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Laboratory 1: Growth Kinetics Study of Microorganism in Shake Flask

1. Abstract

This experiment is carried out to study the growth kinetics of microorganisms in shake flask. E.coli is
grown in a LB broth medium and being fermented for 24 hours. Throughout the fermentation, the cell
culture is taken out for every 3 hours and protein test, glucose test and cell dry weight are being
performed. As for the optical density analysis, the absorbance reading from the spectrophotometer is
taken while for the glucose test, the reading of glucose level is taken from the YSI 2700 Select
Biochemical Analyzer or can also being performed by using DNS reagent and the absorbance value is
taken. These absorbance values will then being compared with the standard curve to get the glucose
concentration inside the shake flask at particular time. 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 viral which
contains the biomass before and after the drying process is recorded to get the dry cell weight.
For the optical density of the cell, the absorbance value showed an increment which indicating that the
cell was growing and number of cell is increased in the shake flask. The glucose concentration,
however, cannot be determined as the absorbance values were increased and decreased unevenly and
comparison cannot be made with the standard curve as the data for the standard curve are not
consistent giving inaccurate curve. Therefore no conclusion can be made about the glucose
concentration in the shake flask. Supposedly, as the number of cell increased, the glucose
concentration would decrease as the glucose consumption by the cells is increased.
The dry cell weight in the other hand can be seen that there is an increment from the beginning of the
cultivation until the 6
th
hour and showed unstable changes until the 24
th
hour. Supposedly, as the
number of cell increased inside the shake flask, the cell dry weight also should be increased.
2. 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.

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In order to prevent any contamination to the culture, shake flask must be plugged. Different plug can
be made of cotton-wool, glass wool, polyurethane foam, gauze or synthetic fibrous material. The plug
has to prevent airborne microorganism from getting into the medium while at the same time allowing
free flow of air into the flask.
The cultures are incubated at certain temperature and shaking frequency 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,
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
growth is inhibited because of the decreasing substrate concentration and the presence of toxic
metabolites.
3. Aims

To study 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

4. 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. 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:




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= x -------------------1
where
x is the concentration of microbial biomass
t is the time, in hours
is the specific growth rate, in hour
-1

on integration, equation (1) gives

------------------2
where

is the original biomass concentration

is the biomass concentration after time interval, t hours

During the exponential phase, the organism is growing at its maximum specific growth rate,

for
the prevailing conditions.
Equation 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 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 and the residual growth-limiting substrate as follows:
=

where

= maximum growth rate

s = residual substrate concentration

= substrate utilization constant

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

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Figure 4.1: Growth curve of microorganism based on cell number analysis

5. Apparatus and material

E.coli
Luria Bertani Broth
Distilled water
Shake flask
Cotton-plugged
Incubator shaker
Cuvettes
Centrifuges
Micropipetor
Pipette tips
Laminar flow
70% ethanol
Lighter and Bunsen burner
Graduated cylinder
Schott bottle
DNS reagent




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6. Procedures


Part 1: Preparation of inoculated fermentation medium

1. 500ml shake flask, bunsen burner, measuring cylinder, LB broth and inoculums are brought
into the laminar flow.
2. Under aseptic technique, 50 ml of media is transferred into 500ml shake flask.
3. Then 6 ml of inoculums is added into the shake flask resulting in final volume of 56ml.
4. The shake flask is plugged with cotton-plugged.
5. The shake flask is swabbed with 70% ethanol.
6. The shake flask is incubated at 350 rpm; T=30C; 24 hours.
Part 2: Sampling for cell dry weight
1. 1ml of biomass concentration is taken out.
2. The 1ml biomass concentration is transferred into micro centrifuge tube. An empty micro
centrifuge tube must be weighted first.
3. The sample is then centrifuged for 10 minutes at 10000 rpm.
4. After that, the supernatant of the sample is taken out carefully without taking out any
biomass.
5. The biomass is then left dried inside an oven at 80C for overnight.
6. The dried biomass is then being placed inside a dessicator to let it cool before rapidly
weighing on an analytical balance.
Part 3: Glucose analysis
1. 1ml of biomass concentration is taken out.
2. The 1ml biomass concentration is transferred into micro centrifuge tube.
3. The sample is then put onto turntable of YSI 2700 Select Biochemical Analyzer for direct
analysis of glucose concentration.
4. Another method of glucose analysis is by using DNS reagent.
5. 1.5ml of DNS reagent is added into 0.5ml of the biomass sample inside a capped test tube
6. The mixture is heated at 90C for 10 minutes to develop the red-brown colour.
7. The heated mixture is then cooled to the room temperature for 2-3 minutes in a cold or ice
water.
8. The mixture is then being diluted with 10ml of distilled water.
9. The absorbance is checked with a spectrophotometer.
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Part 3: Sampling for absorbance analysis/ optical density
1. 2ml of biomass concentration is taken out and being transferred into micro centrifuge tube.
2. The spectrophotometer is calibrated to zero by blank consisting of 2ml LB Broth.
3. The biomass concentration is then being transferred into a cuvette and optical density
measurement is taken with wavelength set at 600nm.
4. More absorbance means higher number of cell.
Part 4: The preparation of glucose standard curve
1. The 20g/L, 40g/L, 60g/L, 80g/L and 100g/L of glucose concentration is prepared by weighing
the suitable amount of glucose and diluted with 10ml of distilled water.
2. 1.5ml of DNS reagent is added with 0.5ml of the glucose sample inside a capped test tube
3. The mixture is heated at 90C for 10 minutes to develop the red-brown colour.
4. The heated mixture is then cooled to the room temperature for 2-3 minutes in a cold or ice
water.
5. The mixture is then being diluted with 10ml of distilled water.
6. The absorbance is checked with a spectrophotometer




















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7. Results

Table 7.1: The results showing the optical density of the cell, cell dry weight and glucose
concentration per time
Hour
Absorbance readings:
Optical density
(spectrometer
wavelength: 600nm)
Biomass concentration: cell dry
weight after 24h (g)
Glucose concentration
(g/L)

Initial Final Net absorbance Glucose
0 0.340 1.052 1.066
0.014
- 0.025
3 1.812 1.153 1.170
0.017
0.071
6 2.597 1.045 1.070
0.025
0.066
9 2.688 1.153 1.175
0.022
0.088
12 2.734 1.167 1.183
0.016
0.079
21 2.801 1.157 1.176
0.019
0.094
24 2.800 1.156 1.175
0.019
0.087


Figure 7.1: The graph showing the absorbance value for optical density
0
0.5
1
1.5
2
2.5
3
0 5 10 15 20 25 30
t
i
m
e

(
h
)

absorbance
absorbance value for optical density
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Figure 7.2: The graph showing the cell dry weight per time

Table 7.2: Data for standard curve of glucose test
Glucose concentration (g/l) Absorbance
0 0.039
20 2.141
40 2.750
60 2.760
80 2.505
100 1.791

0
0.005
0.01
0.015
0.02
0.025
0.03
0 5 10 15 20 25 30
t
i
m
e

(
h
)

cell dry weight (g)
cell dry weight curve
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Figure 7.3: Graph showing the standard curve for the glucose concentration

8. Calculations

Cell dry weight
Net weight = final weight initial weight
= 1.066g 1.052g
= 0.014g






0
0.5
1
1.5
2
2.5
3
0 20 40 60 80 100 120
a
b
s
o
r
b
a
n
c
e

v
a
l
u
e

glucose concentration (g/L)
absorbance value vs glucose concentration
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9. Discussions

This experiment is carried out to study the kinetic growth of microorganism. E.coli is selected as the
cell and being cultivated inside a shake flask. The growth of microorganism in shake flask is a simple
method of fermentation. The nutrients for the microorganism are being supplied by the media which
contain the carbon sources. The flask is shaken during the cultivation to mix the cell and the media;
increase the homogeneity between these two and also to provide aeration for the cells. The culture is
gone through the fermentation process for 24 hours. Within that period, the biomass/cell sample is
taken out for every 3 hours to analyze the concentration of the cell (g/L), the cell dry weight and the
glucose concentration.
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 absorbance reading is
increase from the beginning of the experiment until the 21
st
hour and decrease slightly at the 24
th
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 24
th
hour indicating that the cell growth
has reach its deceleration phase where the growth of the cell is started to slow down. The
decelerating growth phase is where the culture is in a transient state. During this 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. In cell growth, the cell will go through several phases like lag,
exponential, deceleration, stationary and death phase.

In cell cultivation, the cells themselves need food or carbon sources like glucose for growth. In batch
fermentation for example in this experiment, the glucose can be the limiting factor for the cell growth
or we called it as substrate limiting growth. For this condition, the Monod equation can be used to
predict the growth rate and the cell concentration inside the shake flask. In addition, the glucose
concentration can be known by testing the cell sample into the glucose analyzer and the direct glucose
concentration can be obtained. In other way, the glucose concentration is also being obtained by
mixing the sample with DNS reagent. The DNS reagent will be reduced to 3-amino,5-nitrosalicylic
acid in the presence of free carboxyl group (glucose) and absorbance reading can be taken through the
spectrophotometer.


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As for this experiment, the glucose test showed no pattern of changes in absorbance values. These
values increase and decrease unevenly. This might be due to some mistakes occurred during the
glucose test where the volume of sample and DNS reagent that need to be mixed is incorrectly taken.
This has affected the accuracy of the absorbance reading. From the absorbance reading, the
concentration of the glucose can be obtained by referring to the glucose standard curve. The glucose
concentration should be decreased as the number of cell inside the flask is increased. This is because
as the number of organism increases, nutrients are consumed and becoming lesser. However, this
cannot be shown from the results obtained due to some mistakes occurred throughout the experiment.

Figure 9.1: The changes in amount of glucose, lactose and biomass in cell culture per time.
Another analysis that can be performed to analyze the cell sample is 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. The remained cell is then
being dried inside an oven for 24 hours. The dry cell weight is finally taken to know the weight of the
cell that present at particular time during the cultivation. In this experiment, the cell dry weight is
increased from 0
th
hour until 6
th
hour and gradually decreased from the 9
th
hour to 12
th
hour and
increased until the 24
th
hour. The cell dry weight should increase when the number of cell increased
inside the shake flask.


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10. Conclusions

At the end of this experiment, microorganism is suitable to be fermented inside a shake flask and it is
a simple method to investigate the growth kinetics of the microorganism. Knowledge of microbial
growth kinetics is essential to determine when to harvest the culture for different purposes. For a
growth-linked product, it is desirable to harvest the culture at the late exponential growth phase. On
the other hand, for a non-growth-linked product, it would be desirable to harvest the culture at the
stationary growth phase.
As microorganism will go through several phases in their growth, several analyses on the cell need to
be done to know the growth kinetics of the cell and the duration for each phase. This includes the cell
concentration, glucose concentration and also the cell dry weight analyses. This method can be done
in the laboratory before the fermentation or the cultivation of microbes in large scale is performed.
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 is also continuously changing in physical and chemical
conditions.
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.

The batch culture system is still widely used in certain industrial processes for example brewery
industry because of its easy management of feed stocks. These advantages allow the use of unskilled
labour and low risk of financial loss. Low level of microbial contamination in fermented products is at
time tolerable, as long as the microbial contaminants are not pathogenic and do not alter the desired
properties of the product, such as taste, colour and texture.






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11. Recommendations

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.
The supernatant of cell concentration should be taken out carefully without any taking out of
the biomass.
The cap of the viral must be opened to fasten the drying process of the biomass in the oven.
Wash hand after handling the culture.
Disinfect the work area with 70% alcohol before handling the culture.
Dispose of all contaminated materials in appropriate containers.

12. References


PF Stanbury and A Whitaker.,1984. Principles Of Fermentation Technology. Pergamon Press

Crueger W and Crueger A., 1990. Biotechnology: A Textbook of Industrial Microbiology. Sinauer
Associates, Sunderland Massachusetts.

Pirt SJ., 1975. Principles of Microbe and Cell Cultivation. Blackwell Scientific, Oxford.













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13. Appendices


Figure 12.1: The microorganism and media is
cultivated

Figure 12.2: The microorganism is being
incubated inside the incubator.


Figure 12.3: The microorganism is being
transferred into the viral tube.

Figure 12.4: The sample is being centrifuged.

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Figure 12.5: For protein test, the absorbance
reading is taken using spectrophotometer.

Figure 12.6: Glucose analyzer for glucose
concentration reading.

Figure 12.7: The sample for cell dry weight is
being heated overnight using oven.

Figure 12.8: After being heated, the sample is
being cooled inside a desicator.
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Figure 12.9: The sample is tested for glucose
concentration by using DNS reagent.
Figure 12.10: The sample being transferred into
viral tube.