Journal of Applied Phycology

, Volume 31, Issue 1, pp 131–143 | Cite as

Acclimation responses of immobilized N2-fixing heterocystous cyanobacteria to long-term H2 photoproduction conditions: carbon allocation, oxidative stress and carotenoid production

  • Gayathri Murukesan
  • Fiona Lynch
  • Yagut Allahverdiyeva
  • Sergey KosourovEmail author


This study investigates the acclimation of N2-fixing heterocystous cyanobacteria to long-term H2 photoproduction. Wild-type Calothrix sp. 336/3, Anabaena sp. PCC 7120, and the uptake hydrogenase-deficient mutant (ΔhupL) of Anabaena sp. PCC 7120 were entrapped within Ca2+-alginate films and subjected to an argon (Ar) atmosphere containing 6% CO2. Every third day, the atmosphere was changed to Ar + 6% CO2 (control), and air or air + 6% CO2. The air treatments were performed to recover the C/N balance of cells and restore their fitness. After 16–20 h of treatment, the headspace of all vials was again refreshed with Ar + 6% CO2. Cyanobacteria demonstrated strain-specific differences in carbon allocation and antioxidant responses to different treatments. While glycogen accumulation was observed for both Anabaena strains, Calothrix accumulated significantly less. Instead, Calothrix stored other carbohydrates, likely as extracellular polymeric substances (EPS). All alginate-entrapped cultures demonstrated general increases in oxidative stress over the course of the 450-h experiment. However, specific responses differed, with Calothrix accumulating higher total carotenoid and α-tocopherol levels and demonstrating a more diverse carotenoid profile. This strain also showed a relatively stable D1 protein level across different treatments. In general, all H2-photoproducing cyanobacteria demonstrated decreases in echinenone content and a shift toward the accumulation of glycosylated carotenoids: myxol 2′-methylpentoside (likely fucoside) in Calothrix and 4-ketomyxol 2′-fucoside in both Anabaena strains. Thus, long-term H2 photoproduction of immobilized cyanobacteria results in strain-specific acclimation strategies for changing environments.


H2 photoproduction Thin-layer immobilization Carotenoids Glycogen Oxidative stress EPS 



This work was financially supported by the Maj and Tor Nessling Foundation, Kone Foundation, and the Academy of Finland (FCoE program #307335). We are grateful to Professor H. Sakurai for sharing the ΔhupL mutant of Anabaena sp. PCC 7120.

Supplementary material

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ESM 1 (DOCX 127 kb)
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ESM 2 (DOCX 54.9 kb)


  1. BCC Research (2015) The global market for carotenoids. Retrieved from in December 2017
  2. Berglund AH, Nilsson R, Liljenberg C (1999) Permeability of large unilamellar digalactosyldiacylglycerol vesicles for protons and glucose—influence of α-tocopherol, β-carotene, zeaxanthin and cholesterol. Plant Physiol Biochem 37:179–186CrossRefGoogle Scholar
  3. Bothe H, Tennigkeit J, Eisbrenner G (1977) The utilization of molecular hydrogen by the blue-green alga Anabaena cylindrica. Arch Microbiol 114:43–49CrossRefGoogle Scholar
  4. Bothe H, Schmitz O, Yates MG, Newton WE (2010) Nitrogen fixation and hydrogen metabolism in cyanobacteria. Microbiol Mol Biol Rev 74:529–551CrossRefGoogle Scholar
  5. Carrieri D, Wawrousek K, Eckert C, Yu J, Maness P-C (2011) The role of the bidirectional hydrogenase in cyanobacteria. Bioresour Technol 102:8368–8377CrossRefGoogle Scholar
  6. Cerezo J, Zúñiga J, Bastida A, Requena A, Cerón-Carrasco JP, Eriksson LA (2012) Antioxidant properties of β-carotene isomers and their role in photosystems: insights from ab initio simulations. J Phys Chem A 116:3498–3506CrossRefGoogle Scholar
  7. Curatti L, Flores E, Salerno G (2002) Sucrose is involved in the diazotrophic metabolism of the heterocyst-forming cyanobacterium Anabaena sp. FEBS Lett 513:175–178CrossRefGoogle Scholar
  8. Domonkos I, Kis M, Gombos Z, Ughy B (2013) Carotenoids, versatile components of oxygenic photosynthesis. Prog Lipid Res 52:539–561CrossRefGoogle Scholar
  9. Dutta D, De D, Chaudhuri S, Bhattacharya SK (2005) Hydrogen production by Cyanobacteria. Microb Cell Factories 4:36CrossRefGoogle Scholar
  10. Esteves-Ferreira AA, Cavalcanti JHF, Vaz MGMV, Alvarenga LV, Nunes-Nesi A, Araújo WL (2017) Cyanobacterial nitrogenases: phylogenetic diversity, regulation and functional predictions. Genet Mol Biol 40:261–275CrossRefGoogle Scholar
  11. Fay P (1992) Oxygen relations of nitrogen fixation in cyanobacteria. Microbiol Rev 56:340–373PubMedPubMedCentralGoogle Scholar
  12. Hai T, Hein S, Steinbu CA (2001) Multiple evidence for widespread and general occurrence of type-III PHA synthases in cyanobacteria and molecular characterization of the PHA synthases from two thermophilic cyanobacteria: Chlorogloeopsis fritschii PCC 6912 and Synechococcus sp. strain MA19. Microbiology 147:3047–3060CrossRefGoogle Scholar
  13. Hassett DJ (1996) Anaerobic production of alginate by Pseudomonas aeruginosa: alginate restricts diffusion of oxygen. J Bacteriol 178:7322–7325CrossRefGoogle Scholar
  14. Hasunuma T, Kikuyama F, Matsuda M, Aikawa S, Izumi Y, Kondo A (2013) Dynamic metabolic profiling of cyanobacterial glycogen biosynthesis under conditions of nitrate depletion. J Exp Bot 64:2943–2954CrossRefGoogle Scholar
  15. Hinnemann B, Norskov JK (2006) Catalysis by enzymes: the biological ammonia synthesis. Top Catal 37:55–70CrossRefGoogle Scholar
  16. Ibañez E, Cifuentes A (2013) Benefits of using algae as natural sources of functional ingredients. J Sci Food Agric 93:703–709CrossRefGoogle Scholar
  17. Inoue S, Ejima K, Iwai E, Hayashi H, Appel J, Tyystjärvi E, Murata N, Nishiyama Y (2011) Protection by α-tocopherol of the repair of photosystem II during photoinhibition in Synechocystis sp. PCC 6803. Biochim Biophys Acta Bioenerg 1807:236–241CrossRefGoogle Scholar
  18. Jämsä M, Kosourov S, Rissanen V, Hakalahti M, Pere J, Ketoja JA, Tammelin T, Allahverdiyeva Y (2018) Versatile templates from cellulose nanofibrils for photosynthetic microbial biofuel production. J Mater Chem A 6:5825–5835CrossRefGoogle Scholar
  19. Kerfeld CA (2004) Structure and function of the water-soluble carotenoid-binding proteins of cyanobacteria. Photosynth Res 81:215–225CrossRefGoogle Scholar
  20. Knoll AH (2008) Cyanobacteria and earth history. In: Herrero A, Flores E (eds) The cyanobacteria: molecular biology, genomics and evolution. Caister Academic, Norfolk, UK, pp 1–19Google Scholar
  21. Kosourov SN, Seibert M (2009) Hydrogen photoproduction by nutrient-deprived Chlamydomonas reinhardtii cells immobilized within thin alginate films under aerobic and anaerobic conditions. Biotechnol Bioeng 102:50–58CrossRefGoogle Scholar
  22. Kosourov S, Leino H, Murukesan G, Lynch F, Sivonen K, Tsygankov AA, Aro EM, Allahverdiyeva Y (2014) Hydrogen photoproduction by immobilized N2-fixing cyanobacteria: understanding the role of the uptake hydrogenase in the long-term process. Appl Environ Microbiol 80:5807–5817CrossRefGoogle Scholar
  23. Kosourov SN, Murukesan G, Jokela J, Allahverdiyeva Y (2016) Carotenoid biosynthesis in Calothrix sp. 336/3: composition of carotenoids on full medium, during diazotrophic growth and after long-term H2 photoproduction. Plant Cell Physiol 57:2269–2282CrossRefGoogle Scholar
  24. Kosourov S, Murukesan G, Seibert M, Allahverdiyeva Y (2017) Evaluation of light energy to H2 energy conversion efficiency in thin films of cyanobacteria and green alga under photoautotrophic conditions. Algal Res 28:253–263CrossRefGoogle Scholar
  25. Kosourov SN, He M, Allahverdiyeva Y, Seibert M (2018) CHAPTER 15. Immobilization of microalgae as a tool for efficient light utilization in H2 production and other biotechnology applications. In: Seibert M, Torzillo G (eds) Microalgal hydrogen production: achievements and perspectives. The Royal Society of Chemistry, London, pp 355–384CrossRefGoogle Scholar
  26. Kótai J (1972) Instructions for preparation of modified nutrient solution Z8 for algae. NIVA B-11/69Google Scholar
  27. Kusama Y, Inoue S, Jimbo H, Takaichi S, Sonoike K, Hihara Y, Nishiyama Y (2015) Zeaxanthin and echinenone protect the repair of photosystem II from inhibition by singlet oxygen in Synechocystis sp. PCC 6803. Plant Cell Physiol 56:906–916CrossRefGoogle Scholar
  28. Latifi A, Ruiz M, Zhang CC (2009) Oxidative stress in cyanobacteria. FEMS Microbiol Rev 33:258–278CrossRefGoogle Scholar
  29. Leino H, Kosourov SN, Saari L, Sivonen K, Tsygankov AA, Aro E-M, Allahverdiyeva Y (2012) Extended H2 photoproduction by N2-fixing cyanobacteria immobilized in thin alginate films. Int J Hydrog Energy 37:151–161CrossRefGoogle Scholar
  30. Leino H, Shunmugam S, Isojärvi J, Oliveira P, Mulo P, Saari L, Battchikova N, Sivonen K, Lindblad P, Aro E-M, Allahverdiyeva Y (2014) Characterization of ten H2 producing cyanobacteria isolated from the Baltic Sea and Finnish lakes. Int J Hydrog Energy 39:8983–8991CrossRefGoogle Scholar
  31. Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol 148:350–382CrossRefGoogle Scholar
  32. Maksimov EG, Shirshin EA, Sluchanko NN, Zlenko DV, Parshina EY, Tsoraev GV, Klementiev KE, Budylin GS, Schmitt FJ, Friedrich T, Fadeev VV, Paschenko VZ, Rubin AB (2015) The signaling state of orange carotenoid protein. Biophys J 109:595–607CrossRefGoogle Scholar
  33. Masukawa H, Mochimaru M, Sakurai H (2002) Disruption of the uptake hydrogenase gene, but not of the bidirectional hydrogenase gene, leads to enhanced photobiological hydrogen production by the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120. Appl Microbiol Biotechnol 58:618–624CrossRefGoogle Scholar
  34. Mulo P, Sakurai I, Aro E-M (2012) Strategies for psbA gene expression in cyanobacteria, green algae and higher plants: from transcription to PSII repair. Biochim Biophys Acta Bioenerg 1817:247–257CrossRefGoogle Scholar
  35. Newton WE (2007) Physiology, biochemistry and molecular biology of nitrogen fixation. In: Bothe H, Ferguson SJ, Newton WE (eds) Biology of the nitrogen cycle. Elsevier, Amsterdam, pp 109–129CrossRefGoogle Scholar
  36. Nishiyama Y, Allakhverdiev SI, Yamamoto H, Hayashi H, Murata N (2004) Singlet oxygen inhibits the repair of photosystem II by suppressing the translation elongation of the D1 protein in Synechocystis sp. PCC 6803. Biochemistry 43:11321–11330CrossRefGoogle Scholar
  37. Nürnberg DJ, Mariscal V, Bornikoel J, Nieves-Morión M, Krauß N, Herrero A, Maldener I, Flores E, Mullineaux CW (2015) Intercellular diffusion of a fluorescent sucrose analog via the septal junctions in a filamentous cyanobacterium. mBio 6:e02109–e02114CrossRefGoogle Scholar
  38. Pollari M, Rantamäki S, Huokko T, Kårlund-Marttila A, Virjamo V, Tyystjärvi E, Tyystjärvi T (2011) Effects of deficiency and overdose of group 2 sigma factors in triple inactivation strains of Synechocystis sp. strain PCC 6803. J Bacteriol 193:265–273CrossRefGoogle Scholar
  39. Rastogi A, Yadav DK, Szymanska R, Kruk J, Sedlarova M, Pospisil P (2014) Singlet oxygen scavenging activity of tocopherol and plastochromanol in Arabidopsis thaliana: relevance to photooxidative stress. Plant Cell Environ 37:392–401CrossRefGoogle Scholar
  40. Stamatakis K, Tsimilli-Michael M, Papageorgiou GC (2014) On the question of the light-harvesting role of β-carotene in photosystem II and photosystem I core complexes. Plant Physiol Biochem 81:121–127CrossRefGoogle Scholar
  41. Steiger S, Schäfer L, Sandmann G (1999) High-light-dependent upregulation of carotenoids and their antioxidative properties in the cyanobacterium Synechocystis PCC 6803. J Photochem Photobiol B 52:14–18CrossRefGoogle Scholar
  42. Tamagnini P, Axelsson R, Lindberg P, Oxelfelt F, Wünschiers R, Lindblad P (2002) Hydrogenases and hydrogen metabolism of cyanobacteria. Microbiol Mol Biol Rev 66:1–20CrossRefGoogle Scholar
  43. Tamagnini P, Leitão E, Oliveira P, Ferreira D, Pinto F, Harris DJ, Heidorn T, Lindblad P (2007) Cyanobacterial hydrogenases: diversity, regulation and applications. FEMS Microbiol Rev 31:692–720CrossRefGoogle Scholar
  44. Umena Y, Kawakami K, Shen J-R, Kamiya N (2011) Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 Å. Nature 473:55–60CrossRefGoogle Scholar
  45. Vass I (2012) Molecular mechanisms of photodamage in the Photosystem II complex. Biochim Biophys Acta Bioenerg 1817:209–217CrossRefGoogle Scholar
  46. Walsby AE (2007) Cyanobacterial heterocysts: terminal pores proposed as sites of gas exchange. Trends Microbiol 15:340–349CrossRefGoogle Scholar
  47. Zakar T, Laczko-Dobos H, Toth TN, Gombos Z (2016) Carotenoids assist in cyanobacterial photosystem II assembly and function. Front Plant Sci 7:295CrossRefGoogle Scholar
  48. Zhu Y, Graham JE, Ludwig M, Xiong W, Alvey RM, Shen G, Bryant DA (2010) Roles of xanthophyll carotenoids in protection against photoinhibition and oxidative stress in the cyanobacterium Synechococcus sp. strain PCC 7002. Arch Biochem Biophys 504:86–99CrossRefGoogle Scholar

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© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Laboratory of Molecular Plant Biology, Department of BiochemistryUniversity of TurkuTurkuFinland

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