ERT: 314 BIOREACTOR SYSTEM

Experiment 1: Media Development and vessel sterilization

Lee Lay Pei 101140407

Group members :Afifah Fadhilah Binti Rosli 101140027Tan Wan Ting 101141270Moganeswari A/P Arumugam 101140484

Date of experiment : 25th February 2013

1.0 Objectives1.1 To study the media development for yeast fermentation in a bioreactor.1.2 To know the criteria indentify the areas for sterilization before run the experiment.

2.0 IntroductionBioreactor is a vessel or container which carries out biochemical process that involves microorganism. The selection and specific design of a bioreactor system include a series of decision on ranging from basic microbiology and biochemistry to process engineering. Bioreactor provides an optimal and controlled environment for the biological system, operating at minimum cost in order to maximize the productivity and benefit of the process. Fermentation is the conversion of a carbohydrate such as sugar into an acid or an alcohol. More specifically, fermentation can refer to the use of yeast to change sugar into alcohol. All atoms of carbon, hydrogen, oxygen, nitrogen and other elements are consumed by new cells or excreted as product when fermentation. The growth and product synthesis are well formulated by metabolic stoichiometry. This stoichiometry provides information including mass ad energy balance of fermentation, comparison on the theoretical and actual product yields, and consistency of experimental data and lastly the formulation of the nutrient media. The sufficiency of nutrient media, oxygen, temperature and pH is vital to support the cell growth and the produce the desired product. Furthermore, the developed media in the bioreactor has to be sterilized to prevent contamination by the unwanted microorganisms.

Figure 1 : BioreactorFigure 2: Internal stucture of a bioreactor3.0 Apparatus : Bioreactor, Autoclave, Weight balance, Erlenmeyer flask, magnetic stirrer, measuring cylinder. Chemicals : Distilled water, (NH4)2SO4, glucose monohydate, malt extract, yeast extract, KH2PO4, MgSO4

4.0 ProceduresExperiment 1: Media Development4.1.1 The concentration and total amount of glucose and (NH4)2SO4 in the nutrient medium are determined.4.1.2 The required carbon and nitrogen sources are weighted.4.1.3 The prepared media is pour into the 250 ml Erlenmeyer flask.4.1.4 The media is sterilized for 121 C for 15 minutes and let it be cooled upon inoculation of yeast.4.1.5 The media is inoculated and the fermentation process is run for 48 hours.4.2 Experiment 2: Vessel Preparation before AutoclavePreparation of Bioreactor before Autoclave4.2.1 The main power of the bioreactor unit is switched on and filled the water inside the jacket heater to the maximum level.4.2.2 The pH probe is taken out and calibrated it appropriately with pH 7 followed by pH 4 .The probe immerse in KCI solution in a conical flask.4.2.3 Each of the dosing pumps is calibrated to get the correct flow rate during the experiment.4.2.4 The lid is removed and detached all the hosing connected are remove from the main touch panel. All black hosing cannot be autoclaved, remove them before autoclave. Other colour of hosing can be autoclave.4.2.5 Motor all hosing and attached probes are removed from bioreactor.4.2.6 The vessel is rinse with tap water as much time as necessary to make sure it clean and clear.4.2.7 The electrolyte in the pO2 probe is checked whether its still having or not. This is to ensure membrane inside the pO2 probe is protected during autoclave.4.2.8 1L liquid media is prepared and pour into the vessel.4.2.9 The lid reattach back to the vessel. The pH probe and pO2 probe are mounted back.4.2.10 The pH, pO2, antifoam, level, temperature sensor and the hoses are covered with cotton and aluminium foil.4.2.11 The relief valve/filter at the condenser and sampling port are not covered with cotton, just wrap with aluminium foil.

4.3 Maintenance And Safety Precautions4.3.1 All operating instructions is supplied with the unit must be carefully read and understood before attempting to operate the unit.4.3.2 The stirrer, pH electrode, pO2 electrode, antifoam, pump at the bioreactor are turned off.4.3.3 When running the bioreactor must be assisted by trained PLV at all times.4.3.4 All safety appliances are worn when operating a high pressure gas line.

5.4 General Shut Down Procedures5.4.1 The agitator, temperature, dosing pump are turned off.5.4.2 The air sparger valve is closed and the main switch of the touch panel is turned off.5.4.3 The mixer motor is removed from top of the bioreactor and place on top of the apparatus.5.4.4 The pH and pO2 probe are carefully removed and place in the KCI solution for pH probe, while in the distilled water for pO2 probe.5.4.5 Any hosing attached with the bioreactor are removed and the bioreactor carefully bring to the sink.5.4.6 The cover is removed and the cover place on top of the bench so that it will not roll off.5.4.7 The reactor media is pour into the sink and wash thoroughly. The vessel rinse with distilled water.5.4.8 The cover place back to the vessel, and put back the vessel to its position.

5.0 Results and Calculation

No RIGHT WRONG

1All hosing connected from main touch panel is removed before autoclaved.Autoclave without detach all the hosing, especially the black hosing.

2Motor is removed and all probes are removed.Motor and probes is left in the bioreactor.

3The vessel is rinsed with tap water for several times.The vessel is autoclaved without prior rinse with water.

4pO2, antiform, level, pH electrode, temperature sensors and the hoses are covered by cotton and aluminium foil.pO2, antiform, level, pH electrode, temperature sensors and the hose are exposed to air.

5Relief valve or filter, at the condenser is wrapped with aluminium foil only.Relief valve or filter at the condenser is covered with cotton and then wrapped with aluminium foil.

6The tubing which connected with air spurger, sampling pot and from corrective bottles are clipped to prevent liquid from flowing out.The tubing connected with air spurger, sampling pot are not clipped.

Results from Group A1 and A2Time (hour)01516171819202141

OD values0.2660.6900.6940.7490.8350.8751.1240.1870.146

Result from group A3 and A4Time (hr)Optical Density at 495nm

00.182

161.144

171.567

181.582

191.609

201.620

211.629

221.650

241.700

411.726

431.725

441.719

CalculationDetermine the concentration and total amount of glucose and (NH4)2SO4 in the nutrient medium.Concentration of glucose50dw/L=50dw/L= 143.35 g/L glucose.Concentration of (NH4)2SO450dw/L50dw/L=22.9g/L (NH4)2SO4

To prepare 150ml of nutrient media,Molecular weight of NH4)2SO4 = 132g/molMolecular weight of glucose (C6H12O6) = 198g/molMolecular weight of yeast (C6H10NO3) = 144g/molFinal concentration = 50g/LSeed culture 10% from working volume, bioreactor volume capacity is 2L,The working volume = 70% of working volume = 2L X 70% = 1.4LThe growth of beakers yeast on glucose can be written by the following equation: C6H12O6 + 3O2 + 0.48NH3 0.48C6H10NO3 + 4.32H2O + 3.12CO2 YeastWeight of Glucose needed =143.35 g/Lglucose1.4L = 200.7 g glucoseWeight of (NH4)2SO4 needed =22.9g/L (NH4)2SO41.4L = 32g6.0 DiscussionBioreactor is a vessel in which achemical processis carried out which involves organismsorbiochemicallyactivesubstances whichderived from such organisms. This process can either be aerobic or anaerobic. In this experiment, the bioreactor used is stirred tank reactor (STR) which is the most common type used in industry. Typically, it is filled up with 70-80% medium of total volume as during fermentation process, bubbles may form and fill up the headspace of bioreactor. To make thing worst this situation may cause explosion. STR containing stirrer, sparger, 3 flat-blade turbine, baffle and foam breaker.For media preparation, glucose monohydrate and inorganic (NH4)2SO4 act as carbon and nitrogen source respectively. Carbon substrate acts as carbon and energy source, part of it contributes in cell carbon and the rest provide energy. The nutrient media is prepared by using 1 liter of distilled water, macroelement which are 32g of (NH4)2SO4, 200.7g glucose monohydrate, and trace elements which are 1.4g of malt extract, 1.4g of yeast extract, 4.2g of KH2PO4 and 0.28g of MgSO4. They are mixed in a schott bottle.The bioreactor is autoclaved at 121 C before medium is pour into reactor in order to prevent unwanted microorganism remain in it. To autoclave the bioreactor, several preparation steps are needed. Firstly, all the hosing should be removed, especially the black hosing. Then the motor and the electronic probes is also removed, because most of them are not temperature resistant. Besides, the relief valve or filter, male connector and the pH, antifoam, level, temperature sensor are cover with cotton or aluminium foil to avoid spoilage of the probes. Lastly, the tubing is clipped to prevent back flow of liquid. In term of sterility, there are 3 main ways in which a fermentation run may be approached which are firstly no selective condition, fermentation has to perform under strict aseptic conditions. Then partly selective conditions exist, whereby other organisms will proliferate and lastly very selective conditions exist, so there is hardly any chance of contaminating microbes. The longer the duration of the fermentation, more stringent demand on the bioreactor design for aseptic conditions.After sterilizing, STR is cooled down to room temperature and then medium is added into it. The fermentation process is started when the pH is calibrated. This experiment is done for 41 hours and samples are collected and the absorbance values are recorded by using spectrometer at 495nm wavelength. A graph of optical density (OD) versus time is plotted as shown in the result from group A1 and A2. OD values are increasing at the beginning until a certain point then it drops sharply. It can be explained that cells grow rapidly at the beginning and then cells denatured at the end. Most probably is because depletion of nutrients. Compare to the results from group A3 and A4, cells grow rapidly at the beginning then it reached stationary point after 17 hours.

7.0 Question7.1List down the factors that can cause contamination to your culture.The factors that can cause contamination are sterilization is not properly conducted, environmental factors which atmosphere contains many unwanted microorganisms. While weighting the material is contaminated by foreign substances such as dust.7.2Why pH probe needs to be calibrating before autoclave start and pO2 probe after autoclave?Since microbes are very sensitive to pH, 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.7.3What are the difference between impeller design for microbial,mammalian and plant bioreactor and why they are designed in different ways?For microbial bioreactor, their blades are flat and set vertically along an agitation shaft which produces a unidirectional radial flow. All impellers are designed to homogenously mix cells, gases, and nutrients throughout the culture vessel. The mixing action evenly distributes oxygen and nutrients to cells for healthy growth, keeps them from settling to the bottom of the vessel, and helps to maintain a uniform culture temperature. For Mammalian bioreactor, the pitched-blade impeller is used which low shear impeller designed to gently mix the content of culture without causing cell damage. In order to improve the oxygen transfer in a mammalian cell bioreactor, a new type of impeller consisting of a double-screen concentric cylindrical cage impeller was designed and its mass transfer rate evaluated. This new impeller design increases the specific screen area, and the convective mass transfer rate through the annular cage was significantly increased.For plant bioreactor, since plant cells are too fragile and it takes too long to grow in fermentation compared to microbial cells, 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.

7.4Why microsparger is being used in mammalian bioreactor?Mammalian bioreactor is used to improve gas transfer rates, enabling medium oxygenation in the headspace. It provides significantly higher gas medium interface and therefore higher O2 mass transfer coefficients with the same gas flow rate, which effectively reduces the overall superficial gas flow rate.7.5 Give some comment on design for sparger, impeller, baffle and heating element for these three types of bioreactor.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. Mammalian bioreactor, pitched-blade impeller is used. For microbial reactor, Ruston or flat bladed turbine impeller is used. The sparger is used to deliver gas or air which will depend on quantity used of this three bioreactor. The rotation of those ports creates a low-differential pressure at the base of the impeller tube, lifting microbial up through the tube and expelling them out through its ports. This continuous recirculation loop keeps cells uniformly dispersed throughout a vessel. Gases are introduced through a ring sparger, which generates bubbles that pass along the impeller between the exterior of the inner tube and an outer membrane, known as the aeration cage.7.6 How much yeast biomass will be produced theoretically from the stoichiometric equation above? C6H12O6 + 3O2 + 0.48NH3 0.48C6H10NO3 + 4.32H2O + 3.12CO2 0.48mol144g/mol = 69.2g7.7Determine the yield coefficients Yx/s (biomass/glucose) and Yx/o2(biomass/oxygen).(i) Yx/s (biomass/glucose) = (ii)Yx/o2(biomass/oxygen) concentration of O2 = 50dw/L = 50dw/L = 69.4 g/L O2 Yx/o2 = = = 0.72 biomass/gO2

7.8What 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, the air cannot be pass through the hose. If not clipped water will clog in the hose causes the medium cant flow out.

8.0 ConclusionAs a conclusion, media development is done by proper procedures and calculation from the metabolic stoichiometry. Besides that, sterilization process is a very important step to avoid any unwanted microorganisms to contaminate the culture medium.

9.0 ReferenceDoran, P.M. (1995). Bioprocess Engineering Principles. Academic Press, London.http://www.bioprocessintl.com/journal/2009/January/Which-Impeller-Is-Right-for-Your-Cell-Line-183538http://www.ncbi.nlm.nih.gov/pubmed/1860111212