Developing genetically engineered Escherichia coli to biorefine lactic acid from fermented second-generation feedstocks

The exponential global population growth has led to increased consumption of finite resources like oil, gas, and coal, resulting in heightened CO2 emissions and climate change. This has led a number of dependent sectors urgently seeking and implementing sustainable alternatives. Biorefineries, which convert biomass into marketable products and green energy, are one such technology, being considered. Lactic acid (LA) is a platform chemical that can exist in one of two stereoisomers D- and L-LA. Stereospecific LA is primarily produced by fermentation as opposed to chemical synthesis and has applications in various industries, particularly in the production of bioplastics like poly lactic acid (PLA). As commercial LA production through fermentation uses first-generation feedstocks which compete with food production, second generation feedstocks are being sought as a more sustainable alternative. However, issues such as the requirement of costly pretreatments and the formation of racemic mixtures by wild fermentation, exist. To address these challenges, this thesis proposes a bacterial-based biorefining technology based on engineered Escherichia coli strains which can selectively catabolise either D- or L-LA stereoisomer, thus producing an LA solution containing a single stereoisomer from second-generation feedstocks like organic household waste and grass silage leachates. Efforts were also made to remove impurities from wild fermentation LA broths, such as short-chain fatty acids (SCFAs). Genetically modified E. coli strains were developed to selectively remove SCFAs while preserving LA by adapting a wild-type E. coli which could not use butyric acid as a sole carbon and energy source, to produce a strain which was able to remove butyric, valeric and hexanoic acid while leaving LA in solution. As SCFAs are valuable chemicals in their own right, which have uses as building blocks for an array of green chemicals; in this thesis E. coli strains were developed and assessed for their ability to biorefine specific SCFAs in synthetic leachates. While these advancements show promise, further research is needed to optimise and scale up these processes for commercial applications. Nonetheless, the strains resulting from this thesis represent an advancement in the implementation of a microbial-based lactic acid downstream process in a biorefinery context.