Module 6 6.2.1 Biotechnology By Ms Cullen

What is biotechnology? Examples:

Medicine

Food Production and Processing Agriculture & Aquaculture Inherited disease resistance Herbicide / insecticide resistance Increased nutritional value Improved stress tolerance Increased production rate Food Processing Fermentation technology Enzymatic modification Improved process control and efficiency Enhanced characteristics (eg flavour, shelf life, nutritional value) Diagnostic food testing

Environment

Industry

Why are microorganisms used in biotechnology? They have rapid life cycles (30 mins generation time, time taken to double) Reproduce asexually so their populations are genetically identical Can be genetically engineered to produce specific products Simple requirements for growth, can often be grown in fermenters Can often be grown using waste products from industry Do not usually raise ethical issues

Why are microorganisms used in biotechnology? Can be grown anywhere in the world, they are not climate dependent Products generated tend to be purer than those generated from chemical processes Grow well at low temperatures Often produce proteins or chemicals which are given out to surrounding medium and can then be harvested

Investigating population growth Activity 20 Investigating population growth

Haemocytometer Used to count cells. Serial dilution – dilutes sample otherwise too many cells to count accurately. Agitate – to prevent cells clumping. Wait 5 mins – allows time for cells to stop moving and settle. North West rule – prevents counting same cell twice. Count cells in number of squares – to give a more accurate estimate.

The standard growth curve for a culture of microorganisms

Questions: Explain why there is an initial lag in the growth of a microorganism placed into a new culture. Suggest how this lag can be reduced when starting a new culture. Describe the effect on microbial growth of adding fresh nutrients and removing toxic by-products.

Primary metabolites During the log phase many intermediate metabolic products are produced, which are needed by the microbes for growth or to provide energy. These are known as primary metabolites and are produced in excess and will accumulate in the culture. They can then be extracted and purified. Primary metabolites form at same rate as the cells grow, therefore their growth curves are very similar. Example: production of ethanol from yeast

Secondary metabolites These are organic compounds, but they are not necessarily essential for survival of the microorganisms. They are often produced as defence mechanisms or to allow the organism to compete with another. Many secondary metabolites have beneficial uses to humans eg morphine, atropine, penicillin Secondary metabolites are produced after the active phase of growth has ended and the culture is in the stationary phase. A limited number of microorganisms produce secondary metabolites, when 1 or more nutrient is depleted from the growth medium. Many important antibiotics are produced as secondary metabolites from bacteria and fungi.

Growth and product curves showing the production of (a) a primary metabolite and (b) a secondary metabolite 15

Question: Explain why the curve for a primary metabolite production closely resembles the microorganisms growth curve. Explain why this is not the case for production of a secondary metabolite. Explain the differences between a primary and a secondary metabolite in microbial culture.

Generalised diagram showing the features of a large-scale industrial fermenter

Fermenter Read through biofact sheet 33 on fermentation

Asepsis This is the practice of preventing contamination of cultures by unwanted microorganisms. Q Why is this important?

Q What aseptic techniques can be used to prevent contamination?

The Effect of Antibiotics on Bacterial Growth PAG 7.1 The Effect of Antibiotics on Bacterial Growth

Industrial Enzymes

Enzymes Enzymes are important in commercial and industrial processes as they accelerate chemical reactions to produce a useful product or effect. Some enzymes used remain in their cells, others are extracted and purified. In each case if the enzymes are trapped within an insoluble agent their efficiency is increased. This is called enzyme immobilisation.

Enzymes and their uses Enzyme Substrate Uses Proteases Protein Washing powder, leather production, treatment of blood clots Amylases Carbohydrates Sweetners Lipases Lipids Washing powder Cellulases Cellulose Fabric conditioners, fruit juice production – prevents cloudiness Lactases Lactose Producing lactose free milk Pectinases pectin Fruit juice production

Enzymes Enzymes are sourced from a variety of microorganisms, bacteria, fungi and yeast. The bacteria Bacillus and fungi Aspergillus are particularly useful. Microorganisms can be genetically modified to increase the yield of enzymes they produce. The enzymes are purified by grinding cells and/or adding alkalis. The cell debris is then removed by filtration and centrifugation. The enzymes are then precipitated using ammonium sulphate. Further techniques may involve techniques such as filtration, chromatography and electrophoresis.

Immobilising enzymes Enzymes are expensive and therefore being able to recycle them is a cheaper option. The best way to do this is by immobilising them. An enzyme solution is mixed with a solution of sodium alginate. Little droplets of this mixture are added to a solution of calcium chloride. The sodium alginate and calcium chloride react to form a jelly, which turns the droplets into little beads. The enzyme is held in the bead and is therefore immobilised.

Now have a go yourself! Complete steps 1-6 of Activity 22 - Investigating Immobilised Pectinase Whilst you are waiting for 10 minutes: Complete Qs 4 and 5 on P.187 in OCR A2 Biology books

Now complete Activity 22 and graph your results Complete Qs for Activity 22

Activity 23 – Investigating Immobilised Lipase Draw a graph of your results and answer Qs