High-Throughput Screening of Biological Hydrogen Production

Hydrogen (H₂) has the potential to become a sustainable fuel depending on its production process. One possible process involves biological H₂ evolution using enzymes known as hydrogenases. The maximum catalytic activity of [FeFe] hydrogenases can produce H₂ at a turn over frequency (TOF) in the order of 10,000 s^-1. However, these hydrogenases are highly sensitive to oxygen (O₂), and it is not currently possible to combine [FeFe] hydrogenases with solar water splitting where O₂ is a byproduct. For these reasons, there is great interest in using protein engineering to evolve O₂-tolerance in these enzymes. Previous work has discovered a mutant of the Clostridium pasteurianum [FeFe] hydrogenase (CpI) with enhanced O₂-tolerance during H₂ consumption. However, tolerance was not observed when the mutant was producing hydrogen. We therefore sought to develop a high-throughput assay to screen for oxygen tolerance during H₂ production. It has been previously shown that hydrogen molecules can undergo a series of reactions in thin films of Pd-Pt/WO₃ to reduce the WO₃ layer. The reduction is accompanied by a change in color. Our screen uses such a sensor to monitor hydrogen production in the individual wells of a 96-wells plate. A custom fabricated elastomeric gasket provides a seal between the sensor plate and a 96-well plate. A standard CCD camera provides time-lapse images and image analysis software gives a quantitative assessment of the rate of color formation. The device is also designed so that gases of known H₂ partial pressure can be injected below the 96-wells plate for calibration purposes. We will describe the device and show results from initial evaluation of mutated hydrogenases.