Laboratory 1: Growth Kinetics Study of Microorganism in Shake Flask

1. Abstract

This experiment is carried out to study the growth kinetics of microorganisms in shake flask. E.coli is

grown in a LB broth medium and being fermented for 24 hours. Throughout the fermentation, the cell

culture is taken out for every 3 hours and protein test, glucose test and cell dry weight are being

performed. As for the optical density analysis, the absorbance reading from the spectrophotometer is

taken while for the glucose test, the reading of glucose level is taken from the YSI 2700 Select

Biochemical Analyzer or can also being performed by using DNS reagent and the absorbance value is

taken. These absorbance values will then being compared with the standard curve to get the glucose

concentration inside the shake flask at particular time. 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 viral which

contains the biomass before and after the drying process is recorded to get the dry cell weight.

For the optical density of the cell, the absorbance value showed an increment which indicating that the

cell was growing and number of cell is increased in the shake flask. The glucose concentration,

however, cannot be determined as the absorbance values were increased and decreased unevenly and

comparison cannot be made with the standard curve as the data for the standard curve are not

consistent giving inaccurate curve. Therefore no conclusion can be made about the glucose

concentration in the shake flask. Supposedly, as the number of cell increased, the glucose

concentration would decrease as the glucose consumption by the cells is increased.

The dry cell weight in the other hand can be seen that there is an increment from the beginning of the

cultivation until the 6th hour and showed unstable changes until the 24th hour. Supposedly, as the

number of cell increased inside the shake flask, the cell dry weight also should be increased.

2. Introduction

Fermentation can be carried out as batch, continuous and fed-batch processes. In this experiment, the

shake flask fermentation is being used. Shake flask fermentation is the example of batch fermentation.

In shake flask, the culture flask usually Erlenmeyer flask is being used to place and growing the

microorganisms. It is the cheapest and easiest way to culture microorganism aerobically, in small

volumes of nutrient broth. It is a small scale equipment which equivalent to stirred tank bioreactor.

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In order to prevent any contamination to the culture, shake flask must be plugged. Different plug can

be made of cotton-wool, glass wool, polyurethane foam, gauze or synthetic fibrous material. The plug

has to prevent airborne microorganism from getting into the medium while at the same time allowing

free flow of air into the flask.

The cultures are incubated at certain temperature and shaking frequency 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,

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.

3. Aims

– To study 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

4. Theory

Shake flask fermentation is one of the examples of batch fermentation. Batch culture is an example of

a closed culture system which contains an initial, limited amount of nutrient. The inoculated culture

will pass through a number of phases. After an inoculation there is a period during which no growth

appears to take place. This period is referred as the lag phase and may be considered as a time of

adaptation. In a commercial process, the length of the lag phase should be reduced as much as

possible. Following a period during which the cell gradually increases, the cell grows at constant,

maximum rate and this period is known as the log phase or exponential phase. The exponential phase

may be described by the equation below:

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dxdt

= µx -------------------1

where

x is the concentration of microbial biomass

t is the time, in hours

µ is the specific growth rate, in hour -1

on integration, equation (1) gives

x t = xo eµt ------------------2

where

xo is the original biomass concentration

x t is the biomass concentration after time interval, t hours

During the exponential phase, the organism is growing at its maximum specific growth rate, µmax for

the prevailing conditions.

Equation 2 predicts that growth will continue indefinitely. However, growth results in the

consumption of nutrients and the excretion of microbial products. Thus after a certain time the cell

growth rate will decrease until growth ceases. The cessation of growth may be due to the depletion of

some essential nutrient in the medium when there is limitation in substrate.

The decrease in growth rate and the cessation of growth due to the depletion of substrate may be

described by the relationship between µ and the residual growth-limiting substrate as follows:

µ = µmax s

K s+s

where

µmax = maximum growth rate

s = residual substrate concentration

K s = substrate utilization constant

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Figure 4.1: The graph showing the relationship between the parameter of the Monod equation.

The stationary phase in batch culture is the point where the growth rate has declined to zero. In the

other word the growth rate is equivalent to the death rate. The cell death is might due to the nutrient

limitations due to their incorporation into cells during log-phase growth or a build-up of toxins due to

their release of fermentation products also during log-phase growth.

The death phase is the result of the inability of the bacteria to carry out further

reproduction as condition in the medium become less and less supportive of cell

division. The nutrient is extremely insufficient for the growth of the microorganism. Eventually,

the number of viable bacterial cells begins to decline at an exponential rate.

Industrial fermentation is usually interrupted at the end of the exponential

growth phase or before the death phase begins.

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Figure 4.1: Growth curve of microorganism based on cell number analysis

5. Apparatus and material

– E.coli

– Luria Bertani Broth

– Distilled water

– Shake flask

– Cotton-plugged

– Incubator shaker

– Cuvettes

– Centrifuges

– Micropipetor

– Pipette tips

– Laminar flow

– 70% ethanol

– Lighter and Bunsen burner

– Graduated cylinder

– Schott bottle

– DNS reagent

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6. Procedures

Part 1: Preparation of inoculated fermentation medium

1. 500ml shake flask, bunsen burner, measuring cylinder, LB broth and inoculums are brought

into the laminar flow.

2. Under aseptic technique, 50 ml of media is transferred into 500ml shake flask.

3. Then 6 ml of inoculums is added into the shake flask resulting in final volume of 56ml.

4. The shake flask is plugged with cotton-plugged.

5. The shake flask is swabbed with 70% ethanol.

6. The shake flask is incubated at 350 rpm; T=30˚C; 24 hours.

Part 2: Sampling for cell dry weight

1. 1ml of biomass concentration is taken out.

2. The 1ml biomass concentration is transferred into micro centrifuge tube. An empty micro

centrifuge tube must be weighted first.

3. The sample is then centrifuged for 10 minutes at 10000 rpm.

4. After that, the supernatant of the sample is taken out carefully without taking out any

biomass.

5. The biomass is then left dried inside an oven at 80C for overnight.

6. The dried biomass is then being placed inside a dessicator to let it cool before rapidly

weighing on an analytical balance.

Part 3: Glucose analysis

1. 1ml of biomass concentration is taken out.

2. The 1ml biomass concentration is transferred into micro centrifuge tube.

3. The sample is then put onto turntable of YSI 2700 Select Biochemical Analyzer for direct

analysis of glucose concentration.

4. Another method of glucose analysis is by using DNS reagent.

5. 1.5ml of DNS reagent is added into 0.5ml of the biomass sample inside a capped test tube

6. The mixture is heated at 90˚C for 10 minutes to develop the red-brown colour.

7. The heated mixture is then cooled to the room temperature for 2-3 minutes in a cold or ice

water.

8. The mixture is then being diluted with 10ml of distilled water.

9. The absorbance is checked with a spectrophotometer.

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Part 3: Sampling for absorbance analysis/ optical density

1. 2ml of biomass concentration is taken out and being transferred into micro centrifuge tube.

2. The spectrophotometer is calibrated to zero by blank consisting of 2ml LB Broth.

3. The biomass concentration is then being transferred into a cuvette and optical density

measurement is taken with wavelength set at 600nm.

4. More absorbance means higher number of cell.

Part 4: The preparation of glucose standard curve

1. The 20g/L, 40g/L, 60g/L, 80g/L and 100g/L of glucose concentration is prepared by weighing

the suitable amount of glucose and diluted with 10ml of distilled water.

2. 1.5ml of DNS reagent is added with 0.5ml of the glucose sample inside a capped test tube

3. The mixture is heated at 90˚C for 10 minutes to develop the red-brown colour.

4. The heated mixture is then cooled to the room temperature for 2-3 minutes in a cold or ice

water.

5. The mixture is then being diluted with 10ml of distilled water.

6. The absorbance is checked with a spectrophotometer

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7. Results

Table 7.1: The results showing the optical density of the cell, cell dry weight and glucose concentration per time

Hour

Absorbance readings: Optical density (spectrometer

wavelength: 600nm)

Biomass concentration: cell dry weight after 24h (g)

Glucose concentration (g/L)

Initial FinalNet absorbanc

eGlucose

0 0.340 1.052 1.066 0.014 - 0.025

3 1.812 1.153 1.170 0.017 0.071

6 2.597 1.045 1.070 0.025 0.066

9 2.688 1.153 1.175 0.022 0.088

12 2.734 1.167 1.183 0.016 0.079

21 2.801 1.157 1.176 0.019 0.094

24 2.800 1.156 1.175 0.019 0.087

0 5 10 15 20 25 300

0.5

1

1.5

2

2.5

3

absorbance value for optical density

absorbance

time

(h)

Figure 7.1: The graph showing the absorbance value for optical density

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0 5 10 15 20 25 300

0.005

0.01

0.015

0.02

0.025

0.03

cell dry weight curve

cell dry weight (g)

time

(h)

Figure 7.2: The graph showing the cell dry weight per time

Table 7.2: Data for standard curve of glucose test

Glucose concentration (g/l) Absorbance

0 0.039

20 2.141

40 2.750

60 2.760

80 2.505

100 1.791

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0 20 40 60 80 100 1200

0.5

1

1.5

2

2.5

3

absorbance value vs glucose concentration

glucose concentration (g/L)

abso

rban

ce v

alue

Figure 7.3: Graph showing the standard curve for the glucose concentration

8. Calculations

Cell dry weight

Net weight = final weight – initial weight

= 1.066g – 1.052g

= 0.014g

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9. Discussions

This experiment is carried out to study the kinetic growth of microorganism. E.coli is selected as the

cell and being cultivated inside a shake flask. The growth of microorganism in shake flask is a simple

method of fermentation. The nutrients for the microorganism are being supplied by the media which

contain the carbon sources. The flask is shaken during the cultivation to mix the cell and the media;

increase the homogeneity between these two and also to provide aeration for the cells. The culture is

gone through the fermentation process for 24 hours. Within that period, the biomass/cell sample is

taken out for every 3 hours to analyze the concentration of the cell (g/L), the cell dry weight and the

glucose concentration.

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 absorbance reading is

increase from the beginning of the experiment until the 21st hour and decrease slightly at the 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 24 th hour indicating that the cell growth

has reach its deceleration phase where the growth of the cell is started to slow down. The

decelerating growth phase is where the culture is in a transient state. During

this 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. In cell growth, the cell will go through several phases like lag,

exponential, deceleration, stationary and death phase.

In cell cultivation, the cells themselves need food or carbon sources like glucose for growth. In batch

fermentation for example in this experiment, the glucose can be the limiting factor for the cell growth

or we called it as substrate limiting growth. For this condition, the Monod equation can be used to

predict the growth rate and the cell concentration inside the shake flask. In addition, the glucose

concentration can be known by testing the cell sample into the glucose analyzer and the direct glucose

concentration can be obtained. In other way, the glucose concentration is also being obtained by

mixing the sample with DNS reagent. The DNS reagent will be reduced to 3-amino,5-nitrosalicylic

acid in the presence of free carboxyl group (glucose) and absorbance reading can be taken through the

spectrophotometer.

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As for this experiment, the glucose test showed no pattern of changes in absorbance values. These

values increase and decrease unevenly. This might be due to some mistakes occurred during the

glucose test where the volume of sample and DNS reagent that need to be mixed is incorrectly taken.

This has affected the accuracy of the absorbance reading. From the absorbance reading, the

concentration of the glucose can be obtained by referring to the glucose standard curve. The glucose

concentration should be decreased as the number of cell inside the flask is increased. This is because

as the number of organism increases, nutrients are consumed and becoming

lesser. However, this cannot be shown from the results obtained due to some mistakes occurred

throughout the experiment.

Figure 9.1: The changes in amount of glucose, lactose and biomass in cell culture per time.

Another analysis that can be performed to analyze the cell sample is 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. The remained cell is then

being dried inside an oven for 24 hours. The dry cell weight is finally taken to know the weight of the

cell that present at particular time during the cultivation. In this experiment, the cell dry weight is

increased from 0th hour until 6th hour and gradually decreased from the 9th hour to 12th hour and

increased until the 24th hour. The cell dry weight should increase when the number of cell increased

inside the shake flask.

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10. Conclusions

At the end of this experiment, microorganism is suitable to be fermented inside a shake flask and it is

a simple method to investigate the growth kinetics of the microorganism. Knowledge of microbial

growth kinetics is essential to determine when to harvest the culture for different purposes. For a

growth-linked product, it is desirable to harvest the culture at the late exponential growth phase. On

the other hand, for a non-growth-linked product, it would be desirable to harvest the culture at the

stationary growth phase.

As microorganism will go through several phases in their growth, several analyses on the cell need to

be done to know the growth kinetics of the cell and the duration for each phase. This includes the cell

concentration, glucose concentration and also the cell dry weight analyses. This method can be done

in the laboratory before the fermentation or the cultivation of microbes in large scale is performed.

Growth kinetics deals with the rate of cell growth and how it is affected by

various chemical and physical conditions. During the course of growth, the cells

is continuously changing and adapting itself in the media environment, which is

also continuously changing in physical and chemical conditions.

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.

The batch culture system is still widely used in certain industrial processes for

example brewery industry because of its easy management of feed stocks.

These advantages allow the use of unskilled labour and low risk of financial loss.

Low level of microbial contamination in fermented products is at time tolerable,

as long as the microbial contaminants are not pathogenic and do not alter the

desired properties of the product, such as taste, colour and texture.

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11. Recommendations

– 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.

– The supernatant of cell concentration should be taken out carefully without any taking out of

the biomass.

– The cap of the viral must be opened to fasten the drying process of the biomass in the oven.

– Wash hand after handling the culture.

– Disinfect the work area with 70% alcohol before handling the culture.

– Dispose of all contaminated materials in appropriate containers.

12. References

PF Stanbury and A Whitaker.,1984. Principles Of Fermentation Technology. Pergamon Press

Crueger W and Crueger A., 1990. Biotechnology: A Textbook of Industrial Microbiology. Sinauer

Associates, Sunderland Massachusetts.

Pirt SJ., 1975. Principles of Microbe and Cell Cultivation. Blackwell Scientific, Oxford.

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13. Appendices

Figure 12.1: The microorganism and media is

cultivated

Figure 12.2: The microorganism is being

incubated inside the incubator.

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Figure 12.3: The microorganism is being

transferred into the viral tube.

Figure 12.4: The sample is being centrifuged.

Figure 12.5: For protein test, the absorbance

reading is taken using spectrophotometer.

Figure 12.6: Glucose analyzer for glucose

concentration reading.

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Figure 12.7: The sample for cell dry weight is

being heated overnight using oven.

Figure 12.8: After being heated, the sample is

being cooled inside a desicator.

Figure 12.9: The sample is tested for glucose

concentration by using DNS reagent.

Figure 12.10: The sample being transferred into

viral tube.

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