Isolation of strains from a bio-hydrogen plant: Novel [FeFe]-hydrogenases for Exploitation in Biotechnology

Hydrogen is considered a promising fuel that can replace fossil fuels in the future, given the availability of clean and cost effective industrial processes. To this purpose, the exploitation of highly evolved and efficient enzymes such as novel [FeFe]-hydrogenases is of high interest. The microbial consortium responsible for bio-hydrogen production in a specifically engineered pilot plant was characterized. Bacteria were isolated and identified using molecular tools; the ability of each species to produce hydrogen was also tested. Four species belonging to the genus Clostridium (C. butyricum, C. beijerinckii, C. perfringens and C. bifermentans) were directly responsible for H2 production. During the growth, hydrogen evolution was monitored by gas chromatography and the expression of the various [FeFe]-hydrogenases present in C. beijerinckii (6 genes), C. butyricum (4 genes) and C. perfringens (4 genes) was studied by RT-PCR. Subsequently, the genes encoding for five novel previously uncharacterized [FeFe]-hydrogenases were cloned and recombinantly expressed in E. coli. The enzymes showing the highest activity were affinity purified under anaerobic conditions and characterized. In particular, the enzymes encoded by the two genes CPF_2655 from C. perfringens and Cbei_1773 from C. beijerinckii were able to catalyze H2 evolution at high turnover numbers. Also, CPF_2655 was tested as an immobilized catalyst on TiO2 electrodes, resulting in efficiency up to 98%. In conclusion, the isolation and characterisation of the bacterial consortium from a pilot plant allowed the identification and purification of novel [FeFe]-hydrogenases that can be exploited as valuable catalysts with an outstanding applicative potential.