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Advanced Fermentation
and Cell Technology
Advanced Fermentation
and Cell Technology
Volume 1

Byong H. Lee
SportBiomics, CA, USA and Heilenex Pharma Inc., Toronto, Canada
Kangwon National University, Chuncheon, South Korea
Jiangnan University, Wuxi, China
McGill University and AAFC, Quebec, Canada
Advanced Fermentation
and Cell Technology
Volume 2

Byong H. Lee
SportBiomics, CA, USA and Heilenex Pharma Inc., Toronto, Canada
Kangwon National University, Chuncheon, South Korea
Jiangnan University, Wuxi, China
McGill University and AAFC, Quebec, Canada
This edition first published 2022
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Names: Lee, Byong H., author.
Title: Advanced fermentation and cell technology / Byong H. Lee,
SportBiomics, CA, USA and Heilenex Pharma Inc., Toronto, Canada, Kangwon National
University, Chuncheon, South Korea, Jiangnan University, Wuxi, China, McGill University and
AAFC, Quebec, Canada.
Description: Hoboken, NJ, USA : Wiley-Blackwell, 2022. | Includes
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Subjects: LCSH: Fermentation. | Industrial microbiology.
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Contents

Volume 1

Preface ix

Overview on Market Size of Bioproducts and Fundamentals of Cell Technology xiii

Part I 1

1 Microbial Cell Technology 1

1.1 Basic bacterial growth and mode of fermentation 1
1.2 Basic fungal growth 22
1.3 Classical strain improvements and tools 26
1.3.1 Natural selection and mutation 26
1.3.2 Recombination 30
1.4 Modern strain improvement and tools 32
1.4.1 Genome shuffling 33
1.4.2 Recombinant DNA technology 34
Summary 58
1.4.3 RNA interference (RNAi) and CRISPR/Cas technology for genome editing 59
Summary 72
1.4.4 Molecular thermodynamics and down-stream processes on bioproducts 72
Summary 83
1.4.5 Protein engineering 84
Summary 91
1.4.6 Genomics, proteomics, metagenomics, and bioinformatics 91
Summary 99
1.4.7 Systems/synthetic biology and metabolic engineering 100
Summary 106
1.4.8 Quorum sensing and quenching 106
Summary 110
1.5 Bioengineering and scale-up process 111
1.5.1 Microbial and process engineering factors affecting performance and economics 114
1.5.2 Fermenter and bioreactor systems 114
1.5.3 Mass transfer concept 116
1.5.4 Heat transfer concept 120
1.5.5 Mass and heat transfer practice 123
1.5.6 Scale-up and scale-down of fermentations 137
1.5.7 Scale-up challenges 146
Summary 150
vi CONTENTS

1.6 New bioprocesses of fermentation 150

1.6.1 Growth-arrested bioprocesses 150
1.6.2 Integrated bioprocesses 154
1.6.3 Consolidated bioprocessing (CBP) 158
Summary 160
Bibliography 161

Part II 173

2 Applications of Microbial Fermentation to Food Products, Chemicals and

Pharmaceuticals 173
2.1 Fermented dairy products 173
2.1.1 Basic knowledge of manufacture of dairy products 177
2.1.2 Genetic engineering of lactic acid bacteria 186
Summary 197
Bibliography 197
2.2 Fermented meat and fish products 199
2.2.1 Fermented meat products 199
2.2.2 Fermented fish products 204
Summary 207
Bibliography 208
2.3 Fermented vegetable and cereal products 209
2.3.1 Fermented vegetable products 209
2.3.2 Fermented cereal products 214
Summary 219
Bibliography 220
2.4 Organic acids 220
2.4.1 Acetic acid 221
2.4.2 Citric acid 224
2.4.3 Lactic acid 225
2.4.4 Malic acid 227
2.4.5 Fumaric acid 228
Summary 229
Bibliography 230
2.5 Fermentation-derived food and feed ingredients 231
2.5.1 Flavors and amino acids 232
Summary 247
2.5.2 Sweeteners 249
Summary 259
Bibliography 260
2.5.3 Vitamins and pigments 264
Summary 274
Bibliography 274
2.5.4 Microbial polysaccharides and biopolymers 276
Summary 291
Bibliography 293
2.6 Bacteriocins and bacteriophages 296
2.6.1 Bacteriocins 296
2.6.2 Bacteriophage 307
Summary 309
Bibliography 310
2.7 Enzymes 314
Summary 330
Bibliography 331
2.8 Biomass (SCP) and mushrooms 332
 CONTENTS vii

2.8.1 Biomass (SCP) 332

2.8.2 Mushrooms 343
Summary 347
Bibliography 347
2.9 Functional foods and nutraceuticals 349
2.9.1 Probiotics and prebiotics 349
2.9.2 Microbiome 364
Summary 373
Bibliography 374
2.10 Alcoholic beverages 385
2.10.1 Beer 386
2.10.2 Wine 393
Summary 400
Bibliography 401
2.11 Other fermentation chemicals 401
2.11.1 Bioethanol 402
Summary 412
Bibliography 412
2.11.2 Biobutanol 414
Summary 424
2.11.3 Biobutanediol 425
Summary 433
Bibliography 434
2.11.4 Biodiesel 438
Summary 444
Bibliography 445
2.11.5 Biomethane 446
Summary 453
Bibliography 454
2.11.6 Biohydrogen 455
Summary 465
Bibliography 465
2.12 Pharmaceuticals, growth promoters, and biopesticides 467
2.12.1 Antibiotics 467
2.12.2 Antibiotic (or antimicrobial) growth promoters (AGPs) 489
Summary 496
Bibliography 498
2.12.3 Antitumor drugs 503
Summary 531
Bibliography 532
2.12.4 Steroids 536
2.12.5 Statins 551
Summary 561
Bibliography 562
2.12.6 Biopesticides 568
Summary 575
Bibliography 575
Contents

Volume 2

Preface vii

Part III 579

3 Animal Cell Technology 579

3.1 Animal cell culture 579
3.1.1 Introduction 579
3.1.2 Techniques of RNAi and CRISPR 580
3.1.3 Animal cell lines 580
3.1.4 Upstream and downstream bioprocessing 590
3.1.5 Strain development of animal cell cultures 593
3.1.6 Applications of animal cell cultures 595
3.2 Transgenic animal bioreactors 661
3.2.1 Introduction and techniques 661
3.2.2 Applications of transgenic animals 664
Summary 672
Bibliography 673

Part IV 687

4 Plant Cell Technology 687

4.1 Introduction 687
4.2 Plant tissue culture 689
4.3 Applications of plant tissue culture 697
4.3.1 Traditional plant breeding (non-recombinant DNA techniques) 698
4.3.2 General media for plant tissue culture 739
4.3.3 Bioreactor types of plant cell cultures 741
4.3.4 Modern plant breeding or Biotech/GM crops (recombinant DNA techniques) 759
Summary 781
Bibliography 782
vi CONTENTS

Part V 801

5 Safety Issues of New Biotechnologies on Microbial, Animal, and Plant Cells 801
5.1 Introduction 801
5.2 Safety evaluation of novel foods and cell culture products 802
5.2.1 Genetically modified microorganisms and their products 804
5.2.2 Genetically modified animal cell cultures, animals, fishes and their products 807
5.2.3 Genetically modified plants and their products 820
Summary 830
Bibliography 831

Index 835
Preface

The term fermentation derived from the Latin verb fevere (boil) used by humans for the
production of food and beverages since the Neolithic age is the oldest of all biotechno-
logical processes. The fermentation process used in the production of antibiotics, alco-
hol, bread, vinegar and other food or industrial products differs from respiration in that
organic substances rather than molecular oxygen are used as electron acceptors. After suc-
cessful microbial fermentation processes on microbial cells (biomass), microbial enzymes,
microbial metabolites including antibiotics, and other fermented foods, currently more
than 3500 different fermented foods are consumed by humans worldwide. The potential
of fermentation techniques was dramatically increased in the late 1960s and 1970s through
achievements in molecular genetics, cell fusion, and enzyme technology. However, addi-
tional completely novel, powerful techniques such as genetic engineering via recombinant
DNA technology in 1973 and hybridoma technology (monoclonal antibody) in 1975 were
responsible for the current biotechnology boom.
In genetic engineering, a known gene is inserted into a microbial, animal, or plant cell in
order to achieve a desired trait for the overproduction of target compounds, but microor-
ganisms have played a major role in the development of biotechnology. This is due to the
rapid growth of microbes, cheap growth media, massive diversity in the metabolite types
and easy of genetic manipulation. However, mass culture of animal cell lines is also impor-
tant to manufacture viral vaccines and other therapeutic recombinant products such as
enzymes, hormones, immunologicals (monoclonal antibodies, interleukins, lymphokines,
etc), and anticancer agents. Mammalian cells cultivated in bioreactors have surpassed
microbial systems for producing therapeutic recombinant proteins because of their
capacity for proper protein folding, assembly and post-translational modification. Major
therapeutic recombinant proteins are successfully commercialized, but expensive animal
free media, cost of production in large scale with low yield (the tens-of-milligrams/liter),
microbial contamination, and gene regulation are the important issues to be resolved.
Other cell culture research is also underway to produce such complex therapeutic proteins
in insect cell (baculovirus) or in higher plants. Molecular biopharming using transgenic
animals such as goat and pig for rDNA proteins have also successfully commercialized.
Fermentation technology for the production of compounds that find application in food,
biochemical, biomaterial, bioenergy, pharmaceutical sectors encompasses a broad field, not
only including (1) conventional microbial and enzyme systems, (2) genetic and metabolic
engineering, along with systems biology/synthetic biology, and genome editing, but also
including (3) mammalian cell and plant cell systems. Despite a long history of fermentation
processes for generations, the requirement for sustainable production of bioenergy and bio-
materials is also demanding innovation and development of novel fermentation concepts.
Continued introduction of new technology in cell culture systems demands innovation in
new bioreactor process development and scale up processes for cell factory potential.
x PREFACE

This book reflects this transition from traditional fermentation technology to new cell
fermentation technology, that provides equal emphasis on microbial, mammalian as well as
plant cell technologies for new and improved processes and products in today’s biochemical
process industry.
Currently two fermentation textbooks available on the market (1999) are outdated and
do not deal with current progress in fermentation and cell culture technologies and com-
mercial recombinant bioproducts. Most other edited volumes are the work of multiple
contributors and normal didactic criteria for explanation of evolving new techniques and
applications are lacking. Experience in teaching this subject has made clear that the basic
concepts and essential features have not been covered in a typical science curriculum. The
primary objective of writing this book is to relate the food fermentation and cell culture
biopharmaceutical actives using different expression hosts. Product diversity makes fer-
mentation technology a multi -disciplinary expertise associated with microbiology, organic
chemistry, biochemistry, and molecular biology. Remarkable advances in these areas will
help to lift people out of wretched and empower them with new knowledge. The subject
matter is divided into Part V, including microbiology, biotechnology, molecular biology,
biochemical engineering, and global market size of bioproducts, and their applications.
Part I covers microbial cell technology and culture tools (including classical strain
improvements and tools) and modern strain improvement and tools (genome shuffling,
recombinant DNA technology, RNA interference (RNAi) and CRISPR)/Cas technol-
ogy for genome editing; also includes molecular thermodynamics for biotechnology,
protein engineering, genomics, proteomics and bioinformatics, systems/synthetic biol-
ogy and metabolic engineering, quorum sensing and quenching. Other bioengineering
and scale-up processes and new bioprocesses of fermentation such as growth-arrested
bioprocess, integrated bioprocess, and consolidated bioprocessing (CBP) are included.
Part II deals applications of microbial fermentation to food products (dairy, meat/fishes,
vegetable/cereals), organic acids, food ingredients, chemicals, and pharmaceuticals. Food
products/ingredients included flavors and amino acids, sweeteners, vitamins and pig-
ments, microbial polysaccharides/biopolymers, bacteriocins and bacteriophages, enzymes,
biomass (SCP)/mushrooms, functional foods and nutraceuticals such as probiotics and
prebiotics, and microbiome. Others included alcoholic beverages and other fermentation
chemicals (bioethanol, biobutanol/biobutandiol, biodiesel, biomethane, biohydrogen). In
final section, pharmaceuticals such as antibiotics, antibiotic growth promoters, antitumor
drugs, steroids/statins, and biopesticides included.
Part III covers (i) animal cell technology including animal cell culture, bioprocessing,
strain development, applications (monoclonal antibodies, different vaccines (DNA vac-
cines, edible vaccines, zika vaccines, hepatitis vaccines, HIV vaccines, COVID 19 vaccines),
and (ii) transgenic animal bioreactors, and applications in animals and fishes, etc. Part IV on
plant cell technology covered plant tissue and cell culture and applications, bioreactor types
(seed-based bioreactor, plant cell suspension bioreactor, hairy root bioreactor, chloroplast
bioreactors) and modern plant breeding or biotech/GM crops and their applications.
Finally, Part V deals with safety issues of new biotechnologies on microbial, animal, and
plant cells.
This book aims to give readers, general science students, researchers, and industrial
practitioners as well as instructors, an overview of the essential features of advanced fer-
mentation and cell culture technology. I would like to thank my students, post-docs, and
former colleagues at McGill University (Canada), Jiangnan University (China) and Kang-
won National University (Korea), who, for the past 37 years, have helped and suggested
me in teaching this subject course.
 PREFACE xi

Among the over hundres of my former graduate students and post-docs, I would like to
dedicate this book specifically to my beloved former students, Dr. Young J. Choi and Dr.
Marcio Belem in Montreal, whom both passed away suddenly.
Last but not least, I must thank my wife Young for her love and encouragement, together
with the patience of my sons, Edward and his family (wife Maria, son Maxim) in Toronto
and David and his family (wife Ronit, daughter Romy) in Santa Monica, California during
the preparation of this volume.

September, 2021 Byong H. Lee

Toronto, Canada
Overview on Market Size of
Bioproducts and
Fundamentals of Cell
Technology

Biotechnology and global market size of bioproducts

The term “biotechnology” was first used by Karl Ereky in 1919, meaning the production of
products from raw materials with the aid of living cells such as microbe, animal, and plant
in fermenters or bioreactors through process optimization.
Biotechnology is a broad discipline using all living cells, or parts of this to develop
products, and services to meet the needs of humans. New tools and products developed by
biotechnology have been useful in research, agriculture, industry, and the clinic. Industrial
biotechnology experience in fermentation technology started in the field of single-cell
protein production and then moved on to the developments of fermentation technologies
producing antibiotics, amino acids, antitumor agents, statins, vitamins, steroids, and
majority of fermented foods. As a result of this process development activity, microbial
cell culture technology has become one of the leading suppliers of top-quality fermenta-
tion technologies. This domain market is continuously expanding in all continents, with
confidential cooperation involving small and multinational companies. Microbial cell
fermentation technology has been a long-favored organic process because of its simplicity
of reaction, high specificity, low costs, and flexibility of application. Majority industries
have complemented the essential principle of fermentation with advances in techniques
and biotechnology to increase its application to the assembly of a spread of biochemicals,
biomolecules, and biofuels.
Table 1 shows the fermentation biotechnology milestones and some recombinant pro-
teins.
The fermentation’s application areas are broad from food stuffs like wine, cheese,
bread, and beer, into high-value chemicals, pharmaceutical products, and food-related
chemicals. Among many more than 100 recombinant proteins known (Sandez-Garcia
et al., 2016), only few are listed in this table. Further, rising hydrocarbon costs and
depleting fuel reserves have also created a strong case for affordable and easy fermen-
tation processes for manufacturing biofuel chemicals. Microbial cell factories for the
production of bio-based chemicals have been useful for diverse industrial applications
and for achieving a sustainable future, but the overexpression of heterologous enzymes in
xiv OVERVIEW ON MARKET SIZE

Table 1 Fermentation biotechnology milestones and some recombinant proteins

Date Milestone

Old biotechnology
Before 6000 Bread leavening, alcoholic drinks, and fruit vinegars
B.C.
Before 14th Beer and wine, vinegar by the Orleans process
century
1650 Cultivation of French mushrooms
1680 Anton van Leeuwenhoek first observation of yeast cells
1857–1876 Louis Pasteur first discovery of microorganisms causing fermentation
1881 Lactic acid production by microbe
1885 Artificial growing of mushrooms in the USA)
19th century Production of ethanol, acetate, butanol, acetone, and citrate, glycerol, baker’s yeast, and
sewage treatment
1940s Mass cultivation of microbes for antibiotics (penicillin, streptomycin, chlorotetracycline),
bioingredients (amino acids, enzymes, vitamins, polysaccharides), steroids, and vaccines
1953 Discovery of DNA by Watson and Crick
1957 L -Glutamate production by Kinoshita et al.
1955–60 Citric acid production by submerged fermentation
New Biotechnology
1970–1972 Transformation of E. coli by plasmid DNA
1973 First discovery of restriction enzymes and DNA ligases by Cohen and Chang
1974 First expression of heterologous gene in E. coli
1975 Invention of hybridoma technique for monoclonal antibodies by César Milstein and
Georges J. F. Köhler
1978 First discovery of recombinant protein, somatostatin
1980 Cohen/Boyer patent on recombinant DNA technology; Genentech established
1982 Recombinant human insulin (Humulin®) by E. coli
1983 Heterologous plant gene expression; Polymerase Chain Reaction (PCR) by Kary Mullis/Cetus
(Nobel prize)
1985 Recombinant human growth hormone (Protropin®) by E. coli
1986 Recombinant vaccine, Recombivax HB (hepatitis B vaccine by S. cerevisiae
Recombinant gamma−interferon, Roferon A® by E. coli
1987 Recombinant tryptophan, recombinant tissue plasminogen activator, Activase®) by CHO*
1989 Recombinant interleukin-2, Proleukin® by E. coli, Recombinant γ-interferon, Immuneron®
by E. coli
1989–1991 Recombinant chymosin (Gist-Brocades, Genencor, and Pfizer) by E. coli and Aspergillus
oryzae
Recombinant production of vitamin C (ascorbic acid) by Genencor International, lactic acid
bacteria resistant to bacteriophage
1990 Maltase-enhanced baker’s yeast (Gist-Brocades)
1992 Recombinant blood clotting factor VIII (Recombinate® by Genetic Institute/Baxter;
Kogenate® by Bayer, Defacto® (Wyeth) using animal cell lines; Lipase (Unilever),
Amylase (Novomil®)
1993 Site-directed mutagenesis by Michael Smith at UBC (Nobel prize)
1994 Genetically modified Flavr Savr tomato (Calgene, discontinued in 1997), recombinant
bovine somatotropin, BST (Eli Lilly; Monsanto), engineered brewing yeast (Carlsberg
Research Centre), and acetolactate decarboxylase (Maturex)
2003–2013 Epogen (erythropoietin, EPO) by mammalian cells (Amgen), Neupogen (granulocyte
colony-stimulating factor, G-CSF) by E. coli (Amgen), Fabrazyme (human
alpha-galactosidase, Genzyme) by CHO 1996–2015
Over 47 herbicide-resistant (HT) and insect-resistant (BT) crops on market 2018
Golden Rice (a provitamin) approved by US FDA; GM Atlantic salmon developed by
AquaBounty Technologies on market in Canada and USA (FDA approved)
∗ CHO:Chinese Hamster ovaries.
Source: Lee, 2015.
 OVERVIEW ON MARKET SIZE xv

Cell Culture Technology

Microbial cells Animal cells Plant cells

– Fermented foods – Animal breeding – Crop breeding
(beer, yogurt, soy, etc) – Transgenic animals – Transgenic plants
– Bioingredients – Animal (mammalian) – Plant cell culture
(enzyme, amino acid, gum, cell culture
vitamin, etc)
– Pharma products
(antibiotics, steriods,
recombinant proteins, etc)

Figure 1 Concept of cell culture technology derived from microbes, animals, and plants. Source: Nader
Khouri/Getty Images; Toni Barros / https://en.wikipedia.org/wiki/Dolly_(sheep)#/media/File:Dolly_face_
closeup.jpg / CC BY-SA 2.0; Dreamstime; Shutterstock.com; Flickr, Inc.; Thomas Northcut/Getty Images;
maksym yemelyanov/123RF. (See insert for color representation of this figure.)

recombinant strains often leads to metabolic imbalance, resulting in growth retardation

and suboptimal production of the target compounds (Lu et al., 2018). Thus, it is essential
to address metabolic imbalances caused by engineered pathways in microbial hosts. For
establishing a vibrant bio-based economy, multivariate modular metabolic engineering,
modular co-culture engineering, systems biology, and integrative genome-scale metabolic
modeling can be exploited to expedite strain optimization and improve the production
yield of many high-value bio-based chemicals.
Figure 1 shows concept of cell culture technology derived from microbes, animals, and
plants. In transgenic (genetic engineering) technology either in cells of microbes, animals,
or plants, a known gene for a desired trait is inserted into them by recombinant DNA tech-
niques or genome editing by CRISPER/Cas9 techniques. Despite of the potential use of all
living forms, a major player in the development of fermentation biotechnology is microor-
ganisms, because of the ease of mass growth, the rapid growth in cheap waste materials as
media, and the massive diversity of metabolic types, which in turn gives diverse potential
products and the ease of genetic manipulation to improve strains for new products.
It is estimated that the global economic value of industrial biotechnology, renew-
able chemicals and polymers, biofuels, enzymes, and bio-based materials is about
US$355.28 billion, among which US generates 58% of the value, or more than
US$205 billion (www.bio.org/worldcongres). The production of bio-based chemi-
cals alone can yield an annual revenue of US$10–15 billion in the global chemical
industry by 2020 (www.iwbio.de/fileadmin/Publikationen/IWBio-Publikationen/WEF_
Biorefineries_Report_2010.pdf).
xvi OVERVIEW ON MARKET SIZE

Fermentation’s application areas are broad from food stuffs like wine, cheese, bread,
and beer, into high-value chemicals, pharmaceutical products, and food-related chemicals.
Among many more than 100 recombinant proteins (Sandez-Garcia et al., 2016), only few
are listed in this table. Further, rising hydrocarbon costs and depleting fuel reserves have
also created a strong case for affordable and easy fermentation processes for manufacturing
biofuel chemicals. Microbial cell factories for the production of bio-based chemicals have
been useful for diverse industrial applications and for achieving a sustainable future, but the
overexpression of heterologous enzymes in recombinant strains often leads to metabolic
imbalance, resulting in growth retardation and suboptimal production of the target com-
pounds (Lu et al., 2018). Thus, it is essential to address metabolic imbalances caused by
engineered pathways in microbial hosts. For establishing a vibrant bio-based economy,
multivariate modular metabolic engineering, modular co-culture engineering, systems biol-
ogy, and integrative genome-scale metabolic modeling can be exploited to expedite strain
optimization and improve the production yield of many high-value bio-based chemicals.
Genetically modified bacteria such as the Gram-negative Escherichia coli (E. coli) are used
to produce large amounts of proteins for industrial use, but because of the high cost of
extraction and purification, only high value products have been produced at an industrial
scale. Although the recently developed secretary expression system of E. coli using sig-
nal peptide optimization, periplasmic leakage, and chaperones co-expression are reported
(Zhou et al., 2017), the E. coli species exhibits some limitations as the heterologous pro-
teins are typically expressed intracellularly, which results in problems with formation of
inclusion bodies and incorrect protein folding. Thus, Bacillus subtilis has become an indus-
trial workhorse for recombinant protein production due to an easy cultivation, the products
of generally recognized as safe (GRAS), ease of genetic manipulation, well-characterized
expression systems, absence of significant codon bias, and exceptional ability to secrete
heterologous proteins allowing cost-effective downstream processing (Eivind et al., 2018).
However, many recombinant proteins require protein modifications, such as glycosylation
that are available only in eukaryotic cells in that this sometimes leads to the use of yeast,
insect cells, and mammalian cell culture systems. Some medicinal use of recombinant bacte-
ria (E. coli) or yeasts (Saccharomyces cerevisae, Pichia pastoris) was to produce the protein
insulin to treat diabetes, clotting factors to treat hemophilia, human growth hormone to
treat various forms of dwarfism, interferon to treat some cancers, erythropoietin for anemic
patients, and tissue plasminogen activator which dissolves blood clots.
Outside of medicine they have been used to produce recombinant chymosin (cheese
coagulating enzyme), lipase, biofuels, and bioremediation, etc. Besides, with greater under-
standing of the role that the microbiome plays in human health, there is the potential to
treat diseases by genetically altering the bacteria to themselves be therapeutic agents in our
review (Daliri et al., 2018). The main ideas include altering gut bacteria to destroy harm-
ful bacteria or using bacteria to replace or increase deficient enzymes or proteins. One
research focus is to modify Lactobacillus bacteria that naturally provide some protection
against HIV, with genes that will further enhance this protection. However, the microbiome
could also raise safety concerns as interactions between bacteria and the human body are
less well understood than with traditional drugs. There are concerns that horizontal gene
transfer to other bacteria could have unknown effects. As of 2018 there are many clinical
trials underway testing the efficacy and safety of these treatments (Reardon, 2018).
Besides microbial fermented foods, for over a century, bacteria have also been used in
agricultural crops in which Rhizobia (and more recently Azospirillum) have been inoc-
ulated to increase their production or to allow them to be grown outside their original
habitat. It is well known that application of Bacillus thuringiensis (Bt) and other bacteria
can help protect crops from insect infestation and plant diseases. With advances in genetic
 OVERVIEW ON MARKET SIZE xvii

engineering, these bacteria have been manipulated for increased efficiency and expanded
host range as well as tracing the spread of the bacteria by markers. Pseudomonas strains
of bacteria causing frost damage by nucleating water into ice crystals around themselves
led to the development of ice-minus bacteria, that have the ice-forming genes removed.
When applied to crops they can compete with the ice-plus bacteria and confer some frost
resistance (Yetisen et al., 2015). Other applications of genetically modified bacteria include
bioremediation, where the bacteria are used to convert pollutants into a less toxic form such
as removal of petroleum hydrocarbon pollutants by increasing the levels of the enzymes
used to degrade a toxin or to make the bacteria more stable under environmental condi-
tions (Fuentes et al., 2014; Yuniati, 2018).
However, mass culture of animal cell lines is fundamental to the manufacture of viral
vaccines and many biological products produced by recombinant DNA technology in ani-
mal cell cultures include enzymes, synthetic hormones, immunobiologicals such as mono-
clonal antibodies, interleukins, lymphokines, and anticancer agents.
Many simpler proteins can be produced by engineered bacterial cell cultures, but protein
glycosylation (post-translational modification, PTM) is only made in animal cells. As the
production cost of mammalian cell cultures is high, however, other insect cells or higher
plants using single embryonic cell and somatic embryos are using as a source for direct gene
transfer through particle bombardment, and transit gene expression. Mammalian cell-line
products produced from CHO, BHK, NSO, meyloma cells, C127, HEK293) account for
over 70% of currently approved biotherapeutic products including monoclonal antibodies.
Biopharmaceuticals may thus be produced from engineered microbial cells such E. coli
or yeast, mammalian cell culture, plant cell/tissue culture, and moss plants in various biore-
actors or photo-bioreactors. The main issues of mammalian cell culture are cost of produc-
tion and microbial contamination by bacteria, viruses, mycoplasma, or viruses. Potential
safety and ethical issues include in engineered whole plants and animals, which represent
a significant investor risk and risks over consumer acceptance.
Major kinds of recombinant therapeutic proteins (biopharmaceuticals) include: blood
factors (Factor VIII and Factor IX), thrombolytic agents (tissue plasminogen activator),
hormones (insulin, glucagon, growth hormone, gonadotrophins, hematopoietic growth
factors (erythropoietin, colony stimulating factors), interferons (interferons-α, -β, -γ),
interleukin-based products (interleukin-2), vaccines (hepatitis B surface antigen), mon-
oclonal antibodies (various), and additional products (tumor necrosis factor, therapeutic
enzymes).
The important animal cell culture products as monoclonal antibodies are pro-
duced by fusing normal cells with an immortalized tumor cell line. Recent advances
in therapeutic protein drug development are available from: www.researchgate.net/
publication/313463806_Recent_advances_in_therapeutic_protein_
drug_development.
Many other transgenic animals have also been used to secrete human proteins
secreted in the milk of transgenic livestock. Since 1985, several animals, including cow,
goat, pig, horse, cat, rabbits, chickens, and most recently dog as well as fishes, have
been cloned, but the most research has been on cattle. Many applications of animal
biotechnologies are controversial for environmental, health, animal welfare, and social
reasons. For example, only a small percentage of cloning attempts produce live off-spring
and many animal clones are unhealthy. Human-animal chimeras raise safety concerns
about whether new diseases could be transmitted to humans, legal issues about whether
such creatures can be patented and owned, and the troubling possibility that they
could display human-like behavioral characteristics (www.geneticsandsociety.org/topics/
animal-biotechnologies).
xviii OVERVIEW ON MARKET SIZE

Molecular pharming is based on three perceived advantages: the low costs of grow-
ing animals or plants, the immense scalability of agricultural production, and the inherent
safety of animals or plants as hosts for producing pharmaceuticals. This can produce a range
of proteins produced from cloned animals. The main potential benefits of cloned animals
include the production of food, pharmaceuticals (“pharming”), the provision of organs for
xenotransplantation into humans, and the development of models of human diseases. The
benefits to agriculture include resistance to disease, altering the carcass composition such
that it is healthier to consume, improving the pig’s resistance to heat stress, and protecting
the environment. Additional types of genetic modifications will likely provide animals with
characteristics that will benefit humans in currently unimagined ways.
Although the majority of genetically engineered animals are still in the research phase,
some GM animals and fishes already on sale include cloned pet cats, GM ornamental
fish, GM salmon, cloned horses, and at least one rodeo bull. The GM cows by biotech
company SAB Biotherapeutics produced human antibodies, that fight pathogens, that
may one day treat infectious diseases like Ebola, influenza, and Zika and their potential
to address global outbreaks. The US Food and Drug Administration also approved a
GM chicken that makes a drug in its eggs to treat “lysosomal acid lipase deficiency” –
a rare genetic condition that prevents the body from breaking down fatty molecules inside
cells. In 2014, the FDA approved a drug collected from the milk of lab-made rabbits
to treat hereditary angioedema, a genetic disease that causes body swelling and can
be fatal. In 2009, the FDA approved a GM goat that can make a drug in its milk that
prevents fatal blood clots (www.theverge.com/2016/12/3/13819482/genetically-engineered-
animals-drugs-sab-cows-pharming-future).
Despite the ban on cloned animals adopted by the EU, the cloned livestocks would be
used as parents of slaughter pigs, beef cattle, and possibly also milk-producing dairy cows
in China, Canada, USA, and elsewhere. By 2020, the US FDA ruled that food products
derived from cloned animals (cattle, swine, goats, and the offspring of clones of any species
traditionally consumed as food) are safe enough and that labeling is not required because
food products from cloned animals are not materially different (www.fda.gov/animal-
veterinary/animal-cloning/primer-cloning-and-its-use-livestock-
operations).
Human antithrombin, ATryn (brand name) by rEVO Biologics (former GTC Biother-
apeutics) in 2006 was first medicine produced from the milk of genetically modified goats
by microinjection of human antithrombin genes into the cell nucleus of their embryos.
A goat that produces spider’s web protein, stronger (7–10 times) and more flexible than
steel (BioSteel) was successfully produced by a Quebec based Canadian company, Nixia
Biotechnologies, and later by the Randy Lewis lab of the University of Wyoming and Utah
State University (Service, 2002). This company also had created lines of goats to produce
recombinant versions of either the MaSpI (Major ampullate spidroin I) or dragline I (for its
superior elasticity, flexibility and strength) from Nephila clavipes, the golden orb weaver)
or MaSpII (Major ampullate spidroin 2 or dragline 2 from Nephila clavipes) dragline pro-
teins in their milk. When the female goat lactates, the milk containing the recombinant
DNA silk was to be harvested and subjected to chromatographic techniques to purify the
recombinant silk proteins. The purified silk proteins can be dried, dissolved using solvents
(DOPE formation) and transformed into microfibers using wet-spinning fiber production
methods. The spun fibers tend to have tenacities in the range of 2–3 grams/denier and elon-
gation range of 25–45%. The “Biosteel biopolymer” had been transformed into nanofibers
and nanomeshes using the electrospinning technique.
Nexia is the only company which has successfully produced fibers from spider silk
expressed in goat’s milk. The Lewis lab has produced fibers from recombinant spider
 OVERVIEW ON MARKET SIZE xix

silk protein and synthetic spider silk proteins and genetic chimeras produced in both
recombinant E. coli and the milk of recombinant goats, however, no one has been able
to produce the silk in commercial quantities thus far. The company was founded in
1993 by Dr. Jeffrey Turner and Paul Ballard that was sold in 2005 to Pharmathene. In
2018, two transgenic goats were sold to the Canada Agriculture Museum after Nexia
Biotechnologies went bankrupt (https://en.wikipedia.org/wiki/BioSteel). Research has
since continued by Randy Lewis, a professor formerly at the University of Wyoming and
now at Utah State University, where he was also able to successfully breed spider goats
to create artificial silk. There are now about 30 spider goats at a university-run farm.
Applications of artificial spider silk biopolymers include using it for the coating of all
kinds of implants and medical products as well as for artificial ligaments and tendons due
to its elastic tendencies, and also since it is a natural product which will synthesize well
with the body. Furthermore, artificial silk biopolymers can be applicated in personal care
products as well as in textile products.
AquaBounty Technologies originally developed in Canada and later based in Mas-
sachusetts (US) have sold about 4.5 tons of GM salmon fillets in Canada. The engineered
Atlantic salmons developed by AquaBounty are produced by cloning a growth hormone
gene from chinook salmon and a second added gene from the ocean pout accelerates
growth by keeping the hormone gene on permanently. AquaBounty says the salmon grow
twice as fast as typical salmon and consume 20–25% less food per gram of new flesh
(http://aquabounty.com/about). However, the first GM fish to go on sale in the world has
faced fierce opposition from environmental groups, which fear that if they escape from the
tanks where they are reared on Prince Edward Island in eastern Canada, they will upset
natural ecosystems by breeding with native salmon. In the US, the salmon was cleared for
sale in late 2015, but its market debut has been stalled by arguments over how it should be
labelled. Now the salmon is being sold in Canada, other species may eventually follow it
onto the market.
The genetically modified pig (Enviropig™) developed by University of Guelph in
Canada is to excrete less phosphorus in its feces. It will produce the enzyme phytase in
its salivary glands to enable more effective digestion of phytate, the form of phosphorus
found in pig feed ingredients like corn and soybeans.
An ornamental fish that glows in the dark was created by cloning jellyfish DNA into a
zebrafish and is on the market. However, GM fish may escape and cause genetic damage
by hybridizing with native species. Some tropical exotic fish such as piranhas may cause
major problems. The details will be covered in the section of Animal Cell Technology.
Recently, advances have also been made in the production of foreign proteins in trans-
genic plants as alternative to microbial fermentation or mammalian cell culture. Many
therapeutic proteins, including antibodies, blood products, vaccines, hormones, cytokines,
other therapeutic agents, and enzymes are being produced in transgenic plants. The proper
selection of host plant and gene expression system in a food crop or a non-food crop
can act as bioreactors to produce recombinant proteins in larger quantities than those
of mammalian cell systems. Plant systems have the advantages in large-scale production
of recombinant proteins without contaminating human pathogens. Although in the last
two decades, approximately 95 biopharmaceutical products including cytokines, enzymes,
hormones, monoclonal antibodies, and vaccines have been approved for the treatment of
various human diseases, none of the commercial products are currently produced in plants,
mainly due to the low yield and expensive purification costs. However, plant-based DNA
vaccines have potential even for COVID-19 (Rosales-Mendoza et al., 2020).
Genetic modification of plants involves adding a specific stretch of DNA into the plant’s
genome, giving it new or different characteristics. This could include changing the way the
xx OVERVIEW ON MARKET SIZE

plant grows or making it resistant to a particular disease. The new DNA becomes part of
the GM plant’s genome which the seeds produced by these plants will contain. The details
of transgenic plants will be covered in Part IV.
Most biopharmaceutical products are currently manufactured commercially through
various fermentation processes by genetically engineered microorganisms (e.g., E. coli,
yeast, fungi). Some highly successful drugs obtained through fermentation routes are:
human insulin, streptokinase, erythropoietin, hepatitis B vaccine, human growth hormones,
interleukin, granulocyte-colony stimulating factor (GC-SF), granulocyte-macrophage
colony stimulating factor (GM-CSF), interferons (α, β, γ), and many recombinant proteins.
The three domains of microbes, animals, and plants are not only involved in production of
biopharmaceuticals, but also have their applications in foods (Figure 1). Although public
support for new biotechnology is high for new drugs (e.g., insulin, interferon, hormone,
etc.), diagnostics (cancer detection kits), food enzymes (e.g., recombinant rennet and other
enzymes), and vaccines, public perceptions are against GM foods on the whole, because
of their safety issue and because foods are an issue that affect everyone, as everyone eats,
as will be covered in Part V.
Besides the concept of whole cell culture technology derived from microbes, animals,
and plants in Figure 1, a novel cellular agriculture (CA) terminology was first used in
2015. Providing food and healthy diets for growing population, tackling deforestation and
climate change, and reducing negative environmental impacts of conventional agriculture
constitute the grand challenges of current times. By 2050, the global population will
reach to 9.8 billion, demand of our food supplies will be 60% higher than it is today.
At the same time, climate change, urbanization, and soil erosion will decrease of the
availability of arable land and crops account for only about 2% of all plant biomass
including non-food crop (dx.doi.org/10.1038/nature25138). Deforestation is also a direct
consequence of animal agriculture as more and more farmland is required to grow
crops for animal feed. CA will reduce deforestation as the production practices are
decoupled from land use. Although the world currently produces more than enough food
to feed everyone, 815 million people (roughly 11% of the global population) were in a
state of hunger in 2016 (www.fao.org/fileadmin/templates/wsfs/docs/expert_paper/How_
to_Feed_the_World_in_2050.pdf). Not only is providing sufficient food important, but
the low-quality diets causing micronutrient deficiencies and obesity are also currently
problems (dx.doi.org/10.1016/SO140-6736(18)31788-4).
This CA field is not new, as microbial cell production of food protein (single-cell protein),
bioplastics (e.g., biofilm, headset, etc.), leather-like mycelium biomaterials, recombinant
rennet, spider silk protein, bacterial cellulose, and so on, are already commercialized (see
Part II). In vitro animal cell cultures are well established and over 32 therapeutic proteins
including human insulin are commercialized (see Part III). Also, in vitro plant cell culture
has already been used to produce various secondary metabolites and ingredients of phar-
maceutical (e.g. paclitaxel, vinbrastine for anticancer drugs, etc.), cosmetics (pigments),
and foods, etc. (see Part IV). However, recent CA terminology include more for develop-
ing agricultural products, such as cell-based animal products, by cell cultures than living
farm animals (Stephens and Ellis, 2020).
This includes in vitro or cultured meat, and animal products like milk, leather, eggs,
gelatin and silk through tissue engineering and recombinant DNA fermentation-based
industrial biotechnology process for food and materials (Rischer et al., 2020).
In recent years, a number of cellular animal agriculture companies and non-profit orga-
nizations have emerged due to this technological advances and increasing concern over the
animal welfare and rights, environmental, and public health problems associated with con-
ventional animal agriculture. Some examples are: (i) in the US, lab-grown Memphis Meats,
 OVERVIEW ON MARKET SIZE xxi

Bluefin tuna-based Finless Foods, fermentation-based Geltor (gelatin), Clara Foods (egg
white), Perfect Day and New Culture (animal-free dairy products), Modern Meadow (fer-
menting collagen for leather), (ii) in UK, lab-grown hamburger which was cooked and
tasted, (iii) in Netherlands, Mosa Meat and Meatable, (iv) in Israel, Future Meat Technol
and Aleph Farms, (v) in Canada, Appleton Meats, Biosteel (spider web silk), (vi) in Japan,
Integriculture and spider silk, and (vii), in Singapore, Shiok Meats. Although cell-based
meat may be identical to conventional meat, CA technology is hard to develop because
of structural complexity. Moreover, the texture that tastes meat will be hard to mimic.
Biotech-derived ingredients such as tofu and more recently fungal protein Quorn are well
known, but cell-based meat which will taste like animal meat, with the same nutritional
value, requires expensive serum to grow cells and scale up, and safety issue are formidable
challenges to overcome
Despite the many difficult challenges ahead, CA will be a major part of the solution
for three of humanity’s greatest challenges: feeding 10 billion people by 2050, mitigat-
ing climate change, and mitigating the threat of antibiotic-resistant and zoonotic diseases
(www.futurefields.io/post/cellular-agriculture-in-canada).
Figure 2 shows a scheme of microbial bioproducts and global sales (%) of micro-
bial products except for fermented foods and alcohol beverages. Global sales (%)
of bioproducts excluding fermented food/beverages and biorefinery products can be
shown, among which antibiotics (42%) and biopharmaceuticals (25%+ 17%) are the
major bioproducts. The global market for bioproducts is expected to grow to US$700.7
billion by 2018, with a five-year compound annual growth rate (CAGR) of 5.5%. The
non-energetic market, which includes chemicals, pharmaceuticals, and materials, is the
fastest-growing segment overall at a tremendous 14.9% CAGR, reaching US$472.8
billion by 2018, up from US$236.3 billion in 2013 (www.bccresearch.com). The global
market for biorefinery products alone is expected to clinch US$714.6 billion by 2021 from
US$466.6 billion in 2016 at a whopping CAGR of 8.9% (https://tradeessential.com/events/
biorefinery-bio-based-chemical-2).
In the global fermented ingredients market, amino acid has had a significant market
share due to the growing demand for amino acids by food and beverage and animal feed
additive industries from the last few years. The growing preference for fermented ingre-
dients by several personal care product manufactures in their products led to the growth
in demand of fermented ingredients in personal care industry (www.marketwatch.com/
press-release/the-global-fermented-ingredients-market-to-2023-driven-by-the-growing-
demand-of-fermented-ingredients-to-prompt-the-process-of-fermentation---
researchandmarketscom-2018-09-26). Vitamins are also expected to witness substan-
tial growth as key fermented ingredients in the food & beverage sector, especially
due to their increasing application in probiotic drinks such as kefir. Global markets
of biorefinery products are also high with data, from 2010 to 2013, and projections of
CAGRs through 2018. Biorefinery products broken down by product demand include
energetic (ethanol, biodiesel, electricity/heat), non-energetic (chemicals, materials,
herbal/botanicals), and by other segments, such as fuel (transport, mechanical, etc.),
energy (heat, electricity), material (plastics, resins), chemical (alcohols, solvents, acids,
surfactants, etc.), and herbal/botanical (drugs, body care, aroma, etc.). Forecasts for
biomass conversion processes and equipment to produce fuels, power, and chemicals
from biomass are included in the prospects for biorefineries built on different plat-
forms, such as the “sugar platform,” based on fermentation of sugars extracted from
biomass feedstocks, versus the “syngas platform,” based on thermochemical conversion
processes. The non-energetic bioproducts market will reach US$477.0 billion by 2021
from US$281.7 billion in 2016 at a CAGR of 11.1%, from 2016 to 2021. The energetic
xxii OVERVIEW ON MARKET SIZE

Grain

Stach

Industrial enzymes Fructose syrup

- α-amylase Energy of water Glucose syrup Sugar substitute Bio fertilizers
- glucoamylase Biopesticides
- protease - bioinsecticides
- lipase - biofungicides
- cellulase - Nitrogen fixer
- Pectinase etc. - biostimulators
Microbiological industry
Fuel ethanol Chemicals
Vitamins
- riboflavin (B2) - Milk acid
- Ascorbic acid (C) - Lemon acid
- nicotiamide (PP) - 1.3 - propanediol
- kobolamine (B12) Antibodies - Thin products for
Amino Acids pharmaceutical
- L-lysine
Carotenoids industry.
Biomass of live cells - L-threonine
- β-carotene - Biocatalysis in chemistry - L-tryptophane
- astacsantene - Cleaning of soil, water Polisacharides
- lycopene and air - for technical needs
- for food industry

(a)

Other products Organic Acids 3%

(Steroids, immuno drugs Polysaccharides 1%
etc) 17%
Vitamins 1%
Aminoacids 8%

Antibiotics 42%

Biopharmaceuticals 25%

Enzymes 3%
(b)

Figure 2 (a) Scheme of microbial bioproducts except for fermented foods and alcohol beverages and
(b) Global sales (%) of microbial products except for fermented foods and alcohol beverages by 2023
(www.marketresearchfuture.com/sample_request/765).

bioproduct market is estimated to be US$237.6 billion by 2021 from US$184.9 billion

in 2016 at a CAGR of 5.1%, from 2016 to 2021 (www.prnewswire.com/news-releases/
biorefinery-products-global-markets-300426425.html).
Beside the bioproducts as chemicals (or ingredients) and pharmaceuticals described
above, many different types of fermented foods and beverages are also produced in world-
wide such as fermented milks, cereal-based fermented food, and non-alcoholic beverage,
fermented fruits, and vegetables, and fermented meat. The yogurt and fermented milk
 OVERVIEW ON MARKET SIZE xxiii

market in 2014 alone was worth US$55.01 billion, with North America, Europe, and Asia
accounting for 77% of the market. This is largely due to the reported health benefits
of their probiotic properties (so-called functional foods), and their weight management
factor as well as the minimal preparation needed for fermented foods. The health benefits
of fermented foods are predicated on the presence of bacteria in the foods, which served
as probiotics in the human body to positively contribute to naturally occurring intestinal
flora and therefore support gut health. Fermented foods also contain necessary enzymes,
omega-3 fatty acids, and B vitamins among their health repertoire, illustrating vast poten-
tial to boost health of those choosing to eat fermented products. Consumers are also wary
of food additives, rather choosing fermented products because there are inherently less
preservatives in these choices. Since the bioprocess technology depends on the cultivation
and secretion by the live microbial cells, the risk of contamination by the non-desirable
microbes with the consequent toxicity might restraint the objective and success of fermen-
tation technology. Alcoholic drinks gained a significant market share and are expected to
continue and the global alcoholic beverages market was valued at around US$650 billion
in 2017. Asia Pacific was the largest region in the alcoholic beverages market in 2017,
accounting for nearly 45% market share, among which China was the largest country in
the market in 2017, accounting for nearly 25% market share (www.google.com/search?biw
=1280&bih=913&ei=8JpSXPerL4va8QXgjKfwBQ&q=fermented+alcoholic+beverage+
products%3B+global+market+%28%25%29%3B+2019).
In terms of annual world-wide sales, biotechnology-derived products can be divided
into the three categories. First, fine chemicals as low-volume products and as bulk chemical
products usually fall within the 100 kg per year to 100 tons per year range. This category
broadly includes bioproduction of high-value molecules such as vaccines, rDNA products,
5′ -nucleotides, some of the amino acids, enzymes (for medical applications), monoclonal
antibodies, and bioconversion of high-value starting materials such as antibiotics, and
steroids, and so on. A significant fraction of the production costs of these products are
involved in purification and testing of the product to meet the demand of quality and
safety specifications. Intermediate volume chemicals are usually chemicals or ingredients
that are produced microbiologically in the range of 100 to 20,000 tons per year. Such
products have less vigorous quality and safety specifications than do fine chemicals. Some
examples are glutamic acid (monosodium glutamate, MSG), which is used as a flavor
enhancer, antibiotics used for protecting agricultural crops, food and industrial enzymes,
organic acids (citric, lactic, gluconic acids), solvents (acetone, butanol), many fermented
beverages, and food products. Finally, the bulk product sector consists of products that are
usually produced in continuous reactors, exceeding 20,000 tons per year. These products
are marketed on the basis of commodities and overall product performance criteria
rather than on the basis of rigid quality specifications. The microbial products that fall
within this category are single-cell protein (SCP), gasohol (ethanol), biogas (methane), and
biopolymers for enhanced oil recovery.
The use of biopharmaceuticals has grown worldwide in the last few years. In 2016, the
total number of products approved by the Foods and Drugs Administration (FDA) and
European Medicines Agency (EMA) for use in humans reached 1357, of which >130 have
different formulations (reference products), 737 are biosimilars, and the remaining 482 are
classified as biobetters (www.biopharma.com). From 2013 to 2016, 73 biopharmaceuticals
were approved for use in humans. Among them, high prominence was given to mono-
clonal antibodies (23 approvals) widely used in several diagnostic procedures, treatment of
inflammatory diseases, and neoplastic tumors.
The European Medicines Agency (EMA) also licensed two new products based on
gene therapy (insertion of a corrective gene able to produce a normal protein in the
xxiv OVERVIEW ON MARKET SIZE

patient’s genome to cure a genetic disease) for use in human therapeutic protocols. These
products were Glybera, developed by the German company UniQure for the treatment
of lipoprotein lipase deficiency, and Strimvelis, developed by GlaxoSmithKline (GSK)
for the treatment of adenosine deaminase deficiency. Although biopharmaceuticals
can be very effective for disease control or cure, treatment costs can reach up to US$1
million per patient. Jozala et at. (2016) reviewed the biopharmaceuticals from microor-
ganisms: from production to purification. Table 2 lists some recombinant biopharma
products.

Cellular organization and membrane structure

of three domains
Microorganisms were the first living organisms on Earth, and have been around for at least
3.8 billion years – 80% of the Earth’s 4.6 billion’s year history. It has been estimated that
the total number of microbial cells on Earth on the order of 2.5 × 1030 cells, making it the
major fraction of biomass on the planet.
The three-domain system was first introduced by Carl Woese in 1990. This classifica-
tion system is also known as the Six Kingdoms and Three Domains Classification because
it divides the life forms into three domains and six kingdoms. The three domains of Carl
Woese’s Classification system include archaea, bacteria, and eukaryotes, and the six king-
doms are Archaebacteria (ancient bacteria), Eubacteria (true bacteria), Protista, Fungi,
Plantae, and Animalia. This classification system divides life based on the differences in
the 16S ribosomal RNA (rRNA) structure as well as the cell’s membrane lipid structure
and its sensitivity to antibiotics. The main difference from earlier classification systems is
the splitting of archaea from bacteria.
As a result of useful rRNA molecules, perform the identical function throughout nature,
their structure modifications have changed little over time. Therefore, similarities and dis-
similarities in rRNA nucleotide sequences are a good indication of how related or unrelated
different cells and organisms are. There are various hypotheses as to the origin of prokary-
otic and eukaryotic cells. Because all cells are similar in nature, it is generally thought that
all cells came from a common ancestor cell, termed the last universal common ancestor
(LUCA). These LUCAs eventually evolved into three different cell types, each represent-
ing a domain. The three domains based on Carl Woese’s Classification are thus the Archaea,
the Bacteria, and the Eukarya. Figure 3 illustrates a phylogenetic tree based on rRNA data,
showing the separation of bacteria, archaea, and eukaryota domains.
More recently various fusion hypotheses have been proposed, among which one the-
ory is that the diploid or 2N nature of the eukaryotic genome occurred after the fusion of
two haploid or 1N prokaryotic cells. Other hypothesis is that the domains of Archaea and
Eukarya emerged from a common archaeal-eukaryotic ancestor that itself emerged from a
member of the domain Bacteria. Some of the evidence behind this hypothesis is based on a
“superphylum” of bacteria called PVC, members of which share some characteristics with
both archaea and eukaryotes. Growing evidence shows that eukaryotes may have origi-
nated within a subset of archaea. A “superphylum” of bacteria (PVC) refers to the three
members of that superphylum: the Planctomycetes, the Verrucomicrobia, and the Chlamy-
diae. Members of the PVC, while belonging to the domain Bacteria, show some features of
the domains Archaea and Eukarya. Some of these bacteria show that cell compartmen-
talization, wherein membranes surround portions of the cell interior such as groups of
ribosomes or DNA, is close to eukaryotic cells. Some divisions by budding or contain-
ing sterols in their membranes are again similar to eukaryotes. Similar to eukaryotes and
Another random document with
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The Project Gutenberg eBook of Studiën in
Nederlandsche Namenkunde
 This ebook is for the use of anyone anywhere in the United
States and most other parts of the world at no cost and with
almost no restrictions whatsoever. You may copy it, give it away
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Title: Studiën in Nederlandsche Namenkunde

Author: Johan Winkler

Release date: September 19, 2023 [eBook #71689]

Language: Dutch

Original publication: Haarlem: H. D. Tjeenk Willink & Zoon, 1900

Credits: Jeroen Hellingman and the Online Distributed

Proofreading Team at https://www.pgdp.net/ for Project
Gutenberg (This file was produced from images
generously made available by The Internet Archive)

*** START OF THE PROJECT GUTENBERG EBOOK STUDIËN IN

NEDERLANDSCHE NAMENKUNDE ***
[Inhoud]

[Inhoud]

Studiën in Nederlandsche Namenkunde.

[Inhoud]
 STUDIËN
IN
NEDERLANDSCHE
NAMENKUNDE

DOOR
JOHAN WINKLER.
 HAARLEM
H. D. TJEENK WILLINK & ZOON
1900

[Inhoud]
Boeck, ey soo men di wil laecken,
Segg’ dat si yet beters maecken.
Laecken end maecken is groet verscil,
Dye nyet en can maecken magh swigen still.

D’æbarre traeppet plomp yn ’t gnod,

Oer ’t goe kruwd hinne in sykt de Podd’.
Dy hier uwt naet az fuwl op-syckje,
Momme eack, mey rjuecht, by Rea-schonck
lyckje.

Gysbert Japicx.

Wy willen gheerne ’t onse om een beter gheven,

Isser iet ghefaelt, tsy groot oft cleene.
Maer qualick can ment elck te passe gheweven:
Want niemant volmaeckt, dan God alleene.

Marcus van Vaernewyck.

[Inhoud]
 INHOUD.

Bladz.
Inleiding
I. Spotnamen van steden en dorpen 3
II. Nederlandsche plaatsnamen in Frankrijk 91
III. Gentsche geslachtsnamen 136
IV. Helmondsche namen uit de middeleeuwen 171
V. Friesche namen 196
VI. De namen der ingezetenen van Leeuwarden ten
jare 1511 255
VII. De hel in Friesland 280
Register 293

[1]

[Inhoud]
 INLEIDING.

De Namenkunde vormt een belangrijk onderdeel van de Taalkunde

in haren grootsten omvang, en staat tevens in menigvuldige
betrekking tot Geschiedenis en Volkenkunde.

De kennis van de namen in ’t algemeen, wat hun oorsprong,

geschiedenis en beteekenis aangaat, is inderdaad een zeer
bijzonder vak van wetenschap, een tak van studie die mij steeds
bijzonder heeft aangetrokken, en die bij voorkeur door mij beoefend
is geworden. Herhaaldelijk heb ik dan ook het een en ander werk of
werkje geschreven en in ’t licht doen komen, dat de Namenkunde
van Nederland (plaatsnamen) en van Nederlanders (vóórnamen en
geslachtsnamen) in bijzondere onderdeelen behandelt. Ik behoef
hier slechts mijn werk De Nederlandsche Geslachtsnamen in
Oorsprong, Geschiedenis en Beteekenis 📘 (Haarlem, H. D. Tjeenk
Willink, 1885) te noemen en mijne Friesche Naamlijst (Leeuwarden,
Meyer en Schaafsma, 1898), twee uitgebreide, omvangrijke werken,
die mij veel moeitevolle studie hebben gekost, maar die mij evenzeer
veelvuldige voldoening hebben bereid. Buitendien is er nog in
tijdschriften en jaarboekjes 1 menig opstel van mijne hand
verschenen, dat het een of ander gedeelte der Namenkunde tot
onderwerp heeft, dat Nederlandsche namen uit verschillende
tijdperken van ons volksbestaan, en uit verschillende gouwen en
plaatsen behandelt. [2]

Een zestal van die verhandelingen, uit den aard der zaak weinig
bekend, heb ik uitgekozen, en, ten deele aangevuld, vermeerderd,
verbeterd, hier opnieuw doen afdrukken. Een grooter opstel, over de
Spotnamen van steden en dorpen, het hoofdnummer van dezen
bundel, heb ik daarbij gevoegd. Dat verschijnt hier voor ’t eerst in ’t
licht.

Deze verschillende verhandelingen hangen slechts los te zamen;

slechts in zooverre als ze allen een onderwerp van Namenkunde
behandelen. Overigens niet.

Millioenen namen, mans- en vrouwen-vóórnamen in honderderlei

vormen en vervormingen, oorspronkelijk volkseigene en vreemde,
zoowel als geslachts- en plaatsnamen, eveneens in honderderlei
vormen, en die voor een groot deel van die vóórnamen zijn afgeleid
—inderdaad millioenen namen zijn over alle Nederlanden verspreid,
bij het Nederlandsche volk in gebruik. Elke naam heeft zijnen
eigenen, bijzonderen oorsprong, zijne geschiedenis, zijne
beteekenis, en zeer vele namen zijn in hunnen oorsprong, in hunne
geschiedenis en beteekenis belangrijk en merkwaardig. Elke naam
kan met andere soortgelijke in verschillende groepen vereenigd
worden, en al die namengroepen afzonderlijk in wetenschappelijken
zin beoefend en behandeld worden. Welk een arbeidsveld! En, voor
zooveel het onze Nederlandsche namen betreft, is dat veld nog zoo
weinig ontgonnen!

Ik heb slechts hier en daar een greep kunnen doen in deze rijke stof,
die zoo ruimschoots voorhanden, en voor iedereen toegankelijk is;
slechts hier en daar een greep ter verklaring van sommige
namengroepen en namen.

Mogen de volgende studiën, die uit den aard der zaak slechts in zeer
beperkten en beknopten vorm sommige namengroepen behandelen,
den lezer welkom zijn, en zijne belangstelling opwekken! En mogen
velen, door de lezing en de beoefening dezer verhandelingen zich
aangespoord gevoelen om al mede aan dit onderwerp, aan de
Namenkunde, hunne krachten te wijden; en moge onze
vaderlandsche wetenschap daardoor grootelijks verrijkt en gebaat
worden!

Den vriendelijken lezer een vriendelijke groet van

Johan Winkler.

H a a r l e m , 1900. [3]

1 De Navorscher, De Vrije Fries (tijdschrift van het Friesch Genootschap voor

Geschied-, Oudheid- en Taalkunde, Leeuwarden), Rond den Heerd (Brugge),
Ostfriesisches Monatsblatt (Emden), Nomina Geographica Neerlandica (tijdschrift
van het Nederlandsch Aardrijkskundig Genootschap), Belfort (Gent), de Friesche
Volksalmanak (Leeuwarden), de Noordbrabantsche Almanak (Helmond), enz. ↑
[Inhoud]
I
 SPOTNAMEN VAN STEDEN EN DORPEN.

Onderscheid in geaardheid, onderscheid in volkseigene zaken, taal

en tongval, kleeding, zeden en gebruiken, nering en bedrijf bij zee-,
steê- en landvolk, onderscheid in richting en partijschap op
godsdienstig en op staatkundig en maatschappelijk gebied is er
heden ten dage in ons vaderland nog ruimschoots voorhanden,
tusschen de bevolking van het eene en van het andere gewest, van
de verschillende Nederlandsche gewesten onderling.
Niettegenstaande dit onderscheid langzamerhand al minder en
minder wordt, en gedurig uitslijt, vooral door het meerdere en
gemakkelijke verkeer tusschen de lieden uit de verschillende
gewesten van ons land onderling, zoo onderkent men toch den Fries
aan allerlei volkseigene en bijzonder Friesche zaken en
eigenaardigheden nog gemakkelijk uit alle andere Nederlanders.
Maar ook de Groningerlander en de Zeeuw, de Hollander en de
Gelderschman, de Overijsselaar en de Brabander, de Drent en de
Limburger, ja ook de Hollander uit het Noorden (West-Friesland) en
die uit het Zuiden (het Overmaassche) zijn voor den opmerkzamen
man duidelijk en gemakkelijk te kennen, duidelijk en gemakkelijk de
een van den ander te onderscheiden.

Oudtijds traden de kenteekenen die den Fries en den Brabander,

den Gelderschman en den Hollander, den Drent en den Zeeuw
onderscheiden, veel sterker te voorschijn dan heden ten dage. Ja,
allerlei bijzondere kenmerken waren zelfs op te merken [4]bij de
bewoners van verschillende steden en dorpen—kenmerken,
waardoor dezen zich onderscheidden van de ingezetenen van
andere, van naburige of ook van verderaf gelegene plaatsen. Het
onderscheid tusschen de bewoners van twee naburige plaatsen, al
waren die lieden dan ook oorspronkelijk van geheel den zelfden
volksstam, viel juist hen onderling, over en weêr, bijzonder in ’t oog,
klonk juist te duidelijker in hun oor, werd juist door hen te scherper
opgemerkt. Voor den Hollander moge er geen onderscheid zijn te
bespeuren, in spraak noch in voorkomen, noch in eenigerlei andere
volkseigene zaak tusschen eenen burgerman uit Leeuwarden en
eenen uit Dokkum, voor den Leeuwarder en den Dokkumer zelven is
dit onderscheid zeer wel te hooren en te zien. De Friezen mogen de
Noord-Brabanders en Limburgers dooréén werpen, en niet
afzonderlijk onderkennen, Bosschenaren en Maastrichtenaren, die
van Breda en die van Roermond, zijn diep doordrongen van het
verschil dat er tusschen hen onderling bestaat. De Hollander, in ’t
algemeen de Nederlander uit het Westen en het Zuiden des lands
moge al Groningerlanders en Friezen over eenen en den zelfden
kam scheren en niet onderscheiden, de Amsterdamsche
grootstedeling moge die twee gelijkelijk als „buitenlui”, als
„provincialen, uit het Noorden” bestempelen en ze niet
onderscheidenlijk onderkennen, voor den Fries en den
Groningerlander zelven, over en weêr, zijn de bijzondere kenmerken,
die hen onderscheiden, zeer duidelijk en zeer groot, en de
Leeuwarder begrijpt zoo min als de Groninger hoe de Hollander den
een met den ander als in eenen adem kan noemen, hoe hij den een
met den anderen kan verwisselen en verwarren.

In oude tijden, toen de gelegenheden van onderling verkeer

tusschen de verschillende Nederlandsche gewesten, ook tusschen
de verschillende steden en dorpen van het zelfde gewest zoo veel
minder en geringer waren dan thans, kwamen de menschen, over ’t
algemeen genomen, uit de eene plaats vaak weinig of niet, soms
schier nooit in aanraking met die uit eene andere plaats, al ware ’t
ook dat die twee plaatsen, naar ons hedendaagsch begrip, volstrekt
niet verre van elkander af lagen. Natuurlijk bleven, ten gevolge van
dit besloten zijn binnen de muren en wallen en grachten van de
eigene stad, hoogstens binnen de [5]grenzen van de eigene gouw,
de oude volkseigenheden steeds vast en duidelijk in wezen, bleven
scherper begrensd, hielden veel langer stand dan heden ten dage,
nu schier de helft van de Nederlanders niet meer woont in de
plaatsen, waarin ze geboren en groot gebracht zijn, waar hunne
maagschap van oudsher gezeten is.

Het onderlinge verschil tusschen de ingezetenen van de eene plaats

en die van de andere, werd ook wel eene oorzaak van min
vriendelijke verhouding over en weêr, van onderlingen naijver—ja,
als ’t hoog liep, van onderlingen afkeer, zelfs van haat.
Kleingeestigheid, bekrompenheid, uit onkunde geboren, weêrhield,
aan den eenen kant, wederzijdsche erkenning als volks-, als
stamgenooten, en mat, aan de andere zijde, het onderlinge, veelal
onwezenlijke verschil ten breedsten, ten hatelijksten uit.
Leeuwarders en Dokkumers, bij voorbeeld, gevoelden zich niet als
volksgenooten, als Friezen, de eene zoo goed als de andere, maar
als Leeuwarders en Dokkumers op zich zelven, als „L e e u w a r d e r
G a l g e l a p p e r s ” en als „D o k k u m e r G a r n a t e n ”, zoo als
men elkanderen over en weêr betitelde, ja wel uitschold. Tusschen
Amsterdammers en Haarlemmers, al hoe nabij elkanderen hunne
steden ook gelegen zijn, heerschte in de 16e eeuw de grootste
naijver—een naijver die zich onder anderen lucht gaf in de
spotnamen „K o e k e t e r s ” en „M u g g e n ”, die men elkanderen
wederkeerig toevoegde—een naijver die, bij voorbeeld, ook blijkt uit
het min of meer smalende vers, waarmede de blijspeldichter
Gerbrand Adriaense Brederoô, een Oud-Amsterdammer in merg en
been, de Haarlemmers uitdaagde:

„Haerlemsche drooge harten nu,

Toont nu eens wie gy syt!
Wy Amsterdammers tarten u
Te drincken eens om stryt.”
En juist zulk eene verhouding bestond er tusschen den Zwolschen
B l a u w v i n g e r en den Kamper S t e u r , tusschen den
Deventerschman en den Zutfenaar, tusschen den Franeker
K l o k k e d i e f en den Harlinger To b b e d a n s e r , tusschen den
Rotterdammer en den Dordtenaar, tusschen den Emder
P o t s c h ij t e r en den Auriker P o g g e , tusschen den
Antwerpschen S i n j o o r en den Mechelschen
M a n e b l u s s c h e r , tusschen den Gentenaar [6]en den Bruggeling,
tusschen den K e u n e t e r van Duinkerke en den D r i n k e r van St.
Winoksbergen.

Overal in al de Nederlanden, Noord en Zuid, en in aangrenzende

stamverwante gewesten die thans tot Duitschland en Frankrijk
behooren (Oost-Friesland, Bentheim, Munsterland, Fransch-
Vlaanderen en Artesië), had men oudtijds zulke spotnamen voor de
inwoners van steden en dorpen; en al mogen die namen
tegenwoordig al minder sterk op den voorgrond treden als in vorige
tijden het geval geweest is, ze zijn toch heden ten dage nog
geenszins volkomen verdwenen. Oudtijds gaf de onderlinge naijver,
zich vooral ook uitende in het wederkeerig elkander noemen en
schelden met spotnamen, wel aanleiding tot zeer gespannen
verhoudingen, tot wrevel en haat, tot vechtpartijen zelfs, waarbij men
elkanderen wel bloedige koppen sloeg. Dit behoort in onzen tijd tot
het verledene, maar de oude spotnamen zijn nog wel bekend, en
worden nog wel eens gebruikt, zij het dan ook in tamelijk
onschuldige plagerij, of geheel in scherts.

Deze oude spotnamen zijn voor een goed deel belangrijk in menig
opzicht. Velen daarvan zijn reeds zeer oud en dagteekenen uit de
middeleeuwen. Velen ook berusten op het eene of andere
geschiedkundige feit, anderen op het wapen dat eigen is aan stad of
dorp (K l o k k e d i e v e n van Franeker, B a l k e d i e v e n van ’t
Ameland, M o l l e n van Schermerhorn). Anderen weêr danken hun
ontstaan aan het eene of andere bijzondere voorval, waarbij door
den nabuur, den tegenstander, in ’t geven van den spotnaam, juist
de domme, de belachelijke zijde der zaak werd in ’t licht gesteld
(K a l f s c h i e t e r s van Delft, K e i s l e p e r s van Amersfoort,
M a n e b l u s s c h e r s van Mechelen, R o g s t e k e r s van Weert).
Weêr anderen zijn ontleend aan eenen bijzonderen tak van handel,
van nering of bedrijf, die in de eene stad bestond, in de andere niet;
G o r t b u i k e n of G o r t z a k k e n van Alkmaar—te Alkmaar
bestonden oudtijds vele grutterijen, en de Alkmaarsche gort was wijd
vermaard in den lande; B o t e r v r e t e r s van Diksmude en
K a a s m a k e r s van Belle—beide deze Vlaamsche plaatsen zijn
van ouds bekend om hare zuivelbereiding. Sommigen ook zijn
ontstaan door de eene of andere lekkernij, die in de eene of andere
stad bijzonder gemaakt en [7]door de inwoners bij voorkeur gegeten
of gedronken werd. (K o e k e t e r s van Amsterdam,
K l i e n r o g g e n van de Joure, D ú m k e f r e t t e r s van Sneek,
M o l b o o n e n van Groningen, R o o d b i e r d r i n k e r s van
Harelbeke.)

Kieskeurig waren de oude Nederlanders geenszins, in het bedenken

en gebruiken van spotnamen. Van daar dat sommige dezer namen
heden ten dage slechts ternauwernood in beschaafd mannen-
gezelschap genoemd kunnen worden; (Z a n d p i s s e r s van de
Zijpe, G r u p p e n d r i e t e r s van Oldenzaal, P o t s c h ij t e r s van
Emden, L u z e k n i p p e r s van Eernewoude,
M o s t e r d s c h ij t e r s van Diest). Maar, jufferachtig preutsch moet
men niet zijn, als men sommige eigenaardigheden onzer voorouders
in nadere behandeling neemt.

Al deze Oud-Nederlandsche spotnamen te zamen genomen geven

een veelal verrassend, ook leerzaam en soms niet onvermakelijk