The hydrogen metabolism of sulfur deprived Chlamydomonas reinhardtii cells involves hydrogen uptake activities

The hydrogen metabolism of sulfur deprived Chlamydomonas reinhardtii cells involves hydrogen uptake activities

Algal Research 26 (2017) 341–347 Contents lists available at ScienceDirect Algal Research journal homepage: www.elsevier.com/locate/algal The hydro...

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Algal Research 26 (2017) 341–347

Contents lists available at ScienceDirect

Algal Research journal homepage: www.elsevier.com/locate/algal

The hydrogen metabolism of sulfur deprived Chlamydomonas reinhardtii cells involves hydrogen uptake activities

MARK

Alberto Scomaa,b,⁎, Anja Hemschemeierc a

Center for Geomicrobiology, Department of Bioscience, Aarhus University, Ny Munkegade 116, 8000 Aarhus, Denmark Department of Civil, Chemical, Environmental and Materials Engineering (DICAM), School of Engineering and Architecture, Alma Mater Studiorum, University of Bologna, Via U. Terracini 28, I-40131 Bologna, Italy c Ruhr-University of Bochum, Faculty of Biology and Biotechnology, Department of Plant Biochemistry, Working group Photobiotechnology, Universitätsstr. 150, 44801 Bochum, Germany b

A R T I C L E I N F O

A B S T R A C T

Keywords: Green algae H2 uptake Photosynthesis Photobioreactor H2 partial pressure Glycolaldehyde

Several species of unicellular microalgae such as the model species Chlamydomonas reinhardtii possess plastidlocalized [FeFe]-hydrogenases which, via ferredoxin, can accept electrons from photosynthetic electron transport. Thereby, under specific conditions, these algae light-dependently produce molecular hydrogen (H2), which offers a sustainable way to generate a “green” and efficient fuel. Until today, the most common way to induce sustained H2 production is to deprive Chlamydomonas of macronutrients such as sulfur (S) which results in a downregulation of photosynthetic production of molecular oxygen (O2) and of assimilatory processes. These acclimation responses allow the O2 sensitive algal [FeFe]-hydrogenases to become active and serve as an alternative electron sink of photosynthesis. Despite much progress in the field and a general understanding of the underlying mechanisms, many basic and applied aspects of the photosynthetic H2 metabolism of eukaryotic algae remain to be elucidated. One rarely investigated factor is that microalgae have also been reported to consume H2, especially as a response to high H2 concentrations. Here, we analyzed the H2 uptake activities of Sdeprived Chlamydomonas cells incubated in different PBRs providing different gas phase volumes, either in continuous light or in the dark. We show that H2 uptake occurs after prolonged incubation in the light as well as in sudden darkness. Dark-induced H2 uptake can be delayed adding the phosphoribulose kinase inhibitor glycolaldehyde, suggesting a connection to carbohydrate metabolism. The results indicate that PBR setups as well as envisioned outdoor cultivation systems with natural light-dark cycles have to be carefully designed to prevent efficiency losses.

1. Introduction The capability of certain species of unicellular microalgae to generate molecular hydrogen (H2) using electrons derived from photosynthetic electron transport (PET) is a promising way to sustainably generate this clean and efficient fuel [1]. In the model green alga Chlamydomonas reinhardtii (Chlamydomonas in the following) chloroplast localized [FeFe]-hydrogenases accept electrons directly from the photosynthetic ferredoxin PetF [2]. High H2 yields are only observed in the light (e.g., [3]), and photosynthetic electron supply occurs via two main pathways, termed direct and indirect. In the direct pathway, electrons originate from water oxidation at photosystem II (PSII) [4–6]. The indirect pathway utilizes electrons derived from NAD(P)H oxidation by the plastidic NAD(P)H-plastoquinone oxidoreductase Nda2 [7–9] and thus ultimately from the oxidation of organic reserves such as



starch or proteins [4,10]. Chlamydomonas can also produce low amounts of H2 in the dark, probably through PetF reduction by the plastidic fermentative enzyme pyruvate ferredoxin oxidoreductase (PFR) [11,12]. Because of the severe sensitivity of [FeFe]-hydrogenases towards molecular oxygen (O2) [13,14], a H2 metabolism is only established under anoxic or hypoxic conditions. Photoevolution of H2 can be observed in Chlamydomonas cells pre-incubated under anoxic conditions in the dark and then abruptly shifted to illumination (e.g., [15]). Although vital for the reactivation of photosynthesis [16,17], H2 production under this condition is only short-lived due to the accumulation of O2 and the induction of the Calvin-Benson-Bassham (CBB) cycle. The latter is a major competitor of H2 photoproduction [6,18,19], because it consumes the NADPH produced by ferredoxin NADPH reductase (FNR) and thus ultimately competes with electrons delivered by PetF [20,21]. Long-term H2 production can be achieved in dense Chlamydomonas

Corresponding author at: Center for Geomicrobiology, Department of Bioscience, Aarhus University, Ny Munkegade 116, 8000 Aarhus, Denmark. E-mail address: [email protected] (A. Scoma).

http://dx.doi.org/10.1016/j.algal.2017.08.018 Received 17 May 2017; Received in revised form 10 July 2017; Accepted 10 August 2017 2211-9264/ © 2017 Published by Elsevier B.V.

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Nomenclature

(S) (PBRs) (PTOX) (GA) (Chl) (TAP-S)

(PET) photosynthetic electron transport (PSII) photosystem II (PFR) pyruvate ferredoxin oxidoreductase (CBB cycle) Calvin-Benson-Bassham cycle (FNR) ferredoxin NADPH reductase

sulfur photobioreactors plastid terminal oxidase glycolaldehyde chlorophyll a + b sulfur-deprived TAP medium

capacity even after seven days. The application of larger PBR headspaces delays the establishment of a net H2 consumption activity. In the dark, addition of glycolaldehyde (GA), an inhibitor of the CBB cycle [43], delays the onset of H2 consumption activity, suggesting a crosstalk with CO2 fixation.

cultures at low light intensities resulting in balanced photosynthesis and respiration, thereby establishing hypoxia [22]. These conditions favor the accumulation of excess reducing power by the cell, which eventually triggers long-lasting H2 production at low rates, which is primarily based on a direct contribution from PSII [23]. Sustained and prolonged H2 production in the light can also be induced by depriving the cells of macronutrients. For the first time demonstrated by Melis et al. [10] in case of sulfur (S) deprived cells, following studies showed that also nitrogen, phosphorous and magnesium deprivation induce a sustained H2 metabolism [24–26]. All of these approaches have in common that they result in a decreased PET activity and to impaired or ceased growth. Thereby, two important requirements for H2 production are fulfilled, that is decreased O2 concentrations and reduced electron consumption in assimilatory processes. In the first one or two days, macronutrient deprivation stimulates the accumulation of organic reserves such as starch and lipids [10,24–27], and subsequent H2 photoevolution is typically driven by electrons derived both from the direct and indirect pathways [4,5,9,28,29]. Although H2 photoproduction by microalgae represents a sustainable way of generating this fuel gas, it has not been economically established yet. Its theoretical maximum light conversion efficiency is about 10% [1,30], but this has yet to be achieved, although several Chlamydomonas mutant strains with significantly enhanced H2 production capabilities have been described [1,30,31]. Technical issues are related to suitable photobioreactors (PBRs), regarding both design and operating principles, and the light source [32,33]. For example, an inhibiting influence of the H2 partial pressure has been shown [34]. The build-up of a H2 atmosphere is, amongst others, dependent on the bioreactor geometry in that a higher ratio of the gas phase to the cell suspension results in a more dilute gas. It has been discussed that H2 uptake mechanisms are activated in Chlamydomonas cells at H2 partial pressures > 5% [34]. Microalgae have been reported to consume H2 and utilize it either for the photoreduction of carbon dioxide (CO2) in low light or for the so-called oxy-hydrogen reaction in the dark [35–39]. Although the exact pathways are yet unknown, both reactions are proposed to involve the reduction of ferredoxin through the H2 oxidizing reaction of the hydrogenases and subsequent NADPH formation via FNR. During photoreduction, NADPH is probably consumed by the CBB cycle, while during the oxy-hydrogen reaction, NADPH or reduced ferredoxin are assumed to be re-oxidized ultimately by the plastoquinone pool. The latter can be oxidized by plastid terminal oxidase (PTOX) upon the reduction of O2 to H2O ([40] and references therein). However, also the oxy-hydrogen reduction is accompanied by a low rate of CO2 fixation [35,36,39]. An additional issue of large-scale applications for photobiological H2 production by microalgae is that the use of sunlight as a light source would be most desirable. However, analyses of outdoor H2 photoproduction using Chlamydomonas resulted in much lower H2 yields than in the laboratory, and photoinhibition due to the extremely high intensity of sunlight was discussed as one reason [33,41]. Another factor is that cultivation under natural conditions would also involve a phase of darkness in the night which might result in efficiency losses similar to reported biomass decay rates [42]. In the present study, we addressed H2 uptake phenomena and their implications for the biotechnological application of Chlamydomonas subjected to S deprivation both after prolonged incubation in the light and upon sudden darkness, and show that S-deprived cells retain H2 oxidation

2. Materials and methods 2.1. Algal strains and growth conditions Pre-cultures of Chlamydomonas reinhardtii strain CC-124 were grown photomixotrophically in standard TAP medium, pH 7.2 [44] in Erlenmeyer flasks on a rotary (Bologna) or reciprocating (Bochum) shaker. Light of 100 μmol photons·m− 2·s− 1 was provided from the top (Bologna) or from the bottom (Bochum) by cool white fluorescent lamps (Osram, Dulux L). Incident light was measured with a flat quantum radio-photometer (Skye Instruments Ltd). Cultures were kept at 20 °C (Bologna) or at 18 °C (Bochum). A mixture of air and CO2 (98.5:1.5, v:v) was provided during growth in Bologna. 2.2. H2 production and consumption experiments and measurements Cultures were collected by centrifugation (6000 g, 20 °C, 10 min) at the late exponential phase of growth (20 mg chlorophyll a + b [Chl]·L− 1), washed five times in S-free medium (TAP-S; TAP medium in which all sulfate salts were replaced by chloride salts; pH 7.2) and finally resuspended in TAP-S at an initial Chl concentration of 20 ± 2 mg·L− 1. Light was provided on two sides at 65 ± 5 μmol photons·m− 2·s− 1. The PBRs (see below) were kept at 20 °C. Experiments at low gas: liquid phase ratio were conducted in 1.2 L Roux-flask PBRs with a light path of 5 cm (depicted in [45]) (see Supplemental Fig. 1A). The liquid volume was 1.1 L and the gas phase volume was 0.1 L (i.e., a gas:liquid ratio of 0.09). Cultures were mixed using either a stir bar or an impeller. The latter enhances the photosynthetic performance of algal cultures also in S deprivation [45]. In the beginning of the incubation (0 h) the headspace of the PBR including the tubing connecting the gas collection system were flushed with N2 for 5 min. The rates of H2 gas accumulation were measured using the water displacement method described by Melis et al. [10], i.e. recording the volume of gas that accumulated over a given time period (every 4 h, three times a day). Note that these measurements of determining H2 production (termed “H2 accumulation rates” in the following) differ from the small-scale tests of algae withdrawn from the PBRs and incubated in independent vessels described below (termed “in vivo H2 production rates” in the following). Similarly, H2 consumption rates were measured considering the amount of water drawn back in the gastrap over a given time period (every 4 h, three times a day, except for specific time frames when it was recorded every 1 or 2 h), the latter being also evidence of a gas-tight collection system. The composition of the gas (H2 and, if applicable, O2) was analyzed by withdrawing gas samples from the headspace of the reactor and injecting them into a gas chromatograph (GC) (see below). Experiments at a high gas: liquid phase ratio were conducted in 325 mL square glass bottles with a light path of 5 cm sealed with gastight rubber stoppers (Suba Seals® 37, Sigma-Aldrich, www. sigmaaldrich.com) (described in [25]) (see Supplemental Fig. 1B). 342

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by measuring H2 concentration in the headspace of the vials as compared to that in blanks controls with sterile medium. In vitro hydrogenase activity was assayed as reported before [49]. This assay determines the maximal hydrogenase activity in cells lysed by Triton-X-100 and provides excess reductant (sodium dithionite) and the artificial electron donor methylviologen. It is therefore independent from physiological H2 production or uptake rates, but a measure for the total amount of active hydrogenase enzymes in the cells.

2.3. Analytical procedures Chl was extracted by an acetone:water solution (90:10, v:v) from 1 mL samples and quantified spectrophotometrically [50]. Starch was measured as reported before [51]. H2 and O2 were quantified using a Micro GC 3000A (Agilent Technologies, Italy), equipped with a PLOT column (molsieve 5 Å, 10 m by 0.32 mm) from Varian (Palo Alto, CA) and operated with N2 as the carrier gas (Bologna) or a GC-2010 from Shimadzu (www.shimadzu.de) equipped with a PLOT fused silica coating molsieve column [5 Å, 10 m by 0.32 mm] from Varian (Palo Alto, CA) operated using argon as the carrier gas (Bochum). The redox potential in the PBRs was measured with a Hamilton Liq-Glass ORP Platinum electrode.

2.3.1. Statistical analysis Results were analyzed using a nonparametric test (Mann-Whitney test) that considered a two-sided distribution with 95% confidence interval.

Fig. 1. Effect of different mixing systems (stir bar: closed symbols, impeller: open symbols) on H2 production and consumption rates (A) and the redox potential (B) in continuously illuminated S-deprived C. reinhardtii CC-124 cultures incubated in Roux-type PBRs with a low gas: liquid ratio of 0.09. The cultures had initial Chl concentrations of 20 mg L− 1, and were illuminated with 65 μmol photons m− 2·s− 1 from two sides. H2 gas accumulation and consumption rates were determined by measuring the changes of gas volumes through the water displacement method described by Melis et al. [10], while simultaneously determining the gas composition via GC. The values shown are the mean values of two independent biological replicates.

3. Results 3.1. In vivo H2 uptake in PBRs with a low gas:liquid phase ratio upon constant illumination S-deprived C. reinhardtii with an initial Chl content of 20 ± 2 mg L− 1 were incubated in PBRs with a low gas: liquid ratio (0.09) and irradiated from two sides with 65 ± 5 μmol photons m− 2·s− 1 (see Supplemental Fig. 1A). Cultures were mixed with either a stir bar or an impeller, the latter providing an improved mixing regime [45]. Enhanced mixing has the potential to increase photosynthetic yields of Sdeprived cells and lead to larger H2 productivities as compared to standard mixing attained with stir bars, particularly in dense cultures [45]. This was corroborated in the present study, because impellermixed cultures showed a maximum H2 accumulation rate per Chl content of 7.14 ± 1.30 versus 4.30 ± 1.10 nmoles H2·μg Chl− 1·h− 1 in stir bar-mixed cultures (after 40 h, Fig. 1A). The total H2 yields determined before the H2 content in the gas phase started to decrease (ca. 96 h; also see Supplemental Fig. 2A) were 240.2 ± 32.4 and 165.0 ± 52.9 nmoles H2·μg Chl− 1, or 129.2 ± 17.4 and 88.7 ± 28.5 mL·Lculture− 1, in impeller and stir bar-mixed cultures, respectively. H2 production yields attained with the different mixing regime were significantly different (p 0.046). Following the accumulation phase, both cultures initiated to consume the H2 previously produced, with cells mixed with the impeller beginning earlier (ca. 96 h after the beginning of the experiment) as compared to those mixed with the stir bar (after ca. 110 h) (Fig. 1A). After about 116 h, H2 consumption rates did not differ between cultures (− 1.32 ± 0.89 vs. − 1.30 ± 0.84 nmoles H2·μg Chl− 1·h− 1, impeller and stir bar, respectively, Fig. 1A) and stayed on this level for another 24 h. H2 uptake was accompanied by a slightly increasing redox potential, which changed from about − 525 to −425 mV between 96 and 142 h (Fig. 1B). The Chl content increased during the first 2–3 days in sulfur deprivation, and steadily declined thereafter (Supplemental Table 1).

Here, the liquid volume was 115 mL and the gas phase volume was 210 mL (i.e., a gas:liquid ratio of 1.83, ca. 20 times higher than in the previous setup). In the beginning (0 h), the flasks were purged with argon for 5 min. H2 production was assessed by measuring its concentration in the headspace. Algal samples were collected from the 325 mL flasks every 24 h and their starch content was determined. Additionally, 1-h short-term tests measuring H2 production and -uptake rates of the cells as well as in vitro hydrogenase activity was conducted in algal aliquots withdrawn from the PBR vessel. For small-scale in vivo measurements, 2-mL culture samples were transferred to anoxic 8-mL vials sealed gas-tight with red rubber Suba Seals®. For the determination of in vivo H2 production rates in the light, the cells were purged with argon for 30 s to reset the system and then incubated under constant shaking with an irradiation of 100 μmol photons·m− 2·s− 1 for 1 h at 20 °C. The H2 concentration of the headspace was then analyzed by GC. For determining the H2 uptake capacity of the cells, they were supplied with H2 and O2 at final concentration of 29 ± 7% and 2%, respectively, by purging the headspace with 100% H2 for 1 min which resulted in the above mentioned concentration of H2. O2 was introduced by injecting additional 0.4 mL (i.e., 0.08 mL O2). Half of the algal samples were treated with 10 mM glycolaldehyde (GA) shortly before incubation. GA is reported to inhibit the CBB cycle enzyme phosphoribulokinase [43,46,47] and has been applied to analyze the influence of the CBB cycle on H2 evolution by Chlamydomonas before [19,48]. Samples were incubated at 20 °C in the dark under constant shaking for 1 h. Control vessels containing only sterile TAP-S medium were used as a blank and incubated under the same conditions. H2 consumption rates by algal cells (with or without GA) were assessed 343

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Fig. 2. A: Effect of the shift from light to dark conditions on H2 accumulation and consumption rates (open squares) and redox potential (closed circles) in S-deprived C. reinhardtii CC-124 cultures incubated in PBRs with a low gas: liquid ratio. Cultures were mixed with an impeller, and illuminated with ca. 65 μmol photons m− 2·s− 1 on two sides for 50 h, after which the cells were exposed to complete darkness until the end of the experiment. H2 gas accumulation and consumption rates were measured applying the water displacement method described by [10]. B: Zoom of the values between 48 and 58 h. The values shown are the mean value of two independent biological experiments. Fig. 3. H2 metabolism of S-deprived Chlamydomonas cells in PBRs with a high gas: liquid ratio. A: H2 accumulation and consumption rates determined by measuring the H2 content of the headspace (black squares), B: in vivo H2 production rates measured in algal aliquots of 2 mL in independent vials of 8 mL (black squares) and starch content of the cells (white squares), C: in vitro hydrogenase activity measured in lysed cells upon provision of excess reductant and an artificial electron donor (grey squares). The mean values ± standard deviations are from four independent biological experiments.

3.2. In vivo H2 uptake in PBRs with a low gas:liquid phase ratio after a shift to darkness The impact of darkness on H2 oxidation was tested in impellerstirred S-deprived Chlamydomonas cells in the same PBR type and gas:liquid ratio of 0.09. The cells were first illuminated for 50 h to allow H2 to be produced and accumulated. At that time, the H2 partial pressure was 53 ± 13%, and that of O2 about 0.2 ± 0.1%, and the H2 accumulation rates had already started to decrease from their maximum (Fig. 2). Then, the light was switched off and the cells were maintained in the dark until the end of the experiment (Fig. 2). H2 accumulation rates dropped strongly and rapidly upon the shift to darkness. Within 1 h, they decreased from 5 to 0.41 nmoles H2·μg Chl− 1·h− 1, and after an additional hour, a H2 uptake rate of about −2.52 nmoles H2·μg Chl− 1·h− 1 was measured. The decrease in H2 production was accompanied by an equally sudden drop of the redox potential. For the next 6 h, a rather constant H2 consumption activity of − 1.58 ± 0.52 nmoles H2·μg Chl− 1·h− 1 was observed (Fig. 2B). This value was comparable with those previously observed at the end of the H2 consumption phase in the light using either mixing system (compare Figs. 1A and 2B). In the following two days, H2 uptake rates increased to − 2.63 ± 0.42 nmoles H2·μg Chl− 1·h− 1, and then started to decrease again. After 120 h of S deprivation, the last 70 h thereof in darkness, the algae's H2 consumption rates decreased to − 0.76 ± 0.47 nmoles H2·μg Chl− 1·h− 1 (Fig. 2A). H2 oxidation in the following days was accompanied by a slight increase in redox potential, as occurred previously in illuminated cells (compare Figs. 1B and 2A).

3.3. H2 production and uptake in PBRs with a high gas:liquid phase ratio H2 production and uptake rates of S-deprived C. reinhardtii cultures was further analyzed in PBRs providing a high gas:liquid ratio of 1.83 (see Supplemental Fig. 1B), which is about 20-fold higher than in the PBRs described above. The relative volume of the gas phase and the consequential dilution of the H2 gas has been shown to be beneficial for total H2 yields [34]. Although these PBR flasks differed from those used before, it was notable that these cultures produced 4-fold more H2 than the impeller-mixed cultures in the PBRs with the low gas:liquid ratio of 0.09 (compare maximum H2 yields of 1064 ± 76 nmoles H2·μg Chl− 1, or 477 ± 34 mL Lculture− 1 with 240.2 ± 32.4 nmoles H2 μg Chl− 1 or 129.2 ± 17.4 mL Lculture− 1). This was also reflected in H2 accumulation rates higher than those previously observed in impeller-mixed cultures (8.80 ± 1.89 nmoles H2·μg Chl− 1·h− 1, Fig. 3A). Here, H2 uptake activity started only after 144 h (Fig. 3A), with average rates of − 0.44 ± 0.76 nmoles H2·μg Chl− 1·h− 1. In this PBR system, H2 accumulation rates (measured by gas accumulation in the headspace of the PBR) and in vivo H2 production rates (determined in algae aliquots of 2 mL transferred to separate vials of 8 mL) were directly compared. In the first 48 h, the latter were significantly higher than the former (compare Fig. 3A and B). In vivo H2 344

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measured 168 h after the transfer of the cells to S-free medium (Fig. 3C). Cells incubated in PBRs with a low gas:liquid phase ratio started to consume H2 after 96–120 h of S-deprivation (Fig. 1A), while the algae in PBRs with a high gas:liquid phase ratio started about one day later (Fig. 3A). Kosourov et al. [34] reported active H2 uptake of Sdepleted and H2 producing Chlamydomonas cells after H2 was injected into the headspace to reach a concentration of ca. 30% (the exact concentrations that resulted in H2 uptake differed depending on the time-point of injection). Here, the H2 concentrations in the gas phases of the PBRs reached 65% (low gas:liquid phase ratio) and 10% (high gas:liquid phase ratio) after 72 h, and at the time point H2 consumption was detected, they were at 62.5% and 25%, respectively (Supplemental Fig. 2 A and B). This suggests that the high H2 partial pressure, probably in combination with the organic reserves within the algae becoming depleted, was a factor influencing the switch from H2 production to H2 consumption. The lower H2 concentrations in the large headspace of the second type of PBR might also explain that these cells had lower H2 uptake rates (compare Fig. 1A and Fig. 3A). In this context, it was noticeable that there was a difference between the maxima of H2 accumulation rates (measured by analyzing the H2 concentration of the headspace, Fig. 3A) and in vivo H2 production rates (measured in aliquots of the cell suspensions and initially purged with argon to reset the system, Fig. 3B). The much higher rates in the latter small-scale tests might well be due to the higher light intensity reaching each cell, because these rates were determined applying a light intensity of 100 μmol photons m− 2·s− 1 to vessels providing a much shorter light path of ca. 0.5 vs 2.5 cm in the PBR. This, in turn, suggests that the light regime experienced in the PBR did not saturate the photosynthetic H2 production capacity of the cells. An additional fact that distinguished both measurements was that H2 was flushed out from the small-scale vessels to reset the system. Kosourov et al. [34] could show that the H2 concentration in the headspace inhibits ongoing H2 accumulation, and that constant removal of H2 by argon purging resulted in higher H2 production rates especially in the first days (up to 96 h, [34]). Here, a significantly higher in vivo H2 production rate (in small vessels, after argon flushing) than H2 accumulation rate (measuring the differences in H2 amounts in the PBR system) was also observed in the beginning (up to 48 h), while they were similar afterwards (compare Fig. 3A and B). Notably, transferring cells (either the whole PBR, Fig. 2, or Chlamydomonas aliquots, Fig. 4) into darkness at a time-point at which they were still actively producing H2 resulted not only in a predictable drop of H2 accumulation rates, but also in active H2 uptake. This has

production rates and starch content showed a parallel development (Fig. 3B). After 144 h, the in vivo H2 production rate dropped to zero (Fig. 3B), while the in vitro hydrogenase activity, which reflects the total amount of active hydrogenase enzyme in the cells, remained on a relatively high level (> 325 nmoles H2·μg Chl− 1·h− 1, Fig. 3C). 3.4. Short-term H2 uptake in darkness Algal samples from this PBR setup were withdrawn from the incubation flask and further tested in short-term experiments in the dark in the presence of high H2 partial pressures (about 30%) and 2% O2. The algae's H2 uptake capacity in the dark was tested in the absence or presence of the CBB cycle inhibitor GA. As prolonged incubation in the presence of GA decreases the amount of active [FeFe] hydrogenase [19], the Chlamydomonas aliquots were only incubated for 1 h for the analysis of H2 uptake rates. Algal samples withdrawn from the illuminated PBR after 24 h of S deprivation were hardly able to produce H2 in the dark, as opposed to illuminated cells (compare 0.07 ± 0.09 nmoles H2·μg Chl− 1·h− 1 in Fig. 4 to 26.7 ± 3.8 nmoles H2·μg Chl− 1·h− 1 in Fig. 3B). Notably, GA addition to cells incubated in the dark reestablished H2 production (1.11 ± 0.16 nmoles H2·μg Chl− 1·h− 1, Fig. 4). Chlamydomonas samples incubated in the dark started to actively consume H2 after 72 h, when their in vitro hydrogenase activity was the highest (Fig. 3C), but their H2 accumulation rates (Fig. 3A) or in vivo H2 production rates (Fig. 3B) were already remarkably decreasing. H2 uptake rates in cells not treated with GA remained rather constant after 72 h until the end of the experiment (− 0.30 ± 0.17 nmoles H2·μg Chl− 1·h− 1) (Fig. 4). Algal aliquots withdrawn from the PBR at 72 h but treated with GA still produced H2 instead of consuming it. Only in cell aliquots removed after 96 h of S deprivation and later did GA addition result in a very low rate of H2 consumption (− 0.03 ± 0.02 nmoles H2·μg Chl− 1·h− 1) and rates comparable to untreated cells thereafter (p > 0.05). 4. Discussion The capability of Chlamydomonas to produce significant amounts of H2 gas upon macronutrient deprivation has been studied in much detail and from different angles since its discovery [10]. Many studies focus on process optimization in order to use this metabolic pathway for industrial applications [52,53]. The acclimation processes leading to the drastic restructuring of the photosynthetic routes are also investigated to gain insights into the metabolic flexibility of the photosynthetic microalga (e.g., [54–57]). Here, the H2 uptake activity of S-depleted Chlamydomonas cells was analyzed in PBRs providing different mixing regimes and featuring different ratios of the gas phase volume to the algal suspension volume, because both features have an influence on the H2 metabolism of the cells [34,45]. An active consumption of H2 by the algae might have significant impacts on applied approaches, and it concomitantly represents an underinvestigated facet of the integration of hydrogenases into the photofermentative metabolism of Chlamydomonas. H2 uptake could be observed in both PBR types and gas:liquid phase volume ratio regimes, respectively, after prolonged incubation or upon a shift to darkness. In both cases, H2 accumulation rates first followed the typical pattern of reaching a maximum after about two days and then decreasing to almost zero after about 100 h of S deprivation in the light (e.g., [6,52,53]). In the PBRs with a high gas: liquid phase volume ratio, the H2 yields were significantly higher. Comparably high productivities for wild type strains were observed with increasing gas:liquid phase ratios before [34]. Following the decrease of H2 accumulation rates, a net H2 uptake was observed in both PBR systems. Obviously, although the cells stopped to evolve H2 after several days, they retained the capacity to metabolize H2. The enzymatic capacity, i.e., active [FeFe]-hydrogenase was present, because in vitro hydrogenase activity could still be

Fig. 4. Effect of the incubation in the dark at high H2 partial pressures (ca. 30%) plus 2% O2 on the H2 production/consumption activity of S-deprived C. reinhardtii CC-124 cultures. Algal samples were withdrawn from the PBR described in Fig. 3 and tested for 1 h for their H2 oxidation capacity in the absence (white bars) or presence (black bars) of glycolaldehyde (GA) to inhibit the CBB cycle. Results are the mean value of experiments made in four independent replicates.

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Acknowledgements

significant implications for applied processes in natural day-night cycles, as long as the H2 is not removed from the system. The pathways that are responsible for H2 uptake reactions in S-deprived Chlamydomonas cannot be deduced from the results presented here. Light availability apparently did not represent a critical factor, because, 1) different light regimes such as those imposed by mixing did not affect H2 consumption rates in the light, and 2) steady-state H2 uptake rates in the light were comparable to those attained in the dark. This suggests that a pathway similar to the oxy-hydrogen reaction is active, which is light-independent [37,39], whereas the photoreduction of CO2 is dependent on some light [39]. The fact that GA had an influence on dark H2 production/consumption rates (Fig. 4) suggests that the CBB cycle might be operative under this condition and connected to H2 uptake. Although CBB cycle enzymes are activated in the light (e.g., [58]), very low rates of CO2 fixation were observed in the dark and ascribed to a low activity of the CBB cycle [35,37,39]. Upon S depletion, Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) can usually not be detected by immunoblot analyses after a couple of days [51,59,60], although low amounts undetectable by standard techniques may still be present. If the CBB cycle was indeed connected to the H2 uptake observed here, a continuing degradation of Rubisco would explain that in our experiments the effect of GA on H2 production/consumption decreased with time (until 96 h). However, as continued H2 oxidation was noted afterwards, the possibility that multiple pathways utilizing electrons from H2 oxidation may operate concomitantly cannot be excluded. Kessler listed various substrates that might be reduced in the presence of H2 in the dark [61]. In anoxic Chlamydomonas cells, CO2 might also be assimilated by the fermentative enzyme PFR, which is coupled to the [FeFe]-hydrogenase [11]. The enzyme has also been described as pyruvate synthase in other organisms (e.g., [62]), but this has not been studied in Chlamydomonas yet. Not at least, GA might have other effects. The substance has often been applied as a CBB cycle inhibitor [43,46,47], also in the context of analyzing the influence of CO2 fixation on the H2 metabolism of Chlamydomonas [19,48], but other effects of the chemical have been noted, too. GA is an electrophilic substance and can react with negatively charged molecules in the cell, including proteins, and it inhibits fermentation in yeast ([63] and references therein). Likewise, for the time being, we can only speculate on the biological function of H2 uptake in S-depleted Chlamydomonas cells. The cells stop H2 evolution after 3–4 days, even though reserves such as starch and lipids are still present, and this has been explained by a “wearing out” of the cells due to continuous lack of sulfate [64]. It might well be that after prolonged starvation the cells switch to a metabolism that just keeps some functions running. In the green alga Dunaliella salina, which does not have a hydrogenase, a cycling between photosynthesis and respiration has been observed upon S depletion and suggested to be sufficient for ATP generation [65]. Chlamydomonas might employ a permanent degradation and re-synthesis of carbohydrates to fuel photosynthetic electron flow downstream of PSII. In the dark, carbohydrate reserves would also be needed for heterotrophic energy generation.

AS is in debt with Prof. Fabio Fava for his support for the experiments in Bologna. We thank the European Union (Project N°: 212508) (FP7, SolarH2 Consortium) for funding the work conducted in Bochum, Germany. Authors contribution AS designed and performed the experiments, and wrote the manuscript. AH discussed the results and co-wrote the manuscript. Both the authors agree on the final version. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.algal.2017.08.018. References [1] A. Dubini, M.L. Ghirardi, Engineering photosynthetic organisms for the production of biohydrogen, Photosynth. Res. 123 (3) (2014) 241–253. [2] M. Winkler, S. Kuhlgert, M. Hippler, T. Happe, Characterization of the key step for light-driven hydrogen evolution in green algae, J. Biol. Chem. 284 (52) (2009) 36620–36627. [3] R.P. Gfeller, M. Gibbs, Fermentative metabolism of Chlamydomonas reinhardtii: I. Analysis of fermentative products from starch in dark and light, Plant Physiol. 75 (1) (1984) 212–218. [4] V. Chochois, D. Dauvillée, A. Beyly, D. Tolleter, S. Cuiné, H. Timpano, S. Ball, L. Cournac, G. Peltier, Hydrogen production in Chlamydomonas: photosystem IIdependent and -independent pathways differ in their requirement for starch metabolism, Plant Physiol. 151 (2) (2009) 631–640. [5] S. Fouchard, A. Hemschemeier, A. Caruana, J. Pruvost, J. Legrand, T. Happe, G. Peltier, L. Cournac, Autotrophic and mixotrophic hydrogen photoproduction in sulfur-deprived Chlamydomonas cells, Appl. Environ. Microbiol. 71 (10) (2005) 6199–6205. [6] A. Hemschemeier, S. Fouchard, L. Cournac, G. Peltier, T. Happe, Hydrogen production by Chlamydomonas reinhardtii: an elaborate interplay of electron sources and sinks, Planta 227 (2) (2008) 397–407. [7] C. Desplats, F. Mus, S. Cuiné, E. Billon, L. Cournac, G. Peltier, Characterization of Nda2, a plastoquinone-reducing type II NAD (P) H dehydrogenase in chlamydomonas chloroplasts, J. Biol. Chem. 284 (7) (2009) 4148–4157. [8] F. Jans, E. Mignolet, P.-A. Houyoux, P. Cardol, B. Ghysels, S. Cuiné, L. Cournac, G. Peltier, C. Remacle, F. Franck, A type II NAD(P)H dehydrogenase mediates lightindependent plastoquinone reduction in the chloroplast of Chlamydomonas, Proc. Natl. Acad. Sci. U. S. A. 105 (51) (2008) 20546–20551. [9] E. Mignolet, R. Lecler, B. Ghysels, C. Remacle, F. Franck, Function of the chloroplastic NAD(P)H dehydrogenase Nda2 for H2 photoproduction in sulphur-deprived Chlamydomonas reinhardtii, J. Biotechnol. 162 (1) (2012) 81–88. [10] A. Melis, L.P. Zhang, M. Forestier, M.L. Ghirardi, M. Seibert, Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii, Plant Physiol. 122 (1) (2000) 127–135. [11] J. Noth, D. Krawietz, A. Hemschemeier, T. Happe, Pyruvate ferredoxin oxidoreductase is coupled to light-independent hydrogen production in Chlamydomonas reinhardtii, J. Biol. Chem. 288 (6) (2013) 4368–4377. [12] R. van Lis, C. Baffert, Y. Coute, W. Nitschke, A. Atteia, Chlamydomonas reinhardtii chloroplasts contain a homodimeric pyruvate: ferredoxin oxidoreductase that functions with FDX1, Plant Physiol. 161 (1) (2013) 57–71. [13] M.L. Ghirardi, R.K. Togasaki, M. Seibert, Oxygen sensitivity of algal H2-production, Appl. Biochem. Biotechnol. 67 (1–2) (1997) 182. [14] S.T. Stripp, G. Goldet, C. Brandmayr, O. Sanganas, K.A. Vincent, M. Haumann, F.A. Armstrong, T. Happe, How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms, Proc. Natl. Acad. Sci. U. S. A. 106 (41) (2009) 17331–17336. [15] A. Baltz, K.-V. Dang, A. Beyly, P. Auroy, P. Richaud, L. Cournac, G. Peltier, Plastidial expression of type II NAD(P)H dehydrogenase increases the reducing state of plastoquinones and hydrogen Photoproduction rate by the indirect pathway in Chlamydomonas reinhardtii, Plant Physiol. 165 (1) (2014) 1344–1352. [16] T.K. Antal, T.E. Krendeleva, T.V. Laurinavichene, V.V. Makarova, A.A. Tsygankov, M. Seibert, A.B. Rubin, The relationship between the photosystem 2 activity and hydrogen production in sulfur deprived Chlamydomonas reinhardtii cells, Dokl. Biochem. Biophys. 381 (2001) 371–374. [17] B. Ghysels, D. Godaux, R.F. Matagne, P. Cardol, F. Franck, Function of the chloroplast hydrogenase in the microalga Chlamydomonas: the role of hydrogenase and state transitions during photosynthetic activation in Anaerobiosis, PLoS One 8 (5) (2013). [18] R.M. Cinco, J.M. MacInnis, E. Greenbaum, The role of carbon dioxide in light-activated hydrogen production by Chlamydomonas reinhardtii, Photosynth. Res. 38 (1) (1993) 27–33. [19] T. Rühle, A. Hemschemeier, A. Melis, T. Happe, A novel screening protocol for the

5. Conclusions The H2 production process achieved in Chlamydomonas by S deprivation is a complex interplay of electron sources and sinks [6,54,57,64,66], which all need to be addressed to support the design of proper PBRs and the establishment of physiological conditions. The present study indicates that H2 uptake by S-deprived cells can be quickly stimulated in both light and dark conditions. This further supports previous results by Kosourov et al. [34] on the effects of the H2 partial pressure, and indicates the need for counter-measures in applied large-scale systems using natural light (i.e., day-night cycles).

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