Stoichiometrically balanced nutrient management using a newly designed nutrient medium for large scale cultivation of Cyanobacterium aponinum

  • Meghna Rajvanshi
  • Kshipra Gautam
  • Suvarna Manjre
  • G. Raja Krishna Kumar
  • Chitranshu Kumar
  • Sridharan Govindachary
  • Santanu DasguptaEmail author


Cyanobacteria have tremendous potential to produce bioactive molecules, which makes them a highly lucrative organism for use in industrial applications. In the present study, the first commercial nutrient medium for Cyanobacterium aponinum is developed and a process for large scale cultivation of cyanobacteria is established by combining economical medium, pre-emptive nutrient feeding strategy, and semi-continuous mode (SCM) of cultivation. The parameters measured were growth in terms of OD750 biomass, total nitrogen, nitrate, and phosphorous. Results indicated 13% more biomass yield in urea-phosphoric acid medium (UPA), in comparison to blue-green medium (BG-11). Biomass concentration was 5.3 and 4.7 g L−1 with UPA and BG-11 media, respectively. Urea was found to be a preferred nitrogen source for C. aponinum. Nutrient-dosing studies with UPA medium in SCM of operation resulted in an average daily biomass productivity of ~ 0.44 g L−1 day−1, which is significantly higher than those reported in previous studies. Here, the stoichiometric requirement of nitrogen and phosphorous was found to be 31 mg L−1 and 4.5 mg L−1, respectively. Stoichiometric nutrient addition in SCM resulted in a reduction in nutrient loss in blow down. In addition, the outdoor scale-up studies in flat panel photobioreactors further established the efficacy of UPA medium. Cost analysis of media revealed that UPA medium is 4.4 times less expensive than BG-11 and hence is a suitable and economical medium for large scale cultivation of C. aponinum. Further, this nutrient feeding strategy has wider applications which can be extended to other algal strains and cultivation systems.


Biomass CO2 mitigation Cyanobacterium aponinum Growth medium Photobioreactor Urea Cultivation Biofuel Algae 



We sincerely acknowledge Reliance Industries Limited for providing the laboratory resources. We also appreciate Ajit Sapre (Group President, Research & Technology, Reliance Industries Limited) for his support; Saranya Karuppasamy for strain isolation and purification; Vinod Nagle and Akshay Chawande for maintaining Cyanobacterium aponinum; Badrish Soni for providing strain information; Chaitanya Joshi, Rakhi Dixit, and Ashish Waghmare for technical assistance; Debanjan Sanyal and Nishant Saxena for providing sea water analysis data and Uma Shankar Sagaram, G Venkata Subhash, and Tomal Dattaroy for their critical inputs in refining the manuscript.

Funding information

This study received funding from Reliance Industries Limited.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts.

Supplementary material

10811_2019_1851_MOESM1_ESM.docx (16 kb)
ESM 1 (DOCX 16 kb)


  1. Andersen RA, Berges JA, Harrison PJ, Watanabe MM (2005) Appendix A – Recipes for freshwater and seawater media. In: Andersen RA (ed) Algal culturing techniques. Elsevier, Academic Press, Amsterdam, pp 429–538Google Scholar
  2. Belisle BS, Steffen MM, Pound HL, Watson SB, DeBruyn JM, Bourbonniere RA, Boyer GL, Wilhelm SW (2016) Urea in Lake Erie: organic nutrient sources as potentially important drivers of phytoplankton biomass. J Great Lakes Res 42:599–607CrossRefGoogle Scholar
  3. Bellaloui N, Mengistu A (2015) Effects of boron nutrition and water stress on nitrogen fixation, seed δ15N and δ13C dynamics, and seed composition in soybean cultivars differing in maturities. Sci World J 2015:407872CrossRefGoogle Scholar
  4. Bezerra RP, Montoya EYO, Sato S, Perego P, de Carvalho JCM, Converti A (2011) Effects of light intensity and dilution rate on the semicontinuous cultivation of Arthrospira (Spirulina) platensis. A kinetic Monod-type approach. Bioresour Technol 102:3215–3219CrossRefGoogle Scholar
  5. Bolch CJS, Blackburn SI (1996) Isolation and purification of Australian isolates of the toxic cyanobacterium Microcystis aeruginosa Kütz. J Appl Phycol 8:5–13CrossRefGoogle Scholar
  6. Borowitzka MA (1988) Appendix: Algal growth media and sources of algal cultures. In: Borowitzka MA, Borowitzka LJ (eds) Micro-algal biotechnology. Cambridge University Press, Cambridge, pp 456–465Google Scholar
  7. Chaiklahan R, Nattayaporn C, Wipawan S, Kalyanee P, Boosya B (2010) Cultivation of Spirulina platensis using pig wastewater in a semi-continuous process. J Microbiol Biotechnol 20:609–614CrossRefGoogle Scholar
  8. Chisti Y (2008) Biodiesel from microalgae beats bioethanol. Trends Biotechnol 26:126–131CrossRefGoogle Scholar
  9. Collos Y, Mornet F, Sciandra A, Waser N, Larson A, Harrison PJ (1999) An optical method for the rapid measurement of micromolar concentrations of nitrate in marine phytoplankton cultures. J Appl Phycol 11:179–184CrossRefGoogle Scholar
  10. Danesi EDG, de O. Rangel-Yagui C, de Carvalho JCM, Sato S (2002) An investigation of effect of replacing nitrate by urea in the growth and production of chlorophyll by Spirulina platensis. Biomass Bioenergy 23:261–269CrossRefGoogle Scholar
  11. De Philippis R, Vincenzini M (1998) Exocellular polysaccharides from cyanobacteria and their possible applications. FEMS Microbiol Rev 22:151–175CrossRefGoogle Scholar
  12. De Philippis R, Sili C, Paperi R, Vincenzini M (2001) Exopolysaccharide-producing cyanobacteria and their possible exploitation: a review. J Appl Phycol 13:293–299CrossRefGoogle Scholar
  13. Donald DB, Bogard MJ, Finlay K, Leavitt PR (2011) Comparative effects of urea, ammonium, and nitrate on phytoplankton abundance, community composition, and toxicity in hypereutrophic freshwaters. Limnol Oceanogr 56:2161–2175CrossRefGoogle Scholar
  14. Erratt KJ (2017) Urea as an effective nitrogen source for Cyanobacteria. MSc Thesis, The University of Western Ontario, Canada 89 ppGoogle Scholar
  15. Erratt KJ, Creed IF, Trick CG (2018) Comparative effects of ammonium, nitrate and urea on growth and photosynthetic efficiency of three bloom-forming cyanobacteria. Freshw Biol 63:626–638CrossRefGoogle Scholar
  16. Flores E, Herrero A (2005) Nitrogen assimilation and nitrogen control in cyanobacteria. Biochem Soc Trans 33:164–167CrossRefGoogle Scholar
  17. Garcia-Pichel F, López-Cortés A, Nübel U (2001) Phylogenetic and morphological diversity of cyanobacteria in soil desert crusts from the Colorado plateau. Appl Environ Microbiol 67:1902–1910CrossRefGoogle Scholar
  18. Glibert PM, Heil CA (2006) Escalating worldwide use of urea – a global change contributing to coastal eutrophication. Biogeochem 77:441–463CrossRefGoogle Scholar
  19. Gris B, Sforza E, Morosinotto T, Bertucco A, La Rocca N (2017) Influence of light and temperature on growth and high-value molecules productivity from Cyanobacterium aponinum. J Appl Phycol 29:1781–1790CrossRefGoogle Scholar
  20. Grobbelaar JU (2012) Microalgae mass culture: the constraints of scaling-up. J Appl Phycol 24:315–318CrossRefGoogle Scholar
  21. Gudmundsdottir AB, Omarsdottir S, Brynjolfsdottir A, Paulsen BS, Olafsdottir ES, Freysdottir J (2015) Exopolysaccharides from Cyanobacterium aponinum from the Blue Lagoon in Iceland increase IL-10 secretion by human dendritic cells and their ability to reduce the IL-17+RORγt+/IL-10+FoxP3+ ratio in CD4+ T cells. Immunol Lett 163:157–162CrossRefGoogle Scholar
  22. Herrero A, Muro-Pastor AM, Flores E (2001) Nitrogen control in cyanobacteria. J Bacteriol 183:411–425CrossRefGoogle Scholar
  23. Johnson TJ, Jahandideh A, Isaac IC, Baldwin EL, Muthukumarappan K, Zhou R, Gibbons WR (2017) Determining the optimal nitrogen source for large-scale cultivation of filamentous cyanobacteria. J Appl Phycol 29:1–13CrossRefGoogle Scholar
  24. Lau N-S, Matsui M, Abdullah AA-A (2015) Cyanobacteria: photoautotrophic microbial factories for the sustainable synthesis of industrial products. Biomed Res Int 2015:1–9CrossRefGoogle Scholar
  25. Li J, Zhang J, Huang W, Kong F, Li Y, Xi M, Zheng Z (2016) Comparative bioavailability of ammonium, nitrate, nitrite and urea to typically harmful cyanobacterium Microcystis aeruginosa. Mar Pollut Bull 110:93–98CrossRefGoogle Scholar
  26. María A, Guzmán-Murillo MA, López-Bolaños CC, Ledesma-Verdejo T, Roldan-Libenson G, Cadena-Roa MA, Ascencio F (2007) Effects of fertilizer-based culture media on the production of exocellular polysaccharides and cellular superoxide dismutase by Phaeodactylum tricornutum (Bohlin). J Appl Phycol 19:33–41CrossRefGoogle Scholar
  27. Melis A (2009) Solar energy conversion efficiencies in photosynthesis: minimizing the chlorophyll antennae to maximize efficiency. Plant Sci 177:272–280CrossRefGoogle Scholar
  28. Meng F, Cui H, Wang Y, Li X (2018) Responses of a new isolated Cyanobacterium aponinum strain to temperature, pH, CO2 and light quality. J Appl Phycol 30:1525–1532CrossRefGoogle Scholar
  29. Modiri S, Sharafi H, Alidoust L, Hajfarajollah H, Haghighi O, Azarivand A, Zamanzadeh Z, Zahiri HS, Vali H, Noghabi KA (2015) Lipid production and mixotrophic growth features of cyanobacterial strains isolated from various aquatic sites. Microbiol 161:662–673CrossRefGoogle Scholar
  30. Moreno J, Vargas MÁ, Rodrix g H, Rivas J, Guerrero MG (2003) Outdoor cultivation of a nitrogen-fixing marine cyanobacterium, Anabaena sp. ATCC 33047. Biomol Eng 20:191–197CrossRefGoogle Scholar
  31. Mourelle M, Gómez C, Legido J (2017) The potential use of marine microalgae and cyanobacteria in cosmetics and thalassotherapy. Cosmetics 4:46–59CrossRefGoogle Scholar
  32. Probir D, Abdul QM, Kiran CA, Ibrahim TM, Shoyeb K, Ghamza A, Hareb A-J (2018) Outdoor continuous cultivation of self-settling marine cyanobacterium Chroococcidiopsis sp. Ind Biotechnol 14:45–53CrossRefGoogle Scholar
  33. Reichert CC, Reinehr CO, Costa JAV (2006) Semicontinuous cultivation of the cyanobacterium Spirulina platensis in a closed photobioreactor. Braz J Chem Eng 23:23–28CrossRefGoogle Scholar
  34. Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111:1–61Google Scholar
  35. Sakamoto T, Inoue-Sakamoto K, Bryant DA (1999) A novel nitrate/nitrite permease in the marine cyanobacterium Synechococcus sp strain PCC 7002. J Bacteriol 181:7363–7372Google Scholar
  36. Sayre R (2010) Microalgae: the potential for carbon capture. Bioscience 60:722–727CrossRefGoogle Scholar
  37. Scherholz ML, Curtis WR (2013) Achieving pH control in microalgal cultures through fed-batch addition of stoichiometrically-balanced growth media. BMC Biotechnol 13:39–54CrossRefGoogle Scholar
  38. Schuurmans RM, Schuurmans JM, Bekker M, Kromkamp JC, Matthijs HCP, Hellingwerf KJ (2014) The redox potential of the plastoquinone pool of the cyanobacterium Synechocystis species strain PCC6803 is under strict homeostatic control. Plant Physiol 165:463–475CrossRefGoogle Scholar
  39. Setyoningrum Tutik M, Nur MMA (2015) Optimization of C-phycocyanin production from S. platensis cultivated on mixotrophic condition by using response surface methodology. Biocatal Agric Biotechnol 4:603–607CrossRefGoogle Scholar
  40. Sheehan J, Dunahay T, Benemann J, Roessler P (1998) Look back at the U.S. Department of Energy’s aquatic species program: biodiesel from algae – laboratory NRE, Golden, Colorado. NREL/TP-580-24190 pp 1-328Google Scholar
  41. Singh S, Kate BN, Banerjee UC (2005) Bioactive compounds from cyanobacteria and microalgae: an overview. Crit Rev Biotechnol 25:73–95CrossRefGoogle Scholar
  42. Singh JS, Kumar A, Rai AN, Singh DP (2016) Cyanobacteria: a precious bio-resource in agriculture, ecosystem, and environmental sustainability. Front Microbiol 7:1–19Google Scholar
  43. Velu C, Cirés S, Alvarez-Roa C, Heimann K (2015) First outdoor cultivation of the N2-fixing cyanobacterium Tolypothrix sp. in low-cost suspension and biofilm systems in tropical Australia. J Appl Phycol 27:1743–1753CrossRefGoogle Scholar
  44. Walker DA (2009) Biofuels, facts, fantasy, and feasibility. J Appl Phycol 21:509–517CrossRefGoogle Scholar
  45. Wang Q-l, Liu Y-d, Shen Y-w, Jin C-y, Lu J-s, Zhu J-m, Li S-h (1991) Studies on mixed mass cultivation of Anabaena spp. (nitrogen-fixing blue-green algae, cyanobacteria) on a large scale. Bioresour Technol 38:221–228CrossRefGoogle Scholar
  46. Wang Q, Li H, Post AF (2000) Nitrate assimilation genes of the marine diazotrophic, filamentous cyanobacterium Trichodesmium sp. strain WH9601. J Bacteriol 182:1764–1767CrossRefGoogle Scholar
  47. Weyer KM, Bush DR, Darzins A, Willson BD (2010) Theoretical maximum algal oil production. Bioenergy Res 3:204–213CrossRefGoogle Scholar
  48. Winckelmann D, Bleeke F, Bergmann P, Klöck G (2015) Growth of Cyanobacterium aponinum influenced by increasing salt concentrations and temperature. Biotech 5:253–260Google Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Meghna Rajvanshi
    • 1
  • Kshipra Gautam
    • 1
  • Suvarna Manjre
    • 1
  • G. Raja Krishna Kumar
    • 1
  • Chitranshu Kumar
    • 1
  • Sridharan Govindachary
    • 1
  • Santanu Dasgupta
    • 1
    Email author
  1. 1.Reliance Technology Group, Reliance Industries LimitedReliance Corporate ParkNavi MumbaiIndia

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