Strategies to enhance Clostridium thermocellum hydrogen production through hydrogenase overexpression

Biotechnologies are nowadays increasingly present in our life and their applications continue to spread through the use of biological processes, systems or organisms to create products designed to improve the quality of life. One of these products is bio-hydrogen: a promising alternative energy carrier that aligns with the principles of the circular bioeconomy. It is widely used in various industrial sectors, including petroleum refining, ammonia synthesis for fertilizers, hydrogenation processes in the chemical industry, and as a clean fuel for transportation and energy storage. However, most of the hydrogen currently produced comes from fossil fuels via steam methane reforming, a process that generates significant carbon dioxide emissions. To mitigate environmental impact and transition toward sustainable energy sources, it is crucial to develop bio-based hydrogen production strategies. For this reason, this study is focused on the application of Clostridium thermocellum, an anaerobic and cellulolytic bacterium with one of the highest lignocellulose degradation efficiencies. This study explores strategies to enhance its hydrogen yield through genetic engineering approaches. In particular, three C. thermocellum strains (LL345, LL1036, and LL1111) were analyzed to compare their native hydrogen production. Additionally, two plasmids were constructed for the overexpression of hydrogenases: one carrying the endogenous C. thermocellum hydrogenase (clo1313_0554), and the other containing the heterologous Cbeij_1773 hydrogenase gene from Clostridium beijerinckii AM2. These plasmids are intended for future transformation into C. thermocellum via electroporation. Furthermore, in silico structural studies were conducted using AlphaFold 3 and other bioinformatics tools to investigate the structure and catalytic mechanism of the wild-type hydrogenase from C. thermocellum. These findings contribute to advancing microbial biohydrogen production and support the development of engineered strains for sustainable energy applications, thus reducing dependence on fossil fuels and minimizing environmental impact.