Production of Value-added Compounds from Lignocellulosic Biomass

We have become a society that is dependent on fossil resources to cover our energy and carbon needs. To maintain our current lifestyle, incredible amounts of oil, gas and coal are mined and extracted each year, releasing large quantities of greenhouse gases. If left unchanged, the already noticeable effects of climate change will likely create insurmountable problems for humanity. Several solutions have been proposed to address this issue. Behavioural changes, increasing efficiencies, alternative feedstock and energy sources, as well as many others, are proposed. It is currently agreed that a large fraction of the energy
grained from fossil resources can be replaced through electrification. However, in hard-toreplace sectors, such as certain fuels and the chemical industry, alternative carbon feedstocks will still be required. One possible source of renewable carbon is lignocellulose. Through its abundance and ability to regrow, it has the potential to serve as a building block to aid the transition from a linear, fossil-based to a circular, biobased industry. Historically, lignocellulose has played a central role in human development, serving as both building material and energy source. Entire industries have developed around the material, and its
valorisation has been extended to commodity goods such as paper, polymers, and fabric. In recent decades, lignocellulose has gained further attention as a carbohydrate feedstock for biotechnological applications, such as fermentation, thereby extending its use beyond purely chemical and mechanical methods. However, its recalcitrant structure makes its direct use impractical, introducing the need for pretreatment processes. The resulting lignocellulose hydrolysate fractions are amenable to fermentation, albeit with some remaining challenges.

This PhD project aimed to use metabolic strain engineering to improve the fermentative utilisation of such lignocellulose hydrolysates. This thesis presents the results of four projects undertaken during the PhD. They contribute to valorising the cellulose and hemicellulose fractions of lignocellulose, fractionated with the Fabiola organosolv pretreatment process. Each of the fractions displays distinct challenges addressed in the respective chapters.

Chapter 2 explores the possibility of valorising a remarkably clean cellulose hydrolysate to produce aromatic amino acids. With the help of a genome-integrated engineering tool, three chassis strains overproducing the aromatic amino acids L-tyrosine, L-phenylalanine and Ltryptophan were engineered. Comparable titer and yield of each of the amino acids was achieved from the hydrolysate compared to pure glucose, showcasing its broad applicability in fermentation.

During the work for Chapters 3 and 4, strategies for mixed substrates, such as hemicellulose hydrolysates, were investigated. For the first approach, a CRISPRi tool was used to enable co-current aerobic and anaerobic physiologies in a consortium of two strains. The consortium could efficiently valorise a xylose and glucose mixture to xylitol and isobutyric acid.

For Chapter 4, a second consortium of three strains was compared in terms of its performance to a single strain. Through niche partitioning, the efficient valorisation of the three main sugars in hemicellulose hydrolysates, xylose, glucose, and arabinose was achieved.

Acetic acid, a common growth inhibitor, was further co-utilised through an engineered acetic acid auxotrophy. After increasing tolerance to growth inhibitors present in the hydrolysate, the consortium exhibited improved yield and titer values compared to a single co-consuming strain.

Finally, for Chapter 5, the possibility of degrading phenolic growth inhibitors found in hemicellulose hydrolysate was explored. A recombinant pathway derived from soil microbes tolerant to phenolics showed promising results for the degradation of vanillin and syringaldehyde.

The obtained results provide further understanding for the efficient, fermentative valorisation of lignocellulose hydrolysates. Altogether, this thesis contributes to the fractiontailored valorisation of lignocellulose towards a more sustainable, circular economy.