Journal of Microbiology

, Volume 56, Issue 1, pp 56–64 | Cite as

Application of high-salinity stress for enhancing the lipid productivity of Chlorella sorokiniana HS1 in a two-phase process

  • Ramesh Kakarla
  • Jung-Woon Choi
  • Jin-Ho Yun
  • Byung-Hyuk Kim
  • Jina Heo
  • Sujin Lee
  • Dae-Hyun Cho
  • Rishiram Ramanan
  • Hee-Sik KimEmail author
Microbial Ecology and Environmental Microbiology


Increased lipid accumulation of algal cells as a response to environmental stress factors attracted much attention of researchers to incorporate this stress response into industrial algal cultivation process with the aim of enhancing algal lipid productivity. This study applies high-salinity stress condition to a two-phase process in which microalgal cells are initially grown in freshwater medium until late exponential phase and subsequently subjected to high-salinity condition that induces excessive lipid accumulation. Our initial experiment revealed that the concentrated culture of Chlorella sorokiniana HS1 exhibited the intense fluorescence of Nile red at the NaCl concentration of 60 g/L along with 1 g/L of supplemental bicarbonate after 48 h of induction period without significantly compromising cultural integrity. These conditions were further verified with the algal culture grown for 7 days in a 1 L bottle reactor that reached late exponential phase; a 12% increment in the lipid content of harvested biomass was observed upon inducing high lipid accumulation in the concentrated algal culture at the density of 5.0 g DW/L. Although an increase in the sum of carbohydrate and lipid contents of harvested biomass indicated that the external carbon source supplemented during the induction period increased overall carbon assimilation, a decrease in carbohydrate content suggested the potential reallocation of cellular carbon that promoted lipid droplet formation under high-salinity stress. These results thus emphasize that the two-phase process can be successfully implemented to enhance algal lipid productivity by incorporating high-salinity stress conditions into the pre-concentrated sedimentation ponds of industrial algal production system.


Chlorella sorokiniana HS1 high-salinity stress lipid induction microalgae two-phase process 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Supplementary material

12275_2018_7488_MOESM1_ESM.pdf (1010 kb)
Supplementary material, approximately 0.98 MB.


  1. Ahmad, I. and Hellebust, J.A. 1984. Osmoregulation in the extremely euryhaline marine micro-alga Chlorella autotrophica. Plant Physiol. 74, 1010–1015.CrossRefPubMedPubMedCentralGoogle Scholar
  2. Almshawit, H., Pouniotis, D., and Macreadie, I. 2014. Cell density impacts on Candida glabrata survival in hypo-osmotic stress. FEMS Yeast Res. 14, 508–516.CrossRefPubMedGoogle Scholar
  3. Azachi, M., Sadka, A., Fisher, M., Goldshlag, P., Gokhman, I., and Zamir, A. 2002. Salt induction of fatty acid elongase and membrane lipid modifications in the extreme halotolerant alga Dunaliella salina. Plant Physiol. 129, 1320–1329.CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bligh, E.G. and Dyer, W.J. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917.CrossRefPubMedGoogle Scholar
  5. Brennan, L. and Owende, P. 2010. Biofuels from microalgae-a review of technologies for production, processing, and extractions of biofuels and co-products. Renew. Sust. Energ. Rev. 14, 557–577.CrossRefGoogle Scholar
  6. Chen, C.Y., Yeh, K.L., Aisyah, R., Lee, D.J., and Chang, J.S. 2011. Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: a critical review. Bioresour. Technol. 102, 71–81.CrossRefPubMedGoogle Scholar
  7. Cho, D.H., Ramanan, R., Heo, J., Lee, J., Kim, B.H., Oh, H.M., and Kim, H.S. 2015. Enhancing microalgal biomass productivity by engineering a microalgal-bacterial community. Bioresour. Technol. 175, 578–585.CrossRefPubMedGoogle Scholar
  8. Cho, D.H., Ramanan, R., Kim, B.H., Lee, J., Kim, S., Yoo, C., Choi, G.G., Oh, H.M., and Kim, H.S. 2013. Novel approach for the development of axenic microalgal cultures from environmental samples. J. Phycol. 49, 802–810.CrossRefPubMedGoogle Scholar
  9. Church, J., Hwang, J.H., Kim, K.T., McLean, R., Oh, Y.K., Nam, B., Joo, J.C., and Lee, W.H. 2017. Effect of salt type and concentration on the growth and lipid content of Chlorella vulgaris in synthetic saline wastewater for biofuel production. Bioresour. Technol. 243, 147–153.CrossRefPubMedGoogle Scholar
  10. Davis, R., Aden, A., and Pienkos, P.T. 2011. Techno-economic analysis of autotrophic microalgae for fuel production. Appl. Energy 88, 3524–3531.CrossRefGoogle Scholar
  11. El-Kassas, H.Y. 2013. Growth and fatty acid profile of the marine microalga Picochlorum sp. grown under nutrient stress conditions. Egypt. J. Aquat. Res. 39, 233–239.CrossRefGoogle Scholar
  12. Fan, J., Cui, Y., Wan, M., Wang, W., and Li, Y. 2014. Lipid accumulation and biosynthesis genes response of the oleaginous Chlorella pyrenoidosa under three nutrition stressors. Biotechnol. Biofuels 7, 7–17.CrossRefGoogle Scholar
  13. González, L.E., Díaz, G.C., Aranda, D.A.G., Cruz, Y.R., and Fortes, M.M. 2015. Biodiesel production based in microalgae: a biorefinery approach. Nat. Sci. 7, 358.Google Scholar
  14. Habiby, H., Afyuni, M., Khoshgoftarmanesh, A.H., and Schulin, R. 2014. Effect of preceding crops and their residues on availability of zinc in a calcareous Zn-deficient soil. Biol. Fertil Soils 50, 1061–1067.CrossRefGoogle Scholar
  15. Ho, S.H., Chen, C.N.N., Lai, Y.Y., Lu, W.B., and Chang, J.S. 2014. Exploring the high lipid production potential of a thermotolerant microalga using statistical optimization and semi-continuous cultivation. Bioresour. Technol. 163, 128–135.CrossRefPubMedGoogle Scholar
  16. Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M., and Darzins, A. 2008. Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J. 54, 621–639.CrossRefPubMedGoogle Scholar
  17. Kang, Z., Kim, B.H., Ramanan, R., Choi, J.E., Yang, J.W., Oh, H.M., and Kim, H.S. 2015. A cost analysis of microalgal biomass and biodiesel production in open raceways treating municipal waste water and under optimum light wavelength. J. Microbiol. Biotechnol. 25, 109–118.CrossRefPubMedGoogle Scholar
  18. Kim, H.S., Guzman, A.R., Thapa, H.R., Devarenne, T.P., and Han, A. 2016a. A droplet microfluidics platform for rapid microalgal growth and oil production analysis. Biotechnol. Bioeng. 113, 1691–1701.CrossRefPubMedGoogle Scholar
  19. Kim, G.Y., Heo, J., Kim, H.S., and Han, J.I. 2017. Bicarbonate-based cultivation of Dunaliella salina for enhancing carbon utilization efficiency. Bioresour. Technol. 237, 72–77.CrossRefPubMedGoogle Scholar
  20. Kim, B.H., Ramanan, R., Kang, Z., Cho, D.H., Oh, H.M., and Kim, H.S. 2016b. Chlorella sorokiniana HS1, a novel freshwater green algal strain, grows and hyperaccumulates lipid droplets in seawater salinity. Biomass Bioenerg. 85, 300–305.CrossRefGoogle Scholar
  21. Kobayashi, M., Kurimura, Y., and Tsuji, Y. 1997. Light-independent, astaxanthin production by the green microalga Haematococcus pluvialis under salt stress. Biotechnol. Lett. 19, 507–509.CrossRefGoogle Scholar
  22. Kobayashi, N., Noel, E.A., Barnes, A., Rosenberg, J., DiRusso, C., Black, P., and Oyler, G.A. 2013. Rapid detection and quantification of triacylglycerol by HPLC-ELSD in Chlamydomonas reinhardtii and Chlorella strains. Lipids 48, 1035–1049.CrossRefPubMedPubMedCentralGoogle Scholar
  23. Lee, J.Y., Yoo, C., Jun, S.Y., Ahn, C.Y., and Oh, H.M. 2010. Comparison of several methods for effective lipid extraction from microalgae. Bioresour. Technol. 101, S75–S77.CrossRefPubMedGoogle Scholar
  24. Lefebvre, O. and Moletta, R. 2006. Treatment of organic pollution in industrial saline wastewater: a literature review. Water Res. 40, 3671–3682.CrossRefPubMedGoogle Scholar
  25. Li, Z., Sun, M., Li, Q., Li, A., and Zhang, C. 2012. Profiling of carotenoids in six microalgae (Eustigmatophyceae) and assessment of their β-carotene productions in bubble column photobioreactor. Biotechnol. Lett. 34, 2049–2053.CrossRefPubMedGoogle Scholar
  26. Linaric, M., Markic, M., and Sipos, L. 2013. High salinity wastewater treatment. Water Sci. Technol. 68, 1400–1405.CrossRefPubMedGoogle Scholar
  27. Lohman, E.J., Gardner, R.D., Pedersen, T., Peyton, B.M., Cooksey, K.E., and Gerlach, R. 2015. Optimized inorganic carbon regime for enhanced growth and lipid accumulation in Chlorella vulgaris. Biotechnol. Biofuels 8, 82.CrossRefPubMedPubMedCentralGoogle Scholar
  28. Mata, T.M., Martins, A.A., and Caetano, N.S. 2010. Microalgae for biodiesel production and other applications: a review. Renew. Sust. Energ. Rev. 14, 217–232.CrossRefGoogle Scholar
  29. Miller, L. and Houghton, J.A. 1945. The micro-Kjeldahl determination of the nitrogen content of amino acids and proteins. J. Biol. Chem. 169, 373–383.Google Scholar
  30. Minhas, A.K., Hodgson, P., Barrow, C.J., and Adholeya, A. 2016. A review on the assessment of stress conditions for simultaneous production of microalgal lipids and carotenoids. Front. Microbiol. 7, 546.CrossRefPubMedPubMedCentralGoogle Scholar
  31. Mulders, K.J., Lamers, P.P., Martens, D.E., and Wijffels, R.H. 2014. Phototrophic pigment production with microalgae: biological constraints and opportunities. J. Phycol. 50, 229–242.CrossRefPubMedGoogle Scholar
  32. Narala, R.R., Garg, S., Sharma, K.K., Thomas-Hall, S.R., Deme, M., Li, Y., and Schenk, P.M. 2016. Comparison of microalgae cultivation in photobioreactor, open raceway pond, and a two-stage hybrid system. Front. Energy Res. 4, 29.CrossRefGoogle Scholar
  33. Pal, D., Khozin-Goldberg, I., Cohen, Z., and Boussiba, S. 2011. The effect of light, salinity, and nitrogen availability on lipid production by Nannochloropsis sp. Appl. Microbiol. Biotechnol. 90, 1429–1441.CrossRefPubMedGoogle Scholar
  34. Pandit, P.R., Fulekar, M.H., and Karuna, M.S.L. 2017. Effect of salinity stress on growth, lipid productivity, fatty acid composition, and biodiesel properties in Acutodesmus obliquus and Chlorella vulgaris. Environ. Sci. Pollut. Res. 24, 13437–13451.CrossRefGoogle Scholar
  35. Ra, C.H., Kang, C.H., Kim, N.K., Lee, C.G., and Kim, S.K. 2015. Cultivation of four microalgae for biomass and oil production using a two-stage culture strategy with salt stress. Renew. Energy 80, 117–122.CrossRefGoogle Scholar
  36. Ramanan, R., Kim, B.H., Cho, D.H., Ko, S.R., Oh, H.M., and Kim, H.S. 2013. Lipid droplet synthesis is limited by acetate availability in starchless mutant of Chlamydomonas reinhardtii. FEBS Lett. 587, 370–377.CrossRefPubMedGoogle Scholar
  37. Rippka, R., Deruelles, J., Waterbury, J.B., Herdman, M., and Stanier, R.Y. 1979. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Microbiology 111, 1–61.CrossRefGoogle Scholar
  38. Roberts, G.W., Fortier, M.O.P., Sturm, B.S., and Stagg-Williams, S.M. 2013. Promising pathway for algal biofuels through wastewater cultivation and hydrothermal conversion. Energ. Fuel. 27, 857–867.CrossRefGoogle Scholar
  39. Schlagermann, P., Göttlicher, G., Dillschneider, R., Rosello-Sastre, R., and Posten, C. 2012. Composition of algal oil and its potential as biofuel. J. Combust. 201, 285185.Google Scholar
  40. Shurin, J.B., Abbott, R.L., Deal, M.S., Kwan, G.T., Litchman, E., Mc-Bride, R.C., Mandal, S., and Smith, V.H. 2013. Industrial-strength ecology: trade-offs and opportunities in algal biofuel production. Ecol. Lett. 16, 1393–1404.CrossRefPubMedGoogle Scholar
  41. Smith, V.H., Sturm, B.S., de Noyelles, F.J., and Billings, S.A. 2010. The ecology of algal biodiesel production. Trends Ecol. Evol. 25, 301–309.CrossRefPubMedGoogle Scholar
  42. Sung, M.G., Lee, B., Kim, C.W., Nam, K., and Chang, Y.K. 2017. Enhancement of lipid productivity by adopting multi-stage continuous cultivation strategy in Nannochloropsis gaditana. Bioresour. Technol. 229, 20–25.CrossRefPubMedGoogle Scholar
  43. Uduman, N., Qi, Y., Danquah, M.K., Forde, G.M., and Hoadley, A. 2010. Dewatering of microalgal cultures: a major bottleneck to algae-based fuels. J. Renew. Sustain. Energy 2, 012701.CrossRefGoogle Scholar
  44. Wang, H., Pampati, N., McCormick, W.M., and Bhattacharyya, L. 2016. Protein nitrogen determination by Kjeldahl digestion and ion chromatography. J. Pharm. Sci. 105, 1851–1857.CrossRefPubMedGoogle Scholar
  45. Xu, X.Q. and Beardall, J. 1997. Effect of salinity on fatty acid composition of a green microalga from an antarctic hypersaline lake. Phytochemistry 45, 655–658.CrossRefGoogle Scholar
  46. Xu, N., Zhang, X., Fan, X., Han, L., and Zeng, C. 2001. Effects of nitrogen source and concentration on growth rate and fatty acid composition of Ellipsoidion sp. (Eustigmatophyta). J. Appl. Phycol. 13, 463–469.CrossRefGoogle Scholar
  47. Yeesang, C. and Cheirsilp, B. 2011. Effect of nitrogen, salt, and iron content in the growth medium and light intensity on lipid production by microalgae isolated from freshwater sources in Thailand. Bioresour. Technol. 102, 3034–3040.CrossRefPubMedGoogle Scholar
  48. Yoo, G., Park, W.K., Kim, C.W., Choi, Y.E., and Yang, J.W. 2012. Direct lipid extraction from wet Chlamydomonas reinhardtii biomass using osmotic shock. Bioresour. Technol. 123, 717–722.CrossRefPubMedGoogle Scholar

Copyright information

© The Microbiological Society of Korea and Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Ramesh Kakarla
    • 1
  • Jung-Woon Choi
    • 1
  • Jin-Ho Yun
    • 2
  • Byung-Hyuk Kim
    • 3
  • Jina Heo
    • 1
    • 4
  • Sujin Lee
    • 1
    • 4
  • Dae-Hyun Cho
    • 1
  • Rishiram Ramanan
    • 5
  • Hee-Sik Kim
    • 1
    • 4
    Email author
  1. 1.Cell Factory Research CenterKorea Research Institute of Bioscience and Biotechnology (KRIBB)DaejeonRepublic of Korea
  2. 2.Department of Chemical and Biomolecular EngineeringKAISTDaejeonRepublic of Korea
  3. 3.Research Institute of Climate Change and Agriculture, National Institute of Horticultural and Herbal ScienceRDAJejuRepublic of Korea
  4. 4.Environmental BiotechnologyUniversity of Science & TechnologyDaejeonRepublic of Korea
  5. 5.Department of Environmental ScienceCentral University of Kerala, Padannakkad Campus Kasaragod DistrictKasaragodIndia

Personalised recommendations