Omega-7 producing alkaliphilic diatom Fistulifera sp. (Bacillariophyceae) from Lake Okeechobee, Florida

  • Berthold, David Erwin (Department of Earth and Environment, Florida International University) ;
  • Rosa, Nina de la (Department of Earth and Environment, Florida International University) ;
  • Engene, Niclas (Department of Biological Sciences, Florida International University) ;
  • Jayachandran, Krish (Department of Earth and Environment, Florida International University) ;
  • Gantar, Miroslav (Department of Biological Sciences, Florida International University) ;
  • Laughinghouse, Haywood Dail IV (Fort Lauderdale Research and Education Center, University of Florida/IFAS) ;
  • Shetty, Kateel G. (Department of Earth and Environment, Florida International University)
  • Received : 2019.07.19
  • Accepted : 2019.12.16
  • Published : 2020.03.15


Incorporating renewable fuel into practice, especially from algae, is a promising approach in reducing fossil fuel dependency. Algae are an exceptional feedstock since they produce abundant biomass and oils in short timeframes. Algae also produce high-valued lipid products suitable for human nutrition and supplement. Achieving goals of producing algae fuels and high-valued lipids at competitive prices involves further improvement of technology, especially better control over cultivation. Manipulating microalgae cultivation conditions to prevent contamination is essential in addition to promoting optimal growth and lipid yields. Contamination of algal cultures is a major impediment to algae cultivation that can however be mitigated by choosing extremophile microalgae. This work describes the isolation of alkali-tolerant / alkaliphilic microalgae native to South Florida with ideal characteristics for cultivation. For that purpose, water samples from Lake Okeechobee were inoculated into Zarrouk's medium (pH 9-12) and incubated for 35 days. Selection resulted in isolation of three strains that were screened for biomass and lipid accumulation. Two alkali-tolerant algae Chloroidium sp. 154-1 and Chlorella sp. 154-2 were poor lipid accumulators. One of the isolates, the diatom Fistulifera sp. 154-3, was identified as a lipid accumulating, alkaliphilic organism capable of producing 0.233 g L-1 d-1 dry biomass and a lipid content of 20-30% dry weight. Lipid analysis indicated the most abundant fatty acid within Fistulifera sp. was palmitoleic acid (52%), or omega-7, followed by palmitic acid (17%), and then eicosapentanoic acid (15%). 18S rRNA phylogenetic analysis formed a well-supported clade with Fistulifera species.


  1. Adarme-Vega, T. C., Lim, D. K. Y., Timmins, M., Vernen, F., Li, Y. & Schenk, P. M. 2012. Microalgal biofactories: a promising approach towards sustainable omega-3 fatty acid production. Microb. Cell Fact. 11:96.
  2. American Oils Chemists Society. 2005. Fatty acid composition by gas chromatography. AOCS Method Ce 1-62. In Firestone, D. (Ed.) AOCS Official Methods and Recommended Practices of the American Oil Chemists Society. American Oils Chemists Society, Champaign, IL, pp. 1200.
  3. Axelsson, M. & Gentili, F. 2014. A single-step method for rapid extraction of total lipids from green microalgae. PLoS ONE 9:e89643.
  4. Bal, L. M., Meda, V., Naik, S. N. & Satya, S. 2011. Sea buckthorn berries: a potential source of valuable nutrients for nutraceuticals and cosmoceuticals. Food Res. Int. 44:1718-1727.
  5. Barnard, D., Casanueva, A., Tuffin, M. & Cowan, D. 2010. Extremophiles in biofuel synthesis. Environ. Technol. 31:871-888.
  6. Bellinger, E. G. & Sigee, D. C. 2015. Freshwater algae: identification, enumeration, and use as bioindicators. 2nd ed. John Wiley and Sons, Ltd., West Sussex, 290 pp.
  7. Bernstein, A. M., Roizen, M. F. & Martinez, L. 2014. Purified palmitoleic acid for the reduction of high-sensitivity C-reactive protein and serum lipids: a double-blinded, randomized, placebo controlled study. J. Clin. Lipidol. 8:612-617.
  8. Borowitzka, M. A. & Moheimani, N. R. 2013. Algae for biofuels and energy. Vol. 5. Springer, Dordrecht, 288 pp.
  9. Canfield, D. E. Jr. & Hoyer, M. V. 1988. The eutrophication of Lake Okeechobee. Lake Reserv. Manag. 4:91-99.
  10. Chen, W., Zhang, C., Song, L., Sommerfeld, M. & Hu, Q. 2009. A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae. J. Microbiol. Methods 77:41-47.
  11. Chi, Z., O'Fallon, J. V. & Chen, S. 2011. Bicarbonate produced from carbon capture for algae culture. Trends Biotechnol. 29:537-541.
  12. Chiang, I. -Z., Huang, W. -Y. & Wu, J. -T. 2004. Allelochemicals of Botryococcus braunii (Chlorophyceae). J. Phycol. 40:474-480.
  13. Copat, C., Bella, F., Castaing, M., Fallico, R., Sciacca, S. & Ferrante, M. 2012. Heavy metals concentrations in fish from Sicily (Mediterranean Sea) and evaluation of possible health risks to consumers. Bull. Environ. Contam. Toxicol. 88:78-83.
  14. Core Writing Team, Pachauri, R. K. & Meyer, L. A. 2014. Climate change 2014: synthesis report. Contribution of Working Groups I, II and III to the fifth assessment report of the Intergovernmental Panel on Climate Change. Intergovernmental Panel on Climate Change, Geneva, 151 pp.
  15. Costa, M., Costa-Rodrigues, J., Fernandes, M. H., Barros, P., Vasconcelos, V. & Martins, R. 2012. Marine cyanobacteria compounds with anticancer properties: a review on the implication of apoptosis. Mar. Drugs 10:2181-2207.
  16. Davis, F. E. & Marshall, M. L. 1975. Chemical and biological investigations of Lake Okeechobee. January 1973-1974. Interim report. Florida Flood Control District Technical Publication, 75-1. Resource Planning Department, Central Southern Florida Flood Control Distr., West Palm Beach, FL, 91 pp.
  17. Dere, S., Gunes, T. & Sivaci, R. 1998. Spectrophotometric determination of chlorophyll-A, B and total carotenoid contents of some algae species using different solvents. Turk. J. Bot. 22:13-17.
  18. D'Ippolito, G., Sardo, A., Paris, D., Vella, F. M., Adelfi, M. G., Botte, P., Gallo, C. & Fontana, A. 2015. Potential of lipid metabolism in marine diatoms for biofuel production. Biotechnol. Biofuels 8:28.
  19. Edgar, R. C. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32:1792-1797.
  20. Gantar, M., Dhandayuthapani, S. & Rathinavelu, A. 2012. Phycocyanin induces apoptosis and enhances the effect of topotecan on prostate cell line LNCaP. J. Med. Food 15:1091-1095.
  21. Gantar, M. & Svircev, Z. 2008. Microalgae and cyanobacteria: food for thought. J. Phycol. 44:260-268.
  22. Gardner, R., Peters, P., Peyton, B. & Cooksey, K. E. 2011. Medium pH and nitrate concentration effects on accumulation of triacylglycerol in two members of the Chlorophyta. J. Appl. Phycol. 23:1005-1016.
  23. Gimmler, H. & Degenhard, B. 2001. Alkaliphilic and alkalitolerant algae. In Rai, L. C. & Gaur, J. P. (Eds.) Algal Adaptation to Environmental Stresses: Physiological, Biochemical and Molecular Mechanisms. Springer, Berlin, pp. 291-321.
  24. Griffiths, M. J. & Harrison, S. T. L. 2009. Lipid productivity as a key characteristic for choosing algal species for biodiesel production. J. Appl. Phycol. 21:493-507.
  25. Guindon, S. & Gascuel, O. 2003. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52:696-704.
  26. Guiry, M. D. & Guiry, G. M. 2018. AlgaeBase. World-wide electronic publication, National University of Ireland, Galway. Available from: Accessed Jul 2, 2019.
  27. Hagerthey, S. E., Bellinger, B. J., Wheeler, K., Gantar, M. & Gaiser, E. 2011. Everglades periphyton: a biogeochemical perspective. Crit. Rev. Environ. Sci. Technol. 41:309-343.
  28. Han, Y., Wen, Q., Chen, Z. & Li, P. 2011. Review of methods used for microalgal lipid-content analysis. Energy Procedia 12:944-950.
  29. Hannon, M., Gimpel, J., Tran, M., Rasala, B. & Mayfield, S. 2010. Biofuels from algae: challenges and potential. Biofuels 1:763-784.
  30. James, R. T., Smith, V. H. & Jones, B. L. 1995. Historical trends in the Lake Okeechobee ecosystem. 3. Water quality. Arch. Hydrobiol. Suppl. 107:49-69.
  31. Jin, E. S. & Melis, A. 2003. Microalgal biotechnology: carotenoid production by the green algae Dunaliella salina. Biotechnol. Bioprocess Eng. 8:331.
  32. Jones, B. E., Grant, W. D., Collins, N. C. & Mwatha, W. E. 1994. Alkaliphiles: diversity and identification. In Priest, F. G., Ramos-Cormenzana, A. & Tindall, B. J.(Eds.) Bacterial Diversity and Systematics. Springer, Boston, MA, pp. 195-230.
  33. Knothe, G. 2010. Biodiesel derived from a model oil enriched in palmitoleic acid, macadamia nut oil. Energy Fuels 24:2098-2103.
  34. Kolouchova, I., Sigler, K., Schreiberova, O., Masak, J. & Rezanka, T. 2015. New yeast-based approaches in production of palmitoleic acid. Bioresour. Technol. 192:726-734.
  35. Kroll, R. G. 1990. Alkalophiles. In Edwards, C. (Ed.) Microbiology of Extreme Environments. McGraw-Hill, New York, pp. 52-92.
  36. Lange-Bertalot, H. 2001. Navicula sensu stricto. 10 Genera separated from Navicula sensu lato. Frustulia. Diatoms of Europe: diatoms of the European inland waters and comparable habitats. Vol. 2. A.R.G. Gantner Verlag. K.G., Ruggell, 526 pp.
  37. Lenihan-Geels, G., Bishop, K. S. & Ferguson, L. R. 2013. Alternative sources of omega-3 fats: can we find a sustainable substitute for fish? Nutrients 5:1301-1315.
  38. Lewin, J. C. 1955. The capsule of the diatom Navicula pelliculosa. J. Gen. Microbiol. 13:162-169.
  39. Lichtenthaler, H. K. & Wellburn, A. R. 1983. Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochem. Soc. Trans. 11:591-592.
  40. Ma, X.-N., Chen, T.-P., Yang, B., Liu, J. & Chen, F. 2016. Lipid production from Nannochloropsis. Mar. Drugs 14:61.
  41. Matsumoto, M., Mayama, S., Nemoto, M., Fukuda, Y., Muto, M., Yoshino, T., Matsunaga, T. & Tanaka, T. 2014. Morphological and molecular phylogenetic analysis of the high triglyceride-producing marine diatom, Fistulifera solaris sp. nov. (Bacillariophyceae). Phycol. Res. 62:257-268.
  42. Matsumoto, M., Sugiyama, H., Maeda, Y., Sato, R., Tanaka, T. & Matsunaga, T. 2010. Marine diatom, Navicula sp. strain JPCC DA0580 and marine green alga, Chlorella sp. strain NKG400014 as potential sources for biodiesel production. Appl. Biochem. Biotechnol. 161:483-490.
  43. McBride, R. C., Lopez, S., Meenach, C., Burnett, M., Lee, P. A., Nohilly, F. & Behnke, C. 2014. Contamination management in low cost open algae ponds for biofuels production. Ind. Biotechnol. 10:221-227.
  44. Miller, M. A., Pfeiffer, W. & Schwartz, T. 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In 2010 Gatew. Comput. Environ. Workshop (GCE), Institute of Electrical and Electronics Engineers, Piscataway, NJ, pp. 1-8.
  45. Moreno-Garrido, I. & Canavate, J. P. 2001. Assessing chemical compounds for controlling predator ciliates in outdoor mass cultures of the green algae Dunaliella salina. Aquac. Eng. 24:107-114.
  46. Morse, N. 2015. Lipid-lowering and anti-inflammatory effects of palmitoleic acid: evidence from preclinical and epidemiological studies. Lipid Technol. 27:107-111.
  47. Mutanda, T., Ramesh, D., Karthikeyan, S., Kumari, S., Anandraj, A. & Bux, F. 2011. Bioprospecting for hyper-lipid producing microalgal strains for sustainable biofuel production. Bioresour. Technol. 102:57-70.
  48. Muto, M., Tanaka, M., Liang, Y., Yoshino, T., Matsumoto, M. & Tanaka, T. 2015. Enhancement of glycerol metabolism in the oleaginous marine diatom Fistulifera solaris JPCC DA0580 to improve triacylglycerol productivity. Biotechnol. Biofuels 8:4.
  49. Nelson, D. R., Mengistu, S., Ranum, P., Celio, G., Mashek, M., Mashek, D. & Lefebvre, P. A. 2013. New lipid-producing, cold-tolerant yellow-green alga isolated from the Rocky Mountains of Colorado. Biotechnol. Prog. 29:853-861.
  50. Nguyen, H. T., Park, H., Koster, K. L., Cahoon, R. E., Nguyen, H. T. M., Shanklin, J., Clemente, T. E. & Cahoon, E. B. 2015. Redirection of metabolic flux for high levels of omega-7 monounsaturated fatty acid accumulation in camelina seeds. Plant Biotechnol. J. 13:38-50.
  51. Ofosu, F. K., Daliri, E. B., Lee, B. & Yu, X. 2017. Current trends and future perspectives on omega-3 fatty acids. Res. Rev. J. Biol. 5:11-20.
  52. Ronquist, F. & Huelsenbeck, J. P. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572-1574.
  53. Rowan, R. & Powers, D. A. 1991. Molecular genetic identification of symbiotic dinoflagellates (zooxanthellae). Mar. Ecol. Prog. Ser. 71:65-73.
  54. Saleeb, W. F., Yermanos, D. M., Huszar, C. K., Storey, W. B. & Labanauskas, C. K. 1973. The oil and protein in nuts of Macadamia tetraphylla L. Johnson, Macadamia integrifolia Maiden and Betche, and their F1hybrid. J. Am. Soc. Hortic. Sci. 98:453-456.
  55. Sato, R., Maeda, Y., Yoshino, T., Tanaka, T. & Matsumoto, M. 2014. Seasonal variation of biomass and oil production of the oleaginous diatom Fistulifera sp. in outdoor vertical bubble column and raceway-type bioreactors. J. Biosci. Bioeng. 117:720-724.
  56. Satoh, A., Ichii, K., Matsumoto, M., Kubota, C., Nemoto, M., Tanaka, M., Yoshino, T., Matsunaga, T. & Tanaka, T. 2013. A process design and productivity evaluation for oil production by indoor mass cultivation of a marine diatom, Fistulifera sp. JPCC DA0580. Bioresour. Technol. 137:132-138.
  57. Scott, S. A., Davey, M. P., Dennis, J. S., Horst, I., Howe, C. J., Lea-Smith, D. J. & Smith, A. G. 2010. Biodiesel from algae: challenges and prospects. Curr. Opin. Biotechnol. 21:277-286.
  58. Selvarajan, R., Felfoldi, T., Tauber, T., Sanniyasi, E., Sibanda, T. & Tekere, M. 2015. Screening and evaluation of some green algal strains (Chlorophyceae) isolated from freshwater and Soda Lakes for biofuel production. Energies 8:7502-7521.
  59. Shunyu, S., Yongding, L., Yinwu, S., Genbao, L. & Dunhai, L. 2006. Lysis of Aphanizomenon flos-aquae (Cyanobacterium) by a bacterium Bacillus cereus. Biol. Control 39:345-351.
  60. Smith, V. H. & Crews, T. 2014. Applying ecological principles of crop cultivation in large-scale algal biomass production. Algal Res. 4:23-34.
  61. Stemmler, K., Massimi, R. & Kirkwood, A. E. 2016. Growth and fatty acid characterization of microalgae isolated from municipal waste-treatment systems and the potential role of algal-associated bacteria in feedstock production. PeerJ 4:e1780.
  62. Tanaka, T., Maeda, Y., Veluchamy, A., Tanaka, M., Abida, H., Marechal, E., Bowler, C., Muto, M., Sunaga, Y., Tanaka, M., Yoshino, T., Taniguchi, T., Fukuda, Y., Nemoto, M., Matsumoto, M., Wong, P. S., Aburatani, S. & Fujibuchi, W. 2015. Oil accumulation by the oleaginous diatom Fistulifera solaris as revealed by the genome and transcriptome. Plant Cell 27:162-176.
  63. Taylor, J. C., Harding, W. R. & Archibald, C. G. M. 2007. A methods manual for the collection, preparation, and analysis of diatom samples. Report for the water research commission. WRC report TT 281 (v1.0). Water Research Commission, Pretoria, South Africa, 49 pp.
  64. Van Wagenen, J., Miller, T. W., Hobbs, S., Hook, P., Crowe, B. & Huesemann, M. 2012. Effects of light and temperature on fatty acid production in Nannochloropsis salina. Energies 5:731-740.
  65. Vonshak, A. 1993. Microalgae: laboratory growth techniques and the biotechnology of biomass production. In Hall, D. O., Scurlock, J. M. O., Bolhar-Nordenkampf, H. R., Leegood, R. C. & Long, S. P. (Eds.) Photosynthesis and Production in a Changing Environment: A field and Laboratory Mannual. Springer, Dordrecht, pp. 337-355.
  66. Wang, H., Zhang, W., Chen, L., Wang, J. & Liu, T. 2013. The contamination and control of biological pollutants in mass cultivation of microalgae. Bioresour. Technol. 128:745-750.
  67. Ward, O. P. & Singh, A. 2005. Omega-3/6 fatty acids: alternative sources of production. Process Biochem. 40:3627-3652.
  68. Wehr, J. D., Sheath, R. G. & Kociolek, P. J. 2015. Freshwater algae of North America: ecology and classification. 2nd ed. Academic Press, Amsterdam, 1050 pp.
  69. Weis, J. J., Madrigal, D. S. & Cardinale, B. J. 2008. Effects of algal diversity on the production of biomass in homogeneous and heterogeneous nutrient environments: a microcosm experiment. PLoS ONE 3:e2825.
  70. Wensel, P., Helms, G., Hiscox, B., Davis, W. C., Kirchhoff, H., Bule, M. & Chen, S. 2014. Isolation, characterization, and validation of oleaginous, multi-trophic, and haloalkaline-tolerant microalgae for two-stage cultivation. Algal Res. 4:2-11.
  71. Xia, S., Wang, K., Wan, L., Li, A., Hu, Q. & Zhang, C. 2013. Production, characterization, and antioxidant activity of fucoxanthin from the marine diatom Odontella aurita. Mar. Drugs 11:2667-2681.
  72. Yang, B. & Kallio, H. P. 2001. Fatty acid composition of lipids in sea buckthorn (Hippophae rhamnoides L.) berries of different origins. J. Agric. Food Chem. 49:1939-1947.
  73. Zgrundo, A., Lemke, P., Pniewski, F., Cox, E. J. & Latala, A. 2013. Morphological and molecular phylogenetic studies on Fistulifera saprophila. Diatom Res. 28:431-443.
  74. Zhang, Z., Wang, F., Wang, X., Liu, X., Hou, Y. & Zhang, Q. 2010. Extraction of the polysaccharides from five algae and their potential antioxidant activity in vitro. Carbohydr. Polym. 82:118-121.
  75. Zhu, L. D., Li, Z. H. & Hiltunen, E. 2016. Strategies for lipid production improvement in microalgae as a biodiesel feedstock. BioMed Res. Int. 2016:8792548.