Synthesis of polyhydroxyalkanoates (PHAs) by Cupriavidus necator using nanoemulsified oils as a carbon source

Abstract

Rising concern towards the global environmental crisis due to overuse of petroleum plastics and the rapid depletion of fossil fuels has created a renewed interest to biodegradable polymers. Polyhydroxyalkanoates (PHAs) are an example of biodegradable and biocompatible polymers, produced from renewable raw materials mainly by bacteria such as C. necator H16, a non-pathogenic strain that has genetic features to be only dedicated to PHAs and no concomitant products, also has the highest PHAs yields being 90% of cell dry weight. Nonetheless, manufacturing of biosynthesis of PHA is limited by its high cost compared with petroleum-derived plastics. Therefore, several research groups have focused on producing PHA with high productivity and yields to reduce the overall costs. One of these strategies consists in selecting inexpensive alternatives as carbon sources of the fermentation medium such as vegetable oils and supplement them after the adaptation stage of the bacterial culture. This approach has enhanced the intracellular accumulation of PHAs in bacterial cells and the thermomechanical properties of the polymer, but at the same time, the addition of insoluble oil to aqueous growth medium has led to an uneven dispersion of particles that makes this carbon source unavailable to the cells. Hence, this thesis project for a Master’s Program in Nanotechnology as a general objective is to design stable multiphase systems for the synthesis of biopolymers through the formulation of nanoemulsified oils as a strategy of medium optimization. In addition, this study aims to elucidate whether the use of nanoemulsified oils could help microorganisms to have a greater capacity for substrate consumption, due to the unique properties of nanoemulsions such as small droplet size, exceptional stability, transparent appearance and tunable rheology and consequently increase the overall productivity of PHAs and potentiate the thermomechanical properties of the polymer. In the present work, it is theoretically postulated that canola oil nanoemulsion contribution consists in the increase of the incorporation and consequently utilization rate of long-chain fatty acids (LCFA) excess presented on this nanoemulsion. Firstly, forcing Cupriavidus necator to switch to a fermentation pathway for the PHA biosynthesis, using as a fermentation-trigger, the LCFA excess and nutrient restriction (N, O, P, S, Mg). Secondly, shortening the lag time bacterium needs to liberate its biosurfactant in order to emulsify the canola oil, because an already nanoemulsion is being given to C. necator H16, then its lipases can be turned activated immediately by the nano-emulsified LCFA. Tirthly, canola oil nanoemulsion improvement comes with the LCFA incorporation-increase, thanks to the small nanoparticles which increase the total substrate area, it is justified, taking into consideration that bacteria lipases follow a no Michaelis-Menten kinetics, therefore lipase velocity rate is directly proportional to the total substrate area and do not for the substrate concentration. Regarding to the experimental approach, in the present work: formulation and characterization of emulsions were performed considering its further application as growth and PHA production media. The emulsifier that enabled kinetical stability was gum arabic even after autoclave sterilization process. Among the high energy methods used, the effect of the high-pressure disperser (Microfluidizer) on the particle size reduction was studied with respect to the rotor-stator type homogenizing disperser (Ultra-Turrax) achieving the highest (72.19 %) reduction at 4 % of oil and the lowest (38.68 %) one at 3% of oil content. In addition, the lowest particle size diameter of oil droplets was accomplished using the high-pressure disperser which was in the range of a nanoemulsion. With respect of the experimental strategy on biotechnological aspects, a first fed-batch bioreactor fermentation enabled the understanding of kinetic parameters to simulate using a Monod model, thus the next time the fructose concentration was increased 10-fold to and the experimental data adjusted to the predicted 30 g/L of biomass at 24 hours of the fermentation. Finally, the different levels of emulsification were assessed as growth media and microscopic characterization of gram stained C. necator H16 confirmed it, also the pellet area approach evaluating each stage of the biopolymer extraction gave a preliminary idea on a higher PHA production and also suggested suitable steps for the down streaming processing: biomass concentration, oil removal using ice-cold hexane, addition of a concentrated saline solution as emulsifier, and sodium hypochlorite to extract the biopolymer. This research sets a new and wide panorama on the applications of nanoemulsions in the field of biotechnology, in the future it is expected that this line of research includes the use of other microorganisms such as yeasts and fungi that utilize oil substrates. Also, the biosurfactant here explained in the proposed pathway that C. necator undergoes could be characterized.