Done by:
Aman Mangalmurti
Kara Newman
Leong Qi Dong
Soh Han Wei
Rationale
Depletion of nonrenewable 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
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
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)
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
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 Cu2+,Cu3+ ions
Hypothesis
 Ethanol 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
Variables
Constant
Independent
Dependent
• Temperature of
growth of organisms
(30C)
• Initial concentration
of heavy metal ions
(50 ppm)
• Fruit peels used
(AOS: banana, HCI:
mango)
• Organism used (S.
cerevisiae, Z. mobilis)
• Heavy metal ions
(Cu2+ ,Cu4+)
• 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 & Materials
Apparatus
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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
Materials
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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 extracts
To 0.5 ml of extract,
0.5 ml of DNS
(dinitrosalicylic
acid) is added.
The concentration of reducing
sugars in μmol/ml is read from
a maltose standard curve.
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.
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 beads are rinsed
with 0.85% sodium
chloride solution.
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.
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. cerisiae
 To 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)
Repeat for Cu4+
Using a rotary mill to
grind desiccated residue
Allow solution to set for
20 min, preferably at
100rpm to increase
contact time
Sieve to 0.25 mm particle
size.
Add residue powder to
50ppm Cu2+ solution.
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.
The beaker
is drained
and fruit
peel is left to
dry.
25ml of
cellulase is
added to the
beaker.
50 ml water
is added to
beaker
Beaker is left
standing for
1 hour with
continuous
stirring.
Second ethanol fermentation
 Identical to above
 Ethanol 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
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Hossain, A.B.M.S. & Fazliny, A.R. (2010). Creation of alternative energy by bio‐ethanol production
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