Laboratory Experiment to
Determine Batch Ethanol
Production by S. cerevisiae
Benjamin Reves
May 11, 2005
Outline
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Background
Theory
Procedure
Results
Conclusions
Recommendations
Impact
Questions
Background
Need for Ethanol
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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
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Common type of yeast
Eucaryotic
Reproduces by budding
Can grow aerobically or anaerobically
Current Methods
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Batch Reactor
Fed-batch Reactor
Continuous Reactor
Packed Bed Reactor
Theory
Glycolysis
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Breakdown of 6-C glucose into two
molecules of 3-C pyruvate
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Fate of pyruvate
–
–
Aerobic Conditions- TCA cycle
Anaerobic Conditions- Converted to ethanol via
acetaldehyde
Cell Growth
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Substrates + cells
extracellular products + more cells
Batch Growth
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Lag Phase
Exponential Growth Phase
Deceleration Phase
Stationary Phase
Death Phase
Yield Coefficients
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Help to quantify growth kinetics
YX/S=-X/S
YP/S= -P/S
YP/X= P/X
Product Formation
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Growth-associated products
qp 
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1 dP
 YP / X  g
X dt
Non-growth-associated products
qp  
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Mixed-growth-associated products
qp  g  
Temperature Effects
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Optimal temperatures
Product formation affected
Diffusion limitations
Objectives
Objectives
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Study ethanol production and glucose
utilization by Saccharomyces cerevisiae
Study effect of temperature on fermentation
Construct growth curves
Methods
Stages of Experimentation
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Autoclaving
Inoculation
Growth of Culture
Analyzing Samples
Autoclaving
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Sterility is a must!
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Saturated steam at 121oC used to kill all
spores
Autoclave
Preparing Inoculum
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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
Inoculum Preparation
Yeast Extract Broth
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Undefined vs. Defined Media
1 L YEB contains:
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20 grams of glucose
10 grams of yeast extract broth
Glucose is carbon/energy source
Yeast extract provides cofactors and ions
Fermentor
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7.5 L BioFlo 110 Modular Benchtop
Fermentor
Controller and PCU
Temperature Control
Fermentor
PCU
Headplate
Taking Samples
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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
Centrifuge
Centrifuged Samples
Estimating Cell Concentration
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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
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Optical density measured
Washed with 10 mM phosphate buffer
Dried in oven at 35 Celsius
OD Calibration Curve
OD Calibration
0.5
0.45
0.4
X = 1.1435*Abs. + 0.0358
R2 = 0.9966
X (mg/ml)
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0
0.05
0.1
0.15
0.2
Absorbance
0.25
0.3
0.35
0.4
Glucose Determination
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Glucose assay kit ordered from Sigma
Based on UV absorbance of NADH at 340 nm
Glucose + ATP
NAD+
Hexokinase
Glucose-6-Phosphate + ADP
G6PDH
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G6P +
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Samples Diluted
6-Phosphogluconate + NADH
Ethanol Determination
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Ethanol assay kit ordered from R-Biopharm
Based on absorbance of NADH at 340 nm
Sample diluted
ADH
NAD+
acetaldehyde + NADH + H+
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Ethanol +
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Acetaldehyde + NAD+ + H2O
Al-DH
acetic acid + NADH + H+
Results
Growth Curve for 30 Celsius
X (mg cells/ ml)
Growth Curve for 30 Celsius
1.800
1.600
1.400
1.200
1.000
0.800
0.600
0.400
0.200
0.000
X
0
5
10
15
20
Time (h)
25
30
35
Growth Curve for All Runs
X (mg cells/ mL)
Growth Curve
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
30 Celsius
37 Celsius
25 Celsius
0
10
20
30
Time (h)
40
50
Concentration Plot for 30 Celsius
Glucose and Ethanol Concentrations vs. Time for 30 Celsius
45
40
Cocentration (mg/ml)
35
30
25
Glucose
20
Ethanol
15
10
5
0
0
5
10
15
20
Time (h)
25
30
35
Glucose Concentration
Glucose Concentration
(mg/ml)
Glucose Concentration vs. Time
60
50
40
30 Celsius
30
37 Celsius
20
25 Celsius
10
0
0
10
20
30
Time (h)
40
50
Ethanol Production
Ethanol Production
Ethanol Concentration
(mg/ml)
12
10
8
30 Celsius
6
37 Celsius
4
25 Celsius
2
0
0
10
20
30
Time (h)
40
50
Yield Coefficients
YP/S (mg P/mg S)
25oC
30oC
37oC
0.229
0.282
0.247
YX/s (mg cells / mg S)
0.027 0.0378 0.0293
YP/X (mg P/ mg cells)
8.44
7.45
8.44
Conclusions
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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
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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, fedbatch
HPLC
Impact
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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?