BIOTECHNOLOGY
Biotechnology is the use of living organisms
usually microorganisms- to provide us with a
substance or a process.
For example we have already seen how E.coli
can be genetically modified & used to
manufacture human insulin.
People have used biotechnology for thousands
of years . Yeast has been used to make wine &
beer.
Bacteria have been used to make yoghurt &
cheese.
Mining with microorganisms
• Metals are found in earth in the form of orescompounds of the metal that make up the rock .
Many metals that we use widely like iron ,copper
zinc, cobalt & uranium are found in the form of
metal sulphides. These are insoluble in water, which
makes it difficult to extract metal from ore.
• This is where microorganisms can help out. Several
different species of bacteria are able to oxidise the
metal sulphide to a metal sulphate .
• Sulphates are soluble in water ,so they can be
washed out of the rocks using water. This process is
called BIOLEACHING.
Explain why bioleaching is now used on a large scale throughout the world.
1. cheaper (than other methods)
2. does not require energy input
3. does not require other chemicals to be purchased
4. does not require specialist equipment
5. can be done in situ
6. less labour needed
7. bacteria are self-replicating
8. more environmentally friendly than other methods
9. useful for extraction from, low grade ores
State environmental disadvantages of
extracting metals by bioleaching
• takes up large area
• unsightly
• requires, lot of water / continuous water
supply
• contamination of water / pollution due to
acid
• Cu / Fe, toxic to plants
Explain why the production of metallic copper by
bioleaching can be cheaper than using other conventional
mining methods
• low level technology / no sophisticated machinery /
requires less maintenance
• low energy consumption / less fossil fuels used
• few safety hazards / safer
• organism easy to, obtain / culture
• self replicating
• waste less hazardous
• disposal of waste, costs less / is easier
• low grade ores / scrap iron
• less workers needed
• use in situ
• For example a bacterium called Acidithiobacillus
ferrooxidans can change iron sulphide into iron
sulphate
• 26FeS2 + 13O2--------- 13 Fe2 ( SO4)2 + 13 H2SO4
A. Ferrooxidans is an aerobic ,rod shaped
bacterium. It obtains its energy from the
oxidation of iron sulphide. The oxygen required
is obtained from the air. Similar reaction can be
catalysed by other bacteria on other sulphide
ores. Acidithiobacillus thiooxidans &
leptospirillum ferroxidans are two examples.
• About 20% of all the copper that is mined is
extracted by bacterial leaching these ores .
• It is important that the bacteria can survive in
highly acidic condition because sulphuric acid
is produced as a result of reaction.
• They also need to be able to work in a fairly
wide range of temperature, so that they can
be used in different parts of the world and at
different depths underground.
Advantages of using bacteria to mine ores
• It is a way of getting the metals from a low grade ore in
sufficient quantities to make a profit when mining the ore in
a conventional way would not be financially viable.
• It can even be used to extract valuable metals from industrial
waste or ash.
• The bioleaching does not produce sulphur dioxide, which is a
harmful gas produced when metals are extracted from their
sulphides ores.
• Bioleaching can often be used in -situ- that is the bacteria can
do the work underground rather than having to mine the
rock first & then extract the metal from it.
• The only problem is the production of sulphuric acid. Care has
to be taken not to let this flow into the environment.
Large scale production technique
• Many different organisms can be used to make the products that can be
used in medicines & food.
• This normally involves culturing microorganisms in containers called “
Fermenters”.
• The example are below
Manufacturing penicillin
Penicillin is made by a microscopic fungus called “ Penicillium”.
Penicillium does not produce penicillin all the time. Penicillin production
only begins after the fungus has been growing in the medium for a
while. It is said to be as “Secondary Metabolite” which means that we
have to keep on setting up the new fermentations . The fungus is grown
in the fermenter. Secondary metabolite production usually commences
late in the growth of the microbe, often upon entering the stationary or
resting phase .
until the maximum amount of penicillin has been
produced ,then the fermentation is stopped &
the antibiotic harvested. The fermenter is cleaned
out & the process is started all over again.
This is called batch culture.
• In standard batch culture the fermentation is set
up & then left to proceed. Nothing is added or
taken out while the fermentation takes place
,except that waste gases are allowed to escape.
• Penicillin is produced by a variant of this process
called fed batch process. During fermentation, a
carbohydrate source (often corn steep liquor) is
added about every 30 minutes . This can keep
the fermentation going on for a longer time &
therefore can produce more penicillin than the
Standard batch process.
Distinguish between batch culture and continuous
culture of microorganisms.
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batch culture
set up and allowed to proceed ;
nutrients not added or products removed, (during
fermentation) ;
air allowed in/waste gas allowed out ;
at end of each process, product harvested/fermenter
cleaned out ;
continuous culture
nutrients added (all the time) ;
products removed (all the time) ;
no down time
Manufacturing Enzymes
• All living things can produce a huge range of
different enzymes & some of these can have
important uses in the industry, medicine or food
technology.
• For example Protease & lipase enzymes are
added to washing powders to help in the removal
of stains.
• Digestive enzymes are added to cattle feeds to
increase the quantity of nutrients that the cattle
can absorb.
• Enzymes are used in the leather industry to
prepare skin.
• Enzyme production occur in two stages
• First the microorganism is grown. Then the
enzyme is extracted ,purified & concentrated.
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Steps involved in the extraction of β – galactosidase from E.coli
Fermentation– E.coli is fermented by fed batch process.
Heating and cooling process.
Centrifugation---- the cells are harvested by centrifugation.
Disintegration--- The cells are resuspended in buffer & broken
open by being forced at high pressure through a small
opening.
Extraction----The enzyme is extracted and concentrated by
removal of water
Centrifugation---- This removes cell debris, nucleic acids &
proteins larger than the enzymes.
Ultra filtration--- The enzyme concentrate is filtered.
• Many different kinds of bacteria & fungi can
be used to produce enzymes. Often a
thermophilic (heat loving) such as Bacillus
Stearothermophilus is used.
• These bacteria live in hot springs & have
evolved enzymes that are not denatured until
temperature as high as 70°C or more.
• These heat-resistant enzymes are useful in
industrial processes in which higher
temperatures are encountered & also in
products such as biological washing powders.
• To produce the enzymes ,the bacteria are
provided with a carbon & nitrogen source.
• The carbon source is often a waste product from
an agricultural process or industrial process such
as the left- over parts of maize after the grain has
been harvested, remains of sugar cane or meal
made from soya beans or potatoes.
• This helps to keep the cost down
• The nitrogen source can be protein, urea or
ammonium salts , or the remains of the yeast
cells that have been used in other fermentations.
• Usually batch culture is used , as for the
production of penicillin. The bacteria or fungi are
aerobic so the contents of the fermenter are well
aerated .
• Some organisms secrete the enzymes into the
medium around them but in some cases enzymes
remain inside the cells.
• After the fermentation has finished ,the culture is
heated to kill the cells. If the enzyme is still inside
the cell, the cells can be broken open to allow the
enzyme escape from the cells & dissolve in the
culture medium.
• This can then be concentrated & filtered ,leaving
the cell fragments behind & collecting the
enzymes in solution.
• Finally the enzymes can be purified & packed
in a form that can be used.
• Enzymes to be used in washing powders are
formed into tiny capsules covered with nonreactive substances . This is to prevent them
coming into contact with skin of a person
using the washing powder, as they can cause
irritation or allergy in some people.
Manufacturing Mycoproteins
• Mycoprotein means ‘Fungus protein’, eg ‘Quorn’.
• The fungus that is cultured to make mycoprotein is
called “Fusarium”. It is made up of long thin threads
of hyphae.
• The culture medium contains glucose , which is
usually obtained from the starch which has been
hydrolysed by enzymes. This provides the growing
fungus with the respiratory substrate for the release
of energy & also CO2 that can be used to make new
carbohydrates ,protein & fat molecules for growth.
• Ammonium phosphate is added as nitrogen source,
so that the fungus can make protein & nucleic acid
or ammonia gas may also be bubbled in the mixture.
• The temperature , pH & O2 content of the fermenter are kept
constant, providing optimum growing conditions.
• No stirrer is used because it would entangle & break the fungal
hyphae.
• The production of mycoprotein uses continuous culture. This
involves a steady input of the nutrients into the fermenter &
the steady harvest of the fungus from it.
• The liquid culture containing the fungi is run off from the base
of the fermenter & then centrifuged to separate the hyphae
from the liquid.
• The fungal hyphae contains large concentration of RNA , which
would give it an unpleasant taste.
• Enzymes are used to break this down, filtration & steam
treatment complete the process , after which the mycoprotein
can be used in the production of many foods.
• It is an excellent meat substitute , high in protein but low in fat.
Advantages of batch & continuous culture
1 Batch culture---- It is easy to set up the culture ,
we just have to provide the right nutrients in the
right concentration & then allow the
fermentation to continue with the minimal
attention.
• Once the fermenter has been cleaned ,it can used
for a completely different process, if required.
• If something goes wrong eg if culture becomes
contaminated with a different microorganism ,
then only that particular batch need to be thrown
• In continuous culture , the cells can sometimes
clump together & block inlet or outlet pipes, but
this is unlikely in batch culture.
2) Continuous culture----The process is
continuous so there is no “down time “ while
the vessels are cleaned out & set up again.
Relatively small vessels can be used because
the continual input & output means that less
space is needed to grow enough
microorganisms to give a good yield.
How Penicillin works
• Penicillin is an antibiotic (a substance that kills or stops
the growth of bacteria without harming the cells of
the infected organisms).
• Bacterial cells have cell walls made of substances called
peptidoglycans ( these are long molecules containing
peptides & sugars.
• In a bacterial cell wall ,they are held together by cross –
link that form between them.
• Penicillin inhibits the enzymes (glycoprotein
peptidases ) that build up these cross- links ,
• When the newly formed bacterial cell is growing ,it
secretes an enzyme called ‘Autolysins’,(are like
digestive enzymes lysozyme) which make little holes
in its cell wall.
• All the bacteria which have peptidoglycan have
autolysins also.
• The peptidoglycan matrix is very rigid so these
enzymes breakdown the peptidoglycan into small
sections so that the growth and cell division can
occur.
• Autolysins do this by hydrolysing the β 1,4 bond
between N-acetylmuramic acid and
acetylglucomine molecules.
• Autolysis are naturally produced by the
peptidoglycan bacteria , but excessive amount
will degrade the peptidoglycan matrix & causes
cell to burst.
• These holes allow the wall to stretch & new
peptidoglycan chain link up across.
• Penicillin prevents the peptidoglycan chains from
linking up, but there autolysins keep making new
holes.
• The cell wall therefore becomes weaker. Because
the bacteria are always in the watery
environment ,they constantly take up the water
by osmosis & eventually the weakened wall
cannot withstand the pressure exerted on it by
the cell contents and the cell bursts.
• This explains why penicillin does not effect the
human cells. Our cells do not have walls
• It also explains why it does not affect viruses,
which do not have cell walls, alone cell walls.
Causes and effect of antibiotic resistant
• Bacteria are very variable & in the colony of several million
bacteria of a single species ,there may be just by chance
one or two that have resistant genes in their cells.
• The gene that confer resistance to penicillin produces an
enzyme β- lactamase. This enzyme breaks up penicillin
molecules.
• These resistance genes are usually carried on a little
circular piece of DNA called Plasmid. When the bacteria are
exposed to antibiotics , most of them are killed, but the one
or two resistant ones may survive. They can now breed
freely ,producing the whole population of bacteria that
have inherited the resistance disease.
• Sometimes plasmids can even be transferred between
different species of bacteria . This increases the chance that
resistant strains of harmful bacteria may develop.
Immobilising enzymes
• Enzymes which are isolated from cells or tissues are
much useful if they are not in a solution , but instead
they are immobilised by being attached to ,or trapped
within ,an insoluble material.
This has several advantages over the use of dissolved
enzymes.
• Enzymes are much more stable at high temperatures.
• They are more resistant to changes in pH.
• They are less likely to be degraded by organic solvents.
• The products are uncontaminated with the enzymes &
can be collected more easily.
• The enzyme can be retailed and reused
• Use of columns of immobilised enzymes allows
automation of the industrial process.
These advantages are very important for the
industrial processes. Such processes often need
to use high temperature and extreme pH as well
as organic solvents to work efficiently.
Elevated temperatures produce an increased rate
of yield of products . Costs are also minimised if
the enzyme is not lost with the product but
trapped in the immobilised form to be used for
further reaction.
• A number of different substances have been
developed as material for binding enzyme ,
including --- alginate , cellulose , porous glass,
agar gel , nylon, collagen and porous alumina.
There are 5 basic methods of immobilisation
• Adsorption---onto a material such as porous
glass.
• Covalent bonding---onto the solid such as
cellulose
• Crossing linking---between enzyme molecules
using reagents such as Glutaraldehyde.
• Entrapment--- within the internal structure of
polymer such as collagen or alginate.
• Encapsulation--- within the selectively
permeable membrane , such as nylon
• Each method has the advantage and
disadvantage. For example covalent bonding is
commonly used but is expensive .The enzyme
form stable covalent link with the supporting
matrix but it must be ensured that the active
site on each molecule is exposed.
• Entrapment is also widely used, since the
enzyme is trapped rather than bound ,its
catalystic properties are not effected.
• Adsorption is more cheaper methods but the
enzyme can quite easily be washed from the
adsorptive material so more efficient methods
are now preferred.
• Enzyme are expensive. No company wants to
keep buying them over & over again if they can
recycle them in some way. One of the best ways
of doing it is to immobilise the enzymes.
• The enzyme is mixed with a solution of sodium
alginate . Little droplets of this mixture are then
added to a solution of calcium chloride .
• Sodium alginate and calcium chloride instantly
react to form jelly , which turns the droplets into
little beads. The jelly beads contain the enzyme
.The enzyme is held in the beads , or immobilised.
• The beads can be packed gently into a column.
• A liquid containing the enzyme ‘s substrate can be
allowed to tickle , steadily over them.
• As the substrate runs over the surface of the
beads, the enzyme in the beads catalyse a
reaction that convert the substrate into product .
The product continues to tickle down the column
, emerging from the bottom , where it can be
controlled and purified.
• Eg– the enzyme lactase can be immobilised ,&
milk is then allowed to run through the column
of lactase- containing beads. the lactase
hydrolyses the lactose in the milk to glucose &
galactose. The milk is therefore lactose free, &
can be used to make lactose- free dairy products
for the people who cannot digest lactose.
Advantages of Immobilising enzyme
• This process has several advantages compared with
just mixing up the enzyme with the substrate . For eg if
we just mixed lactase with milk ,it would be very
difficult for us to get the lactase back again. And also
the milk will be contaminated with the enzyme.
• Using the immobilised enzymes means that we can
keep & reuse the enzymes , & the product is enzyme
free.
• Another advantage is that immobilised enzymes are
more tolerant of temperature change & pH change.
This may be partly because their molecules are held in
shape by alginate in which they are embedded , & so
do not denature easily
• It may be also because the parts of the molecules that
are embedded in the beads are not fully exposed to
the temperature or pH changes.
Dipsticks & Biosensors.
• Industrially produced enzymes can be used to test for the
levels of different substances in the body fluid. For
example, an enzyme called glucose oxidase (or glucose
dehydrogenase) is used by people with diabetes to test
their blood glucose level, or to check whether their urine
contains any glucose.
• Glucose oxidase is immobilised & stuck to a little pad on
the surface of the dipstick. When it comes to contact with
glucose ,it oxidises glucose , changing it to substance
called Gluconolactone & also producing Hydrogen
peroxide.
• The little pad also contains a colourless chemical
(chromogen). When hydrogen peroxide is produced ,it
changes this chemical to another substance(to its coloured
form) that has brownish colour. The more glucose that
was present ,the darker the colour.
• One of the two enzymes immobilised in the
cellulose pad on the test strip is glucose
oxidase, which catalyses the following
reaction:
• This reaction does not result in the
development of colour by the chromogen.
This is achieved by the activity of the
second immobilised enzyme in the padperoxidase.
• Most people with diabetes , now use a more
reliable method of testing for glucose using a
biosensor. Like dipstick ,this relies on the use
of immobilised glucose oxidase which
converts the glucose into Gluconolactone.
This reaction produces a tiny electric circuit
which is picked up by an electrode on the test
strip. The more glucose that is present the
greater the current. This current is read by a
meter ,which produces a reading for blood
glucose concentration
Monoclonal Antibodies
• Plasma cells manufacture antibodies in response to the
presence of an antigen .
• Antibodies are proteins & are known as Immunoglobulins.
They are very specific ,each one complementary in shape
to a particular antigen. They bind to their antigen and
destroy it directly.
• This specificity of antibodies has made them very desirable
for use in the treatment or diagnosis of diseases.
• For manufacturing antibodies on a large scale we need a
large clone of a particular type B cells, all secreting the
identical or monoclonal antibodies.
• There is a major problem in achieving this because B
lymphocytes which divide do not produce antibodies and
the B lymphocytes(plasma cell) which produce antibodies
do not divide.
• In 1975 a breakthrough was achieved .A small
number of plasma cells producing antibodies
were fused with cancer cells, these cells unlike
other cells go on dividing indefinitely .The cells
produced by this fusion is called “Hybridoma”
. The hybridoma cells divide and secrete
antibodies. The fusion is done by polyethylene
glycol, a virus or by electroporation.
• The myeloma cells are HGPRT- and B cells are HGPRT+(
hypoxanthine –guanine phosphoribosyl transferase.
This is an enzyme involved in the synthesis of
nucleotides from hypoxanthine, an amino acid. The
culture is grown in HAT (hypoxanthine –aminopterin –
thymine)medium, which can sustain only HGPRT+cells.
• The myeloma cells that fuse with other myloma cells
or do not fuse at all die in the HAT medium since they
are HGPRT - . The B cells that fuse with other b cell or
do not fuse at all also die as they are HPRT + and they
do not have the capacity to divide indefinitely . Only
hybridomas between B cells and myeloma cells survive
, being HGPRT + cancerous.
Pregnancy testing kits
• Monoclonal antibodies now have many different uses in
medicine. One such use is in pregnancy testing kits.
• Most pregnancy kits use monoclonal antibodies to test
for the presence of (hCG) Human chorionic
gonadotrophin in her urine.
• Monoclonal antibodies are immunoglobins that are
identical with one another. For pregnancy testing
,antibodies are made that will bind with the hormone .
This is done by injecting hCG into a mouse & then taking
blood from it after its leucocytes have had time to
respond to the hCG as an antigen ,producing antibodies
that are specific to hCG.
• One of these cells is isolated.
• It is then fused with a mouse myeloma cell( a type of
cancer cell) .polyethylene glycol is used to fuse the plasma
membrane of both the cells. This process is hybridoma
• The cell is grown & forms a clone in which all the cells
secrete the one antibody- a monoclonal antibody.
• For one type of kit , these hCG –specific antibodies are
bound to the particles of gold.
• The antibody-gold complexes are then used to coat the end
of a dipstick.
• Another type of a monoclonal antibody is also made ,
which will specifically bind with hCG-antibody complexes.
These are impregented into a region called patient test
region & immobilised.
• To use the dipstick it is dipped into a urine sample.
• Any hCG in the urine will bind to the antibodies at the end
of the stick & will be carried upwards as the urine seeps up
the stick.
• As the hCG- gold complexes reach the test result
region of the stick , they bind to the immobilised
antibodies there & are held firmly in position.
• As more & more particles arrive a pink colour
builds up.
• There is another strip called Procedural control
region, that contains immobilised monoclonal
antibodies.
• These are from goats & they are anti-mouse
antibodies.
• They bind with the antibody –gold complexes
even if these have not encountered & bound
with hCG in the urine sample . This strip therefore
even goes pink even of the result is negative.
Using monoclonal antibodies in diagnosis
• Monoclonal antibodies have many different uses
in medicine . Example --- It can be used to locate
the position of blood clots in the body of a
person thought to have a deep vein thrombosis.
• The antibodies are produced by mouse with
human fibrin( The main protein found in the
blood clots) . The mouse makes many B
lymphocytes that secrete the antibodies against
fibrin, and these plasma cells are collected from
its spleen.
• The plasma cells are fused with cancer cells to
form Hybridomas that secrete the antifibrin
antibody, which is labelled using a radioactive
chemical that produces gamma radiations.
• The labelled antibodies are then introduced
into patient’s body in the blood stream.
• As they are carried around the body in the
bloodstream ,they bind to any fibrin
molecules with which they come into contact .
A gamma –ray camera is then used to detect
the position of antibodies & therefore blood
clots, in the persons body.
• Many biotechnology companies now
manufacture monoclonal bodies to diagnose
hundreds of different medical conditions.
• They can be used to track down cancer cells
,which have different proteins in their cell
surface membrane than normal body cells and
can therefore be picked out by antibodies
• They can also be used to detect exactly which
stain of virus or bacterium is causing an
infection which can speed up the choice of
most appropriate treatment for the patients.
Using monoclonal bodies in treatment
• Monoclonals can also be used to deliver drugs to
the cancer cells . These are called “magic bullets”
because they can pick out just cancer cells
,avoiding harm to other body cells.
• Once the monoclonals have been made to match
up with a protein found on the surface of the
cancer cells, molecules of an anticancer drug are
attached to them .
• The monoclonals are injected to the patients
body, They attach only to cancer cells , avoiding
harm to other cells .
• The drug therefore destroys the cancer cells but
not other cells.
• At the moment , success with this technique
has been limited for a number of reasons.
• It can be difficult to produce antibodies that
bind only to cancer cells and not to other cells.
• Because monoclonal antibodies are produced
by mice , they are recognised as foreign by
human patient’s body & may be destroyed
before they reach their target.