Methods for quantitative determination of microalgal lipid and fatty acids content
- Authors: Morshchinin I.V.1
-
Affiliations:
- ITMO University
- Issue: Vol 8, No 2 (2025)
- Pages: 267-275
- Section: Articles
- URL: https://journal-vniispk.ru/2618-9771/article/view/310364
- DOI: https://doi.org/10.21323/2618-9771-2025-8-2-267-275
- ID: 310364
Cite item
Full Text
Abstract
Microalgae represent a promising feedstock for sustainable biofuel production and high-value lipid-based bioproducts due to their high lipid productivity and rapid growth rates. Accurate and reproducible lipid quantification is essential for strain selection, process optimization, and industrial scaling. This review presents a comprehensive and critical evaluation of contemporary lipid quantification methods applied to microalgae. The methodologies are categorized into screening, quantitative, and profiling approaches, encompassing techniques such as solvent extraction, in situ and direct transesterification, colorimetric assays, spectroscopic tools (NIR, FTIR), and chromatographic techniques (GC, LC–MS/MS). Each method is evaluated across multiple performance axes, including analytical accuracy, throughput, requirement to the sample, technical complexity, and standardization potential. Results are synthesized using the comparative tables. While high-throughput screening tools (e. g., Nile Red, SPV) offer speed and easiness of using, they exhibit limitations in accuracy and reproducibility. Quantitative methods such as acid-catalyzed in situ transesterification coupled with gas chromatography demonstrate a strong balance between precision and scalability. Profiling methods, including LC–MS/MS, provide the highest molecular resolution but are cost- and labor-intensive. The review highlights the need for methodological harmonization and discusses the trade-offs associated with analytical choices in research and industry. Practical recommendations are proposed for selecting the appropriate techniques depending on application context — from early-stage screening to advanced lipidomic profiling.
Keywords
About the authors
I. V. Morshchinin
ITMO University
Author for correspondence.
Email: keshanowak@gmail.com
49, lit. A, Kronverksky pr., St. Petersburg, 197101
References
- Nguyen, H. T. D., Ramli, A., Kee, L. M. (2017). A review on methods used in analysis of microalgae lipid composition. Journal of the Japan Institute of Energy 96(12), 532–537. https://doi.org/10.3775/jie.96.532
- Zhou, J., Wang, M., Saraiva, J. A., Martins, A. P., Pinto, C. A., Prieto, M. A. et al. (2022). Extraction of lipids from microalgae using classical and innovative approaches. Food Chemistry, 384, Article 132236. https://doi.org/10.1016/j.foodchem.2022.132236
- Yao, L., Gerde, J. A., Lee, S. L., Wang, T., Harrata, K. A. (2015). Microalgae lipid characterization. Journal of Agricultural and Food Chemistry, 63(6), 1773–1787. https://doi.org/10.1021/jf5050603
- Challagulla, V., Nayar, S., Walsh, K., Fabbro, L. (2017). Advances in techniques for assessment of microalgal lipids. Critical Reviews in Biotechnology, 37(5), 566–578. https://doi.org/10.1080/07388551.2016.1206058
- Bligh, E. G., Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37(8), 911–917. https://doi.org/10.1139/o59-099
- Morales, M., Aflalo, C., Bernard, O. (2021). Microalgal lipids: A review of lipids potential and quantification for 95 phytoplankton species. Biomass and Bioenergy, 150, Article 106108. https://doi.org/10.1016/j.biombioe.2021.106108
- Byreddy, A., Gupta, A., Barrow, C., Puri, M. (2016). A quick colorimetric method for total lipid quantification in microalgae. Journal of Microbiological Methods, 125, 28–32. https://doi.org/10.1016/j.mimet.2016.04.002
- Yang, M., Fan, Y., Wu, P.-C., Chu, Y.-D., Shen, P. — L., Xue, S. et al. (2017). An extended approach to quantify triacylglycerol in microalgae by characteristic fatty acids. Frontiers in Plant Science, 8, Article 1949. https://doi.org/10.3389/fpls.2017.01949
- Blanco-Llamero, C., García-García, P., Señoráns, F. J. (2024). Efficient green extraction of nutraceutical compounds from nannochloropsis gaditana: A comparative electrospray ionization LC–MS and GC–MS analysis for lipid profiling. Foods, 13(24), Article 4117. https://doi.org/10.3390/foods13244117
- Folch, J., Lees, M., Sloane Stanley, G. H. (1957). A simple method for the isolation and purification of total lipides from animal tissues. The Journal of Biological Chemistry, 226(1), 497–509. https://doi.org/10.1016/S0021-9258(18)64849-5
- Mishra, S. K., Suh, W. I., Farooq, W., Moon, M., Shrivastav, A., Park, M. S. et al. (2014). Rapid quantification of microalgal lipids in aqueous medium by a simple colorimetric method. Bioresource Technology, 155, 330–333. https://doi.org/10.1016/j.biortech.2013.12.077
- Elsey, D., Jameson, D., Raleigh, B., Cooney, M. J. (2007). Fluorescent measurement of microalgal neutral lipids. Journal of Microbiological Methods, 68(3), 639–642. https://doi.org/10.1016/j.mimet.2006.11.008
- Rumin, J., Bonnefond, H., Saint-Jean, B., Rouxel, C., Sciandra, A., Bernard, O. et al. (2015). The use of fluorescent Nile red and BODIPY for lipid measurement in microalgae. Biotechnology for Biofuels, 8(1), Article 42. https://doi.org/10.1186/s13068-015-0220-4
- Wahlen, B.D., Willis, R.M., Seefeldt, L.C. (2011). Biodiesel production by simultaneous extraction and conversion of total lipids from microalgae, cyanobacteria, and wild mixed-cultures. Bioresource Technology, 102(3), 2724–2730. http://dx.doi.org/10.1016/j.biortech.2010.11.026
- Breuer, G., Lamers, P. P., Martens, D. E., Draaisma, R. B., Wijffels, R. H. (2013). The impact of nitrogen starvation on the dynamics of triacylglycerol accumulation in nine microalgae strains. Bioresource Technology, 124, 217–226. https://doi.org/10.1016/j.biortech.2012.08.003
- Meng, Y., Yao, C., Xue, S., Yang, H. (2014). Application of Fourier transform infrared (FT-IR) spectroscopy in determination of microalgal compositions. Bioresource Technology, 151, 347–354. https://doi.org/10.1016/j.biortech.2013.10.064
- Dean, A. P., Sigee, D. C., Estrada, B., Pittman, J. K. (2010). Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae. Bioresource Technology, 101(12), 4499–4507. https://doi.org/10.1016/j.biortech.2010.01.065
- Iverson, S. J., Lang, S. L., Cooper, M. H. (2001). Comparison of the bligh and dyer and folch methods for total lipid determination in a broad range of marine tissue. Lipids, 36(11), 1283–1287. https://doi.org/10.1007/s11745-001-0843-0
- Saini, R. K., Prasad, P., Shang, X., Keum, Y. -S. (2021). Advances in lipid extraction methods — A review. International Journal of Molecular Sciences, 22(24), Article 13643. https://doi.org/10.3390/ijms222413643
- Lewis, T., Nichols, P. D., McMeekin, T. A. (2000). Evaluation of extraction methods for recovery of fatty acids from lipid-producing microheterotrophs. Journal of Microbiological Methods, 43(2), 107–116. https://doi.org/10.1016/s0167-7012(00)00217-7
- Halim, R., Danquah, M. K., Webley, P. A. (2012). Extraction of oil from microalgae for biodiesel production: A review. Biotechnology Advances, 30(3), 709–732. https://doi.org/10.1016/j.biotechadv.2012.01.001
- Chen, Z., Wang, L., Qiu, S., Ge, S. (2018). Determination of microalgal lipid content and fatty acid for biofuel production. BioMed Research International, 2018, Article 1503126. https://doi.org/10.1155/2018/1503126
- Lee, J.-Y., Yoo, C., Jun, S.-Y., Ahn, C.-Y., Oh, H.-M. (2010). Comparison of several methods for effective lipid extraction from microalgae. Bioresource Technology, 101(1, Supplement), S75-S77. https://doi.org/10.1016/j.biortech.2009.03.058
- 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. Journal of Microbiological Methods, 77(1), 41–47. https://doi.org/10.1016/j.mimet.2009.01.001
- Cooper, M. S., Hardin, W. R., Petersen, T. W., Cattolico, R. A. (2010). Visualizing “green oil” in live algal cells. Journal of Bioscience and Bioengineering, 109(2), 198–201. https://doi.org/10.1016/j.jbiosc.2009.08.004
- Brennan, L., Fernández, A.B., Mostaert, A. S., Owende, P. (2012). Enhancement of BODIPY505/515 lipid fluorescence method for applications in biofuel-directed microalgae production. Journal of Microbiological Methods, 90(2), 137–143. https://doi.org/10.1016/j.mimet.2012.03.020
- Khozin-Goldberg, I., Cohen, Z. (2006). The effect of phosphate starvation on the lipid and fatty acid composition of the fresh water eustigmatophyte Monodus subterraneus. Phytochemistry, 67(7), 696–701. https://doi.org/10.1016/j.phytochem.2006.01.010
- Guschina, I. A., Harwood, J. L. (2006). Lipids and lipid metabolism in eukaryotic algae. Progress in Lipid Research, 45(2), 160–186. https://doi.org/10.1016/j.plipres.2006.01.001
- Triebl, A., Trötzmüller, M., Hartler, J., Stojakovic, T., Köfeler, H. C. (2017). Lipidomics by ultrahigh performance liquid chromatography-high resolution mass spectrometry and its application to complex biological samples. Journal of Chromatography B, 1053, 72–80. https://doi.org/10.1016/j.jchromb.2017.03.027
- Li-Beisson, Y., Thelen, J. J., Fedosejevs, E., Harwood, J. L. (2019). The lipid biochemistry of eukaryotic algae. Progress in Lipid Research, 74, 31–68. https://doi.org/10.1016/j.plipres.2019.01.003
- Ryckebosch, E., Bruneel, C., Termote-Verhalle, R., Goiris, K., Muylaert, K., Foubert, I. (2014). Nutritional evaluation of microalgae oils rich in omega-3 long chain polyunsaturated fatty acids as an alternative for fish oil. Food Chemistry, 160, 393–400. https://doi.org/10.1016/j.foodchem.2014.03.087
- Pääkkönen, S., Pölönen, I., Calderini, M., Yli-Tuomola, A., Ruokolainen, V., Vihinen-Ranta, M. et al. (2025). Lipid monitoring of Chlorella vulgaris using non-invasive near-infrared spectral imaging. Journal of Applied Phycology, 37(1), 205–219. https://doi.org/10.1007/s10811-024-03397-6
- Shao, Y., Gu, W., Qiu, Y. A., Wang, S., Peng, Y., Zhu, Y. M. et al. (2020). Lipids monitoring in Scenedesmus obliquus based on terahertz technology. Biotechnology for Biofuels, 13(1), Article 161. https://doi.org/10.1186/s13068-020-01801-0
- Kiyani, D. A., Maryam, S., Amina, S. J., Ahmad, A., Chattha, M. W. A., Janjua, H. A. (2023). Lipid extraction and analysis of microalgae strain pectinodesmus PHM3 for biodiesel production. BMC Biotechnology, 23(1), Article 20. https://doi.org/10.1186/s12896-023-00784-8
- Bouillaud, D., Drouin, D., Charrier, B., Jacquemmoz, C., Farjon, J., Giraudeau, P. et al. (2020). Using benchtop NMR spectroscopy as an online non-invasive in vivo lipid sensor for microalgae cultivated in photobioreactors. Process Biochemistry, 93, 63–68. https://doi.org/10.1016/j.procbio.2020.03.016
- Bouillaud, D., Heredia, V., Castaing-Cordier, T., Drouin, D., Charrier, B., Gonçalves, O. et al. (2019). Benchtop flow NMR spectroscopy as an online device for the in vivo monitoring of lipid accumulation in microalgae. Algal Research, 43, Article 101624. https://doi.org/10.1016/j.algal.2019.101624
- Cheng, F., Cui, Z., Chen, L., Jarvis, J., Paz, N., Schaub, T. et al. (2017). Hydrothermal liquefaction of high- and low-lipid algae: Bio-crude oil chemistry. Applied Energy, 206, 278–292. https://doi.org/10.1016/j.apenergy.2017.08.105
- Harini, A. B., Sarangi, N. V., Nisha, N., Rajkumar, R. (2023). Cultivation of marine diatom, Amphora sp. in municipal wastewater for enhancing lipids toward sustainable biofuel production. South African Journal of Botany, 155, 288–297. https://doi.org/10.1016/j.sajb.2023.02.007
- Akonjuen, B. M., Onuh, J. O., Aryee, A. N. A. (2023). Bioactive fatty acids from non-conventional lipid sources and their potential application in functional food development. Food Science and Nutrition, 11(10), 5689–5700. https://doi.org/10.1002/fsn3.3521
- Jaiswal, K. K., Kumar, V., Vlaskin, M. S., Nanda, M. (2020). Impact of glyphosate herbicide stress on metabolic growth and lipid inducement in Chlorella sorokiniana UUIND6 for biodiesel production. Algal Research, 51, Article 102071. https://doi.org/10.1016/j.algal.2020.102071
- Martínez-Bisbal, M. C., Mestre, N. C., Martínez-Máñez, R., Bauzá, J., Fillol, M. A. (2019). Microalgae degradation follow up by voltammetric electronic tongue, impedance spectroscopy and NMR spectroscopy. Sensors and Actuators, B: Chemical, 281, 44–52. https://doi.org/10.1016/j.snb.2018.10.069
- Mayers, J. J., Flynn, K. J., Shields, R. J. (2013). Rapid determination of bulk microalgal biochemical composition by Fourier-Transform Infrared spectroscopy. Bioresource Technology, 148, 215–220. https://doi.org/10.1016/j.biortech.2013.08.133
- Feng, G. D., Zhang, F., Cheng, L. -H., Xu, X. -H., Zhang, L., Chen, H. -L. (2013). Evaluation of FT-IR and Nile Red methods for microalgal lipid characterization and biomass composition determination. Bioresource Technology, 128, 107–112. https://doi.org/10.1016/j.biortech.2012.09.123
- Czamara, K., Majzner, K., Pacia, M. Z., Kochan, K., Kaczor, A. A., Baranska, M. (2015). Raman spectroscopy of lipids: A review. Journal of Raman Spectroscopy, 46(1), 4–20. https://doi.org/10.1002/jrs.4607
- Wu, H., Volponi, J. V., Oliver, A. E., Parikh, A. N., Simmons, B. A., Singh, S. (2011). In vivo lipidomics using single-cell Raman spectroscopy. Proceedings of the National Academy of Sciences of the United States of America, 108(9), 3809–3814. https://doi.org/10.1073/pnas.1009043108
- Sharma, S. K., Nelson, D. R., Abdrabu, R., Khraiwesh, B., Jijakli, K., Arnoux, M. et al. (2015). An integrative Raman microscopy-based workflow for rapid in situ analysis of microalgal lipid bodies. Biotechnology for Biofuels and Bioproducts, 8, Article 164. https://doi.org/10.1186/s13068-015-0349-1
- Shao, Y., Fang, H., Zhou, H., Wang, Q., Zhu, Y., He, Y. (2017). Detection and imaging of lipids of Scenedesmus obliquus based on confocal Raman microspectroscopy. Biotechnology for Biofuels and Bioproducts, 10(1), Article 300. https://doi.org/10.1186/S13068-017-0977-8
- Bruñas Gómez, I., Casale, M., Barreno, E., Catalá, M. (2022). Near-infrared metabolomic fingerprinting study of lichen thalli and phycobionts in culture: Aquaphotomics of Trebouxia lynnae dehydration. Microorganisms, 10(12), Article 2444. https://doi.org/10.3390/microorganisms10122444
- Beć, K. B., Grabska, J., Huck, C. W. (2020). Near-infrared spectroscopy in bioapplications. Molecules, 25(12), Article 2948. https://doi.org/10.3390/molecules25122948
- Podevin, M., Fotidis, I. A., Angelidaki, I. (2018). Microalgal process-monitoring based on high-selectivity spectroscopy tools: Status and future perspectives. Critical Reviews in Biotechnology, 38(5), 704–718. https://doi.org/10.1080/07388551.2017.1398132
- Cheng, Y.-S., Zheng, Y., Labavitch, J.M., VanderGheynst, J.S. (2011). Rapid quantification of total lipids using a colorimetric method in green microalgae. Lipids, 46(1), 95–103. https://doi.org/10.1007/s11745-010-3494-0
- Khozin-Goldberg, I., Iskandarov, U., Cohen, Z. (2011). LC-PUFA from photosynthetic microalgae: Occurrence, biosynthesis, and prospects in biotechnology. Applied Microbiology and Biotechnology, 91(4), 905–915. https://doi.org/10.1007/s00253-011-3441-x
- Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M. et al. (2008). Microalgal triacylglycerols as feedstocks for biofuel production: Perspectives and advances. The Plant Journal, 54(4), 621–639. https://doi.org/10.1111/j.1365-313X.2008.03492.x
- Lete, M. G., Tripathi, A., Chandran, V., Bankaitis, V. A., McDermott, M. I. (2020). Lipid transfer proteins and instructive regulation of lipid kinase activities: Implications for inositol lipid signaling and disease. Advances in Biological Regulation, 78, Article 100740. https://doi.org/10.1016/j.jbior.2020.100740
- Wagner, H., Jungandreas, A., Fanesi, A., Wilhelm, C. (2014). Surveillance of C-allocation in microalgal cells. Metabolites, 4(2), 453-464. https://doi.org/10.3390/metabo4020453
- Han, Y., Wen, Q., Chen, Z., Li, P. (2011). Review of methods used for microalgal lipid-content analysis. Energy Procedia, 12, 944-950. https://doi.org/10.1016/j.egypro.2011.10.124
Supplementary files
