Journal of Applied Phycology

, Volume 19, Issue 5, pp 417–423 | Cite as

Removal of nitrogen and phosphorus from wastewater using microalgae immobilized on twin layers: an experimental study

  • Jing Shi
  • Björn PodolaEmail author
  • Michael Melkonian


Removal of nitrogen and phosphorus from wastewater by two green microalgae (Chlorella vulgaris and Scenedesmus rubescens) was investigated using a novel method of algal cell immobilization, the twin-layer system. In the twin-layer system, microalgae are immobilized by self-adhesion on a wet, microporous, ultrathin substrate (the substrate layer). Subtending the substrate layer, a second layer, consisting of a macroporous fibrous tissue (the source layer), provides the growth medium. Twin-layers effectively separate microalgae from the bulk of their growth medium, yet allow diffusion of nutrients. In the twin-layer system, algae remain 100% immobilized, which compares favourably with gel entrapment methods for cell immobilization. Both microalgae removed nitrate efficiently from municipal wastewater. Using secondary, synthetic wastewater, the two algae also removed phosphate, ammonium and nitrate to less than 10% of their initial concentration within 9 days. It is concluded that immobilization of C. vulgaris and S. rubescens on twin-layers is an effective means to reduce nitrogen and phosphorus levels in wastewater.

Key words

ammonium Chlorella vulgaris nitrate nutrient removal phosphate Scenedesmus rubescens twin-layer wastewater treatment 


  1. APHA, AWWA, WEF (1998) Standard methods for the examination of water and wastewater. United Book Press, BaltimoreGoogle Scholar
  2. CEN (1996) Water quality determination of phosphorus: ammonium molybdate spectrometric method. European Committee for Standardization, BrusselsGoogle Scholar
  3. Chevalier P, de la Noüe J (1985) Wastewater nutrient removal with microalgae immobilized in carrageenan. Enzyme Microb Technol 7:621–624CrossRefGoogle Scholar
  4. Garbisu C, Gil JM, Bazin MJ, Hall DO, Serra JL (1991) Removal of nitrate from water by foam-immobilized Phormidium laminosum in batch and continuous-flow bioreactors. J Appl Phycol 3:221–234CrossRefGoogle Scholar
  5. Gil JM, Serra JL (1993) Nitrate removal by immobilized cells of Phormidium uncinatum in batch culture and a continuous-flow photobioreactor. Appl Microbiol Biotechnol 39:782–787CrossRefGoogle Scholar
  6. González LE, Bashan Y (2000) Increased growth of the microalga Chlorella vulgaris when coimmobilized and cocultured in alginate beads with the plant-growth-promoting bacterium Azospirillum brasilense. Appl Environ Microbiol 66:1527–1531PubMedCrossRefGoogle Scholar
  7. González LE, Cañizares RO, Baena S (1997) Efficiency of ammonia and phosphorus removal from a Colombian agroindustrial wastewater by the microalgae Chlorella vulgaris and Scenedesmus dimorphus. Bioresour Technol 60:259–262CrossRefGoogle Scholar
  8. Hernandez JP, de-Bashan LE, Bashan Y (2006) Starvation enhances phosphorus removal from wastewater by the microalga Chlorella spp. co-immobilized with Azospirillum brasilense. Enzyme Microb Technol 38:190–198CrossRefGoogle Scholar
  9. Hiscox JD, Israelstam GF (1979) Method for the extraction of chlorophyll from leaf tissue without maceration. Can J Bot 57:1332–1334Google Scholar
  10. Hoffmann JP (1998) Wastewater treatment with suspended and nonsuspended algae. J Phycol 34:757–763CrossRefGoogle Scholar
  11. Hyenstrand P, Burkert U, Pettersson A, Blomqvist P (2000) Competition between the green alga Scenedesmus and the cyanobacterium Synechococcus under different modes of inorganic nitrogen supply. Hydrobiologia 435:91–98CrossRefGoogle Scholar
  12. Jeffrey SW, Humphrey GF (1975) New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton. Biochem Physiol Pflanzen 167:191–194Google Scholar
  13. Lau PS, Tam NFY, Wong YS (1997) Wastewater nutrients (N and P) removal by carrageenan and alginate immobilized Chlorella vulgaris. Environ Technol 18:945–951CrossRefGoogle Scholar
  14. Lau PS, Tam NFY, Wong YS (1998) Operational optimization of batchwise nutrient removal from wastewater by carrageenan immobilized Chlorella vulgaris. Water Sci Technol 38:185–192CrossRefGoogle Scholar
  15. Mallick N (2002) Biotechnological potential of immobilized algae for wastewater N, P and metal removal: a review. Biometals 15:377–390PubMedCrossRefGoogle Scholar
  16. Martínez ME, Sánchez S, Jiménez JM, El Yousfi F, Muñoz L (2000) Nitrogen and phosphorus removal from urban wastewater by the microalga Scenedesmus obliquus. Bioresour Technol 73:263–272CrossRefGoogle Scholar
  17. McFadden GI, Melkonian M (1986) Use of Hepes buffer for microalgal culture media and fixation for electron microscopy. Phycologia 25:551–557Google Scholar
  18. Mehta SK, Gaur JP (2005) Use of algae for removing heavy metal ions from wastewater: progress and prospects. CRC Rev Biotechnol 25:113–152CrossRefGoogle Scholar
  19. Mulbry W, Westhead EK, Pizarro C, Sikora L (2005) Recycling of manure nutrients: use of algal biomass from dairy manure treatment as a slow release fertilizer. Bioresour Technol 96:451–458PubMedCrossRefGoogle Scholar
  20. de la Noüe J, Laliberté G, Proulx D (1992) Algae and waste water. J Appl Phycol 4:247–254CrossRefGoogle Scholar
  21. Nowack ECM, Podola B, Melkonian M (2005) The 96-well twin-layer system: a novel approach in the cultivation of microalgae. Protist 156:239–251PubMedCrossRefGoogle Scholar
  22. Nuñez VJ, Voltolina D, Nieves M, Piña P, Medina A, Guerrero M (2001) Nitrogen budget in Scenedesmus obliquus cultures with artificial wastewater. Bioresour Technol 78:161–164PubMedCrossRefGoogle Scholar
  23. Olguín EJ (2003) Phycoremediation: key issues for cost-effective nutrient removal processes. Biotechnol Adv 22:81–91PubMedCrossRefGoogle Scholar
  24. O’Reilly AM, Scott JA (1995) Defined coimmobilization of mixed microorganism cultures. Enzyme Microb Technol 17:636–646CrossRefGoogle Scholar
  25. Oswald WJ (1988) Microalgae and waste water treatment. In: Borowitzka MA, Borowitzka LJ (eds) Microalgal biotechnology. Cambridge University Press, Cambridge, pp 691–707Google Scholar
  26. Oswald WJ, Gotaas HB, Golueke CG, Kellen WR (1957) Algae in waste treatment. Sewage Ind Wastes 29:437–455Google Scholar
  27. Podola B, Melkonian M (2003) A long-term operating algal biosensor for the rapid detection of volatile toxic compounds. J Appl Phycol 15:415–424CrossRefGoogle Scholar
  28. Stanier RY, Kunisawa R, Mandel M, Cohen-Bazire G (1971) Purification and properties of unicellular blue-green algae (Order Chroococcales). Bacteriol Rev 35:171–205PubMedGoogle Scholar
  29. Syrett PJ (1981) Nitrogen metabolism of microalgae. Can Bull Fish Aquat Sci 210:182–210Google Scholar
  30. Tam NFY, Lau PS, Wong YS (1994) Wastewater inorganic N removal and P removal by immobilized Chlorella vulgaris. Water Sci Technol 30:369–374Google Scholar
  31. Travieso L, Benitez F, Weiland P, Sánchez E, Dupeyrón R, Dominguez AR (1996) Experiments on immobilization of microalgae for nutrient removal in wastewater treatments. Bioresour Technol 55:181–186CrossRefGoogle Scholar
  32. Urrutia I, Serra JL, Llama MJ (1995) Nitrate removal from water by Scenedesmus obliquus immobilized in polymeric foams. Enzyme Microb Technol 17:200–205CrossRefGoogle Scholar
  33. USEPA (1979) Methods for chemical analysis of water and wastes. U.S. Environmental Protection Agency, Cincinnati, OHGoogle Scholar
  34. Voltolina D, Gómez-Villa H, Correa G (2005) Nitrogen removal and recycling by Scenedesmus obliquus in semicontinuous cultures using artificial wastewater and a simulated light and temperature cycle. Bioresour Technol 96:359–362PubMedCrossRefGoogle Scholar
  35. Wang Y, Huang GL (2005) Effect of illumination of nitrate and phosphate removal by coimmobilized Chlorella pyrenoidosa and activated sludge. Artif Cells Blood Substit Immobil Biotechnol 33:357–369PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2007

Authors and Affiliations

  1. 1.Botanisches InstitutUniversität zu Köln, Lehrstuhl 1KölnGermany

Personalised recommendations