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?