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Ethanol yield from fruit peels and adsorption of heavy metal ions
Done by: Aman Mangalmurti
Kara NewmanLeong Qi Dong
Soh Han Wei
RationaleDepletion of non-renewable fossil
fuels due to excessive
consumption as a source of energy
Conversion of renewable
sources, e.g. organic wastes, to
fuel ensures continual energy
supplyPotential for
producing ethanol from fruit peel wastes through fermentation by microorganisms
Ethanol as a renewable,
alternative energy source
RationaleHeavy metal
water contamination of water is rampant
in many countries.
Heavy metal ions accumulate inside
organisms and cause adverse health effects
Biosorption in removal of heavy
metal ions by fruit peel wastes
Literature ReviewDemand for renewable energy resources has
increased due to increased prices for oil and concerns about global warming (Wilkins , Widmer & Grohmann, 2007)
Production of ethanol by Saccharomyces cerevisiae fromMango fruit processing solid and liquid wastes
(Reddy, Reddy & Wee, 2011)Pineapple waste (Hossain & Fazliny, 2010)
Literature ReviewIndustries such as electroplating, mining and
paint contribute to heavy metal pollution in the ambient environment
Heavy metal ions that pollute water include antimony, copper, lead, mercury, arsenic and cadmium (US Environmental Protection Agency, 2011)
Methods of removal of ions include chemical precipitation and solvent extractionExpensive and low efficiency at low metal ion
concentrations
ObjectivesTo prepare extracts of fruit
peel for ethanol fermentation
To determine which fruit peel gives highest ethanol yield from
the fermentation of fruit peel extract
To determine which fruit peel waste gives rise to maximal
adsorption of heavy metal ions of Cu2+
,Cu3+ ions
HypothesisEthanol yield from fermentation of the
banana peel would be higher than that of the mango peel
Zymomonas mobilis produces more ethanol during fermentation as compared to Saccharomyces cerevisiae
The mango peel would adsorb heavy metal ions better as compared to banana peels
Experimental outline
Preparation of fruit peel extract
First ethanol fermentation
Heavy metal ion adsorption for Copper(II), Copper (III) ions
Second ethanol fermentation after treatment of peel residue with
cellulase
VariablesConstant
• Temperature of growth of organisms (30C)
• Initial concentration of heavy metal ions (50 ppm)
Independent
• Fruit peels used (AOS: banana, HCI: mango)
• Organism used (S. cerevisiae, Z. mobilis)
• Heavy metal ions (Cu2+ ,Cu4+)
Dependent
• Initial concentration of reducing sugars in fruit peel extracts
• Ratio of ethanol yield to initial sugar concentration
• Final ethanol yield
• Final concentration of heavy metal ions
Apparatus & MaterialsApparatus Materials Blender Sieve Boiling water bath Spectrophotometer cuvettes Spectrophotometer Centrifuge Glass rod Hot Plate Incubator Dropper Sieve: 0.25mm (60 Mesh) Shaking incubator Fractional distillator Test tubes Filter funnel Filter paper Beaker Volumetric Flask Colorimeter Quincy Lab Model 30 GC hot-air oven Measuring cylinder Magnetic stirrer Rotary mill
Mango Peel Banana Peel Deionised water Dinitrosalicylic acid (DNS acid) Zymomonas mobilis Saccharyomyces cerevisiae Glucose-yeast medium (Yeast malt extract
broth) sodium alginate medium calcium chloride solution sodium chloride solution acidified potassium chromate solution Cu2+ ion solution Cu4+ ion solution MgSO4∙7H2O 0.1 and (magnesium sulfide
hydrate) KH2PO4 0.1 (potassium phosphate) cellulase
Extraction of sugars from fruit peels
30 g of fruit peels are blended in
300 ml of deionised water using a blender.
The liquid is passed through a sieve to remove
the residue.
Determination of sugars in extractsTo 0.5 ml of
extract, 0.5 ml of DNS
(dinitrosalicylic acid) is added.
The mixture is left in a boiling water bath for 5
minutes.
4 ml of water is then added.
The samples are placed in spectrophotometer cuvettes and the absorbance is taken
at 530 nm using a spectrophotometer.
The concentration of reducing sugars in
μmol/ml is read from a maltose standard curve.
Growth of Z. mobilis
Z. mobilis cells are inoculated in 20 ml GY medium (2% glucose, 0.5% yeast extract) and incubated at 30°C for 2 days with shaking.
Immobilisation of cells
The Z. mobilis preculture and S.
cerevisiae preculture are centrifuged at 7000 rpm for 10
minutes and the cell pellets are
resuspended in 7.5 ml of fresh GY medium.
The absorbance of the cultures are taken at 600 nm.
7.5 ml of 2% sodium alginate is added to the cell suspension and
mixed well.
The mixture is dropped into 0.1 mol dm‐3 calcium
chloride solution to form Z. mobilis alginate beads.
The beads are rinsed with 0.85% sodium chloride
solution.
Growth of S. cerevisiae
S. cerevisiae cells are inoculated in 50 ml YM broth medium with the pH adjusted to 5.6 and incubated at 35°C for 1 days with shaking, before being concentrated in a refrigerated centrifuge at 10, 000 rpm.
Ethanol fermentation by immobilized Z. mobilis cells
200 beads are added to 50 ml waste extract.
A control is prepared in
which 200 empty alginate beads
are added to the same volume of waste extract
instead.
All the set‐ups are incubated
with shaking at 30°C for 2 days
for ethanol fermentation to
occur.
The beads are then removed
and the extracts are distilled to obtain ethanol.
Ethanol fermentation by S. cerisiaeTo be added
Back
Determination of ethanol yield with the dichromate test
2.5 ml of acidified
potassium dichromate
solution is added to 0.5 ml of
distillate in a ratio of 5:1.
The samples are placed in a
boiling water bath for 15 minutes.
The absorbance is measured at 590 nm using a
spectrophotometer, and the
concentration of ethanol is read from an ethanol standard curve.
Adsorption of heavy metal ions Desiccate fruit peel
residue, (put the residue in the hot air oven and dry them
at 60 degrees for 23 hours)
Using a rotary mill to grind desiccated
residue
Sieve to 0.25 mm particle size.
Add residue powder to 50ppm Cu2+
solution.
Allow solution to set for 20 min,
preferably at 100rpm to increase
contact time
Repeat for Cu4+
Determination of final ion concentration
Allow solution to set for 20 min, preferably at
100rpm to increase contact time
Remove fruit product, by filtering
the suspension
Using a copper reagent, the remaining
concentration of copper ions will be
found
Treatment of residue with cellulase
Fruit peel particles
are added into the beaker.
50 ml water is added to beaker
25ml of cellulase is added
to the beaker.
Beaker is left
standing for 1 hour
with continuous stirring.
The beaker is drained and fruit
peel is left to dry.
Second ethanol fermentation Identical to aboveEthanol fermentation
Determination of final ethanol yield
2.5 ml of acidified
potassium dichromate
solution is added to 0.5 ml of
distillate in a ratio of 5:1.
The samples are placed in a
boiling water bath for 15 minutes.
The absorbance is measured at 590 nm using a
spectrophotometer, and the
concentration of ethanol is read from an ethanol standard curve.
Applications
Cost-effective method of producing
ethanol
Reduces reliance on non-
renewable fossil fuels
Recycles fruit peels
Viable method in wastewater treatment
Timeline
Finalizing of project
details 12-23 Nov
1st round of experiments 7 Dec - Mar
2nd round of experiments Mar - May
Final round of
experiments and Data Analysis May - Jul
Bibliography Anhwange, T. J. Ugye, T.D. Nyiaatagher (2009). Chemical composition of Musa
sapientum (Banana) peels. Electronic Journal of Environmental, Agricultural and Food Chemistry, 8, 437-442
Retrieved on 29 October 2011 from:http://ejeafche.uvigo.es/component/option,com_docman/task,doc_view/gid,495 Björklund, G. Burke, J. Foster, S. Rast, W. Vallée, D. Van der Hoek, W. (2009,
February 16). Impacts of water use on water systems and the environment (United Nations World Water Development Report 3). Retrieved June 6, 2011, from
www.unesco.org/water/wwap/wwdr/wwdr3/pdf/19_WWDR3_ch_8.pdf US Environmental Protection Agency (2011) .Drinking Water Contaminants.
Retrieved June 6, 2011, From http://water.epa.gov/drink/contaminants/index.cfm Mark R. Wilkins , Wilbur W. Widmer, Karel Grohmann (2007). Simultaneous
saccharification and fermentation of citrus peel waste by Saccharomyces cerevisiae to produce ethanol. Process Biochemistry, 42, 1614–1619.
Retrieved on 29 October 2011 from:http://ddr.nal.usda.gov/bitstream/10113/16371/1/IND44068998.pdf
References Hossain, A.B.M.S. & Fazliny, A.R. (2010). Creation of alternative energy by bio‐ethanol production
from pineapple waste and the usage of its properties for engine. African Journal of Microbiology Research, 4(9), 813‐819. Retrieved October 27, 2011 from http://www.academicjournals.org/ajmr/PDF/Pdf2010/4May/Hossain%20and%20Fazliny.pdf
Mishra, V., Balomajumder, C. & Agarwal, V.K. (2010). Biosorption of Zn(II) onto the surface of non‐living biomasses: a comparative study of adsorbent particle size and removal capacity of three different biomasses. Water Air Soil Pollution, 211, 489‐500. Retrieved October 27, 2011 from http://www.springerlink.com/content/2028u2q551416871/fulltext.pdf
Tanaka, K., Hilary, Z.D. & Ishizaki, A. (1999). Investigation of the utility of pineapple juice and pineapple waste material as low‐cost substrate for ethanol fermentation by Zymomonas mobilis. Journal of Bioscience and Bioengineering, 87(5), 642‐646.
Ban‐Koffi, L. & Han, Y.W. (1990). Alcohol production from pineapple waste. World Journal of Microbiology and Biotechnology, 6(3), 281‐284.
Reddy, L.V., Reddy, O.V.S. & Wee, Y.‐J. (2011). Production of ethanol from mango (Mangifera indica L.) peel by Saccharomyces cerevisiae CFTRI101. African Journal of Biotechnology, 10(20), 4183‐4189. Retrieved October 27, 2011 from http://www.academicjournals.org/AJB/PDF/pdf2011/16May/Reddy%20et%20al.pdf
Isitua, C.C. & Ibeh, I.N. (2010). Novel method of wine production from banana (Musa acuminata) and pineapple (Ananas comosus) wastes. African Journal of Biotechnology, 9(44), 7521‐7524.
Nigam, J.N. (2000). Continuous ethanol production from pineapple cannery waste using immobilized yeast cells. Journal of Biotechnology, 80(2), 189‐193. Saccharomyces cerevisiae ATCC 24553 immobilised in k‐carrageenan