UNIVERSITI TEKNOLOGI MARA

FACULTY OF CHEMICAL ENGINEERING

REACTION ENGINEERING LABORATORY (CHE506)
NAME : 1) MUHAMAD HAZIQ BIN DAUD
2) NUREZLIN AZIERA BINTI ROSIDIN
3) NUR FARAH AMIRAH BINTI MOHD RASHDAN
4) NORASYIKIN BINTI ALIM

STUDENT NO. : 1) 2016238888

2) 2016249968
3) 2016249838
4) 2016249894
GROUP : EH220 5D (GROUP 3)
EXPERIMENT : L6 – GROWTH KINETICS OF MICROORGANISM IN
SHAKE FLASK
DATE PERFORMED : 18th SEPTEMBER 2018
SEMESTER : SEMESTER 5
PROGRAMME / CODE : CHEMICAL ENGINEERING / EH220
SUBMIT TO : MADAM SUHAILA BINTI MOHD SAUID

No. Title Allocated Marks (%) Marks

1 Abstract/Summary 5
2 Introduction 10
3 Aims 5
4 Theory 10
5 Apparatus 5
6 Methodology/Procedure 10
7 Results 10
8 Calculations 10
9 Discussion 20
10 Conclusion 5
11 Recommendations 5
12 Reference / Appendix 5
TOTAL MARKS 100

Remarks:
Checked by:

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Date:
TABLE OF CONTENT

NO CONTENT PAGE NUMBER

1 ABSTRACT 1
2 INTRODUCTION 2
3 OBJECTIVES 3
4 THEORY 3
5 MATERIALS AND APPARATUS 6
6 PROCEDURES 7
7 RESULTS 10
8 SAMPLE CALCULATION 11
9 DISCUSSION 14
10 CONCLUSION 16
11 RECOMMENDATION 17
12 REFRENCES 18
13 APPENDIX 19
1.1 ABSTRACT

The purpose of conducting this experiment is to study the growth of Escherichia coli
(E. Coli) in shake flask. Escherichia coli is a gram-negative, coliform bacterium of the genus
Escherichia that is commonly found in lower intestine of warm-blooded organism.
Erlenmeyer flow is used to observe the growth of microorganism. Terrific Broth (TB)
medium, 350 rpm and 37˚C favour the growth of E. coli. The bacterium is fermented in 24
hours for every 1 hour for 6 hours, 2 hours for 14 hours and 3 hours for the rest of 23 hour.
Meanwhile, value of absorbance is determined by using spectrophotometer. Cell dry weight is
obtained when mass concentration inside flask is dried. Besides, optical density analysis,
absorbance reading from spectrophotometer is taken. In addition, cell dry weight is obtained
after mass concentration is being dried overnight. Weight of tube contain biomass before and
after drying process is recorded to set cell dry weight. Graph is obtained to observe changing
in growth kinetic of the cell. Graph of growth include lag, log, stationary and death phase
other parameter is being considered such as cell concentration, absorbance reading and cell
dry weight.

1
1.2 INTRODUCTION

When microbial cells inoculated in batch reactor contains fresh culture medium, phase of
growth can be observed. The phase of growth is divided into several phase which are initial
lag phase, exponential growth phase that refer as logarithmic phase (determine cell number
and cell dry). Then, stationary phase followed by death phase which where the cell number is
declining.

Fermentation processes are divided into two parts which are continuous and fed-batch
process. In this experiment, the shake flask fermentation was used. The shake flask
fermentation is an example of batch fermentation. In shake flask, usually Erlenmeyer flask is
being used to place and grow the microbes. It is a small equipment which equivalent to stirred
tank bioreactor. Other than that, it is also the cheapest and easiest way to culture
microorganism aerobically, in small volumes of nutrient broth.

The culture is then incubated at certain temperature 37˚C and placed in the incubator
shaker with the shaking frequency set to 350 rpm for 4 hours in order to achieve required
growth rate. The shaking agitates the medium and the culture to keep the mixture
homogenous and ensure aeration create aerobic condition. The medium culture is usually
inoculated with the microorganism. The growth will keep increasing until at certain point, the
growth is inhibited because of decreasing substrate concentration and the present of toxic
metabolites.

Escherichia coli is used as microorganism. Terrific broth is used because it is the most
commonly used medium in molecular biology for E. coli culture. The relationship between
specific growth of microbial population and the substrate concentration is crucial tool in all
fields of microbiology, be it physiology genetics, ecology and biotechnology.
1.3 OBJECTIVES

The objectives for the growth kinetics study of microorganisms in shake flask experiment are
stated as below.

I. To study the growth kinetic of microorganism in shake flask experiment.

II. To construct a growth curve including lag, log, stationary and death phase.

1.4 THEORY

The rate of growth is directly related to cell concentration. The rate of microbial growth is
characterized by the net specific growth rate, and defined as

𝑑𝑋
= 𝜇𝑛𝑒𝑡 𝑋
𝑑𝑡
By rearranging the equation, it becomes

1 𝑑𝑋
𝜇𝑛𝑒𝑡 =
𝑋 𝑑𝑡
𝜇𝑛𝑒𝑡 = 𝜇𝑔 − 𝑘𝑑

Cell growth is the primary response of viable cells to substrates and nutrients. Products
formation is a secondary response.

Where;

X= cell mass concentration (g/l)

t = time (h-1)

𝜇𝑛𝑒𝑡 = 𝑛𝑒𝑡 𝑠𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑔𝑟𝑜𝑤𝑡ℎ 𝑟𝑎𝑡𝑒 (ℎ−1)

𝜇𝑔 = gross specific growth rate

𝑘𝑑 = rate of loss of cell mass due to cell death or endogenous metabolism

Mass doubling time 𝜏𝑑 is calculated based on the cell numbers and the net specific rate of
replication

𝜏𝑑 = ln 2
𝜇𝑛𝑒𝑡 (h)

In order to define more details about the theory of growth kinetics of microorganisms,
the growth curve of the bacteria was analyzed. In the experiment, we placed the bacteria in
the flask, where the bacteria got their nutrients in a controlled condition. Terrific broth was
used as the main supplies for bacteria to keep their growth. If all the nutrients required are
supplied and they grow at a very conducive environmental, the increase in numbers of
bacteria can be measured as they are growth rapidly. This can be further understood in the
graph of growth curve. At certain time, the bacteria will undergo death phase as all the
nutrients needed for them to grow has been used up completely resulting in the bacteria to
have not enough supplies and nutrients to grow. This analyzation can be further explained in
the growth curve versus time that include 4 phases; lag phase, exponential phase, stationary
phase and death phase.

Figure 1: Stages of Bacterial Growth Curve

In the lag phase, fresh cultured bacterium is adjusted to which the media placed on. The
bacteria are let to ‘feel’ the new environment and surroundings in order to adapt themselves.
The cell mass (X) and cell number density (N) are about to increases with time.

The cells can reach up to millions and billions of cells as they are growing rapidly once
they enter the exponential phase. They began to divide themselves in a constant pace.
Exponential phase is also known as logarithmic phase. This is because the number of cells is
calculated in logarithmic functions. Metabolic processes also occurred at constant rate and is
influenced by the conditions of temperature, pH, and properties of medium. Since the
nutrients concentration are higher in this phase, the growth rate is independent to nutrient
concentration.

The stationary phase is marked by a plateau in growth. At this phase, 𝜇𝑛𝑒𝑡 = 0, ie no cell
division, or growth rate is equal to death rate. But cells are still metabolically active and
produce secondary (non-growth related) metabolites. The production of certain metabolites is
enhanced during the stationary phase, due to the metabolite deregulation.

The population begins to decline at the death phase. The growth decelerates due to
either depletion of one or more essential nutrients or the accumulation of toxic-by products.
The results consist of unbalanced growth and also the cell composition and size changed. The
size induced by nutrient depletion or waste accumulation causes the restricting of the cell to
increase the prospect of survival in a hostile environment.
1.5 MATERIALS AND APPARATUS

1 Microbe: Escherichia coli 9 Thermostated rotary shaker/ incubator shaker

2 Shake flask (250 ml and 1000ml flask) 10 Cotton plugged

3 Eppendorf tubes/ falcon tube (1.5ml) 11 Ethanol (70% ethanol for swabbing for
sterility)

4 Cuvettes (spectrophotometer) 12 Graduated flask for measuring media

(1000ml, 100ml, 10ml)

5 Ph meter 13 Bunsen burner for sterility

6 Biochemical analyzer 14 HPLC for product measurement like ethanol

7 Laminar flow hood for sterility 15 Refrigerated centrifuge

8 Media (for specific microbe) 16 Spectrophotometer

1.6 PROCEDURES

Temperature Shaking Shake Filling/ Media Inoculum pH Carbon

(°C) Frequency Flask Working Type Percentage Source
(rpm) Size Volume
(mL)
37 350 500 150 TB 10 7 Glycerol

I. Preparation of media

a) Terrific Broth (TB) preparation

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

2. The media was autoclaved at 121°C for 20 minutes.
3. Glycerol and media have 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 were taken on agar plates and added to the sterilized media
of 150mL in 1000mL shake flask.
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 23 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 was 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 below is referred for planned usage of sample volume:

No. Sample Name Volume ( L) Use 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 was transferred into a cuvette and the optical density measurement
were using a spectrophotometer with the wavelength set at 600nm.
2. The spectrophotometer was 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 are weighted and noted as initial mass. (empty container)
2. 1 mL sample was added to weighted centrifuge tube.
3. The sample was centrifuged at 10,000 rpm and at T of 4°C for 20 minutes.
4. The supernatant was taken out and washed with distilled water and centrifuging may
be repeated.
5. The centrifuge tube is dried (left with biomass only) in oven at 80°C for overnight.
6. The dried centrifuged tubes were 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

VI. Glucose Analysis. (Substrate Concentration) (S) (g/L)

1. Sample of 1.5 mL is transferred into the micro centrifuge tube and refrigerated
centrifuged for 10 minutes at 10,000 rpm.
2. Then, the supernatant is taken out into cuvette and put onto turntable of YSI 2700
Select Biochemical Analyzer for direct analysis of glucose (dextrose) concentration.
3. The glucose analysis is based on Glucose Oxidase that has been immobilized in the
YSI Dextrose Membrane (YSI 2365).
 1.7 RESULTS

A. Seed/Inoculum

No. Time (h) Real OD

1 Initial (0 hour) 0.299
2 Final (4 hour) 1.885

B. Main Experiment

Absorbance Cell Dry

Time Optical Absorbance Empty Dried m2- Weight,X
No (h) Density OD Real OD Centrifuge Centrifuge m1(g) (g/L) ln X Ln(X/Xo)
1 0 0.311 0.311 1.079 1.0814 0.0024 1.2 0.182322 0.000000
2 1 0.945 0.82 1.0666 1.0724 0.0058 2.9 1.064711 0.882389
3 2 1.575 2.2 1.0649 1.0728 0.0079 3.95 1.373716 1.191394
4 3 1.754 2.82 1.0733 1.0823 0.009 4.5 1.504077 1.321756
5 4 1.842 2.8 1.0707 1.0783 0.0076 3.8 1.335001 1.152680
6 5 1.883 3.9 1.0713 1.0688 0.0073 3.65 1.294727 1.112406
7 6 1.894 4.06 1.0615 1.0791 0.0078 3.9 1.360977 1.178655
8 8 1.899 4.15 1.0515 1.0582 0.0067 3.35 1.20896 1.026639
9 10 1.953 3.55 1.0553 1.0634 0.0081 4.05 1.398717 1.216395
10 12 1.984 4.5 1.0705 1.0713 0.0068 3.4 1.223775 1.041454
11 14 2.009 5.35 1.0664 1.078 0.0116 5.8 1.757858 1.575536
12 16 2.04 5.65 1.0648 1.0765 0.0117 5.85 1.766442 1.584120
13 18 2.036 5.47 1.0541 1.0667 0.0126 6.3 1.84055 1.658228
14 21 2.244 1.42 1.0935 1.1032 0.0097 4.85 1.578979 1.396657
1.8 CALCULATIONS

Sample Calculation of Cell Dry Weight, X:

(𝑚2−𝑚1)𝑔
X (g/L) =
0.002 𝐿

(1.0814−1.0790)𝑔
= 0.002 𝐿

0.0024 𝑔
= 0.002 𝐿

= 1.2 g/L

Graph of Absorbance Real OD vs. Time (h)

7
Absorbance Real OD

0
0 5 10 15 20 25
Time (h)

Graph 1: The growth curve of E. Coli plotted using absorbance optical density.
 Cell Dry Weight vs Time
7

6
Cell Dry Weight

0 5 10 15 20 25
Time (h)

Graph 2: Graph of cell dry weight vs time

ln (X/Xo) vs Time
1.6 y = 0.5165x
1.4 R² = 0.8016

1.2
1
ln X/Xo

0.8
0.6
0.4
0.2
0

0 0.5 1 1.5 2 2.5 3 3.5 4

Time (h)

Graph 3: Graph of ln X/Xo vs time

Maximum growth rate, µmax = slope of the graph

= 0.5165 h -1
 ln X vs Time
2
1.8
1.6
1.4
1.2
1
0.8
ln X

0.6
0.4
0.2
0

0 5 10 15 20 25
Time (h)

Graph 4: Graph of ln X vs time

Maximum net growth rate,

ln 𝑋𝑡=18−ln 𝑋𝑡=0
µ ne
= 𝑡=18−𝑡=0
t

1.84055−0.182322
= 18−0

= 0.0921 h -1

Doubling time, td
ln 2
= µ𝑚𝑎𝑥

ln 2
= 0.5165
= 1.3420 h
1.9 DISCUSSION

The experiment of growth kinetics study of microorganisms in the shake flask was
conducted in order to study the growth kinetics of microorganisms in the shake flask. Other
than that, we are also required to construct the growth curve including the lag, log, stationary
and death phases. For this experiment, Escherichia coli (E. Coli) was selected as the cell and
it was being cultivated inside a shake flask. This is because shake flask fermentation is the
cheapest and simplest technique to grow bacteria aerobically. Next, we chose Terrific Broth
as the media used to supply nutrients to the microorganisms since it contains carbon sources
(glycerol). The flask was then shaken during agitation in order to ensure aerations happened
and also to increase the homogeneity between cells and media. The experiment was
conducted for 24 hours long. The cell is taken out for every one hour in order for us to
analyse its cells concentration and cell dry weight.

Next, in order to analyse the cells concentration inside the shake flask, the absorbance
reading for optical density was measured using the spectrophotometer. Theoretically, the
higher the value of the absorbance reading, the higher the concentration of cells. Therefore, as
for this experiment, based on the graph of absorbance (real OD) vs time plotted, we can see
that the absorbance reading fluctuates with time when it was supposed to be increasing
gradually from the beginning of the experiment and then decreasing at the last few hours.
From the graph, the absorbance reading increased from the beginning of the experiment until
the third hour but due to some error occurred, the absorbance reading decreased slightly at the
third hour. Then, the absorbance reading increase gradually from the fourth hour until the
eighth hour. However, due to error occurred while conducting the experiment, the absorbance
reading dropped slightly from the eighth hour to the tenth hour. After that, the absorbance
reading starts to increase steadily at the 10th hour until the 17th hour. The absorbance then
starts to decrease from the 17th hour onwards. Therefore, based on the observations made
from the graph, we can explain that the increment in the absorbance reading indicates that the
concentration of cells is increasing throughout the cultivation thus proving that the cell is
growing. However, the graph was seen to be decreasing at the 17th hour. This indicates that
the cells have already reached its decelerating phase where the cells growth starts to slow
down. This phase is also known as death phase. Death phase occurs due to the depletion of
essentials nutrients needed or the wrong living conditions resulting the bacteria to die.
Therefore, when the bacteria die, the
concentration of cell decrease thus proving the decrement of absorbance reading plotted in
the graph above.

Since we are not required to measure the glucose concentration of the cell sample,
therefore, we can use another type of analysis where the cell sample is analysed by taking the
dry weight of the cell. In this method, the dried centrifuge tubes were first weighted and the
mass for each of the tubes were recorded. The cell then is taken out from the cultivation flask
and transferred into the centrifuge tube. The tubes were then centrifuged at 10,000 rpm to
separate the supernatant and the cells. The remained cell is the being dried inside the oven for
24 hours. The cell dry weight can finally be calculated by subtracting the weight of dried
centrifuge tubes with empty centrifuges tube divided with volume of cell transferred. In order
to meet the objectives of the experiment, a graph of cell dry weight against time was plotted.
Based on the graph plotted, we can see that the cell dry weight varies with time when it was
supposed to increase when the cell concentration increase. Therefore, the results for cell dry
weight are not very dependable since the values are inconsistent in increasing and decreasing.
This might probably due to some error happened while conducting the experiment. Besides
that, the graph of ln (X/Xo) against time was also plotted. From this graph we can determine
maximum growth rate, µmax. The maximum growth rate is also equal to the slope of the
graph, therefore, µmax = 0.5165 h -1. Next, the graph of ln(X) against time plotted helps us to
determine the maximum net growth rate, µnet. The equation used is stated as follows
l n 𝑋𝑡=18−ln 𝑋𝑡=0
Maximum net growth rate, µnet =
𝑡=18−𝑡=0

Therefore, based on the calculation made, µnet = 0.0921 h -1 and the doubling time is 1.342 h.

However, the results obtained from the experiment might be different from the actual
results as there might be some error occurs throughout the experiment. For example, the
students might have extracted and removed the cells along with the supernatant and human
error can contribute to the inaccuracy in weight reading. Besides that, the apparatus used
during conducting the experiment might not be properly cleansed. There might be some
impurities and contaminants inside the beaker and flask used thus affecting the results of the
experiment. Next, fail to handle the apparatus correctly. For example, while using the
spectrophotometer, students sometimes forget to clean the outer part of the cuvette resulting
in the inaccuracy of the absorbance reading taken. Last but not least, students failed to
sterilize the mouth of the shake flask and cotton plugged with flame that later on will cause
contamination.
1.10 CONCLUSION

In conclusion, microorganisms undergo several phases during their growth which

includes lag, log, stationary and also death phases. The cell sample inside the flask was
analysed and from the observation made, we can see that the absorbance reading fluctuates
with time when it was supposed to be increasing gradually from the beginning of the
experiment and then decreasing at the last few hours. Even though we got two reading that
happens to be disobeying the theory, the overall absorbance reading was seen to behave based
on the theory made. This could probably due to the fact that some errors might have occurred
while conducting the experiment. Next, a graph of cell dry weight against time was also
plotted. Based on the graph plotted, we can see that the cell dry weight varies with time when
it was supposed to increase when the cell concentration increase. Therefore, the results for
cell dry weight are not very dependable since the values are inconsistent in increasing and
decreasing. This error occurs probably due to the ways the students handled the separation
process of the supernatant from the samples. They might have extracted and removed the
cells along with the supernatant. Other than that, maximum growth rate, maximum net growth
rate and doubling time were also calculated. Based on the calculation made, µmax = 0.5165 h
-1, µnet = 0.0921 h -1 and the doubling time is 1.342 h. All in all, the experiment was
successfully conducted. The objectives of the experiments were achieved.
1.11 RECCOMENDATION

After conducting the experiments, we come up with a few steps to be taken as a safety
measures and to make sure the experiment will be successful.

I. Before using the spectrophotometer, cleanse the outer surface of the cuvette with
distilled water and ensure that the surface was dried properly before inserting it into
the spectrophotometer.
II. Ensure that all the apparatus used were properly cleansed before conducting the
experiment.
III. Avoid contamination whilst handling the biomass by practising aseptic techniques.
IV. Ensure that the mouth of the shake flask and the cotton plugged is always sterilized
with flame from Bunsen burner in order to avoid contamination.
V. Ensure that the separation process of the supernatant from the sample are handled
properly. The supernatant of the cell concentration should be taken out properly
without removing the cells along.
VI. If the spectrophotometer reading exceeds 0.7, dilution must be made and the optical
reading should be taken again.
VII. The experiment must be conducted under laminar flow in order to prevent the culture
from being contaminated.
1.12 REFRENCES

Fogler, H. (2013). Elements of Chemical Reaction Engineering. Harlow: Pearson.

Bacterial growth curve | Encyclopedia.com. (2018). Retrieved from

https://www.encyclopedia.com/science/dictionaries-thesauruses-pictures-and-press-
releases/bacterial-growth-curve

Kovárová-Kovar, K., & Egli, T. (2018). Growth Kinetics of Suspended Microbial Cells:
From Single-Substrate-Controlled Growth to Mixed-Substrate Kinetics. Retrieved from
https://mmbr.asm.org/content/62/3/646.full

The Bacterial Growth Curve and the Factors Affecting Microbial Growth. (2018). Retrieved
from https://www.thoughtco.com/bacterial-growth-curve-phases-4172692

Shuler, M. (2013). Bioprocess Engineering: Pearson New International Edition. Harlow:

Pearson.