Metabolic and physiological regulation of Chlorella sp. (Trebouxiophyceae, Chlorophyta) under nitrogen deprivation

  • Wai-Kuan Yong
  • Phaik-Eem LimEmail author
  • Vejeysri Vello
  • Kae-Shin Sim
  • Nazia Abdul Majid
  • Emienour Muzalina Mustafa
  • Nik Meriam Nik Sulaiman
  • Kan-Ern Liew
  • Brenna Jia-Tian Chen
  • Siew-Moi Phang


A freshwater green microalgae Chlorella sp., UMACC344 was shown to produce high lipid content and has the potential to be used as feedstock for biofuel production. In this study, photosynthetic efficiency, biochemical profiles and non-targeted metabolic profiling were studied to compare between the nitrogen-replete and deplete conditions. Slowed growth, change in photosynthetic pigments and lowered photosynthetic efficiency were observed in response to nitrogen deprivation. Biochemical profiles of the cultures showed an increased level of carbohydrate, lipids and total fatty acids, while the total soluble protein content was lowered. A trend of fatty acid saturation was observed in the nitrogen-deplete culture with an increase in the level of saturated fatty acids especially C16:0 and C18:0, accompanied by a decrease in proportions of monounsaturated and polyunsaturated fatty acids. Fifty-nine metabolites, including amino acids, lipids, phytochemical compounds, vitamins and cofactors were significantly dysregulated and annotated in this study. Pathway mapping analysis revealed a rewiring of metabolic pathways in the cells, particularly purine, carotenoid, nicotinate and nicotinamide, and amino acid metabolisms. Within the treatment period of nitrogen deprivation, the key processes involved were reshuffling of nitrogen from proteins and photosynthetic machinery, together with carbon repartitioning in carbohydrates and lipids.


metabolic profiling Chlorella sp. nitrogen stress lipid fatty acid 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.



We thank Assoc. Prof. Sanjay Swarup, Dr. Peter Benke and Dr. Shivshankar Umashankar at the Environmental Research Institute, National University of Singapore for their assistance in data analysis.


  1. Allen J W, DiRusso C C, Black P N. 2015. Triacylglycerolsynthesis during nitrogen stress involves the prokaryotic lipid synthesis pathway and acyl chain remodeling in the microalgae Coccomyxa subellipsoidea. Algal Res., 10: 110–120, Scholar
  2. Bligh E G, Dyer W J. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37 (8): 911–917,–099.CrossRefGoogle Scholar
  3. Chu W L, Phang S M, Goh S H. 1994. Studies on the production of useful chemicals, especially fatty acids in the marine diatom Nitzschia conspicua Grunow. Hydrobiologia, 285 (1–3): 33–40, Scholar
  4. Dubois M, Gilles K A, Hamilton J K, Rebers P A, Smith F. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem., 28 (3): 350–356, Scholar
  5. Gao B Y, Yang J, Lei X Q, Xia S, Li A F, Zhang C W. 2016. Characterization of cell structural change, growth, lipid accumulation, and pigment profile of a novel oleaginous microalga, Vischeria stellata (Eustigmatophyceae), cultured with different initial nitrate supplies. J. Appl. Phycol., 28 (2): 821–830,–015–0626–1.CrossRefGoogle Scholar
  6. Gao C F, Wang Y, Shen Y, Yan D, He X, Dai J B, Wu Q Y. 2014. Oil accumulation mechanisms of the oleaginous microalga Chlorella protothecoides revealed through its genome, transcriptomes, and proteomes. BMC Genomics, 15: 582,–2164–15–582.CrossRefGoogle Scholar
  7. Gargouri M, Park J J, Holguin F O, Kim M J, Wang H X, Deshpande R R, Shachar–Hill Y, Hicks L M, Gang D R. 2015. Identification of regulatory network hubs that controllipid metabolism in Chlamydomonas reinhardtii. J. Exp. Bot., 66 (15): 4 551–4 566, Scholar
  8. Gowda H, Ivanisevic J, Johnson C H, Kurczy M E, Benton H P, Rinehart D, Nguyen T, Ray J, Kuehl J, Arevalo B, Westenskow P D, Wang J H, Arkin A P, Deutschbauer A M, Patti G J, Siuzdak G. 2014. Interactive XCMS Online: simplifying advanced metabolomic data processing and subsequent statistical analyses. Anal. Chem., 86 (14): 6 931–6 939, Scholar
  9. Hu Q, Sommerfeld 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 (4): 621–639, 1111/j.1365–313X.2008.03492.x.CrossRefGoogle Scholar
  10. Ito T, Tanaka M, Shinkawa H, Nakada T, Ano Y, Kurano N, Soga T, Tomita M. 2013. Metabolic and morphological changes of an oil accumulating trebouxiophycean alga in nitrogen–deficient conditions. Metabolomics, 9 (S1): 178–187,–012–0463–z.Google Scholar
  11. Juneja A, Ceballos R M, Murthy G S. 2013. Effects of environmental factors and nutrient availability on the biochemical composition of algae for biofuels production: a review. Energies, 6 (9): 4 607–4 638, 3390/en6094607.CrossRefGoogle Scholar
  12. Levitan O, Dinamarca J, Zelzion E, Lun D S, Guerra L T, Kim M K, Kim J, Van Mooy B A S, Bhattacharya D, Falkowski P G. 2015. Remodeling of intermediate metabolism in the diatom Phaeodactylum tricornutum under nitrogen stress. Proc. Natl. Acad. Sci. U. S. A., 112 (2): 412–417, https://doi. org/10.1073/pnas.1419818112.CrossRefGoogle Scholar
  13. Li T T, Gargouri M, Feng J, Park J J, Gao D F, Miao C, Dong T, Gang D R, Chen SL. 2015. Regulation of starch and lipid accumulation in a microalga Chlorella sorokiniana. Bioresour. Technol., 180: 250–257, 1016/j.biortech.2015.01.005.CrossRefGoogle Scholar
  14. Li Y Q, Han F X, Xu H, Mu J X, Chen D, Feng B, Zeng H Y. 2014. Potential lipid accumulation and growth characteristic of the green alga Chlorella with combination cultivation mode of nitrogen (N) and phosphorus (P). Bioresour. Technol., 174: 24–32, biortech.2014.09.142.CrossRefGoogle Scholar
  15. Li Y T, Han D X, Yoon K, Zhu S N, Sommerfeld M, Hu Q. 2013. Molecular and cellular mechanisms for lipid synthesis and accumulation in microalgae: Biotechnological implications. In: Richmond A, Hu Q eds. Handbook of Microalgal Culture: Applied Phycology and Biotechnology. 2 nd edn. John Wiley & Sons, Ltd, Oxford, UK. p.545–565. Scholar
  16. Longworth J, Wu D Y, Huete–Ortega M, Wright P C, Vaidyanathan S. 2016. Proteome response of Phaeodactylum tricornutum, during lipid accumulation induced by nitrogen depletion. Algal Res., 18: 213–224, Scholar
  17. Lu N, Wei D, Chen F, Yang S T. 2013. Lipidomic profiling reveals lipid regulation in the snow alga Chlamydomonas nivalis in response to nitrate or phosphate deprivation. Process Biochem., 48 (4): 605–613, 1016/j.procbio.2013.02.028.CrossRefGoogle Scholar
  18. Martin G J O, Hill D R A, Olmstead I L D, Bergamin A, Shears M J, Dias D A, Kentish S E, Scales P J, Botté C Y, Callahan D L. 2014. Lipid profile remodeling in response to nitrogen deprivation in the microalgae Chlorella sp. (Trebouxiophyceae) and Nannochloropsis sp. (Eustigmatophyceae). PLoS One, 9 (8): e103389, Scholar
  19. Millán–Oropeza A, Fernández–Linares L. 2017. Biomass and lipid production from Nannochloropsis oculata growth in raceway ponds operated in sequential batch mode under greenhouse conditions. Environ. Sci. Pollut. Res. Int., 24 (33): 25 618–25 626,–016–7013–6.CrossRefGoogle Scholar
  20. Msanne J, Xu D, Konda A R, Casas–Mollano J A, Awada T, Cahoon E B, Cerutti H. 2012. Metabolic and gene expression changes triggered by nitrogen deprivation in the photoautotrophically grown microalgae Chlamydomonas reinhardtii and Coccomyxa sp. C–169. Phytochemistry, 75: 50–59, phytochem.2011.12.007.CrossRefGoogle Scholar
  21. Ng F L, Phang S M, Periasamy V, Yunus K, Fisher A C. 2014. Evaluation of algal biofilms on indium tin oxide (ITO) for use in biophotovoltaic platforms based on photosynthetic performance. PLoS One, 9 (5): e97643, https://doi. org/10.1371/journal.pone.0097643.CrossRefGoogle Scholar
  22. Nichols H W, Bold H C. 1965. Trichosarcina polymorpha gen. et sp. nov. J. Phycol., 1 (1): 34–38,–8817.1965.tb04552.x.CrossRefGoogle Scholar
  23. Park J J, Wang H X, Gargouri M, Deshpande R R, Skepper J N, Holguin F O, Juergens M T, Shachar–Hill Y, Hicks L M, Gang D R. 2015. The response of Chlamydomonas reinhardtii to nitrogen deprivation: a systems biology analysis. Plant J., 81 (4): 611–624, Scholar
  24. Recht L, Töpfer N, Batushansky A, Sikron N, Gibon Y, Fait A, Nikoloski Z, Boussiba S, Zarka A. 2014. Metabolite profiling and integrative modeling reveal metabolic constraints for carbon partitioning under nitrogen starvation in the green algae Haematococcus pluvialis. J. Biol. Chem., 289 (44): 30 387–30 403, 1074/jbc.M114.555144.CrossRefGoogle Scholar
  25. Salomon E, Bar–Eyal L, Sharon S, Keren N. 2013. Balancing photosynthetic electron flow is critical for cyanobacterial acclimation to nitrogen limitation. Biochim. Biophys. Acta, 1827 (3): 340–347, 2012.11.010.CrossRefGoogle Scholar
  26. Schmollinger S, Mühlhaus T, Boyle N R, Blaby I K, Casero D, Mettler T, Moseley J L, Kropat J, Sommer F, Strenkert D, Hemme D, Pellegrini M, Grossman A R, Stitt M, Schroda M, Merchant S S. 2014. Nitrogen–sparing mechanisms in Chlamydomonas affect the transcriptome, the proteome, and photosynthetic metabolism. Plant Cell, 26 (4): 1 410–1 435, Scholar
  27. Sharma K K, Schuhmann H, Schenk P M. 2012. High lipid induction in microalgae for biodiesel production. Energies, 5 (5): 1 532–1 553, Scholar
  28. Solovchenko A E, Khozin–Goldberg I, Cohen Z, Merzlyak M N. 2009. Carotenoid–to–chlorophyll ratio as a proxy for assay of total fatty acids and arachidonic acid content in the green microalga Parietochloris incisa. J. Appl. Phycol., 21 (3): 361–366,–008–9377–6.CrossRefGoogle Scholar
  29. Stansell G R, Gray V M, Sym S D. 2012. Microalgal fatty acid composition: Implications for biodiesel quality. J. Appl. Phycol., 24 (4): 791–801,–011–9696–x.CrossRefGoogle Scholar
  30. Stasolla C, Katahira R, Thorpe T A, Ashihara H. 2003. Purine and pyrimidine nucleotide metabolism in higher plants. J. Plant Physiol., 160 (11): 1 271–1 295, 1078/0176–1617–01169.CrossRefGoogle Scholar
  31. Strickland J D H, Parsons T R. 1972. A Practical Handbook of Seawater Analysis. 2 nd edn. Fisheries Research Board of Canada, Ottawa, Canada. 310p.Google Scholar
  32. Wase N, Black P N, Stanley B A, DiRusso C C. 2014. Integrated quantitative analysis of nitrogen stress response in Chlamydomonas reinhardtii using metabolite and protein profiling. J. Proteome Res., 13 (3): 1 373–1 396, https://doi. org/10.1021/pr400952z.CrossRefGoogle Scholar
  33. Worley B, Powers R. 2013. Multivariate analysis in metabolomics. Curr. Metabolomics, 1 (1): 92–107, Scholar
  34. Yang D W, Zhang Y T, Barupal D K, Fan X L, Gustafson R, Guo R B, Fiehn O. 2014. Metabolomics of photobiological hydrogen production induced by CCCP in Chlamydomonas reinhardtii. Int. J. Hydrogen Energy, 39 (1): 150–158, Scholar
  35. Yang Z K, Niu Y F, Ma Y H, Xue J, Zhang M H, Yang W D, Liu J S, Lu S H, Guan Y F, Li H Y. 2013. Molecular and cellular mechanisms of neutral lipid accumulation in diatom following nitrogen deprivation. Biotechnol. Biofuels, 6 (1): 67,–6834–6–67.CrossRefGoogle Scholar

Copyright information

© Chinese Society for Oceanology and Limnology, Science Press and Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Wai-Kuan Yong
    • 1
    • 2
  • Phaik-Eem Lim
    • 1
    Email author
  • Vejeysri Vello
    • 1
    • 3
  • Kae-Shin Sim
    • 3
  • Nazia Abdul Majid
    • 3
  • Emienour Muzalina Mustafa
    • 4
  • Nik Meriam Nik Sulaiman
    • 5
  • Kan-Ern Liew
    • 6
  • Brenna Jia-Tian Chen
    • 7
  • Siew-Moi Phang
    • 1
    • 3
  1. 1.Institute of Ocean and Earth SciencesUniversity of MalayaKuala LumpurMalaysia
  2. 2.Institute of Graduate StudiesUniversity of MalayaKuala LumpurMalaysia
  3. 3.Institute of Biological Sciences, Faculty of ScienceUniversity of MalayaKuala LumpurMalaysia
  4. 4.School of Fisheries and Aquaculture SciencesUniversity of MalaysiaKuala TerengganuMalaysia
  5. 5.Department of Chemical Engineering, Faculty of EngineeringUniversity of MalayaKuala LumpurMalaysia
  6. 6.Airbus Group MalaysiaKuala LumpurMalaysia
  7. 7.Aerospace Malaysia Innovation CentreCyberjayaMalaysia

Personalised recommendations