Biochemical Engineering CEN 551

1. Biochemical EngineeringCEN 551 Instructor: Dr. Christine Kelly Chapter 9

2. Bioreactors • What two type of bioreactors have we discussed in this course? • What are the characteristics of each type of reactor? • Which type is more efficient? • Which type is more common?

3. Reactor Types • Batch and Chemostat (CSTR). • Batch: changing conditions - transient (S, X, growth rate), high initial substrate, different phases of growth. • Chemostat: steady-state, constant low concentration of substrate, constant growth rate that can be set by setting the dilution rate (i.e. the feed flow rate) . • Chemostat more efficient. • Batch more common.

4. Choice of continuous vs. batch production • Productivity • Flexibility • Control • Genetic stability • Operability • Economics • Regulatory What do each of these factors mean?

5. Reactor Choices • Productivity: rate of product per time per volume. Chemostat better for growth associated products. Wasted time in batch process. • Flexibility: ability to make more than one product with the same reactor. Batch better. • Control: maintaining the same conditions for all of the product produced. In theory, chemostat better, steady state. In reality????

6. Genetic stability: maintaining the organism with the desired characteristics. Chemostat selects for fast growing mutants that may not have the desired characteristics. • Operability: maintaining a sterile system. Batch better. • Regulatory: validating the process. Initially, many process batch, too expensive to re validate after clinical trials.

7. Comparison of Productivity: Batch vs. Chemostat Consider production of a growth associated product (like cell mass) in suspension culture F S0 X0 F S X ? air air

8. Batch Reactor Batch cycle time is: where tgrowth is the time required for growth and tl is the lag time + preparation and harvest time. where X0 is the initial concentration and Xmax is the maximum concentration (carrying capacity).

9. Batch Production Rate So net biomass production rate is: Recall the definition of biomass yield: (1)

10. Chemostat For negligible kd, negligible extracellular product formation and steady state, Lec. Notes 16, Eq. (10) gave: (2) For optimum cell productivity (X•D), calculate d(X•D)/dt, set equal to zero, and solve for Dopt: (3)

11. Chemostat Substituting Eq. (2) into Eq. (3) gives the value of X at the maximum production rate. : (4) Optimum productivity is D•X when D=Dopt and X= X (at Dopt): (5)

12. Chemostat Productivity Rate Noting that S0 is usually much larger than KS, we have: (6) Comparing the rates for batch production and production in a chemostat: (7)

13. Comparison Xmax is always larger than X0 and is typically 10-20 times larger, so the chemostat outperforms the batch reactor. For E. coli growing on glucose, µmax is around 1/hr. Using tlag=5 hr and Xmax/X0=20, Even so, most industrial fermentation processes occur in a batch reactor. Why?

14. Reasons for Batch Popularity • Equations were for cell mass (or other growth-associated product). Many industrial applications are for non-growth associated products. • Selective pressure of a chemostat is detrimental to engineered organisms • Batch is more mechanically reliable • Batch system is more more flexible

15. Specialized Reactors • Chemostat with recycle • Multistage chemostat • Fed-batch • Perfusion

16. Chemostat with Recycle Can we operate a chemostat with a dilution rate greater than maximum growth rate? Why or why not? What conditions would we want to operate a chemostat with a dilution rate higher than the maximum growth rate?

17. High dilution rate • No • Because the cell growth cannot keep up with how fast the cells are removed from the reactor, and after some time the cells would washout of the reactor. • We want a high dilution rate when we have a high volume of feed with a low concentration of substrate. Waste water treatment has these characteristics.

18. Operation of Chemostats at High Dilution Rates Chemostats cannot be operated if µmax<D. Higher dilution rates can be achieved with recycle. F S0 X0 (1+a)F S,X F X’ aF S,bX

19. Chemostat with Recycle Biomass balance on the chemostat: (8) where a=volumetric recycle ratio and b=the concentration factor of the separator. At steady state and with X0=0: (9) (10) Note that for b>1, µ<D.

20. Substrate Mass Balance (11) At steady state: (12) (13)

21. Steady-state Values Substituting µ given by Eq. (10) into Eq. (13): (14) We can get the expression for the substrate concentration by equating the expression for µ from Monod kinetics to Eq. (10):

22. Steady-state Values (15) or: (16) So now we can get X entirely as a function of D: (17)

23. Special Cases - Chemostat • Recombinant product under the control of an inducible promoter. • Recombinant strain and wild type grow at the same rate if the recombinant product is not expressed. • If the recombinant product is expressed, the recombinant strain grows much slower. • Design a continuous reactor system to produce this product efficiently.

24. Mulistage chemostat • First chemostat is fed with a non-inducing growth substrate, allowing the recombinant strain to be produced. • The effluent from the first chemostat feeds a second chemostat that is fed inducer, and the product is produced. • Note: new recombinant cells are continually added to the second chemostat not allowing take-over by a fast growing mutant.

25. Fed-batch Operation • Fed-batch reactors gain some advantages of a CSTR, retain some disadvantages of batch. • Reduces substrate inhibition or catabolic repression, allows for high conversion, and the extension of stationary phase. • Semi-batch nature usually leads to higher operations cost and batch variability.

26. Fed-batch Operation F, S0 F, S0 V0, X, S, P V, X, S, P Vw, X, S, P Start fed-batch Fed batch fill Harvest

27. Fed-batch Operation • Fed-batch cultures are started as batch cultures and grown to an initial cell concentration X, after which fed-batch operation begins. • Notation: S0= initial substrate concentration of batch V0= initial volume of batch F= constant flow rate of addition stream during fed-batch X0= initial concentration of batch

28. For a batch culture: (1) Since liquid is being added, the volume is changing: or: (2) If the total amount of biomass (grams) in the reactor is Xt then the concentration X is: (3)

29. So the change in the biomass concentration with time is: (4) Using the definition of the growth rate: ...the dilution rate: ...and the expression for dV/dt: we have: (5)

30. Quasi-steady State • Substrate is consumed at the same rate it is added. Now, consider the case when the fed-batch is started from a culture in the initial substrate concentration was S0 and nutrient feed is begun at flow rate F and concentration S0. Just as nutrient feed begins: (6)

31. At quasi-steady state, for this case we will have: (7) So X is constant (but not Xt). Now we have: (8) Assuming Monod growth kinetics, this gives (just as in the case of a chemostat): (9)

32. If the total amount of substrate in the reactor is St, then a substrate mass balance gives: (10) which, for quasi-steady state gives: (11) Returning to Equation (4), we have, at quasi-steady state: (12)

33. Integrating, we have: (13) since X is constant (dX/dt=0). Therefore, the total biomass in a fed-batch reactor operated as assumed here increases linearly with time. Substituting the appropriate expression for X: (14) Often, S<<S0 and X0<<YX/SS0 and so: (15)

34. Product Output If the specific productivity (g product/g cells/ hr) is constant: or: (16) where Pt is the total product concentration in the reactor: Substituting:

35. we have: (17) Integrating this expression, we have: (18) or in terms of concentration: (19)

36. Repeated Fed-batch Usually, fed-batch cultures are taken through many feeding cycles, with each feeding cycle followed by a harvest cycle during which the volume is drawn back down to V0 and the cycle begun again.

37. For the case of repeated fed-batch cultures: (20) Where Vw is the volume just before harvesting, V0 is the volume after harvesting, Dw=F/Vw and: (21) tw is the cycle time and is given by: (22)

38. With this definition, we now have: (23)

39. Perfusion Culture • Animal Cell Culture • Constant medium flow • Cell retention • Selective removal of dead cells • Removal of cell debris, inhibitory by products • High medium use, costs raw materials and sterilization

40. Immobilized Cell Systems • High cell concentrations • Cell reuse • Eliminates cell washout at high dilution rates • High volumetric productivities • May provide favorable microenvironment • Genetic stability • Protection from shear damage

41. Major Limitation Mass transfer (diffusional) resistances Whole cells provide cofactors, reducing power, energy that many enzymatic reactions require. Advantage over immobilized enzymes

42. Types of Immobilization • Active Immobilization: similar to enzyme immobilization. Entrapment and binding. • Passive Immobilization: Biofilm – multilayer growth on solid surfaces.

43. Diffusional Limitations • Analysis similar to immobilized enzymes • Damkohler number • Effectiveness factor • Thiele modulus

44. Immobilized Bioreactors • Packed-column: feed flows through a column packed with immobilized cells. Similar to a plug flow reactor. Can be recycle chamber. • Fluidized-bed: feed flows up through a bed of immobilized cells, fluidizing the immobilized cell particles. • Airlift: air bubbles suspend the immobilized cell particles in a reactor.

45. Solid-state Fermentations • Fermentations of solid materials • Low moisture levels • Agricultural products or foods • Smaller reactor volume • Low contamination due to low moisture • Easy product separation • Energy efficiency • Differentiated microbiological structures