Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii

Abstract

The work describes a novel approach for sustained photobiological production of H(2) gas via the reversible hydrogenase pathway in the green alga Chlamydomonas reinhardtii. This single-organism, two-stage H(2) production method circumvents the severe O(2) sensitivity of the reversible hydrogenase by temporally separating photosynthetic O(2) evolution and carbon accumulation (stage 1) from the consumption of cellular metabolites and concomitant H(2) production (stage 2). A transition from stage 1 to stage 2 was effected upon S deprivation of the culture, which reversibly inactivated photosystem II (PSII) and O(2) evolution. Under these conditions, oxidative respiration by the cells in the light depleted O(2) and caused anaerobiosis in the culture, which was necessary and sufficient for the induction of the reversible hydrogenase. Subsequently, sustained cellular H(2) gas production was observed in the light but not in the dark. The mechanism of H(2) production entailed protein consumption and electron transport from endogenous substrate to the cytochrome b(6)-f and PSI complexes in the chloroplast thylakoids. Light absorption by PSI was required for H(2) evolution, suggesting that photoreduction of ferredoxin is followed by electron donation to the reversible hydrogenase. The latter catalyzes the reduction of protons to molecular H(2) in the chloroplast stroma.

Figure 1

Absolute activity of oxygenic photosynthesis (P) and oxidative respiration (R) in C. reinhardtii cells suspended in a medium devoid of S. Incubation under S-deprived conditions started at 0 h. Cells were suspended in the presence of 10 mm NaHCO₃, pH 7.6. The rate of cellular respiration was recorded in the dark, followed by a measurement of the rate of light-saturated photosynthesis. Rates of photosynthesis were corrected for the rate of dark respiration.

Figure 2

A, H₂ gas volume accumulated by displacement of water in an inverted graduated cylinder as a function of cell incubation time in the absence of S. B, Quantitation of dissolved CO₂ produced in tandem with H₂ by S-deprived C. reinhardtii. The culture was sealed at about 42 h after suspension of the cells in a S-free medium. Values correspond to 1 L of culture.

Figure 3

Stage 1 → stage 2 temporal separation of photosynthetic O₂ and H₂ gas production by C. reinhardtii cells suspended in a S-free medium. Gases were collected in inverted graduated cylinders by the displacement of water. ○, O₂ (stage 1); ●, H₂ (stage 2).

Figure 4

Chl concentration, cell density, and Chl content per cell in a S-deprived C. reinhardtii culture. Initial values at t = 0 h were: Chl = 7.7 μm, Cell/mL = 2.8 × 10⁶, Chl/cell = 2.8 × 10⁻¹⁵ mol/cell.

Figure 5

Concentration of functional PSII (Q_A), cytochrome b₆-f complex (Cyt f), and PSI (P700) as a function of time in S-deprived C. reinhardtii.

Figure 6

In vivo light-induced absorbance change measurements of P700 (ΔA₇₀₀) in C. reinhardtii S-deprived for 48 h. Cells were suspended in the presence of 20 μm DCMU. The time response of the apparatus was limited through the use of electronic filters to 15 ms. Saturating blue actinic excitation (250 μmol photons m⁻² s⁻¹) came on at 100 ms (white arrow) and went off at 300 ms (black arrow).

Figure 7

Acetate (○), protein (▴), and starch (●, measured as total Glc) contents in C. reinhardtii as a function of time in the absence of S. The absolute values at zero time, corresponding to culture densities of 6 × 10⁶ cells/mL, were: acetate = 15 μmol/mL, starch = 16 nmol Glc/mL, and protein = 150 μg/mL.