Skip to main content
SpringerLink
Log in

Lipid Estimation of Surfactant-Extracted Microalgae Oil Using Nile Red

  • Original Paper
  • Published:
Journal of the American Oil Chemists' Society

Abstract

Nile red staining has been used as a lipid quantification technique in many microalgae growth and oil accumulation studies. However, its application in lysed microalgae cells is limited. Therefore, this study focused on lysed microalgae cells and utilized the Nile red staining technique to evaluate oil content and extraction. This study aims to provide a rapid and high-throughput alternative method particularly in the microalgae extraction screening process. Potential interferences such as chlorophyll, β-carotene, soluble protein, and phospholipids were evaluated. The hydrophobic Nile red dye was found to quench in water, therefore the fluorometric measurement has to be completed immediately or within 5 min of dye addition. The fluorescence intensity was also found to be Nile red concentration dependent. The optimum Nile red concentration of 656 ppb was used throughout the study. In microalgae samples containing chlorophyll and carotenoids (such as Nannochloropsis sp.), Nile red fluorescence intensity was significantly reduced in comparison to non-chlorophyll microalgae (Schizochytrium limacinum). Soluble proteins from defatted microalgae did not fluoresce significantly relative to lipids, therefore did not interfere with the method to a high degree. Comparing the optimized Nile red staining method with the gravimetric lipid quantification method, a good linear correlation was found in all three materials tested (soybean oil, Nannochloropsis sp., and Schizochytrium limacinum).

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11

Similar content being viewed by others

References

  1. Mata TM, Martins AA, Caetano NS (2010) Microalgae for biodiesel production and other applications: a review. Renew Sust Energ Rev 14:217–232

    Article  CAS  Google Scholar 

  2. 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 14:557–577

    Article  CAS  Google Scholar 

  3. Bligh EG, Dyer WJ (1959) A rapid method for total lipid extraction and purification. Can J Biochem Physiol 37:911–917

    Article  CAS  Google Scholar 

  4. Cooksey KE, Guckert JB, Williams SA, Callis PR (1987) Fluorometric determination of the neutral lipid content of microalgal cells using Nile red. J Microbiol Methods 6:333–345

    Article  CAS  Google Scholar 

  5. Halim R, Danquah MK, Webley PA (2012) Extraction of oil from microalgae for biodiesel production: a review. Biotechnol Adv 30:709–732

    Article  CAS  Google Scholar 

  6. Lee JY, Yoo C, Jun SY, Ahn CY, Oh HM (2010) Comparison of several methods for effective lipid extraction from microalgae. Bioresour Technol 101:S75–S77

    Article  CAS  Google Scholar 

  7. Mercer P, Armenta RE (2011) Developments in oil extraction from microalgae. Eur J Lipid Sci Technol 113:539–547

    Article  CAS  Google Scholar 

  8. Greenspan P, Fowler SD (1985) Spectrofluorometric studies of the lipid probe, Nile red. J Lipid Res 26:781–789

    CAS  Google Scholar 

  9. Chen W, Zhang C, Song L, Sommerfield M, Hu Q (2009) A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae. J Microbiol Methods 77:41–47

    Article  CAS  Google Scholar 

  10. Huang GH, Chen G, Chen F (2009) Rapid screening method for lipid production in alga based on Nile red fluorescence. Biomass Bioenerg 33:1386–1392

    Article  CAS  Google Scholar 

  11. Chen W, Sommerfield M, Hu Q (2011) Microwave assisted Nile red method for in vivo quantification of neutral lipids in microalgae. Bioresour Technol 102:135–141

    Article  CAS  Google Scholar 

  12. Doan TTY, Obbard JP (2011) Improved Nile red staining of Nannochloropsis sp. J Appl Phycol 23:895–901

    Article  CAS  Google Scholar 

  13. Pick U, Rachutin-Zalogin T (2012) Kinetic anomalies in the interactions of Nile red with microalgae. J Microbiol Methods 88:189–196

    Article  CAS  Google Scholar 

  14. Gerde J, Montalbo-Lomboy M, Yao L, Grewell D, Wang T (2012) Evaluation of microalgae cell disruption by ultrasonic treatment. Bioresour Technol 125:175–181

    Article  CAS  Google Scholar 

  15. Wang G, Wang T (2012) Characterization of lipid components in two microalgae for biofuel application. J Am Oil Chem Soc 89:135–143

    Article  CAS  Google Scholar 

  16. Johnson MB, Wen Z (2009) Production of biodiesel fuel from the microalga Schizochytrium limacinum by direct transesterification of algal biomass. Energ Fuel 23:5179–5183

    Article  CAS  Google Scholar 

  17. Ethier S, Woisard K, Vaughan D, Wen Z (2011) Continuous culture of the microalgae Schizochytrium limacinum on biodiesel-derived crude glycerol for producing docosahexaenoic acid. Bioresour Technol 102:88–93

    Article  CAS  Google Scholar 

  18. Armenta RE, Valentine MC (2013) Single-cell oils as a source of omega-3 fatty acids: an overview of recent advances. J Am Oil Chem 90:167–182

    Article  CAS  Google Scholar 

  19. Folch J, Lees M, Stanley S (1956) A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226(1):497–509

    Google Scholar 

  20. Bradford M (1976) A rapid and sensitive method for the quantification of microgram of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  21. Lakowicz JR (1999) Principles of fluorescence spectroscopy, 2nd edn. Springer Science, New York

    Book  Google Scholar 

  22. Schoefs B (2002) Chlorophyll and carotenoid analysis in food products. Properties of the pigments and method of analysis. Trends Food Sci Technol 13:361–371

    Article  CAS  Google Scholar 

  23. Cerovic ZG, Ounis A, Cartelat A, Latouche G, Goulas Y, Meyer S, Moya I (2002) The use of chlorophyll fluorescence excitation spectra for the nondestructive in site assessment of UV-absorbing compounds in leave. Plant Cell Environ 25:1663–1676

    Article  CAS  Google Scholar 

  24. Gillbro T, Cogdell R (1989) Carotenoid fluorescence. Chem Phys Lett 158(3,4):312–316

    Article  CAS  Google Scholar 

  25. Archanaa S, Moise S, Suraishkumar GK (2012) Chlorophyll interference in microalgal lipid quantification through the Bligh and Dyer method. Biomass Bioenerg 46:805–808

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Dr. Zhiyou Wen for providing assistance in the cultivation of Schizochytrium limacinum cells. Sincere gratitude is also extended to Dr. Linxing Yao and Dr. Peiyou Qin for their support and technical recommendations.

Author information

Authors and Affiliations

  1. Department of Food Science and Human Nutrition, 2312 Food Science Bldg., Iowa State University, Ames, IA, 50011-3080, USA

    Melissa Montalbo-Lomboy, Meryem Nur Kantekin & Tong Wang

Authors
  1. Melissa Montalbo-Lomboy
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Meryem Nur Kantekin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tong Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tong Wang.

About this article

Cite this article

Montalbo-Lomboy, M., Kantekin, M.N. & Wang, T. Lipid Estimation of Surfactant-Extracted Microalgae Oil Using Nile Red. J Am Oil Chem Soc 91, 665–680 (2014). https://doi.org/10.1007/s11746-013-2395-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11746-013-2395-9

Keywords

Search

Navigation