Applied Microbiology and Biotechnology

, Volume 99, Issue 5, pp 2393–2404 | Cite as

Mixotrophic metabolism of Chlorella sorokiniana and algal-bacterial consortia under extended dark-light periods and nutrient starvation

  • Cynthia Alcántara
  • Carolina Fernández
  • Pedro A. García-Encina
  • Raúl Muñoz
Environmental biotechnology


Microalgae harbor a not fully exploited industrial and environmental potential due to their high metabolic plasticity. In this context, a better understanding of the metabolism of microalgae and microalgal-bacterial consortia under stress conditions is essential to optimize any waste-to-value approach for their mass cultivation. This work constitutes a fundamental study of the mixotrophic metabolism under stress conditions of an axenic culture of Chlorella sorokiniana and a microalgal-bacterial consortium using carbon, nitrogen, and phosphorous mass balances. The hydrolysis of glucose into volatile fatty acids (VFA) during dark periods occurred only in microalgal-bacterial cultures and resulted in organic carbon removals in the subsequent illuminated periods higher than in C. sorokiniana cultures, which highlighted the symbiotic role of bacterial metabolism. Acetic acid was preferentially assimilated over glucose and inorganic carbon by C. sorokiniana and by the microalgal-bacterial consortium during light periods. N-NH4 + and P-PO4 −3 removals in the light stages decreased at decreasing duration of the dark stages, which suggested that N and P assimilation in microalgal-bacterial cultures was proportional to the carbon available as VFA to produce new biomass. Unlike microalgal-bacterial cultures, C. sorokiniana released P-PO4 −3 under anaerobic conditions, but this excretion was not related to polyhydroxybutyrate accumulation. Finally, while no changes were observed in the carbohydrate, lipid and protein content during repeated extended dark-light periods, nutrient deprivation boosted both C-acetate and C-glucose assimilation and resulted in significantly high biomass productivities and carbohydrate contents in both C. sorokiniana and the microalgal-bacterial cultures.


Algal-bacterial consortium Bioremediation C. sorokiniana Extended dark-light periods Mass balances Nutrient deprivation 



This research was supported by the regional government of Castilla y León and the European Social Fund (Contract N° E-47-2011-0053564 and Project Ref. GR76). The financial support of the Ministry of Economy and Competitiveness and the National Institute for Agricultural Research and Technology and Food is also gratefully acknowledged (Project Ref. RTA2013-00056-C03-02).

Supplementary material

253_2014_6125_MOESM1_ESM.docx (45 kb)
ESM 1 (docx 45.1 kb)


  1. Abreu AP, Fernandes B, Vicente AA, Teixeira J, Dragone G (2012) Mixotrophic cultivation of Chlorella vulgaris using industrial dairy waste as organic carbon source. Bioresour Technol 118:61–66CrossRefPubMedGoogle Scholar
  2. Alcántara C, García-Encina PA, Muñoz R (2013) Evaluation of mass and energy balances in the integrated microalgae growth-anaerobic digestion process. Chem Eng J 221:238–246CrossRefGoogle Scholar
  3. Arbib Z, Ruiz J, Álvarez-Díaz P, Garrido-Pérez C, Perales JA (2014) Capability of different microalgae species for phytoremediation processes: wastewater tertiary treatment, CO2 bio-fixation and low cost biofuels production. Water Res 49:465–474CrossRefPubMedGoogle Scholar
  4. Asano T, Burton FL, Leverenz HL, Tsuchihashi R, Tchobanoglous G (2002) Metcalf and Eddy, wastewater engineering: treatment and reuse, 4th edn. McGraw-Hill, New YorkGoogle Scholar
  5. Bajekal SS, Dharmadhikari NS (2008) Use of polyphosphate accumulating organisms (PAO) for treatment of phosphate sludge. The 12th World Lake Conference: 918–922Google Scholar
  6. Cade-Menun BJ, Paytan A (2010) Nutrient temperature and light stress alter phosphorus and carbon forms in culture-grown algae. Mar Chem 121:27–36CrossRefGoogle Scholar
  7. Cea-Barcía G, Buitrón G, Moreno G, Kumar G (2014) A cost-effective strategy for the bio-prospecting of mixed microalgae with high carbohydrate content: diversity fluctuations in different growth media. Bioresour Technol 163:370–373CrossRefPubMedGoogle Scholar
  8. Chu FF, Chu PN, Cai PJ, Li WW, Lam PKS, Zeng RJ (2013) Phosphorus plays an important role in enhancing biodiesel productivity of Chlorella vulgaris under nitrogen deficiency. Bioresour Technol 134:341–346CrossRefPubMedGoogle Scholar
  9. De Godos I, Vargas VA, Blanco S, García-González MC, Soto R, García-Encina PA, Becares E, Muñoz R (2010) A comparative evaluation of microalgae for the degradation of piggery wastewater under photosynthetic oxygenation. Bioresour Technol 101:5150–5158CrossRefPubMedGoogle Scholar
  10. De Philippis R, Ena A, Guastiini M, Sili C, Vincenzini M (1992) Factors affecting poly-β-hydroxybutyrate accumulation in cyanobacteria and in purple non-sulfur bacteria. FEMS Microbiol Lett 103(2–4):187–194Google Scholar
  11. Dean AP, Nicholson JM, Sigee DC (2008) Impact of phosphorus quota and growth phase on carbon allocation in Chlamydomonas reinhardtii: an FTIR microspectroscopy study. Eur J Phycol 43(4):345–354CrossRefGoogle Scholar
  12. De-Bashan LE, Bashan Y (2004) Recent advances in removing phosphorus from wastewater and its future use as fertilizer (1997–2003). Water Res 38(19):4222–4246CrossRefPubMedGoogle Scholar
  13. Eaton AD, Clesceri LS, Greenberg AE (2005) Standard methods for the examination of water and wastewater, 21st edn. American Public Health Association/American Water Works Association/Water Environment Federation, Washington DC, USAGoogle Scholar
  14. Fanta SE, Hill WR, Smith TB, Roberts BJ (2010) Applying the light: nutrient hypothesis to stream periphyton. Freshw Biol 55:931–940CrossRefGoogle Scholar
  15. Feng P, Deng Z, Fan L, Hu Z (2012) Lipid accumulation and growth characteristics of Chlorella zofingiensis under different nitrate and phosphate concentrations. J Biosci Bioeng 114(4):405–410CrossRefPubMedGoogle Scholar
  16. Gómez C, Escudero R, Morales MM, Figueroa FL, Fernández-Sevilla JM, Acién FG (2013) Use of secondary-treated wastewater for the production of Muriellopsis sp. Appl Microbiol Biotechnol 97(5):2239–2249CrossRefPubMedGoogle Scholar
  17. González CV, Cerón Mdel C, Acien FG, Segovia CS, Chisti Y, Fernández JM (2010) Protein measurements of microalgal and cyanobacterial biomass. Bioresour Technol 101:7587–7591CrossRefGoogle Scholar
  18. González-Fernández C, Ballesteros M (2012) Linking microalgae and cyanobacteria culture conditions and key-enzymes for carbohydrate accumulation. Biotechnol Adv 30(6):1655–1661CrossRefPubMedGoogle Scholar
  19. Guieysse B, Plouviez M, Coilhac M, Cazali L (2013) Nitrous oxide (N2O) production in axenic Chlorella vulgaris microalgae cultures: evidence, putative pathways, and potential environmental impacts. Biogeosciences 10(10):6737–6746CrossRefGoogle Scholar
  20. Harold FM (1966) Inorganic polyphosphates in biology: structure, metabolism, and function. Bacteriol Rev 30(4):772–794PubMedCentralPubMedGoogle Scholar
  21. He PJ, Mao B, Lü F, Shao LM, Lee DJ, Chang JS (2013) The combined effect of bacteria and Chlorella vulgaris on the treatment of municipal wastewaters. Bioresour Technol 146:562–568CrossRefPubMedGoogle Scholar
  22. Kamiya A, Kowallik W (1987) Photoinhibition of glucose uptake in Chlorella. Plant Cell Physiol 28:611–619Google Scholar
  23. Kamiya A, Saitoh T (2002) Blue-light-control of the uptake of amino acids and of ammonia in Chlorella mutants. Physiol Plant 116:248–254CrossRefPubMedGoogle Scholar
  24. Kessler E, Oesterheld H (1970) Nitrification and induction of nitrate reductase in nitrogen deficient algae. Nature 228:287–288CrossRefPubMedGoogle Scholar
  25. Kim KH, Choi IS, Kim HM, Wi SG, Bae HJ (2014) Bioethanol production from the nutrient stress-induced microalga Chlorella vulgaris by enzymatic hydrolysis and immobilized yeast fermentation. Bioresour Technol 153:47–54CrossRefPubMedGoogle Scholar
  26. Kulaev I, Vagabov V, Kulakovskaya T (1999) New aspects of inorganic polyphosphate metabolism and function. J Biosci Bioeng 88(2):111–129CrossRefPubMedGoogle Scholar
  27. Li Y, Fei X, Deng X (2012) Novel molecular insights into nitrogen starvation induced triacylglycerols accumulation revealed by differential gene expression analysis in green algae Micractinium pusillum. Biomass Bioenergy 42:199–211CrossRefGoogle Scholar
  28. Liu ZY, Wang GC, Zhou BC (2008) Effect of iron on growth and lipid accumulation in Chlorella vulgaris. Bioresour Technol 99(11):4717–4722CrossRefPubMedGoogle Scholar
  29. Markou G, Nerantzis E (2013) Microalgae for high-value compounds and biofuels production: a review with focus on cultivation under stress conditions. Biotechnol Adv 31(8):1532–1542CrossRefPubMedGoogle Scholar
  30. Mesquita DP, Leal C, Cunha JR, Oehmen A, Amaral AL, Reis MA, Ferreira EC (2013) Prediction of intracellular storage polymers using quantitative image analysis in enhanced biological phosphorus removal systems. Anal Chim Acta 770:36–44CrossRefPubMedGoogle Scholar
  31. Muñoz R, Guieysse B (2006) Algal–bacterial processes for the treatment of hazardous contaminants: a review. Water Res 40(15):2799–2815CrossRefPubMedGoogle Scholar
  32. Panda B, Mallick N (2007) Enhanced poly-β-hydroxybutyrate accumulation in a unicellular cyanobacterium, Synechocystis sp. PCC 6803. Lett Appl Microbiol 44(2):194–198CrossRefPubMedGoogle Scholar
  33. Pérez-García O, de-Bashan LE, Hernández JP, Bashan Y (2010) Efficiency of growth and nutrient uptake from wastewater by heterotrophic, autotrophic, and mixotrophic cultivation of Chlorella vulgaris immobilized with Azospirillum brasilense. J Phycol 46(4):800–812CrossRefGoogle Scholar
  34. Pérez-García O, Escalante FME, de-Bashan LE, Bashan Y (2011a) Heterotrophic cultures of microalgae: metabolism and potential products. Water Res 45(1):11–36CrossRefPubMedGoogle Scholar
  35. Pérez-García RO, Bashan Y, Puente ME (2011b) Organic carbon supplementation of municipal wastewater is essential for heterotrophic growth and ammonium removing by the microalgae Chlorella vulgaris. J Phycol 47(1):190–199CrossRefGoogle Scholar
  36. Powell N, Shilton AN, Pratt S, Chisti Y (2008) Factors influencing luxury uptake of phosphorus by microalgae in waste stabilization ponds. Environ Sci Technol 42(16):5958–5962CrossRefPubMedGoogle Scholar
  37. Powell N, Shilton A, Chisti Y, Pratt S (2009) Towards a luxury uptake process via microalgae—defining the polyphosphate dynamics. Water Res 43(17):4207–4213CrossRefPubMedGoogle Scholar
  38. Prajapati SK, Kaushik P, Malik A, Vijay VK (2013) Phycoremediation coupled production of algal biomass, harvesting and anaerobic digestion: possibilities and challenges. Biotechnol Adv 31(8):1408–1425CrossRefPubMedGoogle Scholar
  39. Sharma L, Mallick N (2005) Accumulation of poly-β-hydroxybutyrate in Nostoc muscorum: regulation by pH, light–dark cycles, N and P status and carbon sources. Bioresour Technol 96(11):1304–1310CrossRefPubMedGoogle Scholar
  40. Sigee DC, Bahrami F, Estrada B, Webster RE, Dean AP (2007) The influence of phosphorus availability on carbon allocation and P quota in Scenedesmus subspicatus: a synchrotron-based FTIR analysis. Phycology 46(5):583–592CrossRefGoogle Scholar
  41. Simionato D, Block MA, La Rocca N, Jouhet J, Maréchal E, Finazzi G, Morosinotto T (2013) The response of Nannochloropsis gaditana to nitrogen starvation includes de novo biosynthesis of triacylglycerols, a decrease of chloroplast galactolipids, and reorganization of the photosynthetic apparatus. Eukaryot Cell 12(5):665–676CrossRefPubMedCentralPubMedGoogle Scholar
  42. Sournia A (1978) Phytoplanton Manual. Museum National d’ Historie Naturelle, París. United Nations Educational. Scientific and Cultural Organization (Unesco)Google Scholar
  43. Subashchandrabose SR, Ramakrishnan B, Megharaj M, Venkateswarlu K, Naidu R (2011) Consortia of cyanobacteria/microalgae and bacteria: biotechnological potential. Biotechnol Adv 29(6):896–907CrossRefPubMedGoogle Scholar
  44. Takagi M, Karseno, Yoshida T (2006) Effect of salt concentration on intracellular accumulation of lipids and triacylglyceride in marine microalgae Dunaliella cells. J Biosci Bioeng 101(3):223–226CrossRefPubMedGoogle Scholar
  45. Venkata Mohan S, Devi MP, Subhash GV, Chandra R (2014) Algae oils as fuels. Biofuels from Algae, Elsevier 8:155–187Google Scholar
  46. Zúñiga C, Morales M, Le Borgne S, Revah S (2011) Production of poly-hydroxybutyrate (PHB) by Methylobacterium organophilum isolated from a methanotrophic consortium in a two-phase partition bioreactor. J Hazard Mater 190(1–3):876–882CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Cynthia Alcántara
    • 1
  • Carolina Fernández
    • 1
  • Pedro A. García-Encina
    • 1
  • Raúl Muñoz
    • 1
  1. 1.Department of Chemical Engineering and Environmental TechnologyValladolid UniversityValladolidSpain

Personalised recommendations