Overview

1. Overview Media Organism P R O C E S S

2. Industrial Microbiology Handling the process What is a bioprocessor (fermenter)?

3. Outline • Industrial batch cultures • Inoculum development • When do we harvest? • Fed batch cultures • Continuous processes • Characteristics of bioprocessors • Aeration and agitation • Ph and temperature control

4. Achieving good volumetric productivity in a batch system • REMINDER • Volumetric Productivity • The amount of product produced per unit volume of production bioprocessor per unit time (or, in crude terms “how fast does the process go”) • NOTE: “Time” includes down time, turn-round time etc. • High Volumetric Productivity minimises the contribution of fixed costs to the cost of the product.

5. What are fixed costs? • Fixed costs are business expenses that are not dependent on the level of product produced. • They tend to be time-related, such as salaries Plant, Power, etc.

6. Product Conc. Time Product formation in a batch culture Fastest production rate

7. Product Conc. Time How to achieve good volumetric productivity • Maximise the proportion of time spent at the fastest production rate by: Fastest production rate

8. Product Conc. Time How to achieve good volumetric productivity • Minimising the lag before maximum production starts • Inoculum development

9. Product Conc. Time How to achieve good volumetric productivity • Avoiding subequent phases of slower/zero production • Choice of harvesting time

10. Product Conc. Time How to achieve good volumetric productivity • Extending the length of time spent in active production • Fed batch can do this

11. Product Conc. Time How to achieve good volumetric productivity • Minimise proportion of time lost as turn-round time • Fed batch • Continuous processes

12. Product Conc. Time How to Achieve Good Volumetric Productivity • Ensure that production is rapid • Choice of medium and organism • High concentration of active organisms • Inoculum development Faster production = steeper slope

13. Key points are: • Inoculum Development • When to Harvest • Extend the Production Phase by Fed- Batch or Continuous cultures

14. Inoculum Development • Inoculum is built up through a series of stages • Production fermenter is inoculated with 3-10% of its total volume • Inoculum contains • A high concentration of active cells • Ready to commence maximum production with a minimal growth requirement

15. Advantages of Proper inoculum Development • High volumetric productivity: • Immediate commencement of production at maximum rate in the production fermenter. • A good concentration of active cells ensures a good production rate..

16. Advantages of Proper Inoculum Development • Balancing growth and production: • Optimise inoculum build-up for growth and production fermenter for production. • Minimise contamination problems. • A large healthy inoculum will out-compete contaminants. • It is economical to discard early stages of build-up which are contaminated. • Correct form of fungal mycelium during production. • Diffuse or pellets.

17. Product Conc. Previous harvest time Time Batch Bioprocesses –Harvesting • When to harvest for best volumetric productivity • Maximum overall rate of product formation (remember to include turn-round time)

18. Product Conc. Previous harvest time Time Batch Bioprocesses –Harvesting • When to harvest for best titre/yield • First point at which maximum concentration is reached

19. Product Conc. Previous harvest time Time Batch Bioprocesses –Harvesting • NOTE that the two potential harvesting points are different

20. Fed batch culture • Substrates are pumped into the fermenter during the process P

21. Fed batch culture • Substrates are pumped into the fermenter during the process P

22. Fed batch culture • What is added? • Medium • Medium component – for example: • Carbon source • Precursor • When is it added? • To a predetermined programme • In response to changes in process variables • pH • O2 concentration

23. Fed batch culture • Can be used to extend the production phase • Substrate may be used as fast as it is added – concentration in the bioprocess is always limiting: • Catabolite repression avoided even with readily used carbon sources (e.g. glucose) • Precursors used efficiently for their correct purpose • Avoid toxicity problems with some substrates • Efficient yeast biomass production on readily used carbohydrates (avoiding the Crabtree effect )

24. The Crabtree Effect. • In the presence of an excess of sugar, yeasts switch from aerobic to anaerobic (alcohol producing) metabolism, even under aerobic conditions. • High Levels glucose accelerates glycolysis, produces ethanol rather than biomass by the TCA cycle

25. Fed batch culture • Rate of addition controls rate of use • Programme changes in metabolic rate i.e. can add slow or fast depending on stage of culture • Avoid oxygen demand outstripping oxygen supply • Status of fed batch culture in industry • Common • More often used than non-fed cultures?

26. Continuous Processes • Pump in medium (or substrates). • Remove culture or spent medium plus product. • Types usually encountered in industry: • Simple mixed system with medium input and culture removal (the Chemostat). • Systems with cell recycle or retention. • Dilution rate (D) is the rate of flow through the system divided by the culture volume. • Units of time-1.

27. The Chemostat • The system will settle to a steady state, where: • Growth rate = dilution rate (μ =D) • Growth is nutrient limited • Growth is balanced by loss of cells through overflow • Unless the dilution rate is too high (D>μmax), when the culture will wash out Chemostat

28. The Chemostat • Not used extensively in industry, • Illustrates the advantages and disadvantages of continuous systems • Disadvantages may be minimised by the use of cell recycle or retention (discussed later)

29. Overview Media Organism P R O C E S S

30. Last Thursday: The Process: • Industrial batch cultures • Productivity and Costs • Inoculum development • When do we harvest? • Fed batch cultures • Started: Continuous processes • Advantages • Disadvantages

31. Today: • Recap advantages and disadvantages of chemostats • Chemostats with recycle • Status of Chemostat Culture in Industry • Industrial and Lab-Scale Bioprocessors

32. Continuous Systems – Industrial Advantages • All the advantages of fed batch Plus • High volumetric productivity: • In theory,operates continuously at the optimum rate. • In practice, re-establishment (turn round) needed at intervals but less often than batch. • Can handle dilute substrates. • Easier to control. • Spreads load on services.

33. Continuous Systems- Problems • Poor yields. • Substrate constantly needed for growth in chemostats. • Unused substrate lost in overflow. • Generate large volumes for downstream processing, often with a poorer titre than batch systems.

34. Continuous Systems- Problems • Constant growth means more chance of mutation/selection. • Chemostats are powerful selection systems for “fitter” mutants or contaminants. • “Fitter” means able to GROW faster under culture conditions. • Greater knowledge/familiarity with batch systems.

35. Continuous Systems- Problems • Existing plant designed for batch operation. • True continuous operation means upstream and downstream processing must also be continuous. • Many (not all) these problems may be minimised by using cell recycle or retention.

36. Continuous Processes with Cell recycle or Retention. • Cells retained in the bioprocessor or removed from the effluent and returned. • Growth rate does not have to equal D for steady state: • Growth rate is less than D. • Growth rate can, in theory be zero with 100% cell retention.

37. Continuous Processes with Cell recycle or Retention. • Compared with chemostats, cell retention or recycle results in: • Higher cell concentrations. • Lower residual substrate concentrations.

38. Cell recycle or Retention – Advantages over Chemostats. • Higher volumetric productivity. • Higher cell concentration. • Better yields/titres. • Less (or no) substrate needed for growth. • Lower residual substrate concentrations means less substrate lost through overflow.

39. Cell Recycle or Retention – Advantages over Chemostats. • Mutation/selection pressures are less. • Low or zero growth. • Less loss of cells in effluent. • Less tendency for culture to wash out. • Growth rate does not have to match D. • Cells are retained.

40. Status of Continuous Cultures in Industry • Not widespread. • Chemostats only suitable for biomass production, but valuable in R & D: • Strain selection. • Physiological studies. • Medium optimisation.

41. Status of Continuous Cultures in Industry • Recycle/retention systems used for: • Biotransformations. • Beverages (with mixed success!). • Effluent treatment: • Continuous supply. • May be dilute. • May be poisonous.

42. What is a bioprocessor? • A vessel and ancillaries designed to facilitate the growth and/or activities of micro-organisms under controlled and monitored conditions

43. Typical Requirements: • Aseptic operation • Agitation and aeration • Measurement and control

44. Aeration and Agitation • Closely related (each helps the other). • Agitation (mixing). • Provides uniform, controllable conditions. • Avoids nutrient depletion and product build-up around cells. • Aeration. • Ensures oxygen supply to the cells.

45. Oxygen Supply to Cultures • Cells can only use dissolved oxygen. • Oxygen is relatively insoluble. • During a process, oxygen must pass from the gas phase (air) to the liquid phase (medium) at a rate which is fast enough to satisfy the culture’s requirements. • The rate of gas to liquid transfer is governed by the gas/liquid interfacial area.

46. Aeration and Agitation in Conventional Bioprocessors • A sparger bubbles air in at the base of the processor • Larger gas/liquid interfacial area • Mixing • Agitators stir the medium • Mixing • Break up bubbles • Larger gas/liquid interfacial area • Increase bubble residence time • Larger gas/liquid interfacial area

47. Sizes of BioprocessorNB: Categories etc. are arbitrary!

48. Production Fermenter • Diagram of 100,000L Fermenter with: • Top drive agitators and foam-breaker

49. Production Fermenter • Diagram of 100,000L Fermenter with: • Internal cooling coils and baffles

50. Production Fermenter • Diagram of 100,000L Fermenter with: • Sparger (air input)