Industrial Microbiology INDM 4005 Lecture 6 17/02/04

1. Industrial MicrobiologyINDM 4005Lecture 617/02/04

2. Questions for today: • 1. What is a fermentation system? • 2. What is the most widely used fermenter? • 3. What are the other types of fermenter? • 4. How do you control a fermentation system? • 5. Why is mass transfer important?

3. Lecture Overview • 1) Basic design criteria and limitations • 2) Stirred Tank Reactor (STR) • 3) Modifications and Industrial Examples

4. Biotechnological processing Types of Process Fermentation Design Fermenter Design Performance Optimisation Construction Configuration Control Stirred Tank Reactor

5. What is a Fermenter? • Vessel or tank in which whole cells or cell-free enzymes transform raw materials into biochemical products and/or less undesirable by-products • Also termed a Bioreactor

6. Fermenter - Basic Function The basic function of a fermenter is to provide a suitable environment in which an organism can efficiently produce a target product that may be - cell biomass, - a metabolite, - or bioconversion product.

7. Fermentation System • In this lecture we will concentrate on fermenters used in traditional microbial, plant and animal cell culture • However with the advent of recombinant DNA technology alternate systems for producing specific cell products are now available

8. Two Types of Fermentation Systems • closed or open. • A closed system implies that all the nutrient components are added at the beginning of the fermentation process and, as a result, the growth rate of the contained organisms will eventually proceed to zero due either to diminishing nutrients or accumulation of toxic waste products. A modification of the batch process is the fed batch system. Here, volumes of nutrients may be added to augment depletion of nutrients. Overall, the system, however, remains closed and there is no continuous flow. • In contrast to the above types, in the open system, organisms and nutrients can continuously enter and leave the fermenter.

9. Fermenter General Functions What it should be capable of; • Biomass concentration must remain high • Maintain sterile conditions • Efficient power consumption • Effective agitation • Heat removal • Correct shear conditions • Sampling facilities

10. Fermenters range from simple stirred tanks to complex integrated systems involving varying levels of computer input. • Fermenter design involves cooperation in Microbiology, Biochemistry, Chemical Engineering, Mechanical Engineering, Economics • There are 3 groups of bioreactor currently used for industrial production; - non-stirred, non-aerated - non-stirred, aerated - stirred, aerated (Beer and wine) (Biomass, eg Pruteen) (Antibiotics)

11. Fermenter construction • All materials must be corrosion resistant to prevent trace metal contamination of the process • Materials must be non-toxic so that slight dissolution of the material or components does not inhibit culture growth • Materials of the fermenter must withstand repeated sterilization with high pressure steam • Fermenter stirrer system and entry ports be sufficiently robust not to be deformed under mechanical stress • Visual inspection of the medium and culture is advantageous, transparent materials should be used

12. Basic fermenter configuration • A microbial fermentation can be viewed as a three-phase system, involving liquid-solid, gas-solid, and gas-liquid reactions. • The liquid phase contains dissolved nutrients, dissolved substrates and dissolved metabolites. • The solid phase consists of individual cells, pellets, insoluble substrates, or precipitated metabolic products. • The gaseous phase provides a reservoir for oxygen supply and for CO2 removal.

13. Optimisation of the Fermenter System • Fermenter should be designed to exclude entrance of contaminating organisms as well as containing the desired organisms • Culture volume should remain constant, • Dissolved oxygen level must be maintained above critical levels of aeration and culture agitation for aerobic organisms • Parameters such as temperature of pH must be controlled, and the culture volume must be well mixed. • Therefore a need for control exists

14. Control of Chemical and Physical Conditions • Intensive properties (cannot be balanced) - temperature, concentration, pressure, specific heat • Extrinsive properties (can be balanced) - mass, volume, entropy and energy • Mass and energy levels should balance at the start and finish of fermentations. • Combining this with determination of thermodynamic properties and rate equations we can build computer and mathematical models to control processes.

15. Basic Fermenter Design Criteria (i). Nature of microbial (or mammalian, plant tissue) cell; (a) Hydrodynamic characteristics (b) Mass and Heat Transfer (c) Kinetics (d) Genotype and Phenotype (ii). Environmental Control and Monitoring of the process; (a) pH, temperature, dissolved oxygen etc. (b) Asepsis and avoidance of contamination (iii). Process factors; (a) Effect on other unit operations (b) Economics (c) Potential for scale-up

16. Types of Fermenter • Aerobic fermenters may be classified depending on how the gas is distributed • Stirred Tank Reactor • Airlift • Loop Reactor • Immobilised System

17. Stirred Tank Reactors • Most commonly fermenter used • Made from stainless steel when over 20 Litres • Height to Diameter ratio 2:1 and 6:1 • Baffles prevent a large central vortex • Also used to carry coolants in large systems

18. Stirred Tank Reactor

19. STR - Control systems • An agitator system • An oxygen delivery system • A foam control system • A temperature control system • A pH control system • Sampling ports • A cleaning and sterilization system. • A sump and dump line for emptying of the reactor.

20. Aeration and agitation • The transfer of energy, nutrients, substrate and metabolite within the bioreactor must be brought about by a suitable mixing device. The efficiency of any one nutrient may be crucial to the efficiency of the whole fermentation. • For the three phases, the stirring of a bioreactor brings about the following: • Dispersion of air in the nutrient solution • Homogenisation to equalise the temperature and the concentration of nutrients throughout the fermenter • Suspension of microorganisms and solid nutrients • Dispersion of immiscible liquids

21. Basic features of a stirred tank bioreactor Agitation system The function of the agitation system is to • provide good mixing and thus increase mass transfer rates through the bulk liquid and bubble boundary layers. • provide the appropriate shear conditions required for the breaking up of bubbles. • The agitation system consists of the agitator and the baffles. • The baffles are used to break the liquid flow to increase turbulence and mixing efficiency.

22. Agitator design and operation Radial flow impellers - Rushton turbine The most commonly used agitator in microbial fermentations Like all radial flow impellers, the Rushton turbine is designed to provide the high shear conditions required for breaking bubbles and thus increasing the oxygen transfer rate.

23. Mass Transfer • One of the most critical factors in the operation of a fermenter is the provision of adequate gas exchange. • Oxygen is the most important gaseous substrate for microbial metabolism, and carbon dioxide is the most important gaseous metabolic product. • For oxygen to be transferred from a air bubble to an individual microbe, several independent partial resistance’s must be overcome

24. Oxygen Mass Transfer Steps Gas bubble Liquid film Microbial cell 1 2 3 4 6 • 1) The bulk gas phase in the bubble • 2) The gas-liquid interphase • 3) The liquid film around the bubble • 4) The bulk liquid culture medium • 5) The liquid film around the microbial cells • 6) The cell-liquid interphase • 7) The intracellular oxygen transfer resistance 7 5

25. Air lift reactors • In such reactors, circulation is caused by the motion of injected gas through a central tube with fluid re-circulating through the head space where excess air and the by-product CO2 disengage. • The degassed liquid then flows down the annular space outside the draught tube

26. Airlift reactors Draught tube

27. Airlift reactors Advantages • Low shear • Easier to maintain sterility • Increased oxygen solubility (KLa) • Can allow large vessels Disadvantages • High capital cost • High energy costs • Hard to control conditions • Foaming hinders gas -liquid separation

28. SOME MODIFICATIONS • (i) Important in tank reactor design: • 1. Continuous flow (activated sludge waste treatment) • · Suitable when substrate at low conc. • · Allows greater control on growth rate\ cell physiology • 2. Immobilised cells - may be membrane (e.g. hollow fibre reactor), immobilised onto support such as ceramic (e.g packed-bed) or in polymers (e.g alginate beads) • · Increases rate of reaction • · Microenvironment created protects cells e.g. from shear damage • 3. Low energy aeration\ mixing Air-lift, draft-tubes, loop reactors etc. • · Increase height to diameter ratio. Increased path length of bubble, improves mass transfer • · Results in decreased shear levels, important in floc systems.

29. SOME MODIFICATIONS • (ii) Industrial examples of modified STR / bioreactors • (i) Waste treatment. - Activated sludge system. • Characterised by: Low substrate conc. Therefore require (a) recycle of biomass, (b) continuous operation, (c) Low cost aeration / mixing. • (ii) Brewing - Cylindro-conical fermenter; • Note no aeration but gas produced by yeast cells contributes to mixing, closed to capture carbon dioxide produced, cone helps sedimentation of yeast, Low shear environment promotes flocculation. • (iii) Tissue culture - low shear, anchored and immobilised systems. • (iv) Solid-state fermentations e.g. silage, mushroom production etc.

30. In Summary Major considerations include 1. Bioreactor size - to provide required production capacity 2. Mass transfer - to provide nutrients to cells, well dispersed, adequate oxygen etc 3. Control systems (a) temperature, pH, etc. (b) sterilisation/ aseptic operation (c) representative sampling (d) heat transfer - example sterilisation of media 4. Requirement for asepsis / containment

31. Critical Concepts or Questions • What are the objectives in fermenter design? • Draw a diagram of a STR • How does a STR relate to structure and function? • How can fermentation systems be controlled?

32. Conclusion • This lecture introduced the various parameters involved in design of an industrial fermenter. • Using a STR it illustrated the optimisation and control of a fermentation system.