A method of batch-purifying microalgae with multiple antibiotics at extremely high concentrations -- Journal of Oceanology and Limnology,2016,34(1):79-85

	Cite this paper:
	HAN Jichang, WANG Song, ZHANG Lin, YANG Guanpin, ZHAO Lu, PAN Kehou. A method of batch-purifying microalgae with multiple antibiotics at extremely high concentrations[J]. Journal of Oceanology and Limnology, 2016, 34(1): 79-85

A method of batch-purifying microalgae with multiple antibiotics at extremely high concentrations

HAN Jichang¹, WANG Song¹, ZHANG Lin¹, YANG Guanpin², ZHAO Lu¹, PAN Kehou¹

1 Laboratory of Applied Microalgae Biology, Key Laboratory of Aquaculture of Ministry of Education, Ocean University of China, Qingdao 266003, China;
2 College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China

Abstract:

Axenic microalgal strains are highly valued in diverse microalgal studies and applications. Antibiotics, alone or in combination, are often used to avoid bacterial contamination during microalgal isolation and culture. In our preliminary trials, we found that many microalgae ceased growing in antibiotics at extremely high concentrations but could resume growth quickly when returned to an antibiotics-free liquid medium and formed colonies when spread on a solid medium. We developed a simple and highly effi cient method of obtaining axenic microalgal cultures based on this observation. First, microalgal strains of diff erent species or strains were treated with a mixture of ampicillin, gentamycin sulfate, kanamycin, neomycin and streptomycin (each at a concentration of 600 mg/L) for 3 days; they were then transferred to antibiotics-free medium for 5 days; and fi nally they were spread on solid f/2 media to allow algal colonies to form. With this method, fi ve strains of Nannochloropsis sp. (Eustigmatophyceae), two strains of Cylindrotheca sp. (Bacillariophyceae), two strains of Tetraselmis sp. (Chlorodendrophyceae) and one strain of Amphikrikos sp. (Trebouxiophyceae) were purifi ed successfully. The method shows promise for batchpurifying microalgal cultures.

Key words: antibiotic|axenic|batch operation|bacterium removal|microalga

Received: 2014-11-23 Revised: 2015-01-08

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References:
Amit K, Sarina E, Yuan X, Ashish S, Zhang Q, Jo D, Xavier M F, Van Langenhove H. 2010. Enhanced CO 2 fi xation and biofuel production via microalgae: recent developments and future directions. Trends Biotechnol ., 28 (7): 371-380.
Azma M, Mohamad R, Rahim R A, Ariff A B. 2010. Improved protocol for the preparation of Tetraselmis suecica axenic culture and adaptation to heterotrophic cultivation. The Open Biotechnol. J ., 4 : 36-46.
Bolch C J S, Blackburn S I. 1996. Isolation and purifi cation of Australian isolates of the toxic cyanobacterium Microcystis aeruginosa Kütz. J. Appl. Phycol ., 8 (1): 5-13.
Brennan L, 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.
Bruckner C G, Kroth P G. 2009. Protocols for the removal of bacteria from freshwater benthic diatom cultures. J .Phycol ., 45 (4): 981-986.
Bumbak F, Cook S, Zachleder V, Hauser S, Kovar K. 2011.Best practices in heterotrophic high-cell-density microalgal processes: achievements, potential and possible limitations. Appl. Microbiol. Biot ., 91 (1): 31-46.
Carvalho A P, Meireles L A, Malcata F X. 2006. Microalgal reactors: a review of enclosed system designs and performances. Biotechnol. Progr ., 22 (6): 1 490-1 506.
Chen G Q, Chen F. 2006. Growing phototrophic cells without light. Biotechnolo. Lett ., 28 (9): 607-616.
Chiovitti D T, Bacic P T, Burke J M, Wetherbee D R. 2003.Heterogeneous xylose-rich glycans are associated with extracellular glycoproteins from the biofouling diatom Craspedostaur o s australis (Bacillariophyceae). Eur. J .Phyco ., 38 : 351-360.
Chisti Y. 2007. Biodiesel from microalgae. Biotechnol. Adv ., 25 (3): 294-306.
Cho J-Y, Choi J-S, Kong I-S, Park S-II, Kerr R G, Hong Y K. 2002. A procedure for axenic isolation of the marine microalga Isochrysis galbana from heavily contaminated mass cultures. J. Appl. Phycol ., 14 (5): 385-390.
Choi G-G, Bae M-S, Ahn C-Y, Oh H-M. 2008. Induction of axenic culture of Arthrospira ( Spirulina ) platensis based on antibiotic sensitivity of contaminating bacteria.Biotechnol. Lett ., 30 (1): 87-92.
Choi G-G, Bae M-S, Ahn C-Y, Oh H-M. 2008. Induction of axenic culture of Arthrospira ( Spirulina ) platensis based on antibiotic sensitivity of contaminating bacteria.Biotechnol. Let ., 30 (1): 87-92.
Chojnacka K, Marquez-Rocha F-J. 2004. Kinetic and stoichiometric relationships of the energy and carbon metabolism in the culture of microalgae. Biotechnol ., 3 (1): 21-34.
Connell L, Cattolico R A. 1996. Fragile algae: axenic culture of fi eld-collected samples of Heterosigma carterae. Mar .Biol ., 125 (2): 421-426.
Cottrell M T, Suttle C A. 1993. Production of axenic cultures of Micromonas pusilla (Prasinophyceae) using antibiotic.J. Phycol ., 29 (3): 385-387.
Divan C L, Schnoes H K. 1982. Production of axenic Gonyaulax cultures by treatment with antibiotics. App .Environ. Microb ., 44 (1): 250-254.
Griesbeck C, Kobl I, Heitzer M. 2006. Chlamydomonas reinhardtii : a protein expression system for pharmaceutical and biotechnological proteins. Mol. Biotechnol ., 34 (2): 213-223.
Guillard R R L, Ryther J H. 1962. Studies of marine planktonic diatoms: I. cyclotella nana hustedt, and detonula confervacea (cleve) gran. Cana. J. Microbiol ., 8 (2): 229- 239.
Huang Z H, Liu X J, Hu Z X, Duan S S. 2007. Eff ects of antibiotics on the growth of Isochrysis zhangjiangensis and axenic culture. Ecologic. Sci ., 26 (2): 120-121. (in Chinese with English abstract)
Katoh H, Furukawa J, Tomita-Yokotani K, Nishi Y. 2012.Isolation and purifi cation of an axenic diazotrophic drought-tolerant cyanobacterium, Nostoc commune, from natural cyanobacterial crusts and its utilization for fi eld research on soils polluted with radioisotopes. Biochimica et Biophysica Acta, 1817 (8): 1 499-1 505.
Lee Y -K. 2001. Microalgal mass culture systems and methods: their limitation and potential. J. Appl. Phycol ., 13 (4): 307- 315.
Li J H, Huang X H, Liu H L. 2009. Sensitivity to antibiotics and axenic culture of Oocystis borgei. J. Guangdong .Ocean University, 29 (3): 37-41. (in Chinese with English abstract)
Lin W, Chen D, Liu X Y. 2000. Marine microalgal axenation and comparison of growth characteristics between natural and axenic marine microalgae. Oceanol. Limnol. Sinica, 31 (6): 647-652. (in Chinese with English abstract)
Lin W. 2000. Axenization of several marine microalgal cultures. Mar. Sci ., 24 (10): 4-6. (in Chinese with English abstract)
Liu X J, Duan S S. 2006. Study on the susceptibility tests of antibiotics and obtain axenic culture of Nannochloropsis ocutala. Ecologic. Sci ., 25 (6): 493-495. (in Chinese with English abstract)
Miao X L, Wu Q Y. 2006. Biodiesel production from heterotrophic microalgal oil. Bioresource Technol ., 97 (6): 841-846.
Perez-Garcia O, Escalante F M E, De-Bashan L E, Bashan Y. 2011. Heterotrophic cultures of microalgae: metabolism and potential products. Water. Res ., 45 (1): 11-36.
Rosenberg J N, Oyler G A, Wilkinson L, Betenbaugh M J. 2008. A green light for engineered algae: redirecting metabolism to fuel a biotechnology revolution. Curr .Opin. Biotech ., 19 (6): 430-436.
Skulberg O M. 2000. Microalgae as a source of bioactive molecules-experience from cyanophyte research. J. Appl .Phycol ., 12 (3-5): 341-348.
Sloth J K, Wiebe M G, Eriksen N T. 2006. Accumulation of phycocyanin in heterotrophic and mixotrophic cultures of the acidophilic red alga Galdieria sulphuraria. Enzyme .Microb. Tech ., 38 (1-2): 168-175.
Spencer C P. 1952. On the use of antibiotics for isolating bacteria-free cultures of marine phytoplankton organisms.J. Mar. Biol. Assoc. UK ., 31 (1): 97-106.
Su J Q, Yang X R, Zheng T L, Hong H S. 2007. An effi cient method to obtain axenic cultures of Alexandrium tamarense —a PSP-producing dinofl agellate. J. Microbiol .Meth ., 69 (3) : 425-430.
Subashchandrabose S R, Ramakrishnan B, Megharaj M,Venkateswarlu K, Naidu R. 2011. Consortia of cyanobacteria/microalgae and bacteria: biotechnological potential. Biotechnol. Adv ., 29 (6): 896-907.
Underwood G J C, Boulcott M, Raines C A. 2004.Environmental eff ects on exopolymer production by marine benthic diatoms: dynamics, changes in composition and pathways of production. J. Phycol ., 40 (2): 293-304.
Vázquez-Martínez G, Rodriguez M H, Hernández-Hernández F, Ibarra J E. 2004. Strategy to obtain axenic cultures from fi eld-collected samples of the cyanobacterium Phormidium animalis. J. Microbiol. Meth ., 57 (1): 115-121.
Wigglesworth-Cooksey B, Berglund D, Cooksey K E. 2001.Cell-cell and cell-surface interactions in an illuminated biofi lm: implications for marine sediment stabilization.Geochem. Trans ., 2 : 75.
Zhang X W, Shi X M, Chen F. 1999. A kinetic model for lutein production by the green microalga Chlorella protothecoides in heterotrophic culture. J. Ind. Microbiol .Biot ., 23 (6): 503-507.
ZoBell C E. 1941. Studies on marine bacteria. I. the cultural requirements of heterotrophic aerobes. J. Mar. Res ., 4 : 42-75.