| Abstract: | 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. |
