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Ethanol Production by S. cerevisiae: A Batch Experiment Study

This laboratory experiment involved studying ethanol production and glucose utilization by Saccharomyces cerevisiae, exploring the impact of temperature on fermentation, and constructing growth curves. Methods included autoclaving, preparing inoculum, using a fermentor for cultivation, taking samples, and using a spectrophotometer for analysis. The experiment revealed that yeast growth was fastest at 30 degrees Celsius, with longer lag times at 25 and 37 degrees Celsius. Recommendations were made to explore other influencing factors, optimize inoculum size, improve sample analysis methods, and vary fermentor operation modes. The experiment contributes valuable insights into ethanol production processes.

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Ethanol Production by S. cerevisiae: A Batch Experiment Study

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  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. Saccharomycescerevisiae • 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 • YX/S=-X/S • YP/S= -P/S • YP/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. Objectives • Study ethanol production and glucose utilization by Saccharomycescerevisiae • 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 121oC 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 30oC • 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 4oC

  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+ + H2O 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

  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?

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