Evaluation of biosensors and flow cytometry as monitoring tools in lignocellulosic bioethanol production

Abstract: The significant environmental impact of the current fossil fuel-based industry is a major concern for society. Consequently, various initiatives are being undertaken to establish a more sustainable industrial model. One example is via the transition from conventional fossil fuel refineries to biorefineries, where renewable raw materials are utilised. Amongst these raw materials, the use of lignocellulosic biomass from agricultural residues or wood has been favoured, as it does not compete with food or land resources. In particular, extensive research has been conducted to produce biofuels such as bioethanol from lignocellulosic biomass, referred to as second-generation (2G) bioethanol.In this thesis work, the goal was to develop and apply new tools to address challenges encountered in 2G bioethanol production. Specifically, the work focused on monitoring the impact of inhibitory compounds and mixed sugars on the fermentation performance of the yeast Saccharomyces cerevisiae.Inhibitory compounds are released during the pretreatment of the lignocellulosic biomass, a crucial step necessary to break down its complex structure and to enhance sugar accessibility This thesis work specifically focused on the redox imbalance induced in cells exposed to furaldehydes such as furfural or HMF. To study this effect, a biosensor for redox imbalance, TRX2p-yEGFP, was introduced into the cells and its fluorescence signal was monitored in real-time using flow cytometry. One potential strategy for enhancing the cells' tolerance to these inhibitors is to prepare them by introducing lignocellulosic hydrolysate in the feed during cell propagation. During this pre-exposure phase, a transient induction of the TRX2p-yEGFP biosensor signal for redox imbalance was observed, which gradually diminished. This indicated that, by the time of cell collection, the cells had adapted to the inhibitor concentration within the culture. To examine whether an increased induction level of the biosensor at the time of cell collection influenced the fermentation performance, an automated control system was devised. This system utilised data from the flow cytometry analysis to control the level of inhibitors in the cultivation feed. Consequently, when the biosensor signal began to decline, higher amounts of inhibitors were added, as long as the addition did not lead to an increase in the number of damaged cells.A second biosensor was used in this thesis work to investigate the sugar signalling response of S. cerevisiae to the presence of xylose. Xylose is the second most abundant sugar in lignocellulosic biomass; however, naturally, S. cerevisiae cannot metabolise it. Genetically modified S. cerevisiae strains have been generated by introducing heterologous pathways such as the XR/XDH or XI pathways to enable xylose consumption. Nevertheless, xylose consumption rates remain lower compared to glucose. Sugar signalling emerged as a potential bottleneck in the efficient utilisation of xylose. In the present work, the response of the SUC2p-yEGFP biosensor for sugar signalling was found to vary significantly depending on the pathway employed. A higher induction for the strains carrying the XI pathway was associated with poorer growth on xylose. Lastly, the effect of introducing a xylose epimerase capable of catalysing the conversion between the two anomers, α-D-xylopyranose and β-D-xylopyranose, as a strategy to improve xylose consumption was studied. The effect was enzyme-specific and proved to be particularly beneficial in strains utilising the xylose isomerase from Lachnoclostridium phytofermentans.In conclusion, the results presented in this thesis demonstrate how biosensors can facilitate the understanding and monitoring of intracellular processes that occur within the cell under stress conditions and be a key tool for improving production processes.