ABSTRACK
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 Terrific Broth (TB) medium at 350 rpm and 37°C. They were fermented for 24 hours. Throughout every 1hour for 4 hour then 2 hours for rest 20 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, absorbance reading, and cell dry weight. This experiment get a a few of human error when taking the reading for absorbance and mass of cell growth. But after we can observe the growth of microorganisms and construct the growth curve and determine the Monod parameters, the value of Monod parameter
of maximum growth gate µmax which is equal to the slope of the graph is 0.2443h-1, maximum net growth rate,µnet is 0.2747 h-1, specific growth rate, µnet is 0.7985 h-1 and mass doubling time, 𝜏𝑑 is 0.8681 h. This experiment was consider succeed .
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INTRODUCTION
When microbial cells are inoculated into a batch reactor containing fresh culture medium and their increase in concentration is monitored, several distinct phases of growth can be observed. There is an initial lag phase, which is of variable duration. This is then followed by the exponential growth phase, where cell number (and dry weight) increases exponentially. This is also referred to as the logarithmic phase, the name arising from the common method of plotting the logarithm of cell number against time. Following this is a short phase of declining growth, and then the stationary phase. Here the cell numbers are highest. Finally the cell numbers decline during the death phase. Fermentation process such as batch, continuous and fed-batch processes. In this experiment, the shake flask fermentation was used. 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. It is 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. In order to prevent any contamination to the culture, shake flask must be plugged. The plug has to prevent airbone microorganism from getting into the medium while at same time allow free flow of air into the flask. Different plug can be made of cotton-wool, glass wool, polyurethane foam, gauze or synthethic fibrous material. 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 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, 2
genetics, ecology, or biotechnology, and therefore it is an important part of the basic teaching of microbiology (Kovárová-Kovar. K, et. al, 1998) .
OBJECTIVE
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.
THEORY
Rate of microbial growth is known as 𝜇 net≅
1 𝑑𝑋 1
[ ]
𝑋 𝑑𝑡 ℎ
Yield coefficients ∆𝑋
𝑌𝑋/𝑆 = − ∆𝑆
[g cells/g substrate]
Mass doubling time (𝜏d) ln 2
𝜏𝑑 = 𝜇
[h]
𝑛𝑒𝑡
Monodequation 𝜇𝑔 =
𝜇𝑚 𝑆 𝐾𝑠 +𝑆
𝜇𝑔 = 𝜇 net when𝐾𝐷 =0 𝐾𝐷 = Endogenous metablosim 𝐾𝑠 = Saturation constant 𝐾𝑠 = 𝑆 𝑤ℎ𝑒𝑛 𝜇𝑔 = When𝜇𝑔 =
𝜇𝑚 𝑆 𝐾𝑠 +𝑆
1 𝜇 2 𝑚
, S<<𝐾𝑠
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Figure 1:Growth curve of microorganism based on cell number analysis
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 the point where the growth rates has declined to zero. 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), number of bacteria became decline because death of bacteria. 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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MATERIAL AND 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 (wavelength: 600nm) 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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(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.
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) and total cell number (biomass concentration: g/L).
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3. Table below is referred for planned usage of sample volume: No.
Sample
Volume (μL)
Use for
Name 1
OD
1000
Optical measurement using spectrophotometer
2
CDW
1500
For Cell Dry Weight measurement
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.
v) Cell Dry Weight. (Biomass Concentration) (X) (g/L) 1. Dried centrifuge tubes is weighted and noted as initial mass.(empty container) 2. 1.5 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
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RESULT AND CALCULATION
Time , t (h)
Absorbance (real OD) Diluted
Not Diluted
Empty
Dried
Cell mass
centrifuge
centrifuge
concentration ,
m1 , (g)
+sample , m2
X=(m2-m1)/0.002L
(g/L)
(g) 0
-0.156
0.066
1.0847
1.0865
0.9
1
-0.109
0.416
1.0680
1.0720
2.0
2
0.188
1.739
1.0877
1.0933
2.8
3
0.433
2.014
1.0272
1.0336
3.2
4
0.484
2.225
1.0818
1.0902
4.2
6
0.583
2.269
0.9493
0.9611
5.9
8
1.542
2.397
1.0193
1.0355
8.1
10
1.607
2.494
1.0731
1.0855
6.2
12
1.863
2.571
1.0771
1.0862
4.6
14
2.063
2.644
1.0724
1.0812
4.4
16
1.135
2.681
1.0741
1.0826
3.6
18
0.996
2.603
1.0677
1.0760
4.2
20
1.334
2.753
1.0699
1.0793
4.7
22
1.210
2.606
1.0631
1.0721
4.5
24
2.377
2.688
1.0820
1.0912
4.6
8
Growth Curve time 0 1 2 3 4 6 8 10 12 14 16 18 20 22 24
Absorbance -0.156 -0.109 0.188 0.433 0.484 0.583 1.542 1.607 1.863 2.063 1.135 0.996 1.334 1.410 2.377
Absorbance nm vs time
3
absorbance (nm)
2.5 2 1.5 1 0.5 0 0
5
10
15
20
25
30
-0.5
time Graph 1: Absorbance (nm) for optical density against time
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Cell mass concentration,X against time time
X 0 1 2 3 4 6 8 10 12 14 16 18 20 22 24
0.9 2.0 2.8 3.2 4.2 5.9 8.1 6.2 4.6 4.4 3.6 4.2 4.7 4.5 4.6
cell mass concentration,X
Cell mass concentration,X against time 9 8 7 6 5 4 3 2 1 0 0
5
10
time,h
15
20
25
30
Graph 2:Cell mass concentration,X against time,h
Cell dry weight = m2 – m1 = 1. 0865g – 1.0847g = 0.0018g Cell mass concentration, X 𝑐𝑒𝑙𝑙 𝑑𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡
𝑋 = 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 =
volume of sample = 0.002L
0.0018𝑔 0.002𝐿
= 0.9g/L 10
Exponential growth phase against time time
Ln(X/X0) 0 0.7985 1.1350 1.2685 1.5404 1.8803 2.1972
0 1 2 3 4 6 8
Ln(X/X0) against time 2.5 y = 0.2443x + 0.4225 R² = 0.9015
2
ln(X/Xo)
1.5 1 0.5 0 0
1
2
3
4
5
6
7
8
9
time Graph 3 : ln(X/Xo) against time
maximum growth rate, μmax the value of µmax which is equal to the slope of the graph is 0.2443h-1
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Growth Rate, µnet time
ln 𝑋 -0.1054 0.6931 1.0296 1.1632 1.4351 1.7750 2.0919 1.8245 1.5261 1.4816 1.2809 1.4351 1.5476 1.5041 1.5261
0 1 2 3 4 6 8 10 12 14 16 18 20 22 24
lnX vs time 2.5 2
y = 0.0311x + 1.0154 R² = 0.2298
lnX
1.5 1 0.5 0 0
5
10
15
20
25
30
-0.5
time Graph 4 : LnX against tim(h)
Maximum net growth rate, µnet = =
𝑙𝑛𝑋𝑡=8−𝑙𝑛𝑋𝑡=0 𝑡=8 − 𝑡=0 ln 8.1−𝑙𝑛0.9 8−0
= 0.2747 h-1
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Growth Rate, 𝜇 net
𝜇= =
𝑙𝑛𝑋1−𝑙𝑛𝑋𝑜 𝑡1−𝑡𝑜 𝑙𝑛2−𝑙𝑛0.9 1−0
= 0.7985 h-1
Mass doubling time, 𝜏𝑑 ln 2
𝜏𝑑 = 𝜇
𝑛𝑒𝑡
ln 2
= 0.7985 = 0.8681 h
DISCUSSION
In this experiment , we selected E.Coli as the cell and it is cultivated in shake flask . 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 first 4 hours, then every 2 hours for the rest 20 hours. We took the cell out to analyze the concentration of the cell (g/L) and cell dry weight. 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 diluted absorbance reading is increase from the beginning of the experiment until the 14st hour and decrease slightly at the 16th hour until 18th hour and increase again until 24thhour. For non-diluted absorbance reading is increasing from begining of the experiment to 24th 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 16th hour for diluted absorbance is indicating that due to error when take the reading of absorbance. During growth 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.for nondiluted absorbance, the higher the absorbance reading means greater number of cells inside the 13
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. In cell growth, the cell will go through several phases like lag, exponential, deceleration, stationary and death phase. 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 for 12 hours and then take the reading of cell dry weight. The cell dry weight is increased from 0th hour until 10th hour and gradually decreased from the 12th hour to 18th hour and 20th hour until 24th hour, 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. The value of Monod parameter of maximum growth gate µmax which is equal to the slope of the graph is 0.2443h-1, maximum net growth rate,µnet is 0.2747 h-1, specific growth rate, µnet is 0.7985 h-1 and mass doubling time, 𝜏𝑑 is 0.8681 h.
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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. we also want to construct a growth curve including lag, log, stationary and death phases. The others parameters that we studied includes cell concentration, absorbance reading, and cell dry weight. From the graph plotted and equations that have been given, we succeeded in calculating the Monod parameter of maximum growth rate, specific growth rate and mass doubling time but the calculating is not well accurate due to error in reading. Some parameters that cannot be calculated because of technical problem that we cannot avoid . This experiment succeed. 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.
RECOMMENDATION –
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.
–
Dispose of all contaminated materials in appropriate containers.
–
Make a dilution if the reading of spectrophotometer is more than 1 and take the Optical Density reading again.
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–
The supernatant of cell concentration should be taken out carefully without any taking out of the biomass.
REFERENCES
1. Kovárová-Kovar, K., & Egli, T. (1998). Growth Kinetics of Suspended Microbial Cells: From Single-Substrate-Controlled Growth to Mixed-Substrate Kinetics. Retrieved on October 4, 2015 from http://mmbr.asm.org/content/62/3/646.full . 2. Suresh, S. et al. (2009) Techniques for oxygen transfer measurement in bioreactors: a review. J. Chem. Technol. Biotechnol. 84, 1091–1103 3. Crueger W and Crueger A., (1990). Biotechnology: A Textbook of Industrial Microbiology. Sinauer Associates, Sunderland Massachusetts. 4. Bernstein, A.M., et al., Dietary protein sources and the risk of stroke in men and women. Stroke, 2012. 43(3): p. 637-44. 5. Manual laboratory of Chemical Reaction Engineering. UiTM Shah Alam.
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APPENDICES
Figure 1: Spectrometer
Figure 2 : Taking sample
Figure 3 : Remove supernatant
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Figure 4: Incubator Shaker YSI
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