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2.6
2.5.7
2.6.3
2.6.4
2.7
3.2
3.4.3
5.4
5.4.1
Example 5.1.
5.5
5.4.2
6.3
5.7.3
5.7.4
5.7.5
7.
6.3.2
Increase in Feed Rate to a Podbielniak
7.2
7.3 Adsorption
7.3.1 Fixed-Bed Adsorption
Example 7.1. Determination of the Mass Transfer Coefficient from Adsorption
Example 7.3. Calculation of the Shock Wave Velocity for a Nonlinear Isotherm
7.4.4 Dispersion Effects in Chromatography
7.4.5 Computer Simulation of Chromatography Considering Axial Dispersion, Fluid-Phase Mass Transfer, Intraparticle Diffusion, and Nonlinear Equilibrium
7.4.6 Gradients and Modifiers
Example 7.5. Equilibrium for a Protein Anion in the Presence of Chloride Ion
7.5 Membrane Chromatography
Example 7.6. Comparison of Time for Diffusion Mass Transfer in Conventional Chromatography and Membrane
7.6
7.7
7.7.4
7.7.5
7.7.6
7.7.7
7.7.8
7.9.1
7.10.1
Example 7.7.
7.10.2
Example 7.8. Scale-up of a
Example 7.10.
8.3
8.4
PREFACE
The field of bioseparations has developed as a significant, separate discipline within the general field of biochemical engineering that involves the separation and purification of compounds of biological origin, which are derived from cells grown in bioreactors or from cells contained in animal or plant tissue. The biotechnology industry, which originated in the late 1970’s, gave added importance to bioseparations, because many of the products of biotechnology are proteins that often are difficult to purify and frequently must be purified to homogeneity or near homogeneity, leading to high costs. The market for biotechnology products is now in the tens of billions of dollars per year and is forecast to more than triple in size over the next twenty-five years. By far the largest sector of biotechnology products is the one for human therapeutics. The increasing demand for human therapeutics has put even more emphasis on the bioseparation processes that lead to very high purity, such as chromatography.
Practitioners of bioseparations include biochemical engineers and biochemists in the pharmaceutical, biotechnology, food, and chemical industries. This text focuses on the science and engineering aspects of bioseparations and is designed for juniors, seniors, and graduate students. The book is also intended to be useful for practitioners in industry.
The first chapter contains introductory material in two parts, the first of which is basic information about biochemistry. Bioprocess engineers need to know about the physical properties of the materials they must purify, which include antibiotics, vitamins, and vaccines, and, in the field of biotechnology, proteins, nucleic acids, antibodies, subcellular particles, and whole cells. An understanding of cell structure and function is necessary for the intelligent choice of early post-fermentation processes. The second part of this chapter is a brief review of engineering analysis, which is used in various forms throughout the book.
Because we believe that good analytical methods are the foundation of any effort to develop, optimize, and then operate and troubleshoot any bioprocess, the introductory chapter is followed by a chapter on analytical methods. A good understanding of these analytical methods is important for anyone involved in bioseparations process development or production.
The important unit operations are thoroughly covered in separate chapters that follow (Chapters 3-12), with the order of the chapters being similar to the order in which the operations are used in a typical bioseparations process. The approach in each of the chapters on unit operations is to start with a qualitative description indicating the importance and general application of the unit operation, then to describe the scientific foundation of the operation, develop the necessary mathematical theory, and finally describe the applications of the theory in engineering practice with an emphasis on design and scale-up.
In deciding on the subject matter in each of the chapters on unit operations, we have been guided by a desire to emphasize those aspects of unit operations most important as applied to bioseparations at the commercial scale. For example, we have not discussed all methods of industrial drying, just those that are the most important in general for drying bioproducts.
A comprehensive treatment of bioseparations process design (Chapter 12) follows the unit operations chapters and focuses on how to integrate the individual unit operations in developing a process design that is the “best” among several plausible alternatives. The use of a process simulator (SuperPro Designer) is illustrated to analyze and evaluate the production of three biological products—citric acid, recombinant human insulin, and monoclonal antibodies. All the problems in this chapter are intended as open-ended assignments, which can be worked either with or without a biochemical process simulator. The use of a bioprocess simulator, however, will greatly facilitate the process analysis, including generating flowsheets, carrying out material balances, and analyzing costs. It is recommended that students have 6 to 8 weeks to complete their problems.
SuperPro Designer is our strong preference for a biochemical process simulator that can handle batch as well as continuous processes. A functional evaluation version of SuperPro Designer can be accessed at the website www.intelligen.com
A site license (one time fee) of SuperPro Designer can be obtained at a discount for courses at universities adopting Bioseparations Science and Engineering (Intelligen, Inc., Scotch Plains, New Jersey).
In the second edition of this text, several topics have received added emphasis. In keeping with the current increased importance of biotechnology products that are proteins, such as monoclonal antibodies, considerable new coverage of chromatography has been added. Compared to the first edition, the following topics were added or major changes were made:
• moment analysis in chromatography
• computer simulation of chromatography
• membrane chromatography
• simulated moving bed chromatography
• bench scale preparative separations using electrophoresis and magnetic separations
• evaporation, a process commonly used for bioproducts such as antibiotics, steroids, and peptides
• updating of the bioprocess design and economics for the production of a therapeutic monoclonal antibody
• updating of all of the costs in the chapter on bioprocess design and economics
Illustrative example problems are included within the text throughout Chapters 1 to 12. The purpose of these examples is often to show how to apply previously presented theory, or in some cases to extend the theory to other situations. One way that these examples can be effectively used in the classroom is as follows: The student reads through the solution of the problem and then (1) formulates a question that someone might have about the problem’s solution and discusses this question and possible answers to the question with another student or students in the course, or (2) discusses a key point in the solution with another student or students in the course. Then, the instructor asks one or more students about the questions or key points that have been raised.
Problems developed for this book are given at the ends of Chapters 1 to 12, now totaling 130 for the second edition. These problems are intended to help the student both understand and be able to apply the material presented. Several of the problems are deliberately underspecified or of an open-ended nature. The problems can be assigned as out-of-class homework, or can be worked in class by students working in groups (of two, three, or four, depending on the complexity of the problem). Groups in class respond to the task in the following way: (1) each student formulates his or her answer, (2) students share their answer with their partner or partners, (3) students listen carefully to partner’s or partners’ answers, and (4) groups create a new answer that is superior to each member’s initial formulation through the process of association, building on each other’s thoughts, and synthesizing.
The practicum (Chapter 13) describes a set of bioseparations experiments that has been thoroughly tested by students (University of Colorado). A few selected experiments, or all of them, could be used in a course on bioseparations.
Additional information supporting this textbook and bioseparations in general can be found at the website http://www.biosep.ou.edu. Material at this website includes (1) a link to the bioprocess simulation software used in the bioprocess design and economics chapter; (2) new problems and examples, which are added periodically; (3) links to useful databases (such as for proteins); and (4) links to manufacturers of bioseparations equipment and supplies.
We are grateful for the contributions that many people made, either directly or indirectly, to this book. University of Oklahoma undergraduate students Emily Burdett and Kevin Dyer contributed new problems in the chapter on liquid chromatography and adsorption. Jean Hunter gave us encouragement and feedback in her review of the prospectus of the second edition. Jean-Bernard Gros read the final draft of the second edition and gave use valuable feedback. We again thank those who contributed in various ways to the first edition, including Ed Cussler, Robert Davis,
Eric Dunlop, Larry Erickson, Antonio Garcia, Jean-Bernard Gros, Dale Gyure, Juan Hong, Harold Monbouquette, Harold Null, Todd Przybycien, Subhas Sikdar, Geoffrey Slaff, and Richard Willson. We appreciate the feedback we have received over several years from students taking courses and the teaching assistants in these courses at the Universities of Colorado and Oklahoma where material on bioseparations was presented.
We are also grateful for being selected by the American Society for Engineering Education in 2006 to receive the Meriam/Wiley Distinguished Author Award for the first edition of Bioseparations Science and Engineering, an award given for writing the outstanding engineering textbook in the previous four years.
Finally, we are thankful for the patience and support from our families during the writing of this book.
Roger G. Harrison
Paul Todd
Scott R. Rudge Demetri Petrides
BIOSEPARATIONS SCIENCE AND ENGINEERING
INTRODUCTION TO BIOPRODUCTS AND BIOSEPARATIONS
Bioproducts—chemical substances or combinations of chemical substances that are made by living things—range from methanol to whole cells. They are derived by extraction from whole plants and animals or by synthesis in bioreactors containing cells or enzymes. Bioproducts are sold for their chemical activity: methanol for solvent activity, ethanol for its neurological activity or as a fuel, penicillin for its antibacterial activity, taxol for its anticancer activity, streptokinase (an enzyme) for its blood clot dissolving activity, hexose isomerase for its sugar-converting activity, and whole Bacillus thuringiensis cells for their insecticide activity, to name a few very different examples. The wide variety represented by this tiny list makes it clear that bioseparations must encompass a correspondingly wide variety of methods. The choice of separation method depends on the nature of the product, remembering that purity, yield, and activity are the goals, and the most important of these is activity.
This first chapter therefore reviews the chemical properties of bioproducts with themes and examples chosen to heighten awareness of those properties that must be recognized in the selection of downstream processes that result in acceptably high final purity while preserving activity. The final part of this chapter is an introduction to the field of bioseparations, which includes a discussion of the stages of downstream processing, the basic principles of engineering analysis as applied to bioseparations, and the various factors involved in developing a bioproduct for the marketplace.
The pharmaceutical, agrichemical, and biotechnology bioproduct industries account for many billion dollars in annual sales—neglecting, of course, commodity foods and beverages. By “bioproduct” we mean chemical substances that are produced in or by a biological process, either in vivo or ex vivo (inside or outside a living organism). Figure 1.1 indicates a clear inverse relationship between bioproduct market size and cost.
FIGURE 1.1 World production levels and prices of bioproducts, showing the inverse relationship between price and production. (Data from sources in the early 1990s time frame.)
TABLE 1.1 Summary of U.S. Biotechnology Product Sales Forecasts (Millions of 2000 Dollars) by Key Market Sectors: 2008–2050a
a Data from reference 2.
Owing to intense competition, cost, price, and value are very closely related, except in the case of completely new products that are thoroughly protected by patents, difficult to copy, and of added value to the end user. Products with these characteristics—“biotechnology products”—have typically been developed at considerable cost (over $800 million for R&D alone in the case of pharmaceutical products requiring clinical trials [1]) and marketed at prices that allow for the recovery of the development, production, and marketing costs. Their total contribution to the bioproduct market is also in the tens of billions of dollars annually (see Table 1.1) [2]. Such products must recover their development costs within a few years of initial sales, owing to potential competing products, expiration of patents, and economic pressures. Eventually such products move “down” the curve of Figure 1.1, but they
stay on the curve. In the future, movement down the curve will require increased scale and reduced cost of production. Therefore, while existing processes meet many of the requirements of the current market, innovation will eventually be required for the economic large-scale production of biotechnology products. Thus, the following chapters are a practical guide to biochemical separations as currently practiced and as might be practiced in the future.
1.1 INSTRUCTIONAL OBJECTIVES
After completing this chapter, the reader should be able to do the following:
• Broadly classify bioproducts as small molecules, macromolecules, and particulate products including cells.
• Explain the differences between the structures of the various bioproducts.
• Explain the difference between a primary metabolite and a secondary metabolite.
• Outline the structure of proteins at four levels and their stability and functions.
• Explain the structures of other macromolecular substances that are commercial bioproducts, such as nucleic acids and polysaccharides.
• Outline the four stages of downstream processing, the objectives of each stage, and typical unit operations for each stage.
• Explain the engineering analysis concepts of material balance, equilibria, and transport phenomena.
• Calculate purity, specific activity, and yield as quality indicators in purification.
1.2 BROAD CLASSIFICATION OF BIOPRODUCTS
Bioproducts can be broadly classified into the following categories: small molecules, consisting of fine chemicals, antibiotics, hormones, amino acids, and vitamins; large molecules, consisting of proteins, polysaccharides, and nucleic acids; and particulate products, consisting of cells, spores, liposomes, and subcellular particles or organelles. Table 1.2 indicates the sizes and masses of each category of product. These three broad categories correspond to three broad categories of separation procedures for purifying them. As we shall see in this book, small molecules cannot be sedimented, but they can be separated by extraction; many large molecules cannot withstand the conditions of solvent extraction, but they are highly adsorptive; and particulate products can be collected by sedimentation or filtration. In addition, separation processes depend on property differences. Therefore it is important to learn as much as we can about the physical properties and chemical characteristics of the categories of products for which we must design separation procedures. The following subsections summarize the bioproduct types in order of increasing molecular size and complexity, which is also in increasing order of complexity of processing.
TABLE 1.2
Broad Categories of Bioproducts and Their Sizes
Bioproduct Examples
Small molecules
Sugars
Large molecules
Particles
200–600 0.5 nm
Amino acids 60–200 0.5 nm
Vitamins 300–600 1–2 nm
Organic acids 30–300 0.5 nm
Proteins 103–106 3–10 nm
Polysaccharides 104–107 4–20 nm
Nucleic acids 103–1010 2–1,000 nma
Ribosomes 25 nm
Viruses 100 nm
Bacteria 1 μ m
Organelles 1 μ m
Yeast cells 4 μ m
Animal cells 10 μ m
a Single pieces of genetic DNA can have end-to-end lengths up to millimeters, depending on method of isolation; the diameter of the DNA double-stranded α helix is 2.5 nm.
1.3 SMALL BIOMOLECULES
Small biomolecules include naturally occurring compounds and metabolites such as citric acid, vitamins, amino acids, and antibiotics. Besides their importance in fermentation processes, many of these compounds are important commercial products, some of them at very large production rates. Small biomolecules can be divided into two categories—primary metabolites and secondary metabolites.
1.3.1 Primary Metabolites
A primary metabolite is one that is formed during the primary growth phase of the organism. Figure 1.2 shows the key central metabolic intermediates of biosynthetic pathways in heterotrophs, organisms that use organic compounds as carbon sources. The intermediates shown in this overview are used in catabolism, the processes by which microorganisms obtain energy from organic compounds, as well as in biosynthesis, also called anabolism.
Sugars
Naturally occurring sugars are found as monosaccharides, disaccharides, or polysaccharides. One of the world’s highest tonnage bioproducts is one of these, sucrose, a disaccharide, whose structure is shown in Figure 1.3. In the food industry, sucrose has been refined from sugarcane for centuries by using an aqueous solution and crystallization process. Mono- and disaccharides are also used in the biochemical
Polysaccharides
Carbohydrates
Other cofactors
Heterotrophs
Intermediates
Glucose 1-phosphate
Glucose 6-phosphate
Ribose 5-phosphate
Organic compounds CO2
NH3 , NO3 , organic N
Erythrose 4-phosphate
Phosphoenolpyruvate
Pyruvate
3-Phosphoglycerate
a-Ketoglutarate
Succinyl-CoA
Oxaloacetate
Dihydroxyacetone
phosphate
Acetyl-CoA
FIGURE 1.2 Overview of the biosynthetic pathways in heterotrophs, organisms that use organic compounds as carbon sources, showing the key central metabolic intermediates.
FIGURE 1.3 Sucrose, or table sugar, is a disaccharide composed of the monosaccharides glucose and fructose.
process industry as solutes for the control of osmotic pressure and, most importantly, as carbon sources for the growth of organisms in fermenters. Glucose, a monosaccharide (Figure 1.3), is the most common fermentation nutrient, and lactose (milk sugar) and maltose are popular commercial disaccharides.
Sugars can also be obtained as products in bioprocesses. For example, the enzyme glucose isomerase can convert glucose to fructose, which is much sweeter than glucose. This latter process is being used commercially to produce large amounts of high-fructose corn syrups, which are used as sweeteners in soft drinks.
Organic Alcohols, Acids, and Ketones
Organic alcohols, acids, and ketones can be produced by the anaerobic fermentation of microorganisms. Many of these organisms proceed from glucose to pyruvic acid and then on to the final bioproduct. Ethanol, isopropanol, acetone, acetic acid, lactic acid, and propionic acid are some of the organic alcohols, acids, and ketones that have been produced in anaerobic fermentations [3]. The most economically important of these processes is the production of ethanol. Another way of producing
organic alcohols, acids, and ketones is by using the acetic acid bacteria to perform partial oxidation. For example, the acetic acid product produced by Acetobacter bacteria with ethanol as a substrate is vinegar.
Vitamins
The name for vitamins was originally derived from “vital amines,” but we have known for decades that not all vitamins are amines, and some are not even vital. Generally, animals do not synthesize vitamins, but generalizations have exceptions. For example, humans and guinea pigs do not synthesize vitamin C (ascorbic acid), while nearly all other mammals do. Vitamin C has many more functions than originally thought. While it is required as a catalyst to support the hydroxylation of proline in collagen, which gives collagen its strength and low solubility, its presence at high concentrations supports antioxidant functions and the sulfation of cholesterol. Biological antioxidants are thought to prolong life and promote health, so vitamin C and its fat-soluble counterparts, vitamins A and E, have become popular consumer products. Both natural and synthetic forms are produced and marketed.
The B vitamins are water soluble and are metabolic precursors of cofactors involved in enzymatically catalyzed reactions. For example, vitamin B6, pyridoxine, becomes phosphorylated inside the cell and serves as a nitrogen atom shuttle in transamination reactions, which convert keto acids into amino acids and vice versa. Niacin becomes nicotinamide adenine dinucleotide (NAD), the cell’s principal redox compound and carrier of hydrogen atoms. The water-soluble vitamins are not phosphorylated because cell membranes cannot transmit organic phosphates; instead, the cell phosphorylates them after they enter. After phosphorylation (and in some cases additional modifications) they become cofactors, or “coenzymes.” Folic acid, also water soluble and named for its origin from leaves, is required in minute quantities for hydrogen atom transfer in very special chemical reactions, such as methyl group transfer in the synthesis of the important amino acid methionine and the important DNA base thymine. Folic acid is present in plant tissues in extremely low quantities and was among the earliest challenges in bioprocessing in terms of purifying usable amounts of a product initially present at minuscule concentrations. Folic acid is now synthesized in bacterial or fungal fermentations and is sold as a common ingredient in daily vitamin tablets.
Vitamin A, a carotenoid, becomes the energy transducer of the pigment proteins of the retina. Vitamin D, perhaps more appropriately called hormone D, is a steroid that regulates the passage of calcium ions in and out of cells. Vitamins A and D are derived from carrots and milk, respectively, and are sold after extraction and purification (or after laboratory synthesis) as over-the-counter consumer products.
Vitamins are an excellent example of a present-day market tension: natural versus synthetic. Most vitamins can be synthesized in organic chemical reactions, but some argue that unnatural solvents and reagents used in these syntheses could be present as impurities, possibly harming the consumer. Extraction from plants and fermentation produces vitamins naturally—at higher cost. This issue is important in
bioseparation technology, because the downstream processes are very different in the two approaches. In the latter case, hundreds of unknown contaminating solutes are present, and the product itself is at a low concentration. In the former case (synthesis), fewer impurities are present, but in some cases these may be more objectionable. The chapters that follow will give much more attention to natural products.
Amino Acids
Amino acids are the building blocks of proteins; understanding their chemistry is critical to an understanding of the stability of protein bioproducts. The general structure of an α-amino acid is given in Figure 1.4, where it is seen that every such amino acid has a side chain, or “R group,” a negatively charged carboxyl group, and a protonated (positively charged) amino group bonded to a carbon atom, which is called the α carbon because it is adjacent to the carboxyl group. At neutral pH, all amino acids are zwitterionic; that is, they carry positive and negative charges simultaneously.
Table 1.3 lists the amino acids, the three-letter and one-letter abbreviations of each, and their respective side chain structures. This table is grouped according to side chain class. The single-letter code allows easy representation of the primary structure of proteins. It can be seen that all the α-amino acids except glycine contain asymmetric carbon atoms, indicating that amino acids, like sugars, should be optically active. With almost no exceptions, the amino acids synthesized by living things are levorotatory and not racemic, so proteins can be studied on the basis of optical rotary dispersion (ORD); each protein, made of a different combination of amino acids, will have a unique ORD spectrum.
When amino acids are polymerized to form proteins, their carboxyl carbons are linked to their neighbors’ amino nitrogens in a classical amide linkage called the peptide bond. This occurs through a dehydration condensation, and equilibrium laws dictate that this reaction is highly unlikely in aqueous solutions. However, living cells solve this problem with a catalytic unit known as a ribosome. This reaction and a resulting peptide backbone are depicted in Figure 1.5.
Specific properties of amino acid side groups can be exploited in purification methods. A protein rich in acidic or basic amino acids on its surface can be adsorbed by ion exchange or separated by electrophoresis. Many aliphatic side chains can result in preferential adsorption onto or extraction into nonpolar separation media. The free–SH (sulfhydryl) group of cysteine can be used to bind proteins to immobilized mercury. Histidine forms coordination complexes with metals, a fact that is being heavily exploited in protein purification by adsorption methods, as we shall see in Chapter 7.
FIGURE 1.4 α-Amino acid structure showing zwitterionic equilibrium at neutral pH.
TABLE 1.3 List of α-Amino Acids Indicating R group (Side Chain) Structure, Abbreviations, pK of R group,a and Class: For general Structure, See Figure 1.4
R Group
—H
—CH3
—CH(CH3)2
—CH2CH(CH3)2
—CHCH3CH2CH3
—CH2OH
—CHOHCH3
—CH2SH
—(CH2)2SCH3
—CH2COOH
—CH2CONH2
—(CH2)2COOH
—(CH2)2CONH2
—(CH2)3CH2NH2
Name
Abbreviations pK of R group Class
Glycine Gly, G Aliphatic
Alanine Ala, A
Valine Val, V
Leucine Leu, L
Isoleucine Ile, I
Serine Ser, S Hydroxyl or sulfur containing
Threonine Thr, T
Cysteine Cys, C 8.3
Methionine Met, M
Aspartic acid Asp, D 3.9 Acids and corresponding amides
Asparagine Asn, N
Glutamic acid Glu, E 4.3
Glutamine Gln, Q
Lysine Lys, K 10.5 Basic
Histidine His, H 6.0
Phenylalanine Phe, F Aromatic
Tyrosine Tyr, Y 10.1
Tryptophan Try, W
—(CH2)3NHCNHNH2 Arginine Arg, R 12.5 CH2 CH2 CH2 CH2 CH2 CH2 SS COOH OH N H N H N NH
Proline Pro, P
Cystine
a See reference 4. pK is the pH at which the R group is half dissociated.
Imino acid
Disulfide
FIGURE 1.5 The formation of a peptide bond (amide linkage) between two amino acids and the resulting peptide backbone of proteins that results from polymerization.
Lipids
The natural fats consist of fatty acids, lipids, steroids, and steroid precursors. This family of bioproducts is highly extractable into nonpolar solvents. Fatty acids are synthesized by cells by building up two-carbon fragments contributed by a precursor compound known as acetyl-coenzyme A (acetyl-CoA), the cell’s principal mechanism of acetyl group transfer. Fatty acids are usually esterified to glycerol to form di- and triglycerides, and diglycerides are usually esterified to a phosphate group (hence “phospholipids”), which may in turn be esterified to ethanolamine or choline, rendering the phosphate “head” zwitterionic or amphoteric (having both charges).
Fatty acids and phospholipids are amphiphilic, having a strongly polar “head” and a strongly nonpolar tail. Such molecules form layers at liquid-liquid interfaces, and just such layers form the membranes that surround living cells and are also found inside eukaryotic cells (fungi, algae, protozoa, and animal and plant cells).
Steroids are heterocyclical compounds, and the most common is cholesterol. Many steroids have hormone activity, partly because they are able to penetrate the nonpolar cell membrane and get inside cells, where they bind and modify the activity of an intracellular protein. It is no surprise that steroids are important bioproducts. Similarly, a family of very potent lipids can profoundly affect the activities of animal cells, namely prostaglandins and leukotrienes; these are also significant commercial bioproducts. Figure 1.6 gives typical structures of four classes of compounds: fatty acid, phospholipid, steroid, and prostaglandin.
Commercial Uses
Many primary metabolites are important commercially; some important examples are given in Table 1.4. The products listed in Table 1.4 are sold in the fermentation, food, and biochemical research marketplaces, and several fine-chemical houses have adopted the sale of primary metabolites as a specialty or as a product line in a warehouse of general chemicals. The biochemical research market demands high variety and low volume. This market is served by a small number of large repackaging or distribution firms who purchase from specialty producers, some of whom also sell directly to customers.
Exemplified by ethanol at the bottom of Figure 1.1, these low molecular weight compounds are the easiest products to purify, mainly owing to their thermal stability. Not surprisingly, they are produced in highest quantity and are used in the beverage, food, feed, solvent, and specialty chemicals industries. Some of the alcohols are traditionally purified using distillation. This is true of none of the other substances discussed in the paragraphs that follow. The highest volume fermentation products in this category include ethanol, acetic acid, butyric acid, lactic acid, citric acid, glutamic acid, tryptophan, and glycine. Two examples are given in Figure 1.7.
1.3.2 Secondary Metabolites
Secondary metabolites are not produced during the primary growth phase of a microorganism, but at or near the beginning of the stationary phase. Antibiotics are the best
(CH3)3NCH2CH2OPOCH2 O CH2OCCH2CH2CH2CH2CH2CH2CH2CH CHCH2CH2CH2CH2CH2CH2CH2CH3 C OCCH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH3
(lonic) polar, hydrophilic “head”
Amphipathic structure
Phosphatidylcholine
Nonpolar, hydrophobic “tail”
Strongly nonpolar hydrocarbon chain of 17 C—C nonpolar bonds + 35 C—H nonpolar bonds Polar terminal group
acid
FIGURE 1.6 Structures of typical compounds: a phospholipid (phosphatidylcholine), a fatty acid (stearic acid), a steroid (estradiol), and a prostaglandin (PGE2).
TABLE 1.4 Some Examples of Primary Metabolites That Are Marketed and Their Uses
Primary metabolite
Citric acid
Acetic acid
Glutamic acid
Lactic acid and
Glycolic acid
Glycerol
Butanol
Fructose
Formic acid
Commercial uses
Beverages
Food (vinegar) and fine chemicals
Food flavoring (as monosodium glutamate, or MSG)
Reactants for production of biodegradable polymers
Solvent
Solvent
Food, fermentation
Fine chemicals
COOH CH2COOH C HO (a)(b) CH2
FIGURE 1.7 Structures of (a) citric acid and (b) glutamic acid (an amino acid).
Stearic
