Park, Jaeyeon
(Environment and Resource Convergence Center, Advanced Institutes of Convergence Technology)
,
Jeong, Hae Jin
(Environment and Resource Convergence Center, Advanced Institutes of Convergence Technology)
,
Yoon, Eun Young
(Environment and Resource Convergence Center, Advanced Institutes of Convergence Technology)
,
Moon, Seung Joo
(Environment and Resource Convergence Center, Advanced Institutes of Convergence Technology)
To develop an easy and rapid method of quantifying lipid contents of marine dinoflagellates, we quantified lipid contents of common dinoflagellate species using a colorimetric method based on the sulpho-phospho-vanillin reaction. In this method, the optical density measured using a spectrophotometer...
To develop an easy and rapid method of quantifying lipid contents of marine dinoflagellates, we quantified lipid contents of common dinoflagellate species using a colorimetric method based on the sulpho-phospho-vanillin reaction. In this method, the optical density measured using a spectrophotometer was significantly positively correlated with the known lipid content of a standard oil (Canola oil). When using this method, the lipid content of each of the dinoflagellates Alexandrium minutum, Prorocentrum micans, P. minimum, and Lingulodinium polyedrum was also significantly positively correlated with the optical density and equivalent intensity of color. Thus, when comparing the color intensity or the optical density of a sample of a microalgal species with known color intensities or optical density, the lipid content of the target species could be rapidly quantified. Furthermore, the results of the sensitivity tests showed that only $1-3{\times}10^5cells$ of P. minimum and A. minutum, $10^4cells$ of P. micans, and $10^3cells$ of L. polyedrum (approximately 1-5 mL of dense cultures) were needed to determine the lipid content per cell. When the lipid content per cell of 9 dinoflagellates, a diatom, and a chlorophyte was analyzed using this method, the lipid content per cell of these microalgae, with the exception of the diatom, were significantly positively correlated with cell size, however, volume specific lipid content per cell was negatively correlated with cell size. Thus, this sulpho-phospho-vanillin method is an easy and rapid method of quantifying the lipid content of autotrophic, mixotrophic, and heterotrophic dinoflagellate species.
To develop an easy and rapid method of quantifying lipid contents of marine dinoflagellates, we quantified lipid contents of common dinoflagellate species using a colorimetric method based on the sulpho-phospho-vanillin reaction. In this method, the optical density measured using a spectrophotometer was significantly positively correlated with the known lipid content of a standard oil (Canola oil). When using this method, the lipid content of each of the dinoflagellates Alexandrium minutum, Prorocentrum micans, P. minimum, and Lingulodinium polyedrum was also significantly positively correlated with the optical density and equivalent intensity of color. Thus, when comparing the color intensity or the optical density of a sample of a microalgal species with known color intensities or optical density, the lipid content of the target species could be rapidly quantified. Furthermore, the results of the sensitivity tests showed that only $1-3{\times}10^5cells$ of P. minimum and A. minutum, $10^4cells$ of P. micans, and $10^3cells$ of L. polyedrum (approximately 1-5 mL of dense cultures) were needed to determine the lipid content per cell. When the lipid content per cell of 9 dinoflagellates, a diatom, and a chlorophyte was analyzed using this method, the lipid content per cell of these microalgae, with the exception of the diatom, were significantly positively correlated with cell size, however, volume specific lipid content per cell was negatively correlated with cell size. Thus, this sulpho-phospho-vanillin method is an easy and rapid method of quantifying the lipid content of autotrophic, mixotrophic, and heterotrophic dinoflagellate species.
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제안 방법
After analyzing lipid content using the SPV method, the lipid content data analyzed for each cell number was calculated using the standard curve, and the lipid content of individual cells was calculated. A t-test and ANOVA were performed to determine if the lipid content of individual cells has any significant difference with the lipid content of the individual cells calculated for each different cell number.
In this study, we applied a modified SPV method for quantifying the lipid content of dinoflagellate species. Furthermore, using this method, we analyzed the lipid content of 9 dinoflagellate species including phototrophic, mixotrophic and heterotrophic species (Amphidinium carterae, Heterocapsa triquetra, Prorocentrum minimum, Alexandrium minutum, Oxyrrhis marina, Scrippsiella trochoidea, Ostreopsis cf.
The direct measurement of lipid content using the SPV reaction in this study was modified from the Inouye and Lotufo protocol (Inouye and Lotufo 2006); in this method, 0.2 mL sample, 0.1 mL sulfuric acid, and 2.4 mL vanillin reagent (1.2 mg vanillin per milliliter of 68% phosphoric acid) were used.
This research was supported by Developing the method of converting food wastes to bioenergy using mass cultured marine protozoa, and the programs “Management of Marine Organisms causing Ecological Disturbance and Harmful Effects” to J. Park and “Useful Dinoflagellate” funded by KIMST/MOF to HJJ.
대상 데이터
Vanillin (≥98%) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
데이터처리
The optical density measured using a spectrophotometer (and equivalent intensity of color) was significantly positively correlated with the known lipid content of the standard oil (i.e., Canola oil) (p < 0.01, linear regression ANOVA) (Fig. 1).
When the lipid contents of the dinoflagellates A. minutum, P. micans, P. minimum, and L. polyedrum were calculated using the standard curve, the lipid amount of each species was also significantly positively correlated with optical density and equivalent intensity of color (p < 0.01 for each of all 4 species, linear regression ANOVA) (Fig. 2).
이론/모형
Lipid content (LC, μg lipid) and cell numbers of Prorocentrum minimum (A), Alexandrium minutum (B), P. micans (C), and Lingulodinium polyedrum (D) as a function of the optical density (OD) obtained from samples when measured using the sulpho-phospho-vanillin method.
Lipid content per cell (LCPC, ng lipid cell-1) of all 11 species (A), the dinoflagellates only (B), and planktonic dinoflagellates only (C) as a function of cell size (equivalent spherical diameter, ESD, μm) when measured using the sulpho-phospho-vanillin method.
7. Lipid content per cell (LCPC, ng lipid cell-1) of all 11 species as a function of the specific growth rate (GR, d-1) when measured using the sulpho-phospho-vanillin method. The equation of the curve was y (LCPC) = -0.
4. Lipid content per cell (ng lipid cell-1) of the 11 experimental species tested when measured using the sulpho-phospho-vanillin method. Bars represent treatment means ± standard deviation (n = 3).
1981), as well as in serum, food, and ecological samples. The SPV assay produces a distinct pink color when reacting with lipids, and the intensity of the color can be quantified by measuring the absorbance at 530 nm using spectrophotometric methods. The advantages of this technique are that it is able to measure lipid content rapidly and simply and requires only a small amount of target sample.
Volume specific lipid content per cell (VLCPC, pg lipid cell-1) of all 11 species (A) and the dinoflagellates only (B) as a function of cell size (ESD, μm) when measured using the sulpho-phosphovanillin method.
성능/효과
6A). Among the dinoflagellate species tested, A. carterae had the highest volume specific lipid content, while L. polyedrum had the lowest. Furthermore, the volume specific lipid content of all dinoflagellates was significantly negatively correlated with cell size (Fig.
2016). Among the dinoflagellates tested in this study, A. carterae had the highest volume specific lipid content even though its absolute lipid content was lower than several other dinoflagellates. Thus, for the use of raw materials as biofuel, determining volume specific lipid content of a microalgae is important.
4). The lipid content per cell of A. minutum measured using the SPV method in this study (0.186-0.345 ng lipid cell-1) was comparable to those measured using other methods (0.288-0.400 ng lipid cell-1) (Table 3). Thus, the SPV method is valid.
The conventional methods of quantifying the lipid contents of microalgae require large amounts of biomass and thus, usually requires a great deal of time and labor; the processing of samples for gas chromatography analysis may also take several days and be laborious work. The results of this study clearly show that both optical density and intensity of color had strong correlations with the lipid content of each of the 4 dinoflagellates tested and thus, the lipid content of a sample containing each species can be measured using spectrophotometry or even the naked eye. Thus, the SPV method applied in this study can be used as an easy and rapid method of quantifying the lipid content of microalgal species.
참고문헌 (40)
Anderson, D. M. 1997. Turning back the harmful red tide. Nature 388:513-514.
Byreddy, A. R., Gupta, A., Barrow, C. J. & Puri, M. 2016. A quick colorimetric method for total lipid quantification in microalgae. J. Microbiol. Methods 125:28-32.
Converti, A., Casazza, A. A., Ortiz, E. Y., Perego, P. & Del Borghi, M. 2009. Effect of temperature and nitrogen concentration on the growth and lipid content of Nannochloropsis oculata and Chlorella vulgaris for biodiesel production. Chem. Eng. Process 48:1146-1151.
de la Jara, A., Mendoza, H., Martel, A. Molina, C., Nordstron, L., de la Rosa, V. & Diaz, R. 2003. Flow cytometric determination of lipid content in a marine dinoflagellate, Crypthecodinium cohnii. J. Appl. Phycol. 15:433-438.
Drevon, B. & Schmit, J. M. 1964. La reaction sulpho-phospho-vanillique dans l'etude des lipides seriques. Bull. Trav. Soc. Pharm. Lyon 8:173-178.
Eppley, R. W. & Sloan, P. R. 1966. Growth rates of marine phytoplankton: correlation with light absorption by cell chlorophyll a. Physiol. Plant. 19:47-59.
Folch, J., Lees, M. & Sloane Stanley, G. H. 1957. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226:497-509.
Fuentes-Grunewald, C., Bayliss, C., Fonlut, F. & Chapuli, E. 2016. Long-term dinoflagellate culture performance in a commercial photobioreactor: Amphidinium carterae case. Bioresour. Technol. 218:533-540.
Fuentes-Grunewald, C., Garces, E., Alacid, E., Sampedro, N., Rossi, S. & Camp, J. 2012. Improvement of lipid production in the marine strains Alexandrium minutum and Heterosigma akashiwo by utilizing abiotic parameters. J. Ind. Microbiol. Biotechnol. 39:207-216.
Fuentes-Grunewald, C., Garces, E., Rossi, S. & Camp, J. 2009. Use of the dinoflagellate Karlodinium veneficum as a sustainable source of biodiesel production. J. Ind. Microbiol. Biotechnol. 36:1215-1224.
Furnas, M. J. 1990. In situ growth rates of marine phytoplankton: approaches to measurement, community and species growth rates. J. Plankton Res. 12:1117-1151.
Grzebyk, D., Bechemin, C., Ward, C. J., Verite, C., Codd, G. A. & Maestrini, S. Y. 2003. Effects of salinity and two coastal waters on the growth and toxin content of the dinoflagellate Alexandrium minutum. J. Plankton Res. 25:1185-1199.
Guerrini, F., Pezzolesi, L., Feller, A., Riccardi, M., Ciminiello, P., Dell'Aversano, C., Tartaglione, L., Dello Iacovo, E., Fattorusso, E., Forino, M. & Pistocchi, R. 2010. Comparative growth and toxin profile of cultured Ostreopsis ovata from the Tyrrhenian and Adriatic Seas. Toxicon 55:211-220.
Guillard, R. R. L. & Ryther, J. H. 1962. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea (Cleve) Gran. Can. J. Microbiol. 8:229-239.
Hansen, P. J. 1992. Prey size selection, feeding rates and growth dynamics of heterotrophic dinoflagellates with special emphasis on Gyrodinium spirale. Mar. Biol. 114:327-334.
Hao, Z., Liu, P., Yang, X., Shi, J. & Zhang, S. 2013. Screening method for lipid-content microalgae based on sulpho-phospho-vanillin reaction. Adv. Mater. Res. 610-613:3532-3535.
Inouye, L. S. & Lotufo, G. R. 2006. Comparison of macrogravimetric and micro-colorimetric lipid determination methods. Talanta 70:584-587.
Jacobson, D. M. & Anderson, D. M. 1996. Widespread phagocytosis of ciliates and other protists by marine mixotrophic and heterotrophic thecate dinoflagellates. J. Phycol. 32:279-285.
Jeong, H. J., Yoo, Y. D., Kang, N. S., Lim, A. S., Seong, K. A., Lee, S. Y., Lee, M. J., Lee, K. H., Kim, H. S., Shin, W., Nam, S. W., Yih, W. & Lee, K. 2012. Heterotrophic feeding as a newly identified survival strategy of the dinoflagellate Symbiodinium. Proc. Natl. Acad. Sci. U. S. A. 109:12604-12609.
Jeong, H. J., Yoo, Y. D., Kim, J. S., Seong, K. A., Kang, N. S. & Kim, T. H. 2010. Growth, feeding, and ecological roles of the mixotrophic and heterotrophic dinoflagellates in marine planktonic food webs. Ocean Sci. J. 45:65-91.
Jeong, H. J., Yoo, Y. D., Park, J. Y., Song, J. Y., Kim, S. T., Lee, S. H., Kim, K. Y. & Yih, W. H. 2005. Feeding by phototrophic red-tide dinoflagellates: five species newly revealed and six species previously known to be mixotrophic. Aquat. Microbial Ecol. 40:133-150.
Kondo, K., Seike, Y. & Date, Y. 1990. Red tides in the brackish Lake Nakanoumi (II). Relationships between the occurrence of Prorocentrum minimum red tide and environmental conditions. Bull. Plankton Soc. Jpn. 37:19-34.
Lee, K. H., Jeong, H. J., Yoon, E. Y., Jang, S. H., Kim, H. S. & Yih, W. 2014a. Feeding by common heterotrophic dinoflagellates and a ciliate on the red-tide ciliate Mesodinium rubrum. Algae 29:153-163.
Lee, S. K., Jeong, H. J., Jang, S. H., Lee, K. H., Kang, N. S., Lee, M. J. & Potvin, E. 2014c. Mixotrophy in the newly described dinoflagellate Ansanella granifera: feeding mechanism, prey species, and effect of prey concentration. Algae 29:137-152.
Lee, S. Y., Jeong, H. J., Kang, N. S., Jang, T. Y., Jang, S. H. & Lim, A. S. 2014b. Morphological characterization of Symbiodinium minutum and S. psygmophilum belonging to clade B. Algae 29:299-310.
Malapascua, J. R., Chou, H.-N., Lu, W.-J. & Lan, J. C.-W. 2012. Development of an indirect method of microalgal lipid quantification using a lysochrome dye, Nile red. Afr. J. Biotechnol. 11:13518-13527.
Mansour, M. P., Volkman, J. K., Jackson, A. E. & Blackburn. S. I. 1999. The fatty acid and sterol composition of five marine dinoflagellates. J. Phycol. 35:710-720.
Mishra, S. K., Suh, W. I., Farooq, W., Moon, M., Shrivastav, A., Park, M. S. & Yang, J. W. 2014. Rapid quantification of microalgal lipids in aqueous medium by a simple colorimetric method. Bioresour. Technol. 155:330-333.
Muscatine, L. 1990. The role of symbiotic algae in carbon and energy flux in reef corals. In Dubinski, Z. (Ed.) Coral reefs: ecosystems of the world. Elsevier, NY, p. 75-87.
Park, J., Jeong, H. J., Yoo, Y. D. & Yoon, E. Y. 2013. Mixotrophic dinoflagellate red tides in Korean waters: distribution and ecophysiology. Harmful Algae 30(Suppl. 1):S28-S40.
Piretti, M. V., Pagliuca, G., Boni, L., Pistocchi, R., Diamante, M. & Gazzotti, T. 1997. Investigation of 4-methyl sterols from cultured dinoflagellate algal strains. J. Phycol. 33:61-67.
Smayda, T. J. 1997. Harmful algal blooms: their ecophysiology and general relevance to phytoplankton blooms in the sea. Limnol. Oceanogr. 42:1137-1153.
Vatassery, G. T., Sheridan, M. A., Krezowski, A. M., Divine, A. S. & Bach, H. L. 1981. Use of the sulpho-phospo-vanillin reaction in a routine method for determining total lipids in human cerebrospinal fluid. Clin. Biochem. 14:21-24.
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