High-throughput biosensor-based platform for semi-quantitative screening of hydrogen-producing microalgae

Green hydrogen (H 2 ) is a promising alternative to carbon-based fuels. However, delivering economically viable large-scale renewable H 2 production remains challenging. Green microalgae, such as Chlamydomonas reinhardtii , offer a sustainable route to H 2 production by coupling water oxidation to the photosynthetic electron transport chain. As a result, considerable effort is being devoted to improving microalgal H 2 yields through engineering of metabolic and photosynthetic pathways. Although microalgal H 2 production has been studied for over two decades, progress towards application remains constrained by the lack of rapid, reproducible and quantitative phenotypic screening tools. Conventional photobioreactor-based assays are low-throughput, labour-intensive and poorly reproducible, limiting comparative assessment across strains and conditions. We developed a cost-effective, plate-based, semi-quantitative, high-throughput assay for screening microalgal H 2 production based on an H 2 -responsive Rhodobacter capsulatus biosensor that reports H 2 via GFP fluorescence. The platform enables simultaneous assessment of H 2 -producing microalgae across multiple strains and conditions in 96-well plates, with a total workflow time of ~ 6 h from setup to data acquisition. Benchmarking the microplate assay against six well-characterised Chlamydomonas reinhardtii strains demonstrated strong concordance with photobioreactor data, while exhibiting good technical precision and biological reproducibility. Chemical-inhibitor treatments revealed pathway-specific contributions to H 2 evolution and highlighted the constraining role of PSII-derived O 2 under illuminated conditions. Importantly, the assay supports assessment of H 2 production under photoautotrophic conditions, which remain challenging to establish in standard photobioreactor workflows. This assay directly addresses the limited throughput and poor reproducibility that currently constrain phenotypic screening in microalgal H 2 research. While not a substitute for larger-scale, long-term photobioreactor-based quantitative studies, it provides a rapid, robust and small-scale upstream screening tool to prioritise strains, mutants, and experimental conditions for downstream analysis, thereby accelerating early-stage phenotypic evaluation in microalgal H 2 -production workflows. In this role, adoption of this assay provides a basis for standardising H 2 -phenotyping workflows and supporting the development of next-generation microalgal H 2 production processes.