Fermentation of Sewage Sludge for Hydrogen Production: Development of Plasmids to Enhance Microbial Efficiency

The transition towards a more sustainable energy system is now a top priority, driven by the urgent need to reduce greenhouse gas emissions and dependence on fossil fuels. Hydrogen stands out as one of the most promising alternatives due to its versatility and high energy potential and clean combustion product. However, its production is still largely reliant on carbon-intensive processes, such as methane reforming, which have a significant environmental impact. For this reason, there is growing interest in more sustainable production methods, including dark fermentation, a biological process that leverages anaerobic microorganisms to convert organic substrates into hydrogen. Among the various feedstocks that can be used for dark fermentation, sewage sludge presents an interesting opportunity. As a byproduct of wastewater treatment, it is widely available and rich in organic matter, making it a suitable substrate for biological hydrogen production. However, several challenges remain, including low hydrogen yields and microbial competition, which can hinder overall efficiency. Additionally, the accumulation of unwanted secondary metabolites, such as lactate and alcohols, can further reduce process performance. The first objective of this study is to compare the hydrogen production capacity of different Clostridium strains grown in sterile sludge, non-sterile sludge, and clostridial medium (CM). Understanding these differences is essential for optimizing biohydrogen production from waste biomass and enhancing microbial efficiency in industrial applications. In the second part of this work, the strains with the highest productive performance were selected to evaluate possible metabolic engineering strategies aimed at improving hydrogen production. The first approach focused on Clostridium perfringens ATCC 13124, a microorganism with a favorable metabolism for hydrogen production but significant pathogenicity to humans. To allow safe handling in the laboratory and potential biotechnological applications, genetic strategies for inactivating the plc gene, responsible for alpha toxin production, were applied. The second approach involved Clostridium beijerinckii NCIMB 8052, a strain known for its metabolic versatility and ability to ferment a wide range of substrates. Here, the overexpression of the glyceraldehyde-3-phosphate dehydrogenase (Gapdh) enzyme was performed. Gapdh plays a crucial role in glycolysis by producing NADH, an essential cofactor for hydrogenase activity. Increasing NADH availability improved hydrogen production efficiency, enhancing the overall fermentation process. The analyses conducted in this study allowed for an evaluation of the impact of these genetic modifications and provided deeper insight into the role of key enzymes in anaerobic fermentation regulation. The results strengthen the case for biohydrogen as a renewable energy carrier and highlight the value of sewage sludge within a circular economy framework. Optimizing bacterial strains and fermentation conditions represents a crucial step toward making biohydrogen production more competitive compared to conventional methods, contributing to the transition to a more sustainable energy system.