Skip to main content
SpringerLink
Log in

Critical assessment of the sulfo-phospho-vanillin method to quantify lipids in freeze-dried microalgae

  • Published:
Journal of Applied Phycology Aims and scope Submit manuscript

Abstract

The sulfo-phospho-vanillin assay is a well-known method to measure the lipid content of microalgae. Following hydrolysis with sulfuric acid, a pink color develops after the addition of sulfo-phospho-vanillin and the absorbance at 530 nm is measured. As any colorimetric method, several factors might affect results consistency such as reaction time, matrix effects and sample background color. In this study, the measurement was carried out on freeze-dried samples of Chlorella and Spirulina to assess these impacts. The results showed the increase of absorbance with time even after the usual 30 min resting period. The main observation is a sample background color following acid hydrolysis and phosphoric acid addition, which questions the generally accepted SPV method reaction mechanism.

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

Similar content being viewed by others

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on request.

References

  • Alonzo F, Mayzaud P (1999) Spectrofluorometric quantification of neutral and polar lipids in zooplankton using Nile red. Mar Chem 67:289–301

    Article  CAS  Google Scholar 

  • Anschau A, Caruso CS, Kuhn RC, Franco TT, Anschau A, Caruso CS, Kuhn RC, Franco TT (2017) Validation of the sulfo-phospho-vanillin (SPV) method for the determination of lipid content in oleaginous microorganisms. Braz J Chem Eng 34:19–27

    Article  CAS  Google Scholar 

  • Bailey LS, Prajapati DV, Basso KB (2022) Optimization of the sulfo-phospho-vanillin assay for total lipid normalization in untargeted quantitative lipidomic LC–MS/MS applications. Anal Chem 94:17810–17818

    Article  CAS  PubMed  Google Scholar 

  • Biller P, Ross AB (2014) Pyrolysis GC–MS as a novel analysis technique to determine the biochemical composition of microalgae. Algal Res 6:91–97

    Article  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Byreddy A, Gupta A, Barrow C, Puri M (2016) A quick colorimetric method for total lipid quantification in microalgae. J Microbiol Meth 125:28–32

    Article  CAS  Google Scholar 

  • Chabrol E, Charonnat R (1937) Une nouvelle réaction pour l’études des lipides: l’oléidémie. La Presse Medicle 45:1713–1714

    CAS  Google Scholar 

  • 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 Meth 77:41–47

    Article  CAS  Google Scholar 

  • Cheng Y-S, Zheng Y, VanderGheynst JS (2011) Rapid quantitative analysis of lipids using a colorimetric method in a microplate format. Lipids 46:95–103

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Cooper MS, Hardin WR, Petersen TW, Cattolico RA (2010) Visualizing “green oil” in live algal cells. J Biosci Bioeng 109:198–201

    Article  CAS  PubMed  Google Scholar 

  • Danielewicz MA, Anderson LA, Franz AK (2011) Triacylglycerol profiling of marine microalgae by mass spectrometry. J Lipid Res 52:2101–2108

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Deshmukh S, Kumar R, Bala K (2019) Microalgae biodiesel: A review on oil extraction, fatty acid composition, properties and effect on engine performance and emissions. Fuel Process Technol 191:232–247

    Article  CAS  Google Scholar 

  • Donot F, Cazals G, Gunata Z, Egron D, Malinge J, Strub C, Fontana A, Schorr-Galindo S (2013) Analysis of neutral lipids from microalgae by HPLC-ELSD and APCI-MS/MS. J Chromatogr B 942–943:98–106

    Article  Google Scholar 

  • Drevon B, Schmit J (1964) La réaction sulfo-phospho-vanillique dans l’étude des lipides sériques. Applications en biochimies clinique et pharmacologique. Bull Trav Soc Pharm Lyon Société de pharmacie de Lyon. Séance du 17 avril 1964. pp 173–178

  • Ellison SLR, Thompson M (2008) Standard additions: myth and reality. Analyst 133:992

    Article  CAS  PubMed  Google Scholar 

  • Elsey D, Jameson D, Raleigh B, Cooney MJ (2007) Fluorescent measurement of microalgal neutral lipids. J Microbiol Meth 68:639–642

    Article  CAS  Google Scholar 

  • Folch J, Lees M, Stanley GHS (1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226:497–509

    Article  CAS  PubMed  Google Scholar 

  • Frings CS, Dunn RT (1970) A colorimetric method for determination of total serum lipids based on the sulfo-phospho-vanillin reaction. Am J Clin Pathol 53:89–91

    Article  CAS  PubMed  Google Scholar 

  • Gao F, Yang H-L, Li C, Peng Y-Y, Lu M-M, Jin W-H, Bao J-J, Guo Y-M (2019) Effect of organic carbon to nitrogen ratio in wastewater on growth, nutrient uptake and lipid accumulation of a mixotrophic microalgae Chlorella sp. Bioresour Technol 282:118–124

    Article  CAS  PubMed  Google Scholar 

  • Hernández-García A, Velásquez-Orta SB, Novelo E, Yáñez-Noguez I, Monje-Ramírez I, Orta Ledesma MT (2019) Wastewater-leachate treatment by microalgae: Biomass, carbohydrate and lipid production. Ecotoxicol Environ Saf 174:435–444

    Article  PubMed  Google Scholar 

  • 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 54:621–639

    Article  CAS  PubMed  Google Scholar 

  • Huang Y, Zhang D, Xue S, Wang M, Cong W (2016) The potential of microalgae lipids for edible oil production. Appl Biochem Biotechnol 180:438–451

    Article  CAS  PubMed  Google Scholar 

  • Inouye LS, Lotufo GR (2006) Comparison of macro-gravimetric and micro-colorimetric lipid determination methods. Talanta 70:584–587

    Article  CAS  PubMed  Google Scholar 

  • Johnson KR, Ellis G, Toothill C (1977) The sulfophosphovanillin reaction for serum lipids: a reappraisal. Clin Chem 23:1669–1678

    Article  CAS  PubMed  Google Scholar 

  • Knight JA, Anderson S, Rawle JM (1972) Chemical basis of the sulfo-phospho-vanillin reaction for estimating total serum lipids. Clin Chem 18:199–202

    Article  CAS  PubMed  Google Scholar 

  • Lang I, 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 11:124

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Laurens LML, Dempster TA, Jones HDT, Wolfrum EJ, Van Wychen S, McAllister JSP, Rencenberger M, Parchert KJ, Gloe LM (2012) Algal biomass constituent analysis: Method uncertainties and investigation of the underlying measuring chemistries. Anal Chem 84:1879–1887

    Article  CAS  PubMed  Google Scholar 

  • MacDougall KM, McNichol J, McGinn PJ, O’Leary SJB, Melanson JE (2011) Triacylglycerol profiling of microalgae strains for biofuel feedstock by liquid chromatography–high-resolution mass spectrometry. Anal Bioanal Chem 401:2609–2616

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • McMahon A, Lu H, Butovich IA (2013) The spectrophotometric sulfo-phospho-vanillin assessment of total lipids in human meibomian gland secretions. Lipids 48:513–525

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Menegazzo ML, Fonseca GG (2019) Biomass recovery and lipid extraction processes for microalgae biofuels production: A review. Renew Sustain Energy Rev 107:87–107

    Article  CAS  Google Scholar 

  • Mishra SK, Suh WI, Farooq W, Moon M, Shrivastav A, Park MS, Yang J-W (2014) Rapid quantification of microalgal lipids in aqueous medium by a simple colorimetric method. Bioresour Technol 155:330–333

    Article  CAS  PubMed  Google Scholar 

  • Palmer JS, Lawton LA, Kindt R, Edwards C (2021) Rapid analytical methods for the microalgal and cyanobacterial biorefinery: Application on strains of industrial importance. Microbiologyopen 10:e1156

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pantami HA, AhamadBustamam MS, Lee SY, Ismail IS, MohdFaudzi SM, Nakakuni M, Shaari K (2020) Comprehensive GCMS and LC-MS/MS metabolite profiling of Chlorella vulgaris. Mar Drugs 18:367

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Park J, Jeong HJ, Yoon EY, Moon SJ (2016) Easy and rapid quantification of lipid contents of marine dinoflagellates using the sulpho-phospho-vanillin method. Algae 31:391–401

    Article  CAS  Google Scholar 

  • Patel A, Antonopoulou I, Enman J, Rova U, Christakopoulos P, Matsakas L (2019) Lipids detection and quantification in oleaginous microorganisms: an overview of the current state of the art. BMC Chem Eng 1:13

    Article  Google Scholar 

  • Pinger C, Copeman L, Stowell M, Cormack B, Fugate C, Rogers M (2022) Rapid measurement of total lipids in zooplankton using the sulfo-phospho-vanillin reaction. Anal Meth 14:2665–2672

    Article  CAS  Google Scholar 

  • Rodas-Zuluaga LI, Castañeda-Hernández L, Castillo-Vacas EI, Gradiz-Menjivar A, López-Pacheco IY, Castillo-Zacarías C, Boully L, Iqbal HMN, Parra-Saldívar R (2021) Bio-capture and influence of CO2 on the growth rate and biomass composition of the microalgae Botryococcus braunii and Scenedesmus sp. J CO2 Util 43:101371

    Article  CAS  Google Scholar 

  • Rodríguez LC, Campaña AMG, Barrero FA, Linares CJ, Ceba MR (1995) Validation of an analytical instrumental method by standard addition methodology. J AOAC Int 78:471–476

    Article  Google Scholar 

  • Sarpal AS, Teixeira CMLL, Silva PRM, Lima GM, Silva SR, Monteiro TV, Cunha VS, Daroda RJ (2015) Determination of lipid content of oleaginous microalgal biomass by NMR spectroscopic and GC–MS techniques. Anal Bioanal Chem 407:3799–3816

    Article  CAS  PubMed  Google Scholar 

  • Sharma KK, Schuhmann H, Schenk PM (2012) High lipid induction in microalgae for biodiesel production. Energies 5:1532–1553

    Article  CAS  Google Scholar 

  • Thompson GA (1996) Lipids and membrane function in green algae. Biochim Biophys Acta - Lipids Lipid Metabol 1302:17–45

    Article  Google Scholar 

  • Voloshin RA, Rodionova MV, Zharmukhamedov SK, Nejat Veziroglu T, Allakhverdiev SI (2016) Review: Biofuel production from plant and algal biomass. Int J Hydrogen Energy 41:17257–17273

    Article  CAS  Google Scholar 

  • Wu S, Zhang B, Huang A, Huan L, He L, Lin A, Niu J, Wang G (2014) Detection of intracellular neutral lipid content in the marine microalgae Prorocentrum micans and Phaeodactylum tricornutum using Nile red and BODIPY 505/515. J Appl Phycol 26:1659–1668

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank Carole Lutz and Marie-Pierre Ottermatte of the ENGEES chemistry laboratory (Laboratoire d’Etude des Eaux, LEE) for their technical help during the analyses.

Funding

This study was supported financially and technically by Strasbourg University and the National Engineering School of Water and Environment of Strasbourg (ENGEES).

Author information

Authors and Affiliations

  1. ICube, UMR 7357, ENGEES, CNRS, Université de Strasbourg, 4 Rue Boussingault, Strasbourg, France

    Julie Farinacci & Julien Laurent

Authors
  1. Julie Farinacci
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Julien Laurent
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Julie Farinacci: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resource, Writing - Original Draft, Writing - Review & Editing, Visualization. Julien Laurent: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing - Review & Editing, Visualization, Supervision.

Corresponding author

Correspondence to Julie Farinacci.

Ethics declarations

Competing interests

The authors declare no competing interests.

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict interest.

Additional information

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Farinacci, J., Laurent, J. Critical assessment of the sulfo-phospho-vanillin method to quantify lipids in freeze-dried microalgae. J Appl Phycol 35, 997–1008 (2023). https://doi.org/10.1007/s10811-023-02930-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10811-023-02930-3

Keywords

Search

Navigation