Commenced in January 2007
Frequency: Monthly
Edition: International
Paper Count: 30011
Statistical Optimization of Medium Components for Biomass Production of Chlorella pyrenoidosa under Autotrophic Conditions and Evaluation of Its Biochemical Composition under Stress Conditions

Authors: N. P. Dhull, K. Gupta, R. Soni, D. K. Rahi, S. K. Soni


The aim of the present work was to statistically design an autotrophic medium for maximum biomass production by Chlorella pyrenoidosa using response surface methodology. After evaluating one factor at a time approach, K2HPO4, KNO3, MgSO4.7H2O and NaHCO3 were preferred over the other components of the fog’s medium as most critical autotrophic medium components. The study showed that the maximum biomass yield was achieved while the concentrations of MgSO4.7H2O, K2HPO4, KNO3 and NaHCO3 were 0.409 g/L, 0.24 g/L, 1.033 g/L, and 3.265 g/L, respectively. The study reported that the biomass productivity of C. pyrenoidosa improved from 0.14 g/L in defined fog’s medium to 1.40 g/L in modified fog’s medium resulting 10 fold increase. The biochemical composition biosynthesis of C. pyrenoidosa was altered using nitrogen limiting stress bringing about 5.23 fold increase in lipid content than control (cell without stress), as analyzed by FTIR integration method.

Keywords: Autotrophic condition, Chlorella pyrenoidosa, FTIR, Response Surface Methodology, Optimization.

Digital Object Identifier (DOI):

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF


[1] D. Klein-Marcuschamer, Y. Chisti, J. R. Benemann, and D. Lewis, “A matter of detail: assessing the true potential of microalgal biofuels,” Biotechnol. Bioeng., vol. 110, pp. 2317–2322, 2013.
[2] O. Pulz, K. Scheibenbogen, and W. Gross, “Biotechnology with cyanobacteria and microalgae,” In Biotechnology, H.-J. Rehm, G. Reed, Ed. vol. 10, VCH: Weinheim, 2001, pp. 105–136.
[3] K. Skjanes, C. Rebours, and P. Lindblad, “Potential for green microalgae to produce hydrogen, pharmaceuticals and other high value products in a combined process,” Crit. Rev. Biotechnol., vol. 1, pp. 44, 2012.
[4] T. M. Mata, A. A. Martins, and N. S. Caetano, “Microalgae for biodiesel production and other applications: a review,” Renew. Sustain. Energy Rev., vol. 14, pp. 217–232, 2010.
[5] A. F. Clarens, E. P. Resurreccion, M. A. White, and L. M. Colosi, “Environmental life cycle comparison of algae to other bioenergy feedstocks,” Environ. Sci. Technol., vol. 44, pp. 1813–1819, 2010.
[6] N. H. Norsker, M. J. Barbosa, M. H. Vermue, and R. H. Wijffels, “Microalgal production - a close look at the economics,” Biotechnol. Adv., vol. 29, pp. 24–27, 2011.
[7] L. F. Razon, and R. R. Tan, “Net energy analysis of the production of biodiesel and biogas from the microalgae: Haematococcus pluvialis and Nannochloropsis,” Appl. Energy, vol. 88, pp. 3507–3514, 2011.
[8] K. Soratana, and A. E. Landis, “Evaluating industrial symbiosis and algae cultivation from a life cycle perspective,” Bioresour. Technol., vol. 102, pp. 6892–6901, 2011.
[9] L. Campenni, B. P. Nobre, C.A. Santos, A. C. Oliveira, M. R. Aires- Barros, and A. M. F. Palavra, “Carotenoid and lipid production by the autotrophicmicroalga Chlorella protothecoides under nutritional, salinity, and luminosity stress conditions,” Appl. Microbiol. Biotechnol., vol. 97, pp. 1383–1393, 2013.
[10] M. A. Carriquiry, X. Du, and G. R. Timilsina, “Second generation biofuels: economics and policies,” Energy Policy, vol. 39, pp. 4222– 4234, 2011.
[11] B. P. Nobre, F. Villalobos, B. E. Barragán, A. C. Oliveira, A. P. Batista, P. A. S. S. Marques, R. L. Mendes, H. Sovová, A. F. Palavra, and L. Gouveia, “A biorefinery from Nannochloropsis sp. microalga - extraction of oils and pigments. Production of biohydrogen from the leftover biomass,” Bioresour. Technol., vol. 135, pp. 128–136. 2013.
[12] A. Singh, P. S. Nigam, and J. D. Murphy, “Mechanism and challenges in commercialisation of algal biofuels,” Bioresour. Technol., vol. 102, pp. 26–34, 2011a.
[13] D. Yasar, “Vitamin E, (α-tocopherol) production by the marine microalgae Nannochloropsis oculata (Eustigmatophyceae) in nitrogen limitation,” Aquaculture, vol. 272, pp. 717–722, 2007.
[14] F. Chen, and Y. Zhang, “High cell density mixotrophic culture of Spirulina platensis on glucose for phycocyanin production using a fedbatch system,” Enzyme Microb. Technol, vol. 20, pp. 221–224,1997.
[15] H. Yu, S. Jia, and Y. Dai, “Growth characteristics of the cyanobacterium Nostoc flagelliforme in photoautotrophic, mixotrophic and heterotrophic cultivation,” J. Appl. Phycol., vol. 21, pp. 127–133, 2009.
[16] R. Gladue, and J. Maxey, “Microalgal feeds for aquaculture,” J. Appl. Phycol., vol., 6, pp. 131-141, 1994.
[17] J. Berges, D. Franklin, and P. Harrison, “Evolution of an artificial seawater medium: improvements in enriched seawater, artificial water over the last two decades,” J. Phycol., vol., 37, pp. 1138–1145, 2001.
[18] G. Markou, and E. Nerantzis, “Microalgae for high-value compounds and biofuels production: A review with focus on cultivation under stress conditions,” Biotechnol. Adv., vol. 31, pp.1532-1542, 2013.
[19] W. Q. Guo, N. Q. Ren, X. J. Wang, W. S. Xiang, J. Ding, and Y. You, “Optimization of culture conditions for hydrogen production by Ethanoligenens harbinense B49 using response surface methodology,” Bioresour. Technol., vol. 100, pp. 1192–1196, 2009.
[20] Y. Li, F. Cui, Z. Liu, Y. Xu, and H. Zhao, “Improvement of xylanase production by Penicillium oxalicum ZH-30 using response surface methodology,” Enzyme Microb. Technol., vol. 40, pp. 1381–1388, 2007.
[21] G. Q. Liu, and X. L. Wang, “Optimization of critical medium components using response surface methodology for biomass and extracellular polysaccharide production by Agaricus blazei,” Appl. Microbiol. Biotechnol., vol. 74, pp. 78–83, 2007.
[22] M. S. Tanyildizi, D. Ozer, and M. Elibol M, “Optimization of -amylase production by Bacillus sp. using response surface methodology,” Process Biochem., vol. 40, pp. 2291–2296, 2005.
[23] T. Heredia-Arroyo, W. Wei, R. Ruan, and B. Hu, “Mixotrophic cultivation of Chlorella vulgaris and its potential application for the oil accumulation from non-sugar materials,” Biomass Bioenergy, vol. 35, pp. 2245–53, 2011.
[24] F. Y. Feng, W. Yang, G. Z. Jiang, Y. N. Xu, and T. Y. Kuang, “Enhancement of fatty acid production of Chlorella sp. (Chlorophyceae) by addition of glucose and sodium thiosulphate to culture medium,” Process Biochem., vol. 40, pp. 1315–1318, 2005.
[25] A. Bhatnagar, S. Chinnasamy, M. Singh, and K. C. Das, “Renewable biomass production by mixotrophic algae in the presence of various carbon sources and waste-waters,” Appl. Energy, vol. 88, pp. 3425– 3431, 2011.
[26] M. Azma, M. S. Mohamed, R. Mohamad, R. A. Rahim, and A. B. Ariff, “Improvement of medium composition for heterotrophic cultivation of green microalgae, Tetraselmis suecica, using response surface methodology,” Biochemical Eng. J., vol.53, pp.187-195, 2010.
[27] Y. Cheng, C. Lu, and W. Q. Gao, “Algae-based biodiesel production and optimization using sugar cane as the feedstock,” Energy Fuels, vol. 23, pp. 4166–4173, 2009.
[28] W. B. Kong, S. F. Hua, H. Cao, Y. W. Mu, H. Yang, H. Song, and C. G. Xia, “Optimization of mixotrophic medium components for biomass production and biochemical composition biosynthesis by Chlorella vulgaris using response surface methodology,” J. Taiwan Int. Chem. Eng., vol. 43, pp. 360-367, 2011.
[29] Z. Li, H. Yuan, J. Yang, and B. Li, “Optimization of the biomass production of oil algae Chlorella minutissima UTEX2341,” Bioresour. Technol., vol. 102, pp. 9128–9134, 2011.
[30] B. Ryu, K. H. Kanfg, D. H. Ngo, Z. J, Qian, and S. K. Kim, “Statistical optimization of microalgae Pavlova Lutheri cultivation conditions and its fermentation conditions by yeast, Candida Rugopelliculosa,” Bioresour. Technol., vol. 107, pp. 307-313, 2011.
[31] T. Xie, Y. Sun, K. Du, B. L, R. cheng, and Y. Zhang, “Optimization of heterotrophic cultivation of Chlorella sp. For oil production,” Bioresour. Technol., vol. 118, pp. 235-242, 2012.
[32] P. Bondioli, L. Della Bella, G. Rivolta, G. Chini Zittelli, N. Bassi, L. Rodolfi, D. Casini, M. Prussi, D. Chiaramonti, and M. R. Tredici, “Oil production by the marine microalgae Nannochloropsis sp. F&M-M24 and Tetraselmis suecica F&M-M33,” Bioresour. Technol., vol. 114, pp. 567–572, 2012.
[33] S. Go, S. J. Lee, G. T. Jeong, and S. K. Kim, “Factors affecting the growth and the oil accumulation of marine microalgae Tetraselmis suecica,” Bioprocess Biosyst. Eng., vol. 35, no. 1–2, pp. 145–150, 2012.
[34] C. H. Su, L. J. Chien, J. Gomes, Y. S. Lin, Y. K. Yu, J. S. Liou, and R. J. Syu, “Factors affecting lipid accumulation by Nannochloropsis oculata in a two-stage cultivation process,” J. Appl. Phycol., vol. 23, no. 5, pp. 903–908, 2011.
[35] J. N. Murdock, D. L. Wetzel, “FTIR microspectroscopy enhances biological and ecological analysis of algae,” Appl. Spectrosc. Rev., vol. 44, pp. 335-361, 2009.
[36] A. M. A. Pistorius, W. J. DeGrip, and T. A. Egorova-Zachernyuk, “Monitoring of biomass composition from microbiological sources by means of FT-IR spectroscopy,” Biotechnol. Bioeng., vol. 103, no. 1, pp. 123-129, 2009.
[37] M. Giordano, M. Kansiz, P. Heraud, J. Beardall,B. Wood, and D. McNaughton, “Fourier transform infrared spectroscopy as a novel tool to investigate changes in intracellular macromolecular pools in the marine microalga Chaetoceros muellerii (Bacillariophyceae),” J. Phycol., vol. 37, pp. 271–279, 2001.
[38] P. Heraud, B. R. Wood, M. J. Tobin, J. Beardall, and D. McNaughton, “Mapping of nutrient-induced biochemical changes in living algal cells using synchrotron infrared microspectroscopy,” FEMS Microbiol. Lett., vol. 249, pp. 219–225, 2005.
[39] K. Stehfest, J. Toepel, and C. Wilhelm, “The application of micro-FTIR spectroscopy to analyze nutrient stress-related changes in biomass composition of phytoplankton algae,” Plant Physiol. Biochem., vol. 43, pp. 717–726, 2005.
[40] A. P. Dean, J. M. Nicholson, and D. C. Sigee, “Impact of phosphorus quota and growth phase on carbon allocation in Chlamydomonas reinhardtii: an FTIR microspectroscopy study,” Eur. J. Phycol., vol. 43, pp. 345–354, 2008.
[41] D. C. Sigee, F. Bahram, B. Estrada, R. E. Webster, and A. P. Dean, “The influence of phosphorus availability on carbon allocation and P quota in Scenedesmus subspicatus: a synchrotron-based FTIR analysis,” Phycologia, vol. 46, pp. 583–592, 2007.
[42] G. E. Fogg, “Algal Culture and Phytoplankton Ecology,” The University of Wisconsin Press, Wisconsin, 1975.
[43] L. Huiping, Z. Guoqun, N. Shanting, and L. Yiguo, “Technologic parameter optimization of gas quenching process using response surface method,” Comput. Mater. Sci., vol. 38, no. 3, pp. 561–570, 2007.
[44] J. Segurola, N. S. Allen, M. Edge, and A. M. Mahon, “Design of eutectic photo initiator blends for UV/curable acrylated printing inks and coatings,” Prog. Org. Coat, vol. 37, no. 1, pp. 23–37, 1999.
[45] M. Muthukumar, D. Mohan, and M. Rajendran, “Optimization of mix proportions of mineral aggregates using Box Behnken design of experiments,” Cem. Concr. Compos., vol. 25, no. 7, pp. 751–758, 2003.
[46] H. L. Liu, Y. W. Lan, and Y. C. Heng, “Optimal production of sulphuric acid by Thiobacillus thiooxidans using response surface methodology,” Process Biochem., vol. 39, no. 12, pp. 1953–1961, 2004.
[47] C. H. Hsieh, and W. T. Wu, “Cultivation of microalgae for oil production with a cultivation strategy of urea limitation,” Bioresour. Technol., vol. 100, pp. 3921-3926, 2009.
[48] Z. Y. Liu, G. C. Wang, and B. C. Zhou, “Effect of iron on growth and lipid lipid accumulation in Chlorella vulgaris,” Bioresour. Technol., vol. 99, pp. 4717-4722, 2008.
[49] V. Ordog, W. A. Stirk, P. Bálint,J. Van Staden, and C. Lovasz, “Changes in lipid, protein and pigment concentrations in nitrogenstressed Chlorella minutissima cultures,” J. Appl. Phycol., vol. 24, pp. 907–914, 2012.
[50] M. J. Behrenfeld, K. Worthington, R. M. Sherrell, F. P. Chavez, P. Strutton, M. McPhaden, and D. M. Shea, “Controls on tropical Pacific Ocean productivity revealed through nutrient stress diagnostics,” Nature, vol. 442, pp. 1025–1028, 2006.
[51] T. Jakob, H. Wagner, K. Stehfest, and C. Wilhelm, “A complete energy balance from photons to new biomass reveals a light- and nutrientdependent variability in the metabolic costs of carbon assimilation,” J. Exp. Bot., vol. 58, no. 8, pp. 2101–2112, 2007.
[52] A. P. Dean, D. C. Sigee, B. Estrada, and J. K. Pittman, “Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae,” Bioresour. Technol., vol. 101, pp. 4499–4507, 2010.
[53] C. T. Evans, A. H. Scragg, and C. Ratledge, “Reguladtion of Citrate Efflux from Mitochondria Oleaginou and Non‐Oleaginous Yeasts by Adenine Nucleotides,” Eur. J. Biochem., vol. 132, no. 3, pp. 609-615, 1983.
[54] M. Siaut, S. Cuine, C. Cagnon, B. Fessler, M. Nguyen, P. Carrier, A. Beyly, F. Beisson, C. Triantaphylides, Y. H. Li-Beisson, and G. Peltier, “Oil accumulation in the model green alga Chlamydomonas reinhardtii: characterization, variability between common laboratory strains and relationship with starch reserves,” BMC Biotechnol., vol. 11, pp. 7-21, 2011.
[55] N. M. D. Courchesne, A. Parisien, B. Wang, and C. Q. Lan, “Enhancement of lipid production using biochemical, genetic and transcription factor engineering approaches,” J. Biotechnol., vol.141, pp. 31–41, 2009.