Bioconversion from inexpensive renewable resource, such as biomass, to liquid fuel is one of the promising technologies to reduce the use of petroleum. We previously reported the genetically engineered Moorella thermoacetica could produce ethanol from the lignocellulosic feedstock. However, it was still unclear which carbon source in the substrate was preferentially consumed to produce ethanol. To identify the hierarchy of the sugar utilization during ethanol fermentation of this strain, we analyzed the sugar composition of lignocellulosic feedstock, and consumption rate of sugars during the fermentation process. The hydrolysates after acid pretreatment and enzymatic saccharification contained glucose, xylose, galactose, arabinose, and mannose. Time course data suggested that xylose was the most preferred carbon source among those sugars during ethanol fermentation. Ethanol yield was 0.40 +/- 0.06 and 0.40 +/- 0.12 g/g-total sugar, from lignocellulosic hydrolysates of Japanese cedar (Cryptomeria japonica) and rice straw (Oryza sativa), respectively. The results demonstrated that the genetically engineered M. thermoacetica is a promising candidate for thermophilic ethanol fermentation of lignocellulosic feedstocks, especially hemicellulosic sugars. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.