3

Biotechnology Revolution


The Role of Bioprocess Engineering in Biotechnology

Bioprocess engineering is the discipline that puts biotechnology to work.

Biotechnology involves using organisms, tissues, cells, or their molecular components (1) to act on living things and (2) to intervene in the workings of cells or the molecular components of cells, including their genetic material (NRC, 2001). Biotechnology evolved as a means of producing food, beverages, and medicines. More than 8,000 years ago, it was used to make leavened bread. Some 5,000 years ago, moldy soybean curd was used to treat skin infections in China. The malting of barley and fermentation of beer was used in Egypt in 2500 BC (Ladisch, 2002).

Biology is central to biotechnology. Louis Pasteur proved in 1857 that yeast is a living cell that ferments sugar to alcohol; in 1877, he showed that some bacteria kill anthrax bacilli. In 1923, Banting and Best showed that insulin from animals could be used to treat people suffering from diabetes. In 1928, Alexander Fleming showed that growing colonies of Penicillium notatum inhibit Staphylococcus cultures. Beginning in 1939, Florey and Chain rediscovered that Fleming’s Penicillium could lyse bacteria, but the yield of penicillin was small; in addition, the penicillin it did produce was unstable (Matales, 1998). They realized that producing Penicillium on a large scale would require isolation and purification procedures that minimized product loss. Early bioprocess engineers found solutions to this problem (Aiba et al., 1973) when researchers in the U.S. Department of Agriculture (USDA) laboratory in Peoria, Illinois, discovered that mold on a cantaloupe (P. chrysogenum) could be grown in large tanks in submerged cultures (Shuler and Kargi, 1991).

During World War II, government incentives encouraged several pharmaceutical companies to develop cost-effective manufacturing processes for penicillin (Hacking, 1986). Chemical engineers, industrial chemists, and microbiologists quickly devised methods of countercurrent extraction, crystallization, and lyophilization to recover penicillin in an active, stable form and established the viability of submerged fermentations (Matales, 1998).

The benefits of biotechnology might be an anomaly if it were not for engineering, specifically bioprocess engineering, the discipline that puts biotechnology to work (NRC, 1992). To quote Louis Pasteur, bioprocess engineering is to biotechnology “as the fruit is to the tree.” Neither can exist without the other. The realization of the benefits of penicillin required the development of methods of transforming microbial growth on the surface of a moldy cantaloupe to cultures grown in large stirred tanks fed by sterile air (Aiba et al., 1973).

It took engineers to design the tanks, impellers, pumps, compressors, columns, pipes, and valves that have made biotechnology products available to large numbers of people. The lifesaving benefits of insulin required engineering for the extraction and purification of insulin from cow and pig pancreas, and later, the large-scale propagation of bacteria engineered to make human insulin, as well as methods of recovery, refolding, and purification to obtain an active molecule (Ladisch, 2001). Biochemical manufacturing and bioseparations have made it possible to purify products derived from biotechnology on a large scale.

High-Fructose Corn Syrup and Bioethanol
In 1957, scientists at USDA reported the discovery of an enzyme with the amazing ability to transform glucose to fructose (although it required arsenic as a cofactor). In 1965, a version of this glucose isomerase enzyme that did not require arsenate was discovered in a species of Streptomyces. Once it was possible to grow this organism using corn-steep liquor to produce a thermally stable enzyme in a cost-effective way, sugars from corn with sweetness similar to sugar from sugar cane became feasible.

Glucose isomerase (which also transformed xylose to xylulose) was used to generate the first commercial shipment of corn syrup containing 42 percent fructose in 1967. Bioprocess engineers invented systems of fixed beds of the glucose isomerase enzyme and demonstrated the utility of biocatalysts for the large-scale industrial production of biochemicals. They also adapted industrial-scale liquid-chromatography separations used in the petrochemical industry to enrich the fructose content in corn syrup from 42 percent to 55 percent (UOP Sarex process), creating 55-percent high fructose corn syrup (HFCS). When a “taste challenge” sponsored by a soft-drink company showed that consumers preferred soft drinks made with 55-percent HFCS, HFCS became a major sweetener in many popular soft drinks. The HFCS industry grew quickly, particularly after 1975, when patent coverage for using xylose (glucose) isomerase to convert glucose to fructose was lost due to a civil action suit (described in Ladisch, 2002).

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The benefits of biotechnology could not be realized
without bioprocess engineering.
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The biotechnology developed for HCFS production intersected with increases in U.S. agricultural productivity to set the stage for the large-scale production of bioethanol as a liquid transportation fuel. In 1988, yields of corn were 85 bushels per acre; by 1990, they had jumped to 119 bushels per acre. Today, yields range from about 130 to 138 bushels per acre. The wet mills that produced HFCS had the infrastructure, integrated processing, biotechnology, and bioprocess engineering expertise to make million-gallon fermenters conceivable. They also had access to glucose from corn to fill these tanks with substrates for the production of fuel ethanol, which was introduced in the 1980s (NRC, 1998). The 81 million acres of corn planted in 2004 will provide renewable raw materials, not only to make sugar, but also to make fuel ethanol and other bioproducts, such as monomers and biodegradable plastics.

DNA, Genetic Engineering, and the Biotechnology Industry
In 1953, Watson and Crick showed that DNA consists of a double helix with a code of triplets of nucleotides that correspond to specific amino acids and the sequence in which they were assembled.1 Spectacular developments followed. Genes in chromosomes were mapped to the genetic bases of diseases. Cellular processing of DNA and other nucleotides derived from it were beginning to be understood. A breakthrough came in 1970 when Smith et al. showed that a restriction endonuclease (i.e., an enzyme that hydrolyzes DNA) from Haemophilis influenzae could recognize specific DNA sequences. Restriction enzymes were then used to cut plasmids (circular DNA found in bacteria) in a way that allowed scientists to insert new genes. By 1973, Cohen et al. had put a plasmid with an inserted gene back into a cell, making it possible to produce a wide range of new products.

One widely used plasmid is PBR 322. Its 4,362 nucleotides were completely sequenced in 1979. PBR 322 contains unique restriction sites and has genes for antibiotic resistance that allows for the selection of transformed bacteria. Thus, specific regions (restriction sites) cleaved by specific restriction enzymes could be identified and foreign genes inserted. In other words, the engineering of genes, or genetic engineering, became possible. When the reconstructed plasmid was reintroduced into a microorganism, E. coli, the molecular machinery in the cell transcribed and translated the instructions to make a human protein in a bacterial cell. Here’s how it worked.

PBR 322 carried instructions (genes) for making enzymes that rendered the antibiotics ampicillin and tetracycline harmless (Old and Primrose, 1994). Only about 1 percent of E. coli cells, however, retain the engineered plasmids inserted into them. Thus, cells that were successfully transformed by inserting the plasmids could be identified by their ability to grow in media containing ampicillin and/or tetracycline. Cells that contained the plasmid had built-in resistance to the antibiotic; cells that did not contain the plasmid were killed. Thus, biologists had found a way to select for transformed cells. Because the plasmid could be “constructed” in a test tube, and because the construction involved genes, the term genetic engineering was used to describe the process.

The stage was now set for the first human protein, human insulin, to be produced in E. coli in a sequence identical to the human pancreatic peptide. In 1978, separate insulin A and B chains were achieved in E. coli K-12, using genes synthesized for the insulin A and B chains and cloned in plasmid PBR 322. Later, human insulin was produced as a preprohormone, using one fermentation instead of two (Chance et al., 1981). Eli Lilly licensed the technology and quickly developed the process, and the first recombinant product, human insulin, was marketed in 1982. By 1991, human insulin provided an estimated 70 percent of the demand for insulin in the United States.

The production of human insulin required 31 major processing steps, 27 of which are associated with product recovery and purification (Prouty, 1991). Bioprocess and bioseparation engineering, which provided technology for carrying out complex, biological processes on a large scale, were critical in bringing human insulin to market.

Monoclonal Antibodies
In 1975, Kohler and Milstein reported that hybrid cells derived from mouse B lymphocytes (which secrete antibodies) fused to mouse myeloma malignant cells will grow in submerged cultures. The fused cells, called hybrid myelomas, or hybridomas (NRC, 2001), had the capability of growing and dividing, and hence producing, monoclonal antibodies in cell culture. The cells derived from the founder cell are identical to it and produce the same antibodies, which are referred to as monoclonals. Here was an example of living things acting on livings things (and with each other) to make a part of a living thing (an antibody) that had therapeutic uses.

Initially, monoclonal antibodies were considered tools for detecting or diagnosing pathogenic microorganisms or cancer cells because of their ability to bind specifically to protein biomarkers that label these cells. When monoclonal antibodies were linked to toxins to deliver them specifically to cancer cells and other therapeutic uses were discovered, demand for their manufacture increased dramatically. Bioprocess engineers are working to scale up processes of cell culture to enable manufacturing facilities to meet that demand.

Biopharmaceuticals and Bioproducts
Biopharmaceuticals (biological molecules with medicinal value) include treatments for cancer, heart disease, and autoimmune diseases. Bioproducts are commodity-scale products that often have a lower molecular weight (e.g., fuel ethanol, monomers for manufacturing biodegradable plastics and carpet fibers, and biocatalysts used in food processing and laundry detergents). Other types of future bioproducts might include functional foods that improve nutrition or contain edible vaccines, biomaterials for paints and coatings, and optical-holographic high-density memories (NRC, 2001). Bioprocess engineering puts biotechnology to work by providing manufacturing systems to generate bioproducts in large volume, at low cost, and with acceptable purity.

Mapping the Human Genome: Genomics
Genomics “provides a means of identifying, in any cell, tissue, or organism, all of the important genes and regulatory regions in the DNA, all of the mRNAs, and all of the proteins in different states of cell and organ function. Genomics has transformed the science of biology by enabling the discovery of new links between protein structure and function” (NRC, 2001). Proteomics addresses “information about RNA and protein products of genes” (NRC, 2001).

The grand challenge of sequencing the human genome required that many existing bioprocessing tools—fermentation, enzymology, and bioseparations—be mapped onto new biotechnologies—cloning, polymerase chain reaction (PCR), and automation of DNA sequence analysis—and used with information-age tools that connected computers through the Internet. The goal was to generate sequences (the order of nucleic acids in DNA) and piece them together to discover genes and the nature of information stored in DNA. To produce enough genetic material, DNA was propagated in microbial cells using bacterial artificial chromosomes.

The human genome consists of chromosomes made of DNA associated with a protein that wraps around it to protect it from the effects of mechanical forces. First, the DNA had to be separated from this protein so that it could be disassembled one nucleic acid at a time. The process was accelerated by a so-called “shotgun” technique. Restriction enzymes were used to break up DNA into small fragments of 300 to 500 base pairs. The fragments were then sequenced and the sequences compared to find overlapping fragments. Computers were used to reassemble the sequences of these overlapping fragments into the original DNA code.

By replicating the procedure in different laboratories, comparing the results against existing databases, and using the Internet and computers to make comparisons, the sequencing task was completed by 2000, several years ahead of schedule. It took another year to determine the number of genes in the human genome (now believed to be about 30,000 but once thought to be 100,000). The engineering of automated instruments and software for analyzing nucleic acid sequencing played a major role in achieving this milestone.

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Future bioproducts might be foods that contain edible
vaccines and optical-holographic high-density memories.
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The cost of sequencing has decreased from $2 per base pair to 20? per base pair. By 2025, perhaps sooner, it may be possible to sequence the entire human genome for $6,000, making it practical to compare a person’s complete DNA sequences with a reference DNA sequence to identify a biological “dog tag” unique to each person. Not only would this preserve the identity of the individual, but it could also lead to the identification of genetic risk factors and an individual’s tolerance to environmental conditions (e.g., heat, cold, high altitude, radiation) or chemotherapy (NRC, 2001).
Identification of differences in DNA is in part made possible by PCR (see Box 1). PCR requires a thermally stable enzyme that catalyzes DNA formation. The enzyme (first isolated from microbes growing in hot springs) enables researchers to make millions of copies of a DNA fragment in as little as an hour, enough to sequence or identify the DNA sequence.

Biotechnology and Agriculture
In the past, plant and animal breeding were used to enhance agriculture by taking advantage of the natural variability of characteristics or inducing mutations (or using natural mutations) in genes. Today, genes from other species can be engineered into plants or animals. For example, antiworm protein from the bacterium Bacillus thuringiensis can be engineered into corn or cotton, thus reducing the need for pesticides. Biotechnology has provided the tools for engineering crops resistant to pests or herbicides and animals capable of producing therapeutic proteins. Another example is sheep that produce human antibodies that can boost the immune system. Organisms with genes transferred from one species to another are called transgenic.

The tools of molecular biology, described in this paper, which were developed largely for medical applications, are being used for cloning genes into microorganisms to produce bioenergy and bioproducts, as well as for the study and modification of metabolic pathways in microorganisms. These studies require gene sequencing and relating protein function to its structure, much as pharmaceutical properties are related to protein structure in the development of new pharmaceuticals. There is a major difference, however. Outputs of bioenergy (such as ethanol) will be measured in tons per day, whereas outputs of biopharmaceuticals may be measured in kilograms per year.

A yeast has been engineered to produce ethanol from xylose, including the formation of xylulose by xylose isomerase (an enzyme that also isomerizes glucose to fructose). The xylulose then enters an ethanol-producing pathway. Glucose has been fermented to ethanol for millions of years, but when xylose isomerase and other enzymes are cloned into yeast, both glucose and xylose can be fermented. Similarly, a genetically engineered E. coli is also capable of fermenting xylose to ethanol. When combined with a suite of other bioprocessing technologies, pentose fermentation increases the yield of ethanol from plant materials by 50 percent, moving us a step closer to the transformation of renewable, agricultural residues into fuel-grade ethanol. Imagine the potential in the United States alone, where an estimated 20 million tons of residues could produce 1.6 billion gallons of ethanol.

The goal of metabolic-pathway engineering is to understand, and ultimately direct, metabolic pathways in microbial cells to make value-added bioproducts. Some transgenic animals and plants have been engineered to produce therapeutically important proteins (although these are not yet commercial). Agriculture could become a producer of large volumes of therapeutic compounds using small amounts of land. Engineering will play an important role in the extraction, recovery, and purification of these products.

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Bioprocess and bioseparation engineering were critical
in bringing human insulin to market.
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Eighty-one percent of U.S. soybean acreage and 15 percent of corn acreage is already planted with genetically engineered, herbicide-tolerant crops. These crops can survive the application of specific herbicides that would otherwise destroy them, as well as targeted weeds. Insect-resistant cotton and corn contain a gene from the soil bacterium, Bacillus thuringiensis, that produces a protein toxic to certain lepidopteran insects (insects that go through a caterpillar stage) and protects the plant over its entire lifetime. Some plants have “stacked” traits (i.e., both insect and herbicide resistance). In 2003, 27 percent of the cotton planted was insect and herbicide resistant. Corn with stacked traits made up only 4 percent of the corn planted in 2003. Corn with the Bacillus thuringiensis trait made up 41 percent in 2003 and appears to be leveling off. Considering that these crops only became available in 1996, their impact has been remarkable. One of the challenges for engineers will be keeping genetically engineered crops separate from other crops, because commercial developers must address consumer resistance to some engineered bioproducts, particularly some types of foods.

On the Horizon
The newest frontiers for bioprocess engineers are biotechnology on the nanoscale. Bionanotechnology involves defining fundamental phenomena for investigating the macroscopic world on a submicroscopic scale. Stem cells, gene therapy, functional foods, edible vaccines, pharmacogenomics, and biosensors are on the horizon for biotechnology. Designing robust manufacturing processes to produce large quantities of these bioproducts will require bioprocess engineering. The absolute scales of these processes will be measured in volumes ranging from nanoliters to cubic meters.
For example, sensors with micron-scale features and nanoliter-sized volumes must be able to sample colloidal fluids with submicron-scale virus particles or microbes and process fluid without becoming plugged up or otherwise inactivated. Sensors used for pathogen detection must be integrated into a system that concentrates a large-volume sample (100 to 200 milliliters) into a small-volume sample (10/?L), perhaps in less than an hour. This will require amplification of the target species by culture or by concentration (filtration) and recovery of the sample. The integrated system will have to be able to concentrate a sample as much as 10,000 fold prior to its being introduced or interrogated by a miniature analytical device. Bioprocess engineering will be required to develop a knowledge base of fluidics at a microscale, self-assembly processes of biological molecules, and microscale bioseparations. The challenge to engineers will be relating properties of the macroscopic world to microscale devices built from submicron, nanoscale components.

Other bioproducts will involve biotechnology (i.e., parts of living things acting on living things) to enhance human performance in emergency situations, to generate electricity from biological molecules or living cells, to use proteins to store holographs of maps, to develop strong, lightweight materials from nanostructured components, to embed physiological monitors, and to stop bleeding. A recent study identified 45 areas of biotechnology that could be useful to the U.S. Army (NRC, 2001).

Summary
Biotechnology is defined by the tools used to practice it. By programming DNA and directing cellular machinery, we can obtain products that were unimaginable even 10 years ago. With biotechnology, we can direct the nanoscale machinery of living cells to produce self-contained factories that perform on a characteristic scale of one micron. To be useful to people, however, bioproducts and bioenergy must be produced in immense quantities. Genetic engineering, for example, is carried out at a molecular scale but is amplified through bioprocess engineering to transfer the technology from the test tube to the bottle through a sequence of integrated steps that generate, recover, purify and package the product (NRC, 1992). The challenge facing bioengineers is to redirect genetic and cellular machinery to make economically important molecules when the cells are placed in controlled environments. Engineers must design, build, and operate hardware and integrated systems that can multiply a cell’s output by a factor of one trillion, as well as recover and purify the products in a cost-effective manner. Bioprocess engineering is the next frontier.

Acknowledgements
The author wishes to thank Professors Daniel I.C. Wang of MIT and Janet Westphaeling of University of Georgia for their contributions over the last 10 years to the concepts of bioprocess engineering and biotechnology presented in this paper, and Roger Brent of the Molecular Sciences Institute, Berkeley, on genomics and Robert Love of the Board on Army Science and Technology, National Research Council, study director for the Army biotechnology study. The author also wishes to thank Professor Nathan Mosier of Purdue University and Carol R. Arenberg, NAE managing editor, for their reviews, suggestions, and input.

References
Aiba, S., A.E. Humphrey, and N.F. Millis. 1973. Biochemical Engineering, 2nd ed. New York: Academic Press.
Arnheim, N., and C.H. Levenson. 1990. Polymerase chain reaction. Chemical & Engineering News 68(40): 38–47.
Chance, R E., E.P. Kroef, and J.A. Hoffmann. 1981. Chemical, Physical, and Biologic Properties of Recombinant Human Insulin in Insulin. Pp. 71–85 in Growth Hormone, and Recombinant Technology, edited by J.L. Gueriguian. New York: Raven Press.
Cohen, S., A.C.Y. Chang, H.W. Boyer, and R.B. Nelling. 1973. Construction of biologically functional bacterial plasmids in vitro. Proceedings of the National Academy of Sciences 70(11): 3240–3244.
Hacking, A.J. 1986. Economic Aspects of Biotechnology. Cambridge, U.K.: Cambridge University Press.
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Box 1 The Polymerase Chain Reaction
The polymerase chain reaction (PCR) is an enzyme-mediated, in vitro amplification of DNA for purposes of analysis. Since about 1985, this method has significantly increased the ease and speed of isolating DNA sequences in vitro. Developed by scientists of Cetus Corporation in 1984 and 1985, PCR is an enzyme-catalyzed reaction that facilitates gene isolation and eliminates the need for the complex process of cloning, which requires the in vivo replication of a target DNA sequence integrated into a cloning vector in a host organism. PCR is initiated by DNA denaturation, followed by primer annealing; a DNA polymerase and deoxynucleoside triphosphates are then added to form a new DNA strand across the target sequence. When this cycle is repeated n times, it produces 2n times as much target sequence as was initially present. Thus 20 cycles of the PCR yields a one million-fold increase or amplification of the DNA. Applications of PCR include comparisons of altered, uncloned genes to cloned genes, diagnoses of genetic diseases, and retrospective analyses of human tissue.

Source: Arnheim and Levenson, 1990.


FOOTNOTES

1The polymers of amino acids are self-assembling, and their sequences determine their three-dimensional conformations. These macromolecules are endowed with catalytic capabilities under atmospheric conditions and temperatures between 10?C and 50?C.

About the Author: Michael Ladisch is Distinguished Professor of Agricultural and Biological Engineering and Biomedical Engineering and Director of the Laboratory of Renewable Resources Engineering at Purdue University.