This experiment is completed to study the progress kinetics of microorganisms in shake flask. E. coli is grown in a LB broth medium and being fermented for 24 hours. Through the entire fermentation, the cell culture is taken out for each 3 hours and health proteins test, glucose ensure that you cell dried weight are being performed. As for the optical density analysis, the absorbance reading from the spectrophotometer is taken while for the blood sugar test, the reading of blood sugar level is taken from the YSI 2700 Select Biochemical Analyzer or can also being performed by using DNS reagent and the absorbance value is considered. These absorbance principles will being weighed against the standard curve to receive the glucose attention inside the tremble flask at particular time. The cell dried up weight, in the other palm, is taken following the mass concentration is being dried immediately in the range. The weight of the viral which provides the biomass before and after the drying process is recorded to find the dried up cell weight.
For the optical density of the cell, the absorbance value showed an increment which indicating that the cell was growing and range of cell is increased in the shake flask. The sugar concentration, however, cannot be identified as the absorbance values were increased and lowered unevenly and evaluation cannot be made using the typical curve as the data for the standard curve aren't consistent supplying inaccurate curve. Therefore no final result can be made about the blood sugar attention in the tremble flask. Supposedly, as the number of cell increased, the glucose concentration would decrease as the sugar intake by the skin cells is increased.
The dry cell weight in the other side can be seen that there is an increment right from the start of the cultivation before 6th hour and confirmed unstable changes before 24th hour. Supposedly, as the amount of cell increased inside the shake flask, the cell dry out weight also should be increased.
Fermentation can be executed as batch, ongoing and fed-batch techniques. In this experiment, the tremble flask fermentation has been used. Tremble flask fermentation is the example of batch fermentation. In shake flask, the culture flask usually Erlenmeyer flask is being used to put and growing the microorganisms. It's the cheapest and simplest way to culture microorganism aerobically, in small amounts of nutritional broth. It is a small level equipment which equal to stirred tank bioreactor.
In order to avoid any contamination to the culture, shake flask must be connected. Different plug can be produced of cotton-wool, cup wool, polyurethane foam, gauze or fabricated fibrous material. The plug has to prevent airborne microorganism from engaging in the medium while at the same time allowing free stream of air into the flask.
The ethnicities are incubated at certain heat range and shaking occurrence within an incubator shaker to attain a required progress rate. The shaking agitates the medium and the culture to keep carefully the blend relatively homogeneous and also to ensure aeration, creating an aerobic condition. In batch culture, you can find neither input supplied nor output produced throughout the fermentation. The medium culture is primarily inoculated with the microorganism. The development keeps increasing until at certain level, the progress is inhibited because of the decreasing substrate focus and the existence of dangerous metabolites.
- To analysis the growth kinetics of microorganism in tremble flask experiment
- To construct a growth curve including lag, log, fixed and loss of life phases
- To determine the Monod parameters
= x -------------------1
x is the attentiveness of microbial biomass
t is the time, in hours
is the precise progress rate, in hour -1
on integration, formula (1) gives
is the original biomass concentration
is the biomass concentration after time interval, t hours
During the exponential phase, the organism keeps growing at its maximum specific growth rate, for the prevailing conditions.
Equation 2 predicts that expansion will continue indefinitely. However, development results in the intake of nutrients and the excretion of microbial products. Thus after having a certain time the cell progress rate will decrease until progress ceases. The cessation of development may be because of the depletion of some essential nutrient in the medium when there may be restriction in substrate.
= maximum progress rate
s = residual substrate concentration
= substrate usage constant
The stationary phase in batch culture is the stage where the development rate has dropped to zero. In the other term the growth rate is the same as the death rate. The cell fatality is might because of the nutrient limitations because of their incorporation into skin cells during log-phase progress or a build-up of contaminants due to their release of fermentation products also during log-phase development.
The death period is the result of the inability of the bacterias to handle further reproduction as condition in the medium become less and less supportive of cell division. The nutrient is incredibly insufficient for the development of the microorganism. Eventually, the number of viable bacterial skin cells begins to drop at an exponential rate. Industrial fermentation is usually interrupted at the end of the exponential expansion phase or before the death phase begins.
Apparatus and material
- E. coli
- Luria Bertani Broth
- Distilled water
- Shake flask
- Incubator shaker
- Pipette tips
- Laminar flow
- 70% ethanol
- Lighter and Bunsen burner
- Graduated cylinder
- Schott bottle
- DNS reagent
Part 1: Prep of inoculated fermentation medium
500ml tremble flask, bunsen burner, measuring cylinder, LB broth and inoculums are brought into the laminar circulation.
Under aseptic technique, 50 ml of media is transferred into 500ml tremble flask.
Then 6 ml of inoculums is added in to the shake flask leading to final level of 56ml.
The shake flask is connected with cotton-plugged.
The tremble flask is swabbed with 70% ethanol.
The shake flask is incubated at 350 rpm; T=30‹C; 24 hours.
Part 2: Sampling for cell dry out weight
1ml of biomass concentration is applied for.
The 1ml biomass attention is moved into micro centrifuge tube. An empty micro centrifuge pipe must be weighted first.
The sample is then centrifuged for ten minutes at 10000 rpm.
After that, the supernatant of the test is applied for carefully without taking right out any biomass.
The biomass is then still left dried inside an oven at 80C for in a single day.
The dried out biomass is then being positioned inside a dessicator to allow it cool before rapidly weighing with an analytical balance.
Part 3: Glucose analysis
1ml of biomass awareness is taken out.
The 1ml biomass attentiveness is moved into micro centrifuge tube.
The test is then put onto turntable of YSI 2700 Select Biochemical Analyzer for direct analysis of sugar concentration.
Another approach to glucose analysis is to apply DNS reagent.
1. 5ml of DNS reagent is added into 0. 5ml of the biomass test in the capped test tube
The mix is heated up at 90‹C for 10 minutes to build up the red-brown colour.
The heated combination is then cooled to the area heat range for 2-3 minutes in a cool or ice water.
The concoction is then being diluted with 10ml of distilled normal water.
The absorbance is examined with a spectrophotometer.
Part 3: Sampling for absorbance evaluation/ optical density
2ml of biomass attentiveness is applied for and being moved into micro centrifuge tube.
The spectrophotometer is calibrated to zero by empty consisting of 2ml LB Broth.
The biomass attention is then being transferred into a cuvette and optical density way of measuring is used with wavelength place at 600nm.
More absorbance means higher quantity of cell.
Part 4: The preparation of glucose standard curve
The 20g/L, 40g/L, 60g/L, 80g/L and 100g/L of glucose concentration is prepared by weighing the best amount of blood sugar and diluted with 10ml of distilled drinking water.
1. 5ml of DNS reagent is added with 0. 5ml of the sugar sample in the capped test tube
The mix is heated up at 90‹C for 10 minutes to build up the red-brown color.
The heated blend is then cooled to the area temperature for 2-3 minutes in a cold or ice drinking water.
The combination is then being diluted with 10ml of distilled normal water.
The absorbance is examined with a spectrophotometer
This test is completed to study the kinetic expansion of microorganism. E. coli is preferred as the cell and being cultivated inside a shake flask. The progress of microorganism in tremble flask is a simple method of fermentation. The nutrition for the microorganism are being given by the media that have the carbon options. The flask is shaken during the cultivation to mix the cell and the press; improve the homogeneity between these two and also to provide aeration for the cells. The culture is fully gone through the fermentation process every day and night. Within that period, the biomass/cell test is applied for for each and every 3 hours to analyze the focus of the cell (g/L), the cell dry weight and the glucose concentration.
In order to analyze the awareness of the cell inside the flask, absorbance reading for the optical density is extracted from the spectrophotometer. The higher the absorbance reading means higher variety of cell occurrence inside the flask at a specific time. As for this test, the absorbance reading is increase right from the start of the test before 21st hour and lower slightly at the 24th hour. It can be explained that the amount of cell increase throughout the cultivation indicating that the cell keeps growing. Within the other hand, the reduction in cell number in 24th hour indicating that the cell progress has reach its deceleration phase where the expansion of the cell is started to decelerate. The decelerating growth phase is where the culture is at a transient status. During this level there are supply/back mechanisms that control the bacterial enzymes involved in key metabolic steps to permit the bacteria to withstand hunger. There is much turnover of proteins for the culture to cope with this era of low substrate supply. In cell expansion, the cell will proceed through several stages like lag, exponential, deceleration, fixed and death phase.
In cell cultivation, the skin cells themselves need food or carbon resources like blood sugar for development. In batch fermentation for example in this test, the glucose can be the limiting factor for the cell expansion or we called it as substrate limiting development. Because of this condition, the Monod equation may be used to predict the growth rate and the cell amount inside the tremble flask. Furthermore, the glucose focus can be known by examining the cell sample into the blood sugar analyzer and the immediate glucose concentration can be acquired. In other way, the blood sugar amount is also being obtained by mixing the test with DNS reagent. The DNS reagent will be reduced to 3-amino, 5-nitrosalicylic acid solution in the occurrence of free carboxyl group (sugar) and absorbance reading can be taken through the spectrophotometer.
As for this experiment, the glucose test proved no structure of changes in absorbance ideals. These beliefs increase and decrease unevenly. This might be due for some mistakes occurred during the glucose test where in fact the volume of test and DNS reagent that need to be blended is incorrectly taken. This has afflicted the precision of the absorbance reading. From your absorbance reading, the awareness of the glucose can be obtained by referring to the glucose standard curve. The blood sugar focus should be reduced as the amount of cell inside the flask is increased. It is because as the amount of organism increases, nutrition are consumed and becoming lesser. However, this cannot be shown from the results obtained scheduled to some mistakes happened throughout the test.
Another analysis that can be performed to analyze the cell sample is by firmly taking the dry out weight of the cell. In this technique, the cell is being taken out from cultivation flask and moved into viral tube. The pipe is the being centrifuged to separate the supernatant with the cell. The continued to be cell is then being dried inside an range every day and night. The dry cell weight is finally taken up to know the weight of the cell that present at particular time during the cultivation. In this experiment, the cell dried weight is increased from 0th hour until 6th hour and slowly but surely lowered from the 9th hour to 12th hour and increased before 24th hour. The cell dried out weight should increase when the number of cell increased inside the tremble flask.
At the finish of this test, microorganism would work to be fermented in the shake flask and it is a simple solution to investigate the growth kinetics of the microorganism. Knowledge of microbial growth kinetics is essential to ascertain when to harvest the culture for different purposes. For any growth-linked product, it is appealing to harvest the culture at the late exponential growth phase. Alternatively, for a non-growth-linked product, it would be suitable to harvest the culture at the stationary growth period.
As microorganism will go through several phases in their development, several analyses on the cell need to be done to learn the expansion kinetics of the cell and the length for each period. This includes the cell focus, glucose concentration as well as the cell dry out weight analyses. This technique can be carried out in the laboratory before the fermentation or the cultivation of microbes in large range is performed. Expansion kinetics handles the speed of cell development and how it is influenced by various chemical and physical conditions. During growth, the cells is consistently changing and adapting itself in the press environment, which is also continuously changing in physical and chemical substance conditions.
In finish, the microbial culture in batch culture system (tremble flask system) undergoes a lag phase, exponential growth stage, decelerating growth stage, stationary phase and sometimes the fatality phase will depend on the finish product desired. The substrate focus in the culture medium and development variables, such as glucose attentiveness changes correspondingly throughout the expansion phases. Thus, the physiology of the microorganisms is usually in a transient level, put through a constantly changing culture conditions. As a result, product formation is restricted to a certain period of cultivation, for example antibiotics would only be stated in the decelerating and fixed growth stages.
The batch culture system is still widely used in certain industrial functions for example brewery industry due to its easy management of feed stocks and shares. These advantages permit the use of unskilled labour and low risk of financial loss. Low level of microbial contamination in fermented products is at time tolerable, so long as the microbial pollutants aren't pathogenic, nor alter the required properties of the merchandise, such as preference, colour and consistency.
Aseptic approach must be practised when controlling biomass amount to avoid any contamination.
Cuvette must be wiped cleanly to avoid any scratch that would influence the spectrophotometer reading during proteins test.
This test must be completed under the laminar move to avoid any contamination to the culture.
The supernatant of cell attention should be taken out carefully without any taking right out of the biomass.
The cap of the viral must be opened to fasten the drying process of the biomass in the range.
Wash hands after controlling the culture.
Disinfect the work area with 70% alcoholic beverages before controlling the culture.
Dispose of all polluted materials in appropriate storage containers.
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