Synthesis of polyhydroxyalkanoates using orange juicing waste as feedstock

Abstract

The cost of polyhydroxyalkanoates (PHA) production currently hinders the wider use of these biopolymers when compare to those of their petroleum-based plastics. Polyhydroxybutyrate (PHB) was the first discovered PHA and is being widely studied over the last four decades as a potential biodegradable replacement for commonly used petroleum-based plastics. This polyester is naturally synthesised by a number of microbial strains, when grown under unbalanced nutritional conditions, such as nitrogen limitation when plenty of carbon source is available. The use of inexpensive, renewable, raw materials is proposed as a feasible strategy to make PHB economically competitive. On the other hand, the orange juicing process discards approximately 50 % (w/w) of the raw material input as waste, becoming a disposal problem for the industries involved. This thesis presents an investigation into the feasibility of a valorisation strategy for orange juicing waste (OJW), via its utilisation as feedstock for PHB production by the microbial strain Cupriavidus necator H16. This constitutes a novel valorisation strategy for OJW, aiming to add value to this material, ensuring a better exploitation of the different fractions that conform it, while at the same time attempting to alleviate the elevate PHB production costs. The characterisation of the OJW demonstrates the potential of its implementation as nutrient source for biotechnology processes, and the naturally low concentration of protein in the material, and consequent low nitrogen concentration in the medium, make it a promising raw material for PHB production. The effects of different extraction ratios and pH conditions on sugar recovery were investigated. A preliminary study showed, for the first time, that C. necator H16 reached similar growth rates using OJW-based medium with a fructose concentration of 3 g/L and a synthetic medium added with 10 g/L of the sugar, 0.15 and 0.1 h-1 respectively. Further research of the sugar extraction process led to an increase in fructose concentrations to levels above 24 g/L. The technical suitability of the OJW based medium for the production of PHB was investigated at two different scales, for the first time. Flask experiments led to a maximum biomass accumulation of 9.5 g/L with a PHB content of 76 % (w/w), corresponding to a PHB concentration of 7.3 g/L. A parallel study on the effect of the inhibitory effect of limonene, the main constituent of orange essential oil, showed that concentrations above 1% (v/v) of the terpene lead to a decrease of the growth rate of 60%, suggesting the necessity of the addition of a limonene extraction stage to the medium production process. Bioreactor experiments were performed in order to gain control over the process parameters; results under batch strategy led to similar results to those found in flask scale. A maximum biomass concentration of 9.4 g/L with a corresponding PHB concentration of 6.62 g/L, 70 % (w/w) of PHB, were achieved when pH and dissolved oxygen control were enabled. The use of a fed batch strategy led to the best results found by this study, reaching biomass and PHB concentrations of 13.3 and 9.8 g/L respectively. Overall, the feasibility of implementation of OJW as a raw material for PHB production has been demonstrated. The results obtained lead to a wider outlook that OJW has the potential to be use as feedstock of a holistic orange biorefinery, taking advantage of the different fractions of the material. Further development of the fermentation process to improve production yields and subsequent integration to the orange juice production process could well be a contribution to the development of a more sustainable industry.

Date of Award	1 Aug 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Konstantinos Theodoropoulos (Supervisor) & James Winterburn (Supervisor)