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The challenges and the opportunities
Modern biotechnology offers new approaches to cleaner industrial products and processes. At its core is the principle of working in harmony, rather than in conflict, with the natural world. Biotechnological solutions can supplant technologies that pollute the biosphere and/or deplete finite resources, but industry, the research community, government, and the public need to work together to
help biotechnology fulfill its potential for industrial sustainability.
Biotechnological processes have already proved their worth in a number of major industries. They have been implemented for various reasons. Market forces may drive their adoption: it may be more economical/ profitable to adopt biotechnological processes. In many cases, government policy (regulation) obliges industry to adopt cleaner technologies, and biotechnology may offer the best
solution, either alone or in conjunction with conventional chemical tools. Science and technology play their part by making biotechnological solutions available, by establishing technical feasibility, and by undertaking R&D; to extend the offerings. Public pressure in favor of better quality of life and environmental protection also
encourages industry to present a "cleaner" image.
Why industry turns to biotechnology
Process economics, including capital investment and compliance costs, underpin industrial decisions on the implementation of new technology. Companies decide to replace end-of-pipe technologies and adopt clean technologies when new production processes reduce costs in comparison to the previous production system or result in better process performance or product quality, or when they are under
pressure to do so from government regulation or public pressure, or a combination of these reasons. To make a decision, industry needs answers to many questions. When considering the possibility of adopting or incorporating biotechnological applications, industrial managers may want to know:
- Is it possible to improve my current process, or a competitor's process, by using biotechnology?
- Do I have to change the entire process or just one step?
- Can I adapt the biology to my process or will I have to adapt to it?
- Are the biological systems available now or is further R&D; necessary?
- Can I incorporate a new biocatalyst to replace a conventional one and will this require radical re-design?
- Can I use conventional organisms or biocatalysts or do they need to be genetically manipulated?
- If the latter, will the public accept my process or the final product?
- How can I be assured that one process is clean or at least cleaner than another?
These are questions that the biotechnology industry and the R&D; sector need to be prepared and able to answer.
Partial changes to plant or processes may be sufficient ...
As current applications have shown, the transition to cleaner industrial manufacturing does not necessarily require completely new and costly plant and equipment. Often, the introduction of biotechnological stages or a modification of existing plant will achieve the desired results.
... but the value and effectiveness of biotechnological solutions must be proved.
Even so, industry will rarely make the investments necessary to develop and incorporate biotechnological processes without some proof that the changes will be beneficial in terms of market niches and/or will be economically competitive. This is especially true for a new technology for which profit margins are uncertain and not readily quantifiable relative to conventional technologies.
Nonetheless, it is not always easy to determine the level of economic benefit to be derived from the adoption of clean processing, because it is difficult, for example, to weigh the costs of adoption against the probability of risk. What value should be placed, for example, on avoiding litigation costs due to the use of hazardous materials or
practices? These are areas that require further research.
Industries may also be concerned about environmental legitimacy and biotechnology may help in this respect.
Establishing environmental legitimacy may also take on strategic importance, and many companies now make considerable efforts to assess their own activities from a broad environmental perspective and take initiatives accordingly. In some, environmental reporting has become a regular practice, in response to shareholder demand and public expectations.
Demonstration activities are essential for bridging the gap between basic research and full-scale implementation.
Because clean biotechnological applications are generally specific to a given process, and even to a type of process within a single company, a bridge is needed between basic research and implementation. Such a bridge can best be provided by partnerships between government, academia and industry that demonstrate the technology's applicability.
| One company's approach to environmental reporting |
In 1974, when Denmark's first Environmental Protection Act took effect, Novo Nordisk, a manufacturer of insulin and enzymes, created an independent environment department to ensure compliance with official protocols. Then, in the late l980s, it increasingly recognized a need to maintain a dialogue with customers, neighbors, environmental organizations, students, investors, employees and
other groups.
In 1996, Novo Nordisk signed the l6-point Business Charter for Sustainable Development drawn up by the International Chamber of Commerce, thereby declaring its willingness to register, control, and report on their environmental performance to the public. The company uses the charter as a guide for its environmental work and policy and publishes an annual Environmental Report so that
interested parties can follow the company's progress in environmental matters. Recent reports have presented detailed pictures of resource consumption and environmental impact in its production plants world-wide, as well as information on compliance with legislation around the world.
The Environmental Report is important both to staff and to the public. It motivates staff by highlighting environmental goals and the results of the work accomplished and helps identify new problem areas, thus serving as a catalyst. The company believes in face-to-face dialogue in discussing environmental issues, especially in complex areas such as biotechnology. Large numbers of
schoolchildren and students visit the company, and its environmental staff appear regularly at conferences and on external courses. |
Biotechnological solutions through R&D;
Achieving greater penetration of biotechnology for clean environmental purposes will require basic research in a variety of exciting areas ...
Despite the current availability of a wide range of biotechnological processes which can be employed for industrial production, there are technical and economic hurdles to be overcome. A number of bottlenecks have limited the penetration of biotechnology for clean industrial processes. These present interesting challenges to the science and technology community. As biotechnology becomes an
increasingly important source of clean industrial products and processes, R&D; development efforts will need to focus on a number of priority areas:
- innovative biocatalyst technology for use in areas where conventional biocatalysts have not yet been exploited;
- wider exploration of biological systems (enzymes, microorganisms, cells, whole organisms);
- novel methodologies for developing biological processes (biomolecular design, bioconsortia, genomics, etc.);
- engineering, especially large-scale engineering, process intensification, measurement, monitoring and control systems;
- development and application of recombinant technology.
... as well as applied research and demonstration projects.
Much also needs to be done in terms of applied research and demonstration projects to show that laboratory and small-scale R&D; activities can be scaled up to industrial production levels.
New developments in molecular biology facilitate exploration of the microbial world and help capitalize on its genetic potential.
The development of novel biocatalysts focuses on: searching in natural habitats, such as geothemal sites, for biocatalysts that can function in difficult and/or in artificial environments; and on modification of existing biocatalysts by genetic or physicochemical methods. Gene probes are being used to identify microorganisms with specific biocatalytic properties that can be detected even when
they are not yet culturable. Genome studies are also revealing the sequences of genes and their functions and are revealing novel enzymatic capabilities.
Among the technical impediments to the application of biocatalysts are their hydrophilic nature and their inactivation at high temperatures...
In the petrochemical industry, for example, the majority of intermediates are water-insoluble, so that reactions currently need to take place in organic solvents. Additional technical problems associated with biological processes include high levels of water use, the need for complex process equipment, and low yields due in part to incomplete understanding of metabolic pathways and physiological
control mechanisms. The production of biopolymers using microorganisms typically requires five times as much water to separate the biopolymer from the biocatalyst than is required for conventional chemical processes. This means that more energy and much higher water processing capacity are needed, creating an environmental impact that goes well beyond simple cost considerations.
| The search for novel biocatalysts |
Organisms with unusual biochemistries are being collected from the very harshest environments on Earth - in the depths of oil wells, in Arctic ice, in desiccating salt marshes, and above steaming heat vents under the ocean. Enzymes from such organisms are of particular interest because enzymes from ordinary microorganisms are often too delicate to be of use in most industrial processes.
For this reason, chemists are looking for "extremozymes" that combine exquisite precision with the toughness needed to survive in industrial processes. The ultimate goal is to obtain enzymes that can perform in environments which would be deadly even to the hardiest of creatures.
The rewards can be high, as the example of the commercial enzyme taq polymerase shows. This enzyme from a thermophilic bacterium is the basis of the polymerase chain reaction (PCR). It has revolutionized entire areas of biochemistry and has resulted in hundreds of millions of dollars in sales. Furthermore, taq polymerase is not considered a true extremozyme. It is hardy enough to survive the
repeated heating and cooling steps of the standard PCR, but it breaks down at a "cool" 80–C temperature. Researchers are now experimenting with hardier enzymes from deep-sea vent bacteria, where the water temperature can reach 120 C.
As a class, biological catalysts are not very stable, and this poses a major problem for their industrial use. In living cells in nature, enzymes are replaced when they wear out, but in a chemical plant replacement is time-consuming and costly. In one effort to solve this problem, Altus Biologics, a subsidiary of Vertex, has commercialized cross-linked enzyme crystals (CLEC(r)). Many enzymes
can be crystallized, so that enzyme molecules occupy repeating points in a regular three-dimensional array. In such an array, the individual enzyme molecules retain their activity, and that activity is often hundreds of times more stable than the same enzyme in solution. Although CLECs can still be improved (for example, enzyme molecules in the center of the crystal do not have access to
substrates), these products have already achieved market acceptance. |
New directions in biotechnology research
... but solutions are being sought.
Enzymes do not necessarily need to be kept in aqueous reaction media to catalyze technologically useful reactions, and this has led to a new form of biotechnology in which enzymes are controlled by water depletion, and persuaded to form chemical bonds they would normally break. In one interesting commercial application, low water organic solvent enzymology has been developed by a DSM/ Japanese
joint venture to produce aspartame in the solvent ethyl acetate. This alternative to chemically synthesized aspartame is already on the market, and is being produced on a large scale. Another example involves enzyme-catalyzed transesterification of monosaccharides with vinyl acrylate using pyridine as solvent. The resultant esters are isolated and subsequently polymerized to form materials
capable of holding 50 times their weight of water. These polymers are largely biodegradable.
Supercritical fluids are providing an important medium for biocatalytic reactions.
Attention has recently turned to supercritical fluids (SCF) as media for biocatalytic reactions. These are non-aqueous materials held above their critical temperature so that they cannot be liquefied. They bridge the gap between the properties of liquids and gases. SCFs offer a number of major advantages for bioprocessing, including increased enzyme reaction rates, protection against microbial
contamination, and options for recycling. Enzyme activity and substrate specificity can be manipulated by modifying the pressure under which the reaction takes place. The enzyme lipase has also been used to catalyze polyester synthesis, and when the reaction takes place in SCFs such as fluoroform, the molecular weight of the polymer can be controlled by the reaction pressure.
Recombinant DNA technology is a powerful tool for industrial biotechnology.
Recombinant DNA technology provides a very powerful means of combining diverse genetic capabilities. It permits the genetic engineering of organisms with specific catalytic capabilities so that they can carry out specific catalytic activities and function at high temperatures, at high solvent concentrations, or other industrial process conditions. In many cases, enzymes from organisms that grow
naturally in extreme environments, such as hot springs or deep-sea thermal vents, are sought. These "extremozymes" are more robust and operate at higher temperatures (but still well below those of conventional petrochemical processes). They can then be transferred into more manageable organisms such as E. coli or yeasts which are traditionally cultured in industrial reactors.
Conversely, the catalytic capabilities of less tolerant organisms can be transferred to those that grow best under harsh environmental conditions. Since such recombinant organisms can be grown under contained good large-scale industrial practices, safety issues relating to environmental release are minimized, although some countries still require the use of non-recombinants for certain
applications, such as food production. Yet the recombinant organisms may have less environmental impact than traditional techniques. For example, enzymes derived from recombinant organisms have higher fermentation yields and therefore reduce the consumption of resources for their production.
Protein engineering has been made possible by discoveries about the behavior of enzymes.
Protein engineering is beginning specifically to alter the amino acid sequence of enzyme proteins in such a way that their folded 3-D structure acquires more or less stability in industrial conditions, and/or confers an altered substrate preference closer to commercial needs. Developments such as these have been made possible by some remarkable discoveries about the behavior of enzymes at the
molecular level. An enzyme, xylose isomerase, is inactivated by a chemical reaction between glucose (its substrate) and the lysine units critical to its three-dimensional structure. Researchers at Gist-brocades have found a way to modify the gene for the enzyme so that the lysine units are replaced by others, which are less reactive but which will still hold the enzyme structure together.
Biochemists at the University of Colorado have designed and produced the first completely synthetic enzyme by creating a molecule that would mimic chymotrypsin. Using a computer program that models the shapes of proteins, the team designed a molecule that would support the key amino acids in the right geometric positions. The investigators then synthesized the molecule which, although not as
fast as the natural enzyme, is more resistant to high temperatures. They are also hoping to anchor artificial enzymes onto a solid matrix. If that effort is successful, artificial enzymes with properties such as superior temperature resistance might constitute a new generation of catalysts.
The use of bioconsortia and metabolic pathway engineering make it possible to combine or to design microorganisms to carry out specific tasks.
In nature, microorganisms rarely if ever live as single species. Instead, several species, each with different functions, act cooperatively. Traditionally, industrial microbiology has adopted the simple technique of working with a single species. It is now realized that by copying nature and bringing selected species together, it is possible to perform reactions that are otherwise difficult or
impossible to carry out. Pathway engineering is a complementary strategy to bring about unusual reactions. This involves the assembly, via recombinant DNA technology, of metabolic sequences from different organisms in a single organism. Early targets were the design of microorganisms to effect the degradation of toxic environmental chemicals such as polychlorinated biphenyls and dioxins. This
technique has recently been successfully employed for the evolution of a metabolic pathway in vitro, a three-gene combination coding for resistance to arsenic. Research is also progressing on the development of so-called "one-pot" multi-enzyme reactors, where mixtures of enzymes are used in cell-free systems, to achieve complex syntheses.
Directed evolution of enzymes offers a way to hasten the development of desirable features in microorganisms...
Chemical engineers who try to design real industrial processes using biological catalysts are constantly stymied by a simple fact: biological systems have evolved over billions of years to perform very specific reactions within particular environments. Some of these features are undesirable when the catalyst is removed from its natural context, and conversely, many properties that are desirable
in nature clash with those needed for industrial processes.
...by "shuffling" DNA sequences.
A strategy in which the properties of natural enzymes or proteins are modified in order to create desirable properties is known as "directed evolution". It complements the technique called "DNA shuffling", which involves taking a set of closely related DNA sequences, fragmenting them randomly, and reassembling the fragments into genes. This process rapidly produces a combination of positive
(desired) mutations as the output of one cycle becomes the input for the next cycle. This reiterative DNA shuffling leads to effective directed evolution. These procedures can be applied to evolve any protein rapidly even if the structure or the catalytic mechanism is unknown. By building enzymes with new features and functions,
enzymes can be tuned to function optimally under specified conditions.
By mimicking key evolutionary processes in the test tube, it is possible to explore enzyme functions without the limitations imposed by a living system. Since the vast majority of proteins remain largely uncharacterized, this represents a huge advantage. The process has been used to achieve improvements in enzyme stability in organic media and substrate specificity. The protease enzyme
subtilisin, which is used as a laundry aid, is stabilized by calcium. Unfortunately, industrial use of this enzyme frequently occurs in the presence of chemicals which bind calcium and consequently destabilize the enzyme.
The part of the enzyme that binds calcium can be deleted, and directed evolution can then be used to develop subtilisin stability that is independent of calcium. An evolved enzyme has been produced that retains the native catalytic activity but has a 1,000-fold increased stability under strong metal-binding conditions. Directed evolution of subtilisin has also produced variants with improved
stability towards hydrogen peroxide, or with enhanced activity, but not both. However, the two variant populations can be recombined by shuffling to create enzymes that are more stable and more catalytically active.
Bioinformatics offers powerful tools for "mining" databases...
Bioinformatics is a new field involving computer science, mathematics, computer and software engineering, and biology, which makes it possible to manipulate and analyze vast quantities of biological information through "data mining" techniques, which may make it possible to undertake experiments in silico, to complement, or even replace, experiments in vitro or in vivo. It is concerned with the
assembly, storage, retrieval and analysis of computer-stored databases, including DNA and protein sequence databases, and phenotypic information.
...on genes and DNA sequences.
A subset of bioinformatics, genomics, is concerned to obtain gene maps and the complete DNA sequences of organisms. The first sequences were achieved in 1995 for two bacterial species, four more were announced in 1996, and by the end of the century, tens of sequences are expected to be available. In addition, mapping and sequence analysis of various animal and plant species are also proceeding
rapidly, particularly the human genome. Data from genomic analyses can be used to address a variety of questions, especially the biotechnological exploitation of novel organisms such as extremophiles.
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Industrial Sustainability
