A new approach for sustained and eﬃcient H 2 photoproduction by Chlamydomonas reinhardtii

Sustained H 2 photoproduction is demonstrated in green algae under a train of strong white light pulses interrupted by longer dark phases. The devised protocol relies on the presence of the [FeFe]-hydrogenase in algal chloroplasts, which is activated within a few seconds after the establishment of anaerobiosis. H 2 photo-production proceeds for up to 3 days with the maximum rate occurring in the first 6 hours.

Photobiological water splitting to molecular hydrogen (H 2 ) and oxygen (O 2 ), also known as direct water biophotolysis, has been considered as one of the most promising and environmentally friendly approaches for generating bulk quantities of clean H 2 biofuel. 1 Many species of cyanobacteria and eukaryotic green algae, including the model organism Chlamydomonas reinhardtii, are capable of catalyzing this reaction. 2,3In green algae, water biophotolysis proceeds in two steps: involving the photosystem II (PSII) water-oxidizing complex at step 1 and the [FeFe]-hydrogenase (H 2 ase) enzyme at step 2 being interconnected via the photosynthetic electron-transport chain (PETC) (Scheme 1, a pathway from A to C). 4 While the splitting of water in PSII results in the release of O 2 , the process catalyzed by the H 2 ase is O 2 -sensitive. 5The O 2 sensitivity issue has been recognized as a major challenge to efficient H 2 production in green algae.Unfortunately, there is no apparent solution allowing the simultaneous production of O 2 and H 2 in algal cultures at full PSII capacity.
H 2 photoproduction in C. reinhardtii also occurs through a mechanism independent of water oxidation.In the indirect process, the reductants derived from the degradation of stored organic substrates, such as starch and proteins, are incorporated into the PETC by a type II NADPH dehydrogenase (Nda2) at the level of the plastoquinone (PQ) pool, 6 thus bypassing the water splitting at PSII (Scheme 1, a pathway from B to C).Similar to the direct process, this pathway requires PSI activity to donate electrons to the H 2 ase.Since both pathways are linked to the H 2 ase via PSI and Fd (Scheme 1, the C pathway), their contribution to the overall H 2 production yield in algal cultures may vary depending on physiological conditions.
Efficient H 2 photoproduction in green algae occurs in the light after a period of dark anaerobic incubation. 7The reaction is transient due to a rapid, within seconds, inhibition of H 2 ase by O 2 , which is co-produced in the water-splitting reaction.One of the approaches to achieve sustained H 2 photoproduction in C. reinhardtii cultures is to deprive them of sulfur. 8Sulfurdeprivation prevents the efficient repair of the light-damaged D1 reaction center protein of PSII, thus leading to a gradual loss of the water-splitting activity in algal cells over time.As a consequence, the actively respiring algae establish an anaerobic environment in the sealed photobioreactor, induce the H 2 ase enzyme(s) and continuously produce H 2 gas for several days. 9Although the loss of active PSII centers sustains H 2 photoproduction in algae, it also downregulates the direct, water oxidation-dependent flow of electrons to the H 2 ase, resulting in low overall efficiency of the process.Sulfurdeprivation requires extensive and time-consuming centrifugations, which make this protocol difficult for application even in laboratory scale projects (yet a few alternatives have been suggested 10,11 ).
In the current work, we demonstrate that efficient H 2 photoproduction can be sustained in growing C. reinhardtii cultures for at least three days by switching the algal suspensions from continuous light to a train of short strong light pulses superimposed on either darkness or permanent low light illumination.The protocol is very simple, non-damaging to algae and reproducible even under strict autotrophic conditions.

Theoretical considerations
At the current state, H 2 photoproduction in algal cultures is only possible via a temporal separation of the O 2 evolving and H 2 producing reactions.C. reinhardtii cultures, dark-adapted in anoxic conditions, produce H 2 upon exposure to light, before the onset of O 2 evolution (Fig. 1A), while sulfur-deprived cells show the opposite behavior (Fig. 1B).Although the maximum specific H 2 photoproduction activity is higher in dark-adapted cells than in sulfur-deprived algae, the latter produce H 2 much longer and yield more H 2 gas.For sustaining the H 2 production process in dark-adapted algae, one could suggest the low light/ high cell density condition that prevents O 2 accumulation in cultures due to active respiration, but at the expense of efficiency. 12Alternatively, H 2 production can be driven at high light intensities by funneling photosynthetic electrons to the H 2 ase (Scheme 1, the C pathway), instead of the Calvin-Benson-Bassham (CBB) cycle (Scheme 1, the D pathway), with simultaneous control of the intracellular O 2 level.Although Rubisco deficiency has been reported to promote H 2 evolution in green algae, the yield of H 2 in the Rubisco-deficient mutant culture was not particularly high, most probably due to the downregulation of the photosynthetic electron flow to the H 2 ase in this strain. 13Nevertheless, the partial inactivation of the CBB cycle did improve the H 2 photoproduction yield. 14he H 2 ase enzyme, induced in algae under dark anaerobic conditions, 15,16 acts as an alternative electron sink upon illumination and promotes the activity of oxygenic photosynthesis by eliminating the accumulation of excess electrons in PETC. 17he light activation of the CBB cycle requires time affecting photosynthetic productivity under fluctuating light. 18We propose that a train of very short light pulses should arrest the algal photosynthesis in the H 2 photoproduction stage, provided the duration of each light pulse is short enough to minimize the electron flow to the CBB cycle and to prevent O 2 accumulation.To test this hypothesis, we subjected C. reinhardtii to a train of short (1-5 s) light pulses interrupted by longer (3-9 s) dark phases.These experiments were subsequently repeated under low background illumination (3 mmol photons m À2 s À1 ) in place of dark phases.

Materials and methods
All experiments were performed with unstressed, actively growing C. reinhardtii cultures either on TAP (photomixotrophic growth) or on a modified TAP medium without acetate (photoautotrophic growth).CC-124, CC-4533 and CC-5128 (hydEF) strains were pregrown under a 14 h photoperiod at 75 mmol photons m À2 s À1 photosynthetic active radiation (PAR) and 25 1C.H 2 photoproduction was analyzed during the active period of photosynthesis, within 5 to 10 h from the beginning of the photoperiod.No centrifugation steps were applied.Growing algal cultures were

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Energy & Environmental Science pipetted into a gas-tight 23 mL GC vial equipped with H 2 and O 2 microsensors (H 2 -NP and OX-NP, Unisense A/S) connected to an amplifier.The electrodes were pierced inside the vial through a Teflon-coated rubber septum.Cells in the vial were sparged with argon (Ar) for 2-3 min in the dark, followed by incubation in the dark for another 1-5 min.Subsequently, a train of light pulses was applied to the culture and the H 2 and O 2 levels were monitored by the OxyHydrogen software via the STM32F103 microcontroller board connected to a high precision 24-bit ADC (ADS1256, Texas Instruments).The white LED light pulses (420 mmol photons m À2 s À1 ) were synchronized through the same microcontroller board.The gas exchange was measured by membrane inlet mass spectrometry (MIMS) using a modified DW1 (Hansatech Instruments) electrode chamber as previously described. 19he long-term H 2 photoproduction experiments were performed with a 10 mL cell suspension in 70 mL gas-tight vials under an Ar atmosphere.The pulses of white light (280 mmol photons m À2 s À1 ) interrupted by dark periods or the constant light of the same intensity were provided by the growth chamber (AlgaeTron AG 130-ECO, PSI).The vials were continuously shaken and H 2 production yields were measured using a gas chromatograph (Clarus 500, PerkinElmer) equipped with a thermal conductivity detector and a molecular sieve 5A column (60/80 mesh).The total Chl content and hydrogenase activity were measured as described previously. 9he average energy of the incident light in the PAR (400-700 nm) region was determined at the surface of the liquid with the STS-VIS spectrometer (Ocean Optics, Inc.).Light energy to hydrogen energy conversion efficiency (LHCE) was calculated using eqn (1), which considers the partial pressure of H 2 gas in the vial headspace at the moment of calculation: 20 Z ð%Þ ¼ 100 where DG1 is the change of the standard Gibb's free energy for the water-splitting reaction (237 200 J mol À1 at 25 1C and 1 atm), R is the universal gas constant, T is the absolute temperature, P1 and P are the standard and observed H 2 pressures (atm), V H is the amount of H 2 photoproduced (mol), E S is the energy of the incident light radiation (J m À2 s À1 ), A is the illuminated surface area (m 2 ) and t is the sum of the illumination periods (s).
For protein analysis, cells were harvested and rapidly frozen in lysis buffer (50 mM Tris pH 8, 2% SDS, 10 mM EDTA, protease inhibitors from Sigma).After thawing, the total protein fraction was isolated and separated in a 12% SDS-PAGE without urea, transferred to a polyvinylidene difluoride membrane (Millipore) and blocked with a 5% blotting grade blocker (Bio-Rad).The samples were loaded on an equal protein basis as determined using a Direct Detect s infrared spectrometer (Merck) and visualized as control with Coomassie Brilliant Blue (Bio-Rad).The accumulation of HydA1/A2 was analyzed by using a specific antibody (Agrisera).As a secondary antibody, antirabbit horseradish peroxidase was used in 1 : 10 000 dilution and HydA1/A2 was visualized with ECL.

A train of light pulses sustains H 2 production in algal cultures
As shown in Fig. 2, a train of 1 s light pulses interrupted by 9 s dark periods induces continuous H 2 photoproduction in algal cultures.The procedure shows reproducibility even at a very low cell density (6-7 mg total Chl L À1 ) and in the absence of acetate (Fig. 3) but requires pre-established anaerobic conditions.Trace quantities of H 2 could be observed almost immediately after starting the light pulse illumination of anaerobic cultures, and thereafter the H 2 level gradually increased with time.The experiments performed in the DW1/AD electrode chamber (Hansatech Instruments) but under high light intensity (B800 mmol photons m À2 s À1 ) pulses produced similar results.The H 2 ase-deficient hydEF mutant did not show the presence of H 2 gas throughout the experiment (Fig. 2A, magenta line), as expected.H 2 photoproduction also occurs in algae exposed to pulses superimposed on low background illumination (Fig. 2A, green line).
During the H 2 production phase, no net O 2 evolution could be detected by either the O 2 electrode (Fig. 2A) or MIMS (Fig. 2C).

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This journal is © The Royal Society of Chemistry 2018 The exposure of algae to a similar train of light pulses in DUAL-PAM, but at a background of measuring light, demonstrated a slight decline of the PSII photochemical efficiency in the course of the experiment (Fig. S1, ESI †).The exposure of the pulseilluminated cells to continuous light induced O 2 evolution, occurring with some delay (Fig. S2, ESI †).The accumulation of O 2 could also be observed on shortening the dark phase to r3 s between the light pulses (Fig. S3, ESI †), thus confirming the presence of functional PSII in algal cells under the illumination system applied here.Gas exchange measurements performed with MIMS showed no signs of CO 2 fixation upon a standard train of light pulses (Fig. 2C).CO 2 fixation occurred only upon accumulation of O 2 in the cultures as a consequence of shortening the dark phase between light pulses (Fig. S3, ESI †).This provides compelling evidence that in the newly established protocol, the algal cells function as a biocatalyst funneling photosynthetic electrons directly to the H 2 ase without the activation of the CBB cycle.

Pulse-illumination shows the presence of H 2 uptake in
As shown in Fig. 2B, transient H 2 production peaks regularly appear upon pulse-illumination of C. reinhardtii, whilst noticeable H 2 consumption takes place between the light pulses.The amplitude of the sawtooth wave, which occurs both in photoheterotrophic (Fig. 2B) and photoautotrophic (Fig. 3A, inset) cultures, became more pronounced in the course of H 2 accumulation in the system.This behavior can be explained by the dependence of the H 2 uptake reaction on the H 2 partial pressure, 21 as well as by the gradual induction of the H 2 ase activity in cells (Fig. 3B).The involvement of passive processes in the overall H 2 uptake, such as a leak of H 2 from the system or H 2 consumption by the microsensor, seems to be very minor since (i) the H 2 trace in cell-free media declines much slower (Fig. 2A, black line) and (ii) the active H 2 consumption does not occur in the hydEF mutant with disrupted H 2 ase (Fig. 2A, magenta line).The H 2 uptake reaction in the dark is very strong, and at the current state we do not know whether H 2 uptake occurs simultaneously with H 2 release in the light.
A switch of pulse-illumination to continuous low light, however, did not lead to any noticeable H 2 consumption in the cultures (Fig. 2A, green line).
Since the reaction balance catalyzed by the reversible H 2 ase is shifted towards H 2 release in the course of pulse-illumination, the involvement of the oxyhydrogen reaction in H 2 uptake is very unlikely or its contribution to the process is minor.A similar conclusion could be applied also to H 2 uptake during the dark phase after the period of pulse illumination (Fig. 2 and 3).Otherwise, flavodiiron proteins might be involved in the oxyhydrogen reaction by donating electrons to O 2 under illumination (Scheme 1, the E pathway). 22,23In principle, H 2 uptake in algae may occur without O 2 consumption: Yet, in such a case, neither the final electron acceptor nor any intermediate players are known.The occurrence of H 2 uptake in the green alga, Scenedesmus sp. was first demonstrated more than 70 years ago. 24Since that time only a little follow-up progress has been made in resolving the metabolic pathways participating in the H 2 uptake reaction.H 2 oxidation has been proposed to provide reducing equivalents for CO 2 fixation, but the reaction requires either a very low level of O 2 (up to 1%) or light illumination in complete anaerobiosis for ATP re-generation. 25he absence of CO 2 fixation either during a train of light pulses or during the dark phase after termination of the protocol (Fig. 2C) suggests that H 2 uptake in algae exposed to pulse-illumination and thereafter is not linked to CO 2 reduction.The presence of the H 2 consumption pathway was also confirmed in sulfur-deprived C. reinhardtii cells, 21,26 harbouring the inactivated Rubisco enzyme. 13,27Since H 2 uptake in both cases occurs upon a shift to darkness, the process seems to be driven by the same catabolic pathway.It is clear that more research is needed to completely understand the mechanism(s) of H 2 consumption in green algae, yet the elimination of this process should dramatically improve the H 2 photoproduction yield in algal cultures.Recently, Liran and co-authors 28 concluded that the entire pool of cellular H 2 ase remains active in air-grown cells, thus allowing algae to produce H 2 even under aerobic conditions, and in particular, on switch from low to high light conditions.On the other hand, there is extensive literature showing the extreme sensitivity of algal H 2 ase to molecular O 2 .For resolving this contradiction, Liran and co-authors suggested the existence of anaerobic niches inside the cells with a high rate of local respiration that protects the H 2 ase from O 2 inactivation.
Our experimental data show that the activation of the H 2 ase enzyme in air-grown cells and the production of H 2 (Fig. 3) occur only after the establishment of anaerobiosis in the culture.As shown in Fig. 3A, the photoautotrophic C. reinhardtii culture is capable of spontaneous establishment of anaerobiosis in the medium under the pulse-illumination if the initial level of O 2 is lowered to below 10 mmol L À1 by Ar purging.Algae start producing H 2 almost immediately after consuming the residual O 2 in the chamber.The reaction, thus, requires strong anaerobiosis and it does not occur in an aerobic environment.The cells pre-grown in air contain HydA1/A2 proteins and the amount does not increase within 15 min of the pulseillumination (Fig. 3B, inset).Nevertheless, the H 2 ase activity (measured in the presence of reduced methyl viologen) rises gradually during this time (Fig. 3B) and correlates with the induction of H 2 photoproduction in the cells (Fig. 3A).The amount of the HydA1/A2 proteins increases later (Fig. 3C, inset).During the long-term cultivation under the train of light pulses, we could detect the rise of H 2 ase in the cells, but continuous high light causes the opposite effect (Fig. 3C).In the latter case, no H 2 production is observed.These experimental data prove that algae express H 2 ase during aerobic growth under moderate light.H 2 ase activation, however, requires strong anaerobiosis, which contradicts the suggestion of Liran and co-authors 28 about the functional [FeFe]-H 2 ase enzyme in an aerobic environment.
A pulse-illumination protocol sustains H 2 production for at least 70 hours and proves the competition between H 2 photoproduction and CO 2 fixation Long-term experiments performed with C. reinhardtii cultures in small anaerobic vials demonstrated that pulse-illuminated algae are capable of producing H 2 continuously for at least 3 days (Fig. 4).The reaction occurs in the absence of acetate and at an extremely low cell density (Fig. 4A), indicating that the self-shading in the suspension is not a reason for the induction of H 2 production in algal cells.C. reinhardtii produces H 2 more efficiently during the first 6 h after which the rate gradually declines.The maximum specific rate exceeds the rate of H 2 photoproduction in sulfur-deprived algae 21,29 and in the best case reaches up to 25 mmol H 2 (mg Chl h) À1 .Under light conditions typical for the original sulfur-deprivation protocol (B200 mmol photons m À2 s À1 ), 8,27 pulse-illuminated cultures yield above 3 mmol H 2 L À1 during the first 48 h (Fig. S4, ESI †), which is very close to the H 2 yield in sulfur-deprived algae. 9owever, due to a much shorter illumination time (Fig. S4, ESI †), the pulse-illuminated cultures produce H 2 more efficiently than the sulfur-deprived cells (0.5% vs. 0.24%, 30 respectively).Sulfurdeprived algae also need an extra 24-48 h (without H 2 production) for PSII inactivation, which is not considered in LHCE calculations.Moreover, the pulse-illuminated algae are capable of producing H 2 at a maximum conversion efficiency of 1.6-1.7%(2-2.2% if the upper H 2 gas combustion energy is assumed) during the first 8 h.
It is important to note that algae do not accumulate biomass under pulse-illumination (Fig. 4C), in contrast to continuous light (Fig. 4D).The inhibition of biomass accumulation under a train of light pulses suggests the successful diversion of photosynthetic reductants from carbon fixation to H 2 photoproduction.These experimental data, thus, bring additional evidence that the re-direction of the photosynthetic electron flow to the [FeFe]-H 2 ase enzyme does improve the H 2 photoproduction activity in algal cells. 31

Conclusions
This research demonstrates that H 2 photoproduction in green algae can be sustained by a simple shift in the light conditions of growing algal cultures from continuous illumination to a train of light pulses interrupted by longer dark phases.In a low O 2 environment, such pulse-illuminated algae can spontaneously establish anaerobiosis and produce H 2 for up to three days.The appearance of H 2 gas in the cultures, almost immediately after the establishment of anaerobiosis, points to an important role of the [FeFe]-H 2 ase enzyme(s) in algal energy metabolism under anaerobic conditions.In addition, the pulse illumination protocol provides strong evidence that CO 2 fixation competes with the [FeFe]-H 2 ase enzyme for the photosynthetic electrons and demonstrates a direct means of eliminating this competition.All the findings together provide new opportunities for metabolic engineering and construction of efficient cell factories with a capacity to re-direct photosynthetic electrons to targeted metabolic pathways and biofuel products, instead of biomass.4 Long-term H 2 photoproduction by photoautotrophic (A) and photoheterotrophic (B) C. reinhardtii cultures.The cultures were exposed to the pulse-illumination protocol under an Ar atmosphere.Algae did not produce H 2 in continuous light (blue lines).The initial total Chl contents were 1 mg mL À1 in (A) and 8.9 mg mL À1 in (B).The cultures did not grow under pulse-illumination (8.8 mg mL À1 ; C) but increased the biomass content under continuous light (26 mg mL À1 ; D).

Fig. 1
Fig. 1 Available protocols for the induction of H 2 production in C. reinhardtii cultures.(A) The dark adaptation protocol was first introduced by Gaffron and Rubin in 1942 and re-produced in our experimental set-up.(B) The sulfur-deprivation protocol was repeated according to Melis and co-authors.

Fig. 2
Fig. 2 Induction of H 2 photoproduction in C. reinhardtii cultures by a train of light pulses.(A) The cultures were flushed with Ar for 3 min in the dark, and H 2 photoproduction was initiated by a train of 1 s light pulses interrupted by 9 s dark periods.(B) A typical trace of H 2 photoproduction shown at higher magnification.(C) Simultaneous monitoring of H 2 , O 2 and CO 2 exchange in algal cultures by MIMS.The sawtooth wave could not be seen in the MIMS due to background noise.Green line (panel A) shows H 2 photoproduction in algae, where 3 mmol photons m À2 s À1 of white light was applied in the background instead of darkness.The kinetics of H 2 diffusion out of the medium (panels A and B, black line) and the suspension of the H 2 ase-deficient hydEF mutant (panels A and B, magenta line) are shown after the injection of a few ml H 2 -saturated medium into the chamber.Downward arrows indicate the injection points.

Fig. 3
Fig. 3 H 2 photoproduction in C. reinhardtii cultures requires anaerobiosis.(A) Accumulation of H 2 under a train of light pulses occurred only after the establishment of anaerobiosis in algal cultures.The pulse-illumination protocol was initiated at a very low O 2 level in the medium, allowing the spontaneous establishment of anaerobiosis in autotrophic algal cultures exposed to a train of light pulses.(B) The activity assay showed an induction of H 2 ase in algal cells under pulse-illumination, but the HydA1/A2 protein level did not change within 15 min.(C) Long-term incubation of autotrophic algae under pulse-illumination led to the accumulation of H 2 in the vial headspace (as detected with GC) and HydA1/A2 proteins in cells, but not under continuous light that caused degradation of H 2 ase enzymes in cells due to the accumulation of O 2 in the vials (not shown).Immunoblots were performed using anti-HydA antibody (Agrisera) that recognizes both H 2 ase proteins in C. reinhardtii cells.

Fig.
Fig.4Long-term H 2 photoproduction by photoautotrophic (A) and photoheterotrophic (B) C. reinhardtii cultures.The cultures were exposed to the pulse-illumination protocol under an Ar atmosphere.Algae did not produce H 2 in continuous light (blue lines).The initial total Chl contents were 1 mg mL À1 in (A) and 8.9 mg mL À1 in (B).The cultures did not grow under pulse-illumination (8.8 mg mL À1 ; C) but increased the biomass content under continuous light (26 mg mL À1 ; D).