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
Aim
• To investigate and compare yield of bioethanol per unit
mass of different substrates
• 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. Lignocellulose contains 4.6% cellulose,
3.6% hemicellulose and 12.3% lignin. (Yong T., Zhao D., Cristhian C., Jiang J., 2011)
Literature Review
• Paper
• The presence of 70% carbohydrates (holocellulose), 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, T. Sathish, Ch.
Subba Rao, R.S. Prakasham, 2009)
• Often discarded when people prepare corn
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 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)
Hypothesis
• Paper produces the greatest yield of bioethanol (cm3/g),
after enzymatic action and fermentation.
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)
• Duration of fermentation (1 day)
MATERIALS AND METHODS
MATERIALS TO BE TESTED ON
• Algae
Ulva sp. (green algae)
• Zea mays (maize)
• Kernel
• Husk
• Waste paper
OTHER MATERIALS USED
• Potato Dextrose Broth
• Cultured Yeast (Sacchromyces 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
glucose
extract
90°C
25°C
00:10:00
Decanting
Centrifugation
Denaturing
• Heated at 90 degrees Celcius to halt enzyme catalysis reaction by inactivating it (Nam
S. W., n.d.)
Methodology
121°C
1L PDB
1L DI Water
24g PDB
(Potato Dextrose Broth)
00:15:00
Preparing yeast broth
Methodology
Yeast
24:00:00
Yeast
30mL
1LPDB
PDB
37°C
Preparing
yeast broth
Inoculation
Methodology
37°C
30mL
6.7mL
PDB
yeast
3.3mL
glucose
extract
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:
Optimal Cellulase Concentration: 0.50%
Optimal 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
0.270
0.237
0.25
0.323
0.5
1
Amylase concentration/%
From the graph we can see that:
Optimal Cellulase Concentration: 1.00%
Optimal Amylase Concentration: 2.00%
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:
Optimal Cellulase Concentration: 1.00%
Optimal 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
0.293
0.275
0.273
0.1
0.247
0.05
0.000
0
1
2
3
Cellulase concentration/%
4
0.25
0.5
1
Amylase concentration/%
From the graph we can see that:
Optimal Cellulase Concentration: 1.00%
Optimal Amylase Concentration: 0.50%
2
Summary
Best Cellulase
Concentration/%
Best Amylase
Concentration/%
Husk
0.50
1.00
Kernel
1.00
2.00
Paper
1.00
0.25
Ulva
1.00
0.50
Data Analysis
• Best amylase concentration varies with each extract.
• However, Mann-Whitney U Test shows that the difference in results are
insignificant.
• Best cellulase concentration for All Starting Materials: 1.00%
• Except husk (0.50%)
Mann Whitney U Test
Conclusion
• Based on best enzymes concentrations, extracts are ranked according to ethanol
yield:
1st
• Paper – 0.480%
2nd
• Husk – 0.373%
3rd
• Kernel – 0.370%
4th
• Ulva – 0.293%
Conclusion
• Converting ethanol yield/% into cm3/g:
Material
Ethanol yield/%
Ethanol/cm3 per setup
Ethanol per gram
Paper
0.480%
0.0480cm3
0.432cm3
Husk
0.373%
0.0373cm3
0.336cm3
Kernel
0.370%
0.0370cm3
0.333cm3
Ulva
0.293%
0.0293cm3
0.264cm3
• “It takes about 20 lb of corn, costing $1.54, to produce a gallon of ethanol” (The Energy
Collective, 2013)
• 0.417cm3/g
Conclusion
• Paper produces the greatest yield of bioethanol (cm3/g),
after enzymatic action and fermentation.
Extensions
• Sargassum sp. can be used to compare with the extracts. Why?
• The brown seaweed Sargassum sp. is a promising feedstock for
ethanol production because of its relatively high content (41.6% dry
basis) of hemicellulose. (Tamayo, J.P. & E.J. Del Rosario, 2014)
• Varying amounts of yeast, temperature and pH for optimal
condition of the enzymatic reaction / fermentation
Possible sources of error and how to
overcome (if applicable)
• 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)
Autoclave solution and do it in sterile environment
References
Alves, T. D. I., Araujo, E. E. C., … Pereira, J. N. (2009). Production of bioethanol from algae. Retrieved from:
http://www.Google.St/patents/WO2009067771A1?Cl=en
Ghosh, S. K., Bannerjee, S. & Aikat K. (2012). In renewable energy, vol. 37, no. 1. Bioethanol production from agricultural wastes: an overview. P. 19 – 27.
Retrieved 19 march 2014, from:
http://www.Unicentro.Br/posgraduacao/mestrado/bioenergia/editais/2012/artigo_anexo%20prova%20de%20profici%c3%aancia%20bioenergia.P
df
Goettemoeller, j. (2007). Sustainable ethanol: biofuels, biorefineries, cellulosic biomass, flex-fuel vehicles, and sustainable farming for energy independence. In
prairie oak publishing, maryville, missouri. P. 42.
Gupta, rachita (2012). Ethanol production from marine algae using yeast fermentation. P. 17- 22. Retrieved 1 march 2014, from:
http://www.Researchdesk.Net
Howard R. L., Abotsi E., Jansen van rensburg E.L. & Howard S. (2003). In african journal of biotechnology vol. 2, no. 12. Lignocellulose biotechnology:
issues of bioconversion and enzyme production. P. 602 – 619. Retrieved 20 march 2014, from:
http://www.Ajol.Info/index.Php/ajb/article/viewfile/14892/61491
Obura, n., Ishida, M., Hamada-sato, N., & Urano, N. (2012). Efficient bioethanol production from paper shredder scrap by a marine derived saccharomyces
cerevisiae c-19 (master's thesis).
References
Pimentel, D., Sarkar, N. , Tad, W. P. (2005). In natural resources research, vol. 14, no. 1. Ethanol production using corn, switchgrass, and wood;
biodiesel production using soybean and sunflower, p. 66-75. Retrieved 16 march 2014, from:
ftp://209.98.98.4/disk2/disk3/neurotic/pimentel2005-etohstudy.Pdf
Rosegrant, M. (2008). Biofuels and grain prices: impacts and policy responses. Retrieved from:
http://www.Grid.Unep.Ch/FP2011/step1/pdf/004_rosegrant_2008.Pdf
Sam, K. (2009) The choice of next-generation biofuels. Retrieved from:
http://www.Originoil.Com/pdf/scotia_capital_algae_excerpt.Pdf
Shetty P. R. (2009) Corn husk as a novel substrate for the production of rifamycin B by isolated amycolatopsis sp. RSP 3 under SSF. Retrieved from:
http://www.Academia.Edu/201890/corn_husk_as_a_novel_substrate_for_the_production_of_rifamycin_b_by_isolated_amycolatopsis_sp._Rsp_3_u
nder_ssf
The Energy Collective. (2013) The US Corn-to-Ethanol Program. Retrieved from:
http://theenergycollective.com/willem-post/287061/us-corn-ethanol-program
United states environmental protection agency (2008) Municipal solid waste generation, recycling, and disposal in the united states: facts and figures
for 2008. Retrieved from:
http://www.Epa.Gov/wastes/nonhaz/municipal/pubs/msw2008rpt.Pdf
References
Yanagisawa M., Nakamura K. , Ariga O. , Nakasaki A. (2011). Production of high concentrations of bioethanol from seaweeds that contain easily
hydrolysable polysaccharides. Retrieved from: http://www.Sciencedirect.Com/science/article/pii/S1359511311002765
Yong T., Zhao D., Cristhian C., Jiang J. (2011) Simultaneous saccharification and cofermentation of lignocellulosic residues from commercial furfural
production and corn kernels using different nutrient media in Biotechnology for Biofuels. Retrieved from:
http://www.biotechnologyforbiofuels.com/content/4/1/22
THANK YOU!
ANY QUESTIONS?