Journal of Applied Phycology

, Volume 27, Issue 5, pp 1833–1843 | Cite as

Design of a continuous milking bioreactor for non-destructive hydrocarbon extraction from Botryococcus braunii

  • Carola GriehlEmail author
  • Christian Kleinert
  • Christoph Griehl
  • Simone Bieler
5th Congress of the International Society for Applied Phycology


The production of low-value products such as biodiesel from microalgae is still too expensive. An alternative to commonly used species is the alga Botryococcus braunii, which excretes high quantities of hydrocarbons into the extracellular matrix. Because of its low growth rate, the common processing procedure (cultivation, harvesting, dewatering, cell disruption, lipid extraction) is not feasible for this alga, and moreover, at present, there is no existing process for the continuous extraction of hydrocarbons from B. braunii during cultivation without an inhibition of cell activity over a longer time period. Therefore, we attempted to develop an in situ extraction system which allows the continuous non-destructive extraction of the extracellular hydrocarbons during the cultivation. With the new extraction system, we were able to show that an average lipid yield of 19.15, 15.32, and 3.80 mg g−1 dry weight (DW) day−1 by the strains SCCAP 1761, CCAP 807/2, and SAG 807/1, respectively, could be achieved during a 5-day experiment. As only B. braunii SAG 807/1 showed a positive biomass yield, the optimization of the new system was conducted with this strain. We were able to show that a continuous extraction was possible for at least 6 weeks. The optimal effective extraction was in the range of 30 to 36 s day−1 with a lipid yield between 1.22 and 2.20 mg g−1 DW day−1.


Botryococcus braunii Hydrocarbons In situ bioreactor Microalgae Milking 



We thank all the contributing members of the workgroup biochemistry and algae biotechnology from the Anhalt University of Applied Sciences, Koethen, Germany.


  1. An JY, Sim SJ, Kim BW, Lee JS (2004) Improvement of hydrocarbon recovery by two-stage cell-recycle extraction in the cultivation of Botryococcus braunii. J Microbiol Biotech 14:932–937Google Scholar
  2. Baba M, Kikuta F, Suzuki I, Watanabe MM, Shiraiwa Y (2012) Wavelength specificity of growth, photosynthesis, and hydrocarbon production in the oil-producing green alga Botryococcus braunii. Bioresour Technol 109:266–270CrossRefPubMedGoogle Scholar
  3. Banerjee A, Sharma R, Chisti Y, Banerjee UC (2002) Botryococcus braunii: a renewable source of hydrocarbons and other chemicals. Crit Rev Biotechnol 22:245–279CrossRefPubMedGoogle Scholar
  4. Borowitzka M (2013) High-value products from microalgae—their development and commercialization. J Appl Phycol 25:743–756CrossRefGoogle Scholar
  5. Borowitzka M, Moheimani N (2013) Sustainable biofuels from algae. Mitig Adapt Strat Glob Change 18:13–25Google Scholar
  6. Brown AC, Knights BA, Conway E (1969) Hydrocarbon content and its relationship to physiological state in the green alga Botryococcus braunii. Phytochemistry 8:543–547CrossRefGoogle Scholar
  7. Casadevall E, Dif D, Largeau C (1985) Studies on batch and continuous cultures of Botryococcus braunii: hydrocarbon production in relation to physiological state, cell ultrastructure, and phosphate nutrition. Biotechnol Bioeng 27:286–295CrossRefPubMedGoogle Scholar
  8. Choi SP, Bahn SH, Sim SJ (2013) Improvement of hydrocarbon recovery by spouting solvent into culture of Botryococcus braunii. Bioprocess Biosyst Eng 36:1977–1985CrossRefPubMedGoogle Scholar
  9. De Boer K, Moheimani N, Borowitzka M, Bahri P (2012) Extraction and conversion pathways for microalgae to biodiesel: a review focused on energy consumption. J Appl Phycol 24:1681–1698CrossRefGoogle Scholar
  10. Du Y, Schuur B, Samori C, Tagliavini E, Brilman DWF (2013) Secondary amines as switchable solvents for lipid extraction from non-broken microalgae. Bioresource Technol 149:253–260CrossRefGoogle Scholar
  11. Eroglu E, Melis A (2010) Extracellular terpenpoid hydrocarbon extraction and quantitation from the green microalgae Botryococcus braunii var. Showa. Bioresource Technol 101:2359–2366CrossRefGoogle Scholar
  12. Fon Sing S, Isdepsky A, Borowitzka MA, Moheimani NR (2013) Production of biofuels from microalgae. Mitig Adapt Strat Glob Change 18:47–72Google Scholar
  13. Frenz J, Largeau C, Casadevall E (1989a) Hydrocarbon recovery and biocompatibility of solvents for extraction from cultures of Botryococcus braunii. Biotechnol Bioeng 34:755–762CrossRefPubMedGoogle Scholar
  14. Frenz J, Largeau C, Casadevall E (1989b) Hydrocarbon recovery by extraction with a biocompatible solvent from free and immobilized cultures of Botryococcus braunii. Enzym Microb Technol 11:717–724CrossRefGoogle Scholar
  15. Griehl C, Bieler S (2011) Algae: raw materials for health, beauty and energy. Nachrichten Chem 59:942–947CrossRefGoogle Scholar
  16. Griehl C, Griehl C, Kleinert C (2014) Verfahren und Vorrichtung zur abtrennung von extrazellulären Lipiden aus Mikroalgen. German Patent DE 10 2014 005 372Google Scholar
  17. Guldhe A, Singh B, Rawat I, Ramluckan K, Bux F (2014) Efficacy of drying and cell disruption techniques on lipid recovery from microalgae for biodiesel production. Fuel 128:46–52CrossRefGoogle Scholar
  18. Hejazi MA, Holwerda E, Wijffels RH (2004) Milking microalga Dunaliella salina for β-carotene production in two-phase bioreactors. Biotechnol Bioeng 85:475–481CrossRefPubMedGoogle Scholar
  19. Hillen LW, Pollard G, Wake LV, White N (1982) Hydrocracking of the oils of Botryococcus braunii to transport fuels. Biotechnol Bioeng 24:193–205CrossRefPubMedGoogle Scholar
  20. Kawachi M, Tanoi T, Demura M, Kaya K, Watanabe MW (2012) Relationship between hydrocarbons and molecular phylogeny of Botryococcus braunii. Algal Res 1:114–119CrossRefGoogle Scholar
  21. Kitazato H, Asaoka S, Iwamoto H (1991) Catalytic cracking of hydrocarbons from microalgae. Int Chem Eng 31:523–529Google Scholar
  22. Kleinert C (2014) Kontinuierliche abtrennung extrazellulärer lipide der grünalge Botryococcus braunii während der kultivierung. Master Thesis, Anhalt University of Applied SciencesGoogle Scholar
  23. Li Y, Horsman M, Wu N, Lan CQ, Dubois-Calero N (2008) Biofuels from microalgae. Biotech Progr 24:815–820Google Scholar
  24. Metzger P, Largeau C (2005) Botryococcus braunii: a rich source for hydrocarbons and related ether lipids. Appl Microbiol Biotechnol 66:486–496CrossRefPubMedGoogle Scholar
  25. Moheimani NR, Cord-Ruwisch R, Raes E, Borowitzka MA (2013) Non-destructive oil extraction from Botryococcus braunii (Chlorophyta). J Appl Phycol 25:1653–1661CrossRefGoogle Scholar
  26. Moheimani NR, Matsuura H, Watanabe MM (2014) Non-destructive hydrocarbon extraction from Botryococcus braunii BOT-22 (race B). J Appl Phycol 26:1453–1463CrossRefGoogle Scholar
  27. Olsson P, Holmbäck J, Herslöf B (2012) Separation of lipid classes by HPLC on a cyanopropyl column. Lipids 47:93–99CrossRefPubMedGoogle Scholar
  28. Ranga Rao A, Sarada R, Ravishankar GA (2007) Influence of CO2 on growth and hydrocarbon production in Botryococcus braunii. J Microbiol Biotech 17:414–419Google Scholar
  29. Rippka R, Deruelles J, Waterbury JB (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111:1–61Google Scholar
  30. Rosello Sastre R, Posten C (2010) The variety of microalgae applications as a renewable resource. Chem Ing Tech 82:1925–1939CrossRefGoogle Scholar
  31. Sim SJ, An JY, Kim BW (2001) Two-phase extraction culture of Botryococcus braunii producing long-chain unsaturated hydrocarbons. Biotechnol Lett 23:201–205CrossRefGoogle Scholar
  32. Singh B, Guldhe A, Rawat I, Bux F (2014) Towards a sustainable approach for development of biodiesel from plant and microalgae. Renew Sust Energy Rev 29:216–245CrossRefGoogle Scholar
  33. Stephens E, Ross IL, Mussgnug JH, Wagner LD, Borowitzka MA, Posten C, Kruse O, Hankamer B (2010) Future prospects of microalgal biofuel production systems. Trends Plant Sci 15:554–564CrossRefPubMedGoogle Scholar
  34. Volkman JK (2014) Acyclic isoprenoid biomarkers and evolution of biosynthetic pathways in green microalgae of the genus Botryococcus. Org Geochem 75:36–47CrossRefGoogle Scholar
  35. Zhang F, Cheng LH, Xu XH, Thang L, Chen HL (2013) Application of membrane dispersion for enhanced lipid milking from Botryococcus braunii FACHB 357. J Biotechnol 165:22–29CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Carola Griehl
    • 1
    Email author
  • Christian Kleinert
    • 1
  • Christoph Griehl
    • 2
  • Simone Bieler
    • 1
  1. 1.Department of Applied Sciences and Process EngineeringAnhalt University of Applied SciencesKoethenGermany
  2. 2.Georg-Cantor-GymnasiumHalleGermany

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