Production of Bioethanol from Different Substrates with Yeast Isolated from Various Sources


Aditya Shah, Sombuddha Sengupta, Tamoghna Das, Shampa Sen*

Department of Biotechnology, VIT University Vellore, Tanilnadu-632014 India

*Corresponding Author E-mail:



Fossil fuels have always been considered as a non-renewable, finite resource of energy. However due to its detrimental effects on the environment, recent research and development has been aimed at finding and improving the production of alternative fuels from other sources. Bioethanol has emerged as one of the most successful product of this endeavour. This study aims to analyze and compare the production of bioethanol by the yeast fermentation process from different substrates which are easy to obtain and economically viable in the Indian subcontinent. The yeast (Saccharomyces cerevisiae) used for fermentation was also isolated from easily available sources. There were four substrates used: Sugarcane juice, Pineapple juice, Grape juice and Sapodilla juice. To isolate the yeast, three sources were used: Sugarcane, Apple and Sweet lime. Bioethanol was separated by fractional distillation and yield was measured using a Hydrometer.


KEYWORDS: Bioethanol, yeast, fermentation, glucose, distillation





During the past decade, there has been a tremendous rise in the pressure on fossil fuels. This poses a threat to their availability in the future. Also, this excessive usage of fossil fuel has resulted in the emission of many greenhouse gases like carbon dioxide and has led to global warming. Thus, an alternative source of energy must be found which is economically and environmentally viable. Biofuel is a good option for such a sustainable source of energy1-2. Bioethanol can be used as a petroleum additive and alternative. It has low emissions, lower octane number and is easy to obtain3. Bioethanol is produced from sugar containing biomass which is subjected to fermentation. Generally, baker’s yeast (Saccharomyces cerevisiae) is used for fermentation. The conditions are kept anaerobic and the sugars are converted to ethanol and carbon dioxide4.


Bioethanol is generally produced from sugar containing crops like sugarcane, grapes and agricultural wastes. India being an agricultural country provides ideal platform for Bioethanol production5. Thus, in this research, bioethanol was produced from different fruit juices readily available and economically viable along with yeast isolated from various sources.



Isolation of Yeast:

Yeast strains were isolated from 3 sources: Sugarcane juice, Apple juice and Sweet lime juice. A quantity of 10mL of each type of freshly prepared juice was taken and kept for 36 hours. From the 10mL, 1ml of juice was taken and serial diluted up to a dilution factor of 10-7 in distilled water. From the serially diluted sample, 0.1ml taken and cultured on YEPD agar media (composition of media: 5g of Yeast Extract Potato Dextrose and 2g of Agar in 100ml distilled water) using the spread plate technique in petri plates. The plates were incubated at 35oC for 36 hours. The colonies obtained were further sub cultured on fresh media by streak plate method. The plates were kept for incubation at 35oC for 36 hours to get isolated colonies. A single isolated colony from each source was taken and inoculated into 20ml of YEPD broth (5g of Extract Potato Dextrose in 100ml distilled water) in sealed test tubes. The test tubes were kept for 48 hours in incubation at 35oC to obtain 3 mother inoculums respective of the 3 different sources. From the 3 mother inoculums, 1ml was taken and further inoculated in 3 flasks containing 50ml of YEPD broth. The flasks were kept 48 hours for incubation at 35oC and 120 rpm to obtain the inoculums further used for fermentation6-7.



4 substrates were used for fermentation: Sugarcane juice, Pineapple juice, Grape juice and Sapodilla juice. 4 air tight flasks of 150ml were prepared for each type of freshly prepared substrate.  Out of the 4 flasks, 1 was kept as control and the rest 3 were inoculated with 10% by volume of yeast inoculums from the 3 different sources. Each flasks contained 150ml of substrate. The fermentation was done for 72 hours at a pH of 4.5-5 at 35ºC8-9. The initial sugar content of the juices was measured using Brix meter. The sugar content of Grape, Sugarcane, Pineapple and Sapodilla were found to be 19ºBx 1611ºBx and 15ºBx respectively10.



The fermented liquid was then distilled at 85o C and the distillate was used to measure the yield of ethanol11.


Measurement of Ethanol yield:

The distillate was used to measure the content of ethanol in the juice. A standard Hydrometer was used to calculate the volume percentage of ethanol12.



The yield of Ethanol in all 16 treatments was measured and tabulated. The yield was measured in grams per liter and then the percentage yield was calculated and tabulated.




Table 1: Yield of Ethanol from different treatments in grams per liter.


Control (g/L)

Inoculated with yeast isolated from Apple (g/L)

Inoculated with yeast isolated from Sugarcane  (g/L)

Inoculated with yeast isolated from Sweet lime (g/L)


Sugarcane Juice






Grape Juice






Sapodilla Juice






Pineapple Juice







Fig 1: Yield of Ethanol from different treatments in grams per liter




Table 2: Percentage yield of Ethanol in different treatments



Inoculated with yeast isolated from Apple

Inoculated with yeast isolated from Sugarcane

Inoculated with yeast isolated from Sweet lime

Sugarcane Juice





Grape Juice





Sapodilla Juice





Pineapple Juice






The average ethanol content as shown in Figure 1 was highest in Grape juice followed by Sugarcane, Sapodilla and Pineapple juice. The average ethanol content was in accordance with the total sugar content of the juices. Higher the sugar content, higher the average ethanol content.


The average percentage yield was greater in fruits containing sugars in simpler forms. The highest percentage yield of Ethanol was found in Grape juice followed by Sugarcane, Pineapple and Sapodilla. The yield and ethanol content of control treatments were significantly less than that in which yeast was inoculated. The source from which yeast is isolated does not have a significant impact on the percentage yield or ethanol content.



Thus, it can be concluded that bioethanol can be successfully made from raw materials easily and economically available. The production of bioethanol is viable regardless the source of yeast. From the substrates used in this study, Grape and Sugarcane are the most appropriate sources for bioethanol. With further research in optimization of conditions for  enzymatic production and ethanol tolerance, bioethanol can be firmly adopted as an alternative source of fuel and a fuel additive.



The authors are grateful to authorities of VIT University, Vellore, for the facilities provided to carry out the research work.



The authors declare no conflict of interest.



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Received on 27.02.2017             Modified on 21.03.2017

Accepted on 05.04.2017           © RJPT All right reserved

Research J. Pharm. and Tech. 2017; 10(4): 1120-1122.

DOI: 10.5958/0974-360X.2017.00202.5