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UNIVERSITI TEKNOLOGI MARA

FAKULTI KEJURUTERAAN KIMIA


BIOPROCESS ENGINEERING LABORATORY(CBE 661)
STUDENT NAME : MEOR AHMAD FAEZ BIN MOHD HASSAN
STUDENT ID : 2013664042
GROUP : EH222 5B
EXPERIMENT : GROWTH KINETICS STUDY OF
MICROORGANISMS IN SHAKE FLASK
DATE PERFORMED : 8 OCTOBER 2015
SEMESTER :5
PROGRAMME/CODE : EH 222
SUBMIT TO : MS. NUR ATIKAH

No. Title Allocated Marks (%) Marks


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

Remarks:
Checked by:
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Date:

1
1.0 ABSTRACT ......................................................................................................................... 3

2.0 INTRODUCTION ............................................................................................................ 3-4

3.0 OBJECTIVES ...................................................................................................................... 4

4.0 THEORY .......................................................................................................................... 5-6

5.0 APPARATUS & MATERIALS .......................................................................................... 7

6.0 PROCEDURES............................................................................................................... 7-10

7.0 RESULTS ..................................................................................................................... 11-18

8.0 DISCUSSIONS .................................................................................................................. 19

9.0 CONCLUSION .................................................................................................................. 20

10.0 RECOMMENDATIONS ................................................................................................. 20

11.0 REFERENCES & Appendices ........................................................................................

............................................................................................................................................. 21-23

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ABSTRACT/SUMMARY

This experiment is to study the growth kinetics of microorganism in shake flask.


Erlenmeyer flask is used in this experiment to grow microorganisms. Escherichia coli (E.Coli) is
grown in Luria Bertani (LB) broth medium and Terrific Broth (TB) medium at 350 rpm and
37°C. They were fermented for 24 hours. Throughout every 2 hours, cell is taken out to obtain
the value of absorbance by using spectrophotometer. Cell dry is then obtained after the mass
concentration inside the flask is dried overnight. As for the optical density analysis, the
absorbance reading from the spectrophotometer is taken. 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 tube
which contains the biomass before and after the drying process is recorded to get the cell dry
weight. Then, the obtained value can be turned into graph to observed the changing in growth
kinetics of the cell . Graph of growth curve including lag, log, stationary and death phases. The
others parameters that we studied includes cell concentration, glucose concentration, absorbance
reading, and cell dry weight (CDW) . This experiment get a a few of human error and technical
problem such as the malfunction of machine to get the reading of glucose . But after we can
observe the growth of microorganisms and construct the growth curve and determine the
Monod parameters , this experiment was consider succeed .

INTRODUCTION

Most industrial bioreactor manufacturers use vessels, sensors and a control


system networked together. Fouling can harm the overall sterility and efficiency of the
bioreactor, especially the heat. To avoid it, the bioreactor must be easily cleaned and as smooth
as possible (therefore the round shape). A heat exchanger is needed to maintain the bioprocess at
a constant temperature. Biological fermentation is a major source of heat, therefore in most cases
bioreactors need refrigeration. They can be refrigerated with an external jacket or, for very large
vessels, with internal coils.

Bioreactor design is quite a complex engineering task. Under optimum conditions the
microorganisms or cells will reproduce at an astounding rate. The vessel's environmental
conditions like gas( such as air , oxygen , nitrogen , carbon dioxide) flow rates, temperature, pH
and dissolved oxygen levels, and agitation speed need to be closely monitored and controlled.

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There are many ways to carried out Fermentation process such as batch, continuous and
fed-batch processes. In this experiment, we decided to use the shake flask fermentation. 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 and it is also the cheapest and easiest
way to culture microorganism aerobically, in small volumes of nutrient broth.

The cultures are incubated at certain temperature 37°C and shaking frequency 350 rpm
for 4hours 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, it is closed environment that means 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 grow this inhibited because of the decreasing substrate concentration and the presence of
toxic metabolites.

The microorganism that we used to study in this experiment is E .coli . There are many
specific media for growth of E. coli but in this experiment we used Luria Bertani (LB) and a
Terrific Broth (TB)media because it is the most commonly used medium in molecular biology
for E. coli cell culture .The relationship between the specific growth rate (μ) of a microbial
population and the substrate concentration (s), is an indispensable tool in all fields of
microbiology, be it physiology, genetics, ecology, or biotechnology, and therefore it is an
important part of the basic teaching of microbiology (Kovárová-Kovar. K, et. al, 1998) .

OBJECTIVES

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 .

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THEORY

Rate of microbial growth is known as


1 𝑑𝑋 1
𝜇 net≅ [ ]
𝑋 𝑑𝑡 ℎ

Yield coefficients
∆𝑋
𝑌𝑋/𝑆 = − ∆𝑆 [g cells/g substrate]

Mass doubling time (𝜏d)


ln 2
𝜏𝑑 = 𝜇 [h]
𝑛𝑒𝑡

Monodequation
𝜇𝑚 𝑆
𝜇𝑔 = 𝐾𝑠 +𝑆

𝜇𝑔 = 𝜇 net when𝐾𝐷 =0

𝐾𝐷 = Endogenous metablosim

𝐾𝑠 = Saturation constant

1
𝐾𝑠 = 𝑆 𝑤ℎ𝑒𝑛 𝜇𝑔 = 𝜇
2 𝑚
𝜇𝑚 𝑆
When𝜇𝑔 = , S<<𝐾𝑠
𝐾𝑠 +𝑆

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1. During lag phase, bacteria adapt themselves to growth conditions. It is the period where
the individual bacteria are maturing and not yet able to divide. During the lag phase of
the bacterial growth cycle, synthesis of RNA, enzymes and other molecules occurs.
2. The log phase is a period characterized by cell doubling. The number of new bacteria
appearing per unit time is proportional to the present population. If growth is not limited,
doubling will continue at a constant rate so both the number of cells and the rate of
population increase doubles with each consecutive time period.
3. The stationary phase is often due to a growth-limiting factor such as the depletion of an
essential nutrient, and/or the formation of an inhibitory product such as an organic acid.
Stationary phase results from a situation in which growth rate and death rate are equal.
The number of new cells created is limited by the growth factor and as a result the rate of
cell growth matches the rate of cell death. The result is a “smooth,” horizontal linear part
of the curve during the stationary phase.
4. At death phase (decline phase), bacteria die. This could be due to lack of nutrients, a
temperature which is too high or low, or the wrong living conditions.

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APPARATUS

1. Microbe:Escherichia Coli .
2. Shake flask (250mL flasks and 1000 mL flasks)
3. Eppendorf tubes / falcon tube (1.5 mL)
4. Cuvettes (spectrophotometer)
5. Incubator Shaker
6. Refrigerated Centrifuge
7. Media (for specific microbe)
8. Ethanol (70% ethanol for swabbing for sterility)
9. Spectrophotometer
10. Bunsen burner for sterility
11. Graduated Flask for measuring media (1000mL, 100mL, 10mL)
12. Laminar Flow hood for sterility
13. Biochemical Analyzer
14. HPLC for product measurement like ethanol
15. Cotton plugged

PROCEDURE

(i) Preparation of media

a) Terrific Broth (TB) preparation

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

2. The media is autoclaved at 121°C 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

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b) Luria Bertani (LB) preparation

1. The recipe as stated at the bottle is followed for LB media.

2. A certain amount of phosphate buffer is added to give a specific pH which is pH 7.Refer to


table below.

3. Glucose solution is prepared and it should be autoclaved separately before cooled down in a
different bottle.

4. Distilled water is autoclaved which will be used for filling up volume to the needed volume
prepared.

5. When both solutions are cooled, then only both solutions are added.

No. Phosphate buffer components Concentration


1 K2HPO4 8 g/l
2 KH2PO4 3 g/l

(ii) Preparation of cell culture

a) Seed culture preparation (inoculums)

1. 5 loops of grown E coli is taken on agar plates and added to the sterilized media of 150mL in
1000mL shake flask. (you may need 2 of 1000mL shake flask to ensure enough inoculums
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.

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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 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 below is referred for planned usage of sample volume:

No. Sample Name Volume (μL) 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 is transferred into a cuvette and the optical density measurement ismade using
a spectrophotometer with the wavelength set at 600nm.

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.

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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 4°C. 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 80°C for overnight.

6. The dried centrifuged tubes is 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).

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RESULT AND CALCULATION

Time , t (h) Absorbance (real OD) Empty Dried Cell dry


Distilled media centrifuge m1 centrifuge weight ,
water , (g) +sample , m2 X=m2-m1
(g) (g/L)
0 0.086 0.138 1.0664 1.0784 0.012
1 0.218 0.074 1.0649 1.0900 0.0251
2 0.320 0.160 1.0804 1.0704 -0.01
3 0.189 0.056 1.0671 1.0776 0.0105
4 0.272 0.135 1.0714 1.0833 0.0114
6 1.873 , 0.242 1.687 , 0.084 1.0648 1.0687 0.0039
8 2.092 , 0.275 1.932 , 0.002 1.1706 1.0751 -0.0955
10 2.001 , 0.351 1.946, 0.2315 1.0616 1.0564 -0.0052
12 2.043 , 0.304 1.918 , 0.169 1.0648 1.0697 0.0049
14 2.045 , 0.349 1.900 , 0.208 1.0725 1.0785 0.0060
16 2.165 , 0.3814 2.011 , 0.198 1.0595 1.0650 0.0055
18 1.999 , 0.342 1.818 , 0.163 0.9658 0.9724 0.0066
20 2.049 , 0.471 1.784 , 0.190 1.0720 1.0751 0.0031
22 2.071 , 0.226 1.795 , 0.733 1.0674 1.0721 0.0047
24 2.119 , 0.822 1.482 , 0.800 1.0637 1.0681 0.0044

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Cell Dry Weight against Time
0.03

0.025

0.02

X (g/l) 0.015
0.01

0.005

0
1 3 4 6 12 14 16 18 20 22 24
Time (h)

Graph of ln X against time which is plotted to construct the growth curve of the cell by using cell
dry weight

ln x vs time
0
1 3 4 6 12 14 16 18 20 22 24
-1

-2

-3
ln x
-4

-5

-6

-7
time , h

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Graph of ln X against time which is plotted to construct the growth curve of the cell by using cell
dry weight.

Optical Density vs time


0.9
0.8
0.7
0.6
0.5
Optical Density
0.4
0.3
0.2
0.1
0
1 2 3 4 6 8 10 12 14 16 18 20 22 24
time , h

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

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Results

Media used: Terrific broth (TB)

Time (h) Absorbance Mass of Mass of m2-m1 Cell Dry


Optical empty centrifuge + Weight, X
(g/L)
Density at centrifuge samples
m2 − m1
600 nm (m1) (m2)
0.002
0 0.028 1.0872 1.0892 0.0020 1.00
1 0.184 1.0822 1.0852 0.0034 1.70
2 0.389 1.0647 1.0702 0.0049 2.45
3 0.553 1.1032 1.1081 0.0062 3.10
4 0.677 1.0832 1.0894 0.0114 5.70
5 0.737 1.0874 1.0988 0.0257 12.85
7 0.764 1.0518 1.0802 0.0284 14.20
9 0.790 1.0663 1.0953 0.0290 14.50
11 0.796 1.0702 1.1002 0.0300 15.00
13 0.821 1.0695 1.0989 0.0294 14.70
15 0.844 1.0768 1.1041 0.0273 13.65
17 0.903 1.218 1.2430 0.0250 12.50
19 0.903 1.0765 1.0985 0.0220 11.00

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Growth Curve

time Absorbance
0 0.028
1 0.194
2 0.389
3 0.553
4 0.677
5 0.737
7 0.764
9 0.790
11 0.796
13 0.821
15 0.844
17 0.903
19 0.903

Absorbance Value for Optical Density vs. Time for TB


1
0.9
0.8
0.7
Absorbance

0.6
0.5
0.4
0.3
0.2
0.1
0
0 2 4 6 8 10 12 14 16 18 20
Time (h)

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Graph Cell Dry Weight against Time

time x
0 1
1 1.70
2 2.45
3 3.10
4 5.70
5 12.85
7 14.20
9 14.50
11 15.00
13 14.70
15 13.65
17 12.50
19 11.00

Cell Dry Weight (gX/L) vs Time for TB


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14
Cell Dry Weight (gX/L)

12
10
8
6
4
2
0
0 5 10 15 20
Time (h)

Sample calculation of Cell Dry Weight (gX/L):

[(𝑀𝑎𝑠𝑠 𝑜𝑓 𝑐𝑒𝑛𝑡𝑟𝑖𝑓𝑢𝑔𝑒 𝑡𝑢𝑏𝑒+𝑠𝑎𝑚𝑝𝑙𝑒)−(𝑀𝑎𝑠𝑠 𝑜𝑓 𝑒𝑚𝑝𝑡𝑦 𝑐𝑒𝑛𝑡𝑟𝑖𝑓𝑢𝑔𝑒 𝑡𝑢𝑏𝑒)]


= 0.002𝐿

(𝑚2−𝑚10
= 0.002𝐿

(1.0892−1.0872)
= 0,002

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= 1.000 gX/L

Graph of Exponential Growth Phase

time ln x/xo
0 0
1 0.53
2 0.89
3 1.13
4 1.74
5 2.55

ln X/X0 vs Time for TB


3

2.5

2
ln X/X0

1.5
y = 0.4626x
1

0.5

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

Maximum growth rate, µmax = slope of the graph

µmax = 0.4626 h-1

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ln X vs Time of TB
3

2.5

2
Ln X

1.5

0.5

0
0 5 10 15 20
Time (h)

𝑙𝑛𝑋𝑡=11−𝑙𝑛𝑋𝑡=0
Maximum net growth rate, µnet = 𝑡=11 − 𝑡=0

ln 15−𝑙𝑛1
= 11−0

= 0.2462 h-1

𝑙𝑛2
Doubling time, td= µ𝑚𝑎𝑥

= 1.4984 h

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DISCUSSION

In this experiment , we selected 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. The duration for this
experiment is 24 hours. The cell is taken out every 1 hour for first4 hours, then every 2 hours
for the rest 20 hours. We took the cell out to analyze the concentration of the cell (g/L), cell dry
weight and glucose concentration.

The concentration of the cell can be calculated by taking the reading of the optical density (OD)
from the spectrophotometer. 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,
the pattern roughly shows an increase from the beginning of the experiment until the end of the
experiment. From this observation , we can explain that the number cell increasing throughout
the cultivation indicating that the cell is growing.

By analyzing the cell sample with the glucose analyzer the glucose concentration can be
measured and the direct glucose concentration can be obtained. For this experiment, the glucose
analysis cannot be performed due to the broken machine. Thus glucose analysis cannot be
performed. The cell sample is analyze 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. Then , the supernatant was removed and the
remaining cell ware dried inside oven and then take the reading of cell dry weight . The results
for cell dry weight are not dependable in this experiments because the values are inconsistent in
increasing and decreasing. This error occurs due to the ways students in separating the
supernatant from the samples . The students might have extracted and removed the cells along
with the supernatant. This human error can cause inaccurate in weight reading .

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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.
Then , we also want to construct a growth curve including lag, log, stationary and death phases.
The others parameters that we studied includes cell concentration, glucose concentration,
absorbance reading, and cell dry weight (CDW). From the graph plotted and equations that have
been given, we succeeded in calculating other Monod parameters even though there is some
parameters that cannot be calculated because of technical problem that we cannot avoid . This
experiment succeed .

RECOMMENDATION

1. Avoid contamination when handling the biomass by practicing excellent aseptic


techniques.
2. This experiment must be carried out under the laminar flow to prevent any contamination
to the culture.
3. Cuvette must be in clean condition to obtain most accurate spectrophotometer reading
during protein test
4. The supernatant of cell concentration should be taken out carefully without any taking
out of the biomass.
5. Always sterilize the mouth of shake flask and cotton plugged with flame from Bunsen
burner to avoid any contamination .
6. Make a dilution if the reading of spectrophotometer is more than 1 and take the Optical
Density reading again.

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REFERENCES/APPENDICES

1. Suresh, S. et al. (2009) Techniques for oxygen transfer measurement in bioreactors: a


review. J. Chem. Technol. Biotechnol. 84, 1091–1103
2. Bernstein, A.M., et al., Dietary protein sources and the risk of stroke in men and
women. Stroke, 2012. 43(3): p. 637-44.
3. Crueger W and Crueger A., (1990).Biotechnology: A Textbook of Industrial Microbiology .
Sinauer Associates, Sunderland Massachusetts.
4. Zwietering M H, Jongenburger I, Rombouts F M, van 'T Riet K (1990). "Modeling of the
Bacterial Growth Curve"

Spectrophotometer

taking samples in each 2 hours

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removing supernatant

samples in centrifuge

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