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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 YX/S=-X/S
YP/S= -P/S
YP/X= P/X
Product Formation
Growth-associated products
Non-growth-associated products
Mixed-growth-associated products
gXPp Ydt
dP
Xq /
1
pq
gpq
Temperature Effects
Optimal temperatures Product formation affected Diffusion limitations
Objectives
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 121oC 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 30oC 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 4oC
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
OD Calibration
X = 1.1435*Abs. + 0.0358R2 = 0.9966
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
Absorbance
X (
mg
/ml)
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+ + H2O acetic acid + NADH + H+
ADH
Al-DH
Results
Growth Curve for 30 Celsius
Growth Curve for 30 Celsius
0.0000.2000.4000.6000.8001.0001.2001.4001.6001.800
0 5 10 15 20 25 30 35
Time (h)
X (
mg
cel
ls/
ml)
X
Growth Curve for All Runs
Growth Curve
00.20.40.60.8
11.21.41.61.8
0 10 20 30 40 50
Time (h)
X (
mg
cel
ls/
mL
)
30 Celsius
37 Celsius
25 Celsius
Concentration Plot for 30 Celsius
Glucose and Ethanol Concentrations vs. Time for 30 Celsius
0
5
10
15
20
25
30
35
40
45
0 5 10 15 20 25 30 35
Time (h)
Co
cen
trat
ion
(m
g/m
l)
Glucose
Ethanol
Glucose Concentration
Glucose Concentration vs. Time
0
10
20
30
40
50
60
0 10 20 30 40 50
Time (h)
Glu
cose
Co
nce
ntr
atio
n
(mg
/ml) 30 Celsius
37 Celsius
25 Celsius
Ethanol Production
Ethanol Production
0
2
4
6
8
10
12
0 10 20 30 40 50
Time (h)
Eth
ano
l C
on
cen
trat
ion
(m
g/m
l) 30 Celsius
37 Celsius
25 Celsius
Yield Coefficients
25oC 30oC 37oC
YP/S (mg P/mg S) 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
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?