Balancing photosynthesis, O2 consumption, and H2 recycling for sustained H2 photoproduction in pulse-illuminated algal cultures

Abstract

Photosynthetic H₂ production in unicellular green alga Chlamydomonas reinhardtii is catalysed by O₂-sensitive [Fe–Fe]-hydrogenase (H₂ase) enzymes located in the chloroplast. The process is difficult to sustain due to (i) the inactivation of H₂ase enzymes by O₂ coevolved in photosynthesis and (ii) the competition of H₂ases with the Calvin–Benson–Bassham (CBB) cycle for photosynthetic reductants. Our previous studies revealed that H₂ production in nutrient-replete algal cultures could be sustained by applying a train of strong but short (1–5 s) light pulses interrupted by longer (3–9 s) dark periods. This limits O₂ accumulation produced by photosystem II, prevents activation of the CBB cycle and redirects photosynthetic electrons to H₂ase. In the present research, we demonstrate that the combination of strong light pulses with continuous low background illumination gives a significant gain in the net H₂ photoproduction yield by pulse-illuminated algae but only for the first 24 h. We bring evidence that the attenuation of H₂ evolution is primarily caused by the accumulation of H₂ in the headspace of vials rather than O₂ inhibition of the H₂ase, whereas an increase in the H₂ partial pressure leads to activation of H₂ recycling and noticeable H₂ uptake, which is accelerated by O₂. We predicted that sustained H₂ production in pulse-illuminated algae, which are additionally exposed to continuous low background light, could be achieved by decreasing the H₂ partial pressure in cultures and preventing excessive accumulation of O₂. Indeed, the application of periodic refreshments of a headspace atmosphere with argon and the introduction of O₂ scavenger L-cysteine allowed the H₂ photoproduction activity in algal cultures to be sustained for more than 10 days both under photoheterotrophic and photoautotrophic conditions, and yielding at least 6-times more H₂ per litre of the culture than the standard pulse-illumination protocol.