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

2. Depletion of non- renewable fossil fuels due to excessive consumption as a source of energy Possible extinction of bananas due to various diseases leads to banana waste Heavy metal water contamination of water is rampant in many countries. Conversion of renewable sources, e.g. organic wastes, to fuel ensures continual energy supply

3. Heavy metal ions accumulate inside organisms and cause adverse health effects Biosorption in removal of heavy metal ions by fruit peel wastes Bananas are threatened by various diseases

4.  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.  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. To prepare extracts of fruit peel for ethanol fermentation To determine which fruit peel gives highest ethanol yield To determine which fruit peel waste adsorbs heavy metal ions best To determine a protocol which maximizes efficiency of fruit waste

7.  Ethanol yield from fermentation would differ for both peels  The efficiency of heavy metal ion adsorption differs for both peels  The order of adsorption and fermentation has an effect on the ethanol yield and the efficiency of adsorption

8. Preparation of fruit peel extract, microbe, heavy metal solution Adsorption of Ions Extraction of sugars Ethanol Fermentation Extraction of sugars Ethanol Fermentation Residue for Adsorption of Ions

9. 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  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) MATERIALS  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. ETHANOL FERMENTATION Growth of Z. mobilisImmobilisation of cellsExtraction of sugars from fruit peelsDetermination of sugars in extracts Ethanol fermentation by immobilized Z. mobilis cells Determination of ethanol yield with the dichromate test ADSORPTION OF HEAVY METAL IONS Adsorption of heavy metal ions Determination of final ion concentration

12. Preparation of Z. mobilis, Fermentation, Determination of Yield

13. 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.

14. 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.

15. 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.

16. 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.

17. 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.

18. 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.

19. Pre-treatment of peel, Creation of heavy metal mixture, Adsorption, Determination of final ion concentration

20.  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 solution is made of 0.5mols of each metal: Pb 2+, Zn 2+, Cu 2+ in 1L of distilled water  Add powder to mixture

21. 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.

22. the % ethanol per g of cells µmol of ethanol per ml reducing sugar in fruit peels The ethanol yield would be evaluate by comparing The ratio of the final concentration of metal ion to the initial concentration The % of heavy metal ions adsorbed The heavy metal ion adsorption efficiency would be evaluated by comparing

23. Cost-effective method of producing ethanol Reduces reliance on non-renewable fossil fuels Using by-product waste Viable method in wastewater treatment

24. 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

25.  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

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