Biodegradable polymer production by bacteria: a bioprocess and mathematical model assessment

The single use plastic polyethylene terephthalate (PET) has greatly contributed to the convenience of our modern lifestyle. However, its widespread use in single use plastic packaging and synthetic fibres, and recalcitrant properties are a major contributor to plastic pollution. Meanwhile, the microbially produced polyesters, polyhydroxyalkanoate (PHA) have received increased attention as a possible solution to the accumulation of plastic waste due to their biodegradable properties. The upcycling of PET waste into a material such as PHA creates an opportunity to improve both resource efficiency and contribute to a circular economy. This is possible as intermediates in the chemical recycling of PET, disodium terephthalate (Na2TA) and ethylene glycol (EG) can be harnessed as cheap microbial substrates for the cultivation of Pseudomonas umsongensis GO16 and polyhydroxyalkanoate (PHA) production. Previous studies have successfully employed Na2TA as a microbial substrate for growth and PHA accumulation. However, issues surrounding its feasibility as a microbial feedstock, such as solubility have not been tackled. This thesis presents the model-based optimisation of a liquid feeding regime with the purpose of improving the automation and operational ease of Na2TA conversion into P. umsongensis GO16 biomass. The model was parameterised and validated using data from dynamic liquid-phase and solid-phase feeding experiments. The validated model identified sodium ion accumulation as a key determinant of bioprocess performance and was used to design a liquid-phase feeding strategy that maximises P. umsongensis GO16 biomass synthesis. The obtained biomass concentrations of 10.5 g/L (liquid-phase feeding) and 15.3 g/L (solid-phase feeding) are the highest ever reported using Na2TA as a sole carbon source for microbial growth. However, even though a solid pulse feeding regime of Na2TA was successful in maximising biomass, it was found to limit PHA accumulation, achieving a 5-fold lower PHA content compared to previously achieved yields using Na2TA as the sole source of carbon and energy. Secondly, this thesis demonstrates a completely biotechnological process for upcycling of PET into PHA. PET was enzymatically hydrolysed to provide Na2TA and EG. Enzymatically hydrolysed PET was then supplied as a sole source of carbon and energy to P. umsongensis GO16. Using nitrogen limiting conditions to stimulate PHA accumulation a biomass of 1.6 g/L was achieved in 24 h with 7% of that biomass representing medium chain length PHA, which was equal to that achieved when a synthetic mixture of both monomers mimicking enzymatically hydrolysed PET was employed. Furthermore, adaptive laboratory evolution carried out on P. umsongensis GO16 was successful in improving the biomass accumulation and total PHA content of the strain when cultivated on enzymatically hydrolysed PET. Recently the genome sequence of P. umsongensis GO16 revealed that it is equipped with the genes required for both medium chain length (mcl) and short chain length (scl) PHA production. The deletion of PHA depolymerases have been known to improve PHA productivity, although its impact is not consistent across different bacterial strains and substrates employed. To date no study has investigated the impact of a PHA depolymerase gene knockout on a scl and mclPHA producer. Accordingly, the final major finding of this study was that the deletion of both scl and mclPHA depolymerases have varying effects on growth and PHA accumulation depending on the depolymerase knocked out and the substrate used. Deleting both scl and/or mclPHA depolymerases significantly impairs growth on sodium octanoate, while the deletion of the sclPHA depolymerase negatively impacted PHA accumulation in P. umsongensis GO16 while also affecting the monomer composition of the polymer with an over 4-fold increase in C4 monomer fraction and over 1.7-fold decrease in PHA content.