Chapter 9: Operating Bioreactors
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1 Chapter 9: Operating Bioreactors David Shonnard Department of Chemical Engineering 1 Presentation Outline: Choosing Cultivation Methods Modifying Batch and Continuous Reactors Immobilized Cell Systems 2 1
2 Choosing the Cultivation Method The Choice of Bioreactor Affects Many Aspects of Bioprocessing. 1. Product concentration and purity 2. Degree of substrate conversion 3. Yields of cells and products 4. Capitol cost in a process (>5% total capital expenses) Further Considerations in Choosing a Bioreactor. 1. Biocatalyst. (immobilized or suspended) 2. Separations and purification processes 3 Batch or Continuous Culture? These choices represent extremes in bioreactor choices Productivity for cell mass or growth-associated products Batch Culture: assume k d = and q p = U E UDWHRIFHOOPDVVSURGXFWLRQLQEDWFKF\FOH U E P < 66 R W F W F EDWFKF\FOHWLPH W F µ PD[ ln X m X o W O Exponential growth time Lag time Harvest & Preparation 4 2
3 Batch or Continuous Culture? (cont.) Continuous Culture: assume k d = and q p = U F UDWHRIFHOOPDVVSURGXFWLRQLQFRQWLQXRXVFXOWXUH U F ' RSW RSW VHW G' G' ' RSW µ PD[ 6 R ). RSW < 6 ' RSW 6 6 R ) < 6 6 R 6 R µ PD[ ' RSW ' RSW RSW < 6 µ PD[ 6 R )6 R 6 R < 6 µ PD[ 6 R ZKHQ 6 R 5 Batch or Continuous Culture? (cont.) Comparing Rates in Batch and Continuous Culture U F U E 6R < 6 < 6µ PD[ 6 R OQ P W O µ PD[ R OQ P R W O µ max $FRPPHUFLDOIHUPHQWDWLRQZLWK P R W O KUDQGµ PD[ KU U F Continuous culture method is ~ 1 times U E more productive for primary products (biomass & growth associated products 6 3
4 Batch or Continuous Culture? (cont.) Why is it that most commercial bioprocess are Batch?? 1. Secondary Product Productivity is > in batch culture (SPs require very low concentrations of S, S << S opt ) 2. Genetic Instability makes continuous culture less productive (revertants are formed and can out-compete highly selected and and productive strains in continuous culture.) 3. Operability and Reliability (sterility and equipment reliability > for batch culture) 4. Market Economics (Batch is flexible can product many products per year) 7 Batch or Continuous Culture? (cont.) Most Bioprocesses are Based on Batch Culture (In terms of number, mostly for secondary, high value products) High Volume Bioprocesses are Based on Continuous Culture (mostly for large volume, lower value, growth associated products -- ethanol production, waste treatment, single-cell protein production) 8 4
5 Modified Bioreactors: Chemostat with Recycle To keep the cell concentration higher than the normal steadystate level, cells in the effluent can be recycled back to the reactor. Advantages of Cell Recycle 1. Increase productivity for biomass production 2. Increase stability by dampening perturbations of input stream properties 9 Chemostat with Recycle: Schematic Diagram Figure 9.1 Mass balance envelope α = recycle ratio C = cell concentration ratio X 1 = cell concentration in reactor effluent X 2 = cell concentration in effluent from separator Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 22 Recycle Stream Centrifuge Microfilter Settling Tank 1 5
6 Chemostat with Recycle: Biomass Balance ) R α)& α ) 9 5 µ 9 5 G GW DWVWHDG\ VWDWH G DQGVWHULOHIHHG R GW α)& α ) 9 5 µ DQGVROYLQJIRUµ µ > α &@' 6LQFH&! DQGα & WKHQµ ' A chemostat can be operated at dilution rates higher than the specific growth rate when cell recycle is used 11 Chemostat with Recycle: Biomass Balance µ > α &@' RQRG(TXDWLRQµ µ max XEVWLWXWHRQRG(TQLQWRDERYHVROYHIRU ' α & µ max ' α & 12 6
7 µx FS o + αfs - (1 + α)fs - V 1 ds R M = V Y R X/S dt at steady - state ( ds =) dt µx FS o + αfs - (1 + α)fs - V 1 R = M Y X/S and solving for X 1 X 1 = D µ Y M X/S(S o - S) But X 1 = Chemostat with Recycle: Substrate Balance Y M X/S (S o -S) [1 + α (1 - C)] = D µ = 1 [1 + α(1 - C)] M Y X/S [1 + α(1 - C)] S o - K S D(1 + α(1 - C)) µ max - D(1 + α(1 - C)) 13 Chemostat with Recycle: Comparison Figure 9.2 X 1 (recycle) Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 22 =DX 1 =DX 1 α=.5, C=2., µ max =1. hr -1, K S =.1 g/l, Y M X/S=.5 X 1 = cell concentration in reactor effluent with no recycle X 1 (recycle) = cell concentration in effluent with recycle 14 7
8 Multiple Chemostat Systems Applicable to fermentations in which growth and product formation need to be separated into stages:. P 1 P 2 Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 22 Growth stage Product formation stage 15 Multiple Chemostat Systems (cont.) 1. Genetically Engineered Cells: Recombinant DNA Translate to protein product Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall,
9 Multiple Chemostat Systems (cont.) Features of Genetically Engineered Cells: have inserted recombinant DNA (plasmids) which allow for the production of a desired protein product. GE cells grow more slowly than original non-modified strain (due to the extra metabolic burden of producing product). Genetic Instability causes the GE culture to (slowly) lose ability to produce product. The non-plasmid carrying cells or the cells with mutation in the plasmid (revertants) grow faster. 17 Multiple Chemostat Systems (cont.) Genetically Engineered Cells (cont.): In the first stage, only cell growth occurs and no inducer is added for product formation. The GE cells grow at the maximum rate and are not out-competed in the first chemostat by revertant cells. When cell concentrations are high, an inducer is added in the latter (or last) chemostat to produce product at a very high rate. 18 9
10 Multiple Chemostat Systems (cont.) 2-Stage Chemostat System Analysis Stage 1 - cell growth conditions, k d =, q p =, steady-state µ µ 1 PD[ 6 ' IURPELRPDVVEDODQFH 6 UHDUUDQJLQJ6 ' ZKHUH' ) µ PD[ ' 9 < 6 6 R 6 IURPVXEVWUDWHEDODQFH 19 Multiple Chemostat Systems (cont.) 2-Stage Chemostat System Analysis Stage 2 - product formation conditions, k d =, F =, steady-state G ) ) 9 µ 2 9 ELRPDVVEDODQFH GW µ µ 2 PD[ 6 ' ZKHUH' ) 6 9 µ )6 )6 9 2 < 9 T 3 G6 9 6 < 36 GW VXEVWUDWHEDODQFH G3 )3 )3 9 T 3 9 GW SURGXFWEDODQFH 2 1
11 Multiple Chemostat Systems (cont.) 2-Stage Chemostat System Analysis Stage 2 - product formation conditions, k d =, F =, steady-state µ µ 2 PD[ 6 ' ELRPDVVEDODQFH 6 µ ' < + T 3 6 ' < 3 6 VXEVWUDWHEDODQFH HTXDWLRQVXQNQRZQV6 )3 )3 9 T 3 9 G3 GW SURGXFWEDODQFH XVH LQSURGXFWEDODQFHWRVROYHIRU3 21 Fed-Batch Operation Useful in Antibiotic Fermentation reactor is fed continuously (or intermittently) reactor is emptied periodically purpose is to maintain low substrate concentration, S useful in overcoming substrate inhibition or catabolic repression, so that product formation increases
12 Fed-Batch Operation (cont.) Before t =, almost all of the substrate, t= S o, in the initial volume, V o, is converted to biomass, X m, with little product formation (X=X m Y X/S S o ) and P. t i At t=, feed is started at a low flow rate n c such that substrate is utilized as fast as r It enters the reactor. Therefore, S remains e very low in the reactor and X continues to a s maintain at Y X/S S o over time. The volume i increases with time in the reactor and n g Product formation continues. t w cycle time Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, Fed-Batch Operation (cont.) Behavior of X, S, P, V, and µ over time Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall,
13 Fed-Batch Operation (cont.) Analysis of Fed-Batch Operation 9ROXPH G9 GW ) 9 9 R )W %LRPDVV) R 9µ G9 GW 9µ G9 GW µ 9 µ ) 9 ) 9 R )W ' R ' R W 9 G GW G9 GW G9 GW ) 9 ' 25 Fed-Batch Operation (cont.) Analysis of Fed-Batch Operation (cont.) 7RWDO%LRPDVV W JFHOOVYVWLPH G GW RU UHDUUDQJLQJ G W GW G W 9 G W 9 GW G9 W GW GW 9 G9 W 9 GW ) < 6 ) P 6 R LQWHJUDWLQJ W WR < 6 6 R )W 9 R )W P 26 13
14 Fed-Batch Operation (cont.) Analysis of Fed-Batch Operation (cont.) 3URGXFW)RUPDWLRQ WRWDOSURGXFW 3 W 39 )RUPDQ\VHFRQGDU\SURGXFWV WKHVSHFLILFUDWHRI SURGXFWIRUPDWLRQLVDFRQVWDQW T 3 G3 W GW T 3 W T 3 9 R )W P LQWHJUDWLQJ 3 W 3 WR T 3 P 9 R )W W RU3 3 9 R R 9 T 9 R 3 P 9 'W W 3 RU3 R 9 R 9 R )W T 9 R 3 P 9 R )W )W 9 R )W W 27 Immobilized Cell Systems 9.4 Restriction of cell mobility within a confined space Potential Advantages: 1. Provides high cell concentrations per unit of reactor volume. 2. Eliminates the need for costly cell recovery and recycle. 3. May allow very high volumetric productivities. 4. May provide higher product yields, genetic stability, and shear damage protection. 5. May provide favorable microenvironments such as cell-cell contact, nutrient-product gradients, and ph gradients resulting in higher yields
15 Immobilized Cell Systems 9.4 Potential Disadvantages/Problems: 1. If cells are growing (as opposed to being in stationary phase) and/or evolve gas (CO 2 ), physical disruption of immobilization matrix could result. 2. Products must be excreted from the cell to be recovered easily. 3. Mass transfer limitations may occur as in immobilized enzyme systems. 29 Methods of Immobilization Active Immobilization: 1. Entrapment in a Porous Matrix: Polymeric Beads: cells Inert/solid core Polymers: agar, alginate κ-carrageenan polyacrylamide gelatin, collagen porous polymer matrix 3 15
16 Methods of Immobilization (cont.) Encapsulation: hollow spherical particle liquid core with cells less severe mass transfer limitations Membrane: nylon, collodion, polystyrene, polylysine-alginate hydrogel Cellulose acetate-ethyl acetate semipermeable membrane 31 Methods of Immobilization (cont.) Hollow Fiber Membrane Reactor: liquid in shell side shell nutrients nutrients products products tube semi-permeable membrane cells 32 16
17 Methods of Immobilization (cont.) 2. Cell Binding to Inert Supports: Micro-porous Supports: mass transfer limitations occur micropores d P >4 d C cells in micropores porous glass, porous silica, alumina ceramics, gelatin, activated carbon Wood chips, poly propylene ion-exchange resins (DEAE-Sephadex, CMC-), Sepharose 33 Methods of Immobilization (cont.) Binding Forces: Electrostatic Attraction ion exchange + support + Hydrogen Bonding cell C-O - O HO support 34 17
18 Methods of Immobilization (cont.) Binding Forces: Covalent Bonding: (review enzyme covalent bonding) Support materials: CMC-carbodiimide support functional groups -OH, -NH 2, -COOH Binding to proteins on cell surface 35 Methods of Immobilization (cont.) Overview of Active Cell Immobilization Methods: Adsorption Adsorption Support Capacity Strength Porous silica low weak Wood chips high weak Ion-exchange resins high moderate CMC high high 36 18
19 Methods of Immobilization (cont.) Passive Immobilization: support liquid phase wastewater treatment mold fermentations fouling of processing equipment Biofilm (biopolymer + polysaccharides) 37 Analysis of Biofilm Mass Transfer Figures 9.1, 9.12 Substrate/product diffusion in biofilms O 2 diffusion in biofilms Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall,
20 Analysis of Biofilm Mass Transfer (cont.) Differential Substrate Balance: Rate of diffusion out through the area x z ' H G6 G\ \ \ x [ ] G6 ' H [ ] z G\ y+ y y y \ Rate of substrate µ consumption in the < 6 PD[ 6 6 [ \ ] volume V = x y z (due to cell growth, or q S 6 [ \ ] product formation) < 36 6 Material volume in biofilm, V = x y z Rate of diffusion in through the area x z 39 Analysis of Biofilm Mass Transfer (cont.) Differential Substrate Balance at Steady-State: Rate of diffusion in through the area x z Rate of diffusion out through the area x z Rate of substrate consumption in the volume V = x y z - - = G6 G6 ' H [ ] ' H G\ \ G\ \ \ [ ] µ 6 PD[ [ \ ] < 6 6 'LYLGHWKURXJKE\ [ \ ]DQGVZLWFKRUGHURIILUVWWHUPV G6 G6 ' H ' H G\ \ \ G\ \ \ µ 6 PD[ <
21 Analysis of Biofilm Mass Transfer (cont.) Differential Substrate Balance at Steady-State: G 6 ' H G\ < µ 6 PD[ 6 6 HTQ %RXQGDU\&RQGLWLRQV 6 6 RL DW\ DWWKHELRILOP OLTXLGLQWHUIDFH G6 DW\ /DWWKHELRILOP VXSSRUWLQWHUIDFH G\ 41 Analysis of Biofilm Mass Transfer (cont.) Dimensionless Substrate Balance at Steady-State: G 6 G\ φ 6 β6 HTQ ZKHUH6 6 6 R,\ \ / β 6 R µ DQGφ / PD[ Å7KLHOHRGXOXVÅ < 6 ' H %RXQGDU\&RQGLWLRQV 6 DW\ DWWKHELRILOP OLTXLGLQWHUIDFH G6 DW\ DWWKHELRILOP VXSSRUWLQWHUIDFH G\ A numerical solution is required Analytical solution is possible for order and 1st order kinetics 42 21
22 Analysis of Biofilm Mass Transfer (cont.) Zero Order Substrate Consumption Kinetics: G 6 φ 6 IRUβ!! DQGφ G\ β 6 G 6 G\ φ ]HUR RUGHUVXEVWUDWHFRQVXPSWLRQNLQHWLFV β G G6 G\ G\ φ G6 G β G\ φ G\ β G6 G\ φ \ & β 43 Analysis of Biofilm Mass Transfer (cont.) Zero Order Substrate Consumption Kinetics: %RXQGDU\FRQGLWLRQÆ DW\ G6 G\ φ & & β φ β G 6 G\ φ φ \ β β LQWHJUDWHDJDLQ G6 φ φ \ G\ β β 6 φ 2β φ \ \ & β 44 22
23 Analysis of Biofilm Mass Transfer (cont.) Zero Order Substrate Consumption Kinetics: %RXQGDU\FRQGLWLRQÆDW\ 6 φ β φ & & β 6 φ φ \ φ \ RU 6 2β β β \ 2 \ φ IRU β 45 Biofilm Effectiveness The effectiveness factor is calculated by dividing the rate of substrate diffusion into the biofilm by the maximum substrate consumption rate. Solve for the Effectiveness Factor, η G6 1 6 $ 6 $ 6 ' H η G\ \ Rate of substrate diffusion into biofilm through an area A S at the surface at y = µ PD[ 6 R ($ 6 / 6 R ) < 6 Volumetric rate of substrate consumption within the biofilm in a volume (A S L) 46 23
24 Effectiveness Factor Biofilm is most effective for β >>1 η increases as φ decreases for any value of β Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, Spherical Particle of Immobilized Cells Figure 9.14 Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 22 V P is particle volume A P is particle area 48 24
25 Analysis of Mass Transfer in Spherical Particle Dimensionless Substrate Balance at Steady-State: G 6 GU G6 U GU φ 6 β6 HTQ ZKHUH6 6 6 R,U U 5 β 6 R DQGφ 5 µ PD[ < 6 ' H Å7KLHOHRGXOXVÅ %RXQGDU\&RQGLWLRQV 6 DWU DWWKHSDUWLFOH OLTXLGLQWHUIDFH G6 DWU DWWKHSDUWLFOHFHQWHU GU 49 Particle Effectiveness If all of the particle cells see substrate at a concentration S o or high enough to grow maximally, then the particle is said to have an effectiveness of 1. Rate of S consumption by a single particle G6 1 6 $ 3 $ 3 ' H η GU U 5 Rate of substrate diffusion into particle through an area A P at the surface at r = R µ PD[ 6 R R ) < 6 Volumetric rate of substrate consumption within the particle in a volume (V P ) 5 25
26 Particle Effectiveness (cont.) Equation 9.58 can be solved analytically for limiting cases: Case 1, for S o <<K S (very dilute substrate) η φ WDQKφ φ φ 9 µ 3 max Å7KLHOHRGXOXVÅ $ 3 < 'H 6 51 Particle Effectiveness (cont.) Equation 9.58 can be solved analytically for limiting cases: Case 2, for S o >>K S (very concentrated substrate) G 6 ' G6 H GU U GU %RXQGDU\&RQGLWLRQV µ PD[ 6 < 6 µ + 6) PD[ < R DWU 5DWWKHSDUWLFOH OLTXLGLQWHUIDFH G6 DWU DWWKHSDUWLFOHFHQWHU GU 52 26
27 Particle Effectiveness (cont.) Equation 9.58 can be solved analytically for limiting cases: Case 2, for S o >>K S Use a variable transformation, S =S/r G 6 U < 6 ' H GU µ PD[ 6ROXWLRQIRU6LV µ 6 6 R PD[ (5 U ) < 6 ' H $WDFULWLFDOUDGLXVU FU 6 µ 6 R PD[ (5 U FU ) < 6 ' H U FU 2 6' 6 < H R 6 5 µ PD[ 5 53 Particle Effectiveness (cont.) Equation 9.58 can be solved analytically for limiting cases: Case 2, for S o >>K S η µ PD[ < π5 U FU ) 4 3 π5 µ PD[ < 6 U FU 5 3 RU 3 6 η ' 6 < 2 H R 6 µ PD[
28 Bioreactors Using Immobilized Cells Figure 9.15 The single particle analysis for η can be used in the analysis of bioreactors having immobilized cells: Consider a plug flow reactor filled with immobilized cell particles Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, Bioreactors Using Immobilized Cells (cont.) A differential balance on a thin slice of particles within the reactor: H S o differential volume element F, S o -ds o z+dz z F, S o z S oi Rate of substrate flow into element )6 R ] )6 R ]G] 1 6 D$G] - Rate of substrate flow out of element = Rate of mass transfer into particles within element 56 28
29 Bioreactors Using Immobilized Cells (cont.) Using the definition of η: 1 6 $ 3 η µ PD[ 6 R. 6 6 R ) 9 3 < 6 ) G6 R G] η µ 6 PD[ 9 R 3 < 6 6 R ) $ 3 D$ ZKHUHD VXUIDFHDUHDRISDUWLFOHSHUXQLWYROXPHRIEHGFP FP EHG $ FURVV VHFWLRQDODUHDRIWKHEHGFP 57 Bioreactors Using Immobilized Cells (cont.) At z =, S o = S oi : integrating assuming η is constant OQ 6 µ RL 6 RL 6 R η PD[ 9 3 D$ 6 R < 6 )$ 3 + IRUORZVXEVWUDWHFRQFHQWUDWLRQ6 RL OQ 6 R 6 RL η µ PD[ 9 3 D$ < 6 )$ 3 + QRWH[ [ 9 3 $ 3 DDYHUDJHFHOOPDVVFRQFLQWKHEHG 58 29
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