Morphology and Growth Monitoring Methods of Aurantiochytrium sp. FIKU018 Cultivated in Flask and Bioreactor
Abstract
The development of growth monitoring method is significant for the biomass production of Aurantiochytrium as a DHA resource. This study aimed to examine the growth measurement methods and morphological observation of Aurantiochytrium cultured in flasks and bioreactors. The batch cultivation of Aurantiochytrium sp. FIKU018 was carried on 50 mL of GYP broth in 250 mL flask. The cultivation was performed at 30 °C on 200 rpm rotary shaker under dark condition. Then, the fed-batch fermentation was examined in 22 L bioreactor contained 6 L GYP broth. The incubation was carried on 30 °C, pH 6.0, 450 rpm of agitation speed and 1.0 VVM of aeration rate. Samples were taken every 4 h for 72 h. In shaking flask, it was found that the batch cultivation of Aurantiochytrium sp. FIKU018 gave specific growth rate (SGR) and maximum dry cell weight (DCW) at 0.11 h-1 and 12.47 ± 0.70 g L-1, respectively. The fed-batch cultivation was performed in 22 L bioreactor contained 6 L of GYP broth. The result showed that cell cultivation during 0 - 20 h showed SGR and maximum DCW at 0.21 h-1 and 38.87 ± 0.71 g L-1, respectively. Fed-batch cultivation during 20 - 42 h and 42 – 72 h showed maximum DCW at 25.17 ± 0.23 g L-1 and 38.87 ± 0.71 g L-1, respectively. Aurantiochytrium sp. FIKU018 morphology changed according to growth and lipid accumulation phases. DCW can be used as growth parameters to monitor the growth of Aurantiochytrium sp. FIKU018 cultured both in flask and in bioreactor. Optical density and cell count can be used to tracking the growth of cells during 0 - 42 h in fed-batch cultivation in bioreactor. Therefore, all growth parameters can be use as growth monitor method during the development of Aurantiochytrium sp. FIKU018 biomass production processes. Until the lipid accumulation phase, fed with broth containing no nitrogen source, DCW associated with morphology are acceptable parameters to monitor the cell growth. Keywords : fed-batch cultivation ; optical density ; cell count ; dry cell weight ; specific growth rateReferences
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Burja, M.A., Radianingtyas, H., Windust, A., & Barrow, J.C. (2006). Isolation and characterization of polyunsaturated fatty acid producing Thraustochytrium species: screening of strains and optimization of omega-3 production. Applied Microbiology and Biotechnology, 72, 1161-1169.
Chi Z., Liu Y., Frear C., & Chen S. (2009). Study of a two-stage growth of DHA-producing marine algae Schizochytrium limacinum SR21 with shifting dissolved oxygen level. Applied Microbiology and Biotechnology, 81(6), 1141-1148.
Coakley, M., Ross, R. P., Nordgren, M., Fitzgerald, G., Devery, R., & Stanton, C. (2003) Conjugated linoleic acid biosynthesis by human-derived Bifidobacterium species. Journal of Applied Microbiology, 91(1),
138-145.
Furlan, M.J.V., Maus, V., Batista, I., & Bandarra, M.N. (2017) Production of docosahexaenoic acid by Aurantiochytrium sp. ATCG PRA-276. Brazilian Journal of Microbiology, 48, 359-365.
Honda, D., Yokochi, T., Nakahara, T., Erata, M., & Higashihara, T. (1998). Schizochytrium limacinum sp. Nov., a new thraustochytrid from a mangrove area in the west Pacific Ocean, Mycological Research, 102(4), 439-448.
Huang, J., Aki, T., Yokochi, T., Nakahara, T., Honda, D., Kawamoto, S., Shigeta, S., Ono, K., & Suzuki, O. (2003). Grouping newly isolated docosahexaenoic acid-producing thraustochytrid based on their polyunsaturated fatty acid profiles and comparative analysis of 18s rRNA genes. Marine Biotechnology, 5, 450-457.
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Kaya, K., Nakazawa, A., Matsuura, H., Honda, D., Inouye, I., & Watanabe, M.M. (2011). Thraustochytrid Aurantiochytrium sp. 18W-13a accumulates high amounts of squalene. Bioscience, Biotechnology, and Biochemistry, 75, 2246-2248.
Kendrick, A., & Ratledge, C. (1992). Lipids of selected molds grown for production of n-3 and n-6 polyunsaturated fatty acids. Lipids, 27, 15-20.
Liu, P., Shen, S.R., Ruan, H., Zhou, Q., Ma, L.L., & He, G.Q. (2011) Production of conjugated linoleic acids by Lactobacillus plantarum strains isolated from naturally fermented Chinese pickles. Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology), 12(11), 923-930.
Mantzouridou, F., Roukas, T., & Kotzekidou, P. (2002). Effect of the aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor: Mathematical modeling. Biochemical Engineering Journal, 10, 123–135.
Miller, L.G. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31(3), 426-428.
Murthy, K., Rajesha, J., Swamy, M., & Ravishankar, G. (2005). Comparative evaluation of hepatoprotective activity of carotenoids of microalgae. Journal of Medicinal Food, 8(4), 523–528.
Raghukumar, S. (2002). Ecology of the marine protists, the labyrinthulomycetes (thraustochytrids and labyrinthulids). European Journal of Protistology, 38, 127–145.
Raghukumar, S. (2008). Thraustochytrid marine Protists: production of PUFAs and other emerging technologies. Marine Biotechnology, 10, 631-640.
Raposo, M.F., de Morais, A.M., & de Morais, R.M. (2015). Carotenoids from marine microalgae: A valuable natural source for the prevention of chronic diseases. Marine Drugs, 13(8), 5128–5155.
Ratledge, C., & Wynn, J.P. (2002). The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. Advance in Applied Microbiology, 5, 1-51.
Ratledge, C. (2004). Fatty acid biosynthesis in microorganisms being used for single cell oil production. Biochimie, 86, 807-815.
Sanders, T.A.B., Gleason, K., Griffin, B., & Miller, G.J. (2006). Influence of an algal triacylglycerol containing docosahexaenoic acid (22:6 n-3) and docosapentaenoic acid (22:5 n-6) on cardiovascular risk factors in healthy men and women. British Journal of Nutrition, 95, 525–531.
Takahata, K., Monobe, K., Tada, M., & Weber, P.C. (1998). The benefits and risks of n-3 polyunsaturated fatty acids. Bioscience, Biotechnology, and Biochemistry, 62, 2079–2085.
Trovão, M., Pereira, H., Costa, M., Machado, A., Barros, A., Soares, M., Carvalho, B., Silva, J, T., Varela, J., & Silva, J. (2020). Lab-Scale Optimization of Aurantiochytrium sp. culture medium for improved growth and DHA production. Applied Sciences, 10(7), 2500.
Yokochi, T., Honda, D., Higashihara, T., & Nakahara, T. (1998). Optimization of docosahexaenoic acid production by Schizochytrium limacinum SR21. Applied Microbiology and Biotechnology, 49, 72–76.
Yokoyama, R., & Honda, D. (2007). Taxonomic rearrangement of the genus Schizochytrium sensu lato based on morphology, chemotaxonomic characteristics, and 18srRNA gene phylogeny (Thraustochytriaceae, Labyrinthulomycetes): emendation for Schizochytrium and erection of Aurantiochytrium and Oblongichytrium gen. nov. Mycoscience, 48, 199-211.
Zhou, Y., Han, L., He, H., Sang, B., Yu, D., Feng, J., & Zhang, X. (2018). Effects of agitation, aeration and temperature on production of a novel glycoprotein GP-1 by Streptomyces kanasenisi ZX01 and scale-up based on volumetric oxygen transfer coefficient. Molecules, 23, 125.
Aki, T., Hachida, K., Yoshinaga, M., Katai, Y., Yamasaki, T., Kawamoto, S., Kakizono, T., Maoka, T., Shigeta, S., Suzuki, O., & Ono, K. (2003). Thraustochytrid as a potential source of carotenoids. Journal of the American Oil Chemists’ Society, 80, 789-794.
Burja, M.A., Radianingtyas, H., Windust, A., & Barrow, J.C. (2006). Isolation and characterization of polyunsaturated fatty acid producing Thraustochytrium species: screening of strains and optimization of omega-3 production. Applied Microbiology and Biotechnology, 72, 1161-1169.
Chi Z., Liu Y., Frear C., & Chen S. (2009). Study of a two-stage growth of DHA-producing marine algae Schizochytrium limacinum SR21 with shifting dissolved oxygen level. Applied Microbiology and Biotechnology, 81(6), 1141-1148.
Coakley, M., Ross, R. P., Nordgren, M., Fitzgerald, G., Devery, R., & Stanton, C. (2003) Conjugated linoleic acid biosynthesis by human-derived Bifidobacterium species. Journal of Applied Microbiology, 91(1),
138-145.
Furlan, M.J.V., Maus, V., Batista, I., & Bandarra, M.N. (2017) Production of docosahexaenoic acid by Aurantiochytrium sp. ATCG PRA-276. Brazilian Journal of Microbiology, 48, 359-365.
Honda, D., Yokochi, T., Nakahara, T., Erata, M., & Higashihara, T. (1998). Schizochytrium limacinum sp. Nov., a new thraustochytrid from a mangrove area in the west Pacific Ocean, Mycological Research, 102(4), 439-448.
Huang, J., Aki, T., Yokochi, T., Nakahara, T., Honda, D., Kawamoto, S., Shigeta, S., Ono, K., & Suzuki, O. (2003). Grouping newly isolated docosahexaenoic acid-producing thraustochytrid based on their polyunsaturated fatty acid profiles and comparative analysis of 18s rRNA genes. Marine Biotechnology, 5, 450-457.
Johnson, E.A., & An, G.H. (1999). Astaxanthin from microbial sources. Critical Reviews in Biotechnology, 11,
297-326.
Kaya, K., Nakazawa, A., Matsuura, H., Honda, D., Inouye, I., & Watanabe, M.M. (2011). Thraustochytrid Aurantiochytrium sp. 18W-13a accumulates high amounts of squalene. Bioscience, Biotechnology, and Biochemistry, 75, 2246-2248.
Kendrick, A., & Ratledge, C. (1992). Lipids of selected molds grown for production of n-3 and n-6 polyunsaturated fatty acids. Lipids, 27, 15-20.
Liu, P., Shen, S.R., Ruan, H., Zhou, Q., Ma, L.L., & He, G.Q. (2011) Production of conjugated linoleic acids by Lactobacillus plantarum strains isolated from naturally fermented Chinese pickles. Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology), 12(11), 923-930.
Mantzouridou, F., Roukas, T., & Kotzekidou, P. (2002). Effect of the aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor: Mathematical modeling. Biochemical Engineering Journal, 10, 123–135.
Miller, L.G. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31(3), 426-428.
Murthy, K., Rajesha, J., Swamy, M., & Ravishankar, G. (2005). Comparative evaluation of hepatoprotective activity of carotenoids of microalgae. Journal of Medicinal Food, 8(4), 523–528.
Raghukumar, S. (2002). Ecology of the marine protists, the labyrinthulomycetes (thraustochytrids and labyrinthulids). European Journal of Protistology, 38, 127–145.
Raghukumar, S. (2008). Thraustochytrid marine Protists: production of PUFAs and other emerging technologies. Marine Biotechnology, 10, 631-640.
Raposo, M.F., de Morais, A.M., & de Morais, R.M. (2015). Carotenoids from marine microalgae: A valuable natural source for the prevention of chronic diseases. Marine Drugs, 13(8), 5128–5155.
Ratledge, C., & Wynn, J.P. (2002). The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. Advance in Applied Microbiology, 5, 1-51.
Ratledge, C. (2004). Fatty acid biosynthesis in microorganisms being used for single cell oil production. Biochimie, 86, 807-815.
Sanders, T.A.B., Gleason, K., Griffin, B., & Miller, G.J. (2006). Influence of an algal triacylglycerol containing docosahexaenoic acid (22:6 n-3) and docosapentaenoic acid (22:5 n-6) on cardiovascular risk factors in healthy men and women. British Journal of Nutrition, 95, 525–531.
Takahata, K., Monobe, K., Tada, M., & Weber, P.C. (1998). The benefits and risks of n-3 polyunsaturated fatty acids. Bioscience, Biotechnology, and Biochemistry, 62, 2079–2085.
Trovão, M., Pereira, H., Costa, M., Machado, A., Barros, A., Soares, M., Carvalho, B., Silva, J, T., Varela, J., & Silva, J. (2020). Lab-Scale Optimization of Aurantiochytrium sp. culture medium for improved growth and DHA production. Applied Sciences, 10(7), 2500.
Yokochi, T., Honda, D., Higashihara, T., & Nakahara, T. (1998). Optimization of docosahexaenoic acid production by Schizochytrium limacinum SR21. Applied Microbiology and Biotechnology, 49, 72–76.
Yokoyama, R., & Honda, D. (2007). Taxonomic rearrangement of the genus Schizochytrium sensu lato based on morphology, chemotaxonomic characteristics, and 18srRNA gene phylogeny (Thraustochytriaceae, Labyrinthulomycetes): emendation for Schizochytrium and erection of Aurantiochytrium and Oblongichytrium gen. nov. Mycoscience, 48, 199-211.
Zhou, Y., Han, L., He, H., Sang, B., Yu, D., Feng, J., & Zhang, X. (2018). Effects of agitation, aeration and temperature on production of a novel glycoprotein GP-1 by Streptomyces kanasenisi ZX01 and scale-up based on volumetric oxygen transfer coefficient. Molecules, 23, 125.
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2022-01-10
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