Optimization of Batch Cultivation of Chlorella sp. Using Response Surface Methodology

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Sandra E. Ezeani
Gideon O. Abu


Optimal biomass production from microalgae using the NPK 20:20:20 medium; a relatively cheaper and locally available medium has been identified as an important factor in the large-scale algal biomass production. In this study, various concentrations (0.3-0.7 g/l) of NPK 20:20:20 were considered as the source of nitrogen in the growth medium for Chlorella sp. Four independent parameters in algae culture (nitrogen concentration, pH, inoculum size and duration of the experiment at varying ranges were studied for maximum biomass and chlorophyll production. Response Surface Methodology (RSM) procedure result that nitrogen concentration and pH level are the dominant factors affecting biomass and chlorophyll production. Maximum biomass was achieved at 0.5 g/l N and 8.5 pH value. Higher N (0.8 g/l) and lower N (0.3 g/l) had minimal effect on biomass and chlorophyll production. There was a linear relationship between chlorophyll and biomass production while the residual nitrogen had an inverse relationship with biomass production. Nitrogen concentration and pH were shown to be limiting factors under the conditions of the study. The inoculum size and duration of the experiment had a minimal effect on biomass production. 

Optimization, batch cultivation, Chlorella sp., optimal biomass production, microalgae.

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How to Cite
Ezeani, S. E., & Abu, G. O. (2019). Optimization of Batch Cultivation of Chlorella sp. Using Response Surface Methodology. Current Journal of Applied Science and Technology, 36(4), 1-14. https://doi.org/10.9734/cjast/2019/v36i430245
Original Research Article


Chisti Y. Biodiesel from nicroalgae. Biotechnol. Adv. 2007;25:294-306.

Al-lwayzy S, Yusaf T and Al-Juboori R. Biofuels from the fresh water microalgae chlorella vulgaris (FWM-CV) for Diesel Engines. Energies. 2014;7:1829-1851.

Sutton M, Skiba U, van Grinsven J, Oenema O, Watson C, Williams J, Hellums D, Maas R, Gyldenkaerne S, Pathak H, Winiwarter W. Green economy thinking and the control of nitrous oxide emissions. Environmental Development. 2014;9,76–85.

Anitha S, Narayanan J. Isolation and identification of microalgal strains and evaluation of their fatty acid profiles for biodiesel production. International Journal of Pharmaceutical & Biological. 2012;3(4):939-944.

Rojan JP, Anisha G, Nampoothiri K, Pandey A. Micro and macroalgal biomass: A renewable source for bioethanol. Bioresource Technology. 2011;102(1):186-193.

Sheehan J, Dunahay T, Benemann J, Roessler P. A look back at the U.S. Department of Energy’s aquatic species program: Biodiesel from algae. National Renewable Energy Laboratory, USA; 1998.

Thurmond W. Algae 2020 study – prospectus; 2010. [Online].
Available: http://www.e m e r g i n g - m a r k e t s . c o m.

Nigam P, Singh A. Production of liquid biofuels from renewable resources. Progress in Energy and Combustion Science. 2011;37(1):52-68.

Liang Y, Sarkany N, Cui Y. Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions. Biotechnology Letters. 2009; 31(7):1043-9.

Prabandono K and Amin S. Biofuel production from microalgae, in Handbook of Marine Microalgae; Biotechnology Advances, London, Academic Press. 1 edition. 2015;145-158.

Vieira Costa J, M Morais M. An open pond system for microalgal cultivation. Biofuels from Algae. 2013;1-22.

Zhu L, Li Z, E Hiltunen E. Strategies for lipid production improvement in microalgae as a biodiesel feedstock. Bio Med Research International. 2016;8.
[Article ID 8792548]

Chen S, Wang L, S Qiu S, GE S. Determination of microalgal lipid content and fatty acid for biofuel production. Bio Med Research International. 2018;1-18.

Sharma J, Kumar S, Bishnoi N, Pugazhendhi A. Enhancement of lipid production from algal biomass through various growth parameters. Journal of Molecular Liquids. 2018;269:712-720.

Chi N, P Duc P, Mathimani T, Pugazhendhi A. Evaluating the potential of green alga Chlorella sp. for high biomass and lipid production in biodiesel viewpoint. Biocatalysis and Agricultural Biotechnology. 2019;17:184-188.

Benemann J, Woertz I, Lundquist T. Autotrophic Microalgae Biomass Production: From niche markets to commodities. Industrial Biotechnology. 2018;14(1).

Sajjadi B, Chen W, Abdul A, Ramanb, Ibrahim S. Microalgae lipid and biomass for biofuel production: A comprehensive review on lipid enhancement strategies and their effects on fatty acid composition. Renewable and Sustainable Energy Reviews. 2018;97:200–232.

Agwa O, Ibe S, Abu G. Economically effective potential of Chlorella sp. for biomass and lipid production. Microbiol. Biotech. Res. 2012;(1):35-45.

Whyte D. Too much of a good thing? Study the effect of fertilizers on algal growth; 2018. [Online].
Available: http:// www.Science Buddies Staff.

AL-Mashhadani M, E Khudhair E. Experimental study for commercial fertilizer NPK (20:20:20+TE N:P: K) in microalgae cultivation at different aeration periods. Iraqi Journal of Chemical and Petroleum Engineering. 2017;18(1)99-110.

Singh G, Sikarwar N. An experimental study: Using plant fertilizer as a potent culture media for chlorella vulgaris. Asian J Pharm Clin Res. 2014;7(1)1-3.

Pittman J, A Dean A, O Osundeko O. The potential of sustainable algal biofuel production using wastewater resources. Bioresource Technol. 2011;102:17–25.

Posadas E, Alcántara C, Gouveia EPAL, Gieysse B, Z Norvill Z, Acién§ F, Markou G, Congestri R, Koreiviene J, R Muñoz R. Microalgae cultivation in wastewater. Woodhead Publishing Series in Energy. 2017;67-91.

Hupfauf B, Süß M, Dumfort A, Fuessl‐Le H. Cultivation of microalgae in municipal wastewater and conversion by hydrothermal carbonization: A review. Chem Bio Eng Reviews. 2016;3(4):186-200.

Shchegolkova N, Shurshin K, S Pogosyan S, Voronova E, D Matorin D, D Karyakin D. Microalgae cultivation for wastewater treatment and biogas production at Moscow wastewater treatment plant. Water Sci Technol. 2018;78(1):69-80.

Sims R, Mabee W, Saddler J, Taylor M. An overview of second generation biofuel technologies. Bioresource Technology. 12010;01:1570–1580.

N Da Silva N, J Garnica J, Batistella C, M Maciel M. Use of experimental design to investigate biodiesel production by multiple-stage ultra shear reactor. Bioresouces Technology. 2010; (102)2672-2677.

Yang F, L Long L, X Sun X, Wu H, Li T, Xiang W. Optimization of medium using response surface methododlogy for Liid production by Scenedesmus sp. Marine Drugs. 2014;12:1245-1257.

Patel A, Suseela M, M Sigh M, Nayaka S. Application of response surface methododlogy for optimization of biomass, carbohydrates and lipid production in BG11 by scenedesmus quadricauda. International Journal of Engineering and Applied Science. 2015;5(6):199-215.

Panjak V, Awasthi M. Optimization and validation of microalgal growth condition by response surface methodology. International Journal of Bio-science and Bio-technology. 2015;(7)199-206.

Anaga A, Abu G. A laboratory-scale cultivation of Chlorella and Spirulina using waste effluent from a fertilizer company in Nigeria. Bioresource Technology. 1996;(58):93-9.

Gopalan R, R Sugumar R, Anand A. A laboratory manual for environmental chemistry. New Delhi: IK International Pvt Ltd; 2008.

Burnison B. Modified Dimethyl Sulfoxide (DMSO) extraction for chlorophyll analysis of phytoplankton. Canadian Journal of Fisheries and Aquatic Sciences. 1980;37(4):729-733.

Emeko H, Olugbogi O, Betiku E. Appraisal of neural network and response surface methodology in modeling and process variable optimization of oxalic acid production from cashew apple juice: A case of surface fermentation. Bioresources. 2015;10(2):2067-208.

Mei X, Wang Z, Miao Y, Wu Z. Recover energy from domestic wastewater using anaerobic membrane bioreactor: Operating parameters optimization and energy balance analysis. Energy. 2016;98:146-154.

María de Lourdes F, R María Dolores Josefina R, Cuauhtémoc Ulises M, Alfredo de Jesús M, Tolerance and nutrients consumption of Chlorella vulgaris growing in mineral medium and real wastewater under laboratory conditions. Open Agriculture. 2017;2:394–400.

Ernst A, M Deicher M, PM Herman PM, Wollenzien UI. Nitrate and phosphate affect cultivability of cyanobacteria from environments with low nutrient levels. Applied and Environmental Microbiology. 2005;71(6):3379–3383.

Chen W, Zhang Q, Dai S. Effects of nitrate on intracellular nitrite and growth of Microcystis aeruginosa. Journal of Applied Phycology. 2009;21(6):701–706.

Gardner R, Peters P, Peyton B, K Cooksey K. Medium pH and nitrate concentration effects on accumulation of triacylglycerol in two members of the chlorophyta. J Appl Phycol. 2011; 23:1005–1016.

Lavens P, Sorgeloos P. Manual on the production and use of live food for aquaculture, Rome: Food and Agriculture Organization of the United Nations; 1996.

Kim J, Lee JY, Lu T. A model for autotrophic growth of Chlorella vulgaris under photolimitation and photoinhibition in cylindrical photobioreactor. Biochemical Engineering Journa. 2015;99:55–60.

Eze VC, Velasquez-Orta SB, Hernández-García A, Monje-Ramírez I, MT Orta-Ledesma MT. Kinetic modelling of microalgae cultivation for wastewater treatment and carbon dioxide sequestration. Algal Research. 2018;(32)131-141.

Da Silveira P, Braz A, Didonet A. Chlorophyll meter to evaluate the necessity of nitrogen in dry beans. Agropecu. Bras. 2003;38:1083-1087.

Soratto R, De Carvalho M, Arf O. Chlorophyll content and grain yield of common bean as affected by nitrogen fertilization. Agropecu. Bras. 2004;39:895-901.

Mencfel R. Relationship among biomass of algae and concentration of chlorophyll a, against the taxonomic structure of phytoplankton in three lakes ON Lublin Polesie Region. Ol pan. 2013;10: 243–249.

Acevedo S, Peñuela GA, Pino N. Biomass production of Scenedesmus sp. and removal of nitrogen and phosphorus in domestic wastewater. Ingenieríay Competitividad. 2017;19(1):185-193.

Abdel-Raouf N, Al-Homaidan AA, Ibraheem IB. Microalgae and wastewater treatment. Saudi Journal of Biological Sciences. 2012;19(3):257–275.