| Microalgae-derived biodiesel from Chlorella protothecoides lipid has attracted intense attention as a renewable biofuel due to the world energy crisis. This study investigated the growth of heterotrophic C. protothecoides on different carbon substrates and the feasibility of biodiesel-derived crude glycerol as an alternative carbon substrate in batch and fed-batch fermentations. In addition, glucose was used as a substrate source in a 7-liter bioreactor performed in fed-batch fermentation for understanding the physical and biological aspects. Furthermore, the mass transfer behavior and the optimal process conditions of supercritical carbon dioxide (SC-CO 2) lipid extraction were discussed.;The specific cell growth rates of C. protothecoides in glucose, pure glycerol and crude glycerol media were 0.83 day-1 , 0.70 day-1, and 0.74 day-1 in batch fermentation, respectively. The biomass and lipid concentration of C. protothecoides cultivated in a crude glycerol medium were 23.53 g/L and 14.59 g/L in a 6-day batch fermentation, separately. The maximum lipid productivity was found in crude-glycerol, fed-batch fermentation (2.99 g/L day). This study showed that a crude glycerol-to-lipid fermentation model potentially provides additional feedstock for biodiesel production while offering a lower-cost carbon substrate and eliminating crude glycerol disposal. In glucose fed-batch fermentation, the biomass/glucose and the lipid/glucose conversion were 43.3% (w/w) and 14.2% (w/w), respectively. The biomass and lipid productivity were 6.28 g/L day and 2.06 g/L day, separately. The requirement of dissolved oxygen level may significantly increase due to the high cell density during the major biomass and lipid production phase.;The mass transfer behavior of SC-CO2 extraction was studied with the use of the Goto model that described the overall extraction curve (OEC) quite well. The major fatty acids were C18:1 (60.0%), C18:2 (18.7%) and C16:0 (11.5%). In addition, no significant differences were found in the C18:1, C18:2 and C16:0 of lipid extracts obtained from hexane Soxhlet extraction and SC-CO2 extraction. Furthermore, response surface methodology (RSM) was sufficient to optimize the SC-CO2 extraction process of lipid yield. The linear and quadratic terms of temperature, pressure and extraction time as well as the interaction between temperature and pressure were significant in the second-order polynomial model for lipid yield. The optimal lipid yield from the model was predicted when the temperature was 59 °C, the pressure was 350.7 bar and the extraction time was 2.83 hours. Under these conditions, the experimental lipid yield (25.05%) was close to the predicted value calculated from the model.From fatty acid methyl ester analysis, C. protothecoides lipid-derived biodiesel was rich in unsaturated fatty acid methyl esters (80.9%). The total amount of linolenic acid methyl ester was 1.9%, and no polyunsaturated fatty acid methyl esters (≥ 4 double bonds) were found.;In summary, this study demonstrated the potential of biodiesel-derived crude glycerol for C. protothecoides fermentation and SC-CO 2 processing for C. protothecoides lipid extraction for biofuel production. |
