Bioresource Technology 153 (2014) 408–412

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Short Communication

Acid-catalyzed hot-water extraction of lipids from Chlorella vulgaris Ji-Yeon Park ⇑, You-Kwan Oh, Jin-Suk Lee, Kyubock Lee, Min-Ji Jeong, Sun-A Choi Department of Clean Fuel, Korea Institute of Energy Research, Daejeon 305-343, Republic of Korea

h i g h l i g h t s  Acid-catalyzed hot-water treatment for efficient lipid extraction from wet microalgae.  Conversion of extracted microalgal lipids to biodiesel by esterification.  Improvement of extraction yield through cell disruption and demulsification by acid.

a r t i c l e

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Article history: Received 24 July 2013 Received in revised form 28 November 2013 Accepted 15 December 2013 Available online 22 December 2013 Keywords: Microalgal lipids Wet extraction Chlorella vulgaris Hot-water treatment Acid catalyst

a b s t r a c t Acid-catalyzed hot-water treatment for efficient extraction of lipids from a wet microalga, Chlorella vulgaris, was investigated. For an initial fatty acids content of 381.6 mg/g cell, the extracted-lipid yield with no heating and no catalyst was 83.2 mg/g cell. Under a 1% H2SO4 concentration heated at 120 °C for 60 min, however, the lipid-extraction yield was 337.4 mg/g cell. The fatty acids content, meanwhile, was 935 mg fatty acid/g lipid. According to the severity index formula, 337.5 mg/g cell of yield under the 1% H2SO4 concentration heated at 150 °C for 8 min, and 334.2 mg/g cell of yield under the 0.5% H2SO4 concentration heated at 150 °C for 16 min, were obtained. The lipids extracted by acid-catalyzed hotwater treatment were converted to biodiesel. The biodiesel’s fatty acid methyl ester (FAME) content after esterification of the microalgal lipids was increased to 79.2% by the addition of excess methanol and sulfuric acid. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Microalgae are photosynthetic microorganisms capable of converting, under light conditions, carbon dioxide and water into macromolecules such as lipids, polysaccharides, and proteins (Fu et al., 2010). Some microalgae boast high oil productivity compared with plant oils, and offer the additional advantage of not competing with food crops. For enhanced economic and environmental feasibilities, moreover, waste water or sea water can be used in place of fresh water in the microalgae production process (Li et al., 2008; Schenk et al., 2008). Conversion of microalgae to biodiesel typically includes the following four steps: microalgae cultivation, cell harvesting, lipid extraction, and biodiesel conversion (Li et al., 2008). Lipid extraction from microalgae can be achieved via a number of methods including solvent extraction, enzymatic hydrolysis, fractionation as well as microwave- and ultrasound-based modalities (Lee et al., 2013; Shin et al., 2011). Polysaccharides in post-extraction cell debris, additionally, can be utilized as a source of sugar for bioethanol production (Sugiyama et al., 1991; Sun and Cheng, 2002). ⇑ Corresponding author. Tel.: +82 42 860 3041; fax: +82 42 860 3495. E-mail address: [email protected] (J.-Y. Park). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.12.065

Recovery of microalgal lipids from dried microalgae has been achieved by many researchers (Demirbas, 2009; Xu et al., 2006); however, given the high costs incurred in the dewatering process, wet extraction has emerged an attractive alternative approach. Several methods of wet lipid extraction have been proposed, though the yields remain unsatisfactory (Cho et al., 2013; Lee et al., 2013). Hot-water treatment facilitates the pressure maintenance necessary to keep water in the liquid state at elevated temperatures. This method has been typically applied to cellulosic biomass pretreatment in order to weaken or disrupt the crystalline structure of cellulose (Mosier et al., 2005; Yu et al., 2013). To improve the pretreatment efficiency, catalysts such as sulfuric acid or sodium hydroxide were used. Given that microalgal cell walls contain cellulose, it is expected that hot-water treatment will become a proven alternative approach to the extraction of lipids from microalgae. In this study, the efficiency of sulfuric-acid-catalyzed hot-water extraction of lipids from wet microalgae was investigated. The relationship between the reaction conditions and the lipid-extraction yield was determined using the severity index formula.

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2. Experimental methods 2.1. Lipid extraction from microalgae Chlorella vulgaris (hereafter: C. vulgaris), a freshwater microalga, was isolated locally and cultured in a nutrient media (constituents: KNO3, 3 mM; KH2PO4, 5.44 mM; Na2HPO4, 1.83 mM; MgSO47H2O, 0.20 mM; CaCl2, 0.12 mM; FeNaEDTA, 0.03 mM; ZnSO47H2O, 0.01 mM; MnCl24H2O, 0.07 mM; CuSO4, 0.07 mM; Al2(SO4)318H2O, 0.01 mM) adjusted to a pH of 6.5. The C. vulgaris was then cultivated at 30 °C in a Pyrex bubble-column reactor (working volume: 6 L) equipped with 12 fluorescent lamps (light intensity: 80 lmol/m2/s) and maintained in a constant-temperature room. The reactor was supplied with 10% (v/v) CO2 in air at a rate of 0.75 L/min. For the purposes of lipid-extraction experiments, cells were harvested by centrifugation (4000 rpm and 10 min) until the cell concentration was 20 g/L. Prior to analyses of fatty acids and polysaccharide contents, cells were harvested by centrifugation, washed with deionized water, and freeze-dried (FD5512, IlShin BioBase Co., Korea) for 4 days. As a control, lipids were extracted from lyophilized C. vulgaris using three organic solvents: hexane (96%, Junsei, Japan), hexane:methanol (99.6%, Junsei, Japan) = 7:3 (v/v), and chloroform (99%, Junsei, Japan): methanol = 2:1 (v/v). The C. vulgaris loading was 5% (w/w). The mixture was stirred at 1000 rpm for 6 h at room temperature, and then separated into the organic-solvent and celldebris layers by 4000 rpm centrifugation for 10 min. Finally, the solvent of lipids-containing organic-solvent layer was removed using a vacuum evaporator (EZ2 PLUS, Genevac, UK), and the lipids were recovered. The lipid-extraction yield was determined by reference to the weight of the recovered lipids. All of the experiments were performed in duplicate. 2.2. Acid-catalyzed hot-water treatment of microalgae Hot-water treatments were carried out in a 100 mL autoclave reactor. To enhance the lipid-extraction efficiency, sulfuric acid (H2SO4; 95%, Junsei, Japan) solution was injected into the harvested cells before heating. The H2SO4 concentration of the culture solution was adjusted to 0%, 0.25%, 0.5%, 0.75%, 1%, 2%, and 3% (w/w), respectively. Various reaction temperatures, namely 120, 130, 140, 150, and 160 °C, were applied. After the reactor was cooled, organic-solvent hexane was mixed with the treatment solution at 1000 rpm for 2 h at room temperature, after which the hexane layer was separated from the cell-debris layer by centrifugation. Finally, the hexane of lipids-containing hexane layer was removed by vacuum evaporation, and the lipids were recovered. The lipid-extraction yield was determined by reference to the weight of the recovered lipid. All of the experiments were performed in duplicate. 2.3. Microalgal lipids conversion to biodiesel

Specifically, cells totaling approximately 10 mg were put in a vial. Two milliliter of chloroform–methanol mixture (2:1, v/v) was added to the cells, which solution was then vigorously agitated for 10 min. One milliliter of chloroform solution containing heptadecanoic acid (Sigma, USA) as an internal standard (500 lg/L), 1 mL of methanol, and 300 lL of H2SO4 were sequentially added to the vial and vortex-mixed for 5 min. The vial was then reacted in a 100 °C water bath for 10 min, after which it was cooled to room temperature, supplemented with 1 mL of distilled water, and intensely mixed for 5 min. After centrifugation, the lower layer (organic phase) was injected into a gas chromatograph. The FAME was analyzed using a gas chromatograph equipped with an automatic injector (Agilent 7890, USA). Mix RM3, Mix RM5, GLC50, and GLC70 (Supelco, USA) were utilized as the standards. The other reagents used were of analytical grade. The fatty acids contents of the microalgal lipids were determined following the modified direct esterification method noted above (Lepage and Roy, 1984). Initially, extracted microalgal lipids (approx. 10 mg per vial) were used instead of cells for esterification. The subsequent procedure was the same as detailed above. The biodiesel’s FAME content was analyzed using a gas chromatograph equipped with an auto-injector (Agilent 6890A, USA). In steps, approximately 250 mg of sample was put in a 10 mL vial. The sample was then mixed with 5 mL of heptane solution containing methyl heptadecanoate (Fluka, Switzerland) as an internal standard (10 mg/mL), and the resultant solution was injected into a gas chromatograph (Park et al., 2008). The cells were hydrolyzed by H2SO4 according to NREL’s Chemical Analysis and Testing Standard Procedures Nos. 001–004 (NREL, 2004); on this basis, the initial polysaccharide content of the C. vulgaris was measured. A UV–VIS Spectrophotometer (Optizen 2120UV, MECASYS, Korea) was used to measure the culture solution’s optical density (600 nm), according to which the cell concentration was then calculated. 2.5. Severity index The severity index is a useful tool for experimental design (Chum et al., 1990). Using this formula, the reaction conditions (reaction time, temperature, and H2SO4 concentration) of H2SO4catalyzed hot-water-based lipid extraction from microalgae can be easily determined. The severity index can be employed to evaluate hot-water treatment as a function of reaction time (t, min) and temperature (T, °C):

Severity Index ðSIÞ ¼ log R0 ¼ logðt expððT  100Þ=14:75ÞÞ

ð1Þ

When treatment is performed under acidic conditions, the effect of pH can be taken into consideration by means of a combined severity index:

Combined Severity Index ðCSÞ ¼ log R1 ¼ log R0  pH The extracted microalgal lipids were converted to biodiesel by esterification. Specifically, the lipids were reacted with methanol under H2SO4 catalysis (concentrations: 1%, 5%, and 10% (w/w) of lipids) at 100 °C for 1 h in a Pyrex-glass tube with a Teflon-sealed screw-cap. The excess methanol (lipids:methanol = 1:1, w/w) was then used to enhance the biodiesel conversion efficiency. Finally, after washing with distilled water and 10,000 rpm centrifugation for 5 min, the fatty acid methyl ester (FAME, biodiesel) content was analyzed. All of the experiments were performed in duplicate.

¼ log R0 þ log½Hþ 

ð2Þ

The severity and combined severity indices, given their assumption that a first-order reaction takes place, can be considered to be proximate methods (Chum et al., 1990; Fernandez-Bolanos et al., 2001; Park et al., 2012). 3. Results and discussion 3.1. Fatty acids and polysaccharide contents of microalgae

2.4. Analyses The fatty acids content of the microalgae was analyzed using the modified direct esterification method (Lepage and Roy, 1984).

The C. vulgaris fatty acids contents and compositions are summarized in Table 1. The total fatty acids content was 381.6 mg/g cell; the palmitic acid content was 100.5 mg/g cell (26.3% of total

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Table 1 Fatty acids contents and compositions of C. vulgaris and its microalgal lipids. Fatty acids

C. vulgaris

Microalgal lipids

Content (mg/g cell)

Composition (%)

Content (mg/g lipid)

Composition (%)

Myristic acid (C14:0) Palmitic acid (C16:0) Palmitoleic acid (C16:1) Stearic acid (C18:0) Oleic acid (C18:1) Linoleic acid (C18:2) Linolenic acid (C18:3) Others

1.0 100.5 0.8 29.2 83.5 81.9 29.0 55.7

0.3 26.3 0.2 7.7 21.9 21.5 7.6 14.6

2.6 244.0 13.8 72.0 208.9 196.0 66.8 131.0

0.3 26.1 1.5 7.7 22.3 21.0 7.1 14.0

Total

381.6

100.0

935.0

100.0

fatty acids), the oleic acid content, 83.5 mg/g cell (21.9% of total fatty acids), and the linoleic acid content, 81.9 mg/g cell (21.5% of total fatty acids). The remaining 55.7 mg/g cell fatty acids were assumed to be polyunsaturated carbon fatty acids or odd-numberchain fatty acids. Lipid extraction from lyophilized cells using organic solvents was examined to confirm the dry-extraction property of C. vulgaris. The lipid-extraction yields and fatty acids contents with the three organic solvents are plotted in Fig. 1. The highest yield, 370.5 mg/g cell, was achieved with the chloroform–methanol solvent. The yields with the hexane-methanol and hexane solvents were 289.5 and 132.0 mg/g cell, respectively. The low extraction yield with hexane was due to the extraction of mainly neutral lipids by this nonpolar solvent. Chloroform–methanol’s highest yield, contrastingly, reflected the high level of impurities due to the additional materials extracted by polar methanol; the conversion to FAME (biodiesel), accordingly in this case, was expected to be limited. The total fatty acids content with the chloroform–methanol solvent, correspondingly, was a low 532.1 mg/g lipid. The total fatty acids contents with the hexane-methanol and hexane solvents, respectively 994.0 and 978.0 mg/g lipid, were considerably higher, owing to the lower impurity levels in their extracted lipids. The C. vulgaris’ total polysaccharide content, an effective sugar source for bioethanol production, was 20.0% (w/w); this consisted of 14.9% (w/w) glucan (hexose) and 5.1% (w/w) xylan–mannan– galactan (pentose).

3.2. Lipid extraction by acid-catalyzed hot-water treatment Cell broth with a cell concentration of 20 g/L was prepared by centrifugation. As a control, lipids were extracted by hexane without heating or catalysis. The broth was loaded with sulfuric acid concentrations of 0%, 0.25%, 0.5%, 0.75%, 1%, 2%, and 3% (120 °C for 60 min). Fig. 2a plots the lipid-extraction yields after acid-catalyzed hot-water treatments. For comparison, the yield was very low with no heating and no catalyst: 83.2 mg/g cell. Under the hot-water condition, contrastingly, the lipid-extraction yield increased with sulfuric acid concentration. Notably, there was a considerable difference between the 0% (no catalyst) and 0.25% sulfuric acid conditions, namely a 2.6 times increase of yield, which reflected the acid catalyst’s remarkable yield-enhancement efficiency owing to cell-wall disruption. With the 0.5% sulfuric acid concentration, the yield became at over 300 mg/g cell, after which the rate of increase slowed. As is apparent, the curve became stationary after the concentration reached 1%. The concentrations of sugars and their decomposition products after the 1% and 3% sulfuric acid treatments at 120 °C for 60 min were analyzed. Whereas it is known that severe acidic conditions lead to production of sugar-decomposition products such as 5-hydroxymethyl furfural (HMF) and furfural (Park et al., 2012; Hendriks and Zeeman, 2009), neither were detected after those treatments. In other words, less-than-3% sulfuric acid concentrations heated at 120 °C for 60 min did not induce microalgae

1000

800

300

600 200 400

100 200

0

Fatty acids content (mg/g lipid)

Lipid extraction yield (mg/g cell)

400

0 Hexane

Hexane-methanol

Chloroform-methanol

Fig. 1. Lipid-extraction yields by room-temperature organic solvents for 6 h (h) and fatty acids contents of microalgal lipids extracted from C. vulgaris (j).

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400

(c) Lipid extraction yield (mg/g cell)

Lipid extraction yield (mg/g cell)

(a) 300

200

100

0

300

200

100

0

0.0

0.5

1.0

1.5

2.0

2.5

3.0

120

130

150

160

400

400

(b)

(d) Lipid extraction yield (mg/g cell)

Lipid extraction yield (mg/g cell)

140

Temperature (C)

Concentration of sulfuric acid (%)

300

200

100

300

200

100

0

0 20

40

60

Reaction time (min)

0.25

0.50

0.75

1.00

Concentration of sulfuric acid (%)

Fig. 2. Lipid-extraction yields after acid-catalyzed hot-water treatment (a) with various sulfuric acid concentrations at 120 °C and 60 min, (b) with reaction times at 1% sulfuric acid concentration and 120 °C, (c) with reaction temperatures according to severity index, and (d) with sulfuric acid concentrations according to severity index.

decomposition into smaller molecules. The glucose concentrations at the 1% and 3% sulfuric acid concentrations were 3.27 and 3.31 g/ L, respectively, and the xylose–mannose–galactose (XMG) concentrations were 0.96 and 1.08 g/L. Pentose and hexose derived from cells can be converted to bioethanol by fermentation. The theoretical maximum concentrations of glucose and XMG are 3.31 and 1.16 g/L. Most of the initial polysaccharides were hydrolyzed to sugars without additional decomposition. For the 1% sulfuric acid concentration (at which, as noted above, the lipid-extraction yield curve became stationary), the reaction time was changed from 20 to 40 and then from 40 to 60 min. As Fig. 2b illustrates, the lipid-extraction yield gradually increased with time. When sulfuric acid was used as a catalyst for hot-water treatment of C. vulgaris, the boundary line between the upper hexane layer (containing lipids) and the lower layer (containing water and sulfuric acid) was very clear. As such, full recovery of the hexane layer, without any loss, was possible. A distinct boundary line reflects demulsifying sulfuric acid’s deterioration of emulsion stability (Dass and Hamdaoui, 2010). Conversely, with no catalyst (0% sulfuric acid), the boundary line between the upper and lower layers was, due to emulsification, unclear. Correspondingly, recovery of the hexane layer was incomplete, and resulted in a low lipidextraction yield (Fig. 2a).

3.3. Hot-water lipid extraction according to severity index Based on a 1% sulfuric acid concentration heated at 120 °C for 60 min, the lipid-extraction yield for which was 337.4 mg/g cell, the reaction temperature and reaction time were varied according to the same severity index. When the sulfuric acid concentration was treated as a constant, the combined severity index was simplified as a function of reaction time (t, min) and temperature (T, C):

t1  expððT 1  100Þ=14:75Þ ¼ t 2  expððT 2  100Þ=14:75Þ

ð3Þ

Because the same severity index represents the same intensity of treatment, a similar lipid-extraction performance was expected. Using 1% sulfuric acid concentration as a constant, the conditions prevailing for the 120–160 °C temperature range were applied with the same severity index (120 °C and 60 min; 130 °C and 31 min; 140 °C and 16 min; 150 °C and 8 min; and 160 °C and 4 min). Fig. 2c shows that between 120 and 150 °C, the lipid-extraction yields were comparable. Therefore, the severity index was a useful tool for reducing the reaction time by means of elevated temperature. At 160 °C, however, a lower yield, due to the too-short contact time (4 min) between the microalgae and the sulfuric acid solution, was observed. According to the

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severity index, the reaction time can be reduced from 60 to 8 min. Using 150 °C as a constant, various sulfuric acid concentrations, namely 0.25%, 0.5%, 0.75%, and 1%, were applied with the same severity index (reaction time: 32, 16, 11, and 8 min, respectively). When the temperature was a constant, the combined severity index was simplified as a function of reaction time (t, min) and sulfuric acid concentration ([H+], %):

t 1  ½Hþ 1 ¼ t2  ½Hþ 2

ð4Þ

At the 0.5%, 0.75% and 1% sulfuric acid concentrations, similar results were observed (Fig. 2d). At 0.25%, a slight lower yield was apparent, due to the weak acid concentration and resultant insufficient damage to cell walls. The sulfuric acid concentration can be reduced from 1% to 0.5% according to the severity index, thereby diminishing the risk of acid-induced reactor corrosion. Under the 1% sulfuric acid concentration heated at 120 °C for 60 min, the total fatty acids content of microalgal lipids, as listed in Table 1, was 935.0 mg/g lipid. The fatty acids compositions of C. vulgaris and the microalgal lipids were almost the same. Therefore, it could be deduced that the lipids that C. vulgaris initially contained were extracted without change incurred to its structure or composition by sulfuric acid or heating, respectively. The compositions of palmitic acid, oleic acid, and linoleic acid were 26.3%, 21.9%, and 21.5% for the microalgae and 26.1%, 22.3%, and 21.0% for the microalgal lipids, respectively.

3.4. Microalgal lipids conversion to biodiesel To examine the feasibility of the conversion of extracted microalgal lipids to biodiesel, esterification was performed under methanol and sulfuric acid conditions. It was found that the conversion reaction was hindered by impurities, such as chlorophyll, in the extracted lipids. Under the 10% sulfuric acid concentration, the biodiesel’s FAME content was 79.2%; under the 1% and 5% concentrations, the FAME content was lower, 51.8% and 72.2%. In order both to increase the FAME content and decrease the amount of catalytic sulfuric acid, pre-esterification upgrading of lipids quality would be required.

4. Conclusion The efficiency of acid-catalyzed hot-water extraction of lipids from C. vulgaris was investigated. From C. vulgaris containing 381.6, 337.4 mg lipids/g cell was extracted under a 1% sulfuric acid concentration heated at 120 °C for 60 min. Sulfuric acid, as a demulsifier, effected a clear phase separation, which resulted in a high upper-solvent-layer recovery yield and induced cell-wall disruption. Although microalgal lipids can be converted to biodiesel by esterification, pre-esterification upgrading of lipids quality, for example by removal of chlorophyll, is required for increased FAME content.

Acknowledgements This work was supported by the New & Renewable Energy Technology Development Program of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) grant funded by the Korea government Ministry of Knowledge Economy (No. 20123010090010) and by the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Ministry of Education, Science and Technology (ABC-2012-053880). References Cho, H.S., Oh, Y.K., Park, S.C., Lee, J.W., Park, J.Y., 2013. Effects of enzymatic hydrolysis on lipid extraction from Chlorella vulgaris. Renew. Energy 54, 156– 160. Chum, H.L., Johnson, D.K., Black, S.K., Overend, R.P., 1990. Pretreatment-catalyst effects and the combined severity parameter. Appl. Biochem. Biotechnol. 24 (25), 1–14. Dass, A., Hamdaoui, O., 2010. Extraction of anionic dye from aqueous solutions by emulsion liquid membrane. J. Hazard. Mater. 178, 973–981. Demirbas, A., 2009. Production of biodiesel from algae oils. Energy Source A31, 163– 168. Fernandez-Bolanos, J., Felizon, B., Heredia, A., Rodriguez, R., Guillen, R., Jimenez, A., 2001. Steam-explosion of olive stones: hemicellulose solubilization and enhancement of enzymatic hydrolysis of cellulose. Bioresour. Technol. 79, 53– 61. Fu, C.C., Hung, T.C., Chen, J.Y., Su, C.H., Wu, W.T., 2010. Hydrolysis of microalgae cell walls for production of reducing sugar and lipid extraction. Bioresour. Technol. 100, 8750–8754. Hendriks, A.T.W.M., Zeeman, G., 2009. Pretreatment to enhance the digestibility of lignocellulosic biomass. Bioresour. Technol. 100, 10–18. Lee, Y.C., Huh, Y.S., Farooq, W., Chung, J., Han, J.I., Shin, H.J., Jeong, S.H., Lee, J.S., Oh, Y.K., Park, J.Y., 2013. Lipid extractions from docosahexaenoic acid (DHA)-rich and oleaginous Chlorella sp. biomasses by organic-nanoclays. Bioresour. Technol. 137, 74–81. Lepage, G., Roy, C.C., 1984. Improved recovery of fatty acid through direct transesterification without prior extraction or purification. J. Lipid Res. 25, 1391–1396. Li, Q., Du, W., Liu, D., 2008. Perspectives of microbial oils for biodiesel production. Appl. Microbiol. Biotechnol. 80, 749–756. Mosier, N., Hendrickson, R., Ho, N., Sedlak, M., Ladisch, M.R., 2005. Optimization of pH controlled liquid hot water pretreatment of corn stover. Bioresour. Technol. 96, 1986–1993. NREL, 2004. Chemical Analysis and Testing Laboratory Analytical Procedures (CAT). National Renewable Energy Laboratory, Golden, CO, USA. Park, J.Y., Kang, M., Kim, J.S., Lee, J.P., Choi, W.I., Lee, J.S., 2012. Enhancement of enzymatic digestibility of Eucalyptus grandis pretreated by NaOH catalyzed steam explosion. Bioresour. Technol. 123, 707–712. Park, J.Y., Kim, D.K., Wang, Z.M., Lu, P., Park, S.C., Lee, J.S., 2008. Production and characterization of biodiesel from tung oil. Appl. Biochem. Biotechnol. 148, 109–117. Schenk, P.M., Thomas-Hall, S.R., Stephens, E., Marx, U.C., Mussgnug, J.H., Posten, C., Kruse, O., Kankamer, B., 2008. Second generation biofuels: high efficiency microalgae for biodiesel production. Bioenerg. Res. 1, 20–43. Shin, H.J., Park, J.H., Jung, W.K., Cho, H., Kim, S.W., 2011. Development of biorefinery process using microalgae. J. Korean Soc. Precis. Eng. 28, 154–167. Sugiyama, J., Vuong, R., Chanzy, H., 1991. Electron diffraction study on the two crystalline phases occurring in native cellulose from an algal cell wall. Macromolecules 24, 4168–4175. Sun, Y., Cheng, J.Y., 2002. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour. Technol. 83, 1–11. Xu, H., Miao, X., Wu, Q., 2006. High quality biodiesel production from a microalga Chlorella protothecoides by heterotrophic growth in fermenters. J. Biotechnol. 126, 499–507. Yu, Q., Zhuang, X., Lv, S., He, M., Zhang, Y., Yuan, Z., Qi, W., Wang, Q., Wang, W., Tan, X., 2013. Liquid hot water pretreatment of sugarcane bagasse and its comparison with chemical pretreatment methods for the sugar recovery and structural changes. Bioresour. Technol. 129, 592–598.

Acid-catalyzed hot-water extraction of lipids from Chlorella vulgaris.

Acid-catalyzed hot-water treatment for efficient extraction of lipids from a wet microalga, Chlorella vulgaris, was investigated. For an initial fatty...
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