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Growth of Escherichia coli in a

5 Litre Batch Fermentation


Vessel

AsAd Mughal
ID: 1421794
University of Birmingham
April 2015
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Abstract
Growth rate, doubling time and final cell yield was measured for a sample of Escherichia
coli which was grown in a 5 litre batch fermenter. A maximum growth rate of 0.966h -1 was
obtained. The results which were obtained have been discussed and evaluated with
methods to improving the process being suggested. The limiting factors that were identifies
have been discussed along with the advantages and disadvantages of the equipment used
for the growth of the sample.

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Contents
1. Introduction...............................................................................................................4
2. Methods....................................................................................................................4
3. Results.......................................................................................................................4
4. Analysis of Results and Discussion...........................................................................5
4.1 Analysis of Experimental Results.........................................................................5
4.1.1 Maximum specific growth rate......................................................................6
4.1.2 Mean Doubling Time......................................................................................6
4.1.3 Dry Cell Weight..............................................................................................7
4.1.4 Final Cell Yield................................................................................................8
4.1.5 Optical Density and Glucose Concentration with Time.................................8
4.1.6 Variation of pH and DOT (%) with Respect to Time.......................................9
4.2 Discussion of Results and Future Improvements.................................................9
5. Conclusion...............................................................................................................11
6. References..............................................................................................................11
7. Appendices..............................................................................................................12
7.1 Equipment and Procedure..................................................................................12
7.1.1 Equipment...................................................................................................12
7.1.2 Procedure.....................................................................................................12
7.2 Experimental Results.........................................................................................13

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1. Introduction
The function of bacteria is vital in everyday life, from the fermentation processed involved
in producing food to the chemical manufacturing of substances such at ethanol, enzymes,
pharmaceuticals etc. Hence it is important to be able to produce bacteria as efficiently as
possible.

The aim of this lab is to grow Escherichia coli in a submerged culture and to monitor
growth, dissolved oxygen, pH and glucose utilisation. By taking samples at hourly intervals,
the aforementioned parameters can be accurately monitored.

The growth rate is monitored by the measurement of the samples optical density, by using
a spectrophotometer, and by the measurement of the dry cell weight at the end of the
process. The glucose concentration is monitored using a glucose meter while the dissolved
oxygen concentration and the pH is monitored by the fermentation controller. After such
readings have been taken, the growth rate, mean doubling time and final cell yield is
calculated and a growth rate curve is plotted.

2. Methods
Refer to the appendices to view a list of the equipment used and the procedure carried
out.

3. Results

Table 1: Changes in the optical density, glucose concentration, pH and DOT (%) with
time.

Time Optical Density Glucose pH DOT (%)


(Hours) (650nm) Concentration
(g/L)
0 0.477 15.67 6.50 98.30
1 1.230 14.59 6.44 91.70
2 4.500 12.25 6.35 70.30
3 11.000 4.63 6.33 21.20
4 22.300 0.00 5.88 65.50
5 25.800 0.00 6.11 95.10
6 23.700 0.00 6.09 96.90
7 24.100 0.00 6.12 98.00
8 20.200 0.00 6.13 98.10

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Table 2: Weights of Eppendorfs with and without cells.

Time = 0 hours

Weight 1 (mg) Weight 2 (mg)


Tube 1 963.2 960.8
Tube 2 961.9 959.9
Tube 3 967.7 965.8
Tube 4 955.5 953.3

Time = Growth rate, doubling time and final cell yield was measured for a sample of
Escherichia coli which was grown in a 5 litre batch fermenter. A maximum growth rate of
0.966h-1 was obtained. The results which were obtained have been discussed and evaluated
with methods to improving the process being suggested. The limiting factors that were
identifies have been discussed along with the advantages and disadvantages of the
equipments used for the growth of the sample.

The function of bacteria is vital in everyday life, from the fermentation processed involved in
producing food to the chemical manufacturing of substances such at ethanol, enzymes,
pharmaceuticals etc. Hence it is important to be able to produce bacteria as efficiently as
possible.

The aim of this lab is to grow Escherichia coli in a submerged culture and to monitor
growth, dissolved oxygen, pH and glucose utilisation. By taking samples at hourly intervals,
the aforementioned parameters can be accurately monitored.

The growth rate is monitored by the measurement of the samples optical density, by using
a spectrophotometer, and by the measurement of the dry cell weight at the end of the
process. The glucose concentration is monitored using a glucose meter while the dissolved
oxygen concentration and the pH is monitored by the fermentation controller. After such
readings have been taken, the growth rate, mean doubling time and final cell yield is
calculated and a growth rate curve is plotted.

Please refer to the appendices to view the equipment used and the procedure carried out

Table 1: Changes in the optical density, glucose concentration, pH and DOT (%) with

Table 2: Weights of Eppendorfs with and without cells.

Time = 0 hours

8 hours

Weight 1 (mg) Weight 2 (mg)


Tube 1 963.2 970.0
Tube 2 958.5 965.0

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Tube 3 958.6 965.7
Tube 4 963.2 969.0

Tubes without cells

Weight pre-dried (mg) Weight dried (mg)


Tube 1 966.3 958.1
Tube 2 957.4 954.9
Tube 3 964.1 961.7

*Weight 1 = Eppendorf only

*Weight 2 = Eppendorf + cells

4. Analysis of Results and Discussion


4.1 Analysis of Experimental Results
The Monod equation is used to model the growth of a bacteria culture (Onyeaka, 2015):

C E=C E 0 e t (1)

Where:

CE = Concentration of Escherichia coli cells (g/dm3)

c= Initial concentration of Escherichia coli cells (g/dm3)

= Specific growth rate (h-1)

t = Time (h)

Equation (1) rearranged:

CE
ln ( )
CE0
=t (2)

Equation (2) can further be simplified to ln(OD) = t (3), by taking the assumption that
CE
C E0 from equation (2) is directly proportional to the optical density (Hall, et al., 2013).

By plotting a ln(OD) vs time graph, the specific growth rate can be calculated by considering
and calculating the gradient of a specific point on the curve.

Graph 1: Growth curve for Escherichia coli culture, ln(OD) vs time

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4.1.1 Maximum specific growth rate

The maximum specific growth rate is located during the exponential phase of the curve, in
this case from time 1 hour to 4 hours, this time period will result in the maximum growth rate
of the culture and therefore the gradient can be calculated to give the growth rate:

(3.105)(0.207)
max = 41

max = 0.966 h-1

4.1.2 Mean Doubling Time

The mean doubling time is calculated using the following equation:

ln 2 td = doubling time at max growth rate (h)


t d=
max max = maximum specific growth rate (h-1)

ln 2 Therefore td= 0.718 hours = 43.05 minutes


t d=
0.966

4.1.3 Dry Cell Weight

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Table 3: Dry cell weights at time=0 to time=8

Tubes without cells:

Weight pre-dried (mg) Weight dried (mg) Weight lost (mg)


Mean 962.60 958.23 4.37

Time 0 hours:

Weight 1 (mg) Weight 2 (mg) Weight 3 (mg) Dry Cell Weight (mg)
Mean 962.08 959.95 964.32 2.24

Time 8 hours:

Weight 1 (mg) Weight 2 (mg) Weight 3 (mg) Dry Cell Weight (mg)
Mean 960.88 967.43 971.79 10.92

*Where:

Weight 1: Weight of Eppendorf only

Weight 2: Weight of Eppendorf + cells

Weight 3: Weight 2 + Weight lost during drying

The dry cell weights are:

Time= 0 hours, Dry cell weight = 2.24 mg

Time = 8 hours, Dry cell weight = 10.92 mg

4.1.4 Final Cell Yield

The final cell yield is calculated using the following equation:

C C DCW at 8 hoursDCW at 0 hours


Y C /S = =
CS Final [ Glucose ] ( mM )Initial [ Glucose ] (mM )

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10.922.24
Y C / S=
087

0.09977 gmol-1

4.1.5 Optical Density and Glucose Concentration with Time

Graph 2: Variation of optical density and glucose concentration with respect to time.

Optical density and glucose concentration vs time

OD650 nm [glucose] (g/L)

The graph above shows the change in concentration of glucose as time passes, it also
shows the change in the optical density as time passes. It clearly shows that as the E.coli
culture grows (exponential growth phase), the concentration of glucose depletes as the cells
use this to grow. As the glucose concentration approaches complete depletion, the optical
density stops increasing (stationary phase) which shows how the growth of the E.coli culture
ceases once the glucose is used up. The death phase is reached after approximately 5
hours.

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4.1.6 Variation of pH and DOT (%) with Respect to Time

Graph 3: The variation of pH and DOT(%) with respect to time.

pH and DOT(%) readings vs Time

DOT (%)
pH

The graph above shows the relationship of pH and DOT(%) as time passes. The pH
gradually decreases to approximately 5.9 from pH 6.5, indicating the formation of acidic
components, which in this case is acetic acid. This decrease in pH lasts up until the 4th hour
whereby thereafter the pH gradually returns to a stable pH 6.1 due to the glucose
concentration being depleted so therefore no more acetic acid is able to be produced.

Given the fact that acetic acid behaves as a metabolic inhibitor (Roe et al., 2002) it is clear
why the DOT(%) gradually drops to roughly 20% due to the fact that as the concentration of

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dissolved is depleted the growth of the culture becomes anaerobic hence leading to the
inhibitor activity of the acid. Again this only lasts until the 4th hour whereby thereafter the
DOT(%) gradually increases back to 100% as the reaction terminates.

4.2 Discussion of Results and Future Improvements

The doubling time for the experimental sample of E.coli was 43 minutes, however the
average expected doubling time of the culture is 20-40 minutes at a constant temperature of
37oC which is an optimum growing temperature (exptec, 2003). The 43minutes to double in
the experiment shows how there is still room for improvement in the overall process in order
to bring the time close to the 20-40minutes ideal. The limiting factors that can be improves is
shown in the table below.

Table 4: Limiting factors that can be addressed to improve overall efficiency.

Factor Improvements
Temperature The optimum temperature for growth is
37oC (exptec, 2003) so therefore in order to
maintain an efficient growth, this
temperature should be achieved and should
also remain constant throughout the
reaction. This constant temperature can be
monitored by using a temperature probe
during the reaction, and the constant
temperature can be achieved by the use of
heat mats and a cooling finger which is
available in the lab.
pH The optimum pH for growth is pH 7 (exptec,
2003), it is important to keep the pH as
close to neutral as possible in order to
achieve high reaction efficiency.
Agitation Agitation increases the metabolic exchange
of the cells with the growth medium (exptec,
2003) and is controlled by the rpm of the
fermenter being used. The higher the rpm
the better the agitation and the aeration.
Nutrients The correct quantities and types of nutrients
are required in order for the culture to grow
properly, these include; metals, minerals
and vitamins. Without the necessary
nutrients the E.coli culture would not grow
properly, so by ensuring the desired

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nutrients are present at any given time, the
expected yield and doubling time would
improve considerably.
Aeration and Oxygen concentration During the production of the culture, acetic
acid is formed which therefore lowers the
pH of the culture during the reaction, this
causes the growth rate to decrease
considerably due to the inhibiting behaviour
of the acetic acid. A perfect balance of
aeration is required otherwise if the aeration
is high, the cells will utilise carbon sources
and increase the pH of culture whereas if
the aeration is low, there would be a
production of acid which will lower pH.
Therefore a controlled aeration environment
is necessary to obtain optimum growth and
efficiency.

A fed-batch system can be utilised instead of the current batch process being used to
grow the culture. This would involve the addition of nutrients and other materials periodically
during the reaction. Ultimately this helps produce a higher cell yield which is desirable in
many cases, with the only limitation being that the growth rate is unaffected.

Table 5: advantages and disadvantages of the equipment used.

Advantages Disadvantages
The ease of control of the temperature of Aeration unable to be controlled or
the equipment. measured.
pH and DOT(%) can be measured and The fermenter is labour intensive and
controlled. requires the need of constant service.
Medium composition can be changed in There is inadequate/incomplete controls of
order to control the buffer concentration metabolism (Onyeaka, 2015).
required and the concentration of nutrients
required.
The agitation can be controlled by changing The effect of changing agitation in the
the rpm of the impellers. system cannot be accurately measured.
The simplicity and ease of use of the Model-based control is inadequate due to
equipment and process involved. the unsteady state responses that are
achieved (Onyeaka, 2015).

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5. Conclusion

During the experiment an E.coli culture was grown in a batch fermenter and then
analysed, it was found that the doubling time of the culture was 43 minutes which shows that
there is room to improve the experimental procedure as the ideal doubling time is 20-40
minutes (exptec, 2003). Understanding the growth of E.coli is important in industry as E.coli
is a commonly used bacteria in many industrial applications. By understanding the details of
its growth it can help advance the understanding of larger scale production in industry.

6. References

Exptec (2003). Available at: http://www.exptec.com/Bacterial%20E.coli%20Growth%20Media.htm (Accessed: 2


April 2015).

Onyeaka, H. (2015). CBP Biochemical Engineering Lecture Notes. Birmingham UK: University of Birmingham

Roe, A. J., OByrne, C., McLaggan, D., Booth, I. R., Roea, A. J., OByrneb, C. and and, D. M. (2002) Inhibition of
Escherichia coli growth by acetic acid: a problem with methionine biosynthesis and homocysteine toxicity. Society
for General Microbiology. Available at: http://mic.sgmjournals.org/content/148/7/2215.full (Accessed: 2 April
2015).

7. Appendices

7.1 Equipment and Procedure

7.1.1 Equipment

- 5 Litre batch fermentation vessel


- Glucose meter
- Agar plates
- Pipettes
- Ethanol
- Bunsen burner
- Spectrophotometer
- Sterile sample tubes
- Sterile Eppendorf tubes
- Virkon
- J cloth

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7.1.2 Procedure

1. Wear a clean lab coat and a pair of safety spectacles

2. Wash hands thoroughly.

3. Sterilize workplace using Virkon and j-cloth and light a Bunsen burner to keep the
surroundings as aseptic as possible.

4. Take the E.coli sample culture from the bath fermenter and note the pH and DO2 (%).
Ethanol should be used to sterilize the sample tubes.

5. Dilute sample and then use the spectrophotometer at 650nm to take readings, while
initially using water as a reference to zero the spectrophotometer.

6. Measure the glucose concentration using the glucose meter.

7. Place some of the E.coli sample in an agar plate and turn the plate upside down in order
to avoid condensation on the agar itself. Then count the number of cultures.

8. Place 1ml sample of E.coli in an Eppendorf tube and place it in a centrifuge at 10000 rpm
for 10 minutes. Carry out procedure 3 times to obtain an average. Place the tubes in an
oven overnight to obtain the dry cell weight.

9. Repeat steps 4-8 again using a second sample 1 hour after taking the initial sample.

10. Sterilize the workplace and wash hand thoroughly when the experiment is concluded.

7.2 Experimental Results

Dry Cell Weight

Sample Measurement(s) Average


1 937.2 946.7 947.2 943.7
2 940.0 950.0 948.6 946.2

Colony Count

Number of
colonies
Sample 1 [7] 64
Sample 2 [7] 237

Sample 1

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Sample 2

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