ISOLATION OF SACCHAROMYCES CEREVISIAE FROM PINEAPPLE AND ORANGE AND STUDY OF METAL’S EFFECTIVENESS ON ETHANOL PRODUCTION

Armanul Naser†, Shafkat Shamim Rahman*†, Md. Mahboob Hossain, Naiyyum ChoudhuryDepartment of Mathematics and Natural Sciences, BRAC University, Bangladesh

*Corresponding author; †Equal contributors

IntroductionThe present study describes the search for potential yeast isolates from fruit peels capable of producing bioethanol, which has a great deal of interest as an alternative to traditional fossil fuels in recent years.

MethodsThe main objective of the work was to isolate & characterize stress tolerant, high potential ethanol producing yeast strains. Two isolates from Pineapple (Pa) and Orange (Or) have been isolated, characterized on the basis of morphological and physic-chemical characters and optimized on ethanol producing capability using sugarcane molasses as substrate. Fermentation was estimated by Conway method.• Isolation and screening of stress tolerance• Fermentation of carbohydrates• Detection of thermotolerance • Detection of ethanoltolerance • Growth at different pH in Liquid Media• Osmotolerance observation• Antibiotic (Chloramphenicol and Nalidixic acid) resistance test• Fermentation of molasses• Effect of sugar concentration• Effect of pH• Estimation of reducing sugars• Effect of Metals• Effect of Agitation

ResultsIsolates were stress (thermo-, pH-, ethanol- and osmo-) tolerant. They were resistant to Chloramphenicol (30µg/disc) and Nalidixic acid (30µg/disc). No killer toxin activity was observed against E. coli. The highest production capacity was found to be 7.39% and 5.02% for Pa & Or respectively at pH 5.0, 30oC temperature in media with, initial reducing sugar concentration 6.5% for Pa and 5.5% for Or isolate in shaking condition. Addition of metal ions increased the rate of ethanol production highest to 10.61%.

• The morphology of the vegetative cells of yeast was observed grown in liquid and on solid media.

• The strain utilized glucose, sucrose, maltose, dextrose, fructose, xylose and trehalose but failed to grow on lactose.

• Both yeast isolates are highly thermotolerant as Or is able to grow up to 40°C and Pa able to grow up to 44°C.

• Or isolate can grow in up to 12% and Pa isolate can grow in up to 15% ethanol containing liquid YEPD media.

• Both Pa and Or isolates can grow in a wide range of pH from 2 to 10, both isolates withstand pH 10.

• Pa yeast isolate showed better tolerance than the Or (12%) yeast isolate at up to 15% sodium chloride equivalent of osmotic pressure.

• No clear zone of inhibition by Yeast was observed against E. coli in Killer toxin test.

• Antibiotic resistance test showed that both the strains Pa and Or were resistant to Chloramphenicol and Nalidixic acid.

• Pa isolate produced 7.39% ethanol with 6.5% sugar conc., 5.0 pH (shaking), after 48 hrs molasses fermentation.

• Under shaking condition (130 rpm), at 30°C temperature using initial reducing sugar conc. of the fermentation media 7.5% and pH 6.0, maximum ethanol production was 7.02% after 96 hrs.

• Highest ethanol production of 10.61% was observed by Pa with KH2PO4 supplements in shaking condition. Or isolate showed highest of 8.04% by MnCl2 supplements in the same condition.

ConclusionsThis study revealed that indigenous yeast isolates could be used to benefit the fuel ethanol, spirit and industrial alcohol industries.

Metals Pa Ethanol % (48 h) Or Ethanol % (48h)MnCl2 9.50 8.04CuSO4 7.58 5.85KH2PO4 10.61 7.98ZnSO4 9.48 7.52

Production of ethanol by Pa & Or at 30°C temp in presence of metals (shaking condition 120 rpm).

Glucose conc. Orange (Shaking) Orange (Non-shaking)

Pineapple (Shaking) Pineapple (Non-shaking)

4.5% 2.03 0.44 3.73 2.275.5% 5.02 2.63 7.22 6.296 % 4.83 4.1 5.19 3.736.5% 3 2.27 7.39 37% 3.73 3.37 5.93 3.377.5% 2.63 3.37 5.56 2.63Alcohol production comparison at different parameters (48-hours)

Ethanol production, pH and sugar depletion by Pa isolate at initial 7.5% sugar conc.