In this study, a novel two-stage hydrothermal liquefaction (HTL) process was employed to produce low-nitrogen bio-crude oils from microalgae, involving pre-treatment of the microalgae with a pre-cooled NaOH/urea solution or a dilute acid and HTL of the pre-treated algal feedstock at 250 degrees C for 10-50 min. The results indicated that the pre-treatment with a pre-cooled NaOH/urea solution effectively removed carbohydrates and protein from the raw microalgae, leading to a decrease in carbohydrates and protein content by 12 wt% and 10 wt% (both absolute values), respectively, while retaining 70 wt% of the solid mass, corresponding to as high as 82% carbohydrates removal efficiency and 40% protein removal efficiency. The two-stage HTL process slightly increased the overall bio-crude oil yields relative to the conventional single-stage HTL process, and the bio-crude oils obtained from the two-stage HTL process have a better quality than those obtained from the single-stage HTL, in terms of lower nitrogen and oxygen levels and higher energy content. (C) 2017 Published by Elsevier Ltd.