UNIVERSITI TEKNOLOGI MARA
FAKULTI KEJURUTERAAN KIMIA
REACTION ENGINEERING LABORATORY
(CHE506)
NAME:
STUDENT NO :
KHAIRUL AMIRIN BIN KHAIRUL ANUAR
2017632082
PUTERA NAJMEEN FARITH BIN ABDUL RAZAK
2017632096
NURUL AMIRAH BINTI MUSDAFA KAMAL
2017632124
NURUL AIDA BINTI MOHAMMAD
2017632132
NURUL KAMILAH BINTI KHAIROL ANUAR
2017632192
NURLINA SYAHIIRAH BINTI MD TAHIR
2017632214
GROUP
: EH2205I
EXPERIMENT
: GROWTH STUDY OF E.COLI IN SHAKE FLASK
DATE PERFORMED
: 18th OCTOBER 2018
SEMESTER
:5
PROGRAMME / CODE : CHEMICAL ENGINEERING / EH220
SUBMIT TO
: MADAM SYAZANA MOHAMAD PAUDZI
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
Title
Abstract/Summary
Introduction
Aims
Theory
Apparatus
Methodology/Procedure
Results
Calculations
Discussion
Conclusion
Recommendations
Reference
Appendix
TOTAL MARKS
Allocated Marks (%)
Marks
5
5
5
5
5
10
10
10
20
10
5
5
5
100
Remarks:
Checked by:
Rechecked by:
---------------------------
---------------------------
Date:
Date:
TABLE OF CONTENT
1.0
ABSTRACT ................................................................................................................... 2
2.0
INTRODUCTION......................................................................................................... 3
3.0
OBJECTIVES ............................................................................................................... 3
4.0
THEORY ....................................................................................................................... 7
5.0
MATERIALS & APPARATUS ................................................................................... 9
6.0
METHODOLOGY ..................................................................................................... 10
7.0
RESULTS .................................................................................................................... 12
8.0
CALCULATIONS ...................................................................................................... 16
9.0
DISCUSSION .............................................................................................................. 16
10.0 CONCLUSION ........................................................................................................... 21
11.0 RECOMMENDATIONS............................................................................................ 22
12.0 REFERENCES ............................................................................................................ 23
13.0 APPENDICES ............................................................................................................. 24
1
1.0
ABSTRACT
The bacterial growth curve is a fundamental part of introductory microbiology. The growth
kinetics of E.coli is studied using the shake flask fermentation experiment by plotting the graph
of real absorbance optical density versus time. The Terrific Broth is prepared by autoclaving
the media at 121⁰C for 20 minutes. The E.coli inoculum is grow for 5 hours at 300 rpm. 10%
of the inoculum which 15 mL is added into sterilized media of 150mL then incubated in
thermostat rotary shaker at required rotational speed and temperature for 16 hours. The
required amount of sample was transferred into the sampling tube with interval time for every
hour or every 2 hours. The reading for the absorbance optical density is measured using the
spectrophotometer. The lag phase occur from t = 0h until t = 1h as the E.coli familiarize
themselves with new environment in the media. There two exponential phase from t = 1 h until
t = 6 h and from t = 8h until t = 14h. The maximum growth rate of the E.coli is believed to be
found from the exponential curve which yield to µmax = 0.016 h-1. The presence of the second
exponential phase is due to the cryptic growth and the possibility of the presence of second
microorganism consuming the E.coli as the food supplies. The deceleration phase cannot be
determined. The death phase occur at t = 14h until t = 16h where the second microorganism is
believed to drastically die due to the abrupt loss of food supplies and their inability in surviving
with their own metabolism. The µnet for the respective phase are µnet, lag phase = 0.6931 h-1, µnet,
exponential phase =
µmax = 0.016 h-1, µnet,
stationary phase
= 0.0000 h-1, µnet,
death phase
= 0.0000 h-1.
Unfortunately, due to limited sources of data, the yield coefficient (YX/S) and saturation
constant (Ks) cannot be determined.
2
2.0
INTRODUCTION
The bacterial growth curve is a fundamental part of introductory microbiology (Monod, 1949).
Escherichia coli are often used both as a model organism to understand fundamental biological
process and as a tool to produce biomolecules, including plasmids and proteins. The growth
and physiology of Escherichia coli cells are studied in a batch cultures. When batch cultures
are used, E. coli cells from an overnight culture are usually inoculated into Erlenmeyer flasks
containing a complex or a defined medium.
Like any other living system, microorganisms also require a source of energy, carbon,
nitrogen, oxygen, iron and other minerals, micronutrients, and water for growth, and
multiplication. All these nutrients that are essential for the growth and multiplication of
microbial organisms are supplied in the form of nutrient media. For commercial purposes, there
commended media should be cheap and available year round. The following are the minimum
components required in a microbial medium for cultivation of microbes in a laboratory:
1) Carbon source
A simple carbon source, which is simple to use and easily available, can be used. Sugars
such as glucose, lactose, sucrose, and complex polysaccharides such as starch, glycogen
cellulose, a mixture of various carbohydrates, and other compounds such as cereal grain
powders, cane molasses, etc., are usually used as carbon sources in microbial culture
media. The main purpose of the carbon source is to provide energy and carbon skeleton
for the synthesis of various other biological compounds.
2) Nitrogen sources
The major types of nitrogen sources used in culture media are ammonium salts, urea,
animal tissue extracts, amino acid mixtures, and plant-tissue extracts.
3) Micro elements or trace elements
Elements required in small amounts or in traces are to be added into the medium as
salts in required amounts. The elements such as copper, cobalt, iron, zinc, manganese,
magnesium, etc., are the microelements.
Typically, to understand and define the growth of a particular microbial isolate, cells are
placed in a liquid medium in which the nutrients and environmental conditions are controlled.
If the medium supplies all nutrients required for growth and environmental parameters are
optimal, the increase in numbers or bacterial mass can be measured as a function of time to
3
obtain a growth curve. Based on Figure 1, several distinct growth phases can be observed
within a growth curve. These include:
1) Lag Phase
2) The Exponential Or Log Phase
3) The Stationary Phase
4) The Death Phase.
Each of these phases represents a distinct period of growth that is associated with typical
physiological changes in the cell culture. As will be seen in the following sections, the rates of
growth associated with each phase are quite different.
Figure 1 - A typical growth curve for bacterial population.
Lag phase represents immediately after inoculation of the cells into fresh medium, the
population remains temporarily unchanged. Although there is no apparent cell division
occurring, the cells may be growing in volume or mass, synthesizing enzymes, proteins, RNA,
etc., and increasing in metabolic activity. The length of the lag phase is apparently dependent
on a wide variety of factors including the size of the inoculum; time necessary to recover from
physical damage or shock in the transfer; time required for synthesis of essential coenzymes or
division factors; and time required for synthesis of new (inducible) enzymes that are necessary
to metabolize the substrates present in the medium.
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, µ and doubling time, d since the growth
4
at this time is the most constant and ideal. The third phase of growth is the stationary phase.
The stationary phase in a batch culture can be defined as a state of no net growth. Although
there is no net growth in stationary phase, cells still grow and divide. Growth is simply balanced
by an equal number of cells dying.
The final phase of the growth curve is the death phase, which is characterized by a net
loss of culturable cells. Even in the death phase there may be individual cells that are
metabolizing and dividing, but more viable cells are lost than are gained so there is a net loss
of viable cells. The death phase is often exponential, although the rate of cell death is usually
slower than the rate of growth during the exponential phase.
LB Media (Luria-Bertani) is common bacterial growth media for Escherichia Coli. Although
already described in the fifties in the early days of phage genetics these media are still widely
used in molecular biology. The two main components of LB media are Tryptone and Yeast
Extract. Tryptone is used in a concentration of 10 gram / litre and Yeast Extract in a
concentration of 5 gram / litre. Many variations of LB medium only differ in the concentration
of NaCl. All LB-Media are listed in order of increasing concentration of NaCl.
Tryptone broth is a moderately rich medium for growth and cultivation of Escherichia
Coli. Terrific Broth is a rich medium compared to LB and Tryptone Media. The medium is
developed for higher density growth of Escherichia Coli cells and higher yield of plasmid DNA
compared to LB and Tryptone broth. Super Broth is an even richer medium developed for
obtaining high yields of lambda bacteriophage in liquid lysates, Botstein, D. et al.
5
3.0
OBJECTIVES
The objectives of the experiment are:
1) To study and 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 of maximum growth rate (µmax), yield of substrate
(YX/S), mass doubling time (td), saturation constant (Ks) and specific growth rate (µnet).
6
4.0
THEORY
Shake flask fermentation is one of the examples of batch fermentation. Batch culture is an
example of closed culture system which contains an initial, limited amount of nutrient. The
inoculated 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 a lag phase and may be
considered as a time adaptation. In a commercial process, the length of lag phase should be
reduced as much as possible. Following a period during which cell gradually increases, the cell
grows at constant, maximum rate and this period is known as the log phase or exponential
phase.
During lag phase dX/dt ans dS/dt are essentially zero. However, as exponential growth
phase begins it is possible to measure dX/dt and dS/dt values which are very useful for defining
important microbial kinetic parameters. Using corresponding observations of dS/dt and dX/dt
obtained just after the onset of exponential growth phase, we can compute the specific growth
rate, μ and yield coefficients, YXS as:
Rate of microbial growth (μnet) is characterized by specific growth rate:
μ net 
1 dX
X dt
Yield coefficients (YX/S) are defined based on the amount of consumption of another material:
YX/S  
ΔX
ΔS
Mass doubling time (τd) is calculated based on cell numbers and the net specific rate of
replication:
τd 
ln2
μ net
The monod equation is a mathematical model for the growth of microorganisms. It is
named for Jacques Monod who proposed using an equation of this form to relate microbial
growth rate in an aqueous environment to the concentration of limiting nutrients.
7
For substrate limited growth Monod equation is applicable in cellular system. Monod equation
is as the following:
μg 
μ mS
KS  S
Where,
μm = maximum specific growth rate when S >> KS
μg = μnet when endogeneous metabolism is unimportant
KS = saturation constant or half-velocity constant
KS = S when μg = 1/2μm
S >> KS, μg = μm
S << KS, μ g 
μ mS
KS  S
8
5.0
MATERIALS & APPARATUS
5.1
Materials
1) Media (for specific microbe)
2) Ethanol (70% ethanol for swabbing for sterility)
3) Microbe: Escherichia Coli
4) Distilled water
5.2
Apparatus
1) Shake flask (250mL flasks and 1000mL flasks)
2) Eppendorf tubes/falcon tube (1.5mL)
3) Cuvettes (spectrophotometer)
4) Thermostated rotary shaker/incubator shaker
5) Refrigerated centrifuge
6) Spectrophotometer
7) Bunsen burner for sterility
8) Graduated Flask for measuring media (1000mL, 100mL, 10mL)
9) Laminar Flow hood for sterility
10) Biochemical Analyzer
11) HPLC for product measurement like ethanol
12) Cotton plugged
13) pH meter
14) Desiccator
9
6.0
METHODOLOGY
6.1
Preparation of media
Media is prepared according to the needs of microorganism used.
6.1.1
Terrific Broth preparation
The recipe is followed as stated at the bottle. The media was autoclaved at 121°C for
20 minutes.
6.2
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.
6.2.1
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.
6.2.2
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 thermostat
rotary shaker at required rotational speed and temperature for 16 hours.
10
6.3
Sampling
1) The required amount of sample was transferred into the sampling tube with interval
time for every hour or every 2 or 3 hours.
2) 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)
6.4
Absorbance Analysis (Optical Density) (OD)
1) 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.
2) 1 mL of chosen media was used as the blank and the spectrophotometer was
calibrated to zero.
3) 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.
6.5
Cell Dry Weight. (Biomass Concentration) (X) (g/L)
1) Dried centrifuge tubes were weighed and these were noted as the initial mass.
2) 1 mL of sample was added to the weighted centrifuge tube.
3) The sample was centrifuged at 10,000 rpm and at T = 4°C for 20 minutes.
4) The supernatant was taken out from the mixture.
5) The centrifuge tube was dried in oven at 80°C for overnight.
6) The dried centrifuged tube was left in the dessicator.
7) The centrifuged tubes were weighed and are noted as the final mass.
Cell Dry Weight = Final mass – Initial Mass
11
7.0
RESULTS
Table 1 – Seed Culture / Inoculum
Time (hour)
Real Optical Density (nm)
0
0.029
4
0.733
Table 2 – Absorbance Optical Density Reading for 16 hours
No
Time (hour)
Absorbance
Dilution ratio
Real Absorbance
optical density,
(Sample : Distilled
Optical density ,
OD
Water)
Real OD
1
0
0.234
-
0.234
2
1
0.426
-
0.426
3
2
0.298
1:9
2.884
4
3
0.388
1:9
3.784
5
4
0.466
1:9
4.564
6
6
0.584
1:9
5.744
7
8
0.620
1:9
6.104
8
10
0.344
1:19
6.784
9
12
0.364
1:19
7.184
10
14
0.389
1:19
7.684
11
16
0.288
1:19
5.664
12
Table 3 – Cell Dry Weight (Concentration of the Biomass)
Empty
No
Time
Centrifuge
(hour)
Tube, m1,
(g)
Dried
Centrifuge
Tube +
Sample,
m2, (g)
Cell
Dry
Weight
(g)
Cell Mass
Concentration
(m1-
Ln X
X (g/L)
Ln
(X/X0)
m2)
1
0
1.084
1.087
0.003
3
1.098612
0
2
1
1.083
1.089
0.006
6
1.791759 0.693147
3
2
1.066
1.073
0.007
7
1.94591
4
3
1.078
1.082
0.004
4
1.386294 0.287682
5
4
1.055
1.060
0.005
5
1.609438 0.510826
6
6
1.087
1.091
0.004
4
1.386294 0.287682
7
8
1.075
1.079
0.004
4
1.386294 0.287682
8
10
1.071
1.075
0.004
4
1.386294 0.287682
9
12
1.054
1.060
0.006
6
1.791759 0.693147
10
14
1.067
1.073
0.006
6
1.791759 0.693147
11
16
1.073
1.079
0.006
6
1.791759 0.693147
0.847298
Table 4 – Net Growth Rate at Different Time
Time (hour)
Cell Mass Concentration, X (g/L)
Ln X
µnet (h-1)
0
3
1.098612
0.0000
1
6
1.791759
0.6931
2
7
1.94591
0.1542
3
4
1.386294
-0.5596
4
5
1.609438
0.2231
6
4
1.386294
-0.1116
8
4
1.386294
0.0000
10
4
1.386294
0.0000
12
6
1.791759
0.2027
14
6
1.791759
0.0000
16
6
1.791759
0.0000
13
Real Optical Density (nm)
Real Absorbance Optical Density vs Time
9
8
7
6
5
4
3
2
1
0
0
2
4
6
8
10
Time (hour)
12
14
16
18
Figure 1 - The Growth Curved Of E. Coli
Figure 1 shows the graph of real absorbance optical density over time. The graph shows
the lag phase from t = 0 until t = 1. The growth curve increases before reaching a stationary
phase at t = 6 h until t = 8 h. The growth curve then increases until t = 14 h before drastically
decreases.
Cell Mass Concentration, X (g/L)
Cell Mass Concentration, X Against Time
8
7
6
5
4
3
2
1
0
0
2
4
6
8
10
Time, t (h)
12
14
16
18
Figure 2 – Graph of Cell Mass Concentration, X versus Time
Figure 2 shows the graph of cell mass concentration, X versus time. The curve shows
rapid increase with peak at X = 7 g/L. Another peak shown is at X = 5 g/L.
14
ln X/X0 vs Time
0.9
0.8
0.7
y = 0.016x + 0.3694
ln X/Xo
0.6
0.5
0.4
0.3
0.2
0.1
0
0
2
4
6
8
10
12
14
16
18
Time
Figure 3 – Graph of ln X/X0 versus Time
Figure 3 shows the plot of ln X/X0 versus time. The slope of this plot will yield to the
value of the maximum growth rate, µmax. From the graph, the slope is 0.016. Thus, µmax = 0.016
h-1.
15
8.0
CALCULATIONS
Sample of Calculation for Determination of Dry Cell Weight (g),
Cell Dry Weight = Final Mass (m2) – Initial Mass (m1)
For 0th hour,
Cell Dry Weight = 1.087g - 1.084g
Cell Dry Weight = 0.003 g
Sample of Calculation for Concentration of Cell Mass, X
Concentration of Cell (g/L) =
Cell Dry Weight (g)
Volume of sample (L)
For 0th hour,
Concentration of Cell (g/L) =
0.001g
0.001L
Concentration of Cell (g/L) = 1g/L
Sample Calculation for ln X/Xo
For 0th hour,
ln (
𝑋
1g/L
) = ln (
)
𝑋0
1g/L
𝑋
ln ( ) = 0
𝑋0
Sample Calculation for the Maximum Growth Rate , Μmax
The value was obtained from the slope of the graph ln X/Xo vs time.
slope = μmax = 0.016 hr-1
Sample Calculation for the Doubling Time, td.
The time required to double the microbial mass is given by equation below:
td =
ln(2)
μmax
t d = 43.32 h
16
Sample Calculation for Net Growth Rate, µnet
μnet =
ln 𝑋2 − ln 𝑋1
t 2 − t1
For Lag Phase, at t = 0h until t = 1h
μnet =
0.6931 − 0.0000
1−0
μnet = 0.6931 h−1
17
9.0
DISCUSSION
This experiment is done to study the growth kinetics of microorganism in shake flask
experiment. In this experiment, the microorganism that are desired to be growth is known as
E.coli. This experiment is done by using batch culture process where there is not inlet of
substrates and outlet of product throughout the fermentation in the given time. The Terrific
broth medium is inoculated with the E.coli which then the growth of E.coli kept increasing
until it reached one point where the growth decelerates due to limited substrates concentration
and the presence of toxic metabolites (Growth Kinetics Study of Microorganism in Shake
Flask, 2017). The other objectives of this experiment is to determine the Monod parameters of
maximum growth rate (µmax), yield of substrate (YX/S), mass doubling time (td), saturation
constant (Ks) and specific growth rate (µnet). In order to determine the parameters, it is
necessary to construct a growth curve including lag, exponential, stationary and death phases.
The growth curve plotted in Figure 1 shows that the lag phase occur during time 0 to
time 1 where the reading of optical density (OD) shows 0.234 and 0.426 respectively. The lag
phase occurs immediately after inoculation of E.coli into the medium. It is known as the period
of the adaptation of cells into new environment where the cells started to embower their habitat.
They started to reorganize their molecular constituent every time they are transferred or
introduced to a new environment. From the graph, it can be seen that the lag phase duration is
short which only occurs for one hour due to the used of inoculum size by 10% from the volume
of the medium. This condition is favourable because it is crucial to obtain high productivity by
maintaining the lag phase as short as possible.
Other than that, the exponential phase can be seen through the growth curve starting
from time 1 to time 6. There is a significant increase in the value from time 1 to time 2 showing
the optical density value from 0.426 to 2.884 respectively followed by time 3, 4, 5 and lastly 6
which is 5.744. At this phase, cell started to grow rapidly along with their maximum growth
rate. In this phase, the cell mass and density increases exponentially with time. The nutrient
concentration contain in the medium is high in this phase resulting in substantive progress
which elevate the growth rate of cell. The exponential growth of this cell is assumed to be first
order. Thus, the maximum growth rate (µmax) of the cell can be determined from the slope of
graph ln X/X0 versus time plotted in Figure 3. The slope shows the value of µmax which are
0.016 hr-1. When the µmax has been obtained, it is possible to find the mass doubling time (td)
18
by using Equation 1. The mass doubling time is the time required to double the microbial mass
and the (td) achieved is 43.32 hour.
Next, the deceleration growth phase should follows the exponential phase. This phase
growth of E.coli decelerates a little bit due to depletion of one or more essential nutrients or
the accumulation of toxic by-products of growth. It occurs for a very short period of time.
However, from the graph in Figure 1, it can be seen that there is no deceleration phase. This is
maybe due to the cell do not detect the depletion of nutrients thus it straight forward carried on
to the stationary phase. It is assumed that the cell were readily restructured themselves to
prepare for cellular survival in the stationary phase due to limited sources of nutrient as their
food to grow.
Figure 1 also shows the stationary phase that can be seen during time 6 to time 8 where
the OD have a small difference and read as 5.744 to 6.104. This phase started when the net
growth rate is said to be zero where there is no cell division. It is also found that in this phase,
the growth rate is equal to the death rate where it is followed by the death phase. The cell were
in the duration approaching death due to lysis. However, even though the net growth rate is
zero and there is no cell division, the cell is still metabolically active and produce secondary
metabolites. This proves the result shown in the graph where there is an increase of optical
density reading showing growth of cell right after the stationary phase. This is because, during
the stationary phase, a second growth rate may occur because cells may grow on lysis products
of lysed cells (Shuler & Kargi, 2002). This is call Cryptic Growth.
Other than that, the condition where there is the second growth rate is also believed that
there may be due to presence of other microorganism known as the 2nd microorganism. The 2nd
microorganism cannot be identified as there is no further investigation being done. The second
growth rate is also believed to be the exponential growth of the 2nd microorganism. Therefore,
in this experiment there is the 2nd exponential phase due to presence of other microorganisms.
The microorganism grew exponentially from time 8 to time 14 where the OD shows reading
of 6.104 to 7.684 by consuming the E.coli as their source of nutrients or food to survive and
grew rapidly throughout the time.
Lastly, the growth curve proceed to the death phase. From Figure 1, it can be seen that
the death phase occur at time 14 to time 16 where the OD shows reading of 7.684 to 5.664
respectively. From the graph, it shows a sudden drop representing a sudden death of the 2nd
19
microorganisms. The sudden death of the 2nd microorganisms is believed to be caused by the
fact that E.coli have been totally consumed resulting to no source of nutrients. The 2nd
microorganisms do not experience stationary phase as it have no strength to undergo cellular
survival and they cannot survive with their own metabolism. That is why when the E.coli have
depleted or consumed, sudden death occur due to insufficient nutrient stocks in the medium.
However, it also assumed that there is presence of toxic product accumulation that lead to the
death of microorganisms.
The µnet for the respective phase are µnet, lag phase = 0.6931 h-1, µnet, exponential phase = µmax =
0.016 h-1, µnet, stationary phase = 0.0000 h-1, µnet, death phase = 0.0000 h-1. The result from calculation
shows inconsistency with theory where the growth rate for the lag phase shows a higher value
compared to the should be higher value of the maximum growth rate for the exponential growth
rate. The error could comes from the incorrect way of handling the experiment or due to human
error in taking the data for the cell dry weight. Based on the data from the absorbance optical
density, the concentration of the cell should be increases over time until t = 14 hour before
decreases until t = 16 hour. However, the calculated data shows inconsistency through out the
time. Thus, the maximum growth rate is suspected to wrongly determined. Thus, another
experiment should be carried out to ensure the consistency of the data. However, limitation of
time restrict the ability of the student to re – conduct the experiment for a better result of the
Monod parameters.
In order to better describe the growth kinetics, the yield coefficient (YX/S) is the best
parameter to be determined. Yields coefficient is define as the rate of formation of cell to the
rate of consumption of the substrates. Another parameter that can be determined is saturation
constant (Ks). Unfortunately, due to limited sources of data, the yield coefficient (YX/S) and
saturation constant (Ks) cannot be determined. This is because, the experiment is lack of
substrates concentration data thus outreach the value of the parameters needed. However, this
experiment have been conducted successfully and most of the objective have been achieved.
20
10.0
CONCLUSION
The growth kinetics of E.coli is studied using the shake flask experiment by plotting the graph
of real absorbance optical density versus time. The lag phase occur from t = 0h until t = 1h as
the E.coli familiarize themselves with new environment in the media. There two exponential
phase from t = 1 h until t = 6 h and from t = 8h until t = 14h. The maximum growth rate of the
E.coli is believed to be found from the exponential curve which yield to µmax = 0.016 h-1. The
presence of the second exponential phase is due to the cryptic growth and the possibility of the
presence of second microorganism consuming the E.coli as the food supplies. The deceleration
phase cannot be determined. The death phase occur at t = 14h until t = 16h where the second
microorganism is believed to drastically die due to the abrupt loss of food supplies and their
inability in surviving with their own metabolism. The µnet for the respective phase are µnet, lag
phase
= 0.6931 h-1, µnet, exponential phase = µmax = 0.016 h-1, µnet, stationary phase = 0.0000 h-1, µnet, death phase
= 0.0000 h-1. Unfortunately, due to limited sources of data, the yield coefficient (YX/S) and
saturation constant (Ks) cannot be determined.
21
11.0
RECOMMENDATIONS
For this experiment, there are certain precaution and recommendation that need to be
considered to ensure to get a more reliable and accurate results. First and foremost, before
starting the experiment the sample must be autoclave first to avoid contamination which can
affect the results. Besides that, the bottles which contain inoculum and media are plugged with
cotton wool and wrap with aluminium foil to avoid contamination. Moreover, the sampling
tube should be put into the refrigerator immediately after taking a sample as well as the flask
should be put immediately in thermostate rotary shaker. Before taking the reading of
absorbance analysis, make sure that the spectrophotometer is calibrated to zero by blank. In
addition, cuvettes must be wipe with kim tissue to clean the sample and prevent any scratch so
that the light from the spectrophotometer can pass through the cuvette and will not affect the
readings. The experiment must be carried out under laminar flow and using aseptic technique
to transfer the inoculum to media so that there is no infections of the bacteria. Last but not least,
wear appropriate PPE such as gloves and mask as well as spray some ethanol on the gloves so
that there is no infections.
22
12.0
REFERENCES
Fogler, H. S. (2006). Element of Chemical Reaction Engineering. Michigan: Prentice Hall.
Growth Kinetics Study of Microorganism in Shake Flask. (February, 2017). Lab Manual
Faculty of Chemical Engineering UiTM Shah Alam. Shah Alam, Selangor: UiTM
Shah Alam.
Jitendra, P. (1 October, 2017). Bacterial Growth Monod Equation. Retrieved from Slide
Share: https://www.slideshare.net/chondu100/bacterial-growth-curve-monodsequation
Microbial Growth. (2016). Retrieved from Bio CS Montana:
https://www.cs.montana.edu/webworks/projects/stevesbook/contents/chapters/chapter
002/section002/black/page001.html
Monod Equation. (n.d.). Retrieved from Wikipedia:
https://en.wikipedia.org/wiki/Monod_equation
Panikov, N. (1995). Microbial Growth Kinetics. Springer Science & Business Media.
Shuler, M. L., & Kargi, F. (2002). How Cells Grow. In M. L. Shuler, & F. Kargi, Bioprocess
Engineering Basic Concepts Second Edition (pp. 155-200). Prentice Hall PTR.
23
13.0
APPENDICES
Figure 4 - Spectrophotometer used to measure the absorbance optical density
(a)
(b)
Figure 5 - (a) 1 mL of sample is taken from the media with 10% innoculum, (b) the sample
is then taken into the cuvette for the reading of absorbance optical density using the
spectrophotometer
24
Figure 6 - The dried sample is further put into the dessicator to remove the remaining
moisture present in the tubes
25