Huynh Xuan Phong * , Pornthap Thanonkeo , Mamoru Yamada , Nguyen Tran Cam Giang , Sukanya Nitiyon and Ngo Thi Phuong Dung

* Correspondence: Huynh Xuan Phong (email:

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Thermotolerant ethanologenic yeasts have highly attracted many scientists due to the current challenges of increasing global temperature and the benefits associated with processing at high temperature as well as reducing cooling cost. In this study, 50 yeast strains were isolated from 35 different materials of cocoa. Based on characteristics of morphology, physiology and biochemistry, 50 yeast isolates were preliminarily classified into genera as Candida, Hanseniaspora, Kluyveromyces, Pichia, and Saccharomyces. There were 17/50 isolates showing their ethanol tolerant ability up to 12% (v/v) of ethanol and 23/50 isolates could grow at 45oC. The two selected isolates (CT2.5D and PD1.6H) were tested for the ethanol fermentation from molasses (22°Brix) at different temperatures. The results of ethanol concentrations obtained as follow: 7.36% (v/v) at 30oC, 4.15% (v/v) at 40oC, 1.45% (v/v) at 42oC by CT2.5D and 7.4% (v/v) at 30oC, 2.93% (v/v) at 40oC, 1.43% (v/v) at 42oC by PD1.6H. The identification results showed that CT2.5D and PD1.6H were Saccharomyces cerevisiae.
Keywords: cocoa, ethanol fermentation, ethanol tolerance, Saccharomyces cerevisiae, thermotolerant yeast

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Abdel-Banat, B.A., Hoshida, H., Ano, A., Nonklang, S., Akada, R., 2010. High-temperature fermentation: how can processes for ethanol production at high temperatures become superior to the traditional process using mesophilic yeast? Applied Microbiology and Biotechnology. 85: 861-867.

Addel-Fattah, W.R., Fadil, M., Nigam, P., Banat, I.M., 2000. Isolation of thermotolerant ethanologenic yeast and use of selected strains in industrial scale fermentation in an Egyptian distillery. Biotechnology Bioengineering. 68(7): 531-535.

Ardhana, M., Fleet, G.H., 2003. The microbial ecology of cocoa bean fermentations in Indonesia. International Journal of Food Microbiology. 86: 87-99.

Banat, I.M., Nigam, P., Singh, D., Marchant, R., McHale, A.P., 1998. Review: Ethanol production at elevated temperatures and alcohol concentrations: Part I - Yeasts in general. World Journal of Microbiology and Biotechnology. 14: 809-821.

Barnett, J.A., Payne, R.W., Yarrow, D., 1983. Yeast: Characteristics and Identification. Cambridge University Press, Cambridge.

Bruce, W.Z., Kenneth, C.F., Barry, H.G., Fred, S.N., 1995. Wine Analysis and Production. Chapman and Hall, New York.

D'Amore, T., Panchal, C.J., Russell, I., Stewart, G.G., 1990. A study of ethanol tolerance in yeast. Critical Reviews in Biotechnology. 9(4): 287-304.

Dung, N.T.P., Thanh, N.N., Phong, H.X., 2013. Microflora composition and isolation of microorganisms in cocoa fermentation. Can Tho University Journal of Science. 25: 271-280 (in Vietnamese).

Kurtzman, P.C., Fell, J.W., Boekhout, T., Robert, V., 2011. Methods for isolation, phenotypic characterization and maintenance of yeast. In “The Yeast, a Taxonomic Study” Vol 1, 5th ed., Elsevier, Amsterdam.

Limtong, S., Sringiew, C., Yongmanitchai, W., 2007. Production of fuel ethanol at high temperature from sugar cane juice by a newly isolated Kluyveromyces marxianus. Bioresource Technology. 98: 3367-3374.

Lin, Y., Zhang, W., Li, C., Sakakibara, K., Tanaka, S., Kong, H., 2012. Factors affecting ethanol fermentation using Saccharomyces cerevisiae BY4742. Biomass and Bioenergy. 47: 395-401.

Pham, L.D., 2006. Industrial Yeasts. Science and Technology Publishing House, Hanoi, Vietnam (in Vietnamese).

Neiman, A.M., 2005. Ascospore formation in the yeast Saccharomyces cerevisiae. Microbiology and Molecular Biology Reviews. 69(4): 565-584.

Nielsen, D.S., Teniola, O.D., Ban-Koffi, L., Owusu, M., Andersson, T.S., Holzapfel, W.H., 2007. The microbiology of Ghanaian cocoa fermentations analysed using culture-dependent and culture-independent methods. International Journal of Food Microbiology. 114 (2): 168-186.

O’Donnell, K., 1993. Fusarium and its near relatives. In: Reynolds D.R. & Taylor J.W. (Eds) “The Fungal Holomorph: Mitotic, Meiotic and Pleomorphic Speciation in Fungal Systematics”. CAB International, Wallingford.

Periyasamy, S., Venkatachalam, S., Ramasamy, S., Srinivasan, V., 2009. Production of bio-ethanol from sugar molasses using Saccharomyces cerevisiae. Modern Applied Science. 3(8): 32-37.

Roehr, M. 2001. The Biotechnology of Ethanol: Classical and Future Applications. 1st edition, Wiley-VHC, Weinheim.

Schwan, R.F., Wheals, A.E., 2004. The microbiology of cocoa fermentation and its role in chocolate quality. Critical Reviews of Food Science Nutrition. 44: 205-221.

Sree, N.K., Sridhar, M., Suresh, K., Banat, I.M., Venkateswar, L.R., 1999. Isolation of thermotolerant, osmotolerant, flocculating Saccharomyces cerevisiae for ethanol production. Bioresource Technology. 72: 43-46.

Ueno, R., Urano, N., Kimura, S., 2002. Effect of temperature and cell density on fermentation by a thermotolerant aquatic yeast strain isolated from a hot spring environment. Fisheries Science. 68: 571-578.