Advertisement

Accumulation of docosahexaenoic acid-rich lipid in thraustochytrid Aurantiochytrium sp. strain T66: effects of N and P starvation and O2 limitation

  • Anita N. JakobsenEmail author
  • Inga M. Aasen
  • Kjell D. Josefsen
  • Arne R. Strøm
Applied Microbial and Cell Physiology

Abstract

Aurantiochytrium sp. strain T66 was grown in batch bioreactor cultures in a defined glutamate- and glycerol-containing growth medium. Exponentially growing cells had a lipid content of 13% (w/w) of dry weight. A fattening of cells fed excess glycerol occurred in the post-exponential growth phase, after the medium was depleted of N or P. Lipid accumulation was also initiated by O2 limitation (below 1% of saturation). N starvation per se, or in combination with O2 limitation, gave the highest lipid content, i.e., 54% to 63% (w/w) of dry weight. The corresponding maximum culture density was 90 to 100 g/l dry biomass. The content of docosahexaenoic acid (22:6n-3) in N starved, well-oxygenated cells reached 29% (w/w) of total fatty acids but increased to 36% to 52% in O2-limited cells, depending on the time span of the limitation. O2-limited cells did not accumulate the monounsaturated fatty acids that were normally present. We inferred that the biological explanation is that O2 limitation hindered the O2-dependent desaturase(s) and favored the O2-independent polyunsaturated fatty acid synthase. The highest overall volumetric productivity of docosahexaenoic acid observed was 93 mg/l/h. Additionally, we present a protocol for quantitative lipid extraction, involving heat and protease treatment of freeze-dried thraustochytrids.

Keywords

Thraustochytrid Aurantiochytrium Docosahexaenoic acid Lipid accumulation Lipid extraction Nutrient starvation 

Notes

Acknowledgements

We are grateful to Kristin B. Antonsen for help with glutamate analysis and Trond E. Ellingsen for valuable comments to the manuscript. This work was supported by grants from the Research Council of Norway.

References

  1. Adl SM, Simpson AGB, Farmer MA, Andersen RA, Anderson OR, Barta JR, Browser SS, Brugerolle G, Fensome RA, Fredericq S, James TY, Karpov S, Kugrens P, Krug J, Lane CE, Lewis LA, Lodge J, Lynn DH, Mann DG, McCourt RM, Mendoza L, Moestrup Ø, Mozley-Standridge SE, Nerad TA, Shearer CA, Smirnov AV, Spiegel FW, Taylor MFJR (2005) The new higher level classification of eukaryotes with emphasis on the taxonomy of protists. J Eukaryot Microbiol 52:399–451CrossRefGoogle Scholar
  2. Arterburn LM, Oken HA, Hoffman JP, Bailey-Hall E, Chung G, Rom D, Hamersley J, McCarthy D (2007) Bioequivalence of docosahexaenoic acid from different algal oils in capsules and in a DHA-fortified food. Lipids 42:1011–1024CrossRefGoogle Scholar
  3. Bailey RB, DiMasi D, Hansen JM, Mirrasoul PJ, Ruecker CM, Veeder GT III, Kaneko T, Barclay WR (2003) Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors. United States Patent 6, 607, 900Google Scholar
  4. Bajpai PK, Bajpai P, Ward OP (1991) Optimization of production of docosahexaenoic acid (DHA) by Thraustochytrium aureum ATCC 34304. JAOCS (J Assoc Oil Chem Soc) 68:509–514Google Scholar
  5. Baykov AA, Evtushenko OA, Avaeva SM (1988) A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay. Anal Biochem 171:266–270CrossRefGoogle Scholar
  6. Bergé JP, Barnathan G (2005) Fatty acids from lipids of marine organisms: molecular biodiversity, roles as biomarkers, biologically active compounds, and economical aspects. Adv Biochem Eng Biotechnol 96:49–125Google Scholar
  7. Birch EE, Garfield S, Castañeda Y, Hughbanks-Wheaton D, Uauy R, Hoffman D (2007) Visual acuity and cognitive outcomes at 4 years of age in a double-blind, randomized trial of long-chain polyunsaturated fatty acid-supplemented infant formula. Early Hum Dev 83:279–284CrossRefGoogle Scholar
  8. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917Google Scholar
  9. Bowles RD, Hunt AE, Bremer GB, Duchars MG, Eaton RA (1999) Long-chain n-3 polyunsaturated fatty acid production by members of the marine protistan group the thraustochytrids: screening of isolates and optimisation of docosahexaenoic acid production. J Biotechnol 70:193–202CrossRefGoogle Scholar
  10. Burja AM, Radianingtyas H, Windust A, Barrow CJ (2006) Isolation and characterization of polyunsaturated fatty acid producing Thraustochytrium species: screening of strains and optimization of omega-3 production. Appl Microbiol Biotechnol 72:1161–1169CrossRefGoogle Scholar
  11. Burja AM, Armenta RE, Radianingtyas H, Barrow CJ (2007) Evaluation of fatty acid extraction methods for Thraustochytrium sp. ONC-T18. J Agric Food Chem 55:4795–4801CrossRefGoogle Scholar
  12. Christie WW (2003) Lipid analysis: isolation, separation, identification and structural analysis of lipids. Oily, BridgewaterGoogle Scholar
  13. Flynn KJ (1988) Some practical aspects of measurements of dissolved free amino acids in natural waters and within microalgae by the use of HPLC. Chem Ecol 3:269–293CrossRefGoogle Scholar
  14. Ganuza E, Izquierdo MS (2007) Lipid accumulation in Schizochytrium G13/2S produced in continuous culture. Appl Microbiol Biotechnol 76:985–990CrossRefGoogle Scholar
  15. Hardy R, Keay JN (1972) Seasonal variations in the chemical composition of Cornish mackerel, Scomber scombrus (L), with detailed reference to the lipids. J Food Technol 7:125–137Google Scholar
  16. Hauvermale A, Kuner J, Rosenzweig B, Guerra D, Diltz S, Metz JG (2006) Fatty acid production in Schizochytrium sp.: involvement of a polyunsaturated fatty acid synthase and a type I fatty acid synthase. Lipids 41:739–747CrossRefGoogle Scholar
  17. Honda D, Yokochi T, Nakahara T, Raghukumar S, Nakagiri A, Schaumann K, Higashihara T (1999) Molecular phylogeny of labyrinthulids and thraustochytrids based on the sequencing of 18S ribosomal RNA gene. J Eukaryot Microbiol 46:637–647CrossRefGoogle Scholar
  18. Huang J, Aki T, Yokochi T, Nakahara T, Honda D, Kawamoto S, Shigeta S, Ono K, Suzuki O (2003) Grouping newly isolated docosahexaenoic acid-producing thraustochytrids based on their polyunsaturated fatty acid profiles and comparative analysis of 18S rRNA genes. Mar Biotechnol 5:450–457CrossRefGoogle Scholar
  19. Jakobsen AN, Aasen IM, Strøm AR (2007) Endogenously synthesized (-)-proto-quercitol and glycine betaine are principal compatible solutes of Schizochytrium sp. strain S8 (ATCC 20889) and three new isolates of phylogenetically related thraustochytrids. Appl Environ Microbiol 73:5848–5856CrossRefGoogle Scholar
  20. Lewis T, Nichols PD, McMeekin TA (2000) Evaluation of extraction methods for recovery of fatty acids from lipid-producing microheterotrophs. J Microbiol Methods 43:107–116CrossRefGoogle Scholar
  21. Lindroth P, Mopper K (1979) High performance liquid chromatographic determination of subpicomole amounts of amino acids by precolumn fluorescence derivatization with o-phthaldialdehyde. Anal Chem 51:1667–1674CrossRefGoogle Scholar
  22. Metcalfe LD, Schmitz AA, Pelka JR (1966) Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Anal Chem 38:514–515CrossRefGoogle Scholar
  23. Metz JG, Roessler P, Facciotti D, Levering C, Dittrich F, Lassner M, Valentine R, Lardizabal K, Domergue F, Yamada A, Yazawa K, Knauf V, Browse J (2001) Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes. Science 293:290–293CrossRefGoogle Scholar
  24. Morita E, Kumon Y, Nakahara T, Kagiwada S, Noguchi T (2006) Docosahexaenoic acid production and lipid-body formation in Schizochytrium limacinum SR21. Mar. Biotechnol. 8:319–327CrossRefGoogle Scholar
  25. Napier JA, Michaelson LV (2001) Genomic and functional characterization of polyunsaturated fatty acid biosynthesis in Caenorhabditis elegans. Lipids 36:761–766CrossRefGoogle Scholar
  26. Perveen Z, Ando H, Ueno A, Ito Y, Yamamoto Y, Yamada Y, Takagi T, Kaneko T, Kogame K, Okuyama H (2006) Isolation and characterization of a novel thraustochytrid-like microorganism that efficiently produces docosahexaenoic acid. Biotechnol Lett 28:197–202CrossRefGoogle Scholar
  27. Qiu X (2003) Biosynthesis of docosahexaenoic acid (DHA, 22:6–4, 7,10,13,16,19): two distinct pathways. Prostaglandins Leukot Essent Fatty Acids 68:181–186CrossRefGoogle Scholar
  28. Qiu X, Hong H, MacKenzie SL (2001) Identification of a Δ4 fatty acid desaturase from Thraustochytrium sp. involved in the biosynthesis of docosahexanoic acid by heterologous expression in Saccharomyces cerevisiae and Brassica juncea. J Biol Chem 276:31561–31566CrossRefGoogle Scholar
  29. Raghukumar S (2002) Ecology of the marine protists, the Labyrinthulomycetes (Thraustochytrids and Labyrinthulids). Eur J Protistol 38:127–145CrossRefGoogle Scholar
  30. Ratledge C (2004) Fatty acid biosynthesis in microorganisms being used for Single Cell Oil production. Biochimie 86:807–815CrossRefGoogle Scholar
  31. Ratledge C, Wynn JP (2002) The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. Adv Appl Microbiol 51:1–51CrossRefGoogle Scholar
  32. Swaaf de ME, Sijtsma L, Pronk JT (2003) High-cell-density fed-batch cultivation of the docosahexaenoic acid producing marine alga Crypthecodinium cohnii. Biotechnol Bioeng 81:666–672CrossRefGoogle Scholar
  33. Unagul P, Assantachai C, Phadungruengluij S, Pongsuteeragul T, Suphantharika M, Verduyn C (2006) Biomass and docosahexaenoic acid formation by Schizochytrium mangrovei Sk-02 at low salt concentrations. Bot Mar 49:182–190CrossRefGoogle Scholar
  34. Ward OP, Singh A (2005) Omega-3/6 fatty acids: alternative sources of production. Process Biochem 40:3627–3652CrossRefGoogle Scholar
  35. Wu G, Truksa M, Datla N, Vrinten P, Bauer J, Zank T, Cirpus P, Heinz E, Qiu X (2005) Stepwise engineering to produce high yields of very long-chain polyunsaturated fatty acids in plants. Nat Biotechnol 23:1013–1017CrossRefGoogle Scholar
  36. Yaguchi T, Tanaka S, Yokochi T, Nakahara T, Higashihara T (1997) Production of high yields of docosahexaenoic acid by Schizochytrium sp. strain SR21. JAOCS (J Assoc Oil Chem Soc) 74:1431–1434Google Scholar
  37. Yokoyama R, Honda D (2007) Taxonomic rearrangement of the genus Schizochytrium sensu lato based on morphology, chemotaxonomic characteristics, and 18S rRNA gene phylogeny (Thraustochytriaceae, Labyrinthulomycetes): emendation for Schizochytrium and erection of Aurantiochytrium and Oblongichytrium gen. nov. Mycoscience 48:199–211CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Anita N. Jakobsen
    • 1
    • 2
    Email author
  • Inga M. Aasen
    • 3
  • Kjell D. Josefsen
    • 3
  • Arne R. Strøm
    • 1
  1. 1.Department of BiotechnologyNorwegian University of Science and TechnologyTrondheimNorway
  2. 2.Department of Food TechnologySør-Trøndelag University CollegeTrondheimNorway
  3. 3.Department of BiotechnologySINTEF Materials and ChemistryTrondheimNorway

Personalised recommendations