Ethanol_17nov - aos-hci-2012-research

Done by:
Aman Mangalmurti
Kara Newman
Leong Qi Dong
Soh Han Wei
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
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,
 Production of ethanol by Saccharomyces cerevisiae
 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
 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
To prepare extracts of fruit peel for ethanol
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+,Zn2+ and Pb2+ ions
To determine which procedure maximizes
usage of fruit peel
 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.
Experimental Outline
Preparation of
fruit peel extract,
microbe, heavy
metal solution
Adsorption of
Cu2+,Zn2+ and
Pb2+ ions
Adsorption of
Cu2+,Zn2+ and
Pb2+ ions
• Fruit peels used (AOS:
banana, HCI: mango)
• Heavy metal ions
• Order of Procedure
• Initial concentration of
reducing sugars in fruit
peel extracts
• Ratio of ethanol yield to
initial sugar
• Final ethanol yield
• Final concentration of
heavy metal ions
• Mass of fruit peel used
for extraction of glucose
• Type of microorganism
• Immobilisation of
• Fermentation
• Initial concentration of
heavy metal ions
• Duration of adsorption
• Mass of fruit peel
particles used for
• Procedure
Apparatus & Materials
Centrifuge tube
Spectrophotometer cuvettes
Glass rod
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
 Lead (II), Copper (II), Zinc (II) ion
 Lead (II). Copper (II), Zinc (II) reagent
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
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.
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
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
The samples are placed in
spectrophotometer cuvettes and
the absorbance is taken at 530 nm
using a spectrophotometer.
Ethanol fermentation by immobilized Z.
mobilis cells
200 beads are added
to 50 ml waste
A control is
prepared in which
200 empty alginate
beads are added to
the same volume of
waste extract
All the set‐ups are
incubated with
shaking at 30°C for 2
days for ethanol
fermentation to
The beads are then
removed and the
extracts are distilled
to obtain ethanol.
Determination of ethanol yield with the
dichromate test
2.5 ml of acidified
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
The absorbance is
measured at 590 nm
using a
and the
concentration of
ethanol is read from
an ethanol standard
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
Determination of final ion
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
method of
producing ethanol
Reduces reliance on
non-renewable fossil
Recycles fruit peels
Viable method in
Finalizing of
project details
12-23 Nov
1st round of
experiments 7
Dec - Mar
2nd round of
Mar - May
Final round of
and Data
Analysis May Jul
 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:,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,
 US Environmental Protection Agency (2011) .Drinking Water Contaminants. Retrieved
June 6, 2011, From
 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:
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
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
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
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

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