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
key technologies of the 21st century. It is the
commercial application of living organisms such
as bacteria, fungi, yeasts, plant cells, viruses, and
mammalian cells or their products, which in-
volves the deliberate manipulation of their DNA
molecules. This article gives an introduction into
the basics in microbiology and provides an ex-
haustive description of the relevant microbial
species and metabolic pathways. Identification,
analysis, and manipulation of the genome, pro-
teome, and metabolome is described, and cul-
tivation requirements as well as process pa-
rameters discussed. Biotransformation and en-
zyme technology plays a central role in indus-
trial biotechnology, and a focus is given on
the development of molecular engineering tech-
niques and new screening methods. Computa-
tional Biochemistry comprises the definition,
monitoring, and modeling of bioprocesses. Ex-
amples of biotechnology applications include
mammalian cell technology, tissue engineering,
and the production of relevant food additives
as well as of various medical and pharmaceu-
tical products. In conclusion, biotechnology of-
fers manifold possibilities in industrial and med-
ical applications.
1. Introduction
Biotechnology can be described as “the com-
mercial application of living organisms or their
products, which involves the deliberate manipu-
lation of their DNA molecules”. This includes
the use of bacteria, fungi, yeasts, plant cells,
viruses, and mammalian cells. This definition
implies a set of laboratory techniques devel-
oped within the last 20 years that have been
responsible for the tremendous scientific and
commercial interest in biotechnology, the found-
ing of many new companies, and the redirec-
tion of research efforts and financial resources
4 Biotechnology
among established companies and universities.
Thus, biotechnology can be regarded as one of
the key technologies of the 21st century. How-
ever, the use of microorganisms and their prod-
ucts is as old as mankind. Fermented products
with yeast such as beer, wine, or sake are known
since several thousands of years (Table 1). The
roots of modern biotechnology are coming from
the era of microbiology, which was developed
in the late 19th century. Cagniard-Latour, Pas-
teur, Koch and Kühne were important scientists
who are decoding the principles of fermentation.
The use of microorganisms for the production of
bulk chemicals such as butanol or acetone and
for pharmaceuticals (antibiotics) was strongly
developed during World War I and II. With the
better understanding of the gene function and its
relation to the metabolisms of cells, the modern
era of biotechnology started in the 70th of the
last century.
Nowadays, biotechnology is a cross-sectoral
technology that has been successfully applied
in many industrial branches. One distinguishes
between several areas or “colors” of biotechnol-
ogy (the so-called red, white, green, and blue
biotechnology).
Red Biotechology. Named as red biotech-
nology are applications in medicine or in the
pharmaceutical industry. Red biotechnology has
already made an impact on healthcare and
will continue to contribute to improving human
health and life expectancy. Today, 20 % of mar-
keted medicines, 50 % of those in clinical tri-
als, and 80 % in early development are biotech-
based products. Forty percent of these candidate
medicines are for the treatment of cancer. Typi-
cal products of red biotechnology are recombi-
nant vaccines, antibodies, blood clotting agents,
and hormones. In addition, tissue engineering is
a part of the area of biotechnology. Tissue en-
gineering aims at the functional regeneration of
tissues through implantation of tissue cultured
in vitro (see Section 9.2).
White Biotechology. The potential and ap-
plications of biotechnology in other sectors
largely pass unnoticed especially in the chemical
industry. The chemical industry has produced
coal, gas, and petroleum-based goods and prod-
ucts for over 150 years. This era is likely to
Table 1. Timetable of biotechnology [1, 2]
Year Biotechnological progress
3000 b.c. Fermentation of sugar containing juices to
various alcoholic beverages
2800 b.c. brewing rooms in Mesopotamia and leaven in
Egypt
1500 b.c. Use of microorganisms for the production of
copper and production of soya sauce
300 b.c. Use of vinegar
1300 Micro algae (Spirulina) as food additives
(Aztecs)
1400 Production of saltpetre (potassium nitrate)
with Nitrosomas sp. and Nitrobacter sp. in
Germany
1676 Anthony van Leuwenhoek observes bacteria
through a microscope
1837 Charles Cagnaird-Latour identifies yeast as
causer of fermentation (one year later
confirmed by Theodor Schwann and Friedrich
Kützing)
1849 Industrial production of bakers yeast
1856 Louis Pasteur separates brewer yeasts from
lactic bacteria
1876 Kühne creates the term “enzymes”
1881 Industrial microbial production of lactic acid
(Boehringer)
1890 Development of first vaccines by Pasteur and
Koch
1893 Industrial production of citric acid using
Aspergillus niger
1897 Starting point of enzyme technology (Eduard
Buchner)
1915 Patent for enzymes in washing powder
1916 Industrial fermentation process for acetone
and n-butanol (Chaim Weizmann)
1928 Alexander Fleming discovers penicillin
1937 Microbial production of vitamin C
1941/42 Industrial production of antibiotics
1957 Use of Corynebacterium glutamicum for the
production of amino acids
1972/1973 Stanley Cohen and Francis Boyer develop a
procedure for in vitro recombination of DNA,
using plasmid vectors
1977 First recombinant proteins from bacteria
1982 First transgenic plants and animals
1985 Development of polymerase chain reaction
(PCR) by Kary Mullis
1990 Start of human genome project (HUGO)
1996 Yeast genome is completely sequenced
2000 Human genome is completely sequenced
(Craig Venter)
end within the next 100 years at the latest. An
extrapolation of oil consumption from 2002 (3.3
billion tonnes), for example, yields a supply pe-
riod of 50 years with estimated stocks of 165
billion tonnes. The underlying reasons for a fu-
ture era of new raw materials are the finite na-
ture of fossil resources on the one hand, and the
connection with many environmental problems
on the other hand. In the past few years condi-
tions for the application of biotechnological pro-
cesses in industrial production have improved

(so-called white biotechnology). New tools such
as screening methods and metabolic engineer-
ing, and also global analysis methods such as ge-
nomics, proteomics, metabolomeics, and bioin-
formatics are gradually becoming more widely
available. These new instruments make it possi-
ble to reduce the time needed to develop and es-
tablish new industrial biotechnological products
and processes; this was hitherto one of the ma-
jor drawbacks of biotechnological, as opposed
to chemical, processes. In addition, they help
to develop biocatalysts (enzymes) and microor-
ganisms which render manufacturing processes
more economical and facilitate new manufactur-
ing processes. For the first time since the begin-
ning of the oil age in the early fifties, one is able
to apply processes with economic potential in
the production of basic chemicals and biopoly-
mers based on biotechnological processes. In
many cases, bioprocesses operate under milder
conditions (lower temperatures and pressures,
etc.) and more selectively than their competitors
(chemical processes) by these means biopro-
cesses conserve resources and improve produc-
tion processes economically and ecologically.
Public debate has often emphasized the advan-
tages for the environment; however, companies
face hard economic competition so that when
a biotechnological process is weighed against
a classical chemical process, only the potential
economic advantages can affect a shift in favour
of biotechnology.
Green Biotechnology. In addition, to over-
come the problems in the production both of
bulk and fine chemicals but also in the produc-
tion of vaccines and pharmaceuticals, the so-
called green biotechnology can offer new so-
lutions. This area of biotechnology involves the
introduction of foreign genes into economically
important plant species, resulting in crop im-
provement and the production of novel products
in plants. Green biotechnology might also pro-
duce more environmentally friendly solutions
than traditional industrial agriculture. An exam-
ple of this is the engineering of a plant to express
a pesticide, thereby eliminating the need for ex-
ternal application of pesticides.
Blue Biotechnology. In a very broad sense,
marine or blue biotechnology can be understood
as the various means of techniques of managing
Biotechnology 5
marine living systems for mankind’s profit. The
domain covered by marine biotechnology is vast
and range over various overlapping disciplines,
from developmental biology to chemistry of nat-
ural substances and bioprocess engineering. Not
all these fields, however, are ready for practical
and industrial applications. Biomass from fish-
ing or aquaculture industry is, in fact, complex,
geographically and seasonally dependent. Fur-
thermore, many “natural substances”, for which
we do not know of any terrestrial counterparts,
and, therefore, present an up most interest, exists
only in tiny amounts in rare biological species
and their exploitation is likely to call for costly
synthesis procedures. Marine natural products
not only display novelty but also complexity in
terms of chemical structures. Isolation and struc-
ture elucidation represents only the emerged part
of an iceberg. The question of the functionality
of new isolated molecules within the perspec-
tive of challenging major public health and en-
vironmental problems is crucial. Likely, in this
domain, ecological and evolutionary approaches
should help the classical screening systems for
determining the right target systems. In addi-
tion, a better understanding of the complex in-
teractions between macro- and microorganisms
is necessary to be able to use these resources
for industrial purposes. Thus, more and more
groups are focussing on the part of bioprocess
engineering and downstream processing in blue
biotechnology [3].
2. Basics in Microbiology
2.1. Microbiology – the Science of
Microscopic Life Forms
Microbiology deals with microscopically tiny
life forms which the resolution of the human
eye (approx. 20 µm) cannot detect. Many of
the single-cell microorganisms, such as yeasts,
algae, or protozoa, do not reach this size. Not
until the invention of the microscope (Antonie
van Leeuwenhoek, 1684) was the detection, and
thus also the characterization, of microorgan-
isms possible [4].
If microorganisms are defined by their size,
many varied life forms come into this category:
single-cell life forms with a real cell nucleus
6 Biotechnology
(e.g., algae, fungi, protozoa) and also some with-
out a real cell nucleus (bacteria and archaea)
which are of particular interest in general mi-
crobiology. Organisms with a genuine cell nu-
cleus are termed eukaryotic organisms, those
without prokaryotic organisms [5]. Today some
6500 species of prokaryotic bacteria and archaea
are known, a relatively small number compared
with the number of the known species of eukary-
otic organisms. However, it is estimated that the
number of actually living species of prokary-
otic organisms is much higher, although most of
them are regarded as not cultivable under labo-
ratory conditions. Analyses of the metagenomes
of the most varied habitats (total DNA assays of
samples from various environments, such as for-
est soil, compost, pond water, etc.) have yielded
estimated values of up to one billion potential
species of prokaryotic organisms worldwide.
For most people, microorganisms have a bad
image. The reason is not hard to find: some
microorganisms can cause disease in man and
animals (pathogenic microorganisms). Thanks
to the achievements of medical microbiology,
the often devastating effects of pathogenic mi-
croorganisms in earlier times have been confined
due to the development of vaccines, antibiotics,
etc. [6]. Nevertheless, microorganisms still re-
present a threat to health (for instance to peo-
ple with a weakened immune system). The fol-
lowing are a few examples of bacteria that are
pathogenic to humans: Bacillus anthracis (an-
thrax), Bordetella pertussis (whooping cough),
Clostridium botulium (botulism), Clostridium
tetani (tetanus), Corynebacterium diphtheriae
(diphtheria), Shigella dysenteriae (shigellosis),
Vibro-cholerae (cholera). The development of
resistance to known antibiotics is a phenomenon
that documents the variability of pathogenic mi-
croorganisms [7]. This also explains why it is
necessary to promote progress in the field of
combating infectious diseases with microbio-
logical methods.
Biotechnology is closely linked to micro-
biology. Biotechnology uses microbiological
metabolism performance to manufacture indus-
trially relevant products. The sector with the
oldest tradition of applying microorganisms for
production purposes is the food industry. The
manufacture of beer, wine, vinegar, bread, and
cheese are early achievements of human history
and pertain to the field of food microbiology.
Microorganisms are used in the production of
sausages (particularly salami), sauerkraut, and
milk products (lactobacilli). In Asia, a variety of
fermented food products are produced on the
basis of rice and soya [8, 9].The typical aro-
matic properties and consistency of many food
products originate from the activity of the mi-
croorganisms used. Nowadays, these organisms
are generally used in the production process as
starter cultures. The fermentation products are
also often used as preservatives (acidification in
lactic acid production by lactobacilli or alcohol
production by yeasts).
The monitoring of food in the framework of
quality control involves excluding undesired mi-
crobial infestation or providing evidence of it.
Microorganisms contribute towards stabiliz-
ing the global natural material cycles (e.g., car-
bon and nitrogen cycles). Furthermore, the waste
flows produced by man are largely reintroduced
into the natural material flows through the ac-
tivity of microorganisms. The field of environ-
mental microbiology addresses the question of
how microorganisms can be utilized for the re-
mediation of contaminated soil, water, and waste
gases. Successful industrial processes have been
developed and are used worldwide for wastewa-
ter and drinking water purification, brownfield
clean-up, composting, and waste fermentation
and also for the purification of contaminated
gases [10 – 12].
Industrial microbiology, or white biotechnol-
ogy, deals with the production of industrially in-
teresting substances involving production vol-
umes of thousands of tons. Effective production
of substances requires on the one hand an effi-
cient production strain to supply as much target
product as possible in the shortest time possi-
ble. On the other hand, it needs a technical fa-
cility where the high-performance microorgan-
isms find optimum living and, therefore, produc-
tion conditions and where in addition monosep-
tic cultivation in large volumes is guaranteed.
Bioreactors fulfill these requirements: they can
be operated with volumes of up to several hun-
dred m3 and are equipped with automatic mea-
surement and control systems adjusted to the
growth needs of the microorganism [13, 14].
Examples of microbially produced industrial
products are diverse. The list begins with mi-
croorganisms that can themselves be the prod-

uct of a biotechnological process and reach
the market as starter cultures [15]. Currently
the quantitatively dominant product is ethanol,
of which over 24 × 106 (metric) tons are pro-
duced worldwide. Ethanol is produced with the
yeast Saccharomyces cerevisiae and is mainly
used as a substitute fuel or in admixture with
other fuels. Other products include pharmaco-
logically active agents (e.g., antibiotics, steroids,
and alkaloids), bulk, special, and fine chemicals,
food and feed additives, chiral intermediates, en-
zymes, antibodies, etc.
The total value of all biotechnologically pro-
duced substances worldwide (including bio-
pharmaceuticals which are often manufactured
with cultures of animal cells) must be in the
realm of > 100 × 109 euros annually.
2.2. Phylogeny and Taxonomy of
Microorganisms
2.2.1. Definition and Survey
Taxonomy is the branch of biology which sub-
sumes individual groups of species into a hier-
archical system. For a long time, the kingdom
was the highest category of creatures. Origi-
nally, a distinction was made between animals
and plants. Later, the bacteria were added and
then the fungi and plants were split up [16]. Fi-
nally the archaea were given their own kingdom.
In recent years, genetic investigations have led
to a new classification with the “domain” super-
imposed over the kingdom. Whereas in earlier
times the aim was to understand and deduce re-
lationships among microorganisms on the basis
of morphological, physiological, and cytologi-
cal similarities, in the last few decades genetic
similarity has been the criterion used to identify
relationships directly from the genetic material.
Nowadays, sequence analysis of ribosomal RNA
(rRNA), which was developed by Carl Woese, is
one of the methods by which phylogenetic trees
can be generated [17].
Probably the best-known gene in the world is
the 16S rRNA (S, Svedberg unit for characteriz-
ing the behavior of sedimentation) of prokary-
otic organisms. This gene is composed of around
1500 base pairs and in the course of evolution
was only subjected to relatively minor changes
through mutations. The reason is that ribosomes
Biotechnology 7
are the sites of protein biosynthesis and the mo-
lecular mechanism taking place is extremely
sensitive to radical mutations. Thus, there are
areas within 16S rRNA that are extremely well
preserved, but also some where base exchanges
have been more frequent. An evolutionary gap
between two organisms can be determined from
the relative similarity of different genes for the
16S rRNA of various prokaryotic organisms;
this can be used as a measure of the degree of
relationship [18].
Analogous to the investigations on prokary-
otic organisms, the characterization of the de-
gree of relationships of eukaryotic organisms is
derived from their 18S rRNA, a molecule with
approximately 2300 bases.
Besides comparing the base sequences of
rRNA, the amino acid sequences of universal
proteins may also be drawn on to establish rela-
tionships. This does not always involve the same
branchings of the phylogenetic tree as with the
comparison of rRNAs. This explains why tax-
onomy, which owes its motivation primarily to
the potential inherent in the new molecular biol-
ogy tools, is currently a highly dynamic area of
science.
Today, all living organisms on earth are clas-
sified into three domains: archaea, bacteria, and
eukaryota. The following representation of the
manifold life forms of microorganisms adheres
to this phylogenetic classification.
2.2.2. Bacteria
Bacterial cells are small, generally less than 2
µm in diameter and 2–5 µm long (there are only
a few giants of 5 × 20 µm dimensions). The cells
do not contain a membrane-enveloped nucleus;
their DNA is rather a circularly closed double
strand lying within the cytoplasm. The bacte-
rial DNA contains the total genetic information
of the cell. In many bacteria there are additional
DNA circles, the plasmids; however, they are not
required for growth under ordinary culture con-
ditions. The ribosomes are small (sedimentation
constant in the ultracentrifuge: 70 S), and their
function is sensitive to various antibiotics that
do not act upon the cytoplasmic ribosomes of
eukaryotic organisms. In contrast to eukaryotic
cells, bacterial cells do not contain organelles,
8 Biotechnology
but often polyphosphates, poly(β-hydroxybu-
tyric acid), or glycogen as storage substances
(Fig. 1). Most bacteria are surrounded by a cell
envelope that contains peptidoglycan (murein).
Many bacteria are motile by either flagellar or
gliding movements.
Figure 1. Longitudinal section through a bacterial cell
ca) capsule; cm) cytoplasmic membrane; cp) cytoplasm;
cw) cell wall; f) flagellum; gly) glycogen; li) lipid droplets;
n) nuclear material or bacterial chromosome; phb) poly(β-
hydroxybutyric acid); pi) pili; pl) plasmid; po) polyphos-
phate; rb) ribosomes; s) sulfur granules
Classification of Bacteria According to the
Phylogenetic System. Originally, the subdivi-
sion of bacteria was based on their morphology
and physiology, in the last few years, however,
their classification has been completely revised
and established on a molecular biology level.
Thus, the taxonomy of bacteria, especially the
higher levels of classification, cannot yet be re-
garded as definitive. Some authorities believe
that the degree of variance between different
bacterial groups is sufficient to give them each
“kingdom status” of their own. Thus, in some
publications reference is made to 13 kingdoms
of bacteria, in others to “phyla” instead of king-
doms.
The classification presented is derived from
Bergey’s Manual of Determinative Bacteriology
[9], which the bacteria are subdivided into the
following phyla:
– Proteobacteria
– Gram-positive bacteria
– Cyanobacteria
– Chlamydia
– Plantomyces
– Bacteroids/Flavobacteria
– Green sulfur bacteria
– Spirochetes
– Deinococci
– Green non-sulfur bacteria
– Hyperthermophiles (3 kingdoms)
Bearing in mind all the postulated, noncul-
tivable species, the actual number of bacterial
phyla (or kingdoms) is probably far higher. Esti-
mates assume that there will ultimately be about
50 phyla (or kingdoms) of bacteria. The cur-
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
proteobacteria is the largest and physiologi-
cally most varied of all bacteria. Representa-
tives of this phylum account for the majority of
known Gram-negative bacteria of medical- and
application-oriented interest [20].
Even the possibly best-known bacterium, Es-
cherichia coli, belongs to this phylum. This ex-
ample is used to demonstrate the taxonomic clas-
sification of an organism:
Domain: Bacteria
Phylum: Proteobacteria
Class: Gamma protobacteria
Order: Enterobacteriales
Family: Enterobacteriaceae
Genera: Escherichia
Species: Escherichia coli
The binomial nomenclature is used through-
out biology. Each organism has a genus name
and a species name and is thus specified unam-
biguously.
2.2.3. Archaea
Several properties distinguish archaea from bac-
teria and eukaryota. Archaea are single-cell or-
ganisms mainly with a closed DNA molecule
lying within the cytoplasm. They are of the
same size as bacteria. Archaea have neither a
cytoskeleton nor cell organelles, they are dis-
tinguished from bacteria by their lack of pepti-
doglycan and the different structure of their ri-
bosomes. With over 200 species they are often
found where extreme conditions prevail.
Archaea have cell walls of pseudopeptido-
glycan and single-layer cell membranes formed
from ether lipids with covalent bonded chains.
Many species of archaea have adapted to their

extreme surroundings, hence some species pre-
fer temperatures of over 80◦ C (thermophile),
others live in salt waters (halophile) or in
very acidic or alkaline environments (aci-
dophile/alkaliphile) [21].
Enzymes from such extremophile organisms,
which are not only found among archaea, are
particularly interesting for industrial applica-
tions; indeed they seem predestined for use in
industrial processes with high temperatures and
salt concentrations [22]. In the area of amylases
thermotolerant enzymes are already applied for
starch saccharification. Moreover the enzymes
used in detergents (proteases, lipases, amylases,
cellulases) are active and fulfill their functions
at temperatures of 60◦ C and above [23].
The phylogenetic system of the archaea that
is valid at present subdivides the domains into
the following phyla [19]:
– Euryarchaeota
– Crenarchaeota
– Korarchaeota
– (Nanoarchaeota is proposed as an additional
phylum)
– Archaeota
The Euryarchaeota are a diverse group.
This phylum comprises not only methanogenic
species, which are strictly anaerobic organisms,
but also strictly aerobic organisms. Furthermore
a large group of hitherto noncultivable organ-
isms belong to this phylum and similarly to that
of the Crenarchaeota, which include both hy-
perthermophilic representatives and mesophilic
marine species. It is only very recently that re-
presentatives of the Korarchaeota phylum have
been cultivated successfully.
2.2.4. Morphological and Physiological
Properties for the Identification of
Prokaryotic Species
The properties of prokaryotic organisms as pre-
sented here permit the identification of species
by conventional methods if molecular biology
techniques are not available or cannot be ap-
plied. The shapes of most species are similar, and
there are only a few morphological characteris-
tics suitable for differentiation. In contrast, the
diversity of metabolic features is tremendous.
Prokaryotic organisms are extremely versatile.
Biotechnology 9
Therefore, a great number of physiological and
biochemical properties have to be determined to
describe and identify a species unambiguously.
Properties and description of prokaryotic or-
ganisms:
1) Shape.
The majority of prokaryotic organisms are
spheres, rods, or helices; these organisms are
called cocci, rods, and spirilla, respectively
(Fig. 2). Some organisms are encapsulated
by proteins or polysaccharides, and some are
combined to form packets, filaments, or con-
sortia.
2) Flagellation.
The kind of flagellation, length, type of undu-
lation, and mode of attachment of flagellae
are important characteristics of prokaryotic
organisms (Fig. 3).
3) Gram Stain.
The Gram stain, originally devised by CHR.
GRAM (1884) to make bacteria visible in an-
imal tissues, is a reliable and fundamental
cellular characteristic by which the bacteria
are divided into two parts: Gram-positive and
Gram-negative bacteria. It is based on the re-
tention or discoloration of bacteria by ethanol
after a staining procedure involving an ani-
line dye and iodine. In Gram-positive bacte-
ria, the dye is retained by a cell wall consisting
of a multilayered network of peptidoglycan;
in Gram-negative bacteria, the dye is readily
washed out because the cell wall consists of
only two peptidoglycan layers and a highly
permeable outer membrane envelope. The ba-
sic structural differences are easily visible in
ultrathin sections (Fig. 4).
4) Endospores.
Endospores are highly refractile, thermoresis-
tant stages in the life cycle of two groups
of Gram-positive bacteria belonging to the
genera Bacillus and Clostridium. Endospores
are easily visible and tolerate pasteurization
(treatment by moist heat for 10 min at 80 ◦ C).
Heating in an autoclave for 20 min at 120 ◦ C
is required to kill the spores.
5) Need for Oxygen.
Many prokaryotic organisms grow only in the
presence of atmospheric oxygen. They are
strictly aerobic organisms. In contrast, strictly
anaerobic organisms can only grow in the ab-
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-

11) Composition of Cellular Macromolecules.
The description of bacteria includes the Gram
type of the cell wall, the type of peptidoglycan
structure, the fraction of cytosine + guanine
in the DNA, the similarity index of the 16 S
rRNA, and data on DNA–DNA hybridization.
12) Antibiotic Resistance.
The characterization of an organism may be
supplemented by its pattern of antibiotic re-
sistance.
Figure 3. Flagellation of bacteria, flagellar movement
Figure 4. Gram-positive and Gram-negative bacterial cell
walls
2.2.5. Eukaryotic Microorganisms
2.2.5.1. Definition
The cells of most eukaryotic organisms are usu-
ally much larger than prokaryotic cells and have
diameters of 10 µm or more [24]. They contain
several structural components, compartments,
and organelles. Figure 5 shows a plant cell as
an example of a eukaryotic cell. The nucleus,
consisting of a set of chromosomes that divide
by mitosis, is surrounded by a double membrane.
Biotechnology 11
The DNA is associated with histone protein. Mi-
tochondria (for respiratory energy conversion)
and chloroplasts (in plants for photosynthetic
energy conversion) contain a prokaryotic type
of DNA and ribosomes. Cytoplasmic ribosomes
are large (80 S). Endoplasmic membranes and
various kinds of membrane vesicles compart-
mentalize the cell; they are involved in the in-
gestion of nutrients (endocytosis) and the pro-
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 5. Longitudinal section of a eukaryotic plant cell
cw) Cell wall; chl) Chloroplast; cm) Cytoplasmic mem-
brane; cp) Cytoplasm; di) Dictyosome; er) Endoplasmic
reticulum; ex) Secretion vesicle for exocytosis; li) Lipid
droplet; mi) Mitochondrion; mt) Microtubule; n) Nucleus
or karyon; rb) Ribosomes; v) Vacuole
2.2.5.2. Fungi
Fungi are aerobic eukaryotic organisms; they
form a separate kingdom within the domain
of the eukaryota [25, 26]. There are unicellu-
lar fungi, but the majority form filaments (hy-
phae), (usually 5–10 µm in diameter) and grow
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-

Ascomycetes. The ascomycetes are named
for their formation of spores within a sac-like
zygote, the ascus. With the exception of a few
unicellular forms they grow as branched mycelia
with open cross walls; they are coenocytic. Many
ascomycetes grow on excrements of animals,
i.e., they are coprophilic, whereas others are par-
asites on plants or insects. Asexual reproduction
occurs by various kinds of conidiospores.
The order Saccharomycetales within the As-
comycetes comprises all yeasts. Typical yeasts,
such as Saccharomyces cerevisiae, are unicellu-
lar. They grow either as diplonts (with diploid
nucleus) or as haplonts (with haploid nucleus),
such as Schizosaccharomyces pombe. All yeasts
are able to grow on sugar substrates, and a few
can even grow on alkanes (Saccharomycopsis,
Candida lipolytica) or on methanol (Candida
boidinii, Hansenula anomala). Yeasts are organ-
isms often used in industrial microbiology.
Zygomycetes. The zygomycetes are lower
fungi and comprise unicellular and mycelial
fungi; they have diverse nutritional habits.
They grow in water, soil, or moist habitats.
There are saprophytic and parasitic species.
The most important group biotechnologically
is that of the Zygomycetales, which include
Mucor mucedo, Rhizopus nigricans, and Phy-
comyces blakesleeanus. Several species are used
for the production of vitamins, carotenoids, or-
ganic acids, and enzymes.
Myxomycetes. The myxomycetes or slime
moulds grow on decaying plant tissue, form
multinuclear plasmodia, and produce fruiting
bodies of yellow, red, or brown color. There is
not yet a consensus on the exact evolutionary
affinities of myxomycetes, but these organisms
constitute a well-defined, homogeneous group
of approximately 900 species.
Deuteromycetes. If a fungus is incapable
of sexual reproduction, it cannot be classi-
fied with either the ascomycetes or basid-
iomycetes, hence a third group of higher fungi
was created. There are many deuteromycetes of
biotechnological importance: Aspergillus, Peni-
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
Biotechnology 13
deuteromycetes will possibly be abandoned in
the future [27, 28].
3. Metabolism
The decisive factor for the development of a suc-
cessful biotechnological production is an effi-
cient biocatalyst. If this biocatalyst is a microbial
production strain, knowledge of its metabolic ca-
pabilities is very important. Such knowledge can
lead to an increase in product concentration and
product variety, because one catalyst can often
be used to produce different products [29].
Metabolism is generally concerned with all
the events occurring within a cell that not only
keep the cell alive but are also manifested in
growth [30]. All synthetic reactions leading to
growth are energy-consuming (endergonic) and
are commonly referred to as anabolic reactions.
In order to carry out these reactions, each indi-
vidual synthesis must be coupled with energy-
producing reactions of the catabolic sequence.
Metabolism therefore consists of an intricate
coupling between anabolism and catabolism
(Fig. 7), whereby energy transactions and trans-
fer mechanisms are based upon the basic laws
of thermodynamics [31, 32].
Furthermore, in order to obtain a functional
biosynthesis, the microbial cell must also pro-
duce a reducing agent, because biosynthesis in-
volves the reduction of small molecules of high
oxidation state to larger molecules of lower ox-
idation states. Both the energy and the reduc-
ing agent can be obtained from any of a number
of diverse reactions depending upon the growth
conditions and the genetic system available [33,
34] within the metabolism. Energy is gener-
ally dependent on adenosine triphosphate (ATP)
and the reductant on nicotinamide dinucleotide
NADH or NADPH.
The cell has a very sophisticated regulatory
control system that avoids oversynthesis of any
chemical compound, thus securing its integrity.
These regulatory control systems start at the
cell membrane (substrate uptake) [35] and are
present throughout the pathways of catabolism
and anabolism.
14 Biotechnology
Figure 7. Anabolic and catabolic reactions in microbial metabolism
3.1. Microbial Systems Biology
In the future, systems biology will enable the
metabolism of some microorganisms to be mod-
eled and mapped in silico to such an ex-
tent that physiological performance and effects
caused by environmental changes will be pre-
dictable. The essential prerequisites include suf-
ficient knowledge of metabolic material flow
and network analysis, reaction kinetic analy-
sis of biochemical networks (enzyme kinetics,
global metabolism regulation), global analy-
sis of gene regulation networks (signal trans-
duction), analysis of populationwide systems
(quorum-sensing networks), and the abstraction
of fundamental regulation patterns. The first
such model that integrates signal transduction,
gene expression and metabolism has already
been introduced for the response of yeast to os-
motic shock [36].
There is no doubt that such models signify
great progress for microbiology and biotechnol-
ogy: quantitative simulations and targeted inter-
ventions in the metabolic network ideally open
up the way to highly specialized, optimized pro-
duction organisms. In order to optimize specific
types of metabolic performance or to acceler-
ate desired biosyntheses, industrial biotechnol-
ogy is already relying to a great extent on the
metabolic engineering of microorganisms. Here,
mention should be made of the synthesis of ami-
no acids [37, 38], optimized riboflavin produc-
tion in B. subtilis [39], and, in the case of ter-
penoids, yield increases by engineering meval-
onate biosynthesis in E. coli [40]. It is likely that
knowledge from systems biology will trigger
tremendous progress in this application-oriented
field. Moreover, strategies based on systems bi-
ology will also be important for biotechnological
process development where an understanding of
the complex regulatory networks of production
organisms will be indispensable for process op-
timization [41].
3.2. Energy Production
Redox reactions are among the most important
chemical reactions in living organisms. These
oxidation – reduction reactions are of special im-
portance for energy production by living organ-
isms:
∆G0 =n F ∆E 0
where ∆G 0 is the standard free energy of the
reaction, n is the number of electrons (or hydro-
gen atoms) involved, F is the Faraday constant,
and ∆E 0 is the potential difference between the
two redox systems. The simplest way to think
about these oxidation – reduction reactions is in
terms of electron donors and acceptors. Thus,
the substrate oxidized has a specific redox po-
tential that is at the lower end of the electrode

potential scale, whereas the final electron accep-
tor has a potential towards the more positive end
of the scale. The difference between the two po-
tentials corresponds to the energy obtainable in
a chemical reaction between them. If the poten-
tials of the substrate (electron donor) and of the
final electron acceptor are known, one can easily
determine the energy production in a microbial
system (Fig. 8) [42, 43].
Figure 8. Redox potentials of various energy-producing mi-
crobial reactions
Because the potential difference between
electron donor and acceptor can be significant,
the energy released could produce so much heat
that the cell would be damaged. In order to avoid
such cell damage, the cell has an energy-release-
controlling cascade system, which makes cer-
tain that energy is released only stepwise. This
cascade system is referred to as the respiratory
chain or the electron transport chain. It is located
in the cellular membrane and is responsible for
the production of energy by electron transfer in-
volving an enzyme called ATPase (which forms
or hydrolyzes ATP) [35, 44]. The ATPase also
plays an important role for the transportation of
substances across the membrane into the cell.
According to the chemiosmotic hypothesis
for ATP production, the electron transfer chains
are coupled to ATP synthesis by a proton elec-
Biotechnology 15
trochemical gradient (∆µ+ H ) across the energy-
transducing membrane. Proton electrochemical
potential is a thermodynamic measure of the
extent to which the proton gradient across the
membrane deviates from equilibrium. This hy-
pothesis is based on two separate proton pumps,
whereby the ∆µ+ H is generated by electron trans-
fer and is used to drive the second pump, in-
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
force are required. Driving forces can be concen-
tration gradients, electrical potentials, metabolic
energy, or a combination of these. Four pro-
cesses are known [45, 46]:
1) In simple diffusion, solutes move passively
across the cell membrane, depending on the
concentration gradient and thermal motion of
the molecules (e.g., water, gases, low molec-
ular hydrophilic compounds, organic acids in
the protonated form).
2) In facilitated diffusion, solutes require a car-
rier for their transport in addition to the con-
centration gradient and thermal motion of the
molecules. In both simple and facilitated dif-
fusion, no metabolic energy or proton motive
force is required. The energy for the transport
stems from existing concentration gradients.
3) The term active transport is applied to a tight
coupling of transport to metabolism in the ion
pumps, which are central to chemiosmotic
energy transduction. There are at least two
16 Biotechnology
distinct classes of active transport systems:
the membrane-bound and the binding protein
transport systems.
4) The group-translocation transport system
comprises processes in which the passage
of the substrate across the membrane occurs
simultaneously with, and as a consequence
of, chemical transformation of the sub-
strate (e.g., phosphoenolpyruvate–glucose–
phosphotransferase system with most anaero-
bic and facultatively anaerobic bacteria). Ac-
tive transport and group-translocation trans-
port are often referred to as “primary trans-
port”, simple and facilitated diffusion as “sec-
ondary transport”. The carrier systems in-
volved can be of the “uniport”, “symport”, or
“antiport” type.
Many bacteria pump natrium ions out of the
cell by taking in two protons per natrium ion in
an antiport system. Sulfate ions, on the other
hand, are only taken in by some transport
systems if protons are simultaneously “sym-
ported”.
3.4. Catabolism
3.4.1. Photosynthesis
Photosynthesis creates living matter out of inor-
ganic material, replenishes the reservoir of oxy-
gen in the atmosphere in the cases of algae and
plants, stores the energy of sunlight to support
the life activities of organisms, and removes car-
bon dioxide:
CO2 +H2 A+light→ (CH2 O) +2 A+chemical energy
If A is taken to be oxygen the process is plant
(or algae or cyanobacteria) photosynthesis; if it
represents a sulfur atom the process is bacterial
photosynthesis [43].
The process of photosynthetic energy conver-
sion is initiated when photons are absorbed by
specific molecules, such as chlorophyll. This ab-
sorption leads to an ejection of electrons, which
are accepted by the compound having the low-
est redox potential in animate nature, namely
ferredoxin. The electrons then move through the
electron transport system, and the energy is fi-
nally converted to ATP. Because the electrons
return to the reaction center (Fig. 9), this sys-
tem is called cyclic photophosphorylation. The
reducing agent is produced separately. In plants
and algae, a second photosystem exists that splits
water into H+ and oxygen, whereby the hydro-
gen ions reduce NADP+ and oxygen is set free.
In bacteria, however, such a second photosystem
does not exist, and inorganic compounds such as
thiosulfate are oxidized, with the electrons run-
ning through the electron transport system to the
reaction center and the proton via ferredoxin to
NAD+ (Fig. 9). This is the reason why plants and
algae possess mainly cyclic and bacteria non-
cyclic photophosphorylation.
3.4.2. Chemosynthesis
In chemosynthesis, all energy and reducing
agents are obtained by catalytic reactions of or-
ganic or inorganic compounds. In the former
case, one refers to chemoorganotrophy and in
the latter to chemolithotrophy. In addition, three
different energy modes, which depend upon the
electron donors and acceptors, are distinguished:
aerobic respiration, anaerobic respiration, and
fermentation [47].
Organic substances are used as an energy
source by the vast majority of microorganisms
that live in natural environments (Fig. 10). Na-
ture provides them with an abundance of large
organic polymers. A polymeric substance con-
sists of series of monomeric units linked to-
gether. These monomeric units can be of the
same kind (e.g., cellulose, starch) or of different
kinds (e.g., pectin, lignin, sucrose). Within the
polymeric structure, the monomeric units can
be linked in a straight chain with identical link-
ages or different linkages or in branched chains;
moreover, they can be linked in a specific se-
quence or at random. The structure of each of the
polymer substrates for microorganisms is very
important, because the enzymes produced by the
microorganisms are specific not only to the types
of monomers, but also in most cases to the way
in which the monomers are linked.
Since microorganisms can only take up
monomers, all enzymes involved in the break-
down of polymeric substances must be extra-
cellular. These enzymes are either membrane-
bound or released into the medium. In gen-
eral terms, Gram-negative bacteria accommo-
date these enzymes in the periplasmic space bet-
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
Gram-positive bacteria and fungi have a ten-
dency to release these enzymes into the medium.
This is one of the reasons why extracellular en-
zyme studies are carried out predominantly with
Gram-positive microorganisms, as well as yeasts
and other fungi.
3.4.3. Carbohydrate Metabolism
Most renewable resources are carbohydrates.
The principal carbohydrate that serves as a car-
bon and energy source for microorganisms is
glucose. However, not all microorganisms fol-
low the same route of glucose utilization. There
are at least four major pathways of glucose
metabolism, of which three lead directly to pyru-
vate:
1) Embden-Meyerhoff (EM) pathway, often re-
ferred to as the glycolytic or fructose bispho-
sphate pathway
2) Hexosemonophosphate (HMP) pathway, of-
ten referred to as the pentose shunt or pentose
phosphate pathway
3) Entner-Doudoroff (ED) pathway
18 Biotechnology
4) Phosphoketolase (PK) pathway, almost en-
tirely specific for heterofermentative lactic
acid bacteria
All four pathways of glucose metabolism
have a great number of intermediates and en-
zymes in common. The details of these pathways
can be found in many textbooks [32, 34, 48].
The EM pathway, for example, provides the
greatest amount of ATP, but it does not produce
ribose-5-phosphate, the important precursor for
RNA and DNA biosynthesis; nor does it produce
erythrose-4-phosphate, which is important for
amino acid biosynthesis. Microorganisms that
are capable of using only the EM pathway for
glucose utilization are therefore not able to grow
on simple media with glucose as the sole carbon
source. They require growth factors or organic
compounds (e.g., yeast extract) for growth and
are referred to as fastidious microorganisms.
In contrast, the HMP pathway produces all
the precursors necessary for both RNA and DNA
as well as for aromatic amino acid biosynthe-
sis, but it produces only half the amount of ATP
energy. This pathway does not produce pyru-
vate directly. The microorganisms must there-
fore possess part of the enzymes of the EM path-
way. It is therefore not surprising that both path-
ways, EM and HMP, are common combinations,
particularly in facultative anaerobic microorgan-
isms.
The ED pathway is unique, however, and
has so far only been found in bacteria. Al-
though this pathway is linked partly to the HMP
pathway in the reverse direction for precur-
sor formation, pyruvate is formed directly be-
cause of the aldolase cleavage of 3-ketodeoxy-
6-phosphogluconate. The ED pathway can exist
on its own and is used by the majority of strictly
aerobic microorganisms. The net result is simi-
lar to the HMP pathway, although one mole of
ATP can be formed only if the carbon atoms go
into pyruvate instead of precursor.
3.4.4. Aerobic Processes
All aerobic microorganisms use the tricarboxyl-
ic acid (TCA) cycle as a main metabolic path-
way. In this cycle, appropriate groups of en-
zymes catalyze a series of consecutive transfor-
mations, including oxidations, which finally re-
sult in the complete oxidation of pyruvate to car-
bon dioxide. The electrons and hydrogen atoms
removed from the individual organic compounds
are accepted by oxygen and form the other two
end products, ATP and water. The tricarboxyl-
ic acid cycle, however, is not only an energy-
producing cycle, but it is also responsible for
the production of precursors for amino acid and
antibiotic biosyntheses [49, 50]: 2-ketoglutarate
and oxalacetate ions. The tricarboxylic acid cy-
cle serves, therefore, as the central pathway for
the production of energy as well as for anabolic
precursors.
Under ideal growth conditions, the interme-
diates of the tricarboxylic acid cycle would be
continuously withdrawn for anabolic reactions.
Then, the formation of oxalacetate at the end of
the cycle became vulnerable. Because oxalac-
etate is required not only as a precursor for an-
abolic amino acid biosynthesis, but also for the
condensation with acetyl-CoA to keep the tricar-
boxylic acid cycle alive, the organism must have
some safety device to ensure that oxalacetate
is always available; otherwise the tricarboxyl-
ic acid cycle would be interrupted. The organ-
ism possesses a very fine control system for en-
ergy production and biosynthetic requirements
in form of replenishment sequence reactions. Al-
together, the microbial world has five enzymes
at its disposal for such sequences, of which
phosphoenolpyruvate carboxylase is probably
the dominant: it catalyzes the formation of ox-
alacetate from phosphoenolpyruvate and carbon
dioxide. Phosphoenolpyruvate, in turn, is the in-
termediate from which pyruvate is formed in the
EM pathway.
This double function of the tricarboxylic acid
cycle as an energy-generating cycle as well as a
biosynthetic precursor-supplying cycle is very
often referred to as amphibolic. This dual func-
tion, of course, must be controlled. There ex-
ists one enzyme in the tricarboxylic acid cycle,
isocitrate dehydrogenase (it catalyzes the reac-
tion: isocitrate → oxalosuccinate), which per-
forms this regulatory control. In the case of an
overproduction of ATP this enzyme is inhibited
by ATP, preventing any further oxidation and
ATP synthesis, until the biosynthetic steps lead-
ing to RNA, DNA, aromatic amino acids, and
fatty acids have caught up and utilized the ex-
cess ATP [33].
The general scheme in Figure 11 outlines the
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-
sary for cellular biosynthesis. Aerobic processes
in biotechnology are used to produce various
products, for example, antibiotics, amino acids,
and organic acids [29, 51, 52].
3.4.5. Fats and Fatty Acid Metabolism
Fats are another major group of naturally occur-
ring compounds (see Fig. 10). The majority of
fats are triglycerides, that is, fatty acids (R1 , R2 ,
R3 ) esterified with glycerol:
These fats have to be converted into the
monomeric components, the fatty acids and
glycerol. This hydrolysis is carried out by en-
zymes called lipases. The glycerol component
is converted to dihydroxyacetone phosphate,
which participates in the EM pathway. The fatty
acids, which vary in their carbon chain length,
have to be activated by the formation of their
respective coenzyme A (CoA) esters; this reac-
tion step requires energy. These fatty-acid-CoA
esters subsequently undergo cyclic oxidation,
eliminating an acetyl unit at each turn. This type
of oxidation is referred to as β-oxidation. The
acetyl-CoA formed after each cycle can then en-
ter the tricarboxylic acid cycle for the formation
of energy and biosynthetic precursors.
However, this type of metabolism leading
to acetyl-CoA does not produce the important
ribose-5-phosphate and erythrose-4-phosphate.
In order to obtain these precursors, all organisms
whose metabolism leads directly into the tricar-
boxylic acid cycle via acetyl-CoA and not via
pyruvate have to build up the carbon chain men-
tioned above and have introduced two pathways
for this biosynthetic purpose:
1) The glyoxylate cycle. As mentioned earlier,
overproduction of ATP inhibits the enzyme
isocitrate dehydrogenase. The organism is
then capable of inducing two new enzymes,
isocitrate lyase and malate synthase, to cir-
cumvent the carbon dioxide-producing steps
of the tricarboxylic acid cycle. This new short-
cut TCA cycle leads to succinate and via gly-
oxylate to malate, and oxalacetate is formed
despite the inhibition of isocitrate dehydro-
genase (see Fig. 10).
2) The gluconeogenic pathways. From the ox-
alacetate formed, the organism is using one of
the earlier mentioned replenishment sequence
reactions to produce phosphoenol-pyruvate
and reverses the EM pathway to glucose-6-
phosphate; from there ribose-5-phosphate and
erythrose-4-phosphate can be formed via the
forward HMP pathway.
A great number of enzymes are com-
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].
3.4.6. Hydrocarbon Metabolism
The metabolism of hydrocarbons has been ex-
amined in detail in the framework of environ-
mental biotechnology in connection with clean-
up of contaminated sites. Aliphatic hydrocar-
bons are good substrates for a large number
of microorganisms. The straight-chain alkanes
are more readily attacked than substituted or
branched-chain alkanes. The attack occurs at ei-
ther one end (monoterminal) or both ends (diter-
minal). The alkane is converted via the primary
alcohol into the corresponding fatty acid and
then via β-oxidation into acetyl-CoA as out-
lined previously. Methyl group oxidation incor-
porates one atom of oxygen; this reaction is cat-
alyzed by a mixed oxidase system consisting of
three proteins: rubredoxin, NADH-rubredoxin
reductase, and -hydroxylase. For the oxidation
of methylene groups, rubredoxin is replaced by
cytochrome P450 and -hydroxylase by methy-
lene hydroxylase [53, 54].
Aromatic hydrocarbon utilization follows a
uniform biochemical concept. The great major-
ity of aromatic hydrocarbons, irrespective of the
number of benzene rings, converge in their ox-
idative metabolism to three major intermediates:
catechol, 3,4-dihydroxybenzoic acid (protocat-
echuic acid), and 2,5-dihydroxybenzoic acid
(gentisic acid):

The benzene nucleus in these intermediates
is cleaved either between adjacent hydroxyl
groups (ortho cleavage) or between a hydroxyl
and a carboxyl group (meta cleavage). In both
cases, the active incorporation of an oxygen
molecule is required. Ortho cleavage, via the 2-
ketoadipate, and meta cleavage, via the keto acid
pathway, lead into the tricarboxylic acid cycle at
the level of acetyl-CoA, succinate, or malate and
fumarate. As in fatty acid metabolism, the organ-
isms use the tricarboxylic acid cycle for energy
production and require the glyoxylate cycle to-
gether with gluconeogenesis to obtain pentose
for RNA and DNA precursors.
3.4.7. Amino Acid Metabolism
The aerobic utilization of amino acids follows
a pattern that is very similar to the metabolism
of other carbon sources [29, 49]. The only dif-
ference is the -amino group. In most cases, the
amino acid is first converted into the correspond-
ing keto acid by either cytochrome-linked oxi-
dases, transaminases, or NAD(P)-linked dehy-
drogenases. The final product of the metabolism
always leads to the intermediates of the tricar-
boxylic acid cycle. This is one of the reasons
why -amino acids are able to function as the
sole source of carbon, nitrogen, and energy for
many microorganisms. As in fatty acid and hy-
drocarbon metabolism, microorganisms grow-
ing on amino acids must possess the glyoxylate
cycle and the gluconeogenesis pathway.
3.4.8. Anaerobic Metabolic Processes
The breakdown, or catabolism, of organic com-
pounds in the absence of oxygen is generally re-
ferred to as fermentation. The microorganisms
that carry out fermentations are either faculta-
tive or obligate anaerobes. The most character-
istic difference between respiratory and fermen-
tative metabolism is in ATP energy production.
Because no electron transport occurs, redox re-
actions of organic compounds play a major role.
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-
ucts are formed, mainly primary alcohols, e.g.,
ethanol or butanol.
Fermentations are normally classified ac-
cording to their principal fermentation products.
Figure 12 summarizes the end products from
carbohydrates.
The distinction between aerobic and anaer-
obic carbohydrate metabolism is made at the
pyruvate level, because in anaerobic fermenta-
tion the organism has to substitute for the tricar-
boxylic acid cycle. In general the organisms are
not able to produce the intermediates that serve
as precursors for amino acid and protein biosyn-
thesis. Therefore, fermentation processes cannot
be carried out on simple defined media, but al-
ways require complex media if the organism is
to grow and maintain its metabolism. Most of
these fermentation processes are rather complex
and details may be obtained from the relevant
literature [32, 34, 55].
3.4.9. Single-Carbon-Compound
Metabolism
A few isolated genera of microorganisms are
capable of oxidizing single-carbon compounds.
Microorganisms are called methylotrophs if they
have the ability to derive both carbon and en-
ergy from the metabolism of methanol, methan-
otrophs if they can do the same with methane
[53, 56 – 58]. Methane and methanol were the
most common substrates used in the production
of single-cell protein. The oxidation of methane
leads to methanol and from there to carbon diox-
ide.
The oxidation of methane or methanol to car-
bon dioxide, however, does not provide the or-
ganism with any precursors for biosynthesis.
These organisms therefore possess an unusual
carbon assimilatory pathway. Depending upon
the enzymic assemblage, formaldehyde is incor-
porated into ribulose-5-phosphate or into the ser-
ine pathway in order to build up the carbon chain
for the biosynthesis of RNA, DNA, and all the
amino acids required for protein biosynthesis.
The absence of a tricarboxylic acid cycle, the
presence of anaplerotic sequence reactions, and
22 Biotechnology
Figure 12. Fermentation end products of carbohydrate metabolism
the glyoxylate cycle demonstrates the biosyn-
thetic capability of these pathways.
3.4.10. Inorganic Metabolism
Some microorganisms are capable of using in-
organic compounds as an energy source in aer-
obic metabolism or as a final electron accep-
tor in anaerobic metabolism (anaerobic respira-
tion). Both of these are combined in nature in
the sulfur cycle and the nitrogen cycle, [32, 34].
Sulfur Cycle. Under anaerobic conditions,
sulfate or thiosulfate can be reduced to sulfide by
sulfate-reducing bacteria. Such processes occur
mainly in water-logged soils or stationary estuar-
ies rich in organic matter, and can cause serious
corrosion of iron and steel pipes in the soil. Hy-
drogen sulfide is an extremely toxic product. If
the soil or estuaries are supplied with oxygen,
hydrogen sulfide can be reoxidized to sulfate
by aerobic sulfur-oxidizing microorganisms, for
example, thiobacilli. The reoxidation of sulfur or
sulfide to sulfate is a microbial process of consid-
erable biotechnological significance. It not only
acidifies the soils and thus makes plant growth
possible, but the sulfuric acid formed can also
be used for ore leaching processes. Sulfuric acid
can form soluble sulfates with a wide range of
metals and by leaching old mining dumps it
helps concentrate these for further utilization of
the ore. Such ore leaching processes have led to
a vastly improved mining industry and increased
the economy of ore mining.
Nitrogen Cycle. Nitrogen is the most abun-
dant gas in the atmosphere. Nitrogen-fixing
organisms are capable of converting nitro-
gen into nitrogen compounds that are accept-
able to plants. Nitrogen-fixing microorganisms
can be divided into free-living (cyanobacteria,
Clostridium, Azotobacter) and symbiotic (Rhi-
zobium). The last group in particular may be-
come of great biotechnological importance, be-
cause they live in association with legume plants
and provide these with the necessary nitrogen
source even in nitrogen-starved soils, thus elim-
inating the need for large amounts of nitrogen
fertilizers. The nitrogen-fixing organisms con-
vert nitrogen to ammonia, which then can be ox-
idized by chemolithotrophs (Nitrosomonas and
Nitrobacter) via nitrite to nitrate. This conver-
sion of nitrogen to nitrate is referred to as ni-
trification. The reverse reaction, called denitri-
fication, is an anaerobic process in which the
nitrate or nitrite serves as electron acceptor. The
product of denitrification is nitrogen gas. Nitrifi-
cation and denitrification are important process
steps in biological wastewater cleaning.
The chemolithotrophs, however, face an en-
ergy problem. The redox potential of the inor-
ganic compounds used as electron donors is well

above that of the NAD/NADH couple that is re-
quired as reducing agent for biosynthesis. Since
the electrons can only enter the redox system
at the cytochrome level, electron transport must
be reversed to obtain NADH. In photosynthesis,
this reversal can be carried out by the reaction
center of the photosynthetic apparatus, but in
chemosynthesis the reversal has to occur with
energy input from the oxidation of the inorganic
source. It is therefore not surprising that such
organisms grow much better in the presence of
organic material, which they are able to assimi-
late directly, thus obviating the reductive biosyn-
thesis and consequently the need for reductant
formation.
3.5. Biosynthesis
The microbial cell consists of five major types
of macromolecules: proteins, polysaccharides,
lipids, RNA, and DNA [32, 34]. These macro-
molecules are built up from relatively few
monomers, which the cell has to provide or
to synthesize: amino acids, sugar phosphates,
fatty acids, ribonucleotides, and deoxyribonu-
cleotides.
Microorganisms are divided into two groups:
autotrophs, which are able to produce all
their cellular requirements from carbon diox-
ide as the sole carbon source, and heterotrophs
– the majority of microorganisms – which pro-
duce the cellular material from organic com-
pounds. Photo- and chemolithotrophs use ATP
and the reducing power produced by photo-
synthesis and/or oxidation of inorganic sub-
strates to reduce carbon dioxide to cellular ma-
terial. The pathway for carbon dioxide reduc-
tion is referred to as the Benson-Calvin cy-
cle. The actual carbon dioxide fixation reac-
tion is catalyzed by ribulose-1,5-bisphosphate
carboxylase; the primary product of this re-
action is 3-phosphoglycerate, which leads to
glyceraldehyde-3-phosphate, a member of the
EM pathway:
3 CO2 +9 ATP+6 NADH2
→glyceraldehyde−3−phosphate
→+9 ADP+8 Pi +6 NAD+
From glyceraldehyde-3-phosphate, these organ-
isms are capable of producing the important
Biotechnology 23
pentoses, trioses, and the dicarboxylic acids
required for macromolecular biosynthesis [46,
47].
3.5.1. Amino Acids
About twenty -amino acids are required for the
biosynthesis of the numerous types of proteins
that provide the catalytic capability of the mi-
croorganisms. These amino acids are produced
from the following precursors:
Erythrose-4-phosphate → tyrosine, tryptophan
Phosphoenolpyruvate → phenylalanine
Ribose-5-phosphate → histidine
3-Phosphoglycerate → serine, glycine, cysteine
Pyruvate → alanine, valine, leucine
2-Oxoglutarate → glutamic acid, glutamine,
arginine, proline
Oxalacetate → aspartic acid, asparagine,
methionine, lysine, threonine,
isoleucine
The most important amino acids are glutamic
acid and glutamine, which figure in transami-
nation reactions during biosynthesis. Therefore,
the availability of these two amino acids is vital
for protein biosynthesis. The detailed pathways
can be found in the literature [32, 34, 46].
Theoretically, each of these -amino acids can
be produced commercially by microorganisms,
some are already produced, but some practical
problems remain to be solved for some of them
[49].
3.5.2. Lipids
Most of the fatty acids that occur in lipids contain
16 or 18 carbon atoms and are saturated or unsat-
urated with one or more double bonds. Acetyl-
CoA is the only precursor for the biosynthesis of
all fatty acids. In order to differentiate between
fatty acid catabolism and fatty acid biosynthe-
sis, the organisms employ CoA activation in the
former and an acyl carrier protein (ACP) in the
latter. The C2 units are added to the acetyl-ACP
in the form of malonyl-ACP. Once the required
chain length is reached, ACP is hydrolyzed and
the appropriate fatty acid is formed. Unsaturated
fatty acids are generally formed by dehydration
of a hydroxy fatty acid.
The fatty acids are then esterified with glyc-
erol, which is readily available from dihydroxy-
acetone phosphate, a member of the EM path-
24 Biotechnology
way. Finally, triglycerides are formed. The intro-
duction of a phosphate group gives a phospha-
tidic acid and a whole range of phospholipids.
If the phosphate group in the phosphatidic acid
is replaced by a carbohydrate, glycolipids are
produced [32, 34, 46].
3.5.3. RNA and DNA
Ribonucleotides consist of a purine or pyrim-
idine derivative, ribose, and phosphate groups.
A purine or pyrimidine base attached to a ri-
bose is referred to as a ribonucleoside; if a phos-
phate group is attached to this ribonucleoside
it is called a ribonucleotide. The most impor-
tant pyrimidine derivatives are uracil, cytosine,
and thymine. The most important purine deriva-
tives are adenine and guanine. The biosynthesis
of pyrimidines and purines starts at the ribose-5-
phosphate level, which also represents the ribose
in the nucleotide. The RNA consists of ribonu-
cleotides. Reduction of the ribonucleotides leads
to deoxyribonucleotides, the building blocks for
DNA biosynthesis [44, 46].
3.6. Regulation
The microbial cell possesses a complex of
catabolic and anabolic capabilities with many
interconnected pathways. The cell is required to
maintain balance among the various parts of this
extremely complex network of reactions [32, 34,
44, 46, 48]. The cell therefore has developed
very intricate and refined devices to streamline
its economy.
Regulation can either be manifested in the
enzyme synthesis or in the enzyme activity. In-
duction and repression of enzyme synthesis oc-
curs at the genetic code, whereas feedback in-
hibition simply regulates the activity of the en-
zyme. The importance for biotechnology is to re-
alize that the first type of regulation is concerned
with substrate, catabolite, and end product in-
hibition, whereas the second mainly involves a
transitional end product inhibition.
More than one enzyme usually is re-
quired to channel a substrate into intermediary
metabolism. The substrate can therefore be re-
sponsible for a coordinate or sequential induc-
tion of enzymes. This activity of the cell nor-
mally occurs during the lag phase of growth and
can be reduced to almost zero by preculture con-
ditions.
A much more complicated regulation is
catabolite repression, which is mainly mani-
fested in the so-called diauxie phenomenon. The
latter is biphasic growth in the presence of more
than one carbon source in the medium. In this
reaction the organism takes up only one sub-
strate at a time and the presence of this partic-
ular substrate represses the enzyme system that
is responsible for the metabolism of the other
substrate. This repression occurs at the operon
level and occurs as soon as the concentration of
the first substrate becomes very small.
Enzyme synthesis can also be inhibited by
end products within a short period of time. For
example, if the medium contains any α-amino
acid, the organism would use this amino acid
directly instead of producing it. The added ami-
no acid therefore represses the synthesis of each
enzyme in its synthesis pathway.
In addition to the regulation of enzyme syn-
thesis, the cell must also adjust the activity
of enzymes to its metabolic requirements. If a
monomer is synthesized in larger amounts than
needed for polymer synthesis, it is not necessary
to stop the synthesis of the enzyme completely,
but it suffices to reduce the activity of that partic-
ular enzyme. This fast control action is referred
to as feedback inhibition. The target enzymes
for feedback inhibition are called allosteric en-
zymes.
4. Metabolic Engineering
The term metabolic engineering refers to the
genetic optimization of prokaryotic or eukary-
otic cells that are used in industrial fermentation
processes. In these processes, they are utilized
for the economically production of proteins or
other fine chemicals. Bailey [59] firstly intro-
duced the definition of metabolic engineering
in 1991 as “the improvement of cellular activi-
ties by manipulation of enzymatic, transport, and
regulatory functions of the cell with the use of
recombinant DNA technology.” However, one
of the early examples of successful genetic en-
gineering of a living organism for production
purposes, namely the production of human in-
sulin in E. coli cells, had already been described

quite some time before a name was given to this
new direction of science.
According to Cameron and Tong [60], there
are five categories of metabolic engineering that
are grouped with respect to the objective of the
genetic modification:
1) Improved production of chemicals already
produced by the host organism.
2) Extended substrate range for growth and
product formation.
3) Introduction of new catabolic activities for the
degradation of toxic materials.
4) Production of chemicals that are new to the
host organism.
5) Modification of cell properties, for instance,
to make them more resistant to the production
conditions.
The process of metabolic engineering is most
often described by a circle of elementary steps
that have to be performed once or several times.
This circular process is demonstrated in Figure
13.
Figure 13. Schematic illustration of the elementary pro-
cesses of metabolic engineering
The elementary processes are
– Analysis of the organisms with respect to the
Proteome and/or metabolome
– Design of a genetic modification that im-
proves/allows the synthesis of the compound
of interest
– Production of a modified strain
– Fermentation and production
Depending on the task at hand, one enters
the engineering cycle at different entry points.
Biotechnology 25
When, for instance, an organism should be mod-
ified to express a recombinant protein, the first
step is to design and create the DNA sequence
that codes for the protein in an appropriate vec-
tor system. Thus, one will enter the cycle at the
design level. The same is true when an existing
metabolic pathway should be extended in the
way that the original end product of that path-
way is metabolized to a compound of interest.
However, when an existing metabolic pathway
shall be used to produce a given compound in
high yields, one will have to start by analyz-
ing the metabolic situation and the metabolic
fluxes in order to identify targets for genetic en-
gineering. In this case, one will enter the cycle
at the analysis level. In particular in the latter
case, it is often necessary to go through the cy-
cle of metabolic engineering more than once,
since complex metabolic networks within living
cells often require several genetic modifications
to provide the compound of interest in improved
quantities.
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
production of a modified strain. Fermentation
will be addressed in other chapters of this series.
(→ Ethanol, Chap. 5, → Antibiotics, Chap. 5)
4.1. Analysis of the Transcriptome,
Proteome, and Metabolome
The bottleneck of all of the traditional methods
of transcriptome and proteome characterization
is the limitation to an analysis of just a few sam-
ples at a time. While conventional methods are
confining themselves to the examination of sin-
gle genes, a DNA or protein chip experiment
26 Biotechnology
delivers a complete gene or protein expression
pattern of the cell. The high degree of paralleliza-
tion realized in biochips is the great advantage
over classic molecular biological approaches.
4.1.1. Gene Expression Analysis using DNA
Microarrays
In the past few years, the complete DNA se-
quences of a number of different microorgan-
isms have been determined and can be ex-
ploited to optimize strains as well as recom-
binant protein production. Strain optimization
involves measurement of genomewide mRNA
levels in wild-type and mutant strains using
DNA microarrays (Fig. 14) Furthermore, mi-
croarray analysis can help identifying previously
unknown genes required for recombinant pro-
tein production.
Bioprocess optimization using microarrays
entails metabolic control analysis, modeling,
and molecular biology to create new mutants
and strains with an, for example, optimized pro-
tein production rate. Recombinant protein ex-
pression exerts a metabolic burden on the host
cell, whereby stress response mechanisms are
triggered on different levels. Microarrays allow
the investigation on a genome scale, which en-
ables the qualitative and quantitative character-
ization of the burden on host cell metabolism.
Thereby, a better understanding of the impact of
recombinant protein production can be achieved
by using the generated “snapshot” of the actual
cellular composition and activity. Additionally,
the knowledge of the interaction of host cell
metabolism with recombinant protein produc-
tion is improved and contributes to process op-
timization.
Chip Technology. Alongside metabolome
analysis, analysis of various components of the
proteome, the transcriptome or the genome will
become increasingly valuable. New biosensors
known as biochips will be required. DNA chip
technology has already opened up new ways
of studying disease in more depth and identi-
fying far more possible targets. A DNA chip
is an array of synthetic DNA sequences repre-
senting different genes. Up to now, membrane
and immune biosensors are used to analyze sub-
stances of the metabolome. One important goal
in chemical and biological sensing is the sen-
sitive detection and selective identification of
biochemical discrete compounds (carcinogens,
metabolites, proteins, etc.) or living systems
(bacteria, viruses, or related components) at low
levels in complex biological matrices, tissues,
and blood. In the future, it will be indispensable
to analyze compounds of the genome and the
proteome. Therefore, new formats of biosen-
sors, so called biochips, have to be developed.
Such biochips or microarrays can be used for
identifying genes as well as changes in their
activity. Furthermore, these biochips allow the
simultaneous detection of several disease end-
points using different bioreceptors such as DNA,
antibodies, enzymes, or cellular probes.
The high parallelization degree of biochips
is a great advantage over classic molecular bi-
ological methods. While conventional methods
are confining themselves to the examination of
single genes, a DNA chip experiment delivers
a complete gene expressions pattern of the cell.
DNA microarrays can be used for the purpose
of monitoring expression levels of thousands
of genes simultaneously. Furthermore, biochips
can solidly simplify and accelerate a number of
long and expensive diagnostic methods. Addi-
tionally, gene expression analysis across various
biological conditions, cell cycle states, tissues,
and subjects may help identify differentially ex-
pressed genes. This type of information is a valu-
able pinpoint in the investigation of biological
processes and functional disorders. Recapitulat-
ing, DNA chips provide a format to profile com-
plex diseases and discover novel disease-related
genes. Applications for this technology include
gene expression monitoring, mutation detection,
metabolic engineering, drug development, tai-
lor made therapeutics, SNP research, GMO de-
tection, and high-throughput screening, among
others. They have a profound impact on biologi-
cal research, industrial production, medicine and
pharmacology and will be used as the biosensors
of the future [63 – 67]. For the understanding of
biological systems with up to 30 000 genes, the
measurement of RNA levels for a complete set
of transcripts of an organism will be necessary.
The use of glass slides as medium enables high
spot densities on microarrays (up to 10 000 spots
per slide). A DNA chip experiment works by hy-
bridizing all of the gene probes on the chip with
the nucleic acid target to be tested. cDNA la-
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
to be analyzed. Hybridizing a sample of nucleic
acid with many complementary gene probes in
parallel on a DNA chip produces a hybridiza-
tion pattern with corresponding hybridization
intensity. DNA chip technology therefore en-
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.
methods made DNA chips affordable for aca-
demic research laboratories. Since 1996, many
DNA arrayers have become available and the
self spotted glass slide DNA arrays are today the
most popular format for gene expression profil-
ing experiments.

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
significant advantages such as an improved an-
alytical speed, a better separation efficiency, re-
duced sample/reagent consumption, contamina-
tion reduction and reduced costs. Protein mi-
croarrays consist of bound antigens or antibodies
as capture molecules for detecting proteins in a
complex mixture. They provide a huge potential
for monitoring protein expression and protein
profiling.
Although the basic construction of such pro-
tein microarrays is similar to DNA microarrays,
the more delicate nature of protein structures has
hindered the development of such devices for the
analysis of proteins for a long time. This is due
to their more complex coupling chemistry, the
instability of the immobilized protein and the
far weaker detection signals. In principal, mi-
croarray technology enables both, the probing
of the genome and the proteome. Today, pro-
tein chip technology focuses basically on the
understanding of molecular pathways which al-
lows conclusions concerning the molecular di-
agnostic, the drug discovery, and metabolic en-
gineering applications. These interferences can
be done because of the capability of protein mi-
croarrays to analyze the protein function of a
whole genome level [69 – 73]. The results of
such high- throughput screening approaches can
change our fundamental understanding of the
cellular processes of life on the molecular level.
However, gene expression analysis does not suf-
ficient enable a reliable prediction of the func-
tion of a protein. Monitoring protein interactions
is an extremely complex matter since the pro-
teome is the quantitative representation of the
complete protein expression pattern of a cell un-
der accurately defined conditions. The proteome
represents the protein equivalent of the genome.
In contrast to the genome which is determined
by the sequence of its nucleotides and therefore
static, the proteome represents an extreme dy-
namic object which is influenced by many pa-
rameters. Not all genes will be switched on at
the same time in a cell and the sensitive balance
between protein synthesis and protein degrada-
tion can vary widely under different metabolic
conditions. Consequently, a repeated analysis of
the proteome will be successful only under ex-
actly defined conditions, for example, cell cul-
ture conditions. In practice, this proves to be
very difficult since conditions to which the cell
severely reacts are often unknown. However, the
sensitive dependence of most different parame-
ters offers the possibility of applying specific
small changes of the protein expression pattern
in order to create sensitive biosensors.
Ideally, the analysis of the proteome delivers
the currently available set of all proteins, if there
is a way to maintain the quantitative relations of
all proteins during the analysis. Such data can-
not be obtained with classical molecular biology
since no strict connection between the amount of
mRNA and the amount of protein exists. Param-
eters such as mRNA stability, posttranslational
modifications, protein degradation, and others
consequently prevent a statement over the cur-
rently available amount of protein. However, this
information is of utmost importance making a
high throughput analysis necessary. One attrac-
tive method is the use of protein microarrays.
Genomewide screens for protein function are of
biological importance for many applications:
– Analyzing protein expression profiles
– Monitoring protein-protein interactions
– Identifying protein posttranslational modifi-
cations
– Screening the substrates of protein kinases
– Examining the protein targets of small
molecules
– Proteomic analysis as a function of bioprocess
cultivation conditions
4.1.4. Metabolome Analysis and Metabolite
flux analysis
In order to improve a given strain of microorgan-
isms or even more complicated a eukaryotic cell
line that is used in a biotechnological produc-
tion process, it is not only necessary to analyze
gene expression patterns or the proteome. Anal-
ysis of metabolic turnover is central for recog-
nizing possible targets for genetic engineering.
Metabolic turnover comprises a large number
of biochemical reactions. For instance, in the
well-examined yeast Saccharomyces cerevisiae,
about 1500 biochemical reactions have been es-
timated involving more than 850 metabolites and
cofactors [74]. Since in most of these reactions
there is more than two educts and more than one
product, not to speak of the necessary cofactors,
the metabolic turnover represents a complex net-
work of biochemical reactions, also referred to

as the metabolic network. The concept of iso-
lated metabolic pathways such as glycolysis, cit-
ric acid cycle, or urea cycle as presented in most
biochemical textbooks is therefore somewhat
misleading since it indicates that these pathways
proceed independently. However, they are all in-
terconnected and represent a huge interdepen-
dent network that can activate or slow down in-
dividual branches. Control over the activity of
individual metabolic branches can be exerted on
the levels of transcription, translation, protein-
protein interaction, or enzyme activity regula-
tion, which adds another level of complexity.
According to Nielsen [77] it is therefore neces-
sary to analyze the metabolic network in three in-
stances (metabolic pathway analysis) in order to
fully oversee the metabolic activities in a living
cells, which is considered as a prerequisite for a
rational optimization of the production strain:
– Network structure (pathway topology).
– Quantification of fluxes through the branches
of the network.
– Identification of control structures.
Much work has been done in the past in
biochemical laboratories around the globe to
identify the metabolic network structure at least
for the more well-known microorganisms – in
particular when they are of industrial impor-
tance. Thus, very often the presence of a cer-
tain metabolic pathway can be deduced from
the available literature. The most powerful ap-
proach to identify a certain pathway when no in-
formation is available is called metabolic label-
ing. Here, the organism is fed with 13 C-labeled
glucose (or any other appropriate fundamental
metabolite) and after a given time that is al-
lowed for metabolization the labeling pattern
of other intracellular components is identified
and reports on the metabolic pathways involved.
From the analytical perspective, analysis of the
metabolite pattern is most often done by GC-MS
(gas chromatography coupled to mass spectrom-
etry) but also by NMR (nuclear magnetic reso-
nance). Later on, biochemical assays addressing
the activity of certain key enzymes can be used
to confirm the presence of a certain pathway and
dig out the cofactor requirements for the individ-
ual steps within the pathway. Pathway identifica-
tion just by enzyme assays is possible, however,
very tedious and time consuming.
Biotechnology 29
Flux Analysis. Once the network topology
has been identified, it is necessary to learn
about the fluxes through each of the network
branches. This kind of analysis requires exper-
iments as well as mathematical modeling. The
most important concept behind flux analysis is
referred to as metabolite balancing. According
to this, the concentration of each metabolite is
stationary inside the cell or in other words: the
generation rate of a given metabolite in one
metabolic pathway (v+ ) equals the rate of its
onward reaction through the sum of all down-
stream metabolic pathways (v− ). Thus, for a
given metabolite the corresponding material bal-
ance can be described as
v+1 +v+2 +v+3 +. . .+v+i =v−1 +v−2 +v−3 +. . .v−i
Such balances are established for all relevant
metabolites, such that a set of algebraic equa-
tions results that describe the flux of molecules
through the different branches of the metabolic
network. When some of the fluxes are experi-
mentally determined, the remaining fluxes can
be deduced mathematically from this set of
equations. The beauty of the metabolic balanc-
ing approach is its simplicity. But reliable pre-
dictions can only be obtained when the cofactor
(NADH, NADPH, . . .) balances are also known
which means that all the pathways that gener-
ate or consume any of these cofactors have to
be considered in the formalism. Feeding the or-
ganisms with 13 C-glucose in combination with
a molecular analysis of the resulting metabolite
labeling downstream of glucose provides, how-
ever, an easier solution. From the labeling pat-
tern of all metabolites it is possible to set up
equations for the balances of individual carbon
atoms. Thus, the number of equations describ-
ing the overall system increases the number of
constraints for the solution and thus, makes bal-
ances of cofactors unimportant.
The final step in analyzing the metabolic
network is to identify the regulatory mecha-
nisms that control the fluxes through each of the
branches [75, 76]. It is very obvious that detailed
knowledge about flux control might immedi-
ately identify possible targets of metabolic en-
gineering, for instance, to redirect the metabolic
flux away from a pathway that does not lead
to the desired product or consumes precursors.
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
the metabolic network [77]. Here the regulatory
instances can be found at several hierarchical
levels as mentioned earlier in this chapter. The
expression level of a given enzyme or any of
its regulatory proteins may be controlled on the
level of transcription, RNA processing, or trans-
lation. DNA and protein chip technology, as de-
scribed above, provide powerful approaches to
unravel whether any control occurs on the level
enzyme expression. Finally, allosteric control
over enzyme activity introduces another level
of metabolic fine-tuning. The kinetic properties
of an enzyme like, for instance, its affinity for
the substrate molecules, the maximum substrate
turnover rate and its sensitivity for product inhi-
bition have to be characterized by biochemical
assays. When these information and the current
concentrations of the relevant metabolites are
available, it should be possible to predict how
the fluxes through individual branches of the
metabolic network will change when the activity
of one enzyme is altered by genetic engineering
or when the feeding situation is changed.
However, it should be emphasized at this
point, that due to the enormous complexity of
the metabolic network and all its regulatory in-
stances, it is very difficult to predict all metabolic
consequences induced by just one genetic mod-
ification. Thus, it may be necessary to com-
pletely analyze the metabolome of the modified
strain and – if the results are not satisfactory – go
through one or several more rounds of metabolic
engineering as sketched in Figure 13. This is par-
ticularly relevant when the organisms respond to
alteration of their genetic make-up by opening
up metabolic side pathways that are silent in the
wild-type strain.
4.2. Design and Production of
Genetically Optimized Strains for
Production – In Vitro Mutagenesis
In order to generate genetically modified organ-
ism that might be superior to wild-type strains
for production purposes, there are basically three
major options:
A) Introduction and expression of a gene that is
not originally expressed by the organism or
only in small copy numbers
B) Silencing of a gene that codes for an en-
zyme or regulatory protein which should be
switched off or reduced in expression level
C) Modifying an existing gene – and hence the
coded protein – that is expressed by the or-
ganism of interest, for instance, to alter the
substrate specificity of an enzyme or engineer
its catalytic performance
In all three cases, the most suitable target for
genetic manipulations is cloned DNA that can
be handled and tailored in the well-defined en-
vironment of a test tube without the complexity
of an entire organism around, in particular the
cellular membranes that may hinder the acces-
sibility of the various reagents. Cloned DNA is
usually handled in form of plasmid DNA. A plas-
mid (cytoplasm + chromatid = plasmid) is a cir-
cular piece of double-stranded DNA that is au-
tonomously replicated in bacteria and may even
be exchanged between them. Plasmid DNA oc-
curs also in wild-type strains since they are reg-
ular extrachromosomal genes that can be easily
shared between certain bacteria. Many molec-
ular tools have been developed that are specif-
ically designed to work with plasmid DNA in
vitro. A particular family of plasmids, the so-
called Expression plasmids or expression vec-
tors as the more general term, additionally con-
tain the necessary nucleic sequences to initi-
ate transcription (promotor) and translation of
a gene that has been correctly inserted into the
plasmid – instead of multiplication only. Expres-
sion plasmids for prokaryotic cells differ from
those for eukaryotic cells [78]. The three differ-
ent strategies to alter gene expression in more
detail:
A) When a new gene should be introduced into
an organism or the expression level of an ex-
isting one should be increased, this particular
gene has to be cloned in a suitable expression
vector controlled by an appropriate promotor
that ensures a sufficiently high copy number
of mRNAs and hence protein. The produc-
tion of human insulin in E. coli is a famous
example for the production of a recombinant
eukaryotic protein in a prokaryotic organism.
B) When an existing gene should be silenced or
reduced in activity, there are several exper-
imental strategies. Today the most promis-
ing technique is RNA interference (RNAi),
a process by which double-stranded RNA si-

lences gene expression. By transfecting cells
with small interfering RNAs (siRNAs) with
100 % homology to a target mRNA sequence,
a specific knock-down of the corresponding
protein can be achieved. Bound by an RNA-
induced silencing complex (RISC), the siR-
NAs induce the degradation of their homol-
ogous mRNA molecules in the cell, thus the
protein expression level decreases. RNAi is
a reverse-genetics approach to identify gene
function by altering the phenotype of a cell
[79 – 82].
C) Until the middle of the last century, tradi-
tional biotechnology concentrated on eluci-
dating new metabolic pathways and improv-
ing the strains used for the production of anti-
biotics and other useful natural products, usu-
ally by exploiting random mutagenesis and
selection. Later in the 1980ies, biologists be-
gan to improve strains through metabolic en-
gineering, that is, through the modification of
single metabolic pathways by means of tar-
geted or site-directed genetic changes. The
two different approaches – random or site-
directed mutagenesis – to improve the genetic
make-up of an organism are still applied to-
day and shall be summarized here. However,
throughout the last years progress in molecu-
lar biology has provided a pool of experimen-
tal approaches for targeted genetic changes
that are way too many and multifaceted to be
reviewed comprehensively. Thus, only a few
principles can be discussed here, and are con-
fined to the best-known models of genetic re-
search, the bacteria. The interested reader is
referred to other chapters of this series or the
literature on molecular biology for more de-
tailed and organism specific information [83,
84] (→ Genetic Engineering).
4.3. Random Mutagenesis, Isolation and
Selection of Mutants
Spontaneous Mutants. In bacterial popula-
tions, mutational events occur at certain rates
without experimental treatment. These sponta-
neous mutations, which result in mutants, are
due to errors that occur during DNA replica-
tion. The frequency of spontaneous mutations in
a bacterial population varies from gene to gene
and species to species and may involve 10−2
Biotechnology 31
to 10−11 of the total cell population. The spon-
taneous mutation rate (probability of a mutation
per cell and generation) for a gene is about 10−5 ,
and for a pair of nucleotides it is 10−8 . The num-
ber of reversions (or functional back-mutations)
is of the same order of magnitude.
Induction of Mutations. The mutant fre-
quency can be considerably increased by treat-
ment of the cells with chemical, physical, or
biologic mutagenic agents. Three classes of mu-
tants can be distinguished with respect to their
genetic structure: 1) replacement of one base
pair by another, e.g., AT by GC; 2) a base pair
can be inserted or eliminated; 3) several base
pairs, DNA fragments, or even genes can be
lost (deletion), change their position within the
chromosome (transposition), or be interrupted
by insertion of foreign DNA (insertion). Class-1
mutants, called point mutants, revert readily to
their original structure.
Various chemical agents are in use to induce
mutations [85, 86]:
1) Incorporation of Base Analogues.
Base analogues are antimetabolites. Some are
sufficiently similar to the natural purine and
pyrimidine bases that they are taken up by
the cells and incorporated into DNA during
replication. They are able to fulfill their func-
tions, but tend to bind a wrong counterpart
during replication, thus introducing a wrong
base pair and causing a point mutation.
2) Chemical Change of Bases.
Several mutagenic agents effect a chemical
change in a base and thus cause an error in
replication. For example, treatment of cells
with nitrite results in the de-amination of
adenine, guanine, and cytosine, and conse-
quently in mispairing. Alkylating agents, such
as ethyl or methyl methanesulfonate, ethyl-
eneimine, nitrogen mustard, and N-methyl-
N-nitro-N-nitrosoguanidine (MNG), belong
to the most effective mutagenic agents. Acri-
dine dyes (proflavin) function by intercalation
and result in insertions or deletions of single
base pairs.
3) Irradiation.
UV irradiation mainly affects pyrimidine
bases and results in replication errors.
4) Transposon Mutagenesis.
By conjugation, transposon-containing plas-
mids (Tn elements) can be transferred from
32 Biotechnology
an appropriate donor bacterium to the bac-
terium to be mutagenized. Transposons are
DNA fragments that tend to “jump” to various
positions in the DNA sequence of the chro-
mosome or plasmid. Their insertion interrupts
genes and results in insertion mutants.
The changes in the DNA described above in-
volve an alteration of its base sequence, called a
premutation. Subsequently, the mutant character
has to be expressed in the phenotype of the cell.
About 10–20 growth cycles may be required for
nuclear segregation, dilution of enzymes, start-
ing or stopping the synthesis of enzymes, and
other functions.
4.4. Types of Mutants and Selection
Principles
A mutation is a rare event, and the number of
mutants in a population is small. Many thou-
sands of Petri dishes would have to be inspected
and many millions of colonies (clones of single
cells) would have to be examined to recognize
and isolate a mutant if there were not a selection
(enrichment) procedure for increasing the num-
ber of the desired mutants within the population.
Enrichment is achieved either by killing part of
the parent cells or by growth under conditions
that confer growth advantage to the mutant cells.
There are many means for enriching mutants. In
fact, designing strategies to select mutants is one
of the most difficult and demanding arts of the
microbiologist, and each type of mutant requires
a special trick.
The recognition of mutants poses another
problem. Mutants that have lost or gained pig-
ment, colony size or characteristics, or that have
become resistant to toxic compounds or bacte-
riophages can be easily detected in a lawn of
parent cell colonies. Some mutants show up af-
ter addition of indicators. The detection of aux-
otrophic mutants requires comparison of growth
on two different nutrient media. One tries to rec-
ognize mutants on an agar plate and to avoid la-
borious test tube assays of thousands of isolated
cell clones. There are, however, many mutant
types that require these efforts.
4.4.1. Auxotrophic Mutants
If the mutation has affected the ability to syn-
thesize, for example, the amino acid leucine,
the mutant will require leucine in the nutri-
ent medium. A mutant that is auxotrophic for
leucine (leu− ) can be recognized by compari-
son of its growth on two agar plates, one with
and the other without leucine. While the pho-
totrophic parent type (leu+ ) will grow on both
plates, the auxotrophic leu− mutant will grow
only on the supplemented agar.
The enrichment of auxotrophic mutants can
be achieved by application of agents such as
penicillin that kill only growing cells and leave
non-growing cells unimpaired. If the population
of parent cells and rare leu− mutants is grown
on a minimal medium (lacking leucine) only the
parent cells will grow and after the addition of
penicillin will be killed within a few hours. The
mutants and some parent cells will survive and
the mutant fraction is increased by a factor of
104 to 106 . Another method involves the prin-
ciple of “lethal synthesis.” Growing cells will,
for example incorporate radioactive phosphate
or antimetabolites and will be killed while the
non-growing mutants will survive.
4.4.2. Regulatory Mutants
Some mutants, which in contrast to the parent
type form a biosynthetic enzyme constitutively,
can be selected directly on the agar plate. For
example, if an antimetabolite is added to the
agar, the parent cells will incorporate it into their
protein and stop growing. Mutants, however,
that overproduce the normal metabolite because
of overproduction of the biosynthetic enzyme
will not incorporate the antimetabolite; they will
grow, and sometimes even show their regulatory
defect by excreting the metabolite, thus initiat-
ing secondary growth of the parent cells in the
surroundings of the mutant colonies. Some pro-
cedures for the selection and recognition of mu-
tants are listed in Table 2.
4.4.3. Other Selection Methods
Various other methods can be used to separate
mutants from their parent cells. Because some
 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
ability to utilize a colonies: the cell suspension is spread on a nutrient agar
special substrate that contains the substrate utilizable by the wild-type
cells at normal concentrations (0.5 vol%), and the
substrate accessible by the desired mutants at a very
low concentration (0.005 vol%). Pinpoint colonies are
transferred and screened.
comparison of colony patterns on agar plates
containing different substrates; mutants unable to
grow on a substrate will need a different nutrient for
growth and are therefore recognized. Excretory
mutants can be recognized by staining reactions (pH
indicators) or by dyes added to the agar or after
colony growth.

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

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-
ties than the average cell mass, the cells can be
incubated under conditions that promote syn-
thesis only in the parent or the mutant cells,
followed by centrifugation in a sucrose gradi-
ent. Cells rich in lipids (poly(β-hydroxybutyr-
ic acid), triglycerides), glycogen, calcium dipi-
colinate, magnetosomes, etc. can thus be sepa-
rated. In other cases tactic responses, such as
migration in light–dark fields (phototaxis), in
gradients of nutrient (chemotaxis), or oxygen
concentrations (aerotaxis), can be used to se-
lect mutants that are defective in transport sys-
tems or in perception mechanisms. Adsorption
on particle surfaces (glycogen or starch gran-
ules, lipid droplets, lignocellulose particles) fol-
lowed by fractional centrifugation can be used to
select for mutants that have altered surface struc-
tures. The separation efficacy can be increased
by the use of column chromatography or by
suicide techniques. Suicide techniques employ
compounds that are converted by the wild types
to toxic compounds; this lethal synthesis will fi-
nally kill the parent cells. Examples are monoflu-
oroacetate, bromopyruvate, or chlorate, which
are converted into fluorocitrate, bromolactate, or
chlorine, respectively. The mutants are unable
to catalyze such conversions and will therefore
survive. With this methods it is possible to se-
lect for cells that are deficient in nitrate reduc-
tase (chlorate, perchlorate), or for fermentative
mutants that have lost the ability to form acids
(bromide, bromate). Successful biotechnology
in many cases depends on the background in-
formation and the ingenuity of the researcher in
selecting, recognizing, and manipulating the de-
sired organism.
In most cases, a single random mutagene-
sis decreases the characteristic of an organism
that is to be changed, such as, for instance, the
catalytic performance of an enzyme. By apply-
ing high-throughput screening technologies, it
34 Biotechnology
is, however, possible to screen large numbers of
strains that are produced by random mutagenesis
and find the valuable ones with beneficial muta-
tions. Thus, by going through several rounds of
random mutagenesis on the one hand and screen-
ing for the desired phenotype on the other, this
process will identify those strains with consid-
erably improved properties for production [87].
4.4.4. Targeted or Site-Directed Mutagenesis
Site-directed mutagenesis allows modifying a
DNA sequence and thus the protein that is coded
by this sequence in a specific and well-controlled
way. Thus, it is possible to exchange one or more
amino acid within the primary structure of the
protein and thereby alter its functionality, for in-
stance the catalytic performance of an enzyme.
Compared to random mutagenesis, this is the
more straightforward and direct approach not
relying on chances that a genetic change may
occur at a site that is useful for strain improve-
ment. However, it requires detailed information
on the three-dimensional shape of the protein
and its primary structure. Many different ap-
proaches for targeted genetic alterations have
been described that cannot be fully reviewed
here. We will summarize the most widely ap-
plied and flexible concept of oligonucleotide-
based mutagenesis.
In oligonucleotide-based mutagenesis, the
gene of interest is cloned in a plasmid. Af-
ter denaturation of the double-stranded circular
DNA, oligonulceotides are added to the system
that share sufficient sequence homology with the
coding strand of the gene to allow for hybridiza-
tion. However, at the site where the mutations
should be introduced the sequence of the olignu-
cleotide is altered. Since this is only a minor
portion of the entire olignucleotide, hybridiza-
tion still occurs but the base exchange has been
introduced. Figure 16 sketches the underlying
principle.
Chain extension by DNA polymerase and lig-
ation results in a double-stranded DNA with one
mutant strand and one wild-type strand. When
these plasmids are then introduced in bacteria
by transformation, semiconservative replication
produces homoduplexes with both strands be-
ing either of the wild-type or the mutant. When
colonies are grown, one can now screen for those
strains in which the mutant plasmid has pre-
vailed.
Many more experimental options exist to in-
troduce site-specific mutations. Many of these
are based on polymerase chain reaction (PCR)
and the application of primers that already hold
the mutation, or insertion or deletion. The inter-
ested reader is referred to Bowen [88].
The aim of modern metabolic engineering is
to study the cell as an integrated system of ge-
netic, protein, metabolite, and pathway events
that are continually changing. This approach
should help to optimize the production of sub-
stances or to produce substances with improved
properties. Historically, metabolic engineering
is the targeted recombination of the DNA for
proteins involved in the metabolism or in reg-
ulation. Today, the objective of metabolic engi-
neering is to quantify the pathway alterations in
response to environmental mediators taking into
account the entire metabolism. Knowledge of in
vivo flux distributions in cells at different physi-
ological states is of increasing importance for the
evaluation of biotechnological processes. Gene
expression data provide information on path-
ways relevant to the metabolic models. Further-
more, the so-called combinatorial biosynthesis
uses techniques from molecular biology to alter
or combine genes from biosynthesis pathways of
the secondary metabolism to generate new prod-
ucts with improved pharmacological profil. The
importance of studying the entire metabolism
rather than one gene or protein at a time has be-
come increasingly relevant with the advent of
high-throughput genomic and proteomic tech-
nologies, especially microarrays. The DNA mi-
croarray technology will gain great importance
in the field of future biological research devel-
oping into a key technology of the 21st cen-
tury hence revolutionizing modern biotechnol-
ogy. DNA chips are used as biosensors in indus-
trial analysis, biomedical diagnosis, and foren-
sic science. Although this technology provides
a powerful tool that is widely utilized for gene
expression, it is just beginning to find appli-
cations outside of genomics. Further develop-
ment of protein, cell, and tissue chips will make
it possible in the future to carry out miniatur-
ized, highly parallel analysis of metabolic path-
ways. The challenge is to transfer these princi-
ples into technically applicable and precise ana-

Figure 16. Site-directed mutagenesis of cloned DNA by oligonucleotide mutagenesis
lytical systems that can be used for many appli-
cations repeatedly.
5. Cultivation and Bioprocesses
In this chapter, only the basic requirements for
bioprocesses are described very shortly. For
any further details concerning different types
of bioprocesses, bioreactors, kinetics, sterility,
and cleaning, the reader is referred to (→ Bio-
chemical Engineering). Very detailed informa-
tion about the monitoring and control of bio-
processes can be found in this contribution in
Chapter 8.
Bioprocesses [89, 90] are used for the trans-
formation of organic matter with the help of bio-
catalysts, such as living cells, dead cells, or their
components, e.g., enzymes [91, 92]. Biopro-
cesses are utilized for chemical syntheses and for
the conversion of waste material to either useful
products (recycling) or effluents harmless to the
environment (waste treatment). The outstanding
feature of bioprocesses is their high synthetic
potential to carry out a series of very compli-
cated chemical reactions in a one-step process.
Biotechnology 35
Classic biocatalysts are yeasts, fungi, and bacte-
ria; animal and plant cell cultures have become
important only in more recent times (see Chap-
ter 9). Process development [93] [94] [95] in-
volves the areas of biology (strain development
and bioregulation), reactor design, process con-
trol [96], and medium design [97, 98] as well as
downstream processing (product recovery, see
Chapter 7).
5.1. Isolation of Microorganisms
Microorganisms can be purchased from cul-
ture collections. But “microbes are everywhere,
the environment selects,” and a desired type
of microorganism can often be isolated from
its most probable natural habitat. For example,
methanogenic bacteria are present in the anoxic
mud sediments of ponds and lakes, hemoglobin-
degrading bacteria in abattoirs, and hydrocar-
bon-oxidizing bacteria around oil fields or leaky
engines of motor cars. From samples of these
materials, bacteria can be isolated by enrich-
ment culture. The technique of enrichment cul-
ture is simple and straightforward [99, 100].
36 Biotechnology
By establishing defined environmental condi-
tions with respect to the energy, carbon, and ni-
trogen sources; hydrogen acceptor; gas atmo-
sphere; temperature; pH value; light; etc., and
by inoculating with a mixed population that con-
tains the desired metabolic type, the best adapted
organism will dominate and overgrow all ac-
companying organisms.
Liquid enrichment culture is recommended
if the fastest growing organism is desired. In this
case, the organism is repeatedly transferred from
the original liquid culture through several sub-
cultures. If, however, a great number of strains
with only slightly differing traits are to be sepa-
rated, the direct plating method is the preferable
technique: the inoculum is diluted and spread
on a solidified enrichment medium (agar dish).
During incubation the cells form colonies and
can be isolated separately.
Whereas formerly these kinds of naturally
pure or well-balanced mixed cultures were used
in industrial practice (e.g., yeast fermentations,
vinegar and cheese production) now only pure
cultures are used. Pure cultures are obtained by
diluting the enrichment culture and spreading
the suspended cells on an agar surface or dis-
tributing the cells in an agar medium. After incu-
bation, single colonies are isolated and streaked
on various media to test for commensalic bac-
teria. Finally, a clone of the desired organism is
obtained as a pure culture.
Preliminary results indicate that previously
noncultivable microorganisms could be the key
to the biosynthesis of active substances. The
genes responsible for the expression of the natu-
ral substance biosynthesis could be inserted into
microorganisms which can be grown easily, a
procedure known as the metagenome approach.
The metagenome is a set of all genetic mate-
rial from organisms which cannot be cultured,
e.g., from the soil or from communities of or-
ganisms. The development of the metagenome
techniques becomes possible with the develop-
ment of high-throughput techniques. The de-
velopment of high-throughput DNA sequenc-
ing began in the early 1990s as simplifications
and automation made it possible to decode the
sequence of entire genomes. In 1995 the first
full genome sequence was published, that of the
Gram-negative bacterium Haemophilus influen-
zae. Of particular interest for biotechnology is
the possibility of sequencing microbial produc-
tion strains developed by repeated random muta-
genesis and screening. A comparison with pre-
cursor strains could identify the mutations re-
sponsible for increased production. That way,
production strains could be designed with just a
minimal number of defined mutations [101].
Designer Bugs. The term used for microor-
ganisms tailored by genetic engineering meth-
ods so that they can carry out desired biochemi-
cal conversions efficiently is designer bugs. Con-
trary to the strategy used in the past to obtain
microorganisms for the production of a desired
product by coincidental mutagenesis and se-
lection, genetic engineering has opened up the
possibility of customized construction of pro-
duction strains by metabolic engineering. The
genome permits insight into the entire metabolic
potential of the organism. This insight is cur-
rently limited because the function of many gene
products is not yet known. Organisms, such as
E. coli, Bacillus subtilis, Corynebacterium glu-
tamicum, Pichia pastoris, or S. cerevisiae, are
preferentially applied in the development of de-
signer bugs. Further important aspects govern-
ing the choice of host organism for a designer
bug are knowledge of metabolism, regulation
mechanisms, growth properties, and hazard po-
tential. Based on methods developed in the last
few years for the analysis of the genome, tran-
scriptome, proteome and metabolome, which
thus permitted a holistic consideration of the or-
ganism (“systems biology”), it will be possible
in future to develop designer bugs for biotech-
nological production of a desired product con-
siderably more quickly and specifically than in
the past.
A list with mostly all culture collec-
tion worldwide can be found in the inter-
net [102] under the URL http://www.bacterio.
cict.fr/collections.html and in ref. [103 – 105].
5.2. Requirements for Growth
The process solution must primarily supply car-
bon and energy. In order to maintain growth,
nutrients, trace elements, and such growth fac-
tors as vitamins must be added to the process
solution. Appropriate selection of the medium
components is essential for proper functioning
and activity of the biocatalysts involved in the
reaction.
 Biotechnology 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
or another microorganism.
5.2.3. Accessory Nutrients
In addition to carbon and energy sources, many
organisms require accessory nutrients (growth
factors), such as vitamins, amino acids, purines,
or pyrimidines (see Table 3).
5.2.4. Sulfur and Nitrogen
These elements can be used in their oxidized
forms as sulfate and nitrate. Ammonium ion is
the most common nitrogen source for microor-
ganisms.
5.2.5. Oxygen
The oxygen atoms of the cellular constituents are
mainly derived from water, substrates, or carbon
dioxide. Atmospheric oxygen serves mainly as
a terminal hydrogen acceptor of aerobic respira-
tion, being reduced to water.
5.2.6. Complex Media
Many microorganisms can grow in a simple nu-
trient medium such as that listed in Table 3 a.
For Leuconostoc mesenteroides such a synthetic
medium contains 40 components. If the nutri-
ent requirements of an organism are not exactly
known, complex nutrient media can be used,
such as yeast extract, yeast autolysate, peptone
(Table 3), meat extract, wort, carrot juice, co-
conut milk, or horse manure extract. In other
cases complex media are used for economical
reasons, for example, whey permeate, corn steep
liquor, soybean extract, or molasses.
5.2.7. Solid Media
For solidification of media, agar is added at a
concentration of 1.5 to 2.0 wt%. Agar melts at
100 ◦ C and solidifies on cooling to below 45 ◦ C.
5.2.8. Hydrogen Ion Concentration
The majority of microorganisms prefer a pH of
about 7.0. However, there are acidophilic and
alkaliphilic microorganisms. Yeasts and other
fungi prefer pH 5.0. Buffers, in most cases phos-
phates, are used to maintain the desired pH, al-
though the CO2 /HCO− 3 system or organic sub-
stances may do as well. The pH value is very im-
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.
5.2.9. Carbon Dioxide
Many microorganisms require a higher concen-
tration of CO2 than exists in the atmosphere,
such as 10 vol%. This requirement applies to
those bacteria that in their natural habitats (in-
testinal tract, tissue, milk, blood, fermenting
juices) are exposed to high CO2 partial pres-
sures.
5.2.10. Aeration
All obligately aerobic microorganisms require
oxygen as an electron acceptor for energy gen-
eration. For growth in thin layers on liquid or
solid media atmospheric oxygen may suffice. In
liquid media aerobic bacteria grow only on the
surface if the medium is not agitated and aer-
ated. Only dissolved oxygen is utilized; its sol-
ubility in water is very low (6.2 mL/L at atmo-
spheric pressure and 20 ◦ C). Therefore oxygen
has to be supplied continuously to the cell sus-
pension growing in submerged culture in flasks
or fermenters. To meet the increasing oxygen
demand in a growing culture, the speed of agita-
tion, the oxygen concentration in the gas phase,
and the total gas pressure can be increased. Var-
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
microorganisms depends on the rate of oxygen
transfer. For fermenter design and scale-up, both
the respiration rate (oxygen uptake) of the mi-
croorganisms and the oxygen transfer rate from
gaseous to liquid phase have to be known. Be-
cause the formation of desired products is highly
dependent on oxygen supply – whether the cells

are able to take up oxygen at their maximum res-
piration rate or only much less – aeration poses
one of the most important problems in fermenter
technology.
5.2.11. Anaerobic Techniques
For the growth of strictly anaerobic bacteria
the complete exclusion of oxygen is required.
Anaerobic techniques involve deaerated nutri-
ent solutions, tightly sealed flasks, incubation in
anaerobic jars, the use of chemical oxygen ab-
sorbers (pyrogallol, dithionite or the “gas pak,”
which contains hydrogen- and carbon dioxide-
evolving agents and a catalyst to promote the
oxyhydrogen reaction), resazurin as a redox in-
dicator, anaerobic hoods, and skilled hands to
practice these anaerobic techniques.
5.2.12. Media Preparation
Substrates for industrial applications are usually
included in complex media, which must be sup-
plemented with special compounds, such as a
nitrogen source, various nutrient salts, or cer-
tain trace elements. Organic precursors are also
added in some cases for efficient product forma-
tion. Many of the feedstocks need appropriate
pretreatment in order to provide for good assimi-
lation by the microorganisms. Starch-containing
materials are prepared by milling or steam treat-
ment for softening and swelling. For many pro-
cesses, especially when yeasts are used, starch
must first be broken down to sugar by treatment
with amylase derived from barley malt or with
microbial amylases. Other feedstocks, such as
wood, must be pretreated with acid or alkali.
Sulfite liquor is stripped from sulfur dioxide by
aeration or neutralization. Molasses is purified
by acidification and centrifugation or filtration.
In some cases heavy metals have to be removed
from feedstocks prior to their use. For processes
using solid substrates, see [107].
In general, the raw materials are dissolved or
suspended in water and the resulting medium
is heated, filtered, and sterilized. The complex
composition of the media used in industry causes
considerable problems. For downstream pro-
cessing (harvest, concentration, and purification
of product) or for analytical assays during the
process, additional pretreatment of the raw ma-
terial is needed to avoid unfavorable side effects.
Biotechnology 39
5.3. Sterilization
The basis of microbiological laboratory meth-
ods and of the conservation of food and feed
products is the killing of microorganisms. Ster-
ilization means the removal of living microor-
ganisms, and can be achieved by moist heat, dry
heat, filtration, irradiation, or chemical means
[108].
5.3.1. Moist Heat
Vegetative cells of bacteria and fungi suspended
in water are killed at temperatures around 60
to 80 ◦ C within 5 to 10 min; yeast and fungal
spores are killed above 80 ◦ C, and endospores
of bacteria at 120 ◦ C within 15 min. Because
endospores of bacteria are highly tolerant to var-
ious environmental conditions, they are ubiqui-
tous and are distributed through the air. Their
presence requires that all nutrient media and
canned foods be sterilized in an autoclave at
about 121 ◦ C for 20 min. If conditions do not al-
low the germination of spores and the growth of
spore-forming bacteria, e.g., in acid fruit juices,
jam, or desserts, heating to 80–100 ◦ C for 10 min
will suffice. This is called partial sterilization or
pasteurization.
5.3.2. Dry Heat
For killing bacterial endospores by dry heat,
longer exposure times and higher temperatures
are required than with moist heat. Glass and
other heat-resistant equipment can be sterilized
for 2 h at 160–180 ◦ C in a dry oven. In any case
appropriate indicators or soil samples that con-
tain bacterial spores are included in the oven to
test whether adequate temperatures have been
reached and even the extremely heat-resistant
spores have been inactivated.
5.3.3. Filtration
Solutions containing thermolabile compounds
can be sterilized by filtration through nitro-
cellulose membranes, kieselguhr, porcelain, as-
bestos, and others. Usually filters with a pore
size <0,2 µm are used.
40 Biotechnology
5.3.4. Irradiation
UV irradiation is used to keep rooms partially
sterile. Bacteria and their spores are killed rather
quickly, but fungal spores are only moderately
sensitive to radiation. Ionizing radiation (X-ray,
gamma radiation) is used to sterilize food and
other compact materials.
5.3.5. Chemical Means
Ethylene oxide is used to sterilize food,
plastics, glassware, and other equipment. β-
Propionolactone and diethyl carbonate are
added to nutrient solutions. The presently used
soaps and detergents applied at moderate tem-
perature kill all vegetative cells and even many
spores. Cleaning by the methods practiced in the
kitchen provides almost sterile glassware.
Remarkably, the killing of microbes is a pro-
cess that follows first-order reaction kinetics.
In a population there are allways some cells or
spores that are more resistant than the majority.
Accordingly, sterilization procedures are most
likely to be effective on relatively clean sub-
strates.
5.4. Types of Bioprocesses
In chemical technology, a choice is to be made
between batch processes or continuously oper-
ated processes. Many parameters usually favor
the second over the first choice. However, in
biotechnology, the batch process still predom-
inates. This specific problem is treated in more
detail (also in regard to engineering aspects) in
→ Biochemical Engineering.
5.4.1. Surface Culture
Surface cultures are mostly used when fungal
mycelia act as catalysts. Mycelium is grown in
shallow pans containing a small volume of the
medium. As the mycelium grows, it eventually
forms a compact mat. This method was typically
used in the production of acids, such as citric
acid. In large-scale production, surface culture
is being abandoned more and more and replaced
by the submerged culture method [109 – 111].
Surface cultivation is usually applied in the
lab scale in fungal research. Large Erlenmeyer
flasks or specially designed vessels, such as the
Fernbach, Roux, or Sakaguchi flasks, containing
small volumes of liquid are used. The flasks
and liquid are sterilized and inoculated with
spore suspensions or small pellets of mycelium.
Growth and product formation are dependent
upon the depth of the medium layer and the
amount of surface provided by the flasks, which
in turn affects the necessary oxygen supply.
In large-scale production large pans are used.
They are filled with substrate, inoculated, and
then kept at controlled temperature and air hu-
midity. Pans are stacked on suitable racks, and
connected with a system of overflowing tubes.
The liquid medium is poured in at the top. In this
way, an entire stack can be loaded at once. If the
stack is designed appropriately, it can be steril-
ized, inoculated, aerated, and maintained under
controlled conditions. The same principle can
be applied for solid substrates, which are first
inoculated and then loaded into the pans. The
material is spread at a thickness of several cen-
timeters onto screens or perforated metal sheets.
Columns filled with carrier material onto
which the microbes are immobilized are also
considered a surface culture as long as the col-
umn is not completely flooded. The medium is
trickled through the column and air is injected at
the bottom. The product is then collected from
the carrier, which is made of wood shavings,
plastics, or ceramics. This type of surface cul-
ture has been used for vinegar production and
sewage treatment. However, it was abandoned
and replaced with submerged culture. Remark-
ably, the immobilization methods that were de-
veloped for such biocatalysts as living cells, rest-
ing cells, enzymes, or organelles are formally
similar to these conventional processes.
5.4.2. Submerged Culture
In this process, the microbes are suspended in the
liquid medium. The simplest example of a sub-
merged culture consists of a vessel without any
agitation device, in which microorganisms settle
at the bottom and form a compact layer. This is
the classic arrangement for all types of ethanol-
producing processes. Some agitation will occur,

however, from the movement of rising carbon
dioxide bubbles.
For submerged, anaerobic processes, simple
agitation devices for gentle mixing will suffice.
This configuration allows for the mixing of liq-
uid and gas. Carbon dioxide or other gases, such
as nitrogen, can be added during the process.
5.5. Process Layout
5.5.1. Reactors
Appropriate reactors in biotechnology have to
meet the requirements for agitation, aeration,
corrosion resistance, and aseptic operation. Var-
ious designs of stirred vessel are therefore used
in the laboratory and in production plants. Con-
tainer construction is based on principles similar
to reactors used in heterogeneous catalysis.
In former times, bioprocesses were carried
out under nonsterile conditions and took place
in vats of wood. Beer today is brewed in large
cubical containers coated with ceramic tiles. For
the more conventional surface processes (citric
acid, acetic acid), small pans were used.
A basic problem in aerobic processes is the
efficient transport of oxygen into the liquid and
the microbial cells. Very efficient devices have
been developed to provide air for aerobic re-
actions. Some examples of more modern reac-
tor configurations for agitation and aeration are
shown in Figures 20 and 21.
5.5.2. Containments for Anaerobic Processes
Large units for anaerobic processes are made
of wood, metal, or coated concrete. They have
no covers and consequently cannot be operated
aseptically. However, submerged anaerobic pro-
cesses can be performed under conditions that
favor the formation of product and inhibit the
accumulation of unwanted organisms by proper
selection of reaction parameters, such as pH and
temperature. Lactic acid, for example, is pro-
duced at 50 ◦ C and low pH, and the development
of competing microbes is negligible.
In other cases, closed vessels are used. They
are usually made of stainless steel, have large
capacities of up to 1000 m3 or more, and are
equipped with agitators and, if necessary, with
Biotechnology 41
cooling and heating coils. Gases that form during
the process (CO2 , H2 , or evaporated solvents)
are collected at the top. These large tanks have
openings for loading, inoculation, harvesting,
and cleaning. Large units are mostly sterilized
with steam.
Extremely large containments with capaci-
ties of thousands of cubic meters are used in
sewage treatment. They are usually constructed
of concrete and allow automatic feed and re-
moval of material. Sludge treatment plants are
often equipped with heating elements to allow
thermophilic processes for methane production.
The gas is collected and stripped of hydrogen
sulfide if necessary.
5.5.3. Reactors for Aerobic Processes
Bioreactors used for aerobic processes are ba-
sically similar to those used for anaerobic pro-
cesses. In addition, they contain devices for aer-
ation and agitation. The capacities used in in-
dustry may vary considerably, from 10 to 200
m3 . Large tanks of 1000 m3 and more are also
used for aerated, sterile processes (e. g., glutamic
acid). For aseptic processes the tank is sterilized
with steam before loading; a slight overpres-
sure is often maintained during cultivation. Ag-
itation principles vary considerably and include
mechanical as well as pneumatic and hydrody-
namic systems.
The most commonly used type of bioreac-
tor is the stirred tank reactor (STR) using a
flat blade turbine (FBT) for agitation (Fig. 17).
This configuration provides excellent mixing
and mass transfer in media of low viscosity.
Drawbacks are high power demand, poor per-
formance with highly viscous liquids, and rather
poor interchange of material between mixing re-
gions around the blades. Air is normally injected
by a sparger. Larger units are equipped for pneu-
matic agitation, in which movement is caused by
injected air. For proper hydrodynamic control,
various kinds of baffles and draft tubes can be
inserted.
5.5.4. Inoculation
After inoculation with the microorganisms, the
process should start immediately and the reac-
tion should proceed fast. The amount of active
42 Biotechnology
cell culture added is therefore critical and de-
pends on the size of the batch. Scaling-up from
the original starter culture to the inoculation
broth is done in several steps in large-scale in-
dustrial processes (Fig. 19). The starter culture
is kept deep frozen (−80 to −90 ◦ C) in sealed
vials or in vials that were previously subjected
to lyophilization (a). They can be stored in this
way for several months or even years without
any damage. For short-term storage at 4 ◦ C, agar
slants are prepared (g) and purity is tested on
Petri dishes (h) before inocula are scaled up for
production.
Figure 17. Stirred tank reactor (STR) equipped with flat
blade turbine (FBT) and baffles for agitation and a sparger
for gas distribution
The ratio of H: D is usually 3 (max. 5).
For scaling-up, the liquid cell culture is
placed in liquid broth, first in test tubes (b), then
in small (c), and subsequently in large Erlen-
meyer flasks (d) containing approximately 200
mL of medium. Proliferation of the culture takes
place by shaking the flasks overnight at constant
temperature. Larger amounts of inocula are then
prepared in agitated and aerated fermenters of
various sizes depending on the volume of the
production plant (e, f). In the early stages of
this procedure (steps a through d) the inoculum
is transferred under sterile conditions in glove
boxes.
In fungal inocula proper wetting of the spores
is achieved by adding small amounts of 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
of the process solution, has shown the highest
production rate. However, practical and biolog-
ical constraints, especially the high contamina-
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
be carried out under aseptic conditions are op-
erated continuously such as sewage treatment,
methane formation, and feed yeast or ethanol
production from sulfite liquor.
The potential advantages of continuous cul-
tivation are substantial because of the efficiency
of the process and the uniformity of the final
product. As a result, much effort is being made
to develop a sound basis of knowledge necessary
for using this process strategy.
In contrast to large-scale industrial processes
continuous cultivation methods are widely used
in the laboratory [113 – 115]. The growth-
controlling factor is either the concentration of
nutrients in the medium (chemostat system) or
the concentration of insoluble biomass sensed by
optical measurement (turbidostat methods). The
purpose of these control systems is to maintain
a constant concentration of nutrients, cells, and
metabolic products in the vessel (steady state).
The kinetics of growth or product formation can

Figure 19. Preparation of inocula.

The individual steps a through h are explained in the text

be kept under precise control by selecting the ap-
propriate dilution rate. All types of variations of
the single-stage continuous cultivation are pos-
sible, such as multistage arrangements, with or
without recycling.
5.6. Process and Product Overview
The potential of biotechnology consists in its
ability to replace classical chemical production
processes and to facilitate the production of new
products. It is undisputed that, particularly in the
area of basic and fine chemicals production, the
use of biotechnological processes is meaningful
since
– biotechnological processes are usually distin-
guished by their high specificity (relating to
the conversion of substrates) and selectivity
(relating to the product spectrum)
– biotechnological processes often use renew-
able resources as raw materials, thus con-
tributing to the much discussed sustainability
of products and processes
– biotechnological processes can be carried out
under mild reaction conditions in terms of
pressure, temperature, and pH.
In view of these facts, in the past hundred
years a multitude of industrial biotechnologi-
cal processes have been developed [116 – 120]
whose efficiency exceeds that of chemical pro-
cesses, and this has helped to establish them in
the long run (Table 5 and 6). One interesting
area is the production of fine chemicals. The
term “fine chemicals products” refers to sub-
stances that are highly functional and for which
world demand is typically quantities of less than
10 000 t/a. From a chemical point of view these
products are usually distinguished by having
several reaction centers and frequently by chi-
rality. Classical syntheses of these substances in-
clude several reaction steps using stoichiometric
quantities of reagents and often deploy extrava-
gant protective group chemistry, expensive no-
ble metal/heavy metal catalysts, and drastic re-
action conditions, e.g., aggressive solvents. Here
biocatalysis allows synthesis under considerably
milder reaction conditions in terms of pressure,
temperature, and acidity. At present, due to the
excellent enantioselectivity of enzymes, white
biotechnology methods are primarily used for
Biotechnology 45
the production of chiral compounds. Straathof
et al. [121] list 134 industrial biotransforma-
tions. Just short of 90 % of the products de-
scribed are chiral fine chemicals. In future, this
field will undoubtedly acquire greater impor-
tance and new reaction sequences for the pro-
duction of compounds with several stereo cen-
tres will be established.
Also bulk chemicals are produced by fer-
mentation or biotransformation processes. Bulk
products mean products exceeding 10 000 t an-
nually. It is expected that, by the year 2010, 6–
12 % of bulk products and polymers produced
by chemical means will already be produced
by biotechnological processes. High-volume,
biotechnologically produced goods are to be
found in the food, livestock feed, and the drinks
and tobacco industries. A prime example of a
bulk product derived by an enzymatic process
is acrylamide. Global biotechnological produc-
tion capacities have continually expanded in the
last few years and are today probably in the re-
gion of 100 000 t/a. Monomers and polymers
produced by biotechnological processes for the
plastics and polymer industry are becoming in-
creasingly interesting. Biotechnologically pro-
duced polymers, such as polylactide (PLA), 1,3-
propandiol (PDO) and poly-3-hydroxybutyrate-
co-3-hydroxyhexanoate (PHBH) could provide
the basis for innovations.
As for the energy industry, besides bioetha-
nol two further products should be mentioned:
biogas and hydrogen. Whereas the technology
for biogas recovery is state-of-the-art, hydro-
gen production is far more complex and requires
long-term research efforts. Biogas recovery is a
recycling method for residues. The product can
be separated comparatively easily, which is cru-
cial to the cost-effectiveness of the process.
6. Biocatalysis and
Biotransformation
6.1. Introduction
Biocatalysis (also called biotransformation) is
the conversion of one substance (substrate) to
another (product) by a microorganism or an iso-
lated enzyme. It is a chemical reaction catalyzed
by a particular cellular enzyme or by an en-
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

Table 5. (continued)
Product/process Annual production t/a
Vitamins
Riboflavin (b2 ) 30 000
Cyanocobalamin (b12 ) 20
Vitamin C 80 000
l-Sorbose (vitamin C 50 000
precursor)
Amino sorbite
Lipids
Phytosphingosine
Animal feed
Galactooligosaccharides 2500
Cosmetics
Dihydroxyacetone
of these enzymes are necessary for the normal
metabolism and reproduction of the cell. In bio-
catalysis, however, these enzymes are simply
used as catalysts for chemical reactions. In ad-
dition to their natural substrates, many enzymes
can utilize other structurally related compounds
as substrates and therefore may catalyze non-
natural reactions upon addition of foreign sub-
strates to the reaction medium. Thus, biocataly-
sis or biotransformation is a specific category of
chemical synthesis.
6.2. Classification of Biocatalysts
Enzymes and whole cells can be applied in a bio-
transformation in many different forms. Usually
a whole cell biotransformation is a growth de-
coupled process, so resting cells (harvested and
washed after fermentation) are employed. The
cells may be intact or permeabilized (e.g., for
better substrate uptake). The different types of
biocatalysts are:
– Whole or treated cells (e.g., lyophilized, per-
meabilized)
– Organelles
– Cell-free multienzyme systems
– Combination of individual cell-free enzymes
– Single cell-free enzyme
Isolated Enzymes may also be applied in dif-
ferent forms. In most cases, it is not necessary
to purify an enzyme to homogeneity, a crude
cell extract or partially purified solution may
also be employed as biocatalyst. In some well-
known technical enzyme preparations, several
isoenzymes are present (e.g., pig liver esterase).
Biotechnology 47
Price EUR/kg Market value 106 EUR Main application
active substance, animal
feed additive
25 000 500 active substance, animal
feed additive
8 640 food, animal feed
cosmetics
3.50 9 prebiotics
suntan preparations
In both whole cells and enzymatic biotransfor-
mations the catalytically active species is the
enzyme. Enzymes can be subdivided into six
classes (classified by the Committee on Enzyme
Nomenclature of the International Union of Bio-
chemistry) depending on the type of reaction
they catalyze (Table 7) and are listed numeri-
cally in a catalogue published by the committee
[125] (→ Enzymes).
6.3. History
Biocatalysis includes some of the oldest human
known chemical transformations such as the use
of yeast for baking or brewing or the use of
chymosin from the stomach of young cattle for
cheese production. Records proving these an-
cient technologies date back to about 7000 to
6000 bc.
Another old and well-known example is vine-
gar production, which has been known since the
dawn of recorded history. It is the oldest example
of microbial oxidation (Fig. 20).
The process has evolved along traditional
lines of fermentation without knowledge of the
underlying biochemistry. In 1862, Pasteur de-
scribed the biochemical nature of vinegar pro-
duction [126]. Subsequently Brown confirmed
in 1886 that the oxidation of alcohol to acetic
acid proceeded through acetaldehyde [127].
Bacterium xylinum, which causes the reaction
to proceed, was first described by Brown and is
the first microbial catalyst.
Today, the oxidation of ethanol to acetic acid
by acetic acid bacteria is well understood; it
proceeds via two successive dehydrogenations,
48 Biotechnology
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
conventional chemical synthesis.
Table 8. Advances in biocatalysis
Biocatalytic synthesis Example reactions
Transformation of steroids and hydroxylation (oxygenase),
sterols dehydrogenation
(dehydrogenase), side-chain
degradation (oxygenase), etc.
Transformation of terpenoids hydroxylation of beta-ionone
with monooxygenase from
Bacillus negaterium [138].
Transformation of alkaloids oxidation of morphine to
2,2 -bimorphin
(pseudomorphine) by
Cylindrocarpon didymium [139];
N-demethylation of codein to
norcodein by Mucus piriformis
[140].
Synthesis of semisynthetic synthesis of semisynthetic
antibiotics penicillins and cephalosporins
(amidase).
Synthesis of organic acids hydration of fumaric acid
(fumarase), diterminal oxidation
of alkanes (oxygenase),
asymmetric hydrolysis of
epoxysuccinic acid (hydrolase),
etc.
Transformation of sugars isomerization of glucose (glucose
isomerase).
Protein synthesis synthesis of plastein (protease),
semisynthesis of human insulin
(protease), etc.
Synthesis of nucleic acid related trans-N-ribosylation and
compounds trans-N-arabinosylation
(phosphorylase),
phosphorylation of nucleosides
(phosphotransferase),
pyrophosphorylation of
nucleotides
(pyrophosphotransferase),
synthesis of sugar nucleotides
(pyrophosphorylase), synthesis
of coenzymes, etc.
Synthesis of amino acids asymmetric hydrolysis of
α-amino-ε-caprolactam, 2-ami-
no-∆2 -thiazoline-4-carboxylic
acid, and 5-substituted
hydantoins (hydrolase),
amination of fumaric acid
(aspartase), synthesis of
l-tyrosine-related amino acids
(β-tyrosinase),
l-tryptophan-related amino acids
(tryptophanase), etc.
Synthesis of amines decarboxylation of amino acids
(decarboxylase).
Synthesis of industrial chemicals synthesis of alkene oxides
(oxygenase), chiral alcohols and
ketones (dehydrogenase),
pyrogallol (decarboxylase),
amides (nitrile hydratase), etc.
Innumerable biocatalytic conversions involv-
ing different types of reactions with organic
compounds and natural products were found
during the following years; some of them are
very useful for chemical synthesis. Advances
in biocatalysis are summarized in Table 8. Al-
though biocatalysis has become an important
technology for synthetic chemistry, it also has
a high potential for many fields of biotechnol-
ogy.
6.4. Characteristics of Enzyme
Reactions Used in Biotransformations
Chemical reactions performed by biocatalysts
(biotransformations) are essentially the same as
those carried out in inorganic or organic chem-
istry.
Enzymes (or biocatalysts) used for microbial
conversions increase the reaction rate by low-
ering the activation energy as normal catalysts
do. The most striking difference between en-
zymes and chemical catalysts, however, lies in
their substrate specificity. They catalyze specific
reactions involving one or only a few structurally
related compounds and they distinguish almost
absolutely between stereoisomers or regioiso-
mers. Therefore, only a very specific change in
a functional group or bond of a compound is ac-
celerated by the enzyme. As a result, one single
product is expected as long as only one enzyme
is involved in a conversion.
In contrast to chemical reactions that often re-
quire high activation energies (high temperature,
pressure, etc.) biotransformations can be per-
formed under considerable mild reaction con-
ditions (Table 9). Due to the low energy in-
put needed for biocatalytic processes and the
high atom utilization (percentage of M r of de-
sired product to sum of M r of all substrates),
biotransformations are considered as an envi-
ronmentally friendly alternative to classical or-
ganic chemistry processes. To compare biocat-
alytic and chemical processes on the basis of the
amount of waste, Roger A. Sheldon [141] de-
vised the “environmental quotient” (EQ), which
is the quotient of the amount of waste product
per kilogram of product (E) and an “unfriendli-
ness quotient” (Q): EQ = E × Q.
High catalytic efficacy is another character-
istic property of enzymes. They increase the
turnover rate without large energy requirements
and only a small amount of enzyme is needed
to catalyze the conversion of a large amount of
substrate.
 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-
ties are required by the conversion or if the na-
ture of the growth medium causes problems for
the isolation of the product, the reaction should
be performed after separating the cells from the
medium, see next section.
6.5.2. Biotransformation Conversion with
Previously Grown Cells
The microorganism is grown under suitable con-
ditions and the cells are then collected by cen-
trifugation or filtration. In this way, the cells re-
tain most of their enzyme activity. For the con-
version they are resuspended either in an in-
complete medium (e.g., one without a nitrogen
source; this prolongs cell viability and activity
as well as possible cofactor regeneration) or in a
simple reaction medium (e.g., a buffer solution
or water containing the substrate). The reaction
is then carried out directly or after suitable treat-
ment of the cells. It is theoretically not important
whether the cells are dead or alive. Vegetative,
washed, and dried cells fall into this category.
The advantages of this method are listed below:
1) Growth and conversion steps are controlled
independently for optimization.
2) The grown cells can be stored for some period
of time until initiation of the reaction.
3) The cell density for the conversion is easily
controlled.
4) The reaction can be carried out under non-
sterile conditions.
5) Product isolation is usually easier because
of the simple composition of the reaction
medium.
In some cases, however, the strict separation
of microbial growth and conversion is consid-
ered to be unfavorable for economic reasons.
Depending on the ability of substrate perme-
ability through the cell membrane these biocat-
alysts may have to be treated differently prior to
their use.
6.5.2.1. Vegetative or Washed Cells
In cases where the cell membrane is permeable
to substrate and product, no special technique is
needed for the preparation of the cells and they
can be used as vegetative or washed cells.
6.5.2.2. Permeabilized Cells
In cases where substrate or product molecules do
not readily permeate the cell membrane, a mod-
ification of the cell becomes necessary. Com-
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
the cell are: using surfactants, organic solvents,
antibiotics or enzymes. Besides these chemical
methods physical procedures, such as osmotic
shock or freezing and thawing may also be use-
ful [142]. The production of high-energy phos-
phate esters, such as adenosine 5-triphosphate,
nicotinamide adenine dinucleotide, flavin ade-
nine dinucleotide, or coenzyme A, is a typical
example [143].
6.5.2.3. Dried Cells
Dried cells are an alternative to permeabilized
cells. Drying causes structural changes in the cell
wall or membrane. Contact between the cell en-
zyme and the substrate is more readily achieved.
In some cases, drying also eliminates undesired
side reactions, because some enzymes are dam-
aged through drying. Dried cells are usually pre-
pared by air-drying, lyophilization and acetone
treatment. The powdered dried cells can retain
their enzyme activities in the frozen state for
years. They can be used as “instant catalysts”
in the same way as normal chemical catalysts.
6.5.3. Biotransformation with Spores
Many fungal spores have as many useful en-
zymes as vegetative or grown cells. The fungi
are cultivated under conditions that induce high
spore yields. The spores are then separated from
the mycelium and used as catalysts in a similar
way as mentioned in the previous section. Pro-
cesses using spores have the same advantages
as those using grown cells. Spores are usually
more stable than cells. Some types of spores can
be stored for several years without loss of con-
version activity. They usually do not germinate
during the reaction, but in some cases germina-
tion is required for maximum conversion activ-
ity. The 11-α-hydroxylation of progesterone by
 Biotechnology 53

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

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

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-
trapped in -carrageenan are viable and reproduce
without loss of activity per unit cells [154]. The
continuous production of ethanol from glucose
by S. cerevisiae in κ-carrageenan [155] and the
∆1 -dehydrogenation of cortisol by Arthrobacter
simplex in calcium alginate [156] are examples
of such successful operations. Further improve-
ment of this technique is now expanding into the
application to complex multistep reactions, such
as the production of antibiotics.
6.5.5. Biotransformation with Cell-free
Enzymes or Purified Enzymes
In general, the use of cell-free enzymes or puri-
fied enzymes for conversions is expensive, be-
cause purification of enzymes is often tedious
and time-consuming. Therefore, they are not
so suitable for large-scale industrial production.
However, there are several cases in which they
are advantageous over systems using cells. Such
cases are:
1) Poor substrate or product permeability
through the cell wall
2) Problems caused by undesired side reactions
3) Commercial availability of the desired en-
zyme at an acceptable price
If an extracellular enzyme is used, cells are
not involved at all.
Cell-free enzymes can also be used as im-
mobilized catalysts [157]. The same techniques
as those used for cell immobilization are avail-
able. Immobilization of penicillin acylase [150],
l-amino acid acylase [146], lactase [152], and
glucose isomerase [151] have been applied to
commercial production. The entrapment of sol-
uble enzymes in a membrane-type reactor with
simultaneous coenzyme regeneration is an alter-
native to the use of cell-free enzymes. l-Amino
acid dehydrogenases in combination with for-
mate dehydrogenase as a coenzyme regenera-
tor and nicotinamide adenine dinucleotide cova-
lently bound to polyethylene glycol in a mem-
brane reactor have been successfully applied to
the continuous amination of keto acids yielding
optically active amino acids [158]. l-Leucine is
already produced commercially by this process.
Whitesides [159] has reviewed the potential
applications of cell-free enzymes in organic syn-
thesis.
6.5.6. Multistep Reactions Using Different
Biocatalysts
In order to perform more than two sequential re-
actions, two or more different biocatalysts are
sometimes required either simultaneously or se-
quentially. Theoretically, any combination of
catalysts can be used for such multistep reac-
tions. Examples of successful applications are:
continuous production of l-alanine from ammo-
nium fumarate via L-aspartate as the interme-
diate by immobilized Escherichia coli (aspar-
tase) and Pseudomonas dacunhae (l-aspartate
β-decarboxylase) cells [149], complete conden-
sation of racemic homocysteine by washed or
dried Pseudomonas putida (racemase) and Al-
caligenes faecalis (S-adenosylhomocysteine hy-
drolase) cells [160], complete hydrolysis of
racemic α-amino-ε-caprolactam (see Section
6.6.2.1), and continuous amination of α-keto
acids to l-amino acids by two types of cell-free
enzymes (see Section 6.5.5).
Another remarkable example is the synthe-
sis of coenzyme A from pantothenic acid, l-
cysteine, and ATP (or AMP), which proceeds
through five sequential steps (see below) and is
commercially performed solely with Brevibac-
terium ammoniagenes cells [161, 162].
Step 1: pantothenic acid + ATP →
phosphopantothenic acid + ADP
Step 2: phosphopantothenic acid +
l-cysteine + ATP →
phosphopantothenoyl-l-cysteine
+ ADP + inorganic phosphate
Step 3: phosphopantothenoyl-l-cysteine
→ phosphopantetheine + CO2
Step 4: phosphopantetheine + ATP →
dephosphocoenzyme A +
inorganic pyrophosphate
Step 5: dephosphocoenzyme A + ATP
→ coenzyme A + ADP
6.5.7. Multiphase Reaction Systems
Since their discovery, enzymes have been
thought to be inert catalysts in organic solvents.
This is basically true, but enzymes have become
available that are catalytically active in water-
miscible organic solvents. Many reactions that
are impossible in water because of kinetic or
thermodynamic reasons can be performed in or-
ganic solvents [163, 164] (e.g., transesterifica-
tion reactions [165]). Chemically modified per-
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
6.6.1. General Considerations
When designing a biocatalytic process many
important aspects require careful consideration.
Besides the essential question of the type of en-
zyme used for biocatalysis there is the selection
of a compound to be synthesized and a survey on
available substrates and routes or reactions that
are of special importance in designing a conver-
sion process (also see Chapter 5).
Figure 22. Synthesis of acetal amino acid (Bristol-Myers
Squibb)
Another interesting example is the produc-
tion of l-tyrosine and l-dopa (dihydroxyphe-
nylalanine) by bacterial β-tyrosinase [169]. This
enzyme has long been known to catalyze the α,
β-elimination of l-tyrosine to phenol, pyruvate,
and ammonia (Eq. 1).

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

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

Product 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.

6.6.1.2. Finding Suitable Enzymes
Occasionally a synthetic scheme has been de-
signed but suitable enzymes or conversion pro-
cesses are yet unknown. In such a case, it be-
comes necessary to assay suitable microbial
strains or enzymes (as described in Section
6.6.2) as to their suitability for catalyzing the de-
sired reaction. The production of l-lysine from
d,l- α- amino- ε-caprolactam (ACL) is another
typical example [172]. This process was de-
signed to utilize cyclohexene, a major byproduct
of nylon production. In this process, the biocat-
alyst is required to hydrolyze only the l-form
of α-amino- ε-caprolactam (l-ACL) to yield l-
lysine (Fig. 23).
Figure 23. Hydrolysation of the l-form of -amino- -capro-
lactam (l-ACL) to yield l-lysine
Two microorganisms were found during the
assay: one of them hydrolyzes the l-form of the
substrate and the other racemizes the remaining
d-form. As a result, all the α-amino- ε-capro-
lactam is converted into l-lysine without requir-
ing any optical resolution.
6.6.1.3. Substrates
Any substrate is theoretically suitable for
biotransformations, provided the substrate
molecules come into contact with the enzyme.
Even gases, such as methane, are suitable
substrates; they are bubbled into the reaction
medium. The substrate should be soluble in the
medium and able to pass through the cell mem-
brane unless the reaction is catalyzed by cell-
free enzymes. The substrate is usually added to
the reaction medium, neat or as a concentrated
Biotechnology 57
solution. Sterilization of the substrate is recom-
mended prior to its use, if required.
Only dissolved substrate is converted. There-
fore, slightly soluble substrates must first be dis-
solved and the products may then crystallize
from the solution after conversion:
Substrate (solid)
Substrate (solution)

Product (solution)
Product (solid)
For example, powdered cortisol in concen-
trations up to 500 g/L is converted to crystalline
prednisolone with a good yield by Arthrobacter
simplex [173]. Substrates may also be dissolved
in water-miscible organic solvents. The lower
alcohols, acetone, dimethylformamide, and di-
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
aqueous reaction medium. This regioselective
oxidation was studied as a biocatalytic alterna-
tive for the chemical synthesis of the antihis-
taminic drug fexofenadine.
In cases where the cell membrane is imper-
meable to the substrate or a cell-free or puri-
fied biocatalyst is required, the permeability of
the cell must be improved, respectively, the cell
wall has to be disrupted. This is accomplished by
air drying, acetone drying, lyophilization, autol-
ysis, lysozyme digestion, surfactant treatment,
osmotic shock, freezing and thawing, or ultra-
sonic treatment. For cell-free preparations, the
cell debris can be removed by centrifugation or
microfiltration.
If the substrate does not suit the desired con-
version, its chemical modification should be
considered. Addition, variation, or removal of
a protecting group or modification of a func-
tional group in the substrate molecule some-
times makes its interaction with the enzyme
more efficient. In addition, these modifications
may prevent undesirable side reactions or degra-
58 Biotechnology
dation. This method is frequently used in steroid
conversions. A systematic assay for a suitable
chloroacetoacetate ester for enantioselective re-
duction yielding the l-form of 4-chloro-3-hy-
droxybutanoate, a promising precursor for the
chemical synthesis of l-carnitine, is an example
[175].
6.6.1.4. Media
Besides performing biocatalysis in aqueous me-
dia, there has developed the so-called “non-
aqueous enzymology” which has become an im-
portant area of research and development dur-
ing the last decades [176]. Enzymes exhibit a
wide array of novel reactivities and selectivi-
ties in nonaqueous solvents. For example, many
reactions that are impossible in water due to
kinetic or thermodynamic reasons can be per-
formed in organic solvents, [163, 164, 177] due
to the suppression of water-induced side reac-
tions. Improved and altered substrate specifici-
ties [178, 179] and selectivities can be observed.
Examples of practical applications are enantios-
elective synthesis [180], chiral resolution [181],
and combinatorial biocatalysis [182]. The pos-
sibility of the solubilization of hydrophobic sub-
strates or products in organic solvents opens
opportunities for the enzymatic production of
poorly water-soluble fine chemicals and phar-
maceuticals. The thermal and storage stability of
enzymes can be significantly enhanced in non-
aqueous media [164, 176, 178].
6.6.2. Selection of Biocatalysts
More than 8000 enzymes are known [125]. Al-
most all types of chemical reactions which are
also catalyzed by normal chemical catalysts are
involved. According to the desired type of con-
version and the classification of the enzymes one
can easily determine which enzyme would be
suitable for the desired conversion by inspecting
the enzyme catalogue published by the commit-
tee [125]. However, the properties of enzymes
from different sources may vary widely, even if
they catalyze the same reaction. Therefore, test-
ing is indispensable. A wide variety of biocata-
lysts are tested for the purpose of determining
their ability to carry out the desired reaction.
Such a test, also called screening, may be one
of the most important steps for a successful bio-
catalytic conversion.
6.6.2.1. Screening
Kitahara and co-workers [183] screened bac-
terial strains from cultures grown aerobically at
30–37◦ C in nutrient broth containing malt ex-
tract. Sodium fumarate and ammonium chloride
were added 24 h after growth started. After in-
cubation for another 24 h the l-aspartate in the
culture broth was analyzed by chromatography.
A strain of E. coli was selected by this screen-
ing as the most promising producer of aspar-
tase. Under suitable reaction conditions, 56 g
of l-aspartate was produced per 100 mL with a
molar yield of 99 % by using 1 g of dried cells.
Subsequently, this process was developed into
large-scale technology by which l-aspartate was
synthesized continuously with E. coli cells im-
mobilized on κ-carrageenan [184].
The screening procedure described above
may be rather simple, but it reflects the essence
of screening. Normal screening procedures can
be more complicated and deal with a great vari-
ety of microorganisms, including bacteria, acti-
nomycetes, molds, yeasts, and basidiomycetes.
6.6.2.2. Enrichment
Another method to find a suitable biocatalyst can
be enrichment. Sometimes it is desired or nec-
essary to find biocatalysts capable of perform-
ing a specific conversion from natural sources.
Soil samples containing mixed microbial popu-
lations are rich sources of test organisms. Be-
cause the enzymes in these microorganisms
modify or degrade a great variety of complex
organic compounds, at least one of the mi-
croorganisms is expected to offer one or several
enzymes that perform the desired conversion.
These microorganisms are selected using the en-
richment technique. A typical enrichment proce-
dure is the isolation of bacterial strains that per-
form the asymmetric hydrolysis of d,l-2-ami-
no- ∆2 -thiazoline-4-carboxylic acid (d,l-ATC),
an intermediate of d,l-cysteine synthesis, to l-
cysteine [185]. Soil samples were incubated in a
medium containing 0.3 % D,L-ATC as the sole

nitrogen source. After spreading each culture
on a plate of the same medium, colonies that
appeared were isolated and grown on nutrient
agar containing 0.2 % d,l-ATC as the enzyme in-
ducer. The resulting cells were then transferred
to a solution containing 1 % d,l-ATC and the
formation of l-cysteine was demonstrated by
chromatographic or microbiological methods.
Among 1975 colonies from 388 soil samples,
31 l-cysteine-producing bacteria were isolated.
One of them, Pseudomonas thiazolinophilum,
was mutagenized. Mutants with inhibited l-
cysteine metabolism were screened from the
mutagenized cultures. One of these mutants con-
verted the added substrate almost stoichiomet-
rically to l-cysteine with a yield of 31.4 mg/mL
[186].
The enzyme system of Pseudomonas thia-
zolinophilum hydrolyzes d,l-ATC in two steps
as shown in Figure 24.
Figure 24. Asymmetric hydrolysis of d,l-2-amino- 2-
thiazoline-4-carboxylic acid (d,l-ATC) to l-cysteine
Although screening sometimes is regarded
as old-fashioned, time-consuming, and even il-
logical, some consider it to be one of the most
promising methods for finding new genes in mi-
croorganisms.
6.6.2.3. Molecular Engineering
Sometimes enzyme yields in wild-type organ-
isms are low or the desired enzyme is not found
in the available microorganisms. In this case,
molecular engineering techniques can lead to an
enzyme catalyzing the desired reaction. There
are two different strategies that represent the
“state-of-the-art” technologies: rational design
and directed evolution [153]. Both technologies
Biotechnology 59
require different knowledge of the target enzyme
as well as laboratory equipment (Table 13).
Table 13. Requirements and molecular methods for rational design
and directed evolution technique
Rational design Directed -evolution
Requirements: 3D structure of the high-throughput-
target enzyme screening/selection
technology to analyze
enzyme properties
knowledge of reaction
mechanism
modeling of
enzyme-substrate
interaction
Molecular methods: site-directed random mutagenesis
mutagenesis
6.7. Improvement of Conversion
Processes
After an adequate process has been designed, it
must be optimized. This includes improvement
of the strain and the search for optimal growth
(highest enzyme yield and maximum activity)
and optimal reaction conditions.
If the enzyme to be used is constitutive, the
amount of biomass containing that enzyme will
have to be increased. This is usually 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-
other structurally related compound, will be re-
quired. If the enzyme is repressive, the repres-
sion factor will either have to be eliminated or the
repressive compounds, which may be present in
the growth medium or formed during the growth,
must be removed.
Catabolites of an easily metabolizable car-
bon source, e.g., glucose or sucrose, and the
end product of a metabolic pathway usually
cause catabolite or product repression, respec-
tively. Therefore, the composition of the growth
medium, growth period, and various physical pa-
rameters of growth, such as temperature, aera-
tion, agitation, and pH, must be carefully con-
trolled. In successful cases, the concentration of
the required enzyme reaches more than 10 % of
the total soluble protein [187].
Optimal physical and chemical conditions for
the biotransformation itself must be determined
with the same care as for the optimization of
growth conditions. This is especially important
60 Biotechnology
if side reactions are observed, because they de-
crease product yield and make product isolation
difficult. Physical or chemical treatment of the
biocatalyst, such as heating, pH change, and the
addition of detergents, organic solvents, or spe-
cific inhibitors may cause specific inactivation of
undesired enzymes. For example, the trans-N-
arabinosylation of adenine from synthetic uracil
arabinoside (Fig. 25) yields adenine arabinoside,
an antiviral agent.
Figure 25. trans-N-arabinosylation of adenine from syn-
thetic uracil arabinoside
This reaction is catalyzed by cells of En-
terobacter aerogenes and takes place only at
temperatures above 60◦ C [188]. The reaction
does not proceed below 50◦ C because hypo-
xanthine is formed by adenine deaminase be-
fore the trans-N-arabinosylation by two kinds
of nucleoside phosphorylases takes place. For-
tunately, these phosphorylases are still active at
60◦ C, whereas the deaminase is completely in-
activated at this temperature. Other examples are
summarized in Table 14.
In the case of immobilized biocatalysts, con-
centration gradients of substrates and products
may occur that could lead to decreased biotrans-
formation rates. These gradients can be reduced
by decreasing the Thiele modulus, e.g., by us-
ing smaller carrier particles or by reducing the
diffusion distance between the free solution and
the immobilized enzymes.
Microbial conversion can also be optimized
by improving the selected strain. Standard meth-
ods of strain improvement include the isolation
of single colonies, optionally after mutagenesis,
and screening of the best suited isolates. Such
conventional genetic manipulation techniques
have been developed during the last decades for
the improvement of useful strains. Recombinant
DNA technology is providing new prospects for
strain improvement. Plasmid or phage vectors
can transfer DNA fragments of microbial, plant,
animal, or even synthetic origin into a suitable
recipient microorganism. These gene cloning
techniques are still restricted to microbial strains
such as E. coli, B. subtilis, and S. cerevisiae, in
which the genetic background and the 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
high chemo-, regio-, and stereoselectivity and
the ability to catalyze reactions under mild con-
ditions.
During the last couple of years, many new
methods and techniques have been developed
to overcome common problems. Some exam-
ples here might be molecular engineering tech-
niques such as rational design or directed evo-
lution to increase low biocatalytic activities
[153], biocatalysis in nonaqueaous reaction me-
dia to bypass solubility problems of substrate
and/or product [179], or the applications of high-
throughput screening methods for the search
of new and suitable biocatalysts. Another field
of interest that will become of great impact is
“metagenomic research” that will enable scien-
tists to find new biocatalysts with desired quali-
ties [192].
To make new biocatalysts attractive for in-
dustrial applications they have to be available
in sufficient amounts. High expression rates of
enzymes can be achieved by genetical and phys-
iological manipulations of the expressing mi-

Table 14. Elimination of side reactions by physical or chemical treatment
Desired reaction
Ammonium fumarate → l-aspartic acid
(Escherichia coli/aspartase)
Fumaric acid → l-malic acid (Brevibacterium
ammoniagenes/fumarase)
l-Aspartic acid → l-alanine (Pseudomonas
dacunhae/aspartate β-decarboxylase)
Cholesterol → 1,4-androstadiene-3,17-dione
(Arthrobacter simplex/multistep conversion)
β-Sitosterol → 17-ketosteroids (Nocardia
species/multistep conversion)
d,l-2-Amino-∆2 -thiazoline-4-carboxylate →
l-cysteine (Pseudomonas
thiazolinophilum/multistep conversion)
croorganism such as optimizing the number of
plasmids per cell or inactivation of “negative”
cell functions (e.g., deletion of protease coding
genes). Apart from deleting unwanted side activ-
ities also novel metabolic pathways can be im-
plemented. These microorganisms, customized
only to fulfill special needs for biocatalytic ap-
plications, are called “designer bugs” or “tailor-
made microorganisms”.
7. Downstream Processing
Downstream processing is one of the most un-
derestimated steps in bioprocesses and it is well
known especially in the pharmaceutical industry
that downstreaming is the most expensive and
unfortunately the most ineffective part of a bio-
process. Thus, one can assume that new devel-
opments are widely described in the literature.
Unfortunately, this is not the case. Only a few
working groups focus on new and more effective
procedures to separate products from fermenta-
tion broths or biotransformations. A chief char-
acteristic of biotechnology is the wide variety
of products. Due to this variety, a broad spec-
trum of separation techniques must be applied.
However, for nearly all products one starts with
a dilute suspension and tries to produce a highly
purified dry product. In the case of extracellular
products, the solids in this suspension may in-
clude intact organisms, other insoluble fractions
of the medium or natural sample, and perhaps in-
soluble products. Concerning intracellular prod-
ucts the solids include in addition fragmented
Biotechnology 61
Side reaction Treatment Reference
fumaric acid → l-malic incubation of culture broth at pH 5 [149]
acid and 45 ◦ C for 1 h
fumaric acid → succinic treatment of immobilized cells with [148]
acid 0.6 % bile extract
l-alanine → d-alanine incubation of culture broth at pH 4.75 [149]
and 30 ◦ C for 1 h
further decomposition of addition of α,α -dipyridyl (1 mM) to [137, 189]
the sterol molecule the culture broth
further decomposition of addition of α,α -dipyridyl (0.3 mM) [190]
steroid ring to the conversion mixture
further degradation of addition of hydroxylamine or [186]
l-cysteine semicarbazide to the reaction mixture
mycelia caused by cell disruption, which is nec-
essary to gain the products. According to this
starting point, nearly each downstream process
consists of the four following steps [193]:
– Removal of insoluble particles
– Isolation of the product
– Purification
– Polishing
With respect to downstreaming in laboratory
scale, one is normally only limited by the avail-
able equipment. However, with regard to upscale
processes one should have in mind that the de-
veloped process should also be applicable in in-
dustry. Thus, Belter et al. [193] have formulated
the following questions, which are also crucial
to biotechnology downstream processes:
– What is the value of the product?
– What is the acceptable product quality?
– Where is the product in each process stream?
– Where are the impurities in each process
stream?
– What are the unusual physicochemical prop-
erties of the product and the principal impu-
rities?
– What are the economics of various alternative
separations?
In addition, it is important to use materials
which are available for all upscale processes,
since the downstream behavior of the product
may change while changing, for example, the
type of chromatographic resin or membrane ma-
terial. Normally the recovery costs exceed the
bioprocess costs; however, some upstream pa-
rameters influence the downstream processing:
62 Biotechnology
– The characteristic properties of the producing
microorganism or cell line
– The location of the product
– The stability of the product
– Byproducts and impurities
– Concentration of the product in the medium
from which it is to be recovered
A general downstream scheme based on these
parameters is given in Figure 26.
Figure 26. General downstream scheme in biotechnology
The main aims of the primary separation step
are to achieve a volume reduction and to make a
first stage purification of the product by remov-
ing dissimilar components from the broth. The
most important techniques in use are:
– Membrane processing
– Ion exchange chromatography
In the membrane process, ultrafiltration and
reverse osmosis are often used for separation,
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-
nium silicate, titanium carbide, etc [197]. The
dispensed sample is introduced into the grind-
ing chamber on a continuous basis. The level of

disruption depends on the variable turning speed
of the bead mill.
By using bead mills, cell disruption can be
achieved in a single run with better temperature
distribution and temperature control. Another
possibility for cell disruption or at least product
liberation is the use of microwaves or ultrasound
which can be combined with sample extraction
by organic solvents. Microwave techniques are
widely used in acid digestion of solid samples.
Their use in the extraction of organic analytes
from environmental samples is less widespread,
despite the availability of commercial devices
for this purpose and their potential for reducing
analysis time and solvent consumption.
A possibility for cell lysis under very mild
conditions is the use of hydrolyzing enzymes.
In addition, enzymes offer selectivity during
product release. Enzymes hydrolyse the walls
of cells, and when sufficient wall has been re-
moved, the internal osmotic pressure bursts the
periplasmic membrane allowing the intracellu-
lar components to be released [195]. The ef-
fect of lytic enzymes is specific to particular
groups of cell types, which is attributed to the
differences in cell wall composition. For exam-
ple, the most efficient lytic enzyme for bacte-
ria is lysozyme from hens’ egg. This enzyme is
also used in large-scale processes for enzyme
production [198]. Even more highly specialized
procedures for sample disruption can be applied
in biotechnology. For example, high yields of in-
tracellular enzymes from yeast can be obtained
by applying a series of electric field pulses [199].
By using this technique, up to 90 % of the total
activity can be released without any further or
previous treatment of the cells. The method is
based on electroinduced changes in the cell en-
velope leading to a leakage of part of the intra-
cellular proteins without the formation of debris
and permits the treatment of large volumes.
7.2. Solid–Liquid Separations
Solid–liquid separation is a very important
procedure during downstream processing in
biotechnology for cell separation, cell debris re-
moval, and also for product recovery. The most
important solid–liquid separation techniques in
use for are filtration and centrifugation. Filtra-
tion separates solids from a liquid by forcing the
Biotechnology 63
liquid through a solid support or filter medium.
Dead-end filtration and cross-flow filtration are
two different designs of filtration which can be
used. In dead-end filtration mode, the total pro-
cess fluid stream flows through the membrane.
The retained solids accumulate on the membrane
and build up a filter cake. The membrane has
to be changed when the membrane pores are
clogged by the solids. When the feed flow is
directed parallel to the membrane surface, the
term cross-flow filtration is used. The tangential
flow of liquid removes any retained molecules or
particles from the membrane surface, which re-
sults in a stable flux for a longer time period. By
using cross-flow filtration, relatively low shear
stress is possible and filter aids are not required.
In addition, scale-up is simple and cell washing
is possible in a single process step.
Over the last 30 years, a number of fur-
ther membrane processes have been developed
for molecular separation. These filtration tech-
niques can be divided into four major groups:
reverse osmosis (hyperfiltration, RO), nanofil-
tration (NF), ultrafiltration (UF), and microfil-
tration (MF) [200]. Membrane processes are
easy to scale up and the possibility of using the
same materials and configurations in different
sizes from laboratory to process scale reduces
the validation effort enormously. However, fil-
tration processes are limited with regard to se-
lectivity. The fractionation of proteins can only
be achieved with large differences in the molec-
ular weight of the proteins and it is important to
keep in mind that a certain difference in the mo-
lecular weight of two proteins does not mean the
same degree of difference in molecular size. Pro-
teins that differ in molecular weight by 10 times
may differ in size by only 3 times when in glob-
ular or folded form. Another problem encoun-
tered when using membrane processes for prod-
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-
teraction of the sample with the membrane ma-
terial. This often depends on unspecific protein
adsorption which is affected by several factors
[201]. Thus, different membrane types have to
be screened to minimize the unspecific binding
of the sample when a new filtration procedure
has to be developed.
64 Biotechnology
7.3. Product Recovery
One of the most important procedures in product
recovery are chromatographic processes. Dur-
ing chromatographic steps the sample is sepa-
rated into several fractions according to inter-
actions between the different molecules in the
liquid phase and the stationary phase (chromato-
graphic resin). The molecules can be separated
according to their size and/or charge and based
on hydrophobic/hydrophilic or affinity interac-
tion with the stationary phase. Column chro-
matography utilizes a vertical column filled with
the solid support with the sample to be separated
placed on top of this support. The rest of the
column is filled with a solvent (eluent) which,
under the influence of gravity, moves the sam-
ple through the column. Differences in rates of
movement through the solid medium are trans-
lated to different exit times from the bottom of
the column for the various elements of the orig-
inal sample. Based on the specific interactions
between the sample in the mobile phase and
the stationary phase the column chromatogra-
phy can be performed as:
– Ion-exchange chromatography,
– Size-exclusion chromatography (gel-
filtration chromatography),
– Reversed-phase chromatography, and
– Affinity chromatography.
Immobilized-metal-ion affinity chromatogra-
phy (IMAC) as a special form of affinity chro-
matography is a popular and powerful way to
purify proteins. It is based on the specific co-
ordinate covalent binding between histidine or
other unique amino acids and various immobi-
lized metal ions (e.g., nickel).
Most of the chromatographic systems are
run in a batch or quasicontinuous mode. Nowa-
days, new developments such as simulated mov-
ing bed chromatography [202] and anular chro-
matography [203] offers the possibility of con-
tinuous processes. One problem in column chro-
matography are the difficulties in upscaling. If
the column radius is increased, unless special
packing techniques are employed, the packing
procedure becomes inefficient and the pack-
ing itself unstable. In addition, to maintain the
optimum mobile phase velocity, the flow rate
will need to be substantially increased and the
consumption of mobile phase will eventually
become economically impractical. Conversely,
if the column length is increased, then the
impedance to flow will become greater lead-
ing to high column pressures. If large column
radii are employed, then the mechanical strength
of the column system will limit the maximum
permissible pressure. Consequently, lengthen-
ing the column will eventually require the parti-
cle diameter to be increased to provide adequate
permeability. Increased particle diameter will, in
turn, reduce the column efficiency, which may
impair the resolution of the compounds of inter-
est [204].
Membrane. Until today, the required pro-
cess steps for the product recovery are carried
out separately, which leads to many energy-
intensive process steps such as filtration or chro-
matography and production of vast wastewa-
ter amounts, respectively. Membrane adsorp-
tion allows the integration of several process
steps into one-unit operation, which means the
downstreaming of biotechnologically produced
proteins can be carried out by saving process
time and resources [205, 206]. In addition to
this, conventional chromatographic techniques
which are based on packed columns often re-
quire lengthy procedures. This can lead to the
degradation of sensitive proteins [207]. Further-
more, packed columns exhibit a high pressure
drop across the column and a slow diffusion-
controlled binding process of solutes within the
matrix [208]. Whereas membrane systems show
several advantages to packed columns, most im-
portant, mass transfer takes place through con-
vection rather than through diffusion. Due to
this fact, membrane adsorbers enable a time-
effective performance with high flow rates with-
out high back pressure [209]. Membranes can
be converted into efficient adsorbers by attach-
ing functional groups to the inner surface of
synthetic microporous membranes. Affinity ad-
sorption, ion exchange, or immobilized-metal
affinity chromatography can be carried out by
these membranes. Membrane ion exchangers
of strong acidic (sulfonic acid), strongly ba-
sic (quarternary ammonium), weakly acid (car-
boxylic acid), and weakly basic (diethylamine)
types are commercially available. A chelating
membrane based on the iminodiacetate (IDA)
group is applicable for IMAC. Membrane ad-
sorber technology has several major advantages
compared to classical separation methods. Due

to the membrane structure, the binding of pro-
teins is not limited by diffusional processes,
therefore loading and elution can be performed
at very high fluxes resulting in very short cycle
times. Compressibility of the membrane under
normal operation conditions can be neglected,
channeling cannot occur, and the pressure dis-
tribution inside the modules is designed to have
plug flow through the module, all of which lead
to sharp breakthrough curves. Scale-up is very
easy, materials and systems allow cleaning in
place (CIP), and the validation of the process
is made easier due to of standard products and
validation service of suppliers.
7.4. Solvent Extraction
Solvent extraction is the most common method
for the recovery of hydrophilic substances and,
therefore, a method for separating well-soluble
metabolites from cultivation media. Classical
extraction processes use organic solvents, which
are often rarely suitable for effective recovery of
the solute. Recently, new extractions have been
developed which form specific adducts with the
metabolite in question and allow its recovery
with high efficiency and selectivity [210]. Sol-
vent extraction in biotechnology focuses on the
recovery both of primary metabolites (e.g., eth-
anol, acetic acid, citric acid, and amino acids)
and of secondary metabolites (e.g., antibiotics
or vitamins). The concentration of secondary
metabolites is usually much lower than that
of primary metabolites. Since most secondary
metabolites are for use as therapeutics, the qual-
ity requirements of the products are high. Sol-
vent extraction can help to fulfill these require-
ments. In addition, the set-up of integrated bio-
processes (production and downstreaming) can
be performed by solvent extraction [211]. Super-
critical fluid extraction (SFE) becomes also an
important tool in biotechnological downstream
processing since it offers important advantages
compared with other solvent extraction meth-
ods. It is possible to work in an oxygen-free
system, which prevents oxidation. The low tem-
peratures applied minimize thermal degradation
and microbes or their spores are not soluble.
In addition, supercritical fluids for extractions
are inexpensive. The successful implementation
of this technique can lead to improved sample
Biotechnology 65
throughput, more efficient recovery of analytes,
cleaner extracts, economic replacement of halo-
genated solvents, and a high level of automa-
tion, compared to conventional sample prepa-
ration procedures [212]. Supercritical fluid pro-
cesses are being commercialized in the polymer,
pharmaceutical, specialty lubricants, and fine-
chemicals industries. Supercritical fluids are ad-
vantageously applied to increase product perfor-
mance to levels that cannot be achieved by tra-
ditional processing techniques.
8. Monitoring and Modeling of
Bioprocesses
The requirement to operate biotechnological
processes in a cost-effective manner by simulta-
neously maintaining a high product quality and
safety is becoming increasingly relevant. Histor-
ically, the most important means of achieving in-
creased productivity from bioprocess plant has
been through time- and resource-consuming ex-
perimental developments. More recently, how-
ever, significant advances have been made in the
area of computer applications for bioprocess su-
pervision, modeling and control, and their intro-
duction in process industry.
Typically, little use is made of the large
amounts of bioprocess online and laboratory as-
say data after an experiment is completed. Re-
cently, attempts have been made using enhanced
automation strategies to improve the quality
of information presented to operators and in-
crease the level of automatic process supervi-
sion, although few industrial applications have
yet been reported. On a plantwide perspective,
the scheduling of process operations is also tra-
ditionally manually undertaken by experienced
personnel by using a trial and error approach
with a planning board. This also appears to be
a potential area for modern bioprocess manage-
ment.
Knowledge-based systems, such as fuzzy
logic systems, can be designed to cope with un-
certainty and allow the coupling of quantitative
information with the qualitative or symbolic ex-
pressions (in the form of heuristics), so as to
reproduce the actions of an experienced process
operator. They might therefore be used as an in-
telligent, online virtual assistant to the process
66 Biotechnology
operator, or, with extra knowledge as a supervi-
sor in running and maintaining bioprocess oper-
ations within optimal operating conditions. The
main objective in this approach is to develop an
expert supervisory system which uses biopro-
cess and control knowledge in the form of rules
in conjunction with bioreactor state estimation
(soft sensing), parametric identification and pro-
cess control algorithms running in real time. In
addition, provision needs to be made for pro-
cess models and known bioprocess behavioral
characteristics to be incorporated in order to en-
hance the overall supervisory control strategy.
The complete system aims to perform process
monitoring, process control, sensor validation,
fault detection, and diagnostic tasks and, in ad-
dition, provide recovery advice so as to achieve
continuous online optimization.
Collateral with the above approach is the on-
line determination of the critical key variables
which govern process production. The measure-
ment and estimation problems are well known
and represent the main drawback concerning an
effective biosystem optimization. Three ways of
reducing the difficulties encountered can be pro-
posed:
– Better sensors
– Better sampling and automatic analysis sys-
tems
– Online estimation of difficult to measure, or
immeasurable, variables.
Although all of these are attracting research
effort, the techniques of online state and parame-
ter estimation are receiving the greatest interest.
Whilst developments in biosensors are in some
cases helping to contribute to the online determi-
nation of bioprocess parameters, they are by no
means at the stage where general applicability
has been achieved. Until this is the case, the de-
velopment of online estimation techniques will
provide the major means by which online closed
loop feedback control of critical bioprocess vari-
ables can be achieved.
Several different approaches can be adopted
in the development of bioprocess estimation al-
gorithms. The easiest but potentially least accu-
rate way of obtaining an estimate of a “difficult
to measure” primary process variable is to estab-
lish a correlation with a measurable secondary
process variable, whilst ignoring measurement
errors, noise, etc. A more robust approach might
be to adopt a numerical estimation technique,
either for estimating the parameters of a prede-
fined model structure, usually of a generalized
linear time series form, or directly to obtain an
estimate of a difficult to measure primary pro-
cess variable (state estimation or software sen-
sor). An alternative approach is to exploit the
nonlinear mechanistic structure of the biopro-
cess, if known, in the form of a state observer.
Neural Computing. A relatively new devel-
opment in artificial intelligence is the area of
neural computing. Neural networks are dynamic
systems composed of highly interconnected lay-
ers of “simple” neuron-like processing elements.
In the field of process engineering, it is their
ability to capture process nonlinearities which
offers potential benefits. Other useful charac-
teristics are their ability to adjust dynamically
to environmental and time-variant changes, to
infer general rules from specific examples and
to recognize invariances from complex, high-
dimensional data. These properties provide neu-
ral networks with the potential to outperform
other “learning” techniques. Given a series of
experimental data the network is able to estab-
lish the governing relationships in these train-
ing data. This ability can be exploited to aid the
nonlinear modeling, control, and optimization
of complex processes, as well as being used in
existing predictive control methods.
8.1. Characteristics of Bioprocesses
8.1.1. System Definition
A biotechnological process consists of a system
of chemical, biochemical, and microbiological
reactions, incorporating process-engineering as-
pects such as mass and energy conservation,
thermodynamics, or heat and mass transfer cor-
relations. (Fig. 27). The biological part of the
system represents the peculiarity of the system
characteristics and causes the great difficulties in
considering it from the system theoretical point
of view.
Common for all system models is their map-
ping of inputs to outputs, of stimuli to responses
in some with the additional introduction of state
variables. Therfore, the task is to define proper
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
those which are chosen by the operator to ma-
nipulate the system in such a way the benefits are
maximized. Typical examples in bioprocess en-
gineering are: temperature trajectories, agitation
rate, or feed rate of key components. Random
forcing functions can be seen as outer distur-
bances of the system whose origin comes from
the system environment and not from internal
uncertainties, e.g., impurities of the inoculums
or the feed medium. To evaluate the system dy-
namics, it is important to define meaningful tra-
jectories of the control variables. Keeping the
values constant will not result in a representative
information retrieval of the biosystem. Only if
characteristic curves – or specific combinations
– are chosen to which the system reacts sensitive
a representative process description is possible.
Therefore, it is evident that the experimental as
well as the theoretical design must be performed
from deep problem awareness.
The state variables −
→
z (t) represent storage el-
ements for mass quantities (e.g., substrate, prod-
ucts, biomass, inhibitors, interferences), infor-
mation, or energy of the system. The state vector
→
−z (t) is composed of any set of quantities suffi-
cient to completely describe the behavior of that
system. Given a state vector at a particular point
in time and a description of the system forcing
functions, in an appropriate uncertainty formu-
lation, and control functions from that point in
time forward, the state at any other time could be
computed. In biotechnological processes state
variables are frequently defined, if the key quan-
tity of interest can not measured directly and
must therefore determined by state estimators
from other process variables [213, 214]. The in-
troduction of state variables is not strictly nec-
essary. But in their absence inner relation can
only be hardly understood and described. Also
a direct mapping of an input vector to an output
vector without consideration of state quantities
in form of a signal model, as it is typical in con-
ventional signal analysis in electrical engineer-
ing, can not be accomplished. Their application
depends on the point of view. A signal model
provides mainly information about the behav-
ior of changing influencing parameters and how
they affect the outputs based upon given inputs.
No information can be provided about the intrin-
sic reactions or the basic mechanism, the focus
is outside of any interpretation purposes [215,
216]. Mostly, they are used for software sensors
[217, 218] or optimization purposes [219, 220].
Both represent black-box applications (see be-
low), where only the outputs are of special inter-
ests, the inner relations or interpretation aspects
are secondary.
As output variables − →y (t), very often quanti-
ties are specified which are accessible by online
measuring systems or which can be estimated
by software algorithms. They can be seen as
the result of a measurement model which relates
the state variables −→z (t) to the output variables
→
−y (t). These quantities represent the values with
whose the system can be evaluated. The output
vector −→y (t) is strongly related to the aspect of
observability (see below).
The division into parameters, state variables,
or output variables is not an immanent property
of a system, but a specific view by the analyzer
of the system. It can be changed in relation to the
mode of process operation, to the process targets
or the simplifying assumptions of the model. A
state variable zi can be exchanged to an output
variable yi and vice versa. A mathematical sys-
tem should provide a coherent description of the
entire bioprocess including all relevant system
aspects. The degree of complexity of the biopro-
cess or the possibility for a simplified modeling
of some parts of the system is determined by
the intended application and by the skills of the
system analyzer.

Parameters p can be seen as fixed quantities
of the system. In the ideal case, they should be
time invariant, as it is indicated by their original
definition. However, sometimes a time depen-
dency of parameters is introduced to gain a bet-
ter system description. Normally, the parameters
are used to interpret the biosystem characteris-
tics. However, the meaning of the parameters
can be quite different depending on the point of
view from which the biosystem is described. In
terms of a mechanistic approach using funda-
mental physico–chemical equations, the deter-
mined parameters, after they are fitted by ex-
perimental data, have a direct physical meaning
and can be used for interpretation purposes and
are comparable between different experiments.
If the behavior is characterized phenomenolog-
ical, by means of descriptive equations, the pa-
rameters determined are only valid under the
specific boundary conditions of the experiments.

They can be quite different in different exper-
iments and possess nearly no general validity.
One must be very careful when comparing dif-
ferent approaches upon the quantities of these
parameters.
8.1.2. System Description
A biotechnological system consists in particular
of a set of chemical, biochemical, and microbi-
ological reactions whose components, reactions
rates, and other characteristics as temperature,
pH-value, or internal energy, are mostly not ex-
actly known. The metabolism of the biological
unit is a very complicated process, not only be-
cause the intracellular reaction network may be
quite complex, but also due to the large num-
ber of inner relations, control loops on reaction
level, regulatory mechanism, and genetic level
that are overlaid to coordinate the elementary re-
actions. So even when the microkinetics could
be measured exactly, it would be impossible to
establish a correct system map in every detail
without the introduction of rigorous simplifica-
tions. Another reason for uncertainty is the inho-
mogenity of the units (e.g., cells) in the whole en-
semble, which is normally not considered when
characterizing biosystems. The relations are not
constant and vary with time. Furthermore, there
might be a morphological differentiation bio-
logical unit which is accompanied generally by
changes in the dynamic behavior.
Therefore, the interesting question is why
these complex systems can often be described
for specific process targets by a few mathemat-
ical equations only. One reason is that the func-
tional blocks of the biosystem operate together
in a network of regulation, and exchange of
mass, charge, and energy and a few bottleneck
processes determine the behavior of the whole
system. Another reason is the tremendous num-
ber of units in the ensemble of a biosystem which
hides individual variations in their growth and
leads to a smoothed average behavior. In most
cases, the problem shifts. It is not the difficulty to
build up the equations, but it is hardly to deter-
mine the characterizing parameters, which are
generally dependent on time and other system
state quantities. Furthermore, a few reactions
can already determine the rates of many others.
When the main inputs and the final overall out-
Biotechnology 69
puts are known, a prediction is often even possi-
ble with incomplete information on the involved
intracellular reaction network [221 – 223].
For analysis and design of those systems, two
aspects have to be considered – the biological re-
actions catalyzed by the bioactive compound,
and the numerous chemical and physical pro-
cesses which precede, accompany, and follow
them. Within the biosystem, the most important
physical processes, which are intimately bound
to the biological reactions, are associated with
transport of material, energy, and information to
and from the location of the bioactive unit. The
dependency of the biological reactions on the
microenvironment around the unit is called mi-
crokinetics. Due to the transport phenomena, the
microenvironment will vary along different lo-
cations in the systems. The integral description
of microkinetics in connection with the trans-
port processes that may be included in a sys-
tem model is called the macrokinetics. Ideally,
one would always attempt to model the transport
phenomena and the microkinetics of the model,
because this model could be combined with
proper reactor models to predict the macroki-
netics of different biosystems. Unfortunately, at
present, the effort for simulation of detailed re-
actor models is rather high and methods for an-
alyzing the microenvironment of the cells are
still rare. Therefore, what is usually observed
is always a kind of macrokinetics. To find a
compromise between these facts and our limited
knowledge about the process, the description
of the biological system is often characterized
by means of so-called formalkinetic approaches.
This means a formal application of system equa-
tions that represent actually microkinetics to a
process, where only averaged macrokinetics can
be measured. As an immediate consequence, the
model parameters will change when the oper-
ating conditions of the reactor are changed, or
even more, if a different reactor with changing
boundary conditions is used. This necessity lim-
its the predictive power of formalkinetic models
for biotechnological processes significantly.
In principle, independent of the available sys-
tem volume, the mass balance of a component i
from the general point of view can be described
using a finite volume approach solving the gov-
erning equations for fluid flow and heat and mass
transfer:
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

biotechnological processes is encountered [232,
233]. The so-called “general reactor model” –
firstly introduced by Bastin and Dochain [232]
– tries to unify chemical, biochemical and mi-
crobiological process using the same approach.
The bioprocess, an n-dimensional system of or-
dinary nonlinear differential equations, is based
on a simple representation of the biotechno-
logical processes, where superfluous details are
omitted. Only the dynamics of those compo-
nents that play an important role in the process
are considered, irrelevant byproducts or nonlim-
ited substrates are neglected. The stoichiometry
of the chemical reactions is only qualitatively
taken into account by means of yield coefficient
Yζ(·)/c(·) or stoichiometric coefficients k∗ij (Eqs.
8 and 9).
M ∗
dςi (t)  ∗
= kij ϕj (ς1 ( t),. . .,ςN (t) ,T (t))
dt j=1
∗
M dςj
dt
J=1
Yςj /c∗ = dc ∗
i i
dt
where ci (·) and ζi (·) are concentrations of the
different (bio-)chemical components, ci (·) are
those substances (i*=1, . . . ,N**) whose re-
actions are calculated by the yield coefficients
Yζ(·)/c(·) , ζi are those components (j=1, . . ., N*)
whose turnover are determined by the dominat-
ing reaction rates ψj .
M∗ Number of the dominating reactions in the
considered volume
ψJ (·) Reaction rate of the j-th reaction
T (·) Temperature
In general, these coefficients do not have any
mechanistic meaning in contrast to the above-
mentioned approach, instead they represent for-
malkinetic quantities, which must be estimated,
determined experimentally, or established by
practical experiences. This allows the inclusion
of chemical, biochemical, and microbiological
processes in a unified approach. However, the
reaction scheme may be inconsistent with the
law of conservation of mass. Also cell-to-cell
heterogeneity, e.g., due to different proliferation
phases, are not considered and simplifies a segre-
gated viewpoint to an unsegregated perspective.
The reactions rates can be often a very com-
plex function of the operating conditions and of
the state of the biosystem. In the case were ψ
Biotechnology 71
is proportional to the specific growth rate of the
organism µ, there exist several possible models
[234]. This approach is very common in liter-
ature (overviews in [235, 236]) and can be as-
signed – in contrast to the above-mentioned ap-
proach – to the class of inverse problems.
It is typical for inverse problems that in many
situations the quantities that we wish to deter-
mine are different from the ones which we are
able to measure. If the measured data depend,
in some way, on the quantities we want, then
the data at least contain some information about
those quantities. Starting with the data that we
have measured, the problem of trying to recon-
struct the quantities that we really want to know
is called an inverse problem; we measure an ef-
fect and want to determine the cause. Typical
applications of inverse problems are model iden-
(8) tification and estimation, image analysis, numer-
ical analysis, or navigation [237, 238].
(9) 8.1.3. Dynamics of Biosystems and
Real-Time Considerations
Real-time considerations play an important rule
dealing with process control strategies. A noto-
rious underestimation of the dynamic properties
of microbial and cellular populations exists and
results mainly from matching the duration of the
respective batch cultivations to the relevant time
constant of the biosystem under investigation.
However, metabolic regulation of enzyme activ-
ities and fluxes often takes place on a time scale
of seconds rather than days although the latter
may also be true for some processes. It is there-
fore in the scope of promoting biotechnological
research to adopt and develop appropriate exper-
imental concepts, methodologies and equipment
in order to consider all relevant time constants
[239].
What is fast? What is slow? What is relevant?
The last question is the most important when
dealing with modeling. The relaxation time con-
cept of Harder and Roels [240] (Fig. 28) maps
typical time constants of microbial and cellular
control on the level of modification of enzymes
(activation, inhibition, dis-/association of sub-
units, covalent modification or digestion) to the
range of milliseconds to seconds, on the level of
regulation of gene expression (induction, repres-
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
evolution to days and larger units. Considering
mainly the growth of microorganisms, typical
time scales of 0.3 to 20 h are relevant. The exam-
ples discussed below will illustrate how bioengi-
neering is facing the individual time constants.
A typical bioprocess, if operated in batch
mode, extends over several hours or a few days.
If operated in continuous mode, it is not rea-
sonable to accept operating times of less than a
month (see, e.g., Heijnen et al. [241]). If an or-
ganism has special physiological features such
as baker’s yeast, a transition from one to the other
domain (e.g., from low to high dilution rates or,
in other words, from purely oxidative to oxidore-
ductive growth) may also result in considerable
changes in the time required to approach a new
steady state. Axelsson et al. [242] reported a
rough estimate for this case: the time constant
for experiments at low dilution rates (D<DR,
where DR=D at which the regulatory switch bet-
ween oxidative and oxidoreductive metabolism
occurs) is, as expected, in the order of the mean
residence time (τ =D−1) however, above DR,
the time constant was predicted to be at least
one order of magnitude greater. In experiments
specifically designed to verify this, either even
greater time constants or no unique stable steady
states at all were found [243].
If excess carbon and energy source is pulsed
to a carbon- and energy-limited culture, the
intracellular ATP concentration initially drops
because of the phosphorylation sink (glucok-
inase and/or hexokinases). Only later, during
catabolism, can the energy provided by this ex-
tra substrate be liberated in terms of new ATP.
The duration of the ATP sink was predicted to be
in the order of a few tens of seconds by Nielsen
et al. [244]. Obviously, such rapid reactions are
under kinetic control and the necessary enzyme
activities are present in sufficient quantities. It
is not clear, at present, whether the fluxes attain
their organism typical maximal values immedi-
ately or some fine tuning of enzymatic control
precedes this event. It is likely that in the latter
case a regulation of (intracellular) enzyme ac-
tivity, if at all necessary, takes place on the level
of enzyme modification (e.g., phosphorylation)
rather than on the level of de novo production
(i.e., control on the transcriptional level) because
the latter would require much more time [245].
The dynamics of microbial cultures have an
important impact on the characteristics of mea-
surement and process control. The “typical time
constant” in a bioprocess is often erroneously
anticipated to be equivalent to the entire duration
of cultivation. However, the quantitative inves-
tigation of substrate uptake requires a time reso-
lution of a few 100 ms, otherwise artifacts must
result [246, 247]. This statement was evidenced
only after a suitable technique had been estab-
lished: glucose metabolism was stopped within
100 ms by spraying the cell suspension from the
overpressurized bioreactor into 60 % methanol
which was pre-chilled to −40◦ C.

The relevant relaxation times of a culture sys-
tem are determined by the actual cell density and
the specific conversion rate (capacity) of the cul-
ture, that is, by one or more operational and state
variables (for instance feed rate, the concentra-
tions or activities of cell mass and of effectors,
if relevant) and inherent characteristic proper-
ties of the biosystem which are represented by
parameters. There are metabolites with a long
lifetime and other (key) metabolites with very
short lifetimes, e.g., molecules representing the
energy currency of cells such as ATP and other
nucleotides. Rizzi et al. [248] and Theobald et
al. [249] have shown that the energy charge re-
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
pulses or shifts are applied to cultures of differ-
ent recent history [245, 250]. Neubauer et al.
[251] found that substrate oscillations greatly af-
fected the growth performance of E. coli. Short
term, that is, less than 2 min, glucose excess, and
starvation were investigated in a looped system
of a stirred tank and a plug flow reactor. The
metabolism changed within some few minutes
totally from anaerobic to aerobic and vice versa.
Similar observations have also been made for
yeast [252].
Two other paradigms demonstrate that the
band width of relaxation times is extremely
broad: 1) the time required to achieve a new
steady state in a chemostat culture is approxi-
mately determined by (µmax −D)−1 . 2) The time
required by a culture to consume a considerable
fraction of small amounts of residual substrate
during sampling – and thus systematically fal-
sify the analytical results if no appropriate inac-
tivation takes place depends, among others, on
the cell density and can also be in the order of a
few seconds only.
Even simple static models are very valuable
for compensation of systematic errors built into
automated analytical procedures. One important
example is the case when sampling requires a
well-known but non-negligible time. A bypass
behaves as a plug flow type reactor fraction
where flow dependent spatial gradients develop
and where no inactivation can take place because
the bulk of the bypassed aliquots are returned
to the reactor. The cells continue to consume
substrate while they are being transported from
the exit of the reactor to the filter. The permeate
Biotechnology 73
recovered is representative for the filter site but
not for the reactor. Knowing the transport time
and some basic kinetic parameters one can easily
compensate online for such errors provided that
a useful estimate of the actual biomass concen-
tration is available. Even though a bypass can
be tuned to operate at a mean residence time
of 5 s or less, this can be enough for a signifi-
cant decrease in substrate concentration in high-
density cultures. Sample buses need a minimal
(dead) tune for transportation of the sample and
in situ filters tend to fail in high density cultures
because of rapid fouling. Hence, the problem is
real and can not be ignored.
8.2. Biotechnological Measurement
Systems
All measurements have the ultimate goal of
creating representative information from the
biosystem involved. Cellular or biochemical
quantities are the primary variables in biopro-
cess engineering. They mainly determine the
performance of the bioprocess and are therefore
of special interest [253, 254]. Measurement and
acquisition of information are building the fun-
dament for all process observations as well as for
the development of new bioprocesses. They re-
present the presupposition for process optimiza-
tion and control. However, they must be avail-
able online to exploit them in a control strategy.
Measurements are not only key issues in modern
process development, they also open the door to
a detailed process supervision and understand-
ing, and avoid restricted views when analyzing
processes just through a keyhole [255, 256].
In comparison to other disciplines such as
physics, mechanical or electrical engineering,
sensors useful for online monitoring of biotech-
nological processes are comparatively few; they
are mostly available for physical and chemical
quantities rather than for biological ones. The
reasons are manifold, but generally biologically
relevant information is much more difficult and
complex to access and interpret than those for
typical physical or chemical quantities [257].
Other important reason derives from restricting
requirements for the sensor equipment, namely
– Sterilization procedures,
– Stability and reliability over extended periods,
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

A series of sensors and methods, that can
be automated and have appeared in the re-
cent decades for the measurement of the to-
tal biomass without discrimination of any seg-
regation (e.g., active or inactive cells). Many
of them rely on optical measuring principles
[259, 260], others exploit filtration character-
istics [261, 262], electrical properties of sus-
pended cells [263, 264], or thermodynamic laws
[265]. One has to decide according to the spe-
cific application, which method seems to be ap-
propriate concerning the boundary conditions,
because all methods reply on indirect measur-
ing principles, and the access of the informa-
tion carrying signal differs in non general ranges.
To get online information about the actual state
of the biomaterial techniques based on fluores-
cence principle, especially the 2-D fluorescence
spectroscopy, should be mentioned. It represents
a noninvasive method to analyze intracellular
compounds. Technically, either intra- or extra-
cellular fluorophores (e.g., NAD+ , Riboflavin,
Tyrosine, ATP) are excited by visible or ultra-
violet light, and the fluorescent light emitted by
the fluorophores at a longer characteristic wave-
length is collected and gives information about
their concentration [266 – 268].
Substrate. The presence of sufficient and
appropriate starting material (substrate) is the
cause of any biotransformation and represents
the supposition of the product formation. One
can solve the inverse problem, namely conclude
that biological activities cease whenever an es-
sential substrate is exhausted, and thus omit
the measurement of the substrate, provided the
progress of the bioprocess and/or product for-
mation is known [269]. But, this is not always
a proper solution because there are many more
plausible, and also probable, reasons for a de-
crease in bioactivities than just their limitation
by depletion of a substrate. One must, then, solve
the direct problem, namely analyze the relevant
quantities of the biological unit. From an engi-
neering point of view this measurement should
be available instantaneously in order to be able to
control the desired process characteristics. In en-
vironmental biotechnology, in particular, the ob-
jective of a bioprocess can be to remove a starting
material as completely as possible rather than
making a valuable product. So, the starting ma-
terial is identical to the process product. In some
Biotechnology 75
other bio- or food technological processes, the
conversion of a substrate from specific state of
aggregation to another is of special interest. For
example, in the case of fouling of heat exchang-
ers where dissolved proteins are transformed to a
solid protein layer, the starting material changes
only its state not its chemical structure. Or, con-
sidering drying or thawing where water changes
its state (or is removed) which strongly influence
the quality and behavior of the biomaterial in-
volved, but no (bio-) chemical conversion takes
place. The classical methods to determine these
quantities are offline laboratory methods so far.
This implies that samples are taken, sometimes
aseptically, pre-treated, and transported to a suit-
able location, where storage of these samples
might be necessary before analyzing manually.
These steps need a lot of human and instrumen-
tal resources.
Product. The product is almost the only rea-
son why a process should run. The main concern
is in maximizing the profit, which depends di-
rectly on the volumetric productivity and/or on
the purity of the resulting product. It is there-
fore of essential interest to know the quantities,
which require measurements. What was said
above about substrate determination and the ap-
plication of classical methods is valid here, too.
In summary, bioprocess science needs sig-
nificantly more quantitative measurements. It is
insufficient to know that something happens, we
need to know almost instantaneously, online and
automatically why, how, and to what extent a
bioprocess behaves [270].
8.2.2. Online Sensing Devices
The development of increasingly sophisticated
equipments for measuring of starting material
and products represents one trend to satisfy
the growing demand for high-quality measure-
ments. In contrast to engineering disciplines
such as mechanical or electrical engineering,
in bioprocess engineering almost no measure-
ment performed is directly, i.e., none is ob-
tained by immediate comparison with a refer-
ence quantity. Most measurements are achieved
by means of some specific physical or chem-
ical property of the process value, often hid-
den in the very complex sample matrix com-
76 Biotechnology
mon to bioprocesses. This very complex ma-
trix, which consists of the numerous substances
and parameters surrounding the analyte and their
mutual influences, makes the task of measuring
difficult and leads to the development of more
and more complicated measurement techniques
[271, 272]. The surrounding equipment, i.e., for
the sample pretreatment, exceeds the pure an-
alytical core [273 – 275]. However, the greater
the measuring system complexity, the more dif-
ficult it is to ensure the accuracy and the reliabil-
ity of the collected data. The need for accuracy
and reliability increases even more if the data of
the process analyzer should be used as input for
a closed-loop controller. When unreliable mea-
surement systems are used, the system opera-
tor can only hardly decide whether an occurring
variation is due to an error of the analyzer or a
result from the bioprocess itself.
The final goal of the measuring unit of a bio-
process is to build up simple online instruments
in order to collect all necessary information from
the biosystem, which is necessary to hold the de-
fined process targets. Therefore, the process tar-
gets should represent the selection criterion for
defining the measuring equipments. The infor-
mation should be available online and no manual
interaction should be necessary to obtain the de-
sired measuring results. This aspect touches the
term “system observability” (see Section 8.4.1),
which is strictly defined for linear systems, but
eludes a definition for nonlinear systems as they
are common for typical biological systems [276,
277]. It is not obvious and definable whether
all system information is available und known
to reach the observability in respect to the pro-
cess goals or which sensors are still necessary
to reach the specified process goals. The conse-
quence is that there are normally one or more
state variables (or linear combinations of them)
that are hidden from the view of the observer
(i.e., measurement equipment).
Concerning the information treatment there
can generally be made different classifications,
which are important for industrial applications.
From the communications engineering point of
view the information can be distinguished bet-
ween online and offline. If a continuous auto-
matic correlation between the signal from the
sensor and the product or process quantity is pos-
sible an online signal is available, otherwise it
is offline. A measurement is called inline, when
a direct interaction between a product property
and sensor exist, otherwise exline; this is more
a technological characterization. Depending on
the site of installation, one discriminates further
between in situ, which means built-in, and ex
situ, synonym for a bypass configuration or for
an exit line. In the latter case, the withdrawn
volumes are lost for the process. A further clas-
sification can be made by the mode of operation
of the sensing device. One can discriminate bet-
ween continuous and discontinuous signal gen-
eration in a predefined time step. This is a very
important fact for planning and developing an
appropriate process controller considering the
time scales of the bioprocess. In the latter casem,
one has to guarantee that the process information
is available in real time.
Only online sensors are of special interest
concerning the process control of biotechno-
logical systems. Indeed there exist a couple of
different, very powerful systems for the offline
analysis of nearly all relevant substances. For
online sensors, the situation is quite different.
In situ instruments exist only for a few quanti-
ties like the measurement of temperature, pH,
pressure, oxygen, carbon dioxide, density, flow
rates, electric power consumption, or redox po-
tential. They use different measuring principles
and are widely introduced in industrial process
engineering.
However, for the most important biologi-
cal quantities in biotechnological systems es-
pecially the measurement of active or inactive
biomass or the specific determination of individ-
ual biochemical substances there exists a deep
lack in online instruments. In the following, prin-
cipal techniques should be presented, which pos-
sess a certain online capability. See also → Bio-
chemical Separations; → Mass Spectrometry;
→ Surface and Thin-Film Analysis.
Chromatographic Methods. A review of
chromatographic methods is beyond the scope
of this contribution. Both (high-pressure) liquid
chromatography and gas chromatography have
been applied in numerous cases to offline anal-
yses of biotechnological samples, but online ap-
plications, especially for industrial processes,
have only recently been reported [278 – 282].
The scope of chromatographic methods is the
separation of the individual constituents of mix-
tures as they pass through columns filled with a

suitable stationary phases. As disadvantages the
consumption of almost expensive reagents must
be mentioned. The samples injected are to be
pretreated in such a way, that they possess nearly
no impurities, like gas bubbles or solid compart-
ments. Any existence of those interferences dis-
turbs the analysis in a significant manner, and
manual service procedures are mostly the con-
sequence. Furthermore the measuring time last
up to 30 or 40 min; a time period which is of-
ten too large for bioprocess supervision. But also
economical aspects must be taken into consider-
ation. Chromatographic systems possess a high
cost price. Furthermore, high-specialized staff
must service them frequently, an aspect, which
represents the major drawback for an industrial
application.
Mass spectrometry. A further measurement
method, in principal suitable for online applica-
tions represents mass spectrometry (MS). Mass
spectrometry has been mainly applied for the on-
line detection and quantification of gases such as
pO2 , pCO2 , pN2 , pH2 , pCH4 , and even H2 S, or
volatile substances such as alcohols, acetoin and
butandiol [283]. Generally, the detection princi-
ple allows simultaneous monitoring and, con-
sequently, control of metabolites. The princi-
ples, sampling systems, control of the measur-
ing device and application of MS for biopro-
cesses have been summarized elsewhere [284].
Of course, mass spectrometry requires very ex-
pensive equipment and needs frequent and com-
plex service procedures. But it should be taken
into account that automatic multiplexing of dif-
ferent sample streams is possible and, in addi-
tion, a great variety of different substances can
be determined simultaneously. Thus, one has to
decide, whether the high economical and appli-
cational expense is justifiable in comparison to
the advantages due to the acquisition of infor-
mation.
Biosensors. The most promising technique
to overcome these problems, represented in the
last decade, is the use of biosensors . Biosensors
consist of a sensing biological module of either
catalytic (e.g., enzymes or microorganism) or
affinity reaction type (antibodies, cell receptors)
in intimate contact with a physical transducer
(electrochemical, optic, calorimetric or acous-
tic sensor). The latter finally converts the (bio-)
Biotechnology 77
chemical into an electrical signal (overviews in
[285 – 287]). In spite the fact, that principally
for nearly all biochemical substances an biolog-
ical recognition unit exists, biosensor applica-
tions in an industrial surrounding often concen-
trate only on a small group of substrates, namely
glucose, ethanol, or lactate [288 – 292]. Gener-
ally, biosensors are tricky to handle and must
in principle be recalibrated in the matrix where
the measurement should take place every time
the process conditions change. Due to the sen-
sible biological element – an enzyme or living
microorganism – they principally cannot be ster-
ilized. They also suffer from changes in the envi-
ronment, for example, changes in pH or forma-
tion or existence of aggressive chemicals such
as H2 O2 [293]. Therefore, they must be used
in a suitable environment. Furthermore, the sur-
rounding must be constant, because in general
an interrelation to the endogenous matrix exists.
If the process media changes significantly, the
biosensor alters its behavior. A compensation of
the changed matrix interferences becomes nec-
essary. This represents one of the most decisive
drawbacks in inline biosensor applications for
bioprocess engineering [294 – 296]. The long-
term stability under working conditions is often
poor; they must be serviced for several hours per
week preferentially by high-specialized staff. In
this way, only limited experience could have
been gained under technical process monitoring
conditions. They possess only in some excep-
tional cases a real industrial potential.
A reasonable way around the problem repre-
sents the measuring of the biotechnological me-
dia after removing a sample from the reactor in
a bypass configuration. Consequently, the sen-
sor is not installed in situ as it would be opti-
mal but the information could be provided online
and can be fed into an automatic process control
system. Depending on the analyte of interest,
i.e., whether it is soluble or (in) the dispersed
phase, one needs to sample either the entire cul-
ture liquid or just a supernatant. The latter can
be acquired using for example a membrane mod-
ule. Concerning sampling, four aspects must be
taken into account.
1) The system must be opened in such a way, that
no infections can enter the reaction space ei-
ther during sampling or between the sampling
events.
78 Biotechnology
2) A representative sample must be provided. So
the overall volume of the sampling chamber
as well as the position of device at the biore-
actor must be determined properly. Addition-
ally, the sample taken can still continue to re-
act on the way to the sensor. In this case the
sample would not be representative for the
interior of the reactor, and appropriate addi-
tional measures or modeling approaches need
to be taken in order to assure representativity.
3) When native samples are applied to the ana-
lytical system, problems can arise from matrix
interferences and matrix effects [297]. There-
fore, the sampling device should be chosen
in that way that no interaction with the matrix
occur which interferes the measurement (e.g.,
absorption of substrate at the membrane). On
the other side, the sample device should with-
hold all the cross-sensitivities for disturbing
co-components.
4) Due to the high number of individual sys-
tem components and their complex interac-
tion (i.e., sample pretreatment and analytical
part), sampling devices provoke a small reli-
ability in regard to the required process time.
Furthermore, the hardware, necessary for the
whole analysis system entails high original
costs. The specific adaptation to one concrete
process condition implies inflexibility. It ex-
cludes the determination of other metabolites
in other process media. Its adaptation is only
possible by time- and money-consuming al-
terations.
Flow Injection Analysis. The most impor-
tant technique of sampling methods in biotech-
nology, behind some conventional devices like
membrane modules, represents the flow injec-
tion analysis (FIA), firstly introduced by Ruz-
icka and Hansen [298]. It can be viewed as a
general solution-handling technique or sampling
device combined with a sensing unit. This com-
bination causes a high flexibility with respect
to the combined analytical procedure. It can be
seen as a principle where a small injected vol-
ume (10-200 µL) is introduced into a continuous
unsegmented stream of carrier. The sample dis-
perses in a well-defined concentration gradient
and is transported by the carrier stream to the
reaction zone and a subsequent detector mod-
ule. FIA cannot generate continuous signals.
But, there are several important advantages like
a high sampling frequency (up to >100 h−1 ),
small sample and reagent consumption, high re-
producibility and nearly total versatility of the
sensing methods.
Software Sensors. The most promising
method to get online access to important key
components of bioprocess quantities are soft-
ware sensors. In general, software sensors sup-
ply the estimation of the missing measurements
by using an appropriate model that relates the
corresponding variable with other physical or
chemical measurements that are correlated to it
in any way. The fact that the model is imple-
mented by means of a software package (hence
the expression soft(ware)-sensor) denotes, that
software sensors provide a software backup for
unavailable sensors, as an alternative to a hard-
ware back-up using spare sensors [299 – 302].
Generally, software sensors are typical solu-
tions of so-called inverse problems (see Section
8.1.2). In a complex biological system, in par-
ticular, the quantities which are normally easiest
to measure are the variables, not the parameters.
In the case of metabolism, the usual parameters
of interest are the enzymatic rate and affinity
constants which are difficult to measure accu-
rately in vitro and virtually impossible in vivo
[303 – 306]. Yet to describe, understand, and
simulate the system of interest we need knowl-
edge of the parameters. In other words, one must
go backwards from variables such as fluxes and
metabolite concentrations, which are relatively
easy to measure, to the parameters.
In other words, when using software sensors
there must always be a model available that re-
liably relates the measured variable with the tar-
get variable or parameter of interest. Normally,
measured variables are easily measurable effects
that are caused and influenced by the target. It
is the special objective of the software sensor to
reach a maximal degree of generalization. How-
ever, this is very difficult – even impossible – to
achieve or furthermore to prove this claim. Thus,
the basic question is: Is the available informa-
tion representative enough to generate a model
for the accurate estimation of the quantity of in-
terest, or is the desired information inaccessible
by the chosen sensor collection? Consequently,
the development of software sensors is often re-
stricted to a specific application; any transfer to

other measuring problems is almost combined
with considerable alterations. This does not con-
cern only the determination of the new param-
eter at an identical model structure. Instead a
complete new system definition can be neces-
sary, where new input variables must be added
or dispensable one can be deleted.
Spectroscopic Methods. In this context the
use of spectroscopic methods [307 – 310], after
optical or sonic stimulation play a dominant rule.
They must be seen as multisensor systems. Their
common property is the detection of absorption
degrees after stimulation at various frequencies.
By this, a set of information is generated, which
possesses more information than the measure-
ment at one specific frequency, the measurement
at one frequency can be seen as the output of one
sensor. The multivariate evaluation in an appro-
priate model should subsequently reveal the ac-
curate determination of the desired quantity. In
such models, normally pure data are used, the
integration of knowledge from well-known fun-
damental equations is only very hardly to real-
ize. The most important advantage of spectro-
scopic methods is their non-invasive character.
The sensing device is not in direct contact with
the media or the process itself, so no interference
is to be expected.
The situation is even more complicated since
things develop in relation to time. The estimation
of the physiological state of a culture involves
more than one (measurable) variable at a time
and the recent history of this set of individual sig-
nal trajectories involved. Consequently, physio-
logical state estimation requires recognition of
complex patterns. Various algorithms used for
this purpose to build up the (data-)model have
in common that it is not always the present values
alone that are evaluated, there is always the re-
cent history of signal trajectories involved. Con-
cerning the models, some authors define soft-
ware sensors only on the basis of neural net-
works but a broader point of view should be
adopted since software-sensor models are also
obtained by using regression or correlation tech-
niques as well as fuzzy logic or first-principle
models or combinations of all of them [306,
311 – 314].
In some cases, the data describing the actual
state and their recent history are compared with
so-called reference patterns: these are data from
Biotechnology 79
historical experiments or runs which an expert
has associated with a typical physiological state.
A physiological state is recognized either if the
actual constellation matches any one of the ref-
erence sets best – in this case, there is always an
identification made – or if the match exceeds a
predefined degree of certainty, e.g., 60 % – then
it can happen that no identification or associa-
tion is possible when the pre-selected threshold
value was not reached. The direct association
with reference data needs normalization (am-
plitude scaling) and, probably, frequency analy-
sis in order to eliminate dependencies on (time)
shifts, biases or drifts.
In other cases, the data trajectories are trans-
lated into trend qualities via shape descriptors.
These combinations of trends of the trajectories
of various state variables or derived variables
define a certain system state; the advantage of
this definition is that the association is no longer
dependent on time and on the actual numerical
values of variables and rates [315].
8.2.3. Further Aspects Concerning
Measuring Systems
Online measurements produced with in situ sen-
sors are difficult to validate. The usual proce-
dure for evaluating the quality of a measure-
ment is restricted to calibration/checking prior
to and after an experimental run. A few sensors
such as the pCO2 - or the Cranfield-glucose sen-
sor [316] allow removal and, therefore, recali-
bration of the transducer during a run. External
chromatographs and FIA systems can be regu-
larly recalibrated but the sterile interface cannot.
Other sensors such as a pH or a pO2 probe can be
mounted via a retractable housing, which allows
either sterile exchange or withdrawal for exter-
nal recalibration during a run. A further possi-
bility to gain information about the reliability of
a measurement is to mount a number of iden-
tical sensors in easy-to-compare positions and
to check the individual signals for equality. This
opportunity has been exploited in particular at
applications where a high process safety must be
guarantied. It is highly desirable to have alterna-
tive principles of measurement at hand, which
operate simultaneously (heterogeneous redun-
dancy) or to introduce a self-supervision op-
80 Biotechnology
tion, like pattern recognition to generally iden-
tify malfunctioning sensors [276].
Elemental balancing permits the determina-
tion of (other) metabolic rates provided that the
stoichiometry is known. Carbon balances are the
most useful but the carbon lost via the exhaust
gas (as CO2 ) and culture liquid (as HCO− 3 ) must
be measured. Heinzle et al. [317] determined
that the state predictions based on experiments
with a small mass spectrometer are not use-
ful due to unacceptable error propagation; for
instance, a 1 % relative offset calibration error
could result in a prediction error for an intracel-
lular storage material (PHB) of >50 %. Instead,
using a highly accurate and precise instrument
(absolute errors <0.02 % gas composition) to-
gether with automatic, repetitive recalibration
resulted, however, in reasonable estimation of
substrates, PHB, and biomass. Others have also
experienced these findings, e.g., [318, 319].
The undelayed evaluation of the state of a cul-
ture by using software sensors and computers,
based on the quantitative analytical information
provided by hardware sensors and intelligent an-
alytical subsystems, constitutes an excellent ba-
sis for targeted process control. Experts – either
human or computer – have the data and the deter-
ministic knowledge to trace observed behavior
back to the physical, chemical, and physiologi-
cal roots thereby gaining a qualitative improve-
ment of bioprocess control, a quantum leap: pro-
cess control can act on the causes of effects rather
than just cure symptoms. A simple standard op-
erating procedure [320] has proven useful in this
concern, namely:
– Measure everything that can be measured at
the very beginning of process development
– Decide whether or not a variable is relevant
– Determine the relevant variables to be mea-
sured, controlled, and/or documented
– Collect all raw data at any time and distin-
guish online between variable and parameter
behavior
– Organize an archive of all these data accord-
ingly and do not discard seemingly useless
data since they contribute to the treasure of
experience
If it is, as some people say, correct that today’s
bioengineering with all its tools and methodolo-
gies is too slow and not efficient enough, then it is
all the more urgent to improve the performance
of the methods, tools, and equipment currently
available and to invent new and better ones. In
essence, techniques of instrumentation, opera-
tion, and causal analytical interpretation of mea-
surements need massive impulses.
8.3. Cognitive Computing
Artificial neural networks (ANNs) are dating
back to 1943, firstly reported by McCulloch
and Pitts [321], then fallen into oblivion, but
started a tremendous comeback in engineering
science with the publications of Rumelhart in
1985, who introduced – again – the backpropa-
gation algorithm and demonstrated its potential
as a learning procedure in neural networks [322,
323]. Fuzzy Logic has been introduced by Zadeh
1965, firstly ignored consequently by techni-
cians because of their undeterministic and un-
certain nature, but provoked a second wave of
interest in the 1980s predominantly in Japan
in respect to their potential in treating highly
complex, nonlinear multiple input multiple out-
put (MIMO) systems in technical applications
[324]. John H. Holland published 1975 his clas-
sic work “Adaption in Natural and Artificial Sys-
tems”; it can be seen as the pioneer work in
evolutionary programming [325]. Compared to
other mathematical principles, these three meth-
ods are fairly new approaches and did not gain
not exclusive acceptance by scientists and engi-
neers. The three methods mentioned above can
be seen as the main representatives of a class
of problem-solving methods taking nature as a
model, especially how it has learned to solve
problems. They can be summarized to the area
of cognitive computing.
It is not within scope of this contribution
to explain these methods in their basic prin-
ciples and functionalities. Rather, their intrin-
sic principles are assumed to be well known;
overviews and extensive explanations can be
found for fuzzy in [326 – 328], for ANN in
[328 – 330] (see also → Process Control En-
gineering, Chap. 13) and for evolutionary pro-
gramming in [331, 332]. In the following, the
focus lies on two methods, on fuzzy logic system
and on ANN, because of their high applicational
potential, especially in bioprocess engineering
[333, 334]. The explanations should show their
potential, typical aspects and also some critical
 Biotechnology 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
attentive processing of stimuli. This leaves unad-
dressed the higher cognitive functions involved
in reasoning, decision making, planning, or con-
trol. The nonlinear neurons and synapses in our
brain perform these functions; they can deal with
ill-posed problems or with a high degree of un-
certainty. Natural selection evolved this capabil-
ity in exemplary manner. Furthermore, it is the
attempt of engineers to copy exactly this capa-
bility to technical problems by developing and
using fuzzy logic systems or artificial neural net-
works.
Both methods try to imitate nature and both
estimate input–output functions, in spite of to-
tally different underlying principles. In contrast
to statistical estimators, they estimate a function
without a mathematical model of how outputs
depend on inputs; this represents their main fea-
ture. They learn “from experience” with numeri-
cal, sometimes, linguistic sample data. Learning
can be accomplished either by changing the sys-
tem structure or by changing the system param-
eter; both principles are described for both algo-
rithms [336 – 339]. Thus, the principle modus
operandi is comparable. After the system prob-
lem is defined, the procedure can be divided into
learning phase and prediction phase. The learn-
ing phase of ANN is quite obvious. Input data
and corresponding output data are provided to
the ANN; the system changes its structure or pa-
rameter, to minimize a predefined cost function.
At first sight, the adaptive and learning character
of fuzzy systems is not obvious. Indeed, there
exist some techniques where the fuzzy system
is built up autonomously based upon available
data sets [339, 340]. On the other hand, in most
heuristic applications a highly experienced tech-
nician does the “learning” step. He collects data,
recognizes the intrinsic information, and formu-
lates upon this information rules upon sets. Thus,
the modeler performs the learning step and its
result is introduced into the fuzzy system.
The most important features intelligent sys-
tems may show are generalization and learn-
ing. All intelligent systems should generalize.
Their behavioral repertoires exceed their learned
repertoire in that way, that intelligent systems
associate similar responses with similar stim-
uli, small input changes produce small out-
put changes. Furthermore, intelligent systems
should learn or adapt. They learn new associ-
ations, new patterns, new functional dependen-
cies. They sample flux of experiences and en-
code new information. They compress the sam-
pled flux into a small but statistically repre-
sentative set of prototypes or exemplars. Sam-
ple data, provided directly or transformed into
linguistic rules, changes the system structure or
parameters. In all cases, learning means chang-
ing. Neural networks learn patterns, functions,
or probability distributions to recognize future
patterns, filter future input streams of data, or
solve future combinatorial optimization prob-
lems. Fuzzy systems can learn or formulate as-
sociative rules to estimate functions or control
systems, either directly by means of data and an
optimization add-on or guided by a highly expe-
rienced supervisor. It can be shown under some
assumption that a neural net can approximate
to any degree of accuracy using a fuzzy expert
system and vice versa [341].
Neural and fuzzy systems ultimatively learn
or estimate some unknown probability function
p(x). The probability density function p(x) de-
scribes the distribution of vector patterns x, a few
of which the neural or fuzzy system samples.
When a neural or fuzzy system estimates a rela-
tion r: X → Y, it in effect estimates the joint prob-
ability densities p(x,y). Then the solution points
(x,r(y)) should reside in high-probability regions
of the input–output product space X × Y. We do
not need to learn if we know p(x,y). We could
proceed directly to our computational task with
the techniques from numerical analysis, combi-
natorial optimization, calculus of variations, or
other mathematical discipline. The need and the
extent to learn varies inversely with the quan-
tity of information available. Supervised learn-
ing uses or creates class–membership functions.
It compares every input with the corresponding
class D and proceeds learning based upon the
difference. Unsupervised learning system pro-
cesses each sample x but does not know that
x belongs – or not – to class D. Neither super-
vised nor unsupervised learning systems assume
knowledge of the underlying probability density
function p(x).
8.3.1. Fuzzy Logic Systems
Is uncertainty the same as randomness? Bay-
esian statistician Dennis Lindley [342] stated

that probability is the only sensible description
of uncertainty and is adequate for all problems
involving uncertainty. Lindley directs his chal-
lenge in large part at fuzzy theory, the theory
that all things admit degrees, but admit them de-
terministically. Although, both expressions are
used simultaneously in the same context, proba-
bility and fuzziness differ conceptually and the-
oretically. In this contribution some important
differences are illustrated, more theoretical as-
pects and proves can be found elsewhere [343].
Fuzzy sets can be more compared with pos-
sibilities than probability. However, probabil-
ity and fuzziness also share many similarities.
Both systems describe uncertainty with num-
bers in the unit interval [0,1], consequently de-
scribe it numerically. Both systems combine sets
and propositions associatively, commutatively,
and distributively. The key distinction concerns
how the systems jointly treat a set A and its op-
posite Ac . Classical set theory demands A∩Ac ,
and probability theory conforms: P(A∩A)=0.
So A∩Ac represents a probabilistically impossi-
ble event. But fuzziness begins when A∩Ac =0,
hence P(A∩A)=0.
Fuzziness measures the degree to which an
event occurs not whether it occurs. Probability
describes the uncertainty of event occurrence.
Whether an event occurs is “random”; to what
degree it occurs is fuzzy. Consider an illustrating
example. The probability this book will be pub-
lished is one thing. The degree to which it will
be published is another. From this book, only
some special chapters may be edited or the whole
entity gets public; this is typical for fuzziness.
Fuzziness is a type of deterministic uncertainty;
it handles with ambiguities. Unlike fuzziness,
probability dissipates with increasing informa-
tion. In fuzziness the uncertainty arises always
form the simultaneous occurrence of two prop-
erties. More formally, does mA (x), the degree to
which element x belongs to fuzzy set A, equal the
probability that x belongs to A? This statement
can be true or not, it depends on the problem
existing, but in fact the point of views are quite
different and so far hardly to compare. The fol-
lowing explanation should not explain the fuzzy
steps in great detail, The focus lies upon two as-
pects. How are the sets defined and which rules
can be determined. This is judged as the cru-
cial aspects for fuzzy application in bioprocess
engineering [344].
Biotechnology 83
Following the introduction of fuzzy sets,
Zadeh began steadily to develop the necessary
inferencing mechanisms and modeling tech-
niques to bring this concept to fruition [345].
In this period, it was recognized that fuzzy logic
could aid in the development of control systems
in which nonlinearities and time variance make
the development of traditional control systems
very difficult. In these cases, a human operator is
often capable of controlling the plant, so a fuzzy
controller can often be designed based on the op-
erator’s expert knowledge. Since that time, con-
trol applications based upon fuzzy logic models
have been extensively investigated [346]. Fuzzy
logic systems are similar to expert systems in
their use of linguistic relationship, but the sub-
stantial difference is that the outputs are in gen-
eral continuous by variation of the inputs [347].
It is exactly this property which causes the high
interest in mathematical modeling, and hence in
engineering science.
Mapping with fuzzy logic is analogous to
classical modeling. The maps have variables
which influence system behavior and relation-
ships among the variables which describe the
system. In classical models, variables have real
number values, the relationships are defined in
terms of mathematical functions. In fuzzy logic
however, the values of variables are expressed
by linguistic terms such as “large, medium, and
small”, the relationships are defined in terms of
“if-then” rules. By using defuzzification tech-
niques, exact numerical outputs are calculated
from fuzzy subsets.
A fuzzy subset A is defined by a membership
function mA (x i ), where x i is the domain, of the
variable on which A is defined.
mA (xi ) ∈[0,1] ∀ xi ∈A (10)
The value of mA (x i ) for each x determines
the degree to which each element in the domain
belongs to A. Although both classical and fuzzy
subsets are defined by membership functions,
the degree to which an element belongs to a clas-
sical subset is limited to being either zero or one.
This means that m(x) may only be a step func-
tion. In fuzzy logic, the degree to which an ele-
ment belongs to a subset may be any value in the
interval [0, 1]. Since mA (x i ) may be defined by
any function, it is easy to see that a classical sub-
set is a special case of a fuzzy subset [348]. The
fuzzification process transforms exact values x
84 Biotechnology
of fuzzy variable vj to mAi (x i ) for all subsets Ai ,
which are defined for the variable vj . More rig-
orous definitions of membership functions may
be found elsewhere [327, 349].
One technique for determining the shape of
membership functions is seeking knowledge of
the system from experts then constructing mem-
bership functions that represent expert opinion.
Observations of many experts should be pre-
ferred. Based upon this observations, member-
ship functions can be constructed using statis-
tical methods accompanied by using the analy-
sis of preferences [350, 351]. Many researchers
have investigated more “rational” techniques
for determining membership functions. One of
these approaches represents one variant of fuzzy
clustering [352, 353]. It requires a set of input
and/or output data from the system to be col-
lected. Patterns of data are found within the input
or output space such that the variance within the
sets is minimized and the variance between sets
is maximized. The prototype, or center point, of
a cluster X is determined and a distance metric is
used to determine the degree of membership that
a value has to X. Another technique for determin-
ing membership functions involves neural net-
works. Neural networks are networks of simple
processors connected by adjustable weighting
factors [328]. The weights are adjusted by using
a backpropagation or other known algorithm to
minimize the difference between the predicted
output and the measured output. The parameters
of membership functions can be “learned” by a
neural net from a set of input–output data [354,
355]. Genetic algorithms have also been used
to determine the optimal shape for membership
functions [356, 357].
For fuzzy subsets to be useful in “modeling”,
there must be a way to define relationships bet-
ween them. This is accomplished by means of
their membership functions mAi (x) with logical
operators and inferences, to extract ambiguities.
There are three basic operators in fuzzy logic, (1)
OR, (2) AND, and (3) NOT, which link two or
more membership functions mAi (x) of different
variables. The activation level of each rule is nor-
mally equal to that of the premise (activation).
All rules are evaluated. If some sets of an output
variable are activated more than once, an aggre-
gation method must be chosen to determine the
final activity. After defining the interferencing
method, the output fuzzy sets are defuzzified to
get a precise value, for further explanation see
[327, 349].
Expert knowledge is the most common tech-
nique for determining rules. The expert is asked
to summarize the knowledge about the system in
the form of cause and effect relationships. From
these the rules are formulated. When no experts
are available or a more analytical approach is
desired, other techniques of system identifica-
tion must be used. One approach is to apply a
set of metarules to adaptively acquire informa-
tion about the system. Linguistic self-organizing
controllers issue control actions and observe the
environment [358]. The metarules evaluate the
performance of the control actions from the ef-
fect of the control actions on the environment.
The metarules then determine whether the con-
trol actions should be modified and perform the
modification, if necessary. This technique has
been applied especially to robotics applications
[359]. Fuzzy classifier systems are another way
to discover rules. Fuzzy classifier systems are
generalizations of genetic classifier systems. A
genetic classifier system uses some of the ideas
from genetic algorithms to develop expert sys-
tems [360, 361]. Standard genetic algorithms
have also been used to discover rules [362].
Sugeno and Yasukawa use the second variant of
fuzzy clustering [fuzzy clustering methods can
also be used to determine the membership func-
tions (see above). Both approaches are addressed
by the term “fuzzy clustering”] on data in the
output space to determine fuzzy rules [363]. The
clusters in the output space are used to induce
clusters in the input space. Then, input space
clusters are projected onto the axes to find the
fuzzy subsets for the input variables. Since the
input clusters are induced from the output clus-
ters, rules can be constructed between the input
subsets and the output subsets. Yoshinori et al.
(1996) discuss another method of fuzzy model
construction based upon clustering techniques
[364]. This method produces a relational as op-
posed to a functional model, and consists of a
series of local, linear models to approximate a
global nonlinear one. Recently; neural networks
have become an active field of interest and have
been used to learn rules as well as member-
ship functions [365, 366]. However, all meth-
ods, which automatically generate rules, have
deficiencies in their interpreting ability. They are
generated to fit available data best and it is not

obvious that the rules express reasoning rela-
tions. This can be seen as a strong disadvantage
compared to the generation of rules by means of
expert knowledge.
The application area of fuzzy systems is
widespread and should only be presented here
for applications in bioprocess engineering. The
fermentation industry was one of the first to
recognize the potential of fuzzy logic for bio-
logical processes [367, 368]. Konstantinov and
Yoshida developed a fuzzy logic system for iden-
tifying physiological states in fermentation pro-
cesses and used it to control distributed an E.
coli fermentation [369]. Meanwhile the range of
applications has enlarged tremendously. In prin-
ciple, they can be divided in two fields. The first
one are those where the knowledge bases is con-
structed such that it is based upon existing (quali-
tative) information, mainly from high-specialist
staff. This represents the area of fuzzy model-
ing [370, 371], fuzzy controller [372, 373], or
estimator for biosystem states or process faults
[374, 375]. One of the intrinsic characteristics is
that significant knowledge engineering effort is
required for setting up a complete and consistent
rule base. The other domain of fuzzy logic is re-
lational model-based systems where the linguis-
tic information is extracted from available data
to build a system model. This second approach
is also directed to the field of bioinformatics
or experimental design [376, 377]. This ap-
proach does not need any preinformation about
the intrinsic relation, however a knowledge base
can arise which is hardly to evaluate or inter-
pret. Additionally, no information can be given
about the generality of the established rules. At
all accounts, when a data driven construction
is used, a subsequent mathematical or manual
evaluation, especially of the rule bases, is nec-
essary after the automatic construction of the
fuzzy system. For the process industry, expert-
knowledge-based approaches possess more in-
terests because they follow a concrete process
target. The second one can be seen as a spe-
cial form of knowledge discovery which enables
to accomplish some linguistic evaluation based
upon data, if no expert knowledge exists. In some
exceptional cases, these clustering techniques
are exploited for control purposes [378]. An ag-
gregation of both principles is also fruitful in the
case where the existing information is too little
to build up a representative “model”. In this case,
Biotechnology 85
a data driven approach can help to complete the
fuzzy structure.
8.3.2. Artificial Neural Networks (ANN)
Artificial neural networks are designed to mimic
the actions of neurons in the human brain. For
detailed information, see [328 – 330]. They re-
present massive collections of interconnected
neurons (nodes), which individually perform a
relative simple signal processing; they are inter-
connected via synaptic links. Like brains, neu-
ral networks recognize patterns or relations bet-
ween inputs and outputs we cannot even define.
They can be seen as pure function approxima-
tors whose abilities depend on the behavior of
the individual nodes, the structure of the net-
work, the learning procedure used, and on a very
strong degree of the quantity of representative
data. Not the total amount of data is crucial but
the information content with small degree of re-
dundancy. Almost the whole quantity space of
the input and output values should be spanned
by the database. Recently, e.g., Cybenko [379]
and Hornik et al. [380], have proved that any
continuous function can be approximated to an
arbitrary degree of exactness on a compact set by
a feedforward neural network comprising two or
more hidden layers and a continuous nonlinear
activation function, providing appropriate data.
Working with neural networks always con-
sists of two phases, a training and a prediction
phase. In the first one, the variable parameters

p (t) of the net are changed in that way, the
ANN fits best to the trainings database. In most

cases, p (t) represents weight parameters of the
intrinsic functions, e.g., the weight how the out-
put of a neuron contributes to the activation of
a subsequent node. On the other hand, there ex-
ist also some nets where the structure of the net
(nodes and their synapses) can be seen as their
parameters which are changed during learning.

The indicator for changing p (t) represents a
formulated performance index J. Based upon the
absolute value J, the parameters are changed in
order to minimize J.


 ∆J ∆J ∆J
min J p = , ,···,  (11)
∆p1 ∆p2 ∆pn
where pi is a parameter i with i = 1 . . . n param-
eters in the ANN.
86 Biotechnology
The definition of J depends on a couple of
different aspects, but in most cases the Euclidian
distance between the desired target and the cal-
culated output integrated over all training data
sets is used. Performing learning should result
in the global optima concerning the specified
problem. However, most training procedures are
local methods, requiring a gradient calculation.
And obviously, any algorithm that relentlessly
crawls downward must have a very lucky start-
ing position if it is to settle into the lowest lo-
cal minimum. Avoiding false minima requires
two separate procedures. First, we must elute in
the initializing phase starting in their vicinity. If
we settle into the neighborhood of a broad mini-
mum, it will be very hard to escape later. Second,
we require a procedure for determining whether
or not we are in a local minimum, and escaping
if we are. This procedure will be constructive in
that we were in a local minimum. Otherwise, we
assume that we have found the global optimum.
In the second phase, the prediction phase, the net
is conditioned to treat new input patterns, hope-
fully gaining the correct corresponding output
values.
The nodes are connected in that way that the
output of one node represents one input of the
adjacent node. A node is stimulated by one or
more inputs − →
x (t) and it generates one output,
a scalar y(t) that is sent to other neurons. The
output y(t) is dependent on the weighted activ-
ities of each input, on the nature all inputs are
transformed within the node and the parameters
constellation −→p (t) in each neuron. The actual
relationship between the inputs and outputs can
be enormously complex, depending on the cho-
sen of entry E(·), aggregation S(·), activation A(·)
and output function O(·):
 
y (t) =O (A( S (E( x (t) , p )) )) (12)
Thus, the resulting behavior of individual
neurons can be modeled by a simple weighted
sum of inputs, a complex collection of interre-
lated or subsequent set of (differential) equa-
tions, or anything in between. There can be
significant time delays in the steady-state out-
put value after stepwise input stimuli. Neurons
do not always respond in the same way to the
same inputs. Even random events can be con-
sidered in the operation of neurons. Luckily,
a large body of research indicates that simple
models, which account for only the most ba-
sic neural processes, can provide excellent so-
lutions to practical problems. The human brain
contains roughly 1011 neurons. As many as 104
synaptic junctions may abut onto single neuron.
That gives roughly 1015 synapses in the human
brain [381]. Consequently, the brain represents
an asynchronous, nonlinear, massively parallel,
feedback dynamical system of nearly “cosmo-
logical” proportions.
The ability of ANN to emulate complex dy-
namical systems stems from the interconnectiv-
ity of neurons. In general an ANN has n neurons
distributed in an input, an hidden, and an output
section. Connections between neurons can be or-
ganized in layers such that information flows in
one direction only, or it can circulate throughout
the network in cyclic patterns. All neurons can
be updated simultaneously or time delays can
be introduced. All responses can be strictly de-
terministic, or random behavior can be allowed;
the variations are nearly endless and should not
be described in detail. Mathematically they can
be formulated in the following vector equation.
  −   
y (t) = h →

o z (t) , x (t) , p )) (13)
where
→
−
y (t) Vector of yi with i = 1 . . . ni outputs of the
ANN
→
−
x (t) Vector of xj with j = 0 . . . nj inputs of the
ANN

p Vector of pm with m = 0 . . . nm variable
trainable parameters of the ANN

z (t) Vector of zj with n = 0 . . . nn for all outputs

of all individual nodes
o (t) Vector function of function om with m = 0 . . .
nm for the calculation of the individual
contribution to the output yi

h (t) Vector function of function hi with i = 0 . . .
ni for the calculations of the real outputs of
the ANN
There also exist some criteria to decide
whether a structure is appropriate for a good
modeling approach. Always, a compromise
must be made between the desire to have a sim-
ple model with fewer parameters and more accu-
rate predictions at the cost of a large number of
parameters. The Akaike’s information criterion
(AIC) uses the number of data points, the over-
all error, and the number of parameters to de-
termine an index, which can be analyzed to fix
the optimal quantity of free parameters [382].
Another concept that may be of interest is the

use of a periodiograms, which gives an indica-
tion regarding the frequency content of the in-
put signals, typical frequency can be character-
ized, which fixes the optimum degree of free-
dom [383]. Another way is the determination of
principal components before fixing the structure
of the net, thus, incorrect or redundant informa-
tion can be rejected before entering in the learn-
ing phase [384]. However, care must be taken
in reducing system complexity or rejecting data
sets, because they can be meaningful and repre-
sent the behavior of the biological system even
though, this may not be obvious from the data
set.
When considering a particular application,
it is to decide what type of network should be
used. Principally, ANN structures are classified
as either global or local. Both possess specific
properties and hence applicational preferences.
Global networks are the most popular und im-
portant ones. Among them, different types exist:
feedforward backpropagation neural networks
(FBNN); recurrent neural networks (RNNs), or
cascade correlation networks (CCNs). Unlike
the FBNN, RNNs are more general in the sense
that connections are allowed both ways between
a pair of neurons and even from a neuron back to
itself on any way. The RNN allows the dynamics
of the network to be considered intrinsically. In
contrast, in FBNN for mapping a dynamic be-
havior the input variables must be supplemented
by their corresponding deviations or time series.
However, the number of weights (for a fully re-
current network) to be determined may easily
become quite large. Therefore structural opti-
mizers are recommended, e.g., the cascade cor-
relation, where connections and nodes are added
as required, [385]. Local networks, such as the
radial basis function network (RBFN) process
data in discrete areas not continuously over the
whole space of the data. They are particularly
suitable for applications in online control and
optimization [386]. In the RBFN, it is necessary
to locate centers among the input/output pairs
such that the sum of the squares of the distance
from the center to the training data set is mini-
mized. These centers are equivalent in concept to
the weights in the FBNN. However, the RBFN
may fail in predicting values if the prediction
space does not contain any center. This must be
seen as a crucial disadvantage, especially for on-
line applications. So, global networks should be
Biotechnology 87
preferred if high uncertainty within the data ex-
ists.
The “explosion” of ANN applications in
nearly all areas of process engineering can be
attributed to the following reasons:
– The tremendous hardware advances in digi-
tal technology over the past decade have en-
abled simulations of neural nets to be made
both economically and with relative ease hand
speed.
– Applications of neural networks for sensor
pattern classification have been found to be
superior to the traditional algorithmic tech-
niques or the expert system approaches.
– Neural networks offer the promise of being
able to extract information from a plant in
an efficient manner with normal availability
of data, especially if severe or unknown non-
linearities and time invariances, typically for
biosystems, exists.
– Some practitioners claim that neural networks
may be easier to use and apply in the real pro-
cess plant, with difficult to handle nonlineari-
ties, as compared with the modeling approach,
which can be subject to various modeling er-
rors.
– Finally, the versatility in structure and appli-
cation of neural networks enables them to be
utilized in the middle ground between con-
ventional model-based approaches and black-
box approaches for solving many classes of
problems. These hybrid-type approaches have
been another factor, which has further at-
tracted their use in chemical process systems
recently.
The applications utilizing neural network
based strategies in bioprocess engineering are
wide ranging. A detailed description of all ap-
plication is outside the scope of this contribu-
tion. Neural networks can be used for classi-
fication, to specify typical classes in the sets
of the data base. Any task that can be done
by traditional classification analysis can be ac-
complished at least as well and almost always
much better by networks. Very important appli-
cations in bioprocess engineering are the multi-
variate exploitation of sensor arrays [387, 388]
or the fault diagnosis [218, 389]. An ANN can be
trained for pattern recognition. These patterns
can be a intervals of time, cultivation states, fer-
mentation series, images et cetera. If a version
88 Biotechnology
of one of these patterns, corrupted by noise, is
presented to a properly trained network, the net-
work can provide the original pattern on which
it was trained [390, 391]. A very common prob-
lem and the most popular application field of
ANN is that of modeling, identification, or esti-
mating the value or the state of a variable, given
historic values of itself and other variables; e.g.,
they were used to estimate the state of microbi-
ological cultures [392], the concentration of un-
measurable mostly intracellular biological key
components [393 – 395], or for the identifica-
tion of bioprocesses [396, 397]. ANNs can also
replace or act as process controllers. The ap-
plications utilizing these neural-network-based
strategies are wide ranging and vary from lin-
ear time invariant to highly nonlinear time vari-
ant systems [398, 399]. Typical advanced re-
presentatives are the model-predictive control,
the inverse model-based and the adaptive con-
trol techniques. These methods have all in com-
mon, that the ANN reacts as a process controller,
modified or expanded in some way by a refer-
ence model or by an online training unit.
8.4. Modeling Aspects of Biological
Systems
By definition, biochemical engineers are con-
cerned with biochemical systems, with reactions
and artifacts of biochemical substances like pro-
teins or with systems employing growing cells.
Even the simplest living cell is a system of such
a forbidding complexity that any forward math-
ematical description of it is – in most cases – an
extremely modest approximation. This situation
prompts for bioprocess modeling the question
from a formal logical viewpoint: “What kind
of relation ship should exist between the un-
derlying physical system and its mathematical
description and which approaches should be ex-
ploited?”.
Contemplation of this question leads, in one
direction, into the labyrinths of philosophy sci-
ence. Guidance into this territory from the learn-
ing guide Rutherford Aris led to John Casti’s
two-volume treatise “Reality Rules”, which ex-
plores the general definitions of a mathemati-
cal model as well as numerous specific exam-
ples of models in different contexts [400, 401].
Gratefully for engineers, who are (or should be)
engaged in discovery and development of use-
ful modeling technology, Casti asserts an inex-
tricable coupling between a model and its in-
tended application: “Basically, the point of mak-
ing models is to be able to bring a measure of
order or probable system performance to our ex-
perience and observations, as well as to make
specific predictions about certain aspects of the
world by experience”. Therefore, mathematical
modeling does not make sense without formu-
lating before making the model what is its use
and what problem it is intended to help to solve.
Thus, a definition of a model can be given as
follows: A model is an image of a real system
that shows analogues behavior in the important
properties, and that allows within a limited re-
gion in respect to the intended purposes a de-
scription of the behavior of the original systems.
The emphasis represents the word important.
A model should be always as simple as possi-
ble; the degree of simplicity depends only on
the model purpose and on the modeler skills.
Modeling the same real system with a differ-
ent focus, the significant properties are mostly
quite different. Single parameters can represent
in one case the most specifying quantities and
in the other case they are completely unimpor-
tant. The focus and the intention of the model
determine not only the parameter, but also the
proceeding in the formulation of the relations.
The modeler must identify the important vari-
ables (inputs, outputs, states) and their separate
effects, which in practice may have a very highly
interactive combined effect on the overall pro-
cess. This is only possible if the modeler hat
a profound technological knowledge. Once the
model is established, it can be used, with rea-
sonable confidence, to predict performances un-
der differing process conditions. But this is only
allowed under specific boundary conditions, un-
der those which were used for the set-up. If you
leave them, the describing ability gets lost. Any
extrapolation should be seen as critical.
8.4.1. Steps in Creating a Model
Model building is always a combination of the-
oretical studies and practical experiments in
a very iterative sequence (Table 15). The de-
scribed sequence should be observed in order

to get proper problem awareness and to mini-
mize the efforts to reach the main targets. The
objective of a scientific investigation is to im-
prove understanding of a system by testing a
hypothesis about that system. The formulation
of the hypothesis is the most important action in
the modeling process. In a scientific process, a
problem or question is posed and stated as a hy-
pothesis or theoretical statement of the problem.
Table 15. Step sequences for model building in bioreaction
engineering. The steps must be seen as a loop whose termination is
determined by the defined cost function
Step Action
1 Proper definition of the problem (hypothesis), the
goals, and the objectives of the study, fixing of
presuppositions, boundary conditions and constraints,
defining the evaluation and error criteria
2 Analysis of the system, determination of the
structural elements, description of the key elements
(variables, processes)
3 Running typical representative experiments,
exploiting the parameter space of the control
variables (experimental design)
4 Establishing the type of model by use of balances,
physical–chemical–biological laws, available data,
empirical equations, uncertainty treatment, problem
formulation in mathematical or linguistic terms
5 Simplifying assumption (e.g. about mixing, process
structure and dynamics, metabolism, kinetics,
neglecting aspects)
6 Choice and definition of the important process
variables: input and output variables, states, and
parameters for the model
7 Simulation of the model, parameter identification,
sensitivity analysis, determination of estimation
properties
8 Evaluation of the model by using test data
considering the evaluation criteria from step 1, if
necessary starting again with step 1
For complex biological systems, however,
the hypothesis may need to be translated into
a mathematical tractable building and the model
predictions are compared to the observed data
as a basis for rejecting or accepting a hypoth-
esis. This step also includes the definitions of
the goals and the objectives. The purpose of the
model usually dictates the form and the detail as
well as the data that are required to develop and
test it. Typical goals can be the identification of
typical parameters or simulations, to predict re-
sponses to a perturbation. If the purpose is to get
a general idea on how the system would behave
under a new condition, the accuracy may not be
important, however, if the purpose is to deter-
mine an “optimal” process trajectory, the model
accuracy may be critical. Finally, the purpose
Biotechnology 89
may be to derive the mechanistic principles un-
derlying the system behavior. So, it is useful to
obtain as much data from the system as possi-
ble. It can be necessary to examine all different
portions of the curves individually. Even, small
deviations can hold large insight into the intrin-
sic physico-chemical effects of the biosystem.
In all real cases of system modeling, also
boundary conditions or system constraints must
be fulfilled. This requirement addresses two dif-
ferent aspects. The first one considers the fact
that the created model is only valid inside typi-
cal boundary limits of the system variables and
inside formulated constraints in respect to re-
alized assumptions. The second one indicates
that building up a model in bioprocess engineer-
ing is always accompanied with an identification
step for optimizing the parameters. But the pa-
rameters are reasonable only within some lim-
its, though they fulfill the mathematical optimal
criteria. Otherwise, they can loose any physi-
cochemical meaning or interpretational ability.
Having more than one parameter to determine
can lead, depending on the complexity of the
system under consideration, to ambiguities. In
this case, although available knowledge, e.g., in
form of experimental data is represented well,
the meaning of the parameters is outside of any
interpretation scope. The extrapolation ability
gets lost.
To accomplish a mathematical treatment of
the model in order to reach the predefined goals,
an evaluation criteria must be formulated. This
can be seen as the mathematical description to
the formulation of the modeling purpose and
is realized by formulating a scalar performance
index. Its quantity gives information about the
model quality. In bioprocess engineering, it is
normally calculated from the results obtained
by the model compared with those of the cor-
responding experimental data.
There are two common approaches to fit mod-
els to data, the least-square approach where
parameter are adjusted to minimize the sum
(over all target values and existing data sets)
of squares of the residuals between the calcu-
lated and experimental data and the maximum-
likelihood approach, which maximizes the prob-
ability of the assumed parameterized probability
density function [234]. Also, the above men-
tioned boundary conditions and system con-
straints must be considered in the performance
90 Biotechnology
index. This aspect is from the logical point of
view strictly necessary, but invokes in some
cases crucial mathematical problems [402].
The system under study may consist of a sin-
gle biomolecule, a cell, or a whole plant. Biolog-
ical systems are normally open systems. They
have inputs and losses, although they may also
be reduced to closed systems as in vitro stud-
ies. Once the system to be studied is defined, the
inputs and outputs are identified. Additionally,
its processes, subsystems, and key compounds
characterize the system. Processes are move-
ments or changes in the system (e.g., absorp-
tion, metabolism, transport, chemical reactions),
subsystems are components of the whole system
(e.g., cells by considering the fermenter as the
system), and compounds/states are the quanti-
ties under consideration (e.g., metabolites).
Almost all modeling approaches in biopro-
cess engineering involve an identification step
of unknown parameters in the model by ex-
perimental data. The aim of the experiment is
to obtain data to confirm or reject a hypothe-
sis. It is essential to decide from which envi-
ronmental and operating conditions measurable
quantities should be gained, and to which preci-
sion and in which measuring range. The phys-
ical, chemical, and biological ones should be
wide ranging, and should also include the qual-
ity of the whole environment and pre-cultures or
pre-states. All measurable bioprocess data must
be treated initially as variables. Afterwards, it
could be decided, whether they remain constant
or not. Automated measurement and control of
bioprocesses, presently an art but – hopefully –
routine in the near future, generates a tremen-
dous amount of data. This requires judgment
of the importance of these data for documen-
tation to reduce data effectively without loss of
valuable information. Experts – either human or
computer – have the data and the deterministic
knowledge to trace observed behavior back to
the physical, chemical, and physiological roots.
Once the biosystem is structured and experi-
ments are done, the model should be fixed. There
exist different approaches to find the best ap-
propriate model type for the underlying prob-
lem (see below). In principle, the model should
represent the theories or hypothesis about how
the system works. Normally, there exist different
structures and parameter constellations which
will fit a particular set of data. The crucial task
for the modeler is to decide which one should be
accomplished from the viewpoint of deep under-
standing of the biosystem. It is important and in
most cases very effective that new approaches
are built up upon information of earlier studies
gained from literature. Differences between the
models should be evaluated to choose the model
most acceptable with current and new biologi-
cal knowledge. Since the problem of parameter
identification and model verification increases
rapidly with model complexity, one should begin
with as far as possible simplified assumptions
and withdraw them step by step, if the model
quality is judged to be not sufficient. In this way,
a most simple initial model grows step by step
in complexity and accuracy, without becoming
too complicated. Modeling includes not only the
selection of correct model structure, but also the
determination of the undefined system parame-
ters.
To give a final statement about the quality of
model, its robustness has to be evaluated. This
concerns sensitivity, identifiability, and stability
aspects. Sensitivity refers to the relative influ-
ence of individual parameters on the solution.
Identifiability refers to the uniqueness of the
model and of its parameters. They should be de-
termined in that way that a data-fitting process
should return the same estimates of parameters
more or less independently of the starting points
for estimation and noise in the data. Stability is
the behavior of a system with respect to a pertur-
bation. Since nearly all real biological processes
are time-variant and highly nonlinear in nature,
the powerful collection of methods concerning
the aspects of controllability, stability, and oper-
ability of linear systems cannot be applied [403].
Sensitivity analysis is one of the most impor-
tant tools to optimized models. It refers to calcu-
lations and analyses performed to describe the
relative dependency of the model parameters. Its
analysis can be used for model validation as well
as for fixing variables to constant values in order
to decrease the degree of freedom. The sensitiv-
ities are also very important for interpretation
purposes, e.g., to detect more or less important
partial processes or quantities of the biosystem.
From a practical perspective, it represents calcu-
lations which reveal the impact of assumptions
made in conjunction with model development.
A distinction can be made between point sensi-

tivity, relative sensitivity, and overall sensitivity.
Considering the model
   time-variant nonlinear system:
y (t) = y (t, p )) (14)
 z (t) =T ∗
with t representing the process time and p the

parameter to identify; y (t) are the measurable
output variables. The relationship between the
state variables and the point sensitivity can be
show using the first two terms in the Taylor ex-
pansion.
   
y (t) = y (t, p 0 )+GT ∂ p +K (15)
 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-
fined as the point sensitivity of yi with respect
to pj . An alternative scale-free sensitivity is the
relative sensitivity S ij
pi ∂yi
Sij = (16)
yi ∂pi
To represent the overall sensitivity of a state
variable as opposed to point sensitivity to pa-
rameter pi over some time interval, S G
ij can be
defined
T2

1 ∂yi
G
Sij = dt (17)
T2 −T1 ∂pj
T1
As applied to modeling, identifiability is a
mathematical concept, which attempts to steer
model development largely on the basis of an
analysis reflecting whether features of a model
for a presumed system can be extracted from a
proposed experiment. Selecting a standardized
method for an experiment directed for elucidat-
ing a model and its features involves selecting
time points for observations, observation sites,
sites for tracer input, as well as forms of tracer
application. In bioprocess engineering, it is sel-
dom possible to sample all units, and the number
of units to which tracer can be applied is limited.
Each choice we make here stands for a chance
of mitigating against identifying aspects of the
model, even in an error-free situation. If the lack
of identifiability makes it impossible to estimate
unique parameters or test relevant hypothesis,
we should like to know this before conducting
the experiment or interpreting the modeling re-
sults. If the experiment is adequate to identify
the model, we are still left with the estimation
problem, that of resolving the parameters amidst
Biotechnology 91
noisy data and to reach the observability. Con-
sider the following general representation of a



 
x t), y (t), p )
with
(18)
0 
z = z (t=t0 )
A system is defined as observable at the time
0
t 0 , if, admitting an arbitrary initial state z , a
finite point of time t 1 >t 0 exist, so that assuming
 
known input x (t) and output y (t) trajectories

tems, this problem involving time-variant non-
linear equations as they arise in bioprocess engi-
neering results in nonlinear algebraic equations
for the solution of which similar powerful meth-
ods do not exist. Consequently, several assump-
tions and individual specifications and transfor-
mations have to be made to make the problem
mathematically tractable with reasonable effort
[404].
8.4.2. Reasons for Making a Model
There is a zoo of mathematical models in the
biochemical engineering and mathematical bi-
ology literature. Many of these appear to naive
as well as to sophisticated reader to have lit-
tle more purpose than calculating numbers and
writing scientific papers which conform reason-
ably to experimental data. This is, in itself, not a
distinguished endeavor; it is not particularly dif-
ficult, and it teaches little. One reason that math-
ematical biology receives so little respect from
biological scientists – and is generally not recog-
nized as a credible research tool in biological sci-
ence and a biotechnological discovery – is that
it represents the failure to communicate clearly
and persuasively the reasons and the motivation
for constructing a model. Sometimes the mod-
eler himself has not clearly asked this question
while doing the work, sometimes biochemical
scientists make one experiment after the other,
not reflecting which mathematical approach ex-
ists to enhance the evaluation or interpretation of
the experiments. However, modeling as a typical
domain of engineering gets increasing interest in
the study of biological systems. The challenge of
92 Biotechnology
modeling biological systems is not to determine
an arbitrary function to fit the data but to use the
modeling process to understand the system.
Modeling can be used to determine the struc-
ture of a system where structure refers to the
relationships between various parts or processes
of a system. A model can be used to determine
the type of relationship as well as the sequence of
events that occur between the various substances
of interest. In some systems, this information
may be known, whereas in others the structure
can only be inferred from the data. Modeling
improves understanding, and it is through un-
derstanding that progress is made. In formulat-
ing a mathematical model, the modeler is forced
to consider the complex cause-and-effect se-
quences of the process in detail, together with
all the complex inter-relationships that may be
involved in the process. The comparison of a
model prediction with actual behavior usually
leads also to an increased understanding of the
process, simply by having to consider the ways
in which the model might be in error. The results
of a simulation can also often suggest reasons as
to why certain observed and apparently inexpli-
cable phenomena occur in practice.
Models can be used to determine parameters
of interest, such as pool sizes clearance rates or
transport rates. If the purpose is to accurately
determine a particular parameter, it is impor-
tant that the model can be uniquely determined.
However, the model must also be consistent with
known biological information. A model may be
well-determined but incorrect.
The purpose of the model may be to deter-
mine the interaction of parts of a system. Models
can integrate information on a number of sub-
systems into an integrated form to represent the
process under consideration in the whole sys-
tem. Because of the interactions, dependencies,
and feedback of the processes, large complex
systems can only be understood by using mod-
eling. An example could be to link metabolism
in one tissue with metabolism in another, or to
link metabolism of a nutrient in one form with
the metabolite in a second form. Large systems
and systems with dynamic properties can only
be understood with the use of models. Because
many processes in large systems occur simulta-
neously and dynamic systems are accompanied
by complex interactions between processes, it is
unlikely that responses to perturbations could be
predicted based solely on intuition and experi-
ence without a model.
Models can be used to simulate inputs into a
system at various sites and to simulate short-term
as well as long-term responses. Experiments can
be simulated rapidly using a model to predict
likely scenarios before undertaking expensive
experimental data collection. While models will
never replace experiments, they can be used to
avoid experiments where insufficient or inappro-
priate data will be collected for testing a certain
hypothesis. Models may be used predictive for
design and control. Once the model has been
established, it should be capable of predicting
performance under differing sets of process con-
ditions. Thus, mathematical models can be used
for the design of relatively sophisticated control
and optimization algorithms, and the model, it-
self, can often form an integral part of the control
algorithm. Optimization usually involves con-
sidering the influence of two or more variables,
often one directly related to profits and one re-
lated to costs. For example, the objective might
be to run a reactor to produce a product at a max-
imum rate, while leaving a minimum amount of
unreacted substrate.
An important use for models is to iden-
tify sites of change in a system when studied
under different conditions. In some cases, the
altered condition may result in large changes
in the kinetic curves and several pathways or
it may result in a subtle change in the data
caused by a large change in only one parame-
ter. A model helps to identify which parameters
change between the conditions and the degree
of change. The conditions may be an untreated
versus treated state, a healthy subject versus a
diseased subject, or a normal versus high intake
of a nutrient.
Models form valuable tools for teaching the
process of scientific inquiry and for challeng-
ing students to think creatively and quantita-
tively about a system. Models can be used to
demonstrate properties of a system, teach princi-
ples (such as feed-back loops, saturation kinetics
etc.), test theories, and design studies. With the
increased speed and convenience of computers,
the availability of modeling software and the ac-
cess to it, the researchers who are developing and
publishing models, need to be cognizant of this.
They need to make their models understandable

and accessible to the biological community at
large as well as to students.
Models help in experimental design. It is im-
portant that experiments be designed in such a
way that the model can be properly tested. Of-
ten, the model itself will suggest the need for
data for certain parameters, which might oth-
erwise be neglected, and hence the need for a
particular type of experiment to provide the re-
quired data. Conversely, sensitivity tests on the
model may indicate that certain parameters may
have a negligible effect, and hence these effects
can be neglected both from the model and from
the experimental program.
8.4.3. Different Types and Basic Approaches
for Building a Model
Incomplete knowledge of the dominant biolog-
ical pathways as well as low availability of sen-
sor information about the current physiological
state is the characteristics of biochemical pro-
cess modeling. From system analysis, as already
described above, the definitions of the describ-
ing variables (e.g., inputs, outputs, or states) can
be given.
In principle, there can be postulated that
these variables are intrinsically interconnected
by means of a set of – indeed real existing – dif-
ferential equations, as it is already described in
previous chapters. But even being aware that
these relations may exist in principal, for math-
ematical treatment they can only be specified
in their structure and intercorrelation as well
as in their parameter constellation in some ex-
ceptional cases with a high degree of accuracy.
Thus, when dealing with modeling of real bio-
logical systems, uncertainty in various aspects
can never be avoided [405]. In bioprocess engi-
neering, uncertainty is an inseparable compan-
ion of any modeling approach.
Uncertainties in engineering systems can be
mainly attributed to ambiguity and vagueness
in defining the architecture, parameters, and
governing prediction models for the systems.
The ambiguity component is generally due to
noncognitive sources. These sources include 1)
physical randomness; 2) statistical uncertainty
due to the use of sampled information to estimate
the characteristics of these parameters; 3) lack of
knowledge; and 4) modeling uncertainty which
Biotechnology 93
is due to simplifying assumptions in analytical
and prediction models, simplified methods, and
idealized representations of real performances.
The vagueness-related uncertainty is due to
cognitive sources that include 1) the definition of
certain parameters, e.g., structural performance
(failure or survival), quality, deterioration, skill,
and experience of construction workers and en-
gineers, environmental impact of projects, con-
ditions of existing structures; 2) other human
factors; and 3) defining the interrelationships
among the parameters of the problems, espe-
cially for complex systems. Other sources of un-
certainty can include conflict in information and
human and organizational errors [406].
Once the systems are defined, the relations
between the outputs and states based upon the
inputs and states have to be formulated to per-
form simulations. Today we have many types
of models, which can be categorized in various
ways such as deterministic versus nondetermin-
istic, logical (linguistic) versus mathematical-
equations-based or data- versus knowledge-
driven. Physical models can be the realization
of the original system in a different (usually
smaller) scale or with structural modifications.
A second type of physical models is obtained
by turning to a different physical system, e.g.,
from the original biosystem to a correspond-
ing electrical circuit [407]. Another group re-
presents verbal models. They give a linguis-
tic representation of our knowledge about the
system, usually formulated as rules, e.g., “IF
this or that happens THEN the system reacts
by. . .”. They are widely applied in the area
of artificial intelligence, namely experts sys-
tem [408, 409]. In the form of fuzzy-based
systems, they gain increasing industrial inter-
est in biotechnological control theory, which is
due to their effective treatment of qualitative
knowledge of experienced technologists in pro-
cess control strategies (see Section 8.3.1), which
could only hardly realized in terms of mathemat-
ical equations [410 – 412]. Mathematical mod-
els form the third class. They can be classified
further depending on the mathematical formal-
ism or the methods for model building. Theo-
retical models as mechanistic models are based
on physical and chemical laws and our knowl-
edge about the inner structure and function of the
system. In contrast, experimental or nonmecha-
nistic mathematical models try to give – without
94 Biotechnology
looking into the interior of the system – only a
description of the observed reaction of the sys-
tem in response to a certain forcing signal. Many
“mechanistic” models in biotechnology are ac-
tually due to their oversimplification quite closer
to black-box models than to real mechanistic
models, although mostly mechanistic interpre-
tations are given [413 – 415].
White-box model. As described above (Sec-
tion 8.3.1), the relation can strictly be mech-
anistic, considering the four differential equa-
tions for mass and heat balance and correspond-
ing transformations. In this so-called white-box
modeling strategy, the model development is
mainly driven by the knowledge of the rel-
evant mechanisms, macroscopic balances and
predefined parameters. Indeed, if all the biosys-
tem underlying mechanism are known and can
be described by means of the above-specified
equations and the accompanied parameters are
known, the resultant white-box model is gen-
erally applicable and show good extrapolation
properties. However, it is not an easy task to re-
veal all the relevant mechanisms and quantify
these mechanisms correctly when dealing with
biological processes. The metabolic modeling
approach is one such approach and has been the
focus of recent attention [416, 417]. Addition-
ally, several attempts have recently been made in
utilizing metabolic information for online iden-
tification and control as a consequence of the
white-box strategy [418, 419].
Black-box model. The black-box model is,
on the other hand, mainly driven by measured
data obtained from the process, either online or
offline. In terms of mathematical expressions,
probability density functions for the important
parameters are specified by means of available
data. Based upon these distributions, the time-
dependent system behavior can be forecasted
[420 – 422]. The earliest stochastic biosystem
model is perhaps that proposed by Yule, to
mimic the evolution of a new species within
a genus [423]. However, they are mainly used
for systems only, where isolated parameters are
treated. It is unrealistic that for a real biological
MIMO system (multiple input multiple output),
all joint probability densities, which are neces-
sary to describe the key behavior, can be approxi-
mated by performed series of experiments. Even
for systems without the existence of any driving-
force function the expense for their determina-
tion is disproportional to the possible result. If
inputs exist, which direct the system in a defined
direction, reaction patterns must be formulated
additionally in terms of probabilistic equations.
Another type of black-box models represents
those where a specific functional structure is as-
sumed, which is combined with a subsequent
parameter estimation to adapt to the specific un-
derlying problem (e.g., polynomial regression,
ANN, chemometric models). However, on ap-
plying this, it has to be considered that the sim-
plest and usually fairly accurate method for pre-
dicting values of the next data point in a time
series from a “well-behaved” biosystem is to
assume that it is the same as the present data
point, a principle known as the first-order trivial
predictor. Similarly, in the second-order trivial
predictor the value of data point n+2 is equal
to that for point n plus twice the signed dif-
ference between the values of point n+1 and
n. If any complex nonlinear predictive model
like ANN can do no better than the first- and
second-order trivial predictors – which are in-
deed very often concurrently – then their devel-
opment is a waste of time. Nevertheless, espe-
cially ANNs have been the focus of much atten-
tion for model development and have already
been applied to various biochemical processes
(Section 8.3.2). Although the main advantage of
the development of black-box models is that a
reasonably accurate model can be obtained with-
out detailed mechanistic system knowledge, it
should be noted that its accuracy depends only
on the quality of the available input and out-
put data. As black-box models are not believed
to have any extrapolation properties, one has to
obtain a large body of data for process identifi-
cation by employing the relevant input variables
in a wide range of fluctuations [218].
Markov chains. Another very attractive way
to use probabilistic approaches are the formu-
lation of the problem by means of so-called
Markov chains [424, 425]. A Markov process
allows the modeling of uncertainties in real-
world systems that evolve dynamically with time
without using mechanistic approaches. The ba-
sic concepts of a Markov process are those of
states of a system and of state transitions. In spe-
cific applications, the modeling “art” is to find

an adequate state description such that the as-
sociated stochastic process has the Markovian
property that the knowledge of the present state
X(t) is sufficient to predict the future stochas-
tic behavior of the system X(t+1). The moving
of i=t to j=t+1 is defined by means of the tran-
sition probability Pij [426]. For bioprocess en-
gineering, controlled Markov chains or Markov
decision processes (MDP) possess a high poten-
tial, for simple modeling as well as for process
optimization [427, 428]. Applied in bioprocess
engineering, they involve the selection of an ac-
tion U t from a set of containing a finite number
of A actions when observing the current state of
the system. The problem is to define the prob-
ability that the system goes to a state j from a
state i under the action of a:
P {Xn+1 =j|Xn =i,Ut =a}=Pij (a) (19)
The controlled Markov chain is completely
defined by the optimality criterion for calcula-
tion of the transition probability matrix, the state
vector X, the action set A, and the decision epoch.
For bioprocess modeling, concentration values
grouped in clusters are often used as state vari-
ables; considering more than one state variable
for each one a separate model is constructed.
One great disadvantage of this approach repre-
sents the huge effort to determine the transition
probability matrix Pij (a) for each control action,
especially if more than one manipulated variable
should be considered [428].
Grey-box model. A grey-box model may be
defined as a suitable combination of a black-
box principle and a white-box model. The ex-
pectation is to obtain good interpolation and
extrapolation properties. Especially, hybrid ap-
proaches, where portions from each principle are
pieced together based upon the amount and the
structure of available knowledge, gain increas-
ing importance. From different points of view,
such hybrid systems can be build up, i.e., ana-
log or discrete consideration, stochastic or deter-
ministic combinations [429 – 431]. Only some
important concepts for bioprocess engineering
should be presented here. In some cases, sta-
tistical methods are included into a mathemat-
ical discrete formulation, this concerns mainly
Kalman filtering. They exploit statistical infor-
mation about the error distribution of the state
Biotechnology 95
and a measurement values, and their propaga-
tion with time to predict online a future state
based upon new measurements and already ex-
isting quantities. Their potential in bioprocess
modeling and control is obvious [432 – 434].
From the structural point of view, hybrids are
described in parallel and serial configurations.
In the former case, the black box is placed in
parallel with a white-box model and its role is
to describe and to weight the difference bet-
ween a white-box model and the real process.
This model has been applied to several pro-
cesses, but its performance might be limited
to some extent to the viewpoint of extrapo-
lation properties [435]. In the serial structure,
the black box is placed in series with a white-
box model. Different applications are described.
Combined with a fuzzy expert system, the hy-
brid was applied to model a real-time fed-batch
culture for baker’s yeast production, a produc-
tion scale beer-brewery fermentation, and mam-
malian cell cultures [431]. To show the better
extrapolation properties of grey-box-model ex-
emplarily, the performance of a serial-type grey-
box model containing a neural network and a
more knowledge-driven white-box model were
compared with that of a more data-driven black-
box model in the enzymatic hydrolysis of peni-
cillin G to 6-amino-penicillanic acid and phenyl
acetic acid by using the enzyme penicillin acy-
lase. The grey-box model was shown to have
significantly more reliable extrapolation prop-
erties than the black-box model [436].
Also linguistic models such as fuzzy models
can be seen as a special type of grey-box models.
Although the intrinsic behavior cannot be for-
mulated by means of mathematical equations, a
priori knowledge is included into the model by
the linkage of various “IF. . ..THEN . . . “ con-
clusions, which is built up by the knowledge
of highly specialized staff. Their fine-tuning is
often accomplished by using data from experi-
ments [437].
Being aware that different approaches ex-
ist how biological systems can be modeled and
treated computationally, an important aspect for
a successful application is the proper choice
which principle fits best to the specific problem
and its boundary conditions. In most cases, more
than one specific approach can be applied in
principal. The most important aspect for choos-
96 Biotechnology
Table 16. Different types of models based upon the system knowledge
High System knowledge
Model deterministic stochastic, statistical expert systems
Examples unstructured, Markov chains,
structured, CFDa maximum likelihood, theory
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-
edge about the underlying problem (Table 16).
Not only the amount of system knowledge is
decisive, but also its structure, the quality and
quantity of accompanied data, and the purpose
for which the model should be used is of special
importance. As a basic principle for building up a
model, it could be postulated “use as much a pri-
ori information as possible”. In mechanical en-
gineering and fluid dynamics, strictly determin-
istic approaches are typical, where the underly-
ing differential equations are “exactly” known.
Thus, there exists no reason to deal with uncer-
tainties and the problem is forwarded. However,
this situation generally does not exist in bio-
process engineering. A biological system con-
sists of a very complex, strongly regulated re-
action network, where only some simple mech-
anisms can be formulated in terms of mathe-
matical discrete forwarded formulations. Thus,
one has to deal in a very strong degree with un-
certainties. If one is able to formulate a set of
differential equations, which represents not an
exact image of the underlying mechanism but is
descriptive enough for the existing problem and
for the modeling purpose, one should formulate
such a model and append a subsequent parame-
ter estimation step where statistical properties or
available data are exploited to consider unknown
or not structural included information. In the
case where a formal-kinetic approach cannot be
formed but qualitative knowledge is available or
can be extracted from available data, expert sys-
tems should be preferred. Especially fuzzy sys-
tems as a special representation of an expert sys-
tem show advantages in modeling approaches
because of their smoothness concerning the sys-
tem output. In the case where a priori knowl-
edge of the system is not available but pure data
from a well-defined experimental design do ex-
ist, approximation or classification approach like
Low
approximation classification
fuzzy models, set neural networks, PLSb , pattern recognition
PCRc
ANN or chemometric models should be used. In
those cases, they are the only alternative to real-
ize appropriate prediction ability. They assume a
system structure, which possesses no causal re-
lation with the underlying real mechanism, but
is able to incorporate and describe the system
dynamics. They possess normally good approx-
imation abilities, but their extrapolation prop-
erty reveals a low reliability especially if outputs
should be forecasted where the corresponding
inputs are outside of the trained data space.
9. Special Applications in
Biotechnology
The following chapters highlight some special
aspects in biotechnology. However, there exist
even more interesting applications in biotech-
nology. The reader should regard the areas cho-
sen by the authors as examples for the wide dis-
tribution of biotechnology in the daily living.
9.1. Mammalian Cell Culture
Technology
9.1.1. Introduction
Mammalian cell culture technology has become
a major field in modern biotechnology, espe-
cially for the area of human health, and fascinat-
ing developments achieved in the last decades
are an impressive example for an interdisci-
plinary interplay between medicine, biology,
and engineering [438, 439]. Among the classi-
cal products from cells, we find viral vaccines,
monoclonal antibodies, interferon, as well as
recombinant therapeutic proteins. Tissue engi-
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
genetically induced proliferation [452, 453].
The following will provide a basic under-
standing of the specific requirements of mam-
malian cells, will describe state of the art process
technology for cultivation of these cells and will
give an outlook to future prospective. A com-
prehensive overview on cell culture technology
is given by Ozturk and Hu [454].
9.1.2. Products from Mammalian Cells
A detailed overview on products from mam-
malian cells is given in [454, 455]. Within “prod-
ucts from cells”, viral vaccines [456] against
polio, hepatitis B, measles, or mumps for hu-
man use, rubella, rabies, or food-and-mouth dis-
ease (FMD) for veterinary use play an impor-
tant role. Viral vaccines are produced very effi-
ciently by means of a cell-based vaccine tech-
nology. For this, mainly primary cells, diploid
cells, or permanent cell lines (e.g., VERO) are
applied, recently even recombinant cell lines. A
breakthrough for the large-scale production of
viral vaccines with anchorage dependent cells
was the development of microcarriers in the late
1960s, allowing cultivation in stirred tanks in
thousand-liter scale. New targets for cell-based
vaccines are human immunodeficiency virus
(HIV), herpes simplex virus, or influenza. Fur-
thermore, new developments include genetically
engineered or DNA vaccines (→ Immunother-
apy and Vaccines).
Monoclonal antibodies [457] have become a
valuable tool for diagnostic purposes as well
as in therapy. Antibodies synthesized by B-
lymphocytes play an important role in the
immunosystem of mammalians. Traditionally,
polyclonal antibodies were isolated from blood
samples. In the 1970s Milstein and Köhler de-
veloped a technology to generate hybridoma
cells producing monoclonal antibodies [458]. As
of the specific binding, monoclonal antibodies
are widely used for diagnostics, where tens of
thousands different monoclonal antibodies are
available. The importance of monoclonal anti-
bodies as therapeutic agents has evolved only re-
cently, as immunogenic mouse antibodies were
replaced by chimeric, humanized, or human an-
tibodies. Fields of application are organ trans-
plantation (OKT3), cancer diagnostic and treat-
ment, rheumatoid arthritis, leukaemia, asthma,
or multiple sclerosis. Nowadays several antibod-
ies are produced in kilogram quantities [440].
Modern recombinant techniques focus on new
antibody formats such as fragmented antibod-
ies (FAbs) or bivalent antibodies with a broad
spectrum of applications.
Glycoproteins are a further important group
of products produced by mammalian cells. Start-
ing with the production of α-interferon as an
anti-infectious drug by (nonrecombinant) Na-
malwa cells in the late 1970s, nowadays a grow-
ing number of glycoproteins for treatment of a
wide variety of diseases are produced by means
of mostly recombinant mammalian cells. Promi-
nent examples are cytokines (e.g., interferons
and interleukins), hematopoetic growth factors
(e.g., erythropoeitin for treatment of anemia),
growth hormones, thrombolytic agents (e.g., tis-
sue plasminogen activator [tPA]), coagulation
factors (factor VII, factor VIII, factor IX etc.),
and recombinant enzymes (DNAse) [440].
Recombinant proteins may be produced by
either bacterial, yeast, or mammalian cells. From
a technological point of view, hosts such as bac-
teria or yeast have an advantage with respect
to growth rate, final cell density, and product
concentration. Nevertheless, mammalian cells
are preferred for those proteins requiring a
specific, humanlike glycosylation pattern [459,
460], which is difficult to obtain in other host
systems. Another problem for microorganisms
is the maximal size of the produced protein
which must be below a molecular mass of ap-
prox. 30.0 kDa. Further, in contrast to extra-
cellular release of most proteins produced in
mammalian cells, products from microorgan-
isms are often accumulated intracellularly in “in-
clusion bodies”. This demands a more complex
downstreaming. Besides this, for mammalian
cells important parameters such as product yield,
medium requirements, and growth characteris-
tics (suspendable, shear resistant) have been sig-
nificantly improved.
Proteins produced in the milk of transgenic
animals have started to compete against “classi-
cal” mammalian cell culture [461 – 464]. The
proteins are usually expressed in enormous
titers, over one order of magnitude higher than
that obtained from cell cultures. The price for
the production in transgenic animals will prob-
ably continue to decrease significantly due to

the development of more efficient reproduction
technologies. These new approaches will signifi-
cantly decrease the time for the development of a
product. Among the disadvantages of transgenic
animals, we find a more sophisticated down-
streaming (high protein loads), the long devel-
opment time, the inability to produce proteins
that might impair the health of the animal (e.g.,
insulin), higher risk of viral contamination and
the possibility of prion contamination (scrapie,
BSE).
The novel field cells as products includes
the development of artificial organs (tissue en-
gineering of liver, kidney) and tissues (skin, car-
tilage, bone), or the expansion of hematopoi-
etic cells for bone marrow transplantation or
gene therapy. The exciting prospects of tissue
engineering [465 – 467] are outlined in Section
9.2. Somatic gene therapy implies transfection
of a specific gene to cells isolated previously
from a patient suffering from a genetic disease
[468 – 470]. The transfected cells are then rein-
troduced into the patient. Gene therapy involves
transfer of genetic material, encoding therapeu-
tic genes and the sequence necessary for expres-
sion to target cells to alter their genetic code
for a desired therapeutic effect. Many diseases
are originated by gene defects. Expression of
the transferred genes can result in the synthesis
of therapeutic proteins or correction of a gene
defect. Gene transfer could also lead to desired
apoptosis or inhibition of cell proliferation. The-
oretically, gene therapy can be applied for repair-
ing single entailed gene defects, acquired gene
defects such as chronic infectious diseases, mul-
tifactor genetic diseases such as cardiovascular
disease, and finally cancer. Since gene therapy
was applied the first time in 1990, the number
of clinical trials increased. Today gene therapy
is widely used in ongoing clinical trials for the
treatment of cancer and infectious diseases such
as human immunodeficiency virus (HIV) infec-
tion.
Cell culture products are currently used
mainly as medicines or in diagnostics. For
medicines, some products have a demand of
500 kg/a and generate $ 1–2 billion dollars in
revenue. Among these are tissue plasminogen
activator (tPA), erythropeithin (EPO) products,
Remicade r
(Centocor) or MAbThera r
[440].
The pipeline for new biopharmaceutical thera-
peutics targets a huge number of diseases (e.g.,
Biotechnology 99
425 for U.S.A in 2002 [471]), most of them for
cancer therapy, infectious diseases, autoimmune
diseases, or AIDS/HIV. It can be expected that
at least some of these will find their way to the
market. Furthermore, new products or medical
protocols can be expected for tissue engineering
and cell therapy. As for some compounds patents
are already expired or will expire in the near fu-
ture, there will be a market chance for biosimilar
or generic products. On the other hand, the costs
for target identification, clinical trials, and pro-
cess development will increase (to date approx.
$ 0.5–1 billion) and only block busters will make
it finally to the market.
9.1.3. Cell Types
In general, mammalian cells relevant for indus-
trial processes can be divided into the following
groups [441, 472]:
– Primary cells have been isolated from a tissue
and than taken into culture (primary culture).
– Permanent or established cell lines originate
from a primary culture, but because of some
transformation became able (at least theoret-
ically speaking) to divide and proliferate in-
definitely. Those cell lines are kept in a cell
bank and are very often used as host cells for
the expression of recombinant proteins.
– Hybridoma cells are cells which are obtained
through the fusion of lymphocytes and tumor
cells and which are able to express mono-
clonal antibodies.
The common procedure for the isolation of a
mammalian cell from a tissue involves the fol-
lowing steps: first, a small piece of tissue is de-
composed into single cells or cell clots mechan-
ically, enzymatically (e.g., typsin [473], colla-
genase), or through a combined procedure. The
cells are separated from the enzymes by cen-
trifugation and resuspended in culture medium.
After that, the cells are inoculated into a spe-
cial glass or plastic vessel with flat bottom. The
anchorage-dependent cells start to adhere to the
bottom surface and, after a lag phase, start to di-
vide by mitosis. Such a cell culture, in which the
cells come from a differentiated tissue, is called
a primary cell culture. The most critical point in
the isolation of primary cells is to avoid external
contamination by practicing aseptic techniques.
100 Biotechnology

When the primary cells have covered the bot-
tom of the culture vessel almost completely, they
are enzymatically cleaved (i.e., trypsin) from
their support and used to inoculate new cultures.
This subculture gives origin to the so-called sec-
ondary cultures. Part of the cells is stored deeply
frozen in liquid nitrogen, to remain as a safety
stock, from which it is possible, at any time it
might be necessary, to get enough “fresh” cells
to start a new series of subcultures for mass pro-
duction. With the primary cells, it is possible to
repeat subcultures several times. However, the
primary cultures have a finite lifespan and there-
transformation and “immortalisation”, as for ex-
ample the treatment with mutagenic substances,
with virus or with oncogenic substances [441].
Transformation of cells in vitro shows some sim-
ilarities with carcinogenese in vivo, but is not
identical to it; for instance, not all the trans-
formed cells are malignant. On the other hand,
all cells that are isolated from tumors (i.e, HeLa
or Namalwa Cells) can be kept in a permanent
culture.
Table 17. Examples of permanent cell lines important for research
and production (data from [441], 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
be easily grown in relatively simple media with-
out addition of expensive growth factors. At first,
transformed cells were identified just by chance,
but nowadays there are techniques to cause cell
Hybridoma cells are “artificial” cells pro-
duced by the fusion of human or mammalian
lymphocytes, which can secret specific antibod-
ies, with a myeloma cell. Usually, lymphocytes
do not survive outside the original organism, i.e.,

cannot be kept alive in an artificial medium. Mil-
stein and Köhler [458] were able to overcome
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-
bodies. The cells generated in this way are hy-
brid cells (also called “hybridoma cells”) and
have the lymphocyte characteristic to produce
antibodies against a certain antigen, as well as
the ability from the myelom cells to survive in
culture.
An alternative expression system for recom-
binant proteins offer insect cells, especially the
insect-cell baculovirus expression system (IC-
BEVS) [476]. Mostly applied are Sf9- or Sf21-
cells isolated from Spodoptera frugiperda. Pro-
duction of heterologous proteins by the IC-
BEVS consists of two stages. Insect cells are
first grown to a desired concentration and then
infected with a recombinant baculovirus con-
taining gene coding for the desired protein.
Meanwhile, several recombinant proteins pro-
duced by this technique have reached the mar-
ket.
The largest and most well known interna-
tional animal cell culture collections are the
American type culture collection (ATCC) [477],
the European collection of animal cell culture
(ECACC) [478] as well as the German resource
centre for biological material (DSMZ) [479].
The application of permanent cell lines for
the production of vaccines faced strong opposi-
tion, mainly in the 1960s, because there was the
suspect that these cell lines, which behave sim-
ilarly to tumor cells, could infect patients with
unknown cancerous substances. Fortunately, no
observation of this kind has been done until
today. In the meanwhile, more and more per-
manent cell lines have been applied for the
production of therapeutic and diagnosis sub-
stances. However, the application of permanent
cell lines, and above all, of recombinant cell
lines, is strictly controlled by supervisory boards
(as the FDA [480] in the USA, and the EMEA
[481] within the EU). During the whole produc-
tion cycle of a pharmaceutics, not only the prod-
uct has to be identical from charge to charge, but
also the phenotypical characteristics of the cell
line has to be maintained. To assure that, before
the beginning of the production, a “master cell
bank” has to be created, from which a “master
Biotechnology 101
working cell bank” is derived, providing the in-
oculation material for the production cultures.
9.1.4. Growth Medium for Cell Culture
Basically, a growth medium for mammalian
cells have to supply all the necessary nutrients
required for growth and product formation [482,
483]. Furthermore, it should have a certain buffer
capacity to stabilize the pH (pH optimum 7.0–
7.3) and should provide an appropriate osmo-
lality in order to avoid damaging of the sensi-
tive cell membrane [484 – 488]. A fundamental
component of all media is a salt solution, which
provides the ions necessary for life, keeps the os-
motic pressure within the desired range, contains
one or more buffer systems (sodium phosphate
buffer, HEPES, and/or carbon dioxide–carbonic
acid buffer) for pH regulation, and contains, in
some cases, a pH indicator (phenol red). Further-
more the media contain glucose and glutamine
as a source of carbon and nitrogen, other ami-
no acids, vitamins, mineral salts, and trace ele-
ments.
A number of medium formulations have
been developed, e.g., Eagle’s minimal essen-
tial medium (MEM), Dulbecco’s enriched (mod-
ified) Eagle’s medium (DMEM), Ham’s F12,
and RPMI 1640, among others. Examples of
medium compositions can be found in the liter-
ature [442, 454, 489]. Traditionally these basic
media were supplied with approx. 5-10 % serum
(e.g., foetal calf serum [FCS] or horse serum
[HS]) in order to supply specific growth factors
and to protect the cells against shear stress. The
disadvantage of media containing serum are the
indefinite composition of such media, the high
serum costs, the difficulty of product purifica-
tion, variations between the charges and the risk
of virus contamination [490].
Serum can be partially substituted by the ad-
dition of transferin, insulin, ethanol amine, albu-
min, or eventually fibronectin as adherence fac-
tor in a serum-free but still protein-containing
medium [491 – 493]. A further step to chemi-
cally defined, protein-free media became possi-
ble by replacing these animal-derived proteins
by iron salts or iron complexes, IGF-1, chem-
ically defined lipid concentrates, precursors or
other stimulating agents such as fatty acids, bi-
otin, cholin, glycerin, ethanolamin, thiole, hor-
102 Biotechnology
mones, and vitamins [494 – 496]. Furthermore,
addition of peptone or yeast extract can be help-
ful [497, 498]. However, the growth rate and pro-
ductivity in a serum-free medium can decrease
[499]. In addition, the sensitivity to shear stress
increases, requiring additives with protecting
characteristics, such as Pluronic F68 [500, 501].
Immobilization can improve the culture stability
of nonanchorage-dependent animal cells grown
in serum-free media [502, 503]
Different cell lines require different compo-
sitions. Adaptation of a cell line to grow with-
out serum is quite time-consuming and not all
cell lines have been adapted to serum-free or
protein-free media [504, 505]. For cultivation
of primary cells, for basic cell-culture research
or for vaccine production still mostly complex,
serum-containing media are common. In indus-
trial production with established, optimized cell
lines, serum-free, bovine-serum and protein-free
as well as chemically defined media are state of
the art [439].
Figure 32. Routine cultivation systems for use in incubators
9.1.5. Small-Scale Culture Systems for
Routine Use
Techniques and culture systems for cultivation
of mammalian cells differ significantly from
those used for microbes. For basic research, a
large number of culture systems have been de-
veloped (Fig. 32), mostly designed for use in
an incubator aerated with 5 % CO2 to main-
tain the pH within the desired range. Multiwell
plates (6–96 wells), T-flasks (25–100 mL), Petri
dishes or roller bottles (50 mL to 5 L) are usu-
ally used for cell maintenance and proliferation,
especially for adherent cells [441, 506 – 509].
These systems allow for sterile handling proce-
dures and are easy to use, disposable, and low-
cost. On the other hand, they require individ-
ual handling, for example in medium exchange
and cell seeding; their usefulness is limited when
large quantities of cells or products are required.
This can be overcome to some extent by using
sophisticated robotics [510]. Furthermore, envi-
ronmental parameters including pH, dissolved
oxygen, and temperature can hardly be con-
trolled within the medium. A further drawback

is the limited increase in cell number (approxi-
mately 10-20 times during cultivation). Alterna-
tively, small well mixed bioreactors (for exam-
ple, shake flasks, stirred vessels, and “super spin-
ner” [511]) can be used, where adherent cells are
grown on microcarriers.
Microcarriers are small beads, either solid or
macroporous, having a diameter of approx. 100-
300 µm and a density slightly higher than the
growth medium. When first developed in the late
1960s [512 – 515], microcarrier culture intro-
duced new possibilities and suspension culture
of anchorage-dependent cells in high density.
Nowadays, beads made of DEAE-Sephadex,
DEAE-polyacrylamide, polyacrylamide, poly-
styrene, cellulose fibers, hollow glass, gelatin,
or gelatin-coated dextran beads are in use [442].
In microcarrier culture, cells grow as monolay-
ers on the surface of small spheres (Fig. 33)
or as multilayers in the pores of macroporous
structures that are usually suspended in culture
medium by gentle stirring. By using microcar-
riers in simple suspension culture, fluidized or
packed-bed systems, yields of up to 200 × 106
cells per milliliter are possible [516]. Microcar-
riers are extensively used for vaccine production
on a larger scale (see below) [517 – 521].
Figure 33. Porcine chondrocytes grown on microcarriers
(Cytodex 3, GE Healthcare)
Biotechnology 103
Microencapsulation is another method for
the immobilization of mammalian cells [441].
Basically, it can be used to protect cells against
hazardous environmental conditions. The three
basic encapsulation systems existing are the
bead, the coated bead, and the membrane-coated
hollow sphere [522 – 524]. Typical size for
beads made of polysine alginate is 300 to 500
µm, and the molecular-mass cutoff of these cap-
sule membranes is 60 to 70 kDa [442]. The
capsules can be cultivated in suspension reac-
tors similar to microcarriers. Detrimental with
respect to scale-up is the fragile nature of the
microcapsule.
In membrane bioreactors, including small
hollow-fiber reactors [525], the miniPerm sys-
tem [526] or the tecnomouse [527], cells are
cultivated at tissuelike densities in a compart-
ment which contains one or several types of
membrane for nutrient and oxygen supply and
removal of toxic metabolites. Hollow-fiber sys-
tems (compare Section 9.1.6) are widely used
in the production of biopharmaceuticals in-
cluding monoclonal antibodies. Several exam-
ples of modified membrane bioreactors exist
for the three-dimensional culture of tissue cells
[528 – 530] including hepatocytes [531 – 533],
skin cells [534], or other human cells [535]. The
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
high-density or heterogeneous systems (fixed-
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 membranes. Selection of a reactor systems
depends to a large extent on the specific pur-
pose such as production of a certain amount of
protein (small amounts for basic scientific stud-
ies up to kilogram quantities for medical appli-
cation), three-dimensional tissue cultivation, or
single-purpose or multipurpose facility.
Design of suspension reactors has to deal
mainly with cell damage due to shear stress
caused by agitation and/or aeration [546 – 548].
Stirred-tank reactors (Fig. 35), which are the
most common type of reactor and are nowa-
days build up to 20 000 L scale, are espe-
cially suited for suspendable cells [549]. Ad-
herent cells can be grown on microcarriers and
by this handled similar to a suspension culture
(see Section 9.1.5). For mixing, standard im-
pellers (e.g., rushton turbine, pitched or marine
impeller) as well as large paddle impellers are
used depending on the shear sensitivity of the
cells, sometimes combined to achieve a homoge-
nous mixing throughout the bioreactor at low-
est possible stirrer speed [441, 549]. For aer-
ation, different methods are shown in Figure
36 [550 – 552]. Surface aeration can be applied
only for low cell densities and volumes (below
1 L). Bubble aeration may cause cell damage
mainly due to foam formation, especially in case
of serum-containing medium. By applying spe-
cial aerators (ring sparger, microsparger) in com-
bination with serum- or protein-free medium,

the detrimental effects of bubble aeration can
be overcome [553 – 556]. Alternatively, bubble-
free membrane aeration systems were developed
[557 – 560]. They are characterized by low shear
rates, but require large membrane areas. On lab-
oratory scale, a tumbling membrane basket can
be used for mixing [511]. Rotating/vibrating
sieves provide a gentle aeration, but are suit-
able mostly for laboratory scale [561]. Bubble-
columns and air-lift reactors were build up to
thousand liter scale, are easy to handle, but re-
quire serum-free media to prevent cell dam-
age through bubbles and foam [549, 562]. For
continuous operation of stirred tank reactors
in perfusion mode (see Section 9.1.7), several
techniques for cell and/or product retention are
in use as shown in Figure 37 (e.g., microfil-
tration and ultrafiltration, sedimentation, cen-
trifugation, spin filters, acoustic filter, dialysis
for cell, and product enrichment [563 – 577]).
Suspension reactors are characterized by ad-
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
the long-term perfusion culture of mammalian
cells for the production of biopharmaceuticals
(e.g., monoclonal antibodies, recombinant drugs
including tPA and EPO). With respect to tis-
sue engineering, they have been investigated
for several applications including the cultiva-
tion of “liver” cells as an extracorporal liver de-
vice [588 – 590] or proliferation of stem cells
[591 – 593]. Furthermore they were success-
fully used for cultivation of cells producing
Biotechnology 105
viruslike particles for gene therapy [594 – 597].
Again, scale-up is critical for both fixed-bed and
fluidized-bed reactors. For fixed-bed reactors, a
radial-flow fixed-bed geometry was suggested
[598 – 600] and successfully applied up to ap-
proximately 25 L fixed-bed volume. At perfu-
sion rates of 10-20 L L−1
FB d
−1
, this would corre-
spond to suspension reactors in the thousand liter
scale. Alternatively, rotating-bed bioreactors are
in use [601]. For fluidized-bed bioreactors, sev-
eral concepts for scale-up have been suggested
as well [514, 585 – 587, 602]. Compared to sus-
pension reactors, fixed-bed and fluidized-bed
reactors have the following advantages: high
volume-specific cell density (approx. 5 × 107
cells/mL reactor) and productivity, low shear
rates, simple medium exchange and cell/product
separation, productivity on a high level during
long-term cultures. Disadvantages are: nonho-
mogeneous cell distribution, difficult determi-
nation of cells and cell harvest.
Figure 35. Multipurpose bioreactor for continuous perfu-
sion culture of mammalian cells (Bioengineering AG, CH-
Wald, with permission)
Within hollow-fiber reactors (compare Sec-
tion 9.1.5), cells are immobilized in the extra-
capillar space of a hollow-fi4ber bundle. Nutri-
ents as well as oxygen pass through the fibers,
metabolites leave the culture chamber by this
way. Cell concentrations comparable to those
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
moderately large quantities of high-molecular-
weight products secreted by human and animal
cells, at high concentrations and with a higher
ratio of product-to-medium-derived impurities
than is generally achieved using homogeneous
(e.g, stirred-tank) culture [603 – 605]. Advan-
tages are: very high cell densities, concentrated
product, lower serum demands (if required),
long-term stability, easy handling, and low cost.
Disadvantages are: limited scale-up, mass-trans-
fer problems/concentration gradients, difficult
determination of the cell number, proteolytic ac-
tivity on the product.
During the last years, a growing number of
disposable or single-use bioreactor systems ap-
peared on the market besides hollow-fiber re-
actors, addressing especially problems related
to early process development such as flexibil-
ity, cost effectiveness, time-to-market as well as
quality and regulatory issues [606 – 608]. These
systems are mostly based on a bag technology
[609, 610]. The bags with volumes up to 250 L
are placed on tilting or rocking devices for mix-
ing and oxygen supply. Advantages of single-
use bioreactors are reduced cleaning procedures,
lack of validation issues (cleaning, sterilization,
etc.), lower investment costs, easier adaptation
to changing process demands, less contamina-
tion risk [611 – 613].
The main characteristics of the culture sys-
tems discussed above are summarized in Table

18. All these systems support growth of mam-
malian cells in one or the other way. On labora-
tory scale, mostly disposable flask, membrane,
or bag systems are the method of choice. Small
suspension reactors as well as fixed bed and flu-
idized bed reactors are mostly used for research
or process development. For larger amounts of
products, only suspension reactors or, up to a
certain scale, fixed-bed or fluidized-bed reactors
have the required scalability. For a detailed dis-
cussion, compare [440, 555, 614 – 618]. Biore-
actor design, in particular, is addressed in [619].
9.1.7. Process Strategies
As for all biotechnological processes, process
strategies for operation of bioreactors can be
classified in discontinuous (batch, repeated-
batch or fed-batch) and continuous modes
(chemostat, perfusion). Batch and fed-batch pro-
cesses are mostly performed in small-scale cul-
ture systems (e.g., flasks, bags) or on larger scale
in suspension reactors. Batch processes are usu-
ally starting with an initial cell density of ap-
proximately 1–2 105 cells mL−1 and last 1–2
weeks. After harvest, the bioreactor has to be
cleaned and refurbished before the cycle is re-
peated [438, 440, 441]. Cell and product yield
can be significantly improved by applying a fed-
batch strategy, were nutrients are added after de-
pletion according to an appropriate feeding strat-
egy [[621 – 634]. Batch and fed-batch strategies
are applied quite frequently on industrial scale
[438, 440, 536].
The drawbacks of discontinuous modes such
as large reactor volumes, high maintenance
(cleaning, sterilization, etc.) can be overcome to
some extend by using continuous mode, espe-
cially perfusion with cell and/or product reten-
tion [438, 563]. Chemostat cultures without cell
retention are a valuable tool for research (e.g.,
kinetic studies) [635 – 639] but not for produc-
tion scale because of low cell and product con-
centration. Continuous perfusion present several
advantages over batch systems [563, 640 – 643].
The advantages include the ability to grow cells
to a very high density, the ease of handling me-
dia exchanges for the purpose of fresh feed and
product harvest, the easy removal of metabo-
lites and other inhibitors, and the prospect of
easy scale-up. When dealing with adherent cells,
Biotechnology 107
perfusion reactor design becomes slightly sim-
pler (e.g., fixed-bed and fluidized-bed) because
of the immobilization of cells within macrop-
orous carriers (compare Section 9.1.6). There is
a growing number of reports on stable perfusion
cultures even on an industrial scale over periods
of several months [563, 644 – 646]. The main
advantage of perfusion cultures can be seen in
the reduced bioreactor size (approx. 1/10 of a
suspension reactor without cell retention). Nev-
ertheless, there are some difficulties in the per-
fusion concept [438, 563]. Further equipment
such as the retention device itself, pumps for
feeding, harvest, and medium circulation, stor-
age tanks for feed and harvest are required. The
amount of media needed to complete a moderate
to long-term run can be excessively large. A fur-
ther possible drawback of continuous cultivation
is the possibility for variability over the time of
the run. Batch or fed-batch cultivations are much
shorter in duration and have fewer chances for
random occurrences to happen. This is impor-
tant in dealing with current manufacturing prac-
tice (cGMP, see below) conditions that dictate
precise reproducibility or at a minimum the evi-
dence of non-effects of minor deviations. In the
meantime, there is a growing number of pharma-
ceutical products in the market, produced suc-
cessfully from perfusion systems. Among these
are block busters such as Kogenate-FS r
(Bayer
Health Care) or Remicade r
(Centocor) [563].
Previous concerns about getting such processes
approved are not longer an issue and it can be ex-
pected that because of an increasing pressure on
production costs more perfusion processes will
be installed in the future.
A process that becomes more and more im-
portant for production of small quantities of
recombinant proteins is transient transfection
[647 – 649]. Usually, nontransfected cells (most
commonly HEK-293, COS, and BHK cells,
compare Section 9.1.2) are first cultivated in
batch mode to a certain cell density and then
transfected with DNA encoding for a certain pro-
tein [441]. Usually, the cells are genetically not
stable and soon lose their expression ability but
can produce a certain amount of protein within
one batch. Originally, it was used just as a pre-
liminary test of gene expression. New develop-
ments show that large scale production up to 100
L is possible [650, 651].
108 Biotechnology
Table 18. Main characteristics of cell culture systems and bioreactors ([620], modified)a
T-flask/roller Membrane reactors
bottles/disposable bags (hollow-fibre, miniPerm,
etc.)
Cell density 1 3
Homogeneity 1 1
Shear stress N N
Product concentration 1 3
Porductivity 1 3
Medium efficiency 3 1
Continuous process N Y
(perfusion)
Control 0 1
Downstream process 1 3
Steam sterilizable N N
Reusable N N
a
0, not possible; 1-3, increasing efficiency; Y, yes; N: no.
b
Additional equipment for cell retention required.
9.1.8. Downstream Processes
The desired protein product, mostly extracellu-
lar, has to be purified after cell cultivation by
a combination of unit processes. From an en-
gineering point of view, the challenges are 1)
low product concentrations in the cell-free su-
pernatant, 2) the complex structures of the pro-
teins, especially the recombinant, glycosylated
proteins, resulting in a high sensitivity against
temperature, shear forces, extreme pH, or prote-
olytic activity, 3) contamination by nucleic acid
(DNA fragments from dead cells), 4) contami-
nation by other proteins (e.g., from serum), 5)
potential contamination by viruses or virus par-
ticles [652, 653]. The main process steps in-
volved are [654]: concentration of supernatant
(e.g., precipitation, centrifugation filtration, or
aqueous two-phase partition), primary purifica-
tion/capture (e.g., chromatographic techniques
such as ion-exchange, gel, or immunoaffinity
chromatography as well as precipitation), fur-
ther purification (e.g., ion-exchange, hydropho-
bic, and gel chromatography), and finally pol-
ishing (gel filtration, diafiltration, ultrafiltration)
and techniques for virus inactivation (acid/base
treatment, nanofiltration or ultraviolet-c (UVc))
[653, 655, 656]. The early stages within a pu-
rification strategy will aim to achieve both con-
centration and purification. The next step will
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,
Suspension (stirred-tank, Fixed-bed/fluidized-bed
air-lift reactors) reactors
2 2–3
3 2
Y N
2 2
2 2
3 2
Yb Y
2 3
2 2
Y Y
Y Y
endotoxins, process chemicals, etc. The impor-
tance of the downstream process is underlined
by the fact, that an estimated 60–80 % of the to-
tal production costs are attributed to purification
[438, 439, 654].
9.1.9. Regulatory and Safety Issues
Pharmaceutical production processes are subject
to strict regulations and regular inspections to
ensure the high quality demands for medicines
[439, 440, 657]. Guidelines for aspects relevant
for the production process are summarized un-
der cGMP and corresponding lists were released
in the United States by the FDA [480] as well as
by the EU [481]. There are some special concern
with the use of mammalian cells for production
of biopharmaceuticals, e.g., 1) risk of contam-
ination by microbes, mycoplasma, or viruses,
2) the genetic stability of the host cell line, or
3) the consistency of the product (glycosyla-
tion pattern, etc.). The documents to be submit-
ted to the authorities include detailed informa-
tion concerning the product, the production pro-
cess, product characterization including analyt-
ical methods, validation procedures, methods,
and results of clinical trials. The efforts to fulfill
these requirements are quite high and bind sig-
nificant resources within the companies [439].

9.2. Tissue Engineering
In this chapter, an overview in the field of “red
biotechnology” is presented. Red biotechnology
comprises three major fields:
– Gene therapy
– Production of proteins, antibodies, and vac-
cines for diagnostics and therapy
– Tissue engineering
Gene therapy is a therapeutic approach based
on the correction of a gene defect causing a
disease. Different techniques exist for achiev-
ing this aim. In most cases, the wild-type gene
is inserted into the genome by special methods
replacing the mutant gene, which leads to the
development of the disease. The most common
vectors for the transport of the correct gene into
the genome are viruses.
In the mid-eighties, several treatments with
the genetically modified cells were reported. The
first gene therapy approach was reported in 1990
on a young girl suffering from severe combined
immunodeficiency (SCID). But the therapy did
not work out, after a few months the procedure
had to be repeated regularly.
A major drawback within this field emerged
when it was evident that a young patient died
after being treated by gene therapy [658]. The
patient was suffering from a rare liver disease
and was a volunteer in a gene therapy program.
Similar incidences occurred in the year of 2000
within a group of boys after gene therapeutic
treatment of SCID. The majority of the boys in
this group developed leukemia. Since then, gene
therapy is only broadly applied within science-
fiction movies. Researchers working in this field
went back a few steps to elucidate the devastat-
ing incidences [659].
Biological active compounds produced from
genetically modified cell cultures have been in
use for several years in the treatment of a whole
range of diseases. In most cases, the active in-
gredients are proteins such as blood clotting
factors (e.g., tissue plasminogen activator, t-
PA), growth factors (e.g., erythropoitin, EPO),
or monoclonal antibodies [660]. All these sub-
stances can be produced in animal cell cultures.
These proteins can also be applied in diagnos-
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.
Biotechnology 109
9.2.1. Application of Tissue Engineering
Tissue engineering is a rather young and rapidly
growing interdisciplinary research field which
combines the principles of engineering, life, and
clinical sciences (Fig. 38). The aim of this re-
search field is the development of biological sub-
stitutes that restore, maintain, or improve tissue
functions [661, 662]. Therefore, the knowledge
of cellular interactions, molecular transport phe-
nomena, interactions of cells with material sur-
face, mechanical loading, and biochemical in-
terventions is applied.
An official definition was presented at a con-
ference of the National Science Foundation in
1988: “. . . the application of principles and
methods of engineering and life sciences to-
ward fundamental understanding of structure–
function relationship in normal and pathologi-
cal mammalian tissues and the development of
biological substitutes to restore, remain, or im-
prove tissue function”.
Over the past two decades, encouraging re-
sults have been achieved. Methods and tech-
niques of tissue engineering have already en-
tered therapeutic practice, e.g., for the treat-
ment of sports injuries and damage to cartilage.
The main approach adopted in these cases is
known as autologous chondrocyte transplanta-
tion (ACT). Cells isolated from cartilage tis-
sue of the patient (autologous) are seeded onto
a collagen matrix and cultivated until a three-
dimensional tissue structure is generated. Then
the construct can be implanted into the defect
[663 – 665]. Also tissue-engineered skin is al-
ready used, e.g., for therapy of severely burned
patients [666 – 669]. Researchers are attempting
to engineer almost every human tissue or organ
because the demand of tissue-engineered cells
and organs is still growing because of the fact of
organ shortage for transplantation. The creation
of three-dimensional tissue constructs which can
mimic all functions and metabolic activity like
organs (e.g., liver [670] or pancreas [671]) is still
a crucial challenge. The first tissue-engineered
heart valves have been implanted to a patient and
show promising results in function and mechan-
ical stability [672, 673].
110 Biotechnology
Figure 38. Disciplines involved in tissue engineering
Figure 39. Principle of tissue engineering
9.2.2. Principle of Tissue Engineering
The principle of tissue engineering can be
demonstrated in the following way: First, cells
have to be isolated. The cells are seeded to a
matrix and are extracorporal cultivated in a suit-
able culture system or bioreactor in medium con-
taining nutrients and growth factors. After cul-
tivation, the resulting tissue is reimplanted into
the patient (Fig. 39).
Tissue engineering is driven by the hopes of
the patients of increased availability of trans-
plantable organs, to ease their suffering, fore-
stall imminent death, and enabling a better qual-
ity of life. Alongside these medical objectives,
there are also huge economic issues at stake
because treatment of tissue and organ damage
or dysfunction often involves long hospitaliza-
tion and thus is very expensive. In contrast to
alternative approaches of xenogenic (from an-

other species) and allogenic (same species but
another individual) organ transplant, tissue en-
gineering involves transplanting cells from the
body of the patient itself. This is the reason for
which such great attention has been focused in
the last few years on this new area of research.
By growing 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-
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.
9.2.3. Strategies
Tissue engineering can be performed by using
one of the three approaches:
In vitro cultivation of autologous cells on or-
ganic, synthetic, or natural matrices
In vitro cultivation of autologous cells on
xenogenic matrices
Iin vitro tissue generation from embryonic
stem cells
Depending on the tissue of interest, either
a close system (e.g., extracorporal device, ar-
tificial liver) or an open system based on a
biodegradable polymer scaffold is developed.
The autologous cells can be isolated tissue-
specific primary cells or stem cells.
9.2.4. The Essentials
The essentials for tissue engineering are shown
in Figure 40. For a successful generation of
three-dimensional tissues, suitable cells have to
be isolated and cultivated on a matrix in an
appropriate medium containing all necessary
growth and differentiation factors by using an
optimized bioreactor system. During cultivation,
Biotechnology 111
the application of a mechanical load (e.g., strain,
compression) is beneficial for the development
of three-dimensional cell–cell contacts resulting
in functional tissue [674 – 682].
Figure 40. The “essentials” for tissue engineering
9.2.5. Cells
For tissue engineering, either tissue-specific
cells can be isolated and cultured or stem cells
can be used for the directed differentiation to the
desired tissue. Due to their potential for differen-
tiation into several different tissue types, nowa-
days the use of stem cells is intensively discussed
[683]. In numerous publications adult stem cells
isolated from bone marrow (bone marrow stro-
mal cells BMSC [684 – 686], fat tissue (adipose-
derived stem cells adSC [687]), and peripheral
blood or umbilical cord blood (both haematopoi-
etic cells [688, 689]) are applied. The potential
for differentiation into several different types of
tissues is already approved. Mesenchymal stem
cells (MSC) play a major role in modern tissue
engineering [690, 691]. These cells are easy to
isolate from bone marrow or fat tissue and their
availability is not so limited compared with that
of the small number of stem cells present in, e.g.,
peripheral blood (0.1–0.2 %). Treatment con-
cept based on the use of autologous stem cells
would eliminate problems of donor site scarcity,
immune rejection and pathogen transfer.
9.2.6. Biomatrices
There has been also huge progress in the de-
velopment of biomaterials for use in tissue-
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
natural occurring compounds or scaffolds, also
synthetic biomaterials are reviewed in the liter-
ature [703 – 705]. Along with polysaccharide-
and protein-based (e.g., hyaluronic acid, colla-
gen) materials, also many synthetic polymers
are used for tissue-engineering application (e.g.,
polylactic acid, polyglycolic acid, polyurethane,
polyesters).
9.2.7. Bioreactors for Tissue Engineering
The design, control, and operation of bioreac-
tors in technical and engineering disciplines are
well-established. Their use in medical sciences
requires an interdisciplinary approach aiming at
the development of functional tissues. There-
fore, frequently the application of mechanical
loading is needed [706 – 709]. The loading can
be disposed in different ways; for example, a
longitudinal straining leads to the formation of
ligaments or tendons; compression is applied
when aiming at the development of, e.g., car-
tilage; pressure (perfusion) can be applied for
bone tissue formation and pulsed bioreactors
are used for blood vessel generation. In Figure
41, a selection of bioreactor schemes for tissue-
engineering applications is presented. Unlike
in conventional mammalian cell culture, where
only a limited number of bioreactor types are
used (e.g., T-flaks, roller bottles, spinner sys-
tems, stirred-tank reactors), in tissue engineer-
ing tailor-made bioreactors have to be devel-
oped and constructed for each specific tissue
type, considering in particular the fluid dynam-
ics which are realized by these different systems.
Figure 41. Selected bioreactor types for tissue engineering
Since human cells are anchorage-dependent,
they must be attached to a matrix in the bioreac-
tor during the cultivation. In a rotating-wall sys-
tem, the cell-seeded matrices are cultured while
the reactor wall is rotated, thus the cells float
through the medium and are affected by mi-
crogravitylike conditions, and the mass-transfer
rates are improved. Moreover, cell-seeded ma-
trices can be cultured while hanging in spinner
flasks, which results also in an improved mass
transfer. Another possibility is the continuous
pumping of the medium through a fixed bed with
cells or the cells can be placed into a chamber
where a mechanical stimulation (here compres-
sion) is applied.
9.2.8. Growing New from Old
The idea of organs one day being freely avail-
able “off the shelf” is still an aspiration today.
The need is great, however, and patients are of
course very eager to have “personalized” treat-
ment from “organ designers” using tissue engi-
neering. The most ambitious concepts involve
applying a sort of “building-block” principle to
the culture of each tissue or organ type: so-
called precursor or stem cells would be used,
whose potential for differentiation could be ex-
ploited through the use of appropriate growth or
differentiation factors. Thanks to this method,
one would “only” need the appropriate “ingre-
dients”: Further advantages of this approach are
the fact that an organ or tissue replacement can
be carried out at any time – in other words it can

be scheduled in advance. Regenerative medicine
has great potential: according to a report in Time
Magazine, over the next 10 to 20 years, tissue en-
gineering will become one of the top careers and
a new era in life sciences and medicine dawns.
The potential applications are highly diverse but
are to be found essentially in the treatment of
cells and tissue which possess little or no capac-
ity to repair themselves such as cartilage, heart
muscle and nerve tissue.
The medicine of tomorrow will strive to de-
velop personalized therapeutic procedures in-
volving the patient in each treatment choice.
There will be a move from a general “one-
size-fits-all” approach to personalized treatment
based on the principle of “one drug, one ther-
apy, one patient”. This will mean using a sys-
tems biology approach to fully restore the origi-
nal healthy state of the patient. This dream could
be realized thanks to the emerging technologies
of regenerative medicine and tissue engineering.
Groups of cells and tissue generated by using
tissue engineering can also serve as test systems
which exhibit characteristics specific to a given
organ. These can be used to test the effective-
ness and duration of certain drugs or to study
their mechanisms and metabolism, thus repre-
senting an alternative to animal experiments, and
a rapid, broadly applicable and cost-effective
way of screening pharmaceutically active sub-
stances.
Over the last ten years, there has been an ex-
plosion of research in tissue engineering, which
shows the enormous importance attached to this
technology. Within Europe, German researchers
are playing a major role. Leading research is also
being carried out by teams in France, Britain,
Italy, and the Netherlands. Large numbers of
small biotechnology companies are working in
tissue engineering and more and more are being
set up all the time.
In the 21st century, major scientific questions
concern modern developments within the field
of regenerative medicine, which includes tissue
engineering, transplantation, prosthesis, as well
as pharmacological interventions stimulating in
situ tissue regeneration.
Biotechnology 113
9.3. Biotechnology and Food
It is not presumptuous to claim that modern
biotechnology has developed from traditional
processes of food preparation. The production of
alcoholic beverages, the coagulation of milk pro-
teins, and the preservation of vegetable food by
fermentation has already been familiar to most
ancient civilizations. Thus, several microorgan-
isms have a very long history of safe use in hu-
man food production. The main focus of this
chapter is not on traditional fermentations but
on recent developments in food biotechnology.
Since this field is remarkably ambitious and
complex, only some techniques of high indus-
trial relevance are covered exemplarily. Genetic
engineering of plants has yielded, for exam-
ple, herbicide tolerant “RoundUp Ready” soy,
Bacillus thuringiensis toxin expressing maize
(Bt maize) and transgenic rice lines with an
increased concentration of β-carotene in the
endosperm (“golden rice”). Genetically engi-
neered animals may produce milk with an al-
tered protein profile, and modification of the
hormone expression of fish imparts accelerated
growth rates. A number of comprehensive re-
views on food biotechnology are available [710].
9.3.1. Production of Food Additives by Cell
Culture Systems
9.3.1.1. Amino Acids
l-Amino acids represent the largest group of
food and feed additives produced by means of
biotechnology [711]. Consequently, the litera-
ture published within the last decade is notably
versatile. l-Glutamic acid ((S)-2-aminoglutaric
acid, E 620) was the first amino acid pro-
duced biotechnologically on an industrial scale,
and is still the leading amino acid by volume.
More than 800 000 t/a of monosodium gluta-
mate, which serves as a flavor enhancer (“umami
taste”), are produced worldwide by cultivation
of Corynebacterium glutamicum and related or-
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-
portant amino acid is l-lysine ((S)-2,6-diami-
nohexanoic acid), which is mainly used to sup-
plement animal feed with this essential and often
limiting amino acid. It is industrially produced
by C. glutamicum or by E. coli species [711]. Ef-
ficient industrial producer strains have been de-
veloped by multiple rounds of mutagenesis. De-
fined l-lysine producing C. glutamicum mutants
have been generated for example by modifica-
tion of the enzyme aspartate kinase (lysC muta-
tion) [715], attenuation of the transcription reg-
ulator tipA gene [716], and the introduction of a
mutation (S361F) into the 6-phosphogluconate
dehydrogenase gene (gnd) [717]. In the latter
case, the modified strain was less sensitive to
allosteric inhibition by intracellular metabolites
than the wild-type strain. Product yields of up
to 100 g/L are attainable within 24 h of culti-
vation with the recombinant production strains.
A third amino acid, l-cysteine ((R)-2-amino-3-
mercaptopropanoic acid), which is applied as a
dough conditioner in the bakery industry, may be
obtained from recombinant E. coli [718 – 720].
9.3.1.2. Organic Acids
Citric acid (2-hydroxy-1,2,3-propanetricarbox-
ylic acid, E 330) is widely used to lower the
pH of food (candy) and beverages (soft drinks,
wine). Furthermore, it acts as an efficient chela-
tor of prooxidative iron and copper ions. Various
fungi and bacteria may be used for the biotech-
nological synthesis of citric acid starting from
numerous substrates. The process has recently
been comprehensively reviewed in [721]. As-
pergillus niger grown as surface cultures on di-
luted molasses has been employed as early as
in the 1920s, and still is the most popular pro-
duction organism. Today, distillers’ grains [722],
palm oil mill effluents [723], or cassava starch
factory wastes [724] may serve as cheap and
abundantly available substrates. By optimiza-
tion of the growth conditions (sufficient oxygen
supply in submerged cultures; media deficient in
manganese, copper, iron, and zinc ions; high glu-
cose concentrations), and by selection of over-
producing A. niger strains, yields of > 100 g/L
are attainable [725]. The yeast Yarrowia lipoly-
tica represents a future potential production or-
ganism. The strain Y. lipolytica 187/1 showed
maximum synthesis rates when grown on raw
agro-industrial fats as substrates [726], while
Rymowicz and Cibis [727] selected the acetate-
negative strain Y. lipolytica AWG-7, cultivated
on glucose syrup, for citric acid production. As
a second important organic acid, l-lactic acid
((S)-2-hydroxypropanoic acid, E 270), has been
used from ancient times as a food preservative
by lowering the pH during fermentation. More
recently, lactic acid has gained increasing in-
terest since the monomer is used for genera-
tion of the biodegradable food packing poly-
mer “polylactic acid (PLA)”. However, only few
PLA-generating processes have been commer-
cialized and the cost of PLA is still not able
to compete with synthetic plastics [728, 729].
Apart from traditional lactic acid bacteria such
as Lactobacillus delbrueckii [730] or L. plan-
tarum [731], a wide range of further microor-
ganisms, especially strains of Rhizopus oryzae,
has been employed for the biosynthesis of l-
lactic acid (for example [732, 733]). A current
review on metabolic engineering approaches of
lactic acid bacteria is available [734].
9.3.1.3. Vitamins
l-Ascorbic acid (vitamin C, E 300) is produced
by a combination of chemical synthesis and en-
zymatic reaction steps. Known as “Reichstein
process”, the procedure was established already
in the 1930s. The key biotransformation step,
the oxidation of d-sorbitol to l-sorbose, is cat-
alyzed by Acetobacter suboxidans [735]. Due
to the worldwide use of l-ascorbic acid as vita-
min in dietary supplements and as antioxidant in
food and feed, the annual need sums up to 60 000
t [711]. Novel approaches use either recom-
binant microorganisms in one-step procedures
[736] or Azotobacter chroococcum strains [737]
for l-ascorbic acid production. For a review
on biotechnological routes to vitamin C, see
[738]. Further water-soluble vitamins, includ-
ing riboflavin (vitamin B2 ) and cyanocobalamin
(vitamin B12 ), are currently produced by mi-
crobial cell cultures. The chemical synthesis of
the yellow-greenish-colored riboflavin has been
largely replaced by biotechnological processes
using either the overproducing fungus Ashbya
gossypii [739] or mutant strains of Bacillus sub-
tilis. The “generally recognized as safe” (GRAS)

organism B. subtilis is widely used in the food in-
dustry [725] and, for example, the mutant strain
CJKB0002 produced 26.5 g/L riboflavin com-
pared to 22.4 g/L obtained from the parent strain
[740]. Detailed mechanistic studies on the ri-
boflavin synthesis in Ashbya gossypii [739] and
in the lactic acid bacterium Lactococcus lac-
tis ssp. cremoris [741] have been published.
Cyanocobalamin is derived from Propionibac-
terium or Pseudomonas denitrificans species
[711]. Recently, a fermented milk product con-
taining Propionibacterium freudenreichii ssp.
shermanii B369 bacteria, capable of produc-
ing cyanocobalamin in situ, was patented [742].
Thus, a vitamin B12 enriched yogurt may be
manufactured as a “nutraceutical” without ex-
ternal vitamin fortification. However, compara-
tively low world-wide annual production rates of
3000 t for vitamin B2 and 10 t for vitamin B12
document the predominant use of theses vita-
mins in dietary supplements and in pharmaceu-
ticals [711]. The main plant-derived carotenoid,
β-carotene (E 160a), serves as provitamin A in
the human diet. Since β-carotene additionally
imparts an intense yellow-orange coloring, it
is widely used as a natural food additive. The
development of biotechnological processes for
the production of β-carotene is mainly driven
by the increasing demand of the food industry.
Besides the extraction from plants (for exam-
ple from lucerne), β-carotene is industrially ob-
tained from the algae Dunaliella salina [743]
or from the fungus Blakeslea trispora [744].
The carotenoid astaxanthin, which is used on
a large scale in aquaculture for the coloring of
salmonids and crustaceae, is formed by the yeast
Phaffia rhodozyma [745].
9.3.1.4. Sweet Compounds
Palatinit (isomalt, E 953) is a non-cariogenic
sugar substitute used in low-calory food,
desserts, ice creams, and diabetic food. It is
a white, crystalline, and non-hygroscopic sub-
stance with a sweetening power of roughly
0.45–0.60 compared to that of sucrose (1.0). In
the first production step, sucrose is converted
enzymatically (by action of a glycosyltrans-
ferase) into palatinose (isomaltulose, O-α-d-
glucopyranosyl-(1–6)-d-fructofuranose) by im-
mobilized cells of Protaminobacter rubrum.
Biotechnology 115
Subsequently, palatinose is reduced to palatinit,
an equimolar mixture of the corresponding alco-
hols (1-O-α-d-glucopyranosyl-d-mannitol and
6-O-α-d-glucopyranosyl-d-sorbitol) by hydro-
genation on a fixed-bed Ni catalyst. In the in-
dustrial process, it is advantageous to use im-
mobilized non-viable cells of P. rubrum in a
packed-bed reactor for the enzymatic step [746].
Apart from P. rubrum, immobilized cells of Ser-
ratia plymuthica [747], Erwinia sp. D12 [748],
or Klebsiella singaporensis LX3T (isolated from
soil) [749] have been used to convert sucrose into
palatinose. In 2005, palatinose was approved ac-
cording to the EU regulation (EC 258/97) con-
cerning novel foods and novel food ingredients,
and the GRAS registration was granted in 2006
by the US Food and Drug Administration (FDA).
Recent inventions suggest a mixture of palati-
nose with cocoa powder for the preparation of in-
stant beverages [750]. Further applications com-
prise the use of palatinose for the production of
low-alcohol beer or beerlike soft drinks [751],
and the preparation of fermented soybean milk
[752].
In the industrial production of the sweet-
ener aspartame (α-l-aspartyl-l-phenylalanine
1-methyl ester, E 951), the methyl ester of the
“DF” dipeptide, both components are produced
by biotechnological processes. l-Phenylalanine
is usually manufactured by large-scale cultiva-
tion of the soil bacteria Serratia marcescens or
C. glutamicum. Immobilized aspartase or alter-
natively intact cells of E. coli are used to syn-
thesize l-aspartic acid starting from the precur-
sors ammonia and fumarate [725]. Usually, amid
formation is performed chemically although an
enzymatic approach exists [753]. Thermolysine,
immobilized on an agarose–aldehyd gel, is used
as biocatalyst for the synthesis of enantiopure
aspartame [754]. Thermolysine is a metallopep-
tidase with a zinc ion in its active site and four
calcium ions, which stabilize the tertiary struc-
ture of the enzyme.
9.3.1.5. Sugar Alcohols
Sugar alcohols such as d-sorbitol (d-glucitol; E
420) and d-xylitol (E 967) have attracted the at-
tention of the food and pharmaceutical industry
because of their sweetening and non-cariogenic
properties. Due to the fact that their metabolism
116 Biotechnology
is insulin-independent, they are well suited for
substituting sugar in diet schemes for diabetics.
Since the biotechnological production of xyli-
tol represents an economically attractive alter-
native to the industrialized chemical process,
various process variants have been published.
Somewhat less literature is available on sor-
bitol, which is mainly formed by cultures of Zy-
momonas mobilis [755, 756]. The synthesis of
xylitol by Candida guilliermondii depends on
several crucial process parameters, one of which
is the concentration of phosphate buffer in the
culture broth (optimum at 0.6 M) [757, 758].
In another approach, the d-xylitol production
of the yeast Debaryomyces hansenii UFV-170
was monitored with respect to temperature and
starting pH [759]. Various substrates, including
for example hydrolysates of corn cobs [760] and
sugarcane bagasse [761] have been used for the
bioconversion of the pentose d-xylose to xylitol.
9.3.1.6. Microbial Saccharides
The most popular polymer currently produced
by means of biotechnology is xanthan gum (E
415). Xanthan gum is a complex bacterial ex-
opolysaccharide structurally closely related to
cellulose. Today it is mainly produced by mu-
tants of Xanthomonas campestris (40 000 t/a
[711, 762]), a Gram-negative bacterium that
is pathogenic to many crops. Due to its out-
standing properties (high salt and pH toler-
ance, thermostability, and unusual rheological
behavior), xanthan is predominantly used as a
thickener in dressings and convenience food
[763]. Sago starch has been suggested as an
alternative carbon source instead of glucose
[764]. Trehalose (α-d-glucopyranosyl-(1-1)-α-
d-glucopyranose) is a nonreducing disaccharide
that is thought to play a role as storage carbo-
hydrate for many microorganisms. It may serve
as an additive to improve the freeze stability
of processed food. In 2000 and 2001, trehalose
has gained the FDA GRAS and the EU novel
food approval, respectively. Trehalose is ob-
tainable inter alia from Brevibacterium [765],
Cellulosimicrobium cellulans [766], or Debary-
omyces hansenii strains. The latter two were cul-
tivated under moderate saline stress for extra-
cellular trehalose production [767]. Literature
[768] reviews trehalose production (with a fo-
cus on the conversion of starch to trehalose) and
potential applications in the food industry.
9.3.1.7. Conjugated Linoleic Acids (CLA)
Lactic acid bacteria play a prominent role in the
traditional fermentation of food. One of the ex-
ceptional features of some lactobacilli is their
ability to isomerize linoleic acid to different
conjugated linoleic acid isomers (CLA). The
Z9,E11- and E9,E11-octadecadienoic acid are
assumed to possess beneficial physiological and
anticarcinogenic properties [735]. Lactobacillus
plantarum AKU 1009a formed up to 40 mg/mL
CLA (33 % molar yield) in 108 h under opti-
mized conditions from 12 % (w/v) linoleic acid
as substrate [769]. Further lactobacilli capable of
CLA production encompass for example L. del-
brueckii ssp. bulgaricus [770] and L. plantarum
JCM 1551 [771]. Castor oil proved to be an ade-
quate substrate for CLA formation. Presumably,
a two-step reaction mechanism, involving hy-
dration of a double bond with subsequent elimi-
nation of water, is leading to different CLA iso-
mers [772].
9.3.1.8. Lactulose
Lactulose acts as a prebiotic compound and thus
may contribute to increased levels of “health-
promoting” bacteria (bifidobacteria and/or lac-
tobacilli) in the human intestinal tract [773].
The term “prebiotics” comprises a wide vari-
ety of oligosaccharides that are not digested in
the stomach and in the small intestine. They
naturally occur in garlic, asparagus, onion, and
others. Apart from soybean oligosaccharides, a
number of uncommon fructo-, galacto-, xylo-,
and isomalto-oligomers are currently in use, es-
pecially in Japan [773]. The disaccharide lac-
tulose (4-O-β-d-galactopyranosyl-d-fructose) is
a non-naturally occurring compound with po-
tential applications in infant milk formulations
and foods [774]. Although lactulose is chiefly
manufactured chemically using boric acid or
aluminates as catalysts, a biotechnological route
starting from lactose (as galactose donor) and
fructose with permeabilized Kluyveromyces lac-
tis yeast cells has been developed [775]. In

another approach, β-galactosidase from As-
pergillus oryzae and a hyperthermostable β-
glycosidase from Pyrococcus furiosus were used
for the transgalactosylation of lactose in the
presence of fructose [776]. Important food ad-
ditives and food supplements produced by cell
culture systems are summarized in Table 19.
9.3.2. Enzyme-Catalyzed Processes
The modification of starch and lipids as well
as the production of cheese and fruit juices are
prime examples of enzyme-catalyzed biopro-
cesses in the food industry. For economic rea-
sons, the necessary enzymes are often employed
as crude products or even as mixtures of en-
zymes with different properties. If required, the
enzymes are purified by means of precipitation,
ultrafiltration and/or fast protein liquid chro-
matography (FPLC). Common FPLC protocols
comprise one ore more of the steps hydropho-
bic interaction chromatography (HIC), ion ex-
change chromatography (IEX), and size exclu-
sion chromatography (SEC). Reusability and
sometimes also stabilization may be achieved
by immobilization of the biocatalyst on an inert
carrier material.
9.3.2.1. Starch-Modifying Enzymes
Traditionally, the degradation of starch to mal-
todextrins and glucose/fructose syrups was per-
formed by acid-catalyzed hydrolysis, using
strong mineral acids such as hydrochloric or
sulfuric acid. The harsh reaction conditions af-
forded a variable spectrum of unwanted by-
and breakdown products. Therefore, the acid
hydrolysis has been replaced more and more by
enzymatic processes. Today, starch-converting
enzymes comprise about 30 % of the enzyme
market worldwide. Amongst the starch con-
verting enzymes, α-amylase (EC 3.2.1.1, 1,4-
α-d-glucan glucanohydrolase) holds the largest
market share with major applications in the
starch and bakery industries [777, 778]. α-
Amylases may be derived from several plant
materials, bacteria, yeasts, and fungi [779].
For commercial applications, mainly strains of
Bacillus amyloliquefaciens and Bacillus licheni-
formis are employed as enzyme producers. α-
Amylase attacks the α-(1-4)-links within the
Biotechnology 117
starch molecule (endoamylase), while the α-
(1-4)-bonds near α-(1-6)-branches are resistant
to hydrolysis. Thus, the end products of ex-
tensive α-amylase treatment are oligosaccha-
rides of varying length with α-configuration
and α-limit dextrins, which constitute branched
oligosaccharides. Since α-amylase of B. licheni-
formis is remarkably thermostable (it remains
active for several hours at temperatures of > 90
◦
C), its application in the industrial starch hydro-
lysis is highly efficient [780].
β-Amylases (EC 3.2.1.2; 1,4-α-d-glucan
maltohydrolase) are present mainly in higher
plants, but also in some microorganisms. Com-
mercial products are available from malt, soy,
and wheat. They hydrolyze α-(1-4)-glucosidic
bonds, effecting successive removal of maltose
units from the nonreducing end (exoamylase).
In contrast to amylose, amylopectin is not com-
pletely hydrolyzed, as all reactions stop before
branch points are reached (β-limit dextrin).
Glucoamylase (EC 3.2.1.3; 1,4-α-d-glucan
glucohydrolase) is formed predominantly by
fungi and yeasts (Aspergillus). It starts at the
nonreducing end of α-(1-4)-d-glucans and suc-
cessively liberates β-d-glucose units. The α-(1-
6)-bonds in amylopectin are also cleaved, but
around 30 times slower than the α-(1-4)-bonds.
At higher concentrations, glucoamylase reforms
all the glycosidic bonds that it hydrolyses. Thus,
it condenses glucose, for example, to isomaltose
(6-O-α-d-glucopyranosyl-d-glucose).
Debranching enzymes exclusively hydrolyze
α-(1-6)-bonds in amylopectin, glycogen, and
pullulan. Linear amylose fragments are formed
from amylopectin. Pullulanase (EC 3.2.1.41;
pullulan α-(1-6)-glucanohydrolase) hydrolyzes
α-(1-6)-bonds in pullulan (a linear polymer of α-
(1-6)-linked maltotriose units) and amylopectin,
while isoamylase (EC 3.2.1.68) is only capable
of the hydrolysis of the α-(1-6)-bonds in amy-
lopectin. Pullulanases have been found in a va-
riety of bacteria; industrially used is an enzyme
derived from Bacillus acidopullulyticus.
Cyclodextrins are produced via an intramo-
lecular transglycosylation reaction in which
the enzyme cyclodextrin glycosyltransferase
(EC 2.4.1.19; 1,4-α-d-glucan 4-α-d-(1,4-α-
d-glucano)-transferase) cleaves the α-(1-4)-
glycosidic bond and concomitantly links the re-
ducing to the nonreducing end.
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-
fructose corn syrups, HFCS) are obtained from
glucose solutions by action of the enzyme xy-
lose (glucose) isomerase (EC 5.3.1.5, d-xylose
ketol isomerase) [725]. Technical enzymes are
derived for example from Streptomyces, Actino-
planes, Arthrobacter, or Bacillus cultures. The
large-scale isomerization of glucose is per-
formed with immobilized enzymes, and product
yields of up to 22 tons of isosugars per kilogram
of catalyst are obtained. Fructose concentrations
of about 90 % are accessible via a subsequent
chromatographic purification step. An excellent
survey of enzymes involved in the industrial pro-
cessing of starch is presented in [781]; and the
most important starch converting enzymes are
summarized in Table 20.
9.3.2.2. Lipases
Lipases (EC 3.1.1.3, triacylglycerol acyl-
hydrolase) are valued biocatalysts with numer-
ous applications in food biotechnology. Many
representatives of this enzyme class are highly
stable in organic solvents, show broad substrate
specificity, and high regio- and/or stereoselec-
tivity [782]. Main fields of application include
the rational design of lipids with specific struc-
tures (“functional oils and fats” [783]), and
the production of volatile fatty acids and fla-
vor esters (“natural food flavorings”). Volatile
fatty acids are the so-called “character impact”
flavor compounds of various types of cheese,
while numerous flavor esters are responsible for
fruity aroma impressions [784]. Industrial high-
level production of lipases requires not only
the efficient overexpression of the correspond-
ing genes, but also a detailed understanding of
the mechanisms governing the folding and se-
cretion of the proteins [785]. Immobilized li-
pases from the yeast Candida rugosa are tra-
ditionally popular, as a wide core in the cat-
alytic centre allows for acceptance of various
fatty acids for trans-esterification or synthesis of
mono- and diacylglycerides, for example, [786].
Especially C. antarctica lipase B features out-
standing properties such as high thermostabil-
ity, sn-2 recognition in hydrolysis of triacylglyc-
erides, and selectivity towards (E)-fatty acids. A
review on applications of the lipases A and B
from Candida antarctica was published recently
[787]. Several strains of another member of the
Candida family, Yarrowia (Candida) lipolytica,
were screened for lipase secretion. The high-
est lipase activity produced on rapeseed oil as
sole carbon and energy source was found to be
2760 U/mL for Y. lipolytica 704 [726]. When
cultured in a 2000 L bioreactor, the lipase activ-
ity reached 1100 U/mL after 53 h [788]. Lipases
with novel catalytic properties are produced for
example by the basidiomycetous fungus Pleuro-
tus sapidus. An extracellular enzyme of the type
B carboxylesterase/lipase family efficiently hy-
drolysed xanthophyll esters [789].
9.3.2.3. Pectin-Degrading Enzymes
Pectin is a complex polysaccharide (α-d-(1-4)-
polygalacturonic acid, esterified with methanol
in varying degree) that represents an essential

Table 20. Starch-converting enzymes
Enzyme Enzyme number
α-Amylase EC 3.2.1.1
β-Amylase EC 3.2.1.2
Glucoamylase EC 3.2.1.3
Pullulanase EC 3.2.1.41
Cyclodextrin glycosyltransferase EC 2.4.1.19
Xylose isomerase EC 5.3.1.5
part of plant cell walls. The group of pecti-
nolytic enzymes comprises pectinases (poly-
galacturonases), lyases, and pectin esterases. A
total market share of 25 % of the global sales
of food enzymes is currently attained by this en-
zyme family [790]. To improve the yields in fruit
and vegetable juice production, and to obtain
clear and concentrated juices, especially pecti-
nases from the mold Aspergillus niger are em-
ployed [791, 792]. For example, the efficiency
of kiwi juice production was increased > 10 %
by addition of 40 mg/L pectinase for 150 min
[793], and the yield of loquat juice was 16 %
higher when 160 mg/L pectinase were added
for 4 h [794]. Under these conditions, the loss
of vitamin C was low and the juice was clar-
ified. Endo- and exopectinases (EC 3.2.1.15,
endopolygalacturonase; EC 3.2.1.67, exopoly-
galacturonase) depolymerize the pectin chain by
hydrolytic cleavage of the glycosidic α-(1-4)-
bonds.
Usually, pectinases are combined with hemi-
cellulases (mixture of cell-wall-degrading en-
zymes) and cellulases (EC 3.2.1.4) to improve
fruit liquefication, leading to high-quality juices
in terms of oxidation, aroma levels, and sta-
bility [795]. As an auxiliary enzyme, α-l-
arabinofuranosidase (EC 3.2.1.55), was sug-
gested for applications in the wine industry and
for clarification of fruit juices [796]. Pectin lyase
(EC 4.2.2.10) and pectate lyase (EC 4.2.2.2)
catalyse the eliminative cleavage of pectin and
pectate, respectively, to oligosaccharides with
4-deoxy-α-d-gluc-4-enuronosyl groups at their
nonreducing ends. Potential production organ-
isms include Erwinia species as well as a num-
ber of phytopathogenic fungi. Pectin esterases
(EC 3.1.1.11) catalyze the hydrolytic cleavage
of the methyl ester group. These enzymes gen-
erally exhibit a high specificity for pectin sub-
strates.
Biotechnology 119
Producer organism Specificity
Bacillus licheniformis endo-α-(1-4)
malt, soy, wheat exo-α-(1-4)
Aspergillus ssp. exo-α-(1-4); exo-α-(1-6)
Bacillus acidopullulyticus endo-α-(1-6)
Bacillus macerans intramolecular transglycosylation
Streptomyces ssp. isomerization of glucose to fructose
9.3.2.4. Chymosin (Aspartic Protease)
Chymosin (EC 3.4.23.4) is a key enzyme with
peptidolytic activity used in the production of
cheese. The casein fraction of milk protein is
coagulated by hydrolysis of κ-casein between
Phe105 und Met106 to form para-κ-casein and
a glycopeptide (κ-casein glycomacropeptide).
From a mechanistic point of view, chymosin be-
longs to the class of aspartate peptidases, which
are characterized by the presence of two aspartic
acid residues in the catalytic center.
Since recombinant chymosin does not exhibit
unwanted side activities and is not contaminated
with microorganisms possibly causing quality
problems, it replaces rennin from the stomachs
of calves (abomasus) to an increasing degree.
The gene encoding chymosin has been cloned
into different microorganisms, and the proper-
ties of the recombinant enzymes expressed in the
yeasts Kluyveromyces lactis and Pichia pastoris
have been compared [797]. Since K. lactis is re-
garded as a safe organism traditionally used in
food processing, it is of outstanding importance
in the biotechnological production of chymosin
on an industrial scale [798]. The technological
characteristics of various enzymes from geneti-
cally modified organisms were nearly identical
to those of natural rennin, and only slight dif-
ferences in the coagulation time and curd firm-
ness were observed [799]. Chymosin obtained
from transgenic sheep milk and the recombinant
protein expressed by K. lactis differed only in
their action on low-molecular-weight substrates,
but not in enzymatic activity on protein sub-
strates [800]. Recently, extracellular acid pep-
tidases from Rhizopus oryzae have been sug-
gested as rennin substitutes in cheese manufac-
turing [801, 802].
120 Biotechnology
9.4. Biotechnology and Health
9.4.1. Individualized Medicine
Single nucleotide polymorphisms (SNPs, a sin-
gle base is exchanged by an alternate) are the
most common form of genetic variant in the hu-
man genome, occurring on average 1 per 1000
base pairs. Since the influence of SNPs on med-
ical therapy or diet is better understood, the SNP
diagnostic is coming in the focus of interest. One
important goal of genome research is studying
genetic variance among individuals to improve
the knowledge and treatment of disease and to
identify the variations in our genes that influence
the risk of an individual of becoming ill. Be-
cause inherited differences influence most com-
mon diseases and many drug responses, the un-
derstanding of existing genetic variations in the
human population is a promising approach in the
development of tailor-made therapeutics. Eighty
percent of the individual variations are SNPs,
which have a direct impact of gene regulation or
of a protein product of a gene. In cases where
one SNP is sufficient to cause disease it is possi-
ble to analyze the causal change and to improve
our understanding of disease [803 – 806]. The
hope is that a complete human SNP map will
increase the efficiency of drug development and
medical treatment of disease. Many genotyping
platforms have been developed to detect many
SNPs at once in a efficient, cost-effective man-
ner including microarrays by the use of allel-
specific oligonucleotides containing the variable
base. Large-scale SNP genotyping should lead
to a fully understanding of the genetic architec-
ture of common traits underlying disease, drug
or diet response. For example, microarray ex-
periments could help to define the ideal dose of
a drug and quantify the impact of this dose on
human health. Since these are the most person-
ally data we know, the access to these data must
be protected so it is not misused by employers
or insurers. The goal of this analysis is to relate
SNP variation to disease or diet.
9.4.2. Clinical Diagnosis as Indicated in
Genetic Anomalies in Cancer
Solid tumors often show alterations in the
genome that lead to changes in DNA sequences
and copy number. These changes can be de-
tected and mapped by using comparative ge-
nomic hybridization (CGH). CGH is a molec-
ular cytogenetic method of screening for ge-
netic alterations. These changes are classified as
DNA gains or losses and show a characteristic
pattern that includes mutations at chromosomal
level. In the past few years, microarray-based
formats for CGH have been developed made
from large genomic clones and cDNAs. These
arrays provide many advantages over conven-
tional CGH, including higher resolution, direct
mapping of changes to the genome sequence,
and high throughput. Together with the analysis
of complex differences in gene expression bet-
ween normal and cancer cells, these data provide
insight into malignancy and reveal genes that
may be useful as diagnostic or prognostic mark-
ers. One problem performing cancer diagnosis
using gene expression profiles from microarray
analysis considers the heterogeneity of a tumor.
Samples taken from different locations in the
tumors are dramatically different. Therefore, bi-
ological classification on the basis of random
sample analysis leads to poor reproducibility and
may not be adequate [807 – 809].
9.4.3. Pharmaceutical Development
Drug biotransformation in man and animals
can lead to a modulation of pharmacodynamic
effects, drug elimination, but also drug tox-
icity. Knowledge of drug biotransformation
pathways at early stages of drug development
could considerably speed up drug development
[810 – 813] and increase the safety of drug de-
velopment in the pharmaceutical industry. Each
year, more than 668 000 animals are used in toxi-
cological drug research in Europe. Animal mod-
els suffer from serious shortcomings regarding
the prediction for a human situation as signif-
icant species differences in enzyme expression
exist between man and animals. Specifically, en-
zyme induction experiments are frequently mis-
leading. 10–20 % of all new drugs are required
to return into preclinical development trials after
first clinical tests because of unforeseen metabo-
lite patterns, whose toxicological relevance is
unclear or untested.
Classical animal-testing strategies are time-
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-
ments
– Gene expression in model systems
– Gene expression patterns in disease
– Gene expression patters in pathogens (host–
pathogen interaction)
9.4.4. Define Molecular Mechanisms of
Toxicity
DNA microarrays can be used to determine the
relative safety of natural or synthetic chemicals
to which humans are exposed. Lots of diseases
are influenced by environmental factors, for ex-
ample, chemicals, radiation, drugs, nutrition,
viruses, carcinogens, reproductive toxins, neu-
rotoxins, and immunotoxins [814 – 819]. Al-
though most of these chemicals are harmless,
it is important to identify potentially hazardous
substances. In the past, toxicological assays have
used rodent, required high doses, were expan-
sive, and take years to complete. Unfortunately,
the information gained from established life an-
imal models such as rodent to investigate en-
zyme induction and toxicity cannot really lead to
correct assumptions of hazards to humans with
respect to species extrapolation. The shortcom-
ing with respect to predictivity of animal models
for human drug biotransformation and toxicity
is based upon a well accepted species difference
in enzyme expression between man and animals.
Nevertheless, at present animal models in drug
metabolism and toxicology are still legally re-
quired in all European countries for a variety
of reasons and legislative bodies are still hes-
itative to recommend specific human liver cell
based tests. In spite of this, modern cell-culture
technology has progressed to the extent that it
is now possible to culture primary human liver
cells under conditions that maintain differenti-
ated functions to perform drug biotransforma-
tion and enzyme induction studies with great
reliability in individual laboratories. These de-
velopments have not yet found entry into legal
recommendations as no prevalidation of such
models on a European scale has been achieved
so far. The widespread use of such alternative
models to animal investigations is being ham-
pered both by insufficient prevalidation and by
further improvements still required in the field
of automation. It would be a significant break-
through in toxicology research if these models
could be sufficiently automated to enter into the
field of accelerated screening as a significant ini-
tiation step towards high-throughput screening.
Microarrays are powerful tools for investigating
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
models [820].
9.4.5. Detection of Genetically Modified
Organisms
The use of genetically modified organisms
(GMOs) in food industry has been rising ex-
ponentially over the past years. In the space of
time from 1996 to 2003, global area of trans-
genic crops increased 40-fold, from 1.7 × 106
hectares in 1996 to 67.7 × 106 hectares in 2003,
with a dominant trait of herbicide tolerance, fol-
lowed by insect resistance (www.isaaa.org, In-
ternational Service for the Acquisition of Agri-
biotech Applications). A GMO is “an organism,
with the exception of human beings, in which the
genetic material has been altered in a way that
does not occur naturally by mating and/or natu-
ral recombination” [821]. Microarrays are a use-
ful tool for GMO detection and the application
of array techniques to GMO detection implies
the capability of sensitivity and standardization
of the assay. Today, commercialized arrays of-
ten do not fulfill these requirements, in spite of
a few promising attempts initialized by biotech-
nical companies and research projects. Never-
theless, microarrays are already being used in
GMO analysis but well established quantitative
real time PCR techniques are state of the art.
GMO detection becomes increasingly important
because by-products of these organisms could
end up in the food chain and affect human health.
10. Concluding Remarks
As it has been demonstrated in the previous
chapters, biotechnology offers manifold possi-
bilities in the production of different products
and in medical applications. It can be used for
better food, better health, and a cleaner environ-
ment. However, in the area of production of fine
and bulk chemicals, biotechnology has still to

Figure 44. Investigation of toxicity through a combined cell-culture and microarray analysis
compete with the chemical industry. The chem-
ical industry owes its success to the principle of
unit construction: from simple basic substances,
like ethylene, carbon monoxide or hydrogen,
more complex precursors can be produced un-
der controlled conditions by chemical reactions;
because of the variety of combination options,
the latter can in turn be converted into inconceiv-
able quantities of derivatives and end products.
Chemistry learned how to produce chemically
pure basic substances from oil that are simple
to handle and exactly defined; this is performed
highly efficiently in refineries. This was the key
to its success. Without exact knowledge of this
functional principle, the triumphant success of
plastics would have been just as impossible as
the production of thousands of other chemical
products that today make our lives safe and com-
fortable. This principle can also be applied to the
so-called ‘biorefinery’ which has similar poten-
tial and could thus provide a further argument for
the concept of material application of renewable
resources and biotechnology. However, a tech-
nically viable separation procedure that would
permit the separate use or further processing
of all the primary products (such as cellulose,
hemicellulose, and lignin) is still in its infancy.
The main focus is on glucose, which can be de-
rived by microbial or chemical methods from
cellulose, since a wide range of biotechnologi-
cal and chemical products can be obtained from
glucose. The potential for the microbial conver-
sion of glucose-based substances is huge and the
reactions are advantageous from the viewpoint
of energy. It is necessary to link the degrada-
tion processes to bulk chemicals via glucose as
closely as possible to the synthesis processes of
further derivatives.
Biotechnology 123
Microorganisms still have a great poten-
tial for inducing new or novel enzyme sys-
tems capable of converting foreign substrates.
It is possible to obtain and cultivate microor-
ganisms that can survive or grow in extraor-
dinary environments, e.g., psychrophilic, ther-
mophilic, acidophilic, and alkaliphilic microor-
ganisms. These microorganisms are capable of
producing unique enzymes stable to heat, alkali,
and acid. In addition, techniques for cultivation
and enzyme purification, and gene and enzyme
technology have made great advances and are
still under development. Certainly biotechno-
logical process development must be founded
on a broader genomic basis. Ten years ago, only
a handful of microorganisms with a completely
sequenced genome existed, today there are sev-
eral hundreds. Hitherto priority was given to se-
quencing pathogenic microorganisms, but it is
now high time to sequence microorganisms that
are relevant to industry. Taking all of these into
consideration, there must be far more ways than
one can imagine in using biotechnology for our
daily life.
11. Acknowledgement
The current update was coordinated by Roland
Ulber.
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