Cell engineering to enhance biohydrogen production from agricultural waste

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