2
The Challenge

The biotechnology industry has been successful in translating basic research in the biological sciences and molecular biology into very high-value-added products. Particular emphasis has been on biopharmaceuticals for the treatment of such catastrophic illnesses as cancer, heart disease, and kidney diseases. A second generation of bioproducts is now being developed whose price-cost difference will be much lower: intermediate-value biopharmaceutical and pharmaceutical products, specialty chemicals, and materials and chemicals derived from renewable resources. Bioprocess engineering is critical for the economical development of the new products, particularly as the difference between price and cost decreases and profitability becomes an important function of production costs.

2.1 TRANSLATING SCIENCE INTO PRODUCTS

''Science and the application of science ... are linked as the fruit is to the tree.''

Louis Pasteur

Engineering innovation and the development of enabling technologies are the essence of translating science into products. The first step of innovation is discovery of a phenomenon, such as the efficacy of a Penicillium culture against Staphylococcus aureus (1928), the ability of xylose isomerase to catalyze formation of fructose from glucose (1957), or the ability of enzymes (known as Type II restriction endonucleases) to cleave specific sites in DNA (1970). Recombinant-DNA technology enables development and manufacture of products that would otherwise not be possible.

The next essential element of engineering innovation is an economic



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Putting Biotechnology to Work Bioprocess Engineering 2 The Challenge The biotechnology industry has been successful in translating basic research in the biological sciences and molecular biology into very high-value-added products. Particular emphasis has been on biopharmaceuticals for the treatment of such catastrophic illnesses as cancer, heart disease, and kidney diseases. A second generation of bioproducts is now being developed whose price-cost difference will be much lower: intermediate-value biopharmaceutical and pharmaceutical products, specialty chemicals, and materials and chemicals derived from renewable resources. Bioprocess engineering is critical for the economical development of the new products, particularly as the difference between price and cost decreases and profitability becomes an important function of production costs. 2.1 TRANSLATING SCIENCE INTO PRODUCTS ''Science and the application of science ... are linked as the fruit is to the tree.'' Louis Pasteur Engineering innovation and the development of enabling technologies are the essence of translating science into products. The first step of innovation is discovery of a phenomenon, such as the efficacy of a Penicillium culture against Staphylococcus aureus (1928), the ability of xylose isomerase to catalyze formation of fructose from glucose (1957), or the ability of enzymes (known as Type II restriction endonucleases) to cleave specific sites in DNA (1970). Recombinant-DNA technology enables development and manufacture of products that would otherwise not be possible. The next essential element of engineering innovation is an economic

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Putting Biotechnology to Work Bioprocess Engineering opportunity arising from a potential societal benefit. The opportunity can be made more attractive by an emergency or economic perturbation that produces unique market conditions, usually for a short period. For instance, World War II provided the incentive in the case of penicillin; high sugar prices and an abundance of corn in 1965–1980 promoted development of the U.S. high-fructose corn syrup industry based on glucose isomerase; and an anticipated decline in U.S. meat consumption, and therefore in the availability of animal pancreas, might have prompted Eli Lilly Co. to convene a meeting in 1976 on applying recombinant-DNA methods to produce insulin. The jump from science to technology involves the convergence of preparation with opportunity, when market forces (and in some cases government policy) present economic incentives for introducing a new product. In the case of penicillin, World War II prompted government to promote industrial research on antibiotic production and other subjects through taxation policies. High-fructose corn syrup production was encouraged by a government-defined minimal sugar price. Recombinant insulin was developed rapidly to meet the needs of millions of Americans. In all three cases, a research infrastructure, a fundamental knowledge of the product, and technically capable scientists and engineers in universities, industry, and national government laboratories were in place when the opportunity arose. The economic impacts have been tremendous. It can be argued that the development of penicillin led to the current U.S. pharmaceutical industry in which the top eight companies had sales of $31.6 billion in 1991. However, 15 years passed between discovery and application, and another 10 years before antibiotic production became an established large-scale endeavor. The lag between discovery of glucose isomerase and the first process was 10 years. Glucose isomerase and, later, application of very-large-scale liquid chromatography resulted in the growth of enzymatically produced 42%–55% high-fructose corn syrups from none in 1966 to 12.7 billion pounds (dry basis) in the United States in 1991 (Antrim et al., 1979; Buzzanell et al., 1992). Recombinant insulin came on the market 12 years after the discovery of Type II restriction endonucleases in 1970; this rapidity reflects not only the technical prowess of the companies involved, but also their ability to deal effectively with the federal government's regulations. Today there are 16 approved biotechnology-produced drugs and vaccines, which would not exist in the absence of molecular biology. Another 120 are in various stages of federal review and approval (Burrill and Lee, 1991). The developments in biopharmaceuticals suggest a clear need for long-term commitment to preparing scientists and engineers to deal with translating science into production. The need is particularly clear in the light of the projected 10-to 20-fold growth of the industry in the next 10 years, developments in bioprocessing of renewable resources, the desire to apply biotechnology to environmental issues, and the nation's goal of a

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Putting Biotechnology to Work Bioprocess Engineering long-term human presence in space. Meeting that commitment will require a strong program for research and education related to bioprocess engineering. Vignette 1 illustrates the key elements of applying bioprocess engineering for "putting biology into the bottle." Tremendous screening efforts coupled with a mechanistic understanding of disease-causing processes are needed to identify new therapeutic agents. After discovery of a new substance, enough of it must be obtained for testing with animals and later, if Vignette 1 The Scaleup of Penicillin Production—Parallels to the New Biotechnology? Alexander Fleming showed that Staphylococcus cultures were inhibited by growing colonies of Penicillium notatum in 1928 and then identified the antimicrobial activity as due to a secreted substance, i.e., penicillin. Florey and Chain rediscovered penicillin in 1939 and found it to be most effective in treating infections. However, the supply was limited, chemical synthesis proved to be difficult, and the war complicated developmental efforts in England. Scaleup to obtain larger amounts was initiated by a joint Anglo-American effort and included the involvement of Merck, Pfizer, Squibb, and U.S. government laboratories. The demand for penicillin in 1941–1943 exceeded the amount that could be produced by surface culturing techniques, even when higher-yielding strains of P. notatum and P. chrysogenum were used. The microorganisms were first grown on the surface of moist bran in milk-bottle-size vessels having a volume of 1–2 L (Shuler and Kargi, 1991). The need for a more efficient manufacturing approach quickly became apparent. The solution to scaleup evolved from a combination of applied microbiology and fermentation engineering. The discovery, on a moldy cantaloupe in Peoria, Ill., of a strain of P. chrysogenum that could be grown in a submerged fermentation was an enabling factor. Scaleup from 1 L to 100,000 L followed (Aiba et al., 1973). Numerous engineering challenges needed to be addressed, from maintaining growing conditions that excluded contaminating organisms to aerating large volumes of fermentation broth. The penicillin itself was labile and thus required development of appropriate purification procedures. As a result of cooperative efforts between government and industry, full-scale production was quickly achieved with microbiologists and chemical engineers working together—i.e., the first bioprocess-engineering teams. Government tax policy further encouraged major industrial involvement in what was considered, at the time, to be the very risky technology of pharmaceutical manufacture by large-scale, submerged, aerated fermentation. Penicillin productivity increased from about 0.001 g/L in 1941 to over 50 g/L by 1970; that led to a decrease in cost by a factor of more than 1,000 and the classification of penicillin as a bulk material. In the meantime, different types of antibiotics, effective against a range of diseases, were developed; 5,500 were identified between 1945 and 1981, of which about 100 reached the market (Hacking, 1986).

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Putting Biotechnology to Work Bioprocess Engineering warranted, for treating humans. Bioprocess engineering is critical in rapidly scaling up production of a promising new therapeutic agent so that sufficient quantities of an ultrapure substance are available for testing. If the trials are successful, manufacturing capability must be developed quickly to make the new drug widely available. Over 100 biopharmaceutical products are in various stages of clinical testing (OTA, 1991). That is almost as many antibiotics as have been brought to market within the last 50 years. The number illustrates the acceleration of pharmaceutical development and the coming need for increased numbers of engineers who have cross-disciplinary training in biology and engineering to scale up the manufacture of new classes of therapeutic compounds. 2.2 BIOPHARMACEUTICALS Total global sales of all pharmaceuticals are about $150 billion a year, of which about one-third is in the United States (Abelson, 1992). The pharmaceutical products of the new rDNA and hybridoma technology currently make up a small but rapidly growing fraction of the total (estimated U.S. sales, $4 billion in 1991). By the end of this century, it is estimated that total U.S. sales of these new products will exceed $30 billion a year, dominated by therapeutic proteins. This growth is anticipated also to include chemotherapeutic compounds, polysaccharides, vaccines, and diagnostics, as well as therapeutic proteins. This exciting potential has spawned hundreds of new biotechnology companies in the United States. In June 1991, the market capitalization of the industry was $35 billion (Burrill and Lee, 1991). Clearly an important new industry has been born. However, the recent purchase of 60% of Genentech, Inc., by Roche Holdings, a Swiss company, and the intensive activity in the Japanese pharmaceutical industry suggest that Americans cannot take for granted our current lead in this important and expanding field. The goal of this section is to assess the role of bioprocess engineering in determining competitive position in the biopharmaceutical industry. 2.2.1 New Technology Required for Biopharmaceuticals The new industry has, to a large extent, required fundamentally new bioprocess technology. A few protein pharmaceuticals had previously been marketed, but RDNA and hybridoma technology has brought such dramatic changes to the manner of production and the range of possible products that a substantially new technology base has had to be developed (see Vignette 2). One characteristic of the new technology is that it enables the design and optimization of the production organism to an unprecedented degree. Another is that the technology must apply to a class of products that, al-

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Putting Biotechnology to Work Bioprocess Engineering Vignette 2 Bioprocess Engineering for Early Biopharmaceuticals engineering: the application of science and mathematics by which the properties of matter and the sources of energy in nature are made useful to people in structures, machines, products, systems, and processes. [From Webster's Ninth New Collegiate Dictionary, Springfield, Mass.: Merriam-Webster Inc., 1984] The tremendous power of rDNA and hybridoma technology has dramatically expanded our capabilities for the development of protein pharmaceuticals. At the same time, it has blurred the boundaries between those who pursue basic biological knowledge and those who apply that knowledge to bring beneficial products and services to society. The definition of "engineering" shown above now describes the design and construction of a new living organism just as well as it describes the design and construction of the bioreactor used to cultivate it. In fact, one of the biggest challenges in achieving optimal benefit from this technology is to bring about the synergistic combination of the skills of the biologist, the biochemist, and the bioprocess engineer. The first rDNA product to be approved, human insulin, provides an illustrative example. By today's standards, the first manufacturing process, developed in the early 1980s, was quite primitive. Insulin is a protein composed of two polypeptide polymers connected by disulfide bonds. The first production process was initiated at Genentech, Inc., and was improved and implemented at Eli Lilly and Co. The process produced each polypeptide separately. First, the individual polypeptides were expressed and accumulated inside an engineered bacterium. However, the polypeptides expressed by themselves were not stable in exposure to the degradative enzymes found inside the bacterium, so they had to be expressed as a small portion of a much larger molecule. The large fusion protein then aggregated into a stable particle inside the cell. To obtain the desired polypeptide, the particle then had to be isolated and solubilized and the fusion protein cleaved with a hazardous chemical that produced many side products. In fact, most of the recombinant protein produced either was not the desired polypeptide or was modified and therefore had to be discarded. The fraction of the total that was the desired polypeptide then had to be purified and combined with its partner (which had been produced from another bacterium in a similar manner), and the resulting insulin molecule was purified again. Implementing that process was an impressive bioprocess-engineering accomplishment. The first process that duplicated all the manufacturing steps was put into mass production in 1982; it was soon improved to use the single-step concept based on proinsulin. The most significant advances were achieved by implementing new ideas, rather than just implementing careful engineering in the traditional sense of scaleup and cost reduction. The new process depends on advances in the biology of protein expression and on new developments in the biochemistry of protein modification. Now both polypeptides are expressed simultaneously in the same bacterium. They are initially connected by another peptide, which guides their assembly. The connecting peptide is then removed by an enzymatic treatment—a step that turned out to be the most difficult task in developing the process. This process is quite similar to the way that insulin is made naturally. Without a strong partnership of

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Putting Biotechnology to Work Bioprocess Engineering biochemists, molecular biologists, microbiologists, and bioprocess engineers, this new and efficient process would not have been achieved. Both scientists and engineers were required to make this important advance in manufacturing, with 300 employees at Eli Lilly laying the groundwork (in 1986) for mass-producing biotechnology products (Eli Lilly and Company, 1986). A similar story can be told about the development of human growth hormone. The first rDNA human growth hormone was made with a process in which the protein accumulates in the bacterium. Large quantities of bioactive growth hormone are obtained in this way, but an artifact of intracellular expression produces a molecule with one extra amino acid (192 instead of 191). Although the enlarged product proved to be safe and effective, a growth hormone with the authentic human amino acid sequence was desired. Both Genentech and Eli Lilly were able to make it. It is not publicly known how Eli Lilly accomplished this for its commercial process, but its publications suggest that it produced a fusion protein in the bacterium and then specifically removed the amino acid extension to produce the authentic growth hormone sequence. Genentech solved the problem by engineering the bacterium to transport the growth hormone from the bacterium's central compartment into another portion. As a result of the process of translocation, the correct molecule is formed. Again, by more closely mimicking the natural production mechanism, a superior production process and an improved product resulted. When the opportunity arose to develop tissue plasminogen activator (t-PA) as an important treatment for heart-attack patients, bacterial expression was again evaluated. This time, the molecule was too large and complex. Even with rapidly developing bacterial technology, a combination of altered expression and protein modification could not reliably produce authentic bioactive t-PA. But the ability to express foreign proteins in mammalian cells had just been developed, and human t-PA was expressed in an active form by cultured, rDNA-modified Chinese hamster ovary cells. The experience with erythropoietin (EPO) was similar, but the technology for large-scale culture of mammalian cells was in its infancy. In fact, the current situation is surprisingly similar to that in the early days of the antibiotic industry 50 years before. Just as the early antibiotic-producing cultures were grown in banks of milk bottles, so the new rDNA mammalian cells were grown in banks of bottles called roller bottles, although robotics (not available in 1943) are used in the modern EPO process. The amount of EPO needed is small, because EPO is effective in minute doses. Hence, EPO production was scaled up simply by automating and expanding the roller-bottle facility. Amgen and its Japanese partners used an approach relying predominantly on traditional engineering. Genentech, however, in developing t-PA, was developing a product that required a much larger dose per treatment. Not only did the cost per unit of protein need to be proportionately lower, but the production requirements were also much larger. Thus, an alternative approach was required. Just as the early antibiotic industry used larger stirred vessels to make more product at a lower cost, so Genentech developed a process that allowed the production of t-PA in large stirred vessels. However, it was not a matter of merely placing the cells in a larger vessel. The first cultured cells grew optimally only when they were attached to a solid surface. Engineering solutions were available, but the best solution was to select new cells that not only made more t-PA, but also grew well when suspended in a liquid culture medium. Many other improvements were needed to produce a workable mammalian cell-culture process for t-PA, and engineering

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Putting Biotechnology to Work Bioprocess Engineering was a critical partner in the scaleup activity. Most of the additional improvements also resulted from the combined efforts of biologists, biochemists, and bioprocess engineers. Those examples focus on the part of the production process that first makes the desired protein. But that is only the beginning. The proteins must be purified, not only from the hundreds of other proteins produced by the RDNA organism, but also from variants of the desired protein that differ in subtle but important ways. It takes a major effort merely to be able to detect and measure the two classes of contaminants. One testimony to the success of the purification and analytical efforts is that the contaminating proteins from the producing organism are often measured in the parts-per-million range—a degree of protein purity that was unheard of before the advent of the RDNA-protein pharmaceutical industry made it possible to manufacture proteins in quantities a million times greater than the amounts found in humans. These processes were developed and implemented with a multidisciplinary team approach that used bioprocess engineering in all phases of product development, manufacturing scaleup, and plant operations. though diverse in biochemical characteristics, must be uniform in its sensitivity to undesired chemical, physical, and enzymatic modification. Furthermore, regulatory and safety demands have required the purification of these protein pharmaceuticals to an unprecedented degree. The early products selected for development either were quite potent or entered established markets. Examples of marketed RDNA-protein pharmaceuticals are listed in Table 2.1. With the possible exception of insulin (which entered a more mature market and for which some process technology was already established), these proteins have a relatively high unit value and have been needed in small quantities. The first challenge for bioprocess engineering was thus to establish the foundation technologies required to produce protein pharmaceuticals of acceptable quality, albeit at a high cost and on a small scale. As the industry continues to develop, however, many of the newer product candidates must be manufactured at much lower cost and higher capacity. They include various therapeutic monoclonal antibodies and IGF-1 and range to such items as human serum albumin and human hemoglobin. The latter two products will probably require selling prices of less than $10/g and manufacturing capacities greater than 10,000 kg/yr. Thus, protein pharmaceuticals have ranges of more than a factor of 105 in unit value and projected production volume. For the lower-unit-value products, effective bioprocess engineering might well spell the difference between success and failure. Expansion of the present technology base by well-coordinated, multidisciplinary bioprocess development will be essential for the required reduction in manufacturing cost and increase in capacity.

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Putting Biotechnology to Work Bioprocess Engineering Table 2-1 Unit Values and Relative Production Quantities for Selected Approved Biopharmaceuticals Product Year Approved Approximate Selling Price, $/g Amount of Product for $200 Million in Sales, kg Human insulin 1982 375 530 Growth hormone 1985 35,000 5.7 Tissue plasminogen activator 1987 23,000 8.7 Erythropoietin 1989 840,000 0.24 Even for products with high unit values, effective bioprocess engineering will be an important competitive factor. Often several companies are in competition to develop the same drug. The company with the first approval or the more attractive product usually has a much stronger competitive position. A distinct advantage will be gained if rapid process development can allow earlier entry into clinical trials. Additional advantage will be gained if the production process results in a product whose biochemical characteristics are more acceptable to the regulatory authorities and to the customer. In summary, the challenge for U.S. biopharmaceutical companies is to develop quickly the technological tools and the processes that produce superior products at acceptable costs and in the required amounts. 2.2.2 Bioprocess Engineering Requires Many Disciplines Bioprocess development for biopharmaceuticals involves all aspects of generating a safe, effective, and stable product. It begins with the biological system, continues with product isolation and purification, and finishes when the product is placed in a stable, efficacious, and convenient form. The product is initially derived either directly or indirectly from a living organism. Thus, process development starts with the development of the biological system. It is usually a living organism that expresses the desired protein; but it might be an enzyme for protein modification or an antibody for immunoaffinity purification. RDNA and hybridoma technology allow the biological system to be optimized for maximal formation of the product, for facilitation of downstream processing, for high product quality, and for improved interaction with the production equipment. In this phase of bioprocess engineering, many disciplines must be applied, including molecular biology, genetics, biochemistry, analytical chemistry, and bioprocess engi-

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Putting Biotechnology to Work Bioprocess Engineering neering. Thus, engineers become full partners with experts trained in the bioscience disciplines in developing and scaling up manufacturing technology for biopharmaceuticals. As the task of process development proceeds through isolation, purification, and formulation, multidisciplinary approaches continue to be advantageous. Bioprocess engineers combine their skills with those of biochemists and analytical biochemists to develop the optimal process. The multidisciplinary task of bioprocess engineering is usually applied to developing processes for the production of biopharmaceutical products, but it can also be applied to the discovery of new products. For example, automated screening methods that use purified cell receptors can be developed. More efficient methods for producing and isolating families of precisely modified proteins can be devised, and the technology required to produce protein pharmaceuticals efficiently with mammalian cells can be applied to develop cell-based assays to screen for product activity or toxicity. 2.2.3 Opportunities The growing biopharmaceutical industry is facing many challenges. Some are technical challenges that can be met by more effective bioprocess engineering or that impede the progress of bioprocess-engineering development. Others are nontechnical, but are strong impediments to the ability of bioprocess engineering to contribute to the competitive position of the U.S. biopharmaceutical industry. The 1991 Office of Technology Assessment report Biotechnology in a Global Economy describes three kinds of research: basic, generic applied, and applied research. From the perspective of the biopharmaceutical industry, the first appears to be well addressed by existing university and government laboratory programs. The last, applied research, is in most cases effectively and appropriately addressed by the private sector during the development of individual products. It is the middle category, generic applied research, that will be most critical for the optimal exploitation of bioprocess engineering in the U.S. biopharmaceutical industry. It is also called "bridge" research, because it bridges the gap between scientific knowledge and practical application. It is often too applied for the scientific disciplines to address, but too risky or requiring too long a period for results for companies to pursue. Table 2.2 lists key technical challenges that the biopharmaceutical industry faces today; most are in the category of generic applied research. Generic applied research has proved to be most valuable for RDNA-pharmaceutical development. It includes biological and biochemical research that a company cannot afford to do but that fits into the mission of the National Institutes of Health (NIH) in the context of a health agency.

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Putting Biotechnology to Work Bioprocess Engineering Table 2.2 Key Technical Challenges in Biopharmaceuticals 1. To develop methods for rapid characterization of biochemical properties, efficacy, and immunogenicity of protein pharmaceuticals (methods should be developed to facilitate rapid development of new products and improvement of processes for existing products). 2. To improve process control and process productivity from genetically altered mammalian cell products. 3. To develop high-resolution protein purification technologies that are relatively inexpensive, are easily scaled up, and have minimal waste-disposal requirements. 4. To expand the range of biopharmaceuticals that can be produced with prokaryotic cells or nonmammalian expression systems, which allow the use of less expensive media and have lower capital requirements than current technologies. 5. To expand technology for stable liquid formulations and nonparenteral administration (or sustained release) of protein pharmaceuticals. 6. To increase knowledge of chemical and biochemical reactions that modify proteins during production and storage. Indeed, in addition to the National Science Foundation (NSF) and other federal agencies that address bioprocess-engineering and biomanufacturing-technology research, NIH has made important contributions to health-product-related, generic applied research in the past, and it is an important component for the future, as indicated in the Federal Coordinating Council for Science, Engineering, and Technology (FCCSET) report (1992). Table 2.3 lists some nontechnical challenges related to the ability of bioprocess engineering to contribute to the competitiveness of the U.S. biopharmaceutical industry. Table 2.3 Key Nontechnical Challenges in Biopharmaceuticals 1. To reduce time required for approval of new protein pharmaceuticals and improved processes by improving communication between industry and Food and Drug Administration, reducing response times after submissions, and ensuring timely resolution of generic safety, efficacy, and manufacturing issues. 2. To increase funding opportunities for intensive and sustained technology development required for developing generic applied technology (includes establishing large manufacturing facilities required for relatively large-volume, low-value-added pharmaceuticals). 3. To provide improved training to enhance integration of many disciplines required for optimal bioprocess development and provide training programs specifically to prepare engineering students for biopharmaceutical industry.

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Putting Biotechnology to Work Bioprocess Engineering 2.3 THE ENVIRONMENT The four major categories of biotechnology applications involved in solving environmental problems are biomaintenance, bioremediation, waste minimization, and environmental monitoring. There are selected opportunities for bioprocess engineering in applying microbe- or enzyme-based treatment protocols to large land masses. Expansion of the knowledge base of different kinds of organisms and their contributions to the ecosystem will help to identify specific mechanisms in the complex, naturally occurring detoxifying activity of our environment. Exploration of biodiversity and use of newly recognized organisms will benefit from this activity. Tremendous engineering challenges must be met if we are to implement rate-enhanced processes in synergy with indigenous phenomena to detoxify the soil, water, and air biologically. The toxins are often dilute and dispersed over a large area. An engineering-bioscience interface will be critical in any biology-based approach, given the need for new genetically designed organisms and systems for cleaning up of site-specific toxins at economically feasible bioremediation facilities. Increased training of microbial and biochemical ecologists, biohydrogeologists, and bioprocess engineers will be useful in providing a cadre of persons to solve these problems. Such an activity, in many cases, would be a logical extension of existing programs and skills in agriculture and civil engineering. It is the committee's opinion that the impact of bioprocess engineering on environmental issues is of great significance and warrants an independent study and analysis. 2.4 CONVERSION OF RENEWABLE AND NONRENEWABLE RESOURCES Most of the applications and potential applications of bioprocessing related to renewable and nonrenewable resources involve large-scale operations and products of relatively low value. The costs of processing have to be low, and the decision to use bioprocessing for such raw materials must be made with care. Precedents for successful (commercial) large-scale bioprocess engineered processes include the corn wet-milling industry, the fuel-alcohol industry, the mining industry (specifically copper extraction), the wastewater-treatment industry, the acetone-butanol fermentation industry (stopped in the West in 1955, but still practiced in China), and fermentation industry that manufactures amino acids and other organic acids (including citric and lactic acids). 2.4.1 Renewable Resources The most abundant renewable material is lignocellulose. Wood, agricultural residue (corn stover, straw, etc.), plants grown deliberately for bio-

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Putting Biotechnology to Work Bioprocess Engineering Vignette 4 Bioprocess Engineering for High-Volume Products: The Case of Corn and the Wet-Milling Industry High-volume products from corn use bioprocess-engineering principles in applying enzymes, fermentation, and separations for production of industrial sugars and fuel alcohol. Fermentation alcohol and enzyme-based production of HFCS predate commercial ventures based on the new biotechnology and provide case studies for large-scale adaptation of bioprocesses in an established field where competitive options are readily accessible. The enabling discovery of xylose (glucose) isomerase was based on classical microbiology and enzymology, rather than recombinant DNA, but the history of its translation into a large-scale manufacturing technology provides useful corollaries for events that might shape the bioprocessing industry in the next 10 years. The glucose-isomerizing capability of xylose isomerase, an enzyme that had been discovered in 1954, was observed at the U.S. Department of Agriculture (USDA) Peoria laboratory in 1957 (Marshall and Kooi, 1957). However, arsenate was required as a cofactor, and this enzyme was later found to be a glucose phosphate isomerase, rather than a glucose isomerase. Other enzymes that did not require arsenate for activity were quickly identified by researchers in Japan, and that opened the possibility of food use. Clinton Corn Processing Company of Iowa, in 1966, entered into an agreement with the Japanese government, commercialized a glucose-isomerizing process, and shipped the first enzymatically produced 42% fructose syrup by 1967. A. E. Staley licensed technology from Clinton and also entered the market. By 1974, U.S. production grew to 500 million pounds a year as sugar prices peaked at $0.30/1b, thereby encouraging investment in new plants. The Japanese technology made it possible to produce xylose isomerase from Streptomyces rubiginosus, using corn bran as an economical source of xylose to induce the microbial production of the enzyme. The resulting enzyme was thermally stable and had high glucose-isomerizing activity at the industrially relevant conditions of 65°C and pH 7.3 (Lloyd and Horwath, 1985). Selection of the appropriate microorganism and adaptation of media composition facilitated the enzyme's economical production. The first processes consisted of adding enzyme to batches of glucose syrup. The recognition that glucose isomerase could be immobilized by sorption on DEAE cellulose or by entrapment in heat-fixed, pelletized cells (the enzyme has a molecular weight of about 170,000) improved production economics by providing stable, reusable enzyme (Lloyd and Horwath, 1985; Lloyd and Khaleeluddin, 1976). The current industrial practice is to fix or entrap the enzyme on a solid material, which, in turn, is packed into a fixed bed. The glucose syrup, derived from enzyme hydrolysis of corn starch, is then passed over a fixed bed of immobilized isomerase, which converts the glucose to fructose. The current practice owes much to bioprocess engineering, including technology for enzyme immobilization, bioreaction engineering and kinetics, development of physically and chemically stable supports (onto which the enzyme is fixed), and optimal design and operation strategies for a protein-based catalyst. The basic patent coverage for use of xylose isomerase to convert glucose to fructose was held invalid in 1975. That provided incentives to develop alternative processes and process improvements and was followed by an industry-wide, major

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Putting Biotechnology to Work Bioprocess Engineering construction boom. Next came overcapacity and low profitability in 1977, when sugar prices fell to $0.12/lb and annual U.S. production had grown to over 3 billion pounds. Those events could find analogies in future developments of the biopharmaceutical industry, where process innovations should be anticipated. Separation technology was also important in the growth of HFCS production. The introduction of very-large-scale liquid chromatography enabled partial separation of glucose from fructose and the production of 55% HFCS (abbreviated HFCS-55). HFCS-55 had sweetening properties nearly equivalent to those of inverted (hydrolyzed) sugar from sucrose and began to appear in soft drinks in 1980. HFCS syrups, based principally on corn grown in North America, now dominate the industrial sugar market, with about 12.5 billion pounds of 42% and 55% HFCS shipped in 1991 in the United States (Buzzanell et al., 1992). Between 1978 and 1990, the industry grew by 350%. The HFCS industry, with its large scale of operation and infrastructure, was largely responsible for the development of large-scale production of ethanol, another product derived from starch, and particularly from corn. Fuel-ethanol production involves fermentation of glucose to ethanol with the yeast Saccharomyces cerevisiae and requires very large fermentors. A cascade system, now operated by most large U.S. plants, evolved in which there is continuous flow through a series of tanks, approximating a continuous fermentation (Hacking, 1986). After fermentation, the solids are separated from the fermentation broth, dried, and sold as animal feed. Recovery of ethanol entails distillation to approximately 92% alcohol followed by azeotropic or extractive distillation. Discovery of corn as a suitable drying agent for alcohol in 1979 (Ladisch and Dyck, 1979) and its later adaptation on a commercial scale by a major alcohol producer in the mid-1980s provided an environmentally acceptable and energy-efficient method of using corn in a manner compatible with a fixed-bed sorption process for large-scale alcohol-drying (Lee et al., 1991). Fundamental research in ethanol production was encouraged through federal funding of research through the Department of Energy (DOE), USDA, and NSF from about 1975 to 1985. That period saw examination of the engineering, enzyme technology, biological science, and economics that would facilitate economic conversion of cellulose to various oxygenated chemicals (including ethanol) via fermentable sugars. Direct fermentation of cellulose to ethanol was proved to be technically feasible (Wang et al., 1983). Complete conversion of cellulose to sugars via enzyme hydrolysis of pretreated biomass was demonstrated (Ladisch et al., 1978). Recombinant methods and applied microbiology resulted in large gains in microbial productivity and cellulose saccharification activity of cellulolytic enzymes (Mandels et al., 1981; Montenecourt et al., 1981). Organosolv pretreatments further enhanced hydrolysis prospects (Holtzapple and Humphrey, 1984; Avgerinos and Wang, 1983). The complex mechanisms of lignin degradation were elucidated (Tien and Kirk, 1984). The potential of xylose isomerase in fermenting pentoses (a major constituent of cellulosic materials) to ethanol was shown in a fermentation process (Gong et al., 1981). Elucidation of the complex metabolism associated with ethanol fermentation resulted in methods for improving ethanol-fermentor design (Maiorella et al., 1984) and bacterial fermentation of pentoses to ethanol (Ingram, 1992). Only a small part of that research, however, found its way into industrial practice, partly because the precipitous decrease in oil prices from $40/barrel to $20/ barrel in 1985–1986 removed many of the visible, short-term economic and strategic

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Putting Biotechnology to Work Bioprocess Engineering incentives for moving rapidly to a renewable-resource-based industry. Nonetheless, the period fostered training of bioprocess engineers, who received a cross-disciplinary education as part of research projects related to renewable resources. Many of them joined the growing biopharmaceutical industry in bioprocess-engineering capacities. 2.4.3 Coupled Synthesis Gas One strategy for using biomass in fuel and chemical production is to reform it thermally to synthesis gas and then to use the synthesis gas in existing petrochemical facilities. The development of appropriate technology for such a process will require elements of bioprocessing and biology. The development of biomass sources that are most appropriate for reforming, converting the biomass to the physical form most appropriate for reforming, and reuse or disposal of the byproducts of the process will require an infusion of bioprocess-engineering concepts. 2.4.4 Enhanced Oil Recovery Microbial polysaccharides (xanthan and guar gums and related polysaccharides) are an important component of enhanced oil recovery (EOR). There is an opportunity to develop new additives for down-hole viscosity Table 2.4 Biochemical Processing Related to the Corn-Refining Industry Product Company Capacity, tons/yr Vitamin C Takeda Chemical Products, United States 5,500 Enzymes Genencor International (Cedar Rapid, Iowa) ($60 million facility) Citric acid Cargill (Eddyville, Iowa) 27,500 Citric acid ADM (Decatur, Ill.) Not known Lysine Heartland Lysine (Eddyville, Iowa) 20,000 Lysine Biokyowa (headquarters at Chesterfield, Mo.) 13,000 Lysine ADM (Decatur, Ill.) 62,500 Tryptophan and threonine (planned) ADM (Decatur, Ill.) Not known Lactic acid, feed-grade penicillin and bacitracin, and biotin (planned) ADM (Decatur, Ill.) 10,000–20,000 (planning stages)   Source: Anonymous, 1991.

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Putting Biotechnology to Work Bioprocess Engineering modification through genetic engineering, to develop on-site production techniques for remote environments, and to develop techniques for other aspects of EOR (surfactants for heavy-crude slurry transport and site remediation). These problems can be approached through bioprocess engineering. 2.4.5 Opportunities The bioprocessing of renewable and nonrenewable resources requires the application of multiple disciplines in a cross-disciplinary approach. As in the case of biopharmaceuticals, products are derived either directly or indirectly from living organisms. Unlike those in the biopharmaceutical sector, however, bioprocess engineers dealing with renewable and nonrenewable resources are involved with high processing volumes and often design and operate large installations. The processes themselves must still be reliable, robust, and cost-effective. The goal is to generate products from renewable resources or to produce microbial and other biocatalysts for use in processing renewable or nonrenewable resources to obtain value-added products. Economies of scale and scalable equipment are important, if not critical, process characteristics. This sector of the biotechnology industry also produces specialty products (enzymes, amino acids, organic acids, and animal-health products) for which the processing technology and regulatory issues are similar to those associated with high-volume biopharmaceutical products, but the ratio of market price to processing cost can be much lower. Production efficiency, as well as product quality, is paramount. A key function of bioprocess engineering is the design, operation, and improvement of manufacturing technology to yield bioproducts at high volume and acceptable costs. Success will require bioprocess engineers to become partners with molecular biologists and geneticists (both plant and microbial), biochemists, carbohydrate chemists, wood chemists, food scientists, analytical chemists, and microbial physiologists. Substantial generic applied research is needed to help foster the development of the U.S. bioprocessing industry for manufacture of specialty products. As listed in Table 2.5, the technical challenges will require cooperative efforts among university researchers, industry, and national government laboratories and research institutions of USDA, DOE, the Environmental Protection Agency, the National Aeronautics and Space Administration (NASA), the National Institute of Standards and Technology, and the Department of Defense (DOD). The challenges listed in Table 2.5 present opportunities related to renewable resources to carry out generic applied research in the universities and government laboratories, because these technologies will have wide impacts on the use of land and natural resources.

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Putting Biotechnology to Work Bioprocess Engineering Table 2.5 Key Technical Challenges in Bioprocessing of Renewable Resources 1. To develop inexpensive cellulose pretreatment and saccharification processes effective with lignocellulosic materials on large scale with environmentally compatible methods. 2. To develop fermentations capable of converting pentoses to value-added products at yields, rates, and extents similar to those obtained for glucose with yeast and to increase product concentrations achievable in both hexose and pentose fermentations. 3. To develop more efficient separations for recovering fermentation products, sugars, and other dissolved materials from water, i.e., lower cost of separating water from product in fermentation broth. 4. To develop processes for large-scale inoculation, control, and propagation of microorganisms in surface culture (e.g., treatment of wood chips and bioremediation of soils) and solid substrate fermentation. 5. To increase knowledge of combinations of chemical, biochemical, and microbial transformations that result in value-added nonfood products from starch and cellulose. 6. To improve fractionation methods for separating oil, starch, and fiber components during corn milling to obtain higher coproduct values with lower capital investment. The universities also have the important task of training bioprocess engineers who will be both competent in engineering fundamentals and conversant in the life sciences. Success will require a cross-disciplinary approach. All three groups—industry, government laboratories, and universities—will need to seek partnerships to build the first pilot units for new bioprocessing technologies. In many instances, the scale required for carrying out worthy fundamental research will be quite large, and realistic feed stocks will be available only at the plant site. As a consequence, special efforts will be needed to transfer technology from the laboratory to the pilot scale and ultimately to full-scale production. Industry involvement and the special facilities and capabilities of government laboratories could rapidly achieve the first adaptation of new bioprocesses on a commercial scale. A historical precedent is found in the development of penicillin (see Vignette 1). Over the last 10 years, the NSF Engineering Research Centers program and more recently the NASA Scientific Centers for research and training have provided working relationships of this type. The committee recommends that these models be examined and applied, in a suitably modified form, to the processes for obtaining value-added products from renewable resources. Incentives for adapting bioprocess technology might be provided by environmental concerns, government tax policies, the possibility of improving product quality, and economic factors. The first investments in new bioprocess technology might appear to yield small unit returns (relative to a high-value-added product), but the overall volume of product will likely be immense. The effects on the U.S. economy would be significant, particularly

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Putting Biotechnology to Work Bioprocess Engineering if biobased products with improved end-use properties begin to supplant products derived from petrochemical sources and methods of producing and processing food are changed through bioprocessing. The committee recommends that a separate study on biobased materials be considered to explore the potential impact of adaptation of bioprocessing technology on the input-output grid of the U.S. economy. 2.5 SPACE Bioprocessing has two major categories of applications related to space: manufacture of biomaterials and bioregenerative life-support systems. Both require bioprocess engineers who are conversant in fundamentals of biochemical engineering, including physical and chemical properties of bio-molecules, bioreactor design principles, separations, fluid flow, heat transfer, and basic biochemistry and biology. Manufacture of biomaterials in space is of little short-term economic interest. The space environment, however, does provide a laboratory for developmental biology and physiology, as discussed in Section 6.1.13. An important feature of bioprocessing in space that distinguishes it from other types of bioprocessing is its application in a systems environment. Reliability and safety are paramount, because of the hostile and remote location of a space station or colony and transportation costs of $8,000–10,000/kg, which limit resupply options. Integration into other subsystems not related to bioprocessing must be carried out. Consequently, bioprocess engineers with a systems background are needed. The scale of staffing needs might be understood if it is considered that several thousand engineers are at work on the space station. Very few have a bioprocess-engineering background, but the space station or a Moon or Mars colony will require bioprocess engineers as strategic members of the various aeronautical-, mechanical-, thermal-, and structural-design teams to foster communication and enable evaluation of bioprocess options for bioregenerative life support. The national space-policy goals are to return to the Moon and establish a permanent human presence. The space station will serve as an international laboratory for study of plants and bioregenerative systems, as well as the ultimate biological system, humans, before long-term trips away from Earth are carried out. The residence time of humans in space will be long, relative to previous experience (e.g., 84 days in the Skylab in 1974). To achieve a Moon base by 2010 and a Mars base by 2020, a manned space station is to be operational by the year 2000. The Moon is intended as a possible staging area for exploration and colonization of Mars. Human travels outside the Earth's biosphere are envisioned to be about 180 days for the Moon base and about 3 years for Mars. Systems are needed that are extremely reliable and function with minimal resupply (because of the risk and ex-

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Putting Biotechnology to Work Bioprocess Engineering pense associated with current space transportation systems). Regenerative life support is a critical need and will likely be based on a combination of physicochemical and biological subsystems. Modeling of the various components of bioregenerative life support is needed to be able to predict responses of an ecosystem to changes in operating conditions, throughputs, or environmental conditions. Bioprocess engineering is a critical element in developing subsystems and integrating biological and mechanical subsystems for bioregenerative life support, for which the criterion of success is reliability, rather than economics. There is no technology base that NASA can rely on to develop bioregenerative life-support systems. Private-sector initiatives are unlikely in the near future, because the market (i.e., NASA) for such a system is so limited. Much of the initial research is being done by plant physiologists and members of allied professions. It is estimated that 50–100 bioprocess engineers will be needed by NASA and NASA contractors by the year 2000 to serve as an interface for life-science developments to be incorporated into component, subsystem, and system designs. 2.6 BIOTECHNOLOGY-RESEARCH INITIATIVE GIVEN BY FEDERAL COORDINATING COUNCIL FOR SCIENCE, ENGINEERING, AND TECHNOLOGY (FCCSET) A recent report of the FCCSET Committee on the Life Sciences and Health describes the objectives, funding levels, and agencies of the U.S. government's biotechnology-research framework (FCCSET, 1992). The goal of the initiative is to sustain and extend U.S. leadership in biotechnology research for the twenty-first century. The strategic objectives (p. 2) are to "extend the scientific and technical foundations for the future development of biotechnology; ensure the development of the human resource foundations for the future development of biotechnology; accelerate the transfer of biotechnology research discoveries to commercial applications; and realize the benefits of biotechnology to the health and well-being of the population and the protection and restoration of the environment." The report suggests directions for future efforts that will draw on advances in modern biotechnology based on fundamental research supported by U.S. federal agencies, most notably NIH. The greatest impact of biotechnology, thus far, is in human health; future developments are viewed as likely to lead to substantial reductions in medical costs through advances in prevention, early diagnosis, and treatment. The report also projects that biotechnology would soon have other impacts: "The next decade should see unprecedented applications of biotechnology to agriculture and aquaculture, to the restoration and protection of the environment, to the production of chemicals and fuels, and to many other ar-

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Putting Biotechnology to Work Bioprocess Engineering eas." The critical role for the federal government, academe, and industry is to ensure "an uninterrupted flow of new ideas and new techniques to U.S. industry," to address the challenge to the U.S. position of international leadership. The FCCSET report gives a comprehensive overview of the biotechnology research programs of 12 federal agencies and how they are related to each other. A partial list of priorities in the report includes maintaining strong support for broadly applicable foundation research to sustain the momentum of progress in all fields of biotechnology and for health-related fields to capitalize on the opportunity for applications to human health, substantially increasing and closely coordinating the federal investment in biotechnology research related to manufacturing and bioprocessing, strengthening and expanding interdisciplinary research, and increasing training programs at all levels to provide essential human resources for biotechnology development. The present committee further addressed those issues as related to bioprocess engineering and the resources required for developing capabilities in manufacture of biotechnology products. The importance of bioprocessing in realizing the commercial benefits of biotechnology are clearly recognized in the FCCSET report, and the priorities identified by FCCSET overlap with the findings in the present report. The bioprocessing research programs of NSF, USDA, DOD, DOE, NASA, the Department of Health and Human Services, the Department of Commerce, and the Department of the Interior are currently projected at $123.8 million for FY 1993. Those programs must be sustained to continue support of the bioprocessing research infrastructure. 2.7 SUMMARY The challenge of bioprocess engineering lies in identifying the needs of the industry and promoting technology transfer and training of engineers who will fit the wide range of markets, activities, and products that are being encompassed by biotechnology and bioprocess-engineering developments. The synthesis and innovation to develop the enabling technologies for industry to exploit the potential of modern biology and chemistry fully is a significant part of this challenge. The vignettes in this chapter illustrate how bioprocessing technology facilitates amplification of existing microbial products, application of new manufacturing techniques for existing products, and the production of proteins derived from mammalian systems that would otherwise not be available in sufficient quantities for practical use as therapeutic agents. The optimistic projection that the biotechnology industry will grow by a factor of at least 10 over the next 10 years is justified by the pervasive influence of biological processes on everyday aspects of life and by the

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Putting Biotechnology to Work Bioprocess Engineering many possible applications of the discoveries emanating from the life sciences. Bioprocesses require manufacturing skills that span products whose prices range over a factor of 107. The definitions of the kinds of expertise that are required for bioprocessing engineering are similarly broad. The basic principles of biotechnology manufacturing processes are based on understanding of the microorganisms or biocatalysts involved: ensuring consistent product quality and product safety, regardless of scale; addressing environmental consequences of the manufacture and use of the products; and continually improving and developing processes in response to the competitive pressures generated by consumer markets. The engineering operations that cut across those requirements include the development of bioreactor technology, the processing of impure dilute product streams into purified and concentrated form, and the ability to apply engineering economics in decision-making processes during the design, development, operation, and improvement of bioprocess facilities. That requires engineers who can solve engineering problems on the basis of an understanding of the biochemistry and genetics of living organisms. A sustained policy is needed to foster development of a fundamental knowledge base for the manufacture of a broad spectrum of bioproducts and the training of bioprocess engineers who will grow with the growing industry. 2.8 REFERENCES Abelson, P. H. 1992. Biotechnology in a global economy [editorial]. Science 255(5043):381. Aiba, S., A. E. Humphrey, and N. F. Millis. 1973. Biochemical Engineering, 2nd Ed. New York: Academic. 434 pp. Anonymous. 1991. Corn refiners push into biochemical processing. Pp. 15–17 in Corn Annual. Washington, D.C.: Corn Refiners Association. Antrim, R. L., W. Colilla, and B. J. Schnyder, 1979. Glucose isomerase production of high fructose syrups. Pp. 97–156 in Applied Biochemistry and Bioengineering, Vol. 2, L. B. Wingard, Jr., E. Katchalski-Katzir, and L. Goldstein, eds. New York: Academic. Avgerinos, G. C., and D.I.C. Wang. 1983. Selective solvent delignification for fermentation enhancement. Biotechnol. Bioeng. 25:67–83. Bungay, H. 1992. Product opportunities for biomass refining. Enzyme Microb. Technol. 14:501–507. Burrill, G. S., and K. B. Lee, Jr. 1991. Biotech '92: Promise to Reality. An Industry Annual Report. San Francisco: Ernst and Young. Buzzanell, P., F. Gray, R. Lord, and W. Moore. 1992. Sugar and Sweetener Situation and Outlook Report. U.S. Department of Agriculture Economic Research Service SSRV17N1. Washington, D.C.: U.S. Department of Agriculture. Eli Lilly and Company. 1986. Leadership in biotechnology—Lilly biosynthetic manufacturing team improves humalin production process. Eli Lilly and Company Second Quarter Report. Indianapolis: Eli Lilly and Company. FCCSET (Federal Coordinating Council for Science, Engineering, and Technology). 1992. Biotechnology for the 21st Century. A Report by the FCCSET Committee on Life Sciences and Health, Office of Science and Technology Policy, Executive Office of the President, Washington, D.C.

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Putting Biotechnology to Work Bioprocess Engineering Finnerty, W. R. 1992. Fossil Energy Biotechnology: A Research Needs Assessment Final Report. DOE Contract 01-91ER30156. Washington, D.C.: U.S. Department of Energy. Glaser, V., and G. Dutton. 1992. Food processors seek to adapt bioproducts for large-scale manufacturing. GEN 12(2):6–8. Gong, C.-S., L.-F. Chen, M. C. Flickinger, L. C. Chiang, and G. T. Tsao. 1981. Production of ethanol from D-xylose using D-xylose isomerase and yeasts. Appl. Environ, Microbiol. 41(2):430–436. Hacking, A. J. 1986. Pp. 214–221 in Economic Aspects of Biotechnology. New York: Cambridge University Press. Holtzapple, M. T., and A. E. Humphrey. 1984. Effect of organosolv pretreatment on the enzymatic hydrolysis of poplar. Biotechnol. Bioeng. 26:670–676. Ingram, L. 1992. Genetic engineering of novel bacteria for the conversion of plant polysaccharides into ethanol. Pp. 507–509 in Harnessing Biotechnology for the 21st Century, M. Ladisch and A. Bose, eds. Washington, D.C.: American Chemical Society. Jain, M. K., R. Datta, and J. G. Zeikus. 1989. High value organic acids fermentation—Emerging products and processes. Pp. 367–389 in Bioprocess Engineering. T.K. Ghose, ed. London: Horwood. Kirk, T. K., and H.-M. Chang, eds. 1990. Biotechnology in Pulp and Paper Manufacture: Applications and Fundamental Investigations. Boston: Butterworth-Heinemann. 696 pp. Ladisch, M. R., and K. Dyck. 1979. Dehydration of ethanol: New approach gives positive energy balance. Science 205:898–900. Ladisch, M. R., and J. A. Svarczkopf. 1991. Ethanol production and the cost of fermentable sugars from biomass. Bioresour. Technol. 36:83–95. Ladisch, M. R., C. M. Ladisch, and G. T. Tsao. 1978. Cellulose to sugars: New path gives quantitative yield. Science 201:743–745. Lee, D. E., and C. D. Scott. 1988. Impact of Biotechnology on Coal Processing. Oak Ridge National Laboratory Report ORNL-6459. Oak Ridge, Tenn.: Oak Ridge National Laboratory. Lee, J. Y., P. J. Westgate, and M. R. Ladisch. 1991. Water and ethanol sorption phenomena on starch. AIChE J. 37:1187–1195. Lloyd, N. E., and R. O. Horwath. 1985. Biotechnology and the development of enzymes for the HFCS industry. Pp. 116–134 in Bio Expo '85, O. Zaborsky, chairman. Stamford, Conn.: Cahners Exposition Group. Lloyd, N. E., and K. Khaleeluddin. 1976. A kinetic comparison of Streptomyces glucose isomerase in free solution and adsorbed on DEAE cellulose. Cereal Chem. 53(2):270–282. Lynd, L. R., J. H. Cushman, R. J. Nichols, and C. E. Wyman. 1991. Fuel ethanol from cellulosic biomass. Science 251:1318–1323. Maiorella, B. L., H. W. Blanch, and C. R. Wilke. 1984. Economic evaluation of ethanol fermentation processes. Biotechnol. Bioeng. 26:1003–1025. Mandels, M., J. E. Medeiros, R. E. Andreotti, and F. H. Bissett. 1981. Enzymatic hydrolysis of cellulose: Evaluation of cellulase culture filtrates under use conditions. Biotechnol. Bioeng. 23:2009–2026. Marshall, R. O., and E. R. Kooi. 1957. Enzymatic conversion of D-glucose to D-fructose. Science 125:648–649. Montenecourt, B. S., S. D. Nhlapo, H. Trimio-Vasquez, S. Cuskey, D.H.J. Schamhart, and D. E. Eveleigh. 1981. Regulatory controls in relation to overproduction of fungal cellulases. Basic Life Sci. 18 (Trends Biol. Ferment. Fuels Chem.):33–53. OTA (Office of Technology Assessment). 1991. Biotechnology in a Global Economy, B. Brown, ed. Office of Technology Assessment, U.S. Congress, Report No. OTA-BA-494. Washington, D.C.: U.S. Government Printing Office.

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Putting Biotechnology to Work Bioprocess Engineering Shuler, M. L., and F. Kargi. 1991. Bioprocess Engineering: Basic Concepts. Englewood Cliffs, N.J.: Prentice Hall. 448 pp. Tien, M., T. K. Kirk. 1984. Lignin-degrading enzyme from Phanerochaete chrysosporium: Purification, characterization, and catalytic properties of a unique H2O2-requiring oxygenase, Proc. Natl. Acad. Sci. USA 81:2280–2284. van Meir, L., and A. Baker. 1992. Revisions in estimates of food and industrial use for feed grains. Pp. 20–25 in Feed Situation and Outlook Yearbook. U.S. Department of Agriculture Economic Research Service FdS-32. Washington, D.C.: U.S. Department of Agriculture. Wang, D.I.C., G. C. Avgerinos, I. Biocic, S.-D. Wang, and H.-Y. Fang. 1983. Ethanol from cellulosic biomass—Direct conversion using a mixed culture of Clostridium thermosac-charolyticum. Philos. Trans. R. Soc. London B 300:323–333. Watson, J. S., and C. D. Scott. 1988. The Impact of Bioprocessing on Enhanced Oil Recovery. Oak Ridge National Laboratory Report ORNL/TM-10676. Oak Ridge, Tenn.: Oak Ridge National Laboratory.