Perry´s chemical engeneers handbook, Notas de estudo de Engenharia Química

Baixe Perry´s chemical engeneers handbook e outras Notas de estudo em PDF para Engenharia Química, somente na Docsity! Section 24 Biochemical EngineeringHenry R. Bungay, P.E., Ph.D., Professor of Chemical and Environmental Engineering, Rensselaer Polytechnic Institute; Member, American Institute of Chemical Engineers, American Chemical Society, American Society for Microbiology, American Society for Engineering Edu- cation, Society for General Microbiology. (Section Editor) Arthur E. Humphrey, Ph.D., Retired, Professor of Chemical Engineering, Pennsylvania State University; Member, U.S. National Academy of Engineering, American Institute of Chem- ical Engineers, American Chemical Society, American Society for Microbiology. George T. Tsao, Ph.D., Director, Laboratory for Renewable Resource Engineering, Pur- due University; Member, American Institute of Chemical Engineers, American Chemical Soci- ety, American Society for Microbiology. Assisted by David T. Tsao.INTRODUCTION TO BIOCHEMICAL ENGINEERING Biological Concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-4 Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-4 Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-4 Algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-4 Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-4 Isolated Plant and Animal Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-4 Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-4 Biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-4 Energy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-5 Photosynthesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-5 Mutation and Genetic Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-6 Additional References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-6 Cell and Tissue Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-6 Mammalian Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-6 Plant Cells and Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-6 Primary Growth Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-6 Secondary Metabolic Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . 24-7 Additional References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-7 RECENT EMPHASES BIOLOGICAL REACTORS Fermenters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-7 Process Considerations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-10 Oxygen Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-10Sparger Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-11 Scale-Up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-11 Sterilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-13 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-14 Additional References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-15 PRODUCT RECOVERY Additional References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-16 PROCESS MODELING Structured Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-17 Continuous Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-17 Mathematical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-17 Computer Aids for Analysis and Design . . . . . . . . . . . . . . . . . . . . . . . . . 24-18 Plant Cell and Tissue Cultures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-18 Additional References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-19 Recycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-19 Mixed Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-19 Bioprocess Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-20 ENZYME ENGINEERING Enzymatic Reaction Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-21 Immobilized Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-21 Enzymatic Reactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-22 Additional References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-2224-1 24-2 BIOCHEMICAL ENGINEERINGNomenclature and Units Symbol Definition SI units U.S. customary units A Empirical constant Dimensionless Dimensionless C Concentration (mass) kg/m3 lb/ft3 C Concentration mol/m3 (lb⋅mol)/ft3 D Diameter m ft D Effective diffusivity m2/s ft2/h D F/V s−1 h−1 DRT Decimal reduction time for s h sterilization E Activation energy cal/mol Btu/(lb⋅mol) F Flow or feed rate m3/s ft3/h H Concentration of host organisms kg/m3 lb/ft3 K Rate coefficient Units dependent on order of reaction Units dependent on order of reaction KM Michaelis constant kg/m3 lb/ft3 Kla Lumped mass-transfer coefficient s−1 h−1 Kd Death-rate coefficient s−1 h−1 Ks Monod coefficient kg/m3 lb/ft3 k Kinetic constants Dependent on reaction order Dependent on reaction order M Coefficient for maintenance energy Dimensionless Dimensionless N Numbers of organisms or spores Dimensionless Dimensionless P Product concentration kg/m3 lb/ft3 QO2 Specific-respiration-rate coefficient kg O2/(kg organism⋅s) lb O2/(lb organism⋅h) R Universal-gas-law constant 8314 J/(mol⋅K) 0.7299 (ft3)(atm)/(lb⋅mol⋅R) r Radial position m ft S Substrate concentration kg/m3 lb/ft3 S Shear N/m2 lbf/ft2 So Substrate concentration in feed kg/m3 lb/ft3 T Temperature K °F t Time s h V Velocity of reaction mol/s (lb⋅mol)/h Vm Maximum velocity of reaction mol/s (lb⋅mol)/h V Air velocity m/s ft/h V Fermenter volume m3 ft3 VVM Volume of air/volume of fermentation Dimensionless Dimensionless broth per minute X Organism concentration kg/m3 lb/ft3 Y Yield coefficient kg/kg lb/lb Greek symbols β Dimensionless Michaelis constant Dimensionless Dimensionless µ Specific-growth-rate coefficient s−1 h−1 µ̂ or µmax Maximum-specific-growth-rate s−1 h−1 coefficient ω Recycle ratio Dimensionless Dimensionless φ Thiele modulus Dimensionless Dimensionless INTRODUCTION TO BIOCHEMICAL ENGINEERING 24-5chain is usually coiled or folded to provide secondary structure to the molecule, and linkages through other functional groups (mainly disul- fide bonds) form the tertiary structure. For some protein molecules, there may be spatial arrangement forming defined aggregates, known as the quarternary structure of proteins. For a polypeptide polymer to have biological activity a certain molecular arrangement is necessary. This requires not only the primary and secondary but also tertiary and sometimes quarternary structure. Such a strict structural requirement explains the high specificity of proteins. In the presence of certain chemical reagents, excessive heat, radiation, unfavorable pH, and so on, the protein structure may become disorganized. This is called denaturation and may be reversible if not too severe. A special class of proteins, the enzymes, are biological catalysts that expedite reactions by lowering the amount of activation energy required for the reactions to go. An enzyme has an active site that may be thought of as an atomic vise that orients a portion of a molecule for its reaction. The rest of the enzyme is not just an inert glob. Regions that are recognized by antibodies enable living systems to identify and inactivate foreign proteins. Immunological reactions involving antibod- ies are a defense against such foreign proteins. Enzymes function in conjunction with another special class of compounds known as coen- zymes. Coenzymes are not proteins; many of the known coenzymes include vitamins, such as niacin and riboflavin, as part of their molecu- lar structure. Coenzymes carry reactant groups or electrons between substrate molecules in the course of a reaction. As coenzymes serve merely as carriers and are constantly recycled, only small amounts are needed to produce large amounts of biochemical product. Hundreds of metabolic reactions take place simultaneously in cells. There are branched and parallel pathways, and a single biochemical may participate in several distinct reactions. Through mass action, concentration changes caused by one reaction may effect the kinetics and equilibrium concentrations of another. In order to prevent accu- mulation of too much of a biochemical, the product or an intermedi- ate in the pathway may slow the production of an enzyme or may inhibit the activation of enzymes regulating the pathway. This is termed feedback control and is shown in Fig. 24-1. More complicated examples are known where two biochemicals act in concert to inhibit an enzyme. As accumulation of excessive amounts of a certain bio- chemical may be the key to economic success, creating mutant cul- tures with defective metabolic controls has great value to the production of a given product.Glucose Lactose40 30 C on ce nt ra tio n, g /L 20 10 0 0 2 4 6 Time, h 8 10 12 Cell mass FIG. 24-2 Computer simulation of typical diauxic behavior.FIG. 24-1 Feedback control. Product inhibits the first enzyme.Cell efficiency is improved by inhibiting or regulating the synthesis of unneeded enzymes, so there are two classes of enzymes—those that are constitutive and always produced and those that are inducible, i.e., synthesized when needed in response to an inducer, usually the initial substrate in a pathway. Enzymes that are induced in one organism may be constitutive in another. Microorganisms exhibit nutritional preferences. The enzymes for common substrates such as glucose are usually constitutive, as are the enzymes for common or essential metabolic pathways. Furthermore, the synthesis of enzymes for attack on less common substrates such as lactose is repressed by the presence of appreciable amounts of com- mon substrates or metabolites. This is logical for cells to conserve their resources for enzyme synthesis as long as their usual substrates are readily available. If presented with mixed substrates, those that are in the main metabolic pathways are consumed first, while the other substrates are consumed later after the common substrates are depleted. This results in diauxic behavior. A diauxic growth curve exhibits an intermediate growth plateau while the enzymes needed for the uncommon substrates are synthesized (see Fig. 24-2). There may also be preferences for the less common substrates such that a mixture shows a sequence of each being exhausted before the start of metabo- lism of the next.Energy Many metabolic reactions, once activated, proceed spon- taneously with a net release of energy. Hydrolysis and molecular re- arrangements are examples of spontaneous reactions. The hydrolytic splitting of starch to glucose, for instance, results in a net release of energy. But a great many biochemical reactions are not spontaneous and therefore require an energy input. In living systems this require- ment is met by coupling an energy-requiring reaction with an energy- releasing reaction. If a sufficient amount of energy is produced by a metabolic reaction, it may be used to synthesize a high-energy com- pound such as adenosine triphosphate (ATP). When the terminal phosphate linkage is broken, adenosine diphosphate (ADP) and inor- ganic phosphate are formed, and energy is provided. When sufficient energy becomes available, ATP is reformed from ADP. In biological systems, the most frequent mechanism of oxidation is the removal of hydrogen, and conversely, the addition of hydrogen is the common method of reduction. Nicotinamide-adenine dinu- cleotide (NAD) and nicotinamide-adenine dinucleotide phosphate (NADP) are two coenzymes that assist in oxidation and reduction. These cofactors can shuttle between biochemical reactions so that one drives another, or their oxidation can be coupled to the formation of ATP. However, stepwise release or consumption of energy requires driving forces and losses at each step such that overall efficiency suffers. Overall redox potential of a system determines the amount of energy that cells can derive from their nutrients. When oxygen is present to be the ultimate acceptor of electrons, complete oxidation of organic molecules yields maximum energy and usually results in the production of H2O and CO2. However, inside animals, in polluted waters, in the benthos (bottom region) of natural waters, and else- where, there is little or no free oxygen. In these environments, organ- isms develop that can partially oxidize substrates or can derive a small amount of energy from reactions where some products are oxidized while others are reduced. The pathways for complete oxidation may be absent and the presence of oxygen can disrupt the mechanisms for anaerobic metabolism so that the cell is quickly killed. The differences in efficiency are striking: Aerobic metabolism of one molecule of glu- cose can generate bond energy as much as 33 molecules of ATP, while anaerobic metabolism can yield as little as two molecules of ATP. Natural anaerobic processes accumulate compounds such as ethanol, acetoin, acetone, butanol, lactate, and malate. Products of natural aerobic metabolism are water and carbon dioxide, cell mass, and sec- ondary metabolic products such as antibiotics. Photosynthesis All living cells synthesize ATP, but only green plants and a few photosynthetic (or phototrophic) microorganisms can drive biochemical reactions to form ATP with radiant energy through the process of photosynthesis. All photosynthetic organisms contain one or more of the group of green pigments called chlorophylls. In plants, these are contained in organelles called chloroplasts. The num- ber per cell of membrane-surrounded chloroplasts varies with species and environmental conditions. In higher plants, numerous chloro- plasts are found in each cell of the mesophyll tissue of leaves, while an algal cell may contain a single chloroplast. A chloroplast has a sand- 24-6 BIOCHEMICAL ENGINEERINGwich of many layers alternating between pigments and enzymatic proteins such that electromagnetic excitation from light becomes chemical bond energy. Prokaryotic organisms have a unique type of chlorophyll and do not possess chloroplasts organelles. Instead, their photosynthetic systems are associated with the cell membrane or with lamellar structures located in organelles known as chromatophores. Chromatophores, unlike chloroplasts, are not surrounded by a mem- brane. The net result of photosynthesis is reduction of carbon dioxide to form carbohydrates. A key intermediate is phosphoglyceric acid, from which various simple sugars are produced and disproportionated to form other carbohydrates. Mutation and Genetic Engineering Exposing organisms to agents such as mustard chemicals, ultraviolet light, and x-rays increases mutation rate by damaging chromosomes. In strain develop- ment through mutagenesis, the idea is to limit the mutagen exposure to kill about 99 percent of the organisms. The few survivors of this intense treatment are usually mutants. Most of the mutations are harmful to the cell, but a very small number may have economic importance in that impaired cellular control may result in better yields of product. The key is to have a procedure for selecting out the useful mutants. Screening of many strains to find the very few worthy of fur- ther study is tedious and expensive. Such screening that was so very important to biotechnology a few decades ago is becoming obsolete because of genetic improvements based on recombinant DNA tech- nology. Whereas mutagenic agents delete or scramble genes, recombinant DNA techniques add desirable genetic material from very different cells. The genes may come from plant, animal, or microbial cells, or in a few instances they may be synthesized in the laboratory from known nucleic acid sequences in natural genes. Opening a chromosome and splicing in foreign DNA is simple in concept, but there are complica- tions. Genes in fragments of DNA must have control signals from other nucleic acid sequences in order to function. Both the gene and its controls must be spliced into the chromosomes of the receiving cul- ture. Bacterial chromosomes (circular DNA molecules) are cut open with enzymes, mixed with the new fragments to be incorporated, and closed enzymatically. The organism will acquire new traits. This tech- nique is referred to as recombinant technology. There are many tricks and some art in genetic engineering. Exam- ples would be using bacteriophage infection to introduce a gene for producing a new enzyme in a cell. Certain strains of E. coli, B. subtilis, yeast, and streptomyces are the usual working organisms (cloning vec- tors) to which genes are added. The reason for this is that the genetics of these organisms is well understood and the methodology has become fairly routine. ADDITIONAL REFERENCES: Murooka, Y. and T. Imanka (ed.), Recombinant Microbes for Industrial and Agricultural Applications, Dekker, NY, 1993. Glick, B. R. and J. J. Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Herndon, VA, 1994. Bajpai, Rakesh K., and Ales Prokop, eds. Recombinant DNA Technology II, Annals of the New York Academy of Sciences, vol. 721, 1993. CELL AND TISSUE CULTURES Mammalian Cells Unlike microbial cells, mammalian cells do not continue to reproduce forever. Cancerous cells have lost this nat- ural timing that leads to death after a few dozen generations and con- tinue to multiply indefinitely. Hybridoma cells from the fusion of two mammalian lymphoid cells, one cancerous and the other normal, are important for mammalian cell culture. They produce monoclonal antibodies for research, for affinity methods for biological separations, and for analyses used in the diagnosis and treatment of some diseases. However, the frequency of fusion is low. If the unfused cells are not killed, the myelomas will overgrow the hybrid cells. The myelomas can be isolated when there is a defect in their production of enzymes involved in nucleotide synthesis. Mammalian cells can produce the necessary enzymes and thus so can the fused cells. When the cells are placed in a medium in which the enzymes are necessary for survival, the myelomas will not survive. The unfused normal cells will die because of their limited life span. Thus, after a period of time, the hybridomas will be the only cells left alive.A hybridoma can live indefinitely in a growth medium that includes salts, glucose, glutamine, certain amino acids, and bovine serum that provides essential components that have not been identified. Serum is expensive, and its cost largely determines the economic feasibility of a particular culture system. Only recently have substitutes or partial replacements for serum been found. Antibiotics are often included to prevent infection of the culture. The pH, temperature and dissolved oxygen, and carbon dioxide concentration must be closely controlled. The salt determines the osmotic pressure to preserve the integrity of the fragile cell. Most glucose is metabolized to lactate because glycolysis is usually much faster than uptake rate of glycolytic intermediates. Glutamine acts as the primary source of nitrogen as well as providing additional carbon and energy. After glutamine is partially oxidized to glutamate, it can enter the TCA cycle and emerge as pyruvate. It has been esti- mated that between 30 and 65 percent of the cell energy requirement is derived from glutamine metabolism when both glucose and gluta- mine are available. Ammonia is produced in the deamination of gluta- mine to form glutamate and in the formation of alpha-ketoglutarate. Plant Cells and Tissues It is estimated that today some 75 per- cent of all pharmaceuticals originate in plants. Typically, these com- pounds are derived from the secondary metabolic pathways of the cells. When plant or animal cells are cultured, concepts from microbi- ology come into play. Only specialized cells are used, and these can be improved with mutation, selection, and recombinant DNA tech- niques. One very major difference between cell and tissue cultures and most microbiological processes is very high susceptibility to con- tamination by foreign organisms. Most microorganisms grow rapidly and compete well; some are aided by their own changes to the envi- ronment. When a microbial process changes the pH to be far from neutrality or when the product such as ethanol is inhibitory to other organisms, growth of contaminants is discouraged. Cell and tissue cul- tures require rich media and are characterized by slow growth rates. There is seldom any protection by the products of the process. Opti- mum conditions for production of the secondary metabolites are not likely to be the same as for growth. Economics may hinge on a good balance of growing sufficient cells and favoring product formation. Only a few biochemicals derived from plant cell and tissue cultures have high volume/low value products, but some have sizeable markets as specialty chemicals such as dyes, fragrances, insecticides, and pesti- cides. These differ from the low volume/very high value compounds that typify life-saving drugs and pharmaceuticals. Examples for both of these categories are listed in Table 24-1 along with the plant species of origin. Because of cell specialization, some products are produced in cul- tures of those cellular types. Three main classifications of the types of plant cell and tissue cultures are: Undifferentiated cell cultures. Aggregate clumps of cells on solid media (callus) or in liquid media (suspension) Protoplast cultures. Cellular tissues devoid of cell wall material in culture Organ cultures. Differentiated tissues of shoots, roots, anthers, ovaries, or other plant organs in culture Primary Growth Requirements Primary growth is defined as the processes in a plant that are essential for the growth of the meri- stematic regions such as the shoot apex, root tip, and axillary meri- stems. Plant cell and tissue cultures have specific optima for their primary growth in terms of lighting, temperature, aeration, a nutrient medium that must supply a carbon source, vitamins, hormones, and inorganic constituents and with pH typically between 5.5 and 6.5. Aer- ation can be critical depending upon the species.TABLE 24-1 Typical Products Derived from Plants Volume/ Compound Application value Source Shikonin Dye High/low Lithospermum erthrorhizon Warfarin Pesticide High/low Sweet clover Gossypol Pesticide/anti-fertility Low/high Cotton Scopolamine Antispasmodic Low/high Daturastramonium Ajmalicine Circulatory agent Low/high Catharanthusroseus Taxol Anticancer Low/high California yew tree BIOLOGICAL REACTORS 24-7Although a few exceptions do exist where glucose, fructose, or galactose is preferred, the majority of the plant cultures use sucrose. Usual trace requirements are thiamine, niacin, riboflavin, pyridoxine, choline, ascorbic acid, and inositol. Hormones such as auxins and cytokinins promote an undifferentiated state or trigger differentiation into specific plant tissues. As with the whole plant, cellular groups, either differentiated or undifferentiated, require a set of inorganic elements such as nitrogen, phosphorus, potassium, magnesium, cal- cium, sulfur, iron, chlorine, boron, manganese, and zinc. The exact compositions and concentrations of these inorganic elements that are optimum for a particular plant species can be highly variable. How- ever, prepackaged formulations of these salts that can even include the carbon source, vitamins, hormones, and pH buffers are commer- cially available. Secondary Metabolic Requirements A difference between the growth and secondary metabolic phase is that the latter gains importance when approaching the reproductive stages. For example, many of the pigments of flowers are secondary metabolites (e.g., shikonin). Secondary mechanisms are typical responses to stress, such as change in pH (e.g., alkaloid production in Hyocyamus muticus cell cultures is optimum at pH 3.5, while growth is best at 5.0). Similarly, carbon-source concentrations affect Morinda citrifolia cell cultures that grow best at 5 percent sucrose but produce the anthraquinonesecondary metabolites optimally at 7 percent. Temperature changes can cause flowering; several plants require a cold treatment to induce flowering. This is called vernalization. Secondary metabolic pathways in plant cell and tissue cultures seem to be highly controlled by the hormone level in the medium. Another method of eliciting secondary metabolites employs the natural defense mechanisms of the plants that have developed through evolution. For example, gossypol pro- duced by Gossypium hirsutum (cotton) cells is a natural response of the plant when subjected to the infections of the wilt-producing fun- gus Verticillium dahliae. ADDITIONAL REFERENCES: Lambert, K. J. and J. R. Birch, “Cell Growth Media” in Animal Cell Biology, vol. 1, 1985, pp. 85–122. van Wezel, A. L., C. A. M. van der Velden-de Groot, H. H. de Haan, N. van der Heuvel, and R. Schasfoort, “Large-Scale Animal Cell Cultivation for Production of Cellular Biologicals,” Dev. Bio. Stand., 60, 229–236 (1985). Altman, D. W., R. D. Stipanovic, D. M. Mitten, and P. F. Heinstein, In Vitro Cell. Dev. Biol., 21, 659 (1985). Toivonen, L., M. Ojala, and V. Kauppinen, Biotechnol. Bioeng., 37, 673 (1991). Calcott, P. H., Continuous Cultures of Cells, vols. 1 and 2, CRC Press, 1981. Maramorosch, K. and A. H. McIntosh, Insect Cell Biotechnology, CRC Press, Boca Raton, 1994. Endress, R., Plant Cell Biotechnology, Springer- Verlag, Berlin, New York 1994. Morgan, S. J. and D. C. Darling, Animal Cell Culture: Introduction to Biotechniques, BIOS Scientific Pub, 1993. Goosen, M., A. Daugulis, and P. Faukner (eds.), Insect Cell Culture Engineering, M. Dekker, New York, 1993.Commercial use of cell and tissue culture continues to expand. Improvement of organisms through recombinant nucleic acid tech- niques has become commonplace. Formerly, a few laboratories were well ahead of most others, but now the methods have been perfected for routine use. Another technique that is widely practiced is culturing of cells that excrete high concentrations of just one antibody protein. The specificity of antibodies and antigens is exploited in medical test- ing procedures using these pure monoclonal antibodies. Environmental issues are driving several aspects of biotechnology. Sites contaminated by toxic wastes can be cleaned by several alterna- tive methods, but all are expensive. The most certain way to remove toxic materials from soil is to excavate it for incineration, but this requires much labor, energy, and money. Bioremediation in situ tends to be much less expensive on one hand but is slow and uncer-tain on the other. Microbial growth rates approach zero as nutrient levels fall to the low concentrations required for approval of the toxic site remediation. This means that rates tend to be unacceptable when striving for complete removal. Many toxic materials do not support growth of microorganisms. However, they may be degraded as the microorganisms grow on other nutrients. This is termed come- tabolism. Materials that are easily biodegraded could substitute for plastics and other organic chemicals that damage the environment. There has been some progress with natural surfactants produced by microorgan- isms; these would be used in detergents if properties were acceptable and costs were competitive. Biodegradable polymers such as poly- beta-hydroxybutyrate or its derivatives should eventually substitute for polyethylene and polypropylene, but costs are still too high.FERMENTERS The term fermentation formerly distinguished processes from which air was absent, but the term has now been extended to aerobic processes. Bioprocessing is usually aseptic (free of unwanted organ- isms) in vessels held under positive pressure of sterile air to resist entry of contaminating microorganisms. A few processes such as the production of pathogenic organisms for medical purposes or for bio- logical warfare operate below atmospheric pressure because safety of the plant operators is more important than the integrity of the prod- uct. Older processes such as manufacture of pickles had no special measures against contamination, but many of these have been con- verted to aseptic operations to prevent impairment of product quality by foreign organisms. Biological waste treatment employs elective cul- tures of microorganisms in relatively crude, open equipment. Activities associated with bioreactors include gas/liquid contacting, on-line sensing of concentrations, mixing, heat transfer, foam control, and feed of nutrients or reagents such as those for pH control. The workhorse of the fermentation industry is the conventional batch fer- menter shown in Fig. 24-3. Not shown are ladder rungs inside the ves- sel, antifoam probe, antifoam system, and sensors (pH, dissolved oxygen, temperature, and the like). Note that coils may lie between baffles and the tank wall or connect to the top to minimize openingsbelow the water level, and bottom-entering mixers are used fre- quently. There is extensive process piping, and copper or brass fittings are taboo for some processes because of highly deleterious effects of copper (more than 50 percent reduction in yield has been noted in penicillin fermentations when a bronze valve was in a feed line). Cool- ing coils must be used for larger tanks because the heat-transfer area of a jacket is inadequate for cooling from sterilization temperature to operating temperature in a reasonable time. Some features of interest for a conventional fermenter are that (1) a bypass valve in the air sys- tem allows diversion of air so that foaming is not excessive and the redox potential is not too high during the early stage of fermentation when the inoculum is becoming established; (2) antifoam is added when excessive foam reaches a conductive or capacitive electronic probe; (3) all piping is sterilized by the use of steam and is protected by steam until put into use; (4) the level of liquid when filling the ves- sel is determined by reference to a calibration chart based on points in the tank such as a rung on the ladder; (5) the weight of the tank con- tents can be determined by the hydrostatic balance against air bub- bled slowly through the sparger; and (6) pumps are very uncommon because it is so easy to force fluid from a pressurized vessel. The need for highly cost-efficient oxygen transfer in fermentations such as those with hydrocarbon feedstocks has led to air-lift fermenters as shown in Fig. 24-4. The world’s largest industrial fermenter wasRECENT EMPHASESBIOLOGICAL REACTORS 24-10 BIOCHEMICAL ENGINEERINGTABLE 24-2 Comparison of Ethanol Fermenters Typical time Typical ethanol System (h) concentration (%) Conventional 72 10 Cell recycle 12 8 Tower fermenter 3 8 Gel immobilization 1 10FIG. 24-13 Sample-line piping. (A valve to the sewer allows bypass of the trap while cooling the line.)PROCESS CONSIDERATIONS Fermentation can be combined with other operations. For example, feedback inhibition of enzymatic hydrolysis of cellulose can be re- lieved by removal of the product glucose by fermentation as it forms. This is termed simultaneous-saccharification-fermentation (SSF). Valves and pumps that have a potential path for contaminating organisms are taboo for aseptic operations. Rising stem valves could bring organisms to the sterile side by the in and out motion as the valve operates. Diaphragm valves are still commonly used, but heat- ing, cooling, and the abrasion by solids in the nutrient media are somewhat severe conditions leading to occasional rupture of a diaphragm and contamination of a run. Ball valves or plug valves do not have an absolute seal to the outside, but the direction of motion does not tend to bring organisms in. Contamination is seldom attrib- uted to these valves; they are designed for easy maintenance in place, and there is the very nice human advantage that a glance at the handle tells easily whether the valve is open or closed. Many runs have been spoiled or impaired because a manual valve was left in the wrong posi- tion. For plant operations, pumps with diaphragms are satisfactory. In the lab or pilot plant, peristaltic pumps (also known as tubing squeez- ers) predominate. Transfer of fluid in a fermentation plant usually makes use of air pressure differences. One or more manifold headers may intercon- nect many vessels. As transfers may have to be aseptic, headers are pressurized with steam until needed. A typical arrangement of steam seals is shown in Fig. 24-12. Sample lines commonly have steam seals too. A typical layout is shown in Fig. 24-13. In the closed position, steam provides an absolute barrier to contamination. To take a sample, the steam line and the trap line are closed, and fermentation medium is flowed to waste until the pipes are cool to the touch so that sensitive products do not give false assays because of thermal destruction. Cooling takes up to 5 liters of medium if not done carefully, and bad practices can waste considerably more. Pilot-sized tanks have less massive fittings that are easier to cool; less medium is wasted, but oversampling to the point where the fermenter volume is low can be a problem. For this reason, alternate sampling methods have been devised. For example, a sterile syringe and needle may be used to sample through a rubber diaphragm in the wall of the tank. Although such methods appear reli- able, there is a tendency to scrap all innovations and to return to tried and true steam seals when the factory encounters any period of con- tamination.FIG. 24-12 Inoculation and harvest header.All piping to a fermenter is flushed with steam during the steriliza- tion period. A clever means for sight-glass cleaning uses steam con- densate that is naturally sterile in a dead leg. Steam pressure behind the condensate forces this water to the sight glass. Without cleaning, splashing and spray can quickly cover the sight glass with a thick coat- ing of microorganisms and medium. While it is easy to add materials to a fermentation, removal is diffi- cult. Membrane devices have been placed in the fermenter or in external recycle loops to dialyze away a soluble component. Cells release wastes or metabolites that can be inhibitory; these are some- times referred to as staling factors. Their removal by dialysis has allowed cell concentrations to reach ten to one hundred times that of control cultures. Solid substrates such as pulverized wood cannot be stirred when slurry concentration exceeds about 5 percent. For saccharification prior to ethanol fermentation, keeping sugar concentration high can avoid an evaporation step. In a batch reactor, mixing limitations with the wood results in a dilute sugar solution. This has been circum- vented by placing the wood in a column and percolating the solution through. As wood dissolves, more is added. Simultaneous fermenta- tion of the sugars formed in the column is possible. Oxygen Transfer Supplying sufficient oxygen can be a very chal- lenging engineering problem for some aerobic fermentations. Oxygen is sparingly soluble in water; saturation with pressurized air at room temperature provides only 6 or 7 milligrams per liter of oxygen. A vig- orous process can deplete the dissolved oxygen in several seconds when aeration is stopped. Mass transfer of gases to liquids is covered in Sec. 5. Emphasis is somewhat different for biological systems that commonly have bubble aeration. Because the number and size of bubbles is very difficult to estimate, transfer area is usually lumped with the mass-transfer coefficient as a Kla term. The “l” subscript in Kl signifies that liquid film resistance should greatly predominate for a sparingly soluble gas such as oxygen. The relationship between oxygen concentration and growth is of a Michaelis-Menten type (see Fig. 24-20). When a process is rate-limited by oxygen, the specific respira- tion rate (QO2) also increases steeply with dissolved oxygen concentra- tion until a plateau is reached. The concentration below which respiration is severely limited is termed the critical oxygen concentra- tion, which typically ranges from 0.5 to 2.0 ppm for well-dispersed bacteria, yeast, and fungi growing at 20 to 30°C (68 to 86°F). Above this critical concentration, the specific oxygen uptake increases only slightly with increasing oxygen concentrations. A plot of the specific respiration rate QO2 versus the specific growth rate coefficient µ is linear, with the intercept on the ordinate equal to the oxygen uptake rate for cell maintenance. A formulation of this is: Uptake rate = uptake for maintenance + uptake for growth or QO2X = (24-1) where X is organism concentration, the subscript M denotes mainte- nance, Yg is yield of cell mass per mass of oxygen, and the Q terms sig- nify oxygen uptake rates in mass O2 per mass of organisms. This type of correlation applies to almost any substrate involved in cellular energy metabolism and is supported by experimental data and energetic considerations. However, it is based on assumptions true at or near the steady-state equilibrium conditions and may not be valid (QO2)MX + µX }} Yg BIOLOGICAL REACTORS 24-11during transient states. The oxygen-uptake equation should be modi- fied when other cellular activities requiring oxygen can be identified. For example, use of oxygen for product formation would be repre- sented by: Uptake rate = maintenance uptake + growth uptake + product uptake or QO2X = (QO2)MX + + (24-2) where P is the product concentration and Y is the yield of product per unit weight of limiting nutrient. Oxygen uptake is distributed between that for growth and that for cellular activities dependent on cell con- centration. As the oxygen transfer rate under steady-state conditions must equal oxygen uptake, Kla may be calculated: Kla = (24-3) where C* = concentration of oxygen in the liquid that would be in equilibrium with the gas-bubble concentration and Kla = the volumet- ric oxygen transfer rate. A convenient method for measuring oxygen transfer rates in micro- bial systems depends on dissolved oxygen electrodes with relatively fast response times. Quite inexpensive oxygen electrodes are available for use with open systems, and steam-sterilizable electrodes are avail- able for aseptic systems. There are two basic types: One develops a voltage from an electrochemical cell based on oxygen, and the other is a polarographic cell whose current depends on the rate at which oxy- gen arrives. See Fig. 24-14. Measurement of oxygen transfer proper- ties requires only a brief interruption of oxygen supply. A mass balance for oxygen is: = rate of supply − uptake rate (24-4) A tracing of the electrode signal during a cycle of turning aeration off and on is shown in Fig. 24-15. The rate of supply is zero (after bub- bles have escaped) in the first portion of the response curve; thus, the slope equals the uptake rate by the organisms. When aeration is resumed, both the supply rate and uptake rate terms apply. The values for C* − C can be calculated from the data, the slope of the response curve at a given point is measured to get dC/dt, and the equation can be solved for K la because all the other values are known. Measurements of the rate of change in concentration of oxidizable chemicals in aerated vessels have questionable value for assessing rates with biological systems. Not only are flow patterns and bubble sizes different for biological systems, but surface active agents and dO } dt overall oxygen uptake rate }}} (C* − C)mean 1 } YP dP } dt 1 } YG dX } dt8 7 D is so lv ed o xy ge n, m g/ L 6 5 4 3 0 1 2 0 20 Time, s 40 60 Air on Air off FIG. 24-15 Computer simulation of response for dynamic measurement of K la.FIG. 24-14 Dissolved-oxygen electrodes: (a) polarographic (impress break- down voltage for oxygen; measure current); (b) voltametric (measure electro- motive force). (a) (b)suspended particles can seriously impair gas transfer. Fig. 24-16 shows the effects of various particles. In general, spherical particles have a small effect, elongated particles have more effect, and entangled par- ticles markedly impair transfer. As many mold cultures are inter- twined and lipids and proteins are present with strong surface activity, oxygen transfer to a fermentation can be much slower than that to simple aqueous solutions. Except as an index of respiration, carbon dioxide is seldom consid- ered in fermentations but plays important roles. Its participation in carbonate equilibria affects pH; removal of carbon dioxide by photo- synthesis can force the pH above 10 in dense, well-illuminated algal cultures. Several biochemical reactions involve carbon dioxide, so their kinetics and equilibrium concentrations are dependent on gas concentrations, and metabolic rates of associated reactions may also change. Attempts to increase oxygen transfer rates by elevating pres- sure to get more driving force sometimes encounter poor process performance that might be attributed to excessive dissolved carbon dioxide. Sparger Systems Gas distributors in tanks are shown in Sec. 6. Large openings are desirable for spargers in industrial fermentations to avoid clogging by microbial growth, but the diameter is usually designed for the acoustic velocity that insures small bubbles. Rela- tively small holes or diffusers are used in activated sludge units for biological waste treatment, but there is commonly a means for swing- ing a section of the aerator out of the vessel for cleaning. Newer designs for fermenters were conceived as answers to the problems of oxygen transfer. The air lift fermenter (Fig. 24-4) creates intimate mixing of air and medium while using the buoyancy of the gas to mix the fluid. Surface active substances also lead to foaming that can be so bad that most of the contents of the fermenter are lost. Mechanical antifoam devices are helpful but cannot function alone except when there is little propensity for foaming. The mechanical foam breakers rupture the large, weak bubbles while allowing tiny, rugged bubbles to accumulate. Surface active antifoam agents tend to reduce elasticity of the bubbles so that mechanical shocks are easily transmitted to encourage rupture. Several antifoam delivery systems are shown in Fig. 24-17. Some lipids used as antifoams are metabolized by the cul- ture and must be replaced. The nutrition supplied by these oils may be beneficial, but they are much more expensive than their equiva- lents in carbohydrate nutrients. Furthermore, it is troublesome to have nutrition coupled to foam control. Several synthetic antifoam agents are not nutrients and tend to persist. Their tendency to be lost by coating solid surfaces in the fermenter means that more must be added occasionally. These synthetic antifoam agents are toxic to some organisms, but one of the many types is usually satisfactory. Scale-Up Fermenters ranging from about two to over 100 liters (0.07–3.5 ft3) have been used for research and development, but the smaller sizes provide too little volume for sampling and are difficult to replicate, while large vessels are expensive and use too much medium. Autoclavable small fermenters that are placed in a water bath for tem- perature control are less expensive than vessels with jackets or coils, but much labor is required for handling them. Pressure vessels that 24-12 BIOCHEMICAL ENGINEERINGFIG. 24-16 Effect of solids on K la. Operating conditions: agitator speed, 800 r/min; air flow, 2.5 min. [M. R. Brierly and R. Steel, Appl. Microbiol., 7, 57 (1959). Courtesy of American Society for Microbiology.]are sterilized in place are more convenient, but initial investment is high. Judgement is needed to select the most economical equipment and to plan for cost-effective experimentation. A suitable means of scale-up for aerobic processes is to measure the dissolved oxygen level that is adequate in small equipment and to adjust conditions in the plant until this level of dissolved oxygen is reached. However, some antibiotic fermentations and the production of fodder yeast from hydrocarbon substrates have very severe require- ments, and designers are hard-pressed to supply enough oxygen. Older methods of fermentation scale up insisted on geometric sim- ilarity based on proportional physical dimensions. It was thought that applying the same power per unit volume as in the pilot equipment would give an equivalent process performance in large fermenters. Antibiotic fermentations aim for mixer power in the range of 0.2 to 4 Kw/m (0.1 to 2 HP/100 gal). As mixing devices have areas (dimen- sions squared) to supply a volume (dimensions cubed), methods based on dimensional similarity are fundamentally unsound. Scale-up based on equivalent oxygen transfer coefficient K la has been reasonably suc- cessful.Impeller Reynolds number and equations for mixing power for par- ticle suspensions are in Sec. 5. Dispersion of gasses into liquids is in Sec. 14. Usually, an increase in mechanical agitation is more effective than is an increase in aeration rate for improving mass transfer. Other scale-up factors are shear, mixing time, Reynolds number, momentum, and the mixing provided by rising bubbles. Shear is max- imum at the tip of the impeller and may be estimated from Eq. (24-5), where the subscripts s and l stand for small and large and Di is impeller diameter [R. Steel and W. D. Maxon, Biotechnol. Bioengr., 4, 231 (1962)]. Ss = Sl 1 2 1/3 (24-5) Some mycelial fermentations exhibit early sporulation, breakup of mycelium, and low yields if the shear is excessive. A tip speed of 250 to 500 cm/s (8 to 16 ft/s) is considered permissible. Mixing time has been proposed as a scale-up consideration, but little can be done to improve it in a large fermenter because gigantic motors would be required to get rapid mixing. Culturing cells from plants or animals is beset by mixing problems because these cell are easily damaged by shear. Constant Reynolds number is not used for fermentation scale-up; it is only one factor in the aeration task. This is also true for considering the impeller as a pump and attempting scale-up by constant momen- tum. As mechanical mixing tends to predominate over bubble effects in improving aeration, scale-up equations including bubble effects have had little use. Fermentation biomass productivities usually range from 2 to 5 g/(l⋅h). This represents an oxygen demand in the range of 1.5 to 4 g O/(l⋅h). In a 500-m fermenter, this means achievement of a volumet- ric oxygen transfer coefficient in the range of 250 to 400 h−1. Such oxy- gen-transfer capabilities can be achieved with aeration rates of the order of 0.5 VVM (volume of air at STP/volume of broth) and mechanical agitation power inputs of 2.4 to 3.2 Kw/m (1.2 to 1.6 HP/ 100 gal). Often heat removal causes design problems for scale-up. Mechani- cal agitation coupled with a metabolic heat from the growing biomass Dis } DilFIG. 24-17 Antifoam systems. PRODUCT RECOVERY 24-15Improvements in fermentation include microcarriers that not only provide support for anchorage-dependent cells but also aid in har- vesting at the end of a run. While microcarriers based on dextran, polystyrene and polyacrylamide beads have been widely used in the past, new materials for microcarriers make separating cells from their growth medium easier. Some are made of collagen that is detached from the cells by immersion in dilute collagenase. However, since some collagen can be left in solution, downstream processing can be made difficult. An alternative is to use plastic beads coated with colla- gen. The plastic can be easily separated after the cells are released enzymatically. Another alternative is glass-coated particles that induce attachment of the cells’ long slender filopodia. At the end of a process, a brief incubation in dilute trypsin gently removes the cells from the beads. Supplying sufficient oxygen can be difficult when dealing with dif- ferentiated plant tissues such as root cultures that can reach lengths of several decimeters and can be highly branched and complex struc-turally. In this case, mechanical agitation is impractical because the mass of roots can occupy approximately 50 percent of the reactor vol- ume. Alternatives are bubble columns, rotating drums, and trickle bed reactors where the medium is recycled and sprayed over the column of roots. Because of the differences in primary and secondary metabolism, a reactor may have a dual-stage fed-batch system. In other words, fed- batch operation optimizes growth with little or no product formation. When sufficient biomass has accumulated, a different fed-batch pro- tocol comes into play. ADDITIONAL REFERENCES: Asenjo, J. A., and J. C. Merchuck, Bioreactor Sys- tem Design, Dekker, New York, 1994. Rehm, H.-J., and G. Reed, Biotechnology, vol. 6b, VCH Verlagsgesellschaft, 1988. Chang, H. N., “Membrane Bioreactors, Engineering Aspects,” Biotechnol. Adv., 5, 129–145 (1987). Cheryan, M. and M. A. Mehaia, “Membrane Bioreactors” in McGregor, W. C. (ed.), Membrane Separations: Biotechnology, Marcel Dekker, New York, 1989. Heath, C. A. and G. Belfort, “Membranes and Bioreactors,” Int. J. Biochem., 22(8), 823–835 (1990).Although most of the purification equipment in a large biotechnolog- ical factory is the same as that used throughout the chemical process industries, there are fewer separations in which the product reaches elevated temperatures. Most biochemicals are destroyed if heated. Recovery of products from the bioprocess fluid can be more difficult and expensive than all of the previous steps. The ratio of recovery costs to cost of creating the product can range from about one to more than ten because the investment for the recovery facilities may be sev- eral times that for the fermenter vessels and their auxiliary equip- ment. As much as 60 percent of the fixed costs of fermentation plants for organic acids or amino acids is attributable to the recovery section. The costs for recovery of proteins based on recombinant DNA tech- niques are particularly high. Research and development for better recovery of existing products may provide diminishing returns as time passes, and some companies focus on new products. Government regulations are different for drugs that are sold in very nearly pure state and for biologicals that may be ill-defined. Little paperwork is required for a process improvement for a pure drug. For biologicals intended for humans, securing government approval makes it unwise to modify the recovery process except when the potential savings are very great. The exten- sive and expensive testing and validation of such therapeutic agents is keyed to the processes for making and purifying them. Only trivial changes are permitted; otherwise the testing must be repeated. Mar- ket forces make it important to have a new product tested and ready for sale as quickly as possible, but this usually means that the process has not been optimized. Whereas competition and process improve- ments resulted in remarkable lowering of prices in the past, the regu- lations now discourage investment in process development other than at the early stages prior to submission of the documents for govern- ment approval. Certain products (e.g., inclusion bodies) are contained inside the cells and are not released or only partially released to the medium. It may be possible to flush impurities from the cells before breaking them to get the product. Cell disruption is a unit operation peculiar to biochemical engineering. The equipment, however, may be borrowed from other industries. Colloid mills and shear devices used for manu- facturing paint and other products effectively rupture walls of many types of cells. Cells with high resistance to shear can be passed through the unit several times, but heat generation from the process can cause loss of product. Shear alone can denature sensitive proteins. Ultrasonic energy is commonly used on a small scale for cell disinte- gration but is impractical for large batches. Grinding with sand or beads, high-pressure pumping through a tiny orifice, freezing and thawing, dessication, adding lytic enzymes, inducing autolysis with a chemical such as chloroform, and various means of creating shear are alternative or synergistic means of rupturing cells. There are encour-aging results with special mutants of cells with impaired ability to form cell walls at temperatures slightly above their normal growth temper- atures. When shifted to the elevated temperature, cell division gives damaged walls and lets the cell contents leak out. The bioprocess operates at its optimum temperature until the temperature is raised shortly before harvest. Cells that form at the elevated temperature release their contents easily. Whenever possible, the fermentation fluid goes directly to ion exchange, solvent extraction, or some other step. However, prior removal of biological cells and other solids is usually necessary. Cen- trifugation can be considered, but rotary drum filters with string dis- charge are commonly used. In the past, large amounts of filter aid were added because many fermentation broths are slimy and hard to filter. Present practice employs polymeric bridging agents to agglom- erate the solids. This allows good filtration with only small amounts of filter aid. The most popular steps for recovery are ion exchange or solvent extraction because selectivity is good, costs are reasonable, and large scale is feasible. Unfortunately, some biochemicals neither exchange ions nor extract well. These and other purification steps can be affected by modifications in fermentation. Adding excess lipids or antifoam oils to a fermentation can aggravate emulsion problems for solvent extraction or impair ion exchange by coating the resin. Stabil- ity of biochemical products can be troublesome. For example, peni- cillin fermentation broth is acidified just prior to contact with the extracting solvent because low pH causes very rapid destruction of penicillin in water. The most popular immiscible solvent is methyliso- butylketone (MIBK); halogenated hydrocarbons are avoided because of their hazard for humans. Penicillin is extracted back into an aque- ous phase at pH 7.5 to 8 using bicarbonate as a buffer because harsher agents are difficult to control. The Podbelniak design of centrifuge (see Sec. 15) is widely used in the United States for extraction of fer- mentation broths, while the Westphalia design is common in Europe. Countercurrent flow through a centrifuge should result in more than one equilibrium contact, but emulsions may carry product into the phase being wasted. Lead-and-trail operation of the centrifuges can improve yields. Extraction back into water is seldom troubled by emulsions, and DeLavalle separators work well. Some products are precipitated from the fermentation broth. The insoluble calcium salts of some organic acids precipitate and are col- lected, and adding sulfuric acid regenerates the acid while forming gypsum (calcium sulfate) that constitutes a disposal problem. An early process for recovering the antibiotic cycloserine added silver nitrate to the fermentation broth to precipitate an insoluble silver salt. This process was soon obsolete because of poor economics and because the silver salt, when dry, exploded easily. Of the various types of purification steps based on sorption to aPRODUCT RECOVERY 24-16 BIOCHEMICAL ENGINEERINGsolid phase, ion exchange is the most straightforward. See Sec. 22 for a discussion of techniques. Ion-exchange resins actually adsorb Vita- min B12 well instead of exchanging it. Carbon adsorption has consid- erable importance to biochemical engineering, primarily for the removal of traces of colored impurities. When neither solvent extrac- tion nor ion exchange can be used as the primary concentration/purifi- cation step because of the chemical properties of the desired product, an alternative may be adsorption on carbon. Although carbon adsorp- tion may be unselective and low in capacity, it can provide a roughing step to get the purification scheme started. Isolation procedures for many biochemicals are based on chroma- tography. Practically any substance can be selected from a crude mix- ture and eluted at relatively high purity from a chromatographic column with the right combination of adsorbent, conditions, and elu- ant. For bench scale or for a small pilot plant, such chromatography has rendered alternate procedures such as electrophoresis nearly obsolete. Unfortunately, as size increases, dispersion in the column ruins resolution. To produce small amounts or up to tens of kilograms per year, chromatography is an excellent choice. When the scale-up problem is solved, these procedures should displace some of the con- ventional steps in the chemical process industries. Affinity chromatography uses ligands with high specificity for certain compounds. There are several types of affinity that can be employed: antigen-antibody, enzyme-substrate, enzyme-cofactor, chelation with metal ions, or special biochemical attractions such as the protein avidin for the vitamin biotin. Numerous purifications have been devised wherein affinity chromatography is able to isolate quite pure product from a very crude mixture. Expensive affinity agents are regenerated and reused many times. In some cases, the attraction is so strong that the adsorbent can be added batchwise. This scales up well but is less convenient than column operations in terms of collection, elution, and regeneration of the affinity agent. Conventional elution chromatography has the serious disadvantage of dilution, and usually a concentration step must follow. The tech- nique of displacement chromatography circumvents dilution and may even result in an eluant more concentrated than the feed. A displacer compound breaks the desired product from the chromatographic material sharply, and a column heavily loaded with several biochemi- cals will release them one at a time depending on their adsorption equilibria. However, the displacers tend to be expensive and can be troublesome to remove from the product. A number of water-soluble polymers will cause phase separation when present together at concentrations of a few percent. The most widely used polymers are polyethylene glycol (PEG) and dextran. Pro- teins, other macromolecules, and cell components such as mitochon- dria distribute in the phases or collect at the interface. Proteins are destabilized at organic solvent/water interfaces, but when each solvent is water, the interfacial tension is negligible. Some salts such as potas- sium phosphate will also induce phase separation when a polymer is present, but the salt concentration must be high. Two-phase aqueous systems provide a mild method for purification of proteins, and scale- up to large volumes presents no engineering problems. The polymerscan have functional groups that improve distribution coefficients of the biochemical products, but the costs for these polymers are high. Although highly promising, two-phase aqueous methods are used only for valuable products because the cost of the polymers is too high and they are not easily recovered for reuse. Another drawback is distribu- tion coefficients not far from 1 for most proteins; several extraction stages are needed to get acceptable yields when the distribution coef- ficients are unfavorable. Surface-active agents and liquids immiscible in water can form tiny dispersed units called reverse micelles. These can extract biochemi- cals from water or permit complexing or reacting in ways not possible in simple aqueous systems. Crystallization is the preferred method of forming many final prod- ucts because very high purification is possible. High purity antibiotic crystals can be produced from colored, rather impure solutions if the fil- ter cake is uniform and amenable to good washing to remove the mother liquor. When a sterile pharmaceutical product is desired, crystals are formed from liquid streams that have been sterilized by filtration. ADDITIONAL REFERENCES: Belter, P. A., E. L. Cussler, and W.-S. Hu, Biosep- arations: Downstream Processing for Biotechnology, Wiley, New York, 1988. Li, N. N., and J. M. Calo (ed.), Separation and Purification Technology, Dekker, New York, 1992. Harrison, R. G. (ed.), Protein Purification Process Engineering, Dekker, New York, 1993. Zaslavsky, B. Y., Aqueous Two-Phase Partitioning: Physical Chemistry and Bioanalytical Applications, Dekker, New York, 1994. Belfort, G., Synthetic Membrane Processes: Fundamentals and Water Applica- tions, Academic Press, New York, 1984. Belfort, G. “Membranes and Bioreac- tors: A Technical Challenge in Biotechnology,” Biotechnol. Bioeng., 33, 1047–1066, 1989. Brandt, S., R. A. Goffe, S. B. Kessler, J. L. O’Connor, and S. E. Zale, Membrane-Based Affinity Technology for Commercial Scale Purifi- cations, Bio/Technology, 6, 779, 1988. Hanisch, W., “Cell Harvesting” in McGregor, W. C. (ed.), Membrane Separations in Biotechnology, Marcel Dekker, New York, 1986. Heath, C. A., and G. Belfort, “Synthetic Membranes in Biotechnology: Realities and Possibilities,” Advances in Biochem. Engr. and Biotechnol., 47, 45–88, 1992. Klein, E., Affinity Membranes, John Wiley & Sons, New York, 1991. Matson, S. L., and J. A. Quinn, “Membrane Reactors in Bioprocessing” in Biochemical Engineering IV, vol. 49, New York Academy of Sciences, New York, 1986. Mattiasson, G., and W. Ramstorp, “Ultrafiltration Affinity Purification” in Biochemical Engineering III: Annals of the New York Academy of Sciences, vol. 413, 1983. Crespo, Jaoa, and Karl Boddeker (eds.), “Membrane Processes” in Separation and Purification, Kluwer Academic Pub- lishers, The Netherlands, 1994. Michaels, A. S., “Membranes, Membrane Processes and Their Applications: Needs, Unsolved Problems and Challenges of the 1990s,” Desalination, 77, 5–34, 1990. Schugerl, K., Solvent Extraction in Biotechnology: Recovery of Primary and Secondary Metabolites, Springer- Verlag, Berlin, New York, 1994. Mattiasson, B., and O. Holst (eds.), Extractive Bioconversions, Marcel Dekker, New York, 1991. Asenjo, J. A. (ed.), Separation Processes in Biotechnology, Marcel Dekker, New York, 1990. Ladisch, M. R. (ed.), Protein Purification: From Molecular Mechanisms to Large-Scale Processes, Am. Chem. Soc. Div. of Biochemical Technol., Washington, DC, 1990. Dechow, F. J., Separation and Purification Techniques in Biotechnology, Noyes Publications, Park Ridge, New Jersey, 1989. Verrall, M. S., and M. J. Hudson (eds.), Separations for Biotechnology, Ellis Harwood, Wiley, New York, 1987. McGregor, W. C. (ed.), Membrane Separations in Biotechnology, Dekker, New York, 1986. Ataai, M. M., and S. K. Sikdar (eds.), New Developments in Bioseparation, AIChE Symposium Series, vol. 88, New York, 1993.It is generally assumed that properties of very large numbers of cells can be treated as continuous functions having average properties because there are so many cell divisions occurring that the overall rates follow smooth curves. There is an exception in which the cells can all be induced to divide at the same time because events such as illumination or temperature changes slow or halt a step in division. The cells can be triggered to proceed together from that point with overall numbers that are stepwise with time. This is termed a syn- chronous culture; the steps are seldom distinct for more than a few generations unless the triggering event continues to be applied peri- odically.Mass balances for common, unsynchronized batch culture give: = µX − KdX (24-10) = − (24-11) µ = f(S) (24-12) Various functional relationships between µ and S have been proposed, but the Monod equation is used almost exclusively: µX } Y dS } dt dX } dtPROCESS MODELING PROCESS MODELING 24-170.5 S pe ci fic g ro w th r at e co ef fic ie nt , h –1 0 0 Concentration of limiting nutrient, g/L 5 FIG. 24-20 Plot of the Monod equation.instability of the culture itself. There is a tendency to revert to less productive strains that quickly replace the finely tuned mutants that achieve high titers of product. The main successes with continuous fermentation have been with rugged strains that are producing either cell mass for cattle feed or a simple enzyme or metabolite. When a sin- gle stage is used for a product that is elaborated from cells that are not growing, it is difficult to optimize simultaneously cell growth, product production, and efficient use of substrates. In view of its few industrial applications, continuous culture gets a disproportionate amount of attention from academicians. As a research tool, batch culture suffers from changing concentrations of products and reactants; varying pH and redox potential; and a compli- cated mix of growing, dying, and dead cells. Data from continuous cul- tures are much easier to interpret because steady states are achieved or there are repeatable excursions from steady state. The usual explana- tions for limited use of continuous culture in industry are: culture instability, difficulty of maintaining asepsis, insufficient knowledge of microbial behavior, and reluctance to convert existing factories. Over- all cost savings can be relatively small for continuous cultivation because productivity of the bioreator is not very important compared to high product concentration. Another factor is the cost of each research station. Rapid progress in research and development requires multiple vessels for screening many variables, but there are usually only one or two continuous fermenters in the lab or pilot plant because the cost of pumps, reservoirs, sterilizers, and controls is relatively high. Conventional means for continuous culturing are the chemostat in which nutrient is fed to a reactor at constant rate and the turbidostat that employs feedback control of pumping rate to maintain a fixed tur- bidity of the culture. Another alternative with feedback control of a nutrient or product concentration has been termed auxostat, nustat, or nutristat. Proportional control of the pumping rate is desirable because continuous cultures can have oscillatory responses induced by turning the feed pump on or off. A chemostat tends to be unstable and erratic at dilution rates that approach the maximum specific growth rate of the organisms. This is explained by the adjustment of growth rate to nutrient concentration in the region where a small change in dilution rate equates to a big change in nutrient concentra- tion. An auxostat has little advantage over a chemostat at moderate dilution rates but is stable at the high dilution rates at which the chemostat is unreliable. MATHEMATICAL ANALYSIS The concept of a limiting nutrient is essential to the theory of contin- uous culture. There will only be exact stoichiometric balance of all the ingredients going into the cells when a very deliberate and time- consuming effort has been made to determine the details of cell nutri- tion. Even then, there may be a different balance if the growth rate is changed or kinetic rather than stoichiometric limitations may apply. The ingredient in short supply relative to the other ingredients will be exhausted first and thus limit cellular growth or product synthesis. The other ingredients may exhibit toxicity or influence cellular activi- ties, but there will not be acute shortage as in the case of the limiting nutrient. Mass balances for one vessel in a series of continuous fermenters give: Rate of change = rate in − rate out + rate of production or = FXn − 1 − FX + VµvXn (24-14) Dividing through by V and substituting D = F/V: = D(Xn − 1 − Xn) + µnXn (24-15) and = FSn − 1 − FSn − − VMXn (24-16) = D(Sn − 1 − Sn) − − MXn (24-17) µnXn } Y dSn } dt VµnXn } Y VdSn } dt dXn } dt VdXn } dtµ = µmax × (24-13) A graph of the Monod equation is shown as Fig. 24-20. The death rate coefficient is usually relatively small unless inhibitory substances accumulate, so Eq. (24-10) shows an exponen- tial rise until S becomes depleted to reduce µ. This explains the usual growth curve (Fig. 24-21) with its lag phase, logarithmic phase, resting phase, and declining phase as the effect of Kd takes over. Structured Models Meaningful detail can be added to culture models in several ways. Cells can be compartmentalized according to biochemical functions, and the components can interact. For exam- ple, there can be a group of equations for carbohydrate metabolism, a group for protein synthesis, another for nucleic acid synthesis, and so on. This permits a much more intricate description of cell activities but at the expense of having so many rate constants that assigning val- ues to them may end up as guesswork. For cells with distinct life cycles, a structured model may have compartments corresponding to each stage in the cycle. In addition, each compartment may be subdi- vided into the biochemical functions mentioned above. Such compli- cated models have had limited practical use but have great value for directing research toward areas where information is lacking. Continuous Culture Continuous culture has been a goal of bio- engineers for several decades because batch culture has inherent down time for cleaning and sterilization and long lags before the organisms enter a brief period of high productivity. Continuous runs can last many weeks, but there must be stoppages for cleaning and maintenance. Bacteria may foul surfaces to a small extent, but molds tend to form thick coatings on the shaft, coils, and any protuberances in the fermenter after several weeks of continuous cultivation, that seriously impair mixing and mass transfer. The nutrition and the product mix can be advantageously manipu- lated as functions of dilution rate. A serious problem, however, is S } Ks + SC on ce nt ra tio n, g /L 40 30 20 10 0 0 Time, h 24 Cells Nutrient FIG. 24-21 Microbial growth curve. dx/dt = µX − KdX; ds/dt = −µX/Y; µ̂ = µmax S/(Ks + S); µmax = 0.35; Kd = 0.025; Ks = 12.0 mg/L; Y = 0.48. 24-20 BIOCHEMICAL ENGINEERINGTABLE 24-5 Mixed Culture Processes Process Types of organisms Commercial Alcoholic beverages Various yeasts, molds, and bacteria Sauerkraut L. plantarum plus other bacteria Pickles L. plantarum plus other bacteria Cheeses Propionibacteria, molds, and possibly many other microorganisms Lactic acid Two lactobacillus species Waste treatment Trickling filters Zoogloea, protozoa, algae, fungi Activated sludge Zoogloea, Sphaerotilus, yeasts, molds, protozoa Sludge digestion Cellulolytic and acid-forming bacteria, methanogenic bacteria Sewage lagoons Many types from most microbial familiesAnother interaction with grave consequences is attack on a species by a phage (microbial virus) that is usually highly specific. Infection of a cell by a virulent phage results in the production of 10 to several hundred new phage particles as phage nucleic acid takes over control of cellular activities. The cell disintegrates and releases phage that infect other cells to reach high phage titers quickly. A few cells of the host species may be resistant to phage; such resistance can be acquired through mutation. These cells have fewer competitors and may thrive. However, mutations also occur in phage, so highly compli- cated behavior occurs as the hosts mutate and mutate further as the phage mutates to counter host resistance. Commercial fermentation groups usually maintain different strains of cultures suitable for production so that phage attacks can be thwarted by substituting a nonsusceptible culture. After a period of time for the phage to dissipate, it may be possible to return the most desirable production strain. Bioprocess Control An industrial fermenter is a fairly sophisti- cated device with control of temperature, aeration rate, and perhaps pH, concentration of dissolved oxygen, or some nutrient concentra- tion. There has been a strong trend to automated data collection and analysis. Analog control is still very common, but when a computer is available for on-line data collection, it makes sense to use it for control as well. More elaborate measurements are performed with research bioreactors, but each new electrode or assay adds more work, addi- tional costs, and potential headaches. Most of the functional relation- ships in biotechnology are nonlinear, but this may not hinder control when bioprocess operate over a narrow range of conditions. Further- more, process control is far advanced beyond the days when the main tools for designing control systems were intended for linear systems. Many of the sensor problems such as those with steam-sterilizable pH electrodes and dissolved oxygen probes have been solved. Perhaps the most important factor for bioprocessing is the concentration of organisms, but there is no practical method for continuous measure- ment. Samples of the process fluid must be filtered and dried to get the mass concentration of cells. Numbers can be obtained by direct counting with a microscope or by counting the colonies that form when samples are cultured with nutrient medium in Petri dishes. In lieu of direct measurement, many other ways to estimate cell concen- tration are tried. Turbidity of the culture fluid can be correlated with cell concentration, but properties and calibration change during the process, and the optical surfaces of the sensors tend to become fouled. Alternatives such as measuring electrical conductivity or capacitance of the fluid sometimes are useful but often are suited only to specific cases. Indirect methods such as measuring protein produced by cells or monitoring nucleic acids are reported, but their proportionality to cell mass may vary during the fermentation. An important advance was made by developing computer models that can interpret mea- sured variables to calculate cell mass or product concentration that may be difficult or impractical to measure on-line. Mounting electrodes in a bioreactor is costly, and there is an addi- tional contamination risk for sensitive cell cultures. Some other sen- sors of practical importance are those for dissolved oxygen and for dissolved carbon dioxide. The analysis of gas exiting from a bioreactor with an infrared unit that detects carbon dioxide or a paramagnetic unit that detects oxygen (after carbon dioxide removal) has been replaced by mass spectrophotometry. Gas chromatographic proce- dures coupled with a mass spectrophotometer will detect all the volatile components. A useful index of process performance is the oxygen uptake rate, OUR, that is calculated from the difference in oxygen concentration of the inlet air and the exiting gas. Also important is the respiration ratio defined as the carbon dioxide evolved divided by the oxygen con- sumed. Although dynamic responses of microbial systems are poorly under- stood, models with some basic features and some empirical features have been found to correlate with actual data fairly well. Real fermen- tations take days to run, but many variables can be tried in a few min- utes using computer simulation. Optimization of fermentation with models and real-time dynamic control is in its early infancy; however, bases for such work are advancing steadily. The foundations for all such studies are accurate material balances.TABLE 24-6 Some Definitions of Microbial Interactions Competition A race for nutrients and space Predation One feeds on another Commensalism One lives off another with negligible help or harm Mutualism Each benefits the other Synergism Combination has cooperative metabolism Antibiosis One excretes a factor harmful to the otherchanges in relative numbers of the various organisms present, but complete takeover by one type is extremely uncommon. Survival of a broad range of species is highly advantageous in natural systems because a needed type will be present should an uncommon nutrient (pollutant?) be added or the conditions change. Prey-predator or host-parasite systems can be analyzed by mass bal- ance equations: = µHH − DH − KHP (24-28) = µPP − DP (24-29) = D(So − S) − (24-30) where H = the concentration of hosts (prey) P = the concentration of predators S = substrate concentration (food for prey) K = a coefficient for killing and µH and µP are Monod functions of S and H respectively. Computer simulation of these equations is shown in Fig. 24-25. Real systems do have this type of oscillating behavior, but frequencies and amplitudes are erratic. µHH } Y dS } dt dP } dt dH } dtC on ce nt ra tio n, g /L 0 0 2 4 6 Time, h 24 Predators Prey FIG. 24-25 Computer simulation of prey-predator kinetics. ENZYME ENGINEERING 24-21The common indices of the physical environment are: temperature, pressure, shaft power input, impeller speed, foam level, gas flow rate, liquid feed rates, broth viscosity, turbidity, pH, oxidation-reduction potential, dissolved oxygen, and exit gas concentrations. A wide variety of chemical assays can be performed; product concentration, nutrient concentration, and product precursor concentration are important. Indices of respiration were mentioned with regard to oxygen transfer and are particularly useful in tracking fermentation behavior. Com- puter control schemes for fermentation can focus on high productiv-ity, high product titer, or minimum cost. Computer systems may per- form on-line optimization of fermentation. Progress has been slow by empirical methods because there is a multiplicity of variables and because statistical techniques suffer from the relatively poor repro- ducibility of fermentations. Careful attention to preparation of inocu- lum, time/temperature factors of sterilization, and the timing of inoculation and feeding can greatly reduce variability of bioprocess performance.ENZYMATIC REACTION KINETICS Enzymes are excellent catalysts for two reasons: great specificity and high turnover rates. With but few exceptions, all reactions in biologi- cal systems are catalyzed by enzymes, and each enzyme usually cat- alyzes only one reaction. For most of the important enzymes and other proteins, the amino-acid sequences and three-dimensional structures have been determined. When the molecular structure of an enzyme is known, a precise molecular weight could be used to state concentration in molar units. However, the amount is usually expressed in terms of catalytic activity because some of the enzyme may be denatured or otherwise inactive. An international unit (IU) of an enzyme is defined as the amount capable of producing one micro- mole of its reaction product in one minute under its optimal (or some defined) reaction conditions. Specific activity, the activity per unit mass, is an index of enzyme purity. Although the mechanisms may be complicated and varied, some simple equations can often describe the reaction kinetics of common enzymatic reactions quite well. Each enzyme molecule is considered to have an active site that must first encounter the substrate (reactant) to form a complex so that the enzyme can function. Accordingly, the following reaction scheme is written: E + SA 2 1 ES →3 P + E (24-31) where E = enzyme, S = substrate, ES = enzyme-substrate complex, and P = product. Reactions 1 and 2 may be assumed to be in equilibrium soon after the enzyme is exposed to its substrate. Rate equations for these reac- tions are: = k2(ES) − k1(E)(S) (24-32) = k1(E)(S) − (k1 + k3)(ES) (24-33) = k3(ES) (24-34) where k1, k2, k3 = kinetic constants shown with the arrows in Eq. (24-31). Analysis leads to the Michaelis-Menten equation: = (24-35) where KM = Michaelis Constant and Vmax = maximum rate of reaction. This equation successfully describes the kinetic behavior of a sur- prisingly large number of reactions of different enzymes. Taking re- ciprocals of both sides gives: = + (24-36) A linear plot of the reciprocal of the reaction rate versus 1/(S) will allow the determination of KM and Vmax from experimental data. Kinetic behavior becomes complicated when there are two chemi- cal species that can both complex with the enzyme molecules. One of the species might behave as an inhibitor of the enzyme reaction with 1 } Vmax KM } (S) Vmax dt } d(P) Vmax (S) } KM + (S) d(P) } dt d(P) } dt d(ES) } dt d(S) } dtthe other as the substrate. Depending upon the nature of the com- plex, different inhibition patterns will yield different kinetic equa- tions. For example: E + SA ES → P + E (24-37) E + IA EI (24-38) Since the EI complex does not yield product P, and I competes with S for E, there is a state of competitive inhibition. By analogy to the Michaelis-Menten equation: = 11 + 2 + (24-39) where I = concentration of the competitive inhibitor and Ki = inhibi- tion constant. Enzyme reactions are also sensitive to pH and temperature changes. In characterizing an enzyme, its optimal pH and optimal temperature are conditions at which the enzyme has its highest cat- alytic activity. For a somewhat more extensive exposure to enzyme reaction kinet- ics, consult standard biochemistry texts and also Dixon, M. and E. C. Webb, Enzymes, 2d ed., Academic Press, 1964; Segal, I. H., Enzyme Kinetics, Wiley, 1975; Gacesa, P. and J. Hubble, Enzyme Technology, Open University Press, England, 1987. Immobilized Enzymes One factor that usually impedes the development of wide industrial application of enzymes is high cost. Immobilization is a technique to retain enzyme molecules for repeated use. The method of immobilization can be adsorption, cova- lent bonding, or entrapment. Semipermeable membranes in the form of flat sheets or hollow fibers are one way to restrain the enzyme while allowing smaller molecules to pass. Polyacrylamide gel, silica gel, and other similar materials have been used for entrapment of biologically active materials including enzymes. Encapsulation is another means of capture by coating liquid droplets containing enzymes with some semipermeable materials formed in situ. Generally speaking, entrap- ment does not involve a chemical or physical/chemical reaction directly with the enzyme molecules; and the enzyme molecules are not altered. Physical adsorption on active carbon particles and ionic adsorption on ion-exchange resins are important for enzyme immobi- lization. A method with a myriad of possible variations is covalent bonding of the enzyme to a selected carrier. Materials such as glass particles, cellulose, silica, and so on, have been used as carriers for immobilization. Enzymes immobilized by entrapment and adsorption may be subject to loss due to leakage or desorption. On the other hand, the chemical treatment in forming the covalent bond between an enzyme and its carrier may permanently damage some enzyme molecules. In enzyme immobilization, two efficiency terms are often used. Immobilization yield can be used to describe the percent of enzyme activity that is immobilized, % yield = 100 × Immobilization efficiency describes the percent of enzyme activity that is observed: percent efficiency = 100 × observed activity}}} activity immobilized activity immobilized }}} starting activity 1 } Vmax [I] } Ki 1 } S KM } Vmax dt } d[P]ENZYME ENGINEERING 24-22 BIOCHEMICAL ENGINEERINGWhen an enzyme molecule is attached to a carrier, its active site might be sterically blocked and thus its activity becomes unobservable (inac- tivated). One of the most important parameters of an immobilized-carrier complex is stability of its activity. Catalytic activity of the complex diminishes with time because of leakage, desorption, deactivation, and the like. The half-life of the complex is often used to describe the activity stability. Even though there may be frequent exceptions, lin- ear decay is often assumed in treating the kinetics of activity decay of an immobilized complex. Immobilization by adsorption or by covalent bonding often helps to stabilize the molecular configurations of an enzyme against alterna- tions including those that may cause thermal deactivation. Immobi- lized enzymes tend to be less sensitive to pH changes than are free enzymes. Although careful choice of the immobilization chemistry can result in stabilized activity, there are some enzymes that are much less stable after immobilization. Most carriers are designed to have high porosity and large internal surface areas so that a relatively large amount of enzymes can be immobilized onto a given volume or given weight of the carrier. Therefore, in an immobilized enzyme-carrier complex, the enzyme molecules are subject to the effect of the micro- environment in the pores of the complex. Surface charges and other microenvironmental effects can create a shift up or down of optimal pH of the enzyme activity. An immobilized enzyme-carrier complex is a special case that can employ the methodology developed for evaluation of a heterogeneous catalytic system. The enzyme complex also has external diffusional effects, pore diffusional effects, and an effectiveness factor. When car- ried out in aqueous solutions, heat transfer is usually good, and it is safe to assume that isothermal conditions prevail for an immobilized enzyme complex. The Michaelis-Menten equation and other similar nonlinear expressions characterize immobilized enzyme kinetics. Therefore, for a spherical porous carrier particle with enzyme molecules immobi- lized on its external as well as internal surfaces, material balance of the substrate will result in the following: 2 + De = (24-40) with also the usual boundary conditions, at r = R, S = S and at r = 0, dS/dr = 0 where R = radius of the sphere, r = distance from sphere center, S = substrate concentration, and De = effective diffusivity. Nor- malizing results in: + − φ2β1 + y2 = 0 (24-41) where y is dimensionless concentration, x is dimensionless distance, and φ and β are dimensionless constants; φ is sometimes referred to as the Thiele modulus of the immobilized enzyme complex. The bound- ary conditions are x = 1, y = 1 and at x = 0, dy/dx = 0. Graphical solu- tions are available in standard tests. Two meaningful asymptotic conditions have analytical solutions. In one extreme, β → 0, meaning S >> Km, and accordingly the Michaelis-Menten equation reduces to a zero-order reaction with V = Vmax. This is the condition of saturation (i.e., the substrate supply is high and saturates all of the active sites of the enzyme molecules). In the other extreme, β → ∞, meaning Km >> S, and accordingly the Michaelis-Menten equation approaches that of a first-order reaction with V = VmaxS/Km. This is the condition of a com- plete substrate control. Enzymatic Reactors Adding free enzyme to a batch reactor is practical only when the value of the enzyme is relatively low. With expensive enzymes, reuse by retaining the enzyme with some type of support makes great economic sense. As some activity is usually lost in tethering the enzyme and the additional operations cost money, sta- bility is very important. However, many enzymes are stabilized by immobilization; thus, many reuses may be possible. Methods of immobilization have already been discussed, and vari- ous reactor configurations are possible. An enzyme immobilized on y } β dy } dx 2 } x d 2y } dx2 VmaxS } KM + S d 2S } dr 2 dS } dr De } r beads of a support material or captured in a gel droplet is essentially a catalytic particle. Mounted in a packed column, there may be upflow or downflow of the feed solution, and a fluidized bed may be feasible except that particle collision often endangers stability of the enzyme. A serious problem is growth of microorganisms on the particles because enzymes are proteins that are nutritious. As immobilized enzymes often have more thermal stability than do free enzymes, the columns can be run at elevated temperatures (50 to 65°C, or 122 to 149°F) to improve reaction rate and to inhibit most but not all con- taminating organisms. Sterile feed solutions and aseptic technique can minimize contamination, but, more commonly, antiseptics are added to the feed, and there is occasional treatment with a toxic chemical to wash organisms from the column. Particles with immobilized enzymes are sometimes added to a reactor and recovered later by filtration or by some trick such as using magnets to collect enzymes attached to iron. Cellulose is hydrolyzed by a complex of several enzymes. The mix of enzyme activities produced by mold cultures can have insufficient amounts of the enzyme beta-glucosidase to maintain a commercially acceptable hydrolysis rate. This enzyme can be produced with a dif- ferent microbial culture and used to supplement the original enzyme mix, but the cost is high. It is logical to immobilize the beta- glucosidase for multiple use. Handling is minimized by circulating fluid from the main reactor through an external packed column of immobilized enzyme. Enzymes can be immobilized in sheets. One design had discs of enzymes fastened to a rotating shaft to improve mass transfer, and an alternate design had the feed stream flowing back and forth through sandwiches of sheets with enzyme. However, volumetric efficiency of such reactors is low because sheets with finite spacing offer less area than that of packed particles. It is possible to add free enzyme and recover it by ultrafiltration, but sufficient membrane surface to get good rates and the required auxiliary equipment are expensive. A hollow fiber device packs a vast amount of membrane area into a small volume. Enzyme may be immobilized inside or outside of the fiber, and it is easy to flush and replace the enzyme. Drawbacks to this design are: (1) The stability of the enzyme is not affected—activity that survives the immobilization step can have enhanced long-term stability; (2) there are two mass- transfer steps, as the substrate must diffuse through the fiber to reach the enzyme and the product must diffuse back; and (3) diffusion is poor on the outside of packed fibers. There is a scheme with the enzyme immobilized on or in the membrane to provide excellent con- tact as the feed is forced through. Although not yet commercialized, this method appears quite attractive. Recent Russian research with quick freezing has produced gels of enzymes that have high activity, good stability, and a temperature range up to the point where the gel collapses. ADDITIONAL REFERENCES: Baldwin, T. O., F. M. Raushel, and A. I. Scott, Chemical Aspects of Enzyme Technology—Fundamentals, Plenum Press, New York, 1990. Various eds., Enzyme Engineering, vols. 2–5, Plenum Press, New York, 1974–1980. Wingard, L. B., I. V. Berezin, and A. A. Klyosov (eds.), Enzyme Engineering—Future Directions, Plenum Press, 1980. ENZYME IMMOBILIZATION: Zaborsky, O. R., Immobilized Enzymes, CRC Press, 1973. Lee, Y. Y. and G. T. Tsao, “Engineering Problems of Immobilized Enzymes,” J. Food Technol., 39, 667 (1974). Messing, R. A., Immobilized Enzymes for Industrial Reactors, Academic Press, 1975. Torry, S., Enzyme Tech- nology, Noyes Data Corp., Park Ridge, New Jersey, 1983. ENGINEERING ASPECTS: Trevan, M. D., Immobilized Enzymes: An Introduc- tion and Applications in Biotechnology, Wiley, 1980. Moo-Young, M., Bioreac- tors: Immobilized Enzymes and Cells: Fundamentals and Applications, Elsevier, London, 1988. REVIEW OF HETEROGENEOUS CATALYSIS: Satterfield, C. N., Mass Transfer in Heterogeneous Catalysis, M.I.T. Press, 1970. Sherwood, T. K., R. L. Pigford, and C. R. Wilke, Mass Transfer, McGraw-Hill, 1975.