Hydrogen photoproduction in green algae Chlamydomonas reinhardtii under magnesium deprivation

Abstract

The effect of Mg-deprivation on green algae Chlamydomonas reinhardtii was studied. It resulted in the decrease of photosynthetic activity, increased respiration and accumulation of starch. After 35 hours anaerobic conditions were established and sustained H₂ evolution (>7 days) was detected.

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Introduction

The quick development and technological shift to clean renewable energy sources is a global challenge. An attractive solution is the development of solar fuels as alternative energy carriers. Molecular hydrogen is one of the most promising clean fuels of the future. Oxygenic photosynthetic organisms are the most advanced users of solar energy among biological species. The remarkable evolutionary achievement of oxygenic photosynthesis is that the reductive electrons, which are necessary for growth and reproduction, are obtained from water, a ubiquitous source of electrons and an attractive substrate for solar fuel production.

Among oxygenic species cyanobacteria and green algae can photoproduce molecular hydrogen (H₂).^1–3 The reaction is catalyzed by hydrogenase enzyme⁴ and is ultimately connected to the photosynthetic electron transport chain.^3,5 Green algae possess the fastest [FeFe]-type of hydrogenase which is irreversibly inhibited by oxygen.^1,4 Sulfur (S)-deprivation is one of the commonly used methods to induce anaerobic conditions in the liquid cultures of C. reinhardtii placed in the gas-tight bioreactors.^6,7 Under these conditions, decrease of photosynthetic activity and establishment of anaerobiosis are caused mainly by decrease of the amount of PSII, O₂ producing protein complex.^8,9 The remaining PSII centers continue to serve as electron donor to hydrogenase with the residual O₂ evolution that is fully consumed by respiration.⁹

Magnesium (Mg) is a major element in any culture medium along with potassium, sulfate and phosphate and plays a key role in many physiological and metabolic processes [ref. 10–17, and references therein]. Photosynthetic reactions have an absolute requirement for this element because Mg occupies the crucial position in the photosynthetic apparatus as a constituent of the Chl molecule. Lack of Mg significantly alters photosynthetic reactions and structure of photosynthetic apparatus.^{13,14,18–24} Mg-deprivation (Mg-D) results in decrease of the photosynthetic activity, which is accompanied by decrease in the electron transport rates, increase in the amount of non Q_B-reducing centers²⁵ and reduction of the PQ-pool.²⁴ It results also in changes in photosynthetic antenna and fluorescence characteristics on both PSII and PSI.^20,26,27 The total amount of Chl always decreases under Mg-D.^{19–21,23,25,28,29}

High rate of respiration and small antenna are important factors to consider the potential H₂ production under the Mg-D conditions. The first one is a prerequisite for creation of the anaerobic state and expression of hydrogenase and the second one could increase the light utilization efficiency by increasing the light penetration in dense cultures.^30–33 In this study, we show for the first time, that cells of C. reinhardtii can photoproduce considerable amounts of H₂ gas under the Mg-D conditions. H₂ evolution takes place over the prolonged period of time of more than 7 days. This is new alternative approach to the photobiological H₂ production in green algae for the generation of H₂ based solar fuels.

Results and discussion

Mg-D results in significant metabolic changes in C. reinhardtii. 7 days in Mg-D media resulted in substantial decrease of total Chl per cell (by 60%) and increase of Chl a/b ratio (from 2.5 to 3.2, Table 1). The lower Chl b content is indicative of the decrease of LHCII during Mg-D.³⁴ Photosynthetic activity measured as F_V/F_M ratio from PSII decreased by 24% during the Mg-D if compared to the control. This result is consistent with drop in the photosynthetic O₂ evolution, measured in the presence of electron acceptors. After seven days the rate of O₂ evolution in Mg-D cells declined by 19% (Table 1). These data show decrease in the amount of functional PSII centers by ∼20%. Similar repression of the photosynthetic activity during Mg-D was reported for Chlorella vulgaris¹⁸ and reflects a combined result of the decreased activity of enzymes involved in CO₂ fixation^14,22 and repair cycle of PSII.^13,14,35 The rate of respiration, measured as O₂ consumption slightly increased by 11% after 7 days of Mg-D (Table 1). This effect is similar to the S-deprivation and is result of the lower photosynthetic activity and accumulation of the endogenous substrate (see below).

Table 1 Effect of initial pH on the physiological characteristics of aerobic C. reinhardtii culture. Cells were grown 7 days in the complete (TAP) and Mg-free (TAP-Mg) medium. Photosynthesis and respiration are shown in μmoles O₂ (mg Chl)⁻¹ × h⁻¹. Data in parenthesis were measured in the presence of 1 mM DCBQ as an electron acceptor

Parameters	Growth medium
	TAP		TAP-Mg
	pH 7.0	pH 7.7	pH 7.0	pH 7.7
pH after 7 days	7.89 ± 0.03	7.91 ± 0.01	7.86 ± 0.04	7.90 ± 0.03
Chl pg per cell	4.93 ± 0.51	4.87 ± 0.32	1.99 ± 0.24	1.91 ± 0.27
Chl a/b	2.49 ± 0.1	2.41 ± 0.1	3.16 ± 0.4	3.14 ± 0.4
Cells, 10^6 ml^−1	8.21 ± 0.2	6.24 ± 0.1	4.28 ± 0.5	3.01 ± 0.4
FV/FM	0.75 ± 0.11	0.77 ± 0.10	0.57 ± 0.31	0.58 ± 0.20
Photosynthesis	84 ± 4 (349 ± 14)	80 ± 2 (328 ± 14)	47 ± 1 (279 ± 20)	39 ± 1 (262 ± 11)
Respiration	11.2 ± 0.9	12.6 ± 0.5	12.5 ± 0.4	16.9 ± 0.5

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Elevated pH of TAP medium was shown to yield in higher H₂ production under S-deprivation.³⁶ At pH 7.7, response of photosynthetic parameters of cells to Mg-D was quite similar (Table 1). However, the respiration rate was increased by >30%. Interestingly, the O₂ evolution rate measured in the absence of electron acceptors was decreased by ∼50% at both pH values, indicating significant limitation of the electron transport after PSII (Table 1). Our flash induced fluorescence and thermoluminescence measurements show significant reduction of the PQ pool after 7 days of Mg-D in C. reinhardtii, see ESI.†

It should be mentioned, that preceding Mg-D incubation for 7 days in open cultures, was found to be necessary in order to observe the H₂ evolution by C. reinhardtii in gas tight bioreactors on the later stage. If the Mg-D culture was transferred to the bioreactors directly without the preliminary incubation, no H₂ formation was observed. The reason for this is that the Mg-D procedure is milder treatment if compared to others reported in the literature and does not inhibit photosynthesis fast enough. 7 days of the preliminary incubation was found to be optimal with respect to changes in the F_V/F_M and photosynthesis/respiration ratio in order to achieve the anaerobic state faster in the closed cultures.

When incubated in the gas-tight bioreactors, Mg-D C. reinhardtii cells undergo dramatic changes in the photosynthetic activity, and consume all produced and dissolved O₂, thus, creating anaerobic conditions. In a few hours after the onset of anaerobiosis, the H₂ production is observed under the Mg-D conditions (Fig. 1). Not surprisingly, the Mg-D culture passed through the same distinct physiological stages that were found for the S-deprived cells: the O₂ evolution phase, the O₂ consumption phase, the anaerobic phase and the H₂ production phase, Fig. 1.^9,36

Fig. 1 Changes in O₂ and H₂ concentrations in the gas phase and in the cell starch concentration during Mg-D of C. reinhardtii in gas-sealed bioreactor. White arrow indicates the time point at which aliquots of the cell culture were taken for the experiments in the presence of DCMU (see Fig. 2). The inset shows changes in the cell and Chl concentrations.

During the O₂ evolution phase, the concentration of O₂ in the gas phase was increased in the first 20 h, indicating that cells were still photosynthetically active (Fig. 1). After that the concentration of O₂ was quickly decreased (O₂ consumption phase), reaching zero level at ∼30 h and remaining at zero level until the end of the entire experiment (up to 231 h, Fig. 1). The reason for this was that the decreased O₂ evolution was completely overtaken by the O₂ consumption. The rate of O₂ evolution in the Mg-D cells after 24 h incubation in the gas-tight vials decreased by two fold (∼19 μmol O₂ (mg Chl)⁻¹ × h⁻¹) if compared with the values obtained at the beginning of the experiment. The rate of respiration however increased to ca. 20 μmol O₂ (mg Chl)⁻¹ × h⁻¹.

The appearance of H₂ in the gas phase of the glass vial was detected ∼5 h after establishment of anaerobiosis and reached the final yield of 6.3 mmol L⁻¹ of culture after about 200 h of the experiment. The Chl concentration at the beginning of experiment was ∼7 μg Chl ml⁻¹ (Fig. 1, inset). Cells produced H₂ with the increasing rate up to ∼70 h (100 h from the beginning of experiment), and then the production declined gradually thereafter. After 7–8 days the amount of produced H₂ stopped to increase and the evolution rate became negligible. Nevertheless, the maximum rate of H₂ evolution during the active phase of production, calculated on the Chl basis, was 17.2 ± 2.1 μmoles H₂ (mg Chl)⁻¹ × h⁻¹. It is nearly two times higher if compared to the H₂ evolution by the S-deprived cells (7.3 ± 1.2 μmoles H₂ (mg Chl)⁻¹ × h⁻¹, Fig. 2A). When Chl loss during Mg-D was taken into account (or comparison is made on the cell basis) the difference was not so significant (20 vs. 16 μmoles H₂ (10⁶ per cells)⁻¹ × h⁻¹, Fig. 2A). More importantly, the Mg-D cells were able to produce twice the amount of H₂ and to sustain the production for much longer than the S-deprived cells (Fig. 2B and C).

Fig. 2 Effect of Mg- and S-deprivation on the H₂ production by C. reinhardtii. The maximum rate of H₂ evolution is shown on the Chl basis in the absence and presence of 20 μM DCMU (at the time point indicated in Fig. 1) and on the cell basis (A). The normalized total yield of the H₂ production (B) and time from the beginning of the H₂ production until reaching the steady state level (C).

We have also tested how this H₂ evolution, induced by Mg-D is dependent on the PSII activity. It was shown that in the case of S-deprivation, the contribution of PSII induced electrons (i.e. electrons obtained from the water oxidation) constitute up to 80% of electrons necessary for the hydrogenase activity [ref. 9 and references therein]. We tested this by addition of DCMU, known PSII inhibitor, to the H₂ cultures after 30 h of anaerobiosis (white arrow in Fig. 1). Addition of DCMU inhibited the H₂ evolution by 74% in the Mg-D culture (Fig. 2A), similar to the 80% inhibition reported for the S-deprived culture. Thus, PSII is indispensable for the H₂ formation in C. reinhardtii also under the Mg-D conditions.

Another important source of electrons to hydrogenase is fermentation process of the endogenous substrate.⁵ The concentration of starch, measured as glucose equivalents, in cells grown in the complete medium (TAP) was around 1.6 μg glucose (10⁶ per cells). In our experiments, cells were grown in the TAP-Mg medium for a week before they have been placed in the gas-tight bioreactors (see Materials and methods). By this time (Fig. 1, t = 0 h) the concentration of starch in the cells increased to 7.6 μg (10⁶ per cells)⁻¹. During the O₂ consumption phase (Fig. 1, t = 24 h) the concentration of starch has reached the maximum of 15 μg (10⁶ per cells)⁻¹ and started to decrease. Subsequently, during the anaerobic and H₂ production phases, the starch content continued to decline to 10.4 μg (10⁶ per cells)⁻¹ at 48 h and 7.2 μg (10⁶ per cells)⁻¹ at 72 h, respectively (Fig. 1). Thus, it is clear that starch catabolism also contribute to the H₂ production under the Mg-D conditions at least in the beginning of the H₂ production phase. We can estimate this contribution to be about 25% since this was the fraction of H₂ formation which was not inhibited by the DCMU addition (Fig. 2A).

Effect of DCMU addition on the H₂ evolution under Mg-D shows that the main flow of electrons for hydrogenase activity originates from the water splitting activity by PSII.⁹ In order to access changes in the functional properties of PSII, fluorescence induction kinetics (OJIP transients) were recorded from cells removed from the bioreactor during different phases of Mg-D (Fig. 3). The kinetic traces measured in the control cells (trace a) demonstrated three well-known transients of the fluorescence changes of the active PSII.^37,38 The first transient (OJ), reflects the reduction of Q_A while the following JIP transition reflects the subsequent reduction of the PQ-pool.^37,38 Lack of Mg in the medium led to increase in both F₀ (transient O) and F_M (transient P) levels and significant changes in the shape of the induction kinetics (Fig. 3). The kinetic trace recorded in the Mg-D cells in the O₂ evolution and O₂ consumption phases (Fig. 3, traces b, c) displayed four-fold and five-fold increased O value if compared to the control. The P level was increased by 20% and 30%, respectively. The amplitude of the JIP phase, which represents the reduction of the PQ-pool,³⁸ was decreased by 30% and 70% in the O₂ evolution and O₂ consumption phases, respectively. In the anaerobic phase this value didn't exceed 14% if compared to control (Fig. 3). The shape of the induction kinetics recorded in the anaerobic phase (Fig. 3, trace d) was very similar to the kinetics measured in the presence of DCMU with JIP transients practically absent, indicating a complete block in the electron transport from Q_A⁻ to Q_B.^37,38 The kinetic trace recorded in the H₂ evolution phase (Fig. 3, trace e) displayed reappearance of the JIP transition indicating partial re-oxidation of PSII acceptor side and the PQ-pool by the electron flow to the activated hydrogenase.

Fig. 3 Effect of Mg-D on Chl fluorescence induction curves measured from C. reinhardtii cells. OJIP transients were recorded in control (a, TAP medium) and Mg-D (b, c, d, e, and f, TAP-Mg medium) cells at pH 7.7. Samples were measured aerobically in a and b (O₂ evolution phase, 3 h) and anaerobically in c (O₂ consumption phase, 21 h), d (anaerobic phase, 30 h) and e (H₂ evolution phase, 50 h), see Fig. 1. Trace f was obtained after aeration of sample from the anaerobic phase (d) for 5 min.

Interestingly, introduction of O₂ to the anaerobic culture (trace f) led to restoration of the typical OJIP transients, similar to that of the control sample (Fig. 1, trace a) within 5 min. This is another confirmation that Mg-D results in the complete reduction of the PQ-pool which is reversible when some kind of the oxidant (hydrogenase) becomes available. Presence of O₂ is known to keep the redox state of the PQ-pool in the mostly oxidized state.³⁹

Conclusions

Mg-D exerts a major effect on metabolic, physiological and biochemical processes in alga and plants affecting cell growth and development. Our study demonstrates that Mg-D alters photosynthetic activity, antenna composition and starch accumulation in C. reinhardtii. These were accompanied with the decrease in the amount of functional PSII by about 20% (Table 1) which is much less if compared to the effect of the S-deprivation where 80% decrease of the PSII centers was reported.⁹

Analysis of the functional status of these remaining after Mg-D PSII centers with the flash-induced fluorescence decay and thermoluminescence measurements (see ESI†) showed that decline in the electron transport occurred due to the limitations on the acceptor side of PSII. This conclusion is supported by the decrease in the O₂ evolution when measured in the absence of the exogenous electron acceptor (Table 1). Taken together, these data indicate to the electron transfer block at the level of PQ-pool which was induced by Mg-D already in the aerobic phase (Fig. 1).

The over-reduction of the PQ-pool was also confirmed by the OJIP transient measurements in cells taken from bioreactors. The number of non-Q_B-reducing PSII centers increased during the O₂ consumption and anaerobic phases Importantly, all fluorescence transients were restored to the control level when the PQ-pool was re-oxidized by introduction of O₂ to the cell culture (Fig. 3, trace f).

More reduced environment and anaerobiosis are necessary preconditions for the potential H₂ formation in the algal cells. Indeed, this was the case during the Mg-D treatment. We were able to detect significant amount of the H₂ gas formation by the Mg-D cultures of C. reinhardtii (Fig. 1). H₂ production was preceded by decline in the O₂ evolution rate and increase in the rate of respiration and, finally after ∼30 h, establishment of the anaerobic conditions (Table 1 and Fig. 1). The H₂ production under Mg-D was more efficient and longstanding (>7 days) if compared to other procedures (Fig. 2C).

The reason for this could be the amount of PSII centers still present when anaerobic conditions were created in the culture.⁹ The Mg-D procedure preserves more functional PSII centers than it has been demonstrated for the S- and N-deprivation.^8,9,40,41 More PSII centers imply that there are more electrons available to hydrogenase for the H₂ formation at the time point when the anaerobic conditions are created in the cell culture. This is also confirmed by experiments with the DCMU addition (Fig. 3A) which showed that 75% of the electrons, utilized by hydrogenase, were originated in the water oxidation process in PSII. Thus, we conclude that PSII is indispensable for the H₂ formation under the Mg-D conditions.

Our results show that the light-induced H₂ formation can be observed under the Mg-deprived cultivation of green algae C. reinhardtii. The H₂ production is sustained for over seven days under these conditions, which is significantly longer than under any other treatment reported so far. We also report significant involvement of PSII, a donor of electrons obtained from water, in the H₂ formation. This is new, potentially very promising approach to the photobiological solar fuel formation in green algae.

Materials and methods

C. reinhardtii wild type strain Dang 137⁺ was grown photoheterotrophically in TAP medium, pH 7.0, in Erlenmeyer flasks at 25 °C under continuous illumination (100 μE m⁻² × s⁻¹) and constant mixing. After reaching the late-logarithmic phase (9–10 × 10⁶ cells per ml), 10 ml aliquots of the culture was centrifuged at 3000 × g for 3 min, the pellet was resuspended in TAP medium without manganese (TAP-Mg, pH 7.0 or 7.7) to a final concentration of 1 × 10⁶ cells per ml. This was followed by 7 days of aerobic incubation in Erlenmeyer flasks at 25 °C under the same light and mixing conditions. TAP-Mg medium was modified from the standard TAP medium by replacing MgSO₄ to Na₂SO₄ at the same concentration. For the H₂ production experiments, the Mg-D aerobic culture (pH 7.7) was collected by centrifugation (3000 × g, 3 min), the pellet was resuspended in fresh TAP-Mg (pH 7.7) and placed into 14 ml gas-tight glass vials (bioreactors, Ø 22 mm) to a final concentration of about 4 × 10⁶ cells per ml (∼7 μg Chl ml⁻¹). The culture was incubated at 25 °C under the light intensity of 80 μE m⁻² × s⁻¹ and constant mixing for the indicated period of time. Cell concentration and Chl content was determined as in ref. 9. Starch content measurement was performed according to (ref. 42). Rates of respiration and photosynthetic O₂ evolution was measured as described in (ref. 9).

The Chl fluorescence induction (OJIP transients) was measured using Plant Efficiency Analyzer (Hansatech, UK). Red light at 650 nm at the intensity of 2000 μE m⁻² s⁻¹ was used as an excitation light. The fluorescence data were sampled at intervals of 0.1 ms for the first 2 ms, 1 ms in the range between 2 ms and 1 s, and 100 ms thereafter. The fluorescence signal at 50 ms, being the earliest measuring point free of the electronic artifacts was considered as F₀.

The amount of H₂ in the gas phase was measured using gas chromatograph Gazokhrom-2000 (Zchrom, Moscow, Russia) completed with the detector of thermoconductivity with argon as a carrier gas. 200 μl of gas sample was taken for the measurements at the indicated time points. The effect of DCMU addition (20 μM) on the H₂ production was measured as described in ref. 9 after 30 h after the anaerobic conditions have been established.

Abbreviations

Chl	Chlorophyll
DCMU	3-(34-Dichlorophenyl)-11-dimethylurea
DCBQ	26-Dichloro-p-benzoquinone
LHC	Light harvesting complex
Mg-D	Mg-deprivation
PQ	Plastoquinone
PSII	Photosystem II
QA and QB	Primary and secondary plastoquinone acceptors in PSII
TAP	Tris-acetate-phosphate

Acknowledgements

This work was supported by the Russian Federal Target Program (8077). A.V. is thankful to the Swedish Institute for the research stipend. M.F. is thankful to the Swedish Energy Agency, the Knut and Alice Wallenberg Foundation, the Carl Tryggers Foundation and the AquaFEED project from the Nordic Energy Research. We also thank Dr L. N. Davletshina for assistance in some measurements and Dr T. Antal and Prof. S. Styring for useful discussions.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra12710b

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