Cell engineering to enhance biohydrogen production from agricultural waste

氢被认为是最有前途的化石燃料替代品之一，是一种可能导致脱碳的绿色能源。它的燃烧只提供水和热能作为反应产物，使其成为无污染的替代品。暗发酵（DF）是一种生物制氢方法，在厌氧条件和缺乏光线的情况下，微生物将复杂的有机物分解成产生生物氢和挥发性脂肪酸（VFAs）的简单化合物。考虑到在商业规模上使用纯碳水化合物作为底物的高成本，人们对使用可再生和更便宜的原料生产生物氢很感兴趣。每年产生超过2200亿吨的农业废弃物，使其成为可获得的可再生资源，可用作暗发酵的原料。因此，利用农业废物生产生物氢是一种循环经济方法，其中有机废物被处理以产生可再生能源，使这些底物的暗发酵既环保又经济。理论上，1摩尔葡萄糖完全氧化最多可得到12摩尔氢气。然而，通过暗发酵每摩尔葡萄糖只能得到4 mol的H2，乙酸和CO2是发酵的其他最终产物，并且在H2的颗粒压力保持足够低的情况下才能得到这个产量。理论上，在发酵制氢的产酸过程中，葡萄糖中三分之一的碳被分解为氢（H2）和二氧化碳（CO2），而其余三分之二的碳以VFAs的形式存在于酵母中，只有不到20%的化学需氧量（COD）被去除。目前，暗发酵产氢的产氢量为1.2 ~ 2.3 mol H2/mol己糖，仅为4 mol H2/mol葡萄糖理论产氢量的30 ~ 50%。生物制氢方法的H2产率低是在用于工业用途之前需要解决的主要挑战之一。在这个项目中，我们将研究哪些菌株、原料和条件最有希望用于制氢。然而，由于暗发酵潜力大但效率低，传统的方法是不够的。在过去的几年里，巨大的基因组测序、功能基因组研究、基因组规模的计算机模型的发展、代谢途径重建和合成生物学方法的可及性都有所提高。这种生物信息学和生物技术方法是提高生物氢产量的关键。本项目旨在通过对暗发酵过程中代谢途径的代谢工程，提高农业废弃物的生物制氢能力。本项目将研究以下问题：(1)哪种菌株和生物质原料更有希望用于生物制氢？为此，我们将测试文献报道的细菌菌株（希瓦氏杆菌MR-1）和从极端环境中分离的新菌株。来自农业废弃物（柳树、干草、小麦和大麦）的不同木质纤维素材料将作为原料进行测试。(2)暗发酵过程中产生生物氢的代谢途径有哪些关键点？多组学方法，考虑基因组学，转录组学，蛋白质组学和代谢组学，将采取解开这些关键点。将使用生物信息学和实验数据。(3)如何优化这一过程？为了将农业废弃物中的碳转化为生物制氢，合成生物学技术将被用于在所选菌株中进行代谢工程，以支持导致氢产量增加的代谢途径。利用实验设计（Design of Experiments, DoE）进行生物处理研究，以探索最佳工艺条件

Hydrogen is considered one of the most promising substitutes for fossil fuels, being a source of green energy that could potentially lead to decarbonization. Its combustion only delivers water and heat energy as reaction products, making it a pollution free alternative. Dark fermentation (DF) is a biological hydrogen production method in which under anaerobic conditions and absence of light, microorganisms break down complex organic matter into simpler compounds producing biohydrogen and volatile fatty acids (VFAs). Given the high cost of using pure carbohydrates as a substrate on a commercial scale, there has been a lot of interest in biohydrogen production using renewable and less expensive feedstocks. Over 220 billion tonnes of agricultural waste are generated yearly, making it an accessible renewable resource to use as feedstock for dark fermentation. Therefore, using agricultural waste for biohydrogen production is a circular economy approach in which organic waste is treated to produce renewable energy, making the dark fermentation of these substrates both environmentally and economically compelling.Theoretically, a maximum of 12 mol of H2 can be obtained from the complete oxidation of one mole of glucose. However, only 4 mol of H2 can be obtained per mole of glucose through dark fermentation, with acetate and CO2 as the other fermentation end products, and this yield is obtained when the particle pressure of H2 is kept adequately low. Theoretically, during the acidogenesis for fermentative hydrogen generation, one-third of carbon from glucose is broken down into hydrogen (H2) and carbon dioxide (CO2), while the remaining two-thirds remain soluble as VFAs in the and less than 20% of the chemical oxygen demand (COD) is removed. Nowadays, the yield of biohydrogen production by dark fermentation is between 1.2 and 2.3 mol H2/mol hexose, which is only 30-50% of the maximum theoretical production of 4 mol H2/mol glucose.The low yield of H2 by biohydrogen production methods is one of the major challenges that needs to be addressed before it can be used for industrial purpose. In this project, we will look into which strains, feedstocks and conditions are the most promising for hydrogen production. However, due to the great potential of dark fermentation but low efficiency, the conventional approach is not enough. The accessibility of huge sequenced genomes, functional genomic studies, the development of in silico models at the genome scale, metabolic pathway reconstruction, and synthetic biology approaches, has risen during the last years. This bioinformatic and biotechnological approaches hold the key for augmentation of biohydrogen production.The aim of this project is to enhance biohydrogen production from agricultural waste through metabolic engineering of the metabolic pathways involved in dark fermentation. The following questions will be investigated during this project:(1) Which strain and biomass feedstocks are more promising for biohydrogen production? For this, we will test bacterial strains reported in the literature (Shewanella oneidensis MR-1) and novel strains isolated from extreme environments. Different lignocellulosic materials from agricultural waste (willow, hay, wheat and barley) will be tested as feedstock.(2) Which are the key points in the metabolic pathways that lead to biohydrogen production during dark fermentation? A multi-omics approach, considering genomics, transcriptomics, proteomics and metabolomics, will be taken to unravel these key points. Bioinformatics and experimental data will be used. (3) How can this process be optimized? To redirect the carbons from the agricultural waste into biohydrogen production, synthetic biology techniques will be used to perform metabolic engineering in the selected strain to favour the metabolic pathway leading to increased hydrogen production. Bioprocessing studies will be done using Design of Experiments (DoE) to explore the most optimal conditio