ERT: 314 BIOREACTOR SYSTEMExperiment 1: Media Development and vessel sterilization Lee Lay Pei 101140407 Group members : Afifah Fadhilah Binti Rosli 101140027 Tan Wan Ting 101141270 Moganeswari A/P Arumugam 101140484 Date of experiment : 25th February 2013 0 nitrogen and other elements are consumed by new cells or excreted as product when fermentation. The sufficiency of nutrient media. Furthermore. fermentation can refer to the use of yeast to change sugar into alcohol. operating at minimum cost in order to maximize the productivity and benefit of the process.1. This stoichiometry provides information including mass ad energy balance of fermentation. 1. comparison on the theoretical and actual product yields. The selection and specific design of a bioreactor system include a series of decision on ranging from basic microbiology and biochemistry to process engineering. oxygen. Fermentation is the conversion of a carbohydrate such as sugar into an acid or an alcohol. the developed media in the bioreactor has to be sterilized to prevent contamination by the unwanted microorganisms.1 To study the media development for yeast fermentation in a bioreactor. Bioreactor provides an optimal and controlled environment for the biological system. All atoms of carbon.2 To know the criteria indentify the areas for sterilization before run the experiment. hydrogen.0 Introduction Bioreactor is a vessel or container which carries out biochemical process that involves microorganism. oxygen. The growth and product synthesis are well formulated by metabolic stoichiometry.0 Objectives 1. Figure 1 : Bioreactor stucture of a bioreactor Figure 2: Internal 1 . temperature and pH is vital to support the cell growth and the produce the desired product. and consistency of experimental data and lastly the formulation of the nutrient media. More specifically. 2. 6 The vessel is rinse with tap water as much time as necessary to make sure it clean and clear.1. 4. KH2PO4. 4.3 Each of the dosing pumps is calibrated to get the correct flow rate during the experiment.5 The media is inoculated and the fermentation process is run for 48 hours. 4.1 The main power of the bioreactor unit is switched on and filled the water inside the jacket heater to the maximum level.2.1.2.The probe immerse in KCI solution in a conical flask. 4. Erlenmeyer flask.2 The pH probe is taken out and calibrated it appropriately with pH 7 followed by pH 4 . pO2.1. Other colour of hosing can be autoclave. 4. 4.4 The lid is removed and detached all the hosing connected are remove from the main touch panel. remove them before autoclave. temperature sensor and the hoses are covered with cotton and aluminium foil. measuring cylinder. 4. glucose monohydate.1.0 Procedures Experiment 1: Media Development 4.2.10 The pH. malt extract.2. 4. 4.0 Apparatus : Bioreactor. Weight balance.5 Motor all hosing and attached probes are removed from bioreactor.2. antifoam.7 The electrolyte in the pO2 probe is checked whether it’s still having or not. level.3 The prepared media is pour into the 250 ml Erlenmeyer flask. 4.8 1L liquid media is prepared and pour into the vessel. All black hosing cannot be autoclaved.3. yeast extract. Chemicals : Distilled water.1 The concentration and total amount of glucose and (NH 4)2SO4 in the nutrient medium are determined.9 The lid reattach back to the vessel.2 Experiment 2: Vessel Preparation before Autoclave Preparation of Bioreactor before Autoclave 4. The pH probe and pO2 probe are mounted back.2. 4.2 The required carbon and nitrogen sources are weighted.4 The media is sterilized for 121 ºC for 15 minutes and let it be cooled upon inoculation of yeast. magnetic stirrer. 4.2. (NH4)2SO4. Autoclave. 2 . This is to ensure membrane inside the pO2 probe is protected during autoclave.1.2. 4.2.2. MgSO4 4. 4. 5. dosing pump are turned off. 4.2. 5.4 All safety appliances are worn when operating a high pressure gas line.7 The reactor media is pour into the sink and wash thoroughly.3 The mixer motor is removed from top of the bioreactor and place on top of the apparatus. 4. 5.4.3.1 The agitator. pH electrode. 5. 5. 5. 5.3. while in the distilled water for pO2 probe. The vessel rinse with distilled water.4. 3 .4.5 Any hosing attached with the bioreactor are removed and the bioreactor carefully bring to the sink. antifoam.3 Maintenance And Safety Precautions 4.11 The relief valve/filter at the condenser and sampling port are not covered with cotton.4. 5. 4.2 The air sparger valve is closed and the main switch of the touch panel is turned off.3. just wrap with aluminium foil.2 The stirrer.4. 4.4 The pH and pO2 probe are carefully removed and place in the KCI solution for pH probe. temperature.6 The cover is removed and the cover place on top of the bench so that it will not roll off. and put back the vessel to its position.3. pump at the bioreactor are turned off.4.4.1 All operating instructions is supplied with the unit must be carefully read and understood before attempting to operate the unit.8 The cover place back to the vessel. pO2 electrode.4.4 General Shut Down Procedures 5.3 When running the bioreactor must be assisted by trained PLV at all times.4. antiform. level. pO2. Results from Group A1 and A2 4 . covered with cotton and then wrapped The tubing which connected with air with aluminium foil. pH electrode. Motor is removed and all probes are especially the black hosing. The vessel is autoclaved without prior 4 several times. level. 5 6 corrective bottles are clipped to prevent liquid from flowing out.5. sampling pot and from sampling pot are not clipped. Relief valve or filter.0 Results and Calculation No 1 RIGHT All hosing connected from main touch WRONG Autoclave without detach all the hosing. pH electrode. pO2. at the condenser is exposed to air. rinse with water. The vessel is rinsed with tap water for bioreactor. 2 panel is removed before autoclaved. Motor and probes is left in the 3 removed. Relief valve or filter at the condenser is wrapped with aluminium foil only. spurger. temperature sensors and the hoses are temperature sensors and the hose are covered by cotton and aluminium foil. antiform. The tubing connected with air spurger. 187 0.2 0 0 5 10 15 20 25 30 Time (hour) 5 35 40 45 .6 0.694 0.266 0.124 0.4 0.2 1 0.Time (hour) OD values 0 15 16 17 18 19 20 21 41 0.690 0.749 0.8 Optical Density 0.146 Graph of Optical Density Versus Time (hour) 1.875 1.835 0. 725 44 1.2 0 0 5 10 15 20 25 30 Time (hr) Calculation 6 35 40 45 50 .Result from group A3 and A4 Time (hr) Optical Density at 495nm 0 0.6 1.4 0.629 22 1.144 17 1.8 1.609 20 1.567 18 1.182 16 1.650 24 1.719 Graph of Optical Density versus Time Optical Density at 495nm 2 1.700 41 1.2 1 0.6 0.620 21 1.582 19 1.726 43 1.8 0.4 1. Molecular weight of NH4)2SO4 = 132g/mol Molecular weight of glucose (C6H12O6) = 198g/mol Molecular weight of yeast (C6H10NO3) = 144g/mol Final concentration = 50g/L Seed culture 10% from working volume.48 mol yeast 198.48 mol NH 3 1mol × × × 50dw/L MW Yeast 0.48 mol yeast = = 143. Concentration of glucose ¿ MW glucose 1 mol glucose × × 50dw/L MW Yeast 0.48 mol NH 3 1mol × × × 50dw/L 144 g / mol 0.Determine the concentration and total amount of glucose and (NH4)2SO4 in the nutrient medium.17 g /mol 1 mol glucose × × 50dw/L 144 g /mol 0.48 mol yeast 2mol ¿ 132 g/mol 0.35 g/L glucose. Concentration of (NH4)2SO4 ¿ MW ( NH 4 ) 2 SO 4 0.32H2O + 3.48NH3 0.9g/L (NH4)2SO4 To prepare 150ml of nutrient media. The working volume = 70% of working volume = 2L X 70% = 1.48C6H10NO3 + 4.12CO2 Yeast 7 .4L The growth of beaker’s yeast on glucose can be written by the following equation: C6H12O6 + 3O2 + 0. bioreactor volume capacity is 2L.48 mol yeast 2mol =22. 28g of MgSO4. The fermentation process is started when the pH is calibrated. After sterilizing. because most of them are not temperature resistant. and trace elements which are 1. there are 3 main ways in which a fermentation run may be approached which are firstly no selective condition. sparger. glucose monohydrate and inorganic (NH 4)2SO4 act as carbon and nitrogen source respectively. 4. To autoclave the bioreactor. Typically. Then partly selective conditions exist. level. For media preparation. 3 flat-blade turbine. They are mixed in a schott bottle. The bioreactor is autoclaved at 121 ºC before medium is pour into reactor in order to prevent unwanted microorganism remain in it. antifoam.4L = 32g 6. baffle and foam breaker. Besides. Lastly.9g/L (NH4)2SO4×1. it is filled up with 70-80% medium of total volume as during fermentation process. the relief valve or filter.4g of malt extract. macroelement which are 32g of (NH 4)2SO4. especially the black hosing. so there is hardly any chance of contaminating microbes. 1. The longer the duration of the fermentation. STR is cooled down to room temperature and then medium is added into it. part of it contributes in cell carbon and the rest provide energy. In term of sterility.7g glucose monohydrate.7 g glucose Weight of (NH4)2SO4 needed =22. This process can either be aerobic or anaerobic.4g of yeast extract. several preparation steps are needed.35 g/Lglucose×1. In this experiment.Weight of Glucose needed =143. the tubing is clipped to prevent back flow of liquid.2g of KH 2PO4 and 0. This experiment is done for 41 8 . Firstly. Then the motor and the electronic probes is also removed. more stringent demand on the bioreactor design for aseptic conditions.0 Discussion Bioreactor is a vessel in which a chemical process is carried out which involves organisms or biochemically active substances which derived from such organisms. 200. all the hosing should be removed. the bioreactor used is stirred tank reactor (STR) which is the most common type used in industry. whereby other organisms will proliferate and lastly very selective conditions exist. temperature sensor are cover with cotton or aluminium foil to avoid spoilage of the probes. The nutrient media is prepared by using 1 liter of distilled water. STR containing stirrer.4L = 200. fermentation has to perform under strict aseptic conditions. Carbon substrate acts as carbon and energy source. male connector and the pH. To make thing worst this situation may cause explosion. bubbles may form and fill up the headspace of bioreactor. the pitched-blade impeller is used which low shear impeller designed to gently mix the content of culture without causing cell damage. All impellers are designed to homogenously mix cells.mammalian and plant bioreactor and why they are designed in different ways? For microbial bioreactor.2 Why pH probe needs to be calibrating before autoclave start and pO2 probe after autoclave? Since microbes are very sensitive to pH. It can be explained that cells grow rapidly at the beginning and then cells denatured at the end. The mixing action evenly distributes oxygen and nutrients to cells for healthy growth. Compare to the results from group A3 and A4. environmental factors which atmosphere contains many unwanted microorganisms. A graph of optical density (OD) versus time is plotted as shown in the result from group A1 and A2. While weighting the material is contaminated by foreign substances such as dust. Most probably is because depletion of nutrients. cells grow rapidly at the beginning then it reached stationary point after 17 hours. For Mammalian bioreactor. The factors that can cause contamination are sterilization is not properly conducted. and helps to maintain a uniform culture temperature.1 List down the factors that can cause contamination to your culture. OD values are increasing at the beginning until a certain point then it drops sharply.0 Question 7. 7. their blades are flat and set vertically along an agitation shaft which produces a unidirectional radial flow. In order to improve the oxygen transfer in a mammalian cell bioreactor. 7. gases. and nutrients throughout the culture vessel. keeps them from settling to the bottom of the vessel.hours and samples are collected and the absorbance values are recorded by using spectrometer at 495nm wavelength.3 What are the difference between impeller design for microbial. 7. therefore pH probe needs to be calibrating before autoclave because need to make sure the pH at neutral which is pH 7 followed by pH4 and the pO2 probe is calibrated after autoclave to get saturated oxygen concentration. a new type of impeller 9 . the blade impeller is used which can be flat or convex to produce an axial flow and used application require gentle mixing without causing cell rupture and damage. pitched-blade impeller is used. known as the aeration cage. since plant cells are too fragile and it takes too long to grow in fermentation compared to microbial cells.consisting of a double-screen concentric cylindrical cage impeller was designed and its mass transfer rate evaluated. Ruston or flat bladed turbine impeller is used. lifting microbial up through the tube and expelling them out through its ports. 7. For microbial reactor. Gases are introduced through a ring sparger. The rotation of those ports creates a low-differential pressure at the base of the impeller tube. and the convective mass transfer rate through the annular cage was significantly increased.5 Give some comment on design for sparger. baffle and heating element for these three types of bioreactor. For plant bioreactor. impeller. This continuous recirculation loop keeps cells uniformly dispersed throughout a vessel. 10 . The sparger is used to deliver gas or air which will depend on quantity used of this three bioreactor. Mammalian bioreactor. It provides significantly higher gas medium interface and therefore higher O2 mass transfer coefficients with the same gas flow rate. which generates bubbles that pass along the impeller between the exterior of the inner tube and an outer membrane.4 Why microsparger is being used in mammalian bioreactor? Mammalian bioreactor is used to improve gas transfer rates. For plant bioreactor. the use of impeller is marine blade impeller. Plant bioreactor the location of the sparger in the flow direction of the impeller guarantees mass and temperature homogeneity and optimal gas dispersion. This new impeller design increases the specific screen area. which effectively reduces the overall superficial gas flow rate. enabling medium oxygenation in the headspace. 7. of mole of yeast 32 g /mol 3 mol O2 × × 50dw/L 144 g /mol 0.32H2O + 3.7 Determine the yield coefficients Yx/s (biomass/glucose) and Yx/o2(biomass/oxygen).48NH3 0.2g 7.6 How much yeast biomass will be produced theoretically from the stoichiometric equation above? C6H12O6 + 3O2 + 0.4 g/L O2 Yx/o2 = biomassg /L O 2 conc g /L 50 g /L = 69.4 g / L = 0. of mole ofO 2 × × 50dw/L MW yeast no .8 What will be happened if you have mistakenly tighten the lid screw and not clipped the hose for aeration during sterization? If mistakenly tighten the lid screw.12CO2 0.72 biomass/gO2 7.7. biomassg/ L 50 g /L = glucose g/ L 130 g /L (i) Yx/s (biomass/glucose) = (ii)Yx/o2(biomass/oxygen) concentration of O2 = = MWO 2 no . 11 .48mol×144g/mol = 69. the air cannot be pass through the hose.48 mol yeast = 69.48C6H10NO3 + 4. If not clipped water will clog in the hose causes the medium can’t flow out. London.nlm.0 Conclusion As a conclusion. P. media development is done by proper procedures and calculation from the metabolic stoichiometry. Academic Press.M.gov/pubmed/18601112 12 . http://www. 9.bioprocessintl.com/journal/2009/January/Which-Impeller-Is-Right-for-Your-CellLine-183538 http://www.0 Reference Doran. Besides that. (1995).8.ncbi. sterilization process is a very important step to avoid any unwanted microorganisms to contaminate the culture medium.nih. Bioprocess Engineering Principles.