1. Laboratory Experiment to Determine Batch Ethanol Production by S. cerevisiae Benjamin Reves May 11, 2005

2. Outline Background Theory Procedure Results Conclusions Recommendations Impact Questions

3. Background

4. Need for Ethanol Ethanol used as raw material and solvent in the chemical, food, and pharmaceutical industries Four million tons of ethanol are produced each year Eighty percent by fermentation Energy Information Administration has predicted annual consumption growth in U.S. of 3.2% each year

5. Saccharomyces cerevisiae Common type of yeast Eucaryotic Reproduces by budding Can grow aerobically or anaerobically

6. Current Methods Batch Reactor Fed-batch Reactor Continuous Reactor Packed Bed Reactor

7. Theory

8. Glycolysis Breakdown of 6-C glucose into two molecules of 3-C pyruvate Fate of pyruvate – Aerobic Conditions- TCA cycle – Anaerobic Conditions- Converted to ethanol via acetaldehyde

9. Cell Growth Substrates + cells extracellular products + more cells

10. Batch Growth Lag Phase Exponential Growth Phase Deceleration Phase Stationary Phase Death Phase

11. Yield Coefficients Help to quantify growth kinetics Y X/S =-  X/  S Y P/S = -  P/  S Y P/X =  P/  X

12. Product Formation Growth-associated products Non-growth-associated products Mixed-growth-associated products

13. Temperature Effects Optimal temperatures Product formation affected Diffusion limitations

14. Objectives

15. Study ethanol production and glucose utilization by Saccharomyces cerevisiae Study effect of temperature on fermentation Construct growth curves

16. Methods

17. Stages of Experimentation Autoclaving Inoculation Growth of Culture Analyzing Samples

18. Autoclaving Sterility is a must! Saturated steam at 121 o C used to kill all spores

19. Autoclave

20. Preparing Inoculum Inoculum is typically 5-10% of total working volume Yeast obtained from microbiology department on plates Inoculating needle used to take yeast from plate into 800 mL of YEB Placed on shaker at 30 o C Importance of inoculum condition

21. Inoculum Preparation

22. Yeast Extract Broth Undefined vs. Defined Media 1 L YEB contains: – 20 grams of glucose – 10 grams of yeast extract broth Glucose is carbon/energy source Yeast extract provides cofactors and ions

23. Fermentor 7.5 L BioFlo 110 Modular Benchtop Fermentor Controller and PCU Temperature Control

24. Fermentor

25. PCU

26. Headplate

27. Taking Samples Fermentor equipped with sample port Sample tubes had been autoclaved Optical density of sample measured Centrifuged for 5 minutes at 2000 rpm Liquid decanted and stored at 4 o C

28. Centrifuge

29. Centrifuged Samples

30. Estimating Cell Concentration Spectrophotometer used to measured absorbance at 650 nm Sterile YEB used as blank Beer’s Law: A=  bc Linear correlation between absorbance and concentration of cells Calibration curve constructed

31. Spectrophotometer

32. Construction of Calibration Curve Optical density measured Washed with 10 mM phosphate buffer Dried in oven at 35 Celsius

33. OD Calibration Curve

34. Glucose Determination Glucose assay kit ordered from Sigma Based on UV absorbance of NADH at 340 nm Glucose + ATP Glucose-6-Phosphate + ADP G6P + NAD + 6-Phosphogluconate + NADH Samples Diluted Hexokinase G6PDH

35. Ethanol Determination Ethanol assay kit ordered from R-Biopharm Based on absorbance of NADH at 340 nm Sample diluted Ethanol + NAD + acetaldehyde + NADH + H + Acetaldehyde + NAD + + H 2 O acetic acid + NADH + H + ADH Al-DH

36. Results

37. Growth Curve for 30 Celsius

38. Growth Curve for All Runs

39. Concentration Plot for 30 Celsius

40. Glucose Concentration

41. Ethanol Production

42. Yield Coefficients 25 o C30 o C37 o C Y P/S (mg P/mg S) 0.2290.2820.247 Y X/s (mg cells / mg S) 0.0270.03780.0293 Y P/X (mg P/ mg cells) 8.447.458.44

43. Conclusions Yeast grew the fastest at 30 Celsius Lag times were longer at 25 and 37 Celsius Glucose was fully used in each run Amount of ethanol produced was almost the same for each run Runs should be allowed to proceed longer

44. Recommendations Determine growth and productivity effects due to other factors such as pH Determine optimal inoculum size and age Investigate better methods of analyzing samples Operate fermentor in other manners: continuous, continuous with recycle, fed- batch

45. HPLC

46. Impact Typically carried out at graduate level CBU has ability to perform at undergraduate level Hope to collaborate with School of Sciences in the future

47. Questions?