Engineering Rhodopseudomonas palustris for enhanced biohydrogen production

化石燃料(石油、煤炭和天然气)在全球能源中占据主导地位，但会排放二氧化碳，从而导致气候变化。它们的开采会损害环境，而且是有限的。向更清洁、可持续的能源过渡至关重要。风能、太阳能、水能和地热等可再生能源加上替代燃料(如氢气和生物燃料)正在获得吸引力。氢气前景看好。它是碳中性的，用途广泛，有助于脱碳。然而，目前的氢气生产依赖于化石燃料，排放二氧化碳。清洁、可再生的选择，如生物氢，使用微生物从有机物中生物产生，提供更低的碳足迹、能源效率和废物管理解决方案。微生物电化学技术，特别是微生物电化学电池(MECs)，用于生物制氢。挑战包括提高产量和生产率。解决这些问题的关键在于了解参与生物制氢的微生物群落。沼泽红假单胞菌或沼泽红假单胞菌是最具吸引力和最具潜力的候选微生物之一，通常用于MEC生物制氢。它可以固定碳和氮。制氢是固氮的副产品。知识差距在于这种细菌中固氮和产氢的代谢模块是如何控制的。在这种背景下，我的项目重点是了解沼泽红假单胞菌中负责产氢的基因之间的相互作用，并对它们进行工程改造，以提高生物产氢能力。工作包1任务1.1--确定不同工业废水流的特性来自这些行业的废水将被探索并确定其有机含量的特性。这将有助于识别富含有机成分的废水溪流。任务1.2-评估不同沼泽杆菌菌株的产氢能力先前研究的沼泽杆菌菌株将被接种到不同的废水中，产氢程度将被记录下来，使我们能够确定最好的产氢菌株。工作包2任务2.1-差异表达/调控途径的鉴定最好的产氢菌株将在氮素限制或非限制条件下生长，并将接受比较蛋白质组学和转录分析。任务2.2-与产氢有关的基因/调控蛋白的验证新确定的途径组件将进行基因失活和后来的功能互补，以阐明它们在氢气生产中的作用。工作包3任务3.1-生物工程一旦确定了负责产氢的最具特征的沼泽杆菌菌株中差异表达基因的调控，潜在的候选基因将受到进一步的遗传修饰(基于质粒的过度表达、基因组整合、解除调控、缺失)，目的是提高产氢能力。工作包4--评估MEC沼泽杆菌工程菌株的产氢能力。任务4.1-MEC建造为此任务，将使用由PEM(Nafion(杜邦))隔开的两室MEC系统。任务4.2-在MEC中评估转基因沼泽红假单胞菌提高产氢能力将在上面构建的MEC中分析转基因菌株和其野生型菌株的产氢情况。工作包5任务5.1-为了提高氢气产量，MEC反应器将根据以下因素进行优化：pH、温度、电极材料。工作包6任务6.1执行生命周期评价以MEC为基础的微生物制氢的环境影响与传统方法相比，可以执行从原料利用到处置的生命周期评价

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