Enhanced hydrogen production by Nostoc sp. CU2561 immobilized in a novel agar bead


A large number of microalgae isolated from Thailand were screened for their hydrogen production capacity. The selected highly efficient microalga, identified as Nostoc sp. CU2561, was investigated for the conditions under which the cells had maximal hydrogen production rate. Nostoc sp. CU2561 showed highest hydrogen production rate when grown in BG11 medium deprived of nitrogen and sulfur (BG11-N-S). To further improve hydrogen production, newly invented agar beads were used as matrix for cell immobilization. The highest hydrogen production rate by 1.5% (w/v) agar bead immobilized cells was obtained using cell concentration of 0.125 mg dry wt mL−1. Agar bead–immobilized cells showed superior hydrogen production rate, 1.5-fold higher when compared with agar cube–immobilized cells, and 5- and 10-fold higher when compared with those in alginate bead–immobilized and alginate bead–suspended cells, respectively. Supplementation of 0.5% (w/v) fructose increased hydrogen production rate of agar bead–immobilized cells approximately 1.7-fold, whereas the reducing agent β-mercaptoethanol increased hydrogen production rate by about 8.2-fold. Overall, Nostoc sp. CU2561 immobilized in agar bead showed the highest hydrogen production rate when incubated in BG11-N-S containing 5 mM β-mercaptoethanol with the highest hydrogen production rate of 18.78 ± 1.44 μmol H2 mg−1 chl a h−1. In addition, agar bead–immobilized cells could continuously produce hydrogen for 3 cycles. The hydrogen production by immobilized cells could be prolonged up to 120 h during the first cycle. This study provides a potential of new immobilization strategy using cyanobacteria immobilized in agar bead for hydrogen production which can be applied to scale up at industrial level in the future.

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  1. Adams MW, Hall DO (1979) Purification of the membrane-bound hydrogenase of Escherichia coli. Biochem J 183:11–22

  2. Altschul SF, Madden LT, Zhang J (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search pro. J Mol Biol 215:403–410

  3. Ananyev G, Carrieri D, Dismukes GC (2008) Optimization of metabolic capacity and flux through environmental cues to maximize hydrogen production by the cyanobacterium Arthrospira (Spirulina) maxima. Appl Environ Microbiol 74:6102–6113

  4. Anjana K, Kaushik A (2014) Enhanced hydrogen production by immobilized cyanobacterium Lyngbya perelegans under varying anaerobic conditions. Biomass Bioenergy 63:54–57

  5. Antal TK, Krendeleva TE, Laurinavichene TV, Makarova VV, Ghirardi ML, Rubin AB et al (2003) The dependence of algal H2 production on photosystem II and O2 consumption activities in sulfur-deprived Chlamydomonas reinhardtii cells. Biochim Biophys Acta 1607:153–160

  6. Antal TK, Kukarskikh GP, Volgusheva AA, Krendeleva TE, Rubina AB (2016) Hydrogen photoproduction by immobilized S-deprived Chlamydomonas reinhardtii: effect of light intensity and spectrum, and initial medium pH. Algal Res 17:38–45

  7. Baebprasert W, Lindblad P, Incharoensakdi A (2010) Response of H2 production and Hox-hydrogenase activity to external factors in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803. Int J Hydrog Energy 35:6611–6616

  8. Baker AM, Paul DF (2017) A review of hydrogen production by photosynthetic organisms using whole-cell and cell-free systems. Appl Biochem Biotechnol 183:503–519

  9. Benemann JR (2000) Hydrogen production by microalgae. J Appl Phycol 12:291–300

  10. Bothe H, Schmitz O, Yates MG, Newton WE (2010) Nitrogen fixation and hydrogen metabolism in cyanobacteria. Microbiol Mol Biol Rev 74:529–551

  11. Chen PC, Fan SH, Chiang CL, Lee CM (2008) Effect of growth conditions on the hydrogen production with cyanobacterium Anabaena sp. strain CH3. Int J Hydrog Energy 33:1460–1464

  12. Hajry HA, Maskry SA, Kharousi LM, Mardi OE, Shayya WH, Goosen FA (1999) Electrostatic encapsulation and growth of plant cell cultures in alginate. Biotechnol Prog 15:768–774

  13. Haury JF, Spiller H (1981) Fructose uptake and influence on growth of and nitrogen fixation by Anabaena variabilis. J Bacteriol 147:227–235

  14. Hellberg RS, Martin KG, Haney CJ, Shen Y, Smiley RD (2013) 16S rRNA partial gene sequencing for the differentiation and molecular subtyping of Listeria species. Food Microbiol 36:231–240

  15. Kayano H, Karube I, Matsunaga T, Suzuki S, Nakayama O (1981) A photochemical fuel cell system using Anabaena N-7363. Eur J Appl Microbiol Biotechnol 12:1–5

  16. Khetkorn W, Lindblad P, Incharoensakdi A (2010) Enhanced biohydrogen production by the N2−fixing cyanobacterium Anabaena siamensis strain TISTR 8012. Int J Hydrog Energy 35:12767–12776

  17. Khetkorn W, Rastogi RP, Incharoensakdi A, Lindblad P, Madamwar D, Pandey A, Larroche C (2017) Microalgal hydrogen production–a review. Bioresour Technol 243:1194–1206

  18. Khosravitabar F, Hippler M (2019) A new approach for improving microalgal biohydrogen photoproduction based on safe & fast oxygen consumption. Int J Hydrog Energy 44:17835–17844

  19. Kim JK, Nhat L, Chun YN, Kim SW (2008) Hydrogen production condition from food waste by dark fermentation with Clostridium beijerinckii KCTC 1785. Biotechnol Bioprocess Eng 13:499–504

  20. Kosourov SN, Seibert M (2009) Hydrogen photoproduction by nutrient-deprive Chlamydomonas reinhardtii cells immobilized within thin alginate films under aerobic and anaerobic conditions. Biotechnol Bioeng 102:50–58

  21. 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 28:253–263

  22. Lang NJ, Krupp JM, Koller AL (1987) Morphological and ultrastructural changes in vegetative cells and heterocysts of Anabaena variabilis grown with fructose. J Bacteriol 169:920–923

  23. Lee BB, Ravindra P, Chan ES (2013) Size and shape of calcium alginate beads produced by extrusion dripping. Chem Eng Technol 36:1627–1642

  24. Leino H, Kosourov SN, Saari L, Sivonen K, Tsygankov AA, Aro EM, Allahverdiyeva Y (2012) Extended H2 photoproduction by N2-fixing cyanobacteria immobilized in thin alginate films. Int J Hydrog Energy 37:151–161

  25. Mackinney G (1941) Absorption of light by chlorophyll solutions. J Biol Chem 140:315–322

  26. Madamwar D, Garg N, Shah V (2000) Cyanobacterial hydrogen production. World J Microb Biot 16:757–767

  27. Maneeruttanarungroj C, Lindblad P, Incharoensakdi A (2010) A newly isolated green alga, Tetraspora sp. CU2551, from Thailand with efficient hydrogen production. Int J Hydrog Energy 35:13193–13199

  28. Márquez-Reyes LA, del Pilar S-SM, Valdez-Vazquez I (2015) Improvement of hydrogen production by reduction of the photosynthetic oxygen in microalgae cultures of Chlamydomonas gloeopara and Scenedesmus obliquus. Int J Hydrog Energy 40:7291–7300

  29. Maswanna T, Phunpruch S, Lindblad P, Maneeruttanarungroj C (2018) Enhanced hydrogen production by optimization of immobilized cells of the green alga Tetraspora sp. CU2551 grown under anaerobic condition. Biomass Bioenergy 111:88–95

  30. Mathews J, Wang G (2009) Metabolic pathway engineering for enhanced biohydrogen production. Int J Hydrog Energy 34:7404–7416

  31. Melis A, Zhang L, Forestier M, Ghirardi ML, Seibert M (2000) Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii. Plant Physiol 122:127–136

  32. Neuer G, Bothe H (1985) Electron donation to nitrogenase in heterocysts of cyanobacteria. Arch Microbiol 143:185–191

  33. Pansook S, Incharoensakdi A, Phunpruch S (2019) Enhanced dark fermentative H2 production by agar-immobilized cyanobacterium Aphanothece halophytica. J Appl Phycol 31:2869–2879

  34. Philips EJ, Mitsui A (1986) Characterization and optimization of hydrogen production by a salt water blue-green alga Oscillatoria sp. Miami BG 7. II. Use of immobilization for enhancement of hydrogen production. Int J Hydrog Energy 11:83–89

  35. Raksajit W, Satchasataporn K, Lehto K, Maenpaa P, Incharoensakdi A (2012) Enhancement of hydrogen production by the filamentous non-heterocystous cyanobacterium Arthrospira sp. PCC 8005. Int J Hydrog Energy 37:18791–18797

  36. Rashid N, Song W, Park J, Jin HF, Lee K (2009) Characteristics of hydrogen production by immobilized cyanobacterium Microcystis aeruginosa through cycles of photosynthesis and anaerobic incubation. J Ind Eng Chem 15:498–503

  37. 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 111:1–61

  38. Sciuto K, Moro I (2016) Detection of the new cosmopolitan genus Thermoleptolyngbya (cyanobacteria, Leptolyngbyaceae) using the 16S rRNA gene and 16S–23S ITS region. Mol Phylogen Evol 105:15–35

  39. Smith R, Hobson S, Ellis I (1987) Evidence for calcium-mediated regulation of heterocyst frequency and nitrogenase activity in Nostoc 6720. New Phytol 105:531–541

  40. Song W, Rashid N, Choi W, Lee K (2011) Biohydrogen production by immobilized Chlorella sp. using cycles of oxygenic photosynthesis and anaerobiosis. Bioresour Technol 102:8676–8681

  41. Taikhao S, Phunpruch S (2017) Increasing hydrogen production efficiency of N2-fixing cyanobacterium Anabaena siamensis TISTR 8012 by cell immobilization. Energy Procedia 138:366–371

  42. Tamagnini P, Leitao E, Oliveira P, Ferreira D, Pinto F, Harris DJ (2007) Cyanobacterial hydrogenase: diversity, regulation and applications. FEMS Microbiol Rev 31:692–720

  43. Vaidyanathan J, Bhathena LZ, Adivarekar RV, Nerurkar M (2012) Production, partial characterization, and use of a red biochrome produced by Serratia sakuensis subsp. nov strain KRED for dyeing natural fibers. Appl Biochem Biotechnol 166:321–335

  44. Wutthithien P, Lindblad P, Incharoensakdi A (2019) Improvement of photobiological hydrogen production by suspended and immobilized cells of the N2-fixing cyanobacterium Fischerella muscicola TISTR 8215. J Appl Phycol 31:3527–3536

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Correspondence to Aran Incharoensakdi.

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Sukrachan, T., Incharoensakdi, A. Enhanced hydrogen production by Nostoc sp. CU2561 immobilized in a novel agar bead. J Appl Phycol (2020).

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  • Hydrogen production
  • Immobilization
  • Agar bead
  • Cyanobacteria
  • Nostoc sp. CU2561