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Alcohol Production from Fruit and Vegetable


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Girisha Malhotra
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International Journal of Applied Engineering Research.
ISSN 0973-4562, Volume 8, Number 15 (2013) pp. 1749-1756
© Research India Publications

Alcohol Production from Fruit and Vegetable Waste

Shilpa C., Girisha Malhotra and Chanchal

Department of Biotechnology, Manav Rachna International University,

Sector - 43, Suraj Kund Badkhal Road, Faridabad, Haryana, India


Fossil fuels create a negative impact on our environment as green-

house gas emissions are harmful. Production of ethanol (as an
alternative fuel) from food and agricultural waste was done in this
research by bio-processing. Wastes from fruits, such as banana,
orange, pineapple and pea peels were subjected to simultaneous
saccharification and fermentation for 7 days by co-culture of
Aspergillus niger and Saccharomyces cerevisiae. The ethanol yield
was determined at 24 hours interval. The results of the study showed
that after 7 days of fermentation, pineapple peels had the highest
biomass yield, followed by banana peels, orange peels, pea peels. The
optimal ethanol yields were 8.34% v/v, 7.45 % v/v, 3.98 % v/v and
2.58 % v/v for pineapple, banana, orange and pea peels respectively.
These indicate that pineapple and banana peels ethanol yields were
significantly higher than orange and pea peel ethanol yield.

Keywords: Saccharomyces cerevisiae, Aspergillus niger, ethanol

production, fruits waste, fermentation, sachcharification.

1. Introduction
Due to rapid exhaustion of fossil fuels there is an urgent need to resort to alternative
fuels e.g. ethanol. The first large scale use of ethanol as a fuel happened during the
early 1900s when petroleum supplies in Europe were short. Though ethanol is
conventionally produced from petroleum by-products, bio ethanol can alternatively be
produced by fermentation technology using renewable raw material.
Saccharomyces cerevisiae is the most popular organism used for ethanol
production due to its high ethanol yield and high tolerance. Now a days, crops are the
main source used for ethanol production. To achieve significant economic and
1750 Girisha Malhotra et al

environmental benefit large amount of food wastes can be utilized to produce ethanol.
Utilization of fruit waste for bioethanol production is one of the best options. One
example of raw material is pineapple waste that is converted to bioethanol (Hossain et
al., 2008). The wastes contain valuable components such as sucrose, glucose, fructose
and other nutrients (Sasaki et al. 1991). Lignocellulose is the major structural
component of woody plants and non woody plants.
The use of mango peel as a source of pectin and fibre production also has been
reported (Pandia et al., 2004). Grohmann et al. (1994; 1995; 1996; 1998) previously
reported ethanol production from orange peel. Ethanol production from banana
(Manikandan et al., 2008) and pineapple peels (Ban-koffi and Han, 1990) were also
investigated. Dried orange peels have a high content of pectin, cellulose and hemi
cellulose, which make it suitable as fermentation substrate when hydrolyzed. Insoluble
carbohydrates are present in the cell walls of the peels, particularly in the form of
pectin, cellulose and hemicellulose.

2. Literature Review
Ethanol was first prepared synthetically in 1826, through the independent effort of
Henry Hennel in Britain and S.G in France. Michael Faraday prepared ethanol by the
acid-catalysed hydration of ethylene in 1828, in a process similar to that used for
industrial synthesis of ethanol today. Almost all ethanol is being produced by
fermentations using S. Cerevisiae. Karsch et al. (1983) evaluated the potential of
Zymomonas mobilis and Saccharomyces cerevisiae for ethanol production from
glucose both aerobically and anaerobically. S. cerevisiae commonly known as Baker’s
yeast has the ability to ferment a sugar solution poorly supplied with oxygen, resulting
in the formation of alcohol and carbon dioxide. Zymomonas mobilis degrades sugars to
pyruvate using the Entner-Doudoroff pathway. The pyruvate is then fermented to
produce ethanol and carbon dioxide as the only products (Farombi and Britton, 1999).
Also A. niger produces enzymes such as amylase, amyloglucosidase, cellulases,
lactase, invertase and pectinases.
Maximum saccharification was achieved by hydrolysing banana-waste cellulose
with a cellulase enzyme from Trichoderma reesei QM 9414. A yield of 1·38% and
0·78% (v/v) and 44·5% and 61·1% ethanol (mg g−1reducing sugars) was achieved
from cellulose and acid hydrolysed (2·5% at 15 psi for 15 min) banana peels,
respectively. Grohmann et al. (1998) observed that E. Chrysanthemi EC16 contains the
PET operon from Zymomonas mobilis on the plasmid pLOI555 which increases the
organism’s ethanol production and decreases the final concentration of co-products
(Beall and Ingram 1993). Escherichia chrysanthemi EC16 fermentations of sugar beet
pulp produced 1.97 % (w/v) ethanol, less than the 2.55 % (w/v) ethanol produced by E.
Coli KO11 on the same substrate. In a study by Jayant Mishra et al. (2012), ethanol
production from fruit peels of pineapple, orange and sweet lime was investigated.
Total amount of sugar in pineapple, sweet lime and orange was 0.5, 1 and 0.8%
respectively. In the solid state fermentation, pineapple agro residue gives a maximum
yield around 2.16% with yeast. With a change of strain to C. albicans, pineapple still
Alcohol Production from Fruit and Vegetable Waste 1751

gives a high yield of 1.08% for group A in 50 ml capacity. Pineapple gives a maximum
yield of 1.87% with S. cerevisae. Lavarack B. P. et al. (2002) tried dilute acid
hydrolysis of bagasse for conversion of hemicellulose to xylose, glucose, arabinose,
acid soluble lignin and furfural.

3. Materials and Methods

3.1 Routine Culture Maintenance
Culture of S.cereviacae was maintained on YEPDA (1% yeast extract, 2% peptone,2%
agar) slant stored at 4°C. The growth of S.cereviacae confirmed plate count methods
(Yeast malt extract medium,at 28°C.incubation period 2-3 days).
Aspergillus niger was cultured on PDA at pH 6.5 and 28C. PDA was prepared
using 20g dextrose, 20g agar and 4g potato extract dissolved completely in 500 ml
water in a conical flask(pH-6.5). The mixture is then sterilized in an autoclave at
121C for 15 minutes. (Kingsley Otulugbu, 2012 )

3.2 Preparation of Pineapple, Banana, Orange and Pea Peels for Ethanol
Pineapple, Banana, orange and pea peels were washed and their outer coats are
removed, cut in small pieces and kept it in the sunlight for few days and then kept in
oven for drying and stored in refrigerator prior to use.

3.3 Preparation of Growth Medium

The growth medium prepared for ethanol production consists 20 g substrate
(Pineapple, orange, banana or pea peel) in 250 ml of conical flask containing 100 ml of
distilled water(pH-5.5). The flasks were autoclaved at 121°C for 20 minutes. This
medium is poured in petriplates and set aside to solidify.

3.4 Preparation of Inoculum

The cells of S.cereviacae were aseptically cultured in Yeast Extract Peptone Dextrose
(YEPD) broth and incubated at 30°C for 24hrs.

3.5 Saccharification of the Fermentation Medium with Aspergillus

The substrate medium as prepared above was inoculated with spores of Aspergillus.
The culture was incubated at 28C for 7days under rigorous aerobic culture. The
samples were taken at regular intervals after every 24 hr for analysis. After 7 days in
culture maximum total sugar was obtained.

3.6 Ethanol Production

Medium (270 ml) was prepared and transferred to a Duran wide mouth bottle. The
media was autoclaved at 121C for 20 minutes and cooled. The culture broth (30 ml)
from the saccharification step was added to the medium. This broth contains the
cellulolytic enzyme complex elaborated by Aspergillus. The bottle was inoculated with
1752 Girisha Malhotra et al

5% v/v of previous activate Saccharomyces cerevisiae. The bottles were cultured in

static conditions. The samples were withdrawn at regular time intervals every 24 h.

3.7 Primary Product Isolation

The samples were centrifuged at 5000 rpm for 5 minutes and stored at -20C for
further analysis. The raw ethanol yield was measured by ethanol assay using potassium
dichromate method.

3.8 Determination of Sugar Concentration by Refractometer

The concentration of sugar in a sample can be determined by measuring the refractive
index of the sample. This measurement is based on the principle that the index of
refraction of a solution containing sugar is proportional to its concentration.

3.9 Estimation of Ethanol

10 ml of sample was drawn in a 250 ml volumetric flask. The water was added to
make the volume up to 250 ml. From this diluted sample 20 ml aliquot was drawn and
drawn in a conical flask. 10 ml of 40% sulphuric acid was added to each flask using a
measuring cylinder. Stopper each flask loosely and heat for 10 minutes in a water bath
at 45–50°C. After 10 minutes the flasks were removed from the bath and 2 g of
potassium iodide was added to each flask. A burette was filled with standard
thiosulphate solution. Titrate the contents of the flask with the thiosulphate solution. 1–
2 ml of starch was added to solution when the brown colour of the solution becomes
green. The equivalence point of the titration is reached when blue colour of the starch
iodine complex just disappears, leaving a clear green colour.

3.10 Qualitative Determination of Amylase Activity

Starch agar plates were prepared using starch(1%w/v), agar(1.5%w/v) and dissolved in
water (pH-6.5) and sterilized by autoclaving. This media was poured into plates and
allowed to set for 1 hr. Vials were bored into the plates. The culture broth (200 µl) was
pipetted into the wells. The plates were incubated at 37C for 4hr. Plates were folded
with iodine solution and destained using saline water. Amylase activity could be
determined by presence of clearing zones around the vials.

3.11 Qualitative Determination of Cellulase Activity

CMC (cellulasemethylcarboxylase) agar plates were prepared using CMC(1%w/v),
agar(1.5%w/v) and dissolved in water (pH-6.5) and sterilized by autoclaving. The
media was poured into plates and allowed to set for 1 hr. Vials were bored into the
plates. The culture broth(200 µl), obtained after culture of Aspergillus niger PDB for 7
days, was pipette into the vials. These were incubated at 37C for 4hrs. Plates were
folded with congo-red solution and destained using saline water. Cellulase activity
could be determined by presence of clearing zones around the vials.
Alcohol Production from Fruit and Vegetable Waste 1753

4. Results & Observations

4.1 Pre-treatment of Vegetable and Fruit peels:-
The media consisting of oven dried substrate (20gms) was used and volume was made
up to 250 ml and pH was adjusted to 5.5. The media was inoculated with spores of
Aspergillus niger. The culture was incubated at 28°C for 7 days under aerobic culture.
The enzyme activity was qualitatively determined by Starch Agar plate assay and
Cellulase Agar plate. The maximum residual sugar was obtained during 192 hr- 216
During the fermentation concentration of reducing sugars was determined by
finding out the refractive index of the solution.

Table 1: Sugar content of different fruit and vegetable waste.

Substrate g sugar/g substrate
Pineapple peel 0.25
Banana peel 0.525
Orange peel 0.4
Pea peel 0.025

The culture was replenished with fresh media and inoculated with an activated
culture of Sacchromyces cerevisiae. The culture was transferred to Duran wide
mouthed bottles. The bottles were sealed and kept static in an incubator at 28°C. The
samples were withdrawn in every 24hrs and the changes in pH, RI and ethanol
concentration are displayed graphically as follws. Similar procedure was carried out
with different substrates. The profile of change in pH, residual sugar and ethanol
production was monitored regularly at 24hr intervals.




%residual sugar
Ethanol g/l

24 48 72 96 120 144 168 192

Figure 1: Progress of ethanol fermentation with pineapple peel as substrate.

1754 Girisha Malhotra et al

15 %residual sugar
10 Ethanol g/l
24 48 72 96 120 144 168 192

Figure 2: Progress of ethanol fermentation with orange peel as substrate.

15 %residual sugar
10 Ethanol g/l
24 48 72 96 120 144 168 192 216

Figure 3: Progress of ethanol fermentation with pea peel as substrate.




20 %residual sugar
Ethanol g/l

24 48 72 96 120 144 168 192

Figure 4: Progress of ethanol fermentation with banana peel as substrate.

Alcohol Production from Fruit and Vegetable Waste 1755

Table 2: Maximum yield of different substrates.

Subsrate Maximum yield
Pineapple 31.53
Orange 37
Banana 32.21
Pea 31.53

The highest ethanol production could be obtained with pineapple peel as substrate
(49.34 g/L) at 28°C.

5. Conclusion
From the experiments, it is proved that the ethanol yield could be produced from fruit
and vegetable waste as the substrates. Maximum activity is obtained by using
pineapple peel as a substrate at 28°C. Bioconversion of vegetable and fruits waste into
ethanol was done. Various analytical tests were performed to estimate total sugar,
fermentable sugar, residual sugars and protein content before and after fermentation.

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