1. Title: The growth curve Homework: complete learning package 1214 January 2016

2. Learning Outcomes Describe, with the aid of diagrams, and explain the standard growth curve of a microorganism in a closed culture.

3. Standard Growth curve

4. Growth curve in a closed culture Lag phase – Bacteria adjusting to new conditions – Takes a while for enzyme production Log phase – Number of bacteria increase rapidly Stationary Phase – Rate of growth is equal to rate of death Decline Phase – Death rate is greater than “birth rate” The first three stages represent a sigmoid growth curve

5. Primary and secondary metabolites Primary metabolites – Substances produced by on organism as part of its growth – Amino acids, proteins, enzymes, nucleic acids, ethanol and lactate – Production matches growth of population of organism Secondary metabolites – Substances produced not as part of normal growth – Antibiotic chemicals are mostly secondary metabolites – Begins after main growth period and does match population growth.

6. Learning Outcomes Explain the importance of manipulating the growing conditions in a fermentation vessel in order to maximise the yield of product required.

7. Large-Scale production Microorganisms are cultured in large containers called fermenters The growing conditions within the fermenter are manipulated and controlled – Precise growing conditions Temperature Type and time of the addition of the nutrient Oxygen concentration pH

8. A batch fermenter

9. Large scale production Three examples are – The production of penicillin – The production of protease enzymes – The production of mycoprotein

10. Learning Outcomes Compare and contrast the processes of continuous culture and batch culture. Describe the differences between primary and secondary metabolites.

11. Metabolism and metabolites Metabolism (process) – Sum total of all the chemical reactions – Processes produce New cell and cell components Chemicals Waste products Metabolites (products) – A substance produced during cell processes

12. Primary and secondary metabolites Primary metabolite – Substance produced by organism as part of it’s normal growth – E.g. amino acids, proteins, enzymes – Production of primary metabolites matches the growth in population Secondary metabolite – A substance only produced at a particular growth phase – No direct involvement in fundamental metabolite processes – Production usually begins after the main growth phase of the micro organisms

13. Batch culture Starter population is mixed with a specific quantity of nutrient solution Allowed to grow for a fixed period Products removed Fermentation tank emptied Examples Penicillin production Enzyme production

14. Continuous Culture Nutrients are added and products are removed from the fermentation tank at regular intervals Examples Insulin production from genetically modified E.Coli Production of mycoprotein

15. Learning outcomes Explain the importance of asepsis in the manipulation of microorganisms

16. Asepsis – absence of unwanted microorganisms Aseptic techniques – Any measure taken during a biotechnological process to prevent contamination by unwanted microorganisms

17. The importance of asepsis Unwanted microorganisms – Compete with the culture microorganisms – Reduce the yield of useful products – Cause spoilage of the product – Produce toxic chemicals – Destroy the culture microorganism or its products.

18. Methods to maintain asepsis Ensure all fermenters and attachments are sterile – Cleaning with pasteurised steam – Chemical sterilisation Sterilise all liquids, solids and gases that enter the reaction vessel Maintain a pressure difference between the air in the room where fermentation is taking place and outside – Maintains a steady airflow out of the room Ensure culture of microorganisms is pure Ensure the workers do not introduce unwanted microorganisms from their skin.

24. Learning Outcomes Describe how enzymes can be immobilised. Explain why immobilised enzymes are used in large-scale production.

25. Immobilising enzymes Enzymes act as catalysts in metabolic reactions Enzymes are useful in industrial processes – Specificity – Temperature of enzyme action Enzymes in solution need to be separated from the products. Immobilised enzymes can be re-used many times and leaves the product enzyme free.

26. Methods for immobilising enzymes Gel entrapment – Example – immobilising lactase in alginate – Stages Enzyme solution is mixed with sodium alginate solution Droplets of this solution are added to a solution of calcium chloride The droplet turns into a bead which contains the enzyme

27. Immobilising lactase in alginate

28. The beads can be tightly packed into a column The liquid substrate can be trickled over the beads The product trickles out of the bottom of the column The product is collected and purified.

29. Methods of immobilising enzymes Adsorption / carrier bound – Enzyme molecules are mixed with immobilising support e.g. glass beads or clay Covalent Bonding / cross-linked – Enzyme molecules covalently bonded to a support

30. Methods of immobilising enzymes Entrapment / inclusion – Enzymes trapped in their natural state in a gel bead – Reaction rate can be reduced as substrate needs to get through the trapping barrier Membrane separation – Substrate separated from the mixture by a partially permeable membrane.

31. Advantages of immobilised enzymes The advantages of using immobilised enzymes over enzymes in solution are – Immobilised enzymes can be reused – Product is enzyme free – Immobilised enzymes are more tolerant to pH and temperature changes