Integration of Biomass Conversion, Cell Factory Optimization and Bioprocess Design to Innovate the Valorization of Lignocellulosic Fibers

If humankind wants to sustain itself on planet earth, a revolution in the way we produce our food, fuels, chemicals, and materials is indispensable. In this context, the replacement of fossil fuel-driven processes by fermentation based biomanufacturing using renewable biomass is one of the key technologies we have at our disposal to realise more sustainable practices. Second-generation biorefineries are likely to play a major role in future bio-manufacturing processes as they do not compete with food crops, but use cheap, widely available and inedible lignocellulosic biomass as feedstock. Despite the vast potential of second-generation biorefineries and years of dedicated research by academia and industry, the realisation of cost-effective commercial-scale second-generation biorefineries has been hampered by interactive technical, economic, and political challenges.

This thesis aims to contribute to overcoming some of the major hurdles associated with second-generation biorefineries, being the toxicity of biomass hydrolysates and the high cost of enzymes required in the process. The main work was done from the perspective of a case study, involving an on-site enzyme production process, using DDGS biomass as a fermentation feedstock. Regardless, the main findings in the specific context can have broader implications for the use of lignocellulose.

Lab-scale experiments were performed to compare and optimise different pretreatment setups. The experimental data was used to develop and compare data- and knowledge-driven computational models to aid the optimisation of pretreatment process parameters (Chapter 2). Next, the individual and combined effects of toxic compounds commonly released during biomass pretreatment were studied for Bacillus subtilis (Chapter 3). Subsequently, adaptive laboratory evolution of B. subtilis was used as a tool to obtain strains with increased tolerance towards biomass-associated, inhibitory compounds. Moreover, evolved strains not only showed increased tolerance, but were also capable of producing significant amounts of protein using biomass hydrolysate media (Chapter 4).

Finally, the cost-effectiveness of DDGS-based on-site enzyme production was explored by performing a techno-economic assessment (Chapter 5).

The presented results lead to an increased understanding of the toxicity of biomass hydrolysates and cost-effective enzyme production. As these are two major hurdles associated with the use of lignocellulose, this thesis contributes to the advancement of future second-generation biorefineries.