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Title: Building a predictive model for PHB production from glycerol
Author: Pérez Rivero, Cristina
ISNI:       0000 0005 0289 4017
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2017
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In recent years, the potential of biological routes to replace fossil fuel-based technologies in the drive towards sustainable production of chemicals and energy has been explored and demonstrated. Biodegradable polymers derived from renewable resources could contribute to the global production of plastics (300 million tons per year) currently derived mainly from crude oil refining. However, an efficient design and optimization is required if bioprocesses are to be competitive with oil-based equivalents and describing microbial kinetics is an essential element of the necessary process development for this transition towards a bio-based economy. Polyhydroxybutyrate (PHB) is a storage compound, which accumulates in various microorganisms, with properties comparable to those of oil-derived plastics. For the case of the wild type bacterial strain Cupriavidus necator, PHB synthesis occurs in situations when there is a lack of an essential nutrient for growth but an excess of carbon. In such cases, carbon is stocked inside insoluble inclusion bodies in the form of PHB. The intracellular polymer can later be mobilized by the cell with favourable energy outcomes. This mechanism of product formation decoupled from growth challenges PHB production in simple batch systems. Thereby a careful evaluation of the feeding of nutrients is essential to enhance productivities. In this study, PHB production from glycerol by C. necator has been investigated with the objective of building a macroscopic model that could assist the process evaluation. Glycerol, an inherent by-product of the biodiesel industry, has been used throughout the research presented in this thesis as it is a potential low-cost industrial feedstock for PHB production. However, cells exhibit a long lag phase and slow growth when cultivated in glycerol for the first time. To overcome these problems, an adaptation procedure was carried out. By the 15th generation of cells grown on glycerol, the doubling time had been reduced from 22 to 1.5 h and the normal preference for glucose over glycerol (catabolic repression effect) was reversed. A further adaptation process with higher glycerol levels showed an improved tolerance up to 100 g/l, which enabled future fermentations with large amounts of glycerol. The fermentation kinetics were determined from batch studies and a relatively simple model was gradually refined and improved with each successive series of experiments. Kinetic constants extracted from the experimental data were treated as absolute constants rather than adopting best fit values each time. The resultant model, a double-substrate multiple-inhibition Monod-type set of equations, was then used to search for the best initial conditions of substrates, solve optimization problems and analyse different scenarios based on these fixed constants. The dual effect of carbon and nitrogen were examined and it was found that absolute amounts, not just ratios, determine the fate of the system. In this way, the fraction of biomass that is not PHB, called in this thesis 'associated biomass', was determined to be linearly related to the amount of nitrogen provided up to a certain level (3 g/l ammonium sulphate). Nitrogen concentrations above a low level were found to inhibit PHB formation while large excesses of carbon were channelled towards by-product (organic acids) formation. Also, the PHB production rate was seen to be influenced by total biomass concentration, to which both PHB and associated biomass contribute. Studies involving forced aeration showed that oxygen availability sets a threshold on the amount of biomass that can be formed in non-aerated systems, e.g. flasks, but did not affect the results in bioreactors as long as the dissolved oxygen was maintained above 20 % of saturation level. Using the model to predict conditions and mode of operation under which PHB production could be maximized, high density cultures (>30 g/l) with high PHB content (almost 80 %) were successfully achieved through nutrient feeding. To maintain an uninterrupted product formation rate, feeding of a complete medium with specified composition was found to be best in both experimental and computational studies. The development of the model and its later application in predicting fermentation profiles have therefore been instrumental in acquiring a macroscopic understanding of a complex system while significantly reducing experimental burden. In an industrial process, separation costs and the cost of PHB itself will ultimately determine whether it is PHB or total biomass that should be maximized. In this way, the work presented in this thesis improves the possibility of the integration of PHB production within a biodiesel plant to reduce the gap between petroleum-based chemicals and the bio-based future.
Supervisor: Webb, Colin ; Theodoropoulos, Konstantinos Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available