In nature, molecular hydrogen is produced by the hydrogenase and the nitrogenase enzymes. Nitrogenase reduces dinitrogen to ammonia and, in this process, it evolves hydrogen. Nitrogenase and hydrogenase are oxygen-sensitive enzymes. We chose to optimize a hydrogen production system based on nitrogenase for four reasons: some organisms carrying nitrogenase simultaneously perform photosynthesis and hydrogen evolution by nitrogenase (direct biophotolysis), thus harvesting solar energy and autonomously converting it into chemical energy in a continuous process; cellular mechanisms exist to protect nitrogenase from oxygen but do not appear to exist for hydrogenase; because nitrogenase couples ATP hydrolysis to hydrogen evolution, this enzyme is able to generate hydrogen against a substantial gas pressure; finally, the biochemistry of the nitrogenase system is well known. The objective of our proposal is to provide new eco-efficient strategies for the biological production of hydrogen. Energy research is a priority theme under the Seventh Research Framework (FP7) cooperation program. The objective of energy research under FP7 is to adapt the current energy system into a more sustainable, competitive and secure one, with emphasis and support given to hydrogen research and renewable fuel production. Our proposal has three major components: (i) in vitro evolution of nitrogenase, in which we generate new nitrogenase variants by metagenomic gene shuffling and random mutagenesis, and select those with increased hydrogen production activity; (ii) the development of a genetic system to select for hydrogen overproducers; and (iii) a biochemical element designed to understand the biochemical requisites for efficient hydrogen production by the molybdenum nitrogenase as a basis for its re-engineering.