Abstract

Interest in the production of bioethanol from inedible biomass is growing worldwide because of its sustainable supply and lack of competition with food supplies. Candida krusei (also known as Pichia kudriavzevii or Issatchenkia orientalis) is one of the most suitable thermotolerant yeasts used in the simultaneous saccharification and fermentation process for bioethanol production. In the production of bioethanol from lignocellulosic biomass as a feedstock, various environmental conditions occur, and the stress tolerance capacity of C. krusei, especially its low pH and tolerance to inhibitors, limits its practical application. In this study, to select a suitable second-generation bioethanol-producing strain, the tolerance capacity of five available C. krusei strains (NBRC0584, NBRC0841, NBRC1162, NBRC1395 and NBRC1664) was characterized. Spot assay and growth experiment results showed that among the five C. krusei strains, C. krusei NBRC1664 showed superior tolerance capacity for low pH and inhibitors. Furthermore, this strain efficiently produced ethanol from glucose under low pH conditions with or without sulfate. A comparative analysis of the draft genome sequences suggested that Opy2, Sln1 and Cdc24 in the HOG pathway are conserved only in C. krusei NBRC1664, which may contribute to its superior tolerance to low pH levels. Moreover, amino acid sequence alignment showed that aldehyde dehydrogenase family proteins, which catalyze the degradation of cyclic aldehydes, are commonly conserved in C. krusei. In addition, the increased transcription levels in C. krusei NBRC1664 could play a role in its higher tolerance to inhibitors. These results suggest that C. krusei NBRC1664 is a more suitable strain for application in industrial processes for second-generation bioethanol production.