1. Abstract: The use of renewable energy sources is becoming increasingly necessary to mitigate global warming. Recently much research has been focused on identifying suitable biomass species, which can provide high-energy outputs, to replace conventional fossil fuels. Here we report an approach for increasing the yield of bio-oil production from fast pyrolysis after manipulating the metabolic pathway in microalgae through heterotrophic growth. The yield of bio-oil (57.9%) produced from heterotrophic Chlorella protothecoides cells was 3.4 times higher than from autotrophic cells by fast pyrolysis. The bio-oil was characterized by a much lower oxygen content, with a higher heating value (41 MJ kg -1 ), a lower density (0.92 kg l -1 ), and lower viscosity (0.02 Pa s) compared to those of bio-oil from autotrophic cells and wood. These properties are comparable to fossil oil. The research could contribute to the creation of a system to produce energy from microalgae, and also could have great commercial potential for liquid fuel production. Xiaoling Miao, Qingyu Wu Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, P.R.China Component (%) AC HC Protein 52.64 10.28 Lipid 14.57 55.20 Carbohydrate 10.62 15.43 Ash 6.36 5.93 Moisture 5.39 1.96 Others 10.42 11.20 Table 1. The contents of main chemical components in cells of autotrophic (AC) and heterotrophic (HC) Chlorella protothecoides. Fig. 1. Culture of autotrophic (AC) and heterotrophic (HC) Chlorella protothecoides. High yield bio-oil production from fast pyrolysis by metabolic controlling of Chlorella protothecoides Properties Typical value Bio-oils Fossil oil Wood AC HC C 56.4% 62.07% 76.22% 83.0-87.0% H 6.2% 8.76% 11.61% 10.0-14.0% O 37.3% 19.43% 11.24% 0.05-1.5% N 0.1% 9.74% 0.93% 0.01-0.7% S n.d. 0.05-5.0% Density (kg/l) 1.20 1.06 0.92 0.75-1.0 Viscosity (Pa s) 0.04-0.20 (at 40 o C) 0.10 (at 40 o C) 0.02 (at 40 o C) 2-1000 Heating value (MJ/kg) 21 30 41 42 StabilityNot as stable as fossil fuelsNot as stable as fossil fuels, But more stable than the bio-oil from wood Table 2. Comparison of elemental compositions and physical properties of fossil oil and bio-oils from fast pyrolysis of wood and autotrophic (AC) and heterotrophic Chllorella protothecoides (HC). N.d., not determined Fig. 2. Cells of photoautotrophic and heterotrophic Chlorella protothecoids under confocal laser scanning microscope (A,B) and under differential interference microscopy (C,D). (A) Autofluorescence of photoautotrophic C. protothecoides cells with chlorophyll; (B) Autofluorescence of chlorophyll disappearing cells of heterotrophic C. protothecoides; (C) Almost no lipid vesicles were observed in photoautotrophic C. protothecoides cells; (D) The cells of heterotrophic C. protothecoides were full of lipid vesicles. Fig. 3. Product yields (on the basis of the dry weight of samples) of fast pyrolysis from microalgae at different pyrolysis temperatures with a heating rate of 600ºC s -1, a sweep gas (N 2 ) flow rate of 0.4 m 3 h -1 and a vapor residence time of 2~3 s. (A) Product yields of photoautotrophic (AC) and heterotrophic (HC) Chllorella protothecoides at 500ºC; (B) Product yields of heterotrophic Chllorella protothecoides at temperatures from 400ºC to 600ºC. The particle size of the sample was Dp < 0.5 mm. ACHC ACHC Fig. 4. Bio-oils from fast pyrolysis of cells of autotrophic (AC) and heterotrophic (HC) Chlorella protothecoides. A B