Chapter 4 Fermentation Monitoring

1. Chapter4 Fermentation Monitoring Common Features of a Fermentor • A large vessel • Made of stainless steel • Equipped with temperature, • pH and dissolved oxygen • measurement and control • systems

2. mechanical agitation fermentor self priming fermentor

3. Tower fermentor Airlift fermentor

4. Unit 1 Fermentation Basics • Four Phases of Bacterial Growth Curve • Lag phase • Log phase • Stationary phase • Death phase

5. Lag Phase • Period of adjustment to new conditions • Cell growth is minimal

6. Log Phase • Cell growth rate and metabolic activity are • the highest • Number of cells produced ﹥Number of cells dying • Cells are most susceptible to adverse • environmental factors (e.g. radiation, antibiotics)

7. Stationary Phase • Population size begins to stabilize • Number of cells produced = Number of cells dying • Factors that slow down microbial growth • Accumulation of toxic waste materials • Acidic pH of media • Limited nutrients • Insufficient oxygen supply

8. Death or Decline Phase • Population size begins to decrease • Number of cells dying > Number of cells produced • Most of the nutrients in the medium have • been consumed

9. Methods for measuring microbial growth Measurement of the changes in number of cells or mass of population.

10. Measurement of Cell Numbers • Direct cell counts - counting chambers • Viable cell counts - plating methods

11. Measurement of Cell Mass • Dry weight- time consuming and not very sensitive • Turbidimetric measures- quick, easy and sensitive • optical density  the number of microbes • E. coli, 1 OD600 = ~ 8 x 108 cells/ml

12. Types of Fermentation Process Batch, continuous and fed batch processes fed batch batch chemostat

13. Batch culture The sterile growth medium is inoculated with the microorganisms and no additional growth medium is added. All the nutrients needed for cell growth will only be added once at the beginning of fermentation.

14. Batch Fermentor

15. Advantages • Optimum levels of product recovery • Simple operation • Disadvantages • The wastage of unused nutrients • Labour and time lost between batches

16. Continuous culture • Maintains cells in log phase at a constant biomass concentration for extended periods. • It can be controlled in two ways • Turbidostatic control (internally controlled) • Chemostatic control (externally controlled)

17. The Turbidostat • Regulates the flow rate of media through vessel to maintain a predetermined turbidity • Maintain the highest growth rate • No limiting nutrient • The Chemostat • Rate of incoming medium = • rate of removal of medium from vessel • Maintain the exponential growth phase • An essential nutrient is in limiting quantities

18. Batch Culture and Continuous Culture

19. Advantages • The growth rate is maintained at optimal levels • Disadvantages • Low biomass and product concentration • Relatively prone to be contaminated • Strain degeneration

20. Q: Use chemostat as bioreactor, the yield is far more than batch , but why batch culture is far more widely used than continuous culture？ 1. Secondary products 2. Genetic instability 3. Operability and reliability 4. Market economics

21. Fed-batch culture A production technique between batch and continuous culture. During fermentation, additional nutrients will be added in a batch way to promote the cell growth or product formation and to avoid nutrient deficiency.

22. Fed-Batch Fermentor Pump Feedstock vessel (sterile)

23. Advantages • Low concentration of specific substrates • (e.g. carbon source) • High concentration of end-product • Providing limiting level of a required nutrient • for an auxotrophic strain

24. Some examples

25. Unit 2 Fermentation Control The success of a fermentation process is highly dependent on environmental factors such as temperature, pH, and dissolved oxygen levels.

26. Fermentation Control Sugar/Oil feed • Sample Analysis • pH • DO • Sugar • Ammonia • Phosphate • Sulphate • Products • Precursors • Contamination Pressure probe Level probe Antifoam pH probe Acid/Base Temp. probe Cooling DO probe Air/agitation

27. Cold Hot Temperature Temperature Control Organisms exhibit distinct cardinal growth temperatures - minimal, maximal, optimal B (Optimum) C (Minimum) A (Maximum)

28. Why? Microbes brings about fermentation by secreting certain enzymes which have an optimum temperature or a temperature range.

29. An energy balance on fermentation yields is the following equation: • Qacc = Qf + Qag – Qevap – Qsurr • Qacc——net heat accumulation • Qf ——heat of fermentation • Qag——heat generated by mechanical agitation • Qevap ——heat of evaporation • Qsurr ——heat dissipated to the surroundings

30. Q: Usually, optimum growth temperature is difference with optimum production temperature, how would you do? Answer Temperature-shifting fermentation

31. Temp. control methods • Cooling water • in cooling jacket or coil • Sterilization- steam • Refrigerator or freezer

32. pH control • Most microorganisms have narrow pH growth • ranges (pH 5 - 7 ) • The buffering in culture media is generally low • Metabolites which released into the medium can • change the pH

33. Q: In many fermentation factories, urea or ammonia water is often used as a fed-batch substance, what’s the function of it ? • Adjusting pH of media • Using as a nitrogen source

34. How to maintain optimum pH? • Addition of base ( NaOH ) or acid ( HCl ) • Addition of physiological acid substance ((NH4)2SO4) or physiological alkali substance (ammonium hydroxide)

35. Dissolved oxygen (DO）Control • Most industrial fermentations are aerobic processes • Supplying oxygen to aerobic cellshas a problem：oxygen is poorly soluble in water • ( 8 mg/L at 4oC , sucrose is soluble to 600 g/L) • Oxygen supplying is the rate limiting step in an aerobic fermentation

36. Factors affecting dissolved oxygen concentration • Temperature • Elevation • Salinity • Turbulence atmosphere o2 o2 o2 o2 o2 o2 Under saturated Super saturated Saturated water

37. Critical dissolved oxygen concentration The minimum concentration of oxygen which has been submerged in water, where oxygen is the limiting factor to the growth of the microbes.

38. The objective is to maintain the optimal dissolved oxygen concentration above a critical concentration to avoid inhibition of the cell growth rate due to lack of oxygen.

39. Q: Use Brevibacterium flavum to produce • Glu and Asp, • dissolved oxygen﹤ critical dissolved oxygen, • yield ↓ • Phe, Val and Leu, • dissolved oxygen﹤ critical dissolved oxygen, yield ↑

41. Exercises 1) In order to control DO correctly, .and should be known for every fermentation production. 2) The optimal dissolved oxygen concentration is related with and.

42. Q: How to maintain a constant dissolved oxygen concentration during the fermentation process? • A: Increase aeration and agitation rate

43. Aeration : by bubbling air through the liquid Agitation: using impellers (agitator)

45. Other influence factors The most important is cell concentration ↑ substrate concentration ↑ fed-batch operation

46. High cell density fermentation • Medium optimization • Fed-batch culture • Increasing DO • Removal of toxic components

47. Oxygen is the most important gaseous substrate for microbial metabolism. • Carbon dioxide is the most important gaseous metabolic product.

48. Foaming control • Foams consist of liquid lamellas filled with gas • How is foam formed? • Surface-active media (peptides) and • foaming components (proteins) • High aeration and agitation rates

49. Excess foaming will……. • displace the fermentation broth and cause it to leak from the vessel. • lead to losses in productivity and culture contamination.

50. Foam control methods • Mechanical defoaming • Foam-breaker （Defoamers） • Defoaming blades in fermentation tank • Separate rotor in the upper area of the culture tank