| Summary: | Yeasts were isolated from four potential sources, sweet sorghum juice, sugar cane juice, grapes and rambutan. The 27 yeast isolates were tested for their ethanol tolerance (15% <i>v</i>/<i>v</i> of ethanol) and ethanol fermentation performance in a synthetic ethanol production medium (200 g/L of total sugar). Only five isolates, SCJ04KKU, SCJ07KKU, SCJ09KKU, SCJ14KKU and SSJ01KKU could tolerate 15% ethanol and produce ethanol at levels higher than 55 g/L. The ethanol production efficiency from sweet sorghum juice under high gravity (HG, 200 and 240 g/L of total sugar) and very high gravity (VHG, 280 g/L of total sugar) conditions of the five isolates was tested. <i>Saccharomyces cerevisiae</i> NP01 and <i>S. cerevisiae</i> ATCC4132 were used as reference strains. The results showed that the SSJ01KKU isolate gave the highest ethanol production efficiency under all conditions. Ethanol concentration (<i>P<sub>E</sub></i>), yield (<i>Y<sub>P/S</sub></i>) and productivity (<i>Q<sub>P</sub></i>) values were 98.89 g/L, 0.50 and 1.18 g/L·h, respectively, with sugar consumption (<i>SC</i>) of 98.96% under the HG condition at 200 g/L of total sugar. Under the HG condition at 240 g/L of total sugar, the <i>P<sub>E</sub></i>, <i>Y<sub>P/S</sub></i> and <i>Q<sub>P</sub></i> values were 118.12 g/L, 0.51 and 1.41 g/L·h, respectively, with the <i>SC</i> of 95.79%. These values were 82.29 g/L, 0.34 and 0.98 g/L·h, respectively, with the <i>SC</i> of 85.59% under the VHG condition. Addition of urea into the sweet sorghum juice under all conditions significantly shortened the fermentation time, resulting in increased <i>Q<sub>P</sub></i> values. Based on molecular taxonomic analysis of the five isolates using sequence analysis of the D1/D2 domain and the ITS1 and ITS2 regions, SSJ01KKU is <i>S. cerevisiae</i>, whereas SCJ04KKU, SCJ07KKU, SCJ09KKU and SCJ14KKU are <i>Pichia caribbica</i>.
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