Sweet sorghum juice supplemented with 0.5% ammonium sulphate was used as a substrate for ethanol production by Saccharomyces cerevisiae TISTR 5048. In batch fermentation, kinetic parameters for ethanol production depended on initial cell and sugar concentrations. The optimum initial cell and sugar concentrations in the batch fermentation were 1 · 10 8 cells ml -1 and 24°Bx respectively. At these conditions, ethanol concentration produced (P), yield (Y ps ) and productivity (Q p ) were 100 g l -1 , 0.42 g g -1 and 1.67 g l -1 h -1 respectively. In fed-batch fermentation, the optimum substrate feeding strategy for ethanol production at the initial sugar concentration of 24°Bx was one-time substrate feeding, where P, Y ps and Q p were 120 g l -1 , 0.48 g g -1 and 1.11 g l -1 h -1 respectively. These findings suggest that fed-batch fermentation improves the efficiency of ethanol production in terms of ethanol concentration and product yield.

Ethanol production at elevated temperatures requires high potential thermotolerant ethanol-producing yeast. In this study, nine isolates of thermotolerant yeasts capable of growth and ethanol production at high temperatures were successfully isolated. Among these isolates, the newly isolated thermotolerant yeast strain, which was designated as Saccharomyces cerevisiae DBKKU Y-53, exhibited great potential for ethanol production from sweet sorghum juice (SSJ) at high temperatures. The maximum ethanol concentrations produced by this newly isolated thermotolerant yeast at 37˝C and 40˝C under the optimum cultural condition were 106.82 g¨L´1 and 85.01 g¨L´1, respectively, which are greater than values reported in the literatures. It should be noted from this study with SSJ at a sugar concentration of 250 g¨L´1 and an initial pH of 5.5 without nitrogen supplementation can be used directly as substrate for ethanol production at high temperatures by thermotolerant yeast S. cerevisiae DBKKU Y-53. Gene expression analysis using real-time RT-PCR clearly indicated that growth and ethanol fermentation activities of the thermotolerant yeast S. cerevisiae DBKKU Y-53 at a high temperature (40˝C) were not only restricted to the expression of genes involved in the heat-shock response, but also to those genes involved in ATP production, trehalose and glycogen metabolism, and protein degradation processes were also involved.

High potential, thermotolerant, ethanol-producing yeasts were successfully isolated in this study. Based on molecular identification and phylogenetic analysis, the isolated thermotolerant yeasts were clustered in the genera of Pichia kudriavzevii, Candida tropicalis, Candida orthopsilosis, Candida glabrata and Kodamea ohmeri. A comparative study of ethanol production using 160 g/L glucose as a substrate revealed several yeast strains that could produce high ethanol concentrations at high temperatures. When sugarcane bagasse (SCB) hydrolysate containing 85 g/L glucose was used as a substrate, the yeast strain designated P. kudriavzevii RZ8-1 exhibited the highest ethanol concentrations of 35.51 g/L and 33.84 g/L at 37 °C and 40 °C, respectively. It also exhibited multi-stress tolerance, such as heat, ethanol and acetic acid tolerance. During ethanol fermentation at high temperature (42 °C), genes encoding heat shock proteins (ssq1 and hsp90), alcohol dehydrogenases (adh1, adh2, adh3 and adh4) and glyceraldehyde-3-phosphate dehydrogenase (tdh2) were up-regulated, suggesting that these genes might play a crucial role in the thermotolerance ability of P. kudriavzevii RZ8-1 under heat stress. These findings suggest that the growth and ethanol fermentation activities of this organism under heat stress were restricted to the expression of genes involved not only in heat shock response but also in the ethanol production pathway.

The application of high-potential thermotolerant yeasts is a key factor for successful ethanol production at high temperatures. Two hundred and thirty-four yeast isolates from Greater Mekong Subregion (GMS) countries, i.e., Thailand, The Lao People's Democratic Republic (Lao PDR) and Vietnam were obtained. Five thermotolerant yeasts, designated Saccharomyces cerevisiae KKU-VN8, KKU-VN20, and KKU-VN27, Pichia kudriavzevii KKU-TH33 and P. kudriavzevii KKU-TH43, demonstrated high temperature and ethanol tolerance levels up to 45 °C and 13% (v/v), respectively. All five strains produced higher ethanol concentrations and exhibited greater productivities and yields than the industrial strain S. cerevisiae TISTR5606 during high-temperature fermentation at 40 °C and 43 °C. S. cerevisiae KKU-VN8 demonstrated the best performance for ethanol production from glucose at 37 °C with an ethanol concentration of 72.69 g/L, a productivity of 1.59 g/L/h and a theoretical ethanol yield of 86.27%. The optimal conditions for ethanol production of S. cerevisiae KKU-VN8 from sweet sorghum juice (SSJ) at 40 °C were achieved using the Box–Behnken experimental design (BBD). The maximal ethanol concentration obtained during fermentation was 89.32 g/L, with a productivity of 2.48 g/L/h and a theoretical ethanol yield of 96.32%. Thus, the newly isolated thermotolerant S. cerevisiae KKU-VN8 exhibits a great potential for commercial-scale ethanol production in the future.

The respiratory chain of the ethanologenic bacterium Zymomonas mobilis was investigated, in which the pyruvate-to-ethanol pathway has been demonstrated to be mainly responsible for NADH oxidation and the tricarboxylic acid cycle is incomplete. Membranes from cells cultivated under aerobic or anaerobic growth conditions showed dehydrogenase and oxidase activities for NADH, D-lactate and D-glucose and ubiquinol oxidase activity. Intriguingly, the NADH oxidase activity level of membrane fractions from cells grown aerobically was found to be higher than that of membrane fractions from Escherichia coli or Pseudomonas putida grown aerobically, indicating a crucial role of the respiratory chain in NADH oxidation in the organism. Cyanide-resistant terminal oxidase activity was observed and appeared to be due to a bd-type ubiquinol oxidase as the only terminal oxidase encoded by the entire genome. The terminal oxidase with a relatively strong ubiquinol oxidase activity exhibited remarkably weak signals of cytochrome d. Considering these findings and the presence of a type-II NADH dehydrogenase but not a type-I, a simple respiratory chain that generates less energymay have evolved in Z. mobilis.