Fed-batch fermentation and supercritical fluid extraction of heterotrophic microalgal Chlorella protothecoides lipids Yen-Hui Chen, Terry H. Walker ⇑ Biosystems Engineering, Clemson University, Biosystems Research Complex, 105 Collings St., Clemson, SC 29634, USA article info Article history: Received 28 October 2011 Received in revised form 16 February 2012 Accepted 7 March 2012 Available online 28 March 2012 Keywords: Chlorella protothecoides Fed-batch fermentation Heterotrophic growth Lipids Supercritical carbon dioxide extraction abstract Lipids obtained from Chlorella protothecoides in heterotrophic cultivation are considered a suitable feed- stock for biodiesel production. In this study, glucose fed-batch fermentation was performed to increase final biomass and lipid production. The biomass productivity and lipid productivity were 6.28 and 2.06 g/L day, respectively. Biomass/glucose conversion and the lipid/glucose conversion were 43.3% and 14.2%, respectively. Extraction of lipids from algae has been identified as a key bottleneck in biopro- cessing operations. Supercritical carbon dioxide (SC-CO 2 ) was applied for neutral lipids extraction and the SC-CO 2 kinetics was investigated by the Goto et al. model. The modeling showed a good fit with exper- imental data. Additionally, neutral lipids extracted by SC-CO 2 displayed a suitable fatty acid profile for biodiesel [mainly C18:1 (60.0%), C18:2 (18.7%) and C16:0 (11.5%)]. Our study demonstrated the ability to produce high levels of neutral lipids through heterotrophic algal culture and subsequent extraction of lipids with SC-CO 2 method developed. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Biodiesel, a mixture of fatty acid methyl/ethyl esters (FAMEs/ FAEEs), made by transesterification of triacylglycerols, is recog- nized as a promising strategy for producing sustainable biofuel (Chisti, 2007). Compared to the petroleum-based diesel, biodiesel exhibits favorable environmental properties, such as biodegrad- ability, lower sulfur content and less carbon monoxide emission (Huang et al., 2010). Microalgal biodiesel is regarded as a second- generation of biofuel because it will not compromise food resources and other products derived from energy crops such as soybean (Cheng et al., 2009). In addition, lipids obtained from Chlorella protothecoides in heterotrophic cultivation have been recommended as a suitable feedstock for sustainable biodiesel pro- duction because of the algae’s shorter growth cycle, less need of land, proper fatty acid composition for biodiesel production (Chisti, 2007; Li et al., 2007; Xiong et al., 2008), and ability to utilize differ- ent carbon sources, such as glucose (Xu et al., 2006), fructose (Gao et al., 2009), sucrose (Gao et al., 2009) and crude glycerol (Chen and Walker, 2011). Microalgal lipids are generally obtained by mechanical pressing or solvent extraction (Fishman et al., 2010; Mercer and Armenta, 2011); however, the application of mechanical pressing is limited because the slow recovery rate for microalgae (Mercer and Armenta, 2011). In addition, solvent extraction can result in poor product quality and requires intensive use of volatile and toxic or- ganic solvents (Mercer and Armenta, 2011). Supercritical fluid extraction (SFE) is considered an important alternative technique to traditional separation methods because the solvent’s density and diffusivity can be controlled by changes in pressure and tem- perature for better solvent power (Machmudah et al., 2007). Super- critical carbon dioxide (SC-CO 2 ) is the most commonly used fluid for SFE, as it is non-flammable, non-toxic, inexpensive and easily separated from the product (Crampon et al., 2011). In addition, SC-CO 2 is a suitable solvent for extracting neutral lipids (triglycer- ides), which are an adequate feedstock for biodiesel applications. Moreover, SC-CO 2 does not solubilize polar lipid, such as phospho- lipids, which eliminates the need for degumming. The use of SC-CO 2 has already been investigated for extraction of b-carotene from Dunaliella salina, Skeletonema costatum, Spirulina pacifica and Synechococcus sp. (Crampon et al., 2011), astaxanthin from Haema- tococcus pluvialis (Krichnavaruk et al., 2008), lutein from Chlorella pyrenoidosa (Wu et al., 2007), c-linolenic acid from Spirulina platensis (Sajilata et al., 2008) and lipids from Chaetomorpha linum, Crypthecodinium cohnii, Chlorococcum sp., Chlorella vulgaris, Nanno- chloropsis sp. and Ochromonas danica (Couto et al., 2010; Crampon et al., 2011; Halim et al., 2011). Mathematical models have been proposed to correlate the experimental overall extraction curve (OEC) during the SFE process (Sousa et al., 2005; Carvalho et al., 2005). The mass transfer behavior related to the rate of solute transferred from solid particles to the supercritical phase is useful for project and process design. The model by Goto et al. (1993) was 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.03.026 ⇑ Corresponding author. Tel.: +1 864 656 0378; fax: +1 864 656 0338. E-mail addresses: ychen@clemson.edu (Y.-H. Chen), walker4@clemson.edu (T.H. Walker). Bioresource Technology 114 (2012) 512–517 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech