Autotrophic and mixotrophic hydrogen photoproduction in sulfur-deprived chlamydomonas cells

In Chlamydomonas reinhardtii cells, H2 photoproduction can be induced in conditions of sulfur deprivation in the presence of acetate. The decrease in photosystem II (PSII) activity induced by sulfur deprivation leads to anoxia, respiration becoming higher than photosynthesis, thereby allowing H2 production. Two different electron transfer pathways, one PSII dependent and the other PSII independent, have been proposed to account for H2 photoproduction. In this study, we investigated the contribution of both pathways as well as the acetate requirement for H2 production in conditions of sulfur deficiency. By using 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a PSII inhibitor, which was added at different times after the beginning of sulfur deprivation, we show that PSII-independent H2 photoproduction depends on previously accumulated starch resulting from previous photosynthetic activity. Starch accumulation was observed in response to sulfur deprivation in mixotrophic conditions (presence of acetate) but also in photoautotrophic conditions. However, no H2 production was measured in photoautotrophy if PSII was not inhibited by DCMU, due to the fact that anoxia was not reached. When DCMU was added at optimal starch accumulation, significant H2 production was measured. H2 production was enhanced in autotrophic conditions by removing O2 using N2 bubbling, thereby showing that substantial H2 production can be achieved in the absence of acetate by using the PSII-independent pathway. Based on these data, we discuss the possibilities of designing autotrophic protocols for algal H2 photoproduction.     

The effect of light intensity on hydrogen production by sulfur-deprived Chlamydomonas reinhardtii

The effect of light intensity on hydrogen production by sulfur-deprived Chlamydomonas reinhardtii was studied in situ using either long- or short-term experiments, or alternatively, with samples withdrawn from the photobioreactor. Overall hydrogen production by S-deprived culture was shown to depend on the light intensity and to exhibit regions of light limitation and light inhibition. The optimal incident light intensity for hydrogen production was independent of the method of sulfur deprivation or the initial acetate concentration in the medium (12-34 mM). However, it varied with the Chl concentration and the thickness of the photobioreactor. To calculate the average light intensity in the photobioreactor under different experimental conditions, a special mathematics approach was developed. The optimal average light intensity for H(2) production appeared to be 30-40 microE m(-2)s(-1) and was independent of the Chl or acetate concentrations and the method of S deprivation. The inhibitory effect of high light intensity was related to the enhanced O(2) evolution activity during the photosynthetic stage of sulfur deprivation and to the high activity of photosystem II at the beginning of the H(2)-production phase. Data support the major role of photosystem II in supplying reductants through photosystem I to the hydrogenase throughout the H(2)-production phase.     

H(2) photoproduction by batch culture of Anabaena variabilis ATCC 29413 and its mutant PK84 in a photobioreactor.

Hydrogen production by Anabaena variabilis ATCC 29413 and of its mutant PK84, grown in batch cultures, was studied in a photobioreactor. The highest volumetric H(2) production rates of native and mutant strains were found in cultures grown at gradually increased irradiation. The native strain evolved H(2) only under an argon atmosphere with the actual rate as high as the potential rate (measured in small vials under optimal conditions). In this case 61% of oxygenic photosynthesis was used for H(2) production. In contrast the mutant PK84 produced H(2) during growth under CO(2)-enriched air. Under these conditions at the maximum rate of H(2) production (10 mL h(-1) L(-1)), 13% of oxygenic photosynthesis was used for H(2) production and the actual H(2) production was only 33% of the potential. Under an atmosphere of 98% argon + 2% CO(2) actual H(2) production by mutant PK84 was 85% of the potential rate and 66% of oxygenic photosynthesis was used for H(2) production. Hydrogen production under argon + CO(2) by the mutant was strictly light-dependent with saturation at about 300 microE m(-2) s(-1). However, the rate of photosynthesis was not saturated at this irradiation. At limiting light intensities (below 250 microE m(-2) s(-1)) 33-58% of photosynthesis was used for H(2) production. Hydrogen evolution by PK84 under air + 2% CO(2) was also stimulated by light; but was not saturated at 332 microE m(-2) s(-1) and did not cease completely in darkness. The rate of oxygen photoevolution was also not saturated. A mechanism for increasing cyanobacterial hydrogen production is proposed.     

A comparison of hydrogen photoproduction by sulfur-deprived Chlamydomonas reinhardtii under different growth conditions

Continuous photoproduction of H(2) by the green alga, Chlamydomonas reinhardtii, is observed after incubating the cultures for about a day in the absence of sulfate and in the presence of acetate. Sulfur deprivation causes the partial and reversible inactivation of photosynthetic O(2) evolution in algae, resulting in the light-induced establishment of anaerobic conditions in sealed photobioreactors, expression of two [FeFe]-hydrogenases in the cells, and H(2) photoproduction for several days. We have previously demonstrated that sulfur-deprived algal cultures can produce H(2) gas in the absence of acetate, when appropriate experimental protocols were used (Tsygankov, A.A., Kosourov, S.N., Tolstygina, I.V., Ghirardi, M.L., Seibert, M., 2006. Hydrogen production by sulfur-deprived Chlamydomonas reinhardtii under photoautotrophic conditions. Int. J. Hydrogen Energy 31, 1574-1584). We now report the use of an automated photobioreactor system to compare the effects of photoautotrophic, photoheterotrophic and photomixotrophic growth conditions on the kinetic parameters associated with the adaptation of the algal cells to sulfur deprivation and H(2) photoproduction. This was done under the experimental conditions outlined in the above reference, including controlled pH. From this comparison we show that both acetate and CO(2) are required for the most rapid inactivation of photosystem II and the highest yield of H(2) gas production. Although, the presence of acetate in the system is not critical for the process, H(2) photoproduction under photoautotrophic conditions can be increased by optimizing the conditions for high starch accumulation. These results suggest ways of engineering algae to improve H(2) production, which in turn may have a positive impact on the economics of applied systems for H(2) production.     

Synechocystis sp PCC 6803 strains lacking photosystem I and phycobilisome function.

To design an in vivo system allowing detailed analysis of photosystem II (PSII) complexes without significant interference from other pigment complexes, part of the psaAB operon coding for the core proteins of photosystem I (PSI) and part of the apcE gene coding for the anchor protein linking the phycobilisome to the thylakoid membrane were deleted from the genome of the cyanobacterium Synechocystis sp strain PCC 6803. Upon transformation and segregation at low light intensity (5 microE m-2 sec-1), a PSI deletion strain was obtained that is light tolerant and grows reasonably well under photoheterotrophic conditions at 5 microE m-2 sec-1 (doubling time approximately 28 hr). Subsequent inactivation of apcE by an erythromycin resistance marker led to reduction of the phycobilin-to-chlorophyll ratio and to a further decrease in light sensitivity. The resulting PSI-less/apcE- strain grew photoheterotrophically at normal light intensity (50 microE m-2 sec-1) with a doubling time of 18 hr. Deletion of apcE in the wild type resulted in slow photoautotrophic growth. The remaining phycobilins in apcE- strains were inactive in transferring light energy to PSII. Cells of both the PSI-less and PSI-less/apcE- strains had an approximately sixfold enrichment of PSII on a chlorophyll basis and were as active in oxygen evolution (on a per PSII basis) as the wild type at saturating light intensity. Both PSI-less strains described here are highly appropriate both for detailed PSII studies and as background strains to analyze site- and region-directed PSII mutants in vivo.     

Autotrophic and Mixotrophic Hydrogen Photoproduction in Sulfur-Deprived Chlamydomonas Cells

In Chlamydomonas reinhardtii cells, H₂ photoproduction can be induced in conditions of sulfur deprivation in the presence of acetate. The decrease in photosystem II (PSII) activity induced by sulfur deprivation leads to anoxia, respiration becoming higher than photosynthesis, thereby allowing H₂ production. Two different electron transfer pathways, one PSII dependent and the other PSII independent, have been proposed to account for H₂ photoproduction. In this study, we investigated the contribution of both pathways as well as the acetate requirement for H₂ production in conditions of sulfur deficiency. By using 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a PSII inhibitor, which was added at different times after the beginning of sulfur deprivation, we show that PSII-independent H₂ photoproduction depends on previously accumulated starch resulting from previous photosynthetic activity. Starch accumulation was observed in response to sulfur deprivation in mixotrophic conditions (presence of acetate) but also in photoautotrophic conditions. However, no H₂ production was measured in photoautotrophy if PSII was not inhibited by DCMU, due to the fact that anoxia was not reached. When DCMU was added at optimal starch accumulation, significant H₂ production was measured. H₂ production was enhanced in autotrophic conditions by removing O₂ using N₂ bubbling, thereby showing that substantial H₂ production can be achieved in the absence of acetate by using the PSII-independent pathway. Based on these data, we discuss the possibilities of designing autotrophic protocols for algal H₂ photoproduction.     

Genomic investigation of the system for selenocysteine incorporation in the bacterial domain

The Photosystem II complex (PSII) is susceptible to inactivation by strong light, and the inactivation caused by strong light is referred to as photoinactivation or photoinhibition. In photosynthetic organisms, photoinactivated PSII is rapidly repaired and the extent of photoinactivation reflects the balance between the light-induced damage (photodamage) to PSII and the repair of PSII. In this study, we examined these two processes separately and quantitatively under stress conditions in the cyanobacterium Synechocystis sp. PCC 6803. The rate of photodamage was proportional to light intensity over a range of light intensities from 0 to 2000 microE m(-2) s(-1), and this relationship was not affected by environmental factors, such as salt stress, oxidative stress due to H2O2, and low temperature. The rate of repair also depended on light intensity. It was high under weak light and reached a maximum of 0.1 min(-1) at 300 microE m(-2) s(-1). By contrast to the rate of photodamage, the rate of repair was significantly reduced by the above-mentioned environmental factors. Pulse-labeling experiments with radiolabeled methionine revealed that these environmental factors inhibited the synthesis de novo of proteins. Such proteins included the D1 protein which plays an important role in the photodamage-repair cycle. These observations suggest that the repair of PSII under environmental stress might be the critical step that determines the outcome of the photodamage-repair cycle.     

Sustained hydrogen photoproduction by Chlamydomonas reinhardtii: Effects of culture parameters

The green alga, Chlamydomonas reinhardtii, is capable of sustained H(2) photoproduction when grown under sulfur-deprived conditions. This phenomenon is a result of the partial deactivation of photosynthetic O(2)-evolution activity in response to sulfur deprivation. At these reduced rates of water-oxidation, oxidative respiration under continuous illumination can establish an anaerobic environment in the culture. After 10-15 hours of anaerobiosis, sulfur-deprived algal cells induce a reversible hydrogenase and start to evolve H(2) gas in the light. Using a computer-monitored photobioreactor system, we investigated the behavior of sulfur-deprived algae and found that: (1) the cultures transition through five consecutive phases: an aerobic phase, an O(2)-consumption phase, an anaerobic phase, a H(2)-production phase and a termination phase; (2) synchronization of cell division during pre-growth with 14:10 h light:dark cycles leads to earlier establishment of anaerobiosis in the cultures and to earlier onset of the H(2)-production phase; (3) re-addition of small quantities of sulfate (12.5-50 microM MgSO(4), final concentration) to either synchronized or unsynchronized cell suspensions results in an initial increase in culture density, a higher initial specific rate of H(2) production, an increase in the length of the H(2)-production phase, and an increase in the total amount of H(2) produced; and (4) increases in the culture optical density in the presence of 50 microM sulfate result in a decrease in the initial specific rates of H(2) production and in an earlier start of the H(2)-production phase with unsynchronized cells. We suggest that the effects of sulfur re-addition on H(2) production, up to an optimal concentration, are due to an increase in the residual water-oxidation activity of the algal cells. We also demonstrate that, in principle, cells synchronized by growth under light:dark cycles can be used in an outdoor H(2)-production system without loss of efficiency compared to cultures that up until now have been pre-grown under continuous light conditions.     

Hydrogen production by Anabaena variabilis PK84 under simulated outdoor conditions

Hydrogen production by autotrophic, vanadium-grown cells of Anabaena variabilis PK84, a cyanobacterial mutant impaired in the utilization of molecular hydrogen, has been studied under simulated outdoor conditions. The cyanobacterium was cultivated in an automated helical tubular photobioreactor (4.35 L) under air containing 2% CO(2), with alternating 12-h light (36 degrees C) and 12-h dark (14 degrees to 30 degrees C) periods. A. variabilis steadily produced H(2) directly in the photobioreactor during continuous cultivation for 2.5 months. The maximum H(2) production by the continuously aerated culture under light of 332 microE. s(-1). m(-2) was 230 mL per 12-h light period per photobioreactor and was observed at a growth density corresponding to 3.6 to 4.6 microgram Chl a. mL(-1) (1.2 to 1.6 mg dry weight. mL(-1)). Replacement of air with an argon atmosphere enhanced H(2) evolution by a factor of 2. This stimulatory effect was caused mainly by N(2) deprivation in the cell suspension. A short-term decrease of the CO(2) concentration in the air suppressed H(2) evolution. Anoxygenic conditions over the dark periods had a negative effect on H(2) production. The peculiarity of hydrogen production and some physiological characteristics of A. variabilis PK84 during cultivation in the photobioreactor under a light-dark regime are investigated.     

Growth condition-dependent sensitivity, photodamage and stress response of Chlamydomonas reinhardtii exposed to high light conditions

Different substrate conditions, such as varying CO(2) concentrations or the presence of acetate, strongly influence the efficiency of photosynthesis in Chlamydomonas reinhardtii. Altered photosynthetic efficiencies affect the susceptibility of algae to the deleterious effects of high light stress, such as the production of reactive oxygen species (ROS) and PSII photodamage. In this study, we investigated the effect of high light on C. reinhardtii grown under photomixotrophy, i.e. in the presence of acetate, as well as under photoautotrophic growth conditions with either low or high CO(2) concentrations. Different parameters such as growth rate, chlorophyll bleaching, singlet oxygen generation, PSII photodamage and the total genomic stress response were analyzed. Although showing a similar degree of PSII photodamage, a much stronger singlet oxygen-specific response and a broader general stress response was observed in acetate and high CO(2)-supplemented cells compared with CO(2)-limited cells. These different photooxidative stress responses were correlated with the individual cellular PSII content and probably directly influenced the ROS production during exposure to high light. In addition, growth of high CO(2)-supplemented cells was more susceptible to high light stress compared with cells grown under CO(2) limitation. The growth of acetate-supplemented cultures, on the other hand, was less affected by high light treatment than cultures grown under high CO(2) concentrations, despite the similar cellular stress. This suggests that the production of ATP by mitochondrial acetate respiration protects the cells from the deleterious effects of high light stress, presumably by providing energy for an effective defense.     

Role of two forms of the D1 protein in the recovery from photoinhibition of photosystem II in the cyanobacterium Synechococcus PCC 7942

The study of turnover of two distinct forms of the photosystem II (PSII) D1 protein in cells of the cyanobacterium Synechococcus PCC 7942 showed that the 'high-light' form D1:2 is degraded significantly faster at 500 microE m(-2) s(-1) as compared with 50 microE m(-2) s(-1) while the degradation rates of the 'low-light' form D1:1 under low and high irradiance are not substantially different. Consequently, the D1:1 turnover does not match photoinactivation of PSII under increased irradiance and therefore the cells containing this D1 form exhibit a decrease in the PSII activity. Monitoring of the content of each D1 form during a recovery from growth-temperature photoinhibition showed a good correlation between the synthesis of D1:2 and restoration of the PSII activity. In contrast, when photoinhibitory treatment was conducted at low temperature, a fast recovery was not accompanied by the D1:2 accumulation. The data suggest that photoinactivation at growth temperature results in a modification of PSII that inhibits insertion of D1:1 and, therefore, for restoration of the photochemical activity in the photoinactivated PSII complexes the D1:2 synthesis is needed. This may represent the primary reason for the requirement of psbAII/psbAIII expression under increased irradiance.     

Outdoor production of Phaeodactylum tricornutum biomass in a helical reactor

The production of microalga Phaeodactylum tricornutum in an outdoor helical reactor was analysed. The influence of temperature, solar irradiance and air flow rate on the yield of the culture was evaluated. Biomass productivities up to 1.5 g l(-1) per day and photosynthetic efficiency up to 14% were obtained by maintaining the cultures below 30 degrees C, dissolved oxygen levels less than 400% Sat. (with respect to air saturated culture) and controlling the cell density in order to achieve an average irradiance within the culture below 250 microE m(-2) s(-1). Under these conditions, the fluorescence parameter, Fv/Fm, which reflects the maximal efficiency of PSII photochemistry, remained roughly 0.6-0.7 and growth rates up to 0.050 h(-1) were achieved. The average irradiance and the light/dark cycle frequency, were the variables determining the behaviour of the cultures. A hyperbolic relationship between growth rate and biomass productivity with the average irradiance was observed, whereas both biomass productivity and photosynthetic efficiency linearly increased with the light/dark cycle frequencies. Optimum design and operational conditions which maximise the production of P. tricornutum biomass in outdoor helical reactors were determined.     

Carotenogenesis of five strains of the algae Dunaliella sp. (Chlorophyceae) isolated from Venezuelan hypersaline lagoons

We evaluated discontinuous cultures (Algal medium at 0.5 mM of NaNO3, and 27% NaCI) of five strains of Dunaliella sp. isolated from Venezuelan hypersaline lagoons (Araya, Coche, Peonia, Cumaraguas. and Boca Chica) and one strain from a reference collection (Dunaliella salina, LB1644). Cultures were maintained to 25+/-1 degrees C, with constant aeration, photoperiod 12:12, and two light intensities (195 and 390 microE.m(-2).s(-1)) during 30 days. Cell count was recorded on a daily basis using a Neubaüer camera. Totals of chlorophyll a and carotenoids were measured at the end of the experiment. The largest cellular densities were measured during the smallest light intensities. The strain with the largest cellular density was isolated from Boca Chica (8 xl0(6) and 2.5 xl0(6) cel.ml(-1) a 390 and 195microE.m(-2).s(-1), respectively). The increment of light intensity produced a significant reduction of growth rates in all strains. Totals of carotenoids by volume were as large as 390 microE.m(-2).s(-1). Strains LB 1644, from Coche and Araya were those that produced the largest amount of carotenoids (38.4; 32.8 and 21.0 microg.ml(-1), respectively). Differences total carotenoids by cell between treatments were significant. The largest concentration was 390 microE.m(-2).s(-1). The strains LB 1644 and Coche produced the highest values of carotenes (137.14 and 106.06 pg.cel(-1), respectively). Differences in the relation carotenoid:chlorophyll a between the strains at various light intensities was significant. Strains LB1644 presented the largest value of the relation carotenoids:chlorophyll a (20:1) at 195 microE.m(-2).s(-1). No significant differences were detected in the strain Coche (15:1). All the other strains showed relations lower than one. Our results suggest that the strains of Coche and Araya show potential to be used in the biotechnology of carotenoids production.     

Limitation in electron transfer in photosystem I donor side mutants of Chlamydomonas reinhardtii. Lethal photo-oxidative damage in high light is overcome in a suppressor strain deficient in the assembly of the light harvesting complex

Strains of Chlamydomonas reinhardtii lacking the PsaF gene or containing the mutation K23Q within the N-terminal part of PsaF are sensitive to high light (>400 microE m(-2) s(-1)) under aerobic conditions. In vitro experiments indicate that the sensitivity to high light of the isolated photosystem I (PSI) complex from wild type and from PsaF mutants is similar. In vivo measurements of photochemical quenching and oxygen evolution show that impairment of the donor side of PSI in the PsaF mutants leads to a diminished linear electron transfer and/or a decrease of photosystem II (PSII) activity in high light. Thermoluminescence measurements indicate that the PSII reaction center is directly affected under photo-oxidative stress when the rate of electron transfer becomes limiting in the PsaF-deficient strain and in the PsaF mutant K23Q. We have isolated a high light-resistant PsaF-deficient suppressor strain that has a high chlorophyll a/b ratio and is affected in the assembly of light-harvesting complex. These results indicate that under high light a functionally intact donor side of PSI is essential for protection of C. reinhardtii against photo-oxidative damage when the photosystems are properly connected to their light-harvesting antennae.     

Effects of cell density,light intensity and mixing on Undaria pinnatifida gametophyte activity in a photobioreactor

An on-line controlled 7 l sterilizable photobioreactor was used for the optimisation of a culture of gametophytes of Undaria pinnatifida. The gametophytes, which had been stored for three years in a culture cabinet at 16 degrees C, could rapidly grow in the photobioreactor under controlled conditions. The rate of increase of dissolved oxygen and pH were used to monitor the photosynthetic activity. Optimal gametophytes density changed varying the light intensity. The optimal cell densities were 3.24 and 3.45 g FW l(-1) when the cultures were exposed to 61.7 and 82.3 microE m(-2) s(-1), respectively. The optimal cell density was higher under a high photon flux density (PFD) than under low PFD. On the other hand, the optimal light intensities were different for different cell density cultures. The light saturation point was higher at high cell density cultures than at low cell density cultures. The optimal rotational speed was 150 rpm for high cell density culture in the photobioreactor.     

Photochemical activity of photosystem II and hydrogen photoproduction in sulfur-deprived Chlamydomonas reinhardtii mutants D1-R323D and D1-R323L

The role of photosystem II in hydrogen photoproduction by Chlamydomonas reinhardtii cells was studied in mutants with modified D1-protein. In D1-R323D and D1-R323L mutants, the replacement of arginine by aspartate or leucine, respectively, resulted in the disruption of electron transport at the donor side of photosystem II. The rate of oxygen evolution in D1-R323D decreased twice as compared to the pseudo-wild type (pWT), and in D1-R323L no oxygen evolution was detected. The latter mutant was not capable of photoautotrophical growth. The dynamics of changes in oxygen content, the reduction of photosystem II active reaction centers (deltaF/F(1)m), and hydrogen production rate in pWT were found to be similar to the wild type if cultivated under sulfur deprivation in a closed bioreactor. The observed gradual decrease in the deltaF/F(1)m value turned to a sharp drop almost to zero followed by a partial recovery during which the production of hydrogen set in. The transition to the anaerobic phase in D1-R323D cultured in a sulfur-deprived medium occurred earlier than it happened in pWt under the same conditions. However, the partial recovery of photosystem II activity and hydrogen production started at a later time, and the rate of hydrogen production was low. The D1-R323L mutant incapable of oxygen evolution entered the rapidly anaerobiosis but produced no hydrogen. The kinetics of photoinduced redox transitions in P700 was similar in all investigated strains and was not affected by diuron addition. This implies that the mutants had a pool of reducers, which could donate electrons through the quinone pool or cytochrome to photosystem I. However, in D1-R323L mutant lacking the active photosystem II, this condition was not sufficient to support hydrogenase activity.     

Hydrogen Production in Chlamydomonas: Photosystem II-Dependent and -Independent Pathways Differ in Their Requirement for Starch Metabolism

Under sulfur deprivation conditions, the green alga Chlamydomonas reinhardtii produces hydrogen in the light in a sustainable manner thanks to the contribution of two pathways, direct and indirect. In the direct pathway, photosystem II (PSII) supplies electrons to hydrogenase through the photosynthetic electron transport chain, while in the indirect pathway, hydrogen is produced in the absence of PSII through a photosystem I-dependent process. Starch metabolism has been proposed to contribute to both pathways by feeding respiration and maintaining anoxia during the direct pathway and by supplying reductants to the plastoquinone pool during the indirect pathway. At variance with this scheme, we report that a mutant lacking starch (defective for sta6) produces similar hydrogen amounts as the parental strain in conditions of sulfur deprivation. However, when PSII is inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, conditions where hydrogen is produced by the indirect pathway, hydrogen production is strongly reduced in the starch-deficient mutant. We conclude that starch breakdown contributes to the indirect pathway by feeding electrons to the plastoquinone pool but is dispensable for operation of the direct pathway that prevails in the absence of DCMU. While hydrogenase induction was strongly impaired in the starch-deficient mutant under dark anaerobic conditions, wild-type-like induction was observed in the light. Because this light-driven hydrogenase induction is DCMU insensitive and strongly inhibited by carbonyl cyanide-p-trifluoromethoxyphenylhydrazone or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, we conclude that this process is regulated by the proton gradient generated by cyclic electron flow around PSI.In the context of economical and environmental concerns around fossil fuel depletion and global warming, the interest in hydrogen as an energy carrier for the future has considerably grown. Because molecular hydrogen is scarce on our planet, the development of a hydrogen economy strongly depends on our ability to propose clean and sustainable technologies of hydrogen production. In this context, the ability of some photosynthetic microorganisms, and particularly cyanobacteria and microalgae, to convert solar energy into hydrogen has been considered as very promising (Ghirardi et al., 2000; Rupprecht et al., 2006). When cells of the unicellular green alga Chlamydomonas reinhardtii are illuminated after adaptation to anaerobic conditions, electrons originating from water splitting at PSII are driven by the photosynthetic electron transport chain to ferredoxin and to a reversible iron hydrogenase, thereby enabling the production of molecular hydrogen from water and solar energy. Because both hydrogenase activity and expression are highly sensitive to the presence of O₂ (Happe et al., 1994; Ghirardi et al., 1997; Happe and Kaminski, 2002) and because O₂ is produced at PSII, hydrogen photoproduction stops after a few minutes of illumination. Melis et al. (2000) proposed an experimental protocol based on sulfur (S) deprivation, allowing long-term hydrogen production. This protocol relies on a two-stage process: during a first stage, oxygenic photosynthesis drives production of biomass and carbohydrate stores, and during a second anaerobic stage, the hydrogenase is induced and hydrogen is produced. Sulfur starvation has two important effects regarding hydrogen production: (1) a massive accumulation of starch that defines a common response to nutrient starvation and (2) a gradual drop in PSII activity (Wykoff et al., 1998). Once the rate of photosynthetic O₂ evolution drops below the rate of respiration, anaerobic conditions are reached, enabling the induction of hydrogenase and the production of significant amounts of hydrogen for several days. In parallel to hydrogen production, starch is degraded (Melis et al., 2000; Melis, 2007).The importance of starch fermentation in hydrogen production has been recognized early from the pioneering work of Gibbs and coworkers (Gfeller and Gibbs, 1984; Gibbs et al., 1986). Based on the observation that starchless C. reinhardtii mutants sta6 and sta7 are strongly affected in their ability to produce hydrogen, Posewitz et al. (2004) proposed that starch metabolism plays a central role in C. reinhardtii hydrogen production. Actually, two different pathways can supply reductants (i.e. reduced ferredoxin) for hydrogen production in the light, a direct pathway involving PSII and an indirect PSII-independent pathway that relies on a nonphotochemical reduction of plastoquinones (PQs; Fouchard et al., 2005; Melis, 2007). Starch catabolism was proposed to play a role in both pathways (Melis, 2007) by (1) sustaining mitochondrial respiration and allowing the maintenance of anaerobic conditions for the PSII-dependent direct pathway and (2) by supplying electrons to the chlororespiratory pathway and to the hydrogenase through a PSI-dependent process during the indirect pathway (Fouchard et al., 2005; Mus et al., 2005; Melis, 2007). Such a dual role of starch was first confirmed by the study of a Rubisco-deficient mutant (CC2653), unable to accumulate starch and to produce hydrogen in conditions of S deprivation (White and Melis, 2006), but was recently challenged by the study of another Rubisco-less mutant (CC2803), which was reported to produce significant amounts of hydrogen in S starvation conditions, although not accumulating starch (Hemschemeier et al., 2008). These conflicting results obtained on two different Rubisco-deficient mutants prompted us to reexamine the contribution of starch to both direct and indirect pathways of hydrogen production. For this purpose, we complemented the initial work of Posewitz et al. (2004) by revisiting the ability of C. reinhardtii mutants deficient in starch metabolism to produce hydrogen. We thus tested the ability to produce hydrogen in a starchless strain carrying defect in the structural gene encoding the small subunit of ADP-Glc pyrophosphorylase (AGPase; sta6; Zabawinski et al., 2001). We found that sta6 mutant produces significant hydrogen amounts in condition of S deprivation but shows a strongly reduced PSII-independent hydrogen production. We conclude that while the PSII-independent hydrogen production pathway strictly relies on starch catabolism, the PSII-dependent pathway may require either starch or acetate as a respiratory substrate to maintain anaerobiosis.     

Biochemical and morphological characterization of sulfur-deprived and H2-producing Chlamydomonas reinhardtii (green alga)

Sulfur deprivation in green algae causes reversible inhibition of photosynthetic activity. In the absence of S, rates of photosynthetic O2 evolution drop below those of O2 consumption by respiration. As a consequence, sealed cultures of the green alga Chlamydomonas reinhardtii become anaerobic in the light, induce the "Fe-hydrogenase" pathway of electron transport and photosynthetically produce H2 gas. In the course of such H2-gas production cells consume substantial amounts of internal starch and protein. Such catabolic reactions may sustain, directly or in directly, the H2-production process. Profile analysis of selected photosynthetic proteins showed a precipitous decline in the amount of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) as a function of time in S deprivation, a more gradual decline in the level of photosystem (PS) II and PSI proteins, and a change in the composition of the PSII light-harvesting complex (LHC-II). An increase in the level of the enzyme Fe-hydrogenase was noted during the initial stages of S deprivation (0-72 h) followed by a decline in the level of this enzyme during longer (t >72 h) S-deprivation times. Microscopic observations showed distinct morphological changes in C. reinhardtii during S deprivation and H2 production. Ellipsoid-shaped cells (normal photosynthesis) gave way to larger and spherical cell shapes in the initial stages of S deprivation and H2 production, followed by cell mass reductions after longer S-deprivation and H2-production times. It is suggested that, under S-deprivation conditions, electrons derived from a residual PSII H2O-oxidation activity feed into the hydrogenase pathway, thereby contributing to the H2-production process in Chlamydomonas reinhardtii. Interplay between oxygenic photosynthesis, mitochondrial respiration, catabolism of endogenous substrate, and electron transport via the hydrogenase pathway is essential for this light-mediated H2-production process.     

Loss of algal Proton Gradient Regulation 5 increases reactive oxygen species scavenging and H2 evolution

We have identified hpm91, a Chlamydomonas mutant lacking Proton Gradient Regulation5(PGR5)capable of producing hydrogen(H2) for 25 days with more than 30-fold yield increase compared to wild type.Thus, hpm91 displays a higher capacity of H2 production than a previously characterized pgr5 mutant. Physiological and biochemical characterization of hpm91 reveal that the prolonged H2 production is due to enhanced stability of PSII, which correlates with increased reactive oxygen species(ROS) scavenging capacity during sulfur deprivation. This anti-ROS response appears to protect the photosynthetic electron transport chain from photooxidative damage and thereby ensures electron supply to the hydrogenase.