Laboratory 1: Growth Kinetics
Laboratory 1: Growth Kinetics Study of Microorg
Study of Microorg anism in Shake
anism in Shake Fla
Flask
sk
1. Introduction
1. Introduction
In the shake flask fermentation, the culture flasks (usually Erlenmeyer) of 250 or 500 mL or In the shake flask fermentation, the culture flasks (usually Erlenmeyer) of 250 or 500 mL or larger are used for growing microorganis
larger are used for growing microorganisms. Shake flask fermentation is the cheapest and simplestms. Shake flask fermentation is the cheapest and simplest technique to grow bacteria or fungi, aerobically, in small volumes of nutrient broth. The broth is technique to grow bacteria or fungi, aerobically, in small volumes of nutrient broth. The broth is poured into Erlenmeyer Flasks equipped with cotton-wool stoppers, and autoclaved. After cooling, poured into Erlenmeyer Flasks equipped with cotton-wool stoppers, and autoclaved. After cooling, some microbes are "seeded" into the flask, and it is placed on a Shaker machine. The shaking some microbes are "seeded" into the flask, and it is placed on a Shaker machine. The shaking agitates the content and so ensures aeration, so that the microbes could breathe. These flasks are agitates the content and so ensures aeration, so that the microbes could breathe. These flasks are shaken, generally, by an incubator shaker at a suitable agitation speed, which is usually in r.p.m. shaken, generally, by an incubator shaker at a suitable agitation speed, which is usually in r.p.m. Shaken cultures are usually applied to aerobic processes. In general, filamentous microorganisms Shaken cultures are usually applied to aerobic processes. In general, filamentous microorganisms are grown for the production of secondary metabolites, which begins 1 to 3 days after inoculation are grown for the production of secondary metabolites, which begins 1 to 3 days after inoculation and continues 3 to 4 days thereafter, for instance. In all such cases, the shaken cultures are used and continues 3 to 4 days thereafter, for instance. In all such cases, the shaken cultures are used for strain improvement as well as for determination of the optimum conditions for the fermentation for strain improvement as well as for determination of the optimum conditions for the fermentation process. In many industrial process
process. In many industrial processes, it is also es, it is also used for the initial stages of used for the initial stages of inoculum developminoculum development.ent. Shaken cultures are a convenient method of growing microorganisms in submerged cultures under Shaken cultures are a convenient method of growing microorganisms in submerged cultures under aerobic conditions created by shaking; it is a small scale equivalent of stirred tank bioreactor. Both aerobic conditions created by shaking; it is a small scale equivalent of stirred tank bioreactor. Both the devices are extensively used with filamentous microorganisms and, often, with other types of the devices are extensively used with filamentous microorganisms and, often, with other types of microorganisms as well.
microorganisms as well.
Usually, complex media are use
Usually, complex media are used for shake flask cultures. However, to enhance the growingd for shake flask cultures. However, to enhance the growing the synthetic medium is being devised for the fermentation process. Studies on inoculum size, the synthetic medium is being devised for the fermentation process. Studies on inoculum size, temperature, agitation, nutrition are initially done using these cultures to monitor their influences temperature, agitation, nutrition are initially done using these cultures to monitor their influences on growth and product formation.
on growth and product formation. Objectives :
Objectives :
•
• To To study/observe the study/observe the growth growth kinetics of kinetics of microorganimicroorganism sm in in shake shake flaskflask experiment
experiment
•
• To To construct a construct a growth growth curve curve including lag, including lag, log, log, stationary and stationary and deathdeath phases.
phases.
•
• To To determine determine the the Monod Monod parameters of parameters of maximum maximum growth growth rate rate ((maxmax),),
yield of substrate (Yx/s), mass doubling time (t
yield of substrate (Yx/s), mass doubling time (tdd), saturation constant), saturation constant
(K
Figure 1 Phases of a typical growth curve of E.coli in a batch culture
In a 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.
Lag phase is the time between inoculation and reaching the maximum growth rate. There are two sub phases in the lag phase. In the first phase, there is no growth identified whereas in the second sub phase which is also known as acceleration phase, there is a constant growth begins. The second phase is exponential phase. The cells begin to proliferate with their maximum growth rate. The doubling time of E.coli is 20 minutes. Exponential phase is important for determining the maximum growth rate, µ and doubling time, d since the growth at this time is the most constant and ideal.
Retardation phase is the period between exponential and stationary phase, or in other words, the phase before the growth becomes stationary. Among the factors that inhibit the growth are reduced dissolved oxygen tension (DOT), substrate concentration, pH changes and presence of inhibiting metabolites. After retardation phase, the growth phase enters stationary phase where the growth becomes constant for a period of time before it declines.
Finally, the growth declines from its stationary phase due to the cells lysation. This is indicated by the decrease of the viable cell number.
There are many specific media for certain microorganisms like Luria Bertani (Lennox) and Terrific Broth media. Bacterial E.coli growth media: LB Miller broth/LB Lennox broth is the most commonly used medium in molecular biology for E.coli cell culture. LB broth contains the enzymatic digestion product of casein commonly known as peptone (some vendors term it Tryptone), yeast extract, and sodium chloride. Peptone is rich in amino acids and peptides. Its amino acid and peptide compositions reflect those of casein. In addition to amino acids and peptides, yeast extract also contains nucleic acids, lipids and other nutrients which are needed for bacterial growth. (LB Miller, Lennox)
incubation conditions. TB is commonly used for protein expression and plasmid production in a laboratory scale. (TB broth)
2. Theories
Rate of microbial growth net is characterized by specific growth rate:
net dt dX X 1 [1/h]
Yield Coefficients (YX/s ) are defined based on the amount of consumption of another material
S X YX s / [g cells/g substrate]
Mass doubling time ( d) is calculated based on cell numbers and the net specific rate of replication
net d 2 ln [h]
For substrate limited growth Monod equation is applicable in cellular system. Monod equation is as the following: S K S s m g [1/h] m
= maximum specific growth rate when S >> Ks
g
= net when endogeneous metabolism is unimportant s
K = saturation constant or half-velocity constant
s K = S when g = ½ m S>> Ks, g= m S<<Ks, S K S s m g
3. Apparatus and Reagent
• Microbe: Escherichia coli
• Shake flask (250mL flasks and 1000 mL flasks)
• Eppendorf tubes/falcon tube (1.5mL)
• Cuvettes (spectrophotometer)
• Thermostated rotary shaker/Incubator shaker
• Refrigerated Centrifuge
• Media (for specific microbe)
• Ethanol (70% ethanol for swabbing for sterility)
• Spectrophotometer
• Bunsen Burner for sterility
• Graduated Flask for measuring media (1000mL, 100mL, 10mL)
• Laminar Flow hood for sterility
• Biochemical Analyzer
• HPLC for product measurement like ethanol
• Cotton plugged
• pH meter
4. Experimental Procedures
The experimental parameters will be as the following
Temperature (oC) Shaking frequency (rpm) Shake flask size Filling/Working Volume (mL) Media Type Inoculum percentage pH Carbon source .37 350 500 150 TB 10 7 Glycerol
Table 1: Experimental Parameters
(i) Preparation of media
Media must be prepared according to the needs of microorganism used.
Microorganism used is Escherichia coli. There are many kinds of media for E coli for instance Luria Bertani broth or Terrific Broth. Terrific Broth is a readied phosphate buffer media.
The example readied recipe for the broths are as the following: Please further read the instruction of bottle
No Name of Brot h Brand Recipe (g/L
1 Terrific Broth SRL Chem
or Merck
47.60 g to be filled up to 1L volume Glycerol as carbon source (4mL/1L)
2 Luria Bertani
(Lennox)
SRL Chem 20 .0 g to be filled up to 1L volume
Glucose as carbon source (10g/L)
Table 2: Brot h a) Terrif ic Brot h preparation
Follow the recipe as stated at the bottle.
Autoclave the media at 121 oC for 20 minutes
Glycerol and media can be autoclaved together.
pH reading should be near 7 as the media is a readied phosphate buffer solution
(ii) Preparation of cell cultu re
Cell culture used must be maintained on an agar plate and liquid broth for the inoculum preparation. A suitable media is needed in order to ensure that the microorganism is growing. Inoculum preparation refers to seed culture which will be the feed for the main experiment.
a) Seed cult ure preparation (inocu lum)
Take 5 loops of grown E coli 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 inoculum needed)
Sterility must be sustained during transfer.
Grow the media at 150 rpm for 4 hours assuming exponential growth of E coli. At this stage, the seed cultures are assumed to be at its most active condition.
Take note the OD for seed culture using spectrophotometer
Temperature (oC) Shaking frequency (rpm) Shake flask size Filling/ Working Volume (mL) Media Type Inoculum percentage pH Carbon source Fermentation time (h) Initial and final OD of seed culture ** 37 350 1000 150 TB 5 loops from agar plate 7 Glycerol 4 OD initial OD final Table 4
Take note: (please prepare enough seeds for all main experiments) ** Please take note the initial OD (after 5 loops inoculation) and final OD (after 4 hours of f ermentation)
b) Main experim ent
Using aseptic technique, transfer 10% of inoculum to the main experiment media. For instance, if the working volume is 150ml, therefore, 10% of inoculum would be 15mL of seed culture needed
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.
The main experiment is stated in Table 1. .
(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. Refer to Table below for planned usage of sample volume:
No Sample
Name
Volume (uL)
Use for
1 OD 1000 Optical measurement using spectrophotometer
2 CDW 1000 For Cell Dry Weight measurement
Table 5: Samplin g v) Absorb ance Analysis (Optical Density) (OD)
1. 1 mL of sample is transferred into a cuvette and the optical density measurement is made 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.
Suggested method:
Certain tenth-time dilution is proposed for the OD measurement by using spectrophotometer. For instance, with 1 mL sample, take only 100 uL sampl e being added to 900 uL of Distilled Water for OD measurement in 1000 uL Cuvette.
vi) Cell Dry Weight. (Biomass Concent ratio n) (X) (g/L)
1. Weigh dried centrifuge tubes and note this 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 oC. for 20 minutes
4. Take out the supernatant and you may repeat washing with distilled water and centrifuging
5. Dry the centrifuge tube (left with biomass only) in oven at 80 oC for overnight
6. Leave the dried centrifuged tubes in dessicator.
7. Weigh the centrifuge tube and note this as final mass (with biomass = Cell Dry Weight)
Alternative method
1. Aluminum weight of boat are dried in an oven at 80C for 6-8 hours and placed in a dessicator containing a drying agent for cooling before weighing (for 30min). 2. The cell pellet (after sample is centrifuged at 10,000 rpm) is suspended in 10 mL
centrifuge tube with distilled water.
3. The cell then transferred to aluminum foil boat. The tube was rinsed with water and placed in an oven at 80C for overnight.
4. The sample is then removed from the oven with tongs and placed in a dessicator to cool and weighed rapidly on an analytical balance. The weight of the cell pellet is recorded.
Page8 of9 vii) Proposed Table Data Collection
Seed/Inoculum
No Time (h)
OD (10 times dilu tio n) Real OD
1 Initial (0 hour) 2 Final (4 hour) Main experiment No Time(h) Abs or banc e Optical Density OD (10 times dilut ion)
OD read
Abs or banc e Real OD (ODread times 10)
Empty Centrifuge m1 Dried Centrifuge tube + sample m2
Cell Dry Weight X (g/L) (m2-m1) 1 0 2 0.5 3 1 4 1.5 5 2 6 2.5 7 3 8 3.5 9 4 10 6 11 8 12 10 13 12 14 16 15 20 16 24
CHE506 Lab 6 Lab Manual
Edited Feb 2017
5. Report: (100M)
Plagiarism is highly prohibited.
1 Abstract/Summary (5M) 2 Introduction (5M) 3 Aims/Objective (5M) 4 Theory (10M) 5 Apparatus (5M) 6 Methodology/Procedure (10M) 7 Results (10M) 8 Calculations (10M) 9 Discussion (20M) 10 Conclusion (10M) 11 Recommendation (5M) 12 Reference/Appendix (5M)
5. Report: (100M)
Plagiarism is highly prohibited.
1 Abstract/Summary (5M) 2 Introduction (5M) 3 Aims/Objective (5M) 4 Theory (10M) 5 Apparatus (5M) 6 Methodology/Procedure (10M) 7 Results (10M) 8 Calculations (10M) 9 Discussion (20M) 10 Conclusion (10M) 11 Recommendation (5M) 12 Reference/Appendix (5M)
6. References
Buffer calculator: http://home.fuse.net/clymer/buffers/phos2.html
Shuler, Michael L., and Fikret Kargi. Bioprocess engineering. New York: Prentice Hall, 2002.
Garvie, Ellen I. "The growth of Escherichia coli in buffer substrate and distilled water." Journal of bacteriology 69.4 (1955): 393.
