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    Livro Bioseparation

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    renata1224

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    © All Rights Reserved
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    eS SSOOC CC COGC OCC OGGGUGCCCUCULCL Wee - 5185+ $450 3/93 — 3412 tHe eaias tas AUihee Yyes- 3027 favre Faian tend f COCOCC( Contents Notation, 1. An Overview of Bioseparations 11 Charactensties of Bioseparations. 2 An idealized Process, 4 14 Corclusions, 8 PART I. REMOVAL OF INSOLUBLES 2. filtration and Microfitration Pretreatment, 17 General Theory for Filtrattin, 22 Continuous Rotary Filters, 30 Laboratory Tests, 35 Microfiltrtion, 39 Conclusions, 41 Equipment for Conventional Filtration, " B W a PART HL PART IN, ti AN YVEeeUOCOCCCCC CCCCOCC Contents Centrifugation 7 3.41 Settling of Solids, 48, 32 Centrifuges, 53 33. Scale-Up of Centrifugation, 62 3.4 Centrifugal Filtration, 68 35° Conclusions, 72 Cell Disruption 7 41 Cell Membranes, 79 42 Chemical Methods, 82 44 Mechanical Disruption, 88 44 Conclusions, 93 ISOLATION 7 Extraction 3.1 The Chemistry of Extraction, 100 Batch Extractions, 109 aged Extractions, 114 Differential Extraction, 122 Fractional Extractions with a Stationary Phase, 128 Fractional Extractions with Two Moving Phases, 132 Conclusions, 140 Adsorption 145 61 The Chemistry of Adsorption, 146 62 Batch Adsorption, 155 63° Adsorption in a Continuous Stirred Tank, 158 6.4 Adsorption in Fixed Beds, 164 65 Conclusions, 176 PRODUCT PURIFICATION 181 Elution Chromatography 183 7.1 Adsorbents, 185 7.2. Yield and Purity, 188 7.3. Discrete Stage Analysis, 194 oreo cee ose COCCCCCC¢ ¢ Contents oad 7.4 Kinetic Analysis, 200 7.5 Scaling-Up Chromatography, 209 7.6 Conclusions, 216 8. Precipitation 81 Precipitation with a Nonsolvent. 222 8.2 Precipitation with Salts, 224 83 Precipitation with Temperature, 227 BA Large Scale Precipitations, 229 85. Conclusions, 235 9. Ulteafiltration and Electrophoresis YT Basic Ideas, 238 9.2 Ultrafiltration, 250 93 Electrophoresis, 260 Ya Mlectrodialysis and Isoelectric Focusing, 45 Conclusions, 268 PART IV. POLISHING 10. n 2, Crystallization TOL Basic Concepts, 274 102 Crystal Size Disteibutions, 103° Batch Crystallization, 292 104 Recnstallization, 299 103 Conclusions, 303, Drying 111 Basic Concepts, 308 11.2. Drying Equipment, 317 113 Conduction Drying, 320 Ha Adiabatic Drying, 324 11.5 Conclusions, 331 Ancillary Operations 121 Water Quality, 336 12.2 Solvent Recovery, 337 123 Waste Disposal, 340 abs C C c¢ 2 237 m wy 335 124 Biosafety, 342 125. Conclusions, 344 Appendix A. Characteristics of Biological Material Appendix B. Limits of the Continuum Approximation Index Contents 347 301 265 Notation (rm) ay deems Surface area per volume Acceleration (Chapter 3) Area Solutes Crystallization feed (Section 10.4) Nucleation rate Concentration of species i Saturation concentration of species i Heat capacity per mass at constant pressure Diameter or other characteristic distance Diffusion coefficient Extraction factor (Chapter 5) or purification factor (Chapter 10) Efficiency of cake washing (Section 2.4) Fraction remaining in position r after n transfers Friction factor Feed Buoyancy and drag forces, respectively Faraday's constant ‘Acceleration due to gravity Gas flow rate Linear crystal growth rate (Chapter 10) Equivalent time, used in centrifuge sign (Section 3.3) Flux of species i, in moles per area per time Solvent flux, in volume per area per time Heat transfer coefficient Heavy phase, usually an aqueous solution Humidity Height of a transfer unit ‘Mass transfer coefficient Boltzmann's constant (R/S) Crystal growth rate constant Nucleation rate constant (Chapter 10) Darcy's law permeabilivy (Chapter 2) ‘Thermal conductivity (Chapter 11) Partition coefficient or equilibrium constant Association constant for weak acid Column length or other characteristic length Length (as a dimension) Light phase, usually a solution in organic solvent Mother liquor from recrystallization (Section 10.4) Exponent in Freundlich isotherm (Section 6.1) Mobility (Section 9.1) Mass Molecular weight Number of stages, centrifuge dises, etc. ‘Number of transfers (Section 5.5) Population density (Chapter 10) ‘Number of crystals per volume (Chapter 10) Avogadro's number ‘Number of transfer units Pressure Rig Fraction extracted or crystallized Partial pressure of species i Probability of N, Amount adsorbed per mass or volume adsorbent (Chapters 6 and 7) Heat Aux (Chapter 11) Maximum amount adsorbed Volumetric flow rate Radius Reaction rate per volume Fraction of solubles remaining after washing (Section 2.4) Mass transfer per volume (Section 5.4 only) Tube or row number (Sections 5.5 and 10.4) Gas constant Fixed radius, for example, of a centrifuge Resistances of filter cake and of filter media, respectively (Chapter 2) Total concentration of charged groups on an ion exchange resin (Section 6.1) Power law exponent for a compressible cake (Chapter 2) Sedimentation coefficient (Chapter 9) Side of cubic crystal (Chapter 10) Pure solvent Time Time to reach half the equilibrium concentration in adsorption or to elute half peak in chromatography Temperature Velocity Velocity under gravity or centrifugal force, respectively (Chapter 3) Velocity of species i Volume Volume to elute half peak in chromatography Partial molar volume of species i Mass of adsorbent ee om Notation Concentration in light phase Concentration in light phase in stage i Mole fraction of species i (Chapters 4 and 9) Crystalline product of recrystallization (Section 10.4) Concentration in heavy phase H Solute concentration in feed Concentration of species i (e.g., when there are several solutes) Concentration in heavy phase in stage é Position Charge on species i (Chapter 9) Specific cake resistance Resistance parameter for a compressible filter cake Fraction of filtration cycle submerged (Chapter 2) Separation factor (Section 10.1) Solubility parameter of species { Void fraction Angle (Chapter 3) Fraction bed loading (Chapter 6) Dimensionless crystal size (Chapter 10) Reaction rate constant Heat of vaporization per mass of water or solvent Viscosity jth moment of crystal population (Section 10.2) Chemical potential of species i Chemical potential of species i in standard state Kinematic viscosity Density, defined variously Mass of solids per volume of liquid (Chapters 2 and 3) Equivalent area used in centrifuge design (Section 3.3) Time constant or dimensionless time Electrochemical potential (Chapter 9) Shape factors (Chapter 10) Dimensionless length (Table 10.1-1) Angular velocity Mass ratio or mass fraction re An Overview of Bioseparations Biotechnology is a rapidly advancing frontier. Hybridoma and DNA tech- nologies are features of scientific publications, and public offerings of zenetic engineering firms are carefully reported on the financial pages. This attention is merited, for the future promised in these areas is bright. ‘The current flurry of attention has tended to obscure the existence of an old and established industry based on biotechnology. By biotechnology. we mean the use of carefully cultured microorganisms, animal cells, and plant cells to produce products useful to humans. By this definition, biotechnol- ogy is as old as history, for the earliest known document (4228 BC) includes 4 description of brewing, Bread, cheese, and yogurt are other early exam- ples of products which depend on biotechnology. Today, we restrict biotechnology largely to those areas where chemically defined species are produced. Under this more restricted definition, biotech- nology is about 100 years old. Originally, those working in this arca produced organic chemicals by fermentation. For example, acetone pro- duced in this way was the basis of the munitions industry in the First World War, Of these early products, only citric and glutamic acids remain. The others are now made from petroleum or other chemicals ‘An Overview of Bioseparations ‘About 40 years ago, antibiotics eclipsed organic chemicals as the prin- cipal products of biotechnology. These secondary metabolites will continue to be of enormous benefit. They have been supplemented by amino acids, enzymes, and yeasts; they will be augmented by other medically useful proteins and such products as microbially based herbicides. Antibiotics do remain a focus of much of our thinking, a focus which we are expanding as the biotechnological frontier continues to advance. This short chapter gives an overview of this evolving area in two separate ways, In Section 1.1, we describe the general characteristics of separation processes in biotechnology. These characteristics reflect the separation of a tremendous diversity of unusually fragile molecules. Distillation, the work- horse of conventional separation technology. is commonly useless; labora- tory methods like chromatography and electrophoresis are frequently im- portant. This diversity makes generalizations dificult In spite of these difficulties, we attempt such generalizations in Section 1.2. We understand that these generalizations are tentative and that they deemphasize some important techniques. However, they do include inher- ent features of bioseparations like the highly dilute feeds and modest production scales. The generalizations provide the format around which the rest of the book is organized, 1.1. CHARACTERISTICS OF BIOSEPARATIONS “A chief characteris of biotechnology 1s the tremendous variety of prod- ucts which are produced. A typical petrochemical company makes about 10 products, but a typical drug company wilmake more than 200, To be sure, all 200 products may not be exclusively made biochemically, but many will be partially biologically converted. The diversity of these products can be illustrated in Table 1.1-1, Even this list is 10 years old, older than many current biotechnology companies. If we made a more current list, we would add genetically engineered insulin, human and bovine growth hormones, and interferons. The list gets longer daily. This diversity of products spawns the broad spectrum. of separation ‘methods used. Comparison of separation processes used in the fermentation industry with more exhaustive classifications gives the result in Table 1.1-2. ‘This table shows that 80% of all the separations classified are practical in biotechnology. All common modes of operation are used: steady and el CA LR LAA KA EL ERR RYN A 141 Characteristics of Bioseparations TABLE 1.1-1, Types of Molecules Manufactured by Fermentation Molecular ‘Number of Type Species Antibiotics 85 ‘Amino acids 18 Enzymes 1s Organic acids and solvents nL Vitamins, yeast, growth factors, nucleotides 6 Miscellaneous —dextrans, steroid biooxidations 8 13 unsteady states: batch and continuous equipment; cocurrent and countes current contacting. The seale of operations varies tremendously. In the early stage of development the objective will be to demonstrate the proposed process. to gain processing information, and to prepare relatively small quantities for subsequent clinical trials and marketing evaluations. This work will be conducted in the laboratory and the pilot plant. Later, the ‘objective will be scale-up and introduction of the process to large seale production. This effort will be conducted in the pilot plant and the commercial facilities. In the face of such diversity, how can we hope to proceed? We ean proceed by recognizing two characteristics of these separations which are widely shared, First. we will usually start with a dilute suspension and try to produce a highly purified dry product. The solids in this suspension may include intact organisms, fragmented mycelia, other insolu- TABLE 1.1-2. Spectrum of Separations Used in Biotechnology Used for All Conventional Used in Classification Chemicals Bioseparations Physical separations 7 7 Equilibrium controlled separations a 8 Rate controlled separations B 10 a 35 COCOECOCOCOCOCCCOEECCECCEC [An Overiew of Bioseparations ble fractions of medium ingredients, residual substrates, and, in some cases, insoluble products. The liquid in the suspension can contain soluble por- tions of residual substrates, metabolic pathway intermediates, and other desired products. In many cases, the final product which we seek will be colorless crystals which are stable indefinitely Second, the tremendous change implied by such a dilute feed and such a pure product means that these separations are elaborate and expensive. The recovery and purification operations may requi € more equipment and labor than all other parts of the process combined, The recovery plant usually represents a major investment and, consequently, the separations cost a substantial fraction of the total cost of the final product. Thus the recovery and the purification processes must be well conceived and well designed. We have found that our own designs have been improved by answering the following questions: What is the value of the product? ‘What is an acceptable product quality? Where is the product in each process stream? Whiere are the impurities in each process stream? What are the unusual physicochemical properties of the product and the principal impurities? 6. What are the economics of various alternative separations? Careful consideration of these questions can provide clues that will lead to optimal processes for the recovery of products of adequate quality coupled with high recovery and minimum effort. 1.2. AN IDEALIZED PROCESS ‘The tremendous diversity of products and separations in biotechnology obscures an overall similarity between many of the processes which are used. This similarity is by no means complete, and has numerous excep- tions. Still, the similarity provides a strategy for thinking, just as the Periodic Table emphasizes parallels between elements. | ¢ Cc ¢ « ax CCeCt 412 An idealized Process 5 From our experience, we find that most bioseparations have four similar steps, which occur sequentially 1, Removal of Insolubles. Filtration and centrifugation are the principal unit operations used in this segment, Relatively little product con. centration ar improvement of product quality occurs, . Isolation of Products. These steps, which are relatively nonspecific, remove materials of widely divergent properties compared to the desired product. Appreciable concentration and product quality in- creases usually occur. Adsorption and solvent extraction are typical. Purification. These processing techniques are highly selective for the product and remove impurities of similar chemical functionality and physical properties. Chromatography, electrophoresis. and precipita- tion are good examples. 4. Polishing. The end use of the product dictates the final sequence utilized, Crystallization is often key. Most products must also be dried. These four steps form the plan around which this book is organized (One way in which these four steps can be appreciated is by considering the product concentration and purity which is involved. A typical processing, profile for a product is given in Table 1.2-1. Note that the biggest incr in concentration comes in the isl jon step, but that quality inereases most Uramatically during purification. Some recent processes simplify this con- ventional sequence by combining the first two steps. These developments, TABLE 1.2-1. Processing Profile, Characteristic of Antibiotics* Product Step Typical Process __Cone.(g/liter) Quality (®) Harvest broth Fermentation 01-5 01-10 Removal of insolubles Filtration 02-20 Isolation Extraction 1-10 Purification ‘Chromatography 50-80 Polishing Crystallization 90-100 1 concentration and quality shown are relative measures, intended a5 illustrations. The ‘quality may refer 19 chemical purty, o activity, oto efcac, [An Overview of Biosepatations whole beer extraction and Muidized ion excl Chapters 5 and 6, respectively. This scheme, fashioned for extracellular products, needs to be modified for the recovery of intracellular materials. In particular, it must be supple- mented with a step to release the desired product from the organism, This release is usually inserted after the initial removal of insolubles, which has reduced the volume. Since a heterogeneous mixture of solubles and insolu- bles still remains after the product release, an additional solids removal operation is necessary to eliminate insoluble cell debris. The liquid effluent containing the desired material as a “solution” is used as feed to the isolation step, and the rest of the process is unaltered How this four-step sequence applies in practice is best seen by consider- ing flow sheets for four typical processes. The individual steps are obvious in the flow diagram for ethanol produc- tion given in Figure 1.2-1. After fermentation, the yeast and residual solid substrate are removed in the “beer” still, where the ethanol and water are vaporized. The more volatile heads are removed in the purifying columns. This sequence represents the isolation segment of the process. The final purification and polishing are obtained by removing the more volatile ethanol from its remaining contaminant, water, in the rectifying unit -—> Heads Conaonse ge, will be discussed in Oz Stlage of ee | Ge) a +s) Figure 1.241. Ethanol from fermentation, In this proces, ethanol and water are removed from 4 fermentation beer. Ths iniil distillate is then further purified. (After Prescott and Dunn, Industrial Microbiology Wiley, 1949) = Hepat rg we 12-2 Cite aed manufacture After fermentation, the fest step is filtration. The product is isolated and puriied by dhe changes in pH. and then given a final purification by Enstalization. cater 1-1. Pepplcr. Micro Technolgy, Acadeane. 1967) The four steps are also clear for citric acid, shown in Figure 1.2-2. After the fermentation, the first step is a filtration to remove insolubles. Adding lime precipitates the calcium product from soluble impuri I of citric acid and serves to isolate the s. The citrate is then purified by conversion back to the aeid and by filtration to remove calcium sulfate, The polishing is achieved by crystallization The flow diagram presented in Figure 1.2-3 for an enzyme produced from bacteria shows a similar sequence. The cells are concentrated by centrifugation, the enzyme is released by cell rupture caused by homogeni- zation, and the cell debris is removed by filtration. The isolation steps consist of a total precipitation followed by a fractional precipitation. This sequence removes many undesired proteins, Enzyme purification centers on ultrafiltration and chromatography. The final polishing includes precipita- tion, centrifugation, and lyophilization Finally, Figure 1.2-4 presents the flow diagram for a penicillin product. As usual, the first step consists of filtration to remove insolubles, The filtered broth is acidified and extracted with an organic solvent. The extract, is stripped with a buffer to isolate a concentrate. The pH of this concentrate is adjusted with acid and the product is purified by a second extraction into organic solvent. The final polishing consists of vacuum concentration, crystallization, and drying of the crystalline penicillin. Again, the four steps are all there, eee eecee cee cceccccc CCC 8 ‘An Overview of Bioseparations cows peels 7 si me is released by Figure 1.23. Enzyme production, In this process. an intracellular ent homogenization, isolated 25-4 precipitate, and puriied by fractional precipitation. The final puntications include chromatography (After P.G. Malby. Proc. Bachem 8, 22,1970) Base hea Sve ‘awn Freeh "phon! pre wast sen Figure 12-4. Peni production. Afr fermentation the biomass separa by Teun whch sce old and pre by extraction th 9 plaid by eotlization and ded a Wastewater 1,3, CONCLUSIONS Because bioseparations are so diverse, we have used an idealized four stage process as a basis for organizing the book. We begin with three chapters on the removal of insolubles. These discuss filtration, centrifugation, and cell disruption. Disruption is necessary when the desired product is intracellu- £ C COCCCECCOCCCCOCCCE Further Reading, c 9 Jar, We then discuss extraction and adsorption as methods of product isolation We are not exhaustive in our treatment of purification. Here, many types of separations are used, yet we have treated in detail only four: chromatog- raphy. precipitation, ultrafitration, and electrophoresis. We have chosen these because they seem to us most suitable for large scale production, We recognize that other methods can and will be useful We center our discussion of the fourth step—polishing—on erystallization, because it is, by far the most important. Final chaptess discuss supporting operations like drying. Organizing the book around these four process steps provides an intel- lectual framework, but at a price. The price is that some separations fit in several places. For example, ultrafiltration can be used for product isolation and extraction can be used for purification, However, because the subject is so diverse, we feel that this price is worth the huge pedagogical gain, FURTHER READING Atkinson, Band Masitana, Feds ‘New York, Macmillan, 1983 Bailey, J. Ba and Olli, D. F. McCraw TB, 1986, Bayurtrom, 126 10985, Buochemecal Engincorne and Buechnuligy Haron Buochemacal Engncering Fundamentals, 2d od, New Vork Biotechnology: fermentation and downstream procesune. Chom Engr 94), Belice. PLA. Recovery processes: past, present, and future, presented at 1MthAmenean Chemical Society Meeting, Kansas City, MO, September, 1942 Duntull P Trends in downsteeam processing in proteins and enasmes, Prneews Bauchemsin 14S), 9 (1983), ulen, EL. Production methods of industrial microbiology. Sr Amer, 28803), 18D (981, Michaels, A S., Adapting moder biology to industrial practice, Chem. Engr, Progress. 90,19 sey Removal of Insolubles As we explained in Chapter 1, we will idealize bioseparations as involving four steps: the removal of insolubles, the isolation of the product, the purification, and the polishing. This fist part of the book is concerned with the removal of insolubles. Later parts will focus on the remaining three steps The removal of insolubles reflects the origin of many biologically synthe- sized materials as dilute solutions produced by fermentation. These solu- tions or “beers” often contain suspended microorganisms. Sometimes the desired product is in the microorganisms; sometimes it is in the surround- ing broth. In either case, we often begin product recovery by separating these microorganisms to produce a clarified beer. Producing a clarified beer usually involves filtration, discussed in Chapter 2. or centrifugation, outlined in Chapter 3. In filtration, the microorganisms are captured in a concentrated cake, which can look like sand, sludge, or paste. In centrifugation microorganisms settle to the bottom of the beer because of centrifugal or gravitational force. In both cases, the separation Produces a clear solution and a solid concentrate. When the desired product is within the cells, we must next rupture the cells in order to begin product isolation. This cell rupture is treated in " 2 Filtration and Microtitration Chapter 4. It is included in this part of the book because the goal of et Iysis is a concentrated stream of product, not pure stream. As such, cl rupture is an additional step in a bioseparation. ; “The discussion in this part of the book differs from that in conventional engineering texts in two ways. Fist, it focuses on clear physical explana- tions of simple cases, and not on the highly specialized analysis available econd, the discussion must reflect aspects for particular chemical products teers ementaton bees These inlet often sins morphology of microorganisms, the compressibility of collected cakes, the danger of con- tamination, and the fragility of many products, especially proteins. These aspects reflect both the challenge and the frustration of bioseparations. | CHAPTER 2 Filtration and.Microfiltration Filtration separates solids from a liquid by forcing the liquid through a solid support or filter medium. It is a straightforward procedure for well defined crystals, However, the small size and deformability of micro- organisms make filtration of fermentation beers or other biological solu- tions considerably more complicated. In fact. if we attempt to filter these beers using purely conventional technology. the filtration is often too slow to be practical As a result, we have two goals in this chapter. First, we must explain in simple terms how conventional filtration is accomplished. Second, we must describe how these procedures are modified for bioseparations. These modifications center on the properties of the cake of solids which accu- rmulates on the filter medium, Only rarely will the solids be removed without additives. Frequently, the solids will be aggregated and removed With another solid, like diatomaceous earth, added as a “filter Sometimes, solid accumulation will be minimal because of rapid “cross flow,” tangential across the surface of the filter medium. It is these modifications which make filtration of biologically produced feeds non- routine, B “4 Filtration and Microfiteation In this chapter, we discuss in detail only filtrations where cake formation is significant. These filtrations usually involve larger particles or micro- organisms, and ate called conventional filtration. Filtrations where cross flow is dominant are effective for smaller microorganisms and so are called are deferred to Chapter 9, where they are discussed in parattel with mote similar processes. “microfiltration.” The We describe the equipment for conventional filtration in Section 2.1 This equipment is used to remove biomass, amorphous precipitates, or crystalline products, Many biological precipitates, which accumulate as dense impermeable cakes, require the unconventional procedures discussed in Section 2.2. The general theory of conventional constant pressure filtra- tion is presented in Section 2.3 and extended to continuous rotary filters in Section 2.4. This theory of filtration is concerned with filtration fluxes and ignores subjects like filtrate clarity and optimization. Laboratory tests which address these questions are covered in Section 2.5. A synopsis of microfiltration is given in Section 2.6. 2.1. EQUIPMENT FOR CONVENTIONAL FILTRATION Equipment for filtration varies widely, from the conventional plate-and frame filter press to rotary vacuum filters. Four types of small filters are illustrated in Figure 2.1-1. The most common, the plate-and-frame press shown in Figure 2.1-1a, contains caulked metal septa and a recessed cake space. Other types have Mushed plates that can be used with a paper or cloth filter medium separated by open frames where the cake is formed. ‘This type of filter is used where a relatively dry cake discharge is desired. It should not be used where there are toxic fumes or biohazards. ‘The other three filters in Figure 2.1-1 are enclosed, and hence can be used with aerosols or biohazards. Because their use is labor intensive, they are used only for relatively small scale work. ‘The horizontal plate filter shown in Figure 2.1-1b is useful for small scale separations. Filtration occurs only from the top of each plate (the bottom side of the cake) so that even with intermittent operation, the deposited cake remains in place. In designs like that shown, the filter leaf assembly is cleaned and the filter medium is added outside of the filter housing. In other variations, the cake may be removed with a sluicing nozzle or discharged by rapidly rotating the leaves. ‘The vertical leaf filter illustrated in Figure 2,1-1c requires only a small floor area but must have sufficient headroom for removal of the leaves and rCCCC CY CCCCCCCE CCK 211 Equipment for Conventional Filtration 5 if nat ‘ 4) Plato and Frame ) Horizontal Pata cane Dscrage ©) Vertes! Lest 4) Candle Type Figure 241, Small ers The conventional plate-and-rame Alte, shown in (a) is less «common for bioseparations than the thice othe types, which are enclosed. These other types are abor intensive, and so used only for small sale fitations the cake. It has a relatively high filtration area per volume. The tubes on the candle type vertical tank filter, shown in Figure 2.1-1d, are suspended from a tube sheet, Filter cake is formed on the external surface of the tubes and filtrate flows through the deposited cake into the head for discharge. The tubes are cleaned by backwashing, ‘We now turn from these small scale, enclosed filters to that workhorse of bioseparations, the rotary vacuum filter. Such filters are common for large Seale operations whenever the solids are difficult to filter. They are used extensively for large scale commercial operations. Because they are often automated, they have a lower labor cost. ‘The designs for rotary vacuum filters vary widely, but are exemplified by that shown schematically in Figure 2.1-2. The filter includes a rotating drum, shown from the end. Pressure outside the drum is atmospheric, but Figure 2:12. Rotary vacuum tee Ths cominuous enc i, shown from he em Fre a forwcpratns The ite rrmved rom the cd ofthe ends drum. ‘The unc shown ae pic values pressure inside the drum is @ partial vacuum, The drum is partially 1 Submerged in the beer and rotates at a low speed during the operation. Liquid is sucked through the filter cloth and solids are retained on the ‘e of the drum, forming, the cake. When the cake rotates out of the it is washed, dried, and removed It is the step of solid removal, shown here with 2 knife, which varies most widely in industrial designs. Methods developed over the years include (1) a simple knife or doctor blade supplemented by an air blowback through the filter medium; (2) a string discharge also assisted by the release of vacuum and a small blowback; (3) a continuous belt discharge where the filter medium is not caulked to the filter drum but moves around a series of rolls; and (4) the continuous rotary vacuum precoat filter. The idea of a filter precoated with some other medium is the first point where bioseparations become different from more conventional technology. ‘After all, this synopsis of equipment is common to many texts, but the idea of precoating is not. What is precoating? 4 Essentially, precoating is the application of a layer of diatomaceous earth ‘or some other inert microporous material to the filter medium itself. This layer is often thick, This coated filter is used with a beer to which still 222 Pretreatment v7 inert material has been added. As the drum rotates, a thin layer of biomass ‘and filter aid accumulates on the surface of the precoat and continues to build during the cake formation segment of the cycle. This cake is washed and dewatered; then, a slowly advancing doctor blade shaves off a thin iayer of accumulated biomass, thereby exposing a fresh surface of precoat for the next cycle, The depth of eut by the doctor blade depends on the penetration of the filtered particles. Vacuum is maintained throughout the entire filtration cycle, When the bed of precoat is depleted, a new layer is applied and further filtration proceeds. The result is a kind of dynamic filter which remains effective much longer than more conventional filters. Its disadvantage is an increased volume of solids, 2.2, PRETREATMENT Fermentation beers and other biological solutions are notoriously hard to liter. They are often hard to filter because of high, non-Newtonian viscosity ‘or because of highly compressible filter cakes. These cakes can deform into an impermeable mat. This is especially true of mycelial microorganisms, but may apply to any bacterial broth. ‘As a result, these biological feeds often require pretreatment. In the following paragraphs, we discuss three such treatments: heating, coagula- tion and flocculation, and adsorption on filter aids. Parenthetically, these treatments are also useful for centrifugation and sedimentation, Heating The simplest pretreatment—and the least expensive—is to heat the feed. Such heating can not only improve the feed's handling characteristics, but also may pasteurize it. The chief constraint of this approach is the thermal stability of the product. Why heating is effective is often chemically complicated. As an example, we consider a dilute ovalbumin solution. If we heat this solution, we will denature this protein and make it much easier to filter. In other words, we are taking a dilute solution of egg white, boiling it, and winding up with threads of cooked egg. The cooked egg threads will be much easier to filter than the original solution, but the ovalbumin has been irreversibly altered. 18 Filtration and Microfitation Coagulation and Flocculation ‘A second pretreatment method is the addition of electrolytes to promote coagulation and flocculation in the initial solution, Useful agents range from simple electrolytes through acids and bases to synthetic potyelectro" Iytes. Simple electrolytes act by screening the electrostatic repulsion which commonly exists between colloidal particles. When this electrostatic repul- sion is reduced, attractive London-van der Waals forces predominate. The {a) Effect of pt Time, minutes Figure 22-1, The effect on filtrate volume of pH and filter aid. For Stepionyces griseus, lowering the pH of adding filter aid accelerates the filtration. (Data from S. Shirato and S. Esumi, J, Ferm. Tech. (Japan), 41,817, 1963) | 22 Pretreatment i) oa Fier din ood Filtrate Volume, cm? (©) Effect of fiter aid Time, minut Figure 22-1. (Coninued colloids can then coagulate as larger, denser particles which are more easily filtered. Acid and bases change the pH and hence the charge on the particles. If this charge is reduced, the particles may coagulate and hence become easier to filter; if the charge is increased, the particles may coagulate still less, and the solution can become more difficult to filter. An example of the effect of pH on filtration rate is given in Figure 22-1. ‘Synthetic polyelectrolytes added as pretreatments can both reduce elec- trostatic repulsion and adsorb on adjacent particles, forming bridges be- 20 Fitration and Microfilteation tween them. As a result, the colloidal particles flocculate as large, less dense aggregates which are more easily filtered, These polyelectrolytes can be anionic, cationic, or nonionic. Commercially available examples include polyacrylamides, polyethylenimines, and polyamine derivatives Preireatments are developed empirically by using past successes and failures as a guide, Frequently, pretreatment chemicals may act simulta- neously as simple electrolytes, as acids and bases, and as polyelectrolytes This is true of ferric chloride and alum, two of the most common water treatment chemicals. These can act as simple electrolytes. can buffer the solution, and can form inorganic polymer bridges between particles. They are often most effective when used in conjunction with synthetic polyelec- trolytes, Adsorption on Filter Aids The third method of improving the filtration characteristics of fermentation broths is the addition of solid filter aids to the broth prior to filtration. Colloids in the broth adsorb on particles of filter aid and hence reduce two of the chief problems in filtration prior to fil jon. The major problem, particularly with mycelial broths, is the compressibility of the accumulated biomass, which dramatically reduces its permeability. The filter aid effects a much less compressible cake. The second problem is penetration of small TABLE 2.2-1, Typical Properties of Filter Aids? Density y Water Sea/m') Adsorption _Relative Grade Dry pit ® Flow Rate Diatomaceous Earths Standard Super-Cel 130 280 70 260 200 512 Hyflo Super-Cel 140 280 100 250 300 335 190 280 100 250 1400 560 310 320 100 220 7500 Perltes Terracel 300 10 260 15 - 300 Terracel 500 130 240 15 = 900 “These data are taken from Johns-Manville brochures. 22 Pretreatment ABLE 22-2. Filler Aid Classification* Relative Flow Relative Visual Clarity Clarity 1000 Bright pectns, sua a sparkle vinegar, alcohol civic acid i azlati, lard, tallow, polymers UB carom FPS 200995 Sparkle Bee. gelatin, fats, ils cider, lacquers i petroleum products. pectins susa, j Vinegar, aleohol, cite acid 4 phosphoric aid, cane sugar. lube oil TE ism FW6 300986 Sparkle Antibitis, beer, caustics, i chemical, cider, enamels gelatin, gle, fruit juices Kelp, lat, ils. petroleum i products TB caom Fw 20 1000960 Clear Acids, citrate, tallow, water, | chemicals, fruit juices, kelp, grape i juiee. apple juice, hemp oi fel oi } oils JB cee Fwso 2500 940, vey Alin, ort bec, aniiae csi j slight rested ies, laequers, polymers, veil syrups, sorghum, tallow, varnish, water, titanium, corn gluten citric juices AB vom Fw 5500927 Sight ——_Algins antibiotics, biochemicals, are charactenstie of commercially avaiable fer aah particles, such as fragmented mycelium or bacterial ces, into the precoat layer in a rotary vacuum filter. This penetration can blind the pores of the recoat matrix. ‘The two types of filter aids which have been found most effective for Pretreatments are the diatomaceous earths and the perlites. The diatoma- cous carths are the skeletal remains of tiny aquatic plants deposited centuries ago. The perlites are volcanic rock processed to yield an expanded form. Properties of both these filter aids are given in Table 2.2-1, and ‘Ypical applications are presented in Table 2.2-2. For special applications where silica-containing materials are undesirable, ground wood pulp and Starch are sometimes used. The vast improvements effected by the addition of filter aids is illustrated by the data in Figure 2.2-1b. There can be disadvantages to using filter acids to enhance filtration. For example, the data in Table 22-2 show that as particle size increases, flow 2 Filtation and Microfitration increases but filtrate clarity decreases. Asa second example, some products like the aminoglycoside antibiotics may irreversibly bind to diatomaceous earth, These examples underscore the importance of experiments in evaluat- ing the use of any pretreatment for filtration 2.3. GENERAL THEORY FOR FILTRATION ‘We now turn to the mathematical description of filtration. We do so in wo steps. First, in this section, we develop general equations for conventional filtration, both for compressible and incompressible cakes. Second, in Section 2.4, we apply these equations to the rotary vacuum filter, for this filter is the most common type in large scale bioseparations. In both of these sections, we will assume that we are using equipment like that described in Section 2.1 and have a feed stream pretreated by the methods of Section 2.2 Our general theory involves three steps. First, we need to review the equations of the fluid mechanies which are important for filtration, @ subjcet conveniently described as Darcy’s law. Second, we solve these equations for incompressible cakes, which is the simplest case, Third, we solve them for compressible cakes, which is the case common for biosep- arations, Darcy's Law Darcy's law relates the flow rate through a porous bed of solids to the pressure drop causing that fow 4£s one (23.1) ue where w is the velocity of the liquid, 4 is a proportionality constant usually called the Darcy's law permeability of the bed, Ap is the pressure drop serous the bed of icine €y amd tatbe Veg GELIe Bad. Lie Ohm’s law. Eq, (2.3-1) states that the flow is directly proportional to the potential Ap and inversely proportional to the resistance (¢/4). Stricily speaking, Darcy's law holds only when dup eee m-9 <> where d is the particle size or pore diameter in the filter cake, p is the liquid | (232) f } 2.1 General Theory for Filtration B density, and e is the void fraction in the cake. For biological separations, the dimensionless quantity (do p/u(1 ~ ¢)}, called the Reynolds number, almost always easily obeys this inequality For a batch filtration, we may rewrite the velocity as av ad (2.33) where V is the total volume of filtrate and ris the time, We may also rewrite the resistance (4/4) (o include explicitly the contributions of cake and filter medium = Ry + R (23-4) where R y is the resistance of the filter medium and R,. is the resistance of the accumulated biomass. Combining Eqs. (2.3-1), (2.3-3), and (2.3-4), we find the basie differential equation for filtration at constant pressure drop lav Ad de Ry + RO The medium’s resistance Ry is a constant, independent of the cake, In contrast, the cake's resistance R. varies with the amount filtered V. The exact nature of this variation depends on whether the cake is incom pressible or not. Incompressible Cakes If the cake is incompressible, the cake thickness is directly proportional to the filtrate volume and inversely proportional to the filter area. As a result, the cake’s resistance R¢ is described by the equation Re= an(=) A (2.36) where a represents the specific cake resistance and py is the mass of cake solids per volume of filtrate, This implies that a has the dimensions of length per mass, Substitution of Eq, (2.3-6) into Eq. (2.3-5) yields la Ap A dt ~ wlapolV7A) + Ry C39) i ss u Filtration and Microiltration This equation is subject to the initial condition r=0, V=0 (23.8) This condition says that atthe start of the experiment, no solution has been fiucred. Equation (2.3-7) is easily integrated and rearranged to give At v le K( (2.3.9) ()-«(a) + where Hep (2.3410) Meas and BRy 2 = (23-1) Oe Thus a plot of (A1/V) versus (V/A) should be linear. The slope K is a function of the pressure drop Ap and of the properties of the cake, represented by @ and p,, The intercept B should be independent of the properties of the cake, but itis proportional to the medium’ resistance R wy Often, this medium resistance is insignificant, so Eq, (2.3-9) becomes simpler still: CBE x ‘These equations, valid for incompressible cakes, will be tested in the ‘examples given later in the section. Compressible Cakes Unfortunately, almost all cakes formed of biological materials are com- pressible, and so cannot be described with the simple analysis just outlined. ‘AS these cakes compress, filtration rates drop. This in turn results in ‘compromised economics. 2 To estimate the effects of compressibility, we assume that the cake resistance a is a function of the pressure drop: a= a(Ap)' Ccccceccecececceccct 23 General Theory for Filtration 25 rae Stope = 5 x Figure 23-1. Cake resistance versus pres x, sure drop. The cake resistance often in fereases as the pressure drop across the ‘ake increases, Such an increase, due to the cake's compressibility, is easily de tog AP seribed by an exponents, where a’ is a constant related largely to the size and shape of the particles forming the cake, and 5, the cake compressibility, varies from zero for a rigid, incompressible cake to near one for a highly compressible cake In practi $ Tanges from 0.1-0.8, Values of 5 and a’ are most easily rmined by plotting the logarithm of « versus the logarithm of p, as shown in Figure 23-1. When values of s are high, one should consider pretreating the feed with filter aids, as described in Section 2.2. Fortunately, the fact that a cake is compressible does not change the basic result for a constant pressure filtration, given by Eq, (2.3-9). It does ‘mean that the value of K measured with this equation may be a more complex function of Ap than first expected. It should not change the value of B, which remains inversely proportional to pressure The implications of these results are illustrated by the examples which follow, Example 23-1. Streptomyces Filtration from an Erythromycin Broth. Using 4 test filter, we find the following data for a broth containing the antibiotic erythromycin and added filter aid: Filtration Time Volume of Filtrate (see) iters) 5 0.040 10 0.055 20 0.080 30 0.095 The filter leaf has a total area of 0.1 ft? and the filtrate has a viscosity of 1.1 eP. The pressure drop is 20 in. of mercury and the feed contains 0.015 kg dry cake per liter. Determine the specific cake resistance a and the medium resistance R.,. v ters A om Finan eho oth The ne 20 np on spt ns Sn ee a inca the ps anarhe e ot Increasing Feistance of Re hte cake Solution. Following Eq. (2.3-9), We plot (At/V) versus (V/A), as shown in Figure 2.3-2. The intercept on this plot is essentially zero, so Ry is zero: ‘The medium has no significant resistance. From the data and Eq, (2.3-10), we see from the slope of the data that Ay? Py 5) Fp O0llg sx) see (eels om 3.39 x 10* 1 in tear He CC CCOCCCCCCOCC CCE CC 23 General Theory for Fitation 5 This gives a= 24x 10" em/g ‘The dimensions of a are consistent with its definition, Example 2.3-2, Filtration of Beer Containing Protease. We have a suspen- sion of Bacillus subtilis fermented to produce the enzyme protease. To separate the biomass, we have added 1.3 times the biomass of a Celatom filter aid, yielding a beer containing 3.6 wt solids, with a viscosity of 6.6 cP. With a Buchner funnel 5 cm in diameter attached to an aspirator, we have found that we can filter 100 cm’ of this beer in 24 min. However, previous studies with this type of beer have had a compressible cake with equal 0 2/3. We now need to filter 3000 liters of this material in one of the pilot plant’s plate-and-frame filter presses. This press has 15 frames, each of area 3520 em®, The spacing between these frames can be made large. This is an advantage, because we can then filter all the beer without emptying the press. and hence reduce the risk of contamination, The resistance of the filters themselves is much smaller than the filter cake, and the total pressure drop which can be used is 65 psi (a) How long will it take to filter this beer at $0 psi? (b) How long will it take at half this pressure drop? Soliion. We first need to calculate the properties of the filter cake. Because the filter medium has a negligible resistance, we can combine Eqs. (2.3-12) and (2.3-13) to find Inserting the values given a'pp _{_100cm? 24 min = —*P ( (14.7 psi)'”? | 274(5 cm) Ha'py = 4.53 min psi!/?/em* ‘The units for wa’py are unusual but convenient. 28 Filtration and Microfittration (a) We now can use this same equation to find the time: ' 433 2(50 psi)” = 82hr : 3000 liters, 1000 em? \? 2% 15 x 3520 em* liter Note that the viseosity and beer concentration do not explicitly enter this calculation: they appear only as the produet je0'Py (b) If the pressure drop is cut in half, the same equation gives 1= 104br use the cake is so compressible. The time increases only slightly be Example 23-3. Filtration of Incompressible Steroid Crystals. We have filtered a slurry of sitosterol at constant pressure medium consisting of a screen support mounted aeross the end of a Pyrex We also through a filtration pipe. We find that the resistance of this new medium is negligible find the following data in a laboratory test Weight of erystals og Pressure of filtration 15 pst Filter diameter 5.08 em Cake depth 12.5em Cake volume 253.3 em" Filtration time 163 min ‘The cake is essentially incompressible. ‘On the basis of this laboratory test, predict the number of frames (G0 in, %30 in, 1 in. thick) needed for a plate-and-frame press (cf. Fig. 2.1-1a), Estimate the time required to filter a 63 kg batch of steroid. In these calculations, assume that the feed pump will deliver 10 psi and that the filtrate from the press must be raised against the equivalent of 15 ft head. ‘Solution, To design this filter, we first recognize that filtration occurs on both sides of the frame. As a result, the area per frame is 254m in. 2 Ve (9m. ) = 1.16 x 10cm? 123 General Theory for Filtration 2» The volume of each frame is 2.54 cm times this area, or 1.48 x 10* em? is area, or en? per frame. The feed contains 63 kg of steroid and the density of the cake is, (62 9/253 em) or 0.245 gem. Thus the total cake volume is 63,000 g 0245 e7em 25.7 x 10%em? The number of frames will then be 25.7 x 10* em’ Ta x10" em ame ~ '74 m8 Thus we need 18 frames, Note that this implies the frames are almost full, So far, we have only made mass balances, with no reference to the laboratory test data, We use these laboratory data to calculate the tration time in the new filter. From Eq, (2.3-12), we see that (ov Whi are not given the feed concentration (as py) oF the volume of filtrate F, we do know the mass of steroid (pV ). Thus. inserting the values, 163 min = U@Z2o)__(62) (15 psi) [274 (5.08 em)*] he Blmia(ps) cat Fhe F Seldom have units been stranger. We now look at the new conditions. While the pump will deliver 10 psi it must also overcome a pressure of 15 ft of water. Thus water. Thus the pressure dro} across the filter will be ‘ ae Ap = 10 psi - ee fe (32.2 tb,, ft/sec* Iby 144in. = 3.5 psi 30 Filtration and Microfiteation Again, from Eq. (2.3-12), [Hey Ceol) - Neal aecai 261 min(psi) em* (63,000 a)” . e 3.5 psif 18(1.16 x 10* em?)| = 68 min A Even though the pressure drop is smaller, the filtration is faster because the area is much larger. 2.4. CONTINUOUS ROTARY FILTERS separations is the continuovs One very common filtration method in bioseparat continua rotary filter. This méthod is used for more large scale antibiotic prowesing than all others combined, It is the preferred choice whenever the filtrati slow and dificult, anit We analyze the operation of these units in this section, AS sh Figure 2.1-2, a filtration cycle on this filter consists of three chief steps 1. cake formation, , 2. cake washing to remove either valuable or unwanted solutes, ani 3 cake discharge Although the type of discharge slete is important 10 the aie su i c of the filter. It is not discusset of the operation, it does not affect the size of 1 i here, but is deed in more specialized monographs. The analysis of the other two steps of this eycle is given in the following paragraphs. Cake Formation i r fi We first consider the cake formation which begins as the foating drum frst drops into the both. We assume thatthe resistance of the filter medium Ry, is negligible, so that we can use Eq, (2.3-5). the basic expressi CC CHO O Ee, 24 Continuous Rotary Filters 3” As before, this is subject to the initial condition that 1-0, v=o (2.4.2) We rewrite R, for @ compressible cake by combining Eqs. (2.3-6) and +19) w(t v = en =)iapy" (2.43) By combining Eq. (2.4-1) with Eq, (2.4-3), we can int jegrate over that part of the drum’s revolution where the cake is forming to find (2.44) where 1, is the cake formation time and ¥, is the volume of filtrate collected during that period. This relationship is sometimes written in terms of the eyele time f,, given by 1 = Bt, (24-5) where B is the fraction of time that the fraction of the cycle devoted to cake for (24-4), we find a relation for fi filter is submerged, that is, the mation, Combining this with Eq, tration flux expressed in terms of cycle time: “ pe} a Harpy! (2.4.6) We clearly see that cake formation can be altere cycle time ¢. of the fraction of the total cycle time devoted to cake formation f. In addition, we note that at constant B, the filtration flux +< inversely proportional to the square root of the cycle time. sd by varying either the total Cake Washing After formation, the cake contains a significant amount of soluteich liquid broth, This broth is usually removed by washing the cake. The washing has ‘0 functions. First it displaces the solute-ich broth trapped in pores in CCCCEC¢ 32 Filtration and Microfitration the cake. Second, it allows diffusion of solute out of the biomass in the cake. Such diffusion will enhance recovery if the desired produet is in the biomass, ‘Two factors are involved in the washing. The first is that fraction of soluble material remaining after the Wash; this fraction governs the volume fof wash liquid required. The second factor is the rate at which wash liquid passes through the cake; this rate of wash controls the time of the eycle devoted to washing, These two factors are discussed sequentially “The fraction of soluble material remaining is often related to the volume of wash liquid by the equation 1-6)" (2.4.7) where + is the ratio of solubles remaining after the wash to that originally present in the cake prior to the wash, is the volume of wash liquid divided by the volume of the liquid retained in the cake, and & is the washing efficiency” of the cake. The fraction r varies from zero to one: lower values of r correspond to more effective washing. An efficiency 6 of zero means that r= | and we get no reduction of solubles, no matter what volume of wash is used. Equation (2.4-7) is an empirical result from a variety of filtrations. We may wonder how well such an empiricism works for bioseparations. One test of the empiricism is given in Figure 24-1 for a beer containing the antibiotic lincomycin in a cake of bacteria. The figure is a plot of the logarithm of r versus m; the straight lines on this plot, for different clliciencies, have slopes of log(1 ~ 6°. Data for measurements at two values of pH show that efficiency does vary with pH. The scatter of these data is typical, and illustrates the accuracy of the empirical Eq. (2.4-7) “Thus we can estimate how much liquid will be needed to wash the cake, which was the first of the key factors here. We now turn to how fast this liquid will flow through the cake, which is the second key factor. “The wash liquid contains no additional solids, so the flow of wash liquid will he constant and equal to the final instantaneous filtrate rate at the end of the cake formation. This rate is i [ueyail2 GT | 2eeeoy [aces (2.48) where V, is the volume of wash water required and 1, is the time required CCC CCCCCC EC 24 Continuous Rotary Fillers 100 = 080 pe q Now gor S| : 5 oxo | : \ “ Zoot [e piso Bd ik ones ee O%0 i 12 WASH LIQUID / RETAINED LIOUID n Figure 2464, Eticeney of wash ‘empincism in Eg. (2.4-7) and tested by these di os eee eine ence Be for the washing. In many cases, we may find it u divide this result by a fi find it useful to divi result by (24-9) fee ¥, is the volume of liquid retained by the cake and f is the ratio of hs Yolume to the volume of filtrate. This form can be easier to use with (2.4-7), as the following example demonstrates. Ek filteation and Microfiteation 25 Laboratory Tests 35 34 sation ot Streptomyces erythens Beer, We want 10 flteh (b) We now turn to the washing step. From (247. sample 2-1 Filtration of Streptomyces we want fe Bea en of 2 bear containing evtvomysin wsing & 100) TT oa ral purchased for another product Our ite ass SO Fite oF ea of 37.2 nf Tt operates under a vacuum of 2 12 1-6)" 1-07)" cake with the resistance in vs pretreated broth forms an incompressible cake with th r e ree ‘Thus from Bq. (2.49) pap, _ 298 Bo 2)0.10 (3.8) es is left, and We want to wash the cake until only 1% of the retained solubles is left, an * os ive expect that the washing efficiency will Pe 70% and that 1% of the filtrate is retained. Calculate the filtration time per cycle. ea : im 2.5. LABORATORY TESTS find the volume of filrate whieh must be removed Pet In the earlier sections of this chapter. we described the equipment and the ‘Solution. (ay We first find the vo! lume analysis of simple filtations, We discussed compressible and incom- cycle pressible cakes, leaf and rotary vacuum filters, and the various methods of 15,000 liters pretreating broths. We unavoidably gave the strong impression that all we = |sa need si intelligence 208 liters This is not true: we need laboratory experiments. We need simple laboratory tests for eake compressibility, for pretreatment, for washing efficiency, and the like. We need to make these tests definitively yet simply “These laboratory tests are the subject of this section, The tests often both fulfill the requirements listed in the preceding paragraph and provide information about other potential troubles absent in the conventional: theory. These include filtrate clarity, tenacity of the cake to adhere to itself or the media, ease of washing, cake cracking, and moisture content of the oe final cake. These factors are vital to the success of a filtration process. cs sata( HL Before we begin this description, we need one word of caution, Be sure 1" Tapla that the experiments are conducted on the same materials as those planned for a joe | 208 x 10? on J the larger scale. Contamination, temperature changes, and cell lysis may ( jrax orem? alter the physical characteristics of the biomass in unexpected ways. Con 312 X10 tamination can produce a biomass of different composition and, con- sequently, of different physical characteristics. Temperature affects the i viscosity of the filtrate and therefore alters the filtration rate, as shown by ‘This is about 1/5 of the cycle time. — sfully design filters is paper, a calculator, and modest We now rearrange Eq, (23-12) ot (244) to find the Siravon tne For an incompressible cakes $ em® = 9 sec Cece cec cece ce cc ce 36 Fitation and Mcofiteation Eqs. (2.3-12) and (2.4-4). For example, if the liquid is water, an increase from 20 to 60°C can double the filtration rate. Make sure that the history of the test sample is as similar as possible to the feed to be processed. Pretreatment We can easily compare different pretreatments with a simple batch settling test. We fill a series of beakers of uniform size, equipped with slowly moving agitators, wth equivalent quantities of broth, as shown at the top of Figure 2.5-1. We add increasing quantities of Rocculant to the beakers. ‘After an arbitrary time, we allow the treated broth to settle. We then measure some criterion of flocculation performance, such as supernatant clarity or filtration rate. The plot in Figure 25-1 shows typical results, Often, we fill find an optimum dosage 10 use im the filtration tests which follow. ° va 12 1 ‘ 5 Ze a8 ge ee 3 as a 5 a log Flocculent Concentration ave ees and Figure 2541 Bach tess of peentnen, Smal doves of acuta ae ie Ip does stn esbiee wspended ods An ntemediat ess vtaly optimal ( Cc COC CCOCCCE 25 Laboratory Tests a TABLE 25-1, Filtration Results for Bacterium subtilis Broth* Filtration Rate Floceulant (%) (ml/min) Filtrate Clarity 00 160 Muddy os uM Slightly turbid 10 32 Slightly turbid 12 6 Clear 1a 46 Clear 16, 2 Slightly turbid “These data illustrate the compromise between filtration rate and filtrate cant Funnel Filtration Tests Using a small Buchner funnel, we can evaluate filtration of new cultures, new fermentation media, and new conditions. Ina typical ease, we just precoat the funnel with the desired filter aid, We then pretreat 100 ml of whole broth and immediately filter the sample for a fixed time under the pressure drop practical for our plant, Typically, we measure the volume and clarity of filtrate collected, As an example, consider the data for the filtrate rate and clarity of a Bacterium subtilis broth shown in Table 2.5-1. These data suggest that a locculent dosage of 1. between filtration rate and filtrate clarity gives a suitable compromise The times determined in such tests can be used to estimate the time required for the large seale filtrations, Although many data must be collected to establish a reliable correlation, we have found that the effort is, well rewarded. Indeed, the same test can also be used as an analytical tool, to determine the best time to harvest a fermenter or to uncover the causes of slow plant filtrations. Filter Leaf Tests If we plan to use a continuous rotary filter, we next need to make filter leat tests, Such tests are the basis of guarantees for most of the rotary filters sold. The procedure consists of manually manipulating a test filter of Known area through the sequence which apes that occurring during a continuous rotary filter cycle. The amount and quality of cake, the amount and quality of filtrate, and the amount and effectiveness of the wash liquid can be determined for each cycle of the drum. This procedure is therefore a Filtration and Microfilteation Ai Bleed Ny Graduated Funnel 6 = SS Fiter Leat to detctmine bow beers will behave on a pment is used 0 ds c i ey aa mane ie ‘key part, the filter leaf, is detailed in Figure 25-3. Tange vontinuous rotary vacuum file direct laboratory simulation of a full scale filtration, necessary because no small rotary filters are available. . 5 ese tests, shown in Figure The equipment for these tests, sho se ts leaf, fitate and wash liquid feeiving vessels, and an adjustable vacuum source. In addition, supplies of the broth tobe filtered and the was aid to be used are needed, The test leaf detailed in Figure 2.5-3 is for precoat operations, but test leaves for nonprecoat operations are similar. consists of the test cake Ring ‘ ‘conditions in an acer ee eee fe 6 et et eS cH : pete i : CCC CCC CCC 26 Microfiteation 3 TABLE 2.5-2. Typical Vacuum Filtration Rates! Filtration Rate Product Microorganisms iters/he my Kanamycin 'S. Kanamycetius ~07 Lincomyein 5. lincolnensis 26-38 Erythromycin S. erthews 29-57 Neomyein 5 fradse ~ 1d Penicillin = 12-16 Protease B. subtilis 09-37 ‘All valves are for vacuum precoat filtration. Values witha filter Kat agree closely with fall seal filters To make a filter leaf test, we first precoat the filter leaf with the selected filter aid. We then place the leaf under vacuum and submerge it in the broth for the time selected for cake formation, During this cake formation, we agitate the broth to give a uniform mixture during the entire test period. After cake formation, we withdraw the leaf and, still under vacuum, allow it to drain for the allotted time, We then submerge the leaf in the wash liquid for the desired washing period, remove it, and dry the accumulated cake. We then advance the cake ring shown in Figure 25-3 to expose the proper thickness of cake and filter aid, and cut off this exposed cake. As a result, we have a fresh precoat surface with which to repeat the test. Measurements of cake mass and filtrate volume are the data needed for the large scale process design implied by the euslier sections of this chapter. Typical values found from these tests are given in Table 2.5-2. Try the test yourself —it works well. 2.6. MICROFILTRATION AL this point, we may wish that we could filter without a filter cake. After all, most of the pressure drop in conventional filtration comes from the cake. When the cake is compressible, the pressure drop is not even linearly related to the flow and, hence, is a significant complication, both for filtration analysis and for filter design. Filtration without a cake might offer real advantages. Filtration without a cake is part of the process called microfiltration. As Figure 2.6-1 suggests it is achieved by using a large cross flow tangential to 40 Filtration and Miceoitation mf. (Goneanasted =| Uh [Pomoc Stra J] iter Meum or “Momerane” cfc sluton i rapidly ree pst he Figure 264. A schematic of micofleaton, The fxd slo titer medlum--the mombraneio miss any cae formation deta nano his tnd pall separation nena Chapt the surface of the filter itself, At the same time, the flow normal to the filter is relatively small, The large ratio of eross flow to flow through the filter minimizes the accumulation of c: Microfiltration is used to concentrate insolubles. It is like ultrafiltration, and is analyzed with similar equations. Parallel equations are used to describe electrophoresis and electrodialysis. As a result, we have deferred a detailed discussion of microfiltration to Chapter 9, where we will describe it along with these purification methods. ‘AL the same time, we want to give a brief synopsis of microfiltration here, just to strengthen the comparison with conventional filtration. This synopsis centers on three points: the filter itself, the requirements for purging, and the filter geometry. Each will be discussed in the following text, ‘The key part of the microfiltration is the filter medium itself, or as itis more commonly called, the membrane. These membranes are thin and microporous. The pores are small and highly monodisperse; they must retain the particles being filtered, but quickly pass liquid and smaller solutes. As a result of the small pores, the membrane has a low Darcy's law permeability and a high resistance to flow. ee ‘These features mean that a microfiltration membrane is different from a more conventional filter medium. Such a conventional medium has a high Darcy's law permeability, that is, a low resistance to flow. Indeed, in the ‘examples of the previous sections, the resistance of the filter medium was often negligible. This 27 Conclusions a ‘The second central point for microfiltration is the necessity of periodic purging. In most cases, the initial microfitration feed is a highly dilute suspension. As liquid is removed, the suspension becomes concentrated. Rapidly pumping this suspension, which is necessary to sustain cross flow, is increasingly difficult, for its viseasity skyrockets. As a rest the con- centrated suspension must be discharged, even when it contains much more liquid than that in a conventional filer cake, As a result, mierotiltration may be followed by conventional filtration or centrifugation to more completely separate the insolubles from the solution The final central point for microfiltration is the shape of the filter. In microfiltration, the flow through the membrane is slower than the flow through a conventional filter cake. Because the flow per area is so slow. microfilter are designed with a larger filter atea per filter volume than conventional filters. Three designs are common: the plate-and.frame, the spiral wound module, and the hollow fiber module. The plate-ande-frame fiter is like that in Figure 2.1-1a, but with @ microfiltration membrane replacing the conventional filter medium. These devices have a modest surface area per volume, but do not often plug and are easy to clean Moreover. if one piece of membrane fails it is easily located and replaced The spiral wound and hollow fiber modules have a much larger area per volume, but plug more easily. The spiral wound device is like a large envelope filled with feed which is filtered through the envelope's wall However, the envelope is loosely rolled. and the filtrate is collected from the end of the roll. The hollow fiber module is like a small shell-and-tube heat exchanger in which the tubes are microporous fiber membranes. The Suspension being filtered flows down the fibers’ interior—the fibers’ lumen —and filtrate is collected from the outside of the fibers on the shell side. Both the spiral wound and the hollow fiber filters are often discarded when ‘even part of the filter fails This synopsis of microfiltration describes how the process occurs and what the equipment will look like. It defers a more det Chapter 9, Take our advice: wait patiently; tailed discussion to it will be clearer the 2.7. CONCLUSIONS Filtration is the best established and most versatile method for removing insolubles from dilute streams like fermentation broths. While filtration has been successfully used for blood fractionation, its value for protein sep- Filtration and Microfiteation| 42 wide variety of arations is unknown, Filtration can be accomplished with a wide varity of equipment, described in Sections 2.1 and 2.5. Some of this equipment, like the plate-and-frame filter press, is common to the chemical industry. Other equipment, especially the continuous rotary precoat filter, is more common for bioseparations. scat analyze most ‘The theory of filtration given in his chapter i sulin! t analyze tos experimental data, but is not sufficient for design without laboratory experiments, This will be a recuring theme in this book: Laboratory experiments are imperative, These experiments, deseribed ae 5. supply the parameters appearing in the theories of Sections 2.3 and 2.4. [At the same time, many biological suspensions are difficult to filter. Their siraton is slow, rodcing Ft aks which a compressible slimy pastes ‘These filtrations can often be improved by filter aids like those a ae Section 22, These improvements produce more slid waste, a major det ment in a time of increased environmental concern. When the filteati remains difficult, we must consider centrifugation, discussed in the next chapter FURTHER READING and ed., New York. Bennett, €.0., and Myers J. E. Momentum, Heat and Mass Transfer. 2od ed. New ¥ ‘McGraw-Hill, 1974, a CCheremisinolf, NP. and Azbel, D.S.. Liga Filtraon, London, utters Jahteis, C. A. Fillzation: advances and guidelines. Chem. Engr, 844), 80 (1976) Wakeman, R.J., Progress in Filiration and Separation, New York, Elsevier, 1982 Wiesmann, U. and Binder, H., Ade. Biochem. Engr 24,119 (1982), PROBLEMS 2.1, A smal et filtration of auromycin crystals in an acetone suspension uses a filter medium of negligible resistance. 1t gives the following data: (sec) V Gliters) 10 0500 200.707 30 0.866 Problems 43 In finding these data, we have used a filter with 89 cm? area and a Pressure drop of 2.6 m of water. We now plan to filter a much larger

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