Planta (2008) 227:397–407 DOI 10.1007/s00425-007-0626-8

O R I G I N A L A R T I CL E

Hydrogen production by Chlamydomonas reinhardtii: an elaborate interplay of electron sources and sinks Anja Hemschemeier · Swanny Fouchard · Laurent Cournac · Gilles Peltier · Thomas Happe

Received: 11 July 2007 / Accepted: 31 August 2007 / Published online: 21 September 2007 © Springer-Verlag 2007

Abstract The unicellular green alga Chlamydomonas reinhardtii possesses a [FeFe]-hydrogenase HydA1 (EC 1.12.7.2), which is coupled to the photosynthetic electron transport chain. Large amounts of H2 are produced in a light-dependent reaction for several days when C. reinhardtii cells are deprived of sulfur. Under these conditions, the cells drastically change their physiology from aerobic photosynthetic growth to an anaerobic resting state. The understanding of the underlying physiological processes is not only important for getting further insights into the adaptability of photosynthesis, but will help to optimize the biotechnological application of algae as H2 producers. Two of the still most disputed questions regarding H2 generation by C. reinhardtii concern the electron source for H2 evolution and the competition of the hydrogenase with alternative electron sinks. We analyzed the H2 metabolism of S-depleted C. reinhardtii cultures utilizing a special mass spectrometer setup and investigated the inXuence of photosystem II (PSII)- or ribulosebisphosphate-carboxylase/oxygenase (Rubisco)-deWciency. We show that electrons for

A. Hemschemeier (&) · T. Happe Fakultät für Biologie, Lehrstuhl für Biochemie der PXanzen, AG Photobiotechnologie, Ruhr Universität Bochum, 44780 Bochum, Germany e-mail: [email protected]

H2-production are provided both by PSII activity and by a non-photochemical plastoquinone reduction pathway, which is dependent on previous PSII activity. In a RubiscodeWcient strain, which produces H2 also in the presence of sulfur, H2 generation seems to be the only signiWcant electron sink for PSII activity and rescues this strain at least partially from a light-sensitive phenotype. The latter indicates that the down-regulation of assimilatory pathways in S-deprived C. reinhardtii cells is one of the important prerequisites for a sustained H2 evolution. Keywords Chlamydomonas reinhardtii · Green algae · Hydrogen production · Photosystem II · Rubisco · Sulfur deprivation Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea FNR Ferredoxin-NADP+-reductase MIMS Membrane inlet mass spectrometer system PQ Plastoquinone PSI Photosystem I PSII Photosystem II S Sulfur Rubisco Ribulosebisphosphate-carboxylase/oxygenase

Introduction S. Fouchard Laboratoire GEPEA, CNRS, Université de Nantes, UMR 6144, Bd de l’Université, CRTT - BP 406, 44602 Saint-Nazaire Cedex, France L. Cournac · G. Peltier CEA Cadarache, DSV, IBEB, SBVME, Laboratoire de Bioénergétique et Biotechnologie des Bactéries et Microalgues, UMR 6191 CNRS/CEA/Université –Aix-Marseille, Saint-Paul-lez-Durance 13108, France

The unicellular green alga Chlamydomonas reinhardtii possesses a highly active [FeFe]-hydrogenase HydA1, which is localized in the chloroplast stroma (Happe and Naber 1993; Happe et al. 1994). HydA1 is extremely sensitive towards molecular O2 and the hydA1-gene is only expressed in the absence of O2 (Happe and Kaminski 2002; Stirnberg and Happe 2004). The enzyme is coupled

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to the reducing site of the photosynthetic electron transport chain and accepts electrons directly from ferredoxin (Florin et al. 2001; Happe and Kaminski 2002). Therefore, H2 evolution competes for electrons with other reductive pathways, CO2 assimilation above all. It was shown that short-term H2 production in C. reinhardtii is highest when the CO2-concentration is low and vice versa (Cinco et al. 1993). A signiWcant H2-production is only observed in the light, demonstrating that electrons originating from photosynthetic light reactions are the main electron source for H+reduction (GaVron and Rubin 1942; Gfeller and Gibbs 1984; Melis and Happe 2004). Earlier studies showed that PSI is indispensable for H2 production in C. reinhardtii, whereas PSII is not (Stuart and GaVron 1972; Redding et al. 1999). Despite the fact that PSII is not essential for H2 photo-evolution, it can be an electron source for the hydrogenase (Bishop and GaVron 1963; Melis et al. 2004), resulting in a simultaneous generation of H2 and O2 (Greenbaum and Lee 1998). However, this process acts only transiently (60–90 s) because the hydrogenase is inhibited by photosynthetically produced O2 (Ghirardi et al. 1997). A second pathway that can provide electrons for H2 photoproduction couples the oxidative degradation of organic substrates with photosynthetic electron transport by transferring electrons from NAD(P)H on the plastoquinone (PQ) pool via an NAD(P)H-plastoquinone-oxidoreductase (Godde and Trebst 1980; Bamberger et al. 1982; Gfeller and Gibbs 1985; Mus et al. 2005). Non-photochemical reduction of the plastoquinone pool has been observed in C. reinhardtii (Bennoun 1982; Peltier and Cournac 2002; Cournac et al. 2002) and in higher plants (Burrows et al. 1998; Horváth et al. 2000). Some years ago, Melis and co-workers showed that fully illuminated C. reinhardtii cells develop a sustained and signiWcant H2 production upon S-deprivation (Melis et al. 2000). The critical aspect of this observation is the activation of a metabolic switch, which causes an algal culture to change its physiology from aerobic photosynthetic growth to an anaerobic resting state in the light. The latter is necessary and suYcient to induce the cell’s H2 metabolism, accompanied by H2 evolution (Melis and Happe 2001; Hemschemeier and Happe 2005). In S-free medium, the cells down-regulate photosynthetic activity particularly at PSII so that O2 evolution decreases dramatically (WykoV et al. 1998; Melis et al. 2000). At the same time, respiratory activity stays high, so that a sealed algal culture establishes anaerobic conditions and starts to accumulate H2 after about 1 day. As has been observed in experiments to analyze shortterm H2-production (Gfeller and Gibbs 1985; Mus et al. 2005), electrons for the reduction of protons in S-deprived and H2-producing C. reinhardtii cells can originate from

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diVerent sources. One possible electron source is the residual water-splitting activity of PSII, the other is the import of electrons into the photosynthetic electron transport chain via non-photochemical PQ-reduction. Melis et al. (2000) proposed that the oxidation of intracellular reserves (starch, protein) is the main source of electrons for the hydrogenase pathway, while others stated PSII activity to be the essential electron donor (Kosourov et al. 2003). Recent results regarding the long-term eVect of DCMU on H2-evolution by S-depleted algal cultures demonstrated that both starch accumulation and H2-production are almost completely prevented in cultures that are treated with DCMU immediately after the transfer to S-free medium (Fouchard et al. 2005). However, C. reinhardtii cultures that were able to accumulate starch before the addition of DCMU did produce H2, indicating that PSII is essential for the build-up of starch, which in turn is indispensable for H2-generation (Fouchard et al. 2005). In this study, by recording the direct eVect of DCMU on in vivo H2-evolution rates, we deWnitely show that H2 production of S-deprived C. reinhardtii is almost equally supplied by the two electron sources PSII activity and nonphotochemical PQ-reduction. However, a fully active PSII in the Wrst hours of S-depletion is the pre-condition for maximal H2 evolution as has been postulated before (Fouchard et al. 2005). The reason for this PSII dependence probably lies in the must for the accumulation of starch, whose oxidation feeds the chlororespiratory pathways. In a Rubisco-deWcient C. reinhardtii strain, however, which also produces signiWcant amounts of H2 in S-containing medium, the hydrogenase seems to replace the Calvin cycle as electron sink. In this mutant strain, which hardly accumulates starch, electrons for H2 evolution are mainly supplied by water splitting at PSII. Analyzing the interplay of electron sources and sinks in algal H2 production is a further contribution to the understanding of the regulation of photosynthetic activity as a reaction to excess light energy and reduced electron consumption, respectively. Furthermore, these two aspects are crucial for a physiological and/or genetical manipulation of green algae as C. reinhardtii for a powerful and easily manageable biotechnological H2 generating system.

Materials and methods Algae strains and growth conditions Wild type strain C. reinhardtii 137C (+) was originally obtained from the Chlamydomonas Culture Collection at Duke University. Strain CC-2803, which is deWcient for the large subunit of Rubisco (RbcL), and the PSII-deWcient

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strain FuD7 were kindly donated by Dr. S. Purton, University College, London. C. reinhardtii cultures were grown photoheterotrophically in Tris–acetate–phosphate (TAP) medium (Harris 1989). Batch cultures were shaken at 20°C under continuous illumination (100
mol photons £ m¡2 £ s¡1). Since strain CC-2803 is light sensitive it was grown under the same conditions as the wild type but covered with paper towels. For S-deprivation, cells were harvested by centrifugation (2 min, 2,500g) in the mid-exponential stage of growth, washed once with TAP-S medium (TAP in which all sulfate compounds are replaced by the chloride counterparts) and resuspended in TAP-S medium to a Wnal density of about 20
g Chl £ ml¡1. Two hundred and ninety milliliter of cells were placed into squared glass bottles with a total volume of 320 ml that were sealed with a gas-tight septum (red rubber Suba seals 37, Sigma–Aldrich, St Louis, MO, USA). These culture vessels were then incubated under continuous one-site illumination of 100
mol photons £ m¡2 £ s¡1. The area of the Xask that directly faced the light-source was about 50 cm2. In some experiments, the Rubisco-deWcient strain CC-2803 was incubated anaerobically in TAP medium. In this case, cells were harvested as described above, resuspended in fresh TAP medium and placed into the same Xasks that were used for S-deprivation experiments. Analysis of the composition of the gas phase above the algal cultures H2 and other gases were analyzed by gas chromatography (Shimadzu GC-2010, Kyoto, Japan; equipped with a capillary column, plot fused silica 10 m £ 0.32 mm, coating mol sieve 5 Å from Varian, Palo Alto, CA, USA) or by mass spectrometry (Prisma QMS 2000, 1–100 amu, quadrupole mass spectrometer from PfeiVer Vacuum, Asslar, Germany; mounted on a turbo-molecular pumping station, and equipped with an inlet made from a UDV 146 metal valve). To analyze the composition of the gas phase, 200
l of gas were taken from the culture vessel by piercing through the septum with a gas-tight syringe (SampleLockTM Syringe, Hamilton, Reno, NV, USA) and injected into the gas chromatograph or the mass spectrometer. Detection of chlorophyll and starch The chlorophyll content of the C. reinhardtii cell suspensions was measured in 80% (v/v) acetone according to the method of Arnon (1949). Starch was determined as described before (Fouchard et al. 2005) utilizing the starch assay kit SA-20 from Sigma–Aldrich following the instructions of the supplier.

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Mass spectrometric analyses of the gas exchange in algal cultures A membrane inlet mass spectrometer system (MIMS) was used to determine the in vivo gas exchange activities of a cell suspension. The merit of this set-up is the possibility to measure evolution and uptake rates of several gases at once and almost without time delay. Therefore, diVerent metabolic processes can be observed at the same time, allowing a direct comparison without the need to take account for diVerent measuring conditions and set-ups (e.g. light intensity, temperature, disturbance of the system by entry of air etc.). For analyses, a cell sample of 1.5 ml was introduced into a measuring chamber (adapted from DW2/2 Electrode Chamber from Hansatech, Norfolk, UK) that was connected to the vacuum of a mass spectrometer (model MM 8–80, VG instruments, Cheshire, UK) by a TeXon membrane at the bottom of the chamber. Gases dissolved in the liquid above the membrane could diVuse through the thin TeXon layer and were directly introduced to the ion source of the mass spectrometer through a vacuum line. The measuring chamber was thermostated by a water jacket at 20°C, and the liquid was continuously mixed by a magnetic stirrer. Light was applied by a Wbre optic illuminator (Schott, Mainz, Germany). In this study, S-deprived C. reinhardtii cultures were prepared as described above. Samples were taken with a gas tight syringe from the incubation bottle by piercing through the septum and transferred to the measuring chamber of the mass spectrometer. To keep the entry of air as low as possible, nitrogen was blown above the cells during injection. After the closure of the chamber, it was waited until the cells had stabilized, that means, until the curves for each recorded gas were stable. Cells were continuously illuminated with 100
moles of photons £ m¡2 £ s¡1. This experimental set-up is ideal for analyzing the photosynthetic and H2 metabolism of C. reinhardtii, since O2 and CO2 producing and consuming activities can be monitored in parallel. By photosynthetic activity, the algae produce 16O2, and respiration generates 12CO2. If the heavy isotopes 18O2 and 13CO2 are added to the cells, their consumption reXects respiratory O2 uptake and photosynthetic CO2 assimilation, respectively. Using a mixture of isotopes, photosynthetic and respiratory activity of C. reinhardtii can be recorded in one sample and upon illumination, whereas in a Clark type oxygen electrode, respiratory activity has to be followed in the dark. Thus, the results obtained by this mass spectrometric set-up are more consistent to the conditions in continuously illuminated S-deprived algal cultures. 13 CO2 was applied as NaH13CO2 (0.3 mM Wnal concentration; 99% 13C isotope content, Euriso-Top, Les Ulys, France). 18O2 (95% 18O2 isotope content, Euriso-Top) was

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applied in low amounts that were just enough to detect a signiWcant signal, but which did not have an obvious eVect on hydrogenase activity. Both isotopes and DCMU (Wnal concentration 2.5 £ 10¡6 M) were injected through the capillary of the plug. Preperation of crude protein extracts and Western blot analyses Three milliliter of cells were taken gas-tightly from the algal culture and harvested by centrifugation (4,000g, 2 min). The cell pellet was resuspended in 50 mM Tris/HCl pH 8.3 plus 1% Triton-X 100 and the suspension was shaken for 30 min in the dark. Afterwards, the cell debris was removed by centrifugation (14,000 rpm, 10 min). Two hundred microliter of the supernatant were mixed with 100
l protein lysis buVer (0.25 M Tris–HCl, pH 8.0; 25% glycerol; 7.5% SDS; 0.25 mg £ ml¡1 bromophenolblue; 12.5% (v/v) 2-mercaptoethanol) and heated for 5 min at 95°C. Warm protein extracts were loaded onto the gels. SDSpolyacrylamide-gelelectrophoresis (SDS-PAGE) was conducted as described before (Laemmli and Favre 1973) using 10% separating and 5% collecting gels. Proteins were transferred to a nitrocellulose membrane and HydA1 protein was detected by polyclonal anti-C. reinhardtii-HydA1 antibodies as described before (Happe et al. 1994). Chemiluminescence was detected by the FluorChem 8800 apparatus from Alpha Innotech, San Leandro, CA, USA.

Results An important parameter aVecting partitioning between PSII-dependent and PSII-independent H2 evolution pathways is the time at which PSII is switched oV (Fouchard et al. 2005). In a PSII-deWcient C. reinhardtii strain (FuD7), or when DCMU was added immediately after the transfer of a wild type culture to S-free medium, no signiWcant H2 production was observed (Fig. 1), although anaerobiosis was reached a few hours after sealing the cultures and the cells showed a high level of hydA1 gene expression and in vitro hydrogenase activity (data not shown). However, when DCMU was added to an S-deprived C. reinhardtii wild type culture at the time point when photosynthesis was already down regulated (as measured by O2 evolution in the light in an O2 electrode), a signiWcant H2 evolution was observed. After 96 h of incubation in S-free medium, the H2 yield of a culture treated with DCMU just at the beginning of the H2 production phase was about 40% of the amount produced by untreated cells (Fig. 1). Obviously, active PSII in the Wrst hours of S-depletion is essential for H2 generation, while it is partially dispensable

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Fig. 1 H2 accumulation of S-deprived C. reinhardtii wild type (dark shaded circle), the wild type treated with DCMU at h = 0 (open diamond), the PSII deWcient strain FuD7 (Wlled diamond) and the wild type treated with DCMU at h = 17 (light shaded circle). The 100% maximum of the y-axis is deWned as the sum of the gases that were detected in the individual samples (mol-%). Cultures were transferred to S-free medium, and DCMU was added in a Wnal concentration of 2.5
M to one wild type culture after 0 h (white arrow) and to another wild type culture after 17 h of incubation in S-free medium (grey arrow). Samples of the gas phase were taken with a syringe and injected into a gas chromatograph. Cultures were of the same chlorophyll content (20
g £ ml¡1). Depicted values show the means of three independent experiments

after H2 evolution has already started. To determine the direct contribution of PSII activity to the H2 production process during a typical S-depletion experiment, the eVect of the PSII inhibitor DCMU on H2-evolution rates was determined by using the MIMS system. C. reinhardtii culture aliquots were sampled at diVerent time intervals after S-depletion (Fig. 2). The respective H2 production rate was Wrst recorded for several minutes. Then DCMU was added. The drop in the H2 production rate in response to DCMU addition indicated the direct contribution of electrons provided by PSII activity to H2 generation. Figure 2 shows that the involvement of PSII in H2 production increased from 20% (24 h) to 35% (48 h) and then declined. This experiment was repeated several times using S-depleted C. reinhardtii cultures with diVerent initial cell densities. The inhibitory eVect of DCMU was found to vary from 0 to 60%, the lowest inhibitory eVect of DCMU being observed in low-density cultures (»17
g Chl £ ml¡1), and the highest in more dense cultures (»27
g Chl £ ml¡1) (data not shown). The experiment shown in Fig. 2 corresponds to the average of three experiments with intermediate cell density (»22
g Chl £ ml¡1). These results show that H2 production depends both on PSII activity and on electron supply by non-photochemical PQ reduction, and that the balance between these two processes strongly depends on parameters (such as light interception by the culture) aVected by cell density. It has been shown previously that the addition of DCMU to S-deprived C. reinhardtii cultures directly after the transfer to S-free medium abolishes starch accumulation (Fouchard et al. 2005). The starch content of S-deprived

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Fig. 2 In-time eVect of DCMU on in vivo hydrogenase activity in Sdepleted C. reinhardtii cultures of 23
g Chl £ ml¡1. Measurements were performed in a MIMS set-up as described in detail in Materials and methods. Cell samples were taken from the incubation bottle with a gas-tight syringe and transferred to the illuminated measuring chamber of the mass spectrometer at the depicted time points. Hydrogenase activity was recorded for several minutes until the rate was stable (dark shaded square). Then, DCMU (2.5
M) was added, and the H2 evolution rate was again recorded for several minutes (light shaded square). Finally, light was shut oV to measure the background of dark H2 production (data not shown). The eVect of DCMU was controlled by recording the O2 evolution rate, which became zero after the application of DCMU (data not shown)

C. reinhardtii wild type cells usually increases signiWcantly (up to tenfold) in the Wrst hours of S-depletion (Melis et al. 2000; Fouchard et al. 2005). Figure 3 shows that the breakdown of the accumulated starch reserves starts coincidentally with the onset of H2 production (Fig. 3), indicating a role of the breakdown of carbon reserves in H2 evolution. To further investigate the role of starch accumulation on H2 generation we studied H2 production in a Rubisco-deWcient strain (CC-2803), which was not expected to accumulate starch. Additionally, the phenotype of strain CC-2803 reXects the physiological state of the C. reinhardtii wild type after 2–3 days of S-starvation. In S-deprived wild type

Fig. 3 Hydrogenase activity (dark shaded square) in nmoles H2 £ h¡1 £
g Chl¡1 and starch content (light shaded square) in
g £
g Chl¡1 of the C. reinhardtii wild type incubated in S-free medium (20
g Chl £ ml¡1). Hydrogenase activity was determined in a sample of cells taken with a syringe and injected in the measuring cell of a mass-spectrometer. Starch content was measured from frozen pellets of cell-samples taken from the incubation bottle with a syringe at the depicted time-points. The displayed data are means of Wve independent series of measurements

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cells, the Rubisco protein is degraded (Zhang et al. 2002) and the culture accumulates CO2. Utilizing the MIMS system, by application of 13CO2, we determined the eVective CO2-Wxing activities of S-depleted C. reinhardtii cells, which decreased in parallel to photosynthetic O2-evolution rates and became undetectable after 2–3 days of S-starvation (data not shown). Therefore, we additionally used the Rubisco-deWcient C. reinhardtii strain to study a possible competition between CO2 assimilation an H2 production. The most remarkable characteristic of strain CC-2803 is its ability to produce H2 in the presence of sulfur (Fig. 4a). As expected, no signiWcant starch accumulation was detected in strain CC-2803, neither in the presence nor in the absence of sulfur. The mutant strain contained a basal level of starch (about 2–3
g starch £
g Chl¡1), which did not change signiWcantly during incubation in S-free or S-containing medium (Fig. 4a). However, both in the presence or the absence of sulfur, H2 evolution occurred very quickly after the culture had been sealed. H2 production rates of strain CC-2803 were quite similar in both media during the Wrst 10 h of incubation, reaching 1.8 nmoles H2 £ h¡1 £
g Chl¡1 at the beginning of the experiment and rising to 4 nmoles H2 £ h¡1 £
g Chl¡1 after 10 h (Fig. 4b). In S-containing medium, H2 production rates increased further to 5.2 nmoles H2 £ h¡1 £
g Chl¡1 at t = 24 h, whereas they started to decrease in the S-starved culture. After this time point, H2 production rates in both cultures of strain CC-2803 decreased steadily. As for the wild type, the eVect of DCMU on H2 production rates was analyzed by taking samples during the time course of these H2 production experiments. In contrast to the partial inhibitory eVect of DCMU on H2 evolution rates in the C. reinhardtii wild type (Fig. 2), the H2 production activities of strain CC-2803 were almost completely inhibited by DCMU both in S-containing and S-free medium and were therefore essentially PSII-dependent (Fig. 4b). Immunodetection analyses performed using HydA1 antibody showed that the CC-2803 mutant strain started synthesizing and accumulating HydA1 protein very early (1 h after the transfer to fresh TAP or TAP-S medium and gastight incubation) (Fig. 4c). Since hydrogenase expression requires anaerobic conditions, the presence of HydA1 protein a couple of hours after the culture vessel had been sealed indicated that the Rubisco-deWcient strain CC-2803 quickly reached anoxia. Indeed, the O2 concentration in the headspace of cultures of the Rubisco-deWcient strain decreased rapidly either in the presence or in the absence of sulfur, whereas more than 40 h were needed to signiWcantly diminish the O2 level in a wild type culture of the same chlorophyll content (Fig. 5). In contrast to the C. reinhardtii wild type (Fouchard et al. 2005), strain CC-2803 passes into anoxia both in the presence and the absence of acetate in the culture medium (data not shown).

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Fig. 5 O2-concentration in the headspace of of the C. reinhardtii wild type (Wlled circle) and the Rubisco-deWcient strain CC-2803 in S-free medium (open triangle), as well as CC-2803 in fresh S-containing medium (Wlled triangle). The experiment was repeated four times and the cultures always had an initial chlorophyll content of 21
g Chl £ ml¡1. Samples of the gas phase were taken at the depicted time points with a gas-tight syringe and analyzed by gas-chromatography

Fig. 4 a H2-accumulation and starch content of cultures of the Rubisco-deWcient C. reinhardtii strain CC-2803 in S-free and in fresh S-containing medium (the depicted values are means of three independent experiments; initial chlorophyll contents were always 20
g £ ml¡1) (dark shaded circle H2-accumulation +S, dark shaded diamond starch content +S, light shaded circle H2-accumulation ¡S, light shaded diamond starch content ¡S). Samples of the gas phases were taken at the depicted time-points and analyzed by gas chromatography. Cell samples for starch detection were taken and treated as described for Fig. 3 and in Materials and methods. b Inhibitory eVect of DCMU on in vivo H2 production in the Rubisco-deWcient strain CC-2803 (20
g Chl £ ml¡1). Samples were analyzed in a MIMS system as described for the wild type in Fig. 2 (dark shaded square H2-evolution rate in +S, light shaded square H2-evolution rate in ¡S, dark shaded triangle H2-evolution rate in +S after addition of DCMU; light shaded triangle H2-evolution rate in ¡S after addition of DCMU). c Western Blot analysis of crude protein extracts of strain CC-2803 in S-containing and S-depleted medium (+S, ¡S). 3 ml of cells were removed with a gas-tight syringe from the incubation Xask at the depicted time-points and harvested by centrifugation. The cells were lysed with Triton X-100, and after removing the cell debris, the supernatant was mixed with protein sample buVer. HydA1 was detected by a polyclonal antibody raised against C. reinhardtii HydA1. Time 0 on the x-axis refers to the time when the cultures were sealed

To analyze O2 Xuxes occurring during the time-course of S-deprivation both in wild type and the Rubisco deWcient strain, we used 18O-enriched O2 and a MIMS (Fig. 6). In contrast to measurements with O2 electrodes, the MIMS set-up allows simultaneous measurement of eVective O2 uptake and O2 evolution rates in the light by following the concentrations of the diVerent O2 species (18O2 and 16O2) in the cell suspension. In the wild type, the actual photosynthetic O2 evolution (which is a true measurement of PSII activity) strongly decreased in response to S-deprivation as previously

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observed for net O2 exchange measurements (WykoV et al. 1998; Melis et al. 2000). The rate of photosynthesis decreased by more than 90% after 24 h and by 97% after 48 h in this set of experiments with an initial chlorophyll content of the cultures of 19
g Chl £ ml¡1 (Fig. 6a). In the following 48 h, there was no further signiWcant decline in the O2 production rate. O2 uptake rates measured in the light also declined in the time course of S-starvation, but respiratory rates decrease signiWcantly less than photosynthetic rates. Because of the diVerent progress of O2 producing and consuming rates, O2 uptake overcame O2 evolution after about 18 h of S-deprivation in this experiment (Fig. 6a). In the Rubisco-deWcient strain CC-2803, O2 uptake rates were higher than O2 evolution rates throughout the time courses of the experiments, in either the presence or the absence of sulfur (Fig. 6b, c). Both O2 producing and consuming activities decreased steadily in the course of the experiment, and they were comparable in S-free and S-containing medium (Fig. 6b, c). The ratio of O2 production to O2 consumption was always lower than one, showing that O2 was removed more eYciently than it was produced. This was in accordance with the development of the O2 concentrations in the headspace of the cultures (Fig. 5). Interestingly, both in the wild type and in the RubiscodeWcient C. reinhardtii strain, O2 uptake seemed to be stimulated by light during the Wrst 40 h of incubation, when O2 uptake rates in the light were 1.2–1.5 times higher than in the dark. Only in the samples taken after 48 h of incubation, O2 uptake rates were more or less equal in the light or in the dark (Fig. 6). Finally, a further remarkable behavior of the RubiscodeWcient C. reinhardtii strain CC-2803 should be noted. Cultures of this strain did not bleach during the experiments in gas-proof bottles, even though the cells encountered the same light intensity than the wild type (100
moles of

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Fig. 7 Photograph of C. reinhardtii CC-2803 cultures after 5 days of anaerobic (left) or aerobic (right) incubation in the light in S-containing TAP-medium. Cells were Wrst grown in aerated and shaded Erlenmeyer Xasks until they had reached a chlorophyll content of 18
g Chl £ ml¡1. Then they were transferred to the depicted squared glass bottles and incubated in the light. The aerated culture shows signiWcant chlorosis after 5 days

Fig. 6 Photosynthetic (white symbols) and respiratory rates detected in the dark (black symbols) or in the light (gray symbols) by measuring 16 O2-production and 18O2-consumption, respectively. a Rates of the C. reinhardtii wild type upon S-deprivation, b rates of strain CC-2803 in S-free medium, c rates of strain CC-2803 in S-containing medium. Cell samples were taken from the incubation bottle at the depicted time points and transferred to the measuring chamber of the MIMS set-up. The cell suspension was continuously illuminated with 100
moles of photons £ m¡2 £ s¡1. O2 evolution and uptake rates were detected by 16 O2 production (photosynthesis) and 18O2 uptake (respiration). 18O2 uptake was Wrst measured in the light and then in the dark. The displayed data are means of three independent experiments. All cultures were of the same initial chlorophyll content (19
g Chl £ ml¡1)

photons £ m¡2 £ s¡1 from one site). In contrast, cultures of this strain did not grow in shaken and aerated Erlenmeyer Xasks in the light unless they were covered with paper towels. Furthermore, already grown cultures of strain CC-2803 that were placed in the light and aerated showed photobleaching very soon (Fig. 7).

Discussion Within one or two days after being deprived of sulfur, cultures of the unicellular green alga C. reinhardtii radically change their physiology from aerobic photosynthetic

growth to an anaerobic resting state. Because H2 evolution by C. reinhardtii is regarded as a promising biotechnological approach for the production of H2 as an energy carrier (Melis and Happe 2001), the question regarding what is the best electron source for H2 production has attracted much interest. Detailed knowledge of these electron pathways would probably allow manipulation and thus enhancement of the electron supply, resulting in a higher H2-yield. Some previous studies and the results obtained in this work show that H2 evolution is both dependent on residual PSII activity and non-photochemical PQ reduction. The latter, however, depends on photosynthetic activity during the initial phase of S-deprivation. A PSII-deWcient strain (FuD7) and the C. reinhardtii wild type treated with DCMU from the beginning of S-deprivation on do not produce signiWcant amounts of H2, although they pass into anoxia immediately after the closure of the incubation vessel and synthesize large amounts of active hydrogenase enzyme as shown by in vitro hydrogenase activity assays (data not shown). These results could be interpreted in terms of PSII being the major electron donor for H2 production, as has been proposed by others (Antal et al. 2003; Kosourov et al. 2003). On the other hand, since S-depleted C. reinhardtii cultures accumulate starch and proteins in the Wrst days and degrade them afterwards, it was postulated that electrons for H2-production are mainly supplied by the oxidation of these organic reserves (Melis et al. 2000). EYcient starch biosynthesis depends on the electrons extracted from water to be used for CO2 Wxation in the Calvin cycle. Therefore, it

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was reasoned that PSII activity in the initial aerobic phase of S-starvation is important for subsequent H2-evolution because of its contribution in starch accumulation. These considerations led to a detailed analysis of the correlation of starch accumulation and H2 production with the time point of DCMU addition (Fouchard et al. 2005). According to predictions, it was shown that the addition of DCMU prevented starch accumulation in S-deprived C. reinhardtii, but if DCMU was added to a culture after 24 or 48 h, starch did accumulate. The amount of H2 produced by S-deprived C. reinhardtii cultures correlated strongly with the amount of starch that had been synthesized before the addition of DCMU (Fouchard et al. 2005). As shown in this study, the addition of DCMU to an S-deprived C. reinhardtii culture, which had just started to produce H2 decreased H2-yields by roughly 60%. The importance of starch for H2 production has also been shown by the impaired H2 evolution rates of C. reinhardtii mutant strains deWcient for isoamlyase or ADP-glucose pyrophosporylase (Posewitz et al. 2004). If these enzymes, which are involved in starch hydrolysis and biosynthesis, respectively, were absent, the initial rate of H2 evolution in anaerobically adapted C. reinhardtii was shown to be signiWcantly lower than in the wild type. Furthermore, the enhanced H2 accumulation of C. reinhardtii mutant strain Stm6 is discussed to rely at least partially on the increased starch content of the cells (Kruse et al. 2005). Though, it was postulated that the degradation of organic reserves could rather be the primary source of reductant for respiratory processes, while residual PSII activity being the major source for H2 generation (Kosourov et al. 2003). The use of a MIMS system in this study, however, oVered the possibility to measure the direct eVect of the PSII inhibitor DCMU on in vivo H2 production by S-depleted C. reinhardtii cells. The prompt inhibitory eVect of DCMU on in vivo H2-production varied between 0 and 60%, showing that 40–100% of in vivo hydrogenase activity are independent of PSII activity and that PSII is only partially involved in ongoing H2 photoproduction by S-depleted C. reinhardtii cells. During several experiments to determine the direct (realtime) eVect of DCMU on H2 evolution rates it turned out that the inhibitory eVect of DCMU varied strongly. The rough trend that could be observed indicates that DCMU inhibition of H2 production was strongest in cultures with a high-chlorophyll content. This can be explained by the reduced accumulation of starch in higher-density cell cultures due to a less eYcient quantum yield of photosynthesis in self-shadowing dense cultures. This phenomenon has yet to be analyzed in more detail. However, it can be concluded from the phenomena described here that C. reinhardtii cells accumulate starch in the Wrst hours of S-deprivation with reductant provided by

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PSII activity. In the H2-producing phase, starch is degraded and the electrons are transferred to the photosynthetic chain by non-photochemical PQ reduction. The data presented in this study support the model that was proposed when the H2 metabolism of S-deprived C. reinhardtii was Wrst published. This special metabolism of the alga was called the “two stage H2 production process”, in which initial (aerobic) photosynthetic assimilation of organic reserves (stage 1) is temporarily separated from (anaerobic) H2 production at the expense of previously synthesized cellular substrates (stage 2) (Melis et al. 2000). Of course, it cannot be excluded that some of the products of starch and protein catabolism which takes place during the H2-production phase of S-deprived C. reinhardtii cells are substrates for respiration as has been suggested by others (Kosourov et al. 2003). However, S-deprived algae that are cultivated autotrophically do also accumulate starch, but they are obviously unable to establish anaerobic conditions and consequently do not produce H2 (Fouchard et al. 2005 and unpublished results). On the other hand, acetate is probably not a direct electron source for H2-production, since above a threshold of 10 mM, initial medium acetate concentrations up to 40 mM do not have an eVect on the establishment of anaerobiosis neither on the H2 yield (our unpublished data). Therefore, we suggest that electrons originating from starch degradation are mainly transferred to the H2-production pathway whereas acetate is the main substrate for respiration. The H2 metabolism of the C. reinhardtii Rubisco-deWcient strain CC-2803 is completely diVerent than that of the wild type. Strain CC-2803 has a generally decreased photosynthetic activity due to the absence of the Rubisco protein, one of the most important electron sinks of photosynthesis. Linear photosynthetic electron transport produces reduced ferredoxin, which can pass the electrons to several further acceptors. Most of the electrons are used to reduce NADP+ to NADPH by ferredoxin-NADP+ reductase (FNR). The majority of the NADPH is consumed by reductive CO2 assimilation in the Calvin cycle via the Rubisco enzyme. It was shown that the absence of FNR or Rubisco leads to signiWcantly lower photochemical quenching and a decrease in overall photosynthetic electron transport in transgenic tobacco (Hajirezaei et al. 2002; Allahverdiyeva et al. 2005). Furthermore, the removal of the most important electron sink leads to increased photoinactivation, a decreased recovery from photoinhibition and overall oxidative stress in tobacco (Palatnik et al. 2003) and C. reinhardtii (Takahashi and Murata 2005). Chlamydomonas reinhardtii strain CC-2803 produces H2 not only in the absence, but also, and at higher levels, in the presence of sulfur. Furthermore, hydrogenase activity and H2 evolution, respectively, start very soon after the

Planta (2008) 227:397–407

transfer of a CC-2803 culture into a gas-proof culture vessel. One of the preconditions for hydrogenase activity, anaerobiosis, is established quite fast in cultures of this C. reinhardtii Rubsico-deWcient strain. The O2 exchange rates of CC-2803 reveal that PSII activity of this strain is very low, even in cultures supplied with sulfur. In contrast, photosynthesis is down regulated as a reaction to nutrientdeprivation in the wild type. O2 evolution in C. reinhardtii CC-2803 is always exceeded by O2 consumption, so that cultures of this Rubisco-deWcient strain become anaerobic as soon as they are cut oV from the air (i.e., O2) supply. The wild type establishes anaerobic conditions only after one or two days of S-deprivation, as respiratory activity exceeds photosynthetic O2-evolution. The low photosynthetic activity of strain CC-2803 is probably the reason for the ability of these cells to pass into anoxia in the absence of acetate, whereas autotrophically cultivated wild type C. reinhardtii cultures are unable to remove oxygen eYciently. As shown by DCMU addition during MIMS measurements, in C. reinhardtii strain CC-2803, H2 production is almost completely dependent on electron supply by PSII. Obviously, the hydrogenase is the only eYcient electron sink for this strain, which is further indicated by the fact that H2 producing CC-2803 cultures are partially rescued from the light-sensitive phenotype usually observed in strain CC-2803. At this point it should be noted that diVerent results regarding H2 production were obtained using strain CC2653 (White and Melis 2006). This strain contains a truncated form of the large subunit of Rubisco, while in strain CC-2803 the rbcL-gene is completely deleted. White and Melis (2006) postulated a role of the catabolism of Rubisco for H2-photoproduction. Degradation products of the Rubisco protein, which exists in large quantities in the chloroplast and is degraded in the wild type during S-depletion (Zhang et al. 2002), could be recycled in some step of the anaerobic metabolism in S-deprived C. reinhardtii cells and have an inXuence on H2 evolution. This might explain the diVerent behavior of the two strains, since one strain (CC2653) produces at least fragments of Rubisco while the other (CC-2803) does not. However, the behavior of the Rubisco-deWcient C. reinhardtii strain CC-2803 suggests that the lack of eYcient electron sinks is one of the parameters that initiate or at least accelerate the drastic physiological changes occurring in S-deprived C. reinhardtii wild type cultures. It was proposed that the decreased activity of PSII in Sdeprived C. reinhardtii cells is not a consequence of an electron “tailback” due to a decreased activity of the Calvin cycle, since it could also be demonstrated in isolated thylakoid membranes in the presence of artiWcial electron acceptors (WykoV et al. 1998). The loss of active PSII reaction

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centers was mainly attributed to an impaired turnover of the D1-protein due to the depletion of sulfurous amino acids (WykoV et al. 1998; Chen et al. 2005). Another study showed that S-depletion causes a slow decrease of the photochemical activity of PSII in the Wrst (aerobic) hours. However, a rapid inactivation of PSII was observed at the onset of anaerobiosis (Antal et al. 2003). The fast inactivation of PSII could be restored by aeration or by addition of external electron acceptors like nitrate or sulfate (Antal et al. 2003, 2004). Summarizing the available literature, PSII inactivation in S-deprived C. reinhardtii cells seems to be a concurrence of decreased PSII repair and PSII photodamage due to less eYcient electron disposal that occurs in anaerobiosis. Studies about the eVect of Calvin cycle inactivation on PSII activity (Palatnik et al. 2003; Takahashi and Murata 2005) and the behavior of the Rubisco-deWcient C. reinhardtii strain CC-2803 analyzed in this study further emphasize the signiWcant role of replenished electron sinks that cause a less eYcient oxidation of the PQ pool and subsequently an accelerated irreversible inactivation of PSII. The exceptional behavior of the Rubisco-deWcient C. reinhardtii strain oVers further clues to the advantages of the hydrogenase pathway for the wild type. This anaerobic plastidic electron “valve” hydrogenase replaces biosynthetic electron sinks and allows the disposal of electrons. Thus, the electron potential within the photosynthetic chain can be relaxed, preventing oxidative and radical damage. The hydrogenase facilitates continuous photosynthetic electron transport, allowing the synthesis of ATP. S-depleted C. reinhardtii cells are able to degrade accumulated starch and transfer the electrons to the hydrogenase pathway. Thereby, the cells can degrade excess carbon reserves and beneWt from a further means of generating ATP. The residual electron transport processes at PSII might also be an instrument to keep some PSII centres active, so that the cells can generate reductant as soon as suYcient nutrients are available again. One interesting by-result arised by utilizing the MIMS technique applied in this work, which allowed to distinguish between O2-uptake in the light and in the dark. It turned out that respiration of S-deprived C. reinhardtii cells is stimulated in the light only in the Wrst two days of Sdepletion. Since this stimulatory eVect can also be observed in the Rubisco-deWcient strain CC-2803, it should not be due to photorespiration. It could either rely on O2 reduction by PSI (Mehler reaction), on stimulation of respiration caused by a higher concentration of O2 or reductant in photosynthesizing cells (Xue et al. 1996), or on stimulation of PQ oxidase activity at the thylakoid level (Cournac et al. 2000). Both the latter explanations would be in line with the stimulation of light respiration only in the Wrst days of S-deprivation, since here, the PSII activity is still relatively high, resulting in the production of reductant and O2.

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Summarizing this study, it provides new insights into the interplay of photosynthetic electron sources and sinks in hydrogenase possessing green algae, and it also contributes to the optimization of the biotechnological application of H2 production by photosynthetic microorganisms. First, it can be reasoned that enhancing the accumulation of intracellular carbon reserves by the cells could result in a higher H2 output. Second, the design of a strain in which the Calvin cycle can be systematically turned oV and on could allow cyclic H2 production without the need to deprive the cells of sulfur. This approach has to be coupled with attempts to inducibly down-regulate PSII activity, to circumvent oxidative stress of the cells by switching oV electrons sinks in the presence of fully active PSII and to allow the establishment of anaerobic conditions by lowering photosynthetic O2 evolution. Acknowledgments This work was supported by DAAD (PPP with France, PROCOPE) and by the European Commission (6th FP, NEST STRP SOLAR-H contract 516510). A. Hemschemeier and T. Happe were further supported by the Deutsche Forschungsgemeinschaft (SFB 480). G. Peltier, L. Cournac and S. Fouchard were also supported by the Agence Nationale de la Recherche (projet PHOTOBIOH2).

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