ArticlePDF Available

Optimization of Bioethanol Production from Fruit Wastes using Isolated Microbial Strains

Authors:
  • Amity University Uttar Pradesh Noida

Abstract and Figures

Production of Ethanol fermented from renewable sources for fuel or fuel additives are known as bioethanol. Bioethanol as a fossil fuel additive to decrease environmental pollution and reduce the stress of the decline in crude oil availability is becoming increasingly popular. Its production mainly utilizes three types of raw materialssugar juice, starchy crops, and lignocellulosic materials. This research work investigate ethanol production from fruit juices of four different fruits such as Vitis vinifera (grapes), Sugarcane (Saccharum officinarum), Citrus cimetta (Mosambi) and Citrullus canatus (Watermelon) using Saccharomyces cerevisiae for fermentation and optimizing several factors that influence the process for bioethanol production such as temperature, pH and sugar concentration.
Content may be subject to copyright.
International Journal of Advanced Biotechnology and Research(IJBR) ISSN 0976-2612, Online ISSN 2278–599X,
Vol5, Issue4, 2014, p598-604 http://www.bipublication.com
Optimization of Bioethanol Production from Fruit Wastes using Isolated
Microbial Strains
Sandesh Babu
1
, K.M.Harinikumar
2
, Ravi Kant Singh
*3
and Aditi Pandey
1
1.3
Department of Biotechnology, IMS Engineering College, Ghaziabad
2
Department of Plant Biotechnology, GKVK Campus, Bengaluru
* Corresponding Author- rksingh.iitr@hotmail.com, raviksingh@imsec.ac.in
[Received 17/08/2014, Accepted-07/10/2014]
ABSTRACT
Production of Ethanol fermented from renewable sources for fuel or fuel additives are known as bioethanol.
Bioethanol as a fossil fuel additive to decrease environmental pollution and reduce the stress of the decline in
crude oil availability is becoming increasingly popular. Its production mainly utilizes three types of raw materials-
sugar juice, starchy crops, and lignocellulosic materials. This research work investigate ethanol production from
fruit juices of four different fruits such as Vitis vinifera (grapes), Sugarcane (Saccharum officinarum), Citrus
cimetta (Mosambi) and Citrullus canatus (Watermelon) using Saccharomyces cerevisiae for fermentation and
optimizing several factors that influence the process for bioethanol production such as temperature, pH and sugar
concentration.
Keywords: Bioethanol production, Agricultural wastes, Process optimization, Fermentation process
1.0 INTRODUCTION
Bioethanol is being widely investigated as a
renewable fuel source because in many respects it is
superior to gasoline fuel [1]. Ethanol provides
energy that is renewable and less carbon intensive
than oil. It is a biofuel produced from biomass via
biochemical procedures. An efficient ethanol
production requires four components: fermentable
carbohydrates, an efficient yeast strain, a few
nutrients and simple culture conditions.
Approximately 80% of world supply of alcohol is
produced by fermentation of sugar and starch
containing crops or byproducts from industries
based on such crops. Among the widely used
substrates for ethanol production are the molasses of
sugarcane and sugar beet. Several studies have
shown that sugarcane-based ethanol reduces
greenhouse gases by 86 to 90% [2, 3]. Sugar-based
bioethanol production- such as sugarcane and sugar
beet- is a simple process and requires one step less
than starch-bioethanol, this is because they are
ready for conversion with limited pre-treatments as
compared with starchy or cellulosic materials.
Generally, the process is based on extraction of
sugars (by means of milling or diffusion), which
may be then taken straight to fermentation. The
Optimization of Bioethanol Production from Fruit Wastes using Isolated Microbial Strains
Ravi Kant Singh, et al. 599
wine is distilled after fermentation, such as in
starch-based production.
The most well-known and commercially significant
yeasts that been primarily used for bioethanol
production are the related species and strains of
Saccharomyces cerevisiae [4]. These organisms
have long been utilized to ferment the sugars of rice,
wheat, barley, and corn to produce alcoholic
beverages and in the backing industry [5]. One yeast
cell can ferment approximately its own weight of
glucose per hour. Sugars from sugar cane, sugar
beets, molasses, and fruits can be converted to
ethanol directly [6].
In order to produce ethanol in large quantities and
reasonable costs, the optimization of various
physico-chemical parameters is important. The
important parameters that could affect ethyl alcohol
fermentation may be mentioned: availability and
ferment ability of the substrate, possible isolation of
new potent strain and improvement of the available
strain towards higher productivity, improvement in
fermentation technology and reduction in by-
product formed during the fermentation process.
2.0 MATERIALS AND METHODOLOGY:
The chemicals that are used in this research are
potassium dichromate, hydrochloric acid,
Saccharomyces cerevisiae strains, different fruits
such as mosambi, sugarcane, watermelon and
grapes. The equipments used are hot plates,
incubators, distillation unit and hydrometer.
Fermenting yeast Saccharomyces cerevisiae
(MTCC 171) procured from MTCC, Institute of
microbial technology, Chandigarh was used in the
present study. The microbial growth media YEPD is
used under aerobic condition at temperature 30
0
C.
2.1 Preparation of the substrate for the
fermentation process
All four fruits (sugarcane, watermelon grape, and
mousambi) were collected from different places.
These fruits were rinsed in water and juice of each
one of them was prepared and collected. The
fermentation system was set up by taking ten
conical flasks and in each set up different substrate
(fruit juice) of 200 mL was used. Hydrochloric acid
was used to adjust the initial pH prior to inoculation
and process was carried out at room temperature.
2.2 Inoculum and inoculation
The yeast inoculum was prepared in YEPD broth.
The fermentation system was inoculated with 2 mL
culture broth /200 mL of substrate. The
fermentation system was left undisturbed for about
a week. The fermentation was carried out at varying
temperature, pH, reducing sugar concentration,
agitation and immobilized condition. During
incubation, specific gravity of the sample was noted
frequently by using a hydrometer. When the specific
gravity reaches a steady value, it indicates the end
of the fermentation process. The incubation period
varies for each fruit juice sample.
2.3 Extraction of Ethanol by Steam Distillation
Fermented broths were removed at 48 hours of
interval and contents were analyzed for total sugar
and ethanol. Simple distillation unit at a temperature
of 78 96°C separated the mixture of ethanol and
hot water. In this method 80% of pure ethanol is
obtained, which rectified by using rectifier units to
obtain 99.2% pure ethanol [7].
2.4 Alcohol Estimation
Samples were first distilled, and the resultant
ethanol concentration was measured using a
dichromate reagent [8].
Table 1: Percentage of Ethanol Obtained
S. No Samples Specific
Gravity Percentage
Ethanol
1 Grape 0.820 11.25
2 Mousambi 0.864 6.23
3 Watermelon 0.835 10.10
4 Sugarcane 0.818 12.15
Table 2: Ethanol Concentration obtained from different
fruit samples
Samples Ethanol Conc
(Mg/Ml)
Ethanol Conc
(µg/Ml)
Sugarcane
0.52 520
Grape
0.47 470
Watermelon
0.39 390
Mousambi
0.32 320
Optimization of Bioethanol Production from Fruit Wastes using Isolated Microbial Strains
Ravi Kant Singh, et al. 600
2.5 Optimization of fermentation process
Fermentation process carried out by yeast is known
to vary with respect to various factors such as-
substrate concentration, temperature, pH, N-source
and inoculum size. It is therefore imperative to
optimize the fermentation conditions for yeast cells
so that the production efficiency increases. Various
factors were investigated affecting ethanol
production from fruits.
2.6 Effect of Temperature
Temperature plays a major role in the production of
ethanol, since the rate of alcoholic fermentation
increases with the increase in temperature. The
fermentation process is always accompanied with
evolution of heat that raises the temperature of the
fermenter. As a result it becomes necessary to cool
the large fermenters in the distilleries. To optimize
the fermentation temperature, fermentation was
carried out at 15, 20, 25, 30 and 35ºC. Fruits diluted
to 20% sugars and supplemented with nitrogen and
phosphorus were used as production media and
fermentation was carried out at different
temperatures. The periodic samples were analyzed
for reducing sugars and ethanol content.
2.7 Effect of pH
pH of solution 5.0, 6.0, 7.0 and 8.0 were tested for
fermentation using fruit sample with 20% sugar
concentration and temperature of 29 ± 1ºC. Low pH
inhibits the yeast multiplication.
2.8 Effect of Sugar concentration
Initial sugar concentration is an important
influencing parameter as it has the direct effect on
fermentation rate and microbial cells. The actual
relationship between initial sugar content and the
fermentation rate is rather more complex. Generally,
fermentation rate will be increased with the increase
in sugar concentration up to a certain level. But
excessively high sugar concentration will exceed the
uptake capacity of the microbial cells leading to a
steady rate of fermentation. In batch fermentation,
increased ethanol productivity and yield can be
obtained at higher initial sugar concentration, but it
takes longer fermentation time and subsequently
increases the recovery cost [9]. To study the effect
of sugar concentration on ethanol production by
S.cerevisiae, the production media was prepared to
sugar concentration of 5, 10, 15, 20, 25, and 30
percent with distilled water and filtered through
ordinary filter paper to remove suspended particles.
Fermentation was carried out in 250 ml conical
flasks. A twenty four hour old inoculum of yeast
was added at the rate of 6 percent to the medium.
Samples were withdrawn after every 12-hour
interval and estimated for residual sugars [10] as
well as ethanol content in the media [11]. GC
method for estimating the percentage of ethanol was
employed. The initial sugar concentration that was
efficiently utilized by the yeast for ethanol
production was selected and maintained in
fermentation media for further use.
2.9 Analytical methods
2.9.1 Spectrophotometric determination of
ethanol [11]
One millilitre of the fermented wash was taken in
500ml Pyrex distillation flask containing 30 ml of
distilled water. The distillate was collected in 50 ml
flask containing 25 ml of potassium dichromate
solution (33.768 g of K
2
Cr
2
O
7
dissolved in 400 ml
of distilled water with 325 ml of sulphuric acid and
volume raised to 1 litre). About 20 ml of distillate
was collected in each sample and the flasks were
kept in a water bath maintained at 62.5ºC for 20
minutes. The flasks were cooled to room
temperature and the volume rose to 50 ml. Five ml
of this was diluted with 5ml of distilled water for
measuring the optical density at 600nm using a
spectrophotometer. A standard curve was prepared
under similar set of conditions by using standard
solution of ethanol containing 2 to 12% (v/v)
ethanol in distilled water. Ethanol content of each
sample was estimated and graph was made.
2.9.2 Estimation of reducing sugars
The DNS method of was used to estimate reducing
sugars. One ml of appropriately diluted solution
(500-1000 µg ml
-1
) sample was taken in a test tube
to which 3 ml of DNS reagent was added. The tubes
were boiled in a boiling water bath for 15 minutes.
Optimization of Bioethanol Production from Fruit Wastes using Isolated Microbial Strains
Ravi Kant Singh, et al. 601
One ml of Rochelle salt was added to these test
tubes and tubes were cooled to room temperature
and used for measuring optical density at 575 nm. A
standard curve of glucose was prepared by using
100-1000 g concentration prepared in distilled water
[10].
2.9.3 Gas chromatography
Ethanol in the fermentation broth was estimated by
gas chromatography method. A computer related
Nucon series gas chromatograph equipped with
flame ionization detector (FID) was employed for
the separation and quantification of ethanol. A
stainless steel column (5m × 2mm) was fitted into
the instrument to provide on column injection. The
column packing was Porapak Q. The detector and
injector temperature was maintained at 200°C. The
gas chromatograph was connected to an integrator
and computer system to determine area of ethanol
and internal standard peak.
3.0 RESULT AND DISCUSSION:
3.1 Growth studies and effect of sugar
concentration
The growth of S.cerevisiae in gradually increasing
concentrations of sugar showed an increase in
optical density upto 20% sugar concentration in
YEPD medium as shown in Table 1. However on
increasing the sugar concentration beyond 20%, the
growth was inhibited as shown by the optical
density measured. Samples were taken every 12
hours for the study of growth kinetics. The growth
was measured at 600nm.
Moaris et al (1996) also studied viability of
Saccharomyces sp. in 50% glucose and reported a
viability of 10-98.8% in different strains of yeast
[12]. The detrimental effect of high sugar
concentration on ethanol production was studied by
Gough et al (1996) in Kluyveromyces marxianus
and a sucrose concentration more than 23% in
molasses was found to affect ethanol production
[13]. Therefore, in the present study growth and
fermentation were carried out with sugar
concentrations upto 20%.
3.2 Effect of temperature on ethanol yield
Temperature is one of the major constraints that
determine the ethanol production. To know the
optimum temperature for ethanol fermentation, the
solutions were kept at 25, 30, 35 and 40°C with
20% initial sugar concentration. Two parameters
were simultaneously studied, the growth of
S.cerevisiae and the ethanol yield. Samples were
withdrawn every 12 hours and the fermentation was
carried out for 48 hours. A low ethanol yield of
6.8% was observed at 25°C in 48 hours. As shown
in Table 2 at 30°C ethanol yield was maximum and
turned out to be 11%. However increasing the
temperature beyond 30°C the growth as well as
concentration of alcohol decreased. This
decrease was pronounced at 40°C so 30°Cwas
selected as optimum temperature for ethanol
production.
Optimization of Bioethanol Production from Fruit Wastes using Isolated Microbial Strains
Ravi Kant Singh, et al. 602
Temperature tolerance was also been found to
depend upon sugar concentrations of the medium as
in the case of, fermentation of molasses at 35°C was
possible when sugar concentration was 20%(w/v)
with no fermentation when sugar concentration was
22%(w/v) [14].
3.3 Effect of pH on ethanol yield
Initial sugar concentration of 20% and optimum
temperature of 30°C was selected for further studies
and subjected to pH treatments 5, 6, 7 and 8. The
results are shown in table 3. At pH 5, fermentation
took place but it gave low ethanol content. Best
results were obtained at pH 6 where maximum
ethanol production was noticed. Yadav et al (1997)
found an increase in alcohol concentration,
productivity as well as efficiency with an increase in
pH from 4.0-5.0 and found that the optimum pH
range for S.cerevisiae strain HAU-1 to be between
pH 4.5-5.0 [15]. Based on fermentation efficiency
the pH 6 was selected for further experimentation.
3.4 Increase in ethanol yield using fermenter
After optimizing the various parameters like pH,
temperature, sugar concentration etc. the experiment
was scaled up from shake flask to fermenter. The
optimum of previous experiments was taken i.e. the
sugar concentration of 20%, pH 6 and temperature
30°C to further carry the experiment on fermenter.
Fermenters are designed to provide best possible
growth and biosynthesis conditions for industrially
important microbial cultures. In fermenter, it is
easier to control various parameters like
temperature, pH that increases the ease of obtaining
the desired product, ethanol in present study. After
carrying out the fermentation, the samples were
analysed using GC and compared with the standard
run of absolute ethanol. From the broadness of peak
it was inferred that there was continuous increase in
ethanol production till 48 hrs.
Optimization of Bioethanol Production from Fruit Wastes using Isolated Microbial Strains
Ravi Kant Singh, et al. 603
4.0 CONCLUSION
In our present study we obtained ethanol from four
ripened fruits Vitis vinifera (grapes), Saccharum
officinarum (Sugarcane), Citrus Cimetta
(Mosambi), Citrullus Canatus (Watermelon)
collected from local markets around Bangalore
.With the aid of yeast strains Saccharomyces
cerevisiae (MTCC NO. 171) procured from MTCC,
Institute of microbial technology, Chandigarh,
ethanol was produced by the process of
fermentation. After two week we could obtain 520
µg/ml and 470µg/ml of ethanol from 100 mL of
fruit juices of Sugarcane, Grape and then followed
by Watermelon and least being the Mousambi after
distillation and maintaining a pH of 4 and
temperature of 35°C. We could infer that more
concentrated form of ethanol could be obtained by
re-distillating the product ethanol obtained initially
using a higher grade of distillation setup. A higher
percentage (v/v) of ethanol could be obtained if the
ethanol tolerance capability of yeast species is
improvised by mutating the yeast species. This more
concentrated form of ethanol could be used as a
biofuel, which releases no toxic gases out in the
environment. This process is environment friendly
and the left over residues after fermentation can be
disposed in the soil acting as a fertilizer for the soil.
So even a common man may develop this process
and produce it on commercial basis.
The fermentation of Sugarcane using S.cerevisiae
(distillery strain) under optimized conditions i.e. pH
6, sugar concentration 20% and temperature 30ºC
revealed an increase in ethanol production with
good fermentation efficiency. However
fermentation efficiency deceases after 48 hours of
fermentation time. This might be due the either
substrate limitations or due to product inhibition. S.
cerevisiae reportedly showed the decrease in growth
with increase in ethanol concentration in the
medium.
5.0 REFERENCES
1. Jones, A.M., Thomas K.C. and Inglew W.M.,
(1994), Ethanolic fermentation of molasses and
sugarcane juice using very high gravity technology.
Journal of Agricultural Chemistry, 42, 1242-1246.
2. Isaias, M., V. Leal, L.M. Ramos and J.A. Da-Silva,
Assessment of greenhouse gas emissions in the
production and use of fuel ethanol in
Brazil.Secretariat of the Environment, Government
of the State of Sao Paulo, 2004.
3. Goettemoeller, J. and A. Goettemoeller, Sustainable
Ethanol: Biofuels, Biorefineries, Cellulosic Biomass,
Flex-Fuel Vehicles, and Sustainable Farming for
Energy Independence. Praire Oak Publishing,
Maryville, Missouri, ISBN: 9780978629304, 2007.
p. 42.
4. Chandel, A.K., Chan, E.S., Rudravaram R., Narasu
M.L., Rao L.V., Ravindra P. (2007), Economics and
Enviromental Impact of Bioethanol Production
Technologies: an Appraisal. Biotechnology and
Molecular Biology Review, 2 (1), 14-32.
5. Tsuyoshi, N., Fudou, R., Yamanaka, S., Kozaki, M.,
Tamang, N., Thapa, S., Tamang, J.P. (2005),
Identification of yeast strains isolated from marcha
in Sikkim, a microbial starter for amylolytic
fermentation. International Journal of Food
Microbiology, 99 (2), 135-146.
6. Janani K., Ketzi M., Megavathi S., Vinothkumar D.,
Ramesh Babu N.G. (2013), Comparative Studies of
Ethanol Production from Different Fruit Wastes
using Saccharomyces cerevisiae." International
Journal of Innovative Research in Science, 2 (12),
7161-7167.
7. Mandal P. and Kathale N. (2012), Production of
Ethanol from Mahua flower (Madhuca Latifolia L.)
using Saccharomyces Cerevisiae-3044 and study of
parameters while fermentation, Abhinav Journal,
1(9), 6-10.
8. Boehringer, P. and Jacob, L. (1964), The
determination of alcohol using chromic acid, Z
Flussiges Abst, 31, 233–236.
9. Zabed H., Faruq G., Sahu J.N., Azirun M.S., Hashim
R., Boyce A.N. (2014), "Bioethanol Production from
Fermentable Sugar Juice.” The Scientific World
Journal, 1-11,
http://dx.doi.org/10.1155/2014/957102.
10. Miller, Gail Lorenz. "Use of dinitrosalicylic acid
reagent for determination of reducing
sugar." Analytical chemistry 31 (3), 1959: 426-428.
11. Arthur, C., Ueda M., and Brown T. (1968),
Spectrophotometric determination of ethanol in
Optimization of Bioethanol Production from Fruit Wastes using Isolated Microbial Strains
Ravi Kant Singh, et al. 604
wine." American Journal of Enology and Viticulture
19 (3), 160-165.
12. Morais, P.B., Rosa, C.A., Linardi, V.R., Carazza, F.,
Nonato E.A., (1996) Production of fuel alcohol by
Saccharomyces strains from tropical habitats,
Biotechnology letters, 18 (11), 1351-1356.
13. Gough S, Flynn O, Hack C.J., Marchant R. (1996),
Fermentation of molasses using a thermotolerant
yeast Kluyveromyces marxianus IMB3: simplex
optimization of media supplements, Applied
Microbiology and Biotechnology, 46 (2), 187-190.
14. Morimura, S., Zhong Y. L., and Kenji K. (1997),
Ethanol production by repeated-batch fermentation
at high temperature in a molasses medium containing
a high concentration of total sugar by a
thermotolerant flocculating yeast with improved salt-
tolerance, Journal of Fermentation and
Bioengineering 83 (3), 271-274.
15. Yadav, A., N. Dilbaghi, and S. Sharma (1997),
Pretreatment of sugarcane molasses for ethanol
production by yeast, Indian Journal of Microbiology,
37 (1), 37-40.
... To optimize the fermentation temperature, fermentation of S. cerevisiae KD2 was carried out at 15, 20, 25, 30 and 35ºC. The periodic samples were analyzed for reducing sugars and ethanol content ( [28]). The effect of pH on bioethanol production was tested by adjusting the production medium pH to 5.0, 6.0, 7.0 and 8.0 before autoclaving. ...
... To study the effect of sugar concentration on ethanol production by S. cerevisiae KD2, the production media was prepared to sugar concentration of 5, 10, 15, 20, 25, and 30 percent with distilled water and filtered through ordinary filter paper to remove suspended particles ( [28]). Fermentation was carried out in 250 ml conical flasks. ...
... The detector and injector temperature was maintained at 200°C. The gas chromatograph was connected to an integrator and computer system to determine area of ethanol and internal standard peak ( [28]). ...
... Total residual sugar was analyzed after 3 subsequent days using the phenol-H 2 SO 4 method as per the procedure used by Nielsen [16]. A calibration equation was developed using dextrose glucose standard solution (5,10,20,40,60, and 80 mg/L). The concentration of total residual sugar was calculated using the calibration equation: residual sugar = 0.012x + 0.034, at R 2 = 0.999, where x is concentration of dextrose glucose (mg/L). ...
... Spectrometric determination of ethanol (%, v/v) using acidified dichromate solution by microdistillation of the samples, which is routinely used in many winery laboratories, was adopted in this study [19]. Using the procedure applied by Babu et al.: into 500 mL fractional distillation flask, 1 mL of wine sample and 30 mL distilled water were placed [20]. During the distillation, 20 mL of the distillate was collected in a 50 mL receiving flask that contained 25 mL of potassium dichromate solution (0.17 M K 2 Cr 2 O 7 dissolved in 5.9 M H 2 SO 4 ). ...
... Further increment of temperature can suppress the growth of yeast cells, as inoculum concentration has shown to have an insignificant effect from 14% to 16%. Babu et al. reported that increasing the temperature beyond 30 °C decreased alcohol content from 9.2 to 6.8% [20]. Also, due to the consumption of substrates in the fermentation process, further production of alcohol was inhibited, which might be attributable to the decline in yeast fermentation activity and population. ...
Article
Full-text available
Cactus pear fruit (Opuntia ficus-indica) has a chemical composition that renders it an attractive substrate for wine fermentation. However, there have been serious post-harvest losses of cactus fruit due to its short shelf life. This study aims to investigate wine production from cactus pear fruit juice by optimizing fermentation temperature, pH, and inoculum concentration (Saccharomyces cerevisiae) to obtain optimum quality-indicative responses. Response surface methodology coupled with central composite rotatable design was adopted in the present study to achieve optimized fermentation process conditions. The fermentation process was carried out for 6 days with varied input variables, and all the models showed significant p-values for interaction of variance (<0.05). Cactus pear fruit wine with a total acidity of 12.39 ± 1.32 g/L equivalent to tartaric acid (TTAE), alcohol content of 9 ± 0.31%, v/v, total antioxidant concentration of 235.3 ± 9.15 mg/L AAE (Ascorbic acid equivalent), and sensory acceptance of 7.74 ± 0.34 was produced at an optimized temperature of 30 °C, pH of 3.9, and inoculum concentration of 16%. The developed models could predict the quality of wine developed from cactus pear fruit.
... The cheapest and easily available source for the production of bioethanol is fruit sugar. It is a potential energy source, from which ethanol can be obtained [8,9]. Approximately 80% of world supply of alcohol is produced by fermentation of sugar and starch containing crops or byproducts from industries based on such crops. ...
... Among the widely used substrates for ethanol production are the molasses of sugarcane and sugar beet. Several studies have shown that sugar based ethanol reduces greenhouse gases by 86 to 90% [6,8]. Sugar-based bioethanol is a simple process and requires one step less than starch-bioethanol, this is because they are ready for conversion with limited pre-treatments as compared with starchy or cellulosic material. ...
... The carrier gas was helium, and the flow rate was 25 ml/min. The oven and detector temperature were 300C [45]. The measurements were carried out at the Egyptian Petroleum Research Institute (EPRI) in Cairo, Egypt. ...
Article
Full-text available
Bioethanol has been classified as the most widely utilized biofuel globally because it helps greatly decrease crude oil consumption and pollution. In this study, bioethanol production improved by 3.6-fold after optimization conditions for commercial Saccharomyces cerevisiae on hydrolysate obtained from enzymatic saccharification of Aspergillus niger to 1% NaOH pretreated wheat straw. 26.0% bioethanol was obtained after 96 h at 30 °C using 10% (W/V) inoculum size of Saccharomyces cerevisiae at pH 5.0 and 2% molasses additives under static condition. After optimization, bioethanol was produced on a large scale, and distillation was carried out, then bioethanol was characterized using Gas chromatography (GC) analysis and ¹H NMR. On large-scale production, one kilogram NaOH pretreated wheat straw was fermented with Aspergillus niger to produce 10 L of hydrolysate that concentrated to 4 L using a rotary evaporator. After concentration, reducing sugar became 35.08 mg/ml, then 2% molasses were added, and the final sugar concentration became 41.7 mg/ml. Finally, reducing sugar was fermented by Saccharomyces cerevesiae to produce one liter of bioethanol. In addition, the obtained bioethanol was blended by the commercial diesel#1/WCO biodiesel commixture with 10% and 20% by volume. The blends of 50%diesel/50%biodiesel, 10% bioethanol/45%diesel/45%biodiesel, and 20%bio ethanol/40%diesel/40%biodiesel were tested as new fuel blends in a single cylinder air-cooled direct injection diesel engine. The engine performance and emission have been recorded at different engine loads and fixed speeds of 1500 rpm. The obtained results reveal that the engine BTE has been enhanced where the engine NOx was reduced if 10% of bioethanol has been added. While increasing bioethanol to 20% by volume base increases the combustion of unburned hydrocarbon and CO emission.
... This result is similar with Sandesh Babu et al. (2014), [10] reported that after two week they obtained 520 µg/ml and 470µg/ml of ethanol from 100 mL of fruit juices of Sugarcane, Grape and then followed by Watermelon and least being the Mousambi after distillation and maintaining a pH of 4 and temperature of 35°C. Starch from starchy crops, such as cereals, to become fermentable needs a pre-treatment composed of three steps: gelatinization, to allow the starch to lose its crystallinity and become an amorphous gel; liquefaction, where starch is hydrolyzed to dextrins by an alfa-amylase and viscosity is reduced; and saccharification, where a gluco-amylase is added to convert dextrins to glucose [11,12]. Saccharification can be managed to be simultaneous to fermentation: this makes the glucose gradually available to microorganisms and reduces contamination risks, process duration, and costs [13,14,15]. ...
Article
Full-text available
Corn (Zea mays L.) is one of the versatile crop which is used as food, feed, fodder and in recent past as a source of bio-fuel. The sub-tropical climate is very favorable for corn cultivation. Traditionally, corn was grown in South and Southeast Asia primarily as a subsistence food crop. Worldwide it is being cultivated in over 170 countries representing an area of 185 million ha with a productivity of 5.62 t ha-1 (FAO, 2017). Out of world corn production of 1037 million MT, SAARC countries comprising of Afghanistan, Bangladesh, Bhutan, India, Maldives, Nepal, Pakistan and Sri Lanka represent 3.2 % with a productivity of 3.8 t ha-1. Among SAARC countries, the highest productivity of 6.9 t ha-1 is reported in Bangladesh. Corn can be an important renewable source for bioethanol production. This research was carried out to evaluate Bangladeshi Corn for optimum bioethanol production. A 100 g of corn flour was mixed with 300 ml distilled water was blended and sterilized. The experiment was conducted with a temperature of 30 oC, pH 6.0 and 20 % sugar concentration. For alcoholic fermentation, 200 ml yeast (Saccharomyces cerevisiae CCD) was added to make the total volume 500 ml. Addition of small amount of 1750 unit α-amylase enzyme to the substrate solution was found to enhance the fermentation process quicker. After 6-days of incubation time corn produces 63.57 ml of ethanol with 13.33 % (v/v) purity. The non-filtered solution produces comparatively more ethanol (63.57 ml with 13.33 % purity) than the filtered solution (53.66 ml with 10 % purity). The purity can be increased by re-distillation process.
... Std, T, Ino.C., L.F.C., Al, TP, and Sens. represents to standard order, temperature, inoculum concentration, Lantana camara fruit concentration, alcohol, total phenol, and sensory, correspondingly. described by Babu et al.[31] using acidified dichromate solution. The distillation process was carried according to the method used by Zenebe et al.[16]. ...
Article
Full-text available
Fermenting blended fruits has been used to improve fruit wine quality. Cactus pear and Lantana camara fruits have well-known nutritive and health benefits. The purpose of this study was to investigate cactus wine quality improvement by applying response surface optimization method of cactus pear and Lantana camara fruits juice fermentation process. Wine quality responses were optimized at an experimental strategy developed using central composite rotatory design by varying fermentation process variable temperature, inoculum, and Lantana camara fruit juice concentration for six days. The developed fermentation models were significant ( p<0.01 ) to predict alcohol, total phenol content, and sensory property of the final wine accurately. From the statistics calculations, fermentation temperature of 24.8°C, inoculum concentration 10.16% ( v/v ), and Lantana camara fruit juice concentration of 10.66% ( v/v ) were the overall optimum values to produce cactus pear fruit wine with alcohol 9.53±0.84% ( v/v ), total phenol content 651.6±54 (mg L ⁻¹ equivalent to gallic acid), and sensory value of 8.83±0.29 . The Lantana camara fruit juice concentration added had shown significant ( p<0.05 ) enhancement on total phenol content and sensory values of the final wine. The results can be used for large-scale wine production in order to reduce its postharvest losses.
... In each wine sample, the ethanol concentration (%, v/v) was determined using acidified dichromate solution according to the procedure described by Babu et al. [30]. Each wine sample was distilled according to the method used by Zenebe et al. [25]. ...
Article
Full-text available
Blending different fruits as well as adding medicinal herbs improves important physicochemical and sensorial properties of fruit wine. The present study aimed at investigating prominent physicochemical and sensory properties of wine produced from cactus pear and Lantana camara fruit juice blend. Both fruit juices were characterized based on pH, sugar, titratable acidity, total phenol, and organic acid contents. The fermentation process was made at previously optimized fermentation temperature of 24.8°C, pH of 3.4, inoculum concentration ( Saccharomyces cerevisiae ) of 10.16% (v/v), and Lantana camara fruit juice concentration of 10.66% (v/v). The final wine was characterized as having pH of 3.47 ± 0.04, 4.6 ± 0.02 g/L sugar equivalent to dextrose, 0.33 ± 0.006% titratable acidity (w/v citric acid), total phenol of 696.1 ± 22.1 mg/L equivalent to gallic acid, and 4.35 ± 0.4 mg/mL organic acid equivalent to citric acid composition. Predominant color intensity, ethanol, methanol, total sulfite, and sensory value of the final wine were measured as 48.07 ± 2.66% of yellowish color, 8.6 ± 0.68% (v/v), 124.4 ± 9.5 mg/L, 129.94 ± 4.04 mg/L, and 8.65 ± 0.92, respectively. The blended Lantana camara fruit enhanced total phenol, color, and sensory value of the final wine. Titratable acidity and methanol and sulfite contents of the final wine are in an acceptable limit compared to standards for commercial wines. Utilizing cactus pear fruit by incorporating Lantana camara fruit for health-enhancing functional food development such as fruit wines could solve the current postharvest loss of both fruits and be a means of alternative beverage.
... Substrate concentration is highly correlated to the reducing sugar content that has great influence on the fermentation process and microbials cell. 59 The relationship of substrate variation and bioethanol yield was tabulated in Table 5. During the fermentation process, the condition of insufficient substrate concentration should be avoided if the bioethanol production is desired to be optimized. ...
Article
Full-text available
Recently, the production of bioethanol is shifted to secondary bioethanol which is produced from nonedible lignocellulosic feedstock to avoid the food versus fuel issue. Mango leaves, a kind of nonedible lignocellulosic material (LCM) that possess a relatively 80.7% of holocellulose (inclusive of cellulose and hemicellulose), appear to be potential candidate to serve as cheap substrate source for bioethanol production. Hence, the objective of this article is to present the current scenario and the potential of mango leaves as a substrate source for bioethanol production. This article also provides an overview on various process parameters such as temperature, pH, substrate concentration, and incubation time that required to be optimized for an efficient fermentation process in the bioethanol production from LCM. Apart from that, several integrated fermentation technologies in bioethanol production which include separate hydrolysis and fermentation, simultaneous saccharification and fermentation, simultaneous saccharification and co‐fermentation, and consolidated bioprocessing will also be discussed in this article. Based on the findings, it is clear that mango leaves have the potential to serve as feedstock for bioethanol production.
... Resultantly, bioethanol productions have been described from diverse waste resources such as market vegetable waste, carrot discard, hydrolyzed agricultural wastes, banana peels, and pulp and peels of mango. [8][9][10][11][12][13]. ...
Chapter
Full-text available
Ground is being prepared all over the world for installation of biofuel plants which can govern the sustainable supply of cleaner fuels at affordable prices and predictable amounts. At the dawn of this century biofuels identified low cost feedstocks, their diverse pretreatments, different methods of saccharifications and fermentations and those for cultivation of biodiesel yielding organisms. Bioalcohols, biohydrogen and biogas represent the biofuels which are derived from microbial work on the biowaste-resources. Extensions in this sector have focused the solar energy captured by the microalgae from which oils can be extracted for biodiesel. Undoubtedly, all forms of available energies on this planet earth had/have been derived, directly or indirectly, from the solar inputs. In this chapter pivotal role of solar insolation will be discussed albeit for regeneration as well as processing of lignocellulosic biomass for obtaining biofuels. Conclusively, biofuels’ sustainable supplies, role of solar energy has been dreamt at various steps of the process; from the collection of biowaste resources through steps of pretreatment, saccharification / fermentation and purification of the product. This chapter discusses the subject matter into two major sub-headings: 1) Biofuels from lignocellulosic / food industrial wastes and 2) Cultivation of microbes for biodiesel.
Article
Full-text available
Bioethanol production from renewable sources to be used in transportation is now an increasing demand worldwide due to continuous depletion of fossil fuels, economic and political crises, and growing concern on environmental safety. Mainly, three types of raw materials, that is, sugar juice, starchy crops, and lignocellulosic materials, are being used for this purpose. This paper will investigate ethanol production from free sugar containing juices obtained from some energy crops such as sugarcane, sugar beet, and sweet sorghum that are the most attractive choice because of their cost-effectiveness and feasibility to use. Three types of fermentation process (batch, fed-batch, and continuous) are employed in ethanol production from these sugar juices. The most common microorganism used in fermentation from its history is the yeast, especially, Saccharomyces cerevisiae, though the bacterial species Zymomonas mobilis is also potentially used nowadays for this purpose. A number of factors related to the fermentation greatly influences the process and their optimization is the key point for efficient ethanol production from these feedstocks.
Article
Full-text available
Bioethanol production from renewable sources to be used in transportation is now an increasing demand worldwide due to continuous depletion of fossil fuels, economic and political crises, and growing concern on environmental safety. Mainly, three types of raw materials, that is, sugar juice, starchy crops, and lignocellulosic materials, are being used for this purpose. This paper will investigate ethanol production from free sugar containing juices obtained from some energy crops such as sugarcane, sugar beet, and sweet sorghum that are the most attractive choice because of their cost-effectiveness and feasibility to use. Three types of fermentation process (batch, fed-batch, and continuous) are employed in ethanol production from these sugar juices. The most common microorganism used in fermentation from its history is the yeast, especially, Saccharomyces cerevisiae, though the bacterial species Zymomonas mobilis is also potentially used nowadays for this purpose. A number of factors related to the fermentation greatly influences the process and their optimization is the key point for efficient ethanol production from these feedstocks.
Article
Full-text available
Contemporary industrial developments and rapid pace of urbanization have called for an environ-mentally sustainable energy sources. Ethanol made from biomass provides unique environmental, economic strategic benefits and can be considered as a safe and cleanest liquid fuel alternative to fossil fuels. There is a copious amount of lignocellulosic biomass worldwide that can be exploited for fuel ethanol production. Significant advances have been made at bench scale towards the fuel ethanol generation from lignocellulosics. However there are still technical and economical hurdles, which make the bioethanol program unsuccessful at commercial scale. This review provides a broad overview on current status of bioethanol production technologies in terms of their economic and environmental viability. These technologies include pretreatment of biomass, the use of cellulolytic enzymes for depolymerisation of carbohydrate polymers into fermentable constituents and the use of robust fermentative microorganisms for ethanol production. Among all the available technologies, dilute acid hydrolysis followed by enzymatic hydrolysis by less expensive and more efficient cellulases has been found more promising towards the potential economics and environmental impact.
Article
Full-text available
Strains of Saccharomyces spp. from tropical substrates tolerated temperatures up to 40 C, sucrose concentrations up to 50% (w/v) and ethanol concentrations up to 20 g/L in fermentation conditions. Strain TD200 tolerated 20 g/l of ethanol. The ethanol produced by strain DR1459 was comparable to that of industrial strain HTYM-81. These strains have potential use for the production of fuel alcohol.
Article
The effect of pretreatment of molasses with H2SO4 and K4Fe(CN)6 on ethanol production by different yeast strains was studied in order to find an effective method to reduce the load of various inhibitory substances and to select a suitable yeast strain for fermentation of pretreated molasses. Pretreatment resulted in decreased level of inhibitory substances like Ca, Cu, Fe in the molasses solution with improved ethanol production. Strain 20 was best among the tested strains with all pretreatments. The inhibitory effect of these constituents was confirmed by supplementation of synthetic medium with residues from different pretreatments and the inhibitory level for various constituents was found to be Ca >0.5%, iron >46 ppm and Cu >5.4 ppm.
Article
The fermentability of blackstrap sugarcane molasses was examined under very high gravity (VHG) conditions. Molasses fermentations were carried out over the range of 10.4-47.6% (w/v) dissolved solids. As the concentration of dissolved solids increased, the percentage of sugar actually converted to ethanol decreased. The suitability of molasses as a carbohydrate adjunct for VHG ethanolic fermentation was also studied; molasses was used to raise the dissolved solids content of both clarified wheat mash base and sugarcane juice to VHG levels. Fermentation of such mashes was 90-93% efficient. In VHG wheat mashes prepared with molasses adjunct, yeast extract accelerated the rate of fermentation but had little effect on the final ethanol concentration. Sugarcane juice was not limiting in assimilable nitrogen since yeast extract or urea failed to stimulate the rate of fermentation of cane juice/molasses worts or to increase the final ethanol concentration achieved. This is the first report of the application of VHG technology to fermentation substrates other than wheat, wort, and grape juice. It is concluded that VHG fermentation of saccharine substrates could lead to moderate increases in alcohol concentration as compared to those presently achieved in industry.
Article
An attempt was made to improve the salt tolerance of the thermotolerant flocculating yeast Saccharomyces cerevisiae strain KF-7 (KF-7) by maintaining a high concentration of KCl in the medium. Among selected strains, K211 had the highest cell viability and ethanol productivity in a molasses medium containing 25% (w/v) total sugar at 35°C. Strain K211 accumulated higher concentrations of glycogen and trehalose than did KF-7, and also remained alive in the stationary phase during batch fermentation in a jar fermentor. As a result of repeated-batch fermentation tests with K211, stable ethanol production was achieved with an ethanol concentration of 92 g/l and a productivity of 3.5 g/l·h at 33°C in 22% molasses medium. With KF-7, stable ethanol production could not be attained under these conditions. In addition, even at the higher temperature of 35°C, strain K211 gave stable ethanol production with an ethanol concentration of 91 g/l and a productivity of 2.7 g/l·h.
Article
The use of molasses as a substrate for ethanol production by the thermotolerant yeast Kluyveromyces marxianus var. marxianus was investigated at 45 degrees C. A maximum ethanol concentration of 7.4% (v/v) was produced from unsupplemented molasses at a concentration of 23% (v/v). The effect on ethanol production of increasing the sucrose concentration in 23% (v/v) molasses was determined. Increased sucrose concentration had a similar detrimental effect on the final ethanol produced as the increase in molasses concentration. This indicated that the effect may be due to increased osmotic activity as opposed to other components in the molasses. The optimum concentration of the supplements nitrogen, magnesium, potassium and fatty acid for maximum ethanol production rate was determined using the Nelder and Mead (Computer J 7:308-313, 1965) simplex optimisation method. The optimum concentration of the supplements were 0.576 g1(-1) magnesium sulphate, 0.288 g1(-1) potassium dihydrogen phosphate and 0.36% (v/v) linseed oil. Added nitrogen in the form of ammonium sulphate did not affect the ethanol production rate.
Article
Marcha or murcha is a traditional amylolytic starter used to produce sweet-sour alcoholic drinks, commonly called jaanr in the Himalayan regions of India, Nepal, Bhutan, and Tibet (China). The aim of this study was to examine the microflora of marcha collected from Sikkim in India, focusing on yeast flora and their roles. Twenty yeast strains were isolated from six samples of marcha and identified by genetic and phenotypic methods. They were first classified into four groups (Group I, II, III, and IV) based on physiological features using an API test. Phylogenetic, morphological, and physiological characterization identified the isolates as Saccharomyces bayanus (Group I); Candida glabrata (Group II); Pichia anomala (Group III); and Saccharomycopsis fibuligera, Saccharomycopsis capsularis, and Pichia burtonii (Group IV). Among them, the Group I, II, and III strains produced ethanol. The isolates of Group IV had high amylolytic activity. Because all marcha samples tested contained both starch degraders and ethanol producers, it was hypothesized that all four groups of yeast (Group I, II, III, and IV) contribute to starch-based alcohol fermentation.