Transcript Document

A COMPARATIVE STUDY OF THE YIELD OF
BIOETHANOL IN ALGAE, CORN AND NEWSPAPER
Group: 01-36
Team Members
Tang 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 energyconsuming
• 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
Literature Review
• 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)
Optimal pH and Temperature
Optimum pH
Optimum Temperature
Cellulase
AlphaAmylase
Cellulase
AlphaAmylase
4.0-5.0
6.0
40-50℃
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 (𝑐𝑚3 /𝑔)
• 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
• Algae
Ulva sp. (green algae)
Sargassum sp. (brown algae)
• Zea mays (maize)
• Kernel
• Husk
• Newspaper
OTHER MATERIALS USED
• Potato Dextrose Broth
• Cultured Yeast (Saccharomyces cerevisiae)
• Cellulase
• Alpha-Amylase
• Deionised Water
APPARATUS
• Rack Shaker
• Incubator
• Weighing Scale
• Water Bath
• Centrifuge machine
• Blender
• Centrifuge tubes
• Ethanol Probe
Methodology
60ml
DI water
60ml
cellulase
60ml
amylase
6g
material
37°C
Enzymatic
action
Homogenisation
24:00:00
• Independent variable – Starting materials (Paper, Ulva sp. , Kernel, Husk)
Methodology
5000 rpm
Supernatant
90°C
25°C
00:10:00
Decanting
Centrifugation
Denaturing
• Heated at 90 degrees Celsius to halt enzyme catalysis reaction by inactivating it (Nam S.
W., n.d.)
60ml
DI water
60ml
pH5.0
Acetate
buffer
Methodology
121°C
1L PDB
1L DI Water
24g PDB
(Potato Dextrose Broth)
00:15:00
Preparing yeast broth
Methodology
Yeast
Yeast
24:00:00
30mL
1LPDB
PDB
37°C
Preparing
yeast broth
Inoculation
Methodology
37°C
30mL
6.7mL
PDB
yeast
3.3mL
Supernatant
extract
24:00:00
Fermentation
Inoculation
Methodology
Reading Results
RESULTS AND ANALYSIS
Results - Husk
Bar chart showing the effect of
concentration of cellulase on ethanol
yield/%
Bar chart showing the effect of
concentration of amylase on ethanol
yield/%
0.25
0.2
0.15
0.1
0.200
0.05
0
0.25
0.245
0.180
0.190
Ethanol Yield/%
Ethanol Yield/%
0.3
0.39
0.38
0.37
0.36
0.35
0.34
0.33
0.32
0.31
0.3
0.29
0.373
0.343
0.370
0.350
0.25
0.5
1
0.5
1
2
Amylase concentration/%
Cellulase concentration/%
From the graph we can see that:
Best Cellulase Concentration: 0.50%
Best Amylase Concentration: 1.00%
2
Results - Kernel
Bar chart showing the effect of
concentration of amylase on ethanol
yield/%
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
0.060
0.070
0.030
0.25
0.030
0.5
1
Cellulase concentration/%
2
Ethanol Yield/%
Ethanol Yield/%
Bar chart showing the effect of
concentration of cellulase on ethanol
yield/%
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0.237
0.25
From the graph we can see that:
Best Cellulase Concentration: 1.00%
Best Amylase Concentration: 2.00%
0.270
0.323
0.5
1
Amylase concentration/%
0.370
2
Results - Paper
Bar chart showing the effect of
concentration of cellulase on ethanol
yield/%
Bar chart showing the effect of
concentration of amylase on ethanol
yield/%
0.6
0.2
0.15
0.1
0.05
0.120
0
0.25
0.150
0.180
0.160
Ethanol Yield/%
Ethanol Yield/%
0.25
0.5
0.4
0.3
0.2
0.480
0.407
0.390
0.387
0.1
0
0.5
1
2
0.25
0.5
1
Cellulase concentration/%
Amylase concentration/%
From the graph we can see that:
Best Cellulase Concentration: 1.00%
Best Amylase Concentration: 0.25%
2
Results – Ulva sp.
Bar chart showing the effect of
concentration of cellulase on ethanol
yield/%
Bar chart showing the effect of concentration
of amylase on ethanol yield/%
0.35
0.3
0.080
0.060
0.040
0.020
0.057
0.060
0.077
0.077
Ethanol Yield/%
Ethanol Yield/%
0.100
0.25
0.2
0.15
0.275
0.293
0.273
0.1
0.247
0.05
0.000
0
1
2
3
Cellulase concentration/%
4
0.25
From the graph we can see that:
Best Cellulase Concentration: 1.00%
Best Amylase Concentration: 0.50%
0.5
1
Amylase concentration/%
2
Data Analysis
• 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%)
Summary
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
Results – Sargassum sp.
Graph showing effect of varying concentration
of cellulase/% on ethanol yield/%
0.40
0.4
0.35
0.35
0.30
0.25
0.20
0.15
0.29
0.10
0.05
0.18
0.22
0.14
Ethanol Yield/%
Ethanol Yield/%
Graph showing effect of varying concentration
of cellulase/% on ethanol yield/%
0.3
0.25
0.2
0.15
0.1
0.05
0.00
0.3
0.18
0.19
0.50
1.00
0.08
0
0.25
0.50
1.00
2.00
0.25
2.00
Results - Paper
Graph showing effect of varying concentration of
cellulase/% on ethanol yield/%
Graph showing effect of varying concentration of
amylase/% on ethanol yield/%
1.2
0.9
0.8
1
0.7
Ethanol Yield/%
0.8
0.6
0.5
Before
Before
0.6
After
After
0.4
0.71
0.95
0.3
0.4
0.59
0.12
0.25
0.15
0.1
0.18
0.24
0.39
0.387
0.25
0.16
0
0
0.25
0.407
0.2
0.2
0.25
0.53
0.48
0.50
1.00
2.00
0.25
0.50
1.00
2.00
Results - Kernel
Graph showing effect of varying concentration of
cellulase/% on ethanol yield/%
Graph showing effect of varying concentration of
amylase/% on ethanol yield/%
0.3
0.45
0.4
0.25
0.35
0.3
Before
0.15
After
0.22
0.1
0.19
0.17
0.25
0.2
0.1
Before
0.37
0.323
0.15
0.05
0.13
0.28
0.27
0.237
0.17
0.16
0.07
0
0.05
0.06
0.25
0.07
0.03
-0.05
0.03
0
0.25
Ethanol Yield/%
Ethanol Yield/%
0.2
0.50
1.00
2.00
-0.1
0.50
1.00
2.00
After
Results - Husk
Graph showing effect of varying concentration of
amylase/% on ethanol yield/%
0.5
0.5
0.45
0.45
0.4
0.4
0.35
0.35
0.3
Before
0.25
0.2
0.38
0.19
0.05
0
0.02
0.25
Before
0.25
0.2
0.373
0.35
0.343
0.42
0.37
0.3
0.1
0.245
0.2
After
0.3
0.15
0.307
0.15
0.1
0.43
Ethanol Yield/%
Ethanol Yield/%
Graph showing effect of varying concentration of
cellulase/% on ethanol yield/%
0.07
0.05
0.11
0
0.25
0.50
0.07
1.00
2.00
0.50
1.00
2.00
After
Results – Ulva sp.
Graph showing effect of varying concentration
of cellulase/% on ethanol yield/%
0.6
0.6
0.5
0.5
0.4
0.4
Before
0.3
After
0.5
0.44
0.2
0.42
Ethanol Yield/%
Ethanol Yield/%
Graph showing effect of varying concentration
of cellulase/% on ethanol yield/%
Before
0.3
After
0.5
0.2
0.35
0.293
0.275
0.273
0.1
0.1
0.065
0.06
0.065
0
0.25
0.50
1.00
0.12
0.1
0.04
2.00
0.247
0.19
0
0.25
0.50
1.00
2.00
Summary
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
Mann-Whitney U Test + Kruskal-Wallis Test (pH 7.0 and
room temperature)
-
Kruskal Wallis Test (pH 5.0 and 45 degrees Celsius)
Data Analysis
• 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.)
COMPARISON OF RESULTS
Before
• Paper – 0.480%
After
• Paper – 0.950%
• Husk – 0.373%
• Husk – 0.430%
• Kernel – 0.370%
• Kernel – 0.280%
• Ulva sp. – 0.293%
• Ulva sp. – 0.500%
• Sargassum sp. – 0.300%
Conclusion
• Converting ethanol yield/% into cm3/g:
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
• “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
• 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).
EXTENSIONS
• Create a bioreactor using
calcium chloride
beads of immobilized
enzymes and yeast
Sources of error and how to overcome them
• Ethanol probe was wet
Clean the probe and calibrate each time before reading results
• Amount of yeast in each set-up was different
Use spectrometer to check turbidity of each PDB for consistency
• Contamination of starting material (Bacteria entering solution)
Micro-filter and do it in sterile environment
Sources of error and how to overcome them
• 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
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