ERT 4163 CHAPTER 8 BIOREACTION DESIGN IN BIOPROCESS

ERT 416/3 CHAPTER 8: BIOREACTION DESIGN IN BIOPROCESS PLANT MISS. RAHIMAH BINTI OTHMAN (Email: rahimah@unimap. edu. my)

COURSE OUTCOME 1 CO 1) CHAPTER 7 : Upstream Processing In Bioprocess Plant. CHAPTER 8 : Bioreaction Design In Bioprocess Plant. DESCRIBE, ILLUSTRATE and COMPARE different types of bioreactors where the conversion of raw materials to desired product takes place. DECIDE and DESIGN the suitable bioreactor based on selected process based on stoichiometry, thermodynamics, separation and reaction engineering principles CHAPTER 9 : Downstream Processing In Bioprocess Plant. CHAPTER 10: Integrated Bioseparation Scheme for Product Isolation, Purification and Formulation Units For Bioprocess.

1. Different types of bioreactors where the conversion of raw materials to desired product takes place. 2. Design process of the suitable bioreactor based on selected process based on stoichiometry, thermodynamics, separation and reaction engineering principles.

GENERALIZED VIEW OF BIOPROCESS RAW MATERIALS UPSTREAM PROCESSES Inoculum Preparation Equipment Sterilization Media Formulation and Sterilization BIOREACTOR - FERMENTER Reaction Kinetics and Bioactivity Transport Phenomena and Fluid Properties Instrumentation and Control DOWNSTREAM PROCESSES Separation Recovery and Purification Waste Recovery, Reuse and Treatment THE BOTTOM LINE REGULATION ECONOMICS HEALTH AND SAFETY

1. Types of Bioreactors v Typical Bioprocess Flow Sheet

TABLE 1. Summary of Bioreactor Systems __________________________ Bioreactor Cell Systems Products used Design __________________________ � Air-Lift Bioreactor Bacteria, Yeast and other fungi SCP, Enzymes, Secondary metabolites, Surfactants � Fluidized-Bed Immobilized bacteria, yeast and other fungi, Activated sludge Ethanol, Secondary metabolites, Wastewater treatment Bioreactor � Microcarrier Bioreactor � Surface Tissue Propagator Immobilized (anchored) Interferons, Growth factors, mammalian cells on Blood factors, Monoclonal solid particles antibodies, Vaccines, Proteases, Hormones mammalian, tissue growth on solid surface, tissue engineering Interferons, Growth factors, Blood factors, Monoclonal antibodies, Vaccines, Proteases, Hormones __________________________

TABLE 1. 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, membranes used, cells Growth factors, Medicinal products Rotorfermentor � l � Modified Stirred Tank Bioreactor Modified Packed. Bed Bioreactor Tower and Loop Bioreactors Immobilized Bacteria, Ethanol, Monoclonal anti. Yeast, Plant cells bodies, Interferons, Growth factors Immobilized Bacteria, Yeasts and other fungi Ethanol, Enzymes, Medicinal products Bacteria, Yeasts Single Cell Protein (SCP) ________________________________________

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

Fig. 1. 1. Schematic diagram of a tower bioreactor system with perforated plates and co-current air liquid flow.

Fig. 1. 2. Schematic diagram of a tower bioreactor system with multiple impellers and liquid down comer and counter-current air liquid flow

FIG. 1. 3. Vacuum Fermenter

2. The Suitable Bioreactor Design Process

TABLE 2. Basic Bioreactor Design Criteria ________________________________ 1. Microbiological and Biochemical Characteristics of the Cell System (Microbial, Mammalian, Plant) 2. Hydrodynamic Characteristics of the bioreactor 3. Aeration and Oxygen, Mass and Heat Transfer Characteristics of the Bioreactor 4. Kinetics of the Cell Growth and Product Formation 5. Genetic Stability Characteristics of the Cell System 6. Aseptic Equipment Design 7. Control of Bioreactor Environment (both macro-and micro-environment) 8. Implications of Bioreactor Design on Downstream Products Separation 9. Capital and Operating Costs of the Bioreactor 10. Potential for Bioreactor Scale-up ________________________

3. Aeration and Oxygen Mass Transfer in Bioreactor Systems

�Living Cells: Bacteria, Yeasts, Plant cells, Fungi, Mammalian Cells �Require Molecular Oxygen O 2 as final Electron Acceptor in Bioxidation of Substrates (Sugars, Fats, Proteins, etc. )

FIG. 2. 1. Bio-oxidation of Substrate with Molecular Oxygen as the Final Electron Acceptor

OXIDATION-REDUCTION REACTION l Glucose is oxidized to make CO 2 �Oxygen is reduced to make H 2 O l Fig. 2. 1. Shows the biochemical pathway for aerobic oxidation of carbohydrates, fatty acids, and amino acids (AA) via the Tricarboxylic acid cycle (T. A. C. ) and electron Transport System. �Molecular oxygen O 2 accepts all the electrons released from the substrates during aerobic metabolism.

OXIDATION-REDUCTION REACTION (CONT’D) l Question: How do we ensure that we provide enough O 2 so that the cell growth in a bioreactor is not limiting? �Answer: Must ensure that O 2 is transferred fast enough from the air bubbles (gas phase) to the liquid phase (usually water) where all cells are present and growing.

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

LIQUID PHASE (CONT’D) � At Steady-state with no O 2 accumulation in the liquid phase: � What are the O 2 requirements of microorganisms?

OXYGEN REQUIREMENTS OF MICROORGANISMS We define: QO 2 = Respiration rate coefficient for a given microorganism. Units of QO 2: (mass of O 2 consumed) ÷ (unit wt. of dry biomass). (time) “Biomass” means the “mass of cells” in a bioreactor vessel. Some units of QO 2: m. M O 2/(g dry wt. of biomass) (hr. ) g. O 2/(g dry wt. ) (hr. ) LO 2/(mg dry wt. ) (hr. )

CONVERSION FACTORS: 1 M O 2 = 32 x 10 -6 g O 2 1 L = 1 x 10 -6 L at S. T. P. 1 mole O 2 = 22. 4 L O 2 at S. T. P. � In general: QO 2 = f(microbial species and type of cell, age of cell, nutrient conc. in liquid medium, dissolved O 2 conc. , temperature, p. H, etc. ) � For a given: 1) type of species of cell 2) age of cell 3) nutrient concentration 4) temperature 5) p. H

and if O 2 concentration, CL, is the limiting factor in cell growth, then QO 2 is a strong function of dissolved O 2 concentration CL (= mg O 2/L). The relationship between QO 2 and CL is of the Monod type. FIG. 2. 4. Respiration coefficient QO 2 as a function of the dissolved oxygen concentration CL.

� where: l l KO 2 = O 2 conc. at QO 2 max/2 CL CRIT. = Critical O 2 conc. beyond which O 2 is not limiting QO 2 = QO 2 max = constant At CLCRIT. respiration enzymes of Electron Transport System are saturated with O 2. When O 2 conc. is the “limiting substrate” then analogous to the Monod equation: µmax. S µ = ____ (S = substrate conc. (g/L) KS + S µ = 1 d. X (h-1) X dt [Ks = S (g/L), at µmax/2]

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

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

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

THE VOLUMETRIC MASS TRANSFER COEFFICIENT k. La AND METHODS OF MEASUREMENT

Mass Balance of Oxygen in Unit Liquid Volume FIG. 2. 7 Schematic diagram of the mass balance of oxygen transfer in unit liquid volume

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

Methods of Measurement of KLa in a Bioreactor

Chemical Methods of KLa Measurement FIG. 2. 8. Schematic diagram of a stirred tank batch reactor

Chemical Methods of KLa Measurement (Cont’d)

Chemical Methods of KLa Measurement (Cont’d) FIG. 2. 9. Concentration of SO 3 -2 as a function of oxidation time

Chemical Methods of KLa Measurement (Cont’d)

EMPIRICAL CORRELATIONS OF KLa

● A large number of Empirical Correlations Exist for KL and KLa for Agitated and Aerated Bioreactor Vessels. ● General Background Reading: Textbook by H. W. Blanch and D. S. Clark “Biochemical Engineering”, Chapter 5. Transport Processes, pp. 343 -415. Publisher: Marcel Dekker, Inc. , New York, 1996. ●Consider a Stirred Tank Bioreactor Vessel at a given:

Q DT HL VL Pg P = Vol. air flow rate @S. T. P. = Tank diameter = Liquid height (un-gassed) = Working Liquid volume (un-gassed) = Gassed power = Un-gassed power FIG. 2. 16. Typical stirred tank bioreactor vessel ● Impeller Speed R. P. M. Aeration Rate Q Working Liquid Volume VL of the Vessel

Most Empirical Correlations for KLa have the following form Where: ● KLa = Vol. mass transfer coefficient ● Pg = Gassed power supplied by mechanical impeller for mixing of bioreactor vessel. ● VL = Liquid working volume of bioreactor vessel

EMPIRICAL CORRELATIONS OF KLa Constants C, m, and k also depend on: ● Temperature, T ● p. H ● Physical properties of the solution ● Presence of other nutrients ● For Pure Water at p. H = 7, T = 25 o. C, the following empirical correlation applies:

AGITATION OF BIOREACTOR SYSTEMS

● Fig. 3. 1 shows the dimensions of what is called a “standard” stirred tank bioreactor vessel with Baffles. FIG. 3. 1. Standard Stirred Tank Bioreactor Geometry [Adopted from S. Aiba, A. E. Humphrey and N. F. Millis. “Bubble Aeration and Mechanical Agitation”. In Biochemical Engineering, 2 nd Ed. , Academic Press, Inc. , New York (1973) 174].

Geometric Ratios for a Standard Bioreactor Vessel Impeller Type Flat-Blade Turbine Di/Dt HL/Dt 0. 33 1. 0 Paddle impeller 0. 3 3 Marine Propeller 0. 33 1. 0 Li/Di 0. 25 - Wi/Di Hb/Di Wb/Dt 0. 2 1. 0 0. 25 1. 0 pitch = D i Where: Dt = tank diameter, HL = liquid height Di = impeller diameter Hb = impeller distance from bottom of vessel Wb = baffle width Li = impeller blade length Wi = impeller blade height 1. 0 No. Baffles 4 4 4

FIG. 3. 2 A. Different Impeller Types. (a) Marine-type propellers; (b) Flatblade turbine, Wi = Di/5. © Disk flat-blade turbine, Wi = Di/5, Di = 2 Dt/3, Li = Di/4; (d) Curved-blade turbine, Wi = Di/3; (e) Pitched-blade turbine, Wi = Di/8; and (f) Shrouded turbine, Wi = Di/8.

FIG. 3. 2 B. Mixing Patterns for Flat-Blade Turbine Impeller. Effect of Baffles. Liquid agitation in presence of a gas-liquid interface, with and without wail baffles: (a) Marine impeller and (b) Disk flat-blade turbines; (c) in full vessels without a gasliquid interface (continuous flow) and without baffles.

3. 1 Mixing and Power Requirements for Newtonian Fluids in a Stirred Tank FIG. 3. 3 NP vs. NRe; the power characteristics are shown by the power number, NP, and the modified Reynolds number, NRe, of single impellers on a shaft. [Adopted from S. Aiba, A. E. Humphrey and N. F. Millis. “Bubble Aeration and Mechanical Agitation”. In Biochemical Engineering, 2 nd Ed. , Academic Press, Inc. , New York (1973) 174].

Thank you Prepared by, MISS RAHIMAH OTHMAN