Light‐Driven Hydrogen Production From Water by An Oxygen‐Sensitive [NiFe] Hydrogenase in a Cyanobacterial Whole‐Cell Biocatalyst

Whole‐cell photobiocatalytic H ₂ production is constrained by hydrogenase O ₂ sensitivity, photosynthetic electron allocation, and cellular physiology. We present a systematic framework to identify and alleviate these interacting limitations using Synechocystis sp. PCC 6803 combined with Pseudomonas taiwanensis VLB120 for extracellular O ₂ microenvironment control. Rapid O ₂ diffusion over micrometer distances enabled efficient extracellular O ₂ scavenging, lowering local O ₂ exposure of the intracellular native [NiFe] hydrogenase and sustaining light‐driven H ₂ production from water. We systematically evaluated genetic, physiological, and reaction parameters affecting H ₂ production. The ndhB mutant lacking functional NDH‐1 complexes achieved the highest rates, indicating that suppressing competing electron sinks, including NDH‐1‐dependent cyclic electron flow and CO ₂ uptake, increases electron availability for H ₂ evolution. Optimization of pH, nitrogen availability, and cellular physiology enhanced performance. Under optimized conditions, H ₂ production was sustained for >72 h, reaching ~1500 µmol H ₂ g _CDW ⁻¹ , averaging ~0.35 µmol H ₂ g _CDW ⁻¹ min ⁻¹ , at least fivefold above a current Synechocystis benchmark, with estimated ~1% light‐to‐H ₂ efficiency. Inhibition of Photosystem II abolished H ₂ formation, demonstrating that water oxidation, rather than carbon metabolism, supplied the predominant electrons. Nitrogen limitation further reduced H ₂ uptake and stabilized net H ₂ accumulation. This workflow provides a transferable methodology for systematically optimizing complex whole‐cell photobiocatalytic systems.