Bioreactor System ERT 314. Sidang /2012
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1 Bioreactor System ERT 314 Sidang /2012
2 Chapter 5: Bioreactor Design
3 Mixing Mixing is a physical operation which reduces nonuniformities in fluid by eliminating gradients of concentration, temperature and other properties It can be accomplished by interchanging material between different location to produce a mingling of components and involves: blending soluble components of the medium i.e sugar dispersing gases i.e air through the liquid in the form of small bubbles maintaining suspension of solid particles i.e cells dispersing immiscible liquids to form an emulsion or suspension fine drops where necessary, promoting heat transfer to or from the liquid
4 Mixing (cont d) Mixing is one of the most important operation in bioprocessing as to create optimal and homogenous environment for fermentation Problem such as increase of nutrient depletion zone might be occur if mixing does not maintain uniform suspension of biomass Other function related to mixing is heat transfer which heat is transferred to or from the broth rapidly The effectiveness of mixing depends in turn on rheological properties of culture fluid such as Newtonian or non-newtonian (viscosity)
5 Mixing Equipment Mixing usually carried out in a stirred tank, which is recommended to have the base is rounded at the edges rather than angled - will eliminate sharp corners and pockets into which fluid current may not penetrate and formation of stagnant region Mixing is achieved by installation of impeller, for Newtonian fluids ratio of tank diameter to impeller diameter is 3:1 The impeller usually installed overhead of stirrer shaft and at the bottom of the vessel Disadvantage of bottom installation is development of leaks if the seal between the shaft and tank is not perfect For efficient mixing with single impeller, the depth of liquid should not be more than times the tank diameter
6 Typical configuration of a stirred tank
7 Mixing Equipment (cont d) Baffles which are the vertical strips of metal mounted against the tank wall, functioned to reduce vortexing and swirling of the liquid Four equally-spaced baffles are sufficient to prevent vortex formation, and its optimum width depends on impeller design and fluid viscosity but in order of 1/10-1/12 of tank impeller
8 For low-viscosity fluids, baffles attached perpendicular to the wall (a), whereas (b) and (c), baffles mounted away from the wall with clearance about 1/50 tank diameter to prevent sedimentation and development of stagnant zone at the inner edge of baffle during mixing viscous liquid.
9 Mixing Equipment (cont d) Many impeller design are available for mixing application. Some impellers have flat blade, propeller and helical screw, which slope of individual blades varies continuously Choice of impeller depends on several factors, including viscosity of the fluid and sensitivity of the culture system to mechanical shear For low-to-medium-viscosity liquids propellers and flat blade turbines The most popular impeller used in industry is 6-flatblade disc-mounted turbine or known as Rushton turbine
10 Impeller design
11 Viscosity ranges for different impeller
12 Flow Patterns in Agitated Tanks The flow pattern in an agitated tank depends on the impeller design, the properties of the fluid, and the size and geometric proportions of the vessel, baffles and agitator. In circular flow, liquid moves in a streamline fashion and there is little mixing between fluid at different heights in the tank. Circular flow should be avoided because it leads to vortex development as it produces high mechanical stresses in the stirrer shaft, bearings and seal at high impeller speed.
13 Circular flow in an unbaffled stirred tank viewed from above (a) Vortex formation during circular flow (b)
14 Radial-flow impellers These impellers have blades which are parallel to the vertical axis of the stirrer shaft and tank such as the sixflat-blade disc turbine (Rushton turbine) Liquid is driven radially from the impeller against the walls of the tank where it divides into two streams, one flowing up to the top of the tank and the other flowing down to the bottom These streams eventually reach the central axis of the tank and are draw back to the impeller Radial flow impellers also set up circular flow which must be reduced by baffles
15 Flow pattern produced by a radial flow impeller in a baffled tank, (a) side view; (b) bottom view
16 Axial-flow impellers These impellers have blades which make an angle of less than 90 to the plane of rotation and promote axial topto-bottom motion such as propellers pinched blade turbine Fluid leaving the impeller is driven downwards until it is deflected from the floor of the vessel Then, it spreads out over the floor and flows up along the wall before being drawn back to the impeller The impellers are useful when strong vertical currents are required i.e if the fluid contain solids, strong axial flow of liquid leaving will discourage settling at the bottom of the tank
17 Pinched blade turbine The angle is <90
18 Flow pattern produced by a axial flow impeller in a baffled tank, (a) side view; (b) bottom view
19 Mechanism of Mixing For mixing to be effective, fluid circulated by the impeller must sweep the entire vessel in reasonable time The velocity of fluid leaving the impeller must be sufficient to carry material into the most remote parts of the tank which development of turbulence is also necessary to ensure better mixing Three physical processes are among important factors in mixing: distribution dispersion diffusion
20 Mechanism of Mixing (cont d) Distribution is called macromixing, diffusion is called micromixing whereas dispersion can be classified as either micro or macromixing depending on the scale of fluid motion In large vessels, the size of circulation paths is large and the time taken to tranverse them is long, therefore distribution is often the slowest step in mixing process If the rotational speed of the impeller is high, distribution step become turbulence where fluid no longer travel in streamlines but move erratically in form of crosscurrent
21 Laminar versus Turbulent flow Eddies
22 The pattern of bulk fluid flow in vessel stirred by a radialflow impeller as shown in the figure. High turbulence region is developed near the impeller where current converge and exchange materials The flow become slower and large streamline is developed as current move away from the impeller which mixing in these region is less intense than near the impeller Flow pattern developed by centrally-positioned radial-flow impeller
23 Mechanism of Mixing (cont d) The process of breaking up bulk flow into smaller eddies is called dispersion, where it facilitates rapid transfer of material The degree of homogeneity of the broth by dispersion is limited by the size of smallest eddies formed in particular fluid The size of eddies can be measured by Kolmogorov scale of turbulence, λ : where: 3 v λ = characteristic dimension of smallest eddies V = kinematic viscosity ε= rate of turbulence energy per unit mass of fluid 1 4
24 Mechanism of Mixing (cont d) According to the equation (previous slide), the greater the power input (ε) to the fluid, the smaller are the eddies Smaller eddies also are produced by low viscosity of fluid (v), i.e water have λ in the range of μm, which the smallest scale of mixing can be achieved by dispersion
25 Mechanism of Mixing (cont d) To achieve mixing on a scale smaller than Kolmogorov scale, diffusion can be applied Molecular diffusion is generally regarded as slow process, but if in small distances it can be accomplished quite rapidly i.e for λ of μm, homogeneity can achieved in 1 second for low viscosity fluids. Mixing time depends variables such as size of tank & impeller, fluid properties such as viscosity, and stirred speed. For rushton turbines, the mixing time, tm, can be determined by the following relationship: N i t m =1.54 V D i 3 Where, Ni = rotational speed of the stirrer, tm = mixing time, V= tank volume, Di = diameter of the stirrer/impeller
26 Power Requirements for Mixing For a given stirrer speed, the power required depends on the resistance offered by the fluid to rotation of the impeller Average power consumption per unit volume for industrial bioreactors Small vessels 10 kwm-3 Large vessels 1 2kWm-3 Friction in the stirrer motor gearbox and seals reduce the energy transmitted to the fluid, therefore the electrical power consumed by the stirrer motor is higher than the mixing power by an amount depending on the efficiency of the drive
27 Ungassed Newtonian Fluids Mixing power for non aerated fluid depends on: the stirrer speed the impeller diameter and geometry the properties of the fluid (i.e density and viscosity) The relationship of these variables is expressed in terms of dimensionless numbers such as Reynolds number, Re and power number, Np
28 Ungassed Newtonian Fluids (cont d) Power number, Np: N P Once value of Np is known, the power required is calculated by: P N P N P 3 i N D 3 i 5 i D where: P = power; ρ = fluid density; N = stirrer speed, D = impeller diameter 5 i
29
30
31 Ungassed Newtonian Fluids (cont d) Laminar regime: Corresponds to Re<10 for many impellers such as anchor and helical ribbon, which laminar flow persist until Re = 100 or greater. N P 1 Re i or P k 1 N Power required for laminar flow is independent of the density but directly proportional to viscosity 2 i D 3 i
32 Ungassed Newtonian Fluids (cont d) Tubulence regime: Power number is independent of Re number in turbulent flow P N ' P N N p for turbines is significantly higher than for most other impellers, indicating turbine transmit more power to the fluid than other design Power required for turbulent flow is independent of the viscosity but proportional to fluid density 3 i D 5 i
33 Constants Impeller type K1 (Re = 1) N p (Re = 10^5) Rushton turbine Paddle 35 2 Marine propeller Anchor Helical Ribbon
34 Ungassed Newtonian Fluids (cont d) Transition regime: Between laminar and turbulent flow regime. Both density and viscosity affect the power requirement in this regime Usually a gradual transition from laminar to fully developed turbulent flow in stirred tanks
35 Gassed fluids Liquids into which gas is sparged have reduced power requirement Gas bubbles decrease the density of the fluid but the influence of density on power requirement does not adequately explain all power characteristic of gas-liquid phase The presence of bubble also affect the hydrodynamic behaviour of fluid around the impeller Ratio gassed to ungassed power: where: P P g F N V i g 0.25 N 2 i D gw V i 4 i 2 / 3 Pg = power with sparging; P0 = power without sparging; 0.2 Fg = volumetric gas flow rate; N = stirrer speed, V = liquid volume; D = impeller diameter; g =gravity; W =impeller width
36 Types of Sparger Large variety of sparger designs: Simple open pipes Perforated tubes Porous diffusers Complex two-phase injector
37 Types of Sparger (cont d) Porous diffuser Perforated tubes Open pipes
38 Types of Sparger (cont d)
39 Flow pattern in stirred aerated bioreactors When air is sparged different gas flow patterns develop depending on the relative rates of gas input and stirring If the agitator speed Ni is low and gas feed rate Fg is high, gas envelopes the impeller without dispersion and the flow pattern dominated by air flow up the stirrer shaft (a). Impeller flooding is occur which the gas handling capacity of the stirrer is smaller than the amount introduced. The flooding should be avoided because an impeller surrounded by gas no longer contacts the liquid properly resulting poor mixing and gas dispersion
40 Flow pattern in stirred aerated bioreactors as a function of impeller speed, N and gas flow rate, Fg
41 Flow pattern in stirred aerated bioreactors (cont d) As the impeller speed increases, gas is captured behind the agitator blades and is dispersed into liquid NiB is the minimum stirrer speed required to just completely disperse the gas, including below the impeller The minimum agitator tip speed for dispersion of air bubbles is about ms-1 Further increase in stirrer speed, small recirculation patterns start to emerge which NiR is the speed at which gross recirculation of gas back to the agitator starts to occur (e).
42 Flow pattern in stirred aerated bioreactors (cont d) Under typical fermenter operating conditions, increasing the stirrer speed improves the value of kla In contrast, except in very low sparging rate, increasing the gas flow is generally considered to exert only a minor influence on kla Increasing number of impeller on the stirrer shaft does not necessarily improved kla even though the power consumption is increased The quantity of gas passing through the upper impellers is small compared with the lower impeller, so that any additional gas dispersion is not significant
43 Dependence of kla on stirrer speed Ni in agitated tank with superficial gas velocity Effect on kla of number of impeller used to mix a viscous mycelial suspension
44 Mass Transfer Mass transfer occurs in mixtures containing local concentration variation Mass is transferred from one location to another under the influence of a concentration difference or concentration gradient in the system For example, in bioprocessing, the supply of oxygen in bioreactors for aerobic culture Concentration of oxygen at the surface of air bubbles is high compared with the rest of the fluid, therefore this concentration difference promotes oxygen transfer from bubbles into the medium
45 Molecular Diffusion Molecular diffusion is the movement of component molecules in a mixture under the influence of a concentration difference in the system It occurs in the direction required to destroy the concentration difference If the gradient is maintained by constantly supplying material to the region of high concentration and removing it from region of low concentration, diffusion will be continue
46 Diffusion Theory
47 Diffusion Theory (cont d) In the previous figure, concentration CA varies from CA1 to CA2 is a function of distance y Molecules A will diffuse away from the region of high concentration until eventually the whole system acquires uniform composition
48 Oxygen Transfer in Cell Cultures Cell in aerobic culture take up oxygen from the liquid. Therefore, the mass transfer of oxygen from gas to liquid is importance, especially at high densities when cell growth is likely to be limited by availablity of oxygen in the medium. The difference (C*AL-CAL) between the maximum possible and actual oxygen concentration represent concentration-difference driving force for mass transfer.
49 Oxygen Transfer in Cell Cultures (cont d) N A K L a C * AL C AL where: NA = rate of oxygen per unit volume (gmol m-3 s-1) kl = liquid phase mass transfer coefficient (ms-1) a= gas-liquid interfacial area per unit volume fluid (m2 m-3) CAL = the oxygen concentration in broth (gmol m-3) C*AL = the oxygen concentration in broth in equilibrium with gas phase (gmol m-3)
50 Oxygen Transfer in Cell Cultures (cont d) The solubility of oxygen in aqueous solution at ambient temperature and pressure is about 10 ppm This amount of oxygen is quickly consumed in aerobic cultures and must be constantly changed by sparging Low solubility of oxygen will show the difference (C*AL- CAL) is very small
51 Factors Affecting Cellular Oxygen The rate at which oxygen is consumed by cells in bioreactors determines the rate which it must be transferred from gas to liquid Many factors influence oxygen demand, the most important of these are: Cell species Culture growth phase Nature of the carbon source in the medium The concentration of cell increases during course of batch culture and the total rate of oxygen uptake is proportional to the no. of cell present
52 Factors Affecting Cellular Oxygen (cont d) The rate of oxygen consumption per cell also known as oxygen uptake rate (OUR): where: Q O Qo = oxygen uptake rate per volume broth (gl-1 s-1) qo = specific oxygen transfer rate (gg-1 s-1) q O x
53 Factors Affecting Cellular Oxygen (cont d) The demand of an organism for oxygen depends primarily on the biochemical nature of the cell and its nutritional environment. To eliminate oxygen limitation and allow cell metabolism to function at its fastest, the dissolved oxygen concentration at every point in the bioreactor must be above Ccrit. The value of Ccrit depends on the organism, but under average operation it usually falls between 5%-10% of air saturation.
54 Oxygen Transfer from Gas Bubble to Cell
55 Oxygen Transfer from Gas Bubble to Cell (cont d) In aerobic fermentation, oxygen molecules must overcome a series of transport resistance before being utilized by the cell Eight mass transfer steps involved in transport of oxygen from the interior of gas bubbles to the site of intracellular reaction 1. transfer from the interior of the bubble to the gas-liquid interface 2. movement across the gas-liquid interface 3. diffusion through the relatively stagnant liquid film surrounding the bubbles 4. transport through the bulk liquid 5. diffusion through the relatively stagnant liquid film surrounding the cells 6. movement across the liquid-cell interface 7. if the cells in a floc, clump or solid particle, diffusion through the solid to the individual cell 8. transport through the cytoplasm to the site of reaction
56 Oxygen Transfer from Gas Bubble to Cell (cont d) For most bioreactors, the analysis of mass transfer is as below 1. Transfer through the bulk gas phase in the bubble is relative fast 2. The gas-liquid interface itself contributes negligible resistance 3. The liquid film around the bubbles is the major resistance to oxygen transfer 4. In a well-mixed bioreactor, concentration gradients in the bulk liquid are minimized and mass transfer resistance in this region is small 5. Because the single cells are much smaller than gas bubbles, the liquid film surrounding each cell is much thinner than around the bubbles and its effect on mass transfer can generally neglected 6. Resistance at the cell liquid interface is generally neglected 7. When cells are in clumps, intraparticle resistance is likely to be significant as oxygen has to diffuse through the solid pellet to reach the interior cells. The magnitude of resistance depends on the size of the clumps 8. Intracellular oxygen transfer resistance is negligible because of small distance involved
57 Oxygen Transfer from Gas Bubble to Cell (cont d) When cell are dispersed in the liquid and the bulk fermentation broth is well mixed, the major resistance to oxygen transfer is the liquid film surrounding the gas bubbles Therefore, transport through this film becomes rate limiting step and controls the overall mass transfer rate At steady state, no accumulation of oxygen at any location in the bioreactor, therefore rate of oxygen transfer from bubbles must be equal to rate of oxygen consumption by cells: K L * C q x a C AL AL O
58 Oxygen Transfer from Gas Bubble to Cell (cont d) If kla is small, the ability of the bioreactor to deliver oxygen is limited The maximum cell concentration can be supported by mass transfer function of the bioreactor is: x max k L a C If xmax is lower than the cell concentration required in fermentation process, kla must be improved Another important parameter is the minimum kla required to maintain CAL>Ccrit in bioreactor k L a crit q O * C AL * AL q O x C crit
59 Oxygen Transfer in Bioreactor Rate of oxygen transfer in fermentation broth is influences by several physical and chemical factors that change the value of kl (liquid-phase-mass-transfer-coefficient) of value of a (transfer area or interfacial area) or driving force (C*AL-CAL). [Oxygen Transfer rate= kl x a x (C*AL-CAL) kl in fermentation liquid is usually in the range of 3-4 x 10-4 ms-1 for bubbles greater than 2-3 mm diameter, but it can be reduced to 1x10-4 ms-1 if smaller bubbles produced If substantial improvement in mass transfer rate is required, it is more productive to increase the interfacial area, a In production scale bioreactor, value of kla is typically in the range of 0.02s-1 to 0.25s-1
60 Bubbles Bubble behavior strongly affects the value of kla, which some may affect mainly on kl, whereas some changes interfacial area, a Bubble in lab-scale bioreactor frequently subjected to severe distortion as they interact with turbulent liquid current, whereas bubble in industrial stirred tanks spend large proportion of their time floating free and impeded through the liquid after initial dispersion at impeller The most important property of air bubbles in bioreactor is their size
61 Bubbles (cont d) Advantages of small bubbles such as: Can produce high level of gas dispersion by providing more interfacial area, a Since it has slow rise velocities, they can stay in the liquid longer, allowing more time for oxygen to dissolve Therefore, small bubble create high gas hold-up defined as fraction of fluid volume in the reactor occupied by gas: where ξ=gas hold up VG = volume of gas bubbles VL= volume of liquid V L V G V G
62 Bubbles (cont d) Disadvantages of small bubbles Bubbles <<1mm can become nuisance in bioreactor where oxygen concentration equilibrates with that in the medium within seconds, so that the gas hold-up no longer reflects the capacity of the system for mass transfer Small bubble in non-newtonian broth (viscous) will remain lodged for long periods due to its velocity is reduced through time Bubble size also affect the value of kl Bubble with diameter less than 2-3 mm, the surface tension effects dominate the behaviour of the bubble surface For bubble with diameter >3 mm, it can develop internal and relatively mobile surface, depending on liquid properties
63 Stirred Tank Bioreactor A: Agitator motor B: Speed reduction unit C: Air inlet D: Air outlet E: Air bypass valve F:Shaft seal G: Sight glass with light H: Sight glass clean-off line I: Manhole with sight glass J: Agitator shaft K: Foam breaker L: Cooling water outlet M: Baffles N: Water jacket (coil) O: Cooling water inlet P: Mixer/ Stirrer Q: Sparger S: Outlet T: Sample valve
64 Stirred Tank Bioreactor Low capital cost and low operating cost. For lab experiments, can be up to 20 L. From glass or stainless steel Height : Diameter, between 2:1 and 6:1 depending on the amount of heat to be removed Stirrer may be top or bottom driven Fitted with baffles to prevent a large central vortex and improve mixing 4 baffles for vessels less than 3 m diameter 6 8 baffles for larger vessels Width of baffle is between T/10 and T/12, T is the tank diameter
65 Estimation for the Dimension of the Bioreactor Bioreactor working volume, V=10m3 with 20% over design as a safety factor The total volume is: V V working m m Volume of bioreactor of cylindrical system is: V 2 D t H L 4 3
66 Estimation for the Dimension of the Bioreactor (cont d) The ratio liquid level to tank diameter HL: Dt = 2:1, therefore: V 2 Dt D 3 t 2D t 12.5m Solving for tank diameter, Dt and liquid height, HL D H t L 2.0m 2 D t 4.0m 3
67 Estimation for the Dimension of the Bioreactor (cont d) Diameter impeller is set to one third of tank diameter: Dt 2.0 Di 0. 67m 3 3 If aeration = 1 vvm, the volumetric flow of air for the bioreactor is defined: 3 3 m 1min 1vvm12m 12 min 60s m 0.21 s F g 3 The gas superficial velocity, Us is defined as ratio of gas flow rate to vessel cross sectional area: 3 m F 12.5 g 60min U S min A 4 m 2 2 1h h 2.0 m
68 Determination of Reynolds Number (cont d) The highest density of the solution has been taken for the determination of the Re number. The broth has viscosity of 0.1 Pa s, kg/m3, and the rotational speed of the impeller is set at 150 rpm = 2.5 rps The Re number is calculated based on the provided data: Re ND i kg 3 m 0.1pa s 2.5rps 0.67m The high Re number represent the turbulent flow regime 2
69 Determination of Power Input If the Re number > 16000, power number = 5 P no N Pg 3 5 i Di Therefore, the ungassed power is calculated by: kg m P m s kg m 1hp P s kg m s rps 0.67m 2.09hp kg m s
70 Determination of Power Input (cont d) The power input power required for one set of impeller. Correction factors for non-geometrical similarity are required to include the effect of known factors in precise calculations i L i L i t i t c D H D H D D D D f
71 Determination of Power Input (cont d) If the design of bioreactor is set at Dt/Di = 3 and HL/Di = 3, upon substitition for the above design, the correction factor is: f c
72 Determination of Power Input (cont d) Therefore, the actual power with correction factor is: * P P hp 2. 96hp Dimensionless aeration rate is defined as follows: N a N F i g D m 0.21 s i rps m From the graph of ratio of gassed power to ungassed power, Pg/P = 0.9, therefore gassed power to motor to rotate is: P hp hp g
73 Determination of Oxygen Transfer Rate The volumetric mass transfer rate coefficient is defined as below: 0.6 P 3 g K La VS V L Where the gas superficial for the above case is calculated as Vs = 0.21 m3/s/4πm2 and the working vessel is defined: V L m Substuting Vs and VL into the above equation determine the volumetric mass transfer coefficient: a K L s 1
74 Determination of Oxygen Transfer Rate (cont d) The simple equation of oxygen transfer rate (OTR): OTR K a C C L The assumption was made that the equilibrium value for oxygen was C*AL = 6 ppm and all oxygen available in liquid phase was used by the organism (CAL = 0), which means the growth was made-transfer limited, and the limited oxygen AL transfer can retard the biological process. OTR is calculated as: 5 1 OTR s kgo m 3 AL s 2
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