1.

0 ABSTRACT

The purpose of this experiment is to study or observe the growth kinetics of microorganism
in shake flask experiment, construct a growth curve including lag, log, stationary and death
phases and to determine the Monod parameters. This experiment was conducted by using
bacteria, E.coli grown in Terrific Broth medium and undergo fermentation for 24 hours with
a rational speed and temperature. The cell culture is taken out every 1 hour from 0 hour to 4th
hour whereas, up to 5th to 20th hour the cell culture is taken out every 2 hours and the absorbance
analysis, glucose analysis and cell dry weight are being performed. Spectrophotometer is used
to read absorbance value for optical density analysis. Meanwhile, for glucose level reading is
taken from the Biochemical analyser. The substrate concentration values will then being
plotted on graph with growth rate. The absorbance value for optical density of cell shows an
increasing with time based on Figure 7.1. This indicate the cell are growing and number of
cell is increased in the shake flask. Based on Figure 7.2 it shows ln X against time, the
exponential phase may occur between 1st and 7th hour. The data from that period will be used
to find the value of µmax. Then, Figure 7.3 which is graph ln X/XO against time shows the
value of µmax which is equal to the slope of the graph is 0.3412. This experiment can be
concluded as successful because all the objectives are fulfilled.

1
2.0 INTRODUCTION

Kinetics of growth refers to the rate at which the number of individual cells (or, more
general, of active biomass) changes in a defined system. However, the area of microbial growth
kinetics consists not just of the simple dynamic recording of biomass increase as a function of
time in a culture but it is more the extraction of parameters that allow to quantitatively
formulate general principles, to construct mathematical models that allow to describe and
predict microbial growth processes and that provide a basis for further experimentation.
Growth kinetics are therefore an indispensable tool not only in the applied fields of industrial
and environmental biotechnology, but also in fundamental areas such as microbial genetics,
physiology, and ecology, including competition, selection, and evolution.

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.

In 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 2.1 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 (D. Rolfe et al, 2011). In the first phase, there is no

2
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. Exponential phase is important for determining the maximum growth

rate, μ 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.

3.0 0BJECTIVES

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

3
4.0 THEORY

Shake flask fermentation is one of the examples of batch fermentation. Batch culture is
an example of a closed culture system which contains an initial, limited amount of nutrient
(Raina, 2008). The inoculated culture will pass through a number of phases. After an
inoculation there is a period during which no growth appears to take place. This period is
referred as the lag phase and may be considered as a time of adaptation. In a commercial
process, the length of the lag phase should be reduced as much as possible. Following a period
during which the cell gradually increases, the cell grows at constant, maximum rate and this
period is known as the log phase or exponential phase. The exponential phase may be
described by the equation below:

𝑑𝑥
= 𝜇𝑥 − − − − − − − − − − 𝑬𝒒𝒖𝒂𝒕𝒊𝒐𝒏 𝟒. 𝟏
𝑑𝑡
Where,

x is the concentration of microbial biomass

t is the time, in hours

μ is the specific growth rate, in hour-1

on integration, Equation 4.1 gives

𝑥𝑡 = 𝑥𝑜 𝑒 𝜇𝑡 − − − − − − − − − − 𝑬𝒒𝒖𝒂𝒕𝒊𝒐𝒏 𝟒. 𝟐

Where,

xo is the original biomass concentration

xt is the biomass concentration after time interval, t hours

During the exponential phase, the organism is growing at its maximum specific growth
rate, 𝝁𝒎𝒂𝒙 for the prevailing conditions. Equation 4.2 predicts that growth will continue
indefinitely. However, growth results in the consumption of nutrients and the excretion of
microbial products. Thus after a certain time the cell growth rate will decrease until growth
ceases. The cessation of growth may be due to the depletion of some essential nutrient in the

4
medium when there is limitation in substrate. The decrease in growth rate and the cessation of
growth due to the depletion of substrate may be described by the relationship between µ and
the residual growth-limiting substrate as follows:

𝜇𝑚𝑎𝑥 𝑠
𝜇=
𝐾𝑠 + 𝑠

Where,

𝝁𝒎𝒂𝒙 = maximum growth rate

s = residual substrate concentration

𝑲𝒔 = substrate utilization constant

Figure 4.1: The graph showing the relationship between the parameter of the Monod equation

The stationary phase in batch culture is the point where the growth rate has declined
to zero. In the other word the growth rate is equivalent to the death rate. The cell death is might
due to the nutrient limitations due to their incorporation into cells during log-phase growth or
a build-up of toxins due to their release of fermentation products also during log-phase growth.

The death phase is the result of the inability of the bacteria to carry out further
reproduction as condition in the medium become less and less supportive of cell division (Lee
Y, 2013). The nutrient is extremely insufficient for the growth of the microorganism.
Eventually, the number of viable bacterial cells begins to decline at an exponential rate.

5
Industrial fermentation is usually interrupted at the end of the exponential growth phase or
before the death phase begins.

Figure 4.2: Growth curve of microorganism based on cell number analysis

6
5.0 MATERIALS AND APPARATUS

Materials:

a) Microbe: Escherichia coli

b) Media (for specific microbe)
c) Ethanol (70% ethanol for swabbing for sterility)

Apparatus:

a) Shake flask (250 mL flasks and 1000 mL flasks)

b) Eppendorf tubes/falcon tube (1.5mL)
c) Cuvettes (spectrophotometer)
d) Thermostated rotary shaker/incubator shaker
e) Refrigerated centrifuge
f) Spectrophotometer
g) Bunsen burner for sterility
h) Graduated flask for measuring media (1000 mL, 100 mL, 10 mL)
i) Laminar flow hood for sterility
j) Biochemical analyser
k) HPLC for product measurement like ethanol
l) Cotton plugged
m) pH meter

7
6.0 PROCEDURES
The experimental parameters will be as the following:

Table 6.1: Experimental Parameters

Shaking Shake Inoculum

Temperature frequency flask Filling/Working Media percentage pH Carbon
(ºC) (rpm) size volume (mL) type (5%) source

37 350 500 150 TB 10 7 Glycerol

I. Preparation of media

Media are prepared according to the needs of microorganism used. Microorganism used
is Escherichia coli. There are many kinds of media for E coli for instance Luria Bertani
Broth or Terrific Broth. Terrific Broth is a readied phosphate buffer media. The Terrific
Broth are prepared according to the recommended formula or recipe stated at the
chemical bottle. The example readied recipe for the broths are as the following: Please
further read the instruction of bottle.

Table 6.2: Broth

No Name of Broth Brand Recipe (g/L)

1. Terrific Broth SRL Chem or Merck 47.60 g to be filled

up to 1 L volume
glycerol as carbon
source (4 mL/1 L)

a) Terrific Broth (TB) preparation

1) The recipe as stated at the bottle is followed.

2) The media is autoclaved at 121ºC 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.

8
 II. Preparation of cell culture

Cell culture used are maintained on an agar plate and liquid broth for the inoculum
preparation. A suitable media is needed in order to ensure that the microorganism is
growing. Inoculum preparation refers to seed culture which will be the feed for the main
experiment.

a) Seed culture preparation (inoculum)

1) 5 loops of grown E coli is taken on agar plates and added to the sterilized media of 150
mL in 1000 mL shake flask. (2 of 1000 mL shake flask is needed to ensure enough
inoculum 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 150 mL, therefore, 10% of inoculum would be
15 mL of seed culture needed).
2) The shake flask is then capped (cotton plugged) and swabbed with 70% ethanol before
incubation in a thermo-stated 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), glucose analysis and total cell number (biomass
concentration: g/L).
3) Table 3 below is referred for planned usage of sample volume.

9
Table 6.3: Sampling
No Sample name Volume (mL) Used for
1. OD 1000 Optical measurement using
spectrophotometer
2. CDW 1000 For cell dry weight measurement
3. S 1500 Glucose analysis

IV. Absorbance analysis (Optical density) (OD)

1) 1 mL of sample is transferred into a cuvette and the optical density measurement is

made using a spectrophotometer with the wavelength set at 600 nm.
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 mL sample is added to weighted centrifuge tube.
3) The sample is centrifuged at 10,000 rpm and at T of 4ºC 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 80ºC for overnight.
6) The dried centrifuged tubes is left in desiccator 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

10
7.0 RESULTS
Seed culture preparation (inoculum)
Initial OD: 0.191
Final OD: 2.002
Table 7.1: Absorbance value at 600nm obtained using Terrific Broth as medium (fermentation)

No. Time (h) Absorbance Optical Density (nm)

1 0 0.406
2 1 2.022
3 2 3.362
4 3 4.746
5 4 4.176
6 5 4.882
7 6 4.896
8 7 5.169
9 8 5.459
10 9 5.342
11 10 5.344

5
Absorbance (OD)

0
0 2 4 6 8 10 12
Time (h)

Figure 7.1: The growth curve of E. coli plotted using absorbance optical density

11
Table 7.2: Result of Cell Dry Weight (CDW)

Time Empty Dried Cell dry Cell mass ln X ln X/X0

(h) Centrifuge Centrifuge weight, m2 concentration,
Tube, m1 (g) Tube + – m1 (g) X (g/L)
Sample, m2 (g)
0 1.0812 1.0811 -0.0001 -0.1 - -
1 1.0618 1.0629 0.0011 1.1 0.0953 0.0000

2 1.0901 1.0934 0.0033 3.3 1.1939 2.5280

3 1.0957 1.0998 0.0041 4.1 1.4110 2.6950

4 1.0809 1.0850 0.0041 4.1 1.4110 2.6950

5 1.0701 1.0741 0.0040 4.0 1.3863 2.6774

7 1.0712 1.0764 0.0052 5.2 1.6487 2.8507

9 1.0967 1.1020 0.0053 5.3 1.6677 2.8622

11 1.0972 1.1024 0.0052 5.2 1.6487 2.8507

13 1.0912 1.0966 0.0054 5.4 1.6864 2.8733

15 1.0830 1.0892 0.0062 6.2 1.8245 2.9520

17 1.0744 1.0795 0.0051 5.1 1.6292 2.8388

12
 2

1.8

1.6

1.4

1.2

1
ln X

0.8

0.6

0.4

0.2

0
0 2 4 6 8 10 12 14 16 18
Time (h)

Figure 7.2: Graph of ln X against Time

From Figure 7.2, it can be determined that exponential phase may occur between 1 st and 7th
hour. Therefore, the data from that period will be used to find the value of µmax.

3.5 y = 0.3412x + 0.9901

2.5
ln (X/Xo)

1.5

0.5

0
0 1 2 3 4 5 6 7 8
Time (h)

Figure 7.3: Graph of ln X/Xo against Time

From the above figure, the value of µmax which is equal to the slope of the graph is 0.3412.

13
8.0 SAMPLE CALCULATIONS

1. Cell dry weight, X:

𝑭𝒊𝒏𝒂𝒍 𝒘𝒆𝒊𝒈𝒉𝒕−𝑰𝒏𝒊𝒕𝒊𝒂𝒍 𝒘𝒆𝒊𝒈𝒉𝒕

X=
𝑽𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝒔𝒂𝒎𝒑𝒍𝒆
1.0629𝑔−1.0618𝑔
=
0.001 𝐿

= 1.1

2. Growth rate, µ:
𝒍𝒏 𝒙𝟏 −𝒍𝒏𝒙𝒐
µ=
𝑻𝒊𝒎𝒆𝟏 −𝑻𝒊𝒎𝒆𝒐

𝑙𝑛1.139−𝑙𝑛0.0953
=
2−1

= 2.481/h

14
9.0 DISCUSSION

In this experiment, we want to observe the growth kinetics of microorganism in shake flask
experiment by selecting E.Coli as the cell and it is cultivated in shake flask. Glycerol and
glucose was being supplied as carbon sources and it both acts as substrate nutrients for the
cells. The flask is shaken during cultivation ensuring it is in homogenous. We took the cell out
to analyze the concentration of the cell (g/L), cell dry weight and glucose concentration in order
to obtain the growth curve of the microorganism.

Based on table 7.1 and graph at figure 7.1 plotted above, we can observe the growth
rate of E.coli. The growth rate of the bacteria is rapidly increase at 0 hour, 1st hour to 8th hour
which the value are increase from 0.406nm to 5.459nm. This phenomenon called as
exponential phase which the growth achieve maximum rate. At this phase, the number of new
bacteria appearing per unit time is proportional to the present population. Since, we cannot see
the lag phase from the graph at the beginning of the growth is because the lag phase occur very
short due to the bacteria can adapt very well with the environment.

Next, we can observe the growth of bacteria undergo stationary phase during 7th to 8th
hour as the value about to constant which are from 5.169nm to 5.459nm. This phase is often
due to a growth-limiting factor such as the depletion of an essential nutrient or the formation
of an inhibitory product such as an organic acid. After that, the value start decreasing at 8 th
hour to 10th hour which are from 5.459nm to 5.344nm. This shows the pattern growth of
bacteria start to decline and finally stop. This phenomenon called as dead phase where bacteria
die. This could be caused by lack of nutrients, environmental temperature above or below the
tolerance condition for the species or other injurious conditions that harm the bacteria.

From the graph plotted at figure 7.1, the absorbance value increase as the time increase.
We can obtain the value of μmax is 0.3412 means the bacteria growth at its highest. The higher
the absorbance reading means greater number of cells inside the flask at a particular time.
Although it did not increase the absorbance reading steadily due to some parallax error might
occur during conducting the experiment that could affect the reading.

15
10.0 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. This include the cell
concentration, glucose concentration and cell dry weight analyses. The aim of this experiment
is to construct a growth curve including lag, log, stationary and death phases. From the graph
plotted and equations that have been given, calculating of the Monod parameter of maximum
growth rate and specific growth rate can be done. But there are some error occurs during the
calculation. This may cause by the error during the experiment such as error during taking the
Absorbance Optical Density (nm) values. During the course of growth, the cells is continuously
changing and adapting itself in the media environment. For overall experiment, it can be
concluded as successful experiment because all the objectives is fulfilled.

As the conclusion, the microbial culture in batch culture system undergoes lag phase,
exponential phase, retardation phase and death phase. At the end of this experiment,
microorganism is suitable to be fermented inside a shake flask.

11.0 RECOMMENDATIONS

There are a few recommendations that should be taken into account in order to obtain more
precise and accurate results.
First, this experiment must be carried out under the laminar flow to prevent any
contamination to the culture and disinfect the work area with 70% alcohol before handling the
culture. Second, aseptic technique must be practised when handling biomass concentration to
avoid any contamination. Third, the supernatant of cell concentration should be taken out
carefully without any taking out of the biomass. Fourth, the cap of the viral must be opened to
fasten the drying process of the biomass in the oven. Fifth, cuvette must be wiped cleanly to
prevent any scratch that would affect the spectrophotometer reading during protein test. Last
but not least, dispose of all contaminated materials in appropriate containers and wash hand
after handling the culture.

16
12.0 REFERENCES

1) CHE 506 Lab Manual 6

2) Matthew D, R., Christopher J, R., Sacha L., Carmen P., Arthur T., Andrew D. S., Cameron
M, A., Michael F. S., Roy P, B., Jozsef, B., Michael W, P., & Jay C. D. H. (2012). Lag
Phase Is a Distinct Growth Phase That Prepares Bacteria for Exponential Growth and
Involves Transient Metal Accumulation. Bacteriology, 194(3), pp 686.
3) Raina M.N. (2008). Bacterial Growth. Basic Microbiological Concepts, pp 37 – 53.
4) Lee Y, K. (2013). Microbial Biotechnology: Principles and Applications Third. 3, pp 32-
33.
5) Varun C N. (2016). Bacterial growth curve. Medical Microbiology.

17
13.0 APPENDIX

18
 Bacteria was put in the oven

ss

Bacteria was put in centrifuge

19