Optimization of biohydrogen production by modifying metabolic pathways in Clostridium Thermocellum

The ever-escalating energy crisis of disruptions in the supply of fossil fuels suggests the need for alternative means with sustainable energy sources. Of these, biohydrogen stands out as promising because it is clean and efficient. Our research aims to provide strategies for fine-tuning the biohydrogen production process using the dark fermentation pathway of Clostridium. Thermocellum since it efficiently degrades cellulose under anaerobic conditions and has high-temperature tolerance, improving hydrogen yield. Sugarcane bagasse enforces this integration by providing abundant resources at little cost, which can support circular economies. However, such problems as ethanol toxicity to C. Thermocellum require genetic modifications to enhance production efficiency. The plasmid design overexpresses NADH-dependent [FeFe] hydrogenase, knocks out ethanol production pathways, and hence maximizes biohydrogen output. The design of the plasmid mainly uses Golden gate assembly and homologous recombination to achieve gene overexpression and deletion, and restriction sites were designed for virtual digestion in plasmid verification.