A sequential hydrothermal liquefaction (SEQHTL) process was evaluated in this work for fractionating different component of yeast biomass. Sugar and protein were separated first at a lower temperature, and the remaining biomass was then converted to bio-oil at a higher temperature. The separated aqueous products were investigated to be recycled as a carbon and nitrogen sources for the yeast culture. In the first step of SEQHTL, the temperature effect on the yield of sugar/protein and inhibitory compounds (acetic acid and 5-hydroxymethyl furfural (5HMF)) was investigated. The highest yields of sugar and protein and a minimal level of inhibitory compounds were obtained at 180 degrees C. At the second step of SEQHTL, the highest bio-oil yield was achieved at 240 degrees C. In comparison to the one-step hydrothermal liquefaction process, SEQHTL produced a higher quality bio-oil with higher fatty acid and lower nitrogen contents. (C) 2014 Elsevier Ltd. All rights reserved.