Journal of Applied Phycology

, Volume 26, Issue 3, pp 1399–1413 | Cite as

Lipid productivity and fatty acid composition-guided selection of Chlorella strains isolated from Malaysia for biodiesel production

  • Vejeysri Vello
  • Siew-Moi Phang
  • Wan-Loy Chu
  • Nazia Abdul Majid
  • Phaik-Eem Lim
  • Soh-Kheang Loh


The need to develop biomass-based domestic production of high-energy liquid fuels (biodiesel) for transportation can potentially be addressed by exploring microalgae with high lipid content. Selecting the strains with adequate oil yield and quality is of fundamental importance for a cost-efficient biofuel feedstock production based on microalgae. This work evaluated 29 strains of Chlorella isolated from Malaysia as feedstock for biodiesel based on volumetric lipid productivity and fatty acid profiles. Phylogenetic studies based on 18S rRNA gene revealed that majority of the strains belong to true Chlorella followed by Parachlorella. The strains were similarly separated into two groups based on fatty acid composition. Of the 18 true Chlorella strains, Chlorella UMACC187 had the highest palmitic acid (C16:0) content (71.3 ± 4.2 % total fatty acids, TFA) followed by UMACC84 (70.1 ± 0.7 %TFA), UMACC283 (63.8 ± 0.7 %TFA), and UMACC001 (60.3 ± 4.0 %TFA). Lipid productivity of the strains at exponential phase ranged from 34.53 to 230.38 mg L−1 day−1, with Chlorella UMACC050 attaining the highest lipid productivity. This study demonstrated that Chlorella UMACC050 is a promising candidate for biodiesel feedstock production.


Algae Biodiesel Biomass productivity Biotechnology Chlorella Fatty acid Lipid 



The first author is grateful to the University of Malaya Bright Scheme Program for the financial support given. Special thanks to the collaborator from the National University of Singapore (NUS), Prof. Dr. Chew Fook Tim, for technical support and advice. The project was funded by the Postgraduate Research Fund (Ref: PS301/2010B and PV032/2011B) and Malaysian Palm Oil Board (MPOB, Ref: 55-02-03-1054).


  1. Becker EW (1994) Microalgae: biotechnology and microbiology. Cambridge University Press, CambridgeGoogle Scholar
  2. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917PubMedCrossRefGoogle Scholar
  3. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  4. Brennan L, Owende P (2010) Biofuels from microalgae—a review of technologies for production, processing, and extractions of biofuels and co-products. Renew Sust Energ Rev 14:557–577CrossRefGoogle Scholar
  5. Canakci M, Sanli H (2008) Biodiesel production from various feedstocks and their effects on the fuel properties. J Ind Microbiol Biot 35:431–441CrossRefGoogle Scholar
  6. Cha TS, Chen JW, Goh EG, Aziz A, Loh SH (2011) Differential regulation of fatty acid biosynthesis in two Chlorella species in response to nitrate treatments and the potential of binary blending microalgae oils for biodiesel application. Bioresour Technol 102:10633–10640PubMedCrossRefGoogle Scholar
  7. Chisti Y (2007) Biodiesel from microalgae. Biotech Adv 25:294–306CrossRefGoogle Scholar
  8. Chu WL, See YC, Phang SM (2009) Use of immobilised Chlorella vulgaris for the removal of colour from textile dyes. J Appl Phycol 21:641–648CrossRefGoogle Scholar
  9. Dahiya A (2012) integrated approach to algae production for biofuel utilizing robust algal species. In: Gordon R, Seckbach J (eds) The science of algal fuels, vol 25. Cellular origin, life in extreme habitats and astrobiology. Springer, Netherlands, pp 83–100Google Scholar
  10. Doan QC, Moheimani NR, Mastrangelo AJ, Lewis DM (2012) Microalgal biomass for bioethanol fermentation: implications for hypersaline systems with an industrial focus. Biomass Bioenerg 46:79–88CrossRefGoogle Scholar
  11. D’oca MGM, Viegas CV, Lemoes JS, Miyasaki EK, Moron Villarreyes JA, Primel EG, Abreu PC (2011) Production of FAMEs from several microalgal lipidic extracts and direct transesterification of the Chlorella pyrenoidosa. Biomass Bioenerg 35:1533–1538CrossRefGoogle Scholar
  12. DuBois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356CrossRefGoogle Scholar
  13. European Committee for Standardization (CEN) (2013) International biodiesel standard for vehicles (EN14214). Accessed 31.01.2013
  14. Feng Y, Li C, Zhang D (2011) Lipid production of Chlorella vulgaris cultured in artificial wastewater medium. Bioresour Technol 102:101–105PubMedCrossRefGoogle Scholar
  15. Gladu PK, Patterson GW, Wikfors GH, Smith BC (1995) Sterol, fatty-acid, and pigment characteristics of UTEX-2341, a marine eustigmatophyte identified previously as Chlorella minutissima (Chlorophyceae). J Phycol 31:774–777CrossRefGoogle Scholar
  16. Griffiths MJ, Harrison STL (2009) Lipid productivity as a key characteristic for choosing algal species for biodiesel production. J Appl Phycol 21:493–507CrossRefGoogle Scholar
  17. Hanagata N, Karube I, Chihara M, Silva PC (1998) Reconsideration of the taxonomy of ellipsoidal species of Chlorella (Trebouxiophyceae, Chlorophyta), with establishment of Watanabea gen. nov. Phycol Res 46:221–229CrossRefGoogle Scholar
  18. Harun R, Danquah MK, Forde GM (2010) Microalgal biomass as a fermentation feedstock for bioethanol production. J Chem Technol Biot 85:199–203Google Scholar
  19. Harun R, Davidson M, Doyle M, Gopiraj R, Danquah M, Forde G (2011) Technoeconomic analysis of an integrated microalgae photobioreactor, biodiesel and biogas production facility. Biomass Bioenerg 35:741–747CrossRefGoogle Scholar
  20. Hempel N, Petrick I, Behrendt F (2012) Biomass productivity and productivity of fatty acids and amino acids of microalgae strains as key characteristics of suitability for biodiesel production. J Appl Phycol 24:1407–1418PubMedCentralPubMedCrossRefGoogle Scholar
  21. Heredia Arroyo T, Wei W, Ruan R, Hu B (2011) Mixotrophic cultivation of Chlorella vulgaris and its potential application for the oil accumulation from non-sugar materials. Biomass Bioenerg 35:2245–2253CrossRefGoogle Scholar
  22. 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 54:621–639PubMedCrossRefGoogle Scholar
  23. Huerlimann R, de Nys R, Heimann K (2010) Growth, lipid content, productivity, and fatty acid composition of tropical microalgae for scale-up production. Biotechnol Bioeng 107:245–257PubMedCrossRefGoogle Scholar
  24. Ichihara K, Fukubayashi Y (2010) Preparation of fatty acid methyl esters for gas–liquid chromatography. J Lipid Res 51:635–640PubMedCentralPubMedCrossRefGoogle Scholar
  25. Knothe G, Matheaus AC, Ryan TW (2003) Cetane numbers of branched and straight-chain fatty esters determined in an ignition quality tester. Fuel 82:971–975CrossRefGoogle Scholar
  26. Krienitz L, Hegewald EH, Hepperle D, Huss VAR, Rohr T, Wolf M (2004) Phylogenetic relationship of Chlorella and Parachlorella gen. nov. (Chlorophyta, Trebouxiophyceae). Phycologia 43:529–542CrossRefGoogle Scholar
  27. Lammens TM, Franssen MCR, Scott EL, Sanders JPM (2012) Availability of protein-derived amino acids as feedstock for the production of bio-based chemicals. Biomass Bioenerg 44:168–181CrossRefGoogle Scholar
  28. Lang IK, Hodac L, Friedl T, Feussner I (2011) Fatty acid profiles and their distribution patterns in microalgae: a comprehensive analysis of more than 2000 strains from the SAG culture collection. BMC Plant Biol 11:124PubMedCentralPubMedCrossRefGoogle Scholar
  29. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) ClustalW and ClustalX version 2.0. Bioinformatics 23:2947–2948Google Scholar
  30. Lim SL, Chu WL, Phang SM (2010) Use of Chlorella vulgaris for bioremediation of textile wastewater. Bioresour Technol 101:7314–7322PubMedCrossRefGoogle Scholar
  31. Luo W, Pröschold T, Bock C, Krienitz L (2010) Generic concept in Chlorella-related coccoid green algae (Chlorophyta, Trebouxiophyceae). Plant Biol 12:545–553PubMedCrossRefGoogle Scholar
  32. Miller R, Wu G, Deshpande RR, Vieler A, Gartner K, Li X, Moellering ER, Zauner S, Cornish AJ, Liu B, Bullard B, Sears BB, Kuo MH, Hegg EL, Shachar-Hill Y, Shiu SH, Benning C (2010) Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism. Plant Physiol 154:1737–1752PubMedCentralPubMedCrossRefGoogle Scholar
  33. Monyem AH, Van Gerpen J (2001) The effect of biodiesel oxidation on engine performance and emissions. Biomass Bioenerg 20:317–325CrossRefGoogle Scholar
  34. Morita M, Watanabe Y, Saiki H (2000) High photosynthetic productivity of green microalga Chlorella sorokiniana. Appl Biochem Biotechnol 87:203–218PubMedCrossRefGoogle Scholar
  35. Mustafa EM, Phang SM, Chu WL (2012) Use of an algal consortium of five algae in the treatment of landfill leachate using the high-rate algal pond system. J Appl Phycol 24:953–963CrossRefGoogle Scholar
  36. Nakamura Y, Ohta H (2010) Phosphatidic acid phosphatases in seed plants. In: Munnik T (ed) Lipid signaling in plants. Springer, Berlin, pp 131–141CrossRefGoogle Scholar
  37. Neustupa J, Němcová Y, Eliáš M, Škaloud P (2009) Kalinella bambusicola gen. et sp. nov. (Trebouxiophyceae, Chlorophyta), a novel coccoid Chlorella-like subaerial alga from Southeast Asia. Phycol Res 57:159–169CrossRefGoogle Scholar
  38. Petkov G, Garcia G (2007) Which are fatty acids of the green alga Chlorella? Biochem Sys Ecol 35:281–285CrossRefGoogle Scholar
  39. Phang SM (1990) Algal production from agroindustrial and agricultural wastes in Malaysia. Ambio 19:415–418Google Scholar
  40. Phang SM, Chu WL (1999) University of Malaya Algae Culture Collection (UMACC) catalogue of strains. Institute of Postgraduate Studies and Research, Kuala LumpurGoogle Scholar
  41. Phang SM, Ong KC (1988) Algal biomass production in digested palm oil mill effluent. Biol Waste 25:177–191CrossRefGoogle Scholar
  42. Phang SM, Miah MS, Yeoh BG, Hashim MA (2000) Spirulina cultivation in digested sago starch factory wastewater. J Appl Phycol 12:395–400CrossRefGoogle Scholar
  43. Phukan MM, Chutia RS, Konwar BK, Kataki R (2011) Microalgae Chlorella as a potential bio-energy feedstock. Appl Energ 88:3307–3312CrossRefGoogle Scholar
  44. Praveenkumar R, Shameera K, Mahalakshmi G, Akbarsha MA, Thajuddin N (2012) Influence of nutrient deprivations on lipid accumulation in a dominant indigenous microalga Chlorella sp., BUM11008: evaluation for biodiesel production. Biomass Bioenerg 37:60–66CrossRefGoogle Scholar
  45. Pribyl P, Cepak V, Zachleder V (2012) Production of lipids in 10 strains of Chlorella and Parachlorella, and enhanced lipid productivity in Chlorella vulgaris. Appl Microbiol Biotechnol 94:549–561PubMedCrossRefGoogle Scholar
  46. Renaud SM, Parry DL, Thinh LV (1994) Microalgae for use in tropical aquaculture I: gross chemical and fatty acid composition of twelve species of microalgae from the Northern Territory, Australia. J Appl Phycol 6:337–345CrossRefGoogle Scholar
  47. Rodolfi L, Zittelli GC, Bassi N, Padovani G, Biondi N, Bonini G, Tredici MR (2009) Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnol Bioeng 102:100–112PubMedCrossRefGoogle Scholar
  48. Safi C, Charton M, Pignolet O, Silvestre F, Vaca-Garcia C, Pontalier P-Y (2013) Influence of microalgae cell wall characteristics on protein extractability and determination of nitrogen-to-protein conversion factors. J Appl Phycol 25:523–529CrossRefGoogle Scholar
  49. Sato N, Tsuzuki M, Kawaguchi A (2003) Glycerolipid synthesis in Chlorella kessleri 11h. I. Existence of a eukaryotic pathway. Biochim Biophys Acta 1633:27–34PubMedCrossRefGoogle Scholar
  50. Schenk P, Thomas Hall S, Stephens E, Marx U, Mussgnug J, Posten C, Kruse O, Hankamer B (2008) Second generation biofuels: high-efficiency microalgae for biodiesel production. Bioenerg Res 1:20–43CrossRefGoogle Scholar
  51. Strickland JDH, Parsons TR (1968) A practical handbook of seawater analysis. Bulletin 167. Fisheries Research Board of Canada, OttawaGoogle Scholar
  52. Swofford DL (2003) PAUP*. Phylogenetic Analysis Using Parsimony (* and other methods). Version 4. Sinauer Associates, Sunderland, MassachusettsGoogle Scholar
  53. Talebi AF, Mohtashami SK, Tabatabaei M, Tohidfar M, Bagheri A, Zeinalabedini M, Hadavand Mirzaei H, Mirzajanzadeh M, Malekzadeh Shafaroudi S, Bakhtiari S (2013) Fatty acids profiling: a selective criterion for screening microalgae strains for biodiesel production. Algal Research 2:258–267CrossRefGoogle Scholar
  54. Tang H, Chen M, Garcia ME, Abunasser N, Ng KY, Salley SO (2011) Culture of microalgae Chlorella minutissima for biodiesel feedstock production. Biotechnol Bioeng 108:2280–2287CrossRefGoogle Scholar
  55. Teoh ML, Chu WL, Marchant H, Phang SM (2004) Influence of culture temperature on the growth, biochemical composition and fatty acid profiles of six Antarctic microalgae. J Appl Phycol 16:421–430CrossRefGoogle Scholar
  56. Thi TYD, Sivaloganathan B, Obbard JP (2011) Screening of marine microalgae for biodiesel feedstock. Biomass Bioenerg 35:2534–2544CrossRefGoogle Scholar
  57. Vairappan C, Yen A (2008) Palm oil mill effluent (POME) cultured marine microalgae as supplementary diet for rotifer culture. J Appl Phycol 20:603–608CrossRefGoogle Scholar
  58. Wu HL, Hseu RS, Lin LP (2001) Identification of Chlorella spp. isolates using ribosomal DNA sequences. Bot Bull Acad Sin 42:115–121Google Scholar
  59. Yamamoto M, Kurihara I, Kawano S (2005) Late type of daughter cell wall synthesis in one of the Chlorellaceae, Parachlorella kessleri (Chlorophyta, Trebouxiophyceae). Planta 221:766–775PubMedCrossRefGoogle Scholar
  60. Zheng H, Yin J, Gao Z, Huang H, Ji X, Dou C (2011) Disruption of Chlorella vulgaris cells for the release of biodiesel-producing lipids: a comparison of grinding, ultrasonication, bead milling, enzymatic lysis, and microwaves. Appl Biochem Biotechnol 164:1215–1224PubMedCrossRefGoogle Scholar
  61. Zhou X, Ge H, Xia L, Zhang D, Hu C (2013) Evaluation of oil-producing algae as potential biodiesel feedstock. Bioresource Technol 134:24–29CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Vejeysri Vello
    • 1
    • 2
  • Siew-Moi Phang
    • 1
    • 2
  • Wan-Loy Chu
    • 3
  • Nazia Abdul Majid
    • 4
  • Phaik-Eem Lim
    • 1
    • 2
  • Soh-Kheang Loh
    • 5
  1. 1.Institute of Biological Sciences, Faculty of ScienceUniversity of MalayaKuala LumpurMalaysia
  2. 2.Institute of Ocean and Earth Sciences (IOES)University of MalayaKuala LumpurMalaysia
  3. 3.International Medical University (IMU)Kuala LumpurMalaysia
  4. 4.Genetics and Molecular Biology Unit, Institute of Biological Sciences, Faculty of ScienceUniversity of MalayaKuala LumpurMalaysia
  5. 5.Malaysian Palm Oil BoardKajangMalaysia

Personalised recommendations