Future Use of Solar Energy by Light-Dependent Hydrogen Production

of the Green Alga Scenedesmus obliquus ?

Röbbe Wünschiers, Kerstin Stangier, Thomas Zinn and Rüdiger Schulz

Philipps-University, FB Biology/Botany, Karl-von-Frisch-Str., D-35032 Marburg, Germany

Tel. +49-6421-282484, Fax +49-6421-282057, E-mail:

schulzr@mailer.uni-marburg.de

After adaptation to anaerobic conditions the eukaryotic unicellular green alga Scenedesmus obliquus shows light-dependent hydrogenase activity. Dependent on the partial pressure of H2 and CO2 hydrogen is either produced or taken up for photoreduction of CO2. Both reactions are mediated by the photosynthetic electron transport chain (1). Inactivation of the enzyme by oxygen in vitro seems to be irreversible, while in vivo a reactivation is possible (2). Preliminary results show that reduced thioredoxin (a typical regulatory component of photosynthetic reactions) has an inhibitory effect on hydrogenase activity. Thus this protein is discussed as a natural regulator of the hydrogenase.

A soluble hydrogenase was purified to homogeneity under strict anaerobic conditions (3). The enzyme consists of at least two subunits with molecular masses of 36 and 55 kDa, respectively. A native molecular weight of 150 kDa was estimated. Physiological, biochemical and spectroscopical investigations gave evidence that the purified hydrogenase from Scenedesmus is of the NiFe-type (4).

Primers designed from prokaryotic conserved sequences published for "Fe-only"- and NiFe-hydrogenases were used for PCR-experiments. The PCR-products and an oligonucleotide, synthesized according to the N-terminal amino acid sequence of the 36 kDa hydrogenase subunit isolated from Scenedesmus, were used for screening of cDNA-libraries. Several cDNA-clones were isolated and are under investigation. By databank search it was shown that the derived amino acid sequence of one cDNA displays significant similarities to "Fe-only"-hydrogenases. These results of sequence comparisions are in good agreement with preliminary biochemical results about a "Fe-only"-hydrogenase in Scenedesmus. For example reduced ferredoxin, purified from Scenedesmus, is able to donate electrons to a hydrogenase preparation of this organism very efficiently, a typical reaction for a "Fe-only"-hydrogenase.

Thus the existence of two hydrogenases, one of the NiFe-type and the other of the "Fe-only"-type, is postulated for Scenedesmus. Southern blot analysis with genomic, chloroplastic and mitochondrial DNA of the alga shows that the hydrogenases are encoded in the nucleus.

The main future goal of our research is the application of hydrogenases in photohydrogen production on a technical scale. Due to the coupling of hydrogenases to photosynthesis, algae are able to use light-energy for photohydrogen production directly and in addition avoid production of CO2. To take advantage of this efficient system, further details of the process have to be collected. The main problem that remains to be solved is the extreme sensitivity of the algal hydrogenase to oxygen. With the help of the cyanobacterium Synechocystis as a genetic tool we hopefully will be able to manipulate the enzyme towards oxygen insensitivity.

Acknowledgements.

We thank Prof. Horst Senger (Marburg) for his continuous support, personal encouragement and many helpful discussions. This work was supported financially by BMFT, DFG, COST, Deutsche Bundesstiftung Umwelt and Studienstiftung des Deutschen Volkes.

References

(1) R. Schulz (1996) J. Mar. Biotechnol., 4:16-22.

(2) T. Urbig, R. Schulz, and H. Senger (1993) Z. Naturforsch., 48c:41-45.

(3) J. Schnackenberg, R. Schulz, and H. Senger (1993) FEBS Lett., 327:21-24.

(4) T. Zinn, J. Schnackenberg, D. Haak, S. Römer, R. Schulz, and H. Senger (1994)

Z. Naturforsch.,

49c:33-38.