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

Outline Background Theory Procedure Results Conclusions Recommendations Impact Questions

Background

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

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

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

Theory

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

Cell Growth Substrates + cells extracellular products + more cells

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

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

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

Temperature Effects Optimal temperatures Product formation affected Diffusion limitations

Objectives

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

Methods

Stages of Experimentation Autoclaving Inoculation Growth of Culture Analyzing Samples

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

Autoclave

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

Inoculum Preparation

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

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

Fermentor

PCU

Headplate

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

Centrifuge

Centrifuged Samples

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

Spectrophotometer

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

OD Calibration Curve

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

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

Results

Growth Curve for 30 Celsius

Growth Curve for All Runs

Concentration Plot for 30 Celsius

Glucose Concentration

Ethanol Production

Yield Coefficients 25 o C30 o C37 o C Y P/S (mg P/mg S) Y X/s (mg cells / mg S) Y P/X (mg P/ mg cells)

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

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

HPLC

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

Questions?