A new in vitro enzymatic pathway for the generation of molecular hydrogen from glucose has been demonstrated. The reaction is based on the oxidation of glucose by Thermoplasma acidophilum glucose dehydrogenase with the concomitant oxidation of NADPH by Pyrococcus furiosus hydrogenase. Stoichiometric yields of hydrogen were produced from glucose with the continuous recycling of cofactor. This simple system may provide a method for the biological production of hydrogen from renewable sources. In addition, the other product of this reaction, gluconic acid, is a high-value chemical commodity.

Using the enzymes glucose dehydrogenase (GDH) and hydrogenase, we have shown that a variety of sugars that are components of renewable resources can be enzymatically converted to molecular hydrogen. The rates at which hydrogen was evolved paralleled the substrate specificity of GDH. The highest rate of hydrogen production measured was 97.8 μmol/h, and the stoichiometric yield of hydrogen was 98% with 50 mM glucose as the substrate. Lactose, sucrose, cellulose, xylan, steam-exploded aspen wood, and starch also served as substrates for hydrogen production when the corresponding enzymes were included in the reaction mixture to generate the appropriate monosaccharide for which GDH has specificity. The data obtained are discussed in the context of the rate-limiting steps of hydrogen production from renewable sugar and the possible applications of enzymatic hydrogen production.