conference poster 2000

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Light, Hydrogen and Green Alga

R. Wünschiers* and R. Schulz**

*Uppsala University, Dept. Plant Physiol., Villavägen 6, 75236 Uppsala, Sweden, wuenschi@yahoo.com
**Botanisches Institut, Christian-Albrechts-Universität, Olshausenstr. 40, 24098 Kiel, Germany

2 H₂O O₂+ 4 e^- + 4 H⁺ 2 H₂

I) WHAT IS IT ABOUT?

Metabolism of molecular hydrogen by microorganisms is known since the turn of the century.
Two different classes of enzyms are responsable for hydrogen metabolism:

Nitrogenases:
they are only present in some bacteria and are well characterized

Hydrogenases:
they were found in a huge number of prokaryotes (bacteria) and some eukaryotes (mainly in algae)
Hydrogenases of prokaryotes are well characterized but there is little known about the eukaryotic enzymes

II) HYDROGENASES VERSUS NITROGENASES

	Hydrogenase	Nitrogenase
H₂-Production	yes	yes
H₂-Uptake	yes	no
reaction energy dependent (ATP)	no	yes
oxygen sensitiv	yes	yes
subunits	1-3	6
catalytic rate	high	low
present in prokaryotes	yes	yes
present in eukaryotes	yes	no

III) METABOLISM INVOLVED IN H₂-EVOLUTION

In all organisms H₂ is produced under anaerobic conditions only.

Nitrogen Fixation:
H₂ is released as an inherent by-product by the nitrogenase reaction performed in some bacteria.

Fermentation:
Organic substrates can be degraded to CO₂ and H₂ in several bacteria.

Anoxygenic Photosynthesis:
Sulfur compounds or organic substrates can serve as electron donors for H₂-production in purple or green bacteria.

Oxygenic Photosynthesis:
Splitting of water can provide reduced substrates for hydrogen production in cyanobacteria and algae.

IV) H₂-EVOLUTION IN PHOTOSYNTHETIC ORGANISMS

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Theoretical capability of hydrogen gas production by photosynthetic microorganisms. Q_B (quinone) and Fd (ferredoxin) indicate the first stable and mobile electron acceptors. On the y-axis the standard redox-potential is drawn. (adapted from Wünschiers et al., 1998)

Purple Bacteria:

are not able to reduce ferredoxin photochemically
although hydrogenases are present, photoproduction of H₂ is mediated by nitrogenases
ferredoxin is reduced via the energy dependent reverse electron flow
organic compounds serve as electron donors

Green Bacteria:

ferredoxin is probably photochemically reduced and hydrogen is evolved by use of reduced sulfur compounds

there are no data on the corresponding enzyme available

Cyanobacteria:

after photochemical reduction of ferredoxin hydrogen can be evolved mainly by the nitrogenase reaction
water is the electron donor
hydrogenases play a minor role in photohydrogen production performed by cyanobacteria

Algae:

as in cyanobacteria water is split and the electrons are excited to the redoxpotential of ferredoxin by the photosynthetic apparatus
photohydrogen production is catalized by a hydrogenase and requires, as in all other organisms mentioned above, anaerobic conditions

V) OUR OWN RESEARCH ON SCENEDESMUS HYDROGENASE

physiological investigations of photohydrogen production:

- light and substrate requirements
- anaerobic adaptation process
- duration of hydrogen evolution

biochemical investigations:

- enzyme localization, regulation and structure
- mechanism of the catalytic process

molecular biological investigations:

- search for functional gens
- regulation, sequencing and cloning of gens

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Our present model of hydrogen metabolism in the green alga Scenedesmus obliquus. (Cyt-b₆f: cytochrome b₆f-complex; DBMIB: 2,5-Dibrom-3-methyl-6-isopropyl-p-benzochinon; DCMU: 3-(3,4-Dichlorphenyl)-1,1'-dimethyl-ureate; Fd: ferredoxin; FNR: ferredoxin-NADP-reduktase; FQR: ferredoxin-plastoquinone-reduktase; H₂ase: hydrogenase; NDH: NAD(P)H-dehydrogenase; PC: plastocyanin; PQ: plastoquinon; PS: photosystem).

In the future we would like to express an oxygen insensitiv hydrogenase.

VI) DEVELOPMENT OF A BIOREACTOR

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Schematic drawing of a future bioreactor for photohydrogen production. (adapted from Wünschiers and Schulz 1998)

Research includes:

monitoring of hydrogen evolution
optimizing illumination of algae cultures
testing of oxygen removing techniques
investigation of factors affecting lifetime of algae

VII) SELECTED LITERATURE

Boichenko & Hoffmann (1994). Photosynthetica 30: 527-552
Schulz (1996). J. Mar. Biotechnol. 4: 16-22
Wünschiers et al. (1996) Biomass for Energy and the Environment. Ed.: P. Chartier, G.J. Ferrero, U.M. Henius, S. Hultberg, J. Sachau, M. Wiinblad. 1. Edition. Elsevier Science Ltd., Oxford, New York, Tokyo, 1668-1673
Schulz et al. (1998) Proceedings of the 12th World Hydrogen Energy Conference 1998. Ed.: J.C. Bolcich und T.N. Veziroglu. 1. Edition. Argentinien, 2069-2078
Appel & Schulz (1998) J. Photochem. Photobiol. 47: 1-11
Wünschiers & Schulz (1998) BIUZ 28: 130-136
Schulz et al. (1998) BioHydrogen, Ed.: O.R. Zarborsky, Plenum Press, New York, 243-251
Wünschiers et al. (1998) Photosynthesis: Mechanisms and Effects, Ed.: G. Garab, 1998, Kluwer Acad. Publ., Dordrecht, Holland, Vol. III, 1951-1954
Wünschiers et al. (1999) FEBS. Lett. 455: 162-164
Wünschiers & Borzner (1999) Chem. Lab. Biotech. 50: 141-146

VIII) ACKNOWLEDGMENTS

Support and encouragement by Prof. H. Senger (Marburg/Germnay) is gratefully acknowledged.
This work was supported financially by BMFT, Germany; MITI, Japan; DFG, Germany; Deutsche Bundesstiftung Umwelt, Germany and Studienstiftung des Deutschen Volkes, Germany.