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Mechanism of lipid extraction from Botryococcus braunii FACHB 357 in a biphasic bioreactor
Journal of Biotechnology 154 (2011) 281–284
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Short communication
Mechanism of lipid extraction from Botryococcus braunii FACHB 357 in a biphasic bioreactor Fang Zhang a , Li-Hua Cheng b,∗ , Wang-Lei Gao c , Xin-Hua Xu b , Lin Zhang a , Huan-Lin Chen a a b c
Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 310027, PR China Department of Environmental Engineering, Zhejiang University, Hangzhou 310058, PR China School of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, PR China
a r t i c l e
i n f o
Article history: Received 16 January 2011 Received in revised form 11 April 2011 Accepted 18 May 2011 Available online 26 May 2011 Keywords: Botryococcus braunii Lipid extraction Biocompatible solvent Biphasic system
a b s t r a c t Algal lipid of Botryococcus braunii could be produced continuously and in situ extracted in an aqueousorganic bioreactor. In this study, the cell ultra-structure and cell membrane permeability of B. braunii FACHB 357 were investigated to understand the mechanism of lipid extraction within the biphasic system. The results showed that biocompatible solvent of tetradecane could induce algal lipid accumulation, enable the cell membrane more active and the cell wall much looser. The exocytosis process was observed to be one of the mechanisms for lipid cross-membrane extraction in the presence of organic solvent. © 2011 Elsevier B.V. All rights reserved.
Algal biodiesel has been considered as one of the most promising renewable transportation fuels due to concerns on recent oil crisis and possible climate change from the greenhouse gases (Chisti, 2007; Greenwell et al., 2010; Hu et al., 2008). During the last decade, although significant advances in microalgal biotechnology have been achieved, challenges still remain in the low cost production of microalgal biodiesel. Among the costly downstream processing steps, it is agreed that harvesting/dewatering and the following extraction of fuel precursors from the biomass consists the most energy intensive steps (Radakovits et al., 2010). As a result, to integrate the steps of harvesting and extraction, thus to allow in situ lipid milking, seems to be a potential solution for cutting the cost of algal-oil production. In this integrated process, algal cells can be cultured in the biphasic bioreactors for lipids production continuously while the lipids are extracted simultaneously from the aqueous phase into the organic phase. This kind of in situ extraction process, also named “milking”, has been found successful in the pigment production from algae (Hejazi and Wijffels, 2003, 2004; Mojaat et al., 2007). As shown in the literature, Botryococcus braunii is one of the most promising algal species for synchronous culture and lipid extraction due to its high lipid content of 25–75% (dry weight biomass) (Mata et al., 2010). The lipid (including hydrocarbons) of this alga could be repeatedly extracted from the wet biomass without the
∗ Corresponding author. Tel.: +86 571 87953802; fax: +86 571 87953802. E-mail address: [email protected] (L.-H. Cheng). 0168-1656/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2011.05.008
usual harvesting and dewatering steps. By exposing B. braunii to organic solvent of hexane, a substantial fraction of hydrocarbons was obtained (Frenz et al., 1989). When an aqueous-dihexyl ether system was adopted, the B. braunii UTEX 572 could be induced to produce more long-chain unsaturated hydrocarbons, and part of those hydrocarbons were extracted into the organic solvent phase after an incubation of 3 days. By recycling of this two-stage system and hence the improvement of mixing, the lipid extractability could be increased from 32% to 60% (An et al., 2004; Sim et al., 2001). Since the organic solvents of both hexane and dehexyl ether were toxic to the algal growth, we had screened the biocompatible solvents for in situ extraction of lipid from B. braunii FACHB 357 (to be published elsewhere). It has been proven that B. braunii FACHB 357 can survive of the 10% (v/v) tetradecane even after 45 days. However, to our best knowledge, the understanding of the extraction process at cell level has not been available in open literature. In this work, we will focus on the effect of tetradecane on cell structure of B. braunii FACHB 357, including the variation of cell membrane, cell wall and lipid accumulation, in order to know more about the mechanism of lipid extraction in the biphasic reactor. In current study, B. braunii FACHB 357 (obtained from Freshwater Algae Culture Collection of the Institute of Hydrobiology, Chinese Academy of Sciences, China) had been cultured in BG 11 medium for 3 weeks. 250 ml Erlenmeyer flask containing 100 ml algae culture was used as bioreactor to perform biphasic experiments. The biocompatible organic phase of tetradecane (Sigma, USA) was added into culture system at the beginning of algal stationary stage. The volume ratio of tetradecane and algal culture was
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F. Zhang et al. / Journal of Biotechnology 154 (2011) 281–284
Fig. 1. The location and accumulation of lipid body in Botryococcus braunii FACHB 357 cells in the absence (A) and presence of 10% (v/v) tetradecane (B) cultured under continuous illumination at 60 mol photons m−2 s−1 and 25 ◦ C for 8 days. A-1, 2 (and B-1, 2) are different cells of the same sample (Py, pyrenoid; Ch, Chloroplast; LB, lipid body; GOM, granular osmiophilic material; CM, cell membrane; CW, cell wall).
et al., 2002), as shown in Fig. 2. For unicellular microalga B. braunii FACHB 357, the cell membrane permeability increased with culture time under both experimental conditions due to the natural aging (Fig. 2). By contrast, this alga which cultured in the presence of tetradecane was apt to absorb more macromolecule Evans Blue into cells. Since the substance cross-membrane transport depended mainly on mechanisms including endocytosis/exocytosis and diffusion (Battey et al., 1999), we deduce that algal cell membrane was stimulated to be more active for exocytosis or more permeable for substance diffusion within the aqueous-organic bioreactor.
Cell MembraneIntegrity ( OD600/ gcell)
10:1. The flask was put in a shaker with 100 rpm under fluorescent light at 25 ◦ C. In this batch culture and the following extraction process, the lipid recovery ratio obtained was about 20% for 96 h of culture time. To locate the lipid in the cell and its accumulation, the cell ultra-structure was observed and photographed with a JEM-1230 transmission electron microscope (JEOL, Japan) after a biphasic culture of 8 days. As shown in Fig. 1, several large dark-staining lipid bodies (LB) were observed at the inner side of the cell membrane of B. braunii FACHB 357 cells while granular osmiophilic material (GOM, Fig. 1A2 and Fig. 1B-2), possible as precursor for lipid biosynthesis, were found near the thylakoid membranes. This indicated that algal lipid was synthesized in chloroplasts and then exported into cytoplasm for energy storage and eventual metabolism (Radakovits et al., 2010). Those cytoplasmic lipid bodies were observed without evident membrane-like structure, although it was reported that some eukaryotic cells produced lipid body consisting of a hydrophobic core of neutral lipids (typically containing TAGs and sterol esters or wax esters) and an envelope of phospholipid–protein membrane (Murphy, 2001). Similar to the effect of environmental changes such as nitrogen limitation, high salinity or high temperature on the lipid accumulation, we found that organic solvent as a stress condition enhanced lipid synthesis for B. braunii FACHB 357. Both the number and size of lipid bodies increased in the presence of tetradecane compared to the control (Fig. 1). Only 1–2 small lipid bodies (diameter <0.3 m) were observed in the control cells (Fig. 1A), while the number of lipid bodies increased to 4–6 and the diameter of LB was around 0.5 m in the algal cells within the biphasic bioreactor (Fig. 1B). The effect of organic solvent on algal cell membrane permeability was further characterized by Evans Blue method (Hamer
0.08
tetradecane control 0.06
0.04
0.02
0.00 0
24
48
72
96
Time(h) Fig. 2. The effect of 10% (v/v) tetradecane on Botryococcus braunii FACHB 357 cell membrane integrity in an aqueous-organic system under continuous illumination at 60 mol photons m−2 s−1 and 25 ◦ C for 96 h.
F. Zhang et al. / Journal of Biotechnology 154 (2011) 281–284
283
Fig. 3. The cell wall (CW) ultrastructure of Botryococcus braunii FACHB 357 in the absence (A) and presence of 10% (v/v) tetradecane (B) under continuous illumination at 60 mol photons m−2 s−1 and 25 ◦ C for 8 days.
As shown in Fig. 3, the variation of cell ultra-structure was also observed in the cell wall of B. braunii FACHB 357. Compared with the control, the cell wall structure of alga cultured in the aqueous-tetradecane system looked much looser. The border of cell wall was found to be blurring, and even part of it collapsed
into pieces (Fig. 3B). Generally, cell wall protected cell from damages caused by outside environment. However, the cell wall was not merely an outer, or an inactive covering of the algal cell. It was critical in the absorption, transport and secretion of substances for cell metabolism. In addition, the soluble substances could diffuse
Fig. 4. The mechanism of lipid cross-membrane transportation for Botryococcus braunii FACHB 357 in the presence of 10% (v/v) tetradecane under continuous illumination at 60 mol photons m−2 s−1 and 25 ◦ C for 8 days.
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F. Zhang et al. / Journal of Biotechnology 154 (2011) 281–284
across the pores of cell wall and then interact with the algal cell membrane. Therefore, adding tetradecane into B. braunii culture system might result in an incompact and porous cell wall, which contributed to nutrient or metabolites such as lipid to transport through this barrier. Based on the aformentioned experimental results and all those references reported, the possible mechanism for in situ lipid extraction by biocompatible organic solvent of tetradecane was proposed and further characterized in the following four steps as shown in Fig. 4. (i) Lipid (mainly fatty acid) was synthesized in chloroplast and then exported into cell cytosol in the shape of lipid body (TAG, triacylglycerol). For microalga B. braunii FACHB 357, most lipid bodies were found to accumulate and locate around the inner side of the cell membrane (Fig. 4A). (ii) Lipid bodies were then translocated across cell membrane by the vesicle-mediated transport mechanism. As shown in Fig. 4B, the vesicles were clearly observed near the lipid body. It had demonstrated lipid globules might be transported from the cytosol to the space between cell membrane and cell wall by the process of exocytosis. Compared to the control, this exocytosis effect was more active in the algal cell membrane with tetradecane treatment, as shown in Fig. 2 that the permeability of cell membrane increased by the characterization of Evans Blue method. (iii) The lipid bodies were further extracted into the space between cell membrane and cell wall (Fig. 4C). The global lipid droplets in this space were found to be sealed with membrane structure, which was different from cytoplasmic LB (no membrane structure as shown in Fig. 1) and probably resulted from the exocytosis process as shown in Fig. 4B. (iv) Finally, lipid was extracted into solvent phase due to the looser structure of the cell wall. As shown in Fig. 4D, the permeation of lipid body across the cell wall was observed. In conclusion, we demonstrated the lipid accumulation of B. braunii FACHB 357 in the presence of the biocompatible solvent of tetradecane. Due to the more active exocytosis of the cell membrane and the much looser cell wall, the algal lipid could be in situ extracted through the multi-layers of the cell structure, and finally into the solvent phase of tetradecane.
Acknowledgments This work is based upon research supported by the National Natural Science Foundation of China (no. 21076177), the Fundamental Research Funds for the Central Universities (2009QNA6007), the Zhejiang Provincial Natural Science Foundation of China (Y4100222) and the Zhejiang Provincial Bureau of Education (Y20101849). References An, J., Sim, S., Kim, B., Lee, J., 2004. Improvement of hydrocarbon recovery by two-stage cell recycle extraction in the cultivation of Botryococcus braunii. J. Microbiol. Biotechnol. 14, 932–937. Battey, N., James, N., Greenland, A., Brownlee, C., 1999. Exocytosis and endocytosis. Plant Cell 11, 643–659. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294–306. Frenz, J., Largeau, C., Casadevall, E., 1989. Hydrocarbon recovery by extraction with a biocompatible solvent from free and immobilized cultures of Botryococcus braunii. Enzyme Microb. Technol. 11, 717–724. Greenwell, H., Laurens, L., Shields, R., Lovitt, R., Flynn, K., 2010. Placing microalgae on the biofuels priority list: a review of the technological challenges. J. R. Soc. Interface 7, 703–726. Hamer, P.W., McGeachie, J.M., Davies, M.J., Grounds, M.D., 2002. Evans Blue Dye as an in vivo marker of myofibre damage: optimising parameters for detecting initial myofibre membrane permeability. J. Anat. 200, 69–79. Hejazi, A.M., Wijffels, R.H., 2003. Effect of light intensity on -carotene production and extraction by Dunaliella salina in two-phase bioreactors. Biomol. Eng. 4-6, 171–175. Hejazi, A.M., Wijffels, R.H., 2004. Milking of microalgae. Trends Biotechnol. 4, 189–194. Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M., Darzins, A., 2008. Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J. 54, 621–639. Mata, T.M., Martins, A.A., Caetano, N.S., 2010. Microalgae for biodiesel production and other applications: a review. Renew. Sust. Energ. Rev. 14, 217–232. Mojaat, M., Foucault, A., Pruvost, J., Legrand, J., 2007. Optimal selection of organic solvents for biocompatible extraction of -carotene from Dunaliella salina. J. Biotechnol. 133, 433–441. Murphy, D.J., 2001. The biogenesis and functions of lipid bodies in animals, plants and microorganisms. Prog. Lipid Res. 40, 325–438. Radakovits, R., Jinkerson, R.E., Darzins, A., Posewitz, M.C., 2010. Genetic engineering of algae for enhanced biofuel production. Eukaryot. Cell 9, 486–501. Sim, S., An, J., Kim, B., 2001. Two-phase extraction culture of Botryococcus braunii producing long-chain unsaturated hydrocarbons. Biotechnol. Lett. 23, 201–205.
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Short communication
Mechanism of lipid extraction from Botryococcus braunii FACHB 357 in a biphasic bioreactor Fang Zhang a , Li-Hua Cheng b,∗ , Wang-Lei Gao c , Xin-Hua Xu b , Lin Zhang a , Huan-Lin Chen a a b c
Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 310027, PR China Department of Environmental Engineering, Zhejiang University, Hangzhou 310058, PR China School of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, PR China
a r t i c l e
i n f o
Article history: Received 16 January 2011 Received in revised form 11 April 2011 Accepted 18 May 2011 Available online 26 May 2011 Keywords: Botryococcus braunii Lipid extraction Biocompatible solvent Biphasic system
a b s t r a c t Algal lipid of Botryococcus braunii could be produced continuously and in situ extracted in an aqueousorganic bioreactor. In this study, the cell ultra-structure and cell membrane permeability of B. braunii FACHB 357 were investigated to understand the mechanism of lipid extraction within the biphasic system. The results showed that biocompatible solvent of tetradecane could induce algal lipid accumulation, enable the cell membrane more active and the cell wall much looser. The exocytosis process was observed to be one of the mechanisms for lipid cross-membrane extraction in the presence of organic solvent. © 2011 Elsevier B.V. All rights reserved.
Algal biodiesel has been considered as one of the most promising renewable transportation fuels due to concerns on recent oil crisis and possible climate change from the greenhouse gases (Chisti, 2007; Greenwell et al., 2010; Hu et al., 2008). During the last decade, although significant advances in microalgal biotechnology have been achieved, challenges still remain in the low cost production of microalgal biodiesel. Among the costly downstream processing steps, it is agreed that harvesting/dewatering and the following extraction of fuel precursors from the biomass consists the most energy intensive steps (Radakovits et al., 2010). As a result, to integrate the steps of harvesting and extraction, thus to allow in situ lipid milking, seems to be a potential solution for cutting the cost of algal-oil production. In this integrated process, algal cells can be cultured in the biphasic bioreactors for lipids production continuously while the lipids are extracted simultaneously from the aqueous phase into the organic phase. This kind of in situ extraction process, also named “milking”, has been found successful in the pigment production from algae (Hejazi and Wijffels, 2003, 2004; Mojaat et al., 2007). As shown in the literature, Botryococcus braunii is one of the most promising algal species for synchronous culture and lipid extraction due to its high lipid content of 25–75% (dry weight biomass) (Mata et al., 2010). The lipid (including hydrocarbons) of this alga could be repeatedly extracted from the wet biomass without the
∗ Corresponding author. Tel.: +86 571 87953802; fax: +86 571 87953802. E-mail address: [email protected] (L.-H. Cheng). 0168-1656/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2011.05.008
usual harvesting and dewatering steps. By exposing B. braunii to organic solvent of hexane, a substantial fraction of hydrocarbons was obtained (Frenz et al., 1989). When an aqueous-dihexyl ether system was adopted, the B. braunii UTEX 572 could be induced to produce more long-chain unsaturated hydrocarbons, and part of those hydrocarbons were extracted into the organic solvent phase after an incubation of 3 days. By recycling of this two-stage system and hence the improvement of mixing, the lipid extractability could be increased from 32% to 60% (An et al., 2004; Sim et al., 2001). Since the organic solvents of both hexane and dehexyl ether were toxic to the algal growth, we had screened the biocompatible solvents for in situ extraction of lipid from B. braunii FACHB 357 (to be published elsewhere). It has been proven that B. braunii FACHB 357 can survive of the 10% (v/v) tetradecane even after 45 days. However, to our best knowledge, the understanding of the extraction process at cell level has not been available in open literature. In this work, we will focus on the effect of tetradecane on cell structure of B. braunii FACHB 357, including the variation of cell membrane, cell wall and lipid accumulation, in order to know more about the mechanism of lipid extraction in the biphasic reactor. In current study, B. braunii FACHB 357 (obtained from Freshwater Algae Culture Collection of the Institute of Hydrobiology, Chinese Academy of Sciences, China) had been cultured in BG 11 medium for 3 weeks. 250 ml Erlenmeyer flask containing 100 ml algae culture was used as bioreactor to perform biphasic experiments. The biocompatible organic phase of tetradecane (Sigma, USA) was added into culture system at the beginning of algal stationary stage. The volume ratio of tetradecane and algal culture was
282
F. Zhang et al. / Journal of Biotechnology 154 (2011) 281–284
Fig. 1. The location and accumulation of lipid body in Botryococcus braunii FACHB 357 cells in the absence (A) and presence of 10% (v/v) tetradecane (B) cultured under continuous illumination at 60 mol photons m−2 s−1 and 25 ◦ C for 8 days. A-1, 2 (and B-1, 2) are different cells of the same sample (Py, pyrenoid; Ch, Chloroplast; LB, lipid body; GOM, granular osmiophilic material; CM, cell membrane; CW, cell wall).
et al., 2002), as shown in Fig. 2. For unicellular microalga B. braunii FACHB 357, the cell membrane permeability increased with culture time under both experimental conditions due to the natural aging (Fig. 2). By contrast, this alga which cultured in the presence of tetradecane was apt to absorb more macromolecule Evans Blue into cells. Since the substance cross-membrane transport depended mainly on mechanisms including endocytosis/exocytosis and diffusion (Battey et al., 1999), we deduce that algal cell membrane was stimulated to be more active for exocytosis or more permeable for substance diffusion within the aqueous-organic bioreactor.
Cell MembraneIntegrity ( OD600/ gcell)
10:1. The flask was put in a shaker with 100 rpm under fluorescent light at 25 ◦ C. In this batch culture and the following extraction process, the lipid recovery ratio obtained was about 20% for 96 h of culture time. To locate the lipid in the cell and its accumulation, the cell ultra-structure was observed and photographed with a JEM-1230 transmission electron microscope (JEOL, Japan) after a biphasic culture of 8 days. As shown in Fig. 1, several large dark-staining lipid bodies (LB) were observed at the inner side of the cell membrane of B. braunii FACHB 357 cells while granular osmiophilic material (GOM, Fig. 1A2 and Fig. 1B-2), possible as precursor for lipid biosynthesis, were found near the thylakoid membranes. This indicated that algal lipid was synthesized in chloroplasts and then exported into cytoplasm for energy storage and eventual metabolism (Radakovits et al., 2010). Those cytoplasmic lipid bodies were observed without evident membrane-like structure, although it was reported that some eukaryotic cells produced lipid body consisting of a hydrophobic core of neutral lipids (typically containing TAGs and sterol esters or wax esters) and an envelope of phospholipid–protein membrane (Murphy, 2001). Similar to the effect of environmental changes such as nitrogen limitation, high salinity or high temperature on the lipid accumulation, we found that organic solvent as a stress condition enhanced lipid synthesis for B. braunii FACHB 357. Both the number and size of lipid bodies increased in the presence of tetradecane compared to the control (Fig. 1). Only 1–2 small lipid bodies (diameter <0.3 m) were observed in the control cells (Fig. 1A), while the number of lipid bodies increased to 4–6 and the diameter of LB was around 0.5 m in the algal cells within the biphasic bioreactor (Fig. 1B). The effect of organic solvent on algal cell membrane permeability was further characterized by Evans Blue method (Hamer
0.08
tetradecane control 0.06
0.04
0.02
0.00 0
24
48
72
96
Time(h) Fig. 2. The effect of 10% (v/v) tetradecane on Botryococcus braunii FACHB 357 cell membrane integrity in an aqueous-organic system under continuous illumination at 60 mol photons m−2 s−1 and 25 ◦ C for 96 h.
F. Zhang et al. / Journal of Biotechnology 154 (2011) 281–284
283
Fig. 3. The cell wall (CW) ultrastructure of Botryococcus braunii FACHB 357 in the absence (A) and presence of 10% (v/v) tetradecane (B) under continuous illumination at 60 mol photons m−2 s−1 and 25 ◦ C for 8 days.
As shown in Fig. 3, the variation of cell ultra-structure was also observed in the cell wall of B. braunii FACHB 357. Compared with the control, the cell wall structure of alga cultured in the aqueous-tetradecane system looked much looser. The border of cell wall was found to be blurring, and even part of it collapsed
into pieces (Fig. 3B). Generally, cell wall protected cell from damages caused by outside environment. However, the cell wall was not merely an outer, or an inactive covering of the algal cell. It was critical in the absorption, transport and secretion of substances for cell metabolism. In addition, the soluble substances could diffuse
Fig. 4. The mechanism of lipid cross-membrane transportation for Botryococcus braunii FACHB 357 in the presence of 10% (v/v) tetradecane under continuous illumination at 60 mol photons m−2 s−1 and 25 ◦ C for 8 days.
284
F. Zhang et al. / Journal of Biotechnology 154 (2011) 281–284
across the pores of cell wall and then interact with the algal cell membrane. Therefore, adding tetradecane into B. braunii culture system might result in an incompact and porous cell wall, which contributed to nutrient or metabolites such as lipid to transport through this barrier. Based on the aformentioned experimental results and all those references reported, the possible mechanism for in situ lipid extraction by biocompatible organic solvent of tetradecane was proposed and further characterized in the following four steps as shown in Fig. 4. (i) Lipid (mainly fatty acid) was synthesized in chloroplast and then exported into cell cytosol in the shape of lipid body (TAG, triacylglycerol). For microalga B. braunii FACHB 357, most lipid bodies were found to accumulate and locate around the inner side of the cell membrane (Fig. 4A). (ii) Lipid bodies were then translocated across cell membrane by the vesicle-mediated transport mechanism. As shown in Fig. 4B, the vesicles were clearly observed near the lipid body. It had demonstrated lipid globules might be transported from the cytosol to the space between cell membrane and cell wall by the process of exocytosis. Compared to the control, this exocytosis effect was more active in the algal cell membrane with tetradecane treatment, as shown in Fig. 2 that the permeability of cell membrane increased by the characterization of Evans Blue method. (iii) The lipid bodies were further extracted into the space between cell membrane and cell wall (Fig. 4C). The global lipid droplets in this space were found to be sealed with membrane structure, which was different from cytoplasmic LB (no membrane structure as shown in Fig. 1) and probably resulted from the exocytosis process as shown in Fig. 4B. (iv) Finally, lipid was extracted into solvent phase due to the looser structure of the cell wall. As shown in Fig. 4D, the permeation of lipid body across the cell wall was observed. In conclusion, we demonstrated the lipid accumulation of B. braunii FACHB 357 in the presence of the biocompatible solvent of tetradecane. Due to the more active exocytosis of the cell membrane and the much looser cell wall, the algal lipid could be in situ extracted through the multi-layers of the cell structure, and finally into the solvent phase of tetradecane.
Acknowledgments This work is based upon research supported by the National Natural Science Foundation of China (no. 21076177), the Fundamental Research Funds for the Central Universities (2009QNA6007), the Zhejiang Provincial Natural Science Foundation of China (Y4100222) and the Zhejiang Provincial Bureau of Education (Y20101849). References An, J., Sim, S., Kim, B., Lee, J., 2004. Improvement of hydrocarbon recovery by two-stage cell recycle extraction in the cultivation of Botryococcus braunii. J. Microbiol. Biotechnol. 14, 932–937. Battey, N., James, N., Greenland, A., Brownlee, C., 1999. Exocytosis and endocytosis. Plant Cell 11, 643–659. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294–306. Frenz, J., Largeau, C., Casadevall, E., 1989. Hydrocarbon recovery by extraction with a biocompatible solvent from free and immobilized cultures of Botryococcus braunii. Enzyme Microb. Technol. 11, 717–724. Greenwell, H., Laurens, L., Shields, R., Lovitt, R., Flynn, K., 2010. Placing microalgae on the biofuels priority list: a review of the technological challenges. J. R. Soc. Interface 7, 703–726. Hamer, P.W., McGeachie, J.M., Davies, M.J., Grounds, M.D., 2002. Evans Blue Dye as an in vivo marker of myofibre damage: optimising parameters for detecting initial myofibre membrane permeability. J. Anat. 200, 69–79. Hejazi, A.M., Wijffels, R.H., 2003. Effect of light intensity on -carotene production and extraction by Dunaliella salina in two-phase bioreactors. Biomol. Eng. 4-6, 171–175. Hejazi, A.M., Wijffels, R.H., 2004. Milking of microalgae. Trends Biotechnol. 4, 189–194. Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M., Darzins, A., 2008. Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J. 54, 621–639. Mata, T.M., Martins, A.A., Caetano, N.S., 2010. Microalgae for biodiesel production and other applications: a review. Renew. Sust. Energ. Rev. 14, 217–232. Mojaat, M., Foucault, A., Pruvost, J., Legrand, J., 2007. Optimal selection of organic solvents for biocompatible extraction of -carotene from Dunaliella salina. J. Biotechnol. 133, 433–441. Murphy, D.J., 2001. The biogenesis and functions of lipid bodies in animals, plants and microorganisms. Prog. Lipid Res. 40, 325–438. Radakovits, R., Jinkerson, R.E., Darzins, A., Posewitz, M.C., 2010. Genetic engineering of algae for enhanced biofuel production. Eukaryot. Cell 9, 486–501. Sim, S., An, J., Kim, B., 2001. Two-phase extraction culture of Botryococcus braunii producing long-chain unsaturated hydrocarbons. Biotechnol. Lett. 23, 201–205.