BiotechnologyThe use of living organisms (usually bacteria) to provide us with a substance or a process.

Uses are generally in industry (manufacturing or food) or in medicine

• Thiobacillus ferrioxidans is used to oxidise the metal sulphides into metal sulphates.

• The sulphate is then soluble in water so it can be washed out of the rocks.

• This is called bioleaching.

• The bacteria can usually survive in highly acidic conditions and in a range of temperatures.

Industrial applications of microorganisms

• Some bacteria are chemoautotrophic (get energy from the breakdown of inorganic chemicals).

• Metals are found in the ground as metal sulphides (heavy metals)

• Bacteria are used to change these in to pure metals

• Metals which can be extracted in this way – copper, uranium, cobalt, lead, nickel and gold.

• Bacteria of the genus Thiobacillus are used

• Advantages of using bacteria to mine ores in this way:

• – Getting metal from low grade ore in sufficient quantities to make a profit

• – Extract valuable wastes from industrial wastes

• – Bioleaching does not produce sulphur dioxide

• – Can often be used in situ• Potential problem:–Production of sulphuric

acid

Large-scale Production Techniques• Penicillin

• Made by Batch Culture.• Penicilium only produces penicillin after it has been

growing for a while.• It is called a secondary metabolite.• Fermentation is carried out in a closed or batch

fermenter. • Microorganisms and a nutrient medium are added to the

fermenter and left for a period of time.• During the process, nothing is added to or removed

from the fermenter. • The penicillin is separated from the mixture at the end. • Temperature is controlled and nutrients are usually

depleted at the end.

• Enzymes can also be manufactured using a batch culture.

• Eg. Protease for washing powders, which “react” in the wash with organic stains

• Bacteria provided with a carbon-source (usually CO2) and a nitrogen-source (usually ammonia).

• After fermentation the culture is heated to kill the cells.(The enzyme can withstand high temperatures)

• The mixture is then filtered and the enzyme comes out in solution, leaving the dead cells behind.

Mycoprotein

Continuous Culture• Fermentation of a fungus, Fusarium, is carried

out in an open fermenter. • Nutrients are added and product removed at a

steady rate throughout the process. • Maintains the microorganisms at the exponential

phase of growth. • Important to monitor pH, temperature and

oxygen concentration as well as levels of nutrients and product.

• All of these should be kept constant.

Comparing Batch and Continuous culture

• In general, batch culture methods have certain advantages over continuous culture.

• These are: 1. easy to set up 2. the environmental conditions are relatively easy to control 3. the types of vessels used can be used for different

processes at different times4. if the culture becomes contaminated, it is only one batch

that is lost5. the level of nutrients drops, which can create the conditions

necessary for the microorganism to manufacture secondary metabolite such as penicillin

Some advantages of continuous culture are:

1. carried out in smaller vessels, given that the microorganisms are maintained in the exponential phase and productivity is therefore high

2. the high productivity for biomass and intra- and extra-cellular enzymes is more cost effective

Disadvantages of continuous culture are: • microbial growth, clumping of cells and

foaming can tend to block up inlet pipes • it can be difficult to control all the

environmental factors – if they are not controlled adequately, there can be a considerable amount of waste

• it is not possible to create the low-nutrient, high-stress conditions under which secondary metabolites such as penicillin are produced

• Antibiotic – a chemical substance, produced by a microorganism (bacterium or fungus) which will inhibit the growth or replication of other microorganisms.

• Antibiotics bring about their effects in a number of ways : • inhibiting protein synthesis – interfering with transcription or translation. • interfering with the synthesis of bacterial cell walls – only effective when the bacteria are growing. • interfering with the functioning of the cell membrane – the bacteria will lose it’s ability to control the uptake or removal of water and other molecules. • inhibiting enzyme activity – this will disrupt metabolism.

• Penicillin is described as a broad spectrum antibiotic

• It works by interfering with the synthesis of new cell walls.

• It inhibits the enzymes involved in the synthesis of cross-links between the peptidoglycan polymers in bacterial cell walls.

The mode of action of penicillin on bacteria

Gram positive bacteria stain purple with Crystal Violet dye, and gram negative ones stain pink with safranin stain. Named after a Danish bacteriologist, Hans Christian Gram, and divides bacteria into two broad groupings

Porins are large protein channels, which allow passive diffusion

• Viruses do not have any form of cell structure or metabolism – hence, antibiotics are ineffective against viruses.

• They replicate only within the living host cells, and make use of the living host cell’s transcription and translation mechanisms. The absence of any sort of cell wall means that penicillin has no effect on viruses.

The causes and effects of antibiotic resistance

• Bacteria are genetically variable. These variations occur by natural mutation giving rise to new alleles of genes.

• Natural selection can change the frequency of these alleles in the population so that most of the bacteria in the population are resistant, by the following steps:

• some bacteria have alleles of genes which give resistance to a particular antibiotic

• an infection leads to treatment of the infected person with an antibiotic

• the antibiotic will kill susceptible bacteria, but resistant bacteria will survive

• only the resistant bacteria will reproduce, resulting in an increase in the frequency of the bacteria that are resistant to that particular antibiotic. An example of directional selection.

• people infected in the future are infected by bacteria more likely to carry the alleles for resistance

• The alleles of genes that cause resistance arise for the first time by mutation and are often located on plasmids, which means that they can rapidly spread from one bacterial species to another since plasmids are naturally exchanged between species. Bacteria of different species may come together, and the plasmids will transfer either by direct contact or through a special channel.

• The plasmids may also contain a number of different antibiotic resistance genes so that species can suddenly acquire resistance to a number of antibiotics when before they had none.

Enzyme immobilisation

• the attachment of enzymes to insoluble materials, which then provide support for the enzymes.

• This allows enzymes to be held in place throughout the reaction, following which they are easily separated from the products and may be used again.

Different ways to immobilize enzymes

The process of immobilization in alginate beads involves the following stages:

– An enzyme is mixed with a solution of sodium

alginate – this mixture is dripped (usually through a

syringe) into a solution of calcium chloride – the sodium ions are displaced by the calcium

ions, resulting in the formation of hard, insoluble beads of calcium alginate, in which are trapped the molecules of amylase

– the alginate beads are left to harden further, and then rinsed, and stored for future use.

Example 1: Using immobilized lactase to prepare lactose free milk for people with lactose intolerance

• Example 2. Producing Maltose from starch

• The beads (having been made to include amylase enzyme) are placed in a suitable container to create a column of beads.

• A suspension of starch can then be trickled down the column and the product is collected in a beaker.

• a solution of maltose, without any starch will be present in the beaker

• By hydrolysing the starch this way, there is no contamination of the product with enzyme – and the amylase remains in the beads, which can then be used again.

advantages of immobilisation:

• enzyme can be recovered after use using a very coarse filter rather than a molecular filter

• enzyme does not contaminate product • immobilisation may enhance stability

(thermostability or pH-stability) of the enzyme molecule as it is supported

• substrate can be easily passed through the enzyme several times

• Industrially produced enzymes are used to test for the different levels of substances in the body.

• Glucose oxidase is used by diabetics to test their blood glucose concentration.

• It is immobilised and stuck on the end of a dipstick.

Biosensors and dipsticks

Dipsticks. These ones test urine for Glucose (diabetes), Blood (kidney problems),Protein (liver problems),PH (metabolic imbalance).

• When it comes into contact with glucose it oxides it to gluconolactone and causes a colour change.

• The more glucose the darker the colour.

• Biosensor - a device which makes use of a biological molecule to detect and measure a chemical compound.

• Most people now use a biosensor which detects an electric current genetrated during this oxidation reaction, which is read by a meter, and displays blood sugar levels.

Biosensor and test-strips

Revision: Antibodies

In our immune system one type of lymphocyte is a B Lymphocyte.

This divides by mitosis to create copies of itself.

Sometimes it divides to become a Plasma Cell, which will produce antibodies in response to a foreign chemical (antigen).

When a foreign cell invades an organism, there are a lot of different types of antigen on its surface membrane, so the B lymphocytes divide to make lots of different plasma cells to counteract each different antigen.

Monoclonal antibodies

Identical antibodies produced to be effective against a single, specific antigen.

• Problem with producing them – B lymphocytes that produce antibodies don’t divide, and B lymphocytes that divide (making plasma cells) don’t produce antibodies!

HAT medium is hypoxanthine aminopterin thymidine. Only hybridomas survive. Myeloma dn normal cells die.

• Plasma cells are fused with cancerous cells which go on dividing indefinitely.

• This formed a hybridoma which divides and secretes antibodies.

Using monoclonal Antibodies

• 1. Pregnancy Tests• The monoclonal antibodies are made that

bind with the human hormone human chorionic gonadotrophin. (hCG)

• The antibody is attached to a dipstick.• The dipstick is dipped into a urine sample.• Any hCG in the urine will bind to the

antibody and will be carried up the stick with the urine.

• Another antibody is made which will bind with the hCG-antibody complex.

• This is placed further up the stick and immobilised.

• As the hCG-antibody reaches the immobilised antibody it binds and a pink colour forms. (or blue, depending on the brand)

As a test, at the top of the stick a different antibody has been imobilized. This comes from cells of a different animal (often a goat), which will naturally react (by binding) to the antibody at the bottom of the stick.

It will only bind with mouse antibodies that have NOT got the hCG hormone attached to it, and again produces a coloured band.

One band: Negative, as there was no hCG.Two bands: Positive, as some of the mouse antibody has hCG bount to it.

Monoclonal antibodies in diagnosis and treatment