Expanding The Algal Hydrogen Toolbox: A Non-GMO Platform Reveals Multiple Physiological Routes To Sustained Hydrogen Production Across Microalgae

Sustainable hydrogen production from microalgae remains limited by intrinsic physiological constraints and the need to preserve biomass value for food and feed applications. Transgenic approaches to overcome these limitations were proven successful, yet result in genetically modified (GMO) strains that face major regulatory and deployment barriers. Here, we present a non-GMO experimental platform that enables systematic isolation of hydrogen-producing phenotypes through high-throughput UV mutagenesis pipline coupled with targeted physiological screening. Applying this approach across phylogenetically distinct algal species, including the industrial strain Chlorella vulgaris and the extremophile Chlorella ohadii, we achieve high discovery efficiency, recovering 0.4-0.6% validated hydrogen-producing mutants and achieving 6.7-25% validation rates among screen-positive candidates, indicating strong enrichment at the primary screening stage. We show that sustained hydrogen production represents a physiologically accessible state emerging across diverse genetic backgrounds. This state is consistently associated with reorganization of photosynthetic electron partitioning, yet arises through multiple distinct configurations that differentially balance hydrogen production, oxygen metabolism, and carbon fixation. This framework provides a scalable route to identify hydrogen-producing strains in industrially relevant algae without introducing foreign DNA and expands the accessible design space for photobiological hydrogen production.