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Biotechnology 1

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, Eidgenössische Technische Hochschule, Zürich, Switzerland
Martijn Griensven, Ludwig Boltzmann Institute, Wien, Austria
Cornelia Kasper, Institute of Technical Chemistry, University of Hannover, Germany
Stephan Lütz, Institute of Biotechnology 2, Research Centre Jülich, Germany
Ralf Pörtner, Institute for Bioprocess Engineering, Technical University of Hamburg-Harburg, Germany
Hans-Günther Schlegel, Institute of Microbiology, University of Göttingen, Göttingen, 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 Münster, Germany
Kerstin Würges, Institute of Biotechnology 2, Research Centre Jülich, Germany
Hideaki Yamada, Department of Agricultural Chemistry, Kyoto University, Kyoto, Japan
Holger Zorn, Institute of Food Chemistry, University of Hannover, Germany

1. Introduction . . . . . . . . . . . . . . 3 3.4.3. Carbohydrate Metabolism . . . . . . 17


2. Basics in Microbiology . . . . . . . 5 3.4.4. Aerobic Processes . . . . . . . . . . . 18
2.1. Microbiology – the Science of 3.4.5. Fats and Fatty Acid Metabolism . . 20
Microscopic Life Forms . . . . . . 5 3.4.6. Hydrocarbon Metabolism . . . . . . 20
2.2. Phylogeny and Taxonomy of 3.4.7. Amino Acid Metabolism . . . . . . . 21
Microorganisms . . . . . . . . . . . 7 3.4.8. Anaerobic Metabolic Processes . . . 21
2.2.1. Definition and Survey . . . . . . . . . 7 3.4.9. Single-Carbon-Compound
2.2.2. Bacteria . . . . . . . . . . . . . . . . . 7 Metabolism . . . . . . . . . . . . . . . 21
2.2.3. Archaea . . . . . . . . . . . . . . . . . 8 3.4.10. Inorganic Metabolism . . . . . . . . . 22
2.2.4. Morphological and Physiological 3.5. Biosynthesis . . . . . . . . . . . . . . 23
Properties for the Identification of 3.5.1. Amino Acids . . . . . . . . . . . . . . 23
Prokaryotic Species . . . . . . . . . . 9 3.5.2. Lipids . . . . . . . . . . . . . . . . . . . 23
2.2.5. Eukaryotic Microorganisms . . . . . 11 3.5.3. RNA and DNA . . . . . . . . . . . . . 24
2.2.5.1. Definition . . . . . . . . . . . . . . . . 11 3.6. Regulation . . . . . . . . . . . . . . . 24
2.2.5.2. Fungi . . . . . . . . . . . . . . . . . . . 11 4. Metabolic Engineering . . . . . . . 24
3. Metabolism . . . . . . . . . . . . . . . 13 4.1. Analysis of the Transcriptome,
3.1. Microbial Systems Biology . . . . 14 Proteome, and Metabolome . . .. 25
3.2. Energy Production . . . . . . . . . . 14 4.1.1. Gene Expression Analysis
3.3. Substrate Transport . . . . . . . . . 15 using DNA Microarrays . . . . . .. 26
3.4. Catabolism . . . . . . . . . . . . . . . 16 4.1.2. Fabrication of DNA microarrays .. 27
3.4.1. Photosynthesis . . . . . . . . . . . . . 16 4.1.3. Proteome Analysis using Protein
3.4.2. Chemosynthesis . . . . . . . . . . . . 16 Microarrays . . . . . . . . . . . . . .. 27

c 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim


10.1002/14356007.a04 107.pub2
2 Biotechnology

4.1.4. Metabolome Analysis and 6.4. Characteristics of Enzyme


Metabolite flux analysis . . . . . . . 28 Reactions Used in
4.2. Design and Production of Biotransformations . . . . . . . . . 50
Genetically Optimized 6.5. Types of Biocatalysts and Reaction
Strains for Production – In Vitro Systems . . . . . . . . . . . . . . . . . 51
Mutagenesis . . . . . . . . . . . . . . 30 6.5.1. Biotransformation with Growing
4.3. Random Mutagenesis, Isolation Cultures . . . . . . . . . . . . . . . . . 51
and Selection of Mutants . . . . . . 31 6.5.2. Biotransformation Conversion with
4.4. Types of Mutants and Selection Previously Grown Cells . . . . . . . 52
Principles . . . . . . . . . . . . . . . . 32 6.5.2.1. Vegetative or Washed Cells . . . . . 52
4.4.1. Auxotrophic Mutants . . . . . . . . . 32 6.5.2.2. Permeabilized Cells . . . . . . . . . . 52
4.4.2. Regulatory Mutants . . . . . . . . . . 32 6.5.2.3. Dried Cells . . . . . . . . . . . . . . . 52
4.4.3. Other Selection Methods . . . . . . . 32 6.5.3. Biotransformation with Spores . . . 52
4.4.4. Targeted or Site-Directed 6.5.4. Biotransformation with
Mutagenesis . . . . . . . . . . . . . . . 34 Immobilized Cells . . . . . . . . . . . 53
5. Cultivation and Bioprocesses . . . 35 6.5.5. Biotransformation with Cell-free
5.1. Isolation of Microorganisms . . . 35 Enzymes or Purified Enzymes . . . 54
5.2. Requirements for Growth . . . . . 36 6.5.6. Multistep Reactions Using Different
5.2.1. Chemical Composition of Bacterial Biocatalysts . . . . . . . . . . . . . . . 54
Cells . . . . . . . . . . . . . . . . . . . 37 6.5.7. Multiphase Reaction Systems . . . . 54
5.2.2. Carbon and Energy Sources . . . . . 37 6.6. Process Design . . . . . . . . . . . . 55
5.2.3. Accessory Nutrients . . . . . . . . . . 38 6.6.1. General Considerations . . . . . . . . 55
5.2.4. Sulfur and Nitrogen . . . . . . . . . . 38 6.6.1.1. Evaluating Enzyme Potential . . . . 55
5.2.5. Oxygen . . . . . . . . . . . . . . . . . . 38 6.6.1.2. Finding Suitable Enzymes . . . . . . 57
5.2.6. Complex Media . . . . . . . . . . . . 38 6.6.1.3. Substrates . . . . . . . . . . . . . . . . 57
5.2.7. Solid Media . . . . . . . . . . . . . . . 38 6.6.1.4. Media . . . . . . . . . . . . . . . . . . . 58
5.2.8. Hydrogen Ion Concentration . . . . 38 6.6.2. Selection of Biocatalysts . . . . . . . 58
6.6.2.1. Screening . . . . . . . . . . . . . . . . 58
5.2.9. Carbon Dioxide . . . . . . . . . . . . 38
6.6.2.2. Enrichment . . . . . . . . . . . . . . . 58
5.2.10. Aeration . . . . . . . . . . . . . . . . . 38
6.6.2.3. Molecular Engineering . . . . . . . . 59
5.2.11. Anaerobic Techniques . . . . . . . . 39
6.7. Improvement of Conversion
5.2.12. Media Preparation . . . . . . . . . . . 39
Processes . . . . . . . . . . . . . . . . 59
5.3. Sterilization . . . . . . . . . . . . . . 39
6.8. Conclusion and Outlook . . . . . . 60
5.3.1. Moist Heat . . . . . . . . . . . . . . . 39
7. Downstream Processing . . . . . . 61
5.3.2. Dry Heat . . . . . . . . . . . . . . . . . 39
7.1. Sample Disruption . . . . . . . . . . 62
5.3.3. Filtration . . . . . . . . . . . . . . . . . 39 7.2. Solid–Liquid Separations . . . . . 63
5.3.4. Irradiation . . . . . . . . . . . . . . . . 40 7.3. Product Recovery . . . . . . . . . . 64
5.3.5. Chemical Means . . . . . . . . . . . . 40 7.4. Solvent Extraction . . . . . . . . . . 65
5.4. Types of Bioprocesses . . . . . . . . 40 8. Monitoring and Modeling
5.4.1. Surface Culture . . . . . . . . . . . . . 40 of Bioprocesses . . . . . . . . . . . . 65
5.4.2. Submerged Culture . . . . . . . . . . 40 8.1. Characteristics of Bioprocesses . 66
5.5. Process Layout . . . . . . . . . . . . 41 8.1.1. System Definition . . . . . . . . . . . 66
5.5.1. Reactors . . . . . . . . . . . . . . . . . 41 8.1.2. System Description . . . . . . . . . . 69
5.5.2. Containments for Anaerobic 8.1.3. Dynamics of Biosystems and
Processes . . . . . . . . . . . . . . . . 41 Real-Time Considerations . . . . . . 71
5.5.3. Reactors for Aerobic Processes . . . 41 8.2. Biotechnological Measurement
5.5.4. Inoculation . . . . . . . . . . . . . . . 41 Systems . . . . . . . . . . . . . . . . . 73
5.5.5. Operation Modes . . . . . . . . . . . 42 8.2.1. Process Requirements Concerning
5.6. Process and Product Overview . . 45 Measuring Quantities . . . . . . . . . 74
6. Biocatalysis and 8.2.2. Online Sensing Devices . . . . . . . 75
Biotransformation . . . . . . . . . . 45 8.2.3. Further Aspects Concerning
6.1. Introduction . . . . . . . . . . . . . . 45 Measuring Systems . . . . . . . . . . 79
6.2. Classification of Biocatalysts . . . 47 8.3. Cognitive Computing . . . . . . . . 80
6.3. History . . . . . . . . . . . . . . . . . 47 8.3.1. Fuzzy Logic Systems . . . . . . . . . 82
Biotechnology 3

8.3.2. Artificial Neural Networks (ANN) . 85 9.2.8. Growing New from Old . . . . . . . 112
8.4. Modeling Aspects of Biological 9.3. Biotechnology and Food . . . . . . 113
Systems . . . . . . . . . . . . . . . . . 88 9.3.1. Production of Food Additives by
8.4.1. Steps in Creating a Model . . . . . . 88 Cell Culture Systems . . . . . . . . . 113
8.4.2. Reasons for Making a Model . . . . 91 9.3.1.1. Amino Acids . . . . . . . . . . . . . . 113
8.4.3. Different Types and Basic 9.3.1.2. Organic Acids . . . . . . . . . . . . . 114
Approaches for Building a Model . 93 9.3.1.3. Vitamins . . . . . . . . . . . . . . . . . 114
9. Special Applications in 9.3.1.4. Sweet Compounds . . . . . . . . . . . 115
Biotechnology . . . . . . . . . . . . . 96 9.3.1.5. Sugar Alcohols . . . . . . . . . . . . . 115
9.1. Mammalian Cell Culture 9.3.1.6. Microbial Saccharides . . . . . . . . 116
Technology . . . . . . . . . . . . . . . 96 9.3.1.7. Conjugated Linoleic Acids (CLA) . 116
9.1.1. Introduction . . . . . . . . . . . . . . . 96 9.3.1.8. Lactulose . . . . . . . . . . . . . . . . 116
9.1.2. Products from Mammalian Cells . . 98 9.3.2. Enzyme-Catalyzed Processes . . . . 117
9.1.3. Cell Types . . . . . . . . . . . . . . . . 99 9.3.2.1. Starch-Modifying Enzymes . . . . . 117
9.1.4. Growth Medium for Cell Culture . 101 9.3.2.2. Lipases . . . . . . . . . . . . . . . . . . 118
9.1.5. Small-Scale Culture Systems for 9.3.2.3. Pectin-Degrading Enzymes . . . . . 118
Routine Use . . . . . . . . . . . . . . . 102 9.3.2.4. Chymosin (Aspartic Protease) . . . 119
9.1.6. Types of Bioreactors . . . . . . . . . 103 9.4. Biotechnology and Health . . . . . 120
9.1.7. Process Strategies . . . . . . . . . . . 107 9.4.1. Individualized Medicine . . . . . . . 120
9.1.8. Downstream Processes . . . . . . . . 108 9.4.2. Clinical Diagnosis as Indicated in
9.1.9. Regulatory and Safety Issues . . . . 108 Genetic Anomalies in Cancer . . . . 120
9.2. Tissue Engineering . . . . . . . . . . 109 9.4.3. Pharmaceutical Development . . . . 120
9.2.1. Application of Tissue Engineering . 109 9.4.4. Define Molecular Mechanisms of
9.2.2. Principle of Tissue Engineering . . 110 Toxicity . . . . . . . . . . . . . . . . . 122
9.2.3. Strategies . . . . . . . . . . . . . . . . 111 9.4.5. Detection of Genetically Modified
9.2.4. The Essentials . . . . . . . . . . . . . 111 Organisms . . . . . . . . . . . . . . . . 122
9.2.5. Cells . . . . . . . . . . . . . . . . . . . 111 10. Concluding Remarks . . . . . . . . 122
9.2.6. Biomatrices . . . . . . . . . . . . . . . 111 11. Acknowledgement . . . . . . . . . . 123
9.2.7. Bioreactors for Tissue Engineering 112 12. References . . . . . . . . . . . . . . . 123

Biotechnology can be regarded as one of the mammalian cell technology, tissue engineering,
key technologies of the 21st century. It is the and the production of relevant food additives
commercial application of living organisms such as well as of various medical and pharmaceu-
as bacteria, fungi, yeasts, plant cells, viruses, and tical products. In conclusion, biotechnology of-
mammalian cells or their products, which in- fers manifold possibilities in industrial and med-
volves the deliberate manipulation of their DNA ical applications.
molecules. This article gives an introduction into
the basics in microbiology and provides an ex-
haustive description of the relevant microbial 1. Introduction
species and metabolic pathways. Identification,
analysis, and manipulation of the genome, pro- Biotechnology can be described as “the com-
teome, and metabolome is described, and cul- mercial application of living organisms or their
tivation requirements as well as process pa- products, which involves the deliberate manipu-
rameters discussed. Biotransformation and en- lation of their DNA molecules”. This includes
zyme technology plays a central role in indus- the use of bacteria, fungi, yeasts, plant cells,
trial biotechnology, and a focus is given on viruses, and mammalian cells. This definition
the development of molecular engineering tech- implies a set of laboratory techniques devel-
niques and new screening methods. Computa- oped within the last 20 years that have been
tional Biochemistry comprises the definition, responsible for the tremendous scientific and
monitoring, and modeling of bioprocesses. Ex- commercial interest in biotechnology, the found-
amples of biotechnology applications include ing of many new companies, and the redirec-
tion of research efforts and financial resources
4 Biotechnology

among established companies and universities. Table 1. Timetable of biotechnology [1, 2]


Thus, biotechnology can be regarded as one of Year Biotechnological progress
the key technologies of the 21st century. How- 3000 b.c. Fermentation of sugar containing juices to
ever, the use of microorganisms and their prod- various alcoholic beverages
ucts is as old as mankind. Fermented products 2800 b.c. brewing rooms in Mesopotamia and leaven in
Egypt
with yeast such as beer, wine, or sake are known 1500 b.c. Use of microorganisms for the production of
since several thousands of years (Table 1). The copper and production of soya sauce
roots of modern biotechnology are coming from 300 b.c. Use of vinegar
1300 Micro algae (Spirulina) as food additives
the era of microbiology, which was developed (Aztecs)
in the late 19th century. Cagniard-Latour, Pas- 1400 Production of saltpetre (potassium nitrate)
teur, Koch and Kühne were important scientists with Nitrosomas sp. and Nitrobacter sp. in
Germany
who are decoding the principles of fermentation. 1676 Anthony van Leuwenhoek observes bacteria
The use of microorganisms for the production of through a microscope
bulk chemicals such as butanol or acetone and 1837 Charles Cagnaird-Latour identifies yeast as
causer of fermentation (one year later
for pharmaceuticals (antibiotics) was strongly confirmed by Theodor Schwann and Friedrich
developed during World War I and II. With the Kützing)
better understanding of the gene function and its 1849 Industrial production of bakers yeast
1856 Louis Pasteur separates brewer yeasts from
relation to the metabolisms of cells, the modern lactic bacteria
era of biotechnology started in the 70th of the 1876 Kühne creates the term “enzymes”
last century. 1881 Industrial microbial production of lactic acid
(Boehringer)
Nowadays, biotechnology is a cross-sectoral 1890 Development of first vaccines by Pasteur and
technology that has been successfully applied Koch
in many industrial branches. One distinguishes 1893 Industrial production of citric acid using
Aspergillus niger
between several areas or “colors” of biotechnol- 1897 Starting point of enzyme technology (Eduard
ogy (the so-called red, white, green, and blue Buchner)
biotechnology). 1915 Patent for enzymes in washing powder
1916 Industrial fermentation process for acetone
and n-butanol (Chaim Weizmann)
Red Biotechology. Named as red biotech- 1928 Alexander Fleming discovers penicillin
nology are applications in medicine or in the 1937 Microbial production of vitamin C
1941/42 Industrial production of antibiotics
pharmaceutical industry. Red biotechnology has 1957 Use of Corynebacterium glutamicum for the
already made an impact on healthcare and production of amino acids
will continue to contribute to improving human 1972/1973 Stanley Cohen and Francis Boyer develop a
procedure for in vitro recombination of DNA,
health and life expectancy. Today, 20 % of mar- using plasmid vectors
keted medicines, 50 % of those in clinical tri- 1977 First recombinant proteins from bacteria
als, and 80 % in early development are biotech- 1982 First transgenic plants and animals
based products. Forty percent of these candidate 1985 Development of polymerase chain reaction
(PCR) by Kary Mullis
medicines are for the treatment of cancer. Typi- 1990 Start of human genome project (HUGO)
cal products of red biotechnology are recombi- 1996 Yeast genome is completely sequenced
nant vaccines, antibodies, blood clotting agents, 2000 Human genome is completely sequenced
(Craig Venter)
and hormones. In addition, tissue engineering is
a part of the area of biotechnology. Tissue en-
gineering aims at the functional regeneration of end within the next 100 years at the latest. An
tissues through implantation of tissue cultured extrapolation of oil consumption from 2002 (3.3
in vitro (see Section 9.2). billion tonnes), for example, yields a supply pe-
riod of 50 years with estimated stocks of 165
White Biotechology. The potential and ap- billion tonnes. The underlying reasons for a fu-
plications of biotechnology in other sectors ture era of new raw materials are the finite na-
largely pass unnoticed especially in the chemical ture of fossil resources on the one hand, and the
industry. The chemical industry has produced connection with many environmental problems
coal, gas, and petroleum-based goods and prod- on the other hand. In the past few years condi-
ucts for over 150 years. This era is likely to tions for the application of biotechnological pro-
cesses in industrial production have improved
Biotechnology 5

(so-called white biotechnology). New tools such marine living systems for mankind’s profit. The
as screening methods and metabolic engineer- domain covered by marine biotechnology is vast
ing, and also global analysis methods such as ge- and range over various overlapping disciplines,
nomics, proteomics, metabolomeics, and bioin- from developmental biology to chemistry of nat-
formatics are gradually becoming more widely ural substances and bioprocess engineering. Not
available. These new instruments make it possi- all these fields, however, are ready for practical
ble to reduce the time needed to develop and es- and industrial applications. Biomass from fish-
tablish new industrial biotechnological products ing or aquaculture industry is, in fact, complex,
and processes; this was hitherto one of the ma- geographically and seasonally dependent. Fur-
jor drawbacks of biotechnological, as opposed thermore, many “natural substances”, for which
to chemical, processes. In addition, they help we do not know of any terrestrial counterparts,
to develop biocatalysts (enzymes) and microor- and, therefore, present an up most interest, exists
ganisms which render manufacturing processes only in tiny amounts in rare biological species
more economical and facilitate new manufactur- and their exploitation is likely to call for costly
ing processes. For the first time since the begin- synthesis procedures. Marine natural products
ning of the oil age in the early fifties, one is able not only display novelty but also complexity in
to apply processes with economic potential in terms of chemical structures. Isolation and struc-
the production of basic chemicals and biopoly- ture elucidation represents only the emerged part
mers based on biotechnological processes. In of an iceberg. The question of the functionality
many cases, bioprocesses operate under milder of new isolated molecules within the perspec-
conditions (lower temperatures and pressures, tive of challenging major public health and en-
etc.) and more selectively than their competitors vironmental problems is crucial. Likely, in this
(chemical processes) by these means biopro- domain, ecological and evolutionary approaches
cesses conserve resources and improve produc- should help the classical screening systems for
tion processes economically and ecologically. determining the right target systems. In addi-
Public debate has often emphasized the advan- tion, a better understanding of the complex in-
tages for the environment; however, companies teractions between macro- and microorganisms
face hard economic competition so that when is necessary to be able to use these resources
a biotechnological process is weighed against for industrial purposes. Thus, more and more
a classical chemical process, only the potential groups are focussing on the part of bioprocess
economic advantages can affect a shift in favour engineering and downstream processing in blue
of biotechnology. biotechnology [3].

Green Biotechnology. In addition, to over-


come the problems in the production both of 2. Basics in Microbiology
bulk and fine chemicals but also in the produc-
tion of vaccines and pharmaceuticals, the so-
2.1. Microbiology – the Science of
called green biotechnology can offer new so-
lutions. This area of biotechnology involves the Microscopic Life Forms
introduction of foreign genes into economically
important plant species, resulting in crop im- Microbiology deals with microscopically tiny
provement and the production of novel products life forms which the resolution of the human
in plants. Green biotechnology might also pro- eye (approx. 20 µm) cannot detect. Many of
duce more environmentally friendly solutions the single-cell microorganisms, such as yeasts,
than traditional industrial agriculture. An exam- algae, or protozoa, do not reach this size. Not
ple of this is the engineering of a plant to express until the invention of the microscope (Antonie
a pesticide, thereby eliminating the need for ex- van Leeuwenhoek, 1684) was the detection, and
ternal application of pesticides. thus also the characterization, of microorgan-
isms possible [4].
Blue Biotechnology. In a very broad sense, If microorganisms are defined by their size,
marine or blue biotechnology can be understood many varied life forms come into this category:
as the various means of techniques of managing single-cell life forms with a real cell nucleus
6 Biotechnology

(e.g., algae, fungi, protozoa) and also some with- and pertain to the field of food microbiology.
out a real cell nucleus (bacteria and archaea) Microorganisms are used in the production of
which are of particular interest in general mi- sausages (particularly salami), sauerkraut, and
crobiology. Organisms with a genuine cell nu- milk products (lactobacilli). In Asia, a variety of
cleus are termed eukaryotic organisms, those fermented food products are produced on the
without prokaryotic organisms [5]. Today some basis of rice and soya [8, 9].The typical aro-
6500 species of prokaryotic bacteria and archaea matic properties and consistency of many food
are known, a relatively small number compared products originate from the activity of the mi-
with the number of the known species of eukary- croorganisms used. Nowadays, these organisms
otic organisms. However, it is estimated that the are generally used in the production process as
number of actually living species of prokary- starter cultures. The fermentation products are
otic organisms is much higher, although most of also often used as preservatives (acidification in
them are regarded as not cultivable under labo- lactic acid production by lactobacilli or alcohol
ratory conditions. Analyses of the metagenomes production by yeasts).
of the most varied habitats (total DNA assays of The monitoring of food in the framework of
samples from various environments, such as for- quality control involves excluding undesired mi-
est soil, compost, pond water, etc.) have yielded crobial infestation or providing evidence of it.
estimated values of up to one billion potential Microorganisms contribute towards stabiliz-
species of prokaryotic organisms worldwide. ing the global natural material cycles (e.g., car-
For most people, microorganisms have a bad bon and nitrogen cycles). Furthermore, the waste
image. The reason is not hard to find: some flows produced by man are largely reintroduced
microorganisms can cause disease in man and into the natural material flows through the ac-
animals (pathogenic microorganisms). Thanks tivity of microorganisms. The field of environ-
to the achievements of medical microbiology, mental microbiology addresses the question of
the often devastating effects of pathogenic mi- how microorganisms can be utilized for the re-
croorganisms in earlier times have been confined mediation of contaminated soil, water, and waste
due to the development of vaccines, antibiotics, gases. Successful industrial processes have been
etc. [6]. Nevertheless, microorganisms still re- developed and are used worldwide for wastewa-
present a threat to health (for instance to peo- ter and drinking water purification, brownfield
ple with a weakened immune system). The fol- clean-up, composting, and waste fermentation
lowing are a few examples of bacteria that are and also for the purification of contaminated
pathogenic to humans: Bacillus anthracis (an- gases [10 – 12].
thrax), Bordetella pertussis (whooping cough), Industrial microbiology, or white biotechnol-
Clostridium botulium (botulism), Clostridium ogy, deals with the production of industrially in-
tetani (tetanus), Corynebacterium diphtheriae teresting substances involving production vol-
(diphtheria), Shigella dysenteriae (shigellosis), umes of thousands of tons. Effective production
Vibro-cholerae (cholera). The development of of substances requires on the one hand an effi-
resistance to known antibiotics is a phenomenon cient production strain to supply as much target
that documents the variability of pathogenic mi- product as possible in the shortest time possi-
croorganisms [7]. This also explains why it is ble. On the other hand, it needs a technical fa-
necessary to promote progress in the field of cility where the high-performance microorgan-
combating infectious diseases with microbio- isms find optimum living and, therefore, produc-
logical methods. tion conditions and where in addition monosep-
tic cultivation in large volumes is guaranteed.
Biotechnology is closely linked to micro- Bioreactors fulfill these requirements: they can
biology. Biotechnology uses microbiological be operated with volumes of up to several hun-
metabolism performance to manufacture indus- dred m3 and are equipped with automatic mea-
trially relevant products. The sector with the surement and control systems adjusted to the
oldest tradition of applying microorganisms for growth needs of the microorganism [13, 14].
production purposes is the food industry. The Examples of microbially produced industrial
manufacture of beer, wine, vinegar, bread, and products are diverse. The list begins with mi-
cheese are early achievements of human history croorganisms that can themselves be the prod-
Biotechnology 7

uct of a biotechnological process and reach are the sites of protein biosynthesis and the mo-
the market as starter cultures [15]. Currently lecular mechanism taking place is extremely
the quantitatively dominant product is ethanol, sensitive to radical mutations. Thus, there are
of which over 24 × 106 (metric) tons are pro- areas within 16S rRNA that are extremely well
duced worldwide. Ethanol is produced with the preserved, but also some where base exchanges
yeast Saccharomyces cerevisiae and is mainly have been more frequent. An evolutionary gap
used as a substitute fuel or in admixture with between two organisms can be determined from
other fuels. Other products include pharmaco- the relative similarity of different genes for the
logically active agents (e.g., antibiotics, steroids, 16S rRNA of various prokaryotic organisms;
and alkaloids), bulk, special, and fine chemicals, this can be used as a measure of the degree of
food and feed additives, chiral intermediates, en- relationship [18].
zymes, antibodies, etc. Analogous to the investigations on prokary-
The total value of all biotechnologically pro- otic organisms, the characterization of the de-
duced substances worldwide (including bio- gree of relationships of eukaryotic organisms is
pharmaceuticals which are often manufactured derived from their 18S rRNA, a molecule with
with cultures of animal cells) must be in the approximately 2300 bases.
realm of > 100 × 109 euros annually. Besides comparing the base sequences of
rRNA, the amino acid sequences of universal
proteins may also be drawn on to establish rela-
2.2. Phylogeny and Taxonomy of tionships. This does not always involve the same
Microorganisms branchings of the phylogenetic tree as with the
comparison of rRNAs. This explains why tax-
2.2.1. Definition and Survey onomy, which owes its motivation primarily to
the potential inherent in the new molecular biol-
Taxonomy is the branch of biology which sub- ogy tools, is currently a highly dynamic area of
sumes individual groups of species into a hier- science.
archical system. For a long time, the kingdom Today, all living organisms on earth are clas-
was the highest category of creatures. Origi- sified into three domains: archaea, bacteria, and
nally, a distinction was made between animals eukaryota. The following representation of the
and plants. Later, the bacteria were added and manifold life forms of microorganisms adheres
then the fungi and plants were split up [16]. Fi- to this phylogenetic classification.
nally the archaea were given their own kingdom.
In recent years, genetic investigations have led
to a new classification with the “domain” super- 2.2.2. Bacteria
imposed over the kingdom. Whereas in earlier
times the aim was to understand and deduce re- Bacterial cells are small, generally less than 2
lationships among microorganisms on the basis µm in diameter and 2–5 µm long (there are only
of morphological, physiological, and cytologi- a few giants of 5 × 20 µm dimensions). The cells
cal similarities, in the last few decades genetic do not contain a membrane-enveloped nucleus;
similarity has been the criterion used to identify their DNA is rather a circularly closed double
relationships directly from the genetic material. strand lying within the cytoplasm. The bacte-
Nowadays, sequence analysis of ribosomal RNA rial DNA contains the total genetic information
(rRNA), which was developed by Carl Woese, is of the cell. In many bacteria there are additional
one of the methods by which phylogenetic trees DNA circles, the plasmids; however, they are not
can be generated [17]. required for growth under ordinary culture con-
Probably the best-known gene in the world is ditions. The ribosomes are small (sedimentation
the 16S rRNA (S, Svedberg unit for characteriz- constant in the ultracentrifuge: 70 S), and their
ing the behavior of sedimentation) of prokary- function is sensitive to various antibiotics that
otic organisms. This gene is composed of around do not act upon the cytoplasmic ribosomes of
1500 base pairs and in the course of evolution eukaryotic organisms. In contrast to eukaryotic
was only subjected to relatively minor changes cells, bacterial cells do not contain organelles,
through mutations. The reason is that ribosomes
8 Biotechnology

but often polyphosphates, poly(β-hydroxybu- Bearing in mind all the postulated, noncul-
tyric acid), or glycogen as storage substances tivable species, the actual number of bacterial
(Fig. 1). Most bacteria are surrounded by a cell phyla (or kingdoms) is probably far higher. Esti-
envelope that contains peptidoglycan (murein). mates assume that there will ultimately be about
Many bacteria are motile by either flagellar or 50 phyla (or kingdoms) of bacteria. The cur-
gliding movements. rent phylogenetic tree, therefore, can only be
regarded as a snapshot.
The bacterial phyla with the highest num-
ber of species known today are the proteobac-
teria, Gram-positive bacteria and cyanobac-
teria. Cyanobacteria are phototrophic organ-
isms closely related to Gram-positive bacte-
ria. Gram-positive bacteria form a group of
chemo-organotrophic bacteria. The phylum of
Figure 1. Longitudinal section through a bacterial cell
proteobacteria is the largest and physiologi-
ca) capsule; cm) cytoplasmic membrane; cp) cytoplasm; cally most varied of all bacteria. Representa-
cw) cell wall; f) flagellum; gly) glycogen; li) lipid droplets; tives of this phylum account for the majority of
n) nuclear material or bacterial chromosome; phb) poly(β- known Gram-negative bacteria of medical- and
hydroxybutyric acid); pi) pili; pl) plasmid; po) polyphos-
phate; rb) ribosomes; s) sulfur granules
application-oriented interest [20].
Even the possibly best-known bacterium, Es-
cherichia coli, belongs to this phylum. This ex-
ample is used to demonstrate the taxonomic clas-
Classification of Bacteria According to the
sification of an organism:
Phylogenetic System. Originally, the subdivi-
sion of bacteria was based on their morphology Domain: Bacteria
and physiology, in the last few years, however, Phylum: Proteobacteria
their classification has been completely revised Class: Gamma protobacteria
Order: Enterobacteriales
and established on a molecular biology level. Family: Enterobacteriaceae
Thus, the taxonomy of bacteria, especially the Genera: Escherichia
higher levels of classification, cannot yet be re- Species: Escherichia coli
garded as definitive. Some authorities believe The binomial nomenclature is used through-
that the degree of variance between different out biology. Each organism has a genus name
bacterial groups is sufficient to give them each and a species name and is thus specified unam-
“kingdom status” of their own. Thus, in some biguously.
publications reference is made to 13 kingdoms
of bacteria, in others to “phyla” instead of king-
doms. 2.2.3. Archaea
The classification presented is derived from
Bergey’s Manual of Determinative Bacteriology Several properties distinguish archaea from bac-
[9], which the bacteria are subdivided into the teria and eukaryota. Archaea are single-cell or-
following phyla: ganisms mainly with a closed DNA molecule
– Proteobacteria lying within the cytoplasm. They are of the
– Gram-positive bacteria same size as bacteria. Archaea have neither a
– Cyanobacteria cytoskeleton nor cell organelles, they are dis-
– Chlamydia tinguished from bacteria by their lack of pepti-
– Plantomyces doglycan and the different structure of their ri-
– Bacteroids/Flavobacteria bosomes. With over 200 species they are often
– Green sulfur bacteria found where extreme conditions prevail.
– Spirochetes Archaea have cell walls of pseudopeptido-
– Deinococci glycan and single-layer cell membranes formed
– Green non-sulfur bacteria from ether lipids with covalent bonded chains.
– Hyperthermophiles (3 kingdoms) Many species of archaea have adapted to their
Biotechnology 9

extreme surroundings, hence some species pre- Therefore, a great number of physiological and
fer temperatures of over 80◦ C (thermophile), biochemical properties have to be determined to
others live in salt waters (halophile) or in describe and identify a species unambiguously.
very acidic or alkaline environments (aci- Properties and description of prokaryotic or-
dophile/alkaliphile) [21]. ganisms:
Enzymes from such extremophile organisms,
1) Shape.
which are not only found among archaea, are
The majority of prokaryotic organisms are
particularly interesting for industrial applica-
spheres, rods, or helices; these organisms are
tions; indeed they seem predestined for use in
called cocci, rods, and spirilla, respectively
industrial processes with high temperatures and
(Fig. 2). Some organisms are encapsulated
salt concentrations [22]. In the area of amylases
by proteins or polysaccharides, and some are
thermotolerant enzymes are already applied for
combined to form packets, filaments, or con-
starch saccharification. Moreover the enzymes
sortia.
used in detergents (proteases, lipases, amylases,
2) Flagellation.
cellulases) are active and fulfill their functions
The kind of flagellation, length, type of undu-
at temperatures of 60◦ C and above [23].
lation, and mode of attachment of flagellae
The phylogenetic system of the archaea that
are important characteristics of prokaryotic
is valid at present subdivides the domains into
organisms (Fig. 3).
the following phyla [19]:
3) Gram Stain.
– Euryarchaeota The Gram stain, originally devised by CHR.
– Crenarchaeota GRAM (1884) to make bacteria visible in an-
– Korarchaeota imal tissues, is a reliable and fundamental
– (Nanoarchaeota is proposed as an additional cellular characteristic by which the bacteria
phylum) are divided into two parts: Gram-positive and
– Archaeota Gram-negative bacteria. It is based on the re-
The Euryarchaeota are a diverse group. tention or discoloration of bacteria by ethanol
This phylum comprises not only methanogenic after a staining procedure involving an ani-
species, which are strictly anaerobic organisms, line dye and iodine. In Gram-positive bacte-
but also strictly aerobic organisms. Furthermore ria, the dye is retained by a cell wall consisting
a large group of hitherto noncultivable organ- of a multilayered network of peptidoglycan;
isms belong to this phylum and similarly to that in Gram-negative bacteria, the dye is readily
of the Crenarchaeota, which include both hy- washed out because the cell wall consists of
perthermophilic representatives and mesophilic only two peptidoglycan layers and a highly
marine species. It is only very recently that re- permeable outer membrane envelope. The ba-
presentatives of the Korarchaeota phylum have sic structural differences are easily visible in
been cultivated successfully. ultrathin sections (Fig. 4).
4) Endospores.
Endospores are highly refractile, thermoresis-
2.2.4. Morphological and Physiological tant stages in the life cycle of two groups
Properties for the Identification of of Gram-positive bacteria belonging to the
Prokaryotic Species genera Bacillus and Clostridium. Endospores
are easily visible and tolerate pasteurization
The properties of prokaryotic organisms as pre- (treatment by moist heat for 10 min at 80 ◦ C).
sented here permit the identification of species Heating in an autoclave for 20 min at 120 ◦ C
by conventional methods if molecular biology is required to kill the spores.
techniques are not available or cannot be ap- 5) Need for Oxygen.
plied. The shapes of most species are similar, and Many prokaryotic organisms grow only in the
there are only a few morphological characteris- presence of atmospheric oxygen. They are
tics suitable for differentiation. In contrast, the strictly aerobic organisms. In contrast, strictly
diversity of metabolic features is tremendous. anaerobic organisms can only grow in the ab-
Prokaryotic organisms are extremely versatile. sence of oxygen. Facultatively anaerobic or-
10 Biotechnology

ganisms can grow under both conditions. The totrophic characterizes the ability to syn-
last group includes aerotolerant species which thesize the majority of cellular constituents
tolerate but do not use oxygen. Others use from carbon dioxide, and heterotrophic, the
oxygen if it is available. Some aerobic species derivation of cellular constituents from or-
are microaerophilic, i.e., they grow at low par- ganic compounds. The designations are usu-
tial pressures of oxygen but not in equilibrium ally combined, e.g., Chromatium okenii is
with air. photolithoautotrophic. Some organisms re-
6) Energy Generation. quire accessory nutrients, such as vitamins or
The production of energy, i.e., regeneration amino acids, for growth.
of adenosine triphosphate (ATP), occurs via 9) Natural Habitats.
three fundamental processes, namely, fermen- The ecological habitat is the place where
tation, respiration, and photosynthesis. The an organism or a population can usually be
strictly anaerobic fermentative prokaryotic found. Examples are marine water, the sed-
organisms rely only on fermentation. All strict iment of a lake, fertile soil, or the intestinal
aerobes regenerate ATP by oxidative phos- tract.
phorylation with oxygen as the electron ac- 10) Symbiotic or Parasitic Relationships.
ceptor. Mechanistically, energy generation by The close relationship between two dissimilar
denitrifying, sulfate-reducing, methanogenic, organisms is called symbiosis. It may be fa-
or acetogenic species is quite similar to aer- vorable for both (mutualistic) or unfavorable
obic respiration and is therefore designated for one (parasitic).
as anaerobic respiration. All photosynthetic
microorganisms contain light-absorbing pig-
ments, 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 ther-
mophilic organisms. The psychrophilic (or
kryophilic) species prefer temperatures below
20 ◦ C. Most organisms tolerate pH values bet-
ween 5 and 8 but prefer neutrality; a few are
acid-tolerant, acidophilic, or alkali-tolerant,
alkaliphilic.
8) Nutritional Types. Figure 2. Shapes of bacteria
All organisms that can use light energy for A) Cocci; B) Diplococci, without and with capsule;
growth are called phototrophic. In contrast, C) Streptococci; D) Tetrads; E) Package cocci (Sarcina);
F) Rods; G) Spore-forming bacteria, differing with respect
the term chemotrophic denotes energy conver- to shape and localization of endospores in the mother
sion by oxidation – reduction reactions that cell; H) Short rods, coccobacilli; I) Coryneform bacte-
involve either fermentation or respiration. ria; J) Mycobacteria; K) Vibrio-like bacteria; L) Spirillum
Organotrophic denotes the utilization of or- (Thiospirillum); M) Spirilla (Aquaspirillum); N) Spindle-
shaped bacteria; O) Trichomes of filamentous cyanobac-
ganic compounds, and lithotrophic the utiliza- teria; P) Filamentous cyanobacteria with heterocyst;
tion of inorganic compounds (ammonia, ni- Q) Filamentous helically shaped cyanobacterium (Spir-
trite, hydrogen sulfide, sulfur, hydrogen, car- ulina); R) Spirochaetes (Spirochaeta plicatilis)
bon monoxide, iron(II)) as reductants. Au-
Biotechnology 11

11) Composition of Cellular Macromolecules. The DNA is associated with histone protein. Mi-
The description of bacteria includes the Gram tochondria (for respiratory energy conversion)
type of the cell wall, the type of peptidoglycan and chloroplasts (in plants for photosynthetic
structure, the fraction of cytosine + guanine energy conversion) contain a prokaryotic type
in the DNA, the similarity index of the 16 S of DNA and ribosomes. Cytoplasmic ribosomes
rRNA, and data on DNA–DNA hybridization. are large (80 S). Endoplasmic membranes and
12) Antibiotic Resistance. various kinds of membrane vesicles compart-
The characterization of an organism may be mentalize the cell; they are involved in the in-
supplemented by its pattern of antibiotic re- gestion of nutrients (endocytosis) and the pro-
sistance. duction and excretion of proteins and particles
(exocytosis).
An internal skeleton, the cytoskeleton, con-
sisting of contractile protein (actin) filaments
and microtubules, gives the cell its shape and
confers ability to move and to transport mem-
brane vesicles within the cell. The cells of eu-
karyotic microorganisms can be motile by either
ameboid or flagellar movement.
Most eukaryotic cells and organisms are aer-
obic. Energy for growth is derived from respira-
tion or photosynthesis.
Eukaryotic microorganisms include the pro-
tozoans, algae, and fungi. Fungi are the most rel-
evant representatives of eukaryotic microorgan-
isms in biotechnology and are used to produce
antibiotics, secondary metabolites, and also or-
ganic acids, vitamins, etc. For this reason, fungi
will be considered here in more detail.
Figure 3. Flagellation of bacteria, flagellar movement

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


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

The cells of most eukaryotic organisms are usu- 2.2.5.2. Fungi


ally much larger than prokaryotic cells and have
diameters of 10 µm or more [24]. They contain Fungi are aerobic eukaryotic organisms; they
several structural components, compartments, form a separate kingdom within the domain
and organelles. Figure 5 shows a plant cell as of the eukaryota [25, 26]. There are unicellu-
an example of a eukaryotic cell. The nucleus, lar fungi, but the majority form filaments (hy-
consisting of a set of chromosomes that divide phae), (usually 5–10 µm in diameter) and grow
by mitosis, is surrounded by a double membrane. as masses of hyphae (mycelia). Many fungi form
12 Biotechnology

fruiting bodies. The shapes of fungi are diverse, eral hundred species and strains of these higher
and fungi are usually identified on a morpholog- fungi, which include many edible mushrooms,
ical basis. They are much less versatile metabol- in submerged culture. Some could be of interest
ically than bacteria, and metabolic properties are in biotechnology.
almost useless for identification. 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 for-
mation, bud formation, or fragmentation. Conid-
iospores are formed on the tips of hyphae (coni-
diophores), e.g., in the genera Aspergillus and
Penicillium. If the spores are formed within spe-
cial vessels, the sporangia, they are called spo-
rangiospores, e.g., in the genera Mucor and Rhi-
zopus. Bud formation is the mechanism of asex-
ual 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 meio-
sis forms a sporangium. In the higher fungi, the
zygote nucleus divides meiotically, and spores
are formed either within a sac (ascus) or by bud-
ding on top of the basidia; the spores formed
are called ascospores and basidiospores, respec-
tively.
The taxonomic classification of fungi is cur-
rently under debate and different suggestions
can be found. The subdivision presented herein
closely follows a traditional classification und
contains the following classes: basidiomycetes,
ascomycetes, zygomycetes, deuteromycetes, Figure 6. Mycelium, conidiophores, and fruiting bodies of
myxomycetes. fungi
A) Mycelium of fungal hyphae; B) Yeast dividing by bud-
Basidiomycetes. In basidiomycetes, the zy- ding; C) Sporangium of Mucor mucedo containing sporan-
gote enlarges to form a club-shaped cell, the giospores; D) Sporophore of Penicillium forming conid-
iospores on the tips of branched hyphae; E) Sporophore of
basidium. The basidiospores are formed from Aspergillus forming conidiospores on the tips of hyphae
projections of the basidium usually resulting in (sterigmata) located on the spherical end of the sporophore
four spores on the tip. Most species are terres- cell; F) Fruiting body (perithecium) of a typical ascomycete
trial fungi and live as saprophytes or parasites; containing asci and ascospores; G) Basidium with four ba-
sidiospores; H) fruiting body of a typical basidiomycete
many form mycorrhizas on trees. Nutrient media
and methods have been developed to grow sev-
Biotechnology 13

Ascomycetes. The ascomycetes are named deuteromycetes will possibly be abandoned in


for their formation of spores within a sac-like the future [27, 28].
zygote, the ascus. With the exception of a few
unicellular forms they grow as branched mycelia
with open cross walls; they are coenocytic. Many 3. Metabolism
ascomycetes grow on excrements of animals,
i.e., they are coprophilic, whereas others are par- The decisive factor for the development of a suc-
asites on plants or insects. Asexual reproduction cessful biotechnological production is an effi-
occurs by various kinds of conidiospores. cient biocatalyst. If this biocatalyst is a microbial
The order Saccharomycetales within the As- production strain, knowledge of its metabolic ca-
comycetes comprises all yeasts. Typical yeasts, pabilities is very important. Such knowledge can
such as Saccharomyces cerevisiae, are unicellu- lead to an increase in product concentration and
lar. They grow either as diplonts (with diploid product variety, because one catalyst can often
nucleus) or as haplonts (with haploid nucleus), be used to produce different products [29].
such as Schizosaccharomyces pombe. All yeasts Metabolism is generally concerned with all
are able to grow on sugar substrates, and a few the events occurring within a cell that not only
can even grow on alkanes (Saccharomycopsis, keep the cell alive but are also manifested in
Candida lipolytica) or on methanol (Candida growth [30]. All synthetic reactions leading to
boidinii, Hansenula anomala). Yeasts are organ- growth are energy-consuming (endergonic) and
isms often used in industrial microbiology. are commonly referred to as anabolic reactions.
In order to carry out these reactions, each indi-
Zygomycetes. The zygomycetes are lower vidual synthesis must be coupled with energy-
fungi and comprise unicellular and mycelial producing reactions of the catabolic sequence.
fungi; they have diverse nutritional habits. Metabolism therefore consists of an intricate
They grow in water, soil, or moist habitats. coupling between anabolism and catabolism
There are saprophytic and parasitic species. (Fig. 7), whereby energy transactions and trans-
The most important group biotechnologically fer mechanisms are based upon the basic laws
is that of the Zygomycetales, which include of thermodynamics [31, 32].
Mucor mucedo, Rhizopus nigricans, and Phy- Furthermore, in order to obtain a functional
comyces blakesleeanus. Several species are used biosynthesis, the microbial cell must also pro-
for the production of vitamins, carotenoids, or- duce a reducing agent, because biosynthesis in-
ganic acids, and enzymes. volves the reduction of small molecules of high
oxidation state to larger molecules of lower ox-
Myxomycetes. The myxomycetes or slime idation states. Both the energy and the reduc-
moulds grow on decaying plant tissue, form ing agent can be obtained from any of a number
multinuclear plasmodia, and produce fruiting of diverse reactions depending upon the growth
bodies of yellow, red, or brown color. There is conditions and the genetic system available [33,
not yet a consensus on the exact evolutionary 34] within the metabolism. Energy is gener-
affinities of myxomycetes, but these organisms ally dependent on adenosine triphosphate (ATP)
constitute a well-defined, homogeneous group and the reductant on nicotinamide dinucleotide
of approximately 900 species. NADH or NADPH.
The cell has a very sophisticated regulatory
Deuteromycetes. If a fungus is incapable control system that avoids oversynthesis of any
of sexual reproduction, it cannot be classi- chemical compound, thus securing its integrity.
fied with either the ascomycetes or basid- These regulatory control systems start at the
iomycetes, hence a third group of higher fungi cell membrane (substrate uptake) [35] and are
was created. There are many deuteromycetes of present throughout the pathways of catabolism
biotechnological importance: Aspergillus, Peni- and anabolism.
cillium, and Cephalosporium. Investigations of
the 18S rRNA revealed that the majority of
deuteromycetes could be assigned to the basid-
iomycetes and ascomycetes so that the group of
14 Biotechnology

Figure 7. Anabolic and catabolic reactions in microbial metabolism

3.1. Microbial Systems Biology tion in B. subtilis [39], and, in the case of ter-
penoids, yield increases by engineering meval-
In the future, systems biology will enable the onate biosynthesis in E. coli [40]. It is likely that
metabolism of some microorganisms to be mod- knowledge from systems biology will trigger
eled and mapped in silico to such an ex- tremendous progress in this application-oriented
tent that physiological performance and effects field. Moreover, strategies based on systems bi-
caused by environmental changes will be pre- ology will also be important for biotechnological
dictable. The essential prerequisites include suf- process development where an understanding of
ficient knowledge of metabolic material flow the complex regulatory networks of production
and network analysis, reaction kinetic analy- organisms will be indispensable for process op-
sis of biochemical networks (enzyme kinetics, timization [41].
global metabolism regulation), global analy-
sis of gene regulation networks (signal trans-
duction), analysis of populationwide systems 3.2. Energy Production
(quorum-sensing networks), and the abstraction
of fundamental regulation patterns. The first Redox reactions are among the most important
such model that integrates signal transduction, chemical reactions in living organisms. These
gene expression and metabolism has already oxidation – reduction reactions are of special im-
been introduced for the response of yeast to os- portance for energy production by living organ-
motic shock [36]. isms:
There is no doubt that such models signify
great progress for microbiology and biotechnol- ∆G0 =n F ∆E 0
ogy: quantitative simulations and targeted inter-
ventions in the metabolic network ideally open where ∆G 0 is the standard free energy of the
up the way to highly specialized, optimized pro- reaction, n is the number of electrons (or hydro-
duction organisms. In order to optimize specific gen atoms) involved, F is the Faraday constant,
types of metabolic performance or to acceler- and ∆E 0 is the potential difference between the
ate desired biosyntheses, industrial biotechnol- two redox systems. The simplest way to think
ogy is already relying to a great extent on the about these oxidation – reduction reactions is in
metabolic engineering of microorganisms. Here, terms of electron donors and acceptors. Thus,
mention should be made of the synthesis of ami- the substrate oxidized has a specific redox po-
no acids [37, 38], optimized riboflavin produc- tential that is at the lower end of the electrode
Biotechnology 15

potential scale, whereas the final electron accep- trochemical gradient (∆µ+ H ) across the energy-
tor has a potential towards the more positive end transducing membrane. Proton electrochemical
of the scale. The difference between the two po- potential is a thermodynamic measure of the
tentials corresponds to the energy obtainable in extent to which the proton gradient across the
a chemical reaction between them. If the poten- membrane deviates from equilibrium. This hy-
tials of the substrate (electron donor) and of the pothesis is based on two separate proton pumps,
final electron acceptor are known, one can easily whereby the ∆µ+ H is generated by electron trans-
determine the energy production in a microbial fer and is used to drive the second pump, in-
system (Fig. 8) [42, 43]. volving the enzyme ATPase, backward in the
direction of ATP synthesis. The final 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, ∆µ+ H , is the key element:
∆µ+
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
Figure 8. Redox potentials of various energy-producing mi- force are required. Driving forces can be concen-
crobial reactions
tration gradients, electrical potentials, metabolic
energy, or a combination of these. Four pro-
Because the potential difference between
cesses are known [45, 46]:
electron donor and acceptor can be significant,
the energy released could produce so much heat 1) In simple diffusion, solutes move passively
that the cell would be damaged. In order to avoid across the cell membrane, depending on the
such cell damage, the cell has an energy-release- concentration gradient and thermal motion of
controlling cascade system, which makes cer- the molecules (e.g., water, gases, low molec-
tain that energy is released only stepwise. This ular hydrophilic compounds, organic acids in
cascade system is referred to as the respiratory the protonated form).
chain or the electron transport chain. It is located 2) In facilitated diffusion, solutes require a car-
in the cellular membrane and is responsible for rier for their transport in addition to the con-
the production of energy by electron transfer in- centration gradient and thermal motion of the
volving an enzyme called ATPase (which forms molecules. In both simple and facilitated dif-
or hydrolyzes ATP) [35, 44]. The ATPase also fusion, no metabolic energy or proton motive
plays an important role for the transportation of force is required. The energy for the transport
substances across the membrane into the cell. stems from existing concentration gradients.
According to the chemiosmotic hypothesis 3) The term active transport is applied to a tight
for ATP production, the electron transfer chains coupling of transport to metabolism in the ion
are coupled to ATP synthesis by a proton elec- pumps, which are central to chemiosmotic
energy transduction. There are at least two
16 Biotechnology

distinct classes of active transport systems: reducing agent is produced separately. In plants
the membrane-bound and the binding protein and algae, a second photosystem exists that splits
transport systems. water into H+ and oxygen, whereby the hydro-
4) The group-translocation transport system gen ions reduce NADP+ and oxygen is set free.
comprises processes in which the passage In bacteria, however, such a second photosystem
of the substrate across the membrane occurs does not exist, and inorganic compounds such as
simultaneously with, and as a consequence thiosulfate are oxidized, with the electrons run-
of, chemical transformation of the sub- ning through the electron transport system to the
strate (e.g., phosphoenolpyruvate–glucose– reaction center and the proton via ferredoxin to
phosphotransferase system with most anaero- NAD+ (Fig. 9). This is the reason why plants and
bic and facultatively anaerobic bacteria). Ac- algae possess mainly cyclic and bacteria non-
tive transport and group-translocation trans- cyclic photophosphorylation.
port are often referred to as “primary trans-
port”, simple and facilitated diffusion as “sec-
ondary transport”. The carrier systems in- 3.4.2. Chemosynthesis
volved can be of the “uniport”, “symport”, or
“antiport” type. In chemosynthesis, all energy and reducing
Many bacteria pump natrium ions out of the agents are obtained by catalytic reactions of or-
cell by taking in two protons per natrium ion in ganic or inorganic compounds. In the former
an antiport system. Sulfate ions, on the other case, one refers to chemoorganotrophy and in
hand, are only taken in by some transport the latter to chemolithotrophy. In addition, three
systems if protons are simultaneously “sym- different energy modes, which depend upon the
ported”. electron donors and acceptors, are distinguished:
aerobic respiration, anaerobic respiration, and
fermentation [47].
3.4. Catabolism Organic substances are used as an energy
source by the vast majority of microorganisms
3.4.1. Photosynthesis that live in natural environments (Fig. 10). Na-
ture provides them with an abundance of large
Photosynthesis creates living matter out of inor- organic polymers. A polymeric substance con-
ganic material, replenishes the reservoir of oxy- sists of series of monomeric units linked to-
gen in the atmosphere in the cases of algae and gether. These monomeric units can be of the
plants, stores the energy of sunlight to support same kind (e.g., cellulose, starch) or of different
the life activities of organisms, and removes car- kinds (e.g., pectin, lignin, sucrose). Within the
bon dioxide: polymeric structure, the monomeric units can
CO2 +H2 A+light→ (CH2 O) +2 A+chemical energy
be linked in a straight chain with identical link-
ages or different linkages or in branched chains;
If A is taken to be oxygen the process is plant moreover, they can be linked in a specific se-
(or algae or cyanobacteria) photosynthesis; if it quence or at random. The structure of each of the
represents a sulfur atom the process is bacterial polymer substrates for microorganisms is very
photosynthesis [43]. important, because the enzymes produced by the
The process of photosynthetic energy conver- microorganisms are specific not only to the types
sion is initiated when photons are absorbed by of monomers, but also in most cases to the way
specific molecules, such as chlorophyll. This ab- in which the monomers are linked.
sorption leads to an ejection of electrons, which Since microorganisms can only take up
are accepted by the compound having the low- monomers, all enzymes involved in the break-
est redox potential in animate nature, namely down of polymeric substances must be extra-
ferredoxin. The electrons then move through the cellular. These enzymes are either membrane-
electron transport system, and the energy is fi- bound or released into the medium. In gen-
nally converted to ATP. Because the electrons eral terms, Gram-negative bacteria accommo-
return to the reaction center (Fig. 9), this sys- date these enzymes in the periplasmic space bet-
tem is called cyclic photophosphorylation. The ween the cell membrane and the cell wall and
Biotechnology 17

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 glucose. However, not all microorganisms fol-
Gram-positive bacteria and fungi have a ten- low the same route of glucose utilization. There
dency to release these enzymes into the medium. are at least four major pathways of glucose
This is one of the reasons why extracellular en- metabolism, of which three lead directly to pyru-
zyme studies are carried out predominantly with vate:
Gram-positive microorganisms, as well as yeasts
1) Embden-Meyerhoff (EM) pathway, often re-
and other fungi.
ferred to as the glycolytic or fructose bispho-
sphate pathway
2) Hexosemonophosphate (HMP) pathway, of-
3.4.3. Carbohydrate Metabolism
ten referred to as the pentose shunt or pentose
Most renewable resources are carbohydrates. phosphate pathway
The principal carbohydrate that serves as a car- 3) Entner-Doudoroff (ED) pathway
bon and energy source for microorganisms is
18 Biotechnology

4) Phosphoketolase (PK) pathway, almost en- bon dioxide. The electrons and hydrogen atoms
tirely specific for heterofermentative lactic removed from the individual organic compounds
acid bacteria are accepted by oxygen and form the other two
All four pathways of glucose metabolism end products, ATP and water. The tricarboxyl-
have a great number of intermediates and en- ic acid cycle, however, is not only an energy-
zymes in common. The details of these pathways producing cycle, but it is also responsible for
can be found in many textbooks [32, 34, 48]. the production of precursors for amino acid and
The EM pathway, for example, provides the antibiotic biosyntheses [49, 50]: 2-ketoglutarate
greatest amount of ATP, but it does not produce and oxalacetate ions. The tricarboxylic acid cy-
ribose-5-phosphate, the important precursor for cle serves, therefore, as the central pathway for
RNA and DNA biosynthesis; nor does it produce the production of energy as well as for anabolic
erythrose-4-phosphate, which is important for precursors.
amino acid biosynthesis. Microorganisms that Under ideal growth conditions, the interme-
are capable of using only the EM pathway for diates of the tricarboxylic acid cycle would be
glucose utilization are therefore not able to grow continuously withdrawn for anabolic reactions.
on simple media with glucose as the sole carbon Then, the formation of oxalacetate at the end of
source. They require growth factors or organic the cycle became vulnerable. Because oxalac-
compounds (e.g., yeast extract) for growth and etate is required not only as a precursor for an-
are referred to as fastidious microorganisms. abolic amino acid biosynthesis, but also for the
In contrast, the HMP pathway produces all condensation with acetyl-CoA to keep the tricar-
the precursors necessary for both RNA and DNA boxylic acid cycle alive, the organism must have
as well as for aromatic amino acid biosynthe- some safety device to ensure that oxalacetate
sis, but it produces only half the amount of ATP is always available; otherwise the tricarboxyl-
energy. This pathway does not produce pyru- ic acid cycle would be interrupted. The organ-
vate directly. The microorganisms must there- ism possesses a very fine control system for en-
fore possess part of the enzymes of the EM path- ergy production and biosynthetic requirements
way. It is therefore not surprising that both path- in form of replenishment sequence reactions. Al-
ways, EM and HMP, are common combinations, together, the microbial world has five enzymes
particularly in facultative anaerobic microorgan- at its disposal for such sequences, of which
isms. phosphoenolpyruvate carboxylase is probably
The ED pathway is unique, however, and the dominant: it catalyzes the formation of ox-
has so far only been found in bacteria. Al- alacetate from phosphoenolpyruvate and carbon
though this pathway is linked partly to the HMP dioxide. Phosphoenolpyruvate, in turn, is the in-
pathway in the reverse direction for precur- termediate from which pyruvate is formed in the
sor formation, pyruvate is formed directly be- EM pathway.
cause of the aldolase cleavage of 3-ketodeoxy- This double function of the tricarboxylic acid
6-phosphogluconate. The ED pathway can exist cycle as an energy-generating cycle as well as a
on its own and is used by the majority of strictly biosynthetic precursor-supplying cycle is very
aerobic microorganisms. The net result is simi- often referred to as amphibolic. This dual func-
lar to the HMP pathway, although one mole of tion, of course, must be controlled. There ex-
ATP can be formed only if the carbon atoms go ists one enzyme in the tricarboxylic acid cycle,
into pyruvate instead of precursor. isocitrate dehydrogenase (it catalyzes the reac-
tion: isocitrate → oxalosuccinate), which per-
forms this regulatory control. In the case of an
3.4.4. Aerobic Processes overproduction of ATP this enzyme is inhibited
by ATP, preventing any further oxidation and
All aerobic microorganisms use the tricarboxyl- ATP synthesis, until the biosynthetic steps lead-
ic acid (TCA) cycle as a main metabolic path- ing to RNA, DNA, aromatic amino acids, and
way. In this cycle, appropriate groups of en- fatty acids have caught up and utilized the ex-
zymes catalyze a series of consecutive transfor- cess ATP [33].
mations, including oxidations, which finally re- The general scheme in Figure 11 outlines the
sult in the complete oxidation of pyruvate to car- intricate interconnections between catabolism
Biotechnology 19

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

and anabolism among all those precursors neces- despite the inhibition of isocitrate dehydro-
sary for cellular biosynthesis. Aerobic processes genase (see Fig. 10).
in biotechnology are used to produce various 2) The gluconeogenic pathways. From the ox-
products, for example, antibiotics, amino acids, alacetate formed, the organism is using one of
and organic acids [29, 51, 52]. the earlier mentioned replenishment sequence
reactions to produce phosphoenol-pyruvate
and reverses the EM pathway to glucose-6-
3.4.5. Fats and Fatty Acid Metabolism phosphate; from there ribose-5-phosphate and
erythrose-4-phosphate can be formed via the
Fats are another major group of naturally occur- forward HMP pathway.
ring compounds (see Fig. 10). The majority of
fats are triglycerides, that is, fatty acids (R1 , R2 , A great number of enzymes are com-
R3 ) esterified with glycerol: mon to both carbohydrate and fatty acid
metabolism, which shows the economy of bacte-
rial metabolism. The principal differences are in
the control systems, which are outlined in detail
in the literature, for example, [32, 34].

These fats have to be converted into the


monomeric components, the fatty acids and 3.4.6. Hydrocarbon Metabolism
glycerol. This hydrolysis is carried out by en-
zymes called lipases. The glycerol component The metabolism of hydrocarbons has been ex-
is converted to dihydroxyacetone phosphate, amined in detail in the framework of environ-
which participates in the EM pathway. The fatty mental biotechnology in connection with clean-
acids, which vary in their carbon chain length, up of contaminated sites. Aliphatic hydrocar-
have to be activated by the formation of their bons are good substrates for a large number
respective coenzyme A (CoA) esters; this reac- of microorganisms. The straight-chain alkanes
tion step requires energy. These fatty-acid-CoA are more readily attacked than substituted or
esters subsequently undergo cyclic oxidation, branched-chain alkanes. The attack occurs at ei-
eliminating an acetyl unit at each turn. This type ther one end (monoterminal) or both ends (diter-
of oxidation is referred to as β-oxidation. The minal). The alkane is converted via the primary
acetyl-CoA formed after each cycle can then en- alcohol into the corresponding fatty acid and
ter the tricarboxylic acid cycle for the formation then via β-oxidation into acetyl-CoA as out-
of energy and biosynthetic precursors. lined previously. Methyl group oxidation incor-
However, this type of metabolism leading porates one atom of oxygen; this reaction is cat-
to acetyl-CoA does not produce the important alyzed by a mixed oxidase system consisting of
ribose-5-phosphate and erythrose-4-phosphate. three proteins: rubredoxin, NADH-rubredoxin
In order to obtain these precursors, all organisms reductase, and -hydroxylase. For the oxidation
whose metabolism leads directly into the tricar- of methylene groups, rubredoxin is replaced by
boxylic acid cycle via acetyl-CoA and not via cytochrome P450 and -hydroxylase by methy-
pyruvate have to build up the carbon chain men- lene hydroxylase [53, 54].
tioned above and have introduced two pathways Aromatic hydrocarbon utilization follows a
for this biosynthetic purpose: uniform biochemical concept. The great major-
1) The glyoxylate cycle. As mentioned earlier, ity of aromatic hydrocarbons, irrespective of the
overproduction of ATP inhibits the enzyme number of benzene rings, converge in their ox-
isocitrate dehydrogenase. The organism is idative metabolism to three major intermediates:
then capable of inducing two new enzymes, catechol, 3,4-dihydroxybenzoic acid (protocat-
isocitrate lyase and malate synthase, to cir- echuic acid), and 2,5-dihydroxybenzoic acid
cumvent the carbon dioxide-producing steps (gentisic acid):
of the tricarboxylic acid cycle. This new short-
cut TCA cycle leads to succinate and via gly-
oxylate to malate, and oxalacetate is formed
Biotechnology 21

The number of cells obtained per mole of sub-


strate in simple defined media is much smaller
than under aerobic conditions. In addition to the
cell material, large amounts of organic end prod-
The benzene nucleus in these intermediates ucts are formed, mainly primary alcohols, e.g.,
is cleaved either between adjacent hydroxyl ethanol or butanol.
groups (ortho cleavage) or between a hydroxyl Fermentations are normally classified ac-
and a carboxyl group (meta cleavage). In both cording to their principal fermentation products.
cases, the active incorporation of an oxygen Figure 12 summarizes the end products from
molecule is required. Ortho cleavage, via the 2- carbohydrates.
ketoadipate, and meta cleavage, via the keto acid The distinction between aerobic and anaer-
pathway, lead into the tricarboxylic acid cycle at obic carbohydrate metabolism is made at the
the level of acetyl-CoA, succinate, or malate and pyruvate level, because in anaerobic fermenta-
fumarate. As in fatty acid metabolism, the organ- tion the organism has to substitute for the tricar-
isms use the tricarboxylic acid cycle for energy boxylic acid cycle. In general the organisms are
production and require the glyoxylate cycle to- not able to produce the intermediates that serve
gether with gluconeogenesis to obtain pentose as precursors for amino acid and protein biosyn-
for RNA and DNA precursors. thesis. Therefore, fermentation processes cannot
be carried out on simple defined media, but al-
ways require complex media if the organism is
3.4.7. Amino Acid Metabolism to grow and maintain its metabolism. Most of
these fermentation processes are rather complex
The aerobic utilization of amino acids follows and details may be obtained from the relevant
a pattern that is very similar to the metabolism literature [32, 34, 55].
of other carbon sources [29, 49]. The only dif-
ference is the -amino group. In most cases, the
amino acid is first converted into the correspond- 3.4.9. Single-Carbon-Compound
ing keto acid by either cytochrome-linked oxi- Metabolism
dases, transaminases, or NAD(P)-linked dehy-
drogenases. The final product of the metabolism A few isolated genera of microorganisms are
always leads to the intermediates of the tricar- capable of oxidizing single-carbon compounds.
boxylic acid cycle. This is one of the reasons Microorganisms are called methylotrophs if they
why -amino acids are able to function as the have the ability to derive both carbon and en-
sole source of carbon, nitrogen, and energy for ergy from the metabolism of methanol, methan-
many microorganisms. As in fatty acid and hy- otrophs if they can do the same with methane
drocarbon metabolism, microorganisms grow- [53, 56 – 58]. Methane and methanol were the
ing on amino acids must possess the glyoxylate most common substrates used in the production
cycle and the gluconeogenesis pathway. of single-cell protein. The oxidation of methane
leads to methanol and from there to carbon diox-
ide.
3.4.8. Anaerobic Metabolic Processes The oxidation of methane or methanol to car-
bon dioxide, however, does not provide the or-
The breakdown, or catabolism, of organic com- ganism with any precursors for biosynthesis.
pounds in the absence of oxygen is generally re- These organisms therefore possess an unusual
ferred to as fermentation. The microorganisms carbon assimilatory pathway. Depending upon
that carry out fermentations are either faculta- the enzymic assemblage, formaldehyde is incor-
tive or obligate anaerobes. The most character- porated into ribulose-5-phosphate or into the ser-
istic difference between respiratory and fermen- ine pathway in order to build up the carbon chain
tative metabolism is in ATP energy production. for the biosynthesis of RNA, DNA, and all the
Because no electron transport occurs, redox re- amino acids required for protein biosynthesis.
actions of organic compounds play a major role. The absence of a tricarboxylic acid cycle, the
presence of anaplerotic sequence reactions, and
22 Biotechnology

Figure 12. Fermentation end products of carbohydrate metabolism

the glyoxylate cycle demonstrates the biosyn- the ore. Such ore leaching processes have led to
thetic capability of these pathways. a vastly improved mining industry and increased
the economy of ore mining.
3.4.10. Inorganic Metabolism Nitrogen Cycle. Nitrogen is the most abun-
dant gas in the atmosphere. Nitrogen-fixing
Some microorganisms are capable of using in-
organisms are capable of converting nitro-
organic compounds as an energy source in aer-
gen into nitrogen compounds that are accept-
obic metabolism or as a final electron accep-
able to plants. Nitrogen-fixing microorganisms
tor in anaerobic metabolism (anaerobic respira-
can be divided into free-living (cyanobacteria,
tion). Both of these are combined in nature in
Clostridium, Azotobacter) and symbiotic (Rhi-
the sulfur cycle and the nitrogen cycle, [32, 34].
zobium). The last group in particular may be-
Sulfur Cycle. Under anaerobic conditions, come of great biotechnological importance, be-
sulfate or thiosulfate can be reduced to sulfide by cause they live in association with legume plants
sulfate-reducing bacteria. Such processes occur and provide these with the necessary nitrogen
mainly in water-logged soils or stationary estuar- source even in nitrogen-starved soils, thus elim-
ies rich in organic matter, and can cause serious inating the need for large amounts of nitrogen
corrosion of iron and steel pipes in the soil. Hy- fertilizers. The nitrogen-fixing organisms con-
drogen sulfide is an extremely toxic product. If vert nitrogen to ammonia, which then can be ox-
the soil or estuaries are supplied with oxygen, idized by chemolithotrophs (Nitrosomonas and
hydrogen sulfide can be reoxidized to sulfate Nitrobacter) via nitrite to nitrate. This conver-
by aerobic sulfur-oxidizing microorganisms, for sion of nitrogen to nitrate is referred to as ni-
example, thiobacilli. The reoxidation of sulfur or trification. The reverse reaction, called denitri-
sulfide to sulfate is a microbial process of consid- fication, is an anaerobic process in which the
erable biotechnological significance. It not only nitrate or nitrite serves as electron acceptor. The
acidifies the soils and thus makes plant growth product of denitrification is nitrogen gas. Nitrifi-
possible, but the sulfuric acid formed can also cation and denitrification are important process
be used for ore leaching processes. Sulfuric acid steps in biological wastewater cleaning.
can form soluble sulfates with a wide range of The chemolithotrophs, however, face an en-
metals and by leaching old mining dumps it ergy problem. The redox potential of the inor-
helps concentrate these for further utilization of ganic compounds used as electron donors is well
Biotechnology 23

above that of the NAD/NADH couple that is re- pentoses, trioses, and the dicarboxylic acids
quired as reducing agent for biosynthesis. Since required for macromolecular biosynthesis [46,
the electrons can only enter the redox system 47].
at the cytochrome level, electron transport must
be reversed to obtain NADH. In photosynthesis,
this reversal can be carried out by the reaction 3.5.1. Amino Acids
center of the photosynthetic apparatus, but in
chemosynthesis the reversal has to occur with About twenty -amino acids are required for the
energy input from the oxidation of the inorganic biosynthesis of the numerous types of proteins
source. It is therefore not surprising that such that provide the catalytic capability of the mi-
organisms grow much better in the presence of croorganisms. These amino acids are produced
organic material, which they are able to assimi- from the following precursors:
late directly, thus obviating the reductive biosyn- Erythrose-4-phosphate → tyrosine, tryptophan
Phosphoenolpyruvate → phenylalanine
thesis and consequently the need for reductant Ribose-5-phosphate → histidine
formation. 3-Phosphoglycerate → serine, glycine, cysteine
Pyruvate → alanine, valine, leucine
2-Oxoglutarate → glutamic acid, glutamine,
arginine, proline
3.5. Biosynthesis Oxalacetate → aspartic acid, asparagine,
methionine, lysine, threonine,
The microbial cell consists of five major types isoleucine

of macromolecules: proteins, polysaccharides, The most important amino acids are glutamic
lipids, RNA, and DNA [32, 34]. These macro- acid and glutamine, which figure in transami-
molecules are built up from relatively few nation reactions during biosynthesis. Therefore,
monomers, which the cell has to provide or the availability of these two amino acids is vital
to synthesize: amino acids, sugar phosphates, for protein biosynthesis. The detailed pathways
fatty acids, ribonucleotides, and deoxyribonu- can be found in the literature [32, 34, 46].
cleotides. Theoretically, each of these -amino acids can
Microorganisms are divided into two groups: be produced commercially by microorganisms,
autotrophs, which are able to produce all some are already produced, but some practical
their cellular requirements from carbon diox- problems remain to be solved for some of them
ide as the sole carbon source, and heterotrophs [49].
– the majority of microorganisms – which pro-
duce the cellular material from organic com-
pounds. Photo- and chemolithotrophs use ATP 3.5.2. Lipids
and the reducing power produced by photo-
synthesis and/or oxidation of inorganic sub- Most of the fatty acids that occur in lipids contain
strates to reduce carbon dioxide to cellular ma- 16 or 18 carbon atoms and are saturated or unsat-
terial. The pathway for carbon dioxide reduc- urated with one or more double bonds. Acetyl-
tion is referred to as the Benson-Calvin cy- CoA is the only precursor for the biosynthesis of
cle. The actual carbon dioxide fixation reac- all fatty acids. In order to differentiate between
tion is catalyzed by ribulose-1,5-bisphosphate fatty acid catabolism and fatty acid biosynthe-
carboxylase; the primary product of this re- sis, the organisms employ CoA activation in the
action is 3-phosphoglycerate, which leads to former and an acyl carrier protein (ACP) in the
glyceraldehyde-3-phosphate, a member of the latter. The C2 units are added to the acetyl-ACP
EM pathway: in the form of malonyl-ACP. Once the required
chain length is reached, ACP is hydrolyzed and
3 CO2 +9 ATP+6 NADH2 the appropriate fatty acid is formed. Unsaturated
→glyceraldehyde−3−phosphate fatty acids are generally formed by dehydration
→+9 ADP+8 Pi +6 NAD+ of a hydroxy fatty acid.
The fatty acids are then esterified with glyc-
From glyceraldehyde-3-phosphate, these organ- erol, which is readily available from dihydroxy-
isms are capable of producing the important acetone phosphate, a member of the EM path-
24 Biotechnology

way. Finally, triglycerides are formed. The intro- can be reduced to almost zero by preculture con-
duction of a phosphate group gives a phospha- ditions.
tidic acid and a whole range of phospholipids. A much more complicated regulation is
If the phosphate group in the phosphatidic acid catabolite repression, which is mainly mani-
is replaced by a carbohydrate, glycolipids are fested in the so-called diauxie phenomenon. The
produced [32, 34, 46]. 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 sub-
3.5.3. RNA and DNA strate at a time and the presence of this partic-
Ribonucleotides consist of a purine or pyrim- ular substrate represses the enzyme system that
idine derivative, ribose, and phosphate groups. is responsible for the metabolism of the other
A purine or pyrimidine base attached to a ri- substrate. This repression occurs at the operon
bose is referred to as a ribonucleoside; if a phos- level and occurs as soon as the concentration of
phate group is attached to this ribonucleoside the first substrate becomes very small.
it is called a ribonucleotide. The most impor- Enzyme synthesis can also be inhibited by
tant pyrimidine derivatives are uracil, cytosine, end products within a short period of time. For
and thymine. The most important purine deriva- example, if the medium contains any α-amino
tives are adenine and guanine. The biosynthesis acid, the organism would use this amino acid
of pyrimidines and purines starts at the ribose-5- directly instead of producing it. The added ami-
phosphate level, which also represents the ribose no acid therefore represses the synthesis of each
in the nucleotide. The RNA consists of ribonu- enzyme in its synthesis pathway.
cleotides. Reduction of the ribonucleotides leads In addition to the regulation of enzyme syn-
to deoxyribonucleotides, the building blocks for thesis, the cell must also adjust the activity
DNA biosynthesis [44, 46]. of enzymes to its metabolic requirements. If a
monomer is synthesized in larger amounts than
needed for polymer synthesis, it is not necessary
3.6. Regulation to stop the synthesis of the enzyme completely,
but it suffices to reduce the activity of that partic-
The microbial cell possesses a complex of ular enzyme. This fast control action is referred
catabolic and anabolic capabilities with many to as feedback inhibition. The target enzymes
interconnected pathways. The cell is required to for feedback inhibition are called allosteric en-
maintain balance among the various parts of this zymes.
extremely complex network of reactions [32, 34,
44, 46, 48]. The cell therefore has developed
very intricate and refined devices to streamline 4. Metabolic Engineering
its economy.
Regulation can either be manifested in the The term metabolic engineering refers to the
enzyme synthesis or in the enzyme activity. In- genetic optimization of prokaryotic or eukary-
duction and repression of enzyme synthesis oc- otic cells that are used in industrial fermentation
curs at the genetic code, whereas feedback in- processes. In these processes, they are utilized
hibition simply regulates the activity of the en- for the economically production of proteins or
zyme. The importance for biotechnology is to re- other fine chemicals. Bailey [59] firstly intro-
alize that the first type of regulation is concerned duced the definition of metabolic engineering
with substrate, catabolite, and end product in- in 1991 as “the improvement of cellular activi-
hibition, whereas the second mainly involves a ties by manipulation of enzymatic, transport, and
transitional end product inhibition. regulatory functions of the cell with the use of
More than one enzyme usually is re- recombinant DNA technology.” However, one
quired to channel a substrate into intermediary of the early examples of successful genetic en-
metabolism. The substrate can therefore be re- gineering of a living organism for production
sponsible for a coordinate or sequential induc- purposes, namely the production of human in-
tion of enzymes. This activity of the cell nor- sulin in E. coli cells, had already been described
mally occurs during the lag phase of growth and
Biotechnology 25

quite some time before a name was given to this When, for instance, an organism should be mod-
new direction of science. ified to express a recombinant protein, the first
According to Cameron and Tong [60], there step is to design and create the DNA sequence
are five categories of metabolic engineering that that codes for the protein in an appropriate vec-
are grouped with respect to the objective of the tor system. Thus, one will enter the cycle at the
genetic modification: design level. The same is true when an existing
1) Improved production of chemicals already metabolic pathway should be extended in the
produced by the host organism. way that the original end product of that path-
2) Extended substrate range for growth and way is metabolized to a compound of interest.
product formation. However, when an existing metabolic pathway
3) Introduction of new catabolic activities for the shall be used to produce a given compound in
degradation of toxic materials. high yields, one will have to start by analyz-
4) Production of chemicals that are new to the ing the metabolic situation and the metabolic
host organism. fluxes in order to identify targets for genetic en-
5) Modification of cell properties, for instance, gineering. In this case, one will enter the cycle
to make them more resistant to the production at the analysis level. In particular in the latter
conditions. case, it is often necessary to go through the cy-
cle of metabolic engineering more than once,
The process of metabolic engineering is most
since complex metabolic networks within living
often described by a circle of elementary steps
cells often require several genetic modifications
that have to be performed once or several times.
to provide the compound of interest in improved
This circular process is demonstrated in Figure
quantities.
13.
Metabolic engineering is to a high degree de-
pendent on novel developments and improve-
ments in chemical analytics as well as molecu-
lar 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 consider-
able progress in metabolic engineering. For fur-
ther information, the interested reader is re-
ferred to the reviews by Nielsen [61] and Oster-
gaard [62] and the website of a scientific 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
Figure 13. Schematic illustration of the elementary pro-
production of a modified strain. Fermentation
cesses of metabolic engineering 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 4.1. Analysis of the Transcriptome,
Proteome and/or metabolome
Proteome, and Metabolome
– Design of a genetic modification that im-
proves/allows the synthesis of the compound The bottleneck of all of the traditional methods
of interest of transcriptome and proteome characterization
– Production of a modified strain is the limitation to an analysis of just a few sam-
– Fermentation and production ples at a time. While conventional methods are
Depending on the task at hand, one enters confining themselves to the examination of sin-
the engineering cycle at different entry points. gle genes, a DNA or protein chip experiment
26 Biotechnology

delivers a complete gene or protein expression sitive detection and selective identification of
pattern of the cell. The high degree of paralleliza- biochemical discrete compounds (carcinogens,
tion realized in biochips is the great advantage metabolites, proteins, etc.) or living systems
over classic molecular biological approaches. (bacteria, viruses, or related components) at low
levels in complex biological matrices, tissues,
and blood. In the future, it will be indispensable
4.1.1. Gene Expression Analysis using DNA to analyze compounds of the genome and the
Microarrays proteome. Therefore, new formats of biosen-
sors, so called biochips, have to be developed.
In the past few years, the complete DNA se- Such biochips or microarrays can be used for
quences of a number of different microorgan- identifying genes as well as changes in their
isms have been determined and can be ex- activity. Furthermore, these biochips allow the
ploited to optimize strains as well as recom- simultaneous detection of several disease end-
binant protein production. Strain optimization points using different bioreceptors such as DNA,
involves measurement of genomewide mRNA antibodies, enzymes, or cellular probes.
levels in wild-type and mutant strains using The high parallelization degree of biochips
DNA microarrays (Fig. 14) Furthermore, mi- is a great advantage over classic molecular bi-
croarray analysis can help identifying previously ological methods. While conventional methods
unknown genes required for recombinant pro- are confining themselves to the examination of
tein production. single genes, a DNA chip experiment delivers
Bioprocess optimization using microarrays a complete gene expressions pattern of the cell.
entails metabolic control analysis, modeling, DNA microarrays can be used for the purpose
and molecular biology to create new mutants of monitoring expression levels of thousands
and strains with an, for example, optimized pro- of genes simultaneously. Furthermore, biochips
tein production rate. Recombinant protein ex- can solidly simplify and accelerate a number of
pression exerts a metabolic burden on the host long and expensive diagnostic methods. Addi-
cell, whereby stress response mechanisms are tionally, gene expression analysis across various
triggered on different levels. Microarrays allow biological conditions, cell cycle states, tissues,
the investigation on a genome scale, which en- and subjects may help identify differentially ex-
ables the qualitative and quantitative character- pressed genes. This type of information is a valu-
ization of the burden on host cell metabolism. able pinpoint in the investigation of biological
Thereby, a better understanding of the impact of processes and functional disorders. Recapitulat-
recombinant protein production can be achieved ing, DNA chips provide a format to profile com-
by using the generated “snapshot” of the actual plex diseases and discover novel disease-related
cellular composition and activity. Additionally, genes. Applications for this technology include
the knowledge of the interaction of host cell gene expression monitoring, mutation detection,
metabolism with recombinant protein produc- metabolic engineering, drug development, tai-
tion is improved and contributes to process op- lor made therapeutics, SNP research, GMO de-
timization. tection, and high-throughput screening, among
Chip Technology. Alongside metabolome others. They have a profound impact on biologi-
analysis, analysis of various components of the cal research, industrial production, medicine and
proteome, the transcriptome or the genome will pharmacology and will be used as the biosensors
become increasingly valuable. New biosensors of the future [63 – 67]. For the understanding of
known as biochips will be required. DNA chip biological systems with up to 30 000 genes, the
technology has already opened up new ways measurement of RNA levels for a complete set
of studying disease in more depth and identi- of transcripts of an organism will be necessary.
fying far more possible targets. A DNA chip The use of glass slides as medium enables high
is an array of synthetic DNA sequences repre- spot densities on microarrays (up to 10 000 spots
senting different genes. Up to now, membrane per slide). A DNA chip experiment works by hy-
and immune biosensors are used to analyze sub- bridizing all of the gene probes on the chip with
stances of the metabolome. One important goal the nucleic acid target to be tested. cDNA la-
in chemical and biological sensing is the sen- beled with fluorescent tags is used, produced by
Biotechnology 27

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

reverse transcription of RNA from the sample methods made DNA chips affordable for aca-
to be analyzed. Hybridizing a sample of nucleic demic research laboratories. Since 1996, many
acid with many complementary gene probes in DNA arrayers have become available and the
parallel on a DNA chip produces a hybridiza- self spotted glass slide DNA arrays are today the
tion pattern with corresponding hybridization most popular format for gene expression profil-
intensity. DNA chip technology therefore en- ing experiments.
ables large numbers of genes to be screened si-
multaneously, 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 specific advantages and dis-
advantages in microarray manufacturing. The Figure 15. Microarray fabrication using an Affymetrix 427
arrayer
photolithographic approach relies on the in situ
synthesis of 25mer oligonucleotides using pho-
tomasks. This means that each probe is indi-
vidually synthesized on the chip surface. Pho-
4.1.3. Proteome Analysis using Protein
tolithography was developed by Fodor et.al. [68]
Microarrays
and commercialized by Affymetrix (Fig. 15).
In contrast, the ink-jetting and contact printing The utilization of protein biochips for the Pro-
methods attach presynthesized DNA probes to teome analysis offers a number of alternatives
the chip surface. While the in situ probe syn- and advantages in metabolic engineering. Pro-
thesis necessitate expensive and sophisticated tein microarrays make use of technological in-
equipment the contact and noncontact spotting novations and enable the proteome analysis in
28 Biotechnology

miniaturized test formats, thereby promising severely reacts are often unknown. However, the
significant advantages such as an improved an- sensitive dependence of most different parame-
alytical speed, a better separation efficiency, re- ters offers the possibility of applying specific
duced sample/reagent consumption, contamina- small changes of the protein expression pattern
tion reduction and reduced costs. Protein mi- in order to create sensitive biosensors.
croarrays consist of bound antigens or antibodies Ideally, the analysis of the proteome delivers
as capture molecules for detecting proteins in a the currently available set of all proteins, if there
complex mixture. They provide a huge potential is a way to maintain the quantitative relations of
for monitoring protein expression and protein all proteins during the analysis. Such data can-
profiling. not be obtained with classical molecular biology
Although the basic construction of such pro- since no strict connection between the amount of
tein microarrays is similar to DNA microarrays, mRNA and the amount of protein exists. Param-
the more delicate nature of protein structures has eters such as mRNA stability, posttranslational
hindered the development of such devices for the modifications, protein degradation, and others
analysis of proteins for a long time. This is due consequently prevent a statement over the cur-
to their more complex coupling chemistry, the rently available amount of protein. However, this
instability of the immobilized protein and the information is of utmost importance making a
far weaker detection signals. In principal, mi- high throughput analysis necessary. One attrac-
croarray technology enables both, the probing tive method is the use of protein microarrays.
of the genome and the proteome. Today, pro- Genomewide screens for protein function are of
tein chip technology focuses basically on the biological importance for many applications:
understanding of molecular pathways which al- – Analyzing protein expression profiles
lows conclusions concerning the molecular di- – Monitoring protein-protein interactions
agnostic, the drug discovery, and metabolic en- – Identifying protein posttranslational modifi-
gineering applications. These interferences can cations
be done because of the capability of protein mi- – Screening the substrates of protein kinases
croarrays to analyze the protein function of a – Examining the protein targets of small
whole genome level [69 – 73]. The results of molecules
such high- throughput screening approaches can – Proteomic analysis as a function of bioprocess
change our fundamental understanding of the cultivation conditions
cellular processes of life on the molecular level.
However, gene expression analysis does not suf-
ficient enable a reliable prediction of the func- 4.1.4. Metabolome Analysis and Metabolite
tion of a protein. Monitoring protein interactions flux analysis
is an extremely complex matter since the pro-
teome is the quantitative representation of the In order to improve a given strain of microorgan-
complete protein expression pattern of a cell un- isms or even more complicated a eukaryotic cell
der accurately defined conditions. The proteome line that is used in a biotechnological produc-
represents the protein equivalent of the genome. tion process, it is not only necessary to analyze
In contrast to the genome which is determined gene expression patterns or the proteome. Anal-
by the sequence of its nucleotides and therefore ysis of metabolic turnover is central for recog-
static, the proteome represents an extreme dy- nizing possible targets for genetic engineering.
namic object which is influenced by many pa- Metabolic turnover comprises a large number
rameters. Not all genes will be switched on at of biochemical reactions. For instance, in the
the same time in a cell and the sensitive balance well-examined yeast Saccharomyces cerevisiae,
between protein synthesis and protein degrada- about 1500 biochemical reactions have been es-
tion can vary widely under different metabolic timated involving more than 850 metabolites and
conditions. Consequently, a repeated analysis of cofactors [74]. Since in most of these reactions
the proteome will be successful only under ex- there is more than two educts and more than one
actly defined conditions, for example, cell cul- product, not to speak of the necessary cofactors,
ture conditions. In practice, this proves to be the metabolic turnover represents a complex net-
very difficult since conditions to which the cell work of biochemical reactions, also referred to
Biotechnology 29

as the metabolic network. The concept of iso- Flux Analysis. Once the network topology
lated metabolic pathways such as glycolysis, cit- has been identified, it is necessary to learn
ric acid cycle, or urea cycle as presented in most about the fluxes through each of the network
biochemical textbooks is therefore somewhat branches. This kind of analysis requires exper-
misleading since it indicates that these pathways iments as well as mathematical modeling. The
proceed independently. However, they are all in- most important concept behind flux analysis is
terconnected and represent a huge interdepen- referred to as metabolite balancing. According
dent network that can activate or slow down in- to this, the concentration of each metabolite is
dividual branches. Control over the activity of stationary inside the cell or in other words: the
individual metabolic branches can be exerted on generation rate of a given metabolite in one
the levels of transcription, translation, protein- metabolic pathway (v+ ) equals the rate of its
protein interaction, or enzyme activity regula- onward reaction through the sum of all down-
tion, which adds another level of complexity. stream metabolic pathways (v− ). Thus, for a
According to Nielsen [77] it is therefore neces- given metabolite the corresponding material bal-
sary to analyze the metabolic network in three in- ance can be described as
stances (metabolic pathway analysis) in order to
v+1 +v+2 +v+3 +. . .+v+i =v−1 +v−2 +v−3 +. . .v−i
fully oversee the metabolic activities in a living
cells, which is considered as a prerequisite for a Such balances are established for all relevant
rational optimization of the production strain: metabolites, such that a set of algebraic equa-
tions results that describe the flux of molecules
– Network structure (pathway topology). through the different branches of the metabolic
– Quantification of fluxes through the branches network. When some of the fluxes are experi-
of the network. mentally determined, the remaining fluxes can
– Identification of control structures. be deduced mathematically from this set of
Much work has been done in the past in equations. The beauty of the metabolic balanc-
biochemical laboratories around the globe to ing approach is its simplicity. But reliable pre-
identify the metabolic network structure at least dictions can only be obtained when the cofactor
for the more well-known microorganisms – in (NADH, NADPH, . . .) balances are also known
particular when they are of industrial impor- which means that all the pathways that gener-
tance. Thus, very often the presence of a cer- ate or consume any of these cofactors have to
tain metabolic pathway can be deduced from be considered in the formalism. Feeding the or-
the available literature. The most powerful ap- ganisms with 13 C-glucose in combination with
proach to identify a certain pathway when no in- a molecular analysis of the resulting metabolite
formation is available is called metabolic label- labeling downstream of glucose provides, how-
ing. Here, the organism is fed with 13 C-labeled ever, an easier solution. From the labeling pat-
glucose (or any other appropriate fundamental tern of all metabolites it is possible to set up
metabolite) and after a given time that is al- equations for the balances of individual carbon
lowed for metabolization the labeling pattern atoms. Thus, the number of equations describ-
of other intracellular components is identified ing the overall system increases the number of
and reports on the metabolic pathways involved. constraints for the solution and thus, makes bal-
From the analytical perspective, analysis of the ances of cofactors unimportant.
metabolite pattern is most often done by GC-MS The final step in analyzing the metabolic
(gas chromatography coupled to mass spectrom- network is to identify the regulatory mecha-
etry) but also by NMR (nuclear magnetic reso- nisms that control the fluxes through each of the
nance). Later on, biochemical assays addressing branches [75, 76]. It is very obvious that detailed
the activity of certain key enzymes can be used knowledge about flux control might immedi-
to confirm the presence of a certain pathway and ately identify possible targets of metabolic en-
dig out the cofactor requirements for the individ- gineering, for instance, to redirect the metabolic
ual steps within the pathway. Pathway identifica- flux away from a pathway that does not lead
tion just by enzyme assays is possible, however, to the desired product or consumes precursors.
very tedious and time consuming. In order to understand flux control, it is nec-
essary to study the regulation of the enzymes
30 Biotechnology

that are active around the branching points of B) Silencing of a gene that codes for an en-
the metabolic network [77]. Here the regulatory zyme or regulatory protein which should be
instances can be found at several hierarchical switched off or reduced in expression level
levels as mentioned earlier in this chapter. The C) Modifying an existing gene – and hence the
expression level of a given enzyme or any of coded protein – that is expressed by the or-
its regulatory proteins may be controlled on the ganism of interest, for instance, to alter the
level of transcription, RNA processing, or trans- substrate specificity of an enzyme or engineer
lation. DNA and protein chip technology, as de- its catalytic performance
scribed above, provide powerful approaches to In all three cases, the most suitable target for
unravel whether any control occurs on the level genetic manipulations is cloned DNA that can
enzyme expression. Finally, allosteric control be handled and tailored in the well-defined en-
over enzyme activity introduces another level vironment of a test tube without the complexity
of metabolic fine-tuning. The kinetic properties of an entire organism around, in particular the
of an enzyme like, for instance, its affinity for cellular membranes that may hinder the acces-
the substrate molecules, the maximum substrate sibility of the various reagents. Cloned DNA is
turnover rate and its sensitivity for product inhi- usually handled in form of plasmid DNA. A plas-
bition have to be characterized by biochemical mid (cytoplasm + chromatid = plasmid) is a cir-
assays. When these information and the current cular piece of double-stranded DNA that is au-
concentrations of the relevant metabolites are tonomously replicated in bacteria and may even
available, it should be possible to predict how be exchanged between them. Plasmid DNA oc-
the fluxes through individual branches of the curs also in wild-type strains since they are reg-
metabolic network will change when the activity ular extrachromosomal genes that can be easily
of one enzyme is altered by genetic engineering shared between certain bacteria. Many molec-
or when the feeding situation is changed. ular tools have been developed that are specif-
However, it should be emphasized at this ically designed to work with plasmid DNA in
point, that due to the enormous complexity of vitro. A particular family of plasmids, the so-
the metabolic network and all its regulatory in- called Expression plasmids or expression vec-
stances, it is very difficult to predict all metabolic tors as the more general term, additionally con-
consequences induced by just one genetic mod- tain the necessary nucleic sequences to initi-
ification. Thus, it may be necessary to com- ate transcription (promotor) and translation of
pletely analyze the metabolome of the modified a gene that has been correctly inserted into the
strain and – if the results are not satisfactory – go plasmid – instead of multiplication only. Expres-
through one or several more rounds of metabolic sion plasmids for prokaryotic cells differ from
engineering as sketched in Figure 13. This is par- those for eukaryotic cells [78]. The three differ-
ticularly relevant when the organisms respond to ent strategies to alter gene expression in more
alteration of their genetic make-up by opening detail:
up metabolic side pathways that are silent in the
wild-type strain. A) When a new gene should be introduced into
an organism or the expression level of an ex-
isting one should be increased, this particular
4.2. Design and Production of gene has to be cloned in a suitable expression
Genetically Optimized Strains for vector controlled by an appropriate promotor
that ensures a sufficiently high copy number
Production – In Vitro Mutagenesis of mRNAs and hence protein. The produc-
tion of human insulin in E. coli is a famous
In order to generate genetically modified organ-
example for the production of a recombinant
ism that might be superior to wild-type strains
eukaryotic protein in a prokaryotic organism.
for production purposes, there are basically three
B) When an existing gene should be silenced or
major options:
reduced in activity, there are several exper-
A) Introduction and expression of a gene that is imental strategies. Today the most promis-
not originally expressed by the organism or ing technique is RNA interference (RNAi),
only in small copy numbers a process by which double-stranded RNA si-
Biotechnology 31

lences gene expression. By transfecting cells to 10−11 of the total cell population. The spon-
with small interfering RNAs (siRNAs) with taneous mutation rate (probability of a mutation
100 % homology to a target mRNA sequence, per cell and generation) for a gene is about 10−5 ,
a specific knock-down of the corresponding and for a pair of nucleotides it is 10−8 . The num-
protein can be achieved. Bound by an RNA- ber of reversions (or functional back-mutations)
induced silencing complex (RISC), the siR- is of the same order of magnitude.
NAs induce the degradation of their homol- Induction of Mutations. The mutant fre-
ogous mRNA molecules in the cell, thus the quency can be considerably increased by treat-
protein expression level decreases. RNAi is ment of the cells with chemical, physical, or
a reverse-genetics approach to identify gene biologic mutagenic agents. Three classes of mu-
function by altering the phenotype of a cell tants can be distinguished with respect to their
[79 – 82]. genetic structure: 1) replacement of one base
C) Until the middle of the last century, tradi- pair by another, e.g., AT by GC; 2) a base pair
tional biotechnology concentrated on eluci- can be inserted or eliminated; 3) several base
dating new metabolic pathways and improv- pairs, DNA fragments, or even genes can be
ing the strains used for the production of anti- lost (deletion), change their position within the
biotics and other useful natural products, usu- chromosome (transposition), or be interrupted
ally by exploiting random mutagenesis and by insertion of foreign DNA (insertion). Class-1
selection. Later in the 1980ies, biologists be- mutants, called point mutants, revert readily to
gan to improve strains through metabolic en- their original structure.
gineering, that is, through the modification of Various chemical agents are in use to induce
single metabolic pathways by means of tar- mutations [85, 86]:
geted or site-directed genetic changes. The
1) Incorporation of Base Analogues.
two different approaches – random or site-
Base analogues are antimetabolites. Some are
directed mutagenesis – to improve the genetic
sufficiently similar to the natural purine and
make-up of an organism are still applied to-
pyrimidine bases that they are taken up by
day and shall be summarized here. However,
the cells and incorporated into DNA during
throughout the last years progress in molecu-
replication. They are able to fulfill their func-
lar biology has provided a pool of experimen-
tions, but tend to bind a wrong counterpart
tal approaches for targeted genetic changes
during replication, thus introducing a wrong
that are way too many and multifaceted to be
base pair and causing a point mutation.
reviewed comprehensively. Thus, only a few
2) Chemical Change of Bases.
principles can be discussed here, and are con-
Several mutagenic agents effect a chemical
fined to the best-known models of genetic re-
change in a base and thus cause an error in
search, the bacteria. The interested reader is
replication. For example, treatment of cells
referred to other chapters of this series or the
with nitrite results in the de-amination of
literature on molecular biology for more de-
adenine, guanine, and cytosine, and conse-
tailed and organism specific information [83,
quently in mispairing. Alkylating agents, such
84] (→ Genetic Engineering).
as ethyl or methyl methanesulfonate, ethyl-
eneimine, nitrogen mustard, and N-methyl-
4.3. Random Mutagenesis, Isolation and N-nitro-N-nitrosoguanidine (MNG), belong
to the most effective mutagenic agents. Acri-
Selection of Mutants dine dyes (proflavin) function by intercalation
Spontaneous Mutants. In bacterial popula- and result in insertions or deletions of single
tions, mutational events occur at certain rates base pairs.
without experimental treatment. These sponta- 3) Irradiation.
neous mutations, which result in mutants, are UV irradiation mainly affects pyrimidine
due to errors that occur during DNA replica- bases and results in replication errors.
tion. The frequency of spontaneous mutations in 4) Transposon Mutagenesis.
a bacterial population varies from gene to gene By conjugation, transposon-containing plas-
and species to species and may involve 10−2 mids (Tn elements) can be transferred from
32 Biotechnology

an appropriate donor bacterium to the bac- 4.4.1. Auxotrophic Mutants


terium to be mutagenized. Transposons are
DNA fragments that tend to “jump” to various If the mutation has affected the ability to syn-
positions in the DNA sequence of the chro- thesize, for example, the amino acid leucine,
mosome or plasmid. Their insertion interrupts the mutant will require leucine in the nutri-
genes and results in insertion mutants. ent medium. A mutant that is auxotrophic for
leucine (leu− ) can be recognized by compari-
The changes in the DNA described above in- son of its growth on two agar plates, one with
volve an alteration of its base sequence, called a and the other without leucine. While the pho-
premutation. Subsequently, the mutant character totrophic parent type (leu+ ) will grow on both
has to be expressed in the phenotype of the cell. plates, the auxotrophic leu− mutant will grow
About 10–20 growth cycles may be required for only on the supplemented agar.
nuclear segregation, dilution of enzymes, start- The enrichment of auxotrophic mutants can
ing or stopping the synthesis of enzymes, and be achieved by application of agents such as
other functions. penicillin that kill only growing cells and leave
non-growing cells unimpaired. If the population
of parent cells and rare leu− mutants is grown
4.4. Types of Mutants and Selection on a minimal medium (lacking leucine) only the
Principles parent cells will grow and after the addition of
penicillin will be killed within a few hours. The
A mutation is a rare event, and the number of mutants and some parent cells will survive and
mutants in a population is small. Many thou- the mutant fraction is increased by a factor of
sands of Petri dishes would have to be inspected 104 to 106 . Another method involves the prin-
and many millions of colonies (clones of single ciple of “lethal synthesis.” Growing cells will,
cells) would have to be examined to recognize for example incorporate radioactive phosphate
and isolate a mutant if there were not a selection or antimetabolites and will be killed while the
(enrichment) procedure for increasing the num- non-growing mutants will survive.
ber of the desired mutants within the population.
Enrichment is achieved either by killing part of
the parent cells or by growth under conditions 4.4.2. Regulatory Mutants
that confer growth advantage to the mutant cells.
There are many means for enriching mutants. In Some mutants, which in contrast to the parent
fact, designing strategies to select mutants is one type form a biosynthetic enzyme constitutively,
of the most difficult and demanding arts of the can be selected directly on the agar plate. For
microbiologist, and each type of mutant requires example, if an antimetabolite is added to the
a special trick. agar, the parent cells will incorporate it into their
The recognition of mutants poses another protein and stop growing. Mutants, however,
problem. Mutants that have lost or gained pig- that overproduce the normal metabolite because
ment, colony size or characteristics, or that have of overproduction of the biosynthetic enzyme
become resistant to toxic compounds or bacte- will not incorporate the antimetabolite; they will
riophages can be easily detected in a lawn of grow, and sometimes even show their regulatory
parent cell colonies. Some mutants show up af- defect by excreting the metabolite, thus initiat-
ter addition of indicators. The detection of aux- ing secondary growth of the parent cells in the
otrophic mutants requires comparison of growth surroundings of the mutant colonies. Some pro-
on two different nutrient media. One tries to rec- cedures for the selection and recognition of mu-
ognize mutants on an agar plate and to avoid la- tants are listed in Table 2.
borious test tube assays of thousands of isolated
cell clones. There are, however, many mutant
types that require these efforts. 4.4.3. Other Selection Methods

Various other methods can be used to separate


mutants from their parent cells. Because some
Biotechnology 33
Table 2. Procedures for selecting and recognizing various mutants

Kinds of mutants Selection or enrichment procedure Recognition of mutants


8
Mutants resistant to about 10 cells spread on a nutrient agar medium only the desired resistant mutants able to grow.
inhibitors, antibiotics, containing the inhibitory or killing agent.
toxic compounds, or
bacteriophages

Auxotrophic mutants penicillin technique or analogous procedures: cells if the cell suspension is spread on a complete
that require accessory grown in a medium lacking the accessory nutrients but medium containing the accessory nutrients and if the
nutrients (vitamins, containing penicillin or another agent that kills only colony pattern is then replicated on a minimal
amino acids, or other growing cells. Auxotrophic mutants not able to grow in medium, colonies that do not grow on the minimal
metabolites) for minimal medium survive. medium are auxotrophs.
growth

Mutants that lack the penicillin technique and/or direct isolation of pinpoint comparison of colony patterns on agar plates
ability to utilize a colonies: the cell suspension is spread on a nutrient agar containing different substrates; mutants unable to
special substrate that contains the substrate utilizable by the wild-type grow on a substrate will need a different nutrient for
cells at normal concentrations (0.5 vol%), and the growth and are therefore recognized. Excretory
substrate accessible by the desired mutants at a very mutants can be recognized by staining reactions (pH
low concentration (0.005 vol%). Pinpoint colonies are indicators) or by dyes added to the agar or after
transferred and screened. colony growth.

Temperaturesensitive penicillin technique to kill the wild-type cells growing comparison of colony pattern on plates that have
(conditional lethal) at their upper temperature limit. been incubated at different growth temperatures.
mutants Only those colonies that grow, e.g., at 25 ◦ C but not
at 37 ◦ C are isolated.

Mutants derepressed 1) continuous culture with the substrate as If the cell suspension is spread on a nutrient agar
or constitutive for growth-limiting factor; 2) alternating growth on two containing a noninducing substrate and after
catabolic enzymes different substrates; 3) growth in the presence of an incubation the colonies are sprayed with a solution
agent suppressing induction (antiinducer). containing the constitutively utilizable substrate +
indicator + inhibitor of enzyme protein synthesis,
only constitutive mutants immediately degrade the
substrate and change the indicator.

Mutants derepressed growth in the presence of an antimetabolite that inhibits only the resistant mutants grow, colonies of a clone
or constitutive for the growth of the wild-type cells. Among the resistant forming the enzymes of a biosynthetic pathway
anabolic enzymes cells are some mutants that are not subject to end constitutively will be surrounded by a halo of
product repression. satellite colonies due to the excretion of a metabolite.

cell constituents have lower or higher densi- compounds that are converted by the wild types
ties than the average cell mass, the cells can be to toxic compounds; this lethal synthesis will fi-
incubated under conditions that promote syn- nally kill the parent cells. Examples are monoflu-
thesis only in the parent or the mutant cells, oroacetate, bromopyruvate, or chlorate, which
followed by centrifugation in a sucrose gradi- are converted into fluorocitrate, bromolactate, or
ent. Cells rich in lipids (poly(β-hydroxybutyr- chlorine, respectively. The mutants are unable
ic acid), triglycerides), glycogen, calcium dipi- to catalyze such conversions and will therefore
colinate, magnetosomes, etc. can thus be sepa- survive. With this methods it is possible to se-
rated. In other cases tactic responses, such as lect for cells that are deficient in nitrate reduc-
migration in light–dark fields (phototaxis), in tase (chlorate, perchlorate), or for fermentative
gradients of nutrient (chemotaxis), or oxygen mutants that have lost the ability to form acids
concentrations (aerotaxis), can be used to se- (bromide, bromate). Successful biotechnology
lect mutants that are defective in transport sys- in many cases depends on the background in-
tems or in perception mechanisms. Adsorption formation and the ingenuity of the researcher in
on particle surfaces (glycogen or starch gran- selecting, recognizing, and manipulating the de-
ules, lipid droplets, lignocellulose particles) fol- sired organism.
lowed by fractional centrifugation can be used to In most cases, a single random mutagene-
select for mutants that have altered surface struc- sis decreases the characteristic of an organism
tures. The separation efficacy can be increased that is to be changed, such as, for instance, the
by the use of column chromatography or by catalytic performance of an enzyme. By apply-
suicide techniques. Suicide techniques employ ing high-throughput screening technologies, it
34 Biotechnology

is, however, possible to screen large numbers of strains in which the mutant plasmid has pre-
strains that are produced by random mutagenesis vailed.
and find the valuable ones with beneficial muta- Many more experimental options exist to in-
tions. Thus, by going through several rounds of troduce site-specific mutations. Many of these
random mutagenesis on the one hand and screen- are based on polymerase chain reaction (PCR)
ing for the desired phenotype on the other, this and the application of primers that already hold
process will identify those strains with consid- the mutation, or insertion or deletion. The inter-
erably improved properties for production [87]. ested reader is referred to Bowen [88].
The aim of modern metabolic engineering is
to study the cell as an integrated system of ge-
4.4.4. Targeted or Site-Directed Mutagenesis netic, protein, metabolite, and pathway events
that are continually changing. This approach
Site-directed mutagenesis allows modifying a should help to optimize the production of sub-
DNA sequence and thus the protein that is coded stances or to produce substances with improved
by this sequence in a specific and well-controlled properties. Historically, metabolic engineering
way. Thus, it is possible to exchange one or more is the targeted recombination of the DNA for
amino acid within the primary structure of the proteins involved in the metabolism or in reg-
protein and thereby alter its functionality, for in- ulation. Today, the objective of metabolic engi-
stance the catalytic performance of an enzyme. neering is to quantify the pathway alterations in
Compared to random mutagenesis, this is the response to environmental mediators taking into
more straightforward and direct approach not account the entire metabolism. Knowledge of in
relying on chances that a genetic change may vivo flux distributions in cells at different physi-
occur at a site that is useful for strain improve- ological states is of increasing importance for the
ment. However, it requires detailed information evaluation of biotechnological processes. Gene
on the three-dimensional shape of the protein expression data provide information on path-
and its primary structure. Many different ap- ways relevant to the metabolic models. Further-
proaches for targeted genetic alterations have more, the so-called combinatorial biosynthesis
been described that cannot be fully reviewed uses techniques from molecular biology to alter
here. We will summarize the most widely ap- or combine genes from biosynthesis pathways of
plied and flexible concept of oligonucleotide- the secondary metabolism to generate new prod-
based mutagenesis. ucts with improved pharmacological profil. The
In oligonucleotide-based mutagenesis, the importance of studying the entire metabolism
gene of interest is cloned in a plasmid. Af- rather than one gene or protein at a time has be-
ter denaturation of the double-stranded circular come increasingly relevant with the advent of
DNA, oligonulceotides are added to the system high-throughput genomic and proteomic tech-
that share sufficient sequence homology with the nologies, especially microarrays. The DNA mi-
coding strand of the gene to allow for hybridiza- croarray technology will gain great importance
tion. However, at the site where the mutations in the field of future biological research devel-
should be introduced the sequence of the olignu- oping into a key technology of the 21st cen-
cleotide is altered. Since this is only a minor tury hence revolutionizing modern biotechnol-
portion of the entire olignucleotide, hybridiza- ogy. DNA chips are used as biosensors in indus-
tion still occurs but the base exchange has been trial analysis, biomedical diagnosis, and foren-
introduced. Figure 16 sketches the underlying sic science. Although this technology provides
principle. a powerful tool that is widely utilized for gene
Chain extension by DNA polymerase and lig- expression, it is just beginning to find appli-
ation results in a double-stranded DNA with one cations outside of genomics. Further develop-
mutant strand and one wild-type strand. When ment of protein, cell, and tissue chips will make
these plasmids are then introduced in bacteria it possible in the future to carry out miniatur-
by transformation, semiconservative replication ized, highly parallel analysis of metabolic path-
produces homoduplexes with both strands be- ways. The challenge is to transfer these princi-
ing either of the wild-type or the mutant. When ples into technically applicable and precise ana-
colonies are grown, one can now screen for those
Biotechnology 35

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

lytical systems that can be used for many appli- Classic biocatalysts are yeasts, fungi, and bacte-
cations repeatedly. ria; animal and plant cell cultures have become
important only in more recent times (see Chap-
ter 9). Process development [93] [94] [95] in-
5. Cultivation and Bioprocesses volves the areas of biology (strain development
and bioregulation), reactor design, process con-
In this chapter, only the basic requirements for
trol [96], and medium design [97, 98] as well as
bioprocesses are described very shortly. For
downstream processing (product recovery, see
any further details concerning different types
Chapter 7).
of bioprocesses, bioreactors, kinetics, sterility,
and cleaning, the reader is referred to (→ Bio-
chemical Engineering). Very detailed informa- 5.1. Isolation of Microorganisms
tion about the monitoring and control of bio-
processes can be found in this contribution in Microorganisms can be purchased from cul-
Chapter 8. ture collections. But “microbes are everywhere,
Bioprocesses [89, 90] are used for the trans- the environment selects,” and a desired type
formation of organic matter with the help of bio- of microorganism can often be isolated from
catalysts, such as living cells, dead cells, or their its most probable natural habitat. For example,
components, e.g., enzymes [91, 92]. Biopro- methanogenic bacteria are present in the anoxic
cesses are utilized for chemical syntheses and for mud sediments of ponds and lakes, hemoglobin-
the conversion of waste material to either useful degrading bacteria in abattoirs, and hydrocar-
products (recycling) or effluents harmless to the bon-oxidizing bacteria around oil fields or leaky
environment (waste treatment). The outstanding engines of motor cars. From samples of these
feature of bioprocesses is their high synthetic materials, bacteria can be isolated by enrich-
potential to carry out a series of very compli- ment culture. The technique of enrichment cul-
cated chemical reactions in a one-step process. ture is simple and straightforward [99, 100].
36 Biotechnology

By establishing defined environmental condi- tion strains developed by repeated random muta-
tions with respect to the energy, carbon, and ni- genesis and screening. A comparison with pre-
trogen sources; hydrogen acceptor; gas atmo- cursor strains could identify the mutations re-
sphere; temperature; pH value; light; etc., and sponsible for increased production. That way,
by inoculating with a mixed population that con- production strains could be designed with just a
tains the desired metabolic type, the best adapted minimal number of defined mutations [101].
organism will dominate and overgrow all ac- Designer Bugs. The term used for microor-
companying organisms. ganisms tailored by genetic engineering meth-
Liquid enrichment culture is recommended ods so that they can carry out desired biochemi-
if the fastest growing organism is desired. In this cal conversions efficiently is designer bugs. Con-
case, the organism is repeatedly transferred from trary to the strategy used in the past to obtain
the original liquid culture through several sub- microorganisms for the production of a desired
cultures. If, however, a great number of strains product by coincidental mutagenesis and se-
with only slightly differing traits are to be sepa- lection, genetic engineering has opened up the
rated, the direct plating method is the preferable possibility of customized construction of pro-
technique: the inoculum is diluted and spread duction strains by metabolic engineering. The
on a solidified enrichment medium (agar dish). genome permits insight into the entire metabolic
During incubation the cells form colonies and potential of the organism. This insight is cur-
can be isolated separately. rently limited because the function of many gene
Whereas formerly these kinds of naturally products is not yet known. Organisms, such as
pure or well-balanced mixed cultures were used E. coli, Bacillus subtilis, Corynebacterium glu-
in industrial practice (e.g., yeast fermentations, tamicum, Pichia pastoris, or S. cerevisiae, are
vinegar and cheese production) now only pure preferentially applied in the development of de-
cultures are used. Pure cultures are obtained by signer bugs. Further important aspects govern-
diluting the enrichment culture and spreading ing the choice of host organism for a designer
the suspended cells on an agar surface or dis- bug are knowledge of metabolism, regulation
tributing the cells in an agar medium. After incu- mechanisms, growth properties, and hazard po-
bation, single colonies are isolated and streaked tential. Based on methods developed in the last
on various media to test for commensalic bac- few years for the analysis of the genome, tran-
teria. Finally, a clone of the desired organism is scriptome, proteome and metabolome, which
obtained as a pure culture. thus permitted a holistic consideration of the or-
Preliminary results indicate that previously ganism (“systems biology”), it will be possible
noncultivable microorganisms could be the key in future to develop designer bugs for biotech-
to the biosynthesis of active substances. The nological production of a desired product con-
genes responsible for the expression of the natu- siderably more quickly and specifically than in
ral substance biosynthesis could be inserted into the past.
microorganisms which can be grown easily, a A list with mostly all culture collec-
procedure known as the metagenome approach. tion worldwide can be found in the inter-
The metagenome is a set of all genetic mate- net [102] under the URL http://www.bacterio.
rial from organisms which cannot be cultured, cict.fr/collections.html and in ref. [103 – 105].
e.g., from the soil or from communities of or-
ganisms. The development of the metagenome
techniques becomes possible with the develop- 5.2. Requirements for Growth
ment of high-throughput techniques. The de-
velopment of high-throughput DNA sequenc- The process solution must primarily supply car-
ing began in the early 1990s as simplifications bon and energy. In order to maintain growth,
and automation made it possible to decode the nutrients, trace elements, and such growth fac-
sequence of entire genomes. In 1995 the first tors as vitamins must be added to the process
full genome sequence was published, that of the solution. Appropriate selection of the medium
Gram-negative bacterium Haemophilus influen- components is essential for proper functioning
zae. Of particular interest for biotechnology is and activity of the biocatalysts involved in the
the possibility of sequencing microbial produc- reaction.
Biotechnology 37
Table 3. Standard nutrient media
sodium, selenium, silicon, tungsten, and a few
a) Minimal medium for bacteria others, that are not required by all organisms.
K2 HPO4 0.5 g/L The compositions of a few simple synthetic nu-
NH4 Cl 1.0 g/L
MgSO4 · 7 H2 O 0.2 g/L
trient media are presented in Table 3.
CaCl2 · 2 H2 O 0.1 g/L
FeSO4 · 7 H2 O 0.01 g/L
Glucose 5.0 g/L
5.2.1. Chemical Composition of Bacterial
Trace element solution 1.0 g/L
Cells
b) Complete medium for bacteria
Peptone 10 g/L Bacterial cells harvested by centrifugation from
Yeast extract 1.0 g/L
NaCl 2.0 g/L
a culture growing in liquid medium contain
MgSO4 · 7 H2 O 0.2 g/L about 70 wt% water. The elemental assay of the
dry mass of E. coli is: approximately 50 % car-
c) Complete medium for fungi (pH 6.0) bon, 20 % oxygen, 14 % nitrogen, 8 % hydro-
Malt extract 10 g/L
Yeast extract 4 g/L gen, 3 % phosphorus, 1 % sulfur, 2 % potassium,
Glucose 2 g/L 0.05 % each of calcium, magnesium, and chlo-
KH2 PO4 0.5 g/L rine, 0.2 % iron, and a total of 0.3 % trace el-
NH4 Cl 1.0 g/L
ements. The organic analysis of the dry mass
d) Vitamin solution for soil and water bacteria of cells is presented in Table 4. It should be
Biotin 0.2 mg noted that the values refer to a specific bacterium
Nicotinic acid 2.0 mg grown in a specific environment and harvested
Thiamin 1.0 mg
Sodium 4-aminobenzoate 1.0 mg
in a specific phase of growth. The results may
Sodium pantothenate 0.5 mg differ if, e.g., the cells encountered limitation of
Pyridoxamine 5.0 mg the nitrogen source and accumulated poly(β-hy-
Cyanocobalamine 2.0 mg droxybutyric acid) (up to 90 wt%) or glycogen
Distilled water 100.0 mL
Two to three mL of this solution is added to 1 L (about 50 %) within their cells.
of nutrient solution.
Table 4. Overall macromolecular composition of Escherichia coli
(mass percent, dry matter basis) [106]
e) Trace element solution Protein 55.0
MnCl2 · 4 H2 O 3 mg/L RNA 20.5
CoCl2 · 6 H2 O 5 mg/L DNA 3.1
CuCl2 · 2 H2 O 1 mg/L Lipid 9.1
NiCl2 · 6 H2 O 2 mg/L Lipopolysaccharide 3.4
Na2 MoO4 · 2 H2 O 3 mg/L Peptidoglycan 2.5
ZnSO4 · 7 H2 O 5 mg/L Glycogen 2.5
H3 BO3 2 mg/L
Total macromolecules 96.1
Soluble pool of building
The growth of microorganisms is dependent blocks, vitamins 2.9
Inorganic ions 1.0
on the presence of water or moisture. Nutrients
to be used as sources of energy and for synthe-
sis of cell constituents are dissolved in water.
The growth requirements for various microor- 5.2.2. Carbon and Energy Sources
ganisms are different, and many recipes for the
composition of nutrient media are known. Ba- Only autotrophic organisms are able to synthe-
sically, all chemical elements that constitute the size all organic cell constituents from carbon
cell substance have to be present in utilizable dioxide as the main carbon source. All oth-
forms. There are ten macroelements that are ers derive the cell carbon from organic com-
constituents of all organisms: carbon, oxygen, pounds, which usually serve as both carbon and
hydrogen, nitrogen, sulfur, phosphorus, potas- energy sources; they are partially assimilated
sium, calcium, magnesium, and iron. In addi- and partially oxidized (dissimilated). Glucose,
tion, there are microelements (trace elements), the monomeric constituent of polysaccharides
such as manganese, nickel, cobalt, molybde- such as cellulose or starch, can be used by the
num, zinc, copper, vanadium, boron, chlorine, majority of microorganisms. Many other natural
38 Biotechnology

compounds can be utilized and degraded by one 5.2.8. Hydrogen Ion Concentration
or another microorganism.
The majority of microorganisms prefer a pH of
about 7.0. However, there are acidophilic and
5.2.3. Accessory Nutrients alkaliphilic microorganisms. Yeasts and other
fungi prefer pH 5.0. Buffers, in most cases phos-
In addition to carbon and energy sources, many phates, are used to maintain the desired pH, al-
organisms require accessory nutrients (growth though the CO2 /HCO− 3 system or organic sub-
factors), such as vitamins, amino acids, purines, stances may do as well. The pH value is very im-
or pyrimidines (see Table 3). portant: the drop from pH 7.0 to pH 6.0 means a
tenfold increase in the concentration of H+ ions,
which is a significant change.
5.2.4. Sulfur and Nitrogen

These elements can be used in their oxidized 5.2.9. Carbon Dioxide


forms as sulfate and nitrate. Ammonium ion is
the most common nitrogen source for microor- Many microorganisms require a higher concen-
ganisms. tration of CO2 than exists in the atmosphere,
such as 10 vol%. This requirement applies to
those bacteria that in their natural habitats (in-
5.2.5. Oxygen testinal tract, tissue, milk, blood, fermenting
juices) are exposed to high CO2 partial pres-
The oxygen atoms of the cellular constituents are sures.
mainly derived from water, substrates, or carbon
dioxide. Atmospheric oxygen serves mainly as
a terminal hydrogen acceptor of aerobic respira- 5.2.10. Aeration
tion, being reduced to water.
All obligately aerobic microorganisms require
oxygen as an electron acceptor for energy gen-
5.2.6. Complex Media eration. For growth in thin layers on liquid or
solid media atmospheric oxygen may suffice. In
Many microorganisms can grow in a simple nu- liquid media aerobic bacteria grow only on the
trient medium such as that listed in Table 3 a. surface if the medium is not agitated and aer-
For Leuconostoc mesenteroides such a synthetic ated. Only dissolved oxygen is utilized; its sol-
medium contains 40 components. If the nutri- ubility in water is very low (6.2 mL/L at atmo-
ent requirements of an organism are not exactly spheric pressure and 20 ◦ C). Therefore oxygen
known, complex nutrient media can be used, has to be supplied continuously to the cell sus-
such as yeast extract, yeast autolysate, peptone pension growing in submerged culture in flasks
(Table 3), meat extract, wort, carrot juice, co- or fermenters. To meet the increasing oxygen
conut milk, or horse manure extract. In other demand in a growing culture, the speed of agita-
cases complex media are used for economical tion, the oxygen concentration in the gas phase,
reasons, for example, whey permeate, corn steep and the total gas pressure can be increased. Var-
liquor, soybean extract, or molasses. ious kinds of fermenters have been designed to
increase the gas transfer from the gaseous to the
liquid phase. The final cell density and yield of
5.2.7. Solid Media microorganisms depends on the rate of oxygen
transfer. For fermenter design and scale-up, both
For solidification of media, agar is added at a the respiration rate (oxygen uptake) of the mi-
concentration of 1.5 to 2.0 wt%. Agar melts at croorganisms and the oxygen transfer rate from
100 ◦ C and solidifies on cooling to below 45 ◦ C. gaseous to liquid phase have to be known. Be-
cause the formation of desired products is highly
dependent on oxygen supply – whether the cells
Biotechnology 39

are able to take up oxygen at their maximum res- 5.3. Sterilization


piration rate or only much less – aeration poses
one of the most important problems in fermenter The basis of microbiological laboratory meth-
technology. ods and of the conservation of food and feed
products is the killing of microorganisms. Ster-
5.2.11. Anaerobic Techniques ilization means the removal of living microor-
ganisms, and can be achieved by moist heat, dry
For the growth of strictly anaerobic bacteria heat, filtration, irradiation, or chemical means
the complete exclusion of oxygen is required. [108].
Anaerobic techniques involve deaerated nutri-
ent solutions, tightly sealed flasks, incubation in
anaerobic jars, the use of chemical oxygen ab- 5.3.1. Moist Heat
sorbers (pyrogallol, dithionite or the “gas pak,”
which contains hydrogen- and carbon dioxide- Vegetative cells of bacteria and fungi suspended
evolving agents and a catalyst to promote the in water are killed at temperatures around 60
oxyhydrogen reaction), resazurin as a redox in- to 80 ◦ C within 5 to 10 min; yeast and fungal
dicator, anaerobic hoods, and skilled hands to spores are killed above 80 ◦ C, and endospores
practice these anaerobic techniques. of bacteria at 120 ◦ C within 15 min. Because
endospores of bacteria are highly tolerant to var-
5.2.12. Media Preparation ious environmental conditions, they are ubiqui-
tous and are distributed through the air. Their
Substrates for industrial applications are usually presence requires that all nutrient media and
included in complex media, which must be sup- canned foods be sterilized in an autoclave at
plemented with special compounds, such as a about 121 ◦ C for 20 min. If conditions do not al-
nitrogen source, various nutrient salts, or cer- low the germination of spores and the growth of
tain trace elements. Organic precursors are also spore-forming bacteria, e.g., in acid fruit juices,
added in some cases for efficient product forma- jam, or desserts, heating to 80–100 ◦ C for 10 min
tion. Many of the feedstocks need appropriate will suffice. This is called partial sterilization or
pretreatment in order to provide for good assimi- pasteurization.
lation by the microorganisms. Starch-containing
materials are prepared by milling or steam treat-
ment for softening and swelling. For many pro- 5.3.2. Dry Heat
cesses, especially when yeasts are used, starch
must first be broken down to sugar by treatment For killing bacterial endospores by dry heat,
with amylase derived from barley malt or with longer exposure times and higher temperatures
microbial amylases. Other feedstocks, such as are required than with moist heat. Glass and
wood, must be pretreated with acid or alkali. other heat-resistant equipment can be sterilized
Sulfite liquor is stripped from sulfur dioxide by for 2 h at 160–180 ◦ C in a dry oven. In any case
aeration or neutralization. Molasses is purified appropriate indicators or soil samples that con-
by acidification and centrifugation or filtration. tain bacterial spores are included in the oven to
In some cases heavy metals have to be removed test whether adequate temperatures have been
from feedstocks prior to their use. For processes reached and even the extremely heat-resistant
using solid substrates, see [107]. spores have been inactivated.
In general, the raw materials are dissolved or
suspended in water and the resulting medium
is heated, filtered, and sterilized. The complex 5.3.3. Filtration
composition of the media used in industry causes
considerable problems. For downstream pro- Solutions containing thermolabile compounds
cessing (harvest, concentration, and purification can be sterilized by filtration through nitro-
of product) or for analytical assays during the cellulose membranes, kieselguhr, porcelain, as-
process, additional pretreatment of the raw ma- bestos, and others. Usually filters with a pore
terial is needed to avoid unfavorable side effects. size <0,2 µm are used.
40 Biotechnology

5.3.4. Irradiation Surface cultivation is usually applied in the


lab scale in fungal research. Large Erlenmeyer
UV irradiation is used to keep rooms partially flasks or specially designed vessels, such as the
sterile. Bacteria and their spores are killed rather Fernbach, Roux, or Sakaguchi flasks, containing
quickly, but fungal spores are only moderately small volumes of liquid are used. The flasks
sensitive to radiation. Ionizing radiation (X-ray, and liquid are sterilized and inoculated with
gamma radiation) is used to sterilize food and spore suspensions or small pellets of mycelium.
other compact materials. Growth and product formation are dependent
upon the depth of the medium layer and the
amount of surface provided by the flasks, which
5.3.5. Chemical Means in turn affects the necessary oxygen supply.
In large-scale production large pans are used.
Ethylene oxide is used to sterilize food, They are filled with substrate, inoculated, and
plastics, glassware, and other equipment. β- then kept at controlled temperature and air hu-
Propionolactone and diethyl carbonate are midity. Pans are stacked on suitable racks, and
added to nutrient solutions. The presently used connected with a system of overflowing tubes.
soaps and detergents applied at moderate tem- The liquid medium is poured in at the top. In this
perature kill all vegetative cells and even many way, an entire stack can be loaded at once. If the
spores. Cleaning by the methods practiced in the stack is designed appropriately, it can be steril-
kitchen provides almost sterile glassware. ized, inoculated, aerated, and maintained under
Remarkably, the killing of microbes is a pro- controlled conditions. The same principle can
cess that follows first-order reaction kinetics. be applied for solid substrates, which are first
In a population there are allways some cells or inoculated and then loaded into the pans. The
spores that are more resistant than the majority. material is spread at a thickness of several cen-
Accordingly, sterilization procedures are most timeters onto screens or perforated metal sheets.
likely to be effective on relatively clean sub- Columns filled with carrier material onto
strates. which the microbes are immobilized are also
considered a surface culture as long as the col-
umn is not completely flooded. The medium is
5.4. Types of Bioprocesses trickled through the column and air is injected at
the bottom. The product is then collected from
In chemical technology, a choice is to be made the carrier, which is made of wood shavings,
between batch processes or continuously oper- plastics, or ceramics. This type of surface cul-
ated processes. Many parameters usually favor ture has been used for vinegar production and
the second over the first choice. However, in sewage treatment. However, it was abandoned
biotechnology, the batch process still predom- and replaced with submerged culture. Remark-
inates. This specific problem is treated in more ably, the immobilization methods that were de-
detail (also in regard to engineering aspects) in veloped for such biocatalysts as living cells, rest-
→ Biochemical Engineering. ing cells, enzymes, or organelles are formally
similar to these conventional processes.

5.4.1. Surface Culture


5.4.2. Submerged Culture
Surface cultures are mostly used when fungal
mycelia act as catalysts. Mycelium is grown in In this process, the microbes are suspended in the
shallow pans containing a small volume of the liquid medium. The simplest example of a sub-
medium. As the mycelium grows, it eventually merged culture consists of a vessel without any
forms a compact mat. This method was typically agitation device, in which microorganisms settle
used in the production of acids, such as citric at the bottom and form a compact layer. This is
acid. In large-scale production, surface culture the classic arrangement for all types of ethanol-
is being abandoned more and more and replaced producing processes. Some agitation will occur,
by the submerged culture method [109 – 111].
Biotechnology 41

however, from the movement of rising carbon cooling and heating coils. Gases that form during
dioxide bubbles. the process (CO2 , H2 , or evaporated solvents)
For submerged, anaerobic processes, simple are collected at the top. These large tanks have
agitation devices for gentle mixing will suffice. openings for loading, inoculation, harvesting,
This configuration allows for the mixing of liq- and cleaning. Large units are mostly sterilized
uid and gas. Carbon dioxide or other gases, such with steam.
as nitrogen, can be added during the process. Extremely large containments with capaci-
ties of thousands of cubic meters are used in
sewage treatment. They are usually constructed
5.5. Process Layout of concrete and allow automatic feed and re-
moval of material. Sludge treatment plants are
5.5.1. Reactors often equipped with heating elements to allow
thermophilic processes for methane production.
Appropriate reactors in biotechnology have to The gas is collected and stripped of hydrogen
meet the requirements for agitation, aeration, sulfide if necessary.
corrosion resistance, and aseptic operation. Var-
ious designs of stirred vessel are therefore used
in the laboratory and in production plants. Con- 5.5.3. Reactors for Aerobic Processes
tainer construction is based on principles similar
Bioreactors used for aerobic processes are ba-
to reactors used in heterogeneous catalysis.
sically similar to those used for anaerobic pro-
In former times, bioprocesses were carried
cesses. In addition, they contain devices for aer-
out under nonsterile conditions and took place
ation and agitation. The capacities used in in-
in vats of wood. Beer today is brewed in large
dustry may vary considerably, from 10 to 200
cubical containers coated with ceramic tiles. For
m3 . Large tanks of 1000 m3 and more are also
the more conventional surface processes (citric
used for aerated, sterile processes (e. g., glutamic
acid, acetic acid), small pans were used.
acid). For aseptic processes the tank is sterilized
A basic problem in aerobic processes is the
with steam before loading; a slight overpres-
efficient transport of oxygen into the liquid and
sure is often maintained during cultivation. Ag-
the microbial cells. Very efficient devices have
itation principles vary considerably and include
been developed to provide air for aerobic re-
mechanical as well as pneumatic and hydrody-
actions. Some examples of more modern reac-
namic systems.
tor configurations for agitation and aeration are
The most commonly used type of bioreac-
shown in Figures 20 and 21.
tor is the stirred tank reactor (STR) using a
flat blade turbine (FBT) for agitation (Fig. 17).
This configuration provides excellent mixing
5.5.2. Containments for Anaerobic Processes
and mass transfer in media of low viscosity.
Large units for anaerobic processes are made Drawbacks are high power demand, poor per-
of wood, metal, or coated concrete. They have formance with highly viscous liquids, and rather
no covers and consequently cannot be operated poor interchange of material between mixing re-
aseptically. However, submerged anaerobic pro- gions around the blades. Air is normally injected
cesses can be performed under conditions that by a sparger. Larger units are equipped for pneu-
favor the formation of product and inhibit the matic agitation, in which movement is caused by
accumulation of unwanted organisms by proper injected air. For proper hydrodynamic control,
selection of reaction parameters, such as pH and various kinds of baffles and draft tubes can be
temperature. Lactic acid, for example, is pro- inserted.
duced at 50 ◦ C and low pH, and the development
of competing microbes is negligible. 5.5.4. Inoculation
In other cases, closed vessels are used. They
are usually made of stainless steel, have large After inoculation with the microorganisms, the
capacities of up to 1000 m3 or more, and are process should start immediately and the reac-
equipped with agitators and, if necessary, with tion should proceed fast. The amount of active
42 Biotechnology

cell culture added is therefore critical and de- For scaling-up, the liquid cell culture is
pends on the size of the batch. Scaling-up from placed in liquid broth, first in test tubes (b), then
the original starter culture to the inoculation in small (c), and subsequently in large Erlen-
broth is done in several steps in large-scale in- meyer flasks (d) containing approximately 200
dustrial processes (Fig. 19). The starter culture mL of medium. Proliferation of the culture takes
is kept deep frozen (−80 to −90 ◦ C) in sealed place by shaking the flasks overnight at constant
vials or in vials that were previously subjected temperature. Larger amounts of inocula are then
to lyophilization (a). They can be stored in this prepared in agitated and aerated fermenters of
way for several months or even years without various sizes depending on the volume of the
any damage. For short-term storage at 4 ◦ C, agar production plant (e, f). In the early stages of
slants are prepared (g) and purity is tested on this procedure (steps a through d) the inoculum
Petri dishes (h) before inocula are scaled up for is transferred under sterile conditions in glove
production. boxes.
In fungal inocula proper wetting of the spores
is achieved by adding small amounts of surfac-
tants to the broth. If inoculation by spare suspen-
sions 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 pro-


cess layout. The standard process is still the
batch operation, which consists of loading, in-
oculation, 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 specific production can be attained by
using semicontinuous or sequential-batch oper-
ations. 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 pro-
duction. Modern vinegar production is operated
in the cyclic manner and maintained by fully au-
tomated process control.
Fully continuous operation, that is, the con-
stant flow of medium into a reaction vessel and
the simultaneous removal of an identical amount
Figure 17. Stirred tank reactor (STR) equipped with flat
of the process solution, has shown the highest
blade turbine (FBT) and baffles for agitation and a sparger production rate. However, practical and biolog-
for gas distribution ical constraints, especially the high contamina-
The ratio of H: D is usually 3 (max. 5). tion risk, have so far prevented the general intro-
duction of continuous processing into biotech-
Biotechnology 43
44 Biotechnology
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
Figure 18. Various bioreactor configurations [112]
a) Gas; b) Motor; c) Draft tube; d) Baffles; e) Mechanical foam breaker; f) Cylinder
A) Multistage flat blade turbine reactor (FBT) and baffles (d) making up the classical stirred tank reactor (STR)
B) Forced agitation with draft tube (c) and ship propeller
C) Combination of flat blade turbine and draft tube, operated with an overflow pattern
D) Combination of flat blade turbine and draft tube, flodded
E) Simple vessel with hollow stirrer element for self-aeration and distribution
F) Same as E, but with draft tube resulting in vortex flow patterns
G) Torus reactor; annular form with ship propeller
H) Tower with multistage flat 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 filled vessel. Forced agitation with
ship propeller.

nology. Therefore, only processes that need not In contrast to large-scale industrial processes
be carried out under aseptic conditions are op- continuous cultivation methods are widely used
erated continuously such as sewage treatment, in the laboratory [113 – 115]. The growth-
methane formation, and feed yeast or ethanol controlling factor is either the concentration of
production from sulfite liquor. nutrients in the medium (chemostat system) or
The potential advantages of continuous cul- the concentration of insoluble biomass sensed by
tivation are substantial because of the efficiency optical measurement (turbidostat methods). The
of the process and the uniformity of the final purpose of these control systems is to maintain
product. As a result, much effort is being made a constant concentration of nutrients, cells, and
to develop a sound basis of knowledge necessary metabolic products in the vessel (steady state).
for using this process strategy. 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 45

be kept under precise control by selecting the ap- the production of chiral compounds. Straathof
propriate dilution rate. All types of variations of et al. [121] list 134 industrial biotransforma-
the single-stage continuous cultivation are pos- tions. Just short of 90 % of the products de-
sible, such as multistage arrangements, with or scribed are chiral fine chemicals. In future, this
without recycling. field will undoubtedly acquire greater impor-
tance and new reaction sequences for the pro-
duction of compounds with several stereo cen-
5.6. Process and Product Overview tres will be established.
Also bulk chemicals are produced by fer-
The potential of biotechnology consists in its mentation or biotransformation processes. Bulk
ability to replace classical chemical production products mean products exceeding 10 000 t an-
processes and to facilitate the production of new nually. It is expected that, by the year 2010, 6–
products. It is undisputed that, particularly in the 12 % of bulk products and polymers produced
area of basic and fine chemicals production, the by chemical means will already be produced
use of biotechnological processes is meaningful by biotechnological processes. High-volume,
since biotechnologically produced goods are to be
– biotechnological processes are usually distin- found in the food, livestock feed, and the drinks
guished by their high specificity (relating to and tobacco industries. A prime example of a
the conversion of substrates) and selectivity bulk product derived by an enzymatic process
(relating to the product spectrum) is acrylamide. Global biotechnological produc-
– biotechnological processes often use renew- tion capacities have continually expanded in the
able resources as raw materials, thus con- last few years and are today probably in the re-
tributing to the much discussed sustainability gion of 100 000 t/a. Monomers and polymers
of products and processes produced by biotechnological processes for the
– biotechnological processes can be carried out plastics and polymer industry are becoming in-
under mild reaction conditions in terms of creasingly interesting. Biotechnologically pro-
pressure, temperature, and pH. duced polymers, such as polylactide (PLA), 1,3-
propandiol (PDO) and poly-3-hydroxybutyrate-
In view of these facts, in the past hundred co-3-hydroxyhexanoate (PHBH) could provide
years a multitude of industrial biotechnologi- the basis for innovations.
cal processes have been developed [116 – 120] As for the energy industry, besides bioetha-
whose efficiency exceeds that of chemical pro- nol two further products should be mentioned:
cesses, and this has helped to establish them in biogas and hydrogen. Whereas the technology
the long run (Table 5 and 6). One interesting for biogas recovery is state-of-the-art, hydro-
area is the production of fine chemicals. The gen production is far more complex and requires
term “fine chemicals products” refers to sub- long-term research efforts. Biogas recovery is a
stances that are highly functional and for which recycling method for residues. The product can
world demand is typically quantities of less than be separated comparatively easily, which is cru-
10 000 t/a. From a chemical point of view these cial to the cost-effectiveness of the process.
products are usually distinguished by having
several reaction centers and frequently by chi-
rality. Classical syntheses of these substances in- 6. Biocatalysis and
clude several reaction steps using stoichiometric Biotransformation
quantities of reagents and often deploy extrava-
gant protective group chemistry, expensive no- 6.1. Introduction
ble metal/heavy metal catalysts, and drastic re-
action conditions, e.g., aggressive solvents. Here Biocatalysis (also called biotransformation) is
biocatalysis allows synthesis under considerably the conversion of one substance (substrate) to
milder reaction conditions in terms of pressure, another (product) by a microorganism or an iso-
temperature, and acidity. At present, due to the lated enzyme. It is a chemical reaction catalyzed
excellent enantioselectivity of enzymes, white by a particular cellular enzyme or by an en-
biotechnology methods are primarily used for zyme originally produced within the cell. Most
46 Biotechnology
Table 5. Products obtained by fermentative processes of biotechnology [122 – 124]

Product/process Annual production t/a Price EUR/kg Market value 106 EUR Main application
Amino acids
l-Glutamate 1500 000 1.20 1800 flavour enhancer
l-Lysine 700 000 2 1400 animal feed additive
l-Threonine 30 000 6 180 animal feed additive
l-Phenylalanine 10 000 10 100 aspartame, medicine
l-Tryptophan 1200 20 24 animal feed, nutrition
l-Arginine 1000 20 20 medicine, cosmetics
l-Cysteine 500 20 30 (incl. extraction) food, pharma
Other amino acids and 3000 pharmaceuticals, cosmetics,
derivates nutrition
Acids
Lactic acid 150 000 1.80 270 food, leather, textiles
Gluconic acid 100 000 1.50 150 food, textiles, metal,
construction
Citric acid 1000 000 0.80 800 medicine, food, metal,
detergents
Acetic acid 190 000 0.50 95 food
Itaconic acid 4000 co-monomer
Solvents
Acetone* 3000 000 – solvent
1-Butanol* 1200 000 – solvent
Bioethanol >18 500 000 0.4 solvent, basic chemical,
energy source
Biomass
Starter cultures 100 food
Baker’s yeast 1800 000 2300
Mineral yeast
Enzymes
Enzymes (total) 1830
Detergents 580 detergents
Food industry 500 starch degradation,
proteases
Textile, leather industry 250 tanning
Animal feed 170 phytases, proteases
Food
Cheese 15 000 000 150 000
Erythritol 30 000 2.25 67 sugar substitute
Drinks and tobacco
Beer 138 000 000 2.5 drinks, tobacco
Wine 27 766 000 drinks, tobacco
Antibiotics
Bacitracin A 4 3000 12 healing of wounds
Bacitracin A >200 <120 24 animal feed additive
Virginiamycin 70 250 17.5 hog feeding
Cyclosporin 3 5200 15.6 organ transplantation
Monensin >3000 8 24 animal feed additive
Penicillins 45 000 300* 13 500 medicine, animal feed
additive
Cephalosporins 30 000 medicine, animal feed
additive
Tetracyclins 5000 50 250
Antibiotics (ca. 160 on 19 000
market)
Other active substances
Acarbose 300 active substance
Biopolymers
Polyhydroxyalkanoate packaging
Polylactide 140 000 2.25 315 packaging
Xanthan 40 000 8.4 336 food, oil production
Scleroglucan oil production
Pullulan film former in foodstuff
applications
Dextran (derivatives) 2600 200** 520 blood substitute
Hyaluronic acid 500
Polyglutamic acid
Biotechnology 47
Table 5. (continued)

Product/process Annual production t/a Price EUR/kg Market value 106 EUR Main application
Vitamins
Riboflavin (b2 ) 30 000 active substance, animal
feed additive
Cyanocobalamin (b12 ) 20 25 000 500 active substance, animal
feed additive
Vitamin C 80 000 8 640 food, animal feed
l-Sorbose (vitamin C 50 000
precursor)
Amino sorbite
Lipids
Phytosphingosine cosmetics
Animal feed
Galactooligosaccharides 2500 3.50 9 prebiotics
Cosmetics
Dihydroxyacetone suntan preparations

of these enzymes are necessary for the normal In both whole cells and enzymatic biotransfor-
metabolism and reproduction of the cell. In bio- mations the catalytically active species is the
catalysis, however, these enzymes are simply enzyme. Enzymes can be subdivided into six
used as catalysts for chemical reactions. In ad- classes (classified by the Committee on Enzyme
dition to their natural substrates, many enzymes Nomenclature of the International Union of Bio-
can utilize other structurally related compounds chemistry) depending on the type of reaction
as substrates and therefore may catalyze non- they catalyze (Table 7) and are listed numeri-
natural reactions upon addition of foreign sub- cally in a catalogue published by the committee
strates to the reaction medium. Thus, biocataly- [125] (→ Enzymes).
sis or biotransformation is a specific category of
chemical synthesis.
6.3. History

6.2. Classification of Biocatalysts Biocatalysis includes some of the oldest human


known chemical transformations such as the use
Enzymes and whole cells can be applied in a bio- of yeast for baking or brewing or the use of
transformation in many different forms. Usually chymosin from the stomach of young cattle for
a whole cell biotransformation is a growth de- cheese production. Records proving these an-
coupled process, so resting cells (harvested and cient technologies date back to about 7000 to
washed after fermentation) are employed. The 6000 bc.
cells may be intact or permeabilized (e.g., for Another old and well-known example is vine-
better substrate uptake). The different types of gar production, which has been known since the
biocatalysts are: dawn of recorded history. It is the oldest example
of microbial oxidation (Fig. 20).
– Whole or treated cells (e.g., lyophilized, per-
The process has evolved along traditional
meabilized)
lines of fermentation without knowledge of the
– Organelles
underlying biochemistry. In 1862, Pasteur de-
– Cell-free multienzyme systems
scribed the biochemical nature of vinegar pro-
– Combination of individual cell-free enzymes
duction [126]. Subsequently Brown confirmed
– Single cell-free enzyme
in 1886 that the oxidation of alcohol to acetic
Isolated Enzymes may also be applied in dif- acid proceeded through acetaldehyde [127].
ferent forms. In most cases, it is not necessary Bacterium xylinum, which causes the reaction
to purify an enzyme to homogeneity, a crude to proceed, was first described by Brown and is
cell extract or partially purified solution may the first microbial catalyst.
also be employed as biocatalyst. In some well- Today, the oxidation of ethanol to acetic acid
known technical enzyme preparations, several by acetic acid bacteria is well understood; it
isoenzymes are present (e.g., pig liver esterase). proceeds via two successive dehydrogenations,
48 Biotechnology
Table 6. Biotechnology products obtained by biocatalysis and biotransformation [122 – 124]

Product/process Annual production t/a Price EUR/kg Market value 106 EUR Main application
Basic chemicals
Acrylamide 100 000 1.40 28 –
Amino acids
l-Aspartic acid 13 000 – aspartame production
l-Methionine 400 20 infusion solutions
l-Dopa 300 – active substance
l-Alanine 500 – infusion solutions
d- and l-Valine 50 – –
l-tert-Leucine 10 500 –
l-Carnitine 200 – –
β-Phenylalanine >1
Food
Glucose 20 000 000 0.30 6000 liquid sugar,fermentation
medium
Fructose sugar from HFCS
Isoglucose, HFCS 8000 000 0.80 6400 liquid sugar
l-Hydroxybutanedioic acid 100 20 acidifying agent
Palatinite sugar substitute
Isomalt 70 000 sugar substitute
Aspartame 10 000 – sweetening agent
Fructooligosaccharide 10 500 2–3 prebiotics
(inulin)
Antibiotics (derivatives)
6-APA 10 000 – –
7-ACA 4000 – –
d-4-Hydroxyphenylglycine 7000 – –
Intermediate products
(S)-2-Chloropropionic acid 2000 – herbicide synthesis
d-Pantolactone 2000 – –
(S)-Methoxyisopropylamineseveral thousands – herbicide synthesis
(Outlook after chiral switch
by Frontier)
(R)-2-(4 -Hydroxyphen- 1000 – –
oxy)propionic acid
(R-HPOPS)
(S)-Phenethylamine etc., 500 – –
optical active amines
d-Mandelic acid >200 ca. 20 chiral auxiliary
Ethyl (S)-4-chloro-3-hy- >150 – cholesterol-reducing drugs,
droxybutyrate e.g., Atorvastatin (Lipitor)
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 – active substance
Ephedrine 1500 60–90 pseudoephedrine, chiral
auxiliary
N-Acetylneuraminic acid sialidase inhibitor (e.g.,
Relenza)
Special products
Cyclodextrins 5000 10 50 domestic, food, stabilizers
Pantothenic acid 11 600 ca. 6 ca. 70
Biotechnology 49

which depend on the cytochrome system for In 1921, a microbial reaction for the stere-
electron transfer to atmospheric oxygen, the ul- ospecific production of d-(−)ephedrine was de-
timate hydrogen acceptor: scribed [131]. A yeast strain, cultivated to pro-
duce acetaldehyde from glucose, was found
Table 7. Classification of enzymes and their reactions
to condense benzaldehyde with acetaldehyde
Class Reaction to form optically active l-phenyl-1-hydroxy-2-
Oxidoreductases: Enzymes catalyzing propanone. This product was in turn converted
oxidation–reduction reactions
involving oxygenation, such as chemically into d-(−)ephedrine (Fig. 21). This
C–H → C–OH, or overall is an example of a successful combination of
removal or addition of hydrogen
atom equivalents, for example
chemical and biological reactions.
CH(OH) → C=O and
CH–CH→C=C.
Dehydrogenases, oxidases,
peroxidases, hydroxylases, and
oxygenases are involved in this
class.
Transferases: Enzymes catalyzing the transfer
of various groups (e.g., aldehyde,
ketone, acyl, sugar, phosphoryl)
from one molecule to another.
Hydrolases: Enzymes catalyzing hydrolytic
cleavage of C–O, C–N, C–C, etc.
Esterases, amidases, peptidases,
phosphatases, glycosidases, etc.
are involved.
Lyases: Enzymes catalyzing cleavage of
C–C, C–O, C–N and other bonds
by elimination, leaving double
Figure 21. Chemoenzymatic synthesis of optically active
bonds, or by adding groups to l-phenyl-1-hydroxy-2-propanone and chemical conversion
double bonds. into d-(-)ephedrine
Isomerases: Enzymes catalyzing a change in
the configuration of a molecule.
Racemases and isomerases are Biocatalyis reached its present significance
involved. much later, when specific modifications of
Ligases: Enzymes catalyzing the joining steroids (or sterols) by microorganisms were
of two molecules with the
accompanying hydrolysis of a discovered. Various steroid reactions, such as
high-energy bond. Such enzymes hydroxylation, epoxidation, dehydrogenation,
are often termed synthetases.
isomerization, and hydrolysis, are performed by
a wide variety of microbial enzymes. These in-
clude the following important reactions: reduc-
tion of androstenedione to testosterone by yeasts
[132], hydroxylation of progesterone to 11-
α-hydroxyprogesterone by Rhizopus arrhizus
Figure 20. Biotransformation of ethanol to acetic acid [133], 11-β-hydroxylation of C21 -steroids by
Curvelaria lunata [134], introduction of a ∆1 -
1 double bond into hydrocortisone by Corynebac-
C2 H5 OH+ O2 →CH3 CHO+H2 O
2 terium simplex [135], 16-α-hydroxylation of
1
9α-fluorohydrocortisone by Streptomyces re-
CH3 CHO+ O2 →CH3 COOH seochromogenes [136], and elimination of the
2
side chain of cholesterol by Arthrobacter sim-
Other historical examples of biocatalytic oxida- plex [137]. Many novel intermediates for the
tion are the oxidation of glucose to gluconic acid chemical synthesis of new steroids became
by Acetobacter aceti [128] and the oxidation available in this way.
of sorbitol to sorbose by Acetobacter species In addition to the considerable practical
[129]. A variety of such early reports on bio- value, the amazing versatility of microorganisms
catalytic conversions was collected by Plimmer in the transformation of steroids provided an im-
[130]. Since then many other books have been portant theoretical background for the applica-
published on this topic. tion of microbial enzymes to chemical synthesis.
50 Biotechnology

Many of these reactions cannot be achieved by in biocatalysis are summarized in Table 8. Al-
conventional chemical synthesis. though biocatalysis has become an important
technology for synthetic chemistry, it also has
Table 8. Advances in biocatalysis
a high potential for many fields of biotechnol-
Biocatalytic synthesis Example reactions ogy.
Transformation of steroids and hydroxylation (oxygenase),
sterols dehydrogenation
(dehydrogenase), side-chain
degradation (oxygenase), etc. 6.4. Characteristics of Enzyme
Transformation of terpenoids hydroxylation of beta-ionone
with monooxygenase from Reactions Used in Biotransformations
Bacillus negaterium [138].
Transformation of alkaloids oxidation of morphine to
2,2 -bimorphin Chemical reactions performed by biocatalysts
(pseudomorphine) by (biotransformations) are essentially the same as
Cylindrocarpon didymium [139]; those carried out in inorganic or organic chem-
N-demethylation of codein to
norcodein by Mucus piriformis istry.
[140]. Enzymes (or biocatalysts) used for microbial
Synthesis of semisynthetic synthesis of semisynthetic
antibiotics penicillins and cephalosporins
conversions increase the reaction rate by low-
(amidase). ering the activation energy as normal catalysts
Synthesis of organic acids hydration of fumaric acid do. The most striking difference between en-
(fumarase), diterminal oxidation
of alkanes (oxygenase), zymes and chemical catalysts, however, lies in
asymmetric hydrolysis of their substrate specificity. They catalyze specific
epoxysuccinic acid (hydrolase), reactions involving one or only a few structurally
etc.
Transformation of sugars isomerization of glucose (glucose related compounds and they distinguish almost
isomerase). absolutely between stereoisomers or regioiso-
Protein synthesis synthesis of plastein (protease), mers. Therefore, only a very specific change in
semisynthesis of human insulin
(protease), etc. a functional group or bond of a compound is ac-
Synthesis of nucleic acid related trans-N-ribosylation and celerated by the enzyme. As a result, one single
compounds trans-N-arabinosylation
(phosphorylase),
product is expected as long as only one enzyme
phosphorylation of nucleosides is involved in a conversion.
(phosphotransferase), In contrast to chemical reactions that often re-
pyrophosphorylation of
nucleotides quire high activation energies (high temperature,
(pyrophosphotransferase), pressure, etc.) biotransformations can be per-
synthesis of sugar nucleotides
(pyrophosphorylase), synthesis formed under considerable mild reaction con-
of coenzymes, etc. ditions (Table 9). Due to the low energy in-
Synthesis of amino acids asymmetric hydrolysis of put needed for biocatalytic processes and the
α-amino-ε-caprolactam, 2-ami-
no-∆2 -thiazoline-4-carboxylic high atom utilization (percentage of M r of de-
acid, and 5-substituted sired product to sum of M r of all substrates),
hydantoins (hydrolase),
amination of fumaric acid
biotransformations are considered as an envi-
(aspartase), synthesis of ronmentally friendly alternative to classical or-
l-tyrosine-related amino acids ganic chemistry processes. To compare biocat-
(β-tyrosinase),
l-tryptophan-related amino acids alytic and chemical processes on the basis of the
(tryptophanase), etc. amount of waste, Roger A. Sheldon [141] de-
Synthesis of amines decarboxylation of amino acids vised the “environmental quotient” (EQ), which
(decarboxylase).
Synthesis of industrial chemicals synthesis of alkene oxides is the quotient of the amount of waste product
(oxygenase), chiral alcohols and per kilogram of product (E) and an “unfriendli-
ketones (dehydrogenase),
pyrogallol (decarboxylase),
ness quotient” (Q): EQ = E × Q.
amides (nitrile hydratase), etc. High catalytic efficacy is another character-
istic property of enzymes. They increase the
Innumerable biocatalytic conversions involv- turnover rate without large energy requirements
ing different types of reactions with organic and only a small amount of enzyme is needed
compounds and natural products were found to catalyze the conversion of a large amount of
during the following years; some of them are substrate.
very useful for chemical synthesis. Advances
Biotechnology 51
Table 9. General characteristics of enzymatic and chemical
reactions
the actual life process of the microorganisms. In
other words, the reaction product results from
Parameter Enzymatic reaction Chemical reaction the complex metabolism of the microorganism
Reaction conditions:
Temperature physiological high from cheap carbon and nitrogen sources. There-
Pressure atmospheric high fore, living cells are required for fermentation,
pH neutral different whereas this is not so important in biotransfor-
Reaction energy enzyme conformation thermal
(van der Waals,
mation. Fermentation products are always nat-
hydrogen bonds, etc.) ural products. Advantages and disadvantages of
Solvent water, (organic water, organic these two microbial processes are briefly sum-
solvents and ionic solvents
liquids under special marized in Table 10. However, it is not always
conditions possible, possible to draw a clear line between the two
e.g., low categories.
wateractivity)
Specificities high low
(substrate, stereo-,
regiospecifity) 6.5. Types of Biocatalysts and Reaction
Concentration of low high
substrate and/or Systems
product
Because biotransformations are essentially cat-
Table 10. Characteristics of biotransformation microbial
alytic chemical reactions, a suitable catalyst for
conversion and fermentative production the desired conversion must be prepared care-
Parameter Biotransformation Fermentative
fully. Although conventional vegetative cell cul-
production tures are most commonly used, there are sev-
Microorganism resting or treated cellsgrowing cells eral alternative methods; some of these have
Reaction simple catalytic life process (multistep already been operated commercially. These al-
reaction (one or few reaction)
steps) ternatives have been evaluated extensively with
Reaction time short long regard to, for example, increased yield, control
Starting materials expensive substrates cheap carbon and of side reactions, simplified processes, and im-
nitrogen sources
Product natural and/or natural proved economy. They have great future poten-
unnatural tial.
Product concentration high low
Various types of biocatalysts are useful for
Product isolation easy tedious
microbial conversions (see Section 6.2. Any
combination of these systems may also be pos-
As a result, enzymes exhibit their activ- sible. Several successful conversions are known
ity under mild reaction conditions, such as at- in which multistep reactions are catalyzed by
mospheric pressure, temperatures around 20– different catalysts (see Section 6.5.6).
40◦ C, and pH values near neutrality, where
normal chemical catalysts are inactive. On the
other hand, enzymes cannot function under dras- 6.5.1. Biotransformation with Growing
tic reaction conditions because of their deli- Cultures
cate protein structures. The unique properties
of enzymes are extremely useful when unstable The substrate is added to the growth medium
molecules are to be converted without undesired during inoculation or an appropriate phase of
side reactions. growth. The preparation and inoculation of the
There are two ways for using biocatalysts in medium, addition of substrate, and incubation
the production of useful compounds: biotrans- are successively carried out in one flask until
formation and fermentation. Fermentation has the reaction is completed. Because of its extreme
been used since ancient times (e.g., alcoholic simplicity, this procedure is frequently used not
fermentation) and is still of industrial impor- only for screening (see Section 6.6), but also in
tance in the production of such compounds as large-scale production. Usually, the substrate is
organic acids, solvents, or antibiotics. In contrast added directly to the medium without steriliza-
to biotransformation, fermentation is considered tion if the fully grown culture is the catalyst.
to be a biologic method, because it results from Continuous chemostat methods may be used to
maintain a steady state of growth, enzyme levels,
52 Biotechnology

and substrate concentration. If high cell densi- 6.5.2.2. Permeabilized Cells


ties are required by the conversion or if the na-
ture of the growth medium causes problems for In cases where substrate or product molecules do
the isolation of the product, the reaction should not readily permeate the cell membrane, a mod-
be performed after separating the cells from the ification of the cell becomes necessary. Com-
medium, see next section. monly used techniques to change the permeabil-
ity of the cell membrane to control the diffusion
of substrate into the cell and of product out of
6.5.2. Biotransformation Conversion with the cell are: using surfactants, organic solvents,
Previously Grown Cells antibiotics or enzymes. Besides these chemical
methods physical procedures, such as osmotic
The microorganism is grown under suitable con- shock or freezing and thawing may also be use-
ditions and the cells are then collected by cen- ful [142]. The production of high-energy phos-
trifugation or filtration. In this way, the cells re- phate esters, such as adenosine 5-triphosphate,
tain most of their enzyme activity. For the con- nicotinamide adenine dinucleotide, flavin ade-
version they are resuspended either in an in- nine dinucleotide, or coenzyme A, is a typical
complete medium (e.g., one without a nitrogen example [143].
source; this prolongs cell viability and activity
as well as possible cofactor regeneration) or in a
simple reaction medium (e.g., a buffer solution 6.5.2.3. Dried Cells
or water containing the substrate). The reaction
is then carried out directly or after suitable treat- Dried cells are an alternative to permeabilized
ment of the cells. It is theoretically not important cells. Drying causes structural changes in the cell
whether the cells are dead or alive. Vegetative, wall or membrane. Contact between the cell en-
washed, and dried cells fall into this category. zyme and the substrate is more readily achieved.
The advantages of this method are listed below: In some cases, drying also eliminates undesired
1) Growth and conversion steps are controlled side reactions, because some enzymes are dam-
independently for optimization. aged through drying. Dried cells are usually pre-
2) The grown cells can be stored for some period pared by air-drying, lyophilization and acetone
of time until initiation of the reaction. treatment. The powdered dried cells can retain
3) The cell density for the conversion is easily their enzyme activities in the frozen state for
controlled. years. They can be used as “instant catalysts”
4) The reaction can be carried out under non- in the same way as normal chemical catalysts.
sterile conditions.
5) Product isolation is usually easier because
of the simple composition of the reaction 6.5.3. Biotransformation with Spores
medium.
In some cases, however, the strict separation Many fungal spores have as many useful en-
of microbial growth and conversion is consid- zymes as vegetative or grown cells. The fungi
ered to be unfavorable for economic reasons. are cultivated under conditions that induce high
Depending on the ability of substrate perme- spore yields. The spores are then separated from
ability through the cell membrane these biocat- the mycelium and used as catalysts in a similar
alysts may have to be treated differently prior to way as mentioned in the previous section. Pro-
their use. cesses using spores have the same advantages
as those using grown cells. Spores are usually
more stable than cells. Some types of spores can
6.5.2.1. Vegetative or Washed Cells be stored for several years without loss of con-
version activity. They usually do not germinate
In cases where the cell membrane is permeable during the reaction, but in some cases germina-
to substrate and product, no special technique is tion is required for maximum conversion activ-
needed for the preparation of the cells and they ity. The 11-α-hydroxylation of progesterone by
can be used as vegetative or washed cells.
Biotechnology 53

Aspergillus ochraceus spores represents a suc- 4) Retention of cells by membranes (microfilti-


cessful spore process [144]. Many other applica- ration, cutoff 0.2–2 µm), e.g., in form of mem-
tions have been reported, e.g., fatty acids conver- brane reactors or hollow fiber modules [153].
sions, triglycerides, carbohydrates, or penicillin In successful cases, the immobilized cells
V [145]. retain their activities over several months and
show a higher operational stability than unsup-
ported cells. Additional advantages are: 1) easy
6.5.4. Biotransformation with Immobilized removal of the cells from the reaction mixture,
Cells 2) repeated use of the cells, 3) continuous op-
eration of the reaction, 4) cell regeneration by
Immobilized cells are cells that have either been immersion into an appropriate nutrient medium,
fixed or bound to a surface, entrapped in a gel and 5) easy product isolation. Some of these fea-
matrix, or retained in a membrane reactor. All of tures are especially advantageous if the collec-
the above-mentioned cells can be immobilized. tion of a sufficient cell mass for the conversion
Numerous immobilization methods have been is costly.
reported; they can be divided into the following However, disadvantages must also be consid-
four groups: ered: 1) The catalytic activity of cells usually
1) Entrapment or encapsulation in a porous poly- decreases during the immobilization procedure
mer network, e.g., polyacrylamide, alginate, because the cells are partly damaged and perme-
κ-carrageenan, gelatine, agar, collagen, cellu- ability is further inhibited. This decreased activ-
lose, polyurethane, poly(vinyl alcohol). ity can be compensated by increasing the cell
2) Covalent, ionic, or physical attachment to density. 2) The immobilization itself sometimes
an appropriate water-insoluble solid support, requires special equipment. The conversion step,
e.g., ion-exchange resins, silica gels, metal especially when operated continuously, also re-
oxides. quires a special engineering design compared to
3) Aggregation of cells by physical or chemical conventional conversion processes. Examples of
cross-linking with glutaraldehyde, polyethyl- successful large-scale applications are listed in
eneimine, or other agents. Table 11.
An advance in this field was the introduc-
tion 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 Applications Year of introduction Ref.


immobilized enzyme
l-Amino acylase from adsorption to optical resolution of 1969 [146]
Aspergillus oryzae DEAE-Sephadex racemic amino acids
Escherichia coli (aspartase) entrapment in continuous production of 1973 [147]
χ-carrageenan gel l-aspartic acid from
ammonium fumarate
Brevibacterium entrapment in continuous production of 1974 [148]
ammoniagenes (fumarase) χ-carrageenan gel l-malic acid from fumaric
acid
Escherichia coli (aspartase) entrapment in continuous production of 1982 [149]
and Pseudomonas dacunhaeχ-carrageenan gel l-alanine from ammonium
(aspartate -decarboxylase) fumarate
Penicillin acylase from entrapment in a cellulose production of 1973 [150]
Escherichia coli triacetate matrix 6-amino-penicillanic acid
from penicillin G
Glucose isomerase from many methods available production of high-fructose 1973 [151]
Bacillus coagulans or syrup from glucose
Streptomyces species
β-Galactosidase from many methods available production of low-lactose 1977 [152]
Lactobacillus bulgaricus, milk and production of
Aspergillus oryzae, etc. sweetenings
Whole cells Arthrobacter retention by microfiltration production of muconic acid [153]
sp. (oxygenase) in cross-flow membrane (raw material for new
reactor resins, pharmaceuticals and
agrochemicals)
54 Biotechnology

into a conversion process. For example, cells en- 6.5.6. Multistep Reactions Using Different
trapped in -carrageenan are viable and reproduce Biocatalysts
without loss of activity per unit cells [154]. The
continuous production of ethanol from glucose In order to perform more than two sequential re-
by S. cerevisiae in κ-carrageenan [155] and the actions, two or more different biocatalysts are
∆1 -dehydrogenation of cortisol by Arthrobacter sometimes required either simultaneously or se-
simplex in calcium alginate [156] are examples quentially. Theoretically, any combination of
of such successful operations. Further improve- catalysts can be used for such multistep reac-
ment of this technique is now expanding into the tions. Examples of successful applications are:
application to complex multistep reactions, such continuous production of l-alanine from ammo-
as the production of antibiotics. nium fumarate via L-aspartate as the interme-
diate by immobilized Escherichia coli (aspar-
tase) and Pseudomonas dacunhae (l-aspartate
6.5.5. Biotransformation with Cell-free β-decarboxylase) cells [149], complete conden-
Enzymes or Purified Enzymes sation of racemic homocysteine by washed or
dried Pseudomonas putida (racemase) and Al-
In general, the use of cell-free enzymes or puri- caligenes faecalis (S-adenosylhomocysteine hy-
fied enzymes for conversions is expensive, be- drolase) cells [160], complete hydrolysis of
cause purification of enzymes is often tedious racemic α-amino-ε-caprolactam (see Section
and time-consuming. Therefore, they are not 6.6.2.1), and continuous amination of α-keto
so suitable for large-scale industrial production. acids to l-amino acids by two types of cell-free
However, there are several cases in which they enzymes (see Section 6.5.5).
are advantageous over systems using cells. Such Another remarkable example is the synthe-
cases are: sis of coenzyme A from pantothenic acid, l-
1) Poor substrate or product permeability cysteine, and ATP (or AMP), which proceeds
through the cell wall through five sequential steps (see below) and is
2) Problems caused by undesired side reactions commercially performed solely with Brevibac-
3) Commercial availability of the desired en- terium ammoniagenes cells [161, 162].
zyme at an acceptable price
If an extracellular enzyme is used, cells are Step 1: pantothenic acid + ATP →
phosphopantothenic acid + ADP
not involved at all. Step 2: phosphopantothenic acid +
Cell-free enzymes can also be used as im- l-cysteine + ATP →
mobilized catalysts [157]. The same techniques phosphopantothenoyl-l-cysteine
+ ADP + inorganic phosphate
as those used for cell immobilization are avail- Step 3: phosphopantothenoyl-l-cysteine
able. Immobilization of penicillin acylase [150], → phosphopantetheine + CO2
l-amino acid acylase [146], lactase [152], and Step 4: phosphopantetheine + ATP →
dephosphocoenzyme A +
glucose isomerase [151] have been applied to inorganic pyrophosphate
commercial production. The entrapment of sol- Step 5: dephosphocoenzyme A + ATP
uble enzymes in a membrane-type reactor with → coenzyme A + ADP

simultaneous coenzyme regeneration is an alter-


native to the use of cell-free enzymes. l-Amino 6.5.7. Multiphase Reaction Systems
acid dehydrogenases in combination with for-
mate dehydrogenase as a coenzyme regenera- Since their discovery, enzymes have been
tor and nicotinamide adenine dinucleotide cova- thought to be inert catalysts in organic solvents.
lently bound to polyethylene glycol in a mem- This is basically true, but enzymes have become
brane reactor have been successfully applied to available that are catalytically active in water-
the continuous amination of keto acids yielding miscible organic solvents. Many reactions that
optically active amino acids [158]. l-Leucine is are impossible in water because of kinetic or
already produced commercially by this process. thermodynamic reasons can be performed in or-
Whitesides [159] has reviewed the potential ganic solvents [163, 164] (e.g., transesterifica-
applications of cell-free enzymes in organic syn- tion reactions [165]). Chemically modified per-
thesis. oxidase exhibits 21 % of its activity in benzene
Biotechnology 55

as compared to that of the unmodified enzyme dehydrogenase from C. boidinii for cofactor re-
in aqueous solution [166]. Polyphenol oxidase generation. Yield (97 %), conversion (97 %), and
converts nearly 100 % of phenol to catechol optical purity of the product (ee > 99 %) are very
when the reaction is carried out in chloroform high.
containing only 1–2 % water [167]. Such liquid–
liquid two-phase systems with low water content
can also be used to shift the hydrolytic equi-
librium toward water elimination. The synthesis
of esters, amides, and peptides by this method
shows great practical potential.
Multiphasic conversion systems using micro-
bial cells as catalysts have also been extensively
studied, especially in the case of lipophilic com-
pounds, which have a limited solubility in water.
In some cases, increased reaction rates and/or
product yields have been achieved.

6.6. Process Design Figure 22. Synthesis of acetal amino acid (Bristol-Myers
Squibb)
6.6.1. General Considerations
Another interesting example is the produc-
When designing a biocatalytic process many tion of l-tyrosine and l-dopa (dihydroxyphe-
important aspects require careful consideration. nylalanine) by bacterial β-tyrosinase [169]. This
Besides the essential question of the type of en- enzyme has long been known to catalyze the α,
zyme used for biocatalysis there is the selection β-elimination of l-tyrosine to phenol, pyruvate,
of a compound to be synthesized and a survey on and ammonia (Eq. 1).
available substrates and routes or reactions that
are of special importance in designing a conver-
sion process (also see Chapter 5).

6.6.1.1. Evaluating Enzyme Potential

When a new conversion or a new enzyme use- Because the enzyme also catalyzes the reverse
ful to biocatalytic conversions is discovered, reaction (Eq. 2) and the β-replacement reaction
the question as to the potential of the new en- of l-tyrosine with phenol derivatives (Eq. 3), it
zyme for practical purposes must be answered did not take long for the application of these re-
first. An example for a high-potential biocatal- actions in the synthesis of l-tyrosine and l-dopa.
ysis with practical application in the phar-
maceutical industry is l-phenylalanine dehy-
drogenase (PheDH, EC 1.4.1.20) from Ther-
moactinomyces intermedius for the conversion
of ketoacid acetals to acetal amino acids [168]
(Fig. 22). The amino acid acetal is a precur-
sor for Omapatrilatr
, an antihypertensive drug,
which inhibits the angiotensin-converting en-
zyme (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 Ther-
moactinomyces intermedius and the formate
56 Biotechnology

Subsequently, similar reactions were found R H+CH3 COCOOH+NH3


to be catalyzed by several pyridoxal phos-
→L (D) −R CH2 CH (NH2 ) COOH+H2 O
phate dependent enzymes, such as trypto-
phanase, cysteine desulfhydrase, and 3-chloro- where for β-tyrosinase R = hydroxyphenyl, –
d-alanine chloride lyase. These enzymes were OH, –SH, –Cl and R = hydroxyphenyl; for tryp-
then successfully used to synthesize l-trypto- tophanase R = indolyl, –OH, –SH, –Cl and R
phan and 5-hydroxy-l-tryptophan, l-cysteine, = indolyl; for cysteine desulfhydrase R = –SH,
and d-cysteine [169 – 171] (see Table 12). These thiol radicals, –Cl and R = –SH, thiol radicals;
reactions can be summarized as follows: and for 3-chloro-D-alanine chloride-lyase R =
–Cl, – SH, thiol radicals and R = –SH.
L (D) −RCH2 CH (NH2 ) COOH+H2 O These successful examples demonstrate the
→RH+CH3 COCOOH+NH3 importance of reassessing the capabilities of
well-known enzymes or reactions.
L (D) −RCH2 CH (NH2 ) COOH+R H
→L (D) −R CH2 CH (NH2 ) COOH+RH

Table 12. Synthesis of l-tyrosine, l-tryptophan, l-cysteine, d-cysteine, and related amino acids by pyridoxal phosphate-dependent
enzymes

Product Yield, Substrates Reaction


Enzyme
g/L mol% Microorganism
l-Tyrosine 58 88 a sodium pyruvate reverse of α,β-elimination
ammonium acetate β-tyrosinase
phenol Erwinia herbicola

l-Tyrosine 53.5 78 b d,l-serine β-replacement


ammonium acetate β-tyrosinase
phenol Erwinia herbicola

l-Dopa 58.5 f
sodium pyruvate reverse of α,β-elimination
ammonium acetate β-tyrosinase
pyrocatechol Erwinia herbicola

l-Dopa 53 71 c d,l-serine β-replacement


ammonium acetate β-tyrosinase
pyrocatechol Erwinia herbicola

l-Tryptophan1 00 100 d sodium pyruvate reverse of α,β-elimination


ammonium acetate tryptophanase
indole Proteus rettgeri

5-Hydroxy-l- 23.3 57 e sodium pyruvate reverse of α,β-elimination


tryptophan ammonium acetate tryptophanase
5-hydroxyindole Proteus rettgeri

l-Cysteine 50 88f 3-chloro-l-alanine β-replacement


sodium sulfide cysteine desulfhydrase
Enterobacter cloacae

d-Cysteine 22 88g 3-chloro-d-alanine β-replacement


sodium hydrogensulfide 3-chloro-d-alanine chloride
lyase
Pseudomonas putida
a
Based on sodium pyruvate.
b
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.
Biotechnology 57

6.6.1.2. Finding Suitable Enzymes solution. Sterilization of the substrate is recom-


mended prior to its use, if required.
Occasionally a synthetic scheme has been de- Only dissolved substrate is converted. There-
signed but suitable enzymes or conversion pro- fore, slightly soluble substrates must first be dis-
cesses are yet unknown. In such a case, it be- solved and the products may then crystallize
comes necessary to assay suitable microbial from the solution after conversion:
strains or enzymes (as described in Section Substrate (solid)  Substrate (solution)
6.6.2) as to their suitability for catalyzing the de-
 Product (solution)  Product (solid)
sired reaction. The production of l-lysine from
d,l- α- amino- ε-caprolactam (ACL) is another For example, powdered cortisol in concen-
typical example [172]. This process was de- trations up to 500 g/L is converted to crystalline
signed to utilize cyclohexene, a major byproduct prednisolone with a good yield by Arthrobacter
of nylon production. In this process, the biocat- simplex [173]. Substrates may also be dissolved
alyst is required to hydrolyze only the l-form in water-miscible organic solvents. The lower
of α-amino- ε-caprolactam (l-ACL) to yield l- alcohols, acetone, dimethylformamide, and di-
lysine (Fig. 23). methylsulfoxide are suitable solvents. Surfac-
tants may also be used to disperse the substrate.
The combination of water-miscible solvents and
surfactants may offer some advantage. In some
cases, chemical modification of the substrate
may improve its solubility.
An example for a process using substrate
with poor solubility is the regioselective oxida-
tion of the tert-butyl group of terfenadine us-
ing Cunninghamela blakesleana in a whole-cell
biotransformation [174]. Due to its low solu-
bility in water, microcrystalline terfenadine is
solved in the water-miscible organic solvent
dimethylfomamide prior to its addition to the
Figure 23. Hydrolysation of the l-form of -amino- -capro- aqueous reaction medium. This regioselective
lactam (l-ACL) to yield l-lysine oxidation was studied as a biocatalytic alterna-
tive for the chemical synthesis of the antihis-
Two microorganisms were found during the taminic drug fexofenadine.
assay: one of them hydrolyzes the l-form of the In cases where the cell membrane is imper-
substrate and the other racemizes the remaining meable to the substrate or a cell-free or puri-
d-form. As a result, all the α-amino- ε-capro- fied biocatalyst is required, the permeability of
lactam is converted into l-lysine without requir- the cell must be improved, respectively, the cell
ing any optical resolution. wall has to be disrupted. This is accomplished by
air drying, acetone drying, lyophilization, autol-
ysis, lysozyme digestion, surfactant treatment,
6.6.1.3. Substrates osmotic shock, freezing and thawing, or ultra-
sonic treatment. For cell-free preparations, the
Any substrate is theoretically suitable for cell debris can be removed by centrifugation or
biotransformations, provided the substrate microfiltration.
molecules come into contact with the enzyme. If the substrate does not suit the desired con-
Even gases, such as methane, are suitable version, its chemical modification should be
substrates; they are bubbled into the reaction considered. Addition, variation, or removal of
medium. The substrate should be soluble in the a protecting group or modification of a func-
medium and able to pass through the cell mem- tional group in the substrate molecule some-
brane unless the reaction is catalyzed by cell- times makes its interaction with the enzyme
free enzymes. The substrate is usually added to more efficient. In addition, these modifications
the reaction medium, neat or as a concentrated may prevent undesirable side reactions or degra-
58 Biotechnology

dation. This method is frequently used in steroid Such a test, also called screening, may be one
conversions. A systematic assay for a suitable of the most important steps for a successful bio-
chloroacetoacetate ester for enantioselective re- catalytic conversion.
duction yielding the l-form of 4-chloro-3-hy-
droxybutanoate, a promising precursor for the
chemical synthesis of l-carnitine, is an example 6.6.2.1. Screening
[175].
Kitahara and co-workers [183] screened bac-
terial strains from cultures grown aerobically at
6.6.1.4. Media 30–37◦ C in nutrient broth containing malt ex-
tract. Sodium fumarate and ammonium chloride
Besides performing biocatalysis in aqueous me- were added 24 h after growth started. After in-
dia, there has developed the so-called “non- cubation for another 24 h the l-aspartate in the
aqueous enzymology” which has become an im- culture broth was analyzed by chromatography.
portant area of research and development dur- A strain of E. coli was selected by this screen-
ing the last decades [176]. Enzymes exhibit a ing as the most promising producer of aspar-
wide array of novel reactivities and selectivi- tase. Under suitable reaction conditions, 56 g
ties in nonaqueous solvents. For example, many of l-aspartate was produced per 100 mL with a
reactions that are impossible in water due to molar yield of 99 % by using 1 g of dried cells.
kinetic or thermodynamic reasons can be per- Subsequently, this process was developed into
formed in organic solvents, [163, 164, 177] due large-scale technology by which l-aspartate was
to the suppression of water-induced side reac- synthesized continuously with E. coli cells im-
tions. Improved and altered substrate specifici- mobilized on κ-carrageenan [184].
ties [178, 179] and selectivities can be observed. The screening procedure described above
Examples of practical applications are enantios- may be rather simple, but it reflects the essence
elective synthesis [180], chiral resolution [181], of screening. Normal screening procedures can
and combinatorial biocatalysis [182]. The pos- be more complicated and deal with a great vari-
sibility of the solubilization of hydrophobic sub- ety of microorganisms, including bacteria, acti-
strates or products in organic solvents opens nomycetes, molds, yeasts, and basidiomycetes.
opportunities for the enzymatic production of
poorly water-soluble fine chemicals and phar-
maceuticals. The thermal and storage stability of 6.6.2.2. Enrichment
enzymes can be significantly enhanced in non-
aqueous media [164, 176, 178]. Another method to find a suitable biocatalyst can
be enrichment. Sometimes it is desired or nec-
essary to find biocatalysts capable of perform-
6.6.2. Selection of Biocatalysts ing a specific conversion from natural sources.
Soil samples containing mixed microbial popu-
More than 8000 enzymes are known [125]. Al- lations are rich sources of test organisms. Be-
most all types of chemical reactions which are cause the enzymes in these microorganisms
also catalyzed by normal chemical catalysts are modify or degrade a great variety of complex
involved. According to the desired type of con- organic compounds, at least one of the mi-
version and the classification of the enzymes one croorganisms is expected to offer one or several
can easily determine which enzyme would be enzymes that perform the desired conversion.
suitable for the desired conversion by inspecting These microorganisms are selected using the en-
the enzyme catalogue published by the commit- richment technique. A typical enrichment proce-
tee [125]. However, the properties of enzymes dure is the isolation of bacterial strains that per-
from different sources may vary widely, even if form the asymmetric hydrolysis of d,l-2-ami-
they catalyze the same reaction. Therefore, test- no- ∆2 -thiazoline-4-carboxylic acid (d,l-ATC),
ing is indispensable. A wide variety of biocata- an intermediate of d,l-cysteine synthesis, to l-
lysts are tested for the purpose of determining cysteine [185]. Soil samples were incubated in a
their ability to carry out the desired reaction. medium containing 0.3 % D,L-ATC as the sole
Biotechnology 59

nitrogen source. After spreading each culture require different knowledge of the target enzyme
on a plate of the same medium, colonies that as well as laboratory equipment (Table 13).
appeared were isolated and grown on nutrient
agar containing 0.2 % d,l-ATC as the enzyme in- Table 13. Requirements and molecular methods for rational design
ducer. The resulting cells were then transferred and directed evolution technique

to a solution containing 1 % d,l-ATC and the Rational design Directed -evolution


formation of l-cysteine was demonstrated by Requirements: 3D structure of the high-throughput-
target enzyme screening/selection
chromatographic or microbiological methods. technology to analyze
Among 1975 colonies from 388 soil samples, enzyme properties
31 l-cysteine-producing bacteria were isolated. knowledge of reaction
mechanism
One of them, Pseudomonas thiazolinophilum, modeling of
was mutagenized. Mutants with inhibited l- enzyme-substrate
cysteine metabolism were screened from the interaction
Molecular methods: site-directed random mutagenesis
mutagenized cultures. One of these mutants con- mutagenesis
verted the added substrate almost stoichiomet-
rically to l-cysteine with a yield of 31.4 mg/mL
[186]. 6.7. Improvement of Conversion
The enzyme system of Pseudomonas thia- Processes
zolinophilum hydrolyzes d,l-ATC in two steps
as shown in Figure 24. 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 sufficient
because the constitutive enzyme is formed in-
dependently of the medium composition. How-
ever, if the enzyme is inducible, an effective in-
ducer, which may be the substrate itself or an-
Figure 24. Asymmetric hydrolysis of d,l-2-amino- 2- other structurally related compound, will be re-
thiazoline-4-carboxylic acid (d,l-ATC) to l-cysteine quired. If the enzyme is repressive, the repres-
sion factor will either have to be eliminated or the
Although screening sometimes is regarded repressive compounds, which may be present in
as old-fashioned, time-consuming, and even il- the growth medium or formed during the growth,
logical, some consider it to be one of the most must be removed.
promising methods for finding new genes in mi- Catabolites of an easily metabolizable car-
croorganisms. bon source, e.g., glucose or sucrose, and the
end product of a metabolic pathway usually
cause catabolite or product repression, respec-
6.6.2.3. Molecular Engineering tively. Therefore, the composition of the growth
medium, growth period, and various physical pa-
Sometimes enzyme yields in wild-type organ- rameters of growth, such as temperature, aera-
isms are low or the desired enzyme is not found tion, agitation, and pH, must be carefully con-
in the available microorganisms. In this case, trolled. In successful cases, the concentration of
molecular engineering techniques can lead to an the required enzyme reaches more than 10 % of
enzyme catalyzing the desired reaction. There the total soluble protein [187].
are two different strategies that represent the Optimal physical and chemical conditions for
“state-of-the-art” technologies: rational design the biotransformation itself must be determined
and directed evolution [153]. Both technologies with the same care as for the optimization of
growth conditions. This is especially important
60 Biotechnology

if side reactions are observed, because they de- Microbial conversion can also be optimized
crease product yield and make product isolation by improving the selected strain. Standard meth-
difficult. Physical or chemical treatment of the ods of strain improvement include the isolation
biocatalyst, such as heating, pH change, and the of single colonies, optionally after mutagenesis,
addition of detergents, organic solvents, or spe- and screening of the best suited isolates. Such
cific inhibitors may cause specific inactivation of conventional genetic manipulation techniques
undesired enzymes. For example, the trans-N- have been developed during the last decades for
arabinosylation of adenine from synthetic uracil the improvement of useful strains. Recombinant
arabinoside (Fig. 25) yields adenine arabinoside, DNA technology is providing new prospects for
an antiviral agent. 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 appropri-
ate host–vector 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.

6.8. Conclusion and Outlook


Biocatalysts have become one of the most im-
portant tools in nearly all fields of industrial
biotechnology. The main advantages of using
biocatalysts are their unique properties such as
Figure 25. trans-N-arabinosylation of adenine from syn- high chemo-, regio-, and stereoselectivity and
thetic uracil arabinoside the ability to catalyze reactions under mild con-
ditions.
This reaction is catalyzed by cells of En- During the last couple of years, many new
terobacter aerogenes and takes place only at methods and techniques have been developed
temperatures above 60◦ C [188]. The reaction to overcome common problems. Some exam-
does not proceed below 50◦ C because hypo- ples here might be molecular engineering tech-
xanthine is formed by adenine deaminase be- niques such as rational design or directed evo-
fore the trans-N-arabinosylation by two kinds lution to increase low biocatalytic activities
of nucleoside phosphorylases takes place. For- [153], biocatalysis in nonaqueaous reaction me-
tunately, these phosphorylases are still active at dia to bypass solubility problems of substrate
60◦ C, whereas the deaminase is completely in- and/or product [179], or the applications of high-
activated at this temperature. Other examples are throughput screening methods for the search
summarized in Table 14. of new and suitable biocatalysts. Another field
In the case of immobilized biocatalysts, con- of interest that will become of great impact is
centration gradients of substrates and products “metagenomic research” that will enable scien-
may occur that could lead to decreased biotrans- tists to find new biocatalysts with desired quali-
formation rates. These gradients can be reduced ties [192].
by decreasing the Thiele modulus, e.g., by us- To make new biocatalysts attractive for in-
ing smaller carrier particles or by reducing the dustrial applications they have to be available
diffusion distance between the free solution and in sufficient amounts. High expression rates of
the immobilized enzymes. enzymes can be achieved by genetical and phys-
iological manipulations of the expressing mi-
Biotechnology 61
Table 14. Elimination of side reactions by physical or chemical treatment

Desired reaction Side reaction Treatment Reference


Ammonium fumarate → l-aspartic acid fumaric acid → l-malic incubation of culture broth at pH 5 [149]
(Escherichia coli/aspartase) acid and 45 ◦ C for 1 h

Fumaric acid → l-malic acid (Brevibacterium fumaric acid → succinic treatment of immobilized cells with [148]
ammoniagenes/fumarase) acid 0.6 % bile extract

l-Aspartic acid → l-alanine (Pseudomonas l-alanine → d-alanine incubation of culture broth at pH 4.75 [149]
dacunhae/aspartate β-decarboxylase) and 30 ◦ C for 1 h

Cholesterol → 1,4-androstadiene-3,17-dione further decomposition of addition of α,α -dipyridyl (1 mM) to [137, 189]
(Arthrobacter simplex/multistep conversion) the sterol molecule the culture broth

β-Sitosterol → 17-ketosteroids (Nocardia further decomposition of addition of α,α -dipyridyl (0.3 mM) [190]
species/multistep conversion) steroid ring to the conversion mixture

d,l-2-Amino-∆2 -thiazoline-4-carboxylate → further degradation of addition of hydroxylamine or [186]


l-cysteine (Pseudomonas l-cysteine semicarbazide to the reaction mixture
thiazolinophilum/multistep conversion)

croorganism such as optimizing the number of mycelia caused by cell disruption, which is nec-
plasmids per cell or inactivation of “negative” essary to gain the products. According to this
cell functions (e.g., deletion of protease coding starting point, nearly each downstream process
genes). Apart from deleting unwanted side activ- consists of the four following steps [193]:
ities also novel metabolic pathways can be im- – Removal of insoluble particles
plemented. These microorganisms, customized – Isolation of the product
only to fulfill special needs for biocatalytic ap- – Purification
plications, are called “designer bugs” or “tailor- – Polishing
made microorganisms”.
With respect to downstreaming in laboratory
scale, one is normally only limited by the avail-
able equipment. However, with regard to upscale
7. Downstream Processing processes one should have in mind that the de-
veloped process should also be applicable in in-
Downstream processing is one of the most un-
dustry. Thus, Belter et al. [193] have formulated
derestimated steps in bioprocesses and it is well
the following questions, which are also crucial
known especially in the pharmaceutical industry
to biotechnology downstream processes:
that downstreaming is the most expensive and
unfortunately the most ineffective part of a bio- – What is the value of the product?
process. Thus, one can assume that new devel- – What is the acceptable product quality?
opments are widely described in the literature. – Where is the product in each process stream?
Unfortunately, this is not the case. Only a few – Where are the impurities in each process
working groups focus on new and more effective stream?
procedures to separate products from fermenta- – What are the unusual physicochemical prop-
tion broths or biotransformations. A chief char- erties of the product and the principal impu-
acteristic of biotechnology is the wide variety rities?
of products. Due to this variety, a broad spec- – What are the economics of various alternative
trum of separation techniques must be applied. separations?
However, for nearly all products one starts with In addition, it is important to use materials
a dilute suspension and tries to produce a highly which are available for all upscale processes,
purified dry product. In the case of extracellular since the downstream behavior of the product
products, the solids in this suspension may in- may change while changing, for example, the
clude intact organisms, other insoluble fractions type of chromatographic resin or membrane ma-
of the medium or natural sample, and perhaps in- terial. Normally the recovery costs exceed the
soluble products. Concerning intracellular prod- bioprocess costs; however, some upstream pa-
ucts the solids include in addition fragmented rameters influence the downstream processing:
62 Biotechnology

– The characteristic properties of the producing first stage purification of the product by remov-
microorganism or cell line ing dissimilar components from the broth. The
– The location of the product most important techniques in use are:
– The stability of the product
– Membrane processing
– Byproducts and impurities
– Ion exchange chromatography
– Concentration of the product in the medium
from which it is to be recovered In the membrane process, ultrafiltration and
A general downstream scheme based on these reverse osmosis are often used for separation,
parameters is given in Figure 26. concentration, and desalting. In addition, po-
lar membranes are used for ion exchange and
desalting. Ion exchange chromatography is ap-
plied in order to remove either major contami-
nants from the broth or the desired product from
the broth. Chromatographic procedures – based
on columns or membranes – are also often used
in the purification step. Other techniques in this
part of the downstream process are precipitation
and liquid–liquid extraction.

7.1. Sample Disruption

The influence 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 de-
stroyed 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 positive-
displacement pump, which supplies the liquid
sample at high pressure through a small nozzle
or an orifice valve. The disruption results from
the combination of shear force and impingement
on the valve. Bead mills use horizontal grinding
chambers filled with glass beads or other resis-
tant materials such as zirconium oxide, zirco-
Figure 26. General downstream scheme in biotechnology
nium silicate, titanium carbide, etc [197]. The
dispensed sample is introduced into the grind-
The main aims of the primary separation step ing chamber on a continuous basis. The level of
are to achieve a volume reduction and to make a
Biotechnology 63

disruption depends on the variable turning speed liquid through a solid support or filter medium.
of the bead mill. Dead-end filtration and cross-flow filtration are
By using bead mills, cell disruption can be two different designs of filtration which can be
achieved in a single run with better temperature used. In dead-end filtration mode, the total pro-
distribution and temperature control. Another cess fluid stream flows through the membrane.
possibility for cell disruption or at least product The retained solids accumulate on the membrane
liberation is the use of microwaves or ultrasound and build up a filter cake. The membrane has
which can be combined with sample extraction to be changed when the membrane pores are
by organic solvents. Microwave techniques are clogged by the solids. When the feed flow is
widely used in acid digestion of solid samples. directed parallel to the membrane surface, the
Their use in the extraction of organic analytes term cross-flow filtration is used. The tangential
from environmental samples is less widespread, flow of liquid removes any retained molecules or
despite the availability of commercial devices particles from the membrane surface, which re-
for this purpose and their potential for reducing sults in a stable flux for a longer time period. By
analysis time and solvent consumption. using cross-flow filtration, relatively low shear
A possibility for cell lysis under very mild stress is possible and filter aids are not required.
conditions is the use of hydrolyzing enzymes. In addition, scale-up is simple and cell washing
In addition, enzymes offer selectivity during is possible in a single process step.
product release. Enzymes hydrolyse the walls Over the last 30 years, a number of fur-
of cells, and when sufficient wall has been re- ther membrane processes have been developed
moved, the internal osmotic pressure bursts the for molecular separation. These filtration tech-
periplasmic membrane allowing the intracellu- niques can be divided into four major groups:
lar components to be released [195]. The ef- reverse osmosis (hyperfiltration, RO), nanofil-
fect of lytic enzymes is specific to particular tration (NF), ultrafiltration (UF), and microfil-
groups of cell types, which is attributed to the tration (MF) [200]. Membrane processes are
differences in cell wall composition. For exam- easy to scale up and the possibility of using the
ple, the most efficient lytic enzyme for bacte- same materials and configurations in different
ria is lysozyme from hens’ egg. This enzyme is sizes from laboratory to process scale reduces
also used in large-scale processes for enzyme the validation effort enormously. However, fil-
production [198]. Even more highly specialized tration processes are limited with regard to se-
procedures for sample disruption can be applied lectivity. The fractionation of proteins can only
in biotechnology. For example, high yields of in- be achieved with large differences in the molec-
tracellular enzymes from yeast can be obtained ular weight of the proteins and it is important to
by applying a series of electric field pulses [199]. keep in mind that a certain difference in the mo-
By using this technique, up to 90 % of the total lecular weight of two proteins does not mean the
activity can be released without any further or same degree of difference in molecular size. Pro-
previous treatment of the cells. The method is teins that differ in molecular weight by 10 times
based on electroinduced changes in the cell en- may differ in size by only 3 times when in glob-
velope leading to a leakage of part of the intra- ular or folded form. Another problem encoun-
cellular proteins without the formation of debris tered when using membrane processes for prod-
and permits the treatment of large volumes. uct recovery can be the slow retentate flux, which
can result in the formation of a thick secondary
membrane. Another possibility is the strong in-
7.2. Solid–Liquid Separations teraction of the sample with the membrane ma-
terial. This often depends on unspecific protein
Solid–liquid separation is a very important adsorption which is affected by several factors
procedure during downstream processing in [201]. Thus, different membrane types have to
biotechnology for cell separation, cell debris re- be screened to minimize the unspecific binding
moval, and also for product recovery. The most of the sample when a new filtration procedure
important solid–liquid separation techniques in has to be developed.
use for are filtration and centrifugation. Filtra-
tion separates solids from a liquid by forcing the
64 Biotechnology

7.3. Product Recovery become economically impractical. Conversely,


if the column length is increased, then the
One of the most important procedures in product impedance to flow will become greater lead-
recovery are chromatographic processes. Dur- ing to high column pressures. If large column
ing chromatographic steps the sample is sepa- radii are employed, then the mechanical strength
rated into several fractions according to inter- of the column system will limit the maximum
actions between the different molecules in the permissible pressure. Consequently, lengthen-
liquid phase and the stationary phase (chromato- ing the column will eventually require the parti-
graphic resin). The molecules can be separated cle diameter to be increased to provide adequate
according to their size and/or charge and based permeability. Increased particle diameter will, in
on hydrophobic/hydrophilic or affinity interac- turn, reduce the column efficiency, which may
tion with the stationary phase. Column chro- impair the resolution of the compounds of inter-
matography utilizes a vertical column filled with est [204].
the solid support with the sample to be separated Membrane. Until today, the required pro-
placed on top of this support. The rest of the cess steps for the product recovery are carried
column is filled with a solvent (eluent) which, out separately, which leads to many energy-
under the influence of gravity, moves the sam- intensive process steps such as filtration or chro-
ple through the column. Differences in rates of matography and production of vast wastewa-
movement through the solid medium are trans- ter amounts, respectively. Membrane adsorp-
lated to different exit times from the bottom of tion allows the integration of several process
the column for the various elements of the orig- steps into one-unit operation, which means the
inal sample. Based on the specific interactions downstreaming of biotechnologically produced
between the sample in the mobile phase and proteins can be carried out by saving process
the stationary phase the column chromatogra- time and resources [205, 206]. In addition to
phy can be performed as: this, conventional chromatographic techniques
– Ion-exchange chromatography, which are based on packed columns often re-
– Size-exclusion chromatography (gel- quire lengthy procedures. This can lead to the
filtration chromatography), degradation of sensitive proteins [207]. Further-
– Reversed-phase chromatography, and more, packed columns exhibit a high pressure
– Affinity chromatography. drop across the column and a slow diffusion-
controlled binding process of solutes within the
Immobilized-metal-ion affinity chromatogra- matrix [208]. Whereas membrane systems show
phy (IMAC) as a special form of affinity chro- several advantages to packed columns, most im-
matography is a popular and powerful way to portant, mass transfer takes place through con-
purify proteins. It is based on the specific co- vection rather than through diffusion. Due to
ordinate covalent binding between histidine or this fact, membrane adsorbers enable a time-
other unique amino acids and various immobi- effective performance with high flow rates with-
lized metal ions (e.g., nickel). out high back pressure [209]. Membranes can
Most of the chromatographic systems are be converted into efficient adsorbers by attach-
run in a batch or quasicontinuous mode. Nowa- ing functional groups to the inner surface of
days, new developments such as simulated mov- synthetic microporous membranes. Affinity ad-
ing bed chromatography [202] and anular chro- sorption, ion exchange, or immobilized-metal
matography [203] offers the possibility of con- affinity chromatography can be carried out by
tinuous processes. One problem in column chro- these membranes. Membrane ion exchangers
matography are the difficulties in upscaling. If of strong acidic (sulfonic acid), strongly ba-
the column radius is increased, unless special sic (quarternary ammonium), weakly acid (car-
packing techniques are employed, the packing boxylic acid), and weakly basic (diethylamine)
procedure becomes inefficient and the pack- types are commercially available. A chelating
ing itself unstable. In addition, to maintain the membrane based on the iminodiacetate (IDA)
optimum mobile phase velocity, the flow rate group is applicable for IMAC. Membrane ad-
will need to be substantially increased and the sorber technology has several major advantages
consumption of mobile phase will eventually compared to classical separation methods. Due
Biotechnology 65

to the membrane structure, the binding of pro- throughput, more efficient recovery of analytes,
teins is not limited by diffusional processes, cleaner extracts, economic replacement of halo-
therefore loading and elution can be performed genated solvents, and a high level of automa-
at very high fluxes resulting in very short cycle tion, compared to conventional sample prepa-
times. Compressibility of the membrane under ration procedures [212]. Supercritical fluid pro-
normal operation conditions can be neglected, cesses are being commercialized in the polymer,
channeling cannot occur, and the pressure dis- pharmaceutical, specialty lubricants, and fine-
tribution inside the modules is designed to have chemicals industries. Supercritical fluids are ad-
plug flow through the module, all of which lead vantageously applied to increase product perfor-
to sharp breakthrough curves. Scale-up is very mance to levels that cannot be achieved by tra-
easy, materials and systems allow cleaning in ditional processing techniques.
place (CIP), and the validation of the process
is made easier due to of standard products and
validation service of suppliers. 8. Monitoring and Modeling of
Bioprocesses
7.4. Solvent Extraction The requirement to operate biotechnological
processes in a cost-effective manner by simulta-
Solvent extraction is the most common method neously maintaining a high product quality and
for the recovery of hydrophilic substances and, safety is becoming increasingly relevant. Histor-
therefore, a method for separating well-soluble ically, the most important means of achieving in-
metabolites from cultivation media. Classical creased productivity from bioprocess plant has
extraction processes use organic solvents, which been through time- and resource-consuming ex-
are often rarely suitable for effective recovery of perimental developments. More recently, how-
the solute. Recently, new extractions have been ever, significant advances have been made in the
developed which form specific adducts with the area of computer applications for bioprocess su-
metabolite in question and allow its recovery pervision, modeling and control, and their intro-
with high efficiency and selectivity [210]. Sol- duction in process industry.
vent extraction in biotechnology focuses on the Typically, little use is made of the large
recovery both of primary metabolites (e.g., eth- amounts of bioprocess online and laboratory as-
anol, acetic acid, citric acid, and amino acids) say data after an experiment is completed. Re-
and of secondary metabolites (e.g., antibiotics cently, attempts have been made using enhanced
or vitamins). The concentration of secondary automation strategies to improve the quality
metabolites is usually much lower than that of information presented to operators and in-
of primary metabolites. Since most secondary crease the level of automatic process supervi-
metabolites are for use as therapeutics, the qual- sion, although few industrial applications have
ity requirements of the products are high. Sol- yet been reported. On a plantwide perspective,
vent extraction can help to fulfill these require- the scheduling of process operations is also tra-
ments. In addition, the set-up of integrated bio- ditionally manually undertaken by experienced
processes (production and downstreaming) can personnel by using a trial and error approach
be performed by solvent extraction [211]. Super- with a planning board. This also appears to be
critical fluid extraction (SFE) becomes also an a potential area for modern bioprocess manage-
important tool in biotechnological downstream ment.
processing since it offers important advantages
compared with other solvent extraction meth- Knowledge-based systems, such as fuzzy
ods. It is possible to work in an oxygen-free logic systems, can be designed to cope with un-
system, which prevents oxidation. The low tem- certainty and allow the coupling of quantitative
peratures applied minimize thermal degradation information with the qualitative or symbolic ex-
and microbes or their spores are not soluble. pressions (in the form of heuristics), so as to
In addition, supercritical fluids for extractions reproduce the actions of an experienced process
are inexpensive. The successful implementation operator. They might therefore be used as an in-
of this technique can lead to improved sample telligent, online virtual assistant to the process
66 Biotechnology

operator, or, with extra knowledge as a supervi- be to adopt a numerical estimation technique,
sor in running and maintaining bioprocess oper- either for estimating the parameters of a prede-
ations within optimal operating conditions. The fined model structure, usually of a generalized
main objective in this approach is to develop an linear time series form, or directly to obtain an
expert supervisory system which uses biopro- estimate of a difficult to measure primary pro-
cess and control knowledge in the form of rules cess variable (state estimation or software sen-
in conjunction with bioreactor state estimation sor). An alternative approach is to exploit the
(soft sensing), parametric identification and pro- nonlinear mechanistic structure of the biopro-
cess control algorithms running in real time. In cess, if known, in the form of a state observer.
addition, provision needs to be made for pro-
cess models and known bioprocess behavioral Neural Computing. A relatively new devel-
characteristics to be incorporated in order to en- opment in artificial intelligence is the area of
hance the overall supervisory control strategy. neural computing. Neural networks are dynamic
The complete system aims to perform process systems composed of highly interconnected lay-
monitoring, process control, sensor validation, ers of “simple” neuron-like processing elements.
fault detection, and diagnostic tasks and, in ad- In the field of process engineering, it is their
dition, provide recovery advice so as to achieve ability to capture process nonlinearities which
continuous online optimization. offers potential benefits. Other useful charac-
Collateral with the above approach is the on- teristics are their ability to adjust dynamically
line determination of the critical key variables to environmental and time-variant changes, to
which govern process production. The measure- infer general rules from specific examples and
ment and estimation problems are well known to recognize invariances from complex, high-
and represent the main drawback concerning an dimensional data. These properties provide neu-
effective biosystem optimization. Three ways of ral networks with the potential to outperform
reducing the difficulties encountered can be pro- other “learning” techniques. Given a series of
posed: experimental data the network is able to estab-
– Better sensors lish the governing relationships in these train-
– Better sampling and automatic analysis sys- ing data. This ability can be exploited to aid the
tems nonlinear modeling, control, and optimization
– Online estimation of difficult to measure, or of complex processes, as well as being used in
immeasurable, variables. existing predictive control methods.

Although all of these are attracting research


effort, the techniques of online state and parame- 8.1. Characteristics of Bioprocesses
ter estimation are receiving the greatest interest.
Whilst developments in biosensors are in some 8.1.1. System Definition
cases helping to contribute to the online determi-
nation of bioprocess parameters, they are by no A biotechnological process consists of a system
means at the stage where general applicability of chemical, biochemical, and microbiological
has been achieved. Until this is the case, the de- reactions, incorporating process-engineering as-
velopment of online estimation techniques will pects such as mass and energy conservation,
provide the major means by which online closed thermodynamics, or heat and mass transfer cor-
loop feedback control of critical bioprocess vari- relations. (Fig. 27). The biological part of the
ables can be achieved. system represents the peculiarity of the system
Several different approaches can be adopted characteristics and causes the great difficulties in
in the development of bioprocess estimation al- considering it from the system theoretical point
gorithms. The easiest but potentially least accu- of view.
rate way of obtaining an estimate of a “difficult Common for all system models is their map-
to measure” primary process variable is to estab- ping of inputs to outputs, of stimuli to responses
lish a correlation with a measurable secondary in some with the additional introduction of state
process variable, whilst ignoring measurement variables. Therfore, the task is to define proper
errors, noise, etc. A more robust approach might inputs and outputs, dependent on the problem
Biotechnology 67

Figure 27. General model structure for biotechnological processes


x = external flow and transport rates; c = concentration of the component (·)I inlet, (·)O outlet; TR = transfer rates; Q =
volumetric reaction rate; q = specific reaction rate; Y= yield coefficient; r = intrinsic reaction rate; (·)i = index for quantity i;
(·)j = index for quantity j


 T
available, to use appropriate signals (trajecto- z (t) = z1 (t) z2 (t) ... zn (t)
ries) to describe the system behavior in their im-  T

portant aspects and to build up the model. x (t) = x1 (t) x2 (t) ... xm (t)
A biotechnological system can be defined as  T (3)

a set of i biochemical reactions involving m in- y (t) = y1 (t) y2 (t) ... yo (t)
put variables −→
x (t), o output variables −
→y (t), m  T

−  
state variables z (t) and r parameters p notated p = p1 p2 ... pr
in operator transformation T(·):

 
z (t) =T x (t) , p (1)
The model does not determine the input vari-
ables −

 
 
y (t) =T ∗ x (t) , z (t) , p (2) x (t). They represent the systems stimuli
and can be divided up into control variables and
with
68 Biotechnology

random forcing functions. Control variables are 216]. Mostly, they are used for software sensors
those which are chosen by the operator to ma- [217, 218] or optimization purposes [219, 220].
nipulate the system in such a way the benefits are Both represent black-box applications (see be-
maximized. Typical examples in bioprocess en- low), where only the outputs are of special inter-
gineering are: temperature trajectories, agitation ests, the inner relations or interpretation aspects
rate, or feed rate of key components. Random are secondary.
forcing functions can be seen as outer distur- As output variables − →y (t), very often quanti-
bances of the system whose origin comes from ties are specified which are accessible by online
the system environment and not from internal measuring systems or which can be estimated
uncertainties, e.g., impurities of the inoculums by software algorithms. They can be seen as
or the feed medium. To evaluate the system dy- the result of a measurement model which relates
namics, it is important to define meaningful tra- the state variables −→z (t) to the output variables
jectories of the control variables. Keeping the →
−y (t). These quantities represent the values with
values constant will not result in a representative whose the system can be evaluated. The output
information retrieval of the biosystem. Only if vector −→y (t) is strongly related to the aspect of
characteristic curves – or specific combinations observability (see below).
– are chosen to which the system reacts sensitive The division into parameters, state variables,
a representative process description is possible. or output variables is not an immanent property
Therefore, it is evident that the experimental as of a system, but a specific view by the analyzer
well as the theoretical design must be performed of the system. It can be changed in relation to the
from deep problem awareness. mode of process operation, to the process targets
The state variables −

z (t) represent storage el- or the simplifying assumptions of the model. A
ements for mass quantities (e.g., substrate, prod- state variable zi can be exchanged to an output
ucts, biomass, inhibitors, interferences), infor- variable yi and vice versa. A mathematical sys-
mation, or energy of the system. The state vector tem should provide a coherent description of the

−z (t) is composed of any set of quantities suffi- entire bioprocess including all relevant system
cient to completely describe the behavior of that aspects. The degree of complexity of the biopro-
system. Given a state vector at a particular point cess or the possibility for a simplified modeling
in time and a description of the system forcing of some parts of the system is determined by
functions, in an appropriate uncertainty formu- the intended application and by the skills of the
lation, and control functions from that point in system analyzer.

time forward, the state at any other time could be Parameters p can be seen as fixed quantities
computed. In biotechnological processes state of the system. In the ideal case, they should be
variables are frequently defined, if the key quan- time invariant, as it is indicated by their original
tity of interest can not measured directly and definition. However, sometimes a time depen-
must therefore determined by state estimators dency of parameters is introduced to gain a bet-
from other process variables [213, 214]. The in- ter system description. Normally, the parameters
troduction of state variables is not strictly nec- are used to interpret the biosystem characteris-
essary. But in their absence inner relation can tics. However, the meaning of the parameters
only be hardly understood and described. Also can be quite different depending on the point of
a direct mapping of an input vector to an output view from which the biosystem is described. In
vector without consideration of state quantities terms of a mechanistic approach using funda-
in form of a signal model, as it is typical in con- mental physico–chemical equations, the deter-
ventional signal analysis in electrical engineer- mined parameters, after they are fitted by ex-
ing, can not be accomplished. Their application perimental data, have a direct physical meaning
depends on the point of view. A signal model and can be used for interpretation purposes and
provides mainly information about the behav- are comparable between different experiments.
ior of changing influencing parameters and how If the behavior is characterized phenomenolog-
they affect the outputs based upon given inputs. ical, by means of descriptive equations, the pa-
No information can be provided about the intrin- rameters determined are only valid under the
sic reactions or the basic mechanism, the focus specific boundary conditions of the experiments.
is outside of any interpretation purposes [215,
Biotechnology 69

They can be quite different in different exper- puts are known, a prediction is often even possi-
iments and possess nearly no general validity. ble with incomplete information on the involved
One must be very careful when comparing dif- intracellular reaction network [221 – 223].
ferent approaches upon the quantities of these For analysis and design of those systems, two
parameters. aspects have to be considered – the biological re-
actions catalyzed by the bioactive compound,
and the numerous chemical and physical pro-
8.1.2. System Description cesses which precede, accompany, and follow
them. Within the biosystem, the most important
A biotechnological system consists in particular physical processes, which are intimately bound
of a set of chemical, biochemical, and microbi- to the biological reactions, are associated with
ological reactions whose components, reactions transport of material, energy, and information to
rates, and other characteristics as temperature, and from the location of the bioactive unit. The
pH-value, or internal energy, are mostly not ex- dependency of the biological reactions on the
actly known. The metabolism of the biological microenvironment around the unit is called mi-
unit is a very complicated process, not only be- crokinetics. Due to the transport phenomena, the
cause the intracellular reaction network may be microenvironment will vary along different lo-
quite complex, but also due to the large num- cations in the systems. The integral description
ber of inner relations, control loops on reaction of microkinetics in connection with the trans-
level, regulatory mechanism, and genetic level port processes that may be included in a sys-
that are overlaid to coordinate the elementary re- tem model is called the macrokinetics. Ideally,
actions. So even when the microkinetics could one would always attempt to model the transport
be measured exactly, it would be impossible to phenomena and the microkinetics of the model,
establish a correct system map in every detail because this model could be combined with
without the introduction of rigorous simplifica- proper reactor models to predict the macroki-
tions. Another reason for uncertainty is the inho- netics of different biosystems. Unfortunately, at
mogenity of the units (e.g., cells) in the whole en- present, the effort for simulation of detailed re-
semble, which is normally not considered when actor models is rather high and methods for an-
characterizing biosystems. The relations are not alyzing the microenvironment of the cells are
constant and vary with time. Furthermore, there still rare. Therefore, what is usually observed
might be a morphological differentiation bio- is always a kind of macrokinetics. To find a
logical unit which is accompanied generally by compromise between these facts and our limited
changes in the dynamic behavior. knowledge about the process, the description
Therefore, the interesting question is why of the biological system is often characterized
these complex systems can often be described by means of so-called formalkinetic approaches.
for specific process targets by a few mathemat- This means a formal application of system equa-
ical equations only. One reason is that the func- tions that represent actually microkinetics to a
tional blocks of the biosystem operate together process, where only averaged macrokinetics can
in a network of regulation, and exchange of be measured. As an immediate consequence, the
mass, charge, and energy and a few bottleneck model parameters will change when the oper-
processes determine the behavior of the whole ating conditions of the reactor are changed, or
system. Another reason is the tremendous num- even more, if a different reactor with changing
ber of units in the ensemble of a biosystem which boundary conditions is used. This necessity lim-
hides individual variations in their growth and its the predictive power of formalkinetic models
leads to a smoothed average behavior. In most for biotechnological processes significantly.
cases, the problem shifts. It is not the difficulty to In principle, independent of the available sys-
build up the equations, but it is hardly to deter- tem volume, the mass balance of a component i
mine the characterizing parameters, which are from the general point of view can be described
generally dependent on time and other system using a finite volume approach solving the gov-
state quantities. Furthermore, a few reactions erning equations for fluid flow and heat and mass
can already determine the rates of many others. transfer:
When the main inputs and the final overall out-
70 Biotechnology
∂ci   dci Hj Reaction enthalpy of j-th reaction
+div ci u −div (Γ gradci ) − =0 (4)
∂t dt cp Averaged specific heat capacity of mass in the
considered volume
where the dependent variable is denoted by the

concentration ci and u represents the velocity. In this model, all the components involved
Both Γ , the diffusion coefficient, and dci /dt, the in the process reactions take part in the mathe-
source term, are specific to a particular mean- matical consideration. Furthermore, Gavalas as-
ing of a component i. From this general descrip- sumes that the j-reactions are independent of
tion, a one-dimensional model can be derived. each other. The mass conservation and more pre-
In the presented approach a dynamic process de- cisely the conservation of atomic species impose
scription is the goal rather than a rigorous three- certain relations between the stoichiometric co-
dimensional simulation of a compartmented sys- efficients k ij .
tem. For describing biological systems a cou- In this case, a so-called forward problem is
pling with the involved chemical, biochemical, considered. A forward problem is one in which
and microbiological reactions is necessary. the parameters and starting conditions of the sys-
In the considered (finite) volume, homoge- tem, and the kinetic or other equations which
neous (bio-)chemical reactions system can be govern its behavior, are known and the equations
analyzed according to Gavalas [224]. He as- can be solved by typical numerical methods.
sumes a detailed knowledge of the chemical be- From a mathematical and physical point of view,
havior of all the system components. The dy- it can be seen as the calculation of the “effect of
namic of those reaction systems is described by a cause” and not to estimate the “cause of an
a set of ordinary differential equations, by us- effect”, as it is accomplished in an inverse prob-
ing the idea that every isolated chemical kinetic lem. In other words, we usually know how to use
system should be consistent with the conserva- mathematics and physical reasoning to describe
tion of atomic species and of internal energy. In what would be measured if conditions were well
principle, this approach can also consider regu- known [225, 226]. Most mathematical models
latory and closed-loop effects at the component in fluid dynamics are of the “forward” type; the
level. A general stoichiometric exact model as relevant properties of the aquifer or reservoirs
a closed homogeneous reaction system of con- are assumed to be known, as well as the initial
stant (finite) volume can be described as follows. and boundary conditions. A model then predicts
M
the resultant flow. This is typically the approach
dci (t)  used to map a scenario caused by lot of complex
= kij ϕj (c1 (t) ,. . .,cN (t) ,T (t)) (5)
dt j=1 interacting partial processes or taken in sensitiv-
where ity studies, which are quite useful, and can show
what the most important features or processes
(·)i Component i with i = 1, . . . , N
(·)j Reaction j with j = 1, . . . , M are likely to be for a site [227 – 229]. Follow-
ci (·) Concentrations of the different ing the ideas of forward problems, structured or
(bio-)chemical components i cybernetic approaches for the description of bi-
jj (·) Reaction rate of the j-th reaction
T (·) Temperature
ological systems can also be mentioned. They
kij Exact stoichiometric coefficient structure the cell mass into several intracellu-
from component i in reaction j lar compounds and functional groups which are
If we consider a reaction in a volume with connected and regulated via an optimal criterion
possible in- and outflow, the above approach to each other and to the environment by fluxes of
must be extended with the balances of the in- material and information. The functional groups
ternal energies may, in one extreme, consist of detailed reaction
 
network considering as many as known reactions
∂T dT and regulatory loops [230, 231].
+div T u −div (Γ gradT ) − =0 (6)
∂t dt Mostly, however, in bioprocess engineering
dT (t) 1 
M when microbiological and biochemical reac-
= Hj ϕj (c1 (t) ,. . .,cN (t) ,T (t)) (7) tions are involved, the exact stoichiometry is un-
dt cp j=1
known. In the engineering literature of the last
where 20 years a less precise mathematical model for
Biotechnology 71

biotechnological processes is encountered [232, is proportional to the specific growth rate of the
233]. The so-called “general reactor model” – organism µ, there exist several possible models
firstly introduced by Bastin and Dochain [232] [234]. This approach is very common in liter-
– tries to unify chemical, biochemical and mi- ature (overviews in [235, 236]) and can be as-
crobiological process using the same approach. signed – in contrast to the above-mentioned ap-
The bioprocess, an n-dimensional system of or- proach – to the class of inverse problems.
dinary nonlinear differential equations, is based It is typical for inverse problems that in many
on a simple representation of the biotechno- situations the quantities that we wish to deter-
logical processes, where superfluous details are mine are different from the ones which we are
omitted. Only the dynamics of those compo- able to measure. If the measured data depend,
nents that play an important role in the process in some way, on the quantities we want, then
are considered, irrelevant byproducts or nonlim- the data at least contain some information about
ited substrates are neglected. The stoichiometry those quantities. Starting with the data that we
of the chemical reactions is only qualitatively have measured, the problem of trying to recon-
taken into account by means of yield coefficient struct the quantities that we really want to know
Yζ(·)/c(·) or stoichiometric coefficients k∗ij (Eqs. is called an inverse problem; we measure an ef-
8 and 9). fect and want to determine the cause. Typical
M ∗ applications of inverse problems are model iden-
dςi (t)  ∗
= kij ϕj (ς1 ( t),. . .,ςN (t) ,T (t)) (8) tification and estimation, image analysis, numer-
dt j=1 ical analysis, or navigation [237, 238].

M dςj
dt
J=1
Yςj /c∗ = dc ∗ (9) 8.1.3. Dynamics of Biosystems and
i i
dt Real-Time Considerations
where ci (·) and ζi (·) are concentrations of the
different (bio-)chemical components, ci (·) are Real-time considerations play an important rule
those substances (i*=1, . . . ,N**) whose re- dealing with process control strategies. A noto-
actions are calculated by the yield coefficients rious underestimation of the dynamic properties
Yζ(·)/c(·) , ζi are those components (j=1, . . ., N*) of microbial and cellular populations exists and
whose turnover are determined by the dominat- results mainly from matching the duration of the
ing reaction rates ψj . respective batch cultivations to the relevant time
constant of the biosystem under investigation.
M∗ Number of the dominating reactions in the However, metabolic regulation of enzyme activ-
considered volume ities and fluxes often takes place on a time scale
ψJ (·) Reaction rate of the j-th reaction
T (·) Temperature of seconds rather than days although the latter
may also be true for some processes. It is there-
In general, these coefficients do not have any fore in the scope of promoting biotechnological
mechanistic meaning in contrast to the above- research to adopt and develop appropriate exper-
mentioned approach, instead they represent for- imental concepts, methodologies and equipment
malkinetic quantities, which must be estimated, in order to consider all relevant time constants
determined experimentally, or established by [239].
practical experiences. This allows the inclusion What is fast? What is slow? What is relevant?
of chemical, biochemical, and microbiological The last question is the most important when
processes in a unified approach. However, the dealing with modeling. The relaxation time con-
reaction scheme may be inconsistent with the cept of Harder and Roels [240] (Fig. 28) maps
law of conservation of mass. Also cell-to-cell typical time constants of microbial and cellular
heterogeneity, e.g., due to different proliferation control on the level of modification of enzymes
phases, are not considered and simplifies a segre- (activation, inhibition, dis-/association of sub-
gated viewpoint to an unsegregated perspective. units, covalent modification or digestion) to the
The reactions rates can be often a very com- range of milliseconds to seconds, on the level of
plex function of the operating conditions and of regulation of gene expression (induction, repres-
the state of the biosystem. In the case were ψ sion, or derepression of transcription) to min-
72 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 inase and/or hexokinases). Only later, during
evolution to days and larger units. Considering catabolism, can the energy provided by this ex-
mainly the growth of microorganisms, typical tra substrate be liberated in terms of new ATP.
time scales of 0.3 to 20 h are relevant. The exam- The duration of the ATP sink was predicted to be
ples discussed below will illustrate how bioengi- in the order of a few tens of seconds by Nielsen
neering is facing the individual time constants. et al. [244]. Obviously, such rapid reactions are
A typical bioprocess, if operated in batch under kinetic control and the necessary enzyme
mode, extends over several hours or a few days. activities are present in sufficient quantities. It
If operated in continuous mode, it is not rea- is not clear, at present, whether the fluxes attain
sonable to accept operating times of less than a their organism typical maximal values immedi-
month (see, e.g., Heijnen et al. [241]). If an or- ately or some fine tuning of enzymatic control
ganism has special physiological features such precedes this event. It is likely that in the latter
as baker’s yeast, a transition from one to the other case a regulation of (intracellular) enzyme ac-
domain (e.g., from low to high dilution rates or, tivity, if at all necessary, takes place on the level
in other words, from purely oxidative to oxidore- of enzyme modification (e.g., phosphorylation)
ductive growth) may also result in considerable rather than on the level of de novo production
changes in the time required to approach a new (i.e., control on the transcriptional level) because
steady state. Axelsson et al. [242] reported a the latter would require much more time [245].
rough estimate for this case: the time constant The dynamics of microbial cultures have an
for experiments at low dilution rates (D<DR, important impact on the characteristics of mea-
where DR=D at which the regulatory switch bet- surement and process control. The “typical time
ween oxidative and oxidoreductive metabolism constant” in a bioprocess is often erroneously
occurs) is, as expected, in the order of the mean anticipated to be equivalent to the entire duration
residence time (τ =D−1) however, above DR, of cultivation. However, the quantitative inves-
the time constant was predicted to be at least tigation of substrate uptake requires a time reso-
one order of magnitude greater. In experiments lution of a few 100 ms, otherwise artifacts must
specifically designed to verify this, either even result [246, 247]. This statement was evidenced
greater time constants or no unique stable steady only after a suitable technique had been estab-
states at all were found [243]. lished: glucose metabolism was stopped within
If excess carbon and energy source is pulsed 100 ms by spraying the cell suspension from the
to a carbon- and energy-limited culture, the overpressurized bioreactor into 60 % methanol
intracellular ATP concentration initially drops which was pre-chilled to −40◦ C.
because of the phosphorylation sink (glucok-
Biotechnology 73

The relevant relaxation times of a culture sys- recovered is representative for the filter site but
tem are determined by the actual cell density and not for the reactor. Knowing the transport time
the specific conversion rate (capacity) of the cul- and some basic kinetic parameters one can easily
ture, that is, by one or more operational and state compensate online for such errors provided that
variables (for instance feed rate, the concentra- a useful estimate of the actual biomass concen-
tions or activities of cell mass and of effectors, tration is available. Even though a bypass can
if relevant) and inherent characteristic proper- be tuned to operate at a mean residence time
ties of the biosystem which are represented by of 5 s or less, this can be enough for a signifi-
parameters. There are metabolites with a long cant decrease in substrate concentration in high-
lifetime and other (key) metabolites with very density cultures. Sample buses need a minimal
short lifetimes, e.g., molecules representing the (dead) tune for transportation of the sample and
energy currency of cells such as ATP and other in situ filters tend to fail in high density cultures
nucleotides. Rizzi et al. [248] and Theobald et because of rapid fouling. Hence, the problem is
al. [249] have shown that the energy charge re- real and can not be ignored.
sponse to a pulse challenge (ATP) of a yeast
culture is a matter of a few seconds only. How-
ever, the responses can differ considerably when 8.2. Biotechnological Measurement
pulses or shifts are applied to cultures of differ- Systems
ent recent history [245, 250]. Neubauer et al.
[251] found that substrate oscillations greatly af- All measurements have the ultimate goal of
fected the growth performance of E. coli. Short creating representative information from the
term, that is, less than 2 min, glucose excess, and biosystem involved. Cellular or biochemical
starvation were investigated in a looped system quantities are the primary variables in biopro-
of a stirred tank and a plug flow reactor. The cess engineering. They mainly determine the
metabolism changed within some few minutes performance of the bioprocess and are therefore
totally from anaerobic to aerobic and vice versa. of special interest [253, 254]. Measurement and
Similar observations have also been made for acquisition of information are building the fun-
yeast [252]. dament for all process observations as well as for
Two other paradigms demonstrate that the the development of new bioprocesses. They re-
band width of relaxation times is extremely present the presupposition for process optimiza-
broad: 1) the time required to achieve a new tion and control. However, they must be avail-
steady state in a chemostat culture is approxi- able online to exploit them in a control strategy.
mately determined by (µmax −D)−1 . 2) The time Measurements are not only key issues in modern
required by a culture to consume a considerable process development, they also open the door to
fraction of small amounts of residual substrate a detailed process supervision and understand-
during sampling – and thus systematically fal- ing, and avoid restricted views when analyzing
sify the analytical results if no appropriate inac- processes just through a keyhole [255, 256].
tivation takes place depends, among others, on In comparison to other disciplines such as
the cell density and can also be in the order of a physics, mechanical or electrical engineering,
few seconds only. sensors useful for online monitoring of biotech-
Even simple static models are very valuable nological processes are comparatively few; they
for compensation of systematic errors built into are mostly available for physical and chemical
automated analytical procedures. One important quantities rather than for biological ones. The
example is the case when sampling requires a reasons are manifold, but generally biologically
well-known but non-negligible time. A bypass relevant information is much more difficult and
behaves as a plug flow type reactor fraction complex to access and interpret than those for
where flow dependent spatial gradients develop typical physical or chemical quantities [257].
and where no inactivation can take place because Other important reason derives from restricting
the bulk of the bypassed aliquots are returned requirements for the sensor equipment, namely
to the reactor. The cells continue to consume
substrate while they are being transported from – Sterilization procedures,
the exit of the reactor to the filter. The permeate – Stability and reliability over extended periods,
74 Biotechnology

– Application over an extended dynamic range, by other measured values. But this implies thor-
– No interferences with the sterile barrier, ough problem awareness and is restricted to spe-
– Insensitivity to protein adsorption, surface cific boundary conditions (see Section 8.2.2).
growth,
– Resistance to degradation or enzymatic break
down, and
– No disturbances from matrix compounds (in-
terferences, matrix effect).
These aspects are in an industrial environ-
ment 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 mea-
surement 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 bio-
compatibility, which often make the construc-
tion of the sensor hardware rather difficult.
Figure 29. Common measurement instruments and control
units of bioreactors as generally accepted as routine equip-
ment (→ measurement only,  measurement and open- or
8.2.1. Process Requirements Concerning closed-loop control)
Measuring Quantities
In modern bioprocess engineering there are un-
doubtedly only a few variables that are generally Biomaterial. The active biomaterial is of
regarded as essential. Among these are several paramount importance to scientists as well as
physical (i.e., temperature), less chemical (pH- to engineers. It is an indicator for the avail-
value) and even less biological quantities (cell able quantity of biocatalysts. It is definitely an
concentration). Figure 29 gives a summary of important key variable, because it determines –
what is nowadays believed to be a minimum set simplifying- the rates of growth and that for bio-
of required measurements in a bioprocess. Such transformation. Almost all process descriptions
equipment is typical for standard production of and optimization tools contain the quantity of
biomaterial [258]. However, the conclusion that biomass as the most important state variable.
these variables are sufficient to characterize the The biomaterial of interest depends on the
microenvironment and activity of the biocompo- system considered: microorganism, enzymes,
nents, of course, is more than questionable. For nucleotides, or simple protein amount and con-
the consideration and compliance of the process stitution. Many control strategies involve the ob-
targets, as described above, besides some envi- jective of optimizing the biomaterial concen-
ronmental and operational quantities, in partic- tration. For automation purposes, the biomass
ular the important biochemical state variables is mostly seen as a homogeneous entity, being
of the biosystems must be known, namely the aware that this represents an uncertainty because
amounts and composition of the active biomass, segregation into different individuals is more re-
of the starting material, of the products (and alistic. An ideal measurement of the bioactive
byproducts) or other metabolites. Modern bio- compound would include their activity, physi-
process observation systems try to overcome ological state, morphology, or other classifica-
the lack in the instrumentations, by building up tions rather than just their mass. But these quan-
functional relationships between these quanti- tities are normally ill-defined and, in general,
ties. The approach is to substitute the direct mea- inaccessible to online measuring devices.
surement by a function based value, calculated
Biotechnology 75

A series of sensors and methods, that can other bio- or food technological processes, the
be automated and have appeared in the re- conversion of a substrate from specific state of
cent decades for the measurement of the to- aggregation to another is of special interest. For
tal biomass without discrimination of any seg- example, in the case of fouling of heat exchang-
regation (e.g., active or inactive cells). Many ers where dissolved proteins are transformed to a
of them rely on optical measuring principles solid protein layer, the starting material changes
[259, 260], others exploit filtration character- only its state not its chemical structure. Or, con-
istics [261, 262], electrical properties of sus- sidering drying or thawing where water changes
pended cells [263, 264], or thermodynamic laws its state (or is removed) which strongly influence
[265]. One has to decide according to the spe- the quality and behavior of the biomaterial in-
cific application, which method seems to be ap- volved, but no (bio-) chemical conversion takes
propriate concerning the boundary conditions, place. The classical methods to determine these
because all methods reply on indirect measur- quantities are offline laboratory methods so far.
ing principles, and the access of the informa- This implies that samples are taken, sometimes
tion carrying signal differs in non general ranges. aseptically, pre-treated, and transported to a suit-
To get online information about the actual state able location, where storage of these samples
of the biomaterial techniques based on fluores- might be necessary before analyzing manually.
cence principle, especially the 2-D fluorescence These steps need a lot of human and instrumen-
spectroscopy, should be mentioned. It represents tal resources.
a noninvasive method to analyze intracellular
compounds. Technically, either intra- or extra- Product. The product is almost the only rea-
cellular fluorophores (e.g., NAD+ , Riboflavin, son why a process should run. The main concern
Tyrosine, ATP) are excited by visible or ultra- is in maximizing the profit, which depends di-
violet light, and the fluorescent light emitted by rectly on the volumetric productivity and/or on
the fluorophores at a longer characteristic wave- the purity of the resulting product. It is there-
length is collected and gives information about fore of essential interest to know the quantities,
their concentration [266 – 268]. which require measurements. What was said
above about substrate determination and the ap-
Substrate. The presence of sufficient and plication of classical methods is valid here, too.
appropriate starting material (substrate) is the In summary, bioprocess science needs sig-
cause of any biotransformation and represents nificantly more quantitative measurements. It is
the supposition of the product formation. One insufficient to know that something happens, we
can solve the inverse problem, namely conclude need to know almost instantaneously, online and
that biological activities cease whenever an es- automatically why, how, and to what extent a
sential substrate is exhausted, and thus omit bioprocess behaves [270].
the measurement of the substrate, provided the
progress of the bioprocess and/or product for-
mation is known [269]. But, this is not always 8.2.2. Online Sensing Devices
a proper solution because there are many more
plausible, and also probable, reasons for a de- The development of increasingly sophisticated
crease in bioactivities than just their limitation equipments for measuring of starting material
by depletion of a substrate. One must, then, solve and products represents one trend to satisfy
the direct problem, namely analyze the relevant the growing demand for high-quality measure-
quantities of the biological unit. From an engi- ments. In contrast to engineering disciplines
neering point of view this measurement should such as mechanical or electrical engineering,
be available instantaneously in order to be able to in bioprocess engineering almost no measure-
control the desired process characteristics. In en- ment performed is directly, i.e., none is ob-
vironmental biotechnology, in particular, the ob- tained by immediate comparison with a refer-
jective of a bioprocess can be to remove a starting ence quantity. Most measurements are achieved
material as completely as possible rather than by means of some specific physical or chem-
making a valuable product. So, the starting ma- ical property of the process value, often hid-
terial is identical to the process product. In some den in the very complex sample matrix com-
76 Biotechnology

mon to bioprocesses. This very complex ma- a direct interaction between a product property
trix, which consists of the numerous substances and sensor exist, otherwise exline; this is more
and parameters surrounding the analyte and their a technological characterization. Depending on
mutual influences, makes the task of measuring the site of installation, one discriminates further
difficult and leads to the development of more between in situ, which means built-in, and ex
and more complicated measurement techniques situ, synonym for a bypass configuration or for
[271, 272]. The surrounding equipment, i.e., for an exit line. In the latter case, the withdrawn
the sample pretreatment, exceeds the pure an- volumes are lost for the process. A further clas-
alytical core [273 – 275]. However, the greater sification can be made by the mode of operation
the measuring system complexity, the more dif- of the sensing device. One can discriminate bet-
ficult it is to ensure the accuracy and the reliabil- ween continuous and discontinuous signal gen-
ity of the collected data. The need for accuracy eration in a predefined time step. This is a very
and reliability increases even more if the data of important fact for planning and developing an
the process analyzer should be used as input for appropriate process controller considering the
a closed-loop controller. When unreliable mea- time scales of the bioprocess. In the latter casem,
surement systems are used, the system opera- one has to guarantee that the process information
tor can only hardly decide whether an occurring is available in real time.
variation is due to an error of the analyzer or a Only online sensors are of special interest
result from the bioprocess itself. concerning the process control of biotechno-
The final goal of the measuring unit of a bio- logical systems. Indeed there exist a couple of
process is to build up simple online instruments different, very powerful systems for the offline
in order to collect all necessary information from analysis of nearly all relevant substances. For
the biosystem, which is necessary to hold the de- online sensors, the situation is quite different.
fined process targets. Therefore, the process tar- In situ instruments exist only for a few quanti-
gets should represent the selection criterion for ties like the measurement of temperature, pH,
defining the measuring equipments. The infor- pressure, oxygen, carbon dioxide, density, flow
mation should be available online and no manual rates, electric power consumption, or redox po-
interaction should be necessary to obtain the de- tential. They use different measuring principles
sired measuring results. This aspect touches the and are widely introduced in industrial process
term “system observability” (see Section 8.4.1), engineering.
which is strictly defined for linear systems, but However, for the most important biologi-
eludes a definition for nonlinear systems as they cal quantities in biotechnological systems es-
are common for typical biological systems [276, pecially the measurement of active or inactive
277]. It is not obvious and definable whether biomass or the specific determination of individ-
all system information is available und known ual biochemical substances there exists a deep
to reach the observability in respect to the pro- lack in online instruments. In the following, prin-
cess goals or which sensors are still necessary cipal techniques should be presented, which pos-
to reach the specified process goals. The conse- sess a certain online capability. See also → Bio-
quence is that there are normally one or more chemical Separations; → Mass Spectrometry;
state variables (or linear combinations of them) → Surface and Thin-Film Analysis.
that are hidden from the view of the observer
(i.e., measurement equipment). Chromatographic Methods. A review of
Concerning the information treatment there chromatographic methods is beyond the scope
can generally be made different classifications, of this contribution. Both (high-pressure) liquid
which are important for industrial applications. chromatography and gas chromatography have
From the communications engineering point of been applied in numerous cases to offline anal-
view the information can be distinguished bet- yses of biotechnological samples, but online ap-
ween online and offline. If a continuous auto- plications, especially for industrial processes,
matic correlation between the signal from the have only recently been reported [278 – 282].
sensor and the product or process quantity is pos- The scope of chromatographic methods is the
sible an online signal is available, otherwise it separation of the individual constituents of mix-
is offline. A measurement is called inline, when tures as they pass through columns filled with a
Biotechnology 77

suitable stationary phases. As disadvantages the chemical into an electrical signal (overviews in
consumption of almost expensive reagents must [285 – 287]). In spite the fact, that principally
be mentioned. The samples injected are to be for nearly all biochemical substances an biolog-
pretreated in such a way, that they possess nearly ical recognition unit exists, biosensor applica-
no impurities, like gas bubbles or solid compart- tions in an industrial surrounding often concen-
ments. Any existence of those interferences dis- trate only on a small group of substrates, namely
turbs the analysis in a significant manner, and glucose, ethanol, or lactate [288 – 292]. Gener-
manual service procedures are mostly the con- ally, biosensors are tricky to handle and must
sequence. Furthermore the measuring time last in principle be recalibrated in the matrix where
up to 30 or 40 min; a time period which is of- the measurement should take place every time
ten too large for bioprocess supervision. But also the process conditions change. Due to the sen-
economical aspects must be taken into consider- sible biological element – an enzyme or living
ation. Chromatographic systems possess a high microorganism – they principally cannot be ster-
cost price. Furthermore, high-specialized staff ilized. They also suffer from changes in the envi-
must service them frequently, an aspect, which ronment, for example, changes in pH or forma-
represents the major drawback for an industrial tion or existence of aggressive chemicals such
application. as H2 O2 [293]. Therefore, they must be used
in a suitable environment. Furthermore, the sur-
Mass spectrometry. A further measurement rounding must be constant, because in general
method, in principal suitable for online applica- an interrelation to the endogenous matrix exists.
tions represents mass spectrometry (MS). Mass If the process media changes significantly, the
spectrometry has been mainly applied for the on- biosensor alters its behavior. A compensation of
line detection and quantification of gases such as the changed matrix interferences becomes nec-
pO2 , pCO2 , pN2 , pH2 , pCH4 , and even H2 S, or essary. This represents one of the most decisive
volatile substances such as alcohols, acetoin and drawbacks in inline biosensor applications for
butandiol [283]. Generally, the detection princi- bioprocess engineering [294 – 296]. The long-
ple allows simultaneous monitoring and, con- term stability under working conditions is often
sequently, control of metabolites. The princi- poor; they must be serviced for several hours per
ples, sampling systems, control of the measur- week preferentially by high-specialized staff. In
ing device and application of MS for biopro- this way, only limited experience could have
cesses have been summarized elsewhere [284]. been gained under technical process monitoring
Of course, mass spectrometry requires very ex- conditions. They possess only in some excep-
pensive equipment and needs frequent and com- tional cases a real industrial potential.
plex service procedures. But it should be taken A reasonable way around the problem repre-
into account that automatic multiplexing of dif- sents the measuring of the biotechnological me-
ferent sample streams is possible and, in addi- dia after removing a sample from the reactor in
tion, a great variety of different substances can a bypass configuration. Consequently, the sen-
be determined simultaneously. Thus, one has to sor is not installed in situ as it would be opti-
decide, whether the high economical and appli- mal but the information could be provided online
cational expense is justifiable in comparison to and can be fed into an automatic process control
the advantages due to the acquisition of infor- system. Depending on the analyte of interest,
mation. i.e., whether it is soluble or (in) the dispersed
phase, one needs to sample either the entire cul-
Biosensors. The most promising technique ture liquid or just a supernatant. The latter can
to overcome these problems, represented in the be acquired using for example a membrane mod-
last decade, is the use of biosensors . Biosensors ule. Concerning sampling, four aspects must be
consist of a sensing biological module of either taken into account.
catalytic (e.g., enzymes or microorganism) or
affinity reaction type (antibodies, cell receptors) 1) The system must be opened in such a way, that
in intimate contact with a physical transducer no infections can enter the reaction space ei-
(electrochemical, optic, calorimetric or acous- ther during sampling or between the sampling
tic sensor). The latter finally converts the (bio-) events.
78 Biotechnology

2) A representative sample must be provided. So a high sampling frequency (up to >100 h−1 ),
the overall volume of the sampling chamber small sample and reagent consumption, high re-
as well as the position of device at the biore- producibility and nearly total versatility of the
actor must be determined properly. Addition- sensing methods.
ally, the sample taken can still continue to re-
act on the way to the sensor. In this case the Software Sensors. The most promising
sample would not be representative for the method to get online access to important key
interior of the reactor, and appropriate addi- components of bioprocess quantities are soft-
tional measures or modeling approaches need ware sensors. In general, software sensors sup-
to be taken in order to assure representativity. ply the estimation of the missing measurements
3) When native samples are applied to the ana- by using an appropriate model that relates the
lytical system, problems can arise from matrix corresponding variable with other physical or
interferences and matrix effects [297]. There- chemical measurements that are correlated to it
fore, the sampling device should be chosen in any way. The fact that the model is imple-
in that way that no interaction with the matrix mented by means of a software package (hence
occur which interferes the measurement (e.g., the expression soft(ware)-sensor) denotes, that
absorption of substrate at the membrane). On software sensors provide a software backup for
the other side, the sample device should with- unavailable sensors, as an alternative to a hard-
hold all the cross-sensitivities for disturbing ware back-up using spare sensors [299 – 302].
co-components. Generally, software sensors are typical solu-
4) Due to the high number of individual sys- tions of so-called inverse problems (see Section
tem components and their complex interac- 8.1.2). In a complex biological system, in par-
tion (i.e., sample pretreatment and analytical ticular, the quantities which are normally easiest
part), sampling devices provoke a small reli- to measure are the variables, not the parameters.
ability in regard to the required process time. In the case of metabolism, the usual parameters
Furthermore, the hardware, necessary for the of interest are the enzymatic rate and affinity
whole analysis system entails high original constants which are difficult to measure accu-
costs. The specific adaptation to one concrete rately in vitro and virtually impossible in vivo
process condition implies inflexibility. It ex- [303 – 306]. Yet to describe, understand, and
cludes the determination of other metabolites simulate the system of interest we need knowl-
in other process media. Its adaptation is only edge of the parameters. In other words, one must
possible by time- and money-consuming al- go backwards from variables such as fluxes and
terations. metabolite concentrations, which are relatively
easy to measure, to the parameters.
Flow Injection Analysis. The most impor- In other words, when using software sensors
tant technique of sampling methods in biotech- there must always be a model available that re-
nology, behind some conventional devices like liably relates the measured variable with the tar-
membrane modules, represents the flow injec- get variable or parameter of interest. Normally,
tion analysis (FIA), firstly introduced by Ruz- measured variables are easily measurable effects
icka and Hansen [298]. It can be viewed as a that are caused and influenced by the target. It
general solution-handling technique or sampling is the special objective of the software sensor to
device combined with a sensing unit. This com- reach a maximal degree of generalization. How-
bination causes a high flexibility with respect ever, this is very difficult – even impossible – to
to the combined analytical procedure. It can be achieve or furthermore to prove this claim. Thus,
seen as a principle where a small injected vol- the basic question is: Is the available informa-
ume (10-200 µL) is introduced into a continuous tion representative enough to generate a model
unsegmented stream of carrier. The sample dis- for the accurate estimation of the quantity of in-
perses in a well-defined concentration gradient terest, or is the desired information inaccessible
and is transported by the carrier stream to the by the chosen sensor collection? Consequently,
reaction zone and a subsequent detector mod- the development of software sensors is often re-
ule. FIA cannot generate continuous signals. stricted to a specific application; any transfer to
But, there are several important advantages like
Biotechnology 79

other measuring problems is almost combined historical experiments or runs which an expert
with considerable alterations. This does not con- has associated with a typical physiological state.
cern only the determination of the new param- A physiological state is recognized either if the
eter at an identical model structure. Instead a actual constellation matches any one of the ref-
complete new system definition can be neces- erence sets best – in this case, there is always an
sary, where new input variables must be added identification made – or if the match exceeds a
or dispensable one can be deleted. predefined degree of certainty, e.g., 60 % – then
it can happen that no identification or associa-
Spectroscopic Methods. In this context the tion is possible when the pre-selected threshold
use of spectroscopic methods [307 – 310], after value was not reached. The direct association
optical or sonic stimulation play a dominant rule. with reference data needs normalization (am-
They must be seen as multisensor systems. Their plitude scaling) and, probably, frequency analy-
common property is the detection of absorption sis in order to eliminate dependencies on (time)
degrees after stimulation at various frequencies. shifts, biases or drifts.
By this, a set of information is generated, which In other cases, the data trajectories are trans-
possesses more information than the measure- lated into trend qualities via shape descriptors.
ment at one specific frequency, the measurement These combinations of trends of the trajectories
at one frequency can be seen as the output of one of various state variables or derived variables
sensor. The multivariate evaluation in an appro- define a certain system state; the advantage of
priate model should subsequently reveal the ac- this definition is that the association is no longer
curate determination of the desired quantity. In dependent on time and on the actual numerical
such models, normally pure data are used, the values of variables and rates [315].
integration of knowledge from well-known fun-
damental equations is only very hardly to real-
ize. The most important advantage of spectro- 8.2.3. Further Aspects Concerning
scopic methods is their non-invasive character. Measuring Systems
The sensing device is not in direct contact with
the media or the process itself, so no interference Online measurements produced with in situ sen-
is to be expected. sors are difficult to validate. The usual proce-
The situation is even more complicated since dure for evaluating the quality of a measure-
things develop in relation to time. The estimation ment is restricted to calibration/checking prior
of the physiological state of a culture involves to and after an experimental run. A few sensors
more than one (measurable) variable at a time such as the pCO2 - or the Cranfield-glucose sen-
and the recent history of this set of individual sig- sor [316] allow removal and, therefore, recali-
nal trajectories involved. Consequently, physio- bration of the transducer during a run. External
logical state estimation requires recognition of chromatographs and FIA systems can be regu-
complex patterns. Various algorithms used for larly recalibrated but the sterile interface cannot.
this purpose to build up the (data-)model have Other sensors such as a pH or a pO2 probe can be
in common that it is not always the present values mounted via a retractable housing, which allows
alone that are evaluated, there is always the re- either sterile exchange or withdrawal for exter-
cent history of signal trajectories involved. Con- nal recalibration during a run. A further possi-
cerning the models, some authors define soft- bility to gain information about the reliability of
ware sensors only on the basis of neural net- a measurement is to mount a number of iden-
works but a broader point of view should be tical sensors in easy-to-compare positions and
adopted since software-sensor models are also to check the individual signals for equality. This
obtained by using regression or correlation tech- opportunity has been exploited in particular at
niques as well as fuzzy logic or first-principle applications where a high process safety must be
models or combinations of all of them [306, guarantied. It is highly desirable to have alterna-
311 – 314]. tive principles of measurement at hand, which
In some cases, the data describing the actual operate simultaneously (heterogeneous redun-
state and their recent history are compared with dancy) or to introduce a self-supervision op-
so-called reference patterns: these are data from
80 Biotechnology

tion, like pattern recognition to generally iden- of the methods, tools, and equipment currently
tify malfunctioning sensors [276]. available and to invent new and better ones. In
Elemental balancing permits the determina- essence, techniques of instrumentation, opera-
tion of (other) metabolic rates provided that the tion, and causal analytical interpretation of mea-
stoichiometry is known. Carbon balances are the surements need massive impulses.
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 8.3. Cognitive Computing
that the state predictions based on experiments
with a small mass spectrometer are not use- Artificial neural networks (ANNs) are dating
ful due to unacceptable error propagation; for back to 1943, firstly reported by McCulloch
instance, a 1 % relative offset calibration error and Pitts [321], then fallen into oblivion, but
could result in a prediction error for an intracel- started a tremendous comeback in engineering
lular storage material (PHB) of >50 %. Instead, science with the publications of Rumelhart in
using a highly accurate and precise instrument 1985, who introduced – again – the backpropa-
(absolute errors <0.02 % gas composition) to- gation algorithm and demonstrated its potential
gether with automatic, repetitive recalibration as a learning procedure in neural networks [322,
resulted, however, in reasonable estimation of 323]. Fuzzy Logic has been introduced by Zadeh
substrates, PHB, and biomass. Others have also 1965, firstly ignored consequently by techni-
experienced these findings, e.g., [318, 319]. cians because of their undeterministic and un-
The undelayed evaluation of the state of a cul- certain nature, but provoked a second wave of
ture by using software sensors and computers, interest in the 1980s predominantly in Japan
based on the quantitative analytical information in respect to their potential in treating highly
provided by hardware sensors and intelligent an- complex, nonlinear multiple input multiple out-
alytical subsystems, constitutes an excellent ba- put (MIMO) systems in technical applications
sis for targeted process control. Experts – either [324]. John H. Holland published 1975 his clas-
human or computer – have the data and the deter- sic work “Adaption in Natural and Artificial Sys-
ministic knowledge to trace observed behavior tems”; it can be seen as the pioneer work in
back to the physical, chemical, and physiologi- evolutionary programming [325]. Compared to
cal roots thereby gaining a qualitative improve- other mathematical principles, these three meth-
ment of bioprocess control, a quantum leap: pro- ods are fairly new approaches and did not gain
cess control can act on the causes of effects rather not exclusive acceptance by scientists and engi-
than just cure symptoms. A simple standard op- neers. The three methods mentioned above can
erating procedure [320] has proven useful in this be seen as the main representatives of a class
concern, namely: of problem-solving methods taking nature as a
model, especially how it has learned to solve
– Measure everything that can be measured at problems. They can be summarized to the area
the very beginning of process development of cognitive computing.
– Decide whether or not a variable is relevant It is not within scope of this contribution
– Determine the relevant variables to be mea- to explain these methods in their basic prin-
sured, controlled, and/or documented ciples and functionalities. Rather, their intrin-
– Collect all raw data at any time and distin- sic principles are assumed to be well known;
guish online between variable and parameter overviews and extensive explanations can be
behavior found for fuzzy in [326 – 328], for ANN in
– Organize an archive of all these data accord- [328 – 330] (see also → Process Control En-
ingly and do not discard seemingly useless gineering, Chap. 13) and for evolutionary pro-
data since they contribute to the treasure of gramming in [331, 332]. In the following, the
experience focus lies on two methods, on fuzzy logic system
If it is, as some people say, correct that today’s and on ANN, because of their high applicational
bioengineering with all its tools and methodolo- potential, especially in bioprocess engineering
gies is too slow and not efficient enough, then it is [333, 334]. The explanations should show their
all the more urgent to improve the performance potential, typical aspects and also some critical
Biotechnology 81

remarks, in particular in respect to bioprocess in our brain, not “out there” in physical reality
engineering applications. on this page. Immanuel Kant called these four
Neural networks and fuzzy theory have been ink stains 1783 noumena “things in themselves”.
underground technologies for many years. They Applied to technical problems, this means that
have had far more critics than supporters for we recognize information or generalize artifacts
most of their brief history. However, in particu- in that way, that we see not only the input–output
lar due to their application potentials in different mapping, but the real relations or laws the data
engineering areas where classical approaches stands for. The data can be seen as facilities or
failed, their acceptance is growing steadily. Neu- indications for the perception of the underlying
ral networks and fuzzy systems estimate func- intrinsic information.
tions from sample data, they map inputs to out-
puts. Statistical and artificial intelligence ap-
proaches also estimate functions. On the other
hand, for each problem statistical approaches re-
quire knowledge how outputs functionally de-
pend on inputs, in other words they need a
mathematical model. Thus, their fundamental
ideas can be more compared to typical conven-
tional mathematical principles. Instead, neural
and fuzzy systems do not require such a mathe-
matical model. They can be seen as model-free
estimators [328].
From another point of view, artificial intelli-
gence expert systems can also be seen as model-
free estimators, they map conditions to actions.
However, experts do not articulate a mathemati-
cal transfer function from the condition space
to the action space and the AI framework is
symbolic. Symbolic processing favors a prepo-
Figure 30. Kanizsa-square illusion
sitional and predicate calculus approach to ma-
chine intelligence. It does not favor numerical
mathematical analysis or hardware implementa- Today, many of the neural mechanisms that
tion. In particular, symbols do not have deriva- Kant could only guess are understood. We take
tives; only sufficiently smooth functions have for granted our high-speed, distributed, nonlin-
derivatives. Symbolic systems may change with ear, massively parallel pre-attentive processing.
time, but they are not properly dynamical sys- In our visual processing, we pay no attention
tems, nor systems of first-order difference or dif- to how we segment images, enhance contrasts,
ferential equations. Therefore, they are unsuit- or discount background luminosity, even if they
able for modeling purposes in bioprocess engi- are the carrier of the real information. When we
neering. process sound, we pay no attention to how our
A small excurse to neural (pre-) attentive cochleas filter out high-frequency signals even
processing in human intelligence should illus- if we collect the data. We likewise ignore our
trate which ideal result a cognitive system pro- real-time pre-attentive processing. We experi-
vides after its learning phase. Take a look at the ence these pre-attentive phenomena, but we ig-
Kanizsa-square illusion (Fig. 30). nore them and cannot control or completely ex-
What do we see when we look at the Kanizsa plain them. Natural selection has ensured only
square [335]. We see a square with bright inte- that we perform them, ceaselessly and fast. But
rior. We see illusory boundaries, or do we? We what we really do, we recognize segmented im-
recognize a bright square. Indeed, technically we age pieces and parsed speech units; we try to
cannot see it, because it is not there. The only extract and introspect the intrinsic carried infor-
things which are shown are four three-quarter mation based upon measured quantities.
circles, nothing more. The square exists only
82 Biotechnology

Neural network studies both pre-attentive and ations, new patterns, new functional dependen-
attentive processing of stimuli. This leaves unad- cies. They sample flux of experiences and en-
dressed the higher cognitive functions involved code new information. They compress the sam-
in reasoning, decision making, planning, or con- pled flux into a small but statistically repre-
trol. The nonlinear neurons and synapses in our sentative set of prototypes or exemplars. Sam-
brain perform these functions; they can deal with ple data, provided directly or transformed into
ill-posed problems or with a high degree of un- linguistic rules, changes the system structure or
certainty. Natural selection evolved this capabil- parameters. In all cases, learning means chang-
ity in exemplary manner. Furthermore, it is the ing. Neural networks learn patterns, functions,
attempt of engineers to copy exactly this capa- or probability distributions to recognize future
bility to technical problems by developing and patterns, filter future input streams of data, or
using fuzzy logic systems or artificial neural net- solve future combinatorial optimization prob-
works. lems. Fuzzy systems can learn or formulate as-
Both methods try to imitate nature and both sociative rules to estimate functions or control
estimate input–output functions, in spite of to- systems, either directly by means of data and an
tally different underlying principles. In contrast optimization add-on or guided by a highly expe-
to statistical estimators, they estimate a function rienced supervisor. It can be shown under some
without a mathematical model of how outputs assumption that a neural net can approximate
depend on inputs; this represents their main fea- to any degree of accuracy using a fuzzy expert
ture. They learn “from experience” with numeri- system and vice versa [341].
cal, sometimes, linguistic sample data. Learning Neural and fuzzy systems ultimatively learn
can be accomplished either by changing the sys- or estimate some unknown probability function
tem structure or by changing the system param- p(x). The probability density function p(x) de-
eter; both principles are described for both algo- scribes the distribution of vector patterns x, a few
rithms [336 – 339]. Thus, the principle modus of which the neural or fuzzy system samples.
operandi is comparable. After the system prob- When a neural or fuzzy system estimates a rela-
lem is defined, the procedure can be divided into tion r: X → Y, it in effect estimates the joint prob-
learning phase and prediction phase. The learn- ability densities p(x,y). Then the solution points
ing phase of ANN is quite obvious. Input data (x,r(y)) should reside in high-probability regions
and corresponding output data are provided to of the input–output product space X × Y. We do
the ANN; the system changes its structure or pa- not need to learn if we know p(x,y). We could
rameter, to minimize a predefined cost function. proceed directly to our computational task with
At first sight, the adaptive and learning character the techniques from numerical analysis, combi-
of fuzzy systems is not obvious. Indeed, there natorial optimization, calculus of variations, or
exist some techniques where the fuzzy system other mathematical discipline. The need and the
is built up autonomously based upon available extent to learn varies inversely with the quan-
data sets [339, 340]. On the other hand, in most tity of information available. Supervised learn-
heuristic applications a highly experienced tech- ing uses or creates class–membership functions.
nician does the “learning” step. He collects data, It compares every input with the corresponding
recognizes the intrinsic information, and formu- class D and proceeds learning based upon the
lates upon this information rules upon sets. Thus, difference. Unsupervised learning system pro-
the modeler performs the learning step and its cesses each sample x but does not know that
result is introduced into the fuzzy system. x belongs – or not – to class D. Neither super-
The most important features intelligent sys- vised nor unsupervised learning systems assume
tems may show are generalization and learn- knowledge of the underlying probability density
ing. All intelligent systems should generalize. function p(x).
Their behavioral repertoires exceed their learned
repertoire in that way, that intelligent systems
associate similar responses with similar stim- 8.3.1. Fuzzy Logic Systems
uli, small input changes produce small out-
put changes. Furthermore, intelligent systems Is uncertainty the same as randomness? Bay-
should learn or adapt. They learn new associ- esian statistician Dennis Lindley [342] stated
Biotechnology 83

that probability is the only sensible description Following the introduction of fuzzy sets,
of uncertainty and is adequate for all problems Zadeh began steadily to develop the necessary
involving uncertainty. Lindley directs his chal- inferencing mechanisms and modeling tech-
lenge in large part at fuzzy theory, the theory niques to bring this concept to fruition [345].
that all things admit degrees, but admit them de- In this period, it was recognized that fuzzy logic
terministically. Although, both expressions are could aid in the development of control systems
used simultaneously in the same context, proba- in which nonlinearities and time variance make
bility and fuzziness differ conceptually and the- the development of traditional control systems
oretically. In this contribution some important very difficult. In these cases, a human operator is
differences are illustrated, more theoretical as- often capable of controlling the plant, so a fuzzy
pects and proves can be found elsewhere [343]. controller can often be designed based on the op-
Fuzzy sets can be more compared with pos- erator’s expert knowledge. Since that time, con-
sibilities than probability. However, probabil- trol applications based upon fuzzy logic models
ity and fuzziness also share many similarities. have been extensively investigated [346]. Fuzzy
Both systems describe uncertainty with num- logic systems are similar to expert systems in
bers in the unit interval [0,1], consequently de- their use of linguistic relationship, but the sub-
scribe it numerically. Both systems combine sets stantial difference is that the outputs are in gen-
and propositions associatively, commutatively, eral continuous by variation of the inputs [347].
and distributively. The key distinction concerns It is exactly this property which causes the high
how the systems jointly treat a set A and its op- interest in mathematical modeling, and hence in
posite Ac . Classical set theory demands A∩Ac , engineering science.
and probability theory conforms: P(A∩A)=0. Mapping with fuzzy logic is analogous to
So A∩Ac represents a probabilistically impossi- classical modeling. The maps have variables
ble event. But fuzziness begins when A∩Ac =0, which influence system behavior and relation-
hence P(A∩A)=0. ships among the variables which describe the
Fuzziness measures the degree to which an system. In classical models, variables have real
event occurs not whether it occurs. Probability number values, the relationships are defined in
describes the uncertainty of event occurrence. terms of mathematical functions. In fuzzy logic
Whether an event occurs is “random”; to what however, the values of variables are expressed
degree it occurs is fuzzy. Consider an illustrating by linguistic terms such as “large, medium, and
example. The probability this book will be pub- small”, the relationships are defined in terms of
lished is one thing. The degree to which it will “if-then” rules. By using defuzzification tech-
be published is another. From this book, only niques, exact numerical outputs are calculated
some special chapters may be edited or the whole from fuzzy subsets.
entity gets public; this is typical for fuzziness. A fuzzy subset A is defined by a membership
Fuzziness is a type of deterministic uncertainty; function mA (x i ), where x i is the domain, of the
it handles with ambiguities. Unlike fuzziness, variable on which A is defined.
probability dissipates with increasing informa-
mA (xi ) ∈[0,1] ∀ xi ∈A (10)
tion. In fuzziness the uncertainty arises always
form the simultaneous occurrence of two prop- The value of mA (x i ) for each x determines
erties. More formally, does mA (x), the degree to the degree to which each element in the domain
which element x belongs to fuzzy set A, equal the belongs to A. Although both classical and fuzzy
probability that x belongs to A? This statement subsets are defined by membership functions,
can be true or not, it depends on the problem the degree to which an element belongs to a clas-
existing, but in fact the point of views are quite sical subset is limited to being either zero or one.
different and so far hardly to compare. The fol- This means that m(x) may only be a step func-
lowing explanation should not explain the fuzzy tion. In fuzzy logic, the degree to which an ele-
steps in great detail, The focus lies upon two as- ment belongs to a subset may be any value in the
pects. How are the sets defined and which rules interval [0, 1]. Since mA (x i ) may be defined by
can be determined. This is judged as the cru- any function, it is easy to see that a classical sub-
cial aspects for fuzzy application in bioprocess set is a special case of a fuzzy subset [348]. The
engineering [344]. fuzzification process transforms exact values x
84 Biotechnology

of fuzzy variable vj to mAi (x i ) for all subsets Ai , get a precise value, for further explanation see
which are defined for the variable vj . More rig- [327, 349].
orous definitions of membership functions may Expert knowledge is the most common tech-
be found elsewhere [327, 349]. nique for determining rules. The expert is asked
One technique for determining the shape of to summarize the knowledge about the system in
membership functions is seeking knowledge of the form of cause and effect relationships. From
the system from experts then constructing mem- these the rules are formulated. When no experts
bership functions that represent expert opinion. are available or a more analytical approach is
Observations of many experts should be pre- desired, other techniques of system identifica-
ferred. Based upon this observations, member- tion must be used. One approach is to apply a
ship functions can be constructed using statis- set of metarules to adaptively acquire informa-
tical methods accompanied by using the analy- tion about the system. Linguistic self-organizing
sis of preferences [350, 351]. Many researchers controllers issue control actions and observe the
have investigated more “rational” techniques environment [358]. The metarules evaluate the
for determining membership functions. One of performance of the control actions from the ef-
these approaches represents one variant of fuzzy fect of the control actions on the environment.
clustering [352, 353]. It requires a set of input The metarules then determine whether the con-
and/or output data from the system to be col- trol actions should be modified and perform the
lected. Patterns of data are found within the input modification, if necessary. This technique has
or output space such that the variance within the been applied especially to robotics applications
sets is minimized and the variance between sets [359]. Fuzzy classifier systems are another way
is maximized. The prototype, or center point, of to discover rules. Fuzzy classifier systems are
a cluster X is determined and a distance metric is generalizations of genetic classifier systems. A
used to determine the degree of membership that genetic classifier system uses some of the ideas
a value has to X. Another technique for determin- from genetic algorithms to develop expert sys-
ing membership functions involves neural net- tems [360, 361]. Standard genetic algorithms
works. Neural networks are networks of simple have also been used to discover rules [362].
processors connected by adjustable weighting Sugeno and Yasukawa use the second variant of
factors [328]. The weights are adjusted by using fuzzy clustering [fuzzy clustering methods can
a backpropagation or other known algorithm to also be used to determine the membership func-
minimize the difference between the predicted tions (see above). Both approaches are addressed
output and the measured output. The parameters by the term “fuzzy clustering”] on data in the
of membership functions can be “learned” by a output space to determine fuzzy rules [363]. The
neural net from a set of input–output data [354, clusters in the output space are used to induce
355]. Genetic algorithms have also been used clusters in the input space. Then, input space
to determine the optimal shape for membership clusters are projected onto the axes to find the
functions [356, 357]. fuzzy subsets for the input variables. Since the
For fuzzy subsets to be useful in “modeling”, input clusters are induced from the output clus-
there must be a way to define relationships bet- ters, rules can be constructed between the input
ween them. This is accomplished by means of subsets and the output subsets. Yoshinori et al.
their membership functions mAi (x) with logical (1996) discuss another method of fuzzy model
operators and inferences, to extract ambiguities. construction based upon clustering techniques
There are three basic operators in fuzzy logic, (1) [364]. This method produces a relational as op-
OR, (2) AND, and (3) NOT, which link two or posed to a functional model, and consists of a
more membership functions mAi (x) of different series of local, linear models to approximate a
variables. The activation level of each rule is nor- global nonlinear one. Recently; neural networks
mally equal to that of the premise (activation). have become an active field of interest and have
All rules are evaluated. If some sets of an output been used to learn rules as well as member-
variable are activated more than once, an aggre- ship functions [365, 366]. However, all meth-
gation method must be chosen to determine the ods, which automatically generate rules, have
final activity. After defining the interferencing deficiencies in their interpreting ability. They are
method, the output fuzzy sets are defuzzified to generated to fit available data best and it is not
Biotechnology 85

obvious that the rules express reasoning rela- a data driven approach can help to complete the
tions. This can be seen as a strong disadvantage fuzzy structure.
compared to the generation of rules by means of
expert knowledge.
The application area of fuzzy systems is 8.3.2. Artificial Neural Networks (ANN)
widespread and should only be presented here
for applications in bioprocess engineering. The Artificial neural networks are designed to mimic
fermentation industry was one of the first to the actions of neurons in the human brain. For
recognize the potential of fuzzy logic for bio- detailed information, see [328 – 330]. They re-
logical processes [367, 368]. Konstantinov and present massive collections of interconnected
Yoshida developed a fuzzy logic system for iden- neurons (nodes), which individually perform a
tifying physiological states in fermentation pro- relative simple signal processing; they are inter-
cesses and used it to control distributed an E. connected via synaptic links. Like brains, neu-
coli fermentation [369]. Meanwhile the range of ral networks recognize patterns or relations bet-
applications has enlarged tremendously. In prin- ween inputs and outputs we cannot even define.
ciple, they can be divided in two fields. The first They can be seen as pure function approxima-
one are those where the knowledge bases is con- tors whose abilities depend on the behavior of
structed such that it is based upon existing (quali- the individual nodes, the structure of the net-
tative) information, mainly from high-specialist work, the learning procedure used, and on a very
staff. This represents the area of fuzzy model- strong degree of the quantity of representative
ing [370, 371], fuzzy controller [372, 373], or data. Not the total amount of data is crucial but
estimator for biosystem states or process faults the information content with small degree of re-
[374, 375]. One of the intrinsic characteristics is dundancy. Almost the whole quantity space of
that significant knowledge engineering effort is the input and output values should be spanned
required for setting up a complete and consistent by the database. Recently, e.g., Cybenko [379]
rule base. The other domain of fuzzy logic is re- and Hornik et al. [380], have proved that any
lational model-based systems where the linguis- continuous function can be approximated to an
tic information is extracted from available data arbitrary degree of exactness on a compact set by
to build a system model. This second approach a feedforward neural network comprising two or
is also directed to the field of bioinformatics more hidden layers and a continuous nonlinear
or experimental design [376, 377]. This ap- activation function, providing appropriate data.
proach does not need any preinformation about Working with neural networks always con-
the intrinsic relation, however a knowledge base sists of two phases, a training and a prediction
can arise which is hardly to evaluate or inter- phase. In the first one, the variable parameters

pret. Additionally, no information can be given p (t) of the net are changed in that way, the
about the generality of the established rules. At ANN fits best to the trainings database. In most

all accounts, when a data driven construction cases, p (t) represents weight parameters of the
is used, a subsequent mathematical or manual intrinsic functions, e.g., the weight how the out-
evaluation, especially of the rule bases, is nec- put of a neuron contributes to the activation of
essary after the automatic construction of the a subsequent node. On the other hand, there ex-
fuzzy system. For the process industry, expert- ist also some nets where the structure of the net
knowledge-based approaches possess more in- (nodes and their synapses) can be seen as their
terests because they follow a concrete process parameters which are changed during learning.
target. The second one can be seen as a spe- 
cial form of knowledge discovery which enables The indicator for changing p (t) represents a
to accomplish some linguistic evaluation based formulated performance index J. Based upon the
upon data, if no expert knowledge exists. In some absolute value J, the parameters are changed in
exceptional cases, these clustering techniques order to minimize J.
  ∆J ∆J ∆J
are exploited for control purposes [378]. An ag- min J p = , ,···,  (11)
gregation of both principles is also fruitful in the ∆p1 ∆p2 ∆pn
case where the existing information is too little where pi is a parameter i with i = 1 . . . n param-
to build up a representative “model”. In this case, eters in the ANN.
86 Biotechnology

The definition of J depends on a couple of sic neural processes, can provide excellent so-
different aspects, but in most cases the Euclidian lutions to practical problems. The human brain
distance between the desired target and the cal- contains roughly 1011 neurons. As many as 104
culated output integrated over all training data synaptic junctions may abut onto single neuron.
sets is used. Performing learning should result That gives roughly 1015 synapses in the human
in the global optima concerning the specified brain [381]. Consequently, the brain represents
problem. However, most training procedures are an asynchronous, nonlinear, massively parallel,
local methods, requiring a gradient calculation. feedback dynamical system of nearly “cosmo-
And obviously, any algorithm that relentlessly logical” proportions.
crawls downward must have a very lucky start- The ability of ANN to emulate complex dy-
ing position if it is to settle into the lowest lo- namical systems stems from the interconnectiv-
cal minimum. Avoiding false minima requires ity of neurons. In general an ANN has n neurons
two separate procedures. First, we must elute in distributed in an input, an hidden, and an output
the initializing phase starting in their vicinity. If section. Connections between neurons can be or-
we settle into the neighborhood of a broad mini- ganized in layers such that information flows in
mum, it will be very hard to escape later. Second, one direction only, or it can circulate throughout
we require a procedure for determining whether the network in cyclic patterns. All neurons can
or not we are in a local minimum, and escaping be updated simultaneously or time delays can
if we are. This procedure will be constructive in be introduced. All responses can be strictly de-
that we were in a local minimum. Otherwise, we terministic, or random behavior can be allowed;
assume that we have found the global optimum. the variations are nearly endless and should not
In the second phase, the prediction phase, the net be described in detail. Mathematically they can
is conditioned to treat new input patterns, hope- be formulated in the following vector equation.
fully gaining the correct corresponding output   −   
y (t) = h →

values. o z (t) , x (t) , p )) (13)
The nodes are connected in that way that the where
output of one node represents one input of the
adjacent node. A node is stimulated by one or →

y (t) Vector of yi with i = 1 . . . ni outputs of the
more inputs − →
x (t) and it generates one output, ANN


x (t) Vector of xj with j = 0 . . . nj inputs of the
a scalar y(t) that is sent to other neurons. The
ANN
output y(t) is dependent on the weighted activ- 
p Vector of pm with m = 0 . . . nm variable
ities of each input, on the nature all inputs are trainable parameters of the ANN

transformed within the node and the parameters z (t) Vector of zj with n = 0 . . . nn for all outputs
constellation −→p (t) in each neuron. The actual 
of all individual nodes
relationship between the inputs and outputs can o (t) Vector function of function om with m = 0 . . .
nm for the calculation of the individual
be enormously complex, depending on the cho- contribution to the output yi

sen of entry E(·), aggregation S(·), activation A(·) h (t) Vector function of function hi with i = 0 . . .
and output function O(·): ni for the calculations of the real outputs of
the ANN
 
y (t) =O (A( S (E( x (t) , p )) )) (12)
There also exist some criteria to decide
Thus, the resulting behavior of individual
whether a structure is appropriate for a good
neurons can be modeled by a simple weighted
modeling approach. Always, a compromise
sum of inputs, a complex collection of interre-
must be made between the desire to have a sim-
lated or subsequent set of (differential) equa-
ple model with fewer parameters and more accu-
tions, or anything in between. There can be
rate predictions at the cost of a large number of
significant time delays in the steady-state out-
parameters. The Akaike’s information criterion
put value after stepwise input stimuli. Neurons
(AIC) uses the number of data points, the over-
do not always respond in the same way to the
all error, and the number of parameters to de-
same inputs. Even random events can be con-
termine an index, which can be analyzed to fix
sidered in the operation of neurons. Luckily,
the optimal quantity of free parameters [382].
a large body of research indicates that simple
Another concept that may be of interest is the
models, which account for only the most ba-
Biotechnology 87

use of a periodiograms, which gives an indica- preferred if high uncertainty within the data ex-
tion regarding the frequency content of the in- ists.
put signals, typical frequency can be character- The “explosion” of ANN applications in
ized, which fixes the optimum degree of free- nearly all areas of process engineering can be
dom [383]. Another way is the determination of attributed to the following reasons:
principal components before fixing the structure – The tremendous hardware advances in digi-
of the net, thus, incorrect or redundant informa- tal technology over the past decade have en-
tion can be rejected before entering in the learn- abled simulations of neural nets to be made
ing phase [384]. However, care must be taken both economically and with relative ease hand
in reducing system complexity or rejecting data speed.
sets, because they can be meaningful and repre- – Applications of neural networks for sensor
sent the behavior of the biological system even pattern classification have been found to be
though, this may not be obvious from the data superior to the traditional algorithmic tech-
set. niques or the expert system approaches.
When considering a particular application, – Neural networks offer the promise of being
it is to decide what type of network should be able to extract information from a plant in
used. Principally, ANN structures are classified an efficient manner with normal availability
as either global or local. Both possess specific of data, especially if severe or unknown non-
properties and hence applicational preferences. linearities and time invariances, typically for
Global networks are the most popular und im- biosystems, exists.
portant ones. Among them, different types exist: – Some practitioners claim that neural networks
feedforward backpropagation neural networks may be easier to use and apply in the real pro-
(FBNN); recurrent neural networks (RNNs), or cess plant, with difficult to handle nonlineari-
cascade correlation networks (CCNs). Unlike ties, as compared with the modeling approach,
the FBNN, RNNs are more general in the sense which can be subject to various modeling er-
that connections are allowed both ways between rors.
a pair of neurons and even from a neuron back to – Finally, the versatility in structure and appli-
itself on any way. The RNN allows the dynamics cation of neural networks enables them to be
of the network to be considered intrinsically. In utilized in the middle ground between con-
contrast, in FBNN for mapping a dynamic be- ventional model-based approaches and black-
havior the input variables must be supplemented box approaches for solving many classes of
by their corresponding deviations or time series. problems. These hybrid-type approaches have
However, the number of weights (for a fully re- been another factor, which has further at-
current network) to be determined may easily tracted their use in chemical process systems
become quite large. Therefore structural opti- recently.
mizers are recommended, e.g., the cascade cor-
relation, where connections and nodes are added The applications utilizing neural network
as required, [385]. Local networks, such as the based strategies in bioprocess engineering are
radial basis function network (RBFN) process wide ranging. A detailed description of all ap-
data in discrete areas not continuously over the plication is outside the scope of this contribu-
whole space of the data. They are particularly tion. Neural networks can be used for classi-
suitable for applications in online control and fication, to specify typical classes in the sets
optimization [386]. In the RBFN, it is necessary of the data base. Any task that can be done
to locate centers among the input/output pairs by traditional classification analysis can be ac-
such that the sum of the squares of the distance complished at least as well and almost always
from the center to the training data set is mini- much better by networks. Very important appli-
mized. These centers are equivalent in concept to cations in bioprocess engineering are the multi-
the weights in the FBNN. However, the RBFN variate exploitation of sensor arrays [387, 388]
may fail in predicting values if the prediction or the fault diagnosis [218, 389]. An ANN can be
space does not contain any center. This must be trained for pattern recognition. These patterns
seen as a crucial disadvantage, especially for on- can be a intervals of time, cultivation states, fer-
line applications. So, global networks should be mentation series, images et cetera. If a version
88 Biotechnology

of one of these patterns, corrupted by noise, is engaged in discovery and development of use-
presented to a properly trained network, the net- ful modeling technology, Casti asserts an inex-
work can provide the original pattern on which tricable coupling between a model and its in-
it was trained [390, 391]. A very common prob- tended application: “Basically, the point of mak-
lem and the most popular application field of ing models is to be able to bring a measure of
ANN is that of modeling, identification, or esti- order or probable system performance to our ex-
mating the value or the state of a variable, given perience and observations, as well as to make
historic values of itself and other variables; e.g., specific predictions about certain aspects of the
they were used to estimate the state of microbi- world by experience”. Therefore, mathematical
ological cultures [392], the concentration of un- modeling does not make sense without formu-
measurable mostly intracellular biological key lating before making the model what is its use
components [393 – 395], or for the identifica- and what problem it is intended to help to solve.
tion of bioprocesses [396, 397]. ANNs can also Thus, a definition of a model can be given as
replace or act as process controllers. The ap- follows: A model is an image of a real system
plications utilizing these neural-network-based that shows analogues behavior in the important
strategies are wide ranging and vary from lin- properties, and that allows within a limited re-
ear time invariant to highly nonlinear time vari- gion in respect to the intended purposes a de-
ant systems [398, 399]. Typical advanced re- scription of the behavior of the original systems.
presentatives are the model-predictive control, The emphasis represents the word important.
the inverse model-based and the adaptive con- A model should be always as simple as possi-
trol techniques. These methods have all in com- ble; the degree of simplicity depends only on
mon, that the ANN reacts as a process controller, the model purpose and on the modeler skills.
modified or expanded in some way by a refer- Modeling the same real system with a differ-
ence model or by an online training unit. ent focus, the significant properties are mostly
quite different. Single parameters can represent
in one case the most specifying quantities and
8.4. Modeling Aspects of Biological in the other case they are completely unimpor-
Systems tant. The focus and the intention of the model
determine not only the parameter, but also the
By definition, biochemical engineers are con- proceeding in the formulation of the relations.
cerned with biochemical systems, with reactions The modeler must identify the important vari-
and artifacts of biochemical substances like pro- ables (inputs, outputs, states) and their separate
teins or with systems employing growing cells. effects, which in practice may have a very highly
Even the simplest living cell is a system of such interactive combined effect on the overall pro-
a forbidding complexity that any forward math- cess. This is only possible if the modeler hat
ematical description of it is – in most cases – an a profound technological knowledge. Once the
extremely modest approximation. This situation model is established, it can be used, with rea-
prompts for bioprocess modeling the question sonable confidence, to predict performances un-
from a formal logical viewpoint: “What kind der differing process conditions. But this is only
of relation ship should exist between the un- allowed under specific boundary conditions, un-
derlying physical system and its mathematical der those which were used for the set-up. If you
description and which approaches should be ex- leave them, the describing ability gets lost. Any
ploited?”. extrapolation should be seen as critical.
Contemplation of this question leads, in one
direction, into the labyrinths of philosophy sci-
ence. Guidance into this territory from the learn- 8.4.1. Steps in Creating a Model
ing guide Rutherford Aris led to John Casti’s
two-volume treatise “Reality Rules”, which ex- Model building is always a combination of the-
plores the general definitions of a mathemati- oretical studies and practical experiments in
cal model as well as numerous specific exam- a very iterative sequence (Table 15). The de-
ples of models in different contexts [400, 401]. scribed sequence should be observed in order
Gratefully for engineers, who are (or should be)
Biotechnology 89

to get proper problem awareness and to mini- may be to derive the mechanistic principles un-
mize the efforts to reach the main targets. The derlying the system behavior. So, it is useful to
objective of a scientific investigation is to im- obtain as much data from the system as possi-
prove understanding of a system by testing a ble. It can be necessary to examine all different
hypothesis about that system. The formulation portions of the curves individually. Even, small
of the hypothesis is the most important action in deviations can hold large insight into the intrin-
the modeling process. In a scientific process, a sic physico-chemical effects of the biosystem.
problem or question is posed and stated as a hy- In all real cases of system modeling, also
pothesis or theoretical statement of the problem. boundary conditions or system constraints must
be fulfilled. This requirement addresses two dif-
Table 15. Step sequences for model building in bioreaction ferent aspects. The first one considers the fact
engineering. The steps must be seen as a loop whose termination is that the created model is only valid inside typi-
determined by the defined cost function
cal boundary limits of the system variables and
Step Action inside formulated constraints in respect to re-
1 Proper definition of the problem (hypothesis), the
goals, and the objectives of the study, fixing of
alized assumptions. The second one indicates
presuppositions, boundary conditions and constraints, that building up a model in bioprocess engineer-
defining the evaluation and error criteria ing is always accompanied with an identification
2 Analysis of the system, determination of the
structural elements, description of the key elements step for optimizing the parameters. But the pa-
(variables, processes) rameters are reasonable only within some lim-
3 Running typical representative experiments, its, though they fulfill the mathematical optimal
exploiting the parameter space of the control
variables (experimental design) criteria. Otherwise, they can loose any physi-
4 Establishing the type of model by use of balances, cochemical meaning or interpretational ability.
physical–chemical–biological laws, available data,
empirical equations, uncertainty treatment, problem
Having more than one parameter to determine
formulation in mathematical or linguistic terms can lead, depending on the complexity of the
5 Simplifying assumption (e.g. about mixing, process system under consideration, to ambiguities. In
structure and dynamics, metabolism, kinetics,
neglecting aspects) this case, although available knowledge, e.g., in
6 Choice and definition of the important process form of experimental data is represented well,
variables: input and output variables, states, and the meaning of the parameters is outside of any
parameters for the model
7 Simulation of the model, parameter identification, interpretation scope. The extrapolation ability
sensitivity analysis, determination of estimation gets lost.
properties To accomplish a mathematical treatment of
8 Evaluation of the model by using test data
considering the evaluation criteria from step 1, if the model in order to reach the predefined goals,
necessary starting again with step 1 an evaluation criteria must be formulated. This
can be seen as the mathematical description to
For complex biological systems, however, the formulation of the modeling purpose and
the hypothesis may need to be translated into is realized by formulating a scalar performance
a mathematical tractable building and the model index. Its quantity gives information about the
predictions are compared to the observed data model quality. In bioprocess engineering, it is
as a basis for rejecting or accepting a hypoth- normally calculated from the results obtained
esis. This step also includes the definitions of by the model compared with those of the cor-
the goals and the objectives. The purpose of the responding experimental data.
model usually dictates the form and the detail as There are two common approaches to fit mod-
well as the data that are required to develop and els to data, the least-square approach where
test it. Typical goals can be the identification of parameter are adjusted to minimize the sum
typical parameters or simulations, to predict re- (over all target values and existing data sets)
sponses to a perturbation. If the purpose is to get of squares of the residuals between the calcu-
a general idea on how the system would behave lated and experimental data and the maximum-
under a new condition, the accuracy may not be likelihood approach, which maximizes the prob-
important, however, if the purpose is to deter- ability of the assumed parameterized probability
mine an “optimal” process trajectory, the model density function [234]. Also, the above men-
accuracy may be critical. Finally, the purpose tioned boundary conditions and system con-
straints must be considered in the performance
90 Biotechnology

index. This aspect is from the logical point of for the modeler is to decide which one should be
view strictly necessary, but invokes in some accomplished from the viewpoint of deep under-
cases crucial mathematical problems [402]. standing of the biosystem. It is important and in
The system under study may consist of a sin- most cases very effective that new approaches
gle biomolecule, a cell, or a whole plant. Biolog- are built up upon information of earlier studies
ical systems are normally open systems. They gained from literature. Differences between the
have inputs and losses, although they may also models should be evaluated to choose the model
be reduced to closed systems as in vitro stud- most acceptable with current and new biologi-
ies. Once the system to be studied is defined, the cal knowledge. Since the problem of parameter
inputs and outputs are identified. Additionally, identification and model verification increases
its processes, subsystems, and key compounds rapidly with model complexity, one should begin
characterize the system. Processes are move- with as far as possible simplified assumptions
ments or changes in the system (e.g., absorp- and withdraw them step by step, if the model
tion, metabolism, transport, chemical reactions), quality is judged to be not sufficient. In this way,
subsystems are components of the whole system a most simple initial model grows step by step
(e.g., cells by considering the fermenter as the in complexity and accuracy, without becoming
system), and compounds/states are the quanti- too complicated. Modeling includes not only the
ties under consideration (e.g., metabolites). selection of correct model structure, but also the
Almost all modeling approaches in biopro- determination of the undefined system parame-
cess engineering involve an identification step ters.
of unknown parameters in the model by ex- To give a final statement about the quality of
perimental data. The aim of the experiment is model, its robustness has to be evaluated. This
to obtain data to confirm or reject a hypothe- concerns sensitivity, identifiability, and stability
sis. It is essential to decide from which envi- aspects. Sensitivity refers to the relative influ-
ronmental and operating conditions measurable ence of individual parameters on the solution.
quantities should be gained, and to which preci- Identifiability refers to the uniqueness of the
sion and in which measuring range. The phys- model and of its parameters. They should be de-
ical, chemical, and biological ones should be termined in that way that a data-fitting process
wide ranging, and should also include the qual- should return the same estimates of parameters
ity of the whole environment and pre-cultures or more or less independently of the starting points
pre-states. All measurable bioprocess data must for estimation and noise in the data. Stability is
be treated initially as variables. Afterwards, it the behavior of a system with respect to a pertur-
could be decided, whether they remain constant bation. Since nearly all real biological processes
or not. Automated measurement and control of are time-variant and highly nonlinear in nature,
bioprocesses, presently an art but – hopefully – the powerful collection of methods concerning
routine in the near future, generates a tremen- the aspects of controllability, stability, and oper-
dous amount of data. This requires judgment ability of linear systems cannot be applied [403].
of the importance of these data for documen- Sensitivity analysis is one of the most impor-
tation to reduce data effectively without loss of tant tools to optimized models. It refers to calcu-
valuable information. Experts – either human or lations and analyses performed to describe the
computer – have the data and the deterministic relative dependency of the model parameters. Its
knowledge to trace observed behavior back to analysis can be used for model validation as well
the physical, chemical, and physiological roots. as for fixing variables to constant values in order
Once the biosystem is structured and experi- to decrease the degree of freedom. The sensitiv-
ments are done, the model should be fixed. There ities are also very important for interpretation
exist different approaches to find the best ap- purposes, e.g., to detect more or less important
propriate model type for the underlying prob- partial processes or quantities of the biosystem.
lem (see below). In principle, the model should From a practical perspective, it represents calcu-
represent the theories or hypothesis about how lations which reveal the impact of assumptions
the system works. Normally, there exist different made in conjunction with model development.
structures and parameter constellations which A distinction can be made between point sensi-
will fit a particular set of data. The crucial task
Biotechnology 91

tivity, relative sensitivity, and overall sensitivity. noisy data and to reach the observability. Con-
Considering the model sider the following general representation of a
   time-variant nonlinear system:
y (t) = y (t, p )) (14)

  
 z (t) =T ∗ x t), y (t), p )
with t representing the process time and p the with
(18)

parameter to identify; y (t) are the measurable 0 
z = z (t=t0 )
output variables. The relationship between the
state variables and the point sensitivity can be A system is defined as observable at the time
show using the first two terms in the Taylor ex- 0
t 0 , if, admitting an arbitrary initial state z , a
pansion. finite point of time t 1 >t 0 exist, so that assuming
     
y (t) = y (t, p 0 )+GT ∂ p +K (15) known input x (t) and output y (t) trajectories

 the state z (t) can uniquely be determined in a
where G is the partial derivative matrix of y (t)
 time interval t 0 <t<t 1 . In contrast to linear sys-
with respect to p (t). An element of G is de- tems, this problem involving time-variant non-
fined as the point sensitivity of yi with respect linear equations as they arise in bioprocess engi-
to pj . An alternative scale-free sensitivity is the neering results in nonlinear algebraic equations
relative sensitivity S ij for the solution of which similar powerful meth-
pi ∂yi ods do not exist. Consequently, several assump-
Sij = (16)
yi ∂pi tions and individual specifications and transfor-
To represent the overall sensitivity of a state mations have to be made to make the problem
variable as opposed to point sensitivity to pa- mathematically tractable with reasonable effort
rameter pi over some time interval, S G [404].
ij can be
defined
T2

1 ∂yi 8.4.2. Reasons for Making a Model
G
Sij = dt (17)
T2 −T1 ∂pj
T1 There is a zoo of mathematical models in the
As applied to modeling, identifiability is a biochemical engineering and mathematical bi-
mathematical concept, which attempts to steer ology literature. Many of these appear to naive
model development largely on the basis of an as well as to sophisticated reader to have lit-
analysis reflecting whether features of a model tle more purpose than calculating numbers and
for a presumed system can be extracted from a writing scientific papers which conform reason-
proposed experiment. Selecting a standardized ably to experimental data. This is, in itself, not a
method for an experiment directed for elucidat- distinguished endeavor; it is not particularly dif-
ing a model and its features involves selecting ficult, and it teaches little. One reason that math-
time points for observations, observation sites, ematical biology receives so little respect from
sites for tracer input, as well as forms of tracer biological scientists – and is generally not recog-
application. In bioprocess engineering, it is sel- nized as a credible research tool in biological sci-
dom possible to sample all units, and the number ence and a biotechnological discovery – is that
of units to which tracer can be applied is limited. it represents the failure to communicate clearly
Each choice we make here stands for a chance and persuasively the reasons and the motivation
of mitigating against identifying aspects of the for constructing a model. Sometimes the mod-
model, even in an error-free situation. If the lack eler himself has not clearly asked this question
of identifiability makes it impossible to estimate while doing the work, sometimes biochemical
unique parameters or test relevant hypothesis, scientists make one experiment after the other,
we should like to know this before conducting not reflecting which mathematical approach ex-
the experiment or interpreting the modeling re- ists to enhance the evaluation or interpretation of
sults. If the experiment is adequate to identify the experiments. However, modeling as a typical
the model, we are still left with the estimation domain of engineering gets increasing interest in
problem, that of resolving the parameters amidst the study of biological systems. The challenge of
92 Biotechnology

modeling biological systems is not to determine predicted based solely on intuition and experi-
an arbitrary function to fit the data but to use the ence without a model.
modeling process to understand the system. Models can be used to simulate inputs into a
Modeling can be used to determine the struc- system at various sites and to simulate short-term
ture of a system where structure refers to the as well as long-term responses. Experiments can
relationships between various parts or processes be simulated rapidly using a model to predict
of a system. A model can be used to determine likely scenarios before undertaking expensive
the type of relationship as well as the sequence of experimental data collection. While models will
events that occur between the various substances never replace experiments, they can be used to
of interest. In some systems, this information avoid experiments where insufficient or inappro-
may be known, whereas in others the structure priate data will be collected for testing a certain
can only be inferred from the data. Modeling hypothesis. Models may be used predictive for
improves understanding, and it is through un- design and control. Once the model has been
derstanding that progress is made. In formulat- established, it should be capable of predicting
ing a mathematical model, the modeler is forced performance under differing sets of process con-
to consider the complex cause-and-effect se- ditions. Thus, mathematical models can be used
quences of the process in detail, together with for the design of relatively sophisticated control
all the complex inter-relationships that may be and optimization algorithms, and the model, it-
involved in the process. The comparison of a self, can often form an integral part of the control
model prediction with actual behavior usually algorithm. Optimization usually involves con-
leads also to an increased understanding of the sidering the influence of two or more variables,
process, simply by having to consider the ways often one directly related to profits and one re-
in which the model might be in error. The results lated to costs. For example, the objective might
of a simulation can also often suggest reasons as be to run a reactor to produce a product at a max-
to why certain observed and apparently inexpli- imum rate, while leaving a minimum amount of
cable phenomena occur in practice. unreacted substrate.
Models can be used to determine parameters An important use for models is to iden-
of interest, such as pool sizes clearance rates or tify sites of change in a system when studied
transport rates. If the purpose is to accurately under different conditions. In some cases, the
determine a particular parameter, it is impor- altered condition may result in large changes
tant that the model can be uniquely determined. in the kinetic curves and several pathways or
However, the model must also be consistent with it may result in a subtle change in the data
known biological information. A model may be caused by a large change in only one parame-
well-determined but incorrect. ter. A model helps to identify which parameters
The purpose of the model may be to deter- change between the conditions and the degree
mine the interaction of parts of a system. Models of change. The conditions may be an untreated
can integrate information on a number of sub- versus treated state, a healthy subject versus a
systems into an integrated form to represent the diseased subject, or a normal versus high intake
process under consideration in the whole sys- of a nutrient.
tem. Because of the interactions, dependencies, Models form valuable tools for teaching the
and feedback of the processes, large complex process of scientific inquiry and for challeng-
systems can only be understood by using mod- ing students to think creatively and quantita-
eling. An example could be to link metabolism tively about a system. Models can be used to
in one tissue with metabolism in another, or to demonstrate properties of a system, teach princi-
link metabolism of a nutrient in one form with ples (such as feed-back loops, saturation kinetics
the metabolite in a second form. Large systems etc.), test theories, and design studies. With the
and systems with dynamic properties can only increased speed and convenience of computers,
be understood with the use of models. Because the availability of modeling software and the ac-
many processes in large systems occur simulta- cess to it, the researchers who are developing and
neously and dynamic systems are accompanied publishing models, need to be cognizant of this.
by complex interactions between processes, it is They need to make their models understandable
unlikely that responses to perturbations could be
Biotechnology 93

and accessible to the biological community at is due to simplifying assumptions in analytical


large as well as to students. and prediction models, simplified methods, and
Models help in experimental design. It is im- idealized representations of real performances.
portant that experiments be designed in such a The vagueness-related uncertainty is due to
way that the model can be properly tested. Of- cognitive sources that include 1) the definition of
ten, the model itself will suggest the need for certain parameters, e.g., structural performance
data for certain parameters, which might oth- (failure or survival), quality, deterioration, skill,
erwise be neglected, and hence the need for a and experience of construction workers and en-
particular type of experiment to provide the re- gineers, environmental impact of projects, con-
quired data. Conversely, sensitivity tests on the ditions of existing structures; 2) other human
model may indicate that certain parameters may factors; and 3) defining the interrelationships
have a negligible effect, and hence these effects among the parameters of the problems, espe-
can be neglected both from the model and from cially for complex systems. Other sources of un-
the experimental program. certainty can include conflict in information and
human and organizational errors [406].
Once the systems are defined, the relations
8.4.3. Different Types and Basic Approaches between the outputs and states based upon the
for Building a Model inputs and states have to be formulated to per-
form simulations. Today we have many types
Incomplete knowledge of the dominant biolog- of models, which can be categorized in various
ical pathways as well as low availability of sen- ways such as deterministic versus nondetermin-
sor information about the current physiological istic, logical (linguistic) versus mathematical-
state is the characteristics of biochemical pro- equations-based or data- versus knowledge-
cess modeling. From system analysis, as already driven. Physical models can be the realization
described above, the definitions of the describ- of the original system in a different (usually
ing variables (e.g., inputs, outputs, or states) can smaller) scale or with structural modifications.
be given. A second type of physical models is obtained
In principle, there can be postulated that by turning to a different physical system, e.g.,
these variables are intrinsically interconnected from the original biosystem to a correspond-
by means of a set of – indeed real existing – dif- ing electrical circuit [407]. Another group re-
ferential equations, as it is already described in presents verbal models. They give a linguis-
previous chapters. But even being aware that tic representation of our knowledge about the
these relations may exist in principal, for math- system, usually formulated as rules, e.g., “IF
ematical treatment they can only be specified this or that happens THEN the system reacts
in their structure and intercorrelation as well by. . .”. They are widely applied in the area
as in their parameter constellation in some ex- of artificial intelligence, namely experts sys-
ceptional cases with a high degree of accuracy. tem [408, 409]. In the form of fuzzy-based
Thus, when dealing with modeling of real bio- systems, they gain increasing industrial inter-
logical systems, uncertainty in various aspects est in biotechnological control theory, which is
can never be avoided [405]. In bioprocess engi- due to their effective treatment of qualitative
neering, uncertainty is an inseparable compan- knowledge of experienced technologists in pro-
ion of any modeling approach. cess control strategies (see Section 8.3.1), which
Uncertainties in engineering systems can be could only hardly realized in terms of mathemat-
mainly attributed to ambiguity and vagueness ical equations [410 – 412]. Mathematical mod-
in defining the architecture, parameters, and els form the third class. They can be classified
governing prediction models for the systems. further depending on the mathematical formal-
The ambiguity component is generally due to ism or the methods for model building. Theo-
noncognitive sources. These sources include 1) retical models as mechanistic models are based
physical randomness; 2) statistical uncertainty on physical and chemical laws and our knowl-
due to the use of sampled information to estimate edge about the inner structure and function of the
the characteristics of these parameters; 3) lack of system. In contrast, experimental or nonmecha-
knowledge; and 4) modeling uncertainty which nistic mathematical models try to give – without
94 Biotechnology

looking into the interior of the system – only a for systems without the existence of any driving-
description of the observed reaction of the sys- force function the expense for their determina-
tem in response to a certain forcing signal. Many tion is disproportional to the possible result. If
“mechanistic” models in biotechnology are ac- inputs exist, which direct the system in a defined
tually due to their oversimplification quite closer direction, reaction patterns must be formulated
to black-box models than to real mechanistic additionally in terms of probabilistic equations.
models, although mostly mechanistic interpre- Another type of black-box models represents
tations are given [413 – 415]. those where a specific functional structure is as-
sumed, which is combined with a subsequent
White-box model. As described above (Sec- parameter estimation to adapt to the specific un-
tion 8.3.1), the relation can strictly be mech- derlying problem (e.g., polynomial regression,
anistic, considering the four differential equa- ANN, chemometric models). However, on ap-
tions for mass and heat balance and correspond- plying this, it has to be considered that the sim-
ing transformations. In this so-called white-box plest and usually fairly accurate method for pre-
modeling strategy, the model development is dicting values of the next data point in a time
mainly driven by the knowledge of the rel- series from a “well-behaved” biosystem is to
evant mechanisms, macroscopic balances and assume that it is the same as the present data
predefined parameters. Indeed, if all the biosys- point, a principle known as the first-order trivial
tem underlying mechanism are known and can predictor. Similarly, in the second-order trivial
be described by means of the above-specified predictor the value of data point n+2 is equal
equations and the accompanied parameters are to that for point n plus twice the signed dif-
known, the resultant white-box model is gen- ference between the values of point n+1 and
erally applicable and show good extrapolation n. If any complex nonlinear predictive model
properties. However, it is not an easy task to re- like ANN can do no better than the first- and
veal all the relevant mechanisms and quantify second-order trivial predictors – which are in-
these mechanisms correctly when dealing with deed very often concurrently – then their devel-
biological processes. The metabolic modeling opment is a waste of time. Nevertheless, espe-
approach is one such approach and has been the cially ANNs have been the focus of much atten-
focus of recent attention [416, 417]. Addition- tion for model development and have already
ally, several attempts have recently been made in been applied to various biochemical processes
utilizing metabolic information for online iden- (Section 8.3.2). Although the main advantage of
tification and control as a consequence of the the development of black-box models is that a
white-box strategy [418, 419]. reasonably accurate model can be obtained with-
out detailed mechanistic system knowledge, it
Black-box model. The black-box model is, should be noted that its accuracy depends only
on the other hand, mainly driven by measured on the quality of the available input and out-
data obtained from the process, either online or put data. As black-box models are not believed
offline. In terms of mathematical expressions, to have any extrapolation properties, one has to
probability density functions for the important obtain a large body of data for process identifi-
parameters are specified by means of available cation by employing the relevant input variables
data. Based upon these distributions, the time- in a wide range of fluctuations [218].
dependent system behavior can be forecasted
[420 – 422]. The earliest stochastic biosystem Markov chains. Another very attractive way
model is perhaps that proposed by Yule, to to use probabilistic approaches are the formu-
mimic the evolution of a new species within lation of the problem by means of so-called
a genus [423]. However, they are mainly used Markov chains [424, 425]. A Markov process
for systems only, where isolated parameters are allows the modeling of uncertainties in real-
treated. It is unrealistic that for a real biological world systems that evolve dynamically with time
MIMO system (multiple input multiple output), without using mechanistic approaches. The ba-
all joint probability densities, which are neces- sic concepts of a Markov process are those of
sary to describe the key behavior, can be approxi- states of a system and of state transitions. In spe-
mated by performed series of experiments. Even cific applications, the modeling “art” is to find
Biotechnology 95

an adequate state description such that the as- and a measurement values, and their propaga-
sociated stochastic process has the Markovian tion with time to predict online a future state
property that the knowledge of the present state based upon new measurements and already ex-
X(t) is sufficient to predict the future stochas- isting quantities. Their potential in bioprocess
tic behavior of the system X(t+1). The moving modeling and control is obvious [432 – 434].
of i=t to j=t+1 is defined by means of the tran- From the structural point of view, hybrids are
sition probability Pij [426]. For bioprocess en- described in parallel and serial configurations.
gineering, controlled Markov chains or Markov In the former case, the black box is placed in
decision processes (MDP) possess a high poten- parallel with a white-box model and its role is
tial, for simple modeling as well as for process to describe and to weight the difference bet-
optimization [427, 428]. Applied in bioprocess ween a white-box model and the real process.
engineering, they involve the selection of an ac- This model has been applied to several pro-
tion U t from a set of containing a finite number cesses, but its performance might be limited
of A actions when observing the current state of to some extent to the viewpoint of extrapo-
the system. The problem is to define the prob- lation properties [435]. In the serial structure,
ability that the system goes to a state j from a the black box is placed in series with a white-
state i under the action of a: box model. Different applications are described.
Combined with a fuzzy expert system, the hy-
P {Xn+1 =j|Xn =i,Ut =a}=Pij (a) (19) brid was applied to model a real-time fed-batch
The controlled Markov chain is completely culture for baker’s yeast production, a produc-
defined by the optimality criterion for calcula- tion scale beer-brewery fermentation, and mam-
tion of the transition probability matrix, the state malian cell cultures [431]. To show the better
vector X, the action set A, and the decision epoch. extrapolation properties of grey-box-model ex-
For bioprocess modeling, concentration values emplarily, the performance of a serial-type grey-
grouped in clusters are often used as state vari- box model containing a neural network and a
ables; considering more than one state variable more knowledge-driven white-box model were
for each one a separate model is constructed. compared with that of a more data-driven black-
One great disadvantage of this approach repre- box model in the enzymatic hydrolysis of peni-
sents the huge effort to determine the transition cillin G to 6-amino-penicillanic acid and phenyl
probability matrix Pij (a) for each control action, acetic acid by using the enzyme penicillin acy-
especially if more than one manipulated variable lase. The grey-box model was shown to have
should be considered [428]. significantly more reliable extrapolation prop-
erties than the black-box model [436].
Grey-box model. A grey-box model may be Also linguistic models such as fuzzy models
defined as a suitable combination of a black- can be seen as a special type of grey-box models.
box principle and a white-box model. The ex- Although the intrinsic behavior cannot be for-
pectation is to obtain good interpolation and mulated by means of mathematical equations, a
extrapolation properties. Especially, hybrid ap- priori knowledge is included into the model by
proaches, where portions from each principle are the linkage of various “IF. . ..THEN . . . “ con-
pieced together based upon the amount and the clusions, which is built up by the knowledge
structure of available knowledge, gain increas- of highly specialized staff. Their fine-tuning is
ing importance. From different points of view, often accomplished by using data from experi-
such hybrid systems can be build up, i.e., ana- ments [437].
log or discrete consideration, stochastic or deter- Being aware that different approaches ex-
ministic combinations [429 – 431]. Only some ist how biological systems can be modeled and
important concepts for bioprocess engineering treated computationally, an important aspect for
should be presented here. In some cases, sta- a successful application is the proper choice
tistical methods are included into a mathemat- which principle fits best to the specific problem
ical discrete formulation, this concerns mainly and its boundary conditions. In most cases, more
Kalman filtering. They exploit statistical infor- than one specific approach can be applied in
mation about the error distribution of the state principal. The most important aspect for choos-
96 Biotechnology
Table 16. Different types of models based upon the system knowledge

High System knowledge Low


Model deterministic stochastic, statistical expert systems approximation classification
Examples unstructured, Markov chains, fuzzy models, set neural networks, PLSb , pattern recognition
structured, CFDa maximum likelihood, theory PCRc
Kalman,
a
CFD, computional fluid dynamics.
b
PLS, partial least
square.
c
PCR, principal com-
ponent regression.

ing the right model should be the system knowl- ANN or chemometric models should be used. In
edge about the underlying problem (Table 16). those cases, they are the only alternative to real-
Not only the amount of system knowledge is ize appropriate prediction ability. They assume a
decisive, but also its structure, the quality and system structure, which possesses no causal re-
quantity of accompanied data, and the purpose lation with the underlying real mechanism, but
for which the model should be used is of special is able to incorporate and describe the system
importance. As a basic principle for building up a dynamics. They possess normally good approx-
model, it could be postulated “use as much a pri- imation abilities, but their extrapolation prop-
ori information as possible”. In mechanical en- erty reveals a low reliability especially if outputs
gineering and fluid dynamics, strictly determin- should be forecasted where the corresponding
istic approaches are typical, where the underly- inputs are outside of the trained data space.
ing differential equations are “exactly” known.
Thus, there exists no reason to deal with uncer-
tainties and the problem is forwarded. However, 9. Special Applications in
this situation generally does not exist in bio- Biotechnology
process engineering. A biological system con-
sists of a very complex, strongly regulated re- The following chapters highlight some special
action network, where only some simple mech- aspects in biotechnology. However, there exist
anisms can be formulated in terms of mathe- even more interesting applications in biotech-
matical discrete forwarded formulations. Thus, nology. The reader should regard the areas cho-
one has to deal in a very strong degree with un- sen by the authors as examples for the wide dis-
certainties. If one is able to formulate a set of tribution of biotechnology in the daily living.
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
9.1. Mammalian Cell Culture
such a model and append a subsequent parame- Technology
ter estimation step where statistical properties or
available data are exploited to consider unknown 9.1.1. Introduction
or not structural included information. In the
case where a formal-kinetic approach cannot be Mammalian cell culture technology has become
formed but qualitative knowledge is available or a major field in modern biotechnology, espe-
can be extracted from available data, expert sys- cially for the area of human health, and fascinat-
tems should be preferred. Especially fuzzy sys- ing developments achieved in the last decades
tems as a special representation of an expert sys- are an impressive example for an interdisci-
tem show advantages in modeling approaches plinary interplay between medicine, biology,
because of their smoothness concerning the sys- and engineering [438, 439]. Among the classi-
tem output. In the case where a priori knowl- cal products from cells, we find viral vaccines,
edge of the system is not available but pure data monoclonal antibodies, interferon, as well as
from a well-defined experimental design do ex- recombinant therapeutic proteins. Tissue engi-
ist, approximation or classification approach like neering or gene therapy open new, challenging
areas [440].
Biotechnology 97

Under “mammalian cell culture” or “ani- were developed that provide the required low-
mal cell culture” is to be understood the cells shear-stress environment by, e.g., introducing
of a mammalian, isolated from specific tissues gentle agitation and aeration with slow moving
(i.e., skin, liver, glands, etc.) and further culti- stirrers in stirred tanks, designing special aera-
vated and reproduced in an artificial medium tors for air-lift reactors, or by separating the cells
(Fig. 31) [441, 442]. During cultivation of mam- from the stressful conditions such as hollow-
malian cells in vitro, outside of a living organ- fiber, fluidized-bed, and fixed-bed reactors. In
ism, some distinct difficulties arise from the ex- industrial scale, adherent cell lines can be cul-
traction of the cells from a “safe” tissue. Slow tivated on microcarriers (e.g., for vaccine pro-
growth rates with doubling times between 18 duction) or are adapted to grow in suspension,
and 28 hours, low productivity, a high sensi- e.g., cell lines derived from baby hamster kid-
tivity against shear stress due to the lack of a ney cells (BHK) or chinese hamster ovary cells
cell wall [443] and high demands in respect to (CHO).
the growth medium challenge the techniques re-
quired 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 multicellu-
lar organisms, mammalian cells still hold the ge-
netic program of inducing their own cell death, a
process called “apoptosis” or “programmed cell
death” [444 – 446]. This can limit culture pro-
ductivity in biotechnological processes. Another
major problem is the finite lifespan of primary
cells, which die after several doublings in vitro.
This problem was solved by transforming pri-
mary cells into immortal “established” or “con-
tinuous” cell lines. The engineering targets re-
lated to mammalian cell culture technology can
be identified as:
– Production of a valuable product (viral vac-
cine, protein) from mammalian cells, devel-
opment of processes for the efficient produc-
tion 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 Figure 31. Morphology of (a) suspendable and (b) adherent
of organic tissues, which can be used as sub- mammalian cells (bar approx. 30 µm)
stitute to original tissues (tissue engineering,
gene therapy, cells as products). Genetic engineering contributes a great deal
Much effort has been put into the develop- to recent progress [449 – 451]. With this tech-
ment of mammalian cell culture technology, and nology, functional proteins can be produced by
great progress was achieved in the last decades introducing recombinant DNA into cell lines,
[447, 448]. Mammalian cells may now be cul- e.g., chimeric (humanized) antibodies are pro-
tured to very large volumes (up to 20 000 L) to duced for in vivo applications in transfectoma
provide the necessary quantities of a desired pro- or recombinant CHO cells. New promoters have
tein. Media that used to contain up to 10 % serum been developed to enhance productivity, and
were continuously improved, and the cultivation product titers over one gram per liter have been
in defined serum-free and even chemically de- reported for some industrial cell lines. Further,
fined, protein-free media is now common for novel cell lines can now be constructed from
most relevant industrial cell lines. Bioreactors
98 Biotechnology

primary cells without loosing functionality by ment, rheumatoid arthritis, leukaemia, asthma,
genetically induced proliferation [452, 453]. or multiple sclerosis. Nowadays several antibod-
The following will provide a basic under- ies are produced in kilogram quantities [440].
standing of the specific requirements of mam- Modern recombinant techniques focus on new
malian cells, will describe state of the art process antibody formats such as fragmented antibod-
technology for cultivation of these cells and will ies (FAbs) or bivalent antibodies with a broad
give an outlook to future prospective. A com- spectrum of applications.
prehensive overview on cell culture technology Glycoproteins are a further important group
is given by Ozturk and Hu [454]. of products produced by mammalian cells. Start-
ing with the production of α-interferon as an
anti-infectious drug by (nonrecombinant) Na-
9.1.2. Products from Mammalian Cells malwa cells in the late 1970s, nowadays a grow-
ing number of glycoproteins for treatment of a
A detailed overview on products from mam- wide variety of diseases are produced by means
malian cells is given in [454, 455]. Within “prod- of mostly recombinant mammalian cells. Promi-
ucts from cells”, viral vaccines [456] against nent examples are cytokines (e.g., interferons
polio, hepatitis B, measles, or mumps for hu- and interleukins), hematopoetic growth factors
man use, rubella, rabies, or food-and-mouth dis- (e.g., erythropoeitin for treatment of anemia),
ease (FMD) for veterinary use play an impor- growth hormones, thrombolytic agents (e.g., tis-
tant role. Viral vaccines are produced very effi- sue plasminogen activator [tPA]), coagulation
ciently by means of a cell-based vaccine tech- factors (factor VII, factor VIII, factor IX etc.),
nology. For this, mainly primary cells, diploid and recombinant enzymes (DNAse) [440].
cells, or permanent cell lines (e.g., VERO) are Recombinant proteins may be produced by
applied, recently even recombinant cell lines. A either bacterial, yeast, or mammalian cells. From
breakthrough for the large-scale production of a technological point of view, hosts such as bac-
viral vaccines with anchorage dependent cells teria or yeast have an advantage with respect
was the development of microcarriers in the late to growth rate, final cell density, and product
1960s, allowing cultivation in stirred tanks in concentration. Nevertheless, mammalian cells
thousand-liter scale. New targets for cell-based are preferred for those proteins requiring a
vaccines are human immunodeficiency virus specific, humanlike glycosylation pattern [459,
(HIV), herpes simplex virus, or influenza. Fur- 460], which is difficult to obtain in other host
thermore, new developments include genetically systems. Another problem for microorganisms
engineered or DNA vaccines (→ Immunother- is the maximal size of the produced protein
apy and Vaccines). which must be below a molecular mass of ap-
Monoclonal antibodies [457] have become a prox. 30.0 kDa. Further, in contrast to extra-
valuable tool for diagnostic purposes as well cellular release of most proteins produced in
as in therapy. Antibodies synthesized by B- mammalian cells, products from microorgan-
lymphocytes play an important role in the isms are often accumulated intracellularly in “in-
immunosystem of mammalians. Traditionally, clusion bodies”. This demands a more complex
polyclonal antibodies were isolated from blood downstreaming. Besides this, for mammalian
samples. In the 1970s Milstein and Köhler de- cells important parameters such as product yield,
veloped a technology to generate hybridoma medium requirements, and growth characteris-
cells producing monoclonal antibodies [458]. As tics (suspendable, shear resistant) have been sig-
of the specific binding, monoclonal antibodies nificantly improved.
are widely used for diagnostics, where tens of Proteins produced in the milk of transgenic
thousands different monoclonal antibodies are animals have started to compete against “classi-
available. The importance of monoclonal anti- cal” mammalian cell culture [461 – 464]. The
bodies as therapeutic agents has evolved only re- proteins are usually expressed in enormous
cently, as immunogenic mouse antibodies were titers, over one order of magnitude higher than
replaced by chimeric, humanized, or human an- that obtained from cell cultures. The price for
tibodies. Fields of application are organ trans- the production in transgenic animals will prob-
plantation (OKT3), cancer diagnostic and treat- ably continue to decrease significantly due to
Biotechnology 99

the development of more efficient reproduction 425 for U.S.A in 2002 [471]), most of them for
technologies. These new approaches will signifi- cancer therapy, infectious diseases, autoimmune
cantly decrease the time for the development of a diseases, or AIDS/HIV. It can be expected that
product. Among the disadvantages of transgenic at least some of these will find their way to the
animals, we find a more sophisticated down- market. Furthermore, new products or medical
streaming (high protein loads), the long devel- protocols can be expected for tissue engineering
opment time, the inability to produce proteins and cell therapy. As for some compounds patents
that might impair the health of the animal (e.g., are already expired or will expire in the near fu-
insulin), higher risk of viral contamination and ture, there will be a market chance for biosimilar
the possibility of prion contamination (scrapie, or generic products. On the other hand, the costs
BSE). for target identification, clinical trials, and pro-
The novel field cells as products includes cess development will increase (to date approx.
the development of artificial organs (tissue en- $ 0.5–1 billion) and only block busters will make
gineering of liver, kidney) and tissues (skin, car- it finally to the market.
tilage, bone), or the expansion of hematopoi-
etic cells for bone marrow transplantation or
gene therapy. The exciting prospects of tissue 9.1.3. Cell Types
engineering [465 – 467] are outlined in Section
9.2. Somatic gene therapy implies transfection In general, mammalian cells relevant for indus-
of a specific gene to cells isolated previously trial processes can be divided into the following
from a patient suffering from a genetic disease groups [441, 472]:
[468 – 470]. The transfected cells are then rein- – Primary cells have been isolated from a tissue
troduced into the patient. Gene therapy involves and than taken into culture (primary culture).
transfer of genetic material, encoding therapeu- – Permanent or established cell lines originate
tic genes and the sequence necessary for expres- from a primary culture, but because of some
sion to target cells to alter their genetic code transformation became able (at least theoret-
for a desired therapeutic effect. Many diseases ically speaking) to divide and proliferate in-
are originated by gene defects. Expression of definitely. Those cell lines are kept in a cell
the transferred genes can result in the synthesis bank and are very often used as host cells for
of therapeutic proteins or correction of a gene the expression of recombinant proteins.
defect. Gene transfer could also lead to desired – Hybridoma cells are cells which are obtained
apoptosis or inhibition of cell proliferation. The- through the fusion of lymphocytes and tumor
oretically, gene therapy can be applied for repair- cells and which are able to express mono-
ing single entailed gene defects, acquired gene clonal antibodies.
defects such as chronic infectious diseases, mul-
tifactor genetic diseases such as cardiovascular The common procedure for the isolation of a
disease, and finally cancer. Since gene therapy mammalian cell from a tissue involves the fol-
was applied the first time in 1990, the number lowing steps: first, a small piece of tissue is de-
of clinical trials increased. Today gene therapy composed into single cells or cell clots mechan-
is widely used in ongoing clinical trials for the ically, enzymatically (e.g., typsin [473], colla-
treatment of cancer and infectious diseases such genase), or through a combined procedure. The
as human immunodeficiency virus (HIV) infec- cells are separated from the enzymes by cen-
tion. trifugation and resuspended in culture medium.
Cell culture products are currently used After that, the cells are inoculated into a spe-
mainly as medicines or in diagnostics. For cial glass or plastic vessel with flat bottom. The
medicines, some products have a demand of anchorage-dependent cells start to adhere to the
500 kg/a and generate $ 1–2 billion dollars in bottom surface and, after a lag phase, start to di-
revenue. Among these are tissue plasminogen vide by mitosis. Such a cell culture, in which the
activator (tPA), erythropeithin (EPO) products, cells come from a differentiated tissue, is called
Remicade r
(Centocor) or MAbThera r
[440]. a primary cell culture. The most critical point in
The pipeline for new biopharmaceutical thera- the isolation of primary cells is to avoid external
peutics targets a huge number of diseases (e.g., contamination by practicing aseptic techniques.
100 Biotechnology

When the primary cells have covered the bot- transformation and “immortalisation”, as for ex-
tom of the culture vessel almost completely, they ample the treatment with mutagenic substances,
are enzymatically cleaved (i.e., trypsin) from with virus or with oncogenic substances [441].
their support and used to inoculate new cultures. Transformation of cells in vitro shows some sim-
This subculture gives origin to the so-called sec- ilarities with carcinogenese in vivo, but is not
ondary cultures. Part of the cells is stored deeply identical to it; for instance, not all the trans-
frozen in liquid nitrogen, to remain as a safety formed cells are malignant. On the other hand,
stock, from which it is possible, at any time it all cells that are isolated from tumors (i.e, HeLa
might be necessary, to get enough “fresh” cells or Namalwa Cells) can be kept in a permanent
to start a new series of subcultures for mass pro- culture.
duction. With the primary cells, it is possible to
repeat subcultures several times. However, the Table 17. Examples of permanent cell lines important for research
primary cultures have a finite lifespan and there- and production (data from [441], modified)

fore, after a certain number of doublings (from Cell Line Origin Application
50 to 100 times), the cells cease to grow and die Baby hamster kidney syrian hamster, 1963 adherent cells, but can
BKH-21 be adapted to
[441]. suspension, FMD
A finite growth capacity is a characteristic of vaccine, rabies
all cells derived from normal mammalian tis- vaccine, recombinant
proteins (Factor VIII)
sue. A cell can be considered as normal when it Chinese hamster ovary of chinese adherent cells, but can
shows a certain set of characteristics [474]: ovary, CHO-K1 hamster, 1957 be adapted to
suspension,
– A diploid number of chromosomes (i.e., 46 recombinant proteins
(HBstg, tPA, Factor
chromosomes for human cells) with which it VIII)
is shown that no gross chromosome damage COS monkey kidney transient protein
has occurred. expression [475]
NAMALWA human lymphatic alpha-interferon
– Adherence: the cells require a surface to tissue
grow attached to (anchorage dependent). The HeLa tumor in a cervical Fast-growing tumor
growth phase extends until the cells reach a vertebra cell line that was
isolated in the
stage of confluence (contact inhibition). beginning of the
– A finite life span in culture. 1950s.
– Non malignant: the cells are not cancerous, HEK-293 human embryonic transient protein
kidney, 1977 expression
i.e., they do not cause tumor in mice. MDCK rabbit kidney adherent cell line with
good growth
In the 1960s, normal cells were required for characteristics, animal
the production of human vaccines in order to vaccines
ensure the safety of these products [441]. MRC-5 human embryonic “normal” cells with a
lung cells finite life span,
Not all cell types produce exclusively pri- vaccine production
mary cell cultures, which after a limited number NS0 and SP2/0 mouse myeloma antibody production
of “passages” die. Some cells acquire an infinite from B-lymphocytes
3T3 mouse connective suspensible, used in
lifespan and such a population is usually called tissue the development of
a “permanent” or “established cell line” (Table the cell culture
technology
17). These cells have undergone a “transforma- WI-38 human embryonic “normal” cells with a
tion”, i.e., they have lost their sensitivity to the lung cells finite life span
growth control mechanisms. Transformed cells vaccine production
Vero long-tailed monkey established cell line,
can also lose the characteristic to grow adhered kidney, 1962 but with some
to a surface and thus become able to grow in sus- characteristics of the
pension. These transformations are also some- normal diploid cells.

times reflected in the chromosomes, changing


the genotype of the cells. Transformed cells can Hybridoma cells are “artificial” cells pro-
be easily grown in relatively simple media with- duced by the fusion of human or mammalian
out addition of expensive growth factors. At first, lymphocytes, which can secret specific antibod-
transformed cells were identified just by chance, ies, with a myeloma cell. Usually, lymphocytes
but nowadays there are techniques to cause cell do not survive outside the original organism, i.e.,
Biotechnology 101

cannot be kept alive in an artificial medium. Mil- working cell bank” is derived, providing the in-
stein and Köhler [458] were able to overcome oculation material for the production cultures.
this problem by fusing a mouse myelom cell sim-
ilar to a tumor cell with a lymphocyte from a
mouse spleen, which was able to produce anti- 9.1.4. Growth Medium for Cell Culture
bodies. The cells generated in this way are hy-
brid cells (also called “hybridoma cells”) and Basically, a growth medium for mammalian
have the lymphocyte characteristic to produce cells have to supply all the necessary nutrients
antibodies against a certain antigen, as well as required for growth and product formation [482,
the ability from the myelom cells to survive in 483]. Furthermore, it should have a certain buffer
culture. capacity to stabilize the pH (pH optimum 7.0–
An alternative expression system for recom- 7.3) and should provide an appropriate osmo-
binant proteins offer insect cells, especially the lality in order to avoid damaging of the sensi-
insect-cell baculovirus expression system (IC- tive cell membrane [484 – 488]. A fundamental
BEVS) [476]. Mostly applied are Sf9- or Sf21- component of all media is a salt solution, which
cells isolated from Spodoptera frugiperda. Pro- provides the ions necessary for life, keeps the os-
duction of heterologous proteins by the IC- motic pressure within the desired range, contains
BEVS consists of two stages. Insect cells are one or more buffer systems (sodium phosphate
first grown to a desired concentration and then buffer, HEPES, and/or carbon dioxide–carbonic
infected with a recombinant baculovirus con- acid buffer) for pH regulation, and contains, in
taining gene coding for the desired protein. some cases, a pH indicator (phenol red). Further-
Meanwhile, several recombinant proteins pro- more the media contain glucose and glutamine
duced by this technique have reached the mar- as a source of carbon and nitrogen, other ami-
ket. no acids, vitamins, mineral salts, and trace ele-
The largest and most well known interna- ments.
tional animal cell culture collections are the A number of medium formulations have
American type culture collection (ATCC) [477], been developed, e.g., Eagle’s minimal essen-
the European collection of animal cell culture tial medium (MEM), Dulbecco’s enriched (mod-
(ECACC) [478] as well as the German resource ified) Eagle’s medium (DMEM), Ham’s F12,
centre for biological material (DSMZ) [479]. and RPMI 1640, among others. Examples of
The application of permanent cell lines for medium compositions can be found in the liter-
the production of vaccines faced strong opposi- ature [442, 454, 489]. Traditionally these basic
tion, mainly in the 1960s, because there was the media were supplied with approx. 5-10 % serum
suspect that these cell lines, which behave sim- (e.g., foetal calf serum [FCS] or horse serum
ilarly to tumor cells, could infect patients with [HS]) in order to supply specific growth factors
unknown cancerous substances. Fortunately, no and to protect the cells against shear stress. The
observation of this kind has been done until disadvantage of media containing serum are the
today. In the meanwhile, more and more per- indefinite composition of such media, the high
manent cell lines have been applied for the serum costs, the difficulty of product purifica-
production of therapeutic and diagnosis sub- tion, variations between the charges and the risk
stances. However, the application of permanent of virus contamination [490].
cell lines, and above all, of recombinant cell Serum can be partially substituted by the ad-
lines, is strictly controlled by supervisory boards dition of transferin, insulin, ethanol amine, albu-
(as the FDA [480] in the USA, and the EMEA min, or eventually fibronectin as adherence fac-
[481] within the EU). During the whole produc- tor in a serum-free but still protein-containing
tion cycle of a pharmaceutics, not only the prod- medium [491 – 493]. A further step to chemi-
uct has to be identical from charge to charge, but cally defined, protein-free media became possi-
also the phenotypical characteristics of the cell ble by replacing these animal-derived proteins
line has to be maintained. To assure that, before by iron salts or iron complexes, IGF-1, chem-
the beginning of the production, a “master cell ically defined lipid concentrates, precursors or
bank” has to be created, from which a “master other stimulating agents such as fatty acids, bi-
otin, cholin, glycerin, ethanolamin, thiole, hor-
102 Biotechnology

mones, and vitamins [494 – 496]. Furthermore, 9.1.5. Small-Scale Culture Systems for
addition of peptone or yeast extract can be help- Routine Use
ful [497, 498]. However, the growth rate and pro-
ductivity in a serum-free medium can decrease Techniques and culture systems for cultivation
[499]. In addition, the sensitivity to shear stress of mammalian cells differ significantly from
increases, requiring additives with protecting those used for microbes. For basic research, a
characteristics, such as Pluronic F68 [500, 501]. large number of culture systems have been de-
Immobilization can improve the culture stability veloped (Fig. 32), mostly designed for use in
of nonanchorage-dependent animal cells grown an incubator aerated with 5 % CO2 to main-
in serum-free media [502, 503] tain the pH within the desired range. Multiwell
Different cell lines require different compo- plates (6–96 wells), T-flasks (25–100 mL), Petri
sitions. Adaptation of a cell line to grow with- dishes or roller bottles (50 mL to 5 L) are usu-
out serum is quite time-consuming and not all ally used for cell maintenance and proliferation,
cell lines have been adapted to serum-free or especially for adherent cells [441, 506 – 509].
protein-free media [504, 505]. For cultivation These systems allow for sterile handling proce-
of primary cells, for basic cell-culture research dures and are easy to use, disposable, and low-
or for vaccine production still mostly complex, cost. On the other hand, they require individ-
serum-containing media are common. In indus- ual handling, for example in medium exchange
trial production with established, optimized cell and cell seeding; their usefulness is limited when
lines, serum-free, bovine-serum and protein-free large quantities of cells or products are required.
as well as chemically defined media are state of This can be overcome to some extent by using
the art [439]. sophisticated robotics [510]. Furthermore, envi-
ronmental parameters including pH, dissolved
oxygen, and temperature can hardly be con-
trolled within the medium. A further drawback

Figure 32. Routine cultivation systems for use in incubators


Biotechnology 103

is the limited increase in cell number (approxi- Microencapsulation is another method for
mately 10-20 times during cultivation). Alterna- the immobilization of mammalian cells [441].
tively, small well mixed bioreactors (for exam- Basically, it can be used to protect cells against
ple, shake flasks, stirred vessels, and “super spin- hazardous environmental conditions. The three
ner” [511]) can be used, where adherent cells are basic encapsulation systems existing are the
grown on microcarriers. bead, the coated bead, and the membrane-coated
Microcarriers are small beads, either solid or hollow sphere [522 – 524]. Typical size for
macroporous, having a diameter of approx. 100- beads made of polysine alginate is 300 to 500
300 µm and a density slightly higher than the µm, and the molecular-mass cutoff of these cap-
growth medium. When first developed in the late sule membranes is 60 to 70 kDa [442]. The
1960s [512 – 515], microcarrier culture intro- capsules can be cultivated in suspension reac-
duced new possibilities and suspension culture tors similar to microcarriers. Detrimental with
of anchorage-dependent cells in high density. respect to scale-up is the fragile nature of the
Nowadays, beads made of DEAE-Sephadex, microcapsule.
DEAE-polyacrylamide, polyacrylamide, poly- In membrane bioreactors, including small
styrene, cellulose fibers, hollow glass, gelatin, hollow-fiber reactors [525], the miniPerm sys-
or gelatin-coated dextran beads are in use [442]. tem [526] or the tecnomouse [527], cells are
In microcarrier culture, cells grow as monolay- cultivated at tissuelike densities in a compart-
ers on the surface of small spheres (Fig. 33) ment which contains one or several types of
or as multilayers in the pores of macroporous membrane for nutrient and oxygen supply and
structures that are usually suspended in culture removal of toxic metabolites. Hollow-fiber sys-
medium by gentle stirring. By using microcar- tems (compare Section 9.1.6) are widely used
riers in simple suspension culture, fluidized or in the production of biopharmaceuticals in-
packed-bed systems, yields of up to 200 × 106 cluding monoclonal antibodies. Several exam-
cells per milliliter are possible [516]. Microcar- ples of modified membrane bioreactors exist
riers are extensively used for vaccine production for the three-dimensional culture of tissue cells
on a larger scale (see below) [517 – 521]. [528 – 530] including hepatocytes [531 – 533],
skin cells [534], or other human cells [535]. The
specific characteristics of hollow-fiber 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, dis-
solved CO2 concentration etc., low levels of
toxic metabolites (ammonia, lactate), high cell
and product concentrations, optimized medium
utilization, surface for adherent cells, and scal-
ability [536 – 545]. Bioreactors for mammalian
cell culture can be divided into two categories
(Fig. 34): Low-density or homogeneous sys-
tems (stirred-tank, bubble-column, air-lift reac-
tor), where cells are cultivated either in sus-
pension or, if required, on microcarriers; and
Figure 33. Porcine chondrocytes grown on microcarriers high-density or heterogeneous systems (fixed-
(Cytodex 3, GE Healthcare)
bed, fluidized-bed, hollow-fibre reactor), where
cells are immobilized either in macroporous sup-
104 Biotechnology

Figure 34. Bioreactor systems for cultivation of mammalian cells

port materials or within a compartment created by this handled similar to a suspension culture
by membranes. Selection of a reactor systems (see Section 9.1.5). For mixing, standard im-
depends to a large extent on the specific pur- pellers (e.g., rushton turbine, pitched or marine
pose such as production of a certain amount of impeller) as well as large paddle impellers are
protein (small amounts for basic scientific stud- used depending on the shear sensitivity of the
ies up to kilogram quantities for medical appli- cells, sometimes combined to achieve a homoge-
cation), three-dimensional tissue cultivation, or nous mixing throughout the bioreactor at low-
single-purpose or multipurpose facility. est possible stirrer speed [441, 549]. For aer-
Design of suspension reactors has to deal ation, different methods are shown in Figure
mainly with cell damage due to shear stress 36 [550 – 552]. Surface aeration can be applied
caused by agitation and/or aeration [546 – 548]. only for low cell densities and volumes (below
Stirred-tank reactors (Fig. 35), which are the 1 L). Bubble aeration may cause cell damage
most common type of reactor and are nowa- mainly due to foam formation, especially in case
days build up to 20 000 L scale, are espe- of serum-containing medium. By applying spe-
cially suited for suspendable cells [549]. Ad- cial aerators (ring sparger, microsparger) in com-
herent cells can be grown on microcarriers and bination with serum- or protein-free medium,
Biotechnology 105

the detrimental effects of bubble aeration can viruslike particles for gene therapy [594 – 597].
be overcome [553 – 556]. Alternatively, bubble- Again, scale-up is critical for both fixed-bed and
free membrane aeration systems were developed fluidized-bed reactors. For fixed-bed reactors, a
[557 – 560]. They are characterized by low shear radial-flow fixed-bed geometry was suggested
rates, but require large membrane areas. On lab- [598 – 600] and successfully applied up to ap-
oratory scale, a tumbling membrane basket can proximately 25 L fixed-bed volume. At perfu-
be used for mixing [511]. Rotating/vibrating sion rates of 10-20 L L−1
FB d
−1
, this would corre-
sieves provide a gentle aeration, but are suit- spond to suspension reactors in the thousand liter
able mostly for laboratory scale [561]. Bubble- scale. Alternatively, rotating-bed bioreactors are
columns and air-lift reactors were build up to in use [601]. For fluidized-bed bioreactors, sev-
thousand liter scale, are easy to handle, but re- eral concepts for scale-up have been suggested
quire serum-free media to prevent cell dam- as well [514, 585 – 587, 602]. Compared to sus-
age through bubbles and foam [549, 562]. For pension reactors, fixed-bed and fluidized-bed
continuous operation of stirred tank reactors reactors have the following advantages: high
in perfusion mode (see Section 9.1.7), several volume-specific cell density (approx. 5 × 107
techniques for cell and/or product retention are cells/mL reactor) and productivity, low shear
in use as shown in Figure 37 (e.g., microfil- rates, simple medium exchange and cell/product
tration and ultrafiltration, sedimentation, cen- separation, productivity on a high level during
trifugation, spin filters, acoustic filter, dialysis long-term cultures. Disadvantages are: nonho-
for cell, and product enrichment [563 – 577]). mogeneous cell distribution, difficult determi-
Suspension reactors are characterized by ad- nation of cells and cell harvest.
vantages such as: conventional reactor systems,
know-how on design and sterile operation, good
mass transfer, homogeneous mixing, possibility
of sampling and determination of the concen-
tration of the cell, and high scale-up potential.
Disadvantages are: difficult oxygen supply at
high cell densities, cell damage by shear and/or
foam (bubble aeration), relatively high demand
for control (temperatur, oxygen, pH, flow rates),
cell retention required for h igh cell densities.
Among high-density systems, we find fixed-
bed and fluidized-bed reactors as well as hollow-
fiber reactors. In fixed-bed and fluidized-bed
bioreactors, cells are immobilized within macro-
porous carriers. The carriers are arranged in
a column either packed (fixed-bed reactor
[578 – 584]) or floating (fluidized-bed reac-
tor [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 efficient for Figure 35. Multipurpose bioreactor for continuous perfu-
the long-term perfusion culture of mammalian sion culture of mammalian cells (Bioengineering AG, CH-
Wald, with permission)
cells for the production of biopharmaceuticals
(e.g., monoclonal antibodies, recombinant drugs
including tPA and EPO). With respect to tis- Within hollow-fiber reactors (compare Sec-
sue engineering, they have been investigated tion 9.1.5), cells are immobilized in the extra-
for several applications including the cultiva- capillar space of a hollow-fi4ber bundle. Nutri-
tion of “liver” cells as an extracorporal liver de- ents as well as oxygen pass through the fibers,
vice [588 – 590] or proliferation of stem cells metabolites leave the culture chamber by this
[591 – 593]. Furthermore they were success- way. Cell concentrations comparable to those
fully used for cultivation of cells producing found in tissues are possible. The use of hollow-
106 Biotechnology

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 ultrafiltration, internal or external; B) Spin filter, internal or external; C) Settler, external or inclined; D) Centrifuge,
external; E) Acoustic filter, 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 fluid
in; f) Dialysing fluid out

fiber culture is a convenient method for making peared on the market besides hollow-fiber re-
moderately large quantities of high-molecular- actors, addressing especially problems related
weight products secreted by human and animal to early process development such as flexibil-
cells, at high concentrations and with a higher ity, cost effectiveness, time-to-market as well as
ratio of product-to-medium-derived impurities quality and regulatory issues [606 – 608]. These
than is generally achieved using homogeneous systems are mostly based on a bag technology
(e.g, stirred-tank) culture [603 – 605]. Advan- [609, 610]. The bags with volumes up to 250 L
tages are: very high cell densities, concentrated are placed on tilting or rocking devices for mix-
product, lower serum demands (if required), ing and oxygen supply. Advantages of single-
long-term stability, easy handling, and low cost. use bioreactors are reduced cleaning procedures,
Disadvantages are: limited scale-up, mass-trans- lack of validation issues (cleaning, sterilization,
fer problems/concentration gradients, difficult etc.), lower investment costs, easier adaptation
determination of the cell number, proteolytic ac- to changing process demands, less contamina-
tivity on the product. tion risk [611 – 613].
During the last years, a growing number of The main characteristics of the culture sys-
disposable or single-use bioreactor systems ap- tems discussed above are summarized in Table
Biotechnology 107

18. All these systems support growth of mam- perfusion reactor design becomes slightly sim-
malian cells in one or the other way. On labora- pler (e.g., fixed-bed and fluidized-bed) because
tory scale, mostly disposable flask, membrane, of the immobilization of cells within macrop-
or bag systems are the method of choice. Small orous carriers (compare Section 9.1.6). There is
suspension reactors as well as fixed bed and flu- a growing number of reports on stable perfusion
idized bed reactors are mostly used for research cultures even on an industrial scale over periods
or process development. For larger amounts of of several months [563, 644 – 646]. The main
products, only suspension reactors or, up to a advantage of perfusion cultures can be seen in
certain scale, fixed-bed or fluidized-bed reactors the reduced bioreactor size (approx. 1/10 of a
have the required scalability. For a detailed dis- suspension reactor without cell retention). Nev-
cussion, compare [440, 555, 614 – 618]. Biore- ertheless, there are some difficulties in the per-
actor design, in particular, is addressed in [619]. fusion concept [438, 563]. Further equipment
such as the retention device itself, pumps for
feeding, harvest, and medium circulation, stor-
9.1.7. Process Strategies age tanks for feed and harvest are required. The
amount of media needed to complete a moderate
As for all biotechnological processes, process to long-term run can be excessively large. A fur-
strategies for operation of bioreactors can be ther possible drawback of continuous cultivation
classified in discontinuous (batch, repeated- is the possibility for variability over the time of
batch or fed-batch) and continuous modes the run. Batch or fed-batch cultivations are much
(chemostat, perfusion). Batch and fed-batch pro- shorter in duration and have fewer chances for
cesses are mostly performed in small-scale cul- random occurrences to happen. This is impor-
ture systems (e.g., flasks, bags) or on larger scale tant in dealing with current manufacturing prac-
in suspension reactors. Batch processes are usu- tice (cGMP, see below) conditions that dictate
ally starting with an initial cell density of ap- precise reproducibility or at a minimum the evi-
proximately 1–2 105 cells mL−1 and last 1–2 dence of non-effects of minor deviations. In the
weeks. After harvest, the bioreactor has to be meantime, there is a growing number of pharma-
cleaned and refurbished before the cycle is re- ceutical products in the market, produced suc-
peated [438, 440, 441]. Cell and product yield cessfully from perfusion systems. Among these
can be significantly improved by applying a fed- are block busters such as Kogenate-FS r
(Bayer
batch strategy, were nutrients are added after de- Health Care) or Remicade r
(Centocor) [563].
pletion according to an appropriate feeding strat- Previous concerns about getting such processes
egy [[621 – 634]. Batch and fed-batch strategies approved are not longer an issue and it can be ex-
are applied quite frequently on industrial scale pected that because of an increasing pressure on
[438, 440, 536]. production costs more perfusion processes will
The drawbacks of discontinuous modes such be installed in the future.
as large reactor volumes, high maintenance A process that becomes more and more im-
(cleaning, sterilization, etc.) can be overcome to portant for production of small quantities of
some extend by using continuous mode, espe- recombinant proteins is transient transfection
cially perfusion with cell and/or product reten- [647 – 649]. Usually, nontransfected cells (most
tion [438, 563]. Chemostat cultures without cell commonly HEK-293, COS, and BHK cells,
retention are a valuable tool for research (e.g., compare Section 9.1.2) are first cultivated in
kinetic studies) [635 – 639] but not for produc- batch mode to a certain cell density and then
tion scale because of low cell and product con- transfected with DNA encoding for a certain pro-
centration. Continuous perfusion present several tein [441]. Usually, the cells are genetically not
advantages over batch systems [563, 640 – 643]. stable and soon lose their expression ability but
The advantages include the ability to grow cells can produce a certain amount of protein within
to a very high density, the ease of handling me- one batch. Originally, it was used just as a pre-
dia exchanges for the purpose of fresh feed and liminary test of gene expression. New develop-
product harvest, the easy removal of metabo- ments show that large scale production up to 100
lites and other inhibitors, and the prospect of L is possible [650, 651].
easy scale-up. When dealing with adherent cells,
108 Biotechnology
Table 18. Main characteristics of cell culture systems and bioreactors ([620], modified)a

T-flask/roller Membrane reactors Suspension (stirred-tank, Fixed-bed/fluidized-bed


bottles/disposable bags (hollow-fibre, miniPerm, air-lift reactors) reactors
etc.)
Cell density 1 3 2 2–3
Homogeneity 1 1 3 2
Shear stress N N Y N
Product concentration 1 3 2 2
Porductivity 1 3 2 2
Medium efficiency 3 1 3 2
Continuous process N Y Yb Y
(perfusion)
Control 0 1 2 3
Downstream process 1 3 2 2
Steam sterilizable N N Y Y
Reusable N N Y Y
a
0, not possible; 1-3, increasing efficiency; Y, yes; N: no.
b
Additional equipment for cell retention required.

9.1.8. Downstream Processes endotoxins, process chemicals, etc. The impor-


tance of the downstream process is underlined
The desired protein product, mostly extracellu- by the fact, that an estimated 60–80 % of the to-
lar, has to be purified after cell cultivation by tal production costs are attributed to purification
a combination of unit processes. From an en- [438, 439, 654].
gineering point of view, the challenges are 1)
low product concentrations in the cell-free su-
pernatant, 2) the complex structures of the pro- 9.1.9. Regulatory and Safety Issues
teins, especially the recombinant, glycosylated
proteins, resulting in a high sensitivity against Pharmaceutical production processes are subject
temperature, shear forces, extreme pH, or prote- to strict regulations and regular inspections to
olytic activity, 3) contamination by nucleic acid ensure the high quality demands for medicines
(DNA fragments from dead cells), 4) contami- [439, 440, 657]. Guidelines for aspects relevant
nation by other proteins (e.g., from serum), 5) for the production process are summarized un-
potential contamination by viruses or virus par- der cGMP and corresponding lists were released
ticles [652, 653]. The main process steps in- in the United States by the FDA [480] as well as
volved are [654]: concentration of supernatant by the EU [481]. There are some special concern
(e.g., precipitation, centrifugation filtration, or with the use of mammalian cells for production
aqueous two-phase partition), primary purifica- of biopharmaceuticals, e.g., 1) risk of contam-
tion/capture (e.g., chromatographic techniques ination by microbes, mycoplasma, or viruses,
such as ion-exchange, gel, or immunoaffinity 2) the genetic stability of the host cell line, or
chromatography as well as precipitation), fur- 3) the consistency of the product (glycosyla-
ther purification (e.g., ion-exchange, hydropho- tion pattern, etc.). The documents to be submit-
bic, and gel chromatography), and finally pol- ted to the authorities include detailed informa-
ishing (gel filtration, diafiltration, ultrafiltration) tion concerning the product, the production pro-
and techniques for virus inactivation (acid/base cess, product characterization including analyt-
treatment, nanofiltration or ultraviolet-c (UVc)) ical methods, validation procedures, methods,
[653, 655, 656]. The early stages within a pu- and results of clinical trials. The efforts to fulfill
rification strategy will aim to achieve both con- these requirements are quite high and bind sig-
centration and purification. The next step will nificant resources within the companies [439].
be the major purification step. The number of
steps included in “further purification and “pol-
ishing” will depend on the intended use of the
protein, as they are mainly designed to reduce
contaminants such as DNA, host cell protein,
Biotechnology 109

9.2. Tissue Engineering 9.2.1. Application of Tissue Engineering


In this chapter, an overview in the field of “red Tissue engineering is a rather young and rapidly
biotechnology” is presented. Red biotechnology growing interdisciplinary research field which
comprises three major fields: combines the principles of engineering, life, and
– Gene therapy clinical sciences (Fig. 38). The aim of this re-
– Production of proteins, antibodies, and vac- search field is the development of biological sub-
cines for diagnostics and therapy stitutes that restore, maintain, or improve tissue
– Tissue engineering functions [661, 662]. Therefore, the knowledge
Gene therapy is a therapeutic approach based of cellular interactions, molecular transport phe-
on the correction of a gene defect causing a nomena, interactions of cells with material sur-
disease. Different techniques exist for achiev- face, mechanical loading, and biochemical in-
ing this aim. In most cases, the wild-type gene terventions is applied.
is inserted into the genome by special methods An official definition was presented at a con-
replacing the mutant gene, which leads to the ference of the National Science Foundation in
development of the disease. The most common 1988: “. . . the application of principles and
vectors for the transport of the correct gene into methods of engineering and life sciences to-
the genome are viruses. ward fundamental understanding of structure–
In the mid-eighties, several treatments with function relationship in normal and pathologi-
the genetically modified cells were reported. The cal mammalian tissues and the development of
first gene therapy approach was reported in 1990 biological substitutes to restore, remain, or im-
on a young girl suffering from severe combined prove tissue function”.
immunodeficiency (SCID). But the therapy did Over the past two decades, encouraging re-
not work out, after a few months the procedure sults have been achieved. Methods and tech-
had to be repeated regularly. niques of tissue engineering have already en-
A major drawback within this field emerged tered therapeutic practice, e.g., for the treat-
when it was evident that a young patient died ment of sports injuries and damage to cartilage.
after being treated by gene therapy [658]. The The main approach adopted in these cases is
patient was suffering from a rare liver disease known as autologous chondrocyte transplanta-
and was a volunteer in a gene therapy program. tion (ACT). Cells isolated from cartilage tis-
Similar incidences occurred in the year of 2000 sue of the patient (autologous) are seeded onto
within a group of boys after gene therapeutic a collagen matrix and cultivated until a three-
treatment of SCID. The majority of the boys in dimensional tissue structure is generated. Then
this group developed leukemia. Since then, gene the construct can be implanted into the defect
therapy is only broadly applied within science- [663 – 665]. Also tissue-engineered skin is al-
fiction movies. Researchers working in this field ready used, e.g., for therapy of severely burned
went back a few steps to elucidate the devastat- patients [666 – 669]. Researchers are attempting
ing incidences [659]. to engineer almost every human tissue or organ
Biological active compounds produced from because the demand of tissue-engineered cells
genetically modified cell cultures have been in and organs is still growing because of the fact of
use for several years in the treatment of a whole organ shortage for transplantation. The creation
range of diseases. In most cases, the active in- of three-dimensional tissue constructs which can
gredients are proteins such as blood clotting mimic all functions and metabolic activity like
factors (e.g., tissue plasminogen activator, t- organs (e.g., liver [670] or pancreas [671]) is still
PA), growth factors (e.g., erythropoitin, EPO), a crucial challenge. The first tissue-engineered
or monoclonal antibodies [660]. All these sub- heart valves have been implanted to a patient and
stances can be produced in animal cell cultures. show promising results in function and mechan-
These proteins can also be applied in diagnos- ical stability [672, 673].
tic 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.
110 Biotechnology

Figure 38. Disciplines involved in tissue engineering

Figure 39. Principle of tissue engineering

9.2.2. Principle of Tissue Engineering Tissue engineering is driven by the hopes of


the patients of increased availability of trans-
The principle of tissue engineering can be plantable organs, to ease their suffering, fore-
demonstrated in the following way: First, cells stall imminent death, and enabling a better qual-
have to be isolated. The cells are seeded to a ity of life. Alongside these medical objectives,
matrix and are extracorporal cultivated in a suit- there are also huge economic issues at stake
able culture system or bioreactor in medium con- because treatment of tissue and organ damage
taining nutrients and growth factors. After cul- or dysfunction often involves long hospitaliza-
tivation, the resulting tissue is reimplanted into tion and thus is very expensive. In contrast to
the patient (Fig. 39). alternative approaches of xenogenic (from an-
Biotechnology 111

other species) and allogenic (same species but the application of a mechanical load (e.g., strain,
another individual) organ transplant, tissue en- compression) is beneficial for the development
gineering involves transplanting cells from the of three-dimensional cell–cell contacts resulting
body of the patient itself. This is the reason for in functional tissue [674 – 682].
which such great attention has been focused in
the last few years on this new area of research.
By growing artificial autologous tissue and or-
gans in vitro, the risk of rejection by the im-
mune system of the body can be avoided. Tis-
sue engineering techniques can be used to gener-
ate replacement tissue for internal organs (liver,
pancreas, heart, kidneys, lungs), sensory organs
(eyes, ears, nose), the skeleton (bones and car-
tilage), the brain and nerves (in particular for
the treatment of Alzheimer’s disease and Parkin-
son’s disease), or the skin (e.g., treatment of se-
vere burns).
A closely related concept to tissue engineer- Figure 40. The “essentials” for tissue engineering
ing is regenerative medicine. Here, the empha-
sis is placed on supporting the body’s natural
healing processes. The potential of stem cells is
crucial for both fields. 9.2.5. Cells
For tissue engineering, either tissue-specific
9.2.3. Strategies cells can be isolated and cultured or stem cells
can be used for the directed differentiation to the
Tissue engineering can be performed by using desired tissue. Due to their potential for differen-
one of the three approaches: tiation into several different tissue types, nowa-
days the use of stem cells is intensively discussed
In vitro cultivation of autologous cells on or- [683]. In numerous publications adult stem cells
ganic, synthetic, or natural matrices isolated from bone marrow (bone marrow stro-
In vitro cultivation of autologous cells on mal cells BMSC [684 – 686], fat tissue (adipose-
xenogenic matrices derived stem cells adSC [687]), and peripheral
Iin vitro tissue generation from embryonic blood or umbilical cord blood (both haematopoi-
stem cells etic cells [688, 689]) are applied. The potential
Depending on the tissue of interest, either for differentiation into several different types of
a close system (e.g., extracorporal device, ar- tissues is already approved. Mesenchymal stem
tificial liver) or an open system based on a cells (MSC) play a major role in modern tissue
biodegradable polymer scaffold is developed. engineering [690, 691]. These cells are easy to
The autologous cells can be isolated tissue- isolate from bone marrow or fat tissue and their
specific primary cells or stem cells. availability is not so limited compared with that
of the small number of stem cells present in, e.g.,
peripheral blood (0.1–0.2 %). Treatment con-
9.2.4. The Essentials cept based on the use of autologous stem cells
would eliminate problems of donor site scarcity,
The essentials for tissue engineering are shown immune rejection and pathogen transfer.
in Figure 40. For a successful generation of
three-dimensional tissues, suitable cells have to
9.2.6. Biomatrices
be isolated and cultivated on a matrix in an
appropriate medium containing all necessary There has been also huge progress in the de-
growth and differentiation factors by using an velopment of biomaterials for use in tissue-
optimized bioreactor system. During cultivation, engineering applications [692, 693]. Research
112 Biotechnology

has concentrated on the development and pro-


duction of biocompatible and biodegradable ma-
terials [694 – 697]. Such biomaterials take over
the task of the extracellular matrix (ECM),
which naturally provides cells with a support-
ive framework of structural proteins, carbohy-
drates, and signaling molecules. The ideal scaf-
fold has to mimic ECM features and would be
made of a biomaterial that provides all the nec-
essary signals for the cells to grow, differenti-
ate and interact, forming the desired structure.
Regardless that general properties and design
features of the biomaterials for discussed pur-
poses have been published in a numerous re-
views [698 – 702], the subject still represents in-
tensive scientific and practical interest. Besides Figure 41. Selected bioreactor types for tissue engineering
natural occurring compounds or scaffolds, also
synthetic biomaterials are reviewed in the liter- Since human cells are anchorage-dependent,
ature [703 – 705]. Along with polysaccharide- they must be attached to a matrix in the bioreac-
and protein-based (e.g., hyaluronic acid, colla- tor during the cultivation. In a rotating-wall sys-
gen) materials, also many synthetic polymers tem, the cell-seeded matrices are cultured while
are used for tissue-engineering application (e.g., the reactor wall is rotated, thus the cells float
polylactic acid, polyglycolic acid, polyurethane, through the medium and are affected by mi-
polyesters). crogravitylike conditions, and the mass-transfer
rates are improved. Moreover, cell-seeded ma-
trices can be cultured while hanging in spinner
9.2.7. Bioreactors for Tissue Engineering flasks, which results also in an improved mass
transfer. Another possibility is the continuous
The design, control, and operation of bioreac- pumping of the medium through a fixed bed with
tors in technical and engineering disciplines are cells or the cells can be placed into a chamber
well-established. Their use in medical sciences where a mechanical stimulation (here compres-
requires an interdisciplinary approach aiming at sion) is applied.
the development of functional tissues. There-
fore, frequently the application of mechanical
loading is needed [706 – 709]. The loading can 9.2.8. Growing New from Old
be disposed in different ways; for example, a
longitudinal straining leads to the formation of The idea of organs one day being freely avail-
ligaments or tendons; compression is applied able “off the shelf” is still an aspiration today.
when aiming at the development of, e.g., car- The need is great, however, and patients are of
tilage; pressure (perfusion) can be applied for course very eager to have “personalized” treat-
bone tissue formation and pulsed bioreactors ment from “organ designers” using tissue engi-
are used for blood vessel generation. In Figure neering. The most ambitious concepts involve
41, a selection of bioreactor schemes for tissue- applying a sort of “building-block” principle to
engineering applications is presented. Unlike the culture of each tissue or organ type: so-
in conventional mammalian cell culture, where called precursor or stem cells would be used,
only a limited number of bioreactor types are whose potential for differentiation could be ex-
used (e.g., T-flaks, roller bottles, spinner sys- ploited through the use of appropriate growth or
tems, stirred-tank reactors), in tissue engineer- differentiation factors. Thanks to this method,
ing tailor-made bioreactors have to be devel- one would “only” need the appropriate “ingre-
oped and constructed for each specific tissue dients”: Further advantages of this approach are
type, considering in particular the fluid dynam- the fact that an organ or tissue replacement can
ics which are realized by these different systems. be carried out at any time – in other words it can
Biotechnology 113

be scheduled in advance. Regenerative medicine 9.3. Biotechnology and Food


has great potential: according to a report in Time
Magazine, over the next 10 to 20 years, tissue en- It is not presumptuous to claim that modern
gineering will become one of the top careers and biotechnology has developed from traditional
a new era in life sciences and medicine dawns. processes of food preparation. The production of
The potential applications are highly diverse but alcoholic beverages, the coagulation of milk pro-
are to be found essentially in the treatment of teins, and the preservation of vegetable food by
cells and tissue which possess little or no capac- fermentation has already been familiar to most
ity to repair themselves such as cartilage, heart ancient civilizations. Thus, several microorgan-
muscle and nerve tissue. isms have a very long history of safe use in hu-
The medicine of tomorrow will strive to de- man food production. The main focus of this
velop personalized therapeutic procedures in- chapter is not on traditional fermentations but
volving the patient in each treatment choice. on recent developments in food biotechnology.
There will be a move from a general “one- Since this field is remarkably ambitious and
size-fits-all” approach to personalized treatment complex, only some techniques of high indus-
based on the principle of “one drug, one ther- trial relevance are covered exemplarily. Genetic
apy, one patient”. This will mean using a sys- engineering of plants has yielded, for exam-
tems biology approach to fully restore the origi- ple, herbicide tolerant “RoundUp Ready” soy,
nal healthy state of the patient. This dream could Bacillus thuringiensis toxin expressing maize
be realized thanks to the emerging technologies (Bt maize) and transgenic rice lines with an
of regenerative medicine and tissue engineering. increased concentration of β-carotene in the
Groups of cells and tissue generated by using endosperm (“golden rice”). Genetically engi-
tissue engineering can also serve as test systems neered animals may produce milk with an al-
which exhibit characteristics specific to a given tered protein profile, and modification of the
organ. These can be used to test the effective- hormone expression of fish imparts accelerated
ness and duration of certain drugs or to study growth rates. A number of comprehensive re-
their mechanisms and metabolism, thus repre- views on food biotechnology are available [710].
senting an alternative to animal experiments, and
a rapid, broadly applicable and cost-effective
way of screening pharmaceutically active sub- 9.3.1. Production of Food Additives by Cell
stances. Culture Systems
Over the last ten years, there has been an ex-
plosion of research in tissue engineering, which 9.3.1.1. Amino Acids
shows the enormous importance attached to this
technology. Within Europe, German researchers l-Amino acids represent the largest group of
are playing a major role. Leading research is also food and feed additives produced by means of
being carried out by teams in France, Britain, biotechnology [711]. Consequently, the litera-
Italy, and the Netherlands. Large numbers of ture published within the last decade is notably
small biotechnology companies are working in versatile. l-Glutamic acid ((S)-2-aminoglutaric
tissue engineering and more and more are being acid, E 620) was the first amino acid pro-
set up all the time. duced biotechnologically on an industrial scale,
In the 21st century, major scientific questions and is still the leading amino acid by volume.
concern modern developments within the field More than 800 000 t/a of monosodium gluta-
of regenerative medicine, which includes tissue mate, which serves as a flavor enhancer (“umami
engineering, transplantation, prosthesis, as well taste”), are produced worldwide by cultivation
as pharmacological interventions stimulating in of Corynebacterium glutamicum and related or-
situ tissue regeneration. ganisms. The production of l-glutamic acid may
be enhanced by overexpression of the glutamate
transporter gene (yhfK) [712], by use of strep-
tomycin resistant Brevibacterium lactofermen-
tum strains [713], and by optimization of the
114 Biotechnology

culture conditions [714]. The second most im- maximum synthesis rates when grown on raw
portant amino acid is l-lysine ((S)-2,6-diami- agro-industrial fats as substrates [726], while
nohexanoic acid), which is mainly used to sup- Rymowicz and Cibis [727] selected the acetate-
plement animal feed with this essential and often negative strain Y. lipolytica AWG-7, cultivated
limiting amino acid. It is industrially produced on glucose syrup, for citric acid production. As
by C. glutamicum or by E. coli species [711]. Ef- a second important organic acid, l-lactic acid
ficient industrial producer strains have been de- ((S)-2-hydroxypropanoic acid, E 270), has been
veloped by multiple rounds of mutagenesis. De- used from ancient times as a food preservative
fined l-lysine producing C. glutamicum mutants by lowering the pH during fermentation. More
have been generated for example by modifica- recently, lactic acid has gained increasing in-
tion of the enzyme aspartate kinase (lysC muta- terest since the monomer is used for genera-
tion) [715], attenuation of the transcription reg- tion of the biodegradable food packing poly-
ulator tipA gene [716], and the introduction of a mer “polylactic acid (PLA)”. However, only few
mutation (S361F) into the 6-phosphogluconate PLA-generating processes have been commer-
dehydrogenase gene (gnd) [717]. In the latter cialized and the cost of PLA is still not able
case, the modified strain was less sensitive to to compete with synthetic plastics [728, 729].
allosteric inhibition by intracellular metabolites Apart from traditional lactic acid bacteria such
than the wild-type strain. Product yields of up as Lactobacillus delbrueckii [730] or L. plan-
to 100 g/L are attainable within 24 h of culti- tarum [731], a wide range of further microor-
vation with the recombinant production strains. ganisms, especially strains of Rhizopus oryzae,
A third amino acid, l-cysteine ((R)-2-amino-3- has been employed for the biosynthesis of l-
mercaptopropanoic acid), which is applied as a lactic acid (for example [732, 733]). A current
dough conditioner in the bakery industry, may be review on metabolic engineering approaches of
obtained from recombinant E. coli [718 – 720]. lactic acid bacteria is available [734].

9.3.1.2. Organic Acids 9.3.1.3. Vitamins

Citric acid (2-hydroxy-1,2,3-propanetricarbox- l-Ascorbic acid (vitamin C, E 300) is produced


ylic acid, E 330) is widely used to lower the by a combination of chemical synthesis and en-
pH of food (candy) and beverages (soft drinks, zymatic reaction steps. Known as “Reichstein
wine). Furthermore, it acts as an efficient chela- process”, the procedure was established already
tor of prooxidative iron and copper ions. Various in the 1930s. The key biotransformation step,
fungi and bacteria may be used for the biotech- the oxidation of d-sorbitol to l-sorbose, is cat-
nological synthesis of citric acid starting from alyzed by Acetobacter suboxidans [735]. Due
numerous substrates. The process has recently to the worldwide use of l-ascorbic acid as vita-
been comprehensively reviewed in [721]. As- min in dietary supplements and as antioxidant in
pergillus niger grown as surface cultures on di- food and feed, the annual need sums up to 60 000
luted molasses has been employed as early as t [711]. Novel approaches use either recom-
in the 1920s, and still is the most popular pro- binant microorganisms in one-step procedures
duction organism. Today, distillers’ grains [722], [736] or Azotobacter chroococcum strains [737]
palm oil mill effluents [723], or cassava starch for l-ascorbic acid production. For a review
factory wastes [724] may serve as cheap and on biotechnological routes to vitamin C, see
abundantly available substrates. By optimiza- [738]. Further water-soluble vitamins, includ-
tion of the growth conditions (sufficient oxygen ing riboflavin (vitamin B2 ) and cyanocobalamin
supply in submerged cultures; media deficient in (vitamin B12 ), are currently produced by mi-
manganese, copper, iron, and zinc ions; high glu- crobial cell cultures. The chemical synthesis of
cose concentrations), and by selection of over- the yellow-greenish-colored riboflavin has been
producing A. niger strains, yields of > 100 g/L largely replaced by biotechnological processes
are attainable [725]. The yeast Yarrowia lipoly- using either the overproducing fungus Ashbya
tica represents a future potential production or- gossypii [739] or mutant strains of Bacillus sub-
ganism. The strain Y. lipolytica 187/1 showed tilis. The “generally recognized as safe” (GRAS)
Biotechnology 115

organism B. subtilis is widely used in the food in- Subsequently, palatinose is reduced to palatinit,
dustry [725] and, for example, the mutant strain an equimolar mixture of the corresponding alco-
CJKB0002 produced 26.5 g/L riboflavin com- hols (1-O-α-d-glucopyranosyl-d-mannitol and
pared to 22.4 g/L obtained from the parent strain 6-O-α-d-glucopyranosyl-d-sorbitol) by hydro-
[740]. Detailed mechanistic studies on the ri- genation on a fixed-bed Ni catalyst. In the in-
boflavin synthesis in Ashbya gossypii [739] and dustrial process, it is advantageous to use im-
in the lactic acid bacterium Lactococcus lac- mobilized non-viable cells of P. rubrum in a
tis ssp. cremoris [741] have been published. packed-bed reactor for the enzymatic step [746].
Cyanocobalamin is derived from Propionibac- Apart from P. rubrum, immobilized cells of Ser-
terium or Pseudomonas denitrificans species ratia plymuthica [747], Erwinia sp. D12 [748],
[711]. Recently, a fermented milk product con- or Klebsiella singaporensis LX3T (isolated from
taining Propionibacterium freudenreichii ssp. soil) [749] have been used to convert sucrose into
shermanii B369 bacteria, capable of produc- palatinose. In 2005, palatinose was approved ac-
ing cyanocobalamin in situ, was patented [742]. cording to the EU regulation (EC 258/97) con-
Thus, a vitamin B12 enriched yogurt may be cerning novel foods and novel food ingredients,
manufactured as a “nutraceutical” without ex- and the GRAS registration was granted in 2006
ternal vitamin fortification. However, compara- by the US Food and Drug Administration (FDA).
tively low world-wide annual production rates of Recent inventions suggest a mixture of palati-
3000 t for vitamin B2 and 10 t for vitamin B12 nose with cocoa powder for the preparation of in-
document the predominant use of theses vita- stant beverages [750]. Further applications com-
mins in dietary supplements and in pharmaceu- prise the use of palatinose for the production of
ticals [711]. The main plant-derived carotenoid, low-alcohol beer or beerlike soft drinks [751],
β-carotene (E 160a), serves as provitamin A in and the preparation of fermented soybean milk
the human diet. Since β-carotene additionally [752].
imparts an intense yellow-orange coloring, it In the industrial production of the sweet-
is widely used as a natural food additive. The ener aspartame (α-l-aspartyl-l-phenylalanine
development of biotechnological processes for 1-methyl ester, E 951), the methyl ester of the
the production of β-carotene is mainly driven “DF” dipeptide, both components are produced
by the increasing demand of the food industry. by biotechnological processes. l-Phenylalanine
Besides the extraction from plants (for exam- is usually manufactured by large-scale cultiva-
ple from lucerne), β-carotene is industrially ob- tion of the soil bacteria Serratia marcescens or
tained from the algae Dunaliella salina [743] C. glutamicum. Immobilized aspartase or alter-
or from the fungus Blakeslea trispora [744]. natively intact cells of E. coli are used to syn-
The carotenoid astaxanthin, which is used on thesize l-aspartic acid starting from the precur-
a large scale in aquaculture for the coloring of sors ammonia and fumarate [725]. Usually, amid
salmonids and crustaceae, is formed by the yeast formation is performed chemically although an
Phaffia rhodozyma [745]. enzymatic approach exists [753]. Thermolysine,
immobilized on an agarose–aldehyd gel, is used
as biocatalyst for the synthesis of enantiopure
9.3.1.4. Sweet Compounds aspartame [754]. Thermolysine is a metallopep-
tidase with a zinc ion in its active site and four
Palatinit (isomalt, E 953) is a non-cariogenic calcium ions, which stabilize the tertiary struc-
sugar substitute used in low-calory food, ture of the enzyme.
desserts, ice creams, and diabetic food. It is
a white, crystalline, and non-hygroscopic sub-
stance with a sweetening power of roughly 9.3.1.5. Sugar Alcohols
0.45–0.60 compared to that of sucrose (1.0). In
the first production step, sucrose is converted Sugar alcohols such as d-sorbitol (d-glucitol; E
enzymatically (by action of a glycosyltrans- 420) and d-xylitol (E 967) have attracted the at-
ferase) into palatinose (isomaltulose, O-α-d- tention of the food and pharmaceutical industry
glucopyranosyl-(1–6)-d-fructofuranose) by im- because of their sweetening and non-cariogenic
mobilized cells of Protaminobacter rubrum. properties. Due to the fact that their metabolism
116 Biotechnology

is insulin-independent, they are well suited for cus on the conversion of starch to trehalose) and
substituting sugar in diet schemes for diabetics. potential applications in the food industry.
Since the biotechnological production of xyli-
tol represents an economically attractive alter-
native to the industrialized chemical process, 9.3.1.7. Conjugated Linoleic Acids (CLA)
various process variants have been published.
Somewhat less literature is available on sor- Lactic acid bacteria play a prominent role in the
bitol, which is mainly formed by cultures of Zy- traditional fermentation of food. One of the ex-
momonas mobilis [755, 756]. The synthesis of ceptional features of some lactobacilli is their
xylitol by Candida guilliermondii depends on ability to isomerize linoleic acid to different
several crucial process parameters, one of which conjugated linoleic acid isomers (CLA). The
is the concentration of phosphate buffer in the Z9,E11- and E9,E11-octadecadienoic acid are
culture broth (optimum at 0.6 M) [757, 758]. assumed to possess beneficial physiological and
In another approach, the d-xylitol production anticarcinogenic properties [735]. Lactobacillus
of the yeast Debaryomyces hansenii UFV-170 plantarum AKU 1009a formed up to 40 mg/mL
was monitored with respect to temperature and CLA (33 % molar yield) in 108 h under opti-
starting pH [759]. Various substrates, including mized conditions from 12 % (w/v) linoleic acid
for example hydrolysates of corn cobs [760] and as substrate [769]. Further lactobacilli capable of
sugarcane bagasse [761] have been used for the CLA production encompass for example L. del-
bioconversion of the pentose d-xylose to xylitol. brueckii ssp. bulgaricus [770] and L. plantarum
JCM 1551 [771]. Castor oil proved to be an ade-
quate substrate for CLA formation. Presumably,
9.3.1.6. Microbial Saccharides a two-step reaction mechanism, involving hy-
dration of a double bond with subsequent elimi-
The most popular polymer currently produced nation of water, is leading to different CLA iso-
by means of biotechnology is xanthan gum (E mers [772].
415). Xanthan gum is a complex bacterial ex-
opolysaccharide structurally closely related to
cellulose. Today it is mainly produced by mu- 9.3.1.8. Lactulose
tants of Xanthomonas campestris (40 000 t/a
[711, 762]), a Gram-negative bacterium that Lactulose acts as a prebiotic compound and thus
is pathogenic to many crops. Due to its out- may contribute to increased levels of “health-
standing properties (high salt and pH toler- promoting” bacteria (bifidobacteria and/or lac-
ance, thermostability, and unusual rheological tobacilli) in the human intestinal tract [773].
behavior), xanthan is predominantly used as a The term “prebiotics” comprises a wide vari-
thickener in dressings and convenience food ety of oligosaccharides that are not digested in
[763]. Sago starch has been suggested as an the stomach and in the small intestine. They
alternative carbon source instead of glucose naturally occur in garlic, asparagus, onion, and
[764]. Trehalose (α-d-glucopyranosyl-(1-1)-α- others. Apart from soybean oligosaccharides, a
d-glucopyranose) is a nonreducing disaccharide number of uncommon fructo-, galacto-, xylo-,
that is thought to play a role as storage carbo- and isomalto-oligomers are currently in use, es-
hydrate for many microorganisms. It may serve pecially in Japan [773]. The disaccharide lac-
as an additive to improve the freeze stability tulose (4-O-β-d-galactopyranosyl-d-fructose) is
of processed food. In 2000 and 2001, trehalose a non-naturally occurring compound with po-
has gained the FDA GRAS and the EU novel tential applications in infant milk formulations
food approval, respectively. Trehalose is ob- and foods [774]. Although lactulose is chiefly
tainable inter alia from Brevibacterium [765], manufactured chemically using boric acid or
Cellulosimicrobium cellulans [766], or Debary- aluminates as catalysts, a biotechnological route
omyces hansenii strains. The latter two were cul- starting from lactose (as galactose donor) and
tivated under moderate saline stress for extra- fructose with permeabilized Kluyveromyces lac-
cellular trehalose production [767]. Literature tis yeast cells has been developed [775]. In
[768] reviews trehalose production (with a fo-
Biotechnology 117

another approach, β-galactosidase from As- starch molecule (endoamylase), while the α-
pergillus oryzae and a hyperthermostable β- (1-4)-bonds near α-(1-6)-branches are resistant
glycosidase from Pyrococcus furiosus were used to hydrolysis. Thus, the end products of ex-
for the transgalactosylation of lactose in the tensive α-amylase treatment are oligosaccha-
presence of fructose [776]. Important food ad- rides of varying length with α-configuration
ditives and food supplements produced by cell and α-limit dextrins, which constitute branched
culture systems are summarized in Table 19. oligosaccharides. Since α-amylase of B. licheni-
formis is remarkably thermostable (it remains
active for several hours at temperatures of > 90
9.3.2. Enzyme-Catalyzed Processes ◦
C), its application in the industrial starch hydro-
The modification of starch and lipids as well lysis is highly efficient [780].
as the production of cheese and fruit juices are β-Amylases (EC 3.2.1.2; 1,4-α-d-glucan
prime examples of enzyme-catalyzed biopro- maltohydrolase) are present mainly in higher
cesses in the food industry. For economic rea- plants, but also in some microorganisms. Com-
sons, the necessary enzymes are often employed mercial products are available from malt, soy,
as crude products or even as mixtures of en- and wheat. They hydrolyze α-(1-4)-glucosidic
zymes with different properties. If required, the bonds, effecting successive removal of maltose
enzymes are purified by means of precipitation, units from the nonreducing end (exoamylase).
ultrafiltration and/or fast protein liquid chro- In contrast to amylose, amylopectin is not com-
matography (FPLC). Common FPLC protocols pletely hydrolyzed, as all reactions stop before
comprise one ore more of the steps hydropho- branch points are reached (β-limit dextrin).
bic interaction chromatography (HIC), ion ex- Glucoamylase (EC 3.2.1.3; 1,4-α-d-glucan
change chromatography (IEX), and size exclu- glucohydrolase) is formed predominantly by
sion chromatography (SEC). Reusability and fungi and yeasts (Aspergillus). It starts at the
sometimes also stabilization may be achieved nonreducing end of α-(1-4)-d-glucans and suc-
by immobilization of the biocatalyst on an inert cessively liberates β-d-glucose units. The α-(1-
carrier material. 6)-bonds in amylopectin are also cleaved, but
around 30 times slower than the α-(1-4)-bonds.
At higher concentrations, glucoamylase reforms
9.3.2.1. Starch-Modifying Enzymes all the glycosidic bonds that it hydrolyses. Thus,
it condenses glucose, for example, to isomaltose
Traditionally, the degradation of starch to mal- (6-O-α-d-glucopyranosyl-d-glucose).
todextrins and glucose/fructose syrups was per- Debranching enzymes exclusively hydrolyze
formed by acid-catalyzed hydrolysis, using α-(1-6)-bonds in amylopectin, glycogen, and
strong mineral acids such as hydrochloric or pullulan. Linear amylose fragments are formed
sulfuric acid. The harsh reaction conditions af- from amylopectin. Pullulanase (EC 3.2.1.41;
forded a variable spectrum of unwanted by- pullulan α-(1-6)-glucanohydrolase) hydrolyzes
and breakdown products. Therefore, the acid α-(1-6)-bonds in pullulan (a linear polymer of α-
hydrolysis has been replaced more and more by (1-6)-linked maltotriose units) and amylopectin,
enzymatic processes. Today, starch-converting while isoamylase (EC 3.2.1.68) is only capable
enzymes comprise about 30 % of the enzyme of the hydrolysis of the α-(1-6)-bonds in amy-
market worldwide. Amongst the starch con- lopectin. Pullulanases have been found in a va-
verting enzymes, α-amylase (EC 3.2.1.1, 1,4- riety of bacteria; industrially used is an enzyme
α-d-glucan glucanohydrolase) holds the largest derived from Bacillus acidopullulyticus.
market share with major applications in the Cyclodextrins are produced via an intramo-
starch and bakery industries [777, 778]. α- lecular transglycosylation reaction in which
Amylases may be derived from several plant the enzyme cyclodextrin glycosyltransferase
materials, bacteria, yeasts, and fungi [779]. (EC 2.4.1.19; 1,4-α-d-glucan 4-α-d-(1,4-α-
For commercial applications, mainly strains of d-glucano)-transferase) cleaves the α-(1-4)-
Bacillus amyloliquefaciens and Bacillus licheni- glycosidic bond and concomitantly links the re-
formis are employed as enzyme producers. α- ducing to the nonreducing end.
Amylase attacks the α-(1-4)-links within the
118 Biotechnology
Table 19. Examples of food additives and food supplements produced by cell cultures

Product Use E number Production organism


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

Syrups high in fructose level (42–44%, high- the efficient overexpression of the correspond-
fructose corn syrups, HFCS) are obtained from ing genes, but also a detailed understanding of
glucose solutions by action of the enzyme xy- the mechanisms governing the folding and se-
lose (glucose) isomerase (EC 5.3.1.5, d-xylose cretion of the proteins [785]. Immobilized li-
ketol isomerase) [725]. Technical enzymes are pases from the yeast Candida rugosa are tra-
derived for example from Streptomyces, Actino- ditionally popular, as a wide core in the cat-
planes, Arthrobacter, or Bacillus cultures. The alytic centre allows for acceptance of various
large-scale isomerization of glucose is per- fatty acids for trans-esterification or synthesis of
formed with immobilized enzymes, and product mono- and diacylglycerides, for example, [786].
yields of up to 22 tons of isosugars per kilogram Especially C. antarctica lipase B features out-
of catalyst are obtained. Fructose concentrations standing properties such as high thermostabil-
of about 90 % are accessible via a subsequent ity, sn-2 recognition in hydrolysis of triacylglyc-
chromatographic purification step. An excellent erides, and selectivity towards (E)-fatty acids. A
survey of enzymes involved in the industrial pro- review on applications of the lipases A and B
cessing of starch is presented in [781]; and the from Candida antarctica was published recently
most important starch converting enzymes are [787]. Several strains of another member of the
summarized in Table 20. Candida family, Yarrowia (Candida) lipolytica,
were screened for lipase secretion. The high-
est lipase activity produced on rapeseed oil as
9.3.2.2. Lipases sole carbon and energy source was found to be
Lipases (EC 3.1.1.3, triacylglycerol acyl- 2760 U/mL for Y. lipolytica 704 [726]. When
hydrolase) are valued biocatalysts with numer- cultured in a 2000 L bioreactor, the lipase activ-
ous applications in food biotechnology. Many ity reached 1100 U/mL after 53 h [788]. Lipases
representatives of this enzyme class are highly with novel catalytic properties are produced for
stable in organic solvents, show broad substrate example by the basidiomycetous fungus Pleuro-
specificity, and high regio- and/or stereoselec- tus sapidus. An extracellular enzyme of the type
tivity [782]. Main fields of application include B carboxylesterase/lipase family efficiently hy-
the rational design of lipids with specific struc- drolysed xanthophyll esters [789].
tures (“functional oils and fats” [783]), and
the production of volatile fatty acids and fla-
vor esters (“natural food flavorings”). Volatile 9.3.2.3. Pectin-Degrading Enzymes
fatty acids are the so-called “character impact”
flavor compounds of various types of cheese, Pectin is a complex polysaccharide (α-d-(1-4)-
while numerous flavor esters are responsible for polygalacturonic acid, esterified with methanol
fruity aroma impressions [784]. Industrial high- in varying degree) that represents an essential
level production of lipases requires not only
Biotechnology 119
Table 20. Starch-converting enzymes

Enzyme Enzyme number Producer organism Specificity


α-Amylase EC 3.2.1.1 Bacillus licheniformis endo-α-(1-4)
β-Amylase EC 3.2.1.2 malt, soy, wheat exo-α-(1-4)
Glucoamylase EC 3.2.1.3 Aspergillus ssp. exo-α-(1-4); exo-α-(1-6)
Pullulanase EC 3.2.1.41 Bacillus acidopullulyticus endo-α-(1-6)
Cyclodextrin glycosyltransferase EC 2.4.1.19 Bacillus macerans intramolecular transglycosylation
Xylose isomerase EC 5.3.1.5 Streptomyces ssp. isomerization of glucose to fructose

part of plant cell walls. The group of pecti- 9.3.2.4. Chymosin (Aspartic Protease)
nolytic enzymes comprises pectinases (poly-
galacturonases), lyases, and pectin esterases. A Chymosin (EC 3.4.23.4) is a key enzyme with
total market share of 25 % of the global sales peptidolytic activity used in the production of
of food enzymes is currently attained by this en- cheese. The casein fraction of milk protein is
zyme family [790]. To improve the yields in fruit coagulated by hydrolysis of κ-casein between
and vegetable juice production, and to obtain Phe105 und Met106 to form para-κ-casein and
clear and concentrated juices, especially pecti- a glycopeptide (κ-casein glycomacropeptide).
nases from the mold Aspergillus niger are em- From a mechanistic point of view, chymosin be-
ployed [791, 792]. For example, the efficiency longs to the class of aspartate peptidases, which
of kiwi juice production was increased > 10 % are characterized by the presence of two aspartic
by addition of 40 mg/L pectinase for 150 min acid residues in the catalytic center.
[793], and the yield of loquat juice was 16 % Since recombinant chymosin does not exhibit
higher when 160 mg/L pectinase were added unwanted side activities and is not contaminated
for 4 h [794]. Under these conditions, the loss with microorganisms possibly causing quality
of vitamin C was low and the juice was clar- problems, it replaces rennin from the stomachs
ified. Endo- and exopectinases (EC 3.2.1.15, of calves (abomasus) to an increasing degree.
endopolygalacturonase; EC 3.2.1.67, exopoly- The gene encoding chymosin has been cloned
galacturonase) depolymerize the pectin chain by into different microorganisms, and the proper-
hydrolytic cleavage of the glycosidic α-(1-4)- ties of the recombinant enzymes expressed in the
bonds. yeasts Kluyveromyces lactis and Pichia pastoris
Usually, pectinases are combined with hemi- have been compared [797]. Since K. lactis is re-
cellulases (mixture of cell-wall-degrading en- garded as a safe organism traditionally used in
zymes) and cellulases (EC 3.2.1.4) to improve food processing, it is of outstanding importance
fruit liquefication, leading to high-quality juices in the biotechnological production of chymosin
in terms of oxidation, aroma levels, and sta- on an industrial scale [798]. The technological
bility [795]. As an auxiliary enzyme, α-l- characteristics of various enzymes from geneti-
arabinofuranosidase (EC 3.2.1.55), was sug- cally modified organisms were nearly identical
gested for applications in the wine industry and to those of natural rennin, and only slight dif-
for clarification of fruit juices [796]. Pectin lyase ferences in the coagulation time and curd firm-
(EC 4.2.2.10) and pectate lyase (EC 4.2.2.2) ness were observed [799]. Chymosin obtained
catalyse the eliminative cleavage of pectin and from transgenic sheep milk and the recombinant
pectate, respectively, to oligosaccharides with protein expressed by K. lactis differed only in
4-deoxy-α-d-gluc-4-enuronosyl groups at their their action on low-molecular-weight substrates,
nonreducing ends. Potential production organ- but not in enzymatic activity on protein sub-
isms include Erwinia species as well as a num- strates [800]. Recently, extracellular acid pep-
ber of phytopathogenic fungi. Pectin esterases tidases from Rhizopus oryzae have been sug-
(EC 3.1.1.11) catalyze the hydrolytic cleavage gested as rennin substitutes in cheese manufac-
of the methyl ester group. These enzymes gen- turing [801, 802].
erally exhibit a high specificity for pectin sub-
strates.
120 Biotechnology

9.4. Biotechnology and Health and copy number. These changes can be de-
tected and mapped by using comparative ge-
9.4.1. Individualized Medicine nomic hybridization (CGH). CGH is a molec-
ular cytogenetic method of screening for ge-
Single nucleotide polymorphisms (SNPs, a sin- netic alterations. These changes are classified as
gle base is exchanged by an alternate) are the DNA gains or losses and show a characteristic
most common form of genetic variant in the hu- pattern that includes mutations at chromosomal
man genome, occurring on average 1 per 1000 level. In the past few years, microarray-based
base pairs. Since the influence of SNPs on med- formats for CGH have been developed made
ical therapy or diet is better understood, the SNP from large genomic clones and cDNAs. These
diagnostic is coming in the focus of interest. One arrays provide many advantages over conven-
important goal of genome research is studying tional CGH, including higher resolution, direct
genetic variance among individuals to improve mapping of changes to the genome sequence,
the knowledge and treatment of disease and to and high throughput. Together with the analysis
identify the variations in our genes that influence of complex differences in gene expression bet-
the risk of an individual of becoming ill. Be- ween normal and cancer cells, these data provide
cause inherited differences influence most com- insight into malignancy and reveal genes that
mon diseases and many drug responses, the un- may be useful as diagnostic or prognostic mark-
derstanding of existing genetic variations in the ers. One problem performing cancer diagnosis
human population is a promising approach in the using gene expression profiles from microarray
development of tailor-made therapeutics. Eighty analysis considers the heterogeneity of a tumor.
percent of the individual variations are SNPs, Samples taken from different locations in the
which have a direct impact of gene regulation or tumors are dramatically different. Therefore, bi-
of a protein product of a gene. In cases where ological classification on the basis of random
one SNP is sufficient to cause disease it is possi- sample analysis leads to poor reproducibility and
ble to analyze the causal change and to improve may not be adequate [807 – 809].
our understanding of disease [803 – 806]. The
hope is that a complete human SNP map will
increase the efficiency of drug development and 9.4.3. Pharmaceutical Development
medical treatment of disease. Many genotyping
platforms have been developed to detect many Drug biotransformation in man and animals
SNPs at once in a efficient, cost-effective man- can lead to a modulation of pharmacodynamic
ner including microarrays by the use of allel- effects, drug elimination, but also drug tox-
specific oligonucleotides containing the variable icity. Knowledge of drug biotransformation
base. Large-scale SNP genotyping should lead pathways at early stages of drug development
to a fully understanding of the genetic architec- could considerably speed up drug development
ture of common traits underlying disease, drug [810 – 813] and increase the safety of drug de-
or diet response. For example, microarray ex- velopment in the pharmaceutical industry. Each
periments could help to define the ideal dose of year, more than 668 000 animals are used in toxi-
a drug and quantify the impact of this dose on cological drug research in Europe. Animal mod-
human health. Since these are the most person- els suffer from serious shortcomings regarding
ally data we know, the access to these data must the prediction for a human situation as signif-
be protected so it is not misused by employers icant species differences in enzyme expression
or insurers. The goal of this analysis is to relate exist between man and animals. Specifically, en-
SNP variation to disease or diet. zyme induction experiments are frequently mis-
leading. 10–20 % of all new drugs are required
to return into preclinical development trials after
9.4.2. Clinical Diagnosis as Indicated in first clinical tests because of unforeseen metabo-
Genetic Anomalies in Cancer lite patterns, whose toxicological relevance is
unclear or untested.
Solid tumors often show alterations in the Classical animal-testing strategies are time-
genome that lead to changes in DNA sequences consuming and therefore rather expensive in
Biotechnology 121

Figure 42. Human liver cells

view of a delayed market penetration and re- nology were brought together by the pharma-
turn of investment. The relevance of a strict con- ceutical industry for preclinical drug research.
trol of development time is immediately expli- Induction phenomena could be correlated with
cable by the fact that preclinical development of microarray chips containing all known genes in-
a novel drug can total up to several hundred mil- volved in drug biotransformation. This informa-
lion Euros. In spite of this, modern cell-culture tion is used to describe the culture models used
technology has progressed to the extent that it is in this study and to develop the tools to finger-
now possible to culture primary human cells un- print interactions of inducing agents with drugs.
der conditions that maintain differentiated func- The use of microarray technology in the field of
tions to perform drug biotransformation and en- drug discovery and development provide four
zyme induction studies with great reliability in significant advantages:
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 significant breakthrough in pharma-
ceutical drug metabolism research was that these
models could be sufficiently automated to enter
into the field of accelerated screening as a sig-
nificant initiation step towards high-throughput
screening. Industrial strategies are aiming at in-
tegrating 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- Figure 43. Bioreactor systems for in vitro testing
122 Biotechnology

– Gene expression in response to drug treat- through in toxicology research if these models
ments could be sufficiently automated to enter into the
– Gene expression in model systems field of accelerated screening as a significant ini-
– Gene expression patterns in disease tiation step towards high-throughput screening.
– Gene expression patters in pathogens (host– Microarrays are powerful tools for investigating
pathogen interaction) the mechanism of toxicity (Fig. 44). A microar-
ray chip for cytochrome P450s and Phase II en-
zymes can be used to evaluate the liver culture
9.4.4. Define Molecular Mechanisms of models [820].
Toxicity

DNA microarrays can be used to determine the 9.4.5. Detection of Genetically Modified
relative safety of natural or synthetic chemicals Organisms
to which humans are exposed. Lots of diseases
are influenced by environmental factors, for ex- The use of genetically modified organisms
ample, chemicals, radiation, drugs, nutrition, (GMOs) in food industry has been rising ex-
viruses, carcinogens, reproductive toxins, neu- ponentially over the past years. In the space of
rotoxins, and immunotoxins [814 – 819]. Al- time from 1996 to 2003, global area of trans-
though most of these chemicals are harmless, genic crops increased 40-fold, from 1.7 × 106
it is important to identify potentially hazardous hectares in 1996 to 67.7 × 106 hectares in 2003,
substances. In the past, toxicological assays have with a dominant trait of herbicide tolerance, fol-
used rodent, required high doses, were expan- lowed by insect resistance (www.isaaa.org, In-
sive, and take years to complete. Unfortunately, ternational Service for the Acquisition of Agri-
the information gained from established life an- biotech Applications). A GMO is “an organism,
imal models such as rodent to investigate en- with the exception of human beings, in which the
zyme induction and toxicity cannot really lead to genetic material has been altered in a way that
correct assumptions of hazards to humans with does not occur naturally by mating and/or natu-
respect to species extrapolation. The shortcom- ral recombination” [821]. Microarrays are a use-
ing with respect to predictivity of animal models ful tool for GMO detection and the application
for human drug biotransformation and toxicity of array techniques to GMO detection implies
is based upon a well accepted species difference the capability of sensitivity and standardization
in enzyme expression between man and animals. of the assay. Today, commercialized arrays of-
Nevertheless, at present animal models in drug ten do not fulfill these requirements, in spite of
metabolism and toxicology are still legally re- a few promising attempts initialized by biotech-
quired in all European countries for a variety nical companies and research projects. Never-
of reasons and legislative bodies are still hes- theless, microarrays are already being used in
itative to recommend specific human liver cell GMO analysis but well established quantitative
based tests. In spite of this, modern cell-culture real time PCR techniques are state of the art.
technology has progressed to the extent that it GMO detection becomes increasingly important
is now possible to culture primary human liver because by-products of these organisms could
cells under conditions that maintain differenti- end up in the food chain and affect human health.
ated functions to perform drug biotransforma-
tion and enzyme induction studies with great
reliability in individual laboratories. These de- 10. Concluding Remarks
velopments have not yet found entry into legal
recommendations as no prevalidation of such As it has been demonstrated in the previous
models on a European scale has been achieved chapters, biotechnology offers manifold possi-
so far. The widespread use of such alternative bilities in the production of different products
models to animal investigations is being ham- and in medical applications. It can be used for
pered both by insufficient prevalidation and by better food, better health, and a cleaner environ-
further improvements still required in the field ment. However, in the area of production of fine
of automation. It would be a significant break- and bulk chemicals, biotechnology has still to
Biotechnology 123

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

compete with the chemical industry. The chem- Microorganisms still have a great poten-
ical industry owes its success to the principle of tial for inducing new or novel enzyme sys-
unit construction: from simple basic substances, tems capable of converting foreign substrates.
like ethylene, carbon monoxide or hydrogen, It is possible to obtain and cultivate microor-
more complex precursors can be produced un- ganisms that can survive or grow in extraor-
der controlled conditions by chemical reactions; dinary environments, e.g., psychrophilic, ther-
because of the variety of combination options, mophilic, acidophilic, and alkaliphilic microor-
the latter can in turn be converted into inconceiv- ganisms. These microorganisms are capable of
able quantities of derivatives and end products. producing unique enzymes stable to heat, alkali,
Chemistry learned how to produce chemically and acid. In addition, techniques for cultivation
pure basic substances from oil that are simple and enzyme purification, and gene and enzyme
to handle and exactly defined; this is performed technology have made great advances and are
highly efficiently in refineries. This was the key still under development. Certainly biotechno-
to its success. Without exact knowledge of this logical process development must be founded
functional principle, the triumphant success of on a broader genomic basis. Ten years ago, only
plastics would have been just as impossible as a handful of microorganisms with a completely
the production of thousands of other chemical sequenced genome existed, today there are sev-
products that today make our lives safe and com- eral hundreds. Hitherto priority was given to se-
fortable. This principle can also be applied to the quencing pathogenic microorganisms, but it is
so-called ‘biorefinery’ which has similar poten- now high time to sequence microorganisms that
tial and could thus provide a further argument for are relevant to industry. Taking all of these into
the concept of material application of renewable consideration, there must be far more ways than
resources and biotechnology. However, a tech- one can imagine in using biotechnology for our
nically viable separation procedure that would daily life.
permit the separate use or further processing
of all the primary products (such as cellulose,
hemicellulose, and lignin) is still in its infancy. 11. Acknowledgement
The main focus is on glucose, which can be de-
rived by microbial or chemical methods from The current update was coordinated by Roland
cellulose, since a wide range of biotechnologi- Ulber.
cal and chemical products can be obtained from
glucose. The potential for the microbial conver-
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