322 Fermentation and Biochemical Engineering handbook must also consider heating and/or cooling requirements as well as to deliver the desired head pressure to overcome hydraulic pressure drop Operating costs must also consider equipment maintenance, cost of cleaning chemicals and labor costs. CFF systems in general, have substan- ially lower maintenance and labor costs compared with other competing technologies. Cleaning chemical costs are typically low and account for only about l to 4%of the total operating costs. 131 6.8 Safety and Environmental Considerations The proper and efficient operation of a cross-flow filtration system requires a design based on sound engineering principles and must rigorously adhere to safe engineering practices. CFF systems must be equipped with high pressure switches to safely diffuse a high pressure situation and must also use materials and design criteria per American Society for Testing and Materials(ASTM) standards. Proper insulation is required in accordance with Occupational Safety and Health Administration(OSHA)regulations for high surface temperatures or hot-spot when operating at elevated tempera tures. For corrosive chemicals, proper handling and disposal procedures must be followed for operator safet Containers approved by OSHa and other regulatory agencies must be used when transporting or transferring hazardous chemicals. In addition, proper procedures must be followed when mixing chemicals, either within the manufacturing process or while handling waste solutions The majority of CFF processes are operated in a closed configuration which minimizes vapor emissions. Some traditional techniques such centrifugal processes may generate aerosol foaming in the air( e.g., patho- gens)which is hig 7.0 APPLICATIONS OVERVIEW Due to the highly proprietary nature of fermentation of biochemical products, the published descriptions on cross-flow filtration performance are very limited. This section will review some of the more important types of applications where cross-flow filtration is used. The performance descrip tions are limited by available published information which is often incom- plete. As a result, at best, only qualitative or general comparisons can be made between the various technology alternative
322 Fermentation and Biochemical Engineering Handbook must also consider heating andor cooling requirements as well as to deliver the desired head pressure to overcome hydraulic pressure drop. Operating costs must also consider equipment maintenance, cost of cleaning chemicals and labor costs. CFF systems in general, have substantially lower maintenance and labor costs compared with other competing technologies. Cleaning chemical costs are typically low and account for only about 1 to 4% of the total operating costs.[3] 6.8 Safety and Environmental Considerations The proper and efficient operation of a cross-flow filtration system requires a design based on sound engineering principles and must rigorously adhere to safe engineering practices. CFF systems must be equipped with high pressure switches to safely diffise a high pressure situation and must also use materials and design criteria per American Society for Testing and Materials (ASTM) standards. Proper insulation is required in accordance with Occupational Safety andHealth Administration (OSHA) regulations for high surface temperatures or hot-spot when operating at elevated temperatures. For corrosive chemicals, proper handling and disposal procedures must be followed for operator safety. Containers approved by OSHA and other regulatory agencies must be used when transporting or transferring hazardous chemicals. In addition, proper procedures must be followed when mixing chemicals, either within the manufacturing process or while handling waste solutions. The majority of CFF processes are operated in a closed configuration which minimizes vapor emissions. Some traditional techniques such as centrifugal processes may generate aerosol foaming in the air (e.g., pathogens) which is highly undesirable. 7.0 APPLICATIONS OVERVIEW Due to the highly proprietary nature of fermentation of biochemical products, the published descriptions on cross-flow filtration performance are very limited. This section will review some of the more important types of applications where cross-flow filtration is used. The performance descriptions are limited by available published information which is often incomplete. As a result, at best, only qualitative or general comparisons can be made between the various technology alternatives
Cross-Flow Filtration 323 7.1 Clarification of Fermentation Broths Fermentation broths tend to be very dilute and contain complex mixtures of inorganic or organic substances. 47 The recovery of a soluble product(MW range 500-2500 dalton)such as an antibiotic, organic acid or animal vaccine from fermentation broth takes several processing steps. The first step is the clarification of broth to separate the low molecular weight soluble product from microorganisms and other particulate matter such as cells. cell debris. husks. colloids and macromolecules from the broth me. dium. 12 (48)In this step, microporous membrane filters(MWCO 10,000 500,000 dalton) compete with pre-coat vacuum filter or centrifuge When membrane filters are used, the soluble product is recovered in the permeate. This step is followed with diafiltration of the concentrate(continu ous or batch) to improve yield. The permeate is then subjected to final concentration Many filtration processes operate in a batch configuration at or near ambient conditions(e. g, 20-30C for penicillin) with some exceptions(e.g 2-5C for certain yeast fermentations and 80C for some higher alcohols) Batch times can range from 12 to 22 hours depending on the desired final concentration and the required number of diafiltration volumes. At the end of a batch run, membranes are chemically cleaned. Cleaning may take up to 3 hours and involve the use of an alkaline or acidic solution, or both, with a final sanitization step(e. g, 200 ppm NaOCI solution, a dilute solution of odium bisulphite or a bactericide/fungicide). In some cases, steam steriliza- tion may be performed at the end of each run especially when using inorganic membrane filters Today many industrial fermentation broth clarifications are performed ing cross-flow MF/UF membrane modules. 2112) The advantage of CFF overtraditional separation processes is not only in superior product flow rates but also in higher yields or lower product losses. Using diafiltration, up to 99%recovery can be obtained. 2 491 7. 2 Purification and Concentration of Enzymes Enzymes are proteins with molecular weights in the range of 20,000 to 200,000 dalton and are predominantly produced in small batch fermenters UF often combined with diafiltration is widely used in the industry to produce a variety of enzymes such as trypsin, proteases, pectinases, penicillinase and carbohydrates. [IJ[14]
Cross-Flow Filtration 323 7.1 Clarification of Fermentation Broths Fermentation broths tend to be very dilute and contain complex mixtures of inorganic or organic sub~tances.[~l[~~] The recovery of a soluble product (MW range 500-2500 dalton) such as an antibiotic, organic acid or animal vaccine from fermentation broth takes several processing steps. The first step is the clarification of broth to separate the low molecular weight soluble product from microorganisms and other particulate matter such as cells, cell debris, husks, colloids and macromolecules from the broth medi~m.[~][~*] In this step, microporous membrane filters (MWCO 10,000 to 500,000 dalton) compete with pre-coat vacuum filter or centrifuge. When membrane filters are used, the soluble product is recovered in the permeate. This step is followed with diafiltration ofthe concentrate (continuous or batch) to improve yield. The permeate is then subjected to final concentration. Many filtration processes operate in a batch configuration at or near ambient conditions (e.g., 20-30°C for penicillin) with some exceptions (e.g., 2-5OC for certain yeast fermentations and 8OoC for some higher alcohols). Batch times can range from 12 to 22 hours depending on the desired final concentration and the required number of diafiltration volumes. At the end of a batch run, membranes are chemically cleaned. Cleaning may take up to 3 hours and involve the use ofan alkaline or acidic solution, or both, with a final sanitization step (e.g., 200 ppm NaOCI solution, a dilute solution of sodium bisulphite or a bactericide/fungicide). In some cases, steam sterilization may be performed at the end of each run especially when using inorganic membrane filters. Today many industrial fermentation broth clarifications are performed using cross-flow MF/UF membrane modules.[21[12] The advantage of CFF over traditional separation processes is not only in superior product flow rates but also in higher yields or lower product losses. Using diafiltration, up to 99% recovery can be 0btained.['*1[~~] 7.2 Purification and Concentration of Enzymes Enzymes are proteins with molecular weights in the range of 20,000 to 200,000 dalton and are predominantly produced in small batch fermenters. UF often combined with diafiltration is widely used in the industry to produce a variety of enzymes such as trypsin, proteases, pectinases, penicillinase and carbohydrate^.[^]['^]
324 Fermentation and Biochemical Engineering Handbook UF offers many advantages over traditional processes such as vacuum evaporation or vacuum evaporation with desalting. These include higher product purity and yields(concentration factor 10 to 50), lower operating costs, ability to fractionate when the molecular sizes of the components differ by a factor of at least 10. The availability of a wide range of Mwco membranes enables the selection of a suitable membrane to maximize flux without substantially compromising retention. UF canalso minimize enzyme inactivation or denaturation by maintaining a constant pH and ionic strength Other techniques such as solvent precipitation, crystallization or solvent extraction may sometimes denature the product owing to phase change UF performance, however, may be influenced by process variables such as pH, nature of ions and ionic strength, temperature and shear. For xample, Melling 50] has reported the effect of pH on the specific enzyme activity of E-coli penicillinase in the ph range 5 to 8. Effects of shear inactivation associated with pumping effects are described by o sullivan et al.(23 Recessed impeller centrifugal pumps or positive displacement pumps may be used to minimize enzyme inactivation due to shear 7.3 Microfiltration for Removal of Microorganisms or Cell Debris In recent years there has been a significant interest in the use of micro- organism-based fermentations for the production of many specialty hemicals (511[531 The product of interest may be produced by either an extracellular or intracellular process relative to the microorganisms. In either of these situations, one of the key steps is the efficient removal of microorgani cell debris from the fermentation broth. 154(551 In biotechnology terminology this step, where cells are separated from the soluble components of the broth, is described as cell harvesting Filtration is often preferred over centrifugation due to problems associated with poor separation which results in either reduced product yield or purity. Aerosol generation during centrifugation could be a maje problem. This can be alleviated in the CfF mode due to the closed nature of system operation. Additionally, centrifuges may require high since there is no appreciable density difference between the bacterial cell walls and the surrounding medium. Pre-coat filtration, when applicable, will suffer from reduced product yield and lower filtration rates (e.g, 0.7 to 16L/hrm2.52
324 Fermentation and Biochemical Engineering Handbook UF offers many advantages over traditional processes such as vacuum evaporation or vacuum evaporation with desalting. These include higher product purity and yields (concentration factor 10 to 50), lower operating costs, ability to fractionate when the molecular sizes of the components differ by a factor of at least 10. The availability of a wide range of MWCO membranes enables the selection of a suitable membrane to maximize flux without substantially compromising retention. UF can also minimize enzyme inactivation or denaturation by maintaining a constant pH and ionic strength. Other techniques such as solvent precipitation, crystallization or solvent extraction may sometimes denature the product owing to phase change.i8] UF performance, however, may be influenced by process variables such as pH, nature of ions and ionic strength, temperature and shear. For example, Melling[sol has reported the effect of pH on the specific enzyme activity of E-coli penicillinase in the pH range 5 to 8. Effects of shear inactivation associated with pumping effects are described by O’Sullivan et a1.[12] Recessed impeller centrifugal pumps or positive displacement pumps may be used to minimize enzyme inactivation due to shear. 7.3 Microfiltration for Removal of Microorganisms or Cell Debris In recent years there has been a significant interest in the use of microorganism-based fermentations for the production of many specialty chemicals. I5 11-[531 The product of interest may be produced by either an extracellular or intracellular process relative to the microorganisms. In either of these situations, one of the key steps is the efficient removal of microorganisms or cell debris from the fermentation br~th.[~~][~~] In biotechnology terminology, this step, where cells are separated from the soluble components of the broth, is described as cell harvesting. Filtration is often preferred over centrifugation due to problems associated with poor separation which results in either reduced product yield or purity. Aerosol generation during centrifugation could be a major problem. This can be alleviated in the CFF mode due to the closed nature of system operation. Additionally, centrifuges may require high energy inputs since there is no appreciable density difference between the bacterial cell walls and the surrounding medium. Pre-coat filtration, when applicable, will suffer from reduced product yield and lower filtration rates (e.g., 0.7 to 16 L/hr-m2).[52]
Cross-Flow Filtration 325 The processing steps differ depending on the location of the product relativeto the microorganisms. For extracellularproducts, maximizing broth recovery by clarification is important since the product is in solution. When the product is located within the cell walls, concentration of cell mass is required followed by cell rupture and recovery of products from the cell debris Cross-flow microfilters, such as microporous hollow fiber or tubular, are preferred over plate and frame or spiral wound which are prone to plugging due to their thin channel geometry. Inaddition, CFFcan be operated in the continuous mode with backwashing or backpulsing which has a beneficial effect on filtration performance. Process economics dictates the use of high cell concentration to maximize product yields but may hinder the recovery of soluble products( e. g, production of penicillin, cephalosporin) In other situations, high bioma y hinder the effici removal(e.g, lactic acid, propionic acid) of inhibitory metabolites 1561 Similar situations exist in the production of acetone-butanol organic acids and amino acids from micro-organism- based fermentations 1431 Concentration of Yeast or E-coli Suspensions. The concentration of east or E-coli is often performed using microporous membrane filters. For example, in the production of ethanol by fermentation, yeast cells are used as biocatalyst. 158 It is necessary to ensure adequate recycling of cell mass to minimize the production of inhibitory products. Typically, a membrane with a pore diameter in the range 0.02 um to 0. 45 um is used, which represents a good compromise between the requirement to maintain relatively high flux at high cell concentrations while minimizing pore plugging and adsorptive surface fouling. These types of microorganisms are not shear sensitive, which allows the use of high shear rates to reduce concentration polarization effects. Initial cell mass concentration may vary from 5 to 25 gm(dry wt )7 L. Final concentrations up to 100 gm (dry wt )L or cell densities up to 10 4 cells/L can be achieved by cross-flow filtration with diafiltration. 41211541 Table ll shows the performance of polymeric and ceramic filters for the separation and concentration of yeast and E-coli suspensi The ceramic filters, due to theirsuperior mechanical resistance, can be backpulsed to reduce flux decline during concentration. This is illustrated in Fig. 19 for the filtration of yeast suspension with 0.45 Hm microporous cellulose triacetate membrane. 4 Polymeric membranes can be backwashed at pres sures up to about 3 bar. The data in Fig. 20 show the flux improvement with backpulsing using 0.2 um microporous alumina membrane. 21)
Cross-Flow Filtration 325 The processing steps differ depending on the location of the product relative to the microorganisms. For extracellular products, maximizing broth recovery by clarification is important since the product is in solution. When the product is located within the cell walls, concentration of cell mass is required followed by cell rupture and recovery of products from the cell debris. Cross-flow microfilters, such as microporous hollow fiber or tubular, are preferred over plate and frame or spiral wound which are prone to plugging due to their thin channel geometry. In addition, CFF can be operated in the continuous mode with backwashing or backpulsing which has a beneficial effect on filtration performance. Process economics dictates the use of high cell concentration to maximize product yields but may hinder the recovery of soluble products (e.g., production of penicillin, cephalosporin). In other situations, high biomass concentrations may hinder the efficient removal (e.g., lactic acid, propionic acid) of inhibitory metabolites.[56] Similar situations exist in the production of acet~ne-butanol[~~] organic acids and amino acids from micro-organism-based fermentations Concentration of Yeast or E-coli Suspensions. The concentration of yeast or E-coli is often performed using microporous membrane filters. For example, in the production of ethanol by fermentation, yeast cells are used as biocatalyst.[58] It is necessary to ensure adequate recycling of cell mass to minimize the production of inhibitory products. Typically, a membrane with a pore diameter in the range 0.02 pm to 0.45 pm is used, which represents a good compromise between the requirement to maintain relatively high flux at high cell concentrations while minimizing pore plugging and adsorptive surface fouling. These types of microorganisms are not shear sensitive, which allows the use of high shear rates to reduce concentration polarization effects. Initial cell mass concentration may vary from 5 to 25 gm (dry wt.)/ L. Final concentrations up to 100 gm (dry wt.)/L or cell densities up to IOl4 cells/L can be achieved by cross-flow filtration with diafiltrati0n.[~][~~1[~~] Table 11 shows the performance of polymeric and ceramic filters for the separation and concentration of yeast and E-coli suspensions. The ceramic filters, due totheir superior mechanical resistance, can be backpulsed to reduce flux decline during concentration. This is illustrated in Fig. 19 for the filtration of yeast suspension with 0.45 pm microporous cellulose triacetate membrane.14] Polymeric membranes can be backwashed at pressures up to about 3 bar. The data in Fig. 20 show the flux improvement with backpulsing using 0.2 pm microporous alumina membrane.[21]
Table 11. Concentration of Yeast and E-coli with Cross-flow MF/UF Feed Pore diameter Type of Membrane L/h Yeast 02 Polymeric Suspension hollow nber (reconstituted) erIc Ceramic multichannel 00 02 Polymeric 54 (reconstituted) hollow nber A concentratlon of organisms expressed In wet wt% concentratlon of organisms expressed in dry g/L
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