Chemo-biotechnological approaches for valorisation of mixed plastic wastes to bioplastics

Tackling the plastic waste crisis is of paramount importance for human and environmental health. Many international scientific and social organizations are working on designing a circular and sustainable plastic economy, e.g., United Nations Sustainable Development Goals, and the Ellen MacArthur foundation. This thesis focuses on chemo-biotechnological approaches to upcycle plastic waste, mixed plastics in particular, to a biodegradable polymer called polyhydroxyalkanoates [PHA] using bacteria belonging to Pseudomonas species. The findings are described in chapters 2, 3 and 4, bookended by an introduction and a general discussion. Plastic waste treatment processes like pyrolysis [heating in an oxygen free environment] on often result in a mixture rich in hydrophobic aromatic plastic monomers such as styrene, toluene, ethylbenzene. The first results chapter focuses on converting these monomers into a single water-soluble substrate, benzoate, to increase bioavailability for the synthesis of PHA in Pseudomonas putida CA-3. Batch, fed batch and continuous bioreactor cultivations at 1 litre scale were evaluated. Fed batch cultivation with a linear feed resulted in the maximum volumetric PHA productivity of 61.67 ± 7.34 mg L-1 h-1; while batch and continuous, at a dilution rate of, d = 0.2 h-1, cultivations resulted in 13.30 ± 0.01 and 4.06 ± 0.01 mg L-1 h-1 of PHA respectively. Chapter 3 focuses on developing a mixed bacterial culture for PHA production using mixed monomers under continuous cultivation. This approach was undertaken as mixed plastic waste could be depolymerized into a mixture of monomers which could be processed by a mixed culture. A mixed culture of P. putida KT2440 ΔΔP14e_dcaAKIJP, P. putida GO16 and P. putida CA-3 ALE18 capable of PHA production was developed. The target waste plastics monomers were terephthalic acid and ethylene glycol from polyethylene terephthalate and polyurethane monomers, adipic acid and 1,4-butanediol. In a 47-day continuous cultivation, the mixed culture achieved a maximum volumetric PHA productivity of 20.6 ± 1.1 mg PHA L-1 from the plastic monomers alone. Adding an additional 5 mM sodium octanoate to the plastic monomer feed, increased PHA accumulation 29.3-fold to 540.6 ± 0.3 mg PHA L-1. Chapter 4 focuses on understanding the changes in genotype that are responsible for the altered phenotype, i.e., improved growth of P. putida CA-3 on 1,4-butanediol, a polyurethane monomer following adaptive laboratory evolution. The evolved strain, P. putida CA-3 ALE18, in 1 litre scale batch bioreactor cultivation, had a growth rate of 0.24 ± 0.02 h-1 which is 3-fold higher than the growth rate of wildtype P. putida CA-3, i.e., 0.07 ± 0.11 h-1. Genomic analysis was carried out on the evolved P. putida CA-3 ALE18 and compared to the wildtype strain.