bioreactor basis

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  • 1.Industrial MicrobiologyIndustrial Microbiology BASIS OF BIOREACTOR FORBASIS OF BIOREACTOR FOR BIOPHARMACEUTICALSBIOPHARMACEUTICALS Angel L. Salamn, PhDAngel L. Salamn, PhD angelsalaman@yahoo.comangelsalaman@yahoo.com

2. Tutorial on BioreactorsTutorial on Bioreactors 1. Introduction1. Introduction 2. O2 uptake and Stoichiometry2. O2 uptake and Stoichiometry 3. Surface aeration3. Surface aeration 4. Methods of aeration4. Methods of aeration 5. Mechanically stirred bioreactors5. Mechanically stirred bioreactors 6. Bubble driven bioreactors6. Bubble driven bioreactors 7. Airlift bioreactors7. Airlift bioreactors 8. Packed bed and trickle flow bioreactors8. Packed bed and trickle flow bioreactors 9. Fluidized bed bioreactors9. Fluidized bed bioreactors 3. Bioreactors- IntroductionBioreactors- Introduction Previous lectures have stress the importance of consideringPrevious lectures have stress the importance of considering process engineering factors when culturing cells.process engineering factors when culturing cells. Biological factors include the characteristics of the cells, theirBiological factors include the characteristics of the cells, their maximum specific growth rate, yield coefficient, pH range andmaximum specific growth rate, yield coefficient, pH range and temperature range.temperature range. We have seen however that the productivity of a fermentation isWe have seen however that the productivity of a fermentation is determined by the mode of operation of the fermentationdetermined by the mode of operation of the fermentation process; eg. the advantages of fed-batch and continuousprocess; eg. the advantages of fed-batch and continuous fermentations over batch fermentations.fermentations over batch fermentations. 4. The oxygen demand of an industrial process isThe oxygen demand of an industrial process is generally satisfied by aeration and agitationgenerally satisfied by aeration and agitation Productivity is limited by oxygen availability andProductivity is limited by oxygen availability and therefore it is important to the factors that affect atherefore it is important to the factors that affect a fermenters efficiency in supplying Ofermenters efficiency in supplying O22 We are going to discuss OWe are going to discuss O22 requirement,requirement, quantification of Oquantification of O22 transfer and factors influencingtransfer and factors influencing the transfer of Othe transfer of O22 into solutioninto solution Bioreactors- IntroductionBioreactors- Introduction 5. Bioreactors- IntroductionBioreactors- Introduction Likewise mass transfer, in particular, oxygenLikewise mass transfer, in particular, oxygen transfer was highlighted as an important factortransfer was highlighted as an important factor which determined how a reactor must bewhich determined how a reactor must be designed and operated.designed and operated. Cost was also described as an importantCost was also described as an important consideration. The larger the reactor or theconsideration. The larger the reactor or the faster the stirrer speed, the greater the costsfaster the stirrer speed, the greater the costs involved.involved. How bioreactors are designed to meet cost,How bioreactors are designed to meet cost, biological and engineering needsbiological and engineering needs 6. MASS TRANSFER and PHASESMASS TRANSFER and PHASES Different phases present -IntroductionDifferent phases present -Introduction Fundamental concept in fermentation technology is transfer of materialsFundamental concept in fermentation technology is transfer of materials (e.g nutrients, products, gases etc.) through different phases (e.g gas into(e.g nutrients, products, gases etc.) through different phases (e.g gas into a liquid).a liquid). Major problem associated with provision of oxygen to the cell - is a rateMajor problem associated with provision of oxygen to the cell - is a rate limiting step and thus serves as a model system to understand masslimiting step and thus serves as a model system to understand mass transfer.transfer. The rate of oxygen transfer = driving force / resistanceThe rate of oxygen transfer = driving force / resistance.. E.gE.g resistance to mass transfer from medium to mo`s are complex and mayresistance to mass transfer from medium to mo`s are complex and may arise from;arise from; Diffusion from bulk gas to gas/liquid interfaceDiffusion from bulk gas to gas/liquid interface Solution of gas in liquid interfaceSolution of gas in liquid interface Diffusion of dissolved gas to bulk of liquidDiffusion of dissolved gas to bulk of liquid Transport of dissolved gas to regions of cellTransport of dissolved gas to regions of cell Diffusion through stagnant region of liquid surrounding the cellDiffusion through stagnant region of liquid surrounding the cell Diffusion into cellDiffusion into cell Consumption by organism (depends on growth/respiration kinetics)Consumption by organism (depends on growth/respiration kinetics) 7. The following diagram serves to illustrate the differentThe following diagram serves to illustrate the different phases and material that are relevant in general transportphases and material that are relevant in general transport processes associated with fermentation technology;processes associated with fermentation technology; Dispersed gases Dissolved nutrients Solid and Immiscible liquid nutrients Floc Cells Products in water MASS TRANSFER 8. Phases present in bioreaction /Phases present in bioreaction / bioreactorbioreactor Non aqueous phase Aqueous phase Solid phase (Reactants / products) Dissolved reactants / products Reaction Gas (O2, CO2, CH4 etc) Cells Liquids (e.g oils) Sugars Organelles Solid (e.g particles of substrate) Minerals Enzymes Enzymes ......... 1 2 .......... 1 = reactant supply and utilisation 2 = product removal and formation 9. One of the most critical factors in the operation of a fermenter is the provision of adequate gas exchange. The majority of fermentation processes are aerobic Oxygen is the most important gaseous substrate for microbial metabolism, and carbon dioxide is the most important gaseous metabolic product. For oxygen to be transferred from a air bubble to an individual microbe, several independent partial resistances must be overcome Mass Transfer 10. 1) The bulk gas phase in the bubble 2) The gas-liquid interphase 3) The liquid film around the bubble 4) The bulk liquid culture medium 5) The liquid film around the microbial cells 6) The cell-liquid interphase 7) The intracellular oxygen transfer resistance 1 2 3 4 5 6 7 Gas bubble Liquid film Microbial cell Oxygen Mass Transfer Steps 11. Stoichiometry of respirationStoichiometry of respiration To consider the Stoichiometry of respiration the oxidation of glucose may be represented as; C6H12O6 + 6O2 = 6H2O + 6CO2 Atomic weight of Carbon Hydrogen Oxygen 12 1 16 Molecular weight of glucose is 180 How many grams of oxygen are required to oxidise 180g of glucose? Answer 192g 12. Solubility of OxygenSolubility of Oxygen Both components oxygen and glucose must be inBoth components oxygen and glucose must be in solution before they become available tosolution before they become available to microorganismsmicroorganisms Oxygen is 6000 times less soluble in water thanOxygen is 6000 times less soluble in water than glucoseglucose A saturated oxygen solution contains only10mgA saturated oxygen solution contains only10mg dmdm-3-3 of oxygenof oxygen Impossible to add enough oxygen to a microbialImpossible to add enough oxygen to a microbial culture to satisfy needs for complete respirationculture to satisfy needs for complete respiration Oxygen must be added during growth at aOxygen must be added during growth at a sufficient rate to satisfy requirementssufficient rate to satisfy requirements 13. Comparison of conc. driving forces and uptakeComparison of conc. driving forces and uptake rates for glucose and oxygen by yeastrates for glucose and oxygen by yeast Problems encountered in oxygen transport can be illustrated byProblems encountered in oxygen transport can be illustrated by comparing transport of glucose vs oxygen;comparing transport of glucose vs oxygen; 1% Sugar (glucose)1% Sugar (glucose) Broth OBroth O22 satsat @ 25@ 25oo CC Conc. in bulk brothConc. in bulk broth 10,000 ppm10,000 ppm approx. 7 ppmapprox. 7 ppm Critical conc .Critical conc . 100 ppm100 ppm 0.8 ppm0.8 ppm (growth stops)(growth stops) Rate of demandRate of demand 2.8 mmoles/ g cells /h2.8 mmoles/ g cells /h 7.7 mmoles/7.7 mmoles/ g cells /hg cells /h 14. MASS TRANSFER and RESPIRATIONMASS TRANSFER and RESPIRATION (a) Mass balance(a) Mass balance StoichiometryStoichiometry of respiration e.g glucose;of respiration e.g glucose; CC66HH1212OO66 + 6O+ 6O22 6H6H22O + 6 COO + 6 CO22 Oxidation ofOxidation of 180 gms Glucose180 gms Glucose requiresrequires 192 gms O192 gms O22 Compare with a hydrocarbon (i.eCompare with a hydrocarbon (i.e 6 CH6 CH22)) 15. The Oxygen requirements ofThe Oxygen requirements of industrial fermentationsindustrial fermentations Oxygen demand dependant on convertion of Carbon (C)Oxygen demand dependant on convertion of Carbon (C) to biomassto biomass Stoichiometry of conversion of oxygen, carbon andStoichiometry of conversion of oxygen, carbon and nitrogen into biomass has been elucidatednitrogen into biomass has been elucidated Use these relationships to predict the oxygen demandUse these relationships to predict the oxygen demand for a fermentationfor a fermentation Darlington (1964) expressed composition of 100g of dryDarlington (1964) expressed composition of 100g of dry yeastyeast CC 3.923.92 HH 6.56.5 OO 1.941.94 16. OO22 RequirementsRequirements 6.67CH6.67CH22O + 2.1OO + 2.1O22 = C= C 3.923.92 HH 6.56.5 OO 1.941.94 + 2.75CO+ 2.75CO22 + 3.42H+ 3.42H22OO 7.14CH7.14CH22 + 6.135O+ 6.135O22 = C= C 3.923.92 HH 6.56.5 OO 1.941.94 + 3.22CO+ 3.22CO22 + 3.89H+ 3.89H22OO where CHwhere CH