Yeast fermentation and process engineering for the upcycling of PET monomers and agricultural waste

Agriculture generates significant amounts of waste (consisting of plant or animal residues that are not processed into food or feed) and represents 3.4% of the global plastic demand. Plastic is used in mulching, covering films, nets, irrigation pipes and in post-harvesting operations, such as food packaging containers and sacks. The extensive use of plastic in the agricultural sector leads to the co-existence of many polymers, leading to serious end-of-life problems and to the accumulation of plastic waste in the environment. Among all plastic, polyethylene terephthalate (PET) was the sixth most used plastic in 2019, representing 5% of the global plastic use, and contributing to 17% of the total plastic waste from packaging (24000 tons of waste PET). Current waste PET management solutions either lead to downcycling or are not sustainable from an economic and environmental point of view. The recent advancements in enzymatic-driven recycling are paving the way for the development of new microbial processes for the upcycling of PET main hydrolysis products, ethylene glycol (EG) and terephthalic acid (TPA). While extensive research has been carried out in bacteria, very scarce knowledge is available for yeasts. In the framework of the European Horizon 2020 project Agro2Circular (A2C), this works aimed at investigating EG and TPA metabolism in yeast for the development of large-scale bioprocesses for their conversion into high value-added organic acids. In parallel, an aqueous side stream from the juicing of lemons (Lemon Extract, LE) was evaluated as a potential growth medium for the production of microbial oil. The first chapters of the thesis focus on EG metabolism in yeast. First, we report a shared ability among yeasts to oxidize EG to glycolic acid (GA) and we demonstrate the inability of Saccharomyces cerevisiae to utilize EG as a carbon source. We then propose a 10 L bioprocess with the yeast Scheffersomyces stipitis for the production of GA, by simulating a side stream of EG derived from PET hydrolysis, as described by one of the partners of the A2C network. To try to elucidate EG metabolism in yeast, the following chapters focused on metabolic engineering of S. cerevisiae and on physiology studies with the red oleaginous yeast Rhodotorula toruloides. Overall, this work is the first in-depth study on EG metabolism in yeast, and we are still moving the first steps into the elucidation of the involved pathways and their regulation. In Chapter 6 we report on our efforts for TPA upcycling with yeast, focusing on the well-known S. cerevisiae for ease of manipulation. In the first part of the study, we show that TPA is not toxic in S. cerevisiae, up to 10 g L-1, and that the efflux transporter Pdr12 might be involved in the resistance to TPA acid stress. In the second half of the chapter, we report our attempt at expressing two enzymes for the conversion of TPA into protocatechuic acid (PCA). No reports regarding the expression of TPA-DO and DCD-DH in eukaryotic are available in literature, and we are laying the ground for future improvements and new expression strategies. Lastly, Chapter 7 is dedicated to the upcycling of LE to microbial oil, exploiting the oleaginous yeast Cutaneotrichosporon oleaginosus. In this chapter we propose a 2 L bioprocess for the production of oil-rich biomass, and we develop a green sustainable extraction method which does not rely on the traditionally used toxic solvents chloroform and methanol. The work presented in this thesis wants to pose itself as a practical example of how industrial biotechnology and biomanufacturing can be used as a powerful tool to foster the transition from a petroleum-based linear economy to greener and circular manufacturing, to accomplish the overarching objective to promote a more sustainable society.