Biochemical Engineering Guide

BIOCHEMICAL
ENGINEERING
BCE4801
DEPARTMENT OF CHEMICAL ENGINEERING
UNISA
Preface
Welcome to Biochemical Engineering (BCE4801), a module that is offered in Unisa’s

Department of Civil and Chemical Engineering. I would like to take this opportunity to wish
you success with your academic year.
Please consult Tutorial Letter 101 for more details about the academic department, the
arrangements relating to this module and assessment.
1. Purpose and outcomes of this module
The purpose of this module is to enable students to apply chemical engineering principles and
concepts to the synthesis and analysis of biologically based processes and products. In
particular, the concepts of thermodynamics, kinetics, and mass and heat transfer will be
applied to enzyme catalysis, microbial growth, bioreactor design, and bio-separations.
Furthermore, students will gain a good understanding of biological concepts, in particular, the
structure and function of cells, enzyme structure and function, and molecular biology.
This module forms part of the Bachelor of Engineering Technology Honours in Chemical
Engineering. The competencies acquired from this module contribute to the development of
skills for the enhancement of the “bioeconomy” of the country.
More specifically, the outcomes of this module are that, after completing the module, you
should be able to
 interrogate cell biology and genetic engineering concepts

 derive rate equations for enzyme-catalysed reactions
 apply concepts of material balances, energy balances, and thermodynamic concepts
to cell metabolism
 apply kinetic, mass and heat transfer principles to the design of biological reactors
 develop strategies for product recovery and purification
The next section will give you a better idea of how the content of the module is structured
and how the various ideas expressed in the learning outcomes are related.
2. How the content of this module is organised
The content of this course covers the main principles of biochemical engineering. The main
topics are as follows:
 History and potential of bioengineering
 Introduction to biological concepts
o Microbiology
o Biochemistry
 Enzymes
o Mechanism and kinetics
o Immobilised enzyme systems
1
o Enzyme inhibition
o Enzyme catalysis (biocatalyst)
o Industrial applications
 Cell cultivation, growth and product formation
o Stoichiometry and yield
o Metabolic pathways and energetics
o Kinetics of growth
 Genetic engineering
 Bioreactor design and operation
o Mass and heat transfer
o Fermenters
o Scale-up
 Product recovery and purification
Now that you have a better idea of how the module is structured, let’s look at what your
studies will involve.
3. Study plan
Use your Study @ Unisa brochure for general time management and planning skills.
This is a semester module offered over 15 weeks, and requires at least 120 hours of study time.
This means that you will have to study at least 8 hours per week for this module.
4. How you should go about studying this module
Distance learning is not easy, and you should not underestimate the time and effort involved.
Your study material will be available online; so, please plan how you will approach and
complete this module. You can use the study plan in the previous section as a guideline to
draw up a reasonable study schedule that can guide you through the whole module.
Remember to take into consideration the due dates of the assignments as given in Tutorial
Letter 101 for this module.
The assignments in this module will take the form of written work, and they should give you
an idea of how well you are progressing in achieving the learning outcomes.
Your work on each learning unit should involve the following:
 Skim through the unit and draw your own basic mind map of the content of the learning
unit. Then expand this map as your knowledge and understanding of the unit increases. If
2
you have internet access, you can learn more about making mind maps on the following
websites:
 http://www.wikihow.com/Make-a-Mind-Map
 http://www.mind-mapping.co.uk/make-mind-map.htm
 Make your own summary of every unit.
As you work, build up your own study and exam preparation file. This file will not be
assessed, but it will be an extremely valuable tool for you in completing your assignments
and revising for the examination.
A study file is a physical or electronic folder or file in which you gather and compile
additional and/or summarised information during the year as you work through the learning
material.
Your study file should comprise
 answers to each activity in each learning unit

 a mind map/summary of each learning unit
 your marked assignments (or a copy you made prior to submitting your assignment)
 where relevant, any extra reading material taken from the internet, additional books,
medical and/or scientific journals
 a new vocabulary of words or glossary of new terms in your own words
Compile and revise the contents of your study file to ensure that you achieve the learning
outcomes of this module.
All the best! I hope you enjoy the course.
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Learning Unit 1: An Overview of Biochemical Engineering
The following are the learning outcomes of this unit:
 Understand what biochemical engineering is.

 Understand the interdisciplinary nature of biochemical engineering and the role of
chemical engineering in biochemical engineering.
1. Introduction
Biochemical engineering is a branch of chemical engineering that incorporates microbiology,

molecular genetics and biochemistry for the production of bioproducts. This is illustrated in figure
1. The key topics of microbiology that are essential to biochemical engineering are microbial cell
structure, metabolism and growth. A knowledge of the cellular process helps the biochemical
engineer to design better biological processes. With regards to biochemistry, enzyme catalysis,
the energetics needed for the synthesis of the microbial cell, and topics of genetics related to
biochemical engineering are molecular genetics and recombinant DNA (deoxyribonucleic acid)
technology that controls and regulates enzyme catalysts and the structure of DNA. DNA plays a
key role in the reproduction of biological organisms. It stores and preserves the genetic
information of biological organisms. The chemical engineering topics that are most relevant to
biochemical engineering are reactor engineering, transport phenomena, heat and mass transfer,
material and energy balance, unit operations, and process design and economics.
The development of a bioproduct is interdisciplinary. Biochemical engineers use the cells and
enzymes developed by microbiologists and biochemists, and genetics to produce bioproducts
such as alcoholic beverages (wine, for example) from fermentation (for example, the fermentation
of grapes), antibiotics, vaccines and blood tissue from microbial and mammalian sources using
modified enzymes. Products such as organic acids, steroids, vitamins and insulin are also
produced from fermentation. The large-scale production of these products was amplified through
advances in recombinant DNA technology. A schematic diagram of this interdisciplinary nature of
bioproduct development using recombinant DNA is illustrated in figure 2.
Chemical
Engineering
Microbiology
Biochemical Biochemistry
Engineering
Molecular
Genetics
Figure 1: Interdisciplinary nature of biochemical engineering
Figure 2: A schematic diagram of the steps involved in the development of a bioproduct
(Source: Doran,1994)
The molecular geneticist and the biochemist are involved in steps 1 to 11 which involve gene
mutation using DNA technology. The microbiologist is involved in the culture of the cells (step 12).
A biochemical engineer is involved in the design of the bioreactor and the upscaling of the
process. He is also involved in the operation, analyses and optimisation of the process (steps 13
to 16). For this to happen, a biochemical engineer needs to have a good understanding of
microbial growth, enzyme kinetics, bioreactor design, bioproduct separation and recovery, and
bioprocess economics and design. These topics will be covered in this module. They have been
covered in different biochemical engineering textbooks and details of these textbooks are
highlighted under the history of biochemical engineering.
2. History of Biochemical Engineering
The development of biochemical engineering started in the early 1940s with the interaction of
microbiology, molecular genetics and biochemistry with engineering (Bailey & Ollis 1986). This
period saw the production of antibiotics, vitamins and amino acids at a commercial scale using
biochemical engineering principles. In the 1960s the large-scale production of single-cell protein
for food or feed supplements followed. During the 1980s advances in recombinant DNA
technology witnessed the large production of therapeutic proteins and steroids. Therapeutic
proteins include plasma protein for blood clotting and insulin for the management of diabetes. The
1990s saw the production of monoclonal antibodies which are used in the detection of cancer.
Also, steroids such as prednisolone, which are used in contraceptives and as anti-inflammatory
and antitumor agents, were produced on a large scale.
The first text book on biochemical engineering was published in 1964 by Prof Webb from the
University College London. It covered chemical engineering topics such as mass and heat
transfer but little or no microbiology and biochemistry. The second text book was written by Aiba,
Humphrey and Milis and was published in 1973. It dealt with microbiology and biochemistry topics
along with chemical engineering topics. In 1986 Bailey and Ollis wrote another biochemical
engineering text book and included molecular genetics with microbiology and chemical
engineering. Subsequently, there have been more text books on biochemical engineering by,
among others, Doran in 1994; Blanch and Clark in 1997 and Shuler and Kurgi in 2002. These text
books can be used by undergraduate and graduate students in biochemical engineering and as
a reference material by practising biochemical engineers.
3. Status of Biochemical Engineering
Biochemical engineering has grown as a discipline. It is now being offered as a course in most
chemical engineering departments at both undergraduate and postgraduate level worldwide. The
chemical engineering departments of most universities are still active in biochemical engineering.
At the University College London, biochemical engineering is now separate from chemical
engineering and the department has been in existence for twenty years.
Biochemical engineering applications has grown from industrial biotechnology and the production
of organic acids, alcohols and ketones, amino acids, polysaccharides, vitamins and enzymes, to
biopharmaceuticals and the production of therapeutic proteins and steroids. This opened the
discovery of new medicines for the treatment of diseases that were not treatable earlier such as
arthritis.
One of the present challenges facing biochemical engineers in the production of these new
medicines is maintaining the structure of the protein during mixing. This has a negative effect on
the large-scale production of these medicines such as increased cost and delay in the period of
production. The challenge of gene rapture also influences the downstream processing of the
proteins cells during either centrifugation or downstream processing.
In South Africa, biochemical engineering has also grown. However, some chemical engineering
departments still do not offer biochemical engineering as a course at undergraduate level. Hence
it is a welcome development that UNISA has introduced biochemical engineering into their
undergraduate curriculum. This would further help in the production of chemical engineering
graduates that would be better placed to use biological agents in the production of therapeutic
protein, antibiotics, enzymes and organic acids. These biological products have a wide application
in the pharmaceutical, food, chemical and energy industries.
4. Review of the Module

In this module, the following will be covered:
Learning unit 2: Introduction to Biological Concepts
o Microbiology
o Biochemistry
Learning unit 3: Enzymes
o Mechanism and kinetics
o Immobilised enzyme systems
o Enzyme inhibition
o Enzyme catalysis (biocatalyst)
o Industrial applications
Learning unit 4: Cell Cultivation, growth and product formation
o Stoichiometry and yield
o Metabolic pathways and energetics
o Kinetics of growth
Learning unit 5: Genetic engineering
Learning unit 6: Bioreactor design and operation
o Mass and heat transfer
o Fermentors
o Scale-up
Learning unit 7: Product Recovery and Purification
Activities
Students are encouraged to read the following articles and also watch videos on biochemical
engineering
The excitement of biochemical engineering.
http://www.ucl.ac.uk/biochemeng/about/excitement.pdf
The importance and future of biochemical engineering

https://onlinelibrary.wiley.com/doi/full/10.1002/bit.27364
Videos about biochemical engineering

https://www.youtube.com/watch?v=7gpLKU7RXiQ
https://www.youtube.com/watch?v=lrMTRgrbRaE
Self-evaluation activity
 List a few bioproducts that would involve the biochemical engineer.
 Classify these products into pharmaceuticals, specialty chemicals, commodity
chemicals, environmental applications, and biodevices.
 Lists the different operations that biochemical engineers would be involved in during the
production of these products, for example, raw pretreatment (hydrolysis/sterilisation),
bioreaction (reactor design), downstream processing (filtration).
References
Aiba, S, Humphrey, AE & Millis, NF. 1964. Biochemical Engineering. 1st edition. New York:
Academic Press.
Bailey, JE & Ollis, DF. 1986. Biochemical Engineering Fundamentals. 2nd edition. Singapore:
McGraw-Hill.
Blanch, HW & Clark, DW. 1997. Biochemical Engineering. 1st edition. New York: Marcel
Dekker.
Doran, PM. 1994. Biochemical Engineering Principles. 1st edition. London: Elsevier
Shuler, ML & Kargi, F. 2002. Biochemical Engineering: Basic Concepts. 2nd edition. NJ:
Prentice Hall
Learning Unit 2: Introduction to Biological Concepts
1. Introduction
In learning unit one, we learnt about the interdisciplinary nature of biochemical engineering as
illustrated in figure 1. In this learning unit, we will learn about microbiology and biochemistry,
two key aspects of biochemical engineering.
Chemical
Engineering
Microbiology
Biochemical Biochemistry
Engineering
Molecular
Genetics
Figure 1: Interdisciplinary nature of biochemical engineering
Microbiology is the study of microbes or microorganisms. Microbes such as bacteria and fungi,
are living organisms that are invisible to the naked eye because they are no more than a few
micrometres in length. They are visible only with the aid of a microscope. This is illustrated in
figure 2. Biochemistry is the study of the chemistry of microbes. Microorganisms contain large
numbers of biomolecules such as proteins and nucleic acids. A study of the chemistry of
biomolecules is essential for understanding the structure of microbes. A knowledge of their
functions are essential for a biochemical engineer because it helps in the design and operation
of biological processes.
Figure 2: Correlation of the size of atomic and microbial cells and the types of
microscope used in measuring them
(Source: http://penpals.web.unc.edu/2013/04/14/what-microscopes-do-you-use-to-see-
microbes/)
Learning outcomes
On successful completion of this learning unit, you should be able to
 describe the main groups of microorganisms

 describe the key components of living cells and their building blocks
 describe and compare the different structures and growth modes of diverse
microorganisms
Activity 1.
Watch this video https://www.youtube.com/watch?v=k7cCBlQARg0 and explain, in your own

words, the importance of studying microorganisms.
2. Microbiology
2.1 Cell theory and living organisms

The smallest unit of a living organism is called a cell. Cells are the building blocks of life. They
contain the structural and functional information of a living organism. Unicellular organisms
such as bacteria, have a single cell whereas multicellular organisms such as plants and
animals, have an incalculable number of cells. Humans, who are also multicellular organisms,
are reported to have more than 100 trillion cells. The study of the cell structure can help in a
better understanding of living organisms.
2.2 Classification of cellular organisms
There are an extensive number of organisms existing. Therefore, it is useful to classify
organisms. Organisms are classified in various ways, for example, according to their structure
and function. Currently, life forms are classified into three domains: bacteria, archaea and
eukarya (eukaryotes). [Previously, archaea and bacteria were classified as prokarya
(prokaryotes)]. This classification is illustrated in figure 3. Prokaryotes includes bacteria and
blue-green (cyanobacteria) algae whereas eukaryotes include protozoa, fungi and algae. All
unicellular and multicellular organisms are classified under the protist kingdom. Non-cellular
protists are non-living microorganisms such as viruses, and are parasitic to other
microorganisms, plants and animals. The major difference between prokaryotes and
eukaryotes is the presence or absence of a membrane-enclosed nucleus. It is present in
eukaryotes and absent in prokaryotes. Other differences between prokaryote and eukaryote
cells are illustrated in figures 4 and 5 and also in table1.
Domains of
Life
Bacteria Archaea Eukarya
Plants Animals Fungi Protists
Figure 3: Classification of living organisms
Table 1: Differences between prokaryote and eukaryote cells
Features Prokaryote cells Eukaryote cells

Membrane-enclosed Absent Present
nucleus
Mitochondria Absent Present
Endoplasmic reticulum Absent Present
Golgi structure Absent Present
Chloroplast Absent Present
Size 0.5 to 3 microns 1 000 to 10 000 times larger
than prokaryote cells
Figure 4: Schematic diagram of a prokaryote
Figure 5: Schematic diagram of an eukaryote
2.3 Prokaryote
2.3.1 Bacteria
Bacteria are important organisms in the production of many bioproducts and in many
biochemical engineering processes in the production of vinegar, antibiotics and animal feed
supplements (lactobacillus probiotic). Bacteria can also be harmful to the human body such
as the fecal bacteria (Enterococcus faecalis) found in polluted drinking water. Examples of
good and bad bacteria in the human gut are given in figure 6.
Useful Bacteria Harmful Bacteria
Clostridium difficile
Lactobacilli
Enterococcus
Escherichia coli
faecalis
Bifidobacteria
Campylobacter
Figure 6: Illustration of useful and harmful bacteria in the human gut
Bacteria are small organisms with rigid cell walls. There are three morphologies of bacteria
according to their shape. Spherical-shaped bacteria are called cocci (coccus in the singular);
cylindrical-/rod-shaped bacteria are called bacilli (bacillus in the singular); and spiral-shaped
bacteria are called spirilla (spirillum in the singular). These forms are illustrated in figure 7.
Figure 7: Different forms of bacteria
Bacteria can also be grouped based on the presence of peptidoglycan in their cell walls. This
is normally determined by the gram stain method. The gram stain method involves the
following: bacteria is, firstly, stained with purple crystal violet, then treated with iodine and,
lastly, washed with alcohol. Gram-positive bacteria remain purple whereas gram-negative
bacteria become colourless. The reason is that the peptidoglycan in the cell walls of gram-
positive bacteria retains crystal violet. Because gram-negative bacteria have no peptidoglycan
in their cell walls, they lose the stain and become colourless.
Bacteria reproduce mainly through binary fission to produce two daughter cells. Binary fission
is a form of asexual production. A cell separates into two daughter cells resulting in the
duplication of the same genetic information in both daughter cells. Sporulation is another form
of asexual production and involves the production and release of spores if conditions are
unfavourable for binary fission.
2.3.2 Blue-green algae

Blue-green algae are also called cyanobacteria. A blue-green alga is, strictly speaking, a
bacterium and not an alga. It is larger than a bacterium and has chlorophyll unlike most
prokaryotes. The presence of chlorophyll makes it possible to produce energy through sunlight
and photosynthesis.
2.4 Eukaryotes
2.4.1 Fungi
The fungus is an important organism in the production of many bioproducts and in many
processes where biochemical engineering is applied. The two types of fungus most used are
yeasts and moulds. They are used in the production of antibiotics and in the baking and
brewing industries. In the chemical industry fungi are used in the production of organic acids.
2.4.1.1 Yeasts
As indicated in figure 1, yeasts are fungi. Yeast can reproduce sexually (by cell fusion)
or asexually (by budding, fission or sporulation). Sporulation is the most common form
of reproduction.
2.4.1.2 Moulds
Moulds are another type of fungus, but differ from yeast in that its filaments are divided
into cells by septa whereas yeasts have one cell. Hence mould is a higher fungus and
yeast a lower fungus. The filaments of moulds are called their mycelium. A long thin
filament of cells in a mycelium is called a hypha (See figure 8). Growth occurs in mould
through the elongation of the tips of the hyphae.
Figure 8: Mycelial structure of moulds
2.4.2 Protozoa and algae
Protozoa and algae are larger eucaroytes than fungi and have a highly organised structure.
Protozoa are mainly used in wastewater treatment and algae have various industrial
applications such as biofuel, food supplements and additives in cosmetics.
2.4.3 Animal and plant cells

Animals and plants are multicellular eucaryotes. Plant cells have cell walls whereas animal
cells do not have cell walls. Only animal cells have cytoplasmic membranes which makes
them more shear sensitive during cultivation than plant cells. Plant and animal cells are now
cultivated commercially in bioreactors as precursors for vaccines and therapeutic medicines.
Previously, the cultivation of plant and animal cells was quite difficult but advances in genetic
engineering have improved their cultivation.
2.5 Viruses
Viruses are small, non-cellular protists, in other words, viruses do not consist of cells. They
are parasitic to hosts such as bacteria, plants and animals. They are not functionally active
except when they are inside the cells of their host. When a virus infects a bacterium, it is called
a bacteriophage. The virus attaches itself to the cell wall of the host bacterium, alters its cell
wall, and injects its viral material into the bacterium. This is illustrated in figure 2.1 in the
prescribed text book.
There are two forms of reproduction in viruses, namely the lytic and the lysogenic cycle. In the
lytic cycle, the phage DNA replicates outside the host bacterium’s DNA; in the lysogenic cycle,
the phage DNA is found in the host DNA and replicates inside it.
Although viruses have a parasitic relationship with their hosts, they are still important in
biotechnology. They are used as a vector in genetic engineering to produce vaccines. In 2018
George Smith and Gregory Winter won the Nobel prize in chemistry for using viruses to infect
bacteria and to produce new types of therapeutic proteins. According to this method, a phage
can produce antibodies and high-efficacy medicine for cancer treatment.
Activity 2
List the various types of microorganisms and describe their different characteristics.
Additional Reading
https://courses.lumenlearning.com/microbiology/chapter/types-of-microorganisms
2.6 Microbial diversity and taxonomy
Most living organisms exist in conditions in which they grow best, conditions with the right
temperature, moisture, nutrients and sufficient oxygen. Organisms that grow at below 20 oC
are called psychrophiles, those that grow between 20 and 50 oC are called mesophiles, and
those that grow above 50 oC are called thermophiles. Those that require oxygen for growth
are called aerobic organisms, those wo do not need any oxygen to grow are called anaerobic
organisms, and those that switch between oxygen and non-oxygen during growth are called
facultative organisms. Organisms that grow in extreme environmental conditions such as the
Arctic and Antarctic, are called extremophiles.
The system for naming organisms is called taxonomy. Organisms can be named according to
either their strain (variation within species), or their species (organisms that are alike) or their
genus (group of related species). Their names are in Latin, for instance, Escherichia coli K12
which is a bacterium. Escherichia is the genus, coli is the species, and K12 is the strain.
2.7 Biochemistry of microorganisms
Biomolecules in microorganisms are the building blocks of the cell unit. These building blocks
are required in the formation of cell components such as ribosomes, nuclei and mitochondria.
The hierarchy of biomolecules in the cell structure is illustrated in figure 9. The major
biomolecules in the cells of microorganisms are proteins, lipids, nucleic acids and
carbohydrates. Microorganisms also contain minor biomolecules such as organic salts,
vitamins and metabolites such as pyruvate and acetate. The cell unit uses these molecules to
produce and store energy used for growth. They also utilise biomolecules to maintain the
stability of their cell structure. The chemical composition of different microorganisms is
illustrated in table 2.
The Cells
Organelles(Nuclei,
Mitochondria,
Chloroplasts)
Supramolecular
Assemblies(Enzyme
complexes, Ribosomes,
Chromosomes,
Membranes)
Nucleic acids Proteins Polysaccharides Lipids
Mononcleotides Amino acids Simple Sugars Fatty Acids
Figure 9: Hierarchy of the cellular structure
Table 2: Chemical composition of different microorganisms
Microorganism Protein (%) Nucleic Acid (%) Lipid (%)
Bacteria 40–50 13–25 10–15
Fungi 10–25 1–3 2–7
Yeasts 40–50 4–10 1–6
Algae 10–60 1–5 40–80

2.7.1 Lipids
Lipids are the major components of the cell membrane. They provide a protective coating to
the cell and act as an energy storage platform. Lipids also help to dissolve and store toxic
organic compounds in an organism. The most common lipids are fats and oils. The major
component of most lipids is fatty acids which occur in an esterified form in cells and tissues.
Fatty acids are made up of hydrophilic carbonyl groups and hydrophobic hydrocarbon chains.
The general formula of a fatty acid is CH3- (CH2)n-COOH. The value of n can be 0,1, 2, 3, ……
If n is 0, it is an acetic acid; when n is 1, it is propionic acid.
Cholesterol, a natural steroid which is found in animal tissue, is also a lipid. Commercial
steroids can now be produced through a biological process using plants as feed stock. Some
of these steroids are used to treat arthritis and other diseases.
2.7.2 Nucleic acids
Nucleic acids contain nucleotides, which are the units that store the genetic information of
cells and which are involved in the reproduction of cells. The parent cell nucleic acids are
passed on to the daughter cells during reproduction. The two major types of nucleic acid are
ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The nucleotide units that make up
DNA and RNA have three key components, namely phosphoric acid, sugar (pentose) and a
nitrogen base (purine or pyrimidine). This is illustrated in figure 2.15 in the textbook. The
pentose sugar can be either a ribose or a deoxyribose, and the nitrogen base can be either a
purine or a pyrimidine. The two major purines present in nucleotides are adenine (A) and
guanine (G), and the three major pyrimidines are thymine (T), cytosine (C), and uracil (U).
DNA contains A, T, G and C, and RNA contains A, U, G, and C as bases.
2.7.3 Proteins and amino acids

Proteins are the most abundant organic molecules in the cell structure. Proteins are found in
the cytoplasm of the cell or in the membrane. Proteins perform different functions in a cell.
They act as catalysts (enzymes) in the isomerisation of glucose to fructose and they transport
lactose across the cell membrane (transport protein). Table 3 details the different functions of
proteins.
Table 3: Selected functions of different proteins
(Source: Baileys & Ollis, 1986)
Proteins Function
Enzymes (catalytic proteins)
Glucose isomerase Isomerises glucose to fructose
Alcohol dehydrogenase Oxidises alcohols to aldehydes
Trypsin Hydrolyses some peptides
Regulatory Proteins (hormones)
Lac repressor Controls RNA Synthesis
Catabolite activator protein Catabolite repression of RNA Synthesis
Interferons Induce virus resistance
Transport Proteins
Lactose permease Transports lactose across cell membrane
Myoglobin Transports oxygen in muscle
Haemoglobin Transports oxygen in blood
Structural Proteins
Collagen Cartilage, tendons
Glycoproteins Cell walls
Elastin Ligaments
Protective Proteins
Antibodies Form complexes with foreign bodies
Proteins are made up of amino acids. There are twenty common amino acids in organisms.
Their different structural configurations are presented in table 4. The general structural
formula of amino acids is given in figure11. The structural formula shows that amino acids
have in their R group acidic (—COOH) and basic (—NH2) groups. The R group forms the
basis of naming the amino acid. For example, when R is H, the amino acid is called glycine
and when R is CH3, the amino acid is called alanine.
The acidic group is neutral at a low pH (—COOH) and negatively charged at a high pH (—
COO-). The pH value at which amino acids have no net charge is called the isoelectric point
and the purification of protein is enhanced at its isoelectric point. In the recovery and
purification of protein as a bioproduct, a higher degree of protein purification is achieved. Then
the pH values of the acidic (—COOH) and basic (—NH2) groups of the protein must have no
net charge.
Table 5: Examples of some amino acids
R-Group Name Abbreviation Symbol Chemical

Group
H Glycine GLY G Aliphatic
CH3 Alanine ALA A Aliphatic
CH(CH3)2 Valine VAL V Aliphatic
CH2CH(CH3)2 Leucine LEU L Aliphatic
CHCH3CH2CH3 Isoleucine ILU I Aliphatic
CH2OH Serine SER S Aliphatic
CHOHCH3 Theronine THR T Aliphatic
CH2SH Cysteine CYS C Aliphatic
(CH2)2SCH3 Methionine MET M Aliphatic
CH2COOH Aspartic Acid ASP A Aliphatic
NH2
R-C-COOH
Figure 11: General structure of an amino acid
2.7.4 Carbohydrates
Carbohydrates help to store compounds in cells. The general formula of carbohydrates is
(CH2O)n, where n is equal to or greater than 3. Carbohydrates are synthesized through
photosynthesis. Monosaccharides are the smallest carbohydrates and contain three to nine
carbon atoms (n is 3 to 9). Glucose is an example of a monosaccharide.
Disaccharides are formed by the condensation of two monosaccharides. Maltose is an

example of a disaccharide. It is formed by the condensation of two glucose molecules through
an a-1,4 glycosidic linkage.
Polysaccharides are formed by the condensation of more than two monosaccharides by

glycosidic bonds. Starch is an example of a polysaccharide.
Activity 3
Draw the general formula of amino acids and list five types of different amino acids.
State the general formula of fatty acids and list the type of acid when n is 1, 2, 3, 4 and 5.
Reflection on this study unit
1. Sketch the diagram of the protists.

2. Briefly compare prokaryotes with eukaryotes in terms of their internal structure and
functions.
3. What is a facultative organism?
4. What are the differences in the cell envelope structure of gram-negative and gram-
positive bacteria?
5. List the different shapes of bacteria and compare them.
6. What are the major classes of fungi? Cite the differences among these classes briefly.
7. Briefly compare protozoa with algae in terms of their cellular structure and functions.
8. Based on the taxonomy of organisms, name the genus, specie and strain of five
organisms.
9. Eukaryotes are generally larger than prokaryotes. Give the characteristic dimensions
of each of these organisms:
 multicellular eukaryotes E. coli
 yeast (S. cerevisiae)
 animal cell
 plant cell
10. What is a bacteriophage and what is its mechanism?
11. List the major biomolecules in microorganisms and the different compositions of these
biomolecules for bacteria, fungi, yeasts and algae.
12. Define lipids, proteins and nucleic acids.
13. List the different types of proteins based on their function and give an example of
each.
14. What are monosaccharides? Mention two examples of monosaccharides and give
their applications.
15. What are the similarities and differences between starch, cellulose and fructose?
Further reading
Introduction to Biochemistry. Available at: https://www.youtube.com/watch?v=CHJsaq2lNjU.

Learning Unit 3: Enzymes
 Study material – Chapter 3 of the prescribed textbook: Bioprocess Engineering:

Basic Concepts by Shuler and Kargi.
1. Introduction
As stated in learning unit two, enzymes are forms of proteins and these macromolecules act
as biocatalysts in transformation processes involving microorganisms. They are used to lower
the activation energy needed for a bioreaction just like conventional chemical catalysts in a
chemical reaction. Also, enzymes do not alter the equilibrium of the bioreaction just like
conventional chemical catalysts. However, enzymes differ from chemical catalysts in terms of
efficiency, specificity, versatility/adaptability and stability. The immobilisation of enzymes can
improve the activity and extend its life span. The activity of enzymes can be reduced by
inhibition. Inhibition occurs when the concentration of the substrate is too high for the enzyme.
 Define how enzymes work and estimate enzyme kinetics from experimental data.
 Explain why enzymes are immobilised and name different methods of enzyme
immobilisation.
 Describe the industrial applications of enzymes and the large-scale production of
enzymes.
2. Mechanism of enzyme catalysis
Enzymes catalyses the substrate(feed) by binding to the substrate before the catalytic process
takes place. The enzyme selects the specific active site of the substrate where the reaction is
expected to take place. This is better illustrated in figure 3.2 in the textbook. Some
enzymes require co-factors and co-enzymes for proper functioning. The co-factors and co-
enzymes are not proteins. In such instances, they are called holoenzyme (apoenzyme and
cofactor) and the protein group is called apoenzyme. Examples of co-factors are metal ions
such as iron, manganese, magnesium and zinc. The coenzymes are complex molecules such
as nicotinamide adenine dinucleotide (NAD), flavin adenine dinucleotide (FAD) and vitamins.
Enzymes have been classified into six types (oxidoreductases, transferases, hydrolases,
lyases, isomerases and ligases). Full details of these enzymes can be found in table 3.1
in the textbook.
A numbering system of enzymes by using codes names was developed by the International
Union of Biochemistry and Molecular Biology. It is done using a four-digit code system. The
first digit determines the function of the enzyme (oxidation-oxidases, transportation-
transferases or isomerization-isomerases), the second and third digits determines the type of
reaction catalysed (2 when electrons or hydrogen are donated, and 3 when electrons or
hydrogen are accepted) and the fourth digit, for instance, EC11127; Lactase dehydrogenase,
L-lactase NAD+ oxidoreductase 1-oxidoreductase, 1-donor group- Lactase 1-acceptor group-
NAD+. A diagram illustrating the six classes of enzymes and how they work is presented in
figure 1 below.
Figure 1: Lock-and-key model of the different types of enzymes
http://www.namrata.co/classification-of-enzymes/
2.1 Enzyme kinetics (the Michaelis-Menten equation)
The rate of reaction (recall from the module on reaction engineering) of enzyme catalysis can
be evaluated according to the Michaelis-Menten equation. This equation was derived by
Michaelis-Menten in 1913 based on experimentally data from batch reactors with constant
liquid volumes in which the initial substrate, enzyme, and concentrations are known. The
enzyme and the substrate form a complex (ES) and dissociate to a product (P) and a free
enzyme (E). This is illustrated in equation 1
E+ S ES E+P Equation 1
The first reaction is assumed to be reversible when the ES complex is formed rapidly and
the rate of the reverse reaction of the second step is negligible.
The assumption of an irreversible second reaction holds only when product accumulation is
negligible at the beginning of the reaction.
At equilibrium, reaction 1 becomes equation 2
𝐾 Equation 2
Km is the dissociation constant of the enzyme-substrate complex (ES). The rate of product
formation is illustrated in equation 3
𝑉 =𝑘 [ES] Equation 3
Since the enzyme acts as a catalyst and is not consumed, a conservation equation for the
total concentration of the enzyme is presented in equation 4
[E]T= [E]+[ES] Equation 4
Where [E]T is the total concentration, [E] is the concentration of the free enzyme, and [ES] is
the concentration of the enzyme-substrate complex.
When we substitute equation 4 into equation 2, it becomes equation 5
𝐸 𝐸𝑆 𝑆
𝐾
𝐸𝑆
Equation 5
Rearranging equation 5 [ES] becomes
𝐸 𝑆
𝐸𝑆
𝐾 𝑆
Equation 6
The rate of product formation in equation 3 can also be expressed in equation 7
𝑑𝑃 𝑘 𝐸 𝑆
𝑉 𝑘 𝐸𝑆
𝑑𝑡 𝐾 𝑆
Equation 7
The maximum rate of product formation; Vm = k2[E]T equation 7 can be rewritten
𝑉 𝑆
𝑉
𝐾 𝑆
Equation 8
The above equation is called Michaelis-Menten Kinetics. The value of Vm changes when more
enzymes are added and does not change when more substrate is added. A low value of 𝐾
indicates that the enzyme has a low affinity for the substrate. 𝐾 can also be related to the
maximum product rate Vm. At half the maximum product rate, the concentration of the
substrate is also the dissociation constant of the enzyme-substrate (𝐾 ). This is illustrated
in figure 3.3 in the textbook.
2.1.1 Estimation of Vm and 𝐾 from Michealis-Menten
Vm and Km can be estimated from experimental data of either substrate or product against time
(𝑉 and plotting V values and S. The kinetic parameters (Vm and 𝐾 ) can then
be estimated from the slope of the curve. In the literature there are different curves used for
estimating Vm and Km and these are presented below.
 Lineweaver-Burk plot – This type of plot involves plotting 1/ V versus 1/S and gives a
linear plot with a slope of 𝐾 /Vm and an intercept of 1/Vm. The Lineweaver-Burk plot
is shown in figure 3.5 in the textbook. There is a lower degree of error in the value
of Vm but a higher degree of error in the value of Km. The governing equation for the
Lineweaver-Burk plot is presented in equation 9.
1 1 𝐾 1
𝑉 𝑉 𝑉 𝑆
Equation 9
 Eddie-Hofstee plot – This plot involves the plotting of V verse V/[S] and gives a linear
plot with a slope of 𝐾 and an intercept of Vm. The Eddie-Hofstee plot is shown in
figure 3.6 in the textbook. The governing equation for the Eddie-Hofstee plot is
presented in equation 10.
𝑉
𝑉 𝑉 𝐾
𝑆
Equation 10
 Hanes-Woolf plot – This involves the plotting of [S]/V verse S and gives a linear plot
with a slope of 1/Vm and an intercept of Km/Vm. The Hanes-Woolf plot is shown in
figure 3.7 in the textbook. The governing equation for the Hanes-Woolf plot is
presented in equation 11.
𝑆 𝐾 1
𝑆
𝑉 𝑉 𝑉
Equation 11
Out of the three plots, the value of Vm estimated from the Hanes-Woolf plot is the most
accurate.
Activity 1
In example 3.2 in the text book, the Hanes-Woolf plot was used to estimate Vm, 𝐾 , and k2
for the given problem. Students are encouraged to use the other plots (Lineweaver-Burk and
Eddie-Hofstee Plots) to estimate the same kinetic parameters Vm, 𝐾 , and k2 and compare
their accuracy.
2.2 Non-equilibrium enzyme kinetics (Briggs-Haldane equation)

The equilibrium assumption in the Michaelis-Menten equation would not hold if the rate of
product formation from the enzyme-substrate complex is similar to the rate of breakdown of
the enzyme-substrate complex and the substrate. This is illustrated in figure 3.4 in the
textbook. Briggs and Haldane introduced a quasi-steady state assumption of the enzyme.
This means that the formation of the enzyme-substrate complex [ES] is at a steady state and
at high concentrations of enzyme or low substrate [E] is close to [S], the assumption of the
steady state of [ES] is no longer valid. The reaction rate is shown in equation 12.
E+ S↔ ESE+P Equation 12
The rate of formation of the enzyme-substrate complex

= k1[E][S]-k2[ES]-k-1[ES]=0 Equation 13
Rearranging equation 13
𝐸 𝑆
𝐸𝑆 𝑘
𝑘 𝑘
Equation 14
Substituting equation 4 into equation 14

𝑘 𝐸 𝐸𝑆 𝑆
𝐸𝑆
𝑘 𝑘
Equation 15
Solve equation 15 for [ES]

𝐸 𝑆
𝐸𝑆
𝑘 𝑘
𝑆
𝑘
Equation 16
Then substitute equation 16 into equation 3
𝑘 𝐸 𝑆
𝑘 𝑘
𝑆
𝑘
Equation 17
v Equation 18
Where Km= (k-1+ k2)/k 1, Vm=k2[E]
2.3 Enzyme activity
Sometimes enzyme contains catalytic and non-catalytic activities. The enzyme catalytic
activity refers to the active concentration of the enzyme which is usually expressed as
“units”. A “unit” is defined as the amount of enzyme that gives a certain amount of catalytic
activity under specified conditions. The unit of enzyme activity can be expressed in equation
19 below.
𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝐸𝑛𝑧𝑦𝑚𝑒 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 Equation 19
A purified enzyme would normally have a higher specific activity than a crude enzyme. How
to estimate the specific enzyme activity is illustrated in example 3.1 in the text book.
2.3.1 Factors that influence enzyme activity
The activity of enzymes can change when there is a change in temperature, the pH or the
mechanical forces. A change in the pH may change the structure of an enzyme or the ionic
form of the active site of the enzyme. These could have a negative affinity of the substrate for
the enzyme and also on the rate of reaction of the enzyme-catalysed reaction.
An increase in temperature leads to greater inactivity and limited reaction. Enzyme activity
slows down as a result of denaturation. This is called the thermal deactivation of the enzyme.
The thermal deactivation of mammalian enzymes normally occurs at a temperature of between
45 and 55 C. The optimal temperature of mammalian enzymes is about 37 C.
A change in the mechanical forces of enzymes in a flow system can exert shear stress on the
enzyme and deactivate it by reducing its activity.
2.4 Enzyme inhibition
Enzyme activity can also be reduced enzyme inhibitors such as metals. The enzyme inhibitor
binds on the active site of the enzyme and makes changes which reduces the activity of the
enzyme. This change could be either reversible or irreversible. Reversible inhibition can be
removed and the activity of the enzyme could recover. With irreversible inhibition it cannot be
done.
There are four types of reversible inhibition, namely competitive, uncompetitive, non-
competitive and substrate inhibition. This is illustrated in figure 3.10 in the textbook. Statins
are examples of enzyme inhibitors that are used to treat cholesterol. Heavy metals can also
inhibit enzyme activity.
2.4.1 Competitive inhibition
Competitive inhibition occurs when an inhibitor directly competes with the substrate in forming
the enzyme complex. Examples of competitive inhibitors are antibiotics such as penicillin, that
is used to block active site of bacteria, and statins that are used to block the action of the liver
enzyme responsible for cholesterol. The equation for competitive inhibition is shown in
equation 19.
𝑉 𝑆
𝑉
𝐼
𝑆 𝐾 1
𝐾
Equation 19
2.4.2 Uncompetitive inhibition
This occurs when the inhibitor competes with the enzyme-substrate complex only. An example
of an uncompetitive inhibitor is nifedipine which is used to block a liver enzyme that is
responsible for high blood pressure in pregnant women. The equation for uncompetitive
inhibition is shown in equation 20.
𝑉 𝑆 𝐾
𝑣 / 𝑆
𝐼 1
1 1
𝐾 𝐾
Equation 20
Where I is the inhibitor concentration and KI is the inhibitor constant.
2.4.3 Non-competitive inhibition
This occurs when the inhibitor binds either to the enzyme or the enzyme-substrate complex.
The equation for non-competitive inhibition is shown in equation 21.
𝑉 𝑆
𝑣
𝐼 𝑆 𝐾
1
𝐾
Equation 21
2.4.4 Substrate inhibition
This occurs at a very high substrate concentration. Some of the substrate may bind at an
alternative site of the active site making the enzyme-substrate complex unreactive. The
equation for substrate inhibition is shown in equation 21.
𝑉 𝑆
𝑣
𝑆
𝐾 𝑆
𝐾
Equation 22
Activity 2
In example 3.3 in the text book, the substrate concentration is doubled for both conditions (0.2
to 0.4; 0.02 to 0.04) but the inhibition concentration is constant. Will the type of inhibition
change? Give a reason. Then find the Vm for the substrate concentrations of 0.4 M and 0.04M.
3.0 Immobilisation of enzymes
Because enzymes are expensive, their reutilisation is important in many biochemical

processes. For example, aminoacylase produced from Aspergillus oryzae is immobilised by
ionic adsorption on cellulose material called DEAE-Sepahdex. The restriction of enzyme
mobility in a fixed space is known as enzyme immobilisation. Immobilised enzymes are
typically macroscopic catalysts that are retained in the reactor. Therefore, they do not have to
be replaced continuously, and the separation of enzymes from other components in the
reaction mixture is simplified.
3.1 Methods of enzyme immobilisation

The two methods of enzyme immobilization are binding and entrapment. Binding can be done
through ionic or adsorptive and covalent bonding and entrapment by using a matrix (fibre and
beads) or a membrane (encapsulation and membrane reactors). This is well illustrated in
figure 3.16 in the textbook. For example, immobilised glucose isomerase is used industrially
to convert glucose to fructose. Fructose is used as a sweetener in carbonated drinks. Another
enzyme used in the production of a sweetener is aspartase. This enzyme converts fumarate
to L-aspartate and the result is a low-calory sweetener called apartame. The aspartase
enzyme is immobilised by entrapment in gelatin treated with glutaraldehyde.
3.2 Diffusion in immobilised enzymes
Mass transfer limitations as a result of resistance from diffusion in immobilised enzymes can
reduce the rate of reaction. The Damköhler number (Da) is used to evaluate if the diffusion
resistance would have a significant effect on the enzymatic reaction rate. It is the relative rate
of the reaction rate compared to the diffusion rate, and it is illustrated in equation 23.
𝑚𝑎𝑥𝑖𝑚𝑢𝑚 𝑟𝑎𝑡𝑒 𝑜𝑓 𝑟𝑒𝑎𝑐𝑡𝑖𝑜𝑛 𝑉𝑚

𝐷
𝑚𝑎𝑥𝑖𝑚𝑢𝑚 𝑟𝑎𝑡𝑒 𝑜𝑓 𝑑𝑖𝑓𝑓𝑢𝑠𝑖𝑜𝑛 𝐾 𝑆
Equation 23
where [Sb] is a substrate concentration in bulk liquid (g/cm3) and KL is the mass-transfer
coefficient (cm/s). The rate of enzymatic conversion may be limited by diffusion of the
substrate or reaction, depending on the value of the Damköhler number. If Da >> 1, the
diffusion rate is limiting. For Da << 1, the reaction rate is limiting, and for Da = 1, the diffusion
and reaction resistances are comparable.
At steady state, the reaction rate is equal to the mass transfer rate. This is illustrated by
equation 24.
𝑉𝑚 𝑆
𝐽 𝐾 𝑆 𝑆
𝐾 𝑆
Equation 24
where Vm is the maximum reaction rate per unit of external surface area and KL is the liquid
mass-transfer coefficient.
Activity 3
In example 3.5 in the textbook, estimate the Damköhler number and state if there are any
diffusional limitations.
4. Industrial applications of enzymes
The industrial application of enzymes would require the large-scale production of enzymes. A
process flow diagram of the large-scale production of an extracellular enzyme is
illustrated in figure 3.23. The process flow diagram shows the cultivation of the organism,
the separation of the enzyme, the removal of cell debris and nucleic acids, the precipitation of
proteins, the ultrafiltration of the desired enzyme, the chromatographic separation
crystallization, and the drying.
The industrial applications of enzymes include food processing, such as cheese making
(rennet), baking, meat tenderisation (papain, trypsin), brewing (trypsin, pepsin); detergents for
the hydrolysis of protein stains (subtilisin Carlsberg); tanning; and the medical treatment of
wounds.
Enzymes are forms of proteins which are used as biological catalysts to increase the rate of
biochemical reactions. They are more effective, specific and versatile than conventional
chemical catalysts but are less stable than conventional catalysts. The thermal deactivation of
mammalian enzymes normally occurs at a temperature range of between 45 and 55 oC. The
optimal temperature for mammalian enzymes is about 37 oC, whereas most conventional
chemical catalysts are still stable at temperature values greater than 55 oC.
Enzymes bind on substrate molecules and reduce the activation energy of biochemical
reactions thereby increasing the rate of the reaction. Enzyme kinetics can be described by
Michaelis-Menten equation and the Briggs-Haldan equation. , Michaelis-Ment equation
assumes equilibrium and non-equilibrium conditions is better described by the Briggs-
Haldane equationequation.
The activity of an enzyme can be reduced by inhibition in four different forms, namely
competitive, non-competitive, uncompetitive and substrate inhibition.
Enzymes require certain optimal conditions such as an optimal temperature and optimal pH,
to achieve its maximum activity. If the optimal temperature and pH are exceeded, an enzyme
loses its activity. Enzyme activity can also be increased by immobilisation. Enzyme
immobilisation helps with enzyme reutilization and eliminates costly enzyme recovery and
purification processes. However, enzyme immobilisation may result in the reduction of mass
transfer owing to diffusion limitations.
Enzymes are used in various industrial applications such as amylases for starch hydrolysis,
rennet in cheese manufacturing, and glucose isomerase in glucose-to-fructose conversion. A
promising future lies ahead for enzyme production worldwide.
1. What are enzymes?

2. What are co-factors and what role do they play in catalysed reactions?
3. Answer questions 3.1 to 3.14 in the textbook.
Learning Unit 4: Cell Cultivation, Growth and Product Formation
 Study material: Chapters 5 to 7 of the prescribed textbook Bioprocess

Engineering: Basic Concepts by Shuler and Kargi.
1. Introduction
In this learning unit, we will study cell cultivation and determine the optimal conditions for cell
growth and product formation. Cell growth and product formation happen under certain
physical, chemical and nutritional conditions. Cells use nutrients for energy production, growth
and product formation. Cell growth is estimated by measuring the cell concentration either
directly or indirectly. The different methods to estimate cell concentration will be detailed. The
product yield of a biological process can be constrained by kinetics or thermodynamics, and
the calculation of product yield and the stoichiometry coefficient will be presented. The
metabolic pathways in terms of catabolism (the process of degrading a compound into smaller
and simpler products) and anabolism (the synthesis of more complex compounds) will be dealt
with. Finally, the bioenergetics and the kinetics of biomass growth will be discussed.
 Define how cells are cultivated and determine the optimal conditions for cell growth
and product formation.
 Calculate the product yield and the stoichiometry coefficient of a biological process.
 Describe metabolic pathways and estimate bioenergetics (the ATP yield coefficient,
the yield coefficient mass of cells or the kcal heat generated during growth).
 Apply both the structured and unstructured model to estimate the kinetics of cell
growth.
2. Cell cultivation and measurement
Cells cultivation is the process of growing cells at different conditions to produce the required
cell concentration and product purity or concentration. These conditions include the
temperature, pH, nutrients and the incubation time. Cells are first cultured in petri dishes and
later transferred to shake flasks and, lastly, to a bioreactor. This is illustrated in figure 1 below.
Shake Flasks Bioreactor

Petri Dishes
Figure 1: Cell cultivation from petri dishes to bioreactors
(Source:
https://www.google.com/search?q=picture+of+bioreactor&tbm=isch&source=hp&sa=X&ved=
2ahUKEwjOmr32nIzgAhVwBGMBHRv2Cq4Q7Al6BAgCECE&biw=1366&bih=651#imgrc=A
ZbenrNNF_t6vM:)
Cell cultivation in bioreactors can be either in batches or in continuous systems. The choice
of cell cultivation can have an impact on productivity in terms of the cell growth or the product
formation. Cell growth is evaluated by measuring the cell concentration.
2.1.1 Estimation of cell concentration
Cell concentration can be estimated directly or indirectly. Direct methods include the
measurement of cell density (plate counting or particle counting) and the measurement of cell
concentration (dry-weight analysis or optical density). Indirect methods include the
measurement of substrate consumption and product formation, and the measurement of the
RNA, DNA and proteins content.
2.1.1.1 Direct methods
Measurement of cell density – Cell density is estimated by counting both the dead and the
viable cells. The number of cells divided per unit volume is equal to the cell density. Cell
counting is done either by hand or an automated counting process.
Manual counting methods include plate counting and a counting chamber (a hemocytometer).
For plate counting, culture samples are diluted and spread on the agar surface, and then the
plates are incubated. The colonies on the agar surface are counted after the incubation period.
The results are expressed in terms of colony-forming units (CFUs). This method is less
suitable for counting dead cells than for counting living (viable) cells. A hemocytometer is a
microscopic slide that has gridded chambers for cell counting. The number of cells are counted
manually in a specified area demarcated by a grid square. There should be at least 20 grid
squares to make the counting statically valid. This method can be used to distinguish between
living and dead cells. However, it is difficult to count mycelial cells such as moulds, with this
method. A video of how this works is shown in the following link:
https://www.youtube.com/watch?v=WWS9sZbGj6A
 Automatic counting methods include particle (coulter) counters, flow cytometry and
image analysis. Flow cytometry is the most effective automated cell counting
method, but is quite expensive.
Measurement of cell concentration – Cell concentration can be quantified by carrying out

a dry-weight analysis or a turbidity measurement.
 A dry-weight analysis is the most used method for measuring cell concentration. It is
used when cells are cultivated in a liquid growth medium system such as shake flasks
and bioreactors. A dry-weight analysis is unsuitable when cells are cultivated in a solid
growth medium in petri dishes. In a dry-weight analysis, liquid samples of the cell
culture is filtered and washed with a buffer solution or water. The washed, wet cell
mass is then dried at 80 oC for 24 hours and the dry cell weight measured.
 Turbidity measurement – According to this method, liquid samples of the cell culture is
centrifuged in an Eppendorf and their absorbance is measured at a certain wave length
by using a spectrometer. A higher turbidity liquid cell culture would have a higher
absorbance than a lower one. A video of how this works is shown in the following link:
https://www.youtube.com/watch?v=EX1tn35AX00
2.1.1.2 Indirect methods

Direct methods might not be suitable for the estimation of cell concentration when there is
inference with the biomass by other solid particles or colour compounds. In such cases, the
substrate consumption and/or product formation is measured to estimate the cell
concentration.
Measurement of intracellular DNA, RNA and protein – Protein measurement methods

include the total amino acids, the Biuret, the Lowry and the Kjeldahl nitrogen measurement
methods. The Total amino acids and the Lowry methods are the most reliable.
Measurement of the ATP concentration – When there is interference from the protein
content in the growth medium and the protein content of the cell, the measurement of ATP is
preferred as an indirect method of cell quantification. The method is based on the luciferase
activity which catalyses oxidation of luciferin at the expense of oxygen and ATP with the
emission of light. When oxygen and luciferin are in excess, the total light emission is
proportional to the total ATP present in the sample. This method is usually used in the
measurement of biological activity in wastewater treatment plants. A video showing the
measurement of ATP in wastewater is available at: https://www.youtube.com/watch?v=7sfE-
lImeBc.
2.2 Batch growth kinetics
There are five phases in bacterial growth for a batch system. These are the:
 lag phase
 exponential phase
 deceleration phase
 stationary phase
 death phase
This is illustrated in figure 6.3 in the textbook. The lag phase occurs after inoculation when
cells adapt to a new environment. The inoculum age and the size affect the duration of the lag
phase. The age of the inoculum refers to how long a culture has been maintained in a growth
medium before it was transferred to a new environment. The lag period increases with the age
of the inoculum. The inoculum size means the volume fraction ratio of the inoculum with
respect to the new growth medium. An inoculum size of 5 to 10% is recommended for an
optimal lag phase.
Exponential phase – As the name of the phase implies, cells grow exponentially since they
have adapted well to the new environment. In this phase, growth is balanced if all
components of the cell grow at the same rate. The growth rate in the exponential phase is
first order and is represented in equation 1 below.
𝑑𝑋
µ 𝑋, 𝑋 𝑋 , 𝑎𝑡 𝑡 0
𝑑𝑡
Equation 1
The integration of equation 1 becomes
𝑋
𝑙𝑛 µ 𝑋, 𝑋 𝑋 𝑒 µ 𝑛𝑒𝑡
𝑋
Equation 2
Where X and X0 are cell concentrations at time t and initial t = 0 respectively.
The time taken to double the cell concentration can be estimated by rearranging equation 2:
𝑙𝑛 2 0.693
Ʈ
µ µ
Equation 3
Deceleration phase – During this phase, the growth rate decreases due to either the depletion
of one or more essential nutrients or the accumulation of the toxic by-products of growth. Cell
growth is unbalanced owing to a change in the environment.
Stationary phase – In this phase, the growth rate is zero or equal to the death rate. Although
there is no growth, metabolism and product formation carry on. This type of metabolism is
called endogenous metabolism. The equation for the stationary phase is as follows:
𝑑𝑋
𝐾 𝑋 𝑜𝑟 𝑋 𝑋 𝑒 𝑘 𝑡
𝑑𝑡
Equation 4
Kd is a first-order rate constant for endogenous metabolism, and Xso is the cell concentration
at the beginning of the stationary phase.
Death phase – In this phase, most cells die. Cell death may start during the stationary phase.
Often dead cells lyse and intracellular nutrients released into the medium are used by the
living organisms during the stationary phase. At the end of the stationary phase, because of
either nutrient depletion or toxic product accumulation, the death phase begins. The equation
for the stationary phase is as follows:
𝑑𝑋
𝐾 𝑁 𝑜𝑟 𝑋 𝑁𝑒 𝑘 𝑡
𝑑𝑡
Equation 5
Ns is the concentration of cells at the end of the stationary phase and Kd is the first-order
death rate constant. A plot of ln N versus t yields a line of slope -Kd.
At the end of the batch growth period, we have growth yield. It is the ratio of the cell formation
to the substrate consumption. This is illustrated in equation 6:
∆𝑌
𝑌
∆𝑆
Equation 6
Yield coefficients may also be based on other substrates such as product formation and
oxygen consumption. They are illustrated in equations 7 and 8:
∆𝑃
𝑌
∆𝑆
Equation 7
∆𝑋
𝑌
∆𝑂
Equation 8
A maintenance coefficient is used to describe the specific rate of substrate uptake
for cellular maintenance. It is represented in equation 9:
𝑑𝑆/𝑑𝑡
𝑚
𝑋
Equation 9
Activity 1
Take example 6.1 in the text book and calculate:

 the specific rate of substrate uptake for cellular maintenance
 the time taken to double the cell concentration
2.3 Continuous-growth kinetics
A material balance on cell growth in a continuously stirred tank reactor (CSTR), which can
also be called a chemostat, is presented in equation 10. A schematic diagram of a
chemostat is illustrated in figure 6.18 in the textbook.
𝑑𝑋
𝐹𝑋 𝐹𝑋 𝑉 µ 𝑋 𝑉 𝐾 𝑋 𝑉
𝑑𝑡
Equation 10
F is the flow rate of a nutrient solution (l/h), VR is the culture volume (l) (assumed constant), X
is the cell concentration (g/l), and µg and Kd are growth and endogenous (or death) rate
constants, respectively (h-1).
Equation 10 can be rearranged
𝑑𝑋
𝐷𝑋 µ 𝐾 𝐷 𝑋
𝑑𝑡
where D is the dilution rate and D = F/VR. D is the reciprocal of residence time.
The growth rate µg is equal to the dilution rate D when at a steady state ( 0) and
endogenous rate Kd is zero and initial cell concentration is zero since the feed media is sterile.
This is presented in equation 11.
µ 𝐷
Equation 11
Since the growth rate is limited by at least one substrate in a chemostat, a simple description
of the chemostat performance can be made by substituting the Monod equation.
µ 𝑠
µ 𝐷
𝐾 𝑆
Equation 12
A plot of 1/µg versus 1/S can be used to estimate values for µm and Ks. We can also relate the
effluent substrate concentration to the dilution rate for D<µm.
𝐾𝐷
𝑆
µ 𝐷
Equation 13
A material balance on the limiting substrate in the absence of endogenous metabolism
yields.
1 1 𝑑𝑆
𝐹𝑆 𝐹𝑆 𝑉 µ 𝑋 𝑉 𝑞 𝑋 𝑉
𝑌𝑀 / 𝑌/ 𝑑𝑡
Equation 14
S0 and S are feed and effluent substrate concentrations (g/l), qP is the specific rate of
extracellular product formation (q P/mg cells h), and YM X/S and Y P/S are yield coefficients (g
cell/g S and g P/g S). The use of the superscript M on Y X/S denotes a maximum value of the
yield coefficient.
When extracellular product formation is negligible, and the system is in a steady state:
µ
𝐷 𝑆 𝑆
/
Equation 15
Inserting equation 11 in equation 15, it becomes
𝑋 𝑌𝑀 / 𝑆 𝑆
Equation 16
𝐾𝐷
𝑋 𝑌𝑀 / 𝑆
𝐷 µ
Equation 17
When the effect of endogenous metabolism is added into equation 11,
µ 𝐷 𝐾
Equation 18
1
𝐷 𝑆 𝑆 𝐷 𝐾 𝑋 0
𝑌𝑀 /
Equation 19
Rearranging equation 19
𝐷 0
/
Equation 20
Equation 21
𝐾
𝑚
𝑌𝑀
Equation 22
where ms is the maintenance coefficient based on substrate S. YAPX/S is the apparent yield.
The values of YM X/S and ms can be obtained from chemostat experiments by plotting 1/YAPX/S
against 1/D. The slope is ms and the intercept is 1/YMX/S.
Activity 2
In example 6.4 in the text book, change the influent concentration from 800 mg/l to 400 mg/l
and calculate the µm, Ks, YM X/S, Kd, and ms.
2.4 Difference in structured and unstructured models for cell growth kinetics
An unstructured model assumes a fixed cell composition, which assumes balanced growth.
The balanced-growth assumption is valid in a single-stage, steady-state continuous culture
and the exponential phase of batch culture. Most of the growth kinetics studied in sections 2.2
and 2.3 is based on the balanced-growth assumption.
An unstructured model assumes transient conditions in which cellular composition and

biosynthetic capabilities change in response to new growth conditions. This is called
unbalanced growth. Some examples of structured models include chemically structured
models and the cybernetic model. Full details of these models can be found in figure 6.14
in the textbook.
3. Stoichiometry and yield
Apart from its kinetics, cell growth and product yield can also be influenced by stoichiometry
(thermodynamic limits). In this section, we will explore ways of estimating biomass and
product yield by using different stoichiometry approaches such as elemental balance and
degree of reduction.
3.1 Elemental balance
An elemental balance on biological reactions can be written if the compositions of substrates,

products, and cellular material are known. Usually, electron-proton balances are required in
addition to elemental balances to determine the stoichiometric coefficients in bioreactions.
A simple biological reaction where there is no product formation is given in equation 23:
CH O aO bNH → cCH O N dH O eCO
Equation 23
CHmOn represents 1 mole of carbohydrate and CHαOβNδ stands for 1 mole of biomass formed.
Elemental balances on C, H, O, and N yield the following equations:
𝐶: 1 𝑐 𝑒
Equation 24
H: m 3b cα 2d
Equation 25
O: n 2a cβ d 2e
Equation 26
N: b cδ
Equation 27
The repository quotient
RQ
Equation 28
Activity 3
Determine the stoichiometric coefficients if the RQ value is 0.43:
C H aO bNH → cCH .. O . N . dH O eCO
3.2 Degree of reduction
Elemental balances do not provide information on the bioenergetics of the bioreaction. Hence
the degree of reduction, which is based on the proton–electron balance, is used to estimate
stoichiometric coefficients when there is product formation. The degree of reduction is
represented by γ, and it is the number of equivalents of available electrons per gram atom C
for any organic compounds. The available electrons are those transferred to oxygen upon
oxidation of a compound to CO2, H2O, and NH3. The number of electrons for some key
elements are C = 4, H = 1, O = -2, P = 5, and S = 6. The number of electrons of N depends
on the source of nitrogen-containing compounds, for Nitrogen is 0, 3 for Ammonia and 5 for
Nitrate.
Take, for example, the substrate CHmOn. The number of electrons is (4+m-2n) and the degree
of reduction is (4+m-2n)/1.
Activity 4
The aerobic degradation of hexadecane by a mixed culture of microorganisms can be

represented by the following reaction:
𝐶 𝐻 𝑎𝑂 𝑏𝑁𝐻 → 𝑐𝐶𝐻 .. 𝑂 . 𝑁 . 𝑑𝐻 𝑂 𝑒𝐶𝑂
(a) Determine aO2, bNH3, 𝑐𝐶𝐻 . 𝑂 . 𝑁 . , dH2O, and eCO2, if the respiratory quotient RQ =
0.43.
(b) Calculate the yield coefficients Y X/S (g dw cell/g substrate), Y X/O2 (g dw cell/g O2).
(c) Determine degree of reduction for the substrate and bacteria.
4 Metabolic pathways and bioenergetics
Metabolic pathways may have an effect on not only the type of products that are formed from
a bioreaction, but also the yield of the product. For example, if Saccharomyces cerevisiae,
also called baker’s yeast, is grown under aerobic conditions, the main product would be
baker’s yeast. If it is grown under anaerobic conditions, the major product would be ethanol.
This indicates that a change in the metabolic pathway is influenced by the environment in
which a bioreaction occurs. Another factor that can influence the change in the metabolic
pathway is concentration of the feed. When baker’s yeast is grown in aerobic conditions at
high glucose concentrations, some ethanol formation is observed. It indicates metabolic
regulation not only by oxygen, but also by glucose. This effect is known as the Crabtree effect.
4.1 Bioenergetics
The energy required for cell growth and product formation is called bioenergetics. In addition,
the metabolic pathway influences the energy required for cell growth and product formation.
Metabolic reactions are divided into three categories and they occur simultaneously. They are
the degradation of nutrients, the biosynthesis of small molecules (eg amino acids) and the
polymerisation of small molecules into large ones (eg amino acids into proteins). This is well
illustrated in figure 5.1 in the textbook.
The energy required to carry out these metabolic reactions are in the form of adenosine
triphosphate (ATP). Energy in biological systems is primarily stored and transferred via ATP.
Biological energy is stored in ATP by reversing this reaction to form ATP from ADP and Pi.
Similarly, ADP dissociates to release energy:
ATP H O ↔ ADP P
𝐴DP H O ↔ AMP P
Nicotinamide adenine dinucleotide phosphate (NADPH) supplies hydrogen in a biosynthetic

reaction. Hydrogen atoms released in biological oxidation-reduction reactions are carried by
nucleotide derivatives, especially by nicotinamide adenine dinucleotide (NADH) and
nicotinamide adenine dinucleotide phosphate (NADPH).
Activity 5
The macromolecular composition of a cell is given below. The ATP requirement for the
biosynthesis of these macromolecules is also provided based on the energy requirements of
the biosynthetic pathways. If the bacterium has a generation time of 30 minutes, determine
the specific rate of ATP and the efficiency of ATP utilisation based on a value of 10.5 g cells/
(mole ATP generated).
Macromolecule ATP requirement (mole ATP/ g

macromolecule) x 104
DNA 330
RNA 373.2
Protein 391.1
Lipid 123.6
Polysaccharides 114.6
Cells can be cultivated in either a batch or a continuous mode. The choice of cell cultivation
in either a batch or a continuous bioreactor can have an impact on productivity either on the
cell growth or on product formation. Cell growth is evaluated by measuring the cell
concentration directly or indirectly. Direct measurement is preferred because it is simple and
cost effective, but the indirect method is often needed when direct measurement is difficult to
achieve.
Cell growth phases under batch conditions includes the lag phase, the exponential phase, the
deceleration phase, the stationary phase and the death phase.
Continuous cell growth is usually evaluated in a continuously stirred tank reactor which is also
be called a chemostat. The net growth rate is equal to the dilution rate which is determined by
the flow rate to the chemostat.
The kinetics of cell growth can be evaluated or predicted by using structured or unstructured
growth models. An unstructured model assumes a fixed cell composition, which assumes
balanced growth, whereas the structured model is based on unbalanced growth.
Apart from its kinetics, cell growth and product yield can also be influenced by stoichiometry
(thermodynamic limits). Stoichiometry calculations are carried out by using the elemental
balance or the degree of reduction or both.
Adenosine triphosphate (ATP) is the key compound used to store and release energy in a
metabolic pathway. Nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine
dinucleotide phosphate (NADPH) are energy carriers and supply hydrogen in a biosynthetic
reaction.
1. Illustrate by means of a diagram the cultivation of the yeast Candida utilis from the petri
dishes to the bioreactor.
2. Describe ways of estimating the concentration of Candida utilis by using either the
direct or the indirect methods of cell measurement for each.
3. State the ATP-consuming and ATP-generating steps in the pathway of glucose
metabolism.
4. Answer questions 6.1 to 6.20 in the textbook.
5. Answer questions 7.1 to 7.7 in the text book.
Learning Unit 5: Genetic Engineering
 Study material – Chapter 8 of the prescribed textbook Bioprocess Engineering:

Basic Concepts by Shuler and Kargi
1. Introduction
In figure 1 in learning unit one, we showed the interdisciplinary nature of biochemical

engineering (chemical engineering, biochemistry, microbiology and molecular genetics). In
this learning unit, we will learn about molecular genetics, also called genetic engineering.
Genetic engineering involves the manipulation of the genetic information of cells for an
enhanced bioprocess, and this can occur through cell mutation. Mutation occurs when there
is an error in the transfer of genetic information, and the cells are called mutants.
Environmental factors such as changes in UV light and exposure to certain chemicals, can
lead to cell mutation. A selectable mutant is a mutant that is desired and advantageous for
better cell growth under certain environmental conditions, and for product formation. The
selection of a desirable mutant is essential in genetic engineering and an understanding of the
mechanism of genetic engineering is needed to achieve it. The different mechanisms for gene
rearrangement include genetic combination, transformation, transduction, conjugation, and so
on. Different techniques of genetic engineering include gene synthesis and polymerase chain
reaction (PCR). There are different applications of genetic engineering and some of them will
be discussed in this learning unit.
 Describe cell mutations. Explain how they occur and how a desirable mutant is
selected.
 Describe the different mechanisms of gene transfer and rearrangement.
 Describe the different techniques of genetic engineering and the applications of
genetic engineering in some biopharmaceutical products.
Further reading
https://www.youtube.com/watch?v=3IsQ92KiBwM
2. Cell mutation and the selection of mutants
Cell mutation occurs when there is an error in the transfer of genetic information. Such cells
are called mutants. The transfer of genetic information in cells is usually carried out through
the reverse transcription of RNA to DNA and the replication of the DNA. The genetic content
of cells (DNA and RNA) is called their genotype. Phenotype is defined as all the observable
characteristics of a cell/organism that result from the interaction of its genotype. The
phenotypic response of a cell culture may change reversibly because of changes in
environmental conditions, whereas the genotype remains constant irrespective of the
environment. A mutation is a genotypic change and is irreversible.
A whole organism or culture can undergo a phenotypic response, whereas only a rare
individual will undergo a genotypic change. For example, a change in the environment can
make some flamingos change their color from white to pink. This is due to the type of food
they eat which changes their phenotype. See figures 1a and 1b.
Figures 1a and 1b
Source: https://evolution.berkeley.edu/evolibrary/article/genovspheno_01
On the other hand, in house cats the genes for ear shapes differ and causes some cats to
have normal ears and others to have curled ears. The genes that produce curved ears are
called mutants and the genes that produce normal ears are called the wild type.
For example, if the colour of a culture changes from white to green when oxygen levels drop,
and from green to white when dissolved oxygen levels rise, the change would be phenotypic.
Now consider an alternative experiment where white cells were removed from a culture and
placed on a plate (a small circular dish filled with nutrients solidified with agar) and allowed to
grow into separate colonies. If only one colony turns green and if the cells obtained from the
green colony remain green when cultured under the original conditions, it would be evidence
of a genotypic change. In this case, the white cells would be the wild type and the green cells
the mutants.
Point mutation is a common form of mutation. It involves a change in a single base (transitions)
of DNA of a wild gene and the insertion of a single base DNA as a result of mutation of the
gene and, lastly, the deletion of a single base DNA. This is well illustrated in figure 8.1 of
the textbook.
2.1 Selection of desirable mutants
The selection of desirable mutants is beneficial because it provides an organism with

characteristics that increase its survival under specific environmental conditions. As a result,
the mutants survive but the wild type does not.
Mutations can be classified as selectable or unselectable. A selectable mutation provides an

organism with characteristics that increase its survival under specific environmental conditions
so that the mutants can survive but the wild type cannot. An example is the survival of
antibiotic-resistant bacteria in the presence of antibiotics. Wild-type bacteria will be killed by
the antibiotic.
An unselectable mutant does not have the survival advantage that a selectable mutant has. It
is difficult to distinguish between a desirable and an unselectable mutant. It would require a
cell-by-cell examination.
Selection can be direct or indirect. Direct selection is used to isolate mutants that have rich
growth factors. If a mutant resistant to an antibiotic compound needs to be selected directly
from a culture, only antibiotic-resistant mutant cells will grow in the growth medium.
Indirect selection is used for the isolation of mutants that are deficient in their capacity to
produce a necessary growth factor. Mutants would not directly grow on such a simple medium
unless it is supplemented with other growth factors. This is illustrated in figure 8.3 in the
textbook.
3.1 Mechanism of gene transfer and rearrangement
Cells are transformed by receiving genes from other organisms in their environment. For
example, some organisms are able to degrade an antibiotic or detoxify a hazardous chemical
in their environment. These alterations cannot be explained by inheritance and small
evolutionary changes in their chromosomes, but arise from a gene transfer from one organism
to another and/or large rearrangements in their chromosomal DNA. In this section, we will
discuss genetic recombination or gene transfer and genetic rearrangement.
3.1 .1 Genetic recombination
Genetic recombination, also called gene reshuffling, is when genetic elements of two different
genomes are transferred into one unit, resulting in new genotypes in the absence of mutations.
The mechanisms for gene recombination are transformation, transduction and conjugation.
3.1.1.1 Transformation
Transformation is a process in which free DNA is taken up by a cell. This is illustrated in

figure 8.4 of the textbook. Transformation is only useful when the information that enters the
cell can be propagated. For transformation, a vector called a plasmid is used. A plasmid is an
autonomous, self-replicating, double-stranded piece of DNA. Plasmids encode genes for
proteins that are non-essential for growth but confer important advantages to their host cells
under certain environmental circumstances. For example, most plasmids encode proteins that
confer resistance to specific antibiotics. Antibiotic resistance is very helpful in selecting cells
that contain a desired plasmid.
3.1.1.2 Transduction
Transduction is a process in which DNA is transferred by a bacteriophage. The most common

type of transduction is called generalised transduction. The infection of a recipient cell results
in fragmentation of the bacterial DNA into a 100 or more pieces. One of these fragments can
be packaged accidentally into a phage particle during formation. This is illustrated in figure
8.5 in the textbook.
Another method of transfer is called specialised transduction. This type of transduction is more
specific with respect to the genes that are transferred. The phage incorporates into specific
sites in the chromosome and the frequency of the transduction of a gene is related to its
distance away from the site of incorporation.
3.1.1.3 Conjugation
Conjugation is similar to sexual reproduction because two cells are involved in the gene
transfer. DNA is transferred between intact cells that are in direct contact with one another.
The other genetic element is called an episome. It is a DNA molecule that is integrated in the
chromosome or exists separately from it. A well-known episome is the F or fertility factor. Such
factors are responsible for the process known as conjugation.
Conjugation, transduction and transformation represent forms of gene transfer from one cell
to another. However, a gene transfer occurs only inside a cell. An example of internal gene
transfer is transposon.
3.1.1.4 Transposon
Transposon involves the tendency of a set of genes to jump from one DNA to another. It is a
powerful tool to alter cellular properties for the following reasons:
 It can induce mutations when inserted in the middle of a gene.
 It can bring separate genes together.
 In combination with plasmid- or viral-mediated gene transfer, it can mediate the
movement of genes between unrelated bacteria (eg multiple antibiotic resistance on
newly formed plasmids).
Activity 1 Develop a model of the rate DNA transcription and translation in a plasmid-encoded
protein.
4. Techniques of genetic engineering
Genetic engineering is also known as recombinant DNA technology. Genetic recombination

is the process of combining genetic elements from two different genomes to form a new
genotype in the absence of mutations.
4.1 Gene synthesis
Gene synthesis involves the joining of nucleotides to a single-stranded DNA molecule in the
right order. Unlike other genetic engineering techniques such as polymerase chain reaction
(PCR), it does not require a template strand for the synthesis.
4.2 Polymerase chain reaction (PCR)
PCR is the preferred method to amplify DNA. Its inventor, Karry Mullis, won a Nobel prize in
1983 for this technique.
According to it, a target sequence of interest is a gene on double-stranded DNA. The original
DNA is heated and complementary strands of double-stranded DNA form because of heat
separation. After separation, two pieces of chemically synthesized DNA (the primers) are
added. Each primer binds to complementary sequences. A heat-stable DNA polymerase from
a bacterium that grows in hot springs (the Taq polymerase) is added and quickly synthesizes
from the primers the complementary DNA strands by using the four nucleotides (A, G, T and
C) which are added to the reaction mixture. This is illustrated in figure 1.
The cycle is repeated from step 2, and the number of DNA molecules doubles each time. It is
called a chain reaction since the number of molecules increases exponentially, like an
explosive chain reaction. Typically, 20 to 30 cycles can be done in less than a day. In one 30-
cycle period, the number of DNA strands amounts to 1 073 741 824.
Figure 1: Procedure of DNA amplification using PCR
(Source: Liu, S. 2017. Bioprocess Engineering, Kinetics, Sustainability and Reactor Design.
Amsterdam: Elsevier. page 801)
5. Applications of genetic engineering
The application of genetic engineering can be grouped into gene products, new phenotypes
and gene therapy. Gene products are divided into protein and non-protein products. The
majority of gene products are protein products made of genetically modified organisms. They
include biopharmaceutical products such as insulin, vaccines and penicillin.
As to new phenotypes, gene technology is applied to modify the characteristics of organisms

in the production of genetically modified crops and animal breeding. This is an extremely
sensitive issue because of public protest. With regard to gene therapy, gene technology is
used to treat human diseases.
Cell mutation occurs when there is an error in the transfer of genetic information. The cells are
called mutants. Examples of mutations are point mutations and deletions. Mutations are either
selectable or unselectable. A selectable mutation provides an organism with characteristics
that increase its survival under specific environmental conditions. An unselectable mutant
does not have the survival advantage of a selectable mutant.
Besides mutation, genes can be transferred from one cell to another through genetic
recombination. The genetic information transferred from another organism may become a
permanent part of the recipient cell. The mechanisms for gene recombination are
transformation, transduction and conjugation.
There are different genetic engineering techniques such as gene synthesis and PCR. PCR is
the preferred method. These techniques are used to produce gene products such as insulin,
penicillium and renin.
1. Would a cell with a point mutation or a deletion mutation be more likely to revert back to
the original phenotype? Why?
2. What is the difference between “transduction” and “transformation” in the context of gene
transfer to bacteria?
3. There are two methods for obtaining donor DNA when performing genetic engineering.
Briefly describe each.
4. Describe how you would engineer E. coli by using PCR to produce a protein.
5. In a table format, list five gene products, their applications and the genetic organism used
in the production of gene products.
Further reading
https://www.khanacademy.org/science/high-school-biology/hs-molecular-genetics/hs-
biotechnology/v/introduction-to-genetic-engineering
References
Blanch, HW & Clark. DW. 1997. Biochemical Engineering. 1st edition. New York: Marcel
Dekker.
Doran, PM. 1994. Biochemical Engineering Principles. 1st edition. London: Elsevier.
Shuler, ML & Kargi, F. 2002. Biochemical Engineering: Basic Concepts. 2nd edition. New
Jersey, USA: Prentice Hall.
Learning Unit 6: Bioreactor Design and Operation

1. Introduction
In this learning unit, we will learn how to design, operate and scale up a bioreactor. There are
different configurations of bioreactor designs. The selection of a bioreactor configuration is
determined by the type of culture (microbial, animal or plant culture) and a low or high rate
shear rate. For example, a bubble bioreactor would be ideal for an animal culture because the
degree of shear is low.
Heat and mass transfer are two key requirements in the design and operation of a bioreactor.
Mass transfer involves the transport of gaseous nutrients such as oxygen, in bioreactors for
cell growth and product formation. High mass transfer rates are needed for high cell growth
and product formation. The mass transfer coefficient (KLa) is estimated through empirical
correlations or determined experimentally. Once the KLa is established, it can be used to
estimate the rate of metabolic heat generation and the total extent of the cooling surface
required.
Heat transfer involves maintaining the temperature when operating a bioreactor. Because
cells generate heat during microbial growth in bioreactors, heat exchangers maintain a
temperature of 1 oC. Heat transfer is also essential in the sterilisation of growth media prior to
the actual bioreaction process to prevent the growth of undesired microorganisms and viruses
due to possible contamination.
Scaling up is a key step in the design of an industrial scale bioreactor. A bioreactor is scaled
up to have a constant height-to-diameter ratio and to keep some design variables constant
such as power-to-volume ratios, KLa, the tip speed and the Reynolds numbers.
 Describe the different types of bioreactors and select the appropriate bioreactor
configuration for a bioprocess.
 Describe how the oxygen supply and temperature control limitations affect the design
and operation of a bioreactor.
 Calculate the oxygen consumption rate through the experimental and empirical
procedure.
 Calculate the power requirement for stirred-tank reactors.
 Describe heat transfer in sterilisers, stirred-tank reactors and bubble column reactors.
 Determine the heat transfer coefficients in stirred-tank reactors and bubble reactors.
 Calculate scale-up parameters for different biological systems.
Further reading on the introduction of a bioreactor can be found in the YouTube link
https://www.youtube.com/watch?v=i7YS5tbHPUk.
2. Bioreactor operation and selection

A bioreactor is at the core of any bioprocess. The function of a bioreactor is to provide a
controlled environment for optimal microbial growth and product formation. Bioreactors are
widely used in the food and pharmaceutical industries, and in wastewater treatment and
bioenergy plants.
As indicated in learning unit 4, there are two types of bioreactors, namely batch and continuous
reactors. A fed-batch reactor is a mode between a batch and a continuous reactor. The mode
of selection of each of these reactors, based on their performance with regards to substrate,
products, by-products, flexibility, and so forth, is presented in table 1.
Table 1: Characteristic of batch, fed-batch and continuous bioreactors
Operational Batch bioreactor Continuous Fed-batch

Mode bioreactor bioreactor
Product Product concentration Ability to The intermittent
increases with promote the desired addition of the
residence product selectivity by substrate improves
time in the reactor controlling the productivity by
substrate concentration maintaining the
substrate
concentration
By-product There is reduced Reduced opportunity
opportunity for product for side product
degradation in the formation with a
desired product controlled substrate
concentration
Substrate Highest concentration Lowest concentration in Start-up period like
in the reactor, the reactor; constant batch, and the bulk
concentration and controllable of the operation
decreases with concentration in the could mimic a
residence time inside reactor continuous reactor
the reactor
Flexibility Distinctive batches, High throughput, easy Distinctive batches,
quality ensured at each to control, consistent Moderate
batch level, easy to quality, difficult to throughput, quality
change operations change operations controllable at each
batch, easy to
change operations
Applications Small-quantity Large-quantity Process requiring
production, high-value production; high strict product quality
substrate/product, demand; consistent control while flexible
multiple products/ substrate supply and operation is
substrates/processes product quality required or catalyst
on demand; catalyst demand; stable catalyst unstable with long
instability (mutation) or controllable through duration of
feed operations
Continuous bioreactors can be divided into two types, namely well-mixed continuous reactors
and plug flow reactors. There are three configurations for well-mixed continuous reactors,
namely stirred-tank reactors, bubble column reactors and airlift loop reactors. This is
illustrated in figure 10.1 in the textbook.
Stirred-tank bioreactors are characterised by high KLa (volumetric mass transfer coefficient)
values for gas transfer. Stirred-tank reactors use impellers and baffles to induce effective
mixing. Gas dispersion is mainly a function of the impeller. The impeller provides adequate
agitation to disperse the gas bubbles in the bioreactor and increase their residence time. The
desire for rapid gas dispersion must, however, be balanced with the impact on the culture.
High-shear impellers are generally utilised on microbial systems, where high gas transfer rates
and cellular growth require rapid dispersion and mass transfer through the vessel. To augment
mixing and gas dispersion, baffles are used to increase the disruption in the vessel and
minimise vortex. Usually, four baffles which are 8 to 10% of the reactor diameter, are used.
As to animal cell cultures, baffles are not used due to shear concerns.
Bubble column bioreactors operate in low-shear environments where animal cultures are
grown. These columns are suitable for low-viscosity Newtonian broths. They have higher
energy efficiencies than agitated vessels in terms of the amount of gas transfer per unit power.
However, bubble column reactors may be limited by foaming and bubble coalescence and are
generally less flexible than stirred-tank bioreactors. The multistage arrangement (separated
by structural packing) alleviates the bubble coalescence problem and increases the mass
transfer efficiency.
Airlift loop bioreactors (ALRs) have intermediate characteristics between bubble columns and
stirred-tank bioreactors. The fast, up-flowing air in the centre draft tube carries the medium
and cells upward. At the top, bubbles break out of the medium and drop cells and liquid
medium. Cells and liquid medium flow down from the annulus on the side, and are then carried
upwards from the centre region (draft tube). Airlift loop reactors can handle more viscous fluids
than bubble columns and are applied where bubble coalescence is generally not a problem.
The key types or configurations of ALR bioreactors include internal-loop split ALR bioreactors,
internal-loop concentric ALR bioreactors and external-loop ALR bioreactors.
As to plug flow reactors, the different configurations are tubular, membrane and packed-bed
reactors. These types of bioreactors are mostly used in immobilised enzyme reactions such
as the isomerisation of glucose to fructose in packed-bed immobilised-enzyme bioreactors.
3.1 Mass transfer
In the design of bioreactors, the oxygen transfer rate (OTR) must be greater than or equal to
the oxygen utilisation rate (OUR) to prevent oxygen limitation and possible cell death.
Oxygen transfer to cells occurs in the gas-liquid interface and oxygen utilisation occurs in the
liquid-cell interface. There are eight mass-transfer steps involved in the transport of oxygen
between the gas phase and the cell. This is illustrated in figure 1 below.
Figure 1: Steps of oxygen transfer to cell
(Source: Doran, PM. 1994. Biochemical Engineering Principles. 1st edition. Elsevier.)
The eight steps of resistance to the mass transfer of oxygen to cells are as follows:
(1) Resistance through the bulk-gas phase in the bubble, which is negligible.
(2) Resistance at the gas-liquid interface, which is negligible.
(3) Resistance in the liquid film surrounding the gas interface, which is significant.
(4) Resistance in the liquid phase, which is negligible in a well-mixed bioreactor.
(5) Resistance in liquid film surrounding the cell, which is significant.
(6) Resistance in the liquid-cell interface, which is negligible.
(7) Resistance is significant especially when the cells are in clumps. The magnitude of the
resistance depends on the size of the cell clumps.
(8) Resistance within the cell, which is significant.
The oxygen concentration decreases in each of the eight steps as illustrated in figure 2.
Figure :2 Oxygen transfer and oxygen concentration
The resistances occur in series and the largest of them is the rate-limiting step, which is
usually resistance number 3. The mass transfer rate of this step is given below.
𝑂𝑇𝑅 𝐾 𝑎 𝐶∗ 𝐶
Equation 1
Where C* is the oxygen solubility, CL the actual dissolved oxygen, and KLa the volumetric
mass transfer coefficient
At steady state, the rate of oxygen transfer from the bubbles must be equal to the oxygen
utilisation rate (OUR) by the cells, and equation 1 becomes:
𝑂𝑇𝑅 𝐾 𝑎 𝐶∗ 𝐶 𝑂𝑈𝑅
Equation 2
The oxygen utilisation rate can also be rewritten

𝑂𝑈𝑅 𝑋𝑞
Equation 3
Where 𝑞 is a specific uptake rate of oxygen and X is the biomass concentration
The maximum concentration of biomass when CL is zero.
𝐾 𝑎𝐶 ∗
𝑋
𝑞
Equation 4
Additional reading
https://www.gelifesciences.com/en/nz/solutions/bioprocessing/knowledge-center/7-
factors-that-affect-oxygen-transfer-to-cells-in-bioreactors
Activity 1
A strain of Azotobacter vinelandii is cultured in a 15 m3 stirred-tank bioreactor for alginate

production. Under current operating conditions KLa is 0.17 s- 1. Oxygen solubility in the broth
is approximately 8 x 10-3 kg m-3. when the specific rate of oxygen uptake is 12.5 mmol g-1 h-1.
Calculate the maximum possible cell concentration.
3.2 Estimation of mass transfer coefficient KLa
The value of the mass transfer coefficient can be estimated from empirical correlations or
determined by measuring the dissolved-oxygen concentration.
3.2.1 Empirical estimation of KLa
There are various correlations for estimation, some of which are presented below.
. . .
𝐾𝑎 𝑘 𝑃 /𝑉 𝑣 𝑁
Equation 5
Where k is an empirical constant, Pg the power requirement in an aerated (gassed)

bioreactor, VR the bioreactor volume, vs the superficial gas exit speed, and N the rotational
speed of the agitator.
A value of Pg can be estimated from the correlation below.
.
𝑃 𝑁𝐷
𝑃 𝐾
𝑄 .
Equation 6
Where K is a constant based on reactor geometry, Pu is the power required in the un-gassed
fermenter, Di is the impeller diameter, and Q is the aeration rate (volume of gas supplied per
minute divided by the liquid volume in the reactor).
The above correlations provide a modest estimation of the parameters for Newtonian systems
and a very poor estimate for non-Newtonian or highly viscous systems. They also neglect the
effects of medium components on KLa. The presence of salts and surfactants can significantly
affect KLa.
Van’t Riet (1979) developed correlations for coalescing clean (free from surfactants) media
and non-coalescing dirty (with surfactants) media based on extensive data from the literature.
They are presented in equations 7 and 8.
For coalescing clean media

. .
𝐾𝑎 2.6 𝑥 10 𝑃/𝑉 𝑣
Equation 7
For non-coalescing dirty media

. .
𝐾𝑎 2.0𝑥 10 𝑃/𝑉 𝑣
Equation 8
3.2.2 Experimental methods
There are four main experimental methods for the measurement of KLa: the unsteady-state,
the steady-state, the dynamic and the sulphite test methods.
3.2.2.1 Unsteady-state method
According to this method, air is introduced and the change in dissolved oxygen (DO) is
monitored until the solution is nearly saturated. The KLa value is evaluated from a plot of log
(C* -𝐶 ) versus time.
𝑑𝐶
𝐾 𝑎 𝐶∗ 𝐶
𝑑𝑡
Equation 9
The integration of the above equation would result in
𝑙𝑛 𝐶 ∗ 𝐶 𝐾 𝑎𝑡
Equation 10
3.2.2.2 Steady-state method
The best way to determine KLa is the steady-state method. According to it a whole reactor is
used as a respirometer. A respirometer is used to measure the rate of respiration by
microorganisms in the form of their oxygen consumption. This approach requires the accurate
measurement of oxygen concentration in all gas exit streams and a reliable measurement
of 𝐶 . A mass balance on O2 in the gas allows the rate of O2 uptake, OUR, to be calculated:
𝑂𝑈𝑅
𝐾𝑎
𝐶∗ 𝐶
Equation 11
3.2.2.3 Dynamic method
This method is similar to the steady-state method but is simpler in its measurement. It uses a
bioreactor with active cells. It requires only a dissolved-oxygen (DO) probe and a chart
recorder instead of off-gas analysers as well as the DO probe required in the steady-state
method. The governing equation for DO levels is
𝑑𝐶
𝑂𝑇𝑅 𝑂𝑈𝑅
𝑑𝑡
Equation 12
3.2.2.4 Sulphite method
This method is conducted in the presence of Cu2+, where sulphite (SO2-3 is oxidized to sulphate
(SO2-4) in a zero-order reaction. The reaction is very rapid; consequently, 𝐶 approaches zero.
The rate of sulphate formation is monitored and is proportional to the O2 consumption. The
governing equation is presented below:
1 𝑑𝐶
𝐾 𝑎 𝐶∗ 0
2 𝑑𝑡
Equation 13
Making KLa the subject of the formula.
1 𝑑𝐶
𝐾𝑎
2 𝑑𝑡
Equation 14
Where CSO4 is the concentration of SO2-4. The factor, ½, requires that CSO4 and C* be
expressed in terms of moles. Oxygen solubility, C*, is a constant dependent on medium
composition, temperature and pressure, and can be measured separately.
Activity 2
A 20 L stirred fermenter containing a Bacillus thuringiensis culture at 30 oC is used for the

production of a microbial insecticide. KLa is determined by using the dynamic method. The air
flow is shut off for a few minutes and the dissolved-oxygen level drops; the air supply is then
re-connected. When steady state is established, the dissolved-oxygen tension is 78% air
saturation. Estimate the KLa from the following results.
Time 5 15
Oxygen Tension (% Air 50 66
saturation)
4.1 Heat transfer
Heat transfer occurs during the sterilisation of the growth media prior to the bioreaction. In the
bioreactor, heat transfer occurs during the cell metabolism. Because heat is generated, there
is a need for temperature control in the bioreactor. Cooling is achieved by circulating water in
a jacketed bioreactor.
4.2 Sterilisation of media
Heating is one of the methods used in the sterilisation of growth media. The liquid growth
medium is normally sterilised in batch mode. The liquid is heated to sterilisation temperature
by introducing steam into the coils. There are three stages in the batch sterilisation cycle:
heating, holding and cooling.
The rate of sterilisation is presented in the governing equation:
𝑑𝑁
𝐾 𝑁
𝑑𝑡
Equation 15
Where N is number of viable cells, t is time and Kd is the specific death constant.
/
𝐾 𝐴𝑒
Equation 16
Where A is the Arrhenius constant or frequency factor, Ed is the activation energy for the
thermal cell death, R is the ideal gas constant, and T is the absolute temperature.
The number of viable cells is determined by considering the extent of cell death during the
heating and cooling periods when the temperature is constant
𝑑𝑁 /
𝐴𝑒 𝑁
𝑑𝑡
Equation 17
The integration of equation 17
For the heating period

𝑁 /
𝑙𝑛 𝐴𝑒
𝑁
Equation 18
For the cooling period

𝑁 /
𝑙𝑛 𝐴𝑒
𝑁
Equation 19
Where t1 is the time at the end of heating, t2 is the time at the end of holding, and tf is the
time at the end of cooling.
It is difficult to complete the integration of these equations until we know how the temperature
varies with time during heating and cooling. An unsteady-state temperature profile during
heating and cooling can be determined from the heat transfer properties of the system.
For the heating period
𝑇 𝑇
𝑇 𝑇 1 𝑒
𝑇
Equation 20
For the cooling period

𝑇 𝑇
𝑇 𝑇 1 𝑒
𝑇
Equation 21
Where
T is temperature
To is the initial medium temperature
Tci is the inlet temperature of cooling water
Ts is the steam temperature
t is time
A is the heat transfer area
Cp is the specific heat capacity of the medium
Cpw is the specific heat capacity of cooling water
H is the enthalpy of steam relative to that of the medium
Mm is the mass of the medium
Mw is the mass flow rate of cooling water
Activity 3
A medium at a flow rate of 2 m3 h-1 is to be sterilised by heat exchange with steam in a steriliser.
The liquid contains bacterial spores at a concentration of 5 x 1012 m-3; the activation energy
and Arrhenius constant for the thermal destruction of these contaminants are 283 kJ gmol-1
and 5.7 x 1039 h-1 respectively. A contamination risk of one organism surviving every 60 days
of operation is considered acceptable. The steriliser pipe has an inner diameter of 0.1 m and
the length of the holding section is 24 m. The density of the medium is 1 000 kg m-3 and the
viscosity is 3.6 kg m-1 h-1. What sterilising temperature is required?
4.3 Heat transfer in bioreactors
The heat exchangers used in bioreactors can be of different configurations. They include
jacketed vessels, external coils, internal helical coils, internal baffle type coils and external
heat exchangers. They are illustrated in the figure 3 below.
Figure 3: Different heat exchanger configurations in bioreactors
(Source: Doran, PM. 1994. Biochemical Engineering Principles. 1st edition. Elsevier.)
4.3.1 Design equation for heat transfer of bioreactors
The rate of heat transfer can be determined by the energy balance in a heat exchanger
bioreactor system.
𝑄 𝑀 𝐶 𝑇 𝑇 𝑀𝐶 𝑇 𝑇
Equation 22
Where Q is the rate of heat transfer, MH is the mass flow rate of the hot fluid, Mc is the mass
flow rate of the cold fluid, Cph is the heat capacity of the hot fluid, Cpc is the heat capacity of
the cold fluid, Thi is the inlet temperature of the hot fluid, Tho is the outlet temperature of the
hot fluid, Tci is the inlet temperature of the cold fluid, and Tco is the outlet temperature of the
cold fluid.
The rate of heat transfer can also be related to the overall heat transfer coefficient and the
heat transfer. It is represented in the equation below:
𝑄 𝑈 𝐴∆𝑇 𝑈𝐴 𝑇 𝑇
Equation 23
Where U is the overall heat transfer coefficient, A is the heat transfer area, Th is temperature
of the hot fluid, and Tc is the temperature of cold fluid.
The overall transfer coefficient U is represented in equation 24:

1 1 ∆𝑟 1
𝑈 ℎ 𝐴 𝑘 𝐴 ℎ 𝐴
Equation 24
Where the first term is the heat transfer coefficient of the hot fluid is hh and Ah is the heat
transfer area of the hot-fluid boundary layer inside the tube or jacket wall. The second term is
the heat resistance through the tube or the jacket wall with a thickness of ∆r and thermal
conductivity of the tube or the jacket wall kw and the heat transfer area of the tube or the jacket
wall. The third term is the heat transfer coefficient of the cold fluid hc and Ac is the heat transfer
area of the cold-fluid boundary layer outside the tube or jacket wall.
The heat transfer coefficient of the hot fluid and the cold fluid can be represented in
ℎ𝐷 𝐷𝑁𝜌 𝐶 µ µ
𝑎
𝑘 µ 𝑘 µ
Equation 25
Where 𝑖𝑠 the Nusselt number and it is related to the Reynolds number and the
µ
µ
Prandtl number and the viscosity ratio of the bulk fluid and the jacket wall
µ
Activity 4
A single-pass shell-and-tube heat exchanger is used to heat a diluted salt solution used in
large-scale protein chromatography. The 25 m 3 h-1 solution passes through 42 parallel tubes
inside the heat exchanger. The internal diameter of the tubes is 1.5 cm and the tube length is
4 m. The viscosity of the bulk salt solution is 10-3 kg m-1 s- 1, the density is 1010 kg m-3, the
average heat capacity is 4 kJ kg-1 C - 1 and the thermal conductivity is 0.64 W m-1 o C-1.
Calculate the heat transfer coefficient.
5. Scaling up of bioreactors
Scaling up is a key step in the design of an industrial-scale bioreactor. In the scale of a

bioreactor, the aim is to have a constant height-to-diameter ratio while also keeping some
design variables constant such as the power-to-volume ratios, KLa, tip speed (ND), and the
Reynolds numbers. For example, an 80 L stirred-tank bioreactor is to be scaled up to 10 000
L. The volume is increased 125-fold and the diameter 5-fold, but the height-to-diameter ratio
is kept constant. The effect of the scale-up on KLa, tip speed, Reynolds numbers and other
variables is illustrated in table 1.
Table 2: Scale-up value from 80 L to 10 000 L
Scale-up Criterion Designation Small Scaled-up Bioreactor 10 000L

bioreactor Po/V N ND Reynolds
80 L number
Energy Po = N3D5 1 125 3125 25 0.2
Energy input / Po/V=N3D2 1 1 25 0.2 0.0016
Volume
Impeller rotation N 1 0.34 1 0.2 0.04
number
Impeller diameter D 1 5 5 5 5
Pump rate of Q=ND3 1 42.5 125 25 5
impeller
Shearing rate ND 1 1.7 5 1 0.2
Reynolds number (N D2𝛒)/µ 1 8.5 25 5 1
Activity 5
Produce a table similar to table 2 above for a 75 L stirred-tank bioreactor which is to be

scaled up to 10 000 L.
There are two main types of bioreactors, namely batch and continuous reactors. A fed-batch
reactor is a mode between a batch and a continuous reactor.
Continuous bioreactors can be grouped into well-mixed continuous reactors and plug-flow
reactors. There are different configurations of the well-mixed continuous reactor, namely
stirred-tank reactors, bubble column reactors, airlift loop reactors, and propeller loop reactors.
In terms of plug flow reactors, the different configurations are tubular, membrane and packed
bed reactors.
The selection of a bioreactor configuration is determined by the type of culture (microbial,

animal or plant culture) and the shear rate (a low or high rate). For example, a bubble
bioreactor would be better suited to an animal culture because it is sensitive to high shear.
Heat and mass transfer are two key requirements in the design and operation of a bioreactor.
Mass transfer is involved in the transport of gaseous nutrients such as oxygen in bioreactors
for cell growth and product formation. A high mass transfer rate is needed for high cell growth
and product formation. The mass transfer coefficient (KLa) is estimated through empirical
correlations or determined experimentally. Once the KLa is established, it can be used to
estimate the rate of metabolic heat generation and the total amount of cooling surface
required.
Heat transfer is a good tool to maintain temperature when operating a bioreactor. Cells
generate heat during microbial growth in bioreactors. Heat exchangers are used to maintain
the temperature at 1 oC. Heat transfer is also essential in the sterilisation of growth media prior
to the actual bioreaction process. The reason is to prevent the growth of undesired
microorganisms and viruses owing to possible contamination.
Scaling up is a key step in the design of an industrial-scale bioreactor. In the scale of a

bioreactor, the aim is to have a constant height-to-diameter ratio while keeping some design
variables constant such as power-to-volume ratios, KLa, tip speed (ND), and the Reynolds
numbers.
1. List the different types of continuous bioreactor and do a comparative analysis of

them.
2. Compare the different methods used to measure KLa.
3. List the different stages in batch sterilisation and state the ones that are relevant in
the determination of the number of viable cells after sterilisation.
4. Answer questions 10.1 to 10.11 in the recommended textbook.
References
Dekker.
Further reading:
https://www.youtube.com/watch?v=B7LfT7BIYSQ – Types of bioreactors
https://www.youtube.com/watch?v=fQOzHC828aM – Types of bioreactors
https://www.youtube.com/watch?v=7SR6okJrPBI – Setting up of a bioreactor
https://www.youtube.com/watch?v=cFzxYNHwOPQ – Setting up of a stirred-tank bioreactor
https://www.youtube.com/watch?v=EVxPihbZDqA – How a stirred-tank bioreactor works

Learning Unit 7: Product Recovery and Purification

1. Introduction
The purification and recovery of desired bioproducts are essential for the successful
commercialisation of an industrial bioprocess. These purification tasks can be quite complex.
In this learning unit, we will learn how to separate and purify a bioproduct. Product recovery
accounts for the high production cost of a bioprocess. Sometimes it represents more than 50%
of the total production cost of a bioproduct. The high cost of product recovery emanates from
the low concentration of the fermentation solution and the intracellular nature of the product in
the cell. Typically, the steps for product purification and recovery include cell removal, primary
isolation, purification and final isolation. The key unit operations of the different steps include
cell removal (sedimentation, filtration and centrifugation), primary isolation (adsorption, liquid
extraction and precipitation), purification (chromatography, ultrafiltration and fractional
precipitation) and final isolation (crystallization and drying). Some of the design parameters of
the various unit operations will be discussed in detail.
 Describe the different types of unit operations used in the purification and recovery of
bioproducts.
 Select the appropriate unit operation for the purification and recovery of a bioproduct.
 Calculate the design parameters of selected unit operations used in the purification
and recovery of a bioproduct.
Further reading
Introduction video on product recovery and purification -

https://www.youtube.com/watch?v=LvG_o5XYVbo
2. Cell removal and separation of insoluble material
Cell removal and the separation of insoluble particles is the first step in the recovery and
purification of a bioproduct from a fermentation broth. These include whole cells, cell debris,
pellets of aggregated proteins and undissolved growth nutrients in the fermentation broth.
Sometimes, the fermentation broth needs to be pretreated by methods such as heat treatment,
pH and ionic strength adjustment, and the addition of coagulants and flocculants to improve
separation.
Further steps would depend on whether the desired bioproduct are cells, such as in the
production of a single-cell product or a specific metabolite in cells. If the product is the cell,
then the separation of solids is the most important step in product recovery. The separation of
solids results in a significant volume reduction. If the product is a soluble compound, solids
need to be separated from liquid before the liquid is further treated to recover and purify the
soluble product. For the recovery of an intracellular product, cells need to be disrupted and
other cellular products need to be separated from the desired product. The major methods for
the separation of cellular material (biomass) are filtration, centrifugation, sedimentation, and
coagulation and flocculation.
2.1 Filtration
Filtration is a cost-effective method for the separation of cells from the fermentation broth. The
fermentation broth is passed through a filter medium and a filter cake is formed because of
the deposition of solids on the filter surface. The most common filtration equipment used in
bioproduct recovery and purification is a rotary vacuum filter. It consists of a perforated
horizontal drum. Prior to filtration, the drum is covered with a filter aid such as diatomaceous
earth, to increase the rate of filtration.
The rate of filtration (the flow of filtrate) for a constant-pressure (vacuum) filtration operation is
determined primarily by the resistance of the cake and filter medium:
𝑑𝑉 𝑔 ∆𝑃𝐴
𝑑𝑡 𝑟 𝑟 µ
Equation 1
Where V is the volume of filtrate, A is the surface area of the filter, ∆P is the pressure drop
through the cake and filter medium, µ is the viscosity of the filtrate, rm is the resistance of the
filter medium, and rc is the resistance of the cake. The value of rm is characteristic of the filter
medium. However, the cake resistance, rc, increases during filtration, and after a start-up
period, rc exceeds rm. The value of rc is given by
𝑊 𝐶𝑉
𝑟 𝛼 𝛼
𝐴 𝐴
Equation 2
Where W is the total weight of the cake on the filter, C is the weight of the cake deposited per
unit volume of filtrate, and A is the average specific resistance of the cake.
The total weight of the cake is related to the total volume of the filtrate by
W= CV
Equation 3
The substitution of equations 2 and 3 into equation 1
𝑑 𝑉/𝐴 𝑔 ∆𝑃𝐴
𝑑𝑡 𝐶𝑉
𝑟 𝛼 µ
𝐴
Equation 4
The integration of equation 4 from V=V to V=V and t =0 to t= t and leads to
V2+2VV0=Kt
Equation 5
Where
𝑟 2𝐴
𝑉 𝐴𝐾 ∆𝑃𝑔
𝛼𝐶 𝛼𝐶µ
The rearrangement of equation 5

𝑡 1
𝑉 2𝑉
𝑉 𝐾
Equation 6
A plot of t/V versus V yields a straight line with a slope of 1/K and an intercept of 2V0/K. The
values for rm and α are calculated from experimentally determined values of K and V0.
If the filter cake is incompressible, the specific cake resistance does not vary with a pressure
drop across the filter. However, cakes from fermentation broths are seldom incompressible.
As these cakes compress, filtration rates decline. For a compressible cake, α can be related
empirically to ∆P as follows:
𝛼 𝛼 ∆𝑃
Equation 7
Activity 1
In example 11.1, when the volume of the filtrate is doubled, estimate the pressure drop across
the filter and comment on the filter medium resistance.
Activity 2
A 30 ml sample of broth from a penicillin fermentation is filtered in the laboratory on a 3 cm2

filter at a pressure drop of 5 psi. The filtration time is 4.5 min. Previous studies have shown
that filter cakes of Penicillium chrysogenum are significantly compressible with s = 0.5. If 500
litres of broth from a pilot scale fermenter must be filtered in one hour, what size filter is
required if the pressure drop is increased to 10 psi? Assume that the resistance due to the
filter medium is negligible.
2.2 Centrifugation and sedimentation
Although centrifugation is more expensive than filtration, it is more effective than filtration in
the separation of small particles. Centrifugation is used to separate particles with a size of
between 100 and 0.1 mm from a liquid. Centrifugation removes cells from a fermentation broth
and eliminates cell debris.
Two common types of centrifuge are used in the recovery and purification of bioproducts,
namely a disc or bowl centrifuge and a tubular-bowl centrifuge. A disc centrifuge consists of
multiple discs. A tubular centrifuge has a much simpler configuration.
In a centrifuge, the major forces acting on a solid particle settling in a liquid are the gravitational
force (FG), the drag force (FD), and the buoyant force (FB). When particles reach a terminal
settling velocity, the forces acting on it balance each other, resulting in a zero net force. This
is presented in the governing equation:
𝐹 𝐹 𝐹
Equation 8
Where
𝜋
𝐹 𝐷 𝜌 𝑔
6
Equation 9
𝐶
𝐹 𝜌 𝑈 𝐴
2
Equation 10
𝜋
𝐹 𝐷 𝜌 𝑔
6
Equation 11
FD is the drag force exerted by the fluid on solid particles, CD is the drag coefficient, 𝛒f is fluid
density, U0 is the relative velocity between the fluid and particle or the terminal velocity of a
particle, and A is the cross-sectional area of the particles
For a sphere, 𝐴 𝐷 .
Equation12
In stokes region, 𝐶
Equation 13
Rep = Reynolds number, 𝑅𝑒

µ
Equation 14
Substituting equations 9 to 14 into 8 and making U0 the subject of the formula
𝑔𝐷 𝜌 𝜌
𝑈
18µ
Equation 15
In a centrifugal field, the terminal separation velocity of particles, U0c, is given by the following
equation, where the centrifugal acceleration is substituted for the gravitational acceleration
𝑟𝜔 𝐷 𝜌 𝜌
𝑈
18µ
Equation 16
The ratio of velocity in the centrifuge to velocity under gravity is called the centrifuge effect or
g-number, and is usually denoted by Z
𝑟𝜔
𝑍
𝑔
Equation 17
Sedimentation occurs in a centrifuge when the cell particles away from the centre of rotation
collide with the walls of the centrifuge bowl. Increasing the velocity of motion will improve the
rate of sedimentation.
The performance of centrifuges of different sizes can be compared by a parameter called the
sigma factor Σ. This parameter represents the cross-sectional area of a gravity settler with the
same sedimentation characteristics as the centrifuge. Σ is related to the feed rate of material:
𝑄
𝛴
2µ
Equation 18
Where Q is the volumetric feed rate and ug is the terminal velocity of the particles in a
gravitational field. If two centrifuges perform with equal effectiveness
𝑄 𝑄
𝛴 𝛴
Equation 19
Activity 3
A continuous-disc stack centrifuge is operated at 5 000 rpm for separation of baker’s yeast.
At a feed rate of 60 l min-1, 50% of the cells are recovered. At a constant centrifuge speed,
solids recovery is inversely proportional to the flow rate.
(a) What flow rate is required to achieve 90% cell recovery if the centrifuge speed is
maintained at 5 000 rpm?
(b) What operating speed is required to achieve 90% recovery at a feed rate of 60 l min-1?
2.3 Coagulation and flocculation
Both coagulation and flocculation are used prior to filtration and centrifugation to improve the
degree of separation. Coagulation involves the use of coagulation agents such as electrolytes,
to form flocs whereas flocculation involves the aggregation of the flocs formed by coagulation.
3.1 Cell disruption
When the desired bioproduct is intracellular in nature, cell disruption is used to release the
intracellular product. The product includes enzymes and recombinant protein which are
normally found in the cell wall and cell membrane. Mechanical and non-mechanical cell
disruption are the two major methods used for cell disruption
3.2 Mechanical method of cell disruption
Mechanical methods of cell disruption include grinding with abrasives (such as grinding in a
ball mill with glass beads), high-pressure pumping and ultrasound. Large-scale cell disruption
is carried out by using a high-pressure homogeniser. This high-pressure pump has an
adjustable valve with an orifice through which cells are forced at pressures of up to 2 000 atm.
An example of this type of homogeniser is the Manton-Gaulin homogeniser. The governing

equation for the evaluation of homogenisers a is given below.
𝑅
𝑙𝑛 𝑘𝑁𝑝
𝑅 𝑅
Where Rm is the maximum amount of protein available for release, R is the amount of protein
released after N passes through the homogeniser, k is a temperature-dependent rate
constant, and p is the operating pressure. The exponent α is a measure of the resistance of
the cells to disruption.
3.3 Non-mechanical method of cell disruption
Non-mechanical methods of cell disruption include osmotic shock, freezing and thawing, the
enzymic digestion of cell walls, and treatment with solvents and detergents. Osmotic shock is
the most commonly used method. A change in the osmotic pressure of the medium may result
in the release of certain enzymes.
Activity 4
Micrococcus bacteria are disrupted at 5oC in a Manton-Gaulin homogeniser operated at

pressures of between 200 and 550 kgf cm-2. Data for protein release as a function of the
number of passes through the homogeniser are as follows:
Pressure drop (kgf cm-2)

200 300 400 500 550
Number of % of protein release
passes
1 5.0 13.5 23.2 36.0 42.0
2 9.5 23.5 40.0 58.5 66.0
3 14.0 33.5 52.5 75.0 83.7
4 18.0 43.0 66.6 82.5 88.5
5 22.0 47.5 73.0 88.5 94.5
6 26.0 55.0 79.5 91.3 -
(a) How many passes are required to achieve an 80% protein release at an operating
pressure of 460 kgf cm-2?
(b) Estimate the pressure required to deliver 70% protein recovery in only two passes.
4.0 Primary isolation of soluble bioproducts
This involves the isolation of the desired products from impurities. These methods include
adsorption, liquid extraction and precipitation. They are used non-selectively for large volumes
of fermentation broth.
4.1 Liquid extraction
Liquid extraction is usually carried out by using organic solvent. The extraction of a bioproduct
from one phase to another is based on the solubility differences in the bioproduct in one phase
relative to another. When a bioproduct is distributed between two immiscible liquids, the ratio
of the concentrations in the two phases is known as the distribution coefficient.
𝑌
𝐾
𝑋
Equation 20
Where KD is the distribution coefficient, YL is the concentration of the light phase (organic
solvent) and XH is the concentration of the heavy phase (soluble fermentation broth).
If KD is constant and the solvents are totally immiscible, then the mass flows of the light and
heavy phases are conserved so that L0 = L1 = L and H0 = H1 = H. A mass balance on the
extracted solute is:
𝐻 𝑋 𝑋 𝐿𝑌
Equation 21
𝐿
𝑋 𝑋 𝑌
𝐻
Equation 22
The substitution of equation 20 into 22 leads to

𝐿
𝑋 𝑋 𝐾 𝑋
𝐻
Equation 23
𝑋 1 1
𝑋 1 𝐿𝐾 /𝐻 1 𝐸
Equation 24
Where E = LKD /H is the extraction factor
For a multistage counter-current liquid extraction operation and the governing equation is
𝐸 𝐸 1
𝑅
𝐸 1
Equation 25
Where R is the rejection ratio, which is the weight ratio of the solute leaving in the light
phase to that leaving in the heavy phase, and n is the number of equilibrium stages.
Activity 6
Streptomycin is extracted from the fermentation broth using an organic solvent in a counter-
current staged extraction unit. The distribution coefficient of streptomycin at pH = 4 is KD = 40,
and the flow rate of the aqueous (H) phase is H = 150 l/min. If only five extraction units are
available to reduce the streptomycin concentration from 10 g/l in the aqueous phase to 0.2 g/l,
determine the required flow rate of the organic phase (L) in the extraction unit.
4.2 Precipitation
Precipitation is commonly used in the purification of antibiotics and industrial enzymes. There
are two methods of protein precipitation, namely salting-out and isoelectric precipitation.
4.2.1 Salting-out precipitation

Salting-out precipitation involves the precipitation of protein at high salt concentrations. The
common salts normally used are (NH4)2 SO4 and Na2 SO4. These salts increase the ionic
strength of the solution and make the protein molecules to precipitate. The solubility of proteins
in a solution as a function of the ionic strength of the solution is given by
S
log K I
S
Equation 26
Where S is the solubility of protein in solution (g/l), S0 is the solubility of protein when I = 0, I
is the ionic strength of solution, and K’s is the salting-out constant, which is a function of
temperature and pH. The ionic strength of a solution is defined as
1
I Σ𝐶 𝑍
2
Equation 27
Where Ci is molar concentration of the ionic species (mol/l), and Zi is the charge (valence) on
ions.
4.2.2 Isoelectric Precipitation
Isoelectric pecipitation is the precipitation of proteins at a pH at which proteins have no net

charge. The governing equation of isoelectric point of a protein is
pI 𝑝𝐾 𝑝𝐾
Equation 28
When pH = pI, proteins are free of charge and precipitate.
5.0 Purification of soluble bioproducts
After the isolation of the bioproducts, there is a need to purify soluble bioproducts of impurities
that have similar chemical and physical properties. Purification methods include
chromatography, ultrafiltration electrophoresis and fractional precipitation.
5.1 Chromatography
Chromatography separates mixtures into components by passing a fluid mixture through a
packed column of adsorbent material. The packed column may be solid, a porous solid, a gel,
or a liquid phase immobilised in or on a solid. The packed column of adsorbent is called the
stationary phase and the fluid that carries solutes through the column is known as the mobile
phase. The different solutes in the mixture migrate at different rates, thereby producing
different peaks in the chromatogram. This is illustrated in figure 11.27 in the textbook. This
type of chromatography is called elution chromatography because the mobile phase is injected
at one end of the column and eluted from the other end of the column.
There are other methods of chromatography such as adsorption chromatography, gel

chromatography, affinity chromatography, ion exchange chromatography, partition
chromatography and hydrophobic chromatography.
 Adsorption chromatography is based on the adsorption of solute molecules onto solid
particles such as alumina, silica gel, diatomaceous earth and charcoal. The chemical
composition of the adsorbents affects the performance.
 Partition chromatography is based on the different partition coefficients of solute
molecules between an adsorbed liquid phase and a passing solution. Often the
adsorbed liquid is non-polar.
 Ion exchange chromatography is based on the adsorption of ion-charged compounds
on ion exchange resin particles by electrostatic forces.
 Gel filtration chromatography is also known as molecular sieving, gel filtration and gel
permeation chromatography. It is based on the penetration of solute molecules in small
pores of packing particles based on their molecular size and the shape of the solute
molecules. It is also known as size exclusion chromatography.
 Affinity chromatography is based on the binding specificity of two molecules, the solute
molecule and ligands. A ligand is the binding molecule and the ligand-solute interaction
is very specific.
 Hydrophobic chromatography is based on hydrophobic interactions between solute
molecules. This method is a type of reverse-phase chromatography which requires
that the stationary phase is less polar than the mobile phase.
5.2 Ultrafiltration
Ultrafiltration is a membrane separation method which separates particles and

macromolecules of 1 000 to 50 000 Daltons. The governing equation of ultrafiltration is given
below:
𝐽 𝐿 ∆ 𝜎∆𝜋
Equation 30
𝐽 𝑃 ∆𝑐 𝐽 1 𝜎 𝑐
Equation 31
Where Jw is the solvent flux [ cm3 (cm2 s)-1], Js is the solute flux [ gm (cm3 s)-1], Lp is the
membrane permeability for the solvent, Ps is the membrane permeability for the solute (cm/s),
∆P is the hydrostatic pressure across the membrane (atm), ∆π is the osmotic pressure, 𝛔 is
the reflection coefficient, and ∆cs is the solute-concentration difference across the membrane
(gm/cm3).
5.3 Electrophoresis
Electrophoresis is used for the separation of charged biomolecules according to their size. In
an electric field, the drag force on a charged particle is balanced by electrostatic forces when
the particle moves with a constant terminal velocity. A force balance on a charged particle
moving with a terminal velocity in an electric field is expressed as follows:
qE 3𝜋µ𝐷 𝑉
Equation 32
𝑞𝐸
𝑉
3𝜋µ𝐷 𝑉
Equation 33
where q is the charge on the particle, E is electric field intensity, Dp is the particle diameter,
µ is the viscosity of the fluid, and Vt is the terminal velocity of the particle.
Activity 7
A pilot scale gel chromatography column packed with sephacryl resin is used to separate
hormones A and B. The column is 5 cm in diameter and 0.3 m high; the void volume is 1.9
x10 -4 m 3. The water regain value of the gel is 3 x10 -3 m 3 kg- 1 dry sephacryl; the density of
wet gel is 1.25 x 103 kg m-3. The partition coefficient for hormone A is 0.38 and the partition
coefficient for hormone B is 0.15. If the eluent flow rate is 0.7 l h-1, what is the retention time
for each hormone?
6. Final purification
High purity is essential for bioproducts, especially for pharmaceutical and therapeutics
products. Further purification such as crystallisation and drying, are some of the unit
operations used in the final separation step.
6.1 Crystallisation
Crystallisation occurs when the solute is supersaturated, in other words, when the solute
exceeds its solubility limit. This leads to the production of a high-purity solid product. Crystal
growth occurs in two steps: first, the transportation of solute to the crystal surface and, second,
the integration into the crystal lattice. The first step is the rate-limiting step and it is represented
in the equation below:
𝑑𝑀
𝑘 𝐴 𝑐 𝑐
𝑑𝑡
Equation 34
Where dMc is the change in mass concentration, dt is the change in time, ks is the mass
transfer coefficient, Ac is the surface area of the crystal and (c-cs) is the difference between
the bulk phase concentration and the concentration of crystallizing solute at saturation.
The mass and the area of the crystal can be related to the length of the crystal
𝑀 𝜌𝑘 𝐿
Equation 35
𝐴 𝑘 𝐿
Equation 36
Where ka and kv are the shape factor characteristics of the crystal geometry. For example, for
a cube ka= 6 an kv=1.
The substitution of equation 35 and 36 into equation 34 leads to
𝑑 𝐿
𝑘 𝜌 𝑘 𝑘 𝐿 𝑐 𝑐
𝑑𝑡
𝑑𝐿
3𝑘 𝜌𝐿 𝑘 𝑘 𝐿 𝑐 𝑐
𝑑𝑡
𝑑𝐿 𝑘 𝑘
𝑐 𝑐
𝑑𝑡 3𝑘 𝜌
6.2 Drying
Drying involves the removal of residual solvent from the wet bioproduct. This is required for
better storage and handling of the bioproduct. Different types of driers are utilised for drying
bioproducts, namely vacuum dryers, freeze dryers, rotary-drum dryers, spray dryers and
pneumatic dryers. Freeze dryers and spray dryers are the two most used dryers of
bioproducts.
 Freeze drying is also called lyophilisation and it involves the sublimation of residual
solvent from the frozen solution. It is the favoured drying method for sensitive biological
materials such as vaccines, pharmaceuticals and diagnostics materials. However, this
method is expensive and complex.
 Spray dryers employ the atomisation and spraying of a product solution into a heated
chamber through a nozzle. Hot gas inside the chamber provides the necessary heat
for evaporation of the liquid. Dried particles are separated from hot gases using
cyclones. Spray dryers are cheaper than freeze dryers and are more suitable for bulk
products such as food and detergents.
The purification and recovery of the desired bioproducts are essential for the successful
commercialisation of an industrial bioprocess. Product recovery accounts for the high
production cost of a bioprocess. The high cost of product recovery emanates from the low
concentration of the fermentation solution and the intracellular nature of products. The steps
for product purification and recovery include cell removal, primary isolation, purification and
final isolation. The key unit operations of the different steps include cell removal
(sedimentation, filtration and centrifugation), primary isolation (adsorption, liquid extraction
and precipitation), purification (chromatography, ultrafiltration and fractional precipitation) and
final isolation (crystallisation and drying).
1. Draw a process flow diagram of the recovery and purification of a special type of
antibiotics that is produced intracellularly by a bacterium.
2. Illustrate, by using a diagram, the correlation between the appropriate unit operation
and separation factors (size, density, solubility, ionic strength).
3. Compare the different types of chromatographic methods in a table format (types of
chromatography, basis of separation, characteristics and application).
4. Answer questions 11.1 to 11.12 in the recommended text book.
References
Dekker.

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BIOCHEMICAL

Welcome to Biochemical Engineering (BCE4801), a module that is offered in Unisa’s

1. Purpose and outcomes of this module

 interrogate cell biology and genetic engineering concepts

2. How the content of this module is organised

 History and potential of bioengineering

 Introduction to biological concepts

o Mechanism and kinetics

o Immobilised enzyme systems

o Enzyme catalysis (biocatalyst)

 Cell cultivation, growth and product formation

o Stoichiometry and yield

o Metabolic pathways and energetics

 Bioreactor design and operation

o Mass and heat transfer

 Product recovery and purification

4. How you should go about studying this module

Your work on each learning unit should involve the following:

Your study file should comprise

 answers to each activity in each learning unit

All the best! I hope you enjoy the course.

The following are the learning outcomes of this unit:

 Understand what biochemical engineering is.

Biochemical engineering is a branch of chemical engineering that incorporates microbiology,

Figure 1: Interdisciplinary nature of biochemical engineering

Figure 2: A schematic diagram of the steps involved in the development of a bioproduct

2. History of Biochemical Engineering

3. Status of Biochemical Engineering

4. Review of the Module

Learning unit 2: Introduction to Biological Concepts

Learning unit 3: Enzymes

o Mechanism and kinetics

o Immobilised enzyme systems

o Enzyme catalysis (biocatalyst)

Learning unit 4: Cell Cultivation, growth and product formation

o Stoichiometry and yield

o Metabolic pathways and energetics

Learning unit 5: Genetic engineering

Learning unit 6: Bioreactor design and operation

o Mass and heat transfer

Learning unit 7: Product Recovery and Purification

The importance and future of biochemical engineering

Videos about biochemical engineering

Figure 1: Interdisciplinary nature of biochemical engineering

On successful completion of this learning unit, you should be able to

 describe the main groups of microorganisms

Watch this video https://www.youtube.com/watch?v=k7cCBlQARg0 and explain, in your own

2.1 Cell theory and living organisms

Bacteria Archaea Eukarya

Plants Animals Fungi Protists

Figure 3: Classification of living organisms

Table 1: Differences between prokaryote and eukaryote cells

Features Prokaryote cells Eukaryote cells

Figure 5: Schematic diagram of an eukaryote

Figure 6: Illustration of useful and harmful bacteria in the human gut

Figure 7: Different forms of bacteria

2.3.2 Blue-green algae

Figure 8: Mycelial structure of moulds

2.4.2 Protozoa and algae

2.4.3 Animal and plant cells

2.6 Microbial diversity and taxonomy

2.7 Biochemistry of microorganisms

Nucleic acids Proteins Polysaccharides Lipids

Mononcleotides Amino acids Simple Sugars Fatty Acids