1. Biotechnology basics State that biotechnology is the industrial use of living organisms (or parts of them) to produce food, drugs or other products. What sort of organisms are involved? See page 159

2. Biotechnology basics Explain why microorganisms are often used in biotechnological processes. Fast growth Often secrete products into growth medium Can be genetically engineered easily Grow well at low temp, lower that chemical processes Not climate dependent, can be grown in culture anywhere Produce purer product than chemical processes Can utilise waste products as growth medium

3. Biotechnology basics What is a culture? What is a mixed culture? What is nutrient broth? How is nutrient broth different from nutrient agar? What is a fermentation tank?

4. © Pearson Education Ltd 2009 This document may have been altered from the original Describe and explain, with the aid of diagrams, the standard growth curve of a population of microorganisms in a closed culture. What are the 4 phases on the graph and why do they occur?

5. Describe the differences between primary and secondary metabolites. What is metabolism? What are primary metabolites? What are secondary metabolites?

6. Growth and product curves showing the production of (a) a primary metabolite and (b) a secondary metabolite Primary metabolite and Secondary metabolite Primary metabolites are made during normal growth, eg proteins, enzymes, nucleic acids, ethanol, lactate etc. Their production rate follows the growth curve Secondary metabolites are not produced during normal growth. Their production usually starts after the normal growth phase Not all microbes produce secondary metabolites

7. Describe how enzymes can be immobilised Adsorption onto clay, resin, glass beads and porous carbon. Enzymes are held on the surface by hydrophobic interactions and ionic links. Good exposure of active sites. Enzyme not tightly bound so may leach off surface and be lost. Bonding may affect active site. Covalent bonding to other enzyme molecules and to a support. Enzyme tightly bound so little leakage from support but cannot bind large amounts of enzyme. Bonding may affect active site. Entrapment within a gel bead or cellulose fibres. Reaction rates can be slowed because substrate molecules have to get through the gel or fibres to get to the active sites. Membrane separation Partially permeable membrane separates enzyme and substrate. Substrate passes through, reaction takes place, products pass back through membrane to be collected. Immobilised Enzymes

8. © Pearson Education Ltd 2009 This document may have been altered from the original a)Adsorption on clay etc b) Covalent linking c) Trapped in fibres

9. © Pearson Education Ltd 2009 This document may have been altered from the original Explain why immobilised enzymes are used in large- scale production. ADVANTAGES Enzymes are kept separate from the products so purification costs are low Enzymes immediately available for re-use, no need for complicated extraction process Immobilised enzymes are more stable because the immobilising matrix protects the molecules (eg from temperature changes or pH changes) Immobilised Enzymes

10. © Pearson Education Ltd 2009 This document may have been altered from the original Explain the problems with immobilised enzymes in large- scale production. DISADVANTAGES Immobilising enzymes is more expensive Immobilised enzymes may give slower reaction rates as molecules do not mix freely with substrate molecules Substrate has to diffuse into bead structures or through mesh Contamination is more difficult to deal with Immobilised Enzymes

11. Immobilised organisms Sometimes microorganisms are immobilised instead of individual enzymes. Advantages Enzymes unlikely to leach out into product Microorganisms make the enzymes necessary Disadvantages Reaction rates may be slower as substrate has to enter through cell membrane Other unwanted products may be made in addition to the required one, these then need separating out.

12. © Pearson Education Ltd 2009 This document may have been altered from the original The production of penicillanic acid using an immobilised enzyme reactor As microbes become resistant to penicillin biochemists try to produce new antibiotics. They put penicillin through immobilised penicillin acyclase. This produces 6 amino penicillanic acid which can be used as a base molecule to produce a range of penicillin- like antibiotics Currently microbes are not resistant to these new molecules

13. © Pearson Education Ltd 2009 This document may have been altered from the original Compare and contrast the processes of continuous and batch culture. Batch Growth slower as nutrients decline Easy to set up and maintain Only one batch lost if contamination occurs Less efficient, fermenter not always in use Useful for producing secondary metabolites Continuous Growth faster as always nutrients there Difficult to set up and maintain Huge volumes lost if contamination occurs More efficient fermenter in use constantly Useful for producing primary metabolites

14. © Pearson Education Ltd 2009 This document may have been altered from the original Manipulating the growing conditions in a fermentation vessel in order to maximise the yield of product required.

15. Explain the importance of asepsis in the manipulation of microorganisms. Asepsis is ensuring there is no entry of any possible contaminating microorganism that could: Compete for the nutrients in the growth medium Compete for space in the fermenter Reduce the yield of product from cultured microbes Cause spoilage of the product Produce toxic chemicals which could be harmful to users of the product Destroy the culture organisms and/or their products

16. Aseptic Technique Lab measures Sterilise all equipment before and after transfers. Dry heat, eg transfer loops; steam/ autoclave, eg liquid or agar medium UV treat, eg plastic petri dishes; ethanol flamed, eg forceps etc Air currents from Bunsen burners or lamina flow cabinets carry airborne contaminants away Cultures kept closed at all times when not in use Industrial measures Wash, disinfect and steam clean fermenter and pipes Surfaces made of easy clean stainless steel High temperature and pressure (autoclave) sterilisation of all culture media before adding to fermenter Micro filters on inlets and outlets prevent unwanted microbes entering or leaving accidentally