1. Done by: Aman Mangalmurti Kara Newman Leong Qi Dong Soh Han Wei

2. Rationale Depletion 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 supply Potential for producing ethanol from fruit peel wastes through fermentation by microorganisms Ethanol as a renewable, alternative energy source

3. Rationale Heavy 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

4. Literature Review Demand 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 from Mango fruit processing solid and liquid wastes (Reddy, Reddy & Wee, 2011) Pineapple waste (Hossain & Fazliny, 2010)

5. Literature Review Industries 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 extraction Expensive and low efficiency at low metal ion concentrations

6. Objectives To 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 Cu 2+,Zn 2+ and Pb 2+ ions To determine which procedure maximizes usage of fruit peel

7. Hypothesis Ethanol yield from fermentation of the banana peel would be higher than that of the mango peel The mango peel would adsorb heavy metal ions better as compared to banana peels Adsorption before fermentation would maximise the use of fruit peel.

8. Experimental Outline Preparation of fruit peel extract, microbe, heavy metal solution Adsorption of Cu 2+, Zn2+ and Pb 2+ ions Ethanol fermentation Adsorption of Cu 2+, Zn2+ and Pb 2+ ions

9. Variables Independent Fruit peels used (AOS: banana, HCI: mango) Heavy metal ions Order of Procedure 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 Constant Mass of fruit peel used for extraction of glucose Type of microorganism used Immobilisation of microorganism Fermentation conditions Initial concentration of heavy metal ions Duration of adsorption Mass of fruit peel particles used for adsorption Procedure

10. Apparatus & Materials Apparatus Materials Centrifuge Centrifuge tube Spectrophotometer Spectrophotometer cuvettes Glass rod Dropper Sieve Blender Boiling water bath Shaking incubator Fractional distillatory Quincy Lab Model 30 GC hot-air oven Rotary Mill Sieve: 0.25mm (60 Mesh) Zymomonas mobilis Glucose-yeast medium Sodium alginate medium Calcium chloride solution Sodium Chloride solution Fruit peel Deionised water Dinitrosalicylic acid Acidified potassium chromate solution Lead (II), Copper (II), Zinc (II) ion solutions Lead (II). Copper (II), Zinc (II) reagent kits

11. 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.

12. 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.

13. 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.

14. Determination of sugars in extracts To 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.

15. 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.

16. 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.

17. 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. A heavy metal mixture with 20ml of each metal ion (zinc, lead and copper ions) is created.. Add powder to mixture

18. Determination of final ion concentration Allow solution to set for 20 min at 100rpm to increase contact time Fruit product is removed by centrifuging Using respective reagent kits, the remaining concentration of lead(II),copper (II) and zinc(II) ions will be found.

19. Applications Cost-effective method of producing ethanol Reduces reliance on non-renewable fossil fuels Recycles fruit peels Viable method in wastewater treatment

20. Timeline Finalizing of project details 12-23 Nov 1 st round of experiments 7 Dec - Mar 2 nd round of experiments Mar - May Final round of experiments and Data Analysis May - Jul

21. 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

22. 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