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Biological conversion of solar energy into hydrogen is naturally realized by some microalgae species due to a coupling between the photosynthetic electron transport chain and a plastidial hydrogenase. While promising for the production of clean and sustainable hydrogen, this process requires improvement to be economically viable. Two pathways, called direct and indirect photoproduction, lead to sustained hydrogen production in sulfur-deprived Chlamydomonas reinhardtii cultures. The indirect pathway allows an efficient time-based separation of O 2 and H 2 production, thus overcoming the O 2 sensitivity of the hydrogenase, but its activity is low. With the aim of identifying the limiting step of hydrogen production, we succeeded in overexpressing the plastidial type II NAD(P)H dehydrogenase (NDA2). We report that transplastomic strains overexpressing NDA2 show an increased activity of nonphotochemical reduction of plastoquinones ( PQ s). While hydrogen production by the direct pathway, involving the linear electron flow from photosystem II to photosystem I, was not affected by NDA2 overexpression, the rate of hydrogen production by the indirect pathway was increased in conditions, such as nutrient limitation, where soluble electron donors are not limiting. An increased intracellular starch was observed in response to nutrient deprivation in strains overexpressing NDA2. It is concluded that activity of the indirect pathway is limited by the nonphotochemical reduction of PQ s, either by the pool size of soluble electron donors or by the PQ -reducing activity of NDA2 in nutrient-limited conditions. We discuss these data in relation to limitations and biotechnological improvement of hydrogen photoproduction in microalgae.
A number of microalgal and cyanobacterial species are able to convert solar energy into hydrogen by photobiological processes and are therefore considered promising organisms for developing clean and sustainable hydrogen production (Benemann, ; Ghirardi et al., ; Rupprecht et al., ). In microalgae, hydrogen photoproduction results from coupling the photosynthetic electron transport chain and a plastidial [FeFe] hydrogenase. Under most conditions, hydrogen photoproduction is a transient phenomenon that lasts from several seconds to a few minutes (Ghirardi et al., ; Melis and Happe, ). It has been considered a relic of evolution that may now serve, under certain environmental conditions, such as induction of photosynthesis in anoxia (Ghysels et al., ), as a safety valve that protects the photosynthetic electron transport chain from photodamage that results from overreduction of electron acceptors (Kessler, ; Tolleter et al., ). A major limitation to sustained hydrogen photoproduction is due to the oxygen sensitivity of the [FeFe] hydrogenase (Happe et al., ; Stripp et al., ). Melis et al. () proposed an elegant way to overcome this oxygen sensitivity through a time-based separation of hydrogen and oxygen production phases occurring, for instance, in response to sulfur deficiency in a closed environment. Another limitation is related to the electron supply for the hydrogenase coming from the photosynthetic electron transport chain (Cournac et al., ). This limitation is partly due to the fact that other metabolic pathways, such as ferredoxin-NADP+ reductase and CO2 fixation, compete with the hydrogenase for the use of reduced ferredoxin (Gaffron and Rubin, ; Hemschemeier et al., ). This is also due to upstream regulation of the electron transport chain, recently evidenced from the study of a Chlamydomonas reinhardtii mutant affected in proton gradient regulation-like1 (PGRL1)-mediated cyclic electron flow (CEF) around PSI. The strong enhancement of hydrogen production rates observed in the pgrl1 mutant was interpreted as the release of a control exerted by the transthylakoidal pH gradient on electron supply to the hydrogenase (Tolleter et al., ).
Two pathways, direct or indirect, can supply electrons to the hydrogenase (Benemann, ; Melis and Happe, ; Chochois et al., ). In the direct pathway, the whole electron transport chain is engaged, with PSII supplying electrons to the plastoquinone (PQ) pool, the cytochrome b6/f complex, and, in turn, PSI, ferredoxin, and the [FeFe] hydrogenase. Due to the high oxygen sensitivity of the [FeFe] hydrogenase and to the fact that O2 is produced during photosynthesis at PSII, the direct pathway only operates when PSII activity is lower than mitochondrial respiration, thereby allowing anaerobiosis to be maintained. Such conditions can be obtained by decreasing PSII activity either by means of sulfur deprivation (Melis et al., ) or by decreasing light intensity in the photobioreactor (Degrenne et al., ). In the indirect pathway, reducing equivalents, stored as starch during the aerobic phase, are subsequently used to fuel hydrogen production. This implies a nonphotochemical reduction of the PQ pool that is at least in part mediated by NDA2, a type II NADH dehydrogenase discovered in C. reinhardtii chloroplasts (Desplats et al., ). RNA interference lines expressing lower levels of NDA2 show lower hydrogen production rates, and it was concluded that NDA2 is involved in hydrogen production by the indirect pathway (Jans et al., ; Mignolet et al., ). The indirect pathway allows for an efficient time-based separation of O2- and H2-producing phases because it does not involve PSII activity and does not produce O2. However, the indirect pathway has a much lower rate than the direct pathway (Cournac et al., ; Antal et al., ; Chochois et al., ). With the aim to identify limiting steps of hydrogen production in microalgae, we attempted to overexpress NDA2 in C. reinhardtii chloroplasts. We report that algal strains displaying a 2-fold increase in NDA2 show an increased nonphotochemical reduction of PQs and an increased rate of hydrogen production by the indirect pathway, the latter being only observed in conditions where stromal reducing equivalents are available in sufficient amounts.
Long-term hydrogen production and intracellular starch contents measured in response to sulfur deficiency. Exponentially growing cells were centrifuged, resuspended in a sulfur-free medium (the initial cellular concentration was 4 × 10 6 cells mL '1 , corresponding to 18 µg chlorophyll mL '1 ), and transferred in illuminated (200 µmol photons m '2 s '1 ) sealed flasks. From 0 to 24 h, the cell concentration increased from 4 × 10 6 to 10 7 cells mL '1 and then remained constant. At 24 h (as indicated by an arrow), the cell suspension was bubbled with N 2 to remove residual O 2 and synchronize hydrogen production. A, Hydrogen production measured in the absence of DCMU . B, Hydrogen production measured in the presence of 20 µm DCMU . C, Intracellular starch measured as Glc equivalents during sulfur deficiency experiments performed in the absence of DCMU . D, Intracellular starch measured as Glc equivalents during sulfur deficiency experiment performed in the presence of 20 µm DCMU . Control (white circles) and CrNDA2 + cells (black circles). Shown are means ± sds (n = 3 for A and B; n = 5 for C and D). chl, Chlorophyll.
Short-term hydrogen photoproduction by the indirect pathway measured in CrNDA2 + C. reinhardtii cells. Hydrogen production was measured using a membrane inlet mass spectrometer following 45-min anaerobic incubation in the dark of exponentially dividing cells (4 × 10 6 cells mL '1 , corresponding to 18 µg chlorophyll mL '1 ). When indicated by the top box, light (800 µmol photons m '2 s '1 PAR ) was switched on. The PSII inhibitor DCMU (final concentration, 20 µm) was added 4 min before the onset of illumination. A and B, Hydrogen production measured in control (A) and CrNDA2 + cells (B) exponentially grown in a TAP medium. C and D, Hydrogen production measured in control (C) and CrNDA2 + cells (D) after 2 d in sulfur-deprived TAP medium. Shown in gray dots are means ± sds (n = 3). chl, Chlorophyll.
Oxidation/reduction kinetics for the primary electron donor to PSI, P700, and CEF measurements in CrNDA2 + cells. Cultured cells shown were poisoned with the PSII inhibitor DCMU (30 μm final concentration) before measurements. Absorption changes were measured at 705 nm during a dark-light-dark transient as indicated by the top boxes. A, Light intensity was 46 µmol photons m '2 s '1 . B, Light intensity was 240 µmol photons m '2 s '1 . C, Light intensity was 750 µmol photons m '2 s '1 . D, Zoom on the P700 + rereduction kinetics in the dark after an illumination at 240 µmol photons m '2 s '1 . Control (gray lines) and CrNDA2 + cells (black lines). The P700-to-chlorophyll ratio, which was determined as described by Johnson and Alric () , was 1,100:1. Shown are means ± sds (n = 3).
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Constructs used for overexpression of CrNDA2 in the C. reinhardtii plastid genome and preliminary characterization of transformants. A, Vectors used for the plastid transformation of the ATPase-deficient mutant FUD50. The vector harbors two homologous recombination regions (HR1 and HR2) and the CrNDA2 gene (or the aadA gene for transformation controls) under control of the psaA 5' UTR promoter region and the 3' UTR rbcL region; putative transformants were screened based on their ability to grow photoautrophically. B, PCR-based characterization of plastid transformants using CpNDA2 primers (shown by black arrows in A), homoplasmy primers (shown by asterisks in A), and 16S primers used as a positive control. C, Immunodetection of a 61-kD protein band using an antibody directed against recombinant CrNDA2; quantitative analysis showed a 1.7 ± 0.2 sd (n = 3) increase in CrNDA2 amounts in CrNDA2 + compared with the control line; Coomassie Blue-stained loading controls are shown at bottom. D, State transitions monitored by low temperature (77 K) chlorophyll fluorescence spectra in control (gray line) and CrNDA2 + cells (black line). Ratios between emission fluorescence signals measured at 685 nm and 715 nm (E 685 /E 715 ) determined in control (gray box) and CrNDA2 + cells (black box) are shown in the insert as means ± sds (n = 3).
It has been shown here that overexpression of the plastidial NDA2 leads to an increased activity of nonphotochemical reduction of PQs and to an enhanced hydrogen production rate by the indirect pathway (measured in the absence of PSII), the latter being essentially observed in conditions of nutrient depletion. For cells grown in optimal conditions (nutrient replete), NDA2 overexpression has a negligible effect on hydrogen photoproduction, thus indicating that the availability of NDA2 electron donors [NAD(P)H or NADH] likely limits the in vivo activity of the enzyme. However, under conditions of nutrient limitation, conditions where the intracellular starch pool is increased and the reducing state of stromal electron carriers is higher (Peltier and Schmidt, ; Grossman, ), higher hydrogen production rates by the indirect pathway are observed (Fig. 3, C and D). Note, however, that in conditions of nutrient deprivation, hydrogen production by the direct pathway (measured in the absence of DCMU) was not increased in CrNDA2+ cells (Fig. 4A), showing that PSII can fully reduce the PQ pool in a photochemical manner without significant additional effect of nonphotochemical reduction by NDA2.
It was recently proposed, from the study of a C. reinhardtii mutant (pgrl1) deficient in PGRL1-mediated CEF around PSI, that the proton gradient generated by CEF strongly inhibits the electron supply to the hydrogenase in wild-type strains (Tolleter et al., ). Based on the fact that hydrogen production measured in conditions of sulfur deficiency, either in the presence or in the absence of DCMU, was enhanced in the pgrl1 mutant, it was proposed that the transthylakoidal proton gradient limits hydrogen production by both the direct and indirect pathway (Tolleter et al., ). The absence of uncoupler effect on short-term hydrogen production measured in the presence of DCMU (Supplemental Fig. S4, E and F) shows, however, that in the conditions of our experiments, the proton gradient does not actually slow down the activity of the indirect pathway. In fact, it is most likely that both pathways (direct and indirect) are similarly sensitive to the proton gradient, because they both contain the proton gradient-sensitive electron carrier (i.e. the cytochrome b6/f complex). The apparent insensitivity of the indirect pathway to FCCP is likely due to the fact that less proton gradient accumulates during the indirect pathway than during the direct one. Unlike PSII, which contributes to the proton gradient by releasing four protons per O2 molecule produced, type 2 NADH dehydrogenases such as CrNDA2 are non-proton-pumping enzymes (Yagi, ; Peltier and Cournac, ). In this context, the long-term stimulation of hydrogen production previously observed in pgrl1 DCMU-treated cells in response to sulfur deprivation may result from an indirect effect, such as an increased activity of nonphotochemical reduction of the PQ pool in the pgrl1 mutant. We therefore conclude that if the direct pathway is controlled by the proton gradient through a modulation of the cytochrome b6/f complex activity (Tolleter et al., ; Fig. 5A), activity of the indirect pathway can be limited by NDA2 activity, but only in conditions (such as nutrient deprivation) in which the stromal donor electron pool of NAD(P)H originating from starch catabolism is not limiting (Fig. 5B).
Schematic views of electron transfer pathways and limiting steps of direct and indirect hydrogen photoproduction. A, In the direct pathway, reducing equivalents generated at PSII are sequentially transferred to PQ, the cytochrome (Cyt) b6/f complex, plastocyanin (Pc), PSI, ferredoxin (Fd), and the hydrogenase (H2ase). The direct pathway has been proposed to be controlled by the proton gradient at the level of the cytochrome b6/f complex (Tolleter et al., ). B, In the indirect pathway, reducing equivalents generated by the photosynthetic electron transport chain during an aerobic phase are transiently stored as starch and, in turn, reinjected in the intersystem electron transport chain by NDA2 reducing PQs. According to this study, NDA2 activity limits the indirect pathway, provided the stromal electron donor pool, i.e. NAD(P)H supplied by starch catabolism, is not limiting. Electron transfer pathways are shown by arrows, and their respective limiting steps (cytochrome b6/f for the direct and NDA2 for the indirect) are surrounded by a gray light.
Long-term sulfur deprivation experiments (Fig. 4) confirm the strong dependence of the indirect pathway upon starch catabolism previously reported from the study of C. reinhardtii mutants either deficient in starch biosynthesis (Posewitz et al., ; Chochois et al., ) or starch breakdown (Chochois et al., ). The faster decline in intracellular starch observed in DCMU-treated CrNDA2+ cells parallels the higher hydrogen production rate observed in the mutant. The higher starch accumulation observed in CrNDA2+ cells, compared with control cells (Fig. 4, C and D), may indicate an involvement of the PQ-reducing state in regulating intracellular starch accumulation. Actually, the reducing state of the PQ pool acts as a cellular sensor regulating several cellular processes, including LHCII phosphorylation during state transition, gene expression or translation, or metabolic adjustments (Dietz and Pfannschmidt, ). Also, starch accumulation generally occurs in conditions, such as high light, nutrient starvation, or the presence of acetate, that provoke higher reduction of the PQ pool. Further studies will be needed to determine whether the PQ-reducing state or the imbalance between the reducing state of stromal carriers and the PQ pool occurring in CrNDA2+ cells is involved in the regulation of starch metabolism.
The indirect pathway of hydrogen production displays interesting features for future biotechnological applications. Due to the O2 sensitivity of the hydrogenase, hydrogen production by the direct pathway is limited by the ability of respiration to consume O2 produced by PSII. The indirect pathway allows an efficient time-based separation of O2- and H2-producing phases. Based on engineering and economic analysis, Benemann () concluded that the most plausible processes for future applications are those coupling stages of carbohydrate synthesis with stages of hydrogen production. However, until recently, the contribution of the indirect pathway to hydrogen photoproduction was considered marginal and was estimated to be between 10% and 20% of the activity of the direct pathway (Antal et al., ; Fouchard et al., ). We have shown here that overexpression of NDA2 may lead to a strong enhancement of the indirect hydrogen photoproduction rate, provided the pool of electron donors is not limiting. In sulfur-deprived CrNDA2+ cells, the rate of the indirect pathway reached, during a few hours, a level close to that of the direct pathway (compare the rate of hydrogen production in Fig. 4, A and B). The fast decline in intracellular starch reserves observed in these conditions in CrNDA2+ cells (Fig. 4D) most likely explains the rapid decline in the hydrogen production rate.
The strong enhancement of the indirect pathway's activity, obtained here by genetic engineering, opens new perspectives and challenges for developing optimized protocols of hydrogen production. An advantage of the indirect pathway is related to its lower quantum requirement. Because the indirect pathway requires only PSI to convert starch into hydrogen, the quantum yield of hydrogen production is divided by two during the hydrogen production phase, compared with that of the direct pathway, which requires two photosystems during the hydrogen production phase. This lower quantum requirement would represent an economic advantage in a two-step protocol, because the size of the hydrogen-tight photobioreactor required to recover hydrogen gas could be divided by two. One could therefore imagine experimental protocols in which algae are grown in low-cost photobioreactors or raceways during the aerobic phase and transferred to hydrogen-tight (more costly) photobioreactors during sulfur deprivation. In this context, increasing the rate of the second step, which converts carbohydrates into hydrogen, would have a significant economic impact. Note however that, as shown by the rapid decline in starch observed in CrNDA2+ cells, a future challenge toward improving microalgae hydrogen production will be the optimization of starch accumulation. Furthermore, relying on the indirect pathway requires PSII activity to be controlled in a reversible manner. In our experiments, PSII activity was inhibited by addition of DCMU. In that context, the use of inducible promoters that have been previously reported to control PSII activity and hydrogen production (Surzycki et al., ) will be particularly helpful.
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