CH3104
Biochemical Engineering
Asst Prof TAN Meng How
N1.2-B2-33
mh.tan@ntu.edu.sg
1
Course Administration
• Lectures:
Fri 11.30am-1.30pm (CBE-LT)
• Tutorials:
Tues 1.30-2.30pm (CBE-SR3) or
Tues 2.30-3.30pm (CBE-SR3) or
Tues 3.30-4.30pm (CBE-SR3) or
Fri 10.30-11.30am (CBE-SR3)
• Textbook:
Michael L. Shuler and Fikret Kargi, Bioprocess
Engineering – Basic Concepts, Pearson
• Grading scheme:
Quiz 1 (20%)
Quiz 2 (20%)
Final Exam (60%)
2
Syllabus (Weeks 1-7)
• Introduction to biochemical engineering
• Biology review (molecular biology, cell biology,
and metabolism)
• Gene regulation
• Alteration of cellular information and cloning
methods
• Genome engineering
• Systems biology
• Synthetic biology
• Genetically engineered organisms
3
Lecture 1 – An Introduction
to Biochemical Engineering
4
What is biochemical engineering?
Biochemical engineering is a process that uses living cells
(such as microorganisms) or biomolecules (such as enzymes)
to carry out a chemical transformation leading to the production
and ultimate recovery of valuable products.
5
A History of “Biochemical Engineering”
Ancient Uses of Microorganisms (Before 1800 A.D.)
•
•
•
•
•
•
•
•
Caveman to Earliest Recorded History --- aging of meats, cheeses, and
alcoholic beverages.
Ancient Chinese and Japanese -- soy sauce from fermented beans.
Ancient Egyptians (2500 B.C.) -- malting of barley and beer fermentation.
Mesopotamian tablet records brewing of wine and beer is established
professions in 2000 B.C.
Columbus lands in North America to find the native people drink beer made
from corn.
Chinese use moldy soy bean curd to clear up skin infections (1000 B.C.)
Central American native peoples use fungi to treat infected wounds.
Middle Ages experimenters learn how to improve the taste of wine, bread,
beer, and cheese.
In the early years, mankind did not know that these fermentation processes were
6
being carried out by microscopic forms of life.
Getting closer to the truth
•
In 1680, Antony Leeuwenhoek designed a microscope
that was powerful enough to see yeast and bacteria
•
A description of these organisms, accompanied by
diagrams, was sent to the Royal Society of London.
•
However, he did not consider the spherical forms to
be living organisms but compared them with starch
granules.
7
A realization in the 1800s
Discovery and isolation of microscopic life
1803 -- A French scientist, L.J. Thenard, announced that yeast was the
cause of fermentation and it was alive, since it contained nitrogen and
yielded ammonia on distillation. Unfortunately, such evidence was not
conclusive of life and his findings were rejected by supporters of the
conventional notion that fermentations were chemical processes only.
1857 -- Louis Pasteur, another French scientist, proved that Thenard was
correct. He showed that the yeast, which produced fermentation, consisted
of living organisms capable of growth and multiplication. He further
demonstrated that oxygen was not required for fermentation. In addition,
Pasteur's research showed that the growth of microorganisms was
responsible for spoiling beverages, such as beer, wine and milk. He further
invented a process in which liquids such as milk were heated to kill most
bacteria and molds already present within them (pasteurization). Pasteur
also showed that certain diseases were caused by microorganisms (germ
theory). The work of Pasteur laid the foundation for modern microbiology and
current-day microbial biotechnology.
8
Rapid development in the 1900s
1901 -- Rudolf Emmerich and Oscar Low, University of Munich,
isolated a primitive antibiotic, pyocyanase, from Pseudomonas
aeruginosa, a bacterium. Several hundred patients were successfully
treated, but quality control was poor and pyocyanase was
abandoned as it was considered to be too hazardous.
World War I -- Chaim Weizmann (considered to be the father of
industrial fermentation) solved a serious British ammunition problem
by using the bacterium Clostridium acetobutylicum (the Weizmann
organism) to produce acetone, which was used in the manufacture of
the explosive cordite.
1923 -- Pfizer opens the first commercial successful plant for citric
acid production from sugar.
1928 -- Alexander Fleming discovers penicillin.
Today, simple organic molecules such as glycerol, lactic acid, and
butanol are produced on an industrial scale by fermentations.
9
CASE STUDY:
Discovery and development of penicillin: How
biologists and engineers work together
A chance discovery
Alexander Fleming's 1928 photo of the plate with
Staphylococcus bacteria and Penicillin mold
Contaminant
Minimal growth
around contaminant
(Why?)
Intended bacteria
Chance favours the prepared mind!
CASE STUDY:
Discovery and development of penicillin: How
biologists and engineers work together
• Fleming noticed “spoiled” experiment — an antimicrobial agent was at work
• WW II — there is a need for huge amounts of antibiotics like penicillin, but how
do we produce them in large quantifies?
• Formidable problems to fermentation:
– media (“food”) that is conducive for growth
– new tank design that guards against entry of undesirable microbes
– large volumes of sterile air required
– separation process for a fragile and unstable product
• No one person had all the necessary skills. Multidisciplinary teams required.
- Biologists: experiments, manipulations of living microorganisms, understanding of
cellular processes
- Engineers: operation of fermentation tanks, theoretical calculations
Result: increased penicillin concentration (up from 0.001g/L to over 50g/L today)
12
The story of penicillin
15+ years
Discovered by
Alexander Fleming
1928
Fermentation
route was chosen
Chemical synthesis
proved to be too difficult
Full scale production
1940’s
Efforts to increase production
•cell line selection
•medium optimization
•process development
Era of Molecular Biology
(Late 1970s - Present)
1973 Herbert Boyer (University of California, San
Francisco) and Stanley Cohen (Stanford
University) establish recombinant DNA
technology
The discovery of recombinant DNA technology and
the birth of genetic engineering allows for the
efficient production of compounds not indigenous
to the host microorganism.
14
The importance of recombinant DNA technology
is recognized by top biomedical awards
Biochemical Engineering as a Discipline
Work in a team environment with chemists, biochemists,
microbiologists, and chemical engineers.
• Steps in the development of a new biochemical process
and roles professionals play.
1. Identify a desired reaction or product (chemist,
biochemist).
2. Identify key enzyme(s) or microorganism (biochemist,
microbiologist).
3. Process development (chemist, biochemist,
microbiologist, chemical engineer).
4. Design of bioreactor and recovery unit operations
(chemical engineer).
5. Metabolic engineering: application of engineering
analysis to metabolic pathways within microorganisms to
improve product yields.
16
Typical chemical processing
A
P
B
A
temperature
flow rate
A+B
P
P
A
B
B
Reactor
Separation
Biocatalyst: cells and enzymes
1-5 mm
10-20 mm
Cell as a bioreactor
A
A
B
C
D
E
P
Reactor
cell
Complex
mixture
?
Products :
Cells
Metabolites
Enzymes
Nucleic acids
Other biomolecules
Separation
(How?)
Bioprocesses: advantages
• Cells will often perform reactions that are too difficult to
do synthetically (e.g. penicillin).
• Cells can turn cheap, basic nutrients (for example,
agricultural waste) into valuable products.
• Enzymes are highly specific catalysts with high catalytic
activity that has been optimized/ evolved by Nature over
thousands of years.
The Challenge
It is difficult to efficiently and economically
recover a high purity biochemical product
from a complex mixture of related and
functional molecules, impurities, and
contaminants, which often have similar
physical and chemical properties.
There are usually trade-offs, e.g. between
purity and yield.
21
Economics of biochemical engineering
22
Workflow
There are
multiple choices
and decisions to
be made.
23
Cell Culture Media Design
1. Nutritional requirements
- Carbon source, specific nutrients (e.g. vitamins,
minerals, amino acids), elemental requirements
2. Environmental requirements
- pH, temperature, oxygen
3. Regulatory constraints
- Qualifications of vendors, potential impurities or
contaminants, consistency
4. Other constraints
- Cost, environmental impact, product inhibition
24
Industry Focus 1: Food & Beverage
Industry Focus 1: Food & beverage
Fermentation Products
•
•
•
•
•
cheese
soy products
yoghurt
wine, beer
bread
Enzymes
• adjust food flavour
• adjust food texture
• improve nutritional quality
Example: high fructose corn syrup
– corn syrup that has
undergone enzymatic
processing to convert some
glucose to fructose to produce a
desired sweetness
Fermentation
• A form of anaerobic respiration occurring in certain
microorganisms (eg. yeasts)
• Alcoholic fermentation is a series of biochemical reactions
by which a simple sugar is converted to ethanol and CO2.
Process for wine-making
•
Wild yeast/ ambient yeast vs cultured yeast
- Wild yeast can produce unique flavored wines, but cultured
yeast is more easily controlled
- Most common cultured yeast: Saccharomyces cerevisiae
(within which, there are hundreds of different strains)
•
Upon addition of yeast:
- Intermediates: three-carbon molecules, acetaldehyde
- Sometimes a small amount of acetic acid is made (vinegar taint)
•
Yeast ceases its activity when:
(1) All the sugar has been exhausted
(2) Concentration has exceed 15% alcohol per unit volume
- Exhausted yeast fall to the bottom of the fermentation tank as
sediment known as lees.
Agricultural Examples
• Recombinant bovine somatotropin (rBST),
a hormone that is injected into cows for
increasing milk production
• Bio-insecticides for crop protection
• Phyto-vanillaTM flavor is produced from
plant cell culture
Industry Focus 2: Textiles
Stone washing denim
•
•
Annual sales of jeans > $700 billion
Jeans are commonly subjected to stone washing to give the fabric a softer, smoother feel.
Indigo dye adheres
to denim surface
traditional
method
new method
Denim is faded by
abrasive action of
pumice stones
Cellulase enzyme
removes some of the dye
by partially hydrolyzing the
cotton surface
• weakens the fabric
• new looks
• lower costs
• shorter treatment times
• less solid waste
Detergents
• Detergent industry is the largest single market for enzymes
at 25 - 30% of total sales
• Dirt comes in many forms and includes proteins, starches
and lipids (fats and oils)
• Proteases, amylases, and lipases are enzymes used in
detergents
• Enzymes allows lower temperatures and less agitation for
washing
• The enzymes used are all produced using Bacillus bacteria
Industry Focus 3: Environment
• Cleanup of hazardous waste sites using
bacteria that feed on pollutants
• Bacteria used for bio-remediation
• Wastewater treatment
• Biosensors: use biological activity to
detect toxic substances
Bioremediation is a waste management technique that involves the use of organisms
to remove or neutralize pollutants from a contaminated site.
According to the EPA, bioremediation is a “treatment that uses naturally occurring
organisms to break down hazardous substances into less toxic or non toxic
substances”. Technologies can be generally classified as in situ or ex situ.
34
Using engineered bacteria to remove
Cadmium from the environment
6His (hexahistidine) is a metal-binding peptide.
35
A whole-cell uranium biosensor
A bacterial strain has been engineered to fluoresce in
the presence of uranium when it is exposed to UV lamp.
36
Industry Focus 4: Pharmaceuticals
6.5 years
Discovery of
a promising
compound
1 year
Preclinical testing
in animals
Phase I clinical trials
in healthy volunteers
3 years
19 months
Phase III clinical trials
in 1000 to 3000
patients
FDA review and
approval
2 years
Phase II clinical trials
in 100
to 300
patients
100-300
patients
Drug may be
prescribed by
physicians
The Drug Development and Approval Process
-The drug discovery and approval process takes and average of
15 years and costs almost $400 million
Products
•
•
•
•
•
Small molecules and metabolites
Antibiotics
Protein drugs
Vaccines
Antibodies, monoclonal antibodies (MAb)
“Recombinant DNA technology enables bacteria
and yeast to produce human proteins such as
insulin”
What can biochemical engineers do?
Case study: Artemisinin production for malaria treatment
Production cost decreased by more than 100-fold!
(Jay Keasling’s lab at UC Berkeley)
40
Contributions from several organizations
41
Synthetic biology – a new frontier in
rational biological engineering
42
iGEM Synthetic Biology Competition
•
An annual international competition for students interested
in the field of synthetic biology
•
NTU participated in 2015, 2016, 2017, and 2018. Each
year, we were awarded a Gold medal for our research.
(http://2015.igem.org/Team:NTU-Singapore)
(http://2016.igem.org/Team:NTU-Singapore)
(http://2017.igem.org/Team:NTU_SINGAPORE)
(http://2018.igem.org/Team:NTU-Singapore)
•
iGEM 2019 will be held from 31 October to 4 November.
•
Please email me if you are interested!
43