Chapter 1
Old vs Modern Biotechnology
Introduction
Biology has changed with time:
- Mistakes were made (proteins are genetic material// age of earth//earth is
flat)
- Ethical concerns have changed (more humane towards animals)
- Human genome was thought to be 100 000 genes instead of 20 000 genes
(same amount as that of flies). The complexity arises from splice variants,
intron regions, and DNA regulation.
Biotechnology-definition and importance
It is the application of scientific and engineering principles to the processing of
material by biological agents to provide goods and services to improve the
lifestyle of humans// the production of commercial products generated by the
metabolic action of microorganisms, plants, and animals.
-env: removing oil spills by
using gen.eng microorganisms
that degrade organic oil spills
-Biomining: microbes to
extract minerals from nature.
NOTES JOANNE
NOTES OMAR
3 Steps of a bioengineered biotechnology process
The process occurs in bioreactants (can go up to 500L): stainless steel vessel
containing organisms and media to make certain products. The organisms could
be naturally producing the product or genetically engineered to produce it.
-Upstream processing: preparation of optimal raw material so
that it can be used as a food source for the microorganism
(oxygen levels; yeasts; temperature; pH). This includes all steps
related to the development of microorganisms, nutrient
preparation, cell culture, cell separation and harvesting.
-Fermentation (microbes) and biotransformation (plants &
animal cells): Growth of the microorganism and production of
the desired compound, carried out by the action of biocatalysts
(enzymes) produced by these microbes.
Note: It is important that the microbe does not produce any
secondary or waste products to ensure the purity of the product.
-Downstream processing: purification of the product from the
medium and from the cells that produce it. This is necessary
before the product is sold to the market.
From the media: collect the media; centrifuge; throw
pellet of microbes; harvest the product (easier purification)
From the microbes: centrifuge; collect the pellet; break
open the cells using detergents; extract the product from the cells; purify it.
Mutagenesis is applied in upstream processing to enhance product yields by using
chemical mutagens or UV radiation we obtain different species that is much
productive (ex: penicillin production).
Note: mutagenesis is different than gene engineering and is less accurate.
Old biotechnology
Earlier, biotechnology relied primarily on the use of microbial tools/ microbial
technology.
The 1st chemical was developed by the French (leaders) and it was alcohol=
ethanol (wine, beer) which was possible after discovering the process of
fermentation by Pasteur. He was the first to demonstrate experimentally
that fermented beverages result from the action of living yeast
(Saccharomyces cerevisiae) transforming glucose into ethanol (anaerobic
pathway// naturally producing)
Note: Pasteur developed the process of Pasteurization which can be used to
sterilize products such as milk to purify it from microbes.
The 2nd biotechnological product was discovered by the Germans (leaders)
is glycerol.
Glycerol is used by the cosmetic, paint, automotive, food, and
pharmaceutical industries and for production of explosives.
-Glycerol and ethanol share the same
precursor which is glucose
-They both rely on glucose fermentation
through several steps: glucose pyruvate
… ethanol or glycerol
-When bisulfide is added to a yeast culture,
glycerol is formed instead of ethanol
The 3rd product is acetone and butanol also by the Germans and British.
To make it, you incubate sugar in the bioreactor in order to create acetone,
in the presence of a microbe, Clostridium acetobutylicum
(aerobic//naturally produced acetone & butanol)
Acetone is produced by semi-continuous fermentation.
Once petroleum became available, they discontinued the production since
you could make acetone and butanol from petroleum.
They used acetone in munitions (for war).
They used butanol in artificial rubber
-Acetone and butanol share the same
precursors
-we first make acetone; if we keep it
longer, we make butanol
-Note that hydrogen gas is produced in
excess => many manufacturing plants
were destroyed by explosions.
A pipeline is required to transfer the H2
out of the bioreactor.
Note:
Block/batch-to-batch fermentation: add sugars and raw material, then purify and
isolate everything in downstream processing, and then start from scratch.
Semi-continuous operation: each time you prepare a new batch of microbial
species, add it to a pre-existing one. For example, from a finished 500L bioreactor,
you take 300L and isolate the product from there, but then add new
microbe/media to the remaining 200L inside the bioreactor.
Advantages: increases the yield of product production because some microbes
are left in bioreactor, so there is no lag period
Disadvantages: higher risk of contamination of all the vessel; possibility of
mutations.
Citric acid:
- Until WW1, it was produced from citrus fruit (stronger muscles and bones)
- After that, it was produced from Aspergillus niger (aerobic) using surface
cultures = no more need to cut trees to obtain citrus fruit.
Penicillin: it was the 1st antibiotic produced from Penicillium notatum
(aerobic) using surface cultures (discovered by Alexander Fleming).
Surface cultures: shelfs of open pans exposed to oxygen which have the media
and microbes in them. They have a high risk of contamination; the solution is
Aseptic techniques - to completely sterilize the environment in question (room I
steamed and then air dried).
It is a very old technology; nowadays, we can pump O2 in large bioreactors.
Single cell protein (revolutionary technology-1960s):
The microbes are dried and sprinkled then injected into food as an additive to
enrich its protein source (protein; CHO; lipids; vitamins):
- Protein supplement in human food
- Food additive for flavor
- Protein supplement for livestock
Example: Algae (Spirulina) eaten by Lake Chad population in Africa// tofu
This technique requires growth of microbes on a large scale. The microbes can be
eukaryotes or prokaryotes as long as they are unicellular.
It is the second largest advancement in industrial microbiology after penicillin
production.
It did not last long since starvation happens in poor countries = there is no money
there to establish these bioreactors = developed countries do not need single cell
proteins.
Risk: we have to use safe microbes to avoid developing infections
Microbial biotechnology
Strain selection//screening of microbes:
Screening: Detecting and isolating commercially important microbes such that
only one microbe is growing in the culture.
Strain selection:
- Mutation (2-Aminopure, methylnitrosoguanidine, UV, x-rays): Once the higher
yield-producing strain is created, we enrich the medium in favor of this strain.
Outdated technique
Example: (P.notatum 2mg/L to P.chrysogenum 20g/L = penicillin)
- Genetic engineering (Rec. DNA technology): introduce genes into microbes
so they make products that they don't naturally make.
Example: insulin is made from E.coli by inserting the human insulin gene into
E.coli.
When I want to isolate my product, we need to purify our culture, for it to be a
desirable culture. It is:
- Pure from other microbes [quality control] (screening/selection)
- Easy to culture
- Vigorous growth
- Produce single product
- Genetically stable (difficult since microbes divide fast and develop
mutations).
Microbial cultures
Establishing pure microbial cultures can only be done starting from a single
colony. To obtain a single colony, we have 2 methods (small scale microbial
culture):
a. Streak plate method:
You take an agar plate and you streak microbes on it with an inoculating loop:
- In quadrant: From quadrant 1, you take a population and spread it onto
quadrant 2, then from 2 until 3, then from 3 until 4. That way, quadrant 1 is
very concentrated; but the culture is diluted in quadrant 4, so a single pure
colony is easy to isolate.
- In continuous: same as above but without quadrants, the end part is dilute
enough.
b. Dilution series method
Small volume from initial culture and place in water and repeat process until
desired diluted concentration is achieved. Then, the final test tube can be poured
on an agar plate and then a single colony will be able to be isolated.
These techniques are used to maximize the ability of attaining an isolated
colony.
Note: Large scale microbial cultures: Bacteria can be grown in large-scale
quantities. The bioreactors (=fermenters) shown here contain several
hundred gallons of bacteria growing in liquid culture.
Industrial fermentation/microbiology
Important microbes:
- Bacillus (bacteria/aerobic): has signaling enzymes that secretes products
into media rather than keeping them within the cell (usually used to make
plants more resistant to insects).
- Streptomyces (bacteria) (Class: Actinomycetes) = antibiotics
- Acetic acid bacteria = vinegar
- Saccharomyces cerevisiae = bread
- Escherichia coli= insulin
- Penicillium= notatum, chrysogenum make penicillin and roquefortii makes
blue cheese
- Rhizopus (fungus) = steroids [used in medicine].
Types of microbes:
- Obligate aerobic/anaerobic
- Facultative aerobic/anaerobic
Note: facultative aerobic are microbes that grow best in anaerobic but can survive
in aerobic when it is needed. We find them at the bottom of a bioreactor.
Facultative anaerobic are found at the top of bioreactor.
Modern biotechnology
It is the deliberate manipulation of DNA molecules to produce commercial
products from living organisms = genetic engineering= recombinant DNA
technology.
It started in early 70s because we discovered in 1972 the restriction enzymes that
are very essential for modern biotech to manipulate DNA.
In 1982, the 1st genetically engineered pharmaceutical product was produced by a
genetically modified E.coli, having the human insulin gene Humulin (human
insulin).
Insulin is a linear and very small protein, so it does not need post-transcription or
post-translation modification in the prokaryotes to activate it (if the protein is
complex, we need to inject in yeast).
Note: before Humulin, they used to extract insulin from pigs; horses; dead
cadavers.
Molecular biotechnology: it is based on the ability of transferring specific units of
genetic information from one organism to another.
Example: “Golden rice” has been genetically modified to contain beta-carotene,
which we use to make vitamin A.
Note:
Insertion of a piece of DNA into a microbial cell =transformation
Insertion of a piece of DNA into an animal cell =transfection
