To combat climate change, clean energy sources must be found. Hydrogen is a good alternative to traditional fossil fuels as it does not produce any greenhouse gases (GHGs) when combusted, however, the majority of global hydrogen production involves the use of fossil fuels. Using fossil fuels such as coal and methane to produce hydrogen releases large amounts of carbon dioxide (CO2) and methods to capture CO2 or using water electrolysis can be expensive. Biohydrogen is an attractive alternative as it does not require a fossil fuel feedstock, but in order to be commercially viable, the biohydrogen yield must first be increased.

In this thesis, hydrogen yield was increased by genetically modifying Escherichia coli. Single knockouts of genes in the mixed acid fermentation pathway were initially investigated by culturing anaerobically in sealed tubes for 48 hours. The headspace gas was then measured using a residual gas analyser. Based on the results, double gene knockouts were made. Four combinations had significantly increased hydrogen production compared to the wildtype strain. Interestingly, all double knockout combinations resulted in significantly lower CO2 production.

The best performing E. coli strains were then confined within a colloidal polymer film (called a biocoating) using a gel coagulation method with MgCl2. This method was found to preserve the highest cell viability after film formation, as was measured by the ATP concentration, and was significantly better than traditional coating methods that did not create colloidal gels. Hydrogen production from these biocoatings was measured in real-time using Unisense hydrogen microsensors and expressed as yield and rate per cell. Compared to suspension, biocoatings produced significantly higher yields of hydrogen and biocoatings also improved upon rates previously observed from photofermentative biocoatings. Overall, this work highlights the potential use of using genetically-modified E. coli confined within biocoatings for the production of more sustainable hydrogen.