A Comparative Study on Different Fruits Wastes Derived Ethanol using Yeast Strains (MTCC 170/ 180): Submerged vs Solid Sate Fermentation - sersc
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International Journal of Advanced Science and Technology
Vol. 29, No. 4s, (2020), pp. 1417-1427
A Comparative Study on Different Fruits Wastes Derived Ethanol using Yeast
Strains (MTCC 170/ 180): Submerged vs Solid Sate Fermentation
Sweety Rajput, Neeraj Saini
Department of Civil Engineering, Faculty of Engineering & Technology, SGT University,
Gurugram 122505, Haryana, India
Abstract
We present the two fermentation methodologies for the yeast induced ethanol production from grape,
orange and sweet lime wastes in the presence/ absence of benzyl penicillin. Different process parameters
were varied such as pH (3.5, 4, 4.5, 5); temperature (20 oC, 25 oC, 30 oC); type of yeast (Saccharomyces
cerevisiae strain MTCC 170 and MTCC 180); type of fermentation (submerged state and solid state) and
Benzyl Penicillin (addition, without addition) to work out the optimized conditions for efficient production
of ethanol. The results concluded that ethanol production was relatively higher in solid state fermentation
as compared to submerged state fermentation. Further, yeast strain MTCC 170 resulted in higher ethanol
production. The optimized experimental condition for maximum ethanol output was pH 5 and 30 oC. The
addition of Benzyl Penicillin further enhanced the ethanol yield. The utmost ethanol production obtained
was 8.9 %, 6.5 % and 3.4 % from grape, orange and sweet lime wastes, respectively.
Keywords: Ethanol, Fermentation, Benzyl penicillin, Reducing Sugar, Fruit wastes.
Introduction
In recent years, tremendous increase in the energy demands has played a key role in geo-political
economics. Given this reality, nations around the world have started investing in alternative sources of
energy, including bioethanol (Carlos et al. 2020, Borah et al. 2011; Mustaffa et al. 2011). But petroleum
derived products face problems of limited stocks and high extraction cost (Agbaji et al. 2005; Hammad et
al. 2011;Jia et al. 2013, Tan et al. 2019).In order to minimize the usage of non-renewable energy
resources, several approaches have been under taken for the production of ethanol from different bio-
wastes (Panneerselvam et al. 2011;Osuntoki et al. 2005, Yusuf et al. 2019). The conversion of bio-wastes
into liquid fuel through biochemical and thermo-chemical processes is an attractive option due to
economical raw material and easier synthetic techniques. This also facilitates in the environmental
pollution remediation (Vishwakarma et al.; Norah et al.).In developed as well as developing countries,
ethanol has been mandated to be used as fuel additive in the gasoline. The fruit and vegetable processing
industries generate 10% to 60% of the raw materials as solid waste (Borah et al. 2011).In fact, the waste
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from one processing plant could become the raw material of another plant (Hua et al. 2012).From
different local markets, large amount of different untreated fruit wastes is being dumped in the water
bodies. Utilization of waste rather than disposal should be the goal of the industry. Amongst these
biofuels, ethanol has great demand as it is widely accepted and it is clean burning (Reddy et al. 2007).
Moreover, ethanol production through fermentation of different fruit wastes is also an attractive
economical option to increase ethanol production and tackling problems of fruit wastes disposal. Solid
state as well as submerged state fermentation methodologies has emerged out as promising approach for
the efficient synthesis of biofuels employing saccharomyces cerevisiae strains (Hari Krishnan et al. 2011;
Gupta et al. 2012). The added advantages of fermentation techniques are simple and stable operations
with lower energy consumption. Further, addition of benzyl penicillin to the medium, results in enhanced
sporulation, thus facilitating in increased ethanol production (Williams et al. 1986).
In the present study, we have synthesized ethanol from inexpensive and easily accessible materials i.e.
raw fruit wastes such as of grapes, oranges and sweet lime. This waste utilization will assist in reducing
the hazardous impact of bio-wastes on the environment. Two, saccharomyces cerevisiae strain’s
(MTCC170 and MTCC180) were used during the fermentation processes. Various parameters such as pH,
temperature, benzyl penicillin, type of yeasts, were analyzed to work out the optimized conditions for the
fermentation of fruit wastes. Maximum ethanol production was obtained at pH 5 and 30 oC. The presence
of benzyl penicillin in the medium was comparatively better for higher ethanol output. Among all, the
maximum ethanol production was incurred in grape residue, due to higher sugar content.
Materials and methods
All experiments were carried out in sterile conditions. Control experiments were carried out to analyze the
results of varying controlling factors at a time. The identification of S.cerevisiae was done through
Stereo-research Microscope procured from Sai Lab Instruments. Laminar Air Flow Chamber (model:
MHL-222 (Toshiba) was used for the addition of inoculum into the YEPD medium. The solution pH was
measured using digital pH meter, model no. 361 (Sysronics).Benzyl Penicillin was added to the different
samples and stored at temperature below 8 oC.Raw fruit wastes i.e. spoiled / rotten grapes, oranges and
sweet limes were collected in plastic bags, each 5 kg, from old sabji mandi market, Kurukshetra, Haryana,
India and kept in sealed plastic bags. At the time of collection, the waste material such as packaging
materials were removed prior to sampling. For the determination of ethanol concentration, chromic acid
solution was prepared. All the chemicals were purchased from Aldrich, U.S.A. and used as supplied
without prior purification. The chromic acid was prepared by dissolving Potassium dichromate solution;
K2Cr2O7 (33.768 g) in 500 mL of distilled water. Later, H2SO4 (325 mL) was added and volume was
raised to 1 L. Further, for the DNS assay; Na-K tartrate (100 g) was added to a well-mixed solution of
NaOH (5 g) in deionized water (250 mL). Then, 3, 5-dinitrosalicylic acid (5 g) was added and
continuously stirred using magnetic stirrer. After that, Na2SO3(0.25 g) and phenol (1 g) was dissolved,
respectively. Finally, the volume was adjusted to 500 mL by deionized water. Spectrophotometer used for
the analytical calculations was Labomed, inc. UVS-2700. Autoclave and hot air oven were bought from
Narang Scientific work Pvt.Ltd. Grinding of the fruit waste was carried out in mixer (Sujata brand)
Sugar and ethanol concentration were calculated by the following equations:
Sugar concentration (%) = (A540− 0.15) / 1.702×100
Ethanol concentration (%) = (A540 − 0.2) / 0.866×100
Micro-organism
Yeast strain, Saccharomyces cerevisiae, was used in the fermentation processes. Two culture of S.
cerevisiae i.e. MTCC170 and MTCC180, in powdered form, were purchased from Institute of Microbial
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Technology, Chandigarh and refrigerated at 4 oC. The cultures were grown on Yeast Extract Peptone
Dextrose (YEPD) medium. These media were stored in the incubators at 25 oC. Plate count methodology
(YEPD, at 25 oC, incubation period 7-8 days) was employed for analysis of the growth pattern of S.
cerevisiae.
Preparation of YEPD medium
Each chemical constituent; yeast extract (1 %, 1.5 g), peptone (2 %, 5 g), dextrose (2 %, 10 g), agar (2 %,
1 g), was dissolved in 100 mL of distilled water. The agar was dissolved in boiling water at temperature
above 50 oC. Later, the volume of solution was made up to 1L by distilled water. Then, 100 mL of the
medium was poured in 10 conical flasks and mounted with cotton plugs. The cotton plug was enwrapped
with butter paper and aluminum foil to keep it away from any contamination. Medium was sterilized in an
autoclave at 15 psi for 20-30 minutes. After sterilizing the medium was left in Laminar Air Flow
Chamber for cooling.
Revival of culture in the medium
Laminar Air Flow Chamber was cleaned with spirit before the revival of the culture. Addition of culture
in the medium was carried out in Laminar Air Flow Chamber to avoid contamination. The medium was
opened in close of spirit lamp and it produced a popping sound ensuring that medium was air tight. After
opening the medium, the culture MTCC 170 was added in four flasks and MTCC180was added in other
four flasks. Two flasks were kept as control. Each flask was then marked and kept for 7 days at 25 oC in
incubator. After 7 days, the growth of culture in medium was observed. The color of the medium changed
from dark yellow to light yellow which ensures the growth of yeast mass in medium.
Sample preparation
Raw fruit wastes were refrigerated at 4 oC, prior to its use, for the preparation of extract.
Grape Sample
Initially, rotten grapes were washed with water to remove the infected parts. Then it was crushed in the
grinder. The grapes juice was used as substrate for submerged state fermentation on the other hand the
whole mass after grinding was used as substrate for solid state fermentation. Later, two different set of 24
samples were prepared for submerged as well as solid state fermentation, without benzyl penicillin, with
separate addition of both inoculums i.e. MTCC170 and MTCC180.Similarly, 48 samples were prepared
with the addition of benzyl penicillin along with inoculums MTCC170 and MTCC180, for submerged
and solid-state fermentation. Each set of 24 samples comprised of 3 subsets of 8 samples for fermentation
at 20 oC, 25 oC and 30 oC. Each subset of 8 samples includes 4 samples at pH 3.5, 4, 4.5 and 5, with
inoculums of MTCC 170 and MTCC 180, respectively. The desired pH of the samples was attained with
1N NaOH/ HCl solution and afterward glucose (2 g) was added. Then the samples were closed with
cotton plugs and covered by aluminum foil. Autoclaving was done at 15 psi pressure for 20 to 30 minutes,
and then the samples were kept for cooling in Laminar Air Flow Chamber.
Orange and Sweet Lime Samples
The peelings of oranges and sweet limes were removed and samples were prepared following the same
procedures as above, under sterile conditions.
Fermentation
After preparing the samples, the inoculum was added for the fermentation of fruit waste. In the
submerged state fermentation process, inoculants (1 mL) were added to each of the sample in close of
spirit lamp in Laminar Air Flow Chamber with micropipette. Later, two set of samples were prepared in
the presence/ absence of benzyl penicillin. After inoculation, the samples were kept in an incubator at 20
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o
C, 25 oC and 30 oC for fermentation. The samples were analyzed after 7 days of fermentation. The
procedure described as above was used for solid state fermentation except that whole mass of fruit waste
after grinding was used as substrate.
Analysis of Samples
1. Reducing Sugar
The reducing sugar was determined through Di-nitro Salicylic Acid Assay (DNS). The sample of each
fruit waste was filtered after fermentation. Then, 0.2 mL of each sample was taken in test tube and 0.3 mL
of distilled water was added to it to make volume to 0.5mL. Incubation was done at room temperature
followed by the addition of DNSA assay (1.5 mL). The samples were kept in hot water bath for 15
minutes. Finally, the absorbance was measured at 540 nm, as compiled in Table S1. The concentration of
reducing sugar was found out through the calibration curve, as depicted in Fig.S1.
Fig. S1. Calibration curve for the determination of sugar concentration
Table S1Optical density was observed in different fruit wastes at different pH for the determination of
initial sugar concentration.
S.No. pH Optical Density
Grape Waste Orange Waste Sweet Lime Waste
1. 3.5 0.4053 0.3202 0.2691
2. 4 0.4733 0.3882 0.3372
3. 4.5 0.5000 0.4563 0.3712
4. 5 0.5755 0.5074 0.4223
2. Ethanol
The ethanol concentration was determined using chromic acid solution. In this method, fermented wash (3
mL) was taken in a pyrex distillation flask containing distilled water (30 mL). The distillate was collected
in a conical flask containing potassium dichromate solution (25 mL). Then, the distillate (20 mL) was
collected for each sample and the flasks were kept in a water bath maintained at 60 ºC for 20 minutes. The
flasks were cooled to room temperature and the volume was raised to 50 mL. Then, the sample (5 mL)
was diluted with distilled water (5 mL) for measuring the optical density at 600 nm. Ethanol content of
each sample was determined through the calibration curve, as shown in Fig.S2.
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Fig. S2. Calibration curve for the determination of ethanol concentration
Result and discussions
In this study, three different fruit wastes were used for the fermentation-based production of ethanol,
employing MTCC 170/ MTCC 180, in the presence/ absence of benzyl penicillin. Different experimental
parameters were studied to conclude the optimized reaction conditions for the efficient synthesis of
ethanol through bio-conversion of raw fruit waste. Among all the explored conditions, at pH 5 and 30 oC,
ethanol production was maximum under ambient conditions. Higher temperature assisted in increased
sporulation of yeast which in turn resulted in enhanced ethanol production. Addition of benzyl penicillin
to the medium further assisted in increased ethanol output. Solid state fermentation was comparatively
better than submerged state due to the availability of concentrated reducing sugar in the medium.
1. Effect of Sugar concentration
Initially, reducing sugar content was determined in the fresh as well as fermented grape, orange and sweet
lime waste for different pH at different temperature conditions, as demonstrated in Table 1. From the
results obtained, it was observed that sugar concentration decreases with the rise in temperature, as shown
in Table S2-S13. In case of grape waste, with MTCC 170, under submerged state fermentation, at pH 3.5,
4, 4.5 and 20oC/ 25 oC / 30 oC, reducing sugar content showed a drastic decrease from 15 % to 7.1 %/ 7.2
%/ 8.4 %,19 % to 7.1 %/ 8.4 %/ 8.8 % and 22 % to 7.8 %/ 8.8 %/ 9.1 %, respectively, in the absence of
benzyl penicillin. However, at pH 5 and 20 oC/ 25 oC/ 30 oC, sugar content was reduced from 25 % to 9.1
%/ 9.3 %/ 9.3 %, respectively. Then, in the solid-state fermentation condition, the observed decrease in
the reducing sugar content was from 15 % to 8.7 %/ 9.1 %/ 9.9 %, 19 % to 9.3 %/ 9.9 %/ 10.6 % and 22
% to 9.9 %, 10.8 %, 15.2 %, respectively. In addition, at pH 5 and 20 oC/ 25 oC/ 30 oC, the sugar content
decreased from 25 % to 11.3 %/ 13.3 %/ 15.9 %, respectively. Then, the experimentation was conducted
in the presence of benzyl penicillin under submerged state fermentation, employing MTCC170, at pH 3.5,
4, 4.5 and 20 oC/ 25 oC/ 30 oC, the deduction in the sugar content was from 15 % to 8.4 %/8.6 %/11.2%,
19 % to 8.6 %, 10 %, 11.6 %, 22 % to 9.9 %, 10.4 %, 11.7 %,respectively. At pH5 and 20 oC/ 25 oC/ 30
o
C, the reducing sugar content fall from 25 % to 10.7 %/ 11.3 %/ 11.7 %, respectively. However, in the
solid-state fermentation condition, at pH 3.5, 4, 4.5 and 20 oC/ 25 oC/ 30 oC, the sugar content diminished
from 15 % to 10.7 %/ 10.5 %/ 11.9 %, 19 % to 11.6 %, 11.9 %, 13.1 %, 22 % to 13.1 %, 15.9 %, 16.4 %,
respectively. Finally, at pH 5 and 20 oC/ 25 oC/ 30 oC, the observed decline in the reducing sugar content
was from 25 % to 15.9 %/ 17.1 %/ 17.9 %, respectively. Similarly, with MTCC180, different experiments
were performed in submerged state/ solid-state fermentation, both in the absence/ presence of benzyl
penicillin and obtained results were analyzed and results were compiled in Table S2-S13. From the
results, it was concluded that maximum reducing sugar content (17.9%) was observed in grape waste
sample in solid state fermentation, in the presence of benzyl penicillin, using MTCC170. For complete set
of samples prepared from different fruit waste, the range of reducing sugar was found to be in the range of
0.52–17.97%.
Table 1 Initial sugar concentration of different fruit wastes at different pH under room temperature
conditions.
S.No. pH Initial Sugar Concentration (%)
Grape Waste Orange Waste Sweet Lime Waste
1. 3.5 15 10 7
2. 4 19 14 11
3. 4.5 22 18 13
4. 5 25 21 16
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2. Effect of pH
In order to study the effect of pH on the ethanol production, various experiments were carried out along
with different fruit wastes under different fermentation processes (submerged/ solid state), type of yeast
strains (MTCC 170/ MTCC 180) and temperature (20 oC/ 25 oC/ 30 oC) for a period of 7 days. The pH
value was varied from 3.5 to5, with an increment of 0.5. The variation in desired pH value was controlled
by NaOH/ HCl solution. The results showed that ethanol production increased with increase in pH from
3.5 to 5 and maximum values were observed at pH 5.The experiments conducted at pH 5 and 30 oC, under
submerged state condition, using MTCC 170 strains, in the presence of benzyl penicillin for grape, orange
and sweet lime wastes resulted in 5.8 %, 5.7 % and 1.6 % ethanol production, respectively. Similarly, in
the absence of benzyl penicillin, under above mentioned conditions, the obtained ethanol yield was 4.9 %,
4.8 % and 1.5 % respectively. However, with MTCC 180, ethanol produced was 5.6 %, 5.5 % and 1.5 %
and 4.6 %, 4.5 % and 1.4 % in the presence/ absence of benzyl penicillin, respectively as shown in Fig. 1.
Following the similar methodology, under solid state fermentation, with MTCC 170, the ethanol output
was 8.9 %, 6.5 %, 3.4 % and 7.9 %, 5.6 % 2.5 %, in the presence/ absence of benzyl penicillin,
respectively. Additionally, with MTCC 180, conversion of reducing sugar to ethanol was 8.6 %, 6.2 %,
3.1 % and 7.2 %, 5.3 %, 2.2 %, in the presence and absence of benzyl penicillin, respectively, as shown in
Fig. 2. Other experiments at varying pH i.e. 3.5, 4 and 4.5 have been performed and obtained percentage
yield of ethanol were demonstrated in the Table S14-S25. The corresponding data was depicted
pictorially in Fig. S3-S6.
Fig. 1 Effect of pH on ethanol production, under submerged state fermentation conditions, in the absence/
presence of benzyl penicillin at 30 oC using yeast; (a)/ (b)MTCC 170 and (c)/ (d) MTCC180, respectively.
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Fig. 2 Effect of pH on ethanol production, under solid state fermentation conditions, in the absence/
presence of benzyl penicillin at 30 oC using yeast; (a)/ (b)MTCC 170 and (c)/ (d) MTCC180, respectively.
3. Effect of Temperature
Further, the effects of variation of temperature on the ethanol production under varying conditions were
studied, as shown in Fig. 3-4, S7-S12. With MTCC 170, ethanol production was higher in the submerged
state fermentation compared to solid state fermentation, in the absence of benzyl penicillin. As shown in
Fig. 3, at pH 5 and 30 oC, under submerged state fermentation, with MTCC 170, the ethanol production in
the presence and absence of benzyl penicillin was 5.9 %, 5.7 %, 1.6 % and 4.9 %, 4.8 %, 1.5 % for grape,
orange and sweet lime wastes, respectively. However, with MTCC 180, the percent ethanol produced was
5.7 %, 5.9 %, 1.5 % and 4.6 %, 4.6 %, 1.4 % for grape, orange and sweet lime wastes, respectively, in the
above similar experimental conditions. Moreover, in the solid-state fermentation conditions, the percent
ethanol obtained was higher than the submerged state fermentation, with MTCC 170/ MTCC 180. As
depicted in Fig. 4, at pH 5 and 30 oC, under solid state fermentation conditions, in the presence and
absence of benzyl penicillin, with MTCC 170 strains, ethanol production was 8.9 %, 6.5 %, 3.4 % and 7.9
%, 5.6 %, 2.5 %, from grape, orange and sweet lime wastes, respectively. Similarly, with MTCC 180,
ethanol obtained was 8.6 %, 6.2 %, 3.1 % and 7.2 %, 5.3 %, 2.2% for grape, orange and sweet lime
wastes, respectively.
On the other side, at low temperature i.e. 20 oC and pH 5, following submerged state fermentation
conditions, with MTCC 170, in the presence as well as absence of benzyl penicillin, ethanol percent was
found to be 5.4 %, 4.3 %, 1.3 % and 4.5 %, 3.6 %, 1.1 % for grape, orange and sweet lime wastes,
respectively. Under similar practices, with MTCC 180, conversion of reducing sugar in grape, orange and
sweet lime wastes resulted into 5.03 %, 4.10 %, 1.03% and 4.13 %, 3.30 %, 0.98 % ethanol, in the
presence as well as absence of benzyl penicillin. For solid state fermentation, with MTCC 170 as well as
MTCC 180, in the presence of benzyl penicillin, percent ethanol content obtained was 7.96 %, 4.32 %,
1.88 % and 6.09 %, 4.11 %, 1.65 % for grape, orange and sweet lime wastes, respectively. Then, in the
absence of benzyl penicillin, the conversion obtained was 5.66 %, 3.55 %, 1.61 % and 5.10 %, 3.32 %,
1.51 % for grape, orange and sweet lime wastes, respectively. It was concluded that the ethanol
production was maximum at 30 oC and minimum at 20 oC. The yeast growth is low at temperature below
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30 oC. However, at 25 oC, under the above said conditions, percent ethanol obtained were compiled in the
Table S14-S25.
Fig.3Effect of temperature on ethanol production, under submerged state fermentation conditions, in the
absence/ presence of benzyl penicillin at pH 5 using yeast; (a)/ (b) MTCC 170 and (c)/ (d) MTCC180,
respectively.
Fig.4Effect of temperature on ethanol production, under solid state fermentation conditions, in the
absence/ presence of benzyl penicillin at pH 5 using yeast; (a)/ (b) MTCC 170 and (c)/ (d) MTCC180,
respectively.
4. Benzyl Penicillin
To study the effect of benzyl penicillin on the quantity of ethanol produced, fermentation experiments
were carried out in two batches: one batch was in the presence, while another batch in the absence of
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benzyl penicillin. The fermentation period was7 days. The effects of Benzyl Penicillin on ethanol
production for varying type of yeast, type of fermentation system, temperature, pH and type of fruit waste
are presented in Fig.5, S13-S15. The ethanol production is more at pH 5 and 30 oC for grape waste as
compared to other fruit wastes, as shown in Fig. 5. The addition of benzyl penicillin in submerged state
fermentation with both yeast strains, MTCC170 and MTCC180, resulted in increased ethanol production.
The results showed that the ethanol production was less using MTCC 180 as compared to MTCC 170,
even after the addition of benzyl penicillin.
Fig.5Effect of benzyl penicillin on ethanol production, under submerged state/ solid state fermentation
conditions, at pH 5 using yeast; (a)/ (b) MTCC 170 and (c)/ (d) MTCC180,respectively.
5. Fermentation system
Under submerged state as well as solid-state fermentation conditions different experiments were carried
out at varying pH, temperature, type of yeast and in the absence /presence of benzyl penicillin for
different fruit wastes. The results showed that ethanol production was relatively higher in solid state
fermentation as compared to submerged state fermentation under similar conditions for all three fruit
wastes.
6. Type of Yeast Strains
Two yeast stains, MTCC 170 and MTCC 180 were used for the production of ethanol from reducing
sugar at varying pH, temperature, type of fermentation, and in the absence/ presence of benzyl penicillin.
The results showed that production of ethanol using MTCC 170 was relatively higher than MTCC 180,
under optimum conditions for all the different fruit wastes. The ethanol production was higher when
benzyl penicillin was added during the fermentation processes.
Conclusion
Two fermentation processes, submerged state as well as solid-state, have been carried out for the
production of ethanol from raw grape, orange and sweet lime waste, in the presence of Saccharomyces
cerevisiae strain’s; MTCC170 and MTCC180. Ethanol production was concluded to be higher in solid
state fermentation system compared to the submerged state fermentation, since in the solid-state, initial
sugar content in the raw fruit waste was relatively high. From the experimentation, it was ascertained that
favorable fermentation conditions for the conversion of reducing sugar to ethanol was pH 5 and 30 oC.
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The ethanol production was low at pH 3.5, 4 and 4.5, since pH values was too low to activate the enzymes
to react. The alcohol production was minimum at 20 oC, as the yeast growth was low at temperature
below 30 oC. Among different substrates, grape waste showed higher ethanol output (4.9-8.9 %)
compared to orange (4.8-6.5 %) and sweet-lime waste (1.5-3.4 %), with MTCC170, under solid-state
fermentation. However, in submerged state fermentation, with MTCC180, the ethanol produced was
relatively less in different fruit wastes. The addition of benzyl penicillin further contributed to enhanced
ethanol production from all the three fruit wastes, under optimized experimental conditions. Benzyl
penicillin assisted in the enhanced sporulation of yeast in a fixed period of time. The ethanol production
was less using MTCC180, even after the addition of benzyl penicillin. Also, due to high sugar content in
the raw grape waste, alcohol production was more compared to orange and sweet-lime waste. Among
three fruit wastes, following order of ethanol production was observed: Grape waste > Orange waste >
Sweet lime waste.
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ISSN: 2005-4238 IJAST
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