CHE506 Lab 6 Lab Manual

Edited Feb 2017

Laboratory 1: Growth Kinetics Study of Microorganism in Shake Flask

Objectives :

To study/observe 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 of maximum growth rate (max),
yield of substrate (Yx/s), mass doubling time (td), saturation constant
(Ks), specific growth rate (net).

1. Introduction

In the shake flask fermentation, the culture flasks (usually Erlenmeyer) of 250 or 500 mL or
larger are used for growing microorganisms. Shake flask fermentation is the cheapest and simplest
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,
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
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
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
for strain improvement as well as for determination of the optimum conditions for the fermentation
process. In many industrial processes, it is also used for the initial stages of inoculum development.
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
the devices are extensively used with filamentous microorganisms and, often, with other types of
microorganisms as well.

Usually, complex media are used for shake flask cultures. However, to enhance the growing
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
on growth and product formation.

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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, 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)

Other media is Bacterial E. coli growth medium TB or Terrific Broth

TB or Terrific broth is a phosphate buffered rich medium. In addition to 20% more peptone and
380% more yeast extract than LB broth, TB also has an added 0.4% glycerol as an extra carbon
source. All these nutrients in TB can support E. coli growth to OD600 5 to 8 under normal shaking

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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:

1 dX
net [1/h]
X dt

Yield Coefficients ( Y X / s ) are defined based on the amount of consumption of another material

X
YX / s [g cells/g substrate]
S

Mass doubling time ( d ) is calculated based on cell numbers and the net specific rate of replication

ln 2
d [h]
net

For substrate limited growth Monod equation is applicable in cellular system. Monod equation is
as the following:

m S
g [1/h]
Ks S
m = maximum specific growth rate when S >> K s
g = net when endogeneous metabolism is unimportant
K s = saturation constant or half-velocity constant

K s = S when g = m
S>> K s , g = m
m S
S<< K s , g
Ks S

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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

Shaking Shake Filling/Working pH

Temperature Media Inoculum Carbon
frequency flask Volume
(oC) Type percentage source
(rpm) size (mL)

TB Glycerol
.37 350 500 150 10 7

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.

Prepare the Terrific Broth according to the recommended formula or recipe stated at
the chemical bottle.

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The example readied recipe for the broths are as the following:
Please further read the instruction of bottle

No Name of Broth Brand Recipe (g/L

1 Terrific Broth SRL Chem 47.60 g to be filled up to 1L volume
or Merck Glycerol as carbon source (4mL/1L)
2 Luria Bertani SRL Chem 20 .0 g to be filled up to 1L volume
(Lennox) Glucose as carbon source (10g/L)

Table 2: Broth

a) Terrific Broth 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 culture

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 culture preparation (inoculum)

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

Shake pH Fermentation Initial

flask Filling/ time and final
Shaking
Temperature size Working Media Inoculum Carbon (h) OD of
frequency
(oC) Volume Type percentage source seed
(rpm)
(mL) culture
**
OD
5 loops
TB Glycerol 4 initial
37 350 1000 150 from agar 7
plate
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 fermentation)

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b) Main experiment
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 Volume Use for

Name (uL)
1 OD 1000 Optical measurement using spectrophotometer
2 CDW 1000 For Cell Dry Weight measurement

Table 5: Sampling

v) Absorbance 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 Concentration) (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)
Cell Dry Weight = Final mass Initial mass

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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.

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vii) Proposed Table Data Collection

Seed/Inoculum

No Time OD (10 times dilution) Real OD

(h)
1 Initial (0 hour)
2 Final (4 hour)

Main experiment

Absorbance
Cell Dry Weight
Optical Density Absorbance Empty Dried Centrifuge
Time X
No OD Real OD Centrifuge tube + sample
(h) (g/L)
(10 times dilution) (ODread times 10) m1 m2
(m2-m1)
OD read
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

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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.

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