Rethinking the dark-to-light transition approach for triggering algal hydrogen photoproduction: A perspective mini-review

Hydrogen (H 2 ) production in green microalgae such as Chlamydomonas reinhardtii presents a renewable, carbon-neutral alternative to fossil-based methods. Traditionally, a dark incubation phase is used to induce anaerobiosis for activating the hydrogenase enzyme, enabling H 2 production upon illumination. However, emerging evidence suggests that this dark-to-light transition may trigger metabolic and photochemical imbalances, such as plastoquinone over-reduction, photosynthetic control mechanisms and photoinhibition that compromise H 2 yield and sustainability. In this perspective mini-review, we revisit the mechanistic rationale for the dark phase and highlight findings showing that real-time monitoring of dissolved O 2 offers a more accurate indicator of intracellular anaerobiosis than headspace-based methods. We further explore how omitting the dark phase can preserve the redox poise of the photosynthetic electron transport chain, improving H 2 production. Finally, we propose integrating these insights into existing systems to develop more photochemically stable platforms and advocate for a re-evaluation of the conventional dark-to-light transition paradigm. Highlights • Re-evaluate the assumed necessity of dark incubation for HydA activation. • Highlight dissolved O 2 as a more reliable metric of functional anoxia. • Discuss how omitting dark incubation may preserve redox homeostasis. • Explore the mechanistic basis of improved H 2 output without a dark phase.