1) Explain the control philosophy for pH, temperature and dissolved oxygen as was used in the fermentation process.
pH – As the bacteria cultured in the fermenter grow, reproduce and carry out metabolic activities, waste materials will be produced. Some of these waste materials that accumulate in the culture broth are acidic and will cause the pH of the broth to decrease. If the pH is allowed to decrease till toxic levels, the bacterial growth will stop or the bacterial cells in the fermenter might die. Depending on the strain and type of the microbe being cultured the waste products might increase or decrease the pH.
Hence to prevent extreme pH deviations from affecting cell growth and survival, acid (for high pH) or bases (for low pH) are pumped into the fermenter via peristaltic pumps to correct the pH back to optimum levels.
Temperature – There is an optimum temperature for the growth of the cultured microbe and the yield of the product it produces. Hence the temperature of the fermenter must be kept at optimum levels for optimum growth and production of the product of interest (GFP). This is done by the cooling jacket of the fermenter where hot or cold water can be pumped into to correct deviations from the optimum temperature.
Increase in temperature could be due to factors such as microbial respiration, movement of the impeller. Decrease in temperature could be due to factors such as cooling from the surroundings (air-conditioning) or endothermic reactions occuring within the cells.
Dissolved Oxygen – The culture requires an optimum amount of dissolved oxygen to provide a sufficient oxygen supply for cellular respiration. If too little dissolved oxygen is present, the growth rate of the culture might slow down. However, if the dissolved oxygen is too high it may become toxic to the cells as high levels of oxygen might generate reactive oxygen species that could damage the cultured cells’ DNA causing cell death.
To regulate the dissolved oxygen levels in our fermenter, the stirrer speed is varied. If the dissolved oxygen is too low, the stirrer speed will increase to break the air bubbles sparged in by the sparger into smaller bubbles. The smaller bubbles provide a larger interfacial area for oxygen to diffuse out of the bubbles and reach the cells in the culture. The reverse happens for high dissolved oxygen levels.
2) Describe the principle of the spectrophotometer which was used to determine the cell density (OD600). Why was 600 nm used?
The spectrophotometer can be used to detect substances that are able to absorb light which in this case are cells by emitting light at a particular wavelength (600nm in this case). This emitted light passes through a cuvette which holds the sample and are absorbed by cells found within the sample. The light which is not absorbed is transmitted across the cuvette into the detector which detects the amount of this transmitted light. The reading is translated into absorbance which is the amount of light absorbed by the sample.
By using Beer-Lambert’s law,
A = ε x l x c
Where,
A is the absorbance,
ε is the extinction coefficient (ml mg-1 cm-1)
l is the path length of light (cm),
c is the concentration (mg/ml),
the cell density can be calculated.
The cell density was measured by using light at wavelength 600nm as it is the best wavelength at which bacterial cells of a certain range of sizes can absorb light. If the wavelength is small, the light might be absorbed by things like proteins and DNA and the spectrophotometer will detect them too. Moreover, if the wavelength is too large, light will not be absorbed by the cells.
Is GFP a primary or secondary metabolite? At which phase should the product be harvested? At which phase was the product actually harvested?
GFP is a secondary metabolite which is produced during the stationary phase. Since GFP is produced at the stationary phase, the product should be harvested at the stationary phase just before the start of the death phase. The product was actually harvested at the stationary phase.
3) What are some advantages of using a computer control system? From the history chart (which will be given to you by your supervisor after the fermentation), comment on the effectiveness of the computer control.
The advantages of using a computer control system are:
- Ability to do data logging, data analysis and process control
- Allow for automatic adjustment of variables to correct deviations from optimum levels.
- Highly efficient in correcting deviations
- Allow the use of lesser manpower as no extra labour is required to constantly monitor and make corrects to deviations from optimum levels of variables.
Computer control in our fermentation is relatively efficient. As soon as a deviation from the set point is detected, the respective corrective action is immediately take. For instance, when the dissolved oxygen decreases below the set point, the pO2 probe will detect this change and relay this information to the computer. The computer will then automatically calculate the degree to which the stirrer speed is to be increased to increase the dissolved oxygen levels in the fermenter. It is these kinds of controls that help keep the media and growth conditions as favourable as possible for cell growth and which makes computer control effective.
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