(660c) High-Throughput Screening of Biological H2 Production

Hydrogenases, ferredoxins (Fd), and ferredoxin-NADP⁺ reductases (FNR) are redox proteins that often mediate electron metabolism in vivo. These proteins are also potential components for biological H₂ production technologies, but have not necessarily evolved for this application. Here, we report a High-throughput H₂ Production Assay Device (H₂PAD) that enables simultaneous evaluation of 96 individual H₂ production reactions. Using a CCD camera, H₂PAD senses the chemo-optical response of Pd/WO₃ thin films to H₂ produced in the individual wells of a 96-well plate. Image analysis software has been developed to interpret these time-lapse photographs. The resulting rates of color change indicate H₂ production rates by the biological assays in the wells. We used H₂PAD to discover hydrogenase and FNR mutants that can enhance biological H₂ production. From a library of 10,080 randomly mutated Clostridium pasteurianum [FeFe] hydrogenase (CpI), we found a mutant with nearly 3-fold higher H₂ production specific activity across a range of pH values. From a library of 400 semi-randomly mutated Oryza sativa (rice root) FNR (RrFNR), the top hit exhibited a 2-fold higher catalytic activity within an NADPH-driven H₂ production pathway. In addition to accelerating the pace of directed evolution in biological H₂-producing systems, H₂PAD has the potential to elucidate fundamental biochemical mechanisms of these systems. As such, it represents a tool to allow research to more rapidly meet the growing global demand for sustainable H₂.