A COMPARATIVE STUDY OF THE YIELD OF BIOETHANOL IN ALGAE, CORN AND NEWSPAPER

Group: 01-36

Team MembersTang Kwan Hou (L) (4S123)

Robin Ho (4S116)Jerroy Chang (4S203)

Content

• Aim

• Hypothesis

• Variables

• Materials and Method

• Results and Analysis

• Conclusions

• Extensions

• References

Problem to be addressed

• Methods of extracting bioethanol too expensive or energy-consuming

• Find out an effective and cheap way to produce bioethanol

oBioethanol is rising in demand across the world

Aim

• To investigate and compare yield of bioethanol per unit mass of different substrates at optimum conditions

• To investigate the optimum concentration of cellulase and amylase to use for each substrate

• Ulva• Macroalgae contain significant amount of sugars (at least 50%) that could

be used in fermentation for bioethanol production (Wi et al., 2009)

• Most green algae can have a cellulose content of up to 70% of dry mass (B. Baldan, P. Andolfo, L. Navazio, C. Tolomio, P. Mariani, 2002)

• Corn• An increase in the ethanol production means an increase in the demand

of corn (Pimental D., 2009)

• Corn kernels contain 75.2% starch and 30% cellulose. (Yong T., Zhao D., Cristhian C., Jiang J., 2011)

Literature Review

• Paper• The presence of 70% cellulose & hemicellulose, α-cellulose

(60%) and lignin (16%) makes it a prospective and renewable biomass for bioethanol production (Alok K.D. et. al, 2012)

• Husk• Corn husks contain 42% cellulose and 13% lignin. (Y.

Mahalaxmi, et. al, 2009)

• Often discarded when people prepare corn

Literature Review

• Sargassum• The brown seaweed Sargassum sp. is a promising feedstock

for ethanol production because of its relatively high content (41.6% dry basis) of holocellulose. It also contains 22.0% of alpha-cellulose and 19.6% of hemicellulose. (Jeylnne P. et. al, 2014)

Literature Review

• Commercial Production• Acid Hydrolysis

• Algae species were hydrolysed in dilute 1.0ml of 0.70% H2SO4 and were heated at

105°C for 6h. (Gupta R. et al, 2012)

• Required 95.103 kWh power which costs $24.42 according to Singapore’s electrical tariff of $0.2568 between 1 July 2014 to 30 Sep 2014

• Wet Milling

• Corn kernel is steeped in water, with or without sulphur dioxide, to soften the seed kernel in order to help separate the kernel’s various components.

• For example, it can separate a 56-pound bushel of corn into more than 31 pounds of corn starch, which in turn can be converted into corn ethanol (J. Womach et al, 2005)

Literature Review

• Cellulase has an optimum pH between 4 to 5 and an optimum temperature between 40 to 50ºC (Carl B. Z., n.d.)

• The optimum temperature of the α-amylase is 50ºC and optimum pH value is 6 (Atiyeh M., Reza H. S., Mehdi R., Vahab J. , 2010)

• Optimum temperature for fermentation by Saccharomyces cerevisiae is at 45ºC but will ethanol yield will drop above that (Lin Y. et al, 2012)

Literature Review

Optimal pH and Temperature

Optimum pH

Cellulase

4.0-5.0

Alpha-Amylas

e

6.0

Optimum Temperature

Cellulase

40-50℃

Alpha-Amylas

e

50℃

Hypothesis

• Paper produces the greatest yield of bioethanol (cm3/g), after enzymatic action and fermentation.

• The usage of pH 5.0 acetate buffer and enzymatic action at 45°C will increase yield of bioethanol (cm3/g).

Variables• Independent:

• Type of starting product

• Concentration of cellulase added (%)

• Concentration of amylase added (%)

• Dependent:

• Yield of bioethanol after a fixed period of time

• Controlled:

• Mass of starting material used (6.0g)

• Temperature of surroundings (Room temperature or 45°C)

• Duration of fermentation (1 day)

• pH value of solution (7.0 or 5.0)

MATERIALS AND METHODS

MATERIALS TO BE TESTED ON

• AlgaeUlva sp. (green algae)Sargassum sp. (brown algae)

• Zea mays (maize)• Kernel

• Husk

• Newspaper

• Potato Dextrose Broth

• Cultured Yeast (Saccharomyces cerevisiae)

• Cellulase

• Alpha-Amylase

• Deionised Water

OTHER MATERIALS USED

APPARATUS

• Rack Shaker

• Weighing Scale

• Centrifuge machine

• Centrifuge tubes

• Incubator

• Water Bath

• Blender

• Ethanol Probe

60mlamylase

60ml cellulase

6g materi

al

60ml DI

water

24:00:00

37°C

• Independent variable – Starting materials (Paper, Ulva sp. , Kernel, Husk)

Methodology

Homogenisation

Enzymatic action

Supernatant

25°C90°C

00:10:00

5000 rpm

Methodology

• Heated at 90 degrees Celsius to halt enzyme catalysis reaction by inactivating it (Nam S. W., n.d.)

Denaturing

CentrifugationDecanting

60ml pH5.0

Acetate buffer

60ml DI

water

1L DI Water

24g PDB(Potato Dextrose

Broth)

1L PDB

00:15:00

121°C

Methodology

Preparing yeast broth

Yeast

Yeast

30mL PDB

30mL PDB1L PDB

37°C

24:00:00

Methodology

Preparing yeast broth

Inoculation

6.7mL

yeast

3.3mL

extract

30mL PDBSupernata

nt

24:00:00

37°C

Methodology

Inoculation

Fermentation

Methodology

Reading Results

RESULTS AND ANALYSIS

Results - Husk

0.25 0.5 1 20

0.05

0.1

0.15

0.2

0.25

0.3

0.2000.245

0.180 0.190

Bar chart showing the effect of concentration of cellulase

on ethanol yield/%

Cellulase concentration/%

Eth

an

ol Y

ield

/%

0.25 0.5 1 20.320.330.340.350.360.370.38

0.343 0.350

0.373 0.370

Bar chart showing the effect of concentration of amylase

on ethanol yield/%

Amylase concentration/%

Eth

an

ol Y

ield

/%From the graph we can see that:Best Cellulase Concentration: 0.50%Best Amylase Concentration: 1.00%

Results - Kernel

0.25 0.5 1 20

0.02

0.04

0.06

0.08

0.030

0.0600.070

0.030

Bar chart showing the effect of concentration of cellulase on

ethanol yield/%

Cellulase concentration/%

Eth

an

ol Y

ield

/%

0.25 0.5 1 20

0.1

0.2

0.3

0.4

0.237 0.2700.323 0.370

Bar chart showing the effect of concentration of amylase on

ethanol yield/%

Amylase concentration/%

Eth

an

ol Y

ield

/%From the graph we can see that:Best Cellulase Concentration: 1.00%Best Amylase Concentration: 2.00%

Results - Paper

0.25 0.5 1 20

0.05

0.1

0.15

0.2

0.1200.150

0.180 0.160

Bar chart showing the effect of concentration of cellulase

on ethanol yield/%

Cellulase concentration/%

Eth

an

ol Y

ield

/%

0.25 0.5 1 20

0.10.20.30.40.50.6

0.480 0.407 0.390 0.387

Bar chart showing the effect of concentration of amylase

on ethanol yield/%

Amylase concentration/%

Eth

an

ol Y

ield

/%From the graph we can see that:Best Cellulase Concentration: 1.00%Best Amylase Concentration: 0.25%

Results – Ulva sp.

0.25 0.5 1 20.22

0.24

0.26

0.28

0.3

0.2750.293

0.273

0.247

Bar chart showing the effect of concentration of amylase on

ethanol yield/%

Amylase concentration/%

Eth

an

ol Y

ield

/%

1 2 3 40.000

0.020

0.040

0.060

0.080

0.100

0.057 0.0600.077 0.077

Bar chart showing the effect of concentration of cellulase on

ethanol yield/%

Cellulase concentration/%

Eth

an

ol Y

ield

/%

From the graph we can see that:Best Cellulase Concentration: 1.00%Best Amylase Concentration: 0.50%

• Best amylase concentration varies with each extract.

• However, Mann-Whitney U and Kruskal-Wallis Test shows that the difference in results are insignificant.

• Best cellulase concentration for All Starting Materials: 1.00%

• Except husk (0.50%)

Data Analysis

Best Cellulase Concentration/%

Best Amylase Concentration/%

Husk 0.50 1.00

Kernel 1.00 2.00

Paper 1.00 0.25

Ulva sp. 1.00 0.50

Summary

Results – Sargassum sp.

0.25 0.50 1.00 2.000.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.140.18

0.22

0.29

Graph showing effect of varying concentration of cellulase/% on

ethanol yield/%

Eth

an

ol Y

ield

/%

0.25 0.50 1.00 2.000

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.08

0.18 0.19

0.3

Graph showing effect of varying concentration of cellulase/% on

ethanol yield/%

Eth

an

ol Y

ield

/%

Results - Paper

0.25 0.50 1.00 2.000

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0.12 0.15 0.18 0.160.25 0.25

0.59

0.95

Graph showing effect of varying concentration of cellulase/% on ethanol

yield/%

BeforeAfter

Eth

anol Yie

ld/%

0.25 0.50 1.00 2.000

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.480.407 0.39 0.387

0.24 0.25

0.53

0.71

Graph showing effect of varying concentration of amylase/% on ethanol

yield/%

BeforeAfter

Results - Kernel

0.25 0.50 1.00 2.000

0.05

0.1

0.15

0.2

0.25

0.030.06 0.07

0.03

0.13

0.170.19

0.22

Graph showing effect of varying concentration of cellulase/% on

ethanol yield/%

BeforeAfter

Eth

anol Yie

ld/%

0.25 0.50 1.00 2.000

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.2370.27

0.3230.37

0.07

0.17 0.16

0.28

Graph showing effect of varying concentration of amylase/% on ethanol

yield/%

BeforeAfter

Eth

anol Yie

ld/%

Results - Husk

0.25 0.50 1.00 2.000

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.343 0.35 0.373 0.37

0.110.07

0.3

0.42

Graph showing effect of varying concentration of amylase/% on ethanol

yield/%

BeforeAfter

Eth

anol Yie

ld/%

0.25 0.50 1.00 2.000

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.20.245

0.307

0.19

0.020.07

0.380.43

Graph showing effect of varying concentration of cellulase/% on

ethanol yield/%

BeforeAfter

Eth

anol Yie

ld/%

Results – Ulva sp.

0.25 0.50 1.00 2.000

0.1

0.2

0.3

0.4

0.5

0.6

0.0650000000000001 0.06 0.065 0.04

0.440.5

0.420.346666666666667

Graph showing effect of varying concentration of cellulase/% on

ethanol yield/%

BeforeAfter

Eth

anol Yie

ld/%

0.25 0.50 1.00 2.000

0.1

0.2

0.3

0.4

0.5

0.6

0.275 0.293 0.273 0.247

0.1

0.5

0.120.19

Graph showing effect of varying concentration of cellulase/% on

ethanol yield/%

BeforeAfter

Eth

anol Yie

ld/%

Best Cellulase Concentration/%

Best Amylase Concentration/%

Sargassum sp. 2.00 2.00

Paper 2.00 2.00

Kernel 2.00 2.00

Husk 2.00 2.00

Ulva sp. 0.50 0.50

Summary

Mann-Whitney U Test + Kruskal-Wallis Test (pH 7.0 and room temperature)

-

Kruskal Wallis Test (pH 5.0 and 45 degrees Celsius)

• Best amylase concentration varies with each extract.

• However, Mann-Whitney U and Kruskal-Wallis Test shows that the difference in results are insignificant.

• Best cellulase concentration for All Starting Materials: 1.00%

• Except husk (0.50%)

Data Analysis

• Most graphs were positive functions

• More enzyme, more ethanol produced

• Best concentration of both cellulase and amylase were 2.00%

• In all cases (except for Ulva sp.)

Data Analysis

COMPARISON OF RESULTS•Paper – 0.480%

•Husk – 0.373%

•Kernel – 0.370%

•Ulva sp. – 0.293%

•Paper – 0.950%

•Husk – 0.430%

•Kernel – 0.280%

•Ulva sp. – 0.500%

•Sargassum sp. – 0.300%

Before

After

• Converting ethanol yield/% into cm3/g:

• “It takes about 20 lb (9.1kg) of corn … to produce a gallon (3.9L) of ethanol” (The Energy Collective, 2013)

• 0.417cm3/g

Conclusion

Material Ethanol yield/%

Ethanol/cm3 per setup

Ethanol per gram (cm3/g)

Paper 0.950 0.0950 0.855

Husk 0.430 0.0430 0.387

Kernel 0.280 0.0280 0.252

Ulva sp. 0.500 0.0500 0.450

Sargassum sp.

0.300 0.0300 0.270

• Paper produces the greatest yield of bioethanol (cm3/g), after enzymatic action and fermentation.

• The usage of pH 5.0 acetate buffer and enzymatic action at 45°C increased yield of bioethanol (cm3/g).

Conclusion

EXTENSIONS

• Create a bioreactor using calcium chloride beads of immobilized enzymes and yeast

Sources of error and how to overcome them

• Ethanol probe was wetClean the probe and calibrate each time before reading

results

• Amount of yeast in each set-up was differentUse spectrometer to check turbidity of each PDB for

consistency

• Contamination of starting material (Bacteria entering solution)Micro-filter and do it in sterile environment

• Ethanol Probe may not be accurate in reading the ethanol yield due to the low yield

• KMnO4 can be added to the ethanol produced and titrated to

get a more accurate concentration

• However, it required a few weeks to prepare the KMnO4 at

the specific concentration required

• Less time-consuming if ethanol probe is used

Sources of error and how to overcome them

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References

THANK YOU!ANY QUESTIONS?