The fermentation ability of thermotolerant Kluyveromyces marxianus BUNL-21 isolated in Laos was investigated. Comparison with thermotolerant K. marxianus DMKU3-1042 as one of the most thermotolerant yeasts isolated previously revealed that the strain possesses stronger ability for conversion of xylose to ethanol, resistance to 2-deoxyglucose in the case of pentose, and tolerance to various stresses including high temperature and hydrogen peroxide. K. marxianus BUNL-21 was found to have ethanol fermentation activity from xylose that is slightly lower and much higher than that of Scheffersomyces stipitis (Pichia stipitis) at 30 °C and at higher temperatures, respectively. The lower ethanol production seems to be due to large accumulation of acetic acid. The possible mechanism of acetic acid accumulation is discussed. In addition, it was found that both K. marxianus strains produced ethanol in the presence of 10 mM hydroxymethylfurfural or furfural, at a level almost equivalent to that in their absence. Therefore, K. marxianus BUNL-21 is a highly competent yeast for high-temperature ethanol fermentation with lignocellulosic biomass.

Candida tropicalis, a xylose-fermenting yeast, has the potential for converting cellulosic biomass to ethanol. Thermotolerant C. tropicalis X-17, which was isolated in Laos, was subjected to repetitive long-term cultivation with a gradual increase in temperature (RLCGT) in the presence of a high concentration of glucose, which exposed cells to various stresses in addition to the high concentration of glucose and high temperatures. The resultant adapted strain demonstrated increased tolerance to ethanol, furfural and hydroxymethylfurfural at high temperatures and displayed improvement in fermentation ability at high glucose concentrations and xylose-fermenting ability. Transcriptome analysis revealed the up-regulation of a gene for a glucose transporter of the major facilitator superfamily and genes for stress response and cell wall proteins. Additionally, hydropathy analysis revealed that three genes for putative membrane proteins with multiple membrane-spanning segments were also up-regulated. From these findings, it can be inferred that the up-regulation of genes, including the gene for a glucose transporter, is responsible for the phenotype of the adaptive strain. This study revealed part of the mechanisms of fermentability at high glucose concentrations in C. tropicalis and the results of this study suggest that RLCGT is an effective procedure for improving multistress tolerance.

Thermotolerant ethanologenic yeasts receive attention as alternative bio-ethanol producers to traditionally used yeast, Saccharomyces cerevisiae. Their utilization is expected to provide several benefits for bio-ethanol production due to their characteristics and robustness. They have been isolated from a wide variety of environments in a number of ASEAN countries: Thailand, Vietnam, Laos, and Indonesia. One of these yeasts, Kluyveromyces marxianus has been investigated regarding characteristics. Some strains efficiently utilize xylose, which is a main component of the 2nd generation biomass. In addition, the genetic basis of K. marxianus has been revealed by genomic sequencing and is exploited for further improvement of the strains by thermal adaptation or gene engineering techniques. Moreover, the glucose repression of K. marxianus and its mechanisms has been investigated. Results suggest that K. marxianus is an alternative to S. cerevisiae in next-generation bio-ethanol production industry. Indeed, we have succeeded to apply K. marxianus for bio-ethanol production in a newly developed process, which combines high-temperature fermentation with simultaneous fermentation and distillation under low pressure. This chapter aims to provide valuable information on thermotolerant ethanologenic yeasts and their application, which may direct the economic bioproduction of ethanol and other useful materials in the future.