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 ores-

compounds 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.

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. Steps involved in the extraction of β – galactosidase from E.coli1) Fermentation– E.coli is fermented by fed batch process.2) Heating and cooling process.3) Centrifugation---- the cells are harvested by centrifugation.4) Disintegration--- The cells are resuspended in buffer & broken

open by being forced at high pressure through a small opening.

5) Extraction----The enzyme is extracted and concentrated by removal of water

6) Centrifugation---- This removes cell debris, nucleic acids & proteins larger than the enzymes.

7) 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 non- reactive 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 pad-peroxidase.

• 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.