Journal of Oceanology and Limnology

, Volume 37, Issue 2, pp 675–684 | Cite as

Characterization of the squalene-rich Botryococcus braunii Abt02 strain

  • Min Cao
  • Fangfang Zhang
  • Yunxiang MaoEmail author
  • Fanna Kong
  • Dongmei Wang


Botryococcus braunii is widely studied due to its high hydrocarbon content. In this study, B. braunii Abt02 was subjected to several analyses, including cytological observation, hydrocarbon composition analysis by gas chromatography mass spectrometry (GC-MS), phylogenetic identification using known races (A, B and L) of B. braunii strains based on their 18S rDNA sequences, and qPCR-based investigation of transcript accumulation levels of hydrocarbon biosynthesis-related enzymes (DXS, MCS, DLS, SQS) during different growth phases (lag phase, log phase, early stationary growth phase, late stationary growth phase) under nitrogen-replete and nitrogen-depleted growth conditions, respectively. Based on cytological observation and on the 18S rDNA phylogenetic analysis, strain Abt02 was assigned to race B. Analysis of the strain’s chemical composition showed that the B. braunii Abt02 contained high levels of hydrocarbons, which accounted for 43.75% of the cell’s dry weight. Of these hydrocarbons, squalene and its derivatives accounted for up to 87.54%. In addition, all four enzymes investigated were expressed at higher levels during the log growth phase under nitrogen depleted conditions than under nitrogen replete conditions.


Botryococcus braunii cytological observation phylogenetic analysis hydrocarbon components qPCR 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Banerjee A, Sharma R, Chisti Y, Banerjee U C. 2002. Botryococcus braunii: a renewable source of hydrocarbons and other chemicals. Critical Reviews in Biotechnology, 22 (3): 245–279.CrossRefGoogle Scholar
  2. Brown A C, Knights B A, Conway E. 1969. Hydrocarbon content and its relationship to physiological state in the green alga Botryococcus braunii. Phytochemistry, 8 (3): 543–547.CrossRefGoogle Scholar
  3. Chiang I Z, Huang W Y, Wu J T. 2004. Allelochemicals of Botryococcus braunii (Chlorophyceae). Journal of Phycology, 40 (3): 474–480.CrossRefGoogle Scholar
  4. Choi G G, Kim B H, Ahn C Y, Oh H M. 2011. Effect of nitrogen limitation on oleic acid biosynthesis in Botryococcus b r aunii. J ournal of Appl ied Phycol ogy, 23 (6): 1 031–1 037.CrossRefGoogle Scholar
  5. Fang L, Sun D Y, Xu Z Y, He J, Qi S Y, Chen X, Chew W, Liu J H. 2015. Transcriptomic analysis of a moderately growing subisolate Botryococcus braunii 779 (Chlorophyta) in response to nitrogen deprivation. Biotechnology for Biofuels, 8: 130.CrossRefGoogle Scholar
  6. Fernandes N V, Yeganehjoo H, Katuru R, DeBose–Boyd R A, Morris L L, Michon R, Yu Z L, Mo H B. 2013. Geranylgeraniol suppresses the viability of human DU145 prostate carcinoma cells and the level of HMG CoA reductase. Experimental Biology and Medicine, 238 (11): 1 265–1 274.CrossRefGoogle Scholar
  7. Hillen L W, Pollard G, Wake L V, White N. 1982. Hydrocracking of the oils of Botryococcus braunii to transport fuels. Biotechnology and Bioengineering, 24 (1): 193–205.CrossRefGoogle Scholar
  8. Huss V A R, Sogin M L. 1990. Phylogenetic position of some Chlorella species within the chlorococcales based upon complete small–subunit ribosomal RNA sequences. Journal of Molecular Evolution, 31 (5): 432–442.CrossRefGoogle Scholar
  9. Ioki M, Baba M, Bidadi H, Suzuki I, Shiraiwa Y, Watanabe M M, Nakajima N. 2012. Modes of hydrocarbon oil biosynthesis revealed by comparative gene expression analysis for race A and race B strains of Botryococcus braunii. Bioresource Technology, 109: 271–276.CrossRefGoogle Scholar
  10. Jarstfer M B, Blagg B S J, Rogers D H, Poulter C D. 1996. Biosynthesis of squalene. Evidence for a tertiary cyclopropylcarbinyl cationic intermediate in the rearrangement of presqualene diphosphate to squalene. Journal of the American Chemical Society, 118 (51): 13 089–13 090.Google Scholar
  11. Kawachi M, Tanoi T, Demura M, Kaya K, Watanabe M M. 2012. Relationship between hydrocarbons and molecular phylogeny of Botryococcus braunii. Algal Research, 1 (2): 114–119.CrossRefGoogle Scholar
  12. Larkin M A, Blackshields G, Brown N P, Chenna R, McGettigan P A, McWilliam H, Valentin F, Wallace I M, Wilm A, Lopez P, Thompson J D, Gibson T J, Higgins D G. 2007. Clustal W and Clustal X version 2.0. Bioinformatics, 23 (21): 2 947–2 948.CrossRefGoogle Scholar
  13. Liao Z H, Chen M, Gong Y F, Miao Z Q, Sun X F, Tang K X. 2006. Isoprenoid biosynthesis in plants: pathways, genes, regulation and metabolic engineering. J ournal of Bio logical Sci ences, 6 (1): 209–219.Google Scholar
  14. Liu X Y. 2013. Studies on the Separation, Identification and Mutation Breeding of Botryococcus braunii. Zhejiang University, Hangzhou. (in Chinese)Google Scholar
  15. Matsushima D, Jenke–Kodama H, Sato Y, Fukunaga Y, Sumimoto K, Kuzuyama T, Matsunaga S, Okada S. 2012. The single cellular green microalga Botryococcus braunii, race B possesses three distinct 1–deoxy–D–xylulose 5–phosphate synthases. Plant Science, 185–186: 309–320.CrossRefGoogle Scholar
  16. Metzger P, Allard B, Casadevall E, BerkaloffC Couté A. 1990. Structure and chemistry of a new chemical race of Botryococcus braunii (Chlorophyceae) that produces lycopadiene, a tetraterpenoid hydrocarbon. Journal of Phycology, 26 (2): 258–266.CrossRefGoogle Scholar
  17. Metzger P, BerkaloffC, Casadevall E, Coute A. 1985. Alkadiene–and botryococcene–producing races of wild strains of Botryococcus braunii. Phytochemistry, 24 (10): 2 305–2 312.CrossRefGoogle Scholar
  18. Metzger P, Casadevall E, Coute A. 1988. Botryococcene distribution in strains of the green alga Botryococcus braunii. Phytochemistry, 27 (5): 1 383–1 388.CrossRefGoogle Scholar
  19. Metzger P, Casadevall E. 1989. Aldehydes, very long chain alkenylphenols, epoxides and other lipids from an alkadiene–producing strain of Botryococcus braunii. Phytochemistry, 28 (8): 2 097–2 104.CrossRefGoogle Scholar
  20. Metzger P, Largeau C. 2005. Botryococcus braunii: a rich source for hydrocarbons and related ether lipids. Applied Microbiology and Biotechnology, 66 (5): 486–496.CrossRefGoogle Scholar
  21. Niehaus T D, Okada S, Devarenne T P, Watt D S, Sviripa V, Chappell J. 2011. Identification of unique mechanisms for triterpene biosynthesis in Botryococcus braunii. Proceedings of the National Academy of Sciences of the United States of America, 108 (30): 12 260–12 265.CrossRefGoogle Scholar
  22. Poulter C D. 1990. Biosynthesis of non–head–to–tail terpenes. Formation of 1'–1 and 1'–3 linkages. Accounts of Chemical Research, 23 (3): 70–77.CrossRefGoogle Scholar
  23. Sato Y, Ito Y, Okada S, Murakami M, Abe H. 2003. Biosynthesis of the triterpenoids, botryococcenes and tetramethylsqualene in the B race of Botryococcus braunii via the non–mevalonate pathway. Tetrahedron Letters, 44 (37): 7 035–7 037.CrossRefGoogle Scholar
  24. Schwender J, Seemann M, Lichtenthaler H K, Rohmer M. 1996. Biosynthesis of isoprenoids (carotenoids, sterols, prenyl side–chains of chlorophylls and plastoquinone) via a novel pyruvate/glyceraldehyde 3–phosphate nonmevalonate pathway in the green alga Scenedesmus obliquus. Biochemical Journal, 316 (Pt 1): 73–80.Google Scholar
  25. Senousy H H, Beakes G W, Hack E. 2004. Phylogenetic placement of Botryococcus braunii (Trebouxiophyceae) and Botryococcus sudeticus isolate UTEX 2629 (Chlorophyceae). Journal of Phycology, 40 (2): 412–423.CrossRefGoogle Scholar
  26. Singh Y, Kumar H D. 1992. Lipid and hydrocarbon production by Botryococcus spp. under nitrogen limitation and anaerobiosis. World Journal of Microbiology and Biotechnology, 8 (2): 121–124.CrossRefGoogle Scholar
  27. Talukdar J, Kalita M C, Goswami B C. 2013. Characterization of the biofuel potential of a newly isolated strain of the microalga Botryococcus braunii Kützing from Assam, India. Bioresource Technology, 149: 268–275.CrossRefGoogle Scholar
  28. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 30 (12): 2 725–2 729.CrossRefGoogle Scholar
  29. Tanoi T, Kawachi M, Watanabe M M. 2011. Effects of carbon source on growth and morphology of Botryococcus braunii. Journal of Applied Phycology, 23 (1): 25–33.CrossRefGoogle Scholar
  30. Wake L V, Hillen L W. 1980. Study of a “bloom” of the oil–rich alga Botryococcus braunii in the Darwin River Reservoir. Biotechnology and Bioengineering, 22 (8): 1 637–1 656.CrossRefGoogle Scholar
  31. Wake L V, Hillen L W. 1981. Nature and hydrocarbon content of blooms of the alga Botryococcus braunii occuring in Australian freshwater lakes. Marine and Freshwater Research, 32 (3): 353–367.CrossRefGoogle Scholar
  32. Wang P Y, Mao Y X, Kong F N, Ma M, Ma F. 2011. Morphological and genetic diversity of Botryococcus braunii. Periodical of Ocean University of China, 41 (5): 63–70. (in Chinese)Google Scholar
  33. White J D, Somers T C, Reddy G N. 1986. The absolute configuration of (–)–botryococcene. Journal of the American Chemical Society, 108 (17): 5 352–5 353.CrossRefGoogle Scholar
  34. White J D, Somers T C, Reddy G N. 1992. Degradation and absolute configurational assignment to C34–botryococcene. The Journal of Organic Chemistry, 57 (18): 4 991–4 998.CrossRefGoogle Scholar
  35. Zhang D L, Poulter C D. 1995. Biosynthesis of non–head–totail isoprenoids. Synthesis of 1'–1 and 1'–3 structures by recombinant yeast squalene synthase. Journal of the American Chemical Society, 117 (5): 1 641–1 642.Google Scholar

Copyright information

© Chinese Society for Oceanology and Limnology, Science Press and Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Min Cao
    • 1
    • 2
  • Fangfang Zhang
    • 1
    • 2
  • Yunxiang Mao
    • 1
    • 2
    • 3
    Email author
  • Fanna Kong
    • 1
    • 2
  • Dongmei Wang
    • 1
    • 2
  1. 1.College of Marine Life SciencesOcean University of ChinaQingdaoChina
  2. 2.Key Laboratory of Marine Genetics and Breeding (OUC)Ministry of EducationQingdaoChina
  3. 3.Laboratory for Marine Biology and BiotechnologyQingdao National Laboratory for Marine Science and TechnologyQingdaoChina

Personalised recommendations