1. What do all these things have in common?

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

3. Overview Problem Statement and Setup Problem Statement and Setup Theory Theory Results and Discussion Results and Discussion Conclusions and Recommendations Conclusions and Recommendations Questions and Answers 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) volumetric mass transfer coefficient (kLa) specific respiration rate, OUR specific respiration rate, OUR yield coefficient Y X/S yield coefficient Y X/S maximum specific growth rate (μ max ) maximum specific growth rate (μ max ) specific glucose uptake rate, R v specific glucose uptake rate, R v

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 Exponential Growth Phase Deceleration Phase Stationary Phase Death Phase

8. Theory: k L a without cells Using Henry’s Law: After re-aeration begins,

9. Theory: k L a without cells, cont. Dividing both sides by C*, separating variables, and integrating:

10. Theory: OUR and k L a with cells During de-aeration, During re-aeration,

11. Theory: OUR and kLa with cells cont. Once OUR is determined, k L a 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 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. Glucose and cell concentrations obtained every half- hour using HPLC and spectrometry (absorbance), respectively.

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

14. Theory: μ max (cont.)

15. If few samples are taken, no asymptotic relationship Because rate of cell growth is Separating variables and integrating gives: Plotting gives a slope of u max.

16. Theory: R v 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: k L a without cells Agitator (RPM) Flow (cc/min) kLa value (hr -1 ) (95%)Variance 50040036.3+/-0.8050.169 50080066.3+/-1.260.415 50080060.8+/-0.7740.156 500120087.0+/-1.690.739 1008007.94+/-0.3210.0270 30080019.9+/-0.2250.132 700800111.7+/-1.710.764 850800158+/-2.631.79

18. Results and Discussion: OUR and k L a with cells Run # OUR(1/hr)OURVariance k L a (hr -1 ) k L a Variance 11.73 +/- 1.32 0.45774.4 +/- 3.39 3.01 212.4 +/- 5.17 6.9695.1 +/- 3.00 2.35

19. Results and Discussion: μ max and Y X/S Run # μ max (hr -1 ) μ max Variance Y X/S Y X/S Variance 10.170 +/- 0.221 0.01280.2360.00871 20.103 +/- 0.163 0.008340.1500.00365

20. Results and Discussion: R v Run # R v (g/L-hr) Variance 1 0.240 +/- 0.472 0.0580 20.319+/-0.2100.0406

21. Conclusions and Recommendations  Start with growth medium as close to growth conditions as possible.  Using dissolved O 2 probe and percent saturation, can’t get accurate k L a with cells growing (should be lower with cells than without)  To increase k L a, 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, 2 nd 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, 2 nd ed., 2002, Prentice Hall, Upper Saddle River, New Jersey.

23. Thank you for listening… Any Questions?