FE 462 BIOCHEMICAL ENGINEERING Cell Kinetics and Fermenter
FE 462 BIOCHEMICAL ENGINEERING Cell Kinetics and Fermenter Design Dr. Ali Coşkun DALGIÇ
Bioreactors Page 2 Dr. Ali Coşkun DALGIÇ
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Stirred Tank Fermenters A bioreactor is a device within which biochemical transformations are caused by the action of enzymes or living cells. The bioreactor is frequently called a fermenter whether the transformation is carried out by living cells or in vivo cellular components (enzymes). For a large scale operation the stirred tank fermenter (STF) is the most widely used design in industrial fermentation. It can be employed for both aerobic or anaerobic fermentation of a wide range of cells including microbial, animal, and plant cells. Page 16 Dr. Ali Coşkun DALGIÇ
Stirred Tank Fermenters Page 17 Dr. Ali Coşkun DALGIÇ
Kinetics of Substrate Utilization, Product Formation, and Biomass Production in Cell Cultures It is difficult to obtain useful kinetic information on microbial populations from reactors that have spatially no uniform conditions. Hence it is desirable to study kinetics in reactors that are well mixed. Ideal Batch Reactor Many biochemical processes involve batch growth of cell populations. After seeding a liquid medium with an inoculum of living cells, nothing (except possibly some gas) is added to the culture or removed from it as growth proceeds. Typically in such reactor, the concentrations of nutrients cells, and products vary with time as growth proceeds. Page 18 Glucose Product Cell biomass Time Dr. Ali Coşkun DALGIÇ
Kinetics of Substrate Utilization, Product Formation, and Biomass Production in Cell Cultures A material balance on moles of component i; Page 19 Dr. Ali Coşkun DALGIÇ
The Ideal Continuous Flow Stirred Tank Reactor (CSTR) The diagram of this process is shown in fig. 2, which is a schematic diagram of completely mixed stirred tank reactor. Such configurations for cultivation of cells are frequently called chemostats. Page 20 Dr. Ali Coşkun DALGIÇ
Continuous Fermentor Flow rate 1 = Flow rate 2 Pump 1 Pump 2 Feedstock vessel (sterile) Collection vessel Page 21 21
Pro and cons of chemostat (+) Excellent experimental tool because µ is defined (-) Low biomass and product concentration Loss of biomass in outflow Relatively prone to be contaminated compare to batch or fed batch reactors Microbial selection for non-producing mutants Page 22 22 Dr. Ali Coşkun DALGIÇ
Characteristics of Continuous (Chemostat) Fermentors Input rate = output rate (volume = const. ) Flow rate is selected to give steady state growth (growth rate = dilution rate) – Dilution rate > Growth rate culture washes out – Dilution rate < Growth rate culture overgrows – Dilution rate = Growth rate steady state culture Stable chemostat cultures can operate continuously for weeks or months. Page 23 23 Dr. Ali Coşkun DALGIÇ
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The Ideal Continuous Flow Stirred Tank Reactor (CSTR) For biomass Page 29 Dr. Ali Coşkun DALGIÇ
The Ideal Continuous Flow Stirred Tank Reactor (CSTR) For Substrate Page 30 Dr. Ali Coşkun DALGIÇ
The Ideal Continuous Flow Stirred Tank Reactor (CSTR) For Product Page 31 Dr. Ali Coşkun DALGIÇ
The Ideal Continuous Flow Stirred Tank Reactor (CSTR) Page 32 Dr. Ali Coşkun DALGIÇ
The Ideal Continuous Flow Stirred Tank Reactor (CSTR) Page 33 Dr. Ali Coşkun DALGIÇ
The Ideal Continuous Flow Stirred Tank Reactor (CSTR) Example- Page 34 Dr. Ali Coşkun DALGIÇ
The Ideal Continuous Flow Stirred Tank Reactor (CSTR) Solution Intercept Slope Page 35 Dr. Ali Coşkun DALGIÇ
The Ideal Continuous Flow Stirred Tank Reactor (CSTR) Solution Page 36 Dr. Ali Coşkun DALGIÇ
Productivity of CSTF Page 37 Dr. Ali Coşkun DALGIÇ
Productivity of CSTF Page 38 Dr. Ali Coşkun DALGIÇ
MULTIPLE FERMENTERS CONNECTED IN SERIES A question arises frequently whether it may be more efficient to use multiple fermenters connected in series instead of one large fermenter. Choosing the optimum fermenter system for maximum productivity depends on the shape of the l/rx versus Cx curve and the process requirement, such as the final conversion. Page 39 Dr. Ali Coşkun DALGIÇ
MULTIPLE FERMENTERS CONNECTED IN SERIES Page 40 Dr. Ali Coşkun DALGIÇ
Cell Recycling For the continuous operation of a PFF or CSTF, cells are discharged with the outlet stream which limits the productivity of fermenters. The productivity can be improved by recycling the cells from the outlet stream to the fermenter. Page 41 Dr. Ali Coşkun DALGIÇ
Considerations in selecting industrial fermenters are: 1. Nature of substrate solid, liquid, suspended slurry, water-immiscible oils 2. Flow behaviour (rheology), broth viscosity and type of fluid (e. g. Newtonian, viscoelastic, pseudoplastic, Bingham plastic). 3. Nature and amount of suspended solids in broth. 4. Whether fermentation is aerobic or anaerobic, and O 2 demand. 5. Mixing requirements. 6. Heat-transfer needs. 7. Shear tolerance of microorganism, substrate and product. 8. Sterility requirements. 9. Process kinetics, batch or continuous operation, single-stage or multistage fermentation. 10. Desired process flexibility. 11. Capital and operational costs. 12. Local technological capability and potential for technology transfer. Page 42
Types of submerged-culture fermenter. (A) Stirred-tank fermenter. (B) Bubble column. 43 (C) Internal-loop airlift fermenter. (D) External-loop airlift fermenter. Page 43 (E) Fluidized-bed fermenter. (F) Trickle-bed fermenter
Bubble Column Although simple, it is not widely used because of its poor performance relative to other systems. It is not suitable for very viscous broths or those containing large amounts of solids. Airlift Fermenters In the internal loop design, the aerated riser and the unaerated downcomer are contained in the same shell. In the external loop configuration, the riser and the downcomer are separate tubes that are linked near the top and the bottom not suitable for viscous broths. Their ability to suspend solids and transfer O 2 and heat is good. The hydrodynamic shear is low. Page 44
Air lift reactors: In such reactors, circulation is caused by the motion of injected gas through a central tube with fluid recirculating through the annulus between the tube and the tower or vice versa. Page 45
Fluidized-bed Fermenter Fresh or recirculated liquid is continuously pumped into the bottom of the vessel, at a velocity that is sufficient to fluidize the solids or maintain them in suspension. Trickle-bed Fermenter a cylindrical vessel packed with support material (e. g. woodchips, rocks, plastic structures). A liquid nutrient broth is sprayed onto the top of the support material, and trickles down the bed. Air may flow up the bed, countercurrent to the liquid flow. These fermenters are used in vinegar production, as well as in other processes. Page 46
Solid-state Fermentations Substrate Characteristics Water Activity Typically, solid state fermentations are carried out with little or no free water. Excessive moisture tends to aggregate the substrate particles, and hence aeration is made difficult. For example steamed rice, a common substrate, becomes sticky when the moisture level exceeds 30– 35% w/w. The low moisture environment of many solid state fermentations favours yeasts and fungi. ( mostly to produce extracellular enzyme by mold on cereals) During fermentation, the water activity is controlled by aeration with humidified air and, sometimes, by intermittent spraying with water. Page 47 Dr. Ali Coşkun DALGIÇ
Advantages of SSF( solid state fermentation) Small fermentor volume, lower capital and operating cost Lower chance of contamination due to low water Easy product seperation Energy efficient Disadvantages of SSF( solid state fermentation) Heterogenous structure due to poor mixing so difficulty in controlling p. H, DO, temperature) Page 48 Dr. Ali Coşkun DALGIÇ
Bubble Column Although simple, it is not widely used because of its poor performance relative to other systems. It is not suitable for very viscous broths or those containing large amounts of solids. Airlift Fermenters In the internal-loop design, the aerated riser and the unaerated downcomer are contained in the same shell. In the external-loop configuration, the riser and the downcomer are separate tubes that are linked near the top and the bottom not suitable for viscous broths. Their ability to suspend solids and transfer O 2 and heat is good. The hydrodynamic shear is low. Page 49
Oxygen Demand Submerged cultures are most commonly aerated by bubbling with sterile air. in small fermenters, the maximum aeration rate ≤ 1 VVM ( 1 VVM volume of air per unit volume of broth per minute). In large bubble columns and stirred vessels, the max superficial aeration velocity < 0. 1 m s− 1. ( Q/A) Page 50
The O 2 requirements of a fermentation depend on the microbial species, the concentration of cells, and the type of substrate. O 2 supply must at least equal O 2 demand, or the fermentation will be O 2 limited. O 2 demand is difficult to meet in viscous fermentation broths and in broths containing a large concentration of O 2 consuming cells. Page 51
The aeration of fermentation broths generates foam. Typically, 20– 30% of the fermenter volume must be left empty to accommodate the foam and allow for gas disengagement. In addition, mechanical ‘foam breakers’ and chemical antifoaming agents are commonly used. Typical antifoams are silicone oils, vegetable oils, polypropylene glycol or polyethylene glycol. Page 52
The excessive use of antifoams may interfere with some downstream separations, such as membrane filtrations hydrophobic silicone antifoams are particularly troubles Page 53 Dr. Ali Coşkun DALGIÇ
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Resistance’s for oxygen transfer from air bubble to the microbial cell 1) resistance within the gas film to the phase boundary 2) penetration of the phase boundary between gas bubble and liquid Page 55 3) transfer from the phase boundary to the liquid 4) movement within the nutrient solution Dr. Ali Coşkun DALGIÇ 5) transfer to the surface of the cell
When oxygen is required as a microbial substrate, it is frequently a limiting factor in fermentation. Because of its low solubility, only 0. 3 m. M O 2, equivalent to 9 mg/l, dissolves in one liter of water at 20 C in an air/water mixture. This amount of oxygen will be depleted in a few seconds by an active and concentrated microbial population unless oxygen is supplied continuously. In contrast, during the same period the amount of other nutrients used is negligible compared to the bulk of concentrations. Therefor most aerobic microbial processes are oxygen limited Page 56
The mass transfer of oxygen into liquid can be characterized by the oxygen tranfer rate (OTR) or by the volumetric oxygen transfer coefficient (k. La). The oxygen transfer rate and the volumetric oxygen transfer coefficient are dependent on the following parameters: the vessel geometry: diameter, capacity mixing properties: power, impeller configuration and size, baffles aeration system: sparger rate, geometry, location the nutrient solution: composition, density, viscosi ty the microorganism: morphology, concentration the antifoam agent used the temperature Page 57
Alternative Fermenters Many alternative fermenters have been proposed and tested. These fermenters were designed to improve either the disadvantages of the stirred tank fermenter high power consumption and shear damage, or to meet a specific requirement of a certain fermentation process, such as better aeration, effective heat removal, cell separation or retention, immobilization of cells, the reduction of equipment and operating costs for inexpensive bulk products, and unusually large designs. Page 58 Dr. Ali Coşkun DALGIÇ
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