1.0 OBJECTIVE1.1To study the media development for yeast fermentation in a bioreactor.1.2To know the criteria and identify the areas of sterilization before run the experiment.

2.0 INTRODUCTIONFermentation is a metabolic process in which bacteria, yeast or other microorganisms break down organic matter to obtain the energy required to stay alive, and produce organic compounds such as alcohol and organic acid, as well as inorganic compounds such as carbon dioxide and hydrogen. Fermentation also refer to the use of yeast to change sugar into alcohol or the use of bacteria to create lactic acid in certain foods. Fermentation is widely applied in industry for the production of wine, mead, cheese and beer. All fermentation process required media for the microorganism to growth. A media must have sufficient nutrient to ensure the microorganism grow in an optimum condition to have high yield of desired products. Sterilization is an important technique in fermentation to prevent the contamination by killing the unwanted microorganism that can affect the product in growth media. Hence, the bioreactor must be autoclaved before the fermentation process start. In order to prepare a suitable medium for the growth of microbes, the main factors to be considered is the sources of carbon and nitrogen as well as vitamins or any other limiting elements and minor minerals which will affect the growth rate of cell.Bioreactor is a vessel where a chemical process which involves organisms or biochemically active substances is carried out. The microorganisms or cells are able to perform their desired function with limited production of impurities under optimum condition. The environmental conditions inside the bioreactor, such as temperature, nutrient concentrations, pH, and dissolved gases (especially oxygen for aerobic fermentations) affect the growth and productivity of the organisms. A properly designed bioreactor is used to provide a controlled environment to achieve optimal growth and product formation in the particular cell system employed.

3.0 PROCEDURES5.0 QUESTIONS5.1 List down the factors that can cause contamination to your culture.If we do not sterilize or autoclave our bioreactor and glassware (like tubes), contamination is easier to occur. Besides, as fermentation occurs, inhibitory compound will be produced which then will mix with the products causing contamination and low yield of the products. Poor aseptic techniques also cause contamination. Improper handling can contaminate the cell line, but this infection is likely to be detected and controlled before inoculation of the bioreactor. Cell culture fermentation is susceptible to microbial contamination due to a longer fermentation process and slower growth rate.5.2 Why pH probe needs to be calibrating before autoclave start and pO2 probe after autoclave?Before the start of autoclaving of the broth for any cultivation experiment, it is essential to calibrate the pH probe. The measured signal is compared with the set point in the controller unit which then activates the acid or alkali to bring the measured value close to the set point. However before the pH probe is used, it needs to be calibrated with two buffers usually in the pH range which is to be used in the bioreactor cultivation experiment. This is because only a narrow pH ranges to culture the cell. For this specific pH controller one has to suitably identify the right control action setting for the addition of certain concentration of acid / alkali in the desired fermentation broth which can give quick control action with-out any oscillations/offset of measured value around the set point.5.3 What are the differences between impeller design for microbial, mammalian, and plant bioreactor and why they are designed in different ways? Microbial Cell (Yeast and bacteria) BioreactorMammalian Cells BioreactorPlant Cells Bioreactor

Rushton turbine is the most common generic term applied to flat-bladed or disk-turbine impellers Their blades are flat and set vertically along an agitation shaft, which produces a unidirectional radial flow. Rushton and Rushton-type impellers are commonly used in fermentations of cell lines that are not considered shear sensitive, including yeasts, bacteria, and some fungi.

Pitch blade impellers. Pitched-blade impellers are low shear impellers designed to gently mix the contents of a culture without causing cell damage. They are most often used with mammalian, insect, or other shear-sensitive cell lines growing in suspension. These impellers are often used in batch or fed-batch cultures, but they can also be used for continuous and perfusion processes.

Large pitch blade impeller.Large, slow-moving axial flow impellers providing good mixing at relatively low tip speeds up to 2.5 m sec-1 are superior for plant cell and tissue cultures. Axial flow impellers are commonly used for more sensitive cultures requiring lower gas dispersion. There is a tendency to develop shear sensitive improved axial flow impellers characterized by profiled large stirring paddles.

Impellers are designed in different patterns because different cells (animal, bacteria or plant) have different cell structures. Therefore, different designs are created to suit the different cell structures.In comparison to microbial cultures, cell cultures which are not protected by a cell wall are much more sensitive to shear stress and foam formation. Therefore for sensitive cell, low shear stress should be achieved by using different impeller which is more flat to avoid cell disruption.

5.4 Why microsparger is being used in mammalian bioreactor?Microsparger which normally have micro-sized (2-30m) pores for aeration and they come from different size and shape. These spargers are made up of sintered metals and sold as cylindrical elements to be connected to a main sparger line. Microsparger line generate very small bubbles (100-400 m), which are extremely efficient for mass transfer of oxygen. Small bubbles give high surface area, for culturing mammalian cell which is animal cell, high oxygen level is needed. Therefore, microsparger is more suitable to be used.

5.5 Give some comment on design for sparger, impeller, baffle and heating element for these three types of bioreactor.To augment mixing and gas dispersion, baffles are employed. They are normally incorporated into agitated vessels of all sizes to prevent vortex and to improve aeration efficiency. Baffles are metal strips roughly one-tenth of vessel diameter and attached radially to the wall of bioreactor. Baffles should be installed in such a way that a gap exist between them and vessel wall, so that there is scouring acting around and behind the baffles thus minimizing microbial growth on the baffles and fermenter walls.

DescriptionMicrobial Cell (Yeast and bacteria) BioreactorMammalian Cells BioreactorPlant Cells Bioreactor

SpargerMicrosparger (2-30m)Macrosparger (also known as ring sparger,0.5-2mm)Macrosparger (also known as ring sparger,0.5-2mm

ImpellerRushtan TurbineLarge pitch blade impellerPitch blade disk turbine

BaffleAdding baffles prevent vortex.Generally four to eight baffles are incorporatedAdding baffles prevent vortex.Generally four to eight baffles are incorporatedWith animal cell culture baffles causes shear damage, instead of baffles bottom drive axial impellers slightly off sight of centre is used.

Heating elementTemperature maintained at 300C by water jacket to maintain its temperature.Temperature maintained at 25-270C by water jacket.Temperature maintained at 29-370C by heat blanket.

5.6 How much yeast biomass will be produced theoretically from the stoichiometric equation above?C6H12O6 + 3O2 + 0.48NH3 0.48C6H10NO3 + 4.32H2O + 3.12CO2Yeast biomass = 0.48[12(6) + 1(10) + 14 + 16(3)]= 69.12Therefore, the yeast biomass will be produced theoretically from the stoichiometric equation is 69.12g dry mass/ g glucose consumed

5.7 Determine the yield coefficients Yx/s (biomass/glucose) and Yx/o2 (biomass/oxygen).Yx/s = = = = 0.384 g biomass/g glucose

Yx/o2 = = = 2.16 g biomass/g oxygen

5.8 What will happen if you have mistakenly tighten the lid screw and not clipped the hose for aeration during sterilization?If the lids screw is tighten before sterilization, the steam inside the vessel will explode since high temperature will cause pressure inside to be higher. Therefore, if the pressure inside the vessels is too high, the steam explosion maybe will cause the vessel to be broken. Besides, the hose normally will be clipped and covered with aluminium foil to be sterilized. If the hose is not clipped and left for autoclave, the hose will easily be damaged and even the media inside the vessels will flow out.

6.0 DISCUSSIONBioreactor also known as fermenter, can be described as a vessel which has provision of cell cultivation under sterile condition and control of environmental conditions such as pH, temperature and dissolved oxygen. It can be used for the cultivation of microbial plant or animal cells. A typical bioreactor consists of following parts: agitator, motor, stirrers, jackets and baffle. Agitator facilitates the mixing of the contents of the reactor which eventually keeps the cells in the perfect homogenous condition for better transport of nutrients and oxygen for adequate metabolisms of cell to the desired products. The agitator can be driven by magnetic /mechanical agitators. Baffle in the reactor is to break the vortex formation in the vessel, which is usually highly undesirable as it changes the centre of gravity of the system and consumes additional power. Sparger is to supply oxygen to the growing cells. Bubbling of air through the sparger not only provide the adequate oxygen to the growing cells but also helps in the mixing of the reactor contents thereby reducing the power consumed to achieve a particular level of (mixing) homogeneity in the culture. Jacket keeps the temperature of the bioreactor at a constant value. The desired temperature of the circulating water is maintained in a separate Chilled Water Circulator. The contact area of jacket provides adequate heat transfer area wherein desired temperature water is constantly circulated to maintain a particular temperature in the bioreactor.In this experiment, we are going to explore with three different types of bioreactor that are plant, mammalian and yeast/bacterial cells. There are some controllers in the bioreactor that are used to control parameter. Temperature, media flow rate, stirrer speed, pO2 detector, pH, antifoaming detectors, level control and gas flow rate(air).If there are any parameter that changes during process, that particular controller will be detected and action will be taken to return the parameter back to normal(set value).For oxygen detector, if the oxygen probe detect there are low concentration of oxygen level in the vessel, controller will take action by injecting more oxygen to the vessel. For the oxygen probe, the bottom part is made up of membrane which the oxygen will dissolve in it. Whole bioreactors have to be autoclaved by covering with aluminium foil under 1200C to kill all the contaminants. Filter in the bioreactor are used to prevent the bacteria out of the bioreactor. The filter got its own lifetime limits. Media can be autoclaved/sterilised while vitamin or minerals cannot be sterilised as it will destroy vitamin itself. While for the yeast, which occupied 10% of working volume is injected into the vessel after the bioreactor has been autoclaved via the largest pore on the upper surface of the bioreactor.For the yeast or bacteria bioreactor, the tank volume capacity is up to 2L.There are four closed tubes which are acid, alkali, media and antifoaming on the upper surface of the reactor. The water jacket is used to maintain the temperature inside the reactor. If we look outside from the reactor, there are 2 layers inside it, which one is jacket and the others is vessel. Jacket temperature is greater than the temperature inside the vessels. The impeller using in the yeast or bacteria reactor is rushtan impeller which have different diameter and size compare to others. Impeller normally contains 6 blades. Their blades are flat and set vertically along an agitation shaft, which produces a unidirectional radial flow. Rushton and Rushton-type impellers are commonly used in fermentations of cell lines that are not considered shear sensitive, including yeasts, bacteria, and some fungi. Inside the reactor which contain level detector and foam detector. Level detector is used to detect the volume of the media culture to prevent overflow while the foam detector is to detect the foam. Baffle is installed in the reactor to prevent vortex. Under the rushton impeller contain microsparger. Microsparger produces small bubbles which gives high surface area to provide more oxygen to the vessels .Besides, there are syringe connected to bio-reactor which function as sucking out the samples in particular time.

While for plant cell cultures bioreactor, the tank volume capacity is bigger than the yeast and mammalian cell. The tank volume is around 5L.There are also some different design of impeller used which is large pitch blade impeller which is suitable for plant (sensitive cell) to provide a low shear sensitivity. Ring spurge is used which the holes is bigger than microsparger. The bubbles produced is bigger than microsparger. The similarity between plant and yeast is both of them using water jacket to maintain temperature constant.

On the other side, for mammalian cell, the size is same like yeasts bioreactor which is 2L.The only difference compare to plant and microbial is mammalian cells bioreactor using heat blanket instead of water jackets. The heat blanket is like the jacket that covered the whole outside vessels. Ring spurge is used which the holes is bigger than microsparger. The bubbles produced is bigger than microsparger. Besides, the speciality of mammalian cells bioreactor compare to the rest two is the bioreactor is put on the weight balance. This weight balance is used to detect and measure the weight before and after the fermentation process.

7.0 CONCLUSION8.0 REFERENCES9.0 APPENDIX9.1 Microbial Cell Bioreactor

9.2 Mammalian Cell Bioreactor

9.3 Plant Cell Bioreactor