To reduce dependence on fossil resources and move closer to a green transition, industrial value chains must shift from carbon-intensive processes to sustainable biomanufacturing methods. Integrating microbial cell factories into biorefineries will enable the valorisation of renewable and waste-derived resources into a diverse portfolio of bio-based products, supporting a circular bioeconomy framework. In this context, oleaginous microorganisms are attractive platform hosts due to their inherent capacity to accumulate increased levels of high-value microbial oil (single-cell oil, SCO) with applications in the biofuel and nutraceutical industries.

This thesis investigates how feedstock chemistry, cultivation strategies, and host metabolism affect growth, lipid accumulation, and product composition in different oleaginous platforms, specifically yeasts, microalgae, and thraustochytrids, aiming for a predictable bioprocess design. Leveraging the key phenotypes of each host, three feedstock classes were examined, namely glucose, volatile fatty acids (VFAs), and hydrophobic substrates. Bioprocess performance was evaluated on both refined and secondary sources, since waste-derived materials often introduce compounds that influence cell growth. The secondary feedstocks investigated included VFAs from the anaerobic digestion of brewer’s spent grain, hydrolysate from lignocellulosic biomass, and waste cooking oil.

The results demonstrated that strategic tuning of bioprocess conditions can redirect intracellular carbon fluxes, thereby determining the biochemical profile of the biomass. Specifically, biodiesel-grade lipids were obtained in nitrogen-limitation from yeast and microalgal cultivation on VFAs, as well as from heterotrophic microalgal cultivation on lignocellulosic hydrolysate. Odd-chain fatty acids (OCFAs), an emerging class of potential specialty lipids, were promoted in yeasts by propionate in nitrogen limitation, and in microalgae on glucose-rich and nitrogen replete conditions, coinciding with enhanced synthesis of nutritionally relevant protein. Marine thraustochytrids, recognised as prominent producers of omega-3 fatty acids, efficiently assimilated hydrophobic substrates, although DHA productivity was reduced. Therefore, to clarify the mechanisms underlying this response, transcriptomic analysis was used to investigate the regulatory pathways from carbon assimilation to lipid biosynthesis.

The findings of this thesis define evidence-based bioprocess outcomes that support the integration of microbial cell factories into biorefineries and provide an industrially relevant foundation for the targeted conversion of heterogeneous waste streams into high-value products.