Environmental Science and Pollution Research

, Volume 25, Issue 23, pp 23018–23032 | Cite as

Cultivation, characterization, and properties of Chlorella vulgaris microalgae with different lipid contents and effect on fast pyrolysis oil composition

  • Ioannis-Dimosthenis Adamakis
  • Polykarpos A. Lazaridis
  • Evangelia Terzopoulou
  • Stylianos Torofias
  • Maria Valari
  • Photeini Kalaitzi
  • Vasilis Rousonikolos
  • Dimitris Gkoutzikostas
  • Anastasios ZouboulisEmail author
  • Georgios ZalidisEmail author
  • Konstantinos S. TriantafyllidisEmail author
Research Article


A systematic study of the effect of nitrogen levels in the cultivation medium of Chlorella vulgaris microalgae grown in photobioreactor (PBR) on biomass productivity, biochemical and elemental composition, fatty acid profile, heating value (HHV), and composition of the algae-derived fast pyrolysis (bio-oil) is presented in this work. A relatively high biomass productivity and cell concentration (1.5 g of dry biomass per liter of cultivation medium and 120 × 106 cells/ml, respectively) were achieved after 30 h of cultivation under N-rich medium. On the other hand, the highest lipid content (ca. 36 wt.% on dry biomass) was obtained under N-depletion cultivation conditions. The medium and low N levels favored also the increased concentration of the saturated and mono-unsaturated C16:0 and C18:1(n-9) fatty acids (FA) in the lipid/oil fraction, thus providing a raw lipid feedstock that can be more efficiently converted to high-quality biodiesel or green diesel (via hydrotreatment). In terms of overall lipid productivity, taking in consideration both the biomass concentration in the medium and the content of lipids on dry biomass, the most effective system was the N-rich one. The thermal (non-catalytic) pyrolysis of Chlorella vulgaris microalgae produced a highly complex bio-oil composition, including fatty acids, phenolics, ethers, ketones, etc., as well as aromatics, alkanes, and nitrogen compounds (pyrroles and amides), originating from the lipid, protein, and carbohydrate fractions of the microalgae. However, the catalytic fast pyrolysis using a highly acidic ZSM-5 zeolite, afforded a bio-oil enriched in mono-aromatics (BTX), reducing at the same time significantly oxygenated compounds such as phenolics, acids, ethers, and ketones. These effects were even more pronounced in the catalytic fast pyrolysis of Chlorella vulgaris residual biomass (after extraction of lipids), thus showing for the first time the potential of transforming this low value by-product towards high added value platform chemicals.


Microalgae Chlorella vulgaris Nitrogen-depleted cultivation Lipids and residual biomass Fast pyrolysis and catalytic fast pyrolysis Aromatic hydrocarbons 


Funding information

This research was funded by ENERGEIA project: a Strategic Co-Funded Project of the European Territorial Cooperation Programme Greece-Bulgaria 2007–2013.


  1. Babich IV, van der Hulst M, Lefferts L, Moulijn JA, O’Connor P, Seshan K (2011) Catalytic pyrolysis of microalgae to high-quality liquid bio-fuels. Biomass Bioenergy 35:3199–3207. CrossRefGoogle Scholar
  2. Beardall J, Roberts S, Raven JA (2005) Regulation of inorganic carbon acquisition by phosphorus limitation in the green alga Chlorella emersonii. Can J Bot 83:859–864. CrossRefGoogle Scholar
  3. Becker EW (2007) Micro-algae as a source of protein. Biotechnol Adv 25:207–210. CrossRefGoogle Scholar
  4. Chagas BME, Dorado C, Serapiglia MJ, Mullen CA, Boateng AA, Melo MAF, Ataíde CH (2016) Catalytic pyrolysis-GC/MS of Spirulina: evaluation of a highly proteinaceous biomass source for production of fuels and chemicals. Fuel 179:124–134. CrossRefGoogle Scholar
  5. Chen C-Y, Yeh K-L, Aisyah R, Lee D-J, Chang J-S (2011) Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: a critical review. Bioresour Technol 102:71–81. CrossRefGoogle Scholar
  6. Chen C-Y, Yeh K-L, Lo Y-C, Wang H-M, Chang J-S (2010) Engineering strategies for the enhanced photo-H2 production using effluents of dark fermentation processes as substrate. Int J Hydrog Energy 35:13356–13364. CrossRefGoogle Scholar
  7. Chioccioli M, Hankamer B, Ross IL (2014) Flow cytometry pulse width data enables rapid and sensitive estimation of biomass dry weight in the microalgae Chlamydomonas reinhardtii and Chlorella vulgaris. PLoS One 9:e97269. CrossRefGoogle Scholar
  8. Chisti Y (2007) Biodiesel from microalgae. Biotechnol Adv 25:294–306. CrossRefGoogle Scholar
  9. D’Oca MGM, Viêgas CV, Lemões JS, Miyasaki EK, Morón-Villarreyes JA, Primel EG, Abreu PC (2011) Production of FAMEs from several microalgal lipidic extracts and direct transesterification of the Chlorella pyrenoidosa. Biomass Bioenergy 35:1533–1538. CrossRefGoogle Scholar
  10. Dibenedetto A, Colucci A, Aresta M (2016) The need to implement an efficient biomass fractionation and full utilization based on the concept of “biorefinery” for a viable economic utilization of microalgae. Environ Sci Pollut Res 23:22274–22283. CrossRefGoogle Scholar
  11. Du Z, Ma X, Li Y, Chen P, Liu Y, Lin X, Lei H, Ruan R (2013) Production of aromatic hydrocarbons by catalytic pyrolysis of microalgae with zeolites: catalyst screening in a pyroprobe. Bioresour Technol 139:397–401. CrossRefGoogle Scholar
  12. Flach B, Lieberz S, Rossetti A (2017) EU Biofuels Annual 2017 Global agricultural information network, USDA Foreign Agricultural Service:
  13. Francavilla M, Kamaterou P, Intini S, Monteleone M, Zabaniotou A (2015) Cascading microalgae biorefinery: fast pyrolysis of Dunaliella tertiolecta lipid extracted-residue. Algal Res 11:184–193. CrossRefGoogle Scholar
  14. Gamliel DP, Cho HJ, Fan W, Valla JA (2016) On the effectiveness of tailored mesoporous MFI zeolites for biomass catalytic fast pyrolysis. Appl Catal A Gen 522:109–119. CrossRefGoogle Scholar
  15. Geider RJ, La Roche J, Greene RM, Olaizola M (1993) Response of the photosynthetic apparatus of Phaeodactylum tricornutum (Bacillariophyceae) to nitrate, phosphate, or iron starvation1. J Phycol 29:755–766. CrossRefGoogle Scholar
  16. Gnaiger E, Bitterlich G (1984) Proximate biochemical composition and caloric content calculated from elemental CHN analysis: a stoichiometric concept. Oecologia 62:289–298. CrossRefGoogle Scholar
  17. Granata T (2017) Dependency of microalgal production on biomass and the relationship to yield and bioreactor scale-up for biofuels: a statistical analysis of 60+ years of algal bioreactor data. BioEnergy Res 10:267–287. CrossRefGoogle Scholar
  18. Herrig R, Falkowski PG (1989) Nitrogen limitation in Isochrysis galbana (Haptophyceae). I. Photosynthetic energy conversion and growth efficiencies1. J Phycol 25:462–471. CrossRefGoogle Scholar
  19. Ho S-H, Chen C-Y, Lee D-J, Chang J-S (2011) Perspectives on microalgal CO2-emission mitigation systems—a review. Biotechnol Adv 29:189–198. CrossRefGoogle Scholar
  20. Ho S-H, Chen C-Y, Yeh K-L, Chen W-M, Lin C-Y, Chang J-S (2010) Characterization of photosynthetic carbon dioxide fixation ability of indigenous Scenedesmus obliquus isolates. Biochem Eng J 53:57–62. CrossRefGoogle Scholar
  21. Hu H, Gao K (2006) Response of growth and fatty acid compositions of Nannochloropsis sp. to environmental factors under elevated CO2 concentration. Biotechnol Lett 28:987–992. CrossRefGoogle Scholar
  22. Ikaran Z, Suárez-Alvarez S, Urreta I, Castañón S (2015) The effect of nitrogen limitation on the physiology and metabolism of chlorella vulgaris var L3. Algal Res 10:134–144. CrossRefGoogle Scholar
  23. Illman AM, Scragg AH, Shales SW (2000) Increase in Chlorella strains calorific values when grown in low nitrogen medium. Enzym Microb Technol 27:631–635. CrossRefGoogle Scholar
  24. Jiménez C, Cossı́o BR, Labella D, Xavier Niell F (2003) The feasibility of industrial production of Spirulina (Arthrospira) in southern Spain. Aquaculture 217:179–190. CrossRefGoogle Scholar
  25. Langley NM, Harrison STL, van Hille RP (2012) A critical evaluation of CO2 supplementation to algal systems by direct injection. Biochem Eng J 68:70–75. CrossRefGoogle Scholar
  26. Li Y, Horsman M, Wang B, Wu N, Lan CQ (2008) Effects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans. Appl Microbiol Biotechnol 81:629–636. CrossRefGoogle Scholar
  27. Liu T, Li Y, Liu F, Wang C (2016) The enhanced lipid accumulation in oleaginous microalgae by the potential continuous nitrogen-limitation (CNL) strategy. Bioresour Technol 203:150–159. CrossRefGoogle Scholar
  28. Lu W, Wang Z, Wang X, Yuan Z (2015) Cultivation of Chlorella sp. using raw dairy wastewater for nutrient removal and biodiesel production: characteristics comparison of indoor bench-scale and outdoor pilot-scale cultures. Bioresour Technol 192:382–388. CrossRefGoogle Scholar
  29. Luque R (2010) Algal biofuels: the eternal promise? Energy Environ Sci 3:254–257. CrossRefGoogle Scholar
  30. Mata TM, Martins AA, Caetano NS (2010) Microalgae for biodiesel production and other applications: a review. Renew Sust Energ Rev 14:217–232. CrossRefGoogle Scholar
  31. Miao X, Wu Q (2004) High yield bio-oil production from fast pyrolysis by metabolic controlling of Chlorella protothecoides. J Biotechnol 110:85–93. CrossRefGoogle Scholar
  32. Mihalcik DJ, Mullen CA, Boateng AA (2011) Screening acidic zeolites for catalytic fast pyrolysis of biomass and its components. J Anal Appl Pyrolysis 92:224–232. CrossRefGoogle Scholar
  33. Minhas AK, Hodgson P, Barrow CJ, Adholeya A (2016) A review on the assessment of stress conditions for simultaneous production of microalgal lipids and carotenoids. Front Microbiol 7:546. CrossRefGoogle Scholar
  34. Miura Y, Sode K, Nakamura N, Matsunaga N, Matsunaga T (1993) Production of γ-linolenic acid from the marine green alga Chlorella sp. NKG 042401. FEMS Microbiol Lett 107:163–167. CrossRefGoogle Scholar
  35. Morales-Sánchez D, Martinez-Rodriguez OA, Martinez A (2017) Heterotrophic cultivation of microalgae: production of metabolites of commercial interest. J Chem Technol Biotechnol 92:925–936. CrossRefGoogle Scholar
  36. Mussgnug JH, Klassen V, Schlüter A, Kruse O (2010) Microalgae as substrates for fermentative biogas production in a combined biorefinery concept. J Biotechnol 150:51–56. CrossRefGoogle Scholar
  37. Negi S, Barry AN, Friedland N, Sudasinghe N, Subramanian S, Pieris S, Holguin FO, Dungan B, Schaub T, Sayre R (2016) Impact of nitrogen limitation on biomass, photosynthesis, and lipid accumulation in Chlorella sorokiniana. J Appl Phycol 28:803–812. CrossRefGoogle Scholar
  38. Nichols HW, Bold HC (1965) Trichosarcina polymorpha gen. et Sp Nov. J Phycol 1:34–38. CrossRefGoogle Scholar
  39. Park S, Li Y (2012) Evaluation of methane production and macronutrient degradation in the anaerobic co-digestion of algae biomass residue and lipid waste. Bioresour Technol 111:42–48. CrossRefGoogle Scholar
  40. Phukan MM, Chutia RS, Konwar BK, Kataki R (2011) Microalgae Chlorella as a potential bio-energy feedstock. Appl Energy 88:3307–3312. CrossRefGoogle Scholar
  41. Pragya N, Pandey KK (2016) Life cycle assessment of green diesel production from microalgae. Renew Energy 86:623–632. CrossRefGoogle Scholar
  42. Přibyl P, Cepák V, Zachleder V (2012) Production of lipids in 10 strains of Chlorella and Parachlorella, and enhanced lipid productivity in Chlorella vulgaris. Appl Microbiol Biotechnol 94:549–561. CrossRefGoogle Scholar
  43. Ramos MJ, Fernández CM, Casas A, Rodríguez L, Pérez Á (2009) Influence of fatty acid composition of raw materials on biodiesel properties. Bioresour Technol 100:261–268. CrossRefGoogle Scholar
  44. Rodolfi L, Chini Zittelli G, Bassi N, Padovani G, Biondi N, Bonini G, Tredici MR (2009) Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnol Bioeng 102:100–112. CrossRefGoogle Scholar
  45. Roessler PG (1990) Environmental control of glycerolipid metabolism in microalgae: commercial implications and future research directions. J Phycol 26:393–399. CrossRefGoogle Scholar
  46. Shekh AY, Shrivastava P, Krishnamurthi K, Mudliar SN, Devi SS, Kanade GS, Chakrabarti T (2016) Stress enhances poly-unsaturation rich lipid accumulation in Chlorella sp. and Chlamydomonas sp. Biomass Bioenergy 84:59–66. CrossRefGoogle Scholar
  47. Singh A, Olsen SI, Nigam PS (2011) A viable technology to generate third-generation biofuel. J Chem Technol Biotechnol 86:1349–1353. CrossRefGoogle Scholar
  48. Singh P, Kumari S, Guldhe A, Misra R, Rawat I, Bux F (2016) Trends and novel strategies for enhancing lipid accumulation and quality in microalgae. Renew Sust Energ Rev 55:1–16. CrossRefGoogle Scholar
  49. Stefanidis SD, Kalogiannis KG, Iliopoulou EF, Michailof CM, Pilavachi PA, Lappas AA (2014) A study of lignocellulosic biomass pyrolysis via the pyrolysis of cellulose, hemicellulose and lignin. J Anal Appl Pyrolysis 105:143–150. CrossRefGoogle Scholar
  50. Stephanidis S, Nitsos C, Kalogiannis K, Iliopoulou EF, Lappas AA, Triantafyllidis KS (2011) Catalytic upgrading of lignocellulosic biomass pyrolysis vapours: effect of hydrothermal pre-treatment of biomass. Catal Today 167:37–45. CrossRefGoogle Scholar
  51. Talukder MMR, Das P, Wu JC (2012) Microalgae (Nannochloropsis Salina) biomass to lactic acid and lipid. Biochem Eng J 68:109–113. CrossRefGoogle Scholar
  52. Thangalazhy-Gopakumar S, Adhikari S, Chattanathan SA, Gupta RB (2012) Catalytic pyrolysis of green algae for hydrocarbon production using H+ZSM-5 catalyst. Bioresour Technol 118:150–157. CrossRefGoogle Scholar
  53. Waghmare AG, Salve MK, LeBlanc JG, Arya SS (2016) Concentration and characterization of microalgae proteins from Chlorella pyrenoidosa. Bioresour Bioprocess 3:16. CrossRefGoogle Scholar
  54. Wang C-Y, Fu C-C, Liu Y-C (2007) Effects of using light-emitting diodes on the cultivation of Spirulina platensis. Biochem Eng J 37:21–25. CrossRefGoogle Scholar
  55. Wang K, Brown RC (2013) Catalytic pyrolysis of microalgae for production of aromatics and ammonia. Green Chem 15:675–681. CrossRefGoogle Scholar
  56. Wang K, Brown RC, Homsy S, Martinez L, Sidhu SS (2013) Fast pyrolysis of microalgae remnants in a fluidized bed reactor for bio-oil and biochar production. Bioresour Technol 127:494–499. CrossRefGoogle Scholar
  57. Wang K, Kim KH, Brown RC (2014) Catalytic pyrolysis of individual components of lignocellulosic biomass. Green Chem 16:727–735. CrossRefGoogle Scholar
  58. Yang C, Li R, Cui C, Liu S, Qiu Q, Ding Y, Wu Y, Zhang B (2016) Catalytic hydroprocessing of microalgae-derived biofuels: a review. Green Chem 18:3684–3699. CrossRefGoogle Scholar
  59. Yeh K-L, Chang J-S (2012) Effects of cultivation conditions and media composition on cell growth and lipid productivity of indigenous microalgae Chlorella vulgaris ESP-31. Bioresour Technol 105:120–127. CrossRefGoogle Scholar
  60. You T, Barnett SM (2004) Effect of light quality on production of extracellular polysaccharides and growth rate of Porphyridium cruentum. Biochem Eng J 19:251–258. CrossRefGoogle Scholar
  61. Zhao C, Bruck T, Lercher JA (2013) Catalytic deoxygenation of microalgae oil to green hydrocarbons. Green Chem 15:1720–1739. CrossRefGoogle Scholar
  62. Zhila NO, Kalacheva GS, Volova TG (2005) Influence of nitrogen deficiency on biochemical composition of the green alga Botryococcus. J Appl Phycol 17:309–315. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Ioannis-Dimosthenis Adamakis
    • 1
  • Polykarpos A. Lazaridis
    • 2
  • Evangelia Terzopoulou
    • 1
  • Stylianos Torofias
    • 2
  • Maria Valari
    • 1
  • Photeini Kalaitzi
    • 1
  • Vasilis Rousonikolos
    • 1
  • Dimitris Gkoutzikostas
    • 1
  • Anastasios Zouboulis
    • 2
    Email author
  • Georgios Zalidis
    • 1
    Email author
  • Konstantinos S. Triantafyllidis
    • 2
    Email author
  1. 1.Laboratory of Applied Soil Science, School of AgricultureAristotle University of ThessalonikiThessalonikiGreece
  2. 2.Laboratory of Chemical and Environmental Technology, Department of ChemistryAristotle University of ThessalonikiThessalonikiGreece

Personalised recommendations