thermostated rotary shaker/incubator shaker

6.

Refrigerated centrifuge

7.

Media (for specific microbe)

8.

Ethanol (70% ethanol for swabbing for sterility)

9.

Spectrophotometer

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

16.

pH meter

PROCEDURE
i Preparation of media
Media is prepared according to the needs of microorganism used.
a

ii

Terrific Broth preparation


The recipe is followed as stated at the bottle.
The media was autoclaved at 121C for 20 minutes.

Preparation of Cell Culture

The cell culture was maintained on an agar plate and liquid broth for the preparation of
inoculum. A suitable media were used to ensure the growth of microorganism.
a

Seed culture preparation (inoculum)


5 loops of grown E Coli were taken from the agar plates and were added to the
sterilized media of 150mL in 1000mL shake flask.
Sterility were being sustained during the transfer.
The media were grown at 300 rpm for 5 hours, assuming the exponential
growth of E Coli. The OD for seed culture was recorded by using the

spectrophotometer.
b Main experiment
10% of inoculum was transferred to the main experiment media by using
aseptic technique. Since 10% of inoculum was needed, thus only 15 mL of
seed culture was needed if the total working volume was 150 mL.
The shake flask was capped by using the cotton plug and swabbed using 70%
of ethanol. It was incubated in a thermostated rotary shaker at required
rotational speed and temperature for 24 hours.

iii
1

Sampling
The required amount of sample was transferred into the sampling tube with interval
time for every hour or every 2 or 3 hours.

5 mL of the sample was taken every time sampling is done during the fermentation
process to measure the optical density (OD), glucose analysis and total cell number
(biomass concentration : g/L)

iv
1

Absorbance Analysis (Optical Density) (OD)


1 mL of the sample was transferred into a cuvette and the spectrophotometer was used

to measure the optical density with the wavelength set at 600nm.


1 mL of chosen media was used as the blank and the spectrophotometer was

calibrated to zero.
This method was used in order to measure the cell growth where high absorbance
indicates high number of cells, which is caused by low transmittance and vice versa.

v
1
2
3
4
5
6
7

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


Dried centrifuge tubes were weighed and this were noted as the initial mass.
1 mL of sample was added to the weighted centrifuge tube.
The sample was centrifuged at 10,000 rpm and at T = 4C for 20 minutes.
The supernatant was taken out from the mixture.
The centrifuge tube was dried in oven at 80C for overnight.
The dried centrifuged tube was left in the dessicator.
The centrifuged tubes were weighed and are noted as the final mass.
Cell Dry Weight = Final mass Initial Mass

RESULT AND CALCULATION


a

Terrific Broth (TB)

Seed/inoculum
Time (h)
0
5

Real OD (nm)
0.214
1.751

Time (h)

Absorbance Optical Density


(nm)

Absorbance Optical
Density (nm)

(not diluted)

(Diluted)
1:9

0.225

-0.067

-0.337

-1.058

0.613

-0.173

0.689

0.122

0.600

-0.534

0.817

-0.822

0.789

-0.795

10

0.762

-0.125

12

1.115

-0.347

14

1.066

-0.187

16

1.186

0.280

18

1.176

0.159

20

1.275

0.177

22

1.448

0.790

24

1.417

0.146

Optical density vs Time


2
1.5
1
0.5
Absorbance Optical density (nm)
0

10

15

20

25

30

-0.5
-1
-1.5
Time (hour)

Graph 1 : The growth curve of E. coli plotted using absorbance optical density against time

Cell Dry Weight (Biomass Concentration)


Time

Initial weight

Final weight (g)

Cell dry

Cell mass

(h)

(g)

(tube+sticker

weight (g)

concentration,

(tube+sticker

label+cell), m2

(m2 m1)

X (g/L)

ln X

ln X/Xo

label), m1
0

1.078

1.079

0.001

0.5

-0.693

0.000

1.083

1.085

0.002

1.0

0.693

1.069

1.074

0.005

2.5

0.916

1.609

1.072

1.076

0.004

2.0

0.693

1.386

1.054

1.059

0.005

2.5

0.916

1.609

1.081

1.087

0.006

3.0

1.099

1.792

1.085

1.091

0.006

3.0

1.099

1.792

10

1.062

1.069

0.007

3.5

1.253

1.946

12

1.066

1.073

0.007

3.5

1.253

1.946

14

1.071

1.080

0.009

4.5

1.504

2.197

16

1.063

1.073

0.010

5.0

1.609

2.303

18

1.076

1.085

0.009

4.5

1.504

2.197

20

1.062

1.070

0.008

4.0

1.386

2.079

22

1.074

1.086

0.012

6.0

1.791

2.485

24

1.071

1.081

0.010

5.0

1.609

2.303

In X vs Time
3
2.5

f(x) = 0.07x + 1.05

2
Ln X

1.5
1
0.5
0

10

15

20

25

30

Time (hr)

Graph 2: Graph of ln X against time which is plotted to construct the growth curve of the cell
by using cell mass concentration, X (g/L) data.
By referring Figure 7.2, it shows that the exponential phase may occur at between 3rd and
12th hour. Thus, the data between that ranges of time will be used to find the value max.

In(X/Xo) vs Time
3
2.5

f(x) = 0.07x + 1.05

2
In(X/Xo)

1.5
1
0.5
0

10

15

20

25

30

Time (hour)

Graph 3 :Graph of ln X/X0 against time is plotted to find the value of max
From graph 3, the value of max is 0.0666 hr-1 which find from the slope of graph ln X/X 0
against time. Thus the doubling time hr.

SAMPLE CALCULATIONS
The value of cell dry weight can be determined by using the formula that will be shown
below:
Cell dry weight = Initial mass Final mass
Terrific Broth (TB)
1. Cell dry weight (g) at time 0th hour

(Final weight of tube with cell) (Initial weight of tube )


1.079 1.078 = 0.001 g

2. Cell mass concentration, X (g/L) at time 0th hour

Cell dry weight sample volume


Volume of sample, 2 mL = 0.002 L

= 0.001 g 0.002 L = 0.5 g/L


3. Maximum growth rate, max

The value is obtained from the slope of the graph of ln X/X0 against time.

max =slope= 0.0666 hr-1

4. Doubling time can be calculated by using formula of:

td = ln 2 max

where max = 0.0666

ln 2 0.0666

= 10.41 hours

DISCUSSION
This experiment is carried out to study the kinetic growth of microorganism. We
choose E.coli as the cell and cultivated it inside a shake flask. The growth of microorganism
in shake flask is one of simple method called fermentation. The media which contain carbon
source is being supplied as the nutrients for the microorganism. The flask was placed in
incubator shaker to mix the cell and media for cultivation. It is to increase the homogeneity
between media and cell and provide aeration for the cell. The culture gone for fermentation
for 24hours.Within that period, the cell sample is taken out for every 2 hours to analyse the
concentration of the cell (g/L) and the cell dry weight.
In order to determine the concentration of the cell inside the flask,t he absorbance
reading of optical density is taken from spectrophotometer using 600m wavelength. The
higher the absorbance reading the higher the number of cell presence in the flask for
particular time. From the graph 1 show the growth E.coli plotted based on absorbance value
optical density (OD) against time. This graph somehow show the value of OD is not same as
theory. The value of OD did not show good pattern as it is increase and decrease at the
particular time but at time 24hours,the cell has reached its deceleration phase where the
growth of cell is started to slow down or in other word start to decay. The optical density
somehow give affect the result this might be during taking measurement of OD, small air
bubbles appeared without notice and it is counted as living cells. The number of microscopic
air bubbles, especially in dense cultures may be quite high. Beside that the error might occur
while conducting experiment is contamination while handling the culture that we are not
aware of.
The graph 2 shows the growth curve of the cell by using cell mass concentration.From
the graph it can be observe the exponential phase is showed between 3 rd to 12th hours.The
exponential phase or log phase of growth is a pattern of balanced growth wherein all the
cells are dividing regularly by binary fission.The rate of exponential growth of a bacterial
cultured is expressed as generation time also the doubling time of the bacterial population.at
time 12th to 22nd hours where the statationary phase occur.Based on the theory,the stationary
phase is where the exponential growth cannot be continued forever in batch culture,it means
that growth of bacteri remain contant and simply stopped growing,however the pattern graph

2 shows at stationary phase there is still increment this is might be,if viable cells are being
counted,it cannot be determined whether some cells are dying and an equal number of cells
are dividing.After time 22hours and 24th hours it is call death phase .During the death
phase,the number of viable cells decreases geometrically (exponentially),essential the reverse
of growth during the log phase. Graph of ln X vs time is plotted in order to find the max.
From the graph, the exponential phase may be occur at time 3 rd and 12th hour. The value of
max is obtained from graph of ln X/X0 vs time. From the graph, the value of max is 0.0666 hr1

. Hence the doubling time is 10.41 hour. As mentioned above, bacterial growth rates during

the phase of exponential growth, under standard nutritional conditions (culture medium,
temperature, pH, etc.), define the bacterium's doubling time. Doubling time for bacteria vary
from about 12 minutes to 24 hours or more.

CONCLUSION
At the end of this experiment,microorganism is suitable to be fermented inside a shake flask
and it is simple methos to investigate the growth of microorganism.The microorganism will
go through several phases in their growth,several analyses on the cell need to be done to now
the growth kinetics of the cell and the duration for each phase.This include the cell
concentration,glucose concentration and cell dry weight analyses. Growth kinetics deals with
the rate of cell growth and how it is affected by various chemical and physical conditions.
During the course of growth, the cells is continuously changing and adapting itself in the
media environment, which can make physical and chemical condition of of cell growth
change. 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

RECOMMENDATION
1. Before start the bioreactor, it must be autoclaved to avoid contamination. The bottles
are plugged with cotton wool and sealed with aluminium foil.
2. Cuvettes must be wiped with Kim Wipe tissues to clean it and prevent any scratch so
that the light from the spectrophotometer can pass through the cuvette and would not
affect the spectrophotometer reading.
3. Wear a glove while conduct experiment and spray on some ethanol before starting
experiment this is to ensure no infections of bacteria.
4. The supernatant of cell concentration should be taken out carefully without any taking
out of the biomass.
5. This experiment must be carried out under the laminar flow to prevent any
contamination to the culture.

REFERENCES
http://www.bioreactors.eu/en/application/optical-density-od-measurement/
http://www.mpi-bremen.de/Binaries/Binary13037/Wachstumsversuch.pdf
http://textbookofbacteriology.net/growth_3.html

https://en.wikipedia.org/wiki/Bacterial_growth

RECOMMENDATION
6. Before start the bioreactor, it must be autoclaved to avoid contamination. The bottles
are plugged with cotton wool and sealed with aluminium foil.
7. Cuvettes must be wiped with Kim Wipe tissues to clean it and prevent any scratch so
that the light from the spectrophotometer can pass through the cuvette and would not
affect the spectrophotometer reading.
8. Wear a glove while conduct experiment and spray on some ethanol before starting
experiment this is to ensure no infections of bacteria.
9. The supernatant of cell concentration should be taken out carefully without any taking
out of the biomass.
10. This experiment must be carried out under the laminar flow to prevent any
contamination to the culture.