Application S
BIOTECHNOLOGY
(a) Outline the use of microorganisms in the extraction of heavy metals from lowgrade ores.
Low-grade ore: naturally occurring solid material from which a small amount of
metal or valuable mineral can be extracted. (vs high-grade ore $$$)
Valuable metals include iron, copper, zinc, cobalt and uranium. They are found
naturally as metal sulphides, which are insoluble in water so are hard to extract.
Some bacteria are described as chemoautotrophic, i.e. they derive their energy by the
breakdown of inorganic chemicals. In some cases, the bacteria break down ores of
heavy metals, and this indirectly releases the metal itself!
Bioaccumulation: occurs when an organism absorbs toxic substance at a rate greater
than that at which the substance is lost. Some microorganisms are able to accumulate
metals; hence they can be exploited in extraction of valuable metals. Pseudomonas
spp. can accumulate mercury, Pseudomonas aeruginosa accumulates uranium while
some Thiobacillus species accumulate silver.
Bioleaching: extraction of specific metals from their ores through the use of bacteria.
They oxidise the metal sulphide to metal sulphates. They are soluble in water, so can
be washed out of the rocks using water. See Figure 22.1, p315, main textbook.
Bioleaching is also known as microbial mining.
Example:
Bacterium: Acidithiobacillus ferooxidans
Work: oxidises iron sulphide to iron sulphate as shown below…
__ FeS2 + __ O2  __ Fe2(SO4)2 + __ H2SO4 (balance this!)
Requirements: aerobic conditions, can survive highly acidic conditions as
______________ is produced in the reaction, preferably bacteria can work in large
range of temperature, so can be exploited in different parts of the world and at
different depths underground (it gets hotter, the deeper down you go).
Traditional methods of extraction: roasting and melting. Both environmentally
unfriendly. Require a sufficient amount of metals to be present in ores.
Advantages of bioleaching:
1. Economical: Bioleaching simpler, and therefore cheaper to operate and maintain.
Fewer specialists required to operate a complex chemical plant/factory.
2. Environmental: More environmentally friendly process. Less landscape damage
since bacteria involved grow naturally, and the mine and surrounding area can be
left relatively untouched as bacteria can work underground. Bacteria can be
recycled.
3. Process: Bacteria can work with low-grade ores, unlike roasting and melting.
Sulphur dioxide, a potentially harmful gas, is also not produced.
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Application S
BIOTECHNOLOGY
Disadvantages of bioleaching:
1. Economical: Bacterial leaching process is very slow! Hence, less profit and
significant delay in cash flow for new plants.
2. Environmental: Toxic chemicals are sometimes produced. Sulphuric acid and H+
ions can leak into the ground and surface water, turning it acidic and causing
environmental damage. Heavy ions such as iron and zinc can leak during acid
mine drainage and can precipitate when the solution is diluted by fresh water. This
may result in “Yellow Boy” pollution.
(b) Explain what is meant by the terms batch culture and continuous culture.
Growing microbes in a fermenter: given suitable nutrient medium and right conditions
(temperature, pH, oxygen levels), it is easy to grow microbes. For commercial
purposes, they are grown in large vessels called fermenters. It can be filled with a
sterile nutrient solution, which is then inoculated with a pure culture of carefully
selected bacterium or fungus. Paddles rotate the mixture so that the suspension is well
mixed. Probes, controlled by computers, monitor the changes in pH, oxygen
concentration and temperature. A water jacket surrounding the fermenter contains fast
flowing cold water to cool the fermenter since fermentation is a heat-generating
process. An exhaust pipe leaves the fermenter to remove any gases produced by cell
metabolism.
Batch culture: a method of culturing organisms in which all the components are
added at the beginning. A batch culture uses a container with a fixed volume of
growing population of a specific organism where there is a limited supply of raw
materials. Population growth follows a sigmoid pattern and is limited to a few
generations. There is a total harvest of the products at the end of the process. E.g. of
batch culture systems are microorganisms suspended in a fermenter or fish in a pond.
During the process, nothing is added to or removed from the fermenter, except for the
venting of waste gases. Product is separated from mixture at the end. Temperature is
controlled and nutrients are usually depleted at the end.
See Fig. 22.2, p 316, main textbook.
Continuous culture: a method of culturing organisms using a container (chemostat)
with a growing population of organisms that is continuously supplied with fresh
raw materials and continuously harvested in order to keep the culture in
exponential growth over long periods of time. E.g. of continuous culture systems
include microorganisms in a fermenter or fish in a commercial pond.
Bacteria grow at the same rate as they are removed from the culture via the overflow.
Nutrients are added and products removed at a steady rate throughout the process.
The rate of addition of fresh medium determines the rate of growth because fresh
medium always contains a limiting amount of an essential nutrient. It is important to
monitor pH, temperature and oxygen concentration as well as levels of nutrients and
products. All of these should be kept constant.
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Application S
BIOTECHNOLOGY
(c) Compare the advantages and disadvantages of batch and continuous culture with
reference to the production of secondary metabolites (e.g. penicillin), enzymes (e.g.
protease) and biomass (e.g. mycoprotein).
Advantages of batch culture methods:
 culture is easy to set up
 easy to control environmental factors
 should a culture become contaminated, only one batch is lost so the loss to the
manufacturer is minimal
 the level of nutrients drop, which can create the conditions necessary for the
microorganism to manufacture secondary metabolite such as penicillin.
Advantages of continuous culture methods (and hence, disadvantageous of batch
culture):
 smaller vessels can be used, given that microorganisms are maintained in the
exponential (log/growth) phase and productivity is therefore high
 high productivity for biomass (e.g. mycoprotein/eukaryotic SCP) and intra- and
extra-cellular enzymes is more cost effective
However, there are some disadvantageous to continuous culture methods in practice:
o very difficult to monitor all environmental factors – if they are not controlled
adequately, there can be considerable amount of waste
o microbial growth, clumping of cells and foaming tend to block up inlet pipes
o not possible to create low-nutrient, high-stress conditions under which secondary
metabolites are produced
Production of secondary metabolites, e.g. penicillin (antibiotic) (fed batch
culture)
Scientists are not sure why microorganisms produce antibiotics. Unlikely that it is to
fight natural enemies, since only a few species make them and only produce them late
in the microorganism’s life cycle. Two theories proposed for antibiotic production:
1. Antibiotics are secondary metabolites. They are produced after the main growth
phase is over. Making a secondary metabolite keeps enzymes active (when
substrates have been depleted) so that the microorganism can quickly take
advantage of any new food supply.
2. Producing antibiotics may be means of getting rid of metabolic waste.
Although the waste is not toxic to the organism itself, it could be highly toxic to
other microorganisms, thus acting as an antibiotic. Furthermore, when a toxin was
added to Penicillium, the production of penicillin increased.
RECALL: penicillin is a secondary metabolite – produced when nutrient level is low
and stress is high.
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Application S
BIOTECHNOLOGY
Penicillin production is done via fed-batch culture and occurs over 6-8 days. In this
process, fresh nutrients are continuously added at a very slow rate. This is because
antibiotics are produced in the greatest quantity when fungus is kept short of
nutrients. Fungus receives too many nutrients, the population grows densely but does
not produce antibiotics. If there is a lack of nutrients, culture will die. PLEASE
READ PAGES 46-47 of Applications Textbook (Microbiology and Biotechnology)
and PAGES 316-317 of main textbook.
Production of enzymes, e.g. proteases (batch culture)
Proteases are added to washing powder to help in the removal of stains. Large-scale
production of enzymes involves two stages:
1. Grow microorganism. (Usually heat-tolerant bacteria are used due to them having
enzymes that are not denatured even at high temperatures)
2. Extract enzyme, purify and concentrate for sale.
Bacteria will be provided with carbon source (usually waste product from agricultural
or other industrial processes, like left over parts of maize, remains of sugar cane, meal
made from soya beans and potatoes – this reduces cost!) and a nitrogen source (e.g.
protein, urea, ammonium salts). Bacteria or fungus are aerobic, so fermenter will be
well aerated.
Batch culture method is used.
Some bacteria or fungus secrete the enzyme produced into the medium. Some retain
the enzymes within the cells, and these will need to be extracted.
At the end, centrifugation is done to remove any microorganisms, disintegration is
done to break open cells (if enzymes are not secreted into medium), enzymes are
concentrated by reducing the water content, leftover bacteria is removed by by
antibacterial agents to prevent contamination, centrifugation is done to remove cell
debris, nucleic acids and proteins larger than the enzyme of interest, ultrafiltration to
obtain concentrated enzyme, quality control to ensure uniformity of product and
finally, packaging.
Factors to consider when selecting microorganisms for enzyme production: simple
nutritional requirements, high growth rate, non-pathogenic, does not produce
toxins/offensive odours, optimum growth temperature (thermophiles can withstand
high temperatures).
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Application S
BIOTECHNOLOGY
Production of biomass, e.g. mycoproteins/eukaryotic SCP (continuous culture)
Mycoprotein = ‘fungus protein’.
Fungus used: Fusarium. Made up of long, thin threads called hyphae.
Culture contains:
Glucose – from starch that has been hydrolysed by enzymes – respiratory substrate to
release energy, carbon source to make new carbohydrate, protein and lipid molecules
for growth
Ammonium phosphate – source of nitrogen to make proteins and nucleic acids
Small amount of zinc and copper – act as cofactors for enzymes that catalyse fungal
metabolic reactions
Ammonia gas bubbled into mixture – could be a source of nitrogen as well to make
amino acids and nucleotides.
Temperature, pH, [O2] kept constant and at optimum levels for high productivity.
No stirrer is used as this would entangle or break the hyphae. Instead, an air lift
causes circulation of the mixture by pumping compressed air into the system. This air
at the base reduces density of the mixture, which therefore rises. Gases (e.g. carbon
dioxide) produced by fungal respiration leave the mixture at the top of the vessel and
now the mixture, with increased density, descends to the bottom in the other limb of
the vessel. Therefore, air supply both agitates the mixture and supplies ocygen for
aerobic respiration.
Continuous culture is used and occurs over a period of 6 weeks. Steady input of
nutrients into fermenter and steady harvest is obtained.
The fungal hyphae contain high concentrations of RNA. Why? __________________
_____________________________________________________________________
Mycoprotein is extracted and used in the production of many different foods.
Advantages of SCP (single cell protein) production:
 Microorganisms grow rapidly when conditions are suitable so production of
biomass can be very high
 They can easily be genetically modified to vary amino acid concentration
 Protein content of microorganisms is generally high in dry mass (40-85%)
 No animal fat, no cholesterol
 Microorganisms can be grown in fermenter vessels by continuous processes
 Microorganisms can utilize a wide range of raw materials, including waste
materials. This allows them to help in removal of waste.
 Ecologically beneficial and low land requirement
Disadvantages of SCP production:
o Ratio of nucleus to cytoplasm in unicells may lead to toxic levels of nucleic acids
being produced (and will need to be broken down by RNAase enzymes)
o Original product is odourless, tasteless and colourless
o Product may be contaminated with substrates (usually cheap/waste materials used
as source of glucose/lipids for microorganisms)
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Application S
BIOTECHNOLOGY
(d) Describe, for penicillin as an example of an antibiotic: the mode of action on
bacteria and why it does not affect viruses, causes and effects of antibiotic resistace.
Antibiotics can be used to treat bacterial infections. Over 4000 different substances
have been isolated by only about 50 are useful as most antibiotics also have high
mammalian toxicity, and thus were useless therapeutically. Some of these are ‘broadspectrum’ antibiotics, while others are effective only against a few pathogenic
bacteria.
In general, antibiotics are bactericidal in a number of ways:
1. inhibit protein synthesis – interfere with transcription/translation of bacteria
2. interfere with synthesis of bacterial cell walls – these antibiotics are only effective
when bacteria are growing
3. interfere with functioning of cell membrane – bacteria will lose its ability to
control uptake or removal of water and other molecules
4. inhibit enzyme activity – this disrupts metabolism of bacteria
How penicillin works against bacteria and why it does not work against viruses:
Penicillin, which has a beta-lactam structure, works by mechanism number ___ (see
above). Is it bactericidal or bacteriostatic? ____________________. Specifically, it
inhibits enzymes involved in the synthesis of cross-links between the peptidoglycan
polymers in bacterial cell walls. The peptidoglycan links are formed using the enzyme
glycoprotein peptidases.
When a newly formed bacterial cell is growing, it secretes enzymes called autolysins,
which make little holes in its cell wall. These holes allow the wall to stretch, and new
peptidoglycan chains link up across it. Penicillin prevents peptidoglycan chains from
linking up, but the autolysins keep making new holes. Cell wall therefore becomes
progressively weaker. As bacteria are always in watery environment, they constantly
take up water by osmosis, and eventually weakened wall cannot withstand the
pressure exerted on it by the cell contents and cell bursts.
Viruses do not have any form of cell structure (or metabolism), therefore antibiotics
are not effective against them. Viruses only replicate within the living host cells, and
make use of the cell’s transcription and translation mechanisms in order to increase in
number antibiotics affect prokaryotic mechanisms only, and not prokaryotic
mechanisms.
Penicillin also does not affect human cells since human cells do not have cell walls.
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Application S
BIOTECHNOLOGY
Causes and effects of antibiotic resistance
Development of antibiotic resistance is an example of the process of natural
selection (remember from Core P?).
Bacteria, like all organisms, are genetically variable. Natural mutation gives rise to
new alleles and causes these variations. Natural selection can change the frequency of
these alleles so that most of the bacteria are resistant:
1. Within natural bacterial populations, some individuals have alleles of genes which
give resistance to a particular antibiotic (e.g. penicillin)
2. Bacteria cause infection, leading to antibiotic treatment
3. Antibiotic kills susceptible individuals, but those that are resistant survive
4. These resistant bacteria survive and will asexually reproduce, passing on the
resistance allele (which codes for penicillinase enzyme or B-lactamase enzyme,
for example), resulting in an increase in the frequency of bacteria that are resistant
to that antibiotic
5. There will be an increase in allele frequency for the allele of the gene that gives
resistance to the bacterial population
6. People infected in the future are infected by bacteria more likely to carry the
alleles for resistance
Increased and widespread use of antibiotics also allowed many strains of bacteria to
become antibiotic-resistant.
Alleles for genes that cause resistance occur due to mutation. They are often located
on plasmids, which are able to spread rapidly from one bacterial species to another
since plasmids are naturally exchanged between species. Since plasmids may also
contain other resistance genes, acquiring a single plasmid can confer resistance to a
number of different antibiotics (multiple resistance).
Effect of antibiotic resistance: extremely difficult to treat bacterial infections. It is also
constantly necessary to try and create new types of antibiotics, which bacteria are not
resistant to. It is proving almost impossible to keep ahead of development of
antibiotic resistance by bacteria!
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Application S
BIOTECHNOLOGY
(e) Immobilize an enzyme in alginate and compare the ease of recovering the enzyme
and ease of purification of the product compared to the same enzyme that has not
been immobilized. (Refer to practical W96, Question 1)
A few methods of immobilizing enzymes are:
a – enzyme non-covalently adsorbed to an
insoluble particle
b – enzyme covalently attached to an insoluble
particle
c – enzyme entrapped within an insoluble
particle by a cross-linked polymer (e.g. alginate)
d – enzyme confined within a semipermeable
membrane
Immobilizing amylase enzyme in an alginate bead:
1. Amylase is mixed with sodium alginate
2. This is then dropped through a syringe into a beaker of calcium chloride solution
3. The calcium ions displaces the sodium ions, forming hard beads of calcium
alginate with amylase trapped inside
4. After the beads have been left to harden, they are rinsed and placed in a column
5. A starch suspension can be trickled over the beads and collected in a beaker at the
bottom of the column.
The original starch solution will test positive with iodine solution but negative for
Benedict’s. However, the solution collected at the bottom of the column will test
negative with iodine solution but positive for Benedict’s, showing that starch has been
hydrolysed to maltose using the amylase enzymes immobilised in the alginate beads.
Advantages of immobilization
Easier to separate enzyme and products.
Enzymes do not contaminate product and
so no purification step required
Increases stability of enzymes over a
wide range of conditions (pH/temp) as
they are embedded so do not denature
easily
Enzymes can be recovered and reused
Disadvantages of immobilization
Immobilisation may alter shape
enzyme
of
May alter catalytic ability
Expensive so immobilizing whole cells
(e.g. yeast cells that have enzymes) are
quicker and cheaper since enzymes do
not have to be purified beforehand
Substrate can be passed through column Enzyme may become detached
of enzymes several times
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Application S
BIOTECHNOLOGY
(f) Explain the principles of operation of dipsticks containing glucose oxidase and
peroxidase enzymes, and biosensors that can be used for quantitative measurements
of glucose.
Dipsticks
Recall Core N: glucose not usually found in urine of healthy mammals although it is
filtered out at the glomeruli. This is because of total absorption at the proximal
convoluted tubules.
Diabetic patients cannot control glucose levels in their blood, hence a variable amount
will be passed out in the urine. One way to determine the quantity of insulin dosage
needed by the patient is to test a sample of urine with a glucose dipstick.
How they work:
1. Dipstick has enzyme glucose oxidase immobilize on a little pad on the surface of
the dipstick – glucose is oxidised by the enzyme  hydrogen peroxide +
gluconolactone.
2. Hydrogen peroxide will oxidise indicator chemical (chromogen which is
colourless) on dipstick via peroxidase enzyme, which causes a change in colour
ranging from green to brown.
3. The exact colour can be related to the [glucose] in the urine sample by comparing
with a colour chart to give a reading.
What kind of measurement is this? Quantitative/Semi-quantitative/Qualitative?
_________________
Biosensor
It is now more common to measure [glucose] of the blood using a glucose biosensor.
Biosensor: an analytical device that converts biological response into an electrical
signal. Often determines concentration of substances.
Benefits: rapid, accurate, simple method of measure [glucose] of blood.
Main components of the biosensor are:
(1) biological
recognition
layer
(enzyme/antibody/organelle/cell/membrane
component/living tissue) converting substrate to products – this layer determines
specificity and sensitivity of biosensor
(2) transducer (electrode) converts biochemical signal from reaction into electrical
signal
(3) output is amplified by amplifier
(4) processed by computer chip
(5) displayed on a screen
For other types of Biosensors, please read p77-79, Microbiology and Biotechnology
Textbook.
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Application S
BIOTECHNOLOGY
(g) Outline the hybridoma method for the production of a monoclonal antibody.
Monoclonal antibodies: clones of single B cells that produce a specific antibody.
Problem: B cells will not grow in culture.
Solution: Fuse them with B myeloma cells, i.e. cancerous B cells.
Myeloma cells: B cells that continue to grow and divide indefinitely, though they do
not produce antibodies.
Myeloma cells fused with B cells are called hybridomas.
Antibodies secreted by hybridomas will be the type of antibody secreted by the
original clone of B cells.
Production of monoclonal antibodies involves (Fig 22.10, p322, main textbook & Fig
5.1, p72, Microbiology and Biotechnology textbook):
1. inject mouse with antigen for which the antibodies are required
2. immune response takes place and mouse plasma cells (B cells) start to make
antibody
3. plasma cells extracted from mouse via removal of spleen 2-3 weeks after injection
4. they are fused with myeloma B cells via a fusogen, which is a chemical (e.g.
polyethene glycol, PEG) that causes cell membranes to join
5. resulting hybridoma cells are separated individually in a plate of wells, allowed to
grow, divide and produce antibodies. Those B cells that do not form hybridoma,
die. Unfused myeloma cells would be killed as well as the special medium can
only support hybridoma cells.
6. some antibodies are removed, tested with relevant antigen, to make sure they are
correct monoclonal antibodies
7. those hybridoma cells which are producing the required antibodies are cultured in
large fermenter
8. monoclonal antibodies are harvested and purified
(h) Evaluate the use of monoclonal antibodies compared to conventional methods for
diagnosis and treatment of disease, and the testing for pregnancy.
Diagnosis - monoclonal antibodies produced from a clone of B cells are all identical,
they can be use to identify macromolecules with a very high degree of specificity.
Example of diagnosis with the use of monoclonal antibodies:
1. blood typing before transfusions
2. tissue typing before transplants
3. identification of pathogens – using monoclonal antibodies, it is now possible to
distinguish between different strains of pathogens, which would, otherwise, be
very difficult
4. identification and location of tumours
5. detection of HIV
6. distinguishing between different types of leukaemia
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Application S
BIOTECHNOLOGY
Treatment – two ways in which monoclonal antibodies are used:
1. Production of passive vaccines: monoclonal antibodies can be injected directly
into the blood to attack a particular pathogen
2. ‘Magic bullets’: monoclonal antibodies can be produced which will combine
specifically with cancer cells. It is now possible to bond cancer drugs to such
antibodies. In this way, the drugs can be delivered directly to the tumour, thereby
reducing the risk of damaging healthy cells.
Disadvantages of monoclonal antibodies in treatment:
 Can be difficult to produce antibodies that bind to cancer cells and not to other
body cells.
 Monoclonal antibodies produced by mice, they are recognised as foreign by
human patient’s immune system and may be destroyed before they reach their
target site.
Pregnancy testing – soon after becoming pregnant, women produce a hormone, called
human chorionic gonadotrophin (HCG). This hormone is produced by the placenta,
so can only be present during pregnancy. Monoclonal antibodies are used to detect
presence of these hormones in urine. This can be done quickly and easily.
How it works:
1. Kit consists of a ‘sampler’, which is a type of dipstick with an absorbent pad.
2. On the surface of the pad, there are monoclonal antibodies embedded, specific to
HCG and to which coloured latex particles are attached – when the pad is
moistened, the molecules of the antibody begin to move.
3. The sampler is dipped into urine – if HCG present, it will bind to the monoclonal
antibodies and will be drawn up the pad.
4. Further up the pad is an area at which there is a line of immobilised HCG
antibodies.
5. Any HCG molecules drawn up the pad will bind with these antibodies and the
latex particles will create a coloured line. This is a positive result.
6. Further along the pad is a second line of immobilised antibodies, to which will
bind any HCG antibodies without HCG. A coloured line in this second area (but
no coloured line in the first area) will confirm that the HCG antibodies have
moved up the pad, but that the result is negative.
Also see Fig 22.11, p323, main textbook.
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