Biodiesel Production from Microalgae: Methods for Microalgal Lipid Assessment with Emphasis on the Use of Flow Cytometry

  • M. Teresa Lopes Da Silva
  • Alberto Reis
Part of the Cellular Origin, Life in Extreme Habitats and Astrobiology book series (COLE, volume 25)


Some microalgae species can accumulate high oil contents which can be used for biodiesel production. When selecting microalgal strains as potential oil producers, improving or scaling-up the microalgal oil production process at an industrial scale, the use of a rapid and accurate technique for lipid production and cell physiological states assessment is crucial. The next chapter will discuss the advantages and drawbacks of the lipid assessment methods currently used.


Nannochloropsis Oculata Microalgal Lipid Crypthecodinium Cohnii Neochloris Oleoabundans Cell Physiological State 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. Adler N, Schmitt M, Altenburger R (2007) Flow cytometry as a tool to study phytotoxic modes-of-action. Environ Toxicol Chem 26:297–306CrossRefGoogle Scholar
  2. Ahlgren G, Uppsala L (1991) Lipid analysis of fresh water microalgae: a method study. Arch Hydrobiol 121:295–303Google Scholar
  3. Bligh E, Dyer WJ (1959) A rapid method for total lipid extraction and purification. Can J Biochem Physiol 37:911–917CrossRefGoogle Scholar
  4. Cabrini L, Landi L, Stefanelli C, Barzantu V, Sechi AM (1992) Extraction of lipids and lipophilic antioxidants from fish tissues – a comparison among different methods. Comp Biochem Physiol 101b:383–386Google Scholar
  5. Cao-Hoang L, Marechal P, Thanh M, Gervais P, Waché Y (2008) Fluorescent probes to evaluate the physiological state and activity of microbial biocatalysts: a guide for prokaryotic and eukaryotic investigation. Biotechnol J 3:890–903CrossRefGoogle Scholar
  6. Carvalho AP, Malcata FX (2005) Preparation of fatty acid methyl esters for gas-chromatographic analysis of marine lipids: insight studies. J Agric Food Chem 53:5049–5059CrossRefGoogle Scholar
  7. Chen W, Zhang C, Song L, Sommerfeld M, Hu Q (2009) A high throughput Nile Red method for quantitative measurement of neutral lipids in microalgae. J Microbiol Methods 77:41–47CrossRefGoogle Scholar
  8. Cheng YS, Zheng Y, VanderGheynst JS (2011) Rapid quantitative analysis using a colorimetric method in a microplate format. Lipids 46:95–103CrossRefGoogle Scholar
  9. Chisti Y (2007) Biodiesel from microalgae. Biotechnol Adv 25:294–306CrossRefGoogle Scholar
  10. Chisti Y (2008) Biodiesel from microalgae beats bioethanol. Trends Biotechnol 26:126–131CrossRefGoogle Scholar
  11. Cid A, Fidalgo P, Herrero C, Abalde J (1996) Toxic effect of copper on the membrane system of a marine diatom measured by flow cytometry. Cytometry 25:32–36CrossRefGoogle Scholar
  12. Cohen Z, Vonshak A, Richmond A (1988) Effect of environmental-conditions on fatty-acid composition of the red alga Porphyridium-cruentum – correlation to growth-rate. J Phycol 4:328–332Google Scholar
  13. Converti A, Casazza A, Oritz E, Perego P, Borghi M (2009) Effect of temperature and nitrogen concentration on the growth and lipid content of Nannochloropsis oculata and Chlorella vulgaris for biodiesel production. Chem Eng Proc 48:1146–1151CrossRefGoogle Scholar
  14. Cooksey K, Guckert J, Williams S, Callis P (1987) Fluorometric determination of the neutral lipid content of microalgal cells using Nile Red. J Microbiol Methods 6:333–345CrossRefGoogle Scholar
  15. Cooney M, Young G, Naggle N (2009) Extraction of bio-oils from microalgae. Sep Purif Rev 38:291–325CrossRefGoogle Scholar
  16. Cooper M, Hardin W, Petersen T, Cattolico R (2010) Visualizing “green oil” in live algal cells. J Biosci Bioeng 109:198–201CrossRefGoogle Scholar
  17. de la Jara A, Mendoza H, Martel A, Molina C, de la Nordstron L, Rosa V, Diaz R (2003) Flow cutometric determination of lipid content in a marine dinoflagellate Crypthecodinium cohnii. J Appl Phycol 15:433–438CrossRefGoogle Scholar
  18. Elsey MI, Raleigh B, Cooney MJ (2007) Fluorescent measurement of microalgal neutral lipids. J Microbiol Methods 68:639–642CrossRefGoogle Scholar
  19. Franklin NM, Stauber JL, Lim RP (2001) Development of flow cytometry-based algal bioassays for assessing toxicity of copper in natural waters. Environ Toxicol Chem 20:160–170CrossRefGoogle Scholar
  20. Franklin NM, Stauber J, Lim R (2004) Development of multispecies algal bioassays using flow cytometry. Environ Toxicol Chem 23:1452–1462CrossRefGoogle Scholar
  21. Franqueira D, Orosa M, Torres E, Herrero C, Cid A (2000) Potential use of flow cytometry in toxicity studies with microalgae. Sci Total Environ 247:119–126CrossRefGoogle Scholar
  22. Fraser S, Green C, Bode H, Giluda N (1987) Selective disruption of gap junctional communication interferes with a patterning process in hydra. Science 3:49–55CrossRefGoogle Scholar
  23. Gao C, Xiong W, Zhang Y, Yuan W, Wu Q (2008) Rapid quantification of lipid in microalgae by time-domain nuclear magnetic resonance. J Microbiol Methods 75:437–440CrossRefGoogle Scholar
  24. Greenspan P, Mayer EP, Fowler SD (1985) Nile Red: a selective fluorescent stain for intracellular lipid droplets. J Cell Biol 100:965–973CrossRefGoogle Scholar
  25. Grima E, Medina A, Giménez A, Pérez J, Camacho F, Sànchez J (1994) Comparison between extraction of lipids and fatty acids from microalgal biomass. J Am Oil Chem Soc 71:955–959CrossRefGoogle Scholar
  26. Haas MJ, Scott KM (2007) Moisture removal substantially improves the efficiency on in situ biodiesel production from soybeans. J Am Oil Chem Soc 84:197–204CrossRefGoogle Scholar
  27. Haas MJ, Scott KM, Foglia TA, Marmer WN (2007) The general applicability on in situ transesterification for the production of fatty acid esters from a variety of feed stocks. J Am Oil Chem Soc 84:963–970CrossRefGoogle Scholar
  28. Hewitt CJ, Nebe-Von-Caron G (2001) An industrial application of multiparameter flow cytometry: assessment of cell physiological state and its application to the study of microbial fermentations. Cytometry 44:179–187CrossRefGoogle Scholar
  29. Lee SJ, Yoon B-D, Oh H-M (1998) Rapid method for the determination of lipid from the green alga Botryococcus braunii. Biotechnol Tech 12:553–556CrossRefGoogle Scholar
  30. Lee J, Yoo C, Jun S, Ahn C, Oh H (2010) Comparison of several methods for effective lipid extraction from microalgae. Bioresour Technol 101:575–577Google Scholar
  31. Lepage G, Roy CC (1984) Improved recovery of fatty acid through direct transesterification without prior extraction or purification. J Lipid Res 25:1391–1396Google Scholar
  32. Liu Z-Y, Wang G, Zhou B-C (2008) Effect of iron on growth and lipid accumulation in Chlorella vulgaris. Bioresour Technol 99:4717–4722CrossRefGoogle Scholar
  33. Luque de Castro MD, Jimenes-Carmons MM, Fernandez-Perez V (1999) Towards more rational techniques for the isolation of valuable essential oils from plants. Trends Anal Chem 18:708–715CrossRefGoogle Scholar
  34. Miao X, Wu Q (2006) Biodiesel production from heterotrophic microalgal oil. Bioresour Technol 97:841–846CrossRefGoogle Scholar
  35. Morowvat M, Rasoul-Amini S, Ghasemi Y (2010) Chlamydomonas as a “new” organism for biodiesel production. Bioresour Technol 101:2059–2062CrossRefGoogle Scholar
  36. Oh S, Han J, Kim Y, Ha J, Kim S, Jeong M, Jeong H, Kim N, Cho J, Yoon W, Lee S, Kang D, Lee H (2009) Lipid production in Porphyridium cruentum grown under different culture conditions. J Biosci Bioeng 108:429–434CrossRefGoogle Scholar
  37. Prado R, García R, Riaboo C, Herrero C, Abalde J, Cid A (2009) Comparison of the sensitivity of different toxicity test endpoints in a microalga exposed to the herbicide paraquat. Environ Int 35:240–247CrossRefGoogle Scholar
  38. Pruvost J, Van Vooren G, Cogne G, Legrand J (2009) Investigation of biomass and lipid production with Neochloris oleoabundans in photobioreactor. Bioresour Technol 100:5988–5995CrossRefGoogle Scholar
  39. Rioboo C, O’Connor J, Prado R, Herrero C, Cid A (2009) Cell proliferation alterations in Chlorella cells under stress conditions. Aquat Toxicol 94:229–237CrossRefGoogle Scholar
  40. Rodriguez-Ruiz J, Belarbi EH, Sanchez JLG, Alonso DL (1998) Rapid simultaneous lipid extraction and transesterification for fatty acid analysis. Biotechnol Tech 12:689–691CrossRefGoogle Scholar
  41. Rudolfi L, Zittelli GC, Bassi N, Padonavi G, Biondi N, Bonini G, Tredici M (2009) Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnol Bioeng 102:100–112CrossRefGoogle Scholar
  42. 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. NREL/TP-580-24190. National Renewable Energy Laboratory, GoldenGoogle Scholar
  43. Silva TL, Santos CA, Reis A (2009a) Multi-parameter flow cytometry as a tool to monitor heterotrophic microalgal batch fermentations for oil production towards biodiesel. Biotechnol Bioprocess Eng 14:330–337CrossRefGoogle Scholar
  44. Silva TL, Reis A, Medeiros R, Oliveira C, Gouveia L (2009b) Monitoring oil production towards biofuel from autotrophic microalgae semi-continuous cultivations by flow cytometry. Appl Biochem Biotechnol 159:568–578CrossRefGoogle Scholar
  45. Stauber JL, Franklin N, Adams M (2002) Applications of flow cytometry to ecotoxicity testing using microalgae. Trends Biotechnol 20:141–143CrossRefGoogle Scholar
  46. Vijayaraghavan K, Hemanathan K (2009) Biodiesel production form freshwater algae. Energy Fuel 23:5448–5453CrossRefGoogle Scholar
  47. Wawrik B, Harriman B (2010) Rapid, colorimetric quantification of lipid from algal cultures. J Microbiol Lipids 80:262–266Google Scholar
  48. Widjaja A, Chien C, Ju Y (2009) Study of increasing lipid production from fresh water microalgae Chlorella vulgaris. J Taiwan Inst Chem Eng 40:13–20CrossRefGoogle Scholar
  49. Xiong W, Li X, Xiang J, Wu Q (2008) High-density fermentation of microalga Chlorella protothecoides in bioreactor for microbio-diesel production. Appl Microbiol Biotechnol 78:29–36CrossRefGoogle Scholar
  50. Xu H, Miaoa X, Wu Q (2006) High quality biodiesel production from a microalga Chlorella protothecoides by heterotrophic growth in fermenters. J Biotechnol 126:499–507CrossRefGoogle Scholar
  51. Yoo C, Jun S-Y, Lee J, Ahn C, Oh H (2010) Selection of microalgae for lipid production under high levels carbon dioxide. Bioresour Technol 101:S71–S74CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  1. 1.Laboratório Nacional de Energia e Geologia, LNEG I.P.Unidade de BioenergiaLisbonPortugal

Personalised recommendations