Transcript Chapter 30

Chapter 30
Biotechnology
Biotechnology is the application of scientific and
engineering principles to the production of
materials by biological agents.
Its origins go back a long way, most were
fermentation processes: e.g.
 food contamination led to improved flavour and
preservation,
 beers was developed from ‘spoilt’ grain while
wine from ‘spoilt’ fruit,
 contaminated alcohol caused the manufacture of
vinegar as food & food preservative,
 contaminated milk led to the production of cheese,
butter & yoghurt.
 Modern biotechnology industry began in the
First World War:
 the Germans used yeast to ferment plant
materials in order to produce glycerol for
explosives;
 the British used bacteria to make acetone &
butanol as part of their war effort.
 In the Second World War, mass production of
penicillin was needed for medical used.
 A major expansion of biotechnology
advancement occurred when recombinant DNA
technology was employed.
30.1 Growth of Microorganisms
30.1.1 Factors affecting growth – applicable to
both plant and animal cell cultures
 Nutrients which include 4 elements: carbon,
oxygen, hydrogen, nitrogen and significant
quantities of phosphorus and sulphur.
 Smaller quantities of nutrients include macronutrients (Ca, K, Mg & Fe) and micronutrients (Mn, Co, Zn, Cu).
 Growth factors like vitamins, amino acids,
purines and pyrimidines are also important.
 Usually greater concentrations of nutrients
will lead to higher rates of growth.
30.1 Growth of Microorganisms
Temperature
All growth is governed by enzymes
which operate within a narrow range
of temperatures.
Groups are classified according to their
preferred temperature ranges:
1 Psychrophiles - optimum growth
temperature below 20℃,
continuing to grow at temperatures
Microorganisms growing on a
down to 0℃.
nutrient medium
2 Mesophiles - optimum growth
temperatures in the range 20 – 40℃
3 Thermophiles - optimum temperature
in excess of 45℃, very few can
survive as high as 90℃
pH
Microorganisms can tolerate a wider range of pH than
plants and animal cells, most prefer acidic conditions.
Oxygen
Most are obligate aerobes, some are facultative aerobes
and obligate anaerobes.
Microaerophiles can tolerate oxygen, nevertheless grow
better when its concentration is low.
Osmotic factors
All microorganisms need water for growth. Most
cannot grow in environments with high solute
concentrations. A few, the halophiles, can survive in
conditions of high salt concentration.
Pressure
Pressure is not a major factor affecting growth
of microorganisms.
A few species inhabiting the ocean depths, the
barophiles, cannot grow in surface waters
where the pressure is too low for their survival.
Light
Photosynthetic microorganisms require an
adequate supply of light to sustain growth.
Water
Water is needed for a variety of cellular
functions. Photosynthetic autotrophs use
hydrogen (from water) to reduce carbon
dioxide and make food. (H2O)
Chemoautotrophs use hydrogen sulphide for
making food.
(H2S)
30.1.2 Growth Patterns
Growth of a population (bacterial growth curve) with
the lag phase, exponential phase, stationary phase
and death phase:
30.1.3 Biotechnology and Food Production
The correct balance of nutrients, an appropriate pH and
a suitable medium are essential to microbial growth.
Agar is seaweed extract which is metabolically inert
and dissolves in hot water but solidifies upon cooling.
Minimal medium: a medium composed to satisfy the
demands of a single species.
Narrow spectrum medium: provides the nutrients for a
small group of microorganisms with similar
requirements, e.g. acidophiles.
Broad spectrum medium: medium for general purposes,
designed to grow as wide a range of microorganisms
as possible.
30.1.4 Aseptic conditions
Pure cultures of single type of microorganism
need to be grown free from contamination
with others.
Sterilizing techniques include
heating,
very fine filters,
UV light illumination or
using disinfectants.
30.2 Industrial fermenters and fermentation
Fermenters enable specific microorganism to grow inside
– a process called fermentation (it might not be anaerobic!)
Fermenter for cloned protein
Batch cultivation: extract products from time to time
with cleaning & sterilization to begin the whole
process again
Continuous cultivation: used medium and products
are continuously removed, raw materials are added
throughout the process
A stirred tank fermenter
Immobilization of cells and enzymes
One problem with the fermentation processes so far is
that at some point the cell culture is removed and
discarded.
Any mechanism for immobilizing the microorganism
and/or the enzymes they produce, improves the
economics of the process.
1 Entrapment – cells or enzyme molecules are trapped in
a suitable meshwork of inert material, e.g. agar,
cellulose, etc
2 Binding – cells or enzyme become physically attached
to the surface of a suitable material, e.g. sand or gravel
3 Cross-linking – cells or enzymes
are chemically bonded to a
suitable chemical matrix
However immobilized, the cells or
enzymes are made into small
beads which are then either
packed into column, or kept in
the nutrient medium.
The nutrient can be continually
added and the product removed
without frequent removal of the
microorganisms/enzymes.
The process cannot be continued Immobilized microbial cell
indefinitely because impurities pellets in a packed reactor
column used to carry out
may accumulate.
biotransformations
30.3 Biotechnology and Food Production
1857: Louis Pasteur showed alcoholic fermentation
by microorganisms
Baking: Use of yeasts in food production (bread)
Beer and wine production: also fermentation by
yeasts
Dairy products: cheese, butter and yoghurt are
produced using various bacteria
Single Cell Protein (SCP)
Single cell protein comprises the cells, or their
products, of microorganisms which are grown
for animal and human consumption.
The product also contains fats, carbohydrates,
vitamins and minerals.
Raw materials: petroleum chemicals, alcohols,
sugars, agricultural & industrial wastes.
Microorganisms: bacteria, filamentous fungi,
algae, yeast.
Others:
Vinegar,
Sauerkraut (salted cabbage),
Olive and cucumber preservation,
Coffee and cocoa beans,
Soy sauce,
Enzymes: lipase (flavour development in cheese),
protease (meat tenderizers),
-amylase (improve flour, breakdown
starch in beer production).
30.4 Biotechnology and Pharmaceuticals
Antibiotics are chemical substances produced by
microorganisms which are effective in dilute
solution in preventing the spread of other
microorganisms.
Most inhibit growth rather than kill microorganisms.
Penicillin – a narrow spectrum antibiotic
Chloremphenicol – a broad spectrum antibiotic
Penicilin fermentation
Comparison of primary
& secondary metabolite
production
Antibiotics are made when growth of the producer organism
is slowing down rather than it is at its maximum.
They are secondary metabolites and their production takes
longer than primary metabolites.
As a result, only batch fermentation can be employed.
Hormones are produced utilizing recombinant
DNA technology, e.g. insulin, growth hormone,
testosterone, oestrogen, etc.
30.5 Biotechnology and Fuel Production
Gasohol production:
Use yeasts to ferment sugar cane into alcohol and produce
a relatively cheap, renewable fuel.
Processes include:
1 Growing & cropping sugar cane
2 Extract the sugars from plants
3 Crystallizing out of the sucrose for sale and leaving a
syrup of glucose & fructose as molasses
4 Fermentation of the molasses by yeast to dilute alcohol
5 Distillate to pure alcohol, using the waste (bagasse) as
a power source
Biogas production
A simple process of utilizing a digester (container) to
hold the wastes with anaerobic bacteria to produce
methane, a gas which is ready for cooking, lighting
and heating.
Biogas generator
30.6 Biotechnology and Waste Disposal
Sewage disposal
Anaerobic digestion: microorganisms break down
organic sewage into acetic acid, carbon dioxide and
hydrogen.
Aerobic digestion: aerobic microbes oxidize organic
compounds in sludge by pumping air through
Sewage farm showing filter beds
Biodegradable plastics
Polythene and polyester polyurethanes of low molecule
mass are developed and can be degraded by
microorganisms, e.g. C. resinae (a fungus).
In general, more flexible plastics are broken down more
easily than rigid ones.
Biodegradable plastic bottles
Disposal of oil
C. resinae can also degrade oil.
Microorganisms, developed from recombinant
DNA technology, have been useful in cleaning
oil spills.
Emulsifier, produced commercially from bacteria,
is used to cause oil to mix with water and so
both disperse it and speed up microbial
breakdown.
Disposal of industrial wastes
An increased awareness of ecological issues and
tighter legislation have prompted safer waste
disposal.
Examples:
Biogas production utilizing many forms of
wastes (paper, cotton);
Brewery waste could be converted to citric acid
by a fungus;
Potato processing plant wastes could be
converted to animal feeds.
Genetically engineered microorganisms are
making the task increasing easy.
Recovery of valuable material from lowlevel sources including wastes
Bacteria can be used to extract copper and
uranium from waste ores, and remove sulphur
from high sulphur coal.
A alga is used to absorb metal ions against a
concentration gradient.
Enhanced oil recovery, utilizes microbes to
extract oil out of the rocks.
30.7 Other products of the biotechnology industry
Examples:
Interferon for treatment of viral diseases,
Vitamin B12 as food supplement,
Protease as detergent additive,
Indigo as a textile dye,
Artificial snow at winter holiday resorts.
Interferon for treatment of viral
diseases
30.8 Cell, tissue and organ culture
Plant and animal cells can also be grown in vitro to
make a variety of products.
The plant meristems retain the growing ability of plant cells.
If a tissue containing meristematic cells, e.g. a bud, root tip,
etc., is removed from the plant and grown aseptically on a
nutrient medium, an undifferentiated mass (callus)
develops in the presence of hormones and growth
regulators.
In vitro: by artificial means outside
the body
Anther callus on agar
Plant tissue culture is the production of
undifferentiated callus.
If the callus is suspended in a liquid medium and
broken into individual cells it forms a plant cell
culture.
These can be maintained indefinitely if sub-cultured
giving rise to a cell-line.
With the help of pectinases & cellulases to dissolve
the cell walls, the protoplasts of different cells can
fuse to form hybrids to grow into new plant
varieties.
Plant organ culture from apical shoot tips growing
with cytokinin can develop a cluster of shoots,
each of which may be grown into new clusters of
genetically identical individuals – a form of
micropropagation.
Micropropagation of plants
growing in medium
Applications:
1 Generation of plants for agricultural or
horticultural use –
Vast quantities of plants can be grown in
sterile controlled conditions ensuring a much
higher survival rate.
A uniform crop with desired characters can be
maintained. Plants are pathogen free and can
develop defence mechanisms against many
diseases.
2 Manufacture of useful chemicals by plant
culture –
atropine (dilation of pupil),
codeine (pain killer),
digoxin (treatment of cardiovascular problems),
jasmine (perfume),
menthol (flavouring).
Animal cell culture
Only in recent years has it proved possible to
culture vertebrate cells on any scale.
With the help of proteolytic enzymes to separate
the cells, a monolayer of cells attach
themselves to the bottom of the container are
capable of cell division – primary culture.
Cells from these can be used to establish
secondary cultures but their life span is
limited, division often ceasing after 50-100
divisions.
It is possible to make these cultures continue to
divide indefinitely by adding chemicals or
viruses to transform them into cancer cells –
neoplastic, which can induce cancers if
transplanted into a related species.
Application:
1 Production of viral vaccines, e.g. polio,
measles, German measles, rabies
2 Pharmaceutical products from cell lines, e.g.
interferon, human growth factor and clotting
factor
3 Monoclonal antibodies production through
recombinant DNA technology to clone viral
vectors