Clostridia from a pilot plant for enhanced hydrogen gas production: isolation, molecular characterization and hydrogenase genes cloning for exploitation in biotechnology

Hydrogen is considered a promising fuel that can replace fossil fuels in the future. To this purpose, the availability of clean and efficient industrial processes is necessary and the ability of microorganisms to produce hydrogen from low cost waste materials is of high interest. In this study a pilot plant was engineered for the fermentative production of hydrogen gas using waste materials (vegetable scraps). The microbial consortium responsible for hydrogen production at the highest efficiency was characterized. Bacteria were isolated and characterized by means of 16S rDNA amplification, RFLP and sequencing, allowing the identification; the ability of each species to produce hydrogen was also tested. The consortium was composed by several non-hydrogen producing bacteria (Enterococcus sp., Lactobacillus plantarum and Staphylococcus hominis) and four species belonging to the genus Clostridium (C. butyricum, C. beijerinckii, C. perfringens and C. bifermentans), that were directly responsible for H2 production. To study the role of the different enzymes responsible for H2 production in each microorganism, C. beijerinckii and C. perfringens were selected for more detailed studies of [FeFe]-hydrogenases gene expression. Subsequently, the [FeFe]-hydrogenase gene hydA from C. perfringens was cloned and the active enzyme (CpHydA) was recombinantly expressed and affinity purified, allowing structural and functional characterization. CpHydA has the spectral features typical of [FeFe]-hydrogenases and it is able to evolve hydrogen at high turnover frequencies (kcat up to 1000 sec-1) and to perform catalysis in immobilized systems on TiO2 electrodes as electron suppliers (efficiency from to 80 to 100%). In conclusion, the isolation and characterisation of the bacterial consortium from an efficient pilot plant fed with waste materials allowed the identification and purification of a novel [FeFe]-hydrogenase that is a valuable catalyst with extended applicative potential.