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CBE 320b BIOCHEMICAL ENGINEERING III COURSE NOTES

CBE 320b BIOCHEMICAL ENGINEERING III COURSE NOTES. Instructor: Dr. A. Margaritis, Ph.D., P.Eng., F.C.I.C. Professor of Biochemical Engineering http://www.eng.uwo.ca/people/amargaritis/ DEPARTMENT OF CHEMICAL AND BIOCHEMICAL ENGINEERING The University of Western Ontario

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CBE 320b BIOCHEMICAL ENGINEERING III COURSE NOTES

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  1. CBE 320b BIOCHEMICAL ENGINEERING III COURSE NOTES Instructor: Dr. A. Margaritis, Ph.D., P.Eng., F.C.I.C. Professor of Biochemical Engineering http://www.eng.uwo.ca/people/amargaritis/ DEPARTMENT OF CHEMICAL AND BIOCHEMICAL ENGINEERING The University of Western Ontario Faculty of Engineering ©A. Margaritis 2006-2007

  2. TABLE OF CONTENTS 1. Introduction  Bioprocess Design  Novel Bioreactor Types  Design Criteria for Bioreactors 2. Aeration and Oxygen MassTransfer in Bioreactor Systems  Oxygen Requirements by Microorganisms  The volumetric Mass Transfer Coefficient KLa and Methods of Measurements  Empirical Correlations of KLa

  3. 3. Agitation of Bioreactor Systems 4. Scale-up of Bioreactor Systems  Scale-up Criteria  Example of Geometric Scale-up 5. Sterilization of Liquid Media  Kinetics of Thermal Death of Microorganisms  Batch Sterilization of Liquid Media  Continuous Sterilization of Liquid Media  Examples of Design for Continuous Liquid Medium Sterilization in a Tubular Sterilizer

  4. Air Sterilization by Fibrous Bed Filters  Mechanisms of Air Filtration and Design of Fibrous Packed Beds  Example of Design of Fibrous Packed Bed for Air Sterilization

  5. 1. Introduction

  6. GENERALIZED VIEW OF BIOPROCESS

  7. TYPICAL BIOPROCESS FLOW SHEET

  8. TABLE 1. Basic Bioreactor Design Criteria ___________________________________________________________________  Microbiological and Biochemical Characteristics of the Cell System (Microbial, Mammalian, Plant)  Hydrodynamic Characteristics of the bioreactor  Mass and Heat Transfer Characteristics of the Bioreactor  Kinetics of the Cell Growth and Product Formation  Genetic Stability Characteristics of the Cell System  Aseptic Equipment Design  Control of Bioreactor Environment (both macro- and micro-environment)  Implications of Bioreactor Design on Downstream Products Separation  Capital and Operating Costs of the Bioreactor  Potential for Bioreactor Scale-up ______________________________________________________________________

  9. TABLE 2. Summary of Bioreactor Systems __________________________________________________________ Bioreactor Cell Systems Products Design used __________________________________________________________  Air-Lift Bioreactor Bacteria, Yeast and SCP, Enzymes, Secondary other fungi metabolites, Surfactants  Fluidized-Bed Immobilized bacteria, Ethanol, Secondary Bioreactor yeast and other fungi, metabolites, Wastewater Activated sludge treatment  Microcarrier Immobilized (anchored) Interferons, Growth factors, Bioreactor mammalian cells on Blood factors, Monoclonal solid particles antibodies, Vaccines, Proteases, Hormones  Surface Tissue mammalian, tissue Interferons, Growth factors, Propagator growth on solid surface, Blood factors, tissue engineering Monoclonal antibodies, Vaccines, Proteases, Hormones __________________________________________________________

  10. TABLE 2. Summary of Bioreactor Systems (Cont’d) ____________________________________________________________________________________________________ Bioreactor Cell Systems used Products Design ________________________________________________________________________________________  Membrane Bioreactors, Bacteria, Yeasts, Ethanol, Monoclonal anti- Hollow fibers and Mammalian cells, Plant bodies, Interferons, Growth membranes used, cells factors, Medicinal products Rotorfermentor  Modified Stirred Immobilized Bacteria, Ethanol, Monoclonal anti- Tank Bioreactor Yeast, Plant cells bodies, Interferons, Growth factors  Modified Packed- Immobilized Bacteria, Ethanol, Enzymes, Medicinal Bed Bioreactor Yeasts and other fungi products  Tower and Loop Bacteria, Yeasts Single Cell Protein (SCP) Bioreactors ________________________________________________________________________________________

  11. TABLE 2. Summary of Bioreactor Systems (Cont’d) ____________________________________________ Bioreactor Cell System used Products design _____________________________________________________________________________________________________________________  Vacuum Bioreactors Bacteria, Yeasts, Fungi Ethanol, Volatile products  Cyclone Bioreactors Bacteria, Yeasts, Fungi Commodity products, SCP • Photochemical Photosynthetic bacteria, SCP, Algae, Medicinal Bioreactors Algae, Cyano bacteria, plant products, Plant Cell culture, r-DNA Monoclonal antibodies, plant cells Vaccines, Interferons ________________________________________________________________________________________

  12. Fig. 1.1. Schematic diagram of a tower bioreactor system with perforated plates and co-current air liquid flow.

  13. Fig. 1.2. Schematic diagram of a tower bioreactor system with multiple impellers and liquid down comer and counter-current air liquid flow

  14. Fig. 1.3. ICI Deep Shaft Unit

  15. FIG. 1.4. EMLICHHEIM FLOWSHEET

  16. FIG. 1.5. Internal circulation patterns of fluidized Ca-alginate beads containing immobilized cells of Z. mobilis. All dimensions in cm.

  17. FIG. 1.6. Vacuum Fermenter

  18. 2. Aeration and Oxygen Mass Transfer in Bioreactor Systems

  19.  Living Cells: Bacteria, Yeasts, Plant cells, Fungi, Mammalian Cells  Require Molecular Oxygen O2 as final Electron Acceptor in Bioxidation of Substrates (Sugars, Fats, Proteins, etc.)

  20. FIG. 2.1. Bio-oxidation of Substrate with Molecular Oxygen as the Final Electron Acceptor

  21. OXIDATION-REDUCTION REACTION • Glucose is oxidized to make CO2  Oxygen is reduced to make H2O • Fig. 2.1. Shows the biochemical pathway for aerobic oxidation of carbohydrates, fatty acids, and amino acids (AA) via the Tri- carboxylic acid cycle (T.A.C.) and electron Transport System.  Molecular oxygen O2 accepts all the electrons released from the substrates during aerobic metabolism.

  22. FIG. 2.2. Aerobic oxidation of carbohydrates, fatty acids, and amino acids via the TCA cycle and the Electron Transport System (ETS) through which electrons are transported and accepted by molecular oxygen (O2). ATP is produced from the phosphorylation of ADP. The ETS is composed of the following: FP1 = NADH; FP2 = succinate dehydrogenase; Q = Co-enzyme Q; Cytochrome b, c, a, and a3. The final electron acceptor O2 is reduced to water. Oxygen comes from the liquid phase and diffuses through the cell.

  23. OXIDATION-REDUCTION REACTION (CONT’D) • Question: How do we ensure that we provide enough O2 so that the cell growth in a bioreactor is not limiting?  Answer: Must ensure that O2 is transferred fast enough from the air bubbles (gas phase) to the liquid phase (usually water) where all cells are present and growing.

  24. LIQUID PHASE FIG. 2.3. The oxygen transport path to the microorganism. Generalized path of oxygen from the gas bubble to the microorganism suspended in a liquid is shown. The various regions where a transport resistance may be encountered are as indicated

  25. LIQUID PHASE (CONT’D)  At Steady-state with no O2 accumulation in the liquid phase:  What are the O2 requirements of microorganisms?

  26. 2.1 OXYGEN REQUIREMENTS OF MICROORGANISMS We define: QO2 = Respiration rate coefficient for a given microorganism. Units of QO2: (mass of O2 consumed) ÷ (unit wt. of dry biomass) . (time) “Biomass” means the “mass of cells” in a bioreactor vessel. Some units of QO2: mM O2/(g dry wt. of biomass) (hr.) gO2/(g dry wt.) (hr.) LO2/(mg dry wt.) (hr.)

  27. CONVERSION FACTORS: 1 M O2 = 32 x 10-6 g O2 1 L = 1 x 10-6 L at S.T.P. 1 mole O2 = 22.4 L O2 at S.T.P.  In general: QO2 = f(microbial species and type of cell, age of cell, nutrient conc. in liquid medium, dissolved O2 conc., temperature, pH, etc.)  For a given: 1) type of species of cell 2) age of cell 3) nutrient concentration 4) temperature 5) pH

  28. and if O2 concentration, CL, is the limiting factor in cell growth, then QO2 is a strong function of dissolved O2 concentration CL (= mg O2/L). The relationship between QO2 and CL is of the Monod type. FIG. 2.4. Respiration coefficient QO2 as a function of the dissolved oxygen concentration CL.

  29.  where: KO2 = O2 conc. at QO2 max/2 CL CRIT. = Critical O2 conc. beyond which O2 is not limiting QO2 = QO2max = constant • At CLCRIT. respiration enzymes of Electron Transport System are saturated with O2. • When O2 conc. is the “limiting substrate” then analogous to the Monod equation: µmax.S µ = ________ (S = substrate conc. (g/L) KS + S µ = 1 dX (h-1) [Ks = S (g/L), at µmax/2] X dt

  30. Table 1 shows typical values of QO2 measured by Warburg respirometer.  Table 2 shows typical data for critical oxygen concentration CL,CRIT. (mmol O2/L).  FIG. 2 shows the variation of QO2 with fermentation time for the microorganism Bacillus subtilis, where QO2 reaches a maximum value during the exponential growth phase.  FIG.3 shows the effect of agitation rate (revolutions per minute) on the value of QO2 for the bacterium Nocardia erythropolis, growing on hexadecane to produce biosurfactants.

  31. TABLE 1. Cell suspensions in glucose. Oxygen uptake determined in constant volume Warburg respirometer

  32. TABLE 2. Typical values of CL CRIT in the Presence of Substrate Adopted from R. K. Finn, P.81 in: N. Blakebrough (ed), Biochemical Engineering Science. Vol. 1, Academic Press, Inc., New York, 1967

  33. FIG. 2. 5a: Oxygen uptake rate, QO2X () and broth viscosity (▲)during batch aerobic fermentation of Bacillus subtilis. b:Respiration rate coefficient,QO2 () and volumetric mass transfer coefficient, KLa (). Taken from A.Richard and A. Margaritis, “Rheology, Oxygen Transfer, and Molecular Weight Characteristics of Poly(glutamic acid) Fermentation by B. subtilis”, Biotechnology and Bioengineering, Vol. 82 No. 3, p. 299-305, (2003)

  34. FIG. 2.6. Effect of agitation on the respiration coefficient (QO2) in a 20 L batch fermentation of Nocardia erythropolis. () 250 r.p.m, () 375 r.p.m, () 500 r.p.m. (Adopted from Kennedy et al. In Dev. Ind. Microbiol., 20 (1978) 623-630)

  35. 2.2 THE VOLUMETRIC MASS TRANSFER COEFFICIENT kLa AND METHODS OF MEASUREMENT

  36. Mass Balance of Oxygen in Unit Liquid Volume FIG. 2.7 Schematic diagram of the mass balance of oxygen transfer in unit liquid volume

  37. Mass Balance of Oxygen in Unit Liquid Volume (Cont’d)

  38. Mass Balance of Oxygen in Unit Liquid Volume (Cont’d)

  39. Mass Balance of Oxygen in Unit Liquid Volume (Cont’d)

  40. Mass Balance of Oxygen in Unit Liquid Volume (Cont’d)

  41. Mass Balance of Oxygen in Unit Liquid Volume (Cont’d)

  42. Methods of Measurement of KLa in a Bioreactor

  43. Chemical Methods of KLa Measurement FIG. 2.8. Schematicdiagram of a stirred tank batch reactor

  44. Chemical Methods of KLa Measurement (Cont’d)

  45. Chemical Methods of KLa Measurement (Cont’d)

  46. Chemical Methods of KLa Measurement (Cont’d)

  47. Chemical Methods of KLa Measurement (Cont’d)

  48. Chemical Methods of KLa Measurement (Cont’d)

  49. Chemical Methods of KLa Measurement (Cont’d)

  50. Chemical Methods of KLa Measurement (Cont’d)

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