International Journal of Scientific & Engineering Research, Volume 5, Issue 1, January-2014 1

ISSN 2229-5518

A comperative study of Bioethanol Production ability of Bacillus subtilis and Sacchromyces cerevisiae using Banana and Orange Peels Amit Ranjan Prasad Singh1

AbstractIn present in present investigation a comparetive analysis of bioethanol production ability of S. cerevisiae and B. subtilis is done on varies parameters such as temperature, pH and incubation time to determine the best optimal conditions of bioethanol production using Banana and orange peels with these micro- organisms. Banana and Orange peels choosen as substrate in solid state fermentation for bioethanol production due to its cheap, easy and whole year availibilty.Collected plants material were dried and converted into fine power, then a pretreatment was given to plant material to delignifie the plant material.The enzymatic hydrolysis of both pretreated substrates were done with cellulase that depolymerized cellulose to monomeric glucose, that converted in to ethanol finally. S. cerevisiae and B. subtilis both exibited maximum yield of bioethanol at 400C, 4.07% (v/v) and

4.10% (v/v) respectively for orange peels. For banana peels S. cerevisiae, B. subtilis both has shown maximum ethanol production at 300C which is

2.86% (v/v) and 3.89% (v/v) repectively. For both orange and banana peels B. subtilis showed maximum ethanol production at pH 7 but S. cerevisiae

shown maximum ethanol production at pH 5 from orange peel and at pH 4 from banana peel. Produced bioethanol purification were done through fractional distillation. Specific-gravity and Iodine values were determined for best optimal conditions to cheak the purity of bioethanol produced. Specific- gravity and iodine values of bioethanol produced through fermentation with S. cerevisiae was found to more close to pure ethanol.

Index TermsS. cerevisiae, B. subtilis, Banana peel, Orange peel, Solid state fermentation.



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Ethanol is one of the most advanced liqid fuel because it is
environment friendly [1]. Its carbon content has a vegetable
origin and as a consequence, when it is released during the
combustion process, it does not contribute to the increase of
carbon dioxide in the atmosphere, reducing global warming
[2] [3] [4]. Biomass is the earth’s most attrative alternative
among fuel sources and sustainable energy resource. As per
the FAO statistics, India is the largest producer of banana in
the world and accounts for nearly 30% of the total world pro-
duction of banana. Though banana peel is a fruit residue, it
accounts for 30–40% of the total fruit weight [5] and contains
carbohydrates, proteins, and fiber in significant amounts.
Since banana peels contain lignin in low quantities [6], it could
serve as a good substrate for production of value-added prod-
ucts like ethanol. Citrus fruits is the one of the major fruit crop
cultivated world wide.In the citrus processing industry citrus
peel is the major solid by-product and compaises around 50%
of the fresh fruit weight and can be utilized as substrate for
bioethanol production. There are major limitations to efficient
ethanol production from agricultural residues. These limita-
tion includes the close physical and chemical association be-
tween lignin and plant cell wall polysaccharides together with
cellulose crystallinity. Lignin froms a protective shield around


1Department of Molecular & Cellular Engineering, Jacob School of Bio- technology & Bioengineering, SHIATS, Allahabad, India.

cellulose and hemicellulose, protecting the polysaccharides from enzymatic degradation. To convert the biomass in to
ethanol, the cellulose must be readily available for cellulose
Thus by removing the lignin, the cellulose becomes vulnerable
to enzymes and allows the yeast to convert the glucose into
ethanol during fermation. Therefore, a pretreatment was ap-
plied to degrate the lignin in the peel residue, decrease cellu-
lose crystallinity and increase the surface area for enzymatic
activity. In present study change in ethanol concentration was
investigated on temperature, pH, and incubation period to
estimate the production ability of S. cerevisiae and B. subtilis
with change in temperature, pH and incubation period.


2.1 Collection of Raw Material

The raw material used in the process of fermantation for the
the production must be economical and its availatity must be
easy to a large degree they determine the economics of the
process, the production method and the quality of the prod-
uct. If the raw material is naturally ocurring subtances it must
be readily available throught most of the year. So kepping all
these things in mind banana and orange peels had been
choosen as substrate for bioethanol production. Banana and
orange peels was collected from local market of Allahabad and
washed twiced with Distled water and wiped with 70% etha-
nol, chopped in to small pieces and dried at room temperature
and stored at 40C for further use.

2.2 Isolation of Bacterial species

Pure culture of B. subtilis and Sacchromycees cerevisiae were
procured and maintained on potato Dextrose agar medium at
40oC. The slants were grown at 280C for 3 days. The spores
were harvested using sterilized water with 0.1% Tween80. For
innoculation 3ml of spore suspension was used. Stock cultures

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of B. subtilis were grown on nutrient agar slants at pH 6.8. They were periodically sub-cultured by transfer onto fresh agar medium. Liquid culture of B. subtilis was prepared in nutrient broth from pure culture of bacterium. For innocula- tion 3ml of bacterial culture was used.

2.2 Preparation of Substrate

Banana and orange peels were dried seperately and powered
in a grider to get the small size of substrate. The substrates
were then treated separately with 2% alkali NaOH. The treted
substrate was subjected to heat treatment by autoclaving for
one houres at 1210C. After the heat treatment, substrates were
washed using distilled water and then neutralized by acetic
acid and sodium hydraoxide. The substrate was dried at 600C
in oven for 12 hours. Enzmatic hydrolysis was carried out in
reaction mixture cotaining 5gm of pretreated substrates (Ba-
nana and orange peel) in 100ml 0.1M citrate buffer with 5µl of
concentrated crude cellulase enzyme, pH was adjusted to 4.5
in all the four flasks. The reaction mixtures were incubated on
rotary shaker at 300C, 75rpm for 24 hours. After the 24 hours
of incubation, reaction mixtures were boiled for 2 minutes to
denature the enzyme and the centrifuge at 5000rpm for 15
minutes. The supernetents collected and used for ferman-

2.3 Effect of pH on Bioethanol Production

Fermantation was carried out by solid state fermantation. The
to 450C, S. cerevisiae will show a high cell growth and ethanol production rate [7]. In the present investigation fermantation process is carried out at 10, 20, 30, and 400C to determine the optimal temperature of Bioethanol production with banana and orange peel using S. cerevisiae and B. sutilis.
As from the results obtained in present study maximum etha-

nol production was obtained at 400C from orange peels by fermantation using S.cerevisiae and Banana peels shows max- imum ethanol production at 300C with S.cerevisiae. S. cereviciae are known to convert sugar in to bioethanol at temperature range of 250C to 300C [8]. The Best optimum temperature for S. cerevisiae is 300C in banana peel [9]. The Best optimum temper- ature found to be 330C using S. cerevisiae mutant strain 4 for bioethanol production using banana peel. [10]. Gomma (2012) founded more ethanol yield than S. cerevisiae using B. subtilis by orange peel fermantation that shows aggriment with re- sults obtained in present study. According to Gomma opti- mum temperatur for bioethanol production is 350C by orange peels using B. subtilis than 400C in present investigation.

Effect of Temperature on


volume of all the four flasks was adjusted to 100ml. The pH of suspention were adjusted. The flasks containing the hydro- lyzed samples were covered with cotton wool, wrapped in aluminium foil, autoclave for 15 minutes at 1210C and allowed to cool at room temperature. A pinch of Ammonium sulphate is added in each flask as a nitrogen source. The sterilized flasks were innculated with 3ml of 24 hours old cultures and incubated under anaerobic condition.

Bioethanol Production

6 Banana peel (B.

4 subtilis)

2 Banana peel (S.

0 cerevisiae)

2.4 Effect of Temperature on Bioethanol Production Optimization of fermantation process had been done at differ- ent parameters to estimate the ethanol production ability of S. cerevisiae and B. subtilis. Various optimization parameters were pH (4.0, 5.0, 6.0 & 7.0), fermantation temperature (100C,

10 20 30 40


hrange peel peel (B. subtilis)

hrange peel (S. cerevisiae)

200C, 300C & 400C), and time of fermentation (48, 72, 96 & 120 hours) to obtained maximum bioethenol production.

2.5 Effect of Incubation Period on Bioethanol Production After different time periods, the fermented substrates were filtered and dispensed into round bottle flask fixed to a distil- lation column enclosed in running tap water. A conical flask was fixed to a distillation column to collect the the distillate. A heating mantle with temperature adjusted to 650C for 6 hours was used to heat the round bottomed flask contaning ferman- ted broth.


3.1 Effect of Temperature on Bioethanol Production Temperature is important factors that effect the fermantation process and product formation. It is commanly belived that

20-350C is the ideal range for fermantation (Wu et al; 1998, Ballestron et al. 2004, Aldiguer et al; 2004, Phisalaphong et al;
2006, Gao et al; 1988) but when the temperature was increased

Fig 1: Effect of Temperature on Bioethanol Production

3.2 Effect of pH on Bioethanol Production

In addition to temperature, pH is also an important factor that affects the ethanol fermentation [11]. In this study change in ethanol concentration was investigated to esti- mate the production ability of S. cerevisiae and B. subtilis with changing pH. Investigation is done on pH 4, 5, 6, 7. Results obtained in present investigation showed that Or- ange peels fermented with S.cerevisiae yield maximum ethanol at pH 4 and Banana peels with S.cerevisiae shown maximum yield at pH 5. B. subtilis show maximum ethanol production at pH 7 with both the substrate. A pH range of

4.0-5.0 regarded as the optimal limit for the anaerobic etha- nol production process using S. cerevisiae and The highest specific ethanol production rate for all the batch experi- ments was achieved at pH 5.0 [7].

The Best optimum pH found to be 4.5 using S. cerevisiae

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mutant strain 4 for bioethanol production using banana peel [10]. S.cerevisiae shown maximum productivity of bi- oethanol at pH 5 for grape waste [12] [13] founded 3.81 as optimul pH for ethanol production from orange juice using S. cerevisiae According to Gomma (2012), highest ethanol production was obtained at pH 7 with orange peels using B. subtilis.

Effect of pH on Bioethanol

cerevisiae fermented hydrolysed sugar extracted from or- ange peel waste and produced ethanol at 72 hours of incu- bation period. Gomma (2012) also recorded highest level of production after 72 hours of incubation period with orange peels using B. subtilis.

Effect of Incubation Period on

Bioehanol Production








4 5 6 7

Banana peel (B. subtilis)

Banana peel (S. cerevisiae)

hrange peel peel

(B. subtilis)

hrange peel(S. cerevisiae)







48 72 96 120

Banana peel (B. subtilis)

Banana peel (S. cerevisiae)

hrange peel (B. subtilis)

hrange peel (S. cerevisiae)

3.4 Specific gravity of produced Bioethanol at optimal

Fig 2: Effect of pH on Bioethanol Production

3.3 Effect of Incubation Period on Bioethanol Production Patil and Dayanand (2006) reported that the period of fer- mentation depends upon the nature of medium, ferment- ing organisms, concentration of nutrients and process physiological conditions.For general ethanol ethanol pro- duction by yeast, the maximum fermentation time in batch process was 72 hours. [14]. Fermention of banana peel and orange peel were done at different incubation time as 48,

72, 96, 120 hours at 300C to estimate the effect of ferman- tation time on the ethanol production activity of S. cere- visiae and B. subtilis. Both S. cerevisiae and B. subtilis shown maximum ethanol production after 72 hours of incubation on pretreated orange peels extract. S. cerevisiae shown max- imum ethanol production after 48 hours of incubation on pretreated banana peels and B. subtilis shown maximum ethanol production on pretreated banana peels. Diluted H2 SO4 prereated banana fruit peels yielded a maximum of

13% ethanol with a fermentation efficiency of 27.13% at 42 hours of incubation with S. cerevisiae [10].The production of ethanol found to be maximum after 48 hours of ferman- tation for grape wastes using S.cerevisiae[12].The concen- tration of bioethanol was found to be increase with respect to time for all temperature [15] [16] [17] which supports resuls obtained in present investigation. Mishra et al (2012) founded increase in quantity of ethanol produced in sub- merged state fermantation as compared to the produced by solid state fermentation and founded optimal incubation period 72 hours for bioethanol production by orange peel using S. cerevisiae. Wikkins et al (2007) reported that S.


The specific gravity of absolute ethanol is 0.79. Specific

gravity of bioethanol produced at different optimization conditions was tabulated. Result shows that specific gravi- ty of bioethanol produced by fermantation is close to spe- cific gravity of absolute ethanol. Specific gravity of ethanol obtained from banana peels fermanted with S.cerevisiae after incubation of 48 hours was fonded more near to absolute ethanol in compare to other substrate combina- tions.

Table:4 Specific gravity of produced Bioethanol at optimal condition:


Banana peel

(B. sutilis)

Banana peel


Orange peel

(B. subtilis)

Orange peel



7.0 (0.949)

5.0 (0.934)

7.0 (0.936)

4.0 (0.920)



( C)

30 (0.961)

30 (0.883)

40 (0.953)

40 (0.893)

Incubation period (in hours)

72 (0.946)

48 (0.824)

72 (0.991)

72 (0.931)

3.5 Iodine value of produced Bioethanol at optimal condition:

The iodine value of absolute ethanol is between 90-100. Iodine value of bioethanol produced by different substrate combinations at different optimization conditions is calcutated and tubulated. Results showed that iodine value

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International Journal of Scientific & Engineering Research, Volume 5, Issue 1, January-2014 4

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of ethanol obtained from banana peels fermanted with S.cerevisiae at 400C is closer to absolute ethanol in compare to other substrate combinations.


Banana peel

(B. sub- tilis)

Banana peel


Orange peel

(B. sub- tilis)

Orange peel



7.0 (79.23)

5.0 (76.07)

7.0 (72.73)

4.0 (73.85)



30 (69.80)

40 (88.23)

40 (80.47)

40 (82.57)

Incubation period (hours)

72 (73.85)

48 (81.34)

72 (74.94)

72 (71.07)


The present study examined the influences of temperature, pH and incubation period on ethanol production ability of S. cerevisiae and B. sutilis using the banana and orange peels as substrate. The results of this study indicates optimal tempera- ture, pH and incubation time for fermantation using S. cere-

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visiae and B. subtilis which may enhance ethanol yield and
minimize the cost of production could be obtained from bana-
na and orange peels. Bioethanol production By B. Subtilis
found more than S. cerevisiae with both the substrates that in-
dicates B. subtlis may be used as sucessful alternative of

S. cerevisiae in bioethanol production.


I am thankful to Er. Krishna Chaitanya and CytoGene Research

& Development, Lucknow for providing me facilities and kind support throughout the research work. I am also thankful to the Almighty.


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