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Ajay Kumar Singh, Et Al
Ajay Kumar Singh, Et Al
Ajay Kumar Singh, Et Al
Ajay Kumar Singh*, Sanat Rath, Yashab Kumar, Harison Masih, Jyotsna K. Peter, Jane
C. Benjamin, Pradeep Kumar Singh, Dipuraj, Pankaj Singh
ABSTRACT
Introduction
Bioethanol as an alternative source of Banana is one of major constitute the
energy has received special attention principal food resources in the world and
world wide due to depletion of fossil fuels. occupy the fourth world rank of the most
In India, sugar cane molasses is the main significant foodstuffs after rice, corn and
raw material for ethanol production. But milk (INIBAP, 2002). Most of the fruit
the short supply and increased cost is the peels/residues are dried, ground,
main hindrance for its use. The cellulosic pelletized, and sold to the feed
materials are cheaper and available in manufacturers at a low price which is not
plenty but their conversion to ethanol considered a highly viable proposition
involves many steps and is therefore (Mamma et al., 2008). As per the FAO
expensive. Under such circumstances a statistics, India is the largest producer of
novel approach is essential to use banana in the world and accounts for
renewable substrates such as fruit waste. nearly 30% of the total world production
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Isolation of microorganisms and its Banana peel wastes were procured from
maintenance local market in Allahabad, Uttar Pradesh,
India. Before processing ripe waste banana
Soil samples were collected randomly peels, it was cleaned, chopped (3-5 cm)
from the top 2 cm of the soil profile at and disinfected with 70% ethanol. It was
three different places. Approximately 50 g sun dried for 7 days and ground to fine
of soils were collected from each site and powder.
put into plastic bags and brought to the
laboratory. Soil samples were air-dried at Simultaneous Saccharification and
room temperature (27±1°C) for 24 to 48 h. Fermentation (SSF) of Banana peels
The dried soil samples were processed to
remove stones and plant residues. 100 mg Ethanol fermentation was carried out in
of each soil samples were transferred to 200 ml flasks containing 5g powdered
labeled test tubes containing five banana peels in 96 ml distilled water. The
milliliters of sterile saline (0.9% NaCl) flasks were sterilized by autoclaving at
(Knudsen et al., 1995). In order to 121°C for 30 min and a 4% (v/v) inoculum
suppress bacterial growth, 30 mg/l of of Aspergillus niger and 3% (w/v)
streptomycin was added. The test tubes inoculum of Saccharomyces cerevisiae
were vortex mixed until. 100 µl of each of was added. Fermentation was done for 7
the suspension was evenly spreaded on days and the ethanol content was
PDA plates with a spreader and incubated measured every 24 hours.
at 27±1°C. Mixed colonies on the plates
were observed after 5 7 days. Pure culture Effect of temperature, pH and yeast
of Aspergillus niger was obtained by inoculum on ethanol production
streak plate method. It was then
maintained on PDA slants at 4oC. Yeast Fermentation of banana peels was carried
strain Saccharomyces cerevisiae (Bakers out at different temperatures (20°C to
yeast) (Kwality, India) was obtained from 50°C) at pH 6 and at different pH (4 to 7)
the local market.It was maintained on at 30°C. The optimum temperature and pH
PDA slants at 4oC. obtained during the course of investigation
was used for fermentation at different
Starch hydrolysis test of isolated strains yeast concentration 3% to 12%.
of Aspergillus niger
Estimation of ethanol content by gas
An inoculum from a pure culture was chromatograph
streaked on a sterile plate of starch agar.
The inoculated plate was incubated at A gas chromatograph (Chemito, 2000)
27°C for 5 to 7 days. Iodine reagent was equipped with a flame ionization detector
then added to flood the growth. Presence (FID) and data acquisition system with
of clear zone surrounding colonies computer software (IRIS 32) was used to
confirmed the positive result and accounts analyze the ethanol concentration. The
for their ability to digest the starch and installed column was a Capillary column
thus indicates presence of alpha-amylase. (30 m). Temperature programming was
implemented for the liquid sample
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Int.J.Curr.Microbiol.App.Sci (2014) 3(5): 84-96
analysis. During the analysis, the oven strain A, strain B and strain C is indicated
temperature was maintained at 80°C. The in table 1 - 3 respectively. The variation in
injector and detector temperatures were ethanol yield from banana peels for
120 and 160°C, respectively. The flow rate different Aspergillus niger strains at pH 6
for carrier gas (Nitrogen) was set at 30 is indicated in table 4. The variation in
ml/min. The injection sample volume was ethanol yield from banana peels with the
0.2 l. The volume of standard ethanol change in pH (4 to 7) for seven days by
used was 0.2 µl. The area of standard Aspergillus niger strain B is indicated in
ethanol was found to be 1500. In each set figure 1. The variation in ethanol yield
of experiments, the data points were from banana peels for different Aspergillus
reported. niger strains at pH 6 is indicated in figure
2. Aspergillus niger strain B was found to
The formula used for the calculation of be efficient strain yielding a higher value
percentage of ethanol is given below. of ethanol production as compared to other
Aspergillus niger strains A and C. The
Conc. of Ethanol = highest bioethanol production was shown
by Aspergillus niger strain B at pH 6
(6.287%) followed by pH 5 (5.638%), pH
7 (2.877%) and pH 4 (2.364%). Mohamed
% of Ethanol =100 - and Reddy (1986) has reported that the
ethanol production from potatoes by
cocultures of Aspergillus niger and
x 100 Saccharomyces cerevisiae was optimal in
the pH range 5 to 6. Neelakandan and
Usharani (2009) has reported that the
maximum ethanol yield from cashew
Results and Discussion apple juice using immobilized yeast cells
by Saccharomyces cerevisiae was
The result of the investigation showed that obtained at pH 6. Shafaghat et al. (2010)
the fermented banana peels produced a has reported that the maximum ethanol
significant amount of ethanol. The production from molasses was achieved at
volumetric production of ethanol was pH 5.6 by Saccharomyces cerevisiae.
varied according to the variations in Togarepi et al. (2010) reported that the
temperature, pH and at different yeast rate of ethanol production was maximum
concentrations. It was also varied at pH 6. Mark et al. (2007) reported that
according to fermentation time and fungal fermentations at initial pH 6.0 produced
strains. the most ethanol. Jannani et al.(2013) also
reported maximum ethanol production at
Effect of pH on ethanol production pH 5.4 from grape fruit waste by using
Saccharomyces cerevisiae. Ado et al.
The ethanol production of inoculated (2009) found maximum ethanol
samples was studied for 7 days regularly production at pH 5 corn cobs using
and the observations were noted down. cocultures of Saccharomyces cerevisiae
The percentage of ethanol production from and Aspergillus niger. Shilpa et al. (2013)
banana peels at 24 hours interval for seven reported maximum ethanol production
days at different pH by Aspergillus niger from banana peels at pH 5.5. Thippareddy
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and Agrawal (2010) also produced from corn flour Aspergillus niger and non
maximum ethanol at pH 5.5 followed by starch-digesting and sugar-fermenting
pH 6 by using Aspergillus niger for Saccharomyces cerevisiae, the optimum
hydrolysis and Saccharomyces cerevisiae value of the temperature was found to be
for fermentation of agriculture waste. 30ºC. Togarepi et al. (2010) reported that
a maximum rate of ethanol production was
Effect of temperature on ethanol achieved at a temperature of 30 ºC.
production Thippareddy and Agrawal (2010) also
observed maximum ethanol at temperature
The ethanol production of samples was 30°C by using Aspergillus niger and
studied for inoculated sample for 7 days Saccharomyces cerevisiae from
regularly and the changes were noted agriculture waste. Magdy et al. (2011)
down. Percentage of ethanol production reported that temperature in the range of
from banana peels at 24 hours interval for 25-30°C is commonly found optimum for
seven days at different temperatures by thermophilic S. cerevisiae strain for
Aspergillus niger strain A, strain B and production of ethanol in SSF of various
strain C is indicated in table 5-7 substrates, i.e. apple pomace (Hang et al.,
respectively. The variation in ethanol yield 1986), carob pod (Roukas, 1994), sweet
from banana peels by different Aspergillus sorghum (Kargi and Curme, 2004).
niger strains at 30ºC is indicated in table 8. Manikandan et al. (2008) reported
The variation in ethanol yield from banana maximum ethanol production at
peels with the change in temperature (20ºC temperature 33°C followed by 30°C.
to 50ºC) for seven days by Aspergillus Jannani et al. (2013) also reported
niger strain B is indicated in figure 3. The maximum ethanol production at
variation in ethanol yield from banana Temperature 30°C from banana waste by
peels by different Aspergillus niger strains using Saccharomyces cerevisiae
at 30ºC is indicated in figure 4.
Aspergillus niger strain B was the most Variation of ethanol production due to
efficient strain yielding a higher value of yeast concentration
ethanol as compared to other Aspergillus
niger strains. It was observed that the The ethanol production of samples was
maximum ethanol production was at studied for inoculated sample for 7 days
temperature 30ºC with 6.434%, followed regularly and the changes were noted
by 40ºC, 20ºC and 50ºC in which down. With the increase in the
bioethanol was decreased to 5.691%, concentration of Saccharomyces
2.637% and 1.957% respectively. Hadeel cerevisiae, the time required for the
et al. (2011) reported that the maximum completion of fermentation decreased
ethanol production from rambutan fruit dramatically. Using a 12%, 9%, 6% and
biomass using yeast Saccharomyces 3% yeast inoculum, maximum ethanol
cerevisiae was at temperature 30ºC. production was completely achieved in 2,
Neelakandan and Usharani (2009) 3, 5, 7 days respectively. The percentage
reported that the maximum ethanol yield of ethanol production from banana peels at
from cashew apple juice using 24 hours interval for seven days at
immobilized yeast cells by Saccharomyces different yeast concentrations by
cerevisiae was obtained at 32ºC. Aspergillus niger strain A, strain B and
Manikandan and Viruthagiri (2010) strain C is indicated in table 9-11
reported that in the ethanol production
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Table.1 Percentage of ethanol production from banana peels at 24 hours interval for seven
days at different pH by Aspergillus niger strain A at 30ºC
Table.2 Percentage of ethanol production from banana peels at 24 hours interval for seven
days at different pH by Aspergillus niger strain B at 30ºC
Table.3 Percentage of ethanol production from banana peels at 24 hours interval for seven
days at different pH by Aspergillus niger strain C at 30ºC
Table.4 Percentage of ethanol production from banana peels at 24 hours interval for seven
days by different Aspergillus niger strains at pH 6 and at 30ºC
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Figure.1 Variation in ethanol yield from banana peels with the change in pH (4 to 7) for
seven days by Aspergillus niger strain B at 30ºC
Figure.2 Variation in ethanol yield from banana peels by different Aspergillus niger strains
at pH 6 and at 30ºC
Table.5 Percentage of ethanol production from banana peels at 24 hours interval for seven
days at different temperatures by Aspergillus niger strain A at pH 6
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Table.6 Percentage of ethanol production from banana peels at 24 hours interval for seven
days at different temperatures by Aspergillus niger strain B at pH 6
Table.7 Percentage of ethanol production from banana peels at 24 hours interval for seven
days at different temperatures by Aspergillus niger strain C at pH 6
Table.8 Percentage of ethanol production from banana peels at 24 hours interval for seven
days by different Aspergillus niger strains at pH 6 and at 30ºC
Figure.3 Variation in ethanol yield from banana peels with the change in temperature (20ºC
to 50ºC) for seven days by Aspergillus niger strain B at pH 6
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Figure.4 Variation in ethanol yield from banana peels by different Aspergillus niger
strains at pH 6 and at 30ºC
Table.9 Percentage of ethanol production from banana peels at 24 hours interval for seven
days at different yeast concentrations by Aspergillus niger strain A at the optimum
temperature (30ºC) and at the optimum pH (pH 6)
Table.10 Percentage of ethanol production from banana peels at 24 hours interval for seven
days at different yeast concentrations by Aspergillus niger strain B at the optimum
temperature (30ºC) and at the optimum pH (pH 6)
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Table.11 Percentage of ethanol production from banana peels at 24 hours interval for seven
days at different yeast concentrations by Aspergillus niger strain C at the optimum
temperature (30ºC) and at the optimum pH (pH 6)
Figure.5 Variation in ethanol yield from banana peels with the change in yeast concentration
(3% to 12%) for seven days by Aspergillus niger strain B at the optimum temperature (30ºC)
and at the optimum pH (pH 6)
respectively. The variation in ethanol yield Ocloo and Ayernor (2010) has reported
from banana peels with the change in yeast that the yeast concentration significantly
concentration (3% to 12%) for seven days affected the time taken for the
by Aspergillus niger strain B at the fermentation to be completed, that is, to
optimum temperature (30ºC) and at the achieve maximum alcohol yield. The
optimum pH (pH 6) is indicated in figure results obtained supported the fact that the
5. Mohamed and Reddy (1986) has speed of fermentation depends on the
reported that the increasing yeast concentration, the higher the
Saccharomyces cerevisiae inoculum in the concentration, the shorter the fermentation
cocultures Aspergillus niger and period required to achieve maximum
Saccharomyces cerevisiae from 4% to alcohol yield (Kordylas, 1990). Ueda et al.
12% gave a dramatic increase in the rate (1981) reported of 5 days fermentation
of ethanol production from potato starch. period for raw cassava root starch using
15% yeast suspension. Togarepi et al. (2010) reported that for the yeast
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concentration the rates increased rapidly (Pawpaw) fruit waste. Asian Journal of
with the increase in the amount of yeast Biochemistry 3(3):188-193.
added, up to the yeast concentration of 8 Benitez, T., Del Casttillo, L., Aguilera, A.,
g/20 g fruit pulp (Fig. 3). Beyond that Conde, J., Oimedo, E.C.(1983)
point the rates no longer significantly Selection of wine yeast for growth and
increased. At this point the substrate fermentation in the presence of ethanol
becomes limiting and increasing the yeast and sucrose, Appl. Environ. Microbiol.
amount does not increase the rate of (45)5:1429-1436.
reaction. Brooks, A.A. (2008). Ethanol production
potential of local yeast strains isolated
The maximum ethanol yield from banana from ripe banana peels, African
peels was 6.540 %. Fungal Strain B gave a journal of Biotechnology, 7(20): 3749-
higher value of ethanol. The amount of 3752.
ethanol content increased with the increase Diwanya, E. L. I., EL-Abyad, M.S., Refai,
in fermentation time. Simultaneous A.H.EL., Sallem, L.A., Allam, R.E.
fermentation of starch to ethanol can be (1992) Effect of some fermentation on
conducted efficiently by using cocultures ethanol production from beet molasses
of the amylolytic fungus Aspergillus Niger by S. cerevisiae, Bioresource
and a non-amylolytic sugar fermenter, technology, 42: 191-195.
Saccharomyces cerevisiae. Therefore the Emaga, T.H., Robert, C., Ronkart, S.N.,
findings of this work suggest that banana Wathelet, B. and Paquot, M.(2008)
peels could be a good substrate for ethanol Dietary fibre component and pectin
production. chemical features of peels during
ripening in banana and plantain
Acknowledgement varieties. Bioresource Technology, 99 :
4346 4354.
The authors thank to the Hon ble Vice Essien, J.P., Akpan, E.J., Essien, E.P.
Chancellor of SHIATS for granting (2005). Studies on mould growth and
approval, and Head, Department of biomass production using waste
Microbiology and Fermentation banana peels, Bioresource Technology,
Technology, SHIATS, Allahabad for 19: 361-363.
providing laboratory facilities to carry out Hadeel, A., Hossain, A. B. M. S. Latifa,
this investigation. K., ALNaqeb, H. Abear, J. and
AlHewiti ,N. (2011) Bioethanol fuel
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