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Journal of Industrial Microbiology & Biotechnology

, Volume 43, Issue 12, pp 1671–1680 | Cite as

Nitrogen deprivation of microalgae: effect on cell size, cell wall thickness, cell strength, and resistance to mechanical disruption

  • Benjamin H. J. Yap
  • Simon A. Crawford
  • Raymond R. Dagastine
  • Peter J. Scales
  • Gregory J. O. Martin
Bioenergy/Biofuels/Biochemicals - Original Paper

Abstract

Nitrogen deprivation (N-deprivation) is a proven strategy for inducing triacylglyceride accumulation in microalgae. However, its effect on the physical properties of cells and subsequently on product recovery processes is relatively unknown. In this study, the effect of N-deprivation on the cell size, cell wall thickness, and mechanical strength of three microalgae was investigated. As determined by analysis of micrographs from transmission electron microscopy, the average cell size and cell wall thickness for N-deprived Nannochloropsis sp. and Chlorococcum sp. were ca. 25% greater than the N-replete cells, and 20 and 70% greater, respectively, for N-deprived Chlorella sp. The average Young’s modulus of N-deprived Chlorococcum sp. cells was estimated using atomic force microscopy to be 775 kPa; 30% greater than the N-replete population. Although statistically significant, these microstructural changes did not appear to affect the overall susceptibility of cells to mechanical rupture by high pressure homogenisation. This is important as it suggests that subjecting these microalgae to nitrogen starvation to accumulate lipids does not adversely affect the recovery of intracellular lipids.

Keywords

Microalgae Cell strength Nitrogen deprivation Cell rupture Atomic force microscopy 

Notes

Acknowledgements

The authors gratefully acknowledge the support of the Particulate Fluids Processing Centre, a Special Research Centre of the Australian Research Council. This work was performed in part at the Melbourne Centre for Nanofabrication (MCN) in the Victorian Node of the Australian National Fabrication Facility (ANFF) and in the Materials Characterization and Fabrication Platform (MCFP) at the University of Melbourne.

References

  1. 1.
    Barsanti L, Gualtieri P (2006) Algae: anatomy, biochemistry, and biotechnology. CRC Press, Taylor & Francis Group, Boca RatonGoogle Scholar
  2. 2.
    Berner T (1993) Ultrastructure of microalgae. CRC Press Inc, Boca RatonGoogle Scholar
  3. 3.
    Blumreisinger M, Meindl D, Loos E (1983) Cell wall composition of chlorococcal algae. Phytochemistry 22:1603–1604. doi: 10.1016/0031-9422(83)80096-x CrossRefGoogle Scholar
  4. 4.
    Bowen WR, Lovitt RW, Wright CJ (2000) Application of atomic force microscopy to the study of micromechanical properties of biological materials. Biotechnol Lett 22:893–903. doi: 10.1023/a:1005604028444 CrossRefGoogle Scholar
  5. 5.
    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. doi: 10.1016/j.biortech.2012.08.003 PubMedGoogle Scholar
  6. 6.
    Converti A, Casazza AA, Ortiz EY, Perego P, Del Borghi M (2009) Effect of temperature and nitrogen concentration on the growth and lipid content of Nannochloropsis oculata and Chlorella vulgaris for biodiesel production. Chem Eng Process 48:1146–1151. doi: 10.1016/j.cep.2009.03.006 CrossRefGoogle Scholar
  7. 7.
    Engler C (1985) Disruption of microbial cells. In: Moo-Young M (ed) Comprehensive biotechnology. Pegamon Press, OxfordGoogle Scholar
  8. 8.
    Fábregas J, Maseda A, Domínguez A, Ferreira M, Otero A (2002) Changes in the cell composition of the marine microalga, Nannochloropsis gaditana, during a light: dark cycle. Biotechnol Lett 24:1699–1703. doi: 10.1023/a:1020661719272 CrossRefGoogle Scholar
  9. 9.
    Goold H, Beisson F, Peltier G, Li-Beisson Y (2015) Microalgal lipid droplets: composition, diversity, biogenesis and functions. Plant Cell Rep 34:545–555. doi: 10.1007/s00299-014-1711-7 CrossRefPubMedGoogle Scholar
  10. 10.
    Greaves GN, Greer A, Lakes R, Rouxel T (2011) Poisson’s ratio and modern materials. Nat Mater 10:823–837CrossRefPubMedGoogle Scholar
  11. 11.
    Griffiths MJ, van Hille RP, Harrison STL (2012) Lipid productivity, settling potential and fatty acid profile of 11 microalgal species grown under nitrogen replete and limited conditions. J Appl Phycol 24(5):989–1001. doi: 10.1007/s10811-011-9723-y CrossRefGoogle Scholar
  12. 12.
    Guihéneuf F, Mimouni V, Ulmann L, Tremblin G (2009) Combined effects of irradiance level and carbon source on fatty acid and lipid class composition in the microalga Pavlova lutheri commonly used in mariculture. J Exp Mar Biol Ecol 369:136–143. doi: 10.1016/j.jembe.2008.11.009 CrossRefGoogle Scholar
  13. 13.
    Guillard RRL, Ryther JH (1962) Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (cleve) Gran. Can J Microbiol 8:229–239CrossRefPubMedGoogle Scholar
  14. 14.
    Guschina IA, Harwood JL (2006) Lipids and lipid metabolism in eukaryotic algae. Prog Lipid Res 45:160–186. doi: 10.1016/j.plipres.2006.01.001 CrossRefPubMedGoogle Scholar
  15. 15.
    Halim R, Gladman B, Danquah MK, Webley PA (2011) Oil extraction from microalgae for biodiesel production. Bioresour Technol 102:178–185. doi: 10.1016/j.biortech.2010.06.136 CrossRefPubMedGoogle Scholar
  16. 16.
    Halim R, Webley PA, Martin GJO (2015) The CIDES process: fractionation of concentrated microalgal paste for co-production of biofuel, nutraceuticals, and high-grade protein feed. Algal Res. doi: 10.1016/j.algal.2015.09.018
  17. 17.
    Hamm CE, Merkel R, Springer O, Jurkojc P, Maier C, Prechtel K, Smetacek V (2003) Architecture and material properties of diatom shells provide effective mechanical protection. Nature 421:841–843CrossRefPubMedGoogle Scholar
  18. 18.
    Hertz H (1881) On the contact of elastic solids. J Reine Angew Math 92:156–171Google Scholar
  19. 19.
    Hu Q, Kurano N, Kawachi M, Iwasaki I, Miyachi S (1998) Ultrahigh-cell-density culture of a marine green alga Chlorococcum littorale in a flat-plate photobioreactor. Appl Microbiol Biotechnol 49:655–662CrossRefGoogle Scholar
  20. 20.
    Hu Q, Sommerfield M, Jarvis E, Ghirardi M, Posewitz M, Seibert M, Darzins A (2008) Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J 54:621–639CrossRefPubMedGoogle Scholar
  21. 21.
    Huerlimann R, de Nys R, Heimann K (2010) Growth, lipid content, productivity, and fatty acid composition of tropical microalgae for scale-up production. Biotechnol Bioeng 107:245–257. doi: 10.1002/bit.22809 CrossRefPubMedGoogle Scholar
  22. 22.
    Hutter JL, Bechhoefer J (1993) Calibration of atomic-force microscope tips. Rev Sci Instrum 64:1868–1873CrossRefGoogle Scholar
  23. 23.
    Jiang PL, Pasaribu B, Chen CS (2014) Nitrogen-deprivation elevates lipid levels in Symbiodinium spp. by lipid droplet accumulation: morphological and compositional analyses. PLoS One 9(1):e87416. doi: 10.1371/journal.pone.0087416 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Johnson KL (1992) Normal contact of elastic solids: Hertz theory. In: Contact mechanics. Cambridge University Press, Cambridge, pp 84–106Google Scholar
  25. 25.
    Lee AK, Lewis DM, Ashman PJ (2013) Force and energy requirement for microalgal cell disruption: an atomic force microscope evaluation. Bioresour Technol 128:199–206CrossRefPubMedGoogle Scholar
  26. 26.
    Liang K, Zhang Q, Gu M, Cong W (2013) Effect of phosphorus on lipid accumulation in freshwater microalga Chlorella sp. J Appl Phycol 25:311–318. doi: 10.1007/s10811-012-9865-6 CrossRefGoogle Scholar
  27. 27.
    Lulevich V, Zink T, Chen HY, Liu FT, Liu G (2006) Cell mechanics using atomic force microscopy-based single-cell compression. Langmuir 22:8151–8155CrossRefPubMedGoogle Scholar
  28. 28.
    Martin GJO, Hill DRA, Olmstead ILD, Bergamin A, Shears MJ, Dias DA, Kentish SE, Scales PJ, Botté CY, Callahan DL (2014) Lipid profile remodeling in response to nitrogen deprivation in the microalgae Chlorella sp. (Trebouxiophyceae) and Nannochloropsis sp. (Eustigmatophyceae). PLoS One 9:e103389. doi: 10.1371/journal.pone.0103389 CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Middelberg APJ (1995) Process-scale disruption of microorganisms. Biotechnol Adv 13:491–551CrossRefPubMedGoogle Scholar
  30. 30.
    Munns R, Greenway H, Setter T, Kuo J (1983) Turgor pressure, volumetric elastic modulus, osmotic volume and ultrastructure of Chlorella emersonii grown at high and low external NaCl. J Exp Bot 34:144–155CrossRefGoogle Scholar
  31. 31.
    Olmstead ILD, Hill DRA, Dias DA, Jayasinghe NS, Callahan DL, Kentish SE, Scales PJ, Martin GJO (2013) A quantitative analysis of microalgal lipids for optimization of biodiesel and omega-3 production. Biotechnol Bioeng 110(8):2096–2104. doi: 10.1002/bit.24844 PubMedGoogle Scholar
  32. 32.
    Olmstead ILD, Kentish SE, Scales PJ, Martin GJO (2013) Low solvent, low temperature method for extracting biodiesel lipids from concentrated microalgal biomass. Bioresour Technol 148:615–619CrossRefPubMedGoogle Scholar
  33. 33.
    Pal D, Khozin-Goldberg I, Cohen Z, Boussiba S (2011) The effect of light, salinity, and nitrogen availability on lipid production by Nannochloropsis sp. Appl Microbiol Biotechnol 90(4):1429–1441. doi: 10.1007/s00253-011-3170-1
  34. 34.
    Rasband WS (1997) ImageJ. National Institute of Health, BethesdaGoogle Scholar
  35. 35.
    Řezanka T, Lukavský J, Nedbalová L, Sigler K (2011) Effect of nitrogen and phosphorus starvation on the polyunsaturated triacylglycerol composition, including positional isomer distribution, in the alga Trachydiscus minutus. Phytochemistry 72:2342–2351. doi: 10.1016/j.phytochem.2011.08.017 CrossRefPubMedGoogle Scholar
  36. 36.
    Richardson B, Orcutt D, Schwertner H, Martinez CL, Wickline HE (1969) Effects of nitrogen limitation on the growth and composition of unicellular algae in continuous culture. Appl Microbiol 18:245–250PubMedPubMedCentralGoogle Scholar
  37. 37.
    Rodolfi L, Chini Zittelli G, Bassi N, Padovani G, Biondi N, Bonini G, Tredici MR (2008) Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnol Bioeng 102:100–112CrossRefGoogle Scholar
  38. 38.
    Roessler PG (1988) Changes in the activities of various lipid and carbohydrate biosynthetic enzymes in the diatom Cyclotella cryptica in response to silicon deficiency. Arch Biochem Biophys 267:521–528. doi: 10.1016/0003-9861(88)90059-8 CrossRefPubMedGoogle Scholar
  39. 39.
    Roessler PG (1990) Environmental control of glycerolipid metabolism in microalgae: commercial implications and future research directions. J Phycol 26:393–399. doi: 10.1111/j.0022-3646.1990.00393.x CrossRefGoogle Scholar
  40. 40.
    Sato T (1968) A modified method for lead staining of thin sections. J Electron Microsc 17:158–159Google Scholar
  41. 41.
    Scott SA, Davey MP, Dennis JS, Horst I, Howe CJ, Lea-Smith DJ, Smith AG (2010) Biodiesel from algae: challenges and prospects. Curr Opin Biotechnol 21:277–286CrossRefPubMedGoogle Scholar
  42. 42.
    Sheehan J, Dunahay T, Benemann J, Roessler P (1998) A look back at the U.S. Department of Energy's Aquatic Species Program—biodiesel from algae. National Renewable Energy Laboratory, Golden, CO. Report NREL/TP-580–24190Google Scholar
  43. 43.
    Simionato D, Block MA, La Rocca N, Jouhet J, Maréchal E, Finazzi G, Morosinotto T (2013) The response of Nannochloropsis gaditana to nitrogen starvation includes de novo biosynthesis of triacylglycerols, a decrease of chloroplast galactolipids, and reorganization of the photosynthetic apparatus. Eukaryot Cell 12:665–676CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Siron R, Giusti G, Berland B (1989) Changes in the fatty acid composition of Phaeodactylum tricornutum and Dunaliella tertiolecta during growth and under phosphorus deficiency. Mar Ecol Prog Ser Oldend 55:95–100CrossRefGoogle Scholar
  45. 45.
    Spiden EM, Scales PJ, Yap BH, Kentish SE, Hill DR, Martin GJ (2015) The effects of acidic and thermal pretreatment on the mechanical rupture of two industrially relevant microalgae: Chlorella sp. and Navicula sp. Algal Res 7:5–10CrossRefGoogle Scholar
  46. 46.
    Spiden EM, Yap BH, Hill DR, Kentish SE, Scales PJ, Martin GJ (2013) Quantitative evaluation of the ease of rupture of industrially promising microalgae by high pressure homogenization. Bioresour Technol 140:165–171. doi: 10.1016/j.biortech.2013.04.074
  47. 47.
    Stenson JD, Thomas CR, Hartley P (2009) Modelling the mechanical properties of yeast cells. Chem Eng Sci 64:1892–1903. doi: 10.1016/j.ces.2009.01.016 CrossRefGoogle Scholar
  48. 48.
    Sukenik A, Carmeli Y (1990) Lipid synthesis and fatty acid composition in Nannochloropsis sp. (Eustigmatophyceae) grown in a light-dark cycle. J Phycol 26:463–469. doi: 10.1111/j.0022-3646.1990.00463.x CrossRefGoogle Scholar
  49. 49.
    Takagi M, Yoshida T (2006) Effect of salt concentration on intracellular accumulation of lipids and triacylglyceride in marine microalgae Dunaliella cells. J Biosci Bioeng 101:223–226CrossRefPubMedGoogle Scholar
  50. 50.
    Thompson GA Jr (1996) Lipids and membrane function in green algae. Biochim Biophys Acta 1302:17–45CrossRefPubMedGoogle Scholar
  51. 51.
    Touhami A, Nysten B, Dufrêne YF (2003) Nanoscale mapping of the elasticity of microbial cells by atomic force microscopy. Langmuir 19:4539–4543. doi: 10.1021/la034136x CrossRefGoogle Scholar
  52. 52.
    Van Donk E, Lurling M, Hessen D, Lokhorst G (1997) Altered cell wall morphology in nutrient-deficient phytoplankton and its impact on grazers. Limnol Oceanogr 42:357–364CrossRefGoogle Scholar
  53. 53.
    Wada H, Murata N (1998) Membrane lipids in cyanobacteria. In: Siegenthaler PA, Murata N (eds) Lipids in photosynthesis: structure, function and genetics. Springer, pp 65–81Google Scholar
  54. 54.
    Warren K, Mpagazehe J, LeDuc P, Higgs C III (2014) Probing the elastic response of microalga Scenedesmus dimorphus in dry and aqueous environments through atomic force microscopy. Appl Phys Lett 105:163701CrossRefGoogle Scholar
  55. 55.
    Wei C, Lintilhac PM (2007) Loss of stability: a new look at the physics of cell wall behavior during plant cell growth. Plant Physiol 145:763–772CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Weldy CS, Huesemann M (2007) Lipid production by Dunaliella salina in batch culture: effects of nitrogen limitation and light intensity. US Dep Energy J Undergrad Res 7:115–122Google Scholar
  57. 57.
    Weng L-C, Pasaribu B, Lin I-P, Tsai C-H, Chen C-S, Jiang P-L (2014) Nitrogen deprivation induces lipid droplet accumulation and alters fatty acid metabolism in symbiotic sinoflagellates isolated from Aiptasia pulchella. Nat Sci Rep 4:5777Google Scholar
  58. 58.
    Wijffels RH, Barbosa MJ, Eppink MH (2010) Microalgae for the production of bulk chemicals and biofuels. Biofuels Bioprod Biorefin 4:287–295CrossRefGoogle Scholar
  59. 59.
    Yap BHJ, Crawford SA, Dumsday GJ, Scales PJ, Martin GJO (2014) A mechanistic study of algal cell disruption and its effect on lipid recovery by solvent extraction. Algal Res 5:112–120CrossRefGoogle Scholar
  60. 60.
    Yap BHJ, Dumsday GJ, Scales PJ, Martin GJO (2015) Energy evaluation of algal cell disruption by high pressure homogenisation. Bioresour Technol 184:280–285. doi: 10.1016/j.biortech.2014.11.049 CrossRefPubMedGoogle Scholar
  61. 61.
    Yap BHJ, Martin GJO, Scales PJ (2016) Rheological manipulation of flocculated algal slurries to achieve high solids processing. Algal Res 14:1–8. doi: 10.1016/j.algal.2015.12.007 CrossRefGoogle Scholar
  62. 62.
    Yu E, Zendejas F, Lane P, Gaucher S, Simmons B, Lane T (2009) Triacylglycerol accumulation and profiling in the model diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum (Baccilariophyceae) during starvation. J Appl Phycol 21:669–681. doi: 10.1007/s10811-008-9400-y CrossRefGoogle Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2016

Authors and Affiliations

  1. 1.Department of Chemical and Biomolecular EngineeringThe University of MelbourneParkvilleAustralia
  2. 2.School of BotanyThe University of MelbourneParkvilleAustralia

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