What do all these things have in common?

1. What do all these things have in common?

2. DETERMINING KINETIC PARAMETERS OF SACCHAROMYCES CEREVISIAEGROWTH IN A BATCH STIRRED-TANK REACTOR Joyanne Schneider CH EN 4903 November 28, 2006

3. Overview • Problem Statement and Setup • Theory • Results and Discussion • Conclusions and Recommendations • Questions and Answers

4. Problem Statement and Setup • Biochemical company wanted to obtain growth kinetics of a genetically modified yeast strain analogous to S. cerevisae for use in recombinant technology: • volumetric mass transfer coefficient (kLa) • specific respiration rate, OUR • yield coefficient YX/S • maximum specific growth rate (μmax) • specific glucose uptake rate, Rv

5. Problem Statement and Setup: New Brunswick BSTR Apparatus

6. Problem Statement and Setup:Conditions • Temperature: 37 degrees Celsius • pH: 6.5 • Agitation Rate: 500 RMP • Air Flow Rate: 800 cc/min • Startup: 1.5 L Deionized water • 40 g/L glucose • 10 g/L of yeast extract • 20 g/L of Bacto Peptone

7. Thoery: Phases • Lag Phase (minimize) • Acceleration Phase • Exponential Growth Phase • Deceleration Phase • Stationary Phase • Death Phase

8. Theory: kLa without cells • Using Henry’s Law: • After re-aeration begins,

9. Theory: kLa without cells, cont. • Dividing both sides by C*, separating variables, and integrating:

10. Theory: OUR and kLa with cells • During de-aeration, • During re-aeration,

11. Theory: OUR and kLa with cells cont. • Once OUR is determined, kLa can be determined by plotting change in percent saturation plus specific respiration rate versus one minus percent saturation, 1-C/C*.

12. Theory: Yield Coefficient • Yield is given by, where ΔX is the change in cell concentration and Δ S is the change in substrate (glucose) concentration. • Glucose and cell concentrations obtained every half-hour using HPLC and spectrometry (absorbance), respectively.

13. Theory:μmax • Monod Equation: • μ is the specific growth rate • μmax is the maximum specific growth rate • S is the substrate (glucose) concentration • Ks is the Monod constant • μmax is the asymptote of μ plotted as a function of S

14. Theory: μmax(cont.)

15. Theory: μmax(cont.) • If few samples are taken, no asymptotic relationship • Because rate of cell growth is • Separating variables and integrating gives: • Plotting gives a slope of umax.

16. Theory: Rv • The volumetric glucose uptake rate (g/(L-hr) is given by: • Since it is just change in glucose concentration per time, can just be calculated from:

17. Results and Discussion: kLa without cells

18. Results and Discussion: OUR and kLa with cells

19. Results and Discussion: μmax and YX/S

20. Results and Discussion: Rv

21. Conclusions and Recommendations • Start with growth medium as close to growth conditions as possible. • Using dissolved O2 probe and percent saturation, can’t get accurate kLa with cells growing (should be lower with cells than without) • To increase kLa, use higher air flow rate and agitation speed • To obtain maximum yield, don’t allow the oxygen to fall below the critical saturation • Use more trials to get more data points

22. References Atkinson, B., Mavituna, F. Biochemical Engineering and Biotechnology Handbook, 2nd ed., 1991, Macmillion Publishers, Hampshire, England. Asenjo, J., Merchuk, J. Bioreactor System Design, 1995, Marcel Dekker, New York. Bailey, J., Ollis, D. Biochemical Engineering Fundamentals, International ed., 1977, McCraw-Hill, Tokyo. Shuler, M., Kargi, F. Bioprocess Engineering: Basic Concepts, 2nd ed., 2002, Prentice Hall, Upper Saddle River, New Jersey.

23. Thank you for listening… Any Questions?