Engineering Rhodopseudomonas palustris for enhanced biohydrogen production

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Fossil fuels (oil, coal, and natural gas) dominate global energy sources but emit CO2, contributing to climate change. Their extraction harms the environment and is finite. Transitioning to cleaner, sustainable energy is essential. Renewables like wind, solar, hydro, and geothermal, plus alternative fuels (e.g., hydrogen and biofuels), are gaining traction. Hydrogen is promising. It's carbon-neutral and versatile, aiding decarbonization. Current hydrogen production, though, relies on fossil fuels, emitting CO2. Clean, renewable options like biohydrogen, produced biologically from organic matter using microbes, offer lower carbon footprints, energy efficiency, and waste management solutions.Microbial electrochemical technologies, notably microbial electrochemical cells (MECs), are used for biohydrogen production. Challenges include enhancing yield and production rate. Addressing these hinges on understanding microbial communities involved in biohydrogen production.Rhodopseudomonas palustris or R. palustris is one of the most attractive and potential candidates commonly utilised in MECs for biohydrogen production. It can fix both carbon and nitrogen. Hydrogen production is the side product of nitrogen fixation. The knowledge gap lies in how the metabolic modules of nitrogen fixation and hydrogen production are controlled in this bacterium. In this context, my project focuses on understanding the interaction among the genes responsible for hydrogen production in R. palustris and engineer them for enhanced biohydrogen production. Work package 1 Task 1.1- Characterisation of different industrial wastewater streams Wastewater from such industries will be explored and characterised for organic content. This will help to identify the wastewater streams that are rich in organic content. Task 1.2- Assessment of different strains of R. palustris that have shown hydrogen productionPreviously studied strains of R. palustris will be inoculated in the different waste streams and the extent of hydrogen production will be documented, enabling us to identify the best hydrogen producing strain. Work package 2Task 2.1- Identification of differentially expressed/regulated pathways The best hydrogen producing strain will be grown under nitrogen limiting or non-limiting conditions and will be subjected to comparative proteomic and transcriptomic analysis.Task 2.2- Validation of genes/regulatory proteins involved in hydrogen production Newly identified pathway components will be subjected to gene inactivation and later functional complementation to elucidate their role in H2 production. Work package 3 Task 3.1- Biological engineering Once the regulation of differentially expressed genes in the best characterised strain of R. palustris responsible for H2 production has been identified, the potential candidate genes will be subjected to further genetic modification (plasmid- based overexpression, genome integration, deregulation, deletion) with the aim of enhancing H2 production. Work package 4- Assessing hydrogen production in engineered R. palustris' strain in MEC. Task 4.1- MEC construction For this task, a two-chambered MEC system separated by a PEM (Nafion (Du Pont) will be used. Task 4.2- Evaluation of genetically engineered R. palustris' strain in MEC for improved hydrogen production The genetically modified strain vs its wildtype will be analysed for hydrogen production in the MEC constructed above. Work package 5 Task 5.1- With the aim to achieve improved hydrogen production, the MEC reactor will be optimised on the following factors:pH, Temperature, Electrode material. Work package 6 Task 6.1 Performing LCA To assess the environmental impact of MEC-based microbial hydrogen production compared to traditional methods, an LCA, from raw material utilisation to disposal, can be executed