Journal of Applied Phycology

, Volume 27, Issue 1, pp 125–140 | Cite as

Nitrogen-depleted Chlorella zofingiensis produces astaxanthin, ketolutein and their fatty acid esters: a carotenoid metabolism study

  • Kim J. M. Mulders
  • Yannick Weesepoel
  • Pierre Bodenes
  • Packo P. Lamers
  • Jean-Paul Vincken
  • Dirk E. Martens
  • Harry Gruppen
  • René H. Wijffels


Natural carotenoids such as astaxanthin, β,β-carotene and lutein are pigments with a high market value. We studied the effects of nitrogen depletion on the carotenoid metabolism of Chlorella zofingiensis (Chlorophyta) and the subsequent treatment with diphenylamine (DPA), an inhibitor of the biosynthesis of secondary ketocarotenoids. Pigments were identified and quantified based on reversed phase ultra-high performance liquid chromatography photodiode array tandem mass spectrometry (RP-UHPLC-PDA-MSn). Nitrogen depletion (without DPA) resulted in a degradation of chlorophylls and primary carotenoids and an accumulation of astaxanthin, ketolutein, canthaxanthin, adonixanthin and β,β-carotene. The DPA treatment decreased the overall production of β,β-carotene derivatives (sum of astaxanthin, canthaxanthin, echinenone and adonixanthin); however, the production of ketolutein and degradation of primary carotenoids were not modified. This suggests that the regulatory mechanisms controlling the flux towards ketolutein and primary carotenoids were not affected by the decreased levels of β,β-carotene derivatives. In addition, DPA increased production of the individual carotenoids, adonixanthin and echinenone. Insight into the regulation of microalgal carotenoid biosynthesis as demonstrated in this paper is essential when a large-scale carotenoid production process is to be optimised or a recombinant C. zofingiensis strain is to be designed with the intention of excessively producing primary or secondary carotenoids.


Chlorella zofingiensis Nitrogen depletion Diphenylamine Enzyme inhibitor Carotenoid metabolism Astaxanthin Ketolutein 



We gratefully thank Tiny Franssen-Verheijen of Wageningen Electron Microscopy Centre for her help with the cryo-SEM. This work was supported by the FeyeCon D&I and by grants from Rijksdienst voor Ondernemend Nederland (Project no. FND09014).

Supplementary material

10811_2014_333_Fig10_ESM.gif (43 kb)
Fig. A1

Time courses of chlorophyll a , chlorophyll b, lutein, 9′cis-neoxanthin, violaxanthin, total astaxanthin (sum of free, mono- and diesters), total ketolutein (sum of free, mono- and diesters), canthaxanthin, adonixanthin, echinenone and β,β-carotene in moles per litre culture volume of nitrogen-depleted C. zofingiensis exposed to no DPA (control) (A) and exposed to repeated additions of DPA resulting each time in a concentration increase of 60 μM (B). Data points represent averages of biological duplicates. For separate data points see Fig. 5 and 6. Solid triangles in B indicate DPA additions. Lines are for visual guidance (GIF 43 kb)

10811_2014_333_MOESM1_ESM.tif (6.8 mb)
High resolution image (TIFF 6933 kb)
10811_2014_333_Fig11_ESM.gif (35 kb)
Fig. A2

Contents (in mg/g DW) of chlorophyll a (A), chlorophyll b (B), lutein (C), 9′cis-neoxanthin (D), violaxanthin (E), total ketolutein (sum of free, mono- and diesters) (F), total astaxanthin (sum of free, mono- and diesters) (G), adonixanthin (H), echinenone (I), canthaxanthin (J) and β,β-carotene (K) of nitrogen-depleted C. zofingiensis exposed to no DPA (control) and exposed to repeated additions of DPA resulting each time in a concentration increase of 60 μM. Triangles indicate DPA additions. Lines are for visual guidance (GIF 35 kb)

10811_2014_333_MOESM2_ESM.tif (4.7 mb)
High resolution image (TIFF 4807 kb)


  1. Bar E, Rise M, Vishkautsan M, Arad S (1995) Pigment and structural changes in Chlorella zofingiensis upon light and nitrogen stress. J Plant Physiol 146:527–534CrossRefGoogle Scholar
  2. Bauch ME (2011) Identifizierung und Quantifizierung der Ketocarotinoide in Dauerstadien von Grünalgen und Ketocarotinoidbiosynthese im Modellorganismus Chlamydomonas reinhardtii. Dissertation, Johannes Gutenberg University, MainzGoogle Scholar
  3. Breuer G, Lamers PP, Martens DE, Draaisma RB, Wijffels RH (2012) The impact of nitrogen starvation on the dynamics of triacylglycerol accumulation in nine microalgae strains. Bioresour Technol 124:217–226PubMedCrossRefGoogle Scholar
  4. Breuer G, Evers WAC, de Vree JH, Kleinegris DMM, Martens DE, Wijffels RH, Lamers PP (2013) Analysis of fatty acid content and composition in microalgae. J Vis Exp 80:e50628Google Scholar
  5. Britton G (1995a) UV/visible spectroscopy. In: Britton G, Liaaen-Jensen S, Pfander H (eds) Carotenoids, vol 1B,Spectroscopy. Birkhäuser, Basel, pp 13–62Google Scholar
  6. Britton G (1995b) Mass spectrometry. In: Britton G, Liaaen-Jensen S, Pfander H (eds) Carotenoids, vol 1B,Spectroscopy. Birkhäuser, Basel, pp 261–317Google Scholar
  7. Britton G, Liaaen-Jensen S, Pfander H (editors) and Mercadante Z, Egeland E (compilers) (2004) Handbook, Carotenoid series. Birkhäuser, BaselGoogle Scholar
  8. Collins AM, Jones HDT, Han D, Hu Q, Beechem TE, Timlin JA (2011) Carotenoid distribution in living cells of Haematococcus pluvialis (Chlorophyceae). PLoS ONE 6(9):e24302PubMedCentralPubMedCrossRefGoogle Scholar
  9. Dall’Osto L, Lico C, Alric J, Giuliano G, Havaux M, Bassi R (2006) Lutein is needed for efficient chlorophyll triplet quenching in the major LHCII antenna complex of higher plants and effective photoprotection in vivo under strong light. BMC Plant Biol 6:32PubMedCentralPubMedCrossRefGoogle Scholar
  10. Del Campo JA, Rodriguez H, Moreno J, Vargas MA, Rivas J, Guerrero MG (2004) Accumulation of astaxanthin and lutein in Chlorella zofingiensis (Chlorophyta). Appl Microbiol Biotechnol 64:848–854PubMedCrossRefGoogle Scholar
  11. Egeland G, Garrido J, Clementson L, Andresen K, Thomas C, Zapata M, Airs R, Llewellyn C, Newman G, Rodríguez F, Roy S (2011) Part VI: Data sheets aiding identification of phytoplankton carotenoids and chlorophylls. In: Roy S, Llewellyn CA, Egeland ES, Johnson G (eds) Phytoplankton pigments: characterization, chemotaxonomy and applications in oceanography. Cambridge University Press, Cambridge, pp 665–822Google Scholar
  12. Enzell CR, Francis GW, Liaaen-Jensen S (1969) Mass spectrometric studies of carotenoids 2. A survey of fragmentation reactions. Acta Chem Scand 23:727–750PubMedCrossRefGoogle Scholar
  13. Falkowski PG, Raven JA (2007) Aquatic photosynthesis, 2nd edn. Princeton University Press, Princeton, 484 ppGoogle Scholar
  14. Fan L, Vonshak A, Gabbay R, Hirshberg J, Cohen Z, Boussiba S (1995) The biosynthetic pathway of astaxanthin in a green alga Haematococcus pluvialis as indicated by inhibition with diphenylamine. Plant Cell Physiol 36:1519–1524Google Scholar
  15. Frassanito R, Cantonati M, Flaim G, Mancini I, Guella G (2008) A new method for the identification and the structural characterisation of carotenoid esters in freshwater microorganisms by liquid chromatography/electrospray ionisation tandem mass spectrometry. Rapid Commun Mass Spectrom 22:3531–3539PubMedCrossRefGoogle Scholar
  16. Fucíková K, Lewis LA (2012) Intersection of Chlorella, Muriella and Bracteacoccus: resurrecting the genus Chromochloris Kol et Chodat (Chlorophyceae, Chlorophyta). Fottea 12:83–93Google Scholar
  17. Goss R, Jakob T (2010) Regulation and function of xanthophyll cycle-dependent photoprotection in algae. Photosynth Res 106:103–122PubMedCrossRefGoogle Scholar
  18. Grünewald K, Hirschberg J, Hagen C (2001) Ketocarotenoid biosynthesis outside of plastids in the unicellular green alga Haematococcus pluvialis. J Biol Chem 276:6023–6029PubMedCrossRefGoogle Scholar
  19. Harker M, Young AJ (1995) Inhibition of astaxanthin synthesis in the green alga Haematococcus pluvialis. Eur J Phycol 30:179–187CrossRefGoogle Scholar
  20. Huang JC, Wang Y, Sandmann G, Chen F (2006) Isolation and characterization of a carotenoid oxygenase gene from Chlorella zofingiensis (Chlorophyta). Appl Microbiol Biotechnol 71:473–479PubMedCrossRefGoogle Scholar
  21. Jahns P, Holzwarth AR (2012) The role of the xanthophyll cycle and of lutein in photoprotection of photosystem II. Biochem Biophys Acta – Bioenerg 1817:182–193CrossRefGoogle Scholar
  22. Jin E, Lee CG, Polle JEW (2006) Secondary carotenoid accumulation in Haematococcus (Chlorophyceae): biosynthesis, regulation, and biotechnology. J Microbiol Biotechnol 16:821–831Google Scholar
  23. Kanehisa Laboratories (2012) KEGG PATHWAY Database. Available at: Accessed 25 Jul 2013
  24. Kliphuis AMJ, Janssen M, Van Den End EJ, Martens DE, Wijffels RH (2011) Light respiration in Chlorella sorokiniana. J Appl Phycol 23:935–947PubMedCentralPubMedCrossRefGoogle Scholar
  25. Lamers PP, Janssen M, De Vos RCH, Bino RJ, Wijffels RH (2008) Exploring and exploiting carotenoid accumulation in Dunaliella salina for cell-factory applications. Trends Biotechnol 26:631–638PubMedCrossRefGoogle Scholar
  26. Lamers PP, Van De Laak CCW, Kaasenbrood PS, Lorier J, Janssen M, De Vos RCH, Bino RJ, Wijffels RH (2010) Carotenoid and fatty acid metabolism in light-stressed Dunaliella salina. Biotechnol Bioeng 106:638–648PubMedCrossRefGoogle Scholar
  27. Lemoine Y, Schoefs B (2010) Secondary ketocarotenoid astaxanthin biosynthesis in algae: a multifunctional response to stress. Photosynth Res 106:155–177PubMedCrossRefGoogle Scholar
  28. Mandalam RK, Palsson BO (1998) Elemental balancing of biomass and medium composition enhances growth capacity in high-density Chlorella vulgaris cultures. Biotechnol Bioeng 59:605–611PubMedCrossRefGoogle Scholar
  29. Mulders KJM, Weesepoel Y, Lamers PP, Vincken J-P, Martens DE, Wijffels RH (2013) Growth and pigment accumulation in nutrient-depleted Isochrysis aff. galbana T-ISO. J Appl Phycol 25:1421–1430CrossRefGoogle Scholar
  30. Mulders KJM, Lamers PP, Martens DE, Wijffels RH (2014) Phototrophic pigment production with microalgae: biological constraints and opportunities. J Phycol 50:229–242CrossRefGoogle Scholar
  31. Orosa M, Torres E, Fidalgo P, Abalde J (2001) Production and analysis of secondary carotenoids in green algae. J Appl Phycol 12:553–556CrossRefGoogle Scholar
  32. Solovchenko AE (2013) Physiology and adaptive significance of secondary carotenogenesis in green microalgae. Russ J Plant Physiol 60:1–13CrossRefGoogle Scholar
  33. Spolaore P, Joannis-Cassan C, Duran E, Isambert A (2006) Commercial applications of microalgae. J Biosci Bioeng 101:87–96PubMedCrossRefGoogle Scholar
  34. Sukenik A, Livne A, Neori A, Yacobi YZ, Katcoff D (1992) Purification and characterization of a light-harversing chlorophyll-protein complex from the marine eustigmatophyte Nannochloropsis sp. Plant Cell Physiol 33:1041–1048Google Scholar
  35. Vidhyavathi R, Venkatachalam L, Sarada R, Ravishankar GA (2008) Regulation of carotenoid biosynthetic genes expression and carotenoid accumulation in the green alga Haematococcus pluvialis under nutrient stress conditions. J Exp Bot 59:1409–1418PubMedCrossRefGoogle Scholar
  36. Vila M, Galván A, Fernández E, León R (2012) Ketocarotenoid biosynthesis in transgenic microalgae expressing a foreign β-C-4-carotene oxygenase gene. Methods Mol Biol 892:283–295PubMedCrossRefGoogle Scholar
  37. Wang Y, Chen T (2008) The biosynthetic pathway of carotenoids in the astaxanthin-producing green alga Chlorella zofingiensis. World J Microbiol Biotechnol 24:2927–2932CrossRefGoogle Scholar
  38. Weesepoel Y, Vincken J-P, Pop R, Liu K, Gruppen H (2013) Sodiation as a tool for enhancing the diagnostic value of MALDI-TOF/TOF-MS spectra of astaxanthin esters in complex mixtures from Haematococcus pluvialis. J Mass Spectrom 48:862–874PubMedCrossRefGoogle Scholar
  39. Zhekisheva M, Zarka A, Khozin-Goldberg I, Cohen Z, Boussiba S (2005) Inhibition of astaxanthin synthesis under high irradiance does not abolish triacylglycerol accumulation in the green alga Haematococcus pluvialis (Chlorophyceae). J Phycol 41:819–826CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Kim J. M. Mulders
    • 1
    • 3
  • Yannick Weesepoel
    • 2
    • 3
  • Pierre Bodenes
    • 4
  • Packo P. Lamers
    • 1
  • Jean-Paul Vincken
    • 2
  • Dirk E. Martens
    • 1
  • Harry Gruppen
    • 2
  • René H. Wijffels
    • 1
  1. 1.Bioprocess Engineering, AlgaePARCWageningen UniversityWageningenThe Netherlands
  2. 2.Laboratory of Food ChemistryWageningen UniversityWageningenThe Netherlands
  3. 3.FeyeCon Development and ImplementationWeespThe Netherlands
  4. 4.Polytech Graduate School of EngineeringNantes UniversitySaint-NazaireFrance

Personalised recommendations