BioEnergy Research

, Volume 5, Issue 4, pp 876–885 | Cite as

Dynamics of Lipid Biosynthesis and Redistribution in the Marine Diatom Phaeodactylum tricornutum Under Nitrate Deprivation

  • Elizabeth H. Burrows
  • Nicholas B. Bennette
  • Damian Carrieri
  • Joseph L. Dixon
  • Anita Brinker
  • Miguel Frada
  • Steven N. Baldassano
  • Paul G. Falkowski
  • G. Charles DismukesEmail author


One approach to achieve continuous overproduction of lipids in microalgal “cell factories” relies upon depletion or removal of nutrients that act as competing electron sinks (e.g., nitrate and sulfate). However, this strategy can only be effective for bioenergy applications if lipid is synthesized primarily de novo (from CO2 fixation) rather than from the breakdown and interconversion of essential cellular components. In the marine diatom, Phaeodactylum tricornutum, it was determined, using 13C-bicarbonate, that cell growth in nitrate (NO 3 )-deprived cultures resulted predominantly in de novo lipid synthesis (60 % over 3 days), and this new lipid consisted primarily of triacylglycerides (TAGs). Nearly complete preservation of 12C occurred in all previously existing TAGs in NO 3 -deprived cultures and thus, further TAG accumulation would not be expected from inhibition of TAG lipolysis. In contrast, both high turnover and depletion of membrane lipids, phosphatidylcholines (PCs), were observed in NO 3 -deprived cultures (both the headgroups and fatty acid chains), while less turnover was observed in NO 3 replete cultures. Liquid chromatography-tandem mass spectrometry mass spectra and 13C labeling patterns of PC headgroups provided insight into lipid synthesis in marine diatoms, including suggestion of an internal pool of glycine betaine that feeds choline synthesis. It was also observed that 16C fatty acid chains incorporated into TAGs and PCs contained an average of 14 13C carbons, indicating substantial incorporation of 13C-bicarbonate into fatty acid chains under both nutrient states.


Algae Biodiesel Nitrate Nutrients Fatty acid metabolism De novo lipid biosynthesis Phaeodactylum tricornutum 



This work was funded by the Air Force Office of Scientific Research, grant # FA9550-05-1-0365. The LC/MS instrument was obtained through National Center for Research Resources (NIH) (grant # RR021120). EHB was additionally funded by the Busch-Waksman Postdoctoral Fellowship, and NBB was funded by the Sustainable Fuels NSF-IGERT program (award #0903675). MF was funded by a gift from James Gibson. We would also like to thank Char Fuller and Kevin Wyman. Elemental analysis was performed by Marshall Otter at the Marine Biological Laboratory Ecosystems Center, Woods Hole, MA, USA.

Supplementary material

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  1. 1.
    Chisti Y (2007) Biodiesel from microalgae. Biotechnol Adv 25(3):294–306PubMedCrossRefGoogle Scholar
  2. 2.
    Dismukes GC, Carrieri D, Bennette N, Ananyev GM, Posewitz MC (2008) Aquatic phototrophs: efficient alternatives to land-based crops for biofuels. Curr Opin Biotechnol 19(3):235–240. doi: 10.1016/j.copbio.2008.05.007 PubMedCrossRefGoogle Scholar
  3. 3.
    Weyer KM, Bush DR, Darzins A, Wilson BD (2010) Theoretical maximum algal oil production. Bioenergy Res 3:204–213CrossRefGoogle Scholar
  4. 4.
    Sheehan J, Dunahay T, Benemann J, Roessler PG (1998) A Look Back at the U.S. Department of Energy's Aquatic Species Program: Biodiesel from Algae. US DoEnergy National Renewable Energy LaboratoryGoogle Scholar
  5. 5.
    Bozarth A, Maier U-G, Zauner S (2009) Diatoms in biotechnology: modern tools and applications. Appl Microbiol Biotechnol 82(2):195–201. doi: 10.1007/s00253-008-1804-8 PubMedCrossRefGoogle Scholar
  6. 6.
    Wijffels RH, Barbosa MJ (2010) Outlook on microalgal biofuels. Science 329:796–799PubMedCrossRefGoogle Scholar
  7. 7.
    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. Plant J 54(4):621–639. doi: 10.1111/j.1365-313X.2008.03492.x PubMedCrossRefGoogle Scholar
  8. 8.
    Frenz J, Largeau C, Casadevall E (1989) Hydrocarbon recovery by extraction with a biocompatible solvent from free and immobilized cultures of Botryococcus braunii. Enzyme Microb Technol 11(11):717–724CrossRefGoogle Scholar
  9. 9.
    Sayre RT (2009) Optimization of biofuel production. United States Patent, International patent number 20090181438Google Scholar
  10. 10.
    Radakovits R, Jinkerson RE, Darzins A, Posewitz MC (2010) Genetic engineering of algae for enhanced biofuel production. Eukaryot Cell 9(4):486–501. doi: 10.1128/ec.00364-09 PubMedCrossRefGoogle Scholar
  11. 11.
    Ramachandra TV, Mahapatra DM, Karthick B, Gordon R (2009) Milking diatoms for sustainable energy: biochemical engineering versus gasoline-secreting diatom solar panels. Ind Eng Chem Res 48(19):8769–8788. doi: 10.1021/ie900044j CrossRefGoogle Scholar
  12. 12.
    Hejazi MA, Wijffels RH (2004) Milking of microalgae. Trends Biotechnol 22(4):189–194PubMedCrossRefGoogle Scholar
  13. 13.
    Suen Y, Hubbard JS, Holzer G, Tornabene TG (1987) Total lipid production of the green-alga Nannochloropsis Sp Qii under different nitrogen regimes. J Phycol 23(2):289–296CrossRefGoogle Scholar
  14. 14.
    Roessler PG (1988) Effects of silicon deficiency on lipid-composition and metabolism in the diatom Cyclotella-Cryptica. J Phycol 24(3):394–400Google Scholar
  15. 15.
    Bowler C, Allen AE, Badger JH, Grimwood J, Jabbari K, Kuo A et al (2008) The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature 456(7219):239–244. doi: 10.1038/nature07410 PubMedCrossRefGoogle Scholar
  16. 16.
    Siaut M, Heijde M, Mangogna M, Montsant A, Coesel S, Allen A et al (2007) Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum. Gene 406(1–2):23–35PubMedCrossRefGoogle Scholar
  17. 17.
    Guillard RR, Ryther JH (1962) Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (cleve) Gran. Can J Microbiol 8:229–239PubMedCrossRefGoogle Scholar
  18. 18.
    Camacho FG, Grima EM, Miron AS, Pascual VG, Chisti Y (2001) Carboxymethyl cellulose protects algal cells against hydrodynamic stress. Enzyme Microb Technol 29(10):602–610CrossRefGoogle Scholar
  19. 19.
    Granum E, Kirkvold S, Myklestad SM (2002) Cellular and extracellular production of carbohydrates and amino acids by the marine diatom Skeletonema costatum: diel variations and effects of N depletion. Mar Ecol Prog Ser 242:83–94CrossRefGoogle Scholar
  20. 20.
    Porra RJ (2002) The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b. Photosynth Res 73(1–3):149–156PubMedCrossRefGoogle Scholar
  21. 21.
    Slayback JRB, Cheung LWY, Geyer RP (1977) Quantitative extraction of microgram amounts of lipid from cultured human cells. Anal Biochem 83(2):372–384PubMedCrossRefGoogle Scholar
  22. 22.
    Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Phys 37(8):911–917CrossRefGoogle Scholar
  23. 23.
    Rodriguez-Ruiz J, Belarbi EH, Sanchez JLG, Alonso DL (1998) Rapid simultaneous lipid extraction and transesterification for fatty acid analyses. Biotechnol Tech 12(9):689–691CrossRefGoogle Scholar
  24. 24.
    Johnson DR, Brinker A, Thackray J, Dixon J Normal phase LC/MS analysis of triglyceride concentration and composition in liver cells. In: 58th ASMS Conference on Mass Spectrometry, 2010. J. Am Society Mass Spectrometry, p S44Google Scholar
  25. 25.
    Homan R, Anderson MK (1998) Rapid separation and quantification of combined neutral and polar lipid classes by high-performance liquid chromatography and evaporative light-scattering mass detection. J Chrom B 708:21–26CrossRefGoogle Scholar
  26. 26.
    Tonon T, Harvey D, Larson TR, Graham IA (2002) Long chain polyunsaturated fatty acid production and partitioning to triacylglycerols in four microalgae. Phytochem 61(1):15–24CrossRefGoogle Scholar
  27. 27.
    Viso A-C, Marty J-C (1993) Fatty acids from 28 marine microalgae. Phytochem 34(6):1521–1533CrossRefGoogle Scholar
  28. 28.
    Chrismadha T, Borowitzka M (1994) Effect of cell density and irradiance on growth, proximate composition and eicosapentaenoic acid production of Phaeodactylum tricornutum grown in a tubular photobioreactor. J Appl Phycol 6(1):67–74. doi: 10.1007/bf02185906 CrossRefGoogle Scholar
  29. 29.
    Parrish CC, Wangersky PJ (1987) Particulate and dissolved lipid classes in cultures of Phaeodactylum tricornutum grown in cage culture turbidostats with a range of nitrogen supply rates. Mar Ecol Prog Ser 35:119–138CrossRefGoogle Scholar
  30. 30.
    Park YI, Buszko ML, Gander JE (1999) Glycine betaine: Reserve form of choline in Penicillium fellutanum in low-sulfate medium. Appl Environ Microbiol 65(3):1340–1342PubMedGoogle Scholar
  31. 31.
    Keller MD, Kiene RP, Matrai PA, Bellows WK (1999) Production of glycine betaine and dimethylsulfoniopropionate in marine phytoplankton. I Batch cultures Mar Bio 135(2):237–248Google Scholar
  32. 32.
    Keller MD, Kiene RP, Matrai PA, Bellows WK (1999) Production of glycine betaine and dimethylsulfoniopropionate in marine phytoplankton. II. N-limited chemostat cultures. Mar Bio 135(2):249–257CrossRefGoogle Scholar
  33. 33.
    Dickson DMJ, Kirst GO (1987) Osmotic adjustment in marine eukaryotic algae—the role of inorganic-ions, quaternary ammonium, tertiary sulfonium and carbohydrate solutes. 1. Diatoms and a Rhodophyte. New Phytol 106(4):645–655CrossRefGoogle Scholar
  34. 34.
    Lomas MW, Glibert PM (2000) Comparisons of nitrate uptake, storage, and reduction in marine diatoms and flagellates. J Phycol 36(5):903–913CrossRefGoogle Scholar
  35. 35.
    Dortch Q, Clayton JR, Thoresen SS, Ahmed SI (1984) Species differences in accumulation of nitrogen pools in phytoplankton. Mar Bio 81(3):237–250CrossRefGoogle Scholar
  36. 36.
    Eppley RW, Coatsworth JL (1968) Nitrate and nitrite uptake by Ditylum brightuellii. Kinetics and mechanisms. J Phycol 4:151–156CrossRefGoogle Scholar
  37. 37.
    Larson TR, Rees TAV (1996) Changes in cell composition and lipid metabolism mediated by sodium and nitrogen availability in the marine diatom Phaeodactylum tricornutum (bacillariophyceae). J Phycol 32(3):388–393CrossRefGoogle Scholar
  38. 38.
    KaiXian Q, Borowitzka MA (1993) Light and nitrogen deficiency effects on the growth and composition of Phaeodactylum tricornutum. Appl Biochem Biotechnol 38(1–2):93–103CrossRefGoogle Scholar
  39. 39.
    Work VH, Radakovits R, Jinkerson RE, Meuser JE, Elliott LG, Vinyard DJ et al (2010) Increased lipid accumulation in the Chlamydomonas reinhardtii sta7-10 starchless isoamylase mutant and increased carbohydrate synthesis in complemented strains. Eukaryot Cell 9:1251–1261PubMedCrossRefGoogle Scholar
  40. 40.
    Yongmanitchai W, Ward OP (1991) Growth of and omega-3-fatty-acid production by Phaeodactylum tricornutum under different culture conditions. Appl Environ Microbiol 57(2):419–425PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Elizabeth H. Burrows
    • 1
  • Nicholas B. Bennette
    • 1
  • Damian Carrieri
    • 2
  • Joseph L. Dixon
    • 3
    • 4
  • Anita Brinker
    • 3
    • 4
  • Miguel Frada
    • 5
  • Steven N. Baldassano
    • 2
  • Paul G. Falkowski
    • 4
    • 5
  • G. Charles Dismukes
    • 1
    Email author
  1. 1.Department of Chemistry and Chemical BiologyRutgers UniversityPiscatawayUSA
  2. 2.Department of ChemistryPrinceton UniversityPiscatawayUSA
  3. 3.Department of Nutritional SciencesRutgers UniversityPiscatawayUSA
  4. 4.Rutgers Center for Lipid ResearchRutgers UniversityPiscatawayUSA
  5. 5.Environmental Biophysics and Molecular Ecology ProgramRutgers UniversityPiscatawayUSA

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