Influence of Different Extraction Methods on Fatty Acid
Composition of Lipid Extracts of Chlorella vulgaris

Beijerinck from Laguna De Bay, Philippines

Rochelle Joie A. Saracanlao1*, June Owen Nacorda2, and Hidelisa P. Hernandez3

1Agricultural Systems Institute, College of Agriculture and Food Science,
University of the Philippines Los Baños (UPLB), College, Laguna 4031 Philippines
2Institute of Biological Sciences, College of Arts and Sciences,
UPLB, College, Laguna 4031 Philippines
3Institute of Chemistry, College of Arts and Sciences,
UPLB, College, Laguna 4031 Philippines

*Corresponding author: This email address is being protected from spambots. You need JavaScript enabled to view it.



Chlorella vulgaris Beijirinck cultures isolated from Laguna de Bay were cultivated and the biomass produced after one month of cultivation was subjected to stepwise selection of solvent system, algae-to-solvent ratio, and extraction method. The optimized procedure for C. vulgaris was found to be sonication with 2:1 (v:v) chloroform:methanol as extractant at 1:20 (g:ml) algae:solvent ratio producing 51% crude lipid (dry-weight basis). Using thin layer chromatography, the C. vulgaris lipid extract showed the following approximate composition: triglycerides (79%), sterols (14%), and 1,3-diglycerides present in substantial amounts. Gas chromatography – flame ionization detection of the fatty acids as fatty acid methyl esters showed the following components: myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, and traces of lauric acid. Analysis of the lipid extracts showed that extraction procedure, along with the solvent system, affects both yield and lipid profile. Total lipid yield and sterol yield increase with increasing polarity of the solvent system. Triglycerides, on the other hand, decreases with the polarity of solvent systems used. A more varied fatty acid class was obtained using more polar solvents (8 fatty acids) compared with the usage of less polar ones (1–3 fatty acids).



Microalgae are regarded as a promising sustainable energy resource due to their capacity to amass huge amounts of lipids that performs similarly to petroleum (Sheehan et al. 1998). This has resulted in researches focusing on the cultivation of algae for lipid production – specifically biodiesel – via lipid transesterification. Lipids can be categorized as polar such as glycolipids and phospholipids and neutral/nonpolar lipids such as mono-, di-, and tri-acylglycerides (Greenwell et al. 2010). . . . read more



ALLEN MM, STAINER RY. 1968. Studies with Cyanidium caldarium, an anomalously pigmented chlorophyte. Arc. Mikrobiol. 32: 270–277.
ARGETE A. 2012. Characterization of the lipid and phenolic content of Chlorella vulgaris Beijerinck cultured in BG-11 media at different nitrogen levels. [BS thesis]. Los Baños (Philippines): University of the Philippines Los Baños. 65 p (Available at UPLB Library).
ARINDUQUE R. 2009. Design, fabrication and preliminary testing of an internally illuminated vertical column photobioreactor for the cultivation of Chlorella vulgaris. [BS thesis]. Los Baños (Philippines): University of the Philippines Los Baños. 100p (Available at UPLB Library).
CHINNASAMY S, RAMAKRISHNAN B, BHATNAGAR A, DAS K. 2009. Biomass production potential of wastewater alga Chlorella vulgaris ARC 1 under elevated levels of CO2 and temperature. Int. J. Mol. Sci. 10(2): 518–532.
DEMORT CI, LOWRY R, TINSLEY I, PHINNEY HK. 1972. The biochemical composition of some estuarine phytoplankton species. I. Fatty acid composition. J. Phycol. 8: 211–216.
DEGEN J, UEBELE A, RETZE A, SCHMID-STAIGER U, TROSCH W. 2001. A novel airflift photobioreactor with baffles for improved light utilization through the flashing light effect. J. Biotechnol 92: 89–94.
DOS SANTOS RR, MOREIRA DM, KUNIGAMI CN, ARANDA DAG, TEIXEIRA CML. 2015. Comparison between several methods of total lipid extraction from Chlorella vulgaris biomass. Ultrasonics Sonochemistry 22: 95–99.
[FAO] Food and Agriculture Organization of the United Nations. 1996. Manual on the production and use of live food for aquaculture. FAO Fisheries Technical Paper No. 361. Rome (Italy): FAO. 295p.
GREENWELL HC, LAURENS LML, SHIELDS RJ, LOVITT RW, FLYNN FK. 2010. Placing microalgae on the biofuels priority list: A review of technological challenges. J. R. Soc. Interface 7: 703–726.
HALIM R, DANQUAH NK, WEBLEY PA. 2012. Extraction of oil from microalgae for biodiesel production: A review. Biotechnol. Adv. 30: 709–732.
HUANG GH, CHEN F, WEI D, ZHANG XW, CHEN G. 2010. Biodiesel production by microalgal biotechnology. Appl. Energy. 87: 38–46.
ICHIHARA K, SHIBAHARA A, YAMAMOTO K, NAKAYAMA T. 1996. An improved method for rapid analysis of the fatty acids for glycerolipids. Lipids 31(5): 535–539.
ILLMAN AM, SCRAGG AH, SHALES SW. 2000. Increase in Chlorella strains calorific values when grown in low nitrogen medium. Enzyme Microb. Technol. 27: 631–635.
LAM MK, LEE KT. 2013. Catalytic esterification of high viscosity crude microalgae lipid to biodiesel: Effect of co-solvent. Fuel Process. Technol. 110: 242–248.
LETELLIER M, BUDZINSKI H. 1999. Microwave assisted extraction of organic compounds. Analysis 27: 259.
LEWIS T, NICHOLS PD, MCMEEKIN TA. 2000. Evaluation of extraction methods for recovery of fatty acids from lipid-producing microheterotrophs. J. Microbiol. Methods 43: 107–116.
LI Y, NAVID RM, SCHENK P. 2012. Current research and perspective of microalgal biofuels in Australia. Biofuels 3: 427–439.
LI Y, NAGHDI FG, GARG S, ADARME-VEGA TC, THURECHT KJ, GHAFOR WA, TANNOCK S, SCHENK P. 2014. A comparative study: The impact of different lipid extraction methods on current microalgal lipid research. Microb. Cell Fact. 13: 14.
LIU ZY, WANG GC, ZHOU BC. 2008. Effect of iron on growth and lipid accumulation in Chlorella vulgaris. Bioresour. Technol. 99(11): 4717–22.
LIANG Y, SARKANY N, CUI Y. 2009. Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions. Biotechnol. Lett. 31: 307–315.
LV JM, CHENG LH, XU XH. 2010. Enhanced lipid production of Chlorella vulgaris by adjustment of cultivation conditions. Bioresour Technol, 101: 6796–6804.
MATA TM, MARTINS AA, CAETANO NS. 2010. Microalgae for biodiesel production and other applications: A review. Renew Sust Energ Rev 14: 217–232.
MOHOLKAR VS, PATIL C, RANJAN A. 2010. Mechanistic Assessment of Microalgal Lipid Extraction. Ind. Eng. Chem. Res. 49(6): 2979–85.
MUBARAK M, SHAIJA A, SUCHITHRA TV. 2015. A review on the extraction of lipid from microalgae for biodiesel production. Algal Research 7: 117–123.
MORILLO A. 2010. A study on the growth response of green algae Chlorella vulgaris in both indoor and outdoor conditions using continuous externally-illuminated vertical-column draft-tube photobioreactor. [BS thesis]. Los Baños (Philippines): University of the Philippines Los Baños. 107 p (Available at UPLB Library).
NETO AMP, DE SOUZA RAS, LEON-NINO AD, COSTA DA, TIBURCIO RS, NUNES TA, DE MELLO TCS, KANEMOTO FT, SALDANHA-CORREA FMP, GIANESELLA SMF. 2013. Improvement in microalgae lipid extraction using sonication-assisted method. Renewable Energy 55(C): 525–531.
PETKOV G, GARCIA G. 2007. Which are fatty acids of the green alga Chlorella? Biochem. Syst. Ecol. 35: 281–285.
QIN J. 2005. Bio-hydrocarbons from algae – Impacts of temperature, light and salinity on algae growth, rural industries research and development. Barton (Australia): Rural Industries Research and Development Corporation.
REDONDO M. 2009. Design fabrication and prelimenary testing of lab-scale externally-illuminated vertical column draft-tube airlift photobioreactor for the cultivation of green algae Chorella vulgaris for biodiesel production. [BS thesis]. Los Baños (Philippines): University of the Philippines Los Baños. 62 p (Available at UPLB Library).
RYCKEBOSCH E, MYUYLAERT K, FOUBERT I. 2012. Optimization of an analytical procedure for extraction of lipids from microalgae. J. Am. Oil. Chem. Soc. 89: 189–198.
SAFI C, ZEBIB B, MERAH O, PONTALIER PY, VACA-GARCIA C. 2014. Morphology, composition, production, processing and application of Chlorella vulgaris: A review. Renew. Sust. Energ. Rev. 35: 265–278.
SEARCHINGER T, HEIMLICH R, HOUGHTON R, DONG F, ELOBEID A, FABIOSA J, TOKGOZ S, HAYES T, YU H. 2008. Use of US croplands for biofuels increases greenhouse gases through emissions from land use change. Science 319: 1238–40.
SCHENK P, THOMAS-HALL S, STEPHENS E, MARX U, MUSSNUG J, KRUSE O, HANKAMER B. 2008. Second generation biofuels: High-efficiency microalgae for biodiesel production. Bioenergy Res. 1: 20–43.
SHEEHAN J, DUNAHAY T, BENEMANN J, ROESSLER P. 1998. A look back at the U.S. Department of Energy’s aquatic species program—Biodiesel from algae. Colorado: US Department of Energy.
SHEN Y, PEI ZJ, YUAN WQ, MAO ER. 2009. Effect of nitrogen and method on algae lipid yield. Int. J. Agric. Biol. Eng. 2: 51–57.
SUALI E, SARBATLY R. 2012. Conversion of microalgae to biofuel. Renew. Sust. Energ. Rev. 16: 4316–42.
TSUZUKI M, OHNUMA E, SATO N, TAKAKU T, KAWAGUCHI A. 1990. Effects of CO2 concentration during growth on fatty acid composition in microalgae. Plant Physiol. 93: 851–856.
VOLKMAN J. 1989. Fatty acids of microalgae used as feedstocks in aquaculture. In: Fats for the Future II. Cambie RC ed. Chichester (UK): Ellis Horwood.
WACKER A, BECKER P, ELERT EV. 2002. Food quality effects of unsaturated fatty acids on larvae of the Zebra mussel Dreissena polymorpha. Limnol. Oceanogr. 47: 1242–48.
ZHUKOVA N, AIZDAICHER N. 1995. Fatty acid composition of 15 species of marine microalgae. Phytochemistry 39(2): 351–356.