Table of Contents
In this experiment, Escherichia coli is used as a sample to study the growth kinetic of microorganism in shake flask. A different volume of E. coli was transferred into 250ml Erlenmeyer/shake flask containing media for the nutrient of microorganism. The different volume of microorganism transferred will give the different effect of reading on the constant volume of media used.
There are three ways to test the growth kinetic rate of microorganism on shake flask, which are by optical density (OD), glucose analysis and cell dry weight (CDW).
Then, from these test the result then used to plot the growth curve graph which next use to compared and analyze the growth curve of microorganism.
Shake flask fermentation is one of the fermentation method which are widely used for screening of high producing strains. The shake flask fermentation is the simplest way to do the fermentation using small amount volume of nutrient broth in laboratory.
A nutritionally rich medium, Lysogeny broth or luria bertani (LB) is used for the growth of bacteria. Usually the shake flask used is between 250ml to 500ml range. From the previous research, it is shown that smaller volume of shake flask give better oxygen transfer rate but it is only suitable for short term fermentation, otherwise the medium will be evaporate. The shake flask have several design which are standard shake flask or Erlenmeyer flask, flying saucer shake flask, shake flask with baffles and flat bed Thompson bottle. In this experiment, the standard shake flask is used.
Erlenmeyer flask which equipped with cotton wool stoppers and autoclaved including the nutrient broth inside the flask. Then, the flask is allowed to cool to the room temperature before some microbes are allowed to grown inside the flask and put into the shaker machine. Most of the shaker machine are suitable for flask fermentation , although there are shaker which can be adjustable to allow the use of Other important component in shake flask is plugs. There are different types of plugs that can be used s such as plugs made from cotton-wool, glass wool, polyurethane foam or synthetic fibrous material.
The function of plugs is to prevent airborne microorganism from getting to the medium while at the same allowing free flow of air into the flask. Shaker machine is designed to assist with oxygen transfer for aerobic respiration microorganism. For more precisely defined environment, incubator shaking cabinet can be very well use. These cabinets can control the temperature, illumination, gaseous levels and humidity. The movement of shaker normally rotary shaking action or reciprocating shaking action is produced. And by increasing the speed of a shaker can increase the oxygen transfer rate of a particular flask.
To study the groth kinetics if microorganism in shake flask experiment. To construct a growth curve including lag, log, stationary and death phases. To determine the Monod parameters.
As the experiment required safety handling and carefully conduct, growth culture in a shake flask will be done through several techniques. The preparation of culture media should be done under laminar flow safety cabinet and the transfer of culture to media and from culture media to cuvette must undergo aseptic technique first to avoid unwanted microorganism enter the culture media. Measuring the sample optical density to develop a rowth curve should be done by using spectrophotometer. The step involve is quick and easy. For sample containing E. coli, the particular wavelength should be set to 600nm. Each reading should be recorded. As the microorganism grow with time, a growth curve can be used to explained by a diagram. The growth curve contains four different phase that are lag phase, exponential phase, stationary phase and death phase. Firstly in the growth of microorganism is the lag phase, which is the slowest part. In this phase there is no increase in the cell number and induction of enzymes to utilized substrate.
It is very important to decrease the lag period in industry to increase the productivity. To decrease the period of lag phase some used to inoculate with exponential phase cell, pre-acclimate inoculum in growth media and use high cell inoculum size. The second phase is the exponential phase. In the exponential phase, cells already adjusted to the surrounding and already begin to develop and increase the number of cells rapidly. Also, the nutrient and substrate concentration are large. The growth rate is independent of nutrient and substrate concentration.
The exponential phase is due to its name which cell number and mass concentration increase exponentially. Between log phase and stationary phase, there is deceleration phase which the depletion of one or more nutrients. This may due to accumulation of toxic byproduct of growth. The growth and metabolism in this phase is unbalanced due to shifts for survival. The third phase is the stationary phase in which in this phase the rate development of new cell & rate of death is same and no net growth of cell number or cell mass which shown no cell division occurs. In this stage, secondary metabolites produce quite high.
Endogeneous metabolism of energy can result in maintaining cell viability. If additional substrate is provided, the inhibitory compound can be remove. The last one is death phase. In this phase, the rate of death is greater than the rate development of new cells. Cell lysis occurs in this phase, and growth can be re-established by transferring to fresh media.
Microbe on agar plate (E. coli). Shake flasks. Sterile loop of wire .Cotton plug (cotton, cotton mesh,aluminium foil) . Incubator shaker. Media for inoculums growth (LB) . Pippettor . Centrifuge tubes. Cuvette. Falcon tubes. Light (bunsen burner) .Centrifuge Ethanol .Distilled water .Sterilized graduated cylinder.
- Luria Bertani (LB) broth (Lennox) is calculated to get concentration for 200ml of broth. LB brpth composition written to make media on the box that stored the LB is 10 g/L. So, 1. 6g of LB broth powder is diluted with 160ml of distilled water to make the media.
- 160ml of the LB broth is prepared inside 250ml Erlenmeyer flask.
- Erlenmeyer flask is closed with cotton covered by gauge and aluminum foil.
- 10 g/L of glucose is diluted with the same amount of distilled water used to dilute media that is 160 ml and is placed in a schott bottle. ) The Erlenmeyer flask containing media with all the experiment is autoclaved for 3 hours.
Sampling for absorbance analysis/optical density:
- 2 ml of inoculums is taken out and being transferred into cuvette.
- 2 ml of blank (LB broth not contain microorganism) is transferred into cuvette.
- The spectrophotometer is calibrated to zero by blank consisting of 2 ml pure LB broth (not contain microorganism).
- Then optical density measurement of the inoculums is measured by setting the wavelength at the range of 600nm.
- More absorbance means higher number of cell detected.
The sampling is repeated 12 times so that we have 12 samples in a time of 24 hours with samples taken in every 2 hours.
- 2 ml of inoculums is transferred into micro centrifuge tube.
- Sample is then centrifuge at 10000 rpm with temperature 4 0C for 10 minutes.
- After centrifuge, cell (solid) will deposited at the bottom of the centrifuge tube and supernatant (liquid) is separated from the cell.
- This supernatant is transfer into another centrifuge tube for glucose analysis; the cell will then followed the next test which is cell dry weight.
- Centrifuge tube containing supernatant is then slotted in the slot of the YTI turntable Biochemical Analyzer in order to analyzer the glucose existed inside the supernatant.
- After 24 hours, the inoculums is transferred back into 135 ml medium prepared making a total of 150 ml of inoculums prepared thus finishing the steps of preparation of inoculums.
Sampling for cell dry weight:
- All 12 centrifuge tube containing cell (solid) from procedure for glucose analysis is arranged on the centrifuge tube rack.
- The cap is opened inserted into the oven.
- Centrifuge tube rack containing centrifuge is put inside the oven at 60 0C for 24 hours.
- The dried biomass is then being placed inside desiccators to let it cool before rapidly weighing on an analytical balance.
Before the experiment is run, we need to do some preparation for the experiment which are devided into three stages , the sterilization preparation, media preparation and inoculum preparation. From the experiment, there is three method that has been used to identify the growth of the bacteria or microorganism which are optical density, cell dry weight and glucose analysis Absorbance analysis or optical density test is conduct using equipment called spectrophotometer which the wavelength is set at 600nm. The 2 ml sample is then diluted and transferred into a cuvette and the reading of optical density then collected.
The reading of OD is then tabulated and graph of optical density is plotted in figure 2. 0. From graph shown at figure 2. 0, the plotted point show that there are rapid increase in reading in the early hour for exact in the first two hour, then the reading quite stabilized until the 22th hour and for the last 2 hour the reading is fall. If we analyze the result from optical density test and compared to microorganism growth curve, it can be conclude that in the first 2 hour is log phase or exponential phase where the cell divide rapidly by binary fission and they also begin to proliferate at a constant rate to their maximum growth rate.
From the growth phase shown at figure 1. 0 , the doubling time instance for E. coli is 20 minutes. In this phase, the maximum growth rate and doubling time is determine since the growth at this time is the most constant and ideal. The first phase in the microorganism growth curve which is lag phase didn’t shown may due to the sample is kept in the incubator for the first 5 hours. After that log phase is the stationary phase where the growth of E. coli is quite slow. It is undetermined whether either the number of cells dividing is equal, some cell are dying or the population of cell simply stop to grow and to divide.
For some cases, at this phase, secondary metabolites are produced for example antibiotics. The stationary phase may occurs due to the lack of nutrient inside the media, the lack of oxygen in the shake flask or due to the number of cell reproduce so rapidly until both oxygen and nutrient is insufficient. In the next test, which is glucose analysis, a certain volume of sample for instance 2 ml of sample is transferred into the centrifuge tube and centrifuge 10 minutes at 10,000 rpm. Then, the supernatant and biomass is separate, the supernatant is taken out to undergo the glucose analysis.
The sample of glucose then separate into two which are diluted and non-diluted to compare the reading between both. The turntable of YSI 2700 is used, then selected the biochemical analyzer for direct analysis of glucose (dextrose) concentration. The glucose analysis is based on glucose oxidase that has been immobilize in the YSI dextrose membrane (YSI 2365). From the figure 2. 1 and 2. 2 the graph of glucose analysis, it can be conclude that the E. coli colony used the glucose as nutrient to grow as we seen that the glucose level is decrease as the time increase.
At high concentrations the specific growth rate is independent of the concentration of nutrient, but at low concentrations the specific growth rate is a strong function of the nutrient concentration. Such a relationship was predicted by Monod; however, Monod’s equation does not predict the relationship over the entire range of nutrient concentration. If parameters of the equation are estimated from the results obtained at low concentrations, then at high concentrations of nutrient, the specific growth rate is significantly higher than that predicted by Monod’s equation.
These results were interpreted on the basis that the rate of growth is controlled by at least two parallel reactions and that the affinities of the enzymes catalyzing these reactions are different. The last test is cell dry weight test, which the sample undergo the centrifuge process and separate into supernatant and biomass. The supernatant is then used in the glucose analysis. The dry weight of biomass produce is then used, the biomass is weighted to identify the massof colony of the E. coli.
The biomass of E. coli is put into the oven to make sure all the water contained is completely absorb. After a few minutes in dissicator which the biomass is let to cooled, it is then weighted by using analytical balance. From the figure 2. 3 the graph of cell dry weight, it can be conclude that the biomass of E. coli is not stable, it may due to the error occurred during this experiment. It is suppose that the cell dry weight of E. coli followed the growth curve of microorganism which shown in the theory section.
The biomass should increase through time with the increase of cell number of E. coli during binary fission. The relationship between specific growth rate and mean cell volume was also measured, and the results indicate that mean cell volume depends not only on the specific growth rate but also on the nature of the limiting nutrient. There are different mean cell volumes at the same specific growth rate established by different limiting nutrients. Therefore, the mean cell volume is not uniquely determined by the specific growth rate.
From the study of growth kinetic curve of E. coli, it can be conclude that this experiment is succeed. If we observe only from the graph of optical density which shown that it similar to the graph of growth curve of microorganism, also the cell dry weight should similar but due to some error the reading from weighted of biomass the biomass is unstable. By using the glucose analysis, we can conclude that all the glucose is completely consume by the E. coli colony. By study the growth curve of microorganism, it can be conclude that E. oli growth is complete in 24 hours is enough using shake flask.
In the future experiment, it better to weigh the mass of empty cuvette first before insert the sample as not all cuvette have the same weight. The second cuvette and first cuvette may varies in weight. Also note that take all weight of sample with cuvette for a better analysis. Aseptic technique should be applied to all steps that require the transfer of culture from one place to another to avoid the enter and present of other microorganism in the culture media.
As for the cuvette, it is suggested that use another type or other large cuvette to insert the 2ml of culture media. The use of small cuvette for 2ml of sampling may lead to unable of cuvette to store it. For the use of spectrophotometer, make sure take the reading of empty medium with no E. coli first before take the reading of culture medium.
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- Microbial Growth Curve Part 2:Stationary PhaseFacts andFallacies,24. . 2012 from url http://fermentationtechnology. blogspot. com/2010/01/stationary-phase-facts-and-fallacies. html
- Talaat E. Shehata and Allen G. Marr, Effect of Nutrient Concentration on the Growth of Escherichia coli (1971) Department of Bacteriology, University of California, Davis, California 95616
- Roger G. Harrison,Paul todd,Bioseparation science and engineering(2003),oxford university press.
- Christie john geankoplis, transport processes and separation process principles (fourth ed),pearson prentice hall (2003)
Cite this Shake Flask Fermentation
Shake Flask Fermentation. (2016, Sep 27). Retrieved from https://graduateway.com/shake-flask-fermentation/