Increased photosystem II stability promotes H2 production in sulfur-deprived Chlamydomonas reinhardtii

Abstract

Photobiological H2 production is an attractive option for renewable solar fuels. Sulfur-deprived cells of Chlamydomonas reinhardtii have been shown to produce hydrogen with the highest efficiency among photobiological systems. We have investigated the photosynthetic reactions during sulfur deprivation and H2 production in the wild-type and state transition mutant 6 (Stm6) mutant of Chlamydomonas reinhardtii. The incubation period (130 h) was dissected into different phases, and changes in the amount and functional status of photosystem II (PSII) were investigated in vivo by electron paramagnetic resonance spectroscopy and variable fluorescence measurements. In the wild type it was found that the amount of PSII is decreased to 25% of the original level; the electron transport from PSII was completely blocked during the anaerobic phase preceding H2 formation. This block was released during the H2 production phase, indicating that the hydrogenase withdraws electrons from the plastoquinone pool. This partly removes the block in PSII electron transport, thereby permitting electron flow from water oxidation to hydrogenase. In the Stm6 mutant, which has higher respiration and H2 evolution than the wild type, PSII was analogously but much less affected. The addition of the PSII inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea revealed that ∼80% of the H2 production was inhibited in both strains. We conclude that (i) at least in the earlier stages, most of the electrons delivered to the hydrogenase originate from water oxidation by PSII, (ii) a faster onset of anaerobiosis preserves PSII from irreversible photoinhibition, and (iii) mutants with enhanced respiratory activity should be considered for better photobiological H2 production.

Fig. 1.

Changes in the concentration of dissolved O₂ (filled circles) and produced H₂ (open circles) during incubation of WT (A, black circles) and the Stm6 mutant (B, red circles) of C. reinhardtii under S-deprived conditions in sealed flasks. Reflecting the O₂ and H₂ concentrations, the time course was divided into four phases: I—the O₂ evolution phase; II—the O₂ consumption phase; III—the anaerobic phase; and IV—the H₂ production phase. The blue stars in each phase indicate the time points for EPR and fluorescence experiments. The sample at t = 0 was taken as a control. (C) The effect of the addition of 20 μM DCMU on the rate of H₂ production. The aliquots of the cell culture were taken as described in the text at the time points indicated by arrows in A and B. The amount of H₂ formed in the respective strains in the absence of DCMU (blue) is set as 100%. The fraction of H₂ formed in the presence of DCMU (red) was similar in both WT and the Stm6 mutant whereas the net amount of H₂ formed was four times greater in the mutant.

Fig. 2.

EPR spectra of tyrosine D^• taken from intact cells of the WT (A) and Stm6 mutant (B) of C. reinhardtii at different stages of S deprivation: control (time = 0, black); the O₂-evolving phase (I, green); the O₂ consumption phase (II, magenta); the anaerobic phase (III, red); and the H₂ production phase (IV, blue). The spectra were normalized to the same Chl concentration. The full induction of the signal was achieved as described in ref. . EPR conditions were: microwave frequency 9.76 GHz, microwave power 8 mW, modulation amplitude of 5 G, and room temperature.

Fig. 3.

Normalized flash-induced fluorescence decay traces from WT (A) and the Stm6 mutant (B) of C. reinhardtii cells in control samples (I, black circles); samples from the anaerobic phase (III, closed red circles); samples from the anaerobic phase in the presence of 20 μM DCMU (III, open red circles); and samples from the H₂ production phase (IV, blue circles). The arrows indicate the time when the flash was applied.