World Journal of Microbiology and Biotechnology

, Volume 26, Issue 10, pp 1785–1793 | Cite as

Hydrogen production by Chlamydomonas reinhardtii revisited: Rubisco as a biotechnological target

  • Julia Marín-NavarroEmail author
  • Maria Gloria Esquivel
  • Joaquín Moreno
Original Paper


Hydrogen production by C. reinhardtii seems a promising alternative as a source of non-polluting biofuel. Hydrogen is generated as a result of combining free protons and electrons (supplied by ferredoxin) through the activity of an oxygen-sensitive hydrogenase. Thus, substantial hydrogen production is only observed in the light under anaerobic conditions. These require a reduced rate of photosynthetic oxygen evolution which is usually achieved by impairing photosystem II through sulphur starvation. Several approaches have been conducted to enhance and extend hydrogen production by addressing problems such as the mechanism of hydrogenase inhibition by oxygen, the stressing impact on the cells of the culture conditions, the use of starch as an alternate source of electrons under reduced photosynthetic activity, and the need of maintaining a balance between oxygen evolution and consumption. The photosynthetic enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) appears as suitable objective for biotechnological optimization of hydrogen production because of its relevance controlling the hydrogenase main competitor electron sink (the Calvin-Benson cycle), as well as starch accumulation and photorespiratory oxygen consumption. Possible strategies for increasing hydrogen generation based on alteration of Rubisco properties and/or catabolism through site-directed mutagenesis are discussed.


Biofuel Chlamydomonas reinhardtii Chloroplast metabolism Hydrogen production Hydrogenase Rubisco 



Work in the authors laboratories are supported by grants BIO2007-67708-C04-02 from MEC (Spain), BFU2009-11965 from MICINN (Spain) and PTDC/EBB-EBI/102728/2008 (Portugal).


  1. Boyd ES, Spear JR, Peters JW (2009) [FeFe] hydrogenase genetic diversity provides insight into molecular adaptation in a saline microbial mat community. Appl Environ Microbiol 75:4620–4623CrossRefGoogle Scholar
  2. Chen HC, Newton AJ, Melis A (2005) Role of SulP, a nuclear-encoded chloroplast sulfate permease, in sulfate transport and H2 evolution in Chlamydomonas reinhardtii. Photosynth Res 84:289–296CrossRefGoogle Scholar
  3. Chochois V, Dauvillee D, Beyly A, Tolleter D, Cuine S, Timpano H, Ball S, Cournac L, Peltier G (2009) Hydrogen production in Chlamydomonas: photosystem II-dependent and -independent pathways differ in their requirement for starch metabolism. Plant Physiol 151:631–640CrossRefGoogle Scholar
  4. Esquivel MG, Pinto TS, Marin-Navarro J, Moreno J (2006) Substitution of tyrosine residues at the aromatic cluster around the betaA-betaB loop of rubisco small subunit affects the structural stability of the enzyme and the in vivo degradation under stress conditions. Biochemistry 45:5745–5753CrossRefGoogle Scholar
  5. Fedorov AS, Kosourov S, Ghirardi ML, Seibert M (2005) Continuous hydrogen photoproduction by Chlamydomonas reinhardtii: using a novel two-stage, sulfate-limited chemostat system. Appl Biochem Biotechnol 121:403–412CrossRefGoogle Scholar
  6. Ferreira RB, Esquivel MG, Teixeira AR (1992) Catabolism of ribulose bisphosphate carboxylase from higher plants. Curr Topics Phytochem 3:129–165Google Scholar
  7. Forestier M, King P, Zhang L, Posewitz M, Schwarzer S, Happe T, Ghirardi ML, Seibert M (2003) Expression of two [Fe]-hydrogenases in Chlamydomonas reinhardtii under anaerobic conditions. Eur J Biochem 270:2750–2758CrossRefGoogle Scholar
  8. Fouchard S, Hemschemeier A, Caruana A, Pruvost J, Legrand J, Happe T, Peltier G, Cournac L (2005) Autotrophic and mixotrophic hydrogen photoproduction in sulfur-deprived Chlamydomonas cells. Appl Environ Microbiol 71:6199–6205CrossRefGoogle Scholar
  9. Frenkel AW (1952) Hydrogen evolution by the flagellate green alga, Chlamydomonas moewusii. Arch Biochem Biophys 38:219–230CrossRefGoogle Scholar
  10. Gaffron H, Rubin J (1942) Fermentative and photochemical production of hydrogen in algae. J Gen Physiol 26:219–240CrossRefGoogle Scholar
  11. Genkov T, Spreitzer RJ (2009) Highly conserved small subunit residues influence rubisco large subunit catalysis. J Biol Chem 284:30105–30112CrossRefGoogle Scholar
  12. Gfeller RP, Gibbs M (1984) Fermentative Metabolism of Chlamydomonas reinhardtii: I. Analysis of Fermentative Products from Starch in Dark and Light. Plant Physiol 75:212–218Google Scholar
  13. Ghirardi ML (2006) Hydrogen production by photosynthetic green algae. J Biochem Biotech 43:201–210Google Scholar
  14. Ghirardi ML, King PW, Posewitz MC, Maness PC, Fedorov A, Kim K, Cohen J, Schulten K, Seibert M (2005) Approaches to developing biological H(2)-photoproducing organisms and processes. Biochem Soc Trans 33:70–72CrossRefGoogle Scholar
  15. Happe T, Kaminski A (2002) Differential regulation of the Fe-hydrogenase during anaerobic adaptation in the green alga Chlamydomonas reinhardtii. Eur J Biochem 269:1022–1032CrossRefGoogle Scholar
  16. Hemschemeier A, Fouchard S, Cournac L, Peltier G, Happe T (2008) Hydrogen production by Chlamydomonas reinhardtii: an elaborate interplay of electron sources and sinks. Planta 227:397–407CrossRefGoogle Scholar
  17. Jans F, Mignolet E, Houyoux PA, Cardol P, Ghysels B, Cuine S, Cournac L, Peltier G, Remacle C, Franck F (2008) A type II NAD(P)H dehydrogenase mediates light-independent plastoquinone reduction in the chloroplast of Chlamydomonas. Proc Natl Acad Sci USA 105:20546–20551CrossRefGoogle Scholar
  18. Kosourov S, Seibert M, Ghirardi ML (2003) Effects of extracellular pH on the metabolic pathways in sulfur-deprived, H2-producing Chlamydomonas reinhardtii cultures. Plant Cell Physiol 44:146–155CrossRefGoogle Scholar
  19. Kruse O, Rupprecht J, Bader KP, Thomas-Hall S, Schenk PM, Finazzi G, Hankamer B (2005) Improved photobiological H2 production in engineered green algal cells. J Biol Chem 280:34170–34177CrossRefGoogle Scholar
  20. Laurinavichene T, Fedorov A, Ghirardi ML, Seibert M, Tsygankov A (2006) Demonstration of sustained hydrogen photoproduction by immobilized, sulfur-deprived Chlamydomonas reinhardtii cells. Int J Hydrogen Energy 31:659–667CrossRefGoogle Scholar
  21. Makarova VV, Kosourov S, Krendeleva TE, Semin BK, Kukarskikh GP, Rubin AB, Sayre RT, Ghirardi ML, Seibert M (2007) Photoproduction of hydrogen by sulfur-deprived C. reinhardtii mutants with impaired photosystem II photochemical activity. Photosynth Res 94:79–89CrossRefGoogle Scholar
  22. Marin-Navarro J, Moreno J (2006) Cysteines 449 and 459 modulate the reduction-oxidation conformational changes of ribulose 1.5-bisphosphate carboxylase/oxygenase and the translocation of the enzyme to membranes during stress. Plant Cell Environ 29:898–908CrossRefGoogle Scholar
  23. Matthew T, Zhou W, Rupprecht J, Lim L, Thomas-Hall SR, Doebbe A, Kruse O, Hankamer B, Marx UC, Smith SM, Schenk PM (2009) The metabolome of Chlamydomonas reinhardtii following induction of anaerobic H2 production by sulfur depletion. J Biol Chem 284:23415–23425CrossRefGoogle Scholar
  24. Maul JE, Lilly JW, Cui L, de Pamphilis CW, Miller W, Harris EH, Stern DB (2002) The Chlamydomonas reinhardtii plastid chromosome: islands of genes in a sea of repeats. Plant Cell 14:2659–2679CrossRefGoogle Scholar
  25. Melis A (2007) Photosynthetic H2 metabolism in Chlamydomonas reinhardtii (unicellular green algae). Planta 226:1075–1086CrossRefGoogle Scholar
  26. 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 122:127–136CrossRefGoogle Scholar
  27. Merchant SS et al (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318:245–250CrossRefGoogle Scholar
  28. Moreno J, Spreitzer RJ (1999) C172S substitution in the chloroplast-encoded large subunit affects stability and stress-induced turnover of ribulose-1, 5-bisphosphate carboxylase/oxygenase. J Biol Chem 274:26789–26793CrossRefGoogle Scholar
  29. Moreno J, Garcia-Murria MJ, Marin-Navarro J (2008) Redox modulation of Rubisco conformation and activity through its cysteine residues. J Exp Bot 59:1605–1614CrossRefGoogle Scholar
  30. Mus F, Dubini A, Seibert M, Posewitz MC, Grossman AR (2007) Anaerobic acclimation in Chlamydomonas reinhardtii: anoxic gene expression, hydrogenase induction, and metabolic pathways. J Biol Chem 282:25475–25486CrossRefGoogle Scholar
  31. Ogren WL (2003) Affixing the O to Rubisco: discovering the source of photorespiratory glycolate and its regulation. Photosynth Res 76:53–63CrossRefGoogle Scholar
  32. Parry MA, Andralojc PJ, Mitchell RAC, Madgwick PJ, Keys AJ (2003) Manipulation of Rubisco: the amount, activity, function and regulation. J Exp Bot 54:1321–1333CrossRefGoogle Scholar
  33. Posewitz MC, Smolinski SL, Kanakagiri S, Melis A, Seibert M, Ghirardi ML (2004) Hydrogen photoproduction is attenuated by disruption of an isoamylase gene in Chlamydomonas reinhardtii. Plant Cell 16:2151–2163CrossRefGoogle Scholar
  34. Posewitz MC, Dubini A, Meuser JE, Seibert M, Ghirardi ML (2008) Hydrogenases, hydrogen production, and anoxia. In: Stern D (ed) The Chlamydomonas sourcebook, vol. 2. Academic Press, Oxford, pp 217–255Google Scholar
  35. Redding KE, Cole DG (2008) Chlamydomonas: a sexually active, light-harvesting, carbon-reducing, hydrogen-belching ‘planimal’. EMBO reports 9:1182–1187CrossRefGoogle Scholar
  36. Rochaix JD (1995) Chlamydomonas reinhardtii as the photosynthetic yeast. Annu Rev Genet 29:209–230CrossRefGoogle Scholar
  37. Ruhle T, Hemschemeier A, Melis A, Happe T (2008) A novel screening protocol for the isolation of hydrogen producing Chlamydomonas reinhardtii strains. BMC Plant Biol 8:107CrossRefGoogle Scholar
  38. Rupprecht J (2009) From systems biology to fuel–Chlamydomonas reinhardtii as a model for a systems biology approach to improve biohydrogen production. J Biotechnol 142:10–20CrossRefGoogle Scholar
  39. Silakov A, Kamp C, Reijerse E, Happe T, Lubitz W (2009) Spectroelectrochemical characterization of the active site of the [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii. Biochemistry 48:7780–7786CrossRefGoogle Scholar
  40. Spreitzer RJ, Salvucci ME (2002) Rubisco: structure, regulatory interactions, and possibilities for a better enzyme. Annu Rev Plant Biol 53:449–475CrossRefGoogle Scholar
  41. Spreitzer RJ, Esquivel MG, Du YC, McLaughlin PD (2001) Alanine-scanning mutagenesis of the small-subunit beta A-beta B loop of chloroplast ribulose-1, 5-bisphosphate carboxylase/oxygenase: substitution at Arg-71 affects thermal stability and CO2/O2 specificity. Biochemistry 40:5615–5621CrossRefGoogle Scholar
  42. Stripp ST, Goldet G, Brandmayr C, Sanganas O, Vincent KA, Haumann M, Armstrong FA, Happe T (2009a) How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms. Proc Natl Acad Sci USA 106:17331–17336CrossRefGoogle Scholar
  43. Stripp ST, Sanganas O, Happe T, Haumann M (2009b) The structure of the active site H-cluster of [Fe-Fe] hydrogenase from the green alga Chlamydomonas reinhardtii studied by X-ray absorption spectroscopy. Biochemistry 48:5042–5049CrossRefGoogle Scholar
  44. Surzycki R, Cournac L, Peltier G, Rochaix JD (2007) Potential for hydrogen production with inducible chloroplast gene expression in Chlamydomonas. Proc Natl Acad Sci USA 104:17548–17553CrossRefGoogle Scholar
  45. Takahashi S, Murata N (2005) Interruption of the Calvin cycle inhibits the repair of Photosystem II from photodamage. Biochim Biophys Acta 1708:352–361CrossRefGoogle Scholar
  46. Vahrenholz C, Riemen G, Pratje E, Dujon B, Michaelis G (1993) Mitochondrial DNA of Chlamydomonas reinhardtii: the structure of the ends of the linear 15.8-kb genome suggests mechanisms for DNA replication. Curr Gen 24:241–247CrossRefGoogle Scholar
  47. White AL, Melis A (2006) Biochemistry of hydrogen metabolism in Chlamydomonas reinhardtii wild-type and a Rubisco-less mutant. Int J Hydrogen Energy 31:455–464CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Julia Marín-Navarro
    • 1
    Email author
  • Maria Gloria Esquivel
    • 2
  • Joaquín Moreno
    • 3
  1. 1.Department of BiotechnologyInstituto de Agroquímica y Tecnología de AlimentosPaternaSpain
  2. 2.Department of Botany and Biological EngineeringTechnical University of LisbonLisbonPortugal
  3. 3.Department of Biochemistry and Molecular BiologyUniversity of ValenciaBurjassotSpain

Personalised recommendations