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Determination of lipid content of oleaginous microalgal biomass by NMR spectroscopic and GC–MS techniques

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Abstract

Direct methods based on 1H NMR spectroscopic techniques have been developed for the determination of neutral lipids (triglycerides and free fatty acids) and polar lipids (glyceroglycolipids/phospholipids) in the solvent extracts of oleaginous microalgal biomasses cultivated on a laboratory scale with two species in different media. The chemical shift assignments observed in the 1H and 13C NMR spectra corresponding to unsaturated (C18:N, N = 1–3, C20:3, C20:5, C22:6, epoxy) and saturated (C14–C18) fatty acid ester components in a complex matrix involving overlapped resonances have been unambiguously confirmed by the application of 2D NMR spectroscopy (total correlation spectroscopy and heteronuclear single quantum coherence–total correlation spectroscopy). The study of the effect of a polar lipid matrix on the determination of neutral lipids by an internal reference blending process by a systematic designed experimental protocol has provided absolute quantification. The fatty acid composition of algal extracts was found to be similar to that of vegetable oils containing saturated (C16–C18:0) and unsaturated (C18:N, N = 1–3, C20:N, N = 3–4, C22:6) fatty acids as confirmed by NMR spectroscopy and gas chromatography–mass spectrometry analyses. The NMR methods developed offer great potential for rapid screening of algal strains for generation of algal biomass with the desired lipid content, quality, and potential for biodiesel and value-added polyunsaturated fatty acids in view of the cost economics of the overall cost of generation of the biomass.

500 MHz full 1H-13C 2D HSQC-TOCSY contour plot of algae extract SNDSPSClMe. b, a= C18:3, C22:6; c, e= epoxy ester; n= C22:6; q= absence of C20:5; f, g= OCH2 (sn1, sn3) and h= OCH (sn2)

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References

  1. Meng X, Yang J, Le XU, Rude MA, Schirmer A (2009) New microbial fuels: a biotech perspective. Curr Opin Microbiol 12:274–281

    Article  Google Scholar 

  2. Zhao C, Brück T, Lercher JA (2013) Catalytic deoxygenation of microalgae oil to green hydrocarbons. Green Chem 15:1720–1739

    Article  CAS  Google Scholar 

  3. Han Y, Wen Q, Chen Z, Li P (2011) Review of methods used for microalgal lipid-content analysis. Energy Procidia 12:944–950

    Article  Google Scholar 

  4. Mercer P, Armenta RE (2011) Development in oil extraction from micro algae. Eur J Lipid Sci Technol 21:1–9

    Google Scholar 

  5. Meng X, Yang J, Xu X, Zhang L, Nie Q (2009) Biodiesel production from oleaginous microorganisms. Renew Energy 34(1):1–5

    Article  Google Scholar 

  6. Schlagermann P (2012) Composition of algal oil and its potential as biofuel. J Combust. doi:10.1155/2012/285185

    Google Scholar 

  7. Danielewicz MA, Anderson LA, Franz AK (2011) Triacylglycerol profiling of marine microalgae by mass spectrometry. J Lipid Res 52(11):2101–2108

    Article  CAS  Google Scholar 

  8. Sarpal AS, Silva PRM (2013) Analytical strategies for determining biodiesel potential of algae. http://www.stle.org/resources/articledetails.aspx?did=1755

  9. dos Santos RR, Moreira DM, Kunigami CN, Aranda DAC, Teixeira CMLL (2015) Comparison between several methods of total lipid extraction from Chorella vulgaris biomass. Ultrason Sonochem 22:95–99

    Article  Google Scholar 

  10. Kumar R, Bansal V, Patel MB, Sarpal AS (2012) NMR determination of iodine value in biodiesel produced from algal and vegetable oils. Energy Fuels 26:7005–7008

    CAS  Google Scholar 

  11. Kumar R, Bansal V, Tiwari AK, Sharma M, Puri SK, Patel MB, Sarpal AS (2011) Estimation of glycerides and free fatty acid in oil extracted from seed for biodiesel production by NMR spectroscopic techniques. J Am Oil Chem Soc 88:1675–1685

    Article  CAS  Google Scholar 

  12. Chopra A, Singh D, Kumar R, Sugmaran V, Sarpal AS (2011) Determination of polyunsaturated fatty esters (PUFE) in biodiesel by GC/GC-MS and NMR techniques. J Am Oil Chem Soc 88:1285–1296

    Article  CAS  Google Scholar 

  13. Oromí-Farrús M, Villorbina G, Eras J, Gatius F, Torres M, Canela R (2010) Determination of the iodine value of biodiesel using 1H NMR with 1,4-dioxane as an internal standard. Fuel 89(11):3489–3349

    Article  Google Scholar 

  14. Sarpal AS, Kapur GS, Mukherjee S, Jayparkash KC, Jain SK (1995) Determination of iodine value of lubricating oils by NMR spectroscopy. Lubr Eng 51(3):209–214

    CAS  Google Scholar 

  15. Gao C, Xiong W, Zhang Y, Yuan W, Wu Q (2008) Rapid quantitation of lipid in microalgae by time-domain nuclear magnetic resonance. J Microbiol Methods 75(3):437–440

    Article  CAS  Google Scholar 

  16. Nuzzo G, Gallo C, d’Ippolito G, Cutignano A, Sardo A, Fontana A (2013) Composition and quantitation of microalgal lipids by ERETIC 1H NMR method. Mar Drugs 11(10):3742–3753

    Article  CAS  Google Scholar 

  17. Pratoomyot J, Srivilas P, Noiraksar T (2005) Fatty acids composition of 10 microalgal species. Songklanakarin J Sci Technol 27(6):1179–1187

    Google Scholar 

  18. Siddiqui N, Sim J, Silwood CJ, Toms H, Iles RA, Grootveld M (2003) Multicomponent analysis of encapsulated marine oil supplements using high-resolution 1H and 13C NMR techniques. J Lipid Res 44:2406–2427

    Article  CAS  Google Scholar 

  19. 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 77:41–47

    Article  CAS  Google Scholar 

  20. Dembitsky VM, Rozentsvet OA, Pechenkina EE (1990) Glycolipids, Phospholipids and fatty acids of brown algae species. Phytochemistry 29:3417–3421

    Article  CAS  Google Scholar 

  21. Jones J, Manning S, Montoya M, Keller K, Poenie M (2012) Extraction of algal lipids and their analysis by HPLC and mass spectrometry. J Am Oil Chem Soc 89:1371–1381

    CAS  Google Scholar 

  22. Sarpal AS, Silva PRM, Cunha VS, Daroda, RJ (2013) Monitoring of biodiesel quality parameters by 1HNMR spectroscopic techniques. http://www.stle.org/resources/articledetails.aspx?did=1836

  23. Beal CM, Webber ME, Ruoff RS, Hebner RE (2010) Lipid analysis of Neochloris oleoabundans by liquid state NMR. Biotechnol Bioeng 106(4):573–583

    Article  CAS  Google Scholar 

  24. Hatzakis E, Agiomyrgianaki A, Kostidis S, Dais P (2011) NMR spectroscopy: an alternative fast tool for qualitative and quantitative analysis of diacylglycerol (DAG) oil. J Am Oil Chem Soc 88:1695–1708

    Article  CAS  Google Scholar 

  25. Kumar R, Bansal V, Patel MB, Sarpal AS (2014) Compositional analysis of algal biomass in a nuclear magnetic resonance (NMR) tube. J Algal Biomass Util 5(3):36–45

    Google Scholar 

  26. Sarpal AS, Silva PRM, Pinto RF, Cunha VS, Daroda RJ, de Souza W (2014) Biodiesel potential of oleaginous yeast biomass by NMR spectroscopic techniques. Energy Fuels 28:3766–3777

    Article  CAS  Google Scholar 

  27. 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 

  28. Han Y, Wen Q, Chen Z, Li P (2011) Review of methods used for microalgal lipid-content analysis. Energy Procedia 12:944–950

    Article  Google Scholar 

  29. Pohnert G, Boland W (2002) The oxylipin chemistry of attraction and defense in brown algae and diatoms. Nat Prod Rep 19:108–122

    Article  CAS  Google Scholar 

  30. Laurens LML, Dempster TA, Jones HDT, Wolfrum EJ, Van Wychen S, McAllister JSP, Rencenberger M, Parchert KJ, Lindsey M, Gloe LM (2012) Algal biomass constituent analysis: method uncertainties and investigation of the underlying measuring chemistries. Anal Chem 84(4):1879–1887

    Article  CAS  Google Scholar 

  31. Algae Biomass Organization (2014) Industrial algae measurements, version 6.0. http://www.algaebiomass.org/wp-content/gallery/2012-algae-biomass-summit/2010/06/IAM-6.0.pdf

  32. Menezes RS, Leles MIG, Soars AT, Franco PIBM, Filho NRA (2013) Evaluation of the potentiality of fresh water microalgae as a source of raw material for biodiesel production. Quim Nova 36(1):10–15

    Article  CAS  Google Scholar 

  33. Bingol K, Bruschweiler-Li L, Li D-W, Brüschweiler R (2014) Customized metabolomics database for the analysis of NMR 1H–1H TOCSY and 13C–1H HSQC-TOCSY spectra of complex mixtures. Anal Chem 86(11):5494–5501

    Article  CAS  Google Scholar 

  34. Davey PT, Hiscox WC, Lucker BF, O’Fallon JV, Chen S, Helms GL (2012) Rapid triacylglyceride detection and quantification in live micro-algal cultures via liquid state 1H NMR. Algal Res 2(1):166–175

    Article  Google Scholar 

  35. Malz F, Jancke H (2005) Validation of quantitative NMR. J Pharm Biomed Anal 38(5):813–823

    Article  CAS  Google Scholar 

  36. Kumar S, Roy R (2012) Quantitative 1H NMR spectroscopy. Trends Anal Chem 35:5–26

    Article  Google Scholar 

  37. Guillard RRL, Lorenzen CJ (1972) Yellow-green algae with chlorophyllide c. J Phycol 8:10–14

    CAS  Google Scholar 

Download references

Acknowledgments

We sincerely thank INMETRO, Brazil, for conducting this research, which is of national importance to Brazil in view of the development of fuels from sustainable resources. We are thank the Instituto Nacional de Tecnologia for collaboration in the project. We also express our thanks and gratitude to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the grant of a fellowship.

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Authors and Affiliations

  1. Instituto Nacional de Metrologia, Qualidade e Tecnologia—INMETRO, Avenida Nossa Senhora das Graças 50, Xerém, Duque de Caxias, RJ, 25250-020, Brazil

    Amarijt S. Sarpal, Paulo R. M. Silva, Samantha R. Silva, Thays V. Monteiro, Valnei S. Cunha & Romeu J. Daroda

  2. Instituto Nacional de Tecnologia, Rio de Janeiro, RJ, Brazil

    Claudia M. L. L. Teixeira & Gustavo M. Lima

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  1. Amarijt S. Sarpal
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Correspondence to Amarijt S. Sarpal.

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Sarpal, A.S., Teixeira, C.M.L.L., Silva, P.R.M. et al. Determination of lipid content of oleaginous microalgal biomass by NMR spectroscopic and GC–MS techniques. Anal Bioanal Chem 407, 3799–3816 (2015). https://doi.org/10.1007/s00216-015-8613-6

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  • DOI: https://doi.org/10.1007/s00216-015-8613-6

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