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IntroductionDeveloping sustainable hydrogen production is critical for advancing renewable energy and reducing reliance on fossil fuels. Cyanobacteria, which harness solar energy through photosynthesis, provide a promising biological platform for hydrogen generation. However, improving hydrogen yields requires strategic metabolic and genetic modifications to optimize energy flow and overcome photosynthetic limitations. MethodsFour cyanobacterial species were evaluated for their hydrogen production capacities under varying experimental conditions. Photosynthesis was partially inhibited using distinct chemical inhibitors, including 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Exogenous glycerol was introduced as a supplementary carbon source. Hydrogen production was monitored over time, and rates were normalized to chlorophyll a content. Genomic analysis of transporter proteins was conducted to identify potential genetic loci for further enhancement of hydrogen output. ResultsNitrogen-fixingDolichospermumsp. exhibited significantly higher hydrogen production compared to the other tested species. Supplementation with glycerol notably increased both the rate and duration of hydrogen evolution, far exceeding previously established benchmarks. The maximum hydrogen production rate forDolichospermumsp. reached 132.3 μmol H₂/mg Chl a/h—representing a 30-fold enhancement over the rates observed with DCMU. Genomic screening revealed key transporter proteins with putative roles in carbon uptake and hydrogen metabolism. DiscussionThese findings underscore the potential of cyanobacteria, particularlyDolichospermumsp., as robust platforms for sustainable hydrogen production. The substantial improvements in hydrogen yield highlight the importance of targeted metabolic engineering and carbon supplementation strategies. Future work focused on optimizing identified transporter proteins and refining genetic interventions could further enhance biohydrogen efficiency. By leveraging the inherent photosynthetic machinery of cyanobacteria, this platform offers a renewable hydrogen source with significant promise for global energy sustainability. 

Serial femtosecond crystallography (SFX) is a powerful technique that exploits X-ray free-electron lasers to determine the structure of macromolecules at room temperature. Despite the impressive exposition of structural details with this novel crystallographic approach, the methods currently available to introduce crystals into the path of the X-ray beam sometimes exhibit serious drawbacks. Samples requiring liquid injection of crystal slurries consume large quantities of crystals (at times up to a gram of protein per data set), may not be compatible with vacuum configurations on beamlines or provide a high background due to additional sheathing liquids present during the injection. Proposed and characterized here is the use of an immiscible inert oil phase to supplement the flow of sample in a hybrid microfluidic 3D-printed co-flow device. Co-flow generation is reported with sample and oil phases flowing in parallel, resulting in stable injection conditions for two different resin materials experimentally. A numerical model is presented that adequately predicts these flow-rate conditions. The co-flow generating devices reduce crystal clogging effects, have the potential to conserve protein crystal samples up to 95% and will allow degradation-free light-induced time-resolved SFX. 

Abstract The world’s first superconducting megahertz repetition rate hard X-ray free-electron laser (XFEL), the European XFEL, began operation in 2017, featuring a unique pulse train structure with 886 ns between pulses. With its rapid pulse rate, the European XFEL may alleviate some of the increasing demand for XFEL beamtime, particularly for membrane protein serial femtosecond crystallography (SFX), leveraging orders-of-magnitude faster data collection. Here, we report the first membrane protein megahertz SFX experiment, where we determined a 2.9 Å-resolution SFX structure of the large membrane protein complex, Photosystem I, a > 1 MDa complex containing 36 protein subunits and 381 cofactors. We address challenges to megahertz SFX for membrane protein complexes, including growth of large quantities of crystals and the large molecular and unit cell size that influence data collection and analysis. The results imply that megahertz crystallography could have an important impact on structure determination of large protein complexes with XFELs.