PROCESS MONITORING AND CONTROL
The demand for new and improved commercial products increasingly will be met through bioprocessing, a type of advanced manufacturing that involves chemical, physical, and biological processes employed by living organisms or their cellular components. Bioprocessing enables the translation of research discoveries into commercial products with unique and highly desirable characteristics and offers new production opportunities for a wide range of items, including
Bioprocessing offers a level of specificity, predictability, and productivity that otherwise would not exist in the manufacture of these products. Moreover, when the raw materials contain certain molecules with complex structures, bioprocesses enable the synthesis of products that cannot be made by any other means. Combined, these capabilities provide for new process designs that are cost effective, energy efficient, product specific, and environmentally benign.
While bioprocessing already is employed in industry, research is needed to define the parameters of the related processes precisely in order to expand use of this approach and derive the maximum possible benefits. The Federal Government plays a critical role in this area because individual manufacturers are unlikely to invest in time-consuming generic research for the benefit of entire industries.
Process design involves the integration and scale-up of a number of operations, including upstream and downstream processing, and process monitoring, optimization, and control. One objective for process design is to quantify process variables and product yields in order to obtain a cost estimate for producing a material of biological origin, and to evaluate alternative designs that minimize the cost of producing the product. An optimal process can be one that minimizes the cost of producing a material, maximizes the profit, or assures that an operation meets special safety or environmental requirements.
A recent National Academy of Sciences report (1) provided a comprehensive assessment of the research and infrastructure necessary to maintain U.S. competitiveness in the bioprocessing arena. This chapter focuses on the Federal role in bioprocessing and provides an overview of opportunities to apply biotechnology in manufacturing. The emphasis is on areas where coordinated Federal research can accelerate markedly the commercialization of new products. Four broad priorities are highlighted:
PRIORITY: Investigate methods to enhance the efficiency and expand the utility of upstream processing technology.
Upstream processing encompasses any technology that leads to the synthesis of a product as well as the fundamental science and engineering needed to understand product formation. Specific areas of opportunity for Federal research include biocatalysis, metabolic engineering, biomass conversion, bioreactor design and cell culturing techniques, and transgenic animals.
Nature's catalysts -- enzymes -- function highly selectively to accelerate the rate of chemical reactions in biological systems. Enzymes also can limit reactivity toward a particular substrate and limit chemical conversion to a single desired product. For many substrates, enzyme-catalyzed reactions exhibit rates that are many orders of magnitude faster than those of uncatalyzed reactions.
In chemical synthesis and other processes of industrial importance, the ability to mimic the selectivity and rate-enhancing properties of enzymes would be of substantial benefit in reducing economic and environmental costs. In some cases, this approach might provide a commercial means of synthesizing a material where only much less desirable methods were available previously.
At present, about 50 enzymes are used in industry. Most of these industrial uses involve breaking down large molecules into simpler ones. Examples include the proteases in laundry detergents, and the hydrolases used in starch processing. The commercial benefits of enzymology would be enhanced if technologists could expand capabilities for creating complex molecules from simpler ones or transforming existing chemical structures into more active compounds. Production of sweetness- enhanced corn syrup by the enzyme glucose isomerase is one noteworthy example of how a chemical transformation can be carried out on the industrial scale. Synthesis routes that can distinguish between right- and left-handed chemical structures are being pursued for pharmaceutical, food, and agricultural applications. Many industrial firms are exploring the unique capabilities of enzymes to carry out such chemical transformations.
Federally sponsored research can facilitate the development and application of new enzymes of industrial importance. Many research efforts are under way, and solid progress has been made using protein engineering, chemical modification, and recombinant monoclonal antibody technology. More recently, recombinant DNA technology has been used to generate catalysts that exhibit the useful characteristics of naturally occurring enzymes but have been designed either to catalyze chemical reactions not found in nature or to alter biological reactions to broaden their specificity and expand their potential for practical application.
Progress in this field will accelerate with an improved understanding of relationships among protein structure, function, and energetics. More efficient methods are being developed to obtain protein structure using X-ray crystallography, nuclear magnetic resonance (NMR), and neutron scattering techniques, coupled with robust algorithms to treat the data and display the results using computer graphics. The use of computer graphics to visualize the dynamics of proteins also will enhance understanding of the mechanisms of enzyme action. Progress in obtaining structural data in real time ultimately will help modelers develop algorithms to predict the dynamics of enzymes. Research also should be supported on the thermodynamics and kinetics of prototypical enzyme-catalyzed reactions of interest to the bulk and commodity chemical markets; examples include the reactions involved in corn processing and the action of laundry detergents.
Metabolic engineering is the use of recombinant DNA technology to enhance the activities of a cell by manipulating its metabolic pathways. To exploit this approach, scientists must develop an improved understanding of cell metabolism. Experimental and mathematical techniques are needed for quantifying the effects of altered uses of raw materials by cells. Techniques such as electrophoresis can be used to distinguish and quantify cellular proteins produced in response to altered metabolism. In addition, non-invasive techniques such as near-infrared spectroscopy and NMR can quantify intracellular concentrations of raw materials. Also of interest is research on thermodynamics and kinetics of prototypical microbial and microalgal systems.
The first applications of metabolic engineering have incorporated genes for pathway enzymes and transport proteins. In this manner, for example, the bacterium E. coli has been induced to produce ethanol. But amplified expression of non-native proteins can affect host cell metabolism -- sometimes interfering with the engineering objective -- because raw material resources are reallocated. The effects may include a slowing of cell growth and possibly cell death. Often, the expression of small amounts of non-native proteins increases the level of stress proteins.
This problem is ubiquitous, occurring in simple organisms such as E. coli and in complex cells, such as yeast cells, human cells, the chinese hamster ovary cell (used commercially for protein synthesis), and an insect cell line that is a possible source of proteins for making AIDS vaccines. Federal support for research in this area can promote efforts to overcome or mitigate these undesirable cellular responses.
Biomass includes organic polymeric material -- such as lignin, starches, celluloses, and oils -- produced by biological processes. Plants and algae are the main sources of biomass, generating many billions of tons annually through photosynthesis. Other sources of biomass derived from human activities include food processing wastes, waste paper, and municipal solid wastes.
The Federal Government should expand its support for research that will facilitate development of commercial products from this abundant raw material. Biomass is a potential source of energy resources such as methane and ethanol, commodity chemicals, animal feed, and specialty products (e.g., flavors, fragrances, pigments). Biomass is an attractive alternative to petroleum- based sources because it not only is a low-cost substrate for production, but also is environmentally friendly; it is renewable and may reduce emissions during processing and improve the biodegradability of end products.
Biotechnology is especially relevant to the commercial use of biomass because it employs natural processes uniquely suited for creating specific products. An example of such a process is fermentation, which converts glucose sugars derived from biomass into commodity chemicals. The components of plant biomass, and products that can be derived from them, include
Fuels and chemicals can be produced not only from oil seed crops (e.g., soybeans, canola, and crambe), but also from unicellular algae and waste fats from animals. The substantial fractions of oils in these resources can be transformed through a chemical process that changes the large molecules in fats and oils into smaller molecules.
Bioreactor Design and Cell Culturing Techniques
If the great variety of potentially commercial bioprocesses are to be developed and applied, then bioreactors must be designed in which the environment can be controlled precisely to maximize process efficiency. Federal support for research in this area can improve the efficiency of bioprocessing and thereby improve prospects for its wide application.
The design of a bioreactor requires a basic understanding of both chemical reactor design and cell biology. First, designers must understand the effects of reaction rates and stoichiometry, mass transfer, heat transfer, and turbulence and mixing on product distribution, reactor productivity and size, and operational characteristics. These phenomena need to be expressed in accurate but tractable models that can be used for design and optimization calculations.
In order to develop effective cell culturing techniques, designers also must have basic knowledge of cellular functions and protein chemistry. They should understand the molecular, genetic, and metabolic processes involved in the growth of cells and the expression of cellular products; and structure/function relationships in the use of proteins for biochemical conversions.
Transgenic animals can be employed either as biofactories for the production of commercial products or as living models for the study of human diseases and evaluation of pharmaceuticals. The use of transgenic animals for these purposes can be more economical or, in the case of human disease and drug models, more realistic than are conventional alternatives. Federally supported research can improve the efficiency of the processes involved and identify new opportunities.
Transgenic animals are being developed for a wide variety of applications. Transgenic cows are used for production of lactoferrin, a naturally occurring milk protein used in products such as baby formula. Several companies plan to market pharmaceuticals from the milk of sheep and goats and the blood of pigs. Anti-clotting factors used to treat cardiovascular diseases have been produced in rabbits, goats, and mice.
Research also is under way to develop transgenic animals -- mice in particular -- that carry genes associated with human diseases. Such animals might enable researchers to identify specific disease genes or produce human antibodies.
PRIORITY: Develop capabilities to recover and purify products from dilute bioprocess streams and develop predictive models to facilitate the design of downstream separations.
Downstream processing includes the cost-effective separation and purification of bioproducts, as well as biorefining. Often, downstream processing is the most expensive phase of producing a substance of biological origin, especially for products with stringent regulatory requirements. A key reason for this high cost is the complex and dilute nature of the aqueous solutions in which bioproducts generally are produced; an inverse relationship has been demonstrated between the price of biological products and the strength of the concentrations from which they must be isolated. This is the case for bulk and commodity chemicals, including ethanol from biological sources.
The high cost of downstream processing means there is significant potential for savings from improved processes. That factor, coupled with the broad applicability of downstream processing research, makes this area a prime target for Federal investment. For regulated bioproducts, such as pharmaceuticals, removal of trace impurities is the expensive step in the purification.
The separation and purification of materials produced in a bioreactor is a critical part of a manufacturing operation. The biological products involved range from high-value-added substances used as pharmaceutical agents (e.g., insulin) to lower-cost products including commodity chemicals (e.g., ethanol).
High-value bioproducts are usually fragile molecules, such as proteins or peptides, that require highly specialized and mild processing conditions and may need to be separated from a complex mixture of molecules, including cell debris. This combination of factors makes separation difficult. At present, most separation schemes are scaled-up laboratory procedures; research is needed to improve their performance.
Biological methods hold promise for improving separations. For example, a cell could be modified genetically to suppress production of undesirable by-products and alter the cell wall so it would be permeable only to the desired product. It is also possible to genetically engineer a product so that it passes through the cell wall, has an affinity for certain separation matrices, or is joined to another molecule with desirable separation characteristics. In addition to improving separations, biological approaches also reduce energy expenses, a major part of the cost of separating biological products.
Biological processes one day may offer economical alternatives to current, petrochemically based methods for manufacturing organic acids and alcohols. However, before these bioprocesses can become commercially viable, nontraditional, lower-cost separation methods need to be developed. Research is under way to develop extracting solvents, resins (separation media), and sorbents that are more selective and have a higher capacity than do current materials. Reversible extraction systems are needed that respond to changes in temperature, pressure, or acidity. Combinations of conventional separation methods and biological methods are being explored to reduce product inhibition, which often occurs in fermentations that produce alcohols and solvents.
In addition, mathematical models of separation steps need to be developed to help reconcile regulatory requirements with basic process conditions during early stages of process development, and to meet the demands of a competitive business environment. Modeling enables scale-up considerations to be estimated very quickly, a capability needed in order to commercialize a bioprocess in an industry where being the "first to market" is a critical element of success.
Biorefining involves the use of microbes in mineral processing systems. Biorefining is environmentally friendly and in some cases enables the recovery of minerals and use of resources that otherwise would not be possible. For example, a significant amount of copper is biorefined from slag heaps. Research now under way or planned addresses the use of microorganisms to bioleach oxide and sulfide ores, and to concentrate metals.
Current research on bioleaching of oxide and sulfide ores addresses treatment of manganese, nickel, cobalt, and precious metal ores. The objective is to identify metabolic pathways in the microorganisms responsible for metal solubilization, and to improve their survival rates and stability. Increased understanding of metabolic pathways will open the door to the manipulation of parameters such as kinetics and metal selectivity, with the aim of enhancing mineral recovery.
An intriguing potential use of biotechnology is in situ bioleaching of ore deposits or waste piles. Research is needed to develop a mechanistic understanding of the bioleaching process and to identify environmental and process factors affecting biosystem performance. In addition, bioprocess monitoring schemes and cell-free leaching systems should be developed. Cell-free or cell component systems, in which cellular components rather than live organisms are used for a specific reaction, eliminate the need to provide an environment conducive to survival of a microorganism. Such approaches also tend to enhance control over the reaction, due to the specificity of the cell component.
Biosorption and metal recovery from dilute aqueous solutions is an emerging field of interest, from both a resource conservation standpoint and an environmental remediation standpoint. Microbes, algae, cell wall material, proteins, and other types of biomass have been investigated for use in this application, but economical methods for selective metal recovery have yet to be developed. The Federal Government should support research in this area. Inexpensive substrates for protein immobilization should be developed, and biomolecules with high metal-binding capacity identified.
Use of microorganisms to remove fine particles from aqueous process streams is also of interest. Laboratory-scale studies have shown that certain microorganisms can cause flocculation of fine mineral suspensions, while others can function as flotation collectors or depressants. Extensive research will be needed before the use of extracellular colloidal molecules (such as polysaccharides and exopolymers released by live biomass) can become a viable alternative to current chemical-based systems.
PROCESS MONITORING AND CONTROL
PRIORITY: Develop methods for monitoring and control of commercial bioprocessing including reliable real- time sensors.
Process monitoring and control requires knowledge of the current state of every step in a bioprocess and the ability to control the process for optimal results. The most efficient use of upstream and downstream processing depends on knowledge of the state of the system and on control algorithms that can optimize the process and maintain it. This is another area where Federal support for generic research can have significant payoffs for multiple industries.
Control and optimization is challenging due to several characteristics of bioprocesses, including their high degree of nonlinearity (meaning that nonlinear differential equations are required for mathematical modeling), especially in batch and fed-batch bioreactors; and their potential for instability when they involve high-yield mutant or recombinant organisms. There are technical obstacles as well, including the unavailability of reliable on-line real-time sensors and realistic models that capture the complexities of biological systems by identifying and quantifying rate-controlling steps in reactions.
Optimization and control methods should be robust, adaptive, and suited to nonlinear processes. Important components of these systems include biosensors and bioelectronics/bionetworks. Because an optimal process often is operating at the limits of process variables, process control can be very difficult; consequently, design methods should consider simultaneously both the economics and the controllability of the process. The affordability of some production processes, particularly for lower-value products, depends on the development of highly accurate sensors and models and the capability to reproduce bioreactor operation precisely.
Sensors are invaluable in the design and operation of automated and environmentally benign manufacturing processes, and in detection, monitoring, and control of food additives, food safety factors, and bioremediation technologies. Biosensors are attractive because they can harness the inherent specificity of many biochemical processes to allow for accurate and highly specific detection of organic and inorganic species in real time. Biotechnology can be used to design, construct, probe, and alter sensitive and highly specific biosensors and incorporate them into transducing and reporting systems.
A biosensor encompasses three major components: a biological component, an interface, and a transducing element. The biological component (e.g., enzyme, immunoprotein, nucleic acids, whole cell) interacts specifically with the substance to be detected. The interface component (e.g., polymeric thick or thin film, chemically modified surface) links the biological component with the transducer. The transducer converts the biochemical interaction into a quantifiable electrical or optical signal.
An example of a biosensor is the plasmon sensor, which consists of a monolayer of immunoproteins immobilized on the surface of a thin film of gold. The analyte -- whatever compound binds to the protein employed -- attaches to the immunoprotein and changes the properties of surface electromagnetic waves that can be excited in the gold film. This change, which signifies the presence of the analyte, can be detected optically.
A vast store of fundamental information is available on the biological components used in biosensors. The key challenge now is to improve the manufacturing process so that reproducible, dependable, low-cost biosensors can be mass produced. Federal support can hasten progress in this area and foster the use of biosensors in a broad range of applications.
There are a number of technical obstacles. Poor adhesion of polymer films to the transducer, and hence poor reliability, is a major problem. The film must be compatible with the biological component and also adhere to the transducer surface. Another problem is that insufficient knowledge of methods and effects of immobilizing biological components in/on polymeric films has led to wide variation in sensor response, complicating the manufacture of commercial biosensors. Non-specific binding must be minimized, because the specificity of analyte detection is degraded when other molecules compete for binding sites.
There are also operational difficulties that preclude the use of biosensors in bioprocessing environments. For example, many sterilization techniques (such as heat) either modify or denature biological molecules. There are also problems with miniaturization, sensitivity, dynamic range, and noise reduction related to specific biosensor fabrication strategies. Finally, there is the question of quality control -- determining quickly and nondestructively whether the biosensor is operating with prescribed precision and accuracy.
Bioelectronics and Bionetworks
Bioelectronics is an emerging technology that employs biological molecules instead of inorganic materials in conventional integrated-circuit technology or in applications involving unconventional architectures, such as optical processors. The driving force for this research is the possibility of constructing devices on the molecular level and thereby achieving extremely high densities of data storage sites and nano-sized computers.
Biological systems are capable of storing information on the molecular level and processing information along pathways defined on a molecular level. Although biological processes function more slowly than do conventional solid-state devices, this penalty is more then offset by a huge increase in the density of operating units. Various biomimetic or biologically based materials, such as the protein bacteriorhodopsin, are being evaluated for use in bioelectronics.
In living things, data processing is achieved by arrays of neurons. Although the operation of single neurons is well understood, the operation of biological neural networks remains largely unexplored. Recent achievements in the culturing of a monolayer of neurons on a micro-electrode array promise to provide some insight into the operation of neuron arrays. Although neuron devices -- bionetworks -- would not operate on a molecular scale, they have the potential to form the basis for new computer architectures, including parallel processors.
Appropriate Federal investments to address key research questions could help make the United States a world leader in this promising new arena. The major technical obstacle to development of practical bioelectronic devices lies in determining how to preserve and control the properties of the active state when the bioactive species is immobilized in a artificial membrane. Research to explore the fundamental process of biological self- assembly, a capability inherent in biological molecules such as lipids, DNA, and proteins, may be useful in resolving some of the organizational problems. The pattern inherent in a bi-lipid layer or an alkane thiol monolayer ("smart" organic compounds) on a metal surface can be used as a template for further organization of biomolecules.
PRIORITY: Expand the development of novel biomaterials, such as biomimetics and replacement tissues, through new tissue engineering and chemical synthesis methods.
Up to this point, this chapter has focused on generic processing technologies used to make a variety of products. This section addresses the processing of one particularly promising category of products -- biomaterials. The tools of biotechnology can be employed to endow materials with properties not achievable using more conventional means. This research area deserves steady Federal support because of the important applications for biomaterials, which include biological substitutes for human tissues.
Due to their diversity, versatility, and unusual combinations of properties, biomolecular materials offer promise for application in virtually all sectors of the economy, including defense, energy, agriculture, health, and environmental technology. Examples of biomolecular materials include silk obtained from spiders, and the ceramics in sea shells.
In addition to their direct use as natural cellular products or modified derivatives, biomolecular materials serve another very important purpose by demonstrating how nature has optimized their physical properties. Research is needed to clarify how higher- order structure is achieved and how it serves to determine macromolecular function in such a variety of forms. With continued advances in modern biology, molecular genetics, and protein engineering, and with rapid improvements in physicochemical characterization, novel biomolecular materials can be designed and tailor-made to meet specific needs. This, in turn, would expand the possibilities for practical applications of these materials.
Standard chemical synthesis has inherent limitations, including the production of compounds with unwanted impurities and by- products. By contrast, biomolecular synthesis -- manufacturing methods based on biological processes -- allows precise control, thereby reducing levels of impurities and by-products. For instance, when a cell produces peptide polymers, control of the amino acid sequence is assured by the fidelity of RNA and DNA replicative mechanisms. Thanks to recombinant DNA technology and continuing advances in molecular biology, that same control over uniformity of composition, length, and sequence now is available to the scientist seeking to synthesize and express natural or tailor-made genes for peptide polymers.
By inserting appropriate DNA sequences into microorganisms, algae, and higher plants, investigators have learned how to induce the cells to synthesize polymers (such as polylysine) that they do not ordinarily produce, in quantities sufficient for further study and eventual application. Research is needed to define the classes and specific types of polymers that can be produced in this manner. Federal support could play a key role in advancing research in this area.
Tissue engineering -- a term coined in 1987 referring to the development of biological substitutes to restore, maintain, or improve human tissue function -- employs the tools of biotechnology and materials science as well as engineering concepts to explore structure-function relationships in mammalian tissues. This emerging technology could provide for substantial savings in health care costs and major improvements in the quality and length of life for patients with tissue loss or organ failure.(3)
Advances in the study of tissue growth and regeneration, at both the cellular and tissue levels, set the stage for practical application of tissue engineering. The culturing of cells in two-dimensional monolayers enabled the study of cellular processes and opened the door to genetic manipulation. Scientists and engineers have begun to view cell culturing as a three-dimensional process, in which external forces on the cells not only may influence cellular products, but also may reawaken the cellular differentiation process. In order to develop living tissue equivalents, it will be important to understand how the cellular environment affects the differentiation process as well as interactions between the engineered tissue and the host.
The first success with differentiated cells came with engineered human skin, now in clinical trials. Scientists also are beginning to explore the potential to grow many tissues in culture. Using stromal cells from human tissue, researchers are developing blood vessels, bone, cartilage, nerve, oral mucosa, bone marrow, liver, and pancreatic cells. Federal support can hasten progress in development of these materials.
Encapsulated cell therapy is an example of a technique under development by industry that employs biomaterials in the treatment of certain serious, chronic diseases. The goal of this approach is to replace cells within the body that have been destroyed by disease in order to augment circulating or local levels of the deficient molecules. Targets for replacement include insulin-producing cells in diabetics and dopamine- secreting cells in patients with Parkinson's disease.
An encapsulated cell implant consists of cells that secrete the desired hormones, enzymes, or neurotransmitters, enclosed within a polymer capsule and implanted into a specific site within the host. Animal studies have shown that the functional activity of secreting cells can be maintained in vivo. The capsule wall is designed to allow passage of small molecules (i.e., glucose, other nutrients, therapeutic molecules) but prevents or retards the passage of large molecules, such as elements of the immune system. Studies suggest that the transplanted cells are protected from destruction and perhaps even recognition by the host's immune system, allowing the use of unmatched or even genetically altered tissue without systemic immunosuppression.
Thus, the use of an encapsulating membrane may overcome two of the difficulties that prevent widespread tissue transplantation into humans: the limited supply of donor human tissue, and the toxic effects of immunosuppressive drugs required to prevent rejection of unencapsulated transplants. This is an important research area where expanded Federal investment could yield significant dividends.
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