International Journal of Hydrogen Energy 27 (2002) 1431 1439 www.elsevier.

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[Fe]-hydrogenases in green algae: photo-fermentation and hydrogen evolution under sulfur deprivation
Martin Winklera;1 , Anja Hemschemeiera;1 , Cecilia Gotorb , Anastasios Melisc , Thomas Happea;
Institut der Universitat Bonn, Karlrobert-Kreiten-Strasse 13, 53115 Bonn, Germany de Bioquimica Vegetal y Fotosintesis, CSIC y Universidad de Sevilla, Seville, Spain c Department of Plant and Microbial Biology, University of California, 111 Koshland Hall, Berkeley, CA 94720-3102, USA
b Instituto a Botanisches

Abstract Recent studies indicate that [Fe]-hydrogenases and H2 metabolism are widely distributed among green algae. The enzymes are simple structured and catalyze H2 evolution with similar rates than the more complex [Fe]-hydrogenases from bacteria. Di erent green algal species developed diverse strategies to survive under sulfur deprivation. Chlamydomonas reinhardtii evolves large quantities of hydrogen gas in the absence of sulfur. In a sealed culture of C. reinhardtii, the photosynthetic O2 evolution rate drops below the rate of respiratory O2 consumption due to a reversible inhibition of photosystem II, thus leading to an intracellular anaerobiosis. The algal cells survive under these anaerobic conditions by switching their metabolism to a kind of photo-fermentation. Although possessing a functional [Fe]-hydrogenase gene, the cells of Scenedesmus obliquus produce no signicant amounts of H2 under S-depleted conditions. Biochemical analyses indicate that S. obliquus decreases almost the complete metabolic activities while maintaining a low level of respiratory activity. ? 2002 International Association for Hydrogen Energy. Published by Elsevier Science Ltd. All rights reserved.
Keywords: Anaerobic adaptation; [Fe]-hydrogenase; Fermentation; Hydrogen evolution; Green algae; sulfur deprivation

1. Introduction Hydrogen gas holds the promise of providing mankind with a clean and renewable energy carrier that does not stress our environment. At present, the production of hydrogen is expensive and hardly reaches a greater output than input of energy. Photobiological H2 production by green algae would generate a renewable fuel from light and water, plentiful resources in nature [1]. [Fe]-hydrogenases were once thought to be limited to a small selection of bacteria and anaerobic living protozoa [2]. Hydrogen metabolism of photosynthetic eukaryotic algae was rstly described in the 1940s [3]. The recent isolation of the hydrogenase-encoding
Corresponding author. Tel.: +49-228-732-075; fax: +49-228731-697. E-mail address: t.happe@uni-bonn.de (T. Happe). 1 M. Winkler and A. Hemschemeier contributed equally to this work.

genes [4 6], as well as the discovery of hydrogen evolution under sulfur deprivation in C. reinhardtii [7,8] gave new impulses for both basic research and biotechnological approaches. Investigation of hydrogen pathways and screening for the most potential algal strains would lead us further on the way to an e ective photobiological hydrogen production [9]. In green algae, the [Fe]-hydrogenases are nuclear encoded, monomeric enzymes that are localized in the chloroplast [10,11]. Ferredoxin PetF, the natural electron donor, links the [Fe]-hydrogenase to the photosynthetic electron transport chain [4,12]. Electrons from reducing equivalents that are generated during fermentation are supplied into the photosynthetic electron transport chain via the plastoquinone pool. Thus, the [Fe]-hydrogenase serves as an electron valve and enables the algae to survive under anaerobic conditions. Under S-depleted and anaerobic conditions, H2 evolution seems to be the only mechanism available for the algae to generate su cient amounts of ATP which is required for

0360-3199/02/$ 22.00 ? 2002 International Association for Hydrogen Energy. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 3 6 0 - 3 1 9 9 ( 0 2 ) 0 0 0 9 5 - 2

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survival [13]. In this study, we summarize the knowledge of hydrogenases and hydrogenase-like sequences in several green algae and compare di erent algal species in their behavior to evolve hydrogen under sulfur deprivation. C. reinhardtii and Scenedesmus obliquus show some remarkable di erences in the hydrogen metabolism and in their strategy to survive S deciency.

cells. The rate of each process was recorded for about 5 min. The photosynthetic measurements were started at 10% O2 saturation and stopped at approximately 50% of O2 saturation. 2.4. Western blot analysis During sulfur depletion, samples were taken at the times indicated. The pelleted cells were boiled in sample bu er [14] for 2 min and the resulting protein extract (10 g protein=lane) was separated onto a SDS-polyacrylamide gel (10%). The proteins were blotted onto nitrocellulose membranes and the blots were incubated with a dilution of anti-spinach antibody against the delta subunit of the chloroplast ATP synthase, an anti-C. reinhardtii O-acetylserine(thiol) lyase antibody and an anti HydA antibody. The polypeptides were detected using the ECL+Plus detection system (Pharmacia Biotech) according to the manufacturers recommendations. 2.5. Detection of fermentative products After centrifugation (12 000 g for 1 min) of 1 ml cell suspension, soluble metabolites were determined in the supernatant. Ethanol was assayed with alcohol-dehydrogenase, formate with formate-dehydrogenase using test kits from Boehringer (Boehringer, Mannheim) following the instructions from the supplier.

2. Materials and methods 2.1. Algae strains, culture conditions, anaerobic adaptation and sulfur depletion Wild-type strain Chlamydomonas reinhardtii 137C (+) was originally obtained from the Chlamydomonas Culture Collection at Duke University. Both Scenedesmus species were obtained from the UTEX culture collection (S. obliquus strain 276-6 UTEX 393, Scenedesmus vacuolatus (Chlorella fusca) UTEX 251).The cells were grown photoheterotrophically in a Tris-Acetate-Phosphate (TAP) medium at 20 C under continuous irradiance of 150 mol photons m2 s1 [10]. For anaerobic adaptation, cells were harvested by centrifugation (8 min; 3500 rpm) in the mid-exponential stage of growth. The pellet was resuspended in 0.1 vol. of fresh TAP medium; the algae were anaerobically adapted by ushing the culture with argon. For sulfur depletion, cells were harvested as described above, washed with TAP-S medium (TAP in which all sulfate salts were replaced by the chloride counterparts) and suspended in TAP-S medium. For the incubation, the cells were placed into 200 ml glass bottles and sealed with suba-seals. 2.2. Hydrogen evolution assay Hydrogen evolution was quantied after separation by gas chromatography using a Hewlett-Packard 5890 A Series II equipped with a thermal conductivity detector [10]. Gas tight sample tubes containing 1:5 ml 50 mM phosphate bu er pH 6,8, 50 mM Na2 S2 O4 ; 10 mM methyl viologen and 200 l algae culture, were incubated in a shaker at 37 C for 20 min. 2.3. Measuring photosynthetic O2 evolution and respiratory oxygen uptake rates Photosynthesis was measured as net O2 evolution in a Clark-type oxygen electrode. Dark respiration was determined as O2 consumption using the same electrode. Two milliliter of the culture were taken from a sealed incubation bottle using a syringe and injected into the reaction chamber of the O2 electrode. Photosynthetic O2 evolution was measured during illumination (200 mol photons m2 s1 ) and respiratory O2 uptake was detected by darkening the

3. Results 3.1. Several algal species possess hydrogenases and a hydrogen metabolism In studies about algal fermentation, several species were described to produce hydrogen upon anaerobic adaptation, such as C. reinhardtii, Chlamydomonas moewusii, Chlorella fusca (Scenedesmus vacuolatus), and Scenedesmus obliquus [15,16]. We examined H2 production of several anaerobically adapted green algae to conrm older studies and to detect further H2 -evolving species (Table 1). The in vitro activities of C. reinhardtii (200 nmol H2 = g Chla h) S. obliquus (150 nmol H2 = g Chla h) and S. vacuolatus (155 nmol H2 = g Chla h) conrmed the values of former investigations [4,6,10], thus demonstrating that anaerobic incubated cultures of S. vacuolatus and S. obliquus generally produce hydrogen with lower rates than C. reinhardtii. Two other Chlorophyceae, Chlorella vulgaris and Dunaliella salina, developed no detectable amounts of hydrogen during anaerobic incubation, indicating the lack of an hydrogenase [6,17]. The largest hydrogenase of 55 kDa was found in the marine green alga Chlorococcum littorale [18], but the specic enzyme activity is very low compared with C. reinhardtii.

M. Winkler et al. / International Journal of Hydrogen Energy 27 (2002) 1431 1439 Table 1 Comparison of hydrogenase activity and summary of known hydrogenase genes and proteins among several green algae Species Chlamydomonas reinhardtii Chlamydomonas moewusii Chlamydomonas noctigama Lobochlamys segnis Scenedesmus obliquus Scenedesmus vacuolatus Chlorococcum littorale Chlorella vulgaris Dunaliella salina In vitro activity nmolH2 =( gChl a h) 200 460 31 96 150 155 52 No activity No activity Mature protein (kDa)=(aa) 49=(441) n.d. n.d. n.d. 44=(413) 45=(415) 55 n.d. n.d. cDNA (kb) 2,4 n.d. n.d. n.d. 2,6 2,4 n.d. n.d. n.d. Ref.

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[5,10] This study This study This study [4] [6] [18] [6] [17]

The in vitro activity represents the maximal H2 -production rate during a 4 h period of anaerobic incubation. n.d.: not determined; Ref.: references.

Algae that have been originally assigned to the genus Chlamydomonas like Chlamydomonas noctigama, and Lobochlamys segnis [19] showed only minor hydrogen production activities (13 and 96 nmol H2 = g Chla h). Interestingly, cultures of C. moewusii evolve in vitro the highest quantities of hydrogen. 3.2. Di erences in the hydrogenase activity between several green algae After anaerobic adaptation, the hydrogenases of C. reinhardtii, S. vacuolatus and S. obliquus are strongly and rapidly induced (Fig. 1A). These three algal species show a similar increase of in vitro hydrogenase activity, whereby C. reinhardtii reaches the highest rates. This data conrmed other experiments that have already shown the quick increase of hydrogenase activity during anaerobic incubation [4,10]. In the absence of sulfur, hydrogenase activity (Fig. 1B) and hydrogen gas accumulation (Fig. 1C) was detected after 24 h in cultures of C. reinhardtii and S. vacuolatus. The in vitro activity of the hydrogenase reached its maximum after 50 h and remained stable for about 120 h. The yield and rate of H2 production under S depletion increased during a 5 day period in sealed cultures of C. reinhardtii. The Western blot of Fig. 3A supports these results since the level of hydrogenase protein in C. reinhardtii remains nearly constant after the anaerobic induction. Interestingly, the in vitro hydrogenase activity under sulfur deprivation is two times higher than under anaerobic adaptation, indicating a higher protein level within the cells. In contrast, S. obliquus shows no hydrogenase activity (Fig. 1B) and no HydA

protein was found under S-depleted conditions (Fig. 3B). Furthermore, no H2 could be detected in the gas phase of the sealed cultures. This indicates that S. obliquus does not induce the hydrogenase pathway and its response to S deprivation is completely di erent from that of the two other green algae. 3.3. Photosynthetic and respiratory activity of S. obliquus compared to C. reinhardtii To gure out the physiological response of S. obliquus, we compared several metabolic features of S-deprived C. reinhardtii cells to those of S. obliquus. Removal of sulfur from the growth medium results in an inhibition of photosystem II (PSII) activity in chloroplasts and a down-regulation of photosynthetic oxygen evolution [7]. It was shown that the D1=32 kDa reaction center protein of PSII declines in C. reinhardtii with time under S deprivation because PSII repair is blocked [8,20]. Fig. 2 shows that in the absence of sulfur the activity of photosynthetic H2 O oxidation and O2 evolution declined in both green algae. H2 O oxidation in S. obliquus is reduced from 13 to about 2 mol O2 = g Chla h in the rst 50 h of S-deciency (Fig. 2A). In the beginning, C. reinhardtii cultures showed a higher photosynthetic activity of 25 mol O2 = g Chla h. Oxygen evolution rates in the light decreased slower than those of S. obliquus. After 50 h of S deprivation, photosynthetic O2 -production rates still had a value of 10 mol O2 = g Chla h and rates similar to those of S. obliquus were not reached before 100 h of S depletion.

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Fig. 2. Absolute activity of oxygenic photosynthesis (A) and mitochondrial respiration (B) under sulfur deprivation. Samples of C. reinhardtii ( ) and S. obliquus (4) cultures were taken with a syringe from the sealed cultures and injected into the chamber of a clark-type O2 -electrode. O2 -evolution was measured in the light (200 mol photons m2 s1 ) and O2 -uptake was recorded in the dark.

3.4. Fermentation and other metabolic activities under S deprivation

Fig. 1. (A) Induction of hydrogenase activity during anaerobic adaptation in C. reinhardtii ( ), S. vacuolatus ( ) and S. obliquus (4). Cells were concentrated and ushed with argon. Samples were removed at the times indicated. After breaking the cells with Triton X-100 the in vitro activity was determined as described in materials and methods. (B) Time course of H2 ase activity upon S-depletion in C. reinhardtii ( ), S. vacuolatus ( ) and S. obliquus (4). At 0 h, TAP medium was replaced by sulfur-free medium and the cultures were sealed. The in vitro H2 ase activity of the cultures was measured every 24 h. (C) H2 gas accumulation in the sealed and S-deprived C. reinhardtii ( ) and S. obliquus (4) cultures. An aliquot of the gas phase was taken and analyzed with the gas chromatograph.

The rate of mitochondrial respiration was not a ected in C. reinhardtii; the respiratory activity remained on a high level of 1216 mol O2 = g Chla h (Fig. 2B). Cellular respiration in S. obliquus starts at 6 mol O2 = g Chla h at t =0 h and declined to 0:5 mol O2 = g Chla h at t = 100 h. Sealed cultures of S. obliquus stayed aerobic (data not shown) while cultures of C. reinhardtii became anaerobic after 30 h when the activity of respiration is higher than the photosynthetic activity.

We compared the amounts of chloroplast ATP synthase and O-acetylserin(thiol)lyase (OASTL) in sulfur-deprived C. reinhardtii and S. obliquus cells. The Western blot of Fig. 3 shows that the level of ATP synthase remains constant in both species (Fig. 3). In the case of OASTL, we observed an reverse regulation of the protein. While OASTL is induced in C. reinhardtii as already described [21], a protein band is visible in the 0 48 h S-depletion period of S. obliquus which disappears after 48 h. In contrast to C. reinhardtii, the HydA protein in S. obliquus is not induced explaining the missing hydrogenase activity under sulfur deprivation. The protein composition of culture extracts from C. reinhardtii and S. obliquus under sulfur-deprived conditions (Fig. 5) showed also remarkable di erences. The protein pattern of the extracts derived from the C. reinhardtii culture indicates only to a slight change in the protein expression. However, the extracts of S. obliquus demonstrate a continuous decline of intensity and quantity in the pattern of the protein bands, suggesting a general reduction of protein expression during the incubation in sulfur-free medium.

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Fig. 3. Western Blot analysis of hydrogenase, chloroplast ATP synthase and OASTL proteins in the time course of S-depletion in C. reinhardtii (A) and S. obliquus (B). S-deprivation was continued for 5 days, samples were taken every day (all 24 h). Proteins were separated onto a SDS-polyacrylamide gel (10%) and blotted onto nitrocellulose membranes. The lters were probed with polyclonal antibodies that were specic against delta-subunit of chloroplast ATP-synthase (anti-spinach), OASTL (anti-C. reinhardtii) and HydA (anti-C. reinhardtii).

detectable in minor amounts (Fig. 4B). In contrast to this, both formate and ethanol showed a constant accumulation in C. reinhardtii cultures up to 2 g ethanol=formate= g chlorophyll.

4. Discussion 4.1. Structural characteristics of [Fe]-hydrogenases from Chlorophyceae [Fe]-hydrogenases are usually divided in two functional domains (Fig. 6) [13,22,23]. The well conserved C-terminal part (H-domain) binds the H-cluster, a catalytically active prosthetic group, consisting of a combination of one [4Fe 4S] subcluster and a uniquely structured 2Fe-subcluster, whose Fe2 -atom is supposed to represent the actual location of the catalytic activity [24]. The H-cluster is connected to the protein chain by four highly conserved cysteine residues and integrated into a hydrophobic niche of the proteins spacial structure, formed by a cascade of essential hydrophobic amino acid residues (Fig. 6) [25]. The N-terminal part of the [Fe]-hydrogenases is rather variable, but contains generally additional [4Fe 4S] or [2Fe2S] clusters (F-clusters) that mediate the electron transport between the H-cluster and the respective e-donor= acceptor. The characterized chlorophycean [Fe]-hydrogenases (Fig. 6) lack the N-terminal domain [1,13], while consisting only of the H-cluster-binding region with a leader sequence, which facilitates the transfer of this nuclear encoded enzyme into the chloroplast stroma. An additional unique feature of all investigated green algal [Fe]-hydrogenases is a polypeptide insertion in the C-terminus. The function of this insertion is still obscure, but might support a direct interaction between the electron donor (ferredoxin) and the H-cluster, therewith making dispensable any accessory FeS-clusters [6,12]. Interestingly, all enzymes that have been characterized from the order Chlorococcales possess with 16 amino residues a denitely smaller insertion

Fig. 4. Accumulation of the fermentative products ethanol (A) and formate (B) in S-deprived cultures of C. reinhardtii ( ) and S. obliquus (4). After centrifugation of 1 ml cell suspension, the supernatant was analysed for the compounds with enzymatic assays described in material and methods.

To examine the question if S. obliquus disposes excess electron power via other fermentative products than H2 , we tested culture probes on several substances that are known to be algal fermentative products, like ethanol, formate and acetate. Ethanol is only a minor product in S. obliquus and reaches amounts of 0:4 g ethanol= g chlorophyll (Fig. 4A). S. obliquus produced formate in relatively high amounts of nearly 2 g formate= g chlrophyll after about 50 h of S deprivation, but thereafter formate is only

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Fig. 5. Protein composition of cellular polypeptides from S-deprived C. reinhardtii (A) and S. obliquus (B) cultures. Gel lanes were loaded with extracts of cells with an equal amount of chlorophyll. Proteins were separated by SDS-PAGE (10%), blotted onto nitrocellulose membranes and stained with Poinceau. The stainings were made from the same blots which afterwards were used for the ATP-synthase detection (compare Fig. 3). Note the generally declining amounts and intensities of protein bands on the stained blot with protein extracts of S. obliquus.

Fig. 6. Schematic amino acid sequence comparison of the functional [Fe]-hydrogenase structure from C. pasteurianum with all characterized green algal hydrogenases (HydA) or putative hydrogenase proteins (HydB). Cysteine residues that are involved in the integration of [FeS]-clusters are symbolized by dark squares. White squares indicate amino acid positions that essentially participate in forming the hydrophobic niche (white circle) that contains the H-cluster (calotta model), or mark residues that mediate the proton transfer. The white triangles mark the cleavage site of the signal peptidase: TP, transit peptide; IR, inserted peptide region.

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than the enzymes coming from C. reinhardtii, a species belonging to the Volvocales. Whether this is a distinct di erence between both orders, or an individual feature remains to be elucidated. Very recently it was demonstrated, that C. reinhardtii [26] as well as S. obliquus [27], beside the physiologically and biochemically characterized [Fe]-hydrogenase (HydA), possess gene sequences that encode for polypeptides with all the essential attributes of an [Fe]-hydrogenase protein. These sequences were termed hydB according to the classication of hydrogenases reported by Vignais et al. [22]. The position of the H-cluster coordinating cysteine residues and other important amino acid residues [24] is highly conserved in HydA and HydB. In the case of C. reinhardtii it could be demonstrated that the expression of both genes hydA and hydB was induced by anoxic conditions and correlated with the hydrogen production activity of the induced culture [26]. To what extent each protein contributes to the physiological activity under anaerobic or sulfur-deprived conditions is still unclear. The expression of HydA from S. obliquus is induced by anaerobiosis and correlates with physiological activity during anaerobic incubation [4]. In contrast to the hydA gene, hydB was constitutively expressed in S. obliquus, independent from anaerobic conditions [27]. The physiological role of the [Fe]-hydrogenases in green algae is still being discussed. The enzymes of the three algal species C. reinhardtii, S. vacuolatus and S. obliquus are anaerobically induced. H2 production can be detected only a few minutes after the beginning of the anaerobic incubation and leads to maximal production rates of comparable intensity. Under anoxic conditions green algae switch their metabolism towards fermentation [16]. The cells of C. reinhardtii carry out a kind of mixed acid fermentation yielding H2 as one of the end products. In the light, anaerobically adapted C. reinhardtii cells produce major amounts of H2 whereas the production rates of other fermentative end products decrease [15,28]. By concentrating on this aspect, hydrogenases and H2 metabolism may be simply interpreted as a feature of fermentative metabolism (photofermentation). Recently, it was observed that sealed cultures of sulfur-deprived C. reinhardtii cells develop a high and long lasting H2 -production activity [7]. This process examined and utilized in two-stage culturing experiments of applied research gave new insights into the physiology of algal [Fe]-hydrogenases, since it indicates a further function of the hydrogenase as part of the metabolic adaptation to conditions of nutrient shortage. Under sulfur-deprivation cells of C. reinhardtii downregulate the photosynthetic O2 -evolution while the respiratory O2 uptake is nearly una ected. Thus, a sealed culture becomes anaerobic within 1 or 2 days of sulfur limitation. Under these conditions, the O2 -sensitive [Fe]-hydrogenase is functionally synthesized for maintaining the anaerobic energy metabolism. However, the reaction of C. reinhardtii

to S deciency is obviously not prototypic for all chlorophycean species. We compared the physiological responses of the three genotypically characterized algal species towards sulfur deprivation. Whereas S. vacuolatus develops a high hydrogenase activity comparable to C. reinhardtii, S. obliquus generates only minor amounts of H2 . However, S. obliquus cells survive more than one week in S-deprived medium (data not shown). There are at least three possibilities to interpret this di erence in adapting to sulfur deprivation. (1) An anoxic surrounding is a precondition for generating a functional [Fe]-hydrogenase. C. reinhardtii down-regulates the photosynthetic O2 -evolution while respiration continues, thus leading to intracellular anaerobiosis. S. obliquus may not down-regulate the photosynthetic H2 O-splitting system and therefore produce oxygen. (2) The hydrogen production of C. reinhardtii is believed to serve as an electron valve [13] which allows an undisturbed electron transport via PSI for a continuous ATP regeneration, securing the survival during a period of nutrient deprivation. Under these conditions the cells stop growing but stay metabolically active, recycling endogenous sulfur resources while generating su cient ATP via photosynthetic electron transport and respiration. S. obliquus may down-regulate the whole metabolism, bridging the time of deciency until the environment changes. (3) Since the hydrogenase is believed to serve as an electron valve for disposing excess redox power, S. obliquus might generate a di erent reduced end product instead of H2 or may even have a strategy to prevent the formation of excess reducing equivalents. As opposed to the rst explanation we could determine that the photosynthetic O2 evolution of S. obliquus was down-regulated, but unlike to C. reinhardtii the respiration rate declines, too. Thus, the culture does not develop anoxic conditions thus preventing the induction of the [Fe]-hydrogenase. In contrast to earlier studies [7] we detected signicant amounts of fermentative end-products like ethanol and formate in the S-depleted medium of C. reinhardtii. This indicates that hydrogen gas does not su ce as electron sink. Additionally, it was suggested that pyruvate resulting from glycolysis is subsequently converted to formate, acetate and ethanol, thus generating further ATP [15,16]. S. obliquus shows only a very low level of fermentative activity. The fact, that in S. obliquus under sulfur deprivation all relevant ATP-generating metabolic processes (photosynthesis, respiration, fermentation) seem to be reduced to a minimum supports the assumption, that this green alga in contrast to C. reinhardtii, down-regulates the whole cellular metabolism. This hypothesis is further supported by comparing the development of the protein composition of culture extracts from C. reinhardtii and S. obliquus during cultivation under sulfur-deprived conditions.

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M. Winkler et al. / International Journal of Hydrogen Energy 27 (2002) 1431 1439 [10] Happe T, Naber JD. Isolation, characterization and N-terminal amino acid sequence of hydrogenase from the green alga Chlamydomonas reinhardtii. Eur J Biochem 1993;214: 47581. [11] Happe T, Mosler B, Naber JD. Induction, localization and metal content of hydrogenase in the green alga Chlamydomonas reinhardtii. Eur J Biochem 1994;222: 76974. [12] Horner DS, Foster PG, Embley TM. Iron hydrogenases and the evolution of anaerobic eukaryotes. Mol Biol Evol 2000;17:1695709. [13] Happe T, Hemschemeier A, Winkler M, Kaminski A. Hydrogenases in green algae: do they save the algaes life and solve our energy problems? Trends Plant Sci 2002;7:24650. [14] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:6805. [15] Gfeller RP, Gibbs M. Fermentative metabolism of Chlamydomonas reinhardtii. 1. Analysis of fermentative products from starch in dark and light. Plant Physiol 1984;75:2128. [16] Kreuzberg KH, Martin W. Oscillatory starch degradation and fermentation in the green alga Chlamydomonas reinhardtii. Biochem Biophys Acta 1984;799:2917. [17] Cao H, Zhang L, Melis A. Bioenergetic and metabolic processes for the survival of sulfur-deprived Dunaliella salina (Chlorophyta). J Appl Phycol 2001;13:2534. [18] Ueno Y, Kurano N, Miyachi S. Purication and characterization of hydrogenase from the marine green alga, Chlorococcum littorale. FEBS Lett 1999;443:1448. [19] Proschold T, Marin B, Schlosser U, Melkonian M. Protist 2001;152:265300. [20] Wyko DD, Davies JP, Melis A. The regulation of photosynthetic electron transport during nutrient deprivation in Chlamydomonas reinhardtii. Plant Physiol 1998;117: 12939. [21] Ravina CG, Barroso C, JM V, Gotor C. Cysteine biosynthesis in Chlamydomonas reinhardtii. Molecular cloning and regulation of O-acetylserine(thiol)lyase. Eur J Biochem 1999;264:84853. [22] Vignais PM, Billoud B, Meyer J. Classication and phylogeny of hydrogenases. FEMS Microbiol Rev 2001;25:455501. [23] Peters JW, Lanzilotta WN, Lemon BJ, Seefeldt LC. X-ray crystal structure of the Fe-only hydrogenase (CPI) from Clostridium pasteurianum to 1.8 Angstrom resolution. Science 1998;282:118538. [24] Peters JW. Structure and mechanism of iron-only hydrogenases. Curr Opin Struct Biol 1999;9:6706. [25] Nicolet Y, Lemon BJ, Fontecilla-Camps J, Peters JW. A novel FeS cluster in Fe-only hydrogenases. Trends Biol Sci 2000;25:13843. [26] Forestier M, Zhang L, King P, Plummer S, Ahmann D, Seibert M, Ghirardi M. The cloning of two hydrogenase genes from the green alga Chlamydomonas reinhardtii. Proceedings of the 12th International Congress on Photosynthesis, 1823 August 2001, Brisbane, Australia, http://www.publish.csiro. au/ps2001, CSIRO Publishing, Brisbane, Australia. [27] Wunschiers R, Stangier K, Senger H, Schulz R. Molecular evidence for a Fe-hydrogenase in the green alga Scenedesmus obliquus. Current Microbiol 2001;42:35360. [28] Gfeller RP, Gibbs M. Fermentative metabolism of Chlamydomonas reinhardtii. 1. Analysis of fermentative

OASTL which is induced in S depleted C. reinhardtii cells incorporates sulde into the O-acetylserine-molecule to form cysteine [29] and is, therefore, an essential enzyme of the sulfur metabolism pathway [21]. The regulation of the level of OASTL activity by the sulfur status has also been reported in plants [30]. A rapid expression of a sulfate transport system and an accumulation of ATP-sulfurylase under sulfur-limiting conditions was shown in C. reinhardtii [31] indicating a fast response of the cells to changes in sulfur nutrients. We could demonstrate signicant di erences between the reactions of the two algal species C. reinhardtii and S. obliquus towards nutrient deprivation. The utilization of algal [Fe]-hydrogenases may be similarly variable as in the prokaryotic domains and depends on the natural environment of the algae. Acknowledgements We thank Professor R. Berzborn (University of Bochum, Germany) for a gift of ATP synthase antibodies. Financial support of the Japanese New Energy and Industrial Technology Development Organization (NEDO-project No. 01GB1) is gratefully acknowledged.

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