Production of Polyhydroxyalkanoate Copolymers by Cupriavidus necator using Spent Coffee Grounds as a Sustainable Feedstock

Abstract

Polyhydroxyalkanoates (PHAs) are biopolymers that have the potential to replace oil-derived plastics, as they are entirely biodegradable and are microbially synthesised from renewable bio-based feedstocks. However, the broader uptake of PHAs is currently being prevented by their high production costs, inconsistent material properties, and processing challenges. Chief among these issues is the cost of production, of which the carbon substrate represents a significant portion (28 – 50 %). Hence, there is substantial interest in using cheap and readily available feedstocks, such as plant oils, biodiesel by-products, and other lipid substrates. Oil extracted from spent coffee grounds – a cheap and readily available feedstock generally discarded as waste – has been demonstrated to be a highly suitable feedstock able to achieve high biopolymer yields. Throughout this thesis, Cupriavidus necator DSM 545 – a bacterium widely renowned for its ability to accumulate high levels of PHAs (≤ 90 %, w/w) from a wide array of carbon substrates – was cultivated for the first time on SCG oil and other plant oils to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) copolymers. The overall aim of the research is to improve PHA production by evaluating and optimising the different factors affecting the consumption of these lipid substrates in submerged fermentations. This thesis is presented in Journal (Alternative) Format and is split over three significant areas of study, presented as separate research articles. Initially, SCG oil was benchmarked against sunflower oil for cell growth and biopolymer production in shake flask and bioreactor (3 L) fermentations. On SCG oil, C. necator DSM 545 produced 8.9 g L−1 of total biomass, of which 89.6 % (w/w) was P(3HB-co-3HV) (6.8 mol% 3HV). In contrast, cells cultivated on sunflower oil reached 9.4 g L−1 of total biomass, with a P(3HB-co-3HV) (0.2 mol% 3HV) content fraction of 88.1 % (w/w). The production of 3HV monomers in the absence of traditional precursors is unusual and was ascribed to the activation of the succinate-propionate metabolic pathway. In doing so, the organism can divert succinyl-CoA away from a dysfunctional tricarboxylic acid (TCA) cycle into propionyl-CoA, which is a 3HV precursor. Including 3HV monomers into the biopolymer structure improves its material properties and thermal processability compared to pure P(3HB). Overall, the ability to synthesise 3HV monomers from a structurally unrelated substrate opens up the potential of eliminating the need for expensive precursor substrates typically required for copolymer synthesis, which will help to significantly reduce production costs. Second, the range of evaluated substrates was expanded to include olive, rapeseed, and sesame seed oils, and the influence of fatty and organic elemental composition on the PHA yields was assessed. During PHA production, the organism was observed to preferentially consume saturated fatty acids (palmitic acid in particular). By analysing the substrate preferences of an organism, it is possible to select the most appropriate substrates for fermentation, including those which are often neglected in industrial use, including SCGs. Among the evaluated plant oils, SCG oil produced the highest concentrations of total biomass (11.3 g L−1) and P(3HB-co-3HV) (10.0 ± 0.6 g L−1; 3.7 mol% 3HV), which was due to the high acid value and palmitic acid content of the oil. The corresponding polymer fractions (as high as 88.9 % (w/w)) are among the highest ever reported values in the literature. As plant oils are immiscible in aqueous fermentation media, they tend to float at the surface, remaining largely out of the organism’s reach. Emulsifying the oils with a surfactant (dimethyl sulfoxide) reduced the duration of the initial lag phase, which can help increase PHA volumetric productivity. Finally, the growth and PHA production of C. necator DSM 545 from SCG oil were optimised by v

Date of Award	31 Dec 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Peter Martin (Supervisor) & James Winterburn (Supervisor)