Biotechnology

Biotechnology
Thomas Becker, Institute of Process Analytics, University of Hohenheim, Germany Dietmar Breithaupt, Institute of Food Chemistry, University of Hohenheim, Germany Horst Werner Doelle, Department of Microbiology, University of Queensland, St. Lucia, Queensland 4067, Australia Armin Fiechter, Institute of Biotechnology, Eidgen ossische Technische Hochschule, Z urich, Switzerland Martijn Griensven, Ludwig Boltzmann Institute, Wien, Austria Cornelia Kasper, Institute of Technical Chemistry, University of Hannover, Germany , Institute of Biotechnology 2, Research Centre J Stephan Lutz ulich, Germany Ralf Portner , Institute for Bioprocess Engineering, Technical University of Hamburg-Harburg, Germany Hans-Gunther Schlegel, Institute of Microbiology, University of G ottingen, G ottingen, Germany Dieter Sell, DECHEMA e. V., Frankfurt, Germany Sakayu Shimizu, Department of Agricultural Chemistry, Kyoto University, Kyoto, Japan Frank Stahl, Institute of Technical Chemistry, University of Hannover, Germany Kirstin Suck, Institute of Technical Chemistry, University of Hannover, Germany Roland Ulber, Institute of Bioprocess Engineering, University of Kaiserslautern, Germany Joachim Wegener, Institute of Biochemistry, University of M unster, Germany Kerstin Wurges , Institute of Biotechnology 2, Research Centre J ulich, Germany Hideaki Yamada, Department of Agricultural Chemistry, Kyoto University, Kyoto, Japan Holger Zorn, Institute of Food Chemistry, University of Hannover, Germany

1. 2. 2.1. 2.2. 2.2.1. 2.2.2. 2.2.3. 2.2.4.

2.2.5. 2.2.5.1. 2.2.5.2. 3. 3.1. 3.2. 3.3. 3.4. 3.4.1. 3.4.2.

Introduction . . . . . . . . . . . . . Basics in Microbiology . . . . . . Microbiology the Science of Microscopic Life Forms . . . . . Phylogeny and Taxonomy of Microorganisms . . . . . . . . . . Denition and Survey . . . . . . . . Bacteria . . . . . . . . . . . . . . . . Archaea . . . . . . . . . . . . . . . . Morphological and Physiological Properties for the Identication of Prokaryotic Species . . . . . . . . . Eukaryotic Microorganisms . . . . Denition . . . . . . . . . . . . . . . Fungi . . . . . . . . . . . . . . . . . . Metabolism . . . . . . . . . . . . . . Microbial Systems Biology . . . Energy Production . . . . . . . . . Substrate Transport . . . . . . . . Catabolism . . . . . . . . . . . . . . Photosynthesis . . . . . . . . . . . . Chemosynthesis . . . . . . . . . . .

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3.4.3. 3.4.4. 3.4.5. 3.4.6. 3.4.7. 3.4.8. 3.4.9. 3.4.10. 3.5. 3.5.1. 3.5.2. 3.5.3. 3.6. 4. 4.1. 4.1.1. 4.1.2. 4.1.3.

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Carbohydrate Metabolism . . . . Aerobic Processes . . . . . . . . . Fats and Fatty Acid Metabolism Hydrocarbon Metabolism . . . . Amino Acid Metabolism . . . . . Anaerobic Metabolic Processes . Single-Carbon-Compound Metabolism . . . . . . . . . . . . . Inorganic Metabolism . . . . . . . Biosynthesis . . . . . . . . . . . . Amino Acids . . . . . . . . . . . . Lipids . . . . . . . . . . . . . . . . . RNA and DNA . . . . . . . . . . . Regulation . . . . . . . . . . . . . Metabolic Engineering . . . . . Analysis of the Transcriptome, Proteome, and Metabolome . . Gene Expression Analysis using DNA Microarrays . . . . . Fabrication of DNA microarrays Proteome Analysis using Protein Microarrays . . . . . . . . . . . . .

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c 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 10.1002/14356007.a04 107.pub2

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Biotechnology
Metabolome Analysis and Metabolite ux analysis . . . . . . . Design and Production of Genetically Optimized Strains for Production In Vitro Mutagenesis . . . . . . . . . . . . . . Random Mutagenesis, Isolation and Selection of Mutants . . . . . . Types of Mutants and Selection Principles . . . . . . . . . . . . . . . . Auxotrophic Mutants . . . . . . . . . Regulatory Mutants . . . . . . . . . . Other Selection Methods . . . . . . . Targeted or Site-Directed Mutagenesis . . . . . . . . . . . . . . . Cultivation and Bioprocesses . . . Isolation of Microorganisms . . . Requirements for Growth . . . . . Chemical Composition of Bacterial Cells . . . . . . . . . . . . . . . . . . . Carbon and Energy Sources . . . . . Accessory Nutrients . . . . . . . . . . Sulfur and Nitrogen . . . . . . . . . . Oxygen . . . . . . . . . . . . . . . . . . Complex Media . . . . . . . . . . . . Solid Media . . . . . . . . . . . . . . . Hydrogen Ion Concentration . . . . Carbon Dioxide . . . . . . . . . . . . Aeration . . . . . . . . . . . . . . . . . Anaerobic Techniques . . . . . . . . Media Preparation . . . . . . . . . . . Sterilization . . . . . . . . . . . . . . Moist Heat . . . . . . . . . . . . . . . Dry Heat . . . . . . . . . . . . . . . . . Filtration . . . . . . . . . . . . . . . . . Irradiation . . . . . . . . . . . . . . . . Chemical Means . . . . . . . . . . . . Types of Bioprocesses . . . . . . . . Surface Culture . . . . . . . . . . . . . Submerged Culture . . . . . . . . . . Process Layout . . . . . . . . . . . . Reactors . . . . . . . . . . . . . . . . . Containments for Anaerobic Processes . . . . . . . . . . . . . . . . Reactors for Aerobic Processes . . . Inoculation . . . . . . . . . . . . . . . Operation Modes . . . . . . . . . . . Process and Product Overview . . Biocatalysis and Biotransformation . . . . . . . . . . Introduction . . . . . . . . . . . . . . Classication of Biocatalysts . . . History . . . . . . . . . . . . . . . . . 6.4. 28 6.5. 30 31 32 32 32 32 34 35 35 36 37 37 38 38 38 38 38 38 38 38 39 39 39 39 39 39 40 40 40 40 40 41 41 41 41 41 42 45 45 45 47 47 6.5.1. 6.5.2. 6.5.2.1. 6.5.2.2. 6.5.2.3. 6.5.3. 6.5.4. 6.5.5. 6.5.6. 6.5.7. 6.6. 6.6.1. 6.6.1.1. 6.6.1.2. 6.6.1.3. 6.6.1.4. 6.6.2. 6.6.2.1. 6.6.2.2. 6.6.2.3. 6.7. 6.8. 7. 7.1. 7.2. 7.3. 7.4. 8. 8.1. 8.1.1. 8.1.2. 8.1.3. 8.2. 8.2.1. 8.2.2. 8.2.3. 8.3. 8.3.1. Characteristics of Enzyme Reactions Used in Biotransformations . . . . . . . . . Types of Biocatalysts and Reaction Systems . . . . . . . . . . . . . . . . . Biotransformation with Growing Cultures . . . . . . . . . . . . . . . . . Biotransformation Conversion with Previously Grown Cells . . . . . . . Vegetative or Washed Cells . . . . . Permeabilized Cells . . . . . . . . . . Dried Cells . . . . . . . . . . . . . . . Biotransformation with Spores . . . Biotransformation with Immobilized Cells . . . . . . . . . . . Biotransformation with Cell-free Enzymes or Puried Enzymes . . . Multistep Reactions Using Different Biocatalysts . . . . . . . . . . . . . . . Multiphase Reaction Systems . . . . Process Design . . . . . . . . . . . . General Considerations . . . . . . . . Evaluating Enzyme Potential . . . . Finding Suitable Enzymes . . . . . . Substrates . . . . . . . . . . . . . . . . Media . . . . . . . . . . . . . . . . . . . Selection of Biocatalysts . . . . . . . Screening . . . . . . . . . . . . . . . . Enrichment . . . . . . . . . . . . . . . Molecular Engineering . . . . . . . . Improvement of Conversion Processes . . . . . . . . . . . . . . . . Conclusion and Outlook . . . . . . Downstream Processing . . . . . . Sample Disruption . . . . . . . . . . SolidLiquid Separations . . . . . Product Recovery . . . . . . . . . . Solvent Extraction . . . . . . . . . . Monitoring and Modeling of Bioprocesses . . . . . . . . . . . . Characteristics of Bioprocesses . System Denition . . . . . . . . . . . System Description . . . . . . . . . . Dynamics of Biosystems and Real-Time Considerations . . . . . . Biotechnological Measurement Systems . . . . . . . . . . . . . . . . . Process Requirements Concerning Measuring Quantities . . . . . . . . . Online Sensing Devices . . . . . . . Further Aspects Concerning Measuring Systems . . . . . . . . . . Cognitive Computing . . . . . . . . Fuzzy Logic Systems . . . . . . . . .

50 51 51 52 52 52 52 52 53 54 54 54 55 55 55 57 57 58 58 58 58 59 59 60 61 62 63 64 65 65 66 66 69 71 73 74 75 79 80 82

4.3. 4.4. 4.4.1. 4.4.2. 4.4.3. 4.4.4. 5. 5.1. 5.2. 5.2.1. 5.2.2. 5.2.3. 5.2.4. 5.2.5. 5.2.6. 5.2.7. 5.2.8. 5.2.9. 5.2.10. 5.2.11. 5.2.12. 5.3. 5.3.1. 5.3.2. 5.3.3. 5.3.4. 5.3.5. 5.4. 5.4.1. 5.4.2. 5.5. 5.5.1. 5.5.2. 5.5.3. 5.5.4. 5.5.5. 5.6. 6. 6.1. 6.2. 6.3.

Biotechnology
8.3.2. 8.4. 8.4.1. 8.4.2. 8.4.3. 9. 9.1. 9.1.1. 9.1.2. 9.1.3. 9.1.4. 9.1.5. 9.1.6. 9.1.7. 9.1.8. 9.1.9. 9.2. 9.2.1. 9.2.2. 9.2.3. 9.2.4. 9.2.5. 9.2.6. 9.2.7. Articial Neural Networks (ANN) . Modeling Aspects of Biological Systems . . . . . . . . . . . . . . . . . Steps in Creating a Model . . . . . . Reasons for Making a Model . . . . Different Types and Basic Approaches for Building a Model . Special Applications in Biotechnology . . . . . . . . . . . . . Mammalian Cell Culture Technology . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . Products from Mammalian Cells . . Cell Types . . . . . . . . . . . . . . . . Growth Medium for Cell Culture . Small-Scale Culture Systems for Routine Use . . . . . . . . . . . . . . . Types of Bioreactors . . . . . . . . . Process Strategies . . . . . . . . . . . Downstream Processes . . . . . . . . Regulatory and Safety Issues . . . . Tissue Engineering . . . . . . . . . . Application of Tissue Engineering . Principle of Tissue Engineering . . Strategies . . . . . . . . . . . . . . . . The Essentials . . . . . . . . . . . . . Cells . . . . . . . . . . . . . . . . . . . Biomatrices . . . . . . . . . . . . . . . Bioreactors for Tissue Engineering 85 88 88 91 93 96 96 96 98 99 101 102 103 107 108 108 109 109 110 111 111 111 111 112 9.2.8. 9.3. 9.3.1. 9.3.1.1. 9.3.1.2. 9.3.1.3. 9.3.1.4. 9.3.1.5. 9.3.1.6. 9.3.1.7. 9.3.1.8. 9.3.2. 9.3.2.1. 9.3.2.2. 9.3.2.3. 9.3.2.4. 9.4. 9.4.1. 9.4.2. 9.4.3. 9.4.4. 9.4.5. 10. 11. 12. Growing New from Old . . . . . . . Biotechnology and Food . . . . . . Production of Food Additives by Cell Culture Systems . . . . . . . . . Amino Acids . . . . . . . . . . . . . . Organic Acids . . . . . . . . . . . . . Vitamins . . . . . . . . . . . . . . . . . Sweet Compounds . . . . . . . . . . . Sugar Alcohols . . . . . . . . . . . . . Microbial Saccharides . . . . . . . . Conjugated Linoleic Acids (CLA) . Lactulose . . . . . . . . . . . . . . . . Enzyme-Catalyzed Processes . . . . Starch-Modifying Enzymes . . . . . Lipases . . . . . . . . . . . . . . . . . . Pectin-Degrading Enzymes . . . . . Chymosin (Aspartic Protease) . . . Biotechnology and Health . . . . . Individualized Medicine . . . . . . . Clinical Diagnosis as Indicated in Genetic Anomalies in Cancer . . . . Pharmaceutical Development . . . . Dene Molecular Mechanisms of Toxicity . . . . . . . . . . . . . . . . . Detection of Genetically Modied Organisms . . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . Acknowledgement . . . . . . . . . . References . . . . . . . . . . . . . . .

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Biotechnology can be regarded as one of the key technologies of the 21st century. It is the commercial application of living organisms such as bacteria, fungi, yeasts, plant cells, viruses, and mammalian cells or their products, which involves the deliberate manipulation of their DNA molecules. This article gives an introduction into the basics in microbiology and provides an exhaustive description of the relevant microbial species and metabolic pathways. Identication, analysis, and manipulation of the genome, proteome, and metabolome is described, and cultivation requirements as well as process parameters discussed. Biotransformation and enzyme technology plays a central role in industrial biotechnology, and a focus is given on the development of molecular engineering techniques and new screening methods. Computational Biochemistry comprises the denition, monitoring, and modeling of bioprocesses. Examples of biotechnology applications include

mammalian cell technology, tissue engineering, and the production of relevant food additives as well as of various medical and pharmaceutical products. In conclusion, biotechnology offers manifold possibilities in industrial and medical applications.

1. Introduction
Biotechnology can be described as the commercial application of living organisms or their products, which involves the deliberate manipulation of their DNA molecules. This includes the use of bacteria, fungi, yeasts, plant cells, viruses, and mammalian cells. This denition implies a set of laboratory techniques developed within the last 20 years that have been responsible for the tremendous scientic and commercial interest in biotechnology, the founding of many new companies, and the redirection of research efforts and nancial resources

Biotechnology
Table 1. Timetable of biotechnology [1, 2] Year 3000 b.c. 2800 b.c. 1500 b.c. 300 b.c. 1300 1400 Biotechnological progress Fermentation of sugar containing juices to various alcoholic beverages brewing rooms in Mesopotamia and leaven in Egypt Use of microorganisms for the production of copper and production of soya sauce Use of vinegar Micro algae (Spirulina) as food additives (Aztecs) Production of saltpetre (potassium nitrate) with Nitrosomas sp. and Nitrobacter sp. in Germany Anthony van Leuwenhoek observes bacteria through a microscope Charles Cagnaird-Latour identies yeast as causer of fermentation (one year later conrmed by Theodor Schwann and Friedrich K utzing) Industrial production of bakers yeast Louis Pasteur separates brewer yeasts from lactic bacteria K uhne creates the term enzymes Industrial microbial production of lactic acid (Boehringer) Development of rst vaccines by Pasteur and Koch Industrial production of citric acid using Aspergillus niger Starting point of enzyme technology (Eduard Buchner) Patent for enzymes in washing powder Industrial fermentation process for acetone and n-butanol (Chaim Weizmann) Alexander Fleming discovers penicillin Microbial production of vitamin C Industrial production of antibiotics Use of Corynebacterium glutamicum for the production of amino acids Stanley Cohen and Francis Boyer develop a procedure for in vitro recombination of DNA, using plasmid vectors First recombinant proteins from bacteria First transgenic plants and animals Development of polymerase chain reaction (PCR) by Kary Mullis Start of human genome project (HUGO) Yeast genome is completely sequenced Human genome is completely sequenced (Craig Venter)

among established companies and universities. Thus, biotechnology can be regarded as one of the key technologies of the 21st century. However, the use of microorganisms and their products is as old as mankind. Fermented products with yeast such as beer, wine, or sake are known since several thousands of years (Table 1). The roots of modern biotechnology are coming from the era of microbiology, which was developed in the late 19th century. Cagniard-Latour, Pasteur, Koch and K uhne were important scientists who are decoding the principles of fermentation. The use of microorganisms for the production of bulk chemicals such as butanol or acetone and for pharmaceuticals (antibiotics) was strongly developed during World War I and II. With the better understanding of the gene function and its relation to the metabolisms of cells, the modern era of biotechnology started in the 70th of the last century. Nowadays, biotechnology is a cross-sectoral technology that has been successfully applied in many industrial branches. One distinguishes between several areas or colors of biotechnology (the so-called red, white, green, and blue biotechnology). Red Biotechology. Named as red biotechnology are applications in medicine or in the pharmaceutical industry. Red biotechnology has already made an impact on healthcare and will continue to contribute to improving human health and life expectancy. Today, 20 % of marketed medicines, 50 % of those in clinical trials, and 80 % in early development are biotechbased products. Forty percent of these candidate medicines are for the treatment of cancer. Typical products of red biotechnology are recombinant vaccines, antibodies, blood clotting agents, and hormones. In addition, tissue engineering is a part of the area of biotechnology. Tissue engineering aims at the functional regeneration of tissues through implantation of tissue cultured in vitro (see Section 9.2). White Biotechology. The potential and applications of biotechnology in other sectors largely pass unnoticed especially in the chemical industry. The chemical industry has produced coal, gas, and petroleum-based goods and products for over 150 years. This era is likely to

1676 1837

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end within the next 100 years at the latest. An extrapolation of oil consumption from 2002 (3.3 billion tonnes), for example, yields a supply period of 50 years with estimated stocks of 165 billion tonnes. The underlying reasons for a future era of new raw materials are the nite nature of fossil resources on the one hand, and the connection with many environmental problems on the other hand. In the past few years conditions for the application of biotechnological processes in industrial production have improved

Biotechnology (so-called white biotechnology). New tools such as screening methods and metabolic engineering, and also global analysis methods such as genomics, proteomics, metabolomeics, and bioinformatics are gradually becoming more widely available. These new instruments make it possible to reduce the time needed to develop and establish new industrial biotechnological products and processes; this was hitherto one of the major drawbacks of biotechnological, as opposed to chemical, processes. In addition, they help to develop biocatalysts (enzymes) and microorganisms which render manufacturing processes more economical and facilitate new manufacturing processes. For the rst time since the beginning of the oil age in the early fties, one is able to apply processes with economic potential in the production of basic chemicals and biopolymers based on biotechnological processes. In many cases, bioprocesses operate under milder conditions (lower temperatures and pressures, etc.) and more selectively than their competitors (chemical processes) by these means bioprocesses conserve resources and improve production processes economically and ecologically. Public debate has often emphasized the advantages for the environment; however, companies face hard economic competition so that when a biotechnological process is weighed against a classical chemical process, only the potential economic advantages can affect a shift in favour of biotechnology. Green Biotechnology. In addition, to overcome the problems in the production both of bulk and ne chemicals but also in the production of vaccines and pharmaceuticals, the socalled green biotechnology can offer new solutions. This area of biotechnology involves the introduction of foreign genes into economically important plant species, resulting in crop improvement and the production of novel products in plants. Green biotechnology might also produce more environmentally friendly solutions than traditional industrial agriculture. An example of this is the engineering of a plant to express a pesticide, thereby eliminating the need for external application of pesticides. Blue Biotechnology. In a very broad sense, marine or blue biotechnology can be understood as the various means of techniques of managing

marine living systems for mankinds prot. The domain covered by marine biotechnology is vast and range over various overlapping disciplines, from developmental biology to chemistry of natural substances and bioprocess engineering. Not all these elds, however, are ready for practical and industrial applications. Biomass from shing or aquaculture industry is, in fact, complex, geographically and seasonally dependent. Furthermore, many natural substances, for which we do not know of any terrestrial counterparts, and, therefore, present an up most interest, exists only in tiny amounts in rare biological species and their exploitation is likely to call for costly synthesis procedures. Marine natural products not only display novelty but also complexity in terms of chemical structures. Isolation and structure elucidation represents only the emerged part of an iceberg. The question of the functionality of new isolated molecules within the perspective of challenging major public health and environmental problems is crucial. Likely, in this domain, ecological and evolutionary approaches should help the classical screening systems for determining the right target systems. In addition, a better understanding of the complex interactions between macro- and microorganisms is necessary to be able to use these resources for industrial purposes. Thus, more and more groups are focussing on the part of bioprocess engineering and downstream processing in blue biotechnology [3].

2. Basics in Microbiology
2.1. Microbiology the Science of Microscopic Life Forms
Microbiology deals with microscopically tiny life forms which the resolution of the human eye (approx. 20 m) cannot detect. Many of the single-cell microorganisms, such as yeasts, algae, or protozoa, do not reach this size. Not until the invention of the microscope (Antonie van Leeuwenhoek, 1684) was the detection, and thus also the characterization, of microorganisms possible [4]. If microorganisms are dened by their size, many varied life forms come into this category: single-cell life forms with a real cell nucleus

Biotechnology and pertain to the eld of food microbiology. Microorganisms are used in the production of sausages (particularly salami), sauerkraut, and milk products (lactobacilli). In Asia, a variety of fermented food products are produced on the basis of rice and soya [8, 9].The typical aromatic properties and consistency of many food products originate from the activity of the microorganisms used. Nowadays, these organisms are generally used in the production process as starter cultures. The fermentation products are also often used as preservatives (acidication in lactic acid production by lactobacilli or alcohol production by yeasts). The monitoring of food in the framework of quality control involves excluding undesired microbial infestation or providing evidence of it. Microorganisms contribute towards stabilizing the global natural material cycles (e.g., carbon and nitrogen cycles). Furthermore, the waste ows produced by man are largely reintroduced into the natural material ows through the activity of microorganisms. The eld of environmental microbiology addresses the question of how microorganisms can be utilized for the remediation of contaminated soil, water, and waste gases. Successful industrial processes have been developed and are used worldwide for wastewater and drinking water purication, browneld clean-up, composting, and waste fermentation and also for the purication of contaminated gases [10 12]. Industrial microbiology, or white biotechnology, deals with the production of industrially interesting substances involving production volumes of thousands of tons. Effective production of substances requires on the one hand an efcient production strain to supply as much target product as possible in the shortest time possible. On the other hand, it needs a technical facility where the high-performance microorganisms nd optimum living and, therefore, production conditions and where in addition monoseptic cultivation in large volumes is guaranteed. Bioreactors fulll these requirements: they can be operated with volumes of up to several hundred m3 and are equipped with automatic measurement and control systems adjusted to the growth needs of the microorganism [13, 14]. Examples of microbially produced industrial products are diverse. The list begins with microorganisms that can themselves be the prod-

(e.g., algae, fungi, protozoa) and also some without a real cell nucleus (bacteria and archaea) which are of particular interest in general microbiology. Organisms with a genuine cell nucleus are termed eukaryotic organisms, those without prokaryotic organisms [5]. Today some 6500 species of prokaryotic bacteria and archaea are known, a relatively small number compared with the number of the known species of eukaryotic organisms. However, it is estimated that the number of actually living species of prokaryotic organisms is much higher, although most of them are regarded as not cultivable under laboratory conditions. Analyses of the metagenomes of the most varied habitats (total DNA assays of samples from various environments, such as forest soil, compost, pond water, etc.) have yielded estimated values of up to one billion potential species of prokaryotic organisms worldwide. For most people, microorganisms have a bad image. The reason is not hard to nd: some microorganisms can cause disease in man and animals (pathogenic microorganisms). Thanks to the achievements of medical microbiology, the often devastating effects of pathogenic microorganisms in earlier times have been conned due to the development of vaccines, antibiotics, etc. [6]. Nevertheless, microorganisms still represent a threat to health (for instance to people with a weakened immune system). The following are a few examples of bacteria that are pathogenic to humans: Bacillus anthracis (anthrax), Bordetella pertussis (whooping cough), Clostridium botulium (botulism), Clostridium tetani (tetanus), Corynebacterium diphtheriae (diphtheria), Shigella dysenteriae (shigellosis), Vibro-cholerae (cholera). The development of resistance to known antibiotics is a phenomenon that documents the variability of pathogenic microorganisms [7]. This also explains why it is necessary to promote progress in the eld of combating infectious diseases with microbiological methods. Biotechnology is closely linked to microbiology. Biotechnology uses microbiological metabolism performance to manufacture industrially relevant products. The sector with the oldest tradition of applying microorganisms for production purposes is the food industry. The manufacture of beer, wine, vinegar, bread, and cheese are early achievements of human history

Biotechnology uct of a biotechnological process and reach the market as starter cultures [15]. Currently the quantitatively dominant product is ethanol, of which over 24 106 (metric) tons are produced worldwide. Ethanol is produced with the yeast Saccharomyces cerevisiae and is mainly used as a substitute fuel or in admixture with other fuels. Other products include pharmacologically active agents (e.g., antibiotics, steroids, and alkaloids), bulk, special, and ne chemicals, food and feed additives, chiral intermediates, enzymes, antibodies, etc. The total value of all biotechnologically produced substances worldwide (including biopharmaceuticals which are often manufactured with cultures of animal cells) must be in the realm of > 100 109 euros annually.

2.2. Phylogeny and Taxonomy of Microorganisms

2.2.1. Denition and Survey Taxonomy is the branch of biology which subsumes individual groups of species into a hierarchical system. For a long time, the kingdom was the highest category of creatures. Originally, a distinction was made between animals and plants. Later, the bacteria were added and then the fungi and plants were split up [16]. Finally the archaea were given their own kingdom. In recent years, genetic investigations have led to a new classication with the domain superimposed over the kingdom. Whereas in earlier times the aim was to understand and deduce relationships among microorganisms on the basis of morphological, physiological, and cytological similarities, in the last few decades genetic similarity has been the criterion used to identify relationships directly from the genetic material. Nowadays, sequence analysis of ribosomal RNA (rRNA), which was developed by Carl Woese, is one of the methods by which phylogenetic trees can be generated [17]. Probably the best-known gene in the world is the 16S rRNA (S, Svedberg unit for characterizing the behavior of sedimentation) of prokaryotic organisms. This gene is composed of around 1500 base pairs and in the course of evolution was only subjected to relatively minor changes through mutations. The reason is that ribosomes

are the sites of protein biosynthesis and the molecular mechanism taking place is extremely sensitive to radical mutations. Thus, there are areas within 16S rRNA that are extremely well preserved, but also some where base exchanges have been more frequent. An evolutionary gap between two organisms can be determined from the relative similarity of different genes for the 16S rRNA of various prokaryotic organisms; this can be used as a measure of the degree of relationship [18]. Analogous to the investigations on prokaryotic organisms, the characterization of the degree of relationships of eukaryotic organisms is derived from their 18S rRNA, a molecule with approximately 2300 bases. Besides comparing the base sequences of rRNA, the amino acid sequences of universal proteins may also be drawn on to establish relationships. This does not always involve the same branchings of the phylogenetic tree as with the comparison of rRNAs. This explains why taxonomy, which owes its motivation primarily to the potential inherent in the new molecular biology tools, is currently a highly dynamic area of science. Today, all living organisms on earth are classied into three domains: archaea, bacteria, and eukaryota. The following representation of the manifold life forms of microorganisms adheres to this phylogenetic classication. 2.2.2. Bacteria Bacterial cells are small, generally less than 2 m in diameter and 25 m long (there are only a few giants of 5 20 m dimensions). The cells do not contain a membrane-enveloped nucleus; their DNA is rather a circularly closed double strand lying within the cytoplasm. The bacterial DNA contains the total genetic information of the cell. In many bacteria there are additional DNA circles, the plasmids; however, they are not required for growth under ordinary culture conditions. The ribosomes are small (sedimentation constant in the ultracentrifuge: 70 S), and their function is sensitive to various antibiotics that do not act upon the cytoplasmic ribosomes of eukaryotic organisms. In contrast to eukaryotic cells, bacterial cells do not contain organelles,

Biotechnology Bearing in mind all the postulated, noncultivable species, the actual number of bacterial phyla (or kingdoms) is probably far higher. Estimates assume that there will ultimately be about 50 phyla (or kingdoms) of bacteria. The current phylogenetic tree, therefore, can only be regarded as a snapshot. The bacterial phyla with the highest number of species known today are the proteobacteria, Gram-positive bacteria and cyanobacteria. Cyanobacteria are phototrophic organisms closely related to Gram-positive bacteria. Gram-positive bacteria form a group of chemo-organotrophic bacteria. The phylum of proteobacteria is the largest and physiologically most varied of all bacteria. Representatives of this phylum account for the majority of known Gram-negative bacteria of medical- and application-oriented interest [20]. Even the possibly best-known bacterium, Escherichia coli, belongs to this phylum. This example is used to demonstrate the taxonomic classication of an organism:
Domain: Phylum: Class: Order: Family: Genera: Species: Bacteria Proteobacteria Gamma protobacteria Enterobacteriales Enterobacteriaceae Escherichia Escherichia coli

but often polyphosphates, poly( -hydroxybutyric acid), or glycogen as storage substances (Fig. 1). Most bacteria are surrounded by a cell envelope that contains peptidoglycan (murein). Many bacteria are motile by either agellar or gliding movements.

Figure 1. Longitudinal section through a bacterial cell ca) capsule; cm) cytoplasmic membrane; cp) cytoplasm; cw) cell wall; f) agellum; gly) glycogen; li) lipid droplets; n) nuclear material or bacterial chromosome; phb) poly( hydroxybutyric acid); pi) pili; pl) plasmid; po) polyphosphate; rb) ribosomes; s) sulfur granules

Classication of Bacteria According to the Phylogenetic System. Originally, the subdivision of bacteria was based on their morphology and physiology, in the last few years, however, their classication has been completely revised and established on a molecular biology level. Thus, the taxonomy of bacteria, especially the higher levels of classication, cannot yet be regarded as denitive. Some authorities believe that the degree of variance between different bacterial groups is sufcient to give them each kingdom status of their own. Thus, in some publications reference is made to 13 kingdoms of bacteria, in others to phyla instead of kingdoms. The classication presented is derived from Bergeys Manual of Determinative Bacteriology [9], which the bacteria are subdivided into the following phyla: Proteobacteria Gram-positive bacteria Cyanobacteria Chlamydia Plantomyces Bacteroids/Flavobacteria Green sulfur bacteria Spirochetes Deinococci Green non-sulfur bacteria Hyperthermophiles (3 kingdoms)

The binomial nomenclature is used throughout biology. Each organism has a genus name and a species name and is thus specied unambiguously. 2.2.3. Archaea Several properties distinguish archaea from bacteria and eukaryota. Archaea are single-cell organisms mainly with a closed DNA molecule lying within the cytoplasm. They are of the same size as bacteria. Archaea have neither a cytoskeleton nor cell organelles, they are distinguished from bacteria by their lack of peptidoglycan and the different structure of their ribosomes. With over 200 species they are often found where extreme conditions prevail. Archaea have cell walls of pseudopeptidoglycan and single-layer cell membranes formed from ether lipids with covalent bonded chains. Many species of archaea have adapted to their

Biotechnology extreme surroundings, hence some species prefer temperatures of over 80 C (thermophile), others live in salt waters (halophile) or in very acidic or alkaline environments (acidophile/alkaliphile) [21]. Enzymes from such extremophile organisms, which are not only found among archaea, are particularly interesting for industrial applications; indeed they seem predestined for use in industrial processes with high temperatures and salt concentrations [22]. In the area of amylases thermotolerant enzymes are already applied for starch saccharication. Moreover the enzymes used in detergents (proteases, lipases, amylases, cellulases) are active and fulll their functions at temperatures of 60 C and above [23]. The phylogenetic system of the archaea that is valid at present subdivides the domains into the following phyla [19]: Euryarchaeota Crenarchaeota Korarchaeota (Nanoarchaeota is proposed as an additional phylum) Archaeota The Euryarchaeota are a diverse group. This phylum comprises not only methanogenic species, which are strictly anaerobic organisms, but also strictly aerobic organisms. Furthermore a large group of hitherto noncultivable organisms belong to this phylum and similarly to that of the Crenarchaeota, which include both hyperthermophilic representatives and mesophilic marine species. It is only very recently that representatives of the Korarchaeota phylum have been cultivated successfully. 2.2.4. Morphological and Physiological Properties for the Identication of Prokaryotic Species The properties of prokaryotic organisms as presented here permit the identication of species by conventional methods if molecular biology techniques are not available or cannot be applied. The shapes of most species are similar, and there are only a few morphological characteristics suitable for differentiation. In contrast, the diversity of metabolic features is tremendous. Prokaryotic organisms are extremely versatile.

Therefore, a great number of physiological and biochemical properties have to be determined to describe and identify a species unambiguously. Properties and description of prokaryotic organisms: 1) Shape. The majority of prokaryotic organisms are spheres, rods, or helices; these organisms are called cocci, rods, and spirilla, respectively (Fig. 2). Some organisms are encapsulated by proteins or polysaccharides, and some are combined to form packets, laments, or consortia. 2) Flagellation. The kind of agellation, length, type of undulation, and mode of attachment of agellae are important characteristics of prokaryotic organisms (Fig. 3). 3) Gram Stain. The Gram stain, originally devised by CHR. GRAM (1884) to make bacteria visible in animal tissues, is a reliable and fundamental cellular characteristic by which the bacteria are divided into two parts: Gram-positive and Gram-negative bacteria. It is based on the retention or discoloration of bacteria by ethanol after a staining procedure involving an aniline dye and iodine. In Gram-positive bacteria, the dye is retained by a cell wall consisting of a multilayered network of peptidoglycan; in Gram-negative bacteria, the dye is readily washed out because the cell wall consists of only two peptidoglycan layers and a highly permeable outer membrane envelope. The basic structural differences are easily visible in ultrathin sections (Fig. 4). 4) Endospores. Endospores are highly refractile, thermoresistant stages in the life cycle of two groups of Gram-positive bacteria belonging to the genera Bacillus and Clostridium. Endospores are easily visible and tolerate pasteurization (treatment by moist heat for 10 min at 80 C). Heating in an autoclave for 20 min at 120 C is required to kill the spores. 5) Need for Oxygen. Many prokaryotic organisms grow only in the presence of atmospheric oxygen. They are strictly aerobic organisms. In contrast, strictly anaerobic organisms can only grow in the absence of oxygen. Facultatively anaerobic or-

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Biotechnology totrophic characterizes the ability to synthesize the majority of cellular constituents from carbon dioxide, and heterotrophic, the derivation of cellular constituents from organic compounds. The designations are usually combined, e.g., Chromatium okenii is photolithoautotrophic. Some organisms require accessory nutrients, such as vitamins or amino acids, for growth. 9) Natural Habitats. The ecological habitat is the place where an organism or a population can usually be found. Examples are marine water, the sediment of a lake, fertile soil, or the intestinal tract. 10) Symbiotic or Parasitic Relationships. The close relationship between two dissimilar organisms is called symbiosis. It may be favorable for both (mutualistic) or unfavorable for one (parasitic).

ganisms can grow under both conditions. The last group includes aerotolerant species which tolerate but do not use oxygen. Others use oxygen if it is available. Some aerobic species are microaerophilic, i.e., they grow at low partial pressures of oxygen but not in equilibrium with air. 6) Energy Generation. The production of energy, i.e., regeneration of adenosine triphosphate (ATP), occurs via three fundamental processes, namely, fermentation, respiration, and photosynthesis. The strictly anaerobic fermentative prokaryotic organisms rely only on fermentation. All strict aerobes regenerate ATP by oxidative phosphorylation with oxygen as the electron acceptor. Mechanistically, energy generation by denitrifying, sulfate-reducing, methanogenic, or acetogenic species is quite similar to aerobic respiration and is therefore designated as anaerobic respiration. All photosynthetic microorganisms contain light-absorbing pigments, such as chlorophyll derivatives and carotenoids. The processes by which ATP is regenerated are similar in respiration and in photosynthesis; both are described as electrophosphorylation. 7) Effects of Temperature and pH. The majority of organisms are mesophilic: they grow at their maximum rate between 20 and 42 C. Thermophilic bacteria and archaea reach their maximum growth rates between 40 and 80 C. A few of them are able to grow above 80 C and up to temperatures of over 100 C; they are called extremely thermophilic organisms. The psychrophilic (or kryophilic) species prefer temperatures below 20 C. Most organisms tolerate pH values between 5 and 8 but prefer neutrality; a few are acid-tolerant, acidophilic, or alkali-tolerant, alkaliphilic. 8) Nutritional Types. All organisms that can use light energy for growth are called phototrophic. In contrast, the term chemotrophic denotes energy conversion by oxidation reduction reactions that involve either fermentation or respiration. Organotrophic denotes the utilization of organic compounds, and lithotrophic the utilization of inorganic compounds (ammonia, nitrite, hydrogen sulde, sulfur, hydrogen, carbon monoxide, iron(II)) as reductants. Au-

Figure 2. Shapes of bacteria A) Cocci; B) Diplococci, without and with capsule; C) Streptococci; D) Tetrads; E) Package cocci (Sarcina); F) Rods; G) Spore-forming bacteria, differing with respect to shape and localization of endospores in the mother cell; H) Short rods, coccobacilli; I) Coryneform bacteria; J) Mycobacteria; K) Vibrio-like bacteria; L) Spirillum (Thiospirillum); M) Spirilla (Aquaspirillum); N) Spindleshaped bacteria; O) Trichomes of lamentous cyanobacteria; P) Filamentous cyanobacteria with heterocyst; Q) Filamentous helically shaped cyanobacterium (Spirulina); R) Spirochaetes (Spirochaeta plicatilis)

Biotechnology 11) Composition of Cellular Macromolecules. The description of bacteria includes the Gram type of the cell wall, the type of peptidoglycan structure, the fraction of cytosine + guanine in the DNA, the similarity index of the 16 S rRNA, and data on DNADNA hybridization. 12) Antibiotic Resistance. The characterization of an organism may be supplemented by its pattern of antibiotic resistance.

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Figure 3. Flagellation of bacteria, agellar movement

The DNA is associated with histone protein. Mitochondria (for respiratory energy conversion) and chloroplasts (in plants for photosynthetic energy conversion) contain a prokaryotic type of DNA and ribosomes. Cytoplasmic ribosomes are large (80 S). Endoplasmic membranes and various kinds of membrane vesicles compartmentalize the cell; they are involved in the ingestion of nutrients (endocytosis) and the production and excretion of proteins and particles (exocytosis). An internal skeleton, the cytoskeleton, consisting of contractile protein (actin) laments and microtubules, gives the cell its shape and confers ability to move and to transport membrane vesicles within the cell. The cells of eukaryotic microorganisms can be motile by either ameboid or agellar movement. Most eukaryotic cells and organisms are aerobic. Energy for growth is derived from respiration or photosynthesis. Eukaryotic microorganisms include the protozoans, algae, and fungi. Fungi are the most relevant representatives of eukaryotic microorganisms in biotechnology and are used to produce antibiotics, secondary metabolites, and also organic acids, vitamins, etc. For this reason, fungi will be considered here in more detail.

Figure 4. Gram-positive and Gram-negative bacterial cell walls

2.2.5. Eukaryotic Microorganisms 2.2.5.1. Denition The cells of most eukaryotic organisms are usually much larger than prokaryotic cells and have diameters of 10 m or more [24]. They contain several structural components, compartments, and organelles. Figure 5 shows a plant cell as an example of a eukaryotic cell. The nucleus, consisting of a set of chromosomes that divide by mitosis, is surrounded by a double membrane.

Figure 5. Longitudinal section of a eukaryotic plant cell cw) Cell wall; chl) Chloroplast; cm) Cytoplasmic membrane; cp) Cytoplasm; di) Dictyosome; er) Endoplasmic reticulum; ex) Secretion vesicle for exocytosis; li) Lipid droplet; mi) Mitochondrion; mt) Microtubule; n) Nucleus or karyon; rb) Ribosomes; v) Vacuole

2.2.5.2. Fungi Fungi are aerobic eukaryotic organisms; they form a separate kingdom within the domain of the eukaryota [25, 26]. There are unicellular fungi, but the majority form laments (hyphae), (usually 510 m in diameter) and grow as masses of hyphae (mycelia). Many fungi form

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Biotechnology eral hundred species and strains of these higher fungi, which include many edible mushrooms, in submerged culture. Some could be of interest in biotechnology.

fruiting bodies. The shapes of fungi are diverse, and fungi are usually identied on a morphological basis. They are much less versatile metabolically than bacteria, and metabolic properties are almost useless for identication. Fungi have cell walls that often consist of chitin or cellulose. The group is rich in species, their number exceeding 100 000. Some estimates assume that 95 % of fungi have not yet been described. Growth and Reproduction. Fungal hyphae grow at their tip (Fig. 6). Each part of the mycelium is potentially able to grow, and a small piece of a mycelium can serve as an inoculum for growth. There are two kinds of reproduction, asexual and sexual. A sexual reproduction occurs by spore formation, bud formation, or fragmentation. Conidiospores are formed on the tips of hyphae (conidiophores), e.g., in the genera Aspergillus and Penicillium. If the spores are formed within special vessels, the sporangia, they are called sporangiospores, e.g., in the genera Mucor and Rhizopus. Bud formation is the mechanism of asexual reproduction among the yeasts. Sexual reproduction involves mating of two nuclei (karyogamy), zygote formation, and meiosis. It usually results in the formation of spores. In the lower fungi the zygote is usually transformed to a durant organ which after meiosis forms a sporangium. In the higher fungi, the zygote nucleus divides meiotically, and spores are formed either within a sac (ascus) or by budding on top of the basidia; the spores formed are called ascospores and basidiospores, respectively. The taxonomic classication of fungi is currently under debate and different suggestions can be found. The subdivision presented herein closely follows a traditional classication und contains the following classes: basidiomycetes, ascomycetes, zygomycetes, deuteromycetes, myxomycetes. Basidiomycetes. In basidiomycetes, the zygote enlarges to form a club-shaped cell, the basidium. The basidiospores are formed from projections of the basidium usually resulting in four spores on the tip. Most species are terrestrial fungi and live as saprophytes or parasites; many form mycorrhizas on trees. Nutrient media and methods have been developed to grow sev-

Figure 6. Mycelium, conidiophores, and fruiting bodies of fungi A) Mycelium of fungal hyphae; B) Yeast dividing by budding; C) Sporangium of Mucor mucedo containing sporangiospores; D) Sporophore of Penicillium forming conidiospores on the tips of branched hyphae; E) Sporophore of Aspergillus forming conidiospores on the tips of hyphae (sterigmata) located on the spherical end of the sporophore cell; F) Fruiting body (perithecium) of a typical ascomycete containing asci and ascospores; G) Basidium with four basidiospores; H) fruiting body of a typical basidiomycete

Biotechnology Ascomycetes. The ascomycetes are named for their formation of spores within a sac-like zygote, the ascus. With the exception of a few unicellular forms they grow as branched mycelia with open cross walls; they are coenocytic. Many ascomycetes grow on excrements of animals, i.e., they are coprophilic, whereas others are parasites on plants or insects. Asexual reproduction occurs by various kinds of conidiospores. The order Saccharomycetales within the Ascomycetes comprises all yeasts. Typical yeasts, such as Saccharomyces cerevisiae, are unicellular. They grow either as diplonts (with diploid nucleus) or as haplonts (with haploid nucleus), such as Schizosaccharomyces pombe. All yeasts are able to grow on sugar substrates, and a few can even grow on alkanes (Saccharomycopsis, Candida lipolytica) or on methanol (Candida boidinii, Hansenula anomala). Yeasts are organisms often used in industrial microbiology. Zygomycetes. The zygomycetes are lower fungi and comprise unicellular and mycelial fungi; they have diverse nutritional habits. They grow in water, soil, or moist habitats. There are saprophytic and parasitic species. The most important group biotechnologically is that of the Zygomycetales, which include Mucor mucedo, Rhizopus nigricans, and Phycomyces blakesleeanus. Several species are used for the production of vitamins, carotenoids, organic acids, and enzymes. Myxomycetes. The myxomycetes or slime moulds grow on decaying plant tissue, form multinuclear plasmodia, and produce fruiting bodies of yellow, red, or brown color. There is not yet a consensus on the exact evolutionary afnities of myxomycetes, but these organisms constitute a well-dened, homogeneous group of approximately 900 species. Deuteromycetes. If a fungus is incapable of sexual reproduction, it cannot be classied with either the ascomycetes or basidiomycetes, hence a third group of higher fungi was created. There are many deuteromycetes of biotechnological importance: Aspergillus, Penicillium, and Cephalosporium. Investigations of the 18S rRNA revealed that the majority of deuteromycetes could be assigned to the basidiomycetes and ascomycetes so that the group of

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deuteromycetes will possibly be abandoned in the future [27, 28].

3. Metabolism
The decisive factor for the development of a successful biotechnological production is an efcient biocatalyst. If this biocatalyst is a microbial production strain, knowledge of its metabolic capabilities is very important. Such knowledge can lead to an increase in product concentration and product variety, because one catalyst can often be used to produce different products [29]. Metabolism is generally concerned with all the events occurring within a cell that not only keep the cell alive but are also manifested in growth [30]. All synthetic reactions leading to growth are energy-consuming (endergonic) and are commonly referred to as anabolic reactions. In order to carry out these reactions, each individual synthesis must be coupled with energyproducing reactions of the catabolic sequence. Metabolism therefore consists of an intricate coupling between anabolism and catabolism (Fig. 7), whereby energy transactions and transfer mechanisms are based upon the basic laws of thermodynamics [31, 32]. Furthermore, in order to obtain a functional biosynthesis, the microbial cell must also produce a reducing agent, because biosynthesis involves the reduction of small molecules of high oxidation state to larger molecules of lower oxidation states. Both the energy and the reducing agent can be obtained from any of a number of diverse reactions depending upon the growth conditions and the genetic system available [33, 34] within the metabolism. Energy is generally dependent on adenosine triphosphate (ATP) and the reductant on nicotinamide dinucleotide NADH or NADPH. The cell has a very sophisticated regulatory control system that avoids oversynthesis of any chemical compound, thus securing its integrity. These regulatory control systems start at the cell membrane (substrate uptake) [35] and are present throughout the pathways of catabolism and anabolism.

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Figure 7. Anabolic and catabolic reactions in microbial metabolism

3.1. Microbial Systems Biology

In the future, systems biology will enable the metabolism of some microorganisms to be modeled and mapped in silico to such an extent that physiological performance and effects caused by environmental changes will be predictable. The essential prerequisites include sufcient knowledge of metabolic material ow and network analysis, reaction kinetic analysis of biochemical networks (enzyme kinetics, global metabolism regulation), global analysis of gene regulation networks (signal transduction), analysis of populationwide systems (quorum-sensing networks), and the abstraction of fundamental regulation patterns. The rst such model that integrates signal transduction, gene expression and metabolism has already been introduced for the response of yeast to osmotic shock [36]. There is no doubt that such models signify great progress for microbiology and biotechnology: quantitative simulations and targeted interventions in the metabolic network ideally open up the way to highly specialized, optimized production organisms. In order to optimize specic types of metabolic performance or to accelerate desired biosyntheses, industrial biotechnology is already relying to a great extent on the metabolic engineering of microorganisms. Here, mention should be made of the synthesis of amino acids [37, 38], optimized riboavin produc-

tion in B. subtilis [39], and, in the case of terpenoids, yield increases by engineering mevalonate biosynthesis in E. coli [40]. It is likely that knowledge from systems biology will trigger tremendous progress in this application-oriented eld. Moreover, strategies based on systems biology will also be important for biotechnological process development where an understanding of the complex regulatory networks of production organisms will be indispensable for process optimization [41].

3.2. Energy Production

Redox reactions are among the most important chemical reactions in living organisms. These oxidation reduction reactions are of special importance for energy production by living organisms:
G0 =n F E 0

where G 0 is the standard free energy of the reaction, n is the number of electrons (or hydrogen atoms) involved, F is the Faraday constant, and E 0 is the potential difference between the two redox systems. The simplest way to think about these oxidation reduction reactions is in terms of electron donors and acceptors. Thus, the substrate oxidized has a specic redox potential that is at the lower end of the electrode

Biotechnology potential scale, whereas the nal electron acceptor has a potential towards the more positive end of the scale. The difference between the two potentials corresponds to the energy obtainable in a chemical reaction between them. If the potentials of the substrate (electron donor) and of the nal electron acceptor are known, one can easily determine the energy production in a microbial system (Fig. 8) [42, 43].

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trochemical gradient (+ H ) across the energytransducing membrane. Proton electrochemical potential is a thermodynamic measure of the extent to which the proton gradient across the membrane deviates from equilibrium. This hypothesis is based on two separate proton pumps, whereby the + H is generated by electron transfer and is used to drive the second pump, involving the enzyme ATPase, backward in the direction of ATP synthesis. The nal result of substrate oxidation is therefore the generation across the cell membrane of gradients of both pH (pH) and electrical potential (E ). Both gradients exert a force on the protons extruded by the respiratory chain, tending to pull them back across the membrane. This proton motive force, + H , is the key element:
+ H =E 2.303 RT /F pH

The numerical value of 2.303 RT/F is 59 mV at 25 C.

3.3. Substrate Transport

Substrates undergoing catabolic reactions must enter the cell before catalysis can occur, because most catalytic enzymes are inside the cell. The cell membrane is a barrier for most ions and other molecules. For an ion to be transported across a membrane both a pathway and a driving force are required. Driving forces can be concentration gradients, electrical potentials, metabolic energy, or a combination of these. Four processes are known [45, 46]: 1) In simple diffusion, solutes move passively across the cell membrane, depending on the concentration gradient and thermal motion of the molecules (e.g., water, gases, low molecular hydrophilic compounds, organic acids in the protonated form). 2) In facilitated diffusion, solutes require a carrier for their transport in addition to the concentration gradient and thermal motion of the molecules. In both simple and facilitated diffusion, no metabolic energy or proton motive force is required. The energy for the transport stems from existing concentration gradients. 3) The term active transport is applied to a tight coupling of transport to metabolism in the ion pumps, which are central to chemiosmotic energy transduction. There are at least two

Figure 8. Redox potentials of various energy-producing microbial reactions

Because the potential difference between electron donor and acceptor can be signicant, the energy released could produce so much heat that the cell would be damaged. In order to avoid such cell damage, the cell has an energy-releasecontrolling cascade system, which makes certain that energy is released only stepwise. This cascade system is referred to as the respiratory chain or the electron transport chain. It is located in the cellular membrane and is responsible for the production of energy by electron transfer involving an enzyme called ATPase (which forms or hydrolyzes ATP) [35, 44]. The ATPase also plays an important role for the transportation of substances across the membrane into the cell. According to the chemiosmotic hypothesis for ATP production, the electron transfer chains are coupled to ATP synthesis by a proton elec-

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Biotechnology reducing agent is produced separately. In plants and algae, a second photosystem exists that splits water into H+ and oxygen, whereby the hydrogen ions reduce NADP+ and oxygen is set free. In bacteria, however, such a second photosystem does not exist, and inorganic compounds such as thiosulfate are oxidized, with the electrons running through the electron transport system to the reaction center and the proton via ferredoxin to NAD+ (Fig. 9). This is the reason why plants and algae possess mainly cyclic and bacteria noncyclic photophosphorylation. 3.4.2. Chemosynthesis In chemosynthesis, all energy and reducing agents are obtained by catalytic reactions of organic or inorganic compounds. In the former case, one refers to chemoorganotrophy and in the latter to chemolithotrophy. In addition, three different energy modes, which depend upon the electron donors and acceptors, are distinguished: aerobic respiration, anaerobic respiration, and fermentation [47]. Organic substances are used as an energy source by the vast majority of microorganisms that live in natural environments (Fig. 10). Nature provides them with an abundance of large organic polymers. A polymeric substance consists of series of monomeric units linked together. These monomeric units can be of the same kind (e.g., cellulose, starch) or of different kinds (e.g., pectin, lignin, sucrose). Within the polymeric structure, the monomeric units can be linked in a straight chain with identical linkages or different linkages or in branched chains; moreover, they can be linked in a specic sequence or at random. The structure of each of the polymer substrates for microorganisms is very important, because the enzymes produced by the microorganisms are specic not only to the types of monomers, but also in most cases to the way in which the monomers are linked. Since microorganisms can only take up monomers, all enzymes involved in the breakdown of polymeric substances must be extracellular. These enzymes are either membranebound or released into the medium. In general terms, Gram-negative bacteria accommodate these enzymes in the periplasmic space between the cell membrane and the cell wall and

distinct classes of active transport systems: the membrane-bound and the binding protein transport systems. 4) The group-translocation transport system comprises processes in which the passage of the substrate across the membrane occurs simultaneously with, and as a consequence of, chemical transformation of the substrate (e.g., phosphoenolpyruvateglucose phosphotransferase system with most anaerobic and facultatively anaerobic bacteria). Active transport and group-translocation transport are often referred to as primary transport, simple and facilitated diffusion as secondary transport. The carrier systems involved can be of the uniport, symport, or antiport type. Many bacteria pump natrium ions out of the cell by taking in two protons per natrium ion in an antiport system. Sulfate ions, on the other hand, are only taken in by some transport systems if protons are simultaneously symported.

3.4. Catabolism
3.4.1. Photosynthesis Photosynthesis creates living matter out of inorganic material, replenishes the reservoir of oxygen in the atmosphere in the cases of algae and plants, stores the energy of sunlight to support the life activities of organisms, and removes carbon dioxide:
CO2 +H2 A+light (CH2 O) +2 A+chemical energy

If A is taken to be oxygen the process is plant (or algae or cyanobacteria) photosynthesis; if it represents a sulfur atom the process is bacterial photosynthesis [43]. The process of photosynthetic energy conversion is initiated when photons are absorbed by specic molecules, such as chlorophyll. This absorption leads to an ejection of electrons, which are accepted by the compound having the lowest redox potential in animate nature, namely ferredoxin. The electrons then move through the electron transport system, and the energy is nally converted to ATP. Because the electrons return to the reaction center (Fig. 9), this system is called cyclic photophosphorylation. The

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Figure 9. Schematic representation of cyclic and noncyclic photophosphorylation in nature

Figure 10. Schematic representation of aerobic catabolism in microorganisms

the enzymes are thus membrane-bound, whereas Gram-positive bacteria and fungi have a tendency to release these enzymes into the medium. This is one of the reasons why extracellular enzyme studies are carried out predominantly with Gram-positive microorganisms, as well as yeasts and other fungi. 3.4.3. Carbohydrate Metabolism Most renewable resources are carbohydrates. The principal carbohydrate that serves as a carbon and energy source for microorganisms is

glucose. However, not all microorganisms follow the same route of glucose utilization. There are at least four major pathways of glucose metabolism, of which three lead directly to pyruvate: 1) Embden-Meyerhoff (EM) pathway, often referred to as the glycolytic or fructose bisphosphate pathway 2) Hexosemonophosphate (HMP) pathway, often referred to as the pentose shunt or pentose phosphate pathway 3) Entner-Doudoroff (ED) pathway

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Biotechnology bon dioxide. The electrons and hydrogen atoms removed from the individual organic compounds are accepted by oxygen and form the other two end products, ATP and water. The tricarboxylic acid cycle, however, is not only an energyproducing cycle, but it is also responsible for the production of precursors for amino acid and antibiotic biosyntheses [49, 50]: 2-ketoglutarate and oxalacetate ions. The tricarboxylic acid cycle serves, therefore, as the central pathway for the production of energy as well as for anabolic precursors. Under ideal growth conditions, the intermediates of the tricarboxylic acid cycle would be continuously withdrawn for anabolic reactions. Then, the formation of oxalacetate at the end of the cycle became vulnerable. Because oxalacetate is required not only as a precursor for anabolic amino acid biosynthesis, but also for the condensation with acetyl-CoA to keep the tricarboxylic acid cycle alive, the organism must have some safety device to ensure that oxalacetate is always available; otherwise the tricarboxylic acid cycle would be interrupted. The organism possesses a very ne control system for energy production and biosynthetic requirements in form of replenishment sequence reactions. Altogether, the microbial world has ve enzymes at its disposal for such sequences, of which phosphoenolpyruvate carboxylase is probably the dominant: it catalyzes the formation of oxalacetate from phosphoenolpyruvate and carbon dioxide. Phosphoenolpyruvate, in turn, is the intermediate from which pyruvate is formed in the EM pathway. This double function of the tricarboxylic acid cycle as an energy-generating cycle as well as a biosynthetic precursor-supplying cycle is very often referred to as amphibolic. This dual function, of course, must be controlled. There exists one enzyme in the tricarboxylic acid cycle, isocitrate dehydrogenase (it catalyzes the reaction: isocitrate oxalosuccinate), which performs this regulatory control. In the case of an overproduction of ATP this enzyme is inhibited by ATP, preventing any further oxidation and ATP synthesis, until the biosynthetic steps leading to RNA, DNA, aromatic amino acids, and fatty acids have caught up and utilized the excess ATP [33]. The general scheme in Figure 11 outlines the intricate interconnections between catabolism

4) Phosphoketolase (PK) pathway, almost entirely specic for heterofermentative lactic acid bacteria All four pathways of glucose metabolism have a great number of intermediates and enzymes in common. The details of these pathways can be found in many textbooks [32, 34, 48]. The EM pathway, for example, provides the greatest amount of ATP, but it does not produce ribose-5-phosphate, the important precursor for RNA and DNA biosynthesis; nor does it produce erythrose-4-phosphate, which is important for amino acid biosynthesis. Microorganisms that are capable of using only the EM pathway for glucose utilization are therefore not able to grow on simple media with glucose as the sole carbon source. They require growth factors or organic compounds (e.g., yeast extract) for growth and are referred to as fastidious microorganisms. In contrast, the HMP pathway produces all the precursors necessary for both RNA and DNA as well as for aromatic amino acid biosynthesis, but it produces only half the amount of ATP energy. This pathway does not produce pyruvate directly. The microorganisms must therefore possess part of the enzymes of the EM pathway. It is therefore not surprising that both pathways, EM and HMP, are common combinations, particularly in facultative anaerobic microorganisms. The ED pathway is unique, however, and has so far only been found in bacteria. Although this pathway is linked partly to the HMP pathway in the reverse direction for precursor formation, pyruvate is formed directly because of the aldolase cleavage of 3-ketodeoxy6-phosphogluconate. The ED pathway can exist on its own and is used by the majority of strictly aerobic microorganisms. The net result is similar to the HMP pathway, although one mole of ATP can be formed only if the carbon atoms go into pyruvate instead of precursor. 3.4.4. Aerobic Processes All aerobic microorganisms use the tricarboxylic acid (TCA) cycle as a main metabolic pathway. In this cycle, appropriate groups of enzymes catalyze a series of consecutive transformations, including oxidations, which nally result in the complete oxidation of pyruvate to car-

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Figure 11. General scheme of interconnections between catabolism and anabolism (cell biosynthesis) P = Phosphate

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Biotechnology despite the inhibition of isocitrate dehydrogenase (see Fig. 10). 2) The gluconeogenic pathways. From the oxalacetate formed, the organism is using one of the earlier mentioned replenishment sequence reactions to produce phosphoenol-pyruvate and reverses the EM pathway to glucose-6phosphate; from there ribose-5-phosphate and erythrose-4-phosphate can be formed via the forward HMP pathway. A great number of enzymes are common to both carbohydrate and fatty acid metabolism, which shows the economy of bacterial metabolism. The principal differences are in the control systems, which are outlined in detail in the literature, for example, [32, 34]. 3.4.6. Hydrocarbon Metabolism The metabolism of hydrocarbons has been examined in detail in the framework of environmental biotechnology in connection with cleanup of contaminated sites. Aliphatic hydrocarbons are good substrates for a large number of microorganisms. The straight-chain alkanes are more readily attacked than substituted or branched-chain alkanes. The attack occurs at either one end (monoterminal) or both ends (diterminal). The alkane is converted via the primary alcohol into the corresponding fatty acid and then via -oxidation into acetyl-CoA as outlined previously. Methyl group oxidation incorporates one atom of oxygen; this reaction is catalyzed by a mixed oxidase system consisting of three proteins: rubredoxin, NADH-rubredoxin reductase, and -hydroxylase. For the oxidation of methylene groups, rubredoxin is replaced by cytochrome P450 and -hydroxylase by methylene hydroxylase [53, 54]. Aromatic hydrocarbon utilization follows a uniform biochemical concept. The great majority of aromatic hydrocarbons, irrespective of the number of benzene rings, converge in their oxidative metabolism to three major intermediates: catechol, 3,4-dihydroxybenzoic acid (protocatechuic acid), and 2,5-dihydroxybenzoic acid (gentisic acid):

and anabolism among all those precursors necessary for cellular biosynthesis. Aerobic processes in biotechnology are used to produce various products, for example, antibiotics, amino acids, and organic acids [29, 51, 52]. 3.4.5. Fats and Fatty Acid Metabolism Fats are another major group of naturally occurring compounds (see Fig. 10). The majority of fats are triglycerides, that is, fatty acids (R1 , R2 , R3 ) esteried with glycerol:

These fats have to be converted into the monomeric components, the fatty acids and glycerol. This hydrolysis is carried out by enzymes called lipases. The glycerol component is converted to dihydroxyacetone phosphate, which participates in the EM pathway. The fatty acids, which vary in their carbon chain length, have to be activated by the formation of their respective coenzyme A (CoA) esters; this reaction step requires energy. These fatty-acid-CoA esters subsequently undergo cyclic oxidation, eliminating an acetyl unit at each turn. This type of oxidation is referred to as -oxidation. The acetyl-CoA formed after each cycle can then enter the tricarboxylic acid cycle for the formation of energy and biosynthetic precursors. However, this type of metabolism leading to acetyl-CoA does not produce the important ribose-5-phosphate and erythrose-4-phosphate. In order to obtain these precursors, all organisms whose metabolism leads directly into the tricarboxylic acid cycle via acetyl-CoA and not via pyruvate have to build up the carbon chain mentioned above and have introduced two pathways for this biosynthetic purpose: 1) The glyoxylate cycle. As mentioned earlier, overproduction of ATP inhibits the enzyme isocitrate dehydrogenase. The organism is then capable of inducing two new enzymes, isocitrate lyase and malate synthase, to circumvent the carbon dioxide-producing steps of the tricarboxylic acid cycle. This new shortcut TCA cycle leads to succinate and via glyoxylate to malate, and oxalacetate is formed

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The benzene nucleus in these intermediates is cleaved either between adjacent hydroxyl groups (ortho cleavage) or between a hydroxyl and a carboxyl group (meta cleavage). In both cases, the active incorporation of an oxygen molecule is required. Ortho cleavage, via the 2ketoadipate, and meta cleavage, via the keto acid pathway, lead into the tricarboxylic acid cycle at the level of acetyl-CoA, succinate, or malate and fumarate. As in fatty acid metabolism, the organisms use the tricarboxylic acid cycle for energy production and require the glyoxylate cycle together with gluconeogenesis to obtain pentose for RNA and DNA precursors. 3.4.7. Amino Acid Metabolism The aerobic utilization of amino acids follows a pattern that is very similar to the metabolism of other carbon sources [29, 49]. The only difference is the -amino group. In most cases, the amino acid is rst converted into the corresponding keto acid by either cytochrome-linked oxidases, transaminases, or NAD(P)-linked dehydrogenases. The nal product of the metabolism always leads to the intermediates of the tricarboxylic acid cycle. This is one of the reasons why -amino acids are able to function as the sole source of carbon, nitrogen, and energy for many microorganisms. As in fatty acid and hydrocarbon metabolism, microorganisms growing on amino acids must possess the glyoxylate cycle and the gluconeogenesis pathway. 3.4.8. Anaerobic Metabolic Processes The breakdown, or catabolism, of organic compounds in the absence of oxygen is generally referred to as fermentation. The microorganisms that carry out fermentations are either facultative or obligate anaerobes. The most characteristic difference between respiratory and fermentative metabolism is in ATP energy production. Because no electron transport occurs, redox reactions of organic compounds play a major role.

The number of cells obtained per mole of substrate in simple dened media is much smaller than under aerobic conditions. In addition to the cell material, large amounts of organic end products are formed, mainly primary alcohols, e.g., ethanol or butanol. Fermentations are normally classied according to their principal fermentation products. Figure 12 summarizes the end products from carbohydrates. The distinction between aerobic and anaerobic carbohydrate metabolism is made at the pyruvate level, because in anaerobic fermentation the organism has to substitute for the tricarboxylic acid cycle. In general the organisms are not able to produce the intermediates that serve as precursors for amino acid and protein biosynthesis. Therefore, fermentation processes cannot be carried out on simple dened media, but always require complex media if the organism is to grow and maintain its metabolism. Most of these fermentation processes are rather complex and details may be obtained from the relevant literature [32, 34, 55]. 3.4.9. Single-Carbon-Compound Metabolism A few isolated genera of microorganisms are capable of oxidizing single-carbon compounds. Microorganisms are called methylotrophs if they have the ability to derive both carbon and energy from the metabolism of methanol, methanotrophs if they can do the same with methane [53, 56 58]. Methane and methanol were the most common substrates used in the production of single-cell protein. The oxidation of methane leads to methanol and from there to carbon dioxide. The oxidation of methane or methanol to carbon dioxide, however, does not provide the organism with any precursors for biosynthesis. These organisms therefore possess an unusual carbon assimilatory pathway. Depending upon the enzymic assemblage, formaldehyde is incorporated into ribulose-5-phosphate or into the serine pathway in order to build up the carbon chain for the biosynthesis of RNA, DNA, and all the amino acids required for protein biosynthesis. The absence of a tricarboxylic acid cycle, the presence of anaplerotic sequence reactions, and

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Biotechnology

Figure 12. Fermentation end products of carbohydrate metabolism

the glyoxylate cycle demonstrates the biosynthetic capability of these pathways. 3.4.10. Inorganic Metabolism Some microorganisms are capable of using inorganic compounds as an energy source in aerobic metabolism or as a nal electron acceptor in anaerobic metabolism (anaerobic respiration). Both of these are combined in nature in the sulfur cycle and the nitrogen cycle, [32, 34]. Sulfur Cycle. Under anaerobic conditions, sulfate or thiosulfate can be reduced to sulde by sulfate-reducing bacteria. Such processes occur mainly in water-logged soils or stationary estuaries rich in organic matter, and can cause serious corrosion of iron and steel pipes in the soil. Hydrogen sulde is an extremely toxic product. If the soil or estuaries are supplied with oxygen, hydrogen sulde can be reoxidized to sulfate by aerobic sulfur-oxidizing microorganisms, for example, thiobacilli. The reoxidation of sulfur or sulde to sulfate is a microbial process of considerable biotechnological signicance. It not only acidies the soils and thus makes plant growth possible, but the sulfuric acid formed can also be used for ore leaching processes. Sulfuric acid can form soluble sulfates with a wide range of metals and by leaching old mining dumps it helps concentrate these for further utilization of

the ore. Such ore leaching processes have led to a vastly improved mining industry and increased the economy of ore mining. Nitrogen Cycle. Nitrogen is the most abundant gas in the atmosphere. Nitrogen-xing organisms are capable of converting nitrogen into nitrogen compounds that are acceptable to plants. Nitrogen-xing microorganisms can be divided into free-living (cyanobacteria, Clostridium, Azotobacter ) and symbiotic (Rhizobium). The last group in particular may become of great biotechnological importance, because they live in association with legume plants and provide these with the necessary nitrogen source even in nitrogen-starved soils, thus eliminating the need for large amounts of nitrogen fertilizers. The nitrogen-xing organisms convert nitrogen to ammonia, which then can be oxidized by chemolithotrophs (Nitrosomonas and Nitrobacter ) via nitrite to nitrate. This conversion of nitrogen to nitrate is referred to as nitrication. The reverse reaction, called denitrication, is an anaerobic process in which the nitrate or nitrite serves as electron acceptor. The product of denitrication is nitrogen gas. Nitrication and denitrication are important process steps in biological wastewater cleaning. The chemolithotrophs, however, face an energy problem. The redox potential of the inorganic compounds used as electron donors is well

Biotechnology above that of the NAD/NADH couple that is required as reducing agent for biosynthesis. Since the electrons can only enter the redox system at the cytochrome level, electron transport must be reversed to obtain NADH. In photosynthesis, this reversal can be carried out by the reaction center of the photosynthetic apparatus, but in chemosynthesis the reversal has to occur with energy input from the oxidation of the inorganic source. It is therefore not surprising that such organisms grow much better in the presence of organic material, which they are able to assimilate directly, thus obviating the reductive biosynthesis and consequently the need for reductant formation.

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pentoses, trioses, and the dicarboxylic acids required for macromolecular biosynthesis [46, 47]. 3.5.1. Amino Acids About twenty -amino acids are required for the biosynthesis of the numerous types of proteins that provide the catalytic capability of the microorganisms. These amino acids are produced from the following precursors:
Erythrose-4-phosphate Phosphoenolpyruvate Ribose-5-phosphate 3-Phosphoglycerate Pyruvate 2-Oxoglutarate Oxalacetate tyrosine, tryptophan phenylalanine histidine serine, glycine, cysteine alanine, valine, leucine glutamic acid, glutamine, arginine, proline aspartic acid, asparagine, methionine, lysine, threonine, isoleucine

3.5. Biosynthesis
The microbial cell consists of ve major types of macromolecules: proteins, polysaccharides, lipids, RNA, and DNA [32, 34]. These macromolecules are built up from relatively few monomers, which the cell has to provide or to synthesize: amino acids, sugar phosphates, fatty acids, ribonucleotides, and deoxyribonucleotides. Microorganisms are divided into two groups: autotrophs, which are able to produce all their cellular requirements from carbon dioxide as the sole carbon source, and heterotrophs the majority of microorganisms which produce the cellular material from organic compounds. Photo- and chemolithotrophs use ATP and the reducing power produced by photosynthesis and/or oxidation of inorganic substrates to reduce carbon dioxide to cellular material. The pathway for carbon dioxide reduction is referred to as the Benson-Calvin cycle. The actual carbon dioxide xation reaction is catalyzed by ribulose-1,5-bisphosphate carboxylase; the primary product of this reaction is 3-phosphoglycerate, which leads to glyceraldehyde-3-phosphate, a member of the EM pathway:
3 CO2 +9 ATP+6 NADH2 glyceraldehyde3phosphate +9 ADP+8 Pi +6 NAD+

The most important amino acids are glutamic acid and glutamine, which gure in transamination reactions during biosynthesis. Therefore, the availability of these two amino acids is vital for protein biosynthesis. The detailed pathways can be found in the literature [32, 34, 46]. Theoretically, each of these -amino acids can be produced commercially by microorganisms, some are already produced, but some practical problems remain to be solved for some of them [49]. 3.5.2. Lipids Most of the fatty acids that occur in lipids contain 16 or 18 carbon atoms and are saturated or unsaturated with one or more double bonds. AcetylCoA is the only precursor for the biosynthesis of all fatty acids. In order to differentiate between fatty acid catabolism and fatty acid biosynthesis, the organisms employ CoA activation in the former and an acyl carrier protein (ACP) in the latter. The C2 units are added to the acetyl-ACP in the form of malonyl-ACP. Once the required chain length is reached, ACP is hydrolyzed and the appropriate fatty acid is formed. Unsaturated fatty acids are generally formed by dehydration of a hydroxy fatty acid. The fatty acids are then esteried with glycerol, which is readily available from dihydroxyacetone phosphate, a member of the EM path-

From glyceraldehyde-3-phosphate, these organisms are capable of producing the important

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Biotechnology can be reduced to almost zero by preculture conditions. A much more complicated regulation is catabolite repression, which is mainly manifested in the so-called diauxie phenomenon. The latter is biphasic growth in the presence of more than one carbon source in the medium. In this reaction the organism takes up only one substrate at a time and the presence of this particular substrate represses the enzyme system that is responsible for the metabolism of the other substrate. This repression occurs at the operon level and occurs as soon as the concentration of the rst substrate becomes very small. Enzyme synthesis can also be inhibited by end products within a short period of time. For example, if the medium contains any -amino acid, the organism would use this amino acid directly instead of producing it. The added amino acid therefore represses the synthesis of each enzyme in its synthesis pathway. In addition to the regulation of enzyme synthesis, the cell must also adjust the activity of enzymes to its metabolic requirements. If a monomer is synthesized in larger amounts than needed for polymer synthesis, it is not necessary to stop the synthesis of the enzyme completely, but it sufces to reduce the activity of that particular enzyme. This fast control action is referred to as feedback inhibition. The target enzymes for feedback inhibition are called allosteric enzymes.

way. Finally, triglycerides are formed. The introduction of a phosphate group gives a phosphatidic acid and a whole range of phospholipids. If the phosphate group in the phosphatidic acid is replaced by a carbohydrate, glycolipids are produced [32, 34, 46]. 3.5.3. RNA and DNA Ribonucleotides consist of a purine or pyrimidine derivative, ribose, and phosphate groups. A purine or pyrimidine base attached to a ribose is referred to as a ribonucleoside; if a phosphate group is attached to this ribonucleoside it is called a ribonucleotide. The most important pyrimidine derivatives are uracil, cytosine, and thymine. The most important purine derivatives are adenine and guanine. The biosynthesis of pyrimidines and purines starts at the ribose-5phosphate level, which also represents the ribose in the nucleotide. The RNA consists of ribonucleotides. Reduction of the ribonucleotides leads to deoxyribonucleotides, the building blocks for DNA biosynthesis [44, 46].

3.6. Regulation
The microbial cell possesses a complex of catabolic and anabolic capabilities with many interconnected pathways. The cell is required to maintain balance among the various parts of this extremely complex network of reactions [32, 34, 44, 46, 48]. The cell therefore has developed very intricate and rened devices to streamline its economy. Regulation can either be manifested in the enzyme synthesis or in the enzyme activity. Induction and repression of enzyme synthesis occurs at the genetic code, whereas feedback inhibition simply regulates the activity of the enzyme. The importance for biotechnology is to realize that the rst type of regulation is concerned with substrate, catabolite, and end product inhibition, whereas the second mainly involves a transitional end product inhibition. More than one enzyme usually is required to channel a substrate into intermediary metabolism. The substrate can therefore be responsible for a coordinate or sequential induction of enzymes. This activity of the cell normally occurs during the lag phase of growth and

4. Metabolic Engineering
The term metabolic engineering refers to the genetic optimization of prokaryotic or eukaryotic cells that are used in industrial fermentation processes. In these processes, they are utilized for the economically production of proteins or other ne chemicals. Bailey [59] rstly introduced the denition of metabolic engineering in 1991 as the improvement of cellular activities by manipulation of enzymatic, transport, and regulatory functions of the cell with the use of recombinant DNA technology. However, one of the early examples of successful genetic engineering of a living organism for production purposes, namely the production of human insulin in E. coli cells, had already been described

Biotechnology quite some time before a name was given to this new direction of science. According to Cameron and Tong [60], there are ve categories of metabolic engineering that are grouped with respect to the objective of the genetic modication: 1) Improved production of chemicals already produced by the host organism. 2) Extended substrate range for growth and product formation. 3) Introduction of new catabolic activities for the degradation of toxic materials. 4) Production of chemicals that are new to the host organism. 5) Modication of cell properties, for instance, to make them more resistant to the production conditions. The process of metabolic engineering is most often described by a circle of elementary steps that have to be performed once or several times. This circular process is demonstrated in Figure 13.

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Figure 13. Schematic illustration of the elementary processes of metabolic engineering

When, for instance, an organism should be modied to express a recombinant protein, the rst step is to design and create the DNA sequence that codes for the protein in an appropriate vector system. Thus, one will enter the cycle at the design level. The same is true when an existing metabolic pathway should be extended in the way that the original end product of that pathway is metabolized to a compound of interest. However, when an existing metabolic pathway shall be used to produce a given compound in high yields, one will have to start by analyzing the metabolic situation and the metabolic uxes in order to identify targets for genetic engineering. In this case, one will enter the cycle at the analysis level. In particular in the latter case, it is often necessary to go through the cycle of metabolic engineering more than once, since complex metabolic networks within living cells often require several genetic modications to provide the compound of interest in improved quantities. Metabolic engineering is to a high degree dependent on novel developments and improvements in chemical analytics as well as molecular biology. In both disciplines, the last decade has brought about powerful new technologies such as protein and DNA chip technology to mention only two that provided considerable progress in metabolic engineering. For further information, the interested reader is referred to the reviews by Nielsen [61] and Ostergaard [62] and the website of a scientic journal that is exclusively devoted to this research area (www.apnet.com/mbe). The following paragraphs will discuss in more detail the three elementary processes of metabolic engineering, analysis, design, and production of a modied strain. Fermentation will be addressed in other chapters of this series. ( Ethanol, Chap. 5, Antibiotics, Chap. 5)

The elementary processes are Analysis of the organisms with respect to the Proteome and/or metabolome Design of a genetic modication that improves/allows the synthesis of the compound of interest Production of a modied strain Fermentation and production Depending on the task at hand, one enters the engineering cycle at different entry points.

4.1. Analysis of the Transcriptome, Proteome, and Metabolome

The bottleneck of all of the traditional methods of transcriptome and proteome characterization is the limitation to an analysis of just a few samples at a time. While conventional methods are conning themselves to the examination of single genes, a DNA or protein chip experiment

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Biotechnology sitive detection and selective identication of biochemical discrete compounds (carcinogens, metabolites, proteins, etc.) or living systems (bacteria, viruses, or related components) at low levels in complex biological matrices, tissues, and blood. In the future, it will be indispensable to analyze compounds of the genome and the proteome. Therefore, new formats of biosensors, so called biochips, have to be developed. Such biochips or microarrays can be used for identifying genes as well as changes in their activity. Furthermore, these biochips allow the simultaneous detection of several disease endpoints using different bioreceptors such as DNA, antibodies, enzymes, or cellular probes. The high parallelization degree of biochips is a great advantage over classic molecular biological methods. While conventional methods are conning themselves to the examination of single genes, a DNA chip experiment delivers a complete gene expressions pattern of the cell. DNA microarrays can be used for the purpose of monitoring expression levels of thousands of genes simultaneously. Furthermore, biochips can solidly simplify and accelerate a number of long and expensive diagnostic methods. Additionally, gene expression analysis across various biological conditions, cell cycle states, tissues, and subjects may help identify differentially expressed genes. This type of information is a valuable pinpoint in the investigation of biological processes and functional disorders. Recapitulating, DNA chips provide a format to prole complex diseases and discover novel disease-related genes. Applications for this technology include gene expression monitoring, mutation detection, metabolic engineering, drug development, tailor made therapeutics, SNP research, GMO detection, and high-throughput screening, among others. They have a profound impact on biological research, industrial production, medicine and pharmacology and will be used as the biosensors of the future [63 67]. For the understanding of biological systems with up to 30 000 genes, the measurement of RNA levels for a complete set of transcripts of an organism will be necessary. The use of glass slides as medium enables high spot densities on microarrays (up to 10 000 spots per slide). A DNA chip experiment works by hybridizing all of the gene probes on the chip with the nucleic acid target to be tested. cDNA labeled with uorescent tags is used, produced by

delivers a complete gene or protein expression pattern of the cell. The high degree of parallelization realized in biochips is the great advantage over classic molecular biological approaches. 4.1.1. Gene Expression Analysis using DNA Microarrays In the past few years, the complete DNA sequences of a number of different microorganisms have been determined and can be exploited to optimize strains as well as recombinant protein production. Strain optimization involves measurement of genomewide mRNA levels in wild-type and mutant strains using DNA microarrays (Fig. 14) Furthermore, microarray analysis can help identifying previously unknown genes required for recombinant protein production. Bioprocess optimization using microarrays entails metabolic control analysis, modeling, and molecular biology to create new mutants and strains with an, for example, optimized protein production rate. Recombinant protein expression exerts a metabolic burden on the host cell, whereby stress response mechanisms are triggered on different levels. Microarrays allow the investigation on a genome scale, which enables the qualitative and quantitative characterization of the burden on host cell metabolism. Thereby, a better understanding of the impact of recombinant protein production can be achieved by using the generated snapshot of the actual cellular composition and activity. Additionally, the knowledge of the interaction of host cell metabolism with recombinant protein production is improved and contributes to process optimization. Chip Technology. Alongside metabolome analysis, analysis of various components of the proteome, the transcriptome or the genome will become increasingly valuable. New biosensors known as biochips will be required. DNA chip technology has already opened up new ways of studying disease in more depth and identifying far more possible targets. A DNA chip is an array of synthetic DNA sequences representing different genes. Up to now, membrane and immune biosensors are used to analyze substances of the metabolome. One important goal in chemical and biological sensing is the sen-

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Figure 14. Interaction of labelled target molecules with probe molecules on a glass array

reverse transcription of RNA from the sample to be analyzed. Hybridizing a sample of nucleic acid with many complementary gene probes in parallel on a DNA chip produces a hybridization pattern with corresponding hybridization intensity. DNA chip technology therefore enables large numbers of genes to be screened simultaneously, giving a comprehensive, detailed picture of changes in gene expression, shedding light on complex regulatory interactions. 4.1.2. Fabrication of DNA microarrays There are three primary technologies used presently in automated microarray fabrication including photolithography, ink-jetting, contact printing, and derivatives thereof. Each of these technologies has specic advantages and disadvantages in microarray manufacturing. The photolithographic approach relies on the in situ synthesis of 25mer oligonucleotides using photomasks. This means that each probe is individually synthesized on the chip surface. Photolithography was developed by Fodor et.al. [68] and commercialized by Affymetrix (Fig. 15). In contrast, the ink-jetting and contact printing methods attach presynthesized DNA probes to the chip surface. While the in situ probe synthesis necessitate expensive and sophisticated equipment the contact and noncontact spotting

methods made DNA chips affordable for academic research laboratories. Since 1996, many DNA arrayers have become available and the self spotted glass slide DNA arrays are today the most popular format for gene expression proling experiments.

Figure 15. Microarray fabrication using an Affymetrix 427 arrayer

4.1.3. Proteome Analysis using Protein Microarrays The utilization of protein biochips for the Proteome analysis offers a number of alternatives and advantages in metabolic engineering. Protein microarrays make use of technological innovations and enable the proteome analysis in

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Biotechnology severely reacts are often unknown. However, the sensitive dependence of most different parameters offers the possibility of applying specic small changes of the protein expression pattern in order to create sensitive biosensors. Ideally, the analysis of the proteome delivers the currently available set of all proteins, if there is a way to maintain the quantitative relations of all proteins during the analysis. Such data cannot be obtained with classical molecular biology since no strict connection between the amount of mRNA and the amount of protein exists. Parameters such as mRNA stability, posttranslational modications, protein degradation, and others consequently prevent a statement over the currently available amount of protein. However, this information is of utmost importance making a high throughput analysis necessary. One attractive method is the use of protein microarrays. Genomewide screens for protein function are of biological importance for many applications: Analyzing protein expression proles Monitoring protein-protein interactions Identifying protein posttranslational modications Screening the substrates of protein kinases Examining the protein targets of small molecules Proteomic analysis as a function of bioprocess cultivation conditions 4.1.4. Metabolome Analysis and Metabolite ux analysis In order to improve a given strain of microorganisms or even more complicated a eukaryotic cell line that is used in a biotechnological production process, it is not only necessary to analyze gene expression patterns or the proteome. Analysis of metabolic turnover is central for recognizing possible targets for genetic engineering. Metabolic turnover comprises a large number of biochemical reactions. For instance, in the well-examined yeast Saccharomyces cerevisiae, about 1500 biochemical reactions have been estimated involving more than 850 metabolites and cofactors [74]. Since in most of these reactions there is more than two educts and more than one product, not to speak of the necessary cofactors, the metabolic turnover represents a complex network of biochemical reactions, also referred to

miniaturized test formats, thereby promising signicant advantages such as an improved analytical speed, a better separation efciency, reduced sample/reagent consumption, contamination reduction and reduced costs. Protein microarrays consist of bound antigens or antibodies as capture molecules for detecting proteins in a complex mixture. They provide a huge potential for monitoring protein expression and protein proling. Although the basic construction of such protein microarrays is similar to DNA microarrays, the more delicate nature of protein structures has hindered the development of such devices for the analysis of proteins for a long time. This is due to their more complex coupling chemistry, the instability of the immobilized protein and the far weaker detection signals. In principal, microarray technology enables both, the probing of the genome and the proteome. Today, protein chip technology focuses basically on the understanding of molecular pathways which allows conclusions concerning the molecular diagnostic, the drug discovery, and metabolic engineering applications. These interferences can be done because of the capability of protein microarrays to analyze the protein function of a whole genome level [69 73]. The results of such high- throughput screening approaches can change our fundamental understanding of the cellular processes of life on the molecular level. However, gene expression analysis does not sufcient enable a reliable prediction of the function of a protein. Monitoring protein interactions is an extremely complex matter since the proteome is the quantitative representation of the complete protein expression pattern of a cell under accurately dened conditions. The proteome represents the protein equivalent of the genome. In contrast to the genome which is determined by the sequence of its nucleotides and therefore static, the proteome represents an extreme dynamic object which is inuenced by many parameters. Not all genes will be switched on at the same time in a cell and the sensitive balance between protein synthesis and protein degradation can vary widely under different metabolic conditions. Consequently, a repeated analysis of the proteome will be successful only under exactly dened conditions, for example, cell culture conditions. In practice, this proves to be very difcult since conditions to which the cell

Biotechnology as the metabolic network. The concept of isolated metabolic pathways such as glycolysis, citric acid cycle, or urea cycle as presented in most biochemical textbooks is therefore somewhat misleading since it indicates that these pathways proceed independently. However, they are all interconnected and represent a huge interdependent network that can activate or slow down individual branches. Control over the activity of individual metabolic branches can be exerted on the levels of transcription, translation, proteinprotein interaction, or enzyme activity regulation, which adds another level of complexity. According to Nielsen [77] it is therefore necessary to analyze the metabolic network in three instances (metabolic pathway analysis) in order to fully oversee the metabolic activities in a living cells, which is considered as a prerequisite for a rational optimization of the production strain: Network structure (pathway topology). Quantication of uxes through the branches of the network. Identication of control structures. Much work has been done in the past in biochemical laboratories around the globe to identify the metabolic network structure at least for the more well-known microorganisms in particular when they are of industrial importance. Thus, very often the presence of a certain metabolic pathway can be deduced from the available literature. The most powerful approach to identify a certain pathway when no information is available is called metabolic labeling. Here, the organism is fed with 13 C-labeled glucose (or any other appropriate fundamental metabolite) and after a given time that is allowed for metabolization the labeling pattern of other intracellular components is identied and reports on the metabolic pathways involved. From the analytical perspective, analysis of the metabolite pattern is most often done by GC-MS (gas chromatography coupled to mass spectrometry) but also by NMR (nuclear magnetic resonance). Later on, biochemical assays addressing the activity of certain key enzymes can be used to conrm the presence of a certain pathway and dig out the cofactor requirements for the individual steps within the pathway. Pathway identication just by enzyme assays is possible, however, very tedious and time consuming.

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Flux Analysis. Once the network topology has been identied, it is necessary to learn about the uxes through each of the network branches. This kind of analysis requires experiments as well as mathematical modeling. The most important concept behind ux analysis is referred to as metabolite balancing. According to this, the concentration of each metabolite is stationary inside the cell or in other words: the generation rate of a given metabolite in one metabolic pathway (v+ ) equals the rate of its onward reaction through the sum of all downstream metabolic pathways (v ). Thus, for a given metabolite the corresponding material balance can be described as
v+1 +v+2 +v+3 +. . .+v+i =v1 +v2 +v3 +. . .vi

Such balances are established for all relevant metabolites, such that a set of algebraic equations results that describe the ux of molecules through the different branches of the metabolic network. When some of the uxes are experimentally determined, the remaining uxes can be deduced mathematically from this set of equations. The beauty of the metabolic balancing approach is its simplicity. But reliable predictions can only be obtained when the cofactor (NADH, NADPH, . . .) balances are also known which means that all the pathways that generate or consume any of these cofactors have to be considered in the formalism. Feeding the organisms with 13 C-glucose in combination with a molecular analysis of the resulting metabolite labeling downstream of glucose provides, however, an easier solution. From the labeling pattern of all metabolites it is possible to set up equations for the balances of individual carbon atoms. Thus, the number of equations describing the overall system increases the number of constraints for the solution and thus, makes balances of cofactors unimportant. The nal step in analyzing the metabolic network is to identify the regulatory mechanisms that control the uxes through each of the branches [75, 76]. It is very obvious that detailed knowledge about ux control might immediately identify possible targets of metabolic engineering, for instance, to redirect the metabolic ux away from a pathway that does not lead to the desired product or consumes precursors. In order to understand ux control, it is necessary to study the regulation of the enzymes

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Biotechnology B) Silencing of a gene that codes for an enzyme or regulatory protein which should be switched off or reduced in expression level C) Modifying an existing gene and hence the coded protein that is expressed by the organism of interest, for instance, to alter the substrate specicity of an enzyme or engineer its catalytic performance In all three cases, the most suitable target for genetic manipulations is cloned DNA that can be handled and tailored in the well-dened environment of a test tube without the complexity of an entire organism around, in particular the cellular membranes that may hinder the accessibility of the various reagents. Cloned DNA is usually handled in form of plasmid DNA. A plasmid (cytoplasm + chromatid = plasmid ) is a circular piece of double-stranded DNA that is autonomously replicated in bacteria and may even be exchanged between them. Plasmid DNA occurs also in wild-type strains since they are regular extrachromosomal genes that can be easily shared between certain bacteria. Many molecular tools have been developed that are specifically designed to work with plasmid DNA in vitro. A particular family of plasmids, the socalled Expression plasmids or expression vectors as the more general term, additionally contain the necessary nucleic sequences to initiate transcription (promotor) and translation of a gene that has been correctly inserted into the plasmid instead of multiplication only. Expression plasmids for prokaryotic cells differ from those for eukaryotic cells [78]. The three different strategies to alter gene expression in more detail: A) When a new gene should be introduced into an organism or the expression level of an existing one should be increased, this particular gene has to be cloned in a suitable expression vector controlled by an appropriate promotor that ensures a sufciently high copy number of mRNAs and hence protein. The production of human insulin in E. coli is a famous example for the production of a recombinant eukaryotic protein in a prokaryotic organism. B) When an existing gene should be silenced or reduced in activity, there are several experimental strategies. Today the most promising technique is RNA interference (RNAi), a process by which double-stranded RNA si-

that are active around the branching points of the metabolic network [77]. Here the regulatory instances can be found at several hierarchical levels as mentioned earlier in this chapter. The expression level of a given enzyme or any of its regulatory proteins may be controlled on the level of transcription, RNA processing, or translation. DNA and protein chip technology, as described above, provide powerful approaches to unravel whether any control occurs on the level enzyme expression. Finally, allosteric control over enzyme activity introduces another level of metabolic ne-tuning. The kinetic properties of an enzyme like, for instance, its afnity for the substrate molecules, the maximum substrate turnover rate and its sensitivity for product inhibition have to be characterized by biochemical assays. When these information and the current concentrations of the relevant metabolites are available, it should be possible to predict how the uxes through individual branches of the metabolic network will change when the activity of one enzyme is altered by genetic engineering or when the feeding situation is changed. However, it should be emphasized at this point, that due to the enormous complexity of the metabolic network and all its regulatory instances, it is very difcult to predict all metabolic consequences induced by just one genetic modication. Thus, it may be necessary to completely analyze the metabolome of the modied strain and if the results are not satisfactory go through one or several more rounds of metabolic engineering as sketched in Figure 13. This is particularly relevant when the organisms respond to alteration of their genetic make-up by opening up metabolic side pathways that are silent in the wild-type strain.

4.2. Design and Production of Genetically Optimized Strains for Production In Vitro Mutagenesis
In order to generate genetically modied organism that might be superior to wild-type strains for production purposes, there are basically three major options: A) Introduction and expression of a gene that is not originally expressed by the organism or only in small copy numbers

Biotechnology lences gene expression. By transfecting cells with small interfering RNAs (siRNAs) with 100 % homology to a target mRNA sequence, a specic knock-down of the corresponding protein can be achieved. Bound by an RNAinduced silencing complex (RISC), the siRNAs induce the degradation of their homologous mRNA molecules in the cell, thus the protein expression level decreases. RNAi is a reverse-genetics approach to identify gene function by altering the phenotype of a cell [79 82]. C) Until the middle of the last century, traditional biotechnology concentrated on elucidating new metabolic pathways and improving the strains used for the production of antibiotics and other useful natural products, usually by exploiting random mutagenesis and selection. Later in the 1980ies, biologists began to improve strains through metabolic engineering, that is, through the modication of single metabolic pathways by means of targeted or site-directed genetic changes. The two different approaches random or sitedirected mutagenesis to improve the genetic make-up of an organism are still applied today and shall be summarized here. However, throughout the last years progress in molecular biology has provided a pool of experimental approaches for targeted genetic changes that are way too many and multifaceted to be reviewed comprehensively. Thus, only a few principles can be discussed here, and are conned to the best-known models of genetic research, the bacteria. The interested reader is referred to other chapters of this series or the literature on molecular biology for more detailed and organism specic information [83, 84] ( Genetic Engineering).

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to 1011 of the total cell population. The spontaneous mutation rate (probability of a mutation per cell and generation) for a gene is about 105 , and for a pair of nucleotides it is 108 . The number of reversions (or functional back-mutations) is of the same order of magnitude. Induction of Mutations. The mutant frequency can be considerably increased by treatment of the cells with chemical, physical, or biologic mutagenic agents. Three classes of mutants can be distinguished with respect to their genetic structure: 1) replacement of one base pair by another, e.g., AT by GC; 2) a base pair can be inserted or eliminated; 3) several base pairs, DNA fragments, or even genes can be lost (deletion), change their position within the chromosome (transposition), or be interrupted by insertion of foreign DNA (insertion). Class-1 mutants, called point mutants, revert readily to their original structure. Various chemical agents are in use to induce mutations [85, 86]: 1) Incorporation of Base Analogues. Base analogues are antimetabolites. Some are sufciently similar to the natural purine and pyrimidine bases that they are taken up by the cells and incorporated into DNA during replication. They are able to fulll their functions, but tend to bind a wrong counterpart during replication, thus introducing a wrong base pair and causing a point mutation. 2) Chemical Change of Bases. Several mutagenic agents effect a chemical change in a base and thus cause an error in replication. For example, treatment of cells with nitrite results in the de-amination of adenine, guanine, and cytosine, and consequently in mispairing. Alkylating agents, such as ethyl or methyl methanesulfonate, ethyleneimine, nitrogen mustard, and N -methylN -nitro-N -nitrosoguanidine (MNG), belong to the most effective mutagenic agents. Acridine dyes (proavin) function by intercalation and result in insertions or deletions of single base pairs. 3) Irradiation. UV irradiation mainly affects pyrimidine bases and results in replication errors. 4) Transposon Mutagenesis. By conjugation, transposon-containing plasmids (Tn elements) can be transferred from

4.3. Random Mutagenesis, Isolation and Selection of Mutants

Spontaneous Mutants. In bacterial populations, mutational events occur at certain rates without experimental treatment. These spontaneous mutations, which result in mutants, are due to errors that occur during DNA replication. The frequency of spontaneous mutations in a bacterial population varies from gene to gene and species to species and may involve 102

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Biotechnology 4.4.1. Auxotrophic Mutants If the mutation has affected the ability to synthesize, for example, the amino acid leucine, the mutant will require leucine in the nutrient medium. A mutant that is auxotrophic for leucine (leu ) can be recognized by comparison of its growth on two agar plates, one with and the other without leucine. While the phototrophic parent type (leu+ ) will grow on both plates, the auxotrophic leu mutant will grow only on the supplemented agar. The enrichment of auxotrophic mutants can be achieved by application of agents such as penicillin that kill only growing cells and leave non-growing cells unimpaired. If the population of parent cells and rare leu mutants is grown on a minimal medium (lacking leucine) only the parent cells will grow and after the addition of penicillin will be killed within a few hours. The mutants and some parent cells will survive and the mutant fraction is increased by a factor of 104 to 106 . Another method involves the principle of lethal synthesis. Growing cells will, for example incorporate radioactive phosphate or antimetabolites and will be killed while the non-growing mutants will survive. 4.4.2. Regulatory Mutants Some mutants, which in contrast to the parent type form a biosynthetic enzyme constitutively, can be selected directly on the agar plate. For example, if an antimetabolite is added to the agar, the parent cells will incorporate it into their protein and stop growing. Mutants, however, that overproduce the normal metabolite because of overproduction of the biosynthetic enzyme will not incorporate the antimetabolite; they will grow, and sometimes even show their regulatory defect by excreting the metabolite, thus initiating secondary growth of the parent cells in the surroundings of the mutant colonies. Some procedures for the selection and recognition of mutants are listed in Table 2. 4.4.3. Other Selection Methods Various other methods can be used to separate mutants from their parent cells. Because some

an appropriate donor bacterium to the bacterium to be mutagenized. Transposons are DNA fragments that tend to jump to various positions in the DNA sequence of the chromosome or plasmid. Their insertion interrupts genes and results in insertion mutants. The changes in the DNA described above involve an alteration of its base sequence, called a premutation. Subsequently, the mutant character has to be expressed in the phenotype of the cell. About 1020 growth cycles may be required for nuclear segregation, dilution of enzymes, starting or stopping the synthesis of enzymes, and other functions.

4.4. Types of Mutants and Selection Principles

A mutation is a rare event, and the number of mutants in a population is small. Many thousands of Petri dishes would have to be inspected and many millions of colonies (clones of single cells) would have to be examined to recognize and isolate a mutant if there were not a selection (enrichment) procedure for increasing the number of the desired mutants within the population. Enrichment is achieved either by killing part of the parent cells or by growth under conditions that confer growth advantage to the mutant cells. There are many means for enriching mutants. In fact, designing strategies to select mutants is one of the most difcult and demanding arts of the microbiologist, and each type of mutant requires a special trick. The recognition of mutants poses another problem. Mutants that have lost or gained pigment, colony size or characteristics, or that have become resistant to toxic compounds or bacteriophages can be easily detected in a lawn of parent cell colonies. Some mutants show up after addition of indicators. The detection of auxotrophic mutants requires comparison of growth on two different nutrient media. One tries to recognize mutants on an agar plate and to avoid laborious test tube assays of thousands of isolated cell clones. There are, however, many mutant types that require these efforts.

Biotechnology
Table 2. Procedures for selecting and recognizing various mutants Kinds of mutants Mutants resistant to inhibitors, antibiotics, toxic compounds, or bacteriophages Auxotrophic mutants that require accessory nutrients (vitamins, amino acids, or other metabolites) for growth Mutants that lack the ability to utilize a special substrate Selection or enrichment procedure about 10 cells spread on a nutrient agar medium containing the inhibitory or killing agent.
8

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Recognition of mutants only the desired resistant mutants able to grow.

penicillin technique or analogous procedures: cells grown in a medium lacking the accessory nutrients but containing penicillin or another agent that kills only growing cells. Auxotrophic mutants not able to grow in minimal medium survive.

if the cell suspension is spread on a complete medium containing the accessory nutrients and if the colony pattern is then replicated on a minimal medium, colonies that do not grow on the minimal medium are auxotrophs.

penicillin technique and/or direct isolation of pinpoint colonies: the cell suspension is spread on a nutrient agar that contains the substrate utilizable by the wild-type cells at normal concentrations (0.5 vol%), and the substrate accessible by the desired mutants at a very low concentration (0.005 vol%). Pinpoint colonies are transferred and screened. penicillin technique to kill the wild-type cells growing at their upper temperature limit.

comparison of colony patterns on agar plates containing different substrates; mutants unable to grow on a substrate will need a different nutrient for growth and are therefore recognized. Excretory mutants can be recognized by staining reactions (pH indicators) or by dyes added to the agar or after colony growth. comparison of colony pattern on plates that have been incubated at different growth temperatures. Only those colonies that grow, e.g., at 25 C but not at 37 C are isolated. If the cell suspension is spread on a nutrient agar containing a noninducing substrate and after incubation the colonies are sprayed with a solution containing the constitutively utilizable substrate + indicator + inhibitor of enzyme protein synthesis, only constitutive mutants immediately degrade the substrate and change the indicator. only the resistant mutants grow, colonies of a clone forming the enzymes of a biosynthetic pathway constitutively will be surrounded by a halo of satellite colonies due to the excretion of a metabolite.

Temperaturesensitive (conditional lethal) mutants

Mutants derepressed or constitutive for catabolic enzymes

1) continuous culture with the substrate as growth-limiting factor; 2) alternating growth on two different substrates; 3) growth in the presence of an agent suppressing induction (antiinducer).

Mutants derepressed or constitutive for anabolic enzymes

growth in the presence of an antimetabolite that inhibits the growth of the wild-type cells. Among the resistant cells are some mutants that are not subject to end product repression.

cell constituents have lower or higher densities than the average cell mass, the cells can be incubated under conditions that promote synthesis only in the parent or the mutant cells, followed by centrifugation in a sucrose gradient. Cells rich in lipids (poly( -hydroxybutyric acid), triglycerides), glycogen, calcium dipicolinate, magnetosomes, etc. can thus be separated. In other cases tactic responses, such as migration in lightdark elds (phototaxis), in gradients of nutrient (chemotaxis), or oxygen concentrations (aerotaxis), can be used to select mutants that are defective in transport systems or in perception mechanisms. Adsorption on particle surfaces (glycogen or starch granules, lipid droplets, lignocellulose particles) followed by fractional centrifugation can be used to select for mutants that have altered surface structures. The separation efcacy can be increased by the use of column chromatography or by suicide techniques. Suicide techniques employ

compounds that are converted by the wild types to toxic compounds; this lethal synthesis will nally kill the parent cells. Examples are monouoroacetate, bromopyruvate, or chlorate, which are converted into uorocitrate, bromolactate, or chlorine, respectively. The mutants are unable to catalyze such conversions and will therefore survive. With this methods it is possible to select for cells that are decient in nitrate reductase (chlorate, perchlorate), or for fermentative mutants that have lost the ability to form acids (bromide, bromate). Successful biotechnology in many cases depends on the background information and the ingenuity of the researcher in selecting, recognizing, and manipulating the desired organism. In most cases, a single random mutagenesis decreases the characteristic of an organism that is to be changed, such as, for instance, the catalytic performance of an enzyme. By applying high-throughput screening technologies, it

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Biotechnology strains in which the mutant plasmid has prevailed. Many more experimental options exist to introduce site-specic mutations. Many of these are based on polymerase chain reaction (PCR) and the application of primers that already hold the mutation, or insertion or deletion. The interested reader is referred to Bowen [88]. The aim of modern metabolic engineering is to study the cell as an integrated system of genetic, protein, metabolite, and pathway events that are continually changing. This approach should help to optimize the production of substances or to produce substances with improved properties. Historically, metabolic engineering is the targeted recombination of the DNA for proteins involved in the metabolism or in regulation. Today, the objective of metabolic engineering is to quantify the pathway alterations in response to environmental mediators taking into account the entire metabolism. Knowledge of in vivo ux distributions in cells at different physiological states is of increasing importance for the evaluation of biotechnological processes. Gene expression data provide information on pathways relevant to the metabolic models. Furthermore, the so-called combinatorial biosynthesis uses techniques from molecular biology to alter or combine genes from biosynthesis pathways of the secondary metabolism to generate new products with improved pharmacological prol. The importance of studying the entire metabolism rather than one gene or protein at a time has become increasingly relevant with the advent of high-throughput genomic and proteomic technologies, especially microarrays. The DNA microarray technology will gain great importance in the eld of future biological research developing into a key technology of the 21st century hence revolutionizing modern biotechnology. DNA chips are used as biosensors in industrial analysis, biomedical diagnosis, and forensic science. Although this technology provides a powerful tool that is widely utilized for gene expression, it is just beginning to nd applications outside of genomics. Further development of protein, cell, and tissue chips will make it possible in the future to carry out miniaturized, highly parallel analysis of metabolic pathways. The challenge is to transfer these principles into technically applicable and precise ana-

is, however, possible to screen large numbers of strains that are produced by random mutagenesis and nd the valuable ones with benecial mutations. Thus, by going through several rounds of random mutagenesis on the one hand and screening for the desired phenotype on the other, this process will identify those strains with considerably improved properties for production [87]. 4.4.4. Targeted or Site-Directed Mutagenesis Site-directed mutagenesis allows modifying a DNA sequence and thus the protein that is coded by this sequence in a specic and well-controlled way. Thus, it is possible to exchange one or more amino acid within the primary structure of the protein and thereby alter its functionality, for instance the catalytic performance of an enzyme. Compared to random mutagenesis, this is the more straightforward and direct approach not relying on chances that a genetic change may occur at a site that is useful for strain improvement. However, it requires detailed information on the three-dimensional shape of the protein and its primary structure. Many different approaches for targeted genetic alterations have been described that cannot be fully reviewed here. We will summarize the most widely applied and exible concept of oligonucleotidebased mutagenesis. In oligonucleotide-based mutagenesis, the gene of interest is cloned in a plasmid. After denaturation of the double-stranded circular DNA, oligonulceotides are added to the system that share sufcient sequence homology with the coding strand of the gene to allow for hybridization. However, at the site where the mutations should be introduced the sequence of the olignucleotide is altered. Since this is only a minor portion of the entire olignucleotide, hybridization still occurs but the base exchange has been introduced. Figure 16 sketches the underlying principle. Chain extension by DNA polymerase and ligation results in a double-stranded DNA with one mutant strand and one wild-type strand. When these plasmids are then introduced in bacteria by transformation, semiconservative replication produces homoduplexes with both strands being either of the wild-type or the mutant. When colonies are grown, one can now screen for those

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35

Figure 16. Site-directed mutagenesis of cloned DNA by oligonucleotide mutagenesis

lytical systems that can be used for many applications repeatedly.

5. Cultivation and Bioprocesses

In this chapter, only the basic requirements for bioprocesses are described very shortly. For any further details concerning different types of bioprocesses, bioreactors, kinetics, sterility, and cleaning, the reader is referred to ( Biochemical Engineering). Very detailed information about the monitoring and control of bioprocesses can be found in this contribution in Chapter 8. Bioprocesses [89, 90] are used for the transformation of organic matter with the help of biocatalysts, such as living cells, dead cells, or their components, e.g., enzymes [91, 92]. Bioprocesses are utilized for chemical syntheses and for the conversion of waste material to either useful products (recycling) or efuents harmless to the environment (waste treatment). The outstanding feature of bioprocesses is their high synthetic potential to carry out a series of very complicated chemical reactions in a one-step process.

Classic biocatalysts are yeasts, fungi, and bacteria; animal and plant cell cultures have become important only in more recent times (see Chapter 9). Process development [93] [94] [95] involves the areas of biology (strain development and bioregulation), reactor design, process control [96], and medium design [97, 98] as well as downstream processing (product recovery, see Chapter 7).

5.1. Isolation of Microorganisms

Microorganisms can be purchased from culture collections. But microbes are everywhere, the environment selects, and a desired type of microorganism can often be isolated from its most probable natural habitat. For example, methanogenic bacteria are present in the anoxic mud sediments of ponds and lakes, hemoglobindegrading bacteria in abattoirs, and hydrocarbon-oxidizing bacteria around oil elds or leaky engines of motor cars. From samples of these materials, bacteria can be isolated by enrichment culture. The technique of enrichment culture is simple and straightforward [99, 100].

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Biotechnology tion strains developed by repeated random mutagenesis and screening. A comparison with precursor strains could identify the mutations responsible for increased production. That way, production strains could be designed with just a minimal number of dened mutations [101]. Designer Bugs. The term used for microorganisms tailored by genetic engineering methods so that they can carry out desired biochemical conversions efciently is designer bugs. Contrary to the strategy used in the past to obtain microorganisms for the production of a desired product by coincidental mutagenesis and selection, genetic engineering has opened up the possibility of customized construction of production strains by metabolic engineering. The genome permits insight into the entire metabolic potential of the organism. This insight is currently limited because the function of many gene products is not yet known. Organisms, such as E. coli, Bacillus subtilis, Corynebacterium glutamicum, Pichia pastoris, or S. cerevisiae, are preferentially applied in the development of designer bugs. Further important aspects governing the choice of host organism for a designer bug are knowledge of metabolism, regulation mechanisms, growth properties, and hazard potential. Based on methods developed in the last few years for the analysis of the genome, transcriptome, proteome and metabolome, which thus permitted a holistic consideration of the organism (systems biology), it will be possible in future to develop designer bugs for biotechnological production of a desired product considerably more quickly and specically than in the past. A list with mostly all culture collection worldwide can be found in the internet [102] under the URL http://www.bacterio. cict.fr/collections.html and in ref. [103 105].

By establishing dened environmental conditions with respect to the energy, carbon, and nitrogen sources; hydrogen acceptor; gas atmosphere; temperature; pH value; light; etc., and by inoculating with a mixed population that contains the desired metabolic type, the best adapted organism will dominate and overgrow all accompanying organisms. Liquid enrichment culture is recommended if the fastest growing organism is desired. In this case, the organism is repeatedly transferred from the original liquid culture through several subcultures. If, however, a great number of strains with only slightly differing traits are to be separated, the direct plating method is the preferable technique: the inoculum is diluted and spread on a solidied enrichment medium (agar dish). During incubation the cells form colonies and can be isolated separately. Whereas formerly these kinds of naturally pure or well-balanced mixed cultures were used in industrial practice (e.g., yeast fermentations, vinegar and cheese production) now only pure cultures are used. Pure cultures are obtained by diluting the enrichment culture and spreading the suspended cells on an agar surface or distributing the cells in an agar medium. After incubation, single colonies are isolated and streaked on various media to test for commensalic bacteria. Finally, a clone of the desired organism is obtained as a pure culture. Preliminary results indicate that previously noncultivable microorganisms could be the key to the biosynthesis of active substances. The genes responsible for the expression of the natural substance biosynthesis could be inserted into microorganisms which can be grown easily, a procedure known as the metagenome approach. The metagenome is a set of all genetic material from organisms which cannot be cultured, e.g., from the soil or from communities of organisms. The development of the metagenome techniques becomes possible with the development of high-throughput techniques. The development of high-throughput DNA sequencing began in the early 1990s as simplications and automation made it possible to decode the sequence of entire genomes. In 1995 the rst full genome sequence was published, that of the Gram-negative bacterium Haemophilus inuenzae. Of particular interest for biotechnology is the possibility of sequencing microbial produc-

5.2. Requirements for Growth

The process solution must primarily supply carbon and energy. In order to maintain growth, nutrients, trace elements, and such growth factors as vitamins must be added to the process solution. Appropriate selection of the medium components is essential for proper functioning and activity of the biocatalysts involved in the reaction.

Biotechnology
Table 3. Standard nutrient media a) Minimal medium for bacteria K2 HPO4 NH4 Cl MgSO4 7 H2 O CaCl2 2 H2 O FeSO4 7 H2 O Glucose Trace element solution b) Complete medium for bacteria Peptone Yeast extract NaCl MgSO4 7 H2 O c) Complete medium for fungi (pH 6.0) Malt extract Yeast extract Glucose KH2 PO4 NH4 Cl 0.5 g/L 1.0 g/L 0.2 g/L 0.1 g/L 0.01 g/L 5.0 g/L 1.0 g/L

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sodium, selenium, silicon, tungsten, and a few others, that are not required by all organisms. The compositions of a few simple synthetic nutrient media are presented in Table 3. 5.2.1. Chemical Composition of Bacterial Cells Bacterial cells harvested by centrifugation from a culture growing in liquid medium contain about 70 wt% water. The elemental assay of the dry mass of E. coli is: approximately 50 % carbon, 20 % oxygen, 14 % nitrogen, 8 % hydrogen, 3 % phosphorus, 1 % sulfur, 2 % potassium, 0.05 % each of calcium, magnesium, and chlorine, 0.2 % iron, and a total of 0.3 % trace elements. The organic analysis of the dry mass of cells is presented in Table 4. It should be noted that the values refer to a specic bacterium grown in a specic environment and harvested in a specic phase of growth. The results may differ if, e.g., the cells encountered limitation of the nitrogen source and accumulated poly( -hydroxybutyric acid) (up to 90 wt%) or glycogen (about 50 %) within their cells.
Table 4. Overall macromolecular composition of Escherichia coli (mass percent, dry matter basis) [106]

10 g/L 1.0 g/L 2.0 g/L 0.2 g/L

10 g/L 4 g/L 2 g/L 0.5 g/L 1.0 g/L

d) Vitamin solution for soil and water bacteria Biotin 0.2 mg Nicotinic acid 2.0 mg Thiamin 1.0 mg Sodium 4-aminobenzoate 1.0 mg Sodium pantothenate 0.5 mg Pyridoxamine 5.0 mg Cyanocobalamine 2.0 mg Distilled water 100.0 mL Two to three mL of this solution is added to 1 L of nutrient solution.

e) Trace element solution MnCl2 4 H2 O CoCl2 6 H2 O CuCl2 2 H2 O NiCl2 6 H2 O Na2 MoO4 2 H2 O ZnSO4 7 H2 O H3 BO3

3 mg/L 5 mg/L 1 mg/L 2 mg/L 3 mg/L 5 mg/L 2 mg/L

Protein RNA DNA Lipid Lipopolysaccharide Peptidoglycan Glycogen Total macromolecules Soluble pool of building blocks, vitamins Inorganic ions

55.0 20.5 3.1 9.1 3.4 2.5 2.5 96.1 2.9 1.0

The growth of microorganisms is dependent on the presence of water or moisture. Nutrients to be used as sources of energy and for synthesis of cell constituents are dissolved in water. The growth requirements for various microorganisms are different, and many recipes for the composition of nutrient media are known. Basically, all chemical elements that constitute the cell substance have to be present in utilizable forms. There are ten macroelements that are constituents of all organisms: carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus, potassium, calcium, magnesium, and iron. In addition, there are microelements (trace elements), such as manganese, nickel, cobalt, molybdenum, zinc, copper, vanadium, boron, chlorine,

5.2.2. Carbon and Energy Sources Only autotrophic organisms are able to synthesize all organic cell constituents from carbon dioxide as the main carbon source. All others derive the cell carbon from organic compounds, which usually serve as both carbon and energy sources; they are partially assimilated and partially oxidized (dissimilated). Glucose, the monomeric constituent of polysaccharides such as cellulose or starch, can be used by the majority of microorganisms. Many other natural

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Biotechnology 5.2.8. Hydrogen Ion Concentration The majority of microorganisms prefer a pH of about 7.0. However, there are acidophilic and alkaliphilic microorganisms. Yeasts and other fungi prefer pH 5.0. Buffers, in most cases phosphates, are used to maintain the desired pH, although the CO2 /HCO 3 system or organic substances may do as well. The pH value is very important: the drop from pH 7.0 to pH 6.0 means a tenfold increase in the concentration of H+ ions, which is a signicant change. 5.2.9. Carbon Dioxide Many microorganisms require a higher concentration of CO2 than exists in the atmosphere, such as 10 vol%. This requirement applies to those bacteria that in their natural habitats (intestinal tract, tissue, milk, blood, fermenting juices) are exposed to high CO2 partial pressures. 5.2.10. Aeration All obligately aerobic microorganisms require oxygen as an electron acceptor for energy generation. For growth in thin layers on liquid or solid media atmospheric oxygen may sufce. In liquid media aerobic bacteria grow only on the surface if the medium is not agitated and aerated. Only dissolved oxygen is utilized; its solubility in water is very low (6.2 mL/L at atmospheric pressure and 20 C). Therefore oxygen has to be supplied continuously to the cell suspension growing in submerged culture in asks or fermenters. To meet the increasing oxygen demand in a growing culture, the speed of agitation, the oxygen concentration in the gas phase, and the total gas pressure can be increased. Various kinds of fermenters have been designed to increase the gas transfer from the gaseous to the liquid phase. The nal cell density and yield of microorganisms depends on the rate of oxygen transfer. For fermenter design and scale-up, both the respiration rate (oxygen uptake) of the microorganisms and the oxygen transfer rate from gaseous to liquid phase have to be known. Because the formation of desired products is highly dependent on oxygen supply whether the cells

compounds can be utilized and degraded by one or another microorganism. 5.2.3. Accessory Nutrients In addition to carbon and energy sources, many organisms require accessory nutrients (growth factors), such as vitamins, amino acids, purines, or pyrimidines (see Table 3). 5.2.4. Sulfur and Nitrogen These elements can be used in their oxidized forms as sulfate and nitrate. Ammonium ion is the most common nitrogen source for microorganisms. 5.2.5. Oxygen The oxygen atoms of the cellular constituents are mainly derived from water, substrates, or carbon dioxide. Atmospheric oxygen serves mainly as a terminal hydrogen acceptor of aerobic respiration, being reduced to water. 5.2.6. Complex Media Many microorganisms can grow in a simple nutrient medium such as that listed in Table 3 a. For Leuconostoc mesenteroides such a synthetic medium contains 40 components. If the nutrient requirements of an organism are not exactly known, complex nutrient media can be used, such as yeast extract, yeast autolysate, peptone (Table 3), meat extract, wort, carrot juice, coconut milk, or horse manure extract. In other cases complex media are used for economical reasons, for example, whey permeate, corn steep liquor, soybean extract, or molasses. 5.2.7. Solid Media For solidication of media, agar is added at a concentration of 1.5 to 2.0 wt%. Agar melts at 100 C and solidies on cooling to below 45 C.

Biotechnology are able to take up oxygen at their maximum respiration rate or only much less aeration poses one of the most important problems in fermenter technology. 5.2.11. Anaerobic Techniques For the growth of strictly anaerobic bacteria the complete exclusion of oxygen is required. Anaerobic techniques involve deaerated nutrient solutions, tightly sealed asks, incubation in anaerobic jars, the use of chemical oxygen absorbers (pyrogallol, dithionite or the gas pak, which contains hydrogen- and carbon dioxideevolving agents and a catalyst to promote the oxyhydrogen reaction), resazurin as a redox indicator, anaerobic hoods, and skilled hands to practice these anaerobic techniques. 5.2.12. Media Preparation Substrates for industrial applications are usually included in complex media, which must be supplemented with special compounds, such as a nitrogen source, various nutrient salts, or certain trace elements. Organic precursors are also added in some cases for efcient product formation. Many of the feedstocks need appropriate pretreatment in order to provide for good assimilation by the microorganisms. Starch-containing materials are prepared by milling or steam treatment for softening and swelling. For many processes, especially when yeasts are used, starch must rst be broken down to sugar by treatment with amylase derived from barley malt or with microbial amylases. Other feedstocks, such as wood, must be pretreated with acid or alkali. Sulte liquor is stripped from sulfur dioxide by aeration or neutralization. Molasses is puried by acidication and centrifugation or ltration. In some cases heavy metals have to be removed from feedstocks prior to their use. For processes using solid substrates, see [107]. In general, the raw materials are dissolved or suspended in water and the resulting medium is heated, ltered, and sterilized. The complex composition of the media used in industry causes considerable problems. For downstream processing (harvest, concentration, and purication of product) or for analytical assays during the process, additional pretreatment of the raw material is needed to avoid unfavorable side effects.

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5.3. Sterilization
The basis of microbiological laboratory methods and of the conservation of food and feed products is the killing of microorganisms. Sterilization means the removal of living microorganisms, and can be achieved by moist heat, dry heat, ltration, irradiation, or chemical means [108]. 5.3.1. Moist Heat Vegetative cells of bacteria and fungi suspended in water are killed at temperatures around 60 to 80 C within 5 to 10 min; yeast and fungal spores are killed above 80 C, and endospores of bacteria at 120 C within 15 min. Because endospores of bacteria are highly tolerant to various environmental conditions, they are ubiquitous and are distributed through the air. Their presence requires that all nutrient media and canned foods be sterilized in an autoclave at about 121 C for 20 min. If conditions do not allow the germination of spores and the growth of spore-forming bacteria, e.g., in acid fruit juices, jam, or desserts, heating to 80100 C for 10 min will sufce. This is called partial sterilization or pasteurization. 5.3.2. Dry Heat For killing bacterial endospores by dry heat, longer exposure times and higher temperatures are required than with moist heat. Glass and other heat-resistant equipment can be sterilized for 2 h at 160180 C in a dry oven. In any case appropriate indicators or soil samples that contain bacterial spores are included in the oven to test whether adequate temperatures have been reached and even the extremely heat-resistant spores have been inactivated. 5.3.3. Filtration Solutions containing thermolabile compounds can be sterilized by ltration through nitrocellulose membranes, kieselguhr, porcelain, asbestos, and others. Usually lters with a pore size <0,2 m are used.

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Biotechnology Surface cultivation is usually applied in the lab scale in fungal research. Large Erlenmeyer asks or specially designed vessels, such as the Fernbach, Roux, or Sakaguchi asks, containing small volumes of liquid are used. The asks and liquid are sterilized and inoculated with spore suspensions or small pellets of mycelium. Growth and product formation are dependent upon the depth of the medium layer and the amount of surface provided by the asks, which in turn affects the necessary oxygen supply. In large-scale production large pans are used. They are lled with substrate, inoculated, and then kept at controlled temperature and air humidity. Pans are stacked on suitable racks, and connected with a system of overowing tubes. The liquid medium is poured in at the top. In this way, an entire stack can be loaded at once. If the stack is designed appropriately, it can be sterilized, inoculated, aerated, and maintained under controlled conditions. The same principle can be applied for solid substrates, which are rst inoculated and then loaded into the pans. The material is spread at a thickness of several centimeters onto screens or perforated metal sheets. Columns lled with carrier material onto which the microbes are immobilized are also considered a surface culture as long as the column is not completely ooded. The medium is trickled through the column and air is injected at the bottom. The product is then collected from the carrier, which is made of wood shavings, plastics, or ceramics. This type of surface culture has been used for vinegar production and sewage treatment. However, it was abandoned and replaced with submerged culture. Remarkably, the immobilization methods that were developed for such biocatalysts as living cells, resting cells, enzymes, or organelles are formally similar to these conventional processes. 5.4.2. Submerged Culture

5.3.4. Irradiation UV irradiation is used to keep rooms partially sterile. Bacteria and their spores are killed rather quickly, but fungal spores are only moderately sensitive to radiation. Ionizing radiation (X-ray, gamma radiation) is used to sterilize food and other compact materials. 5.3.5. Chemical Means Ethylene oxide is used to sterilize food, plastics, glassware, and other equipment. Propionolactone and diethyl carbonate are added to nutrient solutions. The presently used soaps and detergents applied at moderate temperature kill all vegetative cells and even many spores. Cleaning by the methods practiced in the kitchen provides almost sterile glassware. Remarkably, the killing of microbes is a process that follows rst-order reaction kinetics. In a population there are allways some cells or spores that are more resistant than the majority. Accordingly, sterilization procedures are most likely to be effective on relatively clean substrates.

5.4. Types of Bioprocesses

In chemical technology, a choice is to be made between batch processes or continuously operated processes. Many parameters usually favor the second over the rst choice. However, in biotechnology, the batch process still predominates. This specic problem is treated in more detail (also in regard to engineering aspects) in Biochemical Engineering. 5.4.1. Surface Culture Surface cultures are mostly used when fungal mycelia act as catalysts. Mycelium is grown in shallow pans containing a small volume of the medium. As the mycelium grows, it eventually forms a compact mat. This method was typically used in the production of acids, such as citric acid. In large-scale production, surface culture is being abandoned more and more and replaced by the submerged culture method [109 111].

In this process, the microbes are suspended in the liquid medium. The simplest example of a submerged culture consists of a vessel without any agitation device, in which microorganisms settle at the bottom and form a compact layer. This is the classic arrangement for all types of ethanolproducing processes. Some agitation will occur,

Biotechnology however, from the movement of rising carbon dioxide bubbles. For submerged, anaerobic processes, simple agitation devices for gentle mixing will sufce. This conguration allows for the mixing of liquid and gas. Carbon dioxide or other gases, such as nitrogen, can be added during the process.

41

5.5. Process Layout

5.5.1. Reactors Appropriate reactors in biotechnology have to meet the requirements for agitation, aeration, corrosion resistance, and aseptic operation. Various designs of stirred vessel are therefore used in the laboratory and in production plants. Container construction is based on principles similar to reactors used in heterogeneous catalysis. In former times, bioprocesses were carried out under nonsterile conditions and took place in vats of wood. Beer today is brewed in large cubical containers coated with ceramic tiles. For the more conventional surface processes (citric acid, acetic acid), small pans were used. A basic problem in aerobic processes is the efcient transport of oxygen into the liquid and the microbial cells. Very efcient devices have been developed to provide air for aerobic reactions. Some examples of more modern reactor congurations for agitation and aeration are shown in Figures 20 and 21. 5.5.2. Containments for Anaerobic Processes Large units for anaerobic processes are made of wood, metal, or coated concrete. They have no covers and consequently cannot be operated aseptically. However, submerged anaerobic processes can be performed under conditions that favor the formation of product and inhibit the accumulation of unwanted organisms by proper selection of reaction parameters, such as pH and temperature. Lactic acid, for example, is produced at 50 C and low pH, and the development of competing microbes is negligible. In other cases, closed vessels are used. They are usually made of stainless steel, have large capacities of up to 1000 m3 or more, and are equipped with agitators and, if necessary, with

cooling and heating coils. Gases that form during the process (CO2 , H2 , or evaporated solvents) are collected at the top. These large tanks have openings for loading, inoculation, harvesting, and cleaning. Large units are mostly sterilized with steam. Extremely large containments with capacities of thousands of cubic meters are used in sewage treatment. They are usually constructed of concrete and allow automatic feed and removal of material. Sludge treatment plants are often equipped with heating elements to allow thermophilic processes for methane production. The gas is collected and stripped of hydrogen sulde if necessary. 5.5.3. Reactors for Aerobic Processes Bioreactors used for aerobic processes are basically similar to those used for anaerobic processes. In addition, they contain devices for aeration and agitation. The capacities used in industry may vary considerably, from 10 to 200 m3 . Large tanks of 1000 m3 and more are also used for aerated, sterile processes (e. g., glutamic acid). For aseptic processes the tank is sterilized with steam before loading; a slight overpressure is often maintained during cultivation. Agitation principles vary considerably and include mechanical as well as pneumatic and hydrodynamic systems. The most commonly used type of bioreactor is the stirred tank reactor (STR) using a at blade turbine (FBT) for agitation (Fig. 17). This conguration provides excellent mixing and mass transfer in media of low viscosity. Drawbacks are high power demand, poor performance with highly viscous liquids, and rather poor interchange of material between mixing regions around the blades. Air is normally injected by a sparger. Larger units are equipped for pneumatic agitation, in which movement is caused by injected air. For proper hydrodynamic control, various kinds of bafes and draft tubes can be inserted. 5.5.4. Inoculation After inoculation with the microorganisms, the process should start immediately and the reaction should proceed fast. The amount of active

42

Biotechnology For scaling-up, the liquid cell culture is placed in liquid broth, rst in test tubes (b), then in small (c), and subsequently in large Erlenmeyer asks (d) containing approximately 200 mL of medium. Proliferation of the culture takes place by shaking the asks overnight at constant temperature. Larger amounts of inocula are then prepared in agitated and aerated fermenters of various sizes depending on the volume of the production plant (e, f). In the early stages of this procedure (steps a through d) the inoculum is transferred under sterile conditions in glove boxes. In fungal inocula proper wetting of the spores is achieved by adding small amounts of surfactants to the broth. If inoculation by spare suspensions is not optimal, mycelial pellets can be used for start-up. Bacterial spores must be activated by thermal treatment before they can be used for inoculation. During the exponential growth phase of the bioprocess cells can be harvested for following inoculations. 5.5.5. Operation Modes Optimum production depends strongly on process layout. The standard process is still the batch operation, which consists of loading, inoculation, processing, harvest, and cleanup of the vessel. The advantages of this procedure are its adaptability to weekly work hours and small losses in case of contamination or decreases in productivity. Greater specic production can be attained by using semicontinuous or sequential-batch operations. In this method only a partial stream of the broth is withdrawn after completion of the reaction (e.g., 90 %) and new medium is added directly into the remaining volume. This type of operation is also called cyclic continuous production. Modern vinegar production is operated in the cyclic manner and maintained by fully automated process control. Fully continuous operation, that is, the constant ow of medium into a reaction vessel and the simultaneous removal of an identical amount of the process solution, has shown the highest production rate. However, practical and biological constraints, especially the high contamination risk, have so far prevented the general introduction of continuous processing into biotech-

cell culture added is therefore critical and depends on the size of the batch. Scaling-up from the original starter culture to the inoculation broth is done in several steps in large-scale industrial processes (Fig. 19). The starter culture is kept deep frozen (80 to 90 C) in sealed vials or in vials that were previously subjected to lyophilization (a). They can be stored in this way for several months or even years without any damage. For short-term storage at 4 C, agar slants are prepared (g) and purity is tested on Petri dishes (h) before inocula are scaled up for production.

Figure 17. Stirred tank reactor (STR) equipped with at blade turbine (FBT) and bafes for agitation and a sparger for gas distribution The ratio of H: D is usually 3 (max. 5).

Biotechnology

43

44

Biotechnology

Figure 18. Various bioreactor congurations [112] a) Gas; b) Motor; c) Draft tube; d) Bafes; e) Mechanical foam breaker; f) Cylinder A) Multistage at blade turbine reactor (FBT) and bafes (d) making up the classical stirred tank reactor (STR) B) Forced agitation with draft tube (c) and ship propeller C) Combination of at blade turbine and draft tube, operated with an overow pattern D) Combination of at blade turbine and draft tube, odded E) Simple vessel with hollow stirrer element for self-aeration and distribution F) Same as E, but with draft tube resulting in vortex ow patterns G) Torus reactor; annular form with ship propeller H) Tower with multistage at blade turbines I) Vibrating elements in tower J) Pulsated tower K) Horizontal rotating cylinder L) Rotating disks on horizontal axis M) Rotating elements in a bath N) Compact loop reactor with gas liquid separating element. Operated as completely lled vessel. Forced agitation with ship propeller.

nology. Therefore, only processes that need not be carried out under aseptic conditions are operated continuously such as sewage treatment, methane formation, and feed yeast or ethanol production from sulte liquor. The potential advantages of continuous cultivation are substantial because of the efciency of the process and the uniformity of the nal product. As a result, much effort is being made to develop a sound basis of knowledge necessary for using this process strategy.

In contrast to large-scale industrial processes continuous cultivation methods are widely used in the laboratory [113 115]. The growthcontrolling factor is either the concentration of nutrients in the medium (chemostat system) or the concentration of insoluble biomass sensed by optical measurement (turbidostat methods). The purpose of these control systems is to maintain a constant concentration of nutrients, cells, and metabolic products in the vessel (steady state). The kinetics of growth or product formation can

Figure 19. Preparation of inocula. The individual steps a through h are explained in the text

Biotechnology be kept under precise control by selecting the appropriate dilution rate. All types of variations of the single-stage continuous cultivation are possible, such as multistage arrangements, with or without recycling.

45

5.6. Process and Product Overview

The potential of biotechnology consists in its ability to replace classical chemical production processes and to facilitate the production of new products. It is undisputed that, particularly in the area of basic and ne chemicals production, the use of biotechnological processes is meaningful since biotechnological processes are usually distinguished by their high specicity (relating to the conversion of substrates) and selectivity (relating to the product spectrum) biotechnological processes often use renewable resources as raw materials, thus contributing to the much discussed sustainability of products and processes biotechnological processes can be carried out under mild reaction conditions in terms of pressure, temperature, and pH. In view of these facts, in the past hundred years a multitude of industrial biotechnological processes have been developed [116 120] whose efciency exceeds that of chemical processes, and this has helped to establish them in the long run (Table 5 and 6). One interesting area is the production of ne chemicals. The term ne chemicals products refers to substances that are highly functional and for which world demand is typically quantities of less than 10 000 t/a. From a chemical point of view these products are usually distinguished by having several reaction centers and frequently by chirality. Classical syntheses of these substances include several reaction steps using stoichiometric quantities of reagents and often deploy extravagant protective group chemistry, expensive noble metal/heavy metal catalysts, and drastic reaction conditions, e.g., aggressive solvents. Here biocatalysis allows synthesis under considerably milder reaction conditions in terms of pressure, temperature, and acidity. At present, due to the excellent enantioselectivity of enzymes, white biotechnology methods are primarily used for

the production of chiral compounds. Straathof et al. [121] list 134 industrial biotransformations. Just short of 90 % of the products described are chiral ne chemicals. In future, this eld will undoubtedly acquire greater importance and new reaction sequences for the production of compounds with several stereo centres will be established. Also bulk chemicals are produced by fermentation or biotransformation processes. Bulk products mean products exceeding 10 000 t annually. It is expected that, by the year 2010, 6 12 % of bulk products and polymers produced by chemical means will already be produced by biotechnological processes. High-volume, biotechnologically produced goods are to be found in the food, livestock feed, and the drinks and tobacco industries. A prime example of a bulk product derived by an enzymatic process is acrylamide. Global biotechnological production capacities have continually expanded in the last few years and are today probably in the region of 100 000 t/a. Monomers and polymers produced by biotechnological processes for the plastics and polymer industry are becoming increasingly interesting. Biotechnologically produced polymers, such as polylactide (PLA), 1,3propandiol (PDO) and poly-3-hydroxybutyrateco-3-hydroxyhexanoate (PHBH) could provide the basis for innovations. As for the energy industry, besides bioethanol two further products should be mentioned: biogas and hydrogen. Whereas the technology for biogas recovery is state-of-the-art, hydrogen production is far more complex and requires long-term research efforts. Biogas recovery is a recycling method for residues. The product can be separated comparatively easily, which is crucial to the cost-effectiveness of the process.

6. Biocatalysis and Biotransformation

6.1. Introduction
Biocatalysis (also called biotransformation) is the conversion of one substance (substrate) to another (product) by a microorganism or an isolated enzyme. It is a chemical reaction catalyzed by a particular cellular enzyme or by an enzyme originally produced within the cell. Most

46

Biotechnology
Market value 106 EUR 1800 1400 180 100 24 20 30 (incl. extraction)

Table 5. Products obtained by fermentative processes of biotechnology [122 124] Product/process Amino acids l-Glutamate l-Lysine l-Threonine l-Phenylalanine l-Tryptophan l-Arginine l-Cysteine Other amino acids and derivates Acids Lactic acid Gluconic acid Citric acid Acetic acid Itaconic acid Solvents Acetone* 1-Butanol* Bioethanol Biomass Starter cultures Bakers yeast Mineral yeast Enzymes Enzymes (total) Detergents Food industry Textile, leather industry Animal feed Food Cheese Erythritol Drinks and tobacco Beer Wine Antibiotics Bacitracin A Bacitracin A Virginiamycin Cyclosporin Monensin Penicillins Cephalosporins Tetracyclins Antibiotics (ca. 160 on market) Other active substances Acarbose Biopolymers Polyhydroxyalkanoate Polylactide Xanthan Scleroglucan Pullulan Dextran (derivatives) Hyaluronic acid Polyglutamic acid Annual production t/a 1500 000 700 000 30 000 10 000 1200 1000 500 3000 Price EUR/kg 1.20 2 6 10 20 20 20 Main application avour enhancer animal feed additive animal feed additive aspartame, medicine animal feed, nutrition medicine, cosmetics food, pharma pharmaceuticals, cosmetics, nutrition food, leather, textiles food, textiles, metal, construction medicine, food, metal, detergents food co-monomer solvent solvent solvent, basic chemical, energy source 100 2300 food

150 000 100 000 1000 000 190 000 4000 3000 000 1200 000 >18 500 000

1.80 1.50 0.80 0.50

270 150 800 95

0.4

1800 000

1830 580 500 250 170 15 000 000 30 000 138 000 000 27 766 000 4 >200 70 3 >3000 45 000 30 000 5000 50 250 19 000 150 000 67

detergents starch degradation, proteases tanning phytases, proteases

2.25 2.5

sugar substitute drinks, tobacco drinks, tobacco

3000 <120 250 5200 8 300*

12 24 17.5 15.6 24 13 500

healing of wounds animal feed additive hog feeding organ transplantation animal feed additive medicine, animal feed additive medicine, animal feed additive

300

active substance packaging packaging food, oil production oil production lm former in foodstuff applications blood substitute

140 000 40 000

2.25 8.4

315 336

2600 500

200**

520

Biotechnology
Table 5. (continued) Product/process Vitamins Riboavin (b2 ) Cyanocobalamin (b12 ) Vitamin C l-Sorbose (vitamin C precursor) Amino sorbite Lipids Phytosphingosine Animal feed Galactooligosaccharides Cosmetics Dihydroxyacetone Annual production t/a 30 000 20 80 000 50 000 25 000 8 500 640 Price EUR/kg Market value 106 EUR Main application active substance, animal feed additive active substance, animal feed additive food, animal feed

47

cosmetics 2500 3.50 9 prebiotics suntan preparations

of these enzymes are necessary for the normal metabolism and reproduction of the cell. In biocatalysis, however, these enzymes are simply used as catalysts for chemical reactions. In addition to their natural substrates, many enzymes can utilize other structurally related compounds as substrates and therefore may catalyze nonnatural reactions upon addition of foreign substrates to the reaction medium. Thus, biocatalysis or biotransformation is a specic category of chemical synthesis.

In both whole cells and enzymatic biotransformations the catalytically active species is the enzyme. Enzymes can be subdivided into six classes (classied by the Committee on Enzyme Nomenclature of the International Union of Biochemistry) depending on the type of reaction they catalyze (Table 7) and are listed numerically in a catalogue published by the committee [125] ( Enzymes).

6.3. History 6.2. Classication of Biocatalysts

Enzymes and whole cells can be applied in a biotransformation in many different forms. Usually a whole cell biotransformation is a growth decoupled process, so resting cells (harvested and washed after fermentation) are employed. The cells may be intact or permeabilized (e.g., for better substrate uptake). The different types of biocatalysts are: Whole or treated cells (e.g., lyophilized, permeabilized) Organelles Cell-free multienzyme systems Combination of individual cell-free enzymes Single cell-free enzyme Isolated Enzymes may also be applied in different forms. In most cases, it is not necessary to purify an enzyme to homogeneity, a crude cell extract or partially puried solution may also be employed as biocatalyst. In some wellknown technical enzyme preparations, several isoenzymes are present (e.g., pig liver esterase). Biocatalysis includes some of the oldest human known chemical transformations such as the use of yeast for baking or brewing or the use of chymosin from the stomach of young cattle for cheese production. Records proving these ancient technologies date back to about 7000 to 6000 bc. Another old and well-known example is vinegar production, which has been known since the dawn of recorded history. It is the oldest example of microbial oxidation (Fig. 20). The process has evolved along traditional lines of fermentation without knowledge of the underlying biochemistry. In 1862, Pasteur described the biochemical nature of vinegar production [126]. Subsequently Brown conrmed in 1886 that the oxidation of alcohol to acetic acid proceeded through acetaldehyde [127]. Bacterium xylinum, which causes the reaction to proceed, was rst described by Brown and is the rst microbial catalyst. Today, the oxidation of ethanol to acetic acid by acetic acid bacteria is well understood; it proceeds via two successive dehydrogenations,

48

Biotechnology
Market value 106 EUR 28

Table 6. Biotechnology products obtained by biocatalysis and biotransformation [122 124] Product/process Basic chemicals Acrylamide Amino acids l-Aspartic acid l-Methionine l-Dopa l-Alanine d- and l-Valine l-tert -Leucine l-Carnitine -Phenylalanine Food Glucose Annual production t/a 100 000 13 000 400 300 500 50 10 200 >1 20 000 000 Price EUR/kg 1.40 20 500 Main application aspartame production infusion solutions active substance infusion solutions

0.30

6000

Fructose Isoglucose, HFCS 8000 000 l-Hydroxybutanedioic acid 100 Palatinite Isomalt 70 000 Aspartame 10 000 Fructooligosaccharide 10 500 (inulin) Antibiotics (derivatives) 6-APA 10 000 7-ACA 4000 d-4-Hydroxyphenylglycine 7000 Intermediate products (S )-2-Chloropropionic acid 2000 d-Pantolactone 2000 (S )-Methoxyisopropylamineseveral thousands

0.80 20

6400

23

liquid sugar,fermentation medium sugar from HFCS liquid sugar acidifying agent sugar substitute sugar substitute sweetening agent prebiotics

herbicide synthesis herbicide synthesis (Outlook after chiral switch by Frontier)

(R)-2-(4 -Hydroxyphen1000 oxy)propionic acid (R-HPOPS) (S )-Phenethylamine etc., 500 optical active amines d-Mandelic acid >200 Ethyl (S )-4-chloro-3-hy>150 droxybutyrate m-Phenoxybenzaldehyde 100 cyanohydrin (S )-3-Acetyl thioisobutyrate 100 (-)-RAN 50 (R)-Glycidyl butyrate 50 Active substance (precursors) Dilthiazem precursor 50 Nicotinamide 3000 Progesterone 200 Ephedrine 1500 N -Acetylneuraminic acid Special products Cyclodextrins Pantothenic acid

ca. 20

chiral auxiliary cholesterol-reducing drugs, e.g., Atorvastatin (Lipitor)

6090

active substance pseudoephedrine, chiral auxiliary sialidase inhibitor (e.g., Relenza) 50 ca. 70 domestic, food, stabilizers

5000 11 600

10 ca. 6

Biotechnology which depend on the cytochrome system for electron transfer to atmospheric oxygen, the ultimate hydrogen acceptor:
Table 7. Classication of enzymes and their reactions Class Oxidoreductases: Reaction Enzymes catalyzing oxidationreduction reactions involving oxygenation, such as CH COH, or overall removal or addition of hydrogen atom equivalents, for example CH(OH) C=O and CHCHC=C. Dehydrogenases, oxidases, peroxidases, hydroxylases, and oxygenases are involved in this class. Enzymes catalyzing the transfer of various groups (e.g., aldehyde, ketone, acyl, sugar, phosphoryl) from one molecule to another. Enzymes catalyzing hydrolytic cleavage of CO, CN, CC, etc. Esterases, amidases, peptidases, phosphatases, glycosidases, etc. are involved. Enzymes catalyzing cleavage of CC, CO, CN and other bonds by elimination, leaving double bonds, or by adding groups to double bonds. Enzymes catalyzing a change in the conguration of a molecule. Racemases and isomerases are involved. Enzymes catalyzing the joining of two molecules with the accompanying hydrolysis of a high-energy bond. Such enzymes are often termed synthetases.

49

In 1921, a microbial reaction for the stereospecic production of d-()ephedrine was described [131]. A yeast strain, cultivated to produce acetaldehyde from glucose, was found to condense benzaldehyde with acetaldehyde to form optically active l-phenyl-1-hydroxy-2propanone. This product was in turn converted chemically into d-()ephedrine (Fig. 21). This is an example of a successful combination of chemical and biological reactions.

Transferases:

Hydrolases:

Lyases:

Figure 21. Chemoenzymatic synthesis of optically active l-phenyl-1-hydroxy-2-propanone and chemical conversion into d-(-)ephedrine

Isomerases:

Ligases:

Figure 20. Biotransformation of ethanol to acetic acid 1 C2 H5 OH+ O2 CH3 CHO+H2 O 2 1 CH3 CHO+ O2 CH3 COOH 2

Other historical examples of biocatalytic oxidation are the oxidation of glucose to gluconic acid by Acetobacter aceti [128] and the oxidation of sorbitol to sorbose by Acetobacter species [129]. A variety of such early reports on biocatalytic conversions was collected by Plimmer [130]. Since then many other books have been published on this topic.

Biocatalyis reached its present signicance much later, when specic modications of steroids (or sterols) by microorganisms were discovered. Various steroid reactions, such as hydroxylation, epoxidation, dehydrogenation, isomerization, and hydrolysis, are performed by a wide variety of microbial enzymes. These include the following important reactions: reduction of androstenedione to testosterone by yeasts [132], hydroxylation of progesterone to 11-hydroxyprogesterone by Rhizopus arrhizus [133], 11- -hydroxylation of C21 -steroids by Curvelaria lunata [134], introduction of a 1 double bond into hydrocortisone by Corynebacterium simplex [135], 16--hydroxylation of 9-uorohydrocortisone by Streptomyces reseochromogenes [136], and elimination of the side chain of cholesterol by Arthrobacter simplex [137]. Many novel intermediates for the chemical synthesis of new steroids became available in this way. In addition to the considerable practical value, the amazing versatility of microorganisms in the transformation of steroids provided an important theoretical background for the application of microbial enzymes to chemical synthesis.

50

Biotechnology in biocatalysis are summarized in Table 8. Although biocatalysis has become an important technology for synthetic chemistry, it also has a high potential for many elds of biotechnology.

Many of these reactions cannot be achieved by conventional chemical synthesis.

Table 8. Advances in biocatalysis Example reactions hydroxylation (oxygenase), dehydrogenation (dehydrogenase), side-chain degradation (oxygenase), etc. Transformation of terpenoids hydroxylation of beta-ionone with monooxygenase from Bacillus negaterium [138]. Transformation of alkaloids oxidation of morphine to 2,2 -bimorphin (pseudomorphine) by Cylindrocarpon didymium [139]; N -demethylation of codein to norcodein by Mucus piriformis [140]. Synthesis of semisynthetic synthesis of semisynthetic antibiotics penicillins and cephalosporins (amidase). Synthesis of organic acids hydration of fumaric acid (fumarase), diterminal oxidation of alkanes (oxygenase), asymmetric hydrolysis of epoxysuccinic acid (hydrolase), etc. Transformation of sugars isomerization of glucose (glucose isomerase). Protein synthesis synthesis of plastein (protease), semisynthesis of human insulin (protease), etc. Synthesis of nucleic acid related trans-N -ribosylation and compounds trans-N -arabinosylation (phosphorylase), phosphorylation of nucleosides (phosphotransferase), pyrophosphorylation of nucleotides (pyrophosphotransferase), synthesis of sugar nucleotides (pyrophosphorylase), synthesis of coenzymes, etc. Synthesis of amino acids asymmetric hydrolysis of -amino--caprolactam, 2-amino-2 -thiazoline-4-carboxylic acid, and 5-substituted hydantoins (hydrolase), amination of fumaric acid (aspartase), synthesis of l-tyrosine-related amino acids ( -tyrosinase), l-tryptophan-related amino acids (tryptophanase), etc. Synthesis of amines decarboxylation of amino acids (decarboxylase). Synthesis of industrial chemicals synthesis of alkene oxides (oxygenase), chiral alcohols and ketones (dehydrogenase), pyrogallol (decarboxylase), amides (nitrile hydratase), etc. Biocatalytic synthesis Transformation of steroids and sterols

6.4. Characteristics of Enzyme Reactions Used in Biotransformations

Chemical reactions performed by biocatalysts (biotransformations) are essentially the same as those carried out in inorganic or organic chemistry. Enzymes (or biocatalysts) used for microbial conversions increase the reaction rate by lowering the activation energy as normal catalysts do. The most striking difference between enzymes and chemical catalysts, however, lies in their substrate specicity. They catalyze specic reactions involving one or only a few structurally related compounds and they distinguish almost absolutely between stereoisomers or regioisomers. Therefore, only a very specic change in a functional group or bond of a compound is accelerated by the enzyme. As a result, one single product is expected as long as only one enzyme is involved in a conversion. In contrast to chemical reactions that often require high activation energies (high temperature, pressure, etc.) biotransformations can be performed under considerable mild reaction conditions (Table 9). Due to the low energy input needed for biocatalytic processes and the high atom utilization (percentage of M r of desired product to sum of M r of all substrates), biotransformations are considered as an environmentally friendly alternative to classical organic chemistry processes. To compare biocatalytic and chemical processes on the basis of the amount of waste, Roger A. Sheldon [141] devised the environmental quotient (EQ), which is the quotient of the amount of waste product per kilogram of product (E ) and an unfriendliness quotient (Q): EQ = E Q. High catalytic efcacy is another characteristic property of enzymes. They increase the turnover rate without large energy requirements and only a small amount of enzyme is needed to catalyze the conversion of a large amount of substrate.

Innumerable biocatalytic conversions involving different types of reactions with organic compounds and natural products were found during the following years; some of them are very useful for chemical synthesis. Advances

Biotechnology
Table 9. General characteristics of enzymatic and chemical reactions Parameter Reaction conditions: Temperature Pressure pH Reaction energy Enzymatic reaction Chemical reaction

51

Solvent

Specicities (substrate, stereo-, regiospecity) Concentration of substrate and/or product

physiological high atmospheric high neutral different enzyme conformation thermal (van der Waals, hydrogen bonds, etc.) water, (organic water, organic solvents and ionic solvents liquids under special conditions possible, e.g., low wateractivity) high low

the actual life process of the microorganisms. In other words, the reaction product results from the complex metabolism of the microorganism from cheap carbon and nitrogen sources. Therefore, living cells are required for fermentation, whereas this is not so important in biotransformation. Fermentation products are always natural products. Advantages and disadvantages of these two microbial processes are briey summarized in Table 10. However, it is not always possible to draw a clear line between the two categories.

low

high

6.5. Types of Biocatalysts and Reaction Systems

Because biotransformations are essentially catalytic chemical reactions, a suitable catalyst for the desired conversion must be prepared carefully. Although conventional vegetative cell cultures are most commonly used, there are several alternative methods; some of these have already been operated commercially. These alternatives have been evaluated extensively with regard to, for example, increased yield, control of side reactions, simplied processes, and improved economy. They have great future potential. Various types of biocatalysts are useful for microbial conversions (see Section 6.2. Any combination of these systems may also be possible. Several successful conversions are known in which multistep reactions are catalyzed by different catalysts (see Section 6.5.6). 6.5.1. Biotransformation with Growing Cultures The substrate is added to the growth medium during inoculation or an appropriate phase of growth. The preparation and inoculation of the medium, addition of substrate, and incubation are successively carried out in one ask until the reaction is completed. Because of its extreme simplicity, this procedure is frequently used not only for screening (see Section 6.6), but also in large-scale production. Usually, the substrate is added directly to the medium without sterilization if the fully grown culture is the catalyst. Continuous chemostat methods may be used to maintain a steady state of growth, enzyme levels,

Table 10. Characteristics of biotransformation microbial conversion and fermentative production Parameter Fermentative production Microorganism resting or treated cellsgrowing cells Reaction simple catalytic life process (multistep reaction (one or few reaction) steps) Reaction time short long Starting materials expensive substrates cheap carbon and nitrogen sources Product natural and/or natural unnatural Product concentration high low Product isolation easy tedious Biotransformation

As a result, enzymes exhibit their activity under mild reaction conditions, such as atmospheric pressure, temperatures around 20 40 C, and pH values near neutrality, where normal chemical catalysts are inactive. On the other hand, enzymes cannot function under drastic reaction conditions because of their delicate protein structures. The unique properties of enzymes are extremely useful when unstable molecules are to be converted without undesired side reactions. There are two ways for using biocatalysts in the production of useful compounds: biotransformation and fermentation. Fermentation has been used since ancient times (e.g., alcoholic fermentation) and is still of industrial importance in the production of such compounds as organic acids, solvents, or antibiotics. In contrast to biotransformation, fermentation is considered to be a biologic method, because it results from

52

Biotechnology 6.5.2.2. Permeabilized Cells In cases where substrate or product molecules do not readily permeate the cell membrane, a modication of the cell becomes necessary. Commonly used techniques to change the permeability of the cell membrane to control the diffusion of substrate into the cell and of product out of the cell are: using surfactants, organic solvents, antibiotics or enzymes. Besides these chemical methods physical procedures, such as osmotic shock or freezing and thawing may also be useful [142]. The production of high-energy phosphate esters, such as adenosine 5-triphosphate, nicotinamide adenine dinucleotide, avin adenine dinucleotide, or coenzyme A, is a typical example [143]. 6.5.2.3. Dried Cells Dried cells are an alternative to permeabilized cells. Drying causes structural changes in the cell wall or membrane. Contact between the cell enzyme and the substrate is more readily achieved. In some cases, drying also eliminates undesired side reactions, because some enzymes are damaged through drying. Dried cells are usually prepared by air-drying, lyophilization and acetone treatment. The powdered dried cells can retain their enzyme activities in the frozen state for years. They can be used as instant catalysts in the same way as normal chemical catalysts. 6.5.3. Biotransformation with Spores Many fungal spores have as many useful enzymes as vegetative or grown cells. The fungi are cultivated under conditions that induce high spore yields. The spores are then separated from the mycelium and used as catalysts in a similar way as mentioned in the previous section. Processes using spores have the same advantages as those using grown cells. Spores are usually more stable than cells. Some types of spores can be stored for several years without loss of conversion activity. They usually do not germinate during the reaction, but in some cases germination is required for maximum conversion activity. The 11--hydroxylation of progesterone by

and substrate concentration. If high cell densities are required by the conversion or if the nature of the growth medium causes problems for the isolation of the product, the reaction should be performed after separating the cells from the medium, see next section. 6.5.2. Biotransformation Conversion with Previously Grown Cells The microorganism is grown under suitable conditions and the cells are then collected by centrifugation or ltration. In this way, the cells retain most of their enzyme activity. For the conversion they are resuspended either in an incomplete medium (e.g., one without a nitrogen source; this prolongs cell viability and activity as well as possible cofactor regeneration) or in a simple reaction medium (e.g., a buffer solution or water containing the substrate). The reaction is then carried out directly or after suitable treatment of the cells. It is theoretically not important whether the cells are dead or alive. Vegetative, washed, and dried cells fall into this category. The advantages of this method are listed below: 1) Growth and conversion steps are controlled independently for optimization. 2) The grown cells can be stored for some period of time until initiation of the reaction. 3) The cell density for the conversion is easily controlled. 4) The reaction can be carried out under nonsterile conditions. 5) Product isolation is usually easier because of the simple composition of the reaction medium. In some cases, however, the strict separation of microbial growth and conversion is considered to be unfavorable for economic reasons. Depending on the ability of substrate permeability through the cell membrane these biocatalysts may have to be treated differently prior to their use. 6.5.2.1. Vegetative or Washed Cells In cases where the cell membrane is permeable to substrate and product, no special technique is needed for the preparation of the cells and they can be used as vegetative or washed cells.

Biotechnology Aspergillus ochraceus spores represents a successful spore process [144]. Many other applications have been reported, e.g., fatty acids conversions, triglycerides, carbohydrates, or penicillin V [145]. 6.5.4. Biotransformation with Immobilized Cells Immobilized cells are cells that have either been xed or bound to a surface, entrapped in a gel matrix, or retained in a membrane reactor. All of the above-mentioned cells can be immobilized. Numerous immobilization methods have been reported; they can be divided into the following four groups: 1) Entrapment or encapsulation in a porous polymer network, e.g., polyacrylamide, alginate, -carrageenan, gelatine, agar, collagen, cellulose, polyurethane, poly(vinyl alcohol). 2) Covalent, ionic, or physical attachment to an appropriate water-insoluble solid support, e.g., ion-exchange resins, silica gels, metal oxides. 3) Aggregation of cells by physical or chemical cross-linking with glutaraldehyde, polyethyleneimine, or other agents.

53

4) Retention of cells by membranes (microltiration, cutoff 0.22 m), e.g., in form of membrane reactors or hollow ber modules [153]. In successful cases, the immobilized cells retain their activities over several months and show a higher operational stability than unsupported cells. Additional advantages are: 1) easy removal of the cells from the reaction mixture, 2) repeated use of the cells, 3) continuous operation of the reaction, 4) cell regeneration by immersion into an appropriate nutrient medium, and 5) easy product isolation. Some of these features are especially advantageous if the collection of a sufcient cell mass for the conversion is costly. However, disadvantages must also be considered: 1) The catalytic activity of cells usually decreases during the immobilization procedure because the cells are partly damaged and permeability is further inhibited. This decreased activity can be compensated by increasing the cell density. 2) The immobilization itself sometimes requires special equipment. The conversion step, especially when operated continuously, also requires a special engineering design compared to conventional conversion processes. Examples of successful large-scale applications are listed in Table 11. An advance in this eld was the introduction of an immobilized system of growing cells

Table 11. A selection of commercial applications of immobilized biocatalysts: microbial cells or enzymes Microorganism or Methods of immobilization immobilized enzyme l-Amino acylase from adsorption to Aspergillus oryzae DEAE-Sephadex Escherichia coli (aspartase) entrapment in -carrageenan gel entrapment in Brevibacterium ammoniagenes (fumarase) -carrageenan gel Escherichia coli (aspartase) entrapment in and Pseudomonas dacunhae-carrageenan gel (aspartate -decarboxylase) Penicillin acylase from entrapment in a cellulose Escherichia coli triacetate matrix Glucose isomerase from Bacillus coagulans or Streptomyces species -Galactosidase from Lactobacillus bulgaricus, Aspergillus oryzae, etc. Whole cells Arthrobacter sp. (oxygenase) many methods available Applications optical resolution of racemic amino acids continuous production of l-aspartic acid from ammonium fumarate continuous production of l-malic acid from fumaric acid continuous production of l-alanine from ammonium fumarate production of 6-amino-penicillanic acid from penicillin G production of high-fructose syrup from glucose Year of introduction 1969 1973 Ref. [146] [147]

1974

[148]

1982

[149]

1973

[150]

1973

[151]

many methods available

production of low-lactose 1977 milk and production of sweetenings retention by microltration production of muconic acid in cross-ow membrane (raw material for new reactor resins, pharmaceuticals and agrochemicals)

[152]

[153]

54

Biotechnology 6.5.6. Multistep Reactions Using Different Biocatalysts In order to perform more than two sequential reactions, two or more different biocatalysts are sometimes required either simultaneously or sequentially. Theoretically, any combination of catalysts can be used for such multistep reactions. Examples of successful applications are: continuous production of l-alanine from ammonium fumarate via L-aspartate as the intermediate by immobilized Escherichia coli (aspartase) and Pseudomonas dacunhae (l-aspartate -decarboxylase) cells [149], complete condensation of racemic homocysteine by washed or dried Pseudomonas putida (racemase) and Alcaligenes faecalis (S -adenosylhomocysteine hydrolase) cells [160], complete hydrolysis of racemic -amino--caprolactam (see Section 6.6.2.1), and continuous amination of -keto acids to l-amino acids by two types of cell-free enzymes (see Section 6.5.5). Another remarkable example is the synthesis of coenzyme A from pantothenic acid, lcysteine, and ATP (or AMP), which proceeds through ve sequential steps (see below) and is commercially performed solely with Brevibacterium ammoniagenes cells [161, 162].
Step 1: Step 2: pantothenic acid + ATP phosphopantothenic acid + ADP phosphopantothenic acid + l-cysteine + ATP phosphopantothenoyl-l-cysteine + ADP + inorganic phosphate phosphopantothenoyl-l-cysteine phosphopantetheine + CO2 phosphopantetheine + ATP dephosphocoenzyme A + inorganic pyrophosphate dephosphocoenzyme A + ATP coenzyme A + ADP

into a conversion process. For example, cells entrapped in -carrageenan are viable and reproduce without loss of activity per unit cells [154]. The continuous production of ethanol from glucose by S. cerevisiae in -carrageenan [155] and the 1 -dehydrogenation of cortisol by Arthrobacter simplex in calcium alginate [156] are examples of such successful operations. Further improvement of this technique is now expanding into the application to complex multistep reactions, such as the production of antibiotics. 6.5.5. Biotransformation with Cell-free Enzymes or Puried Enzymes In general, the use of cell-free enzymes or puried enzymes for conversions is expensive, because purication of enzymes is often tedious and time-consuming. Therefore, they are not so suitable for large-scale industrial production. However, there are several cases in which they are advantageous over systems using cells. Such cases are: 1) Poor substrate or product permeability through the cell wall 2) Problems caused by undesired side reactions 3) Commercial availability of the desired enzyme at an acceptable price If an extracellular enzyme is used, cells are not involved at all. Cell-free enzymes can also be used as immobilized catalysts [157]. The same techniques as those used for cell immobilization are available. Immobilization of penicillin acylase [150], l-amino acid acylase [146], lactase [152], and glucose isomerase [151] have been applied to commercial production. The entrapment of soluble enzymes in a membrane-type reactor with simultaneous coenzyme regeneration is an alternative to the use of cell-free enzymes. l-Amino acid dehydrogenases in combination with formate dehydrogenase as a coenzyme regenerator and nicotinamide adenine dinucleotide covalently bound to polyethylene glycol in a membrane reactor have been successfully applied to the continuous amination of keto acids yielding optically active amino acids [158]. l-Leucine is already produced commercially by this process. Whitesides [159] has reviewed the potential applications of cell-free enzymes in organic synthesis.

Step 3: Step 4:

Step 5:

6.5.7. Multiphase Reaction Systems Since their discovery, enzymes have been thought to be inert catalysts in organic solvents. This is basically true, but enzymes have become available that are catalytically active in watermiscible organic solvents. Many reactions that are impossible in water because of kinetic or thermodynamic reasons can be performed in organic solvents [163, 164] (e.g., transesterication reactions [165]). Chemically modied peroxidase exhibits 21 % of its activity in benzene

Biotechnology as compared to that of the unmodied enzyme in aqueous solution [166]. Polyphenol oxidase converts nearly 100 % of phenol to catechol when the reaction is carried out in chloroform containing only 12 % water [167]. Such liquid liquid two-phase systems with low water content can also be used to shift the hydrolytic equilibrium toward water elimination. The synthesis of esters, amides, and peptides by this method shows great practical potential. Multiphasic conversion systems using microbial cells as catalysts have also been extensively studied, especially in the case of lipophilic compounds, which have a limited solubility in water. In some cases, increased reaction rates and/or product yields have been achieved.

55

dehydrogenase from C. boidinii for cofactor regeneration. Yield (97 %), conversion (97 %), and optical purity of the product (ee > 99 %) are very high.

6.6. Process Design

6.6.1. General Considerations When designing a biocatalytic process many important aspects require careful consideration. Besides the essential question of the type of enzyme used for biocatalysis there is the selection of a compound to be synthesized and a survey on available substrates and routes or reactions that are of special importance in designing a conversion process (also see Chapter 5). 6.6.1.1. Evaluating Enzyme Potential When a new conversion or a new enzyme useful to biocatalytic conversions is discovered, the question as to the potential of the new enzyme for practical purposes must be answered rst. An example for a high-potential biocatalysis with practical application in the pharmaceutical industry is l-phenylalanine dehydrogenase (PheDH, EC 1.4.1.20) from Thermoactinomyces intermedius for the conversion of ketoacid acetals to acetal amino acids [168] (Fig. 22). The amino acid acetal is a precursor for Omapatrilat r , an antihypertensive drug, which inhibits the angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP). The synthesis is carried out in 16,000 L batch reactions with heat dried E.coli cells containing the cloned and overexpressed PheDH from Thermoactinomyces intermedius and the formate

Figure 22. Synthesis of acetal amino acid (Bristol-Myers Squibb)

Another interesting example is the production of l-tyrosine and l-dopa (dihydroxyphenylalanine) by bacterial -tyrosinase [169]. This enzyme has long been known to catalyze the , -elimination of l-tyrosine to phenol, pyruvate, and ammonia (Eq. 1).

Because the enzyme also catalyzes the reverse reaction (Eq. 2) and the -replacement reaction of l-tyrosine with phenol derivatives (Eq. 3), it did not take long for the application of these reactions in the synthesis of l-tyrosine and l-dopa.

56

Biotechnology
R H+CH3 COCOOH+NH3 L (D) R CH2 CH (NH2 ) COOH+H2 O

Subsequently, similar reactions were found to be catalyzed by several pyridoxal phosphate dependent enzymes, such as tryptophanase, cysteine desulfhydrase, and 3-chlorod-alanine chloride lyase. These enzymes were then successfully used to synthesize l-tryptophan and 5-hydroxy-l-tryptophan, l-cysteine, and d-cysteine [169 171] (see Table 12). These reactions can be summarized as follows:
L (D) RCH2 CH (NH2 ) COOH+H2 O RH+CH3 COCOOH+NH3 L (D) RCH2 CH (NH2 ) COOH+R H L (D) R CH2 CH (NH2 ) COOH+RH

where for -tyrosinase R = hydroxyphenyl, OH, SH, Cl and R = hydroxyphenyl; for tryptophanase R = indolyl, OH, SH, Cl and R = indolyl; for cysteine desulfhydrase R = SH, thiol radicals, Cl and R = SH, thiol radicals; and for 3-chloro-D-alanine chloride-lyase R = Cl, SH, thiol radicals and R = SH. These successful examples demonstrate the importance of reassessing the capabilities of well-known enzymes or reactions.

Table 12. Synthesis of l-tyrosine, l-tryptophan, l-cysteine, d-cysteine, and related amino acids by pyridoxal phosphate-dependent enzymes Product g/L 58 Yield, mol% 88 a Substrates Reaction Enzyme Microorganism reverse of , -elimination -tyrosinase Erwinia herbicola -replacement -tyrosinase Erwinia herbicola reverse of , -elimination -tyrosinase Erwinia herbicola -replacement -tyrosinase Erwinia herbicola reverse of , -elimination tryptophanase Proteus rettgeri reverse of , -elimination tryptophanase Proteus rettgeri -replacement cysteine desulfhydrase Enterobacter cloacae -replacement 3-chloro-d-alanine chloride lyase Pseudomonas putida

l-Tyrosine

sodium pyruvate ammonium acetate phenol d,l-serine ammonium acetate phenol sodium pyruvate ammonium acetate pyrocatechol d,l-serine ammonium acetate pyrocatechol sodium pyruvate ammonium acetate indole sodium pyruvate ammonium acetate 5-hydroxyindole 3-chloro-l-alanine sodium sulde

l-Tyrosine

53.5

78 b

l-Dopa

58.5

l-Dopa

53

71 c

l-Tryptophan1

00

100 d

5-Hydroxy-ltryptophan

23.3

57 e

l-Cysteine

50

88f

d-Cysteine

22

88g

3-chloro-d-alanine sodium hydrogensulde

Based on sodium pyruvate. Based on d,l-serine. c Based on indole. d Based on 5-hydroxyindole. e Based on 3-chloro-l-alanine. f Based on 3-chloro-d-alanine. g Sodium pyruvate was added at 2-h intervals to maintain a concentration of 5 g/L for 48 h.
b

Biotechnology 6.6.1.2. Finding Suitable Enzymes Occasionally a synthetic scheme has been designed but suitable enzymes or conversion processes are yet unknown. In such a case, it becomes necessary to assay suitable microbial strains or enzymes (as described in Section 6.6.2) as to their suitability for catalyzing the desired reaction. The production of l-lysine from d,l- - amino- -caprolactam (ACL) is another typical example [172]. This process was designed to utilize cyclohexene, a major byproduct of nylon production. In this process, the biocatalyst is required to hydrolyze only the l-form of -amino- -caprolactam (l-ACL) to yield llysine (Fig. 23).

57

solution. Sterilization of the substrate is recommended prior to its use, if required. Only dissolved substrate is converted. Therefore, slightly soluble substrates must rst be dissolved and the products may then crystallize from the solution after conversion:
Substrate (solid) Substrate (solution) Product (solid) Product (solution)

Figure 23. Hydrolysation of the l-form of -amino- -caprolactam (l-ACL) to yield l-lysine

Two microorganisms were found during the assay: one of them hydrolyzes the l-form of the substrate and the other racemizes the remaining d-form. As a result, all the -amino- -caprolactam is converted into l-lysine without requiring any optical resolution. 6.6.1.3. Substrates Any substrate is theoretically suitable for biotransformations, provided the substrate molecules come into contact with the enzyme. Even gases, such as methane, are suitable substrates; they are bubbled into the reaction medium. The substrate should be soluble in the medium and able to pass through the cell membrane unless the reaction is catalyzed by cellfree enzymes. The substrate is usually added to the reaction medium, neat or as a concentrated

For example, powdered cortisol in concentrations up to 500 g/L is converted to crystalline prednisolone with a good yield by Arthrobacter simplex [173]. Substrates may also be dissolved in water-miscible organic solvents. The lower alcohols, acetone, dimethylformamide, and dimethylsulfoxide are suitable solvents. Surfactants may also be used to disperse the substrate. The combination of water-miscible solvents and surfactants may offer some advantage. In some cases, chemical modication of the substrate may improve its solubility. An example for a process using substrate with poor solubility is the regioselective oxidation of the tert -butyl group of terfenadine using Cunninghamela blakesleana in a whole-cell biotransformation [174]. Due to its low solubility in water, microcrystalline terfenadine is solved in the water-miscible organic solvent dimethylfomamide prior to its addition to the aqueous reaction medium. This regioselective oxidation was studied as a biocatalytic alternative for the chemical synthesis of the antihistaminic drug fexofenadine. In cases where the cell membrane is impermeable to the substrate or a cell-free or puried biocatalyst is required, the permeability of the cell must be improved, respectively, the cell wall has to be disrupted. This is accomplished by air drying, acetone drying, lyophilization, autolysis, lysozyme digestion, surfactant treatment, osmotic shock, freezing and thawing, or ultrasonic treatment. For cell-free preparations, the cell debris can be removed by centrifugation or microltration. If the substrate does not suit the desired conversion, its chemical modication should be considered. Addition, variation, or removal of a protecting group or modication of a functional group in the substrate molecule sometimes makes its interaction with the enzyme more efcient. In addition, these modications may prevent undesirable side reactions or degra-

58

Biotechnology Such a test, also called screening, may be one of the most important steps for a successful biocatalytic conversion. 6.6.2.1. Screening
Kitahara and co-workers [183] screened bac-

dation. This method is frequently used in steroid conversions. A systematic assay for a suitable chloroacetoacetate ester for enantioselective reduction yielding the l-form of 4-chloro-3-hydroxybutanoate, a promising precursor for the chemical synthesis of l-carnitine, is an example [175]. 6.6.1.4. Media Besides performing biocatalysis in aqueous media, there has developed the so-called nonaqueous enzymology which has become an important area of research and development during the last decades [176]. Enzymes exhibit a wide array of novel reactivities and selectivities in nonaqueous solvents. For example, many reactions that are impossible in water due to kinetic or thermodynamic reasons can be performed in organic solvents, [163, 164, 177] due to the suppression of water-induced side reactions. Improved and altered substrate specicities [178, 179] and selectivities can be observed. Examples of practical applications are enantioselective synthesis [180], chiral resolution [181], and combinatorial biocatalysis [182]. The possibility of the solubilization of hydrophobic substrates or products in organic solvents opens opportunities for the enzymatic production of poorly water-soluble ne chemicals and pharmaceuticals. The thermal and storage stability of enzymes can be signicantly enhanced in nonaqueous media [164, 176, 178]. 6.6.2. Selection of Biocatalysts More than 8000 enzymes are known [125]. Almost all types of chemical reactions which are also catalyzed by normal chemical catalysts are involved. According to the desired type of conversion and the classication of the enzymes one can easily determine which enzyme would be suitable for the desired conversion by inspecting the enzyme catalogue published by the committee [125]. However, the properties of enzymes from different sources may vary widely, even if they catalyze the same reaction. Therefore, testing is indispensable. A wide variety of biocatalysts are tested for the purpose of determining their ability to carry out the desired reaction.

terial strains from cultures grown aerobically at 3037 C in nutrient broth containing malt extract. Sodium fumarate and ammonium chloride were added 24 h after growth started. After incubation for another 24 h the l-aspartate in the culture broth was analyzed by chromatography. A strain of E. coli was selected by this screening as the most promising producer of aspartase. Under suitable reaction conditions, 56 g of l-aspartate was produced per 100 mL with a molar yield of 99 % by using 1 g of dried cells. Subsequently, this process was developed into large-scale technology by which l-aspartate was synthesized continuously with E. coli cells immobilized on -carrageenan [184]. The screening procedure described above may be rather simple, but it reects the essence of screening. Normal screening procedures can be more complicated and deal with a great variety of microorganisms, including bacteria, actinomycetes, molds, yeasts, and basidiomycetes. 6.6.2.2. Enrichment Another method to nd a suitable biocatalyst can be enrichment. Sometimes it is desired or necessary to nd biocatalysts capable of performing a specic conversion from natural sources. Soil samples containing mixed microbial populations are rich sources of test organisms. Because the enzymes in these microorganisms modify or degrade a great variety of complex organic compounds, at least one of the microorganisms is expected to offer one or several enzymes that perform the desired conversion. These microorganisms are selected using the enrichment technique. A typical enrichment procedure is the isolation of bacterial strains that perform the asymmetric hydrolysis of d,l-2-amino- 2 -thiazoline-4-carboxylic acid (d,l-ATC), an intermediate of d,l-cysteine synthesis, to lcysteine [185]. Soil samples were incubated in a medium containing 0.3 % D,L-ATC as the sole

Biotechnology nitrogen source. After spreading each culture on a plate of the same medium, colonies that appeared were isolated and grown on nutrient agar containing 0.2 % d,l-ATC as the enzyme inducer. The resulting cells were then transferred to a solution containing 1 % d,l-ATC and the formation of l-cysteine was demonstrated by chromatographic or microbiological methods. Among 1975 colonies from 388 soil samples, 31 l-cysteine-producing bacteria were isolated. One of them, Pseudomonas thiazolinophilum, was mutagenized. Mutants with inhibited lcysteine metabolism were screened from the mutagenized cultures. One of these mutants converted the added substrate almost stoichiometrically to l-cysteine with a yield of 31.4 mg/mL [186]. The enzyme system of Pseudomonas thiazolinophilum hydrolyzes d,l-ATC in two steps as shown in Figure 24.

59

require different knowledge of the target enzyme as well as laboratory equipment (Table 13).
Table 13. Requirements and molecular methods for rational design and directed evolution technique Rational design 3D structure of the target enzyme Directed -evolution high-throughputscreening/selection technology to analyze enzyme properties

Requirements:

knowledge of reaction mechanism modeling of enzyme-substrate interaction Molecular methods: site-directed random mutagenesis mutagenesis

6.7. Improvement of Conversion Processes

After an adequate process has been designed, it must be optimized. This includes improvement of the strain and the search for optimal growth (highest enzyme yield and maximum activity) and optimal reaction conditions. If the enzyme to be used is constitutive, the amount of biomass containing that enzyme will have to be increased. This is usually sufcient because the constitutive enzyme is formed independently of the medium composition. However, if the enzyme is inducible, an effective inducer, which may be the substrate itself or another structurally related compound, will be required. If the enzyme is repressive, the repression factor will either have to be eliminated or the repressive compounds, which may be present in the growth medium or formed during the growth, must be removed. Catabolites of an easily metabolizable carbon source, e.g., glucose or sucrose, and the end product of a metabolic pathway usually cause catabolite or product repression, respectively. Therefore, the composition of the growth medium, growth period, and various physical parameters of growth, such as temperature, aeration, agitation, and pH, must be carefully controlled. In successful cases, the concentration of the required enzyme reaches more than 10 % of the total soluble protein [187]. Optimal physical and chemical conditions for the biotransformation itself must be determined with the same care as for the optimization of growth conditions. This is especially important

Figure 24. Asymmetric hydrolysis of d,l-2-amino- 2thiazoline-4-carboxylic acid (d,l-ATC) to l-cysteine

Although screening sometimes is regarded as old-fashioned, time-consuming, and even illogical, some consider it to be one of the most promising methods for nding new genes in microorganisms. 6.6.2.3. Molecular Engineering Sometimes enzyme yields in wild-type organisms are low or the desired enzyme is not found in the available microorganisms. In this case, molecular engineering techniques can lead to an enzyme catalyzing the desired reaction. There are two different strategies that represent the state-of-the-art technologies: rational design and directed evolution [153]. Both technologies

60

Biotechnology Microbial conversion can also be optimized by improving the selected strain. Standard methods of strain improvement include the isolation of single colonies, optionally after mutagenesis, and screening of the best suited isolates. Such conventional genetic manipulation techniques have been developed during the last decades for the improvement of useful strains. Recombinant DNA technology is providing new prospects for strain improvement. Plasmid or phage vectors can transfer DNA fragments of microbial, plant, animal, or even synthetic origin into a suitable recipient microorganism. These gene cloning techniques are still restricted to microbial strains such as E. coli, B. subtilis, and S. cerevisiae, in which the genetic background and the appropriate hostvector systems are well known, but their practical application is rare [191]. However, this technology will undoubtedly provide one of the most important keys for the future development of microbial conversions.

if side reactions are observed, because they decrease product yield and make product isolation difcult. Physical or chemical treatment of the biocatalyst, such as heating, pH change, and the addition of detergents, organic solvents, or specic inhibitors may cause specic inactivation of undesired enzymes. For example, the trans-N arabinosylation of adenine from synthetic uracil arabinoside (Fig. 25) yields adenine arabinoside, an antiviral agent.

6.8. Conclusion and Outlook

Biocatalysts have become one of the most important tools in nearly all elds of industrial biotechnology. The main advantages of using biocatalysts are their unique properties such as high chemo-, regio-, and stereoselectivity and the ability to catalyze reactions under mild conditions. During the last couple of years, many new methods and techniques have been developed to overcome common problems. Some examples here might be molecular engineering techniques such as rational design or directed evolution to increase low biocatalytic activities [153], biocatalysis in nonaqueaous reaction media to bypass solubility problems of substrate and/or product [179], or the applications of highthroughput screening methods for the search of new and suitable biocatalysts. Another eld of interest that will become of great impact is metagenomic research that will enable scientists to nd new biocatalysts with desired qualities [192]. To make new biocatalysts attractive for industrial applications they have to be available in sufcient amounts. High expression rates of enzymes can be achieved by genetical and physiological manipulations of the expressing mi-

Figure 25. trans-N -arabinosylation of adenine from synthetic uracil arabinoside

This reaction is catalyzed by cells of Enterobacter aerogenes and takes place only at temperatures above 60 C [188]. The reaction does not proceed below 50 C because hypoxanthine is formed by adenine deaminase before the trans-N -arabinosylation by two kinds of nucleoside phosphorylases takes place. Fortunately, these phosphorylases are still active at 60 C, whereas the deaminase is completely inactivated at this temperature. Other examples are summarized in Table 14. In the case of immobilized biocatalysts, concentration gradients of substrates and products may occur that could lead to decreased biotransformation rates. These gradients can be reduced by decreasing the Thiele modulus, e.g., by using smaller carrier particles or by reducing the diffusion distance between the free solution and the immobilized enzymes.

Biotechnology
Table 14. Elimination of side reactions by physical or chemical treatment Desired reaction Ammonium fumarate l-aspartic acid (Escherichia coli/aspartase) Fumaric acid l-malic acid (Brevibacterium ammoniagenes/fumarase) l-Aspartic acid l-alanine (Pseudomonas dacunhae/aspartate -decarboxylase) Cholesterol 1,4-androstadiene-3,17-dione (Arthrobacter simplex /multistep conversion) -Sitosterol 17-ketosteroids (Nocardia species/multistep conversion) d,l-2-Amino-2 -thiazoline-4-carboxylate l-cysteine (Pseudomonas thiazolinophilum/multistep conversion) Side reaction fumaric acid l-malic acid fumaric acid succinic acid l-alanine d-alanine Treatment incubation of culture broth at pH 5 and 45 C for 1 h treatment of immobilized cells with 0.6 % bile extract incubation of culture broth at pH 4.75 and 30 C for 1 h addition of , -dipyridyl (1 mM) to the culture broth addition of , -dipyridyl (0.3 mM) to the conversion mixture addition of hydroxylamine or semicarbazide to the reaction mixture Reference [149]

61

[148]

[149]

further decomposition of the sterol molecule further decomposition of steroid ring further degradation of l-cysteine

[137, 189]

[190]

[186]

croorganism such as optimizing the number of plasmids per cell or inactivation of negative cell functions (e.g., deletion of protease coding genes). Apart from deleting unwanted side activities also novel metabolic pathways can be implemented. These microorganisms, customized only to fulll special needs for biocatalytic applications, are called designer bugs or tailormade microorganisms.

7. Downstream Processing
Downstream processing is one of the most underestimated steps in bioprocesses and it is well known especially in the pharmaceutical industry that downstreaming is the most expensive and unfortunately the most ineffective part of a bioprocess. Thus, one can assume that new developments are widely described in the literature. Unfortunately, this is not the case. Only a few working groups focus on new and more effective procedures to separate products from fermentation broths or biotransformations. A chief characteristic of biotechnology is the wide variety of products. Due to this variety, a broad spectrum of separation techniques must be applied. However, for nearly all products one starts with a dilute suspension and tries to produce a highly puried dry product. In the case of extracellular products, the solids in this suspension may include intact organisms, other insoluble fractions of the medium or natural sample, and perhaps insoluble products. Concerning intracellular products the solids include in addition fragmented

mycelia caused by cell disruption, which is necessary to gain the products. According to this starting point, nearly each downstream process consists of the four following steps [193]: Removal of insoluble particles Isolation of the product Purication Polishing With respect to downstreaming in laboratory scale, one is normally only limited by the available equipment. However, with regard to upscale processes one should have in mind that the developed process should also be applicable in industry. Thus, Belter et al. [193] have formulated the following questions, which are also crucial to biotechnology downstream processes: What is the value of the product? What is the acceptable product quality? Where is the product in each process stream? Where are the impurities in each process stream? What are the unusual physicochemical properties of the product and the principal impurities? What are the economics of various alternative separations? In addition, it is important to use materials which are available for all upscale processes, since the downstream behavior of the product may change while changing, for example, the type of chromatographic resin or membrane material. Normally the recovery costs exceed the bioprocess costs; however, some upstream parameters inuence the downstream processing:

62

Biotechnology rst stage purication of the product by removing dissimilar components from the broth. The most important techniques in use are: Membrane processing Ion exchange chromatography In the membrane process, ultraltration and reverse osmosis are often used for separation, concentration, and desalting. In addition, polar membranes are used for ion exchange and desalting. Ion exchange chromatography is applied in order to remove either major contaminants from the broth or the desired product from the broth. Chromatographic procedures based on columns or membranes are also often used in the purication step. Other techniques in this part of the downstream process are precipitation and liquidliquid extraction.

The characteristic properties of the producing microorganism or cell line The location of the product The stability of the product Byproducts and impurities Concentration of the product in the medium from which it is to be recovered A general downstream scheme based on these parameters is given in Figure 26.

7.1. Sample Disruption

The inuence of the sample disruption step in upstream and downstream processes cannot be ignored. The disruption is dependent on the properties of the sample. The main focus of the disruption is always to release as much as possible of the product. However, depending on the mechanism, parts of the product may be destroyed during the disruption process. Thus, very effective and short procedures are required. A distinction can be made between mechanical, chemical, and enzymatic procedures for product release. Mechanical methods are often preferred because of short residence time, lower operating costs, and contained operation [194]. The most common mechanical means of disruption are: Homogenizers [195, 196] Bead mills [196]. A homogenizer consists of a positivedisplacement pump, which supplies the liquid sample at high pressure through a small nozzle or an orice valve. The disruption results from the combination of shear force and impingement on the valve. Bead mills use horizontal grinding chambers lled with glass beads or other resistant materials such as zirconium oxide, zirconium silicate, titanium carbide, etc [197]. The dispensed sample is introduced into the grinding chamber on a continuous basis. The level of

Figure 26. General downstream scheme in biotechnology

The main aims of the primary separation step are to achieve a volume reduction and to make a

Biotechnology disruption depends on the variable turning speed of the bead mill. By using bead mills, cell disruption can be achieved in a single run with better temperature distribution and temperature control. Another possibility for cell disruption or at least product liberation is the use of microwaves or ultrasound which can be combined with sample extraction by organic solvents. Microwave techniques are widely used in acid digestion of solid samples. Their use in the extraction of organic analytes from environmental samples is less widespread, despite the availability of commercial devices for this purpose and their potential for reducing analysis time and solvent consumption. A possibility for cell lysis under very mild conditions is the use of hydrolyzing enzymes. In addition, enzymes offer selectivity during product release. Enzymes hydrolyse the walls of cells, and when sufcient wall has been removed, the internal osmotic pressure bursts the periplasmic membrane allowing the intracellular components to be released [195]. The effect of lytic enzymes is specic to particular groups of cell types, which is attributed to the differences in cell wall composition. For example, the most efcient lytic enzyme for bacteria is lysozyme from hens egg. This enzyme is also used in large-scale processes for enzyme production [198]. Even more highly specialized procedures for sample disruption can be applied in biotechnology. For example, high yields of intracellular enzymes from yeast can be obtained by applying a series of electric eld pulses [199]. By using this technique, up to 90 % of the total activity can be released without any further or previous treatment of the cells. The method is based on electroinduced changes in the cell envelope leading to a leakage of part of the intracellular proteins without the formation of debris and permits the treatment of large volumes.

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7.2. SolidLiquid Separations

Solidliquid separation is a very important procedure during downstream processing in biotechnology for cell separation, cell debris removal, and also for product recovery. The most important solidliquid separation techniques in use for are ltration and centrifugation. Filtration separates solids from a liquid by forcing the

liquid through a solid support or lter medium. Dead-end ltration and cross-ow ltration are two different designs of ltration which can be used. In dead-end ltration mode, the total process uid stream ows through the membrane. The retained solids accumulate on the membrane and build up a lter cake. The membrane has to be changed when the membrane pores are clogged by the solids. When the feed ow is directed parallel to the membrane surface, the term cross-ow ltration is used. The tangential ow of liquid removes any retained molecules or particles from the membrane surface, which results in a stable ux for a longer time period. By using cross-ow ltration, relatively low shear stress is possible and lter aids are not required. In addition, scale-up is simple and cell washing is possible in a single process step. Over the last 30 years, a number of further membrane processes have been developed for molecular separation. These ltration techniques can be divided into four major groups: reverse osmosis (hyperltration, RO), nanoltration (NF), ultraltration (UF), and microltration (MF) [200]. Membrane processes are easy to scale up and the possibility of using the same materials and congurations in different sizes from laboratory to process scale reduces the validation effort enormously. However, ltration processes are limited with regard to selectivity. The fractionation of proteins can only be achieved with large differences in the molecular weight of the proteins and it is important to keep in mind that a certain difference in the molecular weight of two proteins does not mean the same degree of difference in molecular size. Proteins that differ in molecular weight by 10 times may differ in size by only 3 times when in globular or folded form. Another problem encountered when using membrane processes for product recovery can be the slow retentate ux, which can result in the formation of a thick secondary membrane. Another possibility is the strong interaction of the sample with the membrane material. This often depends on unspecic protein adsorption which is affected by several factors [201]. Thus, different membrane types have to be screened to minimize the unspecic binding of the sample when a new ltration procedure has to be developed.

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Biotechnology become economically impractical. Conversely, if the column length is increased, then the impedance to ow will become greater leading to high column pressures. If large column radii are employed, then the mechanical strength of the column system will limit the maximum permissible pressure. Consequently, lengthening the column will eventually require the particle diameter to be increased to provide adequate permeability. Increased particle diameter will, in turn, reduce the column efciency, which may impair the resolution of the compounds of interest [204]. Membrane. Until today, the required process steps for the product recovery are carried out separately, which leads to many energyintensive process steps such as ltration or chromatography and production of vast wastewater amounts, respectively. Membrane adsorption allows the integration of several process steps into one-unit operation, which means the downstreaming of biotechnologically produced proteins can be carried out by saving process time and resources [205, 206]. In addition to this, conventional chromatographic techniques which are based on packed columns often require lengthy procedures. This can lead to the degradation of sensitive proteins [207]. Furthermore, packed columns exhibit a high pressure drop across the column and a slow diffusioncontrolled binding process of solutes within the matrix [208]. Whereas membrane systems show several advantages to packed columns, most important, mass transfer takes place through convection rather than through diffusion. Due to this fact, membrane adsorbers enable a timeeffective performance with high ow rates without high back pressure [209]. Membranes can be converted into efcient adsorbers by attaching functional groups to the inner surface of synthetic microporous membranes. Afnity adsorption, ion exchange, or immobilized-metal afnity chromatography can be carried out by these membranes. Membrane ion exchangers of strong acidic (sulfonic acid), strongly basic (quarternary ammonium), weakly acid (carboxylic acid), and weakly basic (diethylamine) types are commercially available. A chelating membrane based on the iminodiacetate (IDA) group is applicable for IMAC. Membrane adsorber technology has several major advantages compared to classical separation methods. Due

7.3. Product Recovery

One of the most important procedures in product recovery are chromatographic processes. During chromatographic steps the sample is separated into several fractions according to interactions between the different molecules in the liquid phase and the stationary phase (chromatographic resin). The molecules can be separated according to their size and/or charge and based on hydrophobic/hydrophilic or afnity interaction with the stationary phase. Column chromatography utilizes a vertical column lled with the solid support with the sample to be separated placed on top of this support. The rest of the column is lled with a solvent (eluent) which, under the inuence of gravity, moves the sample through the column. Differences in rates of movement through the solid medium are translated to different exit times from the bottom of the column for the various elements of the original sample. Based on the specic interactions between the sample in the mobile phase and the stationary phase the column chromatography can be performed as: Ion-exchange chromatography, Size-exclusion chromatography ltration chromatography), Reversed-phase chromatography, and Afnity chromatography. (gel-

Immobilized-metal-ion afnity chromatography (IMAC) as a special form of afnity chromatography is a popular and powerful way to purify proteins. It is based on the specic coordinate covalent binding between histidine or other unique amino acids and various immobilized metal ions (e.g., nickel). Most of the chromatographic systems are run in a batch or quasicontinuous mode. Nowadays, new developments such as simulated moving bed chromatography [202] and anular chromatography [203] offers the possibility of continuous processes. One problem in column chromatography are the difculties in upscaling. If the column radius is increased, unless special packing techniques are employed, the packing procedure becomes inefcient and the packing itself unstable. In addition, to maintain the optimum mobile phase velocity, the ow rate will need to be substantially increased and the consumption of mobile phase will eventually

Biotechnology to the membrane structure, the binding of proteins is not limited by diffusional processes, therefore loading and elution can be performed at very high uxes resulting in very short cycle times. Compressibility of the membrane under normal operation conditions can be neglected, channeling cannot occur, and the pressure distribution inside the modules is designed to have plug ow through the module, all of which lead to sharp breakthrough curves. Scale-up is very easy, materials and systems allow cleaning in place (CIP), and the validation of the process is made easier due to of standard products and validation service of suppliers.

65

throughput, more efcient recovery of analytes, cleaner extracts, economic replacement of halogenated solvents, and a high level of automation, compared to conventional sample preparation procedures [212]. Supercritical uid processes are being commercialized in the polymer, pharmaceutical, specialty lubricants, and nechemicals industries. Supercritical uids are advantageously applied to increase product performance to levels that cannot be achieved by traditional processing techniques.

8. Monitoring and Modeling of Bioprocesses

The requirement to operate biotechnological processes in a cost-effective manner by simultaneously maintaining a high product quality and safety is becoming increasingly relevant. Historically, the most important means of achieving increased productivity from bioprocess plant has been through time- and resource-consuming experimental developments. More recently, however, signicant advances have been made in the area of computer applications for bioprocess supervision, modeling and control, and their introduction in process industry. Typically, little use is made of the large amounts of bioprocess online and laboratory assay data after an experiment is completed. Recently, attempts have been made using enhanced automation strategies to improve the quality of information presented to operators and increase the level of automatic process supervision, although few industrial applications have yet been reported. On a plantwide perspective, the scheduling of process operations is also traditionally manually undertaken by experienced personnel by using a trial and error approach with a planning board. This also appears to be a potential area for modern bioprocess management. Knowledge-based systems, such as fuzzy logic systems, can be designed to cope with uncertainty and allow the coupling of quantitative information with the qualitative or symbolic expressions (in the form of heuristics), so as to reproduce the actions of an experienced process operator. They might therefore be used as an intelligent, online virtual assistant to the process

7.4. Solvent Extraction

Solvent extraction is the most common method for the recovery of hydrophilic substances and, therefore, a method for separating well-soluble metabolites from cultivation media. Classical extraction processes use organic solvents, which are often rarely suitable for effective recovery of the solute. Recently, new extractions have been developed which form specic adducts with the metabolite in question and allow its recovery with high efciency and selectivity [210]. Solvent extraction in biotechnology focuses on the recovery both of primary metabolites (e.g., ethanol, acetic acid, citric acid, and amino acids) and of secondary metabolites (e.g., antibiotics or vitamins). The concentration of secondary metabolites is usually much lower than that of primary metabolites. Since most secondary metabolites are for use as therapeutics, the quality requirements of the products are high. Solvent extraction can help to fulll these requirements. In addition, the set-up of integrated bioprocesses (production and downstreaming) can be performed by solvent extraction [211]. Supercritical uid extraction (SFE) becomes also an important tool in biotechnological downstream processing since it offers important advantages compared with other solvent extraction methods. It is possible to work in an oxygen-free system, which prevents oxidation. The low temperatures applied minimize thermal degradation and microbes or their spores are not soluble. In addition, supercritical uids for extractions are inexpensive. The successful implementation of this technique can lead to improved sample

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Biotechnology be to adopt a numerical estimation technique, either for estimating the parameters of a predened model structure, usually of a generalized linear time series form, or directly to obtain an estimate of a difcult to measure primary process variable (state estimation or software sensor). An alternative approach is to exploit the nonlinear mechanistic structure of the bioprocess, if known, in the form of a state observer. Neural Computing. A relatively new development in articial intelligence is the area of neural computing. Neural networks are dynamic systems composed of highly interconnected layers of simple neuron-like processing elements. In the eld of process engineering, it is their ability to capture process nonlinearities which offers potential benets. Other useful characteristics are their ability to adjust dynamically to environmental and time-variant changes, to infer general rules from specic examples and to recognize invariances from complex, highdimensional data. These properties provide neural networks with the potential to outperform other learning techniques. Given a series of experimental data the network is able to establish the governing relationships in these training data. This ability can be exploited to aid the nonlinear modeling, control, and optimization of complex processes, as well as being used in existing predictive control methods.

operator, or, with extra knowledge as a supervisor in running and maintaining bioprocess operations within optimal operating conditions. The main objective in this approach is to develop an expert supervisory system which uses bioprocess and control knowledge in the form of rules in conjunction with bioreactor state estimation (soft sensing), parametric identication and process control algorithms running in real time. In addition, provision needs to be made for process models and known bioprocess behavioral characteristics to be incorporated in order to enhance the overall supervisory control strategy. The complete system aims to perform process monitoring, process control, sensor validation, fault detection, and diagnostic tasks and, in addition, provide recovery advice so as to achieve continuous online optimization. Collateral with the above approach is the online determination of the critical key variables which govern process production. The measurement and estimation problems are well known and represent the main drawback concerning an effective biosystem optimization. Three ways of reducing the difculties encountered can be proposed: Better sensors Better sampling and automatic analysis systems Online estimation of difcult to measure, or immeasurable, variables. Although all of these are attracting research effort, the techniques of online state and parameter estimation are receiving the greatest interest. Whilst developments in biosensors are in some cases helping to contribute to the online determination of bioprocess parameters, they are by no means at the stage where general applicability has been achieved. Until this is the case, the development of online estimation techniques will provide the major means by which online closed loop feedback control of critical bioprocess variables can be achieved. Several different approaches can be adopted in the development of bioprocess estimation algorithms. The easiest but potentially least accurate way of obtaining an estimate of a difcult to measure primary process variable is to establish a correlation with a measurable secondary process variable, whilst ignoring measurement errors, noise, etc. A more robust approach might

8.1. Characteristics of Bioprocesses

8.1.1. System Denition A biotechnological process consists of a system of chemical, biochemical, and microbiological reactions, incorporating process-engineering aspects such as mass and energy conservation, thermodynamics, or heat and mass transfer correlations. (Fig. 27). The biological part of the system represents the peculiarity of the system characteristics and causes the great difculties in considering it from the system theoretical point of view. Common for all system models is their mapping of inputs to outputs, of stimuli to responses in some with the additional introduction of state variables. Therfore, the task is to dene proper inputs and outputs, dependent on the problem

Biotechnology

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Figure 27. General model structure for biotechnological processes x = external ow and transport rates; c = concentration of the component ()I inlet, ()O outlet; TR = transfer rates; Q = volumetric reaction rate; q = specic reaction rate; Y= yield coefcient; r = intrinsic reaction rate; ()i = index for quantity i; ()j = index for quantity j

available, to use appropriate signals (trajectories) to describe the system behavior in their important aspects and to build up the model. A biotechnological system can be dened as a set of i biochemical reactions involving m in put variables x (t), o output variables y (t), m state variables z (t) and r parameters p notated in operator transformation T():
z (t) =T y (t) =T x (t) , p x (t) , z (t) , p (1) (2)

z (t) = z1 (t) x (t) = x1 (t) y (t) = y1 (t) p = p1 p2

z2 (t) x2 (t) y2 (t) ...

... ... ... pr

T

zn (t) xm (t) yo (t)

T T T

(3)

with

The model does not determine the input vari ables x (t). They represent the systems stimuli and can be divided up into control variables and

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Biotechnology 216]. Mostly, they are used for software sensors [217, 218] or optimization purposes [219, 220]. Both represent black-box applications (see below), where only the outputs are of special interests, the inner relations or interpretation aspects are secondary. As output variables y (t), very often quantities are specied which are accessible by online measuring systems or which can be estimated by software algorithms. They can be seen as the result of a measurement model which relates the state variables z (t) to the output variables y (t). These quantities represent the values with whose the system can be evaluated. The output vector y (t) is strongly related to the aspect of observability (see below). The division into parameters, state variables, or output variables is not an immanent property of a system, but a specic view by the analyzer of the system. It can be changed in relation to the mode of process operation, to the process targets or the simplifying assumptions of the model. A state variable zi can be exchanged to an output variable yi and vice versa. A mathematical system should provide a coherent description of the entire bioprocess including all relevant system aspects. The degree of complexity of the bioprocess or the possibility for a simplied modeling of some parts of the system is determined by the intended application and by the skills of the system analyzer. Parameters p can be seen as xed quantities of the system. In the ideal case, they should be time invariant, as it is indicated by their original denition. However, sometimes a time dependency of parameters is introduced to gain a better system description. Normally, the parameters are used to interpret the biosystem characteristics. However, the meaning of the parameters can be quite different depending on the point of view from which the biosystem is described. In terms of a mechanistic approach using fundamental physicochemical equations, the determined parameters, after they are tted by experimental data, have a direct physical meaning and can be used for interpretation purposes and are comparable between different experiments. If the behavior is characterized phenomenological, by means of descriptive equations, the parameters determined are only valid under the specic boundary conditions of the experiments.

random forcing functions. Control variables are those which are chosen by the operator to manipulate the system in such a way the benets are maximized. Typical examples in bioprocess engineering are: temperature trajectories, agitation rate, or feed rate of key components. Random forcing functions can be seen as outer disturbances of the system whose origin comes from the system environment and not from internal uncertainties, e.g., impurities of the inoculums or the feed medium. To evaluate the system dynamics, it is important to dene meaningful trajectories of the control variables. Keeping the values constant will not result in a representative information retrieval of the biosystem. Only if characteristic curves or specic combinations are chosen to which the system reacts sensitive a representative process description is possible. Therefore, it is evident that the experimental as well as the theoretical design must be performed from deep problem awareness. The state variables z (t) represent storage elements for mass quantities (e.g., substrate, products, biomass, inhibitors, interferences), information, or energy of the system. The state vector z (t) is composed of any set of quantities sufcient to completely describe the behavior of that system. Given a state vector at a particular point in time and a description of the system forcing functions, in an appropriate uncertainty formulation, and control functions from that point in time forward, the state at any other time could be computed. In biotechnological processes state variables are frequently dened, if the key quantity of interest can not measured directly and must therefore determined by state estimators from other process variables [213, 214]. The introduction of state variables is not strictly necessary. But in their absence inner relation can only be hardly understood and described. Also a direct mapping of an input vector to an output vector without consideration of state quantities in form of a signal model, as it is typical in conventional signal analysis in electrical engineering, can not be accomplished. Their application depends on the point of view. A signal model provides mainly information about the behavior of changing inuencing parameters and how they affect the outputs based upon given inputs. No information can be provided about the intrinsic reactions or the basic mechanism, the focus is outside of any interpretation purposes [215,

Biotechnology They can be quite different in different experiments and possess nearly no general validity. One must be very careful when comparing different approaches upon the quantities of these parameters. 8.1.2. System Description A biotechnological system consists in particular of a set of chemical, biochemical, and microbiological reactions whose components, reactions rates, and other characteristics as temperature, pH-value, or internal energy, are mostly not exactly known. The metabolism of the biological unit is a very complicated process, not only because the intracellular reaction network may be quite complex, but also due to the large number of inner relations, control loops on reaction level, regulatory mechanism, and genetic level that are overlaid to coordinate the elementary reactions. So even when the microkinetics could be measured exactly, it would be impossible to establish a correct system map in every detail without the introduction of rigorous simplications. Another reason for uncertainty is the inhomogenity of the units (e.g., cells) in the whole ensemble, which is normally not considered when characterizing biosystems. The relations are not constant and vary with time. Furthermore, there might be a morphological differentiation biological unit which is accompanied generally by changes in the dynamic behavior. Therefore, the interesting question is why these complex systems can often be described for specic process targets by a few mathematical equations only. One reason is that the functional blocks of the biosystem operate together in a network of regulation, and exchange of mass, charge, and energy and a few bottleneck processes determine the behavior of the whole system. Another reason is the tremendous number of units in the ensemble of a biosystem which hides individual variations in their growth and leads to a smoothed average behavior. In most cases, the problem shifts. It is not the difculty to build up the equations, but it is hardly to determine the characterizing parameters, which are generally dependent on time and other system state quantities. Furthermore, a few reactions can already determine the rates of many others. When the main inputs and the nal overall out-

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puts are known, a prediction is often even possible with incomplete information on the involved intracellular reaction network [221 223]. For analysis and design of those systems, two aspects have to be considered the biological reactions catalyzed by the bioactive compound, and the numerous chemical and physical processes which precede, accompany, and follow them. Within the biosystem, the most important physical processes, which are intimately bound to the biological reactions, are associated with transport of material, energy, and information to and from the location of the bioactive unit. The dependency of the biological reactions on the microenvironment around the unit is called microkinetics. Due to the transport phenomena, the microenvironment will vary along different locations in the systems. The integral description of microkinetics in connection with the transport processes that may be included in a system model is called the macrokinetics. Ideally, one would always attempt to model the transport phenomena and the microkinetics of the model, because this model could be combined with proper reactor models to predict the macrokinetics of different biosystems. Unfortunately, at present, the effort for simulation of detailed reactor models is rather high and methods for analyzing the microenvironment of the cells are still rare. Therefore, what is usually observed is always a kind of macrokinetics. To nd a compromise between these facts and our limited knowledge about the process, the description of the biological system is often characterized by means of so-called formalkinetic approaches. This means a formal application of system equations that represent actually microkinetics to a process, where only averaged macrokinetics can be measured. As an immediate consequence, the model parameters will change when the operating conditions of the reactor are changed, or even more, if a different reactor with changing boundary conditions is used. This necessity limits the predictive power of formalkinetic models for biotechnological processes signicantly. In principle, independent of the available system volume, the mass balance of a component i from the general point of view can be described using a nite volume approach solving the governing equations for uid ow and heat and mass transfer:

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Biotechnology
(4)
Hj cp Reaction enthalpy of j-th reaction Averaged specic heat capacity of mass in the considered volume

ci dci +div ci u div ( gradci ) =0 t dt

where the dependent variable is denoted by the concentration ci and u represents the velocity. Both , the diffusion coefcient, and dci /dt, the source term, are specic to a particular meaning of a component i. From this general description, a one-dimensional model can be derived. In the presented approach a dynamic process description is the goal rather than a rigorous threedimensional simulation of a compartmented system. For describing biological systems a coupling with the involved chemical, biochemical, and microbiological reactions is necessary. In the considered (nite) volume, homogeneous (bio-)chemical reactions system can be analyzed according to Gavalas [224]. He assumes a detailed knowledge of the chemical behavior of all the system components. The dynamic of those reaction systems is described by a set of ordinary differential equations, by using the idea that every isolated chemical kinetic system should be consistent with the conservation of atomic species and of internal energy. In principle, this approach can also consider regulatory and closed-loop effects at the component level. A general stoichiometric exact model as a closed homogeneous reaction system of constant (nite) volume can be described as follows.
dci (t) kij j (c1 (t) ,. . .,cN (t) ,T (t)) = dt j =1
M

(5)

where
()i ()j ci () jj () T ( ) kij Component i with i = 1, . . . , N Reaction j with j = 1, . . . , M Concentrations of the different (bio-)chemical components i Reaction rate of the j-th reaction Temperature Exact stoichiometric coefcient from component i in reaction j

If we consider a reaction in a volume with possible in- and outow, the above approach must be extended with the balances of the internal energies
T dT +div T u div ( gradT ) =0 t dt dT (t) 1 = Hj j (c1 (t) ,. . .,cN (t) ,T (t)) dt cp j =1
M

(6)

(7)

where

In this model, all the components involved in the process reactions take part in the mathematical consideration. Furthermore, Gavalas assumes that the j-reactions are independent of each other. The mass conservation and more precisely the conservation of atomic species impose certain relations between the stoichiometric coefcients k ij . In this case, a so-called forward problem is considered. A forward problem is one in which the parameters and starting conditions of the system, and the kinetic or other equations which govern its behavior, are known and the equations can be solved by typical numerical methods. From a mathematical and physical point of view, it can be seen as the calculation of the effect of a cause and not to estimate the cause of an effect, as it is accomplished in an inverse problem. In other words, we usually know how to use mathematics and physical reasoning to describe what would be measured if conditions were well known [225, 226]. Most mathematical models in uid dynamics are of the forward type; the relevant properties of the aquifer or reservoirs are assumed to be known, as well as the initial and boundary conditions. A model then predicts the resultant ow. This is typically the approach used to map a scenario caused by lot of complex interacting partial processes or taken in sensitivity studies, which are quite useful, and can show what the most important features or processes are likely to be for a site [227 229]. Following the ideas of forward problems, structured or cybernetic approaches for the description of biological systems can also be mentioned. They structure the cell mass into several intracellular compounds and functional groups which are connected and regulated via an optimal criterion to each other and to the environment by uxes of material and information. The functional groups may, in one extreme, consist of detailed reaction network considering as many as known reactions and regulatory loops [230, 231]. Mostly, however, in bioprocess engineering when microbiological and biochemical reactions are involved, the exact stoichiometry is unknown. In the engineering literature of the last 20 years a less precise mathematical model for

Biotechnology biotechnological processes is encountered [232, 233]. The so-called general reactor model rstly introduced by Bastin and Dochain [232] tries to unify chemical, biochemical and microbiological process using the same approach. The bioprocess, an n-dimensional system of ordinary nonlinear differential equations, is based on a simple representation of the biotechnological processes, where superuous details are omitted. Only the dynamics of those components that play an important role in the process are considered, irrelevant byproducts or nonlimited substrates are neglected. The stoichiometry of the chemical reactions is only qualitatively taken into account by means of yield coefcient Y ()/c() or stoichiometric coefcients k ij (Eqs. 8 and 9).
di (t) = kij j (1 ( t),. . .,N (t) ,T (t)) dt j =1
M d j dt J =1 Yj /c = dc i i dt M

71

(8)

is proportional to the specic growth rate of the organism , there exist several possible models [234]. This approach is very common in literature (overviews in [235, 236]) and can be assigned in contrast to the above-mentioned approach to the class of inverse problems. It is typical for inverse problems that in many situations the quantities that we wish to determine are different from the ones which we are able to measure. If the measured data depend, in some way, on the quantities we want, then the data at least contain some information about those quantities. Starting with the data that we have measured, the problem of trying to reconstruct the quantities that we really want to know is called an inverse problem; we measure an effect and want to determine the cause. Typical applications of inverse problems are model identication and estimation, image analysis, numerical analysis, or navigation [237, 238]. 8.1.3. Dynamics of Biosystems and Real-Time Considerations Real-time considerations play an important rule dealing with process control strategies. A notorious underestimation of the dynamic properties of microbial and cellular populations exists and results mainly from matching the duration of the respective batch cultivations to the relevant time constant of the biosystem under investigation. However, metabolic regulation of enzyme activities and uxes often takes place on a time scale of seconds rather than days although the latter may also be true for some processes. It is therefore in the scope of promoting biotechnological research to adopt and develop appropriate experimental concepts, methodologies and equipment in order to consider all relevant time constants [239]. What is fast? What is slow? What is relevant? The last question is the most important when dealing with modeling. The relaxation time concept of Harder and Roels [240] (Fig. 28) maps typical time constants of microbial and cellular control on the level of modication of enzymes (activation, inhibition, dis-/association of subunits, covalent modication or digestion) to the range of milliseconds to seconds, on the level of regulation of gene expression (induction, repression, or derepression of transcription) to min-

(9)

where ci () and i () are concentrations of the different (bio-)chemical components, ci () are those substances (i*=1, . . . ,N**) whose reactions are calculated by the yield coefcients Y ()/c() , i are those components (j=1, . . ., N*) whose turnover are determined by the dominating reaction rates j .
M J () T ( ) Number of the dominating reactions in the considered volume Reaction rate of the j-th reaction Temperature

In general, these coefcients do not have any mechanistic meaning in contrast to the abovementioned approach, instead they represent formalkinetic quantities, which must be estimated, determined experimentally, or established by practical experiences. This allows the inclusion of chemical, biochemical, and microbiological processes in a unied approach. However, the reaction scheme may be inconsistent with the law of conservation of mass. Also cell-to-cell heterogeneity, e.g., due to different proliferation phases, are not considered and simplies a segregated viewpoint to an unsegregated perspective. The reactions rates can be often a very complex function of the operating conditions and of the state of the biosystem. In the case were

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Biotechnology

Figure 28. Concept of relevant relaxation times and time constants (according to [240]). Note that the time scale is logarithmic

utes, on the level of population selection and evolution to days and larger units. Considering mainly the growth of microorganisms, typical time scales of 0.3 to 20 h are relevant. The examples discussed below will illustrate how bioengineering is facing the individual time constants. A typical bioprocess, if operated in batch mode, extends over several hours or a few days. If operated in continuous mode, it is not reasonable to accept operating times of less than a month (see, e.g., Heijnen et al. [241]). If an organism has special physiological features such as bakers yeast, a transition from one to the other domain (e.g., from low to high dilution rates or, in other words, from purely oxidative to oxidoreductive growth) may also result in considerable changes in the time required to approach a new steady state. Axelsson et al. [242] reported a rough estimate for this case: the time constant for experiments at low dilution rates (D<DR, where DR=D at which the regulatory switch between oxidative and oxidoreductive metabolism occurs) is, as expected, in the order of the mean residence time ( =D1) however, above DR, the time constant was predicted to be at least one order of magnitude greater. In experiments specically designed to verify this, either even greater time constants or no unique stable steady states at all were found [243]. If excess carbon and energy source is pulsed to a carbon- and energy-limited culture, the intracellular ATP concentration initially drops because of the phosphorylation sink (glucok-

inase and/or hexokinases). Only later, during catabolism, can the energy provided by this extra substrate be liberated in terms of new ATP. The duration of the ATP sink was predicted to be in the order of a few tens of seconds by Nielsen et al. [244]. Obviously, such rapid reactions are under kinetic control and the necessary enzyme activities are present in sufcient quantities. It is not clear, at present, whether the uxes attain their organism typical maximal values immediately or some ne tuning of enzymatic control precedes this event. It is likely that in the latter case a regulation of (intracellular) enzyme activity, if at all necessary, takes place on the level of enzyme modication (e.g., phosphorylation) rather than on the level of de novo production (i.e., control on the transcriptional level) because the latter would require much more time [245]. The dynamics of microbial cultures have an important impact on the characteristics of measurement and process control. The typical time constant in a bioprocess is often erroneously anticipated to be equivalent to the entire duration of cultivation. However, the quantitative investigation of substrate uptake requires a time resolution of a few 100 ms, otherwise artifacts must result [246, 247]. This statement was evidenced only after a suitable technique had been established: glucose metabolism was stopped within 100 ms by spraying the cell suspension from the overpressurized bioreactor into 60 % methanol which was pre-chilled to 40 C.

Biotechnology The relevant relaxation times of a culture system are determined by the actual cell density and the specic conversion rate (capacity) of the culture, that is, by one or more operational and state variables (for instance feed rate, the concentrations or activities of cell mass and of effectors, if relevant) and inherent characteristic properties of the biosystem which are represented by parameters. There are metabolites with a long lifetime and other (key) metabolites with very short lifetimes, e.g., molecules representing the energy currency of cells such as ATP and other nucleotides. Rizzi et al. [248] and Theobald et al. [249] have shown that the energy charge response to a pulse challenge (ATP) of a yeast culture is a matter of a few seconds only. However, the responses can differ considerably when pulses or shifts are applied to cultures of different recent history [245, 250]. Neubauer et al. [251] found that substrate oscillations greatly affected the growth performance of E. coli. Short term, that is, less than 2 min, glucose excess, and starvation were investigated in a looped system of a stirred tank and a plug ow reactor. The metabolism changed within some few minutes totally from anaerobic to aerobic and vice versa. Similar observations have also been made for yeast [252]. Two other paradigms demonstrate that the band width of relaxation times is extremely broad: 1) the time required to achieve a new steady state in a chemostat culture is approximately determined by (max D)1 . 2) The time required by a culture to consume a considerable fraction of small amounts of residual substrate during sampling and thus systematically falsify the analytical results if no appropriate inactivation takes place depends, among others, on the cell density and can also be in the order of a few seconds only. Even simple static models are very valuable for compensation of systematic errors built into automated analytical procedures. One important example is the case when sampling requires a well-known but non-negligible time. A bypass behaves as a plug ow type reactor fraction where ow dependent spatial gradients develop and where no inactivation can take place because the bulk of the bypassed aliquots are returned to the reactor. The cells continue to consume substrate while they are being transported from the exit of the reactor to the lter. The permeate

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recovered is representative for the lter site but not for the reactor. Knowing the transport time and some basic kinetic parameters one can easily compensate online for such errors provided that a useful estimate of the actual biomass concentration is available. Even though a bypass can be tuned to operate at a mean residence time of 5 s or less, this can be enough for a signicant decrease in substrate concentration in highdensity cultures. Sample buses need a minimal (dead) tune for transportation of the sample and in situ lters tend to fail in high density cultures because of rapid fouling. Hence, the problem is real and can not be ignored.

8.2. Biotechnological Measurement Systems

All measurements have the ultimate goal of creating representative information from the biosystem involved. Cellular or biochemical quantities are the primary variables in bioprocess engineering. They mainly determine the performance of the bioprocess and are therefore of special interest [253, 254]. Measurement and acquisition of information are building the fundament for all process observations as well as for the development of new bioprocesses. They represent the presupposition for process optimization and control. However, they must be available online to exploit them in a control strategy. Measurements are not only key issues in modern process development, they also open the door to a detailed process supervision and understanding, and avoid restricted views when analyzing processes just through a keyhole [255, 256]. In comparison to other disciplines such as physics, mechanical or electrical engineering, sensors useful for online monitoring of biotechnological processes are comparatively few; they are mostly available for physical and chemical quantities rather than for biological ones. The reasons are manifold, but generally biologically relevant information is much more difcult and complex to access and interpret than those for typical physical or chemical quantities [257]. Other important reason derives from restricting requirements for the sensor equipment, namely Sterilization procedures, Stability and reliability over extended periods,

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Biotechnology by other measured values. But this implies thorough problem awareness and is restricted to specic boundary conditions (see Section 8.2.2).

Application over an extended dynamic range, No interferences with the sterile barrier, Insensitivity to protein adsorption, surface growth, Resistance to degradation or enzymatic break down, and No disturbances from matrix compounds (interferences, matrix effect). These aspects are in an industrial environment covered by the fact that the operation of the analyzer and its service must be as simple as possible. The aim of applying such a measurement system is to get more information but not to increase the chances of malfunctions of the whole process. Due to the complex nature of many process analyzers this requirement can only be met in some exceptional cases. Finally, material problems can arise from the constraints dictated by sterile process conditions or by biocompatibility, which often make the construction of the sensor hardware rather difcult. 8.2.1. Process Requirements Concerning Measuring Quantities In modern bioprocess engineering there are undoubtedly only a few variables that are generally regarded as essential. Among these are several physical (i.e., temperature), less chemical (pHvalue) and even less biological quantities (cell concentration). Figure 29 gives a summary of what is nowadays believed to be a minimum set of required measurements in a bioprocess. Such equipment is typical for standard production of biomaterial [258]. However, the conclusion that these variables are sufcient to characterize the microenvironment and activity of the biocomponents, of course, is more than questionable. For the consideration and compliance of the process targets, as described above, besides some environmental and operational quantities, in particular the important biochemical state variables of the biosystems must be known, namely the amounts and composition of the active biomass, of the starting material, of the products (and byproducts) or other metabolites. Modern bioprocess observation systems try to overcome the lack in the instrumentations, by building up functional relationships between these quantities. The approach is to substitute the direct measurement by a function based value, calculated

Figure 29. Common measurement instruments and control units of bioreactors as generally accepted as routine equipment ( measurement only, measurement and open- or closed-loop control)

Biomaterial. The active biomaterial is of paramount importance to scientists as well as to engineers. It is an indicator for the available quantity of biocatalysts. It is denitely an important key variable, because it determines simplifying- the rates of growth and that for biotransformation. Almost all process descriptions and optimization tools contain the quantity of biomass as the most important state variable. The biomaterial of interest depends on the system considered: microorganism, enzymes, nucleotides, or simple protein amount and constitution. Many control strategies involve the objective of optimizing the biomaterial concentration. For automation purposes, the biomass is mostly seen as a homogeneous entity, being aware that this represents an uncertainty because segregation into different individuals is more realistic. An ideal measurement of the bioactive compound would include their activity, physiological state, morphology, or other classications rather than just their mass. But these quantities are normally ill-dened and, in general, inaccessible to online measuring devices.

Biotechnology A series of sensors and methods, that can be automated and have appeared in the recent decades for the measurement of the total biomass without discrimination of any segregation (e.g., active or inactive cells). Many of them rely on optical measuring principles [259, 260], others exploit ltration characteristics [261, 262], electrical properties of suspended cells [263, 264], or thermodynamic laws [265]. One has to decide according to the specic application, which method seems to be appropriate concerning the boundary conditions, because all methods reply on indirect measuring principles, and the access of the information carrying signal differs in non general ranges. To get online information about the actual state of the biomaterial techniques based on uorescence principle, especially the 2-D uorescence spectroscopy, should be mentioned. It represents a noninvasive method to analyze intracellular compounds. Technically, either intra- or extracellular uorophores (e.g., NAD+ , Riboavin, Tyrosine, ATP) are excited by visible or ultraviolet light, and the uorescent light emitted by the uorophores at a longer characteristic wavelength is collected and gives information about their concentration [266 268]. Substrate. The presence of sufcient and appropriate starting material (substrate) is the cause of any biotransformation and represents the supposition of the product formation. One can solve the inverse problem, namely conclude that biological activities cease whenever an essential substrate is exhausted, and thus omit the measurement of the substrate, provided the progress of the bioprocess and/or product formation is known [269]. But, this is not always a proper solution because there are many more plausible, and also probable, reasons for a decrease in bioactivities than just their limitation by depletion of a substrate. One must, then, solve the direct problem, namely analyze the relevant quantities of the biological unit. From an engineering point of view this measurement should be available instantaneously in order to be able to control the desired process characteristics. In environmental biotechnology, in particular, the objective of a bioprocess can be to remove a starting material as completely as possible rather than making a valuable product. So, the starting material is identical to the process product. In some

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other bio- or food technological processes, the conversion of a substrate from specic state of aggregation to another is of special interest. For example, in the case of fouling of heat exchangers where dissolved proteins are transformed to a solid protein layer, the starting material changes only its state not its chemical structure. Or, considering drying or thawing where water changes its state (or is removed) which strongly inuence the quality and behavior of the biomaterial involved, but no (bio-) chemical conversion takes place. The classical methods to determine these quantities are ofine laboratory methods so far. This implies that samples are taken, sometimes aseptically, pre-treated, and transported to a suitable location, where storage of these samples might be necessary before analyzing manually. These steps need a lot of human and instrumental resources. Product. The product is almost the only reason why a process should run. The main concern is in maximizing the prot, which depends directly on the volumetric productivity and/or on the purity of the resulting product. It is therefore of essential interest to know the quantities, which require measurements. What was said above about substrate determination and the application of classical methods is valid here, too. In summary, bioprocess science needs signicantly more quantitative measurements. It is insufcient to know that something happens, we need to know almost instantaneously, online and automatically why, how, and to what extent a bioprocess behaves [270]. 8.2.2. Online Sensing Devices The development of increasingly sophisticated equipments for measuring of starting material and products represents one trend to satisfy the growing demand for high-quality measurements. In contrast to engineering disciplines such as mechanical or electrical engineering, in bioprocess engineering almost no measurement performed is directly, i.e., none is obtained by immediate comparison with a reference quantity. Most measurements are achieved by means of some specic physical or chemical property of the process value, often hidden in the very complex sample matrix com-

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Biotechnology a direct interaction between a product property and sensor exist, otherwise exline; this is more a technological characterization. Depending on the site of installation, one discriminates further between in situ, which means built-in, and ex situ, synonym for a bypass conguration or for an exit line. In the latter case, the withdrawn volumes are lost for the process. A further classication can be made by the mode of operation of the sensing device. One can discriminate between continuous and discontinuous signal generation in a predened time step. This is a very important fact for planning and developing an appropriate process controller considering the time scales of the bioprocess. In the latter casem, one has to guarantee that the process information is available in real time. Only online sensors are of special interest concerning the process control of biotechnological systems. Indeed there exist a couple of different, very powerful systems for the ofine analysis of nearly all relevant substances. For online sensors, the situation is quite different. In situ instruments exist only for a few quantities like the measurement of temperature, pH, pressure, oxygen, carbon dioxide, density, ow rates, electric power consumption, or redox potential. They use different measuring principles and are widely introduced in industrial process engineering. However, for the most important biological quantities in biotechnological systems especially the measurement of active or inactive biomass or the specic determination of individual biochemical substances there exists a deep lack in online instruments. In the following, principal techniques should be presented, which possess a certain online capability. See also Biochemical Separations; Mass Spectrometry; Surface and Thin-Film Analysis. Chromatographic Methods. A review of chromatographic methods is beyond the scope of this contribution. Both (high-pressure) liquid chromatography and gas chromatography have been applied in numerous cases to ofine analyses of biotechnological samples, but online applications, especially for industrial processes, have only recently been reported [278 282]. The scope of chromatographic methods is the separation of the individual constituents of mixtures as they pass through columns lled with a

mon to bioprocesses. This very complex matrix, which consists of the numerous substances and parameters surrounding the analyte and their mutual inuences, makes the task of measuring difcult and leads to the development of more and more complicated measurement techniques [271, 272]. The surrounding equipment, i.e., for the sample pretreatment, exceeds the pure analytical core [273 275]. However, the greater the measuring system complexity, the more difcult it is to ensure the accuracy and the reliability of the collected data. The need for accuracy and reliability increases even more if the data of the process analyzer should be used as input for a closed-loop controller. When unreliable measurement systems are used, the system operator can only hardly decide whether an occurring variation is due to an error of the analyzer or a result from the bioprocess itself. The nal goal of the measuring unit of a bioprocess is to build up simple online instruments in order to collect all necessary information from the biosystem, which is necessary to hold the dened process targets. Therefore, the process targets should represent the selection criterion for dening the measuring equipments. The information should be available online and no manual interaction should be necessary to obtain the desired measuring results. This aspect touches the term system observability (see Section 8.4.1), which is strictly dened for linear systems, but eludes a denition for nonlinear systems as they are common for typical biological systems [276, 277]. It is not obvious and denable whether all system information is available und known to reach the observability in respect to the process goals or which sensors are still necessary to reach the specied process goals. The consequence is that there are normally one or more state variables (or linear combinations of them) that are hidden from the view of the observer (i.e., measurement equipment). Concerning the information treatment there can generally be made different classications, which are important for industrial applications. From the communications engineering point of view the information can be distinguished between online and ofine. If a continuous automatic correlation between the signal from the sensor and the product or process quantity is possible an online signal is available, otherwise it is ofine. A measurement is called inline, when

Biotechnology suitable stationary phases. As disadvantages the consumption of almost expensive reagents must be mentioned. The samples injected are to be pretreated in such a way, that they possess nearly no impurities, like gas bubbles or solid compartments. Any existence of those interferences disturbs the analysis in a signicant manner, and manual service procedures are mostly the consequence. Furthermore the measuring time last up to 30 or 40 min; a time period which is often too large for bioprocess supervision. But also economical aspects must be taken into consideration. Chromatographic systems possess a high cost price. Furthermore, high-specialized staff must service them frequently, an aspect, which represents the major drawback for an industrial application. Mass spectrometry. A further measurement method, in principal suitable for online applications represents mass spectrometry (MS). Mass spectrometry has been mainly applied for the online detection and quantication of gases such as pO2 , pCO2 , pN2 , pH2 , pCH4 , and even H2 S, or volatile substances such as alcohols, acetoin and butandiol [283]. Generally, the detection principle allows simultaneous monitoring and, consequently, control of metabolites. The principles, sampling systems, control of the measuring device and application of MS for bioprocesses have been summarized elsewhere [284]. Of course, mass spectrometry requires very expensive equipment and needs frequent and complex service procedures. But it should be taken into account that automatic multiplexing of different sample streams is possible and, in addition, a great variety of different substances can be determined simultaneously. Thus, one has to decide, whether the high economical and applicational expense is justiable in comparison to the advantages due to the acquisition of information. Biosensors. The most promising technique to overcome these problems, represented in the last decade, is the use of biosensors . Biosensors consist of a sensing biological module of either catalytic (e.g., enzymes or microorganism) or afnity reaction type (antibodies, cell receptors) in intimate contact with a physical transducer (electrochemical, optic, calorimetric or acoustic sensor). The latter nally converts the (bio-)

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chemical into an electrical signal (overviews in [285 287]). In spite the fact, that principally for nearly all biochemical substances an biological recognition unit exists, biosensor applications in an industrial surrounding often concentrate only on a small group of substrates, namely glucose, ethanol, or lactate [288 292]. Generally, biosensors are tricky to handle and must in principle be recalibrated in the matrix where the measurement should take place every time the process conditions change. Due to the sensible biological element an enzyme or living microorganism they principally cannot be sterilized. They also suffer from changes in the environment, for example, changes in pH or formation or existence of aggressive chemicals such as H2 O2 [293]. Therefore, they must be used in a suitable environment. Furthermore, the surrounding must be constant, because in general an interrelation to the endogenous matrix exists. If the process media changes signicantly, the biosensor alters its behavior. A compensation of the changed matrix interferences becomes necessary. This represents one of the most decisive drawbacks in inline biosensor applications for bioprocess engineering [294 296]. The longterm stability under working conditions is often poor; they must be serviced for several hours per week preferentially by high-specialized staff. In this way, only limited experience could have been gained under technical process monitoring conditions. They possess only in some exceptional cases a real industrial potential. A reasonable way around the problem represents the measuring of the biotechnological media after removing a sample from the reactor in a bypass conguration. Consequently, the sensor is not installed in situ as it would be optimal but the information could be provided online and can be fed into an automatic process control system. Depending on the analyte of interest, i.e., whether it is soluble or (in) the dispersed phase, one needs to sample either the entire culture liquid or just a supernatant. The latter can be acquired using for example a membrane module. Concerning sampling, four aspects must be taken into account. 1) The system must be opened in such a way, that no infections can enter the reaction space either during sampling or between the sampling events.

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Biotechnology a high sampling frequency (up to >100 h1 ), small sample and reagent consumption, high reproducibility and nearly total versatility of the sensing methods. Software Sensors. The most promising method to get online access to important key components of bioprocess quantities are software sensors. In general, software sensors supply the estimation of the missing measurements by using an appropriate model that relates the corresponding variable with other physical or chemical measurements that are correlated to it in any way. The fact that the model is implemented by means of a software package (hence the expression soft(ware)-sensor) denotes, that software sensors provide a software backup for unavailable sensors, as an alternative to a hardware back-up using spare sensors [299 302]. Generally, software sensors are typical solutions of so-called inverse problems (see Section 8.1.2). In a complex biological system, in particular, the quantities which are normally easiest to measure are the variables, not the parameters. In the case of metabolism, the usual parameters of interest are the enzymatic rate and afnity constants which are difcult to measure accurately in vitro and virtually impossible in vivo [303 306]. Yet to describe, understand, and simulate the system of interest we need knowledge of the parameters. In other words, one must go backwards from variables such as uxes and metabolite concentrations, which are relatively easy to measure, to the parameters. In other words, when using software sensors there must always be a model available that reliably relates the measured variable with the target variable or parameter of interest. Normally, measured variables are easily measurable effects that are caused and inuenced by the target. It is the special objective of the software sensor to reach a maximal degree of generalization. However, this is very difcult even impossible to achieve or furthermore to prove this claim. Thus, the basic question is: Is the available information representative enough to generate a model for the accurate estimation of the quantity of interest, or is the desired information inaccessible by the chosen sensor collection? Consequently, the development of software sensors is often restricted to a specic application; any transfer to

2) A representative sample must be provided. So the overall volume of the sampling chamber as well as the position of device at the bioreactor must be determined properly. Additionally, the sample taken can still continue to react on the way to the sensor. In this case the sample would not be representative for the interior of the reactor, and appropriate additional measures or modeling approaches need to be taken in order to assure representativity. 3) When native samples are applied to the analytical system, problems can arise from matrix interferences and matrix effects [297]. Therefore, the sampling device should be chosen in that way that no interaction with the matrix occur which interferes the measurement (e.g., absorption of substrate at the membrane). On the other side, the sample device should withhold all the cross-sensitivities for disturbing co-components. 4) Due to the high number of individual system components and their complex interaction (i.e., sample pretreatment and analytical part), sampling devices provoke a small reliability in regard to the required process time. Furthermore, the hardware, necessary for the whole analysis system entails high original costs. The specic adaptation to one concrete process condition implies inexibility. It excludes the determination of other metabolites in other process media. Its adaptation is only possible by time- and money-consuming alterations. Flow Injection Analysis. The most important technique of sampling methods in biotechnology, behind some conventional devices like membrane modules, represents the ow injection analysis (FIA), rstly introduced by Ruzicka and Hansen [298]. It can be viewed as a general solution-handling technique or sampling device combined with a sensing unit. This combination causes a high exibility with respect to the combined analytical procedure. It can be seen as a principle where a small injected volume (10-200 L) is introduced into a continuous unsegmented stream of carrier. The sample disperses in a well-dened concentration gradient and is transported by the carrier stream to the reaction zone and a subsequent detector module. FIA cannot generate continuous signals. But, there are several important advantages like

Biotechnology other measuring problems is almost combined with considerable alterations. This does not concern only the determination of the new parameter at an identical model structure. Instead a complete new system denition can be necessary, where new input variables must be added or dispensable one can be deleted. Spectroscopic Methods. In this context the use of spectroscopic methods [307 310], after optical or sonic stimulation play a dominant rule. They must be seen as multisensor systems. Their common property is the detection of absorption degrees after stimulation at various frequencies. By this, a set of information is generated, which possesses more information than the measurement at one specic frequency, the measurement at one frequency can be seen as the output of one sensor. The multivariate evaluation in an appropriate model should subsequently reveal the accurate determination of the desired quantity. In such models, normally pure data are used, the integration of knowledge from well-known fundamental equations is only very hardly to realize. The most important advantage of spectroscopic methods is their non-invasive character. The sensing device is not in direct contact with the media or the process itself, so no interference is to be expected. The situation is even more complicated since things develop in relation to time. The estimation of the physiological state of a culture involves more than one (measurable) variable at a time and the recent history of this set of individual signal trajectories involved. Consequently, physiological state estimation requires recognition of complex patterns. Various algorithms used for this purpose to build up the (data-)model have in common that it is not always the present values alone that are evaluated, there is always the recent history of signal trajectories involved. Concerning the models, some authors dene software sensors only on the basis of neural networks but a broader point of view should be adopted since software-sensor models are also obtained by using regression or correlation techniques as well as fuzzy logic or rst-principle models or combinations of all of them [306, 311 314]. In some cases, the data describing the actual state and their recent history are compared with so-called reference patterns: these are data from

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historical experiments or runs which an expert has associated with a typical physiological state. A physiological state is recognized either if the actual constellation matches any one of the reference sets best in this case, there is always an identication made or if the match exceeds a predened degree of certainty, e.g., 60 % then it can happen that no identication or association is possible when the pre-selected threshold value was not reached. The direct association with reference data needs normalization (amplitude scaling) and, probably, frequency analysis in order to eliminate dependencies on (time) shifts, biases or drifts. In other cases, the data trajectories are translated into trend qualities via shape descriptors. These combinations of trends of the trajectories of various state variables or derived variables dene a certain system state; the advantage of this denition is that the association is no longer dependent on time and on the actual numerical values of variables and rates [315]. 8.2.3. Further Aspects Concerning Measuring Systems Online measurements produced with in situ sensors are difcult to validate. The usual procedure for evaluating the quality of a measurement is restricted to calibration/checking prior to and after an experimental run. A few sensors such as the pCO2 - or the Craneld-glucose sensor [316] allow removal and, therefore, recalibration of the transducer during a run. External chromatographs and FIA systems can be regularly recalibrated but the sterile interface cannot. Other sensors such as a pH or a pO2 probe can be mounted via a retractable housing, which allows either sterile exchange or withdrawal for external recalibration during a run. A further possibility to gain information about the reliability of a measurement is to mount a number of identical sensors in easy-to-compare positions and to check the individual signals for equality. This opportunity has been exploited in particular at applications where a high process safety must be guarantied. It is highly desirable to have alternative principles of measurement at hand, which operate simultaneously (heterogeneous redundancy) or to introduce a self-supervision op-

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Biotechnology of the methods, tools, and equipment currently available and to invent new and better ones. In essence, techniques of instrumentation, operation, and causal analytical interpretation of measurements need massive impulses.

tion, like pattern recognition to generally identify malfunctioning sensors [276]. Elemental balancing permits the determination of (other) metabolic rates provided that the stoichiometry is known. Carbon balances are the most useful but the carbon lost via the exhaust gas (as CO2 ) and culture liquid (as HCO 3 ) must be measured. Heinzle et al. [317] determined that the state predictions based on experiments with a small mass spectrometer are not useful due to unacceptable error propagation; for instance, a 1 % relative offset calibration error could result in a prediction error for an intracellular storage material (PHB) of >50 %. Instead, using a highly accurate and precise instrument (absolute errors <0.02 % gas composition) together with automatic, repetitive recalibration resulted, however, in reasonable estimation of substrates, PHB, and biomass. Others have also experienced these ndings, e.g., [318, 319]. The undelayed evaluation of the state of a culture by using software sensors and computers, based on the quantitative analytical information provided by hardware sensors and intelligent analytical subsystems, constitutes an excellent basis for targeted process control. Experts either human or computer have the data and the deterministic knowledge to trace observed behavior back to the physical, chemical, and physiological roots thereby gaining a qualitative improvement of bioprocess control, a quantum leap: process control can act on the causes of effects rather than just cure symptoms. A simple standard operating procedure [320] has proven useful in this concern, namely: Measure everything that can be measured at the very beginning of process development Decide whether or not a variable is relevant Determine the relevant variables to be measured, controlled, and/or documented Collect all raw data at any time and distinguish online between variable and parameter behavior Organize an archive of all these data accordingly and do not discard seemingly useless data since they contribute to the treasure of experience If it is, as some people say, correct that todays bioengineering with all its tools and methodologies is too slow and not efcient enough, then it is all the more urgent to improve the performance

8.3. Cognitive Computing

Articial neural network s (ANNs) are dating back to 1943, rstly reported by McCulloch and Pitts [321], then fallen into oblivion, but started a tremendous comeback in engineering science with the publications of Rumelhart in 1985, who introduced again the backpropagation algorithm and demonstrated its potential as a learning procedure in neural networks [322, 323]. Fuzzy Logic has been introduced by Zadeh 1965, rstly ignored consequently by technicians because of their undeterministic and uncertain nature, but provoked a second wave of interest in the 1980s predominantly in Japan in respect to their potential in treating highly complex, nonlinear multiple input multiple output (MIMO) systems in technical applications [324]. John H. Holland published 1975 his classic work Adaption in Natural and Articial Systems; it can be seen as the pioneer work in evolutionary programming [325]. Compared to other mathematical principles, these three methods are fairly new approaches and did not gain not exclusive acceptance by scientists and engineers. The three methods mentioned above can be seen as the main representatives of a class of problem-solving methods taking nature as a model, especially how it has learned to solve problems. They can be summarized to the area of cognitive computing. It is not within scope of this contribution to explain these methods in their basic principles and functionalities. Rather, their intrinsic principles are assumed to be well known; overviews and extensive explanations can be found for fuzzy in [326 328], for ANN in [328 330] (see also Process Control Engineering, Chap. 13) and for evolutionary programming in [331, 332]. In the following, the focus lies on two methods, on fuzzy logic system and on ANN, because of their high applicational potential, especially in bioprocess engineering [333, 334]. The explanations should show their potential, typical aspects and also some critical

Biotechnology remarks, in particular in respect to bioprocess engineering applications. Neural networks and fuzzy theory have been underground technologies for many years. They have had far more critics than supporters for most of their brief history. However, in particular due to their application potentials in different engineering areas where classical approaches failed, their acceptance is growing steadily. Neural networks and fuzzy systems estimate functions from sample data, they map inputs to outputs. Statistical and articial intelligence approaches also estimate functions. On the other hand, for each problem statistical approaches require knowledge how outputs functionally depend on inputs, in other words they need a mathematical model. Thus, their fundamental ideas can be more compared to typical conventional mathematical principles. Instead, neural and fuzzy systems do not require such a mathematical model. They can be seen as model-free estimators [328]. From another point of view, articial intelligence expert systems can also be seen as modelfree estimators, they map conditions to actions. However, experts do not articulate a mathematical transfer function from the condition space to the action space and the AI framework is symbolic. Symbolic processing favors a prepositional and predicate calculus approach to machine intelligence. It does not favor numerical mathematical analysis or hardware implementation. In particular, symbols do not have derivatives; only sufciently smooth functions have derivatives. Symbolic systems may change with time, but they are not properly dynamical systems, nor systems of rst-order difference or differential equations. Therefore, they are unsuitable for modeling purposes in bioprocess engineering. A small excurse to neural (pre-) attentive processing in human intelligence should illustrate which ideal result a cognitive system provides after its learning phase. Take a look at the Kanizsa-square illusion (Fig. 30). What do we see when we look at the Kanizsa square [335]. We see a square with bright interior. We see illusory boundaries, or do we? We recognize a bright square. Indeed, technically we cannot see it, because it is not there. The only things which are shown are four three-quarter circles, nothing more. The square exists only

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in our brain, not out there in physical reality on this page. Immanuel Kant called these four ink stains 1783 noumena things in themselves. Applied to technical problems, this means that we recognize information or generalize artifacts in that way, that we see not only the inputoutput mapping, but the real relations or laws the data stands for. The data can be seen as facilities or indications for the perception of the underlying intrinsic information.

Figure 30. Kanizsa-square illusion

Today, many of the neural mechanisms that

Kant could only guess are understood. We take

for granted our high-speed, distributed, nonlinear, massively parallel pre-attentive processing. In our visual processing, we pay no attention to how we segment images, enhance contrasts, or discount background luminosity, even if they are the carrier of the real information. When we process sound, we pay no attention to how our cochleas lter out high-frequency signals even if we collect the data. We likewise ignore our real-time pre-attentive processing. We experience these pre-attentive phenomena, but we ignore them and cannot control or completely explain them. Natural selection has ensured only that we perform them, ceaselessly and fast. But what we really do, we recognize segmented image pieces and parsed speech units; we try to extract and introspect the intrinsic carried information based upon measured quantities.

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Biotechnology ations, new patterns, new functional dependencies. They sample ux of experiences and encode new information. They compress the sampled ux into a small but statistically representative set of prototypes or exemplars. Sample data, provided directly or transformed into linguistic rules, changes the system structure or parameters. In all cases, learning means changing. Neural networks learn patterns, functions, or probability distributions to recognize future patterns, lter future input streams of data, or solve future combinatorial optimization problems. Fuzzy systems can learn or formulate associative rules to estimate functions or control systems, either directly by means of data and an optimization add-on or guided by a highly experienced supervisor. It can be shown under some assumption that a neural net can approximate to any degree of accuracy using a fuzzy expert system and vice versa [341]. Neural and fuzzy systems ultimatively learn or estimate some unknown probability function p(x ). The probability density function p(x ) describes the distribution of vector patterns x, a few of which the neural or fuzzy system samples. When a neural or fuzzy system estimates a relation r : X Y, it in effect estimates the joint probability densities p(x,y). Then the solution points (x,r (y)) should reside in high-probability regions of the inputoutput product space X Y. We do not need to learn if we know p(x,y). We could proceed directly to our computational task with the techniques from numerical analysis, combinatorial optimization, calculus of variations, or other mathematical discipline. The need and the extent to learn varies inversely with the quantity of information available. Supervised learning uses or creates classmembership functions. It compares every input with the corresponding class D and proceeds learning based upon the difference. Unsupervised learning system processes each sample x but does not know that x belongs or not to class D. Neither supervised nor unsupervised learning systems assume knowledge of the underlying probability density function p(x ). 8.3.1. Fuzzy Logic Systems Is uncertainty the same as randomness? Bayesian statistician Dennis Lindley [342] stated

Neural network studies both pre-attentive and attentive processing of stimuli. This leaves unaddressed the higher cognitive functions involved in reasoning, decision making, planning, or control. The nonlinear neurons and synapses in our brain perform these functions; they can deal with ill-posed problems or with a high degree of uncertainty. Natural selection evolved this capability in exemplary manner. Furthermore, it is the attempt of engineers to copy exactly this capability to technical problems by developing and using fuzzy logic systems or articial neural networks. Both methods try to imitate nature and both estimate inputoutput functions, in spite of totally different underlying principles. In contrast to statistical estimators, they estimate a function without a mathematical model of how outputs depend on inputs; this represents their main feature. They learn from experience with numerical, sometimes, linguistic sample data. Learning can be accomplished either by changing the system structure or by changing the system parameter; both principles are described for both algorithms [336 339]. Thus, the principle modus operandi is comparable. After the system problem is dened, the procedure can be divided into learning phase and prediction phase. The learning phase of ANN is quite obvious. Input data and corresponding output data are provided to the ANN; the system changes its structure or parameter, to minimize a predened cost function. At rst sight, the adaptive and learning character of fuzzy systems is not obvious. Indeed, there exist some techniques where the fuzzy system is built up autonomously based upon available data sets [339, 340]. On the other hand, in most heuristic applications a highly experienced technician does the learning step. He collects data, recognizes the intrinsic information, and formulates upon this information rules upon sets. Thus, the modeler performs the learning step and its result is introduced into the fuzzy system. The most important features intelligent systems may show are generalization and learning. All intelligent systems should generalize. Their behavioral repertoires exceed their learned repertoire in that way, that intelligent systems associate similar responses with similar stimuli, small input changes produce small output changes. Furthermore, intelligent systems should learn or adapt. They learn new associ-

Biotechnology that probability is the only sensible description of uncertainty and is adequate for all problems involving uncertainty. Lindley directs his challenge in large part at fuzzy theory, the theory that all things admit degrees, but admit them deterministically. Although, both expressions are used simultaneously in the same context, probability and fuzziness differ conceptually and theoretically. In this contribution some important differences are illustrated, more theoretical aspects and proves can be found elsewhere [343]. Fuzzy sets can be more compared with possibilities than probability. However, probability and fuzziness also share many similarities. Both systems describe uncertainty with numbers in the unit interval [0,1], consequently describe it numerically. Both systems combine sets and propositions associatively, commutatively, and distributively. The key distinction concerns how the systems jointly treat a set A and its opposite Ac . Classical set theory demands AAc , and probability theory conforms: P(AA)=0. So AAc represents a probabilistically impossible event. But fuzziness begins when AAc =0, hence P(AA)=0. Fuzziness measures the degree to which an event occurs not whether it occurs. Probability describes the uncertainty of event occurrence. Whether an event occurs is random; to what degree it occurs is fuzzy. Consider an illustrating example. The probability this book will be published is one thing. The degree to which it will be published is another. From this book, only some special chapters may be edited or the whole entity gets public; this is typical for fuzziness. Fuzziness is a type of deterministic uncertainty; it handles with ambiguities. Unlike fuzziness, probability dissipates with increasing information. In fuzziness the uncertainty arises always form the simultaneous occurrence of two properties. More formally, does mA (x ), the degree to which element x belongs to fuzzy set A, equal the probability that x belongs to A? This statement can be true or not, it depends on the problem existing, but in fact the point of views are quite different and so far hardly to compare. The following explanation should not explain the fuzzy steps in great detail, The focus lies upon two aspects. How are the sets dened and which rules can be determined. This is judged as the crucial aspects for fuzzy application in bioprocess engineering [344].

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Following the introduction of fuzzy sets,

Zadeh began steadily to develop the necessary

inferencing mechanisms and modeling techniques to bring this concept to fruition [345]. In this period, it was recognized that fuzzy logic could aid in the development of control systems in which nonlinearities and time variance make the development of traditional control systems very difcult. In these cases, a human operator is often capable of controlling the plant, so a fuzzy controller can often be designed based on the operators expert knowledge. Since that time, control applications based upon fuzzy logic models have been extensively investigated [346]. Fuzzy logic systems are similar to expert systems in their use of linguistic relationship, but the substantial difference is that the outputs are in general continuous by variation of the inputs [347]. It is exactly this property which causes the high interest in mathematical modeling, and hence in engineering science. Mapping with fuzzy logic is analogous to classical modeling. The maps have variables which inuence system behavior and relationships among the variables which describe the system. In classical models, variables have real number values, the relationships are dened in terms of mathematical functions. In fuzzy logic however, the values of variables are expressed by linguistic terms such as large, medium, and small, the relationships are dened in terms of if-then rules. By using defuzzication techniques, exact numerical outputs are calculated from fuzzy subsets. A fuzzy subset A is dened by a membership function mA (x i ), where x i is the domain, of the variable on which A is dened.
mA (xi ) [0,1] xi A (10)

The value of mA (x i ) for each x determines the degree to which each element in the domain belongs to A. Although both classical and fuzzy subsets are dened by membership functions, the degree to which an element belongs to a classical subset is limited to being either zero or one. This means that m(x ) may only be a step function. In fuzzy logic, the degree to which an element belongs to a subset may be any value in the interval [0, 1]. Since mA (x i ) may be dened by any function, it is easy to see that a classical subset is a special case of a fuzzy subset [348]. The fuzzication process transforms exact values x

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Biotechnology get a precise value, for further explanation see [327, 349]. Expert knowledge is the most common technique for determining rules. The expert is asked to summarize the knowledge about the system in the form of cause and effect relationships. From these the rules are formulated. When no experts are available or a more analytical approach is desired, other techniques of system identication must be used. One approach is to apply a set of metarules to adaptively acquire information about the system. Linguistic self-organizing controllers issue control actions and observe the environment [358]. The metarules evaluate the performance of the control actions from the effect of the control actions on the environment. The metarules then determine whether the control actions should be modied and perform the modication, if necessary. This technique has been applied especially to robotics applications [359]. Fuzzy classier systems are another way to discover rules. Fuzzy classier systems are generalizations of genetic classier systems. A genetic classier system uses some of the ideas from genetic algorithms to develop expert systems [360, 361]. Standard genetic algorithms have also been used to discover rules [362]. Sugeno and Yasukawa use the second variant of fuzzy clustering [fuzzy clustering methods can also be used to determine the membership functions (see above). Both approaches are addressed by the term fuzzy clustering] on data in the output space to determine fuzzy rules [363]. The clusters in the output space are used to induce clusters in the input space. Then, input space clusters are projected onto the axes to nd the fuzzy subsets for the input variables. Since the input clusters are induced from the output clusters, rules can be constructed between the input subsets and the output subsets. Yoshinori et al. (1996) discuss another method of fuzzy model construction based upon clustering techniques [364]. This method produces a relational as opposed to a functional model, and consists of a series of local, linear models to approximate a global nonlinear one. Recently; neural networks have become an active eld of interest and have been used to learn rules as well as membership functions [365, 366]. However, all methods, which automatically generate rules, have deciencies in their interpreting ability. They are generated to t available data best and it is not

of fuzzy variable vj to mAi (x i ) for all subsets Ai , which are dened for the variable vj . More rigorous denitions of membership functions may be found elsewhere [327, 349]. One technique for determining the shape of membership functions is seeking knowledge of the system from experts then constructing membership functions that represent expert opinion. Observations of many experts should be preferred. Based upon this observations, membership functions can be constructed using statistical methods accompanied by using the analysis of preferences [350, 351]. Many researchers have investigated more rational techniques for determining membership functions. One of these approaches represents one variant of fuzzy clustering [352, 353]. It requires a set of input and/or output data from the system to be collected. Patterns of data are found within the input or output space such that the variance within the sets is minimized and the variance between sets is maximized. The prototype, or center point, of a cluster X is determined and a distance metric is used to determine the degree of membership that a value has to X. Another technique for determining membership functions involves neural networks. Neural networks are networks of simple processors connected by adjustable weighting factors [328]. The weights are adjusted by using a backpropagation or other known algorithm to minimize the difference between the predicted output and the measured output. The parameters of membership functions can be learned by a neural net from a set of inputoutput data [354, 355]. Genetic algorithms have also been used to determine the optimal shape for membership functions [356, 357]. For fuzzy subsets to be useful in modeling, there must be a way to dene relationships between them. This is accomplished by means of their membership functions mAi (x ) with logical operators and inferences, to extract ambiguities. There are three basic operators in fuzzy logic, (1) OR, (2) AND, and (3) NOT, which link two or more membership functions mAi (x ) of different variables. The activation level of each rule is normally equal to that of the premise (activation). All rules are evaluated. If some sets of an output variable are activated more than once, an aggregation method must be chosen to determine the nal activity. After dening the interferencing method, the output fuzzy sets are defuzzied to

Biotechnology obvious that the rules express reasoning relations. This can be seen as a strong disadvantage compared to the generation of rules by means of expert knowledge. The application area of fuzzy systems is widespread and should only be presented here for applications in bioprocess engineering. The fermentation industry was one of the rst to recognize the potential of fuzzy logic for biological processes [367, 368]. Konstantinov and Yoshida developed a fuzzy logic system for identifying physiological states in fermentation processes and used it to control distributed an E. coli fermentation [369]. Meanwhile the range of applications has enlarged tremendously. In principle, they can be divided in two elds. The rst one are those where the knowledge bases is constructed such that it is based upon existing (qualitative) information, mainly from high-specialist staff. This represents the area of fuzzy modeling [370, 371], fuzzy controller [372, 373], or estimator for biosystem states or process faults [374, 375]. One of the intrinsic characteristics is that signicant knowledge engineering effort is required for setting up a complete and consistent rule base. The other domain of fuzzy logic is relational model-based systems where the linguistic information is extracted from available data to build a system model. This second approach is also directed to the eld of bioinformatics or experimental design [376, 377]. This approach does not need any preinformation about the intrinsic relation, however a knowledge base can arise which is hardly to evaluate or interpret. Additionally, no information can be given about the generality of the established rules. At all accounts, when a data driven construction is used, a subsequent mathematical or manual evaluation, especially of the rule bases, is necessary after the automatic construction of the fuzzy system. For the process industry, expertknowledge-based approaches possess more interests because they follow a concrete process target. The second one can be seen as a special form of knowledge discovery which enables to accomplish some linguistic evaluation based upon data, if no expert knowledge exists. In some exceptional cases, these clustering techniques are exploited for control purposes [378]. An aggregation of both principles is also fruitful in the case where the existing information is too little to build up a representative model. In this case,

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a data driven approach can help to complete the fuzzy structure. 8.3.2. Articial Neural Networks (ANN) Articial neural networks are designed to mimic the actions of neurons in the human brain. For detailed information, see [328 330]. They represent massive collections of interconnected neurons (nodes), which individually perform a relative simple signal processing; they are interconnected via synaptic links. Like brains, neural networks recognize patterns or relations between inputs and outputs we cannot even dene. They can be seen as pure function approximators whose abilities depend on the behavior of the individual nodes, the structure of the network, the learning procedure used, and on a very strong degree of the quantity of representative data. Not the total amount of data is crucial but the information content with small degree of redundancy. Almost the whole quantity space of the input and output values should be spanned by the database. Recently, e.g., Cybenko [379] and Hornik et al. [380], have proved that any continuous function can be approximated to an arbitrary degree of exactness on a compact set by a feedforward neural network comprising two or more hidden layers and a continuous nonlinear activation function, providing appropriate data. Working with neural networks always consists of two phases, a training and a prediction phase. In the rst one, the variable parameters p (t) of the net are changed in that way, the ANN ts best to the trainings database. In most cases, p (t) represents weight parameters of the intrinsic functions, e.g., the weight how the output of a neuron contributes to the activation of a subsequent node. On the other hand, there exist also some nets where the structure of the net (nodes and their synapses) can be seen as their parameters which are changed during learning. The indicator for changing p (t) represents a formulated performance index J. Based upon the absolute value J, the parameters are changed in order to minimize J.
min J p = J J J , ,, p1 p2 pn (11)

where pi is a parameter i with i = 1 . . . n parameters in the ANN.

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Biotechnology sic neural processes, can provide excellent solutions to practical problems. The human brain contains roughly 1011 neurons. As many as 104 synaptic junctions may abut onto single neuron. That gives roughly 1015 synapses in the human brain [381]. Consequently, the brain represents an asynchronous, nonlinear, massively parallel, feedback dynamical system of nearly cosmological proportions. The ability of ANN to emulate complex dynamical systems stems from the interconnectivity of neurons. In general an ANN has n neurons distributed in an input, an hidden, and an output section. Connections between neurons can be organized in layers such that information ows in one direction only, or it can circulate throughout the network in cyclic patterns. All neurons can be updated simultaneously or time delays can be introduced. All responses can be strictly deterministic, or random behavior can be allowed; the variations are nearly endless and should not be described in detail. Mathematically they can be formulated in the following vector equation.
y (t) = h o z (t) , x (t) , p )) (13)

The denition of J depends on a couple of different aspects, but in most cases the Euclidian distance between the desired target and the calculated output integrated over all training data sets is used. Performing learning should result in the global optima concerning the specied problem. However, most training procedures are local methods, requiring a gradient calculation. And obviously, any algorithm that relentlessly crawls downward must have a very lucky starting position if it is to settle into the lowest local minimum. Avoiding false minima requires two separate procedures. First, we must elute in the initializing phase starting in their vicinity. If we settle into the neighborhood of a broad minimum, it will be very hard to escape later. Second, we require a procedure for determining whether or not we are in a local minimum, and escaping if we are. This procedure will be constructive in that we were in a local minimum. Otherwise, we assume that we have found the global optimum. In the second phase, the prediction phase, the net is conditioned to treat new input patterns, hopefully gaining the correct corresponding output values. The nodes are connected in that way that the output of one node represents one input of the adjacent node. A node is stimulated by one or more inputs x (t) and it generates one output, a scalar y(t ) that is sent to other neurons. The output y(t ) is dependent on the weighted activities of each input, on the nature all inputs are transformed within the node and the parameters constellation p (t) in each neuron. The actual relationship between the inputs and outputs can be enormously complex, depending on the chosen of entry E (), aggregation S (), activation A() and output function O():
y (t) =O (A( S (E ( x (t) , p )) )) (12)

where
y (t) x (t) p z (t) o (t) Vector of yi with i = 1 . . . ni outputs of the ANN Vector of xj with j = 0 . . . nj inputs of the ANN Vector of pm with m = 0 . . . nm variable trainable parameters of the ANN Vector of zj with n = 0 . . . nn for all outputs of all individual nodes Vector function of function om with m = 0 . . . nm for the calculation of the individual contribution to the output yi Vector function of function hi with i = 0 . . . ni for the calculations of the real outputs of the ANN

h (t)

Thus, the resulting behavior of individual neurons can be modeled by a simple weighted sum of inputs, a complex collection of interrelated or subsequent set of (differential) equations, or anything in between. There can be signicant time delays in the steady-state output value after stepwise input stimuli. Neurons do not always respond in the same way to the same inputs. Even random events can be considered in the operation of neurons. Luckily, a large body of research indicates that simple models, which account for only the most ba-

There also exist some criteria to decide whether a structure is appropriate for a good modeling approach. Always, a compromise must be made between the desire to have a simple model with fewer parameters and more accurate predictions at the cost of a large number of parameters. The Akaikes information criterion (AIC) uses the number of data points, the overall error, and the number of parameters to determine an index, which can be analyzed to x the optimal quantity of free parameters [382]. Another concept that may be of interest is the

Biotechnology use of a periodiograms, which gives an indication regarding the frequency content of the input signals, typical frequency can be characterized, which xes the optimum degree of freedom [383]. Another way is the determination of principal components before xing the structure of the net, thus, incorrect or redundant information can be rejected before entering in the learning phase [384]. However, care must be taken in reducing system complexity or rejecting data sets, because they can be meaningful and represent the behavior of the biological system even though, this may not be obvious from the data set. When considering a particular application, it is to decide what type of network should be used. Principally, ANN structures are classied as either global or local. Both possess specic properties and hence applicational preferences. Global networks are the most popular und important ones. Among them, different types exist: feedforward backpropagation neural networks (FBNN); recurrent neural networks (RNNs), or cascade correlation networks (CCNs). Unlike the FBNN, RNNs are more general in the sense that connections are allowed both ways between a pair of neurons and even from a neuron back to itself on any way. The RNN allows the dynamics of the network to be considered intrinsically. In contrast, in FBNN for mapping a dynamic behavior the input variables must be supplemented by their corresponding deviations or time series. However, the number of weights (for a fully recurrent network) to be determined may easily become quite large. Therefore structural optimizers are recommended, e.g., the cascade correlation, where connections and nodes are added as required, [385]. Local networks, such as the radial basis function network (RBFN) process data in discrete areas not continuously over the whole space of the data. They are particularly suitable for applications in online control and optimization [386]. In the RBFN, it is necessary to locate centers among the input/output pairs such that the sum of the squares of the distance from the center to the training data set is minimized. These centers are equivalent in concept to the weights in the FBNN. However, the RBFN may fail in predicting values if the prediction space does not contain any center. This must be seen as a crucial disadvantage, especially for online applications. So, global networks should be

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preferred if high uncertainty within the data exists. The explosion of ANN applications in nearly all areas of process engineering can be attributed to the following reasons: The tremendous hardware advances in digital technology over the past decade have enabled simulations of neural nets to be made both economically and with relative ease hand speed. Applications of neural networks for sensor pattern classication have been found to be superior to the traditional algorithmic techniques or the expert system approaches. Neural networks offer the promise of being able to extract information from a plant in an efcient manner with normal availability of data, especially if severe or unknown nonlinearities and time invariances, typically for biosystems, exists. Some practitioners claim that neural networks may be easier to use and apply in the real process plant, with difcult to handle nonlinearities, as compared with the modeling approach, which can be subject to various modeling errors. Finally, the versatility in structure and application of neural networks enables them to be utilized in the middle ground between conventional model-based approaches and blackbox approaches for solving many classes of problems. These hybrid-type approaches have been another factor, which has further attracted their use in chemical process systems recently. The applications utilizing neural network based strategies in bioprocess engineering are wide ranging. A detailed description of all application is outside the scope of this contribution. Neural networks can be used for classication, to specify typical classes in the sets of the data base. Any task that can be done by traditional classication analysis can be accomplished at least as well and almost always much better by networks. Very important applications in bioprocess engineering are the multivariate exploitation of sensor arrays [387, 388] or the fault diagnosis [218, 389]. An ANN can be trained for pattern recognition. These patterns can be a intervals of time, cultivation states, fermentation series, images et cetera. If a version

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Biotechnology engaged in discovery and development of useful modeling technology, Casti asserts an inextricable coupling between a model and its intended application: Basically, the point of making models is to be able to bring a measure of order or probable system performance to our experience and observations, as well as to make specic predictions about certain aspects of the world by experience. Therefore, mathematical modeling does not make sense without formulating before making the model what is its use and what problem it is intended to help to solve. Thus, a denition of a model can be given as follows: A model is an image of a real system that shows analogues behavior in the important properties, and that allows within a limited region in respect to the intended purposes a description of the behavior of the original systems. The emphasis represents the word important. A model should be always as simple as possible; the degree of simplicity depends only on the model purpose and on the modeler skills. Modeling the same real system with a different focus, the signicant properties are mostly quite different. Single parameters can represent in one case the most specifying quantities and in the other case they are completely unimportant. The focus and the intention of the model determine not only the parameter, but also the proceeding in the formulation of the relations. The modeler must identify the important variables (inputs, outputs, states) and their separate effects, which in practice may have a very highly interactive combined effect on the overall process. This is only possible if the modeler hat a profound technological knowledge. Once the model is established, it can be used, with reasonable condence, to predict performances under differing process conditions. But this is only allowed under specic boundary conditions, under those which were used for the set-up. If you leave them, the describing ability gets lost. Any extrapolation should be seen as critical. 8.4.1. Steps in Creating a Model Model building is always a combination of theoretical studies and practical experiments in a very iterative sequence (Table 15). The described sequence should be observed in order

of one of these patterns, corrupted by noise, is presented to a properly trained network, the network can provide the original pattern on which it was trained [390, 391]. A very common problem and the most popular application eld of ANN is that of modeling, identication, or estimating the value or the state of a variable, given historic values of itself and other variables; e.g., they were used to estimate the state of microbiological cultures [392], the concentration of unmeasurable mostly intracellular biological key components [393 395], or for the identication of bioprocesses [396, 397]. ANNs can also replace or act as process controllers. The applications utilizing these neural-network-based strategies are wide ranging and vary from linear time invariant to highly nonlinear time variant systems [398, 399]. Typical advanced representatives are the model-predictive control, the inverse model-based and the adaptive control techniques. These methods have all in common, that the ANN reacts as a process controller, modied or expanded in some way by a reference model or by an online training unit.

8.4. Modeling Aspects of Biological Systems

By denition, biochemical engineers are concerned with biochemical systems, with reactions and artifacts of biochemical substances like proteins or with systems employing growing cells. Even the simplest living cell is a system of such a forbidding complexity that any forward mathematical description of it is in most cases an extremely modest approximation. This situation prompts for bioprocess modeling the question from a formal logical viewpoint: What kind of relation ship should exist between the underlying physical system and its mathematical description and which approaches should be exploited?. Contemplation of this question leads, in one direction, into the labyrinths of philosophy science. Guidance into this territory from the learning guide Rutherford Aris led to John Castis two-volume treatise Reality Rules, which explores the general denitions of a mathematical model as well as numerous specic examples of models in different contexts [400, 401]. Gratefully for engineers, who are (or should be)

Biotechnology to get proper problem awareness and to minimize the efforts to reach the main targets. The objective of a scientic investigation is to improve understanding of a system by testing a hypothesis about that system. The formulation of the hypothesis is the most important action in the modeling process. In a scientic process, a problem or question is posed and stated as a hypothesis or theoretical statement of the problem.
Table 15. Step sequences for model building in bioreaction engineering. The steps must be seen as a loop whose termination is determined by the dened cost function Step 1 Action Proper denition of the problem (hypothesis), the goals, and the objectives of the study, xing of presuppositions, boundary conditions and constraints, dening the evaluation and error criteria Analysis of the system, determination of the structural elements, description of the key elements (variables, processes) Running typical representative experiments, exploiting the parameter space of the control variables (experimental design) Establishing the type of model by use of balances, physicalchemicalbiological laws, available data, empirical equations, uncertainty treatment, problem formulation in mathematical or linguistic terms Simplifying assumption (e.g. about mixing, process structure and dynamics, metabolism, kinetics, neglecting aspects) Choice and denition of the important process variables: input and output variables, states, and parameters for the model Simulation of the model, parameter identication, sensitivity analysis, determination of estimation properties Evaluation of the model by using test data considering the evaluation criteria from step 1, if necessary starting again with step 1

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For complex biological systems, however, the hypothesis may need to be translated into a mathematical tractable building and the model predictions are compared to the observed data as a basis for rejecting or accepting a hypothesis. This step also includes the denitions of the goals and the objectives. The purpose of the model usually dictates the form and the detail as well as the data that are required to develop and test it. Typical goals can be the identication of typical parameters or simulations, to predict responses to a perturbation. If the purpose is to get a general idea on how the system would behave under a new condition, the accuracy may not be important, however, if the purpose is to determine an optimal process trajectory, the model accuracy may be critical. Finally, the purpose

may be to derive the mechanistic principles underlying the system behavior. So, it is useful to obtain as much data from the system as possible. It can be necessary to examine all different portions of the curves individually. Even, small deviations can hold large insight into the intrinsic physico-chemical effects of the biosystem. In all real cases of system modeling, also boundary conditions or system constraints must be fullled. This requirement addresses two different aspects. The rst one considers the fact that the created model is only valid inside typical boundary limits of the system variables and inside formulated constraints in respect to realized assumptions. The second one indicates that building up a model in bioprocess engineering is always accompanied with an identication step for optimizing the parameters. But the parameters are reasonable only within some limits, though they fulll the mathematical optimal criteria. Otherwise, they can loose any physicochemical meaning or interpretational ability. Having more than one parameter to determine can lead, depending on the complexity of the system under consideration, to ambiguities. In this case, although available knowledge, e.g., in form of experimental data is represented well, the meaning of the parameters is outside of any interpretation scope. The extrapolation ability gets lost. To accomplish a mathematical treatment of the model in order to reach the predened goals, an evaluation criteria must be formulated. This can be seen as the mathematical description to the formulation of the modeling purpose and is realized by formulating a scalar performance index. Its quantity gives information about the model quality. In bioprocess engineering, it is normally calculated from the results obtained by the model compared with those of the corresponding experimental data. There are two common approaches to t models to data, the least-square approach where parameter are adjusted to minimize the sum (over all target values and existing data sets) of squares of the residuals between the calculated and experimental data and the maximumlikelihood approach, which maximizes the probability of the assumed parameterized probability density function [234]. Also, the above mentioned boundary conditions and system constraints must be considered in the performance

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Biotechnology for the modeler is to decide which one should be accomplished from the viewpoint of deep understanding of the biosystem. It is important and in most cases very effective that new approaches are built up upon information of earlier studies gained from literature. Differences between the models should be evaluated to choose the model most acceptable with current and new biological knowledge. Since the problem of parameter identication and model verication increases rapidly with model complexity, one should begin with as far as possible simplied assumptions and withdraw them step by step, if the model quality is judged to be not sufcient. In this way, a most simple initial model grows step by step in complexity and accuracy, without becoming too complicated. Modeling includes not only the selection of correct model structure, but also the determination of the undened system parameters. To give a nal statement about the quality of model, its robustness has to be evaluated. This concerns sensitivity, identiability, and stability aspects. Sensitivity refers to the relative inuence of individual parameters on the solution. Identiability refers to the uniqueness of the model and of its parameters. They should be determined in that way that a data-tting process should return the same estimates of parameters more or less independently of the starting points for estimation and noise in the data. Stability is the behavior of a system with respect to a perturbation. Since nearly all real biological processes are time-variant and highly nonlinear in nature, the powerful collection of methods concerning the aspects of controllability, stability, and operability of linear systems cannot be applied [403]. Sensitivity analysis is one of the most important tools to optimized models. It refers to calculations and analyses performed to describe the relative dependency of the model parameters. Its analysis can be used for model validation as well as for xing variables to constant values in order to decrease the degree of freedom. The sensitivities are also very important for interpretation purposes, e.g., to detect more or less important partial processes or quantities of the biosystem. From a practical perspective, it represents calculations which reveal the impact of assumptions made in conjunction with model development. A distinction can be made between point sensi-

index. This aspect is from the logical point of view strictly necessary, but invokes in some cases crucial mathematical problems [402]. The system under study may consist of a single biomolecule, a cell, or a whole plant. Biological systems are normally open systems. They have inputs and losses, although they may also be reduced to closed systems as in vitro studies. Once the system to be studied is dened, the inputs and outputs are identied. Additionally, its processes, subsystems, and key compounds characterize the system. Processes are movements or changes in the system (e.g., absorption, metabolism, transport, chemical reactions), subsystems are components of the whole system (e.g., cells by considering the fermenter as the system), and compounds/states are the quantities under consideration (e.g., metabolites). Almost all modeling approaches in bioprocess engineering involve an identication step of unknown parameters in the model by experimental data. The aim of the experiment is to obtain data to conrm or reject a hypothesis. It is essential to decide from which environmental and operating conditions measurable quantities should be gained, and to which precision and in which measuring range. The physical, chemical, and biological ones should be wide ranging, and should also include the quality of the whole environment and pre-cultures or pre-states. All measurable bioprocess data must be treated initially as variables. Afterwards, it could be decided, whether they remain constant or not. Automated measurement and control of bioprocesses, presently an art but hopefully routine in the near future, generates a tremendous amount of data. This requires judgment of the importance of these data for documentation to reduce data effectively without loss of valuable information. Experts either human or computer have the data and the deterministic knowledge to trace observed behavior back to the physical, chemical, and physiological roots. Once the biosystem is structured and experiments are done, the model should be xed. There exist different approaches to nd the best appropriate model type for the underlying problem (see below). In principle, the model should represent the theories or hypothesis about how the system works. Normally, there exist different structures and parameter constellations which will t a particular set of data. The crucial task

Biotechnology tivity, relative sensitivity, and overall sensitivity. Considering the model
y (t) = y (t, p )) (14)

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noisy data and to reach the observability. Consider the following general representation of a time-variant nonlinear system:
z (t) =T
0

with t representing the process time and p the parameter to identify; y (t) are the measurable output variables. The relationship between the state variables and the point sensitivity can be show using the rst two terms in the Taylor expansion.
y (t) = y (t, p 0 )+GT p +K (15)

x t), y (t), p )

with (18)

z = z (t=t0 )

A system is dened as observable at the time t 0 , if, admitting an arbitrary initial state z , a nite point of time t 1 >t 0 exist, so that assuming known input x (t) and output y (t) trajectories the state z (t) can uniquely be determined in a time interval t 0 <t <t 1 . In contrast to linear systems, this problem involving time-variant nonlinear equations as they arise in bioprocess engineering results in nonlinear algebraic equations for the solution of which similar powerful methods do not exist. Consequently, several assumptions and individual specications and transformations have to be made to make the problem mathematically tractable with reasonable effort [404]. 8.4.2. Reasons for Making a Model
0

where G is the partial derivative matrix of y (t) with respect to p (t). An element of G is dened as the point sensitivity of yi with respect to pj . An alternative scale-free sensitivity is the relative sensitivity S ij
Sij = pi yi yi pi (16)

To represent the overall sensitivity of a state variable as opposed to point sensitivity to parameter pi over some time interval, S G ij can be dened
G Sij =

1 T2 T1

T2

T1

yi dt pj

(17)

As applied to modeling, identiability is a mathematical concept, which attempts to steer model development largely on the basis of an analysis reecting whether features of a model for a presumed system can be extracted from a proposed experiment. Selecting a standardized method for an experiment directed for elucidating a model and its features involves selecting time points for observations, observation sites, sites for tracer input, as well as forms of tracer application. In bioprocess engineering, it is seldom possible to sample all units, and the number of units to which tracer can be applied is limited. Each choice we make here stands for a chance of mitigating against identifying aspects of the model, even in an error-free situation. If the lack of identiability makes it impossible to estimate unique parameters or test relevant hypothesis, we should like to know this before conducting the experiment or interpreting the modeling results. If the experiment is adequate to identify the model, we are still left with the estimation problem, that of resolving the parameters amidst

There is a zoo of mathematical models in the biochemical engineering and mathematical biology literature. Many of these appear to naive as well as to sophisticated reader to have little more purpose than calculating numbers and writing scientic papers which conform reasonably to experimental data. This is, in itself, not a distinguished endeavor; it is not particularly difcult, and it teaches little. One reason that mathematical biology receives so little respect from biological scientists and is generally not recognized as a credible research tool in biological science and a biotechnological discovery is that it represents the failure to communicate clearly and persuasively the reasons and the motivation for constructing a model. Sometimes the modeler himself has not clearly asked this question while doing the work, sometimes biochemical scientists make one experiment after the other, not reecting which mathematical approach exists to enhance the evaluation or interpretation of the experiments. However, modeling as a typical domain of engineering gets increasing interest in the study of biological systems. The challenge of

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Biotechnology predicted based solely on intuition and experience without a model. Models can be used to simulate inputs into a system at various sites and to simulate short-term as well as long-term responses. Experiments can be simulated rapidly using a model to predict likely scenarios before undertaking expensive experimental data collection. While models will never replace experiments, they can be used to avoid experiments where insufcient or inappropriate data will be collected for testing a certain hypothesis. Models may be used predictive for design and control. Once the model has been established, it should be capable of predicting performance under differing sets of process conditions. Thus, mathematical models can be used for the design of relatively sophisticated control and optimization algorithms, and the model, itself, can often form an integral part of the control algorithm. Optimization usually involves considering the inuence of two or more variables, often one directly related to prots and one related to costs. For example, the objective might be to run a reactor to produce a product at a maximum rate, while leaving a minimum amount of unreacted substrate. An important use for models is to identify sites of change in a system when studied under different conditions. In some cases, the altered condition may result in large changes in the kinetic curves and several pathways or it may result in a subtle change in the data caused by a large change in only one parameter. A model helps to identify which parameters change between the conditions and the degree of change. The conditions may be an untreated versus treated state, a healthy subject versus a diseased subject, or a normal versus high intake of a nutrient. Models form valuable tools for teaching the process of scientic inquiry and for challenging students to think creatively and quantitatively about a system. Models can be used to demonstrate properties of a system, teach principles (such as feed-back loops, saturation kinetics etc.), test theories, and design studies. With the increased speed and convenience of computers, the availability of modeling software and the access to it, the researchers who are developing and publishing models, need to be cognizant of this. They need to make their models understandable

modeling biological systems is not to determine an arbitrary function to t the data but to use the modeling process to understand the system. Modeling can be used to determine the structure of a system where structure refers to the relationships between various parts or processes of a system. A model can be used to determine the type of relationship as well as the sequence of events that occur between the various substances of interest. In some systems, this information may be known, whereas in others the structure can only be inferred from the data. Modeling improves understanding, and it is through understanding that progress is made. In formulating a mathematical model, the modeler is forced to consider the complex cause-and-effect sequences of the process in detail, together with all the complex inter-relationships that may be involved in the process. The comparison of a model prediction with actual behavior usually leads also to an increased understanding of the process, simply by having to consider the ways in which the model might be in error. The results of a simulation can also often suggest reasons as to why certain observed and apparently inexplicable phenomena occur in practice. Models can be used to determine parameters of interest, such as pool sizes clearance rates or transport rates. If the purpose is to accurately determine a particular parameter, it is important that the model can be uniquely determined. However, the model must also be consistent with known biological information. A model may be well-determined but incorrect. The purpose of the model may be to determine the interaction of parts of a system. Models can integrate information on a number of subsystems into an integrated form to represent the process under consideration in the whole system. Because of the interactions, dependencies, and feedback of the processes, large complex systems can only be understood by using modeling. An example could be to link metabolism in one tissue with metabolism in another, or to link metabolism of a nutrient in one form with the metabolite in a second form. Large systems and systems with dynamic properties can only be understood with the use of models. Because many processes in large systems occur simultaneously and dynamic systems are accompanied by complex interactions between processes, it is unlikely that responses to perturbations could be

Biotechnology and accessible to the biological community at large as well as to students. Models help in experimental design. It is important that experiments be designed in such a way that the model can be properly tested. Often, the model itself will suggest the need for data for certain parameters, which might otherwise be neglected, and hence the need for a particular type of experiment to provide the required data. Conversely, sensitivity tests on the model may indicate that certain parameters may have a negligible effect, and hence these effects can be neglected both from the model and from the experimental program. 8.4.3. Different Types and Basic Approaches for Building a Model Incomplete knowledge of the dominant biological pathways as well as low availability of sensor information about the current physiological state is the characteristics of biochemical process modeling. From system analysis, as already described above, the denitions of the describing variables (e.g., inputs, outputs, or states) can be given. In principle, there can be postulated that these variables are intrinsically interconnected by means of a set of indeed real existing differential equations, as it is already described in previous chapters. But even being aware that these relations may exist in principal, for mathematical treatment they can only be specied in their structure and intercorrelation as well as in their parameter constellation in some exceptional cases with a high degree of accuracy. Thus, when dealing with modeling of real biological systems, uncertainty in various aspects can never be avoided [405]. In bioprocess engineering, uncertainty is an inseparable companion of any modeling approach. Uncertainties in engineering systems can be mainly attributed to ambiguity and vagueness in dening the architecture, parameters, and governing prediction models for the systems. The ambiguity component is generally due to noncognitive sources. These sources include 1) physical randomness; 2) statistical uncertainty due to the use of sampled information to estimate the characteristics of these parameters; 3) lack of knowledge; and 4) modeling uncertainty which

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is due to simplifying assumptions in analytical and prediction models, simplied methods, and idealized representations of real performances. The vagueness-related uncertainty is due to cognitive sources that include 1) the denition of certain parameters, e.g., structural performance (failure or survival), quality, deterioration, skill, and experience of construction workers and engineers, environmental impact of projects, conditions of existing structures; 2) other human factors; and 3) dening the interrelationships among the parameters of the problems, especially for complex systems. Other sources of uncertainty can include conict in information and human and organizational errors [406]. Once the systems are dened, the relations between the outputs and states based upon the inputs and states have to be formulated to perform simulations. Today we have many types of models, which can be categorized in various ways such as deterministic versus nondeterministic, logical (linguistic) versus mathematicalequations-based or data- versus knowledgedriven. Physical models can be the realization of the original system in a different (usually smaller) scale or with structural modications. A second type of physical models is obtained by turning to a different physical system, e.g., from the original biosystem to a corresponding electrical circuit [407]. Another group represents verbal models. They give a linguistic representation of our knowledge about the system, usually formulated as rules, e.g., IF this or that happens THEN the system reacts by. . .. They are widely applied in the area of articial intelligence, namely experts system [408, 409]. In the form of fuzzy-based systems, they gain increasing industrial interest in biotechnological control theory, which is due to their effective treatment of qualitative knowledge of experienced technologists in process control strategies (see Section 8.3.1), which could only hardly realized in terms of mathematical equations [410 412]. Mathematical models form the third class. They can be classied further depending on the mathematical formalism or the methods for model building. Theoretical models as mechanistic models are based on physical and chemical laws and our knowledge about the inner structure and function of the system. In contrast, experimental or nonmechanistic mathematical models try to give without

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Biotechnology for systems without the existence of any drivingforce function the expense for their determination is disproportional to the possible result. If inputs exist, which direct the system in a dened direction, reaction patterns must be formulated additionally in terms of probabilistic equations. Another type of black-box models represents those where a specic functional structure is assumed, which is combined with a subsequent parameter estimation to adapt to the specic underlying problem (e.g., polynomial regression, ANN, chemometric models). However, on applying this, it has to be considered that the simplest and usually fairly accurate method for predicting values of the next data point in a time series from a well-behaved biosystem is to assume that it is the same as the present data point, a principle known as the rst-order trivial predictor. Similarly, in the second-order trivial predictor the value of data point n+2 is equal to that for point n plus twice the signed difference between the values of point n+1 and n. If any complex nonlinear predictive model like ANN can do no better than the rst- and second-order trivial predictors which are indeed very often concurrently then their development is a waste of time. Nevertheless, especially ANNs have been the focus of much attention for model development and have already been applied to various biochemical processes (Section 8.3.2). Although the main advantage of the development of black-box models is that a reasonably accurate model can be obtained without detailed mechanistic system knowledge, it should be noted that its accuracy depends only on the quality of the available input and output data. As black-box models are not believed to have any extrapolation properties, one has to obtain a large body of data for process identication by employing the relevant input variables in a wide range of uctuations [218]. Markov chains. Another very attractive way to use probabilistic approaches are the formulation of the problem by means of so-called Markov chains [424, 425]. A Markov process allows the modeling of uncertainties in realworld systems that evolve dynamically with time without using mechanistic approaches. The basic concepts of a Markov process are those of states of a system and of state transitions. In specic applications, the modeling art is to nd

looking into the interior of the system only a description of the observed reaction of the system in response to a certain forcing signal. Many mechanistic models in biotechnology are actually due to their oversimplication quite closer to black-box models than to real mechanistic models, although mostly mechanistic interpretations are given [413 415]. White-box model. As described above (Section 8.3.1), the relation can strictly be mechanistic, considering the four differential equations for mass and heat balance and corresponding transformations. In this so-called white-box modeling strategy, the model development is mainly driven by the knowledge of the relevant mechanisms, macroscopic balances and predened parameters. Indeed, if all the biosystem underlying mechanism are known and can be described by means of the above-specied equations and the accompanied parameters are known, the resultant white-box model is generally applicable and show good extrapolation properties. However, it is not an easy task to reveal all the relevant mechanisms and quantify these mechanisms correctly when dealing with biological processes. The metabolic modeling approach is one such approach and has been the focus of recent attention [416, 417]. Additionally, several attempts have recently been made in utilizing metabolic information for online identication and control as a consequence of the white-box strategy [418, 419]. Black-box model. The black-box model is, on the other hand, mainly driven by measured data obtained from the process, either online or ofine. In terms of mathematical expressions, probability density functions for the important parameters are specied by means of available data. Based upon these distributions, the timedependent system behavior can be forecasted [420 422]. The earliest stochastic biosystem model is perhaps that proposed by Yule, to mimic the evolution of a new species within a genus [423]. However, they are mainly used for systems only, where isolated parameters are treated. It is unrealistic that for a real biological MIMO system (multiple input multiple output), all joint probability densities, which are necessary to describe the key behavior, can be approximated by performed series of experiments. Even

Biotechnology an adequate state description such that the associated stochastic process has the Markovian property that the knowledge of the present state X (t ) is sufcient to predict the future stochastic behavior of the system X (t +1). The moving of i=t to j=t +1 is dened by means of the transition probability Pij [426]. For bioprocess engineering, controlled Markov chains or Markov decision processes (MDP) possess a high potential, for simple modeling as well as for process optimization [427, 428]. Applied in bioprocess engineering, they involve the selection of an action U t from a set of containing a nite number of A actions when observing the current state of the system. The problem is to dene the probability that the system goes to a state j from a state i under the action of a:
P {Xn+1 =j |Xn =i,Ut =a}=Pij (a) (19)

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The controlled Markov chain is completely dened by the optimality criterion for calculation of the transition probability matrix, the state vector X, the action set A, and the decision epoch. For bioprocess modeling, concentration values grouped in clusters are often used as state variables; considering more than one state variable for each one a separate model is constructed. One great disadvantage of this approach represents the huge effort to determine the transition probability matrix Pij (a) for each control action, especially if more than one manipulated variable should be considered [428]. Grey-box model. A grey-box model may be dened as a suitable combination of a blackbox principle and a white-box model. The expectation is to obtain good interpolation and extrapolation properties. Especially, hybrid approaches, where portions from each principle are pieced together based upon the amount and the structure of available knowledge, gain increasing importance. From different points of view, such hybrid systems can be build up, i.e., analog or discrete consideration, stochastic or deterministic combinations [429 431]. Only some important concepts for bioprocess engineering should be presented here. In some cases, statistical methods are included into a mathematical discrete formulation, this concerns mainly Kalman ltering. They exploit statistical information about the error distribution of the state

and a measurement values, and their propagation with time to predict online a future state based upon new measurements and already existing quantities. Their potential in bioprocess modeling and control is obvious [432 434]. From the structural point of view, hybrids are described in parallel and serial congurations. In the former case, the black box is placed in parallel with a white-box model and its role is to describe and to weight the difference between a white-box model and the real process. This model has been applied to several processes, but its performance might be limited to some extent to the viewpoint of extrapolation properties [435]. In the serial structure, the black box is placed in series with a whitebox model. Different applications are described. Combined with a fuzzy expert system, the hybrid was applied to model a real-time fed-batch culture for bakers yeast production, a production scale beer-brewery fermentation, and mammalian cell cultures [431]. To show the better extrapolation properties of grey-box-model exemplarily, the performance of a serial-type greybox model containing a neural network and a more knowledge-driven white-box model were compared with that of a more data-driven blackbox model in the enzymatic hydrolysis of penicillin G to 6-amino-penicillanic acid and phenyl acetic acid by using the enzyme penicillin acylase. The grey-box model was shown to have signicantly more reliable extrapolation properties than the black-box model [436]. Also linguistic models such as fuzzy models can be seen as a special type of grey-box models. Although the intrinsic behavior cannot be formulated by means of mathematical equations, a priori knowledge is included into the model by the linkage of various IF. . ..THEN . . . conclusions, which is built up by the knowledge of highly specialized staff. Their ne-tuning is often accomplished by using data from experiments [437]. Being aware that different approaches exist how biological systems can be modeled and treated computationally, an important aspect for a successful application is the proper choice which principle ts best to the specic problem and its boundary conditions. In most cases, more than one specic approach can be applied in principal. The most important aspect for choos-

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Biotechnology

Table 16. Different types of models based upon the system knowledge High Model Examples System knowledge deterministic unstructured, structured, CFDa Low classication pattern recognition

stochastic, statistical expert systems Markov chains, fuzzy models, set maximum likelihood, theory Kalman,

approximation neural networks, PLSb , PCRc

CFD, computional uid dynamics. PLS, partial least square. c PCR, principal component regression.
b

ing the right model should be the system knowledge about the underlying problem (Table 16). Not only the amount of system knowledge is decisive, but also its structure, the quality and quantity of accompanied data, and the purpose for which the model should be used is of special importance. As a basic principle for building up a model, it could be postulated use as much a priori information as possible. In mechanical engineering and uid dynamics, strictly deterministic approaches are typical, where the underlying differential equations are exactly known. Thus, there exists no reason to deal with uncertainties and the problem is forwarded. However, this situation generally does not exist in bioprocess engineering. A biological system consists of a very complex, strongly regulated reaction network, where only some simple mechanisms can be formulated in terms of mathematical discrete forwarded formulations. Thus, one has to deal in a very strong degree with uncertainties. If one is able to formulate a set of differential equations, which represents not an exact image of the underlying mechanism but is descriptive enough for the existing problem and for the modeling purpose, one should formulate such a model and append a subsequent parameter estimation step where statistical properties or available data are exploited to consider unknown or not structural included information. In the case where a formal-kinetic approach cannot be formed but qualitative knowledge is available or can be extracted from available data, expert systems should be preferred. Especially fuzzy systems as a special representation of an expert system show advantages in modeling approaches because of their smoothness concerning the system output. In the case where a priori knowledge of the system is not available but pure data from a well-dened experimental design do exist, approximation or classication approach like

ANN or chemometric models should be used. In those cases, they are the only alternative to realize appropriate prediction ability. They assume a system structure, which possesses no causal relation with the underlying real mechanism, but is able to incorporate and describe the system dynamics. They possess normally good approximation abilities, but their extrapolation property reveals a low reliability especially if outputs should be forecasted where the corresponding inputs are outside of the trained data space.

9. Special Applications in Biotechnology

The following chapters highlight some special aspects in biotechnology. However, there exist even more interesting applications in biotechnology. The reader should regard the areas chosen by the authors as examples for the wide distribution of biotechnology in the daily living.

9.1. Mammalian Cell Culture Technology

9.1.1. Introduction Mammalian cell culture technology has become a major eld in modern biotechnology, especially for the area of human health, and fascinating developments achieved in the last decades are an impressive example for an interdisciplinary interplay between medicine, biology, and engineering [438, 439]. Among the classical products from cells, we nd viral vaccines, monoclonal antibodies, interferon, as well as recombinant therapeutic proteins. Tissue engineering or gene therapy open new, challenging areas [440].

Biotechnology Under mammalian cell culture or animal cell culture is to be understood the cells of a mammalian, isolated from specic tissues (i.e., skin, liver, glands, etc.) and further cultivated and reproduced in an articial medium (Fig. 31) [441, 442]. During cultivation of mammalian cells in vitro, outside of a living organism, some distinct difculties arise from the extraction of the cells from a safe tissue. Slow growth rates with doubling times between 18 and 28 hours, low productivity, a high sensitivity against shear stress due to the lack of a cell wall [443] and high demands in respect to the growth medium challenge the techniques required for mammalian cells. Furthermore, many cell lines grow adherent, and a suitable surface for attachment must be provided for these cells to proliferate. As of the origin from multicellular organisms, mammalian cells still hold the genetic program of inducing their own cell death, a process called apoptosis or programmed cell death [444 446]. This can limit culture productivity in biotechnological processes. Another major problem is the nite lifespan of primary cells, which die after several doublings in vitro. This problem was solved by transforming primary cells into immortal established or continuous cell lines. The engineering targets related to mammalian cell culture technology can be identied as: Production of a valuable product (viral vaccine, protein) from mammalian cells, development of processes for the efcient production of the desired product from a laboratory to a industrial scale under the restrictions given by the cell properties (products from cells). Development of processes for the cultivation of organic tissues, which can be used as substitute to original tissues (tissue engineering, gene therapy, cells as products). Much effort has been put into the development of mammalian cell culture technology, and great progress was achieved in the last decades [447, 448]. Mammalian cells may now be cultured to very large volumes (up to 20 000 L) to provide the necessary quantities of a desired protein. Media that used to contain up to 10 % serum were continuously improved, and the cultivation in dened serum-free and even chemically dened, protein-free media is now common for most relevant industrial cell lines. Bioreactors

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were developed that provide the required lowshear-stress environment by, e.g., introducing gentle agitation and aeration with slow moving stirrers in stirred tanks, designing special aerators for air-lift reactors, or by separating the cells from the stressful conditions such as hollowber, uidized-bed, and xed-bed reactors. In industrial scale, adherent cell lines can be cultivated on microcarriers (e.g., for vaccine production) or are adapted to grow in suspension, e.g., cell lines derived from baby hamster kidney cells (BHK) or chinese hamster ovary cells (CHO).

Figure 31. Morphology of (a) suspendable and (b) adherent mammalian cells (bar approx. 30 m)

Genetic engineering contributes a great deal to recent progress [449 451]. With this technology, functional proteins can be produced by introducing recombinant DNA into cell lines, e.g., chimeric (humanized) antibodies are produced for in vivo applications in transfectoma or recombinant CHO cells. New promoters have been developed to enhance productivity, and product titers over one gram per liter have been reported for some industrial cell lines. Further, novel cell lines can now be constructed from

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Biotechnology ment, rheumatoid arthritis, leukaemia, asthma, or multiple sclerosis. Nowadays several antibodies are produced in kilogram quantities [440]. Modern recombinant techniques focus on new antibody formats such as fragmented antibodies (FAbs) or bivalent antibodies with a broad spectrum of applications. Glycoproteins are a further important group of products produced by mammalian cells. Starting with the production of -interferon as an anti-infectious drug by (nonrecombinant) Namalwa cells in the late 1970s, nowadays a growing number of glycoproteins for treatment of a wide variety of diseases are produced by means of mostly recombinant mammalian cells. Prominent examples are cytokines (e.g., interferons and interleukins), hematopoetic growth factors (e.g., erythropoeitin for treatment of anemia), growth hormones, thrombolytic agents (e.g., tissue plasminogen activator [tPA]), coagulation factors (factor VII, factor VIII, factor IX etc.), and recombinant enzymes (DNAse) [440]. Recombinant proteins may be produced by either bacterial, yeast, or mammalian cells. From a technological point of view, hosts such as bacteria or yeast have an advantage with respect to growth rate, nal cell density, and product concentration. Nevertheless, mammalian cells are preferred for those proteins requiring a specic, humanlike glycosylation pattern [459, 460], which is difcult to obtain in other host systems. Another problem for microorganisms is the maximal size of the produced protein which must be below a molecular mass of approx. 30.0 kDa. Further, in contrast to extracellular release of most proteins produced in mammalian cells, products from microorganisms are often accumulated intracellularly in inclusion bodies. This demands a more complex downstreaming. Besides this, for mammalian cells important parameters such as product yield, medium requirements, and growth characteristics (suspendable, shear resistant) have been signicantly improved. Proteins produced in the milk of transgenic animals have started to compete against classical mammalian cell culture [461 464]. The proteins are usually expressed in enormous titers, over one order of magnitude higher than that obtained from cell cultures. The price for the production in transgenic animals will probably continue to decrease signicantly due to

primary cells without loosing functionality by genetically induced proliferation [452, 453]. The following will provide a basic understanding of the specic requirements of mammalian cells, will describe state of the art process technology for cultivation of these cells and will give an outlook to future prospective. A comprehensive overview on cell culture technology is given by Ozturk and Hu [454]. 9.1.2. Products from Mammalian Cells A detailed overview on products from mammalian cells is given in [454, 455]. Within products from cells, viral vaccines [456] against polio, hepatitis B, measles, or mumps for human use, rubella, rabies, or food-and-mouth disease (FMD) for veterinary use play an important role. Viral vaccines are produced very efciently by means of a cell-based vaccine technology. For this, mainly primary cells, diploid cells, or permanent cell lines (e.g., VERO) are applied, recently even recombinant cell lines. A breakthrough for the large-scale production of viral vaccines with anchorage dependent cells was the development of microcarriers in the late 1960s, allowing cultivation in stirred tanks in thousand-liter scale. New targets for cell-based vaccines are human immunodeciency virus (HIV), herpes simplex virus, or inuenza. Furthermore, new developments include genetically engineered or DNA vaccines ( Immunotherapy and Vaccines). Monoclonal antibodies [457] have become a valuable tool for diagnostic purposes as well as in therapy. Antibodies synthesized by Blymphocytes play an important role in the immunosystem of mammalians. Traditionally, polyclonal antibodies were isolated from blood samples. In the 1970s Milstein and Kohler developed a technology to generate hybridoma cells producing monoclonal antibodies [458]. As of the specic binding, monoclonal antibodies are widely used for diagnostics, where tens of thousands different monoclonal antibodies are available. The importance of monoclonal antibodies as therapeutic agents has evolved only recently, as immunogenic mouse antibodies were replaced by chimeric, humanized, or human antibodies. Fields of application are organ transplantation (OKT3), cancer diagnostic and treat-

Biotechnology the development of more efcient reproduction technologies. These new approaches will signicantly decrease the time for the development of a product. Among the disadvantages of transgenic animals, we nd a more sophisticated downstreaming (high protein loads), the long development time, the inability to produce proteins that might impair the health of the animal (e.g., insulin), higher risk of viral contamination and the possibility of prion contamination (scrapie, BSE). The novel eld cells as products includes the development of articial organs (tissue engineering of liver, kidney) and tissues (skin, cartilage, bone), or the expansion of hematopoietic cells for bone marrow transplantation or gene therapy. The exciting prospects of tissue engineering [465 467] are outlined in Section 9.2. Somatic gene therapy implies transfection of a specic gene to cells isolated previously from a patient suffering from a genetic disease [468 470]. The transfected cells are then reintroduced into the patient. Gene therapy involves transfer of genetic material, encoding therapeutic genes and the sequence necessary for expression to target cells to alter their genetic code for a desired therapeutic effect. Many diseases are originated by gene defects. Expression of the transferred genes can result in the synthesis of therapeutic proteins or correction of a gene defect. Gene transfer could also lead to desired apoptosis or inhibition of cell proliferation. Theoretically, gene therapy can be applied for repairing single entailed gene defects, acquired gene defects such as chronic infectious diseases, multifactor genetic diseases such as cardiovascular disease, and nally cancer. Since gene therapy was applied the rst time in 1990, the number of clinical trials increased. Today gene therapy is widely used in ongoing clinical trials for the treatment of cancer and infectious diseases such as human immunodeciency virus (HIV) infection. Cell culture products are currently used mainly as medicines or in diagnostics. For medicines, some products have a demand of 500 kg/a and generate $ 12 billion dollars in revenue. Among these are tissue plasminogen activator (tPA), erythropeithin (EPO) products, Remicade r (Centocor) or MAbThera r [440]. The pipeline for new biopharmaceutical therapeutics targets a huge number of diseases (e.g.,

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425 for U.S.A in 2002 [471]), most of them for cancer therapy, infectious diseases, autoimmune diseases, or AIDS/HIV. It can be expected that at least some of these will nd their way to the market. Furthermore, new products or medical protocols can be expected for tissue engineering and cell therapy. As for some compounds patents are already expired or will expire in the near future, there will be a market chance for biosimilar or generic products. On the other hand, the costs for target identication, clinical trials, and process development will increase (to date approx. $ 0.51 billion) and only block busters will make it nally to the market. 9.1.3. Cell Types In general, mammalian cells relevant for industrial processes can be divided into the following groups [441, 472]: Primary cells have been isolated from a tissue and than taken into culture (primary culture). Permanent or established cell lines originate from a primary culture, but because of some transformation became able (at least theoretically speaking) to divide and proliferate indenitely. Those cell lines are kept in a cell bank and are very often used as host cells for the expression of recombinant proteins. Hybridoma cells are cells which are obtained through the fusion of lymphocytes and tumor cells and which are able to express monoclonal antibodies. The common procedure for the isolation of a mammalian cell from a tissue involves the following steps: rst, a small piece of tissue is decomposed into single cells or cell clots mechanically, enzymatically (e.g., typsin [473], collagenase), or through a combined procedure. The cells are separated from the enzymes by centrifugation and resuspended in culture medium. After that, the cells are inoculated into a special glass or plastic vessel with at bottom. The anchorage-dependent cells start to adhere to the bottom surface and, after a lag phase, start to divide by mitosis. Such a cell culture, in which the cells come from a differentiated tissue, is called a primary cell culture. The most critical point in the isolation of primary cells is to avoid external contamination by practicing aseptic techniques.

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Biotechnology transformation and immortalisation, as for example the treatment with mutagenic substances, with virus or with oncogenic substances [441]. Transformation of cells in vitro shows some similarities with carcinogenese in vivo, but is not identical to it; for instance, not all the transformed cells are malignant. On the other hand, all cells that are isolated from tumors (i.e, HeLa or Namalwa Cells) can be kept in a permanent culture.
Table 17. Examples of permanent cell lines important for research and production (data from [441], modied) Cell Line Origin Application Baby hamster kidney syrian hamster, 1963 adherent cells, but can BKH-21 be adapted to suspension, FMD vaccine, rabies vaccine, recombinant proteins (Factor VIII) Chinese hamster ovary of chinese adherent cells, but can ovary, CHO-K1 hamster, 1957 be adapted to suspension, recombinant proteins (HBstg, tPA, Factor VIII) COS monkey kidney transient protein expression [475] NAMALWA human lymphatic alpha-interferon tissue HeLa tumor in a cervical Fast-growing tumor vertebra cell line that was isolated in the beginning of the 1950s. HEK-293 human embryonic transient protein kidney, 1977 expression MDCK rabbit kidney adherent cell line with good growth characteristics, animal vaccines MRC-5 human embryonic normal cells with a lung cells nite life span, vaccine production NS0 and SP2/0 mouse myeloma antibody production from B-lymphocytes 3T3 mouse connective suspensible, used in tissue the development of the cell culture technology WI-38 human embryonic normal cells with a lung cells nite life span vaccine production Vero long-tailed monkey established cell line, kidney, 1962 but with some characteristics of the normal diploid cells.

When the primary cells have covered the bottom of the culture vessel almost completely, they are enzymatically cleaved (i.e., trypsin) from their support and used to inoculate new cultures. This subculture gives origin to the so-called secondary cultures. Part of the cells is stored deeply frozen in liquid nitrogen, to remain as a safety stock, from which it is possible, at any time it might be necessary, to get enough fresh cells to start a new series of subcultures for mass production. With the primary cells, it is possible to repeat subcultures several times. However, the primary cultures have a nite lifespan and therefore, after a certain number of doublings (from 50 to 100 times), the cells cease to grow and die [441]. A nite growth capacity is a characteristic of all cells derived from normal mammalian tissue. A cell can be considered as normal when it shows a certain set of characteristics [474]: A diploid number of chromosomes (i.e., 46 chromosomes for human cells) with which it is shown that no gross chromosome damage has occurred. Adherence: the cells require a surface to grow attached to (anchorage dependent). The growth phase extends until the cells reach a stage of conuence (contact inhibition). A nite life span in culture. Non malignant : the cells are not cancerous, i.e., they do not cause tumor in mice. In the 1960s, normal cells were required for the production of human vaccines in order to ensure the safety of these products [441]. Not all cell types produce exclusively primary cell cultures, which after a limited number of passages die. Some cells acquire an innite lifespan and such a population is usually called a permanent or established cell line (Table 17). These cells have undergone a transformation, i.e., they have lost their sensitivity to the growth control mechanisms. Transformed cells can also lose the characteristic to grow adhered to a surface and thus become able to grow in suspension. These transformations are also sometimes reected in the chromosomes, changing the genotype of the cells. Transformed cells can be easily grown in relatively simple media without addition of expensive growth factors. At rst, transformed cells were identied just by chance, but nowadays there are techniques to cause cell

Hybridoma cells are articial cells produced by the fusion of human or mammalian lymphocytes, which can secret specic antibodies, with a myeloma cell. Usually, lymphocytes do not survive outside the original organism, i.e.,

Biotechnology cannot be kept alive in an articial medium. Mil stein and Kohler [458] were able to overcome this problem by fusing a mouse myelom cell similar to a tumor cell with a lymphocyte from a mouse spleen, which was able to produce antibodies. The cells generated in this way are hybrid cells (also called hybridoma cells) and have the lymphocyte characteristic to produce antibodies against a certain antigen, as well as the ability from the myelom cells to survive in culture. An alternative expression system for recombinant proteins offer insect cells, especially the insect-cell baculovirus expression system (ICBEVS) [476]. Mostly applied are Sf9- or Sf21cells isolated from Spodoptera frugiperda. Production of heterologous proteins by the ICBEVS consists of two stages. Insect cells are rst grown to a desired concentration and then infected with a recombinant baculovirus containing gene coding for the desired protein. Meanwhile, several recombinant proteins produced by this technique have reached the market. The largest and most well known international animal cell culture collections are the American type culture collection (ATCC) [477], the European collection of animal cell culture (ECACC) [478] as well as the German resource centre for biological material (DSMZ) [479]. The application of permanent cell lines for the production of vaccines faced strong opposition, mainly in the 1960s, because there was the suspect that these cell lines, which behave similarly to tumor cells, could infect patients with unknown cancerous substances. Fortunately, no observation of this kind has been done until today. In the meanwhile, more and more permanent cell lines have been applied for the production of therapeutic and diagnosis substances. However, the application of permanent cell lines, and above all, of recombinant cell lines, is strictly controlled by supervisory boards (as the FDA [480] in the USA, and the EMEA [481] within the EU). During the whole production cycle of a pharmaceutics, not only the product has to be identical from charge to charge, but also the phenotypical characteristics of the cell line has to be maintained. To assure that, before the beginning of the production, a master cell bank has to be created, from which a master

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working cell bank is derived, providing the inoculation material for the production cultures. 9.1.4. Growth Medium for Cell Culture Basically, a growth medium for mammalian cells have to supply all the necessary nutrients required for growth and product formation [482, 483]. Furthermore, it should have a certain buffer capacity to stabilize the pH (pH optimum 7.0 7.3) and should provide an appropriate osmolality in order to avoid damaging of the sensitive cell membrane [484 488]. A fundamental component of all media is a salt solution, which provides the ions necessary for life, keeps the osmotic pressure within the desired range, contains one or more buffer systems (sodium phosphate buffer, HEPES, and/or carbon dioxidecarbonic acid buffer) for pH regulation, and contains, in some cases, a pH indicator (phenol red). Furthermore the media contain glucose and glutamine as a source of carbon and nitrogen, other amino acids, vitamins, mineral salts, and trace elements. A number of medium formulations have been developed, e.g., Eagles minimal essential medium (MEM), Dulbeccos enriched (modied) Eagles medium (DMEM), Hams F12, and RPMI 1640, among others. Examples of medium compositions can be found in the literature [442, 454, 489]. Traditionally these basic media were supplied with approx. 5-10 % serum (e.g., foetal calf serum [FCS] or horse serum [HS]) in order to supply specic growth factors and to protect the cells against shear stress. The disadvantage of media containing serum are the indenite composition of such media, the high serum costs, the difculty of product purication, variations between the charges and the risk of virus contamination [490]. Serum can be partially substituted by the addition of transferin, insulin, ethanol amine, albumin, or eventually bronectin as adherence factor in a serum-free but still protein-containing medium [491 493]. A further step to chemically dened, protein-free media became possible by replacing these animal-derived proteins by iron salts or iron complexes, IGF-1, chemically dened lipid concentrates, precursors or other stimulating agents such as fatty acids, biotin, cholin, glycerin, ethanolamin, thiole, hor-

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Biotechnology 9.1.5. Small-Scale Culture Systems for Routine Use Techniques and culture systems for cultivation of mammalian cells differ signicantly from those used for microbes. For basic research, a large number of culture systems have been developed (Fig. 32), mostly designed for use in an incubator aerated with 5 % CO2 to maintain the pH within the desired range. Multiwell plates (696 wells), T-asks (25100 mL), Petri dishes or roller bottles (50 mL to 5 L) are usually used for cell maintenance and proliferation, especially for adherent cells [441, 506 509]. These systems allow for sterile handling procedures and are easy to use, disposable, and lowcost. On the other hand, they require individual handling, for example in medium exchange and cell seeding; their usefulness is limited when large quantities of cells or products are required. This can be overcome to some extent by using sophisticated robotics [510]. Furthermore, environmental parameters including pH, dissolved oxygen, and temperature can hardly be controlled within the medium. A further drawback

mones, and vitamins [494 496]. Furthermore, addition of peptone or yeast extract can be helpful [497, 498]. However, the growth rate and productivity in a serum-free medium can decrease [499]. In addition, the sensitivity to shear stress increases, requiring additives with protecting characteristics, such as Pluronic F68 [500, 501]. Immobilization can improve the culture stability of nonanchorage-dependent animal cells grown in serum-free media [502, 503] Different cell lines require different compositions. Adaptation of a cell line to grow without serum is quite time-consuming and not all cell lines have been adapted to serum-free or protein-free media [504, 505]. For cultivation of primary cells, for basic cell-culture research or for vaccine production still mostly complex, serum-containing media are common. In industrial production with established, optimized cell lines, serum-free, bovine-serum and protein-free as well as chemically dened media are state of the art [439].

Figure 32. Routine cultivation systems for use in incubators

Biotechnology is the limited increase in cell number (approximately 10-20 times during cultivation). Alternatively, small well mixed bioreactors (for example, shake asks, stirred vessels, and super spinner [511]) can be used, where adherent cells are grown on microcarriers. Microcarriers are small beads, either solid or macroporous, having a diameter of approx. 100300 m and a density slightly higher than the growth medium. When rst developed in the late 1960s [512 515], microcarrier culture introduced new possibilities and suspension culture of anchorage-dependent cells in high density. Nowadays, beads made of DEAE-Sephadex, DEAE-polyacrylamide, polyacrylamide, polystyrene, cellulose bers, hollow glass, gelatin, or gelatin-coated dextran beads are in use [442]. In microcarrier culture, cells grow as monolayers on the surface of small spheres (Fig. 33) or as multilayers in the pores of macroporous structures that are usually suspended in culture medium by gentle stirring. By using microcarriers in simple suspension culture, uidized or packed-bed systems, yields of up to 200 106 cells per milliliter are possible [516]. Microcarriers are extensively used for vaccine production on a larger scale (see below) [517 521].

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Microencapsulation is another method for the immobilization of mammalian cells [441]. Basically, it can be used to protect cells against hazardous environmental conditions. The three basic encapsulation systems existing are the bead, the coated bead, and the membrane-coated hollow sphere [522 524]. Typical size for beads made of polysine alginate is 300 to 500 m, and the molecular-mass cutoff of these capsule membranes is 60 to 70 kDa [442]. The capsules can be cultivated in suspension reactors similar to microcarriers. Detrimental with respect to scale-up is the fragile nature of the microcapsule. In membrane bioreactors, including small hollow-ber reactors [525], the miniPerm system [526] or the tecnomouse [527], cells are cultivated at tissuelike densities in a compartment which contains one or several types of membrane for nutrient and oxygen supply and removal of toxic metabolites. Hollow-ber systems (compare Section 9.1.6) are widely used in the production of biopharmaceuticals including monoclonal antibodies. Several examples of modied membrane bioreactors exist for the three-dimensional culture of tissue cells [528 530] including hepatocytes [531 533], skin cells [534], or other human cells [535]. The specic characteristics of hollow-ber reactors are discussed below. 9.1.6. Types of Bioreactors Design and selection of cell culture bioreactors for larger scales have to meet special demands such as gentle agitation and aeration without cell damage, a well controlled environment with respect to pH, temp, dissolved oxygen, dissolved CO2 concentration etc., low levels of toxic metabolites (ammonia, lactate), high cell and product concentrations, optimized medium utilization, surface for adherent cells, and scalability [536 545]. Bioreactors for mammalian cell culture can be divided into two categories (Fig. 34): Low-density or homogeneous systems (stirred-tank, bubble-column, air-lift reactor), where cells are cultivated either in suspension or, if required, on microcarriers; and high-density or heterogeneous systems (xedbed, uidized-bed, hollow-bre reactor), where cells are immobilized either in macroporous sup-

Figure 33. Porcine chondrocytes grown on microcarriers (Cytodex 3, GE Healthcare)

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Figure 34. Bioreactor systems for cultivation of mammalian cells

port materials or within a compartment created by membranes. Selection of a reactor systems depends to a large extent on the specic purpose such as production of a certain amount of protein (small amounts for basic scientic studies up to kilogram quantities for medical application), three-dimensional tissue cultivation, or single-purpose or multipurpose facility. Design of suspension reactors has to deal mainly with cell damage due to shear stress caused by agitation and/or aeration [546 548]. Stirred-tank reactors (Fig. 35), which are the most common type of reactor and are nowadays build up to 20 000 L scale, are especially suited for suspendable cells [549]. Adherent cells can be grown on microcarriers and

by this handled similar to a suspension culture (see Section 9.1.5). For mixing, standard impellers (e.g., rushton turbine, pitched or marine impeller) as well as large paddle impellers are used depending on the shear sensitivity of the cells, sometimes combined to achieve a homogenous mixing throughout the bioreactor at lowest possible stirrer speed [441, 549]. For aeration, different methods are shown in Figure 36 [550 552]. Surface aeration can be applied only for low cell densities and volumes (below 1 L). Bubble aeration may cause cell damage mainly due to foam formation, especially in case of serum-containing medium. By applying special aerators (ring sparger, microsparger) in combination with serum- or protein-free medium,

Biotechnology the detrimental effects of bubble aeration can be overcome [553 556]. Alternatively, bubblefree membrane aeration systems were developed [557 560]. They are characterized by low shear rates, but require large membrane areas. On laboratory scale, a tumbling membrane basket can be used for mixing [511]. Rotating/vibrating sieves provide a gentle aeration, but are suitable mostly for laboratory scale [561]. Bubblecolumns and air-lift reactors were build up to thousand liter scale, are easy to handle, but require serum-free media to prevent cell damage through bubbles and foam [549, 562]. For continuous operation of stirred tank reactors in perfusion mode (see Section 9.1.7), several techniques for cell and/or product retention are in use as shown in Figure 37 (e.g., microltration and ultraltration, sedimentation, centrifugation, spin lters, acoustic lter, dialysis for cell, and product enrichment [563 577]). Suspension reactors are characterized by advantages such as: conventional reactor systems, know-how on design and sterile operation, good mass transfer, homogeneous mixing, possibility of sampling and determination of the concentration of the cell, and high scale-up potential. Disadvantages are: difcult oxygen supply at high cell densities, cell damage by shear and/or foam (bubble aeration), relatively high demand for control (temperatur, oxygen, pH, ow rates), cell retention required for h igh cell densities. Among high-density systems, we nd xedbed and uidized-bed reactors as well as hollowber reactors. In xed-bed and uidized-bed bioreactors, cells are immobilized within macroporous carriers. The carriers are arranged in a column either packed (xed-bed reactor [578 584]) or oating (uidized-bed reactor [585 587]). The column is permanently perfused with a conditioned medium from a medium reservoir, mostly in a circulation loop. These types of reactor are very efcient for the long-term perfusion culture of mammalian cells for the production of biopharmaceuticals (e.g., monoclonal antibodies, recombinant drugs including tPA and EPO). With respect to tissue engineering, they have been investigated for several applications including the cultivation of liver cells as an extracorporal liver device [588 590] or proliferation of stem cells [591 593]. Furthermore they were successfully used for cultivation of cells producing

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viruslike particles for gene therapy [594 597]. Again, scale-up is critical for both xed-bed and uidized-bed reactors. For xed-bed reactors, a radial-ow xed-bed geometry was suggested [598 600] and successfully applied up to approximately 25 L xed-bed volume. At perfu1 1 sion rates of 10-20 L L , this would correFB d spond to suspension reactors in the thousand liter scale. Alternatively, rotating-bed bioreactors are in use [601]. For uidized-bed bioreactors, several concepts for scale-up have been suggested as well [514, 585 587, 602]. Compared to suspension reactors, xed-bed and uidized-bed reactors have the following advantages: high volume-specic cell density (approx. 5 107 cells/mL reactor) and productivity, low shear rates, simple medium exchange and cell/product separation, productivity on a high level during long-term cultures. Disadvantages are: nonhomogeneous cell distribution, difcult determination of cells and cell harvest.

Figure 35. Multipurpose bioreactor for continuous perfusion culture of mammalian cells (Bioengineering AG, CHWald, with permission)

Within hollow-ber reactors (compare Section 9.1.5), cells are immobilized in the extracapillar space of a hollow-4ber bundle. Nutrients as well as oxygen pass through the bers, metabolites leave the culture chamber by this way. Cell concentrations comparable to those found in tissues are possible. The use of hollow-

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Figure 36. Aeration systems for stirred-tank reactors A) Surface aeration; B) Sparging/bubble aeration; C) Rotating or vibrating sieve; D) Bubble-free membrane aeration

Figure 37. Devices for cell or/and product retention A) Micro- or ultraltration, internal or external; B) Spin lter, internal or external; C) Settler, external or inclined; D) Centrifuge, external; E) Acoustic lter, inclined; F) Dialysis, internal or external a) Feed; b) Cell-free harvest; c) Medium from bioreactor; d) Medium back to bioreactor with enriched cells; e) Dialysing uid in; f) Dialysing uid out

ber culture is a convenient method for making moderately large quantities of high-molecularweight products secreted by human and animal cells, at high concentrations and with a higher ratio of product-to-medium-derived impurities than is generally achieved using homogeneous (e.g, stirred-tank) culture [603 605]. Advantages are: very high cell densities, concentrated product, lower serum demands (if required), long-term stability, easy handling, and low cost. Disadvantages are: limited scale-up, mass-transfer problems/concentration gradients, difcult determination of the cell number, proteolytic activity on the product. During the last years, a growing number of disposable or single-use bioreactor systems ap-

peared on the market besides hollow-ber reactors, addressing especially problems related to early process development such as exibility, cost effectiveness, time-to-market as well as quality and regulatory issues [606 608]. These systems are mostly based on a bag technology [609, 610]. The bags with volumes up to 250 L are placed on tilting or rocking devices for mixing and oxygen supply. Advantages of singleuse bioreactors are reduced cleaning procedures, lack of validation issues (cleaning, sterilization, etc.), lower investment costs, easier adaptation to changing process demands, less contamination risk [611 613]. The main characteristics of the culture systems discussed above are summarized in Table

Biotechnology 18. All these systems support growth of mammalian cells in one or the other way. On laboratory scale, mostly disposable ask, membrane, or bag systems are the method of choice. Small suspension reactors as well as xed bed and uidized bed reactors are mostly used for research or process development. For larger amounts of products, only suspension reactors or, up to a certain scale, xed-bed or uidized-bed reactors have the required scalability. For a detailed discussion, compare [440, 555, 614 618]. Bioreactor design, in particular, is addressed in [619]. 9.1.7. Process Strategies As for all biotechnological processes, process strategies for operation of bioreactors can be classied in discontinuous (batch, repeatedbatch or fed-batch) and continuous modes (chemostat, perfusion). Batch and fed-batch processes are mostly performed in small-scale culture systems (e.g., asks, bags) or on larger scale in suspension reactors. Batch processes are usually starting with an initial cell density of approximately 12 105 cells mL1 and last 12 weeks. After harvest, the bioreactor has to be cleaned and refurbished before the cycle is repeated [438, 440, 441]. Cell and product yield can be signicantly improved by applying a fedbatch strategy, were nutrients are added after depletion according to an appropriate feeding strategy [[621 634]. Batch and fed-batch strategies are applied quite frequently on industrial scale [438, 440, 536]. The drawbacks of discontinuous modes such as large reactor volumes, high maintenance (cleaning, sterilization, etc.) can be overcome to some extend by using continuous mode, especially perfusion with cell and/or product retention [438, 563]. Chemostat cultures without cell retention are a valuable tool for research (e.g., kinetic studies) [635 639] but not for production scale because of low cell and product concentration. Continuous perfusion present several advantages over batch systems [563, 640 643]. The advantages include the ability to grow cells to a very high density, the ease of handling media exchanges for the purpose of fresh feed and product harvest, the easy removal of metabolites and other inhibitors, and the prospect of easy scale-up. When dealing with adherent cells,

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perfusion reactor design becomes slightly simpler (e.g., xed-bed and uidized-bed) because of the immobilization of cells within macroporous carriers (compare Section 9.1.6). There is a growing number of reports on stable perfusion cultures even on an industrial scale over periods of several months [563, 644 646]. The main advantage of perfusion cultures can be seen in the reduced bioreactor size (approx. 1/10 of a suspension reactor without cell retention). Nevertheless, there are some difculties in the perfusion concept [438, 563]. Further equipment such as the retention device itself, pumps for feeding, harvest, and medium circulation, storage tanks for feed and harvest are required. The amount of media needed to complete a moderate to long-term run can be excessively large. A further possible drawback of continuous cultivation is the possibility for variability over the time of the run. Batch or fed-batch cultivations are much shorter in duration and have fewer chances for random occurrences to happen. This is important in dealing with current manufacturing practice (cGMP, see below) conditions that dictate precise reproducibility or at a minimum the evidence of non-effects of minor deviations. In the meantime, there is a growing number of pharmaceutical products in the market, produced successfully from perfusion systems. Among these are block busters such as Kogenate-FS r (Bayer Health Care) or Remicade r (Centocor) [563]. Previous concerns about getting such processes approved are not longer an issue and it can be expected that because of an increasing pressure on production costs more perfusion processes will be installed in the future. A process that becomes more and more important for production of small quantities of recombinant proteins is transient transfection [647 649]. Usually, nontransfected cells (most commonly HEK-293, COS, and BHK cells, compare Section 9.1.2) are rst cultivated in batch mode to a certain cell density and then transfected with DNA encoding for a certain protein [441]. Usually, the cells are genetically not stable and soon lose their expression ability but can produce a certain amount of protein within one batch. Originally, it was used just as a preliminary test of gene expression. New developments show that large scale production up to 100 L is possible [650, 651].

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Table 18. Main characteristics of cell culture systems and bioreactors ([620], modied)a T-ask/roller bottles/disposable bags Cell density Homogeneity Shear stress Product concentration Porductivity Medium efciency Continuous process (perfusion) Control Downstream process Steam sterilizable Reusable
a b

1 1 N 1 1 3 N 0 1 N N

Membrane reactors (hollow-bre, miniPerm, etc.) 3 1 N 3 3 1 Y 1 3 N N

Suspension (stirred-tank, air-lift reactors) 2 3 Y 2 2 3 Yb 2 2 Y Y

Fixed-bed/uidized-bed reactors 23 2 N 2 2 2 Y 3 2 Y Y

0, not possible; 1-3, increasing efciency; Y, yes; N: no. Additional equipment for cell retention required.

9.1.8. Downstream Processes The desired protein product, mostly extracellular, has to be puried after cell cultivation by a combination of unit processes. From an engineering point of view, the challenges are 1) low product concentrations in the cell-free supernatant, 2) the complex structures of the proteins, especially the recombinant, glycosylated proteins, resulting in a high sensitivity against temperature, shear forces, extreme pH, or proteolytic activity, 3) contamination by nucleic acid (DNA fragments from dead cells), 4) contamination by other proteins (e.g., from serum), 5) potential contamination by viruses or virus particles [652, 653]. The main process steps involved are [654]: concentration of supernatant (e.g., precipitation, centrifugation ltration, or aqueous two-phase partition), primary purication/capture (e.g., chromatographic techniques such as ion-exchange, gel, or immunoafnity chromatography as well as precipitation), further purication (e.g., ion-exchange, hydrophobic, and gel chromatography), and nally polishing (gel ltration, dialtration, ultraltration) and techniques for virus inactivation (acid/base treatment, nanoltration or ultraviolet-c (UVc)) [653, 655, 656]. The early stages within a purication strategy will aim to achieve both concentration and purication. The next step will be the major purication step. The number of steps included in further purication and polishing will depend on the intended use of the protein, as they are mainly designed to reduce contaminants such as DNA, host cell protein,

endotoxins, process chemicals, etc. The importance of the downstream process is underlined by the fact, that an estimated 6080 % of the total production costs are attributed to purication [438, 439, 654]. 9.1.9. Regulatory and Safety Issues Pharmaceutical production processes are subject to strict regulations and regular inspections to ensure the high quality demands for medicines [439, 440, 657]. Guidelines for aspects relevant for the production process are summarized under cGMP and corresponding lists were released in the United States by the FDA [480] as well as by the EU [481]. There are some special concern with the use of mammalian cells for production of biopharmaceuticals, e.g., 1) risk of contamination by microbes, mycoplasma, or viruses, 2) the genetic stability of the host cell line, or 3) the consistency of the product (glycosylation pattern, etc.). The documents to be submitted to the authorities include detailed information concerning the product, the production process, product characterization including analytical methods, validation procedures, methods, and results of clinical trials. The efforts to fulll these requirements are quite high and bind signicant resources within the companies [439].

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9.2. Tissue Engineering

In this chapter, an overview in the eld of red biotechnology is presented. Red biotechnology comprises three major elds: Gene therapy Production of proteins, antibodies, and vaccines for diagnostics and therapy Tissue engineering Gene therapy is a therapeutic approach based on the correction of a gene defect causing a disease. Different techniques exist for achieving this aim. In most cases, the wild-type gene is inserted into the genome by special methods replacing the mutant gene, which leads to the development of the disease. The most common vectors for the transport of the correct gene into the genome are viruses. In the mid-eighties, several treatments with the genetically modied cells were reported. The rst gene therapy approach was reported in 1990 on a young girl suffering from severe combined immunodeciency (SCID). But the therapy did not work out, after a few months the procedure had to be repeated regularly. A major drawback within this eld emerged when it was evident that a young patient died after being treated by gene therapy [658]. The patient was suffering from a rare liver disease and was a volunteer in a gene therapy program. Similar incidences occurred in the year of 2000 within a group of boys after gene therapeutic treatment of SCID. The majority of the boys in this group developed leukemia. Since then, gene therapy is only broadly applied within sciencection movies. Researchers working in this eld went back a few steps to elucidate the devastating incidences [659]. Biological active compounds produced from genetically modied cell cultures have been in use for several years in the treatment of a whole range of diseases. In most cases, the active ingredients are proteins such as blood clotting factors (e.g., tissue plasminogen activator, tPA), growth factors (e.g., erythropoitin, EPO), or monoclonal antibodies [660]. All these substances can be produced in animal cell cultures. These proteins can also be applied in diagnostic assays (e.g., ELISA). For more simple molecules such as non-glycosylated proteins, peptides and hormones, bacterial or yeast cells can be utilized for production.

9.2.1. Application of Tissue Engineering Tissue engineering is a rather young and rapidly growing interdisciplinary research eld which combines the principles of engineering, life, and clinical sciences (Fig. 38). The aim of this research eld is the development of biological substitutes that restore, maintain, or improve tissue functions [661, 662]. Therefore, the knowledge of cellular interactions, molecular transport phenomena, interactions of cells with material surface, mechanical loading, and biochemical interventions is applied. An ofcial denition was presented at a conference of the National Science Foundation in 1988: . . . the application of principles and methods of engineering and life sciences toward fundamental understanding of structure function relationship in normal and pathological mammalian tissues and the development of biological substitutes to restore, remain, or improve tissue function. Over the past two decades, encouraging results have been achieved. Methods and techniques of tissue engineering have already entered therapeutic practice, e.g., for the treatment of sports injuries and damage to cartilage. The main approach adopted in these cases is known as autologous chondrocyte transplantation (ACT). Cells isolated from cartilage tissue of the patient (autologous) are seeded onto a collagen matrix and cultivated until a threedimensional tissue structure is generated. Then the construct can be implanted into the defect [663 665]. Also tissue-engineered skin is already used, e.g., for therapy of severely burned patients [666 669]. Researchers are attempting to engineer almost every human tissue or organ because the demand of tissue-engineered cells and organs is still growing because of the fact of organ shortage for transplantation. The creation of three-dimensional tissue constructs which can mimic all functions and metabolic activity like organs (e.g., liver [670] or pancreas [671]) is still a crucial challenge. The rst tissue-engineered heart valves have been implanted to a patient and show promising results in function and mechanical stability [672, 673].

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Figure 38. Disciplines involved in tissue engineering

Figure 39. Principle of tissue engineering

9.2.2. Principle of Tissue Engineering The principle of tissue engineering can be demonstrated in the following way: First, cells have to be isolated. The cells are seeded to a matrix and are extracorporal cultivated in a suitable culture system or bioreactor in medium containing nutrients and growth factors. After cultivation, the resulting tissue is reimplanted into the patient (Fig. 39).

Tissue engineering is driven by the hopes of the patients of increased availability of transplantable organs, to ease their suffering, forestall imminent death, and enabling a better quality of life. Alongside these medical objectives, there are also huge economic issues at stake because treatment of tissue and organ damage or dysfunction often involves long hospitalization and thus is very expensive. In contrast to alternative approaches of xenogenic (from an-

Biotechnology other species) and allogenic (same species but another individual) organ transplant, tissue engineering involves transplanting cells from the body of the patient itself. This is the reason for which such great attention has been focused in the last few years on this new area of research. By growing articial autologous tissue and organs in vitro, the risk of rejection by the immune system of the body can be avoided. Tissue engineering techniques can be used to generate replacement tissue for internal organs (liver, pancreas, heart, kidneys, lungs), sensory organs (eyes, ears, nose), the skeleton (bones and cartilage), the brain and nerves (in particular for the treatment of Alzheimers disease and Parkinsons disease), or the skin (e.g., treatment of severe burns). A closely related concept to tissue engineering is regenerative medicine. Here, the emphasis is placed on supporting the bodys natural healing processes. The potential of stem cells is crucial for both elds. 9.2.3. Strategies Tissue engineering can be performed by using one of the three approaches: In vitro cultivation of autologous cells on organic, synthetic, or natural matrices In vitro cultivation of autologous cells on xenogenic matrices Iin vitro tissue generation from embryonic stem cells Depending on the tissue of interest, either a close system (e.g., extracorporal device, articial liver) or an open system based on a biodegradable polymer scaffold is developed. The autologous cells can be isolated tissuespecic primary cells or stem cells. 9.2.4. The Essentials The essentials for tissue engineering are shown in Figure 40. For a successful generation of three-dimensional tissues, suitable cells have to be isolated and cultivated on a matrix in an appropriate medium containing all necessary growth and differentiation factors by using an optimized bioreactor system. During cultivation,

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the application of a mechanical load (e.g., strain, compression) is benecial for the development of three-dimensional cellcell contacts resulting in functional tissue [674 682].

Figure 40. The essentials for tissue engineering

9.2.5. Cells For tissue engineering, either tissue-specic cells can be isolated and cultured or stem cells can be used for the directed differentiation to the desired tissue. Due to their potential for differentiation into several different tissue types, nowadays the use of stem cells is intensively discussed [683]. In numerous publications adult stem cells isolated from bone marrow (bone marrow stromal cells BMSC [684 686], fat tissue (adiposederived stem cells adSC [687]), and peripheral blood or umbilical cord blood (both haematopoietic cells [688, 689]) are applied. The potential for differentiation into several different types of tissues is already approved. Mesenchymal stem cells (MSC) play a major role in modern tissue engineering [690, 691]. These cells are easy to isolate from bone marrow or fat tissue and their availability is not so limited compared with that of the small number of stem cells present in, e.g., peripheral blood (0.10.2 %). Treatment concept based on the use of autologous stem cells would eliminate problems of donor site scarcity, immune rejection and pathogen transfer. 9.2.6. Biomatrices There has been also huge progress in the development of biomaterials for use in tissueengineering applications [692, 693]. Research

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has concentrated on the development and production of biocompatible and biodegradable materials [694 697]. Such biomaterials take over the task of the extracellular matrix (ECM), which naturally provides cells with a supportive framework of structural proteins, carbohydrates, and signaling molecules. The ideal scaffold has to mimic ECM features and would be made of a biomaterial that provides all the necessary signals for the cells to grow, differentiate and interact, forming the desired structure. Regardless that general properties and design features of the biomaterials for discussed purposes have been published in a numerous reviews [698 702], the subject still represents intensive scientic and practical interest. Besides natural occurring compounds or scaffolds, also synthetic biomaterials are reviewed in the literature [703 705]. Along with polysaccharideand protein-based (e.g., hyaluronic acid, collagen) materials, also many synthetic polymers are used for tissue-engineering application (e.g., polylactic acid, polyglycolic acid, polyurethane, polyesters). 9.2.7. Bioreactors for Tissue Engineering The design, control, and operation of bioreactors in technical and engineering disciplines are well-established. Their use in medical sciences requires an interdisciplinary approach aiming at the development of functional tissues. Therefore, frequently the application of mechanical loading is needed [706 709]. The loading can be disposed in different ways; for example, a longitudinal straining leads to the formation of ligaments or tendons; compression is applied when aiming at the development of, e.g., cartilage; pressure (perfusion) can be applied for bone tissue formation and pulsed bioreactors are used for blood vessel generation. In Figure 41, a selection of bioreactor schemes for tissueengineering applications is presented. Unlike in conventional mammalian cell culture, where only a limited number of bioreactor types are used (e.g., T-aks, roller bottles, spinner systems, stirred-tank reactors), in tissue engineering tailor-made bioreactors have to be developed and constructed for each specic tissue type, considering in particular the uid dynamics which are realized by these different systems.

Figure 41. Selected bioreactor types for tissue engineering

Since human cells are anchorage-dependent, they must be attached to a matrix in the bioreactor during the cultivation. In a rotating-wall system, the cell-seeded matrices are cultured while the reactor wall is rotated, thus the cells oat through the medium and are affected by microgravitylike conditions, and the mass-transfer rates are improved. Moreover, cell-seeded matrices can be cultured while hanging in spinner asks, which results also in an improved mass transfer. Another possibility is the continuous pumping of the medium through a xed bed with cells or the cells can be placed into a chamber where a mechanical stimulation (here compression) is applied. 9.2.8. Growing New from Old The idea of organs one day being freely available off the shelf is still an aspiration today. The need is great, however, and patients are of course very eager to have personalized treatment from organ designers using tissue engineering. The most ambitious concepts involve applying a sort of building-block principle to the culture of each tissue or organ type: socalled precursor or stem cells would be used, whose potential for differentiation could be exploited through the use of appropriate growth or differentiation factors. Thanks to this method, one would only need the appropriate ingredients: Further advantages of this approach are the fact that an organ or tissue replacement can be carried out at any time in other words it can

Biotechnology be scheduled in advance. Regenerative medicine has great potential: according to a report in Time Magazine, over the next 10 to 20 years, tissue engineering will become one of the top careers and a new era in life sciences and medicine dawns. The potential applications are highly diverse but are to be found essentially in the treatment of cells and tissue which possess little or no capacity to repair themselves such as cartilage, heart muscle and nerve tissue. The medicine of tomorrow will strive to develop personalized therapeutic procedures involving the patient in each treatment choice. There will be a move from a general onesize-ts-all approach to personalized treatment based on the principle of one drug, one therapy, one patient. This will mean using a systems biology approach to fully restore the original healthy state of the patient. This dream could be realized thanks to the emerging technologies of regenerative medicine and tissue engineering. Groups of cells and tissue generated by using tissue engineering can also serve as test systems which exhibit characteristics specic to a given organ. These can be used to test the effectiveness and duration of certain drugs or to study their mechanisms and metabolism, thus representing an alternative to animal experiments, and a rapid, broadly applicable and cost-effective way of screening pharmaceutically active substances. Over the last ten years, there has been an explosion of research in tissue engineering, which shows the enormous importance attached to this technology. Within Europe, German researchers are playing a major role. Leading research is also being carried out by teams in France, Britain, Italy, and the Netherlands. Large numbers of small biotechnology companies are working in tissue engineering and more and more are being set up all the time. In the 21st century, major scientic questions concern modern developments within the eld of regenerative medicine, which includes tissue engineering, transplantation, prosthesis, as well as pharmacological interventions stimulating in situ tissue regeneration.

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9.3. Biotechnology and Food

It is not presumptuous to claim that modern biotechnology has developed from traditional processes of food preparation. The production of alcoholic beverages, the coagulation of milk proteins, and the preservation of vegetable food by fermentation has already been familiar to most ancient civilizations. Thus, several microorganisms have a very long history of safe use in human food production. The main focus of this chapter is not on traditional fermentations but on recent developments in food biotechnology. Since this eld is remarkably ambitious and complex, only some techniques of high industrial relevance are covered exemplarily. Genetic engineering of plants has yielded, for example, herbicide tolerant RoundUp Ready soy, Bacillus thuringiensis toxin expressing maize (Bt maize) and transgenic rice lines with an increased concentration of -carotene in the endosperm (golden rice). Genetically engineered animals may produce milk with an altered protein prole, and modication of the hormone expression of sh imparts accelerated growth rates. A number of comprehensive reviews on food biotechnology are available [710]. 9.3.1. Production of Food Additives by Cell Culture Systems 9.3.1.1. Amino Acids
l-Amino acids represent the largest group of

food and feed additives produced by means of biotechnology [711]. Consequently, the literature published within the last decade is notably versatile. l-Glutamic acid ((S )-2-aminoglutaric acid, E 620) was the rst amino acid produced biotechnologically on an industrial scale, and is still the leading amino acid by volume. More than 800 000 t/a of monosodium glutamate, which serves as a avor enhancer (umami taste), are produced worldwide by cultivation of Corynebacterium glutamicum and related organisms. The production of l-glutamic acid may be enhanced by overexpression of the glutamate transporter gene (yhfK) [712], by use of streptomycin resistant Brevibacterium lactofermentum strains [713], and by optimization of the

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Biotechnology maximum synthesis rates when grown on raw agro-industrial fats as substrates [726], while Rymowicz and Cibis [727] selected the acetatenegative strain Y. lipolytica AWG-7, cultivated on glucose syrup, for citric acid production. As a second important organic acid, l-lactic acid ((S )-2-hydroxypropanoic acid, E 270), has been used from ancient times as a food preservative by lowering the pH during fermentation. More recently, lactic acid has gained increasing interest since the monomer is used for generation of the biodegradable food packing polymer polylactic acid (PLA). However, only few PLA-generating processes have been commercialized and the cost of PLA is still not able to compete with synthetic plastics [728, 729]. Apart from traditional lactic acid bacteria such as Lactobacillus delbrueckii [730] or L. plantarum [731], a wide range of further microorganisms, especially strains of Rhizopus oryzae, has been employed for the biosynthesis of llactic acid (for example [732, 733]). A current review on metabolic engineering approaches of lactic acid bacteria is available [734]. 9.3.1.3. Vitamins
l-Ascorbic acid (vitamin C, E 300) is produced

culture conditions [714]. The second most important amino acid is l-lysine ((S )-2,6-diaminohexanoic acid), which is mainly used to supplement animal feed with this essential and often limiting amino acid. It is industrially produced by C. glutamicum or by E. coli species [711]. Efcient industrial producer strains have been developed by multiple rounds of mutagenesis. Dened l-lysine producing C. glutamicum mutants have been generated for example by modication of the enzyme aspartate kinase (lysC mutation) [715], attenuation of the transcription regulator tipA gene [716], and the introduction of a mutation (S361F) into the 6-phosphogluconate dehydrogenase gene (gnd) [717]. In the latter case, the modied strain was less sensitive to allosteric inhibition by intracellular metabolites than the wild-type strain. Product yields of up to 100 g/L are attainable within 24 h of cultivation with the recombinant production strains. A third amino acid, l-cysteine ((R)-2-amino-3mercaptopropanoic acid), which is applied as a dough conditioner in the bakery industry, may be obtained from recombinant E. coli [718 720]. 9.3.1.2. Organic Acids Citric acid (2-hydroxy-1,2,3-propanetricarboxylic acid, E 330) is widely used to lower the pH of food (candy) and beverages (soft drinks, wine). Furthermore, it acts as an efcient chelator of prooxidative iron and copper ions. Various fungi and bacteria may be used for the biotechnological synthesis of citric acid starting from numerous substrates. The process has recently been comprehensively reviewed in [721]. Aspergillus niger grown as surface cultures on diluted molasses has been employed as early as in the 1920s, and still is the most popular production organism. Today, distillers grains [722], palm oil mill efuents [723], or cassava starch factory wastes [724] may serve as cheap and abundantly available substrates. By optimization of the growth conditions (sufcient oxygen supply in submerged cultures; media decient in manganese, copper, iron, and zinc ions; high glucose concentrations), and by selection of overproducing A. niger strains, yields of > 100 g/L are attainable [725]. The yeast Yarrowia lipolytica represents a future potential production organism. The strain Y. lipolytica 187/1 showed

by a combination of chemical synthesis and enzymatic reaction steps. Known as Reichstein process, the procedure was established already in the 1930s. The key biotransformation step, the oxidation of d-sorbitol to l-sorbose, is catalyzed by Acetobacter suboxidans [735]. Due to the worldwide use of l-ascorbic acid as vitamin in dietary supplements and as antioxidant in food and feed, the annual need sums up to 60 000 t [711]. Novel approaches use either recombinant microorganisms in one-step procedures [736] or Azotobacter chroococcum strains [737] for l-ascorbic acid production. For a review on biotechnological routes to vitamin C, see [738]. Further water-soluble vitamins, including riboavin (vitamin B2 ) and cyanocobalamin (vitamin B12 ), are currently produced by microbial cell cultures. The chemical synthesis of the yellow-greenish-colored riboavin has been largely replaced by biotechnological processes using either the overproducing fungus Ashbya gossypii [739] or mutant strains of Bacillus subtilis. The generally recognized as safe (GRAS)

Biotechnology organism B. subtilis is widely used in the food industry [725] and, for example, the mutant strain CJKB0002 produced 26.5 g/L riboavin compared to 22.4 g/L obtained from the parent strain [740]. Detailed mechanistic studies on the riboavin synthesis in Ashbya gossypii [739] and in the lactic acid bacterium Lactococcus lactis ssp. cremoris [741] have been published. Cyanocobalamin is derived from Propionibacterium or Pseudomonas denitricans species [711]. Recently, a fermented milk product containing Propionibacterium freudenreichii ssp. shermanii B369 bacteria, capable of producing cyanocobalamin in situ, was patented [742]. Thus, a vitamin B12 enriched yogurt may be manufactured as a nutraceutical without external vitamin fortication. However, comparatively low world-wide annual production rates of 3000 t for vitamin B2 and 10 t for vitamin B12 document the predominant use of theses vitamins in dietary supplements and in pharmaceuticals [711]. The main plant-derived carotenoid, -carotene (E 160a), serves as provitamin A in the human diet. Since -carotene additionally imparts an intense yellow-orange coloring, it is widely used as a natural food additive. The development of biotechnological processes for the production of -carotene is mainly driven by the increasing demand of the food industry. Besides the extraction from plants (for example from lucerne), -carotene is industrially obtained from the algae Dunaliella salina [743] or from the fungus Blakeslea trispora [744]. The carotenoid astaxanthin, which is used on a large scale in aquaculture for the coloring of salmonids and crustaceae, is formed by the yeast Phafa rhodozyma [745]. 9.3.1.4. Sweet Compounds Palatinit (isomalt, E 953) is a non-cariogenic sugar substitute used in low-calory food, desserts, ice creams, and diabetic food. It is a white, crystalline, and non-hygroscopic substance with a sweetening power of roughly 0.450.60 compared to that of sucrose (1.0). In the rst production step, sucrose is converted enzymatically (by action of a glycosyltransferase) into palatinose (isomaltulose, O--dglucopyranosyl-(16)-d-fructofuranose) by immobilized cells of Protaminobacter rubrum.

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Subsequently, palatinose is reduced to palatinit, an equimolar mixture of the corresponding alcohols (1-O--d-glucopyranosyl-d-mannitol and 6-O--d-glucopyranosyl-d-sorbitol) by hydrogenation on a xed-bed Ni catalyst. In the industrial process, it is advantageous to use immobilized non-viable cells of P. rubrum in a packed-bed reactor for the enzymatic step [746]. Apart from P. rubrum, immobilized cells of Serratia plymuthica [747], Erwinia sp. D12 [748], or Klebsiella singaporensis LX3T (isolated from soil) [749] have been used to convert sucrose into palatinose. In 2005, palatinose was approved according to the EU regulation (EC 258/97) concerning novel foods and novel food ingredients, and the GRAS registration was granted in 2006 by the US Food and Drug Administration (FDA). Recent inventions suggest a mixture of palatinose with cocoa powder for the preparation of instant beverages [750]. Further applications comprise the use of palatinose for the production of low-alcohol beer or beerlike soft drinks [751], and the preparation of fermented soybean milk [752]. In the industrial production of the sweetener aspartame (-l-aspartyl-l-phenylalanine 1-methyl ester, E 951), the methyl ester of the DF dipeptide, both components are produced by biotechnological processes. l-Phenylalanine is usually manufactured by large-scale cultivation of the soil bacteria Serratia marcescens or C. glutamicum. Immobilized aspartase or alternatively intact cells of E. coli are used to synthesize l-aspartic acid starting from the precursors ammonia and fumarate [725]. Usually, amid formation is performed chemically although an enzymatic approach exists [753]. Thermolysine, immobilized on an agarosealdehyd gel, is used as biocatalyst for the synthesis of enantiopure aspartame [754]. Thermolysine is a metallopeptidase with a zinc ion in its active site and four calcium ions, which stabilize the tertiary structure of the enzyme. 9.3.1.5. Sugar Alcohols Sugar alcohols such as d-sorbitol (d-glucitol; E 420) and d-xylitol (E 967) have attracted the attention of the food and pharmaceutical industry because of their sweetening and non-cariogenic properties. Due to the fact that their metabolism

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Biotechnology cus on the conversion of starch to trehalose) and potential applications in the food industry. 9.3.1.7. Conjugated Linoleic Acids (CLA) Lactic acid bacteria play a prominent role in the traditional fermentation of food. One of the exceptional features of some lactobacilli is their ability to isomerize linoleic acid to different conjugated linoleic acid isomers (CLA). The Z 9,E 11- and E 9,E 11-octadecadienoic acid are assumed to possess benecial physiological and anticarcinogenic properties [735]. Lactobacillus plantarum AKU 1009a formed up to 40 mg/mL CLA (33 % molar yield) in 108 h under optimized conditions from 12 % (w/v) linoleic acid as substrate [769]. Further lactobacilli capable of CLA production encompass for example L. delbrueckii ssp. bulgaricus [770] and L. plantarum JCM 1551 [771]. Castor oil proved to be an adequate substrate for CLA formation. Presumably, a two-step reaction mechanism, involving hydration of a double bond with subsequent elimination of water, is leading to different CLA isomers [772]. 9.3.1.8. Lactulose Lactulose acts as a prebiotic compound and thus may contribute to increased levels of healthpromoting bacteria (bidobacteria and/or lactobacilli) in the human intestinal tract [773]. The term prebiotics comprises a wide variety of oligosaccharides that are not digested in the stomach and in the small intestine. They naturally occur in garlic, asparagus, onion, and others. Apart from soybean oligosaccharides, a number of uncommon fructo-, galacto-, xylo-, and isomalto-oligomers are currently in use, especially in Japan [773]. The disaccharide lactulose (4-O- -d-galactopyranosyl-d-fructose) is a non-naturally occurring compound with potential applications in infant milk formulations and foods [774]. Although lactulose is chiey manufactured chemically using boric acid or aluminates as catalysts, a biotechnological route starting from lactose (as galactose donor) and fructose with permeabilized Kluyveromyces lactis yeast cells has been developed [775]. In

is insulin-independent, they are well suited for substituting sugar in diet schemes for diabetics. Since the biotechnological production of xylitol represents an economically attractive alternative to the industrialized chemical process, various process variants have been published. Somewhat less literature is available on sorbitol, which is mainly formed by cultures of Zymomonas mobilis [755, 756]. The synthesis of xylitol by Candida guilliermondii depends on several crucial process parameters, one of which is the concentration of phosphate buffer in the culture broth (optimum at 0.6 M) [757, 758]. In another approach, the d-xylitol production of the yeast Debaryomyces hansenii UFV-170 was monitored with respect to temperature and starting pH [759]. Various substrates, including for example hydrolysates of corn cobs [760] and sugarcane bagasse [761] have been used for the bioconversion of the pentose d-xylose to xylitol. 9.3.1.6. Microbial Saccharides The most popular polymer currently produced by means of biotechnology is xanthan gum (E 415). Xanthan gum is a complex bacterial exopolysaccharide structurally closely related to cellulose. Today it is mainly produced by mutants of Xanthomonas campestris (40 000 t/a [711, 762]), a Gram-negative bacterium that is pathogenic to many crops. Due to its outstanding properties (high salt and pH tolerance, thermostability, and unusual rheological behavior), xanthan is predominantly used as a thickener in dressings and convenience food [763]. Sago starch has been suggested as an alternative carbon source instead of glucose [764]. Trehalose (-d-glucopyranosyl-(1-1)-d-glucopyranose) is a nonreducing disaccharide that is thought to play a role as storage carbohydrate for many microorganisms. It may serve as an additive to improve the freeze stability of processed food. In 2000 and 2001, trehalose has gained the FDA GRAS and the EU novel food approval, respectively. Trehalose is obtainable inter alia from Brevibacterium [765], Cellulosimicrobium cellulans [766], or Debaryomyces hansenii strains. The latter two were cultivated under moderate saline stress for extracellular trehalose production [767]. Literature [768] reviews trehalose production (with a fo-

Biotechnology another approach, -galactosidase from Aspergillus oryzae and a hyperthermostable glycosidase from Pyrococcus furiosus were used for the transgalactosylation of lactose in the presence of fructose [776]. Important food additives and food supplements produced by cell culture systems are summarized in Table 19. 9.3.2. Enzyme-Catalyzed Processes The modication of starch and lipids as well as the production of cheese and fruit juices are prime examples of enzyme-catalyzed bioprocesses in the food industry. For economic reasons, the necessary enzymes are often employed as crude products or even as mixtures of enzymes with different properties. If required, the enzymes are puried by means of precipitation, ultraltration and/or fast protein liquid chromatography (FPLC). Common FPLC protocols comprise one ore more of the steps hydrophobic interaction chromatography (HIC), ion exchange chromatography (IEX), and size exclusion chromatography (SEC). Reusability and sometimes also stabilization may be achieved by immobilization of the biocatalyst on an inert carrier material. 9.3.2.1. Starch-Modifying Enzymes Traditionally, the degradation of starch to maltodextrins and glucose/fructose syrups was performed by acid-catalyzed hydrolysis, using strong mineral acids such as hydrochloric or sulfuric acid. The harsh reaction conditions afforded a variable spectrum of unwanted byand breakdown products. Therefore, the acid hydrolysis has been replaced more and more by enzymatic processes. Today, starch-converting enzymes comprise about 30 % of the enzyme market worldwide. Amongst the starch converting enzymes, -amylase (EC 3.2.1.1, 1,4-d-glucan glucanohydrolase) holds the largest market share with major applications in the starch and bakery industries [777, 778]. Amylases may be derived from several plant materials, bacteria, yeasts, and fungi [779]. For commercial applications, mainly strains of Bacillus amyloliquefaciens and Bacillus licheniformis are employed as enzyme producers. Amylase attacks the -(1-4)-links within the

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starch molecule (endoamylase), while the (1-4)-bonds near -(1-6)-branches are resistant to hydrolysis. Thus, the end products of extensive -amylase treatment are oligosaccharides of varying length with -conguration and -limit dextrins, which constitute branched oligosaccharides. Since -amylase of B. licheniformis is remarkably thermostable (it remains active for several hours at temperatures of > 90 C), its application in the industrial starch hydrolysis is highly efcient [780]. -Amylases (EC 3.2.1.2; 1,4--d-glucan maltohydrolase) are present mainly in higher plants, but also in some microorganisms. Commercial products are available from malt, soy, and wheat. They hydrolyze -(1-4)-glucosidic bonds, effecting successive removal of maltose units from the nonreducing end (exoamylase). In contrast to amylose, amylopectin is not completely hydrolyzed, as all reactions stop before branch points are reached ( -limit dextrin). Glucoamylase (EC 3.2.1.3; 1,4--d-glucan glucohydrolase) is formed predominantly by fungi and yeasts (Aspergillus). It starts at the nonreducing end of -(1-4)-d-glucans and successively liberates -d-glucose units. The -(16)-bonds in amylopectin are also cleaved, but around 30 times slower than the -(1-4)-bonds. At higher concentrations, glucoamylase reforms all the glycosidic bonds that it hydrolyses. Thus, it condenses glucose, for example, to isomaltose (6-O--d-glucopyranosyl-d-glucose). Debranching enzymes exclusively hydrolyze -(1-6)-bonds in amylopectin, glycogen, and pullulan. Linear amylose fragments are formed from amylopectin. Pullulanase (EC 3.2.1.41; pullulan -(1-6)-glucanohydrolase) hydrolyzes -(1-6)-bonds in pullulan (a linear polymer of (1-6)-linked maltotriose units) and amylopectin, while isoamylase (EC 3.2.1.68) is only capable of the hydrolysis of the -(1-6)-bonds in amylopectin. Pullulanases have been found in a variety of bacteria; industrially used is an enzyme derived from Bacillus acidopullulyticus. Cyclodextrins are produced via an intramolecular transglycosylation reaction in which the enzyme cyclodextrin glycosyltransferase (EC 2.4.1.19; 1,4--d-glucan 4--d-(1,4-d-glucano)-transferase) cleaves the -(1-4)glycosidic bond and concomitantly links the reducing to the nonreducing end.

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Table 19. Examples of food additives and food supplements produced by cell cultures Product l-Glutamic acid l-Lysine l-Cysteine Citric acid l-Lactic acid l-Ascorbic acid Riboavin Cyanocobalamin -Carotene Palatinit (Isomalt) Aspartame d-Xylitol Xanthan Trehalose Conjugated linoleic acids (CLA) Lactulose Use avour enhancer feed supplement bread enhancer acidity regulator preservative vitamin C; antioxidant food coloring;vitamin B2 vitamin B12 food coloring; provitamin A sugar substitute sweetener sugar substitute thickener; stabilizer cryoprotectant; stabilizer active ingredient of functional food prebiotic E number E 620 E 920 E 330 E 270 E 300 E 101 E 160a E 953 E 951 E 967 E 415 Production organism Corynebacterium glutamicum C. glutamicum Escherichia coli Aspergillus niger Lactobacillus ssp. Acetobacter suboxidans Bacillus subtilis Propionibacterium ssp. Dunaliella salina, Blakeslea trispora Protaminobacter rubrum Serratia marcescens, C. glutamicum Candida guilliermondii Xanthomonas campestris Brevibacterium ssp. Lactobacillus ssp. Kluyveromyces lactis

Syrups high in fructose level (4244%, highfructose corn syrups, HFCS) are obtained from glucose solutions by action of the enzyme xylose (glucose) isomerase (EC 5.3.1.5, d-xylose ketol isomerase) [725]. Technical enzymes are derived for example from Streptomyces, Actinoplanes, Arthrobacter, or Bacillus cultures. The large-scale isomerization of glucose is performed with immobilized enzymes, and product yields of up to 22 tons of isosugars per kilogram of catalyst are obtained. Fructose concentrations of about 90 % are accessible via a subsequent chromatographic purication step. An excellent survey of enzymes involved in the industrial processing of starch is presented in [781]; and the most important starch converting enzymes are summarized in Table 20. 9.3.2.2. Lipases Lipases (EC 3.1.1.3, triacylglycerol acylhydrolase) are valued biocatalysts with numerous applications in food biotechnology. Many representatives of this enzyme class are highly stable in organic solvents, show broad substrate specicity, and high regio- and/or stereoselectivity [782]. Main elds of application include the rational design of lipids with specic structures (functional oils and fats [783]), and the production of volatile fatty acids and avor esters (natural food avorings). Volatile fatty acids are the so-called character impact avor compounds of various types of cheese, while numerous avor esters are responsible for fruity aroma impressions [784]. Industrial highlevel production of lipases requires not only

the efcient overexpression of the corresponding genes, but also a detailed understanding of the mechanisms governing the folding and secretion of the proteins [785]. Immobilized lipases from the yeast Candida rugosa are traditionally popular, as a wide core in the catalytic centre allows for acceptance of various fatty acids for trans-esterication or synthesis of mono- and diacylglycerides, for example, [786]. Especially C. antarctica lipase B features outstanding properties such as high thermostability, sn-2 recognition in hydrolysis of triacylglycerides, and selectivity towards (E)-fatty acids. A review on applications of the lipases A and B from Candida antarctica was published recently [787]. Several strains of another member of the Candida family, Yarrowia (Candida) lipolytica, were screened for lipase secretion. The highest lipase activity produced on rapeseed oil as sole carbon and energy source was found to be 2760 U/mL for Y. lipolytica 704 [726]. When cultured in a 2000 L bioreactor, the lipase activity reached 1100 U/mL after 53 h [788]. Lipases with novel catalytic properties are produced for example by the basidiomycetous fungus Pleurotus sapidus. An extracellular enzyme of the type B carboxylesterase/lipase family efciently hydrolysed xanthophyll esters [789]. 9.3.2.3. Pectin-Degrading Enzymes Pectin is a complex polysaccharide (-d-(1-4)polygalacturonic acid, esteried with methanol in varying degree) that represents an essential

Biotechnology
Table 20. Starch-converting enzymes Enzyme -Amylase -Amylase Glucoamylase Pullulanase Cyclodextrin glycosyltransferase Xylose isomerase Enzyme number EC 3.2.1.1 EC 3.2.1.2 EC 3.2.1.3 EC 3.2.1.41 EC 2.4.1.19 EC 5.3.1.5 Producer organism Bacillus licheniformis malt, soy, wheat Aspergillus ssp. Bacillus acidopullulyticus Bacillus macerans Streptomyces ssp. Specicity endo--(1-4) exo--(1-4) exo--(1-4); exo--(1-6) endo--(1-6) intramolecular transglycosylation isomerization of glucose to fructose

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part of plant cell walls. The group of pectinolytic enzymes comprises pectinases (polygalacturonases), lyases, and pectin esterases. A total market share of 25 % of the global sales of food enzymes is currently attained by this enzyme family [790]. To improve the yields in fruit and vegetable juice production, and to obtain clear and concentrated juices, especially pectinases from the mold Aspergillus niger are employed [791, 792]. For example, the efciency of kiwi juice production was increased > 10 % by addition of 40 mg/L pectinase for 150 min [793], and the yield of loquat juice was 16 % higher when 160 mg/L pectinase were added for 4 h [794]. Under these conditions, the loss of vitamin C was low and the juice was claried. Endo- and exopectinases (EC 3.2.1.15, endopolygalacturonase; EC 3.2.1.67, exopolygalacturonase) depolymerize the pectin chain by hydrolytic cleavage of the glycosidic -(1-4)bonds. Usually, pectinases are combined with hemicellulases (mixture of cell-wall-degrading enzymes) and cellulases (EC 3.2.1.4) to improve fruit liquecation, leading to high-quality juices in terms of oxidation, aroma levels, and stability [795]. As an auxiliary enzyme, -larabinofuranosidase (EC 3.2.1.55), was suggested for applications in the wine industry and for clarication of fruit juices [796]. Pectin lyase (EC 4.2.2.10) and pectate lyase (EC 4.2.2.2) catalyse the eliminative cleavage of pectin and pectate, respectively, to oligosaccharides with 4-deoxy--d-gluc-4-enuronosyl groups at their nonreducing ends. Potential production organisms include Erwinia species as well as a number of phytopathogenic fungi. Pectin esterases (EC 3.1.1.11) catalyze the hydrolytic cleavage of the methyl ester group. These enzymes generally exhibit a high specicity for pectin substrates.

9.3.2.4. Chymosin (Aspartic Protease) Chymosin (EC 3.4.23.4) is a key enzyme with peptidolytic activity used in the production of cheese. The casein fraction of milk protein is coagulated by hydrolysis of -casein between Phe105 und Met106 to form para--casein and a glycopeptide (-casein glycomacropeptide). From a mechanistic point of view, chymosin belongs to the class of aspartate peptidases, which are characterized by the presence of two aspartic acid residues in the catalytic center. Since recombinant chymosin does not exhibit unwanted side activities and is not contaminated with microorganisms possibly causing quality problems, it replaces rennin from the stomachs of calves (abomasus) to an increasing degree. The gene encoding chymosin has been cloned into different microorganisms, and the properties of the recombinant enzymes expressed in the yeasts Kluyveromyces lactis and Pichia pastoris have been compared [797]. Since K. lactis is regarded as a safe organism traditionally used in food processing, it is of outstanding importance in the biotechnological production of chymosin on an industrial scale [798]. The technological characteristics of various enzymes from genetically modied organisms were nearly identical to those of natural rennin, and only slight differences in the coagulation time and curd rmness were observed [799]. Chymosin obtained from transgenic sheep milk and the recombinant protein expressed by K. lactis differed only in their action on low-molecular-weight substrates, but not in enzymatic activity on protein substrates [800]. Recently, extracellular acid peptidases from Rhizopus oryzae have been suggested as rennin substitutes in cheese manufacturing [801, 802].

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Biotechnology and copy number. These changes can be detected and mapped by using comparative genomic hybridization (CGH). CGH is a molecular cytogenetic method of screening for genetic alterations. These changes are classied as DNA gains or losses and show a characteristic pattern that includes mutations at chromosomal level. In the past few years, microarray-based formats for CGH have been developed made from large genomic clones and cDNAs. These arrays provide many advantages over conventional CGH, including higher resolution, direct mapping of changes to the genome sequence, and high throughput. Together with the analysis of complex differences in gene expression between normal and cancer cells, these data provide insight into malignancy and reveal genes that may be useful as diagnostic or prognostic markers. One problem performing cancer diagnosis using gene expression proles from microarray analysis considers the heterogeneity of a tumor. Samples taken from different locations in the tumors are dramatically different. Therefore, biological classication on the basis of random sample analysis leads to poor reproducibility and may not be adequate [807 809]. 9.4.3. Pharmaceutical Development Drug biotransformation in man and animals can lead to a modulation of pharmacodynamic effects, drug elimination, but also drug toxicity. Knowledge of drug biotransformation pathways at early stages of drug development could considerably speed up drug development [810 813] and increase the safety of drug development in the pharmaceutical industry. Each year, more than 668 000 animals are used in toxicological drug research in Europe. Animal models suffer from serious shortcomings regarding the prediction for a human situation as significant species differences in enzyme expression exist between man and animals. Specically, enzyme induction experiments are frequently misleading. 1020 % of all new drugs are required to return into preclinical development trials after rst clinical tests because of unforeseen metabolite patterns, whose toxicological relevance is unclear or untested. Classical animal-testing strategies are timeconsuming and therefore rather expensive in

9.4. Biotechnology and Health

9.4.1. Individualized Medicine Single nucleotide polymorphisms (SNPs, a single base is exchanged by an alternate) are the most common form of genetic variant in the human genome, occurring on average 1 per 1000 base pairs. Since the inuence of SNPs on medical therapy or diet is better understood, the SNP diagnostic is coming in the focus of interest. One important goal of genome research is studying genetic variance among individuals to improve the knowledge and treatment of disease and to identify the variations in our genes that inuence the risk of an individual of becoming ill. Because inherited differences inuence most common diseases and many drug responses, the understanding of existing genetic variations in the human population is a promising approach in the development of tailor-made therapeutics. Eighty percent of the individual variations are SNPs, which have a direct impact of gene regulation or of a protein product of a gene. In cases where one SNP is sufcient to cause disease it is possible to analyze the causal change and to improve our understanding of disease [803 806]. The hope is that a complete human SNP map will increase the efciency of drug development and medical treatment of disease. Many genotyping platforms have been developed to detect many SNPs at once in a efcient, cost-effective manner including microarrays by the use of allelspecic oligonucleotides containing the variable base. Large-scale SNP genotyping should lead to a fully understanding of the genetic architecture of common traits underlying disease, drug or diet response. For example, microarray experiments could help to dene the ideal dose of a drug and quantify the impact of this dose on human health. Since these are the most personally data we know, the access to these data must be protected so it is not misused by employers or insurers. The goal of this analysis is to relate SNP variation to disease or diet. 9.4.2. Clinical Diagnosis as Indicated in Genetic Anomalies in Cancer Solid tumors often show alterations in the genome that lead to changes in DNA sequences

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Figure 42. Human liver cells

view of a delayed market penetration and return of investment. The relevance of a strict control of development time is immediately explicable by the fact that preclinical development of a novel drug can total up to several hundred million Euros. In spite of this, modern cell-culture technology has progressed to the extent that it is now possible to culture primary human cells under conditions that maintain differentiated functions to perform drug biotransformation and enzyme induction studies with great reliability in individual laboratories. Human liver cells are at present the preferred target for drug studies as the use of such cells avoids species extrapolation problems (Figs. 42 and 43). Another signicant breakthrough in pharmaceutical drug metabolism research was that these models could be sufciently automated to enter into the eld of accelerated screening as a signicant initiation step towards high-throughput screening. Industrial strategies are aiming at integrating pharmacokinetics and toxicology into the early phases of drug compound selection to save time, cost, and effort in drug development. Therefore, the cell cultures and the chip tech-

nology were brought together by the pharmaceutical industry for preclinical drug research. Induction phenomena could be correlated with microarray chips containing all known genes involved in drug biotransformation. This information is used to describe the culture models used in this study and to develop the tools to ngerprint interactions of inducing agents with drugs. The use of microarray technology in the eld of drug discovery and development provide four signicant advantages:

Figure 43. Bioreactor systems for in vitro testing

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Biotechnology through in toxicology research if these models could be sufciently automated to enter into the eld of accelerated screening as a signicant initiation step towards high-throughput screening. Microarrays are powerful tools for investigating the mechanism of toxicity (Fig. 44). A microarray chip for cytochrome P450s and Phase II enzymes can be used to evaluate the liver culture models [820]. 9.4.5. Detection of Genetically Modied Organisms The use of genetically modied organisms (GMOs) in food industry has been rising exponentially over the past years. In the space of time from 1996 to 2003, global area of transgenic crops increased 40-fold, from 1.7 106 hectares in 1996 to 67.7 106 hectares in 2003, with a dominant trait of herbicide tolerance, followed by insect resistance (www.isaaa.org, International Service for the Acquisition of Agribiotech Applications). A GMO is an organism, with the exception of human beings, in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination [821]. Microarrays are a useful tool for GMO detection and the application of array techniques to GMO detection implies the capability of sensitivity and standardization of the assay. Today, commercialized arrays often do not fulll these requirements, in spite of a few promising attempts initialized by biotechnical companies and research projects. Nevertheless, microarrays are already being used in GMO analysis but well established quantitative real time PCR techniques are state of the art. GMO detection becomes increasingly important because by-products of these organisms could end up in the food chain and affect human health.

Gene expression in response to drug treatments Gene expression in model systems Gene expression patterns in disease Gene expression patters in pathogens (host pathogen interaction) 9.4.4. Dene Molecular Mechanisms of Toxicity DNA microarrays can be used to determine the relative safety of natural or synthetic chemicals to which humans are exposed. Lots of diseases are inuenced by environmental factors, for example, chemicals, radiation, drugs, nutrition, viruses, carcinogens, reproductive toxins, neurotoxins, and immunotoxins [814 819]. Although most of these chemicals are harmless, it is important to identify potentially hazardous substances. In the past, toxicological assays have used rodent, required high doses, were expansive, and take years to complete. Unfortunately, the information gained from established life animal models such as rodent to investigate enzyme induction and toxicity cannot really lead to correct assumptions of hazards to humans with respect to species extrapolation. The shortcoming with respect to predictivity of animal models for human drug biotransformation and toxicity is based upon a well accepted species difference in enzyme expression between man and animals. Nevertheless, at present animal models in drug metabolism and toxicology are still legally required in all European countries for a variety of reasons and legislative bodies are still hesitative to recommend specic human liver cell based tests. In spite of this, modern cell-culture technology has progressed to the extent that it is now possible to culture primary human liver cells under conditions that maintain differentiated functions to perform drug biotransformation and enzyme induction studies with great reliability in individual laboratories. These developments have not yet found entry into legal recommendations as no prevalidation of such models on a European scale has been achieved so far. The widespread use of such alternative models to animal investigations is being hampered both by insufcient prevalidation and by further improvements still required in the eld of automation. It would be a signicant break-

10. Concluding Remarks

As it has been demonstrated in the previous chapters, biotechnology offers manifold possibilities in the production of different products and in medical applications. It can be used for better food, better health, and a cleaner environment. However, in the area of production of ne and bulk chemicals, biotechnology has still to

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Figure 44. Investigation of toxicity through a combined cell-culture and microarray analysis

compete with the chemical industry. The chemical industry owes its success to the principle of unit construction: from simple basic substances, like ethylene, carbon monoxide or hydrogen, more complex precursors can be produced under controlled conditions by chemical reactions; because of the variety of combination options, the latter can in turn be converted into inconceivable quantities of derivatives and end products. Chemistry learned how to produce chemically pure basic substances from oil that are simple to handle and exactly dened; this is performed highly efciently in reneries. This was the key to its success. Without exact knowledge of this functional principle, the triumphant success of plastics would have been just as impossible as the production of thousands of other chemical products that today make our lives safe and comfortable. This principle can also be applied to the so-called biorenery which has similar potential and could thus provide a further argument for the concept of material application of renewable resources and biotechnology. However, a technically viable separation procedure that would permit the separate use or further processing of all the primary products (such as cellulose, hemicellulose, and lignin) is still in its infancy. The main focus is on glucose, which can be derived by microbial or chemical methods from cellulose, since a wide range of biotechnological and chemical products can be obtained from glucose. The potential for the microbial conversion of glucose-based substances is huge and the reactions are advantageous from the viewpoint of energy. It is necessary to link the degradation processes to bulk chemicals via glucose as closely as possible to the synthesis processes of further derivatives.

Microorganisms still have a great potential for inducing new or novel enzyme systems capable of converting foreign substrates. It is possible to obtain and cultivate microorganisms that can survive or grow in extraordinary environments, e.g., psychrophilic, thermophilic, acidophilic, and alkaliphilic microorganisms. These microorganisms are capable of producing unique enzymes stable to heat, alkali, and acid. In addition, techniques for cultivation and enzyme purication, and gene and enzyme technology have made great advances and are still under development. Certainly biotechnological process development must be founded on a broader genomic basis. Ten years ago, only a handful of microorganisms with a completely sequenced genome existed, today there are several hundreds. Hitherto priority was given to sequencing pathogenic microorganisms, but it is now high time to sequence microorganisms that are relevant to industry. Taking all of these into consideration, there must be far more ways than one can imagine in using biotechnology for our daily life.

11. Acknowledgement
The current update was coordinated by Roland Ulber.

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