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Home , Biochemical engineering, Genetic engineering

BIOCHEMICAL FUNDAMENTALS (revisited)

J.E. BAILEY transformation: the transfer of genetic information into California Institute of a using DNA separated from the cell as a vector. : circular, self-replicating non-chromosomal DNA. Pasadena, CA 91125 (Because the plasmid is small [vs. the major chromo­ D. F. OLLIS somal DNA] it is a useful "shuttle" or vector for moving rDNA into a new cell.) North Carolina State University virus: an infectious agent that requires a host cell in Raleigh, NC 27695-7905 order for it to replicate. It is composed of DNA (or RNA) wrapped in a coat. B IOCHEMICAL ENGINEERING CONCERNS the re- protoplast: a cell (plant, animal, microbial) without a actors and separation systems associated with (structural) wall (cell has "soap bubble" or plasma microbial cells, their enzymes and other products, membrane only). protoplast fusion: a means of achieving genetic transforma­ and with plant and animal cells which can be tion by joining two protoplasts in the laboratory to propagated in an appropriate reactor outside of achieve a viable cell with desirable traits. the whole plant or animal. In 1976, we reported myeloma: tumor (immortal) cells of the antibody-pro­ on a course (1] with an ducing system. outline which appeared in 1977 as a textbook, Bio­ lymphocytes: specialized white blood cells involved in the immune response; each B lymphocyte produces a single Fundamentals. kind of specific antibody. Spectacular changes have occured in this do­ hybridoma: a viable cell hybrid resulting from main over the last ten years. Many of these of a lymphocyte (specific antibody-producing) and a changes concern methods for fusing two cells to­ myeloma ("immortal" or easily propagated cell, result­ gether, to produce a hybrid cell, and for "cutting ing in a cell which can be conveniently cultured (like and pasting" strands of genetic material (DNA) together to form recombinant DNA (rDNA) coding for a desired set of instructions. With the commercialization of processes using these two "new" techniques, a number of new words have appeared in the biological and biopro­ cess literature. For the biochemical engineering teacher and practitioner, the most central vocabu­ lary includes the following [2] : used at the laboratory level to alter the hereditary apparatus of the living cell so that the cell can produce more or different chemicals, or perform completely new functions. DNA: the genetic material found in all living . Jay Bailey is a professor in the chemical engineering department : a group of genetically identical cells or organisms at Caltech. His research interests include the study of structural and produced asexually from a common ancestor. functional effects of immobilization on , altered metabolism : the amplification of segments of DNA, usually anc! multiple product bioconversion in immobilized and , . production of biological polymers by microbial , and rDNA (recombinant DNA) : the hybrid DNA produced by several aspects of kinetics and reactor design for recombinant micro­ ( enzymatically and extracellularly) joining together organisms. (L) pieces of DNA from different sources. David F. Ollis received his BS from Caltech in 1963, his MS from (DNA) vector: a self-replicating DNA molecule (plasmid, Northwestern in 1964, and his PhD from Stanford in 1969. He is virus) that transfers a piece of DNA from one cell currently Distinguished Professor of chemical engineering at North host to another. Carolina State University, where he is engaged in research, teaching, and biotechnology facility development. His hobbies include music © Copyright ChE Division, ASEE, 1985 and raising (mostly redheaded) boys. (R}

168 CHEMICAL The potential impact appears to be endless: virtually any biochemical ... or economically desirable defense mechanism can be produced or incorporated, respectively, in appropriate plant, animal, or microbial cells which can be grown in biological reactors of one sort or another.

microbes) and which produces specific antibodies. cess fermentation/ separation improvements, has be­ monoclonal antibodies (MAb): Homogeneous (all alike) come commercial for gasohol ( <10% ethanol in gaso­ antibodies derived from a single clone of cells; MAb's line) production and is now competitive with ethanol recognize only one kind of antigen (very high specifici­ from ethylene on a new plant basis. ty). • Single cell (microbial) protein has been produced on a mammoth scale (Imperial Chemical Industries) Growth over the last ten years of hoth new using a novel (airlift) configuration and and established commercial efforts in biotechnol­ a major petrochemical (rather than agricultural) ogy has been astonishing. This rate of change is feedstock: methanol. • Bovine has been produced by cul­ nicely chronicled by the following sequence [2] tured rDNA bacteria (E. coli): this product could enhance production in dairy cows and growth 1973 First cloned. rates in beef cattle. 197 4 First expression (production of genetically coded • A soil microbe (Pseudomonas) has been genetically product)' of a gene cloned from a different species engineered to contain bacterial genes coding for a in bacteria. toxin against a nematode root predator, indicating rDNA experiments first discussed in a public forum. potential for a perpetual biocide-generating soil in­ 1975 Science conference at Asilomar, Calif. held; support oculant. for guidelines for recombinant DNA research • Wholly synthetic genes have been constructed by wet voted. biochemical techniques, inserted into a bacterium and First hybrid cell created by the fusion of two animal the newly coded product obtained, thus verifying the cells. ability to "type" any biochemical production instruc­ 1976 First rncombinant DNA-based company founded: tion, by wet chemistry, into the genes of bacteria. Gen en tech. • Plant cells (tobacco) have been manipulated to con­ 1980 New life forms are patentable: Diamond vs. Chakra­ tain microbial (non-plant) genes for a toxin against barty. a specific pest (not commercially useful example, but Wall Street selling record set: Genenetch goes importantly suggestive for engineering plant pest public, stock price moves from $20 to $89/ share resistance). in 20 minutes. United Kingdom, West Germany target biotechnol­ ogy for development The potential impact appears to be endless: Patent (Cohen-Boyer) issued for rDNA methodology virtually any biochemical (enzyme, antibody, hor­ 1981 First test kit marketed mone, vitamin, growth factor) or economically First automated gene synthesizer marketed desirable defense mechanism can be produced or Japan, France target biotechnology Rand D Cetus Corp. raises record $115 million on first public incorporated, respectively, in appropriate plant, offering animal, or microbial cells which can be grown in DuPont dedicates $120 million to life sciences R & D biological reactors of one sort or another. Different 80 new biotechnology companies founded cell types will be preferred for different products; 1982 First rDNA animal (colibacillosis) marketed each will impose specific requirements on biopro­ (Europe) First rDNA pharmaceutical (human / Lilly) cess reaction and separation systems. As new marketed (England) products and new organisms for current products 1983 First plant gene transferred to different species of multiply, the biochemical must provide plant more systematic, predictive guidance to select $500 million raised (total) by new U.S. biotechnol­ among available options and bring optimized or­ ogy companies 1984 First plant cells genetically modified to be resistant ganism-process systems into timely and efficient to a broad spectrum (). production. In our view, these new opportunities and chal­ Additional developments include both the lenges in biotechnology will require biochemical "new" and the 'old' : engineering to move beyond the empirical, macro­ scopic approaches which were adequate in the past. • Renewable resources engineering has demonstrated We believe that biochemical must under­ a number of biological ( enzymatic, microbial) and non-enzymatic means of converting cellulosic ma­ stand, model, and productively exploit the funda­ terials into fermentable feedstocks. mental biological, chemical, and physical mechan­ • Ethanol production, largely through and pro- isms which interact to determine process perform-

FALL 1985 169 ance. Previously, we have often viewed cells only a basis for kinetic models. from a "black-box", "input-output" perspective, 7. Cell kinetics is enlarged to include not only unstruc­ tured models ( constant biomass composition) but and seen aeration primarily in terms of an overall structured (multicomponent) models which allow in­ volumetric mass transfer coefficient. For the fu­ clusion of known internal metabolic details which ture, we need to apply known features of cellular vary with cell environment or culture growth stage. pathways and their regulation to improve reactor Segregated models for kinetics, incorporating cell­ design, and to include detailed studies of hydro­ to-cell heterogeneity (population balances) are also dynamics and interfacial phenomena to enhance included. Similarly, product formation kinetics can be empirically or metabolically modeled. mass transfer rates with minimal damage to cells 8. Transport phenomena (.aeration, agitation, power in­ and products. Armed with quantitative methods put, mixing) is modified to include transport cor­ for reaction engineering and description of multi­ relations for recent configurations (air-lift reactor), component partitioning, the biochemical engineer and materials (viscous fluids) as well as data for CO gas-liquid transfer (coupled to fermentation should seek an increasing role earlier in the pro­ 2 broth pH). Aeration bubble coalescence is discussed, cess research and development sequence, aiding in and both Weber number .and Kolmogoroff definition of organisms and product forms which presented as determinants of bubble size and thus have the characteristics needed to enable or to interfacial area/vol, a, of the mass transfer conduct­ facilitate large-scale manufacture. ance, k1a. The current biochemical engineering lecture 9. Reactor design for pure cultures is revised to include a number of non-ideal reactors (including circulation course additions and alterations, summarized be­ in imperfectly mixed batch reactors). Formulation, low, are driven by our firm conviction that a characterization, and application of immobilized cell major refinement and refocusing of biochemical catalysts are examined. Multiphase reactors, in­ engineering education (and research, but that is cluding packed beds, fluidized beds, and trickle re­ · another story) is needed to meet the technical actors are treated, and an entire section is devoted to reactors for plant and animal cell propagation. challenges ahead and to achieve the most produc­ 10. Instrumentation and Control is an entirely new topic, tive role for the biochemical engineer in future encompassing process for dissolved oxygen, commercial biotechnology enterprises. substrates, temperature, pH, etc., and biological We have, consequently, restructured our course parameters needed for on-line data acquisition and and our text (second edition in press) to include calculation of instantaneous mass and energy bal­ ances. This discussion leads naturally to application the following emphases in the topic sequence indi­ of state estimation and process control. cated 11. Separation and purification processes are also isolated in a new, unified treatment, beginning with the 1. becomes Microbiology and Cell (plant, fermentation broth or biological product source, and animal) Biology. surveying techniques for cell and particle removal 2. includes additional sections on DNA (, sedimentation, coagulation, centrifuga­ structure, , and enzymes need for DNA tion), product concentration (solvent extraction, manipulation via rDNA techniques. aqueous 2-phase extraction, precipitation, adsorp­ 3. Enzyme kinetics includes reversible reactions (e.g., for tion, ultrafiltration, etc.) and product purification high fructose syrup via glucose isomerase) and more (large scale chromatographies, fractional thermal detailed treatment of enzyme deactivation mechan­ and salt-driven precipitations, , isms and kinetics. affinity and immunosorbent 4. Enzyme applications include new immobilized enzyme columns, reverse osmosis). Examples of bulk chemi­ processes, and preparation methods, materials, cata­ cal (ethanol), enzyme, antibiotic, polysaccharide and lyst characteristics including deactivation and mass organic acid recovery are discussed. transport-reaction interactions. 12. Bioprocess economics is introduced as a new subject. 5. Bioenergetics and metabolism is restructured to show A complete case study due to Bartholomew and clearly how energy balances work to give biosyn­ Reisman is first covered, including process inception, thetic engine efficiencies and microbial heat release, flow sheets, equipment sizing, capital equipment, and how stoichiometries of microbial conversions operating, utility, labor, and raw materials costs, are deducible directly from understanding the major and return-on-investment and sensitivity determina­ metabolic flow routes. tions. Subsequent examples include a number of 6. and (microbial) control systems altered major bioproducts (antibiotics, ethanol, enzymes, enormously to indicate techniques of the "new" bio­ proteins from recombinant cells, organic acids, poly­ technology: recombinant DNA manipulations and saccharides, single-cell protein, monoclonal anti­ cell fusion approaches for hybridomas. This section bodies, ) . also emphasizes the need to understand lower (pro­ 13. Mixed populations discussion has been enlarged to in­ caryotic) and higher (eucaryotic) life form bio­ clude mixed cultures arising naturally from propa­ chemical control systems and cell cycles to allow gation of unstable recombinant microbial strains. proper rDNA catalyst preparation and to provide 14. Biological waste treatment continues to be taught be-

170 CHEMICAL ENGINEERING EDUCATION cause: (i) it represents (still) the largest, success­ fully operating microbial reactor systems in exist­ ence, (ii) it provides, in the aer.ated (activated) sludge process, a clearly studied, mixed population [eJb#I letters system (iii) it illustrates beautifully, with an anaerobic reactor simulation, the enormous sensi­ "NONADIABA TIC" A MISNOMER? tivity of actual mixed culture systems to process upsets of flow rate and feed composition. Dear Editor: 15. Homework problems for every topic have been ex­ I noted in a recent issue of the AIChE Journal tensively revised, especially to add a number of brief calculational essays and to edit or remove some un­ (April '85) an article with the word "Nonadia­ duly long problems. batic" in the title. I have always (for the past 25 years) called In spite of the apparent burden of covering such a term simply "diabatic." The term non­ an increasing number of topics in increasing depth, adiabatic is redundant since the a prefix in adia­ the biochemical engineering course has actually batic means nonadiabatic, i.e. without heat been strengthened and streamlined by two peda­ transfer. gogical approaches: (a) the topics build on the The prefixes a, ab, or an in English all infer preceding topics, thus the early material contains a negative connotation as in: primarily those key items needed in latter chap­ ters, and (b) the nomenclature has been shifted aneroid without fluid (in a barometer) and recodified into a vocabulary most familiar to ascorbic acid anti-scorbutic (Vitamin C) anhydrous without water chemical engineers. For example, metabolism anesthesia without feeling leads to stoichiometry and energy balances, bio­ asymmetrical not symmetrical logical products are recovered in separation unit anisotropic not isotropic operations, and bioprocess economics is based on agnostic without knowledge (of God) standard chemical plant cost estimating termin­ atheist does not believe in God abnormal not normal ology. etc. Throughout, original presentations of biologi­ cal background have been pruned and revised to Thus to use the term non-adiabatic, literally present major concepts as clearly and concisely as means non-non-diabatic and the two negatives possible. Introductory summaries are provided for cancel each other to yield the simpler term dia­ all of the less familiar topics (e.g., microbiology batic. No one would think of using non-abnormal and cell biology, biochemistry, enzyme kinetics to replace the word normal, or non-anisotropic for and structure, metabolism, genetics and DNA, bio­ isotropic, so why not diabatic for non-adiabatic? chemical control systems) . As before, no chemical I would appreciate your publishing this letter engineering material not already available to the and maybe someone will know the answer to these chemical engineer by the end of the junior year questions: (stoichiometry, energy balances, transport phe­ nomena, thermodynamics, chemical kinetics) is 1. Have you ever heard of a reactor or a proc­ assumed. Our experience indicates that eager stu­ ess with heat transferred to or from it be­ dents, willing to undertake new vocabularies in ing called "diabatic"? short order and to absorb the considerable amount 2. Any references in books or journals to a of qualitative material prior to "comfortable diabatic reaction or other process? quantitation" in equation form, will find the sub­ ject and its organization fascinating and exciting. In any case, I propose that Chemical Engi­ neers use the simple term "diabatic" to replace this awkward, redundant and more complex ex­ REFERENCE~ pression non-adiabatic, at least for chemical re­ 1. J. E. Bailey and D. F. Ollis, "Biochemical Engineer­ actors. ing Fundamentals," Chem. Eng. Education, Fall, 1976. I have not coined a new word since Merriam­ 2. "Commercial Biotechnology; An International An­ Webster's Unabridged Dictionary lists diabatic alysis" (OTA-BA-214) Office of Technology Assess­ ment, Washington, D.C. 20510 (January, 1984). A and defines it as involving the transfer of heat larger glossary appears on pages 586-597. (For sale ( opposed to adiabatic) . by Superintendent of Documents, U.S. Gov't. Print­ Allen J. Barduhn ing Office, Washington, D.C. 20402.) Syracuse University

FALL 1985 171