ALCOHOL PRODUCTION FROM

MOLASSES BY ETHANOL YEAST,

SACCHAROMYCES CEREVISIAE

INTRODUCTION

HISTORY

First industrial use of ethyl alcohol 1800s

Valuable

as a solvent, germicide, antifreeze, fuel

and chemical raw material
Oil price increases led to interest in alternative
fuels
Molasses (from sugarcane) is the most widely used
feedstock in the commercial production of ethanol
in the Philippines

KEY TERMS

Wort - the sweet

infusion of ground malt
or other grain before
fermentation, used to
produce beer and
distilled malt liquors
Contains sugars to be
fermented

KEY TERMS

Molasses - thick, dark

brown syrup obtained
from raw sugar during
the refining process, a
version of which is used
in baking.

Obtained from sugarcane

(local)

KEY TERMS

Distillation - the action of purifying a liquid by a

process of heating and cooling. (78C EtOH)

KEY TERMS

Ethanol fermentation process where sugars

are converted to ethanol and carbon dioxide by
action of yeasts (sucrase and zymase)

MOLASSES
Liquor left after the crystallization of sugarcane
juice
Dark colored viscous liquid
Contains 79.5% fermentable sugar

MOLASSES - FERMENTATION

C12H22O11 + H2O -> 2C6H12O6

2C6H12O6 -> 4C2H5OH + 4CO2

Theoretical Yield of 37.5 grams of

molasses:
37.50g

1
201.22

4
1

34.34 g ethanol

1
(
)
0.789

43.53mL ethanol

46.068
1

SUCROSE - FERMENTATION

Theoretical Yield of 37.5 grams of

sucrose:
37.50g
1
342.297

4
1

20.19 g ethanol

46.068
1

1
(
)
0.789

25.59 mL ethanol

GLUCOSE - FERMENTATION

C6H12O6 -> 2C2H5OH + 2CO2

Theoretical Yield of 37.5 grams of
glucose:
37.50g

1
180.156

19.178 g

2
46.068
1
1
1
ethanol (
) =
0.789

24.31mL ethanol

METHODOLOGY

MATERIALS NEEDED

Molasses
Glucose
Sucrose
Saccharomyces
cerevisae
MgSO4 x 7H2O
Distilled water
autoclave

Conc. H3PO4
Glucose/yeast/peptone
agar and broth
Erylenmeyer flask
pH meter
Incubator / fermentation
setup

METHODOLOGY A. PREPARATION OF WORT

Weigh 37.5 g
sugar source

+ 1g of urea,
MgSO4 x 7H2O
and KH2PO4

Dissolve in
500 mL

Adjust pH of
worts to 4 using
conc. H3PO4

autoclave

Divide wort to 2
flasks

Autoclave and
store until use

METHODOLOGY B. PREPARATION OF YEAST

INOCULUM AND FERMENTATION

Prepare GYP agar

and broth

Inoculate
Sacccharomyces
cerevisae into GYP
agar for 24 hours

Inoculate yeast
culture from GYP
agar to GYP broth
for 24 hours

Leave to ferment

Transfer broth into

wort flasks

METHODOLOGY C. DISTILLATION OF ETHANOL

Prepare distillation
set up

Distill wort solution

to collect ethanol
Collect ethanol in
vials
Calculate % EtOH
obtained

RESULTS & DISCUSSION

RESULTS
Wort

mL
Collected
Day 7

mL
collected
Day 9

mL
collected
Day 18

Average

Molasses

0.667

Sucrose

2.5

1.167

Glucose

1.000

250mL
wort

Molasses

30mL
Day 7
10/27/2014

Day 9
10/29/2014

Day 18
11/7/2014

Average

Sucrose

30mL

Glucose

Molasses

Sucrose

Glucose

30mL

Vol. collected

0mL

0mL

0mL

mL EtOH

0%

0%

0%

% EtOH

30mL

30mL

30mL

Vol. collected

1mL

2.5mL

2mL

mL EtOH

3.33%

8.33%

6.66%

% EtOH

30mL

30mL

30mL

Vol. collected

1mL

1mL

1mL

mL EtOH

3.33%

3.33%

3.33%

% EtOH

30mL

30mL

30mL

Vol. collected

0.67mL

1.16mL

1mL

mL EtOH

2.23%

3.87%

3.33%

% EtOH

CALCULATIONS EQUATIONS TO USE

% Ethanol =
x 100


Fermentation Yield (Yf) =

0.794
Fermentation Efficiency (Ef) = Yf x
0.511

100
1000

Where:
Vb = volume of wort in flask (L)
Ab = volume of ethanol collected
Mm = mass of sugar used
m = Fermentable sugars content (std value)

% Ethanol =

x 100

CALCULATIONS FOR PERCENT ETHANOL

CALCULATIONS FOR PERCENT ETHANOL

MOLASSES

Day 7
% Ethanol =

% Ethanol =

0
30

x 100

x 100 = 0%

CALCULATIONS FOR PERCENT ETHANOL

MOLASSES

Day 9

% Ethanol =
x 100

1
% Ethanol =
x 100 = 3.33%
30

For 500 mL wort = 16.67 mL ethanol

CALCULATIONS FOR PERCENT ETHANOL

MOLASSES

Day 18

% Ethanol =
x 100

1
% Ethanol =
x 100 = 3.33%
30

For 500 mL wort = 16.67 mL ethanol

CALCULATIONS FOR PERCENT ETHANOL

MOLASSES

AVERAGE

% Ethanol =
x 100

0.67
% Ethanol =
x 100 = 2.23 %
30

For 500 mL wort = 11.17 mL Ethanol

CALCULATIONS FOR PERCENT ETHANOL

SUCROSE

Day 7
%

Ethanol =
x 100

0
% Ethanol =
x 100 = 0%
30

CALCULATIONS FOR PERCENT ETHANOL

SUCROSE
Day 9

% Ethanol =

% Ethanol =

2.5
30

x 100

x 100 = 8.33%

For 500 mL wort = 41.67 mL ethanol

CALCULATIONS FOR PERCENT ETHANOL

SUCROSE

Day 18

% Ethanol =
x 100

1
% Ethanol =
x 100 = 3.33%
30

For 500 mL wort = 16.67 mL ethanol

CALCULATIONS FOR PERCENT ETHANOL

SUCROSE

AVERAGE

% Ethanol =
x 100

1.167
% Ethanol =
x 100 = 3.87 %
30

For 500 mL wort = 19.33 mL Ethanol

CALCULATIONS FOR PERCENT ETHANOL

GLUCOSE

Day 7
%

Ethanol =
x 100

0
% Ethanol = x 100 = 0%
30

CALCULATIONS FOR PERCENT ETHANOL

GLUCOSE

Day 9

% Ethanol =
x 100

2
% Ethanol =
x 100 = 6.67%
30

For 500 mL wort = 33.33 mL ethanol

CALCULATIONS FOR PERCENT ETHANOL

GLUCOSE

Day 18

% Ethanol =
x 100

1
% Ethanol =
x 100 = 3.33%
30

For 500 mL wort = 16.67 mL ethanol

CALCULATIONS FOR PERCENT ETHANOL

GLUCOSE

AVERAGE

% Ethanol =
x 100

1
% Ethanol =
x 100 = 3.33 %
30

For 500 mL wort = 16.67 mL Ethanol

TABLE 1.1 AMOUNT ETHANOL IN ALIQUOT WORT

mL of ethanol collected in 30 mL aliquot of wort
mL of ethanol collected

3
2.5
2
1.5
1
0.5
0

Molasses
Sucrose
Glucose

Day 7
0
0
0

Day 9
1
2.5
2

Day 18
1
1
1

TABLE 1.2 AMOUNT ETHANOL IN WORT

mL of ethanol
collected

mL of Ethanol in 500 mL of wort

80
70
60
50
40
30
20
10
0

Molasses
Sucrose
Glucose

Day 7
0
0
0

Day 9
16.67
71.67
33.33

Day 18
16.67
16.67
16.67

Percent Ethanol Collected (%)

Percent Ethanol Collected from Wort

9
8
7
6
5
4
3
2
1
0

Molasses
Sucrose
Glucose

Day 7
0
0
0

Day 9
3.33
8.33
6.67

Day 18
3.33
3.33
3.33

Average
2.23
3.87
3.33

Fermentation Yield (Yf) =

CALCULATIONS FOR FERMENTATION YIELD

CALCULATIONS FOR FERMENTATION YIELD

MOLASSES

Day 7
Yf =
Yf =

0.5 0
0.0000375 0.795

=0

CALCULATIONS FOR FERMENTATION YIELD

MOLASSES

Day 9
Yf =
Yf =

0.5 0.01667
0.0000375 0.795

Yf = 279.581

0.008335
0.0000298

CALCULATIONS FOR FERMENTATION YIELD

MOLASSES

Day 18
Yf =

Yf =

0.5 0.01667
0.008335
=
0.0000375 0.795 0.0000298

Yf = 279.581

CALCULATIONS FOR FERMENTATION YIELD

MOLASSES

AVERAGE
Yf =

Yf =

0.5 0.01167
0.005835
=
0.0000375 0.795 0.0000298

Yf = 195.740

CALCULATIONS FOR FERMENTATION YIELD

SUCROSE

Day 7
Yf =
Yf =

0.5 0
0.0000375 1.000

=0

CALCULATIONS FOR FERMENTATION YIELD

SUCROSE

Day 9
Yf =

Yf =

0.5 0.04167
0.020835
=
0.0000375 1.000 0.0000375

Yf = 555.600

CALCULATIONS FOR FERMENTATION YIELD

SUCROSE

Day 18
Yf =

Yf =

0.5 0.01667
0.008335
=
0.0000375 1.000 0.0000375

Yf = 222.267

CALCULATIONS FOR FERMENTATION YIELD

SUCROSE

AVERAGE
Yf =

Yf =

0.5 0.01933
0.00967
=
0.0000375 1.000 0.0000375

Yf = 257.733

CALCULATIONS FOR FERMENTATION YIELD

GLUCOSE

Day 7
Yf =
Yf =

0.5 0
0.0000375 1.000

=0

CALCULATIONS FOR FERMENTATION YIELD

GLUCOSE

Day 9
Yf =

Yf =

0.5 0.03333
0.01667
=
0.0000375 1.000 0.0000375

Yf = 444.400

CALCULATIONS FOR FERMENTATION YIELD

GLUCOSE

Day 18
Yf =

Yf =

0.5 0.01667
0.008335
=
0.0000375 1.000 0.0000375

Yf = 222.267

CALCULATIONS FOR FERMENTATION YIELD

GLUCOSE

AVERAGE
Yf =

Yf =

0.5 0.01667
0.008335
=
0.0000375 1.000 0.0000375

Yf = 222.267

DISCUSSION
Fermentation Yield (L/ton)

Fermentation Yield
600
500
400
300
200
100
0

Molasses
Sucrose
Glucose

Day 7
0
0
0

Day 9
279.581
555.6
444.4

Day 18
279.581
222.267
222.267

Comparison of Fermentation Yield of ethanol in worts

Fermentation Yield
(L/ton)

600
500
400
300
200
100
0

Molasses
Sucrose
Glucose

Day 7
0
0
0

Day 9
279.581
555.6
444.4

Day 18
279.581
222.267
222.267

Average
195.74
257.733
222.267

Fermentation Efficiency (Ef) = Yf x

0.794
0.511

100
1000

CALCULATIONS FOR FERMENTATION EFFICIENCY

CALCULATIONS FOR FERMENTATION EFFICIENCY

MOLASSES

Day 7
0.794
100
Ef = Yf x
x
0.511 1000
0.794
100
Ef = 0 x
x
= 0%
0.511 1000

CALCULATIONS FOR FERMENTATION EFFICIENCY

MOLASSES

Day 9

Ef =

0.794
100
Ef = Yf x
x
0.511 1000
0.794
100
279.581 x
x
=
0.511 1000

Ef = 43.44%

43.44%

CALCULATIONS FOR FERMENTATION EFFICIENCY

MOLASSES

Day 18

Ef =

0.794
100
Ef = Yf x
x
0.511 1000
0.794
100
279.581 x
x
=
0.511 1000

Ef = 43.44%

43.44%

CALCULATIONS FOR FERMENTATION EFFICIENCY

MOLASSES

AVERAGE

Ef =

0.794
100
Ef = Yf x
x
0.511 1000
0.794
100
195.740 x
x
=
0.511 1000

Ef = 30.41%

30.41%

CALCULATIONS FOR FERMENTATION YIELD

SUCROSE

Day 7
0.794
100
Ef = Yf x
x
0.511 1000
0.794
100
Ef = 0 x
x
= 0%
0.511 1000

CALCULATIONS FOR FERMENTATION YIELD

SUCROSE

Day 9

Ef =

0.794
100
Ef = Yf x
x
0.511 1000
0.794
100
555.600 x
x
=
0.511 1000

Ef = 86.33%

86.33%

CALCULATIONS FOR FERMENTATION YIELD

SUCROSE

Day 18

Ef =

0.794
100
Ef = Yf x
x
0.511 1000
0.794
100
222.267 x
x
=
0.511 1000

Ef = 34.54%

34.54%

CALCULATIONS FOR FERMENTATION YIELD

SUCROSE

AVERAGE

Ef =

0.794
100
Ef = Yf x
x
0.511 1000
0.794
100
257.733 x
x
=
0.511 1000

Ef = 40.05%

40.05%

CALCULATIONS FOR FERMENTATION YIELD

GLUCOSE

Day 7
0.794
100
Ef = Yf x
x
0.511 1000
0.794
100
Ef = 0 x
x
= 0%
0.511 1000

CALCULATIONS FOR FERMENTATION YIELD

GLUCOSE

Day 9

Ef =

0.794
100
Ef = Yf x
x
0.511 1000
0.794
100
444.400 x
x
=
0.511 1000

Ef = 69.05%

69.05%

CALCULATIONS FOR FERMENTATION YIELD

GLUCOSE

Day 18

Ef =

0.794
100
Ef = Yf x
x
0.511 1000
0.794
100
222.267 x
x
=
0.511 1000

Ef = 34.54%

34.54%

CALCULATIONS FOR FERMENTATION YIELD

GLUCOSE

AVERAGE

Ef =

0.794
100
Ef = Yf x
x
0.511 1000
0.794
100
222.267 x
x
=
0.511 1000

Ef = 34.54%

34.54%

Fermentation Efficiency (%)

Fermentation Efficiency
100
90
80
70
60
50
40
30
20
10
0

Molasses
Sucrose
Glucose

Day 7
0
0
0

Day 9
43.44
86.33
69.05

Day 18
43.44
34.54
34.54

Fermentation Efficiency (%)

Comparison of Fermentation Efficiency in worts

100
90
80
70
60
50
40
30
20
10
0

Molasses
Sucrose
Glucose

Day 7
0
0
0

Day 9
43.44
86.33
69.05

Day 18
43.44
34.54
34.54

Average
30.41
40.05
34.54

CONCLUSION

SUMMARY OF RESULTS - MOLASSES

MOLASSES

% Ethanol

Yf (L/ton)

Ef(%)

Day 7

0%

0%

Day 9

3.33 %

Day 18

3.33 %

0 (L/ton)
279.581
(L/ton)
279.581
(L/ton)

Average

2.23 %

195.540
(L/ton)

43.44%
43.44%
30.41%

PERCENT ERROR - MOLASSES

P.E.=

| |
x

|11.17 43.53|
=
x 100
43.53
32.36
=
x 100
42.53

= 0.7609 x 100
= 76.09% error

100

SUMMARY OF RESULTS - SUCROSE

SUCROSE

% Ethanol

Yf (L/ton)

Ef(%)

Day 7

0%

0%

Day 9

8.33 %

Day 18

3.33 %

0 (L/ton)
555.600
(L/ton)
222.267
(L/ton)

Average

3.87 %

257.733
(L/ton)

86.33 %
34.54 %
40.05%

PERCENT ERROR - SUCROSE

P.E.=

| |
x

|19.33 25.59|
=
x 100
25.59
6.26
=
x 100
25.59

= 0.2446 x 100
= 24.46% error

100

SUMMARY OF RESULTS - GLUCOSE

GLUCOSE

% Ethanol

Yf (L/ton)

Ef(%)

Day 7

0%

0%

Day 9

6.67 %

Day 18

16.67 %

0 (L/ton)
444.400
(L/ton)
222.267
(L/ton)

Average

16.67 %

222.267
(L/ton)

69.05 %
34.54 %
34.54%

PERCENT ERROR - GLUCOSE

P.E.=

| |
x

|16.67 24.31|
=
x 100
24.31
7.64
=
x 100
24.31

= 0.3143 x 100
= 31.43% error

100

CONCLUSION
Based from results, the second day collected
the most ethanol from the three samples
Sucrose ended up producing the most ethanol
from the three wort solutions

REASONS FOR ERROR

For small amount of ethanol produced:

Not

optimum temperature for fermentation (35C

compared to room temperature)
Lack of optimization of nutrients from worts (settle)
High water component (500mL)

Lack of aseptic technique

Entering

of oxygen (aerobic)

REFERENCES
The Sugar Engineers. (2014). Distillery Yields.
Retrieved on November 8, 2014 from
http://www.sugartech.com/alcohol/yields.php
The Fermentation of Sugar and the Isolation of
Ethanol through Distillation. Retrieved on
November 8, 2014 from
http://web.fscj.edu/Milczanowski/ten/ETHANO
L.pdf

REFERENCES
Periyasamy, S., Sivakumar, V., Ramasamy, S.
(2009) Production of Bio-ethanol from Sugar
Molasses Using Saccharomyces cerevisae.
Modern Applied Sciences, Vol. 4, No. 8
Patrascu, ., Rapeanu, R., Hopulele, T. (2009)
Current Approaches to Efficient Biotechnological
Production of Ethanol. Innovative Romanian Food
Technology. Vol. 4

REFFERENCES
Lin, Y., Zhang, W., Li, C., Sakakibara K., Tanaka
S., Kong, H. (2011) Factors Affecting ethanol
fomation using Saccharomyces cervsiae.
Science Direct, 2012
Amin, R., Saquib, H., Sarker, M. (2013)
Simulation of Ethanol Production by
Fermentation of Molasses. Journal of
Engineering, Vol. 1, No. 4 ISSN: 2325-0224

THANK YOU
GROUP 4 : 3A-BIOCHEMISTRY